Next Article in Journal
Sepsis-Associated Muscle Wasting: A Comprehensive Review from Bench to Bedside
Next Article in Special Issue
Synthesis and Evaluation on the Fungicidal Activity of S-Alkyl Substituted Thioglycolurils
Previous Article in Journal
Genetic Variation in Transcription Factor Binding Sites
Previous Article in Special Issue
Obtaining Highly Active Catalytic Antibodies Capable of Enzymatically Cleaving Antigens
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:

[4+2]-Cycloaddition to 5-Methylidene-Hydantoins and 5-Methylidene-2-Thiohydantoins in the Synthesis of Spiro-2-Chalcogenimidazolones

Dmitry E. Shybanov
Maxim E. Kukushkin
Yanislav S. Hrytseniuk
Yuri K. Grishin
Vitaly A. Roznyatovsky
Viktor A. Tafeenko
Dmitry A. Skvortsov
Nikolai V. Zyk
Elena K. Beloglazkina
Department of Chemistry, M. V. Lomonosov Moscow State University, 119991 Moscow, Russia
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(5), 5037;
Submission received: 18 February 2023 / Revised: 3 March 2023 / Accepted: 4 March 2023 / Published: 6 March 2023
(This article belongs to the Special Issue Development and Synthesis of Biologically Active Compounds)


Novel hydantion and thiohydantoin-based spiro-compounds were prepared via theDiels–Alder reactions between 5-methylidene-hydantoins or 5-methylidene-2-thiohydantoins and 1,3-dienes (cyclopentadiene, cyclohexadiene, 2,3-dimethylbutadiene, isoprene). It was shown that the cycloaddition reactions proceed regioselectively and stereoselectively with the formation of exo-isomers in the reactions with cyclic dienes andthe less sterically hindered products in the reactions with isoprene. Reactions of methylideneimidazolones with cyclopentadiene proceed viaco-heating the reactants; reactions with cyclohexadiene, 2,3-dimethylbutadiene, and isoprene require catalysis by Lewis acids. It was demonstrated that ZnI2 is an effective catalyst in the Diels–Alder reactions of methylidenethiohydantoins with non-activated dienes. The possibility of alkylation and acylation of the obtained spiro-hydantoinsat the N(1)nitrogen atoms with PhCH2Cl or Boc2O and the alkylation of the spiro-thiohydantoinsat the S atoms with MeI or PhCH2Cl in high yields have been demonstrated. The preparativetransformation of spiro-thiohydantoins into corresponding spiro-hydantoinsin mild conditions by treating with 35% aqueous H2O2 or nitrile oxide has been carried out. The obtained compounds show moderate cytotoxicity in the MTT test on MCF7, A549, HEK293T, and VA13 cell lines. Some of the tested compounds demonstrated some antibacterial effect against Escherichia coli (E. coli) BW25113 DTC-pDualrep2 but were almost inactive against E. coli BW25113 LPTD-pDualrep2.

1. Introduction

Introducing conformationally rigid lipophilic moieties into a molecule is a common approach in the medicinal chemistry [1,2,3,4] that may lead to significant increases in biological activity [5,6,7,8]. One of the convenient methods for the synthesis of such conformationally rigid polycyclic derivatives is the Diels–Alder reaction of functionalized alkenes containing exocyclic C=C bonds with dienes. Dienesvariation makes it possible to fine-tune the structure of the resulting lipophilic framework [9,10,11], and the presence of a C=C double bond in the [4+2]-cycloaddition adduct opens the way for further modifications of the resulting molecules.
We have recently shown that spiro-2-chalcogenimidazolones, obtained by 1,3-dipolarcycloaddition reactions to 5-methylidene-substituted hydantoins, as well as their 2-thio- and 2-seleno-analogs, possess high cytotoxicity probably due to their ability to inhibit the p53-MDM2 proteins interaction [12,13,14]. However, there were no general methods for the synthesis of 5-methylidene-imidazol-4-onederivatives containing spiro-linked 5- and 6-membered rings described previously, and these compounds have not been previously studied for biological activity.
Diels–Alder reactions of 5-methylene-substituted imidazolones are described in the literature mainly asthe example of highly reactive cyclopentadiene [15,16,17] and as single examples of the reactions with isoprene and 2,3-dimethylbutadiene [16,17] (Scheme 1). In the present article, a systematic study of [4+2]-cycloaddition reactions of cyclopentadiene, cyclohexadiene, 2,3-dimethylbutadiene, and isoprene with 5-methylene-2-chalcogenimidazolone derivatives, both N-unsubstituted and containing substituents of various natures at nitrogen atoms, is carried out. In this work, we first demonstrated the possibility of methylidenethiohydantoins interaction with low active conjugated dienes, which makes it possible to obtain spirocyclic imidazolones containing various lipophilic frameworks. In addition, the possibility of post-synthetic transformations of the formed [4+2]-cycloaddition products using alkylating and acylating agents was demonstrated; these reactions can be an alternative method for the synthesis of those spiro-imidazolones that are formed in the reactions with dienes in low yields or as the inseparable diastereomers mixtures.
Various types of biological activities have been previously described for hydantoins and their spiro derivatives [18,19].In this case, if, for example, anticonvulsants require low toxicity, then inhibition of the growth of bacteria and tumor cells is determined by the cytotoxic effect. Therefore, for the initial assessment of the biological effect on cells of various types, a number of compounds synthesized in this work were tested on cytotoxicity and antibacterial activity on several human cell lines of various etiologies and several strains of E. coli.

2. Results and Discussion

2.1. Synthesis of Dienophiles

Initial dienophiles 1-11, 22, and 23 were prepared according to previously described procedures [20,21,22,23]. Compounds 12-21, containing both N(1)-substituted and N(1)-unsubstituted 2-chalcogenimidazolone moieties with exocyclic C=CH2, C=CHHal, and C=CHAr fragments in five positions, were synthesized according to Scheme 2. The substituents with different electronic and steric effects at the C=C double bond should significantly affect the reactivity of the dienophile, which can allow controlling the rate and selectivity of the Diels–Alder reaction with these substrates. It was found that methylidenehydantoin 1 gives high yields of alkylation and acylation products 12-16, and its reaction with halogens (Br2, I2) in the presence of triethylamine leads to the corresponding halomethylidene-substituted imidazolones 17 and 18 as single isomers (Scheme 2). The configuration of the C=C double bond in compounds 17 and 18 was confirmed by 1H NMR spectra of the products of their reactions with cyclopentadiene (Scheme 3 and Supplementary Information).
Our attempts to introduce methylidenetiohydantoin 2 in the reactions with acetyl chloride and benzyl bromide under the same conditions were unsuccessful. We found that thiohydantoin 2 rapidly degrades in the presence of bases (NEt3, K2CO3), and its alkylation or acylation reactions are not formed even in trace amounts (according to 1H NMR spectra of reaction mixtures). When methylidenethiohydantoin 2 reacted with bromine at 0°C, resinification of the reaction mixture occurred, and the target halogen-substituted methylenethiohydantoin 21 could be isolated from the reaction mixture only in 9% yield (see Scheme 2 and Supplementary Information).
Next, we started studying the Diels–Alder reactions with dienophiles 1-23. It should be noted that for each specific diene (cyclopentadiene 24, cyclohexadiene 25, 2,3-dimethylbutadiene 26, isoprene 27), the optimal reaction conditions turned out to be different and were selected depending on its reactivity.

2.2. Reactions of 5-Methylideneimidazolones 1-6, 9, 12-18, 22, and 23 with Cyclopentadiene

In our previous communication, we demonstrated the possibility of spiro-norbornene derivatives synthesis by the [4+2]-cycloaddition of highly active dienecyclopentadiene with methylideneimidazolones 1 and 2 at an eight-fold excess of the diene in refluxing chloroform [24]. Hydantoins 3-6, 9, and 12-16 were now introduced into the reactions with cyclopentadiene under the same conditions (Scheme 3, Table 1). In all cases, cycloaddition reactions proceeded in high yields with the predominant formation of the exo products 28a-43a.
It should be noted that, unlike exo- and endo-products 29-33a and 29-33b, which do not contain unsubstituted nitrogen atoms, N(1)-unsubstituted compounds 28a, 34a-39a, and 28b, 34b-39b can be separated by column chromatography. Probably, the difference in the chromatographic mobility of the isomers is determined by the ability of the CONH and CSNH fragments of the molecules to form hydrogen bonds with the silica gel surface.
The structures of the resulting spiro-imidazolones 28-43 were determined using 1H and 13C NMR spectroscopy; for compounds 28 and 34, their spectra correspond to those described in the literature [24]. According to 1H NMR spectroscopy data, exo- and endo-products a and b differ in chemical shifts of HC=CH protons of the norbornene framework: the characteristic doublets of the main products are shifted to a weaker field (at 6.20–6.60 ppm) relative to the signals of minor products (at 6.15–6.45 ppm) (Figure 1a), which is consistent with the literature data [15,17,24]. In addition, compounds 28, 34-39 are characterized by a very large difference in the NH protons shifts of exo- and endo-isomers (>1 ppm in CDCl3), which can be explained by the influence of the anisotropy of the double C=C bond, which is quite close to the amide proton in isomer a.
The criterion for assigning exo- and endo-stereoisomers of compounds 28, 34-39, in addition to the chemical shifts of vinyl and NH protons, could be the NOESY spectra shown in the Supplementary Information for compounds 35a and 35b. In the NOESY spectrum of spiro-derivative 35a, the interaction of the NH proton with the double bond proton, as well as with the proton of the nearest methylene group of the bicycle, is manifested, and there is no interaction with the proton at the C(7) atom of the norbornene framework (see Figure 1b). On the contrary, for the minor isomer 35b, when the NH proton was irradiated, the Overhauser effect is observed for the proton at the C(7) atom of norbornene and is absent for the proton at the C=C double bond.
The diastereoselectivity of the cyclopentadiene Diels–Alder reaction with methylenehydantoins was found to be sensitive to the nature of the substituent at position 1 of the imidazolone moiety. Despite the increase in steric hindrance near the exocyclic double bond of the dienophile, the interaction of cyclopentadiene with hydantoins 12-15 containing electron-withdrawing Ac, COOEt, Ts, Boc substituents proceeded less selectively compared to N(1) unsubstituted hydantoin 1. Such results may indicate that the electronic factors of the substituents have a stronger effect on the reactivity of the dienophile than the steric ones. The important role of the substituent in position 1 of the initial heterocycle on the reaction course is also confirmed by the fact that for the imidazolone 16 with a donor benzyl fragment, the yields of the cycloaddition product 33 are low, and only the isomers 33a are formed (see Table 1).
Variation of the exocyclic chalcogen atom of the starting heterocycle does not affect the stereoselectivity of the Diels–Alder reaction (Table 1, compare products 28 and 34). The same stereoselectivity of the Diels–Alder reaction is observed for thiohydantoins 2-6 and 9 with different substituents at N(3) atom. Apparently, the electronic properties of substituents in the C(2) and N(3) positions of the heterocycle have little effect on the reactivity of the dienophile.
An increase in steric hindrance at the C=C bond of methylideneimidazolone should hinder the [4+2]-cycloaddition reaction. Indeed, halomethylidenehydantoins 17, 18 react with cyclopentadiene only when refluxed in ortho-xylene. In the course of these reactions, only exo-isomers 40a and 41a were formed in moderate yields. In the 1H NMR spectra of compounds 40a and 41a, in the region of about 4.7 ppm, there are doublets with J ~3.3 Hz, which confirms the spatial arrangement of the CHBr and CHI groups in the norbornene framework.
Imidazolones 22, 23, containing the bulkier structure and less acceptor compared to halogens aryl substituents at the double C=C bond, did not form the reaction products with cyclopentadiene under similar conditions even in trace amounts (according to 1H NMR spectroscopy of the reaction mixtures), probably for both electronic and steric reasons.

2.3. Reactions of 5-Methylideneimidazolones 1-3, 5, 6, 8-10 with Cyclohexadiene

Reactions of 5-methylideneimidazolones with dienes 25-27, which are less reactive than cyclopentadiene, do not occur even when refluxing in toluene with a 20-fold excess of dienes. Under microwave irradiation, when methylidenehydantoin 1 was heated in benzene to 140°C with an excess of cyclohexadiene 25, the formation of the target products only in trace amounts was detected. The reactions of cyclohexadiene with compounds 2-11 were successfully carried out by catalysis of the reaction with Lewis acids; BF3∙Et2O, AlCl3, and ZnI2 were tested in the target reactions.
It was found that cyclohexadiene 25 rapidly decomposed in the presence of BF3∙Et2O, and no products of its Diels–Alder reaction were formed. In the presence of AlCl3, the decomposition of the diene also occurred, which accelerated upon heating the reaction mixture; however, the target product 44a could be isolated if diene 25 was added slowly into a refluxing solution of dienophile 1 and Lewis acid.
In the presence of 1 equivalent of ZnI2, cyclohexadiene did not react with 5-methylidenehydantoin 1 even when heated, but methylidenetiohydantoines 2, 3, 5, 6, 8-10 reacted with this diene under the same conditions to form the mixtures of diastereomeric products 45a-51a and 45b-51b in a ratio of ~3:1 (Scheme 4, Table 2), which could be separated by column chromatography.
It should be noted that the selectivity of the reaction is practically independent of the substituents in position 3 of the heterocycles 2, 3, 5, 6, 8-10. In the presence of an excess of Lewis acid, the yields of the target products somewhat decreased, presumably due to the acceleration of the decomposition of the diene on the catalyst, and with a submolar amount of ZnI2, the formation of products slowed down.
Different results of the reaction of diene 25 with hydantoin 1 and thiohydantoins 2, 3, 5, 6, 8-10 in the presence of ZnI2 may be due to the fact that ZnI2 as a soft Lewis acid is predominantly coordinated to the sulfur atom of 2-thioimidazolone, and varying the chalcogen (O or S) strongly affects the efficiency of binding the dienophile to the Lewis acid.
The structures of spiro-imidazolones 44-51 were confirmed by 1H and 13C NMR spectroscopy data; the configuration of compound 46a was determined via two-dimensional NMR techniques COSY and gHSQC (see Supplementary Information, Figures S67 and S68).
It may be noted that the 1H, 13C (and 19F NMR in cases where the compounds contained fluorine) spectra of all ortho-phenyl-N(3)-substituted spiro-imidazolones (for example, compounds 35, 46, and 51, see Supplementary Information, Figures S23, S63, and S87 and similar) demonstrate two sets of signals. This can be explained by the hindered rotation around the single bond C(Ar)-N(imidazolone) of the imidazolone fragment and, consequently, the existence of each ortho-phenyl-substituted spiro-compounds as two atropisomers (axially chiral heterocyclic analogs of biaryl derivatives similar to those described for others ortho-substituted 2-thiohydantoins) [25,26,27]. The NMR spectra of these compounds at ambient temperatures do not indicate the presence of intramolecular dynamic processes, apparently due to the high barrier to internal rotation. Thus, weak line broadening in the 19F NMR spectrum of product 35a is observed only at temperatures above 80 °C (Figure S30); this indicates that the rotational barrier is greater than 20 kcal/mol.
As in the reactions with cyclopentadiene, the isomeric exo- and endo-Diels–Alder products of the reactions of methylidenetiohydatoins 2, 3, 5, 6, 8-10 with cyclohexadiene can be distinguished by the chemical shifts of the protons of the CH=CH group: for the main isomers a, a characteristic doublet of doublets observed in the region of 6.62–6.34 ppm, and for minor products b, such doublet of doublets is present in the region of 6.48–6.30 ppm (in CDCl3).
The structure of compound 51a was additionally confirmed by the X-ray diffraction data. The crystal cell shown in Figure 2 includes two spiro-thiohydantoin molecules linked by NHS hydrogen bonds to form an eight-membered cycle. The dihedral angle between the planes of the cycles at the spiro junction is close to 90°; imidazolone five-membered rings are near the planar.

2.4. Reactions of 5-Methylideimidazolones 1-12 with 2,3-Dimethylbutadiene

Reactions of methylideneimidazolones 1-12 with 2,3-dimethylbutadiene also require Lewis acids as the catalysis but can proceed in the presence of both AlCl3 and ZnI2 (Scheme 5, Table 3). The reactions of diene 26 with dienophiles 1 and 2 under the action of AlCl3 proceeded in moderate yields giving the compounds 52 and 53.
As well as in reactions with cyclohexadiene, ZnI2 activates thiohydantoins 2-11 in the reactions with 2,3-dimethylbutadiene with the formation of target compounds 54-62 but does not catalyze the reactions with oxygen analogs, probably due to the selective coordination of the Lewis acid to the C=S bond of the starting heterocycle. A significant increase in the yield of thiohydantoin 53 when AlCl3 was replaced by the softer ZnI2 may be due to a decrease in the rate of the side process of diene polymerization under the action of the Lewis acid.

2.5. Reactions of 5-Methylideneimidazolones 1, 2, 4-6, 8, and 9 with Isoprene

Using isoprene 27, the regioselectivity of the Diels–Alder reaction of methylideneimidazolones 1, 2, 4-6, 8, and 9 were studied. Boiling of hydantoin 1 mixture with a 10-fold excess of this diene in chloroform led to the formation of cycloaddition product 63a in 70% yield and trace amounts of the minor regioisomer 63b. Under similar conditions, thiohydantoins 2, 4-6, 8, and 9 formed an inseparable mixture of regioisomers 64a-69a and 64b-69b in good yields in a ratio of ~87:13 (Scheme 6, Table 4 and Supplementary Information).
The structures of regioisomers were confirmed by1H NOESY1D,1H-13C HSQC, and1H-13C HMBC NMR spectroscopy data for the compounds 67a and 67b (see Supplementary Information, Figures S123–S125). In particular, the position of the methine proton in the six-membered ring of compound 67a was confirmed by the presence of a cross peak in the HMBC spectrum, which is responsible for the vicinal interaction of this proton with the quaternary spiro-carbon.
The results obtained demonstrate that AlCl3 is an effective catalyst for the reactions of low-activity dienes with hydantoins and ZnI2 with thiohydantoins. At the same time, AlCl3 similarly catalyzes reactions with hydantoins and thiohydantoins (see Table 3). It may be supposed that the harder Lewis acid AlCl3 is coordinated to the N(1) atom of the starting imidazolone and, thus, catalyzes both reactions with hydantoins and thiohydantoins, while the softer Lewis acid ZnI2 is coordinated to the S atom and, thus, activates only thiohydantoins; however, exact proof of this assumption requires additional research.

2.6. Alkylation and Acylation of [4+2]-Cycloaddition Reaction Products

Studying the Diels–Alder reactions with methylideneimidazolones 1-18, it was found that in the case of N(1)-unsubstituted dienophiles 1 and 2, the resulting diastereomeric pair of spiroheterocycles can be successfully separated into individual isomers, while N(1)-substituted derivatives are formed either as an inseparable mixture of diastereomers or in extremely low yields (Table 1). Therefore, we studied the possibility of post-modification at the nitrogen atom of N(1)-unsubstituted Spiro derivatives 28a, 34a, 44a, 45a, 45b, and 53.
We found that hydantoins 28a and 44a can be alkylated or acylated with PhCH2Cl or Boc2O in high yields by refluxing in chloroform or acetonitrile (Scheme 7). The spectral data of the resulting products 33a and 34a completely coincided with the spectral data of the main products of the Diels–Alder reaction of methylidenehydantoins 15 and 16 with cyclopentadiene (see Section 2.2).
Thiohydantoins 34a, 45a, 45b, and 54 were alkylated with MeI and PhCH2Cl under milder conditions at room temperature in acetonitrile in the presence of K2CO3, exclusively at the sulfur atom. The formation of S–CH2Ph and S–Me bonds in corresponding imidazolones 72-75 was confirmed by 13C NMR spectroscopy data.

2.7. Desulfurization of Spiro-Thiohydantoins

Since methylidenehydantoin1 reacted with dienes 25 and 26 with low conversion even in the presence of Lewis acids (Section 2.3 and Section 2.4), we proposed an alternative scheme for the synthesis of spirocyclic hydantoins from their thiohydantoin analogs. The procedure described in the literature for the hydrolysis of S-alkylated thiohydantoins under the action of HCl in refluxing ethanol [13,28] applied to compound 72 did not lead to the formation of the corresponding hydantoin 28a. In the 1H NMR spectrum of the reaction mixture, there were no characteristic CH=CH signals of the norbornene skeleton protons, which probably indicates the ongoing processes of cationic polymerization under the reaction conditions.
The desired transformation of spirothiohydantoins 34a and 45a into spirohydantoins 28a and 44a was achieved under milder conditions by treating methanolic solutions of the starting compounds with 35% aqueous H2O2 at room temperature in good yields (Scheme 8).
However, an even more effective way of thiohydantoins desulfurizing turned out to be their interaction with nitrile oxides [29] (Scheme 9). The reactive nitrile oxide was generated in situ from N-hydroxyimidoyl chloride under the action of a tertiary amine. As a result of subsequent N-hydroxyimidoyl chloride 1,3-dipolar cycloaddition at the C=S bond, an unstable oxatriazole was obtained, which then decomposed to form hydantoin, analogously to that described in [30]. For this reaction, we used a recently proposed convenient method of diffusion reagents mixing, which made it possible to suppress undesirable dimerization processes of the 1,3-dipole and introduce thiohydantoin and the dipole precursor into the reaction in a strictly equimolar ratio [31]. Under these conditions, hydantoins 28a, 52, 75-78 were formed in high yields without the side products of dipole addition to the C=C bond. During the synthesis of compound 44a, a certain amount (no more than 10%) of the addition product of nitrile oxide to the C=C bond was formed.

2.8. Cytotoxicity against Human Cell Lines

Some of the obtained spiro-derivatives were tested for cytotoxicity using the standard MTT assay [32]. Cytotoxicity was evaluated using the cell lines of various etiologies: breast cancer MCF7, human lung carcinoma A549, non-cancer human embryonic kidney cell line HEK293T, and non-cancer lung fibroblast VA13 cell line. Lung cancers and breast cancers are among the most common causes of tumor lesions and related death in the world [33], and non-cancerous cells were applied for the specificity of action evaluation. The results are shown in Table 5; the dose-response dependency graphs are available in Supplementary Information.
Generally, the tested compounds show rather low or moderate cytotoxicity to all tested cell lines; their IC50abs values (IC50abs is the concentration resulting in a two-fold decrease in the number of cells in comparison with untreated cells) against A549 and MCF7 cell lines mostly ranging from 20 to 100 µM. Additionally, the tested compounds show little selectivity to all cell lines tested. Despite the nominal IC50abs selectivity of the compounds 55 and 61 against A549 compared to VA13, their IC50 values (IC50 is the concentration resulting in half of the maximal cytotoxic effect) were quite similar (Table S1).
It may also be noticed that the introduction into the tested spiro-imidazolones of halogenated phenyl ring instead of non-halogenated one in most cases increased cytotoxicity against all tested cell lines. It can be seen by comparing the data obtained for compound 63, which is the most cytotoxic of all the tested compounds and has a dihalogenated phenyl ring, with the data obtained for compounds 49a, 55, 58, and 59, which have monohalogenated phenyl ring, and the non-halogenated compounds 28, 34. The compounds with OMe- or Me-substitutient in the phenyl rings at N(3) imidazolone atom had no significant cytotoxicity on the most tested cell lines (Table 5, compounds 37-39, 48, 50, 56, 60, 66).

2.9. Cytotoxicity against E. coli

All compounds investigated for cytotoxicity effects were also analyzed for antibacterial activity. Tests were carried out on E. coli BW25113 DTC-pDualrep2 strain that has a normal cell wall and the E. coli BW25113 LPTD-pDualrep2strain that has damaged cell walls. Some of the tested compounds showed a noticeable antibacterial effect against E. coli BW25113 DTC-pDualrep2 but were almost inactive against E. coli BW25113 LPTD-pDualrep2. All the tested compounds were analyzed for the mechanism of antibacterial activity by means of the pDualrep2 [34] reporter system, which allows for the detection of translational inhibitors and SOS-response inducers, but none of these mechanisms is involved.
Then the minimal inhibitory concentration (MIC) was measured for E. coli BW25113 DTC and E. coli K-12 (WT) for the 12 most bacteriotoxic compounds from the plate test. MIC of compound 77 against E. coli BW25113 DTC is 125 µM, while the other compounds either show MIC ~500 µM, 1 mM, or are inactive in this test (Table 6).
All the tested compounds were completely inactive against E. coli K-12 at concentrations of 1 mM and below. It indicates that the studied compounds are toxic to E. coli but are effectively removed from the cell by efflux systems and, therefore, do not affect the wild type of E. coli.

3. Materials and Methods

All solvents were purified using standard procedures [35]. Starting compounds were purchased from commercial sources (Sigma–Aldrich, ABCR, AKSci, Burlington, VT, USA). Reactions were checked by TLC analysis using silica plates 1H, and 13C NMR spectra were recorded on BrukerAvance or Agilent 400-MR spectrometers (400 MHz for 1H, 100 MHz for 13C). Chemical shifts are reported in parts per million relative to TMS.
Electrospray ionization high-resolution mass spectra were recorded on a TripleTOF 5600+ quadrupole time-of-flight mass spectrometer (ABSciex, Concord, Vaughan, ON, Canada) with DuoSpray ion source in positive ion mode. The capillary voltage was 5.5 kV; nebulizing and curtain gas pressure—15 and 25 psi, respectively; ion source temperature ambient; declustering potential 20 V; m/z range 100–1200. Elemental compositions of the detected ions were determined based on accurate masses and isotopic distributions using Formula Finder software (ABSciex, Concord, ON, Canada).
The X-Ray data for compound 51a were collected via STOE diffractometer Pilatus100K detector, Cu Kα (1.54086Å) radiation, rotation method mode. STOE X-AREA software was used for cell refinement and data reduction. Data collection and image processing were performed with X-Area 1.67 (STOE & Cie GmbH, Darmstadt, Germany, 2013). Intensity data were scaled with LANA (part of X-Area) in order to minimize differences in intensities of symmetry-equivalent reflections (multi-scan method).
The structures were solved and refined with SHELX(1) program [36]. The non-hydrogen atoms were refined by using the anisotropic full matrix least-square procedure. Hydrogen atoms were placed in the calculated positions and allowed to ride on their parent atoms [C-H 0.93–0.98; Uiso 1.2 Ueq(parent atom)]. Hydrogen atoms at nitrogen atoms N2A and N2B (see Figure 2) were localized from Fourier syntheses and refined freely. Molecular geometry calculations were performed with the SHELX program, and the molecular graphics were prepared by using DIAMOND(2) version 4 software [37].
Some crystallographic data for 51a are listed in Table S2; hydrogen bonds are given in Table S3 (see Supplementary part of this article); CCDC-2232689 contain the Supplementary crystallographic data. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via, 1 February 2023.
Experimental procedures and characteristic data for all synthesized compounds are given in the Supplementary Information.
Cell cultures. Human embryonic kidney HEK293T cell line was kindly provided by Dr. E. Knyazhanskaya; immortalized human fibroblasts cell line VA13 was kindly provided by Dr. M. Rubtsova; human breast cancer cell line MCF7 and human lung adenocarcinoma cell line A549 were kindly provided by Dr. S. Dmitriev. MCF7, VA13, A549, and HEK293T cell lines were maintained in DMEM/F-12 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) culture medium containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 50 µg/mL penicillin and 0.05 mg/mL streptomycin at 37°C (Thermo Fisher Scientific, Waltham, Massachusetts, USA) in 5% CO2. Cells were maintained at 37°C in a humidified incubator MCO-18AC (Sanyo, Japan) supplied with 5% CO2. Cell cultures were tested for the absence of mycoplasma.
In Vitro Survival Assay (MTT Assay). The cytotoxicity was tested using the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay [32] with some modifications. A total of 2500 cells per well for the MCF7, HEK293T cell lines, 3000 cells for the A549 cell line, or 4000 cells per well for the VA-13 cell line were plated out in 135 µL of DMEM-F12 media (Gibco, USA) in a 96-well plate and then incubated in the 5% CO2 incubator for first 20 h without treating. After this, 15 µL of media-DMSO solutions of tested substances were added to the cells (final DMSO concentrations in the media were 0,5% or less) and treated for65 h with 45 nM–100 µM (eight dilutions) of our substances (triplicate each) and doxorubicin as a control substance. Then the MTT reagent (Paneco LLC, Moscow, Russia) was added to cells up to the final concentration of 0.5 g/L (10 × stock solution in PBS was used), and cells were incubated for 2 h at 37 °C under an atmosphere of 5% CO2. Then the MTT solution was discarded, and 140 µL of DMSO (PharmaMed LLC, Russia) was added. The plates were swayed on a shaker (200 rpm) to dissolve the formazan. The absorbance was measured using a microplate reader (VICTOR ×5 Light Plate Reader, PerkinElmer, USA) at a wavelength of 555 nm (in order to measure formazan concentration). The results were used to construct dose-response graphs and to estimate IC50absvalues (IC50abs is the concentration resulting in a two-fold decrease in the number of cells in comparison with the untreated cells) and, in some cases, IC50 values (IC50 is the concentration resulting in half of the maximal cytotoxic effect) with GraphPadSoftware, Inc., San Diego, CA, USA.
E. coli cytotoxicity assay with a screening of Mechanism of Action. A total of 50 µL of 20 mM solution of each compound in DMSO and DMSO as a control substance was added into wells punched in an agar plate containing a lawn of the E. coli BW25113 DTC-pDualrep2 or E. coli BW25113 LPTD-pDualrep2, which are hypersensitive to antibiotics. E. coli DTC [38] lacks the tolC gene, which encodes the outer membrane component of several multidrug transporters [39]. E. coli LPTD [40] has the 23-amino-acid deletion in the lptD gene, an inessential protein functioning in the final stages of the assembly of lipopolysaccharides into the outer membrane [41]. After overnight incubation at 37 °C, the plate was scanned with the ChemiDoc (Bio-Rad, Hercules, CA, USA) system with two channels, including “Cy3-blot” (553/574 nm, green pseudocolor) for RFP fluorescence and “Cy5-blot” (588/633 nm, red pseudocolor) for Katushka2S fluorescence. Cells without reporter construction could be detected in both channels. The induction of the expression of Katushka2S is triggered by translation inhibitors, while RFP is up-regulated by the induction of DNA damage and SOS response. In addition, 2 µL of levofloxacin (25 µg/mL) and erythromycin (5 mg/mL) were used as positive controls for DNA biosynthesis and ribosome inhibitors, respectively.
Determination of Minimal Inhibitory Concentration (MIC). MIC values were determined by monitoring growth in 96-well plates of E. coli cultures exposed to serial dilutions of compounds. Specifically, overnight E. coli BW25113 DTC and K12 cultures were diluted in 96-well plates to an OD590 of 0.01 in LB medium. The wells were then supplemented with the solution of each tested compound at concentrations of 1 mM, 500 µM, 250 µM, 125 µM, 62.5 µM, 31.25 µM, 15.63 µM, 7.81 µM, 3.91 µM, and 1.95 µM. Then the plates were incubated with shaking (200 r.p.m.) overnight at 37°C, and cell growth was assessed by scanning OD590 each well with a VictorX5 reader.

4. Conclusions

In the present study, a series of new spiro-derivatives containing hydantoin and thiohydantoin fragments was synthesized by [4+2]-cycloaddition of 1,3-dienes (cyclopentadiene, cyclohexadiene, 2,3-dimethylbutadiene, isoprene)to 5-methylidene-substituted imidazolones (hydantoins or thiohydantoins). It was shown that the studied cycloaddition reactions proceed regioselectively and stereoselectively. Reactions of methylideneimidazolones with cyclopentadiene proceed by co-heating the reactants; reactions with cyclohexadiene, 2,3-dimethylbutadiene, and isoprene require catalysis by Lewis acids. It was demonstrated that ZnI2 is an effective catalyst for the interaction of methylidenethiohydantoins with non-activated dienes. The possibility of alkylation and acylation of the obtained spiro-hydantoinsat the N(1)nitrogen atoms with PhCH2Cl or Boc2O and the alkylation of the spiro-thiohydantoinsat the S atoms with MeI or PhCH2Cl in high yields have been demonstrated. The preparativetransformation of spiro-thiohydantoins into corresponding spiro-hydantoinsin mild conditions by treating with 35% aqueous H2O2 or nitrile oxide has been carried out.
Some synthesized spiro-imidazolones in preliminary biological tests demonstrated moderate cytotoxic activity on MCF7, A549, HEK293T, and VA13 cell lines. They show only little (at concentrations more than 100 µM) antibacterial effects in the experiments with wild type of E. coli.

Supplementary Materials

The following supporting information can be downloaded at: the experimental details.

Author Contributions

Conceptualization, E.K.B. and M.E.K.; methodology, D.E.S.; validation, D.E.S., Y.K.G. and V.A.R.; formal analysis, Y.K.G.; investigation, Y.S.H., D.E.S., V.A.T. and D.A.S.; resources, Y.K.G.; data curation, M.E.K.; writing—original draft, D.E.S.; supervision, E.K.B.; project administration, M.E.K. and N.V.Z.; funding acquisition, E.K.B. and M.E.K. All authors have read and agreed to the published version of the manuscript.


This work was supported by a Grant from the President of the Russian Federation (MK-3748.2022.1.3, dienophiles synthesis, and Diels–Alder reactions), Russian Science Foundation (grant no. 21-13-00023, reactions with nitriloxides; grant no. 22-14-00099, biological tests), and in the part of NMR and X-ray study, by the M.V. Lomonosov Moscow State University Program of Development.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data is available upon request from the authors.

Conflicts of Interest

The authors declare no conflict of interest.


  1. Scammells, P.J.; Baker, S.P.; Luiz Bellardinelli, R.A.O.; Russell, R.A.; Wright, D.M. The Synthesis of the Novel Adenosine Agonists, Exo- and Endo. N6-(5,6-Epoxynorborn-2-Yl)Adenosine. Tetrahedron 1996, 52, 4735–4744. [Google Scholar] [CrossRef]
  2. Lee, W.G.; Lee, S.D.; Cho, J.H.; Jung, Y.; Kim, J.H.; Hien, T.T.; Kang, K.W.; Ko, H.; Kim, Y.C. Structure-Activity Relationships and Optimization of 3,5-Dichloropyridine Derivatives as Novel P2X 7 Receptor Antagonists. J. Med. Chem. 2012, 55, 3687–3698. [Google Scholar] [CrossRef] [PubMed]
  3. Goto, T.; Sakashita, H.; Murakami, K.; Sugiura, M.; Kondo, T.; Fukaya, C. Novel Histamine H3 Receptor Antagonists: Synthesis and Evaluation of Formamidine and S-Methylisothiourea Derivatives. Chem. Pharm. Bull. 1997, 45, 305–311. [Google Scholar] [CrossRef] [Green Version]
  4. Meguro, K.; Tawada, H.; Sugiyama, Y.; Fujita, T.; Kawamatsu, Y. Studies on Antidiabetic Agents. VII Synthesis and Hypoglycemic Activity of 4-Oxazoleacetic Acid Derivatives. Chem. Pharm. Bull. 1986, 34, 2840–2851. [Google Scholar] [CrossRef] [Green Version]
  5. Saha, S.L.; Roche, V.F.; Pendola, K.; Kearley, M.; Lei, L.; Romstedt, K.J.; Herdman, M.; Shams, G.; Kaisare, V.; Feller, D.R. Synthesis and in Vitro Platelet Aggregation and TP Receptor Binding Studies on Bicyclic 5,8-Ethanooctahydroisoquinolines and 5,8-Ethanotetrahydroisoquinolines. Bioorg. Med. Chem. 2002, 10, 2779–2793. [Google Scholar] [CrossRef] [PubMed]
  6. Burmistrov, V.; Morisseau, C.; Karlov, D.; Pitushkin, D.; Vernigora, A.; Rasskazova, E.; Butov, G.M.; Hammock, B.D. Bioisosteric Substitution of Adamantane with Bicyclic Lipophilic Groups Improves Water Solubility of Human Soluble Epoxide Hydrolase Inhibitors. Bioorg. Med. Chem. Lett. 2020, 30, 127430. [Google Scholar] [CrossRef] [PubMed]
  7. Saccomano, N.A.; Vinick, F.J.; Koe, B.K.; Nielsen, J.A.; Whalen, W.M.; Meltz, M.; Phillips, D.; Thadieo, P.F.; Jung, S.; Chapin, D.S.; et al. Calcium-Independent Phosphodiesterase Inhibitors as Putative Antidepressants: [3-(Bicycloalkyloxy)-4-Methoxyphenyl]-2-Imidazolidinones. J. Med. Chem. 1991, 34, 291–298. [Google Scholar] [CrossRef] [PubMed]
  8. Wang, A.; Wang, Y.; Meng, X.; Yang, Y. Design, Synthesis and Biological Evaluation of Novel Thiohydantoin Derivatives as Potent Androgen Receptor Antagonists for the Treatment of Prostate Cancer. Bioorg. Med. Chem. 2021, 31, 115953. [Google Scholar] [CrossRef]
  9. Isizumi, K.; Kojima, A.; Antoku, F.; Saji, I.; Yoshigi, M. Succinimide Derivatives. II. Synthesis and Antipsychotic Activity of N-[4-[4-(1,2-Benzisothiazol-3-Yl)-1-Piperazinyl] Butyl]-1,2-Cis-Cyclohexanedicarboximide (SM-9018) and Related Compounds. Chem. Pharm. Bull. 1995, 43, 2139–2151. [Google Scholar] [CrossRef] [Green Version]
  10. Valderrama, J.A.; Zamorano, C.; González, M.F.; Prina, E.; Fournet, A. Studies on Quinones. Part 39: Synthesis and Leishmanicidal Activity of Acylchloroquinones and Hydroquinones. Bioorg. Med. Chem. 2005, 13, 4153–4159. [Google Scholar] [CrossRef]
  11. Bertilsson, S.K.; Ekegren, J.K.; Modin, S.A.; Andersson, P.G. The Aza-Diels-Alder Reaction Protocol—A Useful Approach to Chiral, Sterically Constrained α-Amino Acid Derivatives. Tetrahedron 2001, 57, 6399–6406. [Google Scholar] [CrossRef]
  12. Kukushkin, M.E.; Skvortsov, D.A.; Kalinina, M.A.; Tafeenko, V.A.; Burmistrov, V.V.; Butov, G.M.; Zyk, N.V.; Majouga, A.G.; Beloglazkina, E.K. Synthesis and Cytotoxicity of Oxindoles Dispiro Derivatives with Thiohydantoin and Adamantane Fragments. Phosphorus Sulfur Silicon Relat. Elem. 2020, 195, 544–555. [Google Scholar] [CrossRef]
  13. Beloglazkina, A.; Barashkin, A.; Polyakov, V.; Kotovsky, G.; Karpov, N.; Mefedova, S.; Zagribelny, B.; Ivanenkov, Y.; Kalinina, M.; Skvortsov, D.; et al. Synthesis and Biological Evaluation of Novel Dispiro Compounds Based on 5-Arylidenehydantoins and Isatins as Inhibitors of P53–MDM2 Protein–Protein Interaction. Chem. Heterocycl. Compd. 2020, 56, 747–755. [Google Scholar] [CrossRef]
  14. 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-4-Ones: Synthesis, Cytotoxicity and Ros Generation Ability. Int. J. Mol. Sci. 2021, 22, 2613. [Google Scholar] [CrossRef] [PubMed]
  15. Cernak, T.A.; Gleason, J.L. Density Functional Theory Guided Design of Exo-Selective Dehydroalanine Dienophiles for Application toward the Synthesis of Palau’amine. J. Org. Chem. 2008, 73, 102–110. [Google Scholar] [CrossRef]
  16. Sankhavasi, W.; Kohmoto, S.; Yamamoto, M.; Nishio, T.; Iida, I.; Yamada, K. Chiral Hydantoin; A New Dienophile for Diels–Alder Reaction. Bull. Chem. Soc. Jpn. 1992, 65, 935–937. [Google Scholar] [CrossRef]
  17. Fraile, J.M.; Lafuente, G.; Mayoral, J.A.; Pallarés, A. Synthesis and Reactivity of 5-Methylenehydantoins. Tetrahedron 2011, 67, 8639–8647. [Google Scholar] [CrossRef] [Green Version]
  18. Soengas, R.G.; Da Silva, G.; Estévez, J.C. Synthesis of Spironucleosides: Past and Future Perspectives. Molecules 2017, 22, 2028. [Google Scholar] [CrossRef] [Green Version]
  19. Wadghane, S.; Bhor, R.; Shinde, G.; Kolhe, M.; Rathod, P. A Review on the Some Biological Activities of the Hydantoin Derivatives. J. Drug Deliv. Ther. 2023, 13, 171–178. [Google Scholar] [CrossRef]
  20. Fujisaki, F.; Shoji, K.; Sumoto, K. A Synthetic Application of β-Aminoalanines to Some New 5-Dialkylaminomethyl-3-Phenylhydantoin Derivatives. Heterocycles 2009, 78, 213–220. [Google Scholar] [CrossRef]
  21. Kukushkin, M.E.; Karpov, N.A.; Shybanov, D.E.; Zyk, N.V.; Beloglazkina, E.K. A Convenient Synthesis of 3-Aryl-5-Methylidene-2-Thiohydantoins. Mendeleev Commun. 2022, 32, 126–128. [Google Scholar] [CrossRef]
  22. Jia, H.H.; Ni, F.; Wang, J.; Wei, P.; Ouyang, P.K. 5-(4-Fluorobenzylidene)Imidazolidine-2,4-Dione. Acta Crystallogr. Sect. E Struct. Rep. Online 2005, 61, 2784–2786. [Google Scholar] [CrossRef]
  23. Maccari, R.; Ettari, R.; Adornato, I.; Naß, A.; Wolber, G.; Bitto, A.; Mannino, F.; Aliquò, F.; Bruno, G.; Nicolò, F.; et al. Identification of 2-Thioxoimidazolidin-4-One Derivatives as Novel Noncovalent Proteasome and Immunoproteasome Inhibitors. Bioorg. Med. Chem. Lett. 2018, 28, 278–283. [Google Scholar] [CrossRef]
  24. Shybanov, D.E.; Kukushkin, M.E.; Tafeenko, V.A.; Zyk, N.V.; Grishin, Y.K.; Roznyatovsky, V.A.; Beloglazkina, E.K. Different Addition Modes of Cyclopentadiene and Furan at Methylidene(Thio)Hydantoins. Mendeleev Commun. 2021, 31, 246–247. [Google Scholar] [CrossRef]
  25. Sarigul, S.; Dogan, I. Atroposelective Synthesis of Axially Chiral Thiohydantoin Derivatives. J. Org. Chem. 2016, 81, 5895–5902. [Google Scholar] [CrossRef] [PubMed]
  26. Kukushkin, M.E.; Kondratieva, A.A.; Karpov, A.; Shybanov, D.E.; Tafeenko, V.A.; Roznyatovsky, V.A.; Grishin, Y.K.; Moiseeva, A.; Zyk, N.V.; Beloglazkina, E.K. Access to Dispiro Indolinone-Pyrrolidine-Imidazolones. R. Soc. Open Sci. 2022, 9. [Google Scholar] [CrossRef]
  27. Sarigul Ozbek, S.; Bacak Erdik, M.; Dogan, I. Aldol Reactions of Conformationally Stable Axially Chiral Thiohydantoin Derivatives. ACS Omega 2021, 6, 27823–27832. [Google Scholar] [CrossRef]
  28. Kukushkin, M.; Novotortsev, V.; Filatov, V.; Ivanenkov, Y.; Skvortsov, D.; Veselov, M.; Shafikov, R.; Moiseeva, A.; Zyk, N.; Majouga, A.; et al. Synthesis and Biological Evaluation of S-, O- and Se-Containing Dispirooxindoles. Molecules 2021, 26, 7645. [Google Scholar] [CrossRef]
  29. Vilková, M.; Prokaiová, M.; Imrich, J. Spontaneous Cyclization of (Acridin-9-Ylmethyl)Thioureas to Spiro [Dihydroacridine-9′(10′H),5-Imidazolidine]-2-Thiones, a Novel Type of Acridine Spirocycles. Tetrahedron 2014, 70, 944–961. [Google Scholar] [CrossRef]
  30. Hewitt, R.J.; Ong, M.J.H.; Lim, Y.W.; Burkett, B.A. Investigations of the Thermal Responsiveness of 1,4,2-Oxathiazoles. Eur. J. Org. Chem. 2015, 2015, 6687–6700. [Google Scholar] [CrossRef]
  31. Shybanov, D.E.; Filkina, M.E.; Kukushkin, M.E.; Grishin, Y.K.; Roznyatovsky, V.A.; Zyk, N.V.; Beloglazkina, E.K. Diffusion Mixing with a Volatile Tertiary Amine as a Very Efficient Technique for 1,3-Dipolar Cycloaddition Reactions Proceeding via Dehydrohalogenation of Stable Precursors of Reactive Dipoles. New J. Chem. 2022, 46, 18575–18586. [Google Scholar] [CrossRef]
  32. Mosmann, T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. J. Lmmunological Methods 1983, 65, 55–63. [Google Scholar] [CrossRef]
  33. Ghoncheh, M.; Mohammadian, M.; Mohammadian-Hafshejani, A.; Salehiniya, H. The Incidence and Mortality of Colorectal Cancer and Its Relationship with the Human Development Index in Asia. Ann. Glob. Health 2016, 82, 726–737. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Osterman, I.A.; Komarova, E.S.; Shiryaev, D.I.; Korniltsev, I.A.; Khven, I.M.; Lukyanov, D.A.; Tashlitsky, V.N.; Serebryakova, M.V.; Efremenkova, O.V.; Ivanenkov, Y.A.; et al. Sorting out Antibiotics’ Mechanisms of Action: A Double Fluorescent Protein Reporter for High-Throughput Screening of Ribosome and DNA Biosynthesis Inhibitors. Antimicrob. Agents Chemother. 2016, 60, 7481–7489. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Tietze, L.F.; Eicher, T. Reaktionen Und Synthesen Im Organisch-Chemischen Praktikum Und Forschungslaboratorium (2. Auflage), 117th ed.; Wiley: Thieme Verlag, Stuttgart, Germany; New York, NY, USA, 1991; ISBN 3527308741. [Google Scholar]
  36. Sheldrick, G.M. A Short History of SHELX. Acta Crystallogr. Sect. A Found. Crystallogr. 2008, 64, 112–122. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Brandenburg, K. DIAMOND, Release 2.1d; Crystal Impact GbR: Bonn, Germany, 2000. [Google Scholar]
  38. Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.; Datsenko, K.A.; Tomita, M.; Wanner, B.L.; Mori, H. Construction of Escherichia Coli K-12 in-Frame, Single-Gene Knockout Mutants: The Keio Collection. Mol. Syst. Biol. 2006, 2, 2006.0008. [Google Scholar] [CrossRef] [Green Version]
  39. Sulavik, M.C.; Houseweart, C.; Cramer, C.; Jiwani, N.; Murgolo, N.; Greene, J.; Didomenico, B.; Shaw, K.J.; Miller, G.H.; Hare, R.; et al. Antibiotic Susceptibility Profiles of Escherichia Coli Strains Lacking Multidrug Efflux Pump Genes. Antimicrob. Agents Chemother. 2001, 45, 1126–1136. [Google Scholar] [CrossRef] [Green Version]
  40. Orelle, C.; Carlson, S.; Kaushal, B.; Almutairi, M.M.; Liu, H.; Ochabowicz, A.; Quan, S.; Pham, V.C.; Squires, C.L.; Murphy, B.T.; et al. Tools for Characterizing Bacterial Protein Synthesis Inhibitors. Antimicrob. Agents Chemother. 2013, 57, 5994–6004. [Google Scholar] [CrossRef] [Green Version]
  41. Wu, T.; McCandlish, A.C.; Gronenberg, L.S.; Chng, S.S.; Silhavy, T.J.; Kahne, D. Identification of a Protein Complex That Assembles Lipopolysaccharide in the Outer Membrane of Escherichia Coli. Proc. Natl. Acad. Sci. USA 2006, 103, 11754–11759. [Google Scholar] [CrossRef] [Green Version]
Scheme 1. Diels–Alder reactions with methylenehydantoins and methylenethiohydantoins [15,16,17].
Scheme 1. Diels–Alder reactions with methylenehydantoins and methylenethiohydantoins [15,16,17].
Ijms 24 05037 sch001
Scheme 2. Starting dienophiles and synthesis of compounds 12-21 from methylideneimidazolones 1,2.
Scheme 2. Starting dienophiles and synthesis of compounds 12-21 from methylideneimidazolones 1,2.
Ijms 24 05037 sch002
Scheme 3. Reactions of methyleneimidazolones with cyclopentadiene.
Scheme 3. Reactions of methyleneimidazolones with cyclopentadiene.
Ijms 24 05037 sch003
Figure 1. (a) Characteristic doublets of doublet set of HC=CH protons of compounds 35a and 35b in their 1H NMR spectra (CDCl3). (b) Characteristic correlations observed in 1H-1H NOESY NMR spectrum of the compounds 35a. (c) 1H NOESY NMR spectrum of compound 35b.
Figure 1. (a) Characteristic doublets of doublet set of HC=CH protons of compounds 35a and 35b in their 1H NMR spectra (CDCl3). (b) Characteristic correlations observed in 1H-1H NOESY NMR spectrum of the compounds 35a. (c) 1H NOESY NMR spectrum of compound 35b.
Ijms 24 05037 g001
Scheme 4. Reactions of methylidenethiohydantoins 1-3, 5, 6, 8-10 with cyclohexadiene.
Scheme 4. Reactions of methylidenethiohydantoins 1-3, 5, 6, 8-10 with cyclohexadiene.
Ijms 24 05037 sch004
Figure 2. Molecular structure of compound 51a. Thermal ellipsoids are given a 30% probability.
Figure 2. Molecular structure of compound 51a. Thermal ellipsoids are given a 30% probability.
Ijms 24 05037 g002
Scheme 5. Reactions of methylideneimidazolones 1-12 with 2,3-dimethylbutadiene.
Scheme 5. Reactions of methylideneimidazolones 1-12 with 2,3-dimethylbutadiene.
Ijms 24 05037 sch005
Scheme 6. Reactions of methylideneimidazolones1, 2, 4-6, 8, and 9 with isoprene.
Scheme 6. Reactions of methylideneimidazolones1, 2, 4-6, 8, and 9 with isoprene.
Ijms 24 05037 sch006
Scheme 7. Alkylation and acylation of spirocyclic imidazolones.
Scheme 7. Alkylation and acylation of spirocyclic imidazolones.
Ijms 24 05037 sch007
Scheme 8. Desulfurization of spiro-thiohydantoins by H2O2.
Scheme 8. Desulfurization of spiro-thiohydantoins by H2O2.
Ijms 24 05037 sch008
Scheme 9. 1 This product was isolated as an inseparable mixture with the addition products of nitrile oxide at the C=C bond. Desulfurization of spiro-thiohydantoins by the nitrile oxide action.
Scheme 9. 1 This product was isolated as an inseparable mixture with the addition products of nitrile oxide at the C=C bond. Desulfurization of spiro-thiohydantoins by the nitrile oxide action.
Ijms 24 05037 sch009
Table 1. Diels–Alder reactions of methylideneimidazolone derivatives with cyclopentadiene.
Table 1. Diels–Alder reactions of methylideneimidazolone derivatives with cyclopentadiene.
ProductsXR1R2R3exo:endo1Yield, %
28a + 28bOPhHH91:981 + 9 2,3 [24]
29a + 29bOPhAcH77:2373 (78:22) 3,4
30a +30bOPhCOOEtH79:2183 (83:17) 3,4
31a +31bOPhTsH87:1391 (89:11) 3,4
32a + 32bOPhBocH85:1573 (85:15) 3,4
33aOPhCH2PhH100:010 3,5
34a + 34bSPhHH91:979 + 8 2,3 [24]
35a + 35bS2-FC6H4HH91:971 + 7 2,3
36a + 36bS2-ClC6H4HH91:968 + 0 2,3
37a + 37bS3-MeC6H4HH91:980 + 8 2,3
38a + 38bS3-MeOC6H4HH91:981 + 9 2,3
39a + 39bS4-MeOC6H4HH91:978 + 8 2,3
40aOPhHBr100:064 6,7
41aOPhHI100:037 6,7
1 Based on 1H NMP spectra of reaction mixture. 2 Isolated yields for major and minor isomers. 3 Conditions: cyclopentadiene (8 equivalents.), CHCl3, reflux, 6h. 4 Isolated yields for the isomers mixture; isomers ratio is indicated in the parentheses. 5 Yield by 1H NMP spectra of reaction mixture. 6 Isolated yields. 7 Conditions: cyclopentadiene (20 equivalents), o-xylene, reflux, 13 h.
Table 2. Diels–Alder reactions of 5-methylideimidazolones with cyclohexadiene.
Table 2. Diels–Alder reactions of 5-methylideimidazolones with cyclohexadiene.
ProductsXLAArYield a 1, %Yield b 1, %
44a + 44bOAlCl3Ph160
ZnI2 00
45a + 45bSZnI2Ph5419
46a + 46bSZnI22-FC6H44415
47a + 47bSZnI23-MeC6H46025
48a + 48bSZnI23-MeOC6H45121
49a + 49bSZnI24-ClC6H45520
50a + 50bSZnI24-MeOC6H45019
51a + 51bSZnI22,6-Me2C6H34415
1 Isolated yields.
Table 3. Diels–Alder reactions of methylideneimidazolones with 2,3-dimethylbutadiene.
Table 3. Diels–Alder reactions of methylideneimidazolones with 2,3-dimethylbutadiene.
ProductXLAArRYield 1, %
ZnI2 0
ZnI2 92
1 isolated yields.
Table 4. Diels–Alder reactions of methylideneimidazolones with isoprene.
Table 4. Diels–Alder reactions of methylideneimidazolones with isoprene.
ProductXLAArYield a + b 1, %
63a + 63bOAlCl3Ph70
64a + 64bSZnI2Ph82
65a + 65bSZnI22-ClC6H461
66a + 66bSZnI23-MeC6H464
67a + 67bSZnI23-MeOC6H476
68a + 68bSZnI24-ClC6H468
69a + 69bSZnI24-MeOC6H471
1 isolated yields.
Table 5. Cytotoxicity effects of the compounds calculated as IC50abs (IC50abs—concentration of compounds, which caused 50% death of cells) for 65-h-treatment of the cell lines of different etiology using the standard MTT assay.
Table 5. Cytotoxicity effects of the compounds calculated as IC50abs (IC50abs—concentration of compounds, which caused 50% death of cells) for 65-h-treatment of the cell lines of different etiology using the standard MTT assay.
Cell line
34a83.0 ± 3.7>>100>>100>>100100µM
35b87.4 ± 9.9>>100>>100>>100100µM
37b115 ± 17 **>>100>>100>>100100µM
38a111 ± 9 **>>100>>100>>100100µM
38b112 ± 8 **>>100>>100>>100100µM
39b120 ± 13 **>>100>>100>>100100µM
40a56.4 ± 2.5>>100>>100>>100100µM
41a69.1 ± 4.8>>100>>100>>100100µM
45a21.3 ± 2.3141 ± 28 **>>10085.2 ± 10.1100µM
48a98.0 ± 9.7>>100>>100>>100100µM
48b108 ± 12 **>>100>>100>>100100µM
49a6.7 ± 0.737.8 ± 6.130.9 ± 3.416.9 ± 1.9100µM
50a41.9 ± 3.8>>100>>100>>100100µM
50b109 ± 12 **>>100>>100>>100100µM
51b87.5 ± 7.2104 ± 11 **>>100140 ± 21 **100µM
5429.6 ± 3122 ± 29 **>>10049.5 ± 5100µM
55 *~5~100>>100~10100µM
5622.0 ± 8.0102 ± 17 **98.7 ± 17.6>>100100µM
5718.3 ± 1.750.6 ± 5.280.8 ± 13.735.4 ± 4.8100µM
5813.7 ± 2.359.0 ± 13.345.0 ± 7.523.2 ± 3.5100µM
594.4 ± 0.676.6 ± 1652.6 ± 7.826.2 ± 3.9100µM
6010.2 ± 1.398.3 ± 19.875.3 ± 1752.2 ± 5.7100µM
61 *~4~100N/A~10100µM
626.8 ± 0.311.1 ± 0.69.8 ± 0.47.9 ± 0.4100µM
66a + 66b44.6 ± 4.9146 ± 22 **110 ± 13 **144 ± 36 **100µM
7260.1 ± 3.4>>100>>100>>100100µM
7599.0 ± 11.1>>100>>100>>100100µM
7648.6 ± 2.486.4 ± 7.3132 ± 17 **135 ± 14 **100µM
Cmax—the highest concentration studied in the test.>>100—compound shows little or no cytotoxicity. IC50abs values could not be calculated. *—approximate IC50abs values are given, see IC50 values in Table 6. **—IC50abs value, calculated by data extrapolation, exceeds Cmax.N/A—not available.
Table 6. Antibacterial effects shown as MIC for E. coli BW25113 DTC and E. coli K-12. MIC values are given in µM.
Table 6. Antibacterial effects shown as MIC for E. coli BW25113 DTC and E. coli K-12. MIC values are given in µM.
E. coli DTC>1000>1000100010001000>1000>1000>10001000500500125
E. coli K12>1000>1000>1000>1000>1000>1000>1000>1000>1000>1000>1000>1000
>1000—compound is inactive in this test.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Shybanov, D.E.; Kukushkin, M.E.; Hrytseniuk, Y.S.; Grishin, Y.K.; Roznyatovsky, V.A.; Tafeenko, V.A.; Skvortsov, D.A.; Zyk, N.V.; Beloglazkina, E.K. [4+2]-Cycloaddition to 5-Methylidene-Hydantoins and 5-Methylidene-2-Thiohydantoins in the Synthesis of Spiro-2-Chalcogenimidazolones. Int. J. Mol. Sci. 2023, 24, 5037.

AMA Style

Shybanov DE, Kukushkin ME, Hrytseniuk YS, Grishin YK, Roznyatovsky VA, Tafeenko VA, Skvortsov DA, Zyk NV, Beloglazkina EK. [4+2]-Cycloaddition to 5-Methylidene-Hydantoins and 5-Methylidene-2-Thiohydantoins in the Synthesis of Spiro-2-Chalcogenimidazolones. International Journal of Molecular Sciences. 2023; 24(5):5037.

Chicago/Turabian Style

Shybanov, Dmitry E., Maxim E. Kukushkin, Yanislav S. Hrytseniuk, Yuri K. Grishin, Vitaly A. Roznyatovsky, Viktor A. Tafeenko, Dmitry A. Skvortsov, Nikolai V. Zyk, and Elena K. Beloglazkina. 2023. "[4+2]-Cycloaddition to 5-Methylidene-Hydantoins and 5-Methylidene-2-Thiohydantoins in the Synthesis of Spiro-2-Chalcogenimidazolones" International Journal of Molecular Sciences 24, no. 5: 5037.

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop