Molecular Modelling Analysis of the Metabolism of Clotrimazole

MATERIALS AND METHODS

This being an entirely theoretical study, only molecular modelling calculations were carried out.

Computational Methods
The geometries of CTZ and its metabolite 2CPBPM have been optimised based on molecular mechanics, semi-empirical and DFT (density functional theory) calculations, using the molecular modelling program Spartan ’04. Molecular mechanics calculations were carried out using MMFF force field. Semi-empirical calculations were carried out using the routine PM3. DFT (Density functional theory) calculations were carried at B3LYP/6-31G* level. In optimization calculations, a RMS gradient of 0.001 was set as the terminating condition. For the optimised structures, single point calculations were carried out to give heat of formation, enthalpy, entropy, free energy, dipole moment, solvation energy, energies for HOMO (Highest occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital). The order of calculations: molecular mechanics followed by semi-empirical followed by DFT ensured that the structure was not embedded in a local minimum. To further check whether the global minimum was reached, some calculations were carried out with improvable structures. It was found that when the stated order was followed, structure corresponding to the global minimum or close to that could ultimately be reached in all cases. Although RMS gradient of 0.001 may not be sufficiently low for vibrational analysis, it is believed to be sufficient for calculations associated with electronic energy levels (Huq and Alsheri, 2006).

RESULTS AND DISCUSSION

Table 1 gives the total energy, heat of formation as per PM3 calculation, enthalpy, entropy, free energy, surface area, volume, dipole moment and energies of HOMO and LUMO as per both PM3 and DFT calculations for CTZ and its metabolite 2CPBPM. Figure 2 and 3 give the regions of negative electrostatic potential (greyish-white envelopes) in (a), HOMOs (where red indicates HOMOs with high electron density) in (b), LUMOs in (c) and density of electrostatic potential on the molecular surface (where red indicates negative, blue indicates positive and green indicates neutral) in (d) as applied to optimised structures of CTZ and its metabolite 2CPBPM.

The LUMO-HOMO energy differences for CTZ and its metabolite 2CPBPM from DFT calculations are respectively 5.05 and 5.88 eV, indicating that both the compounds would be kinetically inert with 2CPBPM being the more inert one.

In the case of CTZ, the electrostatic potential is found to be more negative around nitrogen and chlorine atoms, indicating that the positions may be subject to electrophilic attack. In the case of 2CPBPM, the electrostatic potential is found to be more negative around chlorine and oxygen atoms and above and below the phenyl rings, indicating that the positions may be subject to electrophilic attack.

Table 1:	Calculated thermodynamic and other parameters of CTZ and its metabolite 2CPBPM
*in atomic units from DFT calculations

Fig. 2:	Structure of CTZ giving in: (a) the electrostatic potential (greyish envelope denotes negative electrostatic potential), (b) the HOMOs, (where red indicates HOMOs with high electron density), (c) the LUMOs (where blue indicates LUMOs) and in (d) density of electrostatic potential on the molecular surface (where red indicates negative, blue indicates positive and green indicates neutral)

In the case of CTZ, the HOMOs with high electron density are found to be more localised than the LUMOs whereas in the case of 2CPBPM, they appear to have similar spreads.

The overlap of HOMO with high electron density and region of negative electrostatic potential at some positions, gives further support to the idea that the positions may be subject to electrophilic attack.

The molecular surface of CTZ and 2CPBPM are found to possess negative (yellow and red), neutral (green) and electron-deficient (blue) regions so that they may be subject to electrophilic, lyophilic and nucleophilic attacks. Nucleophilic attacks can be due to glutathione and nucleobases in DNA. Reaction with glutathione can induce cellular toxicity by compromising the antioxidant status of the cell whereas that with nucleobases in DNA can cause DNA damage.

Fig. 3:	Structure of 2CPBPM giving in: (a) the electrostatic potential (greyish envelope denotes negative electrostatic potential), (b) the HOMOs, (where red indicates HOMOs with high electron density), (c) the LUMOs (where blue indicates LUMOs) and in (d) density of electrostatic potential on the molecular surface (where red indicates negative, blue indicates positive and green indicates neutral)

However, as stated earlier, since CTZ and 2CPBPM are expected to be kinetically inert, the rate of such adverse reactions may be low unless speeded up enzymatically. The results of the analyses thus show CTZ and its major metabolite may not induce much cellular toxicity or cause DNA damage.

CONCLUSION

CTZ is an N-subtituted imidazole drug that is used therapeutically as a topical antifungal agent. Molecular modelling analyses based on semi-empirical and DFT calculations show that both CTZ and its metabolite 2CPBPM have large LUMO-HOMO energy differences so that they would be kinetically inert. This means that although molecular surfaces of both the compounds have electron-deficient regions so that they can react with glutathione and nucleobases in DNA, the rates of such adverse reactions may be low, unless speeded up enzymatically. Slightly greater reactivity of CTZ and a greater presence of electron-deficient region on its molecular surface, may make the parent drug more toxic than its metabolite.

ACKNOWLEDGMENT

Fazlul Huq is grateful to the Discipline of Biomedical Science, School of Medical Sciences, The University of Sydney for the time release from teaching.