Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

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

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.

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.

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)

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)-(2S, 4R)-2-phenyl-3-hydroxyimino 4-hydroxymethyl-pyrrolidin-1-yl]-propionic acid methyl ester 7, while alkylation followed by β-elimination afforded (3E)-(2S, 4R)-4-hydroxymethyl-1-methyl-2-phenyl-3-pyrrolidine oxime 6. The identity of the spirocyclic oximes was determined by ¹H and ¹³C nuclear magnetic resonance spectroscopic analysis and the purity by mass spectroscopy based on our previous results¹¹.

The MTT assay is a well-known colorimetric assay for determining cell viability¹². As seen in Figure 2, mitochondrial reductases present in living cells convert the yellow tetrazolium of MTT to an insoluble purple formazan solid. Using a spectrophotometer, the formazan formation is quantified by measuring the absorbance at 600 nm. Cisplatin, which is known to induce cell death at high concentrations, was used as a positive control (Figure 4). As expected, the higher the concentration of cisplatin, the lower the cell viability. Next, the MTT assay was used to test the spirocyclic compounds 6 and 7 and furfurylamine. Furfurylamine was used to determine the effect of the furan ring alone compared to the spirocyclic framework. As depicted in Figure 5, furfurylamine and spirocyclic oxime 6 showed similar cytotoxicity. However, the toxicity of spirocyclic compound 7 was noticeably greater than that of furfurylamine and 6. A library of spirocyclic oximes will be synthesized to fully investigate the cytotoxicity as well as the other anticancer effects of these heterocycles.

Figure 1: Construction of spirocyclic compounds using an updated solid phase synthesis. The orthogonal spirocycic framework is shaded in blue. Note that step (c) is not needed, which avoids using the toxic reagent TBAF. The reaction conditions are as follows: (a) furfurylamine, DMF, (b) β-nitrostyrene, TMSCl, TEA, toluene, (c) TBAF, (d) alkyl halide, DMF, and (e) TEA, DCM. Abbreviations: TBAF = tetrabutylammonium fluoride; DMF = dimethylformamide; TMSCl = trimethylsilyl chloride; TEA = triethylamine; DCM = dichloromethane; ISOC = intramolecular silyoxy olefin cycloaddition. Please click here to view a larger version of this figure.

Figure 2: Mechanism of the MTT assay. Visibly yellow tetrazolium salt of MTT is reduced by mitochondrial reductases in living COS-7 cells to form purple insoluble formazan. Abbreviation: MTT = 3-(4′,5′-dimethylthiazol-2′-yl)-2,5- diphenyltetrazolium bromide. Please click here to view a larger version of this figure.

Figure 3: Monitoring the progress of each solid phase reaction step by infrared spectroscopy. The stretching frequency at 1717 cm^-1 indicated the presence of an unsaturated ester, 1733 cm^-1 depicted a saturated ester, and signal around 3300-3500 cm^-1 indicated the presence of a hydroxyl group. Detectable stretching frequencies for polystyrene are also shown. Abbreviations: REM = Regenerating Michael; ISOC = intramolecular silyoxy olefin cycloaddition. Please click here to view a larger version of this figure.

Figure 4: Effects of cisplatin on COS-7 cell viability in a modified MTT assay. Concentrations of cisplatin ranged from 0 µM to 60 µM. Please click here to view a larger version of this figure.

Figure 5: Effects of test compounds on COS-7 cell viability in a modified MTT assay. Concentrations ranged from 0 µM to 100 µM and were plotted on a log scale. Please click here to view a larger version of this figure.

Table 1: Layout of the 96-well plate. All test data rows were in triplicate. Wells containing only COS-7 cells and medium were used as controls. To ensure that DMSO was not the cause of cytotoxicity in the cisplatin-dosed cells, wells containing only DMSO were used as solvent controls. Wells containing COS-7 cells are highlighted. Abbreviations: DMSO = dimethylsulfoxide; PBS = phosphate-buffered saline. Please click here to view a larger version of this figure.

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^-1). From the previous report, ISOC of 2 produced the tricyclic heterocyclic compound 3, as confirmed by the detection of the TMS group (IR 1214 cm^-1). This is a critical step of the synthesis as ISOC provided the necessary regio- and stereoselectivity of the products. A hydroxyl group stretching frequency of 3500 cm^-1 was observed instead of the frequency of the TMS functional group. This may be because the tricyclic compound is a transient intermediate that leads to the spirocyclic system.

Different types of REM resin were found to limit the synthesis. High-loading polymer (1.00 mmol/g) prevented the synthesis of spirocyclic compounds containing bulky R₂ side chains. Due to the similarities in the functional groups in resins 4 and 5, the results of IR were inconclusive. The success of this step could only be determined by attempting to regenerate the REM linker (5 → 1). Regeneration did not occur in instances when a bulky R₂ group was added. Low-loading resins (0.5 mmol/g or lower) are recommended for successful synthesis. This synthesis method is consistent with procedures described in the literature.

As a preliminary test, a protocol was developed for a cytotoxicity assay using MTT. Over the course of several trials, critical steps and limitations were discovered. For the results to be normalized across all wells, cells had to be evenly seeded across wells, necessitating the measurement of the cell concentration prior to seeding. The assay required plates with flat-bottomed wells, as the absorbance could not be accurately read from round-bottomed wells. Additionally, excess MTT that remained after incubation had to be removed to prevent interference in the readings without disturbing the insoluble formazan.

The absorbance of the dissolved formazan should be read at 590 nm. However, current instrumentation in the lab necessitated taking readings at 600 nm instead. Storage at 0 °C was found to be important for the chemicals used in the assay (cisplatin, spirocyclic molecules, furfurylamine). DMSO-a chemical with known cytotoxicity-was used as the solvent for the test compounds and was used to make dilutions for the assay. The MTT reagent itself had to be prepared, as it was stored as a powder that needed to be dissolved and filtered, as insoluble particles interfered with readings.

Overall, the results for this assay are intended to be preliminary, as only a small number of molecules were tested. An exhaustive test with a battery of molecules is planned, and a full manuscript will be forthcoming. In addition, the synthesis might be applicable for amines derived from pyrrole-2-carbaldehyde. In this case, spirocyclic pyrrolidines can be synthesized and tested for cytotoxic effects on cancer cell lines.

The authors have nothing to disclose.

This work was funded by a grant from the Faculty Research Council to K.S.H. (Office of Research and Grants, Azusa Pacific University-USA). A.N.G. and J.F.M. are recipients of the Scholarly Undergraduate Research Experience (SURE) Fellowship. S.K.M. and B.M.R. are recipients of the STEM Research Fellowship Grants (Center for Research in Science, Azusa Pacific University-USA). We are grateful to Dr. Matthew Berezuk and Dr. Philip Cox for guidance on the bioassays.