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16th November 2014 @ 22:57

Context: 12 compounds were sent for evaluation for metabolic clearance by aldehyde oxidase (Discussion, Compounds sent:

Which Compounds to Send for Aldehyde Oxidase Assay?

Assay performed by: Christine Orozco, Scott Obach, Pfizer, Groton CT.

Results:

Aldehyde Oxidase Results OSM Series 4


Notes:


The compounds were incubated in 1 mg/mL cytosol out to 3 hours. Controls: carbazeran, zoniporide and zaleplon were assayed in each incubation plate to represent high, moderate and low AO clearance respectively. The Open Source Malaria compounds were assayed along with the controls and compared using a relative yardstick approach.

The test compounds had a range of clearances. There was one high clearance compound similar to carbazeran, three moderate clearance compounds similar to zoniporide, two compounds with moderate to low clearance, and the remaining four compounds were stable/low clearance with no turnover within 3 hours.

Two compounds were not included for technical reasons. Compound MMV670250 was not detected under the analytical methods used so new LC/MS methods are being developed for this compound. For MMV672939, there was variability in the later time points of both replicates. These two compounds can be repeated in the future.


More detail on the data, protocol, assay notes and conditions is in the attached spreadsheet.

OpenSource Malaria_Cytosol_Results_Protocol_CCO.xlsx

This post originally authored by Mat Todd


Strings:
MMV668956 FC(F)OC(C=C1)=CC=C1C2=NN=C(N32)C=NC=C3OC4CN(C5=CC(F)=C(F)C=C5)C4 InChI=1S/C21H15F4N5O2/c22-16-6-3-13(7-17(16)23)29-10-15(11-29)31-19-9-26-8-18-27-28-20(30(18)19)12-1-4-14(5-2-12)32-21(24)25/h1-9,15,21H,10-11H2 FFDOLPKXJUAFAH-UHFFFAOYSA-N
MMV669784 FC(F)OC(C=C1)=CC=C1C2=NN=C3C=NC=C(OC4=CC=C(Cl)C=C4)N32 InChI=1S/C18H11ClF2N4O2/c19-12-3-7-13(8-4-12)26-16-10-22-9-15-23-24-17(25(15)16)11-1-5-14(6-2-11)27-18(20)21/h1-10,18H WXNPYOXFQUYIOI-UHFFFAOYSA-N
MMV669844 FC(C=C1)=C(F)C=C1[C@@H](OC)COC2=CN=CC3=NN=C(C4=CC=C(C#N)C=C4)N32 InChI=1S/C21H15F2N5O2/c1-29-18(15-6-7-16(22)17(23)8-15)12-30-20-11-25-10-19-26-27-21(28(19)20)14-4-2-13(9-24)3-5-14/h2-8,10-11,18H,12H2,1H3/t18-/m0/s1 PERKBMZWWUEZNJ-SFHVURJKSA-N
MMV669846 FC1=C(F)C=CC(CCOC2=CN=CC3=NC=C(C4=CC=C(Cl)C=C4)N32)=C1 InChI=1S/C20H14ClF2N3O/c21-15-4-2-14(3-5-15)18-10-25-19-11-24-12-20(26(18)19)27-8-7-13-1-6-16(22)17(23)9-13/h1-6,9-12H,7-8H2 MQHQNFQVEXYAMO-UHFFFAOYSA-N
MMV670947 FC(F)OC(C=C1)=CC=C1C2=NN=C(N32)C=NC=C3OCC(CO)C4=CC=C(F)C(F)=C4 InChI=1S/C21H16F4N4O3/c22-16-6-3-13(7-17(16)23)14(10-30)11-31-19-9-26-8-18-27-28-20(29(18)19)12-1-4-15(5-2-12)32-21(24)25/h1-9,14,21,30H,10-11H2 MKSKANAVVSFYNL-UHFFFAOYSA-N
MMV670246 FC(F)OC(C=C1)=CC=C1C2=NN=C3N2C(C(NC4=CC=C(Cl)C=C4)=O)=CN=C3 InChI=1S/C19H12ClF2N5O2/c20-12-3-5-13(6-4-12)24-18(28)15-9-23-10-16-25-26-17(27(15)16)11-1-7-14(8-2-11)29-19(21)22/h1-10,19H,(H,24,28) CAFUHAVOTBMKAN-UHFFFAOYSA-N
MMV670250 FC1=C(F)C=CC(CCOC2=CN=CC3=CN=C(C4=CC=C(Cl)C=C4)N32)=C1 InChI=1S/C20H14ClF2N3O/c21-15-4-2-14(3-5-15)20-25-11-16-10-24-12-19(26(16)20)27-8-7-13-1-6-17(22)18(23)9-13/h1-6,9-12H,7-8H2 JHORUMGXLQTHAD-UHFFFAOYSA-N
MMV670944 FC(F)OC(C=C1)=CC=C1C2=NN=C3N2C(C(NC4=CC=NC(F)=C4)=O)=CN=C3 InChI=1S/C18H11F3N6O2/c19-14-7-11(5-6-23-14)24-17(28)13-8-22-9-15-25-26-16(27(13)15)10-1-3-12(4-2-10)29-18(20)21/h1-9,18H,(H,23,24,28) UEOZBGRWGZEYED-UHFFFAOYSA-N
MMV670946 FC(F)OC(C=C1)=CC=C1C2=NN=C3N2C=C(OCC4=CC=C(F)C(F)=C4)N=C3 InChI=1S/C19H12F4N4O2/c20-14-6-1-11(7-15(14)21)10-28-17-9-27-16(8-24-17)25-26-18(27)12-2-4-13(5-3-12)29-19(22)23/h1-9,19H,10H2 PPYIQNQWTQZDBZ-UHFFFAOYSA-N
MMV671927 FC(F)OC(C=C1)=CC=C1C2=NN=C3N2C=C(NCCC4=CC=C(F)C(F)=C4)N=C3 InChI=1S/C20H15F4N5O/c21-15-6-1-12(9-16(15)22)7-8-25-17-11-29-18(10-26-17)27-28-19(29)13-2-4-14(5-3-13)30-20(23)24/h1-6,9-11,20,25H,7-8H2 GVXYAEXVJYZTBB-UHFFFAOYSA-N
MMV672727 FC(F)OC(C=C1)=CC=C1C2=NN=C(N32)C=NC=C3OCC(F)(C)C4=CC=C(F)C(F)=C4 InChI=1S/C21H15F5N4O2/c1-21(26,13-4-7-15(22)16(23)8-13)11-31-18-10-27-9-17-28-29-19(30(17)18)12-2-5-14(6-3-12)32-20(24)25/h2-10,20H,11H2,1H3 MHMDPQOWBHVFHN-UHFFFAOYSA-N
MMV672939 FC(F)OC(C=C1)=CC=C1C2=NN=C3C=CC(OCCC4=CC(F)=C(F)C=C4)=NN32 InChI=1S/C20H14F4N4O2/c21-15-6-1-12(11-16(15)22)9-10-29-18-8-7-17-25-26-19(28(17)27-18)13-2-4-14(5-3-13)30-20(23)24/h1-8,11,20H,9-10H2 POGWIRSWBGSGTG-UHFFFAOYSA-N

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31st July 2014 @ 11:54

Update Nov 16th 2014 - results obtained from Pfizer:

Results of Series 4 Aldehyde Oxidase Assay

Update Nov 16th 2014 - the compounds actually sent were as follows

Compounds sent for AO assay

AO Compounds Sent.png

Results:

Update 13th Aug 2014 (original post below)

Final compound list is this, with some reasons (following input from Paul Willis):

Aldehyde Oxidase Shortlist 2

Codes of compounds to be sent:

MMV668956
MMV669784
MMV669844
MMV669846
MMV670246
MMV670250
MMV670944
MMV670946
MMV671927
MMV672727
MMV672939

Plus ONE of these two, whichever has the most sample available:

MMV670947
MMV672942

 

Original Post:

Exciting news. We have a lab willing and able to run the aldehyde oxidase assay, and not just any lab but one of the experts in this field - Scott Obach’s lab at Pfizer, Groton.

(Interestingly Pfizer are the originators of the currently most active series in OSM, Series 4, on which this assay will be deployed).

Scott has previously published on the increasing profile of AO in drug discovery/development. Several Series 4 compounds have shown a susceptibility to metabolic clearance:

Metabolism ID Study for Three Triazolopyrazines

Physiochemical Evaluation and Metabolism of 8 Test Compounds in Human and Mouse Liver Microsomes

There’s a summary on the wiki of all the current metabolic data for this series.

One of the possible problems is that these molecules contain aza-aromatic structures able to be oxidized adjacent to the nitrogen atoms - that is, I believe, a known substrate for AO. If that’s happening the it’s something we can deal with - and was probably something that was briefly looked at in the original work, explaining the synthesis and evaluation of these analogs.

But before we worry too much, we should see if these compounds are substrates for this enzyme. Following our appeal for help, I contacted Scott to see if he’d be interested, and he is. Pfizer have agreed to this, and to be named as contributing to OSM in this way, which is fantastic.

The aim is to run the assay on a set of 6-12 molecules at the outset in a human cytosolic lability assay vs. positive controls to test for AO-mediated consumption (here's the general method). If we see activity and want to examine several compounds further, we could then look at a rodent AO assay, since there are known to be species differences. One step at a time.

Experimental data would allow a comparison with the predictive work performed by Chris Swain.

Scott took a look at the Series 4 wiki and chose the long list of molecules below. Only some of these are available: the list of current stocks is here. Paul Willis added MMV669784, which is available.

AO First Shortlist

So, question. We have space for up to 6 more structures. What should we include, and why? Scott needs 1-2 mg of each. Again, the full list of what’s known about Series 4 metabolism is here, and we’re already including/addressing the most interesting compounds I think.

Some other possibilities from Series 4 are these:

AO Assay Second Set of Possibles

The first two, MMV669846 and MMV670250 seem like obvious choices given their variation of the core heterocycle. MMV670946 is a surrogate for MMV670945 which was on the original list but is no longer available. MMV672727 might be interesting as an ether resistant to benzylic oxidation. We have no good structures containing aliphatic amines, as far as I can tell.

Are we light on the regular ethers, i.e. should we include one more compound analogous to MMV639565 or MMV670437? Maybe we provide one from newly-synthesised stocks in Sydney? Are there other interesting structures available that people feel might ask/answer interesting AO-related questions, particularly alterations in the core triazolopyrazine core?

On the other hand, could we also ship compounds from previous series, to answer questions there too, for publication purposes? (The Series 1 paper is an an advanced state of write-up).

To summarize: which other compounds should go into this assay? We have room for up to 6 more.

Deadline for decision: Morning of Wednesday August 6th 2014. Then we’ll ship.

Places for comments/suggestions: Below (login with Google account to comment) or Github or G+ or Twitter using @O_S_M or (last resort) email (opensourcemalaria@gmail.com) but please make it clear you’re happy to have comments relayed publicly with attribution.

(This post authored by Mat)

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19th December 2013 @ 22:51

Three compounds sourced from MMV in the OSM Series 4 (the triazolopyrazines) were sent for metabolism ID testing at Monash University, to ID how these compounds were being metabolised as a guide for future analog design. Eight other compounds had displayed reasonably rapid clearance and low solubility.

 

Compounds sent for MetID

 

Data received Dec 19th 2013 and are attached to this post.

CDCO_MMV_OSDD_13_006_Hepatocyte stability and Met ID.pdf

Comments from Karen White: "The met ID study for 3 compounds in human and rat hepatocytes has now been completed and the results are summarised in the attached report. Regarding the aldehyde oxidase question, one of the compounds did form an oxygenated metabolite which may or may not be mediated by AO (can also be mediated by CYP enzymes) but we couldn’t definitively tell the site of oxygenation from the available data or the enzyme responsible."

Initial analysis from Paul Willis: "Metabolite ID has shown that the 3 compounds all appear to undergo oxidation on the triazolpyrazine ring.  This could be CYP or aldehyde oxidase mediated, further experiments are planned next year to determine which process is operating.  At this stage the project should evaluate the existing data to see if either changes to the bulk properties (Log P etc.) or remote changes can increase metabolic stability.  This may indicate a strategy for designing new more stable analogues.  Blocking groups could also be introduced into the labile positions of the triazolopyrazine ring to further improve metabolic strategy.  Given the likely synthetic challenges of such an approach, the project is recommended to wait until the full results of the metabolism experiments are available to guide such a strategy."

Results to be discussed in OSM project meeting on Dec 20th: OSM Online Project Meeting Six (20th December 2013)

 

 

Strings

MMV669844

[C@H](COc1cncc2n1c(nn2)c1ccc(cc1)C#N)(c1cc(c(cc1)F)F)OC

MMV669848

c1ncc2nnc(n2c1CN1Cc2c(C1)cccc2)c1ccc(cc1)OC(F)F

MMV670936

C(COc1cncc2n1c(nn2)c1cnc(cc1)C(F)(F)F)c1cc(c(cc1)F)F

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