OSM Series 4 - Next Round of Synthesis

[mattodd]

It’s decision time on the next set of targets.

Alice just mailed the latest set of analogs she has shepherded through (OSM-S-291 to OSM-S-301). This set contains a few compounds that will add to the data surrounding tolerance in the northeast (arising from our 1st year undergrads and Seb Dath’s work (1, 2), and some compounds probing tolerance of a benzyl-linked pyridine in the northwest - we could still explore benzyl substituents more, particularly with blocking groups (as per Chris’s suggestion) - see below for more on this.

But which molecules are next? Generally we need to lower predicted logP, work towards lowering metabolic clearance times while maintaining or improving potency.

Analog design in this series has previously been discussed: June 2015 ELN post, March 2015 ELN Post, December 2014 Xmas Shortlist, and in issue #301.

The most recent biological evaluations were: June 2015 May 2015 Edinburgh November 2014 July 2014 and the most recent online meeting was: July 2015

Summarising the data, and all the many and various community suggestions to date (particularly from the last meeting), we can arrive at some guiding principles:

1) Benzylic blocking groups have not to date resulted in significant improvements in metabolic stability, leading to the suggestion that improvements in gross parameters of the molecules should be improved, most notably logP. cLogP for any proposed analog must now be below 3. This rules out several interesting compounds (red crosses, below) that need to be redesigned, and places a question mark over marginal compounds (orange crosses). For example those suggested by @mrinalkundu would need to be modified with this design criterion in mind - the same goes for the updated set from Mrinal. We do still need to show better the correlation between measured and calculated solubilities, though. There is no clear trend between logD and metabolic stability, for the limited number of compounds we have measured (#333). Note that the cLogP values are only approximate and are in Chemdraw sensitive to whether X-H bonds are drawn out explicitly.

2) However, blocking groups next to oxygens in the northwest linker should be explored, based on the metabolic ID work discussed in the last meeting, since it could be that O-dealkylation is occurring next to the TP ring. See also #334

4) We should probably avoid unsubstituted pyridines, generally, though the Sydney Grammar samples below should be taken through to completion given that we have them!

5) To generate an IVIVC, #351, it might be necessary to re-synthesize specific compounds, but we await input on this point from David Shackleford.

These considerations lead to three groups of compounds - the ones that ought to be made immediately, those that are highly desirable and those that are more speculatively interesting.

The Immediates

Nemesis: Alice is in the process of making this, though it’s being a tough nut.

The CRO: a compound we’ll purchase as a test of the receipt of open data, as described in this background post and in #292.

The NW Aliphatic: The des-methyl alcohol is being prepared by Alice.

Sydney Grammar Pyridines: These are still being purified - Paul King was looking at them. We’re at the stage of attaching the nucleophile in the last step.

The Dimethyl Amine: Needs to be verified based on data from the last round since methylation of that amine appears to have a profound effect on potency.

The Homologous Alcohol: Good to have, but too lipophilic so needs a re-think.

The Homologous Amine: Ditto

The Shackleford: Blocking group next to alcohol, as per last meeting. This compound fails on cLogP, but may be worth making anyway to test clearance rate. Related issue was to search for commercially-available aryl ethanols #302.

The Swain: Blocking group and benzyl ether. Ditto - may be worth it despite the poor cLogP, or structure could be tweaked? Benzyl ethers still badly explored in this series, so the 2-, 3-, and 4-Cl substituted phenethyl analogs could be checked (suggested by Chris) and then drawn by Alice.

The Desirables

Reversed Amides: These have been discussed, e.g. here. The synthetic route to this reversed amide series could be through simple amination of the common chlorotriazolopyrazine intermediate, which both Alice (e.g. here) and Chase are interested in. Chase also suggested azide displacement of Cl followed by reduction to the amine.

The AZ Idea: A group of young scientists at AstraZeneca that includes former OSM-er Matt Tarnowski recently suggested a cyclic analog shown below, and our young contributor Mandrake also suggested some ideas around that molecule. Some cyclic groups have indeed been investigated in this position generally with low potencies - see the summary below. The group joining the side chain to the TP ring (red arrow) can’t be divalent oxygen; we know simple amine linkers are poor choices (e.g. OSM-S-190), yet amines have typically been the attachment point for rings (see below figure). Carbon linkers have occasionally been OK (e.g. MMV 669304, MMV670243). Perhaps a more polar cyclic group at this position would be OK with a carbon-based linker provided the northwest ring is positioned well. Metabolic clearance of carbon-linked compound MMV669304 was atrocious (MMV669304 below), while the related cyclic amine linked MMV668957 was much better (yet much less potent).

The Hypotheticals

Crazy Hemiacetal: Hemiacetals and hemianimals were suggested in the last analog analysis round. They are expected to be too labile, even though acetals themselves can be OK. While exhibiting a blocking group and good predicted hydrophobicity, this compound is probably too risky.

Other Linkers: Some of the SAR data suggests the linker between pyridine and Ar ring in the Northwest doesn’t really matter, leading to suggestions of ketone, sulfoxide and sulfone linkers from Chris and a urea suggested by Chase. We could also investigate a triazole linker (using an alkyne on the pyrazine to avoid a nitrogen-based linker atom on the ring) and an alpha-trifluoromethyl amine as an amide isostere, originally suggested by Chris Burns.

The Tianyi: Rationale for this molecule is that the imidazopyrazine MMV669846 retained potency (vs parent MMV639565) with only a slight deterioration in clearance time (see below), suggesting it might be worth re-examining this compound, but with a blocking group on the imidazole (methyl shown). Named after the student who started looking at this compound, Tianyi Zheng.

Chase Scaffold Hops: suggested here. These will require development of new methods if they’re deemed to be good swaps. Any opinions?

Misc Other Points

1) Now that we have more data in the Master Sheet, I wonder if we might be able to ask you, @wvanhoorn , to re-do your analysis, prioritizing compounds with untried motifs, using data from Series 4, and with an ALogP cutoff of 3. There was also mention of results from a matched pair analysis by Jeremy Besnard - I think the structures are here. It’d be interesting to see what you get now.

2) We will shortly be engaging with the OIDD automated synthesis group, and that some of the above synthesis could be part of that collaboration if, for example, a range of reaction conditions need to be screened or a number of analogs need to be generated. #341

How to Comment on These Suggestions

Please do comment/suggest/criticise. Please do make any of these compounds, provided you share the data so we don't duplicate.

Commenting can be done below, or on Twitter or G+ if you have accounts there. If you have to use email (please don’t) then you can do that.

Suggesting structures: you can paste SMILES, or you can easily drag and drop pictures into comments on Github now.

Deadline

Next shipment of compounds to Syngene: December 15th.

(cheminformatic strings for all molecules mentioned in this post are over on the OSM ELN here)

[MedChemProf]

What is bothering me is that this is technically the second time this amine did not react as expected. Several products seen in the crude NMR when previously reacted with an isocyanate. The HBTU/HOBT conditions are my usual go to for very good yields. Exam week here, so limited lab work right now. Hopefully in the next few weeks we will move more of the amine forward to try a few more conditions.

[MFernflower]

Perhaps you can try something a bit stronger? PyBOP or sodium amide might do the trick (provided the amide doesn't decompose back into the parent amine during treatment or workup)

[MedChemProf]

Don't have any PyBOP available right now. I'll try the reagents we have in the lab first.

[drc007]

HBTU/HOBT is certainly very reliable and usually gives clean products. You could try the acid chloride.

The other possibility is that the amide is very easily hydrolysed on workup? Is it possible the amino heterocylce is a very good leaving group?

On 28 Apr 2016, at 15:24, Chase Smith notifications@github.com wrote:

What is bothering me is that this is technically the second time this amine did not react as expected. Several products seen in the crude NMR when previously reacted with an isocyanate. The HBTU/HOBT conditions are my usual go to for very good yields. Exam week here, so limited lab work right now. Hopefully in the next few weeks we will move more of the amine forward to try a few more conditions.

— You are receiving this because you were mentioned. Reply to this email directly or view it on GitHub https://github.com/OpenSourceMalaria/OSM_To_Do_List/issues/358#issuecomment-215442400

[MedChemProf]

I really had no reason to suspect that the amide product would be that easily hydrolyzed, but I will keep it in mind. Barring the HBTU went bad (which I also think unlikely), the probable failure came with the amine being a weak nucleophile and a more reactive acylating agent or elevated temperatures are required. I may first try to simplify things by just taking the recovered material and attempting to acylate it with acetic anhydride. If that works and the product is stable, then I will invest in creating a hotter derivative of the more expensive 3,4-Difluorophenylacetic acid.

[MFernflower]

@MedChemProf Could one treat the 3,4-DFPA with phosphorus pentoxide to create the needed anhydride??

[MedChemProf]

Just an update on recent lab activity. The following three compounds were synthesized and just sent out for antimalarial testing. Two of the compounds are new and one was synthesized as a control to be able to compare backwards to results from previous screens. The control is compound OSM-W-272 (MMV639565) and its synthesis can be found here. The two new compounds are OSM-W-3 and OSM-W-4. I have entered them into the tracking spreadsheet, please let me know if someone spots any problems. The final synthetic step of OSM-W-3 can be found here and the final synthetic step of OSM-W-4 can be found here. OSM-W-3 was synthesized as part of the effort to lower the LogP and maintain activity. OSM-W-4 was one of the new scaffold hop analogs. The other Tetrazole scaffold hop analog is still in progress (and will be until September when the summer break is over.) Hopefully, we will also have a Reversed Amide synthesized by September also.

[mattodd]

Hi Chase - Fantastic. Where's the evaluation being done? In an ideal world we use similar protocols, and similar controls - @alintheopen could you please point to public domain assay details for e.g. Syngene? Or copy the essentials here.

[MFernflower]

@mattodd @MedChemProf The left hand sulfur gave me an idea - If the sulfur compound proves potent I would like to see a left-hand amide: http://www.sigmaaldrich.com/catalog/product/aldrich/a1800?lang=en&region=US

[alintheopen]

Syngene Assay Details: General assay principle:

"This protocol assesses compound efficacy against Plasmodium falciparum in-vitro. This assay is using [3H]-hypoxanthine incorporation or DNA labeling by SYBR Green as a markers of parasite growth. This procedure is designed for use with culture adapted P. falciparum strains or clones only. On one 96-well plate typically 03 drugs are tested in duplicate. Standard strains: Plasmodium falciparum, NF54 (sensitive to all known drugs), Plasmodium falciparum, K1 (chloroquine and pyrimethamine resistant). The assay can be performed in dose response mode (12 concentrations in duplicate, 24 data points) which allows determining IC50, or in single concentration mode (one concentration in triplicate, 3 data points) which allows determining the percentage of growth inhibition. For more information, see Desjardins et al. (Antimicrob. Agents Chemother., 16(6), 710, 1979)."

[MedChemProf]

@mattodd @alintheopen The assay is being run at the Broad Institute in Cambridge, MA (https://www.broadinstitute.org). Since we have so few compounds, they are generously adding them into the screen that they run regularly. Below is a summary of the screening details that was sent to me:

Drug assays were performed as previously described [Johnson, J.D., et al. "Assessment and Continued Validation of the Malaria SYBR Green I-Based Fluorescence Assay for Use in Malaria Drug Screening", Antimicrobial Agents and Chemotherapy, 2007, 51(6), pp. 1926–1933; doi:10.1128/AAC.01607-06], with modifications for 384-well format. Briefly, synchronized ring-stage parasites were cultured in the presence of 12-point 2-fold serial dilutions of test compounds in 40 µL of RPMI supplemented with 0.5% AlbuMAX II at 1.0% hematocrit and an initial parasitemia of 1.0% in black clear-bottom plates (Greiner Bio-one 781090). Following 72 hours' incubation under standard culture conditions, SYBR Green I dye (Invitrogen S7563) was added to a dilution of 1:5000, and plates were stored at room temperature until fluorescence signal was read on a Spectramax M5 plate reader (Molecular Devices, ex 480 nm, em 530 nM). After background subtraction and normalization, EC50 values were calculated using the Levenberg-Marquardt algorithm as implemented in the Collaborative Drug Discovery database [Hohman M, Gregory K, Chibale K, Smith PJ, Ekins S, Bunin B. Novel web-based tools combining chemistry informatics, biology and social networks for drug discovery. Drug Discov Today 2009;14:261-70.].

The primary contact that I have at the Broad published a paper on some of her screening work here: Amanda K. Lukens, Richard W. Heidebrecht Jr, Carol Mulrooney, Jennifer A. Beaudoin, Eamon Comer, Jeremy R. Duvall, Mark E. Fitzgerald, Daniela Masi, Kevin Galinsky, Christina A. Scherer, Michelle Palmer, Benito Munoz, Michael Foley, Stuart L. Schreiber, Roger C. Wiegand, and Dyann F. Wirth, "Diversity-Oriented Synthesis Probe Targets Plasmodium falciparum Cytochrome b Ubiquinone Reduction Site and Synergizes With Oxidation Site Inhibitors", J Infect Dis. (2015) 211 (7): 1097-1103. doi: 10.1093/infdis/jiu565

[MedChemProf]

@mattodd @alintheopen I just received the screening results for the three compounds shown above in the thread (OSM-W-3, OSM-W-4 and the control OSM-W-272 aka MMV639565). Using the screening protocol (also listed above in the thread), the compounds were screened against two malarial cell lines - 3D7, a generally drug-sensitive strain; and Dd2, a multi-drug resistant strain (Choroquine and folate biosynthesis inhibitors). I have attached a captured image that shows the EC50 curves because for some reason Github was not accepting the pdf upload. The Scaffold Hop analog OSM-W-4 is completely inactive. The p-Sulfone OSM-W-3 (see issue #388 ) retains activity, but is significantly less potent as compared to p-Chloro or p-OCHF2 analogs. The OSM-W-272 / MMV639565 compound was active and comparable to previous results (taking into account different cell lines.) The laboratory that screened the compounds offered to take MMV639565 and do some Target ID work (attempt to evolve in vitro drug resistance to new compounds, then sequence the resistant parasites to see if we can identify mutations in the target of the compound). I was going to send them some more of the compound so they could start this work. I just wanted to note this for OSM community comment and to get any feedback on this option. Please let me know. In about a week, we will restart lab work focusing on the synthesis of the second Scaffold Hop analog (containing the tetrazole, #386 and #398 ) as well as the completing some of the Reversed Amides.

[alintheopen]

Thanks Chase! Some really interesting results here, particularly with respect to the para sulfone - definite food for thought. Target ID work also sounds interesting and will be excellent to compare this data to the other Scaffold Hop analogues. Nice work! Alice 👍

P.s I think that images have to be png format for GitHub

[MFernflower]

@edwintse @alintheopen

Anyone want to make a left-hand para-methoxy? http://www.sigmaaldrich.com/catalog/product/aldrich/154180?lang=en&region=US

Dont think left hand electron donor was ever tested?

[mattodd]

@edwintse is already on the case here - EGT 147-1 and 148-1.

[MedChemProf]

@mattodd Using DataWarrior to run the calculations, OSM-W-272 has a cLogP=3.79 and OSM-W-3 has a cLogP=2.08. The reduced potency aside, is this close enough of a comparison to be used in a MLM / HLM study to determine if the strategy of reducing the LogP is a viable way to attenuate metabolism?

[mattodd]

Hi @MedChemProf - it's probably worth a shot, yes. Do you have a calculated logD - can't remember if DW provides that. (Cross-link to #388 for reference). But we'd probably only do this if the frontrunner set in #400 didn't provide us with enough data on logP/logD vs microsomal stability, I'd guess. It would need to be worth our while you shipping compounds to wherever the frontrunners end up (likely Monash, but not yet confirmed).

[MedChemProf]

@mattodd Should we close this thread as you suggested and begin new ones on any series we are still working on? Thanks.

[mattodd]

It's high time we closed this, but the reason it's not happened yet is that we a) need to ensure that any compounds that have been biologically evaluated have been added to the wiki, and b) that any useful suggestions in this thread have been captured somewhere, which goes to #440. Related to a), Chase, do you have a chemdraw file that we could use to generate pictures to add to the wiki? i.e. structures, codes and potencies, along with the URL for the relevant biological data?

[MedChemProf]

I do have a Chemdraw file with the above compounds. GitHub does not accept chemdraw file types, where is the file going?

[MFernflower]

Dropbox might work

[mattodd]

Yes, this has been a perpetual issue - a shared file store, and which to use? A non-trivial problem. I suspect Dropbox would work, as, perhaps, would Slack. I had hoped we might be able to use Github for this, given that we're all already here. The wiki images are helpful for summarising progress to date. The original intention, 5 years ago or so, was that the writing of the wiki would be essentially the same activity as writing the paper, to avoid duplication. Results would be folded in as they came in. I don't think there's a platform for this, and the problem with the wiki is that it's another sign in.

However, perhaps people could look at the repository I started for the first Series 4 paper which is something we need to re-activate in the coming months. There is a Word document there that can be downloaded, edited and re-uploaded by, I think, anyone in the associated team. One can post chemdraw files there, and, I think, lots of other filetypes. Perhaps it's time for us to step outside of Github as merely a To-Do list and start using its filesharing capabilities more properly for paper writing. I wonder, then, if there's a way we can maintain a Word file as the current status of the project document, with this To Do list acting as a place to discuss things related to the paper specifically. Alternatively the paper is constructed in these windows with markdown, and then converted to a Word document later. I suspect that is a better option, though slightly beyond my technical abilities. Writing the paper while maintaining project status involves a complex web of activities that needs to make sense to everyone, so I'd be grateful for suggestions as how best to do this (in a way that permits open filesharing).

Obviously the size of this question may require me opening a new issue. In fact, let me do that now. Hold on.

[mattodd]

NB update related to mention of Lilly's OIDD above, the platform is closing in Q1 2019. #341