Synthesis and cytotoxic activity of novel 4-amino-5-cyano-2-sulfonylpyrimidines

Article abstract of DOI:10.1016/j.mencom.2020.09.017

Novel 4-amino-5-cyano-2-sulfonylpyrimidines were prepared based on three-component cyclization between isothiouronium salts, benzaldehydes and malononitrile, followed by oxidation of the sulfide moiety with Oxone. The cytotoxic activity of the synthesized compounds, as well as the induction of apoptosis, inhibition of the cell cycle and proliferation tests were performed on selected cancer cell lines A431, A549, A375, HCT 116, MCF7, LNCap and SH-SY5Y.

Full text of DOI:10.1016/j.mencom.2020.09.017

Mendeleev
Communications
Mendeleev Commun., 2020, 30, 604–606
Synthesis and cytotoxic activity
of novel 4-amino-5-cyano-2-sulfonylpyrimidines
Dmitry A. Khochenkov,a,b Yulia A. Khochenkova,b Yulia S. Machkova,b Rovshan E. Gasanov,a
Eugenia V. Stepanovab and Alexander S. Bunev*a
a Medicinal Chemistry Center, Togliatti State University, 445020 Togliatti, Russian Federation.
Fax: +7 848 253 9494; e-mail: a.s.bunev@gmail.com, a.bunev@tltsu.ru
b Laboratory of Biomarkers and Mechanisms of Tumor Angiogenesis, N. N. Blokhin National Medical Research
Center of Oncology, 115478 Moscow, Russian Federation
DOI: 10.1016/j.mencom.2020.09.017
Novel 4-amino-5-cyano-2-sulfonylpyrimidines were prepared
based on three-component cyclization between isothiouro-
nium salts, benzaldehydes and malononitrile, followed by
oxidation of the sulfide moiety with Oxone. The cytotoxic
activity of the synthesized compounds, as well as the
induction of apoptosis, inhibition of the cell cycle and
proliferation tests were performed on selected cancer cell
lines A431, A549, A375, HCT 116, MCF7, LNCap and
SH-SY5Y.
Nꢀ2
N
Cꢀ
ꢀ
Cꢀ
ꢀ
O
O
S
ꢀ
S
ꢀ
O
O
ꢁ
ECꢂ0ꢃAꢄꢅꢆꢇ ꢈ ꢆ.ꢍꢎ ꢉM
ECꢂ0ꢃSꢀꢊSꢋꢂꢋꢇ ꢈ ꢄꢂ0 nM
ECꢂ0ꢃAꢄꢅꢆꢇ ꢈ ꢅ.ꢂꢆ ꢉM
ECꢂ0ꢃSꢀꢊSꢋꢂꢋꢇ ꢈ 2ꢌ0 nM
Cytotoꢏic effect on seven cancer cell lines
Keywords: heterocyclization, pyrimidines, isothiouronic salts, nitriles, sulfones, cytotoxic activity, cancer cell lines.
The development of new anticancer agents with various
+
NH2 X–
mechanisms of cytotoxic effect is an important task of modern
medicinal chemistry. Creation of low molecular weight inhibitors
of kinases,1 inducers of apoptosis,2 antiproliferative agents3–9
should be noted. Heterocyclic pyrimidine-based scaffolds have
shown special results in this respect.10–13 However, data on
5-cyanopyrimidine derivatives are scanty although some of them
manifested antibacterial and antifungal14 as well as immuno-
suppressive15 activities. On the basis of the 5-cyanopyrimidine
scaffold, inhibitors PI3Kd,16 p38a MAP kinase,17 Erk5,18
VEGFR-219 and LSD120 were obtained.
R
NC
CN
+
+
Ar
O
ii
S
NH2
X = Cl, I, HSO4 or MsO
NH2
NH2
N
CN
Ar
CN
Ar
i
N
N
O
R
S
N
S
R
O
Here, we report the synthesis of two series of cytotoxic
5-cyanopyrimidine derivatives (type I and II). Compounds of
type I (Scheme 1) were assembled by the three-component
reaction between aromatic aldehyde, isothiouronic salt and
malononitrile. Further oxidation of the sulfide moiety in the
Oxone/DMF/water system gave sulfones 1a–k. It should be
noted that the reported9 oxidation procedures for pyrimidine
sulfides turned out to be ineffective in our case and led to sulfone/
sulfoxide mixtures.
1a–k
a R = Me, Ar = Ph, 73%
b R = Et, Ar = Ph, 74%
c R = Pri, Ar = Ph, 89%
d R = Bn, Ar = Ph, 91%
e R = Me, Ar = 4-FC6H4, 80% k R = Me, Ar = 4-MeOC6H4, 66%
f R = Me, Ar = 3-FC6H4, 73%
g R = Me, Ar = 2-FC6H4, 87%
h R = Pr, Ar = 4-FC6H4, 86%
i
R = Pr, Ar = 3-FC6H4, 68%
j R = Pri, Ar = 4-FC6H4, 85%
Scheme 1 Reagents and conditions: i, K2CO3, EtOH, reflux, 12 h;
ii, Oxone, DMF/H2O (5:1), 50 °C, 2–4 h.
The preparation of type II pyrimidines 2a–i comprised the
application of ethyl cyanoacetate and required the introduction
of additional stages (Scheme 2). After the pyrimidine core was
assembled, the cycle NH-amide function was converted into
chloroimidoyl one on treatment with POCl3. Chlorine atom in
thus obtained 4-chloro-5-cyanopyrimidines was replaced by
pyrrolidine residue. The final oxidation afforded pyrrolidino
sulfones 2a–i.
The synthesized 5-cyanopyrimidine derivatives 1, 2 were
evaluated for cytotoxic activities by MTT assay against human
lung adenocarcinoma cell line A549, human epidermoid
carcinoma cell line A431, human melanoma cell line A375,
human colorectal cancer cell line HCT-116, human breast cancer
cell line MCF-7, human prostate cancer cell line LNCaP, and
human neuroblastoma cell line SH-SY5Y (Table 1).
The structures of new compounds 1a–k and 2a–i were
confirmed by NMR spectroscopy. As the pyrimidine moieties
The synthesized compounds exhibited stronger anticancer
activities against A431 and SH-SY5Y cell lines. Based on cell
viability data we selected compounds 1c and 2c from each
library for further study. As the compounds manifested cell
cytotoxic activities against A431, A549, and MCF-7 cell lines,
we selected some of them for further tests. To study the
1
were fully substituted, the H NMR spectra were deprived of
their signals. Meanwhile, the 13C NMR spectra contained peaks
for the quaternary pyrimidine carbon atoms (see Online
Supplementary Materials).
© 2020 Mendeleev Communications. Published by ELSEVIER B.V.
on behalf of the N. D. Zelinsky Institute of Organic Chemistry of the
Russian Academy of Sciences.
– 604 –

Mendeleev Commun., 2020, 30, 604–606
+
NH2 X–
O
distinguish healthy cells from apoptotic cells and necrotic cells.
+
+
Ar
R
O
CN
Using the apoptosis assay, we demonstrated increase in apoptotic
and necrotic cells in A431 cells following the treatment with 1c
and 2c. Incubation with 1c led to an increase of early, late apoptotic
and necrotic cells and to reduction of viable cells in A431 cell
lines. The percentage of total apoptotic cells (early and late
apoptotic cells) inA431 increased from 5.60% (control) to 45.09%
(4c) and 18.41% (6c), respectively (Figure S3). No significant
increase in apoptotic and necrotic cells was detected inA549 cells.
After treating A431 and A549 cells with compounds 1c and
2c at different concentrations (EC50 and EC80) for 24 h, cells
were fixed and stained with PI for flow cytometry. Compound 1c
arrested the cell cycle at G2/M phase in a concentration-
dependent manner, accompanied with the substantial decrease of
cells at G0/G1 phase at EC80 concentration (see Figure S3).
To determine the possible mechanisms of the antiproliferative
and cytotoxic action of derivatives 1c and 2c, we performed the
determination of the EGF-induced expression of EGFR and its
downstream signal molecules such as Akt, pAkt, ERK1/2 and
pERK1/2 by In-Cell ELISA assay (ThermoFisher) (Figure S5).
Component 1c caused a decrease in the expression of the
phosphorylated forms of the tyrosine kinase receptor, EGFR and
the intracellular signal molecules pAkt and pERK1/2.
S
NH2
EtO
X = Cl, I, HSO4 or MsO
Cl
N
O
CN
CN
Ar
i
ii
N
HN
R
R
S
Ar
S
N
N
N
N
CN
Ar
CN
Ar
iii
iv
N
N
O
S
R
S
N
R
O
2a–i
a R = Pri, Ar = Ph, 55%
f R = Me, Ar = 3-FC6H4, 85%
g R = Me, Ar = 2-FC6H4, 98%
h R = Et, Ar = Ph, 73%
b R = Bn, Ar = Ph, 85%
c R = Pri, Ar = 4-FC6H4, 77%
d R = Pri, Ar = 3-FC6H4, 56%
e R = Me, Ar = 4-FC6H4, 78%
i R = Pr, Ar = 4-FC6H4, 70%
Scheme 2 Reagents and conditions: i, K2CO3, EtOH, reflux, 8 h; ii, POCl3
(10 equiv.), 1,4-dioxane, reflux, 7–12 h; iii, pyrrolidine (2 equiv.), PriOH,
reflux, 5–8 h; iv, Oxone, DMF/H2O (5:1), 50 °C, 2–4 h.
To sum up, compound 1c exhibits inhibitory activity against
tumor cells of various origins, which suggests the presence of
inhibitory activity against other targets other than EGFR, and is
the subject of our further studies.
antiproliferative activity against A431 and A549 cell lines, we
performed clonogenic cell survival assay with selected
compounds (see Online Supplementary Materials, Figure S1).
Remarkably, compounds 1c and 2c exhibited similar inhibition
of colony formation in A549 and A431 cells contrary to other
selected compounds. Also, compounds 1c and 2c inhibited the
migration of A431 and A549 cells (see Online Supplementary
Materials, Figure S4). To elucidate the mechanisms responsible
for antiproliferative activity of compounds 1c and 2c, we
investigated inhibition of cell cycle and apoptosis induction.
Apoptosis was quantified by double staining of cells treated with
1c or 2c for 24 h with Annexin-V-FITC and PI (Figure S2) to
The study was supported by the Russian Foundation for
Basic Research (project no. 18-015-00321, and partly project
no. 18-33-00666).A. B., D. K. andE. S. designedtheexperiments,
Y. K., Y. M. and R. G. performed the experiments, A. B. and
D. K. analyzed the results, A. B., E. S., Y. K. and E. S. prepared
the manuscript.
Online Supplementary Materials
Supplementary data associated with this article can be found
in the online version at doi: 10.1016/j.mencom.2020.09.017.
Table 1 Cytotoxicity of the synthesized compounds against human cancer cell lines.
EC50/mm
Compound
A431
A549
A375
HCT116
MCF-7
LNCaP
SH-SY5Y
Etoposide
1.63±0.15
139.54±7.05
0.69±0.04
1.73±0.21
10.91±2.1
14.4±3.23
0.75±0.04
1a
1b
1c
1d
1e
1f
9.46±0.39
16.60±1.13
3.51±0.24
24.85±1.19
9.82±0.33
16.44±0.67
29.57±1.17
4.63±0.21
5.69±0.29
3.67±0.25
7.25±0.35
3.75±0.71
2.55±0.22
1.67±0.13
3.34±0.26
4.72±0.44
3.36±0.52
3.92±0.64
5.09±0.31
4.60±0.54
37.64±0.89
26.28±1.61
25.93±1.41
31.86±0.94
40.38±1.03
46.56±2.58
59.06±1.59
29.80±1.47
28.96±2.58
32.90±1.96
35.42±1.39
23.07±1.77
22.33±1.96
16.82±0.81
21.51±1.79
23.37±0.91
21.94±1.08
27.18±2.08
19.06±0.88
23.11±1.07
24.86±1.06
16.48±0.22
16.83±0.44
18.47±0.68
25.63±0.24
35.05±0.43
61.25±1.08
18.58±0.52
19.80±0.65
16.43±0.51
20.02±0.71
13.80±0.49
8.65±0.30
14.66±0.77
8.99±0.19
11.02±0.60
10.73±0.40
13.63±0.26
16.65±0.39
23.86±0.37
10.49±0.45
10.60±0.49
10.13±0.56
10.01±0.49
6.23±0.26
6.41±0.71
3.41±0.11
6.56±0.21
5.06±0.26
5.95±0.40
6.14±0.45
4.51±0.26
5.93±0.27
24.81±1.52
17.55±1.29
16.47±0.84
19.56±1.14
30.98±1.33
34.94±1.75
49.25±2.18
17.49±0.75
18.70±0.79
14.97±0.52
16.90±0.80
17.28±0.97
17.04±1.07
7.37±0.70
12.29±0.44
10.14±1.38
9.82±1.10
13.19±0.65
9.97±0.64
10.28±1.22
16.44±1.59
13.96±1.09
15.07±0.86
12.08±1.15
19.59±1.10
22.52±1.66
30.06±2.44
11.99±1.74
12.68±2.04
12.76±1.11
7.68±0.97
12.42±0.35
7.68±0.51
5.43±0.28
8.79±0.21
9.24±0.24
8.81±027
1.83±0.32
0.79±0.25
0.28±0.14
1.41±0.35
1.72±0.34
3.00±0.50
5.43±0.65
1.02±0.22
1.19±0.28
0.66±0.32
0.28±0.15
0.64±0.51
1.94±0.543
0.45±0.10
0.84±0.36
0.85±0.23
1.25±0.23
0.78±0.43
0.19±0.11
0.97±0.18
1g
1h
1i
1j
1k
2a
2b
2c
2d
2e
2f
5.31±0.14
11.77±0.34
14.57±0.36
12.12±0.31
11.45±0.89
10.47±0.34
15.01±0.32
2g
2h
2i
8.67±0.54
7.87±0.34
9.03±0.20
– 605 –

Mendeleev Commun., 2020, 30, 604–606
13 M. W. N. Deininger and B. J. Druker, Pharmacol. Rev., 2003, 55, 401.
References
14 H. B. Ghodasara, A. R. Trivedi, V. B. Kataria, B. G. Patel andV. H. Shah,
Med. Chem. Res., 2013, 22, 6121.
1 D. Fabbro, Mol. Pharmacol., 2015, 87, 766.
2 Q. Jiao, L. Bi, Y. Ren, S. Song, Q. Wang and Y.-S. Wang, Mol. Cancer,
2018, 17, 36.
15 A. Stella, K. Van Belle, S. De Jonghe, T. Louat, J. Herman, J. Rozenski,
M. Waer and P. Herdewijn, Bioorg. Med. Chem., 2013, 21, 1209.
16 L. Patel, J. Chandrasekhar, J. Evarts, A. C. Haran, C. Ip, J. A. Kaplan,
M. Kim, D. Koditek, L. Lad, E.-I. Lepist, M. E. McGrath, N. Novikov,
S Perreault, K. D. Puri, J. R. Somoza, B. H. Steiner, K. L. Stevens,
J. Therrien, J. Treiberg, A. G. Villasenor, A. Yeung and G. Philips,
J. Med. Chem., 2016, 59, 3532.
17 C. Liu, S. T. Wrobleski, J. Lin, G. Ahmed, A. Metzger, J. Wityak,
K. M. Gilloly, D. J. Shuster, K. W. McIntyre, S. Pitt, D. R. Shen,
R. F. Zhang, H. Zhang, A. M. Doweyko, D. Diller, I. Henderson,
J. C. Barrish, J. H. Dodd, G. L. Schieven and K. Leftheris, J. Med.
Chem., 2005, 48, 6261.
18 S. M. Myers, R. H. Bawn, L. C. Bisset, T. J. Blackburn, B. Cottyn,
L. Molyneux, A.-C. Wong, C. Cano, W. Clegg, R. W. Harrington,
H. Leung, L. Rigoreau, S. Vidot, B. T. Golding, R. J. Griffin,
T. Hammonds, D. R. Newell and I. R. Hardcastle, ACS Comb. Sci.,
2016, 18, 444.
19 L. Anderson, M. Zhou, V. Sharma, J. M. McLaughlin, D. N. Santiago,
F. R. Fronczek, W. C. Guida and M. L. McLaughlin, J. Org. Chem.,
2010, 75, 4288.
3 D.-Y. Ma, L.-L.Wang, Q. Lai, K.-J. Peng, X. Li, Z.-X. Li, L.-J. Liu,
Z.-Y. Luo and S.-Y. Liu, Bioorg. Med. Chem. Lett., 2018, 28, 3346.
4 V. A. Egorov, F. A. Gimalova, Z. R. Zileeva, L. F. Zainullina,
Yu. V. Vakhitova and M. S. Miftakhov, Mendeleev Commun., 2019, 29,
174.
5 L. Botta, S. Filippi, B. M. Bizzarri, R. Meshini, M. Caputo,
L. Proietti-De-Santis, C. Iside, A. Nebbioso, G. Gualandi and
R. Saladino, Bioorg. Med. Chem. Lett., 2019, 29, 78.
6 R. Kh. Bagautdinova, L. I.Vagapova,A.V. Smolobochkin,A. S. Gazizov,
A. R. Burilov, M.A. Pudovik andA. D.Voloshina, Mendeleev Commun.,
2019, 29, 686.
7 N. S. Vostrikov, L. V. Spirikhin, A. N. Lobov, A. M. Gimazetdinov,
Z. R. Zileeva, Yu. V. Vakhitova, Z. R. Macaev, K. K. Pivnitsky and
M. S. Miftakhov, Mendeleev Commun., 2019, 29, 372.
8 A. Uecker, M. Sicker, T. Beckers, S. Mahboobi, D. Hägerstrand,
A. Ostman and F.-D. Böhmer, Anticancer Drugs, 2010, 21, 759.
9 A. D. Zorina, N. S. Nikiforova, V. V. Zarubaev, S. A. Marchenko,
S. I. Selivanov, G. L. Starova, A. R. Mehtiev, E. I. Rodionov,
A. A. Rodionova and R. E. Trifonov, Mendeleev Commun., 2019, 29,
500.
20 L. Ding, Z.-Z. Wang, X.-D. Sun, J. Yang, C.-Y. Ma, W. Li and
H.-M. Liu, Bioorg. Med. Chem. Lett., 2017, 27, 3521.
10 V. L. Rusinov, V. N. Charushin and O. N. Chupakhin, Russ. Chem. Bull.,
Int. Ed., 2018, 67, 573 (Izv. Akad. Nauk, Ser. Khim., 2018, 573).
11 M. Ranson, W. Mansoor and G. Jayson, Expert. Rev. Anticancer Ther.,
2002, 2, 161.
12 D. A. E. Cross, S. E. Ashton, S. Ghiorghiu, C. Eberlein, C. A. Nebhan,
P. J. Spitzler, J. P. Orme, M. R. V. Finlay, R. A. Ward, M. J. Mellor,
G. Hughes, A. Rahi, V. N. Jacobs, M. R. Brewer, E. Ichihara, J. Sun,
H. Jin, P. Ballard, K. Al-Kadhimi, R. Rowlinson, T. Klinowska,
G. H. P. Richmond, M. Cantarini, D.-W. Kim, M. R. Ranson and W. Pao,
Cancer Discov., 2014, 4, 1046.
Received: 24th March 2020; Com. 20/6178
– 606 –