Evaluation of Cytotoxic Properties of N,N'-bis[(1-aryl-3-heteroaryl)propylidene]-hydrazine dihydrochlorides

Article abstract of DOI:10.1007/s11094-020-02274-z

N,N'-bis[(1-aryl-3-heteroaryl)propylidene]hydrazine dihydrochlorides, P1, P4 – P8, and R1 – R7, were assayed against human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4), human promyelocytic leukemia cell line (HL-60), and human normal oral cells (HGF

Full text of DOI:10.1007/s11094-020-02274-z

DOI 10.1007/s11094-020-02274-z
Pharmaceutical Chemistry Journal, Vol. 54, No. 8, November, 2020 (Russian Original Vol. 54, No. 8, August, 2020)
EVALUATION OF CYTOTOXIC PROPERTIES
OF N,N¢-BIS[(1-ARYL-3-HETEROARYL)PROPYLIDENE]-
HYDRAZINE DIHYDROCHLORIDES
K. Kucukoglu,1,* H. I. Gul,2 and H. Sakagami3
Original article submitted February 5, 2020.
N,N¢-bis[(1-aryl-3-heteroaryl)propylidene]hydrazine dihydrochlorides, P1, P4 – P8, and R1 – R7, were as-
sayed against human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4), human promyelocytic leukemia
cell line (HL-60), and human normal oral cells (HGF, HPC, and HPLF) as non-tumor cells to evaluate their
cytotoxic properties. Peplomycin was used as a reference compound. It was found that P- and R-series
hydrazone compounds exhibited cytotoxicity in a range of 11 ± 0.68 – 300 ± 1.0 mM. Compound P1 which is
a non-substituted hydrazone containing piperidine ring and compound R2 which is a 4-methyl hydrazone de-
rivative containing pyrrolidine ring showed the most potent cytotoxic activity. These hydrazone compounds
may serve as promising candidates for further studies.
Keywords: cytotoxic activity, hydrazone, Mannich base, cancer, selectivity, anticancer drugs.
1. INTRODUCTION
antibacterial [10], monoamine oxidase inhibitory [11],
antiplatelet aggregation [12], and antiproliferative or antican-
cer [13 – 18] properties.
Hydrazones having the chemical structure of
R R C=NNH can be derived from a ketone or an aldehyde.
Substrates having at least one active hydrogen atom, an
aldehyde component (generally formaldehyde/paraformal-
dehyde), and an amine reagent react to form compounds gen-
erally known as Mannich bases. Mannich reactions are also
known as aminoalkylation reactions since the obtained prod-
uct is derivative of the substrate with an aminoalkyl moiety
[19, 20]. Mannich bases are important pharmacophores and
it is known that some compounds which are used today for
clinical treatment of various diseases and disorders have this
versatile scaffold, such as cocaine, fluoxetine, atropine,
ethacrynic acid, procyclidine, trihexyphenidyl, ranitidine,
and biperiden [21 – 23]. Furthermore, Mannich bases have
different biological activities including antifungal [24 – 26],
antimicrobial [27 – 29], anti-inflammatory [30 – 32],
anti-HIV [33], carbonic anhydrase inhibition [34 – 40],
cytotoxic and anticancer [18, 37, 40 – 50].
1
2
2
If both R1 and R2 are aryl or alkyl, it indicates that
hydrazone was derived from a ketone. Hydrazones are syn-
thesized by the reaction of hydrazine with a ketone or an al-
dehyde. The oxygen atom of ketone or aldehyde is replaced
with NNH functional group as a result of synthesis reaction
2
[1, 2]. a-Hydrogen atom in hydrazone is 10 times more
acidic than in ketone, therefore, this hydrogen atom is more
nucleophilic [3, 4]. The reactivity of hydrazones comes from
nucleophilicity of hydrogen carbon atom [5]. Furthermore,
two connected nitrogen atoms in hydrazone compounds have
different natures. Both nitrogen atoms of the hydrazone com-
pounds have nucleophilic character and the amino type nitro-
gen is more reactive. The carbon atom in hydrazone com-
pounds shows electrophilic and nucleophilic properties [6].
Hydrazones have been one of the most studied functional
groups because of diverse pharmacological activities includ-
ing anti-inflammatory [7], anti-HIV [8], antituberculosis [9],
Previously, we have designed and synthesized some
hydrazone compounds, N,N¢-bis[(1-aryl-3-heteroaryl)pro-
pylidene]hydrazine dihydrochlorides including P- and R-se-
ries via reaction of mono-Mannich bases, 1-aryl-3-hetero-
aryl-1-propanone, with hydrazine hydrate, evaluated their
cytotoxic activity against human hepatoma (Huh7) and
breast cancer (T47D) cell lines, and got some impressive re-
sults published in [16 – 18]. The present work was aimed to
investigate the cytotoxic effect of synthesized compounds
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Selcuk
University, Konya, Turkey.
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk
University, Erzurum, Turkey.
3
Meikai University Research Institute of Odontology (M-RIO), Sakado,
Saitama 350-0283, Japan
*
e-mail: kucukogluk35@hotmail.com
784
0091-150X/20/5408-0784 © 2020 Springer Science+Business Media, LLC

Evaluation of Cytotoxic Properties
785
Y: Piperidine-1-yl for P-Series, Pyrrolidine-1-yl for R-Series
R1 = R2 = H (P1); R1 = H, R2 = OH (P4); R1 = H, R2 = Cl (P5); R1 = OCH3, R2 = H (P6);
R1 = H, R2 = F (P7); R1 = H, R2 = Br (P8); R1 = R2 = H (R1); R1 = H, R2 = CH3 (R2);
R1 = H, R2 = OCH3 (R3); R1 = H, R2 = OH (R4); R1 = H, R2 = Cl (R5); R1 = OCH3, R2 = H (R6);
R1 = H R2 = F (R7)
Scheme 1. Synthesis of hydrazone compounds, N,N¢-bis[(1-aryl-3-heteroaryl)propylidene]hydrazine dihydrochlorides (P1, P4-P8; and
R1-R7). Reagents and conditions: (a) paraformaldehyde, piperidine HCl/pyrrolidine, HCl (37%) and EtOH, 4 – 9 h reflux for P1m, P4m-P8m;
R1m-R7m; (b) ethanolic acetic acid (3% w/v), hydrazine hydrate stirring for 17 – 26 h for P1, P4-P8; R2-R7 and 3 h reflux for R1.
(Scheme 1) against human oral squamous carcinoma cell
lines (HSC-2, HSC-3, HSC-4) and human leukemia cell line
(HL-60) as tumor cell lines and human promyelocytic nor-
mal oral cells (HGF, HPC, and HPLF) as non-tumor cells. It
was expected to find out new lead compound(s) for treating
dental cancer and leukemia problems.
3. EXPERIMENTAL BIOLOGICAL SECTION
The cytotoxicity of P- and R-series compounds was as-
sayed against three human oral squamous carcinoma cell
lines derived from tongue (HSC-2, HSC-3, HSC-4), human
leukemia cell line (HL-60), and human normal oral cells
(gingival fibroblasts, HGF; periodontal ligament fibroblasts,
HPLF; and pulp cells, HPC) as described in our previous pa-
pers [37, 40 – 47, 49, 50, 52 – 60].
2. EXPERIMENTAL CHEMICAL SECTION
In brief, all cells were cultured in Dulbecco’s modified
Eagle medium (DMEM) supplemented with 10% fetal bo-
vine serum (FBS). Cells (2.5 ´ 103 cells/well) were inocu-
lated and incubated for 48 h to achieve complete adherence.
Near confluent cells were incubated for a further 48 h in the
fresh culture medium containing each test compound (3.12,
6.25, 12.5, 25, 50, 100, 200, 400 mM) or peplomycin (posi-
tive control) (7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1000 mM).
The viable cell numbers were determined by the MTT
method. Cytotoxicity induced by DMSO (0.0078, 0.0156,
0.03125, 0.0625, 0.125, 0.25, 0.5 or 1%) was subtracted from
2.1. Materials and Methods
All commercially available reagents used in the synthesis
of P- and R-series hydrazones were purchased from Merck
AG, Fluka AG, Acros Organics, Riedel-de Haën, J. T. Baker
or Sigma-Aldrich Chemie and used without further purifica-
tion. Melting points were measured on an Electrothermal
9100 melting point apparatus (IA9100, Electrothermal,
Essex, UK). The 1H NMR (400 MHz) and 13C NMR
(100 MHz) spectra were recorded on Varian 400 MHz FT
spectrometer (Danbury, USA) for P- and R-series hydrazone
derivatives, while 1H NMR (60 MHz) spectra were recorded
on a Varian EM-360 spectrometer for Pm and Rm com-
pounds (precursor mono-Mannich bases).
the value for each well. The CC values were determined
50
from the dose-response curves. The tumor selectivity (TS)
has been calculated using the following equation:
mean CC50 against normal cells
TS =
mean CC50 against cancer cells
2.2. Synthesis
Precursor mono-Mannich bases, 1-aryl-3-(heteroaryl)-
1-propanone hydrochlorides (P1m, P4m – P8m, and
R1m – R7m) and their hydrazones; N,N¢-bis[(1-aryl-3-hete-
roaryl)propylidene]hydrazine dihydrochlorides (P1, P4 – P8,
R1 – R7) were freshly synthesized according to Scheme 1 as
described previously [16, 18].
and is shown as B/A in Table 1.
4. RESULTS AND DISCUSSION
In this paper, cytotoxic activities of the hydrazone com-
pounds; P1, P4 – P8, and R1 – R7, N,N¢-bis[(1-aryl-3-hete-

786
K. Kucukoglu et al.
roaryl)propylidene]hydrazine dihydrochlorides were investi-
gated against HSC-2, HSC-3, HSC-4 human oral squamous
carcinoma cells and HL-60 human leukemia cell line as tu-
mor cell lines and HGF, HPC, and HPLF human normal oral
cells as non-tumor cells since some compounds showed
rather impressive results against human hepatoma (Huh7)
and breast cancer (T47D) cell lines [30, 32] with hopes to
find out new lead compounds for treating dental cancer and
leukemia problems. The results are summarized in Table 1.
The results presented in Table 1 show that the
cytotoxicities of P- and R-series compounds against tumor
cell lines vary in the range of 11 ± 0.68 – 300 ± 1.0 mM.
These data suggest that P- and R-series compounds possess
cytotoxic properties. Peplomycin which is a semisynthetic
analog of Bleomycin, a mixture of several basic glycopeptide
antineoplastic antibiotics was used as a reference compound
to compare with the cytotoxic properties of P- and R-series
hydrazones. Compounds P1 (3 times), P5 (1.9 times), P6
(1.6 times), R2 (3.3 times), and R6 (1.9 times) had 1.6 – 3.3
times higher cytotoxicity than peplomycin against HSC-4
cell line. Compound R2 showed cytotoxicity (29 ± 9.0 mM)
comparable with that of peplomycin (28.3 ± 5.9 mM) against
HSC-2 cell line. A similar situation was observed for
cytotoxic activity against HL-60 cell line, where compounds
P7, P8 and the reference compound peplomycin showed al-
most the same activity (28 mM).
The tumor selectivity (TS) of each hydrazone compound
was previously calculated in [41 – 47, 49 – 59]. For this pur-
pose, the average CC value toward a total of four cancer
50
cell lines (TS = B/ A, Table 1) [37, 40, 42, 46, 59]. This cal-
culation pointed out that compounds P4 showed the highest
TS value (1.6). The other hydrazone derivatives having rela-
tively high TS values were compounds P5 and R3 with the
TS values of 1.4.
Thus, compound P1 which is
a non-substituted
hydrazone derivative containing piperidine, and compound
R2 which is a 4-methyl hydrazone derivative containing
pyrrolidine can be chosen as lead compounds for the cancers
mentioned above and used for further investigations. P- and
R-series hydrazone compounds were tested against HSC-2,
HSC-3, HSC-4, and HL-60 cancer cell lines, HGF, HPLF
and HPC normal cells. Compounds P1 and R2 were the
most potent cytotoxic agents with the highest tumor selectiv-
ity in P- and R-series. These compounds may serve as lead
molecules in further search of new drugs for solving cancer
problems under consideration.
ACKNOWLEDGEMENTS
This study was supported by the Research Foundation of
Ataturk University Erzurum (Turkey).
TABLE 1. Cytotoxic Evaluation of N,N¢-bis[(1-aryl-3-heteroaryl)propylidene]hydrazine Dihydrochlorides (P1, P4-P8; and R1-R7)
Hydrazone Compounds.
Compound
CC50 (mM)
Human
leukemia cell
line
Tumor-
specificity
index (TS)
Human oral squamous
carcinoma cell lines
Human oral normal cells
HSC-2
HSC-3
115 ± 40
248 ± 7.5
41 ± 2.6
83 ± 54
HSC-4
12 ± 0.5
78 ± 6.0
19 ± 3.1
23 ± 1.7
HL-60
88 ± 26
90 ± 9.1
61 ± 23
241 ± 87
28 ± 7.2
28 ± 4.0
125 ± 13
33 ± 5.0
125 ± 21
195 ± 26
53 ± 13
47 ± 0.95
98 ± 15
mean (A)
84
HGF
HPLF
HPC
mean (B)
78
B/A
0.9
1.6
1.4
0.7
0.4
1.0
1.2
0.6
1.4
0.6
1.2
0.2
1.2
15.1
P1
121 ± 11
226 ± 8.0
71 ± 6.1
283 ± 37
40 ± 3.8
60 ± 23
79 ± 14
156 ± 3.1
72 ± 14
72 ± 6.0
297 ± 32
92 ± 4.4
167 ± 145
25 ± 7.2
144 ± 41
161 ± 5.7
15.3 ± 3.5
148 ± 5.0
152 ± 1.7
73 ± 4.6
11 ± 1.0
103 ± 46
>400
84 ± 7.8
338 ± 3.5
44 ± 6.0
56 ± 24
P4
161
48
264
69
P5
P6
158
51
106
19
94 ± 63
P7
20 ± 1.0 114 ± 43
78 ± 8.8 300 ± 1.0
20 ± 1.0
138 ± 25
158 ± 1.0
17.3 ± 4.6
292 ± 6
13 ± 1.2
60 ± 9.7
84 ± 1.0
6.2 ± 3.0
264 ± 69
151 ± 41
58 ± 9.1
8.5 ± 1.2
129 ± 17
332 ± 38
P8
117
113
21
114
134
12.9
235
149
77
R1
125 ± 17
29 ± 9.0
231 ± 60
291 ± 23
72 ± 3.5
74 ± 19
110 ± 8
11 ± 6
92 ± 11
11 ± 0.68
163 ± 50
274 ± 17
64 ± 7.5
19 ± 0.95
R2
R3
168
253
64
152 ± 20
251 ± 11
66 ± 4.2
18 ± 10
R4
145 ± 3.8
99 ± 33
R5
R6
40
9.9
10.3 ± 0.55
152 ± 1.7
>400
R7
110
25
128
>377
126 ± 10
28.3 ± 5.9
87 ± 8.1 128 ± 4.4
Peplomycin
6.6 ± 0.80 36.2 ± 2.9 28.0 ± 87

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