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Tetrazole synthesis, Part II: Harnessing the tetrazole-azidoazomethine equilibrium

Welcome to the second part of our Tour de Tetrazoles. In Part I, we saw that tetrazoles, particularly 5-substituted versions, are often made from nitriles and an azide source. Toxic and explosive hydrazoic acid can be formed in this chemistry, but we saw that there are ways to minimize its production.

Now let’s take a look at another general approach to tetrazoles, namely the concerted electrocyclic ring-closure of azidoazomethines. This chemistry will allow us to access a variety of tetrazoles, including fused-bicyclic versions, often under azide-free (and hydrazoic acid-free) conditions.

The tetrazole-azidoazomethine equilibrium

Azidoazomethines, also known as imidoyl azides, can undergo a concerted electrocyclization reaction to tetrazoles:

The tetrazole-azidoazomethine equilibrium

The position of the equilibrium depends on the nature of the substituents: electron-withdrawing groups on the azomethine nitrogen favor the open form, whereas hydrogen and normal organic substituents favor the tetrazole (leading reference and theoretical studies).

Fascinating, but there’s more: This equilibrium is an excellent entry point for tetrazole synthesis. Anything you can do to make the azidoazomethine will, with the right substituents, lead to the tetrazole.

Accessing azidoazomethines, and thus tetrazoles

If you can make the azidoazomethine, you’ll get the tetrazole. So, how does one make azidoazomethines? There are two primary methods, namely (1) the use of an activated amide to acylate sodium azide and, (2) the diazotization of amidrazones, themselves available by acylation of hydrazine with an activated amide:

Routes to azidoazomethines

Notice that the amidrazone method doesn’t require sodium azide or its equivalents, prompting some to call this an “azide-free” method.  A word of caution, though: It’s conceivable that the azidoazomethine could eliminate azide, so hydrazoic acid may still be lurking.  Use caution.

Example: Azidoazomethines (and thus tetrazoles) by Duncia’s method

John Duncia at du Pont reported a nice way to access azidoazomethines (and thus tetrazoles) from amides and TMSN3 using diethyl azodicarboxylate and triphenylphosphine. Shown below is a more recent application of Duncia’s method by discovery chemists Li, Tino, and co-workers at Bristol-Myers Squibb, who synthesized BMS-317180, a GHS agonist.

BMS discovery route using Duncia's method for tetrazole synthesis

The Process R&D group at BMS later felt that the issues of high exothermicity, TMSN3 safety, and PPh3PO removal warranted another approach to the tetrazole for scale-up.

Example: Azidoazomethines (and thus tetrazoles) from amidrazone diazotization

The BMS process group ultimately decided to avoid azide chemistry altogether by using amidrazone diazotization chemistry. Hydrazinolysis of the oxazoline shown below gave an amidrazone that was then diazotized to give the azidoazomethine (not shown) and thus the tetrazole on a 40 kg scale.  Superb.

BMS's Process for Tetrazole Synthesis on Large Scale

By the way, you can also make 5-substituted tetrazoles from amidrazones, though it’s not as common as the nitrile/azide method discussed in Part I of this post. Here’s an example :

Amidrazone route to 5-substituted tetrazoles

Fused bicyclic tetrazoles: An ideal application of the azidoazomethine-tetrazole equilibrium

Azidoazomethines where the azomethine (imine) is part of a ring, e.g. 2-azidopyridines and 4-azidopyrimidines, undergo electrocyclization to provide fused bicyclic tetrazoles. Historically, this is where the azidoazomethine-tetrazole chemistry began, with work by Bülow (1909; azidopyrimidines), Benson (1954, azidopyrimidines), Boyer (1959, azidopyridines), and McKee (1962, azidopyrimidines). The following example is from McKee’s lab at UNC and Montgomery’s lab at SRI (see also here). Both the azide and diazotization methods were used successfully.

McKee and Montgomery fused tetrazole synthesis

Finally, remember that the position of the azidoazomethine-tetrazole equilibrium depends on the electron density around the tetrazole? McKee used that knowledge to develop a new purine synthesis: Converting the above amine (electron donor) to the imidate shown below (electron acceptor) caused the azidoazomethine-tetrazole equilibrium to shift to favor the latter, which upon heating gave a purine. There’s some great stuff in the old literature.

McKee Purine Synthesis by shifting the tetrazole-azidoazomethine equilibrium

McKee, by the way, was the professor that got me interested in heterocyclic chemistry… see an earlier post for that story.

I hope you’ve enjoyed this Tour de Tetrazoles. I know there are some tetrazole experts out there, so please share some of your knowledge in the Comments.

Practical radical aminomethylation of electron-deficient heterocycles

The aminomethylation of arenes and electron-rich aromatic heterocycles typically involves iminium ion chemistry, i.e., Mannich-type reactions.  But when the heterocycle is electron-poor, what then?  Recent work offers an attractive approach involving nucleophilic α-amino radicals.

David Mitchell and co-workers at Lilly recently published the scaleup of LY2784544, a JAK2 inhibitor.  Their paper is chock-full of interesting chemistry and is highly recommended for a read, but let’s look at just one slice: the installation of the morpholinomethyl group using a radical addition reaction.

In a first-generation approach, the intermediate shown below was subjected to Minisci’s method for radical alkylation.  Phthalimide-protected glycine was used as a source of pthalimidomethyl radicals.  Around 3.5 kg of the CH substitution product was obtained, but the route was abandoned due to reproducibility problems, relatively low regioselectivity, insoluble by-products, and the need for large amounts of silver nitrate.

In a second-generation route, iminium ion chemistry was explored, but none of the desired material was formed.  In their survey of iminium ion techniques, however, the Lilly group found one outlier:  Hwang and Uang’s method using N-methylmorpholine-N-oxide and VO(acac)2.  In the Uang work, electron-rich arenes such as phenols and naphthols were aminomethylated; there were no examples of electron-deficient heterocycles.  Nonetheless, after some optimization, the Lilly group was able use the Uang method to produce the desired aminomethylated material shown above in good yield on a 44 kg scale.

Mechanistically, Mitchell and co-workers believe the Uang chemistry is substrate-dependent.  For electrophilic substrates such as the current imidazopyridazine, the reaction proceeds by a radical mechanism involving the addition of a relatively nucleophilic α-amino radical to the pyridazine ring.  With electron-rich systems such as those found in Uang’s work, an iminium ion mechanism is probably operational.  Further mechanistic work is underway.

For those of you in drug discovery, do you think it would be interesting to carrying out such radical aminomethylations on existing drugs or related compounds?  I’m reminded of the recent bevy of direct trifluoromethylation reactions by Baran, MacMillan, and Qing, featured at C&EN and In The Pipeline.  Yes, aminomethyl groups and trifluoromethyl groups serve greatly different ends, but direct introduction of the former at rather unreactive sites would seem to be a nice option.

Fluorescent Indocyanines for Color-Coded Surgery

I always get excited when I see an unexpected glowing spot on a TLC plate.  Fluorescence!  I’ve sent many a graduate student on a wild goose chase to try to identify the source of that glow.

My curiosity about these compounds blossomed during my time in industry, where we worked on fluorescent compounds and quenchers of fluorescence.  Adding to the fun was the fact that most small-molecule fluorophores (as opposed to materials like quantum dots) are heterocyclic compounds.

Let’s look at a current application of fluorescent heterocycles and then a bit of chemistry.

Color-Coded Surgery

Check out this recent TEDMED talk by Quyen T. Nguyen, M.D., a Professor of Surgery at UCSD, who gives an entertaining talk on the use of fluorescent dyes to aid in finding and removing every last bit of a tumor.  Her collaborator, Roger Tsien, received the Nobel Prize in Chemistry in 2008 for his work on green fluorescent protein (GFP).

In this work, cell-penetrating peptides (CPPs) composed of polycationic sequences of 6-12 consecutive arginines are covalently attached to a fluorophore (Cy5, more on that later).  These labeled CPPs can penetrate into pretty much any mammalian cell without requiring specific receptors.  To target them to the tumor, they make activatable CPPs (ACPPs).  Generalized cellular uptake of the CPPs is blocked by fusing them to a polyanionic peptide domain (featuring glutamates) via a protease-cleavable linker, neutralizing the polycation domain via the formation of an intramolecular hairpin.  For specificity, the linker is designed so that it is cleaved by certain matrix metalloproteinases that are overexpressed in tumors.  After injection, the ACPPs travel to the tumor, are cleaved by the proteinases, and accumulate in the adjacent tumor tissue.  Shine some light on the area and you can see the tumor, including the normally difficult-to-find margins.  Great stuff!

What are the fluorescent dyes being used?

Nguyen and Tsien use indocyanines (or simply “cyanines” for our purposes), which have been long-known for their fluorescent properties and are popular for photography, laser printing, non-linear optical materials, and of course labeling biomolecules.  They have the typical push-pull electronic structure of many fluorophores; the conjugated system has an electron donor at one end and an acceptor at the other.

Back to Nguyen and Tsien:  Take a look here and here for some of their work.  For many of their applications, they use Cy5 (originally Pharmacia Biotech, now GE Healthcare), a pentamethine cyanine dye shown in the scheme below, which absorbs at 650 nm and emits at 670 nm in the red region of the visible spectrum.  This is well beyond the region of background fluorescence that is found in tissue, so the signal-to-noise is good.  They’re also quite bright.  Another advantage of these long-wavelength emitters is that the light can travel through quite a bit of tissue.

Structurally, an interesting feature is the presence of the geminal dimethyl groups, which not only dictate the landscape of the conjugated pi-system, they keep the dye molecules from stacking, which would allow energy transfer and thus quenching of fluorescence.  One can vary the number of methine groups in the central conjugated region to tune the wavelengths of absorption and emission (Cy3, Cy7, etc.)

How are cyanine dyes made?

The venerable Fischer indole synthesis is used to make the 2,3,3-trimethyl-(3H)-indole starting materials.  N-alkylation with an alkyl halide produces a salt, which is used in an aldol-like double condensation reaction with 1,1,3-trimethoxy-2-propene, an equivalent of malondialdehyde.

If you would like to learn more about the chemistry of cyanine dyes, take a look at Mojzych and Henary’s review on the synthesis of cyanine dyes, or if you can’t access that, download the thesis of Jamie Gragg, who has a nice historical overview of the discovery and development of cyanines.

If fluorescent heterocycles interest you, check back here for occasional posts; there will be more!


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