Spiro[pyrrolidine-3, 3´-oxindole] as potent anti-breast cancer compounds: Their design, synthesis, biological evaluation and cellular target identification

The spiro[pyrrolidine-3, 3´-oxindole] moiety is present as a core in number of alkaloids with substantial biological activities. Here in we report design and synthesis of a library of compounds bearing spiro[pyrrolidine-3, 3´-oxindole] motifs that demonstrated exceptional inhibitory activity against the proliferation of MCF-7 breast cancer cells. The synthesis involved a one pot Pictet Spengler-Oxidative ring contraction of tryptamine to the desired scaffolds and occurred in 1:1 THF and water with catalytic trifluoroacetic acid and stoichiometric N-bromosuccinimide as an oxidant. Phenotypic profiling indicated that these molecules induce apoptotic cell death in MCF-7 cells. Target deconvolution with most potent compound 5l from the library, using chemical proteomics indicated histone deacetylase 2 (HDAC2) and prohibitin 2 as the potential cellular binding partners. Molecular docking of 5l with HDAC2 provided insights pertinent to putative binding interactions.


Chemistry
All reactions were carried out in flame-dried tubes with magnetic stirring. Unless otherwise noted, all experiments were performed under argon atmosphere. All reagents were purchased from Sigma Aldrich, Acros or Alfa Aesar. Solvents were treated with 4 Å molecular sieves or sodium and distilled prior to use. Purifications of reaction products were carried out by column chromatography using Chem Lab silica gel (230-400 mesh). 1 H NMR and 13 C NMR spectra were recorded with tetramethylsilane (TMS) as internal standard at ambient temperature unless otherwise indicated Bruker 500 MHz for 1 H NMR and 120 MHz for 13 C NMR. Chemical shifts are reported in parts per million (ppm) and coupling constants are reported as Hertz (Hz).
Splitting patterns are designated as singlet (s), broad singlet (bs), doublet (d), triplet (t). Splitting patterns that could not be interpreted or easily visualized are designated as multiple (m). The Mass Spectrometry analysis was done on the 6540 UHD Accurate-Mass Q-TOF LC/MS system (Agilent Technologies) equipped with Agilent 1290 LC system obtained by the Dept. of Chemistry, School of Natural Sciences, Shiv Nadar University, Uttar Pradesh 203207, India.
General procedure for the synthesis of 3, 3´-spiropyrrolooxoindole To a solution of the tryptamine (1.0 eq.) in 1:1 THF/water (20 mL), was added appropriate benzaldehydes (1 eq.), catalytic trifluoroacetic acid (TFA)and the reaction mixture was cooled to 0°C, followed by portion wise addition of N-bromosuccinimide (NBS) (1.1 eq.).The resulting solution was stirred at 0°C for 6h after which it was gradually warmed to room temperature (rt).
Once TLC confirms the total consumption of the starting imine, the reaction was quenched with saturated sodium carbonate solution. The aqueous layer was extracted twice with dichloromethane. The organic extracts were combined and washed with brine, dried over anhydrous magnesium sulphate and was evaporated to provide the crude compound. It was purified by flash column chromatography using a mixture of ethyl acetate and hexane as eluent to afford the desired 3, 3´-spiropyrroloxoindole 5a-m.

Shantani
Background and Overall Goal Step-1) Immobilization of 'Bis-III' on Shantani's proprietary polymer matrix Step-2) Capture and Identification of Targets This note describes the application of the technology in identifying/de-convoluting true positive targets of a non-derivatized 'test' molecule BIS-III.
Bisindolylmaleimide-III (Bis-III) a known inhibitor of GSK3 protein and it induces apoptosis in the cancerous cell-lines. At 1μM, Bis-III inhibits 93% of PKCα kinase activity and also inhibits many other protein kinases including, S6K1, MAPKAP-K1, RSK2 and MSK1 with similar potency. Additionally, it inhibits PDK1, an important kinase in the insulin signaling pathway.
In following experiments Shantani's technology was utilized to identify primary and secondary targets of BIS-III.
1. Based on BIS-III compatibility with the polymer matrix; a 10 ml soluble stock solution of 0.3 mM BIS-III was prepared in HPLC water.
2. The molecule was quoted on the matrix in small amount (1 ml) and allowed to dry at RT. The coating and drying of membrane was carried out till the complete 10 ml solution of Bis-III was coated on the membrane.
The molecule coated matrix was incubated with cell lysate for 2 minute. Excess lysate was removed and proteins were eluted with 2 ml elution buffer (1 mM Bis-III in TBST). Proteins were acetone precipitated, extracted and measured. Two similar experiments were performed -one for western blot analysis and another for Mass spec analysis.
Protein concentration from both control and test experiment was normalized and probed for GSK3-beta protein using western blot method.