Indole Alkaloids from the Sea Anemone Heteractis aurora and Homarine from Octopus cyanea

Article abstract of DOI:10.1002/cbdv.201400406

The two new indole alkaloids 2-amino-1,5-dihydro-5-(1H-indol-3-ylmethyl)-4H-imidazol-4-one (1), 2-amino-5-[(6-bromo-1H-indol-3-yl)methyl]-3,5-dihydro-3-methyl-4H-imidazol-4-one (2), and auramine (3) have been isolated from the sea anemone Heteractis auror

Full text of DOI:10.1002/cbdv.201400406

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Indole Alkaloids from the Sea Anemone Heteractis aurora and Homarine
from Octopus cyanea
a
b
b
b
by Kamel H. Shaker ), Matthias Gçhl ), Tobias Müller ), and Karlheinz Seifert* )
a) Chemistry Department, College of Science, King Khalid University, Abha, Saudia Arabia
b) Department of Organic Chemistry, NW II, University of Bayreuth, DE-95440 Bayreuth
(
phone: þ49-921-553098; fax: þ49-921-555358; e-mail: karlheinz.seifert@uni-bayreuth.de)
The two new indole alkaloids 2-amino-1,5-dihydro-5-(1H-indol-3-ylmethyl)-4H-imidazol-4-one (1),
-amino-5-[(6-bromo-1H-indol-3-yl)methyl]-3,5-dihydro-3-methyl-4H-imidazol-4-one (2), and aura-
2
mine (3) have been isolated from the sea anemone Heteractis aurora. Both indole alkaloids were
synthesized for the confirmation of the structures. Homarine (4), along with uracil (5), hypoxanthine (6),
and inosine (7) have been obtained from Octopus cyanea.
Introduction. – Sea anemones are ocean dwelling, solitary members of the phylum
Cnidaria and the class Anthozoa invertebrates. The phylum Cnidaria includes
anemones, corals, jellyfish, and hydras. Sea anemones have a basic radial symmetry
with tentacles surrounding a central mouth opening. The tentacles are used to catch
food and transfer it to their mouth. At the tentacles and other parts of the sea anemone
are stinging capsules (nematocysts) containing small threads that are forcefully ejected
along with the venom when stimulated mechanically or chemically. Sea anemones use
nematocysts for capturing prey, as well as for defence purposes against predators [1]
and in interspecies aggression [2]. The venom of sea anemones contains a variety of
þ
þ
active compounds like toxins affecting voltage-gated Na and K channels, acid-
sensing ion channels, pore-forming toxins (actinoporins), and protease inhibitors. The
use of toxins affecting voltage-gated ion channels seems logical since these targets are
an important component of the action potential in the signal transduction process of
vertebrates and invertebrates. Actinoporins are highly toxic to fish and crustaceans
which may be the natural prey of sea anemones [1].
The octopuses are cephalopods of the order Octopoda which are characterized by
their eight arms usually bearing suction cups. Octopuses are highly intelligent animals
whose behaviour is easily modified by experience. They live in many diverse regions of
the ocean, especially in holes in rocky areas from the interdidial area to at least 45 m.
Octopus cyanea occurs in the Indo-Pacific [3]. Octopuses use different toxins for prey
acquisition, as well as in defence. Cephalotoxins from posterior salivary glands of
Octopus vulgaris have a strong paralysing effect on crabs [4]. Tetrodotoxin appears to
be actively applied by the blue-ringed octopus Hapalochlaena spp. for acquisition of
prey. Tetrodotoxin, the major neurotoxin involved in envenoming, is secreted by the
two posterior salivary glands and inoculated during the bite. However, it was detected
predominantly in the arms representing about 45% of the body mass by weight,
ꢀ
2015 Verlag Helvetica Chimica Acta AG, Zürich

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1747
followed by the abdomen, where the posterior salivary glands are located, and the
cephalothorax [5].
In this article, the structure determination of the new natural indole alkaloids
1
and 2, together with auramine (3) from the sea anemone Heteractis aurora is
described. The structures of both indole alkaloids 1 and 2 were confirmed by synthesis.
Homarine (4), uracil (5), hypoxanthine (6), and inosine (7) were isolated from
Octopus cyanea.
Results and Discussion. – Isolation and Structure Determination of Compounds 1–
4
. The fresh yellow sea anemones of Heteractis aurora and a frozen octopus of Octopus
cyanea were cut, homogenized, and exhaustively extracted with EtOH. Both EtOH
extracts were concentrated to dryness. The extract of Heteractis aurora was submitted
to CC with SiO to afford three main fractions. The second fraction was chromato-
2
graphed over a further SiO column followed by PTLC (prep. thin layer chromatog-
2
raphy) to yield the compounds 1–3 (Fig.).
The EtOH extract of Octopus cyanea was successively partitioned between H O
2
and hexanes, AcOEt, and BuOH. The BuOH extract was separated over a SiO column
2
followed by PTLC to give homarine (4), uracil (5), inosine (6), and hypoxanthine (7)
(
Fig.).
Compound 1 was found to possess the molecular formula C H N O by HR-ESI-
1
2
12
4
þ
1
13
MS at m/z 229.1083 ([MþH] ). The H- and C-NMR data for 1 (Table 1) showed the
signals of five aromatic H-atoms (d(H) 6.96, 7.05, 7.14, 7.32, 7.53), five quaternary C-
atoms (d(C) 110.1, 127.4, 136.0, 171.9, 188.9), one CH group (d(C) 60.8), and one CH
2
1
13
group (d(C) 27.4). Comparison of the H- and C-NMR data of 1 with those reported
for methyl 2-(1H-indol-3-yl)-2-oxoacetate [6] and 2-amino-5-(3-aminopropyl)-1,5-
dihydro-4H-imidazol-4-one [7] let us propose a 3-substituted indole and a 5-substituted
2
-amino-1,5-dihydro-4H-imidazol-4-one as subunits for 1. The HMBCs HÀC(5’)/C(2’)
13
and HÀC(5’)/C(4’) confirm the C assignments of the imidazolinone ring. The HMBC
Figure. Structures of compounds 1–7

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Table 1. 1H- and 13C-NMR Data of Compounds 1 and 9. d in ppm, J in Hz. Arbitrary numbering, see
Scheme 1.
Position
1a)
9a)
d(H)
d(C)
d(H)
d(C)
2
3
7.14 (d, J¼2.3)
123.3
110.1
127.4
118.5
118.3
120.8
111.2
136.0
27.4
6.75 (d, J¼2.4)
123.8
106.9
127.6
118.4
118.1
120.7
111.2
135.7
24.8
3
4
5
6
7
7
8
a
7.53 ( d, J¼8.0)
7.27 ( d, J¼8.0)
6.96 (ddd, J¼8.0, 8.0, 1.1)
7.05 (ddd, J¼8.0, 8.0, 1.1)
7.32 (d, J¼8.0)
6.83 (ddd, J¼8.0, 8.0, 1.1)
7.00 (ddd, J¼8.0, 8.0, 1.1)
7.20 (d, J¼8.0)
a
2.78 (dd, J¼15.0, 7.2),
3.23 (dd, J¼14.8, 2.3),
3.38 (dd, J¼14.8, 5.5)
3
.11 (dd, J¼15.0, 4.0)
2
4
5
1
2
3
7
’
’
’
’’
’’,6’’
’’,5’’
’’
171.9
188.9
60.8
167.5
184.3
62.2
135.1
128.6
128.6
68.1
3.98–4.03 (m)
4.46–4.49 (m)
7.53–7.56 (m)
7.42–7.46 (m)
5.32 (d, J¼12.1),
5
.43 (d, J¼12.1)
8’’
150.8
a) Measured in (D )DMSO
6
interactions HÀC(5’)/C(8), HÀC(5’)/C(3), CH (8) (d(H) 3.11, 2.78)/C(3a), CH (8)/
2
2
C(2), CH (8)/C(3), and CH (8)/C(4’) revealed the connection between the indole
2
2
moiety in position 3 and the imidazolinone ring in position 5’ through the CH (8) group.
2
Therefore, compound 1 was identified as 2-amino-1,5-dihydro-5-(1H-indol-3-ylmeth-
yl)-4H-imidazol-4-one.
Compound 2 showed two ions at m/z 320 and 322 with relative intensities 100 :98
in the EI-MS suggesting the presence of one Br-atom. The molecular formula was
7
9
þ
established as C H BrN O by HR-ESI-MS (m/z 321.0347 ([MþH] )). The most
1
3
13
4
1
3
remarkable differences in the C-NMR data of the indole part of 2 in comparison with
were the upfield shift for the signals of C(6) (Dd¼ À7.1), the downfield shifts for the
1
signals of C(5) (Dd¼ þ3.0), and C(7) (Dd¼ þ2.7) due to the presence of Br-atom at
1
13
C(6) (Table 2). The H- and C-NMR data of the 6-bromoindole moiety of 2 showed
a good agreement with those reported for methyl 2-(6-bromo-1H-indol-3-yl)-2-
1
3
oxoacetate [6]. The C-NMR shifts in (D )DMSO of the signals C(2’) (d(C) 157.6)
6
and MeÀN(3’) (d(C) 25.7) of the imidazolinone ring of 2 were in good accordance with
those reported for the imidazolidinone ring of 6-bromo-2’-de-N-methylaplysinopsin
C(3’) (d(C) 157.7) and MeÀN(4’) (d(C) 25.6) [8]. The HMBCs MeÀN(3’)/C(2’),
MeÀN(3’)/C(4’), and HÀC(5’)/C(4’), HÀC(5’)/C(2’) confirm the position of the Me
1
3
group at 3’-position and the C assignment of the imidazolinone ring. The connection
between the indole and imidazolinone moieties through the CH (8) of 2 was evident
2
from the HMBCs of CH (8) (d(H) 3.08, 3.22)/C(4’), CH (8)/C(2), CH (8)/C(3),
2
2
2

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1749

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
CH (8)/C(3a), and HÀC(5’)/C(3). Compound 2 was identified as 2-amino-5-[(6-
2
bromo-1H-indol-3-yl)methyl]-3,5-dihydro-3-methyl-4H-imidazol-4-one, an aplysinop-
sin-type alkaloid.
Aplysinopsin has been isolated before from the sponge Aplysinopsis sp. [9].
Aplysinopsin-type indole alkaloids have previously been obtained from the sponge
Dercitus sp. [10], the scleractinian coral Dendrophyllia sp. [8], and the marine sponge
Hyrtios erecta [11]. These alkaloids showed a selective inhibitory activity against the
neuronal isozyme of nitric oxide synthase (nNOS). Nitric oxide (NO) is an important
second messenger and regulates many physiological processes, e.g. inflammation,
regulation of blood pressure, platelet adhesion, and neurotransmission. An excessive
production of NO causes different disease states such as post-ischemic stroke damage,
schizophrenia, development of colitis, tissue damage, and pathological inflammation.
Therefore, a rational control of NO production is assumed to be an efficient approach
to treat these diseases [11].
The molecular formula C H N of compound 3 was determined by HR-EI-MS at
1
7
21
3
þ
13
m/z 267.1736 (M ). The C-NMR data of 3 were in good agreement with those
reported for auramine [12], which is a yellow dye used in the manufacture of paints,
textiles, and rubber products. Auramine, together with rhodamine, is used as
fluorescent dye in the fluorescence microscopy to visualize acid-fast bacteria, like
those of Mycobacteria. Compound 3 shows bacteriostatic activity against Streptococcus
pyogenes and Staphylococcus aureus [13].
þ
The HR-ESI-MS (m/z 138.0549 ([MþH] )) of compound 4 led to a molecular
13
formula of C H NO . Comparison of the C-NMR data of 4 with those of homarine
7
7
2
[
14] showed full accord. Homarine is a fairly ubiquitous metabolite in marine
invertebrates [15] and has been detected before in the mantle, arms, and liver of
Octopus ochellatus [16] and urine of Octopus vulgaris [17]. It has been isolated also
from a lot of other organisms like the echiura Urechis unicintus [18], the ascidian
Eusynstyela latericius [19], and corals [14][15]. Homarine (4) has been suggested to
serve as an active constituent of the innate immune system in the octocoral Leptogorgia
virgulata. It may act synergistically with cofactors in this coral to mount a response to
microbial invasion and disease [14]. Homarine from Leptogorgia virgulata inhibited
significantly the growth of the fouling benthic marine diatom Navicula salinicola at
naturally occurring concentrations [20]. The antarctic soft coral Gersemia antarctica
produces homarine, which is released into the surrounding water column as predator
deterrent compound and antibacterial metabolite [15]. Perhaps homarine of Octopus
cyanea protects its body against bacterial infection.
MS, H- and 13C-NMR data of compounds 5–7 were identical with those of
authentic samples of uracil, hypoxanthine, and inosine. Hypoxanthine has also been
found before as homarine in the urine of Octopus vulgaris [17].
1
Synthesis of the Alkaloids 1 and 2. The preparation of both compounds 1 and 2 and
the comparison of their spectroscopic data with those of the natural products should
confirm the structures. A further aim of the synthesis should be the determination of
the absolute configuration of the two chiral compounds by comparison of the optical
rotations with authentic references.
Compound 1 was prepared through a simple two-step sequence based on a
literature-known procedure [21] (Scheme 1). Cbz-protected (Cbz¼(benzyloxy)car-

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1751
bonyl) l-tryptophan (8) was treated with NHS (N-hydroxysuccinimide) and DCC
N,Nꢀ-dicyclohexylcarbodiimide) to form an activated NHS-ester which was then
(
introduced in an aq. sodium cyanamide solution, furnishing the protected imidazoli-
none 9 (Scheme 1). Deprotection of 9 under hydrogenolytic conditions was carried out
two times independently and gave the desired compound 1 which was NMR
2
3
spectroscopically identical with the natural product 1. The [a] values of the both
D
synthetic samples were determined as À60.0 (MeOH) and–6.1 (MeOH), respectively,
which indicates a partial racemization at the stereogenic center C(5’) with (S)
2
3
configuration of 1. As the optical rotation of the natural compound 1 is [a] ¼ þ3.2
D
(
MeOH), its configuration must be (R). The synthesis of compound (R)-1 has been
described before in an U.S. patent [22].
Scheme 1. Synthesis of Compound 1
a) 1. THF, DCC, NHS, 08, 3 h. 2. NaHNCN, H O, r.t., 16 h; 62%. b) MeOH, Pd/C, H , r.t., 1 h; 83%.
2
2
The synthesis of alkaloid 2 started with a racemic mixture of N-acetyl-6-
bromotryptophan (10) which was treated with the enzyme l-aminoacyclase in a
literature-known procedure [23][24] (Scheme 2). N-Acetyl-6-bromo-d-tryptophan and
6
-bromo-l-tryptophan (11) were obtained, and the amino group of 11 was protected
with Boc CO (Boc¼t-Butyloxycarbonyl) to give 12. Compound 12 reacted in the same
2
way as 9 with NHS and DCC to an activated NHS-ester which formed with sodium
cyanamide the Boc-protected imidazolinone 13. Compound 13 was deprotected to 14
with SnCl in MeCN. The imidazolinone 14 was methylated with MeI in MeCN/EtOH
4
1
:1, and the desired product 2 was obtained. The NMR spectra of the natural and the
synthetic alkaloid 2 were identical. The comparison of the optical rotations of the
2
3
23
natural [a] ¼ þ4.4 (MeOH) and the synthetic alkaloid [a] ¼ À2 (MeOH) with (S)
D
D
configuration showed in the same way as for compound 1 a partial racemization. The
configuration of the natural alkaloid 2 should be (R).
We thank Dr. Michael Müller and Dr. Holm Frauendorf, Institut für Organische und Biomolekulare
Chemie, Georg-August-Universität Gçttingen, and Dr. Jürgen Schmidt, Leibniz Institut für Pflanzen-
biochemie, Halle for ESI spectra measurements. Support of this research by a scholarship of the Dr. Hans
M. Fischer Stiftung for M. G. is gratefully acknowledged.

1
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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Scheme 2. Synthesis of Compound 2
a) Borate buffer, CoCl ·6 H O, pH¼8.5, l-aminoacyclase, 388, 48 h; 88%. b) 1,4-Dioxane, H O, KOH,
2
2
2
Boc O, r.t., 16 h, 94%. c) 1. THF, DCC, NHS, 08, 2 h. 2. NaHNCN, H O, r.t., 16 h; 65%. d) MeCN, SnCl ,
2
2
4
08, under Ar, 45 min; 86%. e) MeCN, EtOH, MeI, r.t., 24 h; 100%.
Experimental Part
General. All reactions were performed in oven-dried glassware. THF was distilled from sodium
diphenyl ketyl. Solvents for chromatography were purchased technical grade and distilled prior to use.
All starting compounds were purchased from commercial sources and used as received. TLC: silica gel 60
F254 precoated plates (Merck); detection with 10% molybdophosphoric acid in EtOH. Column
chromatography (CC): silica gel 60 M (SiO , 40–63 mm; Macherey-Nagel). PTLC: silica gel 60 PF
2
254
precoated plates (Merck). Optical rotations: Jasco P-1020 polarimeter. NMR: Bruker-Avance 300; d in
ppm, J in Hz; (D )DMSO or CD OD as solvent and internal standard ((D )DMSO: d(H) 2.50, d(C)
6
3
6
3
9.5. CD OD: d(H) 3.31, d(C) 49.0). HR-EI-MS and HR-ESI-MS: Bruker APEX IV and Thermofisher
3
Scientific Orbitrap Elite; in m/z. EI-MS: Finnigan MAT-8500; in m/z.
Animal Material. The yellow sea anemones of Heteractis aurora and the octopus of Octopus cyanea
were collected in December 2006, near Bali, Indonesia, at a depth of 3–15 meter, and were identified by
F. Pfeifer, Bayreuth.
Extraction and Isolation. The fresh sea anemones of Heteractis aurora (300 g) were sliced,
homogenized with EtOH, and exhaustively (4Â ) extracted with EtOH. The solvent was removed under
reduced pressure to yield the residue (14 g) which was submitted to CC (SiO ; hexanes, hexanes/AcOEt
2
gradient, CHCl , CHCl /MeOH gradient) to afford three fractions (Frs. I –III). Fr. II (2.0 g) was
3
3
separated by CC (SiO ; CHCl , CHCl /MeOH gradient 18 :1, 18 :3) followed by PTLC (CHCl /MeOH
2
3
3
3
1
8 :3) to give compounds 1 (20 mg), 2 (22 mg), and 3 (30 mg).
The octopus of Octopus cyanea (320 g) were frozen, sliced, homogenized with EtOH, and directly
extracted (4Â ) with EtOH. The extract was evaporated to dryness and the residue (10 g) was

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1753
successively partitioned between hexanes/H O, AcOEt/H O, and BuOH/H O. The BuOH extract (0.8 g)
2
2
2
was subjected to CC (SiO ; CHCl , CHCl /MeOH gradient) to yield two fractions (Frs. A and B). Fr. A
2
3
3
(
(
60 mg) gave, after purification with PTLC (CHCl /MeOH/H O 21:3.5 :0.5), the compounds 5 (3 mg), 6
3 2
2 mg), and 7 (15 mg), and Fr. B (28 mg) gave compound 4 (15 mg).
2
-Amino-1,5-dihydro-5-(1H-indol-3-ylmethyl)-4H-imidazol-4-one (1). [a]23 ¼ þ3.2 (c¼1.00,
D
MeOH). IR (ATR): 3200 (broad), 1693, 1629, 1571, 1482, 1456, 1308, 1265, 1227, 1093, 1009, 741, 718.
1
13
þ
þ
H- and C-NMR: Table 1. EI-MS: 228 (3, M ), 130 (100), 98 (14). HR-ESI-MS: 229.1083 ([MþH] ,
þ
C H N O ; calc. 229.1084).
12
13
4
2
3
2
-Amino-5-[(6-bromo-1H-indol-3-yl)methyl]-3,5-dihydro-3-methyl-4 H-imidazol-4-one (2). [a]
D
¼
1
13
þ
þ
þ4.4 (c¼1.00, MeOH). H- and C-NMR: Table 2. EI-MS: 322 (12.7, M ), 320 (13.0, M ), 208 (100).
þ
79
þ
HR-ESI-MS: 321.0347 ([MþH] , C H BrN O ; calc. 321.0346).
13
14
4
1
3
Auramine (¼4,4’-Carbonimidoylbis[N,N-dimethylbenzenamine]; 3) C-NMR ((D )DMSO): 175.6
6
(
(
C(5)); 154.7 (C(1)); 134.1 (C(3)); 116.7 (C(4)); 111.6 (C(2)); 39.8 (Me NÀC(1)). HR-EI-MS: 267.1736
2
þ
þ
M , C H N
3
; calc. 267.1736).
1
7
21
13
Homarine (¼1-Methylpyridinium-2-carboxylate; 4). C-NMR (CD OD): 165.3 (C(8)); 155.6
3
(
(
C(2)); 147.1 (C(4)); 146.6 (C(6)); 127.7 (C(3)); 127.7 (C(5)); 47.6 (C(7)). HR-ESI-MS: 138.0549
þ
þ
2
[MþH] , C H NO ; calc. 138.0549).
7
8
Synthesis of Benzyl (5S)-2-Amino-4,5-dihydro-5-(1H-indol-3-ylmethyl)-4-oxo-1H-imidazole-1-car-
boxylate (9). To a soln. of N-Cbz protected l-trypthophan 8 (2.000 g, 5.92 mmol) in THF (60 ml) was
added DCC (1.236 g, 6.00 mmol) and NHS (691 mg, 6.00 mmol). The soln. was stirred for 3 h at 08 and
filtered by suction filtration. The filtrate was added to a soln. of NaHNCN (1.135 g, 17.74 mmol) in H O
2
(
60 ml) and stirred for 16 h at r.t. THF was removed under reduced pressure and the soln. was adjusted to
pH 12 with diluted NaOH and washed with CH Cl (2Â50 ml). The aq. layer was acidified to pH 2.2 with
2
2
dil. HCl and extracted with AcOEt (2Â80 ml). The combined org. extracts were dried (MgSO ), and the
4
solvent was removed under reduced pressure. The residue was purified by CC (SiO ; CHCl /MeOH
2
3
9
f 2 3
0 :10) to afford pure 9 (1.329 g, 3.67 mmol, 62%). Amorphous solid. R (SiO , CHCl /MeOH 9 :1) 0.4.
23
[
7
a] ¼ þ45.6 (c¼1.00, MeOH). IR (ATR): 3394, 1710, 1633, 1456, 1392, 1340, 1294, 1228, 1124, 1024,
D
1
13
þ
þ
3
45, 699. H- and C-NMR: Table 1. HR-ESI-MS: 385.1267 ([MþNa] , C H N NaO ; calc.
20
18
4
À
À
3
85.1271), 361.1299 ([MÀH] , C H N O
3
; calc. 361.1306).
20
17
4
2
-Amino-1,5-dihydro-5-(1H-indol-3-ylmethyl)-4H-imidazol-4-one (1). A mixture of 9 (1.000 g,
2
.76 mmol) and Pd/C 10% (300 mg, 10 mol-%) in MeOH (55 ml) was hydrogenated at r.t. under
atmospheric pressure until TLC showed complete consumption of the starting material (1 h). The
catalyst was removed by suction filtration and the filtrate evaporated to dryness. The resultant viscous oil
was purified by prep. MPLC (MN Nucleodur C-18, 100–12; MeCN/H O (1% ammonia) gradient 10% to
2
3
0%, 45 min). Pure alkaloid 1 (0.523 g, 2.29 mmol, 83%) was obtained after lyophilization as a white
23
1
fluffy powder. R (RP-18, H O/MeCN 3 :1, 1% ammonia) 0.3. [a] ¼ À6.1 (c¼1.00, MeOH). H- and
f
2
D
13
þ
þ
C-NMR: Table 1. HR-ESI-MS: 229.1085 ([MþH] , C H N O ; calc. 229.1084), 251.0903 ([Mþ
1
À
2
13
4
þ
þ
À
Na] , C H N NaO ; calc. 251.0903), 227.0936 ([MÀH] , C H N O ; calc. 227.0938).
12
12
4
12 11
4
6
-Bromo-l-tryptophan (¼(2S)-2-Amino-3-(6-bromo-1H-indol-3-yl)propanoic Acid; 11). N-Acetyl-
-bromo-d,l-tryptophan (10, 3.50 g, 10.8 mmol) was dissolved in borate buffer (850 ml) containing
CoCl ·6 H
O (29.73 mg, 0.125 mmol), and the pH was adjusted with borax soln. to pH¼8.5. l-
Aminoacylase (4.15 g, Amano Enzyme Inc., Nagoya, Japan) was added, and the slurry was stirred for
6
2
2
4
5
8 h at 388. The mixture was brought to pH¼5 by addition of 10% HCl and extracted with AcOEt (3Â
00 ml). After drying with MgSO4 and evaporation of the volatiles, crude N-acetyl-6-bromo-d-
tryptophan was obtained. The residual aq. phase was passed through a column filled with Dowex 50Â2–
2
00 resin (70Â35 mm) and eluted with MeOH to get rid of surplus enzyme. The desired amino acid 11
was obtained by eluting the column with MeOH/NH OH 9 :1 (750 ml). After removal of the volatiles,
4
the residue was passed through a plug of SiO (70Â40 mm) by eluting with MeOH/NH OH 9 :1. The
2
4
eluent containing the desired product was concentrated to dryness. The obtained crude product was
purified by MPLC (MN Nucleodur 100–50-RP 18 ec, AcOH/H O 1!100% MeOH) to give 11 (1.34 g,
2
4
.73 mmol, 88%). The yield is based on the maximum of theoretical recovery from the resolution of a
23
1
13
racemic mixture (5.40 mmol). [a]D ¼ À18, c¼1.0, MeOH). H- and C-NMR: Table 2.

1
754
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
(
2S)-3-(6-Bromo-1H-indol-3-yl)-2-[(tert-butoxycarbonyl)amino]propanoic Acid (12). To a suspen-
sion of 6-bromo-l-trytophan (11, 500 mg, 1.77 mmol) in 1,4-dioxane (5 ml) and H O (5 ml), 1m KOH
2
(
1.85 ml, 1.85 mmol) was added. After dissolving of the starting material, Boc O (443 mg, 2.03 mmol) was
2
added in one portion, and the mixture was stirred for 16 h at r.t. The soln. was adjusted to pH¼4 by slow
addition of 1m HCl, diluted with H O (50 ml), and extracted with AcOEt (3Â50 ml). After drying
2
(
MgSO ) and removal of all volatiles, the title compound 12 (634 mg, 1.65 mmol, 94%) was obtained
4
1
13
which can be used without further purification. H- and C-NMR: Table 2. HR-ESI-MS: 405.0419 ([Mþ
þ
79
þ
Na] , C H BrN NaO
4
; calc. 405.0420).
16
19
2
tert-Butyl (5S)-2-Amino-5-[(6-bromo-1H-indol-3-yl)methyl]4,5-dihydro-4-oxo-1H-imidazole-1-car-
boxylate (13). A soln. of 12 (290 mg, 757 mmol) in THF (7.5 ml) was cooled to 08 and DCC (161 mg,
7
80 mmol) and NHS (89.7 mg, 780 mmol) were added. After stirring for 2 h at 08, the soln. was allowed to
warm to r.t. and filtrated into a soln. of NaHNCN (145 mg, 2.27 mmol) in H O (7.5 ml). The mixture was
2
stirred at r.t. for 16 h. THF was removed under reduced pressure, the mixture was basified to pH¼12
with 2m KOH, and H O (50 ml) was added. After washing with CH Cl (2Â25 ml), the aq. phase was
2
2
2
adjusted to pH¼2.2 with 2m HCl and extracted with AcOEt (3Â50 ml). The combined org. extracts
were dried (MgSO ) and all volatiles were removed in vacuo. Purification of the crude product by CC
4
2
3
(
1
4
SiO ; CHCl /MeOH 9 :1) furnished the title compound 13 (199 mg; 490 mmol, 65%). [a] ¼ þ50.1 (c¼
2
3
D
.18, MeOH). 1H- and C-NMR: Table 2. HR-ESI-MS: 407.0711 ([MþH] , C H BrN O ; calc.
13 þ 79
þ
17
20
4
3
07.0713).
(
5S)-2-Amino-5-[(6-bromo-1H-indol-3-yl)methyl]-1,5-dihydro-4H-imidazol-4-one (14). A soln. of
1
3 (60.9 mg, 150 mmol) in MeCN (5 ml) was chilled in an ice bath and SnCl (189 ml, 1.62 mmol) was
4
added under an inert gas atmosphere. Stirring was continued for 45 min at 08, and the mixture was
partitioned between AcOEt (100 ml) and 1m KOH (100 ml). After separation of both phases, the aq.
layer was extracted with AcOEt (3Â100 ml), and the combined org. phases were dried (MgSO ). All
4
volatiles were removed, and the crude product was purified by MPLC (MN Nucleodur C-18, 100–12;
MeCN/H O (1% ammonia) gradient 10% to 35%, 45 min) to obtain the pure compound 14 (39.6 mg,
2
2
3
1
13
1
(
29 mmol, 86%). [a] ¼ À82.0 (c¼1.13, MeOH). H- and C-NMR: Table 2. HR-ESI-MS: 307.0192
D
þ
79
þ
[MþH] , C H BrN O ; calc. 307.0189).
12
12
4
2
-Amino-5-[(6-bromo-1H-indol-3-yl)methyl]-3,5-dihydro-3-methyl-4H-imidazol-4-one (2). To a
soln. of 14 (41.2 mg, 134 mmol) in MeCN (30 ml) and EtOH (10 ml), MeI (2.00 ml, 32.1 mmol) was
added, and the mixture was stirred at r.t. for 24 h. Evaporation of all volatiles yielded compound 2 as
2
3
1
13
hydroiodide (60.2 mg, 134 mmol, 100%). [a] ¼ À2 (c¼0.10, MeOH). H- and C-NMR: Table 2. HR-
D
þ
79
þ
ESI-MS: 321.0348 ([MþH] , C H BrN O ; calc. 321.0346).
13
14
4
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Received November 5, 2014