Pigments of fungi. LXIX.* Total synthesis of (R)-ochratoxin α and the formal total synthesis of ochratoxin A

Article abstract of DOI:10.1071/CH02014

(R)-Ochratoxin α, the monochiral carboxylic acid component of the biologically active dipeptide ochratoxin A, is synthesized for the first time over nine steps from (R)-propylene oxide. The method constitutes a versatile and general route to functionalized dihydroisocoumarins.

Full text of DOI:10.1071/CH02014

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Aust. J. Chem. 2002, 55, 213–217
Pigments of Fungi. LXIX.* Total Synthesis of (R)-Ochratoxin α
and the Formal Total Synthesis of Ochratoxin A
Christopher D. DonnerA and Melvyn GillA,B
A School of Chemistry, The University of Melbourne, Vic. 3010, Australia.
B Author to whom correspondence should be addressed (e-mail: melvyn@unimelb.edu.au).
(R)-Ochratoxin α, the monochiral carboxylic acid component of the biologically active dipeptide ochratoxin A, is
synthesized for the first time over nine steps from (R)-propylene oxide. The method constitutes a versatile and
general route to functionalized dihydroisocoumarins.
Manuscript received: 17 January 2002.
Final version: 31 March 2002.
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Introduction
A. Instead, enantiomerically pure (R)-ochratoxin α (3),
obtained by hydrolysis of the natural product (1), was used as
the substrate in the simple sequence of reactions summarized
in Scheme 1.[9] Thus (R)-ochratoxin α (3) was converted into
the corresponding acid chloride (5) and thence to the acyl
azide (6). Treatment of the azide (6) with L-phenylalanine
gave ochratoxin A (1) in 40% yield over the three steps.
Reaction of the acyl halide (5), rather than the azide (6), with
L-phenylalanine led to extensive epimerization of the amino
acid chiral centre.[9]
Dihydroisocoumarins represent an extensive class of
naturally occurring compounds that display a wide range of
biological activity.[1] The ochratoxins A (1) and B (2) were
first isolated from strains of Aspergillus ochraceus by Steyn
and coworkers in 1965.[2] The chlorinated metabolite
ochratoxin A (1) is responsible for the toxicity of the
organism[3] and has been shown in animal studies to be a
potent nephrotoxin, immunosuppressant, teratogen, and
carcinogen.[4] The problems associated with the presence of
ochratoxin A (1) as a ubiquitous contaminant in infected
agricultural products, such as stored cereal grains, have been
extensively documented.[5,6] Food and feed products show
considerable variation in the levels of contamination by (1)
and the levels also differ from one region to another. High
concentrations of ochratoxin A (1) are associated with
outbreaks of Balkan endemic nephropathy, a disease that
affects rural populations in several areas in the Balkans,[7]
and causes tumours of the urinary tract.[8]
OH
O
O
OH
O
CO2H
H
RO2C
O
N
H
O
Me
Me
H
H
Cl
R
(1) R = Cl
(2) R = H
(3) R = H
(4) R = Me
OH
O
OMe
The less toxic ochratoxin α (3), which contains the intact
isocoumarin nucleus of ochratoxin A (1), has been found,
together with other derivatives, in the culture fluid of a
variety of fungi, including Penicillium viridicatum and P.
verrucosum.[5]
MeO2C
CO2Me
O
R2
R1
(7) R1 = H, R2 = Me
(8) R1 = Me, R2 = H
(20)
The first synthesis of ochratoxin A was reported in 1967
and served to confirm the structure (1).[9] This synthesis
began from 3-bromophenol, which was first converted over
nine straightforward steps into racemic ochratoxin α, (3) and
ent-(3). Partial resolution of the individual enantiomers of
ochratoxin α was achieved by fractional crystallization of the
diastereoisomeric brucine salts which, on acidification, gave
(R)-ochratoxin α (3) and (S)-ochratoxin α ent-(3) in 83 and
79.5% e.e., respectively. Neither of the enantiomer-enriched
synthetic ochratoxins α were carried through to ochratoxin
Later,
Roberts
and
Woollven[10]
synthesized
( )-ochratoxin α (3) and ent-(3) in 0.62% yield over eight
steps from 4-chloro-7-hydroxyindanone. The racemic acid
was partially resolved with L-phenylalanine to give
ochratoxin A with ca. 80% e.e.
Recently, a more concise synthesis of ( )-ochratoxin α
was achieved in a 20% yield over four steps from dimethyl
3-oxopentandicarboxylate.[11]
* Part LXVIII, J. Chem. Soc., Perkin Trans. 1 2002, 938.
© CSIRO 2002
10.1071/CH02014 0004-9425/02/0300213

214
C. D. Donner and M. Gill
hitherto unknown diene (9). After lactonization of the
intermediate benzoate (not shown) appropriate functional
group manipulation should lead to (3).
The alternative disconnection of (3) (Scheme 2, path B)
leads back to methyl propiolate (11) and the highly
functionalized, chiral diene (12). The diene (12) is itself
available from the acetylene (10),[13] making the two routes
nicely complementary.
O
OH
Cl
O
R
O
(i)
(iii)
(3)
(1)
Me
H
(5) R = Cl
(6) R = N3
(ii)
Scheme 1. Reagents and conditions: (i) SOCl2, reflux, 2 h; (ii)
NaN3, 0°C, dimethylformamide, 30 min; (iii) L-phenylalanine, H2O,
Et3N, 5°C, 55 h.
Path A
Our first approach to ochratoxin A (1) required the new
diene (9). Dienes of this type, e.g. the methyl, ethyl, and
propyl substituted systems (17)–(19), respectively, have been
prepared by reaction of ketene dimethyl acetal (16) with the
corresponding acetylenes (13)–(15) (Scheme 3)[14,15]
However, when methyl propiolate (11) and ketene dimethyl
acetal (16) were brought together under similar conditions,
we could find no evidence of the presence of the requisite
diene (9). The major product (20) [electrospray (ES) mass
spectrum: m/z 247, [M + Na]+; C11H12O5], obtained in 40%
yield as a colourless oil after chromatography and
distillation, was characterized from the 1H nuclear magnetic
resonance (NMR) spectrum, which consists of a one-proton
triplet at δ 7.18 (J 7.8 Hz), a two-proton doublet at δ 7.90 (J
7.8 Hz) and a nine-proton singlet at δ 3.91. This product
showed absorption maxima at 207 and 280 nm in the
electronic spectrum, consistent with the presence of a
benzene ring, and the infrared spectrum contained an
absorption at 1724 cm–1 typical of a benzoate ester.
Results and Discussion
It was our plan to extend the methodology we developed
earlier for the synthesis of the monochiral (R)- and
(S)-melleins (7) and (8), respectively,[12] to the synthesis of
ochratoxin A (1). Two closely related retrosynthetic avenues
to ochratoxin A (1) are outlined in Scheme 2. As has already
been discussed, ochratoxin A (1) is available from the
isocoumarin (3) (ochratoxin α) although, strictly speaking
and to date, monochiral ochratoxin A (1) has been the subject
only of a ‘relay’ rather than a ‘total’ synthesis.[9]
O
OH
O
CO2H
H
N
H
O
Me
H
Cl
(1)
Ref. 9
O
OMe
OH
CO2Me
MeO2C
OMe
HO2C
(i)
OMe
O
+
OMe
Me
R
A
B
R
H
Cl
(17) R = Me
(18) R = Et
(19) R = Pr
(13) R = Me
(14) R = Et
(15) R = Pr
(3)
(16)
Path A
Path B
Scheme 3. Conditions: (i) sealed tube, 145–160°C,
OMe
OMe
OMe
20–22 h.[14,15]
CO2Me
CO2Me
+
MeO2C
CO2Me
MeO
+
The formation of the meta-phthalate ester (20) can be
rationalized by initial formation of the requisite diene (9)
followed by its immediate consumption by cycloaddition
with a second molecule of methyl propiolate (11). Shorter
reaction times and lower temperatures failed to deliver the
required diene (9). Other potential methods for preparing the
diene (9) were not pursued, and instead, we moved to explore
path B.
H
Me
OTBDMS
OTBDMS
Me
H
H
(11)
(12)
(9)
(10)
(10)
Scheme 2. Retrosynthesis of ochratoxin A (1).
Path B
The enantiomerically pure (R)-acetylenic ester (10),* [α]D
–5.83, was obtained in 28% yield over three steps from
commercially available (R)-propylene oxide (21), as
described previously.[12]
Following path A in Scheme 2, which closely parallels our
synthesis of (R)-mellein (7),[12] we anticipated that the
isocoumarin (3) would be available by Diels–Alder
cycloaddition between the (R)-acetylenic ester (10) and the
* It should be noted at this point that the chemistry from this point onwards, which is described only in the monochiral series, was first explored
using isochiral compounds. Where differences were observed between the spectroscopic or physical properties of the monochiral and corresponding
isochiral materials, these are recorded in the Experimental section.

Total Synthesis of (R)-Ochratoxin α and the Formal Total Synthesis of Ochratoxin A
215
the isophthalate (20) was also present in the reaction
mixture.
CO2Me
OMe
CO2Me
MeO
Treatment of the chiral benzoate (22) with
para-toluenesulfonic acid in dichloromethane gave the
novel lactone (23) as a colourless oil, [α]D –200 in 82%
O
(ii)
(iv)
3 steps
Ref. 12
(i)
Me
Me
OTBDMS
H
OTBDMS
H
Me
1
H
yield after chromatography. The H NMR spectrum of the
(10)
(21)
(12)
lactone (23) contains resonances from the protons of the
two methoxy groups (δ 3.91 and 3.98) and ortho-coupled
doublets (J 8.0 Hz) at δ 7.02 and 7.89 due to two aromatic
protons. The C(4) methylene protons resonate as a two
proton multiplet at δ 2.92 as the AB portion of an ABX
system coupled to the C(3) methine proton (δ 4.57), with
the protons of the C(3) methyl group appearing as a doublet
(J 6.2 Hz) at δ 1.49. The loss of the tert-butyldimethylsilyl
group is evident from the absence of any signals with δ 1.00
in the 1H NMR spectrum. The 13C NMR spectrum of (23) is
also in full accord with the structure shown and contains
carbonyl carbon resonances at δ 161.5 and 166.0, whilst the
infrared spectrum contains a strong absorption at 1730
cm–1.
OMe
OMe
O
MeO2C
CO2Me
MeO2C
O
(iii)
Me
H
OTBDMS
Me
H
(23)
(22)
OH
O
OH
O
MeO2C
HO2C
O
O
(v), (vi)
Me
Me
H
H
Cl
(3)
(24)
Cleavage of the methyl ether group in (23) was achieved
cleanly in 92% yield with boron trichloride in
dichloromethane at low temperature. The corresponding
phenol (R)-(24) was obtained in this way as an optically
active, colourless crystalline solid, melting point (m.p.)
130–131°C, [α]D –116 in 15% yield over seven steps from
(R)-propylene oxide (21).
The (R)-ester (24) is an intermediate in Takeuchi’s
synthesis of (R)-mellein (7).[16] The physical and
spectroscopic data for our methyl ester (24) are in close
agreement with the literature data.[16]
Scheme 4. Reagents and conditions: (i) ketene dimethyl acetal
(16), sealed tube, 165°C, 23 h; (ii) methyl propiolate (11), sealed
tube, 145°C, 22 h; (iii) p-TsOH, CH2Cl2, room temp., 72 h; (iv) BCl3,
CH2Cl2, 0°C, 10 min; (v) SO2Cl2, CH2Cl2, room temp., 48 h; (vi)
MeOH, LiOH·H2O, reflux, 5 h.
Cycloaddition between the (R)-acetylene (10) and
ketene dimethyl acetal (16) (Scheme 4) gave the diene (12),
which was not purified but was, rather, exposed
immediately to methyl propiolate (11). From the resultant
reaction mixture, the unchanged acetylene (10) was
recovered by distillation under reduced pressure.
Chromatographic purification of the involatile residue gave
the new (R)-benzoate (22) as a colourless, optically active
oil, [α]D –34.7, in a yield of 69% based on the amount of
the acetylene(10) consumed. The molecular formula
C20H32O6Si of (22) was supported by high-resolution
electrospray ionization mass measurement on the
pseudo-molecular ion ([M + Na]+) at m/z 419. The 1H NMR
spectrum of (22) contains ortho-coupled doublets (J 8.1
Hz) at δ 7.06 and 7.82 due to the two aromatic protons, and
three-proton singlets at δ 3.85, 3.91, and 3.92, which
correspond to the protons of the methyl ether and the two
methyl ester groups, respectively. The protons of the
functionalized side chain in (22) give rise to a three-proton
doublet at δ 1.15, an ABX system with components centred
at δ 2.68 [H2(1´)] and δ 4.01 [H(2´)] and two three-proton
singlets (δ –0.26 and –0.10) together with a nine-proton
singlet (δ 0.80) corresponding to the protons of the
tert-butyldimethylsilyl ether group. The infrared spectrum
of (22) shows a strong ester carbonyl absorption at 1727
cm–1 and the 13C NMR spectrum includes ester carbonyl
carbon signals at δ 165.6 and 168.0. The 13C NMR
spectrum also confirms the presence of six aromatic
carbons (δ 121.9–157.5), three methoxy groups (δ 52.2,
52.3, and 63.7), and all of the elements of the
functionalized side chain. All of the spectroscopic data are
fully consistent with the structure (22). A small amount of
To complete the synthesis of (R)-ochratoxin α (3) we
used the conditions developed by Kraus for the
corresponding isochiral ester (24).[11] Thus, exposure of the
(R)-ester (24) to sulfuryl chloride in dichloromethane at
room temperature (Scheme 4) effected halogenation at C(5)
in (24) and delivered the ester (4). The ester (4) was not
isolated but was instead immediately saponified by using
lithium hydroxide in methanol to give, in 68% yield over
two steps, (R)-ochratoxin α (3) as colourless needles, m.p.
213–215°C, from acetone. The mass spectrum of (3) shows
a molecular ion cluster at m/z 256/258 with an isotope
pattern characteristic of the presence of a single chlorine
1
atom in the molecule. The H NMR spectrum contains a
single aromatic resonance at δ 8.41, a methyl doublet at δ
1.63 (J 6.3 Hz), and an ABX multiplet resulting from the
diastereotopic C(4) methylene protons (δ 2.91 and 3.34)
and the C(3) methine proton (δ 4.82) in the heterocyclic
ring in (3).
The spectroscopic and physical properties of synthetic
(R)-ochratoxin α (3) are collected in Table 1 alongside the
corresponding data reported for the product of acid
hydrolysis of natural ochratoxin A (1).[2,17] Since ochratoxin
α (3) is here derived from (R)-propylene oxide (21) without
any intermediate change of stereochemistry, the product (3)
must have the (R)-stereochemistry shown. Accordingly, both
synthetic and natural ochratoxin α (3) are strongly
laevorotatory.

216
C. D. Donner and M. Gill
Table 1. Selected physical and spectroscopic properties of (R)-ochratoxin α (3)
Property
(R)-(–)-ochratoxin α (3)
Lit.[2,17] (R)-(–)-ochratoxin α (3)
(from ochratoxin A (1))
(Synthetic)
m.p. (°C)
[α]D
213–215 (acetone)
–216 (c, 0.08)
239 (subl. 155°C/5 × 10–4 mmHg)
–230 (c, 0.14)A
υ
max (cm–1)
3286(br), 1733, 1703, 1664
218 (4.40), 271 (4.18), 337 (3.78)
1.63, 3H, d, 6.3, 3-Me
2.91, 1H, dd, 17.6, 11.6, H(4ax)
3.34, 1H, dd, 17.6, 3.4, H(4eq)
4.82, 1H, m, H(3)
1735, 1680
λ
max [nm, (log ε)]
212 (4.48), 338 (3.75)
1.63, 3H, d, 6.3, 3-Me
2.91, 1H, dd, 17.6, 11.6, H(4ax)
3.30, 1H, dd, 17.6, 3.5, H(4eq)
4.82, 1H, m, H(3)
1H NMR
(CDCl3)B δ
8.41, 1H, s, H(6)
8.41, 1H, s, H(6)
13.29, 1H, s, 8-OH
10.45, 1H, br, 7-CO2H
13.30, 1H, s, 8-OH
m/z (EI, 70 eV)
258 ([M + 2]+, 17%), 256 (M+, 53),
214 (36), 212 (100), 196 (31), 194 (92)
258 ([M + 2]+, 15%), 256 (M+, 45),
214 (35), 212 (100), 194 (70)
A Conditions are not quoted in the literature.[2]
B 1H NMR data recorded at 400 MHz in CDCl3 for synthetic (3) and 300 MHz in CDCl3 for naturally derived (3).[17]
for 15 h. Flash vacuum pad chromatography (ether/light petroleum 1 : 1,
RF 0.40) followed by Kügelrohr distillation gave the isophthalate ester
(20) (215 mg, 40%) as a colourless oil, b.p. 115–120°C/0.25 mmHg
(lit.[18] 160–170°C/4–5 mmHg). νmax 2952, 1724, 1254, 1209 cm–1.
Conclusions
(R)-Ochratoxin α (3) was obtained in this way in 10% yield
over nine steps from commercially available (R)-propylene
oxide (21). Previous syntheses of the isocoumarin nucleus
(3) of ochratoxin A (1) furnished either isochiral material
[(3) and ent-(3)][10,11] or, at best, anisochiral material [83%
e.e. of the (R)-enantiomer (3)] in a chemical yield of less than
5% over more than 10 steps from 3-bromophenol.[9] The only
previously published work in the monochiral series was
aimed, not at ochratoxin A (1), but rather at mellein (7), and
proceeded only as far as the (R)-ester (24).[16]
The work described here constitutes the first total
synthesis of enantiomerically pure (R)-ochratoxin α (3) and,
since naturally derived ochratoxin α has been previously
converted back to ochratoxin A (1),[9] this report also
constitutes the first formal total synthesis of
enantiomerically pure ochratoxin A (1).
λ
max (MeOH) 207 (log ε 4.33), 272 (3.06), 280 (3.08), 293 nm (3.01).
δ
H (300 MHz) 3.91, 9H, s, 2 × CO2Me and OMe; 7.18, 1H, t, J 7.8 Hz,
H 5; 7.90, 2H, d, J 7.8 Hz, H(4) and H(6). Mass spectrum (ES) m/z 247
([M + Na]+), 225 ([M + H]+), 193 ([M – OMe]+).
Dimethyl (R)-(–)-4-(2-tert-Butyldimethylsilyloxypropyl)-2-methoxy-
benzene-1,3-dicarboxylate (22)
The (R)-hexynoate (10) [1.20 g, 4.68 mmol; prepared in 28% yield over
three steps from (R)-propylene oxide (21) by the method described
previously][12] and ketene dimethyl acetal (16) (0.50 g, 5.68 mmol)
were heated together with stirring in a sealed tube at 165°C for 23 h.
After cooling to room temperature, methyl propiolate (11) (1.5 mL,
16.9 mmol) was added and the mixture was heated at 145°C (sealed
tube) for 22 h. After removal of unchanged acetylene (10) (0.98 g)
under reduced pressure the residue was purified by flash vacuum pad
chromatography using a graded solvent system (ether/light petroleum)
to give; (i) the isophthalate ester (20) (160 mg) (RF 0.40 in ether/light
petroleum, 1 : 1) identical to the product described immediately above,
and (ii) the (R)-benzoate (22) (235 mg, 69%) (RF 0.60 in ether/light
petroleum, 1 : 1) as a colourless oil, [α]D –34.7 (c 0.85, CHCl3) (Found:
[M + Na]+, 419.1862. C20H32NaO6Si requires [M + Na]+, 419.1866).
Experimental
General
max 2949, 1727, 1248, 836 cm–1. δH (400 MHz) –0.26 and –0.10, each
Melting points were determined on a hot-stage apparatus and are
uncorrected. Infrared (IR) spectra were recorded using a Perkin–Elmer
983 G spectrophotometer for samples using potassium bromide disks.
ν
3H, s, SiMe; 0.80, 9H, s, SiCMe3; 1.15, 3H, d, J 5.9 Hz, H3(3´), 2.68,
2H, m, H2(1´), 3.85, 3H, s, OMe; 3.91 and 3.92, each 3H, s, CO2Me;
4.01, 1H, m, H(2´); 7.06, 1H, d, J 8.1 Hz, H(5); 7.82, 1H, d, J 8.1 Hz,
H(6). δC (100 MHz) –5.28 and –4.97, each SiMe; 17.9, SiCMe3; 24.1,
C(3´); 25.8, SiCMe3; 43.8, C(1´); 52.2 and 52.3, each CO2Me; 63.7,
OMe; 68.8, C(2´); 121.9, C(1); 126.9, C(5); 130.8, C(3); 132.2, C(6);
143.1, C(4); 157.5, C(2); 165.6 and 168.0, each CO2Me. Mass
spectrum (ES) m/z 397 ([M + H]+). Mass spectrum (EI) m/z 339
([M – C4H9]+, 84%), 307 (49), 233 (40), 159 (30), 89 (44), 75 (35), 73
(100).
Electronic spectra were recorded on
a Shimadzu UV-2401PC
spectrophotometer using either ethanolic or methanolic solutions in a
10 mm quartz cell. NMR spectra were recorded with JEOL
JNM-GX-400 and Varian Unity 400 spectrometers (1H at 400 MHz and
13C at 100 MHz) for solutions in CDCl3 unless stated otherwise. Mass
spectra were recorded on a Shimadzu GCMS-QP505A spectrometer at
70 eV [probe; electron impact (EI)] and a Micromass QUATTRO II
[electrospray (ES)]. Specific rotations were measured using a JASCO
DIP-1000 polarimeter and are given in units of 10–1 deg cm2 g–1.
Methyl (R)-(–)-8-Methoxy-3-methyl-1-oxo-3,4-dihydro-(1H)-2-
benzopyran-7-carboxylate (23)
Materials
Thin-layer chromatography (TLC) and preparative TLC (PLC) were
performed on Merck pre-coated silica gel 60 F254 and Merck Kieselgel
60 GF254 (20 g silica gel spread on 20x20 cm glass plates), respectively.
Visualization was under UV light (254 or 366 nm).
A
solution of the (R)-benzoate (22) (85 mg, 0.21 mmol) in
dichloromethane (3 mL) containing para-toluenesulfonic acid (5 mg)
was stirred at room temperature for 72 h. After removal of the solvent
under reduced pressure the residue was purified by flash vacuum pad
chromatography using a graded solvent system (ether/light petroleum;
RF 0.38 in ether/light petroleum, 9 : 1) to give the (R)-lactone (23) (44
mg, 82%) as a colourless oil, [α]D –200 (c 0.63, CHCl3) (Found:
[M + Na]+, 273.0735. C13H14NaO5 requires [M + Na]+, 273.0739). νmax
Reaction of Methyl Propiolate (11) with Ketene Dimethyl Acetal (16)
Methyl propiolate (11) (0.20 g, 2.38 mmol) and ketene dimethyl acetal
(16) (0.21 g, 2.38 mmol) were heated together in a sealed tube at 140°C

Total Synthesis of (R)-Ochratoxin α and the Formal Total Synthesis of Ochratoxin A
217
2949, 1730, 1596, 1249, 1217 cm–1. δH (400 MHz) 1.49, 3H, d, J 6.2
Hz, 3-Me; 2.92, 2H, m, H2(4); 3.91 and 3.98, each 3H, s, OMe; 4.57,
1H, m, H(3); 7.02, 1H, d, J 8.0 Hz, H(5); 7.89, 1H, d, J 8.0 Hz, H(6). δC
(100 MHz) 20.6, 3-Me; 36.1, C(4); 52.5, CO2Me; 63.6, OMe(8); 74.0,
C(3); 119.6, C(8a); 122.4, C(5); 126.1, C(7); 135.7, C(6); 145.6, C(4a);
161.5, C(1); 162.1, C(8); 166.0, CO2Me. Mass spectrum (ES) m/z 251
([M + H]+). Mass spectrum (EI) m/z 250 (M+, 17%), 247 (61), 235 (29),
234 (54), 232 (91), 219 (45), 218 (25), 217 (58), 206 (40), 205 (100),
203 (27), 190 (48), 189 (46), 187 (43), 176 (34), 175 (54), 174 (74), 173
(42), 172 (39), 163 (28), 162 (47), 161 (53), 160 (34), 147 (33), 146
(79), 145 (29), 144 (30), 133 (46), 132 (20), 131 (26), 119 (32), 118
(45), 117 (23), 116 (24), 115 (40), 105 (38), 104 (44), 103 (22), 91 (40),
90 (33), 89 (57), 78 (24), 77 (69), 76 (45), 75 (32), 65 (31), 63 (48), 62
(24).
mL). The combined ethyl acetate extracts were dried (MgSO4) and
concentrated under reduced pressure to give (R)-ochratoxin α (3) (9.0
mg, 68%) as a colourless powder from acetone, m.p. 213–215°C (dec.)
[lit.[2] 239°C (dec.)]. ( )-form m.p. 220–222°C [lit.[9] ( )-form 229°C].
See Table 1 for spectroscopic data. νmax ( )-form 3281(br), 1732, 1701,
1665, 1213, 1195, 1178, 1125 cm–1. δH (400 MHz, (D6)DMSO) 1.43,
3H, d, J 6.4 Hz, 3-Me; 2.88, 1H, dd, J 17.2 and 11.8 Hz, H(4ax); 3.19,
1H, dd, J 17.2 and 3.2 Hz, H(4eq); 4.74, 1H, m, H(3); 8.00, 1H, s, H(6).
δC (100 MHz, (D6)DMSO) 20.1, 3-Me; 32.2, C(4); 74.4, C(3); 112.5,
117.7, 120.6, 136.0, 143.4, 160.4, all Ar, all quaternary, except 136.0,
which represents a methine carbon; 165.4, 167.3.
Acknowledgments
C.D.D. is grateful for a Sir John and Lady Higgins Research
Scholarship. M.G. is thankful for generous financial support
from the Australian Research Council.
Methyl (R)-(–)-8-Hydroxy-3-methyl-1-oxo-3,4-dihydro-(1H)-2-
benzopyran-7-carboxylate (24)
To the (R)-methyl ether (23) (40.0 mg, 0.16 mmol) in dichloromethane
(5 mL) at 0°C was added boron trichloride (1M in dichloromethane,
0.32 mL, 0.32 mmol) dropwise. After 10 min, water (5 mL) was added
and the mixture was extracted with dichloromethane (3 × 5 mL), the
combined extracts were dried (MgSO4) and concentrated under reduced
pressure. The residue was purified by flash vacuum pad
chromatography using a graded solvent system (ether/light petroleum;
RF 0.48 in ether) to give the (R)-isocoumarin (24) (34.7 mg, 92%) as
colourless crystals (ether/hexane), m.p. 130–131°C (lit.[16]
134–135°C). {( )-form m.p. 136–139°C [lit.[16] ( )-form
144–145°C]}. [α]D –116 (c, 0.45 in CHCl3) {lit.[16] –117 (c, 1.00 in
CHCl3)}. (Found: M+, 236.0680. C12H12O5 requires M+, 236.0685).
References
[1] R. A. Hill, Prog. Chem. Org. Nat. Prods. 1986, 49, 1.
[2] K. J. van der Merwe, P. S. Steyn, L. Fourie, J. Chem. Soc. C 1965,
7083.
[3] De B. Scott, Mycopathol. Mycol. Appl. 1965, 25, 213.
[4] A. E. Pohl, S. Nesheim, L. Friedman, Pure Appl. Chem. 1992, 64,
1029 and references cited therein.
[5] P. Krogh, in Mycotoxins in Food (Ed. P. Krough) 1987, pp.
97–121 (Academic Press, Inc.: London).
ν
max 3433(br), 2947, 1723, 1655, 1615, 1256, 1237, 1219, 1141 cm–1.
[( )-form 3433(br), 2949, 1722, 1656, 1614, 1257, 1216 cm–1]. δH (400
MHz) 1.54, 3H, d, J 6.3 Hz, 3-Me; 2.97, 2H, d, J 7.1 Hz, H2(4); 3.94,
3H, s, CO2Me; 4.72, 1H, m, H(3); 6.75, 1H, d, J 8.0 Hz, H(5); 8.04, 1H,
d, J 8.0 Hz, H(6); 12.17, 1H, s, 8-OH. δC (100 MHz) 20.6, 3-Me; 35.0,
C(4); 52.3, CO2Me; 75.4, C(3); 110.0, 116.8, 117.5, 137.9, 145.2 and
162.5, all Ar, all quaternary, except 117.5 and 137.9 which represent
methine carbons; 166.3; 168.1. Mass spectrum (EI) m/z 236 (M+, 73%),
205 (63), 204 (21), 192 (29), 189 (21), 187 (22), 161 (20), 160 (100),
77 (27).
[6] Some examples include: (a) L. Stoloff, Carcinogens and
Mutagens in the Environment (Ed. H. Stich) 1982, pp. 97–120
(CRC Press Inc.: Boca Raton, FL); (b) T. Kuiper-Goodman, P.
Scott, Biomed. Environ. Sci. 1989, 2, 179.
[7] (a) T. Vrabcheva, E. Usleber, R. Dietrich, E. Maertlbauer, J.
Agric. Food. Chem. 2000, 48, 2483; (b) P. S. Steyn, M. A.
Stander, J. Toxicol., Toxin Rev. 1999, 18, 229.
[8] J. Bauer, M. Gareis, J. Ve t. Med. 1987, 34, 613.
[9] P. S. Steyn, C. W. Holzapfel, Tetrahedron 1967, 23, 4449.
[10] J. C. Roberts, P. Woollven, J. Chem. Soc. C 1970, 278.
[11] G. A. Kraus, J. Org. Chem. 1981, 46, 201.
[12] C. Dimitriadis, M. Gill, M. F. Harte, Tetrahedron: Asymmetry
1997, 8, 2153.
[13] A. S. Cotterill, M. Gill, N. M. Milanovic, J. Chem. Soc., Perkin
Trans. 1 1995, 1215.
[14] J. Banville, P. Brassard, J. Org. Chem. 1976, 41, 3018.
[15] A. S. Cotterill, M. Gill, A. Gimenez, N. M. Milanovic, J. Chem.
Soc., Perkin Trans. 1 1994, 3269.
[16] N. Takeuchi, K. Goto, Y. Sasaki, T. Fujita, K. Okazaki, K.
Kamata, S. Tobinaga, Heterocycles 1992, 33, 357.
[17] H. Xiao, R. R. Marquardt, A. A. Frohlich, Y. Z. Ling, J. Agric.
Food. Chem. 1995, 43, 524.
[18] T. Naito, E. Kuroda, O. Miyata, I. Ninomiya, Chem. Pharm. Bull.
1991, 39, 2216.
(R)-(–)-5-Chloro-8-hydroxy-3-methyl-1-oxo-3,4-dihydro-(1H)-2-
benzopyran-7-carboxylic Acid (3)
To a solution of the (R)-methyl ester (24) (12.2 mg, 0.052 mmol) in
dichloromethane (0.5 mL) at room temperature was added sulfuryl
chloride (20 µl, 0.249 mmol) and the mixture was stirred under
nitrogen. After 24 h a further aliquot of sulfuryl chloride (20 µL) was
added and the mixture was stirred for a further 48 h. The solution was
concentrated under reduced pressure and the residual oil, which
contained the ester (4), was dissolved in methanol (5 mL). Lithium
hydroxide monohydrate (40 mg, 0.953 mmol) was added and the
suspension was heated at reflux for 5 h. After cooling and removal of
the solvent under reduced pressure the residue was dissolved in water
(5 mL) and extracted with chloroform (5 mL). The aqueous layer was
acidified to pH 2 with 1 M HCl, and extracted with ethyl acetate (3 × 5