Anticancer activities of novel Mannich bases against prostate cancer cells

Article abstract of DOI:10.1007/s00044-019-02426-1

This study was designed to synthesize hybridizing molecules starting from compound of 6-(4-phenyl-piperazin-1-yl)pyridine-3-ylamine by enhancing its biological activity with other heterocycles and to determine anticancer activity of the resulting compound

Full text of DOI:10.1007/s00044-019-02426-1

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02426-1
ORIGINAL RESEARCH
Anticancer activities of novel Mannich bases against prostate cancer
cells
1
Neslihan Demirbaş2
●
Serpil Demirci
Received: 17 June 2019 / Accepted: 14 August 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
This study was designed to synthesize hybridizing molecules starting from compound of 6-(4-phenyl-piperazin-1-yl)
pyridine-3-ylamine by enhancing its biological activity with other heterocycles and to determine anticancer activity of the
resulting compounds. To this end, 6-(4-phenylpiperazin-1-yl)pyridin-3-ylamine (4) was used as the leading compound,
which is known to exert anticancer activities. The synthesis of the leading compound was carried out using 1-(5-nitropyridin-
2-yl)-4-phenylpiperazine (3) which was obtained by a novel method with the reaction of N-phenylpiperazine (2) and 2-
chloro-5-nitropyridine. 6-(4-phenylpiperazin-1-yl)pyridin-3-ylamine (4) was converted to compound 5, an active
intermediate compound, by substitution of one of the amine hydrogens with ethyl bromoacetate. The resulting ester
product (5) followed by the hydrazidation (6) was added arylisocyanate to obtain the active intermediate (8). Then, by a
series of substitution through cyclization and condensation reactions, thiazolidinone (9), 1,3,4-oxadiazole (7), and 1,2,4-
triazole (10) were synthesized. Novel Mannich bases (11a–11f and 12a–12f) were obtained using oxazole (7) and triazole
(10) hetero rings with primer or secondary amine compounds. The characterization of the compounds was completed using
1
FT IR, H-NMR, 13C-NMR, HRMS spectroscopic methods and elemental analysis technique. The chemicals, then, were
tested for their anticancer activities against prostate cancer cell lines PC3 [ATCC, CRL-1435], LNCaP [ATCC, CRL-1740],
and DU145 [ATCC, HTB-81]. The results revealed that the Mannich bases exhibited moderate cytotoxic activity against
cancer cells tested.
●
●
●
Keywords Antitumor activity triazole oxadiazole Mannich reaction
Introduction
hormone-sensitive. The patients with high Gleason scores
generally acquires androgen-refractory phenotype in 1–2
years; hence, combinatorial strategy using surgical removal,
radiotherapy, and chemotherapy is generally used as last
optio (Oudard 2013; Jarvis et al. 2018). Inefficient radio-
therapy, resistance to chemotherapies and significant cyto-
toxic effect of the chemicals used in the treatment bring the
urgent necessity for safer, more efficient, and widely
available approaches.
Since the beginning of cancer therapeutics, along with
enhanced biological knowledge, synthetic chemistry has
had different roles in the anticancer agent discovery (Neidle
and Thurston 2005). In this manner, screening of anticancer
activities of newly synthesized molecules holds great
potential using ex vivo cell lines. One of the best examples
of this potential is the discovery of temozolomide with good
oral bioavailability, uptake, and distribution properties that
is a well-established anticancer drug used to treat gliomas
(Friedman et al. 2000). In the new drug active substance
screening studies, homologous serial design, chain
Prostate cancer (PC) constitutes of nearly 29% of all newly
diagnosed cancer incidents and is the most common cancer
type among men. In 2019, 174,650 new cases are expected
and ~31,620 will die due to PC in US (Siegel et al. 2019).
For clinically localized cancer situations, radiotherapy and
surgery are thought to be the main approaches. However,
patients with advanced and metastatic cancer states are
treated with androgen deprivation therapy if the cancer is
* Serpil Demirci
demirciserpil17@gmail.com
1
Vocational High School of Health Services, Department of
Medical Services and Techniques, Giresun University, 28100
Giresun, Turkey
2
Department of Chemistry, Karadeniz Technical University, 61080
Trabzon, Turkey

Medicinal Chemistry Research
branching, chain-ring transformations, bioisosteric altera-
tions are generally used to identify new analogs with an
augmented therapeutic effect and index compared with a
leader compound. The structural components of these
leading molecules, which interact with the receptors and
are, therefore, responsible for their biological activities,
form their pharmacophores. Once it is defined, various
variations on these pharmacophores are expected to lead
more effective and less toxic analogs than the leading
compound (Silverman et al. 1993).
The Mannich reactions occurs in a single reaction with
three components have become one of the most prominently
used methods in drug developments due to allowing the
synthesis of organic molecules containing different func-
tional groups in a single step. In recent years, screening for
anticancer molecules with stronger activities among Man-
nich bases has become popular (Roman 2015). The struc-
tures of oxadiazole and triazole derivatives containing
active –SH groups allow transferring functional groups to
the leading compounds that increase the biological activity
as a result of aminoalkylation by the Mannich reaction
(Shivarama Holla et al. 2003; Li et al. 2009). Various types
of Mannich bases have been presented in the literature to be
highly reactive and possess various biological roles
including antimicrobial (Idhayadhulla et al. 2014), antic-
ancer (Shivarama Holla et al. 2003), analgesic and anti-
inflammatory (Gökçe et al. 2005), anticonvulsant
(Dimmock et al. 1992), and antimalarial activities (Lopes
et al. 2004). Mannich bases have been evaluated for their
cytotoxicity against lung cancer (Malhotra et al. 2012),
leukemia (Hu et al. 2012), hepatocarcinoma, and breast
cancer cell lines (Kumbhare et al. 2011).
(MI, USA) elemental analyzer. The mass spectra were
obtained at a LC-MS TOF (1200/6210, Agilent) by elec-
trospray ionization.
1-(5-Nitropyridin-2-yl)-4-phenylpiperazine (3)
2-Chloro-4-nitropyridine (2) (0.63 g, 4 mmol) was added
dropwise to N-phenylpiperazine (1) (1.62 g, 10 mmol) at
0–5 °C over 20 min. The temperature was gradually raised
to 135 °C and refluxed for 4 h. The solid obtained upon
adding water into the mixture was filtered off. Crystal-
lization with EtOAc-hexane (1:1) gave a pale brown pro-
duct. Yield, 60% (1.68 g); mp, 155–156 °C; 1H-NMR (400
MHz, CDCl3): δ 9.06 (d, 1H, H-3, J: 2.7 Hz), 8.23 (dd, 1H,
H-5, 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, 2CH2 J:5.14 Hz), 3.42
(d, 4H, 2CH2 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.
6-(4-Phenylpiperazin-1-yl)pyridin-3-amine (4)
The solution of compound 3 in (2.85 g, 10 mmol) n-butanol
in a two necked flask was refluxed in the presence of Pd/C
(0.53 g, 5 mmol) in an oil bath until refluxing started. Then,
hydrazine hydrate (2.5 g, 4.04 mL, 50 mmol) was added
dropwise over 20 min. The mixture was refluxed for 15 h.
The hot reaction mixture was filtered through the celite. The
solvent was removed by evaporation. Crystallization with
EtOAc-hexane (1:1) 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, 2CH2),
3.26–3.16 (m, 4H, 2CH2); 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.
In the present study, novel Mannich bases characterized
1
by elemental analysis, IR, H- and 13C-NMR spectral data
were synthesized and tested for their anticancer activities
against PC cell lines; DU145, PC3, and LNCaP.
Materials and methods
Synthesis
All chemicals were purchased from Fluka Chemie 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. Reac-
tions were monitored by thin-layer chromatography (TLC)
on silica gel 60 F254 aluminum sheets. FT IR spectra were
recorded using a Mattson 1000 FT IR spectrometer.
1H-NMR and 13C-NMR spectra were recorded on Bruker
Avance 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
Ethyl (6-(4-phenylpiperazin-1-yl)pyridin-3-yl)
glycinate (5)
To the solution of the corresponding compound 4 (2.54 g,
10 mmol) in tetrahydrofuran, triethylamine (1.4 ml, 1 g,
10 mmol), and ethyl bromoacetate (1.13 ml, 1.67 g,
10 mmol) were added at 0 °C and the mixture was stirred at

Medicinal Chemistry Research
60 °C for 20 h. The precipitate was removed by filtration
and the resulting solution was evaporated under reduced
pressure to dryness. The solid obtained recrystallized from
EtOH to give the pure compound. Light yellow solid. Yield,
(d, 2H, J: 8.0 Hz), 6.83–6.78 (m, 2H), 5.96 (s, 1H, NH),
4.39 (bs, 2H, CH2), 3.42 (bs, 4H, 2CH2), 3.22 (bs, 4H,
2CH2); APT (13C-NMR)(100 MHz, DMSO-d6): δ 177.88,
162.24, 151.93, 150.91, 136.57, 136.38, 128.93, 125.13,
119.10, 115.66, 109.57, 48.08, 46.05, 38.38; FT IR
(cm–1): 3063 (ar-CH), 1228 (C=S); Elemental analysis
for C18H20N6OS calculated: C, 58.68; H, 5.47; N, 22.81; S,
8.70 found: C, 58.63; H, 5.45; N, 22.84; S, 8.69; HRMS
(APCI): m/z C18H20N6OS calculated (M+ + H): 369.46;
found: 369.15.
1
90% (3.03 g); mp,137 °C; H-NMR (400 MHz, CDCl3): δ
7.75 (s, 1H), 7.28 (t, 2H, J: 7.8 Hz), 7.00–6.97 (m, 3H),
6.88 (t, 1H, J: 7.3 Hz), 6.67 (d, 1H, J: 8.9 Hz), 4.24 (q, 2H,
OCH2, J: 7.1 Hz), 3.86 (s, 2H, CH2), 3.55–3.52 (m, 4H,
2CH2), 3.32–3.31 (m, 4H, 2CH2), 1.29 (t, 3H, CH3, J:
7.1 Hz); APT (13C-NMR)(100 MHz, CDCl3): δ 171,34,
154.54, 151.60, 136.59, 133.61, 129.36, 124.70, 120.16,
116.57, 109.04, 61.68, 49.49, 46.96, 45.19, 14.42; FT IR
(cm–1): 3386 (NH), 2977 (ar-CH), 1724 (C=O); Elemental
analysis for C19H24N4O2 calculated: C, 67.04; H, 7.11; N,
16.46; found: C, 67.19; H, 7.07; N, 16.14; HRMS (APCI):
m/z calculated C19H24N4O2 (M+ + H): 341.43; found:
341.19.
N-phenyl-2-((6-(4-phenylpiperazin-1-yl)pyridin-3-yl)
glycyl)hydrazine-1-carbothioamide (8)
Phenylisothiocyanate (10 mmol) was added to the solution
of compound 6 (10 mmol) in dried DCM dropwise and the
mixture was refluxed for 8 h. The solid precipitate was
collected by filtration and recrystallized from EtOAc to give
the pure compound. Dark pink solid product. Yield 85%
2-((6-(4-Phenylpiperazin-1-yl)pyridin-3-yl)amino)
acetohydrazide (6)
1
(2.35 g); mp, 197 °C; H-NMR (400 MHz, DMSO-d6): δ
10.11 (s, 1H, NH), 9.67 (bs, 1H, NH), 9.53 (bs, 1H, NH),
7.67 (s,1H), 7.44–7.42 (bs, 2H), 7.35 (t, 2H, J: 7.4 Hz),
7.26–7.18 (m, 3H), 6.99–7.06 (m, 3H), 6.80 (t, 2H, J:
9.7 Hz), 5.51 (s,1H, NH), 3.80 (bs, 2H, CH2), 3.41 (bs, 4H,
2CH2), 3.23 (bs, 4H, 2CH2); APT (13C-NMR) (100 MHz,
DMSO-d6): δ 160.43, 158.94, 153.24, 151.57, 139.21,
137.39, 132.62, 129.41, 123.91, 122.17, 119.52, 117.36,
116.15, 109.13, 48.71, 46.70, 38.81; FT IR (cm–1): 3336,
3243 (4NH), 3083 (ar-CH), 1693 (C=O), 1230 (C=S);
Elemental analysis for C24H27N7OS calculated: C, 62.45;
H, 5.90; N, 21.24; S, 6.95 found: C, 62.39; H, 5.60; N,
21.29; S, 6.61; HRMS (APCI): m/z C24H27N7OS calculated
(M+ + H): 462.59; found: 462.20.
A solution of compound 5 (3.4 g, 10 mmol) in ethanol was
refluxed with hydrazine hydrate (25 mmol, 1.21 ml, 1.25 g,
25 mmol l) for 13 h. The resulting solution was evaporated
under reduced pressure to dryness. The solid recrystallized
from EtOAc to give the pure compound. Light yellow solid.
Yield, 88% (2.86 g); mp,154–155 °C; 1H-NMR (400 MHz,
DMSO-d6): δ 9.11 (s, NH), 7.60 (d, 1H, J: 2.4 Hz), 7.23
(t, 2H, J: 7.7 Hz), 7.00–6.96 (m, 4H + NH), 6.79 (dd, 1H, J:
14.8, 8.0 Hz), 5.48 (s, 1H, NH2), 3.60 (d, 2H, CH2, J:
5.6 Hz), 3.44–3.39 (m, 4H, 2CH2 + H2O), 3.22 (bs, 4H,
2CH2); APT (13C-NMR)(100 MHz, DMSO-d6): δ 169.90,
153.02, 151.59, 138.13, 132.34, 129.41, 123.71, 119.51,
116.14, 109.14, 54.39, 48.71, 46.86; FT IR (cm–1): 3390,
3334, 3302 (NH2–NH), 2834 (ar-CH), 1728 (C=O); Ele-
mental analysis for C17H22N6O calculated: C, 62.56; H,
6.79; N, 25.75; found: C, 62.19; H, 6.68; N, 25.72; HRMS
(APCI): m/z C17H22N6O calculated (M+ + H): 327.40;
found: 327.19.
N′-(5-oxo-3-phenylthiazolidin-2-ylidene)-2-((6-(4-
phenylpiperazin-1-yl)pyridin-3-yl)amino)
acetohydrazide (9)
A mixture of compound 8 and ethyl bromoacetate
(10 mmol) in absolute ethanol was allowed to reflux in the
presence of dried sodium acetate (50 mmol) for 15 h. The
reaction mixture was then cooled to room temperature.
After the solvent was removed under reduced pressure and
added water, a solid appeared. This crude product was
recrystallized from EtOAc to give the pure compound.
Light Brown solid product. Yield 99% (4.95 g); mp,
79–80 °C; 1H-NMR (400 MHz, DMSO-d6): δ 10.83
(s, 1H, NH),7.70 (s, 1H), 7.37 (t, 2H, J: 7.6 Hz), 7.24 (t, 2H,
J: 7.6 Hz), 7.14 (t, 1H, J: 7.2 Hz), 7.04 (dd, 1H, J: 8.8,
2.3 Hz), 6.99 (d, 2H, J: 8.4 Hz), 6.92 (d, 2H, J: 7.6 Hz), 6.80
(t, 1H, J: 6.0 Hz), 6.72 (d, 1H, J: 5.3 Hz), 4.15 (s, 2H, CH2),
3.87 (d, 2H, CH2, J: 6.0 Hz), 3.41–3.46 (m, 4H, 2CH2 +
H2O), 3.22–3.23 (m, 4H, 2CH2); APT (13C-NMR)(100
5-(((6-(4-Phenylpiperazin-1-yl)pyridin-3-yl)amino)
methyl)-1,3,4-oxadiazole-2(3H)-thione (7)
CS2 (1.21 ml, 1.52 g, 20 mmol) was added to the solution
of compound 6 (3.26 g, 10 mmol) in ethanol-water (1:1)
and the mixture was refluxed in the presence of KOH
(0.56 g, 10 mmol) for 12 h. Then, the resulting solution was
cooled to room temperature and acidified to pH 4 with
CH3COOH. The solid precipitate was collected by filtration
and recrystallized from EtOAc to give the pure compound.
1
White solid. Yield, 66% (2.41 g); mp, 131–132 °C; H-
NMR (400 MHz, DMSO-d6): δ 9.37 (s, 1H, NH), 7.69
(s, 1H), 7.23 (t, 2H, J: 7.5 Hz), 7.09 (d, 1H, J: 7.5 Hz), 6.99

Medicinal Chemistry Research
MHz, DMSO-d6): δ 169.85, 168.80, 153.13, 152.03,
151.58, 147.78, 137.97, 132.62, 129.78, 129.41, 124.95,
123.98, 121.29, 119.52, 116.15, 108.97, 56.51, 48.69,
46.82, 30.35; FT IR (cm–1): 3278 (2NH), 2831 (ar-CH),
1759, 1733 (C=O), 1598 (N=C); Elemental analysis for
C26H27N7O2S calculated: C, 62.26; H, 5.43; N, 19.55; S,
6.39 found: C, 62.10; H, 5.75; N, 19.29; S, 6.23; HRMS
(APCI): m/z calculated C26H27N7O2S (M+ + H): 502.61;
found: 502.20.
3,3-Dimethyl-7-oxo-6-(((5-(((6-(4-phenylpiperazin-1-
yl)pyridin-3-yl)amino)methyl)-2-thioxo-1,3,4-
oxadiazol-3(2H)-yl)methyl)amino)-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylic acid (11a)
Light yellow solid. Yield, 50% (2.98 g); mp, 194–195°C;
1H-NMR (400 MHz, DMSO-d6): δ 7.91 (s, 1H, NH), 7.41
(s, 1H, NH), 7.23 (bs, 3H), 7.00–6.80 (m, ar-H, 5H),
5.51–5.46 (bs, 1H, CH), 5.29 (bs, 1H, CH), 4.40 (bs, 1H,
CH), 4.22 (bs, 2H, CH2), 3.68 (bs, 2H, CH2), 3.54 (bs, 4H,
2CH2),3.23 (bs, 4H, 2CH2),1.57–1.45(m, 6H, 2CH3); APT
(13C-NMR)(100 MHz, DMSO-d6): δ 175.30, 170.83,
162.58, 157.62, 155.81, 151.59, 137.95, 135.48, 129.71,
129.43, 119.60, 116.05, 108.69, 71.37, 64.21, 63.70, 59.80,
54.99, 48.63, 45.73, 44.98, 28.03, 27.58; FT IR (cm–1):
3386 (NH + OH), 2973 (ar-CH), 1770 (2C=O); Elemental
analysis for C27H32N8O4S2 calculated: C, 54.35; H, 5.41;
N, 18.78; S, 10.75; found: C, 54.37; H, 5.56; N, 18.74; S,
10.53; HRMS (APCI): m/z calculated C27H32N8O4S2 (M+):
595.73; found: 595.37.
4-Phenyl-5-(((6-(4-phenylpiperazin-1-yl)pyridin-3-yl)
amino)methyl)-4H-1,2,4-triazole-3-thiol (10)
The solution of the corresponding compound 8 (4.61 g,
10 mmol) in ethanol:water (1:1) was refluxed in the pre-
sence 100 mL EtOH:H2O (1:1) of 2% NaOH for 3 h. Then
the resulting solution was cooled to room temperature and
acidified to pH 4 with 37% HCl (0.8 mL). The precipitate
formed was filtered off, washed with water, and recrys-
tallized from EtOH:H2O (1:1) to afford the desired com-
pound. White solid. Yield, 98% (3.32 g); mp, 155–156 °C;
1H-NMR (400 MHz, DMSO-d6): δ 13.85 (s, 1H, SH),
7.58–7.52 (m, 5H), 7.45 (d, 2H, J: 7.6 Hz), 7.31 (t, 1H NH,
J: 7.6 Hz), 7.23 (t, 2H, J: 7.2 Hz), 6.99 (d, 2H, J: 8.4 Hz),
6.93 (d, 1H, J: 9.2 Hz), 6.80 (t, 1H, J:7.2 Hz), 6.74 (d, 1H,
J: 8.8 Hz), 5.49 (s,1H, NH), 4.10 (s, 2H, CH2), 3.39–3.40
(m, 4H, 2CH2 + H2O), 3.21 (s, 4H, 2CH2); 13C-NMR (100
MHz, DMSO-d6): δ 156.17, 151.45, 150.73, 141.68,
137.31, 133.95, 129.92, 129.82, 129.49, 128.58, 121.49,
119.59, 117.26, 116.16, 109.79, 48.58, 46.65, 39.69; FT IR
(cm–1): 3224 (NH), 3023 (ar-CH), 2102 (SH); Elemental
analysis for C24H25N7S calculated: C, 64.99; H, 5.68; N,
22.10; S, 7.23 found: C, 64.75; H, 5.87; N, 22.10; S, 7.01;
HRMS (APCI): m/z C24H25N7S calculated (M+ + H):
444.57; found: 444.19.
3-(Acetoxymethyl)-8-oxo-7-(((5-(((6-(4-
phenylpiperazin-1-yl)pyridin-3-yl)amino)methyl)-2-
thioxo-1,3,4-oxadiazol-3(2H)-yl)methyl)amino)-5-
thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
(11b)
1
Light brown solid. Yield, 60% (3.91 g), mp, 198 °C; H-
NMR (400 MHz, DMSO-d6): δ 7.73 (s, 1H, NH),
7.25–7.21 (m, 2H), 7.13 (d, 1H, NH J: 7.2 Hz), 7.01–6.92
(m, 4H), 6.79 (d, 2H, J: 8.4 Hz), 4.94 (s, 3H, CH2 + CH),
4.67 (d, 1H, J = 10.0 Hz), 3.98 (s, 2H, CH2), 3.65 (s, 2H,
CH2), 3.48 (s, 4H, 2CH2), 3.24 (s, 6H, 3CH2), 2.03 (s, 3H,
CH3); APT (13C-NMR)(100 MHz, DMSO-d6): δ 172.49,
170.69, 166.29, 157.95, 153.87, 151.53, 137.99, 135.60,
132.19, 128.89, 124.48, 123.55, 119.58, 116.39, 109.11,
63.41, 63.16, 62.83, 59.32, 50.86, 48.67, 46.52, 45.22,
21.50, 20.50; FT IR (cm–1): 3386 (2NH + OH), 2973 (ar-
CH), 1738 (2C=O); Elemental analysis for C29H32N8O6S2
calculated: C, 53.36; H, 4.94; N, 17.17; S, 9.82; found: C,
53.67; H, 4.67; N, 17.40; S, 9.43; HRMS (APCI): m/z
calculated C29H32N8O6S2 (M+): 652.75; found: 652.31.
General method for the synthesis of compounds
11a–11f, 12a–12f
To a solution of corresponding compound 7 (for 11a–11f)
and 10 (for 12a–12f) (10 mmol) in dimethyl formamide
containing InCl3 (%10 mmol), 6-aminopenicilanic acid (6-
apa) (10 mmol) (for 11a and 12a) (10 mmol), 7-
aminocefalociporanic acid (7-aca) (for 11b and 12b)
(10 mmol), ciprofloxacin (for 11c and 12c) (10 mmol),
norfloxacin (for 11d and 12d) (10 mmol), 4-
aminobenzylpiperidine (10 mmol) (for 11e and 12e), 1-
Methylpiperazine (for 11f and 12f) (10 mmol) were added,
and the mixture were stirred at room temperature in the
presence of formaldehyde (%37, 30 mmol) for 24 h. The
solid precipitate was collected by filtration, washed with
water, and recrystallized from dimethylsulfoxide:water (1:1)
to give the desired compound.
Cyclopropyl-6-fluoro-4-oxo-7-(4-((5-(((6-(4-
phenylpiperazin-1-yl)pyridin-3-yl)amino)methyl)-2-
thioxo-1,3,4-oxadiazol-3(2H)-yl)methyl)piperazin-1-
yl)-1,4-dihydroquinoline-3-carboxylic acid (11c)
1
Light yellow solid. Yield, 37% (2.63 g); mp, 238 °C; H-
NMR (400 MHz, DMSO-d6): δ 8.65 (s, 1H, OH),7.72 (s,
1H), 7.23–7.21 (m, 4H), 7.12 (d, 1H, NH J: 8.4 Hz),
7.00–6.91 (m, 5H), 6.78–6.80 (m, 2H), 4.93 (s, 2H, CH2),
3.97 (bs, 3H, CH2 + CH), 3.46 (bs, 8H, 4CH2), 3.23 (bs,

Medicinal Chemistry Research
8H, 4CH2), 2.88 (bs, 2H, CH2), 1.34 (bs, 2H, CH2); APT
(13C-NMR)(100 MHz, DMSO-d6): δ 181.32, 176.68,
174.45, 166.59, 158.08, 153.40, 148.43, 145.59 and 151.51
(C–F J: 592 Hz), 139.50, 135.91, 135.44, 129.43, 124.14,
119.56, 116.19, 112.68, 111.62, 108.25, 107.30, 106.89,
69.58, 49.68, 48.56, 46.55, 45.70, 44.97, 30.89, 8.03; FT
IR (cm–1): 3326 (OH + NH), 3057 (ar-CH), 1689 (3C=O);
Elemental analysis for C36H38FN9O4S calculated: C,
60.75; H, 5.38; N, 17.71; S, 4.50 found: C, 60.72; H, 5.18;
N, 17.42; S, 4.63; HRMS (APCI): m/z calculated
C36H38FN9O4S (M+): 711.86; found ([M+ + H + Na]):
735.27.
N, 19.92; S, 5.42; HRMS (APCI): m/z calculated
C31H38N8OS (M+ + H): 571.76; found: 571.28.
3-((4-Methylpiperazin-1-yl)methyl)-5-(((6-(4-
phenylpiperazin-1-yl)pyridin-3-yl)amino)methyl)-
1,3,4-oxadiazole-2(3H)-thione (11f)
Recrystallized from EtOH. Brown solid. Yield, 99%
1
(4.75 g); mp, 275 °C; H-NMR (400 MHz, DMSO-d6): δ
7.73–7.71 (m, 1H, NH), 7.23–7.22 (m, 3H), 6.98–6.93 (m,
3H), 6.80 (bs, 2H), 3.48 (bs, 4H, 2CH2), 3.40–3.36 (m,
10H, 5CH2), 3.23 (bs, 6H, 3CH2), 2.50 (s, CH3); APT (13C-
NMR)(100 MHz, DMSO-d6): δ 166.33, 153.84, 153.16,
151.56, 135.51, 131.98, 129.24, 123.18, 119.57, 116.14,
109.10, 63.56, 54.70, 50.88, 48.67, 46.70, 44.98, 14.56; FT
IR (cm–1): 3343 (NH), 3058 (ar-CH), 1772 (C=O); Ele-
mental analysis for C24H32N8OS calculated: C, 59.98; H,
6.71; N, 23.31; S, 6.67 found: C, 59.85; H, 6.55; N, 23.51;
S, 6.96; HRMS (APCI): m/z calculated C24H32N8OS (M+
+ H): 481.64; found: 481.25.
1-Ethyl-6-fluoro-4-oxo-7-(4-((5-(((6-(4-
phenylpiperazin-1-yl)pyridin-3-yl)amino)methyl)-2-
thioxo-1,3,4-oxadiazol-3(2H)-yl)methyl)piperazin-1-
yl)-1,4-dihydroquinoline-3-carboxylic acid (11d)
Dark brown solid. Yield, 40% (2.80 g); mp, degraded at
1
178 °C; H-NMR (400 MHz, DMSO-d6): δ 8.93 (s, 1H,
OH),7.71 (s, 1H), 7.22 (bs, 3H), 7.11 (d, 1H, NH J: 6.8 Hz),
6.99–6.90 (m, 5H), 6.80–6.78 (m, 2H), 4.93 (s, 2H, CH2),
4.66–4.56 (m, 2H, CH2), 3.96 (s, 2H, CH2), 3.46 (bs, 8H,
4CH2), 3.22 (bs, 8H, 4CH2), 1.34–1.41 (m, 3H, CH3); APT
(13C-NMR)(100 MHz, CDCl3): δ 176.63, 166.54, 166.26,
153.87, 151.56, 149.58 and 145.81 (d J: 377 Hz), 148.91,
137.96, 137.67, 136.64, 135.81, 135.60, 131.98, 129.42,
123.29, 119.40, 116.04, 111.60, 108.89, 107.55, 106.27,
63.70, 50.87, 50.28, 50.13, 49.53, 48.54, 46.52, 14.81; FT
IR (cm–1): 3351 (OH + NH), 3054 (ar-CH), 1708 (3C=O);
Elemental analysis for C35H38FN9O4S calculated: C,
60.07; H, 5.47; N, 18.01; S, 4.58; found: C, 60.09; H, 5.50;
N, 18.08; S, 4.58; HRMS (APCI): m/z calculated
C35H38FN9O4S ([M+ + H]): 700.81; found: 700.27.
3,3-Dimethyl-7-oxo-6-(((4-phenyl-3-(((6-(4-
phenylpiperazin-1-yl)pyridin-3-yl)amino)methyl)-5-
thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)methyl)
amino)-4-thia-1-azabicyclo[3.2.0]heptane-2-
carboxylic acid (12a)
1
Light yellow solid. Yield, 81% (5.43 g); mp, 180 °C; H-
NMR (400 MHz, DMSO-d6): δ 9.88 (bs, 1H, OH), 7.91
(bs, 1H, NH), 7.55–7.21 (m, 8H), 6.96–6.79 (m, 5H),
5.45–5.31 (m, 1H), 5.05 (bs, 1H), 4.73 (s, 1H), 4.32–4.26
(m, 2H, CH2), 4.10 (s, 2H, CH2), 3.39 (s, 4H, 2CH2 +
H2O), 3.20 (s, 4H, 2CH2), 1.53–1.41 (m, 6H, 2CH3); APT
(13C-NMR)(100 MHz, DMSO-d6): δ 175.68, 172.09,
156.02, 151.57, 148.66, 141.77, 137.07, 134.13, 129.71,
128.97, 128.13, 126.99, 121.40, 119.57, 117.40, 116.14,
109.31, 107.74, 68.45, 67.26, 67.14, 63.34, 48.68, 46.74,
45.90, 27.08, 26.47; FT IR (cm–1): 3343 (NH + OH), 2919
(ar-CH), 1762 (2C=O); Elemental analysis for
C33H37N9O3S2 calculated: C, 59.00; H, 5.55; N, 18.76; S,
9.54; found: C, 59.04; H, 5.62; N, 18.17; S, 9.64; HRMS
(APCI): m/z calculated C33H37N9O3S2 (M+ + H): 672.84;
found: 672.23.
3-(((1-Benzylpiperidin-4-yl)amino)methyl)-5-(((6-(4-
phenylpiperazin-1-yl)pyridin-3-yl)amino)methyl)-
1,3,4-oxadiazole-2(3H)-thione (11e)
Recrystallized from EtOH. Yield, 35% (1.99 g); mp,
203 °C; 1H-NMR (400 MHz, DMSO-d6): δ 7.93 (d, 1H, J:
2.6 Hz), 7.42 (dd, ar-H + 2NH, J: 9.1, 2.6 Hz), 7.32–7.21
(m, 8H), 6.99 (d, 2H, J: 8.2 Hz), 6.89 (d, 1H, J: 9.2 Hz),
6.80 (t, 1H, J: 7.2 Hz), 5.25 (d, 2H, CH2, J: 16.8 Hz), 4.14
(d, 2H, CH2, J: 14.4 Hz), 3.53 (bs, 4H, 2CH2), 3.33 (s, 8H,
4CH2), 3.21 (d, 4H, 2CH2, J: 4.4 Hz), 2.86 (d, 2H, CH2, J:
3-(Acetoxymethyl)-8-oxo-7-(((4-phenyl-3-(((6-(4-
phenylpiperazin-1-yl)pyridin-3-yl)amino)methyl)-5-
thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)methyl)
amino)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxylic acid (12b)
11.2 Hz); APT (13C-NMR)(100 MHz, DMSO-d6):
δ
174.81, 162.84, 155.65, 151.51, 138.77, 137.91, 135.50,
129.76, 129.42, 129.27, 128.61, 128.38, 119.60, 116.19,
108.45, 63.60, 62.17, 53.61, 52.90, 52.42, 48.58, 45.76,
28.25; FT IR (cm–1): 3100 (NH), 3046 (ar-CH), 1735
(C=O); Elemental analysis for C31H38N8OS calculated: C,
65.24; H, 6.71; N, 19.63; S, 5.62; found: C, 65.45; H, 6.82;
1
Light yellow solid. Yield, 72% (4.83 g); mp, 160 °C; H-
NMR (400 MHz, DMSO-d6): δ 13.84 (s, 1H, OH), 9.85
(s, 1H, NH), 7.56 (d, 3H, J: 7.7 Hz), 7.46 (d, 2H, J: 4.9 Hz),

Medicinal Chemistry Research
7.30 (t, 1H, NH, J: 7.0 Hz), 7.22 (d, 3H, J:6.4 Hz), 6.97 (d,
3H, J: 7.1 Hz), 6.73–6.80 (m, 2H), 5.45 (bs, 1H, CH), 2.03
(s, CH3), 4.86–5.10 (m, 2H, CH2), 4.69 (d, 2H, CH2, J:
13.0 Hz), 4.11 (d, 2H, CH2, J: 20.0 Hz), 3.94 (s, 1H), 3.39
(6H, 3CH2 + H2O), 3.21 (s, 4H, 2CH2); APT (13C-NMR)
(100 MHz, DMSO-d6): δ 170.60, 168.82, 163.46, 156.30,
153.16, 151.17, 150.94, 141.72, 137.28, 134.14, 130.11,
129.69, 128.34, 126.76, 124.12, 121.40, 119.57, 117.18,
116.04, 108.90, 68.42, 63.18, 58.34, 48.69, 46.70, 46.05,
31.17, 26.38, 21.04; FT IR (cm–1): 3316 (OH + NH), 2919
(ar-CH), 1758 (C=O); Elemental analysis for
C35H37N9O5S2 calculated: C, 57.76; H, 5.12; N, 17.32; S,
8.81; found: C, 57.42; H, 5.08; N, 17.53; S, 8.71; HRMS
(APCI): m/z calculated C35H37N9O5S2 (M+ + H): 728.86;
found: 728.23.
2CH2), 2.96–2.89 (m, 8H, 4CH2), 1.39 (t, 3H, CH3, J:
6.8 Hz); APT (13C-NMR)(100 MHz, DMSO-d6):
δ
176.65, 169.59, 167.13, 166.60, 154.61, 153.12, 152.34,
149.53 and 146.16 (d, C–F, J: 337 Hz), 141.66, 137.01,
134.41, 129.89, 129.43, 128.39, 123.90, 121.50, 119.77,
119.53, 117.39, 116.14, 115.71, 111,57, 108.58, 107.64,
106.34, 68.54, 50.00, 49.56, 48.68, 48.48, 46.58, 46.05,
14.82; FT IR (cm–1): 3370 (OH + NH), 2977 (ar-CH),
1712 (C=O); Elemental analysis for C41H43FN10O3S
calculated: C, 63.55; H, 5.59; N, 18.08; S, 4.14; found: C,
63.13; H, 5.43; N, 18.02; S, 4.36. HRMS (APCI): m/z
calculated C41H43FN10O3S (M+ + H): 775.32; found:
775.31.
2-(((1-Benzylpiperidin-4-yl)amino)methyl)-4-phenyl-
5-(((6-(4-phenylpiperazin-1-yl)pyridin-3-yl)amino)
methyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (12e)
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-((4-phenyl-3-(((6-
(4-phenylpiperazin-1-yl)pyridin-3-yl)amino)methyl)-
5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)methyl)
piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic
acid (12c)
Recrystallized from CH2Cl2:Hexane, provided a light purple
1
solid product. Yield, 95% (6.13); mp, 137 °C; H-NMR
(400 MHz, DMSO-d6): δ 7.55–7.22 (m,12H, ar-CH + NH),
6.95–6.80 (m, 6H), 5.44 (bs, 2H, CH2), 4.14–4.11 (m, 2H,
CH2), 3.45 (s, 7H, 3CH2 + CH), 3.34 (s, 8H, 4CH2), 3.17
(s, 4H, 2CH2); APT (13C-NMR) (100 MHz, DMSO-d6): δ
168.71, 155.38, 153.20, 151.54, 147.78, 141.27, 139.06,
136.35, 131.50, 129.80, 129.41, 129.07, 128.47, 127.22,
124.17,119.59, 116.13, 108.68, 107.76, 62.36, 58.22,53.26,
51.92, 48.66, 46.63, 45.47, 31.22; FT IR (cm–1): 3347
(NH), 3027 (ar-CH), 1230 (C=S); Elemental analysis for
C37H43N9S calculated: C, 68.81; H, 6.71; N, 19.52; S, 4.96;
found: C, 68.85; H, 6.74; N, 19.09; S, 4.90; HRMS
(APCI): m/z calculated C37H43N9S (M+): 645.87; found:
(M+ + K + H2O): 702.36.
Light yellow solid. Yield, 97% (7.61 g); mp, 192–193 °C;
1H-NMR (400 MHz, DMSO-d6): δ 15.18 (s, 1H, OH), 9.83
(s, 1H, NH), 8.67–8.50 (m, 2H), 7.91–7.89 (m, 1H),
7.56–7.47 (m, 7H), 7.20–7.14 (m, 2H), 6.93 (bs, 1H),
6.79–6.64 (m, 3H), 5.19–5.23 (m, 4H, CH2), 4.14–4.20 (m,
1H), 3.82 (bs, 2H, CH2), 2.91–3.32 (m, 12H + 6CH2), 2.94
(d, 4H, 2CH2, J: 26.4 Hz), 1.10–1.32 (m, 4H, 2CH2); APT
(13C-NMR)(100 MHz, DMSO-d6): δ 204.40, 176.84,
169.67, 166.33, 154.52, 153.17, 151.80 and 145.69 (d, C–F,
J: 611 Hz), 151.36, 149.86, 148.23, 139.58, 137.08, 134.41,
133.26, 129.89, 129.42, 128.42, 121.48, 119.55, 117.25,
115.81, 111,52, 107.32, 106.91, 68.73, 51.10, 49.93, 48.47,
46.54, 45.89, 36.24, 8.04; FT IR (cm–1): 3440 (OH +
NH),3062 (ar-CH), 1716 (C=O); Elemental analysis for
C42H43FN10O3S calculated: C, 64.10; H, 5.51; N, 17.80; S,
4.07 found: C, 64.26; H, 5.38; N, 17.75; S, 4.05; HRMS
(APCI): m/z calculated C42H43FN10O3S (M+ + H): 787.93;
found: 787.31.
2-((4-Methylpiperazin-1-yl)methyl)-4-phenyl-5-(((6-
(4-phenylpiperazin-1-yl)pyridin-3-yl)amino)methyl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione (12f)
Recrystallized from EtOAc, provided a light green solid
1
product. Yield, 68% (3.77); mp, 94–95 °C; H-NMR (400
MHz, DMSO-d6): δ 7.57–7.54 (m, 5H, ar-CH + NH), 7.45
(m, 2H), 7.23 (t, 2H, J: 7.6 Hz), 6.98 (d, 2H, J: 8.2), 6.89
(d, 1H, J: 8.8 Hz), 6.80 (t, 1H, J: 7.3 Hz), 6.71 (d, 1H, J:
8.9 Hz), 5.03 (s, 2H, CH2), 4.14 (d, 2H, CH2, J: 6.0 Hz),
3.36 (bs, 4H, 2CH2 + H2O), 3.21 (s, 4H, 2CH2), 2.68
(s, 4CH, 2CH2), 2.8 (s, 4H, CH2), 2.12 (s, 3H, CH3); APT
(13C-NMR)(100 MHz, DMSO-d6): δ 169.50, 153.25,
151.56, 149.35, 137.16, 134.43, 132.95, 130.03, 129.89,
129.42, 128.48, 123.99, 119.53, 116.14, 108.90, 69.04,
55.01, 51.34, 50.14, 48.69, 46.68, 46.25; FT IR (cm–1):
3387 (NH), 3027 (ar-CH), 1226 (C=S); Elemental ana-
lysis for C30H37N9S calculated: C, 64.84; H, 6.71; N, 22.68;
S, 5.77; found: C, 64.82; H, 6.14; N, 22.74; S, 5.38; HRMS
1-Ethyl-6-fluoro-4-oxo-7-(4-((4-phenyl-3-(((6-(4-
phenylpiperazin-1-yl)pyridin-3-yl)amino)methyl)-5-
thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)methyl)
piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic
acid (12d)
1
Light gray solid. Yield, 78% (6.03 g); mp, 220 °C; H-
NMR (400 MHz, CDMSO-d6): δ 8.90 (s, 1H, OH),
7.56–7.54 (m, 6H), 7.45–7.43 (m, 1H), 7.32–7.23 (m, 1H,
NH), 7.23–7.13 (m, 4H), 6.99–6.90 (m, 2H), 6.78–6.63
(m, 3H), 5.18 (bs, 2H, CH2), 4.56 (s, 2H, CH2), 4.18 (s,
2H, CH2), 3.34 (bs, 4H, 2CH2 + H2O), 3.21 (s, 4H,

Medicinal Chemistry Research
(APCI): m/z calculated C30H37N9S (M+ + H): 556.75;
found: 556.29.
We showed that the most appropriate method to syn-
thesize the compound (3) was in solvent-free environment
in an oil bath. Then, synthesis of the leading compound (4)
of the study was carried out by reduction of the compound
(3) with hydrazine hydrate in the presence of catalytic Pd/C.
While the molecule is known in the literature and com-
mercially available, synthesis methodology, and spectro-
scopic data were presented in this work for the first time
(Scheme 1).
The synthesis of the hydrazide compound (6) was per-
formed in two steps starting from the amine (4) compound.
This compound (6) was then treated with CS2 in the pre-
sence of KOH to be converted to 1,3,4-oxadiazole (7)
derivative as a result of cyclo-condensation reaction
(Scheme 2). Carbothioamide (8) was synthesized with the
reaction of hydrazide compound (6) and isothiocyanate. The
compound (9), which is a 4-oxo-1,3-thiazolidine derivative,
was obtained from the treatment of the carbothioamide
compound (8) with ethyl bromoacetate.
Anticancer activity
Cell lines and proliferation assay
Human PC cell lines, PC3 (CRL-1435), DU145 (HTB-81),
and LNCaP (CRL-1740), were purchased from ATCC
(Rockville, MD, USA). All cell lines were cultured in
Dulbecco’s Modified Eagle Media (Invitrogen) supple-
mented with 10% fetal bovine serum (Invitrogen) and 1%
penicillin-streptomycin-amphotericin (Invitrogen) in
a
humidified incubator at 37 °C and 5% CO2. Anticancer
activity of newly synthesized hybrid molecules against PC
cell lines were analyzed with the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and
NCI60 response parameters was performed after 24 h
incubation period. The results are given as % cell inhibition,
and optic density for the cells treated with solvent only
(DMSO) was accepted as 100%. Response parameters,
IC50 (50% cell viability inhibition), GI50 (50% growth
inhibition), TGI (total growth inhibition), and LC50 (50%
cell death) were evaluated based on National Institutes of
Health definitions using logarithmic functions (González-
Vallinas et al. 2013).
Elimination of signals in the compound (9) for –NH
group that normally appears in 3336, 3243 cm−1 in FT IR
1
spectra and 10.11–9.53 ppm in H-NMR spectra of the
compound (8), and observation of peaks for the C-2, C-4
and C-5 atoms of the 4-oxo-1,3-thiazolidine ring at the
corresponding chemical shift values in 13C-NMR are con-
firmations for the cyclo-condensation reaction. The chemi-
cal shift values for the compounds are in line with the
previously published data (Küçükgüzel et al. 1999;
Hubschwerlen et al. 2003; Skjemstad et al. 2004; Yolal
et al. 2012). Resonance of the C-5 protons of the thiazoli-
dinone ring at the aliphatic region in 1H-NMR and
13C-NMR as expected is a further proof for the reaction
(Scheme 2).
The 1,2,4-triazole compound (10) was obtained as a
result of the intramolecular ring closure of the correspond-
ing carbothioamide derivative (8) in the presence of base.
As shown in Scheme 3, a tautomeric equilibrium is known
to occur in the 1,2,4-triazole (10) compounds (Küçükgüzel
Results and discussion
Chemistry
To obtain the commercially available 6-(4-phenyl-piper-
azin-1-yl)pyridine-3-ylamine (4) compound, 1-(5-nitropyr-
idin-2-yl)-4-phenylpiperazine (3) was firstly synthesized.
While the synthesis procedure for compound (3) was
described in the literature, it was not possible to obtain the
chemical by following the same protocol.
Scheme 1 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) BrCH2CO2Et in THF, TEA, 60 °C, 20 h; (iv) H2NNH2.H2O in EtOH, reflux, 13 h

Medicinal Chemistry Research
Scheme 2 (i) CS2 in EtOH-H2O, KOH, reflux, 12 h; (ii): C6H5NCS, EtOH, reflux, 18 h; (iv) ethyl bromoacetate, dry CH3COONa, absolute EtOH,
ruflux, 18 h; (v): NaOH, in EtOH:H2O (1:1), reflux, 3 h
Scheme 3 Mechanism of 1,2,4-triazole
et al. 1999; Demirbas et al. 2004; Holla et al. 2005;
Barbuceanu et al. 2009).
compound (7), the spectroscopic data proposed that thiooxo
form (II) was preferred as the dominant tautomeric form. In
FT IR spectrum of this compound, the signal from –C=S
bond was detected in 1228 cm−1. In addition, the [M + 1]
ionic peak of the molecule (7) matches with the appropriate
m/z value and the results of the elemental analysis confirm
the structure.
The Mannich reaction, a three-component reaction in a
single step, that allows synthesis of organic molecules
containing different functional groups in a single molecular
structure has become one of the frequently used methods in
drug design studies.
When X is the sulfur atom, the equilibrium is more
favorable in the form of I, and when X is the oxygen atom,
the equilibrium is shifted in favor of the II form. For the
compound (10) synthesized in this study, the dominant
tautomeric form is the mercapto form represented by I, as
evidenced by FT IR and 1H-NMR data. In the FT IR spectra
of this compound (10), the signal from the voltage of the
S–H bond is seen at 2102 cm−1, while the –SH proton is
1
resonant at 13.85 ppm in the H-NMR spectra.
As in 1,2,4-triazole derivatives, it is also known that
there is a tautomeric balance in 1,3,4-oxadiazole derivatives
as indicated in I and II reactions. For the 1,3,4-oxadiazole
Three components involved in the Mannich reactions are
a non-enolized carbonyl compound, enolizable carbonyl

Medicinal Chemistry Research
Scheme 4 Amine, DMF, HCHO, InCl3, 24 h, rt
compound, and a primary or secondary amine, in which
alkyl (aryl) aminomethylated products can be synthesized
(Roman 2015). In these reactions, any compound contain-
ing active hydrogen can be used instead of the enolable
carbonyl compound. For example, Mannich bases exerting
various biological activities have been obtained using 1,2,4-
triazole derivatives as the active hydrogen component and
methylpiperazine or morpholine as the amine component
(Roman 2015).
In the current study, 1,3,4-oxadiazole compound (7) and
1,2,4-triazole compound (10) were used as the active
hydrogen component in the synthesis of Mannich bases,
11a–11f and 12a–12f, respectively (Scheme 4). For the
synthesis of Mannich bases, Lewis and Bronsted acid cat-
alysts such as p-TSA, FeCl3, InCl3, and HCl are used in
addition to polar solvents. In this study, InCl3 catalyst was
used for Mannich reactions (Mentese et al. 2016).
In the FT IR and NMR spectra of the Mannich bases,
while there was not any peak confirming the presence of
–SH or –NH proton of the compound (7) or (10), the signals
of the amine used were recorded at the corresponding
chemical shift values in the NMR spectra. These com-
pounds also gave elemental analysis results and EI–MS
spectra compatible with their structure.
more sensitive to Mannich bases compared with DU145 and
PC3 cells. In addition, Mannich bases displayed strong
growth inhibitions at relatively low concentrations
(3.43–16.64 µM) to LNCaP cells which is comparable with
control drug treatment (97.63 µM). DU145 cells were found
to be the most resistant cell types against Mannich base
treatment.
Evaluation of hybrid molecules according to
NCI60 screening methodology
Although many anticancer drugs have been developed in
modern chemotherapy, the development of a desirable level
of therapeutic product has not yet been possible. In addition,
due to the mechanisms of resistance against chemother-
apeutic agents and their side effects, their therapeutic
potency is decreasing day by day. Therefore, efforts are
under way to develop new cancer drugs. In this study, an
MTT test was performed to measure the effects of 21 newly
synthesized hybrid molecules on cell proliferation
(NCI60 scanning methodology) and IC50 values. Accord-
ing to this, none of the newly synthesized 3, 5, 11a, and 11b
hybrid molecules, even at a high concentration of 125 µg/
mL, have not shown antiproliferative activity on PC cell
lines (Table 1). However, the fact that the anticancer
activity of these compounds has not been able to demon-
strate does not mean that they are pharmacologically
worthless. A large scale analysis of these molecules, such as
genome, transcriptome, and proteome, may be useful for the
screening of different activities. In addition, when the G50
values of these molecules (2.5–59.5 µg/mL) are examined,
it shows that there is potential for use if they are redesigned
and increased antiproliferative effects. Interestingly, the
Anticancer activity
Newly synthesized Mannich bases were evaluated for their
anticancer activity against PC3, DU145, and LNCaP PC
cell lines in ex vivo conditions. The results revealed that
LC50 levels for all chemicals tested were higher than the
highest concentration used (500 µM) for all cancer cell lines
(Table 1). According to IC50 levels, LNCaP cells were

Medicinal Chemistry Research
Table 1 Life parameters (GI50, TGI, and LC50) and IC50 values determined by MTT test of test molecules
Molecules (µg/mL)
DU145
GI50
PC3
LnCaP
GI50
TGI
LC50
IC50
GI50
TGI
LC50
IC50
TGI
LC50
IC50
3
4.9
>1000
44.5
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
236.8
>1000
44.6
21.3
4.7
>1000
40.8
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
371.7
>1000
40.8
22.5
4.6
4.3
1.2
4.2
4.9
5.6
3.1
3.4
5.5
3.2
2.6
3.4
8.0
3.2
6.5
2.7
3.1
3.5
2.3
12.7
>1000
>1000
>1000
11.5
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
486.2
>1000
>1000
>1000
8.9
4
2.4
5
2.5
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
420.3
35.4
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
418.6
>1000
>1000
88.7
6.0
>1000
122.5
>1000
>1000
402.7
626.7
>1000
>1000
143.6
>1000
16.1
>1000
70.9
6
7.9
6.2
7
>1000
>1000
10.9
16.3
8.2
12.6
10.5
5.2
>1000
>1000
129.8
113.8
>1000
>1000
15.8
40.5
18.5
8
211.3
>1000
82.3
47.2
9
>1000
14.5
10
2.3
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
5-FU
11.3
3.6
>1000
>1000
71.6
>1000
>1000
72.7
59.5
11.2
129.0
6.6
1.6
6.2
>1000
9.7
349.5
725.9
532.3
720.8
805.6
130.6
115.5
49.7
368.2
25.5
1.6
22.7
118.6
12.7
3.4
43.4
4.6
>1000
>1000
>1000
>1000
>1000
>1000
>1000
39.9
>1000
>1000
>1000
>1000
>1000
>1000
>1000
27.8
61.7
31.1
148.3
8.2
113.4
21.2
179.6
2.5
16.6
13.3
3.9
20.7
49.6
2.1
13.8
13.8
10.2
25.9
9.9
53.0
38.7
newly synthesized hybrid molecules 7, 8, 11d, 11f, 12a,
12b, 12d, and 12e showed strong antiproliferative proper-
ties only on LnCaP from the PC cell lines tested (GI50
values 2.6–8.0 µg/mL; TGI values 23.5–805.6 µg/mL, and
IC50 values in the range of 18.5–368.2 µg/mL). However,
the molecule 9 showed antiproliferative properties only on
PC3 from PC cell lines tested (GI50 values was 5.2 µg/mL,
TGI value was 402.7 µg/mL, and IC50 values were
129.8 µg/mL) (Table 1).
The emergence of such a result in different PC cell lines
may have been effective in different molecular mechanisms
of cells as well as newly synthesized hybrid molecules.
When we examine the cells, these cells are susceptible to
LnCaP hormone (androgen, AR+) and are able to secrete
prostate-specific antigen (PSA).
These cells are also sensitive to 5-alpha-dihy-
drotestosterone, which is responsible for cell growth and
modulation of acid phosphatase production. PC3 cells are
hormone (androgen, AR-) insensitive and exhibit low acid
phosphatase and testosterone-5-alpha-reductase activities.
Molecules 6, 10, 11c, and 11e showed antiproliferative
properties similar to 5-fluorouracil (5-FU) which were used
as positive control on both PC3 (G50 values 1.6–6.2 µg/mL,
TGI values 16.1–626.7 µg/mL and IC50 values
9.7–113.8 µg/ml range) and LnCaP (G50 values 1.2–3.4 µg/
mL, TGI values 11.5–725.9 µg/mL, and IC50 values
8.9–72.7 µg/mL) from the PC cell lines tested (Table 1).
While molecules 12c and 12f on the DU145 and LnCaP
of the PC cell lines tested showed potent antiproliferative
properties (DU145 cells, respectively, G50 value 3.4,
2.1 µg/mL and IC50 value 418.6, 88.7 µg/mL, GI50 2.7,
2.3 µg/mL and IC50 value 21.12, 13.8 µg/mL, respectively),
only molecule 4 on both DU145 and PC3 of the PC cell
lines showed antiproliferative (GI50 value 2.4 and 4.7 µg/
mL, TGI value 44.5 and 40.8 µg/mL, respectively, IC50
value 44.6 and 40.8 µg/mL, respectively). Pharmacological
activities of newly synthesized hybrid molecules on cells of
the DU145 PC cell lines were understood to be remarkably
few. This cell is independent of the hormone (androgen,
AR−) and does not secrete the PSA but has a very weak
acid phosphatase activity. We also have to point out that
DU145 cells exhibit quite allogeneic properties.
Generally, when the IC50 data of the test results were
examined, the molecules of the molecule 12c (IC50 values
88.7 21 g/mL) on DU145 cells (Fig. 1), molecules 6, 11c,
11e (IC50 values 70.9, 15.8, and 9.7 µg/mL, respectively)
PC3 on the cells (Fig. 2), 6, 7, 8, 10, 11c, 11e, 11f, 12a, 12c,
12d, 12e, 12f on LnCaP cells (IC50 value in the range of

Medicinal Chemistry Research
3
4
5
6
7
8
9
10
Fig. 1 Determination of
antiproliferative effects of 3-12
test molecules on DU145 cell
line by MTT test. Negative
values on the graph indicate cell
death, positive values indicate
growth inhibition
100
50
0
100
50
0
-50
-50
0.2
0.6
1
1.4
1.8
2.2
0.2
0.6
1
1.4
1.8
2.2
Concentraꢀon (Log10 μg/mL)
Concentraꢀon (Log10 μg/mL)
12a
12d
12b
12e
12c
12f
11a
11d
11b
11e
11c
11f
100
50
0
100
50
0
-50
-50
0.2
0.6
1
1.4
1.8
2.2
0.2
0.6
1
1.4
1.8
2.2
Concentraꢀon (Log10 μg/mL)
Concentraꢀon (Log10 μg/mL
3
4
5
6
7
8
9
10
Fig. 2 Determination of
antiproliferative effects of 3-12
test molecules on PC3 cell line
by MTT test. Negative values on
the graph indicate cell death,
positive values indicate growth
inhibition
100
50
0
100
50
0
-50
-50
0.2
0.6
1
1.4
1.8
2.2
0.2
0.6
1
1.4
1.8
2.2
Concentraꢀon (Log10 μg/mL)
Concentraꢀon (Log10 μg/mL)
11a
11c
11b
11d
12a
12c
12b
12d
100
50
0
100
50
0
-50
-50
0.2
0.6
1
1.4
1.8
2.2
0.2
0.6
1
1.4
1.8
2.2
Concentraꢀon (Log10 μg/mL)
Concentraꢀon (Log10 μg/mL)
8.9–72.7 µg/mL) (Figs. 3 and 4), inhibitory effected on total
growth up to at least 5-FU (control anticancer molecule).
When the TGI values in Table 1 were examined, molecules
4 (TGI value 44.5 µg/ml) on DU145 cells (Fig. 1), molecules 4
and 11e (TGI values were 40.8 and 16.1 µg/ml) on PC3 cells
(Fig. 2), molecules 6, 7, 10, 11c, 12e, 12f (TGI values
respectively 11.5, 40.5, 82.3, 71.6, 49.7, and 13.8 µg/ml)
LnCaP cells on (Fig. 3), inhibitory effected on total growth up
to at least 5-FU.
The lower GI50, TGI, and IC50 values of the said
molecules on cells similar to 5-FU are considered to be
suitable for further clinical studies. Because all of molecules
have very high concentration LC50 value (>1000 µg/mL),
in terms of the concentration values of lethal (lethal,

Medicinal Chemistry Research
10
3
4
5
6
7
8
9
Fig. 3 Determination of MTT
test of antiproliferative effects of
test molecules 3-12 on the
LnCaP cell line. Negative values
on the graph indicate cell death,
positive values indicate growth
inhibition
100
50
100
50
0
0
-50
-100
-50
-100
0.2
0.6
1
1.4
1.8
2.2
0.2
0.6
1
1.4
1.8
2.2
Concentraꢀon (Log10 μg/mL)
Concentraꢀon (Log10 μg/mL)
12a
12d
12b
12e
12c
12f
11a
11b
11c
100
50
100
50
0
0
-50
-100
-50
-100
0.2
0.6
1
1.4
1.8
2.2
0.2
0.6
1
1.4
1.8
2.2
Concentraꢀon (Log10 μg/mL)
Concentraꢀon (Log10 μg/mL)
PC3
100
DU145
LnCaP
50
0
-50
-100
0.5
1
1.5
2
2.5
3
Concentraꢀon(Log10 μg/mL)
Fig. 4 Determination of antiproliferative effects of 5-FU control
anticancer-acting molecules on PC3, DU145, and LnCaP cell line by
the MTT assay
negative values on the graph), the new synthesized mole-
cules not have a very high toxic effect on the cells and
perform better than even positive control (Table 1).
Cancer is the second cause of death in the world, the
amount spent for the treatment of cancer is rapidly
increasing. Research and development studies for the
development of national drugs in the fight against cancer
have also gained speed. in this context, new hybrid mole-
cules developed by us have been added to the science lit-
erature with anticancer properties. The aim of this article is
to study in vitro and then in vivo, to determine the important
pharmacological potentials of molecules, to study “phase 0”
in order to see the availability in medicine and pharmacy. In
addition, it is possible to develop new and more active
molecules based on these molecules.
Fig. 5 DNA laddering test of newly synthesized molecule 4 and 12c.
The test log phase was performed using the DU145 cell line showing
exponential growth and incubation time was limited to 24 h. K-Control
DNA ladder potentials of hybrid molecules
According to the DNA laddering test which determine the
DNA degradation activity, which is an indicator of apop-
tosis, molecules 12c and 4 of the new hybrid molecules
tested on DU145 cells, DU145 cells have been shown to
cause significant DNA breaks (Fig. 5).

Medicinal Chemistry Research
According to the DNA laddering test, no DNA degra-
dation was observed in the control well, whereas DNA
fractures caused by Molecule 12c did not cause very sig-
nificant laddering (Fig. 5). The apopitosis mechanisms seem
to be responsible for part of the anticancer activity of new
hybrid molecules that cause partial breaks on DNA.
References
Barbuceanu S-F, Almajan GL, Saramet I et al. (2009) Synthesis,
characterization and evaluation of antibacterial activity of some
thiazolo[3,2-b][1,2,4]triazole incorporating diphenylsulfone moi-
eties. Eur J Med Chem 44:4752–4757. https://doi.org/10.1016/j.
ejmech.2009.06.021
Demirbas N, Karaoglu SA, Demirbas A, Sancak K (2004) Synthesis and
antimicrobial activities of some new 1-(5-phenylamino-[1,3,4]thia-
diazol-2-yl)methyl-5-oxo-[1,2,4]triazole and 1-(4-phenyl-5-thioxo-
[1,2,4]triazol-3-yl)methyl-5-oxo- [1,2,4]triazole derivatives. Eur J
Med Chem 39:793–804. https://doi.org/10.1016/j.ejmech.2004.
06.007
Dimmock JR, Jonnalagadda SS, Phillips OA et al. (1992) Antic-
onvulsant properties of some Mannich bases of conjugated ary-
lidene ketones. J Pharm Sci 81:436–440
Friedman HS, Kerby T, Calvert H (2000) Temozolomide and treat-
ment of malignant glioma. Clin Cancer Res 6:2585–2597
Gökçe M, Bakır G, Şahin M et al. (2005) Synthesis of new Mannich
bases of arylpyridazinones as analgesic and anti-inflammatory
agents. Arzneimittelforschung 55:318–325. https://doi.org/10.
1055/s-0031-1296866
González-Vallinas M, Molina S, Vicente G et al. (2013) Antitumor
effect of 5-fluorouracil is enhanced by rosemary extract in both
drug sensitive and resistant colon cancer cells. Pharm Res
72:61–68. https://doi.org/10.1016/j.phrs.2013.03.010
Holla BS, Poojary KN, Poojary B et al. (2005) Synthesis, character-
ization and antibacterial activity studies on some fluorine con-
taining quinoline-4-carboxylic acids and their derivatives. Indian
J Chem Sect B 44B:2114–2119
Hu G, Wang G, Duan N et al. (2012) Design, synthesis and antitumor
activities of fluoroquinolone C-3 heterocycles (IV): s-triazole
Schiff–Mannich bases derived from ofloxacin. Acta Pharm Sin B
2:312–317. https://doi.org/10.1016/j.apsb.2011.11.003
Morphological changes of molecules on cells
The effects of the new hybrid molecules used in two
different concentrations (62.5 µg/mL and 125 µg/mL) on
cell morphology were investigated and examined with the
help of phase-contrast microscopy. Control cells show
normal cell morphology, such as fibroblast or epithelial,
and have been a reference for our evaluations. The first
impression we get from phase-contrast microscope ima-
ges is that cells begin to separate from the flask floor in a
concentration-dependent manner. during the separation,
respectively, cells begin to lose their normal forms such
as fibroblast or epithelial, and then turn into rounded
forms, and cytoplasmic bubble and spike formation,
abnormal globular structures and apoptotic bodies
appearing, and ultimately the cells are floating (cells are
dead) and some occurs morphological changes. Findings
from morphological analysis show that new hybrid
molecules cause cell death by using apoptosis pathways,
especially at high concentrations.
Hubschwerlen C, Specklin JL, Sigwalt C et al. (2003) Design,
synthesis and biological evaluation of oxazolidinone-quinolone
hybrids. Bioorg Med Chem 11:2313–2319
Idhayadhulla A, Surendra Kumar R, Abdul Nasser AJ et al. (2014)
Synthesis of some Mannich base derivatives and their anti-
microbial activity study. Arab J Chem 7:994–999. https://doi.org/
10.1016/j.arabjc.2010.12.025
Jarvis C, Nelius T, Martinez-Marin D et al. (2018) Cabazitaxel regi-
mens inhibit the growth of prostate cancer cells and enhances the
anti-tumor properties of PEDF with various efficacy and toxicity.
Prostate 78:905–914. https://doi.org/10.1002/pros.23647
Küçükgüzel ŞG, Rollas S, Küçükgüzel I, Kiraz M (1999) Synthesis
and antimycobacterial activity of some coupling products from 4-
aminobenzoic acid hydrazones. Eur J Med Chem 34:1093–1100.
https://doi.org/10.1016/S0223-5234(99)00129-4
Conclusions
In the present study, the leading compound 6-(4-phenyl-
piperazin-1-yl)pyridine-3-ylamine was used to synthesize
novel Mannich bases. These compounds were characterized
using elemental analysis, IR, H-, and 13C-NMR spectral
1
data and evaluated for their anticancer activities against PC
cell lines. The results showed that the Mannich bases tested
in the current study displayed moderate cytotoxic activities
against PC cells. Further variations in the structure of the
chemicals should be performed to screen more potent
anticancer agent.
Kumbhare RM, Vijay Kumar K, Janaki Ramaiah M et al. (2011)
Synthesis and biological evaluation of novel Mannich bases of 2-
arylimidazo[2,1-b]benzothiazoles as potential anti-cancer agents.
Acknowledgements The support provided by Scientific and Techno-
logical Research Council of Turkey (TUBITAK, Project no: 116Z932)
is gratefully acknowledged. We thank to Selami Demirci for his help
in paper editing.
Eur J Med Chem 46:4258–4266. https://doi.org/10.1016/j.
ejmech.2011.06.031
Li H, Zeng H, Shao H (2009) Bismuth(III) chloride-catalyzed one-pot
Mannich reaction: three-component synthesis of β-amino carbo-
nyl compounds. Tetrahedron Lett 50:6858–6860. https://doi.org/
10.1016/j.tetlet.2009.09.131
Lopes F, Capela R, Gonçaves JO et al. (2004) Amidomethylation of
amodiaquine: antimalarial N-Mannich base derivatives. Tetrahedron
Lett 45:7663–7666. https://doi.org/10.1016/j.tetlet.2004.08.093
Malhotra M, Arora M, Samad A et al. (2012) Synthesis and evaluation
of some novel derivatives of 2-propoxybenzylidene iso-
nicotinohydrazide for their potential antimicrobial activity. J Serb
Chem Soc 77:589–597. https://doi.org/10.2298/JSC110310170M
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.

Medicinal Chemistry Research
Mentese M, Demirci S, Ozdemir SB et al. (2016) Microwave assisted
synthesis and antimicrobial activity evaluation of new hetero-
functionalized norfloxacine derivatives. Lett Drug Des Discov.
https://doi.org/10.2174/1570180813666160712222922
Neidle S, Thurston DE (2005) Chemical approaches to the discovery
and development of cancer therapies. Nat Rev Cancer 5:285–296.
https://doi.org/10.1038/nrc1587
Oudard S (2013) Progress in emerging therapies for advanced prostate
cancer. Cancer Treat Rev 39:275–289. https://doi.org/10.1016/j.
ctrv.2012.09.005
Roman G (2015) Mannich bases in medicinal chemistry and drug
design. Eur J Med Chem 89:743–816. https://doi.org/10.1016/j.
ejmech.2014.10.076
Shivarama Holla B, Veerendra B, Shivananda MK, Poojary B (2003)
Synthesis characterization and anticancer activity studies on some
Mannich bases derived from 1,2,4-triazoles. Eur J Med Chem
38:759–767
Siegel RL, Miller KD, Jemal A (2019) Cancer statistics, 2019. CA
Cancer J Clin 69:7–34. https://doi.org/10.3322/caac.21551
Silverman RB, Tran N, Sinha RK, Neuhaus FC (1993) The oxazoli-
dinone antibacterial agent DuP 105 does not act on cell wall
biosynthesis or on a β-lactamase. Biochem Biophys Res Commun
195:1077–1080. https://doi.org/10.1006/bbrc.1993.2154
Skjemstad JO, Spouncer LR, Cowie B, Swift RS (2004) Calibration of
the Rothamsted organic carbon turnover model (RothC ver. 26.3),
using measurable soil organic carbon pools. Soil Res 42:79.
https://doi.org/10.1071/SR03013
Yolal M, Basoglu S, Bektas H et al. (2012) Synthesis of eperezolid-
like molecules and evaluation of their antimicrobial activities.
Russ J Bioorg Chem 38:539–549