Isoaurostatin: Total synthesis and structural revision

Article abstract of DOI:10.1016/j.tet.2005.01.117

Isoaurostatin, a topoisomerase I inhibitor isolated from Thermomonospora alba has been synthesized for the first time from 2,4-dihydroxyacetophenone via 6-methoxybenzo-2(3H)-furanone in five steps. The E-isomer was converted into Z-isomer, but spectroscopic data of either of these two isomers did not match with those of the natural product. The structure of isoaurostatin has been revised to a known isoflavone, daidzein (2), based on careful analysis of spectroscopic data.

Full text of DOI:10.1016/j.tet.2005.01.117

Tetrahedron 61 (2005) 3013–3017
*
Isoaurostatin: total synthesis and structural revision
Somepalli Venkateswarlu, Gopala K. Panchagnula, Mothukuri Bala Guraiah
*
and Gottumukkala V. Subbaraju
Laila Impex R and D Centre, Unit I, Phase III, Jawahar Autonagar, Vijayawada 520 007, India
Received 7 October 2004; revised 13 January 2005; accepted 28 January 2005
Abstract—Isoaurostatin, a topoisomerase I inhibitor isolated from Thermomonospora alba has been synthesized for the first time from
,4-dihydroxyacetophenone via 6-methoxybenzo-2(3H)-furanone in five steps. The E-isomer was converted into Z-isomer, but spectroscopic
2
data of either of these two isomers did not match with those of the natural product. The structure of isoaurostatin has been revised to a known
isoflavone, daidzein (2), based on careful analysis of spectroscopic data.
q 2005 Elsevier Ltd. All rights reserved.
1
. Introduction
or base catalyzed condensation of substituted 2(3H)-
benzofuranone with aromatic aldehydes. We, therefore,
synthesized the desired 6-methoxybenzo-2(3H)-furanone
(9) as follows. Willgerodt–Kindler reaction of 2-hydroxy-4-
methoxyacetophenone (7) with sulfur and morpholine under
Isoaurostatin, a novel topoisomerase I inhibitor was recently
isolated from the culture filtrate of Thermomonospora alba
1
strain No. 1520. Isoaurostatin inhibited the relaxation
1
1
activity of calf thymus topoisomerase I in a noncompetitive
manner and did not inhibit the relaxation and decatenation
of human placenta topoisomerase II. Structure of isoauro-
statin was determined as 1 based on interpretation of
spectroscopic data. Aurones and isoaurones, are naturally
occurring yellow pigments of plants and are structurally
related to flavonoids. Aurones have limited natural
occurrence and fewer methods of synthesis, and naturally
occurring isoaurones are extremely rare and only two
compounds have been reported so far. Structures of these
compounds have been deduced on the basis of NMR data
and confirmed by partial synthesis. Pterocarposide, the
only isoaurone C-glucoside reported so far, was isolated
from Pterocarpus marsupium and its structure was deduced
phase transfer catalytic conditions gave phenylacetic acid
derivative 8, in 66% yield. Lactonization of 8 in the
1
2
presence of phosphorous oxychloride yielded 6-methoxy-
2(3H)-furanone (9) in 90% yield. Acid catalyzed conden-
sation of 9 with 4-hydroxybenzaldehyde afforded
0
4 -hydroxy-6-methoxyisoaurone (10), which was demethyl-
ated using pyridine hydrochloride to give 4 ,6-dihydroxy-
2
13
0
isoaurone (1) in 63% yield (Scheme 1). The H NMR
3
–5
1
spectrum showed that it is a mixture of two isomers and this
was also confirmed by HPLC (E/Z—90:10). Attempts to
purify further were not successful. In order to assign the
stereochemistry unambiguously, we sought the Z-isomer
also. Therefore, 1 was photoisomerised to 5 using a medium
pressure mercury lamp. Again, it was obtained as a mixture
of two isomers in the ratio of Z/E—90:10, after repeated
crystallizations.
6
,7
8
,9
1
0
from spectroscopic data. In this paper, we wish to report
a total synthesis of the proposed structure of isoaurostatin
(
1) and its structural revision into daidzein (2).
In (E) and (Z)-isoaurones, the configuration was assigned
based on the differences in chemical shifts of olefinic
0
0
protons (H-10) and ortho protons (H-2 and H-6 ) of the
pendant aryl unit. These protons are deshielded by the
carbonyl group and are expected to give downfield
2
. Results and discussion
1
4
0
0
The general synthetic route to isoaurones involves the acid
signals. In E-isomers, H-2 , H-6 protons of the aryl unit
appear as doublet in the range of d 7.0–7.8, whereas in
Z-isomers the corresponding protons appear in the range of
*
Laila Impex communication # 33.
1
4
d 8.0–8.2. The chemical shifts of synthetic 1, gave a
doublet at d 7.65 (H-2 , H-6 ) supporting the E-configuration
Keywords: Isoaurostatin; Thermomonospora alba; Synthesis; Structure
revision; Daidzein.
Corresponding author. Tel.: C91 866 2541303; fax: C91 866 2546216;
e-mail: subbarajugottumukkala@hotmail.com
0
0
0
0
and in 5, these protons resonated at d 8.16 (H-2 , H-6 )
confirming a Z-configuration. The olefinic protons (H-10) in
*
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.01.117

3014
S. Venkateswarlu et al. / Tetrahedron 61 (2005) 3013–3017
Scheme 1. Reagents and conditions: (a) DMS, K
rt, 15 h, 90%; (d) 4-hydroxybenzaldehyde, Ac
2 3 3
CO , acetone, rt, 5 h, 91%; (b) S, morpholine, p-TSA, 20% NaOH, PTC, reflux, 16 h, 66%; (c) POCl , DCE,
O, 90 8C, 3 h, 61%; (e) pyridine HCl, 180–190 8C, 3 h, 63%; (f) UV, THF, rt, 10 h, 70%.
2
1
Table 1. H and C NMR data of 1, 5 and isoaurostatin
13
1
a
a
b
Position
1-E
5-Z
Isoaurostatin
d
H
d
C
d
H
d
C
d
H
d
C
2
3
4
5
6
7
8
9
1
1
2
3
4
169.4
117.8
123.4
111.1
159.9
98.5
154.9
112.9
137.1
124.8
132
166.5
116.9
125.2
111.1
160.3
97.9
152.9
116.3
137.9
120.6
134.3
115.5
159
174.8
123.6
127.3
115.3
162.8
102.2
157.6
116.6
152.9
122.6
130.1
115
7.69 (d, 8.5)
6.58 (d, 8.5)
7.58 (d, 8.3)
6.63 (dd, 2.1, 8.3)
7.94 (d, 8.5)
6.91 (dd, 2.4, 8.5)
6.64 (s)
7.50 (s)
6.59 (d, 2.1)
7.72 (s)
6.83 (d, 2.4)
0
8.25 (s)
0
0
0
0
0
0
,6
7.65 (d, 8.4)
6.92 (d, 8.4)
8.16 (d, 8.7)
6.88 (d, 8.7)
7.36 (d, 8.5)
6.79 (d, 8.5)
,5
115.8
159.8
157.2
a 1
b 1
13
6
H NMR (400 MHz) and C NMR (100 MHz) in DMSO-d .
13
6
H NMR data (500 MHz) and C NMR data (125 MHz) in DMSO-d are taken from Ref. 1.
these isomers gave a singlet at d 7.50 (for E-isomer) and at d
.72 (for Z-isomer).
7
However, the olefinic proton in isoaurostatin was reported to
resonate at d 8.25 and the other proton and carbon NMR
chemical shifts (Table 1) are also not consistent with either
E-isomer 1 or Z-isomer 5. Furthermore, the proton NMR
data of acetyl derivative 4 of the E-isomer are also not
1
consistent with the reported data on isoaurostatin diacetate.
Obviously, isoaurostatin does not posses an isoaurone struc-
ture. So, we have carefully reanalyzed the spectroscopic
Figure 1. Structures of isoaurostatin (1) and daidzein (2).

S. Venkateswarlu et al. / Tetrahedron 61 (2005) 3013–3017
Table 2. H and C NMR data of reassigned isoaurostatin, isoaurostatin diacetate, daidzein (2) and daidzein diacetate (3)
3015
1
13
a
c
Position
Isoaurostatin
Isoaurostatin
b
diacetate
Daidzein (2)
Daidzein
diacetate 3
d
d
H
d
C
d
H
d
H
d
C
d
H
OAc
2.32 (s), 2.36 (s)
8.01 (s)
2.32 (s), 2.37 (s)
8.01 (s)
2
3
4
5
6
7
8
9
8.25 (s)
152.9
123.6
174.8
127.3
115.3
162.8
102.2
157.6
116.6
122.6
130.1
115
8.28 (s)
152.3
122.7
178.6
127.2
115.1
162.6
102.2
157.6
116.9
123.9
130
7.94 (d, 8.5)
6.91 (dd, 2.4, 8.5)
8.32 (d, 8.5)
7.18 (dd, 1.8, 8.5)
7.95 (d, 8.7)
6.92 (dd, 2.1, 8.7)
8.33 (d, 8.7)
e
7.18 (dd)
6.83 (d, 2.4)
7.32 (d, 1.8)
6.84 (d, 2.1)
7.32 (d, 2.1)
1
1
2
3
4
0
0
0
0
0
0
0
,6
7.36 (d, 8.5)
6.79 (d, 8.5)
Not reported
7.17 (d, 8.5)
7.36 (d, 8.6)
6.79 (d, 8.6)
7.59 (d, 8.6)
7.17 (d, 8.6)
,5
115.1
157.3
157.2
a 1
b 1
c 1
d 1
e
13
H NMR (500 MHz) and C NMR (125 MHz) in DMSO-d
6
are taken from Ref. 1 and reassigned.
H NMR (500 MHz) data in CDCl
H NMR (300 MHz) in DMSO-d
H NMR (400 MHz) in CDCl
3
is taken from Ref. 1 and reassigned.
13
6
3 6
and C NMR (15 MHz) in CDCl /DMSO-d are taken from Refs. 15 and 16.
3
.
0
0
Merged with H-3 ,5 doublet.
data reported for natural isoaurostatin and found that the
data agrees well with an isomeric structure 2 (Fig. 1). The
DEPT (100 MHz) spectra were recorded on a Bruker
AMX 400 MHz NMR spectrometer using TMS as internal
reference and the values for chemical shifts (d) being given
in ppm and coupling constants (J) in Hertz (Hz). Mass
spectra were recorded on Agilent 1100 LC/MSD. HPLC
was recorded by a Shimadzu SCL-10A instrument under the
following conditions: column, Altima C18; flow rate,
1 mL/min; detection at 384 nm; mobile phase, 0.1%
phosphoric acid: acetonitrile (65:35, v/v); retention time
for 1, 20.91 and for 5, 22.51 min. Acme silica gel G and
silica gel (100–200 mesh) were used for analytical TLC and
column chromatography, respectively.
carbonyl absorptions in IR for isoaurostatin was reported at
1
K1
630 cm , which is in good agreement with those of
K1
reported for 2, but not to the isoaurones (w1750 cm ).
Further, the singlet at d 8.25 in the H NMR data of
1
isoaurostatin agrees well with the chemical shift reported for
H-2 in 2, but not to the olefinic proton in isoaurones (d 7.0–
10,14
7
.8).
Based on the above, the proton and carbon NMR
data reported for isoaurostatin have been reassigned and the
data are found to be in good agreement with the reported
1
5
16
proton and carbon NMR data of daidzein (2) (Table 2).
Furthermore, the reassigned proton NMR data reported for
isoaurostatin diacetate are also identical with those of
daidzein diacetate, 3 (Table 2) prepared by us. From the
foregoing, the novel inhibitor of topoisomerase I isolated
from the culture filtrate of Thermomonospora alba is, in
fact, daidzein (2), a known isoflavone.
4
.1.1. 2-Hydroxy-4-methoxyacetophenone (7). A mixture
of 6 (6 g, 39.5 mmol), dimethyl sulfate (4.1 mL,
3.4 mmol), potassium carbonate (8.2 g, 59.2 mmol) and
4
acetone (100 mL) was stirred at rt for 5 h. After completion
of reaction, the solid was filtered off and the solvent was
evaporated. The residue obtained was chromatographed
over silica gel column using mixtures of petroleum ether–
ethyl acetate (90:10) as eluent to give 7 (6 g, 91%) as a
3. Conclusions
1
7
white solid, mp 46–48 8C (lit. mp 49–50 8C) and its
spectroscopic data are consistent with those reported in the
literature.
In summary, we have accomplished the first total synthesis
of the proposed structure of isoaurostatin (1), a novel
topoisomerase I inhibitor from Thermomonospora alba in
five steps starting from 2,4-dihydroxyacetophenone. The
E-isomer 1 was converted into Z-isomer 5 and the spectral
data of these isomers did not match with those reported for
isoaurostatin. The reported spectral data of natural product
have been reassigned and found to match well with those
recorded for daidzein (2).
17
4.1.2. 2-(2-Hydroxy-4-methoxyphenyl)acetic acid (8).
A mixture of 7 (1.66 g, 10 mmol), sulfur (0.64 g,
20 mmol), morpholine (3 mL, 30 mmol) and p-toluene-
sulfonic acid (0.06 g, 0.32 mmol) was refluxed under
constant stirring at 120–130 8C for 8 h. After completion
of reaction, the mixture was allowed to cool and 20% NaOH
(
10 mL) and tetrabutylammonium bromide (16 mg,
0.05 mmol) were added and continued hydrolysis for further
h at 100 8C. The cooled reaction mixture was filtered and
4. Experimental
8
4
.1. General
the filtrate was acidified with HCl to pH 2. The precipitated
solid was filtered and chromatographed over silica gel
column using hexane–EtOAc (80:20) as eluents to give 8
Melting points were recorded on a Mel-Temp melting point
apparatus, in open capillaries and are uncorrected. IR
spectra were recorded on a Perkin–Elmer BX1 FTIR
1
8
(1.2 g, 66%) as a light yellow solid, mp 130–132 8C (lit.
mp 130 8C) and its spectroscopic data are consistent with
those reported in the literature.
1
13
18
Spectrophotometer. H NMR (400 MHz), C NMR-

3016
S. Venkateswarlu et al. / Tetrahedron 61 (2005) 3013–3017
4
.1.3. 6-Methoxy-3-hydrobenzo[b]furan-2-one (9). A
mixture of 8 (340 mg) and phosphorous oxychloride
1.5 mL) in dichloroethane (10 mL) was stirred at rt for
5 h. The reaction mixture was diluted with water (50 mL)
(20 mL). The mixture was washed successively with water
(20 mL), dil HCl (20 mL), water (20 mL) and brine (20 mL)
and dried over sodium sulfate. The solution was filtered and
the residue obtained after evaporation of the solvent was
chromatographed over silica gel column using hexane–
EtOAc (80:20) as eluents to give 4 (20 mg) as a light yellow
solid, mp 128–130 8C; nmax (neat): 1767, 1617, 1598, 1195,
(
1
and extracted with chloroform (3!30 mL) and the
combined chloroform layer was washed with water (2!
2
0 mL), sodium bicarbonate (20 mL) and dried over sodium
K1
1
sulfate. The residue obtained after evaporation of the
solvent was chromatographed over silica gel column using
petroleum ether–EtOAc (90:10) as eluents to give 9
(270 mg, 90%) as a light yellow solid, mp 58–60 8C (lit.
mp 55–56 8C) and its spectroscopic data are consistent with
those reported in the literature.
1168, 1118, 1078, 1013 cm ; H NMR (CDCl ): d 2.32
3
(3H, s, OAc), 2.35 (3H, s, OAc), 6.80 (1H, dd, JZ2.2,
8.4 Hz, H-5), 6.96 (1H, d, JZ2.2 Hz, H-7), 7.24 (2H, d, JZ
1
8
0 0 0 0
8.7 Hz, H-3 ,5 ), 7.70 (2H, d, JZ8.7 Hz, H-2 ,6 ), 7.73 (1H,
13
d, JZ8.4 Hz, H-4), 7.81 (1H, s, H-10); C NMR (CDCl ): d
3
1
8
169.1, 168.9, 168.6, 155.0, 152.4, 152.2, 139.7, 131.5,
1
30.7, 123.3, 122.3, 121.6, 119.4, 117.1, 105.8, 21.2, 21.1;
MS (ESI, positive scan): m/z 339 (MCH) . Analysis
C
4
[
4
.1.4. 3-[(4-Hydroxyphenyl)methylene]6-methoxybenzo-
b]furan-2-one (10). A mixture of 9 (0.5 g, 3.05 mmol),
-hydroxybenzaldehyde (0.372 g, 3.05 mmol) in acetic
anhydride (7.5 mL, 79.4 mmol) and triethylamine
0.5 mL) was heated at 90 8C for 2 h. The cooled reaction
found: C, 67.38; H, 4.21%. Calcd for C19
H, 4.17%.
H O : C, 67.45;
14 6
(
4.1.7. 6-Hydroxy-3-[(4-hydroxyphenyl)methylene]benzo-
[b]furan-2-one (5Z). A solution of 1 (100 mg) in THF
(90 mL) was irradiated using a medium pressure mercury
lamp for 10 h and the residue obtained after evaporation of
the solvent was recrystallized from chloroform–methanol to
give 5 (70 mg, 70%) as a yellow solid, Z/E—90:10; mp
mixture was poured into ice-cooled water (50 mL) and
extracted with diethyl ether (3!30 mL). The organic layer
was washed with water (2!20 mL), brine (20 mL) and
dried over sodium sulfate, and the solvent was removed
under vaccum. The residue was dissolved in methanol
1
13
(
10 mL), HCl (20%, 10 mL) and refluxed for 2 h. The
cooled reaction mixture was poured into ice-cooled water
50 mL) and extracted with ethyl acetate (3!30 mL). The
organic layer was washed with water (2!20 mL), brine
20 mL) and dried over sodium sulfate. The residue
258–260 8C; H and C NMR (DMSO-d ): see Table 1; MS
6
K
(ESI, negative scan): m/z 253 (MKH) .
(
4
4
.1.8. Daidzein diacetate (3). A mixture of daidzein (96%,
0 mg), acetic anhydride (1 mL), and pyridine (1 mL) was
(
obtained after evaporation of the solvent was chromato-
graphed over silica gel column using petroleum ether–
EtOAc (80:20) to give 10 (500 mg, 61%), which was
recrystallized from chloroform to give the product as a
yellow crystalls, mp 152–154 8C; nmax (KBr): 3310, 1743,
kept standing at room temperature for 16 h and diluted with
diethyl ether (20 mL). The mixture after usual work-up as
described above, gave diacetate 3 (45 mg, 85%) as a white
1
9
solid, mp 186–188 8C (lit. mp 188–190 8C); n
1750, 1644, 1616, 1222, 1017 cm ; H NMR (CDCl ): see
3
Table 2; MS (ESI, positive scan): m/z 339 (MCH) .
(neat):
max
K1 1
K1
1
C
1
594, 1286, 1226, 1074, 967, 826 cm ; H NMR (DMSO-
d ): d 3.83 (3H, s, –OCH ), 6.76 (1H, d, JZ8.5 Hz, H-5),
6
3
0 0
6
(
.95 (2H, d, JZ8.2 Hz, H-3 ,5 ), 6.96 (1H, s, H-7), 7.60
1H, s, ]CH), 7.70 (2H, d, JZ8.2 Hz, H-2 ,6 ), 7.79 (1H, d,
0 0
Acknowledgements
JZ8.5 Hz, H-5), 10.30 (1H, brs, Ar-OH); MS (ESI,
negative scan): m/z 267 (MKH) . HRMS (m/z): Calcd
K
We sincerely thank Sri G. Ganga Raju, Chairman, and
Mr. G. Rama Raju, Director, of Laila Impex for encourage-
ment and daidzein sample, and Prof. A. Srikrishna, Indian
Institute of Science, Bangalore, India for helpful
discussions.
for C H O (MCNa): 291.0633. Found: 291.0636.
16 12 4
4
.1.5. 6-Hydroxy-3-[(4-hydroxyphenyl)methylene]benzo-
b]furan-2-one (1E). A mixture of 10 (100 mg) and
[
pyridine hydrochloride (1.5 g) was stirred at 180–190 8C
for 3 h. The cooled reaction mixture was diluted with water
(
(
20 mL), acidified with dil HCl and extracted with EtOAc
3!20 mL). The combined EtOAc layer was washed with
brine (20 mL) and dried over sodium sulfate. The residue
obtained after evaporation of the solvent was chromato-
graphed over silica gel column using chloroform–methanol
References and notes
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2001, 64, 204–207.
(
94:6) as eluents to give 1-E (60 mg, 63%), which was
recrystallized from chloroform–methanol to give the
product as a yellow crystalls, E/Z–90:10. Mp 258–260 8C;
nmax (KBr): 3350, 1733, 1582, 1374, 1280, 1241, 1069,
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K1
1
13
9
59 cm ; H and C NMR (DMSO-d ): see Table 1; MS
6
K
ESI, negative scan): m/z 253 (MKH) . HRMS (m/z):
(
Calcd for C H O (MCNa): 277.0477. Found: 277.0484.
1
5 10 4
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5
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4
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7. Schildknecht, H.; Kornig, W.; Siewerdt, R.; Krauss, D. Liebigs
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