Discovery of derivatives of 6(7)-amino-3-phenylquinoxaline-2-carbonitrile 1,4-dioxides: novel, hypoxia-selective HIF-1α inhibitors with strong antiestrogenic potency

Article abstract of DOI:10.1016/j.bioorg.2020.104324

In this article, we describe the synthesis of 3-phenylquinoxaline-2-carbonitrile 1,4-dioxides bearing cyclic diamine residues at positions 6 or 7; the synthesis is based on the nucleophilic substitution of halogens. All synthesized 6(7)-aminoquinoxaline-2-carbonitrile 1,4-dioxides 3–6 demonstrated higher cytotoxicity and hypoxia selectivity compared to the reference agent tirapazamine against breast adenocarcinoma cell lines (MCF7, MDA-MB-231). The structure and position of the diamine residue considerably affects the antiproliferative properties of the quinoxaline-2-carbonitrile 1,4-dioxides. The introduction of a halogen atom at position 7 in the quinoxaline ring of 4a considerably increases the cytotoxicity of compounds 5a and 6a under both normoxic and hypoxic conditions. However, the most hypoxia-selective derivatives were non-halogenated 7-aminosubstituted 3-phenylquinoxaline-2-carbonitrile 1,4-dioxides 3a–j. Of the 32 novel synthesized derivatives, approximately 20 of the 6(7)-amino-3-phenylquinoxaline-2-carbonitrile 1,4-dioxides demonstrated high antiproliferative potency against wild type leukemia cells K562 and drug-resistant subline K562/4 with the expression of p-glycoprotein (p-gp) compared to the reference agent doxorubicin, which exhibited one order of magnitude lower activity towards K562/4 cells than towards K562 cells. Lead compounds 5a and 3f inhibited HIF-1α expression and activity and induced apoptosis in hypoxic tumor cells, which was confirmed by poly(ADP-ribose)polymerase (PARP) cleavage. Moreover, 5a and 3f showed strong antiestrogenic potencies in MCF7 breast cancer cells. Thus, the described series of quinoxaline 1,4-dioxides has high anticancer potential and good aqueous solubility. Therefore, these compounds are promising for further drug development of hypoxia-targeted anticancer agents.

Full text of DOI:10.1016/j.bioorg.2020.104324

Bioorganic Chemistry 104 (2020) 104324
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of derivatives of 6(7)-amino-3-phenylquinoxaline-2-carbonitrile
1,4-dioxides: novel, hypoxia-selective HIF-1
α inhibitors with strong
antiestrogenic potency
,
Galina I. Buravchenkoa b, Alexander M. Scherbakovc, Lyubov G. Dezhenkovaa,
Evgeny E. Bykova, Svetlana E. Solovievaa, Alexander A. Korlukovd, Danila V. Sorokinc,
,
*
Lianet Monzote Fidalgoe, Andrey E. Shchekotikhina
a Gause Institute of New Antibiotics, 11 B. Pirogovskaya Street, Moscow 119021, Russia
b Mendeleyev University of Chemical Technology, 9 Miusskaya Square, Moscow 125190, Russia
c Blokhin National Medical Research Center of Oncology, 24 Kashirskoye sh., Moscow 115522, Russia
d Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 28 Vavilova St., Moscow 119991, Russia
e Department of Parasitology, Pedro Kouri Tropical Medicine Institute, Havana, Cuba
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this article, we describe the synthesis of 3-phenylquinoxaline-2-carbonitrile 1,4-dioxides bearing cyclic
diamine residues at positions 6 or 7; the synthesis is based on the nucleophilic substitution of halogens. All
synthesized 6(7)-aminoquinoxaline-2-carbonitrile 1,4-dioxides 3–6 demonstrated higher cytotoxicity and hyp-
oxia selectivity compared to the reference agent tirapazamine against breast adenocarcinoma cell lines (MCF7,
MDA-MB-231). The structure and position of the diamine residue considerably affects the antiproliferative
properties of the quinoxaline-2-carbonitrile 1,4-dioxides. The introduction of a halogen atom at position 7 in the
quinoxaline ring of 4a considerably increases the cytotoxicity of compounds 5a and 6a under both normoxic and
hypoxic conditions. However, the most hypoxia-selective derivatives were non-halogenated 7-aminosubstituted
3-phenylquinoxaline-2-carbonitrile 1,4-dioxides 3a–j. Of the 32 novel synthesized derivatives, approximately 20
of the 6(7)-amino-3-phenylquinoxaline-2-carbonitrile 1,4-dioxides demonstrated high antiproliferative potency
against wild type leukemia cells K562 and drug-resistant subline K562/4 with the expression of p-glycoprotein
(p-gp) compared to the reference agent doxorubicin, which exhibited one order of magnitude lower activity
Quinoxaline-2-carbonitrile 1,4-dioxide
Antiproliferative activity
Hypoxia selectivity
HIF-1
α
ERK 1/2 signaling pathway
Antiestrogenic potency
towards K562/4 cells than towards K562 cells. Lead compounds 5a and 3f inhibited HIF-1α expression and
activity and induced apoptosis in hypoxic tumor cells, which was confirmed by poly(ADP-ribose)polymerase
(PARP) cleavage. Moreover, 5a and 3f showed strong antiestrogenic potencies in MCF7 breast cancer cells.
Thus, the described series of quinoxaline 1,4-dioxides has high anticancer potential and good aqueous solubility.
Therefore, these compounds are promising for further drug development of hypoxia-targeted anticancer agents.
1. Introduction
[2]. Owing to the effect on various metabolic, molecular–genetic, and
pathophysiological processes (e.g., proliferation, apoptosis, neoangio-
The oxygen status of malignant neoplastic cells is one of the key
factors determining the prognosis of disease and the effectiveness of
treatments [1]. Tumor hypoxia (a decrease in the partial pressure of
oxygen below 10 mm Hg) should be considered because of the imbal-
ance between the high demand of tumor tissues for oxygen and its
inadequate delivery by the abnormally formed tumor vascular network
genesis, and metastasis), hypoxia promotes the development of an
aggressive tumor phenotype [3]. In addition, hypoxia is the main cause
of resistance of neoplasms to various therapeutic agents and radio-
therapy [4]. Thus, hypoxia has stimulated research for the development
of drugs that are activated under hypoxic conditions and promoted the
formation of the concept of hypoxia-selective cytotoxins. Currently, bio-
* Corresponding author.
E-mail addresses: buravchenkogi@gmail.com (G.I. Buravchenko), alex.scherbakov@gmail.com (A.M. Scherbakov), dezhenkovalg@yahoo.com (L.G. Dezhenkova),
dsorokin2018@gmail.com (D.V. Sorokin), monzote@ipk.sld.cu (L. Monzote Fidalgo), shchekotikhin@mail.ru (A.E. Shchekotikhin).
https://doi.org/10.1016/j.bioorg.2020.104324
Received 8 July 2020; Received in revised form 15 September 2020; Accepted 25 September 2020
Available online 28 September 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
reductive prodrugs are essential in chemotherapy. Several classes of
compounds that are activated under hypoxic conditions are known, such
as quinone-containing alkylating anticancer agents (the prototype of
which is mitomycin C [5]), nitroaromatic compounds (e.g., metronida-
zole [6] or nitrofurans [7]) or the recently discovered aromatic and
aliphatic N-oxides [8]. Such hypoxia-selective cytotoxins are activated
by the reduction of the prodrug through one- or two-electron reduction
mediated by cellular reductases [9].
novel water-soluble 6(7)-aminoquinoxaline-2-carbonitrile 1,4-dioxides
3–6, which had promising anticancer properties.
2. Results and discussion
2.1. Chemistry
The synthetic pathway for the preparation of target compounds 3–6
is shown in Scheme 1. Starting halogen derivatives 3-phenyl-2-quinoxa-
linecarbonitrile 1,4-dioxide 2a–d were prepared by a reaction of cor-
responding benzofuroxanes with benzoylacetonitrile in the presence of
triethylamine [15,16]. First, the reactivity of 7-fluoro-3-phenylquinoxa-
line-2-carbonitrile 1,4-dioxide (2a) and its minor isomer 2b, which were
formed via the Beirut reaction [16], in the nucleophilic substitution
reaction was tested. We determined that the substitution of the halogen
in 2a and 2b with amines proceeded under mild conditions and afforded
the corresponding 6(7)-amino derivatives of 3-phenyl-2-quinoxalinecar-
bonitrile 1,4-dioxides. Thus, the treatment of 2a and 2b with an excess
of piperazine in DMF at room temperature afforded 7- and 6-piperazinyl
derivatives 3a and 4a, which were isolated as hydrochlorides in good
yields (72% and 68%, respectively).
The hypoxic agent tirapazamine (TPZ, 1, Fig. 1) is an aromatic het-
erocyclic di-N-oxide, which has been introduced in Phase III clinical
trials in patients with solid tumors [10]. In addition, it is widely used as a
prototype for the generation of novel candidates with advanced anti-
cancer properties. The mechanism of action of this class of hypoxic cy-
totoxins has been well studied for TPZ. Thus, TPZ is a substrate for
intracellular reductases that convert it into cytotoxic species (e.g., hy-
droxyl and benzotriazinyl radicals) under hypoxic conditions. The
cytotoxic effect of these active metabolites results in both single- (SSBs)
and double-stranded breaks (DSBs) of DNA [11]. Many studies have
focused on derivatives of quinoxaline 1,4-dioxide, which are bioisosteric
analogs of TPZ; some of these derivatives exhibit more pronounced ef-
fects on intracellular reductases [12,13,14].
Previously, we described a series of quinoxaline-2-carbonitrile 1,4-
dioxides bearing halogen atoms (F, Cl, Br) at positions 6 and 7 and the
aryl group at position 3 of the heterocyclic nucleus [15,16]. Almost all
compounds exhibited good inhibitory potencies and hypoxia selectivity
profiles against breast adenocarcinoma cell lines and were able to
circumvent the multidrug resistance (MDR) of cancer cells with different
mechanisms of resistance [15]. The most cytotoxic derivatives within
the series were 2a–d (Fig. 1). The revision of regioselectivity of the
Beirut reaction between monosubstituted benzofuroxanes and benzoy-
lacetonitrile allowed to identify the first representatives of 6-substitued
3-phenylquinoxaline-2-carbonitrile 1,4-dioxides [16]. Some of the ob-
tained pairs of isomers (e.g., 6(7)-fluoro derivatives 2a and 2b)
exhibited considerable differences in their anticancer activity and hyp-
oxia selectivity. Thus, 7-fluoro derivative 2a was 5 times more active
under normoxic conditions and 7 times more cytotoxic under hypoxic
conditions than its 6-analogue 2b. Difluoro derivative 2c was the most
potent agent in this series, which has a p53-independent mechanism of
action; it can suppress the growth of tumor cells through the high level of
ROS induced by quinoxaline 1,4-dioxides, which leads to DNA damage,
The structures of isomeric quinoxaline-2-carbonitrile 1,4-dioxides 3a
and 4a were accurately confirmed by spectral studies, and the purity
(>95%) was determined by HPLC. Of note, compared to the starting 7
(6)-fluoroquinoxaline-2-carbonitrile 1,4-dioxides (2a, 2b) [16], the 7-
and 6-piperazinyl derivatives 3a and 4a showed different mobility in
TLC and different retention times (tR) according to the HPLC analysis
[16]. However, similar to the starting compounds 2a and 2b, amino
isomers 3a and 4a have identical 1H NMR spectra (Supplementary
Material, Figs. S1 and S21) but different 13C NMR spectra, which
allowed us to reliably distinguish the isomers (Supplementary Material,
Figs. S2 and S22; Table S1). The analysis of the 13C NMR spectra of
regioisomers 3a and 4a (Supplementary Material, Figs. S2 and S22;
Table S1) showed that the position of amino groups in the heterocyclic
a decrease in HIF-1α expression, and downregulation of VEGF-A tran-
scription [15]. Of note, most previously described quinoxaline-2-
carbonitrile 1,4-dioxide derivatives [8,15–21] possess low aqueous
solubility, which has complicated their further preclinical evaluation.
We assumed that the introduction of amino residues may increase
the aqueous solubility of the quinoxaline-2-carbonitrile 1,4-dioxides and
improve their anticancer potencies. Moreover, despite the considerable
interest and high biological potential of quinoxaline-2-carbonitrile 1,4-
dioxides, their 6(7)-amino derivatives have not been described or
studied yet. Thus, to further evaluate the structure–activity relationships
and improve their chemotherapeutic properties, we explored the effect
of different cyclic diamino residues attached to the quinoxaline ring and
the importance of the distal amino group in the diamine moiety on the
biological properties of these compounds. By applying a set of diamines
to diversify the quinoxaline 1,4-dioxide scaffold, we obtained a series of
Scheme 1. Synthesis of 7(6)-amino derivatives of 3-phenylquinoxaline 1,4-
dioxides 3–6. Reagents and conditions: (a) YH (diamine or N-Boc-protected
diamine), DMF, r.t., 4–8 h; (b) HCl/H2O (for diamines) or HCl/MeOH (for N-
Boc-protected diamines), r.t., 26–93%.
Fig. 1. Structures of hypoxia-selective agents that are based on heterocyclic di-
N-oxides.
2

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
residue considerably affects the chemical shifts of the signals of the
quinoxaline core, whereas the signals of the phenyl, cyano, and piper-
azine residues shifted insignificantly.
However, intermediate Boc-derivatives 3–6 crystallized well from
organic solvents. Thus, the selected crystals of Boc-6a obtained by
recrystallization from a DCM–hexane mixture were suitable for crys-
tallographic analysis, which clearly showed that in Boc-6a, the diamine
residue was located at the 6 position of the quinoxaline ring (Fig. 2,
Supplementary Material, Table S2). According to X-ray studies, all the
bond lengths and angles in Boc-6a were typical values. The quinoxaline
moiety of Boc-6a adopts a planar conformation; the angles between its
mean square plane and the planes of the phenyl and piperazine moieties
are 51.00(14)◦ and 139.35(15)◦, respectively. The chlorine atom and CN
group in the quinoxaline moiety are not coplanar to the heterocyclic
core. The values of the corresponding deviations from the plane of the
quinoxaline moiety are 0.245(7)◦ and 0.151(5)◦. In the crystal, the
molecules of Boc-6a are assembled into a parquet-like motif via in-
teractions between the oxygen atom of the pyrazine 1,4-dioxide moiety
(Supplementary Material, Fig. S96). The O1… C2 [1 ꢀ x, 1/2 + y, z]
distance is 2.765(6) Å, which is 0.45 Å smaller than the sum of the
corresponding van der Waals radii [24].
The comparison of the chemical shifts of the signals in the 13C NMR
spectra of paternal 3-phenylquinoxaline-2-carbonitrile 1,4-dioxide [16]
with the corresponding signals of its 7- and 6-amino derivatives 3a and
4a (Supplementary Material, Figs. S2 and S22; Table S1) showed that
the shifts of the signals correspond (in magnitude and sign) to the in-
crements (IC) of the ternary amino group in the 13C NMR spectra of the
benzene or polyaromatic derivatives [22,23]. The most characteristic
changes in the chemical shifts of the signals of carbon atoms were
observed at positions 3, 10 and 2, 9 of the quinoxaline ring for 3a and
4a, respectively (Table 1). Thus, the introduction of the piperazine
group at position 7 (for 3a) resulted in an upfield shift of the signals for
the para-position of the benzene ring (C-10) and the pyrazine (C-3)
moiety. For isomeric 4a, the signals of the carbon atoms at positions 2
and 9 were more shielded owing to conjugation with the amino group. A
similar strong correlation between the increment values (IC) was
observed for starting fluoro derivatives 2a, 2b, and the other 7(6)-
derived 3-phenylquinoxaline-2-carbonitrile 1,4-dioxides [16]. Thus,
the analysis of the 13C NMR spectra and the values of increment for the
chemical shifts allowed us to undoubtedly determine the position of the
amine residue in the quinoxaline 1,4-dioxides.
In addition, the position of the amino group in the quinoxaline ring
of compounds 5a and 6a was confirmed by the 13C NMR spectra. The
chemical shifts of the signals and the increment values (IC) for the
characteristic carbon atoms at positions 2 and 9 of the quinoxaline core
for compounds 5a and 6a (compared to 3-phenylquinoxaline-2-carbon-
itrile 1,4-dioxide [16,22]) were similar to those for 6-amino derivative
4a (Table 1).
Analogous to derivative 3a, a series of quinoxaline-2-carbonitrile
1,4-dioxides 3b–j was prepared; the obtained compounds varied in the
residues of cyclic diamines at position 7. For diamines f–j, their mono
Boc-protected diamines were used. The obtained intermediate Boc-
derivatives of Boc-3f–j were purified by column chromatography and
crystallization. The deprotection of Boc-derivatives 3f–j with HCl in
methanol afforded the hydrochlorides of quinoxaline-2-carbonitrile 1,4-
dioxides 3f–j. All final compounds 3b–j were further purified by the re-
precipitation of their hydrochloride salts.
The observed differences in the rates of nucleophilic substitution of
the halogens at positions 6 and 7 in quinoxaline-2-carbonitrile 1,4-diox-
ides 2c and 2d are caused by the electron-withdrawing effect of the CN
group at position 2 [25,26]. These experimental data were confirmed by
quantum chemical calculations (B3LYP/6-31Gd). Thus, conjugation
with the CN group increased the positive charge value, Fukui indices,
and the coefficient of LUMO orbital at position 6 compared to those of
the neighboring C7 atom in 2c (Supplementary Material, Fig. S97,
Table S3). Moreover, Meisenheimer’s complex, which formed after the
nucleophilic attack at position 6 of 2c, is considerably more stable (by
~28 kcal/mol) than that produced after the attack at the C7 atom
(Supplementary Material, Fig. S98, Table S4).
In the next step, a similar approach was adopted to modify 6,7-dihal-
ogen derivatives 2c and 2d. The synthesis of 3a and 4a showed that the
reactivity of fluorine atoms at positions 7 or 6 of quinoxaline-2-
carbonitrile 1,4-dioxides 2a and 2b for substitution with piperazine
was similar. However, this reaction exhibited differences in the reac-
tivity of halogens in 6,7-difluoro and 6,7-dichloro derivatives 2c and 2d.
Thus, the treatment of 2c and 2d with diamines primarily afforded the 6-
amino derivatives 5a–e or 6a–e, respectively. Therefore, the halogens at
the 6 position of the heterocyclic core of 2c and 2d were more active for
nucleophilic substitution, which resulted in reasonable regioselectivity
for this reaction. All synthesized compounds 3–6 were characterized by
NMR spectra, HRMS, IR, and UV spectra; the HPLC-determined purity
Finally, the obtained hydrochlorides of the new quinoxaline 1,4-di-
oxides 3–6 bearing diamine residues had improved the aqueous solu-
bility in contrast to the starting water-insoluble quinoxaline-2-
carbonitrile 1,4-dioxides 2a–d. Of note, compared to some quinoxaline
1,4-dioxides, which have been recently described to be unstable in the
solid state and solution [27–30], all obtained amino derivatives 3–6
were not hygroscopic; in addition, these compounds were quite stable
during storage in the solid state and as aqueous solutions.
was
≥
95% (Experimental Section, Supplementary Material,
Figs. S1–95).
The regioselectivity of the nucleophilic substitution reaction was
2.2. Biological screening
clearly confirmed by the structure of Boc-protected derivative 6a, which
was resolved by X-ray analysis. In general, the hydrochlorides of 3–6
were amorphous solids and not suitable for crystallographic studies.
The antiproliferative activity of a series of novel amino derivatives of
the 3-phenylquinoxaline-2-carbonitrile 1,4-dioxides 3–6 was assayed
Table 1
13C chemical shifts (δC, ppm) and characteristic increments (IC) for the 13C chemical shift differences (relative to 3-phenylquinoxaline-2-carbonitrile 1,4-dioxide [16])
for the piperazine group for 3–6a.
Position
δC
IC
δC
IC
δC (J, Hz)
IC
δC
IC
C-2
C-3
C-9
C-10
120.1
139.9
138.0
132.3
ꢀ 0.3
ꢀ 2.8
+0.6
ꢀ 7.1
116.5
142.9
130.0
140.4
ꢀ 3.9
+0.2
ꢀ 7.4
+1.0
118.8
ꢀ 1.6
+0.2
ꢀ 5.5
ꢀ 2.5
119.4
143.3
133.4
138.6
ꢀ 1.0
+0.6
ꢀ 4.0
ꢀ 0.8
142.9
131.9 (10.7)
136.9
3

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
Fig. 2. Structure of Boc-6a according to X-ray studies.
against a panel of cancer cell lines, including two breast adenocarci-
noma cell lines (MCF7 and MDA-MB-231), under normoxic (21% O2)
and hypoxic (1% O2) conditions and against the myeloid leukemia cell
line K562 and its isogenic multidrug-resistant (MDR) counterpart sub-
line K562/4. The cytotoxicity of 3–6 and reference agents tirapazamine
(TPZ), doxorubicin (DOX), and cisplatin (CisPt) are summarized in Ta-
bles 2 and 3.
were similar to those of non-halogenated congeners 3a–j with diamine
residues at position 7; however, the HCR values for 5a–j and 6a–j were
2–10 times smaller than those for 3a–j. Derivatives 6a–j with a chlorine
atom at the 7 position of the quinoxaline ring exhibited strong anti-
proliferative activities against both cell lines, and the activities were
even higher than those of their 7-fluoro-substituted analogues 5a–j.
Thus, most obtained quinoxaline-2-carbonitrile 1,4-dioxides demon-
strated a considerable increase in cytotoxicity under hypoxic conditions,
whereas the activity of doxorubicin and cisplatin, which are traditional
chemotherapeutic agents for breast cancer treatment, did not consid-
erably depend on the oxygen level in malignant cells.
All novel derivatives 3–6 inhibit the growth of tumor cells at sub-
micromolar or low micromolar concentrations. Moreover, the potency
of most of the synthesized compounds against both breast adenocarci-
noma cells increased by approximately 5–40 times under hypoxic con-
ditions (Table 2). The position of the amino group in the structure of 3-
phenylquinoxaline-2-carbonitrile 1,4-dioxide plays a crucial role in the
biological activity of these compounds. Thus, a considerable decrease in
cytotoxicity against both MCF7 and MDA-MB-231 under normoxic (by
9.7 and 18.5 times, respectively) and hypoxic (by 10 and 27.5 times,
respectively) conditions and a decrease in the hypoxic cytotoxicity ratio
(HCR) values (by 2.3 and 3.3 times, respectively) for compound 4a
compared to its regioisomer 3a were observed. Similar differences in
anticancer activity were observed for starting isomeric 7(6)-fluoro de-
rivatives 2a and 2b [16].
Because the development of MDR in tumors frequently limits the
response of many malignancies to chemotherapy [31], the ability of the
obtained quinoxaline-2-carbonitrile 1,4-dioxides to circumvent MDR
mechanisms was investigated. The overexpression of transmembrane
ATP-binding transporters is one of the main mechanisms of MDR, which
often occurs a response of tumor cells to chemotherapy with vinca-
alkaloids, anthracyclines, and some other anticancer agents [32,33].
Thus, K562/4 cells with the expression of the transmembrane efflux
pump P-glycoprotein (P-gp) are more than one order of magnitude less
sensitive to the reference drug doxorubicin and its analogues than the
paternal cell line K562 (resistance index, RI ~ 12, Table 3) [34,35].
However, most new 6(7)-amino-3-phenylquinoxaline-2-carbonitrile
1,4-dioxides have similar cytotoxicity against K562 and the MDR
counterpart K562/4 (Table 3). Of note, the potency of some 6(7)-amino-
3-phenylquinoxaline-2-carbonitrile 1,4-dioxides was higher against the
P-gp-positive subline K562/4 than against parental K562, and the RI was
less than 1. The highest cytotoxicity towards both tested cell lines was
obtained for 6-amino-7-chloro-3-phenylquinoxaline-2-carbonitrile di-
oxide derivatives 6a–j, and the RI values ranged between 0.6 and 2.3
(Table 3).
The most active and hypoxia-selective compound was the 3-(R)-
aminopyrrolidine derivative (R)-3f, which was 7-times more selective
than the reference agent TPZ against MCF7 cells (HCR value of 36.3 and
5.4 for (R)-3f and TPZ, respectively) and 11 times more selective against
triple-negative breast cancer cells MDA-MB-231 (HCR values of 20.3 and
1.8 for (R)-3f and TPZ, respectively). Its stereoisomer (S)-3f was slightly
less active. However, the 6-(S)-aminopyrrolidin-7-chloro derivative (S)-
6f with an HCR value of 11.6 was considerably more selective compared
to its antipode (R)-6f (HCR value of 1.0).
Some correlations between the structure of a cyclic diamine moiety
and cytotoxicity as well as the selectivity of compounds 3–6 under
hypoxic conditions were observed. The structure of the terminal amino
group of the quinoxaline-2-carbonitrile 1,4-dioxides considerably
affected the activity. Generally, the introduction of distal alkyl groups at
the piperazine residue slightly increased hypoxia selectivity but
decreased (by 2–5 times) the cytotoxicity of compounds 3, 5, and 6 with
diamines b, d, and e (with the exception of derivatives 3d, 5b, and 6b).
Overall, halogen atoms at the position 7 of the 6-aminoquinoxaline-
2-carbonitrile 1,4-dioxides considerably increased the cytotoxicity of
compounds 5a–j and 6a–j under normoxic and hypoxic conditions.
Thus, derivatives 5a and 6a exhibited 8–16 higher antiproliferative
potencies than in normoxia and hypoxia compared to their non-
halogenated analog 4a for MCF7 and MDA-MB-231 cell lines. In gen-
eral, the activities of 6-amino-7-halogeno-derivatives 5a–j and 6a–j
2.3. Analysis of the mechanisms of action
To study the effect of the 3-phenylquinoxaline-2-carbonitrile di-
oxides on tumor cells in more detail, two compounds were selected. The
first compound, (R)-3f, showed the highest activity under hypoxic
conditions with an HCR index of 20–36; the other compound, 5a,
exhibited an average HCR value of approximately 5 (Table 2). HIF-1
α
is
inhibition is
considered to be a new approach in anticancer therapy [36]. A decrease
in the oxygen level leads to the stabilization of HIF-1 and its subsequent
activation. HIF-1 supports the expression of genes that are responsible
for resistance to hypoxic conditions. As shown in Fig. 3A, hypoxia
caused a significant increase in HIF-1 expression in MCF7 breast cancer
the main factor in the cell response to hypoxia; HIF-1
α
α
α
α
4

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
Table 2
Structures and cytotoxicity (IC50 a) of 7(6)-amino-3-phenylquinoxaline-2-carbonitrile 1,4-dioxides 3–6 under normoxic and hypoxic conditions.
Compnd
a
R1
R2
IC50
(μ
M)
MCF7
N b
HCR d
MDA-MB-231
N b
HCR d
H c
H c
3a
3b
H
H
1.8 ± 0.3
0.2 ± 0.1
10.6
17.3
2.5 ± 0.1
0.2 ± 0.1
14.8
21.0
5.2 ± 1.0
0.3 ± 0.1
8.2 ± 1.6
0.4 ± 0.1
3c
H
H
H
H
3.3 ± 0.6
10.1 ± 2.0
10.5 ± 2.1
4.7 ± 0.9
0.2 ± 0.1
1.3 ± 0.3
0.8 ± 0.2
0.1 ± 0.03
13.6
7.8
5.0 ± 1.0
8.7 ± 1.7
16.4 ± 3.3
9.9 ± 1.8
0.3 ± 0.1
1.6 ± 0.3
0.9 ± 0.2
0.5 ± 0.07
18.7
5.4
3d
3e
12.9
36.3
17.1
20.3
(R)-3f
(S)-3f
H
H
3.1 ± 0.6
5.4 ± 1.0
0.1 ± 0.06
0.1 ± 0.03
30.8
41.2
7.6 ± 1.5
9.9 ± 2.0
0.3 ± 0.1
0.5 ± 0.1
22.4
19.5
3g
3h
3j
H
H
8.1 ± 1.6
1.9 ± 0.4
0.8 ± 0.1
0.2 ± 0.1
9.8
9.9
8.6 ± 1.7
4.5 ± 0.9
1.2 ± 0.2
0.2 ± 0.1
7.2
18.8
4a
5a
5b
H
F
17.5 ± 1.1
2.0 ± 0.1
2.8 ± 0.1
3.7 ± 0.2
0.4 ± 0.03
2.1 ± 0.1
4.7
4.7
1.3
24.0 ± 2.1
1.5 ± 0.1
4.5 ± 0.5
5.3 ± 1.3
0.4 ± 0.01
0.6 ± 0.1
4.5
4.3
7.4
F
5c
5d
5e
5f
F
F
F
F
4.3 ± 1.6
3.4 ± 0.3
7.9 ± 1.8
2.7 ± 1.1
2.3 ± 0.2
0.7 ± 0.04
0.9 ± 0.04
1.6 ± 1.0
1.9
4.9
8.8
1.7
4.7 ± 1.5
3.2 ± 0.2
10.5 ± 2.3
6.2 ± 3.1
1.9 ± 1.0
0.5 ± 0.01
1.2 ± 0.2
1.3 ± 0.1
2.4
6.4
9.1
4.8
5g
F
1.9 ± 0.1
0.4 ± 0.03
4.5
1.4 ± 0.1
0.3 ± 0.01
4.4
5h
5j
F
F
2.6 ± 0.1
1.8 ± 0.1
0.4 ± 0.05
0.6 ± 0.03
6.5
3.1
1.8 ± 0.5
1.5 ± 0.1
0.4 ± 0.02
0.3 ± 0.02
4.5
5.0
6a
6b
Cl
Cl
0.8 ± 0.05
2.6 ± 0.1
0.2 ± 0.02
0.9 ± 0.1
3.6
2.7
0.9 ± 0.05
1.5 ± 0.1
0.3 ± 0.02
0.4 ± 0.1
3.1
4.2
6c
Cl
2.3 ± 0.6
1.0 ± 0.1
2.2
0.5 ± 0.1
0.7 ± 0.1
0.7
(continued on next page)
5

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
Table 2 (continued)
Compnd
a
R1
R2
IC50 (μM)
MCF7
N b
HCR d
MDA-MB-231
N b
HCR d
H c
H c
6d
Cl
Cl
Cl
2.1 ± 0.1
3.4 ± 0.3
1.9 ± 0.1
0.5 ± 0.05
4.2
1.2 ± 0.1
0.3 ± 0.03
4.0
6e
0.9 ± 0.04
0.2 ± 0.02
12.9
11.6
16.4 ± 3.3
1.7 ± 0.1
0.9 ± 0.2
17.1
3.8
(S)-6f
0.5 ± 0.05
(R)-6f
Cl
Cl
1.3 ± 0.1
1.1 ± 0.1
1.0
2.6
0.9 ± 0.1
0.6 ± 0.05
0.3 ± 0.01
2.0
2.0
6g
0.7 ± 0.01
0.3 ± 0.01
0.6 ± 0.01
6h
6j
Cl
Cl
3.5 ± 1.0
0.6 ± 0.05
0.2 ± 0.03
6.1
2.8
4.7 ± 1.5
0.7 ± 0.02
0.2 ± 0.02
6.7
2.3
0.6 ± 0.08
0.5 ± 0.07
TPZ
–
–
–
–
–
–
24.2 ± 3.3
0.3 ± 0.03
8.3 ± 1.7
4.5 ± 1.1
0.4 ± 0.03
11.4 ± 2.3
5.4
0.7
0.7
39.5 ± 5.4
0.3 ± 0.1
13.0 ± 2.6
22.0 ± 2.1
0.2 ± 0.07
16.0 ± 3.2
1.8
1.5
0.8
DOX
CisPt
HCR, hypoxic cytotoxicity ratio.
a
IC50, μM (mean ± S.D. of 3 experiments).
b
c
N = Normoxia: 21% of oxygen.
H = Hypoxia: 1% of oxygen.
d
HCR, hypoxic cytotoxicity ratio: IC50(N)/IC50(H)
.
cells, while compounds 5a and (R)-3f decreased its expression in a dose-
(R)-3f and 5a reduced cyclin D1 expression in MCF7 cells, which indi-
cated a mechanism of antiproliferative action (Fig. 4).
dependent manner. To analyze the effect of compounds on the HIF-1
α
activity, HRE-Luciferase plasmids containing a luciferase reporter gene
under the hypoxia-responsive element (HRE) were used. Control MCF7
cells were incubated under normoxic conditions for background anal-
An analysis of estrogen receptor activity can be carried out using
plasmids containing the luciferase reporter gene, which is controlled by
a promoter with estrogen response elements (ERE-Luciferase). The
ysis. Both selected compounds considerably inhibited the HIF-1
α
activ-
transfection of such a construct into cells was used to test ER
α
activity by
activation was performed via 10-nM
17β-estradiol treatment, and (R)-3f and 5a were further evaluated as
ER inhibitors. Fig. 5 shows that the 17β-estradiol-induced luciferase
ity in MCF-7 cells (Fig. 3B).
the accumulation of luciferase. ERα
By taking into account the data by Sangi et al. [37] on the devel-
opment of antiestrogenic drugs that are based on 2,3-diarylquinoxalines,
we assumed that the novel 3-phenylquinoxaline-2-carbonitrile dioxides
could also affect steroid signaling pathways in breast cancer cells. MCF7
cells are the most common type of breast carcinoma, luminal A. The
α
activity decreased after treatment with (R)-3f or 5a. Thus, compounds
(R)-3f and 5a inhibited the expression and activity of ERα under hypoxic
conditions.
main growth driver of such cells is estrogen receptor
transcription factor that exists in the unoccupied form in the cytoplasm
[38]. After the binding of estrogens, activation of ER occurs, followed
binds to corre-
α
(ERα
). ER
α
is a
The extracellular signal-regulated kinase (ERK) signaling pathway is
essential for the proliferation and death of cancer cells. Depending on
the stimulus, ERK activity mediates different antiproliferative events
such as apoptosis and autophagy [39–42]. Various cytotoxic agents and
DNA-damaging stimuli (e.g., doxorubicin, etoposide, ionizing irradia-
tion, and ultraviolet irradiation (UV)) activate ERK 1/2 protein kinase in
the cells [40,43]. Compounds with hormonal activity (e.g., 17β-estra-
diol, tamoxifen, apigenin, and quercetin) also affect cell death via ERK
1/2 modulation [40,43]. ERK 1/2 activation (as the ratio of p-ERK 1/2
to ERK 1/2) was analyzed in MCF7 cells using immunoblotting. Both
compounds (R)-3f and 5a showed high ERK 1/2 activation (Fig. 4);
however, 5a was considerably more potent. Besides, both compounds
caused an increase in p-ERK1/2 expression in a dose-dependent manner.
Apoptosis plays an important role in the mechanism of action of the
majority of anticancer drugs. The expression of anti-apoptotic Bcl-2
protein was downregulated in MCF7 cells treated with compound 5a
(Fig. 4). ERK 1/2 activity is known to promote apoptotic pathways by
caspase-8 activation [40]. Then we evaluated the expression of caspase-
α
by ERα transport to the nucleus. In the nucleus, ERα
sponding DNA sequences, or estrogen response elements (EREs), in the
promoter regions of various genes. Thus, the transcription of genes
responsible for cell proliferation is regulated by estrogens. Since the
1970 s, many antiestrogens have been developed that block ERα activ-
ity. The most common among them is tamoxifen, which has become the
“gold” standard of breast cancer therapeutic strategies.
MCF7 cells were incubated with (R)-3f and 5a under hypoxic con-
ditions; after 24 h, the samples were prepared for immunoblotting. Fig. 4
shows that both compounds considerably decreased ER
α
expression.
Moreover, the (R)-3f effects against ER
those of 5a.
α were more pronounced than
The estrogen-mediated activation of ERα in hormone-dependent
cancer cells increases proliferation. Cyclin D1 is among the crucial
markers of the cell cycle that are regulated by estrogens. Compounds
6

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
dioxides by altering the side chain structure afforded (R)-3f and 5a,
Table 3
Circumventing MDR in leukemia cell lines by 7(6)-amino-3-phenylquinoxaline-
which are candidates for further preclinical studies as anticancer agents.
2-carbonitrile 1,4-dioxides 3a–j, 4a, 5a–j, and 6a–j.
Compnd
a
3. Conclusions
IC50
(μM)
K562
K562/4
RI b
Using the SN2Ar reaction, a series of derivatives of 6(7)-amino-3-phe-
nylquinoxaline-2-carbonitrile 1,4-dioxide were synthesized for the first
time. In this reaction, the high regiospecificity of nucleophilic substi-
tution of halogen atoms at position 6 in the 6,7-dihalogeno-3-phenylqui-
noxaline-2-carbonitrile 1,4-dioxides with amines was observed. The
modification at positions 6 or 7 of the quinoxaline ring afforded water-
soluble quinoxaline-2-carbonitrile 1,4-dioxides with improved anti-
cancer properties.
3a
4.8 ± 0.6
9.4 ± 1.3
ntc
14.3 ± 2.1
14.5 ± 2.0
ntc
2.9
1,5
–
1.6
1.7
1.2
>3.5
2.4
–
0.9
–
0.9
0.7
–
3b
3c
3d
13.5 ± 1.3
26.0 ± 2.5
15.5 ± 2.3
14.4 ± 1.7
8.0 ± 1.0
ntc
22.0 ± 2.2
44.0 ± 4.2
19.0 ± 2.7
>50
3e
(R)-3f
(S)-3f
3g
18.8 ± 2.3
3h
ntc
3j
6.5 ± 0.7
6.2 ± 0.6
ntc
The location of the amino group at position 6 in the quinoxaline ring
in derivative 4a considerably decreased the cytotoxicity and hypoxia
selectivity against the breast cancer cell lines compared to its 7-
regioisomer 3a; the introduction of additional halogen atoms at posi-
tion 7 of 4a led to a considerable increase in activity. Thus, fluoro- and
chloro-congeners 5a and 6a were 8–16 times more active under nor-
moxic and hypoxic conditions than non-halogenated 4a with the
piperazine residue at the same position. However, although halogenated
derivatives 5a–j and 6a–j demonstrated high cytotoxicity, in general,
they exhibited modest hypoxia selectivity compared to non-halogenated
analogs 3a–j with amine residues at position 7. The most promising 7-
aminosubstituted quinoxaline-2-carbonitrile 1,4-dioxide (R)-3f,
bearing the 3-aminopyrrolidine moiety, demonstrated superior hypoxic
selectivity for MCF7 and MDA-MB-231 cell lines with HCR values of 36
4a
ntc
5a
4.2 ± 0.5
7.0 ± 0.6
ntc
4.0 ± 0.6
4.8 ± 0.7
ntc
5b
5c
5d
6.0 ± 0.9
11.8 ± 1.1
4.2 ± 0.5
5.2 ± 0.4
5.0 ± 0.5
8.1 ± 0.8
2.0 ± 0.3
4.2 ± 0.5
ntc
4.8 ± 0.7
10.5 ± 1.6
2.8 ± 0.3
12.0 ± 1.2
6.4 ± 0.6
5.0 ± 0.7
1.5 ± 0.2
4.0 ± 0.4
ntc
0.8
0.9
0.6
2.3
1.2
0.6
0.8
0.9
–
0.7
0.6
1.0
1.5
1.8
–
5e
5f
5g
5h
5j
6a
6b
6c
6d
6.2 ± 0.6
13.3 ± 1.3
4.0 ± 0.4
2.8 ± 0.2
2.3 ± 0.2
ntc
4.8 ± 0.7
8.8 ± 1.3
4.1 ± 0.5
4.3 ± 0.4
4.3 ± 0.4
ntc
6e
(S)-6f
(R)-6f
6g
and 20, respectively, and an IC50 < 0.5 μM under hypoxic conditions;
this compound was 7–11 times more potent than the reference hypoxic
cytotoxin TPZ. The alkylation of the piperazine residue (compounds
with diamines b, d, and e, Scheme 1) resulted in a slightly lower
inhibitory potency but higher hypoxia selectivity for the relevant de-
rivatives against adenocarcinoma cell lines.
6h
6j
2.0 ± 0.2
0.3 ± 0.04
1.5 ± 0.3
3.5 ± 0.5
0.7
11.7
DOX
a
IC50
,
μ
M (mean ± S.D. of 3 experiments).
b
c
RI, resistance index: IC50(K562/4)/IC50(K562).
Not tested.
Moreover, most of the 6(7)-amino-3-phenylquinoxaline-2-carboni-
trile 1,4-dioxides circumvented the P-gp-mediated drug efflux and
inhibited the proliferation of MDR cells that are resistant to DOX and
other P-gp-transported drugs. The chosen lead compounds (R)-3f and 5a
7 and caspase-8. As shown in Fig. 6A, treatment of MCF7 cells with (R)-
3f caused increase in expression of cleaved caspase-7 and caspase-8
(minor lower bands). Apoptosis is characterized by marked changes in
cellular morphology and cleavage of poly(ADP-ribose) polymerase
(PARP) [44]. PARP may catalyze the poly(ADP-ribosyl)ation of various
nuclear proteins with NAD as the substrate [45]. PARP (112 kDa) has
been shown to be cleaved into 89- and 24-kDa fragments that contain
the active site and DNA-binding domain of the enzyme. 89-kDa PARP is
considered a proven marker of apoptosis [46]. MCF7 cells were treated
with compounds (R)-3f and 5a at low concentrations for 72 h; then, full
and cleaved PARPs were analyzed by immunoblotting. The antibodies
used could react with both forms of PARP, 112-kDa, and 89-kDa. Fig. 6B
shows that both compounds caused a dose-dependent accumulation of
89-kDa PAPR. The accumulation of 89-kDa PARP was more pronounced
when MCF7 cells were incubated with compound (R)-3f compared to
when they were incubated with compound 5a. Thus, compound (R)-3f
was a strong nanomolar inducer of apoptosis.
inhibited HIF-1
ptotic, anti-HIF-1
dioxides may be considered “first in class” dual HIF-1
α
expression and activity and exhibited strong proapo-
, and antiestrogenic potencies. These quinoxaline 1,4-
/ER blockers
α
α
α
that potently modulate the ERK 1/2 signaling pathway and induce
apoptosis. The development of hypoxia-specific antiestrogens is very
intriguing and represents a new challenge in breast cancer therapy.
Thus, in this study, we discovered a new promising chemotype of water-
soluble hypoxic cytotoxins that was based on a quinoxaline 1,4-dioxide
scaffold and does not yet have analogues in clinical practice. The further
in-depth development of this novel class is expected to afford hypoxia-
specific anticancer agents with improved therapeutic properties.
4. Experimental section
4.1. General methods
Thus, compounds (R)-3f and 5a exhibited pronounced anti-
proliferative effects against breast cancer cells and showed a consider-
NMR spectra of all synthesized compounds were recorded on a
Varian VXR-400 instrument at 400 MHz (1H NMR) and 100 MHz (13
C
able effect on ERα signaling pathways, which support the growth of
NMR). Chemical shifts were measured in DMSO‑d6 using TMS as an
internal standard. Analytical TLC was performed on silica gel F254
plates (Merck) and column chromatography on Silica Gel Merck 60.
Melting points were determined on a Buchi SMP-20 apparatus and are
uncorrected. High-resolution mass spectra were recorded by electron
spray ionization on a Bruker Daltonics micro OTOF-QII instrument. UV
spectra were recorded on a Hitachi-U2000 spectrophotometer. IR
spectra were recorded on Nicolet iS10 Fourier transform IR spectrom-
eter (Nicolet iS10 FT-IR, Madison, WI, USA). HPLC was performed using
Shimadzu Class-VP V6.12SP1 system (GraseSmart RP-18, 6 × 250 mm).
Eluents: A, H3PO4 (0.01 M); B, MeCN. All solutions were evaporated at
these cells. Apoptosis is considered the mechanism of cell death, which
is caused by these compounds. Moreover, ERK 1/2 and HIF-1
α are
involved in the cell response to the compounds. In general, derivatives of
the novel series quinoxaline-2-carbonitrile dioxides could be a useful
scaffold for the development of antiestrogens with high activity under
hypoxic conditions.
Overall, the new 6(7)-amino-3-phenylquinoxaline-2-carbonitrile
1,4-dioxides demonstrated high hypoxia selectivity and anti-
proliferative potency against tumor cells with one of the most common
MDR phenotypes. The diversification of quinoxaline-2-carbonitrile 1,4-
7

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
Fig. 3. HIF-1α expression (A) and activity (B) in MCF7 cells after treatment with compounds (R)-3f and 5a under hypoxic conditions (A – MCF7 cells were treated
with compounds (R)-3f and 5a for 6 h, B - normo – 24 h of normoxia, hypo – 24 h of hypoxia, HRE-Luciferase – plasmid containing the luciferase reporter gene under
the hypoxia-responsive element).
Fig. 4. Immunoblotting analysis of ER
α, Cyclin D1, Bcl-2 and (p-)ERK 1/2 protein levels in MCF7 breast cancer cells in response to 24-h treatment with (R)-3f and
5a. -Tubulin levels in the samples are shown as protein loading controls.
α
reduced pressure on a Buchi-R200 rotary evaporator at a temperature
below 50 ◦C. All products were dried under vacuum at room tempera-
ture. All solvents, chemicals, and reagents were obtained from Sigma-
Aldrich (unless specified otherwise) and used without purification.
The purity of compounds 3a-j, 4a, 5a-j and 6a-j was >95% as deter-
mined by HPLC analysis. All products were dried under vacuum at room
temperature. The corresponding benzofuroxanes were prepared as
described [47]. The starting 3-phenylquinoxaline-2-carbonitrile 1,4-
8

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
[48] program and refined with the ShelXL [49] program. Molecular
graphics were drawn using OLEX2 [50] program. Selected details of X-
ray experiments and crystallographic data for Boc-6a can be found in the
Supplementary material, Fig. 96, Table S2. Crystallographic data for
Boc-6a can be obtained free of charge from the Cambridge Crystallo-
graphic Data Centre via https://www.ccdc.cam.ac.uk/structures.
Quantum chemical calculations were carried out using the software
packages Gaussian-09 (https://www.www.gaussian.com) and Spartan-
09 (https://www.wavefun.com/products/spartan.html) with full opti-
mization of geometric parameters of the structure of 5a by density
functional B3LYP/6–31G. Indices of local electrophilicity (Fukui indices
for the LUMO orbital) are calculated based on the output of the calcu-
lations using the Gaussian-09 software package by means of the special
program UCA-Fukui [51] (provided by Dr J. S. Marquez, Dr D. Z. Cuenca
and Dr A. S. Coronillas, Department of Chemistry-Physics, Faculty of
Sciences, Campus of Puerto Real University of Cadiz (Cadiz, Spain).
Fig. 5. ERα activity in MCF7 cells after treatment with compounds (R)-3f and
4.1.1. 3-Phenyl-7-(piperazin-1-yl)quinoxaline-2-carbonitrile 1,4-dioxide
hydrochloride (3a)
5a under hypoxic conditions (ERE-Luciferase – luciferase gene controlled by the
promoter with estrogen responsive elements).
To a stirring mixture of 7-fluoro-3-phenylquinoxaline-2-carbonitrile
1,4-dioxide (2a, 0.1 g, 0.36 mmol, [16]) in N,N-dimethylformamide (5
ml) the anhydrous piperazine (0.3 g, 3.6 mmol) was added at r.t. The
mixture was stirred for 4 h, poured into cold water (15 ml) and cooled.
The precipitate was filtered and washed with water (30 ml). The residue
was dissolved with heating in a water (2 ml) solution of concentrated
hydrochloric acid (0.3 ml, 4.6 mmol). The hot solution was filtered and
the product was precipitated with acetone-ether mixture (3:1). The red
solid was collected by filtration, successively washed with acetone,
Et2O, n-hexane and dried. The yield of hydrochloride 3a was 91 mg
(72%) as a red solid, m.p.(decomp.) 221–224 ◦C. HPLC (LW = 300 nm,
dioxides 2a-d was synthesized as described [15,16].
Single crystal X-ray studies of Boc-6a were carried out in Center for
molecule composition studies of INEOS RAS with Bruker APEX-II CCD
diffractometer. The structures were solved by direct method and refined
in anisotropic approximation for non-hydrogen atoms. Hydrogens atoms
of methyl, methylene and aromatic fragments were calculated according
to those idealized geometries and refined with constraints applied to
–
–
C
H and N H bond lengths and equivalent displacement parameters
(Ueq(H) = 1.2Ueq(X), X - central atom of XH₂ group; Ueq(H) = 1.5Ueq(Y),
Y - central atom of YH3 group. All structures were solved with the ShelXT
gradient B 20 / 60% (30 min)) tR = 10.25 min, purity 95.0%. λmax.
,
Fig. 6. Immunoblotting analysis of caspase-7, caspase-8, full and cleaved PARP in MCF7 cells in response to treatments with compounds (R)-3f and 5a. (A – 48-h
treatment, B – 72-h treatment).
α-Tubulin levels in the samples are shown as protein loading controls.
9

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
EtOH: 223, 301, 367, 479 nm. IR νmax, (film) cmꢀ 1 3396, 2986, 2913,
2721, 2498, 1712, 1607, 1521, 1502, 1449, 1388, 1335, 1317, 1260,
1195, 1149, 1028, 999, 950. 1H NMR (400 MHz, DMSO‑d6) δ 9.65 (2H,
br.s, NH+2 ); 8.32 (1H, d, J = 9.5, H-5); 7.88 (1H, d, J = 9.5, H-6);
7.70–7.44 (6H, m, C6H5, H-8); 3.82–3.75 (4H, m, CH2); 3.48–3.39 (4H,
m, CH2). 13C NMR (100 MHz, DMSO‑d6) δ 151.90 (7-C); 139.92 (3-C);
138.04 (10-C); 132.25 (9-C); 130.71 (4′-CH); 130.40 (2 × 2′CH); 128.40
(2 × 3′CH); 127.89 (1′-C); 123.56 (5-CH); 121.53 (6-CH); 120.13 (2-C);
111.35 (CN); 98.60 (8-CH); 43.83 (2 × CH2); 42.11 (2 × CH2). HRMS
(ESI) calculated for C19H18N5O+2 [M + H]+ 348.1455, found 348.1442.
(400 MHz, DMSO‑d6) δ 11.16 (1H, br.s, NH+); 8.33 (1H, d, J = 9.5, H-5);
7.90 (1H, dd, J3 = 9.5, J4 = 2.5, H-6); 7.80–7.43 (6H, m, C6H5, H-8);
4.24 (2H, d, J = 13.3, CH2OH); 3.93–3.81 (2H, m, CH2); 3.76–3.28 (9H,
m, CH2, OH). 13C NMR (100 MHz, DMSO‑d6) δ 151.67 (7-C); 139.98 (3-
C); 138.04 (9-C); 132.32 (10-C); 130.74 (4′-CH); 130.41 (2 × 2′CH);
128.40 (2 × 3′CH); 127.89 (1′-C); 123.57 (5-CH); 121.55 (6-CH); 120.16
(2-C); 111.35 (CN); 98.74 (8-CH); 57.67 (CH2-OH); 55.22 (CH2-
CH2OH); 50.68 (2 × CH2); 43.78 (2 × CH2). HRMS (ESI) calculated for
C21H22N5O+3 [M + H]+ 392.1717, found 392.1691.
4.1.6. 7-(3-(R)-Aminopyrrolidine-1-yl)-3-phenylquinoxaline-2-
4.1.2. 7-(Methylpiperazine-1-yl)-3-phenylquinoxaline-2-carbonitrile 1,4-
dioxide hydrochloride (3b)
carbonitrile 1,4-dioxide hydrochloride ((R)-3f)
To a stirring mixture of 7-fluoro-3-phenylquinoxaline-2-carbonitrile
1,4-dioxide (2a, 0.1 g, 0.36 mmol [16]) in N,N-dimethylformamide (5
ml) the (R)-3-(N-Boc-amino)pyrrolidine (0.17 g, 0.9 mmol) was added at
r.t. The mixture was stirred for 8 h, poured into cold water (15 ml) and
cooled. The crude product was filtered, washed with water (30 ml) and
dried. The residue was purified by column chromatography with
chloroform–methanol (1:0 → 5:1) as the eluting solvent. The residue was
crystallized from dichclomethane-n-hexane mixture. Obtained (R)-Boc-
3f dissolved in warm chloroform (5 ml). Solution of HCl in MeOH
(13,3%, 2 ml) was added, the mixture was stirred overnight and evap-
orated. The crystals were dissolved in boiling water (5 ml), filtered and
the product was precipitated with acetone-ether mixture (3:1). The red
solid was collected by filtration, washed with acetone, Et2O, n-hexane
and dried. The yield of hydrochloride (R)-3f was 113 mg (74%) as a red
solid, m.p.(decomp.) 220–222 ◦C. HPLC (LW = 273 nm, gradient B 40 /
65% (30 min)) tR = 10.3 min, purity 95.7%. λmax., EtOH: 225, 311, 368,
422 nm. 1H NMR (400 MHz, DMSO‑d6) δ 8.64 (3H, br.s, NH3); 8.30 (1H,
d, J = 9.5, H-5); 7.76–7.71 (2H, m, C6H5); 7.61–7.57 (3H, m, C6H5); 7.47
(1H, dd, J3 = 9.6, J4 = 2.4, H-6); 7.02 (1H, s, H-8); 3.81–3.45 (5H, m,
CH2, CH); 2.45–2.22 (2H, m, CH2). 13C NMR (100 MHz, DMSO‑d6) δ
149.44 (7-C); 139.13 (3-C); 138.41 (9-C); 131.01 (10-C); 131.42 (4′-
CH); 130.94 (2 × 2′CH); 128.79 (2 × 3′CH); 128.52 (1′-C); 122.54 (5-
CH); 121.95 (6-CH); 120.32 (2-C); 111.89 (CN); 95.31 (8-CH); 51.89
(CH-NH2); 49.88 (CH2); 46.32 (CH2); 29.52 (CH2). HRMS (ESI) calcu-
lated for C19H18N5O+2 [M + H]+ 348.1455, found 348.1475.
This compound was prepared from 2a and 1-methylpiperazine as
described for 3a. A red powder, yield 90%, m.p.(decomp.) 243–244 ◦C.
HPLC (LW = 300 nm, gradient B 20 / 60% (30 min)) tR = 10.6 min,
purity 96.9%. λmax., EtOH: 299, 368, 472 nm. 1H NMR (400 MHz,
DMSO‑d6) δ 8.30 (1H, d, J = 9.7, H-5); 7.87 (1H, dd, J3 = 9.7, J4 = 2.7,
H-6); 7.72 (2H, td, J3 = 3.8, J4 = 2.2, C6H5); 7.61–7.56 (3H, m, C6H5, H-
8); 7.49 (1H, d, J = 2.7, C6H5); 3.55–3.46 (4H, m, CH2); 2.50–2.44 (4H,
m, CH2); 2.25 (3H, s, CH3). 13C NMR (100 MHz, DMSO‑d6) δ 152.62 (7-
C); 139.45 (3-C); 138.20 (9-C); 131.78 (10-C); 130.60 (4′-CH); 130.42
(2 × 2′CH); 128.35 (2 × 3′CH); 128.01 (1′-C); 123.36 (5-CH); 121.33 (6-
CH); 120.00 (2-C); 111.43 (CN); 97.50 (8-CH); 54.04 (2 × CH2); 46.63
(2 × CH2); 45.65 (CH3). HRMS (ESI) calculated for C20H20N5O+2 [M +
H]+ 362.1612, found 362.1636.
4.1.3. 7-(Homopiperazine-1-yl)-3-phenylquinoxaline-2-carbonitrile 1,4-di-
oxide hydrochloride (3c)
This compound was prepared from 2a and homopiperazine as
◦
described for 3a. A red powder, yield 52%, m.p.(decomp.) > 250 C.
HPLC (LW = 305 nm, gradient B 20 / 80% (45 min)) tR = 9.24 min,
purity 98.9%. λmax., EtOH: 223, 307, 361, 506 nm. 1H NMR (400 MHz,
DMSO‑d6) δ 9.54 (2H, br.s, NH+2 ); 8.28 (1H, d, J = 9.8, H-5); 7.76–7.72
(3H, m, C6H5, H-6); 7.59–7.58 (3H, m, C6H5); 7.33 (1H, s, H-8); 3.97
(2H, t, J = 4.7, CH2); 3.74 (2H, t, J = 5.7, CH2); 3.32–3.30 (2H, br.m,
CH2); 3.16–3.14 (2H, br.m, CH2); 2.21–2.20 (2H, m, CH2). 13C NMR
(100 MHz, DMSO‑d6) δ 150.83 (7-C); 138.95 (3-C); 138.15 (9-C); 131.10
(10-C); 130.67 (4′-CH); 130.54 (2 × 2′CH); 128.40 (2 × 3′CH); 128.02
(1′-C); 121.91 (5-CH); 121.53 (6-CH); 119.95 (2-C); 111.51 (CN); 95.27
(8-CH); 47.32 (CH2); 45.20 (CH2); 44.94 (CH2); 44.77 (CH2); 24.00
(CH2). HRMS (ESI) calculated for C20H20N5O+2 [M + H]+ 362.1612,
found 362.1649.
4.1.7. 7-(3-(S)-Aminopyrrolidine-1-yl)-3-phenylquinoxaline-2-carbonitrile
1,4-dioxide hydrochloride ((S)-3f)
This compound was prepared from 2a and (S)-3-(N-Boc-amino)pyr-
rolidine as described for (S)-3f. A red powder, yield 90%, m.p.(decomp.)
229–231 ◦C. HPLC (LW = 273 nm, gradient B 40 / 65% (30 min)) tR
=
10.3 min, purity 96.9. λmax., EtOH: 237, 307, 389, 489 nm. 1H NMR
(400 MHz, DMSO‑d6) δ 8.64 (3H, br.s, NH+3 ); 8.30 (1H, d, J = 9.5, H-5);
4.1.4. 7-(Ethylpiperazine-1-yl)-3-phenylquinoxaline-2-carbonitrile 1,4-di-
oxide hydrochloride (3d)
7.76–7.71 (2H, m, C6H5); 7.61–7.57 (3H, m, C6H5); 7.47 (1H, dd, J3
=
This compound was prepared from 2a and N-ethylpiperazine as
described for 3a. A red powder, yield 92%, m.p.(decomp.) 227–230 ◦C.
HPLC (LW = 300 nm, gradient B 20 / 60% (30 min)) tR = 11.1 min,
purity 95.1%. λmax., EtOH: 222, 300, 367, 476 nm. 1H NMR (400 MHz,
DMSO‑d6) δ 11.20 (1H, br.s, NH+); 8.36 (1H, d, J = 9.5, H-5); 7.91 (1H,
dd, J3 = 9.5, J4 = 2.6, H-6); 7.75–7.59 (6H, m, C6H5, H-8); 4.27 (2H, t, J
= 12.8, CH2); 3.61 (2H, t, J = 12.4, CH2); 3.52–3.47 (2H, m, CH2CH3);
3.23–3.06 (4H, m, CH2); 1.31 (3H, t, J = 7.3, CH3). 13C NMR (100 MHz,
DMSO‑d6) δ 151.69 (7-C); 140.02 (3-C); 138.10 (9-C); 132.41 (10-C);
130.73 (4′-CH); 130.40 (2 × 2′CH); 128.39 (2 × 3′CH); 127.91 (1′-C);
123.58 (5-CH); 121.60 (6-CH); 120.19 (2-C); 111.36 (CN); 98.90 (8-CH);
50.53 (2 × CH2); 49.55 (2 × CH2); 43.22 (CH2); 8.79 (CH3). HRMS (ESI)
calculated for C21H22FN5O2+ [M + H]+ 376.1768, found 376.1737.
9.6, J4 = 2.4, H-6); 7.02 (1H, s, H-8); 3.81–3.45 (5H, m, CH2, CH);
2.45–2.22 (2H, m, CH2). 13C NMR (100 MHz, DMSO‑d6) δ 149.44 (7-C);
139.13 (3-C); 138.41 (9-C); 131.01 (10-C); 131.42 (4′-CH); 130.94 (2 ×
2′CH); 128.79 (2 × 3′CH); 128.52 (1′-C); 122.54 (5-CH); 121.95 (6-CH);
120.32 (2-C); 111.89 (CN); 95.31 (8-CH); 51.89 (2 × CH-NH2); 49.88
(CH2); 46.32 (CH2); 29.52 (CH2). HRMS (ESI) calculated for
C
19H18N5O+2 [M + H]+ 348.1455, found 348.1475.
4.1.8. 7-(4-Aminopiperidine-1-yl)-3-phenylquinoxaline-2-carbonitrile 1,4-
dioxide hydrochloride (3g)
This compound was prepared from 2a and 4-(N-Boc-amino)piperi-
dine as described for (R)-3f. A red powder, yield 88%, m.p.(decomp.)
244–246 ◦C. HPLC (LW = 310 nm, gradient B 20 / 60% (30 min)) tR
=
11.3 min, purity 95.7%. λmax., EtOH: 225, 311, 368, 422 nm. 1H NMR
(400 MHz, DMSO‑d6) δ 8.45 (3H, br.s, NH3); 8.26 (1H, d, J = 9.7, H-5);
7.87 (1H, dd, J3 = 9.5, J4 = 2.3, H-6); 7.77–7.55 (5H, m, C6H5); 7.45
(1H, s, H-8); 4.13 (2H, t, J = 13.0, CH2); 3.36 (1H, m, CH); 3.12 (2H, t, J
= 12.0, CH2); 2.15–2.06 (2H, m, CH2); 1.74–1.60 (2H, m, CH2). 13C
NMR (100 MHz, DMSO‑d6) δ 151.97 (7-C); 139.40 (3-C); 138.19 (9-C);
131.57 (10-C); 130.62 (4′-CH); 130.43 (2 × 2′CH); 128.36 (2 × 3′CH);
4.1.5. 7-(4-(2-Hydroxyethyl)piperazine-1-yl)-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride (3e)
This compound was prepared from 2a and 1-(2-hydroxyethyl)
piperazine as described for 3a. A red powder, yield 83%, m.p.(decomp.)
208–210 ◦C. HPLC (LW = 300 nm, gradient B 20 / 60% (30 min)) tR
=
10.1 min, purity 95.1%. λmax., EtOH: 223, 301, 367, 475 nm. 1H NMR
10

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
127.98 (1′-C); 123.60 (5-CH); 121.43 (6-CH); 119.97 (2-C); 111.39
(CN); 97.49 (8-CH); 47.29 (CH-NH2); 45.28 (2 × CH2); 28.83 (2 × CH2).
HRMS (ESI) calculated for C20H20N5O+2 [M + H]+ 362.1645, found
362.1612.
CH2); 3.30 (4H, br.m, CH2). 13C NMR (100 MHz, DMSO‑d6) δ 157.01 (d,
J1 = 258.4, 7-CF); 144.95 (d, J = 10.7, 6-C); 142.91 (3-C); 136.94 (10-
C); 131.97 (d, J = 10.7, 9-C); 131.09 (4′-CH); 130.17 (2 × 2′-CH);
128.58 (2 × 3′-CH); 127.78 (1′-CH); 118.77 (2-C); 111.25 (CN); 106.88
(d, J = 28.4, 8-CH); 106.74 (5-CH); 46.33 (2 × CH2); 42.32 (2 × CH2).
HRMS (ESI) calculated for C19H17FN5O+2 [M + H]+ 366.1361, found
366.1355.
4.1.9. 7-(3-Aminopiperidine-1-yl)-3-phenylquinoxaline-2-carbonitrile 1,4-
dioxide hydrochloride (3h)
This compound was prepared from 2a and 3-(N-Boc-amino)piperi-
dine as described for (R)-3f. A red powder, yield 65%, m.p.(decomp.) >
250 ◦C. HPLC (LW = 300 nm, gradient B 20 / 80% (45 min)) tR = 9.89
min, purity 94.8%. λmax., EtOH: 223, 300, 361, 496 nm. IR νmax, (film)
cmꢀ 1 3404, 2913, 2237, 1606, 1522, 1461, 1426, 1334, 1246, 1195,
1141, 1097, 1070, 1005, 955. 1H NMR (400 MHz, DMSO‑d6) δ 8.48 (3H,
br.s, NH+3 ); 8.28 (1H, d, J = 9.5, H-8); 7.83 (1H, dd, J3 = 9.7, J4 = 2.1, H-
6); 7.73–7.71 (2H, m, C6H5); 7.58–7.56 (3H, m, C6H5); 7.48 (1H, d, J =
2.1, H-5); 4.15–3.83 (2H, m, CH2); 3.38–3.14 (3H, m, CH2, CH);
2.11–1.60 (4H, m, CH2). 13C NMR (100 MHz, DMSO‑d6) δ 152.14 (7-C);
139.38 (3-C); 138.07 (9-C); 131.70 (10-C); 130.56 (4′-CH); 130.33 (2 ×
2′CH); 128.26 (2 × 3′CH); 127.72 (1′-C); 123.61 (5-CH); 121.44 (6-CH);
119.81 (2-C); 111.09 (CN); 97.97 (8-CH); 49.73 (CH-NH2); 47.05 (CH2);
46.16 (CH2); 27.55 (CH2); 21.96 (CH2). HRMS (ESI) calculated for
4.1.13. 7-Fluoro-6-(methylpiperazine-1-yl)-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride (5b)
This compound was prepared from 2c and N-methylpiperazine as
described for 3a. An orange powder, yield 73%, m.p.(decomp.)
218–220 ◦C. HPLC (LW = 290 nm, gradient B 15 / 70% (35 min)) tR
=
15.7 min, purity 97.7%. λmax., EtOH: 220, 242, 291, 359 nm. 1H NMR
(400 MHz, DMSO‑d6) δ 11.48 (1H, br.s, NH+); 8.27 (1H, d, J = 13.1, H-
5); 7.82 (1H, d, J = 8.1, H-8); 7.74–7.71 (2H, m, C6H5); 7.61–7.60 (3H,
m, C6H5); 3.84 (2H, br.m, CH2); 3.50 (4H, br.m, CH2); 3.27 (2H, br.m,
CH2); 2.82 (3H, s, CH3). 13C NMR (100 MHz, DMSO‑d6) δ 156.99 (d, J1
= 257.9, 7-CF); 144.54 (d, J = 10.7, 6-C); 142.87 (3-C); 136.90 (10-C);
132.07 (d, J = 11.5, 9-C); 131.02 (4′-CH); 130.12 (2 × 2′-CH); 128.52 (2
× 3′-CH); 127.77 (1′-CH); 118.87 (2-C); 111.19 (CN); 107.02 (5-CH);
106.88 (d, J = 28.9, 8-CH); 51.66 (2 × CH2); 46.39 (2 × CH2); 41.90
(CH3). HRMS (ESI) calculated for C20H19FN5O+2 [M + H]+ 380.1491,
found 380.1517.
C
20H19N5O+2 [M + H]+ 362.1612, found 362.1592.
4.1.10. 7-(3-Methylpiperazine-1-yl)-3-phenylquinoxaline-2-carbonitrile
1,4-dioxide hydrochloride (3j)
This compound was prepared from 2a and 1-Boc-2-methylpiperazine
as described for (R)-3f. A red powder, yield 75%, m.p.(decomp.)
4.1.14. 7-Fluoro-6-(homopiperazine-1-yl)-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride (5c)
233–235 ◦C. HPLC (LW = 260 nm, gradient B 20 / 60% (30 min)) tR
=
This compound was prepared from 2c and homopiperazine as
described for 3a. An orange powder, yield 26%, m.p.(decomp.) >
250 ◦C. HPLC (LW = 345 nm, gradient B 20 / 80% (45 min)) tR = 9.76
min, purity 96.1%. λmax., EtOH: 234, 299, 343, 466 nm. 1H NMR (400
MHz, DMSO‑d6) δ 9.64 (2H, s, NH2+); 8.15 (1H, d, J = 13.7, H-8); 7.70
(2H, br.m, C6H5); 7.58 (3H, br. m, C6H5); 7.55 (1H, s, H-5); 3.86 (2H, br.
m, CH2); 3.60 (2H, br.m, CH2); 3.35 (2H, br.m, CH2); 3.21 (2H, br.m,
CH2); 2.24 (2H, br.m, CH2). 13C NMR (100 MHz, DMSO‑d6) δ 155.95 (d,
J1 = 256.9, 7-CF); 145.05 (d, J = 10.7, 6-C); 142.93 (3-C); 137.18 (10-
C); 131.15 (4′-CH); 130.42 (2 × 2′-CH); 129.90 (d, J = 11.5, 9-C);
128.68 (2 × 3′-CH); 128.15 (1′-CH); 117.54 (2-C); 111.64 (CN);
106.95 (d, J = 30.7, 8-CH); 102.73 (5-CH); 49.74 (2 × CH2); 47.65 (2 ×
CH2); 46.10 (2 × CH2); 44.70 (2 × CH2); 24.35 (2 × CH2). HRMS (ESI)
calculated for C20H19FN5O2+ [M + H]+ 380.1517, found 380.1519.
10.1 min, purity 99.1%. λmax., EtOH: 223, 302, 367, 480 nm. 1H NMR
(400 MHz, DMSO‑d6) δ 9.82 (2H, br.s, NH+2 ); 8.30 (1H, d, J = 9.5, H-5);
7.90 (1H, dd, J3 = 9.8, J4 = 2.4, H-6); 7.81–7.48 (6H, m, C6H5, H-8);
4.25–4.09 (2H, m, CH2); 3.36 (4H, m, CH2); 3.23–3.05 (2H, m, CH2);
1.37 (3H, d, J = 6.4, CH3). 13C NMR (100 MHz, DMSO‑d6) δ 151.77 (7-
C); 139.87 (3-C); 138.02 (9-C); 132.17 (10-C); 130.71 (4′-CH); 130.40
(2 × 2′CH); 128.39 (2 × 3′CH); 127.89 (1′-C); 123.58 (5-CH); 121.46 (6-
CH); 120.10 (2-C); 111.33 (CN); 98.54 (8-CH); 49.99 (CH-CH3); 49.77
(CH2); 43.23 (CH2); 41.85 (CH2); 15.45 (CH3). HRMS (ESI) calculated
for C20H19N5O+2 [M + H]+ 362.1610, found 362.1612.
4.1.11. 3-Phenyl-6-(piperazine-1-yl)quinoxaline-2-carbonitrile 1,4-dioxide
hydrochloride (4a)
This compound was prepared from 2b [16] and piperazine as
described for 3a. An orange powder, yield 68%, m.p.(decomp.)
4.1.15. 6-(Ethylpiperazine-1-yl)-7-fluoro-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride (5d)
230–233 ◦C. HPLC (LW = 345 nm, gradient B 20 / 80% (45 min)) tR
=
8.79 min, purity 93.1%. λmax., EtOH: 230, 298, 344, 473 nm. IR νmax
,
This compound was prepared from 2c and N-ethylpiperazine as
described for 3a. An orange powder, yield 63%, m.p.(decomp.)
(film) cmꢀ 1 3371, 2914, 2849, 2522, 2236, 1606, 1451, 1395, 1328,
1269, 1253, 1143, 961, 935. 1H NMR (400 MHz, DMSO‑d6) δ 9.77 (2H,
br.s, NH+2 ); 8.31 (1H, dd, J3 = 9.4, J4 = 1.6, H-8); 7.83 (1H, dd, J3 = 9.4,
J4 = 1.6, H-7); 7.74–7.71 (2H, m, C6H5); 7.64 (1H, s, H-5); 7.60–7.59
(3H, m, C6H5); 3.83 (4H, br. s, CH2); 3.24 (4H, br. s, CH2). 13C NMR
(100 MHz, DMSO‑d6) δ 153.13 (6-C); 142.85 (3-C); 140.38 (10-C);
130.80 (4′-CH); 130.00 (9-C); 130.16 (2 × 2′CH); 128.41 (2 × 3′CH);
128.20 (1′-C); 121.66 (7-CH); 121.40 (8-CH); 116.53 (2-C); 111.58
(CN); 98.71 (5-CH); 43.65 (2 × CH2); 42.03 (2 × CH2). HRMS (ESI)
calculated for C19H18N5O2+ [M + H]+ 348.1455, found 348.1460.
210–211 ◦C. HPLC (LW = 340 nm, gradient B 40 / 60% (30 min)) tR
=
12.3 min, purity 95.6%. λmax., EtOH: 226, 237, 259, 296, 318, 341, 410
nm. 1H NMR (400 MHz, DMSO‑d6) δ 10.74 (1H, br.s, NH+); 8.31 (1H, d,
J = 12.6, H-8); 7.87 (1H, d, J = 8.1, H-5); 7.77–7.69 (2H, m, C6H5);
7.64–7.59 (3H, m, C6H5); 3.99–3.74 (4H, br.m, CH2); 3.47–3.40 (4H, br.
m, CH2); 3.20–3.13 (2H, br.m, CH2CH3); 1.30–1.21 (3H, m, CH3). HRMS
(ESI) calculated for C21H21FN5O+2 [M + H]+ 394.1674, found 395.1674.
4.1.16. 7-Fluoro-6-(1-hydroxyethylpiperazine-1-yl)-3-phenylquinoxaline-
2-carbonitrile 1,4-dioxide hydrochloride (5e)
4.1.12. 7-Fluoro-3-phenyl-6-(piperazine-1-yl)quinoxaline-2-carbonitrile
1,4-dioxide hydrochloride (5a)
This compound was prepared from 2c and 1-(2-hydroxyethyl)
piperazine as described for 3a. An orange powder, yield 85%, m.p.
(decomp.) 185–187 ◦C. HPLC (LW = 340 nm, gradient B 20 / 60% (30
min)) tR = 11.3 min, purity 98.9%. λmax., EtOH: 227, 238, 298, 340, 415
nm. 1H NMR (400 MHz, DMSO‑d6) δ 11.22 (1H, br.s, NH+); 8.29 (1H, d,
J = 12.6, H-8); 7.86 (1H, d, J = 8.1, H-5); 7.73–7.71 (2H, m, C6H5);
7.62–7.61 (3H, m, C6H5); 5.40 (1H, br.s, OH); 3.92–3.83 (4H, m, CH2);
3.68 (2H, d, J = 11.2, CH2); 3.56 (2H, d, J = 12.4, CH2); 3.35 (4H, br.m,
CH2). 13C NMR (100 MHz, DMSO‑d6) δ 156.96 (d, J1 = 258.6, 7-CF);
144.52 (d, J = 10.7, 6-C); 142.86 (3-C); 136.90 (10-C); 132.04 (d, J
This compound was prepared from 2c N-Boc-piperazine as described
◦
for (R)-3f. An orange powder, yield 68%, m.p.(decomp.) 215–217 C.
HPLC (LW = 340 nm, gradient B 20 / 60% (30 min)) tR = 11.5 min,
purity 95.7%. λmax., EtOH: 227, 235, 297, 341, 414 nm. IR νmax, (film)
cmꢀ 1 3434, 2971, 2848, 2731, 2507, 2238, 1616, 1511, 1492, 1455,
1
1388, 1330, 1275, 1215, 1134, 918. H NMR (400 MHz, DMSO‑d6) δ
9.62 (2H, s, NH+2 ); 8.29 (1H, d, J = 12.8, H-8); 7.86 (1H, d, J = 8.1, H-5);
7.74–7.71 (2H, m, C6H5); 7.63–7.61 (3H, m, C6H5); 3.63 (4H, br.m,
11

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
= 11.4, 9-C); 131.00 (4′-CH); 130.10 (2 × 2′-CH); 128.50 (2 × 3′-CH);
127.77 (1′-CH); 118.84 (2-C); 111.18 (CN); 106.93 (d, J = 14.3, 5-CH);
106.79 (d, J = 28.2, 8-CH); 57.72 (CH2-OH); 55.17 (CH2-CH2-OH);
50.78 (2 × CH2); 46.24 (2 × CH2). HRMS (ESI) calculated for
C21H21FN5O+3 [M + H]+ 410.1623, found 410.1610.
4.1.20. 7-Fluoro-6-(3-methylpiperazine-1-yl)-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride (5j)
This compound was prepared from 2c and 1-Boc-2-methylpiperazine
as described for 3f. An orange powder, yield 77%, m.p.(decomp.)
237–238 ◦C. HPLC (LW = 298 nm, gradient B 20 / 60% (30 min)) tR
=
12 min, purity 95.2%. λmax., EtOH: 226, 236, 298, 341, 417 nm. 1H NMR
(400 MHz, DMSO‑d6) δ 8.51 (3H, br.s, NH+3 ); 8.29 (1H, d, J = 12.6, H-8);
7.82 (1H, d, J = 7.9, H-5); 7.73–7.71 (2H, m, C6H5); 7.63–7.60 (3H, m,
C6H5); 3.83 (1H, d, J = 12.2, CH2); 3.53 (1H, d, J = 12.4, CH2); 3.40 (1H,
m, CH); 3.25–3.05 (2H, br.m, CH2); 2.12–1.87 (2H, br.m, CH2);
1.75–1.65 (2H, br.m, CH2). 13C NMR (100 MHz, DMSO‑d6) δ 156.98 (d,
J1 = 257.9, 7-CF); 144.76 (d, J1 = 11.4, 6-C); 142.88 (3-C); 136.95 (10-
C); 132.00 (d, J1 = 11.4, 9-C); 131.00 (4′-CH); 130.12 (2 × 2′CH);
128.54 (2 × 3′CH); 127.81 (1′-C); 118.81 (2-C); 111.21 (CN); 106.97 (d,
J1 = 9.2, 5-CH); 106.81 (d, J1 = 17.6, 8-CH); 52.18 (CH-NH2); 49.99
(CH2); 45.88 (CH2); 41.87 (CH2); 15.38 (CH3). HRMS (ESI) calculated
for C20H19FN5O+2 [M + H]+ 380.1517, found 380.1509.
4.1.17. 6-(3-Aminopyrrolidine-1-yl)-7-fluoro-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride (5f)
This compound was prepared from 2c and (R,S)-3-(Boc-amino)pyr-
rolidine as described for (R)-3f. An orange powder, yield 84%, m.p.
(decomp.) 230–231 ◦C. HPLC (LW = 345 nm, gradient B 20 / 60% (30
min)) tR = 10.8 min, purity 99.7%. λmax., EtOH: 228, 299, 343, 406, 482
nm. 1H NMR (400 MHz, DMSO‑d6) δ 8.62 (3H, br.s, NH+3 ); 8.16 (1H, d, J
= 13.6, H-8); 7.75–7.70 (2H, m, C6H5); 7.62–7.657 (3H, m, C6H5); 7.30
(1H, d, J = 8.5, H-5); 3.98 (2H, d, J = 7.5, CH2); 3.92–3.77 (2H, br.m,
CH2); 3.74–3.64 (1H, m, CH); 3.34–3.28 (2H, br.m, CH2); 2.40–2.15
(2H, br.m, CH2). 13C NMR (100 MHz, DMSO‑d6) δ 154.30 (d, J1 = 257.1,
7-CF); 142.71 (3-C); 141.68 (d, J = 12.2, 6-C); 137.28 (10-C); 130.82
(4′-CH); 130.17 (2 × 2′-CH); 128.88 (d, J = 9.9, 9-C); 128.39 (2 × 3′-
CH); 128.07 (1′-CH); 116.56 (2-C); 111.51 (CN); 106.32 (d, J = 28.9, 8-
CH); 100.27 (d, J = 6.1, 5-CH); 53.54 (CH-NH2); 49.36 (CH2); 48.07
(CH2); 28.70 (CH2). HRMS (ESI) calculated for C19H17FN5O+2 [M + H]+
366.1361, found 366.1355.
4.1.21. 7-Chloro-3-phenyl-6-(piperazine-1-yl)quinoxaline-2-carbonitrile
1,4-dioxide hydrochloride (6a)
This compound was prepared from 2d and N-Boc-piperazine as
described for (R)-3f. An orange powder, yield 79%, m.p.(decomp.)
194–196 ◦C. HPLC (LW = 300 nm, gradient B 20 / 60% (30 min)) tR
=
13.3 min, purity 95.6%. λmax., EtOH: 246, 302, 346, 414 nm. IR νmax
,
4.1.18. 6-(4-Aminopiperidine-1-yl)-7-fluoro-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride (5g)
(film) cmꢀ 1 3373, 2974, 2915, 2849,12230, 1726, 1599, 1513, 1452,
1363, 1303, 1225, 1171, 1095, 982. H NMR (400 MHz, DMSO‑d6) δ
9.68 (2H, br.s, NH+2 ); 8.53 (1H, s, H-8); 7.96 (1H, s, H-5); 7.75–7.61 (5H,
m, C6H5); 3.50 (4H, m, CH2); 3.30 (4H, m, CH2). 13C NMR (100 MHz,
DMSO‑d6) δ 152.87 (6-C); 143.30 (3-C); 138.58 (9-C); 133.44 (10-C);
132.69 (7-CCl); 131.12 (4′-CH); 130.06 (2 × 2′CH); 128.55 (2 × 3′CH);
127.60 (1′-C); 121.67 (8-CH); 119.40 (2-C); 111.05 (CN); 109.22 (5-
CH); 47.47 (2 × CH2); 42.60 (2 × CH2). HRMS (ESI) calculated for
This compound was prepared from 2c and 4-(Boc-amino)piperidine
as described for (R)-3f. An orange powder, yield 42%, m.p.(decomp.)
210–212 ◦C. HPLC (LW = 340 nm, gradient B 20 / 60% (35 min)) tR
=
12.7 min, purity 95.4%. λmax., EtOH: 227, 237, 299, 343, 420, 456 nm.
1H NMR (400 MHz, DMSO‑d6) δ 8.46 (3H, br.s, NH3+); 8.20 (1H, d, J =
12.9, H-8); 7.78 (1H, d, J = 8.3, H-5); 7.73–7.70 (2H, m, C6H5);
7.61–7.59 (3H, m, C6H5); 4.00–3.87 (1H, br.m, NH); 3.37–3.26 (3H, br.
m, CH, CH2); 3.11–2.98 (2H, br.m, CH2); 2.16–2.06 (2H, br.m, CH2);
1.85–1.70 (2H, br.m, CH2). 13C NMR (100 MHz, DMSO‑d6) δ 157.23 (d,
J1 = 258.6, 7-CF); 145.67 (d, J1 = 10.6, 6-C); 142.85 (3-C); 137.01 (10-
C); 131.97 (4′-CH); 131.42 (d, J1 = 11.4, 9-C); 130.19 (2 × 2′CH);
128.55 (2 × 3′CH); 127.90 (1′-C); 118.37 (2-C); 111.74 (CN); 106.68 (d,
J1 = 29.0, 8-CH); 106.21 (d, J1 = 3.8, 5-CH); 47.32 (CH-NH2); 46.95 (2
× CH2N); 29.20 (2 × CH2). HRMS (ESI) calculated for C20H19FN5O+2 [M
+ H]+ 380.1517, found 380.1513.
C
19H17ClN5O+2 [M + H]+ 382.1065, found 382.1046.
4.1.22. 7-Chloro-6-(4-methylpiperazine-1-yl)-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride (6b)
This compound was prepared from 2d and N-methylpiperazine as
described for 3a. An orange powder, yield 93%, m.p.(decomp.)
195–198 ◦C. HPLC (LW = 300 nm, gradient B 20 / 60% (30 min)) tR
=
1
13.2 min, purity 95.7%. λmax., EtOH: 228, 246, 302, 343, 414 nm. H
NMR (400 MHz, DMSO‑d6) δ 8.48 (1H, s, H-8); 7.91 (1H, s, H-5);
7.81–7.54 (5H, m, C6H5); 3.25 (4H, m, CH2); 2.58 (4H, m, CH2); 2.26
(3H, s, CH3). 13C NMR (100 MHz, DMSO‑d6) δ 153.84 (6-C); 143.19 (3-
C); 138.65 (9-C); 133.33 (10-C); 132.13 (7-CCl); 131.03 (4′-CH); 130.06
(2 × 2′CH); 128.50 (2 × 3′CH); 127.72 (1′-C); 121.60 (8-CH); 118.87 (2-
C); 111.13 (CN); 108.46 (5-CH); 54.19 (2 × CH2); 50.47 (2 × CH2);
45.60 (CH3). HRMS (ESI) calculated for C20H19ClN5O+2 [M + H]+
396.1222, found 396.1216.
4.1.19. 6-(3-Aminopiperidine-1-yl)-7-fluoro-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride (5h)
This compound was prepared from 2c and 3-(Boc-amino)piperidine
as described for (R)-3f. An orange powder, yield 54%, m.p.(decomp.)
245–246 ◦C. HPLC (LW = 300 nm, gradient B 20 / 60% (30 min)) tR
=
12.3 min, purity 95.9%. λmax., EtOH: 232, 237, 299, 342, 426 nm. IR
νmax, (film) cmꢀ 1 3341, 3074, 2915, 2849, 1734, 1619, 1528, 1507,
1470, 1386, 1343, 1259, 1199, 1162, 1135, 1020, 983, 949. IR νmax
,
4.1.23. 7-Chloro-6-(homopiperazine-1-yl)-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride (6c)
(film) cmꢀ 1 3341, 3074, 2915, 2849, 1734, 1619, 1528, 1507, 1470,
1386, 1343, 1259, 1199, 1162, 1135, 1020, 983, 949. 1H NMR (400
MHz, DMSO‑d6) δ 8.51 (3H, br.s, NH+3 ); 8.26 (1H, d, J = 12.6, H-8); 7.82
(1H, d, J = 7.9, H-5); 7.73–7.71 (2H, m, C6H5); 7.63–7.60 (3H, m, C6H5);
3.83 (1H, d, J = 12.4, CH2); 3.53 (1H, d, J = 12.4, CH2); 3.40 (1H, m,
CH); 3.25–3.05 (2H, br.m, CH2); 2.12–1.87 (2H, br.m, CH2); 1.75–1.65
(2H, m, CH2). 13C NMR (100 MHz, DMSO‑d6) δ 157.08 (d, J1 = 258.6, 7-
CF); 145.66 (d, J1 = 9.9, 6-C); 142.82 (3-C); 136.97 (10-C); 131.71 (d, J1
= 11.4, 9-C); 130.96 (4′-CH); 130.12 (2 × 2′CH); 128.49 (2 × 3′CH);
127.84 (1′-C); 118.51 (2-C); 111.23 (CN); 106.81 (d, J1 = 9.9, 5-CH);
106.65 (d, J1 = 16.0, 8-CH); 52.01 (CH-NH2); 49.75 (CH2); 46.37
(CH2); 27.27 (CH2); 22.12 (CH2). HRMS (ESI) calculated for
This compound was prepared from 2d and homopiperazine as
described for 3a. An orange powder, yield 34%, m.p.(decomp.) >
250 ◦C. HPLC (LW = 300 nm, gradient B 20 / 80% (45 min)) tR = 11.26
min, purity 91.1%. λmax., EtOH: 236, 303, 349, 420 nm. 1H NMR (400
MHz, DMSO‑d6) δ 9.62 (2H, br.s, NH+2 ); 8.45 (1H, s, H-8); 7.88 (1H, s, H-
5); 7.74–7.71 (2H, m, C6H5); 7.63–7.60 (3H, m, C6H5); 3.79–3.77 (2H,
br.m, CH2); 3.57 (2H, t, J = 5.6, CH2); 3.39–3.38 (2H, br.m, CH2);
3.32–3.28 (2H, br.m, CH2); 2.50 (2H, d, J = 1.9, CH2); 2.25–2.22 (2H, m,
CH2). 13C NMR (100 MHz, DMSO‑d6) δ 153.67 (6-C); 143.16 (3-C);
138.34 (9-C); 131.70 (10-C); 131.22 (7-CCl); 131.03 (4′-CH); 130.10 (2
× 2′CH); 128.51 (2 × 3′CH); 127.76 (1′-C); 121.96 (8-CH); 118.46 (2-C);
111.20 (CN); 107.12 (5-CH); 51.32 (CH2); 48.98 (CH2); 45.55 (CH2);
44.27 (CH2); 24.40 (CH2). HRMS (ESI) calculated for C20H19ClN5O+2 [M
+ H]+ 396.1222, found 396.1252.
C
20H19FN5O+2 [M + H]+ 380.1517, found 380.1511.
12

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
4.1.24. 7-Chloro-6-(ethylpiperazine-1-yl)-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride (6d)
4.1.28. 6-(4-Aminopiperidine-1-yl)-7-chloro-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride (6g)
This compound was prepared from 2d and N-ethylpiperazine as
described for 3a. An orange powder, yield 78%, m.p.(decomp.)
This compound was prepared from 2d and 4-(Boc-amino)piperidine
as described for (R)-3f. An orange powder, yield 63%, m.p.(decomp.)
210–212 ◦C. HPLC (LW = 300 nm, gradient B 20 / 60% (30 min)) tR
=
238–240 ◦C. HPLC (LW = 300 nm, gradient B 20 / 60% (30 min)) tR
=
14.2 min, purity 95.5%. λmax., EtOH: 246, 301, 343, 414 nm. 1H NMR
(400 MHz, DMSO‑d6) δ 11.46 (1H, br.s, NH+); 8.55 (1H, s, H-8); 7.98
(1H, s, H-5); 7.75–7.62 (5H, m, C6H5); 3.89–3.51 (6H, m, CH2, CH2CH3);
3.25–3.11 (4H, m, CH2); 1.31 (3H, t, J = 6.0, CH3). 13C NMR (100 MHz,
DMSO‑d6) δ 152.47 (6-C); 143.32 (3-C); 138.59 (9-C); 133.33 (10-C);
132.77 (7-CCl); 131.13 (4′-CH); 130.07 (2 × 2′CH); 128.56 (2 × 3′CH);
127.60 (1′-C); 121.73 (8-CH); 119.46 (2-C); 111.07 (CN); 109.28 (5-
CH); 50.52 (2 × CH2); 49.96 (2 × CH2); 47.41 (CH2-CH3); 8.81 (CH3).
HRMS (ESI) calculated for C21H21ClN5O+2 [M + H]+ 410.11378, found
410.1395.
14.5 min, purity 95.7%. λmax., EtOH: 239, 303, 350, 414 nm. 1H NMR
(400 MHz, DMSO‑d6) δ 8.51–8.40 (4H, m, NH+3 , H-8); 7.92 (1H, s, H-5);
7.76–7.59 (5H, m, C6H5); 3.64 (2H, t, J = 11.6, CH2); 3.32–3.25 (1H, m,
CH); 2.99 (2H, t, J = 11.2, CH2); 2.13 (2H, t, J = 11.6, CH2); 1.83 (2H, t,
J = 11.2, CH2). 13C NMR (100 MHz, DMSO‑d6) δ 153.89 (6-C); 143.23
(3-C); 138.63 (9-C); 133.61 (10-C); 132.22 (7-CCl); 131.09 (4′-CH);
130.09 (2 × 2′CH); 128.55 (2 × 3′CH); 127.70 (1′-C); 121.55 (8-CH);
118.95 (2-C); 111.14 (CN); 108.78 (5-CH); 49.02 (CH-NH2); 46.95 (2 ×
CH2); 29.48 (2 × CH2). HRMS (ESI) calculated for C20H19ClN5O+2 [M +
H]+ 396.1222, found 396.1279.
4.1.25. 7-Chloro-6-(1-hydroxyethylpiperazine-1-yl)-3-phenylquinoxaline-
2-carbonitrile 1,4-dioxide hydrochloride (6e)
4.1.29. 6-(3-Aminopiperidine-1-yl)-7-chloro-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride (6h)
This compound was prepared from 2d and 1-(2-hydroxyethyl)
piperazine as described for 3a. An orange powder, yield 77%, m.p.
(decomp.) 187–190 ◦C. HPLC (LW = 300 nm, gradient B 20 / 60% (30
min)) tR = 13.0 min, purity 95.1%. λmax., EtOH: 246, 302, 343, 414 nm.
1H NMR (400 MHz, DMSO‑d6) δ 11.14 (1H, br.s, NH+); 8.54 (1H, s, H-8);
7.97 (1H, s, H-5); 7.83–7.45 (5H, m, C6H5); 5.39 (1H, br.s, OH);
4.02–3.62 (7H, m, CH2); 3.59–3.36 (5H, m, CH2). 13C NMR (100 MHz,
DMSO‑d6) δ 152.46 (6-C); 143.31 (3-C); 138.57 (9-C); 133.32 (10-C);
132.75 (7-CCl); 131.11 (4′-CH); 130.05 (2 × 2′CH); 128.54 (2 × 3′CH);
127.60 (1′-C); 121.72 (8-CH); 119.43 (2-C); 111.05 (CN); 109.22 (5-
CH); 57.67 (CH2-OH); 55.18 (CH2-CH2-OH); 51.10 (2 × CH2); 47.29 (2
× CH2). HRMS (ESI) calculated for C21H21ClN5O+3 [M + H]+ 426.1327,
found 426.1327.
This compound was prepared from 2d and 3-(Boc-amino)piperidine
as described for (R)-3f. An orange powder, yield 71%, m.p.(decomp.)
224–226 ◦C. HPLC (LW = 345 nm, gradient B 20 / 80% (45 min)) tR
=
12.10 min, purity 92.7%. λmax., EtOH: 237, 302, 348, 417 nm. IR νmax
,
(film) cmꢀ 1 3406, 2914, 2849, 2232, 1726, 1596, 1513, 1452, 1362,
1304, 1226, 984. 1H NMR (400 MHz, DMSO‑d6) δ 8.49 (3H, br.s, NH+3 );
8.46 (1H, s, H-8); 7.94 (1H, s, H-5); 7.72–7.70 (2H, m, C6H5); 7.60–7.58
(3H, m, C6H5); 3.75–3.72 (1H, m, CH); 3.38–3.34 (2H, m, CH); 3.11 (2H,
m, CH2); 2.13–1.92 (2H, m, CH2); 1.76–1.65 (2H, m, CH2). 13C NMR
(100 MHz, DMSO‑d6) δ 153.61 (6-C); 143.09 (3-C); 138.52 (9-C); 133.79
(10-C); 132.36 (7-CCl); 130.97 (4′-CH); 130.00 (2 × 2′CH); 128.39 (2 ×
3′CH); 127.45 (1′-C); 121.48 (8-CH); 118.99 (2-C); 110.82 (CN); 109.32
(5-CH); 53.17 (CH2); 50.90 (CH2); 46.75 (CH-NH2); 27.30 (CH2); 22.56
(CH2). HRMS (ESI) calculated for C20H19ClN5O+2 [M + H]+ 396.1222,
found 396.1236.
4.1.26. 6-(3-(R)-Aminopyrrolidine-1-yl)-7-chloro-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride ((R)-6f)
This compound was prepared from 2d and (R)-3-(Boc-amino)pyr-
rolidine as described for (R)-3f. An orange powder, yield 81%, m.p.
(decomp.) 213–215 ◦C. HPLC (LW = 350 nm, gradient B 20 / 60% (30
min)) tR = 12.7 min, purity 97.2%. λmax., EtOH: 237, 307, 349, 410, 489
nm. 1H NMR (400 MHz, DMSO‑d6) δ 8.60 (3H, br.s, NH+3 ); 8.34 (1H, s, H-
8); 7.74–7.59 (5H, m, C6H5); 7.44 (1H, s, H-5); 4.09–3.86 (4H, m, CH2);
3.75–3.66 (1H, m, CH); 2.39–2.15 (2H, m, CH2). 13C NMR (100 MHz,
DMSO‑d6) δ 149.00 (6-C); 143.05 (3-C); 138.51 (9-C); 129.38 (10-C);
127.95 (7-CCl); 130.97 (4′-CH); 130.15 (2 × 2′CH); 128.47 (2 × 3′CH);
127.01 (1′-C); 122.45 (8-CH); 116.89 (2-C); 111.42 (CN); 101.69 (5-
CH); 54.67 (CH-NH2); 49.45 (CH2); 49.30 (CH2); 28.87 (CH2). HRMS
(ESI) calculated for C19H17ClN5O+2 [M + H]+ 382.1050, found
382.1065.
4.1.30. 7-Chloro-6-(3-methylpiperazine-1-yl)-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride (6j)
This compound was prepared from 2d and 1-Boc-2-methylpiperazine
as described for (R)-3f. An orange powder, yield 69%, m.p.(decomp.)
209–210 ◦C. HPLC (LW = 300 nm, gradient B 20 / 60% (30 min)) tR
=
14.1 min, purity 95.4%. λmax., EtOH: 246, 302, 346, 414 nm. 1H NMR
(400 MHz, DMSO‑d6) δ 9.74–9.48 (2H, br.s, NH+2 ); 8.54 (1H, s, H-8);
7.97 (1H, s, H-5); 7.73–7.61 (5H, m, C6H5); 3.68 (2H, d, J = 12.2, CH2);
3.32–3.05 (5H, m, CH2, CH); 1.35 (3H, d, J = 6.4, CH3). 13C NMR (100
MHz, DMSO‑d6) δ 152.66 (6-C); 143.31 (3-C); 138.61 (9-C); 133.46 (10-
C); 132.73 (7-CCl); 131.12 (4′-CH); 130.06 (2 × 2′CH); 128.55 (2 ×
3′CH); 127.63 (1′-C); 121.70 (8-CH); 119.43 (2-C); 111.07 (CN); 109.38
(5-CH); 53.29 (CH-CH3); 50.40 (CH2); 47.07 (CH2); 42.20 (CH2). HRMS
(ESI) calculated for C20H19ClN5O+2 [M + H]+ 396.1223, found
396.1222.
4.1.27. 6-(3-(S)-Aminopyrrolidine-1-yl)-7-chloro-3-phenylquinoxaline-2-
carbonitrile 1,4-dioxide hydrochloride ((S)-6f)
This compound was prepared from 2d and (S)-3-(Boc-amino)pyrro-
lidine as described for (R)-3f. An orange powder, yield 80%, m.p.
(decomp.) 225–227 ◦C. HPLC (LW = 350 nm, gradient B 20 / 60% (30
min)) tR = 12.7 min, purity 96.9%. λmax., EtOH: 237, 307, 349, 410, 489
nm. 1H NMR (400 MHz, DMSO‑d6) δ 8.60 (3H, br.s, NH+3 ); 8.34 (1H, s, H-
8); 7.74–7.59 (5H, m, C6H5); 7.44 (1H, s, H-5); 4.09–3.86 (4H, m, CH2);
3.75–3.66 (1H, m, CH); 2.39–2.15 (2H, m, CH2). 13C NMR (100 MHz,
DMSO‑d6) δ 149.00 (6-C); 143.05 (3-C); 138.51 (9-C); 129.38 (10-C);
127.95 (7-CCl); 130.97 (4′-CH); 130.15 (2 × 2′CH); 128.47 (2 × 3′CH);
127.01 (1′-C); 122.45 (8-CH); 116.89 (2-C); 111.42 (CN); 101.69 (5-
CH); 54.67 (CH-NH2); 49.45 (CH2); 49.30 (CH2); 28.87 (CH2). HRMS
(ESI) calculated for C19H17ClN5O+2 [M + H]+ 382.10654, found
382.1092.
4.2. Biology
4.2.1. Cell lines and antiproliferative assay
The MCF7 (ATCC HTB-22) and MDA-MB-231 (ATCC® HTB-26)
human breast cancer cells and K562 (ATCC CCL-243) chronic myelog-
enous leukemia cells were obtained from the ATCC collection. The
multidrug-resistant (MDR) subline K562/4 (kind gift of Dr. Alexander
Shtil, Blokhin N.N. National Medical Research Center of Oncology,
Russia) was obtained by stepwise selection of K562 cells for survival
under the continuous exposure to DOX. This subline expresses the MDR1
gene and functional P-glycoprotein and is characterized by high resis-
tance index for DOX [34]. The MCF7 and MDA-MB-231 cells were
cultured in standard 4.5 g/L glucose DMEM medium (Gibco) supple-
mented with 10% FCS (HyClone), 2 mM L-glutamine, 50 U/mL peni-
cillin, 50 µg/mL streptomycin (PanEco), and 100 µg/mL sodium
pyruvate (Santa Cruz) at 37 ◦C, 5% CO2, and 80–85% humidity in NuAir
13

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
incubator. Suspension myelogenous leukemia cells (К562, К562/4)
were propagated in RPMI-1640 (PanEco) with 5% FCS (HyClone), 2 mM
L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 ◦C,
5% CO2, and 80–85% humidity in Binder incubator. Cells in the loga-
rithmic phase of growth were used in the experiments. The growth
inhibitory activity of compounds was assessed by the MTT test based on
the metabolism of the MTT reagent (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) (Applichem) in living cells, with modi-
fications as described previously in [15].
orthovanadate, as described in [55]. Samples were incubated on ice for
20 min before centrifugation (10000g, 10 min, 4 ◦C). To analyze HIF-1
α,
total MCF-7 cell extracts were prepared as described in [52]. Electro-
phoresis was performed on a 10% polyacrylamide gel followed by pro-
tein transfer to a nitrocellulose membrane and immunoblotting.
Expressions of PARP, (p-)ERK 1/2, cyclin D1, HIF-1α, Bcl-2, caspase-
7, and ER
α
were determined using Cell Signaling Technology antibodies.
Antibodies to caspase-8 were purchased from Affinity Biosciences. An-
tibodies to
α-tubulin are used to normalize and to control the loading of
Briefly (for cells MCF7 and MB-MB-231), the cells were seeded in 24-
samples into a gel. The detection was performed using secondary anti-
bodies to rabbit Ig conjugated with horseradish peroxidase (Jackson
ImmunoResearch) and ImageQuant LAS 4000 (GE HealthCare) imager,
as described in Mruk’s protocol [56].
well plates (Corning) in 900 μL of the standard medium. Compounds at
different concentrations in 100
μL of the appropriate medium were
added, and the cells were grown for 72 h. For assessment of hypoxic
cytotoxicity ratio, hypoxia (1% O2) conditions were simulated in Binder
multigas incubator, as described in [15]. After incubation with com-
pounds, the medium was removed, the MTT reagent that was dissolved
in the medium was added to the final concentration of 0.2 mg/ml to each
well, and the incubation was performed for 2 h. Then the cell superna-
tants were removed and purple formazan crystals were dissolved in
Funding
The study was partially financially supported by Russian Foundation
for Basic Research, grant numbers 19-33-90186 (to G. I. B. for chemical
synthesis), 18-29-09017 (to A. M. S. for gene reporter assay), 18-53-
34005 (to G. I. B., A. M. S., L. G. D. and A. E. S. for chemical and bio-
logical study) and Ministry of Science, Technology and Environment of
the Republic of Cuba (to L. M. F. for biological study).
100% DMSO (350 μL per well). Culture plates were gently shaken, and
the absorbance was measured at 571 nm with a reference wavelength of
630 nm on a MultiScan reader (ThermoFisher). The viability of the cells
was expressed as a percentage of control. Dose-response curves were
analyzed by regression analysis using sigmoid curves (Log(concentra-
tion) vs normalized absorbance). In this study, the half-maximal inhib-
itory concentration (IC50) values were determined using GraphPad
Prism (Version 7.0).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4.2.2. Gene reporter analysis
MCF7 human breast cancer cells were transfected with the HRE-
Luciferase plasmid containing the luciferase reporter gene under the
hypoxia-responsive element (HRE), and co-transfected with β-galacto-
Acknowledgments
The authors thank Dr. A.M. Korolev, N.M. Maliutina, Dr. I.V. Ivanov
and Dr. Y.N. Lusikov (deceased) from Gause Institute of New Antibiotics
for HRMS, HPLS analyses, and NMR spectra, Dr. O.E. Andreeva from
Blokhin National Medical Research Center of Oncology for plasmid
purification and D. I. Salnikova for assistance in biological studies.
sidase plasmid. To analyze ERα activity ERE-Luciferase assay was per-
formed. Cell transfection with reporter gene plasmids was carried out as
described earlier in [15,52]. Briefly, for HRE-Luciferase assay, MCF7
cells were plated (180 000 cells/well) onto 24-well plates containing
DMEM medium supplemented with 10% FCS. For ERE-Luciferase
transfection, MCF7 cells were seeded in phenol red-free DMEM sup-
plemented with 10% dextran-coated charcoal-treated (DCC) serum
(steroid-free conditions). After 24-h incubation, cells were co-
transfected with plasmids containing luciferase reporter gene
controlled by canonical hypoxia-responsive element (HRE-Luciferase)
or by ERE-containing promoter (ERE-Luciferase), and β-galactosidase
(for normalization). The plasmids used in the work were kindly provided
by Prof. Giovanni Melillo [53] and Prof. George Reid [54]. The trans-
fection was carried out for 24 h at 37 ◦C using Metafectene Pro (Biontex
Laboratories GmbH). Following transfection, the medium was changed
and cells were treated with synthesized compounds or vehicle control.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104324.
References
[1] P. Vaupel, A. Mayer, M. Hockel, Tumor hypoxia and malignant progression,
Methods Enzymol. 381 (2004) 335–354, https://doi.org/10.1016/s0076-6879(04)
81023-1.
[2] J.A. Bertout, S.A. Patel, M.C. Simon, The impact of O2 availability on human
cancer, Nat. Rev. Cancer 8 (2008) 967–975, https://doi.org/10.1038/nrc2540.
[3] P. Vaupel, Tumor microenvironmental physiology and its implications for radiation
oncology, Semin. Radiat. Oncol. 14 (2004) 198–206, https://doi.org/10.1016/j.
semradonc.2004.04.008.
To induce HIF-1
oxic conditions for 24 h when control cells were cultured for 24 h in
normoxia. To induce ER activity 10 nM 17β-estradiol was used.
α transcriptional activity cells were incubated in hyp-
¨
[4] M. Hockel, P. Vaupel, Tumor hypoxia: definitions and current clinical, biologic,
α
and molecular aspects, J. Natl. Cancer Inst. 93 (2001) 266–276, https://doi.org/
10.1093/jnci/93.4.266.
The luciferase activity was measured according to a standard pro-
tocol (Promega) using a Tecan Infinite M200 Pro, β-galactosidase ac-
tivity was analyzed by standard colorimetric assay using MultiScan FC.
The luciferase/β-galactosidase activities were normalized by the inter-
nal control values and represented as the mean ± SD for the three in-
dependent experiments. The relative luciferase activity in vehicle
[5] M.M. Paz, X. Zhang, J. Lu, A. Holmgren, Mitomycin C: Mechanism-based inhibition
of thioredoxin reductase, Chem. Res. Toxicol. 25 (2012) 1502–1511, https://doi.
org/10.1021/tx3002065.
[6] W.R. Wilson, M.P. Hay, Targeting hypoxia in cancer therapy, Nat. Rev. Cancer 11
(2011) 393–410, https://doi.org/10.1038/nrc3064.
[7] M. Krasavin, A. Lukin, T. Vedekhina, O. Manicheva, M. Dogonadze,
T. Vinogradova, N. Zabolotnykh, E. Rogacheva, L. Kraeva, P. Yablonsky,
Conjugation of a 5-nitrofuran-2-oyl moiety to aminoalkylimidazoles produces non-
toxic nitrofurans that are efficacious in vitro and in vivo against multidrug-resistant
Mycobacterium tuberculosis, Eur. J. Med. Chem. 157 (2018) 1115–1126, https://
doi.org/10.1016/j.ejmech.2018.08.068.
control-treated cells under hypoxia was taken as 100 units. HIF-1
α
and ERα activities calculated in arbitrary units as the ratio of the lucif-
erase/galactosidase activity.
[8] Y. Hu, Q. Xia, S. Shangguan, X. Liu, R. Sheng, Synthesis and biological evaluation
of 3-aryl-quinoxaline-2-carbonitrile 1,4-di-N-oxide derivatives as hypoxic selective
anti-tumor agents, Molecules 17 (2012) 9683–9696, https://doi.org/10.3390/
molecules17089683.
4.2.3. Immunoblotting
For immunoblotting, the cells at the 80% monolayer stage were lysed
in 150
NaCl, 1 mm ethylenediamine tetraacetate, 1 mm dithiothreitol, 1
aprotinin, leupeptin and pepstatin, 1 mm sodium fluoride and sodium
μ
L buffer: 50 mm Tris⋅HCl pH 7.4, 1% Igepal CA-630, 150 mm
μg/ml
14

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
[9] C. Wigerup, S. Pahlman, D. Bexell, Therapeutic targeting of hypoxia and hypoxia-
inducible factors in cancer, Pharmacol. Ther. 164 (2016) 152–169, https://doi.
org/10.1016/j.pharmthera.2016.04.009.
[32] A. Arora, Y. Shukla, Modulation of vinca-alkaloid induced P-glycoprotein
expression by indole-3-carinol, Cancer Lett. 189 (2003) 167–173, https://doi.org/
10.1016/s0304-3835(02)00550-5.
[10] P.A. DiSilvestro, S. Ali, P.S. Craighead, J.A. Lucci, Y.C. Lee, D.E. Cohn, N.
M. Spirtos, K.S. Tewari, C. Muller, W.H. Gajewski, M.M. Steinhoff, B.J. Monk,
Phase III randomized trial of weekly cisplatin and irradiation versus cisplatin and
tirapazamine and irradiation in stages IB2, IIA, IIB, IIIB, and IVA cervical
carcinoma limited to the pelvis: a Gynecologic oncology group study, J. Clin.
Oncol. 32 (2014) 458–464, https://doi.org/10.1200/jco.2013.51.4265.
[11] G. Cheng, W. Sa, C. Cao, L. Guo, H. Hao, Z. Liu, X. Wang, Z. Yuan, Quinoxaline 1,4-
di-N-Oxides: Biological activities and mechanisms of actions, Front. Pharmacol. 7
(2016) 1–21, https://doi.org/10.3389/fphar.2016.00064.
[33] R. Krishna, L.D. Mayer, Multidrug resistance (MDR) in cancer. Mechanisms,
reversal using modulators of MDR and the role of MDR modulators in influencing
the pharmacokinetics of anticancer drugs, Eur. J. Pharm. Sci. 11 (2000) 265–283,
https://doi.org/10.1016/s0928-0987(00)00114-7.
[34] A.E. Shchekotikhin, L.G. Dezhenkova, O.Y. Susova, V.A. Glazunova, Y.N. Luzikov,
Y.B. Sinkevich, V.N. Buyanov, A.A. Shtil, M.N. Preobrazhenskaya, Naphthoindole-
based analogues of tryptophan and tryptamine: Synthesis and cytotoxic properties,
Bioorg. Med. Chem. 15 (2007) 2651–2659, https://doi.org/10.1016/j.
bmc.2007.01.034.
[12] A. Monge, J.A. Palop, A. Lopez de Cerain, V. Senador, F.J. Martinez Crespo,
Y. Sainz, S. Narro, E. Hamilton, A.J. Barker, New hypoxia-selective cytotoxines
derived from quinoxaline 1,4-dioxides, J. Heterocyclic Chem. 32 (1995)
1213–1217, https://doi.org/10.1002/jhet.5570320420.
[35] Y.L. Volodina, L.G. Dezhenkova, A.S. Tikhomirov, V.V. Tatarskiy, D.N. Kaluzhny,
A.M. Moisenovich, M.M. Moisenovich, A.K. Isagulieva, A.A. Shtil, V.B. Tsvetkov, A.
E. Shchekotikhin, New anthra[2,3-b]furancarboxamides: A role of positioning of
the carboxamide moiety in antitumor properties, Eur. J. Med. Chem. 165 (2019)
31–45, https://doi.org/10.1016/j.ejmech.2018.12.068.
[13] A. Monge, F.J. Martinez-Crespo, A. Lopez de Cerain, J.A. Palop, S. Narro,
V. Senador, A. Marin, Y. Sainz, M. Gonzdlez, E. Hamilton, A.J. Barker, Hypoxia-
selective agents derived from 2-quinoxalinecarbonitrile 1,4-di-N-oxides, J. Med.
Chem. 38 (1995) 4488–4494, https://doi.org/10.1021/jm00022a014.
[14] W. Suter, A. Rosselet, F. Knüsel, Mode of action of quindoxin and substituted
quinoxaline-di-N-oxides on Escherichia Coli, Antimicrob. Agents Chemother. 13
(1978) 770–783, https://doi.org/10.1128/aac.13.5.770.
[36] W. Tang, G. Zhao, Small molecules targeting HIF-1α pathway for cancer therapy in
recent years, 115235–115235, Bioorg. & Med. Chem. 28 (2020), https://doi.org/
10.1016/j.bmc.2019.115235.
[37] D.P. Sangi, M.R. Cominetti, A.B. Becceneri, F.A. Resende, E.A. Varanda, C.
A. Montanari, M.W. Paixao, A.G. Correa, Molecular design, synthesis and
evaluation of 2,3-diarylquinoxalines as estrogen receptor ligands, Med. Chem.
(Shariqah (United Arab Emirates)) 11 (2015) 736–746, https://doi.org/10.2174/
1573406411666150513093039.
[15] A.M. Scherbakov, A.M. Borunov, G.I. Buravchenko, O.E. Andreeva, L.
G. Dezhenkova, A.E. Shchekotikhin, Novel quinoxaline-2-carbonitrile-1,4-dioxide
derivatives suppress HIF1
α
activity and circumvent MDR in cancer cells, Cancer
[38] P. Yas¸ar, G. Ayaz, S. Damla, G. Güpür, M. Muyan, Molecular mechanism of
estrogen–estrogen receptor signaling, reproductive medicine and biology, 16
(2016) 4–20. doi: 10.1002/rmb2.12006.
Invest. 36 (2018) 199–209, https://doi.org/10.1080/07357907.2018.1453072.
[16] G.I. Buravchenko, A.M. Scherbakov, A.A. Korlukov, P.V. Dorovatskii, A.
E. Shchekotikhin, Revision of the regioselectivity of the Beirut reaction of
monosubstituted benzofuroxans with benzoylacetonitrile. 6-Substituted
quinoxaline-2-carbonitrile 1,4-dioxides: structural characterization and estimation
of anticancer activity and hypoxia selectivity, Curr. Org. Chem. 17 (2020) 1–11,
https://doi.org/10.2174/1570179416666191210100754.
[39] F. Llorens, F.A. Miro, A. Casanas, N. Roher, L. Garcia, M. Plana, N. Gomez, E. Itarte,
Unbalanced activation of ERK1/2 and MEK1/2 in apigenin-induced HeLa cell
death, Exp. Cell Res. 299 (2004) 15–26, https://doi.org/10.1016/j.
yexcr.2004.05.006.
[40] S. Cagnol, J.C. Chambard, ERK and cell death: mechanisms of ERK-induced cell
death-apoptosis, autophagy and senescence, The FEBS J. 277 (2010) 2–21, https://
doi.org/10.1111/j.1742-4658.2009.07366.x.
[17] M.M.F. Ismail, K.M. Amin, E. Noaman, D.H. Soliman, Y.A. Ammar, New
quinoxaline 1, 4-di-N-oxides: Anticancer and hypoxia-selective therapeutic agents,
Eur. J. Med. Chem. 45 (2010) 2733–2738, https://doi.org/10.1016/j.
ejmech.2010.02.052.
[41] S. Subramaniam, K. Unsicker, ERK and cell death: ERK1/2 in neuronal death, The
FEBS J. 277 (2010) 22–29, https://doi.org/10.1111/j.1742-4658.2009.07367.x.
[42] A.S. Komendantova, A.M. Scherbakov, A.V. Komkov, V.V. Chertkova, A.
O. Gudovanniy, E.I. Chernoburova, D.V. Sorokin, Y.U. Dzichenka, V.Z. Shirinian, Y.
A. Volkova, I.V. Zavarzin, Novel steroidal 1,3,4-thiadiazines: Synthesis and
biological evaluation in androgen receptor-positive prostate cancer 22Rv1 cells,
103142–103142, Bioorg. Chem. 91 (2019), https://doi.org/10.1016/j.
bioorg.2019.103142.
´
[18] E. Moreno, S. Ancizu, S. Perez-Silanes, E. Torres, I. Aldana, A. Monge, Synthesis
and antimycobacterial activity of new quinoxaline-2-carboxamide 1,4-di-N-oxide
derivatives, Eur. J. Med. Chem. 45 (2010) 4418–4426, https://doi.org/10.1016/j.
ejmech.2010.06.036.
[19] E. Torres, E. Moreno-Viguri, S. Galiano, G. Devarapally, P.W. Crawford,
´
A. Azqueta, L. Arbillaga, J. Varela, E. Birriel, R. Di Maio, H. Cerecetto, M. Gonzalez,
´
I. Aldana, A. Monge, S. Perez-Silanes, Novel quinoxaline 1,4-di-N-oxide derivatives
[43] D. Tang, D. Wu, A. Hirao, J.M. Lahti, L. Liu, B. Mazza, V.J. Kidd, T.W. Mak, A.
J. Ingram, ERK activation mediates cell cycle arrest and apoptosis after DNA
damage independently of p53, J. Biol. Chem. 277 (2002) 12710–12717, https://
doi.org/10.1074/jbc.M111598200.
as new potential antichagasic agents, Eur. J. Med. Chem. 66 (2013) 324–334,
https://doi.org/10.1016/j.ejmech.2013.04.065.
[20] A. Gerpe, L. Boiani, P. Hernandez, M. Sortino, S. Zacchino, M. Gonzalez,
H. Cerecetto, Naftifine-analogues as anti-Trypanosoma cruzi agents, Eur. J. Med.
Chem. 45 (2010) 2154–2164, https://doi.org/10.1016/j.ejmech.2010.01.052.
[21] E. Vicente, L.M. Lima, E. Bongard, S. Charnaud, R. Villar, B. Solano, A. Burguete,
S. Perez-Silanes, I. Aldana, L. Vivas, A. Monge, Synthesis and structure activity
relationship of 3-phenylquinoxaline 1,4-di-N-oxide derivatives as antimalarial
agents, Eur. J. Med. Chem. 43 (2008) 1903–1910, https://doi.org/10.1016/j.
ejmech.2007.11.024.
[44] N.A. Berger, S.J. Petzold, Identification of minimal size requirements of DNA for
activation of poly(ADP-ribose) polymerase, Biochemistry 24 (1985) 4352–4355,
https://doi.org/10.1021/bi00337a015.
[45] A.H. Boulares, A.G. Yakovlev, V. Ivanova, B.A. Stoica, G. Wang, S. Iyer,
M. Smulson, Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis.
Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells,
J. Biol. Chem. 274 (1999) 22932–22940, https://doi.org/10.1074/
jbc.274.33.22932.
[22] E. Pretsch, P. Bullmann, C. Affolter, Structure Determination of Organic
Compounds, fourth, revised and enlarged ed., Springer-Verlag, Berlin Heideberg,
New York, 2000.
[46] P.T. Diamantopoulos, M. Sofotasiou, V. Papadopoulou, K. Polonyfi, T. Iliakis, N.
A. Viniou, PARP1-driven apoptosis in chronic lymphocytic leukemia,
106713–106713, Biomed Res. Int. 2014 (2014), https://doi.org/10.1155/2014/
106713.
[23] P.E. Hansen, 13C NMR of polycyclic aromatic compounds, Organic Magnetic
Resonance 12 (1979) 109–142, https://doi.org/10.1002/mrc.1270120302.
[24] R.S. Rowland, R. Taylor, Intermolecular nonbonded contact distances in organic
crystal structures: comparison with distances expected from van Der Waals radii,
J. Phys. Chem. 100 (1996) 7384–7391, https://doi.org/10.1021/jp953141+.
[25] O.N. Chupakhin, S.K. Kotovskaya, N.M. Perova, Z.M. Baskakova, V.N. Charushin,
Synthesis of new fluorine-containing derivatives of quinoxaline 1,4-dioxides and
condensed systems derived from them, Chem. Of Heterocycl. Comp. 35 (1999)
459–469, https://doi.org/10.1007/BF02319335.
[47] R.J. Gaughran, J.P. Picard, J.V.R. Kaufman, Contribution to the chemistry of
benzfuroxan and benzfurazan derivatives, J. Am. Chem. Soc. 76 (1954)
2233–2236, https://doi.org/10.1021/ja01637a063.
[48] G.M. Sheldrick, SHELXT-Integrated space-group and crystal-structure
determination, Acta Crystallogr. A 71 (2015) 3–8, https://doi.org/10.1107/
S2053273314026370.
[49] C.B. Hübschle, G.M. Sheldrick, B. Dittrich, ShelXle: a Qt graphical user interface for
SHELXL, J. Appl. Cryst. 44 (2011) 1281–1284, https://doi.org/10.1107/
S0021889811043202.
[26] S.K. Kotovskaya, S.A. Romanova, V.N. Charushin, O.N. Chupakhin, Fluorine-
containing heterocycles: Nucleophilic substitution in 6,7-difluoroquinoxalines,
Rus. J. Org. Chem. 38 (2002) 1089–1095, https://doi.org/10.1023/A:
1020870100148.
[50] O.V. Dolomanov, L.J. Bourhis, R.J. Gildea, J.A.K. Howard, H. Puschmann, OLEX2:
a complete structure solution, refinement and analysis program, J. Appl. Cryst. 42
(2009) 339–341, https://doi.org/10.1107/S0021889808042726.
[27] M.J. Haddadin, C.H. Issidorides, The Beirut reaction, Heterocycles 35 (1993)
1503–1525, https://doi.org/10.3987/REV-92-SR(T)8.
´
´
´
[51] J. Sanchez-Marquez, D. Zorrilla, A. Sanchez-Coronilla, D. M. de los Santos, J.
´
´
[28] Y. Kurasawa, R. Miyashita, A. Takada, H.S. Kim, Y. Okamoto, Substituent effects on
the tautomer ratios between the enamine and methylene imine forms in side-
chained quinoxalines, J. Heterocycl. Chem. 32 (1995) 671–674, https://doi.org/
10.1002/jhet.5570310665.
Navas, C. Fernandez-Lorenzo, R. Alcantara, J. Martín-Calleja, Introducing “UCA-
FUKUI” software: reactivity-index calculations, J. Mol. Model. 20 (2014) 2492. doi:
10.1007/s00894-014-2492-1.
[52] A.M. Scherbakov, L.B. Stefanova, D.V. Sorokin, S.E. Semina, L.M. Berstein, M.
A. Krasil’nikov, Snail/beta-catenin signaling protects breast cancer cells from
hypoxia attack, Exp. Cell Res. 319 (2013) 3150–3159, https://doi.org/10.1016/j.
yexcr.2013.08.019.
[29] A.A. Jarrar, Photochemistry of some quinoxaline 1,4-dioxides, J. Heterocycl.
Chem. 15 (1978) 177–179, https://doi.org/10.1002/jhet.5570150201.
´
[30] M. Ortega, M.J. Morancho, F.J. Martínez-Crespo, Y. Sainz, M.E. Montoya, A. Lopez
´
de Cerain, A. Monge, New quinoxalinecarbonitrile 1,4-di-N-oxide derivatives as
[53] A. Rapisarda, B. Uranchimeg, O. Sordet, Y. Pommier, R.H. Shoemaker, G. Melillo,
Topoisomerase I-mediated inhibition of hypoxia-inducible factor 1: mechanism
and therapeutic implications, Cancer Res. 64 (2004) 1475–1482, https://doi.org/
10.1158/0008-5472.can-03-3139.
hypoxic-cytotoxic agents, Eur. J. Med. Chem. 35 (2000) 21–30, https://doi.org/
10.1016/s0223-5234(00)00112-4.
[31] A.A. Shtil, Signal transduction pathways and transcriptional mechanisms as targets
for prevention of emergence of multidrug resistance in human cancer cells, Curr.
Drug Targets 2 (2001) 57–77, https://doi.org/10.2174/1389450013348957.
´
[54] G. Reid, M.R. Hübner, R. Metivier, H. Brand, S. Denger, D. Manu, J. Beaudouin,
J. Ellenberg, F. Gannon, Cyclic, proteasome-mediated turnover of unliganded and
liganded ERalpha on responsive promoters is an integral feature of estrogen
15

G.I. Buravchenko et al.
Bioorganic Chemistry 104 (2020) 104324
signaling, Mol. Cell 11 (2003) 695–707, https://doi.org/10.1016/s1097-2765(03)
pathways, Chem. Biodivers 16 (2019) e1900332, https://doi.org/10.1002/
cbdv.201900332.
00090-x.
[55] A.M. Scherbakov, V.N. Zhabinskii, V.A. Khripach, D.S. Shcherbinin, A.R. Mekhtiev,
Y.Y. Shchegolev, A.P. Savochka, O.E. Andreeva, Biological evaluation of a new
[56] D.D. Mruk, C.Y. Cheng, Enhanced chemiluminescence (ECL) for routine
immunoblotting: An inexpensive alternative to commercially available kits,
Spermatogenesis 1 (2011) 121–122, https://doi.org/10.4161/spmg.1.2.16606.
brassinosteroid: antiproliferative effects and targeting estrogen receptor
α
16