Facile construction of spiroindoline derivatives as potential anti-viral agent via three-component reaction in aqueous with β-cyclodextrin-SO3H as an efficient catalyst

ABSTRACT A convenient and efficient β-cyclodextrin-SO3H-assisted strategy for construction of spiro indoline derivatives in aqueous media was disclosed. The present protocol showed various advantages including short reaction time, broad scope of substituents, simplicity of practice, high yields of products, recyclability of catalysts, safety, and cheapness of benign solvents. Preliminary study indicated that some of spiroindoline derivatives exhibited anti-Tobacco Mosaic Virus (TMV) activity for potential use in plant protection. GRAPHICAL ABSTRACT

To the best of our knowledge, this is the first protocol for the synthesis of this important class of spiro indoline skeletons using not only cheap and easily available starting materials but also cheap, recyclable and easily available β-cyclodextrin-SO 3 H as an efficient catalyst in a green medium.

Results and discussion
Various solvents were first screened for the construction of the spiroindolines at 50 o C in present of β-cyclodextrin-SO 3 H. Dichloroethane (Table 1, entry 1) gave moderate yields (40%) of the product 4. However, no product formed in solvent of alcohols (Table 1, entries 2, 3). Acetonitrile (Table 1, entry 4) could exclusively give the spiroindoline 4 as the spiro-product in promising yields (78%), while excitingly, water was found to significantly improve the yields of 4 (Table 1, entry 5). Further investigations on reaction temperature (Table 1, entries 6-8) and reaction time (Table 1, entries 9-10) indicated that these two factors were also important for the yields of product 4. The lower temperature (Table 1, entry 6) and short time (Table 1 entry 9) could sharply decrease the yields of 4 but the yield of the product could not obviously improve as raising the temperature or prolonging the reaction time (Table 1 entry 8). Thus, the highest yields could be obtained in the reaction for 40 min at 50 o C.
With the optimized reaction conditions in hand ( Table  1, entry 11), we subsequently examined the reaction scopes (Schemes 1 & 2). Reactants with different substituents were first tested for the preparation of the spiro [chromene-4,3'-indoline] derivatives. When 5,5-dimethylcyclohexane-1,3-dione and malononitrile were employed (Scheme 1, 4a to 4q), most of the reactions performed smoothly, both electron-donating and electronwithdrawing groups on benzene ring of isatin were well tolerated with good to excellent yields (Scheme 1, 4a to 4q). The 1-phenylindoline-2,3-dione also gave excellent yields of the product (Scheme 1, 4q). However, an introduction of acetyl (Scheme 1, 4r) could sharply decrease the yields of theproduct due to the strong electron withdrawing property of acetyl. The yields of the products also cut down a lot when the starting material malononitrile switched to ethyl 2-cyanoacetate.
(Scheme 1, 4s & 4t). With the use of cyclohexane-1,3dione (Scheme 1, 4t to 4z, and 4aa to 4af), all the reactions also proceeded smoothly, but the types of the substituents on isatin did not obviously influence the product formation. We randomly investigated the grams-scale reactions for the synthesis of spiro[chromene-4,3'-indoline] derivatives, and the present protocol afforded even higher yields of products (Scheme 1, 4a, 4 m, 4y, and 4ab).
The reusability of the catalyst was illustrated via preparation of 1a using filter liquor with β-cyclodextrin-SO 3 H in it. The results indicated the catalytic system could be used for at least five runs without any significant influence on the yield of the products (Figure 1). Most important of all, the catalyst could be reused directly via using filtrate as the next reaction without any treatment. The proposed mechanism for the construction of spiroindoline derivatives under the acidic conditions has been demonstrated in detail. Just as revealed by Dattatray et al., the low solubility of these starting materials involved in the reaction resulted in quite low yields of the products even undergoing several hours' reaction (38)(39)(40)(41).
We tabulated ( Table 2) the reported catalysts for the synthesis of spiroindolines in aqueous to compare the efficiency of β-CD-SO 3 H. Table 2 indicated that all the catalysts showed excellent efficiencies. However, some of the need assist by ultrasound irradiation ( Table 2, Entries 3 and 5) or add equivalent alcohol as solvent (Table 2, Entries 3, 10, 12 and 15). Except for a few reactions which can be carried out at room temperature (Table 2,  the yields of the products were excellent (greater than 70% and even up to 99%) under a low temperature and in a short time (40 min). The results showed that β-CD-SO 3 H is a better catalyst with respect to reaction.
Obviously, the yields of products improved substantially as the addition of β-cyclodextrin, which could provide a hydrophobic environment via a complexation with isatin and also be a supporter of '-SO 3 H' to yield the solid acid catalysts and to accelerate the process of all of these reactions (18,38,39). The '-SO 3 H' promoted the condensation and cycloaddition reaction via the formation of the intermolecular hydrogen bonding with the isatin and malononitrile respectively. That increased reactivity of isatin and facilitated the condensation reaction with malononitrile to form an isatylidene derivative (44,45). As path B showing ( Figure 2), in β-cyclodextrin's special cavity in which A caught a proton, B could smoothly form and it's carbonyl carbon atom was attacked by isomerized malononitrile H to convert to M. Secondly, transformed from M, N got a proton from β-cyclodextrin and also transferred a proton to β-cyclodextrin to get O possible by removing a molecular of 'H 2 O'. This process demonstrated that β-cyclodextrin could be reused just as Figure 2 showing that the yields of products still reached 89% after the fifth run of β-cyclodextrin. Finally, as the process of Michael addition of enolated dimedone D and O undergoing, a new C-C bond formed to provide isomers J and k, subsequently the spiroindoline products were obtained via the cycloaddition reaction (38,39,45).
Anti-TMV (Tobacco Mosaic Virus) experiments were carried out to demonstrate the practical applications of some of synthesized compounds ( Table 3). The preliminary results indicated that some of the spiroindoline  products exhibited promising inactivation activity in vivo against TMV. For example, compounds 4z and 6g showed higher anti-TMV activities than that of commercially available pesticide Ribavirin.

Conclusions
In summary, this work disclosed a protocol that β-cyclodextrin-SO 3 H catalyzed the synthesis of spiroindoline derivatives via a three-component one-pot condensation of different substituted isatin with diketones and malononitrile (or ethyl 2-cyanoacetate) in aqueous medium in good to excellent yields under mild condition. A broad scope of precursors with different substituents and substitution patterns worked well in this process. Furthermore, the catalyst could be readily recovered and reused for at least five runs without any significant influence on the yields of the products. These results indicated that β-cyclodextrin-SO 3 H showed high catalytic activity. The current approach offered high-purity products in high yields via using a small amount of catalyst, and safe, cheap, and environmentally benign solvent under an easy experimental workup procedure. Interestingly, some synthesized compounds showed promising anti-viral activity against TMV, which could be used as potential agrochemicals. Further investigations into various methods for the construction of spiroindoline derivatives by the catalysis of β-cyclodextrin-SO 3 H and the bio-activities of the afforded products are currently in progress in our laboratory.

General
All the chemicals and reagent involved in the reactions were purchased from commercial supplier and used without purified again. Isatins were purchased from Adamas-beta®, cyclohexane-1,3-dione, dimedone, cyclopentane-1,3-dione were purchased from TCI®, malononitrile and ethyl 2-cyanoacetate were purchased from Sigma-Aldrich, barbituric acid was purchased from Accela®. In addition to special notes, all the reactions were carried out using standard ScLinK technology with magnetic stirrer in the air.
The nuclear magnetic resonance spectra of 1 H NMR at 400 MHz, 13   Add β-cyclodextrin (4.5 mmol) to CH 2 Cl 2 (50 mL) and stir well-distributed, subsequently, chlorosulfonic acid (10 mmol) was add to the mixture at 0 o C for about three hours and stirred for another two hours. Then, the resulting mixture was filtered and washed with methanol and dried to obtain the target products of sulfonated βcyclodextrin as a white powder.

The synthesis of spiro[chromene-4,3'indoline] derivatives 4
A mixture of isatin(1 mmol), 1,3-cyclohexanone(1 mmol) or dimedone(1 mmol) or 1,3-cyclopentanedione(1 mmol) or barbituric acid(1 mmol), malononitrile(1 mmol) or ethyl 2-cyanoacetate(1 mmol) and βcyclodextrin-SO 3 H(10% mmol) in water(10 mL) were stirred at 50°C for the appropriate 40 min. After the reaction is completed, the reaction mixture was filtered and the residue is washed three times with 20 mL water, then dried to obtain a series of pure chromene derivatives. Assignment of structures of the compounds was based on their 1 H NMR, 13 C NMR, 19 F NMR and HR-MS.      Purification of TMV Using Gooding's method (54), the upper leaves of Nicotiana tabacum L. inoculated with TMV were selected, ground in phosphate buffer, and then filtered through a double-layer pledget. The filtrate was centrifuged at 10000 g, treated twice with PEG, and then centrifuged again. The entire experiment was conducted at 4°C. Absorbance value was estimated at 260 nm by ultraviolet spectrophotometer. More details for the protocols of purification of TMV can be found in Supporting Information.
Virus concn = (A 260 × dilution ratio)/E 0.1%,260nm 1cm Inactivation effects of the synthesized compounds against TMV (55)(56)(57)(58) The virus was inhibited by mixing with the compound solution of the same volume for 30 min. Then the mixture was inoculated on the left side, and the virus that is not treated with drugs as the control was inoculated on the right side. After 0.5 h, the leaves were washed with water. The leaves lesion number were counted after inoculation 3-4 days. Do three replicates for each compound.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The National Natural Science Foundation of China (32072445,