Efficient and mild one-pot synthesis of (E)-8′-arylidene-5′,6′,7′,8′-tetrahydrospiro[oxindole-3,4′-pyrano[3,2-c]pyridin] derivatives with potential antitumor activity

1 Introduction

As a well-known and significant structural constituent, the heterocyclic spirooxindole ring skeleton presents in a number of natural products (Cui et al., 1996; Williams and Cox, 2003) and other useful compounds. There are numerous spirooxindole heterocycles; especially, these compounds containing a spirooxindole fused with a monoarylidenepiperidin-4-one ring system possess a variety of highly pronounced biological activity and potential medicinal applications, such as antibacterial activity (Dandia et al., 2013), antimycobacterial activity (Kumar et al., 2008), antitubercular activity (Karthikeyan et al., 2010), anti-tumor activity (Girgis, 2009), and cholinesterase inhibitory activity (Kia et al., 2014a,b). The significance in biological activity may be creditable to their unique structural characteristics, whose structural frameworks assemble two important bioactive heterocycle moieties into a single molecule. To complete the merger of spirooxindole and monoarylidenepiperidin-4-one, intense efforts have been made for the development of various efficient methods during the past few years, and almost all of them are based on 1,3-dipolar cycloaddition reaction (Kumar and Perumal, 2007). Despite these remarkable advances, finding cost-effective, sustainable and creative synthetic methods to reproduce the structural diversity and complexity of biologically important spirooxindole fused with a monoarylidenepiperidin-4-one skeleton would always be a welcome addition, including the design of novel substrate and methodology itself. Accordingly, we make an attempt at accomplishing above-mentioned goal employing another strategy as a candidate reference method, which utilizes a one-pot reaction combining several transformations including preliminary Knoevenagel condensation (Chakrabarty et al., 2009), further addition and final intramolecular cyclization (Chen et al., 2010). If this idea is viable, many novel and diverse (E)-8′-arylidene-5′,6′,7′,8′-tetrahydrospiro[oxindole-3,4′-pyrano[3,2-c]pyridin] derivatives will be formed by using different monoarylidenepiperidin-4-ones, which can be used to identify potential drug candidates.

Recently, this strategy has been successfully applied to the synthesis of structurally complex and diverse heterocyclic products (Liu et al., 2013). To develop more creative methods, the endeavor was centered on the design of new substrates especially novel nucleophiles for further domino transformation of Knoevenagel products, including 1,3-dicarbonyl compounds (Ghahremanzadeh et al., 2010; Karmakar et al., 2012), α-methylenecarbonyl compounds (Elinson et al., 2009; Kamalraja et al., 2014), aromatic phenolic (Heravi et al., 2012; Park et al., 2013), dialkyl acetylenedicarboxylates (Tisseh et al., 2012), and others. The design and synthesis of novel nucleophiles and their use in Multicomponent reactions (MCR) to achieve skeletal diversity might significantly contribute in populating the chemical space. As newly emerged nucleophiles, (E)-3-arylidene-1-methylpiperidin-4-ones were generated in the reaction from the assembly of 1-methylpiperidin-4-one and aldehydes in a Mannich-elimination sequence (Gu et al., 2014). Compared with cyclohexanone (Abdel-Latif and Shaker, 1991; Shanthi et al., 2007), (E)-3-arylidene-1-methylpiperidin-4-ones only have one highly selective and active methylene as reactive site, which was conducive to improve the reaction selectivity and yields. Under the same condition, a comparison between the three-component reaction of isatin, malononitrile, 1-methylpiperidin-4-one and the three-component reaction of isatin, malononitrile, (E)-1-methyl-3-(4-(trifluoromethyl)benzylidene)piperidin-4-one was shown in Scheme 1, and the structure characterization of related compounds was shown in Fig. 1. Besides, a significant exocyclic monoarylidene group was embedded in novel molecular structure.

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Scheme 1 A comparison between 1-methylpiperidin-4-one and monoarylidenepiperidin-4-one.

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Figure 1 The structure characterization of compounds 5a, 5b, 5c.

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Efficiency, sustainability and green operations are of great importance and a demanding challenge in chemical production. Multicomponent reactions (MCR) emerged and offered significant advantages, such as enhancement of atom and energy efficiency, avoidance of intermediate products, retrenchment of effort and resources, and waste minimization (Behr et al., 2014; Khabazzadeh et al., 2012; Mosaddegh and Hassankhani, 2012). As part of our continuing interest in the development of novel synthetic methods in polycyclic heterocycles, we report a mild and efficient three-component cyclization reaction of isatins, malononitrile and (E)-3-arylidene-1-methylpiperidin-4-ones for the synthesis of novel (E)-8′-arylidene-5′,6′,7′,8′-tetrahydrospiro[oxindole-3,4′-pyrano[3,2-c]pyridin] derivatives, using piperidine as an efficient catalyst and ethanol as an environmentally benign solvent at ambient temperature. The in vitro antitumor activity of the synthesized compounds was evaluated according to the National Cancer Institute (NCI) in vitro disease-oriented human cells screening panel assay.

2 Experimental

3 Results and discussion

As shown in Table 1, a three-component reaction of isatin (0.2 mmol), malononitrile (0.2 mmol) and (E)-1-methyl-3-(4-(trifluoromethyl)benzylidene)piperidin-4-one (0.2 mmol) was conducted in methanol using DBU as a catalyst at room temperature. This reaction proceeded smoothly to afford the corresponding spiro compound 4a in 90% yield (Table 1, entry 1). The structure and relative configuration of the product 4a were characterized by a single crystal X-ray crystallographic study (Fig. 2), and a methanol solvate molecule was incorporated in the crystalline structure. To increase the yield of the reaction, we then evaluated other organic bases such as piperidine, pyrrolidine, DIEA and triethylamine, and found that the reaction with piperidine and pyrrolidine showed superior results (Table 1, entries 2–3); the reaction with DIEA and triethylamine showed inferior results (Table 1, entries 4–5) in terms of the yield compared with DBU as the catalyst. The highest yield for product 4a was obtained when piperidine was selected as the catalyst. Consequently, piperidine was applied in the following tests.

Table 1 Optimization of reaction conditions for compound 4a.

Entry	Solvent	Base (equiv)	Time, h	Yield of 4aa (%)
1	MeOH	DBU (1)	1	90
2	MeOH	Piperidine (1)	2	94
3	MeOH	Pyrrolidine (1)	2	93
4	MeOH	DIEA (1)	5	82
5	MeOH	Triethylamine (1)	5	81
6	EtOH	Piperidine (1)	2	95
7	THF	Piperidine (1)	2	94
8	CH3CN	Piperidine (1)	2	89
9	1,4-Dioxane	Piperidine (1)	2	90
10	Toluene	Piperidine (1)	2	84
11	CH2Cl2	Piperidine (1)	2	75
12	EtOH	Piperidine (2)	2	95
13	EtOH	Piperidine (1.5)	2	95
14	EtOH	Piperidine (0.5)	2	93
15	EtOH	Piperidine (0.2)	2	90

a Isolated yield after purification by column chromatography.

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Figure 2 Crystal structure of 4a methanol solvate (the solvent molecule is disordered).

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To further evaluate the efficacy of piperidine, a brief screening of solvents in the formation of 4a was carried out, viz. methanol, ethanol, tetrahydrofuran, acetonitrile, 1,4-dioxane, toluene and dichloromethane (Table 1, entries 2, 6–11). To our delight, all reactions proceeded well especially in ethanol leading to highest yield of 95%. Subsequently, the influence of the amount of piperidine on the yield has been investigated, and the outcomes displayed that 1 equivalent of piperidine was sufficient to complete this reaction (Table 1, entries 12–15). Larger amounts of piperidine did not improve the yields of the reaction. From these results, we can find that 1 equivalent of piperidine and ethanol emerged as the optimized selection of base-solvent combination for this model reaction.

Having optimized the conditions, we explored the substrate scope of the reaction of isatins, nitrilo active methylene components, and monoarylidene cyclic ketones (Table 2). The detailed results show that the approach is compatible with a wide variety of substrates. Firstly, incorporating different protecting groups on the N-1 of isatins was applied to the cyclization reaction (Table 2, entries 1–4). Then, malononitrile was replaced with ethyl cyanoacetate to enrich the diversity of nitrilo active methylene components (Table 2, entries 5–8). Next, many (E)-3-arylidene-1-methylpiperidin-4-one bearing different aromatic substituents were used for the three-component reaction and all reactions had been carried out smoothly with 74–98% isolated yields (Table 2, entries 9–15). In the end, we investigated the reaction of other monoarylidene cyclic ketones having various rings instead of (E)-1-methyl-3-(4-(trifluoromethyl)benzylidene)piperidin-4-one to verify the scope of the three-component one-pot reaction. As expected, these reactions afforded good yields of corresponding spirooxindole derivatives (Table 2, entries 16–20).

Table 2 Synthesis of 2′-aminospiro[indoline-3,4′-pyran]-2-one derivatives.

Entry	R1 (1)	R2 (2)	X/R3 (3)	Products	Yield (%)a
1	H (1a)	CN (2a)	N–CH3/4-CF3C6H4 (3a)	4a	95
2	Me (1b)	CN (2a)	N–CH3/4-CF3C6H4 (3a)	4b	88
3	Et (1c)	CN (2a)	N–CH3/4-CF3C6H4 (3a)	4c	94
4	Bn (1d)	CN (2a)	N–CH3/4-CF3C6H4 (3a)	4d	80
5	H (1a)	COOEt (2b)	N–CH3/4-CF3C6H4 (3a)	4e	66
6	Me (1b)	COOEt (2b)	N–CH3/4-CF3C6H4 (3a)	4f	69
7	Et (1c)	COOEt (2b)	N–CH3/4-CF3C6H4 (3a)	4g	65
8	Bn (1d)	COOEt (2b)	N–CH3/4-CF3C6H4 (3a)	4h	60
9	H (1a)	CN (2a)	N–CH3/4-NO2C6H4 (3b)	4i	91
10	H (1a)	CN (2a)	N–CH3/4-tert-Butylphenyl (3c)	4j	82
11	H (1a)	CN (2a)	N–CH3/3,4-CH3C6H3 (3d)	4k	74
12	H (1a)	CN (2a)	N–CH3/3-CH3C6H4 (3e)	4l	98
13	H (1a)	CN (2a)	N–CH3/2-Naphthyl (3f)	4m	83
14	H (1a)	CN (2a)	N–CH3/2,4-Cl2C6H3 (3g)	4n	92
15	H (1a)	CN (2a)	N–CH3/4-FC6H4 (3h)	4o	97
16	H (1a)	CN (2a)	CH2/4-CF3C6H4 (3i)	4p	81
17	H (1a)	CN (2a)	3jb	4q	65
18	H (1a)	CN (2a)	O/4-CF3C6H4 (3k)	4r	94
19	H (1a)	CN (2a)	S/4-CF3C6H4 (3l)	4s	93
20	H (1a)	CN (2a)	3mc	4t	78

a Isolated yield after purification by column chromatography.

b Compound 3j = (E)-2-(4-(trifluoromethyl)benzylidene)cyclopentanone.

c Compound 3m = (E)-7-(4-(trifluoromethyl)benzylidene)-1,4-dioxaspiro[4.5]decaN-8-one.

All targeted products 4a–4t were characterized by mass spectrometry fragmentation pattern analysis, high-resolution mass spectrometry, ¹H NMR and ¹³C NMR spectroscopy. For instance, the high-resolution mass spectrometry data of compound 4a (Table 2, entry 1) displayed the peak at m/z 465.1531 representing the molecular ion (calculated mass for C₂₅H₁₉F₃N₄O₂ + H⁺: 465.1538 [M + H⁺]). The ¹H NMR spectrum of compound 4a consisted of an N–CH₃ signal 2.09 (s, 3H) and an NH resonance 10.64 (s, 1H). The signals due to the four centrosymmetric aromatic protons were observed around 7.49–7.78 ppm as two doublets: 7.50 (d, J = 8 Hz, 2H) and 7.77 (d, J = 8 Hz, 2H).

A plausible mechanism for the reaction is shown in Scheme 2. The reaction was proposed to proceed through the activation of malononitrile by piperidine to generate a nucleophile, followed by a nucleophilic addition on the C3 carbonyl group in isatin with the generation of α, β-unsaturated nitrile. The electron-deficient α, β-unsaturated dicyano adduct is a potent Michael acceptor for further domino transformation in the presence of nucleophilic (E)-3-arylidene-1-methylpiperidin-4-one. The resulting intermediate undergoes subsequent intramolecular cyclization through [1,3]-sigmatropic proton shift of the iminopyran led to the formation of the final spiro compound.

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Scheme 2 Plausible mechanism for the reaction.

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The synthesized compounds were screened for their in vitro antitumor activity in the full NCI 96 cell panel, including human cervical carcinoma cell line (Hela), human liver hepatocellular carcinoma cell line (HepG2), and human breast carcinoma cell line (MDA-MB-231). During the MTT assay, a common chemotherapeutics drug adriamycin (ADM) was utilized as positive control. In the protocol, all compounds were tested at 20 μmol/L, and their percentage growth inhibition (GI%) was shown in Table 3. The data revealed that compounds 4d, 4k, 4q and 4s exhibited outstanding growth inhibitory activity against the tested subpanel tumor cell lines, which could be used as lead structures for future derivatization or modification to obtain more potent antitumor agents.

4 Conclusion

In summary, an efficient and mild cyclization procedure for the synthesis of (E)-8′-arylidene-5′,6′,7′,8′-tetrahydrospiro[oxindole-3,4′-pyrano[3,2-c]pyridin] derivatives has been identified using piperidine as an efficient catalyst and ethanol as an environmentally benign solvent. The antitumor activity of these compounds was evaluated in human cervical carcinoma cell line (Hela), human liver hepatocellular carcinoma cell line (HepG2), and human breast carcinoma cell line (MDA-MB-231).