Ying Huangab,
Yi-Xin Huangb,
Jing Suna and
Chao-Guo Yan*a
aCollege of Chemistry & Chemical Engineering, Yangzhou University, Yangzhou 225002, China. E-mail: cgyan@yzu.edu.cn
bCollege of Medicine, Yangzhou University, Yangzhou 225001, China
First published on 2nd July 2018
The three-component reaction of 1,2,3,4-tetrahydroisoquinoline, isatins and 3-phenacylideneoxindoles in refluxing ethanol afforded dispiro[indoline-3,1′-pyrrolo[2,1-a]isoquinoline-3′,3′-indolines] (4a–4x) in good yields via 1,3-dipolar cycloaddition of in situ generated azomethine ylide with the exocyclic double bond of 3-phenacylideneoxindoles. 1H NMR spectra and single crystal structures indicated the reaction has high regioselectivity and diastereoselectivity. Furthermore, their biological activities have been preliminarily demonstrated by in vitro evaluation against mouse breast cancer cells 4T1 and human liver cancer cells HepG2 by MTT assay. The results demonstrated that some of the compounds showed cytotoxicities to cell lines of 4T1 and HepG2, and indicated that novel spirooxindoles may become potential lead compounds for further biological screenings of their medicinal applications.
1,3-Dipolar cycloaddition reaction is an efficient and high-yielding, regio- and stereo controlled method for the synthesis of heterocyclic compounds. For the preparation of five-membered nitrogen-containing cyclic compounds, in particular pyrrolidines, dihydropyrroles, and pyrroles, [3+2] cycloaddition of azomethine ylides with alkenes is very effective and has been studied widely.8,9 If the azomethine ylides are generated from isatin derived compounds and α-amino acids through the thermal decarboxylation, pyrrolidine-containing spirooxindoles with high regioselectivity and stereoselectivity will be obtained.10,11
Heteroaromatic N-ylides such as pyridinium, thiazolium, quinolinium, isoquinolinium methylides which are readily available from the alkylation of azaaromatic heterocycles and sequential deprotonation reaction have also been used as one kind of reactive azomethine ylides extensively in cycloadditions for the synthesis of the fused heterocycles with a nitrogen at the point of fusion.12 Our group had reported the green synthetic methods for complex heterocyclic compounds, such as efficient synthesis of spiro[indoline-3,1′-pyrrolo[2,1-a]isoquinolines via 1,3-cycloaddition reactions of 3-phenacylideneoxindoles with aza-aromatic N-ylides generated from isoquinolinium salts.13 Wang's group synthesized some analogues by using 3,4-dihydroisoquinolinium salts.14 In addition, the 1,3-dipolar cycloaddition reactions of isatin, benzylamine and chalcone derivatives or benzylideneacetones are reported, in which azomethine ylides are in situ generated from isatin and benzylamine.15 Using 1,2,3,4-tetrahydroisoquinoline instead of benzylamine, the spiro compounds with key structure of spiro[indoline-3,1′-pyrrolo[2,1-a]isoquinolines could be obtained.16 In this paper, we wish to report an efficient 1,3-dipolar cycloaddition reaction of isatins, 1,2,3,4-tetrahydroisoquinoline and 3-phenacylideneoxindoles for regioselective and diastereoselective synthesis of novel functionalized spirooxindoles. Additionally, their biological activities have been preliminarily demonstrated by in vitro evaluation against mouse breast cancer cells 4T1 and human liver cancer cells HepG2 by MTT assay.
Then, we extended the scope to the reaction under this simple reaction conditions. Various isatins (1a–f) and 3-phenacylideneoxindoles (3a–l) with both electron-withdrawing and electron releasing-substituents were employed in the three-component reaction under same conditions. The corresponding novel dispirooxindoles (4b–x) were successfully synthesized in high yields. The substituents on the both oxindoles showed little effect on the yields of the products. The results are summarized in Table 1. It should be pointed out that the pure products were usually obtained by filtration of the formed precipitates. The products were fully characterized by the spectroscopic methods. For examples, the IR spectrum of 4a showed three peaks of the carbonyl groups at 1725, 1676, 1626 cm−1. Because there are four chiral carbon atoms in the newly formed ring of pyrrolidine, several diastereoisomers might be formed in the 1,3-dipolar cycloaddition reaction. In the 1H NMR spectrum of 4a, two singlets appeared at δ 5.59 and δ 4.89 ppm were the signs of two cyclic CH unit in newly-formed pyrrolidine, and the two characteristic signals appeared at δ 10.38 and δ 10.34 ppm were the corresponding signs of NH protons in two oxindole rings. This result clearly showed that only one diastereoisomer existed in the obtained product. In 13C NMR spectrum of 4a, the two carbonyl group of the oxindole ring showed signs at δ 179.1 and 177.4 ppm, and the carbonyl group in benzoyl group showed sign at δ 196.1 ppm. The mass spectrum displayed a distinguished peak at m/z 554.2447 which further supported the formation of cycloadduct 4a. Other spiro compounds also displayed similar spectroscopy. But the 1H and 13C NMR of the compounds 4t and 4u clearly indicated two diastereoisomers existed in the obtained samples. The major/minor ratios of 4t/4t′ (90:10) and 4u/4u′ (75:25) were determined by integral of signs in the 1H NMR spectra. For determining the relative configuration of the spiro compounds 4a–4x, the single crystal structures of three compounds 4a, 4u and 4v (Fig. 2, 3 and 4) were successfully determined by X-ray diffraction. From the figures, it is clearly seen that the three single crystal structures have same relative configuration, in which two oxindole units existed at trans-position. The two protons in the ring of pyrrolidine also existed in trans-configuration. On the basis of NMR spectra and single crystal structures, we can concluded that this three-component reaction predominately give this kind of the diastereoisomer as major product and the other diastereoisomers were formed as minor product in few cases.
Entry | Compd | R1 | R2 | R3 | R4 | R5 | Yieldb (%) |
---|---|---|---|---|---|---|---|
a Reaction conditions: isatin (0.30 mmol), tetrahydroisoquinoline (0.30 mmol), 3-phenacylideneoxindole (0.25 mmol); reflux, 7 h.b Isolated yields.c Ratio of 4t:4′t = 90:10.d Ratio of 4u:4′u = 75:25. | |||||||
1 | 4a | CH3 | H | CH3 | H | CH3 | 70 |
2 | 4b | CH3 | H | Cl | H | OCH3 | 76 |
3 | 4c | Cl | H | CH3 | H | CH3 | 81 |
4 | 4d | CH3 | H | H | H | H | 90 |
5 | 4e | CH3 | H | CH3 | H | Cl | 80 |
6 | 4f | CH3 | H | Cl | H | CH3 | 50 |
7 | 4g | Cl | H | CH3 | H | Cl | 82 |
8 | 4h | Cl | H | Cl | H | OCH3 | 67 |
9 | 4i | Cl | H | F | H | CH3 | 57 |
10 | 4j | CH3 | H | Cl | CH2Ph | Cl | 57 |
11 | 4k | CH3 | H | H | CH2Ph | H | 58 |
12 | 4l | CH3 | H | CH3 | CH2Ph | CH3 | 62 |
13 | 4m | CH3 | H | CH3 | CH2Ph | Cl | 66 |
14 | 4n | CH3 | H | CH3 | C4H9 | CH3 | 90 |
15 | 4o | Cl | H | Cl | C4H9 | CH3 | 72 |
16 | 4p | H | CH3 | CH3 | H | CH3 | 85 |
17 | 4q | H | CH3 | Cl | H | CH3 | 63 |
18 | 4r | H | CH3 | CH3 | CH2Ph | Cl | 58 |
19 | 4s | CH3 | CH2Ph | CH3 | H | CH3 | 79 |
20 | 4t | CH3 | CH2Ph | Cl | H | OCH3 | 80c |
21 | 4u | CH3 | CH2Ph | Cl | H | CH3 | 62d |
22 | 4v | Cl | CH2Ph | Cl | H | CH3 | 76 |
23 | 4w | CH3 | CH2Ph | CH3 | CH2Ph | Cl | 60 |
24 | 4x | Cl | C4H9 | CH3 | H | CH3 | 50 |
Compound | 4T1 cell death | HepG2 cell death | ||||||
---|---|---|---|---|---|---|---|---|
24 h | 48 h | 24 h | 48 h | |||||
% | Mean ± S.D. | % | Mean ± S.D. | % | Mean ± S.D. | % | Mean ± S.D. | |
a /: no activity. | ||||||||
4a | 2.31 | 0.915 ± 0.011 | 25.80 | 0.870 ± 0.015 | 2.84 | 0.868 ± 0.024 | 32.86 | 0.506 ± 0.004 |
4b | 2.23 | 0.916 ± 0.016 | 15.05 | 0.984 ± 0.014 | 3.99 | 0.858 ± 0.023 | 19.34 | 0.591 ± 0.110 |
4c | 6.99 | 0.871 ± 0.015 | 20.36 | 0.928 ± 0.010 | 5.67 | 0.843 ± 0.016 | 19.71 | 0.589 ± 0.060 |
4d | 6.35 | 0.877 ± 0.040 | 20.82 | 0.923 ± 0.014 | 7.16 | 0.830 ± 0.016 | 66.72 | 0.145 ± 0.046 |
4e | 7.88 | 0.863 ± 0.022 | 20.64 | 0.925 ± 0.060 | 2.97 | 0.867 ± 0.014 | 13.27 | 0.629 ± 0.058 |
4f | 6.45 | 0.876 ± 0.012 | 19.43 | 0.937 ± 0.098 | 4.59 | 0.853 ± 0.013 | 14.22 | 0.623 ± 0.045 |
4g | / | 0.940 ± 0.061 | 27.64 | 0.851 ± 0.034 | 3.71 | 0.861 ± 0.012 | 23.16 | 0.567 ± 0.056 |
4h | 5.23 | 0.887 ± 0.045 | 23.25 | 0.896 ± 0.137 | 6.12 | 0.839 ± 0.013 | 70.11 | 0.098 ± 0.006 |
4i | 10.38 | 0.839 ± 0.025 | 33.31 | 0.791 ± 0.068 | 5.34 | 0.846 ± 0.028 | 73.50 | 0.088 ± 0.004 |
4j | 3.42 | 0.904 ± 0.020 | 17.87 | 0.954 ± 0.051 | 5.02 | 0.849 ± 0.019 | 25.25 | 0.554 ± 0.066 |
4k | 3.43 | 1.329 ± 0.112 | 10.88 | 1.237 ± 0.033 | / | 1.193 ± 0.024 | 2.36 | 1.211 ± 0.106 |
4l | / | 1.423 ± 0.013 | 10.54 | 1.241 ± 0.017 | / | 1.208 ± 0.012 | / | 1.563 ± 0.177 |
4m | 8.16 | 1.268 ± 0.032 | 20.71 | 1.099 ± 0.084 | / | 1.184 ± 0.003 | 4.22 | 1.188 ± 0.012 |
4n | 4.28 | 1.318 ± 0.018 | 15.68 | 1.169 ± 0.057 | 0.66 | 1.170 ± 0.019 | 11.82 | 1.094 ± 0.026 |
4o | 0.23 | 1.370 ± 0.021 | 12.69 | 1.211 ± 0.020 | / | 1.208 ± 0.030 | / | 1.441 ± 0.229 |
4p | / | 1.377 ± 0.011 | 22.86 | 1.070 ± 0.040 | / | 1.180 ± 0.028 | / | 1.393 ± 0.424 |
4q | / | 1.514 ± 0.009 | 4.65 | 1.322 ± 0.018 | 23.9 | 0.897 ± 0.022 | 9.88 | 1.118 ± 0.137 |
4r | / | 1.402 ± 0.027 | 5.11 | 1.316 ± 0.021 | / | 1.217 ± 0.018 | / | 1.285 ± 0.113 |
4s | / | 1.376 ± 0.016 | 8.20 | 1.273 ± 0.007 | / | 1.292 ± 0.025 | 7.23 | 1.151 ± 0.048 |
4t | / | 1.481 ± 0.016 | / | 1.465 ± 0.028 | / | 1.385 ± 0.020 | 10.09 | 1.115 ± 0.069 |
4u | 2.75 | 1.108 ± 0.039 | / | 1.131 ± 0.001 | 2.75 | 1.117 ± 0.154 | 1.053 ± 0.019 | |
4v | / | 1.125 ± 0.065 | / | 1.211 ± 0.021 | / | 1.172 ± 0.018 | / | 1.036 ± 0.010 |
4w | 2.40 | 1.187 ± 0.060 | 5.249 | 1.197 ± 0.005 | 2.40 | 1.121 ± 0.038 | 5.25 | 0.947 ± 0.026 |
4x | / | 1.185 ± 0.033 | / | 1.201 ± 0.002 | / | 1.194 ± 0.042 | / | 1.314 ± 0.110 |
Compound | 200 μg mL−1 | 100 μg mL−1 | 50 μg mL−1 | |||
---|---|---|---|---|---|---|
Cell death | Cell death | Cell death | ||||
% | Mean ± S.D. | % | Mean ± S.D. | % | Mean ± S.D. | |
4d | 67.40 | 0.325 ± 0.018 | 30.93 | 0.691 ± 0.062 | 16.12 | 0.838 ± 0.040 |
4h | 59.74 | 0.402 ± 0.014 | 30.64 | 0.693 ± 0.028 | 20.57 | 0.794 ± 0.028 |
4i | 79.30 | 0.209 ± 0.040 | 70.40 | 0.474 ± 0.056 | 44.08 | 0.559 ± 0.003 |
In detail, compounds 4a–4i which have no substituents on N atom of isatin (R2 = H) and 3-phenacylideneoxindole (R4 = H) inhibited the growth of HepG2 cells at the concentration of 200 μg mL−1, displayed promising cytotoxicity to HepG2 cells with inhibition rates varying from 13.27% to 73.50%. These results were much better than the other compounds (4j–4x) which have N-substituents such as CH3, C4H9 and CH2Ph. Compounds 4j–4x tend to precipitate out during the dilution process. These results would suggest that the polarity or lipo-hydro partition coefficient (logP) of the compound has a significant effect on its activity (Table 2).
As shown in Fig. 5, untreated cells exhibit regular blue colour. In contrast, cells treated with 4i at the concentration of 200 μg mL−1 showed clear red colour. It indicated that large amount of HepG2 cells died after treatment with 4i. Compounds 4d, 4h and 4i showed a tendency of concentration-dependent cytotoxicity because their inhibition rates were greatly enhanced with increasing concentration. Among them, compound 4i (R3 = F) was the most powerful to inhibit the growth of HepG2 cells to 44.08% at the concentration of 50 μg mL−1 (Table 3). The cytotoxicity of compounds 4d, 4h and 4i was tested on 3T3 cells at the concentration of 200 μg mL−1, the results showed that their cytocompatibility were good, which meant that they have cytotoxicity to cancer cells and were not toxic to normal cells (Fig. 6).
Fig. 5 The morphological features of survival status were monitored by fluorescence microscopy after staining with DAPI. |
5,5′′-Dimethyl-2′-(4-methylbenzoyl)-6′,10b′-dihydro-2′H, 5′H-dispiro[indoline-3,1′-pyrrolo[2,1-a]isoquinoline-3′,3′-indoline]-2,2′′-dione (4a). White solid, 70%, mp. 244–245 °C; 1H NMR (400 MHz, DMSO-d6) δ: 10.38 (s, 1H, NH), 10.34 (s, 1H, NH), 7.63 (s, 1H, ArH), 7.23 (s, 1H, ArH), 7.10–7.00 (m, 7H, ArH), 6.88 (brs, 1H, ArH), 6.82–6.77 (m, 2H, ArH), 6.36 (d, J = 7.6 Hz, 1H, ArH), 6.24 (d, J = 7.2 Hz, 1H, ArH), 5.59 (s, 1H, CH), 4.89 (s, 1H, CH), 2.86–2.78 (m, 2H, CH), 2.64–2.60 (m, 2H, CH), 2.29 (s, 3H, CH3), 2.21 (s, 6H, CH3); 13C NMR (150 MHz, DMSO-d6) δ: 196.1, 179.1, 177.4, 142.7, 141.1, 139.1, 135.2, 134.5, 134.2, 130.4, 130.3, 130.2, 129.9, 128.7, 128.5, 127.2, 126.9, 126.3, 126.1, 125.4, 124.8, 123.1, 109.1, 108.6, 70.6, 68.7, 66.1, 58.2, 41.4,29.3, 21.0, 20.9, 20.6; IR (KBr) ν: 3355, 3170, 3029, 2916, 2832, 1725, 1676, 1626, 1607, 1574, 1494, 1428, 1373, 1341, 1296, 1248, 1202, 1166, 1042, 1008, 947, 905, 810, 756, 730 cm−1; MS (m/z): HRMS (ESI) calcd for C36H32N3O3 ([M + H]+): 554.2438, found: 554.2447.
Footnote |
† Electronic supplementary information (ESI) available: 1H and 13C NMR spectra for all new compounds. CCDC 1556207–1556209. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c8ra04375b |
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