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!

 

 

New Book on Computer-Assisted Structure Elucidation

It's been a very long time since my last post. I apologize for that, but I can assure that my posting absence has not been due to lack of quality things to talk about. Hopefully this post will help me get over the activation barrier of ignorning the blog for months!

My motivation for coming back is to update you on some excellent publications that have been published that involve ACD/Labs technology. 

The first one being the exciting release of a new book authored by ACD/Labs employees of past and present entitled, "Contemporary Computer-Assisted Approaches to Molecular Structure Elucidation" 

You can read one of the authors, Tony Williams' comments on the book and the writing process here. 

Mikhail Elyashberg also shared a few words in the official ACD/Labs press release here. 

Enjoy!

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. 

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. 

From FID to Structure in 30 Minutes? Investigating the CASE Limiting Step

In yesterday's post I pointed you to an article that has become the most accessed article of all time in the Journal of Cheminformatics, co-authored by representatives of ACD/Labs.

This article is a very comprehensive outline of the different approaches, developments, and successes of the development of CASE (Computer-Assisted Structure Elucidation) systems . I hope you agree that this technology has come a very, very, very long way, to the point that there are many spectroscopists around the world using ACD/Structure Elucidator routinely to identify unknowns in thier labs whether they be natural products, impurities, degradants, metabolites, etc. 

Anna Codina from Pfizer is an example of just one of those users. She has been a long-time user of Structure Elucidator, and one of the very best in the world. She, along with her group, has really stretched the limits and applications of the software and has also been instrumental in the ongoing improvement of this software over the years. 

At SMASH 2009, Anna shared some of her most recent experiences with the software in a presentation entitled, "From FID to Structure in 30 minutes?" This presentation can be accessed here.

For this presentation, she wanted to see whether it was possible to do a full elucidation starting from the raw data, all the way to the solution in less than 30 minutes. She was able to achieve this with several of her in-house Pfizer compounds, and then, for the purposes of this presentation reproduced these findings with compounds she could share in a pubic forum.

In this presentation she goes through 3 such examples.

The most interesting piece for me was her discussion regarding the CASE limiting step in slides 9 and 10.

In an ideal world, we would be talking about completely automated structure elucidation. Of course, we are not quite there yet, but it is still a dream, as Elyashberg et. al refer to it in the J. Chem. Inf. article.

In the meantime, the process generally involves some manually handling of the data. For this process the general principle applies, "Garbage IN- Garbage OUT"

So the quality of data as well as the peak picking of said data is crucial.

The peak picking of multiple spectra is what Anna was referring to as the CASE-limiting step. We have made tremendous strides over the years to simplify this process as much as possible (Check out the demonstration movie here). But the traditional argument seems to still apply for some:

Why would I spend so much time peak picking all my 2D spectra?

By the time I finish picking and grooming the data, I have already solved the structure, so what's the point?

I will try to address these questions in a different post, but in the meantime, what's Anna's answer?

NMR Workbook, and specifically the NMRSync technology that we developed and released late last year. Note, that NMR Workbook is included with ACD/Structure Elucidator.

Anna proclaims that thanks to NMR Workbook, there is no longer a CASE-limiting step. With syncrhonized peak picking which carries through peak picking applied in one spectrum, to all others associated with the sample, the time spent to prepare the data is drastically reduced and is more consistent with the natural workflow.

For those unfamiliar with NMR Workbook and the NMRSync functionality, I introduced it on the blog about a year ago:

http://acdlabs.typepad.com/my_weblog/2008/12/if-all-of-these-spectra-are-from-the-same-compound-why-am-i-handling-them-all-separately.html

If you want to skip right to the demonstration go here:

http://www.acdlabs.com/products/spec_lab/exp_spectra/nmrworkbook/movie/NMRWorkbookFlash.htm

This product has turned out to be immensely popular and has evolved a long way in just one short year. Admittedly, it's probably been the biggest reason I have not been blogging as much over the past year, I have been touring the world showcasing this product, as well as our other solutions.

I hope over the next little while I can dive a bit more into the details of the product.

Stay Tuned.

Most Accessed Article on Journal of Cheminformatics of All Time!

An article co-authored by several ACD/Labs scientists, the Russian Academy of Scientists, and ChemZoo, Inc. has been generating a lot of interest over the past year.

In fact, this article entitled, "Computer-assisted methods for molecular structure elucidation: realizing a spectroscopist's dream" is the most accessed article of all time, on the Journal of Cheminformatics website.

The news gets better. This article is FREE for your reading pleasure! As per the policy of the Journal:

"The Journal of Cheminformatics offers scientists the opportunity to publish their research rapidly in an open access medium that is freely available online to researchers worldwide. All manuscripts submitted to the journal are subject to rigorous peer review. Published material may include electronic supplementary material, such as animations (e.g. graphical chemical structures) or data (e.g. spectra) that can be downloaded for a reader's use.

The Journal of Cheminformatics aims to work closely with operators of open data repositories such as PubChem. Requests for collaboration are welcome; please contact the Editorial Office."

This motivation for this article was to mark the 40 year anniversary of published developments regarding CASE (computer assisted structure elucidation systems).

It a very interesting read that draws from many meaningful publications on CASE over the last 40 years and tracks history to outline some of the approaches that have been considered, implemented, and successful.

Some of the more significant developments such as fuzzy generation, used to deal with the ambiguity of 2D NMR experiments, as well as the determination of relative stereochemistry of large and rigid molecules with many stereocenters are discussed in this article.

I could spend more time talking about this article, but as I've already mentioned, it's FREE and can be accessed here:

http://www.jcheminf.com/content/1/1/3

Enjoy and feel free to leave any comments for the authors, and I will be sure to get them. 

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.