Selective and Reversible 1,3-Dipolar Cycloaddition of 2-(2-Oxoindoline-3-ylidene)acetates with Nitrones in the Synthesis of Functionalized Spiroisoxazolidines

The 1,3-dipolar cycloaddition of 2-(2-oxoindoline-3-ylidene)acetates with functionalized aldo- and ketonitrones proceeds with good selectivity to provide new highly functionalized 5-spiroisoxazolidines. A characteristic feature of these reactions is reversibility that allows for the control of the diastereoselectivity of cycloaddition. The reduction of obtained adducts using zinc powder in acetic acid leads to 1,3-aminoalcohols or spirolactones. For a number of the spiro compounds obtained, anticancer activity was found.

In the last decade, the number of methodologies for the synthesis of spiroindolin-2-one derivatives has been growing rapidly [14,15]. One of the most effective among them is based on 1,3-dipolar cycloaddition reactions [7,16,17]. The most investigated cycloadditions using indolin-2-one-based dipolarophiles are reactions with azomethine ylides, which make it possible to obtain spiropyrazolines in high yields and selectivity [18][19][20][21][22][23][24]. Promising biological properties were noted for the obtained cycloadducts [25][26][27][28][29][30]. However, the cycloaddition reactions of nitrones to indolin-2-one-based dipolarophiles are represented by only a few examples [31][32][33][34][35][36][37] (Scheme 1). It was found that such reactions can proceed with different regio-and stereoselectivity, depending not only on the structure of the starting compounds but also on the reaction conditions. The prediction of selectivity in the case of 2-(2-oxoindoline-3-ylidene)acetates is complicated due to the reversibility of the nitrone cycloaddition reactions [34,37]. Previously, the reactions with aldonitrones that did not contain additional functional groups were most studied. However, it is known that the transition from aldo-to ketonitrones or the introduction of additional functional groups in the α-position of the nitrone can dramatically affect the regio-and stereoselectivity of the 1,3-dipolar cycloaddition reactions [38][39][40].
In this work, the regio-and stereoselectivity of the 1,3-dipolar cycloaddition reactions of functionalized aldo-and ketonitrones with 2-(2-oxoindoline-3-ylidene)acetates were investigated for the first time. As a result, a series of novel 5-spiroisoxazolidines were synthesized with controllable diastereoselectivity. The introduction of the ester group at the C3 of isoxazolidines makes it possible to redirect the reduction from 1,3-aminoalcohols to spirolactones. In addition, the anticancer activity of the obtained functionalized spiroisoxazolidine derivatives was evaluated.

Synthesis and Structural Characterization
First, we studied the reactions of 2-(2-oxoindoline-3-ylidene)acetates 1 with C-carbamoyl aldonitrones 2. A short optimization of the reaction conditions was carried out for the reaction of 1a with nitrone 2a (Table 1). We obtained the mixtures of isomeric cycloadducts with a predominance of 5-spiroisoxazolidines 3 in all variants of the tested reaction conditions. The stereoselectivity of the reactions was found to be dependent on the conditions. Heating in toluene at 80 °C was optimal for obtaining diastereomer 3a as the main product (Table 1, Entry 2). At a higher temperature, 3a remained the main reaction product, but its yield decreased due to the considerable tarring of the reaction mixture (Table  1, Entry 3). We obtained diastereomer 3′a as the main product of the reactions conducted at room temperature in dichloromethane (DCM). In this case, we had to change the solvent from toluene to DCM due to the low solubility of compounds 1a and 2a in toluene ( Table 1, Entry 4). Table 1. Optimization of cycloaddition reaction conditions. 1 -from 1 H NMR spectra of the crude reaction mixtures; 2 -analysis of the 1 H NMR spectrum of the reaction mixture is difficult due to tarring.
It was found that the reactions of 2-(2-oxoindoline-3-ylidene)acetates 1a,b with Ccarbamoyl nitrones 2a-d proceed regio-and stereoselectively in toluene at 80 °C, predominantly obtaining 5-spiroisoxazolidines 3 ( Table 2). In most experiments, the minor products 3′ and 4 could not be separated chromatographically, and in the table their yields are presented as summary. It was found that the reactions of 2-(2-oxoindoline-3-ylidene)acetates 1a,b with Ccarbamoyl nitrones 2a-d proceed regio-and stereoselectively in toluene at 80 • C, predominantly obtaining 5-spiroisoxazolidines 3 ( Table 2). In most experiments, the minor products 3 and 4 could not be separated chromatographically, and in the table their yields are presented as summary.

Entry
At the same time, it was possible to selectively obtain diastereomers 3 when carrying out the reaction in DCM at room temperature (Table 3). In this case, we observed no signals of regioisomeric products 4 in the 1 H NMR spectra of the crude reaction mixtures. The compound 3 e was also obtained as a major product (yield 54%) in toluene at 55 • C for 7 h.
To confirm the stereochemistry of adducts 3 and 4, X-ray analysis data for compound 3a ( Figure 1) and 1 H-1 H NOESY NMR spectra for compounds 3h, 3 h (Figures S1 and S2 in SI) and 4c ( Figure S6 in SI) were used. To confirm the stereochemistry of adducts 3 and 4, X-ray analysis data for compound 3a ( Figure 1) and 1 H-1 H NOESY NMR spectra for compounds 3h, 3′h (Figures S1 and S2 in SI) and 4c ( Figure S6 in SI) were used. Reversibility was observed for the 1,3-dipolar cycloaddition reactions of nitrones [50,63]. In our case, the ratio of isomeric compounds 3 and 3′ depends on the reaction temperature. Furthermore, the isolated compounds 3′ were noticed to undergo transformation when kept in a CDCl3 solution overnight. The signals of reagents 1 and 2 along with the signals of stereoisomers 3 appeared in the 1 H NMR spectrum of the sample. In this regard, we further inspected the reversibility of the cycloaddition reactions of C-carbamoyl nitrones 2 under the reaction conditions. 1 H NMR spectra were recorded before   At the same time, it was possible to selectively obtain diastereomers 3′ when carrying out the reaction in DCM at room temperature (Table 3). In this case, we observed no signals of regioisomeric products 4 in the 1 H NMR spectra of the crude reaction mixtures. The compound 3′e was also obtained as a major product (yield 54%) in toluene at 55 °C for 7 h.  At the same time, it was possible to selectively obtain diastereomers 3′ when carrying out the reaction in DCM at room temperature (Table 3). In this case, we observed no signals of regioisomeric products 4 in the 1 H NMR spectra of the crude reaction mixtures. The compound 3′e was also obtained as a major product (yield 54%) in toluene at 55 °C for 7 h. Reversibility was observed for the 1,3-dipolar cycloaddition reactions of nitrones [50,63]. In our case, the ratio of isomeric compounds 3 and 3 depends on the reaction temperature. Furthermore, the isolated compounds 3 were noticed to undergo transformation when kept in a CDCl 3 solution overnight. The signals of reagents 1 and 2 along with the signals of stereoisomers 3 appeared in the 1 H NMR spectrum of the sample. In this regard, we further inspected the reversibility of the cycloaddition reactions of C-carbamoyl nitrones 2 under the reaction conditions. 1 H NMR spectra were recorded before and after heating individual compounds 3h and 3 h at 80 • C for 4 h (two times for 2 h) in C 6 D 6 . After heating, the spectra of both samples contained the signals of all three isomers 3h, 3 h, and 4h, as well as the signals of reagents 1b and 2d, and the ratio of the compounds 3h:3 h:4h:2d:1b was 5:1:0.05:1:1 for 3h and 3:1:0.3:1.6:1.6 for 3 h (Figure 2). In both experiments, the product 3h was the main component of the mixture and the ratio of compounds 2d:1b was 1:1. The obtained results provide evidence for the studied reaction to proceed reversibly under the applied conditions. Next, we investigated the reactions of dipolarophiles 1a,b with ketonitrones 5a-c containing two ester groups ( Table 4). As noted in the introduction, the regioselectivity of their cycloadditions can often be different compared to aldonitrones. The reaction with more sterically hindered C,C-bis(methoxycarbonyl)nitrones 5 required more harsh conditions, and the cycloaddition was carried out at 110 °C. The reactions proceeded to obtain only 5-spiro regioisomers 6a-f in good-to-high yields. The signals of regioisomeric products were not observed in the 1 H NMR spectra of crude reaction mixtures. The regioselectivity of the reaction in this case can be explained by steric factors. The structure of the cycloadducts 6 was further confirmed using X-ray analysis data for compound 6e ( Figure  1). Table 4. Cycloaddition of 2-(2-oxoindoline-3-ylidene)acetates 1a,b with ketonitrones 5a-c. Next, we investigated the reactions of dipolarophiles 1a,b with ketonitrones 5a-c containing two ester groups ( Table 4). As noted in the introduction, the regioselectivity of their cycloadditions can often be different compared to aldonitrones. The reaction with more sterically hindered C,C-bis(methoxycarbonyl)nitrones 5 required more harsh conditions, and the cycloaddition was carried out at 110 • C. The reactions proceeded to obtain only 5-spiro regioisomers 6a-f in good-to-high yields. The signals of regioisomeric products were not observed in the 1 H NMR spectra of crude reaction mixtures. The regioselectivity of the reaction in this case can be explained by steric factors. The structure of the cycloadducts 6 was further confirmed using X-ray analysis data for compound 6e (Figure 1). Compounds 6 showed greater stability than compounds 3 and 3 : the 1 H NMR spectrum of 6 did not change when the sample was kept in a CDCl 3 solution for two days at room temperature. However, the noticeable reversibility was indicated under the used cycloaddition reaction conditions. When pure compound 6b was heated in toluene at 110 • C for 2 h, the 1 H NMR spectrum and TLC showed the presence of the starting compounds 1a and 5b in the solution along with the signals of compound 6b. Notably, isomerization or cycloreversion was not observed for solutions of compounds 3h and 6b in DMSO-d 6 at room temperature within two days. Table 4. Cycloaddition of 2-(2-oxoindoline-3-ylidene)acetates 1a,b with ketonitrones 5a-c.
tions, and the cycloaddition was carried out at 110 °C. The reactions proceeded to obtain only 5-spiro regioisomers 6a-f in good-to-high yields. The signals of regioisomeric products were not observed in the 1 H NMR spectra of crude reaction mixtures. The regioselectivity of the reaction in this case can be explained by steric factors. The structure of the cycloadducts 6 was further confirmed using X-ray analysis data for compound 6e ( Figure  1).

Transformations of the Cycloadducts
Next, we studied the possibility of selective opening of the isoxazolidine ring under the action of zinc in acetic acid. The selective conversion to the corresponding amino alcohols was shown for both stereoisomeric adducts with aldonitrones 3 and 3 , at room temperature for 1 h (Scheme 2). Notably, the reduction of diastereomeric isoxazolidines 3e and 3 e made it possible to obtain diastereomeric amino alcohols 7a and 7 a, correspondingly.
The relative configuration of the stereocenters in 7a and 7 a was confirmed by 1 H-1 H NOESY spectra (see Figures S3 and S4 in SI) and the comparison of these data with those of isoxazolidines 3e and 3 e.
A similar reaction of cycloadduct 6d gave spirolactone 8 (Scheme 3). Presumably, in this case, the amino alcohol 9 is formed at the first stage, which is converted to lactone 8 in an acidic medium. The structure of compound 8 was confirmed by 1 H-1 H NOESY spectra (see Figure S5 in SI) and X-ray analysis data (Scheme 3). Compounds 6 showed greater stability than compounds 3 and 3′: the 1 H NMR spec trum of 6 did not change when the sample was kept in a CDCl3 solution for two days a room temperature. However, the noticeable reversibility was indicated under the used cycloaddition reaction conditions. When pure compound 6b was heated in toluene at 110 °C for 2 h, the 1 H NMR spectrum and TLC showed the presence of the starting compounds 1a and 5b in the solution along with the signals of compound 6b. Notably, isomerization or cycloreversion was not observed for solutions of compounds 3h and 6b in DMSO-d6 a room temperature within two days.

Transformations of the Cycloadducts
Next, we studied the possibility of selective opening of the isoxazolidine ring under the action of zinc in acetic acid. The selective conversion to the corresponding amino al cohols was shown for both stereoisomeric adducts with aldonitrones 3 and 3′, at room temperature for 1 h (Scheme 2). Notably, the reduction of diastereomeric isoxazolidines 3e and 3′e made it possible to obtain diastereomeric amino alcohols 7a and 7′a, corre spondingly.
The relative configuration of the stereocenters in 7a and 7′a was confirmed by 1 H-1 H NOESY spectra (see Figures S3 and S4 in SI) and the comparison of these data with those of isoxazolidines 3e and 3′e. Scheme 2. Synthesis of 1,3-aminoalcohols 7 from 3 and 3′.
A similar reaction of cycloadduct 6d gave spirolactone 8 (Scheme 3). Presumably, in this case, the amino alcohol 9 is formed at the first stage, which is converted to lactone 8 in an acidic medium. The structure of compound 8 was confirmed by 1 H-1 H NOESY spec tra (see Figure S5 in SI) and X-ray analysis data (Scheme 3).

Antiproliferative Activity
Given the high potential of spiro derivatives of indolin-2-one in medicinal chemistry it was of keen interest to test the bioactivity potential of the novel synthesized spiro com pounds 3, 3′, and 6, as well as amino alcohols 7. They were evaluated for antiproliferative activity on several tumor cell lines: A549 (lung carcinoma), MCF7 (breast cancer), MDA MB-231 (triple-negative breast cancer), Caki-2 (kidney clear cell carcinoma), and T98G (glioblastoma multiforme) (Figure 3). The highest cytotoxicity was observed for com pound 3b, against the MCF7 (breast cancer) and A549 (lung carcinoma) cell lines. At a concentration of 50 μM, a 32% inhibition for MCF7 was achieved relative to the contro (etoposide). Compounds 3a,d-h, 6, and 7 showed no inhibitory activity on all tested cel lines.

Antiproliferative Activity
Given the high potential of spiro derivatives of indolin-2-one in medicinal chemistry, it was of keen interest to test the bioactivity potential of the novel synthesized spiro compounds 3, 3 , and 6, as well as amino alcohols 7. They were evaluated for antiproliferative activity on several tumor cell lines: A549 (lung carcinoma), MCF7 (breast cancer), MDA-MB-231 (triple-negative breast cancer), Caki-2 (kidney clear cell carcinoma), and T98G (glioblastoma multiforme) (Figure 3). The highest cytotoxicity was observed for compound 3b, against the MCF7 (breast cancer) and A549 (lung carcinoma) cell lines. At a concentration of 50 µM, a 32% inhibition for MCF7 was achieved relative to the control (etoposide). Compounds 3a,d-h, 6, and 7 showed no inhibitory activity on all tested cell lines.

Antiproliferative Activity
Given the high potential of spiro derivatives of indolin-2-one in medicinal chemistry it was of keen interest to test the bioactivity potential of the novel synthesized spiro com pounds 3, 3′, and 6, as well as amino alcohols 7. They were evaluated for antiproliferativ activity on several tumor cell lines: A549 (lung carcinoma), MCF7 (breast cancer), MDA MB-231 (triple-negative breast cancer), Caki-2 (kidney clear cell carcinoma), and T98G (glioblastoma multiforme) (Figure 3). The highest cytotoxicity was observed for com pound 3b, against the MCF7 (breast cancer) and A549 (lung carcinoma) cell lines. At concentration of 50 μM, a 32% inhibition for MCF7 was achieved relative to the contro (etoposide). Compounds 3a,d-h, 6, and 7 showed no inhibitory activity on all tested ce lines.

General Information
All the cycloaddition reactions were performed in anhydrous solvents under an argon atmosphere. Toluene was distilled over sodium. Reaction progress was monitored using thin layer chromatography (TLC) on precoated Silufol UV-254 plates. 1 H and 13 C NMR spectra were recorded in CDCl 3 , benzene-d 6 , DMSO-d 6 using a Bruker Avance 400 spectrometer (see 1 H and 13 C NMR spectra in SI). HRMS spectra were obtained with a Bruker-maXis (QTOF). Xcalibur, Eos diffractometer was used for X-ray analysis. (E)-methyl 2-(2-oxoindolin-3-ylidene)acetates 1a,b and nitrones 2a-d, 5a-c were prepared using known procedures [61,64,65]. washed three times with serum-free PBS. The number of viable cells was determined by trypan blue exclusion.

Antiproliferative Assay
The effects of the synthesized compounds on cell viability were determined using the MTT colorimetric test. All the examined cell lines were diluted with the growth medium to 3.5 × 10 4 cells per mL, and the aliquots (7 × 10 3 cells per 200 µL) were placed in individual wells in 96-well multiplates (Eppendorf, Germany) and incubated for 24 h. The cells were treated with the synthesized compounds separately at a concentration of 50 µM and incubated for 72 h at 37 • C in a 5% CO 2 atmosphere. Each compound was tested in triplicate. The cells were treated with 40 µL of an MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL in PBS) and incubated for 8 h. The medium with the MTT was removed and DMSO (150 µL) was added to dissolve the formazan crystals. The plates were shaken for 10 min. The optical density of each well was determined at 560 nm using a GloMax Multi+ (Promega, Madison, WI, USA) microplate reader. The cytotoxicity of each compound was evaluated in three separate experiments.

Conclusions
It has been shown that the 1,3-dipolar cycloaddition of 2-(2-oxoindoline-3-ylidene)acetates with aldo-and ketonitrones is an effective method for the selective synthesis of new highly functionalized spiroisoxazolidines. The reaction of C-carbamoyl aldonitrones predominantly obtains 5-spiroisoxazolidines. In this case, varying the reaction conditions makes it possible to selectively obtain certain diastereomers in good yields due to the reversibility of the reaction. The reaction of C,C-bis(methoxycarbonyl) ketonitrones obtains only 5-spiroisoxazolidines in good-to-high yields. The reduction of the obtained cycloadducts can obtain aminoalcohols or spirolactones depending on the structure of the starting cycloadduct. Cytotoxicity screening against several cancer cell lines revealed that several cycloadducts exhibit antiproliferative activity.