Makomotindoline from Makomotake, Zizania latifolia infected with Ustilago esculenta

Article abstract of DOI:10.1016/j.bmcl.2012.05.021

Makomotindoline (1) was isolated from Makomotake, Zizania latifolia infected with Ustilago esculenta. The structure was determined by the interpretation of spectroscopic data and synthesis. Makomotindoline (1), its l-Glc isomer (2) and its aglycon (3) wer

Full text of DOI:10.1016/j.bmcl.2012.05.021

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4246–4248
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Bioorganic & Medicinal Chemistry Letters
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Makomotindoline from Makomotake, Zizania latifolia infected
with Ustilago esculenta
Tomohiro Suzuki a, , Jae-Hoon Choi a, , Takumi Kawaguchi b, Kimiko Yamashita a, Akio Morita b,
b
c
d
d
d,
⇑
¯
Hirofumi Hirai , Kaoru Nagai , Tomoyasu Hirose , Satoshi Omura , Toshiaki Sunazuka
,
Hirokazu Kawagishi a,b,
⇑
a Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
b Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
c Department of Epigenetic Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi 409-3898, Japan
d Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Makomotindoline (1) was isolated from Makomotake, Zizania latifolia infected with Ustilago esculenta. The
Received 11 April 2012
Revised 7 May 2012
Accepted 8 May 2012
Available online 17 May 2012
structure was determined by the interpretation of spectroscopic data and synthesis. Makomotindoline
(1), its
L-Glc isomer (2) and its aglycon (3) were synthesized and their effects on rat glioma cells showed
adverse effects on the cell growth.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Makomotake
Ustilago esculenta
Structure determination
Glioma cell
Synthesis
The fungus, Ustilago esculenta, penetrates into the aquatic
perennial grass, Zizania latifolia. After penetration, the fungus in-
cites the formation of an edible gall and inhibits inflorescence
and seed production in the plant. The gall is called Makomotake
in Japanese and Jiaobai in Chinese, and an edible product in Japan,
China and other Asian countries. We have already reported osteo-
clast-forming suppressive nortriterpenes from it1 and in the course
of further search for new compounds from Makomotake, we suc-
ceeded in purifying a novel N-glucoside. Here we describe the iso-
lation, structure determination, synthesis and biological activity of
the compound and its analogues.
Fresh Makomotake was extracted with EtOH and then with ace-
tone. After the solutions had been combined and concentrated un-
der reduced pressure, the concentrate was partitioned between
CH2Cl2 and H2O, and then the H2O soluble part was partitioned be-
tween EtOAc and H2O. The EtOAc soluble part was fractionated by
repeated chromatography, resulting in compound 1.
Figure 1. HMBC correlations in 1.
indicating the presence of seven degrees of unsaturation in the
molecule. The structure of 1 was elucidated by interpretation of
NMR spectra3 including DEPT, COSY, HMQC, and HMBC (Fig. 1).
The complete assignment of the protons and carbons was accom-
plished as shown in Table 1. The presence of the b-glucosyl group
was suggested by the characteristic chemical shifts and coupling
constants: dC 86.2 (C-10), 71.3 (C-20), 77.0 (C-30), 69.8 (C-40), 77.9
(C-50), 61.0 (C-60); dH 4.88 (d, J = 9.2 Hz, H-10), 3.68 (dd, J = 9.2,
Makomotindoline (1)2 was purified as a colorless amorphous
solid. Its molecular formula was determined as C16H21NO7 by HRE-
SIMS m/z 362.1191 [M+Na]+ (Calcd for C16H21NO7Na, 362.1216),3
⇑
Corresponding authors.
E-mail addresses: sunazuka@lisci.kitasato-u.ac.jp (T. Sunazuka), achkawa@ipc.
shizuoka.ac.jp (H. Kawagishi).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.021

T. Suzuki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4246–4248
4247
Table 1
1H and 13C NMR data for 1 and 2 (in D2O)
Position
1
2
1H
13C
1H
13C
(Multiplicity, J in Hz)
d
(Multiplicity, J in Hz)
d
Aglycone
2
3.57 (m)
4.02 (m)
2.33 (m)
67.8
3.59 (m)
4.05 (m)
2.45 (m)
68.3
3
40.6
87.8
39.9
87.8
3a
3b
4
5
6
131.2
124.9
121.6
130.9
109.6
148.5
99.7
130.2
125.3
121.1
131.3
108.2
148.7
98.4
7.33 (d, 7.3)
7.39 (dd, 7.6, 0.9)
6.93 (dd, 7,6, 7.3)
7.29 (ddd, 7.3, 7.9, 0.9)
6.78 (d, 7.9)
6.92 (dd, 7.3, 7.3)
7.25 (dd 7.3, 7.6)
6.83 (d, 7.6)
7
7a
8a
Sugar
10
5.59 (s)
5.51 (s)
4.88 (d, 9.2)
86.2
71.3
77.0
69.8
77.9
61.0
4.89 (d, 9.2)
85.5
70.1
78.0
69.9
78.0
61.2
20
3.68 (dd, 9.2, 9.5)
3.53 (dd, 9.5, 8.9)
3.39 (dd, 8.9, 9.5)
3.42 (m)
3.61 (dd, 12.2, 4.8)
3.73 (dd, 12.2, 1.6)
3.78 (dd, 9.2, 8.9)
3.59 (dd, 8.9, 8.8)
3.48 (dd, 8.8, 9.8)
3.54 (ddd, 9.9, 4.9, 2.1)
3.73 (dd, 12.5, 4.9)
3.83 (dd, 12.5, 2.1)
30
40
Scheme 2. Synthesis of 1 and 2 by N-glucosidation of 3.
50
60
the N-glucosidation of 3 with D- and L
-glucoses11–13 in quantitative
yields, respectively (Scheme 2). The NMR data and the specific
rotation (½a 2D1
ꢁ
ꢀ45, H2O, c 0.07)3 of synthetic 1 were completely
identical with those of natural 1, indicating that the absolute con-
figuration of 1 was as shown.
The effects of the compounds on C6 cell viability were ana-
lyzed.14 The natural product (1) showed no effect. However,
100
at 24 h (112.0 9.8%) and 48 h (114.2 11.0%) treatment. On the
other hand, -glucoside (2) significantly reduced the cell number
at 10 M (92.4 9.1%) and 100 M (83.9 8.0%) in 24 h treated
cells, and at 100 M (87.9 11.1%) in 48 h treated cells. Since dead
cells were hardly observed by the treatment with 2 (data not
shown), the reducing effect of the compound is thought to be
growth retardation. Glycosylated natural products such as sapo-
nins or glycoalkaloids have been reported to show anti-prolifera-
tive activity or apoptotic effect in mammalian cells, especially
cancer cells.15 Although they are expected to be utilized as anti-
cancer agents, some of them show cytotoxicity even in normal
cells. Tomato saponin called as tomatine, has been reported to be
cytotoxic, and its deglycosylation abolished the cytotoxicity.16 It
raises the possibility that each compound used in this study show
distinct effect on mammalian cells.
lM of the aglycone (3) significantly stimulated the cell growth
L
9.5 Hz, H-20), 3.53 (dd, J = 9.5, 8.9 Hz, H-30), 3.39 (dd, Hz, J = 8.9,
9.5 Hz, H-40), 3.42 (m, H-50), 3.61 (dd, J = 12.2, 4.8 Hz, H-6a0), 3.73
(dd, J = 12.2, 1.6 Hz, H-6b0). The presence of a 1,2-substituted phe-
nyl group (C-3b to C-7a) in the aglycon was also suggested by the
NMR data: dC 131.2 (C-3b), 124.9 (C-4), 121.6 (C-5), 130.9 (C-6),
109.6 (C-7), 148.5 (C-7a); dH 7.33 (d, J = 7.3 Hz, H-4), 6.92 (dd,
J = 7.3, 7.3 Hz, H-5), 7.25 (dd J = 7.3, 7.6 Hz, H-6), 6.83 (d,
J = 7.6 Hz, H-7). The structure of the other part, the condensed ring
part, in the aglycon and the linkage position with the phenyl were
elucidated by the correlations in the HMBC data (Fig. 1) and the
molecular formula. The N-glucosidic bond and its position between
the sugar and the aglycon were determined by the HMBC correla-
tions (H-8a/H-10, H-10/H-7a, H-10/H-8a, Fig. 1) and the relatively
high field chemical shift at C-10 (dC 86.2) compared with those of
O-glycosides.4,5
l
l
l
Acknowledgment
To confirm the planar structure and determine the whole abso-
lute configuration of 1, an optically active D-glucoside and L-gluco-
side, 1 and 2,6 were synthesized chemically. Previously, we
completed total synthesis of madindoline A, involving an asym-
metric oxidative ring-closure reaction of tryptophol to give 3 by
modified Sharpless epoxidation [Ti(O-i-Pr)4, (+)-DIPT, t-BuOOH,
CH2Cl2, ꢀ20 to ꢀ40 °C (Scheme 1).7,8 Interest in the synthetic
applications of 3 led us to develop to the asymmetric total synthe-
sis of other optically active indole alkaloid, (ꢀ)-physovenine,9 in a
concise manner.10 Two diastereomers, 1 and 2, were prepared by
We thank V.K. Deo (Shizuoka University) for valuable
discussion.
References and notes
1. Kawagishi, H.; Hota, K.; Masuda, K.; Yamaguchi, K.; Yazawa, K.; Shibata, K.;
Uzuka, N.; Matahira, Y. Biosci. Biotechnol. Biochem. 2006, 70, 2800.
2. The material, Makomotake, was purchased from local market in Shizuoka,
Japan. Fresh Makomotake (16.7 kg) was extracted with EtOH (22 L, 3 times)
and then with acetone (10 L, once). After the solutions had been combined and
concentrated under reduced pressure, the concentrate was partitioned
between CH2Cl2 and H2O, and then the H2O soluble part was partitioned
between EtOAc and H2O. The residue (20.0 g) obtained after removing EtOAc
was fractionated by silica gel flash column chromatography (80% CH2Cl2/
acetone, 50% CH2Cl2/MeOH, MeOH, each 2 L) to obtain seven fractions. Fraction
2 (eluted with 80% CH2Cl2/acetone, 1.28 g) was further separated by reversed-
phase HPLC (Develosil C30-UG, 15% MeOH, 5 mL/tube), and nine fractions were
obtained. Fraction 2–5 (65.7 mg) was further fractionated by reversed-phase
HPLC (Cosmosil cholester, 10% MeOH) to afford compound
1
(1.8 mg; Rt
77.6 min). Compound 1. mp 154–156 °C; ½a D28
ꢁ
ꢀ48 (c 0.30, H2O); IR (neat);
1486, 1608, 3367 cmꢀ1 1H and 13C NMR, see Table 1; ESIMS m/z 362 [M+Na]+;
;
Scheme 1. The enantioselective preparation of 3.
HRESIMS m/z 362.1191 [M+Na]+ (Calcd for C16H21NO7Na, 362.1216).

4248
T. Suzuki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4246–4248
3. 1H NMR spectra (one- and two-dimensional) were recorded on a JEOL lambda-
500 spectrometer at 500 MHz, while 13C NMR spectra were recorded on the
same instrument at 125 MHz. The HRESIMS spectra were measured on a JMS-
T100LC mass spectrometer. A JASCO grating infrared spectrophotometer was
used to record the IR spectra.
and concentrated. Flash chromatography (CHCl3/MeOH = 5/1) gave the
mixture of desired product and slight amount of -glucose, which was
further purified by Sep-Pak Plus C18 Cartridge (CH3CN/H2O = 1/9) to afford 1
(19.9 mg, 100%) as colorless solid. According to the procedure of 1,
(19.9 mg, 100%) was obtained as colorless solid from (10.3 mg,
0.0581 mmol) and -glucose (31.4 mg, 0.174 mmol) with acetic acid (10 L,
0.174 mmol) in EtOH (0.58 mL).
D
a
2
a
3
4. Ogata, M.; Hidari, K. I.; Kozaki, W.; Murata, T.; Hiratake, J.; Park, E. Y.; Suzuki, T.;
Usui, T. Biomacromolecules 2009, 10, 1894.
L
l
5. Ogata, M.; Murata, T.; Murakami, K.; Suzuki, T.; Hidari, K. I.; Suzuki, Y.; Usui, T.
Bioorg. Med. Chem. 2007, 15, 1383.
14. Rat glioma cell line C6 cells were maintained in the Dulbecco’s modified Eagles
medium (D-MEM, SIGMA, USA) supplemented with 10% fetal bovine serum
6. Compound 2. mp 131–132 °C; (½a D28
ꢁ
ꢀ12 (c 0.30, H2O); IR (neat); 1487, 1608,
(FBS), 10 U/mL penicillin, and 100 lg/mL streptomycin at 37 °C in humidified
3387 cmꢀ1
;
1H and 13C NMR, see Table 1; ESIMS m/z 362 [M+Na]+; HRESIMS m/
5% CO2 atmosphere. Cell number analysis was performed by 3-(4,5-dimethyl-
2-thiazolyl) 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. C6 cells were
cultured in 96 well plates at 2000 cells/well cell density. After one-day culture,
the cells were treated with serial concentrations of the compounds in the
medium without FBS. After 24 or 48 h incubation, the treated cells were
subjected to the MTT assay. The treatment medium was replaced to the
medium supplemented with 0.25 mg/mL of MTT without FBS, and the cells
were incubated for 2 h. The incubation was terminated by addition of 20% (w/
v) sodium dodecylsulfate (SDS) and 50% (v/v) dimethylformamide in water.
z 362.1191 [M+Na]+ (Calcd for C16H21NO7Na, 362.1216).
7. Sunazuka, T.; Hirose, T.; Shirahata, T.; Harigaya, Y.; Hayashi, M.; Komiyama, K.;
¯
Omura, S.; Smith, A. B., III J. Am. Chem. Soc. 2000, 122, 2122.
¯
8. Hirose, T.; Sunazuka, T.; Yamamoto, D.; Kaji, E.; Omura, S. Tetrahedron Lett.
2006, 47, 6761.
9. Robinson, B. J. Chem. Soc. 1964, 1503.
10. Sunazuka, T.; Yoshida, K.; Kojima, N.; Shirahata, T.; Hirose, T.; Handa, M.;
¯
Yamamoto, D.; Harigaya, Y.; Kuwajima, I.; Omura, S. Tetrahedron Lett. 2005, 46,
1459.
The absorbance at 570 nm of the reaction mixture was measured by a
11. Chisholm, J. D.; Van Vranken, D. L. J. Org. Chem. 2000, 65, 7541.
12. Messaoudi, S.; Sancelme, M.; Polard-Housset, V.; Aboab, B.; Moreau, P.;
Prudhomme, M. Eur. J. Med. Chem. 2004, 39, 453.
microplate reader (molecular devices, USA). The data were expressed as
means SD. The difference was analyzed by one-way ANOVA followed by
Tukey-Kramer post-hoc test. P values less than 0.05 was considered significant.
15. Christopher, B.; Silke, B.; Mark, S.; Diana, B.; Hendrik, F. Mini-Rev. Med. Chem.
2008, 8, 575.
13. To solution of
3 (9.3 mg, 0.0525 mmol) in dry EtOH (0.53 mL), D-glucose
(28.4 mg, 0.157 mmol) and acetic acid (9.0 mL, 0.157 mmol) at room
temperature were added and the mixture was warmed up to 80 °C with
stirring for 16 h. Then, the reaction mixture was cooled to room temperature
16. Friedman, M.; Levin, C. E.; Lee, S.-U.; Kim, H.-J.; Lee, I.-S.; Byun, J.-O.; Kozukue,
N. J. Agric. Food Chem. 2009, 57, 572.