Monday, June 11, 2018

Pyrone Diterpenoids: A Not-So-Boring Journey 4 Years in the Making

Today, our work on the synthesis of natural products from alpha-pyrone diterpene family is out in JACS. While the paper details our final approach to these compounds, I would like to share the behind the scenes journey towards these molecules. Some of you may recognize that this topic was presented by Phil during his DOC lecture last month.
In the summer of 2014, our long time industrial collaborators  at LEO Pharma asked our lab to synthesize subglutinols A and B due to their reported immunosuppressive properties and potential as therapeutics. At that time, Kevin (postdoc in the lab and now a medicinal chemist at Eli Lilly) and I were charged with accessing these natural products in a concise and divergent manner. Assuming that Suzuki cross-couplings are always supposed to work (perhaps naively), we wanted to append the pyrone to the terpene skeleton via a cross-coupling strategy. To cut a long story short, after 8 months of starting the project (see SI for failures to this intermediate), we prepared the substrate to try our key step. However, it won’t be a good story (only in hindsight) without a catastrophe. Although “borono-subglutinol” could be cross-coupled with simple coupling partners like bromobenzene, the coupling never worked with the required bromo-pyrone, and we spent another 9 months trying to install the pyrone. However, all our efforts with met with spectacular failure. 
With the project in shambles, we decided to come up with a boring route (as Phil calls it) to prepare the natural product so that LEO could do the biological studies. We could in fact synthesize subglutinol A in 25 steps (detailed in the SI), and the natural product was delivered to LEO. Based on our “boring” route, we identified the following key limitations; (1) It took 5 steps to install 5 out of the 6 stereocenters on the molecule (including the core and subglutinol A THF ring) but 16 steps to install 1 stereocenter (C4 stereochemistry) as well as the pyrone; (2) While we had a way to access both subglutinol A and B THF rings selectively, the divergence occurred fairly early on in the sequence, and we wanted to delay this as much as possible; (3) We had a material throughput problem – the polyene cyclization required 3 grams of Mn(OAc)3and 1 gram of Cu(OAc)2for every gram of SM which resulted in purification nightmares!!
I won’t go into detail about how we ended up solving each of these problems ,but the key highlights are:
(1)For the polyene cyclization, electrochemistry allowed us to reoxidize Mn(II) to Mn(III) during the course of the reaction, making the reaction catalytic in Mn salts and resulting in a much simpler workup procedure.
(2)To fix the other problems, we came up with a “revised retrosynthesis”. So far, the sterically hindered C18 methyl group and diortho substituted pyrone had been the bane of my existence. We decided to kill two birds with one stone via a more radical disconnection. Disconnecting C4–C20 bond would allow us to use the sterics of C18 methyl group to our advantage (blocking the top face of the molecule). With regards to the late divergence to access both subglutinols A and B, we believed that under radical cross-coupling conditions, the C12 stereochemistry could be inverted through a stereoablative radical intermediate from a more sterically hindered cis THF ring of subglutinol A to a more stable trans THF ring of subglutinol B. However, the core of the molecule that we could easily access had an extra carbon at both the C12 and C4 position. One way to burn off a carbon to generate an alkyl radical was to perform a decarboxylation transform! This was helped by the fact that our lab was working decarboxylation cross-couplings at the same time and therefore, we thought this would be a good opportunity to develop decarboxylative Giese and alkenylation transformations.
To cut a long story short, we went on to develop a general decarboxylative Giese and alkenylation transformations of redox-active esters, and they could be implemented to the synthesis of subglutinols A and B. Eventually, we could also demonstrate our “revised retrosynthesis template” to prepare sesquicillin A and finish the first total synthesis of higginsianin A. We considered this a less-boring route, but not as exciting as this kind of boring. 
I have been on this project for the past 4 years, and while there have been a lot of ups and downs (definitely more downs), we did eventually reach our desitination! I just want to give a shout out to two extremely talented visiting students, Alex Novak and Yutong Lin, as well as thank our collaborators at LEO for their patience. If you have any questions about the chemistry or natural products themselves, please let us know! Thanks for reading!!

Rohan

Sunday, February 18, 2018

Desulfonylative Arylation: An Academia/Industry Collaborative Success Story

Our most recent work as part of a longstanding industry/academia collaboration between the Baran group at TSRI and Pfizer was published today in Science (on a Sunday!). As far as details of the publication and SI are concerned, we will leave that to you to explore on your own, so this blog post will discuss the origin of the project.

You could say that this project actually began back in 2014 when our lab was initially approached by Pfizer to solve the synthesis of [1.1.1]-bicyclopentylamine on process scale.  This simple idea led to a much greater topic of what we called strain-release amination. A section of this work focused on the use of strained sulfone reagents to access cyclobutanes, and a follow up report showed that we can similarly access enantiopure functionalized cyclopentanes through “housane” strain-release reagents. Although the sulfone functional group was useful for subsequent anionic functionalizations (alkylation, fluorination, etc.), one limitation of this work was that the sulfone had to be removed using strong reducing conditions.

In particular, scientists at Pfizer wanted a way to conduct a desulfonylative cross-coupling. Due to our prior experience in the development of decarboxylative cross-coupling reactions, they approached us and asked if we could help them development a transformation of this type. A key precedent for our work was an elegant comprehensive study from the Denmark Group at UIUC where it was shown that alkyl phenyl sulfones could be desulfonylatively arylated under Fe catalysis with aryl Grignard reagents.
After discussing this potential project with Pfizer, we realized that we could potentially solve a research problem that we were interested in working on; namely, new ways to synthesize (fluoro)alkylated arenes through cross-coupling chemistry. While we wanted to do this in a decarboxylative sense from mono- or difluoro carboxylic acids, these substrates proved recalcitrant, presumably due to the instability of the redox-active ester intermediate.  Fluorinated sulfones, however, are well-established in the literature and have a high degree of stability.
To make a long story short, we found that under Ni-catalysis with arylzinc reagents, we could indeed conduct our desulfonylative arylation reaction. A key to the success of this reaction was the incorporation of the phenyltetrazole group onto the sulfone; in our hands under these and related reaction conditions, this was the only group on the sulfone that permitted the desired reactivity. With the help of two talented visiting students, Monika and Cheng, we were able to quickly realize the scope of the reaction and applied the developed reaction to a variety of examples we found in the patent literature; in many cases, our desulfonylative cross-coupling simplified access to drug-like compounds.
We think the key take away from this work is shown in the scheme below; that is, from a single sulfone intermediate, three analogues (alkyl, monofluoroalkyl, and difluoroalkyl) can be accessed divergently from a common intermediate without the need for toxic and difficult-to-handle deoxyfluorination reagents.

During the course of this work, we identified a few reagents that we thought could be useful to medicinal chemists, shown in the scheme below. In partnership with Asymchem, we prepared these on large scale and have made them available via Twitter for free to chemists interested in trying out our reactions.  Let us know if you’re interested, and we would be happy to ship some to you!



As many of you may know, ChemRXiv, a preprint server for chemistry, was recently established, and the idea of preprints was intriguing to us because it would allow for our work to be viewed and used even prior to publication. We decided to take the plunge and become the Baran Group’s experimental test case in preprints. We really enjoyed the streamlined process by which the work shows up online; within a day, our manuscript was viewable to anyone interested free of charge.

While the manuscript was in review, we were curious about the ranking of alkyl PT sulfones relative to other electrophiles in terms of reactivity. We were pleasantly surprised to find that under the optimized conditions for desulfonylative coupling, the primary RAEs (TCNHPI and NHPI) react preferentially whereas the PT sulfone reacts faster than the primary halides (Cl/Br). There, the qualitative trend would be:
Cl/Br<SO2PT<NHPI/TCNHPI.
We want to thank our collaborators at Pfizer for another successful entry into collaborative work as well as Asymchem for conducting large scale reactions.  Let us know if you have any questions or comments regarding the work! Thanks for reading!

Rohan, Jacob,  and Tian





Tuesday, January 30, 2018

Arylomycins 2.0: Inside the Life of a Joint Graduate Student


As you were likely notified via Twitter, our work on the second-generation synthesis of the arylomycins was recently published in JACS. To tell the truth, saying “our work” is a little complicated and maybe even misleading if you found your way to this post via baranlab.org. As some of you may have noticed, the author list for this publication is a little unusual. Why are there two TSRI professors listed, yet only one student? Whose lab did this work, and where is the inspiration for this project coming from? I hope to answer these questions as well as some other more scientific ones people may have in this blog post about the work and my journey (so far) as a jointly advised graduate student.
Scalable Synthesis of Arylomycin
     It all started in 2014 when I was at a symposium where Prof. Floyd Romesberg was speaking about his lab’s work in antibiotic discovery, specifically regarding a class of molecules called the arylomycins. At the time, I was grad school bound, wanting to do synthetic chemistry yet also having a newly found penchant for the field of microbiology. You can’t always get what you want, but sometimes you do, and I was provided with the opportunity for my graduate studies to involve doing synthetic chemistry in the Baran lab with the purpose of working on antibacterial and microbiological research in the Romesberg lab. The first goal set for this collaborative work was to come up with a more efficient way to make the arylomycins, a still active interest in the Romesberg lab for the use as a chemical probe. On day one of my time at TSRI Floyd, Phil, and I met and the idea of oxidative coupling was born. On day two I had compiled a list of methods available for the oxidative couplings of phenols, and on day three I was in the hood making substrates. The real reason that this came together so fast is that there were so many methods available to perform such a coupling, making the outlook for this project good (at least to a bright-eyed graduate student on day three).
The plan moving forward was to make the desired product of the macrocyclization via the original Suzuki coupling route to have a standard, that once in hand would allow the rapid screening of the plethora of oxidative coupling conditions at our disposal to identify hits. This ultimately ended up providing me with the clearest picture of why this project would be so impactful to the synthesis of the arylomycins. Making the macrocycle via the original route was not only time consuming but extremely low yielding, and the boronic ester substrates that need to be handled turn out to be very sensitive. If banging your head against the wall was considered a synthetic step, it would definitely be included in our first-generation synthesis. Ultimately, the limiting factor (by a large margin) in me starting the screening became the synthesis of the product standard, not the synthesis of the oxidative coupling substrates! I should mention at this point though that the impact of the first-generationsynthesis should never be minimized. It has, up to this point, provided ALL of the material used in ALL of the work that has made the arylomycins the center of an ongoing antibacterial drug discovery campaign.
Once the standard was made, the screening began with really no agenda. We were agnostic to the nature of the oxidant, catalytic or stoichiometric reactions, and operational ease. Early on the usual results were “complete and rapid decomposition” or “no reaction.” You can read more about the discovery of copper being a competent oxidant in the manuscript, but what it won’t tell you is the battle I didn’t know that I was having with ambient water after the 10% yield obtained with complex Nakajima originally reported. A lot of screening was done around this reaction to no avail. Nothing I seemed to do improved the yield, though I did find a few good ways to decrease the yield. Through all of this, I had been working under the assumption that it was totally fine to run the reaction open to air in a “no precautions” manner, since that is what had been reported by Nakajima. When I finally got fed up with having nothing good to write home about I decided to put good old Schlenk technique to work and ran the reaction in an “every precaution I could think of” manner. The result: 65% yield. I honestly didn’t believe the result so I ran it two more times before telling anyone. Doing experiments in which I added water I confirmed that it was the culprit. The rest of the optimization from there you can find in the supporting information.
The optimized conditions led us to scale it up. The large amount of the macrocycle this provided us (see above) naturally led us to want to make analogs. The Romesberg lab had not been involved in making arylomycin analogs since 2013. Since then, RQx (a Romesberg lab derived start-up based on developing the arylomycins as antibiotic therapeutics) had a multi-year research collaboration with Genentech, who combined had presumably made many hundreds of analogs and knew far more about SAR than we did. The only way to not date ourselves would be to go digging in their patents for any hints as to what our new starting point should be. This digging made it clear that they had done a whole lot of work and had probed modification ideas that were both inside and outside of the box. Unfortunately, finding our new starting point from this was not so easy. At the time, we were interested in making analogs with Gram-negative activity, and unbeknownst to us, Genentech had already accomplished this goal and described it in a patent that had not yet become available to the public. With only the older patents to work with, the only Gram-negative MIC data to go off was for a permeablized strain of E. coli. We decided to take a gamble and chose the analog that had the most activity against this permeablized strain, knowing very well this was not necessarily an indicator of having real Gram-negative activity. Luckily this gamble paid off and the set of compounds we based on the Genentech analog have activity against regular Gram-negatives, the data for which you can find in the manuscript. 
The one and only thing that was clear from the arylomycin patents was that overall, there was a lack of diverse chemistry being done for derivatization at the C-terminal region of the arylomycins. Great structural work done by Dr. Mark Paetzel allowed me to spend lots of time scratching my head at this region of the molecule and the way in which it interacts with the active site of SPase. The native carboxylate of the arylomycins have distant interactions with the active site Ser-Lys dyad of SPase that are on the verge of not being H-bonds at all. This isn’t surprising when taken with the fact that the arylomycins are substrate mimics for a protease with Ala-X-Ala recognition and seem to end at Ala-X. I went to Floyd with the idea of extended C-terminal analogs to mimic the missing alanine residue. He was excited and wanted whatever we could put out there. I went to Phil with the idea and he wanted to do new chemistry. We ended up with the best of both worlds and decided on the decarboxylative Giese addition that was being developed in the Baran lab at the time. This would presumably be a far cry from something RQx or Genentech would have already done and would provide new chemical connectivity in this region. While the analogs this gave weren’t the best ones around, it provided valuable information with regards to the SAR, mainly being that this area of the molecule is not inviolate.

LepB (SPase) bound to Arylomycin A2

At the end of all of this, we patented our new technologies and reached out to Genentech who is actively working on developing the arylomycins. Their interest in our work turned into a licensing agreement and collaboration that has so far been a very positive and powerful for both sides. As if being a joint student between two labs at TSRI wasn’t enough for me, I have now added the industry collaboration as well. However, if the added dimension is anything like what I have experienced being a jointly advised student, it will prove to be extremely beneficial to both the science being done and the education of the student; me in this case.
Being a joint student comes with its own unique challenges: going to twice the meetings, presenting the same work to two advisors with different preferences, working on two advisors’ ideas, dealing with twice the personalities in the lab, the list goes on. There was even a dark time at the beginning, which has now passed, when I had to drive to get from one lab to the other. Ultimately, the benefits greatly outweigh the costs and often I find that the challenges listed above are in fact perks. On certain days, I may even go as far as to call them blessings. What the benefit of it really comes down to is the greater number of brilliant people with varying areas of expertise that I get to work with. The science you see in the recent publication is the result of helpful guidance and input from both of my advisors and from both of my sets of labmates. The acknowledgements section of our publication only begins to demonstrate that as members (current and former) of both labs are duly listed. So, if you are still wondering which lab did this work, or which lab is this guy even in, the answer is definitively both.
My Two Families

If you have any questions or comments about the chemistry, the arylomycins, or my unique set of challenges/blessings please let us know. Thanks for reading!

David Peters