Heterocyclic Surgery for Isotopic Labeling

Abstract

Recent developments in the isotopic labeling of heteroarenes may prove to be useful in the realms of biomedical science, materials chemistry, and fundamental organic chemistry. The use of the age-old Zincke reaction, or tactical variants thereof, has become particularly utilitarian in effecting single-atom nitrogen replacement in various azines to generate their desired isotopologues. This chemistry can be synthetically leveraged at an early stage for diversity-oriented heterocyclic labeling of pharmaceuticals and/or natural products. Additionally, given the prevalence of saturated azacycles in biologically relevant molecules, access to these isotopologues becomes relevant through dearomative retrosynthetic analysis from the corresponding ¹⁵N-labeled heteroarenes.

Graphical Abstract

1. Introduction

Heterocyclic chemistry has had a lasting impact on the fields of medicinal chemistry, materials science, and organometallic chemistry, amongst many others. Recently, subfields such as metabolomics and magnetic resonance imaging (MRI) have demanded more precise isotopic labeling as a means of characterizing the behavior of heterocyclic carbogens.^1,2 However, prior to recently, access to valuable isotopically labeled ¹⁵N-heterocycles have remained rare and have limited advances in these translational subfields.³ This Synpact article describes our lab’s and others’ utilization of the Zincke reaction to effect the isotopic labeling of azines. Furthermore, these advances are contextualized and summarized within plethora of useful reactions that expand the palette of heterocyclic functionalizations and transmutations that have greatly expanded practitioners’ ability to easily synthesize and modify useful azine chemotypes.

In 1904 at the University of Marburg, Prof. Theodor Zincke disclosed a seminal discovery regarding an ‘aniline replacement’ in pyridinium salts (Scheme 1A).⁴ Following activation of pyridine with 1-chloro-2,5-dinitrobenzene via nucleophilic aromatic substitution, Zincke discovered a peculiar behavior of these salts (1) upon treatment with 2 equivalents of aniline. A ring opening of the pyridinium by initial dearomative addition of aniline then promoted further fragmentation of the ring followed by further displacement of 2,4-dinitroaniline with aniline to generate iminium 3 as an intermediate. When the reaction was conducted at 100 °C, extrusion of one aniline equivalent was observed concomitant with the rearomatization of 3 to give N-phenylpyridinium chloride (4). This ‘aniline replacement’ was one of the first reactions of its kind, characterizing what was later to be known as an ANRORC-type (addition of nucleophile ring opening ring closing) reaction mechanism in the canon of heterocyclic chemical transforms.⁵ Over 100 years after its discovery, the Zincke reaction has even earned an honored role in complex molecule total synthesis. Perhaps in some of the best examples from this century,⁶ the Vanderwal group leverage a ring-opening reaction of 1 with tryptamine 5 to generate dienal 6 after basic imine hydrolysis (Scheme 1B). This intermediate was then treated with KOt-Bu at elevated temperature to promote a [4+2] cycloaddition and prototropic rearrangement towards enal 7.^7,8 This sequence comprised a facile entryway to the core of various monoterpenoid indole alkaloids, such as strychnine (8)⁹ and more recently isolated natural products like alsmaphorazine B (9)¹⁰ and alstonlarsine A (10).¹¹ From a strategic standpoint, the Zincke salt (1) contained all necessary oxidation to enable a nearly isohypsic synthesis of these indole alkaloid core structures.¹² Given the sustained relevance of Zincke-type aziniums to mainstay heterocyclic motifs, many chemists continued inquiring as to how these malleable intermediates might be manipulated in several adventitious and/or practical synthetic avenues.

Scheme 1.

(A) The original Zincke reaction demonstrating nitrogen replacement. (B) Examples of Zincke salts leveraged in complex molecule synthesis by Vanderwal.

Recently, several research groups have leveraged Zincke-type reactivity for the strategic functionalization of pyridines or conversion to alternative heterocyclic motifs (Scheme 2). For example, McNally and co-workers demonstrated a selective C3-halogenation of pyridines 11 through activation and ring opening to Zincke imines such as 12.¹³ While the parent pyridines are electron-poor and relatively intractable for electrophilic halogenation reactions, the ‘unraveled’ dienamine variant serves as a nucleophilic surrogate with a much higher HOMO. In practice, treatment of 12 with NIS followed by ring closure with NH₄OAc afforded the iodinated pyridine 13 in 68% overall yield. This procedure that involves the activation of azines with triflic anhydride and strategic ring opening with dibenzyl amine to afford imine 12 laid the groundwork for other groups to leverage its adventitious reactivity. For example, one year later, the same intermediate 12 was demonstrated to undergo regiodivergent arylation by Greaney and co-workers.¹⁴ Treatment of 12 with aryl iodoniums 14 promoted substitution at the nucleophilic C5-position to give products like 15. In contrast, Heck-type arylation with 12 gave C4-arylated products 16 following rearomatization with NH₄OAc following with McNally’s reported protocol. Through a similar activation mode, Sarpong and co-workers utilized pyrimidine 17 as precursor to its ‘dehomologated’ pyrazole counterpart 20 in 2022.¹⁵ Following activation of 17 with triflic anhydride and addition of hydrazine, this resulted in the formation of ring-opened intermediate 18. Subsequent condensation of the hydrazine’s terminal N promoted displacement of amidine 19 generating 20 in excellent yield (90%). This formal ring contraction uniquely demonstrated how the pyrimidine could be dearomatized and thus manipulated as if it were a 1,3-dicarbonyl synthon typically employed in classical pyrazole synthesis. Following these contributions, amongst others, it seemed a renaissance of Zincke-type chemistry was already afoot.

Scheme 2.

Zincke chemistry has had a resurgence in recent years, lending itself to novel heterocyclic functionalization and transmutation.

2. Our Lab’s Development of the ¹⁵NRORC Reaction

The inspiration for our lab’s utilization of the Zincke reaction for azine ¹⁵N labeling originated in ongoing efforts to elucidate marine alkaloid biosynthetic pathways. With the aim of generating isotopically labeled substrates for biosynthetic feeding studies, we hypothesized that the ¹⁵N-labeled pyridines 22 might be suitable for identification of downstream metabolites by mass spectrometry and NMR. From a design standpoint, we thought that ‘surgically’ excising the nitrogen of the azine 21 through an ANRORC-type mechanism would enable this formal addition of one neutron to the heterocycle (Scheme 3A).¹⁶ This would require the incorporation of an appropriately functionalized ¹⁵N-labeled amine to generate an intermediate such as 23 that would be poised for a subsequent selective ring closure to deliver 22. If successful, this synthetic process would also open avenues in magnetic resonance imaging (MRI) through a process called SABRE-SHEATH, in which labeled azines 22 can be spin-polarized at the ¹⁵N center to afford MRI-active azines 24.^2,17 Until this work, synthetic access to various labeled azines had been limited, stunting the breadth of this potentially useful technique. At the outset of our work, however, we anticipated that the reaction design was going to be crucial to its success. The main two factors that had to be considered in our lab’s approach were (1) the nature of the activating agent used in the formation of a putative Zincke imine, and (2) the source of ¹⁵N to be used in the replacement of the azine nitrogen.

Scheme 3.

(A) Inspiration and strategic considerations in design of transformation. (B) Initial observation with Tf₂O activation. (C) Optimization of desired labeling reaction. ^a Yield determined by ¹H NMR spectroscopy with internal standard. ^b Isolated yield. ^c Percentage of ¹⁵N incorporation.

Per the aforementioned precedent, azine activation using Tf₂O was first investigated (Scheme 3B).¹³ Upon treatment of isoquinoline (25) with Tf₂O, azinium 28 was easily generated and subject to attack by benzylamine. The desired pathway (path b) would putatively result in a ring-opened intermediate 29 and subsequent condensation to deliver the desired azinium 26. Although some of this product was generated, the competing mechanistic pathway (path a) was just as prevalent resulting in the synthesis of triflimide 27 in a nearly equal quantity. As the intention was to use a labeled benzylamine or variant thereof, activation with Tf₂O was not seen as a viable agent in our hands for attaining high degrees of isotopically labeled azines through this ‘surgical’ N exchange. Notably, while we were not successful in engineering a Tf₂O-based activation protocol, other concurrent contributors in this area including McNally, Sarpong, and Audisio were able to utilize this activation method to successfully implement the same desired ¹⁵N azine labeling (vide infra).

Subsequently, we opted for the more traditional dinitroarene activation and utilized isoquinoline as our test substrate for the desired atomic exchange (see tosylate 30, Scheme 3C). Additionally, with an interest in facilitating fragmentation of the benzyl group attached to the labeled amino unit, three methoxy groups were introduced such that this group would spontaneously ionize in the reaction to directly afford the labeled azine (see 31). Several conditions were surveyed including parameters inclusive of solvent, temperature, time, and equivalency of Et₃N under microwave irradiation. It was initially found that Et₃N was essential to obtaining reasonable yields of 32 (entry 1) in addition to the utilization of either n-BuOH or DCE as a solvent (entries 2–6). A constant temperature of 125 °C proved imperative for cleavage of the TMB moiety, and 1.5 equivalents of 31 were crucial for the reaction to proceed to completion after 23 min (entry 7). Although a higher yield was obtained with n-BuOH as solvent (entry 5), purification issues with this solvent as a contaminant allowed for DCE to emerge as the preferred medium of the reaction. In the end, 32 was cleanly obtained in 75% yield and with 95% isotopic incorporation as determined by HRMS. Furthermore, replacement of 31 with ¹⁵NH₄Cl as the ¹⁵N source under these conditions resulted in poor yield (22%) and ¹⁵N incorporation (56%).

The scope of the ¹⁵NRORC reaction was evaluated under these optimized reaction conditions (Scheme 4A).¹⁶ Activation of various azines 21 with tosylate 33 resulted in the formation of intermediate Zincke salts in a yield range of 33% to 92% depending upon the substrate. Treatment of these salts with amine 31 under the optimized conditions resulted in the generation of a selection of labeled products 22, to which a small selection is shown in Scheme 4A. Various functionalities were tolerated as substituents including an alcohol (22a), an ester (22b), a halogen (22c), a boronic ester (22d), and an acetal (22d). Bicyclic substrates were also successful (22f, 32) in addition to natural products like nicotine (22g) and toddisoquinoline (22h). Importantly, these latter examples demonstrated the ability for later-stage ¹⁵N azine labeling which would likely be applicable to the application of this method to various APIs with azine moieties. To demonstrate the method’s further utility, we employed a dearomative synthesis of ¹⁵N-solifenacin (35) starting from the labeled isoquinoline (32). Activation of 32 with ethyl chloroformate followed by addition of phenylmagnesium bromide afforded an intermediate dihydroisoquinoline (Scheme 4B). Reduction of this dihydroisoquinoline with TFA in the presence of Et₃SiH afforded intermediate 34 in 36% yield over 2 steps. This compound has a known conversion into 35 through direct displacement of the ethoxy group of the carbamate.¹⁸ Beyond the synthesis of labeled compounds such as 35, it is anticipated that this chemistry will be useful in understanding the isoforms of variously formulated APIs through ¹⁵N NMR applications, which is work currently ongoing in our group.

Scheme 4.

(A) Scope of azine-labeling protocol; (B) dearoamative synthesis of the ¹⁵N-labeled API solifenacin

3. Other Recent Azine-Labeling Methods

Several other research groups investigated the ¹⁵N-labeling of azines concurrently with our efforts (Scheme 5). Based on their initial work in pyridine halogenation, McNally and co-workers were able to ¹⁵N label various pyridines through their Tf₂O-based Zincke reactivity platform. Under this method, the same activation of pyridine starting materials 36 was operable to afford their corresponding ring-opened intermediates 37. Following isolation of 37 from the starting pyridine, exposure of this intermediate to ¹⁵NH₄Cl resulted in the formation of the labeled product 38. This two-step procedure has a broad scope of pyridines that could undergo labeling including monocyclic substrates such as 38a, 38b, and 38d that were labeled with 92%, >99%, and >99% ¹⁵N-isotopic incorporations, respectively. Notably, each of these examples are pyridines with electron-withdrawing substituents and/or C2 substitution (38a,d). Furthermore, pyridine rings could be selectively labeled in fused substrates (38g) with high yield and isotopic incorporation in addition to selective pyridine labeling in substrates with multiple heteroaromatic moieties (38e). Lastly, one of the best advantages to this method is its ability to enable late-stage functionalization of drug molecules. To this end, the synthesis of ¹⁵N-loratidine was accomplished in 35% yield and >99% ¹⁵N incorporation over the two steps, and ¹⁵N-vismodegib was also generated in 28% yield and with 98% isotopic incorporation. Also concurrent with McNally’s work, Sarpong and co-workers were able to activate substituted pyrimidines 39 with Tf₂O and perform a nitrogen displacement with ¹⁵N-dimethylaspartic acid in the presence of 2-bromopyridine to form the intermediate pyrimidinium 40.¹⁹ This intermediate underwent mild elimination with DBU to afford the labeled product 41. While the ¹⁵N incorporation for this process was much more modest (Scheme 5B), the broad labeling of pyrimidines was an added benefit to the scope of azines that could undergo this atomic ‘swap’. Thus, pyrimidines such as 41a and 41b were synthesized with 92% and 21% ¹⁵N incorporation, respectively. In applying this protocol to various pyridines, once again triflate activation enabled the successful incorporation of the ¹⁵N nucleus into an azaindole (41d), a fused tricycle pyridine (41c), and two tricyclic bypridines (41e and 41f). Notably, the more sterically hindered of these two substrates (41e) had reduced ¹⁵N incorporation at 16% compared with 77% for the latter. Lastly, the Audisio²⁰ group reported a nearly identical protocol to McNally at the same point in time. While much of the scope is quite similar to that of the McNally group one additional entry of note was their incorporation of a ¹³N radioactive label into a parent azine (Scheme 5C). In the event, 4-phenylpyridine (42) was converted into imine 43 in analogous fashion to McNally followed by treatment with aqueous ¹³NH₃ (generated via cyclotron) at elevated temperature to produce 44 with a 78% radiochemical yield (RCY) after 6 min of reaction.

Scheme 5.

Recently described azine-labeling methods from (A) McNally, (B) Sarpong, and (C) Audisio reported concurrent with the ¹⁵NRORC protocol.

All four of these concurrent azine-labeling methods have important complimentary advantages in their respective scopes. With the ¹⁵NRORC protocol, the two-step procedure tolerates functionality like alcohols and dialkyl amides that may not be amenable to azine activation with triflic anhydride. That being said, there are limitations to the activation of azines with arenes associated with the electronic structure and substitution patterns of the azine inputs. Products such as 38a–g would be inaccessible do to their inability to be activated under classical Zincke conditions due to their electron-poor nature and/or substitution at the 2-position. This scope issue is greatly addressed by the triflate activation and ring-opening popularized by McNally and co-workers. The labeling efforts of Sarpong importantly addressed the isotopic labeling of pyrimidines in addition to pyridines, a useful expansion of the heterocyclic breadth of this tactic. However, as compared with the other methods, not forcing an azine ring opening with an amine like Bn₂NH diminished the degree of isotopic incorporation through this tactic. Sarpong, however, was able to partially circumvent this issue through separation of aziniums of structure 40 followed by subsequent elimination. When separated, azinium 40 could be converted into 41 with ¹⁵N incorporations improving by an average of 45%. With regard to ¹⁵N sourcing, the utilization of ¹⁵NH₄Cl ($74.30/g, Cambridge Isotope Laboratories) by McNally and Audisio has a cost advantage of the designer ¹⁵N surrogate used in the ¹⁵NRORC reaction (31) and the use of the ¹⁵N-dimethylaspartate by Sarpong ($558/g for amino acid, Cambridge Isotope Laboratories).

4. Expanded ANRORC Utilization

Even recently, work from several groups has expanded upon Zincke-inspired ANRORC chemistry for the transmutation of various aromatic moieties (Scheme 6). For example, earlier this year, Sorenson and co-workers were able to generate arenes 47 from their corresponding pyridinium N-oxides 45 through nucleophilic dearomatization of a lithiated sulfoxide anion to generate a ring-opened intermediate such as 46.^21,22 Subseqent condensation of the sulfoxide α-center and extrusion of all the heteroatomic moieties led to a net N to C replacement. Greaney has reported a similar transformation, but through employment of the azine activation with Tf₂O.²³ Following a similar ANRORC pathway, conversion of 48 to intermediate 49 following dearomatization with diethylmalonate proceeds smoothly to then allow for a decarboxylative rearomatization to forge benzoate 50 in good yield. McNally has recently utilized pyrimidines 51 as 1,3-dicarbonyl synthons to transmutate these heterocycles into several other congeners.²³ A selection of the several transformations from this report are shown in Scheme 6. Following activation of 51 and ANRORC substitution with aniline to generate pyrimidinium 52, ring opening with piperidine and condensation with amidine 53 generated pyrimidine 54 in 75% yield (one-pot protocol). The pyrimidinium 52 could also be formally intercepted with hydrazine to synthesize pyrazole 55 and undergo substitution with hydroxylamine to generate oxazole 56 in 40% yield.

Scheme 6.

Recently described chemistry from Sorenson, Greaney, and McNally on ANRORC-based arene transmutation.

5. Conclusion and Outlook

While Zincke set the stage for the power of ANRORC chemistry in the canon of organic synthesis, we have seen its recent renaissance in the 21^st century lay the groundwork for impactful scientific discovery. From total synthesis to heterocyclic transmutation to isotopic labeling, the surgical precision that these transformations offer certainly benefit the toolbox of the academician in addition to the practitioners of translational molecular discovery. It is of little doubt that Zincke’s legacy will continue to earn a considerable role in the future of synthetic heterocyclic chemistry. Indeed, it has enabled access to molecular motifs that, at least in the context of ¹⁵N-labeled azines, have remained largely elusive, or intractable until recently.

Biography

Prof. Joel M. Smith was born and raised in Raleigh, NC as the son of a physician and a secondary school chemistry instructor. He earned his BSc in chemistry and music in 2010 at Furman University in Greenville, SC while performing total synthesis research under the guidance of Prof. Brian C. Goess. His graduate studies were performed as an NSF-GRFP Fellow under the mentorship of Prof. Neil K. Garg at the University of California, Los Angeles and focused on complex alkaloid total synthesis. Following his graduation in 2015, he then conducted postdoctoral research as an Arnold O. Beckman Postdoctoral Scholar in the lab of Prof. Phil S. Baran at Scripps Research where he endeavored in the total synthesis of terpenoid natural products and Ni-catalyzed cross-coupling reactions. He began his independent career as an assistant professor in 2018 at Florida State University where his lab focuses on the concise synthesis of complex molecules and the invention of novel chemical reactions.