Diastereoselective Synthesis of Dispiro[Imidazothiazolotriazine-Pyrrolidin-Oxindoles] and Their Isomerization Pathways in Basic Medium

Highly diastereoselective methods for the synthesis of two series of regioisomeric polynuclear dispyroheterocyclic compounds with five or six chiral centers, comprising moieties of pyrrolidinyloxindole and imidazo[4,5-e]thiazolo[3,2-b]-1,2,4-triazine of linear structure or imidazo[4,5-e]thiazolo[2,3-c]-1,2,4-triazine of angular structure, have been developed on the basis of a [3+2] cycloaddition of azomethine ylides to functionalized imidazothiazolotriazines. Depending on the structure of the ethylenic component, cycloaddition proceeds as an anti-exo process for linear derivatives, while cycloaddition to angular ones resulted in a syn-endo diastereomer. Novel pathways of isomerization for the synthesized anti-exo products upon treatment with sodium alkoxides have been found, which resulted in two more series of diastereomeric dispiro[imidazothiazolotriazine-pyrrolidin-oxindoles] inaccessible with the direct cycloaddition reaction. For the first series, the inversion of the configuration of one stereocenter, i.e., C-4′ atom of the pyrrolidine cycle, (epimerization) was established. For the second one, configuration of the obtained diastereomer formally corresponded to the syn-endo approach of the azomethine ylide in the case of cycloaddition to the ethylenic component.


Introduction
Recent trends of organic and medicinal chemistry consist in constructing rigidly oriented spiroheterocyclic structures with high solubility and bioavailability as well as the ability to interact effectively with various biological targets [1].Special attention is paid to the oxindoles spiro-linked with the pyrrolidine cycle, which have become popular since the discovery of valuable pharmacological properties of a number of natural alkaloids, such as spirotriprostatin B [2], horsfilin [3] and mitraphyllin [4] at the end of the XX century.The antitumor activity of synthetic spiropyrrolidineoxindoles is actively investigated [5][6][7][8][9][10][11] (Figure 1).For example, polymerization inhibitors of actin [8] and tubulin [9], as well as MDM2-p53 protein-protein interaction [10-14] were obtained.
At the same time, spiropyrrolidineoxindole 1 prepared using cycloaddition appeared to be a less active MDM2-p53 protein-protein interaction inhibitor than the MI-888 pre-pared via base-induced isomerization of compound 1 (Scheme 1A) [12].Therefore, development of the methods for the isomerization of spiropyrrolidineoxindole prepared via a cycloaddition reaction are relevant.
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 2 of 24 peared to be a less active MDM2-p53 protein-protein interaction inhibitor than the MI-888 prepared via base-induced isomerization of compound 1 (Scheme 1A) [12].Therefore, development of the methods for the isomerization of spiropyrrolidineoxindole prepared via a cycloaddition reaction are relevant.Scheme 1. Background and purpose of this work.(A).Zhao Y. et al. [12]; (B).Our previous work [28]; (C).This work.
Earlier, we have discovered the skeletal rearrangement of dispiropyrrolidineoxindoles 2 into isomers 3 upon treatment with KOH (see Scheme 1B) [28].Herein, we carried out the cycloaddition of ylidene derivatives of imidazothiazolotriazine and azomethine ylides generated from amino acids and isatins and studied various isomerization pathways of synthesized cycloaddition products 4 in basic medium (see Scheme 1C).
Earlier, we have discovered the skeletal rearrangement of dispiropyrrolidineoxindoles 2 into isomers 3 upon treatment with KOH (see Scheme 1B) [28].Herein, we carried out the cycloaddition of ylidene derivatives of imidazothiazolotriazine and azomethine ylides generated from amino acids and isatins and studied various isomerization pathways of synthesized cycloaddition products 4 in basic medium (see Scheme 1C).
Earlier, we have discovered the skeletal rearrangement of dispiropyrrolidineoxindoles 2 into isomers 3 upon treatment with KOH (see Scheme 1B) [28].Herein, we carried out the cycloaddition of ylidene derivatives of imidazothiazolotriazine and azomethine ylides generated from amino acids and isatins and studied various isomerization pathways of synthesized cycloaddition products 4 in basic medium (see Scheme 1C).

Results and Discussion
Target dispiro[imidazothiazolotriazine-6,3 -pyrrolidin-2 ,3 -oxindoles] 4a-k were prepared according to earlier elaborated procedure [28,29]  The highest yields of cycloadducts 4a, 4b, 4f and 4g (91, 84, 93 and 95%, respectively) were observed when using N-methyl-and N-ethylglycines 7a,b as amino acid and unsubstituted isatin 8a, while application of more sterically hindered N-isopropylglycine 7c and substituted isatins 8b,c led to some decrease in the yields of corresponding dispyrocyclic structures 4c-e and 4h-j to 74-82%.The introduction of the N-methyl norvaline derivative 7d into the reaction, which has an additional substituent at the α-carbon atom in comparison with sarcosine, was accompanied by a significant decrease in the yield of the corresponding product 4k to 31%.The relative configuration of compound rel-(2′R,3aS,4′S,6R,9aR)-4f was unambiguously assigned via single crystal X-ray diffraction and corresponded to the configuration of previously obtained similar compounds [28,29].The configuration of all other products was assigned by analogy.The configuration of the pyrrolidine cycle C-5′ atom in the structure 4k is adopted by analogy with the examples known in the literature [31,32].The absence of signals from other isomers in the 1 H NMR spectra of the evaporated reaction mixtures indicates a high selectivity of the reaction and the formation of single regioisomer and diastereomer 4.
At the same time, the formation of the pyrrolidine cycle during the process of [3+2] cycloaddition of azomethine ylides to the double bond of ethylenic components is ac- The highest yields of cycloadducts 4a, 4b, 4f and 4g (91, 84, 93 and 95%, respectively) were observed when using N-methyl-and N-ethylglycines 7a,b as amino acid and unsubstituted isatin 8a, while application of more sterically hindered N-isopropylglycine 7c and substituted isatins 8b,c led to some decrease in the yields of corresponding dispyrocyclic structures 4c-e and 4h-j to 74-82%.The introduction of the N-methyl norvaline derivative 7d into the reaction, which has an additional substituent at the α-carbon atom in comparison with sarcosine, was accompanied by a significant decrease in the yield of the corresponding product 4k to 31%.The relative configuration of compound rel-(2 R,3aS,4 S,6R,9aR)-4f was unambiguously assigned via single crystal X-ray diffraction and corresponded to the configuration of previously obtained similar compounds [28,29].The configuration of all other products was assigned by analogy.The configuration of the pyrrolidine cycle C-5 atom in the structure 4k is adopted by analogy with the examples known in the literature [31,32].The absence of signals from other isomers in the 1 H NMR spectra of the evaporated reaction mixtures indicates a high selectivity of the reaction and the formation of single regioisomer and diastereomer 4.
At the same time, the formation of the pyrrolidine cycle during the process of [3+2] cycloaddition of azomethine ylides to the double bond of ethylenic components is accompanied with the generation of three new stereocenters.Together with the stereocenters available in the initial compounds (C-3a and C-9a), the number of chiral centers can theo-retically determine the formation of 16 diastereomers (16 enantiomeric pairs).The reasons for decrease in the number of possible diastereomers can be the following: (i) the use of compounds with a Z-configuration of double bonds and rigid cis-junction at the C-3a-C-9a bond as ethylenic components; (ii) the synchronicity of the cyclocondensation process; and (iii) nonequivalence in modes of approach of the azomethine ylide to ethylenic component.
Azomethine ylide generated in situ via condensation of isatin and amino acid (for example, N-methylglycine) followed by thermic decarboxylation of the intermediate lactone (Scheme 3).companied with the generation of three new stereocenters.Together with the stereocenters available in the initial compounds (C-3a and C-9a), the number of chiral centers can theoretically determine the formation of 16 diastereomers (16 enantiomeric pairs).The reasons for decrease in the number of possible diastereomers can be the following: (i) the use of compounds with a Z-configuration of double bonds and rigid cis-junction at the C-3a-C-9a bond as ethylenic components; (ii) the synchronicity of the cyclocondensation process; and (iii) nonequivalence in modes of approach of the azomethine ylide to ethylenic component.Azomethine ylide generated in situ via condensation of isatin and amino acid (for example, N-methylglycine) followed by thermic decarboxylation of the intermediate lactone (Scheme 3).Meanwhile, the nitrogen containing three atom components involved in the [3+2] cycloaddition reaction can be attributed to the dipolar, zwitterionic, pseudo(mono)-or pseudodiradical type [33][34][35][36][37][38].However, recent experimental and theoretical data obtained for azomethine ylides indicate their pseudoradical nature [33][34][35].
Theoretically possible mechanisms of the cycloaddition reaction could include both one-step and stepwise pathways for the formation of the pyrrolidine ring (Scheme 4).During the stepwise reaction of azomethine ylides, which have both a dipole (Path I) and a pseudodiradical (Path II) character, free rotation around the bond brought from the ethylenic component to the target pyrrolidine ring is unlocked within the resulting intermediates.Therefore, a stepwise process should lead to the limitation of stereoselectivity and the formation of two stereoisomeric products 4a and 5a.The absence of signals from other isomers (including epimeric structures 5) in the 1 H NMR spectra of the evaporated reaction mixtures evidence of one-step process (for example, Path III or IV).Recent theoretical investigations (using the topological analysis of the electron localization function (ELF) at the B3LYP/6-31G(d) level of theory) of the cycloaddition reaction of symmetric azomethine ylide prove synchronous concerted transition state structure, and the process may be electronically classified as pseudo-diradical [2n + 2π] process (Path IV) [34].However, the presence of electron-withdrawing carbonyl C=O group in the isatin derivative can modify its reactivity.By taking into account the presence of conjugated C=O group in an ethilenic component, it can be assumed that the cycloaddition take place through a polar non-concerted two-stage one-step mechanism associated with the nucleophilic attack of the least substituted carbon of azomethine ylide on the β-conjugated position of the ethylenic component 9 [27,[35][36][37].Meanwhile, the nitrogen containing three atom components involved in the [3+2] cycloaddition reaction can be attributed to the dipolar, zwitterionic, pseudo(mono)-or pseudodiradical type [33][34][35][36][37][38].However, recent experimental and theoretical data obtained for azomethine ylides indicate their pseudoradical nature [33][34][35].
Theoretically possible mechanisms of the cycloaddition reaction could include both one-step and stepwise pathways for the formation of the pyrrolidine ring (Scheme 4).During the stepwise reaction of azomethine ylides, which have both a dipole (Path I) and a pseudodiradical (Path II) character, free rotation around the bond brought from the ethylenic component to the target pyrrolidine ring is unlocked within the resulting intermediates.Therefore, a stepwise process should lead to the limitation of stereoselectivity and the formation of two stereoisomeric products 4a and 5a.The absence of signals from other isomers (including epimeric structures 5) in the 1 H NMR spectra of the evaporated reaction mixtures evidence of one-step process (for example, Path III or IV).Recent theoretical investigations (using the topological analysis of the electron localization function (ELF) at the B3LYP/6-31G(d) level of theory) of the cycloaddition reaction of symmetric azomethine ylide prove synchronous concerted transition state structure, and the process may be electronically classified as pseudo-diradical [2n + 2π] process (Path IV) [34].However, the presence of electron-withdrawing carbonyl C=O group in the isatin derivative can modify its reactivity.By taking into account the presence of conjugated C=O group in an ethilenic component, it can be assumed that the cycloaddition take place through a polar non-concerted two-stage one-step mechanism associated with the nucleophilic attack of the least substituted carbon of azomethine ylide on the β-conjugated position of the ethylenic component 9 [27,[35][36][37].
Additionally, the complicated structures, both three atom component and ethylenic component, propose nonequivalence in modes of approach of the azomethine ylide (Scheme 5).The addition of sterically bulky azomethine ylides occurs from the less sterically loaded anti-side relative to the imidazolidine cycle and proceeds via an exo-transition state, where the carbonyl groups of the oxindole fragment and the thiazolidinone ring become relative to the pyrrolidine cycle on different sides.
One example shows that the introduction of chiral (R)-2-[(1-phenylethyl)amino]acetic acid 7e into the reaction leads to the formation of a mixture of two diastereomers 4l and 4m in approximately equal amounts instead of racemate (Scheme 6).With an increase in the bulk of the substituent in the reagent, the use of a two-fold excess of amino acid and isatin, as well as a longer reaction time (36 h) are required, and the total yield of the mixture of products 4l and 4m decreased to 41%.It is shown that diastereomers 4l and 4m have different retention times in the chromatographic column and can be isolated individually (see Supplementary Materials).
Scheme 5. Modes of approach of the azomethine ylide.
One example shows that the introduction of chiral (R)-2-[(1-phenylethyl)amino]acetic acid 7e into the reaction leads to the formation of a mixture of two diastereomers 4l and 4m in approximately equal amounts instead of racemate (Scheme 6).With an increase in the bulk of the substituent in the reagent, the use of a two-fold excess of amino acid and isatin, as well as a longer reaction time (36 h) are required, and the total yield of the mixture of products 4l and 4m decreased to 41%.It Scheme 5. Modes of approach of the azomethine ylide.
(R)-2-[(1-phenylethyl)amino]acetic acid 7e into the reaction leads to the formation of a mixture of two diastereomers 4l and 4m in approximately equal amounts instead of racemate (Scheme 6).With an increase in the bulk of the substituent in the reagent, the use of a two-fold excess of amino acid and isatin, as well as a longer reaction time (36 h) are required, and the total yield of the mixture of products 4l and 4m decreased to 41%.It is shown that diastereomers 4l and 4m have different retention times in the chromatographic column and can be isolated individually (see Supplementary Materials).It was noted above that the treatment of structurally related dispiro[imidazothiazolotriazine-pyrrolidin-oxindoles] 2, having an aryl substituent in the pyrrolidine cycle, with KOH is accompanied by hydrolysis of the amide bond and skeletal rearrangement of the thiazolotriazine system, which resulted in regioisomeric products 3 [28].In turn, boiling esters 4a-j in alcohols in the presence of sodium alkoxides mainly led to the formation of a mixture of two new diastereomers 5 and 6 in different ratios (Scheme 7). 1 H NMR monitoring of the reaction showed that the complete conversion of the initial compounds 4 into products 5 and 6 was achieved with the action of 0.25 equivalents of sodium alkoxide for 4 h.It was noted above that the treatment of structurally related dispiro[imidazothiazolotriazine-pyrrolidin-oxindoles] 2, having an aryl substituent in the pyrrolidine cycle, with KOH is accompanied by hydrolysis of the amide bond and skeletal rearrangement of the thiazolotriazine system, which resulted in regioisomeric products 3 [28].In turn, boiling esters 4a-j in alcohols in the presence of sodium alkoxides mainly led to the formation of a mixture of two new diastereomers 5 and 6 in different ratios (Scheme 7). 1 H NMR monitoring of the reaction showed that the complete conversion of the initial compounds 4 into products 5 and 6 was achieved with the action of 0.25 equivalents of sodium alkoxide for 4 h.Herein, an increase in the reaction time had little effect on the yields and the ratio of the products formed, while an increase in the amount of base can accelerate the reaction and slightly change the ratio of products, reducing, nevertheless, their total yield.In some cases, compounds 5c,d,h were isolated from the reaction mixture without impurities of other diastereomers.The signals of their isomers 6c,d,h were observed in the 1 H NMR spectra of evaporated reaction mixtures in trace amounts and were not isolated in Herein, an increase in the reaction time had little effect on the yields and the ratio of the products formed, while an increase in the amount of base can accelerate the reaction and slightly change the ratio of products, reducing, nevertheless, their total yield.In some cases, compounds 5c,d,h were isolated from the reaction mixture without impurities of other diastereomers.The signals of their isomers 6c,d,h were observed in the 1 H NMR spectra of evaporated reaction mixtures in trace amounts and were not isolated in these cases.Each of the isomers 5a-j and 6a,b,e-g,i,j was isolated individually via fractional crystallization from the reaction mixtures or MeCN.The relative configurations of the chiral centers of diastereomers rel-(2 R,3aS,4 R,6R,9aR)-5 and rel-(2 R,3aS,4 R,6S,9aR)-6 were unambiguously determined using single crystal X-ray diffraction for compounds 5b and 6a.For compounds 5, the inversion of the configuration of one stereocenter, i.e., C-4 atom of the pyrrolidine cycle (epimerization), was established in comparison with compounds 4, which was previously described for related spiropyrrolidineindoles on a single example [39].Configuration of diastereomers 6 indicated the inversion of two stereocenters compared to starting compounds 4 (C-3 (C-6) and C-4 atoms of the pyrrolidine cycle) and formally corresponded to the syn-endo approach of the azomethine ylide in the case of cycloaddition to dipolarophile 9 (Scheme 5).
We assumed that the presence of an electron-withdrawing ester group at 4 position of compounds 4 makes hydrogen atom at the corresponding α-carbon atom acidic.Therefore, sodium alkoxide causes the primary deprotonation of structures 4 and the formation of carbanion A (Scheme 8), which transforms into a more stable anion B due to the elimination of the thiolate anion.As a result of the opening of the thiazolidine cycle, it is possible to freely rotate the spiropyrrolidineoxindole fragment of the molecule around a single C-C bond, the further addition of the thiolate anion to a double bond of the Michael acceptor, and finally, the spiro node formation in new syn-endo-diastereomers 6 inaccessible via the direct cycloaddition reaction.The processes occurring in this case do not affect other asymmetric centers present in the molecule (C-3a, C-9a and C-2 ); therefore, the corresponding carbon atoms in isomeric structures 4, 5 and 6 have the same configuration.The anti-exo epimer 5 can be form from carbanion A and alcohol.The structures of the prepared compounds were also proven using spectral methods.In the 1 H NMR spectra, a characteristic signal that allows the resulting compound to be assigned to one of the diastereomeric products 4, 5, or 6 is the signal of the 4′-CH proton of the pyrrolidine ring, which experiences different deshielding effects from neighboring carbonyl groups.Due to its closer spatial arrangement to the atom 4′-CH, deshielding effect of the carbonyl group of the oxindole fragment is higher than that of the carbonyl group of the thiazolidinone ring.As a result, the corresponding signal for the epimeric products 5 downfield shifted (4.40-4.45ppm) compared to its location in the spectra of starting structures 4 (4.03-4.10ppm) (Figure 2).In syn-endo diastereomers 6, the carbonyl groups of the oxindole fragment and the thiazolidine ring are on the same side relative to the pyrrolidine ring, which leads to maximum deshielding of the 4′-CH hydrogen atom by both groups and a strong downfield shift of its signal to the region of 5.02-5.07ppm.The structures of the prepared compounds were also proven using spectral methods.In the 1 H NMR spectra, a characteristic signal that allows the resulting compound to be assigned to one of the diastereomeric products 4, 5, or 6 is the signal of the 4 -CH proton of the pyrrolidine ring, which experiences different deshielding effects from neighboring carbonyl groups.Due to its closer spatial arrangement to the atom 4 -CH, deshielding effect of the carbonyl group of the oxindole fragment is higher than that of the carbonyl group of the thiazolidinone ring.As a result, the corresponding signal for the epimeric products 5 downfield shifted (4.40-4.45ppm) compared to its location in the spectra of starting structures 4 (4.03-4.10ppm) (Figure 2).In syn-endo diastereomers 6, the carbonyl groups of the oxindole fragment and the thiazolidine ring are on the same side relative to the pyrrolidine ring, which leads to maximum deshielding of the 4 -CH hydrogen atom by both groups and a strong downfield shift of its signal to the region of 5.02-5.07ppm.
the carbonyl group of the thiazolidinone ring.As a result, the corresponding signal for the epimeric products 5 downfield shifted (4.40-4.45ppm) compared to its location in the spectra of starting structures 4 (4.03-4.10ppm) (Figure 2).In syn-endo diastereomers 6, the carbonyl groups of the oxindole fragment and the thiazolidine ring are on the same side relative to the pyrrolidine ring, which leads to maximum deshielding of the 4′-CH hydrogen atom by both groups and a strong downfield shift of its signal to the region of 5.02-5.07ppm.To obtain skeletal isomers of compounds 4, three-component [3+2] cycloaddition reaction of azomethine ylides with functionalized imidazothiazolotriazines 10a,b [30] of an angular structure was also carried out by boiling the starting compounds in acetonitrile.The previously unknown regioisomeric dispirocyclic structures 11a-f were synthesized in 41-73% yields (Scheme 9).The relative configuration of the structure rel-(2 S,3aR,4 S,7R,9aS)-11e was determined via X-ray diffraction analysis and appeared to be corresponding to a syn-endo diastereomer.Chemical shifts (4.72-4.82ppm) of the signal for the 4 -CH hydrogen atom of the pyrrolidine ring in the 1 H NMR spectra of compounds 11a-f allow to assign all compounds to the diastereomers of the same structure.To obtain skeletal isomers of compounds 4, three-component [3+2] cycloaddition reaction of azomethine ylides with functionalized imidazothiazolotriazines 10a,b [30] of an angular structure was also carried out by boiling the starting compounds in acetonitrile.The previously unknown regioisomeric dispirocyclic structures 11a-f were synthesized in 41-73% yields (Scheme 9).The relative configuration of the structure rel-(2′S,3aR,4′S,7R,9aS)-11e was determined via X-ray diffraction analysis and appeared to be corresponding to a syn-endo diastereomer.Chemical shifts (4.72-4.82ppm) of the signal for the 4′-CH hydrogen atom of the pyrrolidine ring in the 1 H NMR spectra of compounds 11a-f allow to assign all compounds to the diastereomers of the same structure.

General Information
All standard reagents were purchased from Aldrich or Acros Organics and used without further purification.
Melting points were determined on a Stuart SMP20 apparatus (Stuart (Bibby Scientific), Stone, UK).

General Information
All standard reagents were purchased from Aldrich or Acros Organics and used without further purification.
Melting points were determined on a Stuart SMP20 apparatus (Stuart (Bibby Scientific), Stone, UK).
The starting dipolarophiles 9a,b and 10a,b were prepared according to a procedure described in the literature [30].Amino acid 7e was prepared according to a procedure mentioned in the literature [40].

General Procedure for the Synthesis of Compounds 4a-m
A mixture of corresponding compound 9a,b (1 mmol), aminoacetic acid 7a-d (1.5 mmol) and isatin 8a-c (1.5 mmol) in MeCN (20 mL) was refluxed with stirring for 8 h (24 h for 4e,j).After cooling, the precipitate of compounds 4a-k was filtered off, washed with methanol and dried at 50 • C.
To obtain the target compounds as mixtures of diastereomers 5 and 6, the solvent was evaporated under reduced pressure, and the dry residue was triturated with a small amount of MeCN.The resulting suspension was filtered, and the filter cake was washed with MeCN and dried at 50 • C.
To obtain the individual diastereomers 5 and 6, the resulting precipitate was dissolved in boiling MeCN and the resulting solution was left in an open flask to effect slow crystallization of the precipitate.As the volume of the solution decreased, the crystallizing precipitates were filtered, washed with MeCN, and dried.The filtrate was left in an open flask for further crystallization.This procedure was repeated at least 3-4 times.If necessary, the product contaminated with another isomer could be purified via recrystallization from MeCN.

Scheme 3 .
Scheme 3. The proposed formation mechanism and structure of azomethyne ylide.

Scheme 3 .
Scheme 3. The proposed formation mechanism and structure of azomethyne ylide.

24 Scheme 7 .
Scheme 7. Isomerization of dispirocompounds 4 into diastereomers 5 and 6. a The ratio of compounds 5 and 6 was determined from the 1 H NMR spectrum of the mixture.b Isolated yield.

Scheme 7 .
Scheme 7. Isomerization of dispirocompounds 4 into diastereomers 5 and 6. a The ratio of compounds 5 and 6 was determined from the 1 H NMR spectrum of the mixture.b Isolated yield.

Scheme 8 .
Scheme 8. Plausible mechanism of the isomerization of compounds 4 into isomers 6.

Scheme 8 .
Scheme 8. Plausible mechanism of the isomerization of compounds 4 into isomers 6.