Mellpaladines and dopargimine, novel neuroactive guanidine alkaloids from a Palauan Didemnidae tunicate

Article abstract of DOI:10.1016/j.tet.2016.09.051

Novel guanidine alkaloids dopargimine (1) and mellpaladines A–C (2–4) were isolated from a Palauan Didemnidae tunicate. The structures of 1–4 were elucidated on the basis of spectral data along with chemical reactions. A putative biosynthetic building block of 1–4, 4-guanidinobutyric acid (5), and dimeric polysulfur dopamine, as well as lissoclibadins 11 (6a) and 12 (6b) were also isolated. Compounds 1–3 bound to synaptic receptors, and modulated behavioral profiles of mice after intracerebroventricular injection.

Full text of DOI:10.1016/j.tet.2016.09.051

Accepted Manuscript
Mellpaladines and dopargimine, novel neuroactive guanidine alkaloids from a
Palauan Didemnidae tunicate
Hajime Uchimasu, Ken Matsumura, Masashi Tsuda, Keiko Kumagai, Mai Akakabe,
Masaki J. Fujita, Ryuichi Sakai
PII:
S0040-4020(16)30967-X
10.1016/j.tet.2016.09.051
TET 28122
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 25 August 2016
Revised Date: 20 September 2016
Accepted Date: 21 September 2016
Please cite this article as: Uchimasu H, Matsumura K, Tsuda M, Kumagai K, Akakabe M, Fujita MJ,
Sakai R, Mellpaladines and dopargimine, novel neuroactive guanidine alkaloids from a Palauan
Didemnidae tunicate, Tetrahedron (2016), doi: 10.1016/j.tet.2016.09.051.
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ACCEPTED MANUSCRIPT
Mellpaladines and dopargimine, novel
neuroactive guanidine alkaloids from a Palauan
Didemnidae tunicate.
ꢀ
ꢀ
ꢁ
§
Hajime Uchimasu , Ken Matsumura , Masashi Tsuda , Keiko Kumagai , Mai
§
ꢀ
℃
ꢀ
Akakabe , Masaki J Fujita
, and Ryuichi Sakai *
ꢀ
Faculty and Graduate School of Fisheries Sciences, Hokkaido University, Hakodate
℃
℃
0
41-8611, Japan.
ꢁ
§
Center for Advanced Marine Core Research, Science Research Center Kochi
University, Kochi 783-8502, Japan.
KEYWORDS marine alkaloid, tunicate, neuronal receptor, polysulfur dopamine
1

ACCEPTED MANUSCRIPT
ABSTRACT
Novel guanidine alkaloids dopargimine (1) and mellpaladines A-C (2-4) were isolated
from a Palauan Didemnidae tunicate. The structures of 1-4 were elucidated on the basis
of spectral data along with chemical reactions. A putative biosynthetic building block of
1
-4, 4-guanidinobutyric acid (5), and dimeric polysulfur dopamine, as well as
lissoclibadins 11 (6a) and 12 (6b) were also isolated. Compounds 1-3 bound to synaptic
receptors, and modulated behavioral profiles of mice after intracerebroventricular
injection.
2

ACCEPTED MANUSCRIPT
1
. Introduction
1
,2
3
4
Aromatic guanidine alkaloids are reported from various organisms including plants, a frog, a spider,
5
,6
a sponge and a tunicate. These compounds can be classified into two types; tryptophan-derived
trypargines, and dopamine-derived dopargines. Trypargines are known to possess diverse bioactivities
5
4
7
including antimalarial, insecticidal and neuroactivity. Recently, (+)-7-bromotrypargine was shown to
8
be an antagonist for histamine receptor (H3), and dopamine and norepinephrine transporters. These
results, especially examples of trypargines, suggested potential of these alkaloids as novel neuroactive
scaffold. As part of our continuing research to discover novel neuroactive compounds from marine
9
invertebrates, we isolated four new tetrahydroisoquinolines 1-4 (Chart1). These compounds are
putative biosynthetic products of dopamine and arginine-derived precursors via a Pictet-Spengler
condensation. We also isolated biogenetically related known compounds 5 and 6 from the same extract
(Chart 1). Dopargimine (1) and mellpaladine A (2) modulated behaviors of mice after
intracerebroventricular (i.c.v.) injections, suggesting that they interact with synaptic receptors in the
central nervous system. Here, we describe the isolation, structure elucidation and biological activities of
these novel neuroactive marine alkaloids.
3

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Chart 1
HO
4a
Unit A
6
1
N
Unit B
HO
8a
HO
R4
N+
9
H
N
R4
HO
H N
2
-
-
S
CF COO
CF COO
3
3
H N
2
NH
S
HN
R3
1
H N
2
NH
O
O
R1
COOH
R2
2
; R = R = H, R = R = CH
1 3 2 4 3
HN
3; R = R = R = CH , R = SCH ,
1 2 4 3 3 3
4
; R = R = CH , R = R = H
1
2
3
3
4
H N
2
NH
5
H C
3
CH3
N
OH
OH
OH
O
S
S
O
O
S
S
H C
3
CH3
H C
3
CH3
O
OH
N
N
N
H C
3
CH3
H C
3
CH3
H C
3
CH3
6b
6
a
2
. Results and discussion
We have screened aqueous extracts of marine specimens collected in various sea regions for neuroactive
molecules using primarily mouse behavioral assays. In the mice assay, an i.c.v. injection of the crude
extract of a Didemnidae tunicate collected in Republic of Palau induced dose dependent behavioral
modulation. Mice convulsion accompanied by tremor was observed at 100 µg/mouse. Thus extract of
the specimen (179 g) was subjected to the isolation of its active compounds. The sample was
homogenized with water and the aqueous extract was dialyzed using cellulose membrane to give a
4

ACCEPTED MANUSCRIPT
fraction that passed through the membrane (molecular size smaller than ~50 Å). Since this material
reproduced the behavioral activity in mice, it was separated by chromatography on a Sephadex LH-20
column, eluting with a gradient of aqueous TFA (0.05%) and MeOH. The chemical and biological
profiles of fractions were monitored by TLC, MALDI-TOFMS, and the mouse assay. Fractions with
chemical or biological interest were further separated by a combination of C18 flash column
chromatography and HW40 gel filtration chromatography. Final purification was carried out by
reversed-phase HPLC (C18) to afford pure compounds 1 (25.6 mg), 2 (32.1 mg), 3 (5.1 mg), 4 (1.6 mg),
5
(2.0 mg), 6a (1.2 mg) and 6b (1.9 mg).
+
Dopargimine (1), obtained as trifluoroacetate salt, exhibited a molecular ion at m/z 263 (ESIMS, MH ).
1
3
HRESIMS along with C NMR data established the molecular formula for 1 as C H N O , with seven
1
3
18
4
2
1
degrees of unsaturation. In the H NMR data a total of 13 protons including two aromatic (δ 6.85 and
7
.42) and one hetero atom bearing proton (δ 7.95) were observed. All 13-carbon peaks including eight
2
13
sp carbons appeared in the C NMR spectrum (Table 1). A tetrasubstituted benzene ring with two
hydroxyl groups was assigned on the basis of the one and two dimensional NMR data of 1. Thus, two
1
aromatic H singlets had either HMQC or HMBC correlations with a total of six aromatic carbons
resonating between δ 115.2 and 154.7. This NMR profile suggested a presence of a catechol ring,
supported by dark green coloration on TLC by reaction with FeCl spray reagent. Two oxygen atoms
3
were therefore assigned for the substituent at two lower field carbons at δ 154.7 (C-6) and 145.1 (C-7).
One of the aromatic spin systems CH-5 (δ 115.2/6.85) showed mutual HMBC cross peaks to the CH -4
2
system (δ 24.2/2.92) which was extended to the adjacent methylene CH -3 (δ 40.9/3.71) both by COSY
2
2
and HMBC data. A nitrogen atom (N-2) was connected to the CH -3 system on the basis of the
chemical shift values, thus a dopamine substructure was assembled. Two key HMBC correlations
between both H -3 and H-8 and a quaternary carbon at δ 175.9 (C-1) led us to assign a
2
5

ACCEPTED MANUSCRIPT
3
,4-tetrahydroisoquinoline unit. The other spin system CH
2
-9/CH
2
-10/CH
2
-11/NH-12 was assembled by
both COSY and HMBC data (Scheme 1).
Table 1. NMR data for 1, 1b and dopargine.
a
b
c
1
1b
Dopargine
1
13
1
13
1
13
No
1
H
C
H
C
H
C
175.9
40.9
4.41 t (5.9)
3.31/3.49 m
2.84-2.99 m
54.7
39.4
24.0
4.39 brt (6.4)
3.29/3.46 m
2.77-2.97 m
54.7
3
3.71 t (8.0)
2.92 t (8.0)
39.4
4
24.2
24.0
4
5
6
7
8
8
9
1
1
1
a
132.4
115.2
154.7
145.1
116.8
115.8
29.8
124.1
116.0
143.2
144.2
113.8
123.6
30.2
124.1
116.0
143.2
144.2
113.8
122.6
30.2
6.85 s
7.42 s
6.71 s
6.70 s
6.68 s
6.66 s
a
2.98 t (7.8)
1.84 m
1.93/2.01 m
1.68 m
1.90/1.99 m
1.64 m
0
1
2
3
27.2
24.1
24.1
3.23 m
40.1
3.18 t (6.6)
40.7
3.15 t (6.9)
40.6
7.95 brs
1
156.9
156.9
157.2
a
b
c
Taken in DMSO-d
6
, Taken in D
2
O, In D
2
O, see ref 1a.
1
During the H-NMR measurement of 1 in D O at room temperature, H -9 disappeared slowly,
2
2
suggesting that C-9 positions at α to the imine. An HMBC crosspeak between H -10 and C-1
2
2
definitively connected the CH -9 spin system to the imine carbon. A guanidine group was assigned for
the carbon resonated at δ 156.9 since 1 reacted with Sakaguchi reagent to show orange color. The
6

ACCEPTED MANUSCRIPT
presence of a guanidine in 1 was further confirmed by reacting 1 with pentane-2,4-dione (Scheme 1).
An HRESI mass spectral analysis of the product showed that molecular ion was shifted by 64 mu for
1
13
C H . HNMR showed a new aromatic singlet for pyrimidine hydrogen at δ 6.50. In C NMR, all
5
4
carbons corresponding to the expected pyrimidine (1a) were observed assuring the presence of
guanidine. Finally a nitrogen atom N-12 was readily connected to the guanidine carbon because of an
HMBC correlation between H-11 and C-13. These data together assigned the structure of 1 to be
dopargimine. This conclusion was confirmed by the fact that a treatment of 1 with NaBH gave an
4
1
amine 1b whose NMR data agreed well with known dopargine (Table1).
Scheme 1. Chemical transformations of 1.
Mellpaladine A (2) was obtained as trifluoroacetate showing bright yellow color. The molecular formula
for 2, C H N O S , was deduced on the basis of HRESIMS data which was consistent with the number
2
4
31
5
4 2
1
3
1
of carbons observed in the C NMR data. An overall H NMR spectral profile for 2 shared similarities
to that of 1 to some extent, while extra methyls, methylenes, and an aromatic protons are found for those
of 2. The dopargimine substructure embedded in 2 (Unit A, Chart 1) was identified on the basis of
extensive 2D NMR data analysis: in that, a tetrahydroisoquinoline substructure was first assigned by
COSY and HMBC data as in the case of 1 (Figure 1). The second spin system comprised of
C-9/CH
2
-10/CH
2
-11/NH-12 was also evident by COSY and HMBC data (Table 2, Figure 1). The
7

ACCEPTED MANUSCRIPT
quaternary carbon (C-9, δ 77.5) was then connected to C-1 on the basis of key HMBC correlation
between H -10 and C-1/C-9. A carbon resonated at δ 156.8 (C-13) was assigned as a guanidine carbon
2
on the basis of its chemical shift. HMBC cross peaks between both H -11 and H-12 to C-13 connected
2
the guanidine unit to form 9-subtituted dopargimine substructure for 2 (Figure 1).
9
'
1
1'
H C
3
3
H
HO
HO
N+
COSY
NOE
N
8
'
CH3
10'
1
8
CH2
9
1'
5
'
H
1
2 HN
HO
O
H N
2
NH
CH3
2'
Figure 1. Key NMR correlation data for unit A and B of 2.
Key HMBC
1
The third spin system that forms substituted N,N-dimethylethylamine (Unit B, Chart 1) was identified
on the basis of NMR data as follows. A proton in the aromatic spin system CH-5’ (δ 111.3/6.71) was
connected by HMBC data to four aromatic carbons (C-1’, 3’, 4’, and 6’). A methoxy group was
3
assigned at C-4’ (δ 146.5) because an HMBC between H -12’ and C-4’ was observed. The
N,N-dimethylethylamine system, CH -7’/CH -8’/CH -9’/CH -10’/NH-11’, which was readily assigned
2
2
3
3
by COSY and HMBC data, was connected to C-6’ of the benzene ring by an HMBC between CH -8’
2
and C-6’. In addition, an HMBC correlation between H-5’ and 7’ further supported a covalent bond
between C6 and C7. NOESY cross peak between H-5’ and both H -12’ and H -7’ were consistent with
3
2
the above substitution pattern (Figure 1). A relatively large amount (32 mg) of 2 isolated from the
tunicate allowed us to perform an INADEQUATE experiment successfully to confirm above two carbon
frameworks Units A and B unequivocally (See supporting Figure S25). Thus far, two independent
substructures, a 9-substituted dopargimine and a 1’,2’-disubstituted N,N-dimethyl-4’-O-methyl
dopamine were assigned leaving two sulfur atoms and one unsaturation. We therefore hypothesized a
8

ACCEPTED MANUSCRIPT
presence of a dithioketal (1,3-ditholane) linkage between C-9 and C-1’ and 2’. A treatment of 2 with
mercaptoethanol, expectedly resulted in formation of 1 (See, supporting information Figures S1 and S2).
1
0
An oxidative removal of the dithioketal of 2 with bis(trifluoroacetoxy)iodobenzene, however, was not
successful, resulting in decomposition of the starting material.
Interestingly however, a reduction of 2 with NaBH provided us with an additional support for the
4
dithioketal functionality. A MALDI-TOFMS data for the reaction product showed an ion for the
tetrahydroisoquinoline (7) at m/z 520 as expected, however; amount of 7 decreased over a prolonged
treatment accompanied by a gradual formation of 3,4-dihydroisoquinoline-6,7-diol (8) (See, supporting
information, Figures S3-S5).
Scheme 2. NaBH reduction of 2.
4
HO
HO
HO
N
NH
S
N
N
HO
S
S
HN
S
NaBH4
HN
H N
2
NH
HO
H N
2
O
NH
HO
O
7
: 1,2-dihydromellparadine A:
+
m/z 520(MH )
OH
HO
HO
N H
H+
N
S
S
: m/z 164 (MH+)
8
HO
O
N
NH
HN
S
H N
2
S
O
OH
a: m/z 357 (MH+)
8
9

ACCEPTED MANUSCRIPT
Compound 8, which was likely formed by a C-C bond cleavage between C-1 and C-9 of 7, was actually
1
isolated and its structure was confirmed by H NMR and HRMS data. This result strongly supported the
presence of dithioketal in 2. Thus, the cleavage between C-1 and C-9 bond presumably took place first
by an attack of hydroxide at a secondary amine hydrogen atom. The reductive C-C bond cleavage was
driven by stabilization of building anionic character at C-9 by a dithioketal functionality that allowed
C-9 to take up ambient proton to give 8 as shown in Scheme 2. Moreover, the presence of 8a, the other
half of the cleavage product, was suggested in the MALDI-TOFMS spectrum of the reaction mixture in
+
that a molecular ion corresponding to 8a at m/z 357 (MH ) was found. LC/HRESIMS analysis for the
reaction product further confirmed the presence of these ions whose exact masses and isotope patterns
matched with the corresponding theoretical values (See, supporting information, Figure S4). These data
rigorously supported the presence of dithioketal group, unique for natural products, in 2. Finally, the
fact that a positive ion HRESIMS/MS spectrum for 2 indicating formations of fragment ions A and B
was also supportive of the structure of 2 (Scheme 3). Taken together, the structure of 2 was assigned as
a dopargimine coupled with sulfur-substituted dopamine derivative as depicted in Chart 1.
1
0

ACCEPTED MANUSCRIPT
Table 2. NMR data for 2-4 in DMSO-d6
2
3
4
1
3
1
13
1
13
1
No
1
C
H
C
H
C
H
162.8
46.8
162.4
46.9
162.5
47.0
3
3.60, 2H, t, 7.8
3.61, 2H, t, 7.4
2.53, 2H, t, 7.6
3.60, 2H, dd, 8.6, 6.1
2.53, 2H, m
4
24.9
2.54, 2H, brt, 8.2 24.9
130.7
24.9
4
5
6
7
8
8
9
1
1
1
1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
a
a
130.8
130.6
114.9 6.69, 1H, s
115.0 6.70, 1H, s
114.9 6.69, 1H, s
148.7
148.7
148.5
143.2
143.3
143.2
113.5 7.38, 1H, s
113.2 7.30, 1H, s
113.2 7.29, 1H, s
117.1
77.5
116.9
77.6
117.2
78.0
0
41.4
38.6
2.61, 2H, t, 7.5
41.7
37.3
2.69, 2H, t, 7.2
41.8
37.4
2.65, 2H, t, 7.2
1
3.16, 2H, m
7.53, 1H, brs
3.18, 2H, m
7.61, 1H, brs
3.14, 2H, m
2(NH)
7.42, 1H, brt, 5.0
3
156.8
128.0
124.6
140.7
146.5
156.8
133.0
132.4
147.0
152.4
128.3
129.1
29.6
156.7
128.2
131.8
142.4
150.7
a
a
c
c
'
'
'
'
'
111.3 6.71, 1H, s
121.6
112.3 6.74, 1H, s
a
c
'
127.3
'
29.7
56.1
42.3
42.3
2.86, 2H, m
3.21, 2H, m
2.83, 3H, s
2.83, 3H, s
9.84, 1H, brs
3.78, 3H, s
3.13/3.24, 2H, m 32.9
2.75-2.87, 2H, m
3.00, 2H, m
'
55.2
3.10, 2H, m
2.88, 3H, brs
2.88, 3H, brs
10.08, 1H, brs
3.84, 3H, s
38.3
b
'(NCH
3
)
42.1
42.3
b
0'(NCH3)
1’ (NH)
2'(OCH
7.88, 3H, brs
3.79, 3H, s
3.72, 3H, s
3
)
)
56.2
60.7
60.1
19.0
56.1
59.7
3'(OCH
3
3.81, 3H, s
4'(S CH3)
a , b, c
2.37, 3H, s
Assignments may be interchanged.
1
1

ACCEPTED MANUSCRIPT
1
Mellpaladine B (3) was obtained also as yellow solid. An overall H-NMR profile of 3 suggested a close
similarity between 2 and 3. The molecular formula, C H N O S , secured on the basis of HRESIMS
2
6
35
5
4 3
data as well as NMR data differed from that of 2 by C H S. As suggested from the molecular formula,
2
4
detailed comparison of NMR data between 2 and 3 indicated that they are closely related (Table 2).
Particularly chemical shifts for the dopargimine substructure (C-1 to C-13) for 3 were very similar to
those of 2, indicating that the unit A for 2 and 3 are identical. A reaction of 3 with 2-mercaptoethanol
resulted in formation of 1. This result indicated that 3 shares dopargimine substructure and dithioketal
group with 2. Product ions both A and B were observed in positive ion HRESIMS/MS spectrum which
corresponded well with those of 2 (Scheme 3) indicating that the overall structure of 3 is similar to that
1
13
of 2. The NMR data indicated a presence of two extra methyl groups at δ 3.81/60.1 ( H/ C) and
2
.37/19.0, but instead loss of an aromatic proton (Table 2). Thus, the first methyl group was assigned
tentatively at the oxygen atom at C-3’ and the second one was substituted at C-5’ as methyl thioether.
HMBC correlations from the methyl groups to the aromatic carbons belonging to Unit B supported
these assignments (Figure 2). We therefore assigned that the extra carbon and sulfur atoms in 3 were
resided in Unit B. Small ROESY correlations between C-14’ and H-7’ and H-8’ could specify the
1
3
position of methylsulfide on the aromatic ring of 3. Moreover, comparisons of C NMR data of Unit B
1
1
12
of 3 with lissoclibadins (cf. lissoclibadin 3 and lissoclinotoxins , Supporting Figure S6), supported
the above assignment. Therefore the structure 3 was assigned as depicted in Chart 1.
1
2

ACCEPTED MANUSCRIPT
H C
3
H
N+
NH3+
CH2
COSY
ROE
H C CH3
2
CH2
S CH3
H
H C O
H C O
3
3
O
O
CH3
CH3
13'
Key HMBC
12'
3
4
Figure 2. Key correlation NMR data for unit B of 3 and 4.
Mellpaladine C (4) was isolated as a minor component. Molecular formula for 4 was deduced to be
on the basis of HRESIMS/NMR data. As in the case of 3, dopargimine substructure in 4
was assigned by its NMR (COSY, HSQC, HMBC, ROESY) data with those of 2 and 3 (Table 2). Thus
the difference in the formula between 2 and 4, CH , resides in Unit B. In the NMR data, two resonances
23 29 5 4 2
C H N O S
2
for N-methyl CH -9’ and 10’ groups observed in 2 were missing in 4, instead ammonium protons
3
+
(
N H ) were observed as broad singlet at δ 7.88. COSY data connected the ammonium through CH -7’
3
2
(Figure 2). The connectivity between C-6’ and C-7’ was supported by an HMBC between H-5’ and
1
C-7’and a ROESY cross peak between H-5’ and CH -7’. In the H NMR two O–methyl groups at δ 3.72
2
and 3.79 were evident. Because the later methyl group showed a ROESY cross peak between H-5’, it
was assigned to be CH -12’. C-3’ and C-4’ were assigned on the basis of HMBC cross peaks between
3
3
the CH -13’ and 12’ to each of these carbons, respectively (Figure 2). Thus the dimethyl dopamine
1
,2-disulfide substructure was assigned for Unit B in 4. In a positive ion ESI MS/MS spectrum, only the
product ion A appeared, presumably amino group was less favorable as a leaving group as compared to
dimethylamine group of 2 and 3 (Scheme 3). These data together assigned the structure of 4 as depicted
in Chart 1.
Compounds 2, 3 and 4, possess an asymmetric center at C-9. Determination of the absolute
stereochemistry at this position, however, is not straightforward. Although all three compounds
1
3

ACCEPTED MANUSCRIPT
exhibited optical rotation, their CD data were negligibly low. Attempts to grow single crystals of 2 for
X-ray crystallography in various conditions have unfortunately been failed thus far.
1
4
-Guanidinobutyric acid (5), a putative biosynthetic building block for dopargine and presumably that
for 1-4 was also isolated in the present study. The structure was assigned on the basis of MS and NMR
data and was confirmed by direct comparison with a commercial reagent.
Mellpaladines are interesting in a biosynthetic point of view since they can be envisioned as hybrids
between dopargimine and polysulfur dopamine-related thiols. Polysulfur dopamines, which have more
than one sulfide or disulfide-bridged dopamines are known in some tunicates of family Didemnidae.
1
1,12,13,14
The dithioketal functional group is new in natural products and only close example, cyclic
1
5
14
dithioacetal groups in tunicate products fasmerianamines or lissoclibadin 13 (Chart 2), and cyclic
1
3,16
polysulfides produced by hyperthermophilic archia
are known to the best of our knowledge.
Chart 2
H C O
NOH
3
CH3
H
O
N
O
HO
S
S
CH3
HO
H C
S
S
S
3
O
OH
NH
N
N
H C
3
CH3
fasmerianamine A
lissoclibadin 13
1
4
In the present study, we identified lissoclibadin 11 (6a) and 12 (6b), indicating that this specimen is of
the chemotype that contains polysulfur dopamines. To date, more than 24 ascidian-derived dopamines
1
3
containing multiple sulfur atoms have been identified. Biosynthesis, including origin of the sulfur
atoms, of this peculiar sulfur-containing dopamine unit is not known. The series of compounds 1-6
isolated in the present study may have biogenetic relationships, in that, 1 was formed from dopamine
1
4

ACCEPTED MANUSCRIPT
and 5 via a Pictet-Spengler condensation. Dopamine, on the other hand, is subjected to form dithiol
intermediate 9 which may readily react with the ketone 10 to form mellpaladines (Scheme 4). A
coupling of dithiol 9 (R as methyl) with N,N-dimethyl dopamine would result in the formation of 6ab.
Compounds 1-3 displayed behavioral activities in mice upon i.c.v. administration including convulsion
accompanied by tremor at high doses (50-140 nmol/mouse). At the lower doses (3-14 nmol/mouse)
however, no convulsions occurred while suppression in voluntary movement was observed. ED50 values
determined on the basis of these dose-dependent behavioral changes for 1 and 2 were 19 and 8.0
nmol/mouse, respectively (Supplement Figures S7 and S8). Behavioral changes observed in mouse were
likely resulted from interactions between the drug and synaptic receptors. Thus, target synaptic
receptors for 1 and 2 were surveyed by Psychoactive Drug Screening Program (PDSP). In the primary
binding assay, each compound at fixed concentration (10 µM) was screened for 45 different synaptic
target sites including G-protein coupled receptors, ligand gated ion channels, and transporters (primary
1
5

ACCEPTED MANUSCRIPT
binding assay). Compounds that showed significant binding (>50 % displacement) were further tested in
the secondary binding assay to determine Ki values. Compound 1 showed displacement of radioligands
with Ki value for δ-opioid receptor (DOR 485 nM) and alpha2B adrenergic receptor (5.9 µM).
Compound 2 and 3 showed rather wide spectrum of affinity in the binding assay, but showing affinities
for serotonin receptors with some subtype selectivity (Table 3).
a
Table 3. Ki values (nM) of 1-3 for various receptors and transporters in the competitive binding assay
Comp # 5-HT1A 5-HT1B 5-HT1D 5-HT2A
5-ht5a
-
NA
728
5-HT7
-
757
155
α1A
-
2119
1898
α 1D
NA
-
α 2B
5855
-
α 2C
1
2
3
-
-
-
-
-
-
3895
-
7500
652
NA
489
8800
NA
590
2697
NA
β2
NA
NA
-
D
-
-
1
DAT
-
1641
1208
DOR
485
-
-
GABA
NA
-
A
H
-
-
3
KOR
-
2300
1365
M1
-
7200
5172
NET
-
-
Sigma 2
1
2
3
-
-
1001
NA
1990
1259
570
a
See experimental section for receptor nomenclature. NA not active at >10000nM, “-“ are ones that did
not show 50% inhibition at the primary binding assay.
Biological activities for 4 were not determined due to minute amount of natural compound available.
Several endogenous guanidine metabolites including 5 are known to be neurotoxic, and 5 was
1
7
demonstrated to cause convulsion in rodent models. In our mice assay, 5 (50 µg/mouse) evoked minor
tremor but symptom was transient. Both compound 6a and b did not induce noticeable behavioral
change in mice at (50 µg/mouse).
3
. Conclusion
The present work demonstrated, by isolating new compounds 1-4 and other related metabolites, the
chemical diversity of tunicate-derived dopamine alkaloids and potential of this class of marine alkaloids
for biomedical use in neurological studies. A number of tunicate-derived dopamine derivatives are
1
3
known, however, they are mostly in a form of polysulfur dopamines with a few exceptions including
1
8,19
20
the C-unit of some ecteinascidins
and novel proaporphine alkaloids saldedines. Both of these
1
6

ACCEPTED MANUSCRIPT
compounds form an isoquinoline unit presumably via Pictet-Spengler reaction. Isolation of
mellpaladines, hybrid of isoquinoline alkaloid and polysulfur dopamine, indicated the presence of
interesting biosynthetic machinery in this tunicate that bridges between polysulfur dopamine
biosynthesis and isoquinoline alkaloid biosynthesis.
Interactions between the neuronal receptors and the tunicate-derived alkaloids isolated here supported
9
our idea that marine alkaloids are unique but relatively untapped source of neuroactive compounds.
Although further investigations are required to determine the target receptors that are responsible for the
behavioral phenotypes observed after administrations of 1 and 2 in mice, identification of receptor
groups that compound 1 and 2 preferred to bind provided us with an encouraging start points for further
studies. In vivo behavioral and in vitro functional assays and structure-activity relationship of these
compounds are in progress in our laboratory.
4
. Experimental procedure
4
.1. General.
NMR data were obtained on a JEOL ECZS400 spectrometer. Some of the NMR experiments,
including the INADEQUATE experiment, were performed on a Varian 500 experiments, equipped with
a 5 mm PFG XSens Cold Probe. Samples were dissolved either in deuterium oxide (99.990 atom % D),
1
DMSO-d
residual signals of D
referenced to DMSO-d
6
(99.9 atom % D), or MeOH-d
O and DMSO-d
solvent peak at δ 39.5. The infrared spectra were measured using KBr pellet.
4
(99.8 atom % D). H chemical shifts were referenced to
1
3
2
6
at δ 4.65 and 2.49, respectively. C-NMR chemical shifts were
6
MALDI-TOF MS was measured on an AB4700 spectrometer, using either α-cyano-4-hydroxycinamic
acid (CHCA) or gentisic acid (GA) as a matrix. High resolution electrospray mass spectra (HRESIMS)
were obtained on an Exactive Orbitrap mass spectrometer. Optical rotation was obtained on a JASCO
1
7

ACCEPTED MANUSCRIPT
2
200 polarimeter using 3 x 10 cm cell, and the α value was adjusted against water blank. The ESI
LC-MS analyses (low resolution) were performed in a positive ion mode on a Shimadzu LCMS-8040
system using a COSMOSIL (5C18-AR-II Packed Column, 10 x 250 mm, Nacalai Tesque), or an Inertsil
2
ODS-3 (2.1 × 100 mm, GL-Science) column eluting with a gradient of MeOH-H O (0.2% acetic acid).
The Q-TOF LC-HRESIMS experiments were performed in a positive ESI mode on a Sciex 5600 system
using a YMC reversed-phase column AM12S03 (C18, 4.6 × 75 mm) eluting with a gradient of MeOH
(
0.05% formic acid)-H O (0.1% formic acid). The HRMS/MS data were obtained in an infusion mode
2
with collision energy of 25V. Purity of compound was monitored by using an evaporative light
scattering detector (ELSD) SEDEX 55, or a charged aerosol detector (CAD), Thermo-Fisher Corona
ultra RS. Normal phase (silica gel 60F254) or reversed-phase (RP18F245S) plates were used for thin
layer chromatography (TLC) using (A) pyridine : EtOAc : AcOH : H O, 150:70:33:60, or (B) CHCl :
2
3
MeOH : AcOH : H
2
O, 40:20:2:4 (v/v), for the normal phase; or MeOH : H
2
O, 25:75 for the
reversed-phase separation. HPLC was performed on a system comprised of a pump LC-20AD and a
photodiodearray detector SPD-M20A (Shimadzu) using a COSMOSIL (5C18-AR-II Packed Column,
1
0 mm I.D. × 250 mm), eluting with a gradient of MeOH- H O both containing 0.05% trifluoroacetic
2
acid (v/v).
4
.2. Biological material and extraction.
A sample of tunicate was collected at Republic of Palau in 2007 (7°19’ N, 134°36’ E) by using SCUBA
at a depth of 20 m. The specimen stored in 70 % ethanol was identified to belong to family Didemnidae
by Prof. Teruaki Nishikawa at Toho University. More detailed identification up to genus level, however,
was not possible because tissues degraded in the stored sample. A frozen specimen (179 g) was
lyophilized, and a ground sample was extracted with water. The aqueous extract was dialyzed using a
1
8

ACCEPTED MANUSCRIPT
cellulose tube (40-50 Å pore) against water and the small molecular portion that passed through the
membrane was lyophilized to give an extract (6.97 g).
4
.3. Separations and chemical transformations
The extract was separated by a Sephadex LH-20 column (6 × 70 cm, gradient 0.05% aqueous TFA to
MeOH, 4 mL/min, 10 mL/tube) and tubes were combined according to TLC into a total of 15 fractions
(uc2-81-1~15). A fraction that showed activity in mice, uc2-81-6 (208 mg) was separated on a
reversed-phase open column (Wakogel C18, 63-212 µm, Wako pure chemical, Tokyo), with gradient
elution of 0.05% aqueous TFA to MeOH (1 mL/min, 5 mL/tube) into 12 fractions (uc2-82-1~12).
4
.3.1.
Dopargimine (1). The active fraction uc2-82-3 (60.4 mg, convulsant in mice at 10 µg/mouse) was separated
by a C18 (Wakogel) flush column, and by an HW-40 column to give dopargimine (1, 25.6 mg) as a yellow powder: UV
-1
1
13
(
H O) λ (logε) 245 (3.85), 304 (3.67), 369 (3.72); IR (KBr) νmax 3363, 3174, 1703 cm ; H- and C NMR, See
2 max
+
Table 1; HRESIMS: m/z 263.1504 [MH] (calcd for C13
.3.2. 4-Guanidinobutyric acid (5). Above fraction (uc2-82-2) was chromatographed on an HW-40 column (0.05%
aqueous TFA) into eight fractions (uc3-14-1-8). Fraction 5 was purified by an HPLC (COSMOSIL 5C18-AR-II, 0.05%
19 4 2
H N O , 263.1503).
4
-1
aqueous TFA-MeOH gradient) top give pure 5: UV end absorption, IR (KBr) νmax 3478, 3361, 3202, 1680br, 1183 cm .
+
MALDI-TOFMS m/z 146 [M + H]
4
.3.3.
Reaction of dopargimine (1) with pentane-2,4-dione. A sample of 1 (1.3 mg) was treated with
pentane-2,4-dione (200 µL) with pyridine (200 µL) at 80 ꢂ for 3.5 h. Solvents were removed in vacuo then the
product was purified on an HPLC (gradient of 0.05% aqueous TFA to MeOH, 10 to 100 %) to give 1a (uc3-29-1, 0.8
-1 1
mg, 49%): UV (H
2
O) λmax (logε) 236 (4.18), 302 (3.83), 355 (3.74) nm; IR (KBr) νmax 1683, 1645, 1198 cm ; H NMR
(
D O) δ 7.10 (1H, s), 6.66 (1H, s), 6.51 (1H, s), 3.61, (2H, t, J = 7.8 Hz), 3.37, (2H, t, J = 5.9 Hz), 2.97, (2H, t, J = 6.6
2
13
Hz), 2.81, (2H, t, J = 7.8Hz), 2.20 (6H, s), 2.10, (2H, m), C NMR (D O) δ 178.8, 163.8, 163.5, 154.6, 154.1, 144.3,
2
1
34.1, 118.5, 117.6*, 115.7*, 111.2*, 41.6, 40.5, 30.5, 27.2, 27.1, 25.1 (*peaks split due probably to mixed conformers
+
in water). HRESIMS: m/z 327.1817 [MH] (calcd for C18
H
23
N
4
O
2
, 327.1816).
1
9

ACCEPTED MANUSCRIPT
4
.3.4.
Reduction of 1 with NaBH
4 4
. To an aqueous solution of 1 (6.4 mg) was added NaBH (10 mg/mL, 200 µL)
dropwise until the color of solution disappear. Monitoring on TLC, the reaction was quenched by adding acetic acid
0.5 M). The lyophilized product was purified on an HPLC (COSMOSIL, gradient of 0.05% aqueous TFA to MeOH, 5
to 100%) to give 1b (uc2-52-1, 4.8 mg, 75%; dopargine): UV (H O) λ (logε) 284 (3.46) nm; IR (KBr) νmax 3422,
(
2
max
-
1
1
13
+
1
4
673 cm ; H- and C NMR (D
2
O, 400 MHz) shown in Table 1; MALDI-TOFMS m/z 265 [MH] .
.3.5. Isolation of mellpaladine A (2), B (3), and C (4). One of the Sephadex LH-20 fractions uc2-81-7 (170 mg)
was separated on a C18 flash reversed-phase column with a gradient of 0.05% TFA and methanol into 10 fractions.
th
Each of the 7, 8 and 9 fractions contained compound 2, 4, and 3, respectively, was purified by HPLC (COSMOSIL,
gradient of 0.05% aqueous TFA to MeOH, 3 to 30%) to give 2 (32.1 mg), 3 (5.1 mg) and 4 (1.6 mg).
26
4
.3.6.
Mellpaladine A (2): [α]
D
ꢃ25.1 (c 0.38, H
2
O); UV (H
2
O) λmax (logε) 235 (4.62), 313 (3.96), 420 (3.69) nm;
-1
1
13
+
IR (KBr) νmax 3428, 1672, 1198 cm ; H- and C NMR (See, Table 2); HRESIMS: m/z 518.1896 [MH] (calcd for
+
C H N O S , 518.1890); HRESIMS/MS for m/z 518.1865 [MH] (calcd for C H N O S , 518.1890) : m/z 459.1385
2
4
32
5
4
2
24 32
5
4 2
+
+
[
MH-CH
5
N
3
] (calcd for C23
H
27
N
2
O
4
S
2
, 459.1407) : m/z 414.0807 [MH-CH
5
N
3
2
-C H
7
N] (calcd for C21
H
20NO
4
S
2
,
4
4
14.0828).
.3.7. Mellpaladine B (3): [α]
23 (3.41) nm; IR (KBr) νmax 3404 (br), 3186 (sh), 1680, 1198 cm ; H- and C NMR, See Table 2; HRESIMS: m/z
26
D
2 2
ꢃ1.8 (c 0.37, H O); UV (H O) λmax (logε) 242 (4.42), 268sh (4.05), 311 (3.86),
-1
1
13
4
5
+
+
78.1934 [MH] (calcd for C26
H
36
N
5
O
4
S
3
, 578.1924); HRESIMS/MS for m/z 578.1905 [MH] (calcd for
+
C H N O S , 578.1924): m/z 519.1426 [MH-CH N ] (calcd for C H N O S , 519.1440) : m/z 474.0842 [MH-
2
6
36
5
4
3
5
3
25 31
2
4 3
+
CH N -C H N] (calcd for C H NO S , 474.0862).
5
3
2
7
23 24
4 3
2
6
4
.3.8.
Mellpaladine C (4): [α] ꢃ-2.9 (c 0.14, H O); UV (H O) λ (logε) 235 (4.26), 314 (3.69), 419 (3.15) nm;
D 2 2 max
-1
+
IR (KBr) νmax 3435 (br), 1680, 1198, 1137 cm ; NMR data, see Table 2.; HRESIMS: m/z 504.1741 [MH] (calcd for
+
C H N O S , 504.1734). HRESIMS/MS for m/z 504.1725 [MH] (calcd for C H N O S , 504.1734) : m/z 445.1245
2
3
30
5
4
2
23 30
5
4 2
+
[
M-CH
5
N
3
] (calcd for C22
Treatment of 2 and 3 with mercaptoethanol. An aqueous solution of 2 (or 3) (1mg/mL, 1µl) was mixed with
-mercaptoethanol (1 µl) and heated in boiling water for 1 min. The product was analyzed by LC/MS (MRM mode for
25 2 4 2
H N O S , 445.1250) .
4
2
.3.9.
detection of 1, Figure S1), or by photodiode array detector.
.3.10. Reduction of 2 with NaBH . An aqueous solution of 2 (7.0 mg/200 µL) was treated with NaBH (large
4
4
4
2
0

ACCEPTED MANUSCRIPT
excess). The reaction was quenched by adding 0.5 M acetic acid and the product was passed through a small C18
column with water to remove inorganics. MALDI-TOFMS data of the product showed a presence of ions
+
+
corresponding to 3,4-dihydro isoquinoline-6,7-diol (8, m/z 164, MH ), dihydromellpaladine A (7, m/z 520, MH ),
+
and compound 8a (m/z 357, MH ) (See supporting data Figure S3). This product mixture (9.4 mg) was separated by
1
HPLC to give 8 (0.65 mg). UV λmax 244, 306, 356 nm (from photodiode array detector). H NMR (DMSO-d ) δ 9.73
6
(
1H, brs), 8.79 (1H, brs), 7.18 (1H, brs), 6.76 (1H, brs), 3.72 (2H, t, J = 7.6 Hz), 2.90 (2H, t, J = 7.6 Hz) (See
supporting data Figure S5). LC-HRESIMS analysis of the reaction product indicated that a peak eluted at 6 min
+
contained all the above compounds showing an ion for 7 at m/z 520.2052 [MH] (calcd for C24
H
34
N
5
O
S
4 2
520.2047), 8
+
+
at m/z 164.0701 [MH] (calcd for C H NO , 164.0706), 8a at m/z 357.1416 [MH] (calcd for C H N O S ,
9
10
2
15 25
4
2 2
3
57.1413) (See supporting data Figure S4).
4
.3.11. Treatment of 2 with bis(trifluoroacetoxy)iodobenzene (PIFA) 10
CN-H O was added TFA (0.5 µL) and PIFA (0.27 mg, 1 eq). After 30 min, additional PIFA
0.50 mg, 1.9 eq) was added to the mixture. TLC of the reaction mixture, after 2.5 h, indicated complete
consumption of the 2, but a spot left at origin. MALDI-TOFMS data of the product did not indicate the
presence of expected ion ( 278).
.3.12. Isolation of lissoclibadin 11 (6a) and 12 (6b). A MALDI-TOFMS of a MeOH eluate from the Sephadex
.
Compound 2 (0.32 mg, 0.62 µmol)
in 1 : 1 CH
3
2
(
m/z
4
LH-20 column contained ions correspond to lissoclibadins. Thus the fraction (60 mg) was separated further by C-18
flush column chromatography (0.05% aqueous TFA-MeOH gradient) into nine fractions. HPLC purifications of the
fraction 1 (5.0 mg) using COSMOSIL 5C18-AR-II followed by COSMOSIL π-NAP (0.05% aqueous TFA-MeOH
+
gradient) gave compound 6a (1.2 mg) HRESIMS: m/z 451.1717 [M + H] (calcd for C22
H
31
N
2
O
4
S 451.1720).
2
Compound 6b (1.9 mg) was isolated from the fraction 2 (14.2 mg) by HPLC (Inertsil ODS-3, 0.05% aqueous
+
TFA-MeOH gradient). HRESIMS: m/z 451.1724 [M + H] (calcd for C22
H
31
N
2
O
4
2
S 451.1720).
4
.4. Biological assays
The mouse behavioral assays were performed under approval by the Ethical Committee of Experimental
Animal Care at Hokkaido University. The effects of samples on mice behaviors were assessed as
2
1

ACCEPTED MANUSCRIPT
2
1
described previously.
Briefly, an aqueous solution of sample (10 µL) was injected
intracerebroventricularly (i.c.v.) in male ddY mice of 3 to 4 weeks (Japan SLC Inc, Hamamatsu).
Behaviors were observed for 60 min, and were graded in order of severity as follows: death 7, whole
body convulsion 6, loss of balance 5, violent running-jumping 4, stereotype behaviors 3, loss of
voluntary movement 2, sedating 1, and no effect 0. Data were analyzed by using computer software
GraphPad Prism. Receptor binding profiles and Ki determinations were generously performed by the
National Institute of Mental Health's Psychoactive Drug Screening Program [Contract #
HHSN-271-2008-00025-C (NIMH PDSP)]. The recombinant or native receptors tested for primary
binding assay include serotonin receptors (5-HT , 5-HT , 5-HT , 5-HT , 5-HT , 5-HT , 5-HT ,
1
A
1B
1D
1E
2A
2B
2C
5
3 6 7
-HT , 5-HT5A, 5-HT , and 5-HT ), adrenergic receptors (α1A, α1B, α1D, α2A, α2B, α2C, β1, β2, and
β3), dopamine receptors (D1, D2, D3, D4, D5), dopamine transporter (DAT), GABA (rat brain
A
muscimol binding site), histamine receptors (H1, H2, H3, H4), muscarinic receptors (M1, M2, M3, M4,
M5), Peripheral-type benzodiazepine receptor (PBR), opioid receptors (µ, κ, δ), biogenic amine
transporters (NET, SERT), sigma receptors (σ1, σ2). For experimental details, refer to the PDSP web
site http://pdsp.med.unc.edu/.
5
6
. Supplementary data
Supplementary data associated with this article can be found in the online version, at XXXX
. Note
Authors HU and KM contributed equally. This paper is dedicated to the memory of Tatsuo Higa,
Emeritus professor at University of the Ryukyus, who passed away recently.
2
2

ACCEPTED MANUSCRIPT
7
. Acknowledgement
This research was supported by grant from JSPS to RS (15H0454605). We thank Prof. Teruaki
Nishikawa at Graduate School of Science, Toho University, Japan for identification of tunicate
specimen, and government of Republic of Palau for sample collection permission. Ki determinations,
was generously provided by the National Institute of Mental Health's Psychoactive Drug Screening
Program, Contract # HHSN-271-2013-00017-C (NIMH PDSP), directed by Bryan L. Roth MD, PhD at
the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda
MD, USA.
2
3

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