Development of a new air-stable structure-simplified nafuredin-γ analog as a potent and selective nematode complex I inhibitor

Article abstract of DOI:10.1038/ja.2017.16

Nafuredin-γ, obtained from natural nafuredin, has demonstrated a potent and selective inhibitory activity against nematode complex I. However, nafuredin-γ is unstable in air since its conjugated dienes are oxygen-labile. The instability in air was natural

Full text of DOI:10.1038/ja.2017.16

The Journal of Antibiotics (2017), 1–8
2017 Japan Antibiotics Research Association All rights reserved 0021-8820/17
www.nature.com/ja
&
ORIGINAL ARTICLE
Development of a new air-stable structure-simplified
nafuredin-γ analog as a potent and selective nematode
complex I inhibitor
Masaki Ohtawa1,4, Shiho Arima1,4, Risa Shimizu1, Naomi Hanatani1, Eri Shimizu1, Kazuro Shiomi2,
Kiyoshi Kita3, Satoshi Ōmura1,2 and Tohru Nagamitsu1
Nafuredin-γ, obtained from natural nafuredin, has demonstrated a potent and selective inhibitory activity against nematode
complex I. However, nafuredin-γ is unstable in air since its conjugated dienes are oxygen-labile. The instability in air was
naturally solved by the synthesis of structure-simplified nafuredin-γ analogs without conjugated dienes. However, these modified
analogs showed lower complex I inhibitory activities. Therefore, new air-stable structure-simplified nafuredin-γ analogs were
designed and synthesized herein. Among all analogs synthesized, the one bearing a unique 1-azabicyclo[3.1.0]hexane scaffold
showed the highest inhibitory activity (IC50 = 170 nM) while presenting high selectivity against nematode complex I.
The Journal of Antibiotics advance online publication, 22 February 2017; doi:10.1038/ja.2017.16
INTRODUCTION
chain was changed originating new targets 4–6. Additionally, the
tetrahydrofuran (THF) unit was replaced by pyrrolidine or tetrahy-
drothiophene units to generate new targets 7 and 8 (Figure 2).
Subsequent biological evaluation revealed some of the newly synthe-
sized air-stable nafuredin-γ analogs to be more potent nematode
complex I inhibitors than 3. Herein, we report new structure–activity
relationship studies of air-stable structure-simplified nafuredin-γ
analogs as nematode complex I inhibitors.
Nafuredin (1)1–3 was isolated from the fermentation broth of the
fungal strain Aspergillus niger FT-0554 while screening for selective
complex I inhibitors and proved to be a potent and selective inhibitor
against nematode complex I. In addition, 1 demonstrated anthelmintic
activity against Haemonchus contortus during in vivo trials with
sheeps.1 A total synthetic study of 1 subsequently identified a novel
and structurally simpler γ-lactone compound, nafuredin-γ (2), which
was generated from 1 under mild basic conditions.4–6 Moreover, the
nematode complex I inhibitory activity of 2 was identical to that of 1. RESULTS AND DISCUSSION
We first embarked on the synthesis of new analogs 4–6 by varying the
Therefore, nafuredin (1) and nafuredin-γ (2) holds promise as
a selective antiparasitic agent. However, these compounds are
disadvantageous in that they have poor air stability owing to the
presence of oxygen-labile conjugated diene units (Figure 1). The total
synthesis of 2 has been achieved by our group.7 Several nafuredin-γ
analogs were then synthesized using this total synthesis approach and
their complex I inhibitory activities were examined. Consequently, the
importance of the stereochemistries of C4 and C5 for the complex I
inhibitory activity of these compounds was revealed. Next, we
length of the isoprene unit (Scheme 1) through our previously
developed synthetic route.8 tert-Butyldimethylsilyl protection, deace-
tylation by methanolysis and sharpless asymmetric epoxidation of a
known allyl acetate 99 gave chiral epoxyalcohol 10, which was
subjected to Appel reaction10 to afford iodide 11. Introduction of
the side chain moiety was achieved by coupling the epoxyiodide 11
with the corresponding known sulfones 12–14.11,12 Subsequent Pd-
catalyzed reductive desulfonylation13 afforded 15–17 (the synthesis of
attempted the synthesis of structure-simplified nafuredin-γ analogs intermediate 17 was not optimized. Because our primary interest was
lacking the oxygen-labile conjugated diene units to solve the instability in evaluating the nematode complex I inhibitory activity of the new
of 1 and 2 in air. Thus, we found that a new nafuredin-γ analog analog 6.). Finally, deprotection of the tert-butyldimethylsilyl group
3 obtained by a concise synthesis approach showed moderate complex and acid-catalyzed cyclization gave desired analogs 4–6.
I inhibitory activity while having high air stability (Figure 2).8
The inhibitory activities against nematode complex I of 4–6 were
To improve the nematode complex I inhibitory activity of air-stable evaluated (Table 1, also see experimental section).1,14 Analogs 4 and 5,
nafuredin-γ analogs, additional structure–activity relationship studies possessing shorter side chains (n = 0 or 1) than nafuredin-γ (2)
were carried out over 3. Thus, the length of the isoprene unit as a side and air-stable nafuredin-γ analog 3, exhibited very low complex I
1Graduate School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan; 2Graduate School of Infection Control Sciences, Kitasato University, Tokyo, Japan and 3School of
Tropical Medicine and Global Health, Nagasaki University, Nagasaki, Japan
4These authors contributed equally to this work.
Correspondence: Professor T Nagamitsu, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
E-mail: nagamitsut@pharm.kitasato-u.ac.jp
Received 17 October 2016; revised 27 December 2016; accepted 16 January 2017

Development of a new air-stable nafuredin-γ analog
M Ohtawa et al
2
inhibitory activities as compared with 3. Analog 6 with a longer side
chain (n = 3) showed a moderate inhibitory activity and still lower
than that of 3 (n = 2). Therefore, analog 3 proved to have the
optimum side chain length in terms of complex I inhibitory activity.
Next, we investigated the synthesis of new analogs 7 and 8 bearing
pyrrolidine or tetrahydrothiophene units instead of THF (Scheme 2)
while maintaining the same side chain of 3. Coupling of iodide 9 with
sulfone 1815 followed by Pd(0)-catalyzed desulfonation gave 19, which
OH
O
O
4
O
O
5
HO
O
OH
Nafuredin (1)
(IC50 = 12 nM)
Nafuredin-γ (2)
(IC50 = 6 nM)
unstable in air
Figure 1 Structures and complex I inhibitory activities of nafuredin (1) and nafuredin-γ (2).
Previous work8
· Simplification of lactone
and isoprene units
O
· Lower complex I inhiibitory activity than 2
but still maintains high activity
· Obtained by a concise synthesis
· Air-stable
OH
3
(IC50 = 500 nM)
stable in air
1) Change in the length of isoprene unit
This work
O
O
n
2
OH
OH
4: n = 0
5: n = 1
6: n = 3
3
2) Replacement by pyrrolidine or tetrahydrothiophene
X
2
OH
7 (X = NH)
8 (X = S)
Figure 2 Air-stable structure-simplified nafuredin-γ analogs synthesized and designed by our group.
1)
PhO S
2
1) TBSCl, imidazole
CH2Cl2, rt, quant.
2) aq. K2CO3, MeOH
rt, 97%
n
12 (n = 0) or 13 (n = 1) or 14 (n = 3)
n-BuLi, HMPA, THF, —78 °C
TBSO
HO
OAc
R
O
2) Pd(OAc)2, dppp
3) (–)–DET, Ti(Oi-Pr)4, MS4Å
t-BuOOH, CH2Cl2, –20 °C
90%
9
NaBH4 DMSO, rt
15: 76% (2 steps), 16: 73% (2 steps)
17: 27% (2 steps)
10: R = OH
11: R = I
I2, PPh3
imidazole
CH2Cl2
0 °C, 95%
1) TBAF, THF, rt
2) PPTS, CH2Cl2, rt
4: 97% (2 steps), 5: 94% (2 steps),
6: 71% (2 steps)
TBSO
O
O
n
n
OH
15: n = 0
16: n = 1
17: n = 3
4: n = 0
5: n = 1
6: n = 3
Scheme 1 Synthesis of air-stable structure-simplified nafuredin-γ analogs 4–6.
The Journal of Antibiotics

Development of a new air-stable nafuredin-γ analog
M Ohtawa et al
3
Table 1 Inhibitory activities of 3–6 against nematode complex I
was subjected to deprotection of tert-butyldimethylsilyl ether and
tosylation to afford tosylate 20. SN2 reaction of 20 with potassium
phthalimide and potassium thioacetate yielded phthalimide 21 and
thioacetate 22, respectively.
O
First, phthalimide 21 was exposed to a solution of hydrazine
(4 equiv.) in THF at 50 °C (Scheme 2, condition A) for synthesizing
pyrrolidine analog 7. Surprisingly, unexpected aziridine analog
23 bearing a unique 1-azabicyclo[3.1.0]hexane scaffold was obtained
as a major product (61%) accompanied with desired pyrrolidine
analog 7 (10%) as a minor product. The structure of aziridine analog
23 was determined by NOESY and HMBC spectroscopy. We
speculated that aziridine analog 23 can be stereoselectively derived
from desired pyrrolidine analog 7 via dehydration under high
temperature conditions. This reaction was subsequently carried
out at room temperature and the yield of 7 was slightly improved
(27%) although 23 was also obtained in similar yield (23%) along with
n
OH
4 : n = 0
5 : n = 1
3 : n = 2
6 : n = 3
Compond
IC50 (nM)
4
5
3
6
44.0 ×104
2.3 × 103
5.0 × 102
8.0 × 102
1)
PhO2S
18
n-BuLi, HMPA, THF, —78 °C
TBSO
TBSO
I
O
O
2) Pd(OAc)2, dppp, NaBH4
DMSO, rt
64% (2 steps)
11
19
1) TBAF, THF, rt
TsO
2) TsCl, Et3N, Me3N·HCl
CH2Cl2, 0 °C
93% (2 steps)
O
20
potassium phtalimidate
KSAc
DMF, rt
88%
DMF, rt
83%
O
N
AcS
O
O
O
21
22
Condition A
NH2NH2 · H2O (4 eq.)
THF, 50 ˚C
Condition B
NH2NH2 · H2O (20 eq.)
THF, rt
Condition C
K2CO3, MeOH
0 ˚C to rt
Condition D
DIBAL, CH2Cl2
–78 ºC to 0 ˚C
8: 63%, 24: 6%
7: 10%, 23: 61%
7: 58%, 23: trace
8: 29%, 24: 46%
HN
OH
S
OH
7
8
H
H
HO
H
N
H
S
H
H
24
23
NOESY
HMBC
Scheme 2 Synthesis of air-stable structure-simplified nafuredin-γ analogs 7, 8, 23 and 24.
The Journal of Antibiotics

Development of a new air-stable nafuredin-γ analog
M Ohtawa et al
4
Table 2 Inhibitory activities of 7, 8, 23 and 24 against nematode complex I
O
OH
3
HN
S
OH
H
OH
S
7
8
HO
N
24
23
Compond
IC50 (nM)
3
5.0× 102
3.0× 102
1.7× 102
1.6× 103
1.2× 103
7
23
8
24
the recovered starting material 21 (28%) (data not shown). To
completely consume the starting material, the reaction was carried
out with higher concentrations of hydrazine (20 equiv.) at room
temperature (Scheme 2, condition B) to afford the desired pyrrolidine
analog 7 in 58% yield along with traces of the aziridine analog 23.
With the thioacetate 22 in hand, the synthesis of tetrahydrothio-
In conclusion, novel air-stable structure-simplified nafuredin-γ
analogs, prepared by varying the length of side chain and by replacing
the THF unit by pyrrolidine and tetrahydrothiophene, were designed
and synthesized. In the course of the synthetic studies, we found out
the new aziridine analog 23, which is naturally stable in air and proved
to be the most potent and selective inhibitor against nematode
phene analog 8 was also attempted. Methanolysis of 22 followed by complex I among all analogs synthesized by our group. In vivo tests
of aziridine analog 23 and further structure–activity relationship
studies of the air-stable structure-simplified nafuredin-γ analogs
are currently underway in our laboratory.
epoxide-opening cyclization afforded a separable mixture of desired
tetrahydrothiophene 8 (29%) and tetrahydro-2H-thiopyran 24 (46%)
analogs (Scheme 2, condition C). In contrast to these results,
diisobutylaluminum hydride reduction of 22 gave desired
tetrahydrothiophene analog 8 (63%) as a major product accompanied
with minor amounts of tetrahydro-2H-thiopyran analog 24 (6%)
(Scheme 2, condition D). Tetrahydro-2H-thiopyran analog 24 is
unfavorable according to the Baldwin rule.16 However, the steric
hindrance under condition C might prevail to give 24 as a major
product. On the other hand, the regioselectivity under
condition D would contribute to a greater stabilization of the
partial positive charge at the more hindered end of the epoxide,
resulting by coordination to the oxygen atom of the epoxide by
aluminum species.
EXPERIMENTAL PROCEDURE
General
All reactions were carried out in flame-dried glassware under a nitrogen
atmosphere employing standard techniques for handling air-sensitive materials.
Commercial reagents were used without further purification unless otherwise
indicated. Organic solvents were distilled and dried over 3 or 4 Å molecular
sieves. Cold baths were prepared as follows: 0 °C, wet ice/water; − 78 °C, dry
ice/acetone. Purifications by flash column chromatography were performed
over silica gel 60 N (spherical, neutral, particle size 40–50 μm). TLC was
performed on 0.25 mm Merck silica gel 60 F254 plates and the effluents
were visualized by UV (254 nm) as well as by phosphomolybdic acid and
p-anisaldehyde TLC stains. Yields refer to chromatographically and spectro-
scopically pure compounds, unless otherwise noted. 1H- and 13C-NMR spectra
were recorded using an internal deuterium lock on 400-MR, VNMRS-400 and
UNITY-400 spectrometers (Agilent Technologies, Waldbornn, Germany).
All NMR signals were reported in ppm relative to the internal reference
Table 2 shows the inhibitory activities against nematode complex I
of novel analogs 7, 8, 23 and 24 (also see experimental section). The
complex I inhibitory activity of pyrrolidine analog 7 slightly increased
as compared with THF analog 3. Interestingly, aziridine analog
23 exhibited the most potent complex
I
inhibitory activity standard provided by chloroform (that is, 7.26 or 77.0 ppm for the 1H and
13C spectra, respectively). Multiplicity data were presented as follows:
(IC50 = 170 nM) among all analogs synthesized. On the other hand,
the complex I inhibitory activities of sulfur-containing analogs 8 and
24 greatly decreased as compared with to 3. Aziridine analog 23
exhibited no inhibition activity against a bovine heart NADH oxidase
including complexes I and III (420 μM).14 This result indicated that
aziridine analog 23 is a potent and selective inhibitor against nematode
complex I.
s = singlet, d = doublet, t = triplet, q =quartet, m = multiplet, br= broad,
dd = double doublet and dt= double triplet. Coupling constants (J) were
reported in Hz. IR spectra were recorded on a FT/IR460-plus IR spectrometer
(JASCO, Tokyo, Japan). Absorption data were given in wavenumbers (cm− 1).
Optical rotations were recorded on a JASCO DIP-1000 polarimeter (JASCO,
Tokyo, Japan) and reported as follows: [α]TD, concentration (g per 100 ml),
and solvent. High resolution mass spectra were obtained on JEOL JMS-700
The Journal of Antibiotics

Development of a new air-stable nafuredin-γ analog
M Ohtawa et al
5
Mstation, JEOL JMS-AX505HA and JEOL JMS-T100LP systems (JEOL, Tokyo, tert-Butyl(3-((2R,3S)-3-(iodomethyl)-2-methyloxiran-2-yl)propoxy)
Japan) equipped with FAB, EI and ESI high-resolution mass spectrometers.
dimethylsilane (11)
To a solution of 10 (2.70 g, 10.4 mmol) in CH2Cl2 (100 ml) at 0 °C were
added imidazole (2.04 g, 31.1 mmol), PPh3 (4.08 g, 15.6 mmol) and I2 (3.95 g,
15.6 mmol). The mixture was stirred for 50 min at 0 °C, quenched with
saturated aqueous Na2S2O3 and the aqueous phase was extracted with EtOAc.
The combined organic extracts were dried over anhydrous Na2SO4 and
concentrated under reduced pressure. Flash column chromatography
(30:1 hexane/EtOAc) afforded 11 (3.66 g, 95%) as a colorless oil: [α]30
Enzyme assays
The nematode complex I (NADH-fumarate reductase) activity was measured in
assay mixtures containing 50 mM potassium phosphate (pH 7.2), 10 mM
β-D-glucose, 20 units of glucose oxidase, 26 units of catalase and 200 μM
NADH.1 The reaction is started by the addition of sodium fumarate (5 mM).
The absorbance change at 340 nm (millimolar extinction coefficient of 6.2 for
NADH) was followed. All inhibitory activities against nematode complex I
(IC50 values) in Tables 1 and 2 were the average of three independent
experiments.
− 32.0 (c 1.00, CHCl3); IR (neat) 2953, 2857, 1253, 1101, 776, 616, 474 cm− 1D
;
1H-NMR (400 MHz, CDCl3) δ 3.62–3.59 (td, J = 6.2, 2.3 Hz, 2H), 3.34
(dd, J = 9.7, 5.5 Hz, 1H), 3.07 (dd, J = 8.6, 5.5 Hz, 1H), 2.97 (dd, J = 9.7,
8.6 Hz, 1H), 1.68–1.60 (m, 3H), 1.58–1.48 (m, 1H), 1.26 (s, 3H), 0.87 (s, 9H),
0.031 (s, 6H); 13C-NMR (100 MHz, CDCl3) δ 63.9, 62.7, 62.4, 34.7, 28.5, 26.0,
18.3, 15.7, 2.4, − 5.3; HRMS (FAB, m-NBA) [M+H]+ calcd for C13H28O2SiI
371.0915, found 371.0903.
((2R,3R)-3-(3-((tert-Butyldimethylsilyl)oxy)propyl)-3-methyloxiran-
2-yl)methanol (10)
((3-Methylbut-2-en-1-yl)sulfonyl)benzene (12)
To a solution of 9 (1.09 g, 6.30 mmol) in CH2Cl2 (63 ml) at 0 °C were
added imidazole (515 mg, 7.56 mmol), tert-butyldimethylsilyl chloride
(1.14 g, 7.56 mmol) and dimethylaminopyridine (38.5 mg, 0.320 mmol). The
mixture was stirred for 1.5 h at room temperature, quenched with H2O and the
aqueous phase was extracted with EtOAc. The combined organic extracts were
dried over anhydrous Na2SO4 and concentrated under reduced pressure. Flash
column chromatography (70:1 hexane/EtOAc) afforded (E)-6-((tert-butyldi-
methylsilyl)oxy)-3-methylhex-2-en-1-yl acetate (A) (1.80 g, quant.) as a color-
To
a solution of 1-bromo-3-methyl-2-butene (100 μl, 0.859 mmol) in
dimethylformamide (8.6 ml) at room temperature were added PhSO2Na
(169 mg, 1.03 mmol) and Bu4NI (3.2 mg, 0.00859 mmol). The mixture was
stirred for 1 h at room temperature, quenched with H2O, and the aqueous
phase was extracted with EtOAc. The combined organic extracts were dried
over anhydrous Na2SO4 and concentrated under reduced pressure. Flash
column chromatography (10:1 hexane/EtOAc) afforded 12 (ref. 11; 115 mg,
82%) as a colorless oil: 1H-NMR (400 MHz, CDCl3) δ 7.86–7.83 (m, 2H),
7.62–7.60 (m, 1H), 7.54–7.50 (m, 2H), 5.21–5.15 (m, 1H), 3.76 (d, J = 7.8 Hz,
2H), 1.69 (s, 3H), 1.30 (s, 3H).
less oil: IR (neat) 3055, 1731, 1423, 1265, 741 cm− 1 1H-NMR (300 MHz,
;
CDCl3) δ 5.28–5.24 (m, 1H), 4.48 (d, J = 7.4 Hz, 2H), 3.50 (t, J = 6.5 Hz, 2H),
2.00 (t, J = 7.5 Hz, 2H), 1.94 (s, 3H), 1.61 (s, 3H), 1.58–1.51 (m, 2H), 0.80
(s, 9H), − 0.05 (s, 6H); 13C-NMR (100 MHz, CDCl3) δ 170.1, 141.7, 118.4,
62.3, 59.1, 35.6, 30.6, 26.0, 20.8, 18.2, 16.2, −5.4; HRMS (FAB, m-NBA) [M
+H]+ calcd for C15H31O3Si 287.2029, found 287.2024.
(tert-Butyldimethyl(3-((2R,3R)-2-methyl-3-(4-methylpent-3-en-1-yl)
oxiran-2-yl)propoxy)silane (15)
To a solution of 12 (200 mg, 0.540 mmol) in THF (4.0 ml) at − 78 °C were
added hexamethylphosphoramide (391 μl, 2.25 mmol) and n-BuLi (1.64 M in
n-hexane, 550 μl, 0.900 mmol). After stirring for 1 h at − 78 °C, a solution of 11
(94.6 mg, 0.450 mmol) in THF (2.0 ml) was added dropwise to the above
mixture. The resulting mixture was stirred for 15 min at − 78 °C, quenched
with saturated aqueous NH4Cl and the aqueous phase was extracted with
EtOAc. The combined organic extracts were dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was briefly purified by flash
column chromatography (10:1 hexane/EtOAc) to afford the corresponding
coupling product, which was dissolved in DMSO (4.2 ml) and treated with Pd
(OAc)2 (24.2 mg, 0.108 mmol) and dppp (55.7 mg, 0.135 mmol). After stirring
To a solution of A (3.49 g, 12.2 mmol) in MeOH (61 ml) at 0 °C was added
aqueous K2CO3 (60 ml, 0.2 M). The mixture was stirred for 3 h at room
temperature, diluted with H2O and the aqueous phase was extracted with
CH2Cl2. The combined organic extracts were dried over anhydrous Na2SO4
and concentrated under reduced pressure. Flash column chromatography
(10:1 hexane/EtOAc) afforded (E)-6-((tert-butyldimethylsilyl)oxy)-3-methyl-
hex-2-en-1-ol (B) (2.98 g, 97%) as a colorless oil: IR (neat) 3445, 2953,
1
2857, 2360, 1265, 1097, 743 cm− 1; H-NMR (300 MHz, CDCl3) δ 5.41−5.37
(m, 1H), 4.11 (d, J = 6.6 Hz, 2H), 3.58 (t, J = 6.4 Hz, 2H), 2.03 (t, J = 7.7 Hz,
2H), 1.65 (s, 3H), 1.65–1.58 (m, 2H), 0.87 (s, 9H), 0.023 (s, 6H); 13C-NMR
(100 MHz, CDCl3) δ 139.3, 123.4, 62.7, 59.2, 35.7, 30.9, 25.0, 18.3, 16.2, − 5.3; for 15 min at room temperature, NaBH4 (24.5 mg, 0.648 mmol) was added to
HRMS (FAB, m-NBA) [M+H]+ calcd for C13H29O2Si 245.1927, found
245.1931.
the mixture. The resulting mixture was stirred for 20 min at room temperature,
quenched with H2O and the aqueous phase was extracted with EtOAc. The
combined organic extracts were dried over anhydrous Na2SO4 and concen-
trated under reduced pressure. Flash column chromatography (50:1 hexane/
A mixture of Ti(OiPr)4 (3.60 ml, 12.2 mmol) and 4 Å molecular sieves
(1.19 g) in CH2Cl2 (76 ml) was treated with ( − )-DET (2.09 ml, 12.2 mmol),
and the resulting solution was vigorously stirred at −5 °C for 1 h. tert-Butyl
hydroperoxide (5.0–6.0 M in decane, 4.88 ml, 24.4 mmol) was slowly added to
the mixture, and the solution was stirred at − 20 °C for 1 h. A solution of
B (2.98 g, 12.2 mmol) in CH2Cl2 (46 ml) was added to the above mixture, and
the solution was stirred at − 20 °C for 1.5 h. After Me2S (1.34 ml, 18.3 mmol)
was added, the mixture was further stirred at − 20 °C for 1 h. The resulting
mixture was diluted with CH2Cl2, treated with celite (6.00 g) and
Na2SO4·10H2O (6.00 g), and subsequently stirred for 2 h at room temperature.
The resulting suspension was filtered through a pad of celite, and the filtrate
was concentrated under reduced pressure. The residue was purified by silica gel
flash column chromatography (5:1 hexane/EtOAc) to afford 10 (2.70 g, 90%)
EtOAc) afforded 15 (107 mg, 2 steps, 76%) as a colorless oil: [α]30 − 58.1
D
(c 1.00, CHCl3); IR (neat) 3000, 1635, 1275, 1261, 750 cm− 1
;
1H-NMR
(400 MHz, CDCl3)
δ 5.12 (t, J = 2.0 Hz, 1H), 3.60–3.56 (m, 2H),
2.69 (t, J = 6.3 Hz, 1H), 2.15–2.09 (m, 2H), 1.68 (s, 3H), 1.63–1.44 (m, 6H),
1.59 (s, 3H), 1.23 (s, 3H), 0.88 (s, 9H), 0.022 (s, 6H); 13C-NMR (100 MHz,
CDCl3) δ 132.3, 123.4, 63.2, 62.9, 60.8, 35.1, 28.9, 28.5, 25.9, 25.7, 25.0, 18.3,
17.6, 16.6, − 5.3; HRMS (FAB, m-NBA) [M+H]+ calcd for C18H37O2Si
313.2554, found 313.2563.
(R)-5-Methyl-1-((S)-2-methyltetrahydrofuran-2-yl)hex-4-en-1-ol (4)
To a solution of 15 (81.3 mg, 0.270 mmol) in THF (2.7 ml) at room
temperature was added tetrabutylammonium fluoride (1.0 M in THF, 5.4 ml,
0.540 mmol). After stirring for 1 h at room temperature, the resulting mixture
was quenched with H2O and the aqueous phase was extracted with EtOAc. The
combined organic extracts were dried over anhydrous Na2SO4 and concen-
trated under reduced pressure. This residue was employed in the next reaction
without further purification. The residue was dissolved in CH2Cl2 (2.7 ml) and
treated with pyridinium p-toluenesulfonate (3.4 mg, 0.0135 mmol). After
stirring for 30 min at room temperature, the reaction was quenched with
as a colorless oil: [α]23 +4.26 (c 1.00, CHCl3); IR (neat) 3434, 3021, 1216,
D
769 cm−1 1H-NMR (400 MHz, CDCl3) δ 3.83–3.80 (m, 1H), 3.73–3.70
;
(m, 1H), 3.63–3.59 (m, 2H), 2.70 (dd, J = 6.7, 4.3 Hz, 1H), 1.67–1.52
(m, 4H), 1.30 (s, 3H), 0.88 (s, 9H), 0.039 (s, 6H); 13C-NMR (100 MHz,
CDCl3) δ 62.9, 62.7, 61.3, 61.2, 34.9, 28.3, 26.0, 25.9, 25.8, 18.3, 16.8, − 5.3,
−5.4; HRMS (FAB, m-NBA) [M+H]+ calcd for C13H29O3Si 261.1883, found
261.1886.
The Journal of Antibiotics

Development of a new air-stable nafuredin-γ analog
M Ohtawa et al
6
brine and the aqueous phase was extracted with EtOAc. The combined organic with pyridinium p-toluenesulfonate (8.8 mg, 0.035 mmol). After stirring for
extracts were dried over anhydrous Na2SO4 and concentrated under reduced
pressure. Flash column chromatography (25:1 hexane/EtOAc) afforded
1.5 h at room temperature, the reaction was quenched with H2O and the
aqueous phase was extracted with EtOAc. The combined organic extracts
were dried over anhydrous Na2SO4 and concentrated under reduced
pressure. Flash column chromatography (20:1 hexane/EtOAc) afforded 5
4 (147 mg, 2 steps, 97%) as a colorless oil: [α]30 +32.6 (c 1.00, CHCl3); IR
D
(neat) 3435, 2349, 1635, 1260, 1051, 750 cm− 1; 1H-NMR (400 MHz, CDCl3) δ
5.15–5.11 (m, 1H), 3.91–3.80 (m, 2H), 3.53 (dd, J = 10.5, 2.1 Hz, 1H), 2.36
(brs, 1H), 2.28–2.22 (m, 1H), 2.17–2.07 (m, 1H), 2.05–1.89 (m, 3H), 1.69
(s, 3H), 1.63 (s, 3H), 1.50–1.42 (m, 2H), 1.38–1.30 (m, 1H), 1.11 (s, 3H);
13C-NMR (100 MHz, CDCl3) δ 132.0, 124.2, 85.8, 76.0, 67.9, 31.9, 30.6, 26.3,
25.7, 25.2, 23.1, 17.7; HRMS (FAB, m-NBA) [M+H]+ calcd for C12H23O2
199.1698, found 199.1698.
(175 mg, 2 steps, 94%) as a colorless oil: [α]30 +4.62 (c 1.00, CHCl3);
D
IR (neat) 3452, 3055, 1636, 1422, 1265, 750 cm− 1
;
1H-NMR (400 MHz,
CDCl3) δ 5.16–5.07 (m, 2H), 3.91–3.80 (m, 2H), 3.59 (dd, J = 10.0, 2.0 Hz,
1H), 2.35 (brs, 1H), 2.29–2.21 (m, 1H), 2.17–1.88 (m, 8H), 1.67 (s, 3H),
1.63 (s, 3H), 1.59 (s, 3H), 1.51–1.43 (m, 2H), 1.40–1.30 (m, 1H), 1.11 (s, 3H);
13C-NMR (100 MHz, CDCl3) δ 135.7, 131.3, 124.3, 124.0, 85.7, 76.0, 67.9,
39.7, 31.9, 30.6, 26.7, 26.3, 25.7, 25.0, 23.1, 17.7, 16; HRMS (FAB m-NBA)
[M+H]+ calcd for C17H31O2 267.2327, found 267.2324.
(E)-((3,7-Dimethylocta-2,6-dien-1-yl)sulfonyl)benzene (13)
To a solution of geraniol (200 μl, 1.14 mmol) in Et2O (11 ml) at 0 °C was
added PBr3 (53 μl, 0.570 mmol). After stirring for 1.5 h at 0 °C, the resulting
mixture was quenched with cold water and the aqueous phase was extracted
with a 1:1 Et2O:hexane mixture. The combined organic extracts were dried over
anhydrous Na2SO4 and concentrated under reduced pressure. This residue was
employed in the next reaction without further purification. The residue was
dissolved in DMF (11 ml) and treated with Bu4NI (4.2 mg, 0.0114 mmol) and
PhSO2Na (224 mg, 1.37 mmol). After stirring for 1.5 h at room temperature,
the reaction was quenched with H2O and the aqueous phase was extracted with
EtOAc. The combined organic extracts were dried over anhydrous Na2SO4
and concentrated under reduced pressure. Flash column chromatography
(30:1 hexane/EtOAc) afforded 13 (ref. 11; 279 mg, 2 steps, 88%) as a colorless
oil: 1H-NMR (400 MHz, CDCl3) δ 7.88–7.85 (m, 2H), 7.65–7.61 (m, 1H),
7.55–7.51 (m, 2H), 5.23 (dt, J = 7.8, 1.0 Hz, 1H), 5.03–5.00 (m, 1H),
3.80 (d, J = 7.8 Hz, 2H), 2.00–1.99 (m, 4H), 1.68 (s, 3H), 1.58 (s, 3H), 1.31
(s, 3H).
(((2E,6E,10E)-3,7,11,15-Tetramethylhexadeca-2,6,10,14-tetraen-1-yl)
sulfonyl)benzene (14)
To a solution of geranyl geraniol (43.1 mg, 0.148 mmol) in Et2O (3.0 ml) at
0 °C was added PBr3 (7 μl, 0.0742 mmol). After stirring for 2 h at 0 °C, the
resulting mixture was quenched with cold water and the aqueous phase was
extracted with a 1:1 Et2O:hexane mixture. The combined organic extracts
were dried over anhydrous Na2SO4 and concentrated under reduced pressure.
This residue was employed in the next reaction without further purification.
The residue was dissolved in DMF (3.0 ml) and treated with Bu4NI (0.5 mg,
0.0015 mmol) and PhSO2Na (29.2 mg, 0.178 mmol). After stirring for 3 h at
room temperature, the reaction was quenched with H2O and the aqueous phase
was extracted with EtOAc. The combined organic extracts were dried over
anhydrous Na2SO4 and concentrated under reduced pressure. Flash column
chromatography (70:1 hexanes/EtOAc) afforded 14 (ref. 12; 38.0 mg, 2 steps,
63%) as a colorless oil: 1H-NMR (400 MHz, CDCl3) δ 7.88–7.85 (m, 2H),
7.65–7.61 (m, 1H), 7.55–7.30 (m, 2H), 5.19 (dt, J =8.2, 1.2 Hz, 1H), 5.11–5.03
(m, 3H), 3.80 (d, J = 8.2 Hz, 2H), 2.09–1.92 (m, 12H), 1.67 (s, 3H), 1.65–1.58
(m, 9H), 1.25 (s, 3H).
tert-Butyl(3-((2R,3R)-3-((E)-4,8-dimethylnona-3,7-dien-1-yl)-2-
methyloxiran-2-yl)propoxy)dimethylsilane (16)
To a solution of 13 (147 mg, 0.530 mmol) in THF (7.9 ml) at − 78 °C were
added hexamethylphosphoramide (460 μl, 2.65 mmol) and n-BuLi (1.64 M in
n-hexane, 646 μl, 1.06 mmol). After stirring for 1 h at − 78 °C, a solution of
11 (291 mg, 0.790 mmol) in THF (3.0 ml) was added dropwise to the above
mixture. The resulting mixture was stirred for 30 min at − 78 °C, quenched
with saturated aqueous NH4Cl and the aqueous phase was extracted with
EtOAc. The combined organic extracts were dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was briefly purified by flash
column chromatography (20:1 hexane/EtOAc) to afford the corresponding
coupling product, which was dissolved in DMSO (4.8 ml) and treated with Pd
(OAc)2 (25.9 mg, 0.106 mmol) and dppp (54.6 mg, 0.133 mmol). After stirring
for 15 min at room temperature, NaBH4 (40.1 mg, 1.06 mmol) was added to
the mixture. The reaction was stirred for 20 min at room temperature,
quenched with H2O and the aqueous phase was extracted with EtOAc. The
combined organic extracts were dried over anhydrous Na2SO4 and concen-
trated under reduced pressure. Flash column chromatography (40:1 hexane/
tert-Butyldimethyl(3-((2R,3R)-2-methyl-3-((3E,7E,11E)-4,8,12,16-
tetramethylheptadeca-3,7,11,15-tetraen-1-yl)oxiran-2-yl)propoxy)
silane (17)
To a solution of 14 (32.0 mg, 0.0772 mmol) in THF (2.0 ml) at − 78 °C were
added hexamethylphosphoramide (67 μl, 0.386 mmol) and n-BuLi (1.64 M in
n-hexane, 57 μl, 0.0926 mmol). After stirring for 1 h at −78 °C, a solution of
11 (291 mg, 0.790 mmol) in THF (3.0 ml) was added dropwise to the above
mixture. The resulting mixture was stirred for 30 min at −78 °C, quenched
with saturated aqueous NH4Cl and the aqueous phase was extracted with
EtOAc. The combined organic extracts were dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was purified briefly by flash
column chromatography (10:1 hexane/EtOAc) to afford the corresponding
coupling product, which was dissolved in DMSO (1.0 ml) and treated with Pd
(OAc)2 (3.7 mg, 0.154 mmol) and dppp (7.9 mg, 0.0193 mmol). After stirring
for 15 min at room temperature, NaBH4 (3.5 mg, 0.0926 mmol) was added to
the mixture. The reaction was stirred for 30 min at room temperature,
quenched with H2O and the aqueous phase was extracted with EtOAc. The
combined organic extracts were dried over anhydrous Na2SO4 and concen-
trated under reduced pressure. Flash column chromatography (40:1 hexane/
EtOAc) afforded 16 (129 mg, 2 steps, 73%) as a colorless oil: [α]23 +2.77
D
1
(c 1.00, CHCl3); IR (neat) 3055, 2986, 1636, 1422, 1265, 749 cm−1; H-NMR
(400 MHz, CDCl3) δ 5.14 (dd, J = 7.0, 1.2 Hz, 1H), 5.07 (dt, J =7.0, 1.6 Hz,
1H), 3.60–3.56 (m, 2H), 2.70 (t, J = 6.3 Hz, 1H), 2.20–1.95 (m, 6H), 1.66
(s, 3H), 1.66–1.44 (m, 6H), 1.58 (s, 3H), 1.54 (s, 3H), 1.23 (s, 3H), 0.87 (s, 9H),
0.02 (s, 6H); 13C-NMR (100 MHz, CDCl3) δ 135.9, 131.3, 124.2, 123.2, 63.2,
62.9, 60.8, 39.7, 35.1, 28.9, 28.6, 26.6, 25.9, 25.7, 25.6, 24.8, 18.2, 17.7, 16.6,
16.0, − 5.3; HRMS (EI) [M]+ calcd for C23H44O2Si 380.3113, found 380.3111.
EtOAc) afforded 17 (10.8 mg, 2 steps, 27%) as a colorless oil: [α]30 +0.93
D
1
(c 1.00, CHCl3); IR (neat) 3054, 2986, 1635, 1423, 1265, 748 cm− 1; H-NMR
(400 MHz, CDCl3) δ 5.15–5.09 (m, 4H), 3.62–3.58 (m, 2H), 2.71 (t, J =6.2 Hz
1H), 2.16–2.00 (m, 14H), 1.68 (s, 3H), 1.65–1.46 (m, 6H), 1.62–1.58 (m, 12H),
1.24 (s, 3H), 0.88 (s, 9H), 0.04 (s, 6H); 13C-NMR (100 MHz, CDCl3) δ 135.9,
135.1, 134.9, 131.4, 124.4, 124.2, 124.1, 123.2, 63.2, 62.9, 60.8, 39.7, 35.1, 28.9,
28.5, 26.8, 26.6, 25.9, 25.7, 25.6, 24.9, 18.3, 17.7, 16.6, 16.0, −5.3; HRMS (EI)
[M]+ calcd for C33H60O2Si 516.4366, found 516.4363.
(R,E)-5,9-Dimethyl-1-((S)-2-methyltetrahydrofuran-2-yl)deca-4,
8-dien-1-ol (5)
To a solution of 16 (243 mg, 0.70 mmol) in THF (7.0 ml) at room temperature
was added tetrabutylammonium fluoride (1.0 M in THF, 1.40 ml, 1.40 mmol).
After stirring for 1 h at room temperature, the resulting mixture was quenched
with brine and the aqueous phase was extracted with EtOAc. The combined
organic extracts were dried over anhydrous Na2SO4 and concentrated under
reduced pressure. This residue was employed in the next reaction without
further purification. The residue was dissolved in CH2Cl2 (7.0 ml) and treated
(R,4E,8E,12E)-5,9,13,17-Tetramethyl-1-((S)-2-methyltetra-
hydrofuran-2-yl)octadeca-4,8,12,16-tetraen-1-ol (6)
To a solution of 17 (9.0 mg, 0.0174 mmol) in THF (1.7 ml) at room
temperature was added tetrabutylammonium fluoride (1.0 M in THF, 35 μl,
The Journal of Antibiotics

Development of a new air-stable nafuredin-γ analog
M Ohtawa et al
7
0.0348 mmol). After stirring for 1 h at room temperature, the resulting mixture 1.57–1.48 (m, 4H), 1.19 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 144.7, 138.9,
was quenched with brine and the aqueous phase was extracted with EtOAc. The 136.1, 135.0, 133.1, 131.3, 129.8, 127.9, 124.3, 123.0, 70.3, 63.0, 60.0, 39.7, 34.4,
combined organic extracts were dried over anhydrous Na2SO4 and concen-
trated under reduced pressure. This residue was employed in the next reaction
without further purification. The residue was dissolved in CH2Cl2 (500 μl) and
treated with pyridinium p-toluenesulfonate (0.1 mg, 0.00085 mmol). After
stirring for 1 h at room temperature, the reaction was quenched with H2O
and the aqueous phase was extracted with EtOAc. The combined organic
extracts were dried over anhydrous Na2SO4 and concentrated under reduced
pressure. Flash column chromatography (8:1 hexane/EtOAc) afforded
28.8, 26.7, 26.5, 25.7, 24.8, 24.6, 21.6, 17.7, 16.5, 16.0; HRMS (FAB, m-NBA)
[M+H]+ calcd for C29H45O4S 489.3034, found 489.3039.
2-(3-((2R,3R)-2-Methyl-3-((3E,7E)-4,8,12-trimethyltrideca-3,7,
11-trien-1-yl)oxiran-2-yl)propyl)isoindoline-1,3-dione (21)
To a solution of 20 (381 mg, 0.780 mmol) in DMF (19 ml) was added
potassium phthalimide (173 mg, 0.936 mmol). After stirring for 15 min at
room temperature, the reaction was quenched with saturated aqueous Na2S2O3
and the aqueous phase was extracted with EtOAc. The combined organic
extracts were dried over anhydrous Na2SO4 and concentrated under reduced
pressure. Flash column chromatography (10:1 hexane/EtOAc) afforded 21
(299 mg, 83%) as a colorless oil: [α]23D +4.95 (c 0.100, CHCl3); IR (neat) 3020,
6 (5.0 mg, 2 steps, 71%) as a colorless oil: [α]28 − 9.7 (c 1.00, CHCl3);
D
1
IR (neat) 3441, 3056, 2987, 1634, 1424, 1266, 752 cm− 1; H-NMR (400 MHz,
CDCl3) δ 5.16–5.07 (m, 4H), 3.91–3.80 (m, 2H), 3.53 (dd, J = 10.1, 1.7 Hz,
1H), 2.35 (brs, 1H), 2.29–2.22 (m, 1H), 2.16–1.88 (m, 16H), 1.67 (s, 3H), 1.63
(s, 3H), 1.59 (s, 9H), 1.51–1.43 (m, 2H), 1.40–1.30 (m, 1H), 1.11 (s, 3H);
13C-NMR (100 MHz, CDCl3) δ 135.8, 135.0, 134.9, 131.3, 124.4, 124.2, 124.1,
124.0, 85.8, 76.1, 67.9, 39.8, 39.7, 31.9, 30.6, 26.7, 26.6, 26.3, 25.7, 25.1, 23.1,
17.7, 16.1, 16.0, 15.9; HRMS (FAB, m-NBA) [M+H]+ calcd for C27H47O2
403.3570, found 403.3576.
1712, 1216, 755 cm− 1 1H-NMR (400 MHz, CDCl3) δ 7.96–7.93 (m, 2H),
;
7.83–7.80 (m, 2H), 5.40–5.17 (m, 3H), 3.81–3.76 (m, 2H), 2.82 (t, J = 6.2 Hz,
1H), 2.27–2.03 (m, 10H), 1.90–1.83 (m, 2H), 1.77 (s, 3H), 1.75–1.59 (m, 13H),
1.35 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 168.3, 136.0, 135.0, 133.9, 133.9,
133.8, 132.1, 131.2, 124.3, 124.1, 60.0, 60.2, 39.7, 39.6, 37.8, 35.9, 28.8, 26.7,
26.6, 25.6, 24.3, 24.2, 17.7, 16.5, 16.4, 16.0; HRMS (FAB, m-NBA) [M+H]+
calcd for C30H42O3N 465.3157, found 465.3165.
tert-Butyldimethyl(3-((2R,3R)-2-methyl-3-((3E,7E)-4,8,12-
trimethyltrideca-3,7,11-trien-1-yl)oxiran-2-yl)propoxy)silane (19)
To a solution of 18 (1.25 g, 3.60 mmol) in THF (20 ml) at −78 °C were added
hexamethylphosphoramide (3.1 ml, 18.0 mmol) and n-BuLi (1.64 M in
n-hexane, 4.4 ml, 7.20 mmol). After stirring for 1 h at − 78 °C, a solution of
11 (1.60 mg, 4.30 mmol) in THF (10 ml) was added dropwise to the above
mixture. The resulting mixture was stirred for 40 min at −78 °C, quenched
with saturated aqueous NH4Cl and the aqueous phase was extracted with
EtOAc. The combined organic extracts were dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was briefly purified by flash
column chromatography (20:1 hexane/EtOAc) to afford the corresponding
coupling product, which was dissolved in DMSO (20 ml) and treated with Pd
(OAc)2 (176 mg, 0.720 mmol) and dppp (371 mg, 0.900 mmol). After stirring
for 5 min at room temperature, NaBH4 (272 mg, 7.20 mmol) was added to the
mixture. The reaction was stirred for 50 min at room temperature, quenched
with H2O and the aqueous phase was extracted with EtOAc. The combined
organic extracts were dried over anhydrous Na2SO4 and concentrated under
reduced pressure. Flash column chromatography (70:1 hexane/EtOAc) afforded
(R,4E,8E)-5,9,13-Trimethyl-1-((S)-2-methylpyrrolidin-2-yl)
tetradeca-4,8,12-trien-1-ol (7)
To a solution of 21 (35.7 mg, 0.108 mmol) in THF (2.0 ml) was added
NH2NH2·H2O (67 μl, 2.16 mmol). After stirring for 2 days at room tempera-
ture, flash column chromatography (10:1 CH2Cl2/MeOH (1%NH3 aq.))
afforded 7 (20.9 mg, 58%) as a colorless oil: [α]22 − 3.15 (c 0.50, CHCl3);
D
1
IR (neat) 3437, 3055, 2982, 1634, 1424, 1266, 746 cm− 1; H-NMR (400 MHz,
CDCl3) δ 5.18–5.07 (m, 3H), 3.27 (dd, J = 10.1, 1.9 Hz, 1H), 3.08–3.02
(m, 1H), 2.94–2.88 (m, 1H), 2.34–2.25 (m, 1H), 2.14–1.89 (m, 9H), 1.88–1.72
(m, 3H), 1.68 (s, 3H), 1.63 (s, 3H), 1.59 (s, 6H), 1.56–1.44 (m, 1H),
1.42–1.21 (m, 2H), 0.87 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 135.4,
134.9, 131.3, 124.4, 124.3, 124.2, 76.2, 64.9, 45.7, 39.7, 39.6, 32.2, 30.7, 26.8,
26.6, 25.7, 24.6, 22.6, 17.8, 16.0, 15.9, 15.8; HRMS (FAB, m-NBA) [M+H]+
calcd for C22H40ON 334.3103, found 334.3110.
(5S,6S)-5-Methyl-6-((3E,7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-
yl)-1-azabicyclo[3.1.0]hexane (23)
19 (1.03 g, 2 steps, 64%) as a colorless oil: [α]30 +8.20 (c 1.00, CHCl3);
D
IR (neat) 3051, 2955, 1459, 1260, 739 cm− 1
;
1H-NMR (400 MHz, CDCl3)
To a solution of 21 (42.0 mg, 0.0906 mmol) in THF (1.8 ml) was added
NH2NH2·H2O (11.3 μl, 0.362 mmol). After stirring for 12 h at 50 °C, flash
column chromatography (10:1 CH2Cl2/MeOH (1%NH3 aq.)) afforded 23
δ 5.17–5.07 (m, 3H), 3.69–3.51 (m, 2H), 2.72 (t, J =6.2 Hz, 1H), 2.14–1.96
(m, 10H), 1.68 (s, 3H), 1.65–1.49 (m, 6H), 1.62 (s, 3H), 1.59 (s, 3H), 1.57
(s, 3H), 1.25 (s, 3H), 0.88 (s, 9H), 0.04 (s, 6H); 13C-NMR (100 MHz, CDCl3)
δ 135.9, 135.0, 131.2, 124.3, 124.1, 123.2, 63.2, 62.9, 60.8, 39.7, 35.1, 28.9, 28.6,
26.7, 26.6, 26.0, 25.8, 25.6, 24.8, 18.3, 17.7, 16.6, 16.0, 15.9, −5.3; HRMS (EI)
[M]+ calcd for C28H52O2Si 448.3737, found 448.3737.
(17.4 mg, 61%) and 7 (3.0 mg, 10%) as colorless oils: [α]21 − 6.09 (c 1.00,
D
CHCl3); IR (neat) 3052, 2931, 2856, 1443, 1266, 745 cm− 1
;
1H-NMR
(400 MHz, CDCl3) δ 5.17–5.11 (m, 3H), 2.92–2.87 (m, 1H), 2.61–2.87
(m, 1H), 2.43 (dd, J = 10.5, 2.7 Hz, 1H), 2.20–1.92 (m, 10H), 1.75–1.63
(m, 2H), 1.68 (s, 3H), 1.62 (s, 3H), 1.65–1.58 (m, 1H), 1.60 (s, 6H),
1.53–1.28 (m, 3H), 1.15 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 135.8,
135.0, 131.3, 124.3, 124.1, 123.8, 70.5, 64.5, 43.2, 39.7, 37.8, 29.7, 27.9, 26.7,
26.6, 25.7, 25.3, 23.7, 22.4, 17.7, 16.1, 16.0; HRMS (FAB, m-NBA) [M+H]+
calcd for C22H38N 316.3008, found 316.3004.
3-((2R,3R)-2-Methyl-3-((3E,7E)-4,8,12-trimethyltrideca-3,7,11-trien-
1-yl)oxiran-2-yl)propyl 4-methylbenzenesulfonate (20)
To a solution of 19 (350 mg, 0.780 mmol) in THF (8.0 ml) at room
temperature was added tetrabutylammonium fluoride (1.0 M in THF, 2.3 μl,
3.34 mmol). After stirring for 0.5 h at room temperature, the resulting mixture
was quenched with brine and the aqueous phase was extracted with EtOAc. The
combined organic extracts were dried over anhydrous Na2SO4 and concen-
trated under reduced pressure. This residue was employed in the next reaction
without further purification. The residue was dissolved in CH2Cl2 (8.0 ml) and
treated with Et3N (326 μl, 1.17 mmol), Me3N·HCl (7.50 mg, 0.0780 mmol) and
p-TsCl (223 mg, 1.17 mmol). After stirring for 45 min at 0 °C, the reaction was
quenched with H2O and the aqueous phase was extracted with EtOAc. The
combined organic extracts were dried over anhydrous Na2SO4 and concen-
trated under reduced pressure. Flash column chromatography (10:1 hexane/
S-(3-((2R,3R)-2-Methyl-3-((3E,7E)-4,8,12-trimethyltrideca-3,7,11-
trien-1-yl)oxiran-2-yl)propyl) ethanethioate (22)
To a solution of 20 (84.0 mg, 0.172 mmol) in DMF (1.00 ml) was added KSAc
(98.1 mg, 0.859 mmol). After stirring for 30 min at room temperature, the
reaction was quenched with H2O and the aqueous phase was extracted with
EtOAc. The combined organic extracts were dried over anhydrous Na2SO4
and concentrated under reduced pressure. Flash column chromatography
(30:1 hexane/EtOAc) afforded 22 (59.7 mg, 88%) as
a colorless oil:
[α]21 +0.027 (c 1.00, CHCl3); IR (neat) 2965, 1694, 1448, 1383, 1133,
EtOAc) afforded 20 (355 mg, 93%) as a colorless oil: [α]28 +10.3 (c 1.00,
D
D
625 cm− 1
;
1H-NMR (400 MHz, CDCl3) δ 5.16–5.07 (m, 3H), 2.88–2.84
CHCl3); IR (neat) 3055, 2986, 1600, 1423, 1265, 748 cm− 1
;
1H-NMR
(m, 2H), 2.70 (t, J = 6.4 Hz, 1H), 2.32 (s, 3H), 2.17–1.95 (m, 10H),
(400 MHz, CDCl3) δ 7.79–7.76 (m, 2H), 7.35–7.32 (m, 2H), 5.14–5.07
(m, 3H), 4.07–3.97 (m, 2H), 2.65 (dd, J =12.5, 6.2 Hz, 1H), 2.44 (s, 3H,), 1.74–1.47 (m, 6H), 1.67 (d, J =0.8 Hz, 3H) 1.62 (s, 3H), 1.59 (s, 6H), 1.24
2.16–1.94 (m, 10H), 1.78–1.70 (m, 2H) 1.67 (s, 3H), 1.60 (s, 3H), 1.59 (s, 6H), (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 195.8, 136.2, 135.2, 131.4, 124.5, 124.3,
The Journal of Antibiotics

Development of a new air-stable nafuredin-γ analog
M Ohtawa et al
8
123.3, 63.3, 60.5, 60.5, 39.9, 39.8, 37.9, 30.8, 29.1, 26.9, 26.7, 25.8, 25.6, 25.0, CONFLICT OF INTEREST
17.8, 16.7, 16.2, 14.3; HRMS (ESI+) [M+Na]+ calcd for C24H40NaO2S
The authors declare no conflict of interest.
415.2647, found 415.2655.
(R,4E,8E)-5,9,13-Trimethyl-1-((S)-2-methyltetrahydrothiophen-2-
yl)tetradeca-4,8,12- trien-1-ol (8) and (2R,3R)-3-methyl-2-((3E,7E)-
4,8,12-trimethyltrideca-3,7,11-trien-1- yl)tetrahydro-2H-thiopyran-
3-ol (24)
Methanolysis. To a solution of 22 (22.4 mg, 0.0570 mmol) in MeOH (500 μl)
was added K2CO3 (47.3 mg, 0.342 mmol). After stirring for 1.5 h at room
temperature, the reaction was quenched with H2O and the aqueous phase was
extracted with EtOAc. The combined organic extracts were dried over
anhydrous Na2SO4 and concentrated under reduced pressure. Flash column
chromatography (20:1 hexane/EtOAc) afforded 8 (6.00 mg, 29%) and 24
(8.40 mg, 46%) as colorless oils.
ACKNOWLEDGEMENTS
This research was partially supported by a Grant-in-Aid for Scientific Research
(C) from the ministry of Education, Culture, Sports, Science and Technology
(MEXT) of Japan (SA, 2350132) and Kitasato University research grant
for young researchers (MO). We thank Ms N Sato and Dr K Nagai
(Kitasato University) for kindly measuring NMR and MS spectra.
1
2
3
4
5
Omura, S. et al. An anthelmintic compound, nafuredin, shows selective inhibition of
complex I in helminth mitochondria. Proc. Natl Acad. Sci. USA 98, 60–62 (2001).
Ui, H. et al. Nafuredin, a novel inhibitor of NADH-fumarate reductase, produced by
Aspergillus niger FT-0554. J. Antibiot. 54, 234–238 (2001).
Takano, D. et al. Absolute configuration of nafuredin, a new specific NADH-fumarate
reductase inhibitor. Tetrahedron Lett. 42, 3017–3020 (2001).
Takano, D. et al. Total synthesis of nafuredin, a selective NADH-fumarate reductase
inhibitor. Org. Lett. 3, 2289–2291 (2001).
Nagamitsu, T. et al. Total synthesis of nafuredin-γ, a γ-lactone related to nafuredin with
selective inhibitory activity against NADH-fumarate reductase. Tetrahedron Lett. 44,
6441–6444 (2003).
DIBAL reduction. To a solution of 22 (102 mg, 0.260 mmol) in CH2Cl2
(2.59 ml) was treated with DIBAL (1.03 M in n-hexane, 1.13 ml, 1.17 mmol)
at − 78 °C. After stirring for 1.5 h at 0 °C, MeOH was added dropwise at 0 °C
to the resulting solution until the evolution of gas ceased. The mixture was
diluted with CH2Cl2, treated with Celite (500 mg) and Na2SO4·10H2O
(500 mg), and then stirred for 1 h at 0 °C. The resulting suspension was
filtered through a pad of Celite and the filtrate was concentrated under
reduced pressure. The residue was purified by flash column chromatography
(15:1 hexane/EtOAc) to afford 8 (56.9 mg, 63%) and 24 (5.10 mg, 6%) as
colorless oils.
6
7
8
9
Shiomi, K. et al. A γ-lactone form nafuredin, nafuredin-γ, also inhibits helminth
complex I. J. Antibiot. 58, 50–55 (2005).
Nagamitsu, T. et al. The total synthesis and biological evaluation of nafuredin-γ and its
analogues. Tetrahedron 64, 8117–8127 (2008).
Ohtawa, M. et al. Design, synthesis, and biological evaluation of air-stable nafuredin-γ
analogs as complex I inhibitors. Bioorg. Med. Chem. 23, 932–943 (2015).
Uyanik, M., Ishihara, K. & Yamamoto, H. Catalytic diastereoselective polycyclization of
homo(polyprenyl)arene analogues bearing terminal siloxyvinyl groups. Org. Lett. 8,
5649–5652 (2006).
8. [α]21 +0.028 (c 0.71, CHCl3); IR (neat) 3450, 2927, 1636, 1377,
552 cm− 1D
;
1H-NMR (400 MHz, CDCl3)
δ
5.17–5.07 (m, 3H),
3.48 (dd, J = 1.8, 10.2 Hz, 1H), 2.94–2.81 (m, 2H), 2.32–2.23 (m, 1H),
2.20–1.93 (m, 12H), 1.67 (d, J = 0.8 Hz, 3H), 1.63 (s, 3H), 1.59 (s, 6H),
1.71–1.51 (m, 2H), 1.43–1.34 (m, 1H), 1.38 (s, 3H); 13C-NMR (100 MHz,
CDCl3) δ 135.8, 135.0, 131.3, 124.4, 124.2, 124.0, 76.6, 64.1, 39.7, 37.7, 33.7,
32.1, 31.0, 26.7, 26.7, 26.6, 25.7, 25.6, 17.7, 16.1, 16.0; HRMS (ESI+) [M+Na]+
calcd for C22H38NaOS 373.2541, found 373.2534.
10 Appel, R. Tertiary phosphane/tetrachloromethane, a versatile reagent for chlorination,
dehydration, and P-N Linkage. Angew. Chem. Int. Ed. 14, 801–811 (1975).
11 Hoshino, T., Chiba, A. & Abe, N. Lanosterol biosynthesis: the critical role of the methyl-
29 group of 2,3-oxidosqualene for the correct folding of this substrate and for the
construction of the five-membered D ring. Chem. Eur. J. 18, 13108–13116 (2012).
12 Trost, B. M. & Braslau, R. Tetra-n-butylammonium oxone. Oxidations under anhydrous
conditions. J. Org. Chem. 53, 532–537 (1988).
24. [α]21 −0.167 (c 0.52, CHCl3); IR (neat) 3445, 2925, 1455,
13 Trost, B. M., Dong, G. & Vance, J. A. Cyclic 1,2-diketones as core building blocks:
D
a
strategy for the total synthesis of (− )-terpestacin. Chem. Eur. J. 16,
1286, 548 cm− 1
;
1H-NMR (400 MHz, CDCl3) δ 5.13–5.10 (m, 3H), 2.47
6265–6277 (2010).
(d, J =11.6 Hz, 1H), 2.32–2.27 (m, 2H), 2.13–1.93 (m, 11H), 1.84–1.78
(m, 1H), 1.76–1.70 (m, 1H), 1.69–1.55 (m, 2H), 1.68 (s, 3H), 1.63 (s, 3H),
1.60 (s, 6H), 1.49–1.45 (m, 1H,), 1.25 (s, 3H); 13C-NMR (100 MHz, CDCl3)
δ 136.2, 135.0, 131.3, 124.3, 124.1, 123.4, 69.4, 52.3, 39.7, 39.7, 36.0, 28.4, 26.7,
26.6, 26.4, 25.7, 25.4, 24.5, 23.9, 17.7, 16.1, 16.0; HRMS (ESI+) [M+Na]+ calcd
for C22H38NaOS 373.2541, found 373.2531.
14 Amino, H. et al. Stage-specific isoforms of Ascaris suum complex. II: the
fumarate reductase of the parasitic adult and the succinate dehydrogenase of
free-living larvae share a common iron-sulfur subunit. Mol. Biochem. Parasitol. 106,
63 (2000).
15 Brioche, J. C. R., Goodenough, K. M., Whatrup, D. J. & Harrity, J. P. A. A [3+3]
annelation approach to (+)-rhopaloic acid B. Org. Lett. 9, 3941–3943 (2007).
16 Baldwin, J. E. Rules for ring closure. J. Chem. Soc. Chem. Commun. 734–736 (1976).
Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
The Journal of Antibiotics