Synthesis and absolute configuration of acanthodendrilline, a new cytotoxic bromotyrosine alkaloid from the Thai marine sponge acanthodendrilla sp.

Article abstract of DOI:10.1248/cpb.c15-00901

Acanthodendrilline (1), a new bromotyrosine alkaloid, was isolated from the Thai marine sponge Acanthodendrilla sp. The structure of 1 was fully characterized by spectroscopic analysis, in agreement with the synthesized compound used to resolve the single

Full text of DOI:10.1248/cpb.c15-00901

258
Chem. Pharm. Bull. 64, 258–262 (2016)
Vol. 64, No. 3
Regular Article
Synthesis and Absolute Configuration of Acanthodendrilline, a New
Cytotoxic Bromotyrosine Alkaloid from the Thai Marine Sponge
Acanthodendrilla sp.
Nachanun Sirimangkalakitti,a Masashi Yokoya,b Supakarn Chamni,a Pithi Chanvorachote,c
,b
,a
Anuchit Plubrukrn,d Naoki Saito,* and Khanit Suwanborirux*
a Center for Bioactive Natural Products from Marine Organisms and Endophytic Fungi (BNPME), Department
of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University;
b
Pathumwan, Bangkok 10330, Thailand: Graduate School of Pharmaceutical Sciences, Meiji Pharmaceutical
c
University; 2–522–1 Noshio, Kiyose, Tokyo 204–8588, Japan: Cell-Based Drug and Health Product Development
Research Unit and Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn
d
University; Pathumwan, Bangkok 10330, Thailand: and Department of Pharmacognosy and Pharmaceutical Botany,
Faculty of Pharmaceutical Sciences, Prince of Songkla University; Hat-Yai, Songkhla 90112, Thailand.
Received November 12, 2015; accepted December 8, 2015
Acanthodendrilline (1), a new bromotyrosine alkaloid, was isolated from the Thai marine sponge Acan-
thodendrilla sp. The structure of 1 was fully characterized by spectroscopic analysis, in agreement with the
synthesized compound used to resolve the single chiral center at C-11. Total synthesis of the enantiomers of
1 allowed for the comparison of specific rotation values and hence the determination of the absolute configu-
ration as 11-S. Cytotoxicity evaluation revealed that (S)-1 exhibited approximately three-fold more potent
cytotoxicity against the human non-small cell lung cancer H292 cell line than (R)-1.
Key words bromotyrosine alkaloid; absolute configuration; asymmetric synthesis; cytotoxicity; Acanthoden-
drilla sp.
Bromotyrosine alkaloids are a large group of secondary preparative HPLC to afford 1 (Fig. 2).
metabolites and commonly found in marine sponges.1) The re-
Compound 1, obtained as a colorless amorphous powder,
markable diversities of their chemical structures, together with was optically active [α]D25 +25.8 (c=0.1, MeOH). The appear-
the wide range of bioactivities, including antimicrobial,2–6) ance of the protonated molecular parent ion at m/z 450.9512
antiviral,7) antiprotozoal,8) cytotoxic,3,4,9–12) anti-inflammato- [M+H]+ (Calcd for 450.9504) in the high resolution (HR)-
ry,13) and enzyme inhibitory activities,14–16) make them highly FAB-MS of 1 suggested that the molecular formula was
attractive to both natural product chemists and molecular C14H16Br2N2O5. The 1H-NMR, 13C-NMR, and heteronuclear
biologists. In the course of our search for bioactive second- multiple bond connectivity (HMBC) spectra of 1 (Table 1)
ary metabolites from Thai marine organisms, we recently indicated that 1 possessed a 3,5-dibromotyramine skeleton:
reported the isolation of a series of bromotyrosine alkaloids signals assignable to two aromatic protons [δH 7.36 (s, H-2,
from the Thai marine sponge Acanthodendrilla sp. along with 6)], six aromatic carbons [δC 138.3 (C-1), δC 133.1 (C-2, 6), δC
homoaerothionin, a potent cholinesterase inhibitor17) (Fig. 1). 118.0 (C-3, 5), δC 150.6 (C-4)], and two methylenes [δH 2.75
In addition, several bromotyrosine alkaloids isolated from this (t, J=6.8Hz, H2-7)/δC 34.9 (C-7), δH 3.40 (td, J=6.8, 6.3Hz,
sponge, including verongiaquinol, aerothionin, 11-oxoaero- H2-8)/δC 41.8 (C-8)] were observed. The presence of a meth-
thionin, and 11,19-dideoxyfistularin-3 (Fig. 1), were previously ylcarbamate moiety was suggested by the typical chemical
reported by other research groups as cytotoxic agents against shifts of OCH3 (δC 52.2) and C-9 (δC 157.0) in the 13C-NMR
several cancer cell lines.3,4,12,18)
spectrum and confirmed by the HMBC correlation from the
Further investigation of the crude ethyl acetate (EtOAc) methoxy protons [δH 3.69 (s, OCH3)] to C-9. The connectiv-
extract from this sponge resulted in the isolation of a new ity of the dibromotyramine and carbamate moieties was
1
bromotyrosine alkaloid, acanthodendrilline (1, Fig. 2). In this confirmed by the H–1H correlation spectroscopy (COSY) cor-
work, we describe the structure elucidation of 1 based on relations of H2-7/H2-8/9-NH and the HMBC correlation from
spectroscopic techniques and synthetic protocols. The enantio- H2-8 to C-9. In addition, a 5-methyl-2-oxazolidone moiety [δH
mers of 1 were prepared from commercially available starting 4.21 (d, J=5.1Hz, H2-10)/δC 71.9 (C-10), δH 5.04 (ddt, J=8.7,
materials and used to determine the absolute configuration. 5.9, 5.1Hz, H-11)/δC 74.2 (C-11), δH 3.93 (dd, J=8.7, 5.9Hz,
We also report the cytotoxicity of the enantiomers to human Ha-12) and δH 3.83 (t, J=8.7Hz, Hb-12)/δC 42.6 (C-12), δH 5.64
non-small cell lung cancer H292 and normal human keratino- (br, 13-NH)/δC 159.3 (C-13)] was deduced from the 1H–1H
cyte HaCaT cell lines.
COSY correlations of H2-10/H-11/Ha,b-12 and the HMBC cor-
relations from H-11, Ha,b-12, and 13-NH to C-13. The HMBC
correlation from H2-10 to C-4 suggested the connectivity of
Results and Discussion
The EtOAc extract of the Thai marine sponge Acanthoden- the dibromotyramine and 2-oxazolidone moieties. Selected
drilla sp.17) was repeatedly subjected to silica gel column chro- 1H–1H COSY and HMBC correlations are illustrated in Fig.
matography, Sephadex® LH-20 column chromatography, and 3. Thus, 1 was elucidated as methyl-[2-(3,5-dibromo-4-{[2-
*To whom correspondence should be addressed. e-mail: naoki@my-pharm.ac.jp; khanit.s@pharm.chula.ac.th
© 2016 The Pharmaceutical Society of Japan

Vol. 64, No. 3 (2016)
Chem. Pharm. Bull.
259
oxo-1,3-oxazolidin-5-yl]methoxy}phenyl)ethyl]carbamate and methyl chloroformate at 0°C provided 4 in 56% yield. Com-
named acanthodendrilline. Our spectroscopic studies depicted mercially available (S)- and (R)-epichlorohydrins (5) were
that 1 contained a stereogenic center at C-11. However, the ab- separately transformed in one step into the respective enan-
solute configuration of 1 remained ambiguous. Therefore, we tiomers 5-chloromethyl-2-oxazolidinone [(S)-6 and (R)-6, each
decided to access the S and R enantiomers of 1 by chemical ca. 40% yield] using potassium cyanate as the nucleophile
synthesis. Both synthetic enantiomers could serve as standards to transform the oxirane ring into the oxazolidinone motif.20)
for the comparison of chemical and physical properties.
Both (S)-6 and (R)-6 were finally coupled with 4 by base-
The four-step synthesis of (S)-1 and (R)-1 is shown in Fig. mediated nucleophilic substitution to furnish (S)-1 (48% yield)
4. Tyramine (2) was used as the starting material to prepare and (R)-1 (37% yield), respectively.
1
methyl-[2-(3,5-dibromo-4-hydroxyphenyl)ethyl]carbamate (4)
The H-NMR data of the synthetic enantiomers were identi-
in two steps. First, 2 was brominated by tetrabutylammonium cal to that of natural 1. Determination of the specific rotation
tribromide to give 3,5-dibromotyramine19) (3) in 63% yield. of the enantiomers clarified that (S)-1 {[α]D25 +21.4 (c=0.1,
Subsequent acylation of 3 with a stoichiometric amount of MeOH)} and natural 1 {[α]D25 +25.8 (c=0.1, MeOH)} pos-
sessed almost identical optical rotation values, whereas the
optical rotation of (R)-1 was the opposite {[α]D25 −19.7 (c=0.3,
MeOH)}. Thus, the absolute configuration at C-11 of natural 1
was unambiguously determined as S. Interestingly, measure-
ment of the circular dichroism (CD) spectra of 1 and the syn-
thetic enantiomers yielded no characteristic absorptions.
The cytotoxicity of the synthetic enantiomers was evaluated
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay.21) The results (Table 2) indicated that
(S)-1 (IC50 58.5µM) was approximately threefold more potent
Fig. 2. Structure of Acanthodendrilline (1)
Fig. 1. Structures of Bioactive Bromotyrosine Alkaloids Isolated from
the Thai Marine Sponge Acanthodendrilla sp.
1
Fig. 3. Selected H–1H COSY (
) and HMBC ( ) Correlations of 1
Table 1. 1H- and 13C-NMR Data of 1 in CDCl3
Position
δH (Multiplicity)
δC, Type
HMBC correlation from H to C
C-2,6/C-3,5/C-4/C-7
1
2, 6
3, 5
4
138.3 (C)
133.1 (CH)
118.0 (C)
150.6 (C)
34.9 (CH2)
41.8 (CH2)
157.0 (C)
71.9 (CH2)
74.2 (CH)
42.6 (CH2)
7.36 (2H, s)
7
2.75 (2H, t, J=6.8)
3.40 (2H, td, J=6.8, 6.3)
C-1/C-2,6/C-8
C-1/C-7/C-9
8
9
10
11
12
4.21 (2H, d, J=5.1)
C-4/C-11/C-12
C-13
5.04 (1H, ddt, J=8.7, 5.9, 5.1)
a 3.93 (1H, dd, J=8.7, 5.9)
b 3.83 (1H, t, J=8.7)
C-10/C-11/C-13
13
159.3 (C)
9-NH
13-NH
OCH3
4.82 (1H, br)
5.64 (1H, br)
3.69 (3H, s)
C-11/C-12/C-13
C-9
52.2 (CH3)
Chemical shifts (δ) are expressed in ppm, and coupling constants (J) are presented in Hz.

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Chem. Pharm. Bull.
Vol. 64, No. 3 (2016)
Fig. 4. Synthetic Scheme of (S)-1 and (R)-1
that (S)-1 was approximately threefold more cytotoxic to the
human non-small cell lung cancer H292 cell line than (R)-1.
This biological information clearly emphasized the importance
of the stereochemistry at C-11 of 1 as a key feature related to
the cytotoxicity.
Table 2. Cytotoxicity of Enantiomers of 1 to Cancer (H292) and Normal
(HaCaT) Cells
IC50 S.D. (µM)
Compound
H292
HaCaT
(S)-1
58.5 6.7
173.5 24.7
7.5 0.6
>400
>400
4.6 0.9
Experimental
(R)-1
General Experimental Procedures 1H- and 13C-NMR
spectra were measured on Bruker Avance DPX-300, JEOL
JNM-AL 300, and JEOL JNM-AL 400 NMR spectrometers.
Cisplatin
than (R)-1 (IC50 173.5 µM) against the H292 cell line. Interest- The solvent signals served as the internal standard (CDCl3: δH
ingly, both enantiomers were not cytotoxic to normal HaCaT 7.26/δC 77.0). IR spectra were recorded on a Shimadzu IRAf-
cell line (IC50 >400µM). Moreover, the acetylcholinesterase finity-1 Fourier transform-infrared (FT-IR) spectrophotometer.
(AChE) inhibitory activity of the synthetic enantiomers was HR-MS were acquired with a JEOL JMS 700 mass spectrom-
determined by the modified Ellman’s method.22,23) Both (S)-1 eter. Optical rotation and circular dichroism measurements
and (R)-1 showed weak inhibitory activity toward AChE (23% were carried out on a Horiba SEPA-200 polarimeter and a
and 25% enzyme inhibition at 100µM, respectively).
Jasco J-820 spectropolarimeter, respectively. HPLC was per-
formed on a Shimadzu apparatus equipped with an LC-20AB
binary pump, a Rheodyne 7125 injector port, and an SPD-20A
Conclusion
Acanthodendrilline (1), a new bromotyrosine alkaloid, UV/Vis detector. Silica gel 60F254 aluminum sheets were
was isolated from the Thai marine sponge Acanthodendrilla used for TLC. Spots on TLC chromatograms were detected
sp. It is the first example of a bromotyrosine alkaloid that is under UV light (254nm) and by spraying with p-anisaldehyde
composed of dibromotyramine, methylcarbamate, and 2-oxa- reagent. Silica gel 60 (230–400 and 70–230 mesh) and Sepha-
zolidone moieties in the molecule. The concise synthesis of dex® LH-20 were used for column chromatography.
the enantiomers of 1 was accomplished from commercial ty-
Extraction and Isolation The EtOAc extract (67.81g)
ramine and (S)- and (R)-epichlorohydrins. By comparing the previously prepared from the Thai marine sponge Acantho-
specific rotation values of natural 1 and the synthetic enantio- dendrilla sp.17) was fractionated with the following chro-
mers, the stereocenter at C-11 of natural 1 was assigned the matographic steps: SiO2 (70% EtOAc in n-hexane; then 100%
S configuration. Both enantiomers exhibited weak AChE in- EtOAc), SiO2 (50% EtOAc in n-hexane), Sephadex LH-20
hibitory activity. Interestingly, cytotoxicity evaluation revealed (MeOH), SiO2 (4% MeOH in CH2Cl2), and C18 RP-HPLC

Vol. 64, No. 3 (2016)
Chem. Pharm. Bull.
261
(LiChrospher® 100 RP-18, 10µm, 250×10mm, 65% MeOH in to give a residue (320mg). Chromatography on a silica gel
H2O, flow rate 3.0mL/min, UV detector 230nm), to yield 1 column with n-hexane–EtOAc (1:2) as the eluent gave (R)-6
1
(43.1mg, 0.0006% yield of sponge wet weight).
(272.8mg, 40%) as a colorless powder. H-NMR (300MHz,
Acanthodendrilline (1)
CDCl3) δ: 6.37 (1H, brs, NH), 4.92–4.81 (1H, m), 3.76 (1H,
Colorless amorphous powder. 1H-NMR (300MHz) and td, J=9.2, 0.7Hz), 3.72–3.70 (2H, m), 3.54 (1H, ddd, J=9.2,
13C-NMR (75MHz), see Table 1. IR (KBr) cm−1: 3329, 2950, 5.9, 0.9Hz).20) HR-EI-MS m/z 135.0086 [M]+ (Calcd for
1751, 1705, 1541, 1466, 1449, 1256, 1094, 739. UV λmax C4H6ClNO2: 135.0087). [α]D25 −9.9 (c=1.0, CHCl3).
(MeOH) nm (logε): 214 (4.37). HR-FAB-MS m/z: 450.9512
[M+H]+ (Calcd for C14H17Br2N2O5: 450.9504). [α]D25 +25.8 din-5-yl]methoxy}phenyl)ethyl]carbamate
(c=0.1, MeOH). (21.7mg, 160µmol) in N,N-dimethylformamide (DMF)
Methyl-[2-(3,5-dibromo-4-{[(5S)-2-oxo-1,3-oxazoli-
[(S)-1] (S)-6
4-(2-Aminoethyl)-2,6-dibromophenol (3) Tetrabutylam- (4.0mL) was added to a solution of 4 (43.5mg, 123µmol).
monium tribromide [(n-Bu)4NBr3, 14.1g, 29.9mmol] was K2CO3 (204mg, 1.48mmol) was added, and the reaction
added to a solution of tyramine (2.0g, 14.6mmol) in CH2Cl2 mixture was stirred at 140°C for 45min. The mixture was
(105mL) and MeOH (70mL). The reaction mixture was stirred filtered, and the filtrate was diluted with H2O (5mL) and then
at room temperature for 30min. After the solvent was re- extracted with CHCl3 (3×30mL). The combined extract was
moved in vacuo, the residue was suspended in EtOAc–CHCl3 washed with brine (20mL), dried, and concentrated in vacuo
(1:1), and the precipitate was filtered off and washed with to give a residue (60mg). Chromatography on a silica gel col-
CH2Cl2. Compound 3 was obtained as a pale yellow powder umn with n-hexane–EtOAc (1:2–1:1) as the eluent gave (S)-1
(2.71g, 63%). 1H-NMR (300MHz, CD3OD) δ: 7.43 (2H, s), (26.8mg, 48%) as a colorless powder. 1H-NMR (400MHz,
3.14 (2H, t, J=7.7Hz), 2.86 (2H, t, J=7.7 Hz).19) HR-electron CDCl3) δ: 7.35 (2H, s), 5.79 (1H, br), 5.02 (1H, ddt, J=8.7, 6.2,
ionization (EI)-MS m/z 292.9045 [M]+ (Calcd for C8H9Br2NO: 5.0Hz), 4.82 (1H, br), 4.20 (2H, d, J=5.0Hz), 3.91 (1H, dd,
292.9051).
J=8.7, 6.2Hz), 3.82 (1H, t, J=8.7Hz), 3.67 (3H, s), 3.39 (2H, q,
Methyl-[2-(3,5-dibromo-4-hydroxyphenyl)ethyl]carba- J=6.7Hz), 2.74 (2H, t, J=6.7Hz). HR-FAB-MS m/z 450.9498
mate (4) Methyl chloroformate (ClCO2CH3, 47.7mL, [M+H]+ (Calcd for C14H17Br2N2O5: 450.9504). [α]D25 +21.4
0.59mmol) was added to a solution of 3 (151mg, 0.51mmol) (c=0.1, MeOH).
in tetrahydrofuran (THF) (2mL), H2O (2mL), and NaHCO3
(135mg, 1.61mmol) at 0°C. After stirring for 1.5h, the reac- 5-yl]methoxy}phenyl)-ethyl]carbamate
Methyl-[2-(3,5-dibromo-4-{[(5R)-2-oxo-1,3-oxazolidin-
[(R)-1] (R)-6
tion mixture was diluted with EtOAc (20mL) and then ex- (21.7mg, 160µmol) in DMF (4.0mL) was added to a solu-
tracted with 1N NaOH aq. (2×10mL). The combined alkaline tion of 4 (43.5mg, 123µmol). K2CO3 (204mg, 1.48mmol)
solution was acidified with 1N HCl aq. and then extracted was added, and the reaction mixture was stirred at 140°C for
with CHCl3 (3×10mL). The combined extract was washed 1h. The reaction mixture was filtered, and the filtrate was
with brine (10mL), dried, and concentrated in vacuo to give a diluted with H2O (5mL) and then extracted with chloroform
residue (103mg). Chromatography on a silica gel column with (3×30mL). The combined extract was washed with brine
n-hexane–EtOAc (2:1) as the eluent gave 4 (100.3mg, 56%) as (20mL), dried, and concentrated in vacuo to give a residue
1
a colorless powder. H-NMR (400MHz, CDCl3) δ: 7.28 (2H, (60mg). Chromatography on a silica gel column with n-hex-
s), 4.87 (1H, brs), 3.67 (3H, s), 3.38 (2H, q, J=6.8Hz), 2.71 ane–EtOAc (1:1) as the eluent gave (R)-1 (20.5mg, 37%) as
1
(2H, t, J=6.8Hz). 13C-NMR (100MHz, CDCl3) δ: 157.0 (s), a colorless solid. H-NMR (400MHz, CDCl3) δ: 7.35 (2H, s),
148.1 (s), 133.3 (s), 132.1 (d), 109.9 (s), 52.2 (q), 42.0 (t), 34.7 5.75 (1H, br), 5.02 (1H, ddt, J=8.7, 6.1, 5.1Hz), 4.81 (1H, br),
(t). HR-EI-MS m/z 350.9104 [M]+ (Calcd for C10H11Br2NO3: 4.20 (2H, d, J=5.1Hz), 3.91 (1H, dd, J=8.7, 6.1Hz), 3.82 (1H,
350.9106).
t, J=8.7Hz), 3.67 (3H, s), 3.39 (2H, q, J=6.6Hz), 2.74 (2H,
(5S)-5-(Chloromethyl)-1,3-oxazolidin-2-one [(S)-6] (S)- t, J=6.6Hz). HR-FAB-MS m/z 450.9507 [M+H]+ (Calcd for
Epichlorohydrin [(S)-5, 390µL, 4.99mmol] and MgSO4 (1.2g, C14H17Br2N2O5: 450.9504). [α]D25 −19.7 (c=0.3, MeOH).
10mmol) were added to a solution of potassium cyanate
Cytotoxicity Cytotoxicity to the human non-small cell
(KOCN, 811mg, 10mmol) in H2O (5mL). After stirring at lung cancer NCI-H292 cell line (ATCC, Manassas, VA,
100°C for 5h, the reaction mixture was diluted with H2O U.S.A.) and the normal human keratinocyte HaCaT cell line
(5mL) and then extracted with EtOAc (3×20mL). The com- (Cell Lines Service, Eppelheim, Germany) was investigated
bined extract was washed with brine (20mL), dried, and by performing the MTT assay.21) H292 cells and HaCaT cells
concentrated in vacuo to give a residue (304mg). Chromatog- were seeded onto 96-well plates at the densities of 2.5×103
raphy on a silica gel column with n-hexane–EtOAc (1:2) as and 1×104 cells/well, respectively, and were treated with vari-
the eluent gave (S)-6 (275.7mg, 41%) as a colorless powder. ous concentrations of samples dissolved in the culture medium
1H-NMR (300MHz, CDCl3) δ: 6.37 (1H, brs, NH), 4.92–4.81 containing not more than 0.5% dimethyl sulfoxide (DMSO)
(1H, m), 3.76 (1H, td, J=9.2, 0.7Hz), 3.72–3.70 (2H, m), 3.54 for 72h. The detailed experimental procedure was described
(1H, ddd, J=9.2, 5.9, 0.9Hz).20) HR-EI-MS m/z 135.0087 [M]+ in our previous study.17) Cell viability was calculated with
(Calcd for C4H6ClNO2: 135.0087). [α]D25 +10.0 (c=1.0, CHCl3).
(5R)-5-(Chloromethyl)-1,3-oxazolidin-2-one [(R)-6] (R)- GraphPad Prism (GraphPad Software, U.S.A.).
respect to non-treated control cells. IC50 was determined using
Epichlorohydrin [(R)-5, 390µL, 4.99mmol] and MgSO4
Acetylcholinesterase Inhibitory Activity Inhibitory ac-
(1.2g, 10mmol) were added to a solution of KOCN (811mg, tivity toward acetylcholinesterase from electric eel (Elec-
10mmol) in H2O (5mL). After stirring at 100°C for 5h, the trophorus electricus) was determined by the modified Ell-
reaction mixture was diluted with H2O (5mL) and then ex- man’s method.22,23) Details of the experimental procedure were
tracted with EtOAc (3×20mL). The combined extract was described in our previous report.17)
washed with brine (20mL), dried, and concentrated in vacuo

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Chem. Pharm. Bull.
2936–2937 (1996).
Vol. 64, No. 3 (2016)
Acknowledgments Financial support was provided by
10) Tabudravu J. N., Jaspars M., J. Nat. Prod., 65, 1798–1801 (2002).
11) Tran T. D., Pham N. B., Fechner G., Hooper J. N., Quinn R. J., J.
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the Royal Golden Jubilee Ph.D. Program Grant No.
PHD/0276/2552 (N.Si.), by the Meiji Pharmaceutical Univer-
sity Asia/Africa Center for Drug Discovery (MPU-AACDD),
and by the Thailand Research Fund (A.P.).
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col. Immunotoxicol., 34, 919–924 (2012).
Conflict of Interest The authors declare no conflict of
interest.
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Supplementary Materials The online version of this ar-
1
ticle contains supplementary materials. H-NMR, 13C-NMR,
1H–1H COSY, HSQC, and HMBC spectra, and HR-FAB-MS
1
of natural 1, and H-NMR spectra of synthetic (S)-1 and (R)-1
are available.
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