Synthesis and Characterization of New Spirooxindoles Including Triazole and Benzimidazole Pharmacophores via [3+2] Cycloaddition Reaction: An MEDT Study of the Mechanism and Selectivity

A new series of spirooxindoles based on benzimidazole, triazole, and isatin moieties were synthesized via a [3+2] cycloaddition reaction protocol in one step. The single X-ray crystal structure of the intermediate triazole-benzimidazole 4 was solved. The new chemical structures of these spirooxindole molecules have been achieved for the first time. The final synthesized chemical architecture has differently characterized electronic effects. An MEDT study of the key 32CA reaction between in situ generated azomethine ylide (AY) and chalcones explained the low reaction rates and the total selectivities observed. The supernucleophilic character of AY and the strong electrophilicity of chalcones favor these reactions through a highly polar two-stage one-step mechanism in which bond formation at the β-conjugated carbon of the chalcones is more advanced. The present combined experimental and theoretical study reports the synthesis of new spirooxindoles with potential biological activities and fully characterizes the molecular mechanisms for their formation through the key 32CA reaction step.


Introduction
There are excellent moieties in spiro-heterocyclic compounds that have garnered the attention of researchers due to their numerous biological activities. Among these moieties, the 1,2,3-triazole moiety represents an important class of pharmacophore in medicinal chemistry with a wide range of biological activities, such as antimicrobial, anticancer, anti-inflammatory, and antiviral activity, among others. Because of their higher stability toward light, oxygen, moisture, and metabolism in the body, they are useful building blocks in chemistry and play an important role in pharmacological applications [1][2][3][4]. On the other hand, isatin derivatives have recently drawn considerable attention from researchers worldwide due to their wide applications as anti-HIV, anti-tubercular, sedative, hypnotic, and anticancer agents [5,6]. The important biological activities of both isatin and triazole derivatives as discussed above impelled us to take up the synthesis of these new combined heterocycles, which are likely to have augmented, diverse types of biological activity.
Recently, Barakat et al. reported the synthesis of new spirooxindoles, IX, with the triazole moiety and a ferrocene scaffold using the 32CA reaction approach, and their mechanism was studied via molecular electron density theory (MEDT) [11,12]. Another representative example is the spirooxindole with a benzimidazole scaffold (see SP1 in Figure 1), which has been extensively studied and has shown to be a potent anti-cancer agent [13]. In continuation of our research program about spirooxindoles [14][15][16], we report herein the synthesis of new spiro compounds containing benzimidazole and 1,2,3-triazole scaffolds as well as the theoretical study of the reaction mechanisms of these relevant 32CA reactions based on MEDT [17].
Recently, Barakat et al. reported the synthesis of new spirooxindoles, IX, with the triazole moiety and a ferrocene scaffold using the 32CA reaction approach, and their mechanism was studied via molecular electron density theory (MEDT) [11,12]. Another representative example is the spirooxindole with a benzimidazole scaffold (see SP1 in Figure 1), which has been extensively studied and has shown to be a potent anti-cancer agent [13]. In continuation of our research program about spirooxindoles [14][15][16], we report herein the synthesis of new spiro compounds containing benzimidazole and 1,2,3-triazole scaffolds as well as the theoretical study of the reaction mechanisms of these relevant 32CA reactions based on MEDT [17].

Results and Discussion
1,2,3-Triazoles represent an important class of heterocyclic compounds with a wide range of biological activities, constituting useful building blocks in chemistry and pharmacological applications. In this context, an attempt was made to synthesize a novel series of spiro compounds having a triazole nucleus combined with the benzimidazole scaffold, as depicted in Scheme 1. Scheme 1. Synthesis of chalcones (5a-n) and spiro compounds (8a-n).

Synthesis of Chalcones (5a-n)
The four steps of the synthesis of the target α,β-unsaturated compounds (5a-n) are presented in (Scheme 1). The first step was to synthesize 2-(chloromethyl) benzimidazole (2) via the Phillip's reaction, involving the condensation of o-phenylenediamine with chloroacetic acid in the presence of dilute hydrochloric acid. The second step was the reaction of a mixture of 2-(chloromethyl) benzimidazole (1.0 equiv.) and sodium azide (1.1 equiv.) in DMSO (15 mL), followed by stirring at room temperature. The reaction was completed in 3 h, affording 2-(azidomethyl) benzimidazole (3) in a 75% yield ( 1 HNMR and 13 CNMR data provided in Supplementary materials; Figures S1 and S2). In the third 7.57 and 6.35 ppm due to the presence of aromatic protons. A singlet at δ 5.74 ppm due to -CH 2 protons and two singlets at δ 2.23 ppm and δ 1.98 ppm corresponding to protons of the two -CH 3 groups were also observed. The 13 C-NMR spectrum ( Figure S24) showed the characteristic carbon signals of the proposed compound 8f. The final cycloadduct stereochemistry was aligned with and matched a similar type of [3+2] cycloaddition reaction, which proceeded via complete ortho/endo selectivity [13]. Based on the reported X-ray single-crystal structure of the reported compound in Ref. [13] and a comparison of its 1 H-NMR spectrum with the 1 H-NMR data for compound 8a as an example, we observed that the chemical shifts of the protons for the stereogenic centers totally matched.
ing to the oxindole ring carbonyl and the benzimidazole ring carbonyl, respectively. The strongest absorption band appeared at 3428 cm −1 due to the -NH functionality in the oxindole ring and benzimidazole ring. The 1 H-NMR spectrum ( Figure S23) of compound (8f) showed a singlet at δ 12.48 ppm due to the -NH proton of the benzimidazole ring, a singlet at δ 9.91 ppm due to the -NH proton of the isatin ring, and a multiplet between δ 7.57 and 6.35 ppm due to the presence of aromatic protons. A singlet at δ 5.74 ppm due to -CH2 protons and two singlets at δ 2.23 ppm and δ 1.98 ppm corresponding to protons of the two -CH3 groups were also observed. The 13 C-NMR spectrum ( Figure S24) showed the characteristic carbon signals of the proposed compound 8f. The final cycloadduct stereochemistry was aligned with and matched a similar type of [3+2] cycloaddition reaction, which proceeded via complete ortho/endo selectivity [13]. Based on the reported X-ray single-crystal structure of the reported compound in Ref. [13] and a comparison of its 1 H-NMR spectrum with the 1 H-NMR data for compound 8a as an example, we observed that the chemical shifts of the protons for the stereogenic centers totally matched.

Supramolecular Features
PLATON [19] analysis revealed the presence of both conventional and non-conventional hydrogen bonding [20]. Generally, this analysis showed that N(1)-H2A···N2, C8-H8AB···O1, and C4-H4···O1 inter-molecular interactions were involved in the unit-cell packing. Among them, the N1-H2A···N2 interaction, involved in connecting molecules along the c-axis, is the strongest one, having a bond distance of 2.06 Å. The O1 carbonyl oxygen of the acetaldehyde moiety is responsible for connecting two molecules along the a-axis via C8-H8AB···O1 and C4-H4···O1 interactions, with hydrogen bond distances of 2.50 and 2.58 Å, respectively (see Table 2). Hence, the unit-cell packing was determined to be two-dimensional, as chain elongation occurred in both aand c-axis accordingly (see Figure 3).   In order to understand the experimental formation of spiro compounds 8a-n, the 32CA reaction of chalcone 5a with AY 9, generated in situ through the reaction between (2R)-octahydro-1H-indole-2-carboxylic acid 6 and isatin 7, was theoretically studied from the perspective of MEDT [17].

MEDT Study of the 32CA Reaction between AY 9 and Chalcone 5a
In order to understand the experimental formation of spiro compounds 8a-n, the 32CA reaction of chalcone 5a with AY 9, generated in situ through the reaction between (2R)-octahydro-1H-indole-2-carboxylic acid 6 and isatin 7, was theoretically studied from the perspective of MEDT [17].
AY 9 exhibits an electrophilicity (ω) index [27] of 0.61 eV, categorizing it as a moderate electrophile according to the electrophilicity scale [22,28]. Additionally, it possesses a nucleophilicity (N) index [29] of 5.02 eV, classifying it as a strong nucleophile based on the nucleophilicity scale [22,28]. In fact, its nucleophilic character exceeds 4.0 eV, earning it the title of a supernucleophile [23,28]. On the other hand, chalcone 5a presents electrophilicity Molecules 2023, 28, 6976 8 of 27 (ω) and nucleophilicity (N) indices of 1.25 eV and 2.96 eV, respectively. This characterizes it as a strong electrophile and positions it at the borderline between moderate and strong nucleophiles.
The combination of the supernucleophilic character of AY 9 and the strong electrophilic character of chalcone 5a suggests that the corresponding FEDF 32CA reaction will possess a highly polar character [23]. This heightened polarity enhances reaction rates by reducing activation energies due to the generation of more favorable nucleophilic/electrophilic interactions.

Study of the Competitive Reaction Paths
Due to the non-symmetry of the reagents, the 32CA reaction between AY 9 and chalcone 5a can take place along two ortho/meta regioisomeric reaction paths, two endo/exo stereoisomeric paths, and two facial diastereoisomeric paths, thus leading to up to eight different cycloadducts. However, as the octahydroindole substituent of AY 9 hinders one of its two diastereoisomeric faces, only the less-hindered approach leading to the four isomeric reaction paths depicted in Scheme 2 was studied. For clarity, a reaction mechanism roadmap showing the main isomeric possibilities is provided in Figure S32 in the Supplementary Materials. Note that due to the presence of a methylene (-CH 2 ) in chalcone 5a, the benzimidazole (-BIZ) substituent can be oriented either towards or away from AY 9, thus adding four possible isomeric paths. All of the eight paths were studied, but only the most favourable ones, with the -BIZ fragment situated away from the AY framework, are discussed herein. In addition, a conformational analysis of the reagents and products was performed whenever different conformers were possible in order to consider only the most stable structures. The Gibbs free energy profiles associated with the four competitive reaction paths are represented in Figure 4, while full thermodynamic data are given in Table S1 in the Supplementary Materials. Upon analyzing the stationary points along the four reaction paths, it becomes evident that the 32CA reaction occurs through a one-step mechanism. Each reaction path exhibits a stable molecular complex (MC) formed through weak intermolecular interactions between the reagents. However, due to the thermodynamic equilibrium between the several MCs, only the most stable complex, MC-on, was chosen as the energy reference. The formation of MC-on is slightly exergonic, releasing 1.2 kcal·mol −1 of energy (see Figure 4). Considering the formation of MC-on, the activation Gibbs free energies for the selected isomeric paths range from 11.1 kcal·mol −1 (TS-on) to 16.6 kcal·mol −1 (TS-mx). On the other hand, the reaction Gibbs free energies fall between −18.1 kcal·mol −1 (10a) and −24.0 kcal·mol −1 (8a). The highly exergonic nature of this reaction suggests irreversibility under the experimental conditions, indicating that the reaction is controlled kinetically. the other hand, the reaction Gibbs free energies fall between −18.1 kcal·mol (10a) and −24.0 kcal·mol −1 (8a). The highly exergonic nature of this reaction suggests irreversibility under the experimental conditions, indicating that the reaction is controlled kinetically. Using the Eyring-Polanyi kinetics equation [30], the following product distribution was predicted: 97.0% (8a), 0.1% (10a), 2.8% (11a), and 0.0% (12a). This demonstrates complete ortho/endo selectivity, exclusively yielding the formation of 8a through TS-on, aligning with the experimental data.  Upon analyzing the stationary points along the four reaction paths, it becomes evident that the 32CA reaction occurs through a one-step mechanism. Each reaction path exhibits a stable molecular complex (MC) formed through weak intermolecular interactions between the reagents. However, due to the thermodynamic equilibrium between the several MCs, only the most stable complex, MC-on, was chosen as the energy reference. The formation of MC-on is slightly exergonic, releasing 1.2 kcal·mol −1 of energy (see Figure 4). Considering the formation of MC-on, the activation Gibbs free energies for the selected isomeric paths range from 11.1 kcal·mol −1 (TS-on) to 16.6 kcal·mol −1 (TS-mx). On the other hand, the reaction Gibbs free energies fall between −18.1 kcal·mol −1 (10a) and −24.0 kcal·mol −1 (8a). The highly exergonic nature of this reaction suggests irreversibility under the experimental conditions, indicating that the reaction is controlled kinetically. Using the Eyring-Polanyi kinetics equation [30], the following product distribution was predicted: 97.0% (8a), 0.1% (10a), 2.8% (11a), and 0.0% (12a). This demonstrates complete ortho/endo selectivity, exclusively yielding the formation of 8a through TS-on, aligning with the experimental data. Figure 5 presents the optimized geometries of the four isomeric transition states (TSs) in methanol. The C-C distances between the interacting carbons provide insights into the C-C single-bond formation processes. Except for the most unfavorable TS-mx, the other three TSs exhibit an asynchronous behavior, with the shorter C-C distance involving the most electrophilic β-conjugated C4 carbon of chalcone 5a. The most favorable TSon, characterized by C3-C4 and C1-C5 distances of 2.094 and 2.711 Å, respectively, has the highest degree of asynchronicity. Examining the intrinsic reaction coordinate (IRC) path [31] from the highly asynchronous TS-on to 8a reveals that the 32CA reaction follows a non-concerted two-stage, one-step mechanism [32]. In this mechanism, the formation of the second C1-C5 single bond commences only after the first C3-C4 single bond is fully formed (see Figure S33 in the Supplementary Materials). Figure 5 also provides the GEDT [25] values for the four isomeric TSs. The GEDT taking place in the TS is a measure of the polarity of the 32CA reaction. GEDT values below 0.05 e indicate non-polar processes, while values above 0.20 e indicate polar processes. Among the TSs, the most favorable TS-on exhibits a GEDT value of 0.27 e. This high value arises from the supernucleophilic nature of AY 9 and the strong electrophilic character of chalcone 5a (refer to Table 3). Consequently, the 32CA reaction through TS-on possesses a significant polar character, which accounts for its low activation Gibbs free energy of 11.1 kcal·mol −1 and the overall endo stereoselectivity observed. Note that polar cycloaddition reactions typically exhibit endo stereoselectivity. Furthermore, the positive GEDT sign computed at the AY framework of the TS indicates an electron density flow from AY 9 to chalcone 5a, classifying this 32CA reaction as FEDF, [26] in accordance with the previous analysis of the reactivity indices. most electrophilic β-conjugated C4 carbon of chalcone 5a. The most favorable TS-on, characterized by C3−C4 and C1−C5 distances of 2.094 and 2.711 Å, respectively, has the highest degree of asynchronicity. Examining the intrinsic reaction coordinate (IRC) path [31] from the highly asynchronous TS-on to 8a reveals that the 32CA reaction follows a non-concerted two-stage, one-step mechanism [32]. In this mechanism, the formation of the second C1−C5 single bond commences only after the first C3−C4 single bond is fully formed (see Figure S33 in the Supplementary Materials). 6-311G(d,p) optimized geometries in methanol of the TSs involved in the 32CA reaction of AY 9 with chalcone 5a. Distances between the interacting carbon atoms, highlighted in green, are given in angstroms, Å, while GEDT values, in red, are given in average number of electrons, e. Figure 5 also provides the GEDT [25] values for the four isomeric TSs. The GEDT taking place in the TS is a measure of the polarity of the 32CA reaction. GEDT values below 0.05 e indicate non-polar processes, while values above 0.20 e indicate polar processes. Among the TSs, the most favorable TS-on exhibits a GEDT value of 0.27 e. This high value arises from the supernucleophilic nature of AY 9 and the strong electrophilic character of chalcone 5a (refer to Table 3). Consequently, the 32CA reaction through TSon possesses a significant polar character, which accounts for its low activation Gibbs free energy of 11.1 kcal·mol −1 and the overall endo stereoselectivity observed. Note that polar cycloaddition reactions typically exhibit endo stereoselectivity. Furthermore, the positive GEDT sign computed at the AY framework of the TS indicates an electron density flow from AY 9 to chalcone 5a, classifying this 32CA reaction as FEDF, [26] in accordance with the previous analysis of the reactivity indices. 6-311G(d,p) optimized geometries in methanol of the TSs involved in the 32CA reaction of AY 9 with chalcone 5a. Distances between the interacting carbon atoms, highlighted in green, are given in angstroms, Å, while GEDT values, in red, are given in average number of electrons, e.

Synthesis of Chalcones (5a-n) and Spiro Compounds
In accordance with the Phillip's reaction, a mixture of o-phenylenediamine (10 mmol, 1.08 g) and chloroacetic acid (10 mmol, 0.945 g) was stirred under reflux conditions in the presence of 4N HCl (40 mL) for approximately 4 h. Then, the reaction mixture was cooled at room temperature, and the pH was adjusted to 9 by adding NH 4 OH solution. The obtained precipitate was collected via filtration, washed with water, dried, and recrystallized from ethanol. The pure product was a pale-yellow-colored solid whose melting point was approximately 150-152 • C, and the yield was 92%.

Synthesis of 2-(Azidomethyl)-1H-benzo[d]imidazole 3
NaN 3 (11 mmol, 0.715 g) was added to a solution of 2-(chloromethyl)-1H-benzo[d]imidazole 2 (10 mmol, 1.66 g) in DMSO (10 mL), and the mixture was stirred for 3-4 h. After completion of reaction (as indicated via TLC), water (50 mL) was added with consistent stirring for 10 min. Then, the organic phase was separated using ethyl acetate. The extract was dried over anhydrous sodium sulphate. Evaporation of the solvent gave the crude product which was purified via column chromatography using hexane: ethylacetate (80:20), as an eluent, which was recrystallized from absolute ethanol.
Molecules 2023, 28, x FOR PEER REVIEW 11 of 28 In accordance with the Phillip's reaction, a mixture of o-phenylenediamine (10 mmol, 1.08 g) and chloroacetic acid (10 mmol, 0.945 g) was stirred under reflux conditions in the presence of 4N HCl (40 mL) for approximately 4 h. Then, the reaction mixture was cooled at room temperature, and the pH was adjusted to 9 by adding NH4OH solution. The obtained precipitate was collected via filtration, washed with water, dried, and recrystallized from ethanol. The pure product was a pale-yellow-colored solid whose melting point was approximately 150-152 °C, and the yield was 92%.

Synthesis of 2-(Azidomethyl)-1H-benzo[d]imidazole 3
NaN3 (11 mmol, 0.715 g) was added to a solution of 2-(chloromethyl)-1H-benzo[d]imidazole 2 (10 mmol, 1.66 g) in DMSO (10 mL), and the mixture was stirred for 3-4 h. After completion of reaction (as indicated via TLC), water (50 mL) was added with consistent stirring for 10 min. Then, the organic phase was separated using ethyl acetate. The extract was dried over anhydrous sodium sulphate. Evaporation of the solvent gave the crude product which was purified via column chromatography using hexane: ethylacetate (80:20), as an eluent, which was recrystallized from absolute ethanol.
imidazole 3 (2 mmol, 0.346 g) was added to a solution of (2 mmol, 0.2 g) of acetylacetone and (2 mmol, 0.276 g) of K 2 CO 3 in 10 mL of DMSO. The mixture was stirred for 3 h at 25 • C and poured into ice water, and the precipitate was filtered off and recrystallized from ethylacetate/ethanol. The yield was 0.4 g (78%) of white crystalline compound 4.
imidazole 3 (2 mmol, 0.346 g) was added to a solution of (2 mmol, 0.2 g) of acetylacetone and (2 mmol, 0.276 g) of K2CO3 in 10 mL of DMSO. The mixture was stirred for 3 h at 25 °C and poured into ice water, and the precipitate was filtered off and recrystallized from ethylacetate/ethanol. The yield was 0.4 g (78%) of white crystalline compound 4.

General Procedure for Synthesis of Chalcones 5a-n
A mixture of 1.1 mmol of aromatic aldehydes was added to a solution of acetyl derivative 4 (1 mmol, 0.255 g) in EtOH (20 mL). Then, a 10% solution of KOH was added dropwise at 20 • C with stirring. The reaction mixture was stirred for 10 h. After the completion of the reaction (monitored via TLC), the mixture was poured over crushed ice. The separated precipitate was filtered, washed with water, and dried. The residue was purified via column chromatography (30% ethyl acetate/n-hexane) to afford purely derived chalcones 5a-n.

General Procedure for [3+2] Cycloaddition Reactions for the Synthesis of Spiro Compounds 8a-n
A mixture of the chalcone derivatives 5a-n (0.5 mmol), isatin (0.5 mmol, 73.5 mg), and octahydroindole-2-carboxylic acid (0.5 mmol, 84.5 mg) in methanol (15 mL) was refluxed using an oil bath for an appropriate time of 3-4 h. After completion of the reaction (Monitored using TLC), the solvent volume was removed under vacuum. The crude was purified via column chromatography on silica gel (30% ethyl acetate in n-hexane), yielding the spiro compounds as solids in a pure form.

General Procedure for [3+2] Cycloaddition Reactions for the Synthesis of Spiro Compounds 8a-n
A mixture of the chalcone derivatives 5a-n (0.5 mmol), isatin (0.5 mmol, 73.5 mg), and octahydroindole-2-carboxylic acid (0.5 mmol, 84.5 mg) in methanol (15 mL) was refluxed using an oil bath for an appropriate time of 3-4 h. After completion of the reaction (Monitored using TLC), the solvent volume was removed under vacuum. The crude was purified via column chromatography on silica gel (30% ethyl acetate in n-hexane), yielding the spiro compounds as solids in a pure form.

General Procedure for [3+2] Cycloaddition Reactions for the Synthesis of Spiro Compounds 8a-n
A mixture of the chalcone derivatives 5a-n (0.5 mmol), isatin (0.5 mmol, 73.5 mg), and octahydroindole-2-carboxylic acid (0.5 mmol, 84.5 mg) in methanol (15 mL) was refluxed using an oil bath for an appropriate time of 3-4 h. After completion of the reaction (Monitored using TLC), the solvent volume was removed under vacuum. The crude was purified via column chromatography on silica gel (30% ethyl acetate in n-hexane), yielding the spiro compounds as solids in a pure form.