**2. Results and Discussion**

Dyes **1**–**4** were produced according to the described synthetic procedure [18]. We first applied a Hirshfeld surface analysis [39] to study the intermolecular interactions in the reported crystal structures of **1**–**4** [17]. As a result, a set of 2D fingerprint plots [40] were generated using CrystalExplorer 3.1 [41]. In order to estimate the propensity of two chemical species being in contact, we also calculated the enrichment ratios (*E*) [42] of the intermolecular contacts.

The intermolecular H···H, H···C and H···O contacts occupy a majority of a molecular surface of **1** (Table 1). There is a barely observed splitting of the shortest H···H fingerprint at *d*e + *d*i ≈ 2.4 Å, which is due to the shortest contact being between three atoms, rather than for direct two-atom contact (Figure S1 in Supplementary Materials) [40]. The H···C and H···O shortest contacts are shown at *d*e + *d*i ≈ 2.7 and 2.3 Å, respectively (Figure S1). The latter contacts in the corresponding fingerprint plot are shown as two spikes and mainly correspond to the contacts formed by the carbonyl oxygen atom. The molecular surface of **1** is also characterized by a negligible proportion of the H···N, C···O and O···O contacts, ranging from 0.6% to 2.4% (Table 1).

**Table 1.** Hirshfeld contact surfaces and derived "random contacts" and "enrichment ratios" for the crystal structures of **1–4**.


1 Values are obtained from CrystalExplorer 3.1 [41]. 2 The "enrichment ratios" were not computed when the "random contacts" were lower than 0.9%, as they are not meaningful [42].

Substitution of the 5-hydrogen atom by the MeO function in **2** decreases a proportion of the H···H and H···C contacts by 2.8 and 1.7%, respectively, with the simultaneous increase of a proportion of the H···O contacts up to 30.1% (Table 1). The H···H and H···C shortest contacts are now shown at *d*e + *d*i ≈ 2.2 and 2.9 Å, respectively (Figure S2). The H···O contacts in the corresponding 2D fingerprint plot are also shown as two spikes with the shortest contacts at *d*e + *d*i ≈ 2.3 Å, also being formed by the carbonyl oxygen atom (Figure S2). The Hirshfeld molecular surface of **2** is further described by a very minor proportion of the H···N, C···C, C···O and O···O contacts, varying from 0.3 to 2.4% (Table 1).

In **3,** incorporation of the bromine instead of the hydrogen in **1** or the methoxy group in **2** significantly decreases a proportion of the H··· H and H··· C intermolecular contacts down to 35.4 and 13.7%, respectively. A proportion of the H··· O contact remains almost the same as in **1** (Table 1). The H···H, H···C and H···O shortest contacts are shown in the corresponding 2D fingerprint plots at *d*e + *d*i ≈ 2.2, 2.8, and 2.3 Å, respectively (Figure S3). The molecular surface of **3** is additionally characterized by a remarkable proportion of the H···Br intermolecular contacts of 17.4%, with the shortest contacts at *d*e + *d*i ≈ 3.0 Å (Figure S3). Notably, the molecular surface of **3** is further described by a distinct proportion of the C···C contacts of 4.1%, which is due to intermolecular π··· π stacking interactions between the phenylene rings [17]. These contacts are shown on the 2D plot as a typical area at *d*e = *d*i ≈ 1.7–2.0 Å (Figure S3). Insignificant contributions from H···N, C···N, C···O, C···Br, N···O, O···O, and Br···Br contacts in **3** have also been revealed (Table 1).

Dye **4** contains the NO2 group in the same position as the corresponding substituent in **2** and **3** (Chart 1). The molecular surface of **4** is characterized by a dominant contribution from the H···O contacts of 44.1% with the shortest contacts, shown in the corresponding 2D fingerprint plot at *d*e + *d*i ≈ 2.3 Å mainly formed by both the NO2 and carbonyl oxygen atoms (Figure S4). The H···H and H···C contacts occupy 30.0 and 11.5%, respectively. Notably, the structure of **4**, similar to **3**, also exhibits a distinct proportion of the C···C contacts of 4.9% due to the formation of intermolecular π··· π stacking interactions between the phenylene rings [17]. These contacts are also shown on the 2D plot as a typical area at *d*e = *d*i ≈ 1.7–2.0 Å (Figure S4). Remaining contacts, namely H···N, C···N, C···O, N···O and O···O contacts, occupy a negligible proportion of the molecular surface of **4** (Table 1).

All the H···X (X = C, N, O) contacts are highly favored in **1** and **2** since the corresponding enrichment ratios *E*HX are significantly higher than unity (Table 1). This is explained by a relatively higher proportion of these contacts on the total Hirshfeld surface area over a corresponding proportion of random contacts *R*HX (Table 1). However, only H··· N and H··· O intermolecular contacts are favored in **3** and **4**, while the H··· C contacts on the surface of molecules are much less favored, which is related to a remarkably high enrichment of C··· C contacts in **3** and **4** (Table 1), formed in aromatic stacking. Notably, both **3** and **4** are additionally characterized by highly favored H··· Br and N··· O contacts, respectively, and **3** is also described by less favored Br··· Br contacts (Table 1). The H··· H intermolecular contacts on the molecular surfaces of all compounds are less favored since the corresponding enrichment ratios *E*HH are less than unity ranging from 0.85 to 0.90 (Table 1). The remaining contacts are significantly impoverished with the corresponding enrichment ratios less than 0.50 (Table 1).

We have also examined the optical properties of **1**–**4** in different solvents. We first probed such solvents as cyclohexane (non-polar), THF (polar aprotic), CH3CN (polar aprotic), and EtOH (polar protic) to study the enol-imine and keto-enamine tautomerization in the applied solvents. It is known that the dipole moments for the enol-imine isomers are smaller than those for the keto-enamine derivatives [43,44], which means that the keto-enamine tautomer is more prevalent in polar solvents.

The absorption spectra of **1–3** each contain bands corresponding to n→π\* and π→π\* transitions only in the UV region, regardless of the solvent (Figures S5–S7). Interestingly, the longest wavelength band in the spectra of **2** is remarkably red-shifted (Figures S5–S7). This is due to the electron-donating properties of the MeO fragment in comparison to the H and Br substituents in **1** and **3**, respectively. Furthermore, the spectra of **1**–**3** in EtOH

exhibit an additional low-intense band, centered at about 400–420 nm (Figures S5–S7), which corresponds to the traces of the *cis*-keto-enamine form.

The absorption spectra of **4** in the same solvents, except cyclohexane, contain an intense band in the visible region, which arises from the *cis*-keto-enamine form (Figure 1). Notably, in **4**, the electron-donating OH is involved in a through-resonance effect with the electron-withdrawing NO2 fragment. This leads to nitro–*aci*-nitro tautomerization, yielding an additional quinoid form [45].

**Figure 1.** UV-vis spectra of **4** in the applied solvents.

Optical properties of **4** were also studied in a series of alcohols, namely MeOH, EtOH, *n*PrOH, *i*PrOH, and *n*BuOH. Interestingly, in the absorption spectra of **4** in all alcohols, except *n*PrOH, the band of the *cis*-keto-enamine form was observed with the most remarkable one in MeOH (Figure 2), which is the most polar and acidic within the applied alcohols. The most striking observation is the absence of the band of the *cis*-keto-enamine form in the spectrum of **4** in *n*PrOH (Figure 2). This might be explained by possible specific solute–solvent interactions.

**Figure 2.** UV-vis spectra of **4** in the applied solvents.

Recently, we established that **2** was emissive in EtOH [17]. The resulting emission of **2** in EtOH was due to two emission bands arising from two conformers of the *cis*keto-enamine\* form. Herein we report the emission properties of **4** in different alcohols. Interestingly, **4** was found to be emissive in *i*PrOH and *n*PrOH, while almost no emission was found in EtOH and MeOH (Figure 3).

**Figure 3.** Emission (solid line) and excitation (dashed line) spectra of **4** in MeOH (λexc = 395 nm, λem = 480 nm), EtOH (λexc = 350 nm, λem = 440 nm), *n*PrOH (λexc = 360 nm, λem = 460 nm) and *i*PrOH (λexc = 400 nm, λem = 475 nm).

The emission spectra of **4** in *i*PrOH and *n*PrOH exhibit an intense band at 475 and 460 nm, respectively. Notably, the former band is accompanied with an intense low-energy shoulder (Figure 3). The deconvolution process of the spectrum in *i*PrOH has allowed revealing three single bands at 463, 493, and 526 nm, and a ratio of these bands is 26.4, 30.0, and 43.6%, respectively (Figure S8). The same deconvolution of the emission spectrum of **4** in *n*PrOH also revealed three bands, but with the maxima remarkably shifted to higher energies (444, 466, and 496 nm) and with a ratio of 17.2, 46.2, and 36.6%, respectively (Figure S8). Based on the absorption and excitation spectra of **4** in *i*PrOH we were able to conclude that these three emission bands corresponded to two *cis*-keto-enamine\* and *trans*keto-enamine\* conformers [8–18]. Contrarily, all three bands in the emission spectrum of **4** in *n*PrOH arose exclusively from the emission of different *cis*-keto-enamine\* conformers.

We next studied the solution of **4** upon the gradual addition of two different bases, namely NEt3 and NaOH, to probe the influence of the nature of bases (electronic properties and steric demands). Upon addition of both bases, the absorption band at 313 nm disappeared and two new bands, centered at about 360 and 390 nm, appeared (Figure 4 and Figure S9). Notably, the ratio of intensities of the new bands was in favor of the low-energy band when NaOH was used.

The gradual addition of the methanesulfonic acid into a solution of **4** in EtOH decreased the intensity of the band of the *cis*-keto-enamine form, which was due to protonation of the imine N-atom, while the NO2 group remained unchanged, as seen from the high-energy band, which remained constant during the titration process (Figure 5). Protonation of the imine N-atom prevented the formation of the intramolecular O–H···N bonding.

**Figure 4.** UV-vis spectra of **4** in EtOH upon gradual addition of NaOH up to 2 eqv., with a step of 0.1 eqv.

**Figure 5.** UV-vis spectra of **4** in EtOH upon gradual addition of CH3SO3H up to 2 eqv., with a step of 0.1 eqv.

Since compound **3** also tends to clearly exhibit the *cis*-keto-enamine form in EtOH (Figure S7), although less pronounced than **4,** but more pronounced that **1** and **2**, its optical properties were further studied upon gradual addition of NaOH and CH3SO3H. In general, the behavior of a solution of **3** in EtOH in the presence of NaOH and CH3SO3H was similar to a solution of **4** but much less pronounced; however, emission spectra of **3** differed significantly from those of **4**.

After the addition of NaOH, the absorption band at 330 nm disappeared with the simultaneous increase of a low-intense band in the visible region (Figure 6). However, the addition of CH3SO3H to a solution of **3** in EtOH vanished the same band, arising from the *cis*-keto-enamine tautomer (Figure 7).

**Figure 6.** UV-vis spectra of **3** in EtOH upon gradual addition of NaOH up to 5 eqv., with a step of 0.5 eqv.

**Figure 7.** UV-vis spectra of **3** in EtOH upon gradual addition of CH3SO3H up to 3.5 eqv., with a step of 0.5 eqv.

Upon the addition of NaOH to a solution of **3** in EtOH, an emission band appeared (Figure 8), which deconvolution revealed two bands at 480 and 505 nm and with an area ratio of 30.1 and 69.9%, respectively (Figure S10). Based on the comparison of the absorption (Figure 6) and excitation (Figure 8) spectra, the emission band was assigned to two conformers of the deprotonated form of **3**. Solutions of **3** in EtOH were found to be non-emissive regardless of the added amount of CH3SO3H.

**Figure 8.** Emission (solid line) and excitation (dashed line) spectra of **3** in EtOH gradual addition of NaOH up to 5 eqv., with a step of 0.5 eqv. (λexc = 375 nm, λem = 480 nm).

We have further applied the density functional theory (DFT) calculations to examine the fine features of **1**–**4**. It was established that the calculated values of bond lengths, bond angles, and dihedral angles (Table 2) were in good agreemen<sup>t</sup> with the values recently obtained from single-crystal X-ray diffraction [17]. The observed differences between the calculated and experimental geometrical parameters are obviously explained by the fact that the DFT computations were performed in the gas phase.

**Table 2.** Selected bond lengths (Å) and angles (◦) in the structures of **1–4**, obtained by using the B3LYP/6-311++G(d,p) method 1.


1 The computational results have been compared according to the crystallographic data [17]. 2 The corresponding dihedral angles must be compared by their magnitudes.

According to the DFT calculations, the dipole moments of the fully optimized ground state geometry of the enol-imine forms were 3.279813 Debye for **1**, 4.290475 Debye for **2**, 4.070018 Debye for **3**, and 6.97922 Debye for **4**. Notably, the transformation from the enol-imine to the *trans*-keto-enamine through the *cis*-keto-enamine tautomers was followed by a remarkable increase in the dipole moments for all the structures (Table 3). Significantly higher dipole moments of different tautomers of **4** in comparison to the corresponding values of tautomers of **1**–**3** were obviously explained by the presence of the highly polar NO2 substituent. The energies of the frontier molecular orbitals for the highest occupied molecular orbital (HOMO) and lowest-lying unoccupied molecular orbital (LUMO) are shown in Table 3. Both orbitals were mainly delocalized over the 2-OH(5-X)C6H3CH=N– CH2C(=O) fragment for all the structures except for the LUMO of **4**, where the orbital was mainly spread over the 2-OH(5-X)C6H3 fragment (Figure 9).

**Table 3.** Total energy, dipole moment, frontier molecular HOMO and LUMO orbitals, gap value, and descriptors for **1–4** in gas phase, obtained by using the B3LYP/6-311++G(d,p) method.


**Figure 9.** Energy levels and front views on the electronic isosurfaces of the high occupied and low unoccupied molecular orbitals of the ground state of **1–4**, obtained by using the B3LYP/6-311++G(d,p) method.

The so-called ionization potential (*I*) and the electron affinity (*A*) value of the molecules were established as follows: *I* = −*E*HOMO and *A* = −*E*LUMO (Table 3) [46], which determined the electron-donating ability and the ability to accept an electron, respectively. As such, as lower values of *I* as better donation of an electron, while as higher values of *A* as better ability to accept electrons. Both the *I* and *A* values for all the tautomers of **1**–**4** are remarkably lower than unity (Table 3), indicating that the reported dyes each exhibit high electron-donating and low electron-accepting properties. Notably, the corresponding *cis*keto-enamine and *trans*-keto-enamine tautomers of **1**–**4** are slightly better electron donors and electron acceptors in comparison to their enol-imine derivatives (Table 3).

To estimate the relative reactivity of molecules of **1**–**4**, we have further established values of the so-called global chemical reactivity descriptors derived from the HOMO–LUMO energy gap (Table 3). The values of chemical potential (*μ*) for **1**–**4** were in the range from −0.13135 eV to −0.18025 eV for all tautomers, indicating the poor electron-accepting ability and the strong donating ability, which was further supported with low values of electronegativity, *χ* (Table 3). Notably, the values of electronegativity for all the *cis*-keto-enamine tautomers were lower than those for the enol-imine and *trans*-keto-enamine forms of **1**–**4** (Table 3). Chemical hardness (*η*) describes the resistance towards deformation/polarization of the electron cloud of the molecule upon a chemical reaction, while softness (*S*) is a reverse of chemical hardness [46]. Compounds **1**–**4** are characterized by low values of *η* and high values of *S*, respectively, indicating a remarkable tendency to exchange their electron clouds with the surrounding environment for all the structures (Table 3). It should be noted that the *trans*-keto-enamine tautomers are more pronounced to exchange their electron clouds with the surrounding environment in comparison to the corresponding *cis*-ketoenamine tautomers, and even much more pronounced in comparison to the enol-imine tautomers (Table 3). The electrophilicity index (*ω*) describes the energy of stabilization to accept electrons [46]. The *ω* values for all forms of **1**–**4** were found in the range from about 0.12 eV to 0.25 eV (Table 3). These values are low, indicating the strong nucleophilic nature of **1–4**. Finally, compounds **1**–**4** can accept about 1.77–2.24 electrons for the enol-imine forms, 2.12–2.52 electrons for the *cis*-keto-enamine forms, and 2.30–2.89 electrons for the *trans*-keto-enamine forms, respectively, as evidenced from the ΔNmax values, of which the highest values correspond to **4** (Table 3).

The electrophilic and nucleophilic sites in **1**–**4** were examined using the molecular electrostatic potential (MEP) analysis. The red and blue colors of the MEP surface correspond to electron-rich (nucleophilic) and electron-deficient (electrophilic) regions, respectively. On the MEP surfaces of the enol-imine tautomers of **1**–**4** the most pronounced nucleophilic centers are located on the carbonyl and hydroxyl oxygen atoms, while the other negative electrostatic potential sites in the enol-imine form of **4** are located on the oxygen atoms of the NO2 group (Figure 10). In the *cis*-keto-enamine and *trans*-keto-enamine tautomers of **1**–**4** the carbonyl oxygen atom attached to the aromatic ring is the most remarkable nucleophilic center, alongside with both oxygen atoms of the NO2 group in **4**, while the carbonyl oxygen atom of the carboxyl group becomes a less pronounced nucleophilic center (Figure 10). As the most electrophilic region the CH=N–CH2 fragment for the enol-imine tautomers and the CH–NH–CH2 fragment for the *cis*-keto-enamine and *trans*-keto-enamine tautomers can be highlighted for the structures of **1**–**4** (Figure 10).

The calculated absorption spectra of the fully optimized ground state geometry of all the three tautomers of **1**–**4** (Figures S11–S13) are in agreemen<sup>t</sup> with experimental spectra. In particular, the experimental UV-vis spectra for the enol-imine tautomers exhibited bands at 215–260 and 331–353 nm, and the calculated spectra for the same tautomers contained bands at 225–250 and 302–351 nm (Table S1). The *cis*-keto-enamine tautomers are shown as a band centered at 382–418 nm in both the experimental and calculated absorption spectra (Table S1). The calculated UV-vis spectra for the *trans*-keto-enamine tautomers of **1**–**4** each exhibited a low-energy band at 407–428 nm, while no similar bands were observed in the corresponding experimental spectra (Table S1), thus, testifying to the absence of the *trans*-keto-enamine tautomers of **1**–**4** in the applied solvents.

**Figure 10.** Views of the molecular electrostatic potential surface of **1**–**4**, obtained by using the B3LYP/6-311++G(d,p) method.

The calculated UV-vis spectra of the enol-imine forms of **1**–**4** exhibit absorption bands with three (for **1**) or two (for **2**–**4**) maxima exclusively in the UV region centered at about 220–255 nm and 300–350 nm (Figure S11). These bands mostly corresponded to the HOMO– 1 → LUMO, HOMO → LUMO, and HOMO → LUMO+3 transitions (Table 4, Figure S11). The absorption bands in the spectra of **3** and **4** were additionally supported by the HOMO → LUMO+1 transition and further described by the HOMO–4 → LUMO transition in the spectrum of **3**, and by the HOMO–3 → LUMO+1 and HOMO–1 → LUMO+1 transitions in the spectrum of **4**, respectively (Table 4, Figure S11). As for the calculated UV-vis spectra of the *cis*-keto-enamine and *trans*-keto-enamine tautomers of **1–4**, absorption bands are observed in both the UV and visible regions up to about 500–550 nm (Figure S12) and 600 nm (Figure S13), respectively. These bands are characterized by two maxima centered at about 250–290 nm and 380–430 nm (Figures S12 and S13). Notably, the calculated UV-vis spectrum of the *cis*-keto-enamine form of **4** contained an additional maxima at 320 nm (Figure S12). The corresponding transitions, responsible for the observed bands in the calculated UV-vis spectra of the *cis*-keto-enamine and *trans*-keto-enamine tautomers of **1**–**4**, are shown in Figures S14–S17 and collected in Tables S2 and S3.


**Table 4.** Values for the calculated UV-vis spectra of the ground state of the enol-imine tautomers of **1**–**4**, obtained by using the TD-DFT/B3LYP/6-311++G(d,p) method.



## **3. Materials and Methods**
