Study the Structure and Dynamics of Antifungal Agent Ketoconazole by CSA and Site-Specific Spin-Lattice Relaxation Time Measurements

: An azole antifungal agent, ketoconazole, is widely used in the treatment of mucosal fungal infections related to AIDS immunosuppression, organ transplantation, and cancer chemotherapy. The structure and dynamics of ketoconazole are thoroughly studied by chemical shift anisotropy tensor and site-specific spin-lattice relaxation time measurements. The molecular correlation time at crystallographically different carbon sites is calculated by considering that the spin-lattice relaxation mechanism for the 13 C nucleus is mainly governed by chemical shift anisotropy interaction and hetero-nuclear dipole-dipole coupling. The CSA parameters at the crystallographically distinct sites of ketoconazole are determined by two-dimensional phase adjusted spinning sideband (2D PASS) cross-polarization magic angle spinning (CP-MAS) solid-state NMR experiment. The site-specific spin-lattice relaxation time is measured by the Torchia CP experiment. The spin-lattice relaxation rate is slow for all the carbon nuclei sites except C2, C3, C4, C5, and C26 carbon nuclei reside on the piperazine ring and the methyl group. It suggests the close-pack arrangement of the molecule due to π-π stacking interaction. The molecular correlation time of all the carbon atoms reside on the benzene ring, 1,3-dioxolane ring, imidazole ring, and the 2,4-dichlorobenzene ring is of the order of 10 −4 s, while it is of the order of 10 −7 s for carbon atoms reside on the piperazine ring. The CSA parameters of the carbon nuclei on the piperazine ring (C2, C3, C4, C5), and the methyl group (C26) 2,4-dichlorobenzene ring due to the presence of π-electrons. A huge variation of the spin-lattice relaxation time and the molecular correlation time are observed for numerous carbon nuclei situated on the side-chain of ketoconazole. The spin-lattice relaxation time varies from 500 s to 8 s, and the molecular correlation time varies in the range of 10 −4 s to 10 −7 s. These types of investigations portrayed the correlation between the structure and dynamics of the antifungal drug ketoconazole, which will help to develop the advanced antifungal drugs. Additionally, the CSA information of the drug molecules will be immensely useful for NMR crystallography.

dichlorobenzene ring due to the presence of π-electrons. A huge variation of the spin-lattice relaxation time and the molecular correlation time are observed for numerous carbon nuclei situated on the side-chain of ketoconazole. The spin-lattice relaxation time varies from 500 s to 8 s, and the molecular correlation time varies in the range of 10 −4 s to 10 −7 s. These types of investigations portrayed the correlation between the structure and dynamics of the antifungal drug ketoconazole, which will help to develop the advanced antifungal drugs.
Additionally, the CSA information of the drug molecules will be immensely useful for NMR crystallography.

Introduction:
The azole class compounds are the most potent antifungal drug for its lower toxicity, higher efficacy, and a broad spectrum of activity. Ketoconazole is an azole class compound and a synthetic derivative of phenylpiperazine with antifungal and antineoplastic activity. It is also considered as an antimitotic and a bacteriostatic agent for gram-positive bacteria. Ketoconazole has been used extensively in the treatment of superficial mycoses such as dermatophytosis and candidiasis. [1][2][3][4] This antifungal imidazole agent has also been used for the treatment of histoplasmosis, human systemic fungal infections, against Candida species, Cryptococcus neoformans, tinea corporis, tinea cruris, tinea manuum and tinea pedis, onychomycosis, seborrheic dermatitis. [5][6][7][8][9][10][11][12][13][14][15][16] Ketoconazole prohibits the conversion of lanosterol to ergosterol (which is required to maintain the integrity of the organism's cell membrane) by inhibiting the fungal cytochrome P450 (CYP51). Cytochrome P-450 enzyme is necessary to remove the C-14 methyl group of lanosterol. 17,18 Ketoconazole also can blockage the conversion of lanosterol to cholesterol in mammals. It can block adrenal steroidogenesis by inhibiting the corticoid 11 −hydroxylase.
Because of this property, ketoconazole is used for the treatment of prostate cancer and Cushing's syndrome, respectively. 18 Solid state NMR spectroscopy is an indispensible tool to determine the structure and dynamics of a compound. The chemical shift anisotropy (CSA) tensor is one of the most important parameter which can be measured by applying sophisticated SSNMR techniques. It can provide information about the electronic distribution, molecular orientation, and molecular dynamics surrounding a nucleus. The Larmor precession frequency of the nucleus depends on the orientation of the molecular moiety with respect to the external magnetic field and the electronic distribution around it. The shift of the precession frequency of the nucleus inside the matter is expressed as ( , ) − 0 = + (∆ 2 ⁄ )(3cos 2 − 1 − sin 2 cos2 ), where the anisotropy parameter ∆ measures the deviation of the electron cloud from the spherically symmetric charge distribution, and the asymmetry parameter measures the deviation of electronic charge distribution from axially symmetric shape. In principal axis system (PAS), , and are respectively the polar and azimuthal angles with respect to the direction of the applied magnetic field( ). 19 The effective magnetic field experienced by the nucleus is = (1 ± ) . The dimensionless quantity is the chemical shift anisotropy (CSA) tensor. It is a second rank tensor with nine components. [19][20][21][22][23][24][25][26] The diagonal components of this tensor in the principal axis system (PAS) are 27 52 The molecular correlation time at crystallographically different carbon sites of ketoconazole is also calculated by considering that the spin-lattice relaxation mechanism for 13 C carbon is mainly governed by hetero-nuclear dipole-dipole coupling and CSA-interaction.
These types of investigations will provide a vivid picture about the structure and dynamics of ketoconazole, which will help to develop advanced antifungal drugs.

NMR-Measurements
The active pharmaceutical ingredient of ketoconazole was purchased from Sigma Aldrich. 13

CSA-Measurements
The analysis of static 13 C solid-state NMR spectrum encounters considerable difficulties because anisotropic interactions give rise to a broad powder-pattern and a considerable loss of sensitivity compared to 13

Determination of Chemical Shift Anisotropy Parameter:
The chiral drug ketoconazole is a racemic (1:1) mixture of enantiomers of the cis configuration. 3,4 The chemical formula of this imidazole derivative is cis- . It is insoluble in water. It is a weak base with pKa values of 6.51 and 2.94. Figure 1 (a) shows the chemical structure of ketoconazole molecule, which is associated with the rich aromatic molecular moieties like N-acetylpiperazine phenyl group, 1,3 dioxolane ring, imidazole ring, and dichlorophenyl ring. The piperazine rings have chair conformations, and the benzene ring is distorted because three atoms of the ring are split on the basis of their thermal parameters. 56,57 Phenylpiperazine ring has no contribution to interact with the heme group CYP51, it interact with amino acid residues in the substrate access channel. The conformations of the 1,3-dioxolane rings are halfway between an envelope with flap at the unsubstituted carbon atom. 56,57 Stereochemical orientation of the dioxolane ring plays a vital role in the inhibition of the hedgehog signalling pathway and it is also responsible for inhibition of CYP3A4 to thwart coordination of the molecular oxygen, essential for oxidation. [58][59][60] The imidazole and the 2,4-dichlorobenzene rings have planar conformation. 56,57 The antifungal action of the azole group keteconazole is due to the binding of the imidazole nitrogen with cytochrome P45051. [58][59][60][61] The ketoconazole drug is entrenched in the hydrophobic region. The lipid-drug complex is fabricated by the action of the van der Waals type of interactions and electrostatic interaction. 62 Figure 1 Figure 2 shows the 13 C 2DPASS CP-MAS SSNMR spectrum of ketoconazole. The direct dimension represents the pure isotropic spectrum, and the indirect dimension represents the anisotropic spectrum. Table 1 shows the principal components of CSA parameters at numerous carbon nuclei sites of ketoconazole. Figure 3 and Figure 4 show ] parameter measures the largest separation of spinning CSA sideband pattern from the center of gravity. The sign of the anisotropy parameters says on which side of the certer of gravity, the separation is the largest. Figure 5 shows the bar-diagram of anisotropy and asymmetry parameter at numerous carbon nuclei sites of ketoconazole.
Ketoconazole molecule is chemically related to miconazole. The bioavailability of this antimycotic agent originates from its capability to penetrate the cell membrane due to the presence of the methyl group at C1 position(carbonyl group carbon). 63 The chemical shift is the highest for carbonyl group carbon. One of the reasons of the large value of CSA parameters of carbonyl group carbon is the magnetic anisotropy. There is no symmetry axis in the carbonyl group; naturally, there arise three different magnetic susceptibilities , , along three mutually perpendicular directions of the principal axes system (PAS).
There originate two anisotropic susceptibilities -one parallel to the magnetic field (∆ ǁ = − ) and another perpendicular to the magnetic field (∆ ⊥ = − ). 64 The McConnell equation 65  , where 1 is the angle between the radius vector R and x-axis and θ2 is the angle between radius vector R and z-axis. 66 Large amount of directional dependent magnetic field is generated due to this highly anisotropic magnetic susceptibility. This manifests as a large value of anisotropic chemical shift.

Determination of Spin-lattice Relaxation Time and Molecular Correlation Time:
The dipole-dipole interaction, chemical shift anisotropy (CSA) interaction, spin-rotation interaction, scalar coupling, and the quadrupolar coupling play significant role in the spinlattice relaxation mechanism. But for large molecules the contribution of spin-rotation interaction and scalar coupling is insignificant. Hence, for spin 1 2 ⁄ carbon nuclei, the spin-lattice relaxation mechanism is mainly dominated by chemical shift anisotropy interaction and dipole-dipole coupling interaction. The contribution of chemical shift anisotropy interaction in spin-lattice relaxation mechanism is expressed as 68 ).
The role of dipole dipole interaction in spin-lattice relaxation mechanism is expressed as 70 Where X represents hydrogen, oxygen, chlorine and nitrogen atoms. is the distance between carbon and the nearest neighbour atoms like hydrogen, oxygen, nitrogen, chlorine.
The bond-distance is calculated by X-ray crystallography study. 56,57 Larmor precession frequency of carbon nucleus is = 2 = 2 × 3.14 × 125. The spin-lattice relaxation rate for 13 C can be expressed as Molecular correlation time is calculated by this equation.

Conclusion:
A lipophilic imidazole derivative, ketoconazole, is used for the treatment of mucosal fungal CSA information of the drug molecules will be immensely useful for NMR crystallography.