Dispirooxindoles based on 2-selenoxo-imidazolidin-4-ones: Synthesis, cytotoxicity and ros generation ability

International Journal of

Molecular Sciences

Article

Dispirooxindoles Based on 2-Selenoxo-imidazolidin-4-ones:

Synthesis, Cytotoxicity and ROS Generation Ability

1

1,2

1

Vladimir K. Novotortsev , Maxim E. Kukushkin , Viktor A. Tafeenko , Dmitry A. Skvortsov ,

3

2

4

1

Marina A. Kalinina

, Roman V. Timoshenko , Nelly S. Chmelyuk , Liliya A. Vasilyeva , Boris N. Tarasevich ,

Petr V. Gorelkin ² , Alexander S. Erofeev , Alexander G. Majouga

1,2

1,2,5

, Nikolai V. Zyk ¹

1

,

and Elena K. Beloglazkina *

1

Department of Chemistry, M. V. Lomonosov Moscow State University, Leninskie Gory, 1-3,

19991 Moscow, Russia; vladnov9216@rambler.ru (V.K.N.); lemmingg@mail.ru (M.E.K.);

1

tafeenko-victor@yandex.ru (V.A.T.); skvorratd@mail.ru (D.A.S.); boris-tarasevich@yandex.ru (B.N.T.);

erofeev@polly.phys.msu.ru (A.S.E.); alexander.majouga@gmail.com (A.G.M.); zyk@org.chem.msu.ru (N.V.Z.)

National University of Science and Technology “MISiS”, Leninskiy Prospect, 4, 119049 Moscow, Russia;

timoshenkoroma@mail.ru (R.V.T.); nellichmelyuk@yandex.ru (N.S.C.); peter.gorelkin@gmail.com (P.V.G.)

Skolkovo Institute of Science and Technology, 4 Alfred Nobel Street, Skolkovo, 143025 Moscow, Russia;

Marina.Kalinina@skoltech.ru

2

3

4

Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Leninskie Gory 1,

1

19991 Moscow, Russia; liljavasilyeva@gmail.com

ꢀ

ꢁꢂꢀꢃꢄꢅꢆꢇ

5

D. I. Mendeleev University of Chemical Technology of Russia, Miusskaya sq., 9, 125047 Moscow, Russia

Correspondence: bel@org.chem.msu.ru

ꢀ

ꢁꢂꢃꢄꢅꢆ

*

Citation: Novotortsev, V.K.;

Kukushkin, M.E.; Tafeenko, V.A.;

Skvortsov, D.A.; Kalinina, M.A.;

Timoshenko, R.V.; Chmelyuk, N.S.;

Vasilyeva, L.A.; Tarasevich, B.N.;

Gorelkin, P.V.; et al. Dispirooxindoles

Based on 2-Selenoxo-imidazolidin-

Abstract: A regio- and diastereoselective synthesis of two types of dispiro derivatives of 2-selenoxoim-

idazolidin-4-ones, differing in the position of the nitrogen atom in the central pyrrolidine ring of the

0

00

spiro-fused system—namely, 2-selenoxodispiro[imidazolidine-4,3 -pyrrolidine-2 ,3 -indoline]-2 ,

5

0

00

-diones (5a-h) and 2-senenoxodispiro[imidazolidine-4,3 -pyrrolidine-4 ,3 -indoline]-2 ,5-diones

(6a-m)—were developed based on a 1,3-dipolar cycloaddition of azomethine ylides generated

from isatin and sarcosine or formaldehyde and sarcosine to 5-arylidene or 5-indolidene-2-selenoxo-

tetrahydro-4H-imidazole-4-ones. Selenium-containing dispiro indolinones generally exhibit cytotoxic

activity near to the activity of the corresponding oxygen and sulfur-containing derivatives. Com-

pounds 5b, 5c, and 5e demonstrated considerable in vitro cytotoxicity in the 3-(4,5-dimethylthiazol-

4

-ones: Synthesis, Cytotoxicity and

ROS Generation Ability. Int. J. Mol.

Sci. 2021 , 22 , 2613. https://doi.org/

1

0.3390/ijms22052613

2

-yl)2,5-diphenyl tetrazolium bromide (MTT) test (concentration of compounds that caused 50%

death of cells (CC ) 7.6–8.7 M) against the A549 cancer cell line with the VA13/A549 selectivity

index 5.2–6.9; some compounds ( and ) increased the level of intracellular reactive oxygen species

ROS) in the experiment on A549 and PC3 cells using platinized carbon nanoelectrode. The tests for

Academic Editor: Daniel Plano

Amatriain

µ

50

5

6

(

Received: 13 December 2020

Accepted: 1 March 2021

Published: 5 March 2021

p53 activation for compounds 5 and 6 on the transcriptional reporter suggest that the investigated

compounds can only have an indirect p53-dependent mechanism of action. For the compounds 5b

b, and 6l, the ROS generation may be one of the signiﬁcant mechanisms of their cytotoxic action.

,

6

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in

published maps and institutional afﬁl-

iations.

Keywords: selenohydantoins; spiro compounds; indolinones; azomethine ylides; cytotoxicity;

ROS generation; prooxidant properties

1

. Introduction

Hydantoin and thiohydantoin derivatives containing the spiro-linked indolinone

fragments showed signiﬁcant cytotoxic activity in the in vitro experiments [ ]. The most

potent dispiro-thiohydantoins from this series exhibited an IC₅₀value in the range of

.88–10.46 M against the well-differentiated hepatocellular carcinoma perpetual cell line

HepG2), human embryonic kidney 293 cells (Hek293), breast cancer cell line (MCF-7),

human cervical cancer cells (SiHa), and human colon cancer cells (HCT116); for their

oxo-analogs (dispiro-hydantoins), the IC50 values were in the range of 6.6–24.1 M against

the HEK293, MCF7, and human lung cancer (A549) cell lines. Based on the data presented

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conditions of the Creative Commons

Attribution (CC BY) license (https://

creativecommons.org/licenses/by/

1–3

4

(

µ

4

.0/).

Int. J. Mol. Sci. 2021, 22, 2613. https://doi.org/10.3390/ijms22052613

https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 2613

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in [

1,2

,4

], this activity may be associated with the ability of the dispiro-imidazolone-

oxindoles to affect the p53–MDM2 protein–protein interaction [5–12], as well as nons-

teroidal antiandrogen properties [13–15].

Taking into account that the variation of the exocyclic chalcogen substituent (oxy-

gen or sulfur) of the imidazolone fragment of dispiroimidazolones in the works cited

above [

that selenium-containing compounds are an attractive class of compounds in search for

new cytotoxic anticancer agents [16 19], in this work, we present a further structural

1,2,4] did not lead to a signiﬁcant change in cytotoxicity, and considering the fact

–

modiﬁcation of spiro-linked 2-halcogen-imidazolone derivatives, introducing into their

molecular structure C=Se groups. It may be noted that some organoselenium compounds

are currently evaluated in clinics as anticancer drugs, including L-selenomethionine and

Se-methylselenocysteine, by the National Cancer Institute [20,21]; others [22–29] are being

evaluated in preclinical studies and are showing relevant antiproliferative and proapoptotic

activity in micromolar doses against a number of cancer types (MCF-T [20], FaDu and

A253 [21], some human melanoma cell lines [22], SW480 and RKO colon cancer during

in vitro and in vivo investigations [24], MCF-7 (breast carcinoma), HT-29 (colon carcinoma)

and PC-3 (prostate carcinoma) cells [25], and orthotopic breast cancer model MDA-231-

M2 in vitro and in vivo [28]), including hormone-dependent cancer cell lines LNCaP and

MCF-7 [20]. Note, also, that the selenium-containing derivative S,S-1,4- phenylenebis(1,2-

ethanediyl)bis-isoselenourea (PBISe) has been shown to be a more effective therapeutic

agent than its sulfur analog S,S-1,4-phenylenebis(1,2-ethanediyl)bis-isothiourea (PBIT) dur-

ing in vitro and in vivo studies. PBISe killed melanoma cells >10-fold more effectively than

PBIT, and cultured cancer cells were two- to ﬁve-fold more sensitive than normal cells [30].

Selenium-containing organic compounds may also enhance the chemotherapy/

radiotherapy efﬁcacy [30–32]. Therefore, a highly effective modulation of the therapeutic

efﬁcacy and selectivity of anticancer drugs Taxol and Cisplatin in nude mice bearing human

tumor xenografts of colon carcinoma (HCT-8 and HT-29) and squamous cell carcinoma

of the head and neck (FaDu and A253) using 5-methylselenocysteine or Se-methyl-L-

selenocysteine was demonstrated [31]. Ethaselen (1,2-(bis[1,2-benzisoselenazolone-3(2H)-

ketone])ethane; BBSKE), a selenium-containing thioredoxin reductase inhibitor, was found

to suppress irradiation-induced NF-kB activation when using A549 cells stably transfected

with a NF-kB luciferase reporter [32]; it also showed a radiosensitizing effect in NSCLC cell

lines and tumor models. BBSKE enhanced the efﬁcacy of radiation therapy both in vivo

and in vitro without observable toxicity. It has been shown in experiments on rats bearing

advanced Ward colorectal carcinoma and in nude mice bearing human squamous cell

carcinoma of the head and neck, FaDu, and A253 xenografts that Se-methylselenocysteine

offers selective protection against organ-speciﬁc toxicity induced by clinically active agents

and enhances further the antitumor activity, resulting in an improved therapeutic index,

which allows this selenium compound to be considered as selective modulator of the

antitumor activity and selectivity of anticancer drugs [21].

Moreover, Se-containing compounds were shown to effectively block some angiogenic

factors, thereby suppressing cancer-associated angiogenesis [33

–35]. Se-methylseleno-

cysteine led to a signiﬁcant reduction of intra-tumoral microvessel density in HUVEC

mammary carcinomas [33] and FaDu cell lines [34] in the experiments in vitro and in vivo

methyl Se-containing intermediates, such as methylselenol, demonstrated apoptogenic

activities without genotoxicity and inhibitory activities for angiogenic switching in the

experiments with the HUVEC, MDA-MB-468, MCF-7, and DU145 cell lines [35]. The an-

tiangiogenic attributes reported in this study and the growth arrest make the methylselenol

precursors attractive chemopreventive agents for considerations in humans. Selenium

derivatives have also been demonstrated to inhibit crucial signaling pathways and proteins

deeply implicated in cancer grow and progression; these include calcium-insensitive nitric

oxide synthase and the nitrogen-activated protein kinase [31], Akt3 kinase, and histone

deacetylases [32], as well as melanin biosynthesis by melanocytes [34].

;

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For a long time, Se-containing compounds were mainly tested in relation to protecting

healthy cells from reactive oxygen species (ROS)-induced oxidative damage. However,

recent studies suggest that Se seems to be a potential redox modulator with a dual role in

oxidative stress, acting as a pro-oxidant or antioxidant, depending on the incorporation

type of the organoselenium compound, as well as the dose [17,29].

Based on this data, it can be assumed that the combination of the spiro-linked in-

dolinone and imidazolone with an organoselenium moiety in a single molecule could

favorably affect the antitumor activity of synthesized compounds.

Recently, we synthesized and evaluated a series of novel 2-thioxo-4H-dispiro[imidazo-

lidine-4,3-pyrrolidine-2,3-indole]-2,5(1H)-diones, which have been identiﬁed as potent

anticancer compounds [1,2,4]. Several substances showed a prominent anticancer activity

against the A549, MCF-7, HepG2, SiHa, and HCT116 cell lines associated predominantly

with their effect on the p53–MDM2 interaction. Therefore, one of the key points of the

presented paper was the evaluation of two series of the Se-contained analogs of thiohy-

dantoins described previously to assess their anticancer potency compared to the parent

S-containing molecules. A preliminary study of the cytotoxicity, p53 activation ability, and

pro/antioxidant properties of the obtained compounds was undertaken.

It should be especially noted that even the well-established methods of sulfur-con-

taining compounds cannot be always used directly for the synthesis of their selenium-

containing analogs due to the frequent problems of the precursor synthesis and the lower

stability of selenium-containing derivatives compared to sulfur-containing ones. Thus,

herein, we report a convenient synthetic method for the preparation of dispiro derivatives

of selenoindolinones.

The structural types of the synthesized compounds are shown in Figure 1. The synthe-

sis of the type 1 compounds are ﬁrstly described here; compounds of type 2 were recently

1

presented by us in a short communication of a single example (R = 4-EtO-C H ) [36].

6

4

Figure 1. Structural types of the synthesized dispiroselenohydantoins.

2. Results and Discussion

2.1. Synthesis

A retrosynthetic scheme for the target compounds’ preparation is shown in Scheme 1.

The starting compounds for the synthesis of both types of spiroselenohydantoins was

ethyl 2-isoselenocyanatoacetate

1 synthesized according to the modiﬁed literature proce-

dure [37]. Compound then reacted with commercially available secondary aryl or alkyl

1

amines (substituted anilines, cyclopropylamine, and 4-methoxybenzylamine; Scheme 2). A

catalytic amount (1 mol%) of 4-dimethylaminopyridine was added to the reaction mixtures;

in its absence, the reaction proceeded with a low yield and the formation of signiﬁcant

amounts of byproducts. Diethyl ether was used as a solvent; the reaction products were

practically insoluble in this solvent and crystallize from the reaction mixtures. The reac-

tion products in all cases were selenoureas 2a-f, with the exception of the reaction with

4-methoxybenzylamine, the interaction of which with isoselenocyanate yielded selenohy-

dantoin 2g.

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Scheme 1. The retrosynthetic scheme for the type 1 and type 2 dispiroselenohydantoins.

Scheme 2. Structures and yields of the reaction products of ethyl 2-isoselenocyanatoacetate 1 with amines.

The obtained selenium derivatives 2a-g were further introduced into the Knoevenagel

condensation with substituted benzaldehydes (to obtain 5-arylidene-2-selenoxohydantoins

3

a-d) or isatins (to obtain 5-indolinylidene-2-selenoxohydantoins 4a-m) under KOH cataly-

sis (Scheme 3).

Arylidene-substituted selenohydantoins 3a-d and 5-indoliden-2-selenohydantoins

a-m were obtained according to a modiﬁed procedure described for sulfur analogs [38 40

4

–

]

optimized for maximum yield of target compounds. The conditions for the synthesis of

arylidene and indolinylidene-substituted selenohydantoins 3a-d and 4a-m were similar,

except for the reaction time: during the compounds 3a-d preparations, the reactions

were carried out for three h; for the preparation of compounds 4a-m, one h was enough.

The yields of compounds 3a-d and 4a-m are given in Scheme 3.

All the derivatives 3a-d and 4a-m were obtained as single geometric isomers with a

Z-conﬁguration of the C=C double bonds. The Z-conﬁguration was attributed to products

1

3

a-g based on the chemical shifts of the vinylic protons (6.8–6.7 ppm) in their H NMR

1

13

spectra [41]. For compounds 4a-m, the H and C NMR spectra contain a single set of

signals; the Z-conﬁguration was assigned to products based on the literature analogies [ ].

4

The Z conﬁguration of compound 4b was also indirectly conﬁrmed by the X-ray diffraction

data for the product of its reaction with azomethine ylide (compound 6b [36]).

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Scheme 3. Synthesis of 5-arylidene and 5-indolinylidene-substituted selenohydantoins 3a–d

and 4a–m.

For the synthesis of the target dispiro-selenohydantoine-indolinones 5a-h and 6a-m

the interaction of azomethine ylides, generated from isatins and sarcosine (Scheme 4) or

from formaldehyde and sarcosine (Scheme 5), with dipolarophiles 3a-d and 4a-m were

used. These reactions provided the desired compounds 5a-h and 6a-m in a moderate-to-

high yield.

,

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OEt

OMe

Cl

Br

N

O

Se

O

Se

O

Se

O

Se

NH

Cl

HN

N

HN

N

HN

N

O Me

O Me 5d, 43%

5

a, 55%

5b, 61%

5c, 66%

OMe

Cl

Br

N

O

Se

N

O

Se

O

Se

O

Se

NH

Cl

OMe

HN

N

HN

N

O Me

N

O Me

5

e, 14%

O Me

5

f, 50%

5g, 38%

5h, 15%

Scheme 4. Synthesis of dispiroselenohydantoins 5a-h.

Scheme 5. Synthesis of dispiroselenohydantoins 6.

The reactions of compounds 3a-d with sarcosine and isatins (Scheme 4) were carried

out in boiling ethanol; in most cases, they gave reasonable yields of the target compounds

14–66%). The products precipitated from the reaction mixture; after which, they were

ﬁltered and puriﬁed by ﬂash chromatography. The conversion proceeded with good regio-

(

0

and diastereoselectivity, giving only diastereomeric products with the relative (2 R*,5S*,4 S*)

conﬁguration. The formation of the only diastereomers of compounds 5a-h was conﬁrmed

Int. J. Mol. Sci. 2021, 22, 2613

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1

13

by the presence of a single set of signals in their H and C NMR spectra. For all the

1

compound 5a-h characteristics, there were three pseudo-triplets in their H NMR spectra

at 4.60–3.40 ppm with J~9 Hz, corresponding to three hydrogen atoms of the central

pyrrolidine cycle. We did not observe signals of any minor diastereomers in the NMR

spectra of the reaction mixtures.

The relative conﬁguration of the stereo centers for compound 5b was also proven

using single-crystal X-ray diffraction data. The molecular structure of 5b is shown in

Figure 2. The neighboring spiro-conjugated cycles of the molecule are almost planar and

near perpendicular to each other. A more detailed description of the X-ray data is provided

in the Supplementary Information.

Figure 2. Molecular structure of compound 5b (CCDC 1999058; one crystallographically independent

molecule is shown; the designations of the second independent molecule in the Supplementary

Information are similar (NN_B)).

The mechanism of the spirocyclization reactions is presumably similar to that for the

addition of azomethine ylides to N-substituted 5-arylidenethiohydantoins [4]. It involves

the successive reactions of isatin with sarcosine to form an iminium intermediate

I and fur-

ther a lactone II, which loses the CO molecule to afford dipole III (Scheme 6). This dipole

2

then regio- and diastereoselectively attacks the C=C double bond of 5-arylidenehydantoin 3.

Note that the attack of dipole can proceed from both above and below the double-

bond plane to result in a mixture of enantiomers. Possible ways of the azomethine ylide

approach to the dipolarophile moiety are shown in Scheme 6. Based on the structure of the

products, we can conclude that a cycloaddition reaction takes place only through the exo

transition state, apparently due to an increase in the transition state energy as a result of

electrostatic repulsion in the case of the cis-located carbonyl groups of azomethine.

Compounds 4a-m were reacted with azomethine ylide formed by an interaction of

sarcosine and paraform; as a result, dispiro derivatives 6a-m were obtained (Scheme 5).

The optimal reaction procedure turned the reagents co-boiling in toluene for six h; in

methanol and ethanol, the reaction did not proceed. Sarcosine and paraform were put

into the reaction in an amount of eight molar equivalents, since during the optimization

of the procedure, it was shown that this leads to the highest product yields. The target

compounds 6a-m were isolated by column chromatography.

Compounds 6a-m contained two stereo centers, and their formation proceeded di-

0

astereoselectively with the formation of the products with the (5R*, 4 R*) conﬁguration.

1

For dispiroindolinones

6

, the H NMR spectrum was characteristically in the range of

3

.50–2.90 ppm, in which there were four doublets with J~10 Hz corresponding to the

0

protons of the pyrrolidine fragments (see Supplementary Information). The (5R*, 4 R*)

conﬁguration of compound 6b was proven by the X-ray diffraction data [36].

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For compounds 5a-h:

For compounds 6a-m:

Scheme 6. The supposed mechanism of the formation of dispiro derivatives 5a-h and 6a-m.

The formation of dispiroselenoindolinones 6a-m apparently took place similarly to

compounds 5a-h through the following steps (Scheme 6): (1) the reaction between isatin

and amino acid with the formation of an iminium intermediate, (2) the cyclization of the

Int. J. Mol. Sci. 2021, 22, 2613

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iminium intermediate, followed by the loss of CO and the formation of a 1,3-dipole, and

2

(

3) the regioselective addition of a 1,3-dipole at the 5-indolidene-selenohydantoin C=C

double bond.

Thus, a convenient diastereoselective method for the synthesis of two types of selenohy-

dantoin-dispiroindolinone differing in the position of the N atom in the central pyrrolilidine

cycle were proposed based on the regio- and diastereoselective 1,3-dipolar cycloaddition

of azomethine ylides to electron-deﬁcient alkene systems based on 2-selenoxo-tetrahydro-

4

H-imidazole-4-ones. Therefore, we extended the reaction of 5-methylene substituted

hydantoins and thiohydantoins spirocyclization to their C=Se-containing analogs by the

coupling of 2-selenohydantoins 3a-d and 4a-m with azomethine ylides under easy condi-

tions with reasonable yields.

2.2. Cytotoxicity and p53 Activation

Some of the obtained compounds were tested for cytotoxicity using the standard

-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) test [42]. This study

3

was realized using the cell lines of breast cancer MCF7, human lung carcinoma A549,

non-cancer human embryonic kidney cell line HEK293T, and the noncancer lung ﬁbroblast

VA13 cell line, along with the results obtained for doxorubicin (Dox) as a known cytotoxic

drug used in clinical practice. Lung cancers and breast cancers are the most common causes

of tumor lesions and related deaths in the world, according to the WHO data [43]; thus

they are of great interest for the search for novel anticancer molecules. A549 and MCF7

were selected for the lung tumor model and breast cancer, because (i) these cell lines are

well-studied and often used in cytotoxicity investigations in the literature, and (ii) they

have a high proliferation rate typical for cancer cells. HEK293T and VA13 cells both have

nontumor etiology and were used as noncancer cells. We used immortalized cell lines,

because they are stable and we could compare the data acquired in different experiments

through the years. VA13 is a slow-growth cell line; otherwise, HEK293T has a growth rate

compatible with A549 cells.

This small set of four cell lines allowed us to make a preliminary suggestion about

the selectivity against lung and breast cancer cells and if the selective toxicity is due to

targeting one of the cell division mechanisms. Additionally, it was selected to compare the

obtained data with data from previously investigated O- and S-analogs [

of these assays are shown in Table 1.

1–4]. The results

The most cytotoxic compounds 5b

, 5c, and 5e demonstrated cytotoxicity with IC₅₀

values in the range of 7.6–8.7 M against cell lines A549 and MCF7 (Table 1), which were

µ

signiﬁcantly lower than the cytotoxicity of doxorubicin. However, a relevant selectivity

was observed for the most active compounds among the cell types; therefore, compound

5c turned out to be seven times more cytotoxic in relation to the cancer cell line A549A

compared to the noncancer cell line VA13 (for doxorubicin, this ratio was eight). Com-

paring the obtained data with the previously obtained results for dispirohydantoins and

thiohydantoins of a similar structure [1,2,4], it can be noted that the selenium-containing

analogs generally exhibited cytotoxic activity near to the activity of the corresponding

oxygen and sulfur-containing derivatives.

We measured cytotoxicity of leader compounds using ﬂuorescent cells based cyto-

toxicity assay [1], as well as cytotoxicity of some compounds with high, medium and no

selectivity indexes in MTT assay by CalceinAM staining for key cell lines. Protocols of these

studies and the measured cytotoxicities data are given in the Supplementary Information

(Tables S1 and S2). The results obtained when measuring the cytotoxicity by alternative

methods are generally consistent with the results of the MTT test.

The synthesized compounds were also tested for the activation of p53 in a transcrip-

tional reporter activation test [44] in an effort to elucidate the possible mechanism of

cytotoxic action of dispiroselenohydantoins and compare them with the previously investi-

gated structurally similar hydantoins and thiohydantoins [

1,2,4]. The results are presented

in Table 2. Nutlin-3a, a known inhibitor of p53–MDM2 protein–protein interactions, was

Int. J. Mol. Sci. 2021, 22, 2613

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used as a positive control. We proceeded from the assumption that the mechanisms of

the cytotoxicity of the studied compounds may include p53 activation. It is known that

activated p53 induces the transcription of MDM2, which can directly interact with the

transactivation domain of p53, thereby inhibiting its transcription activity by targeting it

for polyubiquitination and further proteasome-mediated degradation [45]. In many cancer

cells lines, including MCF7 and A549, the overexpression of MDM2 is actually observed,

resulting in signiﬁcant apoptosis attenuation [

p53 via the block of p53–MDM2 interactions and elevates the p53 activity up to 5–10 times

in the reporter assay [46]. Four dilutions of compounds and (100 to 1.56 M) were

tested, and only for compound 6l in a high concentration (>100 M), the treatment of

1]. Nutlin-3a causes a direct activation of

5

6

µ

the cells led to a two-fold p53 activation; it was substantially less than for Nutlin-3a but

comparable with indirect cisplatin action, which suggests that the investigated compounds

can only have an indirect p53-dependent mechanism of action [47]. Consequently, despite

the moderate cytotoxicity of some studied selenoimidazolones, their main toxic effect on

the cell was not due to p53 protein activation.

Table 1. Cytotoxicity,

µM (CC₅₀value—concentration of compounds that caused 50% death of cells), of some synthesized

compounds and 6 against different cell lines after 72 h of treatment, evaluated with the 3-(4,5-dimethylthiazol-2-yl)2,5-

5

diphenyl tetrazolium bromide (MTT) test [42].

Cell Line

Selectivity Indexes

Compound

VA13

HEK293T

A549

MCF7

VA13/A549

HEK293T/A549

5

a

79.9 ± 34.7

45.2 ± 23.3

57.6 ± 24.7

36.1 ± 4.5

31.3 ± 12.4

49 ± 8.4

25.8 ± 3.6

19.8 ± 6

15.6 ± 2.1

8.7 ± 2.5

8.4 ± 1.5

11.5 ± 2.5

10.1 ± 2.3

7.9 ± 0.9

20 ± 3.3

82.5 ± 18.3

20.9 ± 3.6

29.5 ± 9.1

54.3 ± 10.2

24.8 ± 5.1

33.1 ± 6.9

31.6 ± 7.1

20.4 ± 3.6

12.8 ± 2.3

35.1 ± 3

5.1

5.2

6.9

3.1

6.2

1

0.8

1.6

1

1.6

0.9

0.8

2.3

1.1

8.1

1.7

2.3

1.7

2.9

1.7

2.1

1.2

0.9

1.3

1.5

1.2

1.3

1.6

2

5

b

5

c

14.4 ± 5.4

33 ± 10.7

17.3 ± 7.4

16.2 ± 3.3

23.6 ± 3.3

14 ± 1.5

5

d

e

5

f

6

a

20.4 ± 2.2

12.5 ± 1.5

19.3 ± 5.9

21.1 ± 2.4

11.8 ± 4.3

11.1 ± 0.6

8.5 ± 3.2

6

b

15.8 ± 1.5

11.9 ± 1.5

20.4 ± 1.8

7.6 ± 1.6

12 ± 6.7

6

c

10.3 ± 1.3

6

d

e

27.07 ± 4.1

9.7 ± 5.2

20.6 ± 1.4

16.9 ± 11.1

22.1 ± 2.7

17.2 ± 0.3

79.4 ± 5.9

>100

6

g

15.3 ± 12.8

16.6 ± 2.7

13.1 ± 5.1

71.9 ± 27.5

43.2 ± 19

6

h

11.3 ± 1.2

11 ± 0.4

6

i

9.1 ± 0.4

6

k

45.7 ± 15.7

62.2 ± 21.4

16.1 ± 2.7

0.11 ± 0.04

54.9 ± 15

26.6 ± 0.9

14.7 ± 1.5

0.014 ± 0.005

6

l

6

m

28.8 ± 5.1

0.020 ± 0.010

29 ± 5.7

dox

0.037 ± 0.015

1.4

Table 2. p53 reporter activation test at the A549 cell line for some compounds of

5 and 6. The “ONPG/MTT” columns

indicate values of p53 activation normalized to the number of surviving cells (ONPG = ortho-Nitrophenyl-ß-galactoside).

The ONPG/MTT parameter value ≥ 2 may indicate p53 activation [47].

Concentration,

1

00

25

6.25

1.56

100

25

6.25

1.56

µM

Compound

ONPG/MTT

Survived Cells, %

Nutlin-3a

8.62

1.03

0.96

1.57

1.22

1.21

2.08

1.03

6.83

0.96

0.86

1.02

1.31

1.03

0.92

1.15

3.82

0.95

0.97

1.08

1.11

1.16

0.94

1.06

1.97

0.97

0.78

1.12

1.09

0.97

1.15

1.04

77.34

72.94

72.54

73.66

45.20

23.27

50.91

41.91

103.42

81.74

66.33

91.27

52.40

84.28

79.87

79.19

103.41

99.16

68.71

101.07

83.34

90.43

94.15

91.11

106.33

100.89

89.37

105.44

80.78

98.25

93.47

91.02

5

b

c

6

a

6

b

c

6

l

6

m

Int. J. Mol. Sci. 2021, 22, 2613

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2.3. ROS Generation Ability

ROS-mediated nuclease activity [47], mitochondrial membrane depolarization [48],

and apoptosis induction [49] are all well-described cytotoxic mechanisms that are generally

the consequences of reactive oxygen species formation.

We investigated the ROS production ability of the compounds 5b and 5c and 6a-c

and 6m, which were tested for the activation of p53. To detect the ROS production in A549

tumor cells, we used an amperometric nanosensor system previously described in [50 52

, 6l,

–

]

that allowed us to measure the ROS level in single cells using 60–100-nm diameter Pt

nanoelectrodes for the amperometric detection of hydrogen peroxide. This method is low-

invasive and more accurate in comparison with other methods of ROS determination [51].

The setup allows manipulating the nanoelectrode and precisely penetrating it into a single

cell with real-time recording of the differences of the current values between the solution

and cell (Figure 3).

Figure 3. Comparison of the reactive oxygen species (ROS) level inside the A549 cells incubated with

compounds 5 and 6; *, p < 0.05 (one-way ANOVA).

As seen from the obtained data (Figure 3), all the studied compounds, to one degree

or another, turned out to be prooxidants that increased the ROS levels in the tested cells.

Compounds 6b and 6c showed a higher level of ROS generation compared to the other

investigated compounds, increasing the ROS intracellular level by about two times. There

was no correlation between the activation of p53 and the ability to generate reactive oxygen

species; compounds 6b and 6c showed the highest ROS-generating abilities but were not

the best p53 activators.

Thus, at least for compounds 5c, 6a

as the main mechanism of their cytotoxic actions, and this issue requires further study. At

the same time, for compounds 5b 6b, and 6l, ROS generation may be one of the signiﬁcant

, 6c, and 6m, ROS production cannot be accepted

,

mechanisms of their cytotoxic actions.

We also investigated three representatives of each compounds 5a-h and 6a-m (with

the highest. medium and lowest cytotoxicity) for their ability to detect ROS formation in

PC3 tumor cells. These results are presented in the Supplementary Information (Figure S1);

the least cytotoxic compounds 6l showed the weakest ability of ROS generation.

3. Materials and Methods

3.1. General

All starting compounds were commercially available reagents and were purchased

from Sigma-Aldrich. All used solvents were anhydrous and were puriﬁed according to the

procedures described in [53].

1

13

H and C NMR spectra were recorded on a Bruker Avance instrument with an

1

13

operating frequency of 400 MHz for H NMR and 101 MHz for C NMR. Chemical shifts

Int. J. Mol. Sci. 2021, 22, 2613

12 of 26

were given in parts per million on a scale of

internal standard.

δ

relative to hexamethyldisiloxane as an

The IR spectra were measured on a TermoNicolet IR 200 Fourier spectromete (Termo

–1

Scientiﬁc, Waltham, MA, USA) (resolution 4 cm ).

High-resolution mass spectra were recorded on an Orbitrap Elite mass spectrometer

(Thermo Scientiﬁc). To enter solutions with a concentration of 0.1–9 µg/mL (in 1% formic

acid in acetonitrile), direct injection into the ion source using a syringe pump (5

µ

L/min)

◦

was used. Spray voltage ± 3.5 kV, and capillary temperature 275 C.

X-ray diffraction studies were performed on a Syntex P21 diffractometer (SYNTEC

Incorcoration, HsinChu, China) at 293K (graphite monochromator,

-scanning). The absorption was taken into account by measuring the intensities of

the equivalent reﬂections (T_m_i_n/Tmax). The structures were solved by the direct method

SHELXS-97) and reﬁned in full-matrix anisotropic least squares by F2 for all nonhydrogen

λ (MoKα) = 0.71073 Å,

ω

(

atoms (SHELXL-97). All hydrogen atoms were objectively localized and reﬁned in the

isotropic approximation.

3.1.1. Cell Lines

The human breast cancer cell line MCF7 and human lung adenocarcinoma cell line

A549 were kindly provided by Dr. S. Dmitriev, the immortalized human ﬁbroblasts cell

line VA13 was kindly provided by Dr. M. Rubtsova, and the human embryonic kidney

HEK293T cell line was kindly provided by Dr. E. Knyazhanskaya. The MCF7, VA13,

A549, and HEK293T cell lines were maintained in DMEM/F-12 (Thermo Fisher Scientiﬁc)

culture medium containing 10% fetal bovine serum (Thermo Fisher Scientiﬁc), 50-µg/mL

◦

penicillin, and 0.05-mg/mL streptomycin at 37 C (Thermo Fisher Scientiﬁc, USA) in 5%

CO . Medium F-12 (Paneco LLC, Moscow, Russia) containing 10% fetal bovine serum

2

(

Thermo Fisher Scientiﬁc, USA), 50-

Thermo Fisher Scientiﬁc, USA) was used instead of DMEM/F-12 in some cytotoxicity

µg/mL penicillin, and 0.05-mg/mL streptomycin

assays. The PC-3 cell line (ATCC) was cultured in RPMI-1640 medium supplemented

with 10% fetal bovine serum (FBS) and 2-mM L-glutamine (Gibco, Carlsbad, CA, USA).

◦

Cells were maintained at 37 C in a humidiﬁed incubator MCO-18AC (Sanyo, Osaka,

Japan) supplied with 5% CO . After attaining 80% conﬂuence, the cells were harvested

2

with TrypLE (Gibco) and sub-cultured 1:8. Cell cultures were tested for the absence

of mycoplasma.

3.1.2. In Vitro Survival Assay (MTT Assay)

The cytotoxicity of the substances was tested using the MTT (3-(4,5-dimethylthiazol-2-

yl)2,5-diphenyl tetrazolium bromide) assay [42] with some modiﬁcations. Two thousand

ﬁve hundred cells per well for the MCF7, HEK293T, and A549 cell lines or 4000 cells per

well for the VA-13 cell line were plated out in 135 µL of DMEM-F12 media (Gibco) in

9

6-well plates and incubated in a 5% CO incubator for the ﬁrst 16 h without treating.

2

Then, 15

µL of DMSO media solutions of the tested substances was added to the cells

(

ﬁnal DMSO concentrations in the media were 1% or less), and cells were treated 72 h with

5

0 nM–100 µM (eight dilutions) of our substances (triplicate each) and doxorubicin for

the control substance. The MTT reagent (Paneco LLC) was then added to cells up to a

ﬁnal concentration of 0.5 g/L (10x stock solution in PBS was used) and incubated for 2 h

◦

at 37 C in the incubator under an atmosphere of 5% CO . The MTT solution was then

2

discarded, and 140 µL of DMSO (PharmaMed LLC) was added. The plates were swayed

on a shaker (60 rpm) to dissolve the formazan. The absorbance was measured using a

microplate reader (VICTOR X5 Light Plate Reader, PerkinElmer, Waltham, MA, USA) at a

wavelength of 565 nm (in order to measure the formazan concentration). The results were

used to construct a dose–response graph and to estimate the CC₅₀value (concentration of

compounds that caused 50% death of cells) with GraphPad Software [54]. The selectivity

index was calculated according to [55,56].

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3.1.3. Transcriptional Assay for P53 Activation (ONPG-Test)

For testing, we used human adenocarcinoma cells A549 with a recombinant lentiviral

insert LC5 (line A549/LC5 was kindly provided by Professor P. M. Chumakov). The b-

galactosidase reporter construction equipped with the p53 promotor frame [47] was used

to assess the p53 expression level in the p53wt A549 cell line. The compounds were tested

in the concentration range of 1.5–100 µM with four times the dilution steps. The incubation

time was 20 h. To take into account the toxic effect of the molecules, the output signal was

normalized considering the quantity of the cells estimated by the MTT test with the same

incubation tome (20 h) in a parallel assay. The output was signiﬁcant if the background

◦

signal was exceeded two or more times. Cells were cultured at 37 C and 5% CO in

2

DMEM/F-12 medium supplemented with antibiotic serum and L-glutamine.

3.1.4. Intracellular ROS Detection by Pt Nanoelectrodes

Commercially available disk-shaped carbon nanoelectrodes isolated in quartz (ICAP-

PIC Limited, London, UK) with diameters 60–100 nm were used to prepare Pt nanoelec-

trodes for the amperometric detection of ROS. Before the deposition of platinum on the

carbon surface, the disk-shaped carbon was etched in a 0.1-M NaOH, 10-mM KCl solution

during 40 cycles of 10 seconds (from 0 to +2200 mV) to create nanocavities (Figure S2). Fur-

ther electrochemical deposition of platinum in nanocavities was achieved by cycling from

−

to 800 mV with a scan rate of 200 mV s for 4 to 5 cycles in 2-mM H PtCl solution in

2 6

.1-M hydrochloric acid (Figure S3). Cyclic voltammetry in a 1-mM solution of ferrocene in

1

0

methanol in PBS was used to control the electrode surface at all stages of fabrication [50

,

53].

5

A549 (3

×

10 ) cells were seeded in 35-mm Petri dishes and incubated under the conditions

described above, then treated 24 h. The complexes were dissolved in DMSO and diluted

in culture medium. The ﬁnal concentration of the compounds in the culture medium

were IC₅₀with 1 h of incubation time. Untreated cells were used as a control, which was

performed at the beginning and at the end of the experiment. Attached cells in Petri dishes

were washed three times by using Hanks’ Balanced Salt solution to remove the media and

traces of complexes. On average, about 10 cells were measured by 2–4 Pt electrodes in

2

independent Petri dishes for each complex. Prior to the measurements, each platinum

electrode was calibrated using a series of standard H O solutions. Levels of ROS in the

2

cells were determined based on the calibration curve (Figure S4).

The setup for amperometric measurements (Figure 4) included a PC that was con-

nected to a system consisting of an ADC-DAC converter Axon Digidata 1550B (Axon

Instruments, Burlingame, CA, USA) and patch-clamp ampliﬁer MultiClamp 700B (Axon

Instruments, USA). The working head of the ampliﬁer was ﬁxed on a PatchStar Micro-

manipulator (Scientiﬁca, London, UK), which placed near an inverted optical microscope

Nikon Eclipse TI-U (Nikon, Tokyo, Japan). A Pt nanoelectrode was ﬁxed by a special holder

on the working head of the ampliﬁer. Thus, it was possible to change the position of the

electrode with a micromanipulator and carry out an additional assessment in the optical

microscope. A reference electrode (Ag/AgCl) was immersed in the buffer solutions (3-M

KCl). The potential difference between the Pt nanoelectrode and the reference electrode

was recorded with the pClamp 11 software suite (Molecular Devices, Silicon Valley, CA,

USA) and processed with Origin 2018 software [57].

Statistical analyses were conducted using the ANOVA test. All plots show the mean

values

±

SE. All tests assumed a normal distribution, and the statistical signiﬁcance

threshold was set at p < 0.05.

Int. J. Mol. Sci. 2021, 22, 2613

14 of 26

Figure 4. In vitro ROS measurements. (

A

) Scheme showing the setup for intracellular measurements.

(B) ROS intracellular measurements. Recording a difference of the current value between the solution

and a single cell.

3.2. Synthesis

3.2.1. Glycine Ethyl Ester Hydrochloride

In a three-necked ﬂask equipped with a magnetic stirrer, reﬂux condenser, and drop-

◦

ping funnel was 150 mL (2.50 mol) of ethanol cooled to

−

15 C. With vigorous stirring,

2

5 mL (0.35 mol) of SOCl were added dropwise. Then, 25 g (0.33 mol) of glycine were

2

added over 25 min. After this, the cooling was removed, the mixture was brought to a boil

◦

and boiled for 1 h. The hot solution was ﬁltered and cooled to 0 C. The formed precipitate

was ﬁltered off and washed with diethyl ether (2

×

50 mL), then dried to a constant weight

over P O and recrystallized from ethanol-ether mixture (1:1). The reaction yielded 22.5 g

2

5

1

(

δ

48%) of glycine ethyl ester hydrochloride as white crystals. H NMR (400 MHz, DMSO-d ,

6

, ppm): 8.59 (s, 3H, NH₃⁺). 4.18 (q, J = 7.1 Hz, 2H, C(O)OCH₂CH ), 3.74 (s, 2H, CH ), 1.22

3 2

(

t, J = 7.1 Hz, 3H, C(O)OCH CH ).

2 3

3.2.2. N-formylglycine Ethyl Ester

To 90 mL of ethyl formate, 25 g (179.0 mmol) of glycine ethyl ester hydrochloride and

.02 g (0.10 mmol) of p-TsOH were added and heated to boiling. Then, 25 mL (179.0 mmol)

0

of Et N were added, and the resulting solution was boiled within 20 h. Then, the reaction

3

mixture was cooled to room temperature. The precipitate was ﬁltered off, and the ﬁltrate

◦

was evaporated. The ﬁltrate was cooled to

−

15 C. The formed precipitate was ﬁltered

off again. The ﬁltrate was distilled under vacuum. The reaction gave 18.18 g (77%)

◦

1

of N-formylglycine ethyl ester as a clear colorless oil (110 C/0.1 mm Hg). H NMR

400 MHz, CDCl₃, , ppm): 8.22 (s, 1H, CHO), 6.62 (bs, 1H, NH). 4.20 (q, J = 7.2 Hz, 2H,

C(O)OCH CH ), 4.05 (d, J = 5.4 Hz, 2H, CH ), 1.26 (t, J = 7.2 Hz, 3H, C(O)OCH CH ).

(

δ

2

3

2

3

.2.3. Ethyl 2-Isocyanoacetate

To a solution of 14.57 g (111.1 mmol) of N-formylglycine ethyl ester and 38.5 mL

275.5 mmol) of Et N in 115 mL of CH Cl , 10.2 mL (111.1 mmol) of POCl were added

(

3

2

3

◦

dropwise at 0 C, and the reaction mixture was stirred for 1 h at 0 C, then at 20–25 C,

and with vigorous stirring, a solution of 30 g of K CO in 90 mL of H O was slowly

2

3

2

added. After which, the reaction mixture was stirred for 30 min at room temperature. The

organic phase was then separated, and the aqueous phase was diluted to 225 mL and

extracted with CH Cl (2

were combined and dried over Na SO , evaporated, and the residue was distilled under

×

55 mL). After washing all three organic phases with brine, they

2

4

vacuum. The reaction gave 9.86 g (78%) of ethyl 2-isocyanoacetate as a clear yellowish oil

◦

80–82 C/12 mmHg). H NMR (400 MHz, DMSO-d , δ, ppm): 4.64 (s, 2H, CH ), 4.18 (q,

6 2

1

(

J = 7.2 Hz, 2H, C(O)OCH CH ), 1.22 (t, J = 7.1 Hz, 3H, C(O)OCH CH ).

2

3

2

3

.2.4. Ethyl Isoselenocyanatoacetate (1)

.5 g (75.1 mmol) of ethyl 2-isocyanoacetate were added to 240 mL of tetrahydrofuran

in an argon atmosphere. Thirteen point two milliliters (94.8 mmol) of Et N and 6.37 g

8

3

Int. J. Mol. Sci. 2021, 22, 2613

15 of 26

(80.6 mmol) of selenium powder were added then, and the reaction mixture was boiled

for 6 h, then passed through celite, evaporated, and 120 mL of EtOAc were added. The

resulting solution was washed with water (2 70 mL) and dried over Na SO . The product

×

2

4

was isolated by column chromatography in a EtOAc:petroleum ether = 1:9 (v/v) system. As

1

a result of the reaction. 7.64 g (53%) of compound

1

were obtained as a clear red liquid. H

NMR (400 MHz, CDCl₃,

δ

, ppm): 4.35 (s, 2H, CH ), 4.28 (q, J = 7.2 Hz, 2H, C(O)OCH₂CH₃),

2

1.32 (t, J = 7.2 Hz, 3H, C(O)OCH CH ).

2 3

3

.2.5. Selenoureas and 2-Selenoxohydantoins 2a-g (General Procedure)

Ethyl 2-isoselenocyanatoacetate (1 eq.) was dissolved in diethyl ether. Then, amine

1 eq.) was added in portions to the solution. After which, 1 mol% of DMAP was added. The

1

(

reaction mixture was stirred for 6 h. In the case of precipitation, the precipitate was ﬁltered

off and washed with diethyl ether and, if necessary, puriﬁed by column chromatography

in MeOH:CHCl = 1:50 (v/v). If a homogeneous solution formed, it was evaporated, and

3

the product was isolated by column chromatography and yielded 52–86%.

0

N-(4-ethoxyphenyl)-N -carbethoxymethylselenourea (2a)

1

H NMR (400 MHz, CDCl₃, δ, ppm): 8.21 (s, 1H, NH), 7.19 (d, J = 8.8 Hz, 2H,Ar), 6.95

(

d, J = 8.8 Hz, 2H,Ar), 6.68 (s, 1H, NH), 4.50 (d, J = 4.7 Hz, 2H, CH ), 4.21 (q, J = 7.2 Hz, 2H,

2

C(O)OCH₂CH ), 4.04 (q, J = 7.0 Hz, 2H, ArOCH₂CH ), 1.43 (t, J = 7.0 Hz, 3H, ArOCH₂CH₃),

3

1

2

1

6

.28 (t, J = 7.2 Hz, 3H, C(O)OCH₂CH₃). IR (pellet KBr): νmax 3254, 3073, 3042, 3000, 2980,

929, 2881, 1712, 1609, 1588, 1545, 1516, 1471, 1393, 1367, 1338, 1312, 1298, 1283, 1255, 1234,

195, 1173, 1131, 1117, 1045, 1027, 956, 927, 919, 867, 844, 832, 817, 801, 779, 710, 667, 641,

−

1

07, 571, 532 cm

.

0

N-(4-methoxyphenyl)-N -carbethoxymethylselenourea (2b)

1

H NMR (400 MHz, CDCl₃, δ, ppm): 8.26 (s, 1H, NH), 7.21 (d, J = 8.8 Hz, 2H, Ar), 6.97

(

d, J = 8.9 Hz, 2H, Ar), 6.69 (s, 1H, NH), 4.50 (d, J = 4.7 Hz, 2H, CH ), 4.21 (q, J = 7.2 Hz,

2

H, C(O)OCH₂CH ), 3.83 (s, 3H, OCH ), 1.28 (t, J = 7.2 Hz, 3H, C(O)OCH₂CH₃). IR (pellet

3 3

KBr): νmax 3334, 3145, 2976, 2932, 2914, 2836, 1723, 1607, 1549, 1515, 1473, 1444, 1411, 1391,

1

6

369, 1345, 1299, 1251, 1231, 1183, 1171, 1126, 1108, 1028, 966, 938, 865, 843, 831, 792, 744,

−

1

80, 643, 634, 591, 548, 529 cm

.

0

N-(3-chloro-4-ﬂuorophenyl)-N -carbethoxymethylselenourea (2c)

1

H NMR (400 MHz, CDCl₃, δ, ppm): 8.41 (s, 1H, NH), 7.39 (dd, J = 2.2 Hz, J = 6.3 Hz,

1 2

1

H, Ar), 7.26–7.19 (m, 2H, Ar) 6.83 (s, 1H, NH), 4.50 (d, J = 4.6 Hz, 2H, CH ), 4.24 (q, J = 7.2

2

Hz, 2H, C(O)OCH CH ), 1.30 (t, J = 7.2 Hz, 3H, C(O)OCH CH ).

2

3

2

3

0

N-(4-chlorophenyl)-N -carbethoxymethylselenourea (2d)

H NMR (400 MHz, CDCl₃, δ, ppm): 8.44 (s, 1H, NH), 7.44 (d, J = 8.4 Hz, 2H, Ar), 7.26

1

(

d, J = 9.6 Hz, 2H, Ar), 6.95 (s, 1H, NH), 4.50 (d, J = 3.6 Hz, 2H, CH ), 4.22 (q, J = 7.0 Hz, 2H,

2

C(O)OCH₂CH ), 1.30 (t, J = 7.2 Hz, 3H, C(O)OCH₂CH₃). IR (pellet KBr): νmax 3314, 3187,

3

148, 3100, 2993, 2975, 2928, 2909, 2852, 2799, 2363, 2160, 1728, 1692, 1594, 1544, 1525, 1490,

1

8

442, 1417, 1383, 1336, 1321, 1281, 1224, 1159, 1117, 1091, 1022, 1013, 974, 967, 936, 879, 856,

−

1

14, 720, 698, 689, 653, 620, 571, 547 cm

.

0

N-cyclopropyl-N -carbethoxymethylselenourea (2e)

1

H NMR (400 MHz, CDCl₃, δ, ppm): 7.54 (bs, 1H, NH), 7.35 (bs, 1H, NH), 4.51 (s, 2H,

CH ), 4.27 (q, J = 7.2 Hz, 2H, C(O)OCH₂CH ), 2.57 (s, 1H,CHPr), 1.33 (t, J = 7.2 Hz, 3H,

2

3

C(O)OCH CH ), 0.96–0.90 (m, 2H, Pr), 0.85–0.77 (m, 2H, Pr).

2

3

0

N-(3-chlorophenyl)-N -carbethoxymethylselenourea (2f)

H NMR (400 MHz, CDCl₃, δ, ppm): 8.55 (bs, 1H, NH), 7.40 (t, J = 8.6 Hz, 1H,Ar), 7.33

1

(

s, 1H, Ar), 7.33–7.30 (m, 1H, Ar), 7.23 (d, J = 8.3 Hz, 1H, Ar), 7.06 (bs, 1H, NH), 4.51 (s, 2H,

CH ), 4.23 (q, J = 7.1 Hz, 2H, C(O)OCH CH ), 1.30 (t, J = 7.2 Hz, 3H, C(O)OCH CH ).

2

3

2

3

-(4-methoxybenzyl)-2-selenoxoimidazolidin-4-one (2g)

1

H NMR (400 MHz, CDCl₃, δ, ppm): 7.78 (bs, 1H, NH), 7.52 (d, J = 8.7 Hz, 2H, Ar),

6

.85 (d, J = 8.6 Hz, 2H, Ar), 5.05 (s, 2H, CH ), 3.89 (s, 2H, CH ), 3.79 (s, 3H, OCH ).

2 2 3

Int. J. Mol. Sci. 2021, 22, 2613

16 of 26

3

.2.6. 5-Arylidene-2-Selenoxohydantoins 3a-g (General Procedure)

A 2% solution of KOH in ethanol (1 equivalent of KOH) was added to selenourea

or 2-selenoxohydantoin) (1 equiv.). Then, aldehyde (1 equiv.) was added to the so-

lution. The reaction mixture was stirred for 5 h. A dilute hydrochloric acid solution

H O:HCl conc = 9:1) was added to the reaction mixture. The formed precipitate was ﬁl-

(

2

(

2

tered off and washed with water and diethyl ether. If no precipitation occurred, the solvent

was removed from the reaction mixture, and then, the product was puriﬁed by column

chromatography in MeOH:CHCl = 1:50 (v/v) and yielded 65–82%.

3

(

Z)-3-(4-ethoxyphenyl)-5-(4-chlorobenzylidene)-2-selenoxoimidazolidin-4-one (3a)

1

H NMR (400 MHz, DMSO-d₆,

.52 (d, J = 8.5 Hz, 2H, Ar), 7.27 (d, J = 8.9 Hz, 2H, Ar), 7.03 (d, J = 8.9 Hz, 2H, Ar), 6.79

s, 1H, vinyl), 4.08 (q, J = 6.9 Hz, 2H, ArOCH CH ), 1.36 (t, J = 6.9 Hz, 3H, ArOCH CH ).

δ, ppm): 13.12 (s, 1H, NH), 7.89 (d, J = 8.6 Hz, 2H, Ar),

7

(

2

3

2

3

(

Z)-3-(4-methoxyphenyl)-5-(4-chlorobenzylidene)-2-selenoxoimidazolidin-4-one (3b

)

1

H NMR (400 MHz, DMSO-d₆,

δ, ppm): 13.13 (s, 1H, NH), 7.89 (d, J = 8.4 Hz, 2H, Ar),

7

.52 (d, J = 8.5 Hz, 2H, Ar), 7.30 (d, J = 8.8 Hz, 2H, Ar), 7.05 (d, J = 8.9 Hz, 2H,Ar), 6.80

(

s, 1H, vinyl), 3.82 (s, 3H, OCH₃).

Z)-3-(4-methoxyphenyl)-5-(4-methoxybenzylidene)-2-selenoxoimidazolidin-4-one (3c

(

)

1

H NMR (400 MHz, DMSO-d₆,

δ, ppm): 7.88 (s, 1H, NH), 7.29–7.16 (m, 4H, Ar),

7

.05–6.98 (m, 4H, Ar), 6.75 (s, 1H, vinyl), 3.81 (s, 3H, OCH ), 3.80 (s, 3H, OCH ).

3

(

Z)-3-(4-methoxyphenyl)-5-(4-ethylbenzylidene)-2-selenoxoimidazolidin-4-one (3d)

1

H NMR (400 MHz, DMSO-d₆,

H, Ar), 7.31 (d, J = 7.8 Hz, 2H, Ar), 7.29 (d, J = 8.9 Hz, 2H, Ar), 7.05 (d, J = 9.0 Hz, 2H, Ar),

.78 (s, 1H, vinyl), 3.81 (s, 1H, OCH ), 2.64 (q, J = 7.6 Hz, 2H, CH₂CH ), 1.21 (t, J = 7.6 Hz,

δ, ppm): 13.21–12.81 (m, 1H, NH), 7.81 (d, J = 8.1 Hz,

2

6

3

H, CH CH ).

2

3

3.2.7. 5-Indolinyliden-2-Selenoxohydantoins 4a-m (General Procedure)

A 2% solution of KOH in ethanol (1 equivalent of KOH) was added to selenourea

(or 2-selenoxohydantoin) 2 (1 equiv.). Isatin (1 equiv.) was added to the solution. The

reaction mixture was stirred for 1 h; then, a dilute hydrochloric acid solution (H O:HCl

2

conc = 9:1) was added to the reaction mixture. The formed precipitate was ﬁltered off and

washed with water and diethyl ether. If no precipitation occurred, the product is isolated

by column chromatography in MeOH:CHCl = 1:20 (v/v) and yielded 57–91%.

3

(

Z)-3-(3-(4-ethoxyphenyl)-4-oxo-2-selenoxoimidazolidin-5-ylidene)indolin-2-one (4a

)

1

H NMR (400 MHz, DMSO-d₆,

δ, ppm): 12.07–11.66 (m, 1H, NH), 11.21 (bs, 1H, NH),

8

.53 (d, J = 7.7 Hz, 1H, isatin), 7.39–7.33 (m, 1H, isatin), 7.35 (d, J = 8.9 Hz, 2H, Ar), 7.06 (d,

J = 8.9 Hz, 2H, Ar), 7.02 (t, J = 8.2 Hz, 1H, isatin), 6.94 (d, J = 7.8 Hz, 1H, isatin), 4.,09 (q,

J = 6.9 Hz, 2H, ArOCH₂CH ), 1.36 (t, J = 6.9 Hz, 3H, ArOCH₂CH₃). IR (pellet KBr): νmax

3

314, 3189, 2980, 2938, 2885, 2161, 1775, 1727, 1691, 1668, 1643, 1614, 1587, 1516, 1477, 1465,

1

6

414, 1352, 1301, 1258, 1228, 1203, 1172, 1117, 1099, 1046, 975, 926, 886, 820, 793, 767, 748,

−

1

81, 628 cm

Z)-3-(3-(4-ethoxyphenyl)-4-oxo-2-selenoxoimidazolidin-5-ylidene)-5-chloroindolin-2-

one (4b)

¹H NMR (400 MHz, DMSO-d6,

d, J = 1.9 Hz, 1H, isatin), 7.40 (dd, J = 2.0 Hz, J = 8.3 Hz, 1H, isatin), 7.34 (d, J = 8.8 Hz,

.

(

δ

, ppm): 11.95 (bs, 1H, NH), 11.28 (s, 1H, NH), 8.57

(

1

2

H, Ar), 7.07 (d, J = 8.8 Hz, 2H, Ar), 6.94 (d, J = 8.4 Hz, 1H, isatin), 4.10 (q, J = 6.9 Hz, 2H,

ArOCH₂CH ), 1.37 (t, J = 6.9 Hz, 3H, ArOCH₂CH₃). IR (pellet KBr): νmax 3430, 3351, 3288,

3

173, 3110, 3077, 2983, 2933, 1777, 1741, 1732, 1701, 1694, 1636, 1629, 1611, 1588, 1515, 1476,

1

9

458, 1433, 1419, 1395, 1377, 1293, 1258, 1225, 1190, 1171, 1148, 1117, 1073, 1046, 984, 957,

−

1

23, 901, 894, 874, 839, 828, 777, 757, 729, 699, 685, 650, 637, 620, 590, 574 cm

Z)-3-(3-(4-ethoxyphenyl)-4-oxo-2-selenoxoimidazolidin-5-ylidene)-5-bromoindolin-2-

one (4c)

¹H NMR (400 MHz, DMSO-d6,

d, J = 1.9 Hz, 1H, isatin), 7.53 (dd, J = 2.0 Hz, J = 8,4 Hz, 1H, isatin), 7.34 (d, J = 8.9

.

(

δ, ppm): 11.96 (s, 1H, NH), 11.30 (s, 1H, NH), 8.71

(

1

2

Hz, 2H, Ar), 7.07 (d, J = 9.9 Hz, 2H, Ar), 6.90 (d, J = 8.3, 1H, isatin), 4.10 (q, J = 7.0 Hz,

Int. J. Mol. Sci. 2021, 22, 2613

17 of 26

2

3

H, ArOCH₂CH ), 1.37 (t, J = 7.0 Hz, 3H, ArOCH₂CH₃). IR (pellet KBr): νmax 3415, 3349,

287, 3173, 3109, 3075, 2983, 2934, 2884, 2162, 1776, 1741, 1700, 1628, 1609, 1589, 1514, 1475,

3

1

4561435, 1418, 1394, 1292, 1254, 1190, 1171, 1116, 1046, 978, 957, 923, 902, 871, 825, 775, 756,

−

1

7

33, 682, 620, 589 cm

Z)-3-(3-(4-ethoxyphenyl)-4-oxo-2-selenoxoimidazolidin-5-ylidene)-5-nitroindolin-2-

one (4d)

¹H NMR (400 MHz, DMSO-d6,

H, isatin), 8.29 (dd, J = 2.0 Hz, J = 8.8 Hz, 1H, isatin), 7.37 (d, J = 8.8 Hz, 2H, Ar), 7.14 (d,

.

(

δ

, ppm): 12.11 (m, 1H, NH), 11.80 (s, 1H, NH), 9.48 (s,

1

2

J = 8.8 Hz, 1H, isatin), 7.08 (d, J = 8.8 Hz, 2H, Ar), 4.11 (q, J = 6.9 Hz, 2H, ArOCH₂CH₃),

1

.37 (t, J = 6.9 Hz, 3H, ArOCH CH ).

2 3

(

Z)-3-(3-(4-methoxyphenyl)-4-oxo-2-selenoxoimidazolidin-5-ylidene)-5-bromoindolin-

-one (4e)

¹H NMR (400 MHz, DMSO-d6,

d, J = 7.6 Hz, 1H, isatin), 7.29 (d, J = 8.6 Hz, 2H, Ar), 7.05 (d, J = 8.7 Hz, 2H, Ar), 6.81 (d,

J = 8.5 Hz, 1H, isatin), 3.82 (s, 3H, OCH₃).

Z)-3-(3-(4-methoxyphenyl)-4-oxo-2-selenoxoimidazolidin-5-ylidene)-5-nitroindolin-2-

one (4f)

¹H NMR (400 MHz, DMSO-d6,

J = 2.3 Hz, 1H, isatin), 8.29 (dd, J = 2.4 Hz, J = 8.7 Hz, 1H, isatin), 7.39 (d, J = 8.8 Hz, 2H,

2

δ

, ppm): 10.98 (bs, 1H, NH), 8.83 (s, 1H, isatin), 7.43

(

δ

, ppm): 12.04 (bs, 1H, NH), 11.80 (s, 1H, NH), 9.48 (d,

1

2

Ar), 7.15–7.12 (m, 1H, isatin), 7.11 (d, J = 9.1 Hz, 2H, Ar), 3.84 (s, 3H, OCH₃).

Z)-3-(3-(4-chlorophenyl)-4-oxo-2-selenoxoimidazolidin-5-ylidene)-5-bromoindolin-2-

one (4g)

¹H NMR (400 MHz, DMSO-d6,

1.7 Hz, 1H, isatin), 7.65 (d, J = 8,6 Hz, 2H,Ar), 7,55–7,52 (m, 1H,isatin), 7,51 (d, J = 8,7 Hz,

H,Ar), 6,90 (d, J = 8,3 Hz, 1H,isatin).

Z)-3-(3-(4-chlorophenyl)-4-oxo-2-selenoxoimidazolidin-5-ylidene)-5-nitroindolin-2-

one (4h)

¹H NMR (400 MHz, DMSO-d6,

J = 2.2 Hz, 1H, isatin), 8.29 (dd, J = 2.3 Hz, J = 8.7 Hz, 1H, isatin), 7.66 (d, J = 8.6 Hz, 2H,

(

δ, ppm): 12.06 (s, 1H, NH), 11.31 (s, 1H, NH), 8.70 (d, J

=

2

(

δ

, ppm): 12.19 (bs, 1H, NH), 11.81 (s, 1H, NH), 9.46 (d,

1

2

Ar), 7.52 (d, J = 8.6 Hz, 2H, Ar), 7.12 (d, J = 8.7 Hz, 1H, isatin).

Z)-3-(3-(3-chlorophenyl)-4-oxo-2-selenoxoimidazolidin-5-ylidene)indolin-2-one (4i)

(

1

H NMR (400 MHz, DMSO-d6,

7.7 Hz, 1H, isatin), 7.64–7.57 (m, 3H, Ar), 7.50–7.45 (m, 1H, Ar), 7.37 (t, J = 7.7 Hz, 1H,

isatin), 7.02 (t, J = 7.7 Hz, 1H, isatin), 6.95 (d, J = 7.8 Hz, 1H, isatin).

Z)-3-(3-(3-chloro-4-ﬂuorophenyl)-4-oxo-2-selenoxoimidazolidin-5-ylidene)-5-chloro-

indolin-2-one (4j)

¹H NMR (400 MHz, DMSO-d6,

J = 1.8 Hz, 1H, Ar), 7.79 (dd, J = 2.3 Hz, J = 6.9 Hz, 1H, Ar), 7.66 (t, J = 8.9 Hz, 1H, Ar),

δ, ppm): 12.03 (s, 1H, NH), 11.20 (s, 1H, NH), 8.53 (d, J

=

(

δ, ppm): 12.14 (s, 1H, NH), 11.31 (s, 1H, NH), 8.57 (d,

1

2

7

.58–7.51 (m, 1H, Ar), 7.43 (dd, J = 2.1 Hz, J = 8.3 Hz, 1H, Ar), 6.96 (d, J = 8.3 Hz, 1H, Ar).

1 2

(Z)-3-(3-cyclopropyl-4-oxo-2-selenoxoimidazolidin-5-ylidene)-5-bromoindolin-2-one

(4k)

¹H NMR (400 MHz, DMSO-d6,

J = 2.0 Hz, 1H, isatin), 7.52 (dd, J = 2.0 Hz, J = 8.3 Hz, 1H, isatin), 6.87 (d, J = 8.3 Hz, 1H,

δ, ppm): 11.67 (s, 1H, NH), 11.24 (s, 1H, NH), 8.71 (d,

1

2

isatin), 2.90–2.84 (m, 1H, Pr), 1.08–1.01 (m, 4H, Pr).

Z)-3-(3-(4-methoxybenzyl)-4-oxo-2-selenoxoimidazolidin-5-ylidene)indolin-2-one (4l

(

)

1

H NMR (400 MHz, DMSO-d6,

δ

, ppm): 11.85 (bs, 1H, NH), 11.14 (s, 1H, NH), 8.56 (d,

J = 7.8 Hz, 1H, isatin), 7.39–7.34 (m, 1H, isatin), 7.36 (d, J = 8.7 Hz, 2H, Ar), 7.03 (t, J = 7.6 Hz

,

1

H, isatin), 6.91 (d, J = 7.1 Hz, 1H, isatin), 6.90 (d, J = 8.8 Hz, 2H, Ar), 5.08 (s, 2H, CH ), 3.72

2

(

s, 3H, OCH ). IR (pellet KBr): νmax 3254, 2958, 2835, 1772, 1724, 1694, 1635, 1612, 1586,

3

1

7

1

9

514, 1464, 1435, 1405, 1340, 1308, 1248, 1197, 1177, 1146, 1097, 1034, 962, 908, 848, 819, 774,

−

1

55, 681, 631 cm . IR (pellet KBr): νmax 3335, 3195, 3110, 3002, 2958, 2935, 2836, 1777, 1728,

695, 1644, 1612, 1587, 1515, 1456, 1433, 1404, 1342, 1305, 1249, 1197, 1176, 1145, 1108, 1033,

−

1

79, 937, 903, 877, 844, 816, 789, 773, 746, 715, 688, 681, 626, 578 cm

.

Int. J. Mol. Sci. 2021, 22, 2613

18 of 26

(Z)-3-(3-(4-methoxybenzyl)-4-oxo-2-selenoxoimidazolidin-5-ylidene)-5-bromoindolin-

2

-one (4m)

¹H NMR (400 MHz, DMSO-d6,

H, isatin), 7.51 (dd, J = 1.4 Hz, J = 8.4 Hz, 1H, isatin), 7.37 (d, J = 8.5 Hz, 2H, Ar), 6.90 (d,

δ

, ppm): 11.87 (s, 1H, NH), 11.25 (s, 1H, NH), 8.71 (s,

1

2

J = 8.7 Hz, 2H, Ar), 6.87 (d, J = 8.5 Hz, 1H, isatin), 5.08 (s, 2H, CH ), 3.72 (s, 3H, OCH ).

2

3

.2.8. Synthesis of Dispiroindolinones of Type I (Compounds 5a-h; General Procedure)

Ethanol was added to 5-arylidene-2-selenoxohydantoin (1 equiv.) and sarcosine

2 equiv.), and the mixture was brought to a boil. Then, isatin (2 equiv.) was added. The

3

(

reaction mixture was reﬂuxed for 5 h. The precipitate formed was ﬁltered off and puriﬁed

by ﬂash chromatography (eluent MeOH: CHCl = 1:50 (v/v)) and yielded 14–66%.

3

0

3

-(4-ethoxyphenyl)-4 -(4-chlorophenyl)-1 -methyl-2-selenoxodispiro[imidazolidine-

0

00

5

.3 -pyrrolidine-2 .3 -indoline]-2”.4-dione (5a)

R = 0.84 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

δ, ppm): 10.94 (s, 1H, NH), 10.69 (s, 1H, NH), 7.47

(d, J = 8.,6 Hz, 2H, Ar), 7.42 (d, J = 8.6 Hz, 2H, Ar), 7.33 (d, J = 7.5 Hz, 1H, isatin), 7.30 (t,

J = 7.6 Hz, 1H, isatin), 7.01 (t, J = 7.6 Hz, 1H, isatin), 6.88 (d, J = 8.9 Hz, 2H, Ar), 6.84 (d,

J = 7.6 Hz, 1H, isatin), 6.51 (d, J = 8.8 Hz, 2H, Ar), 4.32 (t, J = 9.1 Hz, 1H, pyrrolidine), 4.00 (q,

J = 7.0 Hz, ArOCH₂CH ), 3.98 (t, J = 8.9 Hz, pyrrolidine), 3.47 (t, J = 8.8 Hz, 1H, pyrrolidine),

3

13

2

.13 (s, 3H, NCH ), 1.30 (t, J = 6.9 Hz, 3H, ArOCH₂CH₃). C NMR (101 MHz, DMSO-d6, δ,

3

ppm): 182.2, 174.8, 171.8, 158.6, 142.7, 133.7, 132.3, 131.3, 130.0, 129.5, 128.4, 126.9, 125.7,

23.6, 121.6, 114.3, 109.9, 77.8, 76.9, 63.3, 56.2, 49.5, 34.6, 14.5. IR (pellet KBr): νmax 3203,

085, 2980, 2944, 2878, 2795, 1748, 1713, 1620, 1512, 1469, 1398, 1327, 1296, 1249, 1218, 1160,

1

3

1

−

113, 1090, 1045, 1016, 834, 773, 759 cm . HRMS (ESI, m/z): calculated (C H ClN O Se,

28 25 4 3

1

+

[

M+H] ): 581.0853, found ([M+H] : 581.0860.

0

0 0

-chloro-3-(4-ethoxyphenyl)-4 -(4-chlorophenyl)-1 -methyl-2-selenoxodispiro[imidaz-

5

0

00

olidine-5,3 -pyrrolidine-2 ,3 -indoline]-2 ,4-dione (5b)

R = 0.81 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

δ, ppm): 11.24 (s, 1H, NH), 10.81 (s, 1H, NH), 7.47 (d, J

=

8.6 Hz, 2H, Ar), 7.41 (d, J = 8.3 Hz, 2H, Ar), 7.43–7.39 (m, 1H, isatin), 7.36 (dd, J = 2.1 Hz,

1

J₂= 8.3 Hz, 1H, isatin), 6.90 (d, J = 8.9 Hz, 2H, Ar), 6.86 (d, J = 8.3 Hz, 1H, isatin), 6.54 (d,

J = 8.8 Hz, 2H, Ar), 4.34 (t, J = 9.0 Hz, 1H, pyrrolidine), 4.01 (q, J = 6.9 Hz, ArOCH₂CH₃),

3

.94 (t, J = 9.7 Hz, 1H, pyrrolidine), 3.50 (t, J = 8.6 Hz, 1H, pyrrolidine), 2.16 (s, 3H, NCH ),

3

13

1

.31 (t, J = 6.9 Hz, 3H, ArOCH₂CH₃). C NMR (101 MHz, DMSO-d6, δ, ppm): 182.1, 174.4,

71.5, 158.7, 141.5, 133.5, 132.4, 131.5, 129.9, 129.5, 128.4, 126.9, 125.9, 125.8, 125.7, 114.5,

11.4, 78.0, 77.1, 63.4, 56.0, 49.6, 34.8, 14.6. IR (pellet KBr): νmax 3424, 3249, 2979, 2933, 2867,

716, 1618, 1513, 1495, 1470, 1447, 1300, 1251, 1220, 1167, 1115, 1091, 1043, 831, 820, 778

−

1

+

cm . HRMS (ESI, m/z): calculated (C H Cl N O Se, [M+H] ): 615.0463, found [M+H] :

28

24

2

4

3

6

15.0479.

0

0 0

-bromo-3-(4-ethoxyphenyl)-4 -(4-chlorophenyl)-1 -methyl-2-selenoxodispiro[imida-

5

0

00

zolidine-5,3 -pyrrolidine-2 ,3 -indoline]-2 ,4-dione (5c)

R = 0.82 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

m, 4H, Ar+isatin), 7.41 (d, J = 8.6 Hz, 2H, Ar), 6.90 (d, J = 8.9 Hz, 2H, Ar), 6.81 (d, J = 8.8 Hz

H, isatin), 6.55 (d, J = 8.8 Hz, 2H, Ar), 4.32 (t, J = 9.2 Hz, 1H, pyrrolidine), 4.01 (q, J = 7.0 Hz

δ, ppm): 11.18 (s, 1H, NH), 10.82 (s, 1H, NH), 7.51–7.44

(

1

,

ArOCH₂CH ), 3.95 (t, J = 9.1 Hz, 1H, pyrrolidine), 3.50 (t, J = 9.1 Hz, 1H, pyrrolidine), 2.16

3

13

(

s, 3H, NCH ), 1.31 (t, J = 6.9 Hz, 3H, ArOCH₂CH₃). C NMR (101 MHz, DMSO-d6, δ,

3

ppm): 182.1, 174.2, 171.6, 158.7, 141.9, 133.5, 132.8, 132.4, 131.4, 129.5, 129.4, 128.4, 126.2,

1

25.7, 123.2, 114.5, 113.6, 111.9, 77.0, 63.4, 49.5, 34.7, 14.6. HRMS (ESI, m/z): calculated

+

(

C H BrClN O Se, [M+H] ): 658.9958, found [M+H] : 658.9949.

28 24 4 3

0

-(4-methoxyphenyl)-4’-(4-chlorophenyl)-1 -methyl-2-selenoxodispiro[imidazolidine-

,3’-pyrrolidine-2’,3 -indoline]-2 ,4-dione (5d)

3

0

00

5

R = 0.88 (MeOH:CHCl = 1:50).

f

3

Int. J. Mol. Sci. 2021, 22, 2613

19 of 26

¹H NMR (400 MHz, DMSO-d6,

δ, ppm): 10.96 (s, 1H, NH), 10.69 (s, 1H, NH), 7.47

(d, J = 8.6 Hz, 2H, Ar), 7.42 (d, J = 8.5 Hz, 2H, Ar), 7.32 (d, J = 7.3 Hz, 1H, isatin), 7.30 (t,

J = 7.6 Hz, 1H, isatin), 7.01 (t, J = 7.5 Hz, 1H, isatin), 6.90 (d, J = 9.1 Hz, 2H, Ar), 6.85 (d,

J = 7.6 Hz, 1H, isatin), 6.52 (d, J = 8.8 Hz, 2H, Ar), 4.32 (t, J = 9.1 Hz, 1H, pyrrolidine), 3.98

(

t, J = 9.3 Hz, 1H, pyrrolidine), 3.74 (s, 3H, OCH ), 3.47 (t, J = 8.7 Hz, 1H, pyrrolidine), 2.13

3

s, 3H, NCH₃). ¹³C NMR (101 MHz, DMSO-d6,

δ

, ppm): 182.2, 174.9, 171.9, 159.3, 142.8,

1

5

33.8, 132.3, 131.4, 130.0, 129.6, 128.4, 126.9, 125.9, 123.6, 121.6, 114.0, 109.9, 77.9, 76.9, 56.3,

+

5.4, 49.5, 34.6. HRMS (ESI, m/z): calculated (C H ClN O Se, [M+H] ): 567.0697, found

27

23

4

3

+

[

M+H] : 567.0709.

0

0 0

-chloro-3-(4-methoxyphenyl)-4 -(4-chlorophenyl)-1 -methyl-2-selenoxodispiro[imi-

5

0

00

dazolidine-5,3 -pyrrolidine-2 ,3 -indoline]-2 ,4-dione (5e)

R = 0.84 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6, δ, ppm): 11.24 (s, 1H, NH), 10.81 (s, 1H, NH), 7.47 (d,

J = 8.7 Hz, 2H, Ar), 7.41 (d, J = 8.6 Hz, 2H, Ar), 7.40 (s, 1H, isatin), 7.36 (dd, J = 2.1 Hz,

1

J₂= 8.3 Hz, 1H, isatin), 6.92 (d, J = 9.0 Hz, 2H, Ar), 6.86 (d, J = 8.2 Hz, 1H, isatin), 6.56 (d,

J = 8.8 Hz, 2H, Ar), 4.34 (t, J = 8.8 Hz, 1H, pyrrolidine), 3.94 (t, J = 9.2 Hz, 1H, pyrrolidine),

1

3

.75 (s, 3H, OCH ), 3.50 (t, J = 9.1 Hz, 1H, pyrrolidine), 2.16 (s, 3H, NCH ). C NMR

3

(101 MHz, DMSO-d6,

δ, ppm): 182.0, 174.3, 171.5, 159.4, 141.4, 133.4, 132.4, 131.4, 129.9,

1

29.4, 128.4, 126.8, 125.9, 125.8, 114.1, 111.4, 77.9, 77.1, 56.0, 55.4, 49.6, 34.7. HRMS (ESI,

+

m/z): calculated (C H Cl N O Se, [M+H] ): 601.0307, found [M+H] : 601.0317.

27

22

2

4

3

0

0 0

-bromo-3-(4-methoxyphenyl)-4 -(4-chlorophenyl)-1 -methyl-2-selenoxodispiro[imi-

5

0

00

dazolidine-5,3 -pyrrolidine-2 ,3 -indoline]-2 ,4-dione (5f)

R = 0.87 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

δ, ppm): 11.20 (s, 1H, NH), 10.82 (s, 1H, NH), 7.52–7.45

(

m, 4H, Ar+isatin), 7.41 (d, J = 8.7 Hz, 2H, Ar), 6.92 (d, J = 8.7 Hz, 2H, Ar), 6.81 (d, J = 8.7 Hz

H, isatin), 6.56 (d, J = 8.7 Hz, 2H, Ar), 4.32 (t, J = 8.9 Hz, 1H, pyrrolidine), 3.95 (t, J = 9.2 Hz

,

1

H, pyrrolidine), 3.75 (s, 3H, OCH ), 3.50 (t, J = 9.2 Hz, 1H, pyrrolidine), 2.16 (s, 3H, NCH ).

3

1

3

C NMR (101 MHz, DMSO-d6,

31.5, 129.5, 129.4, 128.4, 126.2, 125.8, 114.1, 114.0, 113.6, 111.9, 78.0, 77.0, 56.1, 55.4, 49.5,

4.7. IR (pellet KBr): νmax 3114, 2866, 1764, 1758, 1709, 1694, 1616, 1512, 1478, 1442, 1411,

δ, ppm): 182.1, 174.2, 171.7, 159.4, 141.9, 133.5, 132.8, 132.4,

1

3

1

−

1

313, 1252, 1217, 1204, 1187, 1164, 1090, 1030, 1014, 941, 895, 831, 819, 775, 733, 694 cm

.

+

HRMS (ESI, m/z): calculated (C H BrClN O Se, [M+H] ): 644.9802, found [M+H] :

2

7

22

4

3

6

44.9789.

0

0 0

-bromo-3-(4-methoxyphenyl)-4 -(4-ethylphenyl)-1 -methyl-2-selenoxodispiro[imi-

5

0

00

dazolidine-5,3 -pyrrolidine-2 ,3 -indoline]-2 ,4-dione (5g)

R = 0.90 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

m, 2H, isatin), 7.33 (d, J = 6.6 Hz, 2H, Ar), 7.19 (d, J = 6.6 Hz, 2H, Ar), 6.91 (d, J = 7.7 Hz,

H, Ar), 6.82 (d, J = 8.2 Hz, 1H, isatin), 6.53 (d, J = 7.5 Hz, 2H, Ar), 4.26 (t, J = 8.8 Hz, 1H,

pyrrolidine), 3.96 (t, J = 8.9 Hz, 1H, pyrrolidine), 3.74 (s, 3H, OCH ), 3.55–3.50 (m, 1H,

δ, ppm): 11.02 (s, 1H, NH), 10.83 (s, 1H NH), 7.52–7.45

(

2

3

pyrrolidine), 2.59 (q, J = 7.2 Hz, 2H, ArCH₂CH ), 2.16 (s, 3H, NCH ), 1.16 (t, J = 7.1 Hz,

3

13

3

H, ArCH₂CH₃). C NMR (101 MHz, DMSO-d6,

δ

, ppm): 182.0, 174.5, 171.8, 159.4, 143.2,

142.0, 132.8, 131.6, 129.6, 129.4, 127.9, 126.5, 126.0, 114.1, 113.6, 112.0, 78.2, 77.2, 56.2, 55.5,

5

0.2, 34.8, 27.9, 15.6. R (pellet KBr): νmax 3183, 2963, 2835, 1751, 1725, 1617, 1605, 1511, 1468,

−

1

1399, 1300, 1250, 1215, 1185, 1163, 1021, 833, 779, 759, 738, 648, 633 cm . HRMS (ESI, m/z):

+

calculated (C H BrN O Se, [M+H] ): 639.0342, found [M+H] : 639.0506.

29

27

4

3

0 0

-(4-methoxyphenyl)-4 -(4-methoxyphenyl)-1 -methyl-2-selenoxodispiro[imidazoli-

3

0

00

dine-5,3 -pyrrolidine-2 ,3 -indoline]-2 ,4-dione (5h)

R = 0.80 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

m, 3H, Ar+isatin), 7.29 (t, J = 7.0 Hz, 1H, isatin), 7.01 (t, J = 6.4 Hz, 1H, isatin), 6.95–6.86 (m,

H, Ar), 6.84 (d, J = 7.2 Hz, 1H, isatin), 6.51 (d, J = 7.5 Hz, 2H, Ar), 4.28 (t, J = 8.3 Hz, 1H,

pyrrolidine), 3.95 (t, J = 8.9 Hz, 1H, pyrrolidine), 3.74 (s, 3H, OCH ), 3.73 (s, 3H, OCH ),

δ, ppm): 10.78 (s, 1H, NH), 10.68 (s, 1H, NH), 7.40–7.32

(

4

3

.43 (t, J = 8.7 Hz, 1H, pyrrolidine), 2.14 (s, 3H, NCH₃). ¹³C NMR (101 MHz, DMSO-d6,

δ,

Int. J. Mol. Sci. 2021, 22, 2613

20 of 26

ppm): 181.9, 175.0, 171.8, 159.4, 142.6, 133.3, 132.5, 130.6, 130.0, 129.6, 127.1, 126.4, 125.6,

1

23.5, 121.6, 114.0, 113.9, 109.9, 78.0, 76.9, 56.5, 55.4, 55.1, 34.8. HRMS (ESI, m/z): calculated

28 26 4 4

+

(

C H N O Se, [M+H] ): 562.1119, found [M+H] : 562.1191.

3.2.9. Synthesis of Dispiroindolinones of Type I (Compounds 6a-m; General Procedure)

Toluene was added to 5-indolinylidene-2-selenoxohydantoin (1 equiv.) and sarcosine

(8 equiv.), and the mixture was brought to a boil. Paraform (8 eq.) was then added. The

reaction mixture was reﬂuxed for 6 h, then evaporated, and the residue was puriﬁed by

column chromatography (eluent MeOH:CHCl = 1:50 (v/v)) and yielded 3–66%.

3

0

00

1

-methyl-3-(4-ethoxyphenyl)-2-selenoxodispiro[imidazolidine-5,3 -pyrrolidine-4 ,3 -

0

indoline]-2 ,4-dione (6a)

R = 0.42 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

.8 Hz, 1H, isatin), 7.13 (d, J = 7.5 Hz, 1H, isatin), 6.95–6.92 (m, 1H, isatin), 6.93 (d, J = 8.6 Hz

H, Ar), 6.86 (d, J = 7.6 Hz, 1H, isatin), 6.73 (d, J = 8.6 Hz, 2H, Ar), 4.03 (q, J = 7.0 Hz, 2H,

δ, ppm): 11.27 (s, 1H, NH), 10.63 (s, 1H, NH), 7.27 (t, J =

7

2

,

ArOCH₂CH ), 3.50 (d, J = 10.0 Hz, 1H, pyrrolidine), 3.34 (m, 1H, pyrrolidine), 3.33–3.29 (m,

3

1

H, pyrrolidine), 3.08 (d, J = 10.1 Hz, 1H, pyrrolidine), 2.48 (s, 3H, NCH ), 1.32 (t, J = 6.9

3

13

Hz, 3H, ArOCH₂CH₃). C NMR (101 MHz, DMSO-d6,

43.0, 129.9, 129.5, 126.0, 125.0, 124.9, 121.4, 114.3, 109.6, 74.5, 63.3, 60.3, 58.8, 57.6, 42.0, 14.6.

IR (pellet KBr): νmax 3284, 3090, 2925, 2853, 2794, 2033, 1748, 1736, 1726, 1699, 1615, 1513,

δ, ppm): 183.5, 176.6, 172.7, 158.6,

1

8

483, 1470, 1402, 1326, 1303, 1246, 1216, 1197, 1160, 1146, 1114, 1105, 1086, 1039, 939, 919,

−

1

70, 825, 789, 756, 735, 694, 674, 637, 615, 579, 554, 529 cm . HRMS (ESI, m/z): calculated

+

(

C H N O Se, [M+H] ): 471.0930, found [M+H] : 471.0923.

22 22 4 3

0

0 0

-chloro-1 -methyl-3-(4-ethoxyphenyl)-2-selenoxodispiro[imidazolidine-5,3 -pyrroli-

5

0

00

dine-4 ,3 -indoline]-2 ,4-dione (6b)

R = 0.34 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

δ, ppm): 11.39 (s, 1H, NH), 10.78 (s, 1H, NH), 7.33

(

dd, J = 2.0 Hz, J = 8,3 Hz, 1H isatin), 7.13 (d, J = 1.8 Hz, 1H, isatin), 6.94 (d, J = 8.8 Hz,

1

2

H, Ar), 6.87 (d, J = 8.3 Hz, 1H, isatin), 6.76 (d, J = 8.6 Hz, 2H, Ar), 4.04 (q, J = 6.9 Hz, 2H,

ArOCH₂CH ), 3.42 (d, J = 11.7 Hz, 1H, pyrrolidine), 3.39 (d, J = 10.7 Hz, 1H, pyrrolidine),

3

.29 (m, 1H, pyrrolidine), 3.10 (d, J = 10.1 Hz, 1H, pyrrolidine), 2.48 (s, 3H, NCH ), 1.33

3

13

(t, J = 6.9 Hz, 3H, ArOCH₂CH₃). C NMR (101 MHz, DMSO-d6,

δ, ppm): 183.3, 175.8,

1

5

72.5, 158.7, 141.8, 129.8, 129.2, 127.4, 126.0, 125.4, 125.0, 114.4, 111.0, 74.5, 63.4, 60.1, 59.1,

7.6, 41.8, 14.6. IR (pellet KBr): νmax 3093, 2982, 2944, 2859, 2799, 1759, 1737, 1699, 1611,

1

591, 1514, 1486, 1475, 1456, 1404, 1333, 1315, 1304, 1280, 1257, 1212, 1197, 1163, 1116, 1090,

−

1

042, 952, 926, 894, 869, 825, 789, 746, 737, 679, 587, 565c m . HRMS (ESI, m/z): calculated

+

(

C H ClN O Se, [M+H] ): 505.0540, found [M+H] : 505.0547.

22 21 4 3

0

0 0

-bromo-1 -methyl-3-(4-ethoxyphenyl)-2-selenoxodispiro[imidazolidine-5,3 -pyrroli-

5

0

00

dine-4 ,3 -indoline]-2 ,4-dione (6c)

R = 0.35 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

δ, ppm): 11.42 (s, 1H, NH), 10.81(s, 1H, NH), 7.46 (dd,

J = 1.4 Hz, J = 8.3 Hz, 1H, isatin), 7.24 (s, 1H, isatin), 6.94 (d, J = 8.6 Hz, 2H, Ar), 6.83 (d,

1

2

J = 8.3 Hz, 1H, isatin), 6.77 (d, J = 8.4 Hz, 2H, Ar), 4.04 (q, J = 6.9 Hz, 2H, ArOCH₂CH₃),

.43 (d, J = 10.3 Hz, 1H, pyrrolidine), 3.39 (d, J = 10.5 Hz, 1H, pyrrolidine), 3.32 (m, 1H,

pyrrolidine), 3.10 (d, J = 10.1 Hz, 1H, pyrrolidine), 2.48 (s, 3H, NCH ), 1.33 (t, J = 6.9 Hz, 3H,

3

13

ArOCH₂CH₃). C NMR (101 MHz, DMSO-d6,

42.3, 142.2, 132.0, 129.8, 127.7, 125.9, 114.4, 113.1, 111.5, 74.4, 63.3, 60.0, 59.0, 57.5, 41.9, 14.6.

IR (pellet KBr): νmax3 089, 2984, 2930, 2852, 2799, 1760, 1738, 1699, 1613, 1591, 1514, 1489,

δ, ppm): 206.9, 183.3, 175.7, 172.5, 158.7,

1

8

471, 1456, 1407, 1315, 1305, 1280, 1256, 1212, 1196, 1165, 1116, 1086, 1042, 951, 929, 893, 865,

−

1

+

24, 788, 736 cm . HRMS (ESI, m/z): calculated (C H BrN O Se, [M+H] ): 549.0035,

22

21

4

3

+

found [M+H] : 549.0037.

0

0 0

-nitro-1 -methyl-3-(4-ethoxyphenyl)-2-selenoxodispiro[imidazolidine-5,3 -pyrroli-

5

0

00

dine-4 ,3 -indoline]-2 ,4-dione (6d)

R = 0.21 (MeOH:CHCl = 1:50).

f

3

Int. J. Mol. Sci. 2021, 22, 2613

21 of 26

¹H NMR (400 MHz, DMSO-d6,

δ, ppm): 11.55 (s, 1H, NH), 11.38 (s, 1H, NH), 8.23 (dd,

J = 2.1 Hz, J = 8.7 Hz, 1H, isatin), 8.06 (s, 1H, isatin), 7.06 (dd, J = 1.6 Hz, J = 8.6 Hz,

1

2

1

2

1

H, isatin), 6.89 (d, J = 8.9 Hz, 2H, Ar), 6.74 (d, J = 8.6 Hz, 2H,Ar), 4.02 (q, J = 6.6 Hz, 2H,

ArOCH₂CH ), 3.50 (d, J = 10.9 Hz, 1H, pyrrolidine), 3.38–3.30 (m, 2H, pyrrolidine), 3.17

3

(

d, J = 9.6 Hz, 1H, pyrrolidine), 2.50 (s, 3H, NCH ), 1.32 (t, J = 6.9 Hz, 3H, ArOCH₂CH₃).

3

13

C NMR (101 MHz, DMSO-d6,

26.5, 125.8, 121.0, 114.3, 109.8, 74.3, 63.3, 60.6, 59.1, 58.4, 41.6, 14.6. IR (pellet KBr): νmax

270, 3103, 2975, 2940, 2865, 2844, 2805, 1739, 1681, 1625, 1600, 1512, 1477, 1455, 1401,

339, 1305, 1274, 1251, 1213, 1197, 1148, 1099, 1042, 979, 935, 916, 836, 788, 757, 741, 701,

δ, ppm): 183.0, 175.9, 172.0, 158.6, 149.1, 141.7, 129.6, 126.9,

1

3

1

6

[

−

1

+

06, 564 cm . HRMS (ESI, m/z): calculated (C H N O Se, [M+H] ): 516.0781, found

22

21

5

+

M+H] : 516.0781.

0

0 0

-bromo-1 -methyl-3-(4-methoxyphenyl)-2-selenoxodispiro[imidazolidine-5,3 -pyrro-

5

0

00

lidine-4 ,3 -indoline]-2 ,4-dione (6e)

R = 0.40 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

8.3 Hz, 1H, isatin), 7.25 (s, 1H, isatin), 6.97 (d, J = 8.4 Hz, 2H, Ar), 6.83 (d, J = 8.4 Hz, 1H,

isatin), 6.79 (d, J = 8.5 Hz, 2H, Ar), 3.78 (s, 3H, OCH ), 3.44 (d, J = 10.1 Hz, 1H, pyrrolidine),

δ, ppm): 11.41 (s, 1H, NH), 10.81 (s, 1H, NH), 7.47 (d, J

=

3

.39 (d, J = 10.4 Hz, 1H, pyrrolidine), 3.31 (m, 1H, pyrrolidine), 3.10 (d, J = 10.5 Hz, 1H,

13

pyrrolidine), 2.48 (s, 3H, NCH₃). C NMR (101 MHz, DMSO-d6,

δ, ppm): 206.9, 184.2,

1

5

1

75.7, 172.5, 159.4, 142.3, 132.0, 129.8, 127.7, 126.1, 114.0, 113.1, 111.5, 74.5, 63.7, 60.0, 59.0,

7.5, 55.4, 41.9. IR (pellet KBr): νmax 3291, 3094, 2936, 2837, 2800, 1734, 1615, 1513, 1477, 1408,

−

301, 1288, 1252, 1221, 1179, 1163, 1129, 1029, 952, 930, 891, 825, 788, 745, 733 cm . HRMS

1

+

(

ESI, m/z): calculated (C H BrN O Se, [M+H] ): 534.9876, found [M+H] : 534.9879.

21 19 4 3

0

0 0

-nitro-1 -methyl-3-(4-methoxyphenyl)-2-selenoxodispiro[imidazolidine-5,3 -pyrroli-

5

0

00

dine-4 ,3 -indoline]-2 ,4-dione (6f)

R = 0.23 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

δ, ppm): 11.48 (bs, 1H, NH), 11.34 (s, 1H, NH), 8.42

(

d, J = 2.4 Hz, 1H, isatin), 8.29 (dd, J = 2.4 Hz, J = 8.6 Hz, 1H, isatin), 7.05 (d, J = 9.0 Hz,

1

2

1

H, isatin), 6.98 (d, J = 9.0 Hz, 2H, Ar), 6.91 (d, J = 9.0 Hz, 2H, Ar), 3.77 (s, 3H, OCH₃),

3.45 (d, J = 11.0 Hz, 1H, pyrrolidine), 3.37 (m, 1H, pyrrolidine), 3.30 (m, 1H, pyrrolidine),

3

.23 (d, J = 9.4 Hz, 1H, pyrrolidine), 2.54 (s, 3H, NCH ). HRMS (ESI, m/z): calculated

3

+

(

C H N O Se, [M+H] ): 502.0624, found [M+H] : 502.0616.

21 19 5 5

0

0 0

-bromo-1 -methyl-3-(4-chlorophenyl)-2-selenoxodispiro[imidazolidine-5,3 -pyrroli-

5

0

00

dine-4 ,3 -indoline]-2 ,4-dione (6g)

R = 0.37 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

δ, ppm): 11.57 (s, 1H, NH), 10.83 (s, 1H, NH), 7.53 (d,

J = 8.8 Hz, 2H, Ar), 7.46 (dd, J = 1.8 Hz, J = 8.3 Hz, 1H, isatin), 7.22 (s, 1H, isatin), 6.92

1

(d, J = 8.5 Hz, 2H, Ar), 6.83 (d, J = 8.3 Hz, 1H, isatin), 47 (d, J = 9.8 Hz, 1H, pyrrolidine),

3

.43 (d, J = 10.2 Hz, 1H, pyrrolidine), 3.31–3.25 (m, 1H, pyrrolidine), 3.14 (d, J = 9.8 Hz, 1H,

13

pyrrolidine), 2.49 (s, 3H, NCH₃). C NMR (101 MHz, DMSO-d6,

δ, ppm): 182.3, 175.6,

1

4

72.1, 142.2, 133.7, 132.5, 132.1, 130.6, 128.9, 127.6, 127.5, 113.1, 111.6, 74.7, 59.9, 59.0, 57.4,

1.9. IR (pellet KBr): νmax 3311, 3088, 2941, 2852, 2798, 1746, 1733, 1616, 1505, 1493, 1478,

406, 1299, 1287, 1260, 1220, 1179, 1165, 1127, 1090, 1016, 950, 890, 826, 819, 789, 742, 734,

1

7

−

1

+

19, 694,648 cm . HRMS (ESI, m/z): calculated (C H BrClN O Se, [M+H] ): 538.9383,

20

16

4

2

+

found [M+H] : 538.9379.

0

0 0

-nitro-1 -methyl-3-(4-chlorophenyl)-2-selenoxodispiro[imidazolidine-5,3 -pyrroli-

5

0

00

dine-4 ,3 -indoline]-2 ,4-dione (6h)

R = 0.21 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

δ, ppm): 11.74 (bs, 1H, NH), 11.48 (s, 1H, NH), 8.25

(

dd, J = 2.4 Hz, J = 8.6 Hz, 1H, isatin), 8.02 (d, J = 2.0 Hz, 1H, isatin), 7.49 (d, J = 8.6 Hz

,

1

2

H, Ar), 7.,09 (d, J = 9.0 Hz, 1H, isatin), 6.92 (d, J = 8.2 Hz, 2H, Ar), 3.72–3.63 (m, 1H,

pyrrolidine), 3.53–3.42 (m, 2H, pyrrolidine), 3.28–3.23 (m, 1H, pyrrolidine), 2.61 (bs, 3H,

NCH₃). ¹³C NMR (101 MHz, DMSO-d6,

, ppm): 206.5, 175.8, 171.8, 149.3, 141.8, 133.7,

32.2, 130.4, 128.9, 126.8, 120.8, 110.0, 74.3, 58.9, 42.1, 30.7. IR (pellet KBr): νmax 3274, 3095,

δ

1

Int. J. Mol. Sci. 2021, 22, 2613

22 of 26

2

9

931, 2853, 2801, 1732, 1623, 1600, 1524, 1495, 1474, 1403, 1339, 1306, 1270, 1201, 1092, 1016,

−

1

15, 833, 785, 754, 735, 698, 561 cm . HRMS (ESI, m/z): calculated (C H ClN O Se,

20

16

5

4

+

[

M+H] ): 506.0129, found [M+H] : 506.0119.

0

0 0 00

-methyl-3-(3-chlorophenyl)-2-selenoxodispiro[imidazolidine-5,3 -pyrrolidine-4 ,3 -

1

0

indoline]-2 ,4-dione (6i)

R = 0.43 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

δ, ppm): 11.47 (s, 1H, NH), 10.67 (s, 1H, NH), 7.54–7.41

(

m, 2H, Ar), 7.28 (m, 1H, Ar), 7.11 (m, 1H, Ar), 6.99–6.76 (m, 4H, Ar), 3.49 (m, 1H, pyrroli-

1

3

dine), 3.44–3.37 (m, 2H, pyrrolidine), 3.09 (m, 1H, pyrrolidine), 2.49 (m, 3H, NCH₃).

C

NMR (101 MHz, DMSO-d6,

28.9, 127.7, 125.0, 124.8, 121.3, 109.7, 74.9, 60.1, 58.9, 57.4, 41.9. IR (pellet KBr): νmax 3499,

406, 3178, 3074, 2933, 2858, 2801, 1756, 1732, 1694, 1619, 1591, 1483, 1471, 1448, 1432,

388, 1352, 1338, 1320, 1306, 1266, 1256, 1217, 1203, 1159, 1147, 1084, 1032, 968, 935, 905,

δ, ppm): 206.9, 176.4, 143.1, 135.0, 132.7, 130.3, 129.5, 129.0,

1

3

1

8

−

1

76, 782, 753, 745, 727, 688, 636, 620, 608, 582, 546 cm . HRMS (ESI, m/z): calculated

+

(

C H ClN O Se, [M + H] ): 461.0278, found [M+H] : 461.0270.

20 17 4 2

0

0 0

-chloro-1 -methyl-3-(3-chloro-4-ﬂuorophenyl)-2-selenoxodispiro[imidazolidine-5,3 -

5

0

00

pyrrolidine-4 ,3 -indoline]-2 ,4-dione (6j)

R = 0.31 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

δ, ppm): 11.62 (s, 1H, NH), 10.81 (s, 1H, NH), 7.57

(

t, J = 9.0 Hz, 1H,Ar), 7.36 (dd, J = 1.8 Hz, J = 8.3 Hz, 1H,Ar), 7.12 (m, 1H,Ar), 7.10

1

2

m, 1H,Ar), 6.96 (m, 1H,Ar), 6.90 (d, J = 8.3 Hz, 1H,Ar), 3.43 (m, 2H, pyrrolidine), 3.32

1

3

m, 1H, pyrrolidine), 3.12 (d, J = 10.2 Hz, 1H, pyrrolidine), 2.49 (s, 3H, NCH ), C NMR

3

101 MHz, DMSO-d6,

δ, ppm): 206.5, 175.7, 158.1, 156.4, 141.9, 130.9, 129.9, 129.2, 127.3,

1

25.3, 124.8, 119.5, 117.1, 111.1, 59.9, 59.2, 57.5, 41.8, 30.7, HRMS (ESI, m/z): calculated

+

(

C H Cl FN O Se, [M+H] ): 512.9794, found [M+H] : 512.9798.

20 15 2 4 2

0

5

-bromo-1 -methyl-3-cyclopropyl-2-selenoxodispiro[imidazolidine-5,3 -pyrrolidine-

0

00

4

,3 -indoline]-2 ,4-dione (6k)

R = 0.48 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

δ, ppm): 11.12 (bs, 1H, NH), 10.70 (s, 1H, NH), 7.40

(

dd, J = 1.2 Hz, J = 8.2 Hz, 1H, isatin), 7.22 (d, J = 1.6 Hz, 1H, isatin), 6.75 (d, J = 8.2 Hz, 1H,

1

2

isatin), 3.32–3.29 (m, 1H, pyrrolidine), 3.29–3.26 (m, 1H, pyrrolidine), 3.25 (d, J = 10.2 Hz

,

1

H, pyrrolidine), 3.09 (d, J = 9.8 Hz, 1H, pyrrolidine), 2.47 (s, 3H, NCH ), 0.91–0.76 (m, 3H,

3

Pr), 0.68–0.60 (m, 1H, Pr), 0.20–0.12 (m, 1H, Pr). ¹³C NMR (101 MHz, DMSO-d6,

δ

, ppm):

1

5

1

7

4

84.2, 175.5, 173.1, 142.0, 131.8, 127.7, 127.5, 112.8, 111.3, 73.4, 60.3, 59.0, 57.8, 41.8, 24.8, 7.5,

.7. R (pellet KBr): νmax 3177, 2953, 2918, 2854, 2821, 2799, 1752, 1730, 1691, 1617, 1472, 1403,

353, 1332, 1316, 1255, 1220, 1202, 1160, 1144, 1132, 1087, 1035, 955, 904, 876, 814, 789, 728,

−

1

+

00, 605, 580, 561, 545 cm . HRMS (ESI, m/z): calculated (C H BrN O Se, [M+H] ):

17

4

2

+

68.9773, found [M+H] : 468.9767.

0

0 0 00

-methyl-3-(4-methoxybenzyl)-2-selenoxodispiro[imidazolidine-5,3 -pyrrolidine-4 ,3 -

1

0

indoline]-2 ,4-dione (6l)

R = 0.42 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

δ, ppm): 11.16 (bs, 1H, NH), 10.55 (s, 1H, NH), 7.16

(t, J = 7.5 Hz, 1H, isatin), 7.06 (d, J = 7.3 Hz, 1H, isatin), 6.88 (d, J = 8.4 Hz, 2H, Ar),

6

4

.77 (d, J = 7.7 Hz, 1H, isatin), 6.73 (d, J = 8.3 Hz, 2H, Ar), 6.70, (t, J = 7.2 Hz, 1H, isatin),

.80 (d, J = 14.9 Hz, 1H, CH ), 4.67 (d, J = 14.9 Hz, 1H, CH ), 3.72 (s, 3H, OCH ), 3.36 (d,

2

3

J = 10.1 Hz, 1H, pyrrolidine), 3.26 (d, J = 9.9 Hz, 1H, pyrrolidine), 3.19 (d, J = 10.1 Hz, 1H,

13

pyrrolidine), 3.06 (d, J = 9.9 Hz, 1H, pyrrolidine), 2.44 (s, 3H, NCH₃). C NMR (101 MHz,

DMSO-d6, , ppm): 182.9, 176.2, 172.6, 158.3, 142.5, 129.0, 128.7, 128.6, 127.6, 125.3, 121.4,

13.5, 109.4, 73.6, 61.4, 58.9, 58.6, 55.0, 44.1, 42.6, 41.8. IR (pellet KBr): νmax 3153, 3015, 2996,

δ

1

2

1

7

948, 2907, 2861, 2833, 2800, 1731, 1690, 1614, 1586, 1515, 1477, 1454, 1430, 1403, 1347, 1322,

308, 1250, 1210, 1185, 1176, 1157, 1148, 1116, 1098, 1037, 964, 896, 849, 836, 823, 791, 777,

−

1

55, 730, 683, 637, 612, 600, 576, 559 cm . HRMS (ESI, m/z): calculated (C H N O Se,

22

4

3

+

[

M+H] ): 471.0930, found [M+H] : 471.0930.

Int. J. Mol. Sci. 2021, 22, 2613

23 of 26

0

0 0

-bromo-1 -methyl-3-(4-methoxybenzyl)-2-selenoxodispiro[imidazolidine-5,3 -pyrro-

5

0

00

lidine-4 ,3 -indoline]-2 ,4-dione (6m)

R = 0.41 (MeOH:CHCl = 1:50).

f

3

1

H NMR (400 MHz, DMSO-d6,

δ, ppm): 11.25 (s, 1H, NH), 10.68 (s, 1H, NH), 7.37

(

dd, J = 2.0 Hz, J = 7.8 Hz, 1H, isatin), 7.34 (s, 1H, isatin), 6.82 (d, J = 7.8 Hz, 2H, Ar),

1

2

6

.75–6.73 (m, 1H, isatin), 6.73 (d, J = 8.8 Hz, 2H, Ar), 4.80 (d, J = 14.7 Hz, 1H, CH ), 4.68 (d,

2

J = 14.7 Hz, 1H, CH ), 3.72 (s, 3H, OCH ), 3.30 (m, 1H, pyrrolidine), 3.20 (d, J = 6.9 Hz, 1H,

2

3

pyrrolidine), 3.17 (d, J = 7.8 Hz, 1H, pyrrolidine), 3.08 (d, J = 9.8 Hz, 1H, pyrrolidine), 2.43

s, 3H, NCH₃). ¹³C NMR (101 MHz, DMSO-d6,

, ppm): 182.6, 175.2, 172.1, 158.3, 141.6,

31.7, 128.7, 128.6, 128.1, 127.5, 113.6, 113.2, 111.3, 73.7, 62.0, 59.7, 59.1, 55.0, 44.1, 41.5. IR

pellet KBr): νmax 3206, 2998, 2934, 2833, 2804, 2030, 1749, 1729, 1689, 1615, 1587, 1515, 1470,

(

δ

1

(

1

6

5

428, 1398, 1342, 1306, 1250, 1203, 1177, 1150, 1120, 1036, 970, 907, 856, 821, 785, 729, 693,

−

1

+

12, 603, 583, 565, 545 cm . HRMS (ESI, m/z): calculated (C H BrN O Se, [M+H] ):

22

21

4

3

+

49.0035, found [M+H] : 549.0045.

4. Conclusions

In the present study, two series of novel 2-selenohydantoin-based dispiro[pyrrolidine-

oxindoles] were synthesized using the regio- and diastereoselective 1,3-dipolar cycload-

ditions of azomethine ylides to 5-arylidene and 5-indolidene-2-seleno-tetrahydro-4H-

imidazole-4-ones. The synthesized seleno-containing oxindoles in the preliminary biotests

demonstrated reasonable cytotoxic activity and the ability to generate ROS. For the com-

pounds of 6, the results obtained by measuring the ROS level in separate cells indicate the

possibility of a correlation between their ability to generate reactive oxygen species and the

effectiveness of their cytotoxic actions.

Supplementary Materials: The following are available online at https://www.mdpi.com/1422-006

7

/22/5/2613/s1 .

Author Contributions: Conceptualization, E.K.B., A.G.M. and N.V.Z.; methodology, V.K.N. and

M.E.K.; validation, E.K.B., V.K.N., M.E.K. and D.A.S.; formal analysis, V.K.N., M.E.K., V.A.T., D.A.S.,

R.V.T., N.S.C., P.V.G. and A.S.E.; investigation, V.K.N., M.E.K., V.A.T., D.A.S., M.A.K., L.A.V., R.V.T.,

N.S.C. and B.N.T.; data curation, V.K.N., M.E.K., V.A.T., D.A.S., R.V.T. and N.S.C.; writing—original

draft preparation, V.K.N., M.E.K., V.A.T., D.A.S., R.V.T., N.S.C., P.V.G. and A.S.E.; writing—review and

editing, E.K.B., A.G.M., R.V.T., P.V.G. and A.S.E.; visualization, V.K.N., R.V.T. and N.S.C.; supervision,

N.V.Z., E.K.B. and A.G.M.; project administration, E.K.B.; and funding acquisition, E.K.B., P.V.G. and

A.S.E. All authors have read and agreed to the published version of the manuscript.

Funding: We are grateful to the Russian Foundation for Basic Research (Projects 19-03-00201 and

2

0-33-90008) for the ﬁnancial support of the synthetical part of this work. The Pt electrodes fabrication

was supported by the Ministry of Education and Science of the Russian Federation in the framework

of increasing the Competitiveness Program of NUST “MISIS”, implemented by a governmental

decree dated 16 March 2013, No. 211. Intracellular ROS measurements were supported by the

Russian Science Foundation Grant 19-79-30062. Evaluation of cytotoxicity and selectivity against

cancer cells and tests of p53 inhibition were funded by the RFBR project 18-29-08060.

Data Availability Statement: The data presented in this study are available in the article or supple-

mentary material.

Acknowledgments: The NMR and X-ray studies of this work were supported by the M.V.Lomonosov

Moscow State University Program of Development.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Article Doi

Dispirooxindoles based on 2-selenoxo-imidazolidin-4-ones: Synthesis, cytotoxicity and ros generation ability

DOI: 10.3390/ijms22052613

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Article abstract of DOI:10.3390/ijms22052613

Full text of DOI:10.3390/ijms22052613

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