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. 2019 Dec 4;4(25):21627–21635. doi: 10.1021/acsomega.9b03558

Investigation of the Detailed Internal Structure and Dynamics of Itraconazole by Solid-State NMR Measurements

Krishna Kishor Dey †, Shovanlal Gayen ‡,*, Manasi Ghosh †,*
PMCID: PMC6921643  PMID: 31867560

Abstract

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The structure and dynamics of itraconazole were investigated by 13C 2DPASS MAS SSNMR and spin-lattice relaxation time measurement to get an insight into its multiple biological activities, e.g., antifungal, antiviral, anticancer activities, etc. The molecular correlation time at chemically different sites of carbon nuclei was calculated by considering that the spin-lattice relaxation mechanism is mainly dominated by chemical shift anisotropy interaction and heteronuclear dipole–dipole interaction. The spin-lattice relaxation time is long for C35, C6, C5, and C34 carbon nuclei that participated in the 1, 2, 4-triazole ring. On the contrary, it is comparatively shorter for C1, C2, C3, and C4 carbon nuclei associated with the sec-butyl group in the triazolane side-chain region. Chemical shift anisotropy (CSA) parameters of C5, C6, C34, and C35 nuclei are much higher than those of C1, C2, C3, C4 nuclei, indicating that the relaxation mechanism at a high value of magnetic field is predominated by chemical shift anisotropy interaction. The molecular correlation time of carbon nuclei residing at the side-chain region is 2–3 orders of magnitude lesser than that of those participated in the 1,2,4-triazole ring. The spin-lattice relaxation time is very long for carbon nuclei C28 and C30 bonded with chlorine. Asymmetry and anisotropy parameters are also very high for the spinning CSA sideband pattern corresponding to the C28 and C30 nuclei. The molecular correlation time is on the order of 10–3 s for C28 and 10–4 s for C30, whereas for side-chain carbon nuclei, it is on the order of 10–6 s. This suggests that the effective magnetic field experienced by C28 and C30 nuclei is affected by the polarization of the chemical bond. A huge variation in molecular correlation time is observed for chemically different sites of carbon nuclei of the itraconazole molecule. These investigations vividly portrayed how the structure is correlated with the dynamics of a valuable drug, itraconazole, with multiple biological activities. This study will enlighten the way of inventing advance medicine for multiple biological activities in the pharmaceutical industry.

1. Introduction

Itraconazole is a trizole-containing drug prescribed for the prevention and treatment of fungal infection.1 The primary structural difference among itraconazole and other azole antifungals is the presence of a triazolone ring (the ring consists of three nitrogens) and a sec-butyl side chain, and these are responsible for its different biological activities like antifungal and anticancer activities, as well as its interesting pharmacokinetic behavior like strong protein binding, tissue penetration, prolonged half-life and bioavailability, etc.1,2 Nitrogen atoms of the azole ring interact with the heme iron of the fungal cytochrome P4503A (CYP3A). As a result, it inhibits the function of the lanosine 14α-demethylase enzyme to stop the synthesis of ergosterol.3 It is the only drug in the azole class of antifungal agents that inhibits the hedgehog (Hh) signaling pathway and angiogenesis, responsible for the anticancer activity.4 It is also used for the treatment of dermatophyte infections, sporotrichosis, penicilliosis, allergic and invasive aspergillosis, histoplasmosis, superficial candidiasis, coccidioidomycosis, blastomycosis, etc. Itraconazole is a well-tolerated drug as the mammalian cytochrome P450 enzyme is less affected even at a high concentration of the drug.3 Hence, the sterol and steroid pathways of the human pituitary–adrenal–testicular axis are less affected by itraconazole.5 It is a weak base (pKa = 3.7). It can be ionized at a low pH. It is available as capsules, intravenous preparations, and oral suspensions. However, it is insoluble in water and dilute acid solutions. Therefore, it is difficult to extract the information about the structure and dynamics of itraconazole in the solution state.

The molecular structure of itraconazole shares a striking similarity with terconazole and ketoconazole. Although terconazole and ketoconazole possess antifungal activity like itraconazole, but they fail to restrain the human umbilical vein endothelial cell (HUVEC) proliferation and to persuade the vascular endothelial growth factor receptor 2 (VEGFR 2) glycosylation defect.6 The range of application of triazoles (itraconazole, fluconazole, voriconazole, and posaconazole) is broader than that of ketoconazole (with an azole ring associated with two nitrogens). Itraconazole can be used for the medication of both superficial and systemic fungal infections. A single drug with multiple biological activities is not so common, and itraconazole is one of them. Hence, it is fascinating to investigate the internal structure and spin dynamics of itraconazole to get an insight into the varying dynamics in different parts of the structure responsible for different biological and pharmacokinetic behaviors (Figure 5a). The structural details and molecular dynamics of this unique azole were investigated by 13C CP-MAS NMR spectral analysis, 13C spin-lattice relaxation time measurements, two-dimensional phase-adjusted spinning sideband (2DPASS) magic-angle-spinning (MAS) nuclear magnetic resonance (NMR) experiment, and calculation of molecular correlation time at numerous carbon nuclei situated at various chemical environments.

Figure 5.

Figure 5

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(a) Activities of different regions of the itraconazole molecule. (b) Molecular correlation time at chemically different sites.

Chemical shift anisotropy provides valuable information about the molecular conformation and internal structure. There are various techniques to determine CSA parameters. They can be measured by the two-dimensional MAS/CSA NMR experiment7 and by SUPER (separation of undistorted powder patterns by effortless recoupling) MAS NMR at a magic-angle-spinning (MAS) of 2.5–5 kHz.8 ROCSA (recoupling of chemical shift anisotropy) pulse sequence was applied to determine CSA parameters at MAS frequencies of 11–20 kHz.9 RNCSA (γ-encoded RNnν-symmetry-based chemical shift anisotropy) recoupling schemes were applied to extract CSA parameters of the system with weak homonuclear dipole–dipole interactions under a wide range of MAS frequencies.10 The two-dimensional magic-angle-flipping (2DMAF) experiment,11−14 two-dimensional magic-angle-turning (2DMAT) experiment,15 and two-dimensional phase-adjusted spinning sideband (2DPASS) magic-angle-spinning (MAS) SSNMR experiment16,17 can extract information about CSA for multiple site compounds at very low MAS speed. The total evolution period in indirect dimension for the 2DMAT experiment is not constant. Consequently, the spin–spin relaxation mechanism makes the spectrum so complicated that it would be difficult to extract the exact information about the relative abundance of chemically different sites of carbon nuclei. Thus, a probe is required for the 2DMAT experiment that can alter the orientation of the spinner during each scan. This type of probe is not commercially available. In these aspects, the two-dimensional phase-adjusted spinning sideband (2DPASS) magic-angle-spinning (MAS) SSNMR technique is more feasible as the total time during five π pulses remains constant, and this experiment can be performed using a standard commercial probe. This technique was employed to investigate the properties of glass compounds18 and biopolymers,19−22 but it is not yet exploited properly to investigate the internal structure and dynamic of such a valuable antifungal drug, itraconazole, with several ancillary biological activities. This study will enlighten the way of inventing advance medicine for fungal infections and design of potent drugs.

2. Experimental Section

2.1. NMR Measurements

An active ingredient of itraconazole, purchased from Sigma Aldrich, was used for solid-state NMR experiments. 13C CP-MAS solid-state NMR experiments were performed using a JEOL ECX 500 NMR spectrometer. The resonance frequency for 13C was 125.721 MHz. All of the experiments were carried out in a 3.2 mm JEOL double resonance MAS probe. The magic-angle-spinning (MAS) speed was 10 kHz for 13C CP-MAS spectrum and spin-lattice relaxation measurements. The condition of cross-polarization (CP) was maintained by keeping contact time 2 ms, and SPINAL-64 1H decoupling was used during acquisition. The 13C spin-lattice relaxation experiment was conducted using the Torchia CP method.23

2.2. CSA Measurements

During the slow MAS speed, the powder pattern breaks into several numbers of sidebands. The spacing among the sidebands is equal to the MAS speed. Using sideband intensities of the spinning CSA sideband pattern, CSA parameters can be measured by the Herzfeld and Berger24 integral method.

The pulse sequence of the 2DPASS MAS NMR experiment with five π pulses was established by Antzutkin et al. in 1995.17 The phase cycling for the desired coherence pathway was done by 13 steps cogwheel phase cycling. In the indirect dimension, data points were sixteen.17 The time evolution of five π pulses was calculated by PASS equations. The 2DPASS experiments were performed at two different values of spinning speed 600 and 2000 Hz. The CP condition for these two spinning speeds was optimized on glycin with 2 ms contact time. The 90° pulse length for 13C was 3 μs.

3. Results and Discussion

3.1. Spin-Lattice Relaxation Measurements

Figure 1a shows that the itraconazole molecule is associated with three prominent regions: triazole-containing dioxolane region, phenyl–piperazine–phenyl linker region, and triazolone side-chain region. Although the phenyl–piperazine–phenyl linker region and triazolone side-chain region are not playing prominent roles in interaction with the heme group CYP51, they interact with amino acid residues in the substrate access channel.25 The side-chain region can easily be replaced by various functional groups like hydrazine carboxamides and meta-substituted amides. The triazole-containing dioxolane region is responsible for inhibition of CYP3A4 to thwart coordination of the molecular oxygen, essential for oxidation.26,27 Stereochemical orientation of the dioxolane ring plays a significant role in inhibition of the hedgehog signaling pathway. The antifungal action is due to the binding of the triazole nitrogen with cytochrome P45051(CYP51).28Figure 1b shows 13C CP-MAS NMR spectrum of itraconazole. Figure 2a–d shows 13C spin-lattice decay curves of various carbon nuclei situated in chemically and crystallographically different environments. The bar diagram of the spin-lattice relaxation time (as shown in Figure 2e) suggests that the spin-lattice relaxation time hugely varied due to the change of the chemical environment surrounding the nuclei. The spin-lattice relaxation time (as shown in Table 1) is very long for C5, C6, C34, and C35 carbon nuclei participated in the 1,2,4-triazole ring. Anisotropy parameters (Table 2) are also comparatively large for these specific sites of carbon nuclei. On the contrary, the relaxation time is shorter and CSA parameters are lower for C1, C2, C3, and C4 carbon nuclei, residing in the side-chain region.

Figure 1.

Figure 1

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(a) Itraconazole molecule fabricated by cis-4[4-4-4][2-(2-4-dichlorophenyl)-2-(1H-1,2,4,triazol-1-methyl)-1,3-dioxolan-4-yl]-1-piperazinylmphenyl]-2,4-dihydro-2-(1-methyl-propyl)-3H-1,2,4-triazol-3-one and (b) 13C CP-MAS NMR spectrum of itraconazole.

Figure 2.

Figure 2

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(a–d) Show 13C spin-lattice decay curves of itraconazole at various resonance peak positions of carbon nuclei. (e) Shows the bar diagram of the spin-lattice relaxation time of carbon nuclei residing in various chemical environments.

Table 1. 13C Spin-Lattice Relaxation Time at Various Sites of Carbon Nuclei of Itraconazole.

13C spin-lattice relaxation time T1) of itraconazole
position of carbon atoms at which relaxation time is measured (ppm) spin-lattice relaxation time (s) position of carbon atoms at which relaxation time is measured (ppm) spin-lattice relaxation time (s)
C35 at 157.31 ppm 142 ± 10 C7 at 155.88 ppm 162 ± 10
C6 at 154.1 ppm 197 ± 10 C20 at 150.18 ppm 192 ± 10
C10 at 147.69 ppm 204 ± 10 C27 at 139.5 ppm 178 ± 10
C34 at 141 ppm 90 ± 10 C17 at 122.83 ppm 35 ± 5 and 2 ± 0.5
C5 at 136.65 ppm 290 ± 10 C30 at 132.73 ppm 196 ± 10
C28 at 131.46 ppm 330 ± 20 and 12 ± 2 C32 at 124.04 ppm 36 ± 5 and 2 ± 0.5
overlap of C18 and C22 at 117.75 ppm 45 ± 5 and 2 ± 0.5 Overlap of line C9 and C11 at 80.6 36 ± 5
overlap of C8 and C12 at 112.04 ppm 210 ± 20 C16 at 55.46 110 ± 10 and 7 ± 2
overlap of line C14 and C33 at 56.52 ppm 240 ± 10 C3 at 56.52 ppm 32 ± 2
C23 at 71.47 ppm 180 ± 10 C4 at 25.39 ppm 47 ± 2 and 2 ± 0.2
C2 at 33.18 ppm 65 ± 2 and 2 ± 0.5 C1 at 15.46 ppm 50 ± 2 and 2 ± 0.5
C25 at 74.322 ppm 73 ± 10 C24 and C26 at 79.46 ppm 116 ± 10
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Table 2. Values of CSA Parameters at Numerous Sites of Carbon Nuclei of Itraconazole.

CSA parameters of itraconazole at different carbon sites
carbon from different chemical environments with isotropic chemical shift (δiso) (ppm) δ11 δ22 δ33 span (ppm) skew anisotropy Inline graphic asymmetry Inline graphic
15.51 (C1) 26.1 10.2 10.2 15.9 –1 15.9 0
25.77 (C4) 43.9 21.1 12.3 31.7 –0.5 27.3 0.5
32.12 (C2) 46.8 25.1 24.5 22.4 –0.9 22.1 0
33.09 47.7 31.3 20.4 27.3 –0.2 21.8 0.8
30.65 42.9 32.3 16.8 26.1 0.2 –20.9 0.8
49.88 (C13) 83.8 50.2 15.7 68.1 0.0 –51.3 1
55.07 (C16) 71.7 58.8 34.7 36.9 0.3 –30.5 0.6
56.78 (C14) 77.8 55.9 36.7 41.1 –0.1 31.5 0.9
57.03 (overlap of C33, C3 and C14) 78.2 55.5 37.4 40.9 –0.1 31.8 0.9
71.19 (C23) 119.3 59.9 34.4 84.8 –0.4 72.1 0.5
73.14 (C25) 100.4 59.5 59.5 40.8 –1 40.8 0
79.25 (C24 and C26) 118.1 66.9 52.8 65.3 –0.6 58.3 0.4
80.23 (C11) 118.4 65.5 56.8 61.5 –0.7 57.2 0.2
82.91 (C9) 123.1 63.2 62.5 60.6 –1 60.3 0.0
111.97 (overlap of C19 and C21) 138.4 113.8 83.7 54.7 0.1 –42.4 0.9
112.46 (overlap of C8 and C12) 138.4 114.9 84.1 54.3 0.1 –42.5 0.8
117.68 (overlap of C18 and C22) 198.9 143.3 10.8 118.1 0.4 –160.3 0.5
122.99 (C17) 235.8 85.4 47.8 188 –0.6 169.2 0.3
124.25 (C32) 235.7 87.9 49.1 186.7 –0.6 167.2 0.4
126.75 (C31) 209 158.6 12.7 196.3 0.5 –171.1 0.4
129.56 (C29) 213.3 144.9 30.5 182.7 0.3 –148.6 0.7
131.12 (C28) 223.4 132.2 37.7 185.7 0.0 –140.1 1
132.69 (C30) 221.48 129.72 46.87 174.6 –0.05 133.2 0.9
134.25 214.4 125.85 62.5 151.9 –0.2 120.2 0.8
136.75 (C5) 234.4 135.8 40.1 194.3 –0.02 146.5 1
139.57 (C27) 224.7 148.9 45 179.7 0.2 –141.8 0.8
141.13 (C34) 237 128.2 58.2 178.8 –0.2 143.8 0.7
147.69 (C10) 197.9 155.5 89.6 108.3 0.2 –87.1 0.7
150.19 (C20) 237.7 131 81.9 155.7 –0.4 131.2 0.6
154.26 (C6) 251.6 136.1 75.1 176.5 –0.3 146 0.6
155.82 (C7) 255.1 135.6 76.7 178.4 –0.3 149 0.6
157.07 (C35) 215.4 171.2 84.6 130.8 0.3 –108.74 0.6
158.01 211.1 175.9 87.1 124 0.4 –106.4 0.5
158.64 227.4 164.7 83.8 143.5 0.1 –112.2 0.8
159.26 228.5 164.7 84.5 144 0.1 –112.1 0.9
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The spin-lattice relaxation time is very long for carbon nuclei C28 and C30 bonded with the chlorine atom. Asymmetry and anisotropy parameters are also very high for the spinning CSA sideband pattern corresponding to the C28 and C30 nuclei. This suggests that the relaxation mechanism is greatly affected by chemical shift anisotropy interaction. The role of chemical shift anisotropy in the spin-lattice relaxation mechanism can be expressed as29−31

3.1. 1

where correlation time τc = 3 τ2, B is the applied magnetic field, S2 = (Δδ)2 (1 + η2/3), and Inline graphic, Inline graphic.

3.2. Chemical Shift Anisotropy

Both isotropic and anisotropic components of the chemical shift are correlated with chemical bonding. The anisotropic component of chemical shift depends on the orientation and conformation of the molecule. Chemical shift anisotropy can be represented by a second-rank tensor with nine components. In the principal axis system (PAS), off-diagonal components are cancel out and three diagonal terms survive. The expressions of these diagonal components of the chemical shift anisotropy tensor (δ11, δ22, and δ33) are given by32,33

3.2. 2
3.2. 3
3.2. 4

where Lx, Ly, and Lz represent the components of angular momentum along x, y, and z directions, respectively. The first part of these three equations generates from those electrons that constitute spherically symmetric charge distribution. There arise distortions in this spherically symmetric charge distribution, when electrons are lifted to the excited state from the ground state. The second term mainly arises for those electrons that reside in the p or d orbital.

The center of gravity of the spinning CSA sideband pattern is represented as an isotropic chemical shift Inline graphic.24,31,34 Changes in the isotropic chemical shift have a great influence on the breadth of the CSA tensor. Generally, small changes in the isotropic chemical shift correspond to a larger change in the chemical shift anisotropy.35 Span (Ω = δ11 – δ33) represents the maximum width of the spinning CSA sideband pattern. According to Haeberlen convention, the anisotropy (Δδ) and asymmetry (η) parameters are defined as Inline graphic and Inline graphic, respectively. Anisotropy represents the largest separation from the center of gravity of the spinning CSA sideband pattern. The sign of the anisotropy tells on which side of the center of gravity one can find the largest separation. When the spinning CSA pattern is axially symmetric (i.e., δ22 is equal to δ11 or δ33), then the value of the asymmetry parameter is zero. Hence, the asymmetry parameter basically shows whether the CSA pattern deviates from its axially symmetric shape or not. As shown in Table 2, the asymmetry parameter is small (<0.5) for C1, C2, C4, C9, C11, C17, C18, C22, C23, C24, C25, C31, and C32 resonance lines, which indicates that the sideband patterns for these resonance lines are axially symmetric. On the contrary, the sideband patterns are highly asymmetric for these resonance lines for which the asymmetry parameter is greater than 0.5, especially for C5, C19, C22, C28, and C30 carbon resonance lines. The orientation of the asymmetry is represented by a parameter referred to as “skew” Inline graphic. The position of δ22 with respect to the center of gravity (δiso) of the spinning CSA sideband pattern determines the sign of “skew”. Skew is zero when δ22 coincides with δiso.

Figure 3 shows the 13C 2DPASS MAS NMR spectrum of itraconazole. The direct dimension and indirect dimension of the 2D spectrum represent, respectively, the infinite spinning speed spectrum and the anisotropic spectrum. The spinning CSA sideband patterns for chemically different carbon sites are also shown in (a) C31, (b) C17, (c) C5, (d) C23, (e) C15, and (f) C27. Table 2 shows that the values of CSA parameters are varied for numerous carbon nuclei situated in different chemical environments. The values of both chemical shift anisotropy parameter (Δδ) and the spin-lattice relaxation time is shorter for carbon nuclei (C1, C2, C3, and C4) associated with the sec-butyl group in the triazolane side-chain region compared to other carbon nuclei. The spinning CSA sideband patterns of C1 and C4 are axially symmetric (η ≈ 0) and span of C1 and C4 is also very low. These data suggest that the CSA parameters of the sec-butyl (C1, C2, C3, and C4) group are greatly influenced by the side-chain conformation and dynamics. On the other hand, anisotropy parameters are very high for carbon nuclei (C35, C6, C5, and C34) situated between two heteroatoms in a five-membered 1,2,4-triazole ring due to the strong deshielding effect. Magnetic shielding and deshielding effects arise due to the existence of the nonbonded electron, which manifest as a large value of anisotropy.36 The electrons revolving along the clockwise direction can generate a magnetic field, which is along the direction of the external magnetic field (paramagnetic current). Consequently, the magnitude of the resultant magnetic field is increased—the deshielding effect. On the contrary, electrons revolving along the counterclockwise direction can generate a magnetic field along the opposite direction of the external magnetic field (diamagnetic current). Hence, the resultant magnetic field is decreased—the shielding effect. As a consequence, the values of magnetic susceptibilities Inline graphic are not the same along the three directions in the principal axis system. Moreover, there exist two components of magnetic susceptibilities—one parallel to the magnetic field Inline graphic and another perpendicular to the magnetic field Inline graphic.38 The magnetic anisotropy in terms of these parallel and perpendicular components of magnetic susceptibilities can be represented by the McConnell equation37

3.2. 5

where θ1 is the angle between the radius vector and x-axis and θ2 is the angle between the radius vector and z-axis. This anisotropic magnetic susceptibility gives rise to the direction-dependent magnetic field. Magnetic shielding/deshielding effect and electrostatic effect are the reasons behind the large value of anisotropy for carbon nuclei surrounded by nonspherical distribution of charges.

Figure 3.

Figure 3

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13C 2DPASS MAS NMR spectrum of itraconazole. The direct dimension of the 2D spectrum represents pure isotropic spectrum, and the indirect dimension represents anisotropic spectrum. (a–f) Spinning CSA sideband pattern for various carbon nuclei situated in chemically different environments.

The stereochemical orientation of the dioxolane ring (C24, C25, and C26 reside on that ring) plays a significant role in Hh-pathway inhibition and compound stability. For carbon nuclei in the dioxolane ring, the CSA parameters are not as high as those of the carbon nuclei associated with the 1, 2, 4-triazole ring.

The electrostatic interaction of a specific molecule with the surrounding molecule generates polarization on the electron density. This polarization particularly influences the strength of the induced magnetic field. As a result, the induced magnetic field is different along different directions.38 As shown in Figure 4, the CSA parameters of C28 and C30 nuclei bonded with a chlorine atom are also high because the effective magnetic field experienced by these nuclei is influenced by the polarization of the chemical bond with which those atoms are attached. The spin-lattice relaxation time is also very long for these nuclei. Even the CSA parameters are also very high for those nuclei (C27, C29, C31, and C32) that reside near the polar bonds because the neighboring polar bonds also polarize the electron cloud surrounding C27, C29, C31, and C32 nuclei. As a result, the local shielding or deshielding become direction-dependent—that means the local field experienced by nuclei may increase in a certain direction or decrease in other directions.39 The effect of the electrostatic polarization of bonds on the shielding tensor is portrayed by this measurement.

Figure 4.

Figure 4

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Magnitude of anisotropy parameter Inline graphicfor chemically different carbon sites of itraconazole.

3.3. Molecular Correlation Time

Heteronuclear dipole–dipole coupling and chemical shift anisotropy interaction play a pivotal role in the relaxation mechanism of 13C nuclei. The role of the chemical shift anisotropy in the relaxation mechanism is expressed in eq 1. The contribution of heteronuclear dipole–dipole coupling to the relaxation mechanism can be expressed as29

3.3. 6

By keeping only the first term

3.3. 7

where X represents 1H, 35Cl, and 14N. The bond distance rCX is represented in Table 4. The Larmor precession frequency (ω) = 2πf= 2 × 3.14 × 125.758 MHz = 789.76024 MHz; B = 11.74T, γC = 10.7084 MHz/T, γH = 42.577 MHz/T, γN = 3.077 MHz/T, and ℏ = 1.054 × 10–34 Js. The crystal structure of itraconazole was collected from the reported structural data.40 The hydrogen atoms were added into the structure. The final structure was optimized using the Hartre–Fock method with the 6–31G (d,p) basis set. In this process, hydrogens in the structure were only optimized by keeping all other atoms frozen as reported earlier.41 The bond distances were derived from the final optimized structure of itraconazole. The whole calculation was performed using the Gaussian 09 package.42

Table 4. Bond Distances of the Itraconazole Molecule.

bond distance(A0) bond distance(A0)
C1–H1 1.095 C16–N4 1.463
C1–H2 1.095 C13–N4 1.463
C1–H3 1.094 C13–H15 1.097
C2–H4 1.097 C13–H16 1.097
C2–H5 1.097 C14–H17 1.097
C3–H6 1.097 C14–H18 1.097
C4–H7 1.096 C15–H19 1.097
C4–H8 1.096 C15–H20 1.097
C4–H9 1.095 C16–H21 1.097
C3–N1 1.493 C16–H22 1.097
N1–N2 1.363 C14–N5 1.463
C5–H10 1.094 C15–N5 1.463
C5–N3 1.349 C17–N5 1.392
C6–N3 1.386 C18–H23 1.087
C6–O1 1.223 C19–H24 1.087
C8–H11 1.084 C21–H25 1.086
C9–H12 1.087 C22–H26 1.087
C11–H13 1.087 C20–O2 1.371
C12–H14 1.081 C23–O2 1.42
C10–N4 1.392 C23–H27 1.098
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The spin-lattice relaxation rate for 13C can be written as

3.3. 8

The relaxation mechanism of nonprotonated carbon nuclei is predominated by chemical shift anisotropy (CSA) in the presence of a high value of magnetic field.30 The molecular correlation time of the itraconazole molecule is calculated using eq 8

Table 3 shows the molecular correlation time of numerous carbon nuclei situated in different chemical environments of itraconazole. It varies in the range of 10–3–10–6 s. It is clear from Tables 1–3 that the spin-lattice relaxation rate, CSA parameters, and molecular correlation time hugely varied for the same carbon nuclei placed in different electronic surroundings and numerous molecular conformations. From Figure 5 and Table 3, it is clear that the molecular correlation time of C1, C2, C3, and C4 carbon nuclei is 2–3 orders of magnitude lesser than that of C5, C6, C34, and C35. The molecular correlation time of the carbon nuclei attached with a chlorine atom is on the order of 10–3 s for C28 and 10–4 s for C30. For side-chain carbon nuclei, the molecular correlation time is on the order of 10–6 s. The spin-lattice relaxation times of C6, C20, C23, C24, and C26 carbon nuclei linked with an oxygen atom are also significantly long. CSA parameters are high for C6 and C20 nuclei. The molecular correlation times for C6, C20, and C23 nuclei are on the order of 10–4 s and for C24, C25, and C26 nuclei on the order of 10–5 s.

Table 3. Molecular Correlation Time of Itraconazole for Various Carbon Nuclei.

carbon nuclei molecular correlation time (s) carbon nuclei molecular correlation time (s)
C1 2 × 10–6 C2 5.1 × 10–6
C3 6.6 × 10–6 C4 6 × 10–6
C5 1.3 × 10–3 C6 7.5 × 10–4
C7 6.4 × 10–4 C8 7.4 × 10–5
C9 2.1 × 10–5 C10 2.9 × 10–4
C11 2.1 × 10–5 C12 7.4 × 10–5
C14 6.6 × 10–6 C16 1.8 × 10–5
C17 1.7 × 10–4 C18 2 × 10–4
C19 7.7 × 10–5 C20 5.9 × 10–4
C21 7.7 × 10–5 C22 2 × 10–4
C23 1.6 × 10–4 C24 and C26 6.6 × 10–5
C25 1.9 × 10–5 C27 6.9 × 10–4
C28 1.4 × 10–3 C29 1.7 × 10–4
C30 7.1 × 10–4 C31 1.7 × 10–4
C32 1.7 × 10–4 C33 6.6 × 10–6
C34 3.5 × 10–4 C35 3 × 10–4
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Perhaps, these substantial variations of CSA parameters and degrees of motion at different regions of this molecule are responsible for its different biological activities like antifungal and anticancer activities, as well as interesting pharmacokinetic behavior like strong protein binding, tissue penetration, and prolonged half-life and bioavailability. In essence, the influences of the local environment on the structure and dynamics of the itraconazole molecule are vividly portrayed by this type of investigation.

4. Conclusions

Extraction of CSA parameters by the 13C 2DPASS MAS NMR experiment, determination of spin-lattice relaxation time by the Torchia CP method, and calculation of molecular correlation time at 35 crystallographically and chemically different sites of carbon nuclei of itraconazole provide the information about the correlation between the structure and dynamics of this valuable antifungal drug. Substantial difference in the spin-lattice relaxation time (shown in Figure 2e), CSA parameters (as shown in Table 2 and Figure 4), and molecular correlation time (as shown in Table 3 and Figure 5b) is observed for different structural parts of this molecule. The spin-lattice relaxation time is long for carbon nuclei (C35, C6, C5, and C34) that participated in the 1, 2, 4-triazole ring. On the contrary, the spin lattice relaxation time is comparatively short for C1, C2, C3, and C4 carbon nuclei that reside at the side-chain region. CSA parameters of C5, C6, C34, and C35 nuclei are much higher than those of C1, C2, C3, and C4 nuclei, indicating that the relaxation mechanism at a high value of magnetic field is predominated by chemical shift anisotropy interaction. The molecular correlation time of C1, C2, C3, and C4 regions is 2–3 orders of magnitude lesser than that of C5, C6, C34, and C35. The spin-lattice relaxation time is very long for carbon nuclei C28 and C30 bonded with chlorine. Asymmetry and anisotropy parameters are also very high of the spinning CSA sideband pattern corresponding to the C28 and C30 nuclei. The molecular correlation time is on the order of 10–3 s for C28 and 10–4 s for C30, whereas for the sec-butyl group carbon nuclei, the molecular correlation time is on the order of 10–6 s. It may be possible that a molecule with different degrees of motions in its structure, like itraconazole, is capable of producing many biological activities by interacting with the enzyme/proteins of different structures and dynamics. This type of investigation will elucidate the way of inventing advanced medicine with multiple biological activities in the pharmaceutical industry.

Acknowledgments

Manasi Ghosh is grateful to the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), government of India (File no. EMR/2016/000249), and UGC-BSR (File no. 30-12/2014 (BSR)) for financial support.

The authors declare no competing financial interest.

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