Emerging Thermosensitive Probes Based on Triamino-Phenazinium Dyes

Abstract

Temperature is an essential physical characteristic that influences all biological processes. Building on previous research on dialkylamino-functionalized rhodamine-based thermo-sensors, we investigate herein the thermosensitive properties of triamino-phenazinium dyes. Through a simple five-step synthetic route, we synthesized amino-phenazinium chromophores 6 and 7, featuring diethylamine substituents at different positions. A comparative analysis of optical properties and thermosensitivity was conducted on these compounds and an isomer, 5, in which butylamine moiety replaced the diethylamine group. The different emissive behaviors of the three fluorophores emphasize that not only the chemical nature but also the specific position of the alkylamine substituent play fundamental roles in the synthesis of highly emissive thermo-probes.

1. Introduction

Temperature is a crucial physical property that plays a role in all biological processes [1]. Fundamental curiosity along with practical emergencies have pushed the research community towards the conceptualization and development of optical nanothermometry as a reliable technique for achieving thermal sensing at the sub-micronic scale. Focusing on the study of the temperature-induced alteration of optical properties in various probes, this field of study comprises a wide variety of methods, such as infrared thermometry, Raman thermal microscopy, and luminescence nanothermometry [2,3]. Due to their superior accuracy, sensitivity, high spatial resolution and the simplicity of the corresponding experimental set-up, luminescent thermometers are frequently preferred, becoming a powerful tool for exploring different cellular processes [2,4,5,6,7]. The versatility of probes (e.g., organic dyes, organo–metallic complexes, quantum dots, polymers) as well as the various parameters related to the radiative deexcitation of luminophores (intensity, band shape, spectral position, polarization, lifetime, bandwidth) establish a dynamic playground for (bio)chemists. The majority of techniques for measuring temperature at the nanoscale rely on analyzing emission intensity. This parameter is significantly influenced by temperature changes, resulting in highly accurate measurements. Moreover, an essential advantage in measuring band intensity as a function of temperature is the facility of practical implementation and data analysis [4] as well as the ability to employ either single-band emission or ratiometric dual fluorescence [7,8].

Small fluorescent molecules, with good cell permeability, reproductible synthesis routes, and fast responses, are among the earliest-used fluorescent sensors for thermal biology [1,9]. As a common guideline, the emission intensity of organic dyes decreases with the increase in temperature, as electrons gain thermal energy and are promoted to higher vibrational energy levels, at which the radiative deexcitation probability is reduced [2,10]. A textbook example of a temperature-sensitive fluorophore is Rhodamine B (Figure 1). This compound, belonging to the class of xanthene dyes, is well known for its bio-imaging properties and high yield of emissions, exhibiting remarkable fluorescence-quenching properties when the solution temperature is increased in the 20–80 °C interval [11]. This behavior is generally agreed to be a consequence of the presence of a diethylamino substituent, whose rotation is facilitated at higher temperatures, leading to a non-radiative relaxation to the ground state via internal conversion [12,13]. Although water solubility along with exceptional temperature sensitivity have supported the wide application of Rhodamine B as a sensor in microfluidic systems, its translation to biological environments is limited by undesired fluctuations in optical response depending on the pH or protein denaturation [14]. The organelle-targeting fluorescent molecular thermometers are particularly attractive for their potential ability to provide a detailed map of temperature variations across different cellular compartments [5]. Mitochondria-staining probes based on rosamine dyes with a temperature response in the 32–37 °C range have been reported (Mito thermo yellow, Figure 1) [15], and so too have modified rhodamines bearing thermosensitive substituents (dialkylamine, or N-methyl piperidine) with a certain freedom of rotation and response covering different segments of the 20–55 °C interval [16,17,18,19]. BODIPY derivatives are another class of thermosensitive probes for cells [20,21,22], but they generally suffer from several drawbacks such as relatively short emission wavelengths, high photosensitivity, and small Stokes shifts, which restrict their broader use in biological sensing and imaging applications [1,23].

Indulines, a well-established family of textile dyes structurally related to rhodamines, have been largely unexplored for biological applications, despite their widespread use in the color industry in the 19–20th century and as electrophotography agents due to their stability and versatile amino-functionalities [24,25,26,27,28,29,30]. More recently, a research study carried by our group uncovered intriguing findings regarding the significant theranostic potential of the triamino-phenazinium-chromophore-type structure 5 depicted in Figure 1. The planar, aromatic cores and cationic nature of these dyes appear to confer a preferential targeting ability and allow high-resolution imaging of mitochondria [31].

Due to our interest in exploring the versatility of triamino-phenazinium dyes and our continuous attempt to create highly fluorescent and stimuli responsive platforms, we decided to introduce a diethylamino unit to the phenazinium core, a thermosensitive group by analogy with Rhodamine B. This functional group was selectively attached to two different positions, and the resulting isomers (6 and 7, Figure 1), together with a derivative bearing only mono-alkylamine groups (5 Figure 1), were comparatively analyzed in terms of their optical properties and fluorescence response to the temperature parameter. Through the study presented herein, we show that not only the nature but also the position of the alkylamine substituent play crucial roles in the synthesis of highly emissive phenazinium thermo-probes. This dual importance is clearly demonstrated by the contrasting behaviors of compounds 6 and 7. Water solubility, excellent emission efficiency (a 68% quantum yield), and the high thermosensitivity of triamino-phenazinium 7 (35% loss of emission intensity) make this derivative a reasonable choice for further biological testing.

2. Results

2.1. Synthesis and Characterization of Phenazinium Dyes

The synthesis of the three phenazinium dyes is illustrated in Scheme 1 and was performed according to a previously reported protocol [31]. Starting from the commercially available 2,4-difluoronitrobenzene, in a simple one- or two-step nucleophilic substitution with propylamine or diethylamine, we obtained the diamino-benzene precursors 2a and 2b in good to excellent yields. Next, the catalytic reduction of the nitro function was carried out, leading to intermediates 3a and 3b, obtained as brown solids in very good yields. For the fluorine substitution performed afterward, we slightly modified the protocol previously employed for 4a type of intermediates, which involved the use of Hünig’s base. Here, we show that precursors 4a−c could be synthesized in quantitative yields without the need for N,N-diisopropylethylamine since the propylamine, butylamine or the diethylamine act as both a nucleophile and a base. Finally, the reduction of the two nitro moieties and the oxidative cyclization of the fully reduced species (non-isolated) led to the formation of the expected phenazinium 5–7 in yields spanning from 27% to 86%.

The synthesized dyes were characterized using HRMS, with the bulk purity confirmed via ¹H and ¹³C NMR. Additionally, the structures of derivatives 6 and 7 were fully established via single-crystal X-ray diffraction. Single crystals of 6 were obtained by the slow evaporation (at room temperature) of a dichloromethane (DCM) solution, in which a few drops of methanol were added. In case of compound 7, the crystals were formed by the slow diffusion of pentane in a chloroform solution of 7, kept at 4 °C.

The molecular structures are shown in Figure 2. Both 6 and 7 crystallized in the triclinic P-1 space group. While the asymmetric unit of 6 contains one molecule, the structural elucidation of 7 unveils two independent units co-crystallized with one molecule of water and two molecules of chloroform (ESI Figure S21). Analysis of the bond lengths and angles of the two dyes confirmed the rigid, tricyclic phenazinium cationic core, with chloride serving as a counterion. Examination of the bond distances within the N(1)–C(1)–C(2)–C(3)–N(2)-C(4)-C(5)-C(6)-N(3) segment shows a full delocalization of the conjugated π system, whereas the “upper part”, i.e., N(5)–C(12)–C(11)–C(10)–N(4)-C(9)-C(8)-C(7), consists of a succession of single and double bonds, implying poorer π delocalization. Additionally, the single-bond character of the C-C bonds between the two segments, C(1)-C(12) in particular, indicates a decrease in the aromaticity of the tricyclic phenazinium core with the stabilization of the cationic charge. The packing motif of 6 reveals a pseudo-dimer unit, stabilized by weak π-π interactions, with a distance of 3.418 Å between the two units (ESI Figure S31). The two molecules are parallelly arranged in a head-to-tail fashion to minimize the positive charge repulsion. In the solid state, dye 7 is packed in a less organized fashion (ESI Figures S32 and S33), with the distances between the molecules (3.495 Å) falling in the range of weak π-π interaction.

The square plane calculated throughout the 14 atoms of the phenazinium core indicates that the skeleton is planar, with a maximum deviation from planarity for 6 determined to be 0.149 Å and, for 7, 0.057 Å. These differences indicate a more rigid structure for derivative 7 and could therefore help explain the remarkable increase in emission efficiency when switching from 6 to 7. The hydrogen bond between the N(5) and the primary amine group, depicted in green in Figure 2, points towards greater freedom of movement for the diethylamine in derivative 7, leading to a gain in thermosensitivity for the latter (vide infra Section 2.2).

2.2. Optical Properties

The optical properties of the structural isomers 5, 6, and 7 were ascertained in acetonitrile (MeCN), dimethylformamide (DMF), and water (H₂O) at room temperature (Figure 3,

Table 1. Optical properties of dyes 5–7 recorded in acetonitrile, dimethylformamide, and water.

Dye	Solvent	λmax (ε, M−1 cm−1)	λem	Φ [a]	Stokes Shift (cm−1)
5	MeCN DMF H2O	552 (33,740) 550 (−) 566 (−)	644 639 668	0.58 0.61 −	2590 2530 2700
6	MeCN DMF H2O	531 (47,960) 545 (−) 538 (−)	642 622 660	0.006 0.006 −	3250 2270 3440
7	MeCN DMF H2O	579 (38,790) 572 (−) 583 (−)	657 651 697	0.68 0.67 −	2050 2120 2800

^[a] Relative fluorescence quantum yields measured with rhodamine B as reference (Φ = 70% in MeOH, λ_ex = 530 nm).

, and ESI Figures S34–S36). The three phenazinium dyes absorb light in the green–yellow region of the electromagnetic spectrum, with maximums centered at 552 nm for 5 and 531 nm for 6 and slightly red-shifted for 7, with a band at 579 nm. For 5 and 7, the absorption band presents a shoulder on the high-energy side, which is absent in the case of isomer 6. The absorption is distinguished by high molar extinction coefficients in the 10 ⁴ M⁻¹ cm⁻¹ region (between 33,000 and 48,000 M⁻¹ cm⁻¹), quite characteristic of triamino-phenazinum chromophores [31]. The emission profiles of 5–7 exhibit a typical, rather wide, featureless fluorescence band in the red region, peaking at approx. 640–650 nm, with an important shift towards the far-red end as acetonitrile is replaced by water. In general, due to their rigid structures, phenazinium dyes are highly emissive, which was further confirmed by the determined quantum yields of 58–61% for 5 and 67–68% for 7. Unexpectedly, the fluorescence of derivative 6 was considerably quenched, with a quantum yield of only 0.6%. In an attempt to explain the very low values of quantum yield for 6, we turned to a previous study we reported [31], in which it was shown that upon the deprotonation of a closely related phenazinium dye, a very poorly emissive quinone was generated. We therefore believe that at very high dilutions, specific for spectroscopy measurements (10⁻⁵–10⁻⁶ M), dye 6 becomes vulnerable to deprotonation by water traces and converts into a quinone [6–H⁺] (ESI Scheme S1). This specific behavior might be due to the presence of a diethylamine unit, which impacts the acidic character of the NH₂ protons in the ortho position. To confirm this hypothesis, we further performed a protonation study (ESI Figure S42), the results of which point towards the formation of a new species, with a considerably improved quantum yield of 34%, as expected for 6.

The calculated Stokes shifts for 5–6 returned nontrivial values in the range of 2000–3000 cm⁻¹. Together with high emission efficiency, this parameter is crucial for the design of biological markers with minimized spectral overlap between absorption and emission, which attenuates the self-quenching phenomena and amplifies the signal-to-background ratio [32].

2.3. Temperature Sensitivity

The thermosensitivity of the three fluorophores was studied in DMF because the higher boiling point allowed a wider range of measurements (from 25 °C to 80 °C), minimizing the erroneous results from solvent evaporation. The fluorescence spectra of 5–7 as a function of temperature are depicted in Figure 4. All three dyes respond to temperature, as displayed by the gradual loss in fluorescence intensity with heating. Derivative 7, nevertheless, seems to have a more advantageous sensitivity, with a 35% loss of signal intensity, which can be compared to the 27% loss in the case of 5 and the 24% loss calculated for 6. A linear relationship was found between the value of the of the emission peak (λ_max) and the temperature (expressed in °C), with very good fitting for 6 (R² = 0.993) and excellent fitting for 5 and 7 (R² = 0.996). Additionally, the thermosensitivity of the three dyes was determined in DMSO, highlighting similar behavior, with linear correlations found for 5 and 7 and a second-degree polynomial fitting for compound 6 (ESI Figures S39–S41). The loss of emission intensity in DMSO was calculated to be 26% for both 5 and 6 and 31% for derivative 7.

3. Discussion

In this study, we tested a hypothesis, derived from literature research, that highlights the thermosensitive properties of dialkylamino-functionalized rhodamines by translating it to the emerging family of triamino-phenazinium dyes. Using a simple pathway and starting from a commercially available building block, we synthesized the phenazinium chromophores 6 and 7, bearing diethylamine substituents at the C12 and C6 positions, respectively (see the X-ray structures in Figure 2), and a monoalkylamine-funcionalized isomer 5, bearing a butylamine group instead. The three dyes were fully characterized via NMR and HMRS as well as single-crystal X-ray techniques for 6 and 7. Furthermore, a comparative analysis of the optical properties and thermosensitivity of the dyes was performed. While the absorption profiles did not reveal important differences between the three compounds, fluorescence measurements indicated an important loss of emission efficiency in derivative 6, in which the diethylamine group is linked to the C12 atom. A possible explanation, derived from the analyzed single-crystal XRD structure, lies in the slightly less rigid structure of 6 compared to 7 as well as an “unfavorable” position of the diethylamino substituent when introduced at the C12 position. The evaluation of the fluorescence response to temperature unveiled a good return for all three dyes, with a decrease in emission intensity upon heating, pointing towards the nontrivial thermosensitivity of the mono-alkylamine substituents as well. Small-molecule dyes display temperature-dependent fluorescence primarily due to the rotational and vibrational freedom of their substituents, leading to nonradiative excited-state decay that can be significantly affected by temperature. The diethylamino group is recognized for inducing such temperature sensitivity, largely because of its ability to rotate freely. We propose that this same principle can be extended to monoalkylamino chains as well, thus explaining the observed important thermosensitivity of molecule 5. However, we found that derivative 7 stands out in terms of both emission efficiency (68% quantum yield) and the highest loss in fluorescence intensity when gradually increasing the temperature from 25 °C to 80 °C (35% loss) as a consequence of a non-hindered rotation of the diethylamine moiety.

In this study, we developed a new family of thermosensitive probes based on triamino-phenazinium dyes 5–7 that could be prepared easily and in just a few steps thanks to the versatility of the synthesis method used. Through a judicious design, i.e., a rigid cationic phenazinium core prompting radiative deexcitation; short alkylamine chains; and the selective introduction of a diethylamine moiety at the C6 position to enhance thermosensitivity, we highlighted that dye 7 (with a 35% loss of emission intensity at 80 °C) possesses promising features for bio-sensing applications. Testing in a biological medium is now envisaged, as we have previously shown that triamino-phenazinium dyes preferentially stain mitochondria organelles [31].

4. Materials and Methods

4.1. General Remarks and Analysis Conditions

Reagents. All reagents and solvents were purchased from Alfa-Aesar (Haverhill, MA, USA) or Sigma-Aldrich (St. Louis, MO, USA)and used as received. When heating was required, oil baths were used. Column chromatography was performed using silica gel (60–120 mesh) and alumina 90 neutral (63–200 μm, Beckmann grade I). Analytical thin-layer chromatography (TLC) was performed on precoated silica gel-60 F254 (0.5 mm) aluminum plate or precoated Al₂O₃ gel-60 neutral (0.2 mm) aluminum plate. Visualization of the spots on TLC plates was achieved via exposure to UV light. Filter aid was performed using Celite AW standard Supercel or Celite type 545. Unless otherwise specified, the desired product was dried under vacuum (<10 mbar) for over 5 h at room temperature.

Analytical Methods and Apparatus. NMR spectra were recorded on a JEOL ECS400 spectrometer (JEOL, Tokyo, Japan) operating at 400 MHz for ¹H and 100 MHz for ¹³C, respectively, at room temperature. NMR chemical shifts are given in ppm (δ) relative to Me₄Si, with solvent resonances used as internal standards (CDCl₃: 7.26 ppm for ¹H and 77.2 for ¹³C; CD₃OD: 3.31 ppm for ¹H and 49.0 for ¹³C). The multiplicity of signals is designated by the following abbreviations: s, singlet; br s, broad singlet; d, doublet; t, triplet; q, quartet; quint, quintet; sext, sextet; m, multiplet; br m, broad multiplet. Coupling constants, J, are reported in Hertz (Hz). High-resolution mass spectrometry (HRMS) analysis was performed using the “Spectropole” of Aix-Marseille University.

Crystallography. Crystals were mounted on a Rigaku Oxford Diffraction SuperNova diffractometer and measured at 293 K using Cu radiation (λ = 1.54184 Å). Data collection, reduction, and multiscan ABSPACK correction were performed with CrysAlisPro version 1.171.43.125 (Rigaku Oxford Diffraction, Tokyo, Japan). Using Olex2 [33], the structures were solved with the ShelXT [34] structure solution program using Intrinsic Phasing and refined with ShelXL [34] using least-squares minimization.

Fluorescence. Emission spectra were measured using a Horiba-Jobin Yvon Fluorolog-3 spectrofluorometer (Horiba, Kyoto, Japan) equipped with a three-slit double-grating excitation and a spectrograph emission monochromator with dispersions of 2.1 nm mm⁻¹ (1200 grooves per mm). A 450 W xenon continuous-wave lamp provided excitation. The luminescence of diluted solutions was detected at right angles using 10 mm quartz cuvettes.

Fluorescence quantum yields Φ were measured in diluted solutions with an optical density lower than 0.1 using the following equation:

$${\displaystyle \frac{{\mathsf{\Phi }}_x}{{\mathsf{\Phi }}_r}}=\left({\displaystyle \frac{A_r\left(\lambda \right)}{A_x\left(\lambda \right)}}\right)\left({\displaystyle \frac{n_x^2}{n_r^2}}\right)\left({\displaystyle \frac{D_x}{D_r}}\right)$$

(1)

where A is the absorbance at the excitation wavelength (λ), n is the refractive index, and D is the integrated intensity. “r” and “x” stand for reference and sample. The fluorescence quantum yields were measured with rhodamine B as a reference (Φ = 70% in MeOH, and λ_ex = 530 nm or, for 6 in acidic acetonitrile, λ_ex = 510 nm) [35].

The spectra of fluorescence as a variation of temperature were recorded using a JASCO Spectrofluorometer FP-8600 (JASCO, Tokyo, Japan) equipped with a Peltier JASCO ETC 815 (JASCO, Tokyo, Japan).

4.2. Synthesis Protocols and Characterization

Compound 1. To a stirred solution of 2,4-difluoronitrobenzene (2 mL, 18.23 mmol, 1 equiv.) in dichloromethane (50 mL) at 0 °C was added 1-propylamine (1.5 mL, 18.23 mmol, 1 equiv.) and then N,N-diisopropylethylamine (3.49 mL, 20 mmol, 1.1 equiv.). The reaction mixture was allowed to warm up to room temperature (over 30 min), after which time the reaction was complete (monitored via TLC). The solvent was evaporated in vacuo to afford yellow residue that was purified over silica gel column gel to afford the desired product as a yellow oil (2.74 g, 13.8 mmol, 76%).

R_f: 0.5 (petroleum ether/ethyl acetate, 96:4). ¹H NMR (400 MHz, CDCl₃): δ = 8.17 (m, 2H), 6.47 (dd, J = 11.6 Hz, J = 2.4 Hz, 1H), 6.35 (ddd, J = 9.6 Hz, 4.8 Hz, 2.4 Hz, 1H), 3.22 (td, J = 7 Hz, J = 5.2 Hz, 2H), 1.76 (sext, J = 7.3 Hz, 2H), 1.06 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ = 167.7 (d, J¹_C–F = 257 Hz, C), 147.7 (d, J³_C–F = 14 Hz, C), 130.1 (d, J³_C–F = 13 Hz, CH), 128.8 (C), 103.9 (d, J²_C–F = 25 Hz, CH), 99.3 (d, J²_C–F = 27 Hz, CH), 45.1 (CH₂), 22.1 (CH₂), 11.6 (CH₃). ¹⁹F NMR (376 MHz, CDCl₃): δ 99.40 (s).

Compound 2a. To a pressure bomb were introduced 2,4-difluoronitrobenzene (2 mL, 18.23 mmol, 1 equiv.), 1-propylamine (6.14 mL, 74.7 mmol, 4.1 equiv.), and N,N-diisopropylethylamine (5.4 mL, 31 mmol, 1.7 equiv.). The bomb was closed with a Teflon seal. The mixture was heated to 145 °C for 3 h. After the mixture had cooled to room temperature, ethanol (5 mL) was added. This suspension was scatted with ultrasound. The resulting solid in the suspension was isolated via filtration, rinsed with hot water, and dried under vacuum to afford the desired product as a yellow powder (4.07 g, 17.14 mmol, 94%).

¹H NMR (400 MHz, CDCl₃): δ = 8.53 (br s, 1H), 8.01 (d, J = 8.8 Hz, 1H), 5.90 (dd, J = 9.2 Hz, 2 Hz, 1H), 5.63 (d, J = 2 Hz, 1H), 4.46 (br s, 1H), 3.18 (m, 4H), 1.75 (sext, J = 7.2 Hz, 2H), 1.68 (sext, J = 7.6 Hz, 2H), 1.05 (t, J = 7.4 Hz, 3H), 1.02 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ = 154.5 (C), 148.7 (C), 129.3 (CH), 123.6 (C), 104.8 (CH), 89.2 (CH), 45.1 (CH₂), 44.8 (CH₂), 22.4 (CH₂), 22.1 (CH₂), 11.8 (CH₃), 11.6 (CH₃).

Compound 2b. To a round-bottom flask equipped with a condenser were introduced 825 mg of precursor 1 (4.16 mmol, 1 equiv.), 4.3 mL of diethylamine (41.6 mmol, 10 equiv.), and 7.2 mL of N,N-diisopropylethylamine (41.6 mmol, 10 equiv.). This mixture was stirred at 80 °C for 60 h. The reaction was left to cool down. The solvent evaporated, and the residue was purified on a silica gel column using petroleum ether/ethyl acetate (96:4) as an eluent to give 717 mg of yellow oily solid (2.83 mmol, 68% yield).

R_f: 0.2 (petroleum ether/ethyl acetate, 96:4). ¹H NMR (400 MHz, CDCl₃): δ = 8.46 (br s, 1H, NH), 8.06 (d, J³ = 9.7 Hz, 1H, CH), 6.08 (dd, J³ = 9.7 Hz, J⁴ = 2.4 Hz, 1H, CH), 5.64 (d, J⁴= 2.2 Hz, 1H, CH), 3.45 (q, J³ = 7 Hz, 4H, CH₂), 3.23 (q, J³ = 7 Hz, 2H, CH₂), 1.79 (sext, J³ = 7.3 Hz, 2H, CH₂), 1.25 (t, J³ = 7 Hz, 6H, CH₃), 1.07 (t, J³ = 7.7 Hz, 3H, CH₃).¹³C NMR (101 MHz, CDCl₃): δ = 153.3 (C), 148.2 (C), 129.5 (CH), 122.9 (C), 102.9 (CH), 90.0 (CH), 45.0 (CH₂), 44.7 (CH₂), 22.2 (CH₂), 12.8 (CH₃), 11.8 (CH₃). HRMS (ESI+) calculated for [C⁺]: 252.1707 (C₁₃H₂₂N₃O₂⁺) found: 252.1707.

Compound 3a. A solution of 2a (1.5 g, 6.3 mmol, 1 equiv.) in 60 mL of tetrahydrofuran was hydrogenated (p = 40 bars) in the presence of Pd/C (5% wt, % 2 mol, 270 mg) overnight. After reducing the pressure, the solution was degassed by bubbling argon in the mixture and keeping it at 0 °C. 1,5-Difluoro-2,4-dinitrobenzene (1.22 g, 6 mmol, 0.95 equiv.) was added at 0 °C while stirring, and the mixture was allowed to warm up to room temperature (over 60 min). Pd/C was then removed via filtration through a Celite^® plug. The crude product was purified on silica gel column chromatography, using dichloromethane as an eluent, and then over a silica gel plug, with petroleum ether/ethyl acetate (9:1) as an eluent, to afford the desired product as a brown-reddish solid (1.8 g, 4.6 mmol, 73% yield).

R_f: 0.5 (dichloromethane/petroleum ether, 6:4). ¹H NMR (400 MHz, CDCl₃): δ = 9.31 (s, 1H), 9.15 (d, J = 8 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 6.55 (d, J = 13.6 Hz, 1H), 6.02 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 5.97 (d, J = 2.4 Hz, 1H), 3.80 (br s, 1H), 3.70 (br s, 1H), 3.12 (t, J = 7 Hz, 2H), 3.09 (m, 2H), 1.69 (sext, J = 7.2 Hz, 2H), 1.59 (sext, J = 7.2 Hz, 2H), 1.03 (t, J = 7 Hz, 3H), 0.92 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ = 161.3 (d, J¹_C–F = 272 Hz, C), 150.6 (d, J²_C–F = 13 Hz, C), 150.3 (C), 145.2 (C), 129.6 (CH), 127.8 (CH), 127.0 (C), 126.9 (C), 110.8 (C), 104.1 (d, J²_C–F = 27 Hz, CH), 101.7 (CH), 95.2 (CH), 45.8 (CH₂), 45.3 (CH₂), 22.8 (CH₂), 22.6 (CH₂), 11.8 (CH₃), 11.7 (CH₃). ¹⁹F NMR (376 MHz, CDCl₃): δ = −105.35 (s).

Compound 3b. A solution of 2b (700 mg, 2.78 mmol, 1 equiv.) in 60 mL of methanol was hydrogenated (p = 40 bars) in the presence of Pd/C (5% wt, % 2 mol, 118 mg) overnight. After reducing the pressure, the solution was degassed by bubbling Argon in the mixture and keeping it at 0 °C. 1,5-Difluoro-2,4-dinitrobenzene (539 mg, 2.64 mmol, 0.95 equiv.) was added at 0 °C while stirring, and the mixture was allowed to warm up to room temperature (over 60 min). Pd/C was then removed via filtration through a Celite^® plug. The crude product was purified on silica gel column chromatography, using dichloromethane as an eluent, and then over a silica gel plug, with petroleum ether/ethyl acetate (9:1) as an eluent, to afford the desired product as a brown-reddish solid (945 mg, 2.33 mmol, 84% yield).

R_f: 0.7 (dichloromethane); 0.2 (petroleum ether/ethyl acetate, 9:1). ¹H NMR (400 MHz, CDCl₃): δ = 9.35 (s, 1H, NH), 9.10 (d, J³ = 7.7 Hz, 1H, CH), 6.88 (d, J³ = 8.6 Hz, 1H, CH), 6.60 (d, J³_H–F = 13.5 Hz, 1H, CH), 6.09 (dd, J³ = 8.6 Hz, J⁴ = 2.1 Hz, 1H, CH), 6.01 (d, J⁴_H–F = 2.4 Hz, 1H, CH), 3.78 (br s, 1H, NH), 3.42 (q, J³ = 6.8 Hz, 4H, CH₂), 3.13 (t, J³ = 6.1 Hz, 2H, CH₂), 1.65 (sext, J³ = 7.3 Hz, 2H, CH₂), 1.24 (t, J³ = 7.2 Hz, 6H, CH₃), 0.97 (t, J³ = 7.2 Hz, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ = 161.1 (d, J¹_C–F = 271 Hz, C), 150.6 (d, J²_C–F = 13 Hz, C), 149.2 (C), 145.0 (C), 128.3 (CH), 127.6 (CH), 126.7 (C), 126.6 (C), 109.4 (C), 104.0 (d, J²_C–F = 27.8 Hz, CH), 100.9 (CH), 94.2 (CH), 45.2 (CH₂), 44.5 (CH₂), 22.5 (CH₂), 12.7 (CH₃), 11.6 (CH₃). ¹⁹F NMR (376 MHz, CDCl₃): δ = −105.73 (s). HRMS (ESI+) calculated for [M+H⁺]: 406.1885 (C₁₉H₂₅FN₅O₄⁺) found: 406.1884.

Compound 4a. In a two-neck flask, 350 mg of 3a (0.89 mmol, 1 equiv.) was dissolved in 15 mL of acetonitrile, and 265 µL of butylamine (3.1 mmol, 3 equiv.) was added. The solution was stirred at room temperature for 2 h. After the evaporation of the solvent, hot ethanol (10 mL) was added to the crude residue. An asphalt-like resulting material was collected via filtration and washed with hot water (30 mL); it was then recovered via dissolution in dichloromethane, dried over anhydrous MgSO₄, and filtered. After the solvent was removed under reduced pressure and the solid was dried under vacuum, the desired product was obtained as a dark asphalt-like solid (396 mg, 0.88 mmol, 99% yield).

¹H NMR (CDCl₃, 400 MHz): δ = δ = 9.27 (s, 1H), 9.12 (s, 1H), 8.21 (t, J = 5.2 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 6.01 (dd, J = 8 Hz, 2.4 Hz, 1H), 5.97 (d, J = 2 Hz, 1H), 5.70 (s, 1H), 3.81 (t, J = 5.2 Hz, 1H), 3.71 (br s, 1H), 3.11 (t, J = 7.2 Hz, 2H), 3.07 (m, 2H), 3.02 (m, 2H), 1.67 (sext, J = 7.2 Hz, 2H), 1.58 (m, 2H), 1.52 (m, 2H), 1.33 (sext, J = 7.4 Hz, 2H), 1.03 (t, J = 7.6 Hz, 3H), 0.92 (t, J = 7.4 Hz, 3H), 0.89 (t, J = 7.4 Hz, 3H). ¹³C NMR (CDCl₃, 101 MHz): δ = 149.7 (C), 149.2 (C), 148.6 (C), 145.3 (C), 129.6 (C), 128.8 (CH), 125.0 (CH), 124.5 (C), 112.1 (C), 101.6 (CH), 95.3 (CH), 93.1 (CH), 46.0 (CH₂), 45.4 (CH₂), 42.8 (CH₂), 30.3 (CH₂), 22.9 (CH₂), 22.7 (CH₂), 20.2 (CH₂), 13.7 (CH₃), 11.8 (CH₃), 11.7 (CH₃).

Compound 4b. In a two-neck flask, 200 mg of 3a (0.51 mmol, 1 equiv.) was dissolved in 15 mL of acetonitrile, and 158 µL of diethylamine (1.53 mmol, 3 equiv.) was added. The solution was stirred at reflux for 16 h. After the reaction was stopped, it was concentrated under low pressure, and the residue was extracted in dichloromethane, washed with 100 mL of distilled water, and extracted with 100 mL of dichloromethane. The organic layers were collected, dried over MgSO₄, and filtered, and the solvent was evaporated. The obtained residue was purified over a silica gel plug with dichloromethane, and after solvent evaporation, it yielded 223 mg of brown solid (0.5 mmol, 99% yield).

¹H NMR (400 MHz, CDCl₃): δ = 9.12 (s, 1H, CH), 8.80 (d, J⁴ = 2.18 Hz, 1H, CH), 6.87 (d, J³ = 8.2 Hz, 1H, CH), 6.01 (d, J³ = 8.3 Hz, 1H, CH), 5.97 (s, 1H, NH), 5.94 (s, 1H, CH), 3.83 (br s, 2H, NH), 3.12 (m, 8H, CH₂), 1.70 (q, J³ = 7.2 Hz, 2H, CH₂), 1.61 (q, J³ = 7.2 Hz, 2H, CH₂), 1.08 (m, 9, CH₃), 0.93 (t, J³ = 7.4 Hz, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ = 149.5 (C), 149.4 (C), 148.2 (C), 145.4 (C), 131.1 (C), 128.8 (CH), 128.6 (CH), 123.6 (C), 112.2 (C), 101.5 (CH), 100.8 (CH), 95.3 (CH), 46.0 (CH₂), 45.9 (2CH₂), 45.3 (CH₂), 22.8 (CH₂), 22.6 (CH₂), 12.0 (2CH₃), 11.7 (CH₃), 11.6 (CH₃). HRMS (ESI+) calculated for [C⁺]: 445.2558 (C₂₂H₃₃N₆O₄⁺) found: 445.2554.

Compound 4c. In a two-neck flask, 280 mg of 3b (0.7 mmol, 1 equiv.) was dissolved in 25 mL of acetonitrile, and 230 µL of 1-propylamine (2.8 mmol, 4 equiv.) was added. The solution was stirred at room temperature for 16 h. After evaporating the solvent, the residue was aliquoted in 100 mL of deionized water, ultrasonicated, and filtered under vacuum. The precipitate was washed with another 100 mL of deionized water and quickly with cold methanol, yielding 306 mg of orange product (0.68 mmol, 98% yield).

¹H NMR (400 MHz, CDCl₃): δ = 9.27 (s, 1H, NH), 9.14 (s, 1H, CH), 8.22 (br s, 1H, NH), 6.91 (d, J³ = 8.8 Hz, 1H, CH), 6.10 (dd, J³ = 8.8 Hz, J⁴ = 2.1 Hz, 1H, CH), 6.02 (d, J⁴ = 2.1 Hz, 1H, CH), 5.73 (s, 1H, CH), 3.78 (br s, NH), 3.41 (q, J³ = 6.8 Hz, 4H, CH₂), 3.09 (br m, 2H, CH₂), 3.03 (q, J³ = 6.5 Hz, 4H, CH₂), 1.64 (m, 4H, CH₂), 1.22 (t, J³ = 6.8 Hz, 6H, CH₃), 0.95 (m, 6H, CH₃). ¹³C NMR (101 MHz, CDCl₃): δ = 149.2 (C), 148.8 (C), 148.6 (C), 145.2 (C), 129.6 (CH), 128.5 (CH), 125.0 (C), 124.5 (C), 110.8 (C), 101.2 (CH), 94.6 (CH), 93.2 (CH), 45.4 (CH₂), 44.8 (CH₂), 44.7 (CH₂), 22.7 (CH₂), 21.6 (CH₂), 12.8 (CH₃), 11.8 (CH₃), 11.5 (CH₃). HRMS (ESI+) calculated for [M + H⁺]: 445.2558 (C₂₂H₃₃N₆O₄⁺) found: 445.2554.

Compound 5. A solution of 4a (344 mg, 0.77 mmol, 1 equiv.) in methanol (40 mL) was hydrogenated (30 bars) in the presence of Pd/C (5 wt. %5 mol, 82 mg) and HCl (12 M, 0.4 mL) for 16 h. Then, the mixture was stirred under air for another 16 h. Pd/C was removed via filtration through a Celite^® plug, which was rinsed multiple times with methanol and dichloromethane. After removal of the solvent under reduced pressure, the resulting solid was purified via an alumina oxide gel chromatography column (neutral, activity I, eluent dichloromethane/methanol, 95:5, solid deposition). The pink fraction was collected, concentrated under vacuum, and repassed on the same type of chromatography column to afford 152 mg of product (0.38 mmol, 50% yield).

R_f: 0.5 (Al₂O₃, dichloromethane/methanol, 95:5). ¹H NMR (400 MHz, CD₃OD,): δ = 7.74 (d, J = 9.2 Hz, 1H), 7.21 (dd, J = 9.2 Hz, 2 Hz, 1H), 6.97 (s, 1H), 6.76 (s, 1H), 6.57 (br s, 1H), 4.55 (t, J = 8 Hz, 2H), 3.33 (m, 2H), 3.27 (m, 2H), 1.97 (quint, J = 7.6 Hz, 2H), 1.76 (sext, J = 7.2 Hz, 4H), 1.56 (sext, J = 7.6 Hz, 2H), 1.20 (t, J = 7.4 Hz, 3H), 1.07 (t, J = 7.4 Hz, 3H), 1.02 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CD₃OD): δ = 154.7 (C), 152.2 (C), 140.6 (C), 139.9 (C), 136.7 (C), 134.1 (C), 132.6 (C), 132.3 (CH), 107.5 (CH), 104.1 (CH), 93.7 (CH), 90.6 (CH), 50.2 (CH₂), 46.0 (CH₂), 44.6 (CH₂), 31.5 (CH₂), 22.9 (CH₂), 21.5 (CH₂), 21.0 (CH₂), 14.2 (CH₃), 11.9 (CH₃), 11.4 (CH₃). HRMS (ESI+) calculated for [C⁺]: 366.2652 (C₂₂H₃₂N₅⁺) found: 366.2650.

Compound 6. A solution of 4b (143 mg, 0.32 mmol, 1 equiv.) in methanol (30 mL) was hydrogenated (30 bars) in the presence of Pd/C (5 wt. %5 mol, 34 mg) and HCl (12 M, 0.3 mL) for 16 h. Then, the mixture was stirred under air for another 24 h. Pd/C was removed via filtration through a Celite^® plug, which was rinsed multiple times with methanol and dichloromethane. After removal of the solvent under reduced pressure, the resulting solid was purified using an alumina oxide gel chromatography column (neutral, activity I, eluent dichloromethane/methanol, 95:5, solid deposition). The pink fraction was collected, concentrated under vacuum, and repassed on the same type of chromatography column to yield 35 mg of product (0.08 mmol, 27% yield).

R_f: 0.55 (Al₂O₃, dichloromethane / methanol, 95:5). ¹H NMR (400 MHz, CD₃OD,): δ = 7.85 (d, J³ = 9.2 Hz, 1H, CH), 7.56 (s, 1H, CH), 7.30 (d, J³ = 9.3 Hz, 1H, CH), 7.06 (s, 1H, CH), 6.66 (s, 1H, CH), 4.60 (t, J³ = 7.7 Hz, 2H, CH₂), 3.42 (t, J³ = 7.1 Hz, 2H, CH₂), 3.24 (q, J³ = 7.1 Hz, 4H, CH₂), 2.04 (sext, J³ = 7.8 Hz, 2H, CH₂), 1.81 (sext, J³ = 7.1 Hz, 2H, CH₂), 1.26 (t, J³ = 7.1 Hz, 3H, CH₃), 1.12 (m, 9H, CH₃). ¹³C NMR (101 MHz, CD₃OD): δ = 157.2 (C), 156.8 (C), 143.1 (C), 137.9 (C), 137.5 (C), 136.3 (C), 134.7 (C), 134.3 (CH), 124.8 (2CH), 93.9 (2CH), 50.0 (CH₂), 47.3 (CH₂), 46.1 (CH₂), 22.9 (CH₂), 20.7 (CH₂), 12.0 (CH₃), 11.9 (CH₃), 11.4 (CH₃). HRMS (ESI+) calculated for [C⁺]: 366.2652 (C₂₂H₃₂N₅⁺) found: 610.4482.

Compound 7. A solution of 4c (225 mg, 0.5 mmol, 1 equiv.) in methanol (40 mL) was hydrogenated (20 bars) in the presence of Pd/C (5 wt. %5 mol, 54 mg) and HCl (12 M, 0.4 mL) for 8 h. Then, the mixture was stirred under air for another 16 h. Pd/C was removed via filtration through a Celite^® plug, which was rinsed multiple times with methanol and dichloromethane. After removal of the solvent under reduced pressure, the resulting solid was purified using an alumina oxide gel chromatography column (neutral, activity I, eluent dichloromethane/methanol, 95:5, solid deposition). The purple fraction was collected, concentrated under vacuum, and precipitated in pentane, and the precipitate was collected via vacuum filtration to afford 173 mg of product (0.43 mmol, 86% yield).

R_f: 0.4 (Al₂O₃, dichloromethane/methanol, 95:5). ¹H NMR (400 MHz, CD₃OD): δ = 7.90 (d, J³ = 9.5 Hz, 1H, CH), 7.48 (dd, J³ = 9.5 Hz, J⁴ = 2.4 Hz, 1H, CH), 7.03 (s, 1H, CH), 6.86 (s, 1H, CH), 6.69 (d, J⁴ = 2.4 Hz, 1H, CH), 4.66 (t, J³ = 7.8 Hz, 2H, CH₂), 3.74 (q, J³= 7 Hz, 4H, CH₂), 3.32 (m, 2H, CH₂), 2.06 (sext, J³ = 7.9 Hz, 2H, CH₂), 1.87 (sext, J³ = 7 Hz, 2H, CH₂), 1.36 (t, J³ = 7.1 Hz, 6H, CH₃), 1.25 (t, J³ = 7.1 Hz, 3H, CH₃), 1.13 (t, J³ = 7.1 Hz, 3H, CH₃). ¹³C NMR (101 MHz, CD₃OD): δ = 152.6 (C), 152.5 (C), 140.7 (C), 140.4 (C), 136.1 (C), 133.6 (CH), 133.0 (C), 132.8 (C), 118.7 (CH), 104.2 (CH), 93.7 (CH), 92.2 (CH), 50.0 (CH₂), 46.7 (CH₂), 46.4 (CH₂), 22.5 (CH₂), 21.2 (CH₂), 12.8 (CH₃), 12.1 (CH₃), 11.4 (CH₃). HRMS (ESI+) calculated for [C⁺]: 366.2652 (C₂₂H₃₂N₅⁺) found: 366.2654.