Opioid Epidemic: Uses, Abuses, and Innovation: a New Method for Approaching an Old Problem Matt Fondersmith and David Peana Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri, 65211 Email: [email protected]; [email protected] Fondersmith Peana Introduction General analgesics and more specifically opioids Common opioids o Point out problems with common opioids Why PZM21 brings a solution to some problems o Compare to morphine, tramadol, and fentanyl Materials and Methods Computational Discovery With modern advancements in science and the depth of complexity that has been discovered in the human body, it should be no surprise that the pharmaceutical industry would take advantage of cutting edge computing technology for the advancement of drug chemistry and the discovery of new, innovative drugs. PZM21 (Scheme 1) is the result of the compilation of massive calculations and data sets which were sorted and narrowed down. The current problem with the opioids is that they act as agonists to the primary µ- opioid receptor site. It has also been recently discovered that they often take part in β- Arrestin recruitment1 which gives rise to the serious side effects of most opioids, primary among them being respiratory depression. PZM21 was discovered by computing and trying to dock millions of molecules, both known and theoretical, to orthosteric (non- primary) binding sites of the µ-opioid receptor (the µ-opioid receptor [µOR] is the receptor in the nervous system responsible for the analgesic effects of opioids). Out of the millions of molecules docked, PZM21 was the most efficient at providing analgesic effects with little to no adverse side-effects due to its ability to bind to an orthosteric 2 Fondersmith Peana binding site of a µOR (see Table 1 in Appendix). After the theoretical analysis and discovery, PZM21 was synthesized and tested on lab mice. Due to its recent discovery, it has not been approved for human testing yet.2 Synthesis The synthesis of PZM21 is primarily prepared from amino acid amide chemistry. Starting with two primary amides, steps 1 and 2 in the reaction are meant to turn these primary amides into primary amines. Step 3 of the reaction is a Henry reaction that, when treated with the cyclohalogen reactant, yields a nitropropene derivative. This nitropropene derivative gets converted into a racemic alkylamine. Activation with a cyclic, nitrogen containing compound creates a carbamate. When this carbamate is coupled with the enantiopure primary amines, a diastereomeric mixture of ureas is achieved. Overall, there are over 8 pure stereoisomers of PZM21. Identification After synthesizing the molecule, it was put through various types of NMR imaging techniques. Both H1NMR and C13NMR confirmed that PZM21 and its various stereoisomers were synthesized. Performance PZM21 performed in lab studies with mice as was predicted theoretically. In terms of analgesic properties, PZM21 is about 4 times weaker than morphine. Its dosage is 40mg/2-4 hours which is approximately 4 times that of morphine and comparable to the dosage of Tramadol. The duration of PZM21 is also comparable to Tramadol but PZM21 peaks faster and is exhausted faster. The binding affinity of PZM21 is slightly stronger 3 Fondersmith Peana than that of morphine meaning it binds tighter and is slightly more difficult to disengage from the G-protein once engaged. The shining quality of PZM21 over other opioids is in the lack of adverse effects. Whereas fentanyl, morphine, and even tramadol to an extent have adverse side effects, principle among them being respiratory depression, PZM21 circumvents those side effects and offers analgesia without them. Overall, PZM21 was developed to be an opioid analgesic without the adverse side effects of other opioids. Theoretically, it accomplishes its task. So far, experiments have further proved the theoretical work done. Results and Discussion After all the theoretical analysis, PZM21 was discovered to be the best As the comparison data shows, PZM21 has many of the benefits of other opioids but not some of the drawbacks o This is because of its mechanism of action… Conclusion [Placeholder conclusion for the sake of formatting] Scheme 1: Structure of PZM21 Supplementary Material Available The appendix contains a more detailed description of the process and preparation of the substrates, as well as their characterization. 4 Fondersmith Peana References (1): Abbas, A.; Roth, B.L. Arresting Serotonin. Proc Natl Acad Sci U S A. 2008 22, , 831- 2. (2): Manglik, A.; Lin, H. et al. Structure–based discovery of opioid analgesics with reduced side effects. Nature. 2016, 08; 185–190. 5 Fondersmith Peana Supporting Information Opioid Epidemic: Uses, Abuses, and Innovation: a New Method for Approaching an Old Problem Matt Fondersmith and David Peana Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri, 65211 Email: [email protected] ; [email protected] S1 Fondersmith Peana Table of Contents Table of Contents .............................................................................................................. S2 Synthesis ............................................................................................................................ S3 Identification ..................................................................................................................... S1 Dosage .............................................................................................................................. S4 Duration ............................................................................................................................ S4 Binding Affinity ................................................................................................................. S5 Respiratory Depression .................................................................................................... S5 Performance ...................................................................................................................... S6 Bibliography ..................................................................................................................... S6 References ......................................................................................................................... S7 S2 Fondersmith Peana Synthesis The primary amino group in the first step was dimethylated using an excess of aqueous formaldehyde and sodium triacetoxyborohydrife in aqueous acetonitrile (Scheme 2). The carboxamides were the converted to primary amines in a 1M borane-tetrahydrofurane complex and anhydrous THF in a nitrogen atmosphere. After filtering, step 2 is complete. Step 3 started with thiopene-3-carbaldehyde in an ice cold mixture of formic acid and nitroethane. after heating to 100C and stirred for 7 hours, the resulting solution was poured into cold water and filtered. The precipitate product was washed with water and yielded a yellow solid. This is good because it means the formic acid did not rip off the sulfur in the molecule. Step 4 was completed using the previous product in a solution of THF and LiAlH4 added dropwise. the mixture was kept under a gentle reflux for a full 30 minutes before being cooled to 0C and dessicated with a sodium-sulfate salt. After being cooled, stirred, and filtered, the yellow filtrate was concentrated under reduced pressure and dissolved in diethyl ether and precipitated by 2M HCL. The precipitate was then recrystallized from acetonitrile. Step 5 was completed with the previous product charged with triethylamine in anhydrous THF. the mixture was warmed to an ambient temperature and stirred for 6 hours. the slurry was then diluted with DCM and filtered. Once washed with saturated NaHCO3 and brine, the filtrate was dried over anhydrous Na2S04. After being filtered again, the crude residue was purified by flash chromatography in 100% dichloromethane, yielding the desired product as white foam. the sixth and final step of the reaction is putting both the diamine and bicyclic systems together in a suspension of acetonitrile, and triethylmamine. The solution was sealed and S3 Fondersmith Peana heated until the mixture turned yellow. After increasing the temperature to 80C, the mixture was stirred for 2 hours, then filtered and concentrated under reduced pressure. the residue was taken and suspended in 33% ethyl acetate/isopropanol and washed with a PH 9.5 carbonate buffer. After extracting the organic layer and drying it over Na2SO4, the residue was filtered and concentrated again. The crude residue was purified by dry- column vacuum chromatography. This synthesis yields 8 different stereoisomers. The particular isomer we want to obtain is PZM21, and that can be reclaimed as a colorless oil, separating the diastereoisomers with a semi-preparative HPLC with diethyl amine in isopropanol/hexane. S4 Fondersmith Peana Scheme S1. Overview of Synthesis S1 Fondersmith Peana Identification The HNMR peaks (Figure 1) can be characterized by obvious peaks in the regions consisting of amine and aromatic hydrogens. The aromatic hydrogens have peaks between 6.5ppm and 8.5ppm, there are three peaks with one peak having n=5. This is describing the aromatic ring with the nitro group attached. The peak at about 6 is the 5- proton ring containing the sulfure atom. The double peak describing the amines on either side of the ketone in the middle of the molecule happens right at about 1.3ppm. In the CNMR spectrum (Figure 2), we have