Beta-carboline alkaloids derived from the ascidian Synoicum sp.

Article abstract of DOI:10.1016/j.bmc.2012.05.002

Six β-carboline alkaloids (1-6) of the eudistomin Y class were isolated from the Korean ascidian Synoicum sp. These compounds were chemically converted to a known compound, eudistomin Y₁ (7) and six new derivatives, designated eudistomins Y₈-Y₁₃ (8-13). Several of these natural and synthetic compounds exhibited moderate to significant antimicrobial activity, weak cytotoxic activity, and inhibitory activities toward sortase A, isocitrate lyase, and Na⁺/K⁺-ATPase. Structure-activity relationships were also deduced.

Full text of DOI:10.1016/j.bmc.2012.05.002

Bioorganic & Medicinal Chemistry 20 (2012) 4082–4087
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Beta-carboline alkaloids derived from the ascidian Synoicum sp.
Tae Hyung Won a, Ju-eun Jeon a, So-Hyoung Lee b, Boon Jo Rho c, Ki-Bong Oh b, , Jongheon Shin a,
⇑
⇑
a Natural Products Research Institute, College of Pharmacy, Seoul National University, San 56-1, Sillim, Gwanak, Seoul 151-742, Republic of Korea
b Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University, San 56-1, Sillim, Gwanak, Seoul 151-921, Republic of Korea
c Department of Biological Science, College of Life Science, Ehwa Womans University, 52 Ehwayeodae-gil, Seodaemun, Seoul 120-750, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Six b-carboline alkaloids (1–6) of the eudistomin Y class were isolated from the Korean ascidian Synoicum
sp. These compounds were chemically converted to a known compound, eudistomin Y1 (7) and six new
derivatives, designated eudistomins Y8–Y13 (8–13). Several of these natural and synthetic compounds
exhibited moderate to significant antimicrobial activity, weak cytotoxic activity, and inhibitory activities
toward sortase A, isocitrate lyase, and Na+/K+ÀATPase. Structure–activity relationships were also
deduced.
Received 23 March 2012
Revised 30 April 2012
Accepted 2 May 2012
Available online 11 May 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
b-Carboline alkaloids
Eudistomin Y class
Synoicum sp.
Chemical modification
Bioactivity
1. Introduction
ate to significant cytotoxicity and antibacterial activity as well as
inhibitory activities toward enzymes, including sortase A (SrtA),
Marine ascidians (phylum Chordata, class Ascidiacea) are widely
recognized as prolific sources of structurally unique and biologi-
cally active alkaloids.1,2 Of these metabolites, b-carboline com-
pounds, represented by eudistomins, are frequently encountered
and have been harvested from various ascidians from several taxo-
nomical groups. Since the first isolation of eudistomins A–Q from
Eudistoma olivaceum,3 about thirty such compounds have been re-
ported from the genera Eudistoma,4–11 Lissoclinum,12 Pseudodistom-
a,13 Ritterella,14 and Synoicum.15 These compounds exhibited
significant bioactivities, including antimicrobial,6,16 antiviral,3,14
calmodulin antagonistic,5 and cytotoxic activities,8,10 and have at-
tracted considerable synthetic and biomedical interests.17–21 In
our search for the bioactive compounds from marine invertebrates,
we encountered purple-colored ascidian Synoicum sp., which was
harvested from Korean coastal waters. The organic extract obtained
isocitrate lyase (ICL), and Na+/K+ÀATPase.
2. Results/discussion
The ascidian specimens were lyophilized, macerated, and
repeatedly extracted with MeOH and CH2Cl2. The combined
extracts were separated by solvent-partitioning followed by
reversed-phase flash chromatography. The polar fractions were
separated by reversed-phase HPLC to yield six compounds as yel-
low amorphous solids. The structures of the major (3, 4, and 5)
and minor compounds (1, 2, and 6) were identified as eudistomins
Y2–Y7 by combined spectroscopic analyses. These compounds were
recently reported as antimicrobial b-carboline alkaloids from the
ascidian Eudistoma sp. The spectroscopic data of these compounds
were in good agreement with those reported in the literature.11
The significant bioactivity of eudistomin extracts prompted us
to synthetically derivatize the major extract constituents. A direct
attempt to debrominate 4 as a means of producing eudistomin Y1
(7), a bromine-lacking eudistomin from Eudistoma sp., using H2
and Pd/C resulted in concomitant debromination and reduction
to yield eudistomin Y8 (8), a new hydroxy-containing derivative.
Compound 7 was subsequently prepared by oxidation of the hy-
droxy group of 8 (Scheme 1). The spectroscopic data of this com-
pound agree with those in the literature.11
from the exhibited moderate cytotoxicity (LC50 39.0 lg/mL) against
the A549 cell line. Bioactivity-guided separation of the extract using
multiple chromatographic methods yielded cytotoxic eudistomins
Y2–Y7 (1–6). The biomedical importance of these compounds
prompted us to pursue chemical derivatization as a means of deter-
mining structure–activity relationships. We report here the isola-
tion and preparation of eudistomins Y1 (7) and Y8–Y13 (8–13).
Several of these natural and synthetic compounds exhibited moder-
Compounds 3–5 were reduced to the corresponding hydroxy-
containing eudistomins Y9–Y11 (9–11) by treating with NaBH4
(Scheme 2). The optical rotations were zero for compounds 8–11,
⇑
Corresponding authors. Tel.: +82 2 880 2484; fax: +82 2 762 8322 (J.S.); tel.: +82
2 880 4646; fax: +82 2 873 3112 (K.-B.O.).
E-mail addresses: ohkibong@snu.ac.kr (K.-B. Oh), shinj@snu.ac.kr (J. Shin).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.002

T. H. Won et al. / Bioorg. Med. Chem. 20 (2012) 4082–4087
4083
N
N
N
a
b
O
OH
O
N
H
N
H
N
H
Br
Br
HO
HO
HO
4
8
7
Scheme 1. For debromination and oxidation. Reagents and conditions: (a) H2, Pd/C, MeOH, Et3N, rt, 18 h; (b) PDC, CH2Cl2/THF, rt, 3 h.
Isocitrate lyase (ICL) is an enzyme that transforms isocitrate
into glyoxylate in the glyoxylate cycle. Relative to the wild type,
the microbial virulence of Candida albicans is significantly reduced
in mutant strains that lack ICL.26 The expression of glyoxylate cycle
genes is detected during specific stages of the interaction between
host and pathogen in a variety of pathogenic bacteria and fungi.
Therefore, the development of specific inhibitors against ICL is an
attractive prospect.27 Compounds 1–13 were evaluated for inhibi-
tion of recombinant C. albicans ICL in accordance with a previous
procedure.28 The inhibitory potencies (IC50) of the tested com-
pounds are shown in Table 1 and are compared to that of a known
R1
H
N
R1
H
N
a
O
OH
N
H
N
H
Br
Br
R2
R2
HO
HO
R1=Br,
R1=H,
R1=Br,
R1=Br,
R1=H,
R1=Br,
3
4
5
R2=H
9
R2=H
R2=Br
R2=Br
R2=Br
R2=Br
10
11
ICL inhibitor, 3-nitropropionic acid (IC50 38.6
7, and 8 showed moderate inhibition, with IC50 values of 50.2,
48.2, and 68.9 M, respectively. Unfortunately, the inhibitory
activities of the other seven compounds could not be determined
(ND in Table 1). It is because the ICL assay spectrophotometrically
measures the formation of glyoxylate phenylhydrazone by
monitoring the solution absorbance at 324 nm in the presence of
phenylhydrazine and isocitrate.29 However, several of the evalu-
ated compounds absorbed strongly at 324 nm, resulting in non-lin-
ear assay absorbance curves.
lM). Compounds 1,
Scheme 2. For reduction. Reagents and conditions: NaBH4, THF, rt, 4 h.
l
which implies a non-stereoselective attack of the hydride at the
carbonyl.
In addition, eudistomin Y13 (13), a new derivative that lacks
substituents on its phenyl ring, was prepared from 9 by a two-step
reaction using Tos-Cl and LiAlH4. The tosylated eudistomin Y12 (12)
was produced as an intermediate (Scheme 3). All the noble syn-
thetic compounds were structurally identified using LRESIMS,
HRFABMS, and NMR analyses. Protons and carbon atoms were ade-
quately assigned by combined 1-D and 2-D NMR methods (Supple-
mentary data).
To assess their potential as therapeutic agents, eudistomins
(1–6) and their derivatives (7–13) were screened for bioactivity.
They were first tested for in vitro cytotoxicity against the cancer
cell line A549 using a MTT assay.22 With the exception of 9 (LC50
In addition, the inhibitory activities of compounds 1–13 against
porcine cerebral cortex Na+/K+ÀATPase were measured fluoromet-
rically.28 The method monitors the formation of fluorescent
3-O-methylfluorescein from the parent compound 3-O-methylfluo-
rescein phosphate.30 The inhibitory potencies (IC50) of the tested
compounds are shown in Table 1 and are compared to that of oua-
bain, a known Na+/K+ÀATPase inhibitor (IC50 6.3
lM). b-Carboline
alkaloids 2–6 were strong inhibitors, with IC50 values of 7.5–
17.9
lM), all of the compounds reported herein exhibited weak
to no activity against this cell line (LC50 3.3
lM for doxorubicin)
22.5 lM. Compound 3 exhibited a similar activity (IC50 7.5 lM) as
(Table 1). However, this indicates that a previous report, which sta-
ted that 1–7 showed no cytotoxicity, may need reconsideration.11
Sortase A (SrtA) is an enzyme that catalyzes the attachment of
surface proteins to the peptidoglycan cell layer in Gram-positive
bacteria, including Staphylococcus aureus.23 Surface proteins not
only promote interactions between invading pathogens and animal
tissues, but also provide strategies for bacterial escape from the
host’s immune response. Therefore, SrtA has been regarded as a
promising target for the development of efficient antibacterial
agents.24 The inhibitory activity of compounds 1–13 against
recombinant SrtA from S. aureus ATCC 6538p were examined.25
Compounds 3 and 4 exhibited marginal inhibitory activity (IC50
that of ouabain. These findings revealed that the bromine group at
the C-13 position of compound 3 was important (Fig. 1). For exam-
ple, compound 7, which does not contain a bromine at the C-13 po-
sition, was inactive with an IC50 of 346.9 lM. Bromination at the C-6
position of compound 3, as in compounds 5, 9, and 11, resulted in
higher inhibitory activities than that of the debrominated com-
pound 10. Also, the presence of a hydroxy group at C-14 was impor-
tant for the bioactivity of 9, which exhibited a higher inhibitory
activity (IC50 33.3
and no substituent, respectively, at this position. These two com-
pounds were inactive with IC50 values of 166.0 and 283.1 M,
respectively. Taken together, these results suggest that the Na+/
lM) than 12 and 13, which had a tosyl group
l
163.2 and 146.4
lM, respectively) (Table 1).
Br
N
Br
N
OH
a
N
H
b
OH
N
H
9
Br
TosO
12
Scheme 3. For tosylation and dehydroxlation. Reagents and conditions: Tos-Cl, CH2Cl2, Et3N, rt, 2 h; LiAlH4, THF, rt, 1 h.
13

4084
T. H. Won et al. / Bioorg. Med. Chem. 20 (2012) 4082–4087
Table 1
Results of bioactivity test
Compound
MIC(
l
g/mL)
A549
SrtA
IC50
ICL
Na+/
K+ÀATPase
Gram (+) bacterium
Gram (À) bacterium
Fungus
I
LC50
(n = 3)
65.6 0.6
(
lM)
(l
M)
IC50 (lM) IC50 (lM)
A
B
C
D
E
F
G
H
J
(n = 3)
(n = 3)
(n = 3)
1
2
3
4
5
6
7
8
9
10
11
12
50
25
25
50
6.25
0.39
25
>100
100
50
100
50
>100
>100
>100
>100
>100
>100
50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
50
>100
>100
>100
>100
>100
>100
100
6.25
>272.3
50.2 0.7 209.3 3.1
>272.3
12.5
3.125 0.78
12.5
12.5
1.56
6.25
0.39
1.56
0.78
0.78
>100
>100
>100
12.5
>100 252.7 0.1 >272.3
22.5 0.6
7.5 0.3
19.8 0.5
10.1 0.4
11.3 0.8
>100 48.4 3.1
>100 85.4 3.3
>100 60.0 3.5
>100 59.0 0.2
163.2 2.4 NDc
146.4 1.9 ND
6.25
1.56
3.125 0.78
>100
100
6.25
25
3.125 3.125 0.78
1.56
1.56
1.56
>100
100
0.39
0.78
>100
>100
>100
12.5
>190.5
>190.5
>346.9
>344.4
>223.2
>223.2
>189.8
>166.0
>283.1
ND
ND
50
>100
100
>100
25
>100
>100
>100
>100
100
>100
>100
50
59.0 2.4
48.2 0.8 >346.9
68.9 1.1 >344.4
ND
ND
ND
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100 >344.4
>100 17.9 1.1
>100 >223.2
>100 >189.8
>100 63.3 1.8
>100 49.0 0.7
>100
12.5
33.3 0.9
122.1 2.2
32.8 1.0
>166.0
6.25
>100
>100
0.39
3.125 3.125 3.125 3.125
>100
100
0.78
>100
100
0.39
>100
>100
0.78
>100
>100
0.39
>166.0
>283.1
13
>283.1
Ampicillin
Amphotericin B
Doxorubicin
pHMBa
3-NPb
6.25
0.78
0.78
0.39 0.39
3.3 0.1
111.6 1.5
38.6 0.9
Ouabain
6.3 0.1
A: Staphylococcus aureus (ATCC 6538p), B: Bacillus subtilis (ATCC 6633), C: Micrococcus luteus (IFO 12708), D: Salmonella typhimurium (ATCC 14028), E: Proteus vulgaris (ATCC
3851), F: Escherichia coli (ATCC 35270), G: Aspergillus fumigatus (HIC 6094), H: Trichophyton rubrum (IFO 9185), I: Trichophyton mentagrophytes (IFO 40996), J: Candida albicans
(ATCC 10231).
a
para-Hydroxymercuribenzoic acid.
3-Nitropropionic acid.
Due to the high initial absorbance, inhibition was not accurately measured.
b
c
3
4
than compounds 9–11, which contain a hydroxy group at the same
position. The reason for this increased activity is not clear. In assays
for antifungal activity against medically important pathogenic fun-
gi, fungal inhibition was observed only with compounds 1 and 7,
which exhibited potent anti-Candida and moderate antifungal
activity, respectively.
5
8
4a
R1
H
R1
R2
6
7
4b
N
N
1
9a
OH
O
10
N
N
8a
H
H12
11
16
R2
R3
13
15
14
R4
R4
R3
HO
3. Conclusion
R1=Br,
R1=H,
R1=Br,
R1=H,
R1=Br,
R1=H,
R1=H,
R3=H,
R4=H
R1=H, R2=H, R3=OH,
R1=Br, R2=Br, R3=OH,
R2=H,
R2=H,
R2=H,
R2=H,
R2=H,
R2=Br,
R2=H,
R4=H
R4=H
R4=Br
R4=Br
1
2
3
4
5
6
7
8
R3=Br, R4=H
R3=Br, R4=H
R3=Br, R4=Br
R3=Br, R4=Br
9
R1=H,
R1=Br,
R1=Br,
R1=Br,
R2=Br,
R2=Br,
R2=Br,
R3=OH,
Six b-carboline alkaloids (1–6) of the eudistomin Y class were
isolated from the Korean ascidian Synoicum sp. These compounds
were chemically converted to a known compound, eudistomin Y1
(7) and six new derivatives, designated eudistomins Y8–Y13
(8–13). Structure–activity relationships were determined for these
compounds during the course of screening for bioactivity. These
findings enhance the potential of these compounds for use as ther-
apeutic leads.
10
11
12
R3=OH,
R3=O-Tos,
4
R =H
R3=Br, R4=Br 13
R2=H, R3=H,
R4=H
R3=H,
R4=H
Figure 1. Structures of compounds 1–13.
K+ÀATPase inhibitory activities of these eudistomins were altered
by bromination at C-6 and C-13 and hydroxylation at C-14. The
replacement of the C-10 carbonyl by a hydroxy group also affected
inhibitory activity. Compounds 3–5, which contain carbonyl groups,
exhibited IC50 values much lower than those of their corresponding
alcohols 9–11.
The in vitro antimicrobial activities of eudistomins (1–13) were
assessed against three Gram-positive bacteria, three Gram-negative
bacteria, and four species of fungi.31 The minimum inhibitory con-
centrations (MICs) of the tested compounds are shown in Table 1.
Compounds 3–6 and 11 exhibited strong inhibitory activities
against Gram-positive and Gram-negative bacteria, with the excep-
4. Experimental
4.1. General experimental procedures
Optical rotations were measured on a JASCO P-1020 polarime-
ter using a 1 cm cell. IR spectra were recorded on a JASCO 4200
FT-IR spectrometer, using a ZnSe cell. UV spectra were acquired
with HITACHI U-3010 spectrophotometer. NMR spectra were re-
corded in DMSO-d6 solutions containing Me4Si as an internal stan-
dard, on Bruker Avance 500, 600, Jeol JNM-LA 300, and Varian
Gemini 2000 spectrometers. Electrospray ionization source (ESI)
low resolution mass spectra were recorded on an Agilent Technol-
ogies 6130 Quadrupole mass spectrometer with an Agilent
Technologies 1200 series HPLC. High resolution mass spectromet-
ric data were obtained at the Korea Basic Science Institute (Daegu,
Korea) and were acquired using a JEOL JMS 700 mass spectrometer
tion of E. coli, with MIC values ranging from 0.39 to 6.25 lg/mL, as
shown compared with ampicillin. In the current series, an increase
in the number of bromine atoms per molecule resulted in an in-
crease in antibacterial activity. Compounds 3–6, 10, and 11, which
contain two or three bromine atoms, were more active than the
other compounds. Interestingly, compounds 3–5, which contain a
carbonyl group at C-10, exhibited higher antibacterial activities
with meta-nitrobenzyl alcohol as
Semi-preparative high performance liquid chromatography (HPLC)
a matrix for the FABMS.

T. H. Won et al. / Bioorg. Med. Chem. 20 (2012) 4082–4087
4085
was performed on a Spectrasystem p2000 equipped with a refrac-
tive index detector (Spectrasystem RI-150) and UV/Vis detector
(Gilson UV/VIS 151). All solvents used were spectroscopic grade
or distilled from glass prior to use.
4.4.1. Preparation of eudistomin Y1 (7)
To a stirred mixture of 3.0 mg of 8 in 1.0 mL of dry CH2Cl2–THF
(99:1) was added 7.5 mg of PDC. The mixture was stirred under N2
for 3 h. Celite was added and the resulting mixture was stirred for
5 min. The mixture was filtered through Celite under vacuum. The
residue was concentrated under reduced pressure and purified by
HPLC (YMC ODS-A column, 1 Â 25 cm, 2.0 mL/min, H2O–MeOH
(23:77 with 0.01% TFA)) to yield the pure compound 7 (2.1 mg)
at retention time 12 min; LRESI-MS m/z 289.2 [M+H]+; HRFABMS
m/z 289.0978 [M+H]+ (calcd for C18H13O2N2, 289.0977).
4.2. Animal material
Specimens of Synoicum sp. (sample number 09CH-11) were col-
lected by hand with scuba equipment at a depth of 20 m off the
coast of Chuja-do, Korea on November 4, 2009. The morphological
features of these purple-colored specimens were identical to those
described for the Synoicum ascidian that was confirmed by B.J.R., an
author of this paper. The colonial tunicates are up to 20 mm high
and up to 50 mm in maximum dimention, rounded, cuchion-
shaped, sessile and fixed by a small part of the basal surface, and
zooids are said to have been bright dark red when alive. Zooids
form circular system of 1 mm diameter with up 6–8 zooids around
a central clonial cavity. Contracted zooids are nearly 7 mm long, of
which the thorax 1 to 1.5 mm and the posterior abdomen is at least
three times that length. Each branchial siphon has 6 lobes and the
atrial siphon protruding, has small pointed lobes around its poster-
ior rim. The thorox is especially large, with 16–18 stigmental rows,
the gut loop is moderately short, the stomach is large almost
spherical and smooth walled. Gonad unknown. The voucher
specimens are deposited at the Natural History Museum, Ehwa
Womans University under the curatorship of B.J.R.
4.4.2. Preparation of eudistomin Y8 (8)
To a stirred mixture of 10.0 mg of 4 and 0.02 mL of Et3N in 2.0 mL
of dry MeOH was added 10.0 mg of Pd/C under ice-cooling. The mix-
ture was stirred and hydrogenated using H2 at room temperature
for 18 h. The mixture was filtered through Celite and silica gel under
vacuum. The residue was concentrated under reduced pressure and
purified by HPLC (YMC ODS-A column, 1 Â 25 cm, 2.0 mL/min, gra-
dient from H2O–MeOH (50:50 with 0.01% TFA) to (0:100 with 0.01%
TFA)) to yield the pure compound 8 (4.5 mg) at retention time
11 min; UV (MeOH) kmax (log
e
) 208 (2.93), 236 (2.93), 290 (2.59),
356 (2.01) nm; IR (ZnSe) mmax 3232, 1509, 1241 cmÀ1
;
1H NMR
(DMSO-d6) d 11.22 (1H, s, NH), 8.22 (1H, d, J = 5.1 Hz, H-3), 8.18
(1H, br d, J = 7.8 Hz, H-5), 7.97 (1H, d, J = 5.1 Hz, H-4), 7.73 (1H, br
d, J = 7.8 Hz, H-8), 7.51 (1H, dd, J = 7.8, 7.8 Hz, H-7), 7.36 (2H, d,
J = 8.7 Hz, H-12, H-16), 7.20 (1H, dd, J = 7.8, 7.8 Hz, H-6), 6.65 (2H,
d, J = 8.7 Hz, H-13, H-15), 6.03 (1H, s, H-10); 13C NMR (DMSO-d6) d
156.3 (C-14), 148.0 (C-1), 140.6 (C-8a), 136.7 (C-3), 134.1 (C-11),
132.0 (C-9a), 128.5 (C-4a), 127.8 (C-7), 127.5 (C-12, C-16), 121.3
(C-5), 120.4 (C-6), 119.0 (C-4b), 114.7 (C-13, C-15), 113.4 (C-4),
112.5 (C-8), 75.6 (C-10); LRESI-MS m/z 291.2 [M+H]+; HRFABMS
m/z 291.1131 [M+H]+ (calcd for C18H15O2N2, 291.1134).
4.3. Chemicals
Chemical reagents were obtained from commercial sources and
used directly without further purification. Sodium borohydride
was purchased from Fluka Organic Chemicals. p-Toluenesulfonyl
chloride was purchased from Junsei Chemical Co. Lithium alumi-
num hydride (1.0 M solution in tetrahydrofuran), pyridinium
dichromate, Pd/C (palladium, 10 wt % [dry basis] on activated car-
bon), and all other chemicals were purchased from Sigma-Aldrich
Chemical Co.
4.4.3. Preparation of eudistomin Y9 (9)
To a stirred mixture of 16.0 mg of 3 in 4.0 mL of THF was added
2.5 mg of NaBH4 under ice-cooling. The mixture was stirred under
N2 gas at room temperature for 4 h. To quench the reaction, NH4Cl
and water were added under ice-cooling. The mixture was parti-
tioned using EtOAc and water, the former was dried over anhy-
drous MgSO4. The EtOAc fraction was filtered and the residue
was concentrated under reduced pressure, purified by HPLC
(YMC ODS-A column, 1 Â 25 cm, 2.0 mL/min, gradient from H2O–
MeOH (50:50 with 0.01% TFA) to (0:100 with 0.01% TFA)) to yield
the pure compound 9 (13.3 mg) at retention time 15 min; UV
4.4. Extraction and isolation
The fresh collected specimens were frozen immediately and
kept at À25 °C until chemical investigations. The specimens were
lyophilized (2.0 kg dry weight), macerated, and extracted repeat-
edly with MeOH (3 L Â 3) and CH2Cl2 (3 L Â 2). The combined or-
ganic extract (160.95 g) was partitioned between n-BuOH and
H2O, and then the n-BuOH layer (15.83 g) was repartitioned be-
tween 15% aqueous MeOH (12.27 g) and n-hexane (3.56 g). The
15% aqueous MeOH layer was subjected to C18 reversed-phase vac-
uum flash chromatography using sequential mixtures of MeOH
and H2O as eluents (6 fractions in gradient, H2O–MeOH, from
50:50 to 0:100 and finally acetone. The H2O–MeOH (20:80)
fraction (0.39 g) was separated by C18 reversed-phase semi-pre-
parative HPLC (YMC ODS-A column, 1 Â 25 cm, 2.0 mL/min,
H2O–MeOH (35:65)) to yield, in order of elution, compounds 4, 5,
6, and 2 at retention times 14, 24, 25, and 69 min, respectively.
The H2O–MeOH (10:90) fraction (0.59 g) was separated by C18 re-
versed-phase semi-preparative HPLC (YMC ODS-A column,
1 Â 25 cm, 2.0 mL/min, H2O–MeOH (20:80)) to yield, in order of
elution, compounds 2, 1, and 3 at retention times 22, 23, and
39 min, respectively. Final purifications of these metabolites were
then accomplished by reversed-phase HPLC (YMC ODS-A column,
1 Â 25 cm, 2.0 mL/min, H2O–MeOH (20:80, 0.01% TFA)) to yield
compounds 1, 2, 3, 4, 5, and 6, at retention times 23, 22, 35, 34,
62, and 58 min, respectively. The overall purified yields were 2.7,
7.6, 119.0, 77.5, 78.3, and 5.0 mg for 1–6, respectively.
(MeOH) kmax (log
e
) 210 (2.91), 234 (2.97), 286 (2.63), 338 (2.12)
nm; IR (ZnSe) mmax 3225, 1482, 1247 cmÀ1
;
1H NMR (DMSO-d6) d
11.43 (1H, s, NH), 8.47 (1H, d, J = 1.8 Hz, H-5), 8.25 (1H, d,
J = 5.1 Hz, H-3), 8.05 (1H, d, J = 5.1 Hz, H-4), 7.70 (1H, d,
J = 8.4 Hz, H-8), 7.69 (1H, d, J = 1.8 Hz, H-12), 7.64 (1H, dd, J = 8.4,
1.8 Hz, H-7), 7.31 (1H, dd, J = 8.4, 1.8 Hz, H-16), 6.84 (1H, d,
J = 8.4 Hz, H-15), 6.03 (1H, s, H-10); 13C NMR (DMSO-d6) d 153.0
(C-14), 147.8 (C-1), 139.3 (C-8a), 137.2 (C-3), 135.8 (C-11), 132.4
(C-9a), 130.5 (C-7), 130.4 (C-12), 127.7 (C-4a), 126.6 (C-16),
124.0 (C-5), 122.3 (C-4b), 115.9 (C-15), 114.6 (C-8), 114.0 (C-4),
111.1 (C-6), 108.8 (C-13), 74.9 (C-10); LRESI-MS m/z 447.0/449.0/
451.0 [M+H]+; HRFABMS m/z 448.9320 [M+H]+ (calcd for
C
18H13O2N279Br81Br, 448.9324).
4.4.4. Preparation of eudistomin Y10 (10)
To a stirred mixture of 3.8 mg of 4 in 1.0 mL of THF was added
0.6 mg of NaBH4 under ice-cooling. The mixture was stirred under
N2 gas at room temperature for 4 h. To quench the reaction, NH4Cl
and water were added under ice-cooling. The mixture was parti-
tioned using EtOAc and water, the former was dried over anhydrous
MgSO4. The EtOAc fraction was filtered and the residue was concen-
trated under reduced pressure, purified by HPLC (YMC ODS-A

4086
T. H. Won et al. / Bioorg. Med. Chem. 20 (2012) 4082–4087
column, 1 Â 25 cm, 2.0 mL/min, gradient from H2O–MeOH (50:50
604.9 [M+H]+; HRFABMS m/z 602.9409 [M+H]+ (calcd for
25H19O4N2S79Br81Br, 602.9413).
with 0.01% TFA) to (0:100 with 0.01% TFA)), to yield the pure com-
C
pound 10 (3.1 mg) at retention time 16 min; UV (MeOH) kmax (log
210 (2.80), 232 (2.68), 288 (2.34), 344 (1.91) nm; IR (ZnSe) mmax
3438, 1627, 1476 cmÀ1 1H NMR (DMSO-d6) d 11.34 (1H, s, NH),
e)
4.4.7. Preparation of eudistomin Y13 (13)
;
To a stirred mixture of 8.0 mg of 12 in 1.0 mL of dry THF was
add 0.25 mL of LiAlH4 under ice-cooling. The mixture was stirred
under N2 for 1 h. To quench the reaction, 0.25 mL of water,
0.25 mL of 15% NaOH, and 0.75 mL of water were added step by
step under ice-cooling and Celite was added. The resulting mixture
was dried over anhydrous MgSO4 and filtered through Celite under
vacuum. The residue was concentrated under reduced pressure
and purified by HPLC (YMC ODS-A column, 1 Â 25 cm, 2.0 mL/
min, gradient from H2O–MeOH (60:40 with 0.01% TFA) to (0:100
with 0.01% TFA)), to yield the pure compound 13 (3.1 mg) at reten-
8.26 (1H, d, J = 5.1 Hz, H-3), 8.21 (1H, br d, J = 7.8 Hz, H-5), 8.01
(1H, d, J = 5.1 Hz, H-4), 7.72 (1H, br d, J = 7.8 Hz, H-8), 7.71 (2H, s,
H-12, H-16), 7.53 (1H, dd, J = 7.8, 7.8 Hz, H-7), 7.21 (1H, dd, J = 7.8,
7.8 Hz, H-6), 6.07 (1H, s, H-10); 13C NMR (DMSO-d6) d 149.8
(C-14), 146.6 (C-1), 140.6 (C-8a), 137.7 (C-11), 137.0 (C-3), 132.1
(C-9a), 129.9 (C-12, C-16), 128.7 (C-4a), 128.0 (C-7), 121.4 (C-5),
120.3 (C-6), 119.2 (C-4b), 113.8 (C-4), 112.5 (C-8), 111.7 (C-13,
C-15), 73.9 (C-10); LRESI-MS m/z 446.9/448.9/450.9 [M+H]+;
HRFABMS m/z 448.9320 [M+H]+ (calcd for C18H13O2N279Br81Br,
448.9324).
tion time 20 min; UV (MeOH) kmax (log
e) 208 (2.67), 236 (2.76),
290 (2.36), 356 (1.80) nm; IR (ZnSe) mmax 3300, 1623, 1274 cmÀ1
;
4.4.5. Preparation of eudistomin Y11 (11)
1H NMR (DMSO-d6) d 11.43 (1H, s, NH), 8.45 (1H, d, J = 1.7 Hz, H-
5), 8.26 (1H, d, J = 5.2 Hz, H-3), 8.04 (1H, d, J = 5.2 Hz, H-4), 7.70
(1H, d, J = 8.7 Hz, H-8), 7.63 (1H, dd, J = 8.7, 1.7 Hz, H-7), 7.59
(2H, d, J = 7.5 Hz, H-12, H-16), 7.28 (2H, dd, J = 7.5, 7.5 Hz, H-13,
H-15), 7.18 (1H, t, J = 7.5 Hz, H-14), 6.14 (1H, s, H-10); 13C NMR
(DMSO-d6) d 148.0 (C-1), 143.5 (C-11), 139.3 (C-8a), 137.1 (C-3),
132.4 (C-9a), 130.4 (C-7), 128.0 (C-12, C-16), 127.7 (C-4a), 127.0
(C-14), 126.2 (C-13, C-15), 124.0 (C-5), 122.3 (C-4b), 114.6 (C-8),
113.9 (C-4), 111.1 (C-6), 76.1 (C-10); LRESI-MS m/z 353.1/355.1
[M+H]+; HRFABMS m/z 353.0287 [M+H]+ (calcd for C18H14O2N279Br,
353.0289).
To a stirred mixture of 3.9 mg of 5 in 1.0 mL of THF was added
1.0 mg of NaBH4 under ice-cooling. The mixture was stirred under
N2 gas at room temperature for 4 h. To quench the reaction, NH4Cl
and water were added under ice-cooling. The mixture was parti-
tioned using EtOAc and water, the former was dried over anhy-
drous MgSO4. The EtOAc fraction was filtered under vacuum, the
residue was concentrated under reduced pressure and purified
by HPLC (YMC ODS-A column, 1 Â 25 cm, 2.0 mL/min, gradient
from H2O–MeOH (40:60 with 0.01% TFA) to (0:100 with 0.01%
TFA)) to yield the pure compound 11 (3.1 mg) at retention time
14 min; UV (MeOH) kmax (log
e
) 208 (2.53), 240 (2.37), 292 (2.02),
358 (1.56) nm; IR (ZnSe) mmax 3418, 1474, 1273 cmÀ1
;
1H NMR
4.5. Biological assays
(DMSO-d6) d 11.47 (1H, s, NH), 9.84 (1H, s, 14-OH), 8.49 (1H, d,
J = 1.8 Hz, H-5), 8.28 (1H, d, J = 5.1 Hz, H-3), 8.08 (1H, d,
J = 5.1 Hz, H-4), 7.71 (2H, s, H-12, H-16), 7.70 (1H, d, J = 8.7 Hz,
H-8), 7.66 (1H, dd, J = 8.7, 1.8 Hz, H-7), 6.68 (1H, d, J = 4.5 Hz,
10-OH), 6.07 (1H, d, J = 4.5 Hz, H-10); 13C NMR (DMSO-d6) d
149.6 (C-14), 147.0 (C-1), 139.3 (C-8a), 137.8 (C-11), 137.3 (C-3),
132.4 (C-9a), 130.5 (C-7), 129.9 (C-12, C-16), 127.8 (C-4a), 124.0
(C-5), 122.2 (C-4b), 114.6 (C-8), 114.2 (C-4), 111.6 (C-13, C-15),
111.2 (C-6), 74.1 (C-10); LRESI-MS m/z 524.9/526.9/528.9/530.9
Cytotoxicity assays were performed in accordance with litera-
ture protocols.22 Antimicrobial assays were performed according
to the method described previously.31 Isocitrate lyase, sortase A,
and Na+/K+ÀATPase inhibition assays were performed according
to previously described methods.25,28,30
Acknowledgments
[M+H]+;
18H11O2N279Br81Br2, 528.8410).
HRFABMS m/z
528.8409
[M+H]+
(calcd
for
We are grateful to the Basic Science Research Institute in Daegu,
Korea, for providing mass data. T.H.W. and J.-e.J. are recipients of a
fellowship from the Ministry of education, Korea, through the Brain
Korea 21 Project. This study was partially supported by National Re-
search Foundation of Korea (NRF)Grants, funded by the Korean gov-
ernment (MEST, Ministry of Education, Science and Technology)
(Nos. 20110030638 and 20110018562).
C
4.4.6. Preparation of eudistomin Y12 (12)
To a stirred mixture of 10.5 mg of 9 and 0.025 mL of Et3N in
3.0 mL of dry CH2Cl2 was added 7.0 mg of Tos-Cl. The mixture
was stirred under N2 for 2 h. After quenched by using 0.1 N HCl,
NaHCO3 and water, the mixture was partitioned using CH2Cl2
and water. The former layer was dried over anhydrous MgSO4
and filtered under vacuum. The residue was concentrated under
reduced pressure and purified by HPLC (YMC ODS-A column,
1 Â 25 cm, 2.0 mL/min, gradient from H2O–MeOH (30:70 with
0.01% TFA) to (0:100 with 0.01% TFA)) to yield the pure compound
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.002.
12 (10.3 mg) at retention time 18 min; UV (MeOH) kmax (log
(2.77), 230 (2.79), 292 (2.29), 358 (1.73) nm; IR (ZnSe) mmax 3425,
1480, 1175 cmÀ1 1H NMR (DMSO-d6) d 11.55 (1H, s, NH), 8.49
e) 208
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