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. 2020 Nov 4;5(45):29373–29381. doi: 10.1021/acsomega.0c04205

Investigation of the Structure and Dynamics of Antiviral Drug Adefovir Dipivoxil by Site-Specific Spin–Lattice Relaxation Time Measurements and Chemical Shift Anisotropy Tensor Measurements

Krishna Kishor Dey †, Manasi Ghosh ‡,*
PMCID: PMC7676337  PMID: 33225168

Abstract

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Adefovir is regarded as a potential antiviral agent. However, it cannot be considered as a valuable drug candidate due to its high polarity that limits its permeability across the human intestinal mucosa. When the ribose phosphate group of adefovir is replaced by the isopolar phosphonomethyl ether functionality, it neutralizes the negative charge of the drug. This makes the drug lipid-soluble and potent to diffuse across the cell membrane. The prodrug adefovir dipivoxil is regarded as a potent antiviral drug against hepatitis B virus (HBV), human immunodeficiency virus (HIV), Rauscher murine leukemia virus (R-MuLV), murine cytomegalovirus (MCMV), herpes simplex virus (HSV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). The correlation between the structure and the dynamics of adefovir dipivoxil is determined by measuring the principal components of chemical shift anisotropy (CSA) tensor, site-specific spin–lattice relaxation time, and molecular correlation time at crystallographically different carbon nuclei sites. The CSA parameters, spin–lattice relaxation time, and molecular correlation time of phosphorous nucleus of the organophosphate group of adefovir dipivoxil molecule are also determined. The spin–lattice relaxation time of carbon nuclei varies from 1 to 107 s. The range of molecular correlation time also varies from 10–4 to 10–8 s. These remarkable diversities of motional dynamics of the molecules imply that there exist various motional degrees of freedom within this valuable drug and these motional degrees of freedom are independent of each other, which may be the reason for the biological activities exhibited by the drug. The correlation between structure and dynamics of such an important antiviral drug adefovir dipivoxil can be visualized by these types of extensive spectroscopic measurements, which will enlighten the path of inventing advanced medicine in the pharmaceutical industry, and it will also illuminate the understanding of the structure–activity relationships of antiviral drug.

1. Introduction

Adefovir dipivoxil is regarded as a stable protected monophosphate nucleoside prodrug that can efficiently cross the biological barrier and reach the targeted cell. It is fabricated by replacing the ribose phosphate group of adefovir by the isopolar phosphonomethyl ether functionality. The acyclic nucleoside phosphonate (ANP) compounds constructed to overcome the first phosphorylation step, which is indispensable for the foundation of the nucleoside analogues such as acyclovir (ACV) or ganciclovir (GCV). The activation of human immunodeficiency virus (HIV) inhibitors zidovudine (AZT), stavudine (D4T), zalcitabine (DDC), didanosine (DDI), and lamivudine (3TC) depends on cellular enzymes. Within the liver cell, adefovir dipivoxil is bisected into adefovir by intracellular esterases. It is phosphorylated by adenylate kinases and then transformed into adefovir diphosphate by nucleoside diphosphate. Adefovir diphosphate is an analogue of deoxyadenosine triphosphate (dATP) and competitive inhibitors of the viral DNA polymerases. It binds with the viral DNA polymerase enzymes of hepatitis, herpes viruses, retroviruses (including HIV), Epstein–Barr virus, human immunodeficiency virus, cytomegalovirus, and other DNA viruses. It assimilates into the growing DNA strand. In this way, it causes chain termination and blocks the replication of viral DNA.1−4

Adefovir dipivoxil is composed of 9-{2-[bis(pivaloyloxymethoxy)phosphinylmethoxy]ethyl}-adenine and falls in the class of acyclic nucleoside phosphonates.5,6,54 It degrades via two pathways: hydrolysis of the pivaloyloxymethyl moiety and formaldehyde-catalyzed dimerization of the adenine ring. The pharmacokinetic of adefovir is altered substantially with moderate and severe renal impairment.8 Adefovir dipivoxil structure is associated with 13 hydrogen bond acceptors and 2 hydrogen bond donors, which makes the structure favorable for cocrystal formation.5,7,8 Adefovir dipivoxil dehydrate is formed by intermolecular interactions via N–H···N, O–H···O, and O–H···N hydrogen bondings. Two adefovir dipivoxil molecules connect by N–H···N hydrogen bonding between adenine rings to form a dimer synthon. Two hetero synthons are also fabricated by O–H···O and O–H···N hydrogen bonding between adefovir dipivoxil and water molecules. Due to the formation of the synthons, adefovir dipivoxil molecules form cocrystal with saccharin in association with hydrogen bond donors and acceptors.9−12,56 The key focus of the present work is to determine the correlation between the structure and dynamics of adefovir dipivoxil by extracting chemical shift anisotropy (CSA) parameters, site-specific spin–lattice relaxation time, and molecular correlation time at crystallographically different carbon sites and phosphorous site of organophosphate group of the molecule. CSA tensor has a huge impact on determining the molecular conformation and electronic distribution surrounding the nucleus. Relaxometry measurements can explore the dynamics of a molecule at an atomic scale. The principal components of the CSA tensor can be also determined by two-dimensional magic-angle spinning (MAS)/CSA NMR experiment;13 separation of undistorted powder patterns by effortless recoupling (SUPER);14 recoupling of chemical shift anisotropy (ROCSA);15 γ-encoded RNnν-symmetry-based chemical shift anisotropy (RNCSA);16 two-dimensional magic-angle flipping (2DMAF) experiment;17−19 two-dimensional magic-angle turning (2DMAT) experiment;20 and 2DPASS CP-MAS (two-dimensional phase-adjusted spinning sideband cross-polarization magic-angle spinning) SSNMR experiment.21,22 Structure and dynamics of biopolymer, drugs molecules, and glass compounds are determined by CSA and site-specific relaxation measurements.24−3113C-CSA parameters of adefovir dipivoxil are calculated by 2DPASS CP-MAS SSNMR experiment. 31P CSA parameters are extracted by the deconvolution of 31P MAS NMR spectrum at the MAS speed of 6 kHz by dmfit.42 Site-specific spin–lattice relaxation time of the carbon-13 nucleus is calculated by the Torchia CP method.23 The spin–lattice relaxation time of phosphorous nucleus is determined by the inversion recovery method. Molecular correlation time of chemically different carbon nuclei sites and phosphorous nucleus site is calculated by considering that the spin–lattice relaxation mechanism is mainly dominated by chemical shift anisotropy interaction and dipole–dipole interactions for spin 1/2 nucleus. These types of in-depth measurements will illuminate the path of inventing advanced antiviral drugs and establishing the structure–activity relationship of drug molecules.

2. Experimental Section

2.1. NMR Measurements

The active pharmaceutical ingredient of adefovir dipivoxil was purchased from Sigma-Aldrich. 13C CP-MAS SSNMR and 31P MAS SSNMR experiments were performed on a JEOL ECX 500 NMR spectrometer with a 3.2 mm JEOL double-resonance MAS probe. The MAS speed for the 13C CP-MAS NMR experiment was 10 kHz. 31P MAS NMR experiments were performed at the MAS speeds of 3, 5, 6, 10, and 13 kHz. The contact time for cross-polarization (CP) was 2 ms, with a repetition interval of 30 s, and SPINAL-64 1H decoupling at 3072 accumulation time. 13C-spin–lattice relaxation experiment was performed using the Torchia CP method23 with a contact time of 2 ms and a relaxation time of 10 s. The spin–lattice relaxation time of the phosphorous nucleus was measured by the inversion recovery method. All solid-state experiments were performed at room temperature.

2.2. CSA Measurements

The information regarding the three-dimensional structure of a molecule and spin dynamics is encoded in anisotropic interactions. The pulse sequences for the 2DPASS CP-MAS SSNMR21,22 experiment is associated with five π pulses of the variable pitch but constant duration. It generates spectra with sidebands of different phases. The time interval among five π pulses is chosen by following the PASS equations. The spacing among the π pulses was reported by Antzutkin et al.21 The 90° pulse length for the 13C nucleus was 3.3 μs. The relaxation delay was 10 s. The number of scans for the 2DPASS CP-MAS NMR experiment was 4030 (integral multiple of 13). The coherence transfer pathway for the 2DPASS CP-MAS NMR experiment is given by Ghosh et al.24 Thirteen steps cogwheel phase cycling was used. As the number of sidebands was less than 16, 16 data points were acquired in the indirect dimension. The anisotropic part of the chemical shift interaction evolves during the PASS sequence. The variable of this evolution is called “pitch,” which varies from 0 to 2π. The principal values of the chemical shift anisotropy tensor can be extracted by evaluating the sideband intensity for few sidebands by the graphical method.3313C 2DPASS CP-MAS SSNMR experiments were performed at MAS frequencies of 600 Hz and 2 kHz.

3. Results and Discussion

3.1. Determination of CSA Parameters

Figure 1a shows that adefovir dipivoxil is fabricated by adenine ring, organophosphate group, and two pivaloyloxymethyl groups. Through a phosphorous–carbon–oxygen bond, the adenine ring is attached to the organophosphate group, which enables the drug to achieve higher degrees of the active metabolites in the cell and to possess antiviral activity against most of the DNA viruses like hepadnaviruses, retroviruses, and herpes viruses. Nucleotides of the nucleic acids are formed by two purine nucleobases adenine and guanine. Adenine binds with thymine of DNA via two hydrogen bonds to bring stability to the structure of nucleic acid. Pivaloyloxymethyl group is a protecting group used to synthesize the prodrugs. The adenine ring exhibits planar conformation. Two pivaloyloxymethyl groups are attached to a negatively charged organophosphate group to neutralize the negative charge of the drug. This is essential to make the drug lipid-soluble and potent to diffusion across the cell membranes into the cell. Hence, two pivaloyloxymethyl ester groups enhanced the bioavailability of the drug. The organophosphate group is a key functional group of DNA, RNA, and ATP. It helps to recognize the viral kinases for phosphorylation and bind with the viral DNA polymerase.57−59

Figure 1.

Figure 1

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(a) Adefovir dipivoxil is composed of an adenine ring, an organophosphate group, and two pivaloyloxymethyl groups. (b) 13C CP-MAS NMR spectrum of adefovir dipivoxil.

Figure 1b represents the 13C CP-MAS SSNMR spectrum of adefovir dipivoxil. The resonance peak position is assigned by following Swain et al.34−36 The isotropic chemical shift of C18 and C24 carbon nuclei of two pivaloyloxymethyl groups is maximum. The electronegative oxygen atoms at the immediate neighborhood of C18 and C24 attract the electron cloud around the carbon nucleus. As a consequence, the nuclear shielding effect is decreased and the effective magnetic field experienced by these nuclei is increased. The Larmor precession frequency corresponding to these nuclei is shifted toward the higher-frequency side. The isotropic chemical shift of carbon nuclei C2, C4, C5, C9, and C7 residing on the adenine ring is substantially high. The isotropic chemical shift is lowest for the methyl group carbon nuclei C28, C30, C31, C32, C33, and C34 residing on the pivaloyloxymethyl group.

Figure 2 shows the 13C 2DPASS CP-MAS SSNMR spectrum of adefovir dipivoxil. The isotropic chemical shift is correlated with the anisotropic chemical shift by this measurement. The direct dimension shows the pure isotropic spectrum, which is also referred to as the infinite spinning speed spectrum. The indirect dimension represents the anisotropic spectrum. Figure 3 shows the spinning CSA sideband pattern at crystallographically different carbon sites of adefovir dipivoxil.

Figure 2.

Figure 2

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13C 2DPASS CP-MAS SSNMR spectrum of adefovir dipivoxil. The direct dimension shows the isotropic spectrum, which is also referred to as an infinite spinning speed spectrum. The indirect dimension shows the anisotropic spectrum.

Figure 3.

Figure 3

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Spinning CSA sideband pattern at crystallographically different carbon nuclei sites of adefovir dipivoxil. For C18, C24, C4, C7, C5, C2, and C9, the CSA pattern is determined by the 2DPASS CP-MAS SSNMR experiment at the MAS frequency of 2 kHz, and for the rest of the carbon nuclei, the spinning CSA sideband pattern is determined by performing the same experiment at the MAS frequency of 600 Hz.

In solid-state NMR spectroscopy, chemical shift interaction, magnetic dipole–dipole interaction, and electrical quadruple interaction are taken into consideration. Each of these interactions has an isotropic and an anisotropic part. The Larmor precession frequency of the nucleus depends on the orientation of the molecular moiety with respect to the direction of the applied magnetic field and the electronic distribution surrounding the nucleus. The shift of the Larmor precession frequency compared to the bare nucleus can be expressed as ω(θ,φ) – ω0 = ωiso + (Δδ/2)(3 cos2 θ – 1 – η sin2 θ cos 2φ), where Δδ is the anisotropy parameter, which measures the deviation of the electron cloud from the spherically symmetric charge distribution, and η is the asymmetry parameter, which measures the deviation from the axially symmetric distribution. θ and φ are the polar and the azimuthal angles with respect to the direction of the applied magnetic field (B0) in the principal axis system (PAS).43 The chemical shift arises due to the interaction of the electron cloud surrounding the nucleus with the applied magnetic field. The effective magnetic field experienced by the nucleus is Beff = (1 ± δ)B0. The dimensionless quantity δ is a second-rank tensor, associated with nine components.40−49 It is called the chemical shift anisotropy (CSA) tensor. It is diagonalized in the principal axis system (PAS), and the expressions of the principal components of CSA tensor are32,33

3.1. 1
3.1. 2
3.1. 3

where Lx, Ly, and Lz are the components of the angular momentum along the x-, y-, and z-axes, respectively. The first part of these three equations arises from the spherical distribution of the electronic changes when the electrons are in the s-orbital state. The second term arises from the distortion of spherical charge distribution when electrons are in the p-orbital state. In liquid-state NMR spectroscopy, due to the tumbling motion of the molecule, the orientation-dependent term of the CSA tensor is averaged out and only the isotropic component of the chemical shift survives.53 The resonance frequency of numerous carbon nuclei differs according to the orientation of the molecular moiety with respect to the external magnetic field. The largest chemical shift (deshielding effect) in the resonance frequency (δ11) occurs when the narrowest part of the electron distribution is oriented along the direction of the external magnetic field (B0), whereas the smallest chemical shift (nuclear shielding effect) in the resonance frequency (δ33) occurs when the widest part of the electron cloud is oriented along the direction of the external magnetic field. The third principal value (δ22) of the CSA tensor arises when the orientation of the molecular moiety is perpendicular to the axes of δ11 and δ33.50−52 The left and right edges of the spinning CSA sideband pattern corresponds to the largest (δ11) and smallest (δ33) chemical shifts, respectively, whereas δ22 corresponds to the position of the maximum intensity of the CSA sideband pattern when δ11 ≥ δ22 ≥ δ33.

Table 1 shows the principal components of the CSA tensor at crystallographically different carbon nuclei sites of adefovir dipivoxil. The CSA parameters of C18 and C24 residing at the pivaloyloxymethyl group are substantially high. As the carbonyl group carbon has no directional symmetry, there arises magnetic anisotropy δanis= {ΔX∥ (3 cos2 θ1 – 1) + ΔX⊥(3 cos2 θ2 – 1)}/3R3, where θ1 and θ2 are the angles subtended by the radius vector with x-axis and z-axis, respectively, and ΔX∥ = Xz – Xx and ΔX⊥ = Xy – Xx are the components of susceptibility parallel and perpendicular to the applied magnetic field, respectively.37 The higher degree of directional specificity due to the presence of magnetic anisotropy leads to the higher values of CSA parameters for C18 and C24 nuclei. The CSA parameters of C2, C4, C5, C7, and C9 residing on the adenine ring are largely due to the presence of π electrons. This induced polarization in the neighborhood region of the carbon nuclei and there induced magnetic anisotropy.

Table 1. Principal Components of the CSA Tensor of Adefovir Dipivoxil at Crystallographically Different Carbon Sites.

CSA parameters of adefovir dipivoxil
carbon nuclei δ11 (ppm) δ22 (ppm) δ33 (ppm) span (ppm) Ω = δ11 – δ33 skew Inline graphic δiso (ppm) anisotropy (ppm) Inline graphic asymmetry Inline graphic
C18 225 225 82.2 142.8 1 177.4 –142.8 0
C24 227.6 225.5 75.4 152.2 1 176.2 –151.1 0.02
C4 252.3 109.5 109.5 142.8 –1 157.1 142.8 0
C7 239.8 176.1 43.6 196.2 0.3 153.2 –164.3 0.6
C5 241 132.4 81 159.9 –0.3 151.5 134.2 0.6
C2 206.1 142 74.9 131.2 0.02 141 –99.2 1
C9 188.4 87.2 79.2 109.2 –0.8 118.3 105.2 0.1
C16 102.9 86.1 60.3 42.6 0.2 83.1 –34.2 0.7
C22 105.6 85.1 56.4 49.2 0.2 82.4 –38.9 0.8
C11 112.8 60.3 48.5 64.3 –0.6 73.8 58.4 0.3
C13 111.4 54.9 40.5 70.9 –0.6 69 63.7 0.3
C10 110 50.8 41.7 68.3 –0.7 67.5 63.7 0.2
C27 60.9 42.6 22.8 38 0.04 42.1 –28.9 0.9
C29 53.3 32.6 30.1 23.2 –0.8 38.7 22 0.2
C28, C30, C31, C32, C33, C34 34.4 30.8 16.4 18 0.6 27.2 –16.2 0.3
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The intermolecular and intramolecular hydrogen bonds O–H···N, O–H···O, and O–H···N are associated with the adenine ring. The adenine ring exhibits a planar conformation because of the orbitals of the nitrogen atoms connected with hydrogen bonds in sp2 hybridization. Two adjacent molecules of adefovir dipivoxil are connected via intermolecular hydrogen bonding through adenine rings. These intermolecular hydrogen bonds reduce the polarity of the molecule and bring stability to the crystal structure. The existence of these hydrogen bonds is another reason for the higher values of the CSA parameters of the adenine ring.

The isotropic chemical shift (δiso = (δ11 + δ22 + δ33)/3) represents the center of gravity of the spinning CSA sideband pattern. The magnitude of the anisotropy parameter (Δδ = δ33 – (δ11 + δ22)/2) indicates the largest separation of the spinning CSA sideband pattern from the center of gravity. The sign of the anisotropy parameters signifies on which side of the center of gravity, the separation is the maximum. Figure 5 shows the variation of asymmetry and anisotropy parameters at numerous carbon sites of adefovir dipivoxil.

Figure 5.

Figure 5

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Bar diagram of (a) asymmetry (η = (δ22 – δ11)/(δ33 – δiso)) and (b) anisotropy (Δδ = δ33 – (δ11 + δ22)/2) parameters at crystallographically different carbon nuclei sites of adefovir dipivoxil.

The asymmetry parameter is defined as η = (δ22 – δ11)/(δ33 – δiso). If δ22 = δ11 or δ22 = δ33, then the spinning CSA sideband pattern is axially symmetric. Table 1 and Figure 5a indicate that the spinning CSA sideband pattern is axially symmetric for C18, C24, and C4. The CSA pattern is nearly axially symmetric for C9, C10, C11, C13, C28, C29, C30, C31, C32, C33, and C34 carbon nuclei when η ≤ 0.3. The CSA pattern is highly asymmetric for C2, C22, and C27 when the asymmetry parameter η ≥ 0.8. Skew Inline graphic represents the orientation of the asymmetric pattern.

Figure 4 shows the 31P MAS SSNMR spectrum of adefovir dipivoxil at the MAS speed of 6 kHz. The line shape is a CSA powder pattern originating from the orientation dependence of the chemical shift, described by the expression δ(α,β)= δ11 sin2 β cos2 α + δ22 sin2 β sin2 α + δ33 cos2 β, where α and β are the Euler angles, and δ11, δ22, and δ33 are the principal components of the CSA tensor in PAS.54 The CSA parameters are determined using dmfit.42 The isotropic chemical shift of phosphorous is 26.69 ppm. The values of asymmetry and anisotropy parameters are −115 ppm and 0.12, respectively. The spinning CSA sideband pattern of phosphorous nucleus residing on the organophosphate group is nearly axially symmetric. The organophosphate functionality allows the drug to penetrate the cell membrane. Within the cell, the bioconversion of adefovir dipivoxil is performed by esterases, and it is transformed into adefovir. Adefovir is then phosphorylated to adefovir monophosphate by various kinases, one of which in lymphoid cells is identified as adenylase kinase. The adefovir monophosphate is then transformed to adefovir diphosphate by second phosphorylation.54−56 Adefivir diphosphate is an aggressive inhibitor of hepatitis B virus (HBV) DNA polymerase (reverse transcriptase) in addition to other viral DNA polymerases. This inhibition results in DNA chain termination and the destruction of viral replication. These make adefovir dipivoxil a highly efficient drug in the treatment of human hepatitis B virus (HBV) infection (Figure 5).

Figure 4.

Figure 4

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31P MAS NMR spectrum of adefovir dipivoxil at the MAS speed of 6 kHz. The red line represents the fitted spectrum.

3.2. Spin–Lattice Relaxation Time and Molecular Correlation Time

The time taken by the nuclear spin system to attain a thermal equilibrium by exchanging energy with the “lattice” is known as the spin–lattice relaxation time. As the lattice is associated with various kinds of degrees of freedom, the spin–lattice relaxation time can probe the entire spectrum of molecular motions. The site-specific spin–lattice relaxation time can provide the detailed features of the nuclear spin dynamics at each chemically distinct nuclei site. Several nuclear interactions like the magnetic dipole–dipole interactions, the electric quadrupole interaction, the spin–rotation interaction, the scalar coupling of the first and second kind, and the chemical shift anisotropy interaction, may simultaneously contribute to the relaxation of the spin system. However, the contribution of the spin–rotation interaction and scalar coupling is insignificant for large molecules. Hence, for spin 1/2 carbon and phosphorous nucleus, the spin–lattice relaxation mechanism is mainly governed by the chemical shift anisotropy interaction and the dipole–dipole coupling interaction. The contribution of chemical shift anisotropy interaction to the spin–lattice relaxation mechanism is expressed as38−41

3.2. 4

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

The role of dipole–dipole interaction in the spin–lattice relaxation mechanism is expressed as41

3.2. 5

By keeping only the first term

3.2. 6

where X represents hydrogen, oxygen, and nitrogen, phosphorous atoms. rCX is the distance between carbon and neighboring atoms hydrogen, oxygen, and fluorine, which is determined by X-ray crystal structural studies.5 Larmor precession frequency of carbon nucleus is ωc = 2πf = 2 × 3.14 × 125.758 MHz = 789.76024 MHz. The Larmor precession frequency of phosphorous nucleus is ωP = 2πf = 2 × 3.14 × 202.457 MHz = 1271.42996 MHz, B = 11.74 T, γC = 10.7084 MHz/T, γH = 42.577 MHz/T, γP = 17.235 MHz/T, and ℏ = 1.054 × 10–34 Js.

The spin–lattice relaxation rate for 13C can be articulated as

3.2. 7

The molecular correlation time is calculated by this equation. Figure 7 represents the bar diagram of the molecular correlation time at crystallographically different carbon sites of adefovir dipivoxil.

Figure 7.

Figure 7

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Molecular correlation time at crystallographically different carbon nuclei sites of adefovir dipivoxil. It varies in the range of 10–4 to 10–8 s.

Table 2 shows the spin–lattice relaxation time and the molecular correlation time of adefovir dipivoxil at crystallographically different sites of carbon nuclei and one phosphorous nucleus. Figure 6 shows the longitudinal magnetization decay curves at (a) C4, (b) C5, and (c) C7 carbon nuclei sites of adefovir dipivoxil. The spin–lattice relaxation time of carbon nuclei residing on the adenine ring is comparatively longer than those residing on the pivaloyloxymethyl ester group. The spin–lattice relaxation time at chemically different carbon nuclei sites varies from 1 to 107 s. Figure 6e shows the bar diagram of the spin–lattice relaxation time at crystallographically different carbon nuclei sites. The spin–lattice relaxation time is maximum at the C5 nuclei site on the adenine ring, and it is minimum for C28, C30, C31, C32, C33, and C34 methyl group carbon nuclei residing on the pivaloyloxymethyl group. The molecular correlation time varies in the range of 10–4 to 10–8 s. For C5, it is 1.7 × 10–4 s and for C28, C30, C31, C32, C33, and C34 methyl group carbon nuclei, it is 2.1 × 10–8 s. Remarkable variation in the spin–lattice relaxation time and the molecular correlation time at different functional groups of the molecule implies that various motional degrees of freedom exist within the molecule and different motional degrees of freedom are independent of each other.

Table 2. Spin–Lattice Relaxation Time and Molecular Correlation Time at Crystallographically Different Carbon Nuclei Sites and Phosphorous Site of Adefovir Dipivoxil.

carbon atom spin–lattice relaxation time(s) molecular correlation time(s)
C18 20 ± 2 3.3 × 10–5
C24 15 ± 2 2.7 × 10–5
C4 85 ± 5 1.4 × 10–4
C7 55 ± 3 1.3 × 10–4
C5 107 ± 5 1.7 × 10–4
C2 62 ± 2 6.5 × 10–5
C9 55 ± 3 4.9 × 10–5
C16 3 ± 0.5 3.3 × 10–7
C22 3 ± 0.5 4.4 × 10–7
C11 42 ± 2 1.2 × 10–5
C13 7 ± 0.5 2.4 × 10–6
C10 7 ± 0.5 2.3 × 10–6
C27 70 ± 3 6.1 × 10–6
C29 12 ± 2 4.6 × 10–7
C28, C30, C31, C32, C33, C34 1 ± 0.5 2.1 × 10–8
P14 15 ± 2 4.1 × 10–5
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Figure 6.

Figure 6

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Longitudinal magnetization decay curves at (a) C4, (b) C5, and (c) C7 carbon nuclei sites of adefovir dipivoxil. (d) Longitudinal magnetization decay curve of the phosphorous nucleus of organophosphate group of adefovir dipivoxil. 13P relaxation time is measured by the inversion recovery method. (e) Bar diagram of the spin–lattice relaxation time at crystallographically different carbon nuclei sites of adefovir dipivoxil.

Within the adenine ring, the molecular correlation times of C2 and C9 nuclei are 1 order faster (10–5 s) than those of the C4, C5, and C7 nuclei (10–4 s). The molecular correlation time of phosphorous nuclei of the organophosphate group is 4.1 × 10–5 s. The pivaloyloxymethyl ester group is responsible for the improved bioavailability of the drug, and there exist various motional degrees of freedom within this molecular moiety. For C18 and C24 carbon double bonded with the oxygen atom, the molecular correlation time is 3.3 × 10–5 and 2.7 × 10–5 s, respectively, but for C16 and C22, the molecular correlation times are 3.3 × 10–7 and 4.4 × 10–7 s, respectively. For methyl group carbon nuclei C28, C30, C31, C32, C33, and C34, the molecular correlation time is 2.1 × 10–8 s and for C27 and C29 it is 6.1 × 10–6 and 4.6 × 10–7 s, respectively. Hence, within this pivaloyloxymethyl group, the molecular correlation time varies from 2.7 × 10–5 to 2.1 × 10–8 s. As mentioned before that the pivaloyloxymethyl group plays a crucial role in neutralizing the organophosphate group and make the drug lipid-soluble and potent to diffuse across the cell membranes. The existence of different degrees of motional freedom within this molecular moiety may be the reason for the biological activity exhibited by this group. The CSA parameters (which reflect the molecular conformation and electronic distribution surrounding the nucleus) of C18 and C24 nuclei are substantially higher than those of other carbon nuclei residing on the pivaloyloxymethyl groups like C16, C22, C27, and C29 nuclei and methyl group carbon nuclei C28, C30, C31, C32, C33, and C34. This implies that various molecular dynamics observed in the pivaloyloxymethyl group are highly correlated with the electronic bonding and molecular conformation (Figure 7).

4. Conclusions

Adefovir dipivoxil is a prodrug of the antiviral agent, adefovir (PMEA). The degradation kinetics of adefovir dipivoxil is administered by two distinct but inter-related degradation pathway hydrolysis of the pivaloyloxymethyl moiety and formaldehyde-catalyzed dimerization of the adenine ring. Formaldehyde is a reactive and cross-linking agent for nucleic acids. Adefovir dipivoxil shows antiviral activity when administered intravenously, intraperitoneally, or intramuscularly against the hepatitis B virus (HBV), human immunodeficiency virus (HIV), Rauscher murine leukemia virus (R-MuLV), murine cytomegalovirus (MCMV), herpes simplex virus (HSV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). The spinning CSA sideband pattern of phosphorous nucleus residing on the organophosphate group, which allows the drug to penetrate the cell membrane, is nearly axially symmetric. The pivaloyloxymethyl group plays a crucial role in neutralizing the organophosphate group and making the drug lipid-soluble. The existence of two pivaloyloxymethyl ester groups is responsible for the improved bioavailability of the drug. The CSA parameters of the carbonyl groups C18 and C24 residing on the pivaloyloxymethyl group are substantially high. As there is no symmetry axis of carbonyl group carbon, the presence of magnetic anisotropy leads to the higher values of CSA parameters for C18 and C24 nuclei. On the other hand, the CSA parameters of the methyl group carbons C28, C30, C31, C32, C33, and C34 residing on the pivaloyloxymethyl group are the lowest. The remarkable variation of the CSA parameters implies a significant difference in the electronic environment and molecular conformation within this molecular moiety. The anisotropy Inline graphic and span (Ω = δ11 – δ33) of C18 and C24 nuclei are substantially higher than those of the C16, C22, C27, and C29 nuclei and methyl group carbon nuclei C28, C30, C31, C32, C33, and C34. The motional degrees of freedom are also different at various portions of the pivaloyloxymethyl group. The molecular correlation time is of the order 10–5 s for C18 and C24; 10–7 s for C16 and C22; and 2.1 × 10–8 s for the methyl group carbon nuclei C28, C30, C31, C32, C33, and C34. The molecular correlation time is 6.1 × 10–6 and 4.6 × 10–7 s for C27 and C29, respectively. This huge variation of the electronic structure and the motional degrees of freedom within this molecular moiety may be the reason for the biological activity exhibited by this group.

The CSA parameters of the carbon nuclei residing on the adenine rings C2, C4, C5, C7, and C9 are substantially high due to the presence of π electrons and hydrogen bonding. Adenine derivatives possess antiviral activity against most of the double-stranded DNA viruses. The adefovir dipivoxil molecules form a chain structure via intermolecular hydrogen bonding through adenine rings. These intermolecular hydrogen bonds reduce the polarity of the molecule and bring stability to the crystal structure. The molecular correlation time of the carbon nuclei of these adenine rings varies in the range of 1.3 × 10–4–6.5 × 10–5 s. The motional degrees of freedom are highly correlated with electronic bonding and molecular conformation. These types of investigations about the structure and dynamics of this valuable antiviral drug will help to illuminate the path of inventing the advanced antiviral drug and to understand the structure–activity relationship of antivirus drugs. These types of studies are also useful in NMR crystallography.

Acknowledgments

The author 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) for financial support. The authors are thankful to SIC, Dr. Harisingh Gour Central University for providing solid state NMR facility.

The authors declare no competing financial interest.

References

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