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 and more specifically

 Common opioids

o Point out problems with common opioids

 Why PZM21 brings a solution to some problems

o Compare to , , and

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 to the primary µ- 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- (the µ-opioid receptor [µOR] is the receptor in the nervous system responsible for the 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

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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

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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.

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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.

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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]

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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

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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

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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.

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Scheme S1. Overview of Synthesis

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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 peaks ranging from 30 to 65 that characterize the carbons attached to amine groups. The double bond between the carbon and the oxygen are responsible for the peaks furthest to the left, aside from the aromatic carbons showing up vividly in the data, with various peaks from 120 to 140ppm.

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Figure S1: HNMR of PZM21

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Figure S2: CNMR of PZM21

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Dosage

Compared to some of the other listed opioids, PZM21 has a comparable dosage to tramadol (Table 1). However, there are fundamental differences. While the PZM21 drug acts as an antagonist for β-Arrestin, tramadol is an NSRI (norepinephrine serotonin reuptake inhibitor) meaning that too much tramadol can be a very dangerous thing because it overloads the brain with norepinephrine and serotonin and the intersynaptic spaces. It is for this reason that it is strongly advised not to take more than 400mg of tramadol daily. PZM21 does not have this same restriction but due to the newness of the drug, further testing must be done to finalize dosage amounts and maximums. However, compared to morphine and fentanyl, it appears that PZM21 dosage follows its performance statistics in a typical manner.1,2

Duration

Another shining aspect of PZM21 is its short onset-to-peak time (around 15 minutes

[Table 1]). A serious drawback of many opioids (including the ones listed) is that they are either taken up so quickly that there can be a shock effect when applied or they take a long time to be taken up which makes it difficult for use in many of the scenarios a breakthrough -killer would be effective. PZM21 appears to be a very promising solution reaching its peak performance within 15 minutes which is second to fentanyl and remaining effective even up to 3 hours after being administered.5

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Binding Affinity

The binding affinity, Ki, represents how tightly the drug molecule binds to the µOR: the lower the number, the stronger the bond holding the molecule to the receptor. Fentanyl has the numerically lowest binding affinity meaning it binds the strongest to the µOR.

This is especially important in a hospital setting for overdose cases. Drugs that act as

µOR antagonists (e.g. and ) are given to counteract opioid agonists such as morphine or tramadol. These drugs work by competitively binding to the same

µOR sites. Because a drug like fentanyl binds so strongly, it is difficult to disengage fentanyl once it has been taken up by the µORs. PZM21 on the other hand has a significantly lighter hold on the µORs so it can be disengaged but it still binds more tightly than morphine (Table 1).3,4

Respiratory Depression

The reason PZM21 was developed in the first place was largely to find a drug that would act as a strong analgesic but not have the respiratory depression that is common in other opioids (Table 1). While tramadol is a bit different because it functions as both an opioid and a non-opioid drug, fentanyl and morphine both cause severe respiratory depression.

When administering morphine or fentanyl, the doctor must always take into account this factor and double check that the patient will make it safely through the dip in respiration.

PZM21 avoids this problem by binding at an orthosteric site and acting as an antagonist to β-Arrestin which prevents the beginning of the signal chain which lead to side effects such as respiratory depression and constipation.1,4,5

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Performance

Table S1: A Comparison of Various Opioids and Their Properties

Because of its prevalence in the medical industry, morphine has been used for the benchmark for analgesic performance (Table 1).

Compared to morphine, fentanyl is 100 times more potent than morphine and PZM21, tramadol are both weaker (4 times and 10 times respectively5). This difference in is effected by many things including but not limited to protein binding, steric hindrance, binding affinity, and additional intermolecular interactions and bonds.2,3,4,5 Bibliography

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Manglik, A; Lin, H; et al. Structure–based discovery of opioid analgesics with reduced side effects. Nature. 2016, September 08, 537(7619): 185–190

References

(1) Duthie, J.R. and tramadol. British Journal of Anaesthesia 1998, 81, 51–

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(2): Buch, J.G. Clinically Oriented Pharmacology, v2; Rajkot: 2010.

(3): Gillen, C; Haurand, M; Kobelt, D.J.; Wnendt, S. Affinity, potency and efficacy of tramadol and its metabolites at the cloned human mu-opioid receptor. Naunyn

Schmiedebergs Arch Pharmacol. 2000, 362), 116-21.

(4): Opioid Pharmacology. David Geffen School of Medicine at UCLA. https://www.ctsi.ucla.edu/education/files/view/training/docs/FERRANTE_M263_Opioid

_Pharmacology.pdf (Accessed 4/6/2018)

(5): Pain Management and Dosing Guide. American Pain Society. http://americanpainsociety.org/uploads/education/PAMI_Pain_Mangement_and_Dosing_

Guide_02282017.pdf (Accessed 4/6/2018)

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