(+)-Cystothiazole G: Isolation and structural elucidation

Article abstract of DOI:10.1016/j.tet.2004.03.067

Palladium-catalyzed cyclization-methoxycarbonylation of (2R,3S)-3-methylpent-4-yne-1,2-diol (6) derived from (2R,3S)-epoxybutanoate 5 followed by methylation gave the tetrahydro-2-furylidene acetate (-)-7, which was converted to the left-half aldehyde (+)-3. A Wittig reaction between (+)-3 and the phosphoranylide derived from the bithiazole-type phosphonium iodide 4 using lithium bis(trimethylsilyl)amide afforded the (+)-cystothiazole G (2), whose spectral data were identical with those of the natural product (+)-2. Thus, the stereochemistry of cystothiazole G (2) was proved to be (4R,5S,6(E)).

Full text of DOI:10.1016/j.tet.2004.03.067

Tetrahedron 60 (2004) 4735–4738
(1)-Cystothiazole G: isolation and structural elucidation
a
a
b
b
Hiroyuki Akita,a Takamitsu Sasaki, Keisuke Kato, Yoshihiro Suzuki, Keiko Kondo,
,
*
Youji Sakagami,b Makoto Ojika,b Ryosuke Fudouc and Shigeru Yamanakac
aSchool of Pharmaceutical Sciences, Toho University, 2-2-1, Miyama, Funabashi, Chiba 274-8510, Japan
bGraduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
cInstitute of Life Science, Ajinomoto Co. Inc., Kawasaki-ku, Kawasaki 210-8681, Japan
Received 16 February 2004; accepted 15 March 2004
Abstract—Palladium-catalyzed cyclization-methoxycarbonylation of (2R,3S)-3-methylpent-4-yne-1,2-diol (6) derived from (2R,3S)-
epoxybutanoate 5 followed by methylation gave the tetrahydro-2-furylidene acetate (2)-7, which was converted to the left-half aldehyde
(þ)-3. A Wittig reaction between (þ)-3 and the phosphoranylide derived from the bithiazole-type phosphonium iodide 4 using lithium
bis(trimethylsilyl)amide afforded the (þ)-cystothiazole G (2), whose spectral data were identical with those of the natural product (þ)-2.
Thus, the stereochemistry of cystothiazole G (2) was proved to be (4R,5S,6(E)).
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
have been isolated by a German group from another
myxobacterium, Myxococcus stipitatus, strain Mx s64,
although the absolute structure of melithiazol H (2) was
not determined.4 This paper describes the isolation,
determination of the absolute structure based on the total
synthesis, and biological activity of cystothiazole G (2)
(Scheme 1).
Recently, we reported the structural elucidation and the total
synthesis of an antifungal substance named cystothiazole A
(1)1,2 from the myxobacterium Cystobacter fuscus strain
AJ-13278 by using an inhibition assay against the
phytopathogenic fungus, Phytophthora capsici. Further
investigation of a large-scale culture of this strain resulted
in the isolation of additional cystothiazole analogs,
cystothiazole G (2).3 This compound showed also inhibitory
activity against P. capsici. More recently, further close
analogues of cystothiazole G (2) named melithiazol H (2)
A further search for additional cystothiazoles afforded
0.5 mg of a new member, cystothiazole G (2) ([a]2D4¼þ108
(c¼0.039, CHCl3)) from the extracts of a large-scale
fermentation. The structure of cystothiazole G (2) was
Scheme 1.
Keywords: (þ)-Cystothiazole G; Cyclization–methoxycarbonylation; Chiral synthesis.
*
Corresponding author. Tel.: þ81-474-72-1805; fax: þ81-474-76-6195; e-mail address: akita@phar.toho-u.ac.jp
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.03.067

4736
H. Akita et al. / Tetrahedron 60 (2004) 4735–4738
Scheme 2.
elucidated by comparison with the spectral data for
1
cystothiazole A (1). The H NMR data were similar to
I2/Ph3P/imidazole provided an iodide 15 in 84% yield. The
reaction of 15 and triphenylphosphine gave a phosphonium
salt 4 in 98% yield, which was condensed with (þ)-3 in the
presence of lithium bis(trimethylsilyl)amide in THF to
afford a mixture ((þ)-(E)-2/(þ)-(Z)-16¼3:1) of olefins in
44% yield. Both isomers were isolated by preparative silica-
gel thin-layer chromatography to provide (þ)-2 (colorless
needles from n-hexane/AcOEt (20:1), mp 121–122 8C,
[a]D21¼þ108.8 (c¼1.025, CHCl3)) and (þ)-16 ([a]2D4¼
þ215.9 (c¼1.09, CHCl3)). The (Z)-geometry of (þ)-16
was confirmed by the NOE enhancement for the olefinic
protons (23%). The physical data of the synthetic (þ)-2
were identical with those ([a]2D4¼þ108 (c¼0.039, CHCl3),
1H NMR(CDCl3)) of the isolated cystothiazole G (þ)-2.
Thus, the stereochemistry of cystothiazole G (2) was proved
to be (4R,5S,6(E)). 1H NMR (CD3OD) data of the synthetic
(þ)-2 were also identical with those of Melithiazole H (2),
while the absolute structure of Melithiazole H (2) was not
determined because of no information with respect to the
specific rotation. The antifungal activity of the natural
cystothiazole G (2) against the phytopathogenic fungus, P.
capsici, was evaluated by using a paper disc assay method
as reported previously.1 The minimum dose applied on a
paper disc to inhibit the fungal growth was 1 mg/disc. The
synthetic cystothiazole G (2) also showed the activity at
a similar level of dosage (0.2 mg/disc). According to our
recent studies on antifungal tests using the phytopathogenic
fungus, P. capsici, synthetic cystothiazole A (1) ((4R,5S)-1)
showed activity up to a dose of 0.04 mg/disc. However, not
only the enantiomer ((4S,5R)-1) but also the two dia-
stereomers ((4S,5S)-1 and (4R,5R)-1) did not show any
antifungal activity up to 100 mg/disc. This result was not
those for cystothiazole A (1) except for the presence of
the signals due to an ethyl group observed at d 1.43
(t, J¼7.6 Hz, 3H) and 3.09 (q, J¼7.6 Hz, 2H) and for the
absence of the signals due to the isopropyl group of
cystothiazole A (1). This NMR information as well as the
molecular formula that is less than that of cystothiazole
A (1) by CH2 suggest that cystothiazole G (2) is
14-demethylcystothiazole A. A similar specific rotation to
that of cystothiazole A (1) suggests that the absolute
stereochemistry of cystothiazole G (2) is the same as that of
cystothiazole A (1). Retrosynthetically, the synthesis of 2
can be achieved by Wittig condensation of the left-half
aldehyde 3 and the right-half phosphonium iodide 4. The
synthesis of chiral aldehyde 3 was achieved in the total
synthesis of cystothiazole A (1).2 Palladium-catalyzed
cyclization–methoxycarbonylation of (2R,3S)-3-methyl-
pent-4-yne-1,2-diol (6) derived from (2R,3S)-epoxybutano-
ate (5) followed by methylation gave the tetrahydro-2-
furylideneacetate 7, which was converted to the left-half
aldehyde (þ)-3.2 The synthesis of the right part 4 is shown
in Scheme 2.
Treatment of propionamide (8) with P4S10 gave a propion-
thioamide (9), which was reacted with a-bromopyruvate to
afford a mono-thiazole ester 10 in 50% overall yield from 8.
Treatment of 10 with NH3/MeOH followed by thioamida-
tion with Lawesson’s reagent yielded a thioamide 12, which
was reacted with a-bromopyruvate to afford a bithiazole
ester 13 in 53% overall yield from 10. LiBH4 reduction
(alcohol 14: 84% yield) of 13 followed by treatment with

H. Akita et al. / Tetrahedron 60 (2004) 4735–4738
4737
expected at all, because all the stereoisomers possess the
b-methoxyacrylate unit that is regarded as the binding site
2.3. 2-Ethylthiazole-4-carboxylic acid ethyl ester (10)
to the target molecules.5
To a solution of phosphorus pentasulfide (P4S10; 5.1 g,
11.47 mmol) in ether (40 mL) was added propionamide 8
(8.39 g, 114.7 mmol) and the whole mixture was stirred for
2 h at room temperature. The reaction mixture was diluted
with brine and extracted with ether. The organic layer was
dried over MgSO4 and evaporated to give a crude 9. A
mixture of the crude 9 and ethyl a-bromopyruvate (22.39 g,
114.5 mmol) in EtOH (100 mL) was stirred at reflux for
15 min. The reaction mixture was evaporated, diluted with
AcOEt, and washed with 7% aqueous NaHCO3. The organic
layer was dried over MgSO4 and evaporated to give a crude
oil, which was chromatographed on silica gel (200 g,
n-hexane/AcOEt¼15:1) to afford colorless compound 10
(10.81 g, 51% overall yield from 8).
In conclusion, palladium-catalyzed cyclization–methoxy-
carbonylation of (2R,3S)-3-methylpent-4-yne-1,2-diol (6)
derived from (2R,3S)-epoxybutanoate 5 followed by
methylation gave the tetrahydro-2-furylidene acetate
(2)-7, which was converted to the left-half aldehyde
(þ)-3. A Wittig reaction between (þ)-3 and the phosphor-
anylide derived from the bithiazole-type phosphonium
iodide 4 using lithium bis(trimethylsilyl)amide afforded
the synthetic (þ)-cystothiazole A (2), whose spectral data
were identical with those of the natural product (þ)-2. The
stereochemistry of cystothiazole G (2) was proved to be
(4R,5S,6(E)).
Compound 10. IR (KBr): 1719 cm21 1H NMR: d 1.41
;
(3H, t, J¼7.2 Hz), 1.41 (3H, t, J¼7.6 Hz), 3.10 (2H, q, J¼
7.6 Hz), 4.42 (2H, q, J¼7.2 Hz), 8.06 (1H, s). 13C NMR: d
14.3, 14.4, 27.1, 61.4, 126.8, 146.8, 161.5, 173.8. MS (FAB)
m/z: 186 (Mþþ1).
2.4. 20-Ethyl[2,40]bithiazolyl-4-carboxylic acid ethyl ester
(13)
2. Experimental
2.1. General
All melting points were measured on a Yanaco MP-3S
micro melting point apparatus and are uncorrected. 1H- and
13C NMR spectra were recorded on JEOL AL 400
spectrometer in CDCl3. Carbon substitution degrees were
established by DEPT pulse sequence. High-resolution mass
spectra (HR-MS) and the fast atom bombardment mass
spectra (FAB-MS) were obtained with a JEOL JMS 600H
spectrometer. IR spectra were recorded with a JASCO
FT/IR-300 spectrometer. Optical rotations were measured
with a JASCO DIP-370 digital polarimeter. All evapo-
rations were performed under reduced pressure. For column
chromatography, silica gel (Kieselgel 60) was employed.
A mixture of 10 (6.0 g, 32.38 mmol) and NH3 saturated
EtOH (100 mL) in a sealed tube was stood for 3 days at
room temperature. After cooling, the reaction mixture was
evaporated to afford a crude amide 11. To a solution of
crude 11 in benzene (100 mL) was added Lawesson’s
reagent (6.47 g, 16 mmol) and the whole mixture was stirred
for 1 h at reflux. The reaction mixture was diluted with H2O
and extracted with AcOEt. The organic layer was washed
with brine and dried over MgSO4. The organic layer was
evaporated to give a crude thioamide 12. To a solution of the
crude thioamide 12 and ethyl a-bromopyruvate (6.05 g,
31 mmol) in absolute EtOH (200 mL) was stirred for 1 h at
reflux. The reaction mixture was evaporated, diluted with
7% aqueous NaHCO3 and extracted with AcOEt. The
organic layer was washed with brine and dried over MgSO4.
The organic layer was evaporated to give a crude residue,
which was chromatographed on silica gel (60 g, n-hexane/
AcOEt¼15:1) to afford 13 (4.6 g, 53%). Recrystallization of
13 from n-hexane provided colorless needles 13.
2.2. Isolation of cystothiazole G (2)
A large-scale fermentation (150 L culture) and fractionation
to obtain the fractions of the first silica gel column
chromatograph were reported in the literature.1 The most
polar part (950–1150 mL) of the hexane/EtOAc (4:1)
fractions and the EtOAc fraction (400 mL) were combined
(2.5 g), and a portion (1.35 g) was subjected to silica gel
medium-pressure liquid chromatography (Develosil Lop 60,
Nomura Chemical; 128 min linear gradient from 1 to 33%
acetone in toluene, 5 mL/min) to give seven fractions. The
first fraction eluted from 1 to 12% acetone (98.2 mg) was
separated by preparative HPLC (Develosil ODS-10 (20
i.d.£250 mm), Nomura Chemical; 60% MeCN, 7 mL/min,
detected at 254 nm) to give cystothiazole G (2) (0.5 mg,
tR¼62.5 min) as a solid. Cystothiazole G (2): [a]2D4¼þ108
(c¼0.039, CHCl3); UV (MeOH) lmax 226 (1 33,400), 244 (1
34,000), 312 (1 12,500) nm, IR (CHCl3): 1717, 1700, 1624,
1
Compound 13. Mp 82–83 8C; IR (KBr): 1712 cm21; H
NMR: d 1.43 (3H, t, J¼7.2 Hz), 1.44 (3H, t, J¼6.8 Hz), 3.09
(2H, q, J¼7.6 Hz), 4.45 (2H, q, J¼7.2 Hz), 8.03 (1H, s),
8.16 (1H, s). 13C NMR: d 14.0, 14.4, 26.9, 61.5, 116.6,
127.6, 147.9, 147.9, 161.5, 163.6, 173.7. Anal. Calcd for
C11H12N2O2S2: C, 49.23; H, 4.51; N, 10.44. Found: C,
49.28; H, 4.49; N, 10.45. MS (FAB) m/z: 269 (Mþþ1).
1
1150, 1094 cm21; H NMR (CDCl3): d 1.22 (3H, d, J¼
2.5. 20-Ethyl[2,40]bithiazolyl-4-methanol (14)
6.9 Hz), 1.43 (3H, t, J¼7.6 Hz), 3.09 (2H, q, J¼7.6 Hz),
3.33 (3H, s), 3.60 (3H, s), 3.66 (3H, s), 3.81 (1H, t, J¼
7.6 Hz), 4.17 (1H, dq, J¼7.6, 6.9 Hz), 4.97 (1H, s), 6.41
(1H, dd, J¼7.6, 15.7 Hz), 6.58 (1H, d, J¼15.7 Hz), 7.09
(1H, s), 7.84 (1H, s). 13C NMR (CDCl3): d 14.1, 14.1, 26.9,
39.8, 50.8, 55.5, 57.0, 84.4, 91.1, 115.0, 115.2, 125.6, 131.7,
148.9, 154.5, 162.4, 167.7, 173.6, 176.7. HR-MS (FAB)
(m/z): calcd for C19H25O4N2 S2 (Mþþ1): 409.1256. Found:
409.1238.
A mixture of 13 (2.0 g, 7.45 mmol) and LiBH4 (0.492 g,
22.6 mmol) in THF (30 mL) was stirred for 50 min at room
temperature. The reaction mixture was diluted with H2O
(10 mL) and the whole was stirred for 5 h at the same
temperature. The reaction mixture was extracted with
AcOEt and washed with brine, and dried over MgSO4.
The organic layer was evaporated to give a crude residue,

4738
H. Akita et al. / Tetrahedron 60 (2004) 4735–4738
which was chromatographed on silica gel (10 g, n-hexane/
AcOEt¼5:1) to afford 14 (1.43 g, 84%). Recrystallization of
14 from n-hexane–AcOEt provided colorless needles 14.
and a colorless oil (þ)-16 (19.8 mg, 11%). Recrystallization
of (þ)-2 from n-hexane/AcOEt (20:1) provided colorless
needles 2.
1
Compound 14. Mp 94–96 8C; IR (KBr): 3223 cm21; H
Compound (þ)-2. Mp 121–122 8C; [a]2D1¼þ108.8
(c¼1.025, CHCl3); IR (KBr): 3110, 2977, 1710, 1618,
NMR: d 1.43 (3H, d, J¼7.6 Hz), 3.08 (2H, q, J¼7.6 Hz),
4.82 (2H, s), 7.20 (1H, s), 7.87 (1H, s). 13C NMR: d 14.1,
26.9, 60.8, 115.2, 115.6, 148.4, 157.1, 163.5, 173.8. Anal.
Calcd for C9H10N2OS2: C, 47.76; H, 4.45; N, 12.38. Found:
C, 47.68; H, 4.51; N, 12.33. MS (FAB) m/z: 227 (Mþþ1).
1
1144, 1079 cm21; H NMR (CDCl3): d 1.22 (3H, d, J¼
6.8 Hz), 1.43 (3H, t, J¼7.6 Hz), 3.09 (2H, q, J¼7.6 Hz),
3.32 (3H, s), 3.60 (3H, s), 3.66 (3H, s), 3.81 (1H, t, J¼
7.8 Hz), 4.17 (1H, dq, J¼7.6, 6.8 Hz), 4.97 (1H, s), 6.41
(1H, dd, J¼7.7, 15.7 Hz), 6.57 (1H, d, J¼15.7 Hz), 7.08
2.6. 20-Ethyl[2,40]bithiazolyl-4-methyleneiodide (15)
(1H, s), 7.84 (1H, s). H NMR (CD3OD): d 1.26 (3H, d,
1
J¼6.8 Hz), 1.46 (3H, t, J¼7.6 Hz), 3.12 (2H, q, J¼7.6 Hz),
3.37 (3H, s), 3.66 (3H, s), 3.66 (3H, s), 3.83 (1H, t, J¼
8.4 Hz), 4.23 (1H, dq, J¼8.4, 6.8 Hz), 5.08 (1H, s), 6.39
(1H, dd, J¼8.4, 15.6 Hz), 6.62 (1H, d, J¼15.6 Hz), 7.40
(1H, s), 8.04 (1H, s). 13C NMR (CD3OD): d 14.6, 15.1, 27.6,
41.2, 51.2, 56.2, 57.1, 85.8, 92.0, 116.7, 117.0, 126.8, 132.3,
149.4, 155.2, 163.8, 169.3, 175.1, 177.5. C19H24N2O4S2: C,
55.86; H, 5.92; N, 6.86. Found: C, 55.85; H, 6.03; N, 6.69.
MS (FAB) m/z: 409 (Mþþ1).
Compound (þ)-16. [a]D24¼þ215.9 (c¼1.09, CHCl3); IR
(CHCl3): 1705, 1620 cm21; 1H NMR (CD3OD): d 1.22 (3H,
d, J¼6.8 Hz), 1.41 (3H, t, J¼7.6 Hz), 3.08 (2H, q, J¼
7.6 Hz), 3.29 (3H, s), 3.34 (3H, s), 3.62 (3H, s), 4.16 (1H,
dq, J¼6.8, 9.6 Hz), 4.96 (1H, s), 5.20 (1H, t, J¼9.6 Hz),
5.49 (1H, dd, J¼9.6, 12.0 Hz), 6.57 (1H, d, J¼12.0 Hz),
7.42 (1H, s), 7.93 (1H, s). 13C NMR (CD3OD): d 14.5, 15.2,
27.6, 40.7, 51.2, 55.7, 56.8, 80.1, 91.8, 116.5, 119.7, 126.4,
133.3, 149.8, 154.6, 162.9, 169.5, 175.4, 177.8. HR-MS
(FAB) (m/z): calcd for C19H25O4N2 S2 (Mþþ1): 409.1256.
Found: 409.1276.
To a mixture of 14 (1.22 g, 5.39 mmol), triphenylphosphine
(1.56 g, 5.95 mmol) and imidazole (0.550 g, 8.1 mmol) in
THF (25 mL) was added I2 (1.51 g, 5.94 mmol) under argon
atmosphere and the whole mixture was stirred for 30 min at
room temperature. The reaction mixture was diluted with
H2O and extracted with AcOEt. The organic layer was
washed with brine and dried over MgSO4. The organic
layer was evaporated to give a crude residue, which was
chromatographed on silica gel (20 g, n-hexane/AcOEt¼
30:1) to afford 15 (1.53 g, 84%). Recrystallization of 22
from n-hexane provided colorless needles 15.
Compound 15. Mp 119–121 8C; IR (KBr): 1533, 1498,
1432 cm21; 1H NMR: d 1.43 (3H, t, J¼7.6 Hz), 3.08 (2H, q,
J¼7.6 Hz), 4.56(2H, s), 7.25(1H, s), 7.86(1H, s). 13C NMR:d
21.53, 14.1, 26.9, 115.6, 116.7, 148.5, 154.0, 163.1, 173.7.
Anal. Calcd for C9H9IN2S2: C, 32.15; H, 2.70; N, 8.33. Found:
C, 32.15; H, 2.90; N, 8.00. MS (FAB) m/z: 337 (Mþþ1).
2.7. 20-Ethyl[2,40]bithiazolyl-4-methylenetriphenyl-
phosphonium iodide (4)
A mixture of 15 (1.40 g, 4.16 mmol) and triphenyl-
phosphine (2.74 g, 10.4 mmol) in benzene (14 mL) was
stirred for 8 h at reflux. After cooling, the resulting colorless
powder 4 (2.46 g, 98%) was obtained by filtration.
Acknowledgements
This work was supported by Grant-in Aid for Scientific
Researchs (C) (No. 14572016 to H. A.) and (B) (No.
12480172 to M. O.) from The Ministry of Education,
Culture, Sports, Science and Technology. The authors
(M. O.) are grateful to Fujisawa Foundation for financial
support.
1
Compound 4. Mp 265–267 8C; H NMR: d 1.40 (3H, t,
J¼7.6 Hz), 3.04 (2H, q, J¼7.6 Hz), 5.51 (2H, d, J¼14 Hz),
7.26 (1H, s), 7.63–7.84 (15H, m), 8.08 (1H, d, J¼3.2 Hz).
Anal. Calcd for C27H24IN2PS2: C, 54.18; H, 4.04; N, 4.68.
Found: C, 54.35; H, 4.11; N, 4.57. MS (FAB) m/z: 471
(Mþ2I).
References and notes
2.8. Wittig condensation of (1)-3 and 4
1. (a) Ojika, M.; Suzuki, Y.; Tsukamoto, A.; Sakagami, Y.; Fudou,
R.; Yoshimura, T.; Yamanaka, S. J. Antibiot. 1998, 51,
275–281. (b) Suzuki, Y.; Ojika, M.; Sakagami, Y.; Fudou,
R.; Yamanaka, S. Tetrahedron 1998, 54, 11399–11404.
To a solution of 4 (0.527 g, 0.88 mmol) in THF (5 mL) was
added lithium bis(trimethylisilyl)amide (1 M solution in
THF, 0.88 mL, 0.88 mmol) at 0 8C under argon atmosphere
and the whole mixture was stirred for 20 min at the same
temperature. A solution of (þ)-3 (0.095 g, 0.44 mmol) in
THF (2 mL) was added to the above reaction mixture at 0 8C
and the whole mixture was stirred for 15 min at the same
temperature. The reaction mixture was diluted with H2O
and extracted with AcOEt. The organic layer was dried over
MgSO4 and evaporated to afford a crude product which was
chromatographed on silica gel (10 g, n-hexane/
AcOEt¼20:1) to give a mixture ((E)/(Z)¼3:1) of 2. This
mixture was subjected to thin-layer chromatography (silica-
gel, n-hexane/AcOEt¼5:1) to afford (þ)-2 (59.3 mg, 33%)
2. (a) Kato, K.; Nishimura, A.; Yamamoto, Y.; Akita, H.
Tetrahedron Lett. 2002, 43, 643–645. (b) Kato, K.; Sasaki,
T.; Takayama, H.; Akita, H. Tetrahedron 2003, 59, 2679–2685.
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Yamanaka, S. 41st Symposium on the Natural Products,
Symposium Paper, Nagoya, Japan, 1999; pp 583–588.
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4. Bohlendrof, B.; Herrmann, M.; Hecht, H.-J.; Sasse, F.; Forche,
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