Biological assay 2023

[dmitrij176]

Good day @wwjvdsande and @MA-Jjingyi. Here is the finalised ketoxime list of the new batch for the assay. The package was dispatched yesterday and according to DHL tracking has beeen successfully delivered to Rotterdam today.

Please find attached supporting information Word document below. In summary: -There are 23 samples in the package. -All of them have an assigned MYOS codes. This includes additional MYOS entries for the separated isomers or E/Z. I have added these to the Master List and will update them with LogD soon. -Oxiconazole nitrate (MYOS0049005_01) salt has new formulation sub-number. Nitrates have an aissgned formulation number 5. Bio assay March 2023.docx

Unfortunately, 2 pyridinium compounds have to be removed from the list due to suspected tautomerisation (DM82-1) and an impurity in DM88-1. Will send the second in the next batch.

[MA-Jjingyi]

@dmitrij176 good afternoon. thanks for your reminder. we will let you know as soon as we get the package. and the MIC test will be performed soon.

[MA-Jjingyi]

@dmitrij176 Hihi, the package with 23 compounds was shipped successfully. just let you know.

[bebi78]

@dmitrij176 Just a note to your remarks about the relevance of possible counteranions. Different counterions may lead to different activities in anticancer active ruthenium complexes (see link below). Maybe it is also reasonable to prepare salts of ketoximes (and fenarimols, benzimidazoles, etc) with bioactive compounds like carboxylates (e.g., COX inhibitor ibuprofen?), or antifungal phenols such as niclosamide.

https://pubs.acs.org/doi/10.1021/ic801159f

[dmitrij176]

@dmitrij176 Just a note to your remarks about the relevance of possible counteranions. Different counterions may lead to different activities in anticancer active ruthenium complexes (see link below). Maybe it is also reasonable to prepare salts of ketoximes (and fenarimols, benzimidazoles, etc) with bioactive compounds like carboxylates (e.g., COX inhibitor ibuprofen?), or antifungal phenols such as niclosamide.

https://pubs.acs.org/doi/10.1021/ic801159f

@bebi78 thank you for the interesting suggestion. The concept outlined in the paper suggests addition of aqueous solution of the appropriate counterion to the organic product. What if we need to prepare an HCL salt formulation (which is quite common with drugs)? Would that be something like adding 10% aq HCL solution to the dissolved mixture? The reason why I am asking is that recently Ive been investigating the Z/E ketoxime isomerisation under specific acid conditions. According to the patents I am working with, addition of specific acid (either strong or weak) promotes isomerisation by shifting equilibrium towards one predominant geometric form. The final product is a salt as well and there is a link to your suggestion Bernard. Since ketoximes are isomers, can addition of aq 10% HCl create potential issue of getting unwanted isomerisation? At least thats why I suspect might happen.

[dmitrij176]

Summary of the concepts Im investigating: For E isomer salt- addition of strong acid like HCl to E/Z mixture (or Z isomer) in a selected solvent, reflux 3h.

For Z isomer salt- addition of weak acid like benzoic acid o E/Z mixture (or E isomer) in a selected solvent, reflux for 15 min to get a benzoate salt enriched with Z form.

2 factors affecting the equilibrium:

1. Acid
2. Temperature (reflux conditions)

[bebi78]

@dmitrij176 Yes, the influence on isomerisation is indeed interesting. I'm not sure if refluxing HCl solution might harm the oxime. Usually, for hydrochloride salt preparation of an organic compound such as pyridines or imidazoles, you dissolve your free base in polar organic solvent such as DCM and add an excess of 2M HCl/diethylether or 4M HCl/dioxane which are commercially available, stirr for some minutes at rt and remove the solvent again. Then, you can recrystallise the residue, your salt product, from proper organic solvent mixtures.
Sometimes I also use sulfonic acids (TosOH or MesOH) for salt preparation in a similar way, with the only difference that I use equimolar amounts (or only a slight excess) of the sulfonic acids. Some approved drugs are mesylates like imatinib. Might be interesting to compare with benzoic acid performance in terms of isomer enrichment.

[wwjvdsande]

@dmitrij176 our technician is preparing the dilutions for your compounds. However there were some duplicates in the bag. For most of the compounds we were able to identify them based on the weight provided. However for MYOS_00_417_00_01 and MYOS 471_01_01 both known as DM74-6 we found the same weight. See the picture below. Which one is which? They seem to be differentiated by a blue line on the lid or two black lines

Another question, just to verify. MYOS_00_489_01_01 is in the list noted as DM91-1, but on the lid we find DM93-2. Is that MYOS489 or 488?

[dmitrij176]

Good day @wwjvdsande . Apologies, I will attach pre-printed labels next time. With regards to DM74-6, they are separate E and Z isomers, which means that they are different entries with unique MycetOS codes. The vial you are holding in the picture is labelled: E isomer, so the other 3 mg vial is Z.

MYOS489 is DM93-1 (E isomer)

[wwjvdsande]

@dmitrij176 is the one with the double lines on the lid for DM74-6 the E isomer, so MYOS-417? E isomer was not written on the vial. It was written on the vial for DM93-1.

[dmitrij176]

@MA-Jjingyi could you please resend an updated postal adress with your name for the upcoming shipment. Since Wendy is on holiday, I will need to dispatch the molecules to you. Also a quick question: any in vitro results coming anytime soon for the ketoximes March 2023 batch ? Thanks

[MA-Jjingyi]

Hi @dmitrij176 , The shipping address is:

ErasmusMC Department of Medical Microbiology & Infectious Diseases, room Nb920 T.a.v Wendy van de Sande and Ma Jingyi Dr Molewaterplein 40 3015 GD Rotterdam The Netherlands

For courier: Wendy van de Sande, Ma Jingyi Tel 0031-10-7033510 The phone number is of the secretary in our department, there should always be somebody during visiting hours.

As we talked last time, the technician performs in vitro susceptibility testing during summer. Due to puzzled information in bottle, she didn't start screening before her holiday. She will start screening after her summer holiday. we will let you know when we get results. Nice weekend.

[dmitrij176]

@dmitrij176 our technician is preparing the dilutions for your compounds. However there were some duplicates in the bag. For most of the compounds we were able to identify them based on the weight provided. However for MYOS_00_417_00_01 and MYOS 471_01_01 both known as DM74-6 we found the same weight. See the picture below. Which one is which? They seem to be differentiated by a blue line on the lid or two black lines

Another question, just to verify. MYOS_00_489_01_01 is in the list noted as DM91-1, but on the lid we find DM93-2. Is that

@wwjvdsande I have finally resolved the issue. The following refers to DM74-6 samples with identical 3 mg mass. MYOS_00417_01_01 is the E-isomer and can be identified by number 8 on the cap/vial MYOS_00471_01_01 is the Z-isomer and can be identified by numbers 5,6 on the ca/vial

The numbers can be clearly seen on the pictures you have taken. I need to know whether your team will be able to differentiate between these entries or I need to resend them again. Let me know as soon as possible. I might be able to include them in the current shipment if not too late

[dmitrij176]

@wwjvdsande @MA-Jjingyi the latest ketoxime batch (including additional 23 molecules from Series 2) has been dispatched. Due for delivery tomorrow. MYOS_samples_210723.xlsx Bio assay August 2023 

[dmitrij176]

Bio assay August 2023 .docx

[MA-Jjingyi]

@wwjvdsande @MA-Jjingyi the latest ketoxime batch (including additional 23 molecules from Series 2) has been dispatched. Due for delivery tomorrow. MYOS_samples_210723.xlsx Bio assay August 2023

Hi @dmitrij176 , will let you know when we get new compounds. thanks for your delivery.

[MA-Jjingyi]

Hi,@dmitrij176. The package arrives at the lab. just let you know.

[MA-Jjingyi]

@dmitrij176 . good morning. a part of compounds list is as following: series 6 compounds list by EMC 21092023.xlsx

the blue one has same MYOS code with the orange one. please give us hands solve this problem.

[MA-Jjingyi]

@wwjvdsande @dmitrij176 Hi Dmitrij, just let you know, I will be in holiday, so, please notice Wendy when you reply to us.

[dmitrij176]

@dmitrij176 . good morning. a part of compounds list is as following: series 6 compounds list by EMC 21092023.xlsx

the blue one has same MYOS code with the orange one. please give us hands solve this problem.

@MA-Jjingyi I am not sure what happened, but the excel file which you mention is incorrect and has mistakes. It has multiple errors. Looking carefully at the data, I came to conclusion that the MYOS codes were duplicated for 2 different series- that is series 2 and series 6. It is likely to have occured because series 2 MYOS codes were never entered into the Master List, whereas my compounds were.

What I suggest:

1. The excel list will have to be corrected or created again. At the moment there are multiple series 6 duplicates and few series 2 which is not the case.
2. My MYOS codes are correct because they are in the Master List database https://docs.google.com/spreadsheets/d/1YhK-3i2KwuVabo1GbZSgVjAUbavEICCMKi5v-EhNq80/edit#gid=954203120 Please check. Same applies to the Word document which I sent you with the shipment. Bio.assay.August.2023.docx Each vial has a printed sticker with the same compound information as in the document.
3. Series 2 compounds will have to be assigned new MYOS codes by whoever was responsible for that list originally. At the moment I would suggest to use the 4 character codes (A4B1-A5B2) from column one. MYOS_samples_210723.xlsx

@wwjvdsande would you kindly help us to resolve the issue and get the right assignment for the assay list?

[wwjvdsande]

@dmitrij176 I will check with the lab. The duplication seems to be in series 6 only. There are big vials and small vials. Can't it simply be that you sent these compounds twice: one with a large amount and one with a small amount? The large vials are in powder, the small ones not but still have the same code. So is that the difference? That these are two duplicates of the same compound but differently purified?

[wwjvdsande]

@dmitrij176 I now understand what problem is. They are the compounds from the two different batches. So indeed they are your compounds but you sent them twice. One in the beginning of summer and one in the end. The only thing which got us puzzled is that although they should be the same, they look different in consistence (oily compared to powder) and a difference in molecular weigth. So the question really is, which version of the molecule do you want us to test? The first batch or the last batch you sent? We are not chemists and are a bit alarmed when it looks different. Usually our drugs always look the same. So amphotericin B is always a yellow powder, itraconazole is always white. Now we get two times the same compound but they look different so that sets of an alarm by us. We just want to know which is the correct one to test.

[dmitrij176]

@dmitrij176 I now understand what problem is. They are the compounds from the two different batches. So indeed they are your compounds but you sent them twice. One in the beginning of summer and one in the end. The only thing which got us puzzled is that although they should be the same, they look different in consistence (oily compared to powder) and a difference in molecular weigth. So the question really is, which version of the molecule do you want us to test? The first batch or the last batch you sent? We are not chemists and are a bit alarmed when it looks different. Usually our drugs always look the same. So amphotericin B is always a yellow powder, itraconazole is always white. Now we get two times the same compound but they look different so that sets of an alarm by us. We just want to know which is the correct one to test.

This is even more confusing. There is no way the compounds could have been sent in duplicates. There are 2 isomers for each entry (option A: seprate E and Z; Option B: one isomer plus E/Z mixture). I checked this thoroughly before sending the last batch. Also, I definitely remember printing only one set of labels (usually 2 labels per entry as I said).

Looking back at Ma's list, rows 86- 105 look totally incorrect. The masses there are enormous. 466 mg, 408 mg, 96.8: I never had anything even close to that in terms of yields and the scale of reactions. There is definitely a mistake.

Compounds highlighed in peach colours and some in blue white (MYOS 578, 584,585, 583) look like the ones that I sent.

Large mass numbers in blue are series 2 aminothiazoles for sure.

[dmitrij176]

MYOS_samples Series 2 corrected.xlsx @wwjvdsande updated Series 2 list with new MYOS codes

[dmitrij176]

@mattodd @wwjvdsande @bebi78 @MA-Jjingyi @OpenSourceMycetoma/corecontrib latest in vitro ketoxime potency results with Stardrop calculated logD values. @wwjvdsande as we discussed earlier, the in vitro results for most tested ketoximes in this batch show complete fungal inhibition. Whenever possible and time permits, could your team please try to get additional data points (IC50, IC90, MIC) and maybe in vivo for very promising candidates. Otherwise, it is impossible to rank these actives.

[wwjvdsande]

@dmitrij176 we are performing them now. hopefully we can upload some of it before the christmas break otherwise it will be before the end of january.

[dmitrij176]

Bio assay December 2023.docx

Good day @wwjvdsande @MA-Jjingyi. I have dispatched my last batch of ketoximes, covering some SAR gaps in the series as well as one missing Fenarimol molecule that was not assessed previously. Could you please test these for us if still possible. The shipment is due tomorrow.

[dmitrij176]

@wwjvdsande the compounds I was asking about are DM91-2F, DM91-2,3,5F and DM91-2,3,5F (dialkylated). These have fully inhibited MAdurella at both concentrations. Any IC50/90, MIC upcoming?

As you may also remeber, commercial Oxiconazole did perform extremely well and to date is the best scoring ketoxime in Series 6. If possible, can you please include it in the new larvae in vivo screening season? Very important molecule; would be interesting to see its influence on larvae survival.

[dmitrij176]

@wwjvdsande @MA-Jjingyi further to the in vitro stage assessment, is there generally a plan to investigate potent ketoximes in vivo? Noticed that a substantial number of series 6 analogues have good IC50/90 values, making them suitable for phase 2. Can you update me on the plans?

[bebi78]

@dmitrij176 @mattodd In addition to CYP51 inhibition, oxiconazole was described as a DNA synthesis inhibitor, maybe like olorofim (DHODH inhibitor)? A close analog, sulconazole, also inhibited DNA and RNA synthesis, but I couldn't find that mechanism for other azoles so far (maybe you can search literature again). Moreover, oxiconazole showed inhibitory effects on Akt/mTOR kinase signaling in cancer cells, but such effects were also described for itraconazole in cancer cells (so probably not unique for oxiconazole). Don't know if pyrifenox has also such effects but combined ergosterol suppression, DNA synthesis inhibition, and Akt signaling downregulation by oxyconazole looks like an excellent combination of mechanisms for an antifungal, and might justify in vivo tests.

https://journals.asm.org/doi/10.1128/cmr.1.2.187 https://www.ijbs.com/v18p3747.htm

[dmitrij176]

I did extensive literature search for ketoximes, including Pyrifenox, but so far never came across these additional properties. Thank you for the suggestions @bebi78. Looks quite interesting.

[dmitrij176]

Ps: will double check that in the literature