Lessons Learned from Organic Synthesis Joshua J. Nyman Howard Hughes Medical Institute Summer Scholar Research Project Mentor: Dr. Yan Zhang, Department of Medicinal Chemistry, VCU School of Pharmacy

Background  Anibamine is a natural product, but recently it has been successfully synthesized in the laboratory.  Anibamine can be used as an anti-HIV drug  It has a unique chemical structure. Anibamine TFA _

The Research Project  The objective of the research project was to synthesize the ring system of anibamine; and then to alter the stereochemistry of its side-chains. Anibamine TFA _

My Synthesis Route acetylacetone + cyanoacetamide K 2 CO 3 3-cyano-4,6-dimethyl-2-hydroxypyridine (2-hydroxy-4,6-dimethyl-nicotinonitrile), TFA, H 2 SO 4 5-bromo-2-hydroxy-4,6-dimethyl-nicotinonitrile CuCN, DMF under nitrogen gas 1,2-dihydro-4,6-dimethyl-2-oxopyridine-3,5-dicarbonitrile Tetrabutylammonium bromide, P 2 O 5 2-bromo-4,6-dimethylpyridine-3,5-dicarbonitrile

Forming the Pyridine Ring  An aqueous solution of potassium carbonate was prepared.  Acetylacetone and cyanoacetamide were added to the solution.  A stir bar was placed into the resulting mixture and the mixture was allowed to stir at room temperature for 24 hours. acetylacetonecyanoacetamide 3-cyano-4,6-dimethyl-2-hydroxypyridine K 2 CO 3

Forming the Pyridine Ring  After 24 hours of stirring, the mixture was vacuum filtrated, yielding a white powder.  Melting point was on the high end of the literature melting point, and the thin-layer chromatography provided a positive presumptive test for the pyridine ring product.  A proton-NMR was also done on the resultant compound, but was later to found to be of little value based on the solvent used to dissolve the sample. Images: Barnard College Organic Chemistry Lab

Brominating the Pyridine Ring  The product of the previous reaction was dissolved in concentrated sulfuric acid and trifluoracetic acid, while in an ice bath. N- bromosuccinimide was then added. **  This reaction was run multiple times with varying degrees of purity.  Melting points taken of the product(s) were 10 to 15 degrees too high in a couple of cases.  Thin-layer chromatography suggested some compounds were impure.  Product appeared to be a light yellow in the more impure products and white in the purer products., TFA, H 2 SO 4

Brominating the Pyridine Ring  It seems the purity of this reaction could have been affected by certain techniques.  For example:  Removing the reaction mixture from the ice bath: Exothermic reaction  Rate of addition: Adding the NBS too quickly likely resulted in an undesired side reaction.  Acid-Base reaction., TFA, H 2 SO 4

Adding a Cyano- Group to the Pyridine Ring  This reaction had some special considerations:  One of the chemicals used in this reaction is a highly toxic compound called cuprous cyanide (a.k.a. copper (I) cyanide).  Handling the cyanide required great care CuCN, DMF under nitrogen gas

Adding a Cyano- Group to the Pyridine Ring  All reactants had to be very dry!!! Oil Pump

Adding a Cyano- Group to the Pyridine Ring  The cuprous cyanide was dissolved in dimethylformamide along with the product from the previous reaction.  The mixture was then refluxed for 48 hours under nitrogen gas. CuCN, DMF under nitrogen gas

Adding a Cyano- Group to the Pyridine Ring  My compound being refluxed under nitrogen protection

Adding a Cyano- Group to the Pyridine Ring  This reaction gave me multiple problems  Proton NMR, Thin-Layer Chromatograhy tests, and melting point tests = impure.  Because of this, I wasn’t able to proceed further before the end of this program. CuCN, DMF under nitrogen gas

Lessons Learned  Organic synthesis can be very tricky, especially when trying to form natural products.  I learned not only to think about the products I am trying to synthesize, but also about which products I do not want to synthesize (i.e. impurities) and how to prevent them.  It’s not just what chemicals you add, but how you add them. Temperature, rate of addition, and other factors can have a considerable impact on the overall synthesis.

References and Acknowledgements  A special thanks to Dr. Yan Zhang (my mentor) for sharing his expertise, his laboratory, and for making this an exceptional research learning experience.  Also a special thanks to Dr. Guo Li and Kendra Haney, for generously sharing their time and for their guidance.  And to all of the others brilliant individuals in Dr. Zhang’s lab whose efforts contributed to my learning experience.

References and Acknowledgements  Literature (the primary source for my research project and background information presented in this presentation):  Guo Li, Karen Watson, Robert W. Buckheit, and Yan Zhang: Total Synthesis of Anibamine, a Novel Natural Product as a Chemokine Receptor CCR5 Antagonist Organic Letters Vol 9. 10:

References and Acknowledgements  Pictures and Graphics:  Title Slide: Erlenmeyer Flasks –  Department of Chemistry & Biochemistry Northern Arizona University  Slide 10: Reflux Apparatus and White Compound in Erlenmeyer Flask –  Barnard College Organic Chemistry Lab