Monday, October 17, 2016

Araiosamines: The Long Journey From Vanuatu to La Jolla

     Today our work on the total synthesis of the araiosamines is out in JACS. While the chemistry (success and unexpected failures) was detailed in the paper and SI, I would like to share some stories from the science behind the construction of these molecules.

     On the first day I joined the lab, I told Phil that my motivation was to get extensive training in natural product total synthesis in a top tier synthetic chemistry lab. Phil immediately suggested the araiosmianes (isolated from a sponge collected from Vanuatu in 2011), saying, “if you want an education in total synthesis, these molecules would be an excellent option.” When I had a first glance at these molecules in the isolation paper, I thought they were only trimers of three bromoindoles, and there were only six carbons in the skeleton, no big deal at all! However, a very talented graduate student, Ming Yan, had been struggling with these molecules for two years. When involved in the project, I found these molecules are really tough to make, to say the least.

     During the first two weeks, we continued to work on the strategy, which employed an Achmatowicz reaction. However, due to the failure of indole installation, Phil decided to abandon this route. We met Phil in his office early on the following morning and tried to come up with new ideas. After one hour of discussion, we left his office without feasible plans in mind. We thought it was the time to move on. Maybe a guanidine related methodology project? Five minutes later, we received an email from Phil, asking, “did we consider the sulfones?” We immediately went to the library to mine the literature and found that α-amido sulfone is actually a stable acylimine precursor for the Mannich reaction. Eventually, the amido sulfone approach saved the project and opened the door to a completely new strategy—stepwise construction of the linear carbon skeleton. 
   The first Mannich reaction gave two diasteremers, however, favoring the undesired one. When performing the DIBAL reduction with either one, we always got the aldehyde as two inseparable diastereomers (poor yields, irreproducible and variable dr). We thought epimerization might be an unavoidable problem of the aldehyde. The next aldol reaction with the aldehyde as a mixture of diastereomers indeed gave the trimer product, but again in irreproducible and variable yields. During the attempts for the second Mannich reaction through nitrile hydrozirconition (SI), we found the nitrile could be reduced to the aldehyde with the Schwartz reagent without epimerization. This tricky reaction (the concentration and stoichiometry are both critical) must be quenched by loading the reaction mixture on the TLC plates (fortunately 1 TLC plate could quench about one gram scale reaction). Trace of EtOAc in the starting material, or quenching the reaction by silica gel powder, or aqueous solution, would induce epimerization.

     With pure aldehydes prepared, we observed their interesting reactivities towards the subsequent aldol reaction: the desired diastereomer 4 worked well to give the trimer product, whereas undesired diastereomer 3 gave a complex mixture, which could not be identified. Fortunately, what we needed to do was to epimerize the undesired nitrile to the desired 2 with t-BuNH2. Moreover, this unusual reactivity also inspired us to solve a key problem of C-H gunidinylation.
     In parallel to the skeleton construction, we also attempted the key C-H guanidinylation by indole oxidation of 6 prepared from the undesired Mannich product as a model substrate, due to the scarcity of the desired, but minor Mannich product. After screening ca. 100 conditions (including DDQ), only mixture of diastereomers in extremely poor yields was observed. When we figured out that the aldol reaction worked only with the desired diastereomer, the question arose: what about this C-N guanidinylation with the desired diastereomer? Surprisingly, DDQ mediated C-N guanidinylaiton worked very well with the desired diastereomer in quantitative yield and with complete stereoselectivity!

     To make a long story short, we combined both a linear and a cyclic strategy to finally make the mesylate 10 for the SN2 reaction. The subsequent reaction sequence of substitution, reduction, and guanidinylation worked very well. It seemed that ariaosmiane C would be conquered very soon. However, two months of effort did not lead to any trace of the natural product. That was the most difficult time for us. We were so close to the natural product: the mass was always correct, but the NMR spectra never matched! We began to doubt the stereochemistry—the only reaction that could give the wrong stereochemistry would be the SN2 reaction, because all other stereogenic centers were confirmed by X-ray crystallography of the precursors. The 2-D NMR spectra did not help very much in this case. Some signals supported the right stereochemistry whereas some did not. Phil said we would definitely need X-ray crystallography, given that Palau’amine’s structure was misassigned by NOE, which was not conclusive for such a complex structure. We decided to determine the stereochemistry of azide 11 by X-ray. However, to grow a crystal of such a late stage intermediate was not an easy task. We spent a lot of effort on the scale up and obtained 20 mg of the azide 11. Unexpectedly, too many bromines hindered the crystallization process, probably due to their too much lipophilicity. After extensive solvent screening, we eventually grew a crystal from a solvent mixture of MeCN, MeOH and H2O. After obtaining the X-ray crystallography, we were completely shocked—the configuration was retained when the azide was introduced in the displacement step. We also found that the six membered N,O-acetal ring has a perfect chair conformation while the unexpected axial bromoindole and azide have an antiperiplanar conformation. Clearly, due to the neighboring group participation of the axial bromoindole, a double inversion took place.
    How could we prevent the neighboring group participation? We tried many approaches including indole protection as the most straightforward, but none of these worked. The final idea was to employ a reductive amination, albeit not anticipated to be stereospecific. Interestingly, hydroxylamine was the only nucleophile that could condense with the ketone. Fortunately, after extensive experimentation the stereo- and chemoselective oxime reduction was achieved with SmI2 in the presence water. We were quite lucky because, initially we used the methoxy-substituted compound 14 as the substrate to investigate the reduction. In a later study, we found OTBS substituted compound 16 completely reversed the stereoselectivity of this reduction. Possibly, OMe directed the protonation from bottom face to give the desired stereochemistry. The desired product 15 showed very broad 1H-NMR peaks (some are missing) compared with the undesired isomer probably due to various conformers. Our tremendous effort spent on characterization of the undesired isomer was quite helpful. Without the X-ray of the undesired azide 11, how could we confirm the stereochemistry of desired amine 15 from its low quality NMR spectra? And the amine 15 was decidedly more difficult to crystallize.

     With the correct stereochemistry established, after guanidinylation we thought the natural product araiosamine C could be obtained immediately after exposure of 18 to TFA. Indeed, we observed a clean conversion to a product showing the mass of araiosamine C. While we were planning our celebration, misery beset us again, but not without company. The NMR spectrum did not match that of the natural product. It was actually the elimination product enamine 19. Hoping to cyclize the guanidine though enamine-iminium equilibrium, we subjected 19 to various acidic conditions. However, no reaction took place. This intermediate’s inertia was confusing. We also attempted cyclization by mesylation of the anomeric alcohol of 22. In addition to the enamine product 23, we unintentionally choreographed an indole dance (see 24)! Apparently, the pesky neighboring group participation happened again, but this time at another position. With this result, we finally came to the conclusion that cyclization via an iminium intermediate would not be possible, because the guanidine could not outcompete the anchimeric indole.
At this time, an idea of ring-chain tautomerization between cyclic hemiaminal 25 and acyclic aldehyde 26 emerged. We proposed that a carefully controlled Boc-deprotection of 22 would equilibrate to araiosamine A. After a discussion on the morning of once de Mayo, 2016, I said to Ming, “maybe today we could actually make the natural product.” But both of us weren’t too optimistic, because we hoped so many times, and were subsequently disappointed. Again, the result was frustrating, as Boc-deprotection in TFA/DCM induced instant dehydration to give again the enamine 19. The hemiaminal ring was not opened to allow equilibration to araiosamine A.

    Maybe Boc-deprotection in an aqueous acidic environment would suppress the dehydration. Thus, in another attempt the deprotection was performed in TFA-MeCN-H2O (1:5:4) at 90 °C. The LCMS showed a very complex mixture. Nevertheless, I still took the crude NMR spectrum, which looked hopelessly complicated. I thought it must be as usual that some isomers had the same mass as natural product but their structures could never be identified. When coming back from the NMR lab and comparing the NMR spectrum with that of natural araiosamine A, I was completely surprised—we made the natural product! (Later we found the crude pruduct was a mixture of three interconvertible compounds, 25, araiosamine A and epi-araiosamine A). Although it was around mid-night, I immediately called Ming to tell him this great news. He drove back to the lab, and we sent Phil an email together. I was too excited to sleep on that night. At 6 am we met Phil in his office. Phil said, in order to confirm it was the natural product we needed to scale up the reaction and get a 13C-NMR spectrum. During the scale-up, the hydrolysis product 22 was originally planned to be isolated before deprotection. Unfortunately, the hydrolysis reaction ended up with being heated up to 90 °C by accident (Another completely different reaction was planned to be performed at 90 °C at the same time. But I was too excited, and heated up this hydrolysis reaction mixture by carelessness). Again, the LCMS showed a major product of dehydration. We were not quite sure if it was the natural product or enamine 19. Meanwhile, the group had been waiting outside the NMR lab for celebrating our success. Much to our relief, we got a clean 1H-NMR spectrum of the dehydration product, which completely matched that of araiosamine C! (The mechanism of this cascade transformation is detailed in our manuscript) With some luck in the final step, we made the entire family of araiosamines in one pot. Employing the Ellman auxiliary, we also achieved the asymmetric synthesis of araiosamine C to establish the absolute stereochemistry of the natural product. Additionally, in stark contrast to the initial isolation report, we have found that these molecules are actually potent broad-spectrum antibacterial agents. This is a rare example of a natural product synthesis enabled discovery of bioactivity after the isolation chemists explicitly stated that this class of alkaloids had no observable activity!
     Lastly, I would say, without the accident in the final step, we would have definitely made araiosmaines C and D after isolation of araiosamine A and epi-araiosamine A, and subsequent subjection to dehydration. However, as 11-step syntheses have been trending in our lab, we were happy to keep it that way.

-Maoqun Tian

Note from Ming: I remembered vividly during my first day of graduate school when Phil described to me the remarkable similarities between araiosamines’ ring-chain tautomerization to that of carbohydrates. These alkaloids can be viewed as having an outward experience of “sea sugar”, though their structures are way more mystifying compared to fellow “sea salt”. This “sugar coating” was rather deceiving, enticing me to this “sweet-looking” project which turned out to be a bitter pill at the outset.
     Our earliest effort in the synthesis attempts to make araiosamine through direct trimerization of indolylacetaldehyde imines/enamines. This aldehyde which rapidly polymerizes in its neat form was later referred to by my colleagues as the “mingaldehyde”. It gave me an early exposure to “interdisciplinary research”: I would be making polymeric materials together with small molecules; the polymers thus produced have translational potentials from bench to the coal-tar industry. Together with Julian Shaw, an extremely talented visiting student, we surveyed an assortment of indolylacetaldehyde surrogates (discussed in the SI). We gained valuable insights on the reactivity and stability of various indole building blocks which would find use in our later efforts. But the end result of trimerization studies may be presented in a highly similar fashion as this legendary publication:

     Thus, half a year into the project, we decided to target the chain topology of araiosamines, embarking on what we dubbed as the “cyclic logic” in the paper. Admittedly, the amount of black tar I produced every day was dramatically reduced but extraneous functional groups present a significant hurdle. This was when we decided to combine lessons from all these approaches and formulate a new strategy to araiosamines. Having worked solo for a while after Julian’s departure, I was fortunate to be joined by Marc.

Monday, August 22, 2016

Cross-Couple While the Iron is Hot

We are delighted to announce that our recent work on Fe-catalyzed cross-couplings of redox-active esters (RAEs) has recently been published in JACSWe'd like to share with you how we started out, developed this project, and how things evolved from the first discovery.

During the past year our group has been involved in using RAEs as coupling partners in cross-coupling. We have developed a variety of coupling reactions to make the route to C–C bonds easy and practical to anyone regardless of skill or level of funding. The reactions with RAEs allowed for the first time the use of any alkyl carboxylic acid as coupling partner. The acid is primed for reaction similar to the activation for amide coupling using simple phthalimide derivatives (NHPI or TCNHPI) or peptide coupling agents (HATU or HBTU). The use of a cheap and abundant Ni catalyst does the rest of the job. These reactions revealed very interesting features which were already described in previous blog posts here and here at Openflask.

We were very aware that our initial reports on Ni still presented some limitations which needed to be addressed. It's always fun to read the comments and concerns on Derek Lowe's blog :) The story with Fe started by trying to address those drawbacks and provide solutions to those chemists out there, which need molecules synthesized easily and in high yield during a coffee break.

Up until now, all RAE couplings we developed were restricted to the use of 10 or 20 mol% loading of Ni (sometimes as low as 5%). FDA considers Ni as class 2A toxic metal impurity, which makes its use in pharma industries less attractive (at least on process scale). In addition, these methodologies posed other issues: occasionally long reaction times for the cross-coupling, the use of isolated RAEs to obtain the very best yields, and in our Suzuki coupling, diluted conditions were also required.

All these issues were then considered in the lab and we decided to tackle these challenges by taking a closer look into the mechanistic aspects of the reaction. We knew this would not be an easy task but we were lucky to gather a team with different backgrounds and expertise to put together this nice story.

Everything started with a simple question, which would solve most of the drawbacks in our Ni-catalyzed reactions: What about Fe? Our lab has been playing around with Fe for quite some time now and we all know the advantages of using Fe over Ni.
What about Fe??
In the past decades chemists have been trying to identify what are the operating mechanisms in Fe-catalyzed cross-couplings. The literature is brimming with papers trying to catch the right intermediate… However, what it seems to emerge as a common theme is that oxidative addition of alkyl bromides and iodides proceeds via SET of a low-valent Fe complex. I believe you all know where I am going here… We simply wondered whether Fe could actually undergo SET to our RAEs, inducing radical fragmentation. If this was possible, a plethora of opportunities would open up by combining the RAEs and known reactivities for Fe catalysis.

Indeed, the answer to the question “What about Fe?” seemed to be “Why not!”. Jacob  tried this reaction using TMEDA and obtained around 30% yield. He graciously shared those results with us before moving to another exciting project (that you can read about in a few months). Fumi (a tremendously talented visiting scientist from Daiichi Sankyo) set up the first reaction using phosphine ligands and 60% yield of product was obtained!! But there is more… it not only afforded good yield of cross-coupling product, but it was extremely fast and proceeded in less than 1 hour! A little bit of tuning identified Bedford’ and Nakamura’s Fe system (Fe(acac)3/dppBz) as optimal for these reactions.

However, it was clear since the very beginning of the project that we did not want to come up with “just the same but with Fe”. What we wanted is to improve our previous work by addressing all the issues we had until now with the Ni chemistry. To make this point very clear, we decided to directly compare Ni and Fe in every compound reported in the paper. In this manner we wanted the reader to contextualize the research and simplify the choice of the appropriate conditions. 

We were really thrilled to observe that a simple activation of the acid by HATU at rt afforded comparable yields as when using the isolated RAE. Unlike our experience with Ni-catalysis, HATU, TCNHPI, NHPI or HBTU all performed similarly. A very interesting feature of this reaction is that you can run the Negishi coupling with pretty much any solvent at reach in the lab. We used toluene, benzene, THF, 1,4-dioxane, DCM, DMF, hexanes, Et2O… and all of them afforded the coupling product in good yields. The difficulty came to select the solvent to carry out the substrate scope. The source of Fe did not matter either… FeCl3, FeCl3 hydrated or Fe(acac)3, valyrian steel, they all performed equally. We bet the reaction would work as well with a piece of the iron throne.

Another interesting feature of this reaction is how fast these couplings are... even at –20 ºC. It is incredible to observe that once you add the zinc reagent into the Fe/ligand/substrate mixture you just have to wait only if you like waiting. The reaction is finished by the time you dispose of the syringe. Simple aqueous workup, evaporation and short chromatography will give you the pure cross-coupling product.
Selected scope of >40 examples

We were very pleased to see that these conditions were very general across a wide range of substrates. Primary and secondary acids performed extremely well. Natural products and drugs were all arylated easily. As in any Baran lab project, pyridines are mandatory :)
During the exploratory scope of the reaction, our “human molecular machine synthesizer” Tie-Gen, also managed to obtain secondary-primary couplings with alkyl zincates. However, we want to warn practitioners that the primary alkyl-alkyl coupling is still beyond the scope of these conditions and more screening is needed. The main byproducts observed when attempting the primary alkyl-alkyl coupling were beta-hydride elimination of both partners in addition to the decarboxylated Barton-type product from the acid. If one wants to perform such coupling, we recommend having a look at our previous method using Ni. It is extremely efficient.

Another important feature of these particular Fe conditions is the possibility of arylating tertiary carboxylic acids. Previously, arylation of such acids was attempted for long time albeit unsuccessfully… We were really happy in the lab when we managed to arylate a [1.1.1] bicyclic system bearing a carboxylic acid in 35% yield. Notably, this is a bond formation that our collaborators at Bristol-Myers Squibb have been eying for years now.

We were thrilled to find that these conditions now allow another type of nucleophile to be introduced in our repertoire: welcome aryl Grignards. We had the idea of using Grignards for the cross-coupling for quite some time now but using Ni, ketone formation with the RAE was always problematic. And we kept failing dramatically… With Fe however, the extremely fast reaction rates observed allow for the integration of aryl Grignards without any ketone formation. It is interesting to mention that Grignards react at faster rates with the Fe catalyst than with the starting RAE. This result is consistent with Fürstner’s cross-coupling with low-valent Fe species where even isocyanates are tolerated.

Grignards work!
At this point, our Austrian postdoc Laurin took a step forward and decide to explore the limits of this system. He decided to subject the terrible beast of cubane to our arylation conditions; cubane is known to be an extremely sensitive material in the presence of transition metals. We were extremely delighted when we saw the GCMS of that reaction… a peak for phenylated product was there! Indeed, Laurin managed to put a phenyl ring into a cubane!!!!

To contextualize this result, a deep search in the literature revealed seldom methods for the synthesis of arylated cubanes. They seem to be restricted to rather bizarre reactions, such as Pb-promoted oxidative Minisci, where the regioselectivity is not controlled. 

Remarkable cubane examples
This modular protocol now allows the introduction of a wide variety of aromatic rings as we demonstrated in a 3-step double arylation of cubane scaffolds. These structures were obtained in reasonable yields and they turned out to be crystalline. We are so proud of these bis-arylated bioisosteres that the X-ray pictures are hanging on the walls in our offices as one of our best achievements :) (Proof of it just below).

Beautiful X-rays
Methods at the Baran lab are conceived with the ultimate goal of being applied at the industrial level. For this reason, the Fe team tackled a question that has been around since we started developing the chemistry of RAEs: can we apply this method in the context of process chemistry (it's already used in medicinal chemistry)?

One of the Figures in the manuscript resumes the work involved in the development of both a medicinal and a process chemistry approach. Since this protocol is easily scalable, no problem was found for the medical chemistry approach when using 1.4 g of starting carboxylic acid. Simple aqueous work-up followed by quick chromatography afforded pure cross-coupling material.

Real scalability
A critique we heard over and over again from process chemists associated with this chemistry was the high catalyst loading of toxic Ni salts, difficulties associated to its removal and dilute conditions for the Ni-catalyzed Suzuki. Our visiting process chemist Fumi gathered the team and said: “I think we can do better”.

Fumi ran the Fe cross-coupling reaction with just 1 mol% Fe and after a series of solvent exchanges and washes, followed by a crystallization, gave us a 61% yield of product. To our excitement, the purity of the compound was 99%. And since we are in the realm of non-toxic Fe, there is no regulatory limit for Fe impurities according to the FDA.

For those process chemistry readers, another feature of this chemistry is that a very small exotherm was obtained when running the reaction at 1.4 g. The internal temperature only rose from 1.5 to 19 ºC (note that most of the temperature increase came from addition of room temperature solution)! So we hope such small temperature change will pose no problem for scaling up cross-couplings with Fe and RAEs in the near future.  We also got the seal of approval from our process chemistry collaborators at BMS (thanks Gardner and Darryl!) who performed several examples in the table.

We hope people will find immediate applications for this interesting cross coupling. We hope you enjoy reading the manuscript and as always, if you have any comments/suggestions/criticism/questions feel free to contact us here directly (anonymous posts are welcome).

Pep and the Fe team

p.s. Check out the SI with photos, FAQ, and even some flow-charts to help practitioners :)