# Synthesis of π-extended CinNapht fluorophores via late-stage Pd-catalyzed C–N and C–C bond formation and application for selective imaging of lipid droplets in living cells

Eléonore Tacke, Philippe Durand, Arnaud Chevalier

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## To cite this version:

Eléonore Tacke, Philippe Durand, Arnaud Chevalier. Synthesis of π-extended CinNapht fluorophores via late-stage Pd-catalyzed C–N and C–C bond formation and application for selective imaging of lipid droplets in living cells. Journal of materials chemistry B, In press, 10.1039/D5TB01877C. hal-05281357

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# Synthesis of π-Extended CinNapht Fluorophores via Late-Stage Pd-Catalyzed C–N and C–C Bond Formation and Application for Selective Imaging of Lipid Droplets in Living Cells

Eléonore Tackea, Philippe Duranda and Arnaud Chevalier*<sup>a</sup>

We report the efficient synthesis of a 4'-brominated CinNapht scaffold, enabling late-stage functionalization via palladium-catalyzed cross-coupling reactions. This strategy granted access to 17 novel derivatives, including the first N-acetyl, N-carbamoyl, and N-aryl analogues, as well as entirely new C–C bonded structures via Suzuki–Miyaura, Heck, and Sonogashira couplings. These unprecedented modifications expand the π-conjugated system and tune the emission properties. Two of these fluorophores were successfully applied as selective probes for lipid droplet imaging in live cells, demonstrating the potential of this approach for biological sensing.

## Introduction

For decades, the organic synthesis of fluorophores has driven the expansion of their applications across various fields. However, their preparation often relies on labor-intensive, multi-step synthetic routes that hinder the widespread development of more sophisticated compounds. To address these challenges, late-stage functionalization methods have emerged as powerful alternatives, enabling the facile introduction of molecular diversity and the fine-tuning of photophysical properties in a more streamlined manner. Among these strategies, palladium-catalyzed cross-coupling reactions have been the most extensively explored. In 2010, Luke Lavis's group made a landmark contribution in this field using Buchwald-Hartwig coupling on a fluorescein di-triflate scaffold to introduce a broad range of amines, thereby expanding the library of rhodamine dyes.¹ Since then, this approach has been successfully applied to various fluorophore families²⁻⁴, aiming either at biological applications⁵⁻⁸ or photophysical properties optimizations.⁹⁻¹² More recently, aromatic nucleophilic substitution (SNAr) has been proposed as an alternative late-stage functionalization strategy.¹³⁻¹⁵ However, this method remains more constrained, primarily allowing the formation of C-N or C-S bonds. Building upon these advances, our team has developed a family of red-emmitting hybrid Naphthalimide/Cinnolines fluorophores that exhibit significant Stokes shifts.¹⁶, ¹⁷ Our extensive work on CinNapht fluorophores has demonstrated their strong potential, particularly for fluorescence microscopy. We have employed late-stage functionalization strategies to introduce amine functionalities, either selectively at the 4' position¹⁸ or simultaneously at both the 4' and 5' positions.¹⁹ However, the

scope of this approach has remained limited to the incorporation of aliphatic and aromatic amines, underscoring the need for further methodological advancements to expand the chemical space accessible for CinNapht derivatives (Figure 1).

<figure><img src="image_3.png" /><figcaption>**Figure 1:** Interest of Pd-Catalyzed late-stage cross-coupling functionalization on CinNapht structural diversification.</figcaption></figure>

To date, no examples of late-stage functionalization of CinNaphts using palladium-catalyzed couplings have been reported in the literature. This study presents an efficient, scalable synthesis of a 4'-brominated CinNapht chromophore, which demonstrates long-term stability under various storage conditions, making it readily available for further applications. It was successfully engaged in various of Pd-catalyzed transformations, enabling both C–C and C–N bond formation, thereby demonstrating the versatility of this late-stage functionalization strategy synthesizing structurally diverse CinNapht derivatives. In this study, we investigate the photophysical characteristics of these newly developed π-
extended analogues and present a first demonstration of their potential utility by demonstrating their suitability as fluorescent probes for selective visualization of lipid droplets in living cells.

## Results and Discussions

### Synthesis of brominated CinNapht and use in palladium catalysed coupling reactions

To enable this, we designed a synthetic route for the selective bromination of CinNapht at the 4'-position (Scheme 1). Initially,

ortho-iodination of aniline 1 followed by a reaction with n-butylamine in refluxing ethanol afforded ortho-iodinated naphthalimide 3 with 93% yield. In parallel, we successfully carried out the bromination of 2-aminophenylboronic acid pinacol ester 4 with NBS at 0 °C in DMF, obtaining the para-brominated derivative 5 in 89% yield. We then leveraged the higher reactivity of iodine over the bromine atom in the Suzuki reaction to efficiently obtain the bis-aryl adduct 6 in 90% yield. A subsequent oxidation step of this dianiline with tert-butyl nitrite, followed by an intramolecular Mills reaction, successfully yielded the brominated CinNapht 7 in 77% yield.

<figure><img src="image_4.png" /><figcaption>**Scheme 1:** Synthesis of 4'-brominated CinNapht 7.</figcaption></figure>

This five-step synthesis can be performed on a gram scale, providing a stable brominated CinNapht 7 that can be stored for extended periods without special storage conditions. As a result, this chromophore can be readily utilized in subsequent palladium-catalyzed cross-coupling reactions for the preparation of multiple new chromophores. To further explore the versatility of this brominated CinNapht, we first examined its potential in Buchwald-Hartwig amination reactions (Scheme 2).

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Scheme 2:** Use of CinNapht 7 for Buchwald-Hartwig coupling reaction to synthesise of 4'-N-Aryl, N-Acyl and N-Carbamate CinNaphts 8a-d. <sup>a</sup>Isolated yield obtained with SNAr late-stage functionalization strategy.</figcaption></figure>

We achieved promising results using Pd-G3-Xantphos, obtaining derivatives 8a-d in satisfactory yields ranging from 58% to 92%. Notably, access to compound 8b, which previously yielded only 12% via SNAr, showed a significant improvement to 58% under

these conditions. More importantly, this strategy unlocked access to N-Phenyl CinNapht (8a) as well as N-acylated CinNapht (8c) and N-carbamoyl CinNapht (8d) derivatives, with remarkable yields of 69%, 92% and 86%, respectively. These results highlight the effectiveness of this approach in overcoming the key limitations of the SNAr strategy,<sup>18</sup> reinforcing its potential as a powerful tool for expanding the structural diversity of CinNapht derivatives. These initial results prompted us to explore the functionalization of the 4'-position via C–C bond formation, a transformation previously unreported for this scaffold. Several Pd-catalyzed coupling strategies could be employed using CinNapht 7. We started by implementing the Suzuki coupling reaction, leveraging its efficiency and versatility to introduce diverse aryl groups onto the CinNapht core.

<figure><img src="image_7.png" /><figcaption>**Scheme 3:** Use of CinNapht 7 for Suzuki coupling reaction to synthesise of 4'-Aryl substituted CinNaphts dyes 9a-e.</figcaption></figure>
Compound **7** was reacted with a selection aryl pinacolboronates (Scheme 3). After testing several catalytic systems (see Table S1), the best results were achieved using palladium tetrakis Pd(PPh₃)₄, leading to the efficient synthesis of aryl derivatives **9a-e** with excellent yields ranging from 68% (**9e**) to 99% (**9a**). Notably, the incorporation of a triphenylamine unit resulted in CinNapht **9c** with a remarkable 92% yield, demonstrating the efficiency of this approach. Furthermore, we successfully coupled CinNapht **7** with a tetraphenylethylene (TPE) unit *via* C-C bond formation (aryl-aryl), yielding a novel CinNapht-TPE hybrid **9e** compound in 68%. We then explored the synthesis of styryl derivatives of CinNaphts using chromophore **7** as a key substrate for Heck reactions (Scheme 4). After optimizing the reaction conditions (Table S2), palladium acetate combined with tri-*ortho*-tolylphosphine emerged as the most effective catalytic system here. This strategy enabled the synthesis of five styryl derivatives with yields ranging from 45% to 74%. We successfully generated a CinNapht-π-TPE hybrid analogue (**10d**) *via* ethylene linkage, achieving an impressive 74% yield.

<figure><img src="image_8.png" /><figcaption>**Scheme 4:** Use of CinNapht **7** for Heck cross-coupling reactions: Synthesis of 4'-Stiryl substituted CinNaphts dyes **10a-e**.</figcaption></figure>

Expanding on this π-conjugation approach, we introduced additional olefin partners to construct A-π-D type fluorophores, where CinNapht serves as the electron acceptor. This led to the synthesis of dimethylaniline (**10b**) and methoxyphenyl (**10c**) derivatives with 62% and 45% yield respectively. Finally, the incorporation of a 7-diethyl-aminocoumarin (DEAC) unit yielded the CinNapht-π-DEAC hybrid fluorophore (**10e**), marking the first reported example of a Coumarin/CinNapht hybrid in the literature. Finally, we explored the extension of the conjugated π-system by introducing an acetylenic function. For this, compound **7** was subjected to Sonogashira cross-coupling with various substrates. This approach led to synthesizing of three additional fluorophores, including the model compound phenylacetylene (**11a**), obtained in an excellent 95% yield. Furthermore, we applied this strategy to the development of an A-π-D fluorophore (**11b**), analogous to compound **10b**, where the ethylene linker was replaced by an acetylenic one.

<figure><img src="image_9.png" /><figcaption>**Scheme 5:** Use of CinNapht **7** for Sonogashira cross-coupling reactions: Synthesis of 4'-Stiryl substituted CinNaphts dyes **11a-c**.</figcaption></figure>

Finally, we successfully synthesized a novel CinNapht-TPE hybrid fluorophore (**11c**) with an excellent 87% yield. In total, this strategy enabled the one-step synthesis of 17 original CinNapht-derived chromophores from brominated precursor **7**.

## Evaluation of Photophysical properties

To evaluate the relevance of this approach from a photophysical perspective, we conducted a comprehensive study of the fluorescence properties of the compounds described in this work. Due to their limited solubility in polar solvents, all photophysical characterizations were performed in toluene ensuring adequate compounds solubilisation. Absorption, excitation, and emission spectra were recorded at 25 °C, allowing determination of the absorption and emission maxima (λ<sub>max</sub>Em and λ<sub>max</sub>Abs), molar extinction coefficient (εmax) and the fluorescence quantum yields (QY<sub>Fl</sub>) as summarized in Table 1. (All data are accessible in Figures S3 and S4). Among the synthesized CinNapht derivatives, only those bearing electron-donating amino substituents exhibited fluorescence. In contrast, compounds lacking such groups showed negligible or no emission, consistent with the absence of intramolecular charge transfer (ICT). Interestingly, although CinNapht-TPE compounds **9e**, **10c**, and **11c** were non-emissive in solution, they demonstrated an orange emission in the solid state (Figure S5). Although not thoroughly investigated here, this phenomenon opens the door to further studies focusing on their solid-state photophysical behavior. No significant emission was observed in solution for the N-acylated compounds **8c** and **8d**. The lack of fluorescence is most likely due to the suppression of internal charge transfer (ICT) caused by acylation of the nitrogen atom. On the other hand, fluorescent properties in solution were measurable for compounds **8a**, **8b**, **9b**, **9c**, **9d**, **10b**, **10e**, and **11b**. The non-extended derivatives **8a** and **8b** exhibit photophysical properties typical of the CinNapht family, with absorption maxima centered around 470 nm and emission in the green-yellow region in toluene. Their fluorescence quantum yields (QY<sub>Fl</sub> = 0.05 and 0.08, respectively) are comparable to the reference CinNapht-*diMe* fluorophore. In contrast, extension of the π-conjugated system via C–C bond formation and introduction of multiple π-links leads to
emission characteristics motivated further evaluation of these compounds in fluorescence imaging by confocal microscopy.

## Imaging lipid droplets in living cells using π-Extended CinNapht Fluorophores

Prior to the microscopy experiments, a preliminary solvatochromic study was performed on compounds **9b**, **10b**, and **11b**. Unlike CinNapht-diMe, these extended structures are

highly sensitive to solvent polarity, with a complete quenching of fluorescence observed in solvents with ET₃₀ values exceeding 36kcal mol⁻¹ (Figure 1b and S6). Such high selectivity for lipophilic environment make these fluorophores well suited for selective imaging of lipophilic cellular compartments, particularly lipid droplets (LDs).²¹⁻²² To evaluate their potential, we investigated the behavior of compounds **9b**, **10b**, and **11b** in live A549 cells.

<figure><img src="image_10.png" /><figcaption>**Figure 2:** Use of π-extended CinNapht Dyes for Imaging LD in living cells. **a.** Structure and emission spectra of CinNapht **9b**, **10b** and **11b** in apolar solvents. **b.** Impact of solvent polarity on fluorescence quantum yields. **c.** Imaging of CinNaphts **10b** and **11b** at 1 µM using a 63× oil immersion objective (green: λ<sub>Ex</sub>: 440 nm, λ<sub>Em</sub>: 460 to 580 nm, red: λ<sub>Ex</sub>: 600 nm, λ<sub>Em</sub>: 630 to 750 nm). Scale bar: 10 µm. Pearson Correlation Coefficient (PCC) was calculated using JaCOP plugin from ImageJ, and the Plot profile was generated using dedicated function of ImageJ software.</figcaption></figure>

Unfortunately, CinNapht **9b** exhibited insufficient solubility in culture medium, preventing its use in cell imaging experiments. Only compounds **10b** and **11b** demonstrated adequate solubility and could be successfully employed for this study. Initial observations revealed a green fluorescence localized in circular vesicles strongly morphologically similar to lipid droplets. The emission spectra of compounds **10b** and **11b**, recorded by confocal microscopy within these vesicles, exhibited maxima at approximately 570 nm and 520 nm, respectively, thereby confirming green emission. Furthermore, the overlay of the emission spectra obtained in various solvents with those recorded in the vesicles showed a strong correspondence with the properties measured in cyclohexane,

thus confirming the highly apolar nature of the observed vesicles. To further validate that these vesicles were indeed lipid droplets, a colocalization study was performed. As shown in Figure 1c, a strong correlation was observed between the green fluorescence of CinNaphts **10b** and **11b** and the red fluorescence of the commercial lipid droplet marker (LipidSpot 610), yielding a yellow signal in the merged image. These findings were corroborated both by the high Pearson's correlation coefficients (PCC) of 0.79 and 0.81 respectively, and by plot profile analyses demonstrating a clear overlap of the two fluorophore signals. Altogether, these results confirm the accumulation of CinNaphts **10b** and **11b** within lipid droplets. This first proof-of-concept application highlights the practical
utility of π-system extension in CinNapht fluorophores via C–C bond formation, enabled by this novel late-stage palladium-catalyzed functionalization strategy.

## Conclusions

In conclusion, we report the synthesis of the first 4'-bromo-CinNapht *via* an efficient and scalable synthetic route. We demonstrated the versatility of this chromophore in various palladium-catalyzed cross-coupling reactions, including Buchwald–Hartwig, Suzuki–Miyaura, Sonogashira, and Heck couplings. This strategy enabled access to previously unattainable *N*-acetyl and *N*-carbamate derivatives. Moreover, we leveraged the Buchwald conditions' efficiency to improve yields signifi-cantly for *N*-arylated compounds. More importantly, this methodology provides a powerful tool for π-extension *via* C–C bond formation, unlocking a library of novel CinNapht-based fluorophores. These new structures exhibit far-red emission in nonpolar solvents and show remarkable selectivity toward apolar environments. This property was successfully exploited for live-cell imaging of lipid droplets, marking a first application of late-stage C–C bond functionalization in this context. We believe this synthetic approach holds great promise for the future development of diverse CinNapht derivatives tailored to a wide range of applications.

## Author contributions

Chemical synthesis and characterization of undescribed compounds as well as photophysical studies were performed by E. T. Cell culture and confocal microscopy experiments were performed by A.C. A. C. supervised the project and wrote the manuscript, with proof reading and suggestion given by E. T. and P. D. All authors have given corrections and approval to the final version of the manuscript.

## Conflicts of interest

There are no conflicts to declare.

## Data availability

The data supporting this article, including absorbance, fluorescence, NMR and MS spectra have been included in the ESI.

## Acknowledgements

This project has received funding by the French National Research Agency under the program CHARMMMAT ANR-11-LABX-0039-grant. We thank Romain Le Bars and Sandrine Lecart from Imagerie Gif light facility for their kind help in microscopy experiments. The present work has benefited from Imagerie-Gif light microscopy core facility supported by l'Agence Nationale de la Recherche (ANR-11-EQPX-0029/Morphoscope, ANR-10-INBS-04/FranceBioImaging ; ANR-11-IDEX-0003-02/ Saclay

Plant Sciences). The University Paris-Saclay and the CNRS are acknowledged for funding.

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