Stereoselective synthesis of (?)-cytoxazone and its unnatural congener (+)-5-epi-cytoxazone

Article abstract of DOI:10.1002/chir.23334

An interesting protocol for stereoselective synthesis of (?)-cytoxazone and its unnatural stereoisomer (+)-5-epi-cytoxazone from d-4-hydroxyphenylglycine in overall yields of 10% and 16%, respectively, is described. The stereoselective addition of cyanide

Full text of DOI:10.1002/chir.23334

Received: 12 September 2020
DOI: 10.1002/chir.23334
Revised: 11 May 2021
Accepted: 28 May 2021
R E S E A R C H A R T I C L E
Stereoselective synthesis of (ꢀ)-cytoxazone and its
unnatural congener (+)-5-epi-cytoxazone
Izabel Luzia Miranda1 | Pedro Henrique Costa dos Santos1 | Markus Kohlhoff2
|
Gislaine Aparecida Purgato3 | Marisa Alves Nogueira Diaz3
| Gaspar Diaz-Muñoz1
1Department of Chemistry, Federal
Abstract
University of Minas Gerais, Belo
Horizonte, Minas Gerais, Brazil
An interesting protocol for stereoselective synthesis of (ꢀ)-cytoxazone and its
unnatural stereoisomer (+)-5-epi-cytoxazone from D-4-hydroxyphenylglycine
in overall yields of 10% and 16%, respectively, is described. The stereoselective
addition of cyanide to an N-Boc protected aminoaldehyde (tert-butyl
((R)-1-(4-methoxyphenyl)-2-oxoethyl)carbamate) (5) constitutes the key step in
this approach, producing a mixture of cyanohydrins 6a and b (1,2-anti and
1,2-syn tert-butyl (2-cyano-2-hydroxy-1-(4-methoxyphenyl)ethyl)carbamate) in
89% yield, with reasonable stereoselectivity (1.0:1.8) in favor of the anti-Felkin
product (1,2-syn). A one-pot sequence of three successive steps from this
mixture produced the oxazolidinone isomers 9a and b ((4R,5R)- and (4R,5S)-
4-(4-methoxyphenyl)-2-oxooxazolidine-5-carboxylate). Chromatographic col-
umn separation and reduction of the ester function of both precursors led to
(ꢀ)-cytoxazone and (+)-5-epi-cytoxazone.
2Chemistry of Bioactive Natural Products,
René Rachou Institute, Oswaldo Cruz
Foundation (Fiocruz), Belo Horizonte,
Minas Gerais, Brazil
3Department of Biochemistry and
Molecular Biology, Federal University of
Viçosa, Viçosa, Minas Gerais, Brazil
Correspondence
Gaspar Diaz-Muñoz, Department of
Chemistry, Federal University of Minas
Gerais, PO Box 31270-901, Belo
Horizonte, Brazil.
Email: gaspardm@qui.ufmg.br
Funding information
~
Fundaçao de Amparo à Pesquisa do
Estado de Minas Gerais; Conselho
Nacional de Desenvolvimento Científico
e Tecnolꢀogico
K E Y W O R D S
D-4-hydroxyphenylglycine, oxazolidinone, stereoisomer, stereoselective addition
1 | INTRODUCTION
cytokines by inhibiting the signaling pathway of T helper
type 2 cells (involved in cell growth and differentiation)
Oxazolidinones constitute a class of natural and synthetic
compounds with multiple biological activities. Naresh
and coworkers1 produced synthetic derivatives of
oxazolidinones with good anticancer activity against lung
A549 and prostate DU145 cancer cells and therapeutic
potential for the treatment of schizophrenia.1 The
biological versatility of oxazolidinones is also made
evident by their neuroleptic,2 psychotropic,3 and anti-
allergic activities.4
but not of T helper type 1 cells (responsible for
hypersensitivity reactions). Research indicates that an
imbalance in the production of cytokines can cause
immunological disorders, such as allergies, progressive
lymphoproliferation, and severe immunodeficiency,6 as
cytokines are directly linked to the production of
antibodies. The promising mechanism of action of
(ꢀ)-cytoxazone aroused the interest of researchers in
investigating its pharmacological potential.
Two oxazolidinones with interesting biological activi-
ties are (ꢀ)-cytoxazone and its unnatural congener
(+)-epi-cytoxazone (Figure 1). (ꢀ)-Cytoxazone was
isolated in 1998 by Kakeya and coworkers5 from bacteria
of the genus Streptomyces. A year later, Kakeya et al.6
observed that (ꢀ)-cytoxazone had a modulating effect on
The absolute configuration (4R,5R) of (ꢀ)-cyto-
xazone was confirmed by nuclear magnetic
resonance (NMR) spectroscopy, circular dichroism
spectroscopy, and X-ray crystallography by Sakamoto
et al.7 and Seki and Mori8 in the first total asymmetric
syntheses.
Chirality. 2021;33:479–489.
wileyonlinelibrary.com/journal/chir
© 2021 Wiley Periodicals LLC.
479

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MIRANDA ET AL.
FIGURE 1 Chemical structure of
(ꢀ)-cytoxazone and its unnatural epimer
(+)-5-epi-cytoxazone
Of note, Kumar et al.9 found (+)-5-epi-cytoxazone
1-methoxy-4-nitromethylbenzene
using
guanidine-
(Figure 1) to have higher antibacterial activity than
(ꢀ)-cytoxazone against Gram-positive Bacillus subtilis
and Gram-negative Escherichia coli. The discoveries made
by these groups encouraged the development of research
aimed at the total synthesis of (ꢀ)-cytoxazone and
congeners.5,6,10
thiourea as chiral catalyst provided nitro alcohol with
high 1,2-syn selectivity (90:10) and 99% enantiomeric
excess (Figure 2, route H). This adduct was the precursor
to (ꢀ)-epi-cytoxazone in the study of Sohtome and
coworkers.16
(ꢀ)-Cytoxazone was also synthesized by George and
coworkers in 200717 (Figure 2, route I). Diastereoselective
bromohydroxylation of α,β-unsaturated carboxamide in
the presence of NaIO4 and LiBr produced 1,2-anti bromo-
hydrin. The stereochemistry of the intermediate allowed
generating the correct stereocenters in (ꢀ)-cytoxazone.
Narina and coworkers18 reported an elegant
enantioselective synthetic route for (ꢀ)-cytoxazone
(Figure 2, route J). Two key steps were required to
introduce stereocenters: L-proline-catalyzed asymmetric
α-aminooxylation and Rh-catalyzed diastereoselective
oxidative C–H amination.
This literature survey revealed that the development
of a large number of synthetic routes for (ꢀ)-cytoxazone
and its congeners was motivated by the relevance of their
bioactivities. From a synthetic point of view, the
construction of the oxazolidinone carbon skeleton is
challenging because, although it is a relatively simple
molecule, it contains a five-membered ring with two
consecutive stereogenic centers. The facts discussed
above motivated us to develop this new study. Some
specific aspects of the project stand out: the key step,
considered of lower complexity than the routes reported
in the literature, was the stereoselective addition of
cyanide to N-Boc aldehyde 5, obtained from the low-cost
chiral substrate D-4-hydroxyphenylglycine. Another step,
no less important, was the chromatographic separation
of a mixture of oxazolidinone isomers 9a and b,
which corresponded to the advanced precursors of
(ꢀ)-cytoxazone and (+)-5-epi-cytoxazone, respectively.
Also noteworthy was the stereoselective synthesis of two
stereoisomers from a single common intermediate.
1.1 | Total syntheses of (ꢀ)-cytoxazone
and congeners: Background
The synthetic approaches for (ꢀ)-cytoxazone and conge-
ners published so far start from simple substrates, such as
cinnamic acid derivatives. Their β-amino acid and
α-hydroxy functionalities are introduced via Sharpless
enantioselective
epoxidation,
hydroxylation,
and
aminohydroxylation or by means of well-known (Evans
oxazolidinone, N-tert-butanesulfinylimines) and less-
known chirality inducers.10 Figure 2 shows a compilation
of some synthetic protocols as well as the key steps for
the construction of the basic skeleton of (ꢀ)-cytoxazone
and congeners.7–9,11–19
Sharpless asymmetric dihydroxylation (Figure 2, route A)
using AD-mix-α in t-BuOH/H2O was the key step in the con-
trol of stereogenic centers at C-4 and C-5 of (ꢀ)-cytoxazone,
according to Sakamoto et al.7 and Seki and Mori.8
Carter et al.12 explored the 1,2-syn aldol reaction
(Figure 2, route D), starting from chiral imide and an
aldehyde and mediated by Bu2BOTf, in the development
of a new enantioselective synthesis of (ꢀ)-cytoxazone.
Auxiliary removal and subsequent steps provided
enantiomerically pure (ꢀ)-cytoxazone in 84% yield.
By applying a new approach based on the Petasis
protocol,20 Sugiyama and collaborators13 developed a route
for the synthesis of (ꢀ)-cytoxazone (Figure 2, route E).
Application of the highly enantioselective Henry
(nitroaldol) reaction between an aldehyde and

MIRANDA ET AL.
481
FIGURE 2 Substrates and key steps involved in some synthetic routes to (ꢀ)-cytoxazone and congeners. A, Sharpless asymmetric
dihydroxylation; B, enzymatic kinetic resolution; C, stereoselective Grignard addition; D, Curtius rearrangement; D0, stereoselective aldol
addition; E, Petasis coupling; F, 1,2-Wittig rearrangement; G, reductive cross-coupling; H, diastereoselective Henry reaction; I,
stereoselective bromohydroxylation; J, stereoselective amination; K, Sharpless kinetic resolution
2 | MATERIALS AND METHODS
2.1 | General information
Foundation (Fiocruz) Natural Products Laboratory (610),
Instituto René Rachou (IRR), Belo Horizonte, MG. The
concentration of the solutions was denoted as c and was
calculated as grams per milliliter (g/100 ml), where the
solvent was indicated as (c, solvent). Purifications by
column chromatography were performed on silica gel,
using normal or flash chromatography, and neutral
alumina. Tetrahydrofuran (THF) was distilled from
sodium metal and benzophenone ketyl under nitrogen.
N,N-Dimethylformamide (DMF) and dichloromethane
were distilled from CaH2. Acetonitrile was dried with
anhydrous MgSO4, distilled, and stored under 3-Å
molecular sieve. Methanol and ethanol were dried using
Mg0 and I2 (cat.) under reflux until Mg0 total consump-
tion, distilled, and stored under 3-Å molecular sieve.
Thin-layer chromatography (TLC) visualization was
achieved in chamber under ultraviolet (UV) light
(λ 254 nm), and by spraying with KMnO4 solution (1.00-g
1
The H and 13C NMR spectra were recorded on a Bruker
Avance spectrometer at 400 and 100 MHz, respectively.
The liquid chromatography–tandem mass spectrometry
(LC–MS/MS) analyses were performed in a Nexera
UHPLC system (Shimadzu) hyphenated to a maXis
ETD high-resolution ESI-QTOF mass spectrometer
(Bruker). The infrared (IR) spectra were recorded on a
Fourier-transform infrared (FTIR) spectrometer Varian
660 with a diamond attenuated total reflectance (ATR)
accessory as a thin film. The melting points were mea-
sured in MQAPF-302 MICROCHEMICAL apparatus and
uncorrected. [α]D Values were measured using Anton
Paar MCP 300 polarimeter equipped with a 589-nm
wavelength sodium vapor lamp from the Oswaldo Cruz

482
MIRANDA ET AL.
KMnO4, 6.66-g K2CO3, and 1.66-ml 5% KOH in 100-ml
distilled water) and heating, and/or resublimated iodine.
All of the chemicals were used as received unless
otherwise stated. Analytical high-performance liquid
chromatography (HPLC) experiments were performed on
(C7), 52.80 (C8), 28.55 (C11). HRMS (ESI, m/z): [M
+ Na]+ calcd for C15H21NO5, 318.1312; found, 318.1320.
2.2.2 | (R)-(2-Hydroxy-1-(4-methoxyphenyl)
ethyl)tert-butyl carbamate (4)
a
Prominence LC-20AR Shimadzu using column
CHIRALCEL OD-R (250 ꢁ 4.6 mm, 10 μm), mobile
phase 100% acetonitrile, and UV–Vis detector. HPLC
grade acetonitrile was degassed and filtered on a 45-μm
Millipore membrane before use. Retention times Rt were
in minutes.
To a 100-ml flask containing ester 3 (0.806 g, 2.73 mmol)
in anhydrous ethanol (55 ml), under an argon atmo-
sphere, were added NaBH4 (0.413 g, 10.92 mmol) and
LiCl (0.463 g, 42.44 mmol). The mixture was kept under
stirring at room temperature for 18 h, an aqueous solu-
tion of HCl (30 ml, 1 mol mlꢀ1) and distilled water
(30 ml) were added, and the mixture was extracted with
ethyl ether (3 ꢁ 30 ml). The organic phase was washed
with a saturated solution of NaHCO3 (30 ml) and brine,
dried over Na2SO4, and concentrated under reduced
pressure. Recrystallization of the crude residue in ethanol
gave alcohol 4 in 92% yield as a white solid, mp
130.4–131.3ꢂC (literature,9 130–131ꢂC). Rf 0.4 (hexane/
2.2 | Synthesis
2.2.1 | (R)-2-((tert-Butoxycarbonyl)amino)-
2-(4-methoxyphenyl)methyl acetate (3)
A
mixture of
D-4-hydroxyphenylglycine
(2.0 g,
11.9 mmol), di-tert-butyldicarbonate (2.59 g, 11.9 mmol),
dioxane (22 ml), water (11 ml), and aqueous Na2CO3
(26 ml, 0.5 mol mlꢀ1) was stirred for 5 min at 0ꢂC under
an argon atmosphere and for a further 24 h at room tem-
perature. An aqueous solution of HCl (2 mol mlꢀ1) was
added slowly to adjust the pH to 2–3, and the mixture
was extracted with EtOAc (3 ꢁ 30 ml). The organic
phases were dried over Na2SO4 and concentrated
under reduced pressure. The crude N-Boc protected
4-hydroxy-D-phenylglycine thus obtained was used with-
out further purification. To a 100-ml flask containing the
residue were added anhydrous DMF (10 ml), anhydrous
K2CO3 (1.99 g, 14.4 mmol), and CH3I (3.6 ml,
57.6 mmol). After stirring for 8 h at room temperature,
the mixture was neutralized with an aqueous solution of
HCl (30 ml, 1 mol Lꢀ1) and treated with a saturated
solution of NaHCO3 (30 ml). The aqueous phase was
extracted with ethyl ether (3 ꢁ 30 ml). The organic phase
was washed with brine, dried over Na2SO4, and concen-
trated under reduced pressure. The residue was purified
by silica gel column chromatography (hexane/EtOAc
85:15), providing ester 3 (94% yield, two steps) as a
yellowish solid, mp 65.5–66.6ꢂC (literature,9 66–67ꢂC). Rf
EtOAc 1:1). [α]D = ꢀ40.1ꢂ (c = 1.0, CHCl3) (literature,9
23
25
[α]D = ꢀ43.59ꢂ [c = 1.09, CHCl3]). IV (neat): vmax 3373,
2983, 2937, 1682, 1613, 1585, 1514, 1462, 1390, 1366,
1350, 1319, 1303, 1284, 1266, 1245, 1167, 1111, 1090,
1
1052, 1030, 934, 910, 871, 837, 822, 782 cmꢀ1. H NMR
(400 MHz, CDCl3, δ) 7.21 (d, J = 8.5 Hz, 2H, Ar H), 6.87
(d, J = 8.55 Hz, 2H, Ar H), 5.27 (s, 1H, NH), 4.71 (s, 1H,
CH), 3.78 (s, 5H, CH2 and CH3), 2.67 (s, 1H, OH), 1.42
(s, 9H, CH3). 13C NMR (100 MHz, CDCl3, δ) 159.24 (C8),
156.27 (C6), 131.86 (C3), 127.84 (C4 e C40), 114.29
(C5 and C50), 80.01 (C9), 66.86 (C1), 56.62 (C2), 55.39
(C7), 28.46 (C10). HRMS (ESI, m/z): [M + Na]+ calcd for
C14H21NO4, 290.1357; found, 290.1367.
2.2.3 | (R)-(1-(4-Methoxyphenyl)-2-oxoethyl)
tert-butyl carbamate (5)
Swern oxidation
To a 50-ml flask containing freshly distilled oxalyl chlo-
ride (0.16 ml, 1.96 mmol) in anhydrous CH2Cl2 (14 ml)
under stirring at ꢀ78ꢂC and an argon atmosphere was
added anhydrous dimethyl sulfoxide (DMSO) (0.14 ml,
2.07 mmol) dropwise. After stirring for 30 min at the
same temperature, a solution of alcohol 4 (0.35 g,
1.31 mmol) in CH2Cl2 (10 ml) was added slowly. The
system was kept under stirring for 1 h, the temperature
was raised to ꢀ35ꢂC, and stirring was maintained for a
further 1 h. Anhydrous diisopropylethylamine (0.5 ml,
2 mmol) was added slowly, and the mixture was stirred
for 10 min at 0ꢂC. Dichloromethane (5 ml) and a satu-
rated solution of NH4Cl (5 ml) were added, the phases
were separated, and the aqueous layer was extracted with
22
0.5 (hexane/EtOAc 70:30). [α]D = ꢀ93.8ꢂ (c = 1.3,
CHCl3) (literature,9 [α]D = ꢀ95.3ꢂ [c = 1.2, CHCl3]). IV
25
(neat): vmax 3375, 2977, 2839, 1743, 1706, 1611, 1586,
1510, 1462, 1438, 1392, 1366, 1305, 1244, 1214, 1158,
1112, 1052, 1028, 914, 894, 852, 832, 797, 781, 763 cmꢀ1
.
1H NMR (400 MHz, CDCl3, δ) 7.27 (d, J = 8.3 Hz, 2H, Ar
H), 6.86 (d, J = 8.5 Hz, 2H, Ar H), 5.50 (s, 1H, NH), 5.25
(s, 1H, CH), 3.78 (s, 3H, CH3), 3.70 (s, 3H, CH3), 1.42
(s, 9H, CH3). 13C NMR (100 MHz, CDCl3, δ) 172.11 (C1),
159.93 (C9), 155.06 (C6), 129.27 (C3), 128.63 (C4 and
C40), 114.54 (C5 and C50), 80.30 (C10), 57.30 (C2), 55.51

MIRANDA ET AL.
483
CH2Cl2 (3 ꢁ 7 ml). The organic phases were combined,
dried over Na2SO4, and concentrated under reduced pres-
sure. The residue was rapidly filtered over silica gel
(hexane/EtOAc 95:5), providing aldehyde 5 as a yellow-
ish oil in 63% yield. The aldehyde was used immediately
after synthesis.
1509, 1364, 1302, 1244, 1158, 1083, 1021, 893, 782 cmꢀ1
.
1H NMR (400 MHz, CDCl3, δ) 7.32–7.28 (m, 4H, Ar H),
6.92–6.89 (m, 4H, Ar H), 5.74 (s, 1H, NH), 5.39 (s, 2H,
NH), 4.91–4.85 (m, 2H, CH), 4.73 (s, 1H, CH), 4.59 (s, 1H,
CH), 3.80 (s, 3H, CH3), 3.79 (s, 3H, CH3), 1.45 (s, 9H,
CH3), 1.43 (s, 9H, CH3). 13C NMR (100 MHz, CDCl3, δ)
160.04 (C9), 159.97 (C9), 157.42 (C7), 156.50 (C7), 128.60
(C5 and C50), 128.17 (C5 and C50), 127.57 (C4), 118.33
(C1), 117.93 (C1), 114.61 (C6 and C60), 114.45 (C6 and
C60), 81.74 (C10), 81.24 (C10), 67.95 (C2), 65.54 (C2),
58.80 (C3), 55.35 (C8), 55.33 (C8), 28.25 (C11), 28.23
(C11). HRMS (ESI, m/z): [M + Na]+ calcd for
C15H20N2O4, 315.1321; found, 315.1313.
Dess–Martin periodinane oxidation
To a 25-ml flask containing Dess–Martin periodinane
(DMP) (0.31 g, 0.74 mmol) in anhydrous CH2Cl2 (3 ml)
under an argon atmosphere were added alcohol solution
4 (0.10 g, 267.15 mmol) in CH2Cl2 (13 ml) and water
(0.013 ml). The reaction mixture was kept under stirring
at room temperature for 90 min. After the addition of
dichloromethane (5 ml), rapid filtration through silica gel
(hexane/EtOAc 95:5) provided aldehyde 5 as a yellowish
oil in 71% yield.
2.2.5 | (4R,5R)- and (4R,5S)-
4-(4-Methoxyphenyl)-2-oxooxazolidine-
5-methyl carboxylate (9a and b)
IBX oxidation
To a 25-ml flask containing a solution of alcohol
4 (0.20 g, 0.75 mmol) in anhydrous CH3CN under an
argon atmosphere was added 2-iodoxybenzoic acid (IBX)
(0.56 g, 2.25 mmol) in a single portion. The mixture was
refluxed for 2 h, cooled to room temperature, and filtered
through Celite. The filtrate was washed with a saturated
solution of NaHCO3 (5 ml) and brine (5 ml). The organic
phase was dried over Na2SO4 and concentrated under
reduced pressure. Then, the residue was rapidly filtered
through silica gel (hexane/EtOAc 95:5), providing alde-
hyde 5 as a yellowish oil in 68% yield.
To a 10-ml flask containing a mixture of cyanohydrins 6a
and b (0.054 g, 0.184 mmol) was added an aqueous solu-
tion of HCl (3 ml, 15%) and refluxed for 1 h. The aqueous
solution was evaporated under reduced pressure. The
mixture of N-unprotected amino acids (7a and b) thus
obtained was used in the next step without any purifica-
tion. Ethyl ether (2 ml) and an aqueous solution of NaOH
(0.086 g, 2.15 mmol, 5 ml) were added to a 10-ml flask
containing 7a and b (0.071 g, 0.28 mmol) under an argon
atmosphere at 0ꢂC and stirred for 10 min. Triphosgene
(0.125 g, 0.42 mmol) was added, and the mixture was
maintained at this temperature. Reaction progress
was monitored by TLC. Upon completion of the reaction,
the pH was adjusted to 2–3 by the addition of an aqueous
solution of HCl (2 mol mlꢀ1), and the mixture was
extracted with EtOAc (3 ꢁ 10 ml). The organic phases
were combined, dried over Na2SO4, and concentrated
2.2.4 | tert-Butyl ((1R,2R)- and (1R,2S)-
2-cyano-2-hydroxy-1-(4-methoxyphenyl)ethyl)
carbamate (6a and b)
A solution containing NaCN (0.019 g, 0.4 mmol), anhy-
drous MeOH (0.2 ml), and acetic acid (0.02 ml,
0.43 mmol) in a 10-ml flask was stirred for 5 min at room
temperature under an argon atmosphere. The tempera-
ture was lowered to 0ꢂC, and a solution of aldehyde
5 (0.042 g, 0.16 mmol) in CH2Cl2 (0.5 ml) was added. The
mixture was kept under stirring at room temperature for
6 h, brine (1 ml) and a saturated solution of NaHCO3
(1 ml) were added, and stirring was maintained for
5 min. After phase separation, the aqueous layer was
extracted with CH2Cl2 (3 ꢁ 5 ml). Organic phases were
combined, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified on a chro-
matographic column (hexane/EtOAc 80:20 ! 70:30),
providing a mixture of cyanohydrins 6a and b in 89%
yield as a yellowish solid (Rf 0.25 and 0.29, hexane/EtOAc
7:3). IV (neat): vmax 3379, 2982, 2932, 2837, 1666, 1613,
under reduced pressure to provide a mixture of
oxazolidinones 8a and b, which was subjected to carboxyl
esterification without further purification. To a 10-ml
flask equipped with a reflux condenser under an argon
atmosphere at 0ꢂC were added a mixture of 8a and
b
(0.082 g, 0.35 mmol), anhydrous MeOH (1 ml),
and freshly distilled SOCl2 (0.076 ml, 1.05 mmol). The
mixture was refluxed until the reaction was complete.
Reaction progress was monitored by TLC. After cooling
to 0ꢂC, the mixture was treated with a saturated solution
of NaHCO3 (3 ml) and extracted with EtOAc (3 ꢁ 10 ml).
Organic phases were combined, dried over Na2SO4, and
concentrated under reduced pressure. The residue was
purified by silica gel chromatography (hexane/EtOAc
75:25 ! 70:30 ! 60:40) to afford, as yellow solids, the
diastereoisomeric oxazolidinones 9a and b in three steps
and yields of 20% and 32%, respectively.

484
MIRANDA ET AL.
Data for oxazolidinone 9a
1H, CH), 3.82 (s, 3H, CH3), 3.48–3.42 (m, 1H, CHb), 3.30–
3.23 (m, 1H, CHa). 13C NMR (100 MHz, CDCl3, δ) 160.21
(C1), 148.12 (C10), 127.89 (C8 and C80), 127.24 (C7),
114.45 (C9 e C90), 80.27 (C5), 62.07 (C6), 57.30 (C4), 55.38
(C11). HPLC column CHIRALCEL OD-R (250 ꢁ 4.6 mm,
10 μm): 100% acetonitrile, detector: 234 nm, flow rate:
Mp 104.2–105.1ꢂC. Rf 0.36 (hexane/EtOAc 25:75).
(c = 0.25,
CH3OH)
(literature,21
22
[α]D = ꢀ82.9ꢂ
23
[α]D = ꢀ94.0ꢂ [c = 1.0, CH3OH]). IV (neat): vmax 3375,
2954, 2841, 2035, 1741, 1612, 1516, 1251, 1182, 1025,
837 cmꢀ1
.
1H NMR (400 MHz, CDCl3, δ) 7.21 (d,
J = 8.7 Hz, 2H, Ar H), 6.88 (d, J = 8.7 Hz, 2H, Ar H), 5.32
(s, 1H, NH), 5.26 (d, J = 9.2 Hz, 1H, CH), 5.17 (d,
J = 9.2 Hz, 1H, CH), 3.80 (s, 3H, CH3), 3.33 (s, 3H, CH3).
13C NMR (100 MHz, CDCl3, δ) 166.75 (C6), 160.38 (C11),
157.73 (C1), 128.17 (C9 and C90), 126.96 (C8), 114.14
(C10 and C100), 78.17 (C5), 57.89 (C4), 55.34 (C12), 52.10
(C7). HRMS (ESI, m/z): [M + Na]+ calcd for C12H13NO5,
274.0686; found, 274.0689.
0.5 ml minꢀ1
,
(4R,5S) = 6.58 min, (4R,5R) = 7.78 min.
HRMS (ESI, m/z): [M + Na]+ calcd for C11H13NO4,
246.0737; found, 246.0735.
2.2.7 | (+)-5-epi-Cytoxazone (2)
(+)-5-epi-Cytoxazone was prepared in a similar manner
to 1 but from oxazolidinone ester 9b. The residue was
purified by silica gel column chromatography (hexane/
EtOAc 25:75) yielding 92% (97.4% de) of 2 as a white
Data for oxazolidinone 9b
22
Mp 91.8–92.6ꢂC. Rf 0.56 (hexane/EtOAc 25:75). [α]D
=
+83.4ꢂ (c = 0.33, CH3OH) (literature,21 [α]D = +90ꢂ
[c = 1.0, CH3OH]). IV (neat): vmax 3245, 3142, 2962, 2925,
2840, 1722, 1614, 1586, 1512, 1414, 1304, 1249, 1229,
solid, mp 161.3–162.3ꢂC. Rf 0.38 (hexane/EtOAc 25:75).
23
25
[α]D = +21.3ꢂ
(c = 0.15,
CH3OH)
(literature,21
25
[α]D = +32.0ꢂ [c = 0.4, CH3OH]). IV (neat): vmax 3328,
1172, 1093, 1021, 954, 830 cmꢀ1
.
1H NMR (400 MHz,
2935, 1733, 1692, 1610, 1514, 1425, 1384, 1299, 1243,
1
CDCl3, δ) 7.29 (d, J = 8.6 Hz, 2H, Ar H), 6.93 (d,
J = 8.6 Hz, 2H, Ar H), 5.48 (s, 1H, NH), 4.92 (d,
J = 5.2 Hz, 1H, CH), 4.75 (d, J = 5.2 Hz, 1H, CH), 3.86 (s,
3H, CH3), 3.82 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3,
δ) 168.99 (C6), 160.55 (C11), 157.43 (C1), 130.86 (C8),
127.52 (C9 and C90), 114.95 (C10 and C100), 80.77 (C5),
58.89 (C4), 55.62 (C12), 53.27 (C7). HRMS (ESI, m/z): [M
+ Na]+ calcd for C12H13NO5, 274.0686; found, 274.0687.
1176, 1028, 831 cmꢀ1. H NMR (400 MHz, acetone-d6, δ)
7.33 (d, J = 8.7 Hz, 2H, Ar H), 6.95 (d, J = 8.7 Hz, 2H, Ar
H), 4.78 (d, J = 6.4 Hz, 1H, CH), 4.35 (t, J = 6.0 Hz, 1H,
OH), 4.29–4.21 (m, 1H, CH), 3.86–3.80 (m, 1H, CHb),
3.79 (s, 3H, CH3), 3.75–3.66 (m, 1H, CHa). 13C NMR
(100 MHz, acetone-d6, δ) 160.71 (C1), 159.13 (C10),
133.98 (C7), 128.49 (C8 and C80), 115.13 (C9 and C90),
85.69 (C5), 62.52 (C6), 57.76 (C4), 55.68 (C11). HPLC col-
umn CHIRALCEL OD-R (250 ꢁ 4.6 mm, 10 μm): 100%
acetonitrile, detector: 234 nm, flow rate: 0.5 ml minꢀ1
,
2.2.6 | (ꢀ)-Cytoxazone (1)
(4R,5S) = 6.57 min, (4R,5R) = 7.79 min. HRMS (ESI, m/z):
[M + Na]+ calcd for C11H13NO4, 246.0737; found,
246.0730.
To a 10-ml flask containing NaBH4 (0.003 g, 0.1 mmol)
and LiCl (0.004 g, 0.1 mmol) in anhydrous MeOH
(0.5 ml) under an argon atmosphere at 0ꢂC was added a
solution of oxazolidinone ester 9a (0.010 g, 0.025 mmol)
in anhydrous MeOH (0.5 ml). The temperature was
raised to room temperature, and the mixture stirred for
2 h until total consumption of the substrate, as monitored
by TLC. Water (1 ml) was added, and the mixture
extracted with EtOAc (3 ꢁ 3 ml). The organic phases
were combined, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by sil-
ica gel chromatography (hexane/EtOAc 25:75), yielding
90% (86.6% de) of 1 as a white solid, mp 120.5–121.3ꢂC. Rf
3 | RESULTS AND DISCUSSION
3.1 | Retrosynthetic analysis of
(ꢀ)-cytoxazone (1) and (+)-5-epi-
cytoxazone (2)
The retrosynthetic analysis of (ꢀ)-cytoxazone (1) and
(+)-5-epi-cytoxazone (2) is presented in Scheme 1. The
formation of the oxazolidinone rings must be achieved
through an N,O-heterocyclization, mediated by
triphosgene, of the respective amino acid precursors,
derived from acid hydrolysis of cyanohydrins 6a and b.
25
0.30 (hexane/EtOAc 25:75). [α]D = ꢀ63.16ꢂ (c = 0.1,
25
[α]D = ꢀ65.7ꢂ
CH3OH)
(literature,21
[c = 0.38,
CH3OH]). IV (neat): vmax 3441, 3238, 2960, 2928, 2841,
Preparation of 6a and
b and separation of the
1709, 1611, 1513, 1401, 1249, 1174, 1047, 1026, 992 cmꢀ1
.
diastereoisomers should be possible through
a
1H NMR (400 MHz, CDCl3, δ) 7.21 (d, J = 8.7 Hz, 2H, Ar
H), 6.91 (d, J = 8.7 Hz, 2H, Ar H), 5.18 (s, 1H, NH), 5.01
(d, J = 8.4 Hz, 1H, CH), 4.90 (ddd, J = 8.4, 7.6 and 4.0 Hz,
stereoselective addition of cyanide to aldehyde 5, which
in turn can be produced from commercially available
D-4-hydroxyphenylglycine.

MIRANDA ET AL.
485
SCHEME 1 Retrosynthetic analysis of (ꢀ)-cytoxazone (1) and (+)-5-epi-cytoxazone (2) from D-4-hydroxyphenylglycine
SCHEME 2 Synthesis of cyanohydrins 6a and b
3.2 | Synthesis of cyanohydrins 6a and b
and 71%, respectively. The aldehyde should be used
immediately after preparation to avoid or minimize race-
mization. Attempts to purify aldehyde 5 by silica gel
chromatography resulted in partial racemization. Thus,
as the diastereoselectivity of the reaction with cyanide
depends on the diastereoisomeric purity of the aldehyde,
5 was used in its crude form in the current study.
The synthesis of cyanohydrins 6a and b, in 89% overall
yield and stereoselectivity (1.0:1.8) toward the anti-Felkin
(1,2-syn) addition product (6b), was performed by reacting
HCN (generated in situ by treatment of a methanolic solu-
tion of NaCN with acetic acid) with N-Boc aldehyde 5 in
The synthesis of 1 and 2 started with the preparation
of N-Boc amino ester 3. N-Boc protection and
methoxylation with concomitant esterification from
D-4-hydroxyphenylglycine produced N-Boc ester 3 in 94%
yield (two steps) (Scheme 2). Reduction of the ester
function of 3 using LiBH4 (generated in situ from NaBH4
and LiCl), followed by DMP oxidation from the resulting
alcohol 4, provided N-Boc aldehyde 5 in 65% yield (two
steps). Three oxidizing agents were tested in this last step,
Swern reagent, IBX, and DMP, giving yields of 63%, 68%,

486
MIRANDA ET AL.
CH2Cl2.22 An acidic medium (HCN) in the nucleophilic
addition to the carbonyl of the aldehyde is necessary,
because, under these conditions, the carbonyl oxygen is
protonated, generating a carboxonium ion that facilitates
the reaction; that is, in an acidic environment, the
thermodynamic balance of the reaction between HCN and
aldehyde is shifted toward the products.23
The stereoisomeric ratio of cyanohydrins 6a to
b (1.0:1.8) was determined by analyzing the 1H NMR
spectrum of the mixture. However, this initial evaluation
did not allow identifying which stereoisomer was formed
preferentially; this was only possible in later steps.
with the synthesis using the diastereoisomeric mixture
(Scheme 3). Acid hydrolysis of the nitrile group in the
presence of a 15% HCl aqueous solution under reflux
led to the removal of the protective N-Boc group and
gave a mixture of carboxylic acids (7a and b). The very
polar crude product was subjected to the next step
without previous purification. N,O-heterocyclization
of 7a and b was performed by using triphosgene
in basic medium to provide
a mixture of the
respective oxazolidinone acid derivatives 8a and b. All
attempts to purify this mixture resulted in product
decomposition.
The crude mixture of 8a and b was subjected to esteri-
fication of the carboxyl function by treatment with SOCl2
and MeOH under reflux, resulting in a mixture of the
respective oxazolidinone esters 9a and b. Purification of
9a and b by column chromatography allowed the
separation of diastereoisomers, obtained in three steps
from cyanohydrins 6a and b in yields of 20% and 32%,
respectively.
3.3 | Synthesis of (ꢀ)-cytoxazone and
(+)-5-epi-cytoxazone from cyanohydrins 6a
and b
Because of the difficulties in separating cyanohydrins
6a and b, after several attempts, we decided to proceed
SCHEME 3 Synthesis of (ꢀ)-cytoxazone (1) and (+)-5-epi-cytoxazone (2) from cyanohydrins 6a and b

MIRANDA ET AL.
487
By separating the oxazolidinone isomers 9a and b, we
practice, as N-Boc favored the five-membered cyclic
conformer A, resulting preferentially in anti-Felkin 6b
(1,2-syn).
Finally, reduction of the respective ester functions of 9a
and b produced (ꢀ)-cytoxazone (1) and (+)-5-epi-cytoxazone
(2) in yields of 90% and 92%, respectively.
were able to determine, through their masses, that the
addition of cyanide to the carbonyl of aldehyde 5 favored
the formation of 1,2-syn cyanohydrin (6b) at a ratio of
1.0:1.8 (Scheme 2).
The stereochemistry of the major cyanohydrin (6b)
can be rationalized by the Felkin–Ahn transition state
model (Figure 3).24 It suggests a balance between
conformers A and B.25 Thus, it was expected that the
use of a bulky N-protective group (Boc) would hinder
the formation of the N–H bond, favoring the formation
of conformer B. However, this was not observed in
Spectroscopic data of 1 and 2 were in agreement with
those reported in the literature,21,26–28 as were their spe-
24
cific rotation values [α]D = ꢀ63.16ꢂ (c = 0.1, CH3OH)
and [α]D = +21.3ꢂ (c = 0.15, CH3OH).21,26–28
24
The relative stereochemistry of 1 and 2 (cis and
trans, respectively) was unequivocally established by
FIGURE 3 Felkin–Ahn transition state model
FIGURE 4 Analysis of the coupling
constant and determination of the relative
stereochemistry of (ꢀ)-cytoxazone (1) and
(+)-5-epi-cytoxazone (2)

488
MIRANDA ET AL.
1
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analyzing their H NMR spectra. The coupling constant
(3JH4–H5 = 8.4 Hz) indicates that these hydrogens are
on the same side of the heterocyclic ring in
cis-oxazolidinone 1 (Figure 4).29,30 The coupling constant
(3JH4–H5 = 6.4 Hz), on the other hand, indicates that the
hydrogens are on opposite faces of the ring in
trans-oxazolidinone 2.29,30
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Information, Appendix S11) showed a correlation signal
between hydrogens 4 and 5, corroborating the cis
configuration of the oxazolidinone ring.
Osada H. Isolation and biological activity of
a
novel
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4 | CONCLUSION
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Commun. 2003;33(16):2907-2916.
In summary, the versatility of our method enabled the
stereoselective synthesis of the oxazolidinones,
(ꢀ)-cytoxazone (1) and (+)-5-epi-cytoxazone (2) in
overall yields of 10% and 16%, respectively, from a single
common chiral intermediate, N-Boc amino aldehyde 5,
derived from D-4-hydroxyphenylglycine. Insertion of the
second oxazolidinone stereocenter was controlled by
adding cyanide to the aldehyde, generating both 1,2-anti
and 1,2-syn diastereoisomeric cyanohydrins (6a and b),
the precursors of 1 and 2, respectively. Independent
synthesis of target molecules was made possible by silica
gel chromatographic separation of the advanced
oxazolidinone diastereoisomers 9a and b. The method is
very advantageous, as it allows the synthesis of two
oxazolidinones via a single protocol.
10. Miranda IL, Lopes IKB, Diaz MAN, Diaz G. Synthesis
approaches to (ꢀ)-cytoxazone, a novel cytokine modulator, and
related structures. Molecules. 2016;21(9):1176-1197.
11. Madhan A, Kumar AR, Rao V. Stereoselective synthesis of
(ꢀ)-cytoxazone.
Tetrahedron:
Asymmetry.
2001;12(14):
2009-2011.
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synthesis of cytoxazone and its diastereomers provides key
initial SAR information. Bioorg Med Chem Lett. 2003;13(7):
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mers of cytoxazone using the Petasis reaction. Tetrahedron:
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rearrangement of hydroximates and its application to synthesis
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15. Lin X, Bentley PA, Xie H. Auxiliary strategies for the prepara-
tion of β-amino alcohols with reductive cross-coupling and a
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7849-7852.
ACKNOWLEDGMENTS
We thank the Brazilian National Council for Scientific
and Technological Development (Conselho Nacional de
Desenvolvimento Científico e Tecnologico [CNPq]) for
granting a doctoral fellowship to ILM and the Minas
Gerais Research Foundation (Fundaçao de Amparo à
Pesquisa do Estado de Minas Gerais [FAPEMIG]) for the
financial support.
ꢀ
~
16. Sohtome Y, Hashimoto Y, Nagasawa K. Diastereoselective and
enantioselective Henry (nitroaldol) reaction utilizing
guanidine-thiourea bifunctional organocatalyst. Eur J Org
Chem. 2006;13:2894-2897.
a
DATA AVAILABILITY STATEMENT
I declare to make my data available.
17. George S, Narina SV, Sudalai A. NaIO4-mediated asymmetric
bromohydroxylation of α, β-unsaturated carboxamides with
high diastereoselectivity: a short route to (ꢀ)-cytoxazone and
droxidopa. Tetrahedron Lett. 2007;48(8):1375-1378.
18. Narina SV, Kumar TS, George S, Sudalai A. Enantioselective
synthesis of (ꢀ)-cytoxazone and (+)-epi-cytoxazone via Rh-
catalyzed diastereoselective oxidative C–H aminations. Tetrahe-
dron Lett. 2007;48(1):65-68.
ORCID
Marisa Alves Nogueira Diaz https://orcid.org/0000-
0002-3370-4149
Gaspar Diaz-Muñoz https://orcid.org/0000-0002-7515-
0420
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SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
How to cite this article: Miranda IL, dos
Santos PHC, Kohlhoff M, Purgato GA, Diaz MAN,
Diaz-Muñoz G. Stereoselective synthesis of
(ꢀ)-cytoxazone and its unnatural congener (+)-5-
epi-cytoxazone. Chirality. 2021;33:479–489. https://
doi.org/10.1002/chir.23334
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