3′,4′-Dihydro-2′H-spiro[indoline-3,1′-isoquinolin]-2-ones as potential anti-cancer agents: synthesis and preliminary screening

Both tetrahydroisoquinolines (THIQs) and oxindoles (OXs) display a broad range of biological activities including anti-cancer activity, and are therefore recognized as two privileged scaffolds in drug discovery. In the present study, 24 3′,4′-dihydro-2′H-spiro[indoline-3,1′-isoquinolin]-2-ones, designed as molecular hybrids of THIQ and OX, were synthesized and screened in vitro against 59 cell lines in the NCI-60 screen. Twenty compounds displayed weak to moderate inhibition of cell proliferation; among them, three compounds displayed at least 50% inhibition of cell proliferation. The compounds appeared to target primarily renal cell cancer lines; however, leukaemia, melanoma, non-small cell lung cancer, prostate, ovarian and even breast cancer cell lines were also affected. Therefore, this class of spirooxindoles may provide useful leads in the search for new anti-cancer agents.


Introduction
The term cancer refers to a group of more than 100 diseases characterized by a common feature of rapid and uncontrolled cell proliferation, invasiveness and metastasis, leading to death. In 2018, worldwide mortality from cancer alone accounted for an estimated 9.6 million deaths, thereby positioning cancer as the number two leading cause of death worldwide [1]. According to statistics from the World Health Organization, five forms of cancer account for the most deaths: lung (1.76 million deaths), liver (782 000 deaths), colorectal (862 000 deaths), stomach (783 000 deaths) and breast (627 000 deaths) [1,2]. In 2010, the total economic cost of cancer was estimated at 1.16 trillion US dollars [1]. When combined with the human cost of the disease, and the increasing incidence of the disease(s), there

Results and discussion
The target compounds were synthesized by the Pictet-Spengler reaction of a substituted phenethylamine with isatin or a substituted isatin. The precursors, 3,4-dimethoxyphenethylamine and 3,4,5trimethoxyphenethylamine were prepared as previously described for related amphetamine analogues [46], while 3-hydroxyphenethylamine was obtained from the demethylation of commercially available 3-methoxyphenethylamine [47]. The N-alkylation of isatin and its analogues has also been reported [48]. For the synthesis of the hydroxylated compounds (1a-q, 2a-q and 3a,b), the phenolic Pictet-Spengler reaction, earlier described by Kametani et al. [47], was found to be quite useful as it proceeded smoothly under relatively mild conditions of heating in ethanol. This reaction generally provided two positional isomers, the 6-hydroxy (2a-q) and 8-hydroxy (1a-q) compounds, in variable ratios. In some instances, only trace amounts of the minor isomer were detected; therefore, only one isomer was isolated. The regioisomers were easily separated by medium pressure liquid chromatography or vacuum liquid chromatography on a silica gel column. To prepare the methoxyl-substituted spirooxindoles (compound series 4, 5 and 6), the Pictet-Spengler reaction of the methoxyphenethylamines with the corresponding isatins was best carried out in polyphosphoric acid at 100°C [49]. The yields were moderate to good. Under these reaction conditions, only a single regioisomer was obtained from the Pictet-Spengler reaction of 3-methoxphenethylamine with the isatins. All the compounds were converted to the corresponding hydrochlorides in methanolic HCl, recrystallized and submitted for biological testing. The target compounds were submitted for anti-cancer screening under the NCI-60 programme of the National Cancer Institute, Bethesda, MD, USA (https://dtp.cancer.gov/discovery_development/nci-60/ methodology.htm). In this protocol, test compounds are screened at a concentration of 10 µM against 59 cancer cell lines. Of 24 compounds tested, 20 were found to display weak to moderate anti-proliferative activity for at least one cancer cell line (table 1).
Three compounds (3b, 4d, 5f ) inhibited cell proliferation by at least 50% and compound 5f appeared to be the most active across a number of cell lines. Furthermore, renal cell cancer appeared to be the most susceptible to this class of compounds as up to 10 analogues displayed some anti-proliferative activity on this cancer cell line. Three compounds exerted anti-proliferative activity on the PC-3 cancer cell line, while the ovarian and breast cancer cell lines were inhibited by two and one compound, respectively.
Among the spirooxindoles showing evidence of anti-proliferative activity, analogues containing the 6,7-dimethoxy-THIQ fragment (series 5) appeared with the highest frequency while those containing the 6-methoxy-THIQ fragment (series 3) appeared with the lowest frequency. Therefore, the 6,7dimethoxy-THIQ fragment appears to be the preferred fragment for subsequent optimization efforts of this hybrid scaffold. Of the 20 compounds showing notable anti-proliferative activity, nine (45%) were unsubstituted at the nitrogen atom of the OX fragment, while the rest featured a substituted benzyl fragment at this position. Therefore, subsequent optimization efforts may concentrate on the synthesis of either N-unsubstituted or N-substituted analogues. In the light of these findings, we conclude that the 3 0 ,4 0 -dihydro-2 0 H-spiro[indoline-3,1 0 -isoquinolin]-2-one scaffold does possess anti-proliferative activity. A recent investigation of the isomeric 2 0 ,4 0 -dihydro-1 0 H-spiro[indoline-3,3 0 -isoquinolin]-2-ones, published while the current study was underway, found that the latter compounds display anti-cancer activity on colon cancer [50]. The most potent compound of that study was found to inhibit Ras-GTP and thereby suppress downstream signalling by MAPK, PI3 K-Akt and Wnt, ultimately resulting in mitochondrial apoptosis [50]. Therefore, while the mode of action of the target 3 0 ,4 0 -dihydro-2 0 Hspiro[indoline-3,1 0 -isoquinolin]-2-ones of the present study is unknown, it does appear that spirofused molecular hybrids of THIQ and OX may be potentially useful leads for anti-cancer drug discovery.
Salsolinol [51] and many other naturally occurring THIQs arise from the Pictet-Spengler type reaction of a biogenic amine with an aldehyde or ketone. The design of the target compounds of this study therefore takes advantage of an endogenous pathway (biomimetic approach) that provides broad flexibility in the construction of compound libraries. Further investigation is underway to optimize the anti-proliferative activity of the target 3 0 ,4 0 -dihydro-2 0 H-spiro[indoline-3,1 0 -isoquinolin]-2-ones against cell lines of interest and to elucidate their mechanism(s) of action.

General
All chemicals were purchased from Sigma Aldrich Chemicals Company, St Louis, MO, USA, and were used as supplied. All solvents were reagent grade. Where necessary, solvents and starting materials were purified using standard procedures. Solvent removal was performed in vacuo using a Buchi rotatory evaporator at temperatures not greater than 60°C. Melting points were measured using the Mel-Temp II apparatus with the use of open capillaries and are uncorrected.
The progress of all reactions was monitored using thin layer chromatography (TLC) on aluminiumbacked sheets of Sigma Aldrich silica gel 60 F 254 plates. Compounds were visualized under UV light at 254 nm or in an iodine chamber. Chromatographic purification of compounds was carried out by medium pressure liquid chromatography using silica gel 60-400 mesh. The solvent mixtures used in specific chromatographic runs are indicated where necessary. High-resolution Fourier transform mass spectrometry electrospray ionization (FTMS-ESI) mass spectra were carried out on an LTQ Orbitrap XL mass spectrometer from Thermo Fisher Scientific (Bremen, Germany). A heated electrospray interface (H-ESI) was operated for ionization of the molecules at a spray voltage of 5 kV, whereas capillary voltage and tube lens voltages were adjusted to 20 and 100 V, respectively. The vaporizer temperature was set at 250°C and the ion transfer capillary temperature to 200°C. Measurements were carried out in the positive ion mode in a mass range of m/z 100-600 at a mass resolution of 60 000 at m/z 200. MS/MS experiments were performed using argon as collision gas in collision-induced dissociation (CID) mode, collision energies were measured at 15, 25 and 35 eV.
Nuclear magnetic resonance spectra were obtained using a Brucker Avance III spectrometer operating at 600 MHz (H 1 ) and 150 MHz ( 13 C). Spectra were recorded in deuterated solvents as indicated in brackets and referenced to residual solvent signals. Chemical shifts (δ) were measured in parts per million ( ppm). Hydrogen and carbon peak assignments were performed using gradient correlation spectroscopy (gCOSY), heteronuclear single quantum correlation (gHSQC) spectroscopy and heteronuclear multiple bond correlation (gHMBC) techniques. For most of the compounds, signals of protons attached to oxygen and nitrogen were not observed and were attributed to exchange with solvent protons. Signal multiplicities are reported as singlet (s), doublet (d), doublet of doublets (dd), doublet of triplets (dt), triplet (t), triplet of doublets (td) and multiplet (m). Coupling constants (J ) are reported in Hertz units.

Synthesis of 3-hydroxyphenethylamine (method A)
This compound was synthesized following the method of Ngo Hanna et al. [52] (scheme 2a). In a typical experiment, a mixture of 3-methoxyphenethylamine (1.0 g, 6.6 mmol), hydrobromic acid (10 ml) and acetic acid (10 ml) was stirred and heated under reflux for 6 h. The cooled mixture was concentrated under reduced pressure to obtain a residue, which was used in subsequent reactions without further purification; crude yield, 97%.

General method for the synthesis of substituted N-benzylisatins (8a-n) (method B)
This was done using the method of Vine et al. [48] with some modifications (scheme 1a). The desired isatin (1 equiv) was taken up in anhydrous acetonitrile or DMF (1 ml per 0.1 mmol of isatin). Solid K 2 CO 3 (1.2 equiv) was added in one portion, and the dark-coloured suspension was stirred at room temperature for 1 h. The appropriate benzyl halide (1.2 equiv) and KI (0.2 equiv) were added, and the reaction mixture was stirred at 80°C for 5-18 h, until the isatin starting material had been consumed as revealed by TLC. The reaction mixture was decanted into HCl (0.5 M, 50 ml) and extracted with methylene chloride (20 ml × 2), dried over anhydrous sodium sulfate and the solvent removed under reduced pressure. The crude product obtained was purified by flash chromatography using isocratic elution with hexane : ethyl acetate giving yellow to red crystals. Yields were between 80 and 95%.
royalsocietypublishing.org/journal/rsos R. Soc. open sci. 7: 191316 3.5. General method for the synthesis of 6 0 -hydroxy The compounds were synthesized by the phenolic Pictet-Spengler reaction reported by Kametani et al. [47] and modified by Ngo Hanna et al. [52] (scheme 2b). In a typical experiment, crude 3hydroxyphenethylamine (0.9 g, 6.6 mmol, 1 equiv), obtained as described in Method A above, and the appropriate isatin (1 equiv, 6.6 mmol) were dissolved in absolute ethanol (10 ml) and triethylamine (1 ml) was added. The reaction mixture was stirred and heated under reflux for 7-10 h. Upon completion, the reaction was concentrated under reduced pressure to remove the solvent. Distilled water was added to the viscous mass and the product which precipitated out was extracted into ethyl acetate (3 × 30 ml). The combined organic extracts were dried over anhydrous sodium sulfate and concentrated to minimum volume. The crude product was further purified by column chromatography using the appropriate solvent system. The final product obtained was recrystallized from absolute ethanol. In most cases, the reaction provided two regioisomers as earlier documented by Kametani et al. [47]: the 8 0 -hydroxy compound, which had a shorter retention time, and the 6 0hydroxy isomer. The combined yields ranged between 40 and 99%.

Synthesis of substituted β-nitrostyrenes (10a, 10b) (method F)
The method of Maresh et al. [46] was employed here with some modification (scheme 1c). A solution of substituted benzaldehyde (1 equiv), nitromethane (2 equiv) and anhydrous ammonium acetate (0.1 equiv) in acetic acid was refluxed for 6 h. The reaction mixture was then diluted slowly while stirring, with 200 ml water. During this time, a heavy yellow crystalline mass was formed. This was removed by filtration, washed with water and sucked as dry as possible, recrystallized from boiling methanol and air dried, yielding the β-nitrostyrene as bright yellow crystals in over 95% yield. The compound was used without further purification.

Synthesis of methoxyphenethylamines (11a, 11b) (method F)
This reaction carried out following the method of Maresh et al. [46] (scheme 1c). Typically, 1.0 mmol of nitrostyrene required 2 ml of methanol, 800 mg of zinc dust (12 mmol) and 2 ml of 37% HCl (24 mmol). Methanol was stirred vigorously in an ice bath maintained between 0 and 5°C (ice/NaCl). Conc. HCl, zinc dust and the appropriate nitrostyrene were slowly added over the course of 30 min in small portions while maintaining the temperature between 0 and 5°C. After complete addition of all starting materials, any solids on the side were washed into the solution with a small amount of methanol. The reaction mixture was further stirred for 1 h until the disappearance of the initial yellow colour of the nitrostyrene was observed. The reaction was further stirred for 4-6 h after the yellow colour had disappeared. Thereafter, the reaction mixture was placed in a 4°C refrigerator overnight. Upon completion of the reaction, the excess solid zinc was removed by filtration through the filter paper. The solution was made basic by dropwise addition of saturated sodium hydroxide in methanol, while maintaining the temperature below 5°C, until the pH was greater than 11 as indicated by the pH paper. The product was then extracted into dichloromethane and the solution was dried over anhydrous sodium sulfate. The insoluble zinc hydroxide which precipitates out during the addition of sodium hydroxide solution was extracted many more times with dichloromethane and filtered. The combined organic extracts were evaporated in vacuo to yield the corresponding phenethylamine as a yellow viscous oil in about 70% yield.