Charge-transfer reaction of 2,3-dichloro-1,4-naphthoquinone with crizotinib: Spectrophotometric study, computational molecular modeling and use in development of microwell assay for crizotinib

Abstract

The reaction of 2,3-dichloro-1,4-naphthoquinone (DCNQ) with crizotinib (CZT; a novel drug used for treatment of non-small cell lung cancer) was investigated in different solvents of varying dielectric constants and polarity indexes. The reaction produced a red-colored product. Spectrophotometric investigations confirmed that the reaction proceeded through charge-transfer (CT) complex formation. The molar absorptivity of the complex was found to be linearly correlated with the dielectric constant and polarity index of the solvent; the correlation coefficients were 0.9567 and 0.9069, respectively. The stoichiometric ratio of DCNQ:CZT was found to be 2:1 and the association constant of the complex was found to be 1.07 × 10(2) l/mol. The kinetics of the reaction was studied; the order of the reaction, rate and rate constant were determined. Computational molecular modeling for the complex between DCNQ and CZT was conducted, the sites of interaction on CZT molecule were determined, and the mechanism of the reaction was postulated. The reaction was employed as a basis in the development of a novel 96-microwell assay for CZT in a linear range of 4-500 μg/ml. The assay limits of detection and quantitation were 2.06 and 6.23 μg/ml, respectively. The assay was validated as per the guidelines of the International Conference on Harmonization (ICH) and successfully applied to the analysis of CZT in its bulk and capsules with good accuracy and precision. The assay has high throughput and consumes a minimum volume of organic solvents thus it reduces the exposures of the analysts to the toxic effects of organic solvents, and significantly reduces the analysis cost.

Keywords: 2,3-Dichloro-1,4-naphthoquinone; Charge–transfer reaction; Crizotinib; High throughput analysis; Microwell assay; Spectrophotometry.

Figure 1

Panel (A): absorption spectra of CZT (1.14 × 10⁻⁴ M) against methanol. Panel (B): absorption spectra of DCNQ (2.28 × 10⁻³ M), and its reaction mixtures with CZT (4.4 × 10⁻⁴ M) measured at varying time intervals (5–90 min) against the reagent blank. Panel (C): absorption spectra of reaction mixtures containing a fixed concentration of DCNQ (2.28 × 10⁻³ M) and varying CZT concentrations (1.78 × 10⁻⁴ to 8.88 × 10⁻⁴ M) against reagent blank. All DCNQ and CZT were prepared in methanol.

Figure 2

Panel (A): absorption spectra of reaction mixtures of DCNQ (2.28 × 10⁻³ M) and CZT (4.44 × 10⁻⁴ M) in different solvents. Solvents were: chloroform (1), 1,4-dioxane (2), dichloroethane (3), dichloromethane (4), propane-2-ol (5) and ethanol (6). Panel (B): the plot of molar absorptivity (ε) of DCNQ-CZT complex versus dielectric constant (●) and polarity index (△) of the solvent and the fitting linear equations.

Figure 3

Energy-minimized CZT and its CT complex with two molecules of DCNQ.

Figure 4

Scheme for the CT reaction pathway of CZT with DCNQ.

Figure 5

Panel (A): the absorption-time curves for the reaction of DCNQ (2.28 × 10⁻³ M) with varying concentrations of CZT; these concentrations were: 0.23 × 10⁻⁴ (●), 0.46 × 10⁻⁴ (△), 0.89 × 10⁻⁴ (▴), 0.91 × 10⁻⁴ (○), and 1.14 × 10⁻⁴ (♦) M. All solutions were prepared in ethanol and the reactions were carried out at room temperature (25 ± 2 °C). Panel (B): linear plot for log C vs. log K for the kinetic reaction of DCNQ (2.28 × 10⁻³ M) with varying concentrations of CZT (0.23 × 10⁻⁴ to 1.14 × 10⁻⁴ M). C and K are the CZT concentration (M) and the reaction rate (s⁻¹), respectively.

Figure 6

Panel A: absorbance-time curves of reaction mixtures containing fixed concentrations of CZT (0.46 × 10⁻⁴ M) with DCNQ (2.28 × 10^–3 M) at different temperatures. Panel B: Arrhenius plot for the reaction of CZT with DCNQ at varying temperatures.

Figure 7

Benesi–Hildebrand plot of CT complex of DCNQ with CZT and the linear fitting equation. [A_o], A^AD and [D₀] are the molar concentration of DCNQ, absorbance of the complex reaction mixture, and molar concentration of CZT, respectively. All solutions were prepared in ethanol and the reactions were carried out at room temperature (25 ± 2 °C).