1. Isolation of Martinella obovata via flash column chromatography Naomi Bryner Dr. David Singleton (advisor) Dr. Kathleen Halligan (advisor)

2. Plant Information • Bignoniaceae family • 120 genera, 800 species • Found in Central and South America • Neotropical liana • Versatile plant • Horticulture, food, crafts, timber, dyes, rituals, and medicine Arevalo, et al. 2011, Gentry and Cook 1984, Gentry 1992, Witherup, et al. 1995

3. Overview • M. Obovata and M. iquitosensis • 2 most known species • 9 species total for Martinella • M. iquitosensis known to produce martinelline and martinellic acid

4. Martinelline & Martinellic acid • Chemically unique • Natural pyrroloquinoline ring structures • Synthesis is sought after • Biologically unique • Natural product (not peptide) that potently antagonize bradykinin receptors (GPCRs) Pyrroloquinoline quinone Davies, et al. 2013, Lovely and Mahmud 1999, , Ma, et al. 2001Ma, et al. 2003 Powell and Batey 2002, Shirai, et al. 2008, Yee, et al. 2006, Zhang, et al. 2007

5. Summer 2013 • Bark removal and extraction • TLC and FCC • Four fractions • IR spectra • 1H and 13C NMR • 2D more helpful… Solvents Used Extraction: CH2Cl2/MeOH FCC: Hex/EtOAC gradient

6. NB1-6-7 IR spectrum

7. Fall 2013 CHM (1 credit) • This proved to be more of a learning experience than productive • HPLC difficulties • Long method time for little purified product • Inconsistent results for first duplicate

8. Fall 2013 BIO • Solubility tests • H2O – no, EtOH(95%) – somewhat, DMSO (99.5%) – yes • Disk diffusion antibiotic sensitivity testing and MIC microtiter assays • Bacteria used • Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus

9. Fall 2013 BIO • Disk diffusion showed that only NB1-6-1, NB1-6-6, and NB1-6-7 merited further investigation • Clear results: • EtOH(10.5%) + 1 mg/mL NB1-6-7 - S. aureus • EtOH (5.25%) + .5 mg/mL NB1-6-7 - B. cereus • DMSO (24.9%) + 1.5 mg/mL NB1-6-7 E. coli • DMSO (24.9%) + 1.5 mg/mL NB1-6-7 S. aureus

10. Loose ends • Will run 2D NMRs overnight this week 2/3/14 • Hope to ID NB1-6-7 • Pyrroloquinoline ring in any sample? • If I had another semester • Bradykinin receptor antagonism investigation • LC-MS analysis to determine mass (no preexisting method)

11. References • (1) Arevalo, C.; Ruiz, I.; Piccinelli, A.; Campone, L.; Rastrelli, L. Phenolic derivatives from the leaves of Martinella obovata (Bignoniaceae). Natural Product Communications, 2011, 6:7, 957-960. • (2) Gentry, A. H. A synopsis of Bignoniaceaeethnobotany and economic botany. Annals of the Missouri Botanical Garden, 1992, 79, 53-64. • (3) Gentry, A.H.; Cook, K. Martinella (Bignoniaceae): A widely used eye medicine of South America. Journal of Ethnopharmacology, 1984, 11, 337-343. • (4) Witherup, K.; Ransom, R.; Graham, A.; Bernard, A.; Salvatore, M.; Lumma, W.; Anderson, P.; Pitzenberger, S.; Varga, S. Martinelline and martinellic acid, novel G-protein linked receptor antagonists from the tropical plant Martinella iquitosensis (Bignoniaceae). Journal of the American Chemical Society, 1995, 117, 6682-6685. • (5) Ma, D.; Xia, C.; Jiang, J.; Zhang, J. First Total Synthesis of Martinellic Acid, a Naturally Occurring Bradykinin Receptor Antagonist. Organic Letters, 2001, 3:14, 2189-2191. • (6) Zhang, Z.; Zhang, Q.; Yan, Z.; Liu, Q. One-Step Synthesis of the Tricyclic Core of Martinellic Acid from 2-(Cyanomethyl)-3-oxo-N-arylbutanamides. Journal of Organic Chemistry, 2007, 72, 9808-9810. • (7) Ma, D.; Xia, C.; Jiang, J.; Zhang, J.; Tang, W. Aromatic Nucleophilic Substitution or CuI-Catalyzed Coupling Route to Martinellic Acid. Journal of Organic Chemistry, 2003, 68, 442-451. • (8) Davies, S.; Fletcher, Ai.; Lee, J.; Lorkin, T.; Roberts, P.; Thomson, J. Asymmetric Synthesis of (-)-Martinellic Acid. Organic Letters, 2013, 15:8, 2050-2053. • (9) Powell, D.; Batey, R. Total Synthesis of the Alkaloids Martinelline and Martinellic Acid via a Hetero Diels-Alder Multicomponent Coupling Reaction. Organic Letters, 2002, 4:17, 2913-2916. • (10) Yee Ng, Pui.; Masse, C.; Shaw, J. Cycloaddition Reactions of Imines with 3-Thiosuccinic Anhydrides: Synthesis of the Tricyclic Core of Martinellic Acid. Organic Letters, 2006, 8:18, 3999-4002. • (11) Shirai, A.; Miyata, O.; Tohnai, N.; Miyata, M.; Procter, D.; Sucunza, D.; Naito, T. Total Synthesis of (-)-Martinellic Acid via Radical Addition-Cyclization-Elimination Reaction. Journal of Organic Chemistry, 2008, 73, 4464-4475. • (12) Lovely, C.; Mahmud, H. An approach to the pyrroloquinoline core of martinelline and martinellic acid. Tetrahedron Letters, 1999, 40, 2079-2082.