Cytotoxicities of novel hydrazone compounds with pyrrolidine moiety: inhibition of mitochondrial respiration may be a possible mechanism of action for the cytotoxicity of new hydrazones

Article abstract of DOI:10.1007/s00044-018-2220-y

N,N’-Bis[1-aryl-3-pyrrolidine-1-yl)propylidene]hydrazine dihydrochlorides (R1–R7) were synthesized by the reaction of 2 mols of 1-aryl-3-(pyrrolidine-1-yl)-1-propanone hydrochlorides with 1 mol of hydrazine hydrate and reported for the first time with their detailed spectral analysis and cytotoxicities towards human hepatoma (Huh7) and breast cancer (T47D) cell lines. Compounds R2, R6, and R7 with the IC₅₀ values of 5.16, 6.96, and 5.96 μM, respectively, showed higher cytotoxic potency than the reference compound 5-FU with 7.0 μM against Huh7 cell line. However, all compounds did not show better cytotoxic activities than 5-FU against T47D cell line at the conditions studied. The representative compound of series, R2, inhibited the mitochondrial respiration at 90, 165, and 265 μM concentrations in a dose dependent manner in liver homogenates, suggesting that mitochondrial respiration may be one of the contributing factor to the cytotoxicity of the compounds synthesized. The compounds R2, R6, and R7 can be chosen as the leader compounds of this study for further studies.

Full text of DOI:10.1007/s00044-018-2220-y

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2220-y
ORIGINAL RESEARCH
Cytotoxicities of novel hydrazone compounds with pyrrolidine
moiety: inhibition of mitochondrial respiration may be a possible
mechanism of action for the cytotoxicity of new hydrazones
Kaan Kucukoglu1 Mustafa Gul Halise Inci Gul Rengul Cetin-Atalay Bernard Geny5
2 ●
3 ●
4 ●
●
Received: 22 March 2018 / Accepted: 7 July 2018
©
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
N,N’-Bis[1-aryl-3-pyrrolidine-1-yl)propylidene]hydrazine dihydrochlorides (R1–R7) were synthesized by the reaction of 2
mols of 1-aryl-3-(pyrrolidine-1-yl)-1-propanone hydrochlorides with 1 mol of hydrazine hydrate and reported for the first time
with their detailed spectral analysis and cytotoxicities towards human hepatoma (Huh7) and breast cancer (T47D) cell lines.
Compounds R2, R6, and R7 with the IC50 values of 5.16, 6.96, and 5.96 μM, respectively, showed higher cytotoxic potency
than the reference compound 5-FU with 7.0 μM against Huh7 cell line. However, all compounds did not show better cytotoxic
activities than 5-FU against T47D cell line at the conditions studied. The representative compound of series, R2, inhibited the
mitochondrial respiration at 90, 165, and 265 µM concentrations in a dose dependent manner in liver homogenates, suggesting
that mitochondrial respiration may be one of the contributing factor to the cytotoxicity of the compounds synthesized. The
compounds R2, R6, and R7 can be chosen as the leader compounds of this study for further studies.
●
●
●
●
●
Keywords Cytotoxic activity Huh7 T47D Hydrazone Mannich base Mitochondrial respiration
Introduction
determined and 8.2 million cancer mortality occurred in
2012 worldwide (Ferlay et al. 2015).
Cancer continues to be an enormous threatening problem
for the human health in the world. According to GLOBO-
CAN’s report (Estimated Cancer Incidence, Mortality and
Prevalence Worldwide) 14.1 million new cancer cases were
Breast cancer is one of the most common type of malig-
nancy and cause of mortality in women worldwide (Rafie-
manesh et al. 2016). It really puts the life of numerous women
with rising morbidity rates continuously at risk (Tang et al.
2016). In 2018, over 30,000 new breast cancer cases will be
diagnosed in women over 80, accounting for 12% of all cases
in the United States (American Cancer Society, Breast Cancer
Facts & Figs. 2017–2018). Breast cancer reportedly accoun-
ted for 29% of all new cancer cases and 14% of all cancer-
related deaths among women in the world up to 2012 (Siegel
et al. 2013). Breast cancer can be characterized as the for-
mation of malignant or cancerous cells in the tissues of the
breast (Giordano et al. 2002). Some factors like age, history of
menopause, inheritancent, environment, diet, and obesity
have been associated with the occurrence of breast cancer
(Meo et al. 2017). Although there are various treatments
choices for breast cancer as surgical removal of cancerous
cells, mastectomy, radiation therapy, and hormone therapy
(Ferlay et al. 2015); chemotherapy is the one of the important
choices for the treatment.
*
Kaan Kucukoglu
kucukogluk35@hotmail.com
Halise Inci Gul
incigul1967@yahoo.com
1
2
3
4
5
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Bülent Ecevit University, Zonguldak, Turkey
Department of Physiology, Faculty of Medicine, Atatürk
University, Erzurum, Turkey
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Atatürk University, Erzurum, Turkey
Cancer Systems Biology Lab. Graduate School of Informatics,
METU, TR-06800 Ankara, Turkey
On the other hand, over 500,000 people are diagnosed
with hepatocellular carcinoma (HCC) every year (El-Serag
Fédération de Médecine Translationnelle, Faculté de Médecine-
EA 3072, Université de Strasbourg, Strasbourg, France

Medicinal Chemistry Research
2
011). Approximately, 80% of patients with cirrhosis his-
et al. 2015; Tugrak et al. 2015; Yerdelen et al. 2015a,
2015b; Gul et al. 2016; Tugrak et al. 2016; Unluer et al.
2016; Yamali et al. 2016; Tugrak et al. 2017; Yamali et al.
2017) activities.
tory are caught HCC, which is the sixth most common
cancer worldwide. Additionally, HCC which is the most
common primary hepatic malignancy ranks at third place in
the list of cancer-related mortality (Bruix and Llovet 2009;
El-Serag 2011; Kalra et al. 2013; Kalra et al. 2015). Sur-
gical removal of part of the liver, liver transplantation,
embolization, ablation, and targeted drugs are used for the
treatment of HCC (Ferlay et al. 2015; Siegel et al. 2015).
An important problem encountered is that most patients
with HCC have already reached metastatic stage at diag-
nosis (Song 2009).
On the basis of afore-mentioned problems, it is very
important to discover new compounds, which can be used
in the treatment of breast cancer and HCC effectively.
Hydrazones are attractive scaffold for medicinal chemist
with wide range of biological activities such as analgesic
and anti-inflammatory, antioxidant, antidepressant, anti-
microbial, anticonvulsant, anticholinesterase (Koçyiğit-
Kaymakçıoğlu et al. 2008; el-Gazzar et al. 2009; Abdel-
Wahab et al. 2011; Kümmerle et al. 2012; Bala et al. 2012;
Gaikwad et al. 2015; Emami and Dadashpour 2015; Roman
In some cases, the cytotoxicities of Mannich bases were
attributed to the produced alfa, beta-unsaturated ketones
from a Mannich bases under the suitable conditions. Pro-
duced enones alkylate cellular thiols (Erciyas et al. 1994;
Gul et al. 2001; Gul et al. 2002; Gul et al. 2005a, 2005b,
2005c). Additionally, inhibition of mitochondrial respira-
tion by Mannich bases (Kucukoglu et al. 2014), inhibition
of topoisomerase enzyme I (Mete et al. 2010) or II
(Yogeeswari et al. 2005), and effects on tubulin polimer-
isation (Emami and Raeesi 2016) were reported as the
possible mechanism of action of Mannich bases.
In our previous reports, we reported the cytotoxicities of
some Mannich bases with dimethylamine and their hydra-
zones against Jurkat cells (Kucukoglu et al. 2011), cyto-
toxicities of hydrazones of Mannich bases with piperidine
(Kucukoglu et al. 2014) and morpholine (Kucukoglu et al.
2016) against breast cancer and human hepatoma cell lines.
Since the results obtained pointed out to go further to get a
final conclusion about this type of hydrazone compounds, in
this study it was aimed: (i) to synthesize hydrazones of
Mannich bases with pyrrolidine having different pKa values
than the previously used amines. Hydrazone compounds
were designed as prodrugs of Mannich base to release it in
an acidic environment of tumor cells since Mannich base is
considered to be responsible from the bioactivity of the
compounds by producing enones (ii) to evaluate their
cytotoxicities against human hepatoma (Huh7) and breast
cancer (T47D) cell lines, and (iii) to test a representative
compound of the series on mitochondrial respiration to see
whether the cytotoxicity of the compounds may result from
inhibition of mitochondrial respiration.
2
015; Mohsen et al. 2015), and antitumoral activities
(
Kucukoglu et al. 2011; Kucukoglu et al. 2014; Kucukoglu
et al. 2016).
There are different considerations regarding the bioac-
tivity of the hydrazone scaffold. First, hydrazone was prone
to form hydrogen bond with molecular target and second,
N=C bond was easy to process additional reaction with the
nucleophile groups (for example, amino –NH and thiol
–
2
SH) in the target molecules. This feature was thought to
enhance antitumor activity of hydrazone scaffold (Zhang
et al. 2012). Furthermore, hydrazone bond is relatively
stable under normal physiological pH of the body such as
plasma (pH 7.4) but in tumor cells where acidity is high
because of the accumulation of acidic metabolites (pH
6
.43–6.83) it is cleavable in endosomes and lysosomes
(
Volk et al. 1993; Bae et al. 2005).
A compound containing at least one reactive hydrogen
Materials and methods
(
ketones, nitroalkanes, β-ketoesters, and β-cyanoacids),
Chemistry
formaldehyde, and primary or secondary amine or ammonia
react with together and give a product known as Mannich
base (Abdulrahman et al. 2008). Some Mannich bases and
their derivatives are already in the market: the anti-
depressant fluoxetine, pain killer tramadol, and trihex-
yphenidyl used in the treatment of Parkinson’s disease
Chemicals used for the synthesis of R-series hydrazone
compounds were as follows: acetophenone, 4’-hydro-
xyacetophenone, paraformaldehyde (Merck, Hohenbrunn,
Germany), 4’-methylacetophenone, 4’-chloroacetophenone,
diethyl ether (Fluka, Steinheim, Germany), 4’-methox-
(
Dimmock et al. 2000). Additionally, Mannich bases are an
yacetophenone,
3’-methoxyacetophenone,
4’-fluor-
important class of compounds in medicinal chemistry due to
wide range of biological activities such as antimicrobial
oacetophenone (Acros, Geel, Belgium), hydrazine hydrate,
ethanol, hydrochloric acid, methanol, acetic acid, ethyl acetate
(Riedel-de Haen, Seelze, Germany), acetone (JT Baker,
Deventer, Holland), pyrrolidine (Sigma-Aldrich Chemie,
Steinheim, Germany), cyclohexan (Merck, Darmstadt, Ger-
many). Melting points were measured on an Electrothermal
(
Gul et al. 2005a, 2005b, 2005c), anti-inflammatory (Gul
et al. 2009), anticonvulsant (Gul et al. 2004; Gul et al.
007), anti-HIV (Pandeya et al. 1999), cytotoxic and
anticancer (Gul et al. 2008; Bilginer et al. 2013; Kucukoglu
2

Medicinal Chemistry Research
9
100 melting point apparatus (IA9100, Electrothermal, Essex,
1
13
UK). H (400 MHz) and C NMR (Nuclear Magnetic
Resonance) (100 MHz) spectra were recorded employing a
Varian 400 MHz FT spectrometer (Danbury, USA) for
1
hydrazones R1–R7 while H NMR (60 MHz) spectra were
recorded on a Varian EM-360 spectrometer for starting
compounds mono Mannich bases R1m–R7m. Chemical
shifts were reported as (δ) ppm. Tetramethyl silane (TMS)
was used as an internal standard. Coupling constants (J) are
reported in Hertz. High resolution mass spectra (HRMS) were
taken on a V6 Waters Micromass ZQ (Waters Corporation,
USA) using TOF method.
General procedure for the synthesis of precursor
mono Mannich bases, 1-aryl-3-(pyrrolidine-1-yl)-1-
propanone hydrochlorides, R1m–R7m
Scheme 1 Synthesis of N,N’-Bis[1-aryl-3-(pyrrolidine-1-yl)propyli-
dene]hydrazine dihydrochlorides, R1–R7Reagents and conditions: a
Paraformaldehyde, pyrrolidine, HCl (37%) and EtOH, 8-9 h reflux b
Ethanolic acetic acid (3% w/v), hydrazine hydrate stirring, exception
R7 and 3 h reflux for R1
The mixture of acetophenone, paraformaldehyde, pyrroli-
dine, and hydrochloric acid (37%) in ethanol in 1: 1.5: 0.9:
1
.5 mol ratio, respectively, was refluxed for sometime and
then solvent was removed in vacuo (Scheme 1). The residue
obtained was crystallized from suitable solvent and, there-
fore, purified. Some-times it was needed to keep the com-
pound at 4 °C for crystallization. Crystals formed were
filtered, washed with diethyl ether, and dried.
precipitated compound was filtered and crystallized from
acetone. Experimental data of R-series hydrazone com-
pounds, R1–R7 (Scheme 1), are shown in Table 1.
The amount of suitable ketone (mmol), heating period
(
h), melting point (°C), crystallization solvent, and yield (%)
N,N’-Bis[1-phenyl-3-(pyrrolidine-1-yl)propylidene]hydrazine
of the compounds R1m–R7m were as follows: R1m
dihydrochlorides (R1)
(
(
(
20 mmol, 9, 155–158 °C, methanol-ether, 28%), R2m
20 mmol, 8, 157–161 °C, methanol-ether, 65%), R3m
20 mmol, 8, 180–183 °C, methanol, 32%), R4m (20 mmol,
A white solid, yield: 35%; mp 158–162 °C; UV (MeOH)
1
λmax(log ε) 244 (4.27) nm; H NMR (CDCl , 400 MHz): δ
3
8
1
, 187–190 °C, methanol, 32%), R5m (20 mmol, 9,
84–186 °C, ethanol, 31%), R6m (20 mmol, 8, 124–129 °C,
= 12.46 (2H, bs, 2HCl), 7.98 (d, 4H, J = 6.8 Hz, C2’-H,
C6’-H), 7.59 (t, 2H, J = 7.2 Hz, C4’-H), 7.47 (t, 4H, J =
8.0 Hz, C3’-H, C5’-H), 3.75 (t, 8H, J = 7.2 Hz, C2”-H2,
C5”-H ), 3.54 (t, 4H, J = 7.2 Hz, C3-H ), 1.75–2.40 (m,
methanol, 65%), R7m (20 mmol, 8, 132–137 °C, methanol,
9%). The precursor Mannich bases were registered in lit-
5
2
2
1
3
eratures with their melting points (Mills 1954; Adamson
et al. 1958; Taylor and Nobles 1960; Gautier et al. 1964;
Lehmann et al. 2005), so NMR data were not reported here.
12H, C3”-H , C4”-H , C2-H );
C NMR (CDCl3,
2
2
2
100 MHz): δ = 196.37 (C=N, C1), 135.73 (C, C1’), 134.33
(CH, C4’), 129.10 (CH, CH, C2’, C6’), 128.52 (CH, CH,
C3’, C5’), 54.13 (CH , CH , C2”, C5”), 50.15 (CH , C3),
2
2
2
General procedure for the synthesis of hydrazones,
N,N’-bis[1-aryl-3-(pyrrolidine-1-yl)propylidene]
hydrazine dihydrochlorides, R1–R7
34.88 (CH , C2), 23.56 (CH , CH , C3”, C4”); TOF ESI-
MS m/z (pos): 404.2906 (M +1, 12), 403.2844 (M , 39),
2
2
+
2
+
205.1413 (M-C H N , 15), 204.1370 (M-C H N , 100),
1
3
19
2
13 19
2
2
02.1440 (M-C H N , 66).
13 17 2
A solution of hydrazine hydrate (1 mol ratio) in ethanol was
added to the solution of mono Mannich base (2 mol ratio) in
ethanolic acetic acid (3% w/v). The mixture was stirred at
room temperature for 22–25 h (Scheme 1). The precipitated
compound was filtered, and dried. The corresponding
hydrazone derivatives were purified by crystallization from
suitable solvent. The crystals formed were filtered and dried
at room temperature. In the case of R1, the reaction mixture
was refluxed for 3 h, then the solvent was removed under
vacuum, and diethyl ether was added to the residue, then the
N,N’-Bis[1-(4-methylphenyl)-3-(pyrrolidine-1-yl)
propylidene]hydrazine dihydrochlorides (R2)
A light yellow solid, yield: 6%; mp 188–193 °C; UV
1
(MeOH) λmax(log ε) 312 (4.34) nm; H NMR (CD OD,
3
400 MHz): δ = 7.93 (d, 4H, J = 8.4 Hz, C2’-H, C6’-H),
7.35 (d, 4H, J = 8.1 Hz, C3’-H, C5’-H), 3.36–3.70 (m, 12H,
C3-H , C2”-H , C5”-H ), 2.42 (s, 6H, Ar–CH ), 1.80–2.40
2
2
2
3
1
3
(m, 12H, C3”-H , C4”-H , C2-H ); C NMR (CD OD,
2
2
2
3

Medicinal Chemistry Research
Table 1 Experimental data of hydrazones synthesized, R1–R7
Compounds
Formula
Aryl
MW
Time (h)
Crystallization solvent
Yield (%)
Melting point (°C)
R1
R2
R3
R4
R5
R6
R7
C26H36Cl2N4
C28H40Cl2N4
C H Cl N O
C6H5
474.23
502.26
534.25
506.22
542.15
534.25
510.21
3a
Acetone
35
6
158–62
188–93
183–5
190–4
186–8
180–3
204–6
4-CH3 C6H4
4-OCH C H
4
22
25
25
24
23
24
Ethanol
Ethanol
34
50
24
7
2
8
40
2
4
2
3
6
C26H36Cl2N4O2
C26H34Cl4N4
4-OH C6H4
4-Cl C6H4
Ethanol
Methanol/Ether
Acetonitril
Methanol/Acetone
C28H40Cl2N4O2
C26H34Cl2F2N4
3-OCH3 C6H4
4-F C6H4
7
aReflux time
1
(
00 MHz): δ = 163.12 (C=N, C1), 141.77 (C, C4’), 133.38
C, C1’), 129.54 (CH, CH, C2’, C6’), 127.36 (CH, CH,
C3’, C5’), 54.10 (CH , CH , C2”, C5”), 51.77 (CH , C3),
N,N’-Bis[1-(4-chlorophenyl)-3-(pyrrolidine-1-yl)propylidene]
hydrazine dihydrochlorides (R5)
2
2
2
2
5.39 (CH , C2), 22.78 (CH , CH , C3”, C4”), 20.22
A yellow solid, yield: 24%; mp 186–188 °C; UV (MeOH)
2
2
2
+
1
(
Ar–CH ); TOF ESI-MS m/z (pos): 432.3189 (M +H, 28),
λmax(log ε) 253 (4.47) nm; H NMR (CD OD, 400 MHz): δ
3
3
+
4
31.3147 (M , 100), 216.1590 (M-C H N , 51).
= 8.04 (d, 4H, J = 8.9 Hz, C2’-H, C6’-H), 7.55 (d, 4H, J =
14
19
2
8
.9 Hz, C3’-H, C5’-H), 3.00–3.80 (m, 12H, C2”-H , C5”-
2
N,N’-Bis[1-(4-methoxyphenyl)-3-(pyrrolidine-1-yl)
propylidene]hydrazine dihydrochlorides (R3)
H , C3-H ), 1.70–2.20 (m, 12H, C3”-H , C4”-H , C2-H );
2 2 2 2 2
1
3
C NMR (CD OD, 100 MHz): δ = 195.80 (C=N, C1),
3
140.06 (C, C4’), 134.64 (C, C1’), 129.80 (CH, CH, C2’,
A light yellow solid, yield: 34%; mp 183–185 °C; UV
C6’), 128.97 (CH, CH, C3’, C5’), 54.44 (CH , CH , C2”,
2 2
1
(
MeOH) λmax(log ε) 277 (4.54) nm; H NMR (CDCl ,
C5”), 50.06 (CH , C3), 34.29 (CH , C2), 22.86 (CH , CH ,
2 2 2 2
3
+
4
8
00 MHz): δ = 12.54 (bs, 2H, 2HCl), 7.96 (d, 4H, J =
.9 Hz, C2’-H, C6’-H), 6.92 (d, 4H, J = 8.9 Hz, C3’-H,
C3”, C4”); TOF ESI-MS m/z (pos): 472.2068 (M +H, 24),
+
471.2089 (M , 49), 239.1573 (M-C H ClNO , 32),
1
3
18
2
C5’-H), 3.85 (s, 6H, Ar–OCH ), 3.40–3.80 (m, 12H, C2”-
238.1291 (M-C H ClNO , 100).
13 18 2
3
H , C3-H , C5”-H ), 2.82–2.90 (m, 4H, C2-H ), 2.08–2.21
2
2
2
2
13
(
m, 8H, C3”-H , C4”-H ); C NMR (CDCl , 100 MHz): δ
N,N’-Bis[1-(3-methoxyphenyl)-3-(pyrrolidine-1-yl)
2
2
3
=
194.70 (C=N, C1), 164.49 (C, C4’), 130.93 (CH, CH,
propylidene]hydrazine dihydrochlorides (R6)
C2’, C6’), 128.80 (C, C1’), 114.24 (CH, CH, C3’, C5’),
5
5.80 (Ar–OCH ), 54.04 (CH , CH , C2”, C5”), 50.26
A light yellow solid, yield: 7%; mp 180–183 °C; UV
3
2
2
1
(
CH , C3), 34.44 (CH , C2), 23.57 (CH , CH , C3”, C4”);
(MeOH) λmax(log ε) 304 (4.22) nm; H NMR (CD OD,
2
2
2
2
3
+
TOF ESI-MS m/z (pos): 464.3128 (M +H, 53), 463.3096
400 MHz): δ = 7.55–7.60 (d, 2H, J = 1.1 Hz, C2’-H), (dd,
2H, J = 8.0 Hz, 1.2 Hz, C4’-H), 7.46 (t, 2H, J = 8.0 Hz,
C5’-H), 7.13 (dd, 2H, J = 8.3 Hz, C6’-H), 3.88 (s, 6H,
Ar–OCH ), 3.35–3.60 (m, 12H, C2”-H , C5”-H , C3-H ),
+
(
M , 24), 235.1519 (M-C H N O, 29), 234.1394 (M-
14 21 2
C H N O, 100).
1
4
21
2
3
2
2
1
2
3
N,N’-Bis[1-(4-hydroxyphenyl)-3-(pyrrolidine-1-yl)
1.90–2.20 (m, 12H, C3”-H , C4”-H , C2-H ); C NMR
2
2
2
propylidene]hydrazine dihydrochlorides (R4)
(CD OD, 100 MHz): δ = 163.06 (C=N, C1), 160.44 (C,
3
C3’), 137.46 (C, C1’), 130.10 (CH, C2’), 119.81 (CH, C4’),
116.38 (CH, C5’), 113.02 (CH, C6’), 54.95 (Ar–OCH3),
54.15 (CH , CH , C2”, C5”), 51.69 (CH , C3), 25.72 (CH ,
A white solid, yield: 50%; mp 190–194 °C; UV (MeOH)
λmax(log ε) 282 (4.51) nm; H NMR (DMSO, 400 MHz): δ
1
2
2
2
2
=
4
10.83 (bs, 2H, 2 HCl), 10.60 (s, 2H, Ar–OH), 7.85 (d,
H, J = 8.8 Hz, C2’-H, C6’-H), 6.89 (d, 4H, J = 8.8 Hz,
C3’-H, C5’-H), 3.36–3.51 (m, 12H, C3-H , C3”-H , C5”-
C2), 22.79 (CH , CH , C3”, C4”); TOF ESI-MS m/z (pos):
2 2
464.3109 (M +H, 19), 463.3077 (M , 78), 232.1561 (M-
+
+
C H N O, 100).
2
2
14 19
2
1
3
H ), 1.65–2.20 (m, 12H, C3”-H , C4”-H , C2-H );
2
C
2
2
2
NMR (DMSO, 100 MHz): δ = 195.42 (C=N, C1), 163.31
C, C4’), 131.26 (CH, CH, C2’, C6’), 128.11 (C, C1’),
N,N’-Bis[1-(4-fluorophenyl)-3-(pyrrolidine-1-yl)propylidene]
hydrazine dihydrochlorides (R7)
(
1
4
16.05 (CH, CH, C3’, C5’), 53.63 (CH , CH , C2”, C5”),
2 2
9.73 (CH , C3), 34.35 (CH , C2), 23.36 (CH , CH , C3”,
A white solid, yield: 7%; mp 204–206 °C; UV (MeOH) λ
2
2
2
2
max
+
1
C4”); TOF ESI-MS m/z (pos): 436.2761 (M +H, 17),
(log ε) 305 (4.25) nm; H NMR (CD OD, 400 MHz): δ =
3
+
4
35.2760 (M , 47), 221.1317 (M-C H N O, 9), 220.1317
8.13 (dd, 4H, J = 9.1 Hz, C2’-H, C2’-F C6’-H, C6’-F), 7.27
(t, 4H, J = 8.8 Hz, C3’-H, C3’-F, C5’-H, C5’-F), 3.30–3.80
13
19
2
(
M-C H N O, 100).
13 19 2

Medicinal Chemistry Research
(
2
1
(
m, 12H, C2”-H , C5”-H , C3-H ), 3.13 (m, 4H, C2-H ),
.07 (m, 8H, C3”-H , C4”-H ); C NMR (CD OD,
2 2 3
00 MHz): δ = 166.09 (C=N, C1), 163.59 (C, C4’), 132.42
C, C1’), 129.95 (CH, CH, C2’, C6’), 115.86 (CH, CH,
bound to cellular proteins, was solubilized by adding 200 ml
of 10 mM Tris-base solution and the absorbance was
acquired at 515 nm. Absorbance values of the compounds
were normalized to their corresponding controls (DMSO)
and then, IC50 values were calculated. The IC50 values
reported the average of two independent determinations,
which differed by < 10% (Shoemaker 2006; Mete et al.
2011; Kucukoglu et al. 2011; Kucukoglu et al. 2014;
Kucukoglu et al. 2016). Cytotoxic activity of hydrazones
R1–R7 (Scheme 1) against Huh7 and T47D cells are shown
in Table 2.
2
2
2
2
13
C3’, C5’), 54.03 (CH , CH , C2”, C5”), 51.44 (CH , C3),
2
2
2
2
5.44 (CH , C2), 22.80 (CH , CH , C3”, C4”); TOF ESI-
2 2 2
+
+
MS m/z (pos): 440.2685 (M +H, 16), 439.2562 (M , 57),
20.1358 (M-C H FN , 100).
2
1
3
16
2
Biological activity
Cytotoxicity of R-series compounds against human
hepatoma (Huh7) and breast cancer (T47D) cells
Inhibition of mitochondrial respiration assay
Cytotoxic activity tests of R-series compounds were per-
formed in KANILTEK screening lab Bilkent University,
Ankara, Turkey. R-Series hydrazone compounds, R1–R7,
were tested with (NCI) anticancer drug screening method
Animals
Experiments were performed on nine adult male Wistar rats
(8-week-old), which were housed in a thermo-neutral
environment (22 ± 2 °C), on a 12:12 h photoperiod. They
were provided food and water ad libitum. This investigation
was carried out in accordance with the Guide for the Care
and Use of Laboratory, which was published by the US
National Institute of Health and approved by the institutional
animal care committee (NIH publication No. 85–23, revised
1996). Animals were submitted to a general anesthesia with
3% isoflurane and oxygen (1 L/min) in an induction cham-
ber (Minerve, Esternay, France). The liver was excised,
cleaned and then immediately used for mitochondrial
respiratory parameters analysis (Kindo et al. 2012).
(
Shoemaker 2006) with the aim of identifying R1–R7 with
growth-inhibitory activity against human hepatoma cells
Huh7) and breast cancer cells (T47D). 5-Fluorouracil (5-
(
FU) was used as the reference compound. T47D and Huh7
cells (5000 or 10000) were inoculated into 96-well micro-
titer plates in 100 µl of standard DMEM (Dulbecco's
Modified Eagle's Medium) medium (Gibco-Invitrogen
Corp., Grand Island, NY) 24 h prior to treatment with
inhibitors. R-Series hydrazone compounds, R1–R7, with
various concentrations (2.5–40 µM) were applied in addi-
tional 100 µl of cell culture medium. After 72 h of treatment
with inhibitors, the cell culture medium was fixed by gentle
addition of 50 µl of ice-cold 10% (w/v) trichloroacetic acid
Study of mitochondrial respiration
(
TCA) for 60 min at 4 °C. Then, cell culture medium was
discarded and washed with distilled water and dried in air.
After this process, 50 µl of a 0.4% (m/v) of sulforhodamine
B (SRB) in 1% acetic acid solution was applied to each well
and incubated for 10 min at room temperature. Extra
unbound dye was washed five times with 200 µl of 1%
acetic acid and air-dried. Sulforhodamine stain, which was
Isolation of liver mitochondria
The liver was maintained on ice finely minced in ice-cold
isolation buffer (50 mM Tris, 1 mM EGTA, 70 mM
Sucrose, 210 mM Mannitol, pH 7.4 at 4 °C), and then,
homogenized with a Potter-Elvehjem device. The obtained
homogenate was centrifuged at 1300 g for 3 min at 4 °C.
Then, the supernatant was ultracentrifuged at 10000 g for
Table 2 Cytotoxic activity of hydrazones R1–R7 (Scheme 1) against
Huh7 and T47D cells (IC , μM)
10 min at 4 °C to sediment mitochondria. Finally, the
50
mitochondrial pellet was washed twice. After that, it was
suspended in 50 mM Tris, 70 mM Sucrose, 210 mM Man-
nitol, pH 7.4 at 4 °C (Guillot et al. 2014; Mansour et al.
Compounds
Huh7
T47D
R1
R2
R3
R4
R5
R6
R7
231.31
5.16
*
34.70
84.48
169.23
28.10
21.34
18.32
15.76
7.0
2
014). Protein content was routinely assayed with a Brad-
ford assay using bovine serum albumin (BSA) as a standard
Bradford 1976). Mitochondria were kept on ice until use.
(
*
126.34
6.96
5.96
42.12
Measurement of the mitochondrial respiratory chain
complexes
5
-FU
Mitochondrial respiration was measured by using a Clark
type electrode (Strathkelvin Instruments, Glasgow, UK) as
5
-FU 5-Fluorouracil, * No inhibition

Medicinal Chemistry Research
mentioned before (Charles et al 2011). Before oxygraph
measurement, 3 ml of solution M (100 mM KCl, 50 mM
Mops, 1 mM EGTA, 5 mM Kpi, 1 mg/ml BSA) was added
to the oxygraph chambers for 10 min then, 0.15 mg of
isolated liver mitochondria were introduced with 10 mM
glutamate and 2.5 mM malate. The temperature was main-
tained at 25 °C. After that, succinate (25 mM) and adeno-
sine diphosphate (2 mM) were added. Mitochondrial
respiration in these conditions allowed to determine the
activities of the complexes I, II, III, IV, and V (Fig. 1). After
that, the representative methyl-bearing compound R2 was
added at different concentrations.
Results are expressed as mean ± SEM. Statistical ana-
lyses were performed using one-way ANOVA followed by
a Newman-Keuls post test (GraphPad Prism 5, Graph Pad
Software, Inc., San Diego, CA, USA). Statistical sig-
nificance required a p < 0.05.
3.75 ppm, which belonged to the protons of methylene
group in pyrrolidine ring, the other triplet at δ 3.54 ppm
belonged to the protons of ethylene chain next to the
nitrogen atom in pyrrolidine ring. 12-Proton-multiplet
observed at δ 1.75–2.40 ppm belonged to the protons in
positions 3 and 4 in pyrrolidine ring according to the
nitrogen atom and the protons connected to the carbon atom
in the ethylene chain next to the imine group. When inter-
1
3
pretating C NMR spectrum of compound R1, the signal
belonged to the carbon atom of imine group, as expected,
was at δ 196.37 ppm. This signal was the most important
signal, which confirmed the structure of compound R1. The
signal of carbon atoms in phenyl ring connected to the
carbon atom of imine group was observed at δ 135.73 ppm.
The other protons of phenyl ring gave signals at δ 134.33, δ
129.10, and δ 128.52 ppm. The signal observed at δ
54.13 ppm in the lowest aliphatic area belonged to the
carbon atoms of methylene groups next to the nitrogen atom
in pyrrolidine ring. The carbon atoms next to the nitrogen
atom in the ethylene chain, which was connected to the
imine group and pyrrolidine ring gave signal at δ
50.15 ppm. The carbons of ethylene chain next to the imine
group gave signal at δ 34.88 ppm. The signal, which was in
the highest area in the spectrum, belonged to the carbon
atoms of methylene groups in the positions of 3 and 4
according to the nitrogen atom in pyrrolidine ring and was
observed at δ 23.56 ppm. In HRMS spectrum of R1,
molecular ion signal, which confirmed the structure of R1
was observed at m/z 403.28. M+1 signal was at m/z
404.28. Two signals that had the highest rate of relative
abundance and confirmed that the fragmentation of the
molecule occurred between the imine groups were at m/z
202.14 and m/z 204.14, respectively.
Results and discussion
New hydrazones, N,N’-Bis[1-aryl-3-pyrrolidine-1-yl)pro-
pylidene]hydrazine dihydrochlorides (R1–R7, Scheme 1),
were successfully synthesized for the first time by this study
1
and their chemical structures were confirmed by H NMR,
1
3
C NMR, and HRMS spectral data. As an example com-
1
pound R1’s spectral data can be summarized. In the H
NMR spectrum of R1, the 2-proton-signal at δ 12.46 ppm,
which was in the lowest area in the spectrum confirmed
dihydrochloride salt of R1. The protons belonged to the
phenyl ring in R1 molecule gave a doublet and two triplets
at δ 7.98 ppm, δ 7.59 ppm and δ 7.20 ppm, respectively. The
protons of methylene group in pyrrolidine ring next to the
nitrogen atom in pyrrolidine ring and the protons of ethy-
lene group next to the nitrogen atom were interacted each
other and gave two triplets. One of which was at δ
Cytotoxic activity results of the R1–R7 against human
hepatoma (Huh7) and breast cancer (T47D) cell lines are
shown in Table 2. The representative compound R2
inhibited the mitochondrial respiration significantly at 165
and, 265 µM concentrations dose dependently in liver
homogenates (Fig. 2).
When the cytotoxicities of the compounds were con-
sidered, the compounds R2 (8.16 times, 4-methyl deriva-
tive), R6 (6.05 times, 3-methoxy derivative), and R7 (7.06
times, 4-fluoro derivative) were 6.05–8.16 times more
cytotoxic than the reference compound 5-Fluorouracil (5-
FU) towards Huh7 (human hepatoma) cell line (Table 2).
However, the compounds had generally presented similar
cytotoxicity towards T47D cell line, however, their cyto-
toxicities were less than 5-FU’s. A substituent on phenyl
ring rather than hydrogen, whether it is electron donating or
electron attracting, was a useful modification in increasing
the cytotoxicity towards Huh7 cell line. On the other hand,
when the cytotoxicity of the compounds towards T47D cell
line were compared, although the compounds R6 and R7
Fig. 1 Schematic representation of the mitochondrial respiratory chain
with specific substrates and inhibitors. CI: complex I, CII: complex II,
CIII: complex III, CIV: complex IV, TMPD: N,N,N’,N’-tetramethyl-
p-phenylenediamine dihydrochloride, H: proton (Thaveau et al. 2007)

Medicinal Chemistry Research
cells used to produce biologically active alkylation center if
the mechanism of action is thiol alkylation. Nevertheless,
the reason of absence or low cytotoxicity for the compounds
of R-series (especially against T47D cells) may be
explained by faster deamination of the compounds than
optimal level needed in the tested cells, which can cause
faster elimination of the active metabolite before reaching
the target. Another explanation for the absence or low
cytotoxicity of the compounds in T47D cells can be the lack
of interaction of the compounds with the suitable receptor/s
for the bioactivity in question. Another explanation for the
absence the cytotoxicity may be the formation of 1-aryl-3-
(3-aryl-4,5-dihydropyrazol-1-yl)propan-1-one, which does
not have a suitable center for thiol alkylation (Dimmock and
Kumar 1997; Gul et al. 2003).
There was no correlation between cytotoxicity of the R-
series and log P-value of the compounds (data not shown).
So, the cytotoxicities of R-series can not be referred to the
solubilities of the compounds.
Fig. 2 In vitro effects of increasing concentrations of compound R2 on
rat-liver mitochondrial respiratory chain. p < 0.05 (one-way ANOVA
followed by Turkey)
R2, with the highest cytotoxic potential against Huh7
cell line, was considered as the representative compound of
the series to evaluate inhibition potential of the series on
mitochondrial respiration. The results of experiment figured
it out that R2 inhibited the mitochondrial respiration at 90,
165, and 265 µM concentrations in a dose dependent man-
ner in liver homogenates. This suggests that inhibition of
mitochondrial respiration may be one of the contributing
factor to the cytotoxicity of the hydrazone compounds of R-
series.
had similar cytotoxicity to each other, they had the lowest
IC50 values among the series towards T47D cell line, i.e.,
they were the most cytotoxic ones in the series. The dif-
ferences in cytotoxicities of the compounds towards dif-
ferent cancer cell lines may result from the divergent
chemical structure of the compounds, the nature of cell lines
used, and different mechanism of action of the compounds
in different cancer cell lines.
N,N’-Bis[1-aryl-3-pyrrolidine-1-yl)propylidene]hydra-
zine dihydrochlorides, R-series compounds, were designed
as acid sensitive compounds targeting cancer cells, which
have lower pH than normal tissues since hydrazones are
acid sensitive compounds. They can show their cytotoxi-
cities as suggested in the previous paper (Gul et al. 2003): it
can briefly be summarized as: a hydrazone derivative may
undergo a deamination process and may produce a N,N’-bis
Conclusions
N,N’-Bis[1-aryl-3-pyrrolidine-1-yl)propylidene]hydrazine
dihydrochlorides, R-series, were reported with their synth-
esis, spectral analysis, and cytotoxic activities against Huh7
and T47D cell lines for the first time here. Of the com-
pounds, R2 (8.16 times), R6 (6.05 times), and R7 (7.06
times) were more cytotoxic than 5-FU towards Huh7 cell
line while the most cytotoxic compound in series towards
T47 cell line was R7. The compound R2 inhibited the
mitochondrial respiration in dose dependent manner. This
suggets that hydrazones may show their cytotoxicity by
inhibition of mitochondrial respiration.
(
1-aryl-propylidene)hydrazine, then this can interact with
cellular thiol to form cytotoxicity. Another possibility, a
hydrazone compound in the acidic medium of the cancer
can hydrolize and form 2 mols of mono Mannich bases
(
Rm Series) from 1 mol of a hydrazone (R Series) first, then
the produced Mannich base may deaminate to generate
biologically active α, β-unsaturated ketone moiety, which
has the ability to alkylate cellular thiols to produce cyto-
toxicity. One of the reported possibility for Mannich bases
cytotoxicities is inhibition of mitochondrial respiration.
Mannich bases of styryl ketones inhibited mitochondrial
respiration in rat-liver cells and lead cytotoxicity (Hamon
et al. 1978; Dimmock et al. 1985).
Acknowledgements This study was supported by the Research
Foundation of Atatürk University and the KANILTEK anticancer
biomolecule screening facility, Bilkent University.
Compliance with ethical standards
Observed cytotoxicities with R-series of compounds
against cancer cell lines used may be explained with the
optimum deamination ratio of these compounds in cancer
Conflict of interest The authors declare that they have no conflict of
interest.

Medicinal Chemistry Research
References
Erciyas E, Erkaleli HI, Cosar G (1994) Antimicrobial evaluation of
some styryl ketone derivatives and related thiol adducts. J Pharm
Sci 83:545–548
Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M,
Parkin DM, Forman D, Bray F (2015) Cancer incidence and
mortality worldwide: sources, methods and major patterns in
GLOBOCAN 2012. Int J Cancer 136:E359–E386
Gaikwad DD, Chapolikar AD, Devkate CG, Warad KD, Tayade AP,
Pawar RP, Domb AJ (2015) Synthesis of indazole motifs and
their medicinal importance: an overview. Eur J Med Chem
Abdel-Wahab BF, Awad GE, Badria FA (2011) Synthesis, antimicrobial,
antioxidant, anti-hemolytic and cytotoxic evaluation of new
imidazole-based heterocycles. Eur J Med Chem 46:1505–1511
Abdulrahman M, Balakrishina A, Kalluraya M (2008) Synthesis,
characterization, antimicrobial and anthelmintic activity of some
sydnone-N-Mannich bases. J Ind Counc Chem 25:10–14
Adamson DW, Barrett PA, Billinghurst JW, Jones TSG (1958) Ami-
noalkyl tertiary carbinols and derived products. Part VI. The
stereochemistry of some 1-phenyl-1-(2-pyridyl)prop-1-enes, and
of some 3-(tertiaryamino)-1-phenyl-1-(2-pyridyl)prop-1-enes
carrying additional substituents J Chem Soc 312–324
American Cancer Society, Breast Cancer Facts & Figs. 2017–2018.
https://www.cancer.org/content/dam/cancer-org/research/cancer-
facts-and-statistics/breastcancer-facts-and-figures/breast-cancer-fa
cts-and-figures-2017-2018.pdf. Accessed 21 Nov 2017
Bae Y, Nishiyama N, Fukushima S, Koyama H, Yasuhiro M, Kataoka
K (2005) Preparation and biological characterization of polymeric
micelle drug carriers with intracellular pH-triggered drug release
property: tumor permeability, controlled subcellular drug dis-
tribution, and enhanced in vivo antitumor efficacy. Bioconjug
Chem 16:122–130
9
0:707–731
Gautier JA, Miocque M, Quan DQ (1964) Mannich reaction on p-
hydroxyacetophenone. Compt Rend 258:3731–3734
Giordano SH, Buzdar AU, Hortobagyi GN (2002) Breast cancer in
men. Ann Intern Med 137:678–687
Guillot M, Charles AL, Chamaraux-Tran TN, Bouitbir J, Meyer A,
Zoll J, Schneider F, Geny B (2014) Oxidative stress precedes
skeletal muscle mitochondrial dysfunction during experimental
aortic cross-clamping but is not associated with early lung, heart,
brain, liver, or kidney mitochondrial impairment. J Vasc Surg
6
0:1043–1051.e5
Gul HI, Calis U, Vepsalainen J (2004) Synthesis of some mono-Mannich
bases and corresponding azine derivatives and evaluation of their
anticonvulsant activity. Arzneimittelforschung 54:359–364
Gul HI, Calls U, Ozturk Z, Tutar E, Calikiran L (2007) Evaluation of
anticonvulsant activities of bis(3-aryl-3-oxo-propyl) ethylamine
hydrochlorides and 4-aryl-3-arylcarbonyl-1-ethyl-4-piperidinol
hydrochlorides. Arzneimittelforschung 57:133–136
Gul HI, Gul M, Vepsalainen J, Erciyas E, Hanninen O (2003) Cyto-
toxicity of some azines of acetophenone derived mono-mannich
bases against jurkat cells Biol Pharm Bull 26:631–637
Gul HI, Sahin F, Gul M, Ozturk S, Yerdelen KO (2005a) Evaluation of
antimicrobial activities of several Mannich bases and their deri-
vatives. Arch Pharm (Weinh) 338:335–338
Bala S, Uppal G, Kamboj S, Saini V, Prasad DN (2012) Design,
characterization, computational studies, and pharmacological
evaluation of substituted-N’-[(1E) substituted-phenylmethyli-
dene]benzohydrazide analogs. Med Chem Res 22:2755–2767
Bilginer S, Gul HI, Mete E, Das U, Sakagami H, Umemura N, Dim-
mock JR (2013) 1-(3-Aminomethyl-4-hydroxyphenyl)-3-pyr-
idinyl-2-propen-1-ones:
A novel group of tumour-selective
cytotoxins. J Enzym Inhib Med Chem 28:974–980
Bradford MM (1976) A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 72:248–254
Gul HI, Suleyman H, Gul M (2009) Evaluation of the anti-
inflammatory activity of N,N’-bis(3-dimethylamino-1-phenyl-
propylidene) hydrazine dihydrochloride. Pharm Biol 47:968–972
Gul HI, Tugrak M, Sakagami H (2016) Synthesis of some acrylo-
phenones with N-methylpiperazine and evaluation of their cyto-
toxicities. J Enzym Inhib Med Chem 31:147–151
Bruix J, Llovet JM (2009) Major achievements in hepatocellular car-
cinoma. Lancet 373:614–616
Charles AL, Guilbert AS, Bouitbir J, Goette-Di Marco P, Enache I,
Zoll J, Piquard F, Geny B (2011) Effect of postconditioning on
mitochondrial dysfunction in experimental aortic cross-clamping.
Br J Surg 98:511–516
Dimmock JR, Kandepu NM, Nazarali AJ, Motaganahalli NL,
Kowalchuk TP, Pugazhenthi U, Prisciak JS, Quail JW, Allen TM,
Le Clerc R, Santos CL, De Clercq E, Balzarini J (2000)
Sequential cytotoxicity: a theory evaluated using novel 2-[4-(3-
aryl-2-propenoyloxy)phenylmethylene]cyclohexanones and rela-
ted compounds. J Med Chem 43:3933–3940
Dimmock JR, Kumar P (1997) Anticancer and cytotoxic properties of
mannich bases. Curr Med Chem 4:1–22
Dimmock JR, Shyam K, Hamon NW, Patil SA, Smith PJ (1985) bis-
Mannich bases of styryl ketones as antileukemic agents. Neo-
plasma 32:85–91
el-Gazzar AB, Hafez HN, Nawwar GA (2009) New acyclic nucleo-
sides analogues as potential analgesic, anti-inflammatory, anti-
oxidant and anti-microbial derived from pyrimido[4,5-b]quino-
lines. Eur J Med Chem 44:1427–1436
El-Serag HB (2002) Hepatocellular carcinoma: an epidemiologic view.
J Clin Gastroenterol 35:72–78
Gul HI, Yerdelen KO, Das U, Gul M, Pandit B, Li PK, Dimmock JR
(2008) Synthesis and cytotoxicity of novel 3-aryl-1-(3’-dibenzy-
laminomethyl-4’-hydroxyphenyl)-propenones and related com-
pounds. Chem Pharm Bull (Tokyo) 56:1675–1681
Gul M, Atalay M, Gul HI, Nakao C, Lappalainen J, Hӓnninen O
(2005b) The effects of some Mannich bases on heat shock pro-
teins HSC70 and GRP75, and thioredoxin and glutaredoxin levels
in Jurkat cells. Toxicol Vitr 19:573–580
Gul M, Gul HI, Das U, Hanninen O (2005c) Biological evaluation and
structure-activity relationships of bis-(3-aryl-3-oxo-propyl)-
methylamine hydrochlorides and 4-aryl-3-arylcarbonyl-1-methyl-
4
-piperidinol hydrochlorides as potential cytotoxic agents and
their alkylating ability towards cellular glutathione in human
leukemic T cells. Arzneimittelforschung 55:332–337
Gul M, Gul HI, Hӓnninen O (2002) Effects of Mannich bases on
cellular glutathione and related enzymes of Jurkat cells in culture
conditions. Toxicol Vitr 16:107–112
Gul M, Gul HI, Vepsalainen J, Erciyas E, Hanninen O (2001) Effect of
acetophenone derived Mannich bases on cellular glutathione level
in Jurkat cells. Arzneimittelforschung 51:679–682
El-Serag HB (2011) Hepatocellular carcinoma. N Engl J Med
3
65:1118–1127
Emami S, Dadashpour S (2015) Current developments of coumarin-
based anti-cancer agents in medicinal chemistry. Eur J Med Chem
Hamon NW, Bassendowski DL, Wright DE, Dimmock JR, Noble LM
(1978) Effect of antineoplastic and cytotoxic Mannich Bases
1
02:611–630
derived from conjugated styryl ketones on mitochondrial
respiration in rat liver cells. J Pharm Sci 67:1539–1542
Emami S, Raeesi M (2016) Synthesis of ciprofloxacin-isatin con-
jugates as potential cytotoxic agents. J Mazandaran Univ Med Sci
Inci Gul H, Yamali C, Tugce Yasa A, Unluer E, Sakagami H, Tanc M,
Supuran CT (2016) Carbonic anhydrase inhibition and
2
6:161–169

Medicinal Chemistry Research
cytotoxicity studies of Mannich base derivatives of thymol. J
Enzym Inhib Med Chem 31:1375–1380
Kalra N, Gupta P, Chawla Y, Khandelwal N (2015) Locoregional
treatment for hepatocellular carcinoma: the best is yet to come.
World J Radiol 28:306–318
Kalra N, Kang M, Bhatia A, Duseja AK, Dhiman RK, Arya VK,
Rajwanshi A, Chawla YK, Khandelwal N (2013) Role of radio-
frequency ablation in unresectable hepatocellular carcinoma: An
Indian experience. Indian J Radiol Imaging 23:139–144
Kindo M, Gerelli S, Bouitbir J, Charles AL, Zoll J, Hoang Minh T,
Monassier L, Favret F, Piquard F, Geny B (2012) Pressure
overload-induced mild cardiac hypertrophy reduces left ven-
tricular transmural differences in mitochondrial respiratory chain
activity and increases oxidative stress. Front Physiol 3:332
Koçyiğit-Kaymakçıoğlu B, Elçin Oruç-Emre E, Unsalan S, Rollas S
Pandeya SN, Sriram D, Nath G, De Clercq E (1999) Synthesis, anti-
bacterial, antifungal and anti-HIV evaluation of Schiff and Man-
nich bases of isatin derivatives with 3-amino-2-methylmercapto
quinazolin-4(3H)-one. Pharm Acta Helv 74:11–17
Rafiemanesh H, Salehiniya H, Lotfi Z (2016) Breast cancer in Iranian
woman. Incidence by age group, morphology and trends. Asian
Pac J Cancer Prev 17:1393–1397
Roman G (2015) Mannich bases in medicinal chemistry and drug
design. Eur J Med Chem 89:743–816
Shoemaker RH (2006) The NCI60 human tumour cell line anticancer
drug screen. Nat Rev Cancer 6:813–823
Siegel RL, Miller KD, Jemal A (2015) Cancer statistics, 2015. A
Cancer Journal of Clinicians. CA Cancer J Clin 65:5–29
Siegel R, Naishadham D, Jemal A (2013) Cancer statistics, 2013. CA
Cancer J Clin 63:11–30
(
2008) Antituberculosis activity of hydrazones derived from 4-
Song IH (2009) Molecular targeting for treatment of advanced hepa-
tocellular carcinoma. Korean J Hepatol 15:299–308
Tang X, Li X, Li Z, Liu Y, Yao L, Song S, Yang H, Li C (2016)
Downregulation of CXCR7 inhibits proliferative capacity and
stem cell-like properties in breast cancer stem cells. Tumour Biol
37:13425–13433
fluorobenzoic acid hydrazide. Med Chem Res 18:277–286
Kucukoglu K, Gul M, Atalay M, Mete E, Kazaz C, Hanninen O, Gul
HI (2011) Synthesis of some Mannich bases with dimethylamine
and their hydrazones and evaluation of their cytotoxicity against
Jurkat cells. Arzneimittelforschung 61:366–371
Kucukoglu K, Gul HI, Cetin-Atalay R, Baratli Y, Charles AL,
Sukuroglu M, Gul M, Geny B (2014) Synthesis of new N,N’-bis
Taylor ED, Nobles WL (1960) Some ketonic Mannich bases. J Am
Pharm Assoc Am Pharm Assoc 49:317–319
[
1-aryl-3-(piperidine-1-yl)propylidene]hydrazine dihydrochlorides
Thaveau F, Zoll J, Rouyer O, Chafke N, Kretz JG, Piquard F, Geny B
(2007) Ischemic preconditioning specifically restores complexes I
and II activities of the mitochondrial respiratory chain in ischemic
skeletal muscle. J Vasc Surg 46:541–547
and evaluation of their cytotoxicity against human hepatoma and
breast cancer cells. J Enzym Inhib Med Chem 29:420–426
Kucukoglu K, Gul HI, Gul M, Cetin-Atalay R, Baratli Y, Geny B
(
2016) Cytotoxicity of hydrazones of morpholine bearing Man-
Tugrak M, Gul HI, Sakagami H (2015) Synthesis and cytotoxicities of
2-[4-hydroxy-(3,5-bis-aminomethyl)-benzylidene]-indan-1-ones.
Lett Drug Des Discov 12:806–812
Tugrak M, Gul HI, Sakagami H, Mete E (2017) Synthesis and antic-
ancer properties of mono Mannich bases containing vanillin
moiety. Med Chem Res 26:1528–1534
Tugrak M, Yamali C, Sakagami H, Gul HI (2016) Synthesis of mono
Mannich bases of 2-(4-hydroxybenzylidene)-2,3-dihydroinden-1-
one and evaluation of their cytotoxicities. J Enzym Inh Med
Chem 31:818–823
Unluer E, Gul HI, Demirtas A, Sakagami H, Umemura N, Tanc M,
Kazaz C, Supuran CT (2016) Synthesis and bioactivity studies of
1-aryl-3-(2-hydroxyethylthio)-1-propanones. J Enzym Inh Med
Chem 31:105–109
Volk T, Jӓhde E, Fortmeyer HP, Glüsenkamp KH, Rajewsky MF
(1993) pH in human tumour xenografts: effect of intravenous
administration of glucose. Br J Cancer 68:492–500
Yamali C, Gul HI, Sakagami H, Supuran CT (2016) Synthesis and
bioactivities of halogen bearing phenolic chalcones and their
corresponding bis Mannich bases. J Enzym Inh Med Chem
31:125–131
nich bases towards Huh7 and T47D cell lines and their effects on
mitochondrial respiration. Lett Drug Des Discov 13:734–741
Kucukoglu K, Mete E, Cetin-Atalay R, Gul HI (2015) Synthesis of 3-
aroyl-4-aryl-1-isopropylamino-4-piperidinols and evaluation of
the cytotoxicities of the compounds against human hepatoma and
breast cancer cell lines. J Enzym Inhib Med Chem 30:564–568
Kümmerle AE, Schmitt M, Cardozo SV, Lugnier C, Villa P, Lopes AB,
Romeiro NC, Justiniano H, Martins MA, Fraga CA, Bourguignon
JJ, Barreiro EJ (2012) Design, synthesis, and pharmacological
evaluation of N-acylhydrazones and novel conformationally con-
strained compounds as selective and potent orally active
phosphodiesterase-4 inhibitors. J Med Chem 55:7525–7545
Lehmann F, Currier EA, Olsson R, Hacksell U, Luthman K (2005)
Isochromanone-based urotensin-II receptor agonists. Bioorg Med
Chem 13:3057–3068
Mansour Z, Charles AL, Kindo M, Pottecher J, Chamaraux-Tran TN,
Lejay A, Zoll J, Mazzucotelli JP, Geny B (2014) Remote effects
of lower limb ischemia-reperfusion: impaired lung, unchanged
liver, and stimulated kidney oxidative capacities. Biomed Res Int
2
014:392390
Meo SA, Suraya F, Jamil B, Al Rouq F, Meo AS, Sattar K, Ansari MJ,
Alasiri SA (2017) Association of ABO and Rh blood groups with
breast cancer. Saudi J Biol Sci 24:1609–1613
Mete E, Gul HI, Canturk P, Topcu Z, Pandit B, Gul M, Li PK (2010)
Biological activity of 1-aryl-3-phenethylamino-1-propanone
hydrochlorides and 3-aroyl-4-aryl-1-phenethyl-4-piperidinols on
PC-3 cells and DNA topoisomerase I enzyme. Z Naturforsch C
Yamali C, Ozgun DO, Gul HI, Sakagami H, Kazaz C, Okuidara N
(2017) Synthesis and structure elucidation of 1-(2,5/3,5-difluor-
ophenyl)-3-(2,3/2,4/2,5/3,4-dimethoxyphenyl)-2-propen-1-ones
as anticancer agents. Med Chem Res 26:2015–2023
Yerdelen KO, Gul HI, Sakagami H, Umemura N (2015a) Synthesis
and biological evaluation of 1,5-bis(4-hydroxy-3-methox-
yphenyl)penta-1,4-dien-3-one and its aminomethyl derivatives. J
Enzym Inh Med Chem 30:383–388
65:647–652
Mete E, Gul HI, Cetin-Atalay R, Das U, Sahin E, Gul M, Kazaz C,
Dimmock JR (2011) The design and cytotoxic evaluation of some
Yerdelen KO, Gul HI, Sakagami H, Umemura N, Sukuroglu M (2015b)
Synthesis and cytotoxic activities of a curcumin analogue and its
bis-Mannich derivatives. Lett Drug Des Discov 12:643–649
Yogeeswari P, Sriram D, Kavya R, Tiwari S (2005) Synthesis and in-
vitro cytotoxicity evaluation of gatifloxacin Mannich bases.
Biomed Pharmacother 59:501–510
1
-aryl-3-isopropylamino-1-propanone hydrochlorides towards
human Huh-7 hepatoma cells. Arch Pharm (Weinh) 344:333–339
Mills J (1954) 1-(p-Chlorophenyl)-2-phenyl-4-(pyrrolidino)butenes.
U.S. 2,655,509. Chem Abstr 48:P13726c. 13 Oct. 1953
Mohsen UA, Koçyiğit-Kaymakçıoğlu B, Oruç-Emre EE, Kaplancıklı
ZA, Rollas S (2015) Studies on hydrazide-hydrazones derivatives
as acetylcholinesterase inhibitors. MÜSBED 1:10–14
Zhang B, Zhao YF, Zhai X, Fan WJ, Ren JL, Wu CF, Gong P (2012)
Design, synthesis and antiproliferative activities of diaryl urea
derivatives bearing N-acylhydrazone moiety. Chin Chem Lett
2
3:915–918