Synthesis and Anti-Cancer Evaluation of Spiro-indolinone Derivatives

A series of spiro-indolin-2-one derivatives were designed and synthesized as p53-MDM2 binding inhibitors. Though p53-MDM2 binding inhibitory and activities against p53 wild-type cell lines of most compounds were not that promising, some obtained structures showed moderate to strong inhibitory activities (IC50<0.08 μM) against p53 mutant cell lines (SW620), suggesting that these compounds may have different modes of action to p53 pathway, further studies on treatment of p53 mutant tumors are under investigation.cc


Introduction
The spiro-indolin-2-one compounds were synthesized by a series of compounds [1][2][3] with good p53-MDM2 binding inhibitory activity found in the structure-based design strategy of Wang and his group [4]. From the result of p53-MDM2 complex crystal structure [5], tryptophan residues on the indole ring of p53 is the most critical binding element for p53 binding MDM2 protein, which was buried in the hydrophobic pocket, and indole on the NH and MDM2 formed a hydrogen bond. Wang and his team members used computer-assisted drug screening to find compounds that mimic the indole ring and found that the structural properties of 2-indolinone were most consistent with that of indole. 6-chloro-2-indolinone was identified as a predominant fragment on the basis of previous work on peptide compounds [6,7] and Nutlin (Trp pocket with key chlorine atoms capable of occupying MDM2 protein) [8].
The Spiro cyclic backbone provides the necessary platform for introducing side chains so that the hydrophobic side chains can be inserted into Leu and Phe pockets. The crystal structures of the resulting compounds MI-63 and MDM2 were recently reported by Popowicz et al. [8]. Interestingly, the MI-63 binding pattern is mirrored as previously assumed [9][10][11]. As previously assumed, 6-chloro-2indolinone occupies Trp pockets but the combination of neopentyl and substituted phenyl Mode and the previous hypothesis were reversed, suggesting that the conformation of the compound has been reversed, but the two states of MDM2 have a better inhibition. Based on the above studies, we designed a class of spiro-indolin-2-one compounds, in order to improve the class of compounds on MDM2 binding activity, and have a better anti-cancer effect ( Figure 1). First, the hydrophobic groups such as methyl group, propionyl group and piperidine carboxyl group were introduced into pyrrole N to study the influence of different length or volume of hydrophobic groups on the activity. Then the effect of chlorine atom on the activity of 2 -indolinone was investigated. The effect of chlorine atom on the activity of benzene ring at the 4' position was investigated. And last the nitrogen-containing molecular fragments were introduced into the 4 'position of the pyrrole to enhance the watersolubility of the compounds, and the effects on the activity were also investigated.

Chemistry
The synthesis route of the spiro-indolin-2-one compounds was designed via systematic literature (Scheme 1).
A total of 27 spiro-indolin-2-one compounds were synthesized by the method described above. The structures of the new compounds were confirmed by 1 H NMR MS and elemental analysis.

Experiments Biology
Protein inhibitory test assay: Measurements were performed using an EnVision Multilabel Plate Reader with a 480 nM excitation filter and a 535 nM emission filter. The fluorescence polarization value FP was measured by incubating the test compound (DMSO <4%) at a concentration of 10 μM with the protein (final concentration of 10 nM) -fluorescence substrate (final concentration 1 nM) for 1 h. Simultaneously, DMSO (<4%) with the same dosing volume was incubated with the protein (final concentration of 10 nM) solution and the fluorescent substrate (final concentration of 1 nM), respectively, to test FPmax and FPmin. The inhibition of p53-MDM2 binding at the concentration of 10 µM was calculated according to the following formula:

Inhibition%=1-(FP-FP min )/(FP max -FP min )
Cell viability assay (SRB assay): 100 μL of cell suspension (A549, HCT116, SW620, and PC3 cells) was added to each well in a 96-well cell culture plate. The plate was incubated in a CO 2 incubator for 24 1 hour. Drain the fixative, wash with deionized water 5 times per well, dry and air dry. 100 μL SRB solution was added to each well. After 10 minutes at room temperature, SRB bound to protein was washed 5 times with 1% acetic acid and air-dried. The bound SRB was lysed with 150 μL of 10 mmol/L unbuffered Tris alkaline solution (pH 10.5).
The cell inhibition rate was calculated by the following formula: Inhibitory%=(A515 control cells −A515 treated cells )/A515 control cells × 100% (A515: OD under wavelength of 515 nm) hours. After the culture plate was removed, a complete culture solution of the same concentration of each sample was added to each well. Three parallel wells were set up at each concentration, and A cells were added to the normal control wells (C) without drug-containing complete culture. After that, the suction hole in the culture medium, deionized water washed 5 times. The plates were shaken on a microplate shaker and incubated in a CO 2 incubator for 72 hours. The plate was removed and 50 μL of 50% trichloroacetic acid (TCA) was added to each well. After standing for 5 minutes, the plate was allowed to stand at 4°C for The nitrogen-containing molecular fragments were introduced to improve the water-solubility of the compounds, and the effects on the activity were also investigated.

Synthesis of 6-chloro-1-(4-methoxybenzyl)indoline-2,3dione (5)
6-Chloroindole (10.0 g, 55 mmol) was dissolved in DMSO (20 mL) in an ice bath, NaOH (2.7 g, 66 mmol) was added, after stirring under ice bathe for 30 min, 4-methoxybenzylchloride (9.0 mL, 66 mmol) was added, after 3 h in room temperature stirring, water was added to quench the reaction. The obtained red solid was recrystallized with ethyl estate and petroleum ether, and get a red solid 6-chloro-1-(4-methoxybenzyl) indoline; The obtained solid (5.0 g, 16 mmol) was dissolved in a mixture of acetone/Acetic acid (3: 1, 13 mL) in an ice bath, then a solution of CrO 3 (4.6 g, 46 mmol) in water (15 mL) was added slowly, and after stirring for 30 min, the reaction mixture was diluted with water and extracted with ethyl estate, the obtained reddish oil was separated with column chromatography to get a poppy solid 5

General method for the synthesis of compounds 26-27 or 29-30
Compounds were obtained by two steps. First, the obtained 11-13 (0.47 mmol) were dissolved in methylene chloride (30 mL), and triethylamine (0.4 mL) was added, while stirring, a solution of bis(trichloromethyl)carbonate (0.63 mmol) in dry methylene chloride (10 mL) was added in a period of 15 min. The reaction mixture was stirred at 0°C for 30 min and solvent was removed to dryness. The residue was dissolved in dry methylene chloride (10 mL), and a solution of correspondingly amines (2.88 mmol) in methylene chloride (10 mL) was added. The reaction was stirred for 20 min till it was done. The solvent was removed, extracted with methylene chloride and water, keep the organic, and washed with brine (2 × 20 mL), dried with sodium sulfate. The solvent was removed to dryness and the obtained solid was crystallized with ethyl estate and petroleum ether to get a white solid 26-27 or 29-30. -1-ylcarbonyl)-2,3,3a,9b- ,4.91;N,9.30;Found: C,63.89;H,4.99;N,9. ,4.91;N,9.30;Found: C,63.81;H,4.89;N,9.27. General method for the synthesis of compounds 31-43 25-30 (0.16 mmol) was dissolved in THF (6 mL), corresponding amine (0.48 mmol) was added, after refluxing for min, the solvent was removed by rotary evaporation and the residue was extracted by ethyl estate and water, the organic layer was kept and washed with brine (3×10 mL), dried with sodium sulfate, then removed the solvent, the obtained solid was recrystallized in ethyl estate/petroleum ether and get a white solid 31-43.