Antiproliferative Activity of 2-Aroyland 2-Heteroyl-1,1,3,3-Tetracyanoprop-2-en-1-ides

Article abstract of DOI:10.1007/s11094-020-02170-6

The influence of previously synthesized 2-aroyl-1,1,3,3-tetracyanoprop-2-en-1-ides on the growth of conditionally normal and tumor cells was studied in continuation of a search for new anticancer drugs. Cytotoxicities of the compounds were studied with respect to human tumor cell lines from the ATCC. All compounds were ineffective against melanoma and lung and ovary cancer cell lines and exhibited moderate activity in the other cases. The tested compounds exhibited highly selective effects because they were safe for conditionally normal skin fibroblasts.

Full text of DOI:10.1007/s11094-020-02170-6

DOI 10.1007/s11094-020-02170-6
Pharmaceutical Chemistry Journal, Vol. 54, No. 2, May, 2020 (Russian Original Vol. 54, No. 2, February, 2020)
ANTIPROLIFERATIVE ACTIVITY OF 2-AROYL-
AND 2-HETEROYL-1,1,3,3-TETRACYANOPROP-2-EN-1-IDES
M. A. Mar’yasov,1,* Ya. S. Kayukov,1 and O. E. Nasakin1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 54, No. 2, pp. 40 – 42, February, 2020.
Original article submitted April 9, 2019.
The influence of previously synthesized 2-aroyl-1,1,3,3-tetracyanoprop-2-en-1-ides on the growth of condi-
tionally normal and tumor cells was studied in continuation of a search for new anticancer drugs.
Cytotoxicities of the compounds were studied with respect to human tumor cell lines from the ATCC. All
compounds were ineffective against melanoma and lung and ovary cancer cell lines and exhibited moderate
activity in the other cases. The tested compounds exhibited highly selective effects because they were safe for
conditionally normal skin fibroblasts.
Keywords: 1,1,3,3-tetracyanopropenide, carbonitrile, cancer, antiproliferative activity, MTT assay.
According to the World Health Organization, 18.1 mil-
lion new cases of cancer and 9.6 million deaths from it were
recorded in 2018 [1]. Modern chemotherapy uses drugs that
typically have low efficacy and high toxicity. Therefore, the
search for new antitumor drugs is a crucial problem of con-
temporary healthcare.
EXPERIMENTAL CHEMICAL PART
The studied compounds were prepared by the previously
reported method.
Potassium
2-(4-methylbenzoyl)-1,1,3,3-tetracyano-
prop-2-en-1-ide (1a). 4-Methylacetophenone (0.1 mol) and
NaBr (5.0 g, 0.048 mol) were dissolved in DMSO (50 mL)
in a 150-mL beaker. The mixture was heated carefully to
85°C with stirring and treated quickly with ~5 mL of conc.
H SO . Foam formed because of the generation of Me S gas.
Polycarbonitrile compounds have been searched by us
over many years for new biologically active compounds with
antiproliferative activity as potential chemotherapy agents
[2 – 5].
2
4
2
In continuation of efforts in this direction, the antiproli-
ferative activity of previously synthesized 2-aroyl- and 2-he-
teroyl-1,1,3,3-tetracyanopro-2-en-1-ides 1a-j [6 – 7] was
studied.
The reaction temperature also increased. It was important to
keep the reaction temperature in the range 100 – 115°C.
When the reaction was finished (5 – 7 min), the bubbles of
Me S rapidly disappeared and the mixture became viscous.
2
1
The yellowish-orange oil that formed on cooling was dis-
solved in EtOH (50 mL). The resulting solution was used in
I. N. Ul’yanov Chuvash State University, Cheboksary, 428010 Russia.
*
e-mail: marsikprovisor@mail.ru
R = 4-CH3C6H4 (1a), 2-((CH3)2CHOOC)C6H4 (1b), 3,4-(OCH3)2C6H3 (1c), 4-NO2C6H4 (1d), 4-CH3OC6H4 (1e), 2-thienyl (1f), 3-ClC6H4 (1g), 2-furyl (1h),
4-BrC6H4 (1i), Ph (1j).
142
0091-150X/20/5402-0142 © 2020 Springer Science+Business Media, LLC

Antiproliferative Activity
143
the next step. Br (16.0 g, 0.1 mol) was dissolved in distilled
tion was finished (TLC monitoring). The obtained yellow so-
lution was quickly filtered and left to cool. The resulting
precipitate was filtered off and recrystallized from
i-PrOH—EtOH (1:1) [7].
2
H O (600 mL) with stirring. Malononitrile (13.2 g, 0.2 mol)
2
was dissolved in EtOH (50 mL). The mixture was poured
into the Br solution. A mixture containing 4-methylphenyl-
2
glyoxal was added dropwise to the resulting solution with
vigorous stirring for 30 min. The resulting white precipitate
was filtered off, rinsed with chilled EtOH, and used in the
next step without further purification.
EXPERIMENTAL BIOLOGICAL PART
Cytotoxicity of compounds was evaluated using the
MTT proliferation assay [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] (Promega, USA) [8 – 10].
MTT reagent is converted by mitochondrial dehydrogenases
of living cells into insoluble violet-colored formazan. The
formazan crystals are readily dissolved by organic solvents
such as i-PrOH or DMSO upon lysis of the cells. The activity
of mitochondrial dehydrogenases and; therefore, the viability
of the cells can be determined from the optical density of the
formazan solution (500 – 600 nm). [11].
3-(4-Methylbenzoyl)-cyclopropane-1,1,2,2-tetracarbonit
rile (0.05 mol) was added to a mixture of KOAc (0.07 mol)
and EtOH (20 mL) and stirred at 45 – 50°C until the solids
dissolved. The resulting dark-yellow solution was filtered,
cooled, and poured into KCl solution (10%, 75 mL). The
mixture was left for 30 min at 5 – 10°C. The resulting pre-
cipitate was filtered off, rinsed with Et O, and dried in air
2
[6].
Compounds 1c-j were prepared analogously.
Potassium 2-[2-(isopropylcarbonyl)benzoyl]-1,1,3,3-
tetracyanoprop-2-en-1-ide (1b). Ninhydrin monohydrate
(1.96 g, 0.01 mol) was dissolved in DMF (15 mL) with gen-
tle heating (50 – 55°C). The solution was treated with
malononitrile (1.32 g, 0.02 mol) and Py (5 drops, ~0.1 g),
stirred at 50 – 55°C for 5 – 7 min, and cooled to room tem-
perature. The dark-brown solution was neutralized by adding
H SO solution (25%), cooled again, treated dropwise with
Cytotoxicity of the compounds was studied using human
tumor cell lines from the ATCC (MCF7 breast adenocarcino-
ma; PC-3 prostate adenocarcinoma; A-498 kidney carci-
noma; SNB-19 glioblastoma; M-14 melanoma; OVCAR-4
ovary adenocarcinoma; NCI-H322M non-small-cell bronchi-
oloalveolar carcinoma; MDA-MB-231 metastatic breast
adenocarcinoma; HCT-116 colon carcinoma) and condition-
ally normal primary human skin fibroblasts. The investiga-
tion included construction of a dose—effect curve and deter-
mination of the half-maximum growth inhibition concentra-
2
4
Br (1.6 g, 0.01 mol), stirred at room temperature for 10 min,
2
and poured into distilled H O (150 mL). The resulting solid
2
(1¢,3¢-dioxo-1¢,3¢-dihydrospiro[cyclopropane-1,2¢-indene]-2,
2,3,3-tetracarbonitrile) was filtered off and recrystallized
from i-PrOH—MeCN (3:1). A suspension of 1¢,3¢-dioxo-
1¢,3¢-dihydrospiro[cyclopropane-1,2¢-indene]-2,2,3,3-tetraca
rbonitrile (0.27 g, 0.001 mol) and anhydrous KOAc (0.1 g,
0.001 mol) in i-PrOH (1.5 mL) was refluxed until the reac-
tion for tumor cells (IC ) and conditionally normal cells
50
(CC ).
50
Human skin fibroblasts were cultivated in DMEM me-
dium with added fetal bovine serum (10%), L-glutamine
(2 mM), penicillin (100 mg/mL), and streptomycin
(100 U/mL). Cells were inoculated in a 96-well plate with
TABLE 1. Half-maximum Cell Growth Inhibitory Concentration, Mean ± Standard Deviation
Cell line
Compound
MDA-MB
231
MCF-7
HSF
M-14
PC-3
NCI-H322M
SNB-19
A-498
HCT-116
OVCAR
1a
1b
1c
1d
1e
1f
35 ± 1.84 >250 ± 3.54 >250 ± 3.52 19 ± 0.42 192 ± 1.51
22 ± 0.27
48 ± 0.32
18 ± 0.41
39 ± 0.50
61 ± 0.58
46 ± 0.37
59 ± 0.45
73 ± 0.66
28 ± 0.34
35 ± 0.31
23 ± 0.28
22 ± 0.32
49 ± 0.41
37 ± 0.32
64 ± 0.54
37 ± 0.39
42 ± 0.51
56 ± 0.48
46 ± 0.32
59 ± 0.53
78 ± 0.62
91 ± 0.38
12 ± 0.25 >250 ± 3.03
33 ± 0.32 >250 ± 2.72
15 ± 0.30 >250 ± 3.11
37 ± 1.02 >250 ± 2.09 242 ± 1.67 97 ± 1.01
25 ± 0.74 >250 ± 2.98 168 ± 1.34 22 ± 0.50
41 ± 1.35 >250 ± 3.12 191 ± 1.82 31 ± 0.53
48 ± 1.01 >250 ± 3.35 248 ± 1.93 111 ± 0.99
57 ± 1.22 >250 ± 2.81 189 ± 2.02 35 ± 0.47
112 ± 0.94 >250 ± 2.66 240 ± 1.12 127 ± 1.12
36 ± 1.12 >250 ± 3.14 191 ± 1.24 35 ± 0.63
32 ± 1.31 >250 ± 3.04 178 ± 1.17 27 ± 0.48
72 ± 1.58 >250 ± 2.94 217 ± 1.55 98 ± 1.05
49 ± 0.62
34 ± 0.35
29 ± 0.45
59 ± 0.42
57 ± 0.55
71 ± 0.67
49 ± 0.53
36 ± 0.48
55 ± 0.52
31 ± 0.29
211 ± 2.08
47 ± 0.52 >250 ± 3.24
34 ± 0.36 >250 ± 3.13
63 ± 0.54 >250 ± 2.89
59 ± 0.49 >250 ± 3.10
44 ± 0.31 >250 ± 3.07
69 ± 0.63 >250 ± 2.93
1g
1h
1i
47 ± 0.39 115 ± 0.75
66 ± 0.57 101 ± 0.92 128 ± 1.02
79 ± 0.71 145 ± 1.07 151 ± 1.30
1j
Doxo-
rubicin
0.13 ± 0.05
0.8 ± 0.01 0.14 ± 0.09 0.1 ± 0.06 0.15 ± 0.07 0.1 ± 0.02 0.1 ± 0.00 0.08 ± 0.03 0.2 ± 0.10
0.15 ± 0.07

144
M. A. Mar’yasov et al.
3000 cells per well and cultivated in the presence of a phos-
other compounds were moderately active. Compound 1c [po-
tassium 2-(3,4-dimethoxybenzoyl)-1,1,3,3-tetracyanoprop-2-
en-1-ide] was more active, possibly because of the larger
lipophilicity of the radical. The proposed mechanism ex-
plaining the role of the carbonitrile in producing the activity
is related to the formation of H-bonds with various amino-
acid sequences in the enzymes [13].
phonium salt solution (1 mg/mL to 1 mg/mL) for 72 h at
37°C and 5% CO [12]. Grown cells were rinsed with fresh
2
medium and assayed by MTT as usual.
The reference drug was doxorubicin, currently one of the
most efficacious and frequently used drugs.
A mixture of growth medium (9 mL) and MTT reagent
(1 mL) (5 mg/mL in Hank’s solution) was prepared in a res-
ervoir for a multichannel pipette and used to fill a 96-well
plate. The prepared reaction mixture (100 mL) was placed
An analysis of the results showed that the synthesized
compounds, despite the weaker cytotoxicities for tumor
cells, had highly selective action because they were safe for
conditionally normal skin fibroblasts. This indicated that fur-
ther searching in this compound class was advisable.
into each plate well and incubated in a CO incubator for
2
3.5 h. Growth medium with the tested compounds was re-
moved carefully with a vacuum aspirator, trying not to
loosen the cells. When the incubation time was reached, the
growth medium with the reagent was removed with a vac-
uum aspirator. Each plate well was treated with DMSO
(100 mL) and incubated for 5 – 10 min. Violet color that ap-
peared was detected on a Tecan plate reader at 555 nm
(650 nm reference wavelength).
REFERENCES
1. International Agency for Research on Cancer, Press-release
No. 263, World Health Organization, (2018), 3 pp; https: //
www.who.int / cancer / PRGlobocanFinal.pdf.
2. O. E. Nasakin, A. N. Lyschikov, Ya. S. Kayukov, and V. P. She-
verdov, Pharm. Chem. J., 34(4), 170 – 185 (2000).
Results were processed using OriginPro 8 software. The
percent surviving cells in each test well was calculated rela-
tive to wells with the positive control, the survival of which
was taken as 100%. Next, cell viability expressed in percent
was plotted as a function of the decimal logarithm of the con-
centrations of added compounds. The resulting curve was an-
alyzed using the Fit Sigmoidal – DoseResp program in
OriginPro 8. The logarithm of the concentration at 50% cell
survival was found. The half-maximum growth inhibitory
3. V. P. Sheverdov, O. V. Ershov, and O. E. Nasakin, Pharm.
Chem. J., 42(12), 670 – 673 (2008).
4. V. P. Sheverdov,
A. Yu. Andreev,
O. E. Nasakin,
and
V. L. Gein, Pharm. Chem. J., 48(6), 379 – 382 (2014).
5. M. A. Mar’yasov, V. P. Sheverdov, V. V. Davydova, and
O. E. Nasakin, Pharm. Chem. J., 50(12), 519 – 522 (2017).
6. S. V. Karpov, A. A. Grigor’ev, Ya. S. Kayukov, et al., J. Org.
Chem., 81(15), 6402 – 6408 (2016).
7. S. V. Karpov, Ya. S. Kayukov, A. A. Grigor’ev, et al., Org.
Biomol. Chem., 14, 3758 – 3764 (2016).
concentrations of conditionally normal and tumor cells (IC ,
50
mM, Table 1) were calculated.
8. R. I. Freshney (ed.), Culture of Animal Cells: A Manual of Basic
Technique and Specialized Applications, 6th Ed., Wiley-Liss,
Inc., (2010).
The biological targets for cyano-containing drugs could
be various structures in transformed cells. The target could
be DNA molecules themselves and amino-acid residues such
as serine and arginine because cyano groups tend to form
H-bonds [13]. Blocking of the serine residue causes com-
plete inhibition of enzymatic catalysis (e.g., acetylcholine
esterase) and disruption of intercellular signal transduction,
which leads eventually to the death of the tumor cells.
Blocking the arginine residues disturbs nitrogen metabolism,
which also negatively affects cell growth.
9. MTT Cell Proliferation Assay. Instruction Guide. American
Type Culture Collection (2011); http: // atcc.org / ~ / media /
DA5285A1F52C414E864C966FD78C9A79.ashx.
10. A. H. Cory, T. C. Owen, J. A. Barltrop, and J. G. Cory, Cancer
Commun., 3(7), 207 – 212 (1991).
11. E. K. Anderberg, C. Nystrom, and P. Artursson, J. Pharm. Sci.,
81(9), 879 – 887 (1992).
12. M. V. Pugachev, N. V. Shtyrlin, L. P. Sysoeva, et al., Bioorg.
Med. Chem., 21, 4388 – 4395 (2013).
All compounds were ineffective against melanoma and
lung and ovary cancer (three independent experiments). The
13. F. F. Fleming, L. Yao, P. C. Ravikumar, L. Funk, and
B. C. Shook, J. Med. Chem., 53, 7902 – 7917 (2010).