Organocatalytic Asymmetric Synthesis of Spiro-oxindole Piperidine

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Cite This: Org. Lett. 2017, 19, 6752−6755

Organocatalytic Asymmetric Synthesis of Spiro-oxindole Piperidine Derivatives That Reduce Cancer Cell Proliferation by Inhibiting MDM2−p53 Interaction Ming-Cheng Yang,†,§ Cheng Peng,*,† Hua Huang,† Lei Yang,‡ Xiang-Hong He,‡ Wei Huang,† Hai-Lei Cui,§ Gu He,*,‡ and Bo Han*,† †

School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China § Laboratory of Asymmetric Synthesis, Chongqing University of Arts and Sciences, Chongqing 402160, China ‡

S Supporting Information *

ABSTRACT: Asymmetric synthesis of pharmacologically interesting piperidinefused spiro-oxindole derivatives has been achieved via an organocatalytic Michael/aza-Henry/hemiaminalization cascade reaction. Chiral compounds synthesized by this strategy potently inhibited the proliferation of several breast cancer cell lines. Mechanistic studies suggest that the most potent compound 9e can directly interfere with MDM2−p53 interactions and elevate protein levels of p53 and p21, thereby inducing cell cycle arrest and mitochondrial apoptosis.

D

Scheme 1. (a) Racemic Spiro-piperidine Oxindole Derivatives for in Vitro Activity Assays and (b) Strategies for Asymmetric Synthesis of Chiral Spiro-piperidine Oxindoles

esign and synthesis of small-molecule inhibitors to block the MDM2−p53 interaction is an active research area in medicinal chemistry because these inhibitors may have strong anticancer potential. By liberating the tumor suppressor p53 from MDM2, these inhibitors allow p53 to exert its antiproliferative effects.1 Diverse privileged scaffolds for preparing potential inhibitors have been described,2 including derivatives of imidazoline, piperidinone, benzodiazepine, chromenotriazolopyrimidine, terphenyl, isoindolinone, and pyrrolidine, as well as C3-spirocyclic oxindoles.3 C3-modified spiro-oxindoles with a rigid nitrogen heterocyclic system are more effective at inhibiting cancer cell proliferation than oxygen- or sulfur-containing heterocyclic C3-substituted molecules.4 C3 modifications have been reported using fivemembered N-heterocyclic rings (such as pyrrolidine,5 thiazolidine,6 isoxazoline7); the purpose of this modification is to provide a hydrophobic moiety to insert into the Leu26 and Phe19 pockets of the binding site on MDM2. Recently, several six-membered piperidine- or piperidone-based spiro-oxindole derivatives have been shown to inhibit MDM2−p53 interaction with submicromolar IC50 values,8 although so far, only racemic mixtures have been screened (Scheme 1a). Wang’s group has demonstrated that stereochemistry was critical to the therapeutic efficacy of spirocyclic oxindoles: the least and most potent stereoisomers of a given spirocyclic oxindole may bind to MDM2 with affinities varying by >100-fold.9 This highlights the need to develop convenient, efficient asymmetric syntheses of chiral spiro-oxindole derivatives bearing six-membered N-heterocycle moieties. Several groups have achieved enantioselective synthesis of spiro-piperidine oxindoles using metal catalysis,10 phosphoric acid catalysis,11 bifunctional hydrogen-bonding catalysis,12 or © 2017 American Chemical Society

NHC catalysis13 (Scheme 1b). Inspired by these successes and motivated by our continuing interest in constructing medicinally relevant scaffolds via chiral amine catalysis,14,15 we envisaged a three-step organocatalytic relay cascade involving a Michael/azaHenry/hemiaminalization relay reaction to achieve the asymmetric synthesis of spirocyclic oxindole skeletons containing a densely functionalized piperidine with diverse substituents and contiguous stereogenic centers (Scheme 2). Our approach took advantage of the fact that the chiral γ-nitroaldehyde intermediate 3, easily obtained from the amine-catalyzed asymmetric Michael reaction of nitroalkene 1 and aldehyde 2, could react with ketimine substrate 4 to generate the chiral spiro-piperidine framework.16 We also hoped to use our approach to create a chiral heterocycle library for identifying the lead compound inhibitors of MDM2−p53. Received: November 13, 2017 Published: December 6, 2017 6752

DOI: 10.1021/acs.orglett.7b03516 Org. Lett. 2017, 19, 6752−6755

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Organic Letters Scheme 2. Asymmetric Construction of Pharmacologically Interesting Scaffolds via [2 + 2 + 2] Annulation

To realize this proposed organocatalytic cascade, we selected (E)-nitrostyrene 1a, n-propanal 2a, and N-Bn-isatin ketimine 4a as substrates. The first Michael reaction proceeded in the presence of Hayashi−Jørgensen secondary amine catalyst (10 mol %) in toluene at room temperature for 3 h. Adding a toluene solution of 4a and triethylamine in a one-pot operation resulted in successive aza-Henry and hemiaminalization reactions. The cascade process proceeded smoothly to afford the desired products 5a, albeit in low yield. Direct dehydroxylation of the hemiaminal using Et3SiH and TFA at −60 °C gave the corresponding stable spiro-piperidine oxindole 6a with good diastereoselectivity and excellent enantioselectivity (Scheme 3a). Scheme 3. Optimization of the Model Reaction

To improve the yield and stereocontrol, more reaction parameters were screened (Table S1 in Supporting Information). Conducting the reaction in MeCN at 40 °C with DBU as base delivered the product 6a in 61% yield, 94% ee, and 90:10 dr (Scheme 3b). These optimal conditions were used for the substrate scope investigation (Figure 1). We evaluated the tolerance of the reaction to various functional groups at position R1 on β-aryl-substituted nitroalkenes 1. The reaction proceeded well for substituents with different electronic properties (Cl, Br, F, Me, iPr, MeO) at different positions (ortho, meta, para); the expected domino adducts 6b−6i were obtained efficiently. Using naphthyl, furyl, or thienyl groups led to the corresponding target products 6j, 6k, or 6l, albeit with slightly lower ee or dr values. Linear aliphatic aldehydes and branched α,β-unsaturated aldehydes proved to be suitable substrates 2, giving the corresponding products 6m and 6n with excellent stereoselectivities. The reaction tolerated electronically different substituents at positions C5−C7 of the isatin ketimines, but the alkyl-subsituted substrate afforded lower yield than halogen-substituted analogues (6o vs 6p and 6q). Benzyl, acetyl, and Boc protecting groups were well tolerated (6r and 6s). To demonstrate that our approach could be combined with efforts to increase “druglikeness” based on Lipinski’s rule, we eliminated the hydroxy of the hemiaminal moiety and deprotected the resulting molecule, thereby ensuring a desirable

Figure 1. Substrate scope of the cascade reaction. Yield was calculated from the isolated pure diastereomer; dr value was determined by 1H NMR analysis of the crude reaction mixture; ee value was determined by chiral HPLC analysis. The absolute configuration of 6p was determined by X-ray analysis (CCDC 1582063), and other products were assigned by analogy. 5 was dehydrated with 3 equiv of p-Tos acid at −60 °C to yield 7. 6a was treated with TFA in CH2Cl2 at rt for 24 h to yield 8a. 6a was treated with TFA in CH2Cl2 at rt for 10 min to yield 9a.

lipo-hydro partition coefficient and number of hydrogen bond donors/acceptors. In this way, we generated the spirotetrahydropyridine oxindole derivatives (7a−7g) and regioselectively deprotected products (8a and 9a). Having synthesized a series of chiral six-membered Nheterocycle-fused spiro-oxindoles, we evaluated their ability to bind to MDM2 and inhibit proliferation of two breast cancer lines expressing wild-type p53 (MCF-7, ZR-75-1) and a breast cancer line expressing mutated p53 (BT-474) (Figure 2a and Table S2). In these experiments, the compound concentration was always 20 μM. Compounds 6a−6s and 7a−7g showed little bioactivity, while 8a and 9a showed moderate inhibitory activity against MCF-7 and ZR-75-1 cells. Thus, a series of regioselectively deprotected derivatives 8 and 9 were synthesized and screened (Figure 2b,c). Compounds 9a−9f were more 6753

DOI: 10.1021/acs.orglett.7b03516 Org. Lett. 2017, 19, 6752−6755

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Organic Letters

oxindole can interact with MDM2 via hydrogen-bonding and hydrophobic interactions analogously to p53 residues Trp23, Phe19, and Leu25 (Figure S1). The main hydrogen-bonding interaction between 9e and MDM2 appears to involve residue Leu54 (Table S3). To begin to understand how 9e inhibits proliferation of breast cancer cells, we examined its ability to promote apoptosis in MCF-7 cells. Incubating cells for 24 h with 2.0 μM 9e led to apoptotic nuclear fragmentation (Figure 3a). Proportions of cells

Figure 3. (a) MCF-7 breast cancer cells were treated with 2.0 μM 9e, and nuclear morphology was examined using Hoechst 33258. Arrows indicate fragmented nuclei. (b) Apoptosis in MCF-7 cells treated with 9e was quantitated based on dual staining with Annexin V and propidium iodide, followed by flow cytometry. (c) Cell cycle analysis of MCF-7 cells treated with 9e. (d) Effects of 9e on expression levels of MDM2, p53, p21, and apoptosis-related proteins after 24 h incubation.

Figure 2. (a) Heat map of mean inhibitory ratios of compounds 6−9 against MDM2 and against breast cancer cell lines. The ability of compounds to inhibit the MDM2−p53 interaction was measured using a homogeneous time-resolved fluorescence assay, and their cytotoxicity to cells was assessed using the MTT method. In all assays, compound concentration was 20 μM. The mean inhibitory ratio was calculated from duplicate measurements in three independent experiments. (b) Structures of 8a−8f and 9a−9f. (c) Mean IC50 values of 8a−8f and 9a− 9f against MDM2 after 2 h incubation. (d) Mean IC50 values of 9e against a panel of breast cancer cell lines after 24 h incubation. Orange indicates WT p53 cell lines, blue indicates MUT p53 cell lines. (e) Mainchain rmsd fluctuations in a MD simulation of a 9e−MDM2 complex. (f) Potential modes of 9e binding to MDM2. The binding mode of p53 peptide to MDM2 is shown in green as a reference. (g) 2D schematic of the potential binding mode of 9e to MDM2.

in early or late stages of apoptosis were measured based on double staining with Annexin V and propidium iodide (PI). The proportion of cells in late apoptosis (Annexin-V+, PI+) was significantly higher after incubation with 5 μM 9e (10.8 ± 2.10%) than after incubation with 2 μM 9e (2.93 ± 0.4%, p < 0.05) or no inhibitor (0.33 ± 0.1%, p < 0.05; Figure 3b). However, similar proportions of cells were found to be in early stages of apoptosis (Annexin V+, PI−) after incubation with 5 μM 9e (24.6%) or 2 μM 9e (27.5%). In addition, 9e at submicromolar concentration induced cell cycle arrest in the G0/G1 phase rather than apoptosis (Figure 3c). Incubating MCF-7 cells with 9e also increased levels of p53 and p21 but slightly reduced levels of MDM2, based on Western blotting. This suggests that 9e inhibits MDM2-mediated p53 degradation, and 9e is not a specific MDM2 inhibitor since it fails to increase MDM2 protein. At the same time, 9e treatment led to higher levels of the apoptosis activator Bax, cleavage of caspase-9, and activation of PARP (Figure 3d). These results suggest that 9e may activate the mitochondrial apoptosis pathway. Herein, we describe an organocatalytic, asymmetric cascade reaction of nitroalkenes, aldehydes, and ketimines in the presence of chiral secondary amine. This reaction efficiently generates spiro-piperidine oxindole derivatives in good yields with high diastereoselectivities and excellent enantioselectivities. Compound 9e in this library displayed the most potent MDM2 inhibition and antiproliferative effects on breast cancer cells expressing wild-type p53. Molecular dynamics simulations suggest that 9e binds to MDM2 analogously to p53. The compound may induce cell cycle arrest at G0/G1 as well as

bioactive than 8a−8f despite sharing similar substitutions (see Supporting Information for the preliminary SAR discussion). None of the compounds we tested showed cytotoxicity against BT-474 cells, suggesting that the p53 status determines much of the efficacy of these potential inhibitors. We suspected that an N-Boc group on the piperidine ring could strengthen bioactivity, so we tested 9e for bioactivity. It inhibited the interaction between MDM2 and p53 peptide with an IC50 of 0.91 ± 0.12 μM, and it suppressed proliferation of five breast cancer lines: MCF-7 (IC50 2.5 μM), ZR-75-1 (3.3 μM), BT-474 (35.5 μM), MDA-MB-231 (47.3 μM), and SKBR-3 (41.8 μM) (Figure 2d). To begin to understand how 9e and potentially other molecules with a spiro-oxindole scaffold may bind to MDM2, we performed a 100 ns molecular dynamics simulation, which showed that the complexes of 9e and MDM2 reached equilibrium after 5 ns and that main-chain rmsd, which fluctuated less than 0.05 nm, averaged 0.25 nm for 9e on its own and 0.12 nm for the complex (Figure 2e). Binding modes along the trajectory between 6 and 100 ns were analyzed in detail (Figure 2f,g). It appears that N-Boc and 4-chlorophenyl groups on the 6754

DOI: 10.1021/acs.orglett.7b03516 Org. Lett. 2017, 19, 6752−6755

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Organic Letters

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mitochondrial apoptosis by up-regulating p53 and p21. Further chemical modification and biological exploration of this lead compound are underway in our laboratory.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b03516. Experimental details and analytical data (PDF) Accession Codes

CCDC 1582063 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_ [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Gu He: 0000-0002-1536-8882 Bo Han: 0000-0003-3200-4682 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for financial support from the National Natural Science Foundation of China (81573588, 81630101, 81773889, and 21772131), the Science & Technology Department of Sichuan Province (2017JQ0002).

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REFERENCES

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DOI: 10.1021/acs.orglett.7b03516 Org. Lett. 2017, 19, 6752−6755