Semi-synthesis and biological evaluation of analogues of UK-2A, a novel antifungal antibiotic from Streptomyces sp. 517-02

Article abstract of DOI:10.1016/j.bmcl.2005.02.065

Several analogues of UK-2A, a novel antifungal antibiotic isolated from Streptomyces sp. 517-02, were semi-synthesized for structure-activity studies. In vitro antifungal activities of these compounds against Saccharomyces cerevisiae IFO 0203 were evaluat

Full text of DOI:10.1016/j.bmcl.2005.02.065

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2011–2014
Semi-synthesis and biological evaluation of analogues of UK-2A,
a novel antifungal antibiotic from Streptomyces sp. 517-02
Yoshinosuke Usuki,a,* Koichi Mitomo,c Noriko Adachi,a Xu Ping,b Ken-Ichi Fujita,b
Osamu Sakanaka,c Katsuharu Iinuma,d Hideo Iioa and Makoto Taniguchib,*
aDepartment of Material Science, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto,
Sumiyoshi-ku, Osaka 558-8585, Japan
bDepartment of Biology, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan
cCMC Research Laboratories, Meiji Seika Kaisha Ltd, Kayama, Odawara-shi, Kanagawa 250-0852, Japan
dFuji Amide Chemical Co., Ltd, Ukima, Kita-ward, Tokyo 115-0051, Japan
Received 17 January 2005; revised 17 February 2005; accepted 19 February 2005
Abstract—Several analogues of UK-2A, a novel antifungal antibiotic isolated from Streptomyces sp. 517-02, were semi-synthesized
for structure–activity studies. In vitro antifungal activities of these compounds against Saccharomyces cerevisiae IFO 0203 were evalu-
ated by the conventional paper disk method. Several derivatives exhibited growth inhibitory activity similar to UK-2A.
Ó 2005 Elsevier Ltd. All rights reserved.
UK-2A is an antifungal antibiotic produced by Strepto-
myces sp. 517-02, and is similar to antimycin A3 (AA) in
both structure and inhibitory activity toward electron
transport at complex III in mitochondria.1–4 Both UK-
2A and AA consist of nine-membered dilactone rings
linked via an amide bond to an aromatic acid moiety
(Fig. 1): UK-2A possesses a 3-hydroxy-4-methoxypicol-
inic moiety, while the AAs have 3-formamidosalicylic
moieties, reported to be essential for binding to complex
III protein and the inhibition of electron transfer be-
tween cytochromes b and c1 in the mitochondrial respi-
ratory chain.5 The benzyl group at the C2 position in
UK-2A has not been reported in antimycins and a
methyl group is lacking at the C8 position. Further-
more, UK-2A was less cytotoxic than AA against sev-
Figure 1. The structures of UK-2A and AA, showing the focus of
SAR.
eral mammalian cell lines, including murine leukemia
P388, murine melanoma B16, porcine renal proximal tu-
bule LLC-PK1, human oral epidermoid carcinoma KB,
and human colon adenocarcinoma COLO201.6 As both
UK-2A and AA inhibited mitochondrial electron trans-
port, the difference in cytotoxicity between UK-2A and
AA could be attributed to their ability to produce reac-
tive oxygen species.6,7 In continuing studies on UK-2A,8
we have been interested in establishing structure–activity
relations among UK-2A analogues.9–11 This study fo-
cuses on (1) the 30-OH group, (2) the benzene moiety,
and (3) selective cleavage of the amide bond as shown
in Figure 1, and reports the results of preliminary stud-
ies on the semi-synthetic preparation and biological
evaluation of UK-2A analogues.
Keywords: Antifungal activity; Respiratory inhibition; Structure–
activity relationship; Nine-membered dilactone; UK-2A.
*
Corresponding authors. Tel.: +81 6 6605 2563; fax: +81 6 6605 2522
(Y.U.); e-mail addresses: usuki@sci.osaka-cu.ac.jp; makoto@sci.
osaka-cu.ac.jp
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.065

2012
Y. Usuki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2011–2014
Scheme 1. Alkylation and acylation of the 30-OH group of UK-2A.
To examine the role of the 30-OH group in analogue
activity, the chemically modified analogues 3–7 were
prepared as shown in Scheme 1. Treatment of UK-2A
with iodomethane and ethyl bromoacetate under alka-
line conditions afforded the corresponding 30-O-alkyl-
ated compounds 3 and 4, respectively, in modest
yields. Acylation of UK-2A with acetic anhydride and
pyridine proceeded smoothly to afford compound 5 in
quantitative yield. Heptanedioyl dichloride was treated
with an equimolar amount of benzyl alcohol and triethyl
amine in dichloromethane; addition of a UK-2A solu-
tion in dichloromethane afforded the corresponding
diester derivative 6 in 62% yield. Hydrogenation of 6
over Pd/C in MeOH gave monoacid 7 in 66% yield.
tained was hydrogenated over Pd/C in EtOH to give
amine 9 in 72% yield. Treatment of 9 with Ac2O and for-
mic acid in dichloromethane afforded N-formated 10. In
the presence of aqueous HCHO, 9 was hydrogenated
over Pd/C in EtOH to give N,N-dimethyl amine 11 in
66% yield.
The chemo-selective amide bond cleavage of UK-2A
was expected to afford the nine-membered dilactone
amine B, which is a useful building block for UK-2A
derivatives. However, the acidic or basic hydrolysis of
amide bonds requires prolonged heating that could
decompose UK-2A. A survey of mild hydrolysis re-
agents led to the choice of phosphorus pentachloride
(PCl5), which has been applied to the removal of amide
side chains in the industrial scale manufacture of semi-
synthetic cephalosporins12 and penicillins.13
Then we were interested in introducing nitrogen func-
tionality into the benzene moiety of UK-2A (Scheme
2). Treatment of UK-2A with fumic HNO3 in dichloro-
methane at ꢀ20 °C achieved para-selective nitration of
the benzene ring in good yield. Compound 8 thus ob-
Treatment of 30-O-acetylated UK-2A (5) with PCl5–pyr-
idine (2 equiv of each) in dichloromethane, followed by
Scheme 2. Introduction of nitrogen functionality of the benzene ring of UK-2A.

Y. Usuki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2011–2014
2013
Scheme 3. Chemo-selective cleavage of the amide bond and condensation with o-hydroxyl aromatic acids.
heating under reflux for 1.5 h, afforded the imidoyl chlo-
ride intermediate, which was then converted to the imi-
no ether with MeOH. Further aqueous work-up gave B
as a p-toluenesulfonic acid salt in 51% yield along with
methyl picolinate derivative A in 21% yield. Amide form-
ation of B with various o-hydroxyl aromatic acids was
achieved in THF–pyridine with EDCÆHCl and HOBt in
modest to good yields. The structures of UK-2A deriva-
tives (12–17) prepared according to this method are
presented in Scheme 3.
UK-2A, suggesting that substitutions at the 50- and/or
60-position of picolinic acid are not effective promoters
of growth inhibitory activity.
In summary, chemical modifications of the 30-OH group
revealed that the free hydroxyl group at the 30 position is
necessary for the inhibitory activity in UK-2A as well as
AAs. Nitration at the para position of the benzene moi-
ety and chemo-selective cleavage of the amide bond were
achieved in good yields without decomposition of the
nine-membered dilactone ring of UK-2A. p-Amino deri-
vative 9 is a suitable precursor for photoaffinity-labeled
Compounds 3–1714 were then evaluated for in vitro anti-
fungal activity against Saccharomyces cerevisiae IFO
0203 by the conventional paper disk method using a test
medium composed of 1% yeast extract, 2% polypeptone,
3% glycerol, and 2% Bacto-agar. Results are summa-
rized in Table 1.
Table 1. Antifungal activity of UK-2A, AA, and their derivatives (3–
17)a
Compounds
Dose (lg/disc)
0.05 0.125
0.025
0.25
O-Alkylation products (3 and 4) did not inhibit the
growth of S. cerevisiae, while the O-acylated derivatives
5–7 showed inhibitory activity less than that exhibited
by UK-2A, suggesting that in vivo hydrolysis of 5–7
regenerates UK-2A, and the carboxylic group in 7 facili-
tated smooth reaction. The ether bonds in 3 and 4 are
chemically and enzymatically stable and may prevent
the regeneration of UK-2A in vivo. The free 30-OH
group is thus essential for the growth inhibitory activity
as reported earlier for AA.5 Among derivatives with a
substituent at the para position of the benzene ring (8–
11), the p-nitro derivative 8 showed the strongest inhibi-
tory activity; no relation between the electronegativity
or basicity of substituents and growth inhibition was
found. The replacement of a picolinic acid moiety by
salicylic acid derivatives resulted in a slight decrease in
the inhibition. 3-Hydroxy-2-quinoxalinecarboxylic acid
derivative 12 and 3-hydroxy-6-methoxypicolinic acid
derivative 13 showed inhibitory activity similar to
UK-2A (1)
AA (2)
19
16
0
22
18
0
26
19
0
26
26
0
3
4
0
0
0
0
5
10
8
12
10
16
15
0
14
15
21
18
8
18
18
24
20
10
0
6
7
12
12
0
8
9
10
11
12
13
14
15
16
17
0
0
0
0
11
18
19
12
8
15
20
24
16
11
16
12
19
24
27
17
12
17
14
14
16
0
8
8
12
8
8
a Values are stated in diameter of inhibitory zone around the discs
(mm).

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Y. Usuki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2011–2014
4. Shibata, K.; Hanafi, M.; Fujii, J.; Sakanaka, O.; Iinuma,
M.; Ueki, M.; Taniguchi, M. J. Antibiot. 1998, 51, 1113.
5. Tokutake, N.; Miyoshi, H.; Satoh, T.; Hatano, T.;
Iwamura, H. Biochim. Biophys. Acta 1994, 1185, 271.
6. Takimoto, H.; Akimoto, H.; Machida, K.; Ueki, M.;
Tanaka, T.; Taniguchi, M. J. Antibiot. 1999, 52, 480.
7. Machida, K.; Takimoto, H.; Miyoshi, H.; Taniguchi, M.
J. Antibiot. 1999, 52, 748.
8. For a review, see: Ueki, M.; Machida, K.; Taniguchi, M.
Curr. Opin. Anti-infective Invest. Drugs 2000, 2, 387.
9. Usuki, Y.; Tani, K.; Fujita, K.-I.; Taniguchi, M. J.
Antibiot. 2001, 54, 600.
analogues designed for elucidating the UK-2A binding
site on mitochondria.
Acknowledgements
This research was partially supported by Grant-in-Aids
for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology of Japan
(nos. 12556017 and 14580615), which are gratefully
appreciated.
10. Usuki, Y.; Goto, K.; Kiso, T.; Tani, K.; Ping, X.; Fujita,
K.-I.; Taniguchi, M. J. Antibiot. 2002, 55, 607.
11. Fujita, K.-I.; Kiso, T.; Usuki, Y.; Tanaka, T.; Taniguchi,
M. J. Antibiot. 2004, 57, 687.
References and notes
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2. Hanafi, M.; Shibata, K.; Ueki, M.; Taniguchi, M. J.
Antibiot. 1996, 49, 1226.
3. Ueki, M.; Taniguchi, M. J. Antibiot. 1997, 50, 1052.
13. Weissenburger, H. W. O.; van der Hoeven, M. G. Recl.
Trav. Chim. Pays-Bas 1970, 89, 1081.
14. The identity and purity of all new compounds were
1
established by H NMR and MS.