Synthesis of a Carbazole Intermediate, a Dihydronaphthalene For Analysis of Antimicrobial and Anti-tumor Effects Tiffany Layport, Department of Biology, York College Naturally occurring carbazoles isolated from the stem bark of Glycosmis mauritiana have demonstrated antimicrobial and anti- tumor activity. (Kumar et al. 1989) Regiocontrolled carbazole synthetic strategies have been developed for cost efficiency of product yield. Several carbazoles, Glycomaurrol and its derivatives Eustifolines A-D have been successfully synthesized. (Lebold and Kerr 2007) Medicinal benefits of the synthetic carbazoles are unknown. Effect of synthetic dihydronaphthalene derivative, an intermediate of Glycomaurrol, in antimicrobial and anti-tumor bioassays is unknown. This study examines the antimicrobial and anti-tumor activity of the dihydronaphthalene derivative at varying concentrations. Introduction Hypotheses Antimicrobial Assays H 0 : The dihydronaphthalene derivative will have no effect on microbial growth inhibition. H A : The dihydronaphthalene derivative with a concentration range of 0-25mg/mL will show a difference in microbial growth inhibition from the untreated organisms. Anti-tumor Assays H 0 : There is no difference between the effect of the dihydronaphthalene derivative with a concentration range of M and the untreated U87-MG cells on cell viability. H A : There is a difference between the effect of the dihydronaphthalene derivative within a concentration range of M and the untreated U87-MG cells on cell viability. Scheme 1: Preparation of diene Scheme 2: Preparation of dienophile Scheme 3: Diels-Alder Reaction Methods Abdel-Rahman, Taha M Synthesis, Reactions, and Anticancer Activity of Some 1,3,4- Thiadiazole/Thiadiazine Derivatives of Carbazole. Phosphorus, Sulfur, and Silicon. 181: Asche, Christian Antitumor Carbazoles. Anti-Cancer Agents in medicinal Chemistry. 7: 247. Eflora.org England, Dylan B. and Kerr, Michael A Synthesis and Cross- Coupling Reactions of Substituted 5-Triflyoxyindoles. Journal of Organic Chemistry. 70: Greger, H. and Zechner, G Bioactive Amides from Glycosmis Species. Journal of Natural Products 59: Kamal, Ahmed; Reddy, S. J.; Bharathi, E. V.; Dastagiri, D Base-free Monosulfonylation of Amines using Tosyl or Mesyl Chloride in Water. Tetrahedron Letters 49: Kasai, Yuki, Shindo, Kazutoshi, Harayama, Shigeaki, and Misawa, Norihiko Molecular Characterization and Substrate Preference of a Polycyclic Aromatic Hydrocarbon Dioxygenase from Cycloclasticus sp. Strain A5. Applied and Environmental Microbiology 69: Knolker, Hans-Joachim; Reddy, Kethiri R Isolation and Synthesis of Biologically Active Carbazole Alkaloids. Chemistry Reviews. 102: Lebold, Terry P. and Kerr, Michael A Total Synthesis of Eustifolines A-D and Glycomaurrol via a Divergent Diels-Alder Strategy. Organic Letters. 9: McBride, James, Ingram, Paul, Henriquez, Fiona, and Roberts, Craig Development of Colorimentric Microtiter Plate Assay for Assessment of Antimicrobials against Acanthamoeba. Journal of Clinical Microbiology 43: Zhong, Yong-Li; Shing, Tony K. M Efficient and Facile Glycol Cleavage Oxidation Using Improved Silica Gel-Supported Sodium Metaperiodate. Journal of Organic Chemistry 62: Literature Cited Results Conclusions Acknowledgements Organism Control Minimum Inhibitory Concentration DHN no growth inhibition DMSO no growth inhibition Untreated no growth inhibition AMP >0.0098mg/mL inhibits growth MPA >0.001mg/mL inhibits growth Table 1. Minimum concentration of Dihydronaphthalene, DMSO, Ampicillin, Mycophenolic Acid, and no treatment required to inhibit organismal growth. I would like to thank Dr. Thompson and Dr. Halligan for mentoring this research. I would also like to thank Dr. Singleton and Dr. Mathur for their guidance and contributions. Thank you to the Biology Department and faculty. Glycosmis Mauritiana. Eflora.org The compound that was synthesized is a derivative of dihydronaphthalene, a naturally occurring carbazole intermediate. This key intermediate of Glycomaurrol can be extracted from the bark of Glycosmis mauritiana. The dihydronaphthalene was synthesized in order to test for anti-tumor and antimicrobial properties present at the intermediate level. The key intermediate was synthesized via a Diels-Alder strategy following preparation of the diene and dienophile. The target dihydronaphthalene derivative was synthesized in 5 steps. The final product was then exposed to E. coli, S.aureus, P. mirabilis, B. cereus, P. stuartii, C. albicans, S. cerevisiae and U87-MG Human Glioblastoma cells to determine medicinal benefits. The dihydronaphthalene derivative had no effect on microbial growth inhibition, however it showed 85% U87-MG cell death at maximum concentration. Abstract Concentration (M) % Viable Cells a n b SEM c SD d a mean b sample size c standard error of the mean d standard deviation Figure 1. Mean percent viable U87-MG Human Glioblastoma cells from absorbance values at 490nm after exposure to a dihydronaphthalene derivative at varying concentrations of a 2-fold serial dilution. Data are means and standard errors from five replicates for each concentration. P= Concentration (M) % Viable Cells a SD b SEM c Minimum (0.0) Maximum (0.044) Table 3. Percent of viable cells present at minimum and maximum concentrations of a dihydronaphthalene derivative. a mean b standard deviation c standard error of the mean Table 2. Percent of viable U87-MG Human Glioblastoma cells after exposure to a dihydronaphthalene derivative at varying concentrations. Antimicrobial The results showed that the dihydronaphthalene derivative had no effect on the growth inhibition for the organisms being tested. Therefore, the null hypothesis must be accepted, stating that there is no significant difference between the effect of the dihydronaphthalene derivative and the untreated organisms that were used as a control group. It is implied that the minimum inhibitory concentration if any is greater than the tested range of drug concentration. Anti-tumor The P value= indicating that there is a significant difference in the mean percent of cell viability between maximum dihydronaphthalene derivative concentration and the untreated cells. Therefore, the null hypothesis can be rejected. 100 uL of appropriate media was placed in all rows for columns uL of DHN was placed in rows A, B, E, and F for column 1 only. 200 uL of DMSO was placed in rows D and H for column 1 only. 200 uL of specified control (AMP or MPA) was placed in rows C and G for column 1 only. Each row was diluted via a 2-fold serial dilution in columns uL of appropriate organism was placed in all rows from column Column 11 contained organism and media only. Column 12 contained media only. Plate was allowed to incubate at 37 o C for 48 hours before results were recorded. Antimicrobial 150 uL U87-MG cells were placed in all rows for columns The cells set for 24 hours to allow reattachment. 100 uL of DHN was placed in rows A, B, C, and D for column 1 only. 100 uL of DMSO was placed in rows E and F for column 1 only. 100 uL of cycloheximide was placed in rows G and H for column 1 only. Each row was diluted via a 2-fold serial dilution in columns uL of U87-MG cell media was placed in all rows for columns Column 12 contained U87-MG cells and media only. The cells were allowed to incubate at 37 o C for 48 hours. Celltiter 96 aqueous reagent proliferation assay was added to all wells. The absorbance for each well was recorded at 490nm. Anti-tumor Antimicrobial and anti-tumor bioassays were performed in a 96 well microtiter plate. The synthesis of the dihydronaphthalene was monitored via thin layer chromatography (TLC) and purified via flash column chromatography (FCC). The success of the reactions was confirmed by Fourier Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance Spectroscopy (NMR). Part II Part I Dihydronaphthalene Derivative Figure 2. U87-MG Human Glioblastoma cells treated with the dihydronaphthalene derivative in rows A, B, C, D, and E, columns Cells were treated with DMSO in row E and F, columns Cells were treated with cyclohexamine in row G, columns 1-4. Column 12 contained untreated cells. Row G, columns 5-12 were blank.