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DOI: 10.1039/D4CC00490F

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Synthesis of Sydnonimines from Sydnones and their use for
bioorthogonal release of isocyanates in cells.

Judith Baudet,ª Emilie Lesur,ª Maxime Ribéraud,ª Arnaud Chevalier,ᵇ Timothée D'Anfray,ª Pierre Thuéry,ᶜ Davide Audisioª and Frédéric Taranª*

Received 00th January 20xx,
Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

In this article, we report the synthesis of sydnonimines from sydnones and their use as dipoles for fast click-and-release reactions. The process relies on nucleophilic aromatic substitution of aliphatic and aromatic amines with triflated sydnones. This new methodology allowed the preparation of functionalised sydnonimines probes otherwise difficult to prepare. Those probes were then used to release a drug and a fluorescent aromatic isocyanate inside living cells.

Sydnonimines (SI) belong to the mesoionic family, closely related to sydnones with the key distinction of containing a nitrogen atom at position 6. Discovered in the 1950s¹ and further developed in the 1970s for their biological properties, some of them have been approved as drugs.² Renewed interest in SI has recently emerged due to their ability to undergo chemoselective cycloaddition reactions with strained alkynes, termed SPSIC (for Strained Promoted SydnonImine Cyclooctyne Cycloaddition).³ This reaction mechanism involves a two-step process: a [3+2] cycloaddition followed by a retro-Diels-Alder, yielding a pyrazole clicked product and an isocyanate released product. SPSIC exhibits promise as a novel bioorthogonal click-and-release tool applicable in target fishing,⁴ bioconjugation,⁵ cell imaging⁶ and drug release in complex biological media.⁷ The speed of the reaction is remarkably affected by the substituents at position 6 of the SI core: the rate constant of the SPSIC reaction can be enhanced by 6 orders of magnitude simply by replacing electron-withdrawing groups by electron donating groups at this position (Scheme 1).⁸ Our group has recently reported on novel SI bearing alkyl or aryl groups at position 6 exhibiting remarkable kinetics for the SPSIC reaction (k up to 1000 M⁻¹

s⁻¹).⁸ The synthesis of this compounds involves either reductive amination or cyclodehydration of nitrosoamides as key steps (Scheme 1). To expand the scope of synthetically accessible SI, we aimed to develop a method enabling direct modification at position 6 via nucleophilic aromatic substitution (S<sub>N</sub>Ar) of sydnones with amines, which are accessible both synthetically and commercially. Such method would enable the SI to be obtained in a single step with the possibility of significant structural diversity.

<figure><img src="image_1.png" /><figcaption>Scheme 1. SPSIC reaction and synthetic routes to 6-N-Alkyl and 6-N-Aryl-SI.</figcaption></figure>

The oxygen at position 6 of sydnones is known for its poor nucleophilicity and only very strong electrophiles are able to react with it.⁹ Nonetheless, aromatic nucleophilic substitutions at this position were reported in 1985 after activation of this oxygen atom by Tf₂O and addition of malononitrile carbanion.¹⁰ This reaction was recently extended to other activated methylene nucleophiles by the group of A. Schmidt to synthesise and study a series of sydnone methides compounds.¹¹ However, to date no implementation of this methodology has been reported for the introduction of alternative nucleophiles. Inspired by this work, we hypothesised that amines could serve as nucleophiles in this reaction, offering a new route to SI (Scheme 1). According to literature data,¹⁰ the reaction of sydnones with Tf₂O yields a mixture of sydnone 5-triflates I

<footnote>ªDr. J. Baudet, Dr. E. Lesur, Dr. M. Riberaud, T. D'Anfray, Dr. D. Audisio, Dr. F. Taran
Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SCBM, 91191 Gif-sur-Yvette, France
E-mail: frederic.taran@cea.fr</footnote>

<footnote>ᵇDr. A. Chevalier
Université Paris-Saclay, CNRS, Institut de Chimie des Substances Naturelles, UPR 2301, 91198, Gif-sur-Yvette, France.</footnote>

<footnote>ᶜDr. P. Thuéry
Université Paris-Saclay, CEA, CNRS, NIMBE, 91191, Gif-sur-Yvette, France
Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/x0xx00000x</footnote>
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and bis-sydnone ethers II, found to be very sensitive towards minute traces of water. Both species are in principle able to react with nucleophiles. Prior to investigating the reaction with amines, we initiated a study to elucidate this activation step through quenching experiments with isotopically labelled H₂¹⁸O (Scheme 2).

<figure><img src="image_2.png" /><figcaption>**Scheme 2.** Reaction of sydnones with Tf₂O. I.E.: isotopic enrichment</figcaption></figure>

The activation of sydnone 1 was carried out using 0.5 equiv. of Tf₂O to favour the formation of intermediate II. As a consequence, after quenching with H₂¹⁸O a maximum of 50% isotopic enrichment (I.E) of labelled sydnone [¹⁸O]1 or Tf¹⁸OH could be expected. ¹⁹F-NMR monitoring showed complete consumption of Tf₂O before addition of H₂¹⁸O, while ¹H-NMR indicated the formation of two new sydnone species (Figure S1). ¹⁹F-NMR and HRMS analysis just after addition of H₂¹⁸O showed Tf¹⁸OH as the major labelled product which can be generated only from the hydrolysis of I (Figure S2). ¹⁸O-labelled sydnone [¹⁸O]1, formed by S<sub>N</sub>Ar reaction either on I or II, represented only a quarter of the total labeled compounds. Control experiments confirmed that no ¹⁸O exchange occurred neither on sydnone nor on triflate salt under the reaction conditions (Figure S3). These results proved that I is the main active species formed during the reaction and, as expected, that the S<sub>N</sub>Ar reaction on I competes with the nucleophilic attack on the sulphur atom of the triflate moiety. This side reaction could pose a significant challenge when amine nucleophiles are used on I. From these findings, we explored the reaction with aliphatic and aromatic amines using benzylamine and aniline as model substrates (Table 1). The S<sub>N</sub>Ar reactions of both model amines on triflate I were found very fast: completion of the reactions were observed after 10 min even at room temperature showing the high reactivity of triflate I. Gentle heating at 40 °C allowed a significant increase of the yield, but higher temperatures were detrimental to the reaction due to the degradation of substrates. The presence of a base is beneficial to the reaction, TEA being the most effective. Finally, the influence of solvent was investigated revealing CH₃CN as the most suitable. Despite these efforts, yields plateaued around 50% due to the side reaction generating triflated amines 2 and to purification issues. Attempts to use alternative activation methods for sydnones besides Tf₂O (Ts₂O, NS₂O...) to minimize the formation of 2 were unsuccessful.

Despite the challenges, we were intrigued by the potential synthetic utility of this reaction and explored its scope further (Scheme 3).

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<figure><img src="image_3.png" /><figcaption>**Table 1.** Optimization of the reaction.</figcaption></figure>

DOI: 10.1039/D4CC00490F

<table><thead><tr><th>Entry</th><th>Solvent</th><th>RNH<sub>2</sub></th><th>Base</th><th>Temp.</th><th>SI (yield)<sup>a</sup></th><th>2 (yield)<sup>b</sup></th></tr></thead><tbody><tr><td>1</td><td>MeCN</td><td>BnNH<sub>2</sub></td><td>-</td><td>25 °C</td><td>30%</td><td>40%</td></tr><tr><td>2</td><td>MeCN</td><td>BnNH<sub>2</sub></td><td>DiPEA</td><td>25 °C</td><td>31%</td><td>24%</td></tr><tr><td>3</td><td>MeCN</td><td>BnNH<sub>2</sub></td><td>DiPEA</td><td>40 °C</td><td>39%</td><td>39%</td></tr><tr><td>4</td><td>MeCN</td><td>BnNH<sub>2</sub></td><td>DiPEA</td><td>60 °C</td><td>n.d</td><td>n.d</td></tr><tr><td>5</td><td>MeCN</td><td>BnNH<sub>2</sub></td><td>DABCO</td><td>40 °C</td><td>47%</td><td>3%</td></tr><tr><td>6</td><td>MeCN</td><td>BnNH<sub>2</sub></td><td>Pyridine</td><td>40 °C</td><td>n.d</td><td>n.d</td></tr><tr><td>7</td><td>MeCN</td><td>BnNH<sub>2</sub></td><td>TEA</td><td>40 °C</td><td>50%</td><td>36%</td></tr><tr><td>8</td><td>DMF</td><td>BnNH<sub>2</sub></td><td>TEA</td><td>40 °C</td><td>n.d</td><td>n.d</td></tr><tr><td>9</td><td>THF</td><td>BnNH<sub>2</sub></td><td>TEA</td><td>40 °C</td><td>45%</td><td>n.d.</td></tr><tr><td>10</td><td>CHCl<sub>3</sub></td><td>BnNH<sub>2</sub></td><td>TEA</td><td>40 °C</td><td>42%</td><td>11%</td></tr><tr><td>11</td><td>Toluene</td><td>BnNH<sub>2</sub></td><td>TEA</td><td>40 °C</td><td>50%</td><td>n.d.</td></tr><tr><td>12</td><td>MeCN</td><td>PhNH<sub>2</sub></td><td>TEA</td><td>40 °C</td><td>44%</td><td>19%</td></tr><tr><td>13</td><td>MeCN</td><td>PhNH<sub>2</sub></td><td>TEA</td><td>60 °C</td><td>41%</td><td>25%</td></tr><tr><td>14</td><td>MeCN</td><td>PhNH<sub>2</sub></td><td>TEA</td><td>80 °C</td><td>40%</td><td>17%</td></tr><tr><td>15</td><td>THF</td><td>PhNH<sub>2</sub></td><td>NaH</td><td>40 °C</td><td>n.d</td><td>n.d</td></tr><tr><td>16</td><td>THF</td><td>PhNH<sub>2</sub></td><td>tBuOK</td><td>40 °C</td><td>n.d</td><td>n.d</td></tr></tbody></table>

<sup>a</sup> crude yields determined by NMR for entries 1-11 and isolated yields for entries 12-16. n.d = not detected. <sup>b</sup> crude yields determined by UPLC-MS

<figure><img src="image_4.png" /><figcaption>**Scheme 3.** Scope of the reaction. Reaction conditions: 1) Tf₂O 1.2 equiv., MeCN – 25 °C, 2) RR'NH 2.0 equiv. TEA 4 equiv. MeCN – 40 °C. n.d = not detected. The trifluoroacetate anion comes from the reversed phase purification.</figcaption></figure>
Smooth S<sub>N</sub>Ar reactions were observed with both aliphatic and aromatic amines bearing electron rich or electron-poor substituents. To compare the S<sub>N</sub>Ar reaction (method A) with our previously reported cyclodehydration approach (method B),<sup>8</sup> we synthesized a series of **SI** using both methods. The S<sub>N</sub>Ar procedure proved to be a valuable complement to method B and allowed the synthesis of **SI** otherwise difficult or impossible to prepare. Although the S<sub>N</sub>Ar approach gave low to moderate yields, it tolerates a variety of functional groups such as phenol, azide and carbamate which are incompatible with method B. Interestingly, secondary amines are also good substrates for the S<sub>N</sub>Ar reaction, yielding unprecedented compounds bearing an exocyclic tertiary amine at position 6 of the sydnonimine core. These derivatives, inaccessible *via* method B, are not mesoionics and thus represent novel chemical species for exploration in cycloaddition reactions with strained alkynes. We thus conducted kinetic experiments with model substrates **SI5** and **SI6**, which differ only in the presence of a methyl group, in presence of the cyclooctyne DBCO (Scheme 4).

<figure><img src="image_5.png" /><figcaption>**Scheme 4.** Reaction of sydnonimines **SI5** and **SI6** with DBCO. Proposed mechanism for the formation of **P1** and **P2** from **SI6**. The rate constants were determined by UV monitoring, [**SI5**] = [**SI6**] = 100 µM, DBCO 1.0 equiv. in 0.1 M PBS pH = 7.4 or in DMSO. Yields of pyrazoles were estimated by ¹H NMR monitoring in DMSO-d₆.</figcaption></figure>

As anticipated, the kinetic behaviour of the two compounds exhibited drastic differences. Compound **SI6** reacted 923 times slower in reaction with DBCO in PBS compared to **SI5**. In addition, the reaction of DBCO with **SI6** produced not just one but two pyrazole products **P1** and **P2**. Both pyrazoles were formed at the same speed with a kinetic constant of k = 0.22 ± 0.05 M⁻¹·s⁻¹ in PBS, suggesting that the mechanism involves a common intermediate whose formation is the rate determining step. We postulate that the formation of intermediate **Int** resulting from a [3+2] cycloaddition on **SI6** can undergo a rDA reaction to form pyrazole **P1**. On the other hand, **Int** can also evolve after proton abstraction to generate pyrazole **P2** (Scheme 4). As the formation of pyrazole **P1** is concomitant with the release of the secondary amine attached in position 6 of the **SI** core, a possible application of this chemistry may lie in the release of drugs attached to this position. To evaluate this hypothesis, we conducted proof-of-concept experiments. **SI19**, obtained in 20% yield using the S<sub>N</sub>Ar reaction, was designed to release

the antidepressant drug Paroxetin upon reaction with DBCO (Scheme 5). The SPSIC reaction was conducted in aqueous conditions at pH 7.4, 25 °C and monitored by UPLC. The results confirmed a significant rate constant of k = 1.12 ± 0.21 M⁻¹·s⁻¹ but incomplete release of the drug over time (~13% release, Figure S13). This lack of release efficiency may be attributed to the formation of **Int₂** that cannot undergo the rDA-promoted release of the Paroxetin. To avoid this, we then synthesised **SI20** and **SI21** substituted by a Cl atom and a methyl group respectively at position 4 of the **SI** core in order to prevent from the proton abstraction step. Although **SI21** proved to be unreactive, **SI20** allowed slow but efficient drug release upon reaction with DBCO (Schemes 5 and S16). Although improvements are clearly required, these **SI**-protected drugs might be interesting in future bioorthogonal decaging strategies.

<figure><img src="image_6.png" /><figcaption>**Scheme 5.** Release of Paroxetin from **SI19-21**, monitoring was carried out by UPLC, 4-Nitro-2-fluorophenol as internal standard. Reaction conditions: [**SI**] = 100 µM, DBCO 1.0 equiv. in PBS pH = 7.4, 25 °C.</figcaption></figure>

We then focused our interest in the coumarin-containing compound **SI18**, which should generate a fluorescent isocyanate upon SPSIC reaction. Interestingly, we found that **SI18** reacted as a fluorogenic probe with DBCO to form an isocyanate which fluorescence is slightly higher than the starting sydnonimine (turn-on around x 5, Figure 1).

<figure><img src="image_7.png" /><figcaption>**Figure 1.** SPSIC reaction of probe **SI18** with DBCO under physiological conditions. Kinetics were monitored by fluorescence, λ<sub>ex</sub> = 404 nm, λ<sub>em</sub> = 510 nm. Reaction conditions: 37 °C, [**SI18**] = 10 µM, DBCO 1.5 equiv. in PBS pH = 7.4.</figcaption></figure>

<figure><img src="image_8.png" /></figure>
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The pKa of compound **SI18** was determined at 4.8 ± 0.1 (Figures S8 and S10). Accordingly, **SI18** is mainly deprotonated at physiological pH generating a highly reactive mesoionic species. This was confirmed by fluorescence monitoring of the SPSIC reaction of **SI18** with DBCO: the kinetic constant was found to be k = 533 ± 27 M⁻¹.s⁻¹ meaning complete complete click-and-release reaction within minutes when compounds are used in the µM concentrations (Figure S12). We then studied whether this probe may be used to release fluorescent isocyanates inside living cells (Figure 2A). A549 cells were incubated for 2 h with **SI18**. After washing, SPSIC reaction was initiated by addition of DBCO (250 µM). Careful monitoring using wide field microscopy (Figures S17 and S18) over 2 h showed a 4-fold increase in fluorescent signal intensity achieved after 30 min reaction time (Figure 2B). The confocal microscopy images shown in Figure 2D indicates a significant increase in fluorescence intensity after addition of DBCO. Interestingly, this labelling was resistant to several successive washing steps (Figures 2C and S19), suggesting irreversible fluorescent labelling of the cells. In the meantime, the same washing process induced a significant drop of the fluorescence signal intensity in the case of untreated **SI18**. Altogether, these results confirmed the effective biorthogonal release of the coumarin-isocyanate derivative inside the cells leading to persistent labeling of the cells.

<figure><img src="image_9.png" /><figcaption>**Figure 2.** SPSIC reaction inside living A549 cells. Cells were washed before addition of DBCO. A) Schematic representation of isocyanate release. B) Fluorescence intensity evolution with time in A549 cells after addition of DBCO. C) Fluorescence intensity after 2 h in presence or absence of DBCO and with or without cell washing. D) Confocal microscopy images of cells treated with probe **SI18** in presence or absence of DBCO. Conditions: [**SI18**] = 50 µM, incubation 2 h then wash PBS, addition of [DBCO] = 20 µM during 1 h.</figcaption></figure>

In summary, we have developed a new method for synthesizing sydnonimines that were previously difficult to obtain. Although yields need improvements, this method's simplicity and tolerance to functional groups make it

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advantageous compared to existing methods. We have successfully synthesized unique **SI** with tertiary amines at position 6, tested them in cycloaddition reactions with DBCO, and explored potential applications in drug release. Additionally, the S<sub>N</sub>Ar reaction allowed access to a fluorogenic probe for efficient fluorescence labeling inside living cells. Such compounds allow the release of fluorescent isocyanates in cells which can be exploited to visualize bionucleophiles and to label permanently living cells holding promise for advancing our understanding of cellular processes and protein dynamics.

Acknowledgements

This work was supported by the Agence Nationale de la Recherche (ANR-23-CE07-0041-01). The project benefited from Imagerie-Gif core facility supported by l'Agence Nationale de la Recherche (ANR-11-EQPX-0029/Morphoscope, ANR-10-INBS-04/FranceBiolmaging; ANR-11-IDEX-0003-02/ Saclay Plant Sciences). The authors thank David-Alexandre Buisson, Sabrina Lebrequier and Amélie Goudet for excellent analytical support

Conflicts of interest

"There are no conflicts to declare".

Notes and references

1. P. Brookes, J. Walker, *J. Chem. Soc. 1957*, 4409-4416.

2. E. Y. Khmel'nitskaya, V. I. Levina, L. A. Trukhacheva, N. B. Grigoriev, V. N. Kalinin, I. A. Cherepanov, S. N. Lebedev, V. G. Granika. *Russ. Chem. Bull*. **2004**, *53*, 2840-2844.

3. S. Bernard, D. Audisio, M. Riomet, S. Bregant, A. Sallustrau, L. Plougastel, E. Decuypere, S. Gabillet, R. A. Kumar, J. Elyian, M. N. Trinh, O. Koniev, A. Wagner, S. Kolodych, F. Taran, *Angew. Chem. Int. Ed.*, **2017**, *56*, 15612-15616

4. S. Saidjalolov, E. Braud, Z. Edoo, L. Iannazzo, F. Rusconi, M. Riomet, A. Sallustrau, F. Taran, M. Arthur, M. Fonvielle, M. Etheve-Quelquejeu. *Chem. Eur. J.* **2021**, *27*, 7687-7695.

5. I. Dovgan, A. Hentz, O. Koniev, A. Ehkirch, S. Hessmann, S. Ursuegui, S. Delacroix, M. Riomet, F. Taran, S. Cianférani, S. Kolodych, A. Wagner, *Chem. Sci.*, **2020**, *11*, 1210–1215.

6. M. Riomet, K. Porte, A. Wijkhuisen, D. Audisio, F. Taran. *Chem. Commun.* **2020**, *56*, 7183 – 7186.

7. a) Z. Shao, W. Liu, H. Tao, F. Liu, R. Zeng, P. A. Champagne, Y. Cao, K. N. Houk, Y. Liang, *Chem. Commun.*, **2018**, *54*, 14089–14092; b) M. Feng, L. Madegard, M. Riomet, M. Louis, P. A. Champagne, G. Pieters, D. Audisio, F. Taran. *Chem. Commun.* **2022**, *58*, 8500–8503.

8. M. Ribéraud, K. Porte, A. Chevalier, L. Madegard, A. Rachet, A. Delaunay-Moisan, F. Vinchon, P. Thuéry, G. Chiappetta, P. A. Champagne, G. Pieters, D. Audisio, F. Taran. *J. Am. Chem. Soc.* **2023**, *145*, 4, 2219-2229.

9. S. Wiechmann, T. Freese, M. H. H. Drafz, E. G. Hübner, J. C. Namyslo, M. Niegerb, A. Schmidt. *Chem. Commun.*, **2014**, *50*, 11822–11824.

10. S. Araki, J. Mizuya, Y. Butsugan, *J. Chem. Soc. PerkinTrans.* **1** **1985**, 2439 – 2441.

11. S. Mummel, F. Lederle, E. G. Hübner, J. C. Namyslo, M. Nieger, A. Schmidt. *Angew.Chem.Int.Ed.* **2021**, *60*, 18882–18887.

<figure><img src="image_10.png" /></figure>