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).
1. Solid phase synthesis of spirocyclic heterocycles 6 and 7
NOTE: Synthesis was based on previously published work11,12. 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.
<ol>
	<li>Perform Michael addition of furfurylamine to the REM linker (duration: 25 min setup + 24 h reaction time).
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Perform tandem Michael addition/1,3-dipolar cycloaddition (duration: 25 min setup + 48 h reaction time).
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Securely cap the reaction vessel, and agitate at room temperature for 48 h. 
		NOTE: Ensure thorough mixing of the resin with the reagents.</li>
		<li>Use 5 mL of methanol to quench the reaction.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Perform N-alkylation of the resin-bound heterocycle to form the quaternary amine (duration: 10 min setup + 24 h reaction time).
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Perform &#946;-elimination of the quaternary amine for cleavage from the polymer support (duration: 15 min setup + 24 h reaction time).
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Triturate with methanol to purify the spirocyclic oxime. Dry the resin thoroughly in the reaction vessel following washes for reuse in future experiments.</li>
	</ol>
	</li>
</ol>
2. Cytotoxicity assay using MTT 14
<ol>
	<li>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 &#176;C. Then, prepare a 1:1 dilution of the MTT solution from step 2.1 in serum-free cell culture medium (DMEM).</li>
	<li>Prepare 1 mL each of stock solutions in 1.5 mL microcentrifuge tubes of 100 mM, 10 mM, 1 mM, 100 &#181;M, 10 &#181;M, 1 &#181;M, 0.1 &#181;M, and 0.01 &#181;M of test compounds in dimethyl sulfoxide (DMSO). Store at -20 &#176;C. Prepare 200 &#181;L per dose of the working solutions of test compounds by diluting stock concentrations 1:1000 in serum-free medium in 1.5 mL tubes.</li>
	<li>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 &#215; 103 cells/200 &#181;L 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.</li>
	<li>Incubate COS-7 cells for 24 h at 37 &#176;C in an atmosphere containing 5% CO2.</li>
	<li>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.</li>
	<li>Aspirate the supernatant from the wells. Add 200 &#181;L of MTT solution to each well. Incubate at 37 &#176;C in an atmosphere containing 5% CO2 for 4 h.</li>
	<li>Gently aspirate the supernatant from the wells without disturbing the purple formazan crystals. Add 200 &#181;L of DMSO to each well to dissolve the purple formazan crystals. Incubate at room temperature for 15 min.</li>
	<li>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)</li>
</ol>

<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 authors have nothing to disclose.

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-1). From the previous report, ISOC of&#160;2&#160;produced the tricyclic heterocyclic compound&#160;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&#160;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&#160;side chains. Due to the similarities in the functional groups in resins&#160;4&#160;and&#160;5, the results of IR were inconclusive. The success of this step could only be determined by attempting to regenerate the REM linker (5&#160;&#8594;&#160;1). Regeneration did not occur in instances when a bulky R2&#160;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 &#176;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.

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><tbody><tr><td>&lt;strong&gt;CELLS&lt;/strong&gt;</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>&lt;strong&gt;CHEMICALS&lt;/strong&gt;</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&amp;nbsp;</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>&lt;em&gt;trans&lt;/em&gt;-&amp;beta;-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>&lt;strong&gt;GLASSWARE/INSTRUMENTATION&lt;/strong&gt;</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-[(3E)-(2S, 4R)-2-phenyl-3-hydroxyimino 4-hydroxymethyl-pyrrolidin-1-yl]-propionic acid methyl ester 7, while alkylation followed by &#946;-elimination afforded (3E)-(2S, 4R)-4-hydroxymethyl-1-methyl-2-phenyl-3-pyrrolidine oxime 6. The identity of the spirocyclic oximes was determined by&#160;1H and&#160;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&#160;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&#160;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&#160;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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61950/61950fig1.jpg" /> 
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) &#946;-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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61950/61950fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61950/61950fig2.jpg" /> 
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&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61950/61950fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61950/61950fig3.jpg" /> 
Figure 3: Monitoring the progress of each solid phase reaction step by infrared spectroscopy. The stretching frequency at 1717 cm-1&#160;indicated the presence of an unsaturated ester, 1733 cm-1&#160;depicted a saturated ester, and signal around 3300-3500 cm-1&#160;indicated the presence of a hydroxyl group. Detectable stretching frequencies for polystyrene are also shown. Abbreviations: REM = Regenerating Michael; ISOC = intramolecular silyoxy olefin cycloaddition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61950/61950fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61950/61950fig4.jpg" /> 
Figure 4: Effects of cisplatin on COS-7 cell viability in a modified MTT assay. Concentrations of cisplatin ranged from 0 &#181;M to 60 &#181;M. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61950/61950fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61950/61950fig5.jpg" /> 
Figure 5: Effects of test compounds on COS-7 cell viability in a modified MTT assay. Concentrations ranged from 0 &#181;M to 100 &#181;M and were plotted on a log scale. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61950/61950fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/61950/61950table1.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61950/61950table1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

functionalized-spirocyclic-heterocycle-synthesis-cytotoxicity

Nanyang Technological University

Research

JoVE Journal

Biology

1.5K Views.  Azusa Pacific University. Dit protocol maakt gebruik van een recyclebare en gemodificeerde methode bij het produceren van spirocyclische verbindingen. De MTT-test helpt bij het beantwoorden van de vraag met betrekking tot de cytotoxiciteit van deze nieuwe moleculen. Deze techniek produceert nieuwe aanpasbare moleculen met behulp van een regenererend uitgangsmateriaal.De MTT-test biedt relatief snelle en gemakkelijk te analyseren biologische resultaten Begin met het uitvoeren van solide fasesynthese van spirocyc...

Video: Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

Chemistry

Synthesis of Antiviral Tetrahydrocarbazole Derivatives by Photochemical and Acid-catalyzed C-H Functionalization via Intermediate Peroxides (CHIPS)

The direct functionalization of C-H bonds is an important and long standing goal in organic chemistry. Such transformations can be very powerful in order to streamline synthesis by saving steps, time and material compared to conventional methods that require the introduction and removal of activating or directing groups. Therefore, the functionalization of C-H bonds is also attractive for green chemistry. Under oxidative conditions, two C-H bonds or one C-H and one heteroatom-H bond can be transformed to C-C and C-heteroatom bonds, respectively. Often these oxidative coupling reactions require synthetic oxidants, expensive catalysts or high temperatures. Here, we describe a two-step procedure to functionalize indole derivatives, more specifically tetrahydrocarbazoles, by C-H amination using only elemental oxygen as oxidant. The reaction uses the principle of C-H functionalization via Intermediate PeroxideS (CHIPS). In the first step, a hydroperoxide is generated oxidatively using visible light, a photosensitizer and elemental oxygen. In the second step, the N-nucleophile, an aniline, is introduced by Br&#248;nsted-acid catalyzed activation of the hydroperoxide leaving group. The products of the first and second step often precipitate and can be conveniently filtered off. The synthesis of a biologically active compound is shown.

Synthesis of Antiviral Tetrahydrocarbazole Derivatives by Photochemical and Acid-catalyzed C-H Functionalization via Intermediate Peroxides CHIPS

A two-step procedure for the synthesis of pharmaceutically active indole-derivatives by C-H functionalization with anilines is described, using photo- and Br&#248;nsted acid catalysis.

The direct functionalization of C-H bonds is an important and long standing goal in organic chemistry. Such transformations can be very powerful in order ...

Accessing Valuable Ligand Supports for Transition Metals: A Modified, Intermediate Scale Preparation of 1,2,3,4,5-Pentamethylcyclopentadiene

A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that derive from initial 2-bromo-2-butene lithiation followed by acid mediated dienol cyclization. The revised synthesis and purification of the ligand avoids the use of mechanical stirring while still permitting access to significant quantities (39 g) of Cp*H in good yield (58%). The procedure offers other additional benefits, including a more controlled quench of excess lithium during the production of the intermediate heptadienols and a simplified isolation of Cp*H of sufficient purity for metallation with transition metals. The ligand was subsequently used to synthesize [Cp*MCl2]2 complexes of both iridium and ruthenium to demonstrate the utility of the Cp*H prepared and purified by our method. The procedure outlined herein affords substantial quantities of a ubiquitous ancillary ligand support used in organometallic chemistry while minimizing the need for specialized laboratory equipment, thus providing a simpler and more accessible entry point into the chemistry of 1,2,3,4,5-pentamethylcyclopentadiene.

Reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented. The revised protocol for the synthesis and purification of the ligand minimizes the need for specialized laboratory equipment while simplifying reaction workups and product purification. Use of Cp*H in the synthesis of [Cp*MCl2]2 complexes (M = Ru, Ir) is also described.

A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that ...

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of solid-phase synthesis, regenerating Michael (REM) linker strategies, and 1,3-dipolar cycloaddition, a library of structurally similar heterocycles, both with and without a spirocyclic center, can be constructed. The main advantages of the solid-support synthesis are as follows: first, each reaction step can be driven to completion using a large excess of reagents resulting in high yields; next, the use of commercially available starting materials and reagents keep the costs low; finally, the reaction steps are easy to purify via simple filtration. The REM linker strategy is attractive because of its recyclability and traceless nature. Once a reaction scheme is completed, the linker can be reused multiple times. In a typical solid-phase synthesis, the product contains either a part of or the whole linker, which can prove undesirable. The REM linker is &#34;traceless&#34; and the point of attachment between the product and the polymer is indistinguishable. The high diastereoselectivity of the intramolecular 1,3-dipolar cycloaddition is well documented. Limited by the insolubility of the solid support, the reaction progression can only be monitored by a change in the functional groups (if any) via infrared (IR) spectroscopy. Thus, the structural identification of intermediates cannot be characterized by conventional nuclear magnetic resonance (NMR) spectroscopy. Other limitations to this method stem from the compatibilities of the polymer/linker to the desired chemical reaction scheme. Herein we report a protocol that allows for the convenient production of spirocyclic heterocycles that, with simple modifications, can be automated with high-throughput techniques.

Here we present a protocol to demonstrate an efficient method for the synthesis of spirocyclic heterocycles. The five-step process utilizes solid-phase synthesis and regenerating Michael linker strategies. Generally difficult to synthesize, we present a customizable method for the synthesis of spirocyclic molecules otherwise inaccessible to other modern approaches.

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of ...

Efficient Construction of Drug-like Bispirocyclic Scaffolds Via Organocatalytic Cycloadditions of &#945;-Imino &#947;-Lactones and Alkylidene Pyrazolones

Bispirocyclic scaffolds are one of the important structural subunits in many natural products that exhibit diverse and attractive biological activities. Recently, we have developed an efficient organocatalytic strategy, which provides facile access to a variety of enantiomerically enriched bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons. In this paper, we demonstrate a detailed protocol for the asymmetric synthesis of drug-like bispirocyclic compounds with two spirocyclic carbon centers via an organocatyltic 1,3-dipolar cycloaddition reaction. Spirocyclization synthons &#945;-imino &#947;-lactones and alkylidene pyrazolones are prepared first, which are then subjected to a cycloaddition reaction in the presence of a bifunctional squaramide organocatalyst to afford the desired bispirocycles in high yields and excellent stereoselectivities. Chiral high-performance liquid chromatography (HPLC) is carried out to determine the enantiomeric purity of the products, and the d.r. value is examined by proton nuclear magnetic resonance (1H NMR). The absolute configuration of the product is assigned according to an X-ray crystallographic analysis. This synthetic strategy allows scientists to prepare a diversity of bispirocyclic scaffolds in high yields and excellent diastereo- and enantioselectivities.

Enantiomerically enriched bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons are asymmetrically synthesized through a simple organocatalytic 1,3-dipolar cycloaddition reaction.

Bispirocyclic scaffolds are one of the important structural subunits in many natural products that exhibit diverse and attractive biological activities. ...

Design, Synthesis, and Photochemical Properties of Clickable Caged Compounds

Caged compounds enable the photo-mediated manipulation of the cell physiology with high spatiotemporal resolution. However, the limited structural diversity of currently available caging groups and the difficulties in synthetic modification without sacrificing their photolysis efficiencies are obstacles to expanding the repertoire of caged compounds for live cell applications. As the chemical modification of coumarin-type photo-caging groups is a promising approach for the preparation of caged compounds with diverse physical and chemical properties, we report a method for the synthesis of clickable caged compounds that can be modified easily with various functional units via the copper(I)-catalyzed Huisgen cyclization. The modular platform molecule contains a (6-bromo-7-hydroxycoumarin-4-yl)methyl (Bhc) group as a photo-caging group, which exhibits a high photolysis efficiency compared to those of the conventional 2-nitrobenzyls. General procedures for the preparation of clickable caged compounds containing amines, alcohols, and carboxylates are presented. Additional properties such as the water solubility and cell targeting ability can be readily incorporated into clickable caged compounds. Furthermore, the physical and photochemical properties, including the photolysis quantum yield, were measured and were found to be superior to those of the corresponding Bhc caged compounds. The described protocol could therefore be considered a potential solution for the lack of structural diversity in the available caged compounds.

A protocol for the synthesis and measurement of the photochemical properties of modular caged compounds with clickable moieties is presented.

Caged compounds enable the photo-mediated manipulation of the cell physiology with high spatiotemporal resolution. However, the limited structural ...

Cercosporin-Photocatalyzed [4+1]- and [4+2]-Annulations of Azoalkenes Under Mild Conditions

The interest on nitrogen-containing heterocycles has expanded rapidly in the synthetic community since they are important motifs for new drugs. Traditionally, they were synthesized through thermal cycloaddition reactions, whereas today, photocatalysis is preferred due to the mild and efficient conditions. With this focus, a new photocatalytic method for the synthesis of nitrogen-containing heterocycles is highly desired. Here, we report a protocol for the biosynthesis of cercosporin, which could function as a metal-free photocatalyst. We then illustrate cercosporin-photocatalyzed protocols for the synthesis of nitrogen-containing heterocycles 1,2,3-thiadiazoles through annulation of azoalkenes with KSCN, and synthesis of 1,4,5,6-tetrahydropyridazines [4+2] through cyclodimerization of azoalkenes under mild conditions, respectively. As a result, there is a new bridge between the microbial fermentation method and organic synthesis in a mild, cost-effective, environmentally friendly and sustainable manner.

New routes for the synthesis of nitrogen-containing heterocycles utilizing cercosporin as a metal-free photocatalyst were developed.

The interest on nitrogen-containing heterocycles has expanded rapidly in the synthetic community since they are important motifs for new drugs. ...

Synthesis of pH Dependent Pyrazole, Imidazole, and Isoindolone Dipyrrinone Fluorophores using a Claisen-Schmidt Condensation Approach

Methine-bridged conjugated bicyclic aromatic compounds are common constituents of a range of biologically relevant molecules such as porphyrins, dipyrrinones, and pharmaceuticals. Additionally, restricted rotation of these systems often results in highly to moderately fluorescent systems as observed in 3H,5H-dipyrrolo[1,2-c:2',1'-f]pyrimidin-3-ones, xanthoglows, pyrroloindolizinedione analogs, BODIPY analogs, and the phenolic and imidazolinone ring systems of Green Fluorescent Protein (GFP). This manuscript describes an inexpensive and operationally simple method of performing a Claisen-Schmidt condensation to generate a series of fluorescent pH dependent pyrazole/imidazole/isoindolone dipyrrinone analogs. While the methodology illustrates the synthesis of dipyrrinone analogs, it can be translated to produce a wide range of conjugated bicyclic aromatic compounds. The Claisen-Schmidt condensation reaction utilized in this method is limited in scope to nucleophiles and electrophiles that are enolizable under basic conditions (nucleophile component) and non-enolizable aldehydes (electrophile component). Additionally, both the nucleophilic and electrophilic reactants must contain functional groups that will not inadvertently react with hydroxide. Despite these limitations, this methodology offers access to completely novel systems that can be employed as biological or molecular probes.

The Claisen-Schmidt condensation reaction is an important methodology for the generation of methine-bridged conjugated bicyclic aromatic compounds. Through utilizing a base-mediated variant of the aldol reaction, a range of fluorescent and/or biologically relevant molecules can be accessed through a generally inexpensive and operationally simple synthetic approach.

Methine-bridged conjugated bicyclic aromatic compounds are common constituents of a range of biologically relevant molecules such as porphyrins, ...

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as a consequence, they are now used clinically. It is, therefore, critical to seek effective strategies for synthesizing cyclic peptides to promote their application in the field of drug discovery. This paper reports a detailed protocol for the efficient synthesis of cyclic peptides using on-resin or intramolecular (intermolecular) bisalkylation. Using this protocol, linear peptides were synthesized by taking advantage of solid-phase peptide synthesis with cysteine (Cys) and methionine (Met) coupled simultaneously on the resin. Further, cyclic peptides were synthesized via bisalkylation between Met and Cys using a tunable tether and an on-tether sulfonium center. The whole synthetic route can be divided into three major processes: the deprotection of Cys on the resin, the coupling of the linker, and the cyclization between Cys and Met in a trifluoroacetic acid (TFA) cleavage solution. Furthermore, inspired by the reactivity of the sulfonium center, a propargyl group was attached to the Met to trigger thiol-yne addition and form a cyclic peptide. After that, the crude peptides were dried and dissolved in acetonitrile, separated, and then purified by high-performance liquid chromatography (HPLC). The molecular weight of the cyclic peptide was confirmed by liquid chromatography-mass spectrometry (LC-MS), and the stability of the cyclic peptide combination with the reductant was further confirmed using HPLC. In addition, the chemical shift in the cyclic peptide was analyzed by 1H nuclear magnetic resonance (1H NMR) spectra. Overall, this protocol aimed to establish an effective strategy for synthesizing cyclic peptides.

This protocol presents the synthesis of cyclic peptides via bisalkylation between cysteine and methionine and the facile thiol-yne reaction triggered by the propargyl sulfonium center.

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

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