Synthesis of the Original Hit "RA-0001351" and the subsequent cysteine adduct.

[qxsml]

Plan：

Step 1. Make a lot of the original hit RA-0001351:

Step 2. deliberately synthesise the adduct of cysteine to the iminoquinone RA-0001351. Obtain NMR spectra to confirm the structure and HPLC trace to record the retention time of the adduct.

Step 3. Deliberately synthesise the adduct of the cysteine to commercially available iminoquinones using the optimised conditions developed in step 2.

[qxsml]

Step 1 completed: synthesized 800 mg intermediate 1 (600 mg remaining) and 300 mg of RA-0001351 as a yellow needle crystal

[qxsml]

Step 2 in progress:

Rationalisation:

1. I choose ethanol as the solvent as it can dissolve both starting materials, and is less nucleophilic than methanol.

2. The reaction was started at room temperature, tlc (please see below) 5 min and 24 h showed almost no reaction. Heat the reaction at 80 degree is able to completely consume the iminoquinone starting material.

3. The polarity of the develoing solvents was increased to move the new spot above the bottom line (DCM/MeOH 1:1). The t.l.c. (please see below) was visualised using UV and Iodine in slica bath.

The reaction was attempted to purify using normal phase Biotage, failed. Details was explained below:

First, It's out of my expectations and odd that the new spot is lower running than N-acetyl cysteine. Could this be due to the acetyl group falling off? Will need to isolate the spot and if it is, do a 40-degree reaction later.

Second, in the first trial of purification, 10% MeOH to 50% MeOH in DCM (gradient), the desired spot eluted together with other spots at ~28% MeOH and could be detected by t.l.c.. However, I couldn't detect it again in the second trial, not even after using 100% MeOH.

Correction to the above: **I concentrated two different fractions after purification, submitted H NMR in CDCl3 and found no interesting signals, so I thought somehow I lost the material. However, the other day, I looked at the RBFs which were used to concentrate these fractions, and I could see something in them. I then submitted H NMRs using DMSO-d6. I'm attaching their H NMR spectra below.

The quantity of the material was very small, so I misjudged and thought they had been dissolved in CDCl3, but in fact, not.**

Below is the H NMR spectrum for the higher running spot, Rf~0.15, which I think indicates 4-chlorobenzylsulfone group falls off.

Below are the H NMR spectrum and LCMS spectra for the lower running spot Rf ~0.1, whose structure remains to be confirmed.

Current progress a 60 mg scale reaction under purification in order to get enough material for a decent full NMR analysis.

[mattodd]

I wonder if you want to watch the reaction by 1H NMR first, to see what's what, and to use a solvent that is not remotely nucleophilic. What are the chances that your alcohol is competing off the thiol just because there's a lot more of it?

[drc007]

The structure you have drawn is not N-acetyl Cysteine

[qxsml]

I wonder if you want to watch the reaction by 1H NMR first, to see what's what, and to use a solvent that is not remotely nucleophilic. What are the chances that your alcohol is competing off the thiol just because there's a lot more of it?

Thank you very much for your advice! I wonder do you mean to submit a crude NMR or to use deuterated solvent and follow the reaction by NMR?

Yeah, I agree the solvent may compete -SH, that's why I choose ethanol over methanol. I'll try other solvents since I'm heating the reaction so both starting materials may dissolve in solvents that are unable to dissolve them at room temperature.

[qxsml]

The structure you have drawn is not N-acetyl Cysteine

Thank you very much for pointing out, my mistake, I'll correct it.

[mattodd]

Yes, deuterated solvent, just to establish that a reaction is taking place, and one that looks reasonable.

[ahsgc]

Step 2 in progress:

Rationalisation:

1. I choose ethanol as the solvent as it can dissolve both starting materials, and is less nucleophilic than methanol.
2. The reaction was started at room temperature, tlc (please see below) 5 min and 24 h showed almost no reaction. Heat the reaction at 80 degree is able to completely consume the iminoquinone starting material.
3. The polarity of the develoing solvents was increased to move the new spot above the bottom line (DCM/MeOH 1:1). The t.l.c. (please see below) was visualised using UV and Iodine in slica bath.

The reaction was attempted to purify using normal phase Biotage, failed. Details was explained below:

First, It's out of my expectations and odd that the new spot is lower running than N-acetyl cysteine. Could this be due to the acetyl group falling off? Will need to isolate the spot and if it is, do a 40-degree reaction later.

Second, in the first trial of purification, 10% MeOH to 50% MeOH in DCM (gradient), the desired spot eluted together with other spots at ~28% MeOH and could be detected by t.l.c.. However, I couldn't detect it again in the second trial, not even after using 100% MeOH.

so the current plan would be:

Repeat the reaction on a larger scale (50 mg) and isolate the new spot to see what it is, then determine the next step.

I was too rushed to do the reaction on a 10 mg scale because I thought if this one worked, then I could simply repeat the same procedure for the other two purchased (25 mg) iminoquinone substrates.

Hi @qxsml do you want to monitor the pH of your reaction mixture and then add some base to mimic any biochemical assay? Also, I recommend running a control reaction with a known cysteine-capturing agent to compare your reaction.

[qxsml]

Step 2 in progress: Rationalisation:

1. I choose ethanol as the solvent as it can dissolve both starting materials, and is less nucleophilic than methanol.
2. The reaction was started at room temperature, tlc (please see below) 5 min and 24 h showed almost no reaction. Heat the reaction at 80 degree is able to completely consume the iminoquinone starting material.
3. The polarity of the develoing solvents was increased to move the new spot above the bottom line (DCM/MeOH 1:1). The t.l.c. (please see below) was visualised using UV and Iodine in slica bath.

The reaction was attempted to purify using normal phase Biotage, failed. Details was explained below: First, It's out of my expectations and odd that the new spot is lower running than N-acetyl cysteine. Could this be due to the acetyl group falling off? Will need to isolate the spot and if it is, do a 40-degree reaction later. Second, in the first trial of purification, 10% MeOH to 50% MeOH in DCM (gradient), the desired spot eluted together with other spots at ~28% MeOH and could be detected by t.l.c.. However, I couldn't detect it again in the second trial, not even after using 100% MeOH. so the current plan would be: Repeat the reaction on a larger scale (50 mg) and isolate the new spot to see what it is, then determine the next step. I was too rushed to do the reaction on a 10 mg scale because I thought if this one worked, then I could simply repeat the same procedure for the other two purchased (25 mg) iminoquinone substrates.

Hi @qxsml do you want to monitor the pH of your reaction mixture and then add some base to mimic any biochemical assay? Also, I recommend running a control reaction with a known cysteine-capturing agent to compare your reaction.

Thank you! Excellent idea.

[mattodd]

Hi @qxsml - for the above, is more discussion needed, or can we close this issue? I've linked to this issue in the wiki. Is the discussion of the identity of this adduct now moved to another issue, like #4? If yes, close this issue with a comment. If something more is needed here, what is it? (Thinking about the above reactions, running the control with nothing added (to check for thermal decomp in the solvent) and with something similar like N-Ac-alanine etc, might help resolve what changes are occurring because of the presence of the thiol).