Different Behavior of 2-Substituted 3-Nitro-2H-chromenes in the Reaction with Stabilized Azomethine Ylides Generated from α-Iminoesters

The AgOAc-catalysed reaction of 3-nitro-2-phenyl-2H-chromenes with stabilized azomethine ylides generated from the imines based on methyl glycinate and arylaldehydes leads to a mixture of endo and endo’ isomers of the corresponding chromeno[3,4-c]pyrrolidines in a ratio of 2.0–2.3:1 in 85–93% total yields as a result of a Michael addition/Mannich reaction sequence. In a similar reaction involving 2-trifluoromethyl-3-nitro-2H-chromenes, only endo chromeno[3,4-c]pyrrolidines are formed in 85–94% yields. 3-Nitro-2-(trichloromethyl)-2H-chromenes under the same conditions react with these azomethine ylides to give the corresponding Michael adducts as individual anti-isomers with the cis,trans-configuration of the chromane ring in 40–67% yields. Some 4-CF3-substituted chromano[3,4-c]pyrrolidines exhibited high cytotoxic activity against HeLa human cervical carcinoma cells.


Introduction
The chromeno [3,4-c]pyrrolidine scaffold is the main structural element of a number of bioactive molecules with important pharmaceutical properties. For example, the transchromeno [3,4-c]pyrrolidine derivative S33138 is a dopamine D 3 receptor antagonist and a potential drug for the treatment of CNS disorders such as schizophrenia and Parkinson's disease [1], while its cis-derivative, fiduxosin, is an α 1 -adrenoceptor antagonist and a promising drug for the treatment of benign prostatic hyperplasia [2] (Figure 1). It was recently reported that spirooxindole derivatives of chromenopyrroli(zi)dines 1 and 2 show high antitumor activity against human cervical carcinoma and human rhabdomyosarcoma cancer cells along with low cytotoxicity against normal human dermal fibroblast [3][4][5]. Fused prolinates 3 have been successfully tested as antimycobacterial agents against the M. tuberculosis H37Rv strain [6]. Therefore, the development of regio-and stereoselective methods for the synthesis of novel ∆ 3 -fused chromenopyrrolidine derivatives is an urgent task.
A convenient one-pot atom-economical method for the synthesis of functionalized pyrrolidines is based on the reaction of electron-deficient alkenes with stabilized azomethine ylides generated in situ from Schiff bases [7][8][9][10][11][12]. Due to the high regio-and stereoselectivity of reactions involving these ylides, this approach is an indispensable tool in the synthesis of complex heterocyclic molecules containing up to four new chiral centers with the required arrangement and spatial orientation of substituents from relatively simple and commercially available precursors. When amino acid esters are used as the amino component of the Schiff base, prolinates are formed as reaction products. These reactions are usually carried out in the presence of catalytic amounts of a Brønsted base and transition metal or lithium salt as a Lewis acid. Lewis acids increase the stereoselectivity of the process by stabilizing the W-conformation of the ylide [13,14]. Being readily available and highly reactive substrates, conjugate nitroalkenes are widely used as dipolarophiles for the stereoselective synthesis of nitroprolinates [15][16][17][18]. In the reactions of trans-nitrostyrene with iminoesters, four diastereomers can be formed, classified as endo, exo, endo' and exo' isomers (Scheme 1).
Due to the presence of a β-nitrostyrene moiety, 3-nitro-2H-chromenes can also react with azomethine ylides to form chromenopyrroli(zi)dine derivatives [28][29][30]. There are only four reports on the reactions of nitrochromenes with stabilized azomethine ylides Being readily available and highly reactive substrates, conjugate nitroalkenes are widely used as dipolarophiles for the stereoselective synthesis of nitroprolinates [15][16][17][18]. In the reactions of trans-nitrostyrene with iminoesters, four diastereomers can be formed, classified as endo, exo, endo' and exo' isomers (Scheme 1). the amino component of the Schiff base, prolinates are formed as reaction products These reactions are usually carried out in the presence of catalytic amounts of a Brønsted base and transition metal or lithium salt as a Lewis acid. Lewis acids increase the stere oselectivity of the process by stabilizing the W-conformation of the ylide [13,14]. Being readily available and highly reactive substrates, conjugate nitroalkenes are widely used as dipolarophiles for the stereoselective synthesis of nitroprolinates [15][16][17][18] In the reactions of trans-nitrostyrene with iminoesters, four diastereomers can be formed classified as endo, exo, endo' and exo' isomers (Scheme 1).
Our group's science research is focused on the development of methods for Δ 3 -carbo-and heteroannulation of 2-trifluoromethyl-substituted 3-nitro-2H-chromenes using available ambiphilic reagents [3][4][5]34,35]. The introduction of the electron-withdrawing CF3-group in position 2 of 3-nitro-2H-chromene not only activates the double bond but also increases the stereoselectivity of their reactions with nucleophiles and ambiphiles. Furthermore, the replacement of the methyl group by the trifluoromethyl one in the bioactive molecule can lead to an increase in pharmacological properties due to enhanced lipophilicity and metabolic stability [36][37][38].

Results and Discussion
To obtain 4-phenyl-substituted chromeno [3,4-c]pyrrolidines 8, we used the Nyerges group's method [31], but the amount of AgOAc was reduced from 150 to 10 mol%. The reaction between chromene 4a and imine ester 5b (Ar = 4-MeOC6H4) in the presence of Et3N and AgOAc in toluene at room temperature for 5 h led to the mixture of endo-8b and endo'-8b isomers in a ratio of 2.1:1 in 60% total yield (Scheme 3, Table 1, entry 1). It motivated us to optimize the conditions for this reaction. Replacing toluene with acetonitrile or tetrahydrofuran led to an increase in the yield of the target product to 91-92% (Table 1, entries 2-3). The best yield of adduct 8b (93%) was achieved when the reaction was carried out in dichloromethane (DCM) ( Table 1, entry 4). The use of CuI as a catalyst proved Scheme 2. Reactions of 2-aryl-substituted 3-nitro-2H-chromenes 4 with azomethine ylides generated from amino acid esters and arylaldehydes [6,31].
Our group's science research is focused on the development of methods for ∆ 3 -carboand heteroannulation of 2-trifluoromethyl-substituted 3-nitro-2H-chromenes using available ambiphilic reagents [3][4][5]34,35]. The introduction of the electron-withdrawing CF 3group in position 2 of 3-nitro-2H-chromene not only activates the double bond but also increases the stereoselectivity of their reactions with nucleophiles and ambiphiles. Furthermore, the replacement of the methyl group by the trifluoromethyl one in the bioactive molecule can lead to an increase in pharmacological properties due to enhanced lipophilicity and metabolic stability [36][37][38].

Results and Discussion
To obtain 4-phenyl-substituted chromeno [3,4-c]pyrrolidines 8, we used the Nyerges group's method [31], but the amount of AgOAc was reduced from 150 to 10 mol%. The reaction between chromene 4a and imine ester 5b (Ar = 4-MeOC 6 H 4 ) in the presence of Et 3 N and AgOAc in toluene at room temperature for 5 h led to the mixture of endo-8b and endo'-8b isomers in a ratio of 2.1:1 in 60% total yield (Scheme 3, Table 1, entry 1). It motivated us to optimize the conditions for this reaction. Replacing toluene with acetonitrile or tetrahydrofuran led to an increase in the yield of the target product to 91-92% (Table 1, entries 2-3). The best yield of adduct 8b (93%) was achieved when the reaction was carried out in dichloromethane (DCM) ( to be less efficient (Table 1, entries [5][6][7][8]. Regardless of the nature of the catalyst a vent, the ratio of stereoisomers remained unchanged.  Under optimized conditions, chromeno [3,4-c]pyrrolidines 8a−g were obtai mixtures of endo and endo' isomers in a 2.0-2.3:1 ratio with 85-93% total yields (Sch Table 2). The donor-acceptor properties of substituents in chromene 4 and in t fragment of the α-iminoester 5 had no significant effect on the yields of product the diastereoselectivity of the reaction. Individual isomers endo-8a−g and endo'-8 54-61% and 20-27% yields, respectively, were prepared after the purification o products by column chromatography.  Under optimized conditions, chromeno [3,4-c]pyrrolidines 8a−g were obtained as mixtures of endo and endo' isomers in a 2.0-2.3:1 ratio with 85-93% total yields (Scheme 4, Table 2). The donor-acceptor properties of substituents in chromene 4 and in the aryl fragment of the α-iminoester 5 had no significant effect on the yields of products 8 and the diastereoselectivity of the reaction. Individual isomers endo-8a−g and endo'-8a−g in 54-61% and 20-27% yields, respectively, were prepared after the purification of crude products by column chromatography.  Under optimized conditions, chromeno [3,4-c]pyrrolidines 8a−g were obtained as mixtures of endo and endo' isomers in a 2.0-2.3:1 ratio with 85-93% total yields (Scheme 4, Table 2). The donor-acceptor properties of substituents in chromene 4 and in the aryl fragment of the α-iminoester 5 had no significant effect on the yields of products 8 and the diastereoselectivity of the reaction. Individual isomers endo-8a−g and endo'-8a−g in 54-61% and 20-27% yields, respectively, were prepared after the purification of crude products by column chromatography. Next, we examined 2-trifluoromethyl-substituted chromenes 4d−i in the reaction with iminoesters 5a−e. It was found that under the same conditions, adducts endo-9a−j are formed in 85-94% yields as the only reaction products (Scheme 5, Table 3). Other isomers were not detected in the reaction mixtures by 19 F NMR spectroscopy. The product yields also did not depend on the nature of the substituents in the starting chromenes 4 and Schiff bases 5. Next, we examined 2-trifluoromethyl-substituted chromenes 4d−i in the reaction with iminoesters 5a−e. It was found that under the same conditions, adducts endo-9a−j are formed in 85-94% yields as the only reaction products (Scheme 5, Table 3). Other isomers were not detected in the reaction mixtures by 19 F NMR spectroscopy. The product yields also did not depend on the nature of the substituents in the starting chromenes 4 and Schiff bases 5.     3-Nitro-2-(trichloromethyl)-2H-chromenes 4j−n under the same conditions react with iminoesters 5a,b,e to give the corresponding Michael adducts 10a−g in 40-67% yields as individual anti isomers with the cis,trans configuration of the chromane ring (Scheme 6, Table 4). The lowest yield (40%) was observed in the reaction involving nitrochromene 4n with the MeO group in position 6. The isomers syn-10 were not observed in the reaction mixtures.  To understand the reason for such different stereoselectivity of the reactions involving 2-Ph-and 2-CF3-substituted chromenes 4, additional experiments have been performed. If the reaction between chromene 4d and iminoesters 5b was carried out at −20 °C, the mixture of the products endo-9b, endo'-9b and exo-9b was obtained in a ratio of 65:27:8, respectively (Scheme 7). When this process was carried out at room temperature without AgOAc, the content of the isomer endo'-9b increased to 38% (endo-9b:endo'-9b:exo-9b = 51:38:11), and the total yield decreased to 42%. If the crude mixture of compounds endo-8b endo'-8b was heated in toluene for 12 h, the content of the endo' isomer was reduced to 11%.  To understand the reason for such different stereoselectivity of the reactions involving 2-Ph-and 2-CF 3 -substituted chromenes 4, additional experiments have been performed. If the reaction between chromene 4d and iminoesters 5b was carried out at −20 • C, the mixture of the products endo-9b, endo'-9b and exo-9b was obtained in a ratio of 65:27:8, respectively (Scheme 7). When this process was carried out at room temperature without AgOAc, the content of the isomer endo'-9b increased to 38% (endo-9b:endo'-9b:exo-9b = 51:38:11), and the total yield decreased to 42%. If the crude mixture of compounds endo-8b endo'-8b was heated in toluene for 12 h, the content of the endo' isomer was reduced to 11%. trochromene 4n with the MeO group in position 6. The isomers syn-10 were not observe in the reaction mixtures.  To understand the reason for such different stereoselectivity of the reactions i volving 2-Ph-and 2-CF3-substituted chromenes 4, additional experiments have bee performed. If the reaction between chromene 4d and iminoesters 5b was carried out −20 °C, the mixture of the products endo-9b, endo'-9b and exo-9b was obtained in a ratio 65:27:8, respectively (Scheme 7). When this process was carried out at room temperatu without AgOAc, the content of the isomer endo'-9b increased to 38% (e do-9b:endo'-9b:exo-9b = 51:38:11), and the total yield decreased to 42%. If the crude mi ture of compounds endo-8b endo'-8b was heated in toluene for 12 h, the content of th endo' isomer was reduced to 11%. Thus, chromeno [3,4-c]pyrrolidines endo-8,9 and exo-9 are formed as a result of the Michael addition of W-shaped ylides to chromenes 4 followed by Mannich cyclization through intermediates A and C. (Scheme 8). A similar process involving S-shaped ylides leads to minor products endo'-8,9 through intermediate B. Apparently, S-ylides are formed in the presence of a slight excess of Et3N relative to AgOAc in the reaction mixture. If chromene 4 contains a trichloromethyl group at position 2, closing the pyrrolidine ring is impossible due to steric repulsions between the CCl3 and Ar substituents. In this case, the end products of the reaction are Michael adducts anti-10.
The addition of azomethine ylides to chromenes 4 occurs reversibly. The isomers endo'-8,9 and exo-9 are kinetic control products (KC) and convert into the thermodynamically more stable isomers endo-8,9 at higher temperatures (TC). In the case of more reactive 2-CF3-chromenes 4, the reverse reaction proceeds even at room temperature under the reaction conditions. Scheme 8. Proposed mechanism for the AgOAc-catalyzed reaction of chromenes 4 with iminoesters 5.
The structure and relative configuration of compounds 8-10 were confirmed by 1D and 2D NMR spectroscopy and X-ray single-crystal analysis. In the 1 H NMR spectra of chromeno [3,4-  The addition of azomethine ylides to chromenes 4 occurs reversibly. The isomers endo'-8,9 and exo-9 are kinetic control products (KC) and convert into the thermodynamically more stable isomers endo-8,9 at higher temperatures (TC). In the case of more reactive 2-CF 3 -chromenes 4, the reverse reaction proceeds even at room temperature under the reaction conditions.
The structure and relative configuration of compounds 8-10 were confirmed by 1D and 2D NMR spectroscopy and X-ray single-crystal analysis. In the 1 H NMR spectra of chromeno [3,4-     The relative configuration of endo chromeno[3,4-c]pyrrolidines 8 and 9 was unambiguously confirmed by single crystal X-ray diffraction analysis of compounds endo-8b and endo-9a (Figures 4 and 5). In both molecules, the H-1, H-3, and H-4 atoms are located on one side of the condensed tricyclic system, with the 4-Ph or 4-CF 3 -group occupying the axial position, while the nitro group is in the equatorial positions. The pyran and pyrrolidine rings have half-chair and twist conformations, respectively.  The relative configuration of endo chromeno[3,4-c]pyrrolidines 8 and 9 was unambiguously confirmed by single crystal X-ray diffraction analysis of compounds endo-8b and endo-9a (Figures 4 and 5). In both molecules, the H-1, H-3, and H-4 atoms are located on one side of the condensed tricyclic system, with the 4-Ph or 4-CF3-group occupying the axial position, while the nitro group is in the equatorial positions. The pyran and pyrrolidine rings have half-chair and twist conformations, respectively.        In the 1 H NMR spectra of 2-CCl 3 -substituted chromanes 10a−g, the signals of the H-2', H-3' and H-4' protons of the chromane ring in the range of 5.18-5.30, 6.25-6.29 and 4.08-4.14 ppm, respectively, with spin-spin coupling constants 3 J 2',3' ≈ 3 J 3',4' ≈ 1.0-1. 8 Hz, and a singlet of the vinylic proton at 7.87-7.99 ppm are observed (see Supplementary Materials for NMR spectra).
The structure and relative configuration of chromane 10c was unambiguously confirmed by single crystal X-ray diffraction analysis ( Figure 6). In this molecule, the nitro and CF 3 groups are located on the same side of the pyran ring, with the latter occupying the equatorial position. The iminoester fragment and the nitro group are arranged trans-diaxially. The pyran ring has a distorted half-chair conformation. 4.08-4.14 ppm, respectively, with spin-spin coupling constants 3 J2',3' ≈ 3 J3',4' ≈ 1.0-1. 8 Hz, and a singlet of the vinylic proton at 7.87-7.99 ppm are observed (see Supplementary Materials for NMR spectra). The structure and relative configuration of chromane 10c was unambiguously confirmed by single crystal X-ray diffraction analysis ( Figure 6). In this molecule, the nitro and CF3 groups are located on the same side of the pyran ring, with the latter occupying the equatorial position. The iminoester fragment and the nitro group are arranged trans-diaxially. The pyran ring has a distorted half-chair conformation. For representative chromeno [3,4-c]pyrrolidines endo-8b,e−g and endo-9e,f−j, their in vitro cytotoxic activity against HeLa cervical cancer and human dermal fibroblast cells (HDF) was evaluated. The known cytotoxin camptothecin [39] was used for comparison (Table 5). Of all the tested 2-Ph-substituted chromeno [3,4-c]pyrrolidines 8, only endo-8e bearing a benzo[d][1,3]dioxol-5-yl substituent at position 3 showed noticeable cytotoxic activity against HeLa cells. Compound endo-9j with a p-methoxyphenyl substituent at position 3 and the EtO group at position 6 is cytotoxic to HeLa and HDF cells. Compound endo-9b with a p-methoxyphenyl group at position 3 exhibited a high antitumor activity along with low toxicity and is a promising drug candidate.  For representative chromeno [3,4-c]pyrrolidines endo-8b,e−g and endo-9e,f−j, their in vitro cytotoxic activity against HeLa cervical cancer and human dermal fibroblast cells (HDF) was evaluated. The known cytotoxin camptothecin [39] was used for comparison (Table 5). Of all the tested 2-Ph-substituted chromeno [3,4-c]pyrrolidines 8, only endo-8e bearing a benzo[d][1,3]dioxol-5-yl substituent at position 3 showed noticeable cytotoxic activity against HeLa cells. Compound endo-9j with a p-methoxyphenyl substituent at position 3 and the EtO group at position 6 is cytotoxic to HeLa and HDF cells. Compound endo-9b with a p-methoxyphenyl group at position 3 exhibited a high antitumor activity along with low toxicity and is a promising drug candidate. In summary, it has been found that the addition of azomethine ylides derived from α-iminoesters to 2-Ph-and 2-CF 3 -substituted 3-nitro-2H-chromenes proceeds as a reversible Michael addition/Mannich reaction sequence. The reaction of these ylides with 2-CCl 3chromenes stops at the Michael addition step. The stereochemistry of chromenoprolinates can be controlled by varying the temperature and solvent. One-pot stereoselective approaches to the synthesis of 4-(trifluoromethyl)-substituted chromeno [3,4-c]pyrrolidines and methyl 2-(arylideneamino)-2-(2-(trichloromethyl)chroman-4-yl)acetates from available reagents have been developed. Some 4-CF 3 -substituted chromeno [3,4-c]pyrrolidine derivatives have shown high antitumor activity and are of undoubted interest in medicinal chemistry.

Assessment of In Vitro Cytotoxic Activity
The cells were seeded in 96-well microplates at a seeding density of 2 × 10 5 cells per mL and cultured for 24 h in DMEM medium with glutamine (1%) in the presence of 10% fetal bovine serum and gentamicin (50 mg/L) at 37 • C in a humidified atmosphere containing 5% CO 2 . Then the tested compounds were added to the wells in various concentrations (10 −7 M, 10 −6 M, 10 −5 M, 10 −4 M). Cells with compounds were incubated for 72 h, after which cell viability was assessed using the standard MTT test [43] based on the reduction of the yellow tetrazole salt by living cell mitochondrial dehydrogenases to formazan crystals, soluble in DMSO. Experiments were performed in triplicates with negative control (culture medium), positive control (camptothecin, 3 mM) and solvent control (DMSO). The results of the MTT test were evaluated by comparing the optical density of the formazan solution measured on a flatbed scanner Tecan Infinite M200 PRO (Tecan Austria GmbH, Austria) at a wavelength of 570 nm in the experimental and control wells and control wells and calculating the cytotoxicity index (IC). The cytotoxicity index was determined for each concentration of the studied substances by AAT Bioquest-calculator: https:// www.aatbio.com/tools/ic50-calculator (accessed on 15 November 2022). The parameters of the arithmetic mean value and the standard error were calculated. The differences in the average values according to the Mann-Whitney U test with p < 0.05 were considered reliable. For the statistical analysis, Microsoft Excel 2019 (Microsoft corp., Redmond, DC, USA) and Statistika 13.3 (Tibco, Palo Alto, CA, USA) were used.