The synthesis and preliminary cytotoxicity evaluation of hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1 H ),4″-dione compounds

Abstract

Seven hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione compounds were synthesized by a catalyst-free 1,3-dipolar cycloaddition reaction in a one-pot three-component system containing 3,5-diarylidene-1-benzylpiperidin-4-one, isatin and l-pipecolic acid. The structures of these compounds were characterized by nuclear magnetic resonance, high-resolution mass spectrometry and X-ray data analysis. These chemical entities were evaluated for their cytotoxicity (brine shrimp assay), and five compounds were found potential in this bioassay. Compound 2e was the most prominent target against Artemia salina with an LC₅₀ value of 7.27 μg/mL.

Natural nitrogenous heterocyclic compounds have attracted great attention of chemical and pharmaceutical researchers for their various structures and extensive bioactivities.^1,2 The synthesis of these compounds or new creations from these skeletons has become an important research direction,^3–5 and many new molecules with novel structure^6,7 or high bioactivity^6,8 have been constructed. Among them, spiro-oxindole compounds are an important family with diverse biological activities such as anti-tumour (I and II),^9,10 anti-microbe (III), ¹¹ and inhibiting cholinesterase (IV) ¹² (Figure 1), which show its prospect in pharmaceutical research.

Figure 1.

Potential synthetic bioactive spiro-oxindole compounds.

The research and development of anti-neoplastic drugs has been the focus of pharmaceutical researchers in recent years. Facing the large number of chemical monomers and extracts, the brine shrimp lethality test is often used for the preliminary evaluation of cytotoxicity.^13,14

Based on our strong interest in the bioactivities of spiro-oxindole heterocyclic compounds and the further study of this type of compounds, ¹⁵ we synthesized a new group of hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione compounds through 1,3-dipolar cycloaddition reaction in a one-pot three-component system (Scheme 1). By screening on the brine shrimp bioassay, these compounds exhibited shrimp lethality effect with LC₅₀ valves ranging from 7.27 to 174.59 µg/mL. The most potential compound had an LC₅₀ of 7.27 g/mL. The LC₅₀ value of positive control podophyllotoxin is 2.16 µg/mL. The strong shrimp lethality suggests it has promising cytotoxicity.

Scheme 1.

Synthesis of hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione compounds.

Results and discussion

Chemistry

The 1-benzyl-3,5-diarylmethylidene-piperidin-4-one (1) compounds were prepared efficiently according to the reported procedures. ¹⁶ The 1,3-dipolar cycloaddition reaction of 1 to an azomethine ylide from in situ reaction of isatin and l-pipecolic acid yielded hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione compounds 2. This cycloaddition process is conservative on regioselectivity to afford only one isomer, which was evidenced from thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) analyses.

The possible way of the azomethine ylide approaching the dipolarophile 1 is shown in Scheme 2. The approaching direction from the least hindered side via path A results in the regioselectivity of the formation of 2 as evidenced by single crystal analysis. The other regioisomer via path B is not founded probably due to static repulsion (Scheme 2). The structures of 2a–g were initially established by analysis of high-resolution mass spectrometry (HRMS), infrared (IR), 1D and 2D NMR data. The positive electrospray ionization (ESI)-HRMS of 2a exhibited a quasi-molecular ion peak at m/z 716.1402 ([M+H]⁺), implying a molecular formula of C₃₉H₃₃Cl₄N₃O₂. The IR spectrum of 2a displayed ν_N-H and ν_C=O at 3421 and 1691 cm⁻¹, respectively. In the upfield of ¹H NMR spectrum, 16 proton signals can be observed. Among them, seven methane signals can be easy recognized by analysis of the HSQC (heteronuclear single quantum coherence spectroscopy) spectrum of 2a. The remaining two upfield methylene protons at δ 4.64 and δ 3.86 were also located at C-4 (δ 49.00) and C-4a (δ 63.41) by HSQC correlations, respectively. The benzylic proton H4 occurred as a doublet peak instead of a singlet one, which proved the regiochemistry of the cycloaddition reaction. Clean ¹H-¹HCOSY correlations ensured the sequential connectivity of H4, H4a, H5 (δ 1.71 and 1.43) and H6 (δ 2.29 and 2.10). The ¹³C NMR spectrum revealed two sp³ spiro carbons at δ 77.17 (C2) and δ 61.80 (C3) and two sp² amide and ketone carbonyl carbons at δ 177.20 (C2″) and δ 196.44 (C4′), respectively.

Scheme 2.

A plausible mechanism for the formation of hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione compounds.

In heteronuclear multiple bond correlation (HMBC), the correlations from characteristic H-4 to C2′ (δ 57.81), C4′, C2 and C5 (δ 30.66) can be easily observed. The HMBC showed the key correlations from H-2′ (δ 3.23 and 1.87) to C6′ (δ 54.29), C4′ and spiro carbons C3 and C2. Protons of –NH (δ 7.92) and H-4″ (δ 7.11) correlate with spiro carbon C2. Additional HMBC correlations from the olefin proton (δ 7.55) of 2,4-dichlorobenzylidene moiety to C4′ and C6′ and from the CH₂ protons (δ 3.53 and 3.03) of N-benzyl group to C2′ and C6′ are observed. All the HMBCs supported the dispiro heterocycle structure of 2a (Figure 2). Furthermore, an integrated crystal of 2g·EtOAc was obtained, and the relative structure of product 2 was confirmed by X-ray data analysis, which is shown in Figure 3.

Figure 2.

Partial HMBC correlations of 2a.

Figure 3.

ORTEP diagram of 2g·EtOAc with 50% probability (H atoms have been omitted for clarity).

Bioassay

All the synthesized hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione compounds were evaluated for their cytotoxicity (brine shrimp bioassay). ¹⁴ Five products showed different cytotoxic effects against Artemia salina. In particular, compound 2e has the most significant cytotoxic biological activity with an LC₅₀ value of 7.27 µg/mL on brine shrimp. In contrast, compounds 2a and 2f are considered inactive owing to the LC₅₀ values larger than 100 µg/mL.

Experimental

All the chemical reagents are commercially available without further purification. All of the solvents were dried and redistilled before use. The TLC analysis was performed with silica gel GF254 plates. The column chromatography was carried out using silica gel (200–300 mesh). All NMR spectra were recorded on a Bruker AV-II 500 MHz NMR spectrometer – ¹H at 500 MHz and ¹³C at 125 MHz. Transcranial magnetic stimulation (TMS) was used as an internal reference for ¹H and ¹³C chemical shifts, and CDCl₃ was used as a solvent. All mass spectra were obtained using a Waters ACQUITY UPLC XEVO Q-TOF mass spectrometer. IR spectra were recorded on a Perkin-Elmer spectrometer (Spectrum One). Melting points were measured with a Yanaco MP500 melting point apparatus and are uncorrected. X-ray analysis was performed on a Bruker Apex-II CCD diffractometer. The crystallographic data for compound 2g·EtOAc are summarized in Table 1. CCDC 1863987 contains the supplementary crystallographic data for this paper, which can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Table 1.

Crystallographic data for 2g·EtOAc.

Empirical formula	C43H43N3O4F2
Mr	703.80
Temperature	296(2) K
Crystal system	Monoclinic
Space group	C2
Unit cell dimensions	a = 25.270 (18) A, α = 90° b = 9.627 (8) A, β = 97.155 (15)° c = 30.86 (2) A, γ = 90°
Volume	7449(10) Å3
Z	8
Calculated density	1.225 g m−3
Absorption coefficient μ	0. mm−1
F(000)	2976
Reflections collected	6435 reflections, 4217 with I > 2σ(I) for 472 parameters
Goodness of fit on F2	1.049
Final R indices (I > 2σ (I))	R1 = 0. 0709, wR2 = 0.1722
R indices (all data)	R1 = 0.1069, wR2 = 0.1995

General procedure for the synthesis of hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]dione compounds (2a–g)

A mixture of (3E,5E)-1-benzyl-3,5-bis(2,4-dichlorobenzylidene)piperidin-4-one (1a) (200 mg, 0.40 mmol), isatin (65 mg, 0.44 mmol) and l-pipecolic acid (62 mg, 0.48 mmol) was dissolved in EtOH (10 mL). The solution was refluxed (approx. 6 h) to produce the cycloadduct and monitored by TLC analysis. After completion of the reaction, the solvent was evaporated under reduced pressure. The residue was subjected to silica gel (50 g) column chromatography eluted with petroleum ether–ethyl acetate (3.5:1, V/V) to afford the compound 2a (231 mg) with 81% yield. Following a similar procedure, the other six hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]dione compounds (2b–g) were obtained in 63%–85% yields.

(1′RS,2′SR,3′RS,8a′SR)-1″-benzyl-5″-(2,4-dichlorobenzylidene)-1′-(2,4-dichlorophenyl)-1′,5′,6′,7′,8′,8a′-hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione (2a)

White solid, yield 81%; m.p.: 237–239 °C; IR (KBr) (v cm⁻¹): 3421, 1691; ¹H NMR (500 MHz, CDCl₃) δ 7.92 (s, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.56 (s, 1H), 7.38 (d, J = 1.6 Hz, 1H), 7.32 (d, J = 9.1 Hz, 2H), 7.16 (m, 4H), 7.11 (d, J = 7.5 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.92 (t, J = 7.6 Hz, 1H), 6.89 (m, 2H), 6.75 (d, J = 7.6 Hz, 1H), 6.60 (d, J = 8.4 Hz, 1H), 4.64 (d, J = 9.4 Hz, 1H), 3.86 (t, J = 9.5 Hz, 1H), 3.53 (d, J = 13.5 Hz, 1H), 3.23 (d, J = 13.1 Hz, 1H), 3.20 (d, J = 16.1 Hz, 1H), 3.03 (d, J = 13.5 Hz, 1H), 2.70 (d, J = 15.2 Hz, 1H), 2.29 (d, J = 10.0 Hz, 1H), 2.10 (td, J = 10.5, 3.2 Hz, 1H), 1.87 (d, J = 12.6 Hz, 1H), 1.71 (m, 2H), 1.42 (m, 2H), 1.22 (m, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 196.44, 177.20, 142.03, 136.97, 135.69, 135.15, 134.78, 134.13, 133.60, 132.95, 131.93, 131.65, 130.30, 129.59, 129.15, 128.68, 128.60, 128.31 × 2, 128.11, 128.07 × 2, 127.04, 127.00, 126.73, 126.52, 122.79, 108.76, 77.17, 63.41, 62.70, 61.80, 57.81, 54.29, 49.00, 45.71, 30.66, 25.25, 24.05. ESI-HRMS calc. for [C₃₉H₃₄Cl₄N₃O₂]⁺ ([M+H]⁺): 716.1400, Found: 716.1402.

(1′RS,2′SR,3′RS,8a′SR)-1″-benzyl-5″-(4-chlorobenzylidene)-1′-(4-chlorophenyl)- 1′,5′,6′,7′,8′,8a′-hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione (2b)

White solid, yield 68 %; m.p.: 213–215 °C; IR (KBr) (v cm⁻¹): 3220, 1694; ¹H NMR (500 MHz, CDCl₃) ¹H NMR (500 MHz, CDCl₃) δ 8.20 (s, 1H), 7.33–7.30 (m, 2H), 7.29 (m, 2H), 7.23–7.16 (m, 3H), 7.13 (d, J = 8.1 Hz, 2H), 7.07 (d, J = 8.2 Hz, 2H), 7.01 (d, J = 7.3 Hz, 2H), 6.95–6.90 (m, 2H), 6.77 (d, J = 8.2 Hz, 2H), 6.67 (d, J = 7.6 Hz, 1H), 4.25 (d, J = 10.4 Hz, 1H), 3.79 (t, J = 8.8 Hz, 1H), 3.68 (d, J = 13.3 Hz, 1H), 3.55 (d, J = 12.3 Hz, 1H), 3.33 (d, J = 14.6 Hz, 1H), 3.15 (d, J = 13.2 Hz, 1H), 2.65 (dd, J = 14.5, 1.9 Hz, 1H), 2.34 (t, J = 6.6 Hz, 2H), 2.25 (d, J = 6.6 Hz, 2H), 1.86 (dt, J = 12.5, 6.2 Hz, 2H), 1.78 (d, J = 12.5 Hz, 1H), 1.37 (m, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 198.52, 177.82, 141.95, 136.60, 136.49, 136.12, 134.49, 133.83, 133.37, 132.57, 131.66, 130.91 × 2, 128.72 × 2, 128.44 × 2, 128.41 × 2, 128.31, 128.12 × 2, 127.84, 127.75, 127.12, 121.96 × 2, 108.65, 75.62, 64.74, 62.01, 61.38, 56.97, 53.52, 52.31, 45.39, 30.78, 25.55, 23.76. ESI-HRMS calc. for [C₃₉H₃₆Cl₂N₃O₂]⁺ ([M+H]⁺): 648.2179, Found: 648.2180.

(1′RS,2′SR,3′RS,8a′SR)-1″-benzyl-5″-(4-tert-butylbenzylidene)-1′-(4-tert-butylphenyl)-1′,5′,6′,7′,8′,8a′-hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione (2c)

Pale yellow syrupy, yield 75%; IR (KBr) (v cm⁻¹): 3264, 1696; ¹H NMR (500 MHz, CDCl₃) δ 7.82 (s, 1H), 7.29 (m, 3H), 7.21 (d, J = 8.3 Hz, 3H), 7.17 (d, J = 7.2 Hz, 2H), 7.15 (d, J = 6.6 Hz, 1H), 7.12 (d, J = 8.2 Hz, 1H), 7.08 (t, J = 7.7 Hz, 1H), 7.01 (d, J = 6.7 Hz, 3H), 6.94 (t, J = 7.5 Hz, 1H), 6.85 (d, J = 8.2 Hz, 2H), 6.64 (d, J = 7.7 Hz, 1H), 4.24 (d, J = 10.3 Hz, 1H), 3.83 (t, J = 8.5 Hz, 1H), 3.64 (d, J = 13.4 Hz, 1H), 3.53 (d, J = 12.7 Hz, 1H), 3.39 (d, J = 14.5 Hz, 1H), 3.27 (d, J = 13.4 Hz, 1H), 2.82 (d, J = 14.6 Hz, 1H), 2.34 (t, J = 6.6 Hz, 1H), 2.26 (t, J = 8.5 Hz, 1H), 1.86 (m, 1H), 1.81 (d, J = 12.5 Hz, 1H), 1.72 (m, 1H), 1.38 (m, 2H), 1.31 (s, 9H), 1.26 (m, 2H), 1.25 (s, 9H). ¹³C NMR (125 MHz, CDCl₃) δ 198.86, 177.82, 151.77, 149.36, 141.80, 137.22, 136.56, 134.85, 132.90, 132.39, 129.90 × 2, 128.94 × 2, 128.53, 128.22, 127.97 × 2, 127.97, 126.90 × 2, 126.01, 125.16 × 2, 125.02 × 2, 122.02, 108.47, 75.67, 65.05, 61.78, 61.37, 56.42, 53.68, 52.81, 45.47, 34.68, 34.41, 31.40 × 3, 31.12 × 3, 30.93, 25.67, 23.86. ESI-HRMS calc. for [C₄₇H₅₄N₃O₂]⁺ ([M+H]⁺): 692.4211, Found: 692.4211.

(1′RS,2′SR,3′RS,8a′SR)-1″-benzyl-5″-(4-methylthiobenzylidene)-1′-(4-methylthiophenyl)-1′,5′,6′,7′,8′,8a′-hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione (2d)

White solid, yield 83 %; m.p.: 222–224 °C; IR (KBr) (v cm⁻¹): 3258, 1695; ¹H NMR (500 MHz, CDCl₃) δ 7.30 (m, 2H), 7.20 (d, J = 7.1 Hz, 4H), 7.18–7.14 (m, 1H), 7.09 (d, J = 7.9 Hz, 1H), 7.08–7.04 (m, 1H), 7.03–6.98 (m, 5H), 6.91 (t, J = 7.5 Hz, 1H), 6.81 (d, J = 8.2 Hz, 2H), 6.64 (d, J = 6.5 Hz, 1H), 4.24 (d, J = 10.3 Hz, 1H), 3.80 (t, J = 9.0 Hz, 1H), 3.68 (d, J = 13.3 Hz, 1H), 3.54 (d, J = 12.5 Hz, 1H), 3.38 (d, J = 14.6 Hz, 1H), 3.16 (d, J = 13.3 Hz, 1H), 2.76–2.69 (m, 1H), 2.48 (s, 3H), 2.41 (s, 3H), 2.24 (d, J = 6.5 Hz, 2H), 1.83 (d, J = 12.6 Hz, 1H), 1.77 (d, J = 8.4 Hz, 1H), 1.71 (s, 1H), 1.49–1.43 (m, 1H), 1.42–1.36 (m, 1H), 1.33 (d, J = 12.2 Hz, 1H), 1.26 (m, 1H). ¹³C NMR (125 MHz, CDCl₃) δ 198.59, 177.67, 141.88, 139.96, 136.99, 136.88, 136.40, 135.01, 132.63, 131.53, 130.40 × 2, 128.71 × 2, 128.59, 128.08 × 2, 127.97, 127.81, 127.00 × 2, 126.57 × 2, 125.37 × 2, 123.61, 121.90, 108.47, 75.70, 64.71, 62.15, 61.41, 56.94, 53.93, 52.60, 45.43, 30.83, 25.60, 23.82, 15.93, 15.06. ESI-HRMS calc. for [C₄₁H₄₂N₃O₂S₂]⁺ ([M+H]⁺): 672.2713, Found: 672.2708.

(1′RS,2′SR,3′RS,8a′SR)-1″-benzyl-5″-(3,4,5-trimethoxybenzylidene)-1′-(3,4,5-trimethoxyphenyl)-1′,5′,6′,7′,8′,8a′-hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione (2e)

White solid, yield 66 %; IR (KBr) (v cm⁻¹): 3308, 1708; m.p.: 231–233 °C; ¹H NMR (500 MHz, CDCl₃) δ 7.68 (s, 1H), 7.29 (s, 1H), 7.18 (t, J = 7.3 Hz, 2H), 7.16 (s, 2H), 7.10 (t, J = 7.5 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 7.04 (d, J = 7.6 Hz, 2H), 7.00 (s, 1H), 6.93 (t, J = 7.5 Hz, 1H), 6.69 (d, J = 7.7 Hz, 1H), 6.11 (s, 2H), 4.18 (d, J = 9.8 Hz, 1H), 3.90 (s, 6H), 3.85 (s, 3H), 3.79 (s, 3H), 3.75 (d, J = 11.5 Hz, 1H), 3.71 (d, J = 6.9 Hz, 1H), 3.64 (s, 6H), 3.60 (d, J = 12.5 Hz, 1H), 3.42 (d, J = 14.7 Hz, 1H), 3.19 (d, J = 12.9 Hz, 1H), 2.76 (d, J = 14.6 Hz, 1H), 2.28 (d, J = 5.2 Hz, 2H), 1.89 (d, J = 12.6 Hz, 1H), 1.84 (d, J = 9.2 Hz, 1H), 1.73 (m, 2H), 1.51 (m, 1H), 1.33–1.28 (m, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 198.56, 177.46, 152.93 × 2, 152.67 × 2, 141.85, 138.50, 137.51, 136.87, 136.74, 136.31, 133.78, 132.80, 132.64, 130.50, 128.91 × 2, 128.50, 128.13 × 2, 127.88, 127.24, 121.97, 108.47 × 2, 107.28 × 2, 75.71, 64.58, 62.48, 61.97, 60.86 × 2, 56.32, 56.23, 55.86 × 2, 53.86, 53.46, 45.46, 30.94, 29.71, 25.61, 23.85. ESI-HRMS calc. for [C₄₅H₅₀N₃O₈]⁺ ([M+H]⁺): 760.3592, Found: 760.3591.

(1′RS,2′SR,3′RS,8a′SR)-1″-benzyl-5″-(4-methoxybenzylidene)-1′-(4-methoxyphenyl)-1′,5′,6′,7′,8′,8a′-hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione (2f)

White solid, yield 63 %; m.p.: 225–227 °C; IR (KBr) (v cm⁻¹): 3430, 1693; ¹H NMR (500 MHz, CDCl₃) δ 7.92 (s, 1H), 7.31 (m, 2H), 7.23–7.13 (m, 3H), 7.10 (d, J = 7.5 Hz, 1H), 7.06 (d, J = 7.9 Hz, 2H), 7.04 (d, J = 7.7 Hz, 2H), 6.91 (d, J = 7.6 Hz, 1H), 6.88 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.2 Hz, 2H), 6.70 (d, J = 8.5 Hz, 2H), 6.64 (d, J = 7.7 Hz, 1H), 4.23 (d, J = 10.4 Hz, 1H), 3.80 (s, 3H), 3.74 (s, 3H), 3.70 (d, J = 13.3 Hz, 1H), 3.53 (d, J = 12.5 Hz, 1H), 3.38 (d, J = 14.5 Hz, 1H), 3.16 (d, J = 13.3 Hz, 1H), 2.94 (s, 1H), 2.88 (s, 1H), 2.74 (dd, J = 14.4, 1.9 Hz, 1H), 2.24 (d, J = 6.3 Hz, 2H), 1.84 (d, J = 12.6 Hz, 1H), 1.79–1.67 (m, 2H), 1.45 (d, J = 12.3 Hz, 1H), 1.34–1.28 (m, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 198.77, 177.66, 162.60, 159.85, 158.35, 141.91, 137.33, 137.11, 131.89 × 2, 131.38, 130.15, 128.75 × 2, 128.47, 128.14, 128.06 × 2, 127.84, 127.81, 126.93 × 2, 121.85, 113.69 × 2, 113.61 × 2, 108.40, 75.81, 64.61, 62.29, 61.67, 57.00, 55.22, 54.11, 52.42, 45.47, 30.87, 29.72, 25.66, 23.90. ESI-HRMS calc. for [C₄₁H₄₂N₃O₄]⁺ ([M+H]⁺): 640.3170, Found: 640.3173.

(1′RS,2′SR,3′RS,8a′SR)-1′-benzyl-5′-(4-fluorobenzylidene)-1′-(4-fluorophenyl)-1′,5′,6′,7′,8′,8a′-hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione (2g)

White solid, yield 85 %; m.p.: 243–245 °C; IR (KBr) (v cm⁻¹): 3426, 1711; ¹H NMR (500 MHz, CDCl₃) δ 7.95 (s, 1H), 7.35 (m, 2H), 7.22–7.14 (m, 3H), 7.09 (t, J = 6.1 Hz, 2H), 7.00 (t, J = 8.0 Hz, 4H), 6.96 (s, 1H), 6.93 (t, J = 7.6 Hz, 1H), 6.87–6.80 (m, 4H), 6.66 (d, J = 7.8 Hz, 1H), 4.26 (d, J = 10.4 Hz, 1H), 3.79 (t, J = 8.8 Hz, 1H), 3.68 (d, J = 13.3 Hz, 1H), 3.54 (d, J = 12.5 Hz, 1H), 3.34 (d, J = 14.4 Hz, 1H), 3.16 (d, J = 13.3 Hz, 1H), 2.67 (dd, J = 14.5, 2.2 Hz, 1H), 2.24 (d, J = 6.5 Hz, 2H), 1.77 (d, J = 12.6 Hz, 4H), 1.44 (m, 1H), 1.35–1.28 (m, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 198.70, 177.68, 163.50, 162.82, 161.51, 160.87, 141.93, 136.70, 136.33, 133.65, 133.23, 131.63 × 2, 131.11, 128.74 × 2, 128.66, 128.09 × 2, 127.81, 127.09 × 2, 121.96, 115.28 × 2, 115.11 × 2, 108.55, 75.65, 64.72, 62.02, 61.60, 56.99, 53.58, 52.23, 45.42, 30.80, 25.59, 23.80. ESI-HRMS calc. for [C₃₉H₃₆F₂N₃O₂]⁺ ([M+H]⁺): 616.2770, Found: 616.2770.

Cytotoxicity study protocol (brine shrimp assay)

Fifty milligrams of brine shrimp (Artemia salina Leach) eggs were hatched in a 500-mL flask filled with artificial seawater under constant aeration. After 48 h, the nauplii were collected by pipette from the lighted side. Ten nauplii were transferred to each well of 96-well plates. Each well was made up to 195 μL with artificial seawater. Sample solution was prepared with dimethyl sulfoxide (DMSO) at the initial concentration of 4.0 mg/mL. Being diluted with DMSO, sample solutions at five concentration gradients, 4.0, 2.0, 1.0, 0.5 and 0.25 mg/mL, were obtained. The initial concentration of positive control, podophyllotoxin, was 2.0 mg/mL, and six concentration gradients were prepared. From each sample, 5 μL of sample solution was transferred to each well, and each sample was repeated three times (LC₅₀ is the mean of three values). For each sample, one well was kept as control by adding only 5 μL of DMSO. After 24 h, the number of survivors were counted and analysed by Bliss ¹⁷ method to determine the LC₅₀ values. The brine shrimp bioassay results of hexahydrodispiro[indole-3,3′-indolizine-2′,3′′-piperidine]-2(1H),4″-dione compounds and positive control podophyllotoxin are listed in Table 2.

Table 2.

Brine shrimp bioassay data of dispiro heterocyclic compounds and podophyllotoxin.

Compound	LC50 (μg/mL)
2a	174.59
2b	59.09
2c	97.60
2d	61.08
2e	7.27
2f	126.28
2g	69.63
Podophyllotoxin	2.16

Conclusion

Seven region-selective hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione compounds were synthesized by a catalyst-free one-pot 1,3-dipolar cycloaddition reaction in a three-component system containing 1-benzyl-3,5-diarylidenepiperidin-4-one, isatin and l-pipecolic acid. In the brine shrimp bioassay, five pure products showed cytotoxicity effect. The most cytotoxic compound is (1′RS,2′SR,3′RS,8a′SR)-1″-benzyl-5″-(3,4,5-trimethoxybenzylidene)-1′-(3,4,5-trimethoxyphenyl)-1′,5′,6′,7′,8′,8a′-hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]-2(1H),4″-dione, which owned an LC₅₀ value of 7.27 µg/mL. The research data have shown that hexahydrodispiro[indole-3,3′-indolizine-2′,3″-piperidine]dione compounds possess potent cytotoxic activity and may help the clinical researchers to pursuit the effective anti-tumour drugs.