Application of spectroscopic methods (FT-IR, Raman, ECD and NMR) in studies of identification and optical purity of radezolid

Article abstract of DOI:10.1016/j.saa.2017.04.038

In the presented study, N-{[(5S)-3-(2-fluoro-4′-{[(1H-1,2,3-triazol-5-ylmethyl)amino]methyl}biphenyl-4-yl)-2-oxo-1,3-oxazolidin-5-yl]methyl}acetamide (radezolid) was synthesized and characterized using FT-IR, Raman, ECD and NMR. The aim of this work was t

Full text of DOI:10.1016/j.saa.2017.04.038

Accepted Manuscript
Application of spectroscopic methods (FT-IR, Raman, ECD and
NMR) in studies of identification and optical purity of radezolid
Katarzyna Michalska, Ewa Gruba, Mikołaj Mizera, Kornelia
Lewandowska, Elżbieta Bednarek, Wojciech Bocian, Judyta
Cielecka-Piontek
PII:
DOI:
Reference:
S1386-1425(17)30306-2
doi: 10.1016/j.saa.2017.04.038
SAA 15091
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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13 April 2017
18 April 2017
Please cite this article as: Katarzyna Michalska, Ewa Gruba, Mikołaj Mizera, Kornelia
Lewandowska, Elżbieta Bednarek, Wojciech Bocian, Judyta Cielecka-Piontek ,
Application of spectroscopic methods (FT-IR, Raman, ECD and NMR) in studies of
identification and optical purity of radezolid. The address for the corresponding author was
captured as affiliation for all authors. Please check if appropriate. Saa(2017), doi: 10.1016/
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ACCEPTED MANUSCRIPT
Application of spectroscopic methods (FT-IR, Raman, ECD and NMR) in
studies of identification and optical purity of radezolid
Katarzyna Michalska1, Ewa Gruba1, Mikołaj Mizera2, Kornelia Lewandowska3, Elżbieta
Bednarek4, Wojciech Bocian4, Judyta Cielecka-Piontek2
1 Department of Antibiotics and Microbiology, National Medicines Institute, Chełmska 30/34,
00-725 Warsaw, Poland
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Poznan University of
Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
3 Department of Molecular Crystals, Institute of Molecular Physics of the Polish Academy of
Sciences, Poznan, Poland
4 Nuclear Magnetic Resonance Laboratory, National Medicines Institute, Chełmska 30/34, 00-
725 Warsaw, Poland
Correspondence: Judyta Cielecka-Piontek
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Poznan University of Medical
Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
Tel.: +48 8546649, fax + 48 61 864 66 52
e-mail: jpiontek@ump.edu.pl
Katarzyna Michalska
Department of Antibiotics and Microbiology, National Medicines Institute, Chełmska 30/34, 00-725
Warsaw, Poland
Tel.: +48 22 851 52 15; fax: +48 22 851 52 15
e-mail: k.michalska@nil.gov.pl
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Abstract:
In the
presented
study,
N-{[(5S)-3-(2-fluoro-4′-{[(1H-1,2,3-triazol-5-
ylmethyl)amino]methyl}biphenyl-4-yl)-2-oxo-1,3-oxazolidin-5-yl]methyl}acetamide
(radezolid) was synthesized and characterized using FT-IR, Raman, ECD and NMR. The aim
of this work was to assess the possibility of applying classical spectral methods such as FT-IR,
Raman, ECD and NMR spectroscopy for studies on the identification and optical purity of
radezolid. The experimental interpretation of FT-IR and Raman spectra of radezolid was
conducted in combination with theoretical studies. Density functional theory (DFT) with the
B3LYP hybrid functional was used for obtaining radezolid spectra. Full identification was
1
1
carried out by COSY, H {13C} HSQC and H {13C} HMBC experiments. The experimental
NMR chemical shifts and spin-spin coupling constants were compared with theoretical
calculations using the DFT method and B3LYP functional employing the 6-311++G(d,p) basis
set and the solvent polarizable continuum model (PCM). The experimental ECD spectra of
synthesized radezolid were compared with experimental spectra of the reference standard of
radezolid. Theoretical calculations enabled us to conduct HOMO and LUMO analysis and
molecular electrostatic potential maps were used to determine the active sites of
microbiologically active form of radezolid enantiomer. The relationship between results of ab
initio calculations and knowledge about chemical-biological properties of S-radezolid and
other oxazolidinone derivatives are also discussed.
Introduction
Studies concerning new entities at the early stage of R&D, subsequent in-process controls and
finally control of the finished product, broadly defined as quality control, are often based on
application of spectroscopic methods. UV and FT-IR, according to pharmacopeia guidelines,
are the most frequently used for identification of selected drugs [1]. In the case of innovative
drugs for which the effects of interactions with solvents have not been yet investigated, it is
important to start studying them by characterization in the solid state. Hence spectroscopic
methods are recommended for identification of novel drugs. FT-IR, Raman and NMR
spectroscopy are simple and reproducible techniques for the analysis of drug racemates at
different stages of development. The chiral analysis of drugs has also been extended by the
connection of successive spectroscopic techniques with electronic circular dichroism (ECD)
[2–5]. The possibility of studying enantiomers without using chiral selectors and organic
solvents may be considered as a promising solution for quality control, especially in the field
of optical drug isomerism. However, it should be stressed out, that the above-mentioned
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spectroscopic techniques do not permit selective determination of the principal analyte in the
presence of its impurities. This is only possible when suitable chemometric solutions are
applied, allowing for the assessment of the type and concentration of a particular impurity
based on predefined descriptors differentiating the API sample from its impurities.
The oxazolidinones are a novel class of synthetic antimicrobial agents, unrelated to any other
class, showing a comprehensive spectrum of activity against the major and nosocomial Gram-
positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA),
enterococci and pneumococci [6]. The first oxazolidinone introduced into treatment was
linezolid in 2000; the next agent – tedizolid – was approved in 2014. Radezolid (RX-1741)
(Fig. 1) is a novel biaryloxazolidinone antibacterial agent which is in clinical development;
two phase-2 clinical trials have been completed, the first in uncomplicated skin and skin-
structure infections (uSSSI) and the second in community-acquired pneumonia (CAP) [7–9].
Radezolid emerged from the Rχ-01 discovery programme at Rib-X Pharmaceuticals, Inc., and
revealed excellent antibacterial activity against linezolid-susceptible staphylococci,
enterococci and pneumococci as well as against linezolid-resistant isolates, especially
enterococci and pneumococci. Unlike linezolid, radezolid also showed proper antibacterial
activity against the causative agents of CAP, such as Haemophilus influenzae and Moraxella
catarrhalis [6].
In the oxazolidinone derivatives group, HPLC has been most frequently used for analysing
linezolid and tedizolid in different matrices [10–12]. Capillary electrophoresis has also played
a significant role in the analysis of linezolid and tedizolid [13–15]. Previously published
studies confirmed the applicability of FT-IR and Raman spectra for identification of
oxazolidinone derivatives and assessment of their chiral purity based on the analysis of ECD
spectra [16–18]. Theoretical analysis of linezolid and tedizolid supported by density
functional theory (DFT) calculations with 6-31G(d,p), 6-311G(d,p) and M06-2X/6-31G(d,p)
basis sets allowed identification of the differences in molecular electrostatic potential (MEP)
maps, which may be connected with their varied antimicrobial activity, and regions that
indicate differing localization of electrons, which are connected to the chemical reactivity.
Regarding spectroscopic bands important for identification of radezolid, such domains as
triazole, biphenyl (including phenyl and 2-fluorophenyl moieties) and oxazolidinone rings as
well as a methylacetamide substituent can be distinguished.
The aim of this study was identification of radezolid by application of FT-IR, Raman and
NMR spectra, while ECD spectra were used for studies of the optical purity of radezolid.
During the identification of the appropriate bands or chemical shifts and evaluation of their
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intensity, FT-IR, Raman and NMR spectra analysis was supported by quantum chemical
calculations using a Becke, 3-parameter, Lee-Yang-Parr (B3LYP) hybrid functional with a 6-
31G(d,p) or 6-311++G(d,p) standard basis set. Based on the performed identification, a
comparative analysis of radezolid enantiomers was executed, in regards to further evaluation
of chiral purity. Additional characteristics were obtained to those previously achieved from
spectra of radezolid by performing electronic properties tests such as HOMO-LUMO orbitals
and MEPs for both (R)- and (S)- radezolid enantiomers.
Experimental methods
Substance for studies
Radezolid was synthesized as described in the supplementary information with slight
improvements to the literature methods [19–21]. During experiments, as a reference material
for comparison and of purity studies, single isomer (S)-radezolid from ApexBio Technology
LLC (Houston, TX, USA) with 98% purity was used.
Spectroscopic methods
The vibrational infrared spectra of radezolid were recorded between 4000 and 100 cm−1 in
powder, at room temperature, with a Bruker Equinox 55 FT-IR spectrometer equipped with a
Bruker Hyperion 1000 microscope. Raman scattering spectra were obtained with a LabRAM
HR800 spectrometer (HORIBA Jobin Yvon) with laser excitation λexc = 1064 nm (He-Ne
laser). In each case the power of the laser beam focused on the sample was less than 1 mW to
avoid damage of the sample. (S)-radezolid was established using a Jasco J-715 circular
dichroism spectrometer. The ECD spectra of synthesized (S)-radezolid and its reference
standard were recorded in water with the concentrations of 2.3 x 10-4 mol/L and 1.8 x 10-4
mol/L, respectively. The range of the ECD spectra was within 180–450 nm, with cell length
0.1 cm, scanning speed 100 nm/min, while the detector response was obtained every 0.5 s. All
1
measurements were conducted at room temperature. The H and 13C NMR spectra of
radezolid were recorded in D2O (1.9 mg/mL) and DMSO-d6 (8.0 mg/mL) solutions at 303 K
1
13
using a Varian VN-MRS 500 spectrometer operated at 499.8 and 125.7 MHz for H and C,
respectively. The full assignment of proton and carbon signals was accomplished using COSY,
1H {13C} HSQC and 1H {13C} HMBC experiments.
1H{13C}HSQCAD: an acquisition time of 0.15 s, relaxation delay of 1.0 s and spectral
windows of 6000 Hz in F2 and 22600 Hz in F1. 512 complex data points were collected in the
indirectly detected dimension (13C) with 4 scans and 2048 points per increment. The data
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were linearly predicted to 1K and zero filled to 4K complex data points in F1 and processed
using the cosine window function in both dimensions prior to Fourier transformation. The
proton and carbon π/2 pulse lengths were 6.75 and 14.8 µs, respectively.
1H{13C}-gHMBCAD: sweep width of 6000 Hz in F2 and 25150 Hz in F1, with an acquisition
time of 0.17 s, relaxation delay of 1.0 s and nJ(C,H) = 8 Hz. Overall, 512 complex data points
were collected in the indirectly detected dimension (13C) with 32 scans and 2048 points per
increment. The data were linearly predicted to 1K and zero filled to 4K complex data points in
F1 and processed using sine-bell square multiplication in F2 and Gaussian window function in
F1 dimensions prior to Fourier transformation.
Computation details
The harmonic vibrational frequencies for spectroscopic analysis (for FT-IR and Raman
spectra) were carried out with DFT using a B3LYP hybrid functional with a 6-31G(d,p)
standard basis set and its variations with diffuse basis functions [22]. Obtained frequencies for
FT-IR and Raman spectra were scaled by a factor of 0.961. Theoretical computation of
optimal geometry, frontier molecular orbitals (FMOs) and MEP maps were calculated at the
same level of theory for optimized structures. The NMR magnetic shielding and spin-spin
coupling constants were calculated using the gauge-independent atomic orbital (GIAO)
method [23] under DFT. The B3LYP functional employing the standard 6-311++G(d,p) basis
set was used. The polarizable continuum model (PCM) using the standard integral equation
formalism variant (IEFPCM) was used to mimic the influence of water as a solvent [24]. The
GIAO calculations were preceded by precise searching for the lowest energy conformers
conducted at the same level of theory. The NMR chemical shifts were calculated by a linear
least squares method of fitting averaged values of NMR shieldings for the two lowest energy
radezolid conformers (differing by mutual twisting of the fluorophenyl ring). All calculations
were performed using Gaussian 09 software and visualized using GaussView [22].
Results and discussion
Identification of radezolid based on FT-IR and Raman spectra
For analysis of FT-IR and Raman spectra for detailed identification of the vibrational modes
of radezolid, the following domains were separated: triazole ring, biphenyl ring (including the
phenyl and 2-fluorophenyl moieties), oxazolidinone ring and methylacetamide substituent.
The identification of characteristic spectral regions for radezolid was supported by comparing
with theoretical spectra obtained based on DFT with the B3LYP hybrid functional and 6-
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31G(d,p) basis set (Figs. 2 and 3). The calculated vibrational frequencies were scaled in order
to improve agreement with experimental values. It is relevant to discuss the vibrational
spectra of radezolid in terms of characteristic spectral regions as described below. The most
characteristic bands associated with vibrations of the bonds present in the radezolid are
located between 2000 and 500 cm−1. The bands assignment of a vibrational model for the
above-mentioned domains in the molecule in the FT-IR and Raman region are summarized in
Table 1. In FT-IR and Raman spectra of radezolid, we observed many C–H bonds, and many
bands related to them. Heteroaromatic organic compounds (in the case of triazole and
biphenyl rings) and their derivatives are structurally very close to benzene and commonly
exhibit multiple weak bands in the region 3100–3000 cm−1 due to the presence of C–H
stretching vibrations [25]. For radezolid, bands related to the stretching vibration of C–H
bonds were observed above 2500 cm−1. In our study the bands were not as well resolved, that
could be a result of intermolecular interaction. Besides that, characteristic bands were also
observed related to the vibrations of the following bonds: C=O, C–O, C=C, C–C, C–N, N–N
and C–F. Moreover, bands corresponding to the deformation of C–H bonds were also located
in this range. In the FT-IR spectrum of radezolid there were better exposed and more intense
bands than in the Raman spectrum. For example, in the FT-IR spectrum a quite strong band at
838 cm−1 is related to wagging vibration of the C–H bond in the triazole ring, while a strong
band at 1136 cm−1 mainly corresponds to scissoring vibrations of the C–H bonds in the F-
phenyl ring, but both of them have an additional component related to stretching vibration of
the C–F bond. In contrast, the bands located on the slopes of wider bands at 1253 and 1486
cm−1 are related to twisting vibrations of the C–H bond in the methylacetamide substituent,
scissoring vibrations of the C–H bonds in the oxazolidinone ring and rocking vibration of the
C–H bonds in the phenyl and F-phenyl rings. In the Raman scattering spectrum bands
corresponding to the vibration of C–H bonds were also observed; for example, at 553, 818,
900 and 1197 cm−1, however they were very low in intensity. In contrast, very strong bands
were observed in the Raman spectrum at 1617 and 1304 cm−1, the first is associated with
stretching vibration of the C=C bonds in the phenyl and F-phenyl rings, and the second is
related to stretching vibration of the C–C bond between these rings and stretching vibration of
this bond. The band at 1272 cm−1 is also related to stretching vibration of the C–C bond
between the phenyl and F-phenyl rings. In the Raman spectrum the bands corresponding to
stretching vibrations of the C–C bonds were also located at 1020 and 1217 cm−1. The first
band is related to vibration in the oxazolidinone ring, and the second is associated not only
with stretching vibration of the C–C bond, but has additional components related to stretching
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vibration of the C–N bond in the oxazolidinone ring, stretching vibration of the C–F bond and
twisting vibration of the C–H bonds in the oxazolidinone ring, too. In the FT-IR spectrum we
also observed stretching vibration of the C=C and C–C bonds; for example, the bands at 1293,
1530, 1561 and 1577 cm−1 correspond to stretching vibration of the C–C bonds in the phenyl
and F-phenyl rings, vibrations between these rings and stretching vibration of the C=C and C–
C bonds in the F-phenyl ring ring, respectively.
The characteristic peaks in the FT-IR spectrum at 1676 and 1754 cm−1 are related to stretching
vibrations of the C=O bonds in the methylacetamide group and oxazolidinone ring,
respectively. The other characteristic bands are those associated with stretching vibration of
the C–N bonds. They are observed in the FT-IR spectrum at 1202, 1329 and 1379 cm−1. The
first band is mainly related to C–N vibrations in the oxazolidinone ring, but in this range we
also observed additional components associated with rocking vibration of the C–H and N–H
bonds in the triazole ring. The second band is related to stretching vibration of the C–N bonds
between the triazole and oxazolidinone rings and in the oxazolidinone ring, and the last band
corresponds to C–N vibration in both the oxazolidinone and triazole rings. In the lower
frequencies bands related to bending vibration of the C–N–C bond in the
[(methyl)amino]methyl group (for example 975 cm−1) were also observed. Bands
corresponding to N–H vibrations are also characteristic for radezolid. In our study bands
related to stretching vibrations of those bonds were not easily visible; they were located above
3300 cm−1, whereas at lower frequencies bands associated with the deformation vibration of
these bonds were effortlessly observed. The bands of deformations in plane bending are
assigned at 1417, 1442 and 1501 cm−1, and are related to rocking vibrations in triazole ring
and [(methyl)amino]methyl and methylacetamide substituents, respectively. It is worth
pointing out, that vibrations belonging to C–X (F) bonds, which are formed between the ring
and halogen atoms, are a mixture of vibrations. This is possible due to lowering of the
molecules’ symmetry and the presence of halogen atoms. C–F stretching vibrations appeared
in the lower range of frequencies at 1400–1000 and 760–505 cm−1 and C–F deformations
occurred in the range of 700–500 and 300–140 cm−1. The bands which are mainly related to
stretching and bending vibrations of C–F were located at 1199 and 480 cm−1, respectively.
Those bands were very often seen as an additional components in other bands (Table 1).
The assignments made in this study are also supported by the literature [26]. Bands
characteristic of the key domains in molecules were defined for both of the oxazolidinone
derivatives studied (linezolid, tedizolid) and attention was paid to signals registered for
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carbon–fluorine bonds [16–18]. The number of bands and their intensity were greater in the
case of FT-IR spectra.
NMR spectra
The results of NMR experiments in D2O and DMSO-d6 solutions are summarized in Table 2.
1
In addition to H and 13C NMR chemical shifts, the values of selected spin-spin coupling
constants (homo- and heteronuclear) are included. The presented data allowed us to fully
confirm the molecular structure of radezolid and provide its full spectral characteristics in
solutions. Additionally, theoretical calculations were made of both NMR chemical shifts and
spin-spin coupling constants. Calculations were carried out using DFT with the B3LYP hybrid
functional and 6-311++G(d,p) basis set. Simulations took into account the effect of the
solvent by performing a reaction field calculation using IEFPCM. As the calculations were
performed for the liquid state, it turned out to be necessary to consider the conformational
mobility of the radezolid molecule. Sufficiently accurate results were obtained by averaging
the calculated NMR data obtained for the two lowest energy radezolid conformers. These
conformers differ only by torsional orientation of the fluorophenyl ring. The results of
calculations are also presented in Table 2. Generally, the calculated and experimental values
of NMR chemical shifts were in very good agreement. Some difficulties were observed only
during comparison of the nuclear spin-spin couplings, which is not surprising, due to the lack
of sophistication in available calculation methods. In the Table 2. are included only
heteronuclear proton–fluorine and carbon–fluorine coupling constants as they seem to be the
most characteristic and were calculated accurately enough.
Studies of chiral purity based on ECD spectra
An ECD technique was chosen as a reference measurement method for studies of the chiral
purity of a radezolid enantiomer (Fig. 4). Comparison of the ECD spectra of a synthesized
(S)-radezolid to an (S)-isomer serving as a reference standard, both dissolved in distilled water,
confirmed that the differences found in the shapes of spectra curves were not significant.
Those experiments also proved the stereoselective synthesis of (S)-radezolid. Radezolid
showed a positive Cotton band with its maximum at 270 nm and a negative band with the
maximum at 215 nm. Those observations suggest that the synthesized enantiomer did not
contain any significant level of impurities of the chromophore structure, which could possibly
lead to a change in the position of the absorption maxima and the shape of the spectra.
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Frontier molecular orbitals
The HOMO (highest occupied molecular orbital) energy characterizes the ability of donating
electrons while the LUMO (lowest unoccupied molecular orbital) energy characterizes the
ability of accepting electrons [27–29]. The possibility of donating and withdrawing electrons
by the triazole ring and the methylacetamide substituent was not observed. The HOMO is
localized vertically on the biphenyl system whereas the LUMO is localized horizontally (Fig.
5). For the chiral centre at position C5 of the oxazolidinone structure no significant
differences were found in the localization of electrons for the analysed FMOs. For radezolid
EHOMO and ELUMO were −6.097 eV and −1.188 eV, respectively. Since the chemical potential
of radezolid is negative, the compound is chemically stable and not prone to spontaneous
degradation [30].
Molecular electrostatic potentials
Variations in electrostatic potential are largely responsible for the drug binding to receptor
binding sites and hydrogen bonding interactions [31,32]. The MEP maps and their contours
for radezolid generated with optimized geometry of the title molecule using GaussView
software are shown in Fig. 6. The red and blue colours refer to electron-rich and electron-poor
regions while the green area suggests almost neutral potentials. As a consequence, it is
possible to predict the binding sites for electrophilic or nucleophilic attack in the molecule.
Analysis of the MEP maps showed that two positive regions (blue) localized in the triazole
and oxazolidinone rings correspond to the nucleophilic reactivity of radezolid. In the structure
of oxazolidinone the nucleophilic area also comprises the chiral centre at position C5. It
should be stressed out that the carbonyl groups in the methylacetamide substituent and the
oxazolidinone structure are the sole negative regions responsible for the electrophilic
reactivity of radezolid. Similar results were obtained for tedizolid and linezolid [16–19]. The
differences in the MEP maps were observed at the 1,3-oxazolidin-2-one structure, which
contains an asymmetric carbon atom, and is linked to the antibacterial activity of
oxazolidinone derivatives [6].
Conclusions
The study involved experimental and theoretical spectroscopic analysis of (S)-radezolid – a
novel oxazolidinone antibacterial agent – using FT-IR, Raman, NMR and ECD techniques
with the support of the DFT theory. Theoretical analysis allowed to determination of charge
transfer within the molecule and the calculated HOMO and LUMO energies demonstrated
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that the molecule. was chemically stable. A plot of the MEPs showed the location of
nucleophilic and electrophilic reactivity sites. The application of FT-IR, Raman and NMR
techniques can be recommended for identification of radezolid both in solid and in solution
states whereas the use of ECD spectra was found to be suitable for evaluating the chiral purity
of microbiologically active (S)-radezolid. However, each of the proposed spectroscopic
methods should be considered as a preliminary step valuable for principal identification of the
analyte but not allowing for its assay in the presence of related sustances.
Acknowledgments
This study was supported by a SONATA grant from the National Science Centre, Poland
(UMO-2013/11/D/NZ7/01230).
This research was supported in part by PL-Grid Infrastructure.
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Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N.
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R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S.
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Gaussian, Inc., Wallingford CT, 2009.
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Mol. Biomol. Spectrosc. 138 (2015) 711–722.
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(2015) 184–197.
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[32] F.J. Luque, J.M. Lopez, M. Orozco, Theor. Chem. Acc. 103 (2000) 343–345.
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Fig. 1. Chemical structure of (S)-radezolid.
13

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reference standard
synthesized sample
theoretical spectrum of (S)-radezolid
400
800
1200
1600
2500 3000 3500 4000
Wavenumber (cm-1)
Fig. 2. The calculated (black), synthesized sample (blue), and reference standard (red) FT IR
absorption spectra in room temperature of (S)-radezolid.
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reference standard
synthesized sample
theoretical spectrum of (S)-radezolid
200 400 600 800 1000 1200 1400 1600 1800
3000
3500
Raman Shift (cm-1)
Fig. 3. The calculated (black), synthesized sample (blue) and reference standard (red) Raman
scattering spectra in room temperature of (S)-radezolid.
15

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Fig. 4.
Experimental of UV-CD spectra for synthetized (S)-radezolid (blue) and ApexBio
reference standard of (S)-radezolid (green).
16

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A.
B
Fig. 5 The frontier molecular orbitals for (S)-radezolid: HOMO (A) and LUMO (B)
17

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A
B
Fig. 6. Spatial (A) and contour (B) molecular electrostatic potential map (MEP) for (S)-
radezolid.
18

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Table 1. Comparison of the selected vibrations observed experimental and calculated
(B3(LYP/6-31G(d,p) spectra of radezolid

Bands assignment
exp.IR

410
def. all molecule
C-F b
405
473
509
564
615
698
480
515
N-H w in triazole ring
C-H w
553
600
724
597
722
def 2-fluorophenyl ring
N-H w in triazole ring
C-C-O b in oxazolidinone ring + C-C-N b in triazole ring and in
(methyl)amino group
N-H w in triazole ring
743
744
777
786
750
779
797
818
N-H w in (methyl)amino group + C-H w in 2-fluorophenyl ring
Breathing phenyl ring + C-H w phenyl ring + N-H w in
(methyl)amino group
C-H w in phenyl ring
C-H w in triazole ring
811
832
851
838
Breathing 2-fluorophenyl ring and oxazolidinone ring + C-N s in
oxazolidinone ring + C-C s in o oxazolidinone ring + C-F s in 2-
fluorophenyl + C-H r in oxazolidinone ring
839
860
885
900
976
C-H r in oxazolidinone ring ring
975
C-N-C b in łącznik + C-H t łącznik + C-C s in triazole ring
Breathing phenyl and 2-fluorophenyl ring (C-C s)
C-C s in oxazolidinone ring
969
1005
1020
1109
1121
984
1020
1014
1068
1083
1130
1156
1164
1173
1186
C-N-C b + C-H t in (methyl)amino group
C-H r in 2-fluorophenyl ring + C-N s in oxazolidinone ring
C-H sc in 2-fluorophenyl ring + C-F s in 2-fluorophenyl ring
C-H r in 2-fluorophenyl ring + C-F s in 2-fluorophenyl ring
C-H w in phenyl ring
1136
1161
1197
1199
1202
C-F s in 2-fluorophenyl ring + C-H w in oxazolidinone ring
C-N s in oxazolidinone ring + C-H r + N-H r in triazole ring
C-C s between phenyl ring and (methyl)amino group + C-N s
in oxazolidinone ring + C-F s in 2-fluorophenyl ring + C-H t in
oxazolidinone ring
1217
1192
N-N r in methylacetamid group+ C-C s in methylacetamid
group + C-H r 2-fluorophenyl ring + C-H w in methylacetamid
group
1225
1253
1216
1251
1257
C-H t in methylacetamid group
1272
1304
C-C s between phenyl and 2-fluorophenyl ring
C-C s in phenyl and 2-fluorophenyl ring + C-H w in phenyl ring
and (methyl)amino group
C-C s between phenyl and 2-fluorophenyl ring + C-C s in phenyl
and 2-fluorophenyl ring
1293
1273
1277
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C-N s between 2-fluorophenyl and oxazolidinone ring + C-N s in
1329
1379
1326
triazole ring + C-H r in phenyl ring
C-N s in oxazolidinone ring and triazole ring + C-H w in
oxazolidinone ring
1307
1423
1416
1376
1433
1443
1417
1442
N-H r in triazole ring + (methyl)amino group
N-H r in (methyl)amino group
C-H sc in oxazolidinone ring ring + C-H r in phenyl and 2-
fluorophenyl ring
1486
1501
1530
1476
1492
N-H r in methylacetamid group
C-C between phenyl and 2-fluorophenyl ring + C-H r in phenyl
and 2-fluorophenyl ring
C=C s + C-C s in phenyl and 2-fluorophenyl ring
C-C s in phenyl and 2-fluorophenyl ring
C=C s in phenyl and 2-fluorophenyl ring
C=C s in phenyl and 2-fluorophenyl ring
C=O s in methylacetamid group
1530
1502
1538
1557
1598
1607
1703
1786
1561
1577
1629
1629
1676
1754
1617
1617
C=O s in oxazolidinone ring
All positions in cm-1; s-stretching, b-bending, r-rocking, w-wagging, sc-scissoring, oop-out of the plane.
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Table 2. The experimental and calculated NMR data (δ -chemical shifts [ppm] of 1H and 13C atoms and J coupling constants[Hz}) of Radezolid
synthesized (R)-radezolid –D2O
APEX-BIO -(S)-radezolid –D2O
synthesized (S)-radezolid –DMSO
Calculated DFT PCM(H2O)
1H
13C
1H
13C
1H
13C
1H
13C (15N)
2C=O
4CH2
5CH
-
159.42
50.93
-
159.49
50.96
-
153.90
47.10
-
156.48
3.88 (dd, 1H, J = 6.0, 9.4
3.93 (dd, 1H, J = 6.0, 9.4
3.79 (dd, 1H, J = 6.4, 9.3
Hz)
Hz)
Hz)
3.96
4.02
49.12
77.01
41.47
4.25 (dd, 1H, J = 8.9, 9.4
4.28 (dd, 1H, J = 8.9, 9.4
4.17 (dd, 1H, J = 8.8, 9.3
Hz)
Hz)
Hz)
4.89 – 4.94 (m, 1H)
3.59 (dd, 1H, J= 5.4, 14.9
75.79
44.67
4.91 – 4.96 (m, 1H)
3.61 (dd, 1H, J = 5.4, 14.9
75.82
44.66
4.75 – 4.80 (m, 1H)
3.44 (dd, 2H, J = 5.5 Hz)
71.71
41.29
4.96
4.01
Hz)
Hz)
6CH2
3.63 (dd, 1H, J = 3.9, 14.9
3.65 (dd, 1H, J = 3.9, 14.9
Hz)
Hz)
-
-
-
-
8.26 (t, 1H, J = 6 Hz,
NHCOCH3)
-
-
7NH
8C=O
9CH3
5.83
-
-
-
177.79
24.69
-
177.82
24.69
169.94
22.35
(173.01)
23.89
2.01 ( s, 3H)
2.01 ( s, 3H)
1.84 ( s, 3H)
2.24
-
141.39
-
141.40
-
139.51
(JCF = 11.1 Hz)
105.48
142.34
1’C
-
(JCF = 9.3 Hz)
7.48 (dd, 1H, J = 2.3, 12.9
110.08
(JCF =28.3
Hz)
7.50 (dd, 1H, J = 2.3, 12.9
110.16
7.63 ( 1H)
106.12
2’CH
Hz)
Hz)
(JCF = 28.3 Hz
(JCF = 28.6 Hz)
7.16 (JHF=14.9 Hz)
(JCF = 28.9 Hz)
-
-
162.34
-
-
162.32
(JCF = 243.7
Hz)
-
158.92
163.74
3’CF
(JCF = 244.9 Hz)
-
(JCF =-316.2 Hz)
126.73
133.90
118.04
139.15
132.46
133.09
126.75
-
122.02
(JCF = 13.5 Hz)
130.78
126.76
(JCF = 10.9 Hz)
133.45
4’C
5’CH
6’CH
1”C
-
7.57 (dd, 1H, J = 8.6, 8.6
7.59 (dd, 1H, J = 8.6, 8.6
133.93
(J= 4.7 Hz)
118.12
(J= 3.5 Hz)
139.17
7.60 (1H)
7.59 (JHF= 8.2 Hz)
Hz)
Hz)
7.36 (dd, 1H, J = 2.3, 8.6
Hz)
(JCF = 4.5 Hz)
113.84
(JCF = 2.6 Hz)
115.50
7.34 (dd, 1H, J = 2.3, 8.6
7.44 (dd, 1H, J = 2.2, 8.6
7.88 (JHF=-1.1 Hz)
Hz)
-
Hz)
-
(JCF = 3.2 Hz)
135.23
(JCF = 4.3 Hz)
138.16
-
(JCF = 1.8 Hz)
128.75
(JCF =-2.0 Hz)
131.00
2”CH
/ 6”CH
3”CH
7.68 (dd, 2H, J = 1.6, 8.3
Hz)
7.57 (d, 2H, J = 8.3 Hz)
7.69 (dd, 2H, J = 1.6, 8.3
Hz)
7.58 (d, 2H, J = 8.3 Hz)
132.48
133.09
7.62 (2H)
7.66 (2H)
7.72
7.77
(JCF = 3.1 Hz)
130.33
(JCF =6.5, 0.0 Hz)
131.73
/ 5”CH
21

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4”C
4’”CH
5’”C
-
132.82
n.d.
-
132.85
n.d.
-
130.96
n.d.
-
143.45
132.84
141.94
45.79
8.07 (br. s, 1H, triazole)
8.07 (br. s, 1H, triazole)
7.98 (br. s, 1H, triazole)
7.72
-
-
140.11
43.65
-
140.10
43.67
-
138.12
40.73
4.50 (br. s, 2H)
4.50 (br. s, 2H)
4.33 (br. s, 2H)
6’” CH2
4.21
4.37 (br. s, 2H)
-
53.10
-
4.38 (br. s, 2H)
-
53.11
-
4.27 (br. s, 2H)
49.35
-
8’” CH2
4.20
-
57.80
9.51 (br. s, 2H,
ArCH2N+H2)
N+H2
(156.48)
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Graphical abstract
23

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Highlights



Raman, FT-IR, NMR and UV-CD spectra of radezolid were established
Geometric structure of molecules, HOMO and LUMO orbitals and MEP were determined
Benefits of applying FT-IR, Raman, NMR and UV-CD spectroscopy were signed
24