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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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January 8, 2013

Metal Impurities Will Waste Your Time

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Posted by Derek

Here's a paper on a high-throughput screening issue that everyone in the field should be aware of: metal impurities in your compounds. The group (from Roche) describes a recent experience, and I think that many readers will shiver in recognition:

The hits were resynthesized, and close analogues were prepared for early structure−activity relationship (SAR) exploration. All three series lacked conclusive SAR. Most exemplifying are the activities of different batches of the very same compounds that exhibited very different activities from being low micromolar to inactive with IC50 values greater than one millimolar (Table 1). Additionally, the SAR of close analogues was either flat or very steep as indicated by compounds with minimal structural changes losing all activity (data not shown).

For these particular hits, we investigated these findings further. It was discovered that for one series, different routes of synthesis were used for the original preparation of the HTS library compound and its resynthesis. The historic synthesis made use of a zinc/titanium reduction step, whereas the new synthesis leading to inactive compounds did not. The schemes to prepare compounds of the other series also had steps involving zinc. Elemental analysis of the samples to determine the zinc content revealed that the active batches contained different amounts of zinc of up to 20% of total mass, whereas the inactive batches only had traces. . .

I think that many of us have been burned by this in the past, but it's something that should be out there in the literature so that it's easier to make the case to those who haven't heard about it. The Roche group suggests a counterscreen using a zinc chelator (TPEN) that will get rid of zinc-based effects. They pulled out 90 of their hits based on that work, and checking those against past assays showed that they had unusually high hit rates across the years. Some of them had, in fact, been considered hits for current Roche projects, and checking those assays showed that they were sensitive to zinc as well.

I can tell you from personal experience that the stuff can be a real problem. In that case, "impurity" was a relative term - the compound from the files turned out to be a 1:1 zinc complex, not that this little fact was noted anywhere in its (rather ancient) documentation from inside the company.

And I've seen copper do the same sort of thing. I would very much recommend checking out any active compound that looks to have been made by Ullmann chemistry or the like. I mean, I like the Ullmann reaction (and it looks like I may be setting some of them up soon), but there's a lot of copper in those things, and some assays are very sensitive to it. In extreme cases, I've seen compounds come in from custom synthesis houses that were colored green from the stuff, and that's just not going to work out. There are regrettably few lead-like compounds that come by a green tint honestly: you're looking at copper, maybe chromium, or perhaps even nickel, none of which will help you generate reliable assay numbers. Don't even let the green stuff into your collection, if you can - clean it up first, and complain to the people who sent it to you. (Note, by contrast, that zinc complexes tend to show no added colors).

Jonathan Beall speculated to me in an e-mail that maybe this is one way that frequent-hitter compounds can get on such lists, by coordinating metals. It's certainly possible. Ignore metals at your peril!

Comments (23) + TrackBacks (0) | Category: Drug Assays


COMMENTS

1. Anonymous on January 8, 2013 9:58 AM writes...

I used to work in Crop protection research.
We definitely avoided any tin reagents in the final chemical steps: many compounds would tend to be anti-fungi actives otherwise...

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2. anon the II on January 8, 2013 10:05 AM writes...

As one of those comby chem people who is responsible for destroying the industry, I have to say "Shame on you, Roche". We knew early on, before we even got started, that one should never go forward with analog synthesis from an HTS hit until the hit had been absolutely confirmed by re-synthesis and full characterization. For some reason, it's a simple economics lesson that's really hard to beat into the heads of some people.

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3. imatter on January 8, 2013 10:26 AM writes...

Not just the metal contamination--it's any possible chelating moeity in the drug lead needs to be looked at closely before moving ahead. What was that procaspase-3 activating small molecule?

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4. marcello on January 8, 2013 10:52 AM writes...

definitely passing compounds through resin-based metal-capturing cleanup reagents would be a good practice

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5. Quintus on January 8, 2013 10:58 AM writes...

They should have talked to the guys in chemical development. They know all about metal contamination.

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6. Ken on January 8, 2013 11:26 AM writes...

Obviously costly to analyze HTS compounds for metals, but what is more costly, the time/money spent on analysis or the time/money spent on a program making decisions based on GIGO (garbage in, garbage out) data?

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7. RO Beef Supreme on January 8, 2013 11:49 AM writes...

I've encountered Zn problems in a hERG screen:

I was half way through a scale up when "we" discovered the compound of interest had significant hERG inhibition. Fortunately, I was able to push through some final compound before being re-tasked. My re-synthesized material did not show significant hERG inhibition. Turns out my esteemed colleague had prepared a sample full of Zn salts which tweaked out the hERG assay.

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8. Dennis on January 8, 2013 12:13 PM writes...

Does anyone have any suggestions for chromium scavenging?

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9. anon on January 8, 2013 12:16 PM writes...

The wonder antibacterial that was made with sodium cyanide as the last step - it was polar and in the days before routine 13C nmr - took a while to NFI

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10. Joel on January 8, 2013 12:49 PM writes...

Hey Derek- your RSS feed mangles your formatting on block quotes. The first paragraph is always formatted correctly, but then the rest of your pullquote looks like you wrote it. Might want to get someone to have a look.

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11. Magpie on January 8, 2013 1:01 PM writes...

This is exactly why elemental analysis can't be totally ignored.

@Dennis: You should take a look at Quadrasil scavengers. There is probably one that works for Cr.

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12. anon on January 8, 2013 1:24 PM writes...

@Dennis: I've had a similar problem recently and had some success with a scavenger from Silicycle.

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13. z on January 8, 2013 2:31 PM writes...

I always resynthesize HTS hits to confirm activity prior to making analogs, but I typically use the same scheme that was used in the original synthesis. I do make sure the compound is clean, but I only characterize it by NMR and LC-MS. I've never checked for the presence of metals (which sometimes do have a way of getting carried through unnoticed). Shame on me, maybe, but I'm just curious what most people do? Elemental analysis on everything?

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14. milkshake on January 8, 2013 3:11 PM writes...

EDTA aqueous wash does wonders for transition metal carry-overs. I only once encountered workup where EDTA failed me (diaminopyrimidine-acetylenes cyclized with superstoechiometric CuI) and I resorted to cyanide in the end.
Thiosulfate is also good for some metals.

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15. sd1 on January 8, 2013 5:45 PM writes...

How do you get to % Zn?!

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16. Nick K on January 8, 2013 9:52 PM writes...

Just to echo #11, this episode merely underlines the folly of neglecting microanalysis. Under the "old" regime (pre-HTS, pre-Combichem) these compounds would never even make it to registration, let alone screening as the metal contamination would have showed up immediately in the combustion analysis. In their obsession with increasing "productivity" Pharma senior management has blundered badly.

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17. Ted on January 8, 2013 10:07 PM writes...

Hi all:

Don't be shocked if it comes in with your ligand. Many metal coordinating ligands are constructed via metal catalyzed chemistry - you can see the obvious purification problem! I had to re-purify a multi-kilo batch a few years ago when I figured out the client's use test problem. We re-engineered our synthesis to avoid the problem. The lab that developed these ligands and the chemistry had no idea... One of our competitors figured it out a few months later - we found out when we were sent a 'complimentary' replacement for one of our reference standards...

Fortunately, ICP/MS has gotten much more reliable in recent years. Still, it never hurts to send out a blinded standard.

As for removal of metals:

EDTA mostly sucks
There is a world of variation amongst charcoals - test them all. Darco KBB and G-60 usually come out on top, but there's always an outlier.
Charcoal doesn't work very well if you don't heat the solution and let it stir. One hour at 50degC is a good starting point.
Charcoal adsorbs metals differently in different solvents
Save the expensive stuff (smopex, deloxan) until after you've gotten down to double digit ppm levels
If your compound can be crystallized from an alcohol, Repic's approach works well - you can tie a bunch of Pd up in solution using N-acetyl cysteine (a dietary supplement!)
ammonia does a good job with copper, but you usually need to mix it with brine - otherwise you'll have a bright blue lava lamp...

-t

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18. z on January 8, 2013 10:29 PM writes...

@Nick K. #16, I assume that the "increasing 'productivity'" you refer to includes making more compounds faster, which is probably a good thing and I think it's here to stay. So it comes down to a cost-benefit analysis involving the frequency of false positives due to metal contamination (and the extent to which this sort of thing typically derails a project before it gets noticed and sorted out) versus the cost of more comprehensive routine characterization. I don't know the numbers on either side, and I suspect there's no good way to measure of the total impact of the false positives. I'm curious (because I have no idea), how much does elemental analysis cost?

My current workflow is to characterize all final compounds by NMR and LC-MS (and this seems to be typical, at least among my colleagues). I only get elemental analysis on compounds undergoing pretty advanced profiling. Maybe it would be worth at least spot checking a few more compounds here and there, maybe once per change in overall synthetic scheme?

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19. Nick K on January 8, 2013 10:48 PM writes...

#18: I actually don't understand the current reluctance to use CHN analysis. With a modern automated Carlo Erba apparatus and an electronic microbalance an experienced analyst can run a sample in twenty minutes or less. Perhaps the bottleneck is at the Chemistry stage - getting the compound really clean and solvent-free takes time and effort.

PS: Another advantage of CHN is that it tells you if you have a solvate or not.

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20. Tom on January 9, 2013 4:25 AM writes...

@Dennis - and all, sorry for self promotion, but we at Phosphonics (www.phosphonics.com) can supply scavenger kits for a variety of base and precious metals, Cr, Cu, Os and all PGMs.

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21. istvan on January 9, 2013 5:00 PM writes...

Recently, an organomercury compound was found in certain commercial lipase inhibitor samples:
http://www.nature.com/neuro/journal/v10/n1/full/nn0107-134a.html
The culprit was bis(methylthio)mercuran that contaminated the 6-methyl-2-p-tolylaminobenzo[d]oxazin-4-one (URB754)sample obtanied by the desulfurative amination of a methylthiobenzoxazine using red mercury. It turns out (http://www.ncbi.nlm.nih.gov/pubmed/17970304) that Hg(SCH3)2 is a potent inhibitor of the sulfhydryl-contaning enzyme and could only be removed by chromatography.

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22. WB on January 9, 2013 8:56 PM writes...

For solution-based libraries, a quick check with ICP can't hurt.

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23. D.J. on February 5, 2013 6:37 PM writes...

There are regrettably few lead-like compounds that come by a green tint honestly: you're looking at copper, maybe chromium, or perhaps even nickel, none of which will help you generate reliable assay numbers.

It probably says bad things about me that I read 'lead-like' as 'containing the element lead'. But what color /would/ a compound with lead in it have? Would such a thing ever be in a drug?

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