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Article

Pyrrolizine- and Indolizine-Derived Spirooxindoles: Synthesis, Antibacterial Activity and Inverse Docking Analysis

by
Pablo Romo
1,2,
María del Pilar Crespo
3,
Mauricio Barreto
3,
María Elena Burbano
3,
Melissa Mejia-Gutierrez
4,
Jairo Quiroga
1,* and
Rodrigo Abonia
1,*
1
Heterocyclic Compounds Research Group, Department of Chemistry, Universidad del Valle, Cali 760042, Colombia
2
Research Group on Chemistry and Biotechnology, Universidad Santiago de Cali, Cali 760035, Colombia
3
Microbiology and Infectious Diseases Research Group, Department of Microbiology, Universidad del Valle, Cali 760042, Colombia
4
Pharmaceutical and Nutrition Sciences Research Group, Department of Chemistry, University of Saskatchewan, Saskatoon, SK S7N 5C9, Canada
*
Authors to whom correspondence should be addressed.
Chemistry 2025, 7(1), 18; https://doi.org/10.3390/chemistry7010018
Submission received: 27 December 2024 / Revised: 24 January 2025 / Accepted: 26 January 2025 / Published: 1 February 2025
(This article belongs to the Section Medicinal Chemistry)
Browse Figures
Versions Notes

Abstract

:
Spirooxindoles are a family of heterocyclic compounds which bear the oxindole nucleus in their structures, which have a considerable pharmaceutical potential and which have been linked to various drugs for the treatment of diverse diseases. In this work, a wide variety of spirooxindoles bearing a pyrrolizinic nucleus were obtained by a 1,3-dipolar cycloaddition reaction between substituted isatins, trans-3-benzoyl acrylic acid and L-proline. In this approach, the target products 9a–m were obtained in 40–86% yields under heating to reflux in methanol over 2 h. Similarly, spirooxindoles containing an indolizinic nucleus 11a–j were obtained in 45–69% yields by switching L-proline for pipecolic acid under heating to reflux in acetonitrile for 8 h. The antibacterial activity of the obtained products was evaluated against P. aeruginosa, K. pneumoniae, E. coli, S. aureus, and N. gonorrhoeae, also including an inverse docking analysis. Results show that 9f and 11i, were the most active compounds against S. aureus, while compounds 9d and 9m displayed the higher activity against N. gonorrhoeae. Inverse docking analysis showed that compounds 9b, 11a 11e, and 11i displayed high affinity to the target protein 6TYM and 7Q6S, which are involved in biological pathways of diverse cancer and Parkinson diseases.

1. Introduction

Spirooxindoles are a family of compounds that have two important characteristics: they have a spiro carbon that gives them structural rigidity and an oxindole nucleus associated with their pharmacophoric properties. In this sense, spirooxindoles are widely distributed in nature (Figure 1a) [1], like the alkaloids elacomine [2,3] and horsfiline [4]; spirotryprostatin A and B, with inhibitory activity of the cellular cycle [5]; and rhynchophylline, with antihypertensive, hypotensive and neuroprotective properties [6,7,8,9].
Considering the pharmaceutical potential, and their ability to act as protein inhibitors that have been linked to various diseases, representing a growing area of research [10,11,12,13], these compounds could pave the way for novel therapeutic approaches and drug developments. Some outstanding examples are shown in Figure 1b: MI-888 is a potent antitumor agent [14], the compound 1 is a HIV-1 reverse transcriptase inhibitor [15], and compound 2 is active against the bacterium Mycobacterium tuberculosis (MTB) with an MIC of 2.8 µM [16]. On the other hand, compound 3 is an acetylcholinesterase inhibitor (AchE) with an IC50 of 0.11 µM [17] and the spirooxindole 4 [18] exhibited anticancer activity against cell lines of prostate cancer DU145 (IC50: 8.02 µM), esophageal cancer line EC109 (IC50: 6.62 µM), human gastric cancer cell line MGC803 (IC50: 9.49 µM), and breast adenocarcinoma line MCF-7 (IC50: 17.65 µM). Additionally, compound 5 displayed antibacterial activity against Bacillus subtilis and Staphylococcus aureus with an MIC of 0.291 µM (for both strains), and against Pseudomonas aeruginosa, Salmonella typhimurium and Proteus vulgaris with an MIC of 0.146 µM (for the three strains) [19].
On the other hand, isatin is used as a building block for spirooxindoles, usually through multicomponent reactions such as cyclocondensation [20,21,22,23,24,25], and 1,3-dipolar cycloaddition [26,27,2829,30,31,32,33,34,35], with the latter allowing one to obtain stereospecific products with high structural complexity. A fundamental feature of this process is the generation of azomethine ylides, as discovered by Grigg and co., using secondary amino acids and carbonylic compounds. In this approach, the azomethine ylide is formed in situ after NH to C=O addition, condensation/dehydration and a decarboxylation process of the CO2H functionality coming from the amino acid reagent. Finally, the azomethine ylide-formed (1,3-dipole species) reacts with different dipolarophiles to achieve highly substituted cycloaddition products [36,37].
For instance, in this research work we aimed to use this tricomponent methodology as a key strategy for creating new spirooxindoles with potential biological activity using substituted isatins, trans-3-benzoyl acrylic acid and two amino acids (i.e., L-proline and pipecolic acid) as starting materials, followed by the evaluation, both in vivo and in silico, of the antibacterial and toxicological properties of the obtained compounds, as well as their inverse docking analysis.

2. Materials and Methods

2.1. Chemistry

The reagents and solvents in this work were obtained from commercial sources such as Aldrich (St. Louis, MO, USA), Alfa Aesar (Haverhill, MA, USA), Merck (St. Louis, MO, USA), etc., the reactions were followed by thin layer chromatography (TLC) on silica gel plates 60GF254 (Merck, St. Louis, MO, USA). The melting points were measured using a Stuart SMP3 melting point apparatus (Cole-Parmer Instrument Company LLC, Burlington, MA, USA) and are uncorrected. FT-IR spectra were taken on a SHIMADZU IRAffinity-1 spectrophotometer (Shimadzu, Kyoto, Japan) with an ATR probe. 1H and 13C NMR spectra were measured with a BRUKER DPX 400 spectrophotometer (Bruker, Bruker BioSpin GmbH, Rheinstetten, Germany) operating at 400 and 100 MHz, respectively, using dimethylsulfoxide–d6 and chloroform–d as solvents. Mass spectra were obtained on a SHIMADZU-GCMS-QP2010 spectrometer (Shimadzu, Kyoto, Japan) operating between 20 and 70 eV. Microanalysis was performed on a Thermo Electron Flash EA 1112 analyzer (Thermo Fisher Scientific, Waltham, MA, USA).

2.1.1. General Procedure for the Synthesis of Spirooxindoles 9a–m

Equimolar amounts of the corresponding substituted isatins 6, L-proline 7a and trans-3-benzoyl acrylic acid 8 were dissolved in 10 mL of MeOH in a flat bottom flask and subjected to reflux for 2 h (TLC control). The solid formed was isolated by filtration on vacuum and washed with cold MeOH. The products did not require further purification.
(1′RS,2′RS,3SR,7a′SR)-2′-Benzoyl-1-benzyl-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid 9a: White solid. Yield: 61%. M.p.: 228–229 °C (Decomp). FT-IR (ATR) υ (cm−1): 2957, 2864, 1726, 1678, 1697. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.67–1.79 (m, 1 H, H-6′), 1.80–1.94 (m, 2 H, H-6′, H-7′), 2.01–2.13 (m, 1 H, H-7′), 2.24–2.34 (m, 1 H, H-5′), 2.34–2.44 (m, 1 H, H-5′), 3.62 (t, J = 10.4 Hz, 1 H, H-1′), 3.92–3.97 (m, 1 H, H-7a’), 4.22 (d, J = 15.8 Hz, 1 H, N-CH), 4.73 (d, J = 15.8 Hz, 1 H, N-CH), 4.87 (d, J = 11.3 Hz, 1 H, H-2′), 6.50 (d, J = 7.8 Hz, 1 H), 6.91–6.98 (m, 1 H), 7.00–7.11 (m, 3 H), 7.15 (d, J = 7.4 Hz, 1 H), 7.18–7.26 (m, 3 H), 7.30 (d, J = 3.9 Hz, 4 H), 7.52 (br. s., 1 H), 12.71 (br.s., COOH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 27.4 (C-6′), 31.3 (C-7′), 43.2 (N-CH2), 47.5 (C-5′), 51.4 (C-1′), 59.9 (C-2′), 67.4 (C-7a’), 71.7 (C-spiro), 109.6 (CH), 122.5 (CH), 124.4 (C), 127.3 (CH), 127.4 (CH), 127.8 (CH), 127.9 (CH), 128.8 (CH), 129.1 (CH), 129.9 (CH), 133.7 (CH), 136.3 (C), 136.8 (C), 142.7 (C), 173.4 (C), 178.0 (C), 197.2 (C). MS (EI, 70 eV) m/z: 466 (M+•, 2), 430 (5), 404 (66), 375 (11), 339 (28), 290 (78), 223 (25), 91 (100). Anal. Calcd. for C29H26N2O4: C: 74.66; H: 5.62; N: 6.00. Found: C: 74.54; H:5.61; N: 6.02.
(1′RS,2′RS,3SR,7a′SR)-2′-Benzoyl-1-methyl-2-oxo-1′,2′,5′,6′,7′,7a’-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid 9b: White solid. Yield: 44%. M.p.: 214–216 °C (Decomp). FT-IR (ATR) υ (cm−1): 3071, 2978, 2936, 2864, 1730, 1672. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.65–1.78 (m, 1 H, H-6′), 1.79–1.93 (m, 2 H, H-6′, H-7′), 2.00–2.10 (m, 1 H, H-7′), 2.23–2.30 (m, 1 H, H-5′), 2.35–2.44 (m, 1 H, H-5′), 2.64 (s, 3 H, N-CH3), 3.51 (t, J = 10.5 Hz, 1 H, H-1′), 3.88 (dt, J = 9.6, 6.1 Hz, 1 H, H-7a’), 4.73 (d, J = 11.3 Hz, 1 H, H-2′), 6.56 (d, J = 7.8 Hz, 1 H), 6.94–7.02 (m, 1 H), 7.07–7.20 (m, 4 H), 7.28 (t, J = 7.6 Hz, 2 H), 7.49 (t, J = 7.3 Hz, 1 H), 12.65 (br. s., 1 H, COOH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 26.3 (CH3), 27.5 (C-6′), 31.4 (C-7′), 47.7 (C-5′), 50.9 (C-1′), 61.2 (C-2′), 67.6 (C-7a’), 71.8 (C-spiro), 108.9 (CH), 122.4 (CH), 124.3 (C), 126.9 (CH), 127.6 (CH), 128.6 (CH), 130.0 (CH), 133.3 (CH), 136.9 (C), 143.5 (C), 173.4 (C), 177.8 (C), 197.5 (C). MS (EI, 70 eV) m/z: 390 (M+•, 2), 354 (7), 299 (46), 263 (31), 241 (44), 214 (100). Anal. Calcd. for C23H22N2O4: C: 70.75; H: 5.68; N: 7.18. Found: C: 70.82; H: 5.71; N: 7.14.
(1′RS,2′RS,3SR,7a′SR)-2′-Benzoyl-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid 9c: White solid. Yield:72%. M.p.: 211–212 °C (Decomp). FT-IR (ATR) υ (cm−1): 3310, 2874, 1699, 1678. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.64–1.76 (m, 1 H, H-6′), 1.78–1.91 (m, 2 H, H-7′, H-6′), 1.99–2.09 (m, 1 H, H-7′), 2.24–2.31 (m, 1 H, H-5′), 2.42 (q, J = 8.3, 7.4 Hz, 1 H, H-5′), 3.56 (t, J = 10.5 Hz, 1 H, H-1′), 3.88 (dt, J = 9.7, 6.2 Hz, 1 H, H-7a’), 4.77 (d, J = 11.3 Hz, 1 H, H-2′), 6.46 (d, J = 8.0 Hz, 1 H), 6.87 (t, J = 7.5 Hz, 1 H), 7.01–7.10 (m, 2 H), 7.26–7.39 (m, 4 H), 7.49 (t, J = 6.8 Hz, 1 H), 10.17 (s, 1 H, NH), 12.63 (s, 1 H, COOH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 27.2 (C-6′), 31.2 (C-7′), 47.6 (C-5′), 51.4 (C-1′), 59.7 (C-2′), 67.3 (C-7a’), 72.0 (C-spiro), 110.1 (CH), 121.6 (CH), 124.9 (C), 127.6 (CH), 127.9 (CH), 128.8 (CH), 129.8 (CH), 133.6 (CH), 136.9 (C), 142.3 (C), 173.5 (C), 179.5 (C), 197.4 (C). MS (EI, 70 eV) m/z: 376 (M+•, 1), 314 (100), 285 (97), 257 (20), 200 (30), 105 (74), 77 (49). Anal. Calcd. for C22H20N2O4: C: 70.20; H: 5.36; N: 7.44. Found: C: 69.99; H: 5.32; N: 7.41.
(1′RS,2′RS,3SR,7a′SR)-2′-Benzoyl-1-hexyl-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid 9d [38]: White solid. Yield: 60%. M.p.: 196–197 °C. FT-IR (ATR) υ (cm−1): 2957, 2932, 2866, 1738, 1676, 1604. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 0.81 (t, J = 6.7 Hz, 3 H, CH3), 1.03–1.24 (m, 9 H), 1.65–1.78 (m, 1 H), 1.79–1.92 (m, 2 H), 1.99–2.10 (m, 1 H), 2.24 (ddd, J = 10.0, 6.8, 4.1 Hz, 1 H), 2.36 (td, J = 8.7, 6.3 Hz, 1 H), 3.07 (dt, J = 13.6, 6.1 Hz, 1 H), 3.57 (dd, J = 11.1, 10.0 Hz, 1 H, H-1′), 3.89 (dt, J = 9.7, 6.2 Hz, 1 H, H-7a’), 4.79 (d, J = 11.3 Hz, 1 H, H-2′), 6.65 (d, J = 7.8 Hz, 1 H), 6.96 (t, J = 7.5 Hz, 1 H), 7.10–7.19 (m, 2 H), 7.22 (d, J = 7.4 Hz, 2 H), 7.29 (t, J = 7.7 Hz, 2 H), 7.47 (t, J = 7.4 Hz, 1 H), 12.66 (br. s., 1 H, COOH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 14.3 (CH3), 22.4 (CH2), 26.2 (CH2), 26.8 (CH2), 27.5 (C-6′), 31.3 (CH2), 31.4 (C-7′), 39.7 (CH2), 47.4 (C-5′), 51.2 (C-1′), 60.1 (C-2′), 67.5 (C-7a’), 71.6 (C-spiro), 109.1 (CH), 122.1 (CH), 124.4 (C), 127.3 (CH), 127.8 (CH), 128.7 (CH), 130.0 (CH), 133.5 (CH), 137.0 (C), 143.0 (C), 173.4 (C), 177.5 (C), 197.3 (C). MS (EI, 70 eV) m/z: 460 (M+•, 3), 398 (100), 369 (12), 333 (18), 284 (44), 105 (40), 77 (27). Anal. Calcd. for C28H32N2O4: C: 73.02, H: 7.00, N: 6.10. Found: C: 73.10, H: 7.10, N: 6.20.
(1′RS,2′RS,3SR,7a′SR)-2′-Benzoyl-5-chloro-1-methyl-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3’-pyrrolizine]-1’-carboxylic acid 9e: White solid. Yield: 58%. M.p.: 218–219 °C (Decomp.). FT-IR (ATR) υ (cm−1): 3404, 3053, 1698, 1553. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.67–1.79 (m, 1 H, H-6′), 1.82–1.97 (m, 2 H, H-6′, H-7′), 1.98–2.08 (m, 1 H, H-7′), 2.23–2.32 (m, 1 H, H-5′), 2.36–2.44 (m, 1 H, H-5′), 2.64 (s, 3 H, N-CH3), 3.56 (t, J = 10.6 Hz, 1 H, H-1′), 3.88 (dt, J = 9.7, 6.3 Hz, 1 H, H-7a’), 4.74 (d, J = 11.5 Hz, 1 H, H-2′), 6.60 (d, J = 8.4 Hz, 1 H), 7.14–7.19 (m, 3 H), 7.24 (dd, J = 8.4, 2.0 Hz, 1 H), 7.31 (t, J = 7.7 Hz, 2 H), 7.46–7.52 (m, 1 H). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 26.5 (N-CH3), 27.7 (C-6′), 31.1 (C-7′), 47.6 (C-5′), 50.7 (C-1′), 61.3 (C-2′), 67.7 (C-7a’),71.8 (C-spiro), 110.4 (CH), 126.3 (C), 126.8 (CH), 126.8 (C), 127.6 (CH), 128.8 (CH), 129.9 (CH), 133.6 (CH), 136.8 (C), 142.4 (C), 173.3 (C), 177.5 (C), 197.4 (C). MS (EI, 70 eV) m/z: 424 (M+•, 3), 362 (100), 313 (4), 297 (11), 248 (49), 105 (92), 77 (89). Anal. Calcd. for C23H21ClN2O4: C: 65.02; H: 4.98; N: 6.59. Found: C: 65.08; H: 4.96; N: 6.57.
(1′RS,2′RS,3SR,7a′SR)-2’-Benzoyl-1-benzyl-5-chloro-2-oxo-1’,2’,5’,6’,7’,7a′-hexahydrospiro[indoline-3,3’-pyrrolizine]-1’-carboxylic acid 9f: White solid. Yield: 41%. M.p.: 222–224 °C. FT-IR (ATR) υ (cm−1): 3169, 2860, 1662, 1585. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.68–1.83 (m, 1 H), 1.83–1.98 (m, 2 H), 2.00–2.11 (m, 1 H), 2.26–2.35 (m, 1 H), 2.35–2.44 (m, 1 H), 3.67 (t, J = 10.4 Hz, 1 H, H-1′), 3.94 (dt, J = 9.6, 6.3 Hz, 1 H, H-7a’), 4.27 (d, J = 15.8 Hz, 1 H, N-CH), 4.72 (d, J = 15.8 Hz, 1 H, N-CH), 4.89 (d, J = 11.3 Hz, 1 H, H-2′), 6.54 (d, J = 8.4 Hz, 1 H), 6.97–7.04 (m, 2 H), 7.17 (d, J = 8.4 Hz, 1 H), 7.19–7.25 (m, 4 H), 7.30–7.38 (m, 4 H), 7.51–7.59 (m, 1 H). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 27.7 (C-6′), 31.0 (C-7′), 43.3 (N-CH2), 47.4 (C-5′), 51.2 (C-1′), 60.0 (C-2′), 67.5 (C-7a’), 71.6 (C-spiro), 111.1 (CH), 126.5 (C), 126.9 (C), 127.3 (CHx2), 127.9 (CH), 129.0 (CH), 129.1 (CH), 129.9 (CH), 134.0 (CH), 136.0 (C), 136.6 (C), 141.6 (C), 173.3 (C), 177.7 (C), 197.1 (C). MS (EI, 70 eV) m/z: 500 (M+•, 1), 438 (30), 387 (9), 347 (15), 324 (48), 257 (16), 105 (86), 91 (100). Anal. Calcd. for C29H25ClN2O4: C: 69.53; H: 5.03; N: 5.59. Found: C: 69.48; H: 5.04; N: 5.62.
(1′RS,2′RS,3SR,7a′SR)-2′-Benzoyl-5,7-dichloro-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid 9g: White solid. Yield: 58%. M.p.: 228–229 °C. FT-IR (ATR) υ (cm−1): 2956, 2949, 2855, 1682, 1584, 1510. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.68–1.81 (m, 1 H, H-6′), 1.81–1.96 (m, 2 H, H-6′, H-7′), 1.96–2.07 (m, 1 H, H-7′), 2.28–2.36 (m, 1 H, H-5′), 2.36–2.45 (m, 1 H, H-5′), 3.60 (t, J = 10.5 Hz, 1 H, H-1′), 3.87 (dt, J = 9.6, 6.3 Hz, 1 H, H-7a′), 4.79 (d, J = 11.5 Hz, 1 H, H-2′), 7.15 (s, 1 H), 7.30–7.39 (m, 5 H), 7.54 (t, J = 6.9 Hz, 1 H), 10.76 (s, 1 H, NH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 27.7 (C-6′), 30.9 (C-7′), 47.4 (C-5′), 50.9 (C-1′), 60.7 (C-2′), 67.6 (C-7a’), 72.6 (C-spiro), 115.3 (C), 126.1 (CH), 126.5 (C), 127.7 (CH), 128.3 (C), 129.0 (CH), 129.3 (CH), 133.8 (CH), 136.8 (C), 139.2 (C), 173.2 (C), 179.3 (C), 197.4 (C). MS (EI, 70 eV) m/z: 444 (M+•, 2), 382 (100), 353 (87), 325 (12), 268 (16), 105 (53), 77 (53). Anal. Calcd. for C22H18Cl2N2O4: C: 59.34; H: 4.07; N: 6.29. Found: C: 59.39; H: 4.08; N: 6.31.
(1′RS,2′RS,3SR,7a′SR)-2′-Benzoyl-5-methyl-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid 9h: White solid. Yield: 49%. M.p.: 222–224 °C. FT-IR (ATR) υ (cm−1): 2956, 2939, 2854, 1683, 1583, 1510. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.64–1.77 (m, 1 H), 1.78–1.93 (m, 2 H), 1.98–2.09 (m, 1 H), 2.21 (s, 3 H, 5-CH3), 2.23–2.31 (m, 1 H), 2.38–2.48 (m, 1 H), 3.56 (t, J = 10.5 Hz, 1 H, H-1′), 3.87 (dt, J = 9.9, 6.0 Hz, 1 H, H-7a′), 4.75 (d, J = 11.3 Hz, 1 H, H-2′), 6.35 (d, J = 7.7 Hz, 1 H), 6.86 (d, J = 9.4 Hz, 2 H), 7.27–7.39 (m, 4 H), 7.49 (t, J = 7.1 Hz, 1 H), 10.06 (s, 1 H, NH), 12.62 (s, 1 H, COOH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 21.2 (5-CH3), 27.3 (C-7′), 31.1 (C-6′), 47.6 (C-5′), 51.4 (C-1′), 59.7 (C-2′), 67.3 (C-7a′), 72.1 (C-spiro), 109.8 (CH), 125.0 (C), 127.9 (CH), 128.0 (CH), 128.8 (CH), 130.0 (CH), 130.3 (C), 133.5 (CH), 137.0 (C), 139.9 (C), 173.6 (C), 179.4 (C), 197.5 (C). MS (EI, 70 eV) m/z: 368 (4), 328 (100), 299 (92), 277 (17), 223 (14), 105 (43), 77 (30). Anal. Calcd. for C23H22N2O4: C: 70.75; H: 5.68; N: 7.18. Found: C: 70.87; H: 5.65; N: 7.14.
(1′RS,2′RS,3SR,7a′SR)-2′-Benzoyl-5-nitro-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid 9i: White solid. Yield: 40%. M.p.: 161–162 °C. FT-IR (ATR) υ (cm−1): 3595, 3234, 1734, 1678, 1520. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.68–1.82 (m, 1 H, H-6′), 1.82–1.96 (m, 2 H, H-6′, H-7′), 2.01–2.13 (m, 1 H, H-7′), 2.29–2.37 (m, 1 H, H-5′), 2.41–2.48 (m, 1 H, H-5′), 3.63 (t, J = 10.5 Hz, 1 H, H-1′), 3.94 (dt, J = 9.8, 6.2 Hz, 1 H, H-7a′), 4.85 (d, J = 11.1 Hz, 1 H, H-2′), 6.69 (d, J = 8.6 Hz, 1 H), 7.30–7.40 (m, 4 H), 7.49–7.56 (m, 1 H), 7.83 (d, J = 2.2 Hz, 1 H), 8.06 (dd, J = 8.8, 2.2 Hz, 1 H), 10.98 (br. s., 1 H, NH). 13C NMR (100 MHz, DMSO–d6, DEPT-135 experiment) δ (ppm): 27.4 (C-6′), 30.9 (C-7′), 47.7 (C-5′), 51.4 (C-1′), 59.9 (C-2′), 67.4 (C-7a′), 110.5 (CH), 122.7 (CH), 127.2 (CH), 127.9 (CH), 129.1 (CH), 134.1 (CH). MS (EI, 70 eV) m/z: 359 (100), 330 (70), 284 (22), 254 (10), 105 (24), 77 (18). Anal. Calcd. for C22H19N3O6: C: 62.70; H: 4.54; N: 9.97. Found: C: 62.77; H: 4.51; N: 9.92. The 13C spectrum could not be recorded due to solubility problems.
(1′RS,2′RS,3SR,7a′SR)-2′-Benzoyl-1-benzyl-5,7-dichloro-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid 9j: White solid. Yield: 86%. M.p.: 226–227 °C. FT-IR (ATR) υ (cm−1): 2968, 2872, 1728, 1705, 1676. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.74–1.87 (m, 1 H, H-6′), 1.88–2.00 (m, 2 H, H-6′, H-7′), 2.00–2.10 (m, 1 H, H-7′), 2.32–2.40 (m, 1 H, H-5′), 2.41–2.48 (m, 1 H, H-5′), 3.68 (t, J = 10.5 Hz, 1 H, H-1′), 3.93 (dt, J = 9.5, 6.5 Hz, 1 H, H-7a′), 4.60 (d, J = 16.8 Hz, 1 H, N-CH), 4.90 (d, J = 11.3 Hz, 1 H, H-2′), 4.99 (d, J = 16.6 Hz, 1 H, N-CH), 6.91 (d, J = 7.2 Hz, 2 H), 7.16–7.26 (m, 3 H), 7.27–7.33 (m, 4 H), 7.41 (t, J = 7.6 Hz, 2 H), 7.61 (t, J = 7.3 Hz, 1 H), 12.75 (br. s., 1 H, COOH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 28.1 (C-6′), 30.9 (C-7′), 44.7 (N-CH2), 47.1 (C-5′), 50.6 (C-1′), 61.4 (C-2′), 67.8 (C-7a’), 71.0 (C-spiro), 115.6 (C), 126.0 (CH), 126.4 (CH), 127.5 (CH), 127.7 (C), 127.7 (CH), 129.0 (CH), 129.1 (CH), 129.3 (C), 131.5 (CH), 134.0 (CH), 136.9 (C), 137.4 (C), 137.7 (C), 173.1 (C), 178.8 (C), 197.3 (C). MS (EI, 70 eV) m/z: 368 (2), 313 (3), 236 (6), 83 (52), 69 (100). Anal. Calcd. for C29H24Cl2N2O4: C: 65.06; H: 4.52; N: 5.23. Found: C: 65.22; H: 4.54; N: 5.21.
(1′RS,2′RS,3SR,7a′SR)-2′-Benzoyl-1-benzyl-5-fluoro-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid 9k: White solid. Yield: 80%. M.p.: 228–229 °C. FT-IR (ATR) υ (cm−1): 2963, 2864, 1724, 1703, 1678. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.68–1.83 (m, 1 H, H-6′), 1.83–1.99 (m, 2 H, H-6′, H-7′), 1.99–2.11 (m, 1 H, H-7′), 2.27–2.35 (m, 1 H, H-5′), 2.35–2.45 (m, 1 H, H-5), 3.69 (t, J = 10.5 Hz, 1 H, H-1′), 3.94 (dt, J = 9.6, 6.4 Hz, 1 H, H-7a′), 4.23 (d, J = 16.0 Hz, 1 H), 4.73 (d, J = 16.0 Hz, 1 H), 4.90 (d, J = 11.5 Hz, 1 H, H-2′), 6.51 (dd, J = 8.7, 4.2 Hz, 1 H), 6.95 (td, J = 9.0, 2.2 Hz, 1 H), 6.99–7.05 (m, 2 H), 7.13 (dd, J = 8.3, 2.2 Hz, 1 H), 7.18–7.25 (m, 3 H), 7.30–7.39 (m, 4 H), 7.55 (t, J = 6.6 Hz, 1 H), 12.70 (br. s., 1 H, COOH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 27.7 (C-6′), 31.1 (C-7′), 43.3 (N-CH2), 47.4 (C-5′), 51.1 (C-1′), 60.0 (C-2′), 67.5 (C-7a’), 71.9 (C-spiro), 110.5 (d, JC-F = 8.0 Hz, CH), 115.3 (d, JC-F = 24.8 Hz, CH),116.4 (d, JC-F = 23.3 Hz, CH), 126.3 (d, JC-F = 7.3 Hz, C) (C), 127.3 (CH), 127.8 (CH), 127.9 (CH), 129.0 (CH), 129.1 (CH), 133.9 (CH), 136.1 (C), 136.7 (C), 138.9 (C), 157.3 (C), 159.7 (C), 173.4 (C), 177.9 (C), 197.1 (C). MS (EI, 70 eV) m/z: 422 (38), 371 (10), 331 (14), 308 (21), 105 (32), 91 (100). Anal. Calcd. for C29H25FN2O4: C: 71.89; H: 5.20; N: 5.78. Found: C: 71.78; H: 5.22; N: 5.76.
(1′RS,2′RS,3SR,7a′SR)-2′-Benzoyl-5-fluoro-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid 9l: White solid. Yield:71%. M.p.: 227–228 °C. FT-IR (ATR) υ (cm−1): 3321, 2862, 1711, 1692, 1676. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.64–1.80 (m, 1 H, H-6′), 1.80–1.96 (m, 2 H, H-6′, H-7′), 1.96–2.08 (m, 1 H, H-7′), 2.24–2.36 (m, 1 H, H-5′), 2.36–2.46 (m, 1 H, H-5′), 3.61 (t, J = 10.5 Hz, 1 H, H-1′), 3.87 (dt, J = 9.6, 6.2 Hz, 1 H, H-7a′), 4.78 (d, J = 11.3 Hz, 1 H, H-2′), 6.45 (dd, J = 8.4, 4.3 Hz, 1 H), 6.91 (td, J = 9.0, 2.2 Hz, 1 H), 7.00 (dd, J = 8.4, 2.0 Hz, 1 H), 7.34 (t, J = 7.6 Hz, 2 H), 7.40 (d, J = 7.4 Hz, 2 H), 7.52 (t, J = 7.1 Hz, 1 H), 10.21 (s, 1 H, NH), 12.63 (br. s., 1 H, COOH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 27.5 (C-6′), 31.0 (C-7′), 47.5 (C-5′), 51.2 (C-1′), 59.8 (C-2′), 67.4 (C-7a′), 72.3 (C-spiro), 110.8 (d, JC-F = 8.0 Hz, CH), 115.2 (d, JC-F = 24.8 Hz, CH), 116.3 (d, JC-F = 22.6 Hz, CH), 126.7 (d, JC-F = 7.3 Hz, C), 127.9 (CH), 128.9 (CH), 133.8 (CH), 136.8 (C), 138.5 (C), 156.7 (C), 159.1 (C), 173.4 (C), 179.5 (C), 197.2 (C). MS (EI, 70 eV) m/z: 394 (M+•, 1), 368 (2), 332 (83), 303 (75), 275 (10), 218 (29), 105 (46), 77 (33), 73 (55), 44 (100). Anal. Calcd. for C22H19FN2O4: C: 67.00; H: 4.86; N: 7.10. Found: C: 66.96; H: 4.83; N: 7.05.
(1′RS,2′RS,3SR,7a′SR)-2′-Benzoyl-5-chloro-1-hexyl-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid 9m: White solid. Yield: 78%. M.p.: 167–170 °C. FT-IR (ATR) υ (cm−1): 2930, 2855, 1724,1680. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 0.81 (t, J = 6.9 Hz, 3 H, CH3), 0.99–1.20 (m, 8 H, CH2 × 4), 1.27 (d, J = 15.8 Hz, 1 H), 1.68–1.80 (m, 1 H), 1.82–1.95 (m, 2 H), 1.97–2.10 (m, 1 H), 2.22–2.30 (m, 1 H), 2.32–2.41 (m, 1 H), 3.05–3.17 (m, 1 H), 3.62 (dd, J = 11.2, 9.9 Hz, 1 H, H-1′), 3.89 (dt, J = 9.7, 6.4 Hz, 1 H, H-7a′), 4.81 (d, J = 11.5 Hz, 1 H, H-2′), 6.71 (d, J = 8.4 Hz, 1 H), 7.19 (d, J = 1.95 Hz, 1 H), 7.22–7.29 (m, 3 H), 7.30–7.35 (m, 2 H), 7.46–7.53 (m, 1 H), 12.65 (br.s., 1 H, COOH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 14.3 (CH3), 22.4 (CH2), 26.1 (CH2), 26.7 (CH2), 27.8 (CH2), 31.1 (CH2), 31.2 (CH2), 39.9 (CH2), 47.3 (CH2), 51.0 (C-1′), 60.2 (C-2′), 67.5 (C-7a′), 71.6 (C-spiro), 110.6 (CH), 126.5 (C), 126.5 (C), 127.2 (CH), 127.8 (CH), 128.9 (CH), 129.9 (CH), 133.7 (CH), 136.8 (C), 142.0 (C), 173.3 (C), 177.2 (C), 197.2 (C). MS (EI, 40 eV) m/z: 494 (M+•, 1), 458 (2), 432 (30), 367 (25), 318 (100), 105 (67), 77 (39), 43 (62). Anal. Calcd. for C28H31ClN2O4: C: 67.94; H: 6.31; N: 5.66. Found: C: 67.82; H: 6.35; N: 5.61.

2.1.2. General Procedure for the Synthesis of Spirooxindoles 11a–j

Equimolar amounts of the corresponding substituted isatin 6, pipecolic acid 7b and trans-3-benzoyl acrylic acid 8 were dissolved in 10 mL of acetonitrile in a flat bottom flask and subjected to reflux for 8 h, (TLC control). The solid formed was isolated by filtration on vacuum and washed with cold acetonitrile. The products did not require further purification.
(1′RS,2′RS,3SR,8a′SR)-2′-Benzoyl-5-methyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro[indoline-3,3′-indolizine]-1′-carboxylic acid 11a [39]: White solid. Yield: 48%. M.p.: 256–257 °C. FT-IR (ATR) υ (cm−1): 3364, 3225, 2957, 1741, 1704, 1668. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.10–1.20 (m, 2 H, H-8′, H-7′), 1.21–1.32 (m, 1 H, H-6′), 1.43–1.50 (m, 1 H, H-7′), 1.67–1.74 (m, 1 H, H-8′), 2.05–2.18 (m, 5H, 5-CH3, H-5′, H-6′), 2.23 (td, J = 10.9, 2.8 Hz, 1 H, H-5′), 3.22–3.29 (m, 1 H, H-8a′), 3.44 (t, J = 9.8 Hz, 1 H, H-1′), 4.47 (d, J = 9.6 Hz, 1 H, H-2′), 6.30 (d, J = 7.8 Hz, 1 H), 6.57 (s, 1 H), 6.71 (d, J = 7.8 Hz, 1 H), 7.28 (t, J = 7.7 Hz, 2 H), 7.38 (d, J = 7.3 Hz, 2 H), 7.43 (t, J = 7.3 Hz, 1 H), 10.35 (s, 1 H, NH), 12.62 (s, 1 H, COOH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 21.0 (CH3), 23.8 (C-8′), 25.5 (C-7′), 31.8 (C-6′), 45.6 (C-5′), 50.5 (C-1′), 54.9 (C-2′), 61.4 (C-8a′), 71.4 (C-spiro), 109.1 (CH), 126.7 (CH), 127.5 (C), 127.6 (CH), 128.8 (CH), 129.7 (CH), 130.9 (C), 133.4 (CH), 137.2 (C), 140.3 (C), 173.6 (C), 179.3 (C), 197.6 (C). MS (EI, 70 eV) m/z: 404 (M+•, 9), 368 (5), 332 (39), 315 (15), 271 (30), 255 (17), 227 (69), 141 (33), 105 (100), 77 (69). Anal. Calcd. for C24H24N2O4: C: 71.27; H: 5.98; N: 6.93. Found: C: 71.18; H: 5.96; N: 6.89.
(1′RS,2′RS,3SR,8a′SR)-2′-Benzoyl-5-fluoro-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro[indoline-3,3′-indolizine]-1′-carboxylic acid 11b [39]: White solid. Yield: 68%. M.p.: 235–236 °C. FT-IR (ATR) υ (cm−1): 3478, 3096, 2937, 1715, 1676. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.11–1.23 (m, 2 H, H-8′. H-7′), 1.23–1.33 (m, 1 H, H-6′), 1.42–1.54 (m, 1 H, H-7′), 1.66–1.79 (m, 1 H, H-8′), 2.05–2.19 (m, 2 H, H-5′, H-6′), 2.20–2.32 (m, 1 H, H-5′), 3.25 (t, J = 10.0 Hz, 1 H, H-8a′), 3.45 (t, J = 9.7 Hz, 1 H, H-1′), 4.50 (d, J = 9.7 Hz, 1 H, H-2′), 6.41 (dd, J = 8.6, 4.3 Hz, 1 H), 6.54 (dd, J = 8.2, 2.7 Hz, 1 H), 6.78 (td, J = 9.0, 2.7 Hz, 1 H), 7.32 (t, J = 7.6 Hz, 2 H), 7.40–7.51 (m, 3 H), 10.52 (s, 1 H, NH), 12.69 (s, 1 H, COOH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 23.7 (C-8′), 25.5 (C-7′), 31.8 (C-6′), 45.6 (C-5′), 50.5 (C-1′), 55.1 (C-2′), 61.5 (C-8a′), 71.6 (C-spiro), 110.4 (d, JC-F = 6.9 Hz, CH), 113.5 (d, JC-F = 24.7 Hz, CH), 116.0 (d, JC-F = 23.6 Hz, CH), 127.6 (CH), 129.0 (CH), 129.4 (d, JC-F = 7.4 Hz, C), 133.8 (CH), 136.9 (C), 139.0 (C), 159.5 (C), 173.4 (C), 179.3 (C), 197.4 (C). MS (EI, 70 eV) m/z: 408 (M+•, 10), 368 (17), 336 (37), 313 (12), 275 (36), 259 (16), 231 (39), 141 (22), 105 (49), 77 (34). Anal. Calcd. for C23H21FN2O4: C: 67.64; H: 5.18; N: 6.86. Found: C: 67.54; H: 5.19; N: 6.84.
(1′RS,2′RS,3SR,8a′SR)-2′-Benzoyl-1-methyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro[indoline-3,3′-indolizine]-1′-carboxylic acid 11c [39]: White solid. Yield: 49%. M.p.: 219–220 °C. FT-IR (ATR) υ (cm−1): 2941, 2356, 1709, 1677. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.09–1.21 (m, 2 H, H-8′, H-7′), 1.21–1.36 (m, 1 H, H-6′), 1.38–1.52 (m, 1 H, H-7′), 1.66–1.81 (m, 1 H, H-8′), 2.01–2.18 (m, 2 H, H-5′, H-6′), 2.23 (t, J = 10.2 Hz, 1 H, H-5′), 2.98 (s, 3 H, N-CH3), 3.22–3.29 (m, 1 H, H-8a′), 3.42 (t, J = 10.0 Hz, 1 H, H-1′), 4.47 (d, J = 9.8 Hz, 1 H, H-2′), 6.52 (d, J = 7.8 Hz, 1 H), 6.79–6.90 (m, 2 H), 7.01 (t, J = 7.5 Hz, 1 H), 7.20–7.31 (m, 4 H), 7.37–7.47 (m, 1 H), 12.66 (s, 1 H, COOH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 23.7 (C-8′), 25.4 (C-7′), 26.2 (CH3), 31.8 (C-6′), 45.6 (C-5′), 50.4 (C-1′), 55.9 (C-2′), 61.8 (C-8a′), 71.1 (C-spiro), 108.2 (CH), 122.9 (CH), 125.6 (CH), 127.0 (C), 127.4 (CH), 128.7 (CH), 129.6 (CH), 133.5 (CH), 137.0 (C), 144.0 (C), 173.4 (C), 177.4 (C), 197.3 (C). MS (EI, 70 eV) m/z: 404 (M+•, 1), 393 (12), 368 (22), 339 (26), 313 (70), 264 (34), 236 (16), 57 (100). Anal. Calcd. for C24H24N2O4: C: 71.27; H: 5.98; N: 6.93. Found: C: 71.09; H: 5.97; N: 6.91.
(1′RS,2′RS,3SR,8a′SR)-2′-Benzoyl-5-chloro-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro[indoline-3,3′-indolizine]-1′-carboxylic acid 11d: White solid. Yield: 68%. M.p.: 225 °C (Decomp). FT-IR (ATR) υ (cm−1): 3370, 3218, 2955, 2360, 1742, 1707. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.12–1.22 (m, 2 H, H-8′, H-7′), 1.22–1.33 (m, 1 H, H-6′), 1.40–1.53 (m, 1 H, H-7′), 1.64–1.76 (m, 1 H, H-8′), 2.09–2.19 (m, 2 H, H-5′, H-6′), 2.21–2.31 (m, 1 H, H-5′), 3.24 (t, J = 9.3 Hz, 1 H, H-8a′), 3.44 (t, J = 9.8 Hz, 1 H, H-1′), 4.50 (d, J = 9.6 Hz, 1 H, H-2′), 6.43 (d, J = 8.3 Hz, 1 H), 6.71 (s, 1 H), 6.99 (dd, J = 8.3, 2.3 Hz, 1 H), 7.32 (t, J = 7.6 Hz, 2 H), 7.41 (d, J = 7.3 Hz, 2 H), 7.47 (t, J = 7.3 Hz, 1 H), 10.66 (s, 1 H, NH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 23.7 (C-8′), 25.5 (C-7′), 31.8 (C-6′), 45.7 (C-5′), 50.5 (C-1′), 55.2 (C-2′), 61.6 (C-8a′), 71.4 (C-spiro), 111.0 (CH), 126.0 (CH), 126.2 (C), 127.6 (CH), 129.1 (CH), 129.4 (CH), 129.7 (C), 133.8 (CH), 137.0 (C), 141.7 (C), 173.4 (C), 179.0 (C), 197.5 (C). MS (EI, 70 eV) m/z: 424 (M+•,19), 368 (14), 352 (73), 335 (30), 313 (38), 291 (65), 247 (71), 105 (100), 77 (76). Anal. Calcd. for C23H21ClN2O4: C: 65.02; H: 4.98; N: 6.59. Found: C: 65.20; H: 4.96; N: 6.57.
(1′RS,2′RS,3SR,8a′SR)-2′-Benzoyl-1-benzyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro[indoline-3,3′-indolizine]-1′-carboxylic acid 11e: White solid. Yield: 45%. M.p.: 203–205 °C. FT-IR (ATR) υ (cm−1): 3225, 2962, 1738, 1681. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.13–1.32 (m, 3 H, H-8′, H-7′, H-6′), 1.43–1.51 (m, 1 H, H-7′), 1.67–1.78 (m, 1 H, H-8′), 2.04–2.13 (m, 1 H, H-5′), 2.15–2.22 (m, 1 H, H-6′), 2.21–2.30 (m, 1 H, H-5′), 3.33 (t, J = 10.0 Hz, 1 H, H-8a′), 3.49 (t, J = 9.8 Hz, 1 H, H-1′), 4.53 (d, J = 9.5 Hz, 1 H, H-2′), 4.63 (d, J = 15.6 Hz, 1 H, CH), 4.89 (d, J = 15.6 Hz, 1 H, CH), 6.54 (d, J = 7.8 Hz, 1 H), 6.83 (d, J = 4.4 Hz, 2 H), 6.95 (dq, J = 8.1, 4.1, 3.6 Hz, 1 H), 7.13 (t, J = 7.7 Hz, 2 H), 7.23–7.37 (m, 7H), 7.39 (t, J = 7.4 Hz, 1 H).13C NMR (100 MHz, DMSO–d6) δ (ppm): 23.7 (C-8′), 25.5 (C-7′), 31.9 (C-6′), 43.3 (CH2), 45.6 (C-5′), 50.6 (C-1′), 55.2 (C-2′), 61.6 (C-8a′), 71.1 (C-spiro), 109.0 (CH), 123.0 (CH), 126.1 (CH), 126.7 (C), 127.6 (CH), 128.0 (CH), 128.8 (CH), 129.2 (CH), 129.6 (CH), 133.5 (CH), 136.7 (C), 136.9 (C), 143.3 (C), 173.5 (C), 177.6 (C), 197.3 (C). MS (EI, 70 eV) m/z: 480 (M+•, 9), 444 (12), 418 (95), 389 (10), 339 (80), 331 (100), 141 (28), 105 (39), 91 (35), 77 (18). Anal. Calcd. for C30H28N2O4: C: 74.98; H: 5.87; N: 5.83. Found: C: 75.07; H: 5.89; N: 5.80.
(1′RS,2′RS,3SR,8a′SR)-2′-Benzoyl-1-hexyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro[indoline-3,3′-indolizine]-1′-carboxylic acid 11f [39]: White solid. Yield: 48%. M.p.: 176–177 °C. FT-IR (ATR) υ (cm−1): 2931, 2858, 1723, 1684, 1662. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 0.79–0.91 (m, 3 H, CH3), 1.08–1.22 (m, 2 H), 1.20–1.33 (m, 7H), 1.35–1.57 (m, 3 H), 1.66–1.78 (m, 1 H), 2.00–2.10 (m, 1 H), 2.11–2.27 (m, 2 H), 3.23–3.29 (m, 1 H, H-8a′), 3.37–3.51 (m, 2 H, NCH, H-1′), 3.60 (dt, J = 14.5, 7.4 Hz, 1 H, NCH), 4.48 (d, J = 9.7 Hz, 1 H, H-2′), 6.60 (d, J = 7.8 Hz, 1 H), 6.74–6.91 (m, 2 H), 7.01 (t, J = 7.5 Hz, 1 H), 7.19–7.32 (m, 4 H), 7.42 (t, J = 7.2 Hz, 1 H), 12.68 (s, 1 H, COOH). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 14.3 (CH3), 22.5 (CH2), 23.7 (C-8′), 25.5 (C-7′), 26.4 (CH2), 27.4 (CH2), 31.3 (CH2), 31.8 (C-6′), 39.7 (CH2), 45.5 (C-5′), 50.5 (C-1′), 55.4 (C-2′), 61.6 (C-8a′), 70.9 (C-spiro), 108.4 (CH), 122.7 (CH), 126.0 (CH), 126.9 (C), 127.5 (CH), 128.8 (CH), 129.6 (CH), 133.5 (CH), 143.5 (C), 173.5 (C), 177.2 (C), 197.4 (C). MS (EI, 70 eV) m/z: 475 (M+•+ H, 3), 368 (22), 339 (35), 313 (75), 264 (39). Anal. Calcd. for C29H34N2O4: C: 73.39; H: 7.22; N: 5.90. Found: C: 73.55; H: 7.25; N: 5.93.
(1′RS,2′RS,3SR,8a′SR)-2′-Benzoyl-5-chloro-1-methyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro[indoline-3,3′-indolizine]-1′-carboxylic acid 11g [39]: White solid. Yield: 69%. M.p.: 224–226 °C. FT-IR (ATR) υ (cm−1): 3378, 3227, 2957, 1744, 1708, 1668. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.11–1.22 (m, 2 H), 1.22–1.36 (m, 1 H), 1.40–1.50 (m, 1 H), 1.66–1.79 (m, 1 H), 2.05–2.19 (m, 2 H), 2.19–2.29 (m, 1 H), 2.97 (s, 3 H, CH3), 3.24 (td, J = 10.2, 2.5 Hz, 1 H, H-8a′), 3.43 (t, J = 10.0 Hz, 1 H, H-1′), 4.48 (d, J = 9.9 Hz, 1 H, H-2′), 6.56 (d, J = 8.3 Hz, 1 H), 6.79 (d, J = 2.1 Hz, 1 H), 7.09 (dd, J = 8.3, 2.2 Hz, 1 H), 7.23–7.35 (m, 4 H), 7.45 (td, J = 7.0, 1.6 Hz, 1 H). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 23.6 (C-8′), 25.4 (C-7′), 26.4 (CH3), 31.7 (C-6′), 45.7 (C-5′), 50.2 (C-1′), 56.1 (C-2′), 62.0 (C-8a′), 71.1 (C-spiro), 109.9 (CH), 125.5 (CH), 127.0 (C), 127.4 (CH), 128.9 (CH), 129.2 (C), 129.5 (CH), 133.8 (CH), 136.8 (C), 142.9 (C), 173.3 (C), 177.0 (C), 197.3 (C). MS (EI, 70 eV) m/z: 438 (M+•, 1), 336 (17), 313 (18), 275 (17), 231 (30), 141 (41), 105 (91), 77 (55), 57 (87), 43 (100). Anal. Calcd. for C24H23ClN2O4: C: 65.68; H: 5.28; N: 6.38. Found: C: 65.56; H: 5.29; N: 6.35.
(1′RS,2′RS,3RS,8a′SR)-2′-Benzoyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro[indoline-3,3′-indolizine]-1′-carboxylic acid 11h: White solid. Yield: 53%. M.p.: 225–226 °C. FT-IR (ATR) υ (cm−1): 3479, 3380, 2957, 1741, 1706. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.11–1.20 (m, 2 H, H-8′, H-7′), 1.21–1.32 (m, 1 H, H-6′), 1.43–1.52 (m, 1 H, H-7′), 1.68–1.76 (m, 1 H, H-8′), 2.08–2.19 (m, 2 H, H-5′, H-6′), 2.20–2.29 (m, 1 H, H-5′), 3.22–3.30 (m, 1 H, H-8a′), 3.45 (t, J = 9.9 Hz, 1 H, H-1′), 4.48 (d, J = 9.6 Hz, 1 H, H-2′), 6.41 (d, J = 7.6 Hz, 1 H), 6.76 (d, J = 4.3 Hz, 2 H), 6.92 (dt, J = 8.2, 4.4 Hz, 1 H), 7.28 (t, J = 7.5 Hz, 2 H), 7.39 (d, J = 7.8 Hz, 2 H), 7.44 (t, J = 7.3 Hz, 1 H), 10.47 (s, 1 H, NH), 12.63 (s, 1 H, COOH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 23.7 (C-8′), 25.5 (C-7′), 31.8 (C-6′), 45.5 (C-5′), 50.6 (C-1′), 55.0 (C-2′), 61.4 (C-8a′), 71.3 (C-spiro), 109.4 (CH), 122.2 (CH), 126.2 (CH), 127.5 (C), 127.6 (CH), 128.9 (CH), 129.5 (CH), 133.5 (CH), 137.0 (C), 142.7 (C), 173.6 (C), 179.3 (C), 197.5 (C). MS (EI, 70 eV) m/z: 390 (M+•, 1), 318 (7), 257 (13), 213 (23), 141 (44), 105 (100), 77 (68). Anal. Calcd. for C23H22N2O4: C: 70.75; H: 5.68; N: 7.18. Found: C:70.54; H: 5.68; N: 7.16.
(1′RS,2′RS,3SR,8a′SR)-2′-Benzoyl-1-benzyl-5-chloro-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro[indoline-3,3′-indolizine]-1′-carboxylic acid 11i: White solid. Yield: 67%. M.p.: 218–219 °C. FT-IR (ATR) υ (cm−1): 3225, 2944, 1735, 1677. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 1.10–1.24 (m, 2 H, H-8′, H-7′), 1.24–1.35 (m, 1 H, H-6′), 1.44–1.52 (m, 1 H. H-7′), 1.69–1.79 (m, 1 H, H-8′), 2.07–2.13 (m, 1 H, H-5′), 2.14–2.22 (m, 1 H, H-6′), 2.22–2.30 (m, 1 H, H-5′), 3.32 (t, J = 9.9 Hz, 1 H, H-8a′), 3.49 (t, J = 9.7 Hz, 1 H, H-1′), 4.55 (d, J = 9.5 Hz, 1 H, H-2′), 4.64 (d, J = 15.5 Hz, 1 H. CH), 4.88 (d, J = 15.6 Hz, 1 H, CH), 6.57 (d, J = 9.5 Hz, 1 H), 6.78 (s, 1 H), 7.04 (d, J = 8.3 Hz, 1 H), 7.17 (t, J = 7.6 Hz, 2 H), 7.25–7.37 (m, 7H), 7.42 (t, J = 7.4 Hz, 1 H). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 23.6 (C-8′), 25.5 (C-7′), 31.8 (C-6′), 43.4 (CH2), 45.8 (C-5′) 50.5 (C-1′), 55.3 (C-2′), 61.7 (C-8a′), 71.1 (C-spiro), 100.0 (C), 110.6 (CH), 126.0 (CH), 127.2 (C), 127.6 (CH), 128.0 (CH),128.1 (CH) 128.9 (C), 129.0 (CH), 129.2 (CH), 129.5 (CH), 133.4 (CH), 136.3 (C), 136.8 (C), 142.2 (C), 173.4 (C), 177.1 (C), 197.3 (C). MS (EI, 70 eV) m/z: 514 (M+•, 19), 478 (9), 452 (55), 373 (81), 365 (86), 141 (27), 105 (55), 91 (100), 77 (64). Anal. Calcd. for C30H27ClN2O4: C: 69.97; H: 5.28; N: 5.44. Found: C: 70.07; H: 5.33; N: 5.40.
(1′RS,2′RS,3SR,8a′SR)-2′-Benzoyl-5-chloro-1-hexyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro[indoline-3,3′-indolizine]-1′-carboxylic acid 11j: White solid. Yield: 65%. M.p.: 226–228 °C. FT-IR (ATR) υ (cm−1): 2932, 2857, 1711, 1701, 1678. 1H NMR (400 MHz, DMSO–d6) δ (ppm): 0.80–0.88 (m, 3 H, CH3), 1.14–1.31 (m, 9H), 1.33–1.52 (m, 3 H), 1.66–1.77 (m, 1 H), 2.02–2.11 (m, 1 H), 2.11–2.26 (m, 2 H), 3.21–3.29 (m, 1 H, H-8a′), 3.36–3.49 (m, 2 H, NCH, H-1′), 3.59 (dt, J = 14.6, 7.5 Hz, 1 H, NCH), 4.49 (d, J = 9.8 Hz, 1 H, H-2′), 6.65 (d, J = 8.4 Hz, 1 H), 6.78 (s, 1 H), 7.09 (dd, J = 8.3, 2.3 Hz, 1 H), 7.24–7.36 (m, 4 H), 7.44 (d, J = 7.1 Hz, 1 H), 12.71 (s, 1 H, COOH). 13C NMR (100 MHz, DMSO–d6) δ (ppm): 14.3 (CH3), 22.5 (CH2), 23.6 (C-8′), 25.4 (C-7′), 26.4 (CH2), 27.3 (CH2), 31.3 (CH2), 31.7 (C-6′), 45.6 (C-5′), 50.3 (C-1′), 55.5 (C-2′), 61.8 (C.8a′), 70.9 (C-spiro), 110.1 (CH), 125.9 (CH), 126.9 (C), 127.5 (CH), 129.0 (CH), 129.1 (C), 129.5 (CH), 133.7 (CH), 142.4 (C), 173.3 (C), 176.8 (C), 197.4 (C). MS (EI, 70 eV) m/z: 508 (M+•, 13), 472 (6), 446 (41), 367 (65), 359 (69), 332 (16), 141 (100), 105 (74), 77 (34). Anal. Calcd. for C29H33ClN2O4: C: 68.43; H: 6.53; N: 5.50. Found: C: 68.47; H: 6.57; N: 5.53.

2.2. Biology

2.2.1. Antibacterial Activity

The novel compounds were screened for bacterial activity using the agar diffusion method as described before [40]. Briefly, 10 µL of each compound stock solution (100 mg/mL) were added into 6 mm wells in Mueller Hinton agar (MHA) inoculated with the selected bacterial strain and incubated 24 h at 35–37 °C. The solvent (dimethyl sulfoxide (DMSO)) was included as negative control and known antibiotics such as gentamicin and trimethoprim sulfamethoxazole were positive controls for inhibition. To test N. gonorrhoeae, this method was performed by inoculating 200 µL of bacterial suspensions (1.5 × 108 CFU/mL) onto enriched gonococcal agar (GC) before adding the compounds into each well, then the plates were incubated up to 48 h at 35–37 °C in 5% CO2 atmosphere. Tetracycline and penicillin were included as growth inhibition controls. Controls were interpreted using the quality ranges established by the standards [38], assays were considered valid as all the controls achieved the standard values.
Compounds inducing growth inhibition in more than one experiment and an inhibition diameter >13 mm were subjected to microdilution test to determine the minimum inhibitory concentration (MIC) according with the standard [38]. The final readings were performed using resazurin (125 µg/mL). Known inhibitors were included as controls (gentamicin and tetracycline), assays were done at least twice. To define the MIC for N. gonorrhoeae the method was performed following the standard with modifications [40]. A 1% oxidase solution was used to determine endpoints of bacterial growth. Experiments were conducted in two different days.

2.2.2. Toxicity Studies Using the In Vivo Model

The level of toxicity was determined for the potentially active compounds. Six- instar larvae of Galleria mellonella were inoculated with 10 µL of the selected compound up to 2000 mg/kg [41,42]. The larvae viability was checked every 24 h over 5 days and the half lethal dose (LD50) was determined.

2.2.3. Hemolytic Activity

The ability to induce hemolysis in human red blood cells (huRBCs) of compounds with antibacterial effect was assessed using the spectrophotometry method [43]. Briefly, 240 μL of 5% huRBC in phosphate buffer saline (PBS) were placed into 96-well plates and subsequently exposed to 200 μg/mL of the selected compounds (i.e., 10 μL of 5 mg/mL working solution of each compound in Mueller Hinton broth (MHB) with 5% DMSO 0.1% Tween-80). As positive control 10 μL SDS 1% was added. For a negative control, only medium with no chemicals was added to huRBC. Measurements were performed at 540 nm in a microplate spectrophotometer. Experiments were performed at least twice.

2.3. Molecular Modeling

In Silico Analysis of Toxicity and Inverse Docking Analysis

Chemical structures from the most active compounds were examined using Protox II [44]. Protox II uses several models by comparison of similar structures to predict hepatotoxicity, immunotoxicity, carcinogenicity, mutagenicity, cytotoxicity and metabolic interactions [44].
Inverse docking analysis was performed in triplicate using AutoDock Vina.(autodock vina 1.1.2) Crystallographic protein structures were downloaded from the Protein Data Bank. Target optimization was executed using a python script (Python 3.10.9) which removed water molecules, heteroatoms and non-polar hydrogens. A Chimera X command line was launched to minimize the energy and add the corresponding charges (AMBER). Compounds’ 3D structures were converted into the “sdf” format and using Gaussian 09, they were optimized with the B3LYP multiparameter functional and a 6–31 G Gaussian basis set. An Open Babel 2.4.1 command line was executed to add polar hydrogens, Gasteiger charges, and free bond rotation modes into the corresponding “pdbqt” format files. A bash script was designed in order to perform each protein docking. The docking site was settled up into a box located in the coordinates of the co-crystalized ligand found on the PDB protein file. Each run was executed with a total of 10 number modes, an exhaustiveness of 100, and using a total of 8 cpus for each target. A python script was designed in order to analyze, compare and sort the docking results [45]. Physicochemical descriptors were calculated using SwissADME online server (http://www.swissadme.ch, accessed on 24 June 2024) [46].

3. Results and Discussion

3.1. Synthetic Approaches

The 1,3-CD tricomponent reaction between N-benzylisatin 6 (R1 = Bn, R2 = H), L-proline 7a and trans-3-benzoylacrylic acid 8 was performed as a model reaction (Scheme 1) under different reaction conditions (Table 1).
All reactions were followed by TLC. Initially, the reaction was evaluated in ethanol as solvent under reflux for 2 h with the formation of a small amount of a new product along with unreacted precursors (Entry 1, Table 1). The isolation and preliminary characterization of this product by mass spectrometry (showing a molecular ion peak with m/z = 466), indicated that it could correspond to either structure 9a or its regioisomeric analogue 10a (in any case with a yield of 26%). In an attempt to improve the yield of the new product 9a/10a, the reaction was carried out in acetonitrile, affording an improvement of the reaction yield (56%) (Entry 2, Table 1). When water and methanol were used as solvents we observed a significant increase in the reaction yields (i.e., 59% and 61%, respectively) (Entries 3 and 4, Table 1). Finally, when the reaction was performed in refluxing THF the isolated product 9a/10a was obtained in 45% yield (Entry 5, Table 1). According to the above results, the refluxing methanol afforded the better yield for which this reaction condition was adopted as the optimal to continue with the experiments.
Regarding the structure of the isolated solid 9a/10a, its elucidation was achieved by (1D, 2D)–NMR, FT-IR, and mass spectrometry. The mass spectrum of this compound showed a molecular ion peak with m/z = 466 which could be assigned to either spirooxindole 9a or 10a but served as initial evidence for us of the formation of the product of interest. Preliminary analysis of the NMR spectrum allowed us to rule out the formation of a mixture of both regioisomers (9a and 10a), as no signal duplication was observed. In the 1H NMR spectrum of product 9a/10a (see Supplementary Data), the signals corresponding to the formation of the bicyclic pyrrolizinic system (i.e., H–2′, H–1′ and H–7a′ protons) are highlighted (the numbering of the pyrrolizinic moiety is shown in Figure 2).
Independent of the 9a/10a regioisomer formed, the H–2′ proton is observed as a doublet (by its neighboring with H–1′ proton) at 4.87 ppm with J = 11.3 Hz, the H–1′ proton appears as a false triplet at 3.62 ppm with J = 10.4 Hz, while the H–7a′ proton appears as a multiplet in the region of 3.92–3.97 ppm. The signals of the six protons belonging to the three methylene groups were observed at high field in the region of 1.60–2.45 ppm. The signals of the diastereotopic protons of CH2-Ph (Bn) susbtituent were observed at 4.22 ppm and 4.73 ppm as doublets with J = 15.8 Hz for each one, while the proton signal of the CO2H group is observed as a singlet at 12.71 ppm. The signals for the fourteen aromatic protons were observed in the region between 6.50–7.60 ppm. In the 13C NMR spectrum and DEPT-135 of the product 9a or 10a (see Supplementary Data) the characteristic signals that gave us the confirmation of the formation of the pyrrolizinic ring were observed. In the 13C NMR spectrum and DEPT-135 of the product 9a or 10a (see Supplementary Data) the characteristic signals that allowed us to confirm the formation of the bicyclic pyrrolizinic moiety are highlighted. Signals for the three methylene carbon atoms C–7′, C–6′ and C–5′ are observed at high field at 31.3, 27.4, and 47.5 ppm, respectively. The spiro-carbon atom signal C–3′ was observed at 71.7 ppm. The C–1′, C–2′ and C–7a′ carbon atom signals were observed at 51.4, 59.9 and 67.4 ppm, respectively. The ten signals belonging to the CH aromatic carbon atoms were also observed at 109.6, 122.5, 127.3, 127.4, 127.8, 127.9, 128.8, 129.1, 129.9 and 133.7 ppm, and the three C=O carbon atoms at 173.4, 178.0 and 197.2 ppm. In the FT-IR spectrum is highlighted some relevant signals like the C-H stretching bands at 2957 and 2864 cm−1, and the C=O stretching bands at 1726, 1697 and 1678 cm−1. Resuming, the above spectroscopic data and the long range 2′-H–C correlations in the HMBC experiment suggests that the correct structure for the isolated solid is more likely associated to regioisomer 9a than 10a.
Once an acceptable structure of the isolated solid 9a was established, the generality and scope of this reaction was extended to various N-alkyl- and aromatic ring-substituted isatins 6, affording product 9 in 40–86% yields (Table 2).
All products were fully characterized by NMR, FT-IR, and MS (see Supplementary Data). Additionally, the growth of single crystals of product 9d suitable for X-ray diffraction unequivocally confirmed the regioselective formation of the spiro-derivative 9 instead of 10, as shown in Figure 3 [38].
Subsequently, the synthetic utility of the established reaction conditions was extended to the synthesis of a new series of spirooxindoles, 11/12, switching the L-proline 7a by pipecolic acid 7b (Table 3). Initially, a model reaction starting with pipecolic acid 7b, 5-methylisatin 6 (R1 = H, R2 = 5-Me) and trans-3-benzoylacrylic acid 8 was subjected to the previously established reaction conditions (i.e., methanol under reflux for 6 h), but unfortunately, without any evident change in precursors. This drawback suggested that a detailed revision of the reaction conditions for this new approach must also be undertaken. Repeating the same reaction in refluxing ethanol for 6 h showed a similar behavior and no change in precursors was observed. When refluxing toluene and xylenes were used as solvents (8 h of heating), complex mixtures of reagents and products were observed. When the reaction was performed in an ethanol/acetic acid mixture (heating for 8 h), the formation of a small amount of a new product was observed, along with unreacted starting materials. After its isolation the corresponding structure could be assigned to either regioisomer 11a or 12a (25% yield in any case), as confirmed by mass spectrometry showing a molecular ion with m/z = 404.
While looking for an improvement of the reaction yield, additional solvents such as acetonitrile (8 h, 48%) and glacial acetic acid (8 h, 36%) under reflux were evaluated, indicating that acetonitrile afforded the best result. In addition to the mass spectrum, the NMR spectrum of the obtained solid showed the formation of a single regioisomer, 11a or 12a, as no duplicate signals were observed. Moreover, signals for the protons assigned to H–2′ (doublet at 4.47 ppm with J = 9.6 Hz), H–1′ (false triplet at 3.44 ppm with J = 9.8 Hz) and H–8a′ (multiplet at 3.22–3.29 ppm), corroborated the formation of the bicyclic spiro-indolizinic moiety, as shown in Figure 4. The previous structural findings for the spiro-derivative 9a (Figure 3), and the long range 2′-H–C correlations in the HMBC experiment suggests that the correct structure for the isolated solid is more likely associated with regioisomer 11a.
Nevertheless, it was not possible to discriminate conclusively by NMR techniques the correspondence of the structure of the isolated solid to either the 11a or 12a regioisomer. Fortunately, the growth of single-crystals of this compound suitable for X-ray diffraction unequivocally confirmed the regioselective formation of the spiro-derivative 11a instead of 12a, as shown in Figure 5 [39].
The scope of this reaction under the new established reaction conditions (i.e., reflux in acetonitrile), was extended to the diversely substituted isatin 6, affording a new series of spirooxindolindolizine 11 in 45–69% yields (Table 3). Additionally, the growth of single crystals for some other derivatives of 11 and their X-ray diffraction analysis re-confirmed the regioselectivity of this reaction.
The possible mechanistic route for the formation of products 9/11 begins with the addition of the amino acid (i.e., L-proline 7a or pipecolic acid 7b) over the substituted isatin 6 at C-3, thus forming the additional products I, which, after cyclization and elimination of water, form the lactonic intermediates II. Subsequently, II may undergo decarboxylation process to generate the key azomethine ylides AY-1 or AY-2 (see Scheme 2).
In a second step, the azomethine ylides AY-1 or AY-2 react with the dipolarophile 8 via two possible transition states TS-1 or TS-2 (see Figure 6). The results obtained by X-ray diffraction allow us to confirm that the reaction passes through the TS-2 for the two products obtained, 9 and 11 [38,39]. Structures 9*/11* are stereoisomeric products of 9/11 but there is no evidence of their formation. Products 9 and 11 were obtained as enantiomeric pairs (1′R,2′R,3S,7a′S)/(1′S,2′S,3R,7a′R) and (1′R,2′R,3S,8a′S)/(1′S,2′S,3R,8a′R), respectively.

3.2. Biological Evaluation

3.2.1. Antibacterial Activity

The antibacterial activities of the obtained compounds against Pseudomonas aeruginosa, Klebsiella. pneumoniae, Escherichia coli, Staphylococcus aureus, and Neisseria gonorrhoeae (see Table 4 and Table 5) were evaluated. The compounds showing inhibitory activity against S. Aureus were 9f, 9j, 11i, and 11j. Among these, the most active were 11i (S. aureus ATCC 25923 MIC: 4 µg/mL, S. aureus ATCC 43300 MIC: 16.12 µg/mL) and 9f (S. aureus ATCC 25923 MIC: 8 µg/mL, S. aureus ATCC 43300 MIC: 8 µg/mL) (see Figure 7). Interestingly, the most active ones contain in their structures the N-Bn group and 5-Cl substituent on the benzene ring. Whereas the compounds active against N. gonorrhoeae were the products 9d, 9f, 9j, 9m, 11f, 11i, 11j, with the most active compounds being 9d (MIC: 62.5 µg/mL) and 9m (MIC: 62.5 µg/mL). These compounds stand out for presenting an N-hexyl group, and, in the case of 9m, a 5-Cl substituent on the benzene ring, and were more active than their 9f homologues (N-Bn and 5-Cl), and 9j (N-Bn and 5,7-di-Cl) homologues.

3.2.2. Toxicological Activity

Toxicity parameters, median lethal dose (LD50) and toxicity classification were predicted for the active compounds using Protox II [44]. All compounds (6/6) showed predicted neurotoxicity, respiratory and clinical toxicity and, though less likely, nephrotoxicity and immunotoxicity. Some compounds showed alerts for blood–brain barrier toxicity (9f, 9j, 11i, 11j) and GABA receptor events (9d, 9m, 11j), though with a predicted low likelihood (CS < 0.7), as well as metabolic interference by cytochrome inhibition, as shown in Table 6. Most compounds (5/6) were classified as toxicity class 4.
Consensus score is indicated as follows: <0.3 not good prediction, 0.3–0.7 marginal prediction. > 0.7 strong predictions. Toxicity alerts with consensus scoring (CS) > 0.7 are highlighted in bold. Toxicity classification: Class I: fatal if swallowed (LD50 ≤ 5 mg/kg), II: fatal if swallowed (5 < LD50 ≤ 50), III: toxic if swallowed (50 < LD50 ≤ 300), IV: harmful if swallowed (300 < LD50 ≤ 2000), V: may be harmful if swallowed (2000 < LD50 ≤ 5000), VI: non-toxic (LD50 > 5000). Toxicity in vivo of the compounds was also tested in the Galleria mellonella model. Five out of six compounds were at least at toxicity class 4 as no larval killing was observed when larvae were inoculated with 500 mg/kg. The toxicity class was the same in vivo and in silico for five out of six compounds. Although larval killing was observed at 500 mg/Kg for 11i and 9d, they showed lower LD50 in vivo than toxicity predicted in silico.
The ability to induce membrane damage was tested in vitro by assessing the hemolytic activity of the compounds against human red blood cells (RBCs). The hemolytic activity of the active compounds ranged from 2.4% to 100%. The spirooxindoles containing the pyrrolizine moiety showed higher hemolytic activity, with the compounds 9j and 9f having 2.4% and 16.6% hemolytic activity, respectively. By contrast, the spirooxindol-indolizines had lower hemolytic effects, and compounds 11f and 11i induced 2.3% and 8% hemolysis, respectively, suggesting that they have lower membrane interactions and toxicity.

3.3. Inverse Docking Analysis and Computing the Physicochemical Descriptors

The new spirooxindoles 9a–m and spirooxindoles 11a–j were docked against a total of 129 biological pathways and 26 human diseases reported in the Kyoto Encyclopedia of Genes and Genomes (KEGG) data base (see the list on the Supporting Information). The compounds were docked into the protein crystallographic structures available in the PDB database, at the exact position where the native ligands were coupled. The best docking results are shown in Table 7 (see the Supporting Information for more details). Comparison with the native ligands is shown in bold. While it is generally expected that native ligands exhibit high affinity for their respective protein targets, this assumption was only validated for the target protein 7Q6S.
Additionally, physicochemical descriptors, pharmacokinetics and druglikeness were assessed using the SwissADME database tool [46]. A summary is shown in Table 8 (see Supplementary Data). From these results, the best molecules were 9b, 9c, 9d, 9g, 9l, and 11j, as these molecules were predicted as noninhibitors of CYP3A4 and CYP2D6, which are the two most abundant cytochrome P450 enzymes that are involved in drug metabolism.

4. Conclusions

New and diversely substituted spirooxindoles 9 and 11 were synthesized by a 1,3-dipolar cycloaddition reaction between substituted isatins with two amino acids (i.e., L-proline, and pipecolic acid) and trans-3-benzoyl acrylic acid as dipolarophile. Spirooxindoles 9a–m were obtained in 40–86% yields by reflux in methanol, while spirooxindoles 11a–j were obtained in 45–69% yields by reflux in acetonitrile.
The most active products against S. Aureus were 11i (S. aureus ATCC 25923 MIC: 4 µg/mL, S. aureus ATCC 43300 MIC: 16.12 µg/mL) and 9f (S. aureus ATCC 25923 MIC: 8 µg/mL, S. aureus ATCC 43300 MIC: 8 µg/mL) and the most active compounds against N. gonorrhoeae were 9d (MIC: 62.5 µg/mL) and 9m (MIC: 62.5 µg/mL). Toxicological analysis in vivo and in silico of active compounds 9d, 9f, 9j, 9m, 11i, and 11j showed that 5/6 compounds were at toxicity class 4, and compounds 11i and 9d showed lower LD50 in vivo than in silico. The hemolytic activity of the active compounds ranged from 2.4% to 100%. Overall, the most active compounds against S. aureus 9f and 11i and N. gonorrhoeae 9m and 9d may be starting points for further improvement of the activity and lowering of potential toxicity.
Inverse docking analysis showed that compounds 11e, 11i, 11a, and 9b displayed high affinity toward the protein targets 6TYM and 7Q6S (both are a KEAP1, INrf2, KLHL19), these proteins are involved in biological pathways such as those associated with Parkinson’s disease, different pathways in cancer, chemical carcinogenesis with reactive oxygen species, hepatocellular carcinoma, and fluid shear stress and atherosclerosis. On the other hand, compounds 9c and 9l reported a high affinity to the protein 5KIR (a human cyclooxygenase-2) displaying higher affinity than the Rofecoxib, a COX-2-selective nonsteroidal anti-inflammatory drug (NSAID).

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/chemistry7010018/s1, Chemistry: 1H NMR, 13C NMR, DEPT-135 (1D NMR) spectra for compounds 9a–m and 11a–j (Figures S1–S76), 2D NMR spectra for compounds 9a and 11a (Figures S4–S7, Figures S46–S49); FT-IR spectra of compounds 9a–m and 11a–j (Figures S77–S99). Antibacterial activity: Antimicrobial screening of the novel heterocycles 9a–m and 11a–j (Table S1). Inverse docking analysis: Tested biological pathways and diseases, KEGG database (Table S2). Predicted physicochemical descriptors, pharmacokinetic properties, druglike nature and medicinal chemistry friendliness of spirooxindoles 9a–m and spirooxindoles 11a–j (Table S3). Docking scores of the evaluated spirooxindoles 9a–m and spirooxindoles 11a–j (Table S4).

Author Contributions

Data curation, synthesis, characterization, writing—original draft preparation, P.R.; antibacterial evaluation, toxicology analysis, M.d.P.C., M.B. and M.E.B.; inverse docking analysis, M.M.-G.; writing—review and editing, R.A.; writing—review and editing, supervision, project administration, J.Q. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Vicerrectoría de Investigaciones, Universidad del Valle, grant number CI71374.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Data, further inquiries can be directed to the corresponding author.

Acknowledgments

P.R., R.A., M.d.P.C. and J.Q. wish to acknowledge Universidad del Valle for financial support.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. (a) Spirooxindole derivatives found in nature. (b) Pharmacologically active spirooxindole derivatives obtained by synthetic methods.
Figure 1. (a) Spirooxindole derivatives found in nature. (b) Pharmacologically active spirooxindole derivatives obtained by synthetic methods.
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Scheme 1. Model reaction for the synthesis of spirooxindoles containing the pyrrolizine moiety.
Scheme 1. Model reaction for the synthesis of spirooxindoles containing the pyrrolizine moiety.
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Figure 2. Numbering of positions in the pyrrolizine moiety.
Figure 2. Numbering of positions in the pyrrolizine moiety.
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Figure 3. ORTEP diagram for the obtained product 9d.
Figure 3. ORTEP diagram for the obtained product 9d.
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Figure 4. Numbering of positions in the indolizine moiety.
Figure 4. Numbering of positions in the indolizine moiety.
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Figure 5. ORTEP diagram for the obtained product 11a.
Figure 5. ORTEP diagram for the obtained product 11a.
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Scheme 2. Plausible mechanistic route for the formation of azomethine ylides AY-1/AY-2.
Scheme 2. Plausible mechanistic route for the formation of azomethine ylides AY-1/AY-2.
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Figure 6. Plausible transition states for the 1,3-dipolar cycloaddition between the in situ generated azomethine ylides AY-1 and AY-2 and the dipolarophile 8.
Figure 6. Plausible transition states for the 1,3-dipolar cycloaddition between the in situ generated azomethine ylides AY-1 and AY-2 and the dipolarophile 8.
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Figure 7. Structure–activity relationship of spirooxindoles 9d, 9f, 9j, 9m, 11f, 11i, and 11j against S. aureus and N. gonorrhoeae.
Figure 7. Structure–activity relationship of spirooxindoles 9d, 9f, 9j, 9m, 11f, 11i, and 11j against S. aureus and N. gonorrhoeae.
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Table 1. Setting up the reaction conditions for the synthesis of the spirooxindole 9a/10a.
Table 1. Setting up the reaction conditions for the synthesis of the spirooxindole 9a/10a.
EntryCondition (i)Time (h)Yield of 9a/10a (%)
1EtOH, reflux226
2ACN, reflux256
3H2O, reflux359
4MeOH, reflux261
5THF, reflux445
Table 2. Synthesis of the series of spirooxindoles containing the pyrrolizine moiety 9a–m.
Table 2. Synthesis of the series of spirooxindoles containing the pyrrolizine moiety 9a–m.
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CompoundR1R2Yield of 9 (%)
9aBnH61
9bCH3H44
9cHH72
9d *n-HexylH60
9eCH35-Cl58
9fBn5-Cl41
9gH5,7-di-Cl58
9hH5-CH349
9iH5-NO240
9jBn5,7-di-Cl86
9kBn5-F80
9lH5-F71
9mn-Hexyl5-Cl78
* Structure of product 9d re-confirmed by single-crystal X-ray diffraction analysis [45].
Table 3. Synthesis of the series of spirooxindoles containing the indolizine moiety 11a–j.
Table 3. Synthesis of the series of spirooxindoles containing the indolizine moiety 11a–j.
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CompoundR1R2Yield of 11 (%)
11a *H5-CH348
11b *H5-F68
11c *CH3H49
11dH5-Cl68
11eBnH45
11f *n-HexylH48
11g *CH35-Cl69
11hHH53
11iBn5-Cl67
11jn-Hexyl5-Cl65
* Structure of products of 11 re-confirmed by single-crystal X-ray diffraction analysis [39].
Table 4. Antibacterial activity of spirooxindoles 9 containing the pyrrolizine moiety.
Table 4. Antibacterial activity of spirooxindoles 9 containing the pyrrolizine moiety.
CompoundR1R2Result
9aBnHNot measured
9bCH3HNot active against P. aeruginosa, K. pneumoniae, E. coli, S. aureus, N. gonorrhoeae
9cHHNot active against P. aeruginosa, K. pneumoniae, E. coli, S. aureus, N. gonorrhoeae
9dHexylHNot active against P. aeruginosa, K. pneumoniae, E. coli, S. aureus,
Active against N. gonorrhoeae MIC. 62.5 µg/mL
9eCH35-ClNot active against P. aeruginosa, K. pneumoniae, E. coli, S. aureus, N. gonorrhoeae
9fBn5-ClNot active against P. aeruginosa, K. pneumoniae, E. coli
Active against S. aureus ATCC 25923
MIC: 8 µg/mL
S. aureus ATCC 43300
MIC: 8 µg/mL
N. gonorrhoeae MIC: 250 µg/mL
9gH5,7-di-ClNot active against P. aeruginosa, K. pneumoniae, E. coli, S. aureus, N. gonorrhoeae
9hH5-CH3
9iH5-NO2Not measured
9jBn5,7-di-ClNot active against P. aeruginosa, K. pneumoniae, E. coli
Active against S. aureus ATCC 25923
MIC: >500 µg/mL
S. aureus ATCC 43300
MIC > 500 µg/mL
N. gonorrhoeae MIC. 125 µg/mL
9kBn5-FNot active against P. aeruginosa, K. pneumoniae, E. coli, S. aureus, N. gonorrhoeae
9lH5-F
9mHexyl5-ClNot active against P. aeruginosa, K. pneumoniae, E. coli, S. aureus,
Active against N. gonorrhoeae MIC. 62.5 µg/mL
Table 5. Antibacterial activity of spirooxindoles 11 containing the indolizine moiety.
Table 5. Antibacterial activity of spirooxindoles 11 containing the indolizine moiety.
ProductR1R2Result
11aH5-CH3Not active against P. aeruginosa, K. pneumoniae, E. coli, S. aureus, N. gonorrhoeae
11bH5-F
11cCH3H
11dH5-Cl
11eBnH
11fHexylHNot active against P. aeruginosa, K. pneumoniae, E. coli, S. aureus
Active against N. gonorrhoeae MIC: 1mg/mL
11gCH35-ClNot active against P. aeruginosa, K. pneumoniae, E. coli, S. aureus, N. gonorrhoeae
11hHH
11iBn5-ClNot active against P. aeruginosa, K. pneumoniae, E. coli.
Active against S. aureus ATCC 25923 MIC: 4 µg/mL
S. aureus ATCC 43300 MIC: 16.12 µg/mL
N. gonorrhoeae MIC: 500 µg/mL
11jHexyl5-ClNot active against P. aeruginosa, K. pneumoniae, E. coli.
Active against S. aureus ATCC 25923 MIC: >500 µg/mL
S. aureus ATCC 43300 MIC > 500 µg/mL
N. gonorrhoeae MIC 250 µg/mL
Table 6. Toxicological data predicted with Protox II for active compounds: 9d, 9f, 9j, 9m, 11i, and 11j.
Table 6. Toxicological data predicted with Protox II for active compounds: 9d, 9f, 9j, 9m, 11i, and 11j.
Compound9d9f9j9m11i11j
HepatotoxicityInactiveInactiveInactiveInactiveInactiveInactive
Consensus scoring (CS)0.770.700.700.720.700.71
NeurotoxicityActiveActiveActiveActiveActiveActive
Consensus scoring (CS)0.620.730.730.750.770.77
CardiotoxicityInactiveInactiveInactiveInactiveInactiveInactive
Consensus scoring (CS)0.800.790.790.800.800.81
NephrotoxicityActiveActiveActiveActiveActiveActive
Consensus scoring (CS)0.600.630.630.600.630.59
Respiratory toxicityActiveActiveActiveActiveActiveActive
Consensus scoring (CS)0.750.720.720.770.780.79
ImmunotoxicityActiveInactiveInactiveActiveInactiveActive
Consensus scoring (CS)0.710.760.650.880.850.81
CarcinogenicityInactiveInactiveInactiveInactiveInactiveInactive
Consensus scoring (CS)0.560.610.610.590.620.60
MutagenicityInactiveInactiveInactiveInactiveInactiveInactive
Consensus scoring (CS)0.670.680.680.630.680.63
CytotoxicityInactiveInactiveInactiveInactiveInactiveInactive
Consensus scoring (CS)0.620.580.580.580.610.59
Clinical toxicityActiveActiveActiveActiveActiveActive
Consensus scoring (CS)0.690.740.740.670.780.70
Blood brain barrier toxicityInactiveActiveActiveInactiveActiveActive
Consensus scoring (CS)0.570.540.540.560.530.57
GABA receptor eventActiveInactiveInactiveActiveInactiveActive
Consensus scoring (CS)0.570.610.610.530.590.52
Cytochrome inhibition (P450 2C9)InactiveActiveActiveActiveActiveActive
Consensus scoring (CS)0.500.570.570.600.550.57
LD50(mg/Kg)208769769500729325
Toxicity class344444
LD50: 50% lethal dose. GABA: gamma aminobutyric acid.
Table 7. Highest docking affinities, spirooxindoles 9a–m and spirooxindoles 11a–j.
Table 7. Highest docking affinities, spirooxindoles 9a–m and spirooxindoles 11a–j.
Protein TargetCompoundDocking Score (Kcal/mol) a
6TYM11e−10.4
6TYM11i−10.6
6TYMNative ligand b (08A)−10.7
4JYG9b−10.5
4JYGNative ligand (1NY)−9.7
5KIR9c−10.6
5KIR9l−10.2
5KIRNative ligand (RCX)−6.4
5UZK9g−10
5UZKNative ligand (504)−9.8
7Q6S9b−10
7Q6S11e−10.1
7Q6S11a−10.1
7Q6S9h−10
7Q6SNative ligand (91M)−12.6
a Docking score (kcal/mol) calculated using AutoDock Vina. b Reported native ligand co-crystallized, PDB database.
Table 8. Physicochemical descriptors of spirooxindoles 9a–m and spirooxindoles 11a–j.
Table 8. Physicochemical descriptors of spirooxindoles 9a–m and spirooxindoles 11a–j.
CompoundGI AbsorptionMW#H-Bond Acceptors#H-Bond DonorsLog PCYP2D6 InhibitorCYP3A4 Inhibitor
9aHigh466.53512.74YesYes
9bHigh390.43511.47NoNo
9cHigh376.41521.37NoNo
9dHigh460.56513.28NoNo
9eHigh424.88512.10YesYes
9fHigh500.97513.24YesYes
9gHigh445.3522.48NoNo
9hHigh390.43521.69YesYes
9iHigh421.4720.63YesYes
9jHigh535.42513.72NoNo
9kHigh484.52613.01YesYes
9lHigh394.4621.68NoNo
9mHigh495.01513.76YesNo
11aHigh404.46521.98YesYes
11bHigh408.42621.98YesYes
11cHigh404.46511.82YesYes
11dHigh424.88522.16YesYes
11eHigh480.55512.91YesYes
11fHigh474.59513.55YesNo
11gHigh438.9512.41YesYes
11hHigh390.43521.62YesYes
11iHigh515513.51YesYes
11jHigh509.04514.02NoNo
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Romo, P.; Crespo, M.d.P.; Barreto, M.; Burbano, M.E.; Mejia-Gutierrez, M.; Quiroga, J.; Abonia, R. Pyrrolizine- and Indolizine-Derived Spirooxindoles: Synthesis, Antibacterial Activity and Inverse Docking Analysis. Chemistry 2025, 7, 18. https://doi.org/10.3390/chemistry7010018

AMA Style

Romo P, Crespo MdP, Barreto M, Burbano ME, Mejia-Gutierrez M, Quiroga J, Abonia R. Pyrrolizine- and Indolizine-Derived Spirooxindoles: Synthesis, Antibacterial Activity and Inverse Docking Analysis. Chemistry. 2025; 7(1):18. https://doi.org/10.3390/chemistry7010018

Chicago/Turabian Style

Romo, Pablo, María del Pilar Crespo, Mauricio Barreto, María Elena Burbano, Melissa Mejia-Gutierrez, Jairo Quiroga, and Rodrigo Abonia. 2025. "Pyrrolizine- and Indolizine-Derived Spirooxindoles: Synthesis, Antibacterial Activity and Inverse Docking Analysis" Chemistry 7, no. 1: 18. https://doi.org/10.3390/chemistry7010018

APA Style

Romo, P., Crespo, M. d. P., Barreto, M., Burbano, M. E., Mejia-Gutierrez, M., Quiroga, J., & Abonia, R. (2025). Pyrrolizine- and Indolizine-Derived Spirooxindoles: Synthesis, Antibacterial Activity and Inverse Docking Analysis. Chemistry, 7(1), 18. https://doi.org/10.3390/chemistry7010018

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