Synthesis of [¹⁸F]Fluoroarenes via Nucleophilic Radiofluorination of N-Arylsydnones

Abstract

A practical method for radiofluorination of anilines with [¹⁸F]fluoride via N-arylsydnone intermediates is described. These precursors are stable, easy to handle and facilitate direct and regioselective ¹⁸F-labeling to prepare [¹⁸F]fluoroarenes. The value of this methodology is further highlighted by successful application to prepare an ¹⁸F-labeled neuropeptide.

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A practical radiofluorination of anilines with [¹⁸F]fluoride is achieved via N-arylsydnone intermediates. This method displays broad functional group tolerance, is compatible to automation on a commercial radiosynthesis module and can facilitate rapid ¹⁸F-labeling of peptides.

Positron emission tomography (PET) is emerging as a critically important, non-invasive in vivo imaging technique used for diagnosing, staging and therapy monitoring of disease.^[1] The convenient half-life (110 min), high positron yield (97%), and broad availability of fluorine-18 (¹⁸F) continue to make it the preferred radionuclide for clinical PET.^[2] For decades the landscape of molecules easily accessible for widespread PET research has been limited by chemistry. Early approaches to afford [¹⁸F]fluoroarenes from [¹⁸F]fluoride were based on the Balz-Schiemann reaction via aryldiazonium salts.^[3] Poor yields, competing fluoride incorporation from the BF₄⁻ counterion, and precursor instability has impeded wide adoption of this methodology for ¹⁸F radiotracer preparation.^[4] Recent advances in fluorination chemistry, particularly transition-metal-mediated methods to afford fluoroarenes, have soared dramatically in the last 5 years.^[5] Fluorination with the radioisotope fluorine-18 has in turn significantly expanded the scope and diversity of ¹⁸F-labeled small molecules.^{[4, 6]} These metal-mediated methods offer approaches to previously inaccessible ¹⁸F-labeled aromatics, thus increasing the potential radiochemical space for pre-clinical or clinical PET.^[7] However, many methods rely on stoichiometric quantities of transition metals to forge the carbon-fluorine bond, require complex starting materials, or suffer from competing protonation side-reactions. Such characteristics pose practical challenges for automation on a commercial radiosynthesis platform and require difficult purification steps that affect the overall yield and reproducibility of these methods.

Despite the remarkable, recent developments in radiofluorination, ¹⁸F-labeled arenes are most frequently synthesized via nucleophilic aromatic substitution (S_NAr).^[4] Trimethylanilinium triflates, triarylsulfonium salts,^[8] diarylsulfoxides,^[9] and diarylselenones^[10] are typical precursors used in S_NAr reactions (Scheme 1). Diaryliodonium salts^[11] are also viable precursors which give moderate to good selectivity that can be significantly improved with a copper catalyst.^[12] Alternatively, spirocyclic iodonium ylides have demonstrated regioselective ¹⁸F-fluorination with a broad substrate scope.^[13] Recently, a concerted S_NAr reaction was reported to enable ¹⁸F-deoxyfluorination of phenols via uronium intermediates.^[14] However, competing formation of volatile [¹⁸F]fluoromethane,^[15] regioisomeric mixtures of ¹⁸F-labeled products,^[8a] challenging precursor preparation or requirement for expensive reagents can be limiting for these methods.

Scheme 1.

Modern, metal-free methods for ¹⁸F fluorination of arenes.

Sydnones are remarkably stable mesoionic heterocycles composed of an azomethine imine 1,3-dipole that, upon cycloaddition with alkynes and expulsion of CO₂, afford substituted pyrazole products.^[16] From our previous experience constructing functionalized sydnones, we hypothesized that the electron-withdrawing nature of the 1,2,3-oxadiazole motif might enable activation of the attached aromatic ring for metal-free S_NAr chemistry. Arylsydnones are particularly attractive precursors for PET radiotracers as they can be readily prepared from inexpensive, commercially available anilines. Additionally, the mesoionic ring is stable to most chemical group manipulations, which allows for construction of functionally diverse precursors.

We report the development of a new method for the nucleophilic ¹⁸F-fluorination of a broad range of aniline-derived N-arylsydnones (Scheme 1). These shelf-stable precursors can be handled in air, without special precautions, and their radiofluorination readily proceeds with [¹⁸F]fluoride, without expensive stoichiometric reagents or transition-metal catalysts. In addition, we apply this methodology towards the synthesis of a sydnone prosthetic group and demonstrate rapid and facile ¹⁸F-labeling of a commercial peptide via [3+2] cycloaddition.

N-Arylsydnones are prepared in two steps from the appropriate anilines via initial formation of N-aryl glycines (Scheme 2).^[17] Subsequent N-nitrosation and cyclodehydration of the resulting nitrosamines affords the mesoionic compounds in good yields.

Scheme 2.

Preparation of N-arylsydnone precursors. Reagents and conditions: (a) ethyl bromoacetate, NaOAc, EtOH, reflux; LiOH, H₂O:THF (1:1), 0 °C; (b) NaOAc, AcOH, glyoxylic acid monohydrate, NaBH₃CN, MeOH, RT; (c) tButyl nitrite, THF, trifluoroacetic anhydride, RT. THF = tetrahydrofuran.

We initially investigated ¹⁸F-fluorination of 4-nitro-phenyl sydnone 4 because of the strongly activating nature of para-nitro substituents for S_NAr reactions. Radiofluorination of 4 proceeded in excellent radiochemical conversion (RCC) to afford [¹⁸F]fluoro-4-nitrobenzene 4a after 5 min at 150 °C (Scheme 3). Ortho substitution was well tolerated, affording 5a – 7a in >92% RCC. For 5 and 6, radiofluorination can also proceed at mild temperature (70 °C) in 68% and 72% RCC, respectively. Of note, formation of 4-[¹⁸F]fluorophenyl sydnone 4b via displacement of the nitro group was not detected, whereas sydnone displacement occurred readily. This selectivity resulted in a single radioactive product, and very clean HPLC traces, in all cases examined.

Scheme 3.

Radiofluorination of para-nitro substituted N-arylsydnones. RCC was determined by radio-TLC. ^an = 4. ^bn = 3. DMSO = dimethyl sulfoxide.

To understand the origin of this selectivity, we performed density functional theory (DFT) calculations at the M06-2X/6-311+G(d,p) level of theory,^[18] using Gaussian 09 (Figure 1).^[19] The SMD solvation model^[20] for DMSO was used throughout the calculations. For the reaction of 4 with fluoride, a typical S_NAr mechanism is found, whether attack of F⁻ occurs on C(1) (sydnone-bearing) or C(4) (nitro-bearing). In both cases, a barely stable Meisenheimer σ-complex (int) is located after the rate-determining fluoride addition transition state (TS1). Addition at C(1) is predicted to be 3.9 kcal/mol more favorable than addition at C(4), which is consistent with exclusive substitution of the sydnone group from 4.

Figure 1.

Calculated free energy profile for addition of fluoride to either C(1) (pathway a, in red) or C(4) (pathway b, in blue) of 4 (M06-2X/6-311+G(d,p)/SMD(DMSO) level of theory).

This reactivity difference can be explained by the electronic structure of 4. Natural bond orbital (NBO) analysis shows that C(1) bears a partial positive charge (+0.18) double in magnitude to that of C(4) (+0.07).^[19b] This indicates that a sydnone is inductively more electron-withdrawing than a nitro. This also explains why the formation of 4a is more exergonic (by 10.0 kcal/mol) than 4b, following the elimination transition state (TS2). Indeed, the sydnone anion ejected upon C(1) substitution is an excellent leaving group, much more stable than the nitrite that would result from reaction at C(4).^[19b]

Although a sydnone is inductively more electron-withdrawing than a nitro, it cannot however achieve full resonance with the aromatic ring, due to unfavorable peri hydrogen contacts that prohibit the two groups from being coplanar. In 4, the sydnone plane lies 40° away from the aryl plane (Figure 1), limiting its ability to stabilize an incoming negative charge by resonance delocalization. As such, we have estimated the Hammett σ⁻_p constant^[21] of sydnone (0.79)^[22] to be much smaller than that of nitro (1.24).^[19b] This indicates that a nitro is a better anion stabilizer than a sydnone, and its positioning para to the site of attack is preferred during an S_NAr reaction. Overall, fluoride addition at C(1) of 4 is strongly favored, because this pathway enjoys greater stabilizing interactions as the reaction progresses. This was confirmed by a distortion/interaction analysis along the IRC.^{[19b, 23]}

With these results in hand, the optimized reaction conditions were applied to a series of diverse N-arylsydnones to establish the scope of the reaction. As summarized in Figure 2, the radiofluorination proceeds in high RCC with substrates bearing electron-withdrawing substituents on the aryl ring and is compatible with numerous functional groups, such as nitro, cyano, ester, chloro, trifluoromethyl, ether, sulfone, sulfonamide, pyrrolidinyl, morpholinyl and amide. Several heterocycles, which are often problematic for metal-mediated radiofluorination methods, underwent radiofluorination with high RCC to afford thiophene (15a), benzofuran (16a) and pyridine (17a) derivatives. Of particular note is the successful preparation of ethyl 3-[¹⁸F]fluorobenzofuran-2-carboxylate 16a in 45% RCC which is, to the best of our knowledge, the first report of an [¹⁸F]benzofuran derivative. Synthesis of methyl 3-[¹⁸F]fluorothiophene-2-carboxylate 15a in 33% RCC is also significant as thiophenes are consistently difficult to radiofluorinate.^[4] Unactivated or electron rich substituents failed to give detectable [¹⁸F]fluorinated products. ^[19b]

Figure 2.

Scope of sydnone-mediated ¹⁸F fluorination. Reaction conditions: precursor (25 μmol), Et₄NHCO₃ (50 μmol), DMSO (200 μL), [¹⁸F]fluoride (300 μCi), 150 °C, 5 min. RCC was determined by radio-TLC. The identity of each labeled product was confirmed by radio-HPLC; ^a10 min. ^b15 min. ^c20 min. ^dRCC reported as radio-TLC conversion multiplied by radio-HPLC fraction of identified product.

While sydnones are valuable precursors for the synthesis of functionalized pyrazoles^[24] and biologically interesting molecules,^[16c] their most popular utility in the last few years has been their role in bioorthogonal reactions.^{[17b, 25]} Previously, we disclosed the bioorthogonal reaction of phenyl sydnones with dibenzocyclooctyne derivatives DIBAC and BARAC to undergo [3+2] cycloaddition for applications in fluorescence protein labeling.^[25c] Encouraged by these results, we sought to utilize this ligation to rapidly incorporate [¹⁸F]fluorine into peptides and other biomolecules for applications in PET molecular imaging. Thus, we applied our radiofluorination method towards the synthesis of 4-[¹⁸F]fluorophenyl sydnone 4b via the phenyl bissydnone precursor 21 and optimized the fully automated process using an ELIXYS radiosynthesis module (Tables S3 and S4). Unlike prosthetic groups such as [¹⁸F]fluoroethylazide and PEGylated counterparts,^[26] sydnone 4b is stable, non-volatile and displays strong UV absorbance. These properties make isolation and handling significantly less challenging and also simplify identification and quantification of the radiolabeled product via HPLC analysis.

Using 4b as a prosthetic group, we radiolabeled the 5 amino acid (H-Tyr-Ala-Gly-Phe-Leu-OH) neuropeptide, [D-Ala², D-Leu⁵]-Enkephalin 23 (Scheme 4). Peptide 23 is a selective delta-opioid receptor agonist which displays protective effects towards the preservation of neurologic function and neuronal damage against hypoxic or ischemic induced brain injury.^[27] Peptide 23 was functionalized with succinimidyl ester 22 to afford DIBAC-peptide conjugate 24. The desired 4-[¹⁸F]fluorophenyl sydnone 4b (6–10 mCi) was purified and isolated within 62 min from the end of bombardment in 21 ± 3% non-decay-corrected radiochemical yield (RCY) (n=17) with a specific activity of 1.3 Ci μmol⁻¹. Purified 4b was stirred with 24 in 1:1 DMSO:PBS at a concentration typically reported for [¹⁸F]peptide labeling.^[28] Remarkably, clean conversion (>97%) to pyrazole 25 was observed after 15 min at 40 °C, as determined by HPLC analysis of the reaction mixture (Figures S24 and S25). Notably, in all cases investigated, cycloaddition with 4b proceeded at 37 °C to >98% conversion after 30 min; at 50 °C, complete conversion was achieved within 8 min (Table S6).^[19b] Our application of [¹⁸F]sydnones as bioorthogonal prosthetic groups for the production of ¹⁸F radiotracers is related to Taran and coworkers’ recent Pd(II)-mediated formation of 4-[¹⁸F]fluoro-N-(p-tolyl)-sydnone, which is also an efficient cycloaddition partner.^[29] Indeed, our method compares advantageously as it uses simple reagents for one-step, direct radiofluorination without transition metals, gives improved RCY and is fully automatable.

Scheme 4.

Preparation and application of [¹⁸F]sydnone 4b towards ¹⁸F labelling of peptide [D-Ala², D-Leu⁵]-Enkephalin 23.

In conclusion, the first practical nucleophilic radiofluorination of anilines with [¹⁸F]fluoride is achieved by the initial modification of ArNH₂ to an N-arylsydnone precursor via a two-step process. This simple transformation was successfully automated on a commercial radiochemistry synthesis module to provide an efficient synthesis of 4-[¹⁸F]fluorophenyl sydnone which was utilized to prepare an ¹⁸F-labeled neuropeptide via bioorthogonal cycloaddition. We anticipate this methodology will complement the expanding radiofluorination methods and, due to its operational simplicity, find extensive applications in the production of radiopharmaceuticals for PET molecular imaging.