CBZE was successfully synthesized by a two-step process. In summary, benzoyl chloride initially reacted with hydroperoxide ion to give peroxybenzoic acid which was then used to effect the epoxidation of the azepine residue of CBZ (Scheme 1).

Synthesis afforded CBZE in moderate yield of 50.1% and with a purity of 97.11%w/w when analyzed by LC–MS. The chromatographic conditions employed involved gradient elution on a C-18 column using 0.1% formic acid in water: 0.1% acetic acid in methanol (70:30) at a flow rate of 1 mL/min. The color and melting point of the product were consistent with those found in the literature [23]. Although the mass spectral analysis of the product did not yield a molecular ion, as is often the case with epoxides which are unstable to ionization energy, the mass spectrum revealed the most abundant peaks at m/z of 179.7, 191.7 and 207.8 [24]. This fragmentation pattern was consistent with some of the top peaks reported for CBZE in the literature [25].

Color: white; mp: 190–193 °C Yield: 50.1%; ATR-FTIR cm−1: 3431.0 (N–H), 1673.5 (C=O), 1096.1 (C–O); 1H NMR (DMSO-d6 60 MHz) \(\delta :\) 7.31–7.47 (m, 8H Aromatic H), 6.92 (s, 2H CH–O–CH), 1.23 (s, 2H NH2); 13C NMR (DMSO-d6 60 MHz) \(\delta :\) 172.37 (C-15), 143.0 (C-1 and C-14), 130.87 (C-3 and C-12), 128.75 (C-5, C-10, C-6 and C-9), 126.62 (C-4 and C-11), 122.15 (C-2 and C-13), 70.69 (C-7 and C-8); negative ion ESI: m/z molecular peak not found, Calculated M 252.27.

The changes in the 278 nm band of BSA with increasing concentrations of CBZ or CBZE are depicted in Fig. 1. Absorption measurements of the band showed progressive hyperchromic changes with increasing mole ratios of CBZ and CBZE. In addition, the band position of the CBZE-BSA complex showed a very small hypsochromic shift (\(\Delta \lambda = - 1\,\, }\)).

Absorption spectra of BSA with increasing concentrations (0–20 µM) of (a) CBZ (b) CBZE

The formation constants of complexes following the binding of small ligands to a protein molecule can be estimated from Eq. 1 [22].

$$\frac }} = \frac - A_ }} + \frac - A_ } \right)}}.\frac}} \right]}}$$

(1)

where \(A_\) and A are the absorbance values of BSA before and after the addition of increasing concentrations of the ligands, respectively. \(A_\) is the final absorbance of the ligand–protein complex.

The double reciprocal plots obtained are depicted in Figs. 2 and 3 for CBZ and CBZE, respectively. A linear relationship (R2 > 0.9) between \(1/A - A_\) and reciprocal of ligand concentration can be observed in both instances. The formation constants of CBZ-BSA and CBZE-BSA complexes are depicted in Table 1. The values were determined from the ratio of the intercept to the slope of the respective plots. The thermodynamic parameters associated with the binding of the ligands with BSA are also shown in Table 1.

Benesi–Hilderbrand plots for CBZ-BSA system in the absence and presence of competing drugs at 298 K

Benesi–Hilderbrand plots for CBZE-BSA system in absence and presence of competing drugs at 298 K

The effects of the presence of paracetamol and ascorbic acid on the albumin binding affinities of CBZ and CBZE were also examined. The electronic absorption spectra of a fixed concentration of BSA in the presence of increasing mole ratios of ligands and a fixed concentration of the competing drug showed progressive changes in the absorptivity of the band. As shown in the representative plots, incremental addition of CBZE in the presence of paracetamol and ascorbic acid respectively caused hyperchromic and hypochromic changes in the 278 nm band of BSA (Fig. 4).

Absorption of BSA with increasing concentrations of CBZE in presence of (a) paracetamol (b) ascorbic acid

The calculated binding constants and number of binding sites of the CBZ-BSA and CBZE-BSA systems in the presence of the competing drugs are depicted in Table 2. The number of binding sites of albumin involved in the formation and stabilization of the complexes were estimated using a modified Scatchard plot derived from Eq. 2 [3, 4]

$$\log \frac - A}} = \log K_ + n\log \left[ }} \right]$$

(2)

where Ao and A are the absorbance values of protein–ligand systems in the absence and presence of competing drug, Ka is binding constant, n is number of binding sites in BSA molecule and [ligand] is the concentration of the ligand.

The difference spectrum of free albumin and CBZ-albumin complex are depicted in Fig. 5. The spectrum of free BSA showed the characteristic Amide I and II bands at 1677 and 1593 cm−1, respectively. Following complexation with CBZ, a substantial decrease in intensity and position (from 1677 to 1623 cm−1) of the Amide I band was observed. A similar decrease in band intensity of the CBZ-albumin complex was observed in the presence of interfering paracetamol or ascorbic acid. This was accompanied by a shift in Amide I band position to 1617 and 1624 cm−1, respectively.

Difference spectrum of (a) free BSA, (b) CBZ-BSA complex, (c) CBZ-BSA-paracetamol (d) CBZ-BSA-ascorbic acid

The difference spectrum of CBZE-albumin complex in the absence and presence of interfering drugs are also depicted in Fig. 6.

Difference spectrum of CBZE-BSA complex (a) alone (b) with paracetamol (c) with ascorbic acid

The reduction in the Amide I band was further confirmed by the quantitative estimation of the secondary structure of the protein using infra-red self-deconvolution and curve fitting methods. The α-helical content of albumin decreased from 67.34% in free BSA to 42.56 and less drastically, 56.43% upon interaction with CBZ and CBZE, respectively. In the presence of paracetamol and ascorbic acid interference, the CBZ-BSA complex showed a further decrease in the α-helical content of BSA to 37.76 and 34.87%, respectively. Similarly, the α-helical content of BSA in CBZE-BSA complex decreased to 46.74% in the presence of ascorbic acid while paracetamol induced more extensive perturbations in the secondary structure of BSA as the helical content was reduced to 22.78%.