A Solid-State NMR Study of the Antibacterial Drug Cefpodoxime Proxetil


 The structure-activity relationship of various molecular moieties of cefpodoxime proxetil is described by measuring Chemical shift anisotropy (CSA) tensor, spin-lattice relaxation time and molecular correlation time at twenty one crystallographically different carbon nuclei sites. Cefpodoxime proxetil molecule is associated with three rings, the aminothiazole ring, β-lactam ring, and the dihydrothiazine ring, which provide stability to the drug molecule towards β-lactamases and increase the affinity of the drug to PBPs receptors. A large variation of CSA parameters and motional degrees of freedom are observed among carbon nuclei reside on these three rings, which implies that the electronic environment, molecular conformation, and molecular dynamics are altered substantially within the ring. The substitution at the C7 position of the β-lactam ring (like acyl side chain, oxime group, and aminothiazole ring) is responsible for semi-synthetic incorporation. It also influences the antibacterial activity and the binding affinity of the drug with β-lactamase. A huge variation of the spin-lattice relaxation time and molecular correlation time is observed in this region. These types of description of the structure-activity relationship of the antibacterial drug cefpodoxime proxetil will be beneficial for developing the advanced antibacterial drugs, and it will also be useful in NMR crystallography.


Introduction:
The -lactam antibiotics exhibit antibacterial activity by inhibiting the cell membrane synthesis in microorganisms. The basic action method of the -lactam antibiotics is to interrupt the transpeptidation process that combines the individual peptidoglycan components of the bacterial cell wall. 1 These types of antibiotics are bind with the inner surface of the bacterial cell membrane, the penicillin-binding proteins (PBPs). The PBPs enzymes are necessary to assemble the bacterial cell wall and to reshape the cell wall during the growth and the division of the bacterial cell. The biochemical modification of the basic structure of the -lactam (a four-membered -lactam ring fused to a second ring) is incorporated to increase bacterial cell wall penetration and binding to PBPs. In penicillins (the -lactam antibiotics), the -lactam ring is fused to a five-membered thiazolidine ring, whereas in cephalosporins, the -lactam ring is attached to a six-membered dihydrothiazine ring. The cephalosporin is more active against -lactamase-producing bacteria compared to penicillin. 1 It also offer more sites for biochemical modifications. Therapeutically important modifications have been made at the acyl group attached to the -lactam ring and the moiety attached with the dihydrothiazine ring. Modifications in the acyl side chain change the antibacterial activity, while modifications of the dihydrothiazine ring side chain alter the pharmacokinetics and metabolic parameters of the antibiotics. Cefpodoxime proxetil is a third-generation cephalosporin antibacterial drug. It is used for the treatment of infections due to Gram-positive and Gram-negative bacteria. It showed great efficacy to treat skin and soft tissue infections, acute otitis media, pharyngitis, bronchitis, pneumonia, respiratory and urinary tracts infection, and primarily gastrointestinal tract disturbances of pediatric patients. 2 The current work is targeted to determine the structure-activity relationship of cefpodoxime proxetil by solid sate NMR spectroscopic measurements. High-resolution solid state NMR spectroscopy is an indispensable tool to determine the information about local electronic structure and symmetries encoded in chemical shift anisotropy (CSA) interactions. The detailed structure and molecular dynamics at various molecular moieties of cefpodoxime proxetil is portrayed by measuring principal components of CSA tensor at carystallographically different carbon nuclei sites by two-dimensional phase adjusted spinning sideband (2DPASS) cross-polarization (CP) magic angle spinning (MAS) solid-state NMR experiment. 32,33 The dynamics at crystallographically different carbon nuclei sites are determined by site-specific spin-lattice relaxation time measurements by the Torchia CP method. 47 The molecular correlation time at various carbon nuclei sites is also calculated.  Where , , denote the Cartesian coordinates. The ψ are the electronic wavefunctions; ψ 0 and ψ k are the ground state and the excited state electronic wavefunction respectively. , are the coordinates of the th electron along the and directions respectively. ̂,̂ are the components of the electronic orbital angular momentum operator along the and directions respectively. , 0 are the energy of the electron in the excited state and the ground state respectively. 19 The detailed picture of the correlation between the structure and dynamics of charge transferred cocrystal, biopolymer, drug molecules, and glass, were given by 2DPASS CP-MAS SSNMR experiment and Torchia CP experiment. [34][35][36][37][38][39][40][41][42][43][44][45][46]57,58 The structure-activity relationship of cefpodoxime proxetil are investigated by 2DPASS CP-MAS SSNMR experiment 32,33 and Torchia-CP experiment, 47 which will enlighten the path of developing the advanced antibacterial drug, and will be useful in NMR crystallography.

NMR Measurements
Active pharmaceutical ingredient of cefpodoxime proxetil was purchased from Sigma Aldrich. 13  temperature. The referencing for the 13 C spectrum is done by using tetramethylsilane.

CSA Measurements
The resonances lines in liquid state NMR spectra are very narrow because the anisotropic parts of the chemical shift, dipole-dipole, and the quadrupolar interactions are averaged out due to the rapid tumbling motions of the molecules. On the contrary, the resonance lines in reported by Ghosh et al. 34 Thirteen steps cogwheel phase cycling was used. Sixteen data points were acquired in the indirect dimension as the numbers of sidebands were less than sixteen. The anisotropic part of the chemical shift interaction in natural abundance 13 C spin-1 2 ⁄ nuclei (for those nuclei where the homonuclear dipole-dipole coupling is much less than the rotor frequency) evolves during the PASS sequence under the rotor pitch evolution in t1 dimension.

Solid State NMR Spectral Analysis:
Cefpodoxime proxetil is an ester modified prodrug of cefpodoxime. It is composed of aminothiazole ring, oxime group, acyl side chain, beta-lactam ring, dihydrothiazine ring, and ester group side chain. The beta-lactam ring is the core structure of antibiotics like cephalosporins, penicillins, carbapenems, and monobactams, which are also known as -

CSA Measurements:
The values of the principal components of the chemical shift anisotropy tensor depend on the distribution of electron density around nuclei. The electron density distribution is correlated with the geometry of chemical bonds, i.e. with the structure of the molecular moiety. The chemical shift anisotropy tensor can be represented by an ellipsoid centred on the nucleus it applies to. The axes of the ellipsoid coincide with the axes of the PAS of the CSA tensor. The length of each principal axis of the ellipsoid is proportional to the principal components of the CSA tensor associated with that principal axis. Hence, the orientation of the CSA tensor changes with the orientation of the molecule. 19 The CSA tensor at a nuclear site is axially symmetric in PAS if the principal axis coincides with the symmetry axis and the principal components of the CSA tensor is such that 11 = 22 . ) is defined as the center of gravity of the spinning CSA sideband pattern. 23 According to the Haeberlen convention anisotropy parameter is defined as(∆ = 33 −      spinning CSA sideband pattern deviates from its axially symmetric shape. Figure 5 shows the bar-diagram of (a) anisotropy parameter and (b) asymmetry parameter at crystallographically different sites of cefpodoxime proxetil. If 11 = 22 or 11 = 33 , then the value of the asymmetry parameter is zero, and the shape of the spinning CSA sideband pattern is axially symmetric. Figure 5(b), Figure 3, Figure 4, and Table 1 show that the CSA pattern of C4, C5, and C6 is axially symmetric. On the contrary, the CSA pattern is highly asymmetric for C10, C16, C2, C3, C18, C7, C9, C14, C17, C20, and C21. The spinning CSA sideband pattern for C1, C8, and C15 is nearly axially symmetric.
Where 1 is the angle between the radius vector and x-axis and 2 is the angle between radius vector and z-axis. 54 The anisotropic magnetic susceptibility is the source of the directional dependent magnetic field, which manifests as large values of chemical shift anisotropy at carbonyl site. The carbonyl group contains a polar bond. Hence, there arises a polarization on the electron cloud surrounding the carbon nucleus due to the electrostatic interaction of the central molecule with other molecules. The strength of the induced magnetic field is different at different directions due to this polarization, which is also a reason behind the large CSA parameters of carbonyl group carbon. 54 The aminothiazole ring, -lactam ring, dihydrothiazine ring brings stability towards the most commonly found plasmid-mediated -lactamases and increase the affinity of the drug to the penicillin binding proteins (PBPs) receptors. 2

Spin-lattice Relaxation Measurements:
The fluctuating local magnetic field experienced by the nucleus is the source of nuclear spin relaxation. The relaxation mechanism is governed by homonuclear dipole-dipole interaction, ).
The role of heteronuclear dipole dipole coupling on spin-lattice relaxation mechanism is articulated as 52 By keeping only the first term, Where X represent hydrogen, oxygen and nitrogen atoms. is the distance between carbon and neighbouring atoms hydrogen, oxygen, nitrogen. As the contribution of hetero-nuclear dipole-dipole interaction on spin-lattice relaxation mechanism is inversely proportional to the sixth power of the distance between the carbon and other nuclei, hence only the nearest neighbour distances is taken into consideration. Larmor precession frequency = 2 = 2 × 3.14 × The expression of the spin-lattice relaxation rate for 13 C carbon is    Table 1 shows that the CSA parameters are also different for these three nuclei. 55 The spin-lattice relaxation time of oxime group C4 nuclei is 55s and it is 112 s for carbonyl group carbon C6. The CSA parameters of these two nuclei are not so different. Both C4 and C6 nuclei are in sp2 hybridized state, and double bonded with electronegative atoms nitrogen and oxygen respectively. The oxime and methyl group improve the stability oflactamases. 55 The carbonyl group carbon C8 reside on the -lactam ring is responsible for nucleophilic attack. The spin-lattice relaxation time and the molecular correlation time of C8 are respectively 94 s and 1.6 × 10 −4 s. Figure 2 and Table 1

Conclusion:
Cefpodoxime proxetil is a prodrug of cefpodoxime, which has a broad spectrum of antibacterial activity encompassing both Gram-negative and Gram-positive bacteria. The structure and dynamics of cefpodoxime proxetil at atomic scale resolution is described by measuring CSA tensor, site-specific spin-lattice relaxation time, and calculating the molecular correlation time. The structure-activity relationship of various molecular moieties of the drug is also described by these measurements. Cefpodoxime proxetil molecule is associated with three rings, the aminothiazole ring, -lactam ring, and the dihydrothiazine ring, which provide stability of the drug towards -lactamases and increase the affinity of the  proxetil will definitely enlighten the path of developing the advanced antibacterial drugs. It will also enrich the data of NMR crystallography of the drug molecules.