Spectroscopic identification of intermediates and final products of the chiral pool synthesis of sutezolid

Article abstract of DOI:10.1016/j.molstruc.2020.128396

Sutezolid is a new oxazolidinone derivative currently in clinical trials to determine its safety and efficacy towards highly drug-resistant tuberculosis. The aim of the study was the spectroscopic identification of selected key intermediate products of th

Full text of DOI:10.1016/j.molstruc.2020.128396

Journal of Molecular Structure 1217 (2020) 128396
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Spectroscopic identification of intermediates and final products of the
chiral pool synthesis of sutezolid
,
Katarzyna Michalska a *, Kornelia Lewandowska b, Mikołaj Mizera c, Wojciech Bocian d,
Barbara Pałys e, Judyta Cielecka-Piontek c
a Department of Synthetic Drugs, National Medicines Institute, Chelmska 30/34, 00-725, Warsaw, Poland
b Department of Molecular Crystals, Institute of Molecular Physics of the Polish Academy of Sciences, Smoluchowskiego, 17, 60-179, Poznan, Poland
c Department of Pharmacognosy, Faculty of Pharmacy, Poznan University of Medical Sciences, 60-781, Poznan, Poland
d Department of Counterfeit Medicinal Products and Drugs, National Medicines Institute, Chełmska 30/34, 00-725, Warsaw, Poland
e Laboratory of Electrochemistry, Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093, Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Sutezolid is a new oxazolidinone derivative currently in clinical trials to determine its safety and efficacy
towards highly drug-resistant tuberculosis. The aim of the study was the spectroscopic identification of
selected key intermediate products of the chiral pool synthesis of sutezolid: (S1) (4-(2-fluoro-4-
nitrophenyl)thiomorpholine), (S2) 4-[2-fluoro-4-(benzyloxycarbonyl)aminophenyl] thiomorpholine,
(S4) (R)-[[3-[3-fluoro-4-(4-thiomorpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-p-toluenesulfonate,
(S5) (R)-[[3-(3-fluoro-4-(4-thiomorpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]azide, as well as (S6)
sutezolid, the final synthesis product. Spectroscopic identification was carried out based on infrared
spectra analysis (FT-IR) and Raman scattering. This part of the analysis was supported by quantum
chemical calculations based on the density functional theory (DFT) utilizing the B3LYP hybrid functional
and the 6-31G(d,p) basis set to obtain reference standards for intermediates and sutezolid. Additionally,
the sutezolid configuration was confirmed by analysing the electronic circular dichroism (ECD) spectra of
(S)- and (R)- isomers, combined with the time-dependent DFT calculations to simulate ECD spectra
depending on the solvent and type of conformers.
Received 9 October 2019
Received in revised form
20 March 2020
Accepted 5 May 2020
Available online 10 May 2020
Keywords:
ECD
FT-IR
Raman scattering
Sutezolid
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
drug-resistant tuberculosis. Studies on STD are important because
of the serious, multilateral clinical problem associated with long-
Sutezolid (STD), N-({(5S)-3-[3-fluoro-4-(thiomorpholin-4-yl)
phenyl]-2-oxo-oxazolidin-5-yl}methyl)acetamide, is the next
analogue after linezolid (LIN), tedizolid and radezolid (currently at
the clinical trial stage) (Fig. 1), which belongs to a truly new class of
antibacterial agents known as oxazolidinones [1]. This is un-
doubtedly the most anticipated class of antimicrobial agents
against Gram-positive bacteria, considering the pressing concern of
(i) the decrease in efficacy of already known antibacterial agents
simultaneously with (ii) the relatively fast decline in efficacy of new
chemical molecules used in the clinic due to the spread of
antibiotic-resistant pathogenic bacteria.
term treatment of multi-drug and extensively drug-resistant
tuberculosis (MDR-TB), and a small armamentarium containing
only a few viable or effective solutions in this regard [3].
STD as a completely synthetic drug may be synthesised by
different pathways, thus, full characterisation of key intermediates
as well as STD are crucial for quality control, therapeutic safety and
drug efficacy. It should also be emphasized that in order to fully and
comprehensively characterize the current state of knowledge in the
field of STD analysis, it is necessary to mention the results described
by Guo et al. [4], where an achiral degradation products of STD have
recently been characterized using LC-MSn, LC-H RMS and nuclear
magnetic resonance (NMR) spectroscopy. Moreover, enantiose-
lective recognition of STD by cyclodextrin modified non-aqueous
capillary electrophoresis and explanation of complex formation
by means of infrared spectroscopy, NMR and molecular modelling
was performed by our group [5], entering it into a large project
concerning the mechanistic study on the enantioseparation of
Sutezolid is currently in a Phase 4 clinical trial (NCT03237182,
study in progress) [2] for the individualized treatments of M(X)
* Corresponding author.
E-mail address: k.michalska@nil.gov.pl (K. Michalska).
https://doi.org/10.1016/j.molstruc.2020.128396
0022-2860/© 2020 Elsevier B.V. All rights reserved.

2
K. Michalska et al. / Journal of Molecular Structure 1217 (2020) 128396
Fig. 1. Molecular structures of antibacterial agents of the oxazolidinone class: linezolid, tedizolid, radezolid and sutezolid.
novel oxazolidinones, like LIN [6,7], tedizolid [8e10] and radezolid
[11e13].
spectroscopy have demonstrated great potential in the rapid
identification of drug molecules in the solid state. In the IR spectra,
strong absorption bands are derived from vibrations of polar
groups, capable of solvating and forming hydrogen bonds and
dipole-dipole interactions. The Raman active vibrations are intense
for nonpolar moieties, while the vibrations of a highly polar moiety
are usually weak. Hence, Raman scattering complements IR spec-
troscopy, so parallel analysis using the distinguishable potential of
both techniques guarantee the most complete overall view of the
molecule. Importantly, both FT-IR and Raman spectroscopy also
provide information on the fundamental vibrational bands, also
known as the fingerprint region, offering a high degree of analytical
specificity [14,15], thus becoming interesting tools that have
created opportunities to integrate real-time process analytical
technology into synthesis or manufacturing processes [16], as well
as to characterize and distinguish counterfeit drugs [17].
From the chemical point of view, the molecular structure of STD
can be divided into the following building units, as seen in Fig. 1:
the oxazolidine-2-one ring with a (methyl)acetamide substituent, a
fluorophenyl ring and a thiomorpholine ring. STD possesses a single
stereogenic center at position C-5 of the oxazolidine-2-one ring,
which is associated with the antibacterial activity of the drug.
Substitution of the morpholine ring (as in LIN) by a thiomorpholine
ring is the only difference between STD and its predecessor, LIN.
This change involves differences in their physicochemical proper-
ties, such as the low water solubility of STD (0.2 mg/mL). Thus, the
use of a solid state in spectral analysis methods to identify com-
pounds with low water solubility circumvents the solubility prob-
lem and appears to be the method of choice.
In recent years, vibrational spectroscopic techniques such as FT-
IR (Fourier transform infrared) spectroscopy and Raman
Because STD is a chiral molecule, a comprehensive analysis of

K. Michalska et al. / Journal of Molecular Structure 1217 (2020) 128396
3
sutezolid should also include the assessment of chiral
identification.
toluenesulfonyl chloride (S4), sodium azide (S5) and thioacetic acid
(S6) were purchased from Sigma-Aldrich (Steinheim, Germany); 4-
methoxybenzyl chloride and benzyl chloroformate were purchased
from TCI (Tokyo, Japan); sodium bicarbonate, magnesium sulphate,
sodium sulphate, potassium carbonate, sodium chloride, ammo-
nium chloride, hexane, ethyl acetate, methanol (MeOH), acetone,
tetrahydrofuran, dimethylformamide, hydrochloric acid, ethanol,
toluene, dichloromethane and chloroform were purchased from
POCH (Gliwice, Poland) and all were of analytical grade.
So far, spectral analysis of the purity of oxazolidinone de-
rivatives by electronic circular dichroism (ECD), including the
assessment of their optical purity as well as distinguishing between
polymorphic forms, has been carried out with respect to LIN,
tedizolid and radezolid [9,12,13,18]. The advantages of optical
analysis performed by ECD and its complies with the green
approach to pharmaceutical analysis was presented by Okuom et al.
[19]. However, for the qualitative evaluation of enantiopurity by
ECD, in accordance with the above-mentioned protocol [19], both
spectra of (S)-, and (R)-enantiomers should be determined, and the
3. Instrumentation
differential extinction coefficient (
Dε) should be plotted versus the
3.1. Spectroscopic analysis, FT-IR
enantiopurity at the wavelength of maximum amplitude. However,
the appropriate quantities of both (S)- and (R)-enantiomers of STD
at the proper chiral purity are needed. Another approach, this time
using chromatographic methods to assess the enantiopurity of a
particular compound, even when only one enantiomer is available
as reference, is based on the inverted chirality columns approach
(ICCA), which involves the use of chiral stationary phases available
in both enantiomeric forms [20]. The ICCA method was later used,
among others, to track an asymmetric synthetic reaction [21].
In our experiments, we compared synthesised (R)-STD to the
reference material of the (S)-isomer, and based on the degree of
spectral compatibility, the proper course of the synthesis was
confirmed and the appropriate spectrum assigned to the corre-
sponding (S)- or (R)- enantiomer. The same strategy was previously
proposed by us for other antibacterial agents, tedizolid [9] and
radezolid [12,13].
However, the existence of single bond between sutezolid rings is
responsible for the lack of conformational stability of STD, resulting
in a low Cotton effect in both aqueous and organic environments in
ECD spectra. Using the time-dependent density functional theory
(TD-DFT), theoretical UV and ECD spectra for STD were calculated
as a weighted average of the conformers’ contribution according to
their Boltzmann equilibrium populations based on free energy
values. The calculations were carried out taking into account the
effects of the solvent and using the protocol proposed by Sardella
et al. [22].
The vibrational infrared spectra of intermediates and final
products of STD synthesis were recorded using a Thermo Scientific
Nicolet 8700 spectrometer between 7000 and 400 cmꢀ1
.
3.2. Spectroscopic analysis, Raman scattering
Raman scattering spectra were obtained with a LabRAM HR800
spectrophotometer (HORIBA Jobin Yvon) with laser excitations at
lexc ¼ 633 nm (Stabillite 2017 Ar laser and HeeNe laser). In each
case, the power of the laser beam at the sample was less than 1 mW
to avoid damage to the samples.
3.3. Electronic circular dichroism
The spectra of (S)- and (R)-STD were measured in water and
MeOH, using the Jasco J-715 (Easton, MD, USA) Circular Dichroism
spectrometer and were collected using a 50 nm/min scanning
speed, a step size of 0.2 nm and an integration time of 0.5 s in the
range selected according to the solvent used. (S)-STD was measured
in MeOH at a concentration of 0.28 mmol/L, (R)-STD in MeOH at a
concentration of 0.26 mmol/L, (S)-STD in water at a concentration
of 0.37 mmol/L and (R)-STD in water at a concentration of
0.12 mmol/L.
3.4. Theoretical approach
Considering the challenges related to the chemical and optical
identification of STD in relation to the six-step of chiral pool syn-
thesis of STD (Scheme 1), the aim of this work was to develop a
reliable, reproducible and low-cost approach to confirm the iden-
tity of intermediates and STD (Fig. 2) by FT-IR, Raman scattering and
ECD spectra. Due to a lack of reference material, the theoretical
spectra obtained as a result of quantum chemical calculations based
on the DFT, calculations utilizing the B3LYP hybrid functional and
the 6-311G(d,p) basis set were applied as the reference spectra for
experimental FT-IR and Raman scattering, while TD-DFT calcula-
tions were proposed to ECD spectra.
3.4.1. FT-IR and Raman spectroscopy
In order to interpret the experimental results of FT-IR absorption
and Raman scattering investigations, the quantum chemical cal-
culations were performed using the Gaussian 09 package [27].
Theoretical FT-IR and Raman spectra of STD were obtained with the
use of density functional theory calculations using Becke’s three-
parameter hybrid functional (B3LYP) with 6e311G(d,p) basis set.
The GaussView program was utilized to propose an initial geometry
of the investigated molecules and for visual inspection of the
normal modes. The molecular geometries were optimized using the
Density Functional Theory (DFT) method with the B3LYP hybrid
functional and the 6e311G(d,p) basis set. The calculated vibrational
frequencies were scaled by 0.964 in order to improve the agree-
ment with experimental values. Although the theoretical spectra of
STD were calculated for an isolated molecule in a gas state, differ-
ences between experimental and scaled wavenumber values were
small; therefore, a detailed interpretation of FT-IR and Raman
spectra of STD was possible.
2. Materials and methods
2.1. Synthesis procedures
(R)-STD was synthesised in our laboratory as described in the
patent [23] with slight modifications [24e26] from step 1 to step 6
(Scheme 1), while (S)-STD was purchased from Axon Medchem B.V.
(The Netherlands, EU). The following materials and reagents were
used for synthesis: silica gel 60 (70e230 mesh) was purchased from
Merck GmbH (Darmstadt, Germany); 3,4-difluoronitrobenzene
(S1), N,N-diisopropylethyl amine (S1), thiomorpholine (S1), so-
dium hydrosulfite (S2), N,N-dimethylaniline (S2), benzyl-
chloroformate (95% purity) (S2), (S)-glycidyl butyrate (S3), n-
butyllithium (1.6 M in hexane) (S3), trimethylamine (S4), p-
3.4.2. Chiroptical spectroscopy
Stereochemical characterization by time-dependent density
functional theory (TD-DFT) calculations were determined for (R)-
sutezolid using the standard protocol [22]. Because STD is a rather
simple molecule, but with many single rotational bonds, all of its
stable, low-energy 180 conformers have been generated and

4
K. Michalska et al. / Journal of Molecular Structure 1217 (2020) 128396
Scheme 1. Reaction pathways of sutezolid synthesis.
optimized by DFT methods using the B97D functional in combi-
nation with the TZVP basis set and the IEFPCM salvation model for
water and MeOH. All DFT and TD-DFT calculations were carried out
using the Gaussian 09 software [24]. The PBE0 (PBE1PBE) func-
tional [28] was used with the TZVP basis set and the IEFPCM sol-
vation model for TD-DFT calculations. Theoretical values of
oscillator strength (fj), rotational strength in dipole velocity
4. Results and discussion
4.1. Vibrational characterization of sutezolid
Spectral methods such as FT-IR and Raman scattering due to the
low cost of analysis, simple sample preparation procedures and the
ability to test in glass packaging are important for intraoperative
quality control of API synthesis. Therefore, the identities of key
intermediates and final products of STD synthesis were confirmed
by analysis of their FT-IR and Raman spectra. In the first stage, the
locations of characteristic STD bands (types and intensities) were
determined. The bands were identified in accordance with the
theoretical spectra. Assignment of the vibrational model for the
abovementioned domains in the molecule in the FT-IR and Raman
regions is summarized in Table 1. Ultimately, it was possible to
interpret the bands for the following STD molecules: (methyl)
acetamide group and 3-flurophenyl, thiomorpholine and oxazoli-
dine-2-one rings (Table 1, Figs. 3e4). Bearing in mind the consistent
complementation of FT-IR and Raman techniques in the following
analysis, the authors refer to those bands that were visible on the
spectra of STD registered with both techniques.
formalism (Rj) and excitation energy (expressed as wavelength, lj)
were calculated for 50 lowest energy electronic transitions of each
optimized conformer. The theoretical spectra of the optimized
conformers were then derived by approximation of fj and Rj values
to Gaussian bands with a Ds value of 0.25 eV [29]. The theoretical
ECD and UV spectra of the STD were finally derived as the weighted
average of the contribution of conformers according to their
Boltzmann equilibrium populations at 298.15 K and 1 atm, based on
free energy values (cG). Table 1S (in the supplementary material)
presents all optimized unique 120 conformers of STD including free
energy values and Boltzmann populations.

K. Michalska et al. / Journal of Molecular Structure 1217 (2020) 128396
5
Fig. 2. Optimized geometry of the key intermediates of the sutezolid (DFT) method with the B3LYP hybrid functional and the 6-311G(d,p) basis set.
The most characteristic two bands related to the stretching vi-
bration of the C]O bonds in the (methyl)acetamide group and
oxazolidine-2-one ring were clearly visible in the Raman scattering
spectrum, and these bonds were located above 1662 and
1750 cmꢀ1. The second band was also visible in the IR absorption
spectrum at 1749 cmꢀ1. The stretching vibration frequencies of the
CeO bonds in the oxazolidine-2-one ring were located between
1105 and 1013 cmꢀ1 and were visible in both Raman scattering and
FT-IR spectra. These bands were less intense and also had addi-
tional components associated with the stretching vibration of the
CeC bonds in the 3-fluorophenyl ring and stretching vibration of
the CeN bond in the thiomorpholine ring. Bands corresponding to
the stretching vibration of the CeN bond had similar intensities in
the oxazolidine-2-one ring and were located at 1205 cmꢀ1 in the
Raman scattering spectrum and at 1324/1322 and 1427/1422 cmꢀ1
in the FT-IR/Raman scattering spectra, respectively. The first and
the second bands at 1205 and 1322 cmꢀ1 had additional compo-
nents associated with the stretching vibration of the CeF bond. The
last band was also associated with the CeH bond vibrations, while
the band in the IR absorption spectrum at 1518 cmꢀ1 corresponded
to the stretching vibration of the CeN bond in the (methyl)acet-
amide group. A rather strong band observed in the Raman scat-
tering spectrum at 749 cmꢀ1 and in the IR at 747 cmꢀ1 was related
to the bending vibration out of the plane of CeN]O bonds in the
oxazolidin-2-one ring. One of the most intense bands in the Raman
scattering spectrum, located at 1629 cmꢀ1, corresponded to the
stretching vibration of the C]C bond in the 3-fluorophenyl ring. A
band related to the stretching vibration with respect to the CeC in
the 3-fluorophenyl ring was also observed in the IR absorption
spectrum. For this ring, bands related to the wagging and stretching
vibration of the CeH bonds (Table 1) were also observed. For the
thiomorpholine ring, mainly bands associated with bending and
stretching of CeH bonds were observed, whereas at approximately
1037 cmꢀ1 a band related to the stretching vibration of the CeC
bonds was noted. Bands corresponding to the stretching vibration
of the CeC and CeN bonds were located at 1125 and 1260 cmꢀ1 in
the IR and Raman scattering spectra, respectively. In the FT-IR ab-
sorption spectrum at 3477 cmꢀ1, a band related to the stretching
vibration of the NeH bond in the (methyl)acetamide group was
also visible. At approximately 3100e2800 cmꢀ1, bands related to
the stretching vibration of the CeH bonds were observed. At lower
frequencies (900-500 cmꢀ1, as seen in Table 1) bands associated
with the breathing and deformation of all rings were noted. In this
region of the Raman scattering spectrum at 665 cmꢀ1, the band
corresponding to the stretching vibration of the CeS bond in the
thiomorpholine ring was also observed.
4.2. Vibrational characterization of intermediate products of
sutezolid synthesis
Spectral analysis to identify key intermediates of STD synthesis
was carried out with reference to S1, S2, S4 and S5 (Figs. 5 and 6). As
in the case of STD, the theoretical spectra were generated for all
products, indicating the bands that could be considered as refer-
ence bands. The spectra of key intermediates and STD in the study
were described in terms of the characteristic positions and in-
tensities of their bands. In all spectra of all key intermediates,
characteristic bands associated with the same vibration of bonds
were observed. Most of such bands were observed in the interval
between 1700 and 1400 cmꢀ1. Most bands were associated with
stretching vibrations CeN, CeC, C]C and C]O. Obviously, the
teams (bonding pairs) were moved and had different intensities
and shapes, but they were related to the same vibration in all
products. For instance, the bands associated with the stretching
vibration of the C]O bonds in the oxazolidine-2-one ring of the IR
absorption spectra were located at approximately 1706, 1735, 1749
and 1749 cmꢀ1 for S2, S4, S5 and STD, respectively, whereas the
bands corresponding to the stretching vibration of the C]C bonds
in 3-fluorophenyl were observed at 1604, 1621, 1631, 1626 and
1641 cmꢀ1 for S1, S2, S4, S5 and STD, respectively. Another char-
acteristic feature was the band associated mainly with the
stretching vibration of the CeN bond in the thiomorpholine ring,
stretching vibration of the CeC bonds and bending vibration of the
CeH bonds in the 3-fluorophenyl ring and scissoring vibration of
the CeH bonds in the oxazolidine-2-one ring. They were observed
at approximately 1512, 1529, 1518, 1520 and 1518 cmꢀ1 for S1, S2,
S4, S5 and STD, respectively. For STD, this band has additional
components related to the stretching vibration of the CeN bond in

6
K. Michalska et al. / Journal of Molecular Structure 1217 (2020) 128396
Table 1
Selected characteristic vibriations features of all intermediate products and sutezolid in theory with application of 6e31G(d,p) basis and experiment bands; s-stretching, w-
wagging, t-twisting, r-rocking, sc-scissoring, b-bending, oop-out of plane, def.-deformation.
S1
S2
S4
S5
S6-sutezolid
IR/Raman
Bands assignment
IR/Raman
DFT
IR/Raman
DFT
IR/Raman
DFT
IR/Raman
DFT
DFT
-/391
-/444
391
444
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
breathing thiomorpholine ring
def. thiomorpholine ring
SO2 sc in SO3CH3 þ CeH oop in 3-
fluorophenyl
e
e
477/-
481
e
509/510
515
e
e
e
e
e
e
e
e
e
e
e
e
e
e
def. thiomorpholine ring and 3-
fluorophenyl
CeS s between oxazolidine-2-one and
SO3CH3 þ SeO sc þ CeOeS b
NeH oop
e
531/-
535
e
e
e
533/536
541
e
e
e
e
e
e
e
e
e
e
e
e
e
e
-/547
550
def. 3-fluorophenyl þ CeH w in
thiomorpholine
548/548
565
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
CeCeC oop in 3-fluorophenyl
rocking ring 3-fluorophenyl and
oxazolidine-2-one
-/576
580
e
595/595
598
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
rocking ring 3-fluorophenyl
def. all rings
-/610
608
e
e
615/615
637
e
e
e
e
e
e
def. benzyloxycarbonyl
e
639/639
658
e
669/669
e
657
e
655/-
e
658
e
669/668
e
655
e
e
e
def. thiomorpholine and 3-fluorophenyl
CeS s in thiomorpholine þ def. 3-
fluorophenyl and oxazolidine-2-one
CeS s in thiomorpholine
SeO s þ CeS s between oxazolidine-2-
one and SO3CH3 þ def. oxazolidine-2-
one
-/665
661
e
-/662
688
e
696/700
e
687
e
e
e
e
e
e
e
e
e
e
e
670/-
693
e
718/-
714
713/711
-/723
716
736
717/-
723
719/719
725
-/725
725
CeH oop in benzyloxycarbonyl and def.
all rings
CeOeC b in benzyloxycarbonyl þ def.
thiomorpholine and 3-fluorophenyl
OeNeO oop
CeN]O oop in oxazolidine-2-one þ def.
3-fluorophenyl þ rot. C]O in
oxazolidine-2-one
e
e
e
e
e
e
e
e
744/747
742
e
e
e
e
e
e
e
e
e
e
e
733/-
756
745/752
756
747/749
756
e
e
e
e
e
754/-
772
e
e
e
e
e
e
e
e
CeS s in SO3CH3
e
e
781/778
785
e
e
def. 3-fluorophenyl and
benzyloxycarbonyl
806/805
826/-
824
839
e
e
e
e
e
e
e
e
e
OeNeO sc
CeH oop in 3-fluorophenyl
CeCeO b in
820/-
837/839
813
835
815/-
e
810
e
812/-
e
809
e
805/-
e
810
e
e
e
e
benzyloxycarbonyl þ breathing
benzene ring in benzyloxycarbonyl
breathing oxazolidine-2-one ring ¼ C
eN s in (methyl)acetamide þ CeC s in
(methyl)acetamide
CeF s in 3-fluorophenyl þ NeCeO b in
oxazolidine-2-one þ breathing ring
oxazolidine-2-one þ def.
e
e
e
e
e
e
e
e
e
e
-/850
852
862/-
871
864/862
869
e
e
thiomorpholine and 3-fluorophenyl
breathing thiomorpholine ring and
oxazolidine-2-one þ CeO s in
oxazolidine-2-one þ def. 3-
fluorophenyl and oxazolidine-2-one
CeH oop in 3-fluorophenyl
def. thiomorpholine, 3-fluorophenyl
and oxazolidine-2-one þ CeN s in azide
CeH oop in benzyloxycarbonyl
880/-
903
895/-
905
908/-
907
881/-
900
914/914
912
892/892
911
e
e
e
e
e
e
e
e
e
e
e
e
e
928/-
926
920/-
922
e
e
e
937/-
935
e
e
e
e
e
e
e
e
e
e
e
e
e
928/-
943
CeN s in azide þ rot. N]N and N^N
CeN s in 3-fluorophenyl
rot. N]N and N^N
e
938/943
955
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
952/953
965
e
963/-
985
973/-
988
970/-
980
973/969
986
970/973
987
CeSeC b in thiomorpholine þ CeC s in
thiomorpholine þ CeN s in
thiomorpholine þ CeO s between
oxazolidine-2-one and SO3CH3
CeH t in thiomorpholine
e
e
e
e
e
e
e
e
e
-/998
e
1010
e
e
e
e
-/997
e
1020
e
e
e
e
e
breathing benzyloxycarbonyl
982/-
1018
1011/1010
1025
1013/-
1027
CeO s in oxazolidine-2-one and
SO3CH3 þ CeC s in 3-fluorophenyl
CeC s in thiomorpholine
1013/-
1025/1028
1043
1054
-/1009
e
1045
e
e
e
e
e
e
e
1039/-
1056
1035/1034
1055
1037/1036
1056
CeH r in thiomorpholine þ breathing 3-
fluorophenyl þ CeC s in
thiomorpholine

K. Michalska et al. / Journal of Molecular Structure 1217 (2020) 128396
7
Table 1 (continued )
e
e
1089
1068/-
1067
e
e
e
e
e
e
e
CeO s in benzyloxycarbonyl þ CeH w in
thiomorpholine and 3-fluorophenyl
CeC s in oxazolidine-2-one þ CeH r in
oxazolidine-2-one
e
e
e
e
e
1070/-
e
1079
e
1064/-
e
1078
e
e
e
1076/1072
e
e
e
CeN s in 3-fluorophenyl þ def. CeH in
3-fluorophenyl
e
e
1101/-
1112
1089/1093
1101
1082/1084
1101
CeO s in oxazolidine-2-one þ CeH w in
3-fluorophenyl þ CeC s in 3-
fluorophenyl
e
e
e
1113
1136
e
e
e
e
e
e
CeH b in benzyloxycarbonyl
e
1123/-
1139
1116/1116
1113/-
1129
-/1112
1128
1105/1105
1128
CeN s in thiomorpholine þ CeH sc in
thiomorpholine and 3-fluorophenyl þ C
eH t in 3-fluorophenyl þ CeF s in 3-
fluorophenyl þ CeO s in oxazolidine-2-
one þ CeC in all rings
1146/
1166
1138/-
1160
e
e
e
e
e
e
CeN s in thiomorpholine þ CeH sc in
thiomorpholine and 3-fluorophenyl þ C
eH t in 3-fluorophenyl þ CeF s in 3-
fluorophenyl
e
e
e
1154/-
1174
1154/-
1173
1157/-
1173
CeH w in thiomorpholine and 3-
fluorophenyl and oxazolidine-2-
one þ CeF s in 3-fluorophenyl þ CeN s
in 3-fluorophenyl þ CeC s in 3-
fluorophenyl
e
e
1172/-
1188
e
e
e
e
e
e
e
CeF s in 3-fluorophenyl þ CeH b in 3-
e
fluorophenyl
1165/1166
1194
e
e
e
e
e
1173/-
1194
e
-/1176
1197
e
CeN s in thiomorpholine þ CeH t in
thiomorpholine
CeF s in 3-fluorophenyl þ CeN s in 3-
fluorophenyl and oxazolidine-2-
one þ CeH w in 3-fluorophenyl and 2-
oxazolidinone
e
1202/-
1213
e
e
e
e
e
1203/-
1218
e
e
e
e
e
e
CeN s in 3-fluorophenyl þ CeH t in
thiomorpholine þ CeNeH b in 3-
fluorophenyl
CeN s in oxazolidine-2-one þ CeH t in
thiomorpholine and oxazolidine-2-
one þ CeH sc in thiomorpholine and 3-
fluorophenyl
e
e
e
e
1219/-
1222
1200/-
1221
1201/-
1222
1204/1203
1232
1216/-
1231
e
e
e
e
-/1205
1232
CeH t in thiomorpholine þ CeF s in 3-
fluorophenyl þ CeN s in
thiomorpholine and 3-fluorophenyl
CeN s in thiomorpholine and 3-
fluorophenyl and oxazolidine-2-
one þ CeH t in thiomorpholine and 3-
fluorophenyl and oxazolidine-2-one
CeF s in 3-fluorophenyl þ CeH t in
thiomorpholine and 3-fluorophenyl þ C
eN s in thiomorpholine and 3-
fluorophenyl and oxazolidine-2-
one þ CeC s in thiomorpholine
CeH t in thiomorpholine þ breathing 3-
fluorophenyl
CeH w in thiomorpholine and 3-
fluorophenyl
CeN s in thiomorpholine and 3-
fluorophenyl and oxazolidine-2-
one þ CeH w in all molecule
CeH t in (methyl)acetamide
e
e
e
1221/-
1246
1216/1215
1245
e
e
e
1228/-
1253
1235/1231
1257
1230/-
1258
1224/-
1258
1225/-
1257
1233/1236
1255/1254
1261
1281
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
-/1262
1285
1260/1261
1290
-/1260
1290
e
e
e
e
e
e
e
e
e
e
e
e
e
e
1288/-
1295
1283/1298
1297
e
e
CeC s in 3-fluorophenyl þ CeN s in 3-
fluorophenyl þ CeO s in
benzyloxycarbonyl
1299/-
1324
1307/1301
1321
1293/-
1322/-
1322
1334
e
e
-/1297
1322
CeH t in thiomorpholine and
oxazolidine-2-one þ S]O s in SO3CH3
CeN s in thiomorpholine, 3-
fluorophenyl, oxazolidine-2-one and
azide þ CeC s in thiomorpholine and 3-
fluorophenyl þ CeH w in
e
e
e
1330/1335
1328
e
e
e
thiomorpholine and oxazolidine-2-one
CeH w in thiomorpholine þ CeC s in 3-
fluorophenyl
1325/-
1340
-/1331
1340
e
e
e
e
e
e
e
e
e
e
e
e
e
1341/1346
1344
CeN s in 3-fluorophenyl þ CeF s in 3-
fluorophenyl þ N]N s in azide þ CeC s
in 3-fluorophenyl
e
(continued on next page)

8
K. Michalska et al. / Journal of Molecular Structure 1217 (2020) 128396
Table 1 (continued )
e
e
e
e
e
e
e
e
e
e
e
e
1322/1324
1356
CeN s in oxazolidine-2-one þ CeF s in
3-fluorophenyl þ CeH w in all molecule
N¼N s in azide þ CeN s in 3-
fluorophenyl þ CeC s in 3-fluorophenyl
and 3
e
e
1367/1363
1358
e
e
e
1369
e
e
1359/-
1362
e
e
e
e
CeH sc in CH3 and in SO3CH3 þ CeN s in
e
3-fluorophenyl
1332/1322
e
e
e
e
e
e
e
e
e
e
e
e
e
e
CeN s in 3-fluorophenyl
CeC s between oxazolidine-2-one and
azide
e
1382/1381
1407
e
1416/1426
1424
1420/1410
1422
1421/-
1447/-
1419
1437
1422/-
-/1425
1420
1432
1418/-
1417
1431
CeH w in thiomorpholine and
oxazolidine-2-one þ CeN s in
thiomorpholine and oxazolidine-2-one
CeN s in thiomorpholine and 3-
fluorophenyl and oxazolidine-2-
one þ CeH sc in thiomorpholine and þ
CeH w in oxazolidine-2-one
CeC s in 3-fluorophenyl þ CeN s in 3-
fluorophenyl and benzyloxycarbonyl
and in oxazolidine-2-one þ CeH sc in
thiomorpholine and 3-fluorophenyl
CeH sc in thiomorpholine and
benzyloxycarbonyl
e
e
e
1427/1422
e
1453/1452
1492/1497
1478
1497
1452/1452
1489/1487
1463
1490
1454/-
1487/-
1471
1497
1450/1449
-/1486
1471
1495
1455/1456
1476/1477
1472
1496
e
e
e
1505/-
1518/-
1529
1544
1499/1496
1520/1527
1532
1544
1492/-
1518/-
1530
1542
CeH sc in oxazolidine-2-one
e
1512/-
1535
1529/1525
1556
CeN s between thiomorpholine and 3-
fluorophenyl and
benzyloxycarbonyl þ CeC s in 3-
fluorophenyl þ CeH b in 3-
fluorophenyl þ CeH sc in oxazolidine-
2-one þ CeN s in (methyl)acetamide
NeO s þ CeC s in 3-fluorophenyl
C¼C s in 3-fluorophenyl þ NeO s in 3-
fluorophenyl
1533/-
1585
e
e
e
e
e
e
e
e
e
1589/1586
1622
1571/-
1602
1575/-
1602
1564/-
1602
e
1604/1601
1644
1621/1622
1667
1631/-
1666
1626/1630
1665
1641/1629
1665
C¼C s in 3-fluorophenyl þ NeO s in 3-
fluorophenyl
e
e
e
e
e
e
e
e
e
-/1662
1749/1750
e
1763
1846
e
C¼O s in (methyl)acetamide
C¼O s in oxazolidine-2-one
N^N s þ N]N s in azide
CeH s in thiomorpholine
CeH s in thiomorpholine
CeH s in oxazolidine-2-one
e
e
1706/1707
1792
e
1735/-
1850
e
1749/1745
-/2439
1851
2255
e
e
e
2676/-
2740/-
2966
2978
e
2689/-
2757/-
e
2936
2953
e
2638/-
2750/-
e
2938
2955
e
2681/-
2755/-
2909/2904
2938
2955
2991
e
e
2760/2757
e
2955
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
-/2915
3021
e
e
e
CeH s in oxazolidine-2-one and azide
CeH s in (methyl)acetamide
e
e
-/2838
-/2905
3025
3045
e
CeH s in (methyl)acetamide
e
2846/2849
3050
e
e
e
e
e
2839/-
e
3050
e
e
e
e
e
e
e
CeH s in thiomorpholine
CeH s in oxazolidine-2-one and azide
2937/2937
3095
e
2974/2975
3111
e
2950/2952
-/3059
3106
3192
2970/-
3082/-
3106
3192
2960/2963
3016/3016
3106
3193
e
e
CeH s in thiomorpholine
CeH s in 3-fluorophenyl
3079/-
3194
e
S1
S2
S4
S5
S6-sutezolid
IR/Raman
Bands assignment
IR/Raman
IR/Raman
DFT
IR/Raman
DFT
IR/Raman
DFT
IR/Raman
DFT
DFT
3084/3087
3232
e
3088/3086
3339/3340
3242
3626
3114/-
e
3246
e
3101/3099
e
3245
e
3243/-
e
3244
e
CeH s in 3-fluorophenyl
NeH s in benzyloxycarbonyl
e
e
e
e
e
e
e
e
e
3477/-
3638
NeH s in (methyl)acetamide
the (methyl)acetamide group. Another characteristic feature was
the band related to the stretching vibration of the CeC bonds in the
3-fluorophenyl ring and that of the CeN bond in 3-fluorophenyl,
the oxazolidine-2-one ring and S2 in benzyloxycarbonyl. This band
in the IR spectra was located at 1453, 1452, 1454, 1450 and
1455 cmꢀ1 for S1, S2, S4, S5 and STD, respectively. In the IR ab-
sorption spectra and the Raman scattering spectra of all products,
there were also many bands associated with the wagging, twisting,
scissoring or rocking vibration of the CeH bonds. In this range, for
example, bands at approximately 1420 and 1490 cmꢀ1 were
observed, which were mainly related to the wagging and scissoring
vibration of the CeH bonds in thiomorpholine and the oxazolidine-
2-one ring. Bands related to the vibration of the CeH bonds were
also visible at a lower frequency, for example, a band at approxi-
mately 820e800 cmꢀ1, which was associated with the out-of-plane
vibration of the CeH bonds in the 3-fluorophenyl ring, whereas the
band at approximately 720 cmꢀ1 corresponded to the deformation
of the all rings and out-of-plane vibration of the CeH bonds in
benzyloxycarbonyl in S2. The bands related to the breathing of the
thiomorpholine and oxazolidine-2-one rings and deformation of all
rings were located at 880, 895, 908, 881 and 914 cmꢀ1 for S1, S2, S4,
S5 and STD, respectively. These bands also had an additional
component related to the stretching vibration of the CeO bond in
the oxazolidine-2-one ring. The most common characteristic of all
products was the presence of bands at approximately 963, 973, 970,
973 and 970 cmꢀ1 for S1, S2, S4, S5 and STD, respectively, mainly

K. Michalska et al. / Journal of Molecular Structure 1217 (2020) 128396
9
stretching vibration of the CeO bond between the oxazolidine-2-
one ring and the SO3CH3 group. Another characteristic feature
was a band at approximately 1230e1225 cmꢀ1, mainly related to
the stretching vibration of the CeF bond in the 3-fluorophenyl ring
and that of the CeC and CeN bonds in the 3-fluorophenyl, thio-
morpholine and oxazolidine-2-one rings. Similarly, a band at
approximately 1125e1105 cmꢀ1 was associated with a similar vi-
bration, which was at approximately 1225 cmꢀ1. Obviously, above
2500 cmꢀ1, there were many bands associated with the stretching
vibration of the CeH bonds in all products.
FT-IR and Raman scattering spectra also have characteristic
bands for individual intermediates. For S1, the most common
characteristic bands were associated with the out-of-plane vibra-
tion and scissoring vibration of the OeNeO bonds that were
located at 744 and 806 cmꢀ1. A band related to the stretching vi-
bration of the NeO and CeC bonds was also observed at 1533 cmꢀ1
.
In this product, bands associated with the stretching vibration of
the CeN bonds were also observed, for example, at 1076 and
1332 cmꢀ1. For S1, several bands were also recorded corresponding
to the deformation, rocking and breathing of the 3-fluorophenyl,
and thiomorpholine rings, especially at a lower frequency.
Fig. 3. Calculated (black 1) and experimental (red 2) FT-IR absorption spectra of STD at
room temperature, in polycrystalline powder.
For S2, the most common characteristic bands were related to
the vibration of the bands in the benzyloxycarbonyl group. The
bands associated with the deformation and breathing of the ben-
zene ring were observed here, for example, the bands at 615, 781
and 997 cmꢀ1. The bands at 723 and 837 cmꢀ1, related to the
bending vibration of the CeOeC and CeCeO bonds in the benzy-
loxycarbonyl group, were also characteristic features. In addition,
bands corresponding to the out-of-plane vibration of NeH and CeH
bonds at 533 and 937 cmꢀ1, respectively, were also observed. The
band related to the stretching vibration of the NeH bond was
located at 3339 cmꢀ1
.
In the case of S4, bands related to the vibration of the SeO and
CeS bonds can be distinguished. For example, the bands at 477 and
670 cmꢀ1 were related to the scissoring vibration of the SO2 and
stretching vibration of the SeO and CeS bonds. The bands at 531
and 754 cmꢀ1 were also related to the stretching vibration of the
CeS bond between the oxazolidine-2-one ring and SO3CH3, but the
first of these has additional components related to the bending
vibration of the CeOeS bonds and scissoring vibration of the SeO
bonds, while the band at 1359 cmꢀ1 corresponded to the scis-
soring vibration of the CeH bonds in the CH3 group.
For S5, the most common characteristic bands were related to
the vibration of the N]N and N^N bonds. For example, the bands
at 928 and 952 cmꢀ1 were associated with the rotation of these
bonds. The first band also had additional components related to the
stretching vibration of the CeN bond in azide, whereas the bands at
1341 and 1367 cmꢀ1 were related to the stretching vibration of the
Fig. 4. Calculated (black 1) and experimental (red 2) Raman scattering spectra of STD
at room temperature, in polycrystalline powder.
related to the bending vibration of the CeSeC bond in the thio-
morpholine ring. Clearly, they have additional components corre-
sponding to the stretching vibration of the CeC and CeN bonds in
the thiomorpholine ring. For S4, this band was also related to the
Fig. 5. FT-IR theoretical (A) and experimental (B) of S1, S2, S4 and S5.

10
K. Michalska et al. / Journal of Molecular Structure 1217 (2020) 128396
Fig. 6. Raman scattering theoretical (A) and experimental (B) of S1, S2, S4 and S5.
N]N bond. These two bands were also additional components
corresponding to the stretching vibration of the CeN, CeC and CeF
bonds in the 3-fluorophenyl ring. However, an approximately
2439 cmꢀ1 band was observed related only to the stretching vi-
bration of the N]N and N^N bonds, whereas the band related to
the stretching vibration of the CeC bond between the oxazolidine-
2-one ring and azide was located at approximately 1382 cmꢀ1
Bands related to the stretching vibration of the CeH bonds in the
benzyloxycarbonyl group were also observed above 2900 cmꢀ1
.
.
4.3. The spatial structure of sutezolid
As mentioned earlier, STD is characterized by a lack of confor-
mational stability, therefore its single structure cannot be defined.
However, DFT calculations suggest that two main conformations
with the lowest free energies can be identified (Table 1S in the
supplementary material). Both make up about 16% in the Boltz-
mann population. They have non-linear conformations with an
amide group directed at the aromatic ring (Fig. 7, conformer 1 and
conformer 2). They differ only by twisting the molecule in the
middle by 180ꢁ around the CeN bond (between the oxazolidine-2-
one and 3-fluorophenyl rings).
Fig. 8. Experimental ECD and UV spectra of (S)- and (R)-sutezolid in aqueous solution
(blue, black lines) and in methanol (red, green lines).
The solid state structure of X-ray crystallography is known for
LIN, a compound that differs from STD only by the presence of an
oxygen atom instead of sulfur in the morpholine ring [30]. There
are two crystallographically independent molecules in the crystal.
Fig. 7. DFT calculated structures of (R)-sutezolid.

K. Michalska et al. / Journal of Molecular Structure 1217 (2020) 128396
11
Fig. 9. Theoretical ECD and UV spectra of (R)-sutezolid calculated by TD-DFT.
One of them is very similar to the conformation 4 calculated for STD
and the other is similar to conformation 8 (Fig. 7 and Table 1S in the
supplementary material). In fact, they become identical after con-
verting oxygen atoms to sulfur and minimizing their structures.
probably caused by the fact that a relatively simplified solvent
model was used in the calculations. Unfortunately, the use of better
solvent models goes beyond our current computational capabil-
ities. Experimental and calculated UV spectra also show great
similarity.
4.4. Confirmation of the configuration of sutezolid
5. Conclusions
Optical identification of STD isomers was carried out by ana-
lysing electronic circular dichroism spectra. As a test sample for this
stage of research, the ECD spectrum of the (R)-isomer of STD ob-
tained as a result of synthetic modification was adopted. (R)-STD
was prepared from (S)-glycidyl butyrate as a chiral carrier to use
with the reaction of a carbamate to form the oxazolidine-2-one
ring, which enabled only one enantiomerically pure, desirable
oxazolidinone derivative. Assuming a mirror image of both (R)- and
(S)-enantiomers, as a reference material (S)-STD, commercially
available isomer was used.
Since it is well known that the ECD spectra depend on the sol-
vent used [31], we tested aqueous and nonaqueous (MeOH) solu-
tions. Initially, the ECD spectra of both enantiomers of STD were
determined in water (Fig. 8). Two Cotton effects (CEs) with opposite
signs were observed at approximately 206 nm and 255 nm: posi-
tive and negative sequence of CEs for the (R)-isomer and vice versa
for the (S)-enantiomer. The ECD spectrum of (R)-STD recorded in
MeOH exhibited two negative, not fully resolved CEs at around
212 nm and 220 nm and one negative, broad band at 260 nm (Fig. 8,
pink lines).
The application of FT-IR with Raman techniques can be recom-
mended for the identification of STD and products of its synthesis.
Moreover, experimental ECD spectra together with TD-DFT theo-
retical spectra were found to be suitable for confirmatory evalua-
tion of the configuration of STD. All of the presented approaches to
chemical and optical identification of STD are valuable in terms of
the cost of analysis, reliability, reproducibility and time to imple-
mentation. Considering the difficulty, cost and unfavorable envi-
ronmental impact of analyses allowing the assessment of optical
purity, attention should be paid to the possibility of applying a
spectral approach in the analytical screening of chiral analogues of
oxazolidinone derivatives.
Acknowledgments
This study was supported by a SONATA grant from the Poland-
UMO-2013/11/D/NZ7/01230.
Thanks to Ewa Gruba for the synthesis of sutezolid from the
second (S2) to the sixth (S6) stages. Ewa was employed at the
Department of Antibiotic and Microbiology of the National Medi-
cines Institute until November 2017.
The (R)-isomer of STD recorded in MeOH showed identically
shaped ECD bands with negative signs for all Cotton effects com-
parison with (S)-STD, which ultimately confirms the sutezolid
configuration of the synthesised (R)-STD. Moreover, for (R)- and (S)-
STD, the comparison between the experimental ECD spectra of the
two enantiomeric fractions and the TD-DFT theoretical spectrum
(Fig. 9) allowed to clearly confirm the absolute configuration.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2020.128396.
Twenty-four conformers with the lowest free energy DG and a total
Boltzmann population of 90% were used for theoretical calculations
of the spectra. The use of a larger number of conformations has a
practically insignificant effect on the spectral shape, as shown by
simulating the spectrum for 58 cumulative conformations and 99%
of the Boltzmann population (Fig. 1S in the supplementary
material).
Regardless of the number of conformations compared, only one
stereoisomer always provides the ECD spectrum to bands of the
same sign in the calculated and experimental spectrum. The
calculated ECD spectrum shows only a slight solvent effect, it is
Author statement
Katarzyna Michalska: work concept, article preparation, data
analysis and interpretation of ECD spectra, synthesis of sutezolid at
stage S1, S2a, S2b, preparation of scheme 1sch1, revision of the
manuscript, Writing - review & editing. Kornelia Lewandowska -
conducting the FT-IR and Raman experiment. Mikołaj Mizera -
calculations using the DFT method - B3LYP. Wojciech Bocian - data
analysis and interpretation of TD-DFT calculations, Writing - review
& editing. Barbara Pałys - conducting Raman experiments; Judyta

12
K. Michalska et al. / Journal of Molecular Structure 1217 (2020) 128396
spectroscopy as
a process analytical technology for pharmaceutical
Cielecka-Piontek - analysis of the results of the FT-IR research,
Raman, conducted a sketch of the manuscript in part FT-IR and
Raman, response to the comments of the editor and reviewer.
manufacturing and bioprocessing, Anal. Bioanal. Chem. 409 (2017) 637e649,
https://doi.org/10.1007/s00216-016-9824-1.
[17] D. Custers, T. Cauwenbergh, J.L. Bothy, P. Courselle, J.O. De Beer, S. Apers,
E. Deconinck, ATR-FTIR spectroscopy and chemometrics: an interesting tool to
discriminate and characterize counterfeit medicines, J. Pharmaceut. Biomed.
Anal. 112 (2015) 181e189, https://doi.org/10.1016/j.jpba.2014.11.007.
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