Antimicrobial Activity and In Silico Molecular Docking Studies of Pentacyclic Spiro[oxindole-2,3’-pyrrolidines] Tethered with Succinimide Scaffolds

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Herein, and in continuation of our research in the domain of one-pot MCRs of azomethine ylides [33][34][35][36][37][38][39][40][41], we report on the synthesis of various spiro[oxindole-2,3 -pyrrolidines] tethered with succinimide derivatives.The latter were assembled by a one-pot threecomponent [3+2]-cycloaddition reaction of isatin itself or halogenated isatin derivatives 1, L-valine 2 and (E)-3-arylidene-1-methyl-succinimides 4. Note that isatin and its analogues also constitute an important class of organic compounds featuring biological and pharmacological proprieties and serve as building blocks for further transformation.To valorize the synthetic results, some selected heterocyclic compounds were screened in vitro to evaluate their antimicrobial activity and the structure-activity relationship (SAR) was supported by a molecular docking analysis.

Synthetic Chemistry
At the onset of our work, 5-chloroisatin 1a, L-valine 2 and dipolarophile 4d were probed as model substrates to optimize the reaction conditions leading to pentacyclic compound 5d (Scheme 2).The effects of solvents and temperature on the reaction time and yield were examined and the results are summarized in Table 1.
Herein, and in continuation of our research in the domain of one-pot MCRs of azomethine ylides [33][34][35][36][37][38][39][40][41], we report on the synthesis of various spiro[oxindole-2,3′-pyrrolidines] tethered with succinimide derivatives.The latter were assembled by a one-pot threecomponent [3+2]-cycloaddition reaction of isatin itself or halogenated isatin derivatives 1, L-valine 2 and (E)-3-arylidene-1-methyl-succinimides 4. Note that isatin and its analogues also constitute an important class of organic compounds featuring biological and pharmacological proprieties and serve as building blocks for further transformation.To valorize the synthetic results, some selected heterocyclic compounds were screened in vitro to evaluate their antimicrobial activity and the structure-activity relationship (SAR) was supported by a molecular docking analysis.

Synthetic Chemistry
At the onset of our work, 5-chloroisatin 1a, L-valine 2 and dipolarophile 4d were probed as model substrates to optimize the reaction conditions leading to pentacyclic compound 5d (Scheme 2).The effects of solvents and temperature on the reaction time and yield were examined and the results are summarized in Table 1.  a The reaction of 1a (0.5 mmol), 2 (0.6 mmol) and 4d (0.5 mmol) carried out in various solvents (5.0 mL).b Yield of the isolated product after purification.
Having once established the optimal conditions (Table 1, entry 4), we next examined the scope of the reaction in regard to diverse α-arylidene-succinimides 4, as well as with isatin1c and various halogenated isatin derivatives 1a,b (Schema 3, Table 2).
The electronic properties and position of the substituent on the aromatic cycle of dipolarophiles 4 had no substantial influence on the efficiency of this reaction.As summarized in a The reaction of 1a (0.5 mmol), 2 (0.6 mmol) and 4d (0.5 mmol) carried out in various solvents (5.0 mL).b Yield of the isolated product after purification.

Spectroscopic and Crystallographic Characterization of the Cycloadducts 5
The structure and the relative configuration of the spiro-adducts 5 was deduced by NMR spectroscopy.As a representative example, 5b has been chosen and selected 1 H and 13 C chemical shifts along with some characteristic coupling constants are shown in Figure 2. The aromatic protons give rise to a multiplet in the region δ 6.77-7.31ppm and a broad singlet at δ 10.62 ppm is attributed to the NH proton of the oxindole motif.In addition, a doublet at δ 3.80 ppm can be assigned to the NH proton of the pyrrolidine ring.The regio-and stereochemical outcome of the reaction was finally ascertained by an X-ray study, allowing elucidation of the molecular structure of 5b, and confirming that the isopropyl and aryl group stemming from the dipolarophile are in a transoid configuration.
The crystallographic data and the structural refinement details are reported in Table S1 (Supplementary Materials).The asymmetric unit contains two independent molecules.A thermal ellipsoid plot with the atom numbering is shown in Figure 3, and selected bond distances and angles are listed in Table S2.The 1 H NMR spectrum of 5b shows a singlet at δ 2.64 ppm due to the N-methyl protons.Two mutually coupled doublets are observed at δ 2.10 and 2.31 ppm (J = 16 Hz) corresponding to the diastereotopic4 -CH 2 group of the pyrrolide-2,5-dione cycle.A doublet at δ 3.67 ppm (J = 8 Hz) and quadruplet-like multiplet at δ 4.15-4.19ppm (J = 6.8 Hz, J = 14 Hz) are assigned to the pyrrolidine H-4 and H-5 protons, respectively.The multiplicity of these signals clearly demonstrates the regiochemistry of the 1,3-dipolar cycloaddition reaction.If the alternative other regioisomer 6b (Scheme 3) would have been formed, the pyrrolidinyl H-3 and H-5 protons should give rise to a singlet and doublet of doublet pattern in the 1 H NMR spectrum.
The aromatic protons give rise to a multiplet in the region δ 6.77-7.31ppm and a broad singlet at δ 10.62 ppm is attributed to the NH proton of the oxindole motif.In addition, a doublet at δ 3.80 ppm can be assigned to the NH proton of the pyrrolidine ring.
The proton decoupled 13 C NMR spectrum of 5b exhibits signals at δ 41.1 and 72.8 ppm corresponding to the C-4 and C-5 carbons.The two spiranic carbons C-3 and C-2 resonate at δ 60.2 and 78.8 ppm, respectively.In addition, the oxindole carbonyl carbon is recognized at 178.8 and two further signals resonating at δ 184.0 and 184.4 ppm are attributed to the succinimide carbonyl carbons.
The regio-and stereochemical outcome of the reaction was finally ascertained by an X-ray study, allowing elucidation of the molecular structure of 5b, and confirming that the isopropyl and aryl group stemming from the dipolarophile are in a transoid configuration.
The crystallographic data and the structural refinement details are reported in Table S1 (Supplementary Materials).The asymmetric unit contains two independent molecules.A thermal ellipsoid plot with the atom numbering is shown in Figure 3, and selected bond distances and angles are listed in Table S2.
The solid-state structure of compound 5b is stabilized through significant intermolecular interactions such as N−H S2).Within the supramolecular crystal structure of 5b (Figure 4), the two independent molecules are linked together via weak non-conventional intermolecular H-bonding interactions.Thus, the N2 atom of the pyrrolidine ring of the first molecule is involved in a N4−H4•••N2 H-bond with the H4 atom of the pyrrolidine ring of the second molecule with a separation of 2.871(2) Å.The H5A atom of the pyrrolidine ring in the second molecule is also involved in another N5−H5A . . .O6 H-bond with the O6 oxygen atom of pyrrolidine-2,5-dionering of another nearby molecule with a length of 2.957(2) Å (Figure 4a).The regio-and stereochemical outcome of the reaction was finally ascertained by an X-ray study, allowing elucidation of the molecular structure of 5b, and confirming that the isopropyl and aryl group stemming from the dipolarophile are in a transoid configuration.
The crystallographic data and the structural refinement details are reported in Table S1 (Supplementary Materials).The asymmetric unit contains two independent molecules.A thermal ellipsoid plot with the atom numbering is shown in Figure 3, and selected bond distances and angles are listed in Table S2.The solid-state structure of compound 5b is stabilized through significant intermolecular interactions such as N−H S2).Within the supramolecular crystal structure of 5b (Figure 4), the two independent molecules are linked together via weak non-conventional intermolecular H-bonding interactions.Thus, the N2 atom of the pyrrolidine ring of the first molecule is involved in a N4−H4•••N2 H-bond with the H4 atom of the pyrrolidine ring of the second molecule with a separation of 2.871(2) Å.The H5A atom of the pyrrolidine ring in the second molecule is also involved in another N5−H5A…O6 H-bond with the O6 oxygen atom of pyrrolidine-2,5-dionering of another nearby molecule with a length of 2.957(2) Å (Figure 4a).A similar contact also occurs between the H46 atom of the phenyl ring of one molecule and the p-chloroaryl substituent of a neighbored molecule (C46-H46••••Cl1, 2.871 Å (Figure 4b).Further details of the intermolecular interactions present in 5b are listed in Table S2.
First, the in situ decarboxylative condensation of isatin derivatives 1 with L-valine 2 generates the (Z,E)-dipole 3. Then the azomethine ylide subsequently undergoes a 1,3dipolar cycloaddition reaction across the dipolarophile 4. A priori, two regioisomeric and A similar contact also occurs between the H46 atom of the phenyl ring of one molecule and the p-chloroaryl substituent of a neighbored molecule (C46-H46••••Cl1, 2.871 Å (Figure 4b).Further details of the intermolecular interactions present in 5b are listed in Table S2.
refer to the approach of the dipolarophile with its electron-withdrawing group (carbonyl group) oriented toward the EWG group of the dipole (carboxamide group of oxindole) or away from it, respectively.The presence of a secondary orbital interaction (SOI) [42], via path A, favors more the formation of the exo-regioisomer 5, which occurs between the oxygen atom of the carbonyl of the diketone and the carbon atom of the carbonyl succinimide acting as dipolarophile according to Scheme 4. The formation of the alternative regioisomers or diastereoisomers 5′ or 6/6′ via pathways B, C and D is probably less favorable because of steric or electronic repulsion occurring in their corresponding transition states.We are aware that, apart from SOI interactions, other parameters, such as reaction temperature [35,40] or the choice of the solvent [43], may also impact the stereochemical outcome.To confirm our mechanistic proposal, DFT computing is intended in prospective work.Scheme 4. Suggested mechanism for the 1,3-dipolar cycloaddition of azomethine ylides across (E)-3-arylidene-1-metyl-pyrrolidine-2,5-diones 4.

Electronic Properties: UV-Visible Absorption Spectroscopy
The absorption spectral data recorded in DMSO as solvent for the spiro[pyrrolidin-2,3-oxindoles]5 are summarized in Table 3.As representative example, the superposition of the electronic absorption spectra of 5c, 5g and 5h using a dilute concentration around 10 −5 M are depicted in Figure 5. First, the in situ decarboxylative condensation of isatin derivatives 1 with L-valine 2 generates the (Z,E)-dipole 3. Then the azomethine ylide subsequently undergoes a 1,3dipolar cycloaddition reaction across the dipolarophile 4. A priori, two regioisomeric and two stereoisomeric pathways can be taken in consideration.The exoand endo-approaches refer to the approach of the dipolarophile with its electron-withdrawing group (carbonyl group) oriented toward the EWG group of the dipole (carboxamide group of oxindole) or away from it, respectively.The presence of a secondary orbital interaction (SOI) [42], via path A, favors more the formation of the exo-regioisomer 5, which occurs between the oxygen atom of the carbonyl of the diketone and the carbon atom of the carbonyl succinimide acting as dipolarophile according to Scheme 4. The formation of the alternative regioisomers or diastereoisomers 5 or 6/6 via pathways B, C and D is probably less favorable because of steric or electronic repulsion occurring in their corresponding transition states.We are aware that, apart from SOI interactions, other parameters, such as reaction temperature [35,40] or the choice of the solvent [43], may also impact the stereochemical outcome.To confirm our mechanistic proposal, DFT computing is intended in prospective work.

Electronic Properties: UV-Visible Absorption Spectroscopy
The absorption spectral data recorded in DMSO as solvent for the spiro[pyrrolidin-2,3-oxindoles]5 are summarized in Table 3.As representative example, the superposition of the electronic absorption spectra of 5c, 5g and 5h using a dilute concentration around 10 −5 M are depicted in Figure 5.The broad absorption band with λabs in the range 290-340 nm can be assigned due to π-π* transitions.Additionally, in the case of p-hydroxyspiro[pyrrolidin-2,3-oxindoles 5fh and 5n-p, the appearance of a second high intensity band within the 409-454 nm region is noticeable.The intense absorptions can be attributed to an intramolecular charge transfer (ICT) transition involving to hydroxy group [44] (Figure 5) and (Table 3).The broad absorption band with λ abs in the range 290-340 nm can be assigned due to π-π* transitions.Additionally, in the case of p-hydroxyspiro[pyrrolidin-2,3-oxindoles 5f-h and 5n-p, the appearance of a second high intensity band within the 409-454 nm region is noticeable.The intense absorptions can be attributed to an intramolecular charge transfer (ICT) transition involving to hydroxy group [44] (Figure 5) and (Table 3).
This CT-band is red-shifted for compounds 5h and 5o, where the methoxy group is attached at 3,5-positions.The bathochromic effect on the charge transfer band is mainly due to the electron-withdrawing propensity of OCH 3 in an inductive sense [45].In contrast, a hypsochromic shift is noticed for p-hydroxyspiro[pyrrolidin-2,3-oxindoles 5h and 5p bearing bromo substituents.
The optical gap (E gap-op ) refers to the energetic separation between the HOMO and the LUMO frontier orbitales of the spiropyrrolidine oxindoles.To determine the optical bandgap, we used the UV-visible spectrum, employing the Tauc plot method [46].The E gap values in DMSO solution are 2.44 eV and 2.69 eV for 5i and 5p, respectively (Table 3).
The determinations of the minimal inhibition concentrations (MIC) and the minimal bactericidal/fungicidal concentrations (MBC/MFC) were conducted following the technique of dilution in liquid medium, as described by Snoussi et al. [47].
When tested on different microorganism strains, the compounds were found to display significant activity.The potencies were qualitatively assessed by the presence or the absence of an inhibition zone diameter (Figures 6 and 7).The MIC, MBC and MFC values are presented in Tables 4-6, respectively.The determinations of the minimal inhibition concentrations (MIC) and the minimal bactericidal/fungicidal concentrations (MBC/MFC) were conducted following the technique of dilution in liquid medium, as described by Snoussi et al. [47].
When tested on different microorganism strains, the compounds were found to display significant activity.The potencies were qualitatively assessed by the presence or the absence of an inhibition zone diameter (Figures 6 and 7).The MIC, MBC and MFC values are presented in Tables 4-6, respectively.
The highest antibacterial activity of the cycloadducts is display against gram-positive bacteria (Staphylococcus aureus ATCC 25,923 and Micrococcus luteus NCIMB 8166), whereas towards gram-negative bacteria (Escherichia coli ATCC 25,922 and Pseudomonas aeroginosa ATCC 27853), the probed compounds are in general less active compared to gram-positive ones.
When testing against Micrococcus luteus NCIMB 8166, compounds 5f, 5g, 5n and 5o were equipotent as Tetracycline.Thus, 5h and 5p were found to be the most active, with MIC values of 31.5 and 62.5 µ g/mL, respectively (Table 4).Concerning the evaluation of the inhibition zone, the compounds 5d, 5o and 5p were found to be the most sensitive, with 17mm to 14 mm.Additionally, the different compounds were evaluated against two gram-negative bacteria (Escherichia coli ATCC 25,922 and Pseudomonas aeroginosa ATCC 27853).In this case, 5e was found to be the most sensitive, with an inhibition zone of 15 mm.All compounds were found to be equal or less active (125->1000 µ g/mL) concerning the MIC or MBC compared to standard antibiotic Tetracycline.
On the other hand, the in vitro antifungal activity data reveal that 5a, 5f and 5i display the highest activity with MIC 7.812 µ g/mL against Amphotericin B. In the first screening phase against Staphylococcus aureus ATCC 25923, 5a was noted to be the most sensitive derivative, with an inhibition zone of 31 mm, the MIC being 3.9 µg/mL and the MBC being 31.5 µg/mL, respectively (Tables 4-6).Then, against the same pathogenic bacteria, four compounds 5d, 5e, 5h and 5l showed an excellent activity with MIC values in the range of 31.5-62.5 µg/mL as MBC with an inhibition zone between 11 and 20 mm, compared to standard antibiotic Tetracycline.
When testing against Micrococcus luteus NCIMB 8166, compounds 5f, 5g, 5n and 5o were equipotent as Tetracycline.Thus, 5h and 5p were found to be the most active, with MIC values of 31.5 and 62.5 µg/mL, respectively (Table 4).Concerning the evaluation of the inhibition zone, the compounds 5d, 5o and 5p were found to be the most sensitive, with 17mm to 14 mm.Additionally, the different compounds were evaluated against two gram-negative bacteria (Escherichia coli ATCC 25,922 and Pseudomonas aeroginosa ATCC 27853).In this case, 5e was found to be the most sensitive, with an inhibition zone of 15 mm.All compounds were found to be equal or less active (125->1000 µg/mL) concerning the MIC or MBC compared to standard antibiotic Tetracycline.
On the other hand, the in vitro antifungal activity data reveal that 5a, 5f and 5i display the highest activity with MIC 7.812 µg/mL against Amphotericin B.
Compounds 5d, 5k and 5q showed a somewhat less efficient activity, when compared to compounds 5i and 5p with MIC 31.5 µg/mL relating to Candida albicans ATCC 90028.Additionally, regarding the results of antifungal screening, the MFC is noted to have good antifungal activity on 5a, 5i and 5c with 7.81, 31.5 and 62.5 µg/mL, respectively.Furthermore, 5b appeared to be the most sensitive heterocycle (14 mm).In contrast, as indicated in Table 5, 5n was revealed to be the least sensitive heterocycle, with an inhibition zone of 6 mm.Finally, we discuss the last yeast strains is Candida krusei ATCC 6258.In fact, 5g, 5i and 5p showed a superior sensitivity with an inhibition zone of 14-12 mm.It is also worth mentioning the excellent antifungal activity of compounds 5a, displaying both a MIC and MFC of 31.5 µg/mL (Table 6).

Molecular Docking Studies
In order to provide a comprehensive rationale for the obtained biological activities, we docked the most active compounds,5a and 5i, into glucosamine 6-phosphate synthase (GlcN6P) and methionyl-trna-synthetase (1PFV).GlcN6P catalyzes a multifarious reaction comprising both sugar isomerization (fructosamine-6-phosphate to glucosamine-6phosphate) and ammonia transfer (L-glutamine to Fru-6-P) [48][49][50].This reaction initiates a pathway that finally drives the production of uridine 5 -diphospho-N-acetyl-d-glucosamine.This nucleotide sugar is used to assemble the macromolecules essential for constructing cell walls, such as the macromolecules with several amino sugar units, like chitin or proteins in fungi, and lipopolysaccharides and peptide glycan in bacteria.GlcN6P synthase inhibition is fatal, even for a short time, in prokaryotic cells but not in human cells.Therefore, it is considered as a promising target for developing antibacterial and antifungal agents.
The compounds, except 5a enantiomer (which has the most potent antibacterial activity among all the synthesized compounds), exhibit interactions with several amino acid residues at the active site of GlcN6P and reveal low binding energy.They also share several amino acids with the positive controls, Ampicillin and Tetracycline (Table 7) and (Figure 8).
Infections caused by antibiotic-resistant pathogens have raised markedly in recent years.Over 60% of staphylococcal infections in intensive care units in the United States, for example, are caused by methicillin-resistant bacterial strains.The incidence of the emerging methicillin-resistant staphylococcus aureus strains in community has raised many concerns.
These strains were found to express a wide range of virulence factors (https://en.wikipedia.org/wiki/Virulence_factor,accessed on 15 January 2021) and have an enhanced tendency toward progression into invasive ailments.The action takes place over two-step reactions.In the first step, methionine and ATP bind together to the enzyme's active site, resulting in the formation of methionyl adenylate, releasing pyrophosphate (PPi).In the following reaction, the activated methionyl adenylate is moved to tRNAMetat and its 3 end, releasing an AMP molecule.MetRS has the unique ability to identify and load two tRNA substrates; namely, tRNAmMet and tRNAfMet [51].For these reasons, the design of alternative antibacterial agents exhibiting an activity against drug-resistant staphylococci represents an important area for the development of novel drugs.Again, our in silico study results revealed that the screened compounds, except the 5a enantiomer, exhibited low binding energy when docked into methionyl-trna-synthetase (1PFV) and shared similar interactions with several amino acids similar to the positive control, Ampicillin and Tetracycline (Table 7) and Figure 9.
Interestingly, all compounds obeyed to the parameters of the rule of 5 (Lipinski rule) [52].These include molecular weight, number of hydrogen bonds, accepting (lip_acc) and donating (lip_don) groups, and the drug-likeness potential (Table 8).

General Experimental Methods
The 1 H NMR spectra were recorded at 400 MHz using a Bruker Avance III-400 machine (Rheinstetten, Germany).The chemical shifts were recorded in ppm relative to TMS and with the solvent resonance as the internal standard.Data were reported as follows: chemical shift, multiplicity (bs = broad singlet, s = singlet, d = doublet, dd = doublet of doublet, m = multiplet), coupling constants (Hz), and integration. 13C{ 1 H} NMR data were collected at 100 MHz with complete proton decoupling with the solvent resonance as the internal standard.Optical absorption spectra were recorded at an ambient temperature, using a Shimadzu UV-3600 double-beam UV-visible spectrophotometer and the concentrations of spiro[pyrrolidin-2,3 -oxindole] derivatives were controlled to be of 10 −5 M in DMSO.The materials used were: thin-layer chromatography (TLC): TLC plates (Merck, silica gel 60 F254 0.2 mm 200× 200 nm), and substances were detected using UV light at 254 nm.High Resolution Mass Spectra (ES-HRMS) were obtained with Micromass LCT (ESI technique, positive mode) spectrometers.

X-ray Crystallography
X-ray suitable crystal of 5b for the diffraction study were obtained by slow evaporation of a diethyl ether solution containing 5b.The crystallographic data were collected at 193(2)K on a Bruker-AXS D8-Venture diffractometer equipped with a Photon III-C14 detector using MoK α radiation (λ = 0.71073Å).Phi-and omega-scans were used.Space group was determined on the basis of systematic absences and intensity statistics.Semi-empirical absorption correction was employed [53].The structure was solved using an intrinsic phasing method (SHELXT) [54] and refined using the least-squares method on F [55].All non-H atoms were refined with anisotropic displacement parameters.Hydrogen atoms were refined isotropically at calculated positions using a riding model with their isotropic displacement parameters constrained to be equal to 1.5 times the equivalent isotropic displacement parameters of their pivot atoms for terminal sp 3 carbon and 1.2 times for all other carbon atoms.H on nitrogen atoms were located by difference Fourier maps.
CCDC-2063918 contains the supplementary crystallographic data for this paper.These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via https://www.ccdc.cam.ac.uk/structures (accessed on 19 Mars 2021).
Crystal data for 5b:C 25

Antimicrobial Screening
First, the optical density of each microorganism suspensions was adjusted to 0.1 at OD600 for bacteria and 0.4 at OD540 for yeasts.Then, 500 µL of inoculums were dropped onto adequate agar plates.Sterile filter discs (diameter 6 mm, Biolife, Italy) were placed at the surface of the appropriate agar mediums and 10 mg/disc of the product dissolved in 10% of dimethyl sulfoxide was dropped onto each disc.Tetracycline (10 mg/mL; 10 µL/disc) and Amphotericin B (10 mg/mL; 10 µL/disc), were used as reference drugs.After incubation for 18-24 h at 37 • C, the antibacterial activities were evaluated by measuring an inhibition zone formed around the disc.Each assay was performed in triplicate.
For this, the microbial inoculums were prepared from 12 h broth cultures and spectrophotometrically adjusted to 10 7 UFC/mL.
Serial two-fold dilutions of the different amounts of the compounds (1000 to 0.09 µg/mL) were prepared in adequate broth.Then, 10µL of the inocula of each reference strain were added to the plates containing the serial dilution and were incubated aerobically at 37 • C for 24 h.MIC was defined as the lowest concentration that completely inhibited visible cell growth during 24 h incubation period at 37 • C.

20 J
= 14 Hz)  are assigned to the pyrrolidine H-4 and H-5 protons, respectively.The multiplicity of these signals clearly demonstrates the regiochemistry of the 1,3-dipolar cycloaddition reaction.If the alternative other regioisomer 6b (Scheme 3) would have been formed, the pyrrolidinyl H-3 and H-5 protons should give rise to a singlet and doublet of doublet pattern in the 1 H NMR spectrum.

Figure 2 .
Figure 2. 1 H (black) chemical shifts with coupling constants and 13 C NMR (pink) data (ppm) of 5b.The proton decoupled 13 C NMR spectrum of 5b exhibits signals at δ 41.1 and 72.8 ppm corresponding to the C-4 and C-5 carbons.The two spiranic carbons C-3 and C-2 resonate at δ 60.2 and 78.8 ppm, respectively.In addition, the oxindole carbonyl carbon is recognized at 178.8 and two further signals resonating at δ 184.0 and 184.4 ppm are attributed to the succinimide carbonyl carbons.The regio-and stereochemical outcome of the reaction was finally ascertained by an X-ray study, allowing elucidation of the molecular structure of 5b, and confirming that the isopropyl and aryl group stemming from the dipolarophile are in a transoid configuration.The crystallographic data and the structural refinement details are reported in TableS1(Supplementary Materials).The asymmetric unit contains two independent molecules.A thermal ellipsoid plot with the atom numbering is shown in Figure3, and selected bond distances and angles are listed in TableS2.

Figure 3 .
Figure 3.The molecular structure of one of the two independent molecules of 5b, showing the atomnumbering scheme.The displacement ellipsoids drawn at the 50% probability level.Only relevant H atoms are shown for clarity.
In addition, this crystal packing is reinforced by several weak C−H••••O interactions between the H16A atom of the pyrrolidine-2,5-dione group of one molecule and the O5 oxygen atom of the same group of another molecule, with a C16-H16A••••O5 distance of 2.943 Å. recognized at 178.8 and two further signals resonating at δ 184.0 and 184.4 ppm are attributed to the succinimide carbonyl carbons.

Figure 3 .
Figure 3.The molecular structure of one of the two independent molecules of 5b, showing the atomnumbering scheme.The displacement ellipsoids drawn at the 50% probability level.Only relevant H atoms are shown for clarity.

Figure 3 .
Figure 3.The molecular structure of one of the two independent molecules of 5b, showing the atom-numbering scheme.The displacement ellipsoids drawn at the 50% probability level.Only relevant H atoms are shown for clarity.
In addition, this crystal packing is reinforced by several weak C−H••••O interactions between the H16A atom of the pyrrolidine-2,5-dione group of one molecule and the O5 oxygen atom of the same group of another molecule, with a C16-H16A••••O5 distance of 2.943Å.

Figure 4 .
Figure 4. (a) Representation of the N-H••••N and N−H••••O bonding between two adjacent molecules of 5b.Intermolecular interactions contacts are shown as dashed lines.(b) Presentation of the intermolecular C-H••• Cl and C-H••• O contacts occurring in 5b.

Figure 4 .
Figure 4. (a) Representation of the N-H••••N and N−H••••O bonding between two adjacent molecules of 5b.Intermolecular interactions contacts are shown as dashed lines.(b) Presentation of the intermolecular C-H••• Cl and C-H••• O contacts occurring in 5b.

Table 1 .
Optimization of the reaction parameters for the synthesis of 5d a.

Table 1 .
Optimization of the reaction parameters for the synthesis of 5d a .

Table 2 .
Synthesis of the spirooxindole pyrrolidines 5 a .

Table 2 .
Synthesis of the spirooxindole pyrrolidines 5 a .

Table 5 .
Antibacterial and antifungal activity of series 5 (Inhibition zone in mm).