First total synthesis of the antifungal antibiotic thiobutacin

Article abstract of DOI:10.1016/j.tetlet.2008.06.036

The first total synthesis of thiobutacin, a butanoic acid with antifungal activity recently isolated from the culture broth of a soil actinomycete, Lechevalieria aerocolonigenes strain VK-A9, is described. The five-step procedure involves readily availabl

Full text of DOI:10.1016/j.tetlet.2008.06.036

Tetrahedron Letters 49 (2008) 5056–5058
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Tetrahedron Letters
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First total synthesis of the antifungal antibiotic thiobutacin
a
a
b
Narayan Chakor a, , Sabrina Dallavalle , Loana Musso , Maddalena Moretti
*
a Dipartimento di Scienze Molecolari Agroalimentari, Università di Milano, Via Celoria 2, 20133 Milano, Italy
b Istituto di Patologia Vegetale, Università di Milano, Via Celoria 2, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The first total synthesis of thiobutacin, a butanoic acid with antifungal activity recently isolated from the
culture broth of a soil actinomycete, Lechevalieria aerocolonigenes strain VK-A9, is described. The five-step
procedure involves readily available and cheap starting materials and can easily be transposed to the
large scale. Fungal growth inhibition of thiobutacin is mediated by the pH of the growth medium.
Maximum inhibitory activity was obtained between pH 6 and 7.
Received 24 April 2008
Revised 3 June 2008
Accepted 9 June 2008
Available online 12 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Antifungal
Thiobutacin
Synthesis
Natural product
Thiobutacin (1, 4-(2-aminophenyl-4-oxo-2-methylthiobutanoic
acid)) was recently isolated by Hwang and co-workers from the
culture broth of a soil actinomycete, Lechevalieria aerocolonigenes
strain VK-A9.1 Although thiobutacin presents a stereogenic centre
at carbon C-2, no optical data were reported for the natural com-
pound, most probably due to the easy racemization. It is well
known that mercapto-bearing carbons adjacent to an electron-
withdrawing group like an acid, ester, amide or nitrile are extre-
mely sensitive to racemization, either complete or partial.2 Thio-
butacin showed actinomycete activity against Phytophthora
In this Letter, we describe a preparatively simple five-step route
to thiobutacin that is depicted in Scheme 1.
Crotonic condensation of 2-nitroacetophenone with glyoxylic
acid monohydrate afforded compound 3 in 77% yield. The reaction
was accomplished by heating at 96 °C under reduced pressure in
the presence of water and a catalytic amount of concd H2SO4, using
a modified version of the procedures described by Bianchi4a and
Kameo.4b Michael addition of thioacetic acid to 3 provided 2-acet-
ylsulfanyl-4-(2-nitrophenyl)-4-oxobutyric acid 45 that was easily
converted to 5 (92% yield) by refluxing with concd H2SO4 and
AcOH. Subsequently, compound 5 was methylated with methyl
iodide to furnish 2-methylsulfanyl-4-(2-nitrophenyl)-4-oxobutyric
acid 6 in good yield.
capsici in microtiter broth dilution assay (MIC 10
fungal activity against Botrytis cinerea (MIC 50
l
l
g/mL) and anti-
g/mL) and the
yeast Saccharomyces cerevisiae (MIC 30 l
g/mL).1 In a following
paper, the same authors confirmed the in vitro actinomycete activ-
ity of thiobutacin against P. capsici and its control efficacy against
Phytophthora blight in vivo.3
To verify the regioselectivity of the Michael addition, compound
6 was converted to the methyl ester and reduced with NaBH4 in
ethanol/water giving the corresponding diol 8 as a mixture of dia-
stereomers. Analysis of the 1H NMR spectra of the two ( )-diaste-
reomers 8a and 8b confirmed that the thioacetate was bound at
O
SCH3
COOH
the
a position with respect to the carboxylic acid group. In fact
the hydrogen on C-4, which bears the hydroxy group, gives in both
compounds a double doublet at 5.45 ppm in 8a (J = 2.6, 9.6 Hz), and
at 5.55 ppm in 8b (J = 2.6, 9.9 Hz), thus indicating the presence of 2
hydrogens on C-3. Moreover, the multiplicity of H-3A (in 8a: ddd,
J = 2.6, 6.3, 14.7 in 8b: ddd, J = 2.6, 11.4, 14.7, Hz), and H-3B (in 8a:
ddd, J = 6.6, 9.6, 14.7 in 8b: ddd, J = 5.5, 9.9, 14.7 Hz) clearly
demonstrates the proposed structure.
The final step of the synthesis was the reduction of the nitro
group. This was first attempted using Na2S2O4 in a mixture of diox-
ane/H2O (1.2:1)6 but the troublesome workup and the unsatisfac-
tory yield prompted us to search for an alternative route. After
NH2
1
As part of our studies on natural compounds with antifungal
activity, we became interested in developing a general method
for synthesizing 1, which might also be amenable to the synthesis
of analogues.
* Corresponding author. Tel.: +39 0250316016; fax: +39 0250316801.
E-mail address: narayan.chakor@unimi.it (N. Chakor).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.06.036

N. Chakor et al. / Tetrahedron Letters 49 (2008) 5056–5058
5057
O
O
O
SCOCH3
COOH
O
SH
COOH
a
c
b
COOH
NO2
NO2
NO2
NO2
2
3
4
5
d
O
SCH3
O
SCH3
COOH
e
COOH
NH2
NO2
6
1
f
OH SCH3
OH
O
SCH3
O
OCH3
g
NO2
NO2
8
7
Scheme 1. Reagents and conditions: (a) glyoxylic acid monohydrate, 96 °C, reduced pressure, concd H2SO4, H2O, 1.5 h, 77%; (b) thioacetic acid, CH2Cl2, rt, 3.5 h, 94%; (c) AcOH,
concd H2SO4, H2O, reflux, 2 h, 92%; (d) MeI, TFA, CH2Cl2, rt, 3 h, 69%; (e) HI, 90 °C, 3 h, 70%; (f) MeOH, H2SO4, rt, 14 h, 83%; (g) NaBH4, ethanol/water, rt, 1.5 h, 72%.
several other attempts, it was found that the use of 57% HI7 at non-
refluxing conditions (90 °C) for 3 h yielded the corresponding
amine thiobutacin (1) with good chemoselectivity, that is, without
affecting the carbonyl group.8
pared to the one reported by Lee et al.1 led us to hypothesize that
the medium pH, and consequently thiobutacin charge, could influ-
ence its biological activity. There are examples in the literature of
such effects.10 Therefore, we tested the effectiveness on B. cinerea
growth at 5 different pH conditions, from 4 to 8 (Fig. 1). We found
that growth inhibition was strongly affected by the pH of the cul-
ture medium, as reported in Figure 1, being higher at pH between 6
and 7 and steeply decreasing at higher or lower pH conditions.
These results could explain the discrepancy of our data with those
of Lee et al.
In conclusion, we have accomplished the first synthesis of the
natural antifungal thiobutacin; the proposed route is concise and
modular, making it convenient for large scale preparation and
rapid synthesis of analogues. Antifungal tests against B. cinerea
highlighted that the pH of the medium strongly affects the
growth inhibition activity, this latter being highest at pH between
6 and 7.
The spectroscopic data, including 1H NMR, 13C NMR, HMBC,
COSY and MS spectra of the synthetic thiobutacin matched with
those reported in the literature1 for the natural compound, thus
confirming its structure.
Thiobutacin was then tested for its biological activity. As we
were unable to secure an authentic sample for direct comparison,
the experiments were performed only on the synthetic sample.
The antifungal activity was evaluated against B. cinerea, Penicil-
lium sp., Mucor mucedo, Alternaria alternata and the yeast S. cerevi-
siae.9 The higher inhibitory effect was observed on the growth of S.
cerevisiae, with a 30% inhibition at a dose of 250 lg/mL. A 10% inhi-
bition was observed on B. cinerea and M. mucedo at the same dose,
whilst the compound was ineffective on A. alternata and Penicillium
sp. The lower antifungal activity of synthetic thiobutacin as com-
Acknowledgement
60
50
40
30
20
10
0
600
500
400
300
This work was supported by the University of Milano (FIRST
funds).
Supplementry data
Supplementary data (synthetic procedures, characterization
data and copies of 1H, 13C and HMBC NMR spectra of thiobutacin)
associated with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2008.06.036.
200
100
0
References and notes
1. Lee, J. Y.; Moon, S. S.; Yun, B. S.; Yoo, I. D.; Hwang, B. K. J. Nat. Prod. 2004, 67,
2076–2078.
-10
3
4
5
6
7
8
9
2. Strijtveen, B.; Kellog, R. M. J. Org. Chem. 1986, 51, 3664–3671.
3. Lee, J. Y.; Sherman, D. H.; Hwang, B. K. Pest Manag. Sci. 2008, 64, 172–177.
4. (a) Bianchi, M.; Butti, A.; Christidis, Y.; Perronet, J.; Barzaghi, F.; Cesana, R.;
Nencioni, A. Eur. J. Med. Chem. 1988, 23, 45–52; (b) Kameo, K.; Asami, Y.;
Ogawa, K.; Matsunaga, T.; Saito, S.; Tomisawa, K.; Sota, K. Chem. Pharm. Bull.
1989, 37, 1260–1267.
growth
(absorbance)
Inhibition (%)
Figure 1. Effect of the pH of the culture medium on the growth of B. cinerea
(measured from absorbance at 492 nm, left scale) and on its growth inhibition
5. Tomisawa, K.; Kameo, K.; Matsunaga, T.; Saito, S.; Hosoda, K.; Asami, Y.; Sota, K.
Chem. Pharm. Bull. 1985, 33, 2386–2394.
induced by 100 lg/mL thiobutacin (right scale).

5058
N. Chakor et al. / Tetrahedron Letters 49 (2008) 5056–5058
6. Gante, J.; Weitzel, R. Tetrahedron Lett. 1988, 29, 181–184.
7. Dileep Kumar, J. S.; Ho, M. M.; Toyokuni, T. Tetrahedron Lett. 2001, 42, 5601–
5603.
3.86 (1H, dd, H-2, J = 4.4, 10.3 Hz), 3.84 (3H, s, OCH3), 3.51 (1H, dd, H-3A,
J = 10.3, 18.0 Hz), 3.21 (1H, dd, H-3B, J = 4.4, 18.0 Hz), 2.20 (3H, s, SCH3). 13C
NMR (CDCl3) d: 199.41, 171.97, 145.46, 136.98, 134.34, 128.65, 124.60, 52.77,
45.14, 41.52, 14.33.
8. Spectral data: Compound 3: IR (film) 3060, 3010, 1720, 1540, 1430, 1370, 1280,
1230, 750, 720 cmꢀ1
;
1H NMR (DMSO-d6) d: 13.25 (br s, 1H, –COOH), 8.21 (d,
Compound 8a: IR (film) 3350, 3060, 3000, 1530, 1440, 1330, 1275, 1100, 900,
1H, H-30, J = 8.2 Hz), 7.90 (dd, 1H, H-50, J = 8.2, 8.2 Hz), 7.82 (dd, 1H, H-40,
J = 8.2 Hz), 7.65 (d, 1H, H-60, J = 8.2 Hz), 7.20 (d, 1H, J = 16.0 Hz), 6.38 (d, 1H,
J = 16.0 Hz). 13C NMR (acetone-d6) d: 191.92; 165.32; 146.70; 139.28; 134.86;
134.71; 133.15; 131.74; 128.96; 124.63. HRMS (ESIꢀ) calcd for C10H6NO5
[MꢀH]ꢀ 220.02515, found 220.02488; calcd for C20H13N2O10 [2MꢀH]ꢀ
441.05757, found 441.05630; calcd for C20H12N2O10Na [2Mꢀ2H+Na]ꢀ
463.03951, found 463.03822.
750, 720 cmꢀ1 1H NMR(CDCl3) d: 7.91 (1H, d, H-30, J = 8.5 Hz ) 7.86 (1H, d, H-60,
;
J = 8.1 Hz), 7.64 (1H, dd, H-50, J = 8.1, 8.1 Hz), 7.41 (1H, dd, H-40, J = 8.5, 8.1 Hz),
5.45 (1H, dd, H-4, J = 2.6, 9.6 Hz), 3.77 (1H, dd, H-1A, J = 6.3, 11.4 Hz), 3.67 (1H,
dd, H-1B, J = 6.3, 11.4 Hz), 3.00 (1H, m, H-2), 2.15 (1H, ddd, H-3A, J = 2.6, 6.3,
14.7 Hz), 2.10 (3H, s, SCH3), 2.01 (1H, ddd, H-3B, J = 6.6, 9.6, 14.7 Hz); 13C NMR
(CDCl3) d: 147.36, 139.68, 132.65, 128.12 (x2), 124.43, 68.33, 63.49, 48.50,
39.99, 12.72; HRMS (ESI+) calcd for C11H15NO2S [M+Na]+ 280.06195, found
280.06219.
Compound 4: IR (film) 3060, 3000, 1720, 1540, 1430, 1355, 1280, 750,
720 cmꢀ1
;
1H NMR (CDCl3) d: 8.13 (1H, d, H-30, J = 8.2 Hz), 7.75 (1H, dd, H-50,
Compound 8b: IR (film) 3350, 3060, 3000, 1530, 1440, 1330, 1275, 1100, 900,
J = 7.4, 7.4 Hz), 7.62 (1H, dd, H-40, J = 7.4, 8.2 Hz), 7.49 (1H, d, H-60, J = 7.4 Hz),
4.76 (1H, dd, H-2, J = 7.4, 4.8 Hz), 3.56 (1H, H-3A, J = 7.4, 18.2 Hz), 3.40 (1H, dd,
H-3B, J = 4.8, 18.2 Hz), 2.42 (3H, s, COCH3). 13C NMR (acetone-d6) d: 192.68;
170.93; 146.34; 135.79; 134.10; 131.57; 128.16; 124.33; 43.88; 39.79; 28.74.
HRMS (ESIꢀ) calcd for C12H10NO6S [MꢀH]ꢀ 296.02343, found 296.02304; calcd
750, 720 cmꢀ1 1H NMR (CDCl3) d: 7.91 (1H, d, H-30, J = 8.5 Hz) 7.85 (1H, d, H-60,
;
J = 8.1 Hz), 7.64 (1H, dd, H-50, J = 8.1, 8.1 Hz), 7.49 (1H, dd, H-40, J = 8.5, 8.1 Hz),
5.55 (1H, dd, H-4, J = 2.6, 9.9 Hz), 3.68 (2H, d, H-1, J = 6.3), 3.09 (1H, m, H-2),
2.10 (3H, s, SCH3), 2.04 (1H, ddd, H-3A, J = 2.6, 11.4, 14.7 Hz), 1.91 (1H, ddd,
H-3B, J = 5.5, 9.9, 14.7 Hz). 13C NMR (CDCl3) d:147.41, 139.65, 133.69, 128.23,
128.12, 124.38, 67.43, 63.22, 46.57, 39.58, 11.84. HRMS (ESI+) calcd for
for
C
C
24H21N2O12S2 [2MꢀH]ꢀ 593.05414, found 593.05140; calcd for
24H20N2O12S2Na [2Mꢀ2H+Na]ꢀ 615.03608, found 615.03414.
C
11H15NO2S [M+Na]+ 280.06195, found 280.06176.
Compound 5: IR (film) 3060, 3000, 1720, 1560, 1430, 1280, 750, 720 cmꢀ1
;
1H
9. Bioassays procedure: Antimicrobial activity of the newly synthesized
thiobutacin was at first assessed on B. cinerea, Penicillium sp., Alternaria
alternata, Mucor mucedo and Saccharomyces cerevisiae in 96-well microtiter
NMR (acetone-d6) d: 8.11 (1H, d, H-30, J = 8.2 Hz), 7.89 (1H, dd, H-50, J = 7.4,
7.4 Hz), 7.79 (1H, dd, H-40, J = 7.4, 8.2 Hz), 7.75 (1H, d, H-60, J = 7.4 Hz), 3.93 (1H,
ddd, H-2, J = 4.0, 9.3, 9.3 Hz), 3.63 (1H, H-3A, J = 18.2, 9.3 Hz), 3.38 (1H, dd,
H-3B, J = 18.2, 4.8 Hz), 2.76 (1H, d, –SH, J = 9.3 Hz). 13C NMR (acetone-d6) d:
198.73; 172.88; 146.37; 135.89; 134.10; 131.53; 128.10; 124.33; 47.25; 34.67.
HRMS (ESIꢀ) calcd for C10H8NO5S [MꢀH]ꢀ 254.01287, found 254.01356; calcd
dishes with the modified method described by Lee et al.1 Briefly 50
lL of fungal
spore (2 ꢁ 103 spores/mL) and yeast cell (2 ꢁ 102 CFU/mL) suspensions in
potato dextrose broth PDB (Difco) were added to each well containing 50
lL of
PDB amended with thiobutacin at different concentrations from 0 to 250
l
g/
for
C
C
20H17N2O10S2 [2MꢀH]ꢀ 509.03301, found 509.03234; calcd for
mL. To assess the influence of pH on thiobutacin activity, an experiment was
performed with B. cinerea grown at different pH conditions, from 4 to 8, adding
an adequate amount of HCl or KOH to PDB. The absorbance was measured with
a microplate reader at 492 nm wavelength twice, the first just after filling
the plates and the second at the end of the experiment after incubation
for 2 days at 25 °C. Final absorbance values were calculated subtracting the
values of the first reading from those of the second reading. Percent growth
inhibition at the different thiobutacin doses was calculated with respect to the
control wells.
20H16N2O10S2Na [2Mꢀ2H+Na]ꢀ 531.01495, found 531.01523.
Compound 6: IR (film) 3060, 3000, 1720, 1540, 1430, 1350, 1270, 910, 750,
720 cmꢀ1 1H NMR (CDCl3) d: 8.13 (1H, d, H-30, J = 8.2 Hz), 7.75 (1H, dd, H-50,
;
J = 7.4, 7.4 Hz), 7.62 (1H, dd, H-40, J = 7.4, 8.2 Hz), 7.48 (1H, d, H-60, J = 7.4 Hz),
3.87 (1H, dd, H-2, J = 4.5, 10.1 Hz), 3.48 (1H, H-3A, J = 10.1, 18.2 Hz), 3.24 (1H,
dd, H-3B, J = 18.2, 4.5 Hz), 2.27 (3H, s, –SCH3). 13C NMR (CDCl3) d: 199.24,
177.35, 145.44, 136.90, 134.45, 130.88, 127.52, 124.49, 44.05, 41.66, 14.78.
HRMS (ESIꢀ) calcd for C11H10NO5S [MꢀH]ꢀ 268.02852, found 268.02861; calcd
for C22H21N2O10S2 [2MꢀH]ꢀ 537.06431, found 537.06278; calcd for
10. (a) Futagawa, M.; Wedge, D. E.; Dayan, F. E. Pestic. Biochem. Physiol. 2002, 73,
87–93; (b) Milewski, S.; Mignini, F.; Prasad, R.; Borowski, E. Antimicrob. Agents
Chemother. 2001, 45, 223–228; (c) Krasowska, A.; Chmielewska, L.; Luczynski,
J.; Witek, S.; Sigler, K. Cell. Mol. Biol. Lett. 2003, 8, 111–120.
C
22H20N2O10S2Na [2Mꢀ2H+Na]ꢀ 559.04625, found 559.04489.
Compound 7: IR (film) 3060, 2990, 1740, 1540, 1440, 1430, 1360, 1280, 750,
720 cmꢀ1
;
1H NMR (CDCl3) d: 8.12 (1H, d, H-30, J = 8.1 Hz ) 7.74 (1H, dd, H-50,
J = 7.7, 7.7 Hz), 7.61 (1H, dd, H-40, J = 7.7, 8.1 Hz), 7.49 (1H, d, H-60, J = 7.7 Hz),