Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

Summary

Here, we describe a bioassay using 3-(4′,5′-dimethylthiazol-2′-yl)-2,5- diphenyltetrazolium bromide (MTT) to test previously synthesized spirocyclic oximes.

Abstract

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring systems is challenging. An efficient methodology to synthesize these compounds was recently published that described the solid phase synthesis in four steps rather than the previously reported five steps. The advantage of this shorter synthesis is the elimination of the use of toxic reagents. Low-loading Regenerating Michael (REM) linker-based resin was found to be crucial in the synthesis as high-loading versions prevented the addition of reagents containing bulky phenyl and aromatic side chains. The colorimetric 3-(4′,5′-dimethylthiazol-2′-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to examine the cytotoxicity of micromolar concentrations of these novel spirocyclic molecules in vitro. MTT is readily available commercially and produces relatively fast, reliable results, making this assay ideal for these spirocyclic heterocycles. Orthogonal ring structures as well as furfurylamine (a precursor in the synthesis method containing a similar 5-member ring motif) were tested.

Introduction

Small-molecule inhibition of the interaction of E3 ubiquitin-ligase mouse double minute 2 homolog (MDM2) with p53 is known to restore p53-mediated induction of tumor cell apoptosis^1,2,3. MDM2 is a negative regulator of the p53 pathway and is often overexpressed in cancer cells^4,5,6,7,8,9. Recent crystallographic and biochemical studies have revealed that small molecules containing a spirocyclic framework can effectively inhibit MDM2-p53 interactions¹⁰. The spirocyclic framework (Figure 1, shaded in blue) is considered a privileged motif as derivatization of this rigid orthogonal ring system has led to the discovery of novel therapeutic drugs. Accessing this interesting architecture poses a challenge when using traditional organic synthesis techniques. Although the therapeutic effects of spirocyclic molecules in biological systems have been investigated, synthesis of these molecules is still a cumbersome process. Unwanted side products, using harsh conditions, and hazardous transition metals are often problematic.

The potential use of the spirocyclic motif in drug development led to the development of a protocol utilizing solid-phase synthesis to generate a library of molecules with the motif in addition to other interchangeable functional groups^11,12. The separation of products and reactants between steps could be achieved by simply utilizing an REM linker attached to a resin bead and a solid-phase filter vessel. This would cut down steps and potentially increase yields. This synthetic approach could produce a large array of potential drug candidates. However, the effectiveness of these molecules in a biological system would require further investigation.

To determine the cytotoxicity of these spirocyclic compounds, the MTT assay^13,14 was employed. This method measures cell viability and can be used to indirectly determine cell cytotoxicity. Different concentrations of the inhibitors were added to cultured cells in a 96-well plate, and the proportion of living cells was measured by colorimetric analysis of the extent of reduction of yellow MTT by mitochondrial dehydrogenases to the purple formazan compound (Figure 2). The activity is most often reported as an IC₅₀ value-the concentration at which cell growth is inhibited by 50% relative to an untreated control. This paper describes the protocol for the MTT assay and the preliminary results of these novel spirocyclic molecules.

Protocol

NOTE: Several chemicals and biological reagents used in this protocol are toxic and carcinogenic. Consult relevant material safety data sheets (MSDS) prior to use. Use appropriate personal protective gears (Occupational Safety and Health Administration-approved safety goggles, proper gloves, lab coats, full-length pants, and closed-toe shoes) prior to starting the experiment. In addition, adopt appropriate safety practices when performing synthesis and handling toxic chemicals and reagents (fume hood).

1. Solid phase synthesis of spirocyclic heterocycles 6 and 7

NOTE: Synthesis was based on previously published work^11,12. The updated protocol reveals that the tetrabutylammonium fluoride-catalyzed ring opening of the tricyclic heterocycle was not needed, and thus its elimination shortens the synthetic procedure.

1. Perform Michael addition of furfurylamine to the REM linker (duration: 25 min setup + 24 h reaction time).
   1. Add 1 g (1 equivalents [equiv.]) of REM resin, 20 mL (20 equiv.) of dimethylformamide (DMF), and 2.4 mL of furfurylamine to a 25 mL solid-phase reaction vessel. Agitate the reaction vessel at room temperature for 24 h following the reaction initiation.
      NOTE: Ensure thorough mixing so that the resin does not sit at the bottom of the vessel.
   2. Wash the resin with DMF 1x after the reaction is complete. Then, wash 4x, alternating between dichloromethane (DCM) and methanol. Dry the resin thoroughly in the reaction vessel following washes.
2. Perform tandem Michael addition/1,3-dipolar cycloaddition (duration: 25 min setup + 48 h reaction time).
   1. To the dry resin, add 1.48 mL (5 equiv.) of triethylamine (TEA), 0.637 g (2 equiv.) of nitro-olefin, and 10 mL of dry toluene to the reaction vessel.
   2. Then, add 1.085 mL (4 equiv.) of trimethylsilyl chloride (TMSCl) to the reaction vessel in a well-ventilated fume hood.
      NOTE: As this reaction produces HCl gas, do not cap the reaction vessel until the gas has been released under a fume hood.
   3. Securely cap the reaction vessel, and agitate at room temperature for 48 h.
      NOTE: Ensure thorough mixing of the resin with the reagents.
   4. Use 5 mL of methanol to quench the reaction.
   5. Drain the vessel to remove the solution, and then wash 4x, alternating between DCM and methanol. Dry the resin thoroughly in the reaction vessel following washes.
3. Perform N-alkylation of the resin-bound heterocycle to form the quaternary amine (duration: 10 min setup + 24 h reaction time).
   1. To the dry resin in the reaction vessel, add 5 mL of DMF and 10 equiv. of alkyl halide, and agitate at room temperature for 24 h.
      NOTE: Ensure thorough mixing of the reagents with the resin.
   2. Wash the resin with DMF 1x after the reaction is complete. Then, use DCM and methanol alternately to wash 4x. Dry the resin in the reaction vessel following washes.
4. Perform β-elimination of the quaternary amine for cleavage from the polymer support (duration: 15 min setup + 24 h reaction time).
   1. To the dry resin in the reaction vessel, add 3 mL of DCM and 1.49 mL (5 equiv.) of TEA to cleave the heterocycle from the polymer support.
   2. Agitate the reaction mixture for 24 h to ensure thorough mixing of the resin with the solution. Wash 4x, alternating between DCM and methanol. Collect the elution from all the washes, and concentrate via rotatory evaporation.
   3. Triturate with methanol to purify the spirocyclic oxime. Dry the resin thoroughly in the reaction vessel following washes for reuse in future experiments.

2. Cytotoxicity assay using MTT ¹⁴

1. Prepare 20 mL of a 5 mg/mL MTT solution using sterile phosphate-buffered saline (PBS, 0.9% NaCl in water) as the diluent. Filter and store at -20 °C. Then, prepare a 1:1 dilution of the MTT solution from step 2.1 in serum-free cell culture medium (DMEM).
2. Prepare 1 mL each of stock solutions in 1.5 mL microcentrifuge tubes of 100 mM, 10 mM, 1 mM, 100 µM, 10 µM, 1 µM, 0.1 µM, and 0.01 µM of test compounds in dimethyl sulfoxide (DMSO). Store at -20 °C. Prepare 200 µL per dose of the working solutions of test compounds by diluting stock concentrations 1:1000 in serum-free medium in 1.5 mL tubes.
3. In the tissue culture hood, seed COS-7 cells (African green monkey kidney cells, Cercopithecus aethiops kidney) in complete medium [DMEM with 10% fetal bovine serum (FBS)] onto flat-bottom, tissue-culture-treated 96-well plates at a concentration of 4 × 10³ cells/200 µL per well using a multi-channel pipettor. COS-7 cells were chosen because (1) these are commonly used cells for cytotoxicity assays and (2) these were already available in the institution.
4. Incubate COS-7 cells for 24 h at 37 °C in an atmosphere containing 5% CO₂.
5. Aspirate the supernatant from the wells using a glass Pasteur pipette attached to a vacuum pump. Dose the cells in triplicate with the test compounds using the working solutions prepared in step 2.2 (See Table 1). Incubate cells as described in step 2.4.
6. Aspirate the supernatant from the wells. Add 200 µL of MTT solution to each well. Incubate at 37 °C in an atmosphere containing 5% CO₂ for 4 h.
7. Gently aspirate the supernatant from the wells without disturbing the purple formazan crystals. Add 200 µL of DMSO to each well to dissolve the purple formazan crystals. Incubate at room temperature for 15 min.
8. Measure absorbance at 590 nm¹⁴ or 600 nm for each well using a 96-well plate reader. Use wells with no cells as background and average the absorbance value. Subtract the averaged absorbance background value from the absorbance value of each treated well. Normalize the data as a percentage of the average zero dosage value (average the three zero-dose values). Plot data on the y-axis: linear (% relative cell viability); x-axis: log (concentration). Plot each series as an individual curve (e.g., triplicate data should have 3 curves)

Results

Spirocyclic oximes 6 and 7 were synthesized using a modified protocol (Figure 1). Michael addition of furfurylamine to an REM linker 1b afforded polymer-bound resin 2. The progress of the reaction was monitored by infrared (IR) spectroscopy by detecting the disappearance of the α,β-unsaturated ester at 1722 cm^-1 (Figure 3). Spirocyclic-bound resin 4 was formed from 2 via a transient intermediate 3. Methanolic hydrolysis of 4 produced 3-[(3E)-...

Discussion

The synthesis of the spirocyclic compounds was based on previous research conducted by this laboratory, but with some modifications (Figure 1)^11,12. The progress of each reaction step was monitored by IR spectroscopy. Michael addition of the REM linker 1 with furfurylamine afforded polymer-bound 2 (IR 1722 cm^-1 → 1731 cm^-...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CELLS
COS-7 cells (ATCC CRL-1651)	ATCC	CRL-1651	African green monkey kidney cells
CHEMICALS
1-Bromooctane	Sigma-Aldrich	152951	Alkyl-halide
Allylbromide	Sigma-Aldrich	337528	Alkyl-halide
Benzylbromide	Sigma-Aldrich	B17905	Alkyl-halide
Cisplatin	Cayman Chemical	13119	Cytotoxicity control
Dichloromethane (DCM)	Sigma-Aldrich	270997	Solvent
Dimethylformamide (DMF)	Sigma-Aldrich	227056	Solvent
Dimethylsulfoxide (DMSO)	Sigma-Aldrich	276855	Solvent
DMEM, high glucose, with L-glutamine	Genesee Scientific	25-500	Cell culture media
FBS (Fetal bovine serum)	Sigma-Aldrich	F4135	Cell culture media
Furfurylamine	Acros Organics	119800050	reagent
Iodomethane	Sigma-Aldrich	289566	Alkyl-halide
Methanol	Sigma-Aldrich	34860	Solvent
MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)	EMD Millipore	Calbiochem 475989-1GM	Reagent
Phosphate-buffered Saline (PBS)	Genesee Scientific	25-507	Cell culture media
REM Resin	Nova Biochem	8551010005	Polymer support; 0.500 mmol/g loading
trans-β-nitrostyrene	Sigma-Aldrich	N26806	Nitro-olefin reagent
Toluene	Sigma-Aldrich	244511	Solvent
Triethylamine (TEA)	Sigma-Aldrich	T0886	Reagent for beta-elimination
Trimethylsilyl chloride (TMSCl)	Sigma-Aldrich	386529	Reagent; CAUTION - highly volatile; creates HCl gas
GLASSWARE/INSTRUMENTATION
25 mL solid-phase reaction vessel	Chemglass	CG-1861-02	Glassware with filter
96 Well plate reader	Promega (Turner Biosystems)	9310-011	Instrument
AVANCE III NMR Spectrometer	Bruker	N/A	Instrument; 300 MHz; Solvents: CDCl3 and CD3OH
Thermo Scientific Nicole iS5	Thermo Scientific	IQLAADGAAGFAHDMAZA	Instrument
Wrist-Action Shaker	Burrell Scientific	757950819	Instrument