Monday, May 14, 2012

#BadBosutinib: It's not paranoid if the molecules are really out to get you

Would you have caught this problem?
Credit: Chemical and Engineering News
a.k.a. Don't take the bad bosutinib!

From the pages of Chemical and Engineering News, a synthetic mystery story by Bethany Halford that is so freaky, you'll go back to your lab and try not to put every bottle through a full characterization study:
Some bad bosutinib is going around. Scientists who have purchased the compound may have actually received an isomer rather than the genuine molecule. 
Bosutinib, also known as SKI-606, is currently in Phase III clinical trials to treat chronic myeloid leukemia. Officials at Pfizer, the company sponsoring the clinical trials, say that only genuine bosutinib has been administered to patients. However, as a selective kinase inhibitor, the compound is also used in medical and basic research. The news that researchers may have unwittingly been using an isomer of bosutinib instead of using the genuine compound threatens to invalidate research efforts around the world. 
The first inklings that there may be problems in the bosutinib supply were discovered in December, when Nicholas M. Levinson, a postdoc at Stanford University, noticed something funny in the Protein Data Bank. A structure recently deposited by Stefan Knapp, Frank von Delft, and coworkers at England’s Oxford University was supposed to show bosutinib bound to a kinase, but the arrangement of one of the ligand’s atoms was wrong. 
It was a subtle difference. Bosutinib has a chloro group on the 2-position of its aniline subunit, but this was missing in the Oxford structure. Instead, this molecule appeared to have a chlorine in the 3-position, where bosutinib has only a hydrogen. (Emphasis CJ's)  
Levinson had been working on the structure of bosutinib bound to a different kinase, and when he saw this new structure, it convinced him that what had been a nagging inconsistency in his own work was actually a widespread problem. 
Levinson and Boxer put their publication on hold, ordered bosutinib from a different vendor, and did a battery of tests to determine which material was the genuine bosutinib. They soon figured out the original compound they had done all their research on turned out not to be bosutinib. “We had wasted a huge amount of time and money on the wrong isomer,” Boxer says. On the basis of multidimensional nuclear magnetic resonance (NMR) experiments, Boxer and Levinson believe that this isomer not only has a chlorine at the 3-position rather than the 2-position, but also that the chloro and methoxy groups that appear in the 4- and 5-positions, respectively, in bosutinib’s aniline moiety have been switched. (Emphasis CJ's)
What I find interesting is that the relevant company (LC Pharmaceuticals) that sold the research sample to Stanford has started blogging about this issue, and in a February report, he raises questions about Wyeth/Pfizer's own characterization of the compounds:
 Bosutinib (of empirical formula C26H29Cl2N5O3) could be taken to be one or more of:
(i) a particular arrangement of those atoms and bonds depicted graphically, such as in Figure 2, as was published in the 2001 J. Med. Chem. article. 
(ii) a substance in a bottle at Wyeth-Ayerst (the company that originated bosutinib and was later acquired by Pfizer) at the time that company first made the compound;
(iii) a substance made by a particular synthetic sequence and reaction conditions, such as in J. Med. Chem., op. cit. or by one of the three other published synthetic routes that we are aware of; 
(iv) a substance with (i) certain specific inhibitory potencies or inactivities when tested on a panel of eleven rat and human kinases [Cancer Res. 66: 11314-11322 (2006)] or (ii) other activity profiles as measured in other set(s) of bioassays;
or, a new, very important alternative: 
(v) the substance that is being administered to human patients in Pfizer-sponsored clinical trials.
In Bethany Halford's article, Pfizer makes very clear statements that only the correct bosutinib has been given to patients. Boy, for their sake, I sure hope they're right.

As a synthetic chemist (and someone who used to make molecules for sale to researchers), I'm in profound sympathy with LC Pharmaceuticals. I would like to think that I would have caught the weird NMR issues (Figure S2, Word document) and I would have noticed that things that weren't symmetrical were acting symmetrically. I hope that I am right.

Finally, I believe that this mistake could have happened to anyone. However, the fact that the incorrect compounds came to LC Pharmaceuticals by way of Chinese manufacturers cannot escape anyone's notice nor improve their reputation (or lack thereof) for quality.

Readers, would you have caught this? What, if anything, would you do to make sure this didn't happen to you?


  1. Typo-Watch: Stanford needs another "o" (middle), and your asterisk at the bottom doesn't seem to lead anywhere...

  2. I work at a large paint/coatings company's corporate analytical lab, and we've received a lot of work requesting us to characterize raw materials from China. The results of these projects have varied wildly. Sometimes the Chinese material is indistinguishable from our references; other times the two aren't even close. I can't say I was surprised to hear that a Chinese manufacturer has been implicated as the origin of this issue.

    I sympathize with the parties involved. I have the luxury of knowing if I missed this kind of detail in my work that it wouldn't affect products that are meant to be ingested or impact human health.

  3. mistakes of this kind are common, especially if you are synthesizing the molecule for a bio catalog on a smal scale and do it only few times, and if you source the building blocks from Asia. This sort of stuff happened at SUGEN - they got material custom made and instead of dimedone someone used isomeric 4,4-dimethyl-1,3-cyclohexadione. The material was incorporated into final compounds (which turnd out to be active) and noone notices, i suppose because the spectra of final compounds are quite busy. A J Med Chem paper and couple patents ended up with wrong (isomeric) structures in it and the broblem was discovered only few years later once they got a co-crystal. It became a source of embarasment and insider joke, about the migrating methyls. As far as I know the patents and publications were never corrected/retracted.

  4. Readers, would you have caught this? What, if anything, would you do to make sure this didn't happen to you?

    I don't know. I mean, it's easy to think that I would have (the number of aromatic hydrogen peaks doesn't even match, nevermind the shift differences), but it's all after the fact. I'm fairly sure that if the molecule were something like tobramycin there's no way I would have spotted an isomer/diastereomer via nmr, just because the peaks are so crowded.

    If you were a supply company, trying to confirm the structure of everything that comes in, I guess you could compare retention times on a chiral column and/or MS/MS. But both methods are probably going to require comparison to a known sample...

  5. Sympathy? I'm not so sure - I mean someone really effed up at some point and used wrong aryl. And that was easy to check, and should've been checked.

  6. Offline, CJ asked me to comment on whether a PHOSITA (Person having ordinary skill in the art) would have caught the bosutinib problem. This is what I said:

    When you look at those 1H NMR shifts in Boxer’s Supporting Info, it’s pretty clear you’re looking at two different things. So, on the surface, I’d say yes, a good analyst would have caught this.

    But there are, as the PKC Pharma page points out, unusual circumstances with the NMR of this compound. The patent, with no solvent reported, lists totally different chemical shifts from the J. Med. Chem paper. Plus, there’s the complication of the recent Molecules paper on bosutinib (there are a few PHOSITAs that you would think would have caught it and didn’t). They now believe they’ve made the isomer, but it’s a literature source that reports that isomer as bosutinib. So, if you’re going to the literature to check, you could easily get confused.

    Personally, I’m not sure I could just look at the proton spectrum of the isomer and know it was symmetrical, and at one time anyway, I would have counted myself as a PHOSITA.

    Also, you can’t catch the problem if you’re not doing 1H NMR and not everyone does that analysis on the stuff they buy and resell. Did you see The Scientist story? ( Selleckchem posted the spectrum for the isomer on their website. There’s another PHOSITA who didn’t catch it.

  7. In development we had to make all possible isomers, especially when the compound contained aromatic rings, for analytical purposes. The incoming SM was checked for their presence or rather absence before it was released for use.

  8. Unstable IsotopeMay 15, 2012 at 2:35 PM

    Personally I think this is something they could've caught. Since there was disagreement in the literature they should have followed up to determine what the actual NMR spectrum looked and the NMRs of common isomers looked like. I really don't think they have a good excuse. This was not unknowable.

  9. At ACD/Labs, we thought it would be interesting to see if an Automated Structure Verification (ASV) system such as ours would be able to flag the original bosutinib structure for review and/or pass the isomer when compared with experimental NMR data. We are working with Phil Keyes of Lexicon Pharmaceuticals, and you can see our initial results here:

  10. AK Scientific did not communicate to me that they had sold me the bogus isoform and i stumbled upon the LC Labs article only in September 2012. caveat emptor certainly. but for them not to communicate to me during the three months after the published article stinks. they refused a request for a modest store credit to compensate for the significant damages i incurred because of their egregious carelessness or willful negligence. caveat emptor AKS.
    Charles D.

  11. we just published a paper where we made the accidental discovery of Bosutinib-isomer. We were screening for kinase inhibitors for off target activities that can be repurposed for inhibiting the DNA damage checkpoint. Here is our story:


looks like Blogger doesn't work with anonymous comments from Chrome browsers at the moment - works in Microsoft Edge, or from Chrome with a Blogger account - sorry! CJ 3/21/20