Chemistry of renieramycins. Part 14: Total synthesis of renieramycin i and practical synthesis of cribrostatin 4 (renieramycin H)

Article abstract of DOI:10.3390/md13084915

The first total synthesis of (±)-renieramycin I, which was isolated from the Indian bright blue sponge Haliclona cribricutis, is described. The key step is the selenium oxide oxidation of pentacyclic bis-p-quinone derivative (3) stereo- and regioselectively. We also report a large-scale synthesis of cribrostatin 4 (renieramycin H) via the C3-C4 double bond formation in an early stage based on the Avenda?o's protocol, from readily available 1-acetyl-3-(3-methyl-2,4,5-trimethylphenyl)methyl-piperazine-2,5-dione (8) in 18 steps (8.3% overall yield). The synthesis provides unambiguous evidence supporting the original structure of renieramycin I.

Full text of DOI:10.3390/md13084915

Mar. Drugs 2015, 13, 4915-4933; doi:10.3390/md13084915
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Chemistry of Renieramycins. Part 14: Total Synthesis of
Renieramycin I and Practical Synthesis of Cribrostatin 4
(Renieramycin H)
Masashi Yokoya, Keiichiro Kobayashi, Mitsuhiro Sato and Naoki Saito *
Graduate School of Pharmaceutical Sciences, Meiji Pharmaceutical University, 2-522-1 Noshio,
Kiyose, Tokyo 204-8588, Japan; E-Mails: yokoya@my-pharm.ac.jp (M.Y.);
k.koba0222@gmail.com (K.K.); m146212@std.my-pharm.ac.jp (M.S.)
*
Author to whom correspondence should be addressed; E-Mail: naoki@my-pharm.ac.jp;
Tel./Fax: +81-(0)-42-495-8794.
Academic Editor: Orazio Taglialatela-Scafati
Received: 27 June 2015/ Accepted: 22 July 2015/ Published: 6 August 2015
Abstract: The first total synthesis of (±)-renieramycin I, which was isolated from the
Indian bright blue sponge Haliclona cribricutis, is described. The key step is the selenium
oxide oxidation of pentacyclic bis-p-quinone derivative (3) stereo- and regioselectively.
We also report a large-scale synthesis of cribrostatin 4 (renieramycin H) via the C3-C4
double bond formation in an early stage based on the Avendaño’s protocol, from readily
available 1-acetyl-3-(3-methyl-2,4,5-trimethylphenyl)methyl-piperazine-2,5-dione (8) in
1
8 steps (8.3% overall yield). The synthesis provides unambiguous evidence supporting the
original structure of renieramycin I.
Keywords: cribrostatin 4 (renieramycin H); renieramycin I; total synthesis; structural
determination; selenium oxide oxidation; marine natural product
1
. Introduction
Many tetrahydroisoquinoline antitumor natural products, such as renieramycins, saframycins, and
ecteinascidins, have attracted considerable interest due to their extraordinary structures and meager
availability in nature, as well as their potent antitumor activity [1,2]. Among them, renieramycins H
(
1h) and I (1i) were isolated from the methanol extract of the Indian bright blue sponge Haliclona

Mar. Drugs 2015, 13
4916
cribricutis collected from the intertidal region of Okha, Gujarat, in 1988 [3]. Original structures 1h and
i were given the names renieramycins H and I, respectively. Thereafter, we revised the structure of
renieramycin H to that of cribrostatin 4 (2) [4–6], which was independently isolated from the blue
1
1
3
sponge Cribrochalina sp. collected from reef passages in the Republic of Maldives, based on C NMR
studies of several semi-synthetic models (Figure 1) [7,8]. Cribrostatin 4 (2) has attracted the interest of
several medicinal chemistry experts because of its unique structure and cytotoxicity despite the lack of
the hemiaminal or aminonitrile function at C-21. Three total syntheses of 2 have been
reported [9–11]. Recently, we completed a 21-step stereocontrolled total synthesis of (±)-2 from
1-acetyl-3-(3-methyl-2,4,5-trimethylphenyl)methyl-piperazine-2,5-dione (8) in 3.4% overall yield [12,13].
Furthermore, we have accomplished the total synthesis of renieramycin G (1g) [14,15]. The
availability of 1g and 2 has enabled us to prepare several renieramycin derivatives having a lactam
carbonyl to understand the molecular basis of their impressive cytotoxicity profiles. We present herein
an alternative large-scale approach for the total synthesis of 2. This approach might yield a variety of
novel analogs of cribrostatin 4 (2), as well as C3-C4 unsaturated bis-p-quinone derivatives, such as
renieramycin I (1i), for detailed studies of structure activity relationships (SARs) of these classes of
antitumor marine natural products.
Figure 1. Structures of bis-1,2,3,4-tetrahydroisoquinoline marine natural products.
2
. Results
The most serious problem in our previous cribrostatin 4 (2) synthesis was that 1-epi-pentacyclic
alcohol (4) (Chart 1) might be formed, and the undesired stereochemistry had to be converted into the
natural one at C-1 position via enolate formation through several cycles. Avendaño et al. reported that
the stereocenter at C-3 of 1,3-trans-compound 5 [16] could be transformed into corresponding
1
,3-cis-compound 7 via unsaturated compound 6 through regioselective radical bromination, followed
by hydrogenation from the less hindered α-face in good yield [17]. They applied this protocol to the
preparation of pentacyclic phthalascidin analogs [18]. We were very interested in this procedure for
constructing the 1,3-cis relationships of renieramycins (Scheme 1) [19].

Mar. Drugs 2015, 13
4917
Chart 1. Structure of 1-epi-pentacyclic alcohol.
Scheme 1. Epimerization at C-3 through regioselective bromination at C-3 position and
reduction sequences by Avendaño and co-workers.
Based on Avendaño’s protocol, we designed an alternative synthetic plan that involves the key
transformations outlined in Scheme 2: (1) construction of tricyclic compound 9 having an
α,β-unsaturated amide carbonyl from readily available compound 8 [20]; (2) condensation of 9 with
benzaldehyde derivative and subsequent regio- and stereospecific hydrogenation leading to
compound 11; (3) construction of pentacyclic framework and conversion of ester into our intermediate
1
2, which can be transformed into cribrostatin 4 intermediate 3 [13] (Scheme 2).
Scheme 2. Strategy for practical synthesis of compound 3, which will be converted into 1i and 2.
According to the results of our previous studies [21,22], treatment of 8 with trimethylsilyl chloride
(
TMSCl) in the presence of triethylamine (TEA) in CH
2
Cl
2
gave O-trimethylsilyl lactim
intermediate 14, which was treated with 2,2-diethoxyethyl benzoate in the presence of trimethylsilyl

Mar. Drugs 2015, 13
4918
triflate (TMSOTf) and acetic anhydride to give 15 as an inseparable mixture of diastereomers
15a:15b = 10:3) in 92% yield. After exerting a great deal of effort to separate this mixture by column
(
chromatography several times, we obtained both isomers in their pure forms, and detailed 2D NMR
studies confirmed the structures of 15a (minor) and 15b (major). The NMR spectrum of 15a displayed
H-1 and H-3 proton signals at δ 6.20 and δ 4.70, respectively, whereas the NMR spectrum of 15b
showed H-1 and H-3 proton signals appearing at δ 6.15 and δ 4.07, respectively. An observable nuclear
Overhauser enhancement (NOE) between H-3 and H-22 revealed that compound 15a has the
trans form (Scheme 3).
Scheme 3. 8 → 9: (1) TMSCl, TEA, CH
2
Cl
2
; (2) (EtO)
i
2
CHCH
2
OBz, TMSOTf, Ac O,
2
CH Cl ; (3) NBS, CCl , 60 °C, 3 h; (4) H
2
2
4
2
, Pd/C, PrOH/DMF, 25 °C, 11 h.
We then studied the conversion of 15 into unsaturated compound 9, which is the first key step of
our synthesis. A preliminary experiment was carried out using major isomer 15a. According to the
typical conditions of Avendaño et al. [17], 15a was treated with N-bromosuccinimide (NBS: 1.0
equiv.) and 2,2′-azobisisobutyronitrile (AIBN: 0.1 equiv.) in CCl
4
at 80 °C for 6 h to generate 9 (55%)
1
and 16 (4%) plus unreacted 15a (33%). The H NMR spectrum of 9 showed an H-4 olefinic proton
1
signal that appeared as a singlet at δ 7.47. The H NMR spectrum of 16 showed characteristic AB type
doublet proton signals at δ 4.60 and δ 4.57 along with the H-4 olefinic singlet proton signal at δ 7.41.
Accordingly, 16 might be a product of over-reaction product at C-6 aromatic methyl group. After
extensive investigation of the reaction conditions, we found that the yield of our target 9 could be
improved by slightly lowering the reaction temperature (60 °C) and excluding AIBN. Thus, the
reaction of 15a with NBS (2 equiv.) in CCl at 60 °C for 6 h gave 9 (69%) and 16 (13%). It was
4
extremely difficult to separate 9 and 16 in a large scale using silica gel column chromatography.
However, catalytic reduction of the above mixture using 10% Pd/C in 2-propanol and DMF at 25 °C
for 11 h gave 9 as the sole product in 82% overall yield. Accordingly, the transformation of 15a into 9
without any purification of the intermediates was found to be the best choice in terms of overall yield
(
9 in 71% yield in four steps).
With key intermediate 9 in hand, we next looked into ways to design a practical transformation of 9
into 12, which was the key intermediate in our previous total synthesis of cribrostatin 4 (2) (Schemes 4
and 5). Condensation of 9 with benzaldehyde derivative 17 [20] in the presence of potassium
tert-butoxide gave (Z)-arylidenepiperazinedione 10 in 70% yield. Catalytic hydrogenation of the

Mar. Drugs 2015, 13
4919
trisubstituted double bond of 10 over 10% Pd on carbon in MeOH at 25 °C proceeded
chemoselectively to give desired 11a (72%) along with 11b (21%). Detailed 2D NMR studies were
performed to confirm the structures of 11a and 11b. The NMR spectrum of 11a displayed H-1 and
H-13 proton signals at δ 6.53 and δ 4.34, respectively, whereas the NMR spectrum of 11b had H-1 and
H-13 proton signals appearing δ 6.43 and δ 4.45, respectively. An NOE between H-1 and H-13 proton
signals was observed in 11a but not 11b. Thus, the hydrogenation of 10 obviously occurred
stereoselectively from the α-face to generate H-1 and H-13 cis isomer 11a. It is proposed that the steric
hindrance due to the C-8 methoxy group and the C-21 carbonyl group was responsible for the
β-axial orientation of C-1 substituent as shown in conformer X of 10.
Scheme 4. Preparation of key intermediate 11a.
The piperazinedione ring of 11a was activated by introducing a 2-propyloxycarbonyl group to give
imide 18 in 96% yield. Chemoselective reduction of 18 in the conventional manner afforded a
hemiaminal, which was treated with formic acid at 25 °C for 0.5 h to afford 19 [23] in 82% yield.
Deprotection of 19 with TFA and H
reductive methylation in high yield. Hydrolysis of 21 with 10 N aqueous LiOH in THF/MeOH at
5 °C for 8 h gave primary alcohol 12 in 97% yield, which is identical to the intermediate in our
previous total synthesis [13].
2
SO
4
gave secondary amine 20, which was transformed into 21 by
2
Scheme 5. Construction of pentacyclic primary alcohol 12.

Mar. Drugs 2015, 13
4920
The conversion of 12 into 22 was accomplished by partial demethylation with boron tribromide
BBr ), followed by oxidative demethylation to give bis-p-quinone 22 in 67% yield (Scheme 6).
Acylation of 22 with in situ prepared angeloyl chloride in dichloromethane gave common intermediate
3 in 84% yield. Encouraged by the results of our extensive model studies, including the
transformation of several natural products [24–26], the introduction of a methoxy group to the C-14
position of 23 was achieved using 10 equiv. of SeO in a mixture of methanol and dioxane at 100 °C
(
3
2
2
for six days to give 1i in 43% yield along with secondary alcohol 24 in 29% yield. The orientation of
the methoxy group of 1i was assigned on the basis of the signal of 14-H (δ 4.34, d, J = 1.4 Hz).
The spectroscopic properties of synthetic 1i were in complete accord with those of natural
renieramycin I (1i) [27].
Scheme 6. Transformation of compound 12 into renieramycin I (1i) and cribrostatin 4 (2)
through compound 23.
3. Experimental Section
1
IR spectra were obtained with a Shimadzu Prestige 21/IRAffinity-1 FT-IR spectrometer. H- and
1
3
1
C-NMR spectra were recorded on a JEOL JNM-ECA 500 FT NMR spectrometer at 500 MHz for H
1
3
1
and 125 MHz for C; a JEOL JNM-AL 400 NMR spectrometer at 400 MHz for H and 100 MHz for
1
3
1
13
C; and a JEOL JNM-AL 300 NMR spectrometer at 300 MHz for H and 75 MHz for C (ppm, J in
Hz with TMS as internal standard). All proton and carbon signals were assigned by extensive NMR
measurements 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. Elemental analyses were conducted
on a YANACO MT-6 CHN CORDER elemental analyzer.

Mar. Drugs 2015, 13
4921
3
.1. ((6R*,11aR*)-2-Acetyl-7,8,10-trimethoxy-9-methyl-1,4-dioxo-1,3,4,6,11,11a-hexahydro-2H-
pyrazino-(1,2-b)isoquinolin-6-yl)methyl Benzoate (15a) and ((6R*,11aS*)-2-acetyl-7,8,10-trimethoxy-
-methyl- 1,4-dioxo-1,3,4,6,11,11a-hexahydro-2H-pyrazino(1,2-b)isoquinolin-6-yl)methyl Benzoate
15b)
9
(
TMSCl (498 μL, 3.9 mmol) was added to a stirred solution of 8 (1.05 g, 3.0 mmol) in
dichloromethane (18 mL) and TEA (544 μL, 3.9 mmol), and stirring was continued at 25 °C for 2 h. A
solution of 2,2-diethoxyethyl benzoate [28] (785.8 mg, 3.3 mmol) in dichloromethane (12 mL)
followed by TMSOTf (2.71 mL, 15 mmol) was added dropwise for 5 min each, and then Ac O
2
(
283.6 μL, 3.0 mmol) was added in one portion at 25 °C and the reaction mixture was stirred for 4 h.
The reaction mixture was diluted with saturated NaHCO
3
solution (100 mL) and extracted with CHCl
3
(
100 mL × 3). The combined extracts were washed with brine (100 mL), dried, and concentrated in
vacuo. The residue was subjected to column chromatography with ethyl acetate–hexane (1:2) to give
5 (1.37 g, 92%, 15a:15b = 10:3) as a colorless amorphous powder, which was an inseparable mixture
1
of diastereomers. Each authentic sample was obtained by chromatography on preparative layer silica
gel plates (Merck 5715).
1
Compound 15a: H-NMR (400 MHz, CDCl
3
) δ: 7.99 (2H, m, Ar-H), 7.57 (1H, m, Ar-H), 7.44 (2H,
m, Ar-H), 6.20 (1H, dd, J = 9.5, 3.9 Hz, C6-H), 4.75 (1H, dd, J = 11.7, 9.5 Hz, C12-H), 4.70 (1H, dd,
J = 9.8, 5.4 Hz, C11a-H), 4.52 (1H, dd, J = 11.7, 3.9 Hz, C12-H), 4.36 (1H, d, J = 18.0 Hz, C3-H),
4
.31 (1H, d, J = 18.0, C3-H), 3.95 (3H, s, C7-OMe), 3.80 (3H, s, C8-OMe), 3.72 (3H, s, C10-OMe),
.40 (1H, dd, J = 16.6, 5.4 Hz, C11-Hα), 3.14 (1H, dd, J = 16.6, 9.8 Hz, C11-Hβ), 2.59 (3H, s,
3
13
COCH
3
), 2.21 (3H, s, C9-Me). C-NMR (100 MHz, CDCl
3
) δ: 171.7 (s, COCH ), 167.9 (s, C1), 166.6
3
(
s, OCOPh), 163.2 (s, C4), 152.4 (s, C10), 150.6 (s, C8), 146.2 (s, C7), 133.3 (d, Ph), 129.7 (d, Ph × 2),
129.6 (s, Ph), 128.5 (d, Ph × 2), 125.9 (s, C9), 122.6 (s, C6a), 121.4 (s, C10a), 63.6 (t, C12), 60.6 (q,
C7-OCH
3
), 60.3 (q, C10-OCH ), 60.0 (q, C8-OCH ), 54.0 (d, C11a), 48.3 (d, C6), 45.7 (t, C3), 27.2 (q,
3
3
−1
COCH
3
), 26.0 (t, C11), 9.5 (q, C9-CH
3
). FT-IR (KBr) cm : 1717, 1686, 1672, 1275, 714. EI-MS
+
m/z (%): 496 (M , 11), 361 (100), 319 (13), 234 (15), 204 (8), 105 (7). HR-EI-MS: calcd for
C
26
H
28
N
2
O
8
, 496.1846, found: 496.1841.
1
Compound 15b: H-NMR (400 MHz, CDCl
3
) δ: 7.93 (2H, m, Ar-H), 7.54 (1H, m, Ar-H), 7.42 (2H,
m, Ar-H), 6.15 (1H, dd, J = 7.0, 4.6 Hz, C6-H), 5.10 (1H, d, J = 16.0 Hz, C3-H), 4.55 (1H, dd,
J = 11.5, 7.0 Hz, C12-H), 4.37 (1H, dd, J = 11.5, 4.6 Hz, C12-H), 4.07 (1H, dd, J = 12.2, 4.9 Hz,
C11a-H), 3.94 (3H, s, C7-OMe), 3.79 (3H, s, C8-OMe), 3.78 (1H, d, J =16.0 Hz, C3-H), 3.68 (1H, dd,
J =15.9, 4.9 Hz, C11-Hα), 3.64 (3H, s, C10-OMe), 3.05 (1H, dd, J = 15.9, 12.2 Hz, C11-Hβ), 2.59
1
3
(
3H, s, COMe), 2.22 (3H, s, C9-Me). C-NMR (100 MHz, CDCl
66.4 (s, OCOPh), 166.1 (s, C4), 151.7 (s, C10), 150.6 (s, C8), 146.2 (s, C7), 133.2 (d, Ph × 2), 129.7
s, Ph), 129.6 (d, Ph), 128.4 (d, Ph × 2), 126.1 (s, C9), 123.6 (s, C6a), 121.8 (s, C10a), 66.1 (t, C12),
3
) δ: 170.9 (s, COCH ), 168.7 (s, C1),
3
1
(
6
2
1
1.0 (q, C10-OCH
3
), 60.7 (q, C7-OCH
3
), 60.0 (q, C8-OCH ), 56.3 (d, C11a), 48.5 (d, C6), 45.6 (t, C3),
3
−1
6.9 (q, COCH
3
), 22.8 (t, C11), 9.4 (q, C9-CH
3
). FT-IR (KBr) cm : 1721, 1707, 1692, 1412, 1368,
+
273. EI-MS m/z (%): 496 (M , 10), 361 (100), 319 (11), 234 (13), 204 (7), 105 (7). HR-EI-MS: calcd
for C26
H
28
N
2
8
O , 496.1846, found: 496.1841.

Mar. Drugs 2015, 13
.2. (2-Acetyl-7,8,10-trimethyl-1,4-dioxo-1,3,4,6-tetrahydro-2H-pyrazino(1,2-b)Isoquinoline-6-
4922
3
yl)methyl Beozoate (9) and (2-acetyl-9-(bromomethyl)-,7,8,10-trimethoxy-1,4-dioxo-1,3,4,6-
tetrahydro-2H-pyrazino(1,2-b)isoquinolin-6-yl)methyl Beozoate (16)
NBS (106.8 mg, 0.6 mmol) was added to a stirred solution of 15a (150.0 mg, 0.3 mmol) in dry
CCl , and the reaction mixture was heated at 60 °C for 6 h. The reaction mixture was filtered through a
short pad of Celite, and the filtrate was washed with CCl . The combined filtrates were concentrated
in vacuo, and the residue was purified by flash chromatography on silica gel with ethyl acetate–hexane
4
4
(
1:3) as solvent to give 9 (102.0 mg, 69%) and 16 (17.7 mg, 13%).
1
Compound 9: H-NMR (400 MHz, CDCl
3
) δ: 7.91 (2H, m, Ar-H), 7.55 (1H, m, Ar-H), 7.47 (1H, s,
C11-H), 7.42 (2H, m, Ar-H), 6.47 (1H, dd, J = 7.7, 3.8 Hz, C6-H), 4.93 (1H, d, J = 17.6 Hz, C3-H),
4
3
2
.52 (1H, dd, J = 11.7, 7.7 Hz, C12-H), 4.27 (1H, dd, J = 11.7, 3.8 Hz, C12-H), 3.96 (3H, s, C7-OMe),
.92 (1H, d, J =17.6 Hz, C3-H), 3.85 (3H, s, C8-OMe), 3.71 (3H, s, C10-OMe), 2.61 (3H, s, COMe),
1
3
.21 (3H, s, C9-Me). C-NMR (100 MHz, CDCl
3
) δ: 171.8 (s, COCH ), 166.3 (s, OCOPh), 162.7 (s,
3
C4), 160.7 (s, C1), 154.1 (s, C8), 152.4 (s, C10), 146.0 (s, C7), 133.3 (d, Ph), 129.6 (d, Ph × 2), 129.4
s, Ph), 128.5 (d, Ph × 2), 126.7 (s, C9), 125.8 (s, C11a or C10a), 121.5 (s, C6a), 118.9 (s, C11a or
C10a), 114.7 (d, C11), 64.3 (t, C12), 62.1 (q, C10-OCH ), 60.8 (q, C7-OCH ), 60.2 (q, C8-OCH ),
). FT-IR (KBr) cm : 1701, 1416, 1393,
(
3
3
3
−
1
4
1
2
7.9 (d, C6), 45.2 (t, C3), 26.7 (q, COCH
368, 1269, 1211, 1088, 1072, 1043, 1028, 1007, 964, 459. EI-MS m/z (%): 494 (M , 14), 359 (100),
60 (84), 232 (38), 217 (6), 202 (5), 105 (6). HR-EI-MS: calcd for C26 , 494.1689,
3
), 9.4 (q, C9-CH
3
+
H
26
N
2
O
8
found: 494.1686.
1
Compound 16: H-NMR (400 MHz, CDCl
m, Ar-H), 7.41 (1H, s, C11-H), 6.49 (1H, dd, J = 7.7 Hz, 3.8 Hz, C6-H), 4.94 (1H, d, J =17.6 Hz,
C3-H), 4.60 (1H, d, J =9.1 Hz, C9-CH Br), 4.57 (1H, d, J = 9.1, C9-CH Br), 4.55 (1H, dd, J = 11.6
Hz, 7.7 Hz, C12-H), 4.30 (1H, dd, J = 11.6 Hz, 3.8 Hz, C12-H), 4.03 (3H, s, C8-OMe), 3.97 (3H, s,
C7-OMe), 3.90~3.95 (1H, overlapped, C3-H), 3.90 (3H, s, C10-OMe), 2.62 (3H, s, COCH ).
13C-NMR (100 MHz, CDCl
) δ: 171.8 (s, COCH ), 166.3 (s, OCOPh), 162.7 (s, C4), 160.4 (s, C1),
53.9 (s, C8), 152.1 (s, C10), 146.0 (s, C7), 133.4 (d, Ph), 129.6 (d, Ph × 2), 129.3 (s, Ph), 128.5 (d,
Ph × 2), 127.3 (s, C9), 126.3 (s, C10a or C11a), 125.1 (s, C6a), 119.4 (s, C10a or C11a), 113.6 (d,
C11), 64.2 (t, C12), 63.4 (q, C10-OCH ), 60.7 (q, C7-OCH and C8-OCH ), 47.9 (d, C6), 45.2 (t, C3),
Br). FT-IR (KBr) cm : 1701, 1368, 1292, 1269, 1215. EI-MS m/z
3
) δ: 7.90 (2H, m, Ar-H), 7.55 (1H, m, Ar-H), 7.42 (2H,
2
2
3
3
3
1
3
3
3
−1
26.7 (q, COCH
3
), 21.5 (t, C9-CH
2
+
(
%): 572 (M , 15), 493 (9), 439 (100), 417 (14), 393 (13), 375 (5), 371 (8), 364 (8), 359 (24), 339 (11),
3
10 (21), 274 (9), 260 (27), 231 (10), 105 (21). HR-EI-MS: calcd for C26
H25BrN
2
8
O , 572.0794,
found: 572.0796.
3
.3. (2-Acetyl-7,8-10-trimethyl-1,4-dioxo-1,3,4,6-tetrahydro-2H-pyrazino(1,2-b)isoquinolin-6-
yl)methyl Benzoate (9) from 8 in Four Steps
TMSCl (498 μL, 3.9 mmol) was added to a stirred solution of 8 (1.05 g, 3.0 mmol) in
dichloromethane (18 mL) and TEA (544 μL, 3.9 mmol), and stirring was continued at 25 °C for 2 h. A
solution of 2,2-diethoxyethyl benzoate (785.8 mg, 3.3 mmol) in dichloromethane (12 mL) followed by
TMSOTf (2.71 mL, 15 mmol) was added dropwise respectively over 5 min. Then, Ac O (283.6 μL,
2

Mar. Drugs 2015, 13
4923
3.0 mmol) was added at 25 °C and the reaction mixture was stirred for 4 h. The reaction mixture was
diluted with saturated NaHCO solution (100 mL) and extracted with CHCl (100 mL × 3). The
3
3
combined extracts were washed with brine (100 mL), dried, and concentrated in vacuo. The residue
was subjected to column chromatography with ethyl acetate–hexane (1:2) to give 15 (1.37 g, 92%,
1
5a:15b = 10:3) as a colorless amorphous powder. Diastereomeric mixture 15 was dissolved in CCl
90 mL) and NBS (106.8 mg, 5.46 nmol) was added at 25 °C, and the reaction mixture was heated at
0 °C for 3 h. The reaction mixture was filtered through a short pad of Celite, and the filtrate was
washed with CCl . The combined filtrates were concentrated in vacuo, and the residue was used in the
next step without further purification. The above residue was dissolved in 2-propanol/ DMF (1:0.2)
72 mL) and was hydrogenated over 10% Pd/C (980 mg) at 25 °C for 11 h. The catalyst was removed
by filtration and washed with CHCl and MeOH. The combined filtrates were diluted with H
100 mL) and extracted with CHCl (100 mL × 3). The combined extracts were washed with
4
(
6
4
(
3
2
O
(
3
brine (100 mL), dried, and concentrated in vacuo. The residue was subjected to column
chromatography with ethyl acetate–hexane (1:4) to give 9 (1.05 g, 71% overall yield, 4 steps) as a
colorless amorphous powder.
3
.4. (Z)-(7,8,10-Trimethoxy-9-methyl-1,4-dioxo-3-(2,4,5-trimethoxy-3-methylbenzylidene)-1,3,4,6-
tetra-hydro-2H-pyrazino(1,2-b)isoquinolin-6-yl)methyl Benzoate (10)
t-
t-
A solution of BuOK in BuOH (1 M, 2.4 mL, 2.4 mmol) was added to a solution of 9 (988 mg,
.0 mmol) and 2,4,5-trimethoxybenzaldehyde (17) (420 mg, 2.0 mmol) in CH Cl (20 mL) over 1 h at
°C, and the reaction mixture was stirred at 25 °C for 2 h. The reaction mixture was diluted with
Cl (100 mL) and extracted with CH Cl (100 mL × 3). The combined extracts were
2
0
2
2
saturated NH
4
2
2
washed with brine (100 mL), dried, and concentrated in vacuo. The residue was subjected to column
chromatography with ethyl acetate–hexane (1:2) to give 10 (901 mg, 70%) as a pale yellow
amorphous powder.
1
Compound 10: H-NMR (400 MHz, CDCl
3
) δ: 9.40 (1H, s, N-H), 7.93 (2H, m, Ar-H), 7.49 (1H, m,
Ar-H), 7.36 (2H, m, Ar-H), 7.31 (1H, s, C11-H), 6.94 (1H, s, C3a-H), 6.68 (1H, dd, J = 6.8, 3.9 Hz,
C6-H), 6.62 (1H, s, C6′-H), 4.52 (1H, dd, J = 11.7, 6.8 Hz, C12-H), 4.45 (1H, dd, J = 11.7, 3.9 Hz,
C12-H), 3.98 (3H, s, C7-OMe), 3.84 (3H, s, C8-OMe), 3.83 (3H, s, C4′-OMe), 3.83 (3H, s, C5′-OMe),
1
3
3
.69 (3H, s, C10-OMe), 3.58 (3H, s, C2′-OMe), 2.24 (3H, s, C3′-Me), 2.19 (3H, s, C9-Me). C-NMR
100 MHz, CDCl ) δ: 166.1 (s, OCOPh), 157.4 (s, C4), 156.5 (s, C1), 153.3 (s, C8), 151.8 (s, C10),
49.6 (s, C5′), 149.2 (s, C2′), 149.0 (s, C4′), 146.0 (s, C7), 132.9 (d, Ph), 129.7 (s, Ph), 129.7 (d,
(
3
1
Ph × 2), 128.2 (d, Ph × 2), 126.6 (s, C3′), 126.4 (s, C9), 126.2 (s, C11a), 125.4 (s, C3), 121.6 (s, C1′),
20.6 (s, C6a), 119.1 (s, C10a), 114.0 (d, C3a), 111.9 (d, C6′), 110.8 (d, C11), 65.0 (t, C12), 62.0
q, C10-OCH ), 60.9 (q, C2′-OCH ), 60.7 (q, C7-OCH ), 60.4 (q, C4′-OCH ), 60.1 (q, C8-OCH ), 55.9
q, C5′-OCH ), 48.7 (d, C6), 9.5 (q, C3′-CH ), 9.3 (q, C9-CH ). FT-IR (KBr) cm : 3246, 1724, 1688,
628, 1489, 1468, 1452, 1414, 1387, 1369, 1323, 1269, 1248, 1090, 1067, 1003, 712. EI-MS
1
(
(
3
3
3
3
3
−1
3
3
3
1
+
m/z (%): 644 (M , 7), 509 (100), 481 (5), 232 (10). HR-EI-MS: calcd for C35
H
36
2
N O10, 644.2370,
found: 644.2369.

Mar. Drugs 2015, 13
4924
3
.5. ((3S*,6R*)-7,8,10-Trimethoxy-9-methyl-1,4-dioxo-3-(2,4,5-trimethoxy-3-methylbenzyl)-1,3,4,6-
tetrahydro-2H-pyrazino(1,2-b)isoquinolin-6-yl)methyl Benzoate (11a) and ((3R*,6R*)-7,8,10-
trimethoxy-9-methyl-1,4-dioxo-3-(2,4,5-trimethoxy-3-methylbenzyl)-1,3,4,6-tetrahydro-2H-pyrazino-
(
1,2-b)isoquinolin-6-yl)methyl Benzoate (11b)
A solution of 10 (644.0 mg, 1.0 mmol) in MeOH (50 mL) was hydrogenated over 10% Pd/C
(
213.0 mg) at 25 °C for 22 h. The catalyst was removed by filtration and washed with CHCl and
3
MeOH. The combined filtrate was concentrated in vacuo. The residue was subjected to column
chromatography with ethyl acetate–hexane (1:2) to give 11b (137.0 mg, 21%) as a pale yellow
amorphous powder, and with ethyl acetate–hexane (2:1) to give 11a (463.0 mg, 72%) as a pale yellow
amorphous powder.
1
Compound 11a: H-NMR (400 MHz, CDCl
3
) δ: 8.00 (2H, m, Ar-H), 7.53 (1H, m, Ar-H), 7.42
(
2H, m, Ar-H), 7.25 (1H, s, C11-H), 6.53 (1H, dd, J = 7.5, 4.2 Hz, C6-H), 6.51 (1H, s, C6′-H), 6.22
1H, s, N-H), 4.45 (1H, dd, J = 11.4, 7.5 Hz, C12-H), 4.34 (1H, dd, J = 10.3, 3.3 Hz, C3-H), 4.31 (1H,
(
dd, J =11.4, 4.2 Hz, C12-H), 3.97 (3H, s, C7-OMe), 3.82 (3H, s, C8-OMe), 3.80 (3H, s, C5′-OMe),
.78 (3H, s, C4′-OMe), 3.67 (3H, s, C10-OMe), 3.64 (3H, s, C2′-OMe), 3.52 (1H, dd, J =13.8, 3.3 Hz,
3
1
3
C3a-H), 2.73 (1H, dd, J =13.8, 10.3 Hz, C3a-H), 2.20 (3H, s, C3′-Me), 2.19 (3H, s, C9-Me). C-NMR
100 MHz, CDCl ) δ: 166.1 (s, OCOPh), 165.4 (s, C4), 160.7 (s, C1), 153.2 (s, C8), 151.8 (s, C10),
50.9 (s, C2′), 149.7 (s, C5′), 147.6 (s, C4′), 146.0 (s, C7), 133.1 (d, Ph), 129.9 (d, Ph × 2), 129.8
s, Ph), 128.3 (d, Ph × 2), 126.4 (s, C9), 126.3 (s, C3′), 126.3 (s, C11a), 123.7 (s, C1′), 121.2 (s, C6a),
(
3
1
(
1
19.1 (s, C10a), 111.5 (d, C6′), 110.7 (d, C11), 64.6 (t, C12), 62.0 (q, C10-OCH
0.6 (q, C2′-OCH ), 60.2 (q, C4′-OCH ), 60.1 (q, C8-OCH ), 56.0 (q, C5′-OCH
), 9.4 (q, C9-CH ).
3
), 60.8 (q, C7-OCH
3
),
6
3
3
3
3
) 55.3 (d, C3), 47.8 (d,
C6), 33.5 (t, C3a), 9.7 (q, C3′-CH
3
3
−1
FT-IR (KBr) cm : 3391, 1724, 1690, 1628, 1489, 1468, 1458, 1414, 1368, 1337, 1269, 1240, 1121,
+
1088. EI-MS m/z (%): 646 (M , 14), 511 (100), 509 (13), 483 (36), 260 (8), 232 (17), 195 (10).
HR-EI-MS: calcd for C35
H
38
N
2
O10, 646.2527, found: 646.2523.
1
Compound 11b: H-NMR (400 MHz, CDCl ) δ: 7.93 (2H, m, Ar-H), 7.52 (1H, m, Ar-H), 7.39 (2H,
3
m, Ar-H), 6.91 (1H, s, C11-H), 6.43 (1H, dd, J = 7.1, 4.4 Hz, C6-H), 6.39 (1H, s, C6′-H), 4.45 (1H, dd,
J = 8.9, 5.2 Hz, C3-H), 4.36 (1H, dd, J = 11.7, 7.1 Hz, C12-H), 4.31 (1H, dd, J =11.7, 4.4 Hz,
C12-H), 3.98 (3H, s, C7-OMe), 3.83 (3H, s, C8-OMe), 3.66 (3H, s, C4′-OMe), 3.62 (3H, s, C5′-OMe),
3
.61 (3H, s, C10-OMe), 3.57 (3H, s, C2′-OMe), 3.08 (1H, dd, J = 14.5, 8.9 Hz, C3a-H), 3.06 (1H, dd,
1
3
J = 14.5, 5.2 Hz, C3a-H), 2.17 (3H, s, C9-Me), 2.05 (3H, s, C3′-Me). C-NMR (100 MHz, CDCl
δ: 166.2 (s, OCOPh), 165.0 (s, C4), 160.6 (s, C1), 153.0 (s, C8), 151.5 (s, C10), 151.5 (s, C2′), 149.1
s, C5′), 147.7 (s, C4′), 145.9 (s, C7), 133.0 (d, Ph), 129.7 (s, Ph), 129.7 (d, Ph × 2), 128.3 (d, Ph × 2),
26.2 (s, C9), 125.8 (s, C3′), 125.4 (s, C11a or C10a), 121.9 (s, C1′), 120.7 (s, C6a), 119.0 (s, C11a or
C10a), 112.0 (d, C6′), 109.3 (d, C11), 64.9 (t, C12), 61.9 (q, C10-OCH ), 60.7 (q, C7-OCH ), 60.5 (q,
C2′-OCH ), 60.2 (q, C4′-OCH ), 60.1 (q, C8-OCH ), 57.0 (d, C3), 55.8 (q, C5′-OCH ), 47.9 (d, C6),
6.5 (t, C3a), 9.2 (q, C9-CH and C3′-CH
3
)
(
1
3
3
3
3
3
3
−
1
3
1
3
3
). FT-IR (KBr) cm : 3300, 1724, 1692, 1628, 1487, 1468,
+
414, 1379, 1321, 1271, 1119, 1088. EI-MS m/z (%): 646 (M , 11), 511 (100), 483 (39), 232 (16), 195
(
9). HR-EI-MS: calcd for C35
H
38
2
N O10, 646.2527, found: 646.2521.

Mar. Drugs 2015, 13
4925
3
1
.6. Isopropyl ((3S*,6R*)-7,8,10-Trimethoxy-9-methyl-1,4-dioxo-3-(2,4,5-trimethoxy-3-methylbenzyl)-
,3,4,6-tetrahydro-2H-pyrazino(1,2-b)isoquinolin-6-yl)methyl Benzoate (18)
A solution of 11a (16.47 g, 25 mmol), TEA (28.10 mL, 200 mmol), and DMAP (12.21 g,
1
00 mmol) in dichloromethane (400 mL) was cooled with ice water, and isopropyl chloroformate
(
40.08 mL, 350 mmol) was added dropwise over 30 min. The reaction mixture was stirred at 25 °C for
h. The organic layer was diluted with dichloromethane (500 mL), washed with 1 M aqueous HCl
500 mL ×2) and then water (500 mL), dried, and concentrated in vacuo to give a residue. The residue
4
(
was subjected to column chromatography with ethyl acetate–hexane (2:5) to give 18 (17.5 g, 96%) as a
yellow amorphous powder.
1
Compound 18: H-NMR (400 MHz, CDCl
3
) δ: 8.00 (2H, m, Ar-H), 7.48 (1H, m, Ar-H), 7.34 (2H,
m, Ar-H), 7.19 (1H, s, C11-H), 6.53 (1H, t, J = 3.4 Hz, C6-H), 5.24 (1H, t, J = 6.1 Hz, C3-H), 4.96
1H, sept, J = 6.3 Hz, CO CH(CH ), 4.65 (1H, dd, J = 11.5, 3.4 Hz, C12-H), 4.36 (1H, dd, J = 11.5,
.4 Hz, C12-H), 3.92 (3H, s, C7-OMe), 3.80 (3H, s, C4′-OMe or C5′-OMe), 3.78 (3H, s, C8-OMe),
.63 (3H, s, C4′-OMe or C5′-OMe), 3.59 (3H, s, C2′-OMe), 3.44 (3H, s, C10-OMe), 3.34 (1H, dd,
(
2
)
3 2
3
3
J = 13.7, 6.1 Hz, C3a-H), 3.17 (1H, dd, J = 13.7, 6.1 Hz, C3a-H), 2.10 (3H, s, C9-Me), 2.09 (3H, s,
1
3
C3′-Me), 1.28 (3H, d, J = 6.3 Hz, CO
100 MHz, CDCl ) δ: 165.7 (s, OCOPh), 163.4 (s, C4), 157.6 (s, C1), 153.4 (s, C8), 151.7 (s,
CO CH(CH ), 151.6 (s, C10), 151.4 (s, C2′), 149.0 (s, C4′ or C5′), 147.4 (s, C4′ or C5′), 145.8 (s,
C7), 133.0 (d, Ph), 129.7 (d, Ph × 2), 129.6 (s, Ph), 128.3 (d, Ph × 2), 126.8 (s, C11a or C10a), 126.2
s, C9), 125.6 (s, C3′), 122.5 (s, C1′), 120.0 (s, C6a), 119.3 (s, C11a or C12), 112.0 (d, C6′), 111.6 (d,
C11), 71.8 (d, CO CH(CH ), 66.3 (t, C12), 61.8 (q, C10-OCH ), 60.6 (q, C2′-OCH ), 60.5 (q,
C7-OCH ), 60.0 (q, C4′-OCH or C5′-OCH ), 60.0 (q, C8-OCH ), 59.4 (d, C3), 55.9 (q, C4′-OCH or
C5′-OCH ), 49.9 (d, C6), 35.8 (t, C3a), 21.6 (q, CO CH(CH ), 21.5 (q, CO CH(CH ), 9.7 (q,
C3′-CH ), 9.2 (q, C9-CH
2
CH(CH
3
)
2
), 1.20 (3H, d, J = 6.3 Hz, CO
2
CH(CH
3
2
) ). C-NMR
(
3
2
)
3 2
(
2
3
)
2
3
3
3
3
3
3
3
3
2
3
)
2
2
3 2
)
−1
3
3
). FT-IR (KBr) cm : 2938, 1722, 1684, 1468, 1418, 1391, 1375, 1344, 1271,
+
1
252, 1107, 1092, 1072, 712. EI-MS m/z (%): 732 (M , 12), 597 (100), 569 (19), 555 (6), 511 (8), 483
(21), 415 (9), 260 (13), 232 (19), 195 (13). HR-EI-MS: calcd for C39
H
44
2
N O12, 732.2894,
found: 732.2889.
3
.7. Isopropyl (6S*,9R*,15R*)-9-((benzoyloxy)methyl)-1,2,4,10,11,13-hexamethoxy-3,12-dimethyl-7-
oxo-6,7,9,15-tetrahydro-5H-6,15-epiminobenzo(4,5)azocino(1,2-b)isoquinoline-16-carboxylate (19)
A stirred solution of 18 (402.3 mg, 0.55 mmol) in THF (36 mL) was cooled with ice water and
lithium tri-tert-butoxyaluminohydride (1.12 g, 4.4 mmol) was added over 10 min. After continued
stirring at 25 °C for 30 min, anhydrous Na
quenched with water. The reaction mixture was filtered through Celite pad and then, the filtrate was
diluted with brine (200 mL) and extracted with CHCl (3 × 200 mL). The combined extracts were
2
SO
4
(2 g) was added and the reaction mixture was
3
washed with brine (200 mL), dried, and concentrated in vacuo to give a residue, which was used in the
next step without further purification. A solution of the residue as above in formic acid (36 mL) was
stirred at 25 °C for 30 min. The reaction mixture was concentrated in vacuo, and the residue was
diluted with saturated aqueous NaHCO
3
solution (80 mL) and extracted with CHCl (3 × 80 mL). The
3
combined extracts were washed with brine (80 mL), dried, and concentrated in vacuo to give a residue.

Mar. Drugs 2015, 13
4926
The residue was subjected to column chromatography with ethyl acetate–hexane (1:3) to give 19
(
322.7 mg, 82%) as a pale yellow amorphous powder.
1
Compound 19: H-NMR (400 MHz, DMSO, 140 °C) δ: 7.58 (2H, m, Ar-H), 7.54 (1H, m, Ar-H),
7
.41 (2H, m, Ar-H), 6.26 (1H, s, C14-H), 6.22 (1H, dd, J = 8.1, 4.2 Hz, C9-H), 5.93 (1H, d, J = 1.5,
C15-H), 4.98 (1H, m, 1.5 Hz, C6-H), 4.86 (1H, sept, J = 6.2 Hz, CO CH(CH )), 3.92 (1H, dd, J = 11.5,
.2 Hz, C16-H), 3.87 (3H, s, C2-OMe or C11-OMe), 3.87 (3H, s, C10-OMe), 3.85 (1H, dd, J = 11.5,
.1 Hz, C16-H), 3.75 (3H, s, C4-OMe or C13-OMe), 3.73 (3H, s, C2-OMe or C11-OMe), 3.67 (3H, s,
2
3
4
8
C4-OMe or C13-OMe), 3.51 (3H, s, C1-OMe), 3.09 (1H, br d, J =17.6 Hz, C5-H), 3.04 (1H, dd,
J =17.6, 3.9 Hz, C5-H), 2.15 (3H, s, C3-Me or C12-Me), 1.98 (3H, s, C3-Me or C12-Me), 1.23 (3H, d,
13
J = 6.2 Hz, CO
40 °C) δ: 165.4 (s, C7), 164.4 (s, OCOPh), 152.1 (s, CO
C11), 149.2 (s, C4 and C13), 145.0 (s, C2 or C11), 144.7 (s, C10), 132.3 (s, C14a), 131.9 (d, Ph),
28.7 (s, Ph), 128.3 (d, Ph × 2), 127.5 (d, Ph × 2), 124.5 (s, C3 or C12), 124.0 (s, C3 or C12), 123.7 (s,
C4a), 119.9 (s, C15a), 118.8 (s, C13a), 118.2 (s, C9a), 100.0 (d, C14), 68.8 (d, CO CH(CH ), 62.6 (t,
C16), 60.3 (q, C4-OCH or C13-OCH ), 59.8 (q, C2-OCH or C10-OCH or C11-OCH ), 59.2
or C11-OCH ), 59.1 (q, q, C2-OCH or C10-OCH or C11-OCH ), 58.9
), 58.6 (q, C1-OCH ), 52.5 (d, C6), 48.9 (d, C15), 45.5 (d, C9), 26.9 (t, C5),
), 20.9 (q, CO CH(CH ), 8.24 (q, C3-CH or C12-CH ), 8.18 (q, C3-CH or
2
CH(CH
3
)
2
), 1.21 (3H, d, J = 6.2 Hz, CO
2
CH(CH
3
)
2
). C-NMR (100 MHz, DMSO,
1
2
3 2
CH(CH )
), 151.7 (s, C1), 150.2 (s, C2 or
1
2
)
3 2
3
3
3
3
3
(
q, C2-OCH
q, C4-OCH
3
or C10-OCH
or C13-OCH
CH(CH
3
3
3
3
3
(
3
3
3
2
0.9 (q, CO
2
3
)
2
2
3
)
2
3
3
3
−1
C12-CH
3
). FT-IR (KBr) cm : 1717, 1707, 1686, 1647, 1466, 1414, 1362, 1344, 1298, 1269, 1109,
+
1
070, 1007, 964, 712. EI-MS m/z (%): 716 (M , 22), 581 (100), 553 (67), 234 (21). HR-EI-MS: calcd
for C39
H
44
2
N O11, 716.2945, found: 716.2942.
3
1
.8. ((6S*,9R*,15R*)-1,2,4,10,11,13-hexamethoxy-3,12-dimethyl-7-oxo-6,7,9,15-tetrahydro-5H-6,
5-epiminobenzo(4,5)azocino(1,2-b)isoquinolin-9-yl)methyl Benzoate (20)
Concentrated H
34 mL) at 0 °C over 5 min, and the reaction mixture was stirred at 25 °C for 4 h. The reaction mixture
was poured into water (40 mL) at 0 °C, basified with concentrated NH OH, and then extracted with
CHCl (3 × 100 mL). The combined extracts were washed brine (100 mL), dried, and concentrated in
2
SO (1.7 mL) was added to a stirred solution of 19 (322.7 mg, 0.45 mmol) in TFA
4
(
4
3
vacuo to give a residue. The residue was subjected to column chromatography with ethyl
acetate–hexane (1:3) to give 20 (267.6 mg, 94%) as a pale yellow amorphous powder.
1
Compound 20: H-NMR (400 MHz, CDCl
3
) δ: 7.70 (2H, m, Ar-H), 7.49 (1H, m, Ar-H), 7.38 (2H,
m, Ar-H), 6.41 (1H, dd, J = 8.1, 5.2 Hz, C9-H), 6.26 (1H, s, C14-H), 4.99 (1H, s, C15-H), 4.12 (1H, br
d, J = 6.3 Hz, C6-H), 3.95~3.89 (2H, overlapped, C16-H), 3.90 (3H, s, C10-OMe), 3.87 (3H, s,
C1-OMe), 3.76 (3H, s, C13-OMe), 3.76 (3H, s, C11-OMe), 3.67 (3H, s, C2-OMe), 3.44 (3H, s,
C4-OMe), 3.19 (1H, dd, J = 17.0, 1.3 Hz, C5-H), 3.07 (1H, dd, J = 17.0, 6.3 Hz, C5-H), 2.19 (3H, s,
13
C12-Me), 1.88 (3H, s, C3-Me). C-NMR (100 MHz, CDCl
3
) δ: 168.5 (s, C7), 166.0 (s, OCOPh),
152.5 (s, C4), 150.8 (s, C11), 149.9 (s, C2), 149.8 (s, C13), 146.3 (s, C1), 146.0 (s, C10), 136.1 (s,
C13a or C14a), 132.5 (d, Ph), 129.6 (d, Ph), 129.4 (s, Ph), 128.3 (d, Ph), 125.7 (s, C12), 125.3 (s,
C15a), 125.0 (s, C3), 121.5 (s, C4a), 120.4 (s, C13a or C14a), 119.5 (s, C9a), 99.8 (d, C14), 63.4 (t,
C16), 61.4 (q, C13-OCH
3
), 60.8 (q, C10-OCH
3
), 60.2 (q, C1-OCH
3
), 60.1 (q, C11-OCH ), 59.9
3

Mar. Drugs 2015, 13
4927
(
q, C2-OCH
3
), 59.3 (q, C4-OCH
3
), 53.9 (d, C6), 50.1 (d, C15), 45.7 (d, C9), 29.0 (t, C5), 9.3 (q,
−1
C12-CH ), 9.1 (q, C3-CH
3
3
). FT-IR (KBr) cm : 2938, 1722, 1680, 1638, 1466, 1412, 1362, 1271,
+
1
248, 1115, 1070, 1009, 964, 712. EI-MS m/z (%): 630 (M , 22), 495 (48), 467 (100), 234 (39), 204
(
18). HR-EI-MS: calcd for C35
H
38
N O
2 9
, 630.2577, found: 630.2581.
3
1
.9. ((6S*,9R*,15R*)-1,2,4,10,11,13-hexamethoxy-3,12,16-trimethyl-7-oxo-6,7,9,15-tetrahydro-5H-6,
5-epiminobenzo(4,5)azocino(1,2-b)isoquinolin-9-yl)methyl Benzoate (21)
A 37% aqueous solution of formaldehyde (6 mL) was added to a stirred solution of 20 (251.2 mg,
0
.4 mmol) in formic acid (7.0 mL) at 60 °C, and the reaction mixture was heated at 70 °C for 1 h. The
reaction mixture was diluted with 5% aqueous NaHCO
3
solution (80 mL) and extracted with CHCl
3
(
3 × 80 mL). The combined extracts were washed brine (80 mL), dried, and concentrated in vacuo to
give a residue. The residue was subjected to column chromatography with ethyl acetate–hexane (1:1)
to give 21 (247.3 mg, 96%) as a colorless amorphous powder.
1
Compound 21: H-NMR (400 MHz, CDCl
3
) δ: 7.71 (2H, m, Ar-H), 7.49 (1H, m, Ar-H),
7
.38 (2H, m, Ar-H), 6.43 (1H, t, J = 6.4 Hz, C9-H), 6.30 (1H, s, C14-H), 4.63 (1H, s, C15-H),
.92 (3H, s, C10-OMe), 3.91 (2H, d, J = 6.4 Hz, C17-H), 3.87 (3H, s, C1-OMe), 3.76 (6H, s,
3
C11-OMe and C13-OMe), 3.68 (1H, overlapped, C6-H), 3.67 (3H, s, C2-OMe), 3.43 (3H, s, C4-OMe),
.15 (1H, dd, J = 17.6, 4.1 Hz, C5-H), 3.11 (1H, br d, J = 17.6 Hz, C5-H), 2.55 (3H, s, N-Me), 2.20
3H, s, C12-Me), 1.89 (3H, s, C3-Me). 13C-NMR (100 MHz, CDCl
) δ: 168.1 (s, C7), 166.0 (s,
OCOPh), 152.4 (s, C4), 150.7 (s, C11), 149.8 (s, C2), 149.5 (s, C13), 146.1 (s, C1), 145.9 (s, C10),
3
(
3
1
32.7 (s, C14a), 132.5 (d, Ph), 129.6 (d, Ph × 2), 129.3 (s, Ph), 128.3 (d, Ph × 2), 126.0 (s, C15a),
25.7 (s, C12), 124.7 (s, C3), 121.1 (s, C4a), 119.9 (s, C13a), 119.6 (s, C9a), 102.7 (d, C14), 63.6 (t,
1
C17), 61.3 (q, C11-OCH
q, C11-OCH or C13-OCH
), 29.2 (t, C5), 9.2 (q, C12-CH
3
or C13-OCH
3
), 60.7 (q, C10-OCH
3
), 60.5 (d, C6), 60.1 (q, C1-OCH
3
), 60.0
(
3
3
), 59.8 (q, C2-OCH
3
), 59.2 (q, C4-OCH ), 56.8 (d, C15), 45.5 (d, C9),
3
−1
4
1
1
1.6 (q, N-CH
3
3
), 9.0 (q, C3-CH ). FT-IR (KBr) cm : 1722, 1678,
3
638, 1464, 1412, 1358, 1341, 1271, 1126, 1113, 1099, 1067, 1007, 712. EI-MS m/z (%): 644 (M+,
7), 509 (20), 481 (100), 248 (37), 218 (15). HR-EI-MS: calcd for C36
H
40
N
2
9
O , 644.2734,
found: 644.2733.
3
.10. (6S*,9R*,15R*)-9-(hydroxymethyl)-1,2,4,10,11,13-hexamethoxy-3,12,16-trimethyl-5,6,9,15-
tetrahydro-7H-6,15-epiminobenzo(4,5)azocino(1,2-b)isoquinolin-7-one (12)
A 10 M aqueous solution of lithium hydroxide monohydrate (77 μL, 0.77 mmol) was added to a
stirred solution of 21 (226.3 mg, 0.35 mmol) in THF (2.0 mL) and MeOH (0.7 mL), and stirring was
continued at 25 °C for 8 h. The reaction mixture was diluted with water (80 mL), and the mixture was
extracted with CHCl (3 × 80 mL). The combined extracts were washed with brine (80 mL), dried, and
3
concentrated in vacuo to give a residue. The residue was subjected to column chromatography with
ethyl acetate–hexane (2:1) to give 12 (184.1 mg, 97%) as a colorless amorphous powder.
1
Compound 12: H-NMR (400 MHz, CDCl
3
) δ: 6.27 (1H, s, C14-H), 6.08 (1H, dd, J = 7.9, 5.1 Hz,
C9-H), 4.70 (1H, brs, C15-H), 3.88 (3H, s, OMe), 3.86 (3H, s, OMe), 3.78 (3H, s, OMe), 3.77 (3H, s,
OMe), 3.75 (3H, s, OMe) 3.72 (1H, br t, C6-H), 3.66 (3H, s, OMe), 3.32 (1H, dt, J = 11.5, 5.1 Hz,

Mar. Drugs 2015, 13
4928
C17-H), 3.22 (1H, dt, J = 11.5, 7.9 Hz, C17-H), 3.21 (2H, d, J = 4.4 Hz, C5-H), 2.57 (3H, s, N-Me),
1
3
2
.19 (3H, s, C12-Me), 2.18 (3H, s, C3-Me), 1.37 (1H, t, J = 6.1 Hz, -OH). C-NMR (100 MHz,
CDCl ) δ: 169.1 (C7), 152.6 (C4), 150.7 (C11), 150.1 (C2), 149.7 (C13), 146.3 (C1), 145.8 (C10),
32.8 (C14a), 126.0 (C15a), 125.4 (C12), 125.1 (C3), 121.1 (C4a), 120.6 (C9a), 119.5 (C13a), 102.9
14), 64.6 (C17), 61.3 (-OCH ), 60.6 (-OCH ), 60.6 (C6), 60.1 (-OCH ), 60.1 (-OCH ), 60.0 (-OCH ),
9.8 (-OCH ), 56.5 (C15), 49.1 (C9), 41.6 (N-CH ), 29.3 (C5), 9.4 (-CH ), 9.2 (-CH ). FT-IR
3
1
(
3
3
3
3
3
5
3
3
3
3
−1
(
KBr) cm : 3468, 2940, 1672, 1636, 1466, 1412, 1341, 1248, 1113, 1065, 1007, 964. EI-MS m/z (%):
+
5
40 (M , 9), 509 (27), 481 (100), 248 (51). HR-EI-MS: calcd for C29
H
36
N
2
8
O , 540.2472,
found: 540.2473.
3
1
.11. ((6S*,9R*,15R*)-1,2,4,10,11,13-hexamethoxy-3,12,16-trimethyl-7-oxo-6,7,9,15-tetrahydro-5H-6,
5-epiminobenzo(4,5)azocino(1,2-b)isoquinolin-9-yl)methyl Benzoate (22)
To a stirred solution of 12 (216.0 mg, 0.4 mmol) in CH
solution of BBr (1.0 M, 2.40 mL, 2.4 mmol) over 5 min. Stirring was continued at the same
temperature for 1 h, and then at −20 °C for 14.5 h. The reaction mixture was diluted with water
200 mL) and extracted with 5% MeOH in CHCl (4 × 200 mL). The combined extracts were washed
with 5% NaHCO solution (200 mL), dried, and concentrated in vacuo to give a residue. A solution of
the above residue in 10 N HNO (5.0 mL) was stirred at 25 °C for 30 min. The reaction mixture was
2
Cl
2
(24 mL) at −78 °C was added a CH
2
Cl
2
3
(
3
3
3
diluted with water (150 mL) and extracted with ethyl acetate (3 × 200 mL). The combined extracts
were washed with brine (200 mL), dried, and concentrated in vacuo. The residue was subjected to
purification by silica gel chromatography with ethyl acetate to give 22 (130.0 mg, 67%) as a dark
purple amorphous powder.
1
Compound 22: H-NMR (500 MHz, CDCl
3
) δ: 6.26 (1H, s, 14-H), 5.96 (1H, dd, J = 7.1, 4.5 Hz,
), 3.97 (3H, s, OCH ), 3.74 (1H, dt, J = 6.5, 1.5 Hz, 6-H),
.50 (1H, dd, J = 11.4, 4.5 Hz, 17-H), 3.36 (1H, dd, J = 11.4, 7.1 Hz, 17-H), 2.96 (1H, dd, J = 19.8, 6.5
), 1.96 (3H, s, 3-CH or
), 1.62 (1H, br s, OH). C-NMR (125 MHz, CDCl ) δ: 186.6
9
-H), 4.55 (1H, s, 15-H), 4.02 (3H, s, OCH
3
3
3
Hz, 5-Hα), 2.89 (1H, dd, J = 19.8, 1.5 Hz, C5-Hβ), 2.51 (3H, s, N-CH
3
3
1
3
1
2-CH
3
), 1.94 (3H, s, 3-CH
3
or 12-CH
3
3
(
s, C-4), 185.0 (s, C-13), 180.5 (s, C-10 and C-1), 168.0 (s, C-7), 156.0 (s, C-11), 155.4 (s, C-2), 140.7
s, C-14a), 140.0 (s, C-4a), 136.5 (s, C-15a), 134.5 (s, C-13a), 129.1 (s, C-3), 127.6 (s, C-12), 125.0 (s,
(
C-9a), 101.8 (d, C-14), 62.9 (t, C-17), 61.1 (q, OCH
3
), 61.0 (q, OCH
3
), 59.6 (d, C-6), 54.4 (d, C-15),
−1
4
3
8.4 (d, C-9), 41.2 (q, N-CH
3
), 28.7 (t, C-5), 8.8 (q, Ar-CH
3
), 8.7 (q, Ar-CH
3
). FT-IR (KBr) cm :
+
+
347, 2951, 2855, 1654, 1616, 1568, 1450, 1373, 1310. LR-MS (FAB ): 481 [M + H] . HR-MS
+
(FAB ): calcd for C25
H
25
N
2
8
O , 481.1611, found: 481.1623.
3
.12. ((6S*,9R*,15R*)-2,11-dimethoxy-3,12,16-trimethyl-1,4,7,10,13-pentaoxo-1,5,6,7,9,10,13,15-
octa-hydro-4H-6,15-epiminobenzo(4,5)azocino(1,2-b)isoquinolin-9-yl)methyl (Z)-2-methylbut-2-
enoate (23)
A solution of angelic acid (601.0 mg, 6.0 mmol) in ether (30 mL) was cooled with ice water, and a
solution of oxalyl chloride (0.5 mL, 5.9 mmol) in DMF (46.0 μL, 592 mmol) was added dropwise over
5
min. The resulting solution was stirred at 25 °C for 2 h. Then, a solution of 22 (142.0 mg, 0.3 mmol)

Mar. Drugs 2015, 13
4929
in CH
2
Cl
.0 mL with a stream of argon, and CH
5 °C for 21 h. The reaction mixture was directly purified by silica gel chromatography with ethyl
2
(15 mL) was added over 5 min. The reaction mixture was concentrated to approximately
3
2
2
Cl (8.0 mL) was added. The resulting mixture was stirred at
2
acetate–hexane (2:1) to afford 23 (139.0 mg, 84%) as a dark purple film.
1
Compound 23: H-NMR (500 MHz, CDCl
3
) δ: 6.24 (1H, s, 14-H), 6.12 (1H, dd, J = 5.7, 2.9 Hz,
9
4
-H), 5.92 (1H, qq, J = 7.4, 1.4 Hz, 21-H), 4.50 (1H, br s, 15-H), 4.21 (1H, dd, J = 11.9, 5.7 Hz, 17-H),
.05 (3H, s, C11-OCH ), 4.01 (3H, s, 2-OCH ), 4.01 (1H, dd, J = 11.9, 2.9 Hz, 17-H), 3.72 (1H, dt,
3
3
J = 6.8, 1.4 Hz, 6-H), 2.95 (1H, dd, J = 19.8, 6.8 Hz, 5-Hβ), 2.84 (1H, dd, J = 19.8, 1.4 Hz, 5-H), 2.47
(
(
(
(
(
(
3H, s, N-CH
3
), 1.96 (3H, s, 12-CH
3
), 1.92 (3H, s, 3-CH
3
), 1.75 (3H, dq, J = 7.4, 1.4 Hz, 21-CH
3
), 1.57
1
3
1H, quint, J = 1.4 Hz, 20-CH
3
). C-NMR (125 MHz, CDCl ) δ: 186.5 (s, C-4), 184.9 (s, C-13), 180.5
3
s, C-1), 180.1 (s, C-10), 167.1 (s, C-7 and C-19), 156.2 (s, C-11), 155.2 (s, C-2), 140.6
s, C-14a), 139.8 (s, C-4a), 139.3 (d, C-21), 136.2 (s, C-15a), 134.6 (s, C-13a), 128.5 (s, C-3), 127.3
s, C-12), 126.8 (s, C-20), 124.2 (s, C-9a), 101.3 (d, C-14), 62.4 (t, C-17), 61.1 (q, 11-OCH
3
), 61.0
q, 2-OCH ), 59.5 (d, C-6), 54.3 (d, C-15), 47.1 (d, C-9), 41.1 (q, N-CH ), 28.3 (t, C-5), 20.2 (q, C-23),
3
3
−1
1
1
5.5 (q, C-22), 8.7 (q, 3-CH
3
), 8.6 (q, 12-CH
3
). FT-IR (KBr) cm : 2949, 1653, 1616, 1570, 1458,
+
309, 1228, 1153. EI-MS m/z (%): 562 (M , 5), 421 (100), 218 (40). HR-EI-MS: calcd for
C
30
H
30
N
2
9
O , 562.1951, found: 562.1952.
3
.13. Renieramycin I (1i)
A suspension of 23 (15.0 mg, 0.027 mmol) and SeO (29.6 mg, 0.27 mmol) in dioxane (3.0 mL)
2
and MeOH (1.0 mL) was heated at 100 °C for 6 days. The reaction mixture was filtered and the filter
cake was washed with ethyl acetate. The combined filtrates were concentrated in vacuo to give a
residue. Flash column chromatography on silica gel with ethyl acetate–hexane (2:3) afforded 1i
(
6.8 mg, 43%) as a dark red film and 24 (4.4 mg, 29%).
1
Renieramycin I (1i): H-NMR (400 MHz, CDCl
3
) δ: 6.26 (1H, s, 4-H), 6.07 (1H, dd, J = 5.5, 2.7
Hz, 1-H), 5.92 (1H, qq, J = 7.3, 1.4 Hz, 26-H), 4.54 (1H, d, J = 0.9 Hz, 11-H), 4.34 (1H, d, J = 1.4 Hz,
1
4-H), 4.16 (1H, dd, J = 12.1, 5.5 Hz, 22-H), 4.06 (3H, s, 7-OCH
2-H), 3.98 (3H, s, 17-OCH ), 3.74 (1H, br t, J = 1.4 Hz, 13-H), 3.62 (3H, s, 14-OCH
), 1.96 (3H, s, 6-CH ), 1.94 (3H, s, 16-CH ), 1.73 (3H, dq, J = 7.3, 1.4 Hz, 26-CH
). C-NMR (100 MHz, CDCl ) δ: 185.8 (s, C-15), 184.7 (s, C-5), 180.9 (s,
C-18), 180.1 (s, C-8), 167.1 (s, C-24), 164.1 (s, C-21), 156.1 (s, C-7), 155.2 (s, C-17), 139.2 (d, C-26),
38.6 (s, C-3), 137.4 (s, C-19), 137.0 (s, C-20), 134.2 (s, C-10), 129.3 (s, C-16), 127.4 (s, C-6), 126.7
s, C-25), 124.9 (s, C-9), 102.2 (d, C-4), 73.4 (d, C-14), 64.7 (d, C-13), 62.6 (t, C-22), 61.2
3
), 4.02 (1H, dd, J = 12.1, 2.7 Hz,
), 2.55 (3H, s,
), 1.55 (3H,
2
3
3
N-CH
3
3
3
3
1
3
quint, J = 1.4 Hz, 25-CH
3
3
1
(
(
q, 7-OCH
3
), 60.9 (q, 17-OCH
3
), 59.4 (q, 14-OCH
3
), 54.5 (d, C-11), 47.3 (d, C-1), 41.7 (q, N-CH
3
),
−1
2
1
0.2 (q, 25-CH ), 15.5 (q, 26-CH
3
3
), 8.9 (q, Ar-CH
3
), 8.7 (q, Ar-CH
3
). FT-IR (KBr) cm : 3429, 2949,
717, 1684, 1655, 1614, 1570, 1454, 1342, 1306, 1233, 1209, 1153, 1096. EI-MS m/z (%): 594
+
+
+
(
[M + 2H] , 0.7), 593 ([M + H] , 2), 592 (M , 6), 479 (19), 452 (26), 451 (100), 421 (20), 248 (25),
18 (11). HR-EI-MS: calcd for C30 10, 592.2057, found: 592.2056.
2
H
30
2
N O

Mar. Drugs 2015, 13
4930
3
1
.14. ((6S*,9R*,15R*)-5-hydroxy-2,11-dimethoxy-3,12,16-trimethyl-1,4,7,10,13-pentaoxo-
,5,6,7,9,10,13,15-octahydro-4H-6,15-epiminobenzo(4,5)azocino(1,2-b)isoquinolin-9-yl)
methyl (Z)-2-methylbut-2-enoate (24)
1H-NMR (500 MHz, CDCl
) δ: 6.28 (1H, s, 4-H), 6.09 (1H, dd, J = 6.0, 2.9 Hz, 1-H), 5.93 (1H, qq,
3
J = 7.3, 1.5 Hz, 26-H), 4.86 (1H, br d, J = 7.0 Hz, 14-H), 4.52 (1H, d, J = 1.1 Hz, 11-H), 4.19 (1H, dd,
J = 12.0, 6.0 Hz, 22-H), 4.06 (3H, s, 7-OCH ), 4.01 (3H, s, 17-OCH ), 3.99 (1H, dd, J = 12.0, 2.9 Hz,
2-H), 3.75 (1H, dd, J = 1.7, 1.1 Hz, 13-H), 2.88 (1H, br d, J = 7.0 Hz, OH), 2.55 (3H, s, N-CH ), 1.96
3H, s, 6-CH ), 1.93 (3H, s, 16-CH ), 1.75 (3H, dq, J = 7.3, 1.5 Hz, 26-CH ), 1.56 (3H, quint,
J = 1.5 Hz, 25-CH ). C-NMR (125 MHz, CDCl ) δ: 186.7 (s, C-15), 184.7 (s, C-5), 181.0 (s, C-18),
80.0 (s, C-8), 167.1 (s, C-24), 163.9 (s, C-21), 156.1 (s, C-7), 155.4 (s, C-17), 139.3 (d, C-26), 138.4
s, C-10 or C-20), 138.3 (s, C-10 or C-20), 136.9 (s, C-19), 134.3 (s, C-3), 128.8 (s, C-16), 127.4 (s,
C-6), 126.7 (s, C-25), 124.8 (s, C-9), 102.2 (d, C-4), 67.1 (d, C-13), 64.8 (d, C-14), 62.3 (t, C-22), 61.1
3
3
2
3
(
3
3
3
1
3
3
3
1
(
(
q, OCH
3
), 61.0 (q, OCH
3
), 54.7 (d, C-11), 47.2 (d, C-1), 41.5 (q, N-CH
3
), 20.2 (q, 25-CH ), 15.5 (q,
3
−1
2
6-CH
3
), 8.7 (q, Ar-CH
3
), 8.7 (q, Ar-CH
3
). FT-IR (KBr) cm : 3446, 2930, 2857, 1654, 1616, 1570,
+
1
456, 1307, 1233, 1211, 1153. EI-MS m/z (%): 578 (M , 5), 437 (100), 421 (48), 218 (20). HR-EI-MS:
calcd for C30
.15. Cribrostatin 4 (2) via 24
A suspension of 23 (112.4 mg, 0.2 mmol) and SeO
H
30
N
2
O10, 578.1900, found: 578.1899.
3
2
(110.9 mg, 1.0 mmol) in dioxane (30 mL) and
water (3.0 mL) was heated at 80 °C for 6 h. The reaction mixture was filtered and the filter cake was
washed with ethyl acetate (200 mL). The combined filtrates were concentrated in vacuo to give a
residue (248.3 mg). Flash column chromatography on silica gel (70 g) with hexane–ethyl acetate (1:1)
afforded 24 (72.5 mg, 63%) along with recovered 23 (16.2 mg, 14%). Dess-Martin periodinane (DMP,
4
45.3 mg, 1.05 mmol) was added to a stirred solution of 24 (57.8 mg, 0.1 mmol) in dichloromethane
15 mL), and the mixture was stirred at 25 °C for 3 h. The reaction mixture was diluted with THF
50 mL), saturated aqueous Na solution (50 mL) was added, and the mixture was stirred at 25 °C
(
(
2
S
2
O
3
for 2 h. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate
100 mL × 3). The combined extracts were washed with brine (100 mL), dried, and concentrated
(
in vacuo. The residue (230 mg) was purified by silica gel (9 g) flash column chromatography with
hexane–ethyl acetate (1:2) to give cribrostatin 4 (2: 81.0 mg, 70.0% from 23) as a dark red film.
1
H-, 13C NMR, and also IR spectral charts of synthetic renieramycin I and cribrostatin 4 are available
in the supplementary information.
4
. Conclusions
In summary, we have succeeded in reducing the number of steps in our first version of the total
synthesis of cribrostatin 4 through key intermediate 12, from 21 steps in 3.4% overall yield to 18 steps
in 8.3% overall yield. The main point of this alternative total synthesis is based on the Avendano’s
protocol introducing C3-C4 double bond in the early stage. We have completed the first total synthesis
of renieramycin I (1i), and the spectroscopic data provide unambiguous evidence that supports the
original structure of the natural product. The development of ways to utilize this approach for the

Mar. Drugs 2015, 13
4931
synthesis of other members of the C3-C4 unsaturated renieramycin family, and the examination of
their biological activities to evaluate the mechanism of action, are undergoing in our laboratory.
Acknowledgments
This work supported by Japan Society for the Promotion of Science (JSPS) Grant-in Aid for
Scientific Research (C) (No. 15K07873).
Author Contributions
Masashi Yokoya Keiichiro Kobayashi, and Mitsuhiro Sato: conducted the experimental.
Naoki Saito and Masashi Yokoya: wrote the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References and Notes
1.
2.
3.
Scott, J.D.; Williams, R.M. Chemistry and Biology of the Tetrahydroisoquinoline Antitumor
Antibiotics. Chem. Rev. 2002, 102, 1669–1730.
Avendaño, C.; de la Cuesta, E. Synthetic Chemistry with N-Acyliminium Ions Derived from
Piperazine-2,5-Diones and Related Compounds. Curr. Org. Synth. 2009, 6, 143–168.
Parameswaran, P.S.; Naik, C.G.; Kamat, S.Y.; Pramanik, B.N. Renieramycins H and I, Two
Novel Alkaloids from the Sponge Haliclona cribricutis Dendy. Indian J. Chem. 1998, 37B,
1
258–1263.
4.
5.
6.
7.
8.
9.
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. Isolation and Structure of Cribrostatins 1 and 2 from the Blue
Marine Sponge Cribrochalina sp. Can. J. Chem. 1992, 70, 1170–1175.
Pettit, G.R.; Knight, J.C.; Collins, J.C.; Herald, D.L.; Pettit, R.K.; Boyd, M.R.; Young, V.G.
Antineoplastic Agents 430. Isolation and Structure of Cribrostatins 3, 4, and 5 from the Republic
of Maldives Cribrochalina Species. J. Nat. Prod. 2000, 63, 793–798.
Pettit, G.R.; Collins, J.C.; Knight, J.C.; Herald, D.L.; Nieman, R.A.; Williams, M.D.; Pettit, R.K.
Antineoplastic Agents. 485. Isolation and Structure of Cribrostatin 6, a Dark Blue Cancer Cell
Growth Inhibitor from the Marine Sponge Cribrochalina sp. J. Nat. Prod. 2003, 66, 544–547.
Kubo, A.; Saito, N.; Sakai, H.; Takai, E.; Muranaka, R.; Itabashi, M.; Yazawa, K.; Mikami, Y.
Synthesis and Antitumor Evaluation of Octahydro-5-hydroxy-1,5-imino-3-benzazocin-4,7,10-triones.
Heterocycles 1997, 46, 309–320.
Saito, N.; Sakai, H.; Suwanborirux, K.; Pummangura, S.; Kubo, A. 13C-NMR Spectral
Assignment of 5-Hydroxy-1,5-imino-3-benzazocin-4,7,10-trione Derivatives: The Revised
Structure of Renieramycin H. Heterocycles 2001, 55, 21–28.
Chan, C.; Heid, R.; Zheng, S.; Guo, J.; Zhou, B.; Furuuchi, T.; Danishefsky, S.J. Total Synthesis
of Cribrostatin IV: Fine-tuning the Character of an Amide Bond by Remote Control. J. Am. Chem.
Soc. 2005, 127, 4596–4598.

Mar. Drugs 2015, 13
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1
1
1
0. Vincent, G.; Williams, R.M. Asymmetric Total Synthesis of (−)-Cribrostatin 4 (Renieramycin H).
Angew. Chem. Int. Ed. 2007, 46, 1517–1520.
1. Chen, X.; Zhu, J. Total Synthesis of the Marine Natural Product (−)-Cribrostatin 4
(
Renieramycin H). Angew. Chem. Int. Ed. 2007, 46, 3962–3965.
2. Yokoya, M.; Ito, H.; Saito, N. Synthesis of Renieramycins: Construction of the Core Ring System of
Cribrostatin 4 through Modified Pictet-Spengler Cyclization of 3,6-Bisarylpiperazine-2,5-diones with
Diethoxyethyl Benzoate. Chem. Pharm. Bull. 2011, 59, 787–792.
1
1
1
3. Yokoya, M.; Ito, H.; Saito, N. Chemistry of Renieramycins. Part 11: Total Synthesis
of (±)-Cribrostatin 4. Tetrahedron 2011, 67, 9185–9192.
4. Yokoya, M.; Shinada-Fujino, K.; Saito, N. Chemistry of Renieramycins. Part 9: Stereocontrolled
Total Synthesis of (±)-Renieramycin G. Tetrahedron Lett. 2011, 52, 2446–2449.
5. Yokoya, M.; Shinada-Fujino, K.; Yoshida, S.; Mimura, M.; Takada, H.; Saito, N. Chemistry of
Renieramycins. Part 12: An Improved Total Synthesis of (±)-Renieramycin G. Tetrahedron 2012,
6
8, 4166–4181.
1
1
1
1
6. For simplicity, natural product numbering was used in the manuscript, but IUPAC names and
numbers were used in the Experimental.
7. González, J.F.; de la Cuesta, E.; Avendaño, C. Short Stereocontrolled Synthesis of trans and
cis-Tetrahydro-pyrazinoisoquinolinediones. Tetrahedron Lett. 2003, 44, 4395–4398.
8. González, J.F.; Salazar, L.; de la Cuesta, E.; Avendaño, C. Synthesis of Phthalascidin Analogs.
Tetrahedron 2005, 61, 7447–7455.
9. Ortín, I.; González, J.F.; de la Cuesta, E.; Avendaño, C. Reactions Promoted by
(Hydroxy(tosyloxy)iodo)benzene in Pyrazino(1,2-b)isoquinolines. Tetrahedron 2010, 66,
6
46–652.
2
0. Kubo, A.; Saito, N.; Yamato, H.; Yamauchi, R.; Hiruma, K.; Inoue, S. Synthesis of Saframycins.
II. Preparations and Reactions of N-Methyl-2,5-piperazinediones. Chem. Pharm. Bull. 1988, 36,
2
607–2614.
2
1. Yokoya, M.; Kawachi, O.; Saito, N. Synthesis of Tetrahydroisoquinoline Antitumor Natural
Products: Construction of Tricyclic Lactams through Pictet-Spengler-type Cyclization of
N-methyl-3-arylmethylpiperazine-2,5-dione with Ethyl diethoxyacetate. Heterocycles 2008, 76,
1
497–1509.
2
2
2
2. Nakai, K.; Kubo, K.; Yokoya, M.; Saito, N. Preparation of Renieramycin Left-half Model
Compounds. Tetrahedron 2014, 70, 6529–6545.
3. The signal in the NMR spectrum (CDCl
3
, at 20 °C) of 19 was not split, which was a mixture of
two rotational isomers.
4. Saito, N.; Ōhira, Y.; Wada, N.; Kubo, A. Synthesis of saframycins. V. Selenium Oxide Oxidation
of Hexahydro-1,5-imino-3-benzazocin-7,10-dione; a Useful Method for Constructing Saframycins
C and D from Saframycin B. Tetrahedron 1990, 46, 7711–7728.
2
5. Saito, N.; Harada, S.; Inouye, I.; Yamaguchi, K.; Kubo, A. Synthesis of Saframycins. XII. Total
Synthesis of (−)-N-Acetylsaframycin Mx 2 and its epi-(+)-Enantiomer. Tetrahedron 1995, 51,
8
231–8246.

Mar. Drugs 2015, 13
4933
2
6. Mori, M.; Daikuhara, N.; Yamada, J.; Saito N. Selenium Oxide Oxidation of
Hexahydro-1,5-imino-3-benzazocine-7,10-dione in Aliphatic Alcohol for Conversion of
Renieramycin Marine Natural Products. Heterocycles 2012, 86, 317–330.
1
3
2
7. We cannot obtain any C NMR spectral data of natural renieramycin I (1i); see reference [3].
8. Du, J.; Watanabe, K.A. Facile Preparation of α-Acyloxyacetaldehyde, a Versatile Intermediate in
the Synthesis of Antiviral Nucleosides. Synth. Commun. 2004, 34, 1925–1930.
2
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