Chemistry of renieramycins. Part 13: Isolation and structure of stabilized renieramycin type derivatives, renieramycins W-Y, from Philippine blue sponge Xestospongia sp., pretreated with potassium cyanide

Article abstract of DOI:10.1016/j.tet.2012.06.067

Three new bistetrahydroisoquinoline marine natural products, renieramycins W (1w), X (1x), and Y (1y), along with two known renieramycins M (1m) and T (1t), were isolated from the pretreated Philippine blue sponge Xestospongia sp. with KCN and their structures were elucidated by comparing their spectral data with those of 1m, 1t, and N-acetylsafracin B (11). Renieramycins W (1w) and X (1x) are the first examples of tiglic acid ester derivatives at the C-1 side chain. Renieramycin Y (1y) possesses a characteristic substitution pattern in A-ring and isolation of it from marine organism strongly evidences to link the possible precursor 3-hydroxy-5-methyl-O-methyltyrosine with both renieramycin and ecteinascidin marine natural products.

Full text of DOI:10.1016/j.tet.2012.06.067

Tetrahedron 68 (2012) 7422e7428
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Chemistry of renieramycins. Part 13: Isolation and structure of stabilized
renieramycin type derivatives, renieramycins WeY, from Philippine blue sponge
Xestospongia sp., pretreated with potassium cyanide
a
b
b
b
~
Mari Tatsukawa , Louvy Lynn C. Punzalan , Hilbert D.S. Magpantay , Irene M. Villasenor ,
Gisela P. Concepcion c , Khanit Suwanborirux , Masashi Yokoya , Naoki Saito
,
d
a
a,
*
*
a Graduate School of Pharmaceutical Sciences, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan
b Institute of Chemistry, University of the Philippines, Diliman, Quezon City 1101, Philippines
c Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines
d Department of Pharmacognosy and Pharmaceutical Botany and Center for Bioactive Natural Products from Marine Organisms and Endophytic Fungi (BNPME), Faculty of
Pharmaceutical Sciences, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 May 2012
Received in revised form 16 June 2012
Accepted 18 June 2012
Available online 4 July 2012
Three new bistetrahydroisoquinoline marine natural products, renieramycins W (1w), X (1x), and Y (1y),
along with two known renieramycins M (1m) and T (1t), were isolated from the pretreated Philippine
blue sponge Xestospongia sp. with KCN and their structures were elucidated by comparing their spectral
data with those of 1m, 1t, and N-acetylsafracin B (11). Renieramycins W (1w) and X (1x) are the first
examples of tiglic acid ester derivatives at the C-1 side chain. Renieramycin Y (1y) possesses a charac-
teristic substitution pattern in A-ring and isolation of it from marine organism strongly evidences to link
the possible precursor 3-hydroxy-5-methyl-O-methyltyrosine with both renieramycin and ecteinascidin
marine natural products.
Keywords:
Renieramycin
Tetrahydroisoquinoline
Isolation
Ó 2012 Elsevier Ltd. All rights reserved.
Marine natural product
Structure elucidation
1. Introduction
produce 2 and 3.10 After several attempts, the oxidation of 1e with
selenium oxide and p-toluenesulfonic acid in 1,4-dioxane at 80 ꢀC
Renieramycins and ecteinascidins are tetrahydroisoquinoline
marine natural products that are structurally and biologically re-
lated to other isoquinoline natural products, including saframycin,
naphthyridinomycin, and quinocarcin antibiotics.1e3 These tetra-
hydroisoquinoline natural products are relatively unstable and
available in only trace amounts because they are easily decom-
posed during both extraction and isolation procedures. We solved
this problem by converting the original natural products having
for 14 h was found to be the best choice in terms of yield (2: 46.4%
and 3: 43.9%) (Fig. 1). We also found that the selenium oxide oxi-
dation of jorumycin (4)11 furnished renierol acetate (5)12 and 3 in
37.2% and 20.5% yields, respectively. These findings indicate that
such simple isoquinoline compounds may be oxidative degradation
products and/or artifacts of the isolation procedure.
To our knowledge, no renieramycin-related products have been
isolated from Philippine marine organisms. Nevertheless, we are
very interested in several Philippine marine organisms because of
reports of the discovery of 3 along with simple isoquinoline com-
pounds from them. For example, in 1996, Edrada et al.13 found a new
N-ethylene methyl ketone derivative of renierone 6,14 which was
isolated from an unknown blue Philippine marine sponge belonging
to the genus Xestospongia, along with three known compounds 2, 3,
and 7.15,16 In addition, new isoquinoline marine natural product 8
was isolated by Rashid et al. from an aqueous extract of the Philip-
pine marine sponge Haliclona sp. along with 3 (Fig. 2).17,18
a relatively unstable
a-amino alcohol functional group at C-21
position into stable -aminonitriles by pretreatment with KCN. We
a
have succeeded in identifying a number of biologically active tet-
rahydroisoquinolines from several Thai marine organisms, such as
the tunicate Ecteinascidia thurstoni,4 the blue sponge Xestospongia
sp.,5e7 and the nudibranch Jorunna funebris.8
In 1989, He and Faulkner suggested that renieramycin E (1e)
underwent oxidative cleavage to furnish renierone (2) and mim-
osamycin (3).9 We have obtained evidence that 1e decomposed to
As part of our search for new metabolites via the isolation and
characterization of biologically active compounds, we have suc-
ceeded in finding three new compounds from the Philippine blue
sponge Xestospongia sp., growing in the vicinity of Puerto Galera,
* Corresponding authors. Tel./fax: þ81 42 495 8794 (N.S.); tel.: þ63 3 433 2990
(G.P.C.); e-mail addresses: gpconcepcion@gmail.com (G.P. Concepcion), naoki@
my-pharm.ac.jp (N. Saito).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.06.067

M. Tatsukawa et al. / Tetrahedron 68 (2012) 7422e7428
7423
OMe
O
H
Me
O
O
O
E
H
3
OMe
Me
Me
D
N
Me
O
N
Me
O
A
B
C
N
N
O
MeO
H
MeO
21
1
H
+
O
O
OH
O
22
Me
O
R
O
O
R
Me
Me
H
H
mimosamycin (3)
renierone (2): R =
renieramycin E (1e): R =
jorumycin (4): R = Me
Me
Me
renierol acetate (5): R = Me
Fig. 1. Oxidative degradation of renieramycin E and jorumycin.
O
O
Me
Me
N
O
Me
N
MeO
MeO
O
OMe
O
O
Me
O
Me
HO
MeO
O
N
Me
O
O
H
N
7
Me
O
OH
Me
O
O
6
O
Me
8
N
MeO
mimosamycin (3)
O
Me
An unidentified Philippine
Marine Sponge of the
O
H
The Philippine marine
Me
sponge Haliclona sp.
Genus Xestospongia sp.
renierone (2)
Fig. 2. Simple isoquinoline marine natural products from Philippine marine organisms.
Oriental Mindoro, Mindoro Island. We present here the isolation
and structure elucidation of three new stable renieramycins WeY
(1wey) along with two known renieramycins M (1m) and T (1t)7
from the KCN-pretreated Xestospongia sp.
1m(Fig. 3). The 1Hand 13C NMR spectraldata of 1w wereverysimilar
to those of 1m except for the signals assigned to the unsaturated
tiglic acid ester in 1was opposed tothose assigned tothe angelic acid
ester in 1m (Table 1). The characteristic NMR signals of the tiglate
appeared at dC 11.9 ppm (20-CH3) and dH 6.48 ppm (30-H).19
2. Results and discussion
Treatment of jorunnamycin A (9) with tigloyl chloride in
dichloromethane and DMF at 25 ꢀC for 66 h gave 1w in 31% yield,20
which had identical data with those of a natural sample (Chart 1).
Renieramycin X (1x) was isolated as a pale yellow amorphous
solid. The molecular formula of 1x was determined to be C31H33N3O8
by HRFABMS, and was also the same as that of 1m. Due to the limited
availability of the sample, many quaternary carbon signals could not
be detected. Comparison of the 1H NMR data of 1x with those of
renieramycin T (1t) revealed that the two molecules were identical in
The blue sponge Xestospongia sp. (0.2 kg, wet weight) was col-
lected by scuba divers in the vicinity of Puerto Galera, Oriental
Mindoro, Mindoro Island (13.50818ꢀ N & 120.95877ꢀ E) at depths of
3e5m on23September2011. Thecollectedspongewashomogenized
with phosphate buffer solution to adjust the pH to 7. After adding
aqueous KCN solution (1.5 mL), the reaction mixture was macerated
with methanol and the extract was collected by filtration. The con-
centrated extract (9.52 g) was partitioned between ethyl acetate and
water and the organic layer was concentrated in vacuo to give a resi-
due (560 mg), which was subjected to flash column chromatography
on silica gel to give renieramycin M (1m) mainly in 33.1% yield based
on the weight of the ethyl acetate extract. We also isolated known
renieramycin T (1t: 0.20% yield based on the weight of the ethyl ac-
etate extract), the identification of which was accomplished by
comparing all spectroscopic data (1H NMR, 13C NMR, MS, IR). In ad-
dition, we found three new compounds and named them renier-
amycins WeY (1wey) in 0.25%, 0.20%, and 0.16% yields, respectively.
New compound (1w), named renieramycin W, was confirmed to
have the molecular formula C31H33N3O8 on the basis of high-
resolution MS, and its molecular formula was the same as that of
that they both had a characteristic pair of doublets at
5.92 ppm (J¼2 Hz) in addition to the D2O exchangeable proton signal
at 4.30 ppm. The presence of only one methoxy proton signal at
4.00 ppm indicated that one of the quinone rings should be reduced
to form an aromatic ring in 1x. It was also evidence that the diagnostic
homoallylic coupling (approx. 3 Hz) between 1-H and 4-H through
d 5.86 and
d
d
b
five bonds was negligible in 1x (Table 1).21 The major differences in
the 1H NMR data between 1x and 1t were similar to those detected
between the unsaturated tiglic acid ester of 1w and the angelic acid
esterof1m(Table 1).ThecharacteristicNMRsignalsofthetiglatein1x
were detected at dC 12.0 ppm (20-CH3) and dH 6.58 ppm (30-H).
Renieramycin Y (1y) was isolated as a pale yellow solid. The mo-
lecular formula of 1y was determined to be C31H35N3O7 by HRFABMS,

7424
M. Tatsukawa et al. / Tetrahedron 68 (2012) 7422e7428
OMe
OMe
OMe
O
H
Me
O
O
H
Me
O
O
H
Me
O
O
E
HO
O
H
N
H
N
H
N
O
Me
D
Me
Me
N
Me
N
Me
A
B
N
Me
C
H
MeO
H
H
MeO
O
H
H
H
CN
R1
O
CN
R1
CN
HO
O
O
Me
2'
2'
3'
O
R2
1'
3'
O
R2
1'
O
H
Me
Me
Me
renieramycins
renieramycins
renieramycin Y (1y)
M (1m): R1 = Me, R2 = H
W (1w): R1 = H, R2 = Me
T (1t): R1 = Me, R2 = H
X (1x): R1 = H, R2 = Me
Fig. 3. Structures of new renieramycins W, X, and Y along with known renieramycins M and T.
Table 1
1H and 13C NMR data of renieramycins W (1w) and X (1x) compared with known renieramycins M (1m) and T (1t) in CDCl3
Atom
Chemical shift (d
), multiplicitya (J in Hz)
1m
1t
1w
1x
d
dC
dH
dC
dH
dC
dH
dC
dH
1
3
4
56.3 d
54.1 d
25.4 t
3.99 (m)b
3.11 (dt, 11, 3)
2.89 (dd, 17, 8)
1.36 (ddd, 17, 11, 3)
56.4 d
56.2 d
26.8 t
4.16 (dd, 5, 4)
56.4 d
54.3 d
25.4 t
4.00 (m)b
3.09 (dt, 11, 2)
2.91 (dd, 17, 2)
1.31 (ddd, 17, 11, 3)
56.6 d
56.4 d
26.8 t
4.16 (dd, 5, 3)
3.23 (dt, 12, 3)
2.87 (dd, 15, 3)
1.63 (dd, 15, 12)
3.24 (ddd, 12, 3, 2)
2.87 (dd, 15, 2)
1.67 (dd, 15, 12)
5
6
7
8
185.4 s
128.6 s
155.8 s
180.9 s
135.7 s
141.3 s
54.2 d
54.6 d
21.3 d
144.7 s
106.2 s
144.9 s
136.8 s
112.1 s
113.1 s
54.9 d
54.8 d
21.2 d
185.5 s
128.4 s
155.7 s
181.0 s
135.6 s
141.6 s
54.2 d
54.5 d
21.1 d
nd
nd
nd
nd
nd
nd
54.9 d
54.8 d
21.2 t
9
10
11
13
14
4.01 (dd, 3, 2)
3.40 (ddd, 8, 3, 2)
2.76 (dd, 21, 8)
2.30 (d, 21)
4.00 (dd, 3, 1)
3.37 (ddd, 7, 2, 1)
2.75 (dd, 21, 7)
2.30 (d, 21)
4.01 (dd, 2, 1)
3.38 (ddd, 8, 3, 1)
2.74 (dd, 21, 8)
2.29 (d, 21)
3.99 (dd, 2, 1)
3.35 (ddd, 8, 3, 1)
2.73 (dd, 21, 8)
2.27 (d, 21)
15
16
17
18
19
20
21
22
185.9 s
128.6 s
155.2 s
182.5 s
135.0 s
142.0 s
58.5 d
62.0 t
186.1 s
129.0 s
155.4 s
182.8 s
135.7 s
141.8 s
59.8 d
186.0 s
128.5 s
155.1 s
182.5 s
134.8 s
142.1 s
58.6 d
nd
nd
nd
nd
nd
nd
59.8 d
65.1 t
4.07 (d, 3)
4.10 (dd, 12, 3)
4.53 (dd, 12, 3)
4.11 (d, 2)
3.99 (dd, 11, 5)
4.41 (dd, 11, 4)
4.05 (d, 3)
4.01 (dd, 12, 3)
4.59 (dd, 12, 4)
4.06 (d, 3)
3.95 (dd, 11, 5)
4.41 (dd, 11, 3)
64.6 t
62.5 t
10
166.5 s
126.3 s
140.5 d
15.7 q
20.4 q
8.7 q
167.1 s
126.8 s
139.7 d
15.7 q
20.5 q
8.8 q
166.9 s
128.5 s
137.9 d
14.3 q
11.9 q
8.8 qc
167.1 s
nd
139.8 d
14.4 q
12.0 q
8.8 q
20
30
5.96 (qq, 7, 2)
1.82 (dq, 7, 2)
1.58 (dq, 2, 2)
1.94 (s)
1.90 (s)
4.02 (s)
6.00 (qq, 7, 2)
1.85 (dq, 7, 2)
1.69 (dq, 2, 2)
2.11 (s)
6.48 (qq, 8, 1)
1.66 (qq, 8, 1)
1.58 (qq, 1, 1)
1.93 (s)
1.96 (s)
4.02 (s)
6.58 (qq, 7, 1)
1.72 (qq, 7, 1)
1.67 (qq, 1, 1)
2.12 (s)
40
20-CH3
6-CH3
16-CH3
7-OCH3
OCH2O
8.6 q
8.7 q
1.94 (s)
8.7 qc
8.8 q
1.96 (s)
60.9 qc
61.0 qc
101.1 t
5.85 (d, 2)
5.92 (d, 2)
3.98 (s)
101.1 t
5.86 (d, 2)
5.92 (d, 2)
4.00 (s)
17-OCH3
N-CH3
CN
61.0 qc
41.5 q
116.9 s
3.99 (s)
2.28 (s)
60.9 q
41.4 q
117.4 s
61.1 qc
41.5 q
116.9 s
4.01 (s)
2.27 (s)
61.0 q
41.5 q
nd
2.29 (s)
2.29 (s)
5-OH
4.55 (br s)
4.30 (br s)
nd¼not detected.
a
Proton assignments were based on COSY and homonuclear decoupling experiments. Carbon multiplicities were determined on the basis of either DEPT or HMQC data.
The signal overlapped with the methyl singlet.
Assignments are interchangeable.
b
c
d
Carbon resonances were observed through proton resonances by HMQC experiment due to the limited amount of sample available.
and was 14 mass units less than those of 1m and 1t. All proton and
Observed were the characteristic D2O exchangeable proton signal at
5.80 ppm, two carbonyl resonances ( 186.2 and 182.8 ppm) in-
dicative of a quinone ring, and an aromatic CH carbonyl resonance (
120.9 ppm). These data revealed that 1y might have one quinone ring
and a penta-substituted phenol ring. As the diagnostic homoallylic
carbon signals of 1y were assigned after extensive NMR measure-
ments using COSY, HMQC, and HMBC techniques (Table 2). The mo-
lecular formula indicated 16 degrees of unsaturation and the detected
13C carbon resonance attributable to 12 olefinic carbons, three car-
bonyl groups, and one nitrile carbon in 1y accounted for 13 degrees of
unsaturation. This compound was presumed to have five rings.
d
d
d
coupling (approx. 3 Hz) between 1-H and 4-Hb through five bonds
was negligible in 1y, 1y might have a p-quinone at the E ring. The

M. Tatsukawa et al. / Tetrahedron 68 (2012) 7422e7428
7425
OMe
OMe
Cl
H
O
H
Me
O
O
H
Me
O
O
O
O
O
O
Me
H
N
O
H
N
Me
Me
Me
N
Me
N
Me
CH2Cl2, 25oC
H
H
MeO
MeO
H
H
CN
CN
OH
H
O
Me
jorunnamycin A (9)8,10
Me
renieramycin W (1w)
Chart 1. Preparation of renieramycin W (1w) from jorunnamycin A (9).
Table 2
1H and 13C data of renieramycin Y (1y) and related N-acetylsafracin B (11) in CDCl3
Atom no.
1y
N-Acetylsafracin B (11)23,a
dC
dH
HMBC correlation (from C)
3-H, 21-H, 22-Ha, 22-Hb
dC
dH
1
3
4
56.4 d
56.5 d
33.1 t
4.30 (dd, 6, 3)
3.27 (dt, 12, 3)
2.56 (dd, 15, 3)
1.95 (dd, 15, 12)
6.43 (s)
58.2 d
50.4 d
25.4 t
4.20 (m)
1-H, 4-H
a, 4-Hb, 21-H
5-H, 11-H
2.90 (dd, 17, 4)
1.50 (ddd, 17, 11, 2.8)
5
6
7
8
120.9 d
129.3 s
143.8 s
145.1 s
116.7 s
131.7 s
54.9 d
54.9 d
21.3 t
4-H
a
, 4-H
b, 6-CH3
187.5 s
128.9 s
156.3 s
182.3 s
134.1 s
142.2 s
53.8 d
50.1 d
24.8 t
6-CH3
5-H, 6-CH3, 7-OCH3, 8-OH
1-H, 8-OH
9
1-H, 4-H
1-H, 4-H
a
a
, 4-H
, 4-H
b
b
, 5-H, 8-OH
10
11
13
14
3.97 (dd, 3, 2)
3.35 (ddd, 8, 3, 2)
2.75 (dd, 21,8)
2.28 (d, 21)
4-H
a
, 4-H
b, 13-H
4.00 (d, 1.6)
3.15 (dd, 7.7, 2)
2.85 (dd, 19, 7.7)
2.28 (d, 19)
11-H, 14-H
13-H, 21-H
a
, 14-H
b, 21-H
15
16
17
18
19
20
21
22
186.2 s
129.1 s
155.3 s
182.8 s
135.7 s
141.9 s
60.1 d
14-H
16-CH3
16-CH3, 17-OCH3
11-H
11-H, 14-H
11-H, 13-H, 14-H
1-H, 3-H, 13-H, 14-H
1-H
a
, 14-H
b
, 16-CH3
121.9 d
130.9 s
137.2 s
144.7 s
112.1 s
118.0 s
82.0 d
6.73 (s)
a, 14-H
b
a
, 14-H
b
4.09 (d, 3)
4.00 (dd, 11, 6)
a
, 14-H
b
4.40 (d, 2)
2.90 (d, 16)
3.70 (dd, 16, 4)
65.8 t
42.8 t
4.37 (dd, 11, 3)
167.2 s
126.9 s
139.6 d
15.8 q
20.6 q
15.7 q
60.9 q
8.7 q
61.0 q
41.5 q
117.7 s
5.80 (s)
10
22-Ha, 22-Hb, 20-CH3, 30-H
173.1 s
50.3 d
17.8 q
20
20-CH3, 40-H3, 30-H
3.38 (q, 7)
0.80 (d, 7)
30
6.02 (dq,7, 2)
1.87 (dq, 7, 1)
1.73 (dq, 2, 1)
2.24 (s)
3.77 (s)
1.94 (s)
20-CH3, 40-H3
30-H
40
20-CH3
6-CH3
7-OCH3
16-CH3
17-OCH3
N-CH3
CN
30-H
5-H
9.6 q
1.86 (s)
4.03 (s)
2.23 (s)
3.76 (s)
2.31 (s)
61.4 q
16.4 q
60.5 q
40.8 q
3.99 (s)
2.28 (s)
11-H, 13-H
11-H, 21-H
8-OH
a
N-Acetyl chemical shifts (dC) are 22.8 and 175.2 ppm (COCH3).
substituents on phenol ring A were assigned as follows: the chemical
B (11),23 which was prepared from 10b and acetic anhydride in
methanol. As the key EI mass fragment peak of 1y, m/z 204þ
(fragment A), was negligible, both 448þ and 218þ ion peaks were
assigned to fragments B and C, respectively. These data confirm the
locations of the p-quinone ring and the penta-substituted phenol
ring with respect to each other and eliminate the possibility of
reversal (Chart 2).
shifts of C-7 (
substitution, and the former was observed to have long-range 1He13C
correlations to 6-CH3 protons ( 2.24 ppm) and 7-OCH3 protons (
3.77 ppm). Thelong-range 1He13C correlations of the characteristic 5-
H singletproton( 6.43 ppm)toC-4( 33.1 ppm), C-6 ( 129.3ppm), C-
7, and C-9 ( 116.7 ppm) revealed the positions of these carbons.
d 143.8 ppm) and C-8 (d 145.1 ppm) indicated oxygen
d
d
d
d
d
d
While renieramycin Y (1y) is the first example of a penta-
substituted phenol at the A-ring, it is well known antitumor anti-
biotic, safracins A (10a)22 and B (10b) have a same phenol at the
E-ring22,23 (Fig. 4). The structure of 1y was elucidated by comparing
its spectral data to those of safracins A, B and cyanosafracin A (10c).
Safracins possess a penta-substituted phenol at the E-ring, which is
3. Conclusion
Three new bistetrahydroisoquinoline marine natural products,
renieramycins W (1w), X (1x), and Y (1y), along with two known
renieramycins M (1m) and T (1t), were isolated from the Philippine
blue sponge Xestospongia sp. It is much better source to take
renieramycin M (1m) to an approximately 3-fold increasing in the
the same as that of ecteinascidin 743.4,24 In Table 2, the 1H and 13
NMR data of 1y were listed together with those of N-acetylsafracin
C

7426
M. Tatsukawa et al. / Tetrahedron 68 (2012) 7422e7428
OMe
OMe
HO
HO
H
Me
X
HO
H
Me
O
E
O
E
N
H
H
MeO
H
Me
Me
N
Me
N
Me
O
A
A
OMe
N
O
N
O
H
HO
H
Me
MeO
H
MeO
S
H
H
H
AcO
Y
O
OH
NHAc
Me
O
O
Me
N
Me
NH2
O
O
N
H
Me
O
OH
O
H
safracins
11
A (10a): X = Y = H
B (10b): X = H, Y = OH
10c: X = H, Y = CN
ecteinascidin 743
(Et 743)
Fig. 4. Structures of safracins and ecteinascidins.
OMe
OMe
Me
OMe
HO
O
H
O
Me
Me
H
N
Me
O
N
Me
O
1y
N
N
H
MeO
HO
Me
Me
CN
m/z 218
(fragment C)
m/z 204
m/z 448 (fragment B)
(fragment A)
Chart 2. EI mass spectral fragmentation of 1y.
yields of 1m from the Thai blue sponge. Both 1w and 1x are the first
examples of tiglic acid ester derivatives and are the geometrical
isomers at the C-1 unsaturated ester of known angelate derivatives
1m and 1t, respectively. We also found renieramycin Y (1y), which
is the first example having a penta-substituted phenol in the A-ring
in this series.
One study of the biosynthesis of antitumor isoquinoline natural
products has demonstrated that 3-hydroxy-5-methyl-O-methyl-
tyrosine, a nonproteinogenic amino acid, may be the precursor of
the tetrahydroisoquinoline core.25 Although several groups have
proposed biosynthetic schemes for these natural products,26e28 no
solid experimental evidence has been offered regarding the bio-
synthetic intermediates or the enzymatic conversion in vitro. In-
depth studies of the biological activities of new renieramycins for
the evaluation of antitumor activity, and the isolation of an addi-
tional amount of 1y, which is necessary to understand its bio-
synthetic pathway, are underway (Fig. 5).29e31
4. Experimental section
4.1. General experimental procedure
Optical rotations were measured on a Horiba-SEPA polarimeter.
Circular dichroism (CD) measurements were carried out on a Jasco
820 spectropolarimeter. IR spectra were obtained with a Shimadzu
Prestige 21/IRA Affinity-1 FT-IR spectrometer. 1H and 13C NMR
spectra were recorded on a JEOL-JNM-ECA500 FT NMR spectrom-
eter at 500 MHz for 1H and 125 MHz for 13C, on a JEOL-JNM-AL400
NMR spectrometer at 400 MHz for 1H and 100 MHz for 13C, and on
a JEOL-JNM-AL300 NMR spectrometer at 300 MHz for 1H and
75 MHz for 13C (ppm, J in Hz with TMS as internal standard). All
proton and carbon signals were assigned by extensive NMR mea-
surements using COSY, HMBC, and HMQC techniques. Mass spectra
were recorded on a JEOL JMS 700 instrument with a direct inlet
system operating at 70 eV.
OMe
OMe
HO
H
Me
HO
Me
H
O
E
E
H
H
H
Me
Me
Me
OH
N
H
H
N
H
H
A
A
NH2
N
O
N
MeO
MeO
MeO
H
H
OH
HO
OH
HO
HO
NH
H
N
H
N
CnH2n+1
C13H27
O
O
3-hydroxy-5-methyl-
O
Me
O
O-methyltyrosine
SH
31
30
12a
12b
Fig. 5. Structure of the fatty-acid-bound pentacyclic framework that acts as the scaffold of the natural products.

M. Tatsukawa et al. / Tetrahedron 68 (2012) 7422e7428
7427
4.2. Animal material
4.5. Renieramycin T (1t)
a 2D1
The sponge sample (OMID-312-B) was identified as Xestospongia
sp. (Family Petrosidae) by Ms. Fleurdeliz Panga and deposited at the
University of the Philippines Marine Science Institute Museum
(UPMSI). Thissponge takes a digitate to fused lobate habit. It grows on
live coral or rock at depths of 2e4 m. It has a fine micropunctipore
surface. At the tips of the tubes are oscules with diameters of ap-
proximately 6 mm. General consistency is hard, although the sponge
is brittle and easilycrumbles. Fresh specimens are bright blue in color,
but turn brown to orange in alcohol. Megascleres are lightly bent
½
ꢂ
ꢃ19.5 (c 0.04, CHCl3); CD
D
3
(c: 52
m
M, methanol, 22 ꢀC) ꢃ4.8
(345), þ7.1 (268), ꢃ0.2 (235), þ9.6 (212); IR (KBr) 3435, 2926, 2855,
1713,1653,1616,1460,1437, 1375, 1308,1234,1190,1152, 1030 cmꢃ1
;
1H NMR (CDCl3, 500 MHz), see Table 1; 13C NMR (CDCl3,125 MHz) see
Table 1; FABMS m/z 576 [MþH]þ; HRFABMS m/z 576.2350 (Mþþ1,
calcd for C31H34N3O8, 576.2346).
4.6. Renieramycin W (1w)
oxeas (range: 212.80
mmꢁ7.6
mme296.40
mmꢁ7.6
mm, average:
½
a 2D5
ꢂ
ꢃ55.1 (c 0.03, CHCl3); CD
D
3 (c: 63
m
M, methanol, 22 ꢀC) ꢃ2.2
234.84 m). No microscleres are noted. The skeleton is nei-
mmꢁ7.6
m
(353), ꢃ1.1 (306), ꢃ5.1 (281), þ3.0 (257), ꢃ1.4 (228), þ2.3 (206); IR
(KBr) 3429, 2941, 2852,1710,1645,1616,1449,1375,1312,1263,1234,
1190,1151,1080 cmꢃ1; 1H NMR (CDCl3, 300 MHz), see Table 1; FABMS
m/z 576 [MþH]þ; HRFABMS m/z 576.2341 (Mþþ1, calcd for
C31H34N3O8, 576.2346).
ther specialized nor distinct apart from a mesh of free oxeas or end-
ings of choasomal tracts. The choasomal skeleton is a reticulation of
pauci- to multi-spicular (6e10 spicules) forming polygonal meshes.
4.3. Collection and extraction of Xestospongia sponge
4.7. Transformation of jorunnamycin A (9) into renieramycin
W (1w)
The sponge Xestospongia sp. was collected by scuba divers of
MPMSI in the vicinity of Puerto Galera, Oriental Mindoro, Mindoro
Island (13.50818ꢀ N & 120.95877ꢀ E) at depths of 3e5 m on 23
September 2011 and frozen until used. The collected sponge (0.2 kg,
wet weight) was homogenized and phosphate buffer solution was
added to the resulting homogenized solution (200 mL) to adjust pH
to 7. After 10% potassium cyanide solution (1.5 mL) was added
dropwise to the suspension, the reaction mixture was stirred for
A solution of tigloyl chloride was prepared by stirring tiglic acid
(45.3 mg, 0.45 mM) in dry ether (2.3 mL) and mixing it with oxalyl
chloride (38.1 mL, 0.45 mM) in dry DMF (6.9
for 2 h. A solution of 9 (11.0 mg, 22.3 M) in dichloromethane
m
L, 0.082 mM) at 25 ꢀC
m
(1.5 mL) was added to the above acid chloride solution at 0 ꢀC for
5 min and the mixture was concentrated in vacuo with a stream of
argon gas to give a residue. Dichloromethane (0.8 mL) was added to
the residue and the resulting mixture was stirred at 25 ꢀC for 66 h.
After concentration, the crude product was subjected to column
chromatography on silica gel (4 g) with ethyl acetate/hexane (1:2) to
5
h. The reaction mixture was macerated with methanol
(3ꢁ200 mL) and the combined extracts were filtered. The com-
bined filtrates were concentrated in vacuo to give a residue (9.52 g).
This residue was diluted with water (150 mL) and extracted with
ethyl acetate (300, 200, 100 mL). The combined extracts were
washed with brine (200 mL), dried, and concentrated in vacuo to
give a residue (560 mg). This residue was subjected to step-gradient
silica gel (50 g) column chromatography with a solvent system
consisting of 0e100% hexane/ethyl acetate to yield five fractions
(fractions AeE). Fraction B (138.1 mg) yielded dark brown crystals
and recrystallization from ethyl acetate/ether gave renieramycin M
(1m: 77.7 mg). The mother liquor was concentrated in vacuo to
afford fraction B1 (58.7 mg). Fraction B1 was combined with frac-
tion C (84.9 mg) and the whole was chromatographed on a silica gel
column (CH2Cl2/MeOH¼250:1) to give 1m (fraction F1: 106.8 mg,
total 184.5 mg, 33.1% yield based on the weight of the ethyl acetate
extract) and fraction F2 (6.7 mg). Fraction F2 was subjected to silica
gel column chromatography (hexane/ethyl acetate¼5:1) to give
renieramycin W (1w, 1.4 mg, 0.25% yield based on the weight of the
ethyl acetate extract) and renieramycin X (1x, 1.1 mg, 0.20% yield
based on the weight of the ethyl acetate extract). Fraction D
(5.0 mg) was subjected to silica gel column chromatography
(hexane/ethyl acetate¼5:1) to give renieramycin Y (1y, 0.9 mg,
0.16% yield based on the weight of the ethyl acetate extract) and
renieramycin T (1t, 1.1 mg, 0.20% yield based on the weight of the
ethyl acetate extract).
give 1w (4.0 mg, 31%) as a pale yellow amorphous powder. CD
D 3 (c:
63
m
M, methanol, 22 ꢀC) ꢃ4.8 (353), ꢃ2.2 (306), ꢃ12.4 (281), þ7.0
(257), ꢃ2.3 (228), þ4.4 (204); IR (KBr) 3429, 2941, 2852, 1710, 1645,
1616, 1449, 1375, 1312, 1263, 1234, 1190, 1151, 1080 cmꢃ1
;
1H NMR
(CDCl3, 500 MHz)
d
1.31 (1H, ddd, J¼17, 11, 3 Hz, 4-H
b), 1.58 (3H, dq,
J¼2, 1 Hz, 20-CH3), 1.66 (3H, dq, J¼7, 1 Hz, 40-H3), 1.93 (3H, s, 6-CH3),
1.96 (3H, s, 16-CH3), 2.27 (3H, s, NCH3), 2.29 (1H, d, J¼21 Hz, 14-H
b
),
2.74 (1H, dd, J¼21, 8 Hz, 14-H
a), 2.91 (1H, dd, J¼17, 2 Hz, 4-H
a), 3.09
(1H, dt, J¼11, 2 Hz, 3-H), 3.38 (1H, ddd, J¼8, 3,1 Hz,13-H), 4.00 (3H, s,
17-OCH3), 4.00 (1H, overlapped,1-H), 4.01 (1H, dd, J¼12, 3 Hz, 22-H),
4.01 (1H, dd, J¼2, 1 Hz, 11-H), 4.02 (3H, s, 7-OCH3), 4.05 (1H, d,
J¼3 Hz, 21-H), 4.59 (1H, dd, J¼12, 4 Hz, 22-H), 6.48 (1H, qq, J¼8,1 Hz,
30-H); 13C NMR (CDCl3, 125 MHz) 8.7 (ArCH3), 8.8 (ArCH3), 11.9 (20-
d
CH3), 14.3 (C40), 21.1 (C14), 25.4 (C4), 41.5 (NCH3), 54.2 (C11), 54.3
(C3), 54.5 (C13), 56.4 (C1), 58.6 (C21), 61.0 (OCH3), 61.1 (OCH3), 62.5
(C22), 116.9 (CN), 128.5 (C20), 128.4 (C6), 128.5 (C16), 134.8 (C19),
135.6 (C9),137.9 (C30),141.6 (C10),142.1 (C20),155.1 (C17),155.7 (C7),
166.9 (C10), 181.0 (C8), 182.5 (C18), 185.5 (C5), 186.0 (C15); EIMS m/z
575 (Mþ,12), 243 (8), 221 (15), 220 (100), 219 (15), 218 (26); EIHRMS
m/z 575.2271 (Mþ, calcd for C31H33N3O8, 575.2268).
4.8. Renieramycin X (1x)
½
a 2D1
ꢂ
ꢃ12.5 (c 0.04, CHCl3); CD
D
3 (c: 52
m
M, methanol, 22 ꢀC) ꢃ2.4
4.4. Renieramycin M (1m)
(348), þ1.9 (262), ꢃ0.4 (236), þ4.8 (209); IR (KBr) 3442, 2926, 2855,
1707, 1653, 1618, 1456, 1379, 1307, 1263, 1236, 1150, 1091 cmꢃ1 1H
;
Mp 196.5e197 ꢀC (lit.,5 mp 194.5e197 ꢀC); ½a 2D5
ꢂ
ꢃ49.5 (c 0.2,
NMR (CDCl3, 500 MHz), see Table 1; 13C NMR (CDCl3, 125 MHz),
see Table 1; FABMS m/z 576 [MþH]þ; HRFABMS m/z 576.2340 (Mþþ1,
calcd for C31H34N3O8, 576.2346).
CHCl3); CD
D
3
(c: 60
m
M, methanol, 22 ꢀC) ꢃ4.5 (355), ꢃ1.6
(304), ꢃ10.3 (281), þ8.9 (257), ꢃ3.7 (228), þ6.2 (211); IR (KBr)
3441, 2922, 2851, 2230w, 1713, 1699, 1655, 1616, 1449, 1412, 1375,
1348, 1312, 1234, 1190, 1152, 1103, 1080 cmꢃ1
;
1H NMR (CDCl3,
4.9. Renieramycin Y (1y)
500 MHz), see Table 1; 13C NMR (CDCl3, 125 MHz), see Table 1;
EIMS m/z 575 (Mþ, 9), 260 (12), 243 (8), 221 (21), 220 (100), 219
(14), 218 (23); EIHRMS m/z 575.2269 (Mþ, calcd for C31H33N3O8,
575.2268).
½
a 2D1
ꢃ1.9 (355), þ2.6 (262), ꢃ1.3 (234), þ7.9 (207); IR (KBr) 3435, 2941,
1714, 1653, 1616, 1420, 1387, 1373, 1310, 1234, 1152 cmꢃ1 1H NMR
ꢂ
þ4.2 (c 0.05, CHCl3); CD
D
3
(c: 52 m
M, methanol, 22 ꢀC)
;

7428
M. Tatsukawa et al. / Tetrahedron 68 (2012) 7422e7428
(CDCl3, 500 MHz), see Table 2; 13C NMR (CDCl3, 125 MHz), see Table
2; EIMS m/z 561 (Mþ, 12), 448 (28), 229 (11), 221 (18), 220 (100),
219 (22), 218 (21); FABMS m/z 562 [MþH]þ; HRFABMS m/z
562.2562 (Mþþ1, calcd for C31H36N3O7, 562.2553).
11. Fontana, A.; Cavaliere, P.; Wahidulla, S.; Naik, C. G.; Cimino, G. Tetrahedron 2000,
56, 7305e7308.
12. De Silve, E. D.; Gulavita, N. K. Abstracts of Papers, 16th IUPAC International
Symposium on the Chemistry of Natural Products, Kyoto, May 1988. Also
see, Kubo, A.; Kitahara, Y.; Nakahara, S. Chem. Pharm. Bull. 1989, 37,
1184e1186.
13. Edrada, R. A.; Proksch, P.; Wray, V.; Christ, R.; Witte, L.; Van Soest, R. W. M. J.
Nat. Prod. 1996, 59, 973e976.
Acknowledgements
14. We have also found compound 6 from the Thai blue sponge, Xestospongia sp.5
15. Frincke, J. M.; Faulkner, D. J. J. Am. Chem. Soc. 1982, 104, 265e269; Also see,
McIntyre, D. E.; Faulkner, D. J.; Engen, D. V.; Clardy, J. Tetrahedron Lett. 1979, 20,
4163e4166.
This work was supported by the Japan Society for Promotion of
Science(JSPS) Asiaand AfricaScience PlatformProgram(2010e2012),
and was also partially supported by a grant from the High-Tech Re-
search Center Project from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT), Japan (No. S080104). This work also
supported by the PharmaSeas program at UPMSI funded by the
Philippine Department of Science and Technology.
16. Pettit, G. R.; Collins, J. C.; Herald, D. L.; Doubek, D. L.; Boyd, M. R.; Schmidt, J. M.;
Hooper, J. N. A.; Tackett, L. P. Can. J. Chem. 1992, 70, 1170e1174; Also see, Pettit,
G. R.; Knight, J. C.; Collins, J. C.; Herald, D. L.; Pettit, R. K. J. Nat. Prod. 2000, 63,
793e798.
17. Rashid, M. A.; Gustafson, K. R.; Boyd, M. R. J. Nat. Prod. 2001, 64, 1249e1250 A
new isoquinolinequinone, cribrostatin 7 was isolated from the Philippines
marine sponge Petrosia sp. along with two known compounds renierone and O-
demethylrenierone, See, Ref.18..
18. Sandoval, I. T.; Davis, R. A.; Bugni, T. S.; Concepcion, G. P.; Harper, M. K.; Ireland,
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Supplementary data
19. Brouwer, H.; Stothers, J. B. Can. J. Chem. 1972, 50, 601e611.
20. Charupant, K.; Daikuhara, N.; Saito, E.; Amnuoypol, S.; Suwanborirux, K.; Owa,
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21. Kubo, A.; Saito, N.; Yamato, H.; Masubuchi, K.; Nakamura, M. J. Org. Chem. 1988,
53, 4295e4310.
Color picture of Philippine Xestospongia sp. along with a map of
collecting place is available. Supplementary data associated with
this article can be found, in the online version, at http://dx.doi.org/
10.1016/j.tet.2012.06.067.
22. Ikeda, Y.; Matsuki, H.; Ogawa, T.; Munakata, T. J. Antibiot. 1983, 36, 1284e1289.
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