Synthesis, characterization and solvatochromic behaviour of new water-soluble 8-hydroxy-3,6-disulphonaphthyl azohydroxynaphthalenes

A series of five tetracyclic mono azo dyes based on the diazotization of 1-amino-8-hydroxynaphthalene-3,6-disulphonic acid and subsequent coupling with substituted (un)sulphonated naphthalene derivatives have been synthesized. The chemical structures of the dyes were established using UV–visible, IR, NMR as well as mass spectrometry. Based on the number of sulphonic acid and other hydrophilic groups they contain, the compounds showed varying extent of water solubility. Spectroscopic characterization revealed the existence of azo-hydrazone tautomerism and the influence of the structures of solvating solvents on the equilibrium. Statistical analysis of single, dual and multiparametric equations of Kamlet Abboud and Taft parameters showed that solvent polarity was the most significant contributor to the observed spectral patterns of the dyes in pure solvents. The compounds could find useful applications as solvatochromic probes, food and drug colour additives.


Introduction
Azo dyes are widely used in the textile, printing, plastics, cosmetics and food industries [1,2]. Of these industrial dyes, sulphonated azoaromatic compounds are the most commonly utilized. For example, nearly 70% of dyes employed in textile processing belong to this chemical class [3]. The number and position of sulphonic acid groups per azo molecule have been shown to have effects on their colour, solvatochromic properties and by extension their applications as photochromic or solvatochromic probes [4][5][6]. In addition, sulphonic acid group substitution in azoaromatic structures improves their water solubility. This increases both the applicability of the dyes in terms of application media, as well as the ecofriendly bacterial degradation of the dyes under certain conditions [7][8][9]. A prominent route of sulphonation involves the azo coupling of diazotized aromatic amines with naphthalene sulphonic acids, such as 1-amino-8-hydroxynaphthalene-3,6-disulphonic acid, which is perhaps the most commonly utilized dye intermediate in that regard [10]. 1-amino-8-hydroxynaphthalene-3,6-disulphonic acid has therefore been employed as a coupling component in the synthesis of azo dyes of varying functionality [11,12]. Chemically, 1-amino-8hydroxynaphthalene-3,6-disulphonic acid contains free hydroxyl and amino groups, each of which can dictate the position of electrophilic aromatic substitution by an incoming diazonium ion during coupling reactions. Consequently, diazo coupling to 1-amino-8-hydroxynaphthalene-3,6-disulphonic acid can occur at para positions to either its free hydroxyl or amino groups in alkaline or acidic pH respectively.
Nevertheless, both coupling reaction conditions will yield azo products with sulphonic groups at ortho position to the azo linkage. This chemical feature might be undesirable in certain instances such as the reported reduced ability of aerobic azo reductase from Xenophilus azovorans to degrade ortho sulpho azo compounds [13]. The alternative use of 1-amino-8hydroxynaphthalene-3,6-disulphonic acid as an amine precursor for diazotization will therefore provide more options as to the chemical structures that can be incorporated into 1-amino-8-hydroxynaphthalene-3,6disulphonic acid derived dyes. In particular, it will provide an additional chemical route to preclude having a sulphonic acid group at ortho position to the azo linkage in this series of compounds. However, to the best of our knowledge, little or no report exists as regards the diazotization of 1-amino-8-hydroxynaphthalene-3,6-disulphonic acid and its use as a diazonium ion precursor in the synthesis of azo compounds. This might partly be due to its poor solubility in mineral acids. The objective of the study was therefore to successfully diazotize 1-amino-8-hydroxynaphthalene-3,6-disulphonic acid and couple it with hydroxynaphthalenes residues to afford new tetracyclic azo compounds containing two to four sulphonic acid groups per dye molecule.

General procedure for synthesis of 8-hydroxy-3,6-disulphonaphthalene-1-diazonium (1)
A mixture of 1-amino-8-hydroxynaphthalene-3,6disulphonic acid monosodium salt (0.10 g, 0.30 mmol) dissolved in 10 mL of 0.5% w/v aqueous NaHCO 3 and 2 mL aqueous solution of Na 2 SO 4 (0.02 g, 0.14 mmol) were continuously stirred at 0°C as a cold aqueous solution of NaNO 2 (0.3 g, 4.35 mmol) was added dropwise over 10 min. After stirring for an additional 10 min, the resultant solution was added drop by drop to a continuously stirred 10 mL solution of 1M HCl maintained at 0°C. The reaction mixture was covered with aluminium foil and allowed to proceed for 90 min at 0°C after which urea (2.5 g, 42.0 mmol) was added. The resultant golden yellow diazonium solution (1) was used without isolation.

General procedure for the synthesis of 8-hydroxy-3,6-disulphonaphthyl azohydroxynaphthalenes (3a-e)
A batch of 8-hydroxy-3,6-disulphonaphthalene-1-dia zonium (1) as prepared in 2.3.1 was added dropwise into continuously stirred solutions of substituted naphthol compounds (2a-e) (0.30 mmol) in optimized coupling solutions as depicted in Table 1. In each case, the reaction was stirred for 3 h at 0°C and the progress monitored with TLC. The TLC conditions employed included silica gel stationary phase with the following mobile phases, butanol:ethanol:water:25% ammonia (3:3:1:1); methanol:N,N-dimethylformamide:ethyl acetate (5:2:3); ethyl acetate:1-propanol:ethanol:25% ammonia (4:1:4:1) and reversed-phase silica with 40% aqueous methanolic solution as mobile phase. After completion of the reaction, the reaction mixture was acidified (pH 1-2) with concentrated HCl and sufficient sodium chloride added to precipitate out the compound. The crude product was collected by filtration and dried at 70°C. The product was re-dissolved in 50 mL water and filtered, after which a sufficient amount of sodium chloride was added to the filtrate to initiate salting out of the dye. The mixture was allowed to stand overnight at 4°C before the product was collected by filtration.
Final recrystallization was done by refluxing using the optimized solvents and conditions as depicted in Table 1 before the solid products (3a-e) were collected by filtration and dried at 70°C.
The NMR spectra of the compounds (3a-e) were recorded at room temperature in DMSO-d 6 solutions and their chemical shifts with respect to residual protons of the solvent are given below.

Solvatochromic assessment
The UV-visible spectra of the dyes dissolved at 1.44 − 2.01 × 10 −5 M concentrations in neat organic solvents were acquired at 25°C.

Results and discussion
The overall reaction and structures of the five new tetracyclic azo compounds are shown in Scheme 1. The optimized reaction conditions for synthesis are indicated in Table 1.

Water solubility
The water solubility of the compounds increased with the number of sulphonic acid groups per dye molecule. Thus, water solubility increased from disulphonated 3a and 3b (10 and 14 mg/10 mL respectively), trisulphona ted 3d and 3e (20 mg/mL) to tetrasulphonated 3c (40 mg/mL). The sulphonic acid group improves water solubility via ion-dipole interactions and by its ability to serve as both hydrogen bond donors and acceptors [14]. Hydroxyl and amino groups, which are also present in the compounds, have been estimated to possess carbon-solubilizing potential of five to seven carbon atoms each [15]. An organic molecule becomes water-soluble when the combined solubilizing potential of the functional groups present in it exceeds the total number of carbon atoms it contains. The enhanced water solubility of the synthesized dyes can increase their applicability in textile and printing industries. More importantly, the combination of high water solubility, extreme polarity and high molecular weights will hinder lipophilicity, bioaccumulation and consequently reduce the toxicity of the compounds [16]. These are particularly critical attributes for clinical marker dyes or food/drug colour additives that are not expected to reach systemic circulation. It is noteworthy that the most commonly used food and drug additives such as amaranth, tartrazine, and sunset yellow all contain varying number of sulphonic acid groups [17].

Azo-hydrazone tautomerism
The five compounds, 3a-e, are capable of forming hydrazone tautomers as depicted in Scheme 2. However, formation of the hydrazone of 3b, which is an ortho hydroxyl azo compound, involves the intramolecular rearrangement of the ortho hydroxyl group with the azo linkage in such a manner that an extra cyclic structure is formed [18]. The proton is therefore unavailable for intermolecular interactions. On the other hand, the formation of hydrazone tautomers of 3a, 3c, 3d and 3e involves the para hydroxyl group, which is labile and available for interactions with solvent milieu [19]. Nevertheless, the predominance of both types of hydrazone tautomers is associated with the formation of the ketonic group as shown in Scheme 2. The azohydrazone tautomerism of 3a-e was therefore investigated using IR, NMR and UV-visible spectroscopy.

1 H and 13 C NMR characterization of azo-hydrazone tautomerization
The 13 C NMR shifts of azo forms are usually found around 160 ppm due to the carbon bearing the hydroxyl while chemical shifts of hydrazone forms are routinely found around 170 ppm due to the formation of the ketonic group [18,22]. The 13 C NMR spectra of the five dyes are depicted in Figure 1. The spectra of 3b-e revealed low field chemical shifts which correspond to the ketonic group. Consequently, the peak at 177.71 ppm was assigned to C12 in 3b while the chemical shifts of 180.71, 176.77 and 181.10 ppm were assigned to C14 of 3c, 3d and 3e respectively.
This confirmed that 3b-e exist predominantly as the hydrazone tautomers. However, no peak at such low field could be observed in 13 C NMR of 3a, which is suggestive, the compound exists majorly in the azo form. The chemical shifts of the protons of the naphthalene rings of the five compounds can be easily assigned as they occur in the aromatic region and by using the splitting patterns as shown in Figure 2. Hydrazone formation is also associated with low field peaks in 1 HNMR [18]. As depicted in the representative 1 HNMR of 3c and 3d, the active peaks found at 16.431 and 16.788 ppm and with an integral height of 0.71 and 1.0 respectively were therefore attributed to the N-H bonds of the hydrazone form of the compounds (Figure 3). We have also attributed the peak in 1 HNMR of 3c found at 5.467 ppm and with an integral height of 0.3 to its azo form. Thus the hydrazone tautomeric ratios of 3c and 3d, calculated from the peak heights, are 71% and 100% respectively. The predominance of the hydrazone forms of 3c and 3d is anticipated as the presence of electron-withdrawing sulpho groups at meta position to C-14 hydroxyl group (Scheme 2) increases its propensity for proton release and the subsequent formation of the ketonic group [23]. The increased azo ratio of 3a could therefore be attributed to its lack of electronwithdrawing groups. The most deshielded protons in the 1 HNMR of 3b and 3e were found at 11.703 and 11.528 ppm respectively and were therefore assigned to the NH of their hydrazone tautomers. The peaks with the respective integral of 0.88 and 1.0 confirmed that both compounds exist majorly in the hydrazone form.

pH-driven azo-hydrazone tautomerism
The UV-visible spectra of aqueous solutions of the compounds at concentrations of 2.11 × 10 −5 (3a and 3b), 1.48 × 10 −5 (3c) and 1.69 × 10 −5 (3d and 3e) were acquired at room temperature. The effect of pH on the azo-hydrazone equilibrium of the compounds was investigated by the addition of various volumes of 2 M HCl or NaOH to the solutions of the dyes. The results are depicted in Figure 4. An aqueous solution of 3a has a pH of 6.80 and 3 UV-vis absorption bands. The absorption bands at 351 and 546 nm are due to π − π * transition of its hydrazone form [18,24]. A decrease in pH from 6.8 to 3.1 will lead to a shift of the equilibrium towards imino or hydrazone formation. This is associated with a loss of conjugation as depicted in Scheme 2. Expectedly, a large hypsochromic shift ( λ max = 40 nm) was observed with a decrease in pH. On the other hand, the addition of NaOH to the neutral solution of 3a produced a maximum bathochromic shift of λ max = 10 nm only as the compound already exists majorly in azo form. This, therefore, agrees with NMR data that 3a exists predominantly in the azo tautomeric form.
The pH of an aqueous solution of 3b is 6.8 and its UV-visible spectra showed three bands at 225, 297 and 513 nm with the latter two ascribed to the π − π * transition of the hydrazone tautomer. As deduced from the NMR data, 3b exists predominantly in the hydrazone form in which the imino group is protonated. Expectedly, the addition of HCl did not change the position of the 513 nm band. However, when pH increased from 6.8 to 10.48, the peaks at 297 and 513 nm disappeared and in their stead, two new red-shifted peaks could be observed at 308 and 544 nm respectively. In addition, a new band at 379 nm was seen with the increase in pH. The bathochromic shifts are suggestive of a shift of equilibrium towards azo formation and the naphtholate anion. The increased electron density of the azo linkage and naphtholate anion will lead to increased conjugation and the observed bathochromic shifts [14]. Similarly, the addition of HCl to aqueous solutions of 3c, 3d and 3e did not induce any shift in the position of its bands but on the contrary, increase in pH produced bathochromic shifts of λ max = 20 nm, λ max = 31 nm and λ max = 22 nm respectively. Thus, it can be concluded that the hydrazone form of 3c, 3d and 3e dominate in neutral and acidic conditions as suggested by NMR data [25].

Solvatochromism of dyes
The various colours exhibited by the dyes when introduced into neat organic solvents are depicted in Table  2. The representative electronic absorption spectra of the five dyes in selected solvents: ethanol, acetonitrile, acetone, ethyl acetate and DMF are shown in Figure  5(a-e) respectively.
The high-energy bands consisted of high-intensity peaks at around 230-259 nm and relatively less intense bands with peaks in the range of 292-314 nm. The highenergy absorption bands were due to the π-π * transitions of the aromatic naphthalene residues. The positions of the low-energy absorption bands (which arises from the π − π * electronic transitions of interactions between azo chromophoric units and surrounding solvent molecules) varied from 460 to 592 nm and bore a relationship with the chemical structures of the solutes, solvents characteristics and the predominant solute-solvent interactions including non-specific (polarity) and specific (intermolecular) interactions at play. The variation of the molar transition energies   Table 3.
With respect to ethyl acetate, progressive batho chromic shifts of λ max = 23 nm, λ max = 39 nm, and λ max = 79 nm can be observed in the visible bands of 3a as the solvent polarity increased from dioxane (π =0.48), propan-1-ol (π =0.52) to water (π =1.09). Similar increase in the positions of the visible bands was observed in the spectral patterns of the other dyes. This type of non-specific interaction is found when the excited state of a solute is better stabilized than the ground state in polar solvents. However, a cursory examination of the spectra data of 3a in DMF and DMSO revealed higher bathochromic shifts than what was obtained in water despite its higher polarity. In contrast, no such bathochromic shifts were observed in the spectra of 3b in either proton acceptor solvents. This is suggestive that a second or alternative solute-solvent interaction (other than polarity effects) was at play when solvated in DMF or DMSO [5]. Azo form is stabilized in hydrogen bond acceptor solvents while the basic imino group of the hydrazone form is stabilized in proton donating solvents [26]. Thus, when compared with 3b, pronounced bathochromic shifts and hyperchromic changes were observed in the visible bands of 3a, due to the availability of its C14 proton for hydrogen bond donation to DMF or DMSO. Similar bathochromic shifts were also observed in the spectra of 3c and 3e both of which contain free labile C14 proton available for donation to DMF. In addition, only a single visible band was observable in the electronic absorption spectra of 3b while two visible bands were present in the spectra of 3a when examined in both hydrogen bond acceptor solvents. Azo compounds such as 3b that contain hydroxyl groups at ortho position to their azo bonds have been reported to exhibit a lone visible spectral band because of the involvement of the proton in hydrazone formation and its consequent unavailability for solute-solvent interactions [5]. On the other hand, when present at para position to the azo linkages, azo-hydrazone equilibrium is more pronouncedly influenced by external environment including solutesolvent interactions [23], concentrations of co-solutes [27,28], charge and surfactant action [29]. For example, azo-hydrazone switching and consequent changes in the non-linear optical properties of methyl red were reported with increasing concentrations of amino acids and ionic surfactants [27].
The optical band gap energy associated with transition in the visible region of the compounds were determined from Tauc's plots using Equation (1): where hv is the photon energy, E g is optical energy gap, A is a constant that depends on transition probability while n can take up values of 1 2 , 2, 3/2 and 3 for direct allowed, indirect allowed, direct forbidden and indirect forbidden transitions respectively [30]. The four power probabilities were plotted against hv and the plot in which n = 1 2 was found to best satisfy the lineal condition at the absorption edge. The optical energy gap of the compounds in organic solvents was determined by extrapolating the linear ascending part of the absorption edge to the x-axis of the Tauc's plots. Representative plots of compounds 3a-e in acetone and ethyl acetate are depicted in Figure 6(a and b) respectively. The results showed that a change in solvent milieu from ethyl acetate to acetone led to an increase in the optical energy gap of 3b, 3c and 3e. These compounds also  showed the lowest band gap in the solvents and are thus most suitable for optoelectronic devices [30].

Multiparametric correlation studies
In non-chlorinated solvents, the contributory roles of specific and non-specific interactions to the total molar transition energies of a solute can be estimated by single, dual and multiparametric statistical analysis of Equation (2).
where A • is the intercept of the regression equation, a, b and p are the regression coefficients that measure the Scheme 1. Synthesis of 8-hydroxy-3,6-disulphonaphthyl azohydroxynaphthalenes (3a-e) with yields.

Scheme 2.
Azo-hydrazone tautomerism of 3a-e. magnitude of the solvent acidity (α), basicity (β) and polarizability (π * ) respectively to the regression model. The Kamlet-Taft parameters with the highest contributions to molar transition energies of the dyes are depicted in Table 4. A single parametric equation containing solvent polarity produced the best fit for the molar transition energies of the five dye compounds. An inverse relationship between the solvent polarity and the molar transition energy was observed for the compounds. Expectedly, the contribution of solvent polarity to the observed spectral patterns was least with 3b. This is consistent with the explanation that the intramolecular rearrangement of its hydrazone preclude interaction with solvents.

Conclusion
A new series of five tetracyclic azo dyes containing varying types and number of water-solubilizing groups have been synthesized and characterized using UV-visible, IR, NMR and mass spectral analyses. The solvatochromic properties of the compounds in organic solvents have also been elucidated. The compounds could find useful applications as solvatochromic probes, food and drug additives.