Ryan's Blog on NMR Software

Bosutinib: A Case of What Could Have Been?

For many years I have been looking for a high profile public case that represents a good example of why an automated NMR structure verification system can help prevent bad things from happening. We all know these types of examples exist, but they don't generally become public knowledge. 

Yes, I've blogged about this topic many times over the  years. To name a few:

Back to Reality #1- LC/MS vs. NMR

The Realities of NMR in the New(ish) Discovery Environment

Are We There Yet? Automated Structure Verification (ASV)

The Black Swan of Compound Libraries

Recently, an article in C&E News was published. It can be read here:

Bosutinib Buyer Beware

In short, there appears to be a lot of bottles of bosutinib out there that actually contains the wrong isomer. 

Bosutunib is a tyrosine kinase inhibitor that Pfizer is hoping will be one of their next big drugs. Also known as SKI-606, it is currently in Phase III trials to treat chronic myeloid leukemia. 

Naturally, for such a high profile compound there are a fair amount of research groups who have been conducting their own studies on this compound. Problem is, there is a lot of the incorrect stuff floating around out there. The latest update on the PKC Pharmaceuticals, Inc. website have reported that up to 18 vendors have actively sold to one or more customers in the biochemical reagent marketplace, an incorrect bosutinib isomer.

Derek Lowe at In the Pipeline has also sounded off on the issue.

Naturally, the first thing that came to my mind when reading this story was, could an automated NMR software system have caught this mistake. From the C&E News article:

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.The difference between the two molecules is subtle. Mass spectrometry and elemental analysis would be the same for both compounds. There are differences in the aromatic region of the ¹H NMR, but one wouldn’t necessarily pick up on them unless they compared the two compounds side by side. It’s only when you use ¹³C NMR that the symmetrical nature of the aniline group in the isomer becomes clear.

 

Here are the two molecules in question:

Much of the discussion has been that the average chemist would let the incorrect isomer through with just the LC-MS information and quick glance at the ¹H. Of course the ¹³C NMR would clearly point out the difference but this isn't standard practice for many bench chemists. 

But would one have even needed to acquire a ¹³C NMR spectrum for this?

Would a ¹H NMR have been enough? At the very worst, a HSQC experiment? The supporting documentation for this article suggests clearly yes to the latter. 

Phil Keyes from Lexicon Pharmaceuticals and I are working on this right now and should have some results showing how our automated NMR verification system would have handled this case. 

More details to follow...stay tuned!

 

 

Back to Reality #1- LC/MS vs. NMR

Reality #1:

LC/MS has become the primary analytical check for the medicinal chemist. I Love NMR, but it's the reality

So how did we get here? Clearly there a lot of reasons, but one of my hypotheses is based on the evolution of open access and the walk-up environment. No doubt that moving away from the traditional analytical service departments has been a great thing from a productivity standpoint.

LC-MS lends itself really well to the open access environment. There are good systems out there that can auto-process data well and provide a nice report that gives the chemist a simple answer. In most cases, the chemists can get the answer they are looking for with a quick glance at the report. In addition, for this kind of routine analysis, the interpretation of the results is very easy. 

Contrast that to NMR. How often are the printouts from the instrument good enough? Not nearly as often. More and more it is becoming common practice for the chemist to import their raw or processed data into the processing software on the instrument or in some offline processing software. Within these applications, they can get a better look at their data, zoom in on areas of interest, re-integrate the spectrum so it makes sense, perhaps even generate a multiplet report for a potential patent down the road.

Add up those tasks alone, and the chemists may be forced to spend 5-10 more minutes on a routine NMR than an LC-MS when you factor in interpretation as well! Unfortunately, the NMR instrument is not printing out an answer, it is printing out a spectrum that needs to be interpreted by the chemists which on a routine basis is a lot more difficult and complex task than checking an LC-MS for reaction completion. Furthermore, let's keep in mind that intrepreting analytical data is not the medicinal chemists job. They are evaluated, compensated, etc. for making molecules. The more molecules the better so the inherent risk here is obvious and I don't think it's fair to simply "blame the chemists"

Given the above, is there any wonder why LC-MS has become the primary analytical check?

The Realities of NMR in the New(ish) Discovery Environment

Reality #1:

LC/MS has become the primary analytical check for the medicinal chemist. I Love NMR, but it's the reality

Reality #2: 

More often than not, NMR spectra are merely glanced at:

• Sometimes interpretation is based on the presence or absence of one peak (did my reaction go to completion?)
• How often are NMR spectra fully assigned by a medicinal/synthetic chemist? Not very often, it's not their job. 
• Is all the information a ¹H NMR spectrum can provide being effectively used? Some chemists are extremely good at NMR, other couldn't be bothered with the intimate details, and I can't say I blame them. It's largely a training/education issue
• How often are 2-dimensional experiments acquired and interpreted? Of course this varies chemist by chemists, organization..but despite strong advances in hardware, acquisition, etc, I believe the safe answer is, "not enough"

Reality #3:

In many organizations, upwards of 95% of compounds are being registered by synthetic/medicinal chemists without having undergone any analytical review by an analytical specialist (NMR spectroscopist). 

***

How and why did we get here? I have some hypotheses, and will write about them in the coming days. 

Are We There Yet? Automated Structure Verification (ASV)

When I created this blog several years ago, one of the first links I added to my blog was to Stan's NMR Blog. Over the years I have had the pleasure to meet Stan who is a wonderful individual. His blog is very interesting, Stan is incredibly knowledgeable, and I really enjoy his writing style. 

Recently, Stan posted about a topic, those readers who follow my blog know I am very passionate about; Automated Structure Verification (ASV). The initial entry was very thought-provoking and while I think the numbers Stan posts regarding the number of spectra chemists interpret below are too high, I think he's put together an interesting argument nonetheless about the need and application of ASV in the scientific community:

The typical application area for such a product is drug discovery, meaning Pharmas. I was told that these Companies employ masses of chemists who have to interpret some 50 spectra a day, having only 8h/50 = 9:36 minutes per spectrum. Clearly, they don't wag their tails, nor wish to pursue more balls. They were reduced to the echelon of routine workers and they hope that the new software might give them a few minutes more time to maybe ponder again that strange spectral peak that looked like an interesting challenge. Illusion, of course, because their top CEO's hope that the software will make half of the chemists redundant, leaving the rest doing a spectrum every 4:48 minutes (guess who will prevail).

Stan's entry definitely triggered some interesting comments from the community as well, in addition to a follow up letter from John Hollerton, GSK.

My take is that we don't need to be waiting 3-5 years for this technology to become more relevant...it already is! It is already being used in the industry. However, the secret is finding and recognizing the right application for it within the industry, and that application will vary considerably from lab to lab. 

It seems the main discussion about this completely surrounds whether or not ASV can replace the NMR interpretation of the medicinal or synthetic chemist. Of course this is the ultimate goal, but it should not be considered the only goal. John eludes to some other applications in his letter and there are other uses as well. 

I will blog more later about my thoughts on the "Reality of the New(ish) Discovery Environment" as it pertains to NMR and more thoroughly justify why I feel the way I do about this technology. But for me, the bottom line is that this is not just a concept, it's not a dream that we need to wait be realized in the next 3-5 years. It is also not a new technology on the horizon. Our first publication on this topic was back in 2006 and we've continued to build on those approaches in 2007 and 2008. Work was done by Griffiths et. al in the late 90s and early 2000s. The techonology has only gotten better and better. We have been working diligently with industry partners for over the last decade on this concept. It is not new and it is not an unsolved area. 

Just to be clear, am I saying this technology is ready to completely replace the NMR interpretation responsibilities of the average medicinal/synthetic chemist? Definitely not. 

Can it be used to help chemist make better, faster, and more independent decisions? Yes. Can it be used in complimentary way to the chemists existing interpretation workflow? Yes. Can it used to be validate the quality of registration libraries? Yes. Can it be used to verify compounds purchased from external sources, whether this are assay materials or simple starting materials? Yes.  The list of applications go on for a while. 

I believe current ASV applications can improve all the above workflows. The key is identifying the best applications and practicing caution in how this technology is deployed and used by chemists. 

In conclusion, my advice is that if we continue to treat this technology as only applicable to the ultimate goal, perhaps it will take longer than 3-5 years, or perhaps NMR will become irrelevant before we get there. The goal of NMR scientists, hardware, and software vendors should be to work hard to continue to make NMR technology relevant in all industries. If we can do this, we all win.

The Black Swan of Compound Libraries

Recently I finished a great book that made me think about different things on a lot of different levels. The book is called The Black Swan and the author is Nassim Nicholas Taleb. 

In short, a black swan is defined as an event, positive or negative, that is deemed improbable yet causes massive consequences. 

From wikipedia:

What we call here a Black Swan (and capitalize it) is an event with the following three attributes. First, it is an outlier, as it lies outside the realm of regular expectations, because nothing in the past can convincingly point to its possibility. Second, it carries an extreme impact. Third, in spite of its outlier status, human nature makes us concoct explanations for its occurrence after the fact, making it explainable and predictable.

Anyone who follows this blog knows that I have a passion around the concept of automated structure verification by software methods and the different applications it can play within the pharmaceutical industry. 

Reading through this book, one of the things that popped in my mind was the black swan impact as it pertains to compound registration libraries in pharma.

I spend many hours in my role discussing the concepts of automated structure verification (by NMR of course) to spectroscopists, chemists, directors, VPs, Managers of compound management, etc. 

In many of these conversations, the risk of having an incorrect compound in the registration library is always a fruitful and interesting discussion. However, this conversation is almost always dominated by low probabilities. The low probability that the compound being registered is wrong in the first place. I agree, this is a low probability. After all, a trained chemist has synthesized said material and has used a variety of analytical methods to confirm it's identity. In some organizations, the compounds in this library will be validated further either before they are accepted in the store, or before they are sent off to assay. 

So the argument goes that incorrect compounds in the registration library are low probability. And furthermore, there are steps being taken in some organizations to proactively catch incorrect compounds (most often LC-MS). Finally, perhaps the lowest probability event of them all is the compound being identified as a hit during the assay. 

So in the end, what are the chances that a meaningful compound that is identified as a "hit" is actually an incorrect compound? Furthermore, in this instance what's the chances that this compound will make it too far downstream without it's false identity being exposed?

In short, it's a pretty low probability event, and because in most cases it will eventually be caught it's a stretch to really call this a massive consequence or impact. 

That's not the black swan I am talking about in this post. 

In my opinion, the Black Swan concept is more relevant to those incorrect compounds that lie in the registration library and never get cherry picked out. A compound that doesn't produce anything interesting from the assay. A compound that effectively hides in the back of the shelves in a compound management cold room, that has been documented as "tested" but never advanced any further because of poor assay results.

Of course this compound is not the Black Swan. However, it's possible that the compound that it was supposed to be is. The compound that the chemist thought they made, and the compound that was referenced in the inventory.   

While it's low probability, there are probably tens of thousands of misrepresented compounds that haven't truly been assayed over the last 20 years. Of these thousands could any of them turned into the highly coveted, blockbuster drug?

Sure, it's doutbful, but the infinitely small possibility exists. 

And further, this very idea contains one of the core components of a Black Swan; "nothing in the past can convincingly point to it's possibility"

I am not certain that there is an example of a blockbuster drug that was originally missed because of a mistake in synthesis, or a misrepresented registrant. My guess is "not really" And if it did, perhaps as Taleb suggests, it would have been rationalized by hindsight. 

How do Medicinal and Synthetic Chemists use NMR Software?- Part 6

This is a continuation of a series of posts that began here.

In the previous post of this series, I discussed how chemists can extract multiplet reports in journal format on the fly using the same procedure to re-cut integrals in the spectrum. Well in addition to having this nice reporting tool, the software can make valuable use out of this multiplicity information.

The software has the ability to assist the chemist with the interpretation of their spectrum. It does this by using the NMR prediction algorithms embedded in the software. With the use of these algorithms structure interpretation and verification can be employed.

Essentially when a chemical structure is attached to a spectrum, the software will predict a spectrum in the background and automatically compare the predicted spectrum to the experimental spectrum. To do this automatic evaluation, the software will compare three properties between the spectra:

1) Chemical Shift
2) Integration
3) Multiplicity information

As a result, by re-cutting your integrals in the software (and hence defining multiplet patterns and coupling constants) the software will use this information to help a chemist interpret their spectrum. Specifically, it will use this information to provide the chemist with immediate feedback as to what atoms in the chemical structure a multiplet is consistent with. See the movie below:

How do Medicinal and Synthetic Chemists use NMR Software?- Part 5

This is a continuation of a series of posts that began here.

In my last post, I promised to address how, by simply spending 2 minutes re-cutting their integrals, a chemist can extract much more valuable information from their spectra.

The #1 thing in our NMR software that blows chemists away is the production of the following:

¹H NMR (DMSO-d6) Shift d: 7.90 - 7.95 (m, 2H), 7.79 (s, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.33 (dd, J = 8.6, 1.8 Hz, 1H), 4.30 (q, J = 7.1 Hz, 2H), 4.21 (q, J = 7.1 Hz, 2H), 2.53 (s, 1H), 1.25 (t, J = 7.0 Hz, 3H), 1.24 (t, J = 7.1 Hz, 3H)

To elucidate and format this information for experimental sections in patents, reports, and publications, this is a very time consuming process by hand.

Can software make it easier?

This capability has been available in our software for a long time. However, in older versions of our software, while it was much easier than doing it by hand, it was a little time-consuming and not obvious how to do this. For example you would have to first pick your peaks in peak picking mode, then you would have to integrate those peaks in integration mode, and finally you would enter Multiplet Mode and create those multiplets. 

As I've already mentioned, Chemists like to re-cut their integrals to make them look nice and normalized. Well now, when chemists do this procedure they are also peak picking and defining multiplet patterns and extracting coupling constant on the fly. Below is an 11 second video clip showing this capability in action. 

Prior to the implementation of this feature, using our software to create these reports was something that was done around patent-writing time. If a patent was being written and experimental sections were needed, the chemist would go back to find their old spectra, and create these reports then.

Now that this process is so quick, easy, and intuitive, I've talked to many chemists around the world who are creating multiplet reports for virtually every spectrum they acquire. They can then paste their multiplet information in a report or ELN, and it will save them time in the patent writing process down the road. And that's not the only benefit chemists can potentially get from extracting this information each time they acquire a spectrum. I'll talk about another, next time.

I wish all the Moms out there a Happy Mother's Day!

How do Medicinal and Synthetic Chemists use NMR Software?- Part 4

This is a continuation of a series of posts that began here.

Here's a short and sweet one.

In my last post, I talked about how having immediate access to the processed data is something that most chemists like. From there, for a lot of chemists the workflow is very simple. Let's just get the integrals looking nice.  Note: They don't generally look nice from automated processing off the instrument unless multiplets are really well resolved and separated.

So for some chemists, re-cutting their integrals and then printing off the spectrum, just might be the peak of their use of an NMR software package.

But what if this very simple process that generally takes no more than 2 minutes could also provide the chemist with much more valuable information about their spectrum beyond integrals?

I'll address that on my next post. 

How do Medicinal and Synthetic Chemists use NMR Software?- Part 3

This is a continuation of a series of posts that began here.

I learned an interesting lesson a few years ago when I was presenting our software on-site to a group of chemists. I began the demonstration by opening up an FID. The response from most of the room was a mix of confusion as well as horror! What is that squigly line you have up on the screen?

It was then I learned that most chemists don't need fancy processing, nor go through the manual steps of FT, baseline correction, phasing, etc. Provided that the sample was prepared and acquired well, the chemist generally doesn't need to do anything fancy to make their ¹H NMR spectrum look reasonable.

This is why in many open access environments that I have visited, chemists are not required to process their data at all. No need to FT their data, nor baseline correct it, nor phase it.

This can be accomplished in a couple of different ways.

You can supply the chemists with processed data from the instrument. In Bruker-speak that's the 1r file. In Varian-speak, it's a phasefile. This is really useful for a chemist because it saves them the additional mouse-clicks for processing data every single time. The only drawback of this approach is that the integrals are usually not all that nice. So a standard workflow for this, is deleting the integrals and re-cutting them a small price to pay, in my opinion.  And of course there are always going to be some cases where the phasing is off, or the spectrum will need some additional baseline correction.

Some other ways, for example, in our software, is to use macros to automatically process the data with one button click. Essentially, you need only create a macro with all the steps set to your liking. Once complete, you can use this macro on all your data. Simply import the FID and click the button. Of course you can also import processed Bruker or Varian files and work with them straightaway in the software. Finally, one of the things we added in the software back in version 10 was Shortcut Mode. With this mode, when you import or drop an FID into the software, it will auto-execute a set of pre-defined processing steps. For example FT, phasing, baseline correction, weighting functions, etc.

All in all, there are a variety of ways to greatly simplify the software interaction for a chemist. I recommend if you are a chemist, or the NMR person responsible for setting up the instrument, that you investigate the possibility of getting direct access to processed data. From there, it's just a matter of cutting integrals, and you're away!

How do Medicinal and Synthetic Chemists Use NMR Software?- Part 2

This is the continuation of a series of posts that began here.

Have you ever worked in an environment where there was a walk-up NMR lab?

If yes, were you fortunate enough to have software in your office or lab where you could view, process, and print out your own plots at your convenience?

If no, then you can relate to having to walk down to the NMR lab to get your print-out only to realize that all you really need is a zoomed in plot from 6.4 ppm -7.3 ppm. Or the processing done off the instrument didn't do the integrals the way you like them.

But John the Chemist, is ahead of you in the queue at the only workstation that has NMR software that allows you to do this. So you have to wait around until he's done his analysis, and he just happens to be one of those chemists who loves to use all the bells and whistles in the software :) Did I mention it was 6:30 PM on Friday?

Having software on the computer in your office or lab (or on your laptop) can result in a huge productivity boost. You may not think it, but I have had many NMR people in companies tell me that this where they get most of their ROI from a deployment of software.

The simple fact that chemists don't have to walk down to the lab for every spectrum, or wait in line to use the software, can save a lot of time and grief!