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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.


Aldehyde Oxidase Results OSM Series 4


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

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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Attached Files
AO Results from Pfizer.cdxml
AO Results from Pfizer.png
OpenSource Malaria_Cytosol_Results_Protocol_CCO.xlsx
Re: Results of Series 4 Aldehyde Oxidase Assay by Matthew Todd
19th November 2014 @ 11:45
Scott Obach (by email) noted the following 3 points:

1) The AO reaction involves nucleophilic attack on the carbons alpha to a nitrogen (or at electronically equivalent system) on the azaheterocycle. Thus the more electron–deficient the carbon, the faster the rate. The compounds substituted with an electron withdrawing amide would be expected to be better substrates than those with an electron donating group like an ether oxygen.

2) The electron density on the rings is just part of the story. How the enzyme binds substrates and orients them — i.e. the interaction with the protein — will also be important but that is not yet understood.

3) Would you expect the AO product (i.e. the lactams) to retain target activity? Maybe these metabolites would be new leads?

In reply from Mat: With regards this last point, a lactam has been evaluated (MMV669025) at 4 micromolar, whereas the corresponding non-oxo analog (OSM-S-260, MMV675960) is 0.26 micromolar. This strongly implies that the azaheterocyclic N needs to retain its basic character, in turn implying that these metabolites (certainly if oxidised at that position) will not possess activity.

There is also the comparison to be made with the chloro-subst analog MMV668823, which is potent. As Scott subsequently noted, if that site is the site of oxidation, then the Cl will block that site, but such substitution would likely make the ring more susceptible to AO elsewhere, perhaps the other side of the N on that ring. This is on top of any possible inherent chemical reactivity of the chloro analog.

These issues will be discussed at the next online meeting: