Synthesis and structure-activity relationship study of lamellarin derivatives

Article abstract of DOI:10.1021/np0104525

Ten derivatives (3-12) of marine alkaloid lamellarin D (1) were synthesized and evaluated for cytotoxicity against a HeLa cell line in an effort to examine their structure-activity relationships. It appeared that the hydroxyl groups at positions C-8 and C-20 of 1 were important structural requirements for cytotoxic activity, while the hydroxyl group at C-14 and the two methoxy groups at C-13 and C-21 were not necessary for the activity.

Full text of DOI:10.1021/np0104525

500
J . Nat. Prod. 2002, 65, 500-504
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip Stu d y of La m ella r in Der iva tives
Fumito Ishibashi,*,† Shinji Tanabe,† Tatsuya Oda,† and Masatomo Iwao‡
Division of Marine Life Science and Biochemistry, Faculty of Fisheries, and Department of Applied Chemistry,
Faculty of Engineering, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, J apan
Received September 14, 2001
Ten derivatives (3-12) of marine alkaloid lamellarin D (1) were synthesized and evaluated for cytotoxicity
against a HeLa cell line in an effort to examine their structure-activity relationships. It appeared that
the hydroxyl groups at positions C-8 and C-20 of 1 were important structural requirements for cytotoxic
activity, while the hydroxyl group at C-14 and the two methoxy groups at C-13 and C-21 were not necessary
for the activity.
Lamellarins are a group of polycyclic pyrrole alkaloids
that were first isolated from the marine prosobranch
mollusc Lamellaria sp. in 1985.1 Since that time, over 30
compounds belonging to this group have been isolated from
ascidians.2-7 Early findings on the biological activities of
lamellarins included inhibition of cell division of sea urchin
egg,1 cytotoxicity,3,5 and immunomodulatory activity.3 In
recent years, two important findings on the biological
activity of lamellarins have been reported. First, some
lamellarins showed equally good antitumor activity against
both multidrug-resistant (MDR) and their corresponding
parental cell lines.8 It was also revealed that lamellarin I
reverted multidrug-resistance (MDR) at nontoxic doses by
inhibiting P-glycoprotein (P-gp)-mediated drug efflux and
that the potency of lamellarin I as an MDR modulator was
9- to 16-fold higher than that of verapamil.8 Second, the
newly isolated lamellarin R 20-sulfate was reported to
inhibit HIV-1 integrase in vitro, with IC50 values of 16 µM
for terminal cleavage activity and 22 µM for strand transfer
activity.7 The same compound was also shown to inhibit
replication of the HIV-1 virus in cell cultures (IC50, 8 µM).7
In this paper, we report on the synthesis and cytotoxicity
of 10 derivatives (3-12) of lamellarin D (1) with varying
substituents on the aromatic rings and a study of their
structure-activity relationships.
quent lactonization. Thus, additions of excess triethylamine
to methanol solutions of 30-37 formed deep purple solu-
tions of the corresponding N-ylides, which, on refluxing,
underwent dehydrative cyclization with simultaneous aro-
matization and lactonization to yield compounds 11, 12,
and 38-43. Finally, lamellarin derivatives 3-8 were
obtained by hydrogenolysis of the corresponding benzyl
ethers over Pearlman’s catalyst.12 The tetrahydroxy deriva-
tive 9 was synthesized from 11 by cleavage of the methyl
ethers using boron tribromide.
The cytotoxicities of 1 and 11 were measured in terms
of the inhibition of colony formation (the colony assay)
using two normal (Vero and MDCK) and two tumor (HeLa
and XC) cell lines. IC50 values of 1 and 11 estimated from
dose-response curves are shown in Table 1. Although no
selectivity was observed with these cell lines, 1 showed
quite high activity with IC50 values of 10-23 nM, compa-
rable to that of mitomycin C. We also tested the cytotoxicity
of 1 by the MTT tetrazolium cytotoxic assay in MCDK cells;
however, it was found to be completely inactive, even at a
concentration of 1 mM (data not shown).
The cytotoxicity of lamellarin derivatives evaluated by
the colony assay in HeLa cells is shown in Table 2. Most
derivatives possessing hydroxyl groups at both the C-8 and
C-20 positions (1, 3, 6, 7) showed quite high activity, with
IC50 values of 10.5-70.0 nM. The low toxicity of 2 might
be partly due to its low solubility in the assay medium.
The hydroxyl functional group at C-20 appears to be an
essential prerequisite for activity since the activity of 4
(IC50, 0.85 µM), which lacks the hydroxyl group, decreased
markedly compared with that of 1 (IC50, 1.05 × 10-3 µM).
The importance of the 20-hydroxyl group for bioactivity is
also apparent when comparing the activity of 5 (IC50, 2.5
µM) with those of 11 (IC50, 5.7 µM) and 12 (IC50, >100 µM).
The hydroxyl group at C-8 might also be important for the
activity since methylation of both hydroxyl groups at C-8
and C-14 of 1 as 5 leads to severe decrease in activity,
whereas compound 7, which has the 8-hydroxy group but
lacks the 14-hydroxyl group of 1, still maintains high
activity. The presence of a hydroxyl group at C-14 and
methoxy groups at C-13 and C-21 appears not to affect the
activity since the 14-dehydroxy, 13-demethoxy, and 21-
demethoxy derivatives (7, 6, and 3, respectively) displayed
only a slight decrease in activity relative to their parent
compound.
Resu lts a n d Discu ssion
A synthetic route to the lamellarins of the 5,6-dehydro
type by assembly of benzylisoquinoline and 2-meth-
oxymethoxybenzoate was developed earlier in the total
synthesis of lamellarins D (1) and H (2).9 This method is
useful for the synthesis of lamellarin analogues since a
variety of compounds can be obtained by the appropriate
combination of readily available reagents including ben-
zylisoquiolines and benzoates. The synthesis of lamellarin
derivatives is outlined in Scheme 1. Condensation of
benzylisoquinolines 13-16, prepared by a standard pro-
cedure,10 with benzoates 17-21 in the presence of lithium
diisopropylamide (LDA) afforded adducts 22-29. N-Alky-
lation of these compounds with ethyl bromoacetate followed
by methanolysis of the methoxymethyl (MOM) protecting
group yielded the corresponding quaternary ammonium
salts 30-37. The lamellarin ring system (6H[1]benzopyrano-
[4′,3′:4′,5]pyrrolo[2,1-a]isoquinolin-6-one) was constructed
by N-ylide-mediated pyrrole ring formation11 and subse-
In conclusion, this study provided basic information
regarding the structure-activity relationship of lamellarin
D (1). The presence of hydroxyl groups at the C-8 and C-20
positions of 1 appears to be essential for cytotoxicity, while
* To whom correspondence should be addressed. Tel: +81-95-847-1111,
ext. 3178. Fax: +81-95-844-3516. E-mail: fumito@net.nagasaki-u.ac.jp.
† Division of Marine Life Science and Biochemistry, Faculty of Fisheries.
‡ Department of Applied Chemistry, Faculty of Engineering.
10.1021/np0104525 CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/14/2002

Lamellarin Derivatives
J ournal of Natural Products, 2002, Vol. 65, No. 4 501
Sch em e 1. Synthesis of Lamellarin Derivativesa
a
Reagents and conditions: (a) LDA, THF, -78 °C; (b) BrCH2COOEt, 70 °C; (c) cat. HCl, MeOH, reflux; (d) Et3N, MeOH, reflux; (e) H2, 10% Pd(OH)2-C,
EtOAc, rt, 1 atm; (f) BBr3, CH2Cl2, -78 °C then rt, 84% yield. bOverall yields from compound 22-29, respectively.
Ta ble 1. Cytotoxicities of Lamellarin D (1) and Compound 11
against Several Cell Lines, IC50 (nM)
Gemini 200, Gemini 300, and UNITY plus 500 instruments
at nearly 200, 300, and 500 MHz, respectively. EIMS and
HREIMS were recorded on a J EOL J MS-DX303 spectrometer.
Tetrahydrofuran (THF) was distilled from sodium metal/
benzophenone ketyl under an atmosphere of dry nitrogen prior
to use.
compound
HeLa
XC
Vero
MDCK
lamellarin D (1)
11
mitomycin C
10.5
5700
68.0
12.4
5600
NDa
10.5
4700
ND
22.5
5600
ND
Ben zylisoqu in olin es 13-16. Benzylisoquinolines 13-16
were prepared via Bishler-Napieralski reactions of corre-
sponding 2-methoxy-2-arylethylamides of aryl acetates (for a
detailed procedure, see ref 9). Benzylisoquinoline 16, papav-
erine, was obtained from Tokyo Chemical Industry Co. Ltd.
Ben zoa tes 17-21. Benzoates 17, 18, 19, 20, and 21 were
prepared by a standard methoxymethylation (MOMCl, NaH,
THF) of methyl 4-benzyloxy-5-methoxy-2-hydroxybenzoate,9
a
ND: not determined.
the hydroxyl group at C-14 and the two methoxy groups
at C-13 and C-21 appear to be less important.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points (mp)
were uncorrected. H NMR spectra were recorded on Varian
1

502 J ournal of Natural Products, 2002, Vol. 65, No. 4
Ishibashi et al.
Ta ble 2. Cytotoxic Activity of Lamellarin Derivatives on HeLa
Cells
16 and 17 in 50% yield by the same procedure as that for 22:
yellow amorphous powder; 1H NMR (300 MHz) δ 2.73 (3H, s,
OCH3), 3.75 (3H, s, ArOCH3), 3.82 (3H, s, ArOCH3), 3.87 (6H,
s, ArOCH3 × 2), 4.00 (3H, s, ArOCH3), 4.28 (1H, d, J ) 7.7
Hz, CH3OCH-O), 4.33 (1H, d, J ) 7.7 Hz, CH3OCH-O), 5.10
(1H, d, J ) 11.0 Hz, PhCH-O-), 5.13 (1H, d, J ) 11.0 Hz,
PhCH-O-), 6.63 (1H, s, H-1′), 6.67 (1H, s, H-5), 6.77-6.88 (3H,
m, H-2′′, H-5′′, H-6′′), 7.03 (1H, s, H-8), 7.25-7.42 (7H, m, C6H5-
CH2O, H-4, H-3′′′), 7.73 (1H, s, H-6′′′), 8.26 (1H, d, J ) 7.6 Hz,
H-3); anal. C 68.99%, H 5.89%, N 2.15%, calcd for C37H37NO9,
C 69.47%, H 5.83%, N 2.19%.
6,7-Dim eth oxy-1-[1-(3,4-d im eth oxyp h en yl)-2-(2-m eth -
oxym eth oxyp h en yl)-2-oxoeth yl]isoqu in olin e (25). The
title compound was synthesized from 16 and 18 in 71% yield
by the same procedure as that for 22: yellow amorphous
powder; 1H NMR (500 MHz) δ 2.92 (3H, s, OCH3), 3.76 (3H, s,
ArOCH3), 3.83 (3H, s, ArOCH3), 3.88 (3H, s, ArOCH3), 3.99
(3H, s, ArOCH3), 4.64 (1H, d, J ) 6.9 Hz, CH3OCH-O), 4.66
(1H, d, J ) 6.9 Hz, CH3OCH-O), 6.68 (1H, s, H-1′), 6.80 (1H,
d, J ) 8.0 Hz, H-5′′), 6.83-6.90 (2H, m, H-2′′, H-6′′), 7.02-
7.06 (2H, m, H-3′′′, H-5′′′), 7.03 (1H, s, H-5), 7.33-7.37 (1H,
m, H-4′′′), 7.35 (1H, d, J ) 5.7 Hz, H-4), 7.39 (1H, s, H-8), 8.02
(1H, dd, J ) 7.8, 1.6 Hz, H-6′′′), 8.26 (1H, d, J ) 5.7 Hz, H-3);
anal. C 68.96%, H 5.92%, N 2.79%, calcd for C29H29NO7, C
69.17%, H 5.81%, N 2.78%.
compound R1
R2
OMe OMe OH
OH OH OH
OMe OMe OH
OMe OMe OH
R3
R4
R5
OMe
OH
H
R6 IC50 (µM)
1
2
3
4
OH
OH
OH
OH
OH
0.0105
>100
0.0395
0.8500
2.5
0.0380
0.0700
4.0
OH
OH
H
OH
OH
OH
H
OMe
5
OMe OMe OMe OMe OMe
6
7
8
9
OH
OH
OH
OH
OMe
OMe OMe
H
OH
H
OMe
OMe
H
OMe OMe OH
OH OH OH
H
H
1.1
10
11
12
OAc OAc OAc OAc OAc
OMe OMe OMe OMe
OMe OMe OMe OMe -OCH2O-
OAc 11.0
H
H
5.7
>100
6,7-Dim eth oxy-1-[1-(3,4-d im eth oxyp h en yl)-2-(2-m eth -
oxym eth oxy-4,5-m eth ylen ed ioxyp h en yl)-2-oxo]eth yliso-
qu in olin e (26). The title compound was synthesized from 16
and 21 in 56% yield by the same procedure as that for 22:
yellow amorphous powder; 1H NMR (200 MHz) δ 2.79 (3H, s,
OCH3), 3.76 (3H, s, ArOCH3), 3.82 (3H, s, ArOCH3), 3.89 (3H,
s, ArOCH3), 4.00 (3H, s, ArOCH3), 4.43 (2H, s, CH3OCH2-O),
5.96 (2H, s, OCH2O), 6.62 (1H, s, H-1′), 6.63 (1H, s, H-3′′′),
6.76-6.90 (3H, m, H-2′′, H-5′′, H-6′′), 7.03 (1H, s, H-5), 7.32-
7.40 (2H, m, H-4, H-6′′′), 7.59 (1H, s, H-8), 8.27 (1H, d, J )
5.6 Hz, H-3); anal. C 65.38%, H 5.37%, N 2.75%, calcd for
methyl 2-hydroxybenzoate (Tokyo Chemical Industry Co. Ltd.),
methyl 4-benzyloxy-2-hydroxybenzoate,9 methyl 5-methoxy-2-
hydroxybenzoate prepared by methylation (MeI, K2CO3, ace-
tone) of methyl 2,5-dihydroxybenzoate (Aldrich Chemical Co.),
and methyl 2-hydroxy-4,5-methylenedioxybenzoate obtained
by MnO2-KCN-MeOH oxidation of 2-hydroxy-4,5-methyl-
enedioxybenzaldehyde,13 respectively.
6-Ben zyloxy-7-m eth oxy-1-[1-(4-ben zyloxy-3-m eth oxy-
ph en yl)-2-(4-ben zyloxy-2-m eth oxym eth oxyph en yl)-2-oxo-
eth yl]isoqu in olin e (22). To a solution of lithium diisopro-
pylamide prepared from diisopropylamine (0.290 mL, 2.06
mmol) and a 1.57 M hexane solution of butyllithium (0.79 mL,
1.24 mmol) in dry THF (8 mL) at -10 °C for 20 min was added
dropwise a solution of benzylisoquinoline 13 (405 mg, 0.824
mmol) in THF (10 mL) over 5 min at -78 °C under Ar. The
solution was gradually warmed to -10 °C and kept at that
temperature for 1 h. A solution of benzoate 19 (375 mg, 1.24
mmol) in THF (3 mL) was then added, and the mixture was
stirred at -10 °C for 1 h before being quenched with 10% NH4-
Cl solution (20 mL). The mixture was extracted twice with
EtOAc, dried (Na2SO4), and concentrated. The oily residue was
chromatographed on silica gel eluted with hexanes-EtOAc (2:
1-1:2), giving 22 (218 m g, 0.287 mmol) in 35% yield as an
yellow amorphous powder: 1H NMR (200 MHz) δ 2.72 (3H, s,
OCH3), 3.76 (3H, s, ArOCH3), 3.84 (3H, s, ArOCH3), 4.48 (1H,
d, J ) 6.9 Hz, CH3OCH-O), 4.55 (1H, d, J ) 6.9 Hz, CH3OCH-
O), 5.03 (2H, s, PhCH2O), 5.09 (2H, s, PhCH2O), 5.26 (2H, s,
PhCH2O), 6.62 (1H, s, H-1′), 6.64 (1H, s, H-5), 6.74-6.88 (3H,
m, H-2′′, H-5′′, H-6′′), 7.05 (1H, s, H-8), 7.20-7.50 (18H, m,
C6H5 × 3, H-4, H-3′′′, H-5′′′), 8.11 (1H, d, J ) 8.4 Hz, H-6′′′),
8.23 (1H, d, J ) 5.5 Hz, H-3); anal. C 75.71%, H 5.89%, N
1.95%, calcd for C48H43NO8, C 75.67%, H 5.69%, N 1.84%.
6-Ben zyloxy-7-m eth oxy-1-[1-(4-ben zyloxy-3-m eth oxy-
p h en yl)-2-(5-m eth oxy-2-m eth oxym eth oxyp h en yl)-2-oxo-
eth yl]isoqu in olin e (23). The title compound was synthesized
from 13 and 20 in 54% yield by the same procedure as that
for 22: yellow amorphous powder; 1H NMR (300 MHz) δ 2.85
(3H, s, OCH3), 3.77 (3H, s, ArOCH3), 3.78 (3H, s, ArOCH3),
3.85 (3H, s, ArOCH3), 4.52 (2H, s, OCH2OCH3), 5.11 (2H, s,
PhCH2O), 5.26 (2H, s, PhCH2O), 6.68 (1H, s, H-1′), 6.81 (1H,
s, H-5), 6.82-6.89 (3H, m, H-2′′, H-5′′, H-6′′), 7.05 (1H, s, H-8),
7.24-7.48 (12H, m, C6H5 x 2, H-4, H-3′′′, H-4′′′), 7.58 (1H, d,
J ) 2.8 Hz, H-6′′′), 8.23 (1H, d, J ) 5.3 Hz, H-3). anal. C
73.51%, H 5.84%, N 2.16%, calcd for C42H39NO8, C 73.56%, H
5.73%, N 2.04%.
C
30H29NO8, C 65.81%, H 5.34%, N 2.56%.
6-Ben zyloxy-7-m eth oxy-1-[1-(4-ben zyloxyp h en yl)-2-(4-
ben zyloxy-5-m eth oxy-2-m eth oxym eth oxyp h en yl)-2-oxo-
eth yl]isoqu in olin e (27). The title compound was synthesized
from 14 and 17 in 12% yield by the same procedure as that
for 22: yellow amorphous powder; 1H NMR (200 MHz) δ 2.68
(3H, s, OCH3), 3.86 (3H, s, ArOCH3), 3.81 (3H, s, ArOCH3),
4.31 (2H, s, PhCH2O), 4.99 (2H, s, CH3OCH2O), 5.11 (2H, s,
PhCH2O), 5.26 (2H, s, PhCH2O), 6.63 (1H, s, H-1′), 6.66 (1H,
s, H-5), 6.90 (2H, d, J ) 8.7 Hz, H-3′′, H-5′′), 7.04 (1H, s, H-6′′′),
7.21 (2H, d, J ) 8.7 Hz, H-2′′, H-6′′), 7.20-7.50 (18H, m, C6H5
× 3, H-4, H-8, H-3′′′), 8.27 (1H, d, J ) 5.6 Hz, H-3); anal. C
75.73%, H 5.68%, N 2.03%, calcd for C48H43NO8, C 75.67%, H
5.69%, N 1.84%.
6-Ben zyloxy-7-m et h oxy-1-[1-(3-m et h oxyp h en yl)-2-(4-
ben zyloxy-5-m eth oxy-2-m eth oxym eth oxyp h en yl)-2-oxo-
eth yl]isoqu in olin e (28). The title compound was synthesized
from 15 and 17 in 19% yield by the same procedure as that
for 22: yellow amorphous powder; 1H NMR (300 MHz) δ 2.96
(3H, s, OCH3), 3.71 (3H, s, ArOCH3), 3.87 (3H, d, ArOCH3),
3.88 (3H, d, ArOCH3), 4.32 (2H, s, CH3OCH2O), 5.13 (2H, s,
PhCH2O), 5.27 (2H, s, PhCH2O), 6.66 (1H, s, H-1′), 6.68 (1H,
s, H-5), 6.73-6.90 (3H, m, H-2′′, H-4′′, H-6′′), 7.20-7.50 (14H,
C6H5 × 2, H-4, H-5′′, H-3′′′, H-6′′′), 7.73 (1H, s, H-8), 8.24 (1H,
d, J ) 5.7 Hz, H-3).
6-Ben zyloxy-7-m eth oxy-1-[1-(4-ben zyloxy-3-m eth oxy-
ph en yl)-2-(2-m eth oxym eth oxyph en yl)-2-oxoeth yl]isoqu in -
olin e (29). The title compound was synthesized from 13 and
18 in 48% yield by the same procedure as that for 22: pale
1
crystals; mp 147-150 °C; H NMR (300 MHz) δ 2.86 (3H, s,
OCH3), 3.77 (3H, s, ArOCH3), 3.86 (3H, s, ArOCH3), 4.62 (1H,
d, J ) 7.5 Hz, CH3OCH-O), 4.68 (1H, d, J ) 7.5 Hz, CH3OCH-
O), 5.11 (2H, s, PhCH2O), 5.26 (2H, s, PhCH2O), 6.66 (1H, s,
H-1′), 6.81 (1H, s, H-5), 6.84 (1H, d, J ) 14 Hz, H-5′′), 6.95-
7.08 (2H, m, H-3′′′, H-5′′′), 7.05 (1H, s, H-8), 7.24-7.49 (14H,
m, C6H5 × 2, H-4, H-2′′, H-6′′, H-4′′′), 8.02 (1H, dd, J ) 7.9,
2.0 Hz, H-6′′′), 8.23 (1H, d, J ) 5.6 Hz, H-3); anal. C 74.64%,
H 5.84%, N 5.01%, calcd for C41H37NO7, C 75.10%, H 5.69%,
N 2.14%.
6,7-Dim eth oxy-1-[1-(3,4-d im eth oxyp h en yl)-2-(4-ben zyl-
oxy-5-m eth oxy-2-m eth oxym eth oxyp h en yl)-2-oxoeth yl]-
isoqu in olin e (24). The title compound was synthesized from

Lamellarin Derivatives
J ournal of Natural Products, 2002, Vol. 65, No. 4 503
3,11-Bis(ben zyloxy)-12-m eth oxy-14-(4-ben zyloxy-3-m eth -
oxyp h en yl)-6H[1]ben zop yr a n o[4,3;4,5]p yr r olo[2,1-a ]iso-
qu in olin e-6-on e (38). A mixture of 22 (201.7 mg, 0.2647
mmol) was heated together with ethyl bromoacetate (1 mL)
at 70 °C for 22 h under Ar. The mixture was poured into dry
ether, and the precipitated quaternary ammonium salt was
collected by filtration. The salt was dissolved in MeOH (20 mL)
containing 0.2 mL of concentrated HCl and heated under
reflux for 30 min. After the mixture had been cooled to room
temperature, Et3N (1.0 mL) was added and the resulting deep
purple solution was heated under reflux for 2 h, at which time
white crystals of 38 appeared. The mixture was then concen-
trated, and the residue was chromatographed on silica gel
eluted with CH2Cl2-EtOAc (1:1) to give 38 (53.5 mg, 0.0723
mmol) in 27% yield as white crystals: mp 227-228 °C; 1H
NMR (300 MHz) δ 3.31 (3H, s, OCH3), 3.88 (3H, s, OCH3), 5.09
(2H, s, PhCH2O), 5.24 (2H, s, PhCH2O), 5.28 (1H, d, J ) 12.6
Hz, PhCH-O), 5.38 (1H, d, J ) 12.6 Hz, PhCH-O), 6.71 (1H,
dd, J ) 2.6, 8.8 Hz, H-6′), 6.97 (1H, d, J ) 7.4 Hz, H-1), 7.01
(1H, d, J ) 2.6 Hz, H-2′), 7.05 (1H, s, H-10), 7.08 (1H, s, H-13),
7.15 (1H, dd, J ) 6.3, 8.9 Hz, H-5), 7.30-7.55 (18H, m, C6H5
× 3, H-4, H-9, H-5′), 9.19 (1H, d, J ) 7.4 Hz, H-8); EIMS m/z
739 (M+, 1.7), 648 (1.1), 557 (4.1), 483 (1.9), 453 (2.6), 354 (4.1),
278 (3.1), 105 (2.4), 91 (7.2), 69 (3.8), 57(4.5), 55 (4.1) 44 (100),
43 (9.0); HREIMS m/z 739.2620 (calcd for C48H37NO7, 739.2570).
Hz, H-8); anal. C 78.23%, H 5.15%, N 2.01%, calcd for C48H37-
NO7, C 77.93%, H 5.04%, N 1.89%.
3,11-Bis(ben zyloxy)-2,12-dim eth oxy-14-(3-m eth oxyph en -
yl)-6H[1]-ben zopyr an o[4,3;4,5]pyr r olo[2,1-a ]isoqu in olin e-
6-on e (42): 17% yield from 28; white crystals; mp 270-271
°C; 1H NMR (300 MHz) δ 3.44 (3H, s, OCH3), 3.46, 3H, s,
OCH3), 3.84 (3H, s, OCH3), 5.20 (2H, s, PhCH2O), 5.26 (2H, s,
PhCH2O), 6.72 (1H, s, H-4), 6.95 (1H, s, H-1), 6.99 (1H, dd, J
) 7.5, 0.7 Hz, H-4′), 7.11 (1H, s, H-10), 7.09-7.18 (3H, m, H-13,
H-2′, H-6′), 7.22-7.48 (11H, m, C6H5 × 2, H-9, H-10), 7.57 (1H,
t, J ) 8.1 Hz, H-5′), 9.20 (1H, d, J ) 7.2 Hz, H-8); EIMS m/z
663 (M+, 10), 572 (9), 105 (28), 91 (31), 77 (18), 69 (10), 57
(11), 55 (14), 51 (8), 44 (100), 43 (21); HREIMS m/z 663.2220
(calcd for C42H33NO7, 663.2257).
11-(Ben zyloxy)-12-m eth oxy-14-(4-ben zyloxy-3-m eth ox-
yph en yl)-6H[1]ben zopyr an o[4,3;4,5]pyr r olo[2,1-a ]isoqu in -
olin e-6-on e (43): 15% yield from 29; white crystals; mp 223-
225 °C; 1H NMR (300 MHz) δ 3.32 (3H, s, OCH3), 3.88 (3H, s,
OCH3), 5.23 (2H, s, PhCH2O), 5.28 (1H, d, J ) 12.5 Hz, PhCH-
O), 5.39 (1H, d, J ) 12.5 Hz, PhCH-O), 6.97 (1H, d, J ) 7.5
Hz, H-5′), 7.02-7.18 (7H, m, ArHs), 7.25-7.58 (12H, m, ArHs),
9.22 (1H, d, J ) 7.3 Hz, H-8); anal. C 77.96%, H 5.14%, N
2.13%, calcd for C41H31NO6, C 77.71%, H 4.93%, N, 2.21%.
La m ella r in D (1), La m ella r in H (2), a n d Hexa a ceta te
of 2 (10). See ref 9.
3,11-Dih yd r oxy-12-m eth oxy-14-(4-ben zyloxy-3-m eth ox-
yph en yl)-6H[1]ben zopyr an o[4,3;4,5]pyr r olo[2,1-a ]isoqu in -
olin e-6-on e (3). Compound 38 (48.5 mg, 0.0656 mmol) was
hydrogenated over 20% Pd(OH)2-C (20 mg) in EtOAc (80 mL)
at room temperature at atmospheric pressure for 8 h. The
mixture was then filtered, and the filtrate was concentrated
in vacuo. The residue was chromatographed on silica gel eluted
with hexanes-EtOAc (1:2) to give 3 (17.8 mg, 0.0379 mmol)
in 58% yield as a light gray amorphous powder: 1H NMR (300
MHz, acetone-d6) δ 3.47 (3H, s, OCH3), 3.90 (3H, s, OCH3),
6.64 (1H, dd, J ) 2.4, 8.7 Hz, H-6′), 6.86 (1H, d, J ) 2.4 Hz,
H-2′), 7.03-7.28 (7H, m, ArHs), 8.01 (1H, br s, OH), 8.36 (1H,
br s, OH), 8.98 (lH, br s, OH), 9.14 (1H, d, J ) 7.4 Hz, H-8);
EIMS m/z 469 (M+, 1.2), 199 (2.5), 111 (1.6), 98 (2.5), 97 (2.8),
95 (2.1), 85 (2.4), 84 (2.1), 83 (3.4), 81 (2.2), 71 (3.4), 69 (5.6),
57 (6.6), 55 (5.4), 45 (3.3), 44 (100), 43 (7.6); HREIMS m/z
469.1174 (calcd for C27H19NO7, 469.1162).
11-Hyd r oxy-2,12-d im et h oxy-14-(4-h yd r oxy-3-m et h ox-
yph en yl)-6H[1]ben zopyr an o[4,3;4,5]pyr r olo[2,1-a ]isoqu in -
olin e-6-on e (4): 76% yield from 39; light gray amorphous
powder; 1H NMR (300 MHz, acetone-d6) δ 3.49 (3H, s, OCH3),
3.53 (3H, s, OCH3), 3.92 (3H, s, OCH3), 6.89 (1H, d, J ) 3.0
Hz, H-1), 6.99 (1H, dd, J ) 9.0, 3.0 Hz, H-3), 7.02-7.28 (6H,
m, H-9, H-10, H-13 and 14-ArHs), 8.02 (1H, s, OH), 8.37 (1H,
s, OH), 9.19 (1H, d, J ) 7.2 Hz); EIMS m/z 484 [(M + 1)+, 28],
483 (M+, 86), 86 (26), 84 (41), 58 (23), 49 (41), 44 (100), 43
(54), 42 (23); HREIMS m/z 483.1313 (calcd for C28H21NO7,
483.1318).
3-Hyd r oxy-14-(3,4-d im eth oxyp h en yl)-2,11,12-tr im eth -
oxy-6H[1]ben zopyr an o[4,3;4,5]pyr r olo[2,1-a]isoqu in olin e-
6-on e (5): 81% yield from 40; pale yellow amorphous powder;
1H NMR (300 MHz, acetone-d6) δ 3.42 (3H, s, OCH3), 3.47 (3H,
s, OCH3), 3.90 (3H, s, OCH3), 3.95 (3H, s, OCH3), 3.96 (3H, s,
OCH3), 6.78 (1H, s, H-4), 6.88 (1H, s, H-1), 7.20 (1H, s, H-10),
7.20-7.35 (4H, m, 14-ArHs and H-9), 7.36 (1H, s, H-13), 8.25
(1H, br s, OH), 9.16 (1H, d, J ) 7.4 Hz, H-8); anal. C 68.00%,
H 5.09%, N 2.39%, calcd for C30H25NO8, C 68.30%, H 4.78%,
N 2.66%.
3,11-(Dih ydr oxy)-2,12-dim eth oxy-14-(4-h ydr oxyph en yl)-
6H[1]ben zop yr a n o-[4,3;4,5]p yr r olo[2,1-a ]isoqu in olin e-6-
on e (6): 75% yield from 41; light gray amorphous powder;
1H NMR (200 MHz, acetone-d6) δ 3.48 (3H, s, OCH3), 3.49 (3H,
s, OCH3), 6.79 (1H, s, H-4), 6.88 (1H, s, H-1), 7.17 (1H, d, J )
7.4 Hz, H-9), 7.20 (2H, d, J ) 8.6 Hz, H-3′ and H-5′), 7.22 (1H,
s, H-10), 7.25 (1H, s, H-13), 7.49 (2H, d, J ) 8.6 Hz, H-2′ and
H-6′), 8.26 (1H, br s, OH), 8.35 (1H, br s, OH), 8.76 (1H, br s,
OH), 9.13 (1H, d, J ) 7.4 Hz, H-8); EIMS m/z 470 [(M + 1)+,
7], 469 (M+, 20), 97 (8), 83 (9), 78 (48), 73 (8), 71 (9), 69 (12),
63 (54), 44 (100); HREIMS m/z 469.1158 (calcd for C27H19NO7,
469.1162).
11-(Ben zyloxy)-2,12-dim eth oxy-14-(4-ben zyloxy-3-m eth -
oxyp h en yl)-6H[1]ben zop yr a n o[4,3;4,5]p yr r olo[2,1-a ]iso-
qu in olin e-6-on e (39): 33% yield from 23; white crystals; mp
1
239-241 °C; H NMR (300 MHz) δ 3.35 (3H, s, OCH3), 3.44
(3H, s, OCH3), 3.90 (3H, s, OCH3), 5.23 (1H, d, J ) 3.8 Hz,
PhCH-O), 5.24 (2H, s, PhCH2O), 5.32 (1H, d, J ) 3.8 Hz,
PhCH-O), 6.77 (1H, d, J ) 3.0 Hz, H-1), 6.91 (1H, dd, J ) 3.0,
7.4 Hz, H-3), 7.50 (1H, d, J ) 7.4 Hz, H-4), 7.10-7.20 (3H, m,
H-2′, H-5′, H-6′), 7.13 (1H, s, H-10), 7.250-7.65 (12H, m, C6H5
× 2, H-9, H-13), 9.24 (1H, d, J ) 7.6 Hz, H-8); EIMS m/z 664
[(M + 1)+, 9], 663 (M+, 19), 572 (12), 481 (5), 354 (8), 272 (9),
207 (5), 149 (6), 91 (31), 58 (26), 44 (100), 43 (77); HREIMS
m/z 663.2279 (calcd for C42H33NO7, 663.2257).
3-Ben zyloxy-14-(3,4-dim eth oxyph en yl)-2,11,12-tr im eth -
oxy-6H[1]ben zopyr an o[4,3;4,5]pyr r olo[2,1-a ]isoqu in olin e-
6-on e (40): 28% yield from 24; white crystals; mp 253-254
°C; 1H NMR (300 MHz) δ 3.48 (3H, s, OCH3), 3.50 (3H, s,
OCH3), 3.92 (3H, s, OCH3), 3.93 (3H, s, OCH3), 4.00 (3H, s,
OCH3), 5.04 (2H, s, PhCH2O), 6.75 (1H, s, H-4), 6.81 (1H, s,
H-1), 6.95-7.05 (3H, s, H-2′, H-5′, H-6′), 7.12-7.43 (8H, m,
C6H5, H-9, H-10, H-13), 9.12 (1H, d, J ) 8.2 Hz, H-8); anal. C
71.93%, H 5.30%, N 2.15%, calcd for C37H31NO8, C 71.85%, H
5.06%, N 2.27%.
11,12-Dim eth oxy-14-(3,4-dim eth oxyph en yl)-6H[1]ben zo-
pyr an o-[4′,3′;4,5]pyr r olo[2,1-a ]isoqu in olin e-6-on e (11): 33%
1
yield from 25; white crystals; mp 254-256 °C; H NMR (300
MHz) δ 3.47 (3H, s, OCH3), 3.87 (3H, s, OCH3), 3.99 (3H, s,
OCH3), 4.03 (3H, s, OCH3), 7.03-7.20 (6H, m, H-9, H-10, H-13,
H-2′′, H-5′′, H-6′′), 7.26-7.46 (4H, m, H-1, H-2, H-3, H-4), 9.28
(d, 1H, J ) 7.4 Hz, H-8); EI-MS m/z 482 [(M + 1)+, 33%], 481
(M+, 100%); anal. C 71.59%, H 4.82%, N 2.93%, calcd for
C
29H23NO6, C 72.34%, H 4.81%, N 2.91%.
11,12-Dim eth oxy-14-(3,4-d im eth oxyp h en yl)-2,3-m eth -
ylen ed ioxy-6H[1]ben zop yr a n o[4′,3′;4,5]p yr r olo[2,1-a ]iso-
qu in olin e-6-on e (12): 28% yield from 26; white crystals; mp
1
267-268 °C; H NMR (200 MHz) δ 3.45 (3H, s, OCH3), 3.87
(3H, s, OCH3), 3.98 (3H, s, OCH3), 4.02 (3H, s, OCH3), 5.97
(2H, s, OCH2O), 6.69 (1H, s, H-4), 6.94-7.15 (7H, m, H-1, H-9,
H-10, H-13, H-2′, H-5′, H-6′), 9.24 (1H, d, J ) 7.24, H-8); anal.
C 67.88%, H 4.44%, N 2.68%, calcd for C30H23NO8, C 68.57%,
H 4.41%, N 2.67%.
3,11-Bis(b en zyloxy)-2,12-d im et h oxy-14-(4-b en zyloxy-
p h en yl)-6H[1]ben zop yr a n o[4,3;4,5]p yr r olo[2,1-a ]isoqu in -
olin e-6-on e (41): 58% yield from 27; white crystals; mp 198
°C; 1H NMR (200 MHz) δ 3.37 (3H, s, OCH3), 3.40 (3H, s,
OCH3), 5.19 (2H, s, PhCH2O), 5.23 (2H, s, PhCH2O), 5.25 (2H,
s, PhCH2O), 6.68 (1H, s, H-4), 6.94 (1H, s, H-1), 6.96 (2H, d, J
) 7.0 Hz, H-3, H-5), 7.10 (2H, d, J ) 7.0 Hz, H-2, H-6), 7.40-
7.57 (18H, m, C6H5 × 3, H-9, H-10, H-13), 9.18 (1H, d, J ) 7.2

504 J ournal of Natural Products, 2002, Vol. 65, No. 4
Ishibashi et al.
3,11-(Dih ydr oxy)-2,12-dim eth oxy-14-(3-m eth oxyph en yl)-
6H[1]-ben zop yr a n o[4,3;4,5]p yr r olo[2,1-a ]isoqu in olin e-6-
on e (7): 72% yield from 42; light gray amorphous powder;
1H NMR (200 MHz, acetone-d6) δ 3.42. (6H, s, OCH3 × 2), 3.78
(3H, s, OCH3), 5.72 (1H, br s, OH), 5.81 (1H, br s, OH), 6.60
(1H, s, H-4), 6.95 (1H, s, H-1), 6.90-7.16 (4H, m, 14-ArHs),
7.03 (1H, s, H-10), 7.12 (1H, s, H-13), 7.48 (1H, d, J ) 7.4 Hz,
H-9), 9.15 (1H, d, J ) 7.4 Hz, H-8); EIMS m/z 484 [(M + 1)+,
24], 483 (M+, 74), 264 (10), 256 (9), 236 (7), 129 (11), 111 (11),
97 (21), 83 (26), 81 (21), 69 (40), 57 (46), 55 (31), 44 (100), 43
(56), 41 (55); HREIMS m/z 483.1329 (calcd for C28H21NO7,
483.1318).
11-Hydr oxy-12-m eth oxy-14-(4-h ydr oxy-3-m eth oxyph en -
yl)-6H[1]ben zop yr a n o[4,3;4,5]p yr r olo[2,1-a ]isoqu in olin e-
6-on e (8): 50% yield from 43; light gray crystals; mp 300 °C;
1H NMR (200 MHz, DMSO-d6) δ 3.38 (3H, s, OCH3), 3.77 (3H,
s, OCH3), 6.95-7.50 (10H, m, ArHs), 9.08 (1H, d, J ) 7.5 Hz,
H-8), 9.37 (1H, s,OH), 9.94 (1H, s, OH); EIMS m/z 454 [(M +
1)+, 16], 453 (M+, 41), 86 (17), 84 (26), 58 (13), 51 (12), 49 (30),
44 (100), 43 (43); HREIMS m/z 453.1218 (calcd for C27H19NO6,
453.1213).
acetate by a standard acetylation (Ac2O, pyridine) followed by
recrystallization from aqueous EtOH: anal. C 66.38%, H
4.25%, N 2.32%, calcd for C33H23NO10, C 66.78%, H 3.91%, N,
2.32%.
Cytotoxicity Assa y. The cytotoxicity of test compounds
was determined by the inhibitory activity of colony formation
of HeLa cells as described previously.14 In brief, 100-150 cells
per well in
a 24-well plate were cultured with varying
concentrations of each test compound in the growth medium
(MEM containing 10% fetal calf serum) for 5-8 days at 37 °C
under 5% CO2/95% air. The number of colonies formed was
counted after staining with 1% methylene blue in 50%
methanol. Clusters of 40 or more cells were considered as
colonies.
Ack n ow led gm en t. Financial support of this work by a
Grant-in-Aid for Scientific Research No. 11660207 from the
Ministry of Education, Science and Culture, J apan, is acknowl-
edged.
Refer en ces a n d Notes
(1) Andersen, R. J .; Faulkner, D. J .; Cun-heng, H.; Van Duyne, G. D.;
Clardy, J . J . Am. Chem. Soc. 1985, 107, 5492-5495.
(2) Lindquist, N.; Fenical, W.; Van Duyne, G. D.; Clardy, J . J . Org. Chem.
1988, 53, 4570-4574.
11,12-D ih y d r o x y -14-(3,4-d ih y d r o x y p h e n y l)-6H [1]-
ben zop yr a n o-[4′,3′;4,5]p yr r olo[2,1-a ]isoqu in olin e-6-on e
(9). To a solution of 11 (206 mg, 0.426 mmol) in dry CH2Cl2
(20 mL) was added dropwise a solution of BBr3 (0.162 mL, 1.71
mmol) in CH2Cl2 (5 mL) at -10 °C. The mixture was gradually
warmed to room temperature and stirred for 4 h. The reaction
was quenched with a 4.7 M NH4OH solution (3 mL), and the
aqueous layer was made acidic (pH ) 5) with a 2 M HCl
solution. The mixture was extracted three times with a
mixture of EtOAc and THF (30 mL × 3), dried (Na2SO4), and
concentrated. The residue was chromatographed on silica gel
eluted with hexane-THF (1:2) to give 213 mg of crude 9, which
was recrystallized from THF-CH2Cl2 to give 9 (152 mg, 0.358
mmol, 84%) as pale yellow crystals: mp not determined (over
(3) Carroll, A. R.; Bowden, B. F.; Coll, J . C. Aust. J . Chem. 1993, 46,
489-501.
(4) Urban, S.; Capon, R. J . Aust. J . Chem. 1996, 49, 711-713.
(5) Reddy, M. V. R.; Faulkner, D. J .; Venkateswarlu, Y.; Rao, M. R.
Tetrahedron 1997, 53, 3457-3466.
(6) Davis, R. A.; Carroll, A. R.; Pierens, G. K.; Quinn, R. J . J . Nat. Prod.
1999, 62, 419-424.
(7) Reddy, M. V. R.; Rao, M. R.; Rhodes, D.; Hansen, M. S. T.; Rubins,
K.; Bushman, F. D.; Venkateswarlu, Y.; Faulkner, D. J . J . Med. Chem.
1999, 42, 1901-1907.
(8) Quesada, A. R.; Garc´ıa Gra´valos, M. D.; Ferna´ndez Puentes, J . L.
Br. J . Cancer 1996, 74, 677-682.
(9) Ishibashi, F.; Miyazaki, Y.; Iwao, M. Tetrahedron 1997, 53, 5951-
5962.
1
(10) For example: Chen, C.-H.; Soine, T. O.; Lee, K.-H. J . Pharm. Sci.
1971, 60, 1634-1638.
(11) Iwao, M.; Kuraishi, T. Bull. Chem. Soc. J pn. 1980, 53, 297-298.
(12) Pearlman, W. M. Tetrahedron Lett. 1967, 1663-1664.
(13) Fukui, K.; Nakayama, M. Bull. Chem. Soc. J pn. 1962, 35, 1321.
(14) Oda, T.; Tsuru, M.; Hatakeyama, T.; Nagatomo, H.; Muramatsu, T.;
Yamasaki, N. J . Biochem. 1997, 121, 560-567.
300 °C); H NMR (acetone-d6, 300 MHz) δ 6.91 (1H, dd, J )
8.1, 1.8 Hz, H-6′), 7.04 (1H, d, J ) 1.8 Hz, H-2′), 7.04-7.11
(2H, m, ArHs), 7.12 (1H, d, J ) 8.1 Hz, H-5′), 7.17 (1H, d, J )
7.5 Hz, H-9), 7.26 (1H, s, H-10), 7.29 (1H, s, H-13), 7.36-7.40
(2H, m, ArHs), 8.24 (1H, br s, OH), 8.36 (1H, br s, OH), 8.67
(1H, br s, OH), 8.74 (1H, br s, OH), 9.13 (1H, d, J ) 7.5 Hz,
H-8). An analytically pure sample was obtained as its tetra-
NP0104525