Construction of Spirooxindole Analogues Engrafted with Indole and Pyrazole Scaffolds as Acetylcholinesterase Inhibitors

Abstract

Twenty-five new hits of spirooxindole analogs 8a–y engrafted with indole and pyrazole scaffolds were designed and constructed via a [3+2]cycloaddition (32CA) reaction starting from three components: new chalcone-based indole and pyrazole scaffolds 5a–d, substituted isatins 6a–c, and secondary amines 7a–d. The potency of the compounds were assessed in modulating cholinesterase (AChE) activity using Ellman’s method. Compounds 8i and 8y showed the strongest acetylcholine esterase inhibition (AChEI) with IC₅₀ values of 24.1 and 27.8 μM, respectively. Molecular docking was used to study their interaction with the active site of hAChE.

Introduction

Neurodegeneration is a key aspect of a large number of diseases that come under the umbrella of neurodegenerative disease. Of these different disorders, the most notable are Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease (AD). Alzheimer’s disease (AD) symptoms are memory loss, impairment of cognitive functions, and dementia. AD involves two major neuropathological hallmarks causing neuronal dysfunctions and cell death: the presence of extracellular amyloid β-peptide (Aβ) deposits (senile plaques) and aggregates of the hyperphosphorylated tau protein (neurofibrillary tangles)¹ along with the tau hyperphosphorylation are the most proposed pathogenetic mechanisms,² and mitochondrial cascade hypothesis has attracted much interest recently.³ Other much debatable AD hypotheses are the tau hypothesis,⁴ cholesterol hypothesis,⁵ inflammatory hypothesis,⁶ oxidative stress hypothesis,⁷ metal hypothesis,⁸ vascular hypothesis,⁹ and cell cycle hypothesis.¹⁰ Up to date, rivastigmine, galantamine, and donepezil represent the only ChE inhibitors approved for AD treatment, differing in chemical structures and pharmacologic and pharmacokinetic profiles. To design and discover a new agent that might work as ChE inhibitors is a challenge.

Heterocycles having azoles as a core structure have been discovered for several applications.¹¹ Pyrazoles are one of the important heterocycles, which exhibited significant properties in material sciences,¹² agriculture development,¹³ medicine,¹⁴ and pharmacological applications.¹⁵ Among pharmacological applications are antibiotic,¹⁶ sensors,¹⁷ pesticide,¹⁸ antibacterial,¹⁹ and antifungal activities.²⁰ On the other hand, several molecules engrafted with the pyrazole scaffold have shown high efficacy toward antiviral,²¹ antitumor,²² anti-inflammatory,²³ antioxidant,²⁴ and antidepressant activities.²⁵ Indeed, many drugs incorporated the pyrazole moiety employed for the treatment of metabolic disorder diseases such as Alzheimer’s,²⁶ Parkinson’s,²⁷ and neuroprotective,²⁸ which makes this pharmacophore very attractive for drug discovery. One representative example of advanced glycation inhibitors reported by Han et al. is that this agent is based on the pyrazole-5-carboxamide as a core structure.²⁹ However, Turkan et al. have reported substituted pyrazole derivatives, which have been discovered as potent cholinesterase inhibitors.³⁰

Spirooxindoles exhibit a broad range of biological effects and are well-tolerated in biomedical applications.³¹ Their applications use AChEs.³² Kia et al. reported representative examples, including spirooxindoles engrafted with piperidine and pyrrolizine scaffolds, which are found to be beneficial for AChE (compound III; IC₅₀ = 3.36 μM or 2.28 ± 0.07 μg/mL) (Figure 1),^32d and another representative example based on mono- and bis-spiro-pyrrolidines, where the hit IV shows high efficacy against AChE with an IC₅₀ value of 2.35 μM (Figure 1).^32f Chigurupati et al. reported indolopyrazolines with the high biochemical application against AChE inhibition (V; IC₅₀ = 13.77 ± 0.25 lM), respectively.³³ Extending our recent efforts on the development of cholinesterase inhibition, Barakat et al. reported a new series of spirooxindoles engrafted with the benzo[b]thiophene scaffold, which were found to exhibit moderate potential against AD (Figure 1).^32h,34 The above reports inspired the investigation of several pharmacophores inside the rigid spirooxindole privileged structure, such as indole and pyrazole scaffolds, which might act as better AChE inhibitors.

Figure 1.

Significant acetylcholinesterase (AChE) inhibitory activity of representative spirooxindole analogues.

Several progresses have been devoted toward the synthesis of spirooxindoles recently.³⁵ In between these approaches, the [3 + 2] cycloaddition reaction protocol was efficient and promising to afford the spirooxindole privileged structures with several stereogenic centers.³⁶

In this paper, we described the [3 + 2] cycloaddition reaction approach for the synthesis of new spirooxindoles based on a new chalcone engrafted with indole and pyrazole motifs. Many substituted isatins and amino acids were also investigated. The biochemical potential of AChE was also studied.

Results and Discussion

Synthesis of Spirooxindole Analogs 8a–y Engrafted with Indole and Pyrazole Scaffolds

The indole and pyrazole scaffolds as interesting pharmacophores are combined into the spirooxindole analogs for exploring the acetylcholinesterase (AChE) inhibitory activity. Initially, we synthesized the new chalcones 5a–d by aldol condensation of acetyl-pyrazole and substituted indole-3-carbaldehyde in basic condition under reflux for 72 h. The new chalcone-based indole and pyrazole scaffolds, which act as a synthon for the [3 + 2] cycloaddition (32CA) reaction, are depicted in Scheme 1. Subsequently, the new series of spirooxindole analogues tethered indole and pyrazole scaffolds were constructed via a one-pot multicomponent reaction approach³⁷ in MeOH under reflux conditions for 2–24 h (Scheme 2). Twenty-five examples were achieved by variation of many substituted isatins 6a–c with different electronic effects (isatin 6a; 6-chloroisatin 6b; 5-nitroisatin 6c) with four different amino acids 7a–d (thioproline 7a, octahydro-1H-indole-2-carboxylic acid 7b; l-proline 7c; sacrosine 7d). The spirooxindole analogues engrafted with indole and pyrazole scaffolds were isolated in a single regio- and diastereoselective isomer in acceptable to excellent chemical yield up to 86%. The proposed mechanism, as shown in Scheme 3, proceeded via an ortho/endo 32CA approach.³⁸ The optical rotation of the synthesized compounds was measured, and the regio- and diastereoselectivity of the cycloadducts were confirmed. Single-crystal X-ray diffraction analysis of compound 8c confirmed that our hypothesis belongs to the final stereoselectivity of the cycloadducts of the final compounds. The absolute configurations of products 8a–y were assigned based on the obtained x-ray diffraction analysis as follows for the 4 stereogenic centers as R, S, R, S.

Scheme 1. Synthesis of the Chalcone Engrafted with Indole and Pyrazole Scaffolds 5a–d.

Scheme 2. Synthesis of the Spirooxindole Analogues Engrafted with Indole and Pyrazole Scaffolds 8a–y.

Scheme 3. Proposed Approach for the [3 + 2] Cycloaddition Reaction, Explaining the Regio- and Diastereoselective Synthesis.

Crystal Structure Description

The X-ray structure of 8c showing atom numbering and thermal ellipsoids drawn at a 30% probability level is shown in Figure 2. The structure agreed very well with the spectral analyses and revealed the presence of asymmetric centers at C9, C21, C10, and C13. The crystal data and structure refinement details are depicted in Table 1. The compound crystallized in the triclinic system and the P1̿ space group with unit cell parameters of a = 12.71740(10) Å, b = 15.72830(10) Å, c = 19.3853(2) Å, and β = 105.9640(10)°. The unit cell volume is 3727.97(6) ų with Z = 4. The asymmetric unit comprised one molecule and two chloroform molecules as the crystal solvent. The selected bond distances and angles are listed in Table S1 (Supporting Information).

Figure 2.

Atom numbering and thermal ellipsoids at a 30% probability level for 8c.

Table 1. Crystal Data.

	8c
CCDC	2105913
empirical formula	C34H28BrCl6N5O2S
fw	863.28
temp (K)	120(2)
λ (Å)	1.54184
cryst syst	monoclinic
space group	P21/n
a (Å)	12.71740(10)
b (Å)	15.72830(10)
c (Å)	19.3853(2)
β (deg)	105.9640(10)
V (Å3)	3727.97(6)
Z	4
ρcalc (Mg/m3)	1.538
μ (Mo Kα) (mm–1)	6.305
no. reflns.	38891
unique reflns.	7824
completeness to θ = 67.684°	100.0%
GOOF (F2)	1.024
Rint	0.0352
R1a (I ≥ 2σ)	0.0354
wR2b (I ≥ 2σ)	0.0861

^aR₁ = Σ||F_o| – |F_c||/Σ|F_o|.

^bwR₂ = [Σ[w(F_o² – F_c²)²]/ Σ[w(F_o²)²]]^1/2.

The molecular packing in 8c is controlled by strong N–H...O and N–H...N hydrogen bonds as well as weak C––H...X interactions (X=Cl, Br, N, or O). The corresponding hydrogen bond parameters are listed in Table S2 (Supporting Information) and are shown in the left part of Figure 3. The hydrogen bond network is shown in the right part of the same figure.

Figure 3.

Hydrogen bond contacts (left) and packing of molecular units via hydrogen bonding interactions (right).

Acetylcholine Esterase Inhibitory Activity

The ability of the synthesized compounds to inhibit acetylcholine esterase (AChE) was evaluated using Ellman’s method.³⁹ Compounds 8i and 8y showed the strongest acetylcholine esterase inhibition (AChEI) with IC₅₀ values of 24.1 and 27.8 μM, respectively.

Four Compounds; 8c, 8d, 8f, 8h, 8j, 8w, and 8x showed moderate activity inhibitory activity (with IC₅₀ ≤ 50 μM). Compounds 8m, 8o, 8p, 8q, and 8s had weak activity (with IC₅₀ values 65–90 μM), while compounds 8b, 8e, 8q, 8k, 8e, 9r, 8r, 8t, 8u, and 8v were in active with IC₅₀ > 100 μM (Table 2).

Table 2. AChE Inhibitory Activity of the Synthesized Spirooxindole Analogues Engrafted with Indole and Pyrazole Scaffolds 8a–y.

Molecular Docking Study

Molecular docking has been used extensively to identify and explain the molecular mechanism of several lead compounds in drug discovery.⁴⁰ The software validation revealed that it was able to reproduce the experimental pose with root-mean-square deviation (RMSD) equal to 0.39 (Figure 4). Since in vitro enzyme inhibition showed that compounds 8i, 8h, and 8y are the most active among the synthesized compounds with moderate inhibition activity, we used molecular docking to gain insights into the molecular interaction of the compound with the active site of hACHE.

Figure 4.

Donepezil (Red) docked in the active site of ACHE (4EY7) and overlaid with cocrystallized ligand (Green) RMSD = 0.39.

In the context of the total energy required for binding, the cocrystallized ligand achieved much better binding affinity than the 3 compounds, yet their binding energy was found to be reasonable with respect to their moderate activity in the enzyme inhibition assay, as presented in Table 3. Post docking analysis showed that compound 8i has different binding modes rather than compounds 8h and 8y, as the first one tends to bind in the middle of the gorge of hACHE. On the other hand, compounds 8h and 8y showed most of their interactions with amino acids located at the entrance of the gorge. For example, compound 8i was able to interact with amino acids in diverse sites in the enzyme such as TRP-86 and TYR-337 in the anionic site, Ser-203 and HIS-447 in the catalytic site, PHE-295 and PHE-297 in the acyl binding site, and formed 2 hydrogen bonds with SER-125 and GLU-202, as depicted in Figure 5, these interactions are well reported to be important for achieving good inhibitory activity.⁴¹

Table 3. Binding Energy of Compound Docked in the Binding Site of the hACHE Active Site^a.

	compound	total energy	VDW	H-bond
1.	8i	–127.335	–121.335	–6
2.	8y	–126.589	–118.253	–8.336
3.	8h	–123.962	–118.983	–4.979
4.	cocrystallized ligand	–149.55	–139.4	–10.15

^aVDW = Van der Waals force and H-Bond = Hydrogen bond.

Figure 5.

8i docked in the active site of ACHE (4EY7) and corresponding two-dimensional (2D) presentation. The H-bond is represented by green dotted lines, hydrophobic interactions are represented by magenta dotted lines, and Pi-sulfur interaction is represented by orange dotted lines.

On the other hand, compound 8y showed interactions only with TYR-337 and PHE-295 from the anionic and acyl binding sites, respectively, but with less hydrophobic interaction than 8i. Nevertheless, they were able to access the peripheral anionic site by interacting with amino acids, such as TYR-124, TRY-341, and TRP-286, as shown in Figures 6 and 7, which might explain their ability to achieve the moderate inhibitory effect as observed in the enzyme inhibition assay.

Figure 6.

8y docked in the active site of ACHE (4EY7) and corresponding 2D presentation. The H-bond is represented by green dotted lines, hydrophobic interactions are represented by magenta dotted lines, and Pi-sulfur interaction is represented by orange dotted lines.

Figure 7.

8h docked in the active site of ACHE (4EY7) and corresponding 2D presentation. The H-bond is represented by green dotted lines, hydrophobic interactions are represented by magenta dotted lines, and Pi-sulfur interaction is represented by orange dotted lines.

Conclusions

We summarized in this paper that we had synthesized 25 hits based on a spirooxindole scaffold engrafted with two other pharmacophores, including indole and pyrazole moieties. The results of the AChE assay show that compounds 8i and 8y show the strongest acetylcholine esterase inhibition (AChEI) with IC₅₀ values of 24.1 and 27.8 μM, respectively. The AChE activity exhibited promising results, which make them candidates for further research.

Experimental Section

General information

Phenylhydrazine, acetylacetone, N,N-dimethylformamide-dimethyl acetal (DMF-DMA), Piperidine, and NaOH were purchased from Aldrich and used as received. All of the indole derivatives were purchased from Aldrich and used as is. All bases were used as received (in air) or dried under vacuum at 100 °C (under an inert atmosphere). All solvents were used as received when experiments were conducted in air. Flash chromatography was performed on 100–200 mesh silica gel. ¹H- and ¹³C-nuclear magnetic resonance (NMR) spectra were recorded on JEOL-700 MHz spectrometers at ambient temperature in CDCl₃ & DMSO-d₆, which were purchased from Sigma-Aldrich. Chemical shifts (ppm) are referenced to the residual solvent peak. Coupling constants, J, are given in hertz. Abbreviations used in the designation of the signals: s = singlet, d = doublet, dd = doublet of doublets, ddd = doublet of doublet of doublets, dt = doublet of triplets, t = triplet, td = triplet of doublets, and m = multiplet. All melting points were measured on a Gallenkamp melting point apparatus in open glass capillaries and are uncorrected. IR Spectra were measured as KBr pellets on a Nicolet 6700 FT-IR spectrophotometer. Specific rotations were recorded in ‘A KRÜSS Optronic GmbH P8000 polarimeter.

Synthesis of 1-(5-Methyl-1-phenyl-1H-pyrazol-4-yl)ethanone (3)

N,N-Dimethylformamide-dimethyl acetal (DMF-DMA) (17.85 g, 0.15 mol) was added to acetylacetone 1 (0.1 mol) and stirred for 10 min at ambient temperature, followed by the addition of phenylhydrazine derivative 2 (0.1 mol), and the reaction mixture was heated at 70–80 °C for 24 h. The completion of the reaction was monitored by thin-layer chromatography (TLC) (20% EA/n-hexane). The reaction mixture was then allowed to cool and kept in a fridge for 24 h and a white solid precipitated out, which was isolated by simple filtration and washed with diethyl ether to afford pure white product 3 (5 g, 25 mmol, 25% yield). The MLs part was concentrated and purified using a column to afford another 6 g of pure white product 3 (30 mmol, 30%). The overall yield of acetyl-pyrazole-3 (11.0 g, 55%).⁴²

m.p.: 88–90 °C; ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 7.99 (s, 1H, pyrazole-H), 7.52–7.46 (m, 2H, Ar-H), 7.44 (d, J = 6.8 Hz, 1H, Ar-H), 7.41–7.36 (m, 2H, Ar-H), 2.56 (s, 3H, COCH₃), 2.47 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) = 193.56 (CO), 143.05, 141.97, 138.58, 129.36, 128.89, 125.61, 121.16, 28.75 (COCH₃), 12.44 (CH₃); IR (KBr, cm^–1): 3060, 3001, 1660, 1597, 1545, 1502, 1457, 1399, 1382, 1363, 1276, 1238, 1196, 1171, 1011, 937, 884, 866, 769, 718, 690, 662, 637, 558; [anal. calcd. for C₁₂H₁₂N₂O: C, 71.98; H, 6.04; N, 13.99; found: C, 72.07; H, 6.01; N, 13.94]; LC/MS (ESI, m/z): found 201 [M + H]⁺; exact mass 200.09 for C₁₂H₁₂N₂O. All of the analytical data are in accordance with the reported literature.⁴²

Synthesis of Chalcones (5a–d) from Acetyl-pyrazole (3) and Substituted Indole-3-Carboxyaldehyde (4a–d) (GP1)

Compound 3 (1 g, 5 mmol) and substituted indole-3-carboxyaldehyde 4a–d (5 mmol) were dissolved in ethanol (20 mL) in a 100 mL round bottom flask. Piperidine (950 mg, 2 equiv) was added to the reaction mixture and refluxed at 80 °C 48–72 h. The completion of the reaction was monitored by TLC (30% EA/n-hexane). Then, the solid precipitated out, which was isolated by simple filtration and washed with ethanol to afford pure white/yellow product 5a–d (70–80% yield).

Synthesis of (E)-3-(1H-Indol-3-yl)-1-(5-methyl-1-phenyl-1H-pyrazol-4-yl)prop-2-en-1-one (5a)

Following the general procedure (GP1), acetyl-pyrazole 3 (1.0 g, 5.0 mmol) and indole-3-carboxyaldehyde 4a (0.87 g, 6.0 mmol) produce pyrazolenone-5a (yield 0.9 g, 55%); m.p.: 222–224; ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) = δ 11.85 (s, 1H, NH), 8.59 (s, 1H, Ar-H), 8.17–8.11 (m, 1H, Ar-H), 8.08 (s, 1H, Ar-H), 8.00 (d, J = 15.4 Hz, 1H, CH=CH), 7.58 (d, J = 4.3 Hz, 4H, Ar-H), 7.54–7.41 (m, 3H, Ar-H & CH=CH), 7.29–7.17 (m, 2H, Ar-H), 2.62 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) = δ 184.19 (CO), 142.69, 141.53, 138.53, 137.47, 136.61, 132.48, 129.30, 128.56, 125.41, 125.20, 122.58, 121.61, 121.00, 120.51, 118.15, 112.55, 112.33, 12.20 (CH₃); IR (KBr, cm^–1) ν_max = 3158, 3106, 3047, 2877, 1649, 1638, 1581, 1521, 1502, 1453, 1384, 1347, 1279, 1255, 1229, 1217, 1190, 1140, 1115, 1072, 1064, 1036, 1034, 1004, 974, 945, 893, 879, 852, 834, 762, 734, 714, 696, 656, 641, 603, 593, 558, 507; LC/MS (ESI, m/z): 328.2 [M + H]⁺, exact mass 327.14 for C₂₁H₁₇N₃O.

Synthesis of (E)-1-(5-Methyl-1-phenyl-1H-pyrazol-4-yl)-3-(1-methyl-1H-indol-3-yl)prop-2-en-1-one (5b)

Following the general procedure (GP1), acetyl-pyrazole 3 (1.0 g, 5.0 mmol) and indole-1-methyl-3-carboxyaldehyde 4b (0.95 g, 6.0 mmol) produce pyrazolenone-5b (yield 1.0 g, 56%); m.p.: 182–183; ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) = δ 8.58 (s, 1H, Ar-H), 8.15 (d, J = 7.8 Hz, 1H, Ar-H), 8.04 (s, 1H, Ar-H), 7.95 (d, J = 15.5 Hz, 1H, CH=CH), 7.58 (d, J = 4.4 Hz, 3H, Ar-H), 7.57–7.49 (m, 3H, Ar-H), 7.46 (d, J = 15.5 Hz, 1H, CH=CH), 7.33–7.25 (m, 2H, Ar-H), 3.85 (s, 3H, CH₃), 2.62 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) = δ 184.09 (CO), 142.71, 141.53, 141.49, 138.51, 137.93, 135.98, 135.89, 129.30, 128.56, 125.63, 125.40, 122.66, 121.59, 121.26, 120.67, 120.61, 118.17, 118.10, 111.56, 110.75, 110.66, 32.97 (CH₃), 12.15 (CH₃); IR (KBr, cm^–1) ν_max = 3108, 3045, 1643, 1569, 1524, 1501, 1472, 1464, 1386, 1373, 1341, 1281, 1259, 1219, 1187, 1178, 1157, 1132, 1075, 1039, 1003, 937, 855, 841, 821, 771, 751, 699, 681, 654, 540; LC/MS (ESI, m/z): found 342.2 [M + H]⁺, exact mass 341.15 for C₂₂H₁₉N₃O.

Synthesis of (E)-3-(5-Bromo-1H-indol-3-yl)-1-(5-methyl-1-phenyl-1H-pyrazol-4-yl)prop-2-en-1-one (5c)

Following the general procedure (GP1), acetyl-pyrazole 3 (1.0 g, 5.0 mmol) and 5-bromoindole-3-carboxyaldehyde 4c (1.34 g, 6.0 mmol) produce pyrazolenone-5c (yield 0.85 g, 42%); m.p.: 214–215; ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) = δ 12.02 (s, 1H, NH), 8.63 (s, 1H, Ar-H), 8.26 (d, J = 1.9 Hz, 1H, Ar-H), 8.14 (s, 1H, Ar-H), 7.95 (d, J = 15.5 Hz, 1H, CH=CH), 7.60–7.55 (m, 4H, Ar-H + CH=CH), 7.53–7.49 (m, 1H, Ar-H), 7.46 (d, J = 3.9 Hz, 1H, Ar-H), 7.45 (d, J = 3.1 Hz, 1H, Ar-H), 7.35 (dd, J = 8.6, 1.9 Hz, 1H, Ar-H), 2.61 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) = δ 184.22 (CO), 142.77, 141.76, 138.50, 136.07, 135.67, 132.99, 129.30, 128.56, 127.02, 125.39, 125.18, 122.39, 121.52, 119.03, 114.25, 113.74, 112.17, 12.19 (CH₃); IR (KBr, cm^–1) ν_max = 3153, 3077, 3030, 2934, 2900, 1643, 1559, 1500, 1454, 1433, 1394, 1370, 1299, 1273, 1243, 1223, 1182, 1136, 1095, 1038, 1022, 1007, 956, 938, 881, 851, 824, 789, 758, 689, 660, 636, 609, 554; LC/MS (ESI, m/z): found 406.6 [M(₇₉Br) + H]⁺, 408.1 [M(₈₁Br) + H]⁺; exact mass 405.05 for C₂₁H₁₆BrN₃O.

Synthesis of (E)-3-(5-Chloro-1H-indol-3-yl)-1-(5-methyl-1-phenyl-1H-pyrazol-4-yl) prop-2-en-1-one (5d)

Following the general procedure (GP1), acetyl-pyrazole 3 (1.0 g, 5.0 mmol) and 5-chloroindole-3-carboxyaldehyde 4d (1.07 g, 6.0 mmol) produce pyrazolenone-5d (yield 0.9 g, 50%); m.p.: 237–238; ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) = δ 11.95 (bs, 1H, NH), 8.65 (s, 1H, Ar-H), 8.20–8.05 (m, 2H, Ar-H), 7.95 (d, J = 15.4 Hz, 1H, CH=CH), 7.57 (d, J = 4.4 Hz, 4H, Ar-H), 7.50 (dd, J = 8.7, 4.6 Hz, 2H, Ar-H), 7.46 (d, J = 15.7 Hz, 1H, CH=CH), 7.23 (dd, J = 8.2, 2.1 Hz, 1H, Ar-H), 2.61 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) = δ 184.24 (CO), 142.78, 141.80, 138.52, 135.86, 135.76, 133.34, 129.30, 128.56, 126.35, 125.73, 125.40, 122.60, 121.53, 119.55, 118.92, 113.81, 112.30, 12.19 (CH₃); IR (KBr, cm^–1) ν_max = 3107, 2896, 1648, 1637, 1576, 1518, 1500, 1453, 1395, 1383, 1373, 1314, 1251, 1212, 1142, 1123, 1072, 1047, 1005, 973, 944, 897, 863, 837, 794, 765, 740, 697, 651, 643, 609, 594, 575; LC/MS (ESI, m/z): found 362.1 [M(₃₅Cl) + H]⁺, 364.1 [M(₃₇Cl) + H]⁺; exact mass 361.09 for C₂₁H₁₆ClN₃O.

Synthesis of Spirooxindole Derivatives 8a–y

General Procedure (GP2)

Chalcones 5a–d (0.25 mmol), isatin derivatives 6a–c (0.25 mmol), and amino acids 7a–d (1.5 equiv, 0.37 mmol) were dissolved in methanol (20 mL), and the reaction mixture was refluxed for 2–4 h. Finally, the products were isolated by flash column chromatography, using 1–3% MeOH/DCM to afford pyrazole spirooxindole 8a–y.

(3R,6′S,7′R,7a’S)-7′-(1H-Indol-3-yl)-6′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-1′,6′,7′,7a’-tetrahydro-3′H-spiro[indoline-3,5′-pyrrolo[1,2-c]thiazol]-2-one (8a)

Following the general procedure (GP2), chalcone 5a (82 mg, 0.25 mmol), isatin 6a (37 mg, 0.25 mmol), and thioproline 7a (50 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8a; yield (85 mg, 62%); m.p.: 138–139 °C; [α]_D²⁵ = −16.19° (c 0.13, MeOH); ¹H NMR (700 MHz, CDCl₃) δ (ppm) = 8.59 (s, 1H, NH), 8.33 (s, 1H, NH), 8.10 (d, J = 7.1 Hz, 1H, Ar-H), 7.92 (s, 1H, Ar-H), 7.72 (d, J = 7.6 Hz, 1H, Ar-H), 7.40–7.33 (m, 4H, Ar-H), 7.28 (d, J = 2.5 Hz, 1H, Ar-H), 7.20–7.17 (m, 2H, Ar-H), 7.17–7.14 (m, 3H, Ar-H), 7.04–7.01 (m, 1H, Ar-H), 6.64 (d, J = 7.7 Hz, 1H, Ar-H), 4.79 (d, J = 11.8 Hz, 1H, CHCO), 4.66–4.61 (m, 1H, NCH), 4.25 (dd, J = 11.8, 9.7 Hz, 1H, NCHCH), 3.94 (d, J = 10.5 Hz, 1H, NCH₂), 3.61 (d, J = 10.5 Hz, 1H, NCH₂), 3.11–3.03 (m, 2H, SCH₂), 1.92 (s, 3H, CH₃); ¹³C NMR (176 MHz, CDCl₃) δ (ppm) = 191.09 (CO), 180.75 (CO), 143.50, 141.28, 141.03, 138.31, 136.74, 129.76, 129.29, 129.15, 128.88, 126.46, 125.47, 124.08, 123.02, 122.28, 122.27, 121.00, 119.93, 119.84, 113.65, 111.58, 109.65, 75.12, 73.75, 63.86, 55.16, 43.58, 37.41, 11.75 (CH₃); IR (KBr, cm^–1) ν_max = 3288, 3058, 2921, 1733, 1722, 1717, 1699, 1694, 1682, 1674, 1668, 1661, 1652, 1619, 1615, 1597, 1538, 1532, 1504, 1470, 1456, 1393, 1337, 1222, 1119, 935, 875, 807, 749, 695; [anal. calcd. for C₃₂H₂₇N₅O₂S: C, 70.44; H, 4.99; N, 12.83; found: C, 70.31; H, 5.05; N, 12.91]; LC/MS (ESI, m/z): found 546.4 [M + H]⁺, exact mass 545.19 for C₃₂H₂₇N₅O₂S.

(3R,6′S,7′R,7a’S)-6′-(5-Methyl-1-phenyl-1H-pyrazole-4-carbonyl)-7′-(1-methyl-1H-indol-3-yl)-1′,6′,7′,7a’-tetrahydro-3′H-spiro[indoline-3,5′-pyrrolo[1,2-c]thiazol]-2-one (8b)

Following the general procedure (GP2), chalcone 5b (85 mg, 0.25 mmol), isatin 6a (37 mg, 0.25 mmol), and thioproline 7a (50 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8b; yield (101 mg, 72%); m.p.: 186–187 °C; [α]_D²⁵ = −12.53° (c 0.13, MeOH); ¹H NMR (700 MHz, CDCl₃) δ (ppm) = 8.80 (s, 1H, NH), 8.09 (d, J = 7.24 Hz, 1H, Ar-H), 7.92 (s, 1H, Ar-H), 7.72 (d, J = 7.63 Hz, 1H, Ar-H), 7.39–7.35 (m, 3H, Ar-H), 7.29 (ddd, J = 8.1, 1.4, 0.7 Hz, 1H, Ar-H), 7.25 (dd, J = 6.9, 1.3 Hz, 1H, Ar-H), 7.21 (dd, J = 5.0, 1.4 Hz, 1H, Ar-H), 7.19 (s, 1H, Ar-H), 7.19–7.16 (m, 2H, Ar-H), 7.16–7.12 (m, 2H, Ar-H), 7.03 (td, J = 7.6, 1.1 Hz, 1H, Ar-H), 6.65 (d, J = 7.73 Hz, 1H, Ar-H), 4.77 (d, J = 11.8 Hz, 1H, CHCO), 4.67–4.61 (m, 1H, NCH), 4.22 (dd, J = 11.8, 9.8 Hz, 1H, NCHCH), 3.94 (d, J = 10.6 Hz, 1H, NCH₂), 3.74 (s, 3H, CH₃), 3.61 (d, J = 10.5 Hz, 1H, NCH₂), 3.07 (d, J = 4.3 Hz, 2H, SCH₂), 1.92 (s, 3H, CH₃); ¹³C NMR (176 MHz, CDCl₃) δ (ppm) = 191.09 (CO), 180.96 (CO), 143.43, 141.25, 141.21, 138.27, 137.40, 129.70, 129.23, 129.03, 128.81, 127.60, 126.88, 125.45, 124.02, 122.13, 121.80, 120.97, 119.90, 119.38, 112.03, 109.75, 109.58, 75.20, 73.86, 63.89, 55.20, 43.45, 37.35, 32.80 (CH₃), 11.67 (CH₃); IR (KBr, cm^–1) ν_max = 3237, 2923, 1738, 1733, 1722, 1699, 1694, 1682, 1674, 1668, 1661, 1652, 1645, 1634, 1622, 1615, 1597, 1557, 1538, 1532, 1505, 1470, 1456, 1398, 1329, 1229, 1180, 1156, 1115, 1069, 1013, 934, 801, 741, 695; [anal. calcd. for C₃₃H₂₉N₅O₂S: C, 70.82; H, 5.22; N, 12.51; found: C, 71.01; H, 5.15; N, 12.39]; LC/MS (ESI, m/z): found 560.4 [M + H]⁺, exact mass 559.14 for C₃₃H₂₉N₅O₂S.

(3R,6′S,7′R,7a’S)-7′-(5-Bromo-1H-indol-3-yl)-6′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-1′,6′,7′,7a’-tetrahydro-3′H-spiro[indoline-3,5′-pyrrolo[1,2-c]thiazol]-2-one (8c)

Following the general procedure (GP2), chalcone 5c (102 mg, 0.25 mmol), isatin 6a (37 mg, 0.25 mmol), and thioproline 7a (50 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8c; yield (59 mg, 38%); m.p.: 180–181 °C; [α]_D²⁵ = −12.53° (c 0.11, MeOH); ¹H NMR (700 MHz, CDCl₃) δ (ppm) = 9.05 (s, 1H, NH), 8.66 (s, 1H, NH), 8.18 (s, 1H, Ar-H), 7.98 (s, 1H, Ar-H), 7.69 (d, J = 7.5 Hz, 1H, Ar-H), 7.38–7.35 (m, 4H, Ar-H), 7.20–7.18 (m, 1H, Ar-H), 7.15–7.13 (m, 2H, Ar-H), 7.03 (d, J = 7.6 Hz, 1H, Ar-H), 6.62 (d, J = 7.7 Hz, 1H, Ar-H), 4.63 (d, J = 11.6 Hz, 1H, CHCO), 4.50–4.49 (ddd, J = 9.4, 6.2, 2.5 Hz, 1H, NCH), 4.21 (m, 1H, NCHCH), 3.92 (d, J = 10.4 Hz, 1H, NCH₂), 3.60 (d, J = 10.4 Hz, 1H, NCH₂), 3.08–3.04 (m, 1H, SCH₂), 3.03–3.98 (m, 1H, SCH₂), 1.91 (s, 3H, CH₃); ¹³C NMR (176 MHz, CDCl₃) δ (ppm) = 191.02 (CO), 180.91 (CO), 143.59, 141.14, 138.23, 135.18, 129.84, 129.33, 128.96, 128.58, 127.75, 127.12, 125.47, 125.19, 124.00, 123.78, 122.33, 122.04, 120.91, 113.74, 113.18, 113.03, 109.80, 75.06, 74.25, 65.41, 54.90, 42.63, 37.28, 11.70 (CH₃); IR (KBr, cm^–1) ν_max = 3273, 2924, 1721, 1668, 1617, 1597, 1537, 1503, 1470, 1455, 1396, 1331, 1283, 1223, 1179, 1099, 934, 884, 807, 795, 762, 752, 694, 656, 604; [anal. calcd. for C₃₂H₂₆BrN₅O₂S: C, 61.54; H, 4.20; N, 11.21; found: C, 61.43; H, 4.35; N, 11.27]; LC/MS (ESI, m/z): found 624.7 [M(₇₉Br) + H]⁺, 626.1 [M(₈₁Br) + H]⁺; exact mass 623.10 for C₃₂H₂₆BrN₅O₂S.

(3R,6′S,7′R,7a’S)-7′-(5-Chloro-1H-indol-3-yl)-6′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-1′,6′,7′,7a’-tetrahydro-3′H-spiro[indoline-3,5′-pyrrolo[1,2-c]thiazol]-2-one (8d)

Following the general procedure (GP2), chalcone 5d (91 mg, 0.25 mmol), isatin 6a (37 mg, 0.25 mmol), and thioproline 7a (50 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8d; yield (76 mg, 52%); m.p.: 210–212 °C; [α]_D²⁵ = −12.03° (c 0.11, MeOH); ¹H NMR (700 MHz, CDCl₃) δ (ppm) = 8.61 (s, 1H, NH), 8.38 (s, 1H, NH), 8.00 (s, 1H, Ar-H), 7.86 (s, 1H, Ar-H), 7.64 (d, J = 7.6 Hz, 1H, Ar-H), 7.33 (dd, J = 10.7, 7.0 Hz, 3H, Ar-H), 7.23–7.18 (m, 2H, Ar-H), 7.09 (dd, J = 15.4, 8.3 Hz, 4H, Ar-H), 7.00–6.96 (m, 1H, Ar-H), 6.60 (d, J = 7.8 Hz, 1H, Ar-H), 4.58 (d, J = 11.6 Hz, 1H, CHCO), 4.50–4.42 (m, 1H, NCH), 4.16 (t, J = 10.6 Hz, 1H, NCHCH), 3.88 (d, J = 10.4 Hz, 1H, NCH₂), 3.55 (d, J = 10.4 Hz, 1H, NCH₂), 3.06–2.94 (m, 2H, SCH₂), 1.86 (s, 3H, CH₃); ¹³C NMR (176 MHz, CDCl₃) δ (ppm) = 190.95 (CO), 180.78 (CO), 143.60, 141.17, 141.01, 138.27, 134.95, 129.88, 129.35, 129.06, 128.97, 127.87, 125.71, 125.50, 124.00, 122.75, 122.40, 120.91, 119.13, 113.90, 112.54, 109.76, 74.12, 64.53, 54.95, 42.77, 37.30, 11.72 (CH₃); IR (KBr, cm^–1) ν_max = 3284, 2926, 1720, 1668, 1652, 1616, 1597, 1538, 1504, 1470, 1397, 1329, 1283, 1268, 1223, 1180, 1103, 934, 893, 806, 796, 763, 752, 694, 683, 658, 605; [anal. calcd. for C₃₂H₂₆ClN₅O₂S: C, 66.26; H, 4.52; N, 12.07; found: C, 66.14; H, 4.63; N, 12.15]; LC/MS (ESI, m/z): found 580.6 [M(₃₅Cl) + H]⁺, 582.3 [M(₃₇Cl) + H]⁺; exact mass 579.15 for C₃₂H₂₆ClN₅O₂S.

(3R,6′S,7′R,7a’S)-6-Chloro-7′-(1H-indol-3-yl)-6′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-3′,6′,7′,7a’-tetrahydro-1′H-spiro[indoline-3,5′-pyrrolo[1,2-c]thiazol]-2-one (8e)

Following the general procedure (GP2), chalcone 5a (82 mg, 0.25 mmol), 6-chloroisatin 6b (46 mg, 0.25 mmol), and thioproline 7a (50 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8e; yield (44 mg, 30%); m.p.: 188–190 °C; [α]_D²⁵ = −18.78° (c 0.11, MeOH); ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) = 11.00 (s, 1H, NH), 10.68 (s, 1H, NH), 7.87 (d, J = 7.8 Hz, 1H, Ar-H), 7.84 (d, J = 2.5 Hz, 1H, Ar-H), 7.54 (d, J = 8.2 Hz, 1H, Ar-H), 7.51–7.45 (m, 4H, Ar-H), 7.38–7.32 (m, 3H, Ar-H), 7.11–7.06 (m, 2H, Ar-H), 7.04 (d, J = 8.2 Hz, 1H, Ar-H), 6.67 (s, 1H, Ar-H), 4.73 (d, J = 11.8 Hz, 1H, CHCO), 4.33–4.29 (m, 1H, NCH), 4.13–4.09 (m, 1H, NCHCH), 3.78 (d, J = 10.3 Hz, 1H, NCH_2(a)), 3.43 (d, J = 8.2 Hz, 1H, NCH_2(b)), 3.07–2.99 (m, 2H, SCH₂), 1.92 (s, 3H, CH₃); ¹³C NMR (176 MHz, DMSO-d₆): δ (ppm) =190.34 (CO), 178.87 (CO), 143.68, 142.64, 140.80, 137.91, 136.59, 133.90, 129.93, 129.31, 128.87, 128.07, 126.65, 126.45, 126.21, 125.26, 123.43, 122.45, 121.19, 120.54, 118.77, 112.02, 109.44, 73.87, 73.77, 62.86, 54.93, 53.92, 43.19, 36.70, 11.22 (CH₃); IR (KBr, cm^–1) ν_max = 3295, 2928, 1723, 1717, 1699, 1668, 1645, 1615, 1598, 1538, 1533, 1504, 1483, 1456, 1398, 1379, 1338, 1325, 1282, 1244, 1224, 1183, 1125, 1096, 1072, 926, 852, 811, 764, 743, 694, 658, 594, 529; [anal. calcd. for C₃₂H₂₆ClN₅O₂S: C, 66.26; H, 4.52; N, 12.07; found: C, 66.12; H, 4.67; N, 12.22]; LC/MS (ESI, m/z): found 580.5 [M(₃₅Cl) + H]⁺, 582.3 [M(₃₇Cl) + H]⁺; exact mass 579.15 for C₃₂H₂₆ClN₅O₂S.

(3R,6′S,7′R,7a’S)-7′-(1H-Indol-3-yl)-6′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-5-nitro-1′,6′,7′,7a’-tetrahydro-3′H-spiro[indoline-3,5′-pyrrolo[1,2-c]thiazol]-2-one (8f)

Following the general procedure (GP2), chalcone 5a (82 mg, 0.25 mmol), 5-nitroisatin 6c (48 mg, 0.25 mmol), and thioproline 7a (50 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield yellow solid compound 8f; yield (49 mg, 33%); m.p.: 213–214 °C; [α]_D²⁵ = −36.24° (c 0.15, MeOH); ¹H NMR (700 MHz, DMSO-d₆) δ (ppm) = 11.26 (s, 1H, NH), 11.04 (s, 1H, NH), 8.46 (s, 1H, Ar-H), 8.17 (d, J = 8.2 Hz, 1H, Ar-H), 7.89–7.84 (m, 2H, Ar-H), 7.55 (s, 1H, Ar-H), 7.50–7.47 (m, 2H, Ar-H), 7.46 (d, J = 7.5 Hz, 1H, Ar-H), 7.36 (d, J = 6.9 Hz, 1H, Ar-H), 7.32–7.30 (m, 2H, Ar-H), 7.09–7.10 (m, 2H, Ar-H), 6.88 (d, J = 8.6 Hz, 1H, Ar-H), 4.79 (d, J = 12.0 Hz, 1H, CHCO), 4.41–4.36 (m, 1H, NCH), 4.14–4.09 (m, 1H, NCHCH), 3.80 (d, J = 10.8 Hz, 1H, NCH_2(a)), 3.50 (d, J = 10.8 Hz, 1H, NCH_2(b)), 3.08 (t, J = 3.6 Hz, 2H, SCH₂), 1.86 (s, 3H, CH₃); ¹³C NMR (176 MHz, DMSO-d₆) δ (ppm) = 190.24 (CO), 179.44 (CO), 148.63, 142.79, 141.34, 140.76, 137.81, 136.64, 129.34, 128.93, 128.08, 126.92, 126.46, 126.07, 125.26, 124.34, 123.83, 121.25, 120.47, 118.89, 118.54, 111.42, 109.90, 74.15, 73.58, 62.93, 54.48, 43.59, 36.77, 11.25 (CH₃); IR (KBr, cm^–1) ν_max = 3252, 2924, 2853, 1736, 1729, 1665, 1652, 1626, 1598, 1526, 1504, 1478, 1455, 1398, 1338, 1300, 1248, 1223, 1198, 1180, 1124, 1098, 1069, 932, 907, 828, 807, 763, 743, 694, 557; [anal. calcd. for C₃₂H₂₆N₆O₄S: C, 65.07; H, 4.44; N, 14.23; found: C, 64.91; H, 4.56; N, 14.04]; LC/MS (ESI, m/z): found 591.7 [M + H], exact mass 590.17 for C₃₂H₂₆N₆O₄S.

(3R,6′S,7′R,7a’S)-6-Chloro-6′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-7′-(1-methyl-1H-indol-3-yl)-1′,6′,7′,7a’-tetrahydro-3′H-spiro[indoline-3,5′-pyrrolo[1,2-c]thiazol]-2-one (8g)

Following the general procedure (GP2), chalcone 5b (85 mg, 0.25 mmol), 6-chloroisatin 6b (46 mg, 0.25 mmol), and thioproline 7a (50 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8g; yield (55 mg, 37%); m.p.: 164–165 °C; [α]_D²⁵ = −41.94° (c 0.12, MeOH); ¹H NMR (700 MHz, CDCl₃) δ (ppm) = 9.23 (s, 1H, NH), 8.07 (d, J = 7.9 Hz, 1H, Ar-H), 7.94 (s, 1H, Ar-H), 7.64 (d, J = 8.1 Hz, 1H, Ar-H), 7.40 (d, J = 6.2 Hz, 2H, Ar-H), 7.36 (s, 1H, Ar-H), 7.29 (d, J = 8.2 Hz, 1H, Ar-H), 7.25–7.22 (m, 1H, Ar-H), 7.21–7.18 (m, 2H, Ar-H), 7.16 (d, J = 8.1 Hz, 2H, Ar-H), 7.00 (d, J = 8.1 Hz, 1H, Ar-H), 6.51 (s, 1H, Ar-H), 4.75 (d, J = 11.8 Hz, 1H, CHCO), 4.63–4.60 (m, 1H, NCH), 4.21–4.15 (m, 1H, NCHCH), 3.93 (d, J = 10.6 Hz, 1H, NCH_2(a)), 3.74 (s, 3H, NCH₃), 3.56 (d, J = 10.7 Hz, 1H, NCH_2(b)), 3.09–3.02 (m, 2H, SCH₂), 1.99 (s, 3H, CH₃); ¹³C NMR (176 MHz, CDCl₃) δ (ppm) = 190.90 (CO), 180.77 (CO), 143.66, 141.19, 138.14, 137.42, 135.36, 130.05, 129.39, 129.11, 128.65, 127.59, 127.10, 126.86, 125.53, 122.49, 121.90, 120.90, 119.84, 119.50, 111.84, 110.17, 109.63, 74.78, 73.80, 63.95, 55.17, 43.58, 37.44, 32.85 (NCH₃), 11.71 (CH₃); IR (KBr, cm^–1) ν_max = 3234, 2922, 2849, 1733, 1717, 1699, 1667, 1652, 1612, 1598, 1544, 1537, 1532, 1503, 1483, 1454, 1401, 1377, 1327, 1261, 1223, 1182, 157, 1123, 1071, 1012, 925, 808, 765, 738, 694, 612, 529; [anal. calcd. for C₃₃H₂₈ClN₅O₂S: C, 66.71; H, 4.75; N, 11.79; found: C, 66.85; H, 4.62; N, 12.03]; LC/MS (ESI, m/z): found 594.5 [M(₃₅Cl) + H]⁺, 596.0 [M(₃₇Cl) + H]⁺; exact mass 593.17 for C₃₃H₂₈ClN₅O₂S.

(3R,6′S,7′R,7a’S)-6′-(5-Methyl-1-phenyl-1H-pyrazole-4-carbonyl)-7′-(1-methyl-1H-indol-3-yl)-5-nitro-3′,6′,7′,7a’-tetrahydro-1′H-spiro[indoline-3,5′-pyrrolo[1,2-c]thiazol]-2-one (8h)

Following the general procedure (GP2), chalcone 5b (85 mg, 0.25 mmol), 5-nitroisatin 6c (48 mg, 0.25 mmol), and thioproline 7a (50 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 16 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield yellow solid compound 8h; yield (41 mg, 27%); m.p.: 172–173 °C; [α]_D²⁵ = −22.87° (c 0.13, MeOH); ¹H NMR (700 MHz, CDCl₃) δ (ppm) = 9.78 (s, 1H, NH), 8.73 (s, 1H, Ar-H), 8.14 (dd, J = 27.8, 8.4 Hz, 2H, Ar-H), 7.99 (s, 1H, Ar-H), 7.47–7.41 (m, 3H, Ar-H), 7.40 (d, J = 4.3 Hz, 1H, Ar-H), 7.35 (d, J = 8.2 Hz, 1H, Ar-H), 7.30 (d, J = 8.2 Hz, 1H, Ar-H), 7.23–7.19 (m, 1H, Ar-H), 7.16 (dd, J = 6.7, 3.1 Hz, 2H, Ar-H), 6.69 (d, J = 9.2 Hz, 1H, Ar-H), 4.89 (d, J = 11.5 Hz, 1H CHCO), 4.73 (m, 1H NCH), 4.30 (t, J = 10.7 Hz, 1H, NCHCH), 3.99 (d, J = 10.4 Hz, 1H, NCH_2(a)), 3.79 (s, 3H, CH₃), 3.51 (d, J = 10.5 Hz, 1H, NCH_2(b)), 3.16 (d, J = 4.4 Hz, 2H, SCH₂), 2.01 (s, 3H, CH₃); ¹³C NMR (176 MHz, CDCl₃) δ (ppm) = 190.04 (CO), 180.99 (CO), 146.95, 143.91, 142.89, 141.14, 137.92, 137.52, 129.47, 129.26, 128.66, 128.04, 127.74, 127.12, 126.55, 125.38, 125.06, 124.92, 121.96, 120.90, 119.75, 111.02, 109.77, 73.37, 63.71, 55.11, 44.29, 37.47, 32.86 (CH₃), 11.86 (CH₃); IR (KBr, cm^–1) ν_max = 3208, 2926, 2859, 1736, 1716, 1699, 1682, 1678, 1668, 1652, 1622, 1615, 1598, 1524, 1504, 1475, 1455, 1404, 1337, 1221, 1177, 1123, 1103, 932, 833, 805, 765, 741, 694, 556; [anal. calcd. for C₃₃H₂₈N₆O₄S: C, 65.55; H, 4.67; N, 13.90; found: C, 65.67; H, 4.81; N, 14.04]; LC/MS (ESI, m/z): found 605.6 [M + H], exact mass 604.19 for C₃₃H₂₈N₆O₄S.

(3R,6′S,7′R,7a′S)-7′-(5-Bromo-1H-indol-3-yl)-6′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-5-nitro-3′,6′,7′,7a’-tetrahydro-1′H-spiro[indoline-3,5′-pyrrolo[1,2-c]thiazol]-2-one (8i)

Following the general procedure (GP2), chalcone 5c (102 mg, 0.25 mmol), 5-nitroisatin 6c (48 mg, 0.25 mmol), and thioproline 7a (50 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 16 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield yellow solid compound 8i; yield (82 mg, 49%); m.p.: 250–251 °C; [α]_D²⁵ = −126.29° (c 0.19, MeOH); ¹H NMR (700 MHz, DMSO-d₆) δ (ppm) = 11.30 (d, J = 2.6 Hz, 1H, NH), 11.25 (s, 1H, NH), 8.45 (d, J = 2.4 Hz, 1H, Ar-H), 8.17 (dd, J = 8.6, 2.4 Hz, 1H, Ar-H), 7.98 (d, J = 1.9 Hz, 1H, Ar-H), 7.84 (s, 1H, Ar-H), 7.67 (d, J = 2.6 Hz, 1H, Ar-H), 7.51–7.48 (m, 2H, Ar-H), 7.48–7.45 (m, 1H, Ar-H), 7.35–7.31 (m, 3H, Ar-H), 7.21 (dd, J = 8.5, 1.9 Hz, 1H, Ar-H), 6.87 (d, J = 8.6 Hz, 1H, Ar-H), 4.67 (d, J = 11.9 Hz, 1H, CHCO), 4.37–4.33 (m, 1H, NCH), 4.10 (dd, J = 11.8, 9.5 Hz, 1H, NCHCH), 3.79 (d, J = 10.7 Hz, 1H, NCH_2(a)), 3.50 (d, J = 10.6 Hz, 1H, NCH_2(b)), 3.09–3.04 (m, 2H, SCH₂), 1.86 (s, 3H, CH₃); ¹³C NMR (176 MHz, DMSO-d₆) δ (ppm) = 190.22 (CO), 179.25 (CO), 148.59, 142.75, 141.34, 140.74, 137.77, 135.16, 129.31, 128.90, 128.07, 126.90, 125.42, 125.21, 124.29, 123.85, 123.69, 120.73, 120.38, 113.84, 111.50, 111.42, 109.86, 73.95, 73.48, 63.46, 54.14, 42.83, 36.56, 11.21 (CH₃); IR (KBr, cm^–1) ν_max = 3383, 3101, 2855, 1747, 1729, 1649, 1622, 1598, 1530, 1504, 1463, 1454, 1412, 1337, 1290, 1253, 1222, 1199, 1173, 1097, 933, 880, 830, 799, 753, 693, 607; [anal. calcd. for C₃₂H₂₅BrN₆O₄S: C, 57.40; H, 3.76; N, 12.55; found: C, 57.36; H, 3.84; N, 12.59]; LC/MS (ESI, m/z): found 669.6 [M(₇₉Br) + H]⁺, 671.5 [M(₈₁Br) + H]⁺; exact mass 668.08 for C₃₂H₂₅BrN₆O₄S.

(3R,6′S,7′R,7a’S)-7′-(5-Chloro-1H-indol-3-yl)-6′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-5-nitro-3′,6′,7′,7a’-tetrahydro-1′H-spiro[indoline-3,5′-pyrrolo[1,2-c]thiazol]-2-one (8j)

Following the general procedure (GP2), chalcone 5d (91 mg, 0.25 mmol), 5-nitroisatin 6c (48 mg, 0.25 mmol), and thioproline 7a (50 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield yellow solid compound 8j; yield (53 mg, 40%); m.p.: 196–197 °C; [α]_D²⁵ = −53.73° (c 0.10, MeOH); ¹H NMR (700 MHz, DMSO-d₆) δ (ppm) = 11.30 (s, 1H, NH), 11.27 (s, 1H, NH), 8.46 (s, 1H, Ar-H), 8.18 (d, J = 8.6 Hz, 1H, Ar-H), 7.85 (s, 1H, Ar-H), 7.69 (s, 1H, Ar-H), 7.53–7.46 (m, 2H, Ar-H), 7.48 (d, J = 5.5 Hz, 1H, Ar-H), 7.39 (d, J = 8.6 Hz, 1H, Ar-H), 7.34–7.31 (m, 3H, Ar-H), 7.12 (d, J = 8.6 Hz, 1H, Ar-H), 6.88 (d, J = 8.7 Hz, 1H, Ar-H), 4.68 (d, J = 11.7 Hz, 1H, CHCO), 4.39–4.35 (m, 1H, NCH), 4.12 (t, J = 12.1 Hz, 1H, NCHCH), 3.80 (d, J = 10.9 Hz, 1H, NCH_2(a)), 3.51 (d, J = 10.9 Hz, 1H, NCH_2(b)), 3.11–3.05 (m, 2H, SCH₂), 1.87 (s, 3H, CH₃); ¹³C NMR (176 MHz, DMSO-d₆) δ (ppm) = 190.27 (CO), 179.33 (CO), 148.63, 142.79, 141.39, 140.77, 137.80, 134.98, 129.35, 128.94, 127.38, 126.94, 126.66, 126.45, 125.62, 124.33, 123.50, 121.22, 120.41, 117.74, 113.42, 111.62, 109.90, 74.00, 73.49, 62.93, 54.94, 42.87, 36.62, 11.23 (CH₃); IR (KBr, cm^–1) ν_max = 3344, 3270, 2932, 2861, 1725, 1657, 1623, 1598, 1530, 1503, 1477, 1454, 1339, 1296, 1224, 1180, 1101, 931, 892, 797, 764, 693, 614, 553; [Anal. Calcd. for C₃₂H₂₅ClN₆O₄S: C, 61.49; H, 4.03; N, 13.44; found: C, 61.35; H, 3.91; N, 13.62]; LC/MS (ESI, m/z): found 625.8 [M (₃₅Cl) + H]⁺, 627.2 [M (₃₇Cl) + H]⁺; exact mass 624.13 for C₃₂H₂₅ClN₆O₄S.

(1′R,2′S,3R,9a’R)-1′-(1H-Indol-3-yl)-2′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-1′,2′,4a’,5′,6′,7′,8′,8a’,9′,9a’-decahydrospiro[indoline-3,3′-pyrrolo[1,2-a]indol]-2-one (8k)

Following the general procedure (GP2), chalcone 5a (82 mg, 0.25 mmol), isatin 6a (37 mg, 0.25 mmol), and octahydro-1H-indole-2-carboxylic acid 7b (64 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8k; yield (125 mg, 86%); m.p.: 200–201 °C; [α]_D²⁵ = −18.76° (c 0.12, MeOH); ¹H NMR (700 MHz, CDCl₃) δ (ppm) = 8.33 (s, 1H, NH), 8.04 (d, J = 1.2 Hz, 1H, Ar-H), 8.04–8.02 (m, 1H, Ar-H), 7.95 (s, 1H, NH), 7.42 (d, J = 7.4 Hz, 1H, Ar-H), 7.40–7.34 (m, 3H, Ar-H), 7.33–7.29 (m, 1H, Ar-H), 7.20–7.14 (m, 5H, Ar-H), 7.13–7.09 (m, 1H, Ar-H), 7.06–7.02 (m, 1H, Ar-H), 6.57 (d, J = 7.7 Hz, 1H, Ar-H), 4.96 (d, J = 11.9 Hz, 1H, CHCO), 4.57–4.52 (m, 1H, NCH), 4.20 (dd, J = 12.0, 10.0 Hz, 1H, NCHCH), 3.27 (q, J = 3.8 Hz, 1H, NCH), 2.19–2.14 (m, 1H, NCHCH), 1.93 (s, 3H, CH₃), 1.85–1.78 (m, 2H, CH₂), 1.59–1.44 (m, 4H, CH₂), 1.20–1.15 (m, 1H, CH₂), 1.07–1.01 (m, 1H, CH₂), 1.01–0.95 (m, 2H, CH₂); ¹³C NMR (176 MHz, CDCl₃) δ (ppm) = 191.46 (CO), 181.76 (CO), 143.42, 141.62, 140.65, 138.43, 136.60, 129.25, 128.94, 128.74, 128.53, 127.06, 125.41, 125.10, 121.99, 121.78, 121.74, 121.26, 120.04, 119.48, 114.40, 111.35, 109.61, 72.74, 70.23, 66.94, 57.75, 45.57, 41.98, 38.40, 28.59, 27.78, 24.86, 20.13, 11.88 (CH₃); IR (KBr, cm^–1) ν_max = 3291, 2926, 2854, 1716, 1699, 1694, 1683, 1668, 1660, 1652, 1617, 1597, 1557, 1538, 1505, 1475, 1456, 1398, 1373, 1330, 1221, 1179, 1099, 1011, 934, 870, 790, 741, 694; [anal. calcd. for C₃₇H₃₅N₅O₂: C, 76.40; H, 6.06; N, 12.04; found: C, 76.28; H, 6.14; N, 12.11]; LC/MS (ESI, m/z): found 582.5 [M + H]⁺, exact mass 581.28 for C₃₇H₃₅N₅O₂.

(1′R,2′S,3R,9a’R)-6-Chloro-1′-(1H-indol-3-yl)-2′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-1′,2′,4a’,5′,6′,7′,8′,8a’,9′,9a′-decahydrospiro[indoline-3,3′-pyrrolo[1,2-a]indol]-2-one (8l)

Following the general procedure (GP2), chalcone 5a (82 mg, 0.25 mmol), 6-chloroisatin 6b (46 mg, 0.25 mmol), and octahydro-1H-indole-2-carboxylic acid 7b (64 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8l; yield (131 mg, 85%); m.p.: 205–206 °C; [α]_D²⁵ = −20.56° (c 0.11, MeOH); ¹H NMR (700 MHz, CDCl₃) δ (ppm) = 8.55 (s, 1H, NH), 8.32 (s, 1H, NH), 8.10 (d, J = 2.8 Hz, 1H, Ar-H), 8.00 (d, J = 7.6 Hz, 1H, Ar-H), 7.42–7.38 (m, 3H, Ar-H), 7.32 (d, J = 8.4 Hz, 2H, Ar-H), 7.19–7.11 (m, 5H, Ar-H), 7.01 (d, J = 8.0 Hz, 1H, Ar-H), 6.41 (d, J = 2.3 Hz, 1H, Ar-H), 4.96 (d, J = 12.0 Hz, 1H, CHCO), 4.56–4.52 (m, 1H, NCH), 4.15 (dd, J = 12.0, 10.0 Hz, 1H, NCHCH), 3.22 (q, J = 3.8 Hz, 1H, NCH), 2.19–2.14 (m, 1H, NCHCH), 2.00 (s, 3H, CH₃), 1.82–1.76 (m, 2H, CH₂), 1.59–1.55 (m, 1H, CH₂), 1.54–1.46 (m, 3H, CH₂), 1.20–1.16 (m, 1H, CH₂), 1.07–0.96 (m, 3H, CH₂); ¹³C NMR (176 MHz, CDCl₃): δ (ppm) = 191.36 (CO), 181.77 (CO), 143.61, 142.07, 141.60, 138.28, 136.59, 134.55, 129.39, 129.03, 128.68, 126.99, 125.46, 123.59, 122.06, 121.81, 121.56, 121.16, 119.95, 119.55, 114.15, 111.40, 110.13, 72.39, 70.19, 67.06, 57.81, 45.69, 41.98, 38.35, 28.65, 27.77, 24.81, 20.14, 11.86 (CH₃); IR (KBr, cm^–1) ν_max = 3298, 2926, 2851, 1725, 1668, 1652, 1614, 1598, 1538, 1532, 1504, 1484, 1455, 1447, 1398, 1373, 1323, 1284, 1243, 1222, 1179, 1130, 1072, 1011, 937, 924, 869, 795, 764, 741, 694, 660, 597, 584, 524; [anal. calcd. for C₃₇H₃₄ClN₅O₂: C, 72.12; H, 5.56; N, 11.37; found: C, 71.96; H, 5.63; N, 11.28]; LC/MS (ESI, m/z): found 616.6 [M(₃₅Cl) + H]⁺, 618.3 [M(₃₇Cl) + H]⁺; exact mass 615.24 for C₃₇H₃₄ClN₅O₂.

(1′R,2′S,3R,9a’R)-1′-(1H-Indol-3-yl)-2′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-5-nitro-1′,2′,4a’,5′,6′,7′,8′,8a’,9′,9a’-decahydrospiro[indoline-3,3′-pyrrolo[1,2-a]indol]-2-one (8m)

Following the general procedure (GP2), chalcone 5a (82 mg, 0.25 mmol), 5-nitroisatin 6c (48 mg, 0.25 mmol), and octahydro-1H-indole-2-carboxylic acid 7b (64 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 4 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield yellow solid compound 8m; yield (103 mg, 66%); m.p.: 210–211 °C; [α]_D²⁵ = −15.63° (c 0.13, MeOH); ¹H NMR (700 MHz, CDCl₃) δ (ppm) = 9.19 (s, 1H, NH), 8.27–8.22 (m, 2H, Ar-H), 8.11 (s, 1H, NH), 8.06 (d, J = 8.6 Hz, 1H, Ar-H), 8.02 (d, J = 7.9 Hz, 1H, Ar-H), 7.42–7.37 (m, 3H, Ar-H), 7.34 (d, J = 7.0 Hz, 1H, Ar-H), 7.20 (s, 1H, Ar-H), 7.18–7.14 (m, 2H, Ar-H), 7.10 (d, J = 7.5 Hz, 2H, Ar-H), 6.42 (d, J = 8.7 Hz, 1H, Ar-H), 5.03 (d, J = 12.0 Hz, 1H, CHCO), 4.63–4.58 (m, 1H, NCH), 4.21 (t, J = 11.0 Hz, 1H, NCHCH), 3.24 (q, J = 3.7 Hz, 1H, NCH), 2.23–2.18 (m, 1H, NCHCH), 1.95 (s, 3H, CH₃), 1.90–1.86 (m, 1H, CH₂), 1.84–1.81 (m, 1H, CH₂), 1.61–1.56 (m, 1H, CH₂), 1.53–1.46 (m, 3H, CH₂), 1.22–1.17 (m, 1H, CH₂), 1.08–0.98 (m, 2H, CH₂), 0.93–0.89 (m, 1H, CH₂); ¹³C NMR (176 MHz, CDCl₃): δ (ppm) = 190.53 (CO), 182.11 (CO), 146.67, 143.82, 142.71, 141.51, 138.12, 136.64, 129.48, 129.20, 127.14, 126.80, 126.02, 125.38, 123.96, 122.21, 121.96, 121.13, 120.02, 119.71, 113.58, 111.43, 109.26, 72.52, 70.05, 67.02, 57.95, 46.02, 42.11, 38.27, 28.56, 27.72, 24.77, 20.05, 11.95 (CH₃); IR (KBr, cm^–1) ν_max = 3341, 2926, 2854, 1733, 1668, 1653, 1623, 1598, 1533, 1503, 1477, 1455, 1397, 1338, 1221, 1178, 1125, 1096, 1072, 1011, 930, 833, 764, 742, 695, 660, 555; [anal. calcd. for C₃₇H₃₄N₆O₄: C, 70.91; H, 5.47; N, 13.41; found: C, 71.06; H, 5.39; N, 13.49]; LC/MS (ESI, m/z): found 627.5 [M + H]⁺; exact mass 626.26 for C₃₇H₃₄N₆O₄.

(1′R,2′S,3R,9a’R)-6-Chloro-2′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-1′-(1-methyl-1H-indol-3-yl)-1′,2′,4a’,5′,6′,7′,8′,8a’,9′,9a’-decahydrospiro[indoline-3,3′-pyrrolo[1,2-a]indol]-2-one (8n)

Following the general procedure (GP2), chalcone 5b (85 mg, 0.25 mmol), 6-chloroisatin 6b (46 mg, 0.25 mmol), and octahydro-1H-indole-2-carboxylic acid 7b (64 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8n; yield (134 mg, 85%); m.p.: 172–173 °C; [α]_D²⁵ = −19.29° (c 0.10, MeOH); ¹H NMR (700 MHz, CDCl₃) δ (ppm) = 8.06 (s, 1H, NH), 8.01 (d, J = 7.9 Hz, 1H, Ar-H), 7.44–7.40 (m, 3H, Ar-H), 7.31 (d, J = 8.0 Hz, 1H, Ar-H), 7.27 (s, 1H, Ar-H), 7.23–7.20 (m, 1H, Ar-H), 7.17 (d, J = 7.7 Hz, 3H, Ar-H), 7.10 (s, 1H, Ar-H), 7.02 (d, J = 8.0 Hz, 1H, Ar-H), 6.43 (s, 1H, Ar-H), 4.93 (d, J = 12.0 Hz, 1H, CHCO), 4.56–4.52 (m, 1H, NCH), 4.16–4.11 (m, 1H, NCHCH), 3.73 (s, 3H, NCH₃), 3.23 (q, J = 3.8 Hz, 1H, NCH), 2.20–2.15 (m, 1H, NCHCH), 2.02 (s, 3H, CH₃), 1.84–1.79 (m, 2H, CH₂), 1.60–1.56 (m, 1H, CH₂), 1.55–1.48 (m, 3H, CH₂), 1.21–1.17 (m, 1H, CH₂), 1.08–0.96 (m, 3H, CH₂); ¹³C NMR (176 MHz, CDCl₃): δ (ppm) = 191.30 (CO), 181.64 (CO), 143.59, 142.02, 141.57, 138.33, 137.27, 134.52, 129.38, 128.99, 127.44, 127.12, 126.40, 125.46, 123.59, 121.66, 121.56, 121.16, 120.13, 119.09, 112.69, 110.05, 109.36, 72.29, 70.32, 67.20, 57.79, 45.60, 42.00, 38.38, 32.83 (NCH₃), 28.64, 27.78, 24.84, 20.11, 11.88 (CH₃); IR (KBr, cm^–1) ν_max = 3057, 2926, 2849, 1733, 1668, 1652, 1615, 1538, 1532, 1504, 1484, 1455, 1404, 1373, 1328, 1282, 1224, 1179, 1130, 1071, 1012, 935, 924, 869, 796, 764, 740, 694; [anal. calcd. for C₃₈H₃₆ClN₅O₂: C, 72.43; H, 5.76; N, 11.11; found: C, 72.52; H, 5.71; N, 11.03]; LC/MS (ESI, m/z): found 630.6 [M(₃₅Cl) + H]⁺, 632.3 [M(₃₇Cl) + H]⁺; exact mass 629.26 for C₃₈H₃₆ClN₅O₂.

(1′R,2′S,3R,9a’R)-2′-(5-Methyl-1-phenyl-1H-pyrazole-4-carbonyl)-1′-(1-methyl-1H-indol-3-yl)-5-nitro-1′,2′,4a’,5′,6′,7′,8′,8a’,9′,9a’-decahydrospiro[indoline-3,3′-pyrrolo[1,2-a]indol]-2-one (8o)

Following the general procedure (GP2), chalcone 5b (85 mg, 0.25 mmol), 5-nitroisatin 6c (48 mg, 0.25 mmol), and octahydro-1H-indole-2-carboxylic acid 7b (64 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 4 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield yellow solid compound 8o; yield (119 mg, 74%); m.p.: 185–186 °C; [α]_D²⁵ = −32.74° (c 0.14, MeOH); ¹H NMR (700 MHz, CDCl₃) δ (ppm) = 11.03 (s, 1H, NH), 8.16 (d, J = 7.6 Hz, 2H, Ar-H), 7.98 (s, 1H, Ar-H), 7.86 (d, J = 7.9 Hz, 1H, Ar-H), 7.52–7.50 (m, 2H, Ar-H), 7.49–7.45 (m, 2H, Ar-H), 7.38–7.33 (m, 3H, Ar-H), 7.16–7.13 (m, 1H, Ar-H), 7.10–7.07 (m, 1H, Ar-H), 6.85 (d, J = 9.1 Hz, 1H, Ar-H), 4.92 (d, J = 12.1 Hz, 1H, CHCO), 4.25–4.12 (m, 1H, NCH), 4.13 (dd, J = 12.1, 9.9 Hz, 1H, NCHCH), 3.71 (s, 3H, NCH₃), 3.22 (q, J = 3.5 Hz, 1H, NCH), 2.19–2.13 (m, 1H, NCHCH), 2.05–1.99 (m, 1H, CH₂), 1.81 (s, 3H, CH₃), 1.65–1.61 (m, 1H, CH₂), 1.55–1.50 (m, 1H, CH₂), 1.48–1.43 (m, 1H, CH₂), 1.39–1.27 (m, 2H, CH₂), 1.15–1.10 (m, 1H, CH₂), 1.04–0.97 (m, 1H, CH₂), 0.96–0.90 (m, 1H, CH₂), 0.80–0.74 (m, 1H, CH₂); ¹³C NMR (176 MHz, CDCl₃): δ (ppm) = 190.37 (CO), 181.03 (CO), 148.21, 142.53, 141.42, 141.08, 137.89, 136.71, 129.31, 128.82, 128.03, 127.10, 126.54, 126.41, 125.20, 125.07, 123.11, 121.17, 120.49, 119.19, 118.56, 111.66, 109.68, 71.51, 70.53, 66.16, 56.87, 44.37, 41.52, 37.53, 32.33 (NCH₃), 27.69, 27.27, 24.37, 19.27, 11.08 (CH₃); IR (KBr, cm^–1) ν_max = 3056, 2927, 2854, 1738, 1733, 1716, 1699, 1694, 1682, 1674, 1668, 1661, 1652, 1645, 1634, 1622, 1615, 1598, 1557, 1538, 1524, 1519, 1505, 1475, 1464, 1456, 1436, 1427, 1398, 1373, 1338, 1223, 1178, 1127, 1072, 1011, 930, 833, 795; [anal. calcd. for C₃₈H₃₆N₆O₄: C, 71.23; H, 5.66; N, 13.12; found: C, 71.31; H, 5.74; N, 13.27]; LC/MS (ESI, m/z): found 641.5 [M + H]⁺; exact mass 640.26 for C₃₈H₃₆N₆O₄.

(1′R,2′S,3R,9a’R)-1′-(5-Bromo-1H-indol-3-yl)-6-chloro-2′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-1′,2′,4a’,5′,6′,7′,8′,8a’,9′,9a’-decahydrospiro[indoline-3,3′-pyrrolo[1,2-a]indol]-2-one (8p)

Following the general procedure (GP2), chalcone 5c (102 mg, 0.25 mmol), 6-chloroisatin 6b (46 mg, 0.25 mmol), and octahydro-1H-indole-2-carboxylic acid 7b (64 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8p; yield (155 mg, 89%); m.p.: 199–200 °C; [α]_D²⁵ = −26.47° (c 0.12, MeOH); ¹H NMR (700 MHz, CDCl₃) δ (ppm) = 8.72 (s, 1H, NH), 8.63 (s, 1H, NH), 8.12 (s, 1H, Ar-H), 8.05 (s, 1H, Ar-H), 7.43–7.39 (m, 3H, Ar-H), 7.31 (d, J = 8.0 Hz, 1H, Ar-H), 7.23 (d, J = 1.5 Hz, 2H, Ar-H), 7.15–7.12 (m, 2H, Ar-H), 7.11 (d, J = 2.5 Hz, 1H, Ar-H), 7.02 (dd, J = 8.0, 1.9 Hz, 1H, Ar-H), 6.35 (s, 1H, Ar-H), 4.79 (d, J = 12.0 Hz, 1H, CHCO), 4.38–4.33 (m, 1H, NCH), 4.09 (dd, J = 12.1, 10.1 Hz, 1H, NCHCH), 3.21 (q, J = 3.7 Hz, 1H, NCH), 2.22–2.17 (m, 1H, NCHCH), 1.98 (s, 3H, CH₃), 1.85–1.78 (m, 2H, CH₂), 1.60–1.56 (m, 1H, CH₂), 1.54–1.45 (m, 3H, CH₂), 1.22–1.17 (m, 1H, CH₂), 1.09–0.97 (m, 3H, CH₂); ¹³C NMR (176 MHz, CDCl₃): δ (ppm) = 191.05 (CO), 181.83 (CO), 143.75, 142.03, 141.48, 138.22, 135.08, 134.65, 129.46, 129.36, 129.17, 129.15, 128.70, 127.14, 125.45, 125.03, 123.42, 122.38, 122.12, 121.67, 121.05, 114.32, 112.87, 110.15, 72.37, 70.86, 67.65, 57.77, 44.84, 41.96, 38.35, 28.57, 27.77, 24.77, 20.11, 11.80 (CH₃); IR (KBr, cm^–1) ν_max = 3273, 2926, 2853, 1733, 1699, 1674, 1652, 1615, 1557, 1538, 1532, 1504, 1483, 1456, 1398, 1373, 1322, 1278, 1220, 1179, 1130, 1102, 1072, 936, 932, 884, 869, 797, 765, 693, 670, 595, 525; [anal. calcd. for C₃₇H₃₃BrClN₅O₂: C, 63.94; H, 4.79; N, 10.08; found: C, 64.09; H, 4.86; N, 10.13]; LC/MS (ESI, m/z): found 695.0 [M(₃₅Cl/₇₉Br) + H]⁺, 696.3 [M(₃₇Cl/₈₁Br) + H]⁺, 698.1 [M(₃₇Cl + ₈₁Br) + H]⁺; exact mass 693.15 for C₃₇H₃₃BrClN₅O₂.

(1′R,2′S,3R,9a’R)-1′-(5-Bromo-1H-indol-3-yl)-6-chloro-2′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-1′,2′,4a’,5′,6′,7′,8′,8a’,9′,9a’-decahydrospiro[indoline-3,3′-pyrrolo[1,2-a]indol]-2-one (8q)

Following the general procedure (GP2), chalcone 5c (102 mg, 0.25 mmol), 5-nitroisatin 6c (48 mg, 0.25 mmol), and octahydro-1H-indole-2-carboxylic acid 7b (64 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 4 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield yellow solid compound 8q; yield (120 mg, 68%); m.p.: 190–199 °C; [α]_D²⁵ = −167.95° (c 0.10, MeOH); ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) = 11.17 (d, J = 2.7 Hz, 1H, NH), 11.01 (s, 1H, NH), 8.26 (d, J = 2.3 Hz, 1H, Ar-H), 8.15 (dd, J = 8.6, 2.4 Hz, 1H, Ar-H), 8.04 (s, 1H, Ar-H), 8.03 (d, J = 1.9 Hz, 1H, Ar-H), 7.57 (d, J = 2.6 Hz, 1H, Ar-H), 7.53–7.50 (m, 2H, Ar-H), 7.48–7.45 (m, 1H, Ar-H), 7.36 (d, J = 7.1 Hz, 2H, Ar-H), 7.29 (d, J = 8.6 Hz, 1H, Ar-H), 7.18 (dd, J = 8.5, 1.9 Hz, 1H, Ar-H), 6.84 (d, J = 8.6 Hz, 1H, Ar-H), 4.88 (d, J = 12.0 Hz, 1H, CHCO), 4.17–4.11 (m, 2H, NCH + NCHCH), 3.23 (q, J = 3.4 Hz, 1H, NCH), 2.18–2.10 (m, 2H NCHCH₂), 1.81 (s, 3H, CH₃), 1.63–1.58 (m, 1H, CH₂), 1.54–150 (m, 1H, CH₂), 1.49–1.45 (m, 1H, CH₂), 1.38–1.28 (m, 2H, CH₂), 1.15–1.10 (m, 1H, CH₂), 1.03–0.97 (m, 1H, CH₂), 0.96–0.90 (m, 1H, CH₂), 0.77–0.72 (m, 1H, CH₂); ¹³C NMR (176 MHz, DMSO-d₆): δ (ppm) = 190.50 (CO), 180.99 (CO), 148.17, 142.49, 141.54, 141.20, 137.91, 134.91, 129.31, 128.92, 128.81, 128.03, 126.51, 126.40, 125.20, 125.06, 123.62, 123.52, 123.33, 121.21, 120.55, 113.43, 112.57, 111.14, 109.61, 71.50, 70.72, 66.35, 56.77, 43.80, 41.56, 37.06, 27.65, 27.29, 24.42, 19.25, 11.08 (CH₃); IR (KBr, cm^–1) ν_max = 3363, 2930, 2854, 1734, 1762, 1621, 1599, 1528, 1503, 1478, 1454, 1398, 1337, 1221, 1178, 1126, 1098, 1081, 928, 884, 866, 831, 795, 765, 753, 693, 659, 598, 553; [anal. calcd. for C₃₇H₃₃BrN₆O₄: C, 63.94; H, 4.79; N, 10.08; found: C, 64.09; H, 4.86; N, 10.13]; LC/MS (ESI, m/z): found 705.8 [M(₇₉Br) + H]⁺, 707.5 [M(₈₁Br) + H]⁺, exact mass 704.17 for C₃₇H₃₃BrN₆O₄.

(1′R,2′S,3R,9a’R)-6-Chloro-1′-(5-chloro-1H-indol-3-yl)-2′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-1′,2′,4a’,5′,6′,7′,8′,8a’,9′,9a’-decahydrospiro[indoline-3,3′-pyrrolo[1,2-a]indol]-2-one (8r)

Following the general procedure (GP2), chalcone 5d (91 mg, 0.25 mmol), 6-chloroisatin 6b (46 mg, 0.25 mmol), and octahydro-1H-indole-2-carboxylic acid 7b (64 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8r; yield (148 mg, 91%); m.p.: 168–169 °C; [α]_D²⁵ = −121.51° (c 0.12, MeOH); ¹H NMR (700 MHz, CDCl₃) δ (ppm) = 8.77 (d, J = 11.7 Hz, 1H, NH), 8.63 (s, 1H, NH), 8.13 (d, J = 2.0 Hz, 1H, Ar-H), 7.91 (d, J = 2.0 Hz, 1H, Ar-H), 7.45–7.39 (m, 3H, Ar-H), 7.31 (d, J = 8.0 Hz, 1H, Ar-H), 7.28–7.26 (m, 1H, Ar-H), 7.15–7.12 (m, 3H, Ar-H), 7.11 (dd, J = 8.6, 2.0 Hz, 1H, Ar-H), 7.02 (dd, J = 8.0, 1.9 Hz, 1H, Ar-H), 6.35 (s, 1H, Ar-H), 4.81 (d, J = 12.0 Hz, 1H, CHCO), 4.39–4.36 (m, 1H, NCH), 4.10 (dd, J = 11.9, 10.0 Hz, 1H, NCHCH), 3.22 (q, J = 3.7 Hz, 1H, NCH), 2.19–2.14 (m, 1H, NCHCH), 1.99 (s, 3H), 1.83–1.76 (m, 2H, CH₂), 1.58–1.53 (m, 1H, CH₂), 1.52–1.44 (m, 3H, CH₂), 1.19–1.15 (m, 1H, CH₂), 1.07–0.95 (m, 3H, CH₂); ¹³C NMR (176 MHz, CDCl₃): δ (ppm) = 191.10 (CO), 181.85 (CO), 143.75, 142.06, 141.49, 138.22, 134.82, 134.65, 129.46, 129.16, 128.69, 128.47, 127.14, 125.45, 123.43, 122.60, 122.47, 121.65, 121.05, 119.07, 114.34, 112.48, 110.16, 72.39, 70.79, 67.65, 57.78, 44.90, 41.96, 38.35, 28.58, 27.77, 24.77, 20.12, 11.79 (CH₃); IR (KBr, cm^–1) ν_max = 3287,2925, 2854, 1723, 1661, 1652, 1615, 1538, 1533, 1504, 1484, 1398, 1373, 1320, 1277, 1221, 1178, 1131, 1104, 1072, 736, 926, 893, 869, 796, 763, 694, 606, 525; [anal. calcd. for C₃₇H₃₃Cl₂N₅O₂: C, 68.31; H, 5.11; N, 10.76; found: C, 68.25; H, 5.07; N, 10.84]; LC/MS (ESI, m/z): found 650.8 [M(₃₅Cl) + H]⁺, 652.0 [M(₃₇Cl) + H]⁺, 654.0 [M(₃₇Cl + ₃₇Cl) + H]⁺; exact mass 649.2 for C₃₇H₃₃Cl₂N₅O₂.

(1′R,2′S,3R,9a’R)-1′-(5-Chloro-1H-indol-3-yl)-2′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-5-nitro-1′,2′,4a’,5′,6′,7′,8′,8a’,9′,9a’-decahydrospiro[indoline-3,3′-pyrrolo[1,2-a]indol]-2-one (8s)

Following the general procedure (GP2), chalcone 5d (91 mg, 0.25 mmol), 5-nitroisatin 6c (48 mg, 0.25 mmol), and octahydro-1H-indole-2-carboxylic acid 7b (64 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 4 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield yellow solid compound 8s; yield (132 mg, 80%); m.p.: 172–173 °C; [α]_D²⁵ = −11.15° (c 0.10, MeOH); ¹H NMR (700 MHz, DMSO-d₆): δ (ppm) = 11.15 (d, J = 2.6 Hz, 1H, NH), 11.01 (s, 1H, NH), 8.25 (d, J = 2.3 Hz, 1H, Ar-H), 8.15 (dd, J = 8.6, 2.3 Hz, 1H, Ar-H), 8.03 (s, 1H, Ar-H), 7.89 (d, J = 2.1 Hz, 1H, Ar-H), 7.58 (d, J = 2.5 Hz, 1H, Ar-H), 7.53–7.49 (m, 2H, Ar-H), 7.49–7.45 (m, 1H, Ar-H), 7.38–7.35 (m, 2H, Ar-H), 7.33 (d, J = 8.6 Hz, 1H, Ar-H), 7.07 (dd, J = 8.6, 2.0 Hz, 1H, Ar-H), 6.84 (d, J = 8.6 Hz, 1H, Ar-H), 4.88 (d, J = 12.0 Hz, 1H, CHCO), 4.15 (m, 2H, NCH + NCHCH), 3.23 (q, J = 3.4 Hz, 1H, NCH), 2.19–2.09 (m, 2H, CH₂), 1.81 (s, 3H, CH₃), 1.62–1.59 (m, 1H, CH₂), 1.55–1.50 (m, 1H, CH₂), 1.49–1.44 (m, 1H, CH₂), 1.39–1.27 (m, 2H, CH₂), 1.14–1.10 (m, 1H, CH₂), 1.03–0.97 (m, 1H, CH₂), 0.97–0.91 (m, 1H, CH₂), 0.77–0.72 (m, 1H, CH₂); ¹³C NMR (176 MHz, DMSO-d₆): δ (ppm) = 190.50 (CO), 180.99 (CO), 148.17, 142.49, 141.53, 141.20, 137.91, 134.69, 129.31, 128.81, 128.18, 128.03, 126.51, 126.40, 125.20, 123.81, 123.15, 121.00, 120.54, 118.20, 112.96, 112.64, 109.61, 71.51, 70.67, 66.34, 56.78, 43.83, 41.56, 37.09, 27.65, 27.29, 24.42, 19.25, 11.07 (CH₃); IR (KBr, cm^–1) ν_max = 3349, 2928, 2853, 1732, 1661, 1623, 1598, 1537, 1525, 1504, 1455, 1398, 1337, 1220, 1178, 1128, 1099, 1078, 931, 892, 867, 833, 798, 766, 755, 694, 552; [anal. calcd. for C₃₇H₃₃ClN₆O₄: C, 67.22; H, 5.03; N, 12.71; found: C, 67.34; H, 12.63; N, 12.56]; LC/MS (ESI, m/z): found 661.6 [M(₃₅Cl) + H]⁺, 663.3 [M(₃₇Cl) + H]⁺, exact mass 660.23 for C₃₇H₃₃ClN₆O₄.

(1′R,2′S,3R,7a’R)-6-Chloro-1′-(1H-indol-3-yl)-2′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-1′,2′,5′,6′,7′,7a’-hexahydrospiro[indoline-3,3′-pyrrolizin]-2-one (8t)

Following the general procedure (GP2), chalcone 5a (82 mg, 0.25 mmol), 6-chloroisatin 6b (46 mg, 0.25 mmol), and l-proline 7c (43 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8t; yield (103 mg, 73%); m.p.: 165–166 °C; [α]_D²⁵ = −32.67° (c 0.12, MeOH); ¹H NMR (700 MHz, CDCl₃): δ (ppm) = 9.13 (s, 1H, NH), 8.33 (s, 1H, NH), 8.09–8.04 (m, 1H, Ar-H), 7.93 (s, 1H, Ar-H), 7.41–7.35 (m, 3H, Ar-H), 7.32–7.30 (m, 1H, Ar-H), 7.29 (d, J = 8.1 Hz, 1H, Ar-H), 7.23 (d, J = 2.4 Hz, 1H, Ar-H), 7.19–7.13 (m, 4H, Ar-H), 6.99 (dd, J = 8.0, 1.9 Hz, 1H, Ar-H), 6.63 (d, J = 1.9 Hz, 1H, Ar-H), 4.89 (d, J = 11.4 Hz, 1H, CHCO), 4.49–4.46 (m, 1H, NCH), 4.19 (dd, J = 11.5, 9.9 Hz, 1H, NCHCH), 2.80–2.75 (m, 1H, NCH_2(a)), 2.70–2.66 (m, 1H, NCH_2(b)), 2.09 (s, 3H, CH₃), 2.06–2.02 (m, 1H, CH₂), 1.97–1.91 (m, 1H CH₂), 1.90–1.85 (m, 1H CH₂), 1.79–1.73 (m, 1H CH₂); ¹³C NMR (176 MHz, CDCl₃): δ (ppm) =191.91 (CO), 181.47 (CO), 143.56, 142.53, 141.27, 138.24, 136.68, 134.93, 129.33, 128.97, 128.86, 126.77, 125.58, 124.44, 122.32, 122.10, 121.88, 121.27, 119.90, 119.67, 114.21, 111.44, 110.70, 73.77, 70.74, 65.90, 48.38, 44.74, 31.06, 27.28, 12.00 (CH₃); IR (KBr, cm^–1) ν_max = 3273, 3056, 2962, 2867, 1733, 1661, 1653, 1613, 1536, 1531, 1502, 1486, 1453, 1396, 1324, 1285, 1244, 1222, 1185, 1131, 1072, 936, 922, 813, 794, 763, 741, 694, 526; [anal. calcd. for C₃₃H₂₈ClN₅O₂: C, 70.52; H, 5.02; N, 12.46; found: C, 70.66; H, 4.91; N, 12.53]; LC/MS (ESI, m/z): found 562.4 [M(₃₅Cl) + H]⁺, 564.1 [M(₃₇Cl) + H]⁺; exact mass 561.19 for C₃₃H₂₈ClN₅O₂.

(1′R,2′S,3R,7a’R)-6-Chloro-2′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-1′-(1-methyl-1H-indol-3-yl)-1′,2′,5′,6′,7′,7a’-hexahydrospiro[indoline-3,3′-pyrrolizin]-2-one (8u)

Following the general procedure (GP2), chalcone 5b (85 mg, 0.25 mmol), 6-chloroisatin 6b (46 mg, 0.25 mmol), and l-proline 7c (43 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8u; yield (124 mg, 87%); m.p.: 150–151 °C; [α]_D²⁵ = −23.35° (c 0.11, MeOH); ¹H NMR (700 MHz, CDCl₃): δ (ppm) = 9.02 (s, 1H, NH), 8.07 (d, J = 7.9 Hz, 1H, Ar-H), 7.89 (s, 1H, Ar-H), 7.41–7.35 (m, 3H, Ar-H), 7.30–7.26 (m, 2H, Ar-H), 7.23–7.20 (m, 1H, Ar-H), 7.17 (dd, J = 16.0, 7.6 Hz, 3H, Ar-H), 7.13 (s, 1H, Ar-H), 6.99 (dd, J = 8.0, 1.9 Hz, 1H, Ar-H), 6.65 (d, J = 1.9 Hz, 1H, Ar-H), 4.85 (d, J = 11.5 Hz, 1H, CHCO), 4.50–4.45 (m, 1H, NCH), 4.16 (dd, J = 11.5, 10.0 Hz, 1H, NCHCH), 3.73 (s, 3H, NCH₃), 2.82–2.76 (m, 1H, NCH_2(a)), 2.70–2.65 (m, 1H, NCH_2(b)), 2.09 (s, 3H, CH₃), 2.07–2.01 (m, 1H, CH₂), 1.98–1.92 (m, 1H, CH₂), 1.90–1.86 (m, 1H, CH₂), 1.78–1.73 (m, 1H, CH₂); ¹³C NMR (176 MHz, CDCl₃): δ (ppm) = 191.89 (CO), 181.46 (CO), 143.51, 142.51, 141.24, 138.29, 137.38, 134.91, 129.32, 128.93, 128.86, 127.21, 126.92, 125.58, 124.47, 121.86, 121.71, 121.29, 120.07, 119.21, 112.71, 110.64, 109.44, 73.67, 70.80, 66.02, 48.43, 44.66, 32.82 (NCH₃), 30.96, 27.19, 12.01 (CH₃); IR (KBr, cm^–1) ν_max = 3216, 3058, 2958, 2872, 1733, 1662, 1614, 1536, 1503, 1484, 1455, 1398, 1326, 1285, 1227, 1184, 1130, 1172, 1012, 935, 922, 815, 795, 765, 740, 694; [anal. calcd. for C₃₄H₃₀ClN₅O₂: C, 70.89; H, 5.25; N, 12.16; found: C, 71.11; H, 5.16; N, 12.01]; LC/MS (ESI, m/z): found 576.5 [M(₃₅Cl) + H]⁺, 578.2 [M(₃₇Cl) + H]⁺; exact mass 575.21 for C₃₄H₃₀ClN₅O₂.

(1′R,2′S,3R,7a’R)-1′-(5-Bromo-1H-indol-3-yl)-2′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-1′,2′,5′,6′,7′,7a’-hexahydrospiro[indoline-3,3′-pyrrolizin]-2-one (8v)

Following the general procedure (GP2), chalcone 5c (102 mg, 0.25 mmol), isatin 6a (37 mg, 0.25 mmol), and l-proline 7c (43 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8v; yield (136 mg, 90%); m.p.: 178–179 °C; [α]_D²⁵ = −37.86° (c 0.10, MeOH); ¹H NMR (700 MHz, DMSO-d₆) δ (ppm) = 11.16 (s, 1H, NH), 10.35 (s, 1H, NH), 8.10 (s, 1H, Ar-H), 7.85 (d, J = 3.0 Hz, 1H, Ar-H), 7.51–7.46 (m, 3H, Ar-H), 7.44 (d, J = 9.1 Hz, 1H, Ar-H), 7.39 (d, J = 7.9 Hz, 1H, Ar-H), 7.34–7.29 (m, 3H, Ar-H), 7.19 (d, J = 10.5 Hz, 1H, Ar-H), 7.14–7.10 (m, 1H, Ar-H), 6.98–6.94 (m, 1H, Ar-H), 6.65 (d, J = 10.3 Hz, 1H, Ar-H), 4.68 (d, J = 11.7 Hz, 1H, CHCO), 4.09 (t, J = 12.0 Hz, 1H, NCHCH), 4.05–4.01 (m, 1H, NCH), 2.62–2.57 (m, 1H, NCH_2(a)), 2.39–2.35 (m, 1H, NCH_2(b)), 1.94–1.90 (m, 1H, CH₂), 1.89 (s, 3H, CH₃), 1.87–1.84 (m, 1H, CH₂), 1.80–1.75 (m, 1H, CH₂), 1.75–1.69 (m, 1H, CH₂); ¹³C NMR (176 MHz, DMSO-d₆) δ (ppm) = 191.33 (CO), 179.87 (CO), 142.37, 141.96, 140.86, 138.03, 135.10, 129.29, 129.08, 128.74, 128.70, 127.85, 125.27, 125.23, 123.96, 123.53, 121.32, 120.99, 120.78, 113.57, 113.33, 111.21, 109.43, 73.20, 71.04, 65.39, 47.42, 43.25, 30.49, 27.08, 11.21 (CH₃); IR (KBr, cm^–1) ν_max = 3270, 2958, 2928, 2869, 1717, 1662, 1653, 1616, 1597, 1538, 1504, 1470, 1457, 1397, 1330, 1268, 1221, 1190, 1104, 936, 884, 793, 754, 694, 678, 659, 610; [anal. calcd. for C₃₃H₂₈BrN₅O₂: C, 65.35; H, 4.65; N, 11.55; found: C, 65.24; H, 4.77; N, 11.49]; LC/MS (ESI, m/z): found 606.7 [M(₇₉Br) + H]⁺, 608.4 [M(₈₁Br) + H]⁺; exact mass 605.14 for C₃₃H₂₈BrN₅O₂.

(1′R,2′S,3R,7a’R)-1′-(5-Chloro-1H-indol-3-yl)-2′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)-5-nitro-1′,2′,5′,6′,7′,7a’-hexahydrospiro[indoline-3,3′-pyrrolizin]-2-one (8w)

Following the general procedure (GP2), chalcone 5d (91 mg, 0.25 mmol), 5-nitroisatin 6c (48 mg, 0.25 mmol), and l-proline 7c (43 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 6 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield yellow solid compound 8w; yield (74 mg, 49%); m.p.: 225–226 °C; [α]_D²⁵ = −67.91° (c 0.13, MeOH); ¹H NMR (700 MHz, DMSO-d₆) δ (ppm) = 11.18 (s, 1H, NH), 11.16 (s, 1H, NH), 8.14 (dd, J = 8.6, 2.3 Hz, 1H, Ar-H), 8.12 (d, J = 2.3 Hz, 1H, Ar-H), 7.93 (d, J = 2.1 Hz, 1H, Ar-H), 7.84 (s, 1H), 7.55 (d, J = 2.5 Hz, 1H, Ar-H), 7.51–7.48 (m, 2H, Ar-H), 7.47–7.44 (m, 1H, Ar-H), 7.36 (d, J = 8.6 Hz, 1H, Ar-H), 7.31 (d, J = 8.6 Hz, 2H, Ar-H), 7.08 (dd, J = 8.6, 2.0 Hz, 1H, Ar-H), 6.88 (d, J = 8.6 Hz, 1H, Ar-H), 4.75 (d, J = 11.4 Hz, 1H, CHCO), 4.15–4.09 (m, 2H, NCH + NCHCH), 2.70–2.65 (m, 1H, NCH_2(a)), 2.46–2.42 (m, 1H, NCH_2(b)), 1.99–1.94 (m, 1H, CH₂), 1.92 (s, 3H, CH₃) 1.90–1.88 (m, 1H, CH₂), 1.84–1.76 (m, 2H, CH₂); ¹³C NMR (176 MHz, DMSO-d₆) δ (ppm) = 191.15 (CO), 180.27 (CO), 148.48, 142.63, 141.67, 140.69, 137.82, 134.92, 129.30, 128.86, 127.70, 126.56, 126.22, 125.19, 124.53, 123.23, 122.78, 121.03, 120.59, 118.14, 113.15, 112.52, 109.86, 72.44, 70.67, 65.39, 47.41, 43.52, 30.02, 27.09, 11.21 (CH₃); IR (KBr, cm^–1) ν_max = 3408, 3378, 3111, 3058, 2973, 2861, 2836, 1748, 1645, 1622, 1598, 1525, 1503, 1479, 1455, 1417, 1392, 1373, 1338, 1293, 1253, 1221, 1188, 1174, 1137, 1100, 1076, 986, 938, 930, 887, 860, 850, 832, 798, 763, 755, 694, 655, 610, 554; [anal. calcd. for C₃₃H₂₇ClN₆O₄: C, 65.29; H, 4.48; N, 13.84; found: C, 65.39; H, 4.56; N, 13.87]; LC/MS (ESI, m/z): found 607.5 [M (₃₅Cl) + H]⁺, 609.2 [M (₃₅Cl) + H]⁺; exact mass 606.18 for C₃₃H₂₇ClN₆O₄.

(2′R,3′S,4′R)-6-Chloro-4′-(1H-indol-3-yl)-1′-methyl-3′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)spiro[indoline-3,2′-pyrrolidin]-2-one (8x)

Following the general procedure (GP2), chalcone 5a (82 mg, 0.25 mmol), 6-chloroisatin 6b (46 mg, 0.25 mmol), and sacrosine 7d (34 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8x; yield (48 mg, 35%); m.p.: 140–141 °C; [α]_D²⁵ = −19.37° (c 0.10, MeOH); ¹H NMR (700 MHz, CDCl₃): δ (ppm) = 8.51 (s, 1H, NH), 8.19 (s, 1H, NH), 8.08 (d, J = 9.5 Hz, 1H, Ar-H), 7.73 (s, 1H, Ar-H), 7.45–7.38 (m, 3H, Ar-H), 7.33–7.30 (m, 1H, Ar-H), 7.26 (d, J = 1.2 Hz, 1H, Ar-H), 7.20–7.14 (m, 5H, Ar-H), 6.92 (dd, J = 8.0, 1.9 Hz, 1H, Ar-H), 6.49 (d, J = 1.8 Hz, 1H, Ar-H), 4.80–4.73 (m, 1H, NCH₂CH), 4.45 (d, J = 9.6 Hz, 1H, CHCO), 3.86–3.81 (m, 1H, NCH_2(a)), 3.49–3.45 (m, 1H, NCH_2(b)), 2.29 (s, 3H, NCH₃), 2.17 (s, 3H, CH₃); ¹³C NMR (176 MHz, CDCl₃): δ (ppm) = 192.45 (CO), 180.34 (CO), 143.46, 142.23, 140.55, 138.20, 136.60, 134.48, 129.44, 129.18, 127.71, 126.87, 126.77, 125.71, 122.53, 122.21, 122.16, 121.44, 120.04, 119.75, 115.62, 111.30, 109.77, 73.69, 64.47, 59.54, 35.99, 35.30 (NCH₃), 11.91 (CH₃); IR (KBr, cm^–1) ν_max = 3278, 3072, 2934, 2826, 2797, 1726, 1666, 1654, 1612, 1597, 1534, 1503, 1485, 1552, 1398, 1322, 1284, 1240, 1216, 1179, 1119, 1094, 1069, 936, 910, 882, 814, 798, 766, 718, 693, 592; [anal. calcd. for C₃₁H₂₆ClN₅O₂: C, 69.46; H, 4.89; N, 13.07; found: C, 69.31; H, 4.97; N, 12.93]; LC/MS (ESI, m/z): found 536.4 [M(₃₅Cl) + H]⁺, 538.2 [M(₃₇Cl) + H]⁺; exact mass 535.18 for C₃₁H₂₆ClN₅O₂.

(2′R,3′S,4′R)-4′-(5-Bromo-1H-indol-3-yl)-6-chloro-1′-methyl-3′-(5-methyl-1-phenyl-1H-pyrazole-4-carbonyl)spiro[indoline-3,2′-pyrrolidin]-2-one (8y)

Following the general procedure (GP2), chalcone 5c (102 mg, 0.25 mmol), 6-chloroisatin 6b (46 mg, 0.25 mmol), and sacrosine 7d (34 mg, 0.37 mmol) in methanol (20 mL) were refluxed for 3 h and purified by column chromatography 100–200 mesh silica gel and MeOH/CH₂Cl₂ (3:97) to yield light yellow solid compound 8y; yield (73 mg, 47%); m.p.: 158–159 °C; [α]_D²⁵ = −35.63° (c 0.12, MeOH); ¹H NMR (700 MHz, CDCl₃): δ (ppm) = 8.99 (s, 1H, NH), 8.42 (s, 1H, NH), 8.18 (s, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 7.44–7.38 (m, 3H, Ar-H), 7.22 (dd, J = 8.7, 2.0 Hz, 2H, Ar-H), 7.18–7.11 (m, 4H, Ar-H), 6.92 (dd, J = 8.0, 1.8 Hz, 1H, Ar-H), 6.54 (d, J = 1.8 Hz, 1H, Ar-H), 4.70 (q, J = 9.8 Hz, 1H, NCH₂CH), 4.36 (d, J = 9.4 Hz, 1H, CHCO), 3.75 (t, J = 9.5 Hz, 1H, NCH_2(a)), 3.46 (t, J = 9.5 Hz, 1H, NCH_2(b)), 2.27 (s, 3H, NCH₃), 2.19 (s, 3H, CH₃); ¹³C NMR (176 MHz, CDCl₃): δ (ppm) = 192.35 (CO), 180.67 (CO), 143.63, 142.34, 140.51, 138.14, 135.16, 134.58, 129.47, 129.24, 128.70, 127.64, 126.53, 125.71, 125.00, 123.27, 122.55, 122.51, 121.38, 115.62, 113.00, 112.75, 110.02, 73.75, 64.57, 59.54, 35.58, 35.26 (NCH₃), 11.93 (CH₃); IR (KBr, cm^–1) ν_max = 3276, 3071, 2936, 2843, 2795, 1722, 1668, 1652, 1613, 1598, 1537, 1504, 1484, 1554, 1397, 1321, 1280, 1244, 1219, 1181, 1115, 1096, 1068, 938, 916, 883, 813, 793, 765, 714, 694, 593; [anal. calcd. for C₃₁H₂₅BrClN₅O₂: C, 60.55; H, 4.10; N, 11.39; found: C, 60.41; H, 4.15; N, 11.47]; LC/MS (ESI, m/z): found 614.8 [M(₃₅Cl/₇₉Br) + H]⁺, 616.1 [M(₃₇Cl/₈₁Br) + H]⁺, 618.0 [M(₃₇Cl + ₈₁Br) + H]⁺, exact mass 613.09 for C₃₁H₂₅BrClN₅O₂.

Acetylcholine Esterase (AChE) Inhibitory Assay (AChEI)

AChEI activity was measured using Ellman’s method as previously described and provided in the Supporting Information.^39b,39c

Molecular Docking Study

The protocol for the molecular docking study to investigate the binding mode of the most active compounds are provided in the Supporting Information.⁴³

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c03978.

• AChE assay; molecular docking study protocol; copies of NMR and MS spectrum; and also X-ray single-crystal analysis of compound 8c (PDF)

The authors declare no competing financial interest.

Notes

^∇ The current research was carried out in the current affiliation.