Synthesis of podoscyphic acid

Article abstract of DOI:10.1016/S0040-4039(01)02264-X

Podoscyphic acid 1, a fungal metabolite inhibiting RNA-directed DNA polymerases, was synthesised in four steps starting from dodecanal. The concluding ester hydrolysis of 4 was not feasible with chemical reagents, only enzymatic hydrolysis was mild enough to release the highly reactive natural product in good yields.

Full text of DOI:10.1016/S0040-4039(01)02264-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 891–893
Synthesis of podoscyphic acid
Johan Eriksson, Martin Johansson and Olov Sterner*
Department of Organic and Bioorganic Chemistry, Lund University, PO Box 124, SE-221 00 Lund, Sweden
Received 28 September 2001; revised 25 October 2001; accepted 28 November 2001
Abstract—Podoscyphic acid 1, a fungal metabolite inhibiting RNA-directed DNA polymerases, was synthesised in four steps
starting from dodecanal. The concluding ester hydrolysis of 4 was not feasible with chemical reagents, only enzymatic hydrolysis
was mild enough to release the highly reactive natural product in good yields. © 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
study its chemical reactivity and facilitate the prepara-
tion of suitable derivatives, a synthetic procedure to
obtain the natural product in larger amounts was
required.
The fungal metabolite podoscyphic acid 1 was origi-
nally isolated from the mycelium of the basidiomycete
Podoscypha petalodes, during a screening of fungal
extracts for new inhibitors of avian myeloblastosis virus
and murine leukemia virus.1 It is an effective and
selective inhibitor of reverse transcription, catalysed by
RNA-directed DNA polymerase, with an IC50 value of
15 mg/ml for avian myeloblastosis virus reverse tran-
scriptase.1,2 Podoscyphic acid 1 contains a highly
unusual g,d-dioxoacrylate moiety, and we have recently
discovered that the chemical reactivity of the com-
pound is very suitable for the preparation of small
libraries of novel compounds of which some have
shown interesting biological properties. In order to
further survey the biological activities of 1, as well as
2. Results and discussion
The synthetic route developed, depicted in Scheme 1,
starts from the commercially available aldehyde dode-
canal. The coupling of dodecanal with triethyl 4-phos-
phonocrotonate
using
Wadsworth–Emmons
conditions3,4 proceeded smoothly, and the diene 2 was
obtained in 72% yield (as a 1:9 mixture of the cis and
trans isomers). The diene 2 was thereafter subjected to
catalytic dihydroxylation with osmium tetroxide
(according to Sharpless’ dihydroxylation protocol) for
O
EtO
EtO
O
P
OEt
O
O
a
b
OEt
H
2
OH
O
O
O
c
OEt
OEt
OH
O
3
4
O
d
CO2H
O
1
Scheme 1. Reagents and conditions: (a) LDA, THF, −78 to 20°C (72 %); (b) OsO4, K3Fe(CN)6, K2CO3, quinuclidine,
t-BuOH/H2O, stirred for 4 days (51 %); (c) TEMPO, CH2Cl2, 0°C (100%); (d) Novozyme 435, isopropyl ether, 2 h (70%).
Keywords: podoscyphic acid; synthesis lipase; enzymatic hydrolysis.
* Corresponding author. Tel.: +46-46-222 8213; fax: +46-46-222 8209; e-mail: olov.sterner@bioorganic.lth.se
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)02264-X

892
J. Eriksson et al. / Tetrahedron Letters 43 (2002) 891–893
four days, and the 4,5-dihydroxylated product 3 was
obtained in 51% yield.6,7 The 1,2-diketo functionality was
then introduced by oxidation of 3 with TEMPO,
(2,2,6,6)-tetramethyl-1-piperidinyloxy, resulting in the
ethyl ester of podoscyphic acid 4 in quantitative yield.5
Surprisingly, other mild oxidation procedures, e.g. the
Swern oxidation, did not give a satisfactory result. The
final step, the hydrolysis of the ester 4 to give free
podoscyphic acid 1, was anticipated to be the easiest but
turned out to be difficult. All commonly employed
chemical procedures for ester hydrolysis, both acidic and
basic, were tried, but none gave a satisfactory result. The
reason for this is the chemical reactivity of the g,d-
dioxoacrylate moiety in podoschyphic acid, as soon as
1 is formed by chemical hydrolysis it is affected by the
hydrolytic conditions to yield, at best, modest amounts
of 1. An attempt to reverse the order of transformations
and first hydrolyse the ester 3 to the corresponding acid
and then oxidise the diol to podoscyphic acid 1 failed,
as the oxidation was not possible to perform even with
TEMPO. However, with a lipase,8 a 70% conversion of
the ester 4 to podoscyphic acid 1 could be achieved in
a few hours at room temperature. The spectroscopic data
of the synthetic podoscyphic acid were identical with
those reported for the natural product.1
mixture of the (2E,4E) and (2E,4Z) isomers of hexadeca-
2,4-dienoic acid ethyl ester (2). The NMR data for the
1
major isomer is given: H NMR (400 MHz, CDCl3): l
0.87 (t, J=7.5 Hz, 3H), 1.23–1.33 (m, 16H), 1.29 (t,
J=7.1 Hz, 3H); 1.42 (m, 2H), 2.16 (t, J=7.5 Hz, 2H),
4.28 (q, J=7.0 Hz, 2H), 5.78 (d, J=15.4 Hz, 1H), 6.12
(dt, J=15.3 and 6.4 Hz, 1H), 6.16 (dd, J=10.0 and 15.3
Hz, 1H), 7.26 (dd, J=10.0 and 15.4 Hz, 1H); 13C NMR
(100 MHz, CDCl3): l 14.5, 14.7, 23.1, 29.1, 29.5, 29.6,
29.7, 29.8, 29.95, 30.1, 32.3, 33.4, 60.5, 119.6, 128.8,
145.1, 145.6, 167.7. HRFABMS: 281.2480 (M+H+,
C18H33O2 requires 281.2480).
3.1.2. (2E)-4,5-Dihydroxy-2-hexadecenoic acid ethyl ester
3. K3Fe(CN)6 (2.14 mmol, 3.0 equiv.), K2CO3 (2.14
mmol, 3.0 equiv.) and quinuclidine (0.035 mmol, 5 wt%)
were added to a 1:1 solution of water: t-BuOH (4 mL)
under stirring. OsO4 in t-BuOH (0.021 mmol, 3 wt.%)
was then added to the solution and the mixture was
cooled to 0°C. Methanesulfone amide (0.71 mmol, 1.0
equiv.) was then added to the cooled solution followed
by hexadeca-2,4-dienoic acid ethyl ester (2) (0.71 mmol,
1.0 equiv.). The reaction was stirred for 4 days under a
nitrogen atmosphere and followed with TLC. Sodium
metabisulphite (1.1 g) was then added to the reaction
mixture and the suspension was stirred for 30 min. The
reaction was diluted with brine (10 mL) and extracted
with CH2Cl2 (4×20 mL), the organic phase was dried with
MgSO4 and concentrated under reduced pressure. Chro-
matography on SiO2 with EtOAc/heptane (1:8) resulted
in 115 mg (51%) of (2E)-4,5-dihydroxy-2-hexadecenoic
acid ethyl ester 3 and 33 mg (16%) recovery of the starting
material 2. 1H NMR (400 MHz, CDCl3): l 0.89 (t, J=7.0
Hz, 3H), 1.15–1.33 (m, 18H), 1.29 (t, J=7.0 Hz, 3H), 1.62
(m, 2H), 2.03 (d, J=3.8 Hz, 1H), 2.40 (d, J=5.2 Hz, 1H),
3.57 (brs, 1H), 4.15 (d, J=3.6 Hz, 1H), 4.28 (q, J=7.0
Hz, 2H), 6.16 (d, J=15.9 Hz, 1H), 6.94 (dd, J=15.6 Hz,
1H); 13C NMR (100 MHz, CDCl3): l 14.1, 14.3, 22.6,
22.7, 25.5, 29.3, 29.4, 29.5, 29.6, 29.6, 31.9, 33.0, 60.6,
74.0, 74.1, 122.3, 147.0, 166.4. HRFABMS: 315.2549
(M+H+, C18H35O4 requires 315.2535).
3. Experimental
3.1. General procedures
Unless otherwise noted, chemicals were of p.a. quality
and obtained from commercial suppliers and used with-
out further purification. TLC analyses were made on
Merck DC–Alufolien Kiselgel 60 F254 SiO2 plates,
visualised by spraying with anisaldehyde/sulphuric acid
and warming to 120°C. The MS spectra (FAB ionisation)
was recorded with a JEOL SX102 spectrometer, and
NMR spectra (in CDCl3) with a Bruker ARX 500
spectrometer at 500 MHz (1H) and 125 MHz (13C). The
chemical shifts are reported in ppm with the solvent
signals (lH=7.26 and lC=77.0) as reference. COSY,
HMQC and HMBC experiments were recorded with
gradient enhancements using sine shaped gradient pulses.
3.1.3. (2E)-4,5-Dioxo-2-hexadecenoic acid ethyl ester 4.
(2E)-4,5-Dihydroxy-2-hexadecenoic acid ethyl ester (0.3
mmol, 1 equiv.) was dissolved in CH2Cl2 (5.0 mL) and
cooled to 0°C. To the solution, TEMPO (0.015 mmol,
0.05 equiv.), KBr (0.15 mmol, 0.5 equiv.) and water (0.3
mL) was added, followed by saturated NaHCO3 (4 mL)
and saturated NaOCl (4 mL). After stirring for 10 min
the phases were separated and the water phase was
extracted with CH2Cl2 (2×20 mL). The combined organic
phase was dried with MgSO4 and concentrated resulting
in a quantitative yield of (2E)-4,5-dioxo-2-hexadecenoic
3.1.1. Hexadeca-2,4-dienoic acid ethyl ester 2. To a
solution of diisopropylamine (1.99 mmol, 1.22 equiv.) in
dry THF (3 mL) at −78°C under a nitrogen atmosphere,
BuLi (1.63 mmol, 1.11 equiv.) was added gently to
generate LDA. After 10 min, triethyl 4-phosphonocroto-
nate (2.17 mmol, 1.11 equiv.) was added dropwise, and
after an additional 15 min, dodecanal (1.63 mmol, 1.0
equiv.) dissolved in dry THF (3 mL) was cannulated into
the reaction mixture. The reaction was allowed to reach
0°C and stirred for 1 h. The reaction was then stirred at
room temperature for an additional hour, whereafter the
reaction mixture was diluted with saturated NH4Cl (10
mL) and extracted with diethyl ether (3×20 mL). The
combined ether layers was dried with MgSO4 and the
solvent removed under reduced pressure. The remaining
oily extract was purified by SiO2 chromatography,
EtOAc/heptane (1:20), resulting in 330 mg (72%) of a 9:1
1
acid ethyl ester (100%). H NMR (400 MHz, CDCl3): l
0.87 (t, J=7.5 Hz, 3H), 1.15–1.42 (m, 16H), 1.29 (t,
J=7.1 Hz, 3H), 1.63 (m, 2H), 2.81 (t, J=7.5 Hz, 2H),
4.28 (q, J=7.0 Hz, 2H), 6.92 (d, J=16.4 Hz, 1H), 7.78
(d, J=16.4 Hz, 1H); 13C NMR (100 MHz, CDCl3): l
14.1, 14.3, 22.7, 22.9, 29.1, 29.3, 29.4, 29.5, 29.6, 29.7,
31.9, 36.4, 62.0, 132.9, 135.6, 165.3, 187.4, 199.9.
HRFABMS: 311.2227 (M+H+, C18H31O4 requires
311.2222).

J. Eriksson et al. / Tetrahedron Letters 43 (2002) 891–893
893
3.1.4. (2E)-4,5-Dioxo-2-hexadecenoic acid 1, podo-
scyphic acid. (2E)-4,5-Dioxo-2-hexadecenoic acid ethyl
ester (0.3 mmol, 1 equiv.) was dissolved in methyl
t-butyl ether saturated with water (10 mL) and immo-
bilised Candida antarctica Lipase B (Novozyme 435)
(20 mg) was added. The slurry was stirred at room
temperature for 2 h whereafter the Novozyme 435
pellets were removed by filtration and the organic sol-
vent evaporated under reduced pressure. Purification by
chromatography on silica gel with EtOAc/heptane (1:6)
containing 1% HOAc yielded 60 mg (70%) of (2E)-4,5-
dioxo-2-hexadecenoic acid and 17 mg (18%) recovery of
the starting material 3. The spectroscopic data were
T. Anke, University of Kaiserslautern (FRG), for a
sample of natural podoscyphic acid 1, and Novo
Nordisk AS for the gift of Novozyme 435.
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1
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1.15–1.42 (m, 16H), 1.63 (m, 2H), 2.81 (t, J=7.5 Hz,
2H), 6.92 (d, J=16.4 Hz, 1H), 7.78 (d, J=16.4 Hz,
1H); 13C NMR (100 MHz, CDCl3): l 14.1, 22.7, 22.9,
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Acknowledgements
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This work was supported financially by the Swedish
Natural Science Research Board. We thank Professor
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