3-(4-Ferrocenyl-1H-1,2,3-triazol-1-yl)cholic Acid

Surfactants are functional compounds able to modify the interfacial energy, i.e., the energy necessary to modify the extent of the contact area of the interface formed by immiscible phases. A large interface area is required for numerous chemical and industrial applications, such as heterogeneous reactions occurring at liquid–liquid boundaries or the development of detergents and emulsifying systems. Thus, the increase in interfacial area and the decrease in interfacial energy become crucial and can indeed be accomplished by surfactants that, due to their structure, show compatibility with each phase involved. A relevant feature of such amphiphiles is their tendency to self-assemble into micelles and their large variety of nanostructures, such as tubules, wires, fibres, or lamellae. Surfactants occur in biological organisms, like phospholipids in cell membranes, but they are also industrially produced on a massive scale (~1.6 × 10⁷ t per year) as detergents, emulsifying and coating agents, or phase transfer catalysts. The typical structure of a surfactant involves a hydrophilic head, namely a charged or polar group, and a hydrophobic alkyl chain, known as the tail; the surface properties depend on the relative proportions of such different regions. Indeed, the aggregation of amphiphilic compounds can be ascribed to the so-called hydrophobic effect, which is basically the tendency of the system to minimize the contact area between water and the apolar domain in the surfactant structure [1,2,3]. In the last thirty years, the structure of classical surfactants (i.e., the ones whose aggregation was mainly ruled by the interplay between hydrophilic and lipophilic domains) was tailored with the aim of giving them additional properties, such as the ability to respond to several external stimuli like pH changes or photochemical, magnetic, and thermal variations [4,5,6]. Such stimuli could be used to modulate the surfactant self-assembly pattern or even to trigger the aggregation/disaggregation process. Another possible approach to a responsive surfactant is its functionalization with a redox-active group, which can reversibly modify its charge upon a suitable stimulus. This would cause a drastic change in the hydrophobic–hydrophilic balance of the molecule, thus affecting both the critical micellar concentration and the self-assembly properties. The most widespread unit used for this purpose is ferrocene, whose preparation methods, synthetic manipulation, redox, and physico-chemical properties are very well known [7,8,9]. A plethora of ferrocene-based classical surfactants have been described in the literature, together with their solution properties. They show variable structures, with the metal subunit embedded in many different combinations of cationic, anionic or nonionic polar head groups and single-chain or double-tailed lipophilic domains [10]. In the surfactant panorama, a non-negligible space has been occupied by bile acids, natural compounds with a steroidal skeleton [11]. Due to the rigidity of their polycyclic structure and the presence of distinct facial hydrophobic and hydrophilic regions, they exhibit a peculiar aggregation behaviour, mostly driven by hydrophobic interactions [12,13]. The possibility of controlling their tensioactive properties by functionalizing specific sites in their structure made bile acids very popular. An important example of this approach was recently reported with the introduction of aromatic subunits onto C-3 of bile acids. Probably due to additional intermolecular pi-stacking interactions, such derivatives were able to self-assemble, affording more complex aggregates than simple micelles [14], the former being particularly useful for the preparation of innovative supracolloidal structures [15,16]. This suggested the functionalization of cholic acid at its C-3 position with the ferrocene system, this latter being able to play both the roles of aromatic moiety and redox-sensitive unit. Thus, in this paper, we report the synthesis in two steps of 3-(4-ferrocenyl-1H-1,2,3-triazol-1-yl)cholic acid 4 and its spectral characterization. The redox potential was determined by cyclic voltammetry and critical micelle concentration (c.m.c.) by surface tension measurements.

The synthesis of the novel bile acid derivative 4 was carried out by a two-step procedure, starting from two readily attainable subunits, namely the 3-azido derivative of cholic acid methyl ester 1 [17] and ethynylferrocene 2 [18], each of them prepared in three steps according to procedures from the literature from cholic acid and ferrocene, respectively. Firstly, a copper-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction [19,20] in a 7:3 t-BuOH/H₂O mixture, in the presence of CuSO₄·5H₂O and sodium ascorbate for in situ reduction of Cu²⁺ to Cu⁺, was employed to link the steroidic and the redox-active moieties. The choice of such a reaction was motivated by our purpose to create a linker unit that could expand the aromatic region onto the steroidic C-3 position. In this sense, the 1,4-disubstituted triazole ring arising from the CuAAC reaction was ideal. This reactive step furnished the ester intermediate 3 in an 82% yield after chromatographic purification. The subsequent hydrolysis of this compound in basic conditions, using a 2 N LiOH aqueous solution, afforded the target molecule 4, which was obtained in a 63% yield after purification by column chromatography. The two-step synthetic path starting from 1 and 2 is shown in Scheme 1.

The structure of ferrocene derivatives 3 and 4 was inferred through ¹H- and ¹³C-NMR spectroscopy. The proton spectrum of the ester intermediate 3 shows, in the region between 0.7 and 2.5 ppm, several overlapped multiplets, ascribed to protons belonging to the steroidic skeleton and lateral chain of the bile acid moiety, while the multiplet at 2.96 ppm is attributable to the steroidic H-3 proton. Hydrogen atoms attached to the other two carbinolic carbons on the backbone, namely C-12 and C-7, give two multiplets at 3.91 and 4.02 ppm, respectively, whereas the singlet at 3.67 ppm, accounting for three protons, is ascribed to the methoxy ester group. As far as the ferrocene moiety is concerned, the signal arising from the five protons on the unsubstituted cyclopentadienyl ring is visible as a slightly broad singlet around 4.09 ppm, while those of the ring linked to the triazole subunit give two different multiplets, at 4.31 and 4.75 ppm, for protons in β and in α to the substituent, respectively. Finally, the singlet at 7.48 ppm is relative to the triazolic proton. The ¹H-NMR spectrum of the carboxylic acid 4 shows obvious similarities with the corresponding ester derivative, the main difference being the disappearance of the sharp singlet at 3.67 ppm arising from the methoxy protons of 3, while the other signals do not undergo significant shifts. As regards the ¹³C-NMR spectra, peaks between 10 and 50 ppm are relative, for both compounds, to carbons on the steroidic backbone and lateral chain, while, in the case of the ester intermediate 3, the methoxy carbon yields a peak at 51.5 ppm. For heteroatom-bearing carbons of the bile acid, a signal is visible at 56.6 ppm for C-3, while C-7 and C-12 give rise to peaks at 66.6 and 68.6 ppm. Signals belonging to the ferrocenyl subunit can be identified as those at 69.1, 69.7, 73.0 and 80.3 ppm, while carbons on the triazole moiety account for peaks at 118.2 and 146.0 ppm for the C-H and the quaternary carbon, respectively. Finally, the ester carbonyl of intermediate 3 produces a signal at 174.8 ppm. The ¹³C-NMR spectrum of target compound 4 shows analogous signals, with negligible shifts with respect to the ester 3, for the steroidic skeleton, the triazole and ferrocenyl subunits, but the disappearance of the methoxy peak and the deshielding of the carbonyl carbon, whose signal is visible in this case at 178.4 ppm, provides further evidence of the successful achievement of the desired product.

Once the title compound had been prepared, its c.m.c. was determined through surface tension measurements. As reported in Figure 1, plotting the surface tension values at different bile salt concentrations (in logarithmic scale), the c.m.c. value was identified as the intersection between the two linear fits encountering the breaking point of the recorded data. This method provided a c.m.c. value of 7 × 10⁻⁵ M. In agreement with the results reported in the literature for other bile salt derivatives, bearing themselves aromatic groups on the C-3 position of the steroidal backbone [14], the estimated c.m.c. for 4 is relevantly lower than that of sodium cholate [21], due to the more hydrophobic nature of the molecule.

In order to study the optical properties of derivative 4, its UV-Vis spectrum was recorded in the range between 360 and 610 nm, detecting a maximum of absorption at 430 nm related to a forbidden d-d transition of the iron metal centre [22], as shown in Figure 2.

Taking into account that micelles formation occurs at 7 × 10⁻⁵ M concentration, for the considered absorption band at 430 nm, we observed absorbance vs. concentration linearity in the concentration range between 0.1 and 4 mM. From the linear fit (see Supplementary Materials), an extinction coefficient ε of (2.96 ± 0.09) × 10² M⁻¹ cm⁻¹ was calculated.

In order to determine the electrochemical properties, cyclic voltammetry (CV) measurements were performed at different scan rates, as reported in Figure 3. The trend of the current as a function of the applied potential for the title compound was compared with the one for ferrocene. A redox potential value of 0.560 V vs. Ag/Ag⁺ as the reference electrode was determined for the couple Fc/Fc⁺ embedded in derivative 4. This value is lower than the observed potential of the classical ferrocene/ferrocenium redox couple, with a potential of 0.629 V. It has been previously reported that the couple Fc⁺/Fc in dichloromethane (DCM) presents a formal potential oscillating between 0.46 and 0.48 vs. SCE depending on the nature of the supporting electrolyte [23]. Our value of 0.629 V differs from that reported in the literature because it refers to a different reference electrode (Ag/AgNO₃ in non-aqueous CH₃CN) for which the activity coefficient of the Ag⁺ cation is expected to be quite different from that in the aqueous ambient that defines SCE potential [24]. Moreover, in our experimental conditions, an additional potential difference in the membrane can also arise through the septum of fritted glass, which separates the CH₃CN solution of our reference electrode from the bulk of the DCM solution containing ferrocene. The electrochemical characterization was conducted in disaggregating conditions, using dichloromethane (DCM) as a solvent for properly solubilizing the bile salt derivative in the monomeric state. The decrease in the redox potential of 4 with respect to simple ferrocene revealed its stronger oxidizing tendency. This could be attributed to an electron-donor effect arising from the extended π-conjugation between the cyclopentadienyl rings and the 1,2,3-triazole ring, directly linked to the cholic acid moiety.

In this work, the synthesis of a novel ferrocene-functionalized cholic acid derivative, in which the surfactant is linked to the redox-active moiety by a triazole ring, has been described. Compound 4 was obtained in two steps from the azido derivative of cholic acid methyl ester and ethynylferrocene, with an overall 52% yield. Its aggregation behaviour was studied by surface tension measurements, and a c.m.c. of 7 × 10⁻⁵ M was assessed, confirming the amphiphile properties of this derivative. Its UV-Vis optical absorption properties were clarified. CV experiments allowed us to determine its redox potential, with a result equal to 0.560 V vs. Ag/Ag⁺, showing a stronger oxidation tendency with respect to ferrocene alone.