Absolute configuration of nafuredin, a new specific NADH-fumarate reductase inhibitor

Article abstract of DOI:10.1016/S0040-4039(01)00355-0

Nafuredin, a new specific NADH-fumarate reductase inhibitor, was isolated from the culture broth of a fungal strain Aspergillus niger FT-0554. The stereoselective synthesis of three degradation products obtained by ozonolysis of nafuredin allowed elucidation of the absolute configuration of nafuredin.

Full text of DOI:10.1016/S0040-4039(01)00355-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 3017–3020
Absolute configuration of nafuredin, a new specific
NADH-fumarate reductase inhibitor
Daisuke Takano,a Tohru Nagamitsu,b,c Hideaki Ui,b Kazuro Shiomi,b Yuuichi Yamaguchi,b
b,
Rokuro Masuma,b Isao Kuwajimab,c and Satoshi Omura *
(
aSchool of Pharmaceutical Science, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan
bResearch Center for Biological Function, The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan
cCREST, The Japan Science and Technology Corporation (JST), 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan
Received 2 March 2001; accepted 5 March 2001
Abstract—Nafuredin, a new specific NADH-fumarate reductase inhibitor, was isolated from the culture broth of a fungal strain
Aspergillus niger FT-0554. The stereoselective synthesis of three degradation products obtained by ozonolysis of nafuredin allowed
elucidation of the absolute configuration of nafuredin. © 2001 Elsevier Science Ltd. All rights reserved.
NADH-fumarate reductase (NFRD) is a terminal elec-
tron transport system of anaerobic energy metabolism.
Some kinds of parasites use this metabolism to generate
ATP instead of the classical glycolysis, TCA cycle and
electron transport systems. So an inhibitor of NFRD is
potentially a selective antiparasitic agent. In the course
of our screening for NFRD inhibitors, nafuredin (1)
was obtained from the fermentation broth of a fungal
strain Aspergillus niger FT-0554 isolated from a marine
sponge.1,2 Nafuredin (1) inhibited NFRD of A. suum
with an IC50 value of 12 nM without cytotoxicity and
showed good specificity to NFRD of helminths.
Although the planar structure of 1 was elucidated by
spectroscopic analyses and an additional NOE experi-
ment showed the relative configuration of the b,g-
epoxy-d-lactone moiety in 1,2 the stereochemistry at
C(10) and C(16) was unknown. Herein we report the
elucidation of the absolute configuration of 1 by degra-
dation and synthetic studies.
Our strategy for the elucidation of the absolute configu-
ration of 1 is shown in Fig. 1. Ozonolysis of 1 would
afford three degradation products, which would be
compared with the analytical data of the optically
O
O
4
CHO
Ozonolysis
3
5
10
16
1
+
O
+
O
OHC
O
OHC
HO
2
HO
O
O
Nafuredin (1)
2
4
3
OBn
O
OBn
HO
HO
AllylO
AllylO
Glucose
O
O
BzO
OBn
OBn
5
6
Figure 1.
Keywords: NADH-fumarate reductase (NFRD); nafuredin; absolute configuration.
* Corresponding author. Tel.: +81-3-5791-6101; fax: +81-3-3444-8360; e-mail: omura-s@kitasato.or.jp
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00355-0

3018
D. Takano et al. / Tetrahedron Letters 42 (2001) 3017–3020
active synthetic compounds (2–4). The optically active
lactone 2 would be derived from glucose via selective
protection and deprotection of the hydroxyl groups,
stereoselective 1,2-addition at C(4), and epoxidation.
Compound 1 protected as TBS ether was subjected to
ozonolysis and quenched with dimethyl sulfide; the
resulting aldehyde was treated with sodium borohy-
dride in one pot to produce lactone 11 in 75% overall
yield from 1.
D
-Glucose as the starting material was used to synthe-
size the optically active 2, and converted to the known
diol 73 (Scheme 1). Regioselective and reductive open-
ing of benzylidene acetal4 after protection of diol 7 as
allyl ether followed by Dess–Martin oxidation5
afforded ketone 6 without complication. Treatment of
ketone 6 with MeLi furnished the desired tert-alcohol 8
(66% yield) together with its epimer (19% yield).6
Deprotection of the allyl group and subsequent regiose-
lective benzoylation at C(2)7 gave diol 5. Mesylation of
5 followed by epoxidation by treating with sodium
hydride yielded epoxy alcohol 9 after quenching with
methanol. Compound 9 was protected with t-butyl-
dimethylsilyl (TBS) chloride and the corresponding
TBS ether was converted to lactol 10 by hydro-
genolysis.8 Fetizon oxidation9 of 10 led to lactone 11,10
which was subjected to Dess–Martin oxidation to afford
aldehyde 12. Though the structure of aldehyde 12 was
Fortunately, lactone 11 synthesized from D-glucose was
identical with that derived from natural nafuredin (1) in
1
all respects ([h]D, H and 13C NMR, IR, and FABMS).
This result shows that the absolute configuration of the
lactone moiety in 1 is (2R,3S,4S,5R) as depicted in
Scheme 2.
We next synthesized the optically active and racemic
degradation products 3 and 4 as shown in Scheme 3. In
order to synthesize the optically active (R)-3, (S)-(+)-3-
hydroxyisobutyric acid methyl ester 1311 was converted
to nitrile 14 in four steps: (1) protection of the primary
alcohol with t-butyldiphenylsilyl (TBDPS) chloride; (2)
reduction of the ester with DIBAL; (3) mesylation of
the resulting primary alcohol; and (4) nucleophilic dis-
placement with NaCN. Compound 14 was treated with
MeLi to furnish the corresponding ketone after acidic
work-up, which was subjected to deprotection of the
TBDPS ether and subsequent nitroxyl radical
oxidation12 to yield the optically active (R)-3. Next,
2-benzyloxyethanol 1511 was subjected to Dess–Martin
oxidation and the resulting aldehyde was treated with
1-triphenylphosphoranylidene-2-propanone to yield
1
confirmed by H NMR of the resulting mixture, it was
very unstable and did not allow purification with silica
gel as it possessed a b,g-epoxy aldehyde unit. So, we
realized it was difficult to isolate 12 and 2 for compari-
son of their analytical data. Next we expected that
lactone 11 would be also derived from 1 via ozonolysis
and reduction.
Ph
HO
OH
O
OBn
OBn
O
O
HO
HO
HO
AllylO
AllylO
AllylO
HO
HO
d
e, f
a - c
See ref. 3
3
1
O
O
O
OBn
8
O
2
AllylO
OH
OBn
OBn
7
6
D-Glucose
OH
OBn
OBn
OH
HO
O
O
O
O
CHO
HO
g, h
l
i, j
k
O
O
O
O
O
TBSO
TBSO
BzO
HO
TBSO
O
O
OBn
OBn
OH
10
9
5
11
12
Scheme 1. (a) AllylBr, NaH, DMF, quant.; (b) NaBH3CN, HCl (in Et2O), THF, 86%; (c) Dess–Martin periodinane, CH2Cl2,
98%; (d) MeLi, Et2O, 66%; (e) Pd/C, cat. p-TsOH, MeOH–H2O, quant.; (f) Bu2SnO, benzene, then BzCl, Et3N, dioxane, 91%;
(g) MsCl, i-Pr2NEt, CH2Cl2, 94%; (h) NaH, THF, then MeOH, 95%; (i) TBSCl, imidazole, DMF, 90%; (j) H2, Pd(OH)2, EtOH,
quant.; (k) Ag2CO3, Celite, benzene, 80%; (l) Dess–Martin periodinane, CH2Cl2, quant.
OH
O
S
O
a, b
S
R
O
O
HO
R
TBSO
O
O
Nafuredin (1)
[α]D22= –34.4˚ (c = 0.18, CHCl3)
[α]D22= –32.9˚ (c = 0.14, CHCl3)
11 (Synthetic)
11 (Natural)
Scheme 2. (a) TBSCl, imidazole, THF; (b) O3, MeOH, Me2S, then NaBH4, 75% (two steps).

D. Takano et al. / Tetrahedron Letters 42 (2001) 3017–3020
3019
R
O
h
e-g
a-d
O
R
HO
TBDPSO
CN
CO2Me
OHC
O
O
(R)-3
(R)-3A
13
14
O
k, l, g
h
i, j
O
BnO
BnO
OH
OHC
O
O
O
15
16
(±)-3
(±)-3A
S
O
h
g
S
HO
OHC
O
17
(S)-4
(S)-4A
Scheme 3. (a) TBDPSCl, i-Pr2NEt, CH2Cl2, quant.; (b) DIBAL, CH2Cl2, 99%; (c) MsCl, i-Pr2NEt, CH2Cl2, 93%; (d) NaCN,
DMSO, 80%; (e) MeLi, Et2O, then aq. H2SO4, quant.; (f) TBAF, THF, quant.; (g) TEMPO, NaClO, KBr, NaHCO3, CH2Cl2,
50–60%; (h) 1,2-ditrimethylsiloxymethylbenzene, TMSOTf, CH2Cl2, 80–85%; (i) Dess–Martin periodinane, CH2Cl2, 70%; (j)
1-triphenylphosphoranylidene-2-propanone, benzene, 72%; (k) Me2Cu(CN)Li, Et2O, 85%; (l) H2, Pd/C, EtOH, 80%.
Figure 2.
a,b-unsaturated ketone 16. Treatment of 16 with
Me2Cu(CN)Li followed by hydrogenolysis and nitroxyl
radical oxidation led to racemic 3. The optically active
(S)-4 was derived from (S)-(−)-2-methyl-1-butanol 1711
by nitroxyl radical oxidation.
propanol (3:7) (Senshu HPLC system; 10 ml injection;
UV, 207 nm; flow rate, 0.3 ml/min), each enantiomer
proved to be separated completely as shown in Fig.
2(A). Fig. 2(B) and (C) show HPLC analytical data of
( )-3A and ( )-4A, respectively. Next, (R)-3A and (S)-
4A were also subjected to HPLC analysis to show that
their retention times are 127 and 183 min, respectively,
as shown in Fig. 2(D) and (E).
We next analyzed the degradation products ( )-3 and
( )-411 with various chiral HPLC columns; however, we
could not find the conditions to separate each enan-
tiomer. Therefore, 3 and 4 were converted to acetals 3A
and 4A, respectively. When the 1:1 mixture of acetals
( )-3A and ( )-4A was injected to a Daicel-OJ column
(0.46 cm ƒ×25 cm), and eluted with hexane–iso-
Nafuredin (1) was subjected to ozonolysis and
quenched with dimethyl sulfide; the resulting aldehydes
3 and 4 were converted to acetals 3A and 4A in one
pot, whose TLC behaviors in several solvent systems

3020
D. Takano et al. / Tetrahedron Letters 42 (2001) 3017–3020
were identical with those of synthetic acetals 3A and
4A. After standard work-up, HPLC analysis of the
resulting mixture showed clearly that the absolute
configuration of the side chain in 1 was (10R) and
(16S) as shown in Fig. 2(F).
1982, 108, 97–101.
5. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48,
4155–4156.
6. Miljkovic, M.; Gligorijevic, M.; Satoh, T.; Miljkovic, D.
J. Org. Chem. 1979, 39, 1379–1384.
7. Munavu, R. M.; Szmant, H. H. J. Org. Chem. 1976, 41,
1832–1836.
8. The stereochemistry of monobenzyl compound 10%
obtained by hydrogenolysis (30 min) of TBS protected 9
was confirmed by X-ray crystallographic analysis as fol-
lows. This X-ray data has been deposited with the CCDC
(CCDC 157565).
In summary, the absolute configuration of nafuredin (1)
has been elucidated as (2R,3S,4S,5R,6E,8E,10R,
12E,14E,16S)-3,4-epoxy-2-hydroxy-4,10,12,16-tetrame-
thyl-6,8,12,14-octadecatetraeno-5-lactone by stereose-
lective synthesis and HPLC analysis of the degradation
products obtained by ozonolysis of 1. Further studies
for the total synthesis of nafuredin are in progress.
Acknowledgements
We thank Professor H. Takayanagi (Kitasato Univer-
sity) for kindly performing the X-ray crystal analysis.
H.U. acknowledges a Grant-in-Aid for Encouragement
of Young Scientists from the Japan Society for the
Promotion of Science (12771373).
9. Fetizon, M.; Golfier, M.; Mourgues, P.; Louis, J.-M.
Organic Syntheses by Oxidation with Metal Compounds;
Plenum Press: New York, 1986; pp. 503–567.
10. 11: mp=135–137°C; [h]2D5=−34.4 (c=0.18, CHCl3); IR
(KBr) cm−1=3466, 3460, 2929, 2858, 1745, 1392, 1250,
1188, 1124, 1076, 835, 787, 584; 1H NMR (270 MHz,
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