Nafuredin

1. Discovery, producing organism and structure1-7)
Nafuredin was isolated from the culture broth of Aspergillus niger strain FT-0554 and recognized
as an inhibitor of helminth NADH-fumarate reductase. Its target was revealed to be complex I(NADH-quinone oxidoreductase), and it was identified as a selective inhibitor of helminth complex I. Nafuredin showed anthelmintic activity against Haemonchus contortus in in vivo studies.
The structure of nafuredin was elucidated as β,γ-epoxy-δ-lactone with a branched side chain, andits absolute configuration was revealed synthetically.

Conidiophore and conidiogenous cells		
Bar: 20 µm

O

CH3CH3 CH3CH3CH3OHOO

Nafuredin

Conidia Bar: 2 µm
Aspergillus niger FT-0554

2. Physical data
White powder. C22H32O4; mol wt 360.50. Sol. in DMSO, EtOH, EtOAc, CHCl3. Insol. in H2O,
hexane.

3. Biological activity1,4)1) Effects on electron transport enzymes

IC50 (nM)

Ascaris suum Ascaris suum Haemonchus			
contortus (adult)		
Complex(adult)(L2)
NADH-fumarate reductase
I+II
NADH-ubiquinone reductase
I
NADH-rhodoquinone reductase IRhodoquinol-fumarate reductase IISuccinate-ubiquinone reductase IINT : not tested

12824
80,000>100,000NT
8.99.0NTNTNT86195NTNTRatliver

1,000
10,000>100,000
NT>100,000

Many adult parasites live in low oxygen environments. Such organisms generate ATP differently from aerobic mammals. Anaerobic parasites fix CO2 to phosphoenolpyruvate producing
oxaloacetate, which is converted into malate and transported to the mitochondria. Malate is thenconverted to fumarate, and reduced to succinate by complex II which uses NADH as a reducingagent. This system, composed of complexes I and II, is called NADH-fumarate reductase. Thequinone used in this system is not ubiquinone but rhodoquinone. In a screening of NADH-fumarate reductase inhibitors for new anthelmintics, we found nafuredin and atpenin (See p. 92).

2) Kinetic analysis of nafuredin inhibition of A. suum complex I
The inhibition is noncompetitive with NADH (Ki = 8.1 nM) and competitive with rhodoquinone(Ki = 8.3 nM).

3) Effect on Haemonchus contortus infected sheep
8000
Number of eggs in 1 g feces

TreatedControl60004000

2000

0–4–2024681012Day
Eggs per gram of feces from H. contortus infected sheep on different days before and after oral treatment with 2 mg/kg nafuredin compared to untreated infected sheep are shown.
Values are the mean of two trials (control) or three trials (treated animals) ± S.D.

4) Toxicity
There were no signs of side effects or reduction of body weight during the testing period in either sheep (2 mg/kg p.o.) or mice (50 mg/kg p.o. and i.p.).

5) Antimicrobial activity (10 µg/6 mm disc, paper disc method)		
Test organism

Inhibitory 		zone (mm) Test organism

Bacillus subtilis ATCC6633—
Staphylococcus aureus ATCC6538p—
Micrococcus luteus ATCC9341—
Mycobacterium smegmatis ATCC607—
Escherichia coli NIHJ—
Pseudomonas aeruginosa IFO3080+
Xanthomonas campestris pv. oryzae KB88 —Inhibitoryzone (mm)Bacteroides fragilis ATCC23745
Acholeplasma laidlawii PG8
Pyricularia oryzae KF180
Candida albicans KF1
Saccharomyces cerevisiae KF26
Aspergillus niger ATCC6275
Mucor racemosus IFO4581——±12——104. Biosynthesis2)
Nafuredin is biosynthesized from a nonaketide and four methionines (branched methyl carbons).
5. Nafuredin- γ 8)
Nafuredin is easily converted to a compound named nafuredin-γ by weak alkaline treatment. The
structure of nafuredin-γ was elucidated as a γ-lactone form of nafuredin with keto-enol tautomerism. Nafuredin-γ shows similar complex I inhibitory activity as nafuredin, and it also possesses
anthelmintic activity in vivo.

HOO

CH 3CH3CH3CH3CH3CH3CH3O

CH3OHOOO

CH 3OHnafuredin-γCH36. Total synthesis9,10) (See Appendix-I)
1) Total synthesis of nafuredin
1) Ac2O, Et 3N, cat. DMAP
2) Diethyl malonate, NaH,cat. Pd(PPh 3)41) TENPO, NaClO, KBr2) Ph3P=CHCO2Me
3) DIBAL

HO4) MnO2(Ss )-(–)-2--methyl- 5) Ph3P=C(Me)CO2Et
1-butanol
6) DIBAL
31% (6 steps)
1) PivCl, Et3N, then BuLi,
BnR)-4-Bn-2-oxazolidinone(R2) NaHMDS, MeION85% (2 steps)
OOHO1) LiBH4, EtOH
2) Dess-Martin periodinane

OORef. 3

HO58%(7 steps)O

HOA
1) H2, Pd(OH)2
2) TBSCl, ii-Pr2NEt
3) TIPSOTf, 2,6-lutidine
4) TBAF, BF 3·Et2O

BOH

70% (4 steps)

OH
O

BzON

HOMsO

96% (2 steps)

SO2

O

BzO

OTIPS

N

NN
Ph

KHMDS, then A
79%OTIPS
1) NaH
2) DIBAL
3) AllocCl, cat. DMAP, Py.

HOMsO
O

BzO

1) Dess-Martin periodinane
2) (EtO)2P(O)CH2CO2allyl,ii-Pr2NEt, LiCl3) cat. Pd(PPh 3)4, NaBH44) MeO2CCl, Et3N, thenLiAlH(tt-BuO)
53% (4 steps)

OTIPS

1) DEAD, PBu 3,1-phenyl-1H-tetrazole-5-thiol
2) H2O2, cat. Mo 7O24(NH4)6·4H2O

O

BzO

HOMsO

OBnOBn
HOMsO

OBnO

BzO

OHC

85% (2 steps)

Bn
HOMsO

HO2C

3) KOH
4) Cu2O
71% (4 steps)O4) HF·Py
HO5) Dess-Martin Periodinane
6) HCO2H, Et3N, cat. Pd(PPh 3)4OTIPS

62% (6 steps)

OO

Nafuredin

Overall yield 10 % in 24 steps (from B)

2) Total synthesis of nafuredin-γOH1) PMBzCl, Et3N, cat. DMAP
2) (DHQ)2 PHAL, K2OsO4·2H 2O,2) K 3Fe(CN)6, K 2CO 3
95% (2 steps)

1) (+)-DET, Ti(Oi-Pr)4, TBHP
2) NCS, PPh3, 2-methyloxirane
94% (2 steps)

OTBS

OO

OTBS

HOO

1) cat. AgNO 3, NBS
2) HF·pyridine
3) Bu3SnH,
3) cat. PdCl2(PPh3)271% (3 steps)

O
O

HOCl

OPMBz

O

86% (3 steps)

HOO

HO

cat. PdCl 2(MeCN)2 , NMP
72%OOH1) cat. TEMPO,
1) trichloroisocyanuric acid
CO2Me
2) LHMDS, (MeO)2P(O)OTBS75% (2 steps)

OTBSISnBu3OH

4) Ph 3P=CHCO 2Et
5) DIBAL
68% (5 steps)

1) n-BuLi
2) TBSOTf, 2,6-lutidine3) 70% aq. AcOHOHO

1) cat. Sc(OTf)3, 1,1-dimethoxycyclopentane
2) NaOMe, MeOH
3) Dess-Martin periodinane

OHOOOH
Nafuredin-γ
Overall yield 20% in 18 steps

7. Close structural relatives11)
Analyses of type I polyketide synthase gene clusters of Streptomyces aculeolatus NRRL 18422
and Streptomyces sp. Eco86 predicted three 5-alkenyl-3-furanones, one (E-837) from the former
and two (E-492, E-975) from the latter strain. They were isolated and their inhibitory activity
against NADH-fumarate reductase were evaluated, because they were structurally similar to nafuredin-γ. However their showed only moderate inhibitory activity (IC50 = 1-4 µg/ml).
O
HO
H3C

HOOH

OH

E-837

O
O

H3 C

CH3OH

CH3OH

CH3

CH3CH3OH

E-492 (R=H)
R E-975 (R=CH3)O
CH3CH3CH3

8. References
1. [768] S. Ōmura et al., Proc. Natl. Acad. Sci. USA 98, 60-62 (2001)
2. [772] H. Ui et al., J. Antibiot. 54, 234-238 (2001)
3. [776] D. Takano et al., Tetrahedron Lett. 42, 3017-3020 (2001)
4. [785] K. Shiomi et al., Bioscience and Industry 59, 37-38 (2001)
5. [868] K. Shomi & S. Ōmura, Proc. Jpn. Acad., Ser. B 80, 245-258 (2004)
6. [946] K. Kita et al., Trends Parasitol. 23, 223-229 (2007)
7. [947] S. Ōmura & K. Shiomi, Pure Appl. Chem. 79, 581-591 (2007)
8. [877] K. Shiomi et al., J. Antibiot. 58, 50-55 (2005)
9. [787] D. Takano et al., Org. Lett. 3, 2289-2291 (2001)
10. [837] T. Nagamitsu et al., Tetrahedron Lett. 44, 6441-6444 (2003)
11. [953] A. H. Banskota et al., J. Antibiot. 59, 168-176 (2006)