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.

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).
<p class="jove...

<ol>
	<li>Shangary, S., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+inhibitors+of+the+MDM2-p53+protein-protein+interaction+to+reactivate+p53+function:+a+novel+approach+for+cancer+therapy.">Small-molecule inhibitors of the MDM2-p53 protein-protein interaction to reactivate p53 function: a novel approach for cancer therapy.</a> Annual Review of Pharmacology and Toxicology. 49, 223-241 (2009).</li><li>Zhao, Y., Aguilar, A., Bernard, D., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+inhibitors+of+the+MDM2-p53+protein-protein+interaction+(MDM2+inhibitors)+in+clinical+trials+for+cancer+treatment.">Small-molecule inhibitors of the MDM2-p53 protein-protein interaction (MDM2 inhibitors) in clinical trials for cancer treatment.</a> Journal of Medicinal Chemistry. 58 (3), 1038-1052 (2015).</li><li>Paolo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+effective+virtual+screening+protocol+to+identify+promising+p53-MDM2+inhibitors.">An effective virtual screening protocol to identify promising p53-MDM2 inhibitors.</a> Journal of Chemical Information and Modeling. 56 (6), 1216-1227 (2016).</li><li>Shieh, S. Y., Ikeda, M., Taya, Y., Prives, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage-induced+phosphorylation+of+p53+alleviates+inhibition+by+MDM2.">DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2.</a> Cell. 91 (3), 325-334 (1997).</li><li>Hwang, B. J., Ford, J. M., Hanawalt, P. C., Chu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+p48+xeroderma+pigmentosum+gene+is+p53+dependent+and+is+involved+in+global+genomic+repair.">Expression of the p48 xeroderma pigmentosum gene is p53 dependent and is involved in global genomic repair.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (2), 424-428 (1999).</li><li>Oliner, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncoprotein+MDM2+conceals+the+activation+domain+of+tumor+suppressor+p53.">Oncoprotein MDM2 conceals the activation domain of tumor suppressor p53.</a> Nature. 362, 857-860 (1993).</li><li>Nag, S., Qin, J., Srivenugopal, K. S., Wang, M., Zhang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MDM2-p53+pathway+revisited.">The MDM2-p53 pathway revisited.</a> Journal of Biomedical Research. 27 (4), 254-271 (2013).</li><li>Bond, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+nucleotide+polymorphism+in+the+MDM2+promoter+attenuates+the+p53+tumor+suppressor+pathway+and+accelerates+tumor+formation+in+humans.">A single nucleotide polymorphism in the MDM2 promoter attenuates the p53 tumor suppressor pathway and accelerates tumor formation in humans.</a> Cell. 119 (5), 591-602 (2004).</li><li>Isobe, M., Emanuel, B. S., Givol, D., Oren, M., Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+gene+for+human+p53+tumor+antigen+to+band+17p13.">Localization of gene for human p53 tumor antigen to band 17p13.</a> Nature. 320 (6057), 84-85 (1986).</li><li>Gupta, A. K., Bharadwaj, M., Kumar, A., Mehrotra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiro-oxindoles+as+a+promising+class+of+small+molecules+inhibitors+of+p53-MDM2+interaction+useful+in+targeted+cancer+therapy.">Spiro-oxindoles as a promising class of small molecules inhibitors of p53-MDM2 interaction useful in targeted cancer therapy.</a> Topics in Current Chemistry. 375 (1), 1-25 (2017).</li><li>Griffin, S. A., Drisko, C. R., Huang, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricyclic+heterocycles+as+precursors+to+functionalized+spirocyclic+oximes.">Tricyclic heterocycles as precursors to functionalized spirocyclic oximes.</a> Tetrahedron Letters. 58, 4551-4553 (2017).</li><li>Drisko, C. R., Griffin, S. A., Huang, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-phase+synthesis+of+[4.4]spirocyclic+oximes.">Solid-phase synthesis of [4.4]spirocyclic oximes.</a> Journal of Visualized Experiments. (144), e58508 (2019).</li><li>Lawrence, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linked+parallel+synthesis+and+MTT+bioassay+screening+of+substituted+chalcones.">Linked parallel synthesis and MTT bioassay screening of substituted chalcones.</a> Journal of Combinatorial Chemistry. 3 (5), 421-426 (2001).</li><li>. MTT assay protocol Available from: <a target="_blank" href="https://www.abcam.com/kits/mtt-assay-protocol">https://www.abcam.com/kits/mtt-assay-protocol</a> (2020)</li></ol>

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&#160;1&#160;with furfurylamine afforded polymer-bound&#160;2&#160;(IR 1722 cm-1&#160;&#8594; 1731 cm-...

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 apoptosis1,2,3. MDM2 is a negative regulator of the p53 pathway and is often overexpressed in cancer cells4,5,6,7,8,9. 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 groups11,12. 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 assay13,14 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CELLS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>COS-7 cells (ATCC CRL-1651)</td>
			<td>ATCC</td>
			<td>CRL-1651</td>
			<td>African green monkey kidney cells</td>
		</tr>
		<tr>
			<td>CHEMICALS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-Bromooctane</td>
			<td>Sigma-Aldrich</td>
			<td>152951</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Allylbromide</td>
			<td>Sigma-Aldrich</td>
			<td>337528</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Benzylbromide</td>
			<td>Sigma-Aldrich</td>
			<td>B17905</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Cisplatin</td>
			<td>Cayman Chemical</td>
			<td>13119</td>
			<td>Cytotoxicity control</td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Dimethylformamide (DMF)</td>
			<td>Sigma-Aldrich</td>
			<td>227056</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, with L-glutamine</td>
			<td>Genesee Scientific</td>
			<td>25-500</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>FBS (Fetal bovine serum)</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>Furfurylamine</td>
			<td>Acros Organics</td>
			<td>119800050</td>
			<td>reagent&#160;</td>
		</tr>
		<tr>
			<td>Iodomethane</td>
			<td>Sigma-Aldrich</td>
			<td>289566</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)</td>
			<td>EMD Millipore</td>
			<td>Calbiochem 475989-1GM</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline (PBS)</td>
			<td>Genesee Scientific</td>
			<td>25-507</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>REM Resin</td>
			<td>Nova Biochem</td>
			<td>8551010005</td>
			<td>Polymer support; 0.500 mmol/g loading</td>
		</tr>
		<tr>
			<td>trans-&#946;-nitrostyrene</td>
			<td>Sigma-Aldrich</td>
			<td>N26806</td>
			<td>Nitro-olefin reagent</td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Sigma-Aldrich</td>
			<td>244511</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Triethylamine (TEA)</td>
			<td>Sigma-Aldrich</td>
			<td>T0886</td>
			<td>Reagent for beta-elimination</td>
		</tr>
		<tr>
			<td>Trimethylsilyl chloride (TMSCl)</td>
			<td>Sigma-Aldrich</td>
			<td>386529</td>
			<td>Reagent; CAUTION - highly volatile; creates HCl gas</td>
		</tr>
		<tr>
			<td>GLASSWARE/INSTRUMENTATION</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL solid-phase reaction vessel</td>
			<td>Chemglass</td>
			<td>CG-1861-02</td>
			<td>Glassware with filter</td>
		</tr>
		<tr>
			<td>96 Well plate reader</td>
			<td>Promega (Turner Biosystems)</td>
			<td>9310-011</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>AVANCE III NMR Spectrometer</td>
			<td>Bruker</td>
			<td>N/A</td>
			<td>Instrument; 300 MHz; Solvents: CDCl3 and CD3OH</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nicole iS5</td>
			<td>Thermo Scientific</td>
			<td>IQLAADGAAGFAHDMAZA</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Wrist-Action Shaker</td>
			<td>Burrell Scientific</td>
			<td>757950819</td>
			<td>Instrument</td>
		</tr>
	</tbody>
</table>

functionalized spirocyclic heterocycle synthesis and cytotoxicity assay

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&#8242;,5&#8242;-dimethylthiazol-2&#8242;-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.

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 &#945;,&#946;-unsaturated ester at 1722 cm-1&#160;(Figure 3). Spirocyclic-bound resin 4 was formed from 2 via a transient intermediate 3. Methanolic hydrolysis of 4 produced 3-...

Watch this Scientific Journal Video about Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay at JoVE.com

Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

Here, we describe a bioassay using 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-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 ...

This protocol utilizes a recyclable and modified method in producing spirocyclic compounds. The MTT assay helps answer the question pertaining to the cytotoxicity of these novel molecules. This technique producers novel modifiable molecules using a regenerating starting material.The MTT assay provides relatively quick, and easy to analyze biological results Begin by performing solid phase synthesis of spirocyclic hetero cycles six and seven, as previously demonstrated. Prepare 20 milliliters of a five milligrams per milliliter MTT solution using sterile PBS as the diluent. Filter and store the solution at minus 20 degrees Celsius.Then, prepare a one-to-one dilution of the MTT solution in DMEM. Prepare one milliliter each of stock solutions in 1.5 milliliter micro centrifuge tubes of test compounds in DMSO. Store them at minus 20 degrees Celsius.Prepare 200 microliters per dose of the working solutions of test compounds by diluting stock concentrations. One to 1000 in serum free medium. In the tissue culturehood seeds, COS seven cells in complete medium onto flat bottom tissue culture treated 96 well plates at a concentration of 4, 000 cells per 200 microliters per well.Using a multi-channel pipeter. Incubate the cells for 24 hours at 37 degrees Celsius in an atmosphere containing 5%carbon dioxide. On the next day, aspirate the supernatant from the wells using a glass pasture pipette attached to a vacuum pump.Dose the cells in triplicate with the test compounds using the previously prepared working solutions. Then return the cells to the incubator. After 24 hours, aspirate the supernatant from the wells and add 200 microliters of MTT solution to each well.Incubate the plate for four hours. Gently aspirate the supernatant from the wells without disturbing the purple formazane crystals. Add 200 microliters of DMSO to each well to dissolve the crystals.Then incubate the plate at room temperature for 15 minutes. Measure absorbance for each well at 590 or 600 nanometers using a 96 well plate reader. Use wells with no cells as background and average the absorbance value.Spirocyclic gox seams six and seven were synthesized using a modified protocol. The progress of the reaction was monitored by detecting the disappearance of the alpha beta unsaturated Esther using infrared spectroscopy. The MTT assay was used for determining cell viability.Cisplatin, which is known to induce cell death at high concentrations, served as a positive control. As expected, the higher the concentration of cisplatin the lower is the cell viability. The MTT assay was then used to test the spirocyclic compounds six and seven and furfurylamine.Furfurylamine and spirocyclic gox seam six showed similar cytotoxicity. However, the toxicity of spirocyclic compound seven was noticeably greater than that of furfurylamine and six. When attempting this protocol, keep in mind that N-alkylation performed using bulky acyl halides was found to reduce the rate of beta elimination thus reducing the product yield.

functionalized-spirocyclic-heterocycle-synthesis-cytotoxicity

Research

JoVE Journal

Biology

1.4K vistas.  Azusa Pacific University. Este protocolo utiliza un método reciclable y modificado en la producción de compuestos espirocíclicos. El ensayo MTT ayuda a responder la pregunta relacionada con la citotoxicidad de estas nuevas moléculas. Esta técnica produce nuevas moléculas modificables utilizando un material de partida regenerador.El ensayo MTT proporciona resultados biológicos relativamente rápidos y fáciles de analizar Comience realizando la síntesis en fase sólida de los ciclos hetero espirocíclico...