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StructuralGenomicsConsortium / CNP8-Nsp13-Indoles

An Open Chemistry Network dedicated to finding hits vs SARS-CoV-2 Nsp13 based on a hit containing an indole.
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Purification issue #1

Open alvaro-magalhaes opened 1 year ago

alvaro-magalhaes commented 1 year ago

Synthesis of 3 out of the 4 molecules we are interested in was achieved successfully. However, the last one has been posing some purification issues. The entire synthetic plan begins with a low yielding reaction (Scheme below), which limits the amounts going forward but the problem arises at the ethyl ester hydrolysis.

The first time I attempted this reaction, I got a small amount (around 20 mg) of an orange solid with one impurity (LC-MS). The run in the LC shows the two peaks slightly overlapping at 100 % acetonitrile, which makes separating both challenging. I decided to repeat the synthesis and, yet again, I reached an orange solid which is not pure.

The ester hydrolysis was performed in methanol, with 1 M NaOH (5 equiv.), at 60 ºC for 15 minutes. After that time, a small amount of ice was added and the mixture was acidified with 1 M HCl, until pH 3 was reached. The suspension was filtered and the solid washed with water and hexane. The solid showed poor purity by NMR. The following triturations were attempted: DCM and diethyl ether. The residue was also dissolved in acetone and I tried to crystallise it with water. The solid obtained from this was also not pure and mostly containing the impurities. The filtrate solution was left at room temperature overnight and some precipitate formed - this was separated and washed with water. This last solid showed LC trace equal to the first attempt of making this molecule. LC-MS document here.

SMILES: O=C(/C=C(C(O)=O)\O)/C=C/C1=CN(C2=CC=C(F)C=C2)C3=CC=CC=C31 MW: 351.33; Exact Mass: 351.09 Structure: image

The difference between this molecule (1b) and the other one I made before (1c) is a CH2 between the indole and the phenyl.

Giving the proximity of the LC trace and that the MS trace only shows one peak, maybe having the phenyl directly attached to the indole creates some weird electronic effect and a mixture of cis/trans is formed. We can test some conditions to see if we can get better separation on the C18 column, otherwise a C8 column might help the separation.

mattodd commented 1 year ago

...so the evidence for E/Z is that the two peaks in the LCMS have the correct mass, right? How does the crude NMR spectrum compare with the spectrum for the ethyl ester? All the right peaks in there that you'd expect?

I would definitely run normal phase prep LC to see if you can nail this. Always difficult to crystallise small amounts.

Maybe you could reach out to Costi and Tramontano to ask how they did it if it's not described in full in the paper?

alvaro-magalhaes commented 1 year ago

This issue was discussed recently in person. A few more details on the data can be found here.

The mass on the LCMS is the correct mass (both positive and negative mode). The comparison of ester/acid NMR's is in the file above. Ethyl ester looks nice and pure and the NMR of the acid has some peaks that are not well resolved, not allowing to conclude if it's a mixture of E/Z.

The plan is to try to separate both compounds by prep HPLC and elucidate both structures by NMR. It was also raised the question of, if it's a mixture of E/Z, could they be in equilibrium? If that's the case, separating them might not lead anywhere but still worth to try it.

mattodd commented 1 year ago

Yes VT NMR might help answer the isomer-mixture question.