Post #3, EXP Fun Fun

I have around 2 more weeks left at the Ballatore Lab at UPenn, and so far I've had a very good experience. Although my lab is small, with only 4 people working at the moment, there is a very unique dynamic at play, and always an opportunity to learn something new (or make a mistake). 

Goals

So, as I explained in my previous blog post, we completed the reaction to create: 

5-Chloro-6-(2,4,6-trifluorophenyl)-N-(2,2,2-trifluoroethyl)-[1,2,4]triazolo[1,5-a]pyrimidine-7-amine (C₁₃H₆ClF₆H₅)

Overall, the yield was 100mg, or above around 30% yield, which was the goal for this reaction. Since the compound is a possible drug candidate, the goal was to produce >100mg, the amount required to conduct advanced drug testing. Our lab is unique in that Dr. Ballatore is a professor in both the Department of Chemistry and the Department of Pathology and Laboratory Medicine; this means that there is close collaboration between the Ballatore Lab (Chemistry) and the Center for Neurodegenerative Research (Drug Testing/Biology) in the Perelman school of Medicine. Whereas labs elsewhere sometimes spend months collaborating in order to mediate drug synthesis and testing, compounds created in our lab are able to be quickly tested for pathological "activity" (whether they display stabilization of tubulin, a key factor in Alzheimer's research), with results within a week or two. 

With this in mind, Dr. Carlo decided that the next step would be to 

1.Create a new, previously un-synthesized compound

2. Visit the Perelman School of Medicine and observe drug testing

Reaction Overview

Although the reaction sounds confusing, it was almost identical to the previous reaction I previously finished; the only difference was the amine that we used, the functional group that was added to the starting compound in the substitution. (The products of both reactions belong to the same family of chemical analogues known as "imidazoles"). Another minor difference was the scale of the reaction. Since the reaction had not been done before, we used approximately 4x less reagent than in a normal reaction, in order to minimize possible loss of compounds.

The new compound I would synthesize was 5-Chloro-6-(2,4,6-trifluorophenyl)-N-[1,3-Dimethylbutylamine][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (C₁₇H₆ClF₆) through a "substitution reaction", a reaction where "one functional group in a chemical compound is replaced by another functional group" (Wikipedia). Simplified, this meant I would remove a functional group off the starting compound (a chlorine atom) and replace it with an amine group, in this case "1,3-Dimethylbutylamine". 

1.     5,7-dichloro-6-(2,4,6-trifluorophenyl) [1,2,4] triazolo [1,5-a] pyrimidine (starting compound)
 2.     1,3-Dimethylbutylamine (amine group)
3.      5-Chloro-6-(2,4,6-trifluorophenyl)-N-[1,3-Dimethylbutylamine][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (final compound)

The process of synthesizing the compound was relatively straightforward, as I had already successfully completed a very similar reaction. The steps were exactly the same: dissolution of the starting compound in solvent (DMF), followed by the quick addition of the amine and base (DiEA), before leaving the reaction to complete overnight. 

LC-MS (It takes samples from the test tubes)

Afterwards, we took the reaction mixture and did an LC-MS scan on it (Liquid Chromatography-Mass Spectrometry) in order to determine whether the reaction had been completed or not. 
(Mass-Spectrometry reveals the molecular weight of the compounds present in a sample and based on the molar weight of the predicted compound, we can tell whether the reaction is successful or not). 
Since we saw a large peak of the predicted compound, and no peaks for the reagents, we concluded that the reaction has concluded and began the purification, first completing a "workup" to purify the reaction mixture into a still relatively crude mixture, then using the HPLC (High-performance liquid chromatography) machine. 





Quick Digression (HPLC Summary):

HPLC (It expels the constituent compounds into test tubes)

The machine works according to the basic principles of liquid chromatography, the same way that a TLC or column chromatography would work. By running a certain "eluent" (a liquid mixture of a specific ratio between a polar and non-polar solvent) literally through a sample, the constituent compounds of the sample will separate, as they "adsorb"/adhere to the "eluent" at different times, based on their differing polarities. The HPLC machine, therefore, is a very precise and effective way to purify a compound (>95%), but usually takes a very long to conduct. In order to be sure of the precision of the machine, each trial/run can only handle up to 50mg of a sample, with each trial taking between 10-15 minutes (not including setting up the machine, throwing away tubes, etc). Since the scale of the reaction this time was relatively small though, this time the HPLC was quick and painless :). 

Finishing the HPLC, we were collected the tubes that contained the desired product (based on computer) and collected them in a single vial. Since the product was still dissolved in the eluent, we used a combination of Rotovapor (a machine that simply dissolves solvent) and the freeze-dryer (low pressure-vacuum) to get rid of the excess solvent and completely purify the product. 

Plans:
Having successfully conducted the reaction, the next step will be to bring the sample to the Perelman School of Medicine (Center for Neurodegenerative Disease Research) and see the drug tested on cells, specifically an ELISA test that will test for the sample's tubulin stabilizing abilities. Whether or not the compound will undergo further testing (transgenic mice, clinical testing) will be based on the results of the test (!).