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Animal Testing

A Toxicological Flag



Here’s a caution from a new paper out of Manchester. The group had been synthesizing inhibitors of PARG (poly-ADP ribose glycohydrolase), an enzyme involved in DNA repair. The general chemotype is shown at right, but there are a number of variations. That fluorine is a new addition, though. The corresponding cyclopropylmethyl series came from their earlier paper, but they found that the series wasn’t particularly stable to metabolic oxidation. The major metabolites proved to be the primary sulfonamides in each case, suggesting that oxidation of the cyclopropylmethyl itself (which would be most likely at the methyl carbon) was the problem. Indeed, fluorination as shown increased the microsomal stability substantially (difluorination decreased the affinity for the PARG target itself, and deuteration, interestingly, had no real effect).

So far, so good. But trouble hit when the compounds were dosed in mice – there were obvious signs of toxicity, and deaths at the higher doses. This had never been seen with the earlier compounds, and dosing a fluoromethyl analog that had significantly reduced PARG potency showed the same effects. The trouble was noted at around the five-hour point, and only after oral dosing. Necropsy showed obvious signs of liver damage, which fits with that observation as well.

That all adds up to off-target tox brought on by metabolic activation of the fluoromethylcyclopropyl group. What the active species might be is not clear yet, nor is the specific liver target. But these observations alone are enough to flag that modification as a serious potential problem, which is the whole reason this new paper was published. Monofluoroalkyls can be a little odd – terminal monofluoroalkanes get cleaved down to fluoroacetic acid, for example, which is toxicological bad news. And fluorobenzyls (monofluoromethyl aryls) are reactive as electrophiles, despite the fluorocarbon reputation for stability. But this appears to be something different from either of those cases.

It’s true that this may vary according to the rest of the molecule, but just seeing it show up this strongly in one series is cause for concern. And since cyclopropyl groups and fluorinated methyls are both pretty common motifs in medicinal chemistry, it’s important to realize that the combination could be bad news. A quick Reaxys search of the fluoromethylcyclopropylsulfonamide side chain gave 183 hits, but those are all from two patents filed by the Manchester group themselves.  But if you search for the corresponding amide, you also pick up a couple of compounds from Gilead as IRAK-4 inhibitors. The amine is a perfectly reasonable-looking building block, and it would be a good thing if it did not spread through the med-chem literature without people knowing that there may be a red flag attached to it.

28 comments on “A Toxicological Flag”

  1. Mark says:

    Looks to me like a substrate for a Brady- Johnson olefin formation type reaction

  2. I have to say my first thought skimming this was “WHY DO YOU WANT TO INHIBIT AN ENZYME INVOLVED IN DNA REPAIR” but I suppose there’s some good reason. I’m just a dumb retired software guy.

    1. AGMMGA says:

      Long story short: in some situations cancer cells are slightly more sensitive to DNA damage than normal cells.
      Partly this is because cancer cells divide faster than your normal cells, and usually have lost a lot of checks to their replication. Thus cancer cells accumulate DNA damage and die, whereas normal cells take things slow, repair their DNA and survive; or don’t replicate at all and stay put.
      In some situations, cancer cells might also have lost some DNA repair pathways, whereas normal cells have more redundancy at their disposal. Therefore a normal cell will repair their DNA with a pathway that we have not inhibited, and the cancer cell dies.

      Or so goes the theory. In practice, things are messy, so we just try and see what happens?

      1. MTK says:

        More than just in theory as PARP inhibitors have shown some exceptionally good results in BRCA deficient tumors through synthetic lethality, the concept that shutting down to components of homologous recombination DNA repair causes cell death. There are now 4 (I think) approved PARP inhibitors.

        I’m not as familiar with PARG but I’ll assume they work in the same pathway and the thought is the same.

        1. That is very cool! Thanks, guys!

    2. Genentech Guy says:

      PARG likely regulates DNA repair by enzymatically-degrading the poly ADP ribose synthesized by PARP 1 an enzyme that is, as pointed out, also involved in DNA repair. PARP 1 has been shown to be an efficacious target in, as also pointed out, BRCA-deficient tumors.

  3. Captain Obvious says:

    I’m going with aziridine. Let’s try to remember our sophomore O-Chem…

    More surprised that this compound is stable enough to be handled, but I guess F is just nonreactive enough. I’ll bet Cl would decompose to all kinds of crap on the bench.

  4. dasf says:

    Does it maybe react a bit like an allylic halide? These cyclopropyl groups have plenty of sp2 character. I don’t know about the amino-substituted ones, though.

  5. MTK says:

    Fluorines are pretty terrible leaving groups and the C-F bond is a very strong bond. Seems like sort of a bad combination of a nitrogen and the fluoromethyl that could result in a particularly reactive cyclopropyl ring.

  6. cb says:

    Formation of reactive 1-azaspiro-2.2.-pentane moiety by intramolecular SN2 reaction…!?

  7. CM says:

    Cyclopropylcarbinyl radical or cation as an intermediate? The bond strength and the leaving group ability depend in part on the stability of the resulting radical or cation; if memory serves, the cyclopropylcarbinyl versions are considerably more stable than the classical localized radical and cation.

  8. Hap says:

    Can the sulfonyl group attack via O? That would yield a cyclopropylmethyl sulfoximinate, which should be a better leaving group than F.

    1. Nick K says:

      A third possibility is anion formation of the sulfonamide followed by a 1,2-shift of the cyclopropyl to displace the fluoride, giving a reactive N-sulfonylcyclobutaneimine.

  9. radical clock says:

    cyclopropylcarbinyl radical formed by CYP mediated oxidation will pop open the cycloproane to give the sulfonamido substituted but-2-eneyl system with an allylic terminal radical on one end and the fluoromethyl on the other end. Radical is quenched one way or another (time dependent CYP inhibitor?) In any event, the examine system can hydrolase to the alpha fluormethyl ketone.

  10. radical clock says:

    Meant to say: cyclopropylcarbinyl radical formed by CYP mediated oxidation will pop open the cyclopropane to give the 1-fluoro-2-sulfonamido substituted but-1-enyl system with a terminal radical at the 4-position. Radical is quenched one way or another (time dependent CYP inhibitor?) In any event, the examine system can hydrolase to the alpha fluormethyl ketone.

  11. Med chemist says:

    Interesting. Regardless of mechanism, good reminder that the med chemists’ intuition to put fluorine everywhere you want to block metabolism isn’t always the magic trick to generating quality drug candidates.

    1. radiochemist says:

      It’s unfortunate, with a fluorine in your drug you also have a perfect candidate for F-18 radiotracer development…

  12. Peter Kenny says:

    Possibly some neighboring group participation with displacement of fluorine linked to formation of cyclobutyl cation. Sulfonamide then deprotonates to give what would be a very electrophilic imine (alternatively sulfonamide deprotonation could be synchronized with displacement of fluorine). On a semi-related note the weaker activity of the CF2 and CF3 analogs may be due to effect on sulfonamide NH rather than steric.

    1. Peter Kenny says:

      Should have written displacement of fluoride (rather than fluorine).

  13. Wile E. Coyote, Genius says:

    Really a shame that they didn’t do histopath on the liver, only gross at necropsy. There can be clues there as to mechanism of toxicity under the microscope.

  14. John Wayne says:

    I’ve been burned by cyclopropanes more than once. It seems like they are much less stable when there is a functional group psuedoallylic to the ring. Makes chemical sense that that would be so.

  15. Professor Electron says:

    The fluorine would make chasing metabolism by NMR the obvious step – fluorine NMR on unpurified samples to see what modifications had happened near the fluorine. Modern spectrometers can detect less than a microgramme.

  16. Gen J. Ripper says:

    Do you realize that in addition to fluoridating water, why, there are studies underway to fluoridate salt, flour, fruit juices, soup, sugar, milk, PARG INHIBITORS, ice cream? Children’s ice cream!…You know when fluoridation began?…1946. 1946, How does that coincide with your post-war Commie conspiracy, huh? It’s incredibly obvious, isn’t it? A foreign substance is introduced into our precious bodily fluids without the knowledge of the individual, and certainly without any choice.
    That’s the way your hard-core Commie works. First PARG Inhibitors, tomorrow the World!

    1. loupgarous says:

      Obviously, we don’t use any of this compound’s derivatives on commanders of Global Strike Command bomb wings.

  17. Istvan Ujvary says:

    Interestingly, they do not seem to have tried the good old vinylcyclopropyl group.

  18. winampdfx says:

    The hydrolysis metabolite of the compound which is 1-(fluoromethyl)cyclopropanol will be unstable and will undergo rearrangement to 1-fluorobutan-2-one. The last one may be responsible for the toxicity. For, example, fluoroacetone is a highly toxic compound.

    Further metabolism may lead to fluoroacetate or structurally similar compounds. Fluoroacetate is known to be highly toxic because it disrupts the Krebs cycle by combining with coenzyme A (

    1. winampdfx says:

      Cyclopropanol is unstable, it rearranges to propionaldehyde (; So, 1-(fluoromethyl)cyclopropanol will most probably be unstable too.

    2. winampdfx says:

      LD50 (for fluoroacetate, oral route, in mouse) = 0,1 mg/kg

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