2023 York University project

[P-Rayner]

Please accept mine and @JuliaSarju's apologies for the lack of update. This semester has been quite the... challenge.

We have been making progress on synthesising a range of phenothiazines and derivatives. Here is an updated summary. If any of the compounds are of interest we can send to people ASAP.

SMILES Synthesised Compounds CC(N1C2=CC=CC=C2SC3=C1C=CC=C3)=O O=C(C1=CC=CC=C1)N2C3=C(C=CC=C3)SC4=CC=CC=C42 CCN1C2=C(C=CC=C2)SC3=CC=CC=C31 C12=CC=CC=C1N(CC3=CC=CC=C3)C4=C(C=CC=C4)S2 O=C(CCl)N1C2=C(C=CC=C2)SC3=CC=CC=C31 C12=CC=CC=C1N(CCC3=CC=CC=C3)C4=C(C=CC=C4)S2 O=S(N1C2=C(C=CC=C2)SC3=CC=CC=C31)(C4=CC=C(N+=O)C=C4)=O O=S(N1C2=C(C=CC=C2)SC3=CC=CC=C31)(C4=CC=CC=C4N+=O)=O CCCCCN1C2=C(C=CC=C2)SC3=CC=CC=C31

Purification Required O=C(CN1CCCC1)N2C3=C(C=CC=C3)SC4=CC=CC=C42 O=C(C)N1C2=C(C=CC(C(F)(F)F)=C2)SC3=CC=CC=C31 CCCCCN1C2=C(C=CC=C2)OC3=CC=CC=C31 O=C(C1=CC=CC=C1)N2C3=C(C=CC=C3)OC4=CC=CC=C42 O=C(C)N1C2=C(C=CC=C2)OC3=CC=CC=C31 CCN1C2=C(C=CC=C2)OC3=CC=CC=C31

Synthesis in Progress O=C(CN(CC)CC)N1C2=C(C=CC=C2)SC3=CC=CC=C31 O=C(CN1CCCCC1)N2C3=C(C=CC=C3)SC4=CC=CC=C42 O=C(CN1CCN(C)CC1)N2C3=C(C=CC=C3)SC4=CC=CC=C42 CCN(CC)CCN1C2=C(C=CC=C2)SC3=CC=CC=C31 C12=CC=CC=C1N(CCN3CCCCC3)C4=C(C=CC=C4)S2 C12=CC=CC=C1N(CCN3CCCC3)C4=C(C=CC=C4)S2 CN(CC1)CCN1CCN2C3=C(C=CC=C3)SC4=CC=CC=C42

Chemdraw file PhenothaizinesUoY.zip

If you could let me know what to do next, I would greatly appreciate the guidance! Hope you all have a great Christmas break.

[MFernflower]

@P-Rayner - This looks fantastic!!!!!!!!!

I do think we are still waiting on test results from another class of tricyclics derived from curaxin 137 - so could be another core to look into if they prove potent (carbazoles and carbolines) @lferrins @wwjvdsande @MA-Jjingyi

[MFernflower]

@P-Rayner

I don't think this is too much of a concern but a few are pretty close to promethazine and therefore might have sedative effects (the ones I posted below) - I wonder if you have not made these yet if you could change up the head and tail regions a bit (I posted an example on the end of this comment?)

[P-Rayner]

Thanks @MFernflower. I think you are probably right and we will look to change things up abit. The students are busy with exams until Feb now so we can explore synthesis options if any exciting biological data arises beforehand. :relaxed: I am also happy to post any compounds early in the new year if any are of interest.

[MFernflower]

@MOUSEY007 would your lab be interested as well in some of these compounds? @lferrins

[MFernflower]

@P-Rayner

Some interesting ideas from searching pubchem:

https://pubchem.ncbi.nlm.nih.gov/compound/352306

https://pubchem.ncbi.nlm.nih.gov/compound/85792721

https://pubchem.ncbi.nlm.nih.gov/compound/826961

[MFernflower]

Actually turns out the ones with the free hydroxy are listed as inactive in some pubchem yeast assay so perhaps something like 2-ethoxyethyl chloride or 2-methoxyethyl chloride could be used to attach interesting tails to the tricyclic cores @P-Rayner

[P-Rayner]

Thanks @MFernflower and Happy New Year! We can certainly make those derivatives. We have 8 days left in the lab after the exam period so we should get through a fair bit more synthesis.

[MFernflower]

@P-Rayner might be worth trying different cores - Dibenzazepine is pretty cheap

[MFernflower]

Actually scrap that the hydrogenated version is cheaper and can be delivered to the uk: https://www.tcichemicals.com/GB/en/p/D0269 @P-Rayner - also we are still waiting on the test results from #6

[bebi78]

@MFernflower @P-Rayner Some papers calculated/determined the ´´butterfly angle´´ of bioactive N-substituted phenothiazines. Might be interesting if there is a correlation of butterfly angle with antifungal activity.

[MFernflower]

Yeah there's a rich amount of stuff to be explored in the tricyclic space - for example swapping out phenothiazine for so-called "Iminodibenzyl" would get us closer to compounds like the NEU Curaxin ones of which we are still waiting for potency data on to be posted @OpenSourceMycetoma/corecontrib

[MFernflower]

@JuliaSarju @P-Rayner Are you able to share plans for what next your group wants to make or should I wait for the schools meeting?

[P-Rayner]

@MFernflower Yes! I will upload something this afternoon. We are pretty flexible and can discuss further at the schools meeting.

[MFernflower]

Sounds good @p-rayner

[P-Rayner]

Here is the current state of play for the phenothaizines we have made in York to date along with somethings that are ongoing as the students get towards the end of their projects. I will also post an update of the aminothiazoles on the corresponding series 2 page.

We are also looking to make analogues of these with different cores but these are probably going to be when the next group of students start and we have some more biological data to target what might be useful. Judging by the recent biological data it looks like the tails might have more of an effect that then cores but happy to discuss on Monday so we can target efforts going forward. We are looking forward to see how the testing on the ones we have sent get on.

[MFernflower]

Looks super good! I was just wondering that since the tail seems important - do you have time or space available to have a student reflux isovaleryl chloride, phenothiazine, and toluene for a few hours? - I will try to attend the meeting tomorrow @P-Rayner

[P-Rayner]

@MFernflower Yes - I will ask one of the students to do this and let you know how it goes.

[P-Rayner]

Hope everyone is well. Following the recent meeting, here are the general procedures we have used to make our phenothiazine derivatives. All pretty straight forward. We have another batch of compounds that are ready to send once I am back from holiday after Easter.

Acylation General Procedure Acetyl chloride (1.2 equiv) was added to a stirred solution of phenothiazine (1.0 equiv.) in toluene (60 mL) at room temperature. The mixture was heated to reflux for 3 h (monitored by TLC (Petroleum-EtOAc 9:1). Then the solution was allowed to cool to rt and saturated sodium bicarbonate (25 mL) was added. The mixture was extracted with ethyl acetate (2 x 25 mL) and the combined organic layers were washed with 3 M HCl (25 mL), water (2 x 50 mL), dried (MgSO4) and concentrated under reduced pressure to give the crude product. The crude product was recrystallised from ethanol to give the pure compound.

Alkylation General Procedure Phenothiazine (1.00 g, 5 mmol, 10 equiv.) was added to a stirred solution of toluene (ca. 5 mL) and 12 M aq. NaOH (7 mL). To this, tetrabutylammonium iodide (0.17 g, 0.5 mmol, 1 equiv.) and 1-bromobutane (2.2 mL, 20 mmol, 40 equiv.) was added. The reaction mixture was heated to 110 °C with stirring and monitored by TLC (9:1 n-hexane/EtOAc) with stirring until the reaction was observed to be complete (ca. 3 h.). The reaction mixture was cooled to 30 °C (ca. 20 minutes). The reaction mixture was extracted with ethyl acetate (2 x 10 mL) and the combined organic layers were dried (MgSO4), and the solvent was evaporated under reduced pressure, giving the crude product as a yellow-brown solid. Purification by column chromatography using petroleum ether as an eluent yielding the product.

Reductive Amination General Procedure Phenoxazine (1.00 g, 5.45 mmol, 1 equiv.), Triflouroacetic acid (4.05 mL, 6.03 g, 52.87 mmol, 9.7 equiv.), triethyl silane (8.71 mL, 6.34 g, 54.50 mmol, 10.0 equiv.) and phenylacetaldehyde (1.21 mL, 1.31 g, 10.90 mmol, 2 equiv.) was charged in CH2Cl2 (50 mL) under N2 at 0 °C. The reaction was monitored by TLC (9:1 petrol/EtOAc). Upon completion (ca. 4 hours) the reaction mixture was washed with NaOH (1 x 50 mL) and extracted by EtOAc (3 x 30 mL). The organic layer was then dried in vacuo to give an emerald solid and recrystalised in hot EtOH to give the purified compound.

Sulfonylation General Procedure Dry pyridine (5 mL) was added to a solution of phenothiazine (1.00 g, 0.005 mol) in toluene (30 mL). The reaction mixture was warmed to 50 °C and stirring commenced. 4-nitrobenzenesulfonyl chloride (1.32 g, 0.0052 mol) was added slowly to produce a dark red solution that was heated to 90 °C for 30 minutes. The mixture was then left to stand at room temperature overnight. The resulting solution was filtered to remove solid impurities and then concentrated to produce a black viscous mass. The crude product was recrystallised from hot ethanol to form the final product as a yellow solid.

[MFernflower]

@P-Rayner I changed a little bit of this for clarity and put it in the wiki - can you look over it some time? https://github.com/OpenSourceMycetoma/Series-3-Phenothiazines/wiki/Synthetic-Chemistry-Overview

Have a nice holiday

[P-Rayner]

Hi @MFernflower. Thanks - all looks good to me.

[MFernflower]

@P-Rayner I am just curious - since these molecules are made in a few hundred mg batches every-time. I'm going to ping @lferrins because her paper with @MOUSEY007 was what inspired me to push this whole project forward

[MFernflower]

to see maybe if samples can be shared for there research into trypanosomes and ameobae

[lferrins]

I can facilitate a connection to our tryps collaborator just let me know? I will let Chris comment on whether he can screen for amoeba.

[P-Rayner]

@P-Rayner I am just curious - since these molecules are made in a few hundred mg batches every-time. I'm going to ping @lferrins because her paper with @MOUSEY007 was what inspired me to push this whole project forward

We have plenty of most of the compounds available here in York so happy to send things off if desired.

[bebi78]

@MFernflower @P-Rayner Yes, drugs against advanced sleeping sickness that can enter the brain/cross the BBB are sought! I think tricyclic compounds would fit well for that purpose.

[MFernflower]

Was thinking more towards Naegleria as sleeping sickness technically has a cure (fexinidazole)

[bebi78]

@MFernflower Monotherapy poses a high risk of resistance formation. Cross-resistance with nifurtimox is also considered as a problem. DNDi is developing acoziborole as a second drug with different mode of action now.

[P-Rayner]

Hi everyone - hope you are all well.

I have just posted a final batch of phenothiazines that we finally purified from the project that ran with students last academic year.

When the students return after the summer holidays we will have some more students in the lab with the aim to change the tricycle but keep the N-ethylisopropylamine tail as suggested by @MFernflower.

[KlementineJBS]

Hello Peter - how many compounds have you sent? I'm guessing they would be the ones with MYOS codes 738-768? Also, just wondering if you saw that there are now MIC values for the batch with MYOS codes 657 - 668.

[P-Rayner]

Hi @KlementineJBS, yes they are the ones, though there are some remaining series 2 compounds in there as well. I had seen the other results - unfortunately disappointing but at least gives us some direction of where to go next. I am working with some students to pull together the data visually in a similar way to the series 2 compounds.

[MFernflower]

@P-Rayner this would be fantastic

I am quite curious as to where the stock of extra phenothiazines are going to go now that they are found inactive in eumycetoma? Do they go into a compound library at the university? If shipping is not too costly might be worth submitting them to CO-ADD @mattodd Just a random shower thought

[P-Rayner]

They are currently all just in our chemical store along with any intermediates in their synthesis etc.

[KlementineJBS]

Hey @P-Rayner - I don't have official triplicate results to share yet but having looked at the plates I suspect that the initial results will be replicated and unfortunately only a handful of the latest batch had any promising activity - namely compounds MYOS741, 752, 771 and 772 (initial results here). I'll be doing MIC determinations for them from next week but the rest failed to inhibit fungal growth even at high concentrations.

[MFernflower]

Very interesting information: MYOS741 and 752 are rather different from the expected sar for series 3

MYOS771 and 772 aren't that much different from the original series 2 hit save for the fact that methyl group of the pyridine has been replaced by bromine

SEP23_2024.CSV

@OpenSourceMycetoma/corecontrib

[P-Rayner]

@KlementineJBS Great news. Thank you very much for the update!

[MFernflower]

Cross ref to https://github.com/OpenSourceMycetoma/Monthly-zoom-meetings/issues/26

[KlementineJBS]

Hello - just wondering what happened to these compounds? I don't see testing results for them, were they made in the end or not?

[P-Rayner]

We have most of these compounds but I didn't deem them pure enough for testing. We will either purify them or resynthesise them, and send them with the next batch of compounds. Peter

On Fri, 11 Oct 2024 at 08:49, Klementine Burrell-Sander < @.***> wrote:

Hello - just wondering what happened to these compounds? I don't see testing results for them, were they made in the end or not?

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[KlementineJBS]

Hello, I've finished the high concentration testing and unfortunately we've only had 3 good hits - MYOS752, 771 and 772. I'm running MICs for these at the moment.

MYOS741 and 746 did inhibit at higher concentration (100 uM) but unfortunately not at lower concentrations (25 uM).

All the results are in the masterlist now, or you can find it on the second page of this file: High-low results Aug - Oct 2024.xlsx

[P-Rayner]

Hi @KlementineJBS This is great. Thank you for all the information. It be worth making a few analogous of 752 with different tricycles on there. Peter