Synthesis of (-)-cytoxazone and (+)-epi-cytoxazone: The chiral pool approach

Article abstract of DOI:10.1081/SCC-200048953

Immunomodulator (-)-cytoxazone and its epimer (+)-epi-cytoxazone were synthesized starting from (D)-(-)-4-hydroxyphenylglycine. Homologation of the amino acid was achieved via the corresponding aldehyde, by a cyanohydrin reaction. The racemization of high

Full text of DOI:10.1081/SCC-200048953

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Synthesis of (-)‐Cytoxazone and
(+)‐epi‐Cytoxazone: The Chiral
Pool Approach
Zorana Tokic‐Vujosevic a , Goran Petrovic b , Bojana
Rakic b , Radomir Matovic c & Radomir N. Saicic b c
a Faculty of Pharmacy , University of Belgrade ,
Belgrade, Serbia and Montenegro
b Faculty of Chemistry , University of Belgrade ,
Studentski trg 16, P. O. B. 158, 11000, Belgrade,
Serbia and Montenegro
c ICTM‐Centre for Chemistry , Belgrade, Serbia and
Montenegro
Published online: 28 Jul 2007.
To cite this article: Zorana Tokic‐Vujosevic , Goran Petrovic , Bojana Rakic ,
Radomir Matovic & Radomir N. Saicic (2005) Synthesis of (-)‐Cytoxazone and
(+)‐epi‐Cytoxazone: The Chiral Pool Approach, Synthetic Communications: An
International Journal for Rapid Communication of Synthetic Organic Chemistry, 35:3,
435-447, DOI: 10.1081/SCC-200048953
To link to this article: http://dx.doi.org/10.1081/SCC-200048953
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Synthetic Communicationsw, 35: 435–447, 2005
Copyright # Taylor & Francis, Inc.
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1081/SCC-200048953
Synthesis of (2)-Cytoxazone and (1)-epi-
Cytoxazone: The Chiral Pool Approach
Zorana Tokic-Vujosevic
Faculty of Pharmacy, University of Belgrade, Belgrade,
Serbia and Montenegro
Goran Petrovic and Bojana Rakic
Faculty of Chemistry, University of Belgrade, Belgrade,
Serbia and Montenegro
Radomir Matovic
ICTM-Centre for Chemistry, Belgrade, Serbia and Montenegro
Radomir N. Saicic
Faculty of Chemistry, University of Belgrade, Belgrade, Serbia
and Montenegro and ICTM-Centre for Chemistry, Belgrade,
Serbia and Montenegro
Abstract: Immunomodulator (2)-cytoxazone and its epimer (þ)-epi-cytoxazone were
synthesized starting from (D)-(2)-4-hydroxyphenylglycine. Homologation of the
amino acid was achieved via the corresponding aldehyde, by a cyanohydrin reaction.
The racemization of highly sensible amido aldehyde was efficiently suppressed when
the oxidation of the parent aminoalcohol was performed by a modified Dess-Martin
procedure.
Keywords: Amino alcohols, cyanohydrins, cytoxazone, oxazolines
Received in Poland October 20, 2004
Address correspondence to Radomir N. Saicic, Faculty of Chemistry, University of
Belgrade, Studentski trg 16, P. O. B. 158, 11000, Belgrade, Serbia and Montenegro.
E-mail: rsaicic@chem.bg.ac.yu

436
Z. Tokic-Vujosevic et al.
INTRODUCTION
Owing to its strong, yet selective, immunosuppressive activity, cytoxazone
1—a secondary metabolite of Streptomyces sp.—has attracted attention of
synthetic chemists. Since its isolation in 1998,[1,2] several syntheses of the
compound,[3–12] as well as of its epimer epi-cytoxazone 2,[9–11] have been
reported. These approaches involved asymmetric synthesis,[3–12];) as well
as the separation of the racemic compound.[5,8] No attempt has been made,
however, to prepare the optically pure 1 starting from an amino acid, using
a chiral pool approach, which is the subject of this study.
RESULTS AND DISCUSSION
We considered using (D)-(2)-4-hydroxyphenylglycine 4—a commercially
available, nonexpensive amino acid—as a starting compound for the
synthesis of both (2)-cytoxazone 1 and (þ)-epi-cytoxazone 2 (Scheme 1).
Amino acids are routinely used as chiral starting material for syntheses of
optically pure compounds,[13] and their homologation into amino alcohols,
via the corresponding amino aldehydes,[14] is a well-known process.
Addition of vinyl organo metalics,[15] acetylide anion,[16] as well as cyano-
hydrin reaction,[17] were usually used for this purpose (when one-carbon
Scheme 1.

Synthesis of (2)-Cytoxazone and (1)-Cytoxazone
437
homologation is required, the first two methods are used in combination with
subsequent oxidative fragmentation). However, the aminoaldehyde-type inter-
mediates can easily epimerize and require a careful choice of protective group
on the nitrogen atom; most often, these include carbamates, sulfonamides, and
alkyl substituted amine derivatives. We intended to perform the homologation
using a cyanohydrin reaction. Rather than using its more sophisticated
variants, such as trimethylsilyl cyanide,[18] tributylstannyl cyanide,[19] with
or without added Lewis acids, or diethylaluminum cyanide (Nagata’s
reagent),[20] we opted for performing the cyanohydrin reaction under opera-
tionally simple conditions, with cheap and environmentally least-harmful
sodium cyanide. The diastereoselectivity of such a reaction is reagent con-
trolled and would not be expected to be high; therefore, the homologated inter-
mediate should be amenable to configurational inversion at the newly created
stereocenter. Previously, it was shown that this transformation can be con-
veniently effected via oxazoline intermediates. In order to minimize protective
group manipulations, this approach would require the use of amido aldehyde 3
as a homologation intermediate. However, due to their extreme proclivity
towards epimerization, these species have not found synthetic application.
Thus, the feasibility of the approach was to be tested experimentally.
The course of synthesis is displayed in Scheme 2. (D)-(2)-4-Hydroxy-
phenylglycine was converted into the corresponding amido alcohol 7 via a
three-step procedure (71% overall).1 Given the high propensity of a-amido
aldehydes toward racemization, we performed a short study on the
oxidation of 7 under a variety of conditions. The optical purity of the
aldehyde 3 was determined by its reduction with sodium borohydride back
to 7, and the comparison of the optical rotation value with the original
sample. Swern oxidation on a closely related system was reported to give
amido aldehyde with a high level of optical purity.[15,10] In our hands,
however, the enantiomeric excess of the amido aldehyde 3 was 83%. Initial
experiments with TEMPO mediated oxidation were quite disappointing, as
the product completely racemized.[21] After careful optimization (time
of the reaction; solvent; concentration; stoichiometry), we were able to
obtain the product with 85% ee, which was still unsatisfactory. Dess-Martin
oxidation in THF afforded the aldehyde with 67% ee; in acetonitrile the
optical purity improved to 75%.2 Recently, the mechanism of the Dess-
Martin oxidation was studied in more detail and it was found that the water
accelerated oxidation with freshly prepared periodinane can afford amido
aldehydes with excellent levels of optical purity.[22] Indeed, this protocol
1It should be noted that (D)-(2)-N-benzoyl-4-hydroxyphenylglycine 5 easily epi-
merizes: when the benzoylation was performed in refluxing dichloromethane using tri-
ethylamine as a base, the racemic N,O-dibenzoylated product was obtained.
2Dichloromethane could not be used as a solvent due to the insolubility of the
starting amidoalcohol 7.

438
Z. Tokic-Vujosevic et al.
Scheme 2. a) BzCl, NaOH, H2O, 08C-rt, 2.5 h (88%); b) Me2SO4, K2CO3, acetone,
reflux, 3 h (89%); c) NaBH4, MeOH, rt, 3 h (91%); d) DMP, CH2Cl2, H2O (cat.),
12 min (94%); e) KCN, CH2Cl2, MeOH, AcOH (92%, 8:9 ¼ 1:1.5); f) separation by
column chromatography; g) 15% HCl, 1108C. 3 h h) triphosgene, NaOH, H2O, 08C,
1 h; i) CH2N2, THF (65% from 8, 71% from 9); j) NaBH4, THF, H2O, 08C, 2 h (83%).
allowed us to obtain the amido aldehyde 3 in 94% yield, with 98% ee. Cyano-
hydrin reaction of 3 proved also to be potentially epimerizing step (as with the
amido aldehyde, the optical purity of the cyanohydrins 8 and 9 was determined
by their reduction with sodium borohydride to the starting amidoalcohol).
Thus, when the cyanohydrin reaction with optically pure 3 was performed
under basic conditions according to described procedures,[23] partial racemiza-
tion occured. Neutral conditions proved more suitable for this

Synthesis of (2)-Cytoxazone and (1)-Cytoxazone
439
transformation:[22] with in situ generated methanolic hydrogen cyanide the
reaction was very fast and afforded essentially optically pure cyanohydrins
8 and 9 in 92% yield. The product was obtained as a diastereomeric
mixture in a ratio 8 : 9 ¼ 1 : 1.5–2.0 and the isomers were separated by
flash chromatography.
The anti-isomer 8 was hydrolyzed into a-hydroxy-b-amino acid 10,
which was converted into (2)-cytoxazone 1 by a previously reported three-
step procedure (54% from 8).[7] The physical data of the obtained product
were in agreement with those of the natural compound; in addition to the
optical rotation value, its optical purity was confirmed by HPLC analysis on
a chiral column.[5] By the same sequence of reactions the syn-isomer 9 was
converted into enantiopure (þ)-epi-cytoxazone 2. However, this compound
could also be used for (2)-cytoxazone synthesis: treatment of 9 with
triphenylphosphine/DEAD afforded oxazoline 16 with the inversion of
configuration at the a-carbon atom (73%; Scheme 3). Acidic hydrolysis of
16, followed by the aforementioned protocol, furnished (2)-cytoxazone
identical in all respects to the natural sample (56% from 16). Conversely,
anti-cyanohydrin 8 could be converted into (þ)-epi-cytoxazone, by an
analogous set of reactions (43% overall yield).
To summarize, optically pure (2)-cytoxazone 1 and (þ)-epi-cytoxazone
2 were synthesized from (D)-(2)-4-hydroxyphenylglycine 4. It is shown that
the homologation of amino acids can be effected without epimerization via
amido aldehydes, under carefully controlled conditions, where N-benzoyl
group serves both as a protective group and the internal nucleophile in the
configurational inversion step.
EXPERIMENTAL
All chromatographic separations were performed on silica, 10–18, 60A, ICN
Biomedicals. Standard techniques were used for the purification of reagents
and solvents. NMR spectra were recorded on a Varian Gemini 200, 1H
NMR at 200 MHz, 13C NMR at 50 MHz. Chemical shifts are expressed in
ppm using tetramethylsilane as internal standard, coupling constants (J) are
Scheme 3. a) Ph3P, DEAD, C6H6, rt, 30 min (73%); b) 15% HCl, 1108C, 1.5 h.

440
Z. Tokic-Vujosevic et al.
in Hz. IR spectra were recorded on a Perkin-Elmer 457 grating FT instrument,
and are expressed in cm21. Mass spectra were obtained on a Finnigan ITDS
700 instrument. Microanalyses were performed at the Vario EL III instrument
CHNOS Elementar Analyzer, Elemantar Analysensysteme GmbH, Hanau-
Germany. Melting points were determined on a Kofler hot-stage apparatus
and are uncorrected. Optical rotation measurements were performed on
Perkin Elmer 141 MC polarimeter. HPLC analyses were performed on the
Hewlett Packard 1100 Agilent Technologies instrument, with HP 1100
UV-VIS detector. Measurements were performed at 235 nm wavelength, at
408C, at a flow rate of 1 mL/min. Chiracel OD column (200 ꢀ 4.6 mm) was
used with a solvent mixture: n-hexane/iso-propanol ¼ 80/20 as a mobile
phase for the determination of cytoxazone, and n-hexane/ethanol ¼ 85/15
for epi-cytoxazone.
(R)-N-Benzoyl-4-hydroxyphenylglycine (5). To a cold (08C), vigor-
ously stirred solution of (D)-(2)-4-hydroxyphenylglycine
4 (9.00 g;
53.8 mmol) in aqueous NaOH (156 mL of 2 M solution) was added benzoyl
chloride (15.6 mL; 134 mmol), drop-wise, over 30 min. After the addition
was complete, the reaction mixture was stirred for 2.5 h at room temperature
(rt), followed by the addition of aqueous NaOH (30 mL of 4 M solution) and
another 30 min stirring at rt, after which time no starting compound could be
detected in the reaction mixture (the course of the reaction was monitored by
TLC, eluent: toluene/ethyl acetate ¼ 7/3, with 2 drops of formic acid). The
reaction mixture was filtered with suction and the product was precipitated
from the cold filtrate by the addition of concentrated HCl (ca. 18 mL) until
pH 1-2. The precipitate was separated on a Buchner funnel, washed with
water until pH 4 (5 ꢀ 10 mL), dried in a vacuum dessicator over P2O5. The
amorphous solid was first recrystallized from toluene and then from water,
to give 12.8 g (88%) of the title compound 5 as white crystals.
Physical data for 5: m.p. 2238C; [a]D 2 137.98 (c 1.0, THF, 188C);
Anal. calcd. for C15H13NO4: C 66.41, H 4.83, N 5.16; found: C 66.72, H
4.87, N 4.88; IRKBr: 3412, 3358, 1738, 1614, 1567, 1539, 1518, 1491,
1
1378, 1360, 1270, 1209; H NMR (DMSO) d: 5.46 (d, J ¼ 7.2, 1H); 6.77
(d, J ¼ 8.4, 2H); 7.30 (d, J ¼ 8.4, 2H); 7.41–7.58 (m, 3H); 7.94
(d, J ¼ 1.2, 2H); 8.89 (d, J ¼ 7.2, NH); 9.52 (bs, COOH); 13C NMR
(DMSO) d: 56.67; 115.38; 127.38; 127.98; 128.44; 129.70; 131.69; 134.08;
157.48; 166.56; 172.66.
Methyl (R)-N-benzoyl-4-methoxyphenylglycinate (6). To a solution
of (R)-N-benzoyl-4-hydroxyphenylglycine 5 (12.00 g; 44 mmol) in acetone
(420 mL) was added anhydrous K2CO3 (18.30 g; 133 mmol) and dimethyl
sulphate (13.95 g; 10.52 mL; 111 mmol). The resulting suspension was
stirred and heated to reflux for 3 h (the course of the reaction was moni-
tored by TLC, eluent: toluene/ethyl acetate ¼ 7/3). The mixture was
cooledto rt, filtered with suction and the filtrate was concentrated under
reduced pressure to give amorphous, ochre-colored crude ester 6 (12.80 g).

Synthesis of (2)-Cytoxazone and (1)-Cytoxazone
441
The solid was crystallized from ethyl acetate with the addition of activated
carbon. The second crystallization from ethyl acetate/petroleum ether
afforded 11.80 g (89%) of the title compound 6, as colorless needles.
Physical data for 6: m.p. 1448C; [a]D 2 1238 (c 1.0, CHCl3, 218C); Anal.
calcd. for C17H17NO4: C 68.21, H 5.72, N 4.68; found: C 68.08, H 5.95, N
1
4.52; IRKBr: 3314, 1741, 1638, 1516, 1320, 1277, 1249, 1186, 1027; H
NMR (CDCl3) d: 3.76 (s, 3H); 3.78 (s, 3H); 5.71(d, J 6.8, 1H); 6.86–6.93
(m, 2H); 7.18 (d, J ¼ 6.8, 1H); 7.26–7.51 (m, 5H); 7.79–7.84 (m, 2H); 13C
NMR (CDCl3) d: 52.79; 55.24; 56.19; 114.36; 127.10; 128.54; 128.58;
131.80; 133.59; 159.75; 171.73.
(R)-N-Benzoyl-4-methoxyphenylglycinol (7). Sodium borohydride
(5.42 g; 143 mmol) was added in small portions to a solution of Methyl (R)-
N-benzoyl-4-methoxyphenylglycinate 6 (7.16 g; 24 mmol) in dry methanol
(400 mL). After the addition was complete (ca 30 min) the reaction mixture
was stirred at rt for 3 h, when the reaction was complete (the course of the
reaction was monitored by TLC, eluent toluene/ethyl acetate ¼ 6 : 4). The
reaction mixture was concentrated under reduced pressure to a volume of ca
60 mL, cooled to 08C, carefully acidified with 2M HCl (ca 18 mL) to a pH
2 and extracted with 4 ꢀ 150 mL of ethyl acetate. The combined organic
extract was washed successively with 10% aqueous NaOH (3 ꢀ 50 mL)
until pH 9, then with 2 M HCl, water, and brine until pH 7. The organic
layer was dried over anhydrous MgSO4 and concentrated under reduced
pressure to give the crude product (6.7 g). Purification by dry-flash chromato-
graphy (gradient elution with toluene/ethyl acetate ¼ 7/3-1/1) afforded
5.90 g (91%) of the title compound 7 as colorless crystals.
Physical data for 7: m.p. 1548C; [a]D þ 16.78 (c 1.0, AcOEt, 208C); Anal.
calcd. for C16H17NO3: C 70.83, H 6.32, N 5.16; found: C 71.09, H 6.30, N
5.34; IRKBr: 3431, 3331, 2923, 1634, 1532, 1491, 1300, 1248, 1180, 1033;
1H NMR (DMSO) d: 3.39–3.69 (m, 2H); 3.72 (s, 3H); 4.91 (t, J ¼ 5.7,
1H); 5.03 (dd, J1 ¼ 5.7, J2 ¼ 8.0, 1H); 6.88 (d, J ¼ 8.8, 2H); 7.32
(d, J ¼ 8.8, 2H); 7.42–7.50 (m, 3H); 7.90 (d, J ¼ 8.0, 2H); 8.65
(d, J ¼ 8.0, 1H); 13C NMR (DMSO) d: 55.24; 55.56; 64.79; 113.78;
127.64; 128.38; 128.44; 131.37; 133.65; 134.99; 158.48; 166.34.
(R)-N-Benzoyl-4-methoxyphenylglycinal (3). Freshly prepared DMP[24]
(2.62 g; 619 mmol) was added with stirring to a suspension of (R)-N-benzoyl-
4-methoxyphenylglycinol 7 (0.80 g; 295 mmol) in dichloromethane saturated
with water (16 mL). After 2 min the reaction mixture became clear, and
after 12 min the reaction was complete (TLC: toluene/ethyl acetate ¼ 3/2).
The reaction was quenched by the addition of diethyl ether (12 mL) and
the solution of Na2S2O3 (8.05 g; 32 mmol; dissolved in 10 mL of the
saturated aqueous solution of NaHCO3), and the mixture was stirred at rt
until it cleared up completely, with the separation of layers. The organic
layer was separated, the aqueous layer was extracted with diethyl ether
(3 ꢀ 20 mL), and the combined extract was washed successively with

442
Z. Tokic-Vujosevic et al.
saturated aqueous Na2S2O3 (2 ꢀ 10 mL), NaHCO3 (2 ꢀ 5 mL), water and
brine. Drying over anhydrous MgSO4, followed by solvent evaporation under
reduced pressure gave 0.75g (94%) of (R)-N-benzoyl-4-methoxyphenylglycinal
3, as pale-yellow crystals, which was used in the next step without further
purification.
Physical data for 3: IRfilm in CHCl3: 3318, 2927, 1713, 1620, 1516, 1320,
1
1249, 1184, 1027, 829; H NMR (CDCl3) d: 3.80 (s, 3H); 5.71 (d, J ¼ 5.8,
1H); 6.94 (d, J ¼ 8.8, 2H); 7.30 (d, J ¼ 8.8, 2H); 7.38–7.56 (m, 4H); 7.87
(d, J ¼ 8.0, 2H); 9.62 (s, 1H); 13C NMR (CDCl3) d: 55.29; 63.24; 114.87;
124.97; 127.14; 128.62; 129.51; 131.93; 133.49; 160.06; 166.72; 194.96.
The optical purity of (R)-N-benzoyl-4-methoxyphenylglycinal 3 was
determined by its’ reduction into (R)-N-benzoyl-4-methoxyphenylglycinol
7, and the comparison of the optical rotation value with the original sample:
Sodium borohydride (20 mg; 0.529 mmol) was added to the solution of (R)-
N-benzoyl-4-methoxyphenylglycinal 3 (30 mg; 0.112 mmol) in ethanol
(3 mL) and THF (1 mL). The mixture was stirred at rt for 30 min, after
which time the reaction was complete. The reaction mixture was carefully
acidified to pH ꢀ4 by the addition of 1.5 M HCl and extracted with ethyl
acetate (3 ꢀ 10 mL). The combined organic extract was washed with
saturated aqueous NaHCO3 (2 ꢀ 5 mL), water and brine, dried over
anhydrous MgSO4, and concentrated under reduced pressure. The solid
residue was purifies by dry-flash chromatography (gradient elution with
toluene/ethyl acetate ¼ 7/3-1/1) afforded 25 mg (83%) of (R)-N-benzoyl-
4-methoxyphenylglycinol 7 as colorless crystals. m.p. 1548C; [a]D þ 16.5
(c 1.0, AcOEt, 248C) ) 98.2% ee.
(2R,3R)-2-Hydroxy-3-(4-methoxyphenyl)-3-N-benzoylaminopropio-
nitrile (8) and (2S,3R)-2-Hydroxy-3-(4-methoxyphenyl)-3-N-benzoylami-
nopropionitrile (9) (CAUTION: Hydrogen cyanide is highly toxic! All
operations with hydrogen cyanide should be performed in a well venti-
lated hood). Acetic acid (0.43 g; 0.41 mL; 7.167 mmol) was added drop-
wise to a suspension of potassium cyanide (0.43 g; 6.603 mmol) in dry
methanol (3.52 mL), with stirring. After 5 min stirring at rt, the clear methano-
lic solution of HCN was added drop-wise (with a syringe) to a cold (08C) sus-
pension of (R)-N-benzoyl-4-methoxyphenylglycinal 3 (0.71 g; 2.636 mmol) in
dichloromethane (8.8mL), with vigorous stirring, under an argon atmosphere.
After 10min TLC indicated the complete conversion of the starting material
(eluent: toluene/ethyl acetate ¼ 7/3). The reaxtion mixture was quenched by
the addition of the mixture of water (18mL), brine (14.4 mL) and saturated
aqueous NaHCO3 (3.6mL) and the stirring was continued for 5 more min.
The organic layer was separated and the aqueous layer was extracted with
ether (3 ꢀ 15mL). The combined organic extract was washed with a mixture
of brine and saturated aqueous NaHCO3 in a 4 : 1 ratio (10 mL) and water,
dried over anhydrous MgSO4, and concentrated under reduced pressure. Puri-
fication by dry-flash chromatography (eluent: toluene/ethyl acetate ¼ 4/1)

Synthesis of (2)-Cytoxazone and (1)-Cytoxazone
443
afforded 0.71g (92%) of the mixture of diastereoisomeric cyanohydrins 8 (anti):
9 (syn) ¼ 1 : 1.5–1 : 2, as a colorless foam. The isomers were separated by flash-
chromatography (eluent: chloroform/methanol ¼ 50/1).
Physical data for the mixture of 8 and 9: m.p. 458C; IRfilm in CHCl3: 3327,
3068, 2053, 1898, 1642, 1613, 1579, 1515, 1488, 1464, 1306, 1251, 1182,
1081, 1029, 832; NMR spectra of pure compounds 8 and 9: (2R,3R)-2-
1
Hydroxy-3-(4-methoxyphenyl)-3-N-benzoylaminopropionitrile (8): H NMR
(CDCl3): 3.82 (s, 3H); 4.74 (br s, 1H); 5.32 (dd, J1 ¼ 2.2, J2 ¼ 2.5, 1H);
6.32 (br s, 1H); 6.85 (d, J ¼ 5.1, NH); 6.98 (d, J ¼ 8.7, 2H); 7.26–7.59
(m, 5H); 7.78 (d, J ¼ 6.9, 2H); 13C NMR (CDCl3) d: 55.33; 59.12; 67.73;
114.85; 117.89; 127.31; 128.25; 128.81; 132.58; 160.28; 169.94. (2S,3R)-2-
1
Hydroxy-3-(4-methoxyphenyl)-3-N-benzoylaminopropionitrile (9): H NMR
(CDCl3): 1H NMR (CDCl3) d: 3.77 (s, 3H); 4.84 (br s, 1H); 5.37 (dd,
J1 ¼ 5.4, J2 ¼ 6.2, 1H); 5.50 (d, J ¼ 5.4, 1H); 6.89 (d, J ¼ 8.8, 2H); 7.27–
7.47 (m, 5H); 7.48 (d, J ¼ 7.0, 1H) 7.75 (d, J ¼ 6.8, 2H); 13C NMR
(CDCl3) d: 55.26; 56.65; 64.82; 114.41; 118.49; 127.25; 127.72; 128.67;
132.20; 133.19; 159.89; 168.60.
The optical purity of the cyanohydrins 8 and 9 was determined by their
reduction with sodium borohydride to (R)-N-benzoyl-4-methoxyphenylglyci-
nol 7 and the comparison of the optical rotation data to those of the original
sample, as described for (R)-N-benzoyl-4-methoxyphenylglycinal 3. The
obtained sample had m.p. 1548C and the optical rotation value [a]D þ 16.3
(c 1.0, AcOEt, 248C). Similarly, to verify that cyanohydrins 8 and 9 do not
racemize on silica-gel, the purified samples of the separated cyanohydrins 8
and 9 were also submitted to the reduction. The obtained alcohol had the
physical properties identical to the reference compound 7.
(2R,3R)-3-Amino-3-(4-methoxyphenyl)-2-hydroxypropanoic
acid
hydrochloride (10). A mixture of (2R,3R)-2-Hydroxy-3-(4-methoxyphe-
nyl)-3-N-benzoylaminopropionitrile 8 (72 mg; 0.243 mmol) and 15% HCl
(7.5 mL; 34 mmol) was heated to reflux with stirring. After 3 h TLC (eluent:
toluene/ethyl acetate ¼ 3/2, with 3 drops of formic acid) indicated the full
consumption of the starting material. The reaction mixture was concentrated
to dryness under reduced pressure, dry ethanol (8 mL) was added to the
viscous residue, and the mixture was evaporated to dryness. This procedure
was repeated twice, and then three times by substituting acetone for
ethanol. Ether (20 mL) was added, heated to the boiling point and the
etheral extract was decanted. This operation was repeated one more time,
with the same amount of ether, when benzoic acid was completely
removed. Drying under vacuum (0.1 mmHg) afforded 62 mg of the title
compound 10 as the amorphous, colorless powder, which was used in the
next step without further purification.
(4R,5R)-4-(4-Methoxyphenyl)-1,3-Oxazolidine-2-one-5-carboxylic
acid (11) (CAUTION: Phosgene is highly toxic! All operations with
trichloromethyl chloroformate should be performed in a well ventilated

444
Z. Tokic-Vujosevic et al.
hood). (2R,3R)-3-Amino-3-(4-methoxyphenyl)-2-hydroxypropanoic acid
hydrochloride 10 (62 mg; 0.243 mmol) was added to a cold (08C) solution
of NaOH (75 mg; 1.875 mmol) in water (4 mL) and the mixture was stirred
for 10 min in an ice bath. Trichloromethyl chloroformate (45 mL; 73.8 mg;
0.373 mmol) was added drop-wise and the mixture was stirred for 90 min at
that temperature; pH of the reaction mixture should be 9-10. The progress
of the reaction was monitored by TLC (eluent: toluene/ethyl acetate ¼ 3/2,
with 3 drops of formic acid). As TLC indicated the presence of starting
material (about 20%), additional NaOH (25 mg) and trichloromethyl chloro-
formate (15 mL) were added and stirring was continued for 1 h.The reaction
was quenched by the addition of 1.5 M HCl until pH 2 (with stirring and
cooling). The reaction mixture was extracted with ethyl acetate (3 ꢀ 15 mL),
the combined organic extract was washed with water (2 ꢀ 5 mL), brine and
dried over anhydrous MgSO4. Evaporation of solvent under reduced pressure
afforded 45 mg of the title compound 11 as beige-colored, amorphous
powder, which was used in the next step without further purification.
Methyl (4R,5R)-4-(4-methoxyphenyl)-1,3-oxazolidine-2-one-5-car-
boxylate (12) (CAUTION: Diazomethane is explosive!). Nitrosomethylurea
(80 mg) was added in small portions to a cold (08C) solution of KOH (200 mg)
in water (0.2 mL) and ether (4.8 mL). The yellow etheral solution was
decanted, dried over anhydrous Na2SO4 and added drop-wise to a cold
solution of (4R,5R)-4-(4-methoxyphenyl)-1,3-oxazolidine-2-one-5-carboxylic
acid 11 (45 mg, from the previous reaction) in THF (2.5 mL), with stirring,
while the progress of the reaction was monitored by TLC (eluent: toluene/
ethyl acetate ¼ 4/1). Evaporation of solvent under reduced pressure,
followed by purification by dry-flash chromatography (eluent: toluene/ethyl
acetate ¼ 4/1) afforded 39 mg (65% from cyanohydrin 8) of the title
compound 12, as colorless, star-shaped crystals.
Physical data for 12: m.p. 1068C; recrystallization from ethyl acetate/n-
hexane gives colorless crystals m.p. 110–110.58C; [a]D 2 94.0 (c 1.0, MeOH,
238C); Anal. calcd. for C12H13NO5: C 57.37, H 5.22, N 5.58; found: C 57.10,
H 5.31, N 5.54; IRKBr: 3465, 3271, 3012, 1757, 1738, 1517, 1458, 1280, 1217,
1
1029, 935; H NMR (CDCl3) d: 3.31 (s, 3H); 3.80 (s,3H); 5.18 (d, J ¼ 9.3;
1H); 5.26 (d, J ¼ 9.3, 1H); 6.09 (br s, 1H); 6,88 (d, J ¼ 8.8, 2H); 7.21
(d, J ¼ 8.8, 2H); 13C NMR(CDCl3) d: 52.00; 55.26; 57.88; 78.13; 114.05;
126.99; 128.14; 158.24; 160.28; 166.85.
(4R,5R)-4-(4-Methoxyphenyl)-5-Hydroxymethyl-1,3-oxazolidine-2-
one (cytoxazone, 1). Sodium borohydride (36.4 mg; 0.963 mmol) was added
in one portion to a cold (08C) solution of methyl (4R,5R)-4-(4-methoxyphe-
nyl)-1,3-oxazolidine-2-one-5-carboxylate 12 (29.1 mg; 0.116 mmol) in THF
(1.9 mL) and water (0.2 mL), and the reaction mixture was stirred for 1 h
while monitoring the progress of the reaction by TLC (eluent: toluene/ethyl
acetate ¼ 7/3). Additional sodium borohydride (10 mg; 0.264 mmol) was
added and stirring was continued for one more hour, when no starting

Synthesis of (2)-Cytoxazone and (1)-Cytoxazone
445
compound could be detected in the reaction mixture. 1 M HCl was added until
pH 6-7 and the mixture was extracted with ethyl acetate (3 ꢀ 10 mL). The
combined extract was washed with 5% aqueous NaOH (2 ꢀ 4 mL), water
and brine, dried over anhydrous MgSO4 and concentrated under reduced
pressure. The solid residue was purified by dry-flash chromatography
(eluent: toluene/ethyl acetate ¼ 45/55) to give 22 mg (83%) of (2)-cytoxa-
zone 1 as shiny, colorless crystals, m.p. 120–1218C (lit.[2] m.p. 118–
1218C); Anal. calcd. for C11H13NO4: C 59.20, H 5.87, N 6.27; found: C
58.90, H 5.76, N 6.27; [a]D 2 65.7 (c 0.38, MeOH, 258C); (lit.[2]
[a]D 2 75.7 (c 1.0, MeOH, 268C)); Spectral data identical to those reported
in the literature.[2] HPLC analysis on Chiracel OD column showed the
compound to be .98% ee.
(4R,5R)-2-Phenyl-4-(4-methoxyphenyl)-1,3-oxazoline-5-carbonitrile
(16; isomerization of syn-cyanohydrin 9). Diethyl azodicarboxylate
(394 mg; 352 mL; 2.263 mmol) was added over 4 min to a solution
of (2S,3R)-2-Hydroxy-3-(4-methoxyphenyl)-3-N-benzoylaminopropionitrile 9
(100 mg; 0.338 mmol), triphenylphosphine (594 mg; 2.263 mmol) and p-nitro-
benzoic acid (101.7 mg; 0.608 mmol), with stirring, at rt, under an argon
atmosphere. The course of the reaction was monitored by TLC (eluent:
toluene/ethyl acetate ¼ 9/1). After 3 h the mixture was filtered through a
sintered glass funnel, diluted with dichloromethane (50 mL), washed with
saturated aqueous NaHCO3 (2 ꢀ 5 mL), water and brine. The organic
extract was dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The pale-yellow viscous residue was purified by dry-flash chromato-
graphy (eluent: toluene/ethyl acetate ¼ 39/1) to give 68 mg (73%) of the title
compound 16, which crystallizes from ethyl acetate/n-hexane as shiny
needles.
Physical data for 16: m.p. 1158C; [a]D þ 3.1 (c 0.38, EtOAc, 238C); Anal.
calcd. for C17H14N2O2: C 73.37, H 5.07, N 10.07; found: C 73.01, H 5.08, N
10.06; IRKBr: 2965, 2930, 1667, 1581, 1513, 1456, 1327, 1271, 1249, 1176,
1
1059, 1028, 787; H NMR (CDCl3) d: 3.81 (s, 3H); 5.52 (d, J ¼ 10.0, 1H);
5.66 (d, J ¼ 10.0, 1H); 6.94 (d, J ¼ 8.8, 2H); 7.27 (d, J ¼ 8.8, 2H); 7.43–
7.61 (m, 3H); 8.04 (d, J ¼ 6.5, 2H); 13C NMR (CDCl3) d: 55.20; 71.59;
72.75; 114.24; 114.70; 125.77; 128.28; 128.61; 128.64; 128.70; 132.46;
160.04; 163.37.
Hydrolysis of 16 into (2R,3R)-3-Amino-3-(4-methoxyphenyl)-2-hydro-
xypropanoic acid hydrochloride 10 was carried out according to the
procedure described above for the conversion of 8 to 10. Conversion of 10
into cytoxazone was accomplished by a three-step procedure, as described
above. The cytoxazone thus obtained was identical in all respect to the
sample obtained from 8.
Methyl
(4R,5S)-4-(4-methoxyphenyl)-1,3-oxazolidine-2-one-5-car-
boxylate (15). This compound was prepared from 9 by a three-step
procedure, as described for the preparation of methyl (4R,5R)-4-(4-

446
Z. Tokic-Vujosevic et al.
methoxyphenyl)-1,3-oxazolidine-2-one-5-carboxylate 12 from 8. Starting
from 84 mg of 9, 51 mg (71% over three steps) of 15 was obtained, as
colorless crystals.
Physical data for 15: m.p. 91–928C; [a]D þ 90.0 (c 1.0, MeOH, 208C);
Anal. calcd. for C12H13NO5: C 57.37, H 5.22, N 5.58; found: C 57.51, H
4.99, N 5.54; IRKBr: 3230, 2966, 1784, 1729, 1613, 1513, 1251, 1192,
1
1069, 938; H NMR (CDCl3) d: 3.82 (s, 3H); 3.87 (s, 3H); 4.75 (d, J ¼ 5.3,
1H); 4.93 (d, J ¼ 5.3, 1H); 5.76 (br s, 1H); 6.94 (d, J ¼ 8.8, 2H); 7.29
(d, J ¼ 8.8, 2H); 13C NMR (CDCl3) d: 53.02; 55.33; 58.70; 80.46; 114.61;
127.25; 130.73; 157.80; 160.17; 168.83.
(4R,5S)-epi-Cytoxazone 2. According to the procedure described for
cytoxazone 1. Starting from 38.1 mg of Methyl (4R,5S)-4-(4-methoxy-
phenyl)-1,3-oxazolidine-2-one-5-carboxylate 15, 36 mg (93%) of (4R,5S)-
epi-cytoxazone 2 was obtained in form of colorless crystals, m.p.
161–1628C, (lit.[11] m.p. 161.5–162.58C); [a]D þ 32.0 (c 0.4, MeOH,
258C); (lit.[11] for the enantiomeric, (4S,5R)-epi-cytoxazone: [a]D 2 30.4 (c
1.0, MeOH, 208C)); Spectral data identical to those previously reported.[11]
Determination of optical purity by HPLC analysis on Chiracel OD column
showed the compound to be .96% ee.
ACKNOWLEDGMENT
This work was financially supported by the Ministry of Science, technology
and development of the Republic of Serbia (project No. 1719).
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