Lamellarin-inspired potent topoisomerase I inhibitors with the unprecedented benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one scaffold

Article abstract of DOI:10.1016/j.bmc.2018.11.037

A new class of topoisomerase I inhibitors containing the unprecedented benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one (abbreviated as BBPI) ring system have been developed based on structure-activity relationship studies of the cytotoxic marine alkaloid la

Full text of DOI:10.1016/j.bmc.2018.11.037

Accepted Manuscript
Lamellarin-inspired potent topoisomerase I inhibitors with the unprecedented
benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one scaffold
Tsutomu Fukuda, Yusuke Nanjo, Masahiro Fujimoto, Kenyu Yoshida, Yuko
Natsui, Fumito Ishibashi, Fumiyasu Okazaki, Hideto To, Masatomo Iwao
PII:
S0968-0896(18)31523-2
https://doi.org/10.1016/j.bmc.2018.11.037
BMC 14641
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 August 2018
26 November 2018
27 November 2018
Please cite this article as: Fukuda, T., Nanjo, Y., Fujimoto, M., Yoshida, K., Natsui, Y., Ishibashi, F., Okazaki, F.,
To, H., Iwao, M., Lamellarin-inspired potent topoisomerase I inhibitors with the unprecedented benzo[g]
[1]benzopyrano[4,3-b]indol-6(13H)-one scaffold, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/
10.1016/j.bmc.2018.11.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Lamellarin-inspired potent topoisomerase I inhibitors Leave this area blank for abstract info.
with the unprecedented benzo[g][1]benzopyrano[4,3-
b]indol-6(13H)-one scaffold
Tsutomu Fukuda, Yusuke Nanjo, Masahiro Fujimoto, Kenyu Yoshida, Yuko Natsui, Fumito Ishibashi,
Fumiyasu Okazaki, Hideto To, Masatomo Iwao

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Lamellarin-inspired potent topoisomerase I inhibitors with the unprecedented
benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one scaffold
Tsutomu Fukuda a, Yusuke Nanjo a, Masahiro Fujimoto a, Kenyu Yoshida a, Yuko Natsui a, Fumito
a,*
Ishibashi b, Fumiyasu Okazaki c, Hideto To c, Masatomo Iwao
aDivision of Chemistry and Materials Science, Graduate School of Engineering, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
bDivision of Marine Life Science and Biochemistry, Graduate School of Fisheries and Environmental Sciences, Nagasaki University, 1-14 Bunkyo-machi,
Nagasaki 852-8521, Japan
cDepartment of Medical Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani, Toyama
930-0194, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A
new class of topoisomerase
I
inhibitors containing the unprecedented
benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one (abbreviated as BBPI) ring system have been
developed based on structure-activity relationship studies of the cytotoxic marine alkaloid
lamellarin D. The pentacyclic BBPI scaffold was constructed from N-tert-butoxycarbonylpyrrole
by sequential and regioselective functionalization of the pyrrole core using directed lithiation,
conventional electrophilic substitution, and palladium-catalyzed cross-coupling reactions.
Further N-alkylation of the scaffold followed by selective deprotection of the O-isopropyl group
produced a range of N-substituted BBPI derivatives. The BBPIs thus prepared exhibited potent
topoisomerase I inhibitory activity in DNA relaxation assays. The activities of BBPIs were
higher than those of lamellarin D and camptothecin; they showed potent and selective
antiproliferative activity in the panel of 39 human cancer cell lines established by Japanese
Foundation for Cancer Research. COMPARE analyses indicated that the inhibition patterns of
the BBPIs correlated well with those of the known topoisomerase I inhibitors such as SN-38 and
TAS-103. The water-soluble valine ester derivative exhibited antitumor activity in vivo against
murine colon carcinoma colon 26. The activity was comparable to that of the approved
anticancer agent irinotecan.
Available online
Keywords:
Topoisomerase I inhibitors
Lamellarin D
BBPI
Scaffold hopping
Antitumor activity
2009 Elsevier Ltd. All rights reserved.
1. Introduction
water-soluble prodrug that is used to facilitate the administration
of the parental potent inhibitor SN-38 (4) (Fig. 1). After the
success of these drugs, structurally unique non-camptothecin-
Topoisomerase I is a nuclear enzyme that mediates the
relaxation of supercoiled DNA during replication, transcription,
and other nuclear processes via reversible DNA single strand
cleavage and religation.1–3 Mechanistically, this process involves
a simple transesterification between the phosphodiester bond of
DNA and the tyrosine 723 hydroxyl group of topoisomerase I.
Topoisomerase I inhibitors stabilize the transient topoisomerase
I–DNA covalent complex and inhibit the subsequent religation
step. This action of the inhibitors causes single-strand DNA
breakages that are then transformed into double-strand breakages
lethal for growing cells. Therefore, topoisomerase I inhibitors can
be regarded as effective anticancer agents. In fact, the plant
alkaloid camptothecin (1)-derived topoisomerase I inhibitors
such as topotecan (2) and irinotecan (3) have been approved for
the treatment of a wide range of cancers.4 Compound 3 is a
type topoisomerase
indenoisoquinolines,
I
inhibitors such as indolocarbazoles,
benzophenanthridines,
dibenzonaphthylidines have been developed5 and some of them
are even being clinically tested.
_________
* Corresponding author.
Fig. 1. Camptothecin family of topoisomerase I inhibitors.
E-mail address: iwao@nagasaki-u.ac.jp (M. Iwao).

Lamellarins are biologically active marine alkaloids that
contain common 14-phenyl-6H-
panel of 39 human cancer cell lines established by the Japanese
Foundation for Cancer Research (JFCR39).16 The COMPARE
analyses17 of the inhibition patterns suggested that the molecular
target of these analogues was also topoisomerase I.
a
[1]benzopyrano[4′,3′:4,5]pyrrolo[2,1-a]isoquinoline scaffold.6–8
The anticancer activity of lamellarins was first discovered by
Quesada et al. in 1996.9 They showed that the triacetates of
lamellarins D (5), K, and N exhibit potent cytotoxicity against a
range of cancer cell lines including the multidrug-resistant
(MDR) phenotype. In 1997, we achieved the first total synthesis
of 5 via N-ylide-mediated cyclization as a key reaction.10
Subsequently, we prepared several non-natural analogues of 5
and conducted a structure–activity relationship (SAR) study.11 It
was confirmed that 5 is quite cytotoxic (IC50 = 10.5 nM in colony
formation assay using HeLa cell line) and its activity is
dependent on the substitution pattern of the oxygen
functionalities on the lamellarin scaffold. Namely, the hydroxy
groups at C8 and C20 are essential for realizing potent
cytotoxicity, whereas the hydroxy group at C14 is less important.
Later, Ploypradith and coworkers performed a more precise SAR
study using more lamellarins and various cancer cell lines, and
confirmed our results.12
Based on these fundamental studies, we initiated a project to
generate a new class of potent topoisomerase I inhibitors via
scaffold hopping.18 Among several potential candidates, we
selected the benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one
(BPPI) scaffold. This scaffold can be regarded as a regioisomer
of the pentacyclic core (ABCDE-ring) of lamellarin D (5) with
respect to the position of the ring-nitrogen (Fig. 2). Therefore, it
was presumed that the BBPI derivatives 7 having two hydroxy
groups at C3 and C10 and two methoxy groups at C2 and C11,
respectively, could maintain the potent activity of 5. In addition,
it was anticipated that a variety of R groups could be readily
introduced to the ring-nitrogen (N13) by simple alkylation and
they could modulate the physical and biological properties of 7.
Based upon these assumptions, the synthesis and biological
evaluation of BBPI derivatives were investigated.
2. Results and discussion
In 2003, Bailly and coworkers reported that lamellarin D (5)
was a potent inhibitor of topoisomerase I.13 The good correlation
between the cytotoxicity and topoisomerase I inhibitory activities
of lamellarin D analogues suggested that the principal cellular
target of 5 is topoisomerase I.14 Moreover, Bailly et al. proposed
a theoretical model of lamellarin D (5)–DNA–topoisomerase I
ternary complex that explained the results of the SAR studies.13,14
According to this model, 5 intercalates at the +1 (C·G) and
−1 (A·T) cleavage site of the DNA–topoisomerase I covalent
complex and is stabilized by hydrogen bonding interactions with
three specific amino acid residues of topoisomerase I. The
hydroxy groups at C8 and C20 and the carbonyl oxygen of the
lactone ring are at a hydrogen bonding distance from Asn722,
Glu356, and Arg364, respectively. On the other hand, the aryl
ring at C1 (F-ring) is directed toward the major groove cavity and
does not have any direct interaction with the enzyme. This model
suggests that the F-ring is probably not essential for
topoisomerase I inhibition and it may be possible to replace the
F-ring with other groups without affecting the biological activity.
We confirmed this speculation by synthesizing a series of F-ring-
defected lamellarin D analogues 6 (X = H, CH3, CH2NMe2,
CHO, F, Cl, Br) (Fig. 2).15 Antiproliferative activities of 6 were
found to be as potent as that of the parent compound 5 in the
2.1. Synthesis of BBPIs
The synthesis of N-unsubstituted BBPI 20 is shown in
Scheme 1. The basic strategy to construct the unprecedented
BBPI ring system comprises sequential and regioselective
functionalization of the central pyrrole core corresponding to the
C-ring via directed lithiation, conventional electrophilic
substitution, and palladium-catalyzed cross-coupling reactions.
Thus, directed lithiation of N-Boc-pyrrole (8) with LDA followed
by the reaction with trimethyl borate afforded pyrrole-2-boronic
acid 9 in 75% yield.19 This compound was easily isolated on a
preparative scale (>15 g) after a simple purification step of
hexane washing of the crude product. Suzuki–Miyaura coupling
of 9 with the known bromide 1020 produced 11 in 94% yield. The
D-ring moiety of the BBPI scaffold was constructed in two steps
from 11 by applying the method utilized for the synthesis of
polyaromatic hydrocarbons.21 Thus, Wittig reaction of 11 with
commercially available (methoxymethyl)triphenylphosphonium
chloride in the presence of t-BuOK produced the enol ether 12 in
93% yield as a mixture of E- and Z-isomers. Acid-catalyzed
cyclization of 12 led to D-ring formation and the tricyclic
benzo[g]indole compound 13 was obtained in 92% yield.
Conventional electrophilic bromination of 13 with N-
bromosuccinimide (NBS) in DMF afforded an approximately 1:1
mixture of 14 and the unwanted 3-bromo analogue. On the other
hand, directed lithiation of 13 with t-BuLi followed by a reaction
with 1,2-dibromo-1,1,2,2-tetrafluoroethane provided the 2-bromo
compound 14 selectively in 95% yield. Suzuki–Miyaura coupling
of 14 with the known boronic acid 1522 afforded 16 in 97% yield.
Subsequent electrophilic bromination of 16 with NBS gave 17
regioselectively in 99% yield. Bromine–lithium exchange of 17
with 2 equiv. of t-BuLi followed by a reaction with methyl
chloroformate produced the methoxycarbonylated compound 18
in 81% yield. Simultaneous deprotection of the tert-
butoxycarbonyl (Boc) and methoxymethyl (MOM) protecting
groups with hydrochloric acid led to concomitant lactonization
and the formation of the key BBPI intermediate 19 in 99% yield.
Final deprotection of the O-isopropyl groups with BCl3 afforded
N-unsubstituted BBPI 20. The overall yield of 20 from
commercially available 8 was 39% in ten steps. The modular
nature of this synthesis warrants easy access to a wide range of
BBPIs with different substitution patterns at the A- and E-rings
by simple modification of the aromatic building blocks
corresponding to 10 and 15.
Fig. 2. Design of a new class of topoisomerase I inhibitors BBPIs 7 by
structural modification of lamellarin D (5).

Scheme 1. Synthesis of N-unsubstituted BBPI 20. Reagents and conditions: (a) (1) LDA, THF, −78 °C, 1 h, (2) B(OMe)3, −78 °C, 1 h, then rt, 15 h (75%); (b)
10, Pd(PPh3)4, Na2CO3, THF, water, reflux, 18 h (94%); (c) MeOCH2P+Ph3·Cl−, t-BuOK, THF, 0 °C, 3 h (93%); (d) CH3SO3H, CH2Cl2, 0 °C, 23 h (92%); (e) (1)
t-BuLi, THF, −78 °C, 1 h, (2) BrCF2CF2Br, −78 °C, 1 h (95%); (f) 15, Pd(PPh3)4, Na2CO3, DME, water, reflux, 23 h (97%); (g) NBS, DMF, 0 °C, 6.5 h (99%);
(h) (1) t-BuLi, THF, −78 °C, 1 h, (2) ClCO2Me, −78 °C, 1 h (81%); (i) HCl, MeOH, CHCl3, 50 °C, 24.5 h (99%); (j) BCl3, DCM, −78 °C to rt (88%).
Scheme 2. Synthesis of N-substituted BBPIs 22a–e.
The synthesis of N-substituted BBPIs 22a–e is shown in
Scheme 2. N-Alkylation of 19 with a range of commercially
available alkylating agents such as methyl iodide, ethyl iodide,
allyl bromide, propargyl bromide, and 2-(dimethylamino)ethyl
chloride in the presence of sodium hydride in DMF afforded
21a–e in good to modest yields. Care must be taken in the
synthesis of 21e to preclude water from the hygroscopic 2-
(dimethylamino)ethyl chloride hydrochloride. The yield was
considerably decreased when the wet reagent was employed
probably because of hydrolytic ring opening of the lactone
moiety of the starting material. The O-isopropyl groups of 21a–d
were selectively deprotected by BCl3 to give 22a–d, respectively.
An attempt to deprotect 21e with BCl3 produced 22e in very low
yield (15%) together with an unidentified poorly soluble solid.
Fortunately, however, 22e was obtained in good yield by
deprotection with AlCl3.
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC·HCl) in the presence of 4-(N,N-dimethylamino)pyridine
The BBPIs 20 and 22 thus synthesized were essentially
insoluble in water, except for the trifluoroacetic acid (TFA) salt
22e. The aqueous insolubility is a major concern for in vivo
biological evaluations of BBPIs. Bailly and coworkers have
previously reported that the cationic amino acid ester derivatives
of lamellarin D (5) maintain the topoisomerase I inhibitory and
cytotoxic activities.23 Especially, the activities of the valine ester
are equally potent as those of the parent compound 5. Based on
these important findings, we prepared the valine ester 24 in two
steps (Scheme 3). Condensation of 22a and N-Boc-L-valine with
Scheme 3. Synthesis of the water-soluble valine ester derivative 24.
Reagents and conditions: (a) N-Boc-L-valine, EDC·HCl, DMAP, rt, 24 h
(66%); (b) TFA, rt, 1 h (quant).

(DMAP) produced bis-O-valinate 23 in 66% yield. Subsequent
acid-promoted cleavage of the N-Boc groups with TFA gave the
desired compound 24 in quantitative yield. This cationic species
was readily soluble in water and suitable for in vivo biological
evaluations.
tested BBPIs induced the intense bands corresponding to the
nicked + relaxed DNA and the reduced number of the bands
corresponding to the relaxed topoisomers (Fig. 4a). The enzyme
inhibitory activities of the BBPIs appear to be higher than those
of camptothecin (1) and lamellarin D (5).
Finally, we performed the X-ray crystallographic analysis of
the N-methylated BBPI 21a. The ORTEP drawing shows that the
fused pentacyclic core of 21a is essentially planar as expected
(Fig. 3). However, it is interesting to note that the methyl group
at N13 is slightly bent (ca. 9.5°) from the plane of the BBPI core.
This unexpected structure may be a result of severe steric
hindrance between the methyl group at N13 and the peri-
hydrogens at C1 and C12.
Next, we performed the agarose gel electrophoresis in the
presence of ethidium bromide in order to exclude the possibility
that the BBPIs act as non-specific inhibitors of the enzyme.25 It
has been established that the bands corresponding to the nicked
DNA and the relaxed DNA can be clearly separated in the
presence of ethidium bromide.24 As shown in Fig. 4b, the intense
nicked DNA bands were induced by all BBPIs as well as by the
reference compounds. The result indicates that the BBPIs are the
specific inhibitors, like camptothecin (1) and lamellarin D (5), to
stabilize the DNA–topoisomerase I cleavable complex and
induce single-strand DNA breakage.
2.3. Docking study
In order to speculate how BBPIs interfere with the enzymatic
action of topoisomerase I, we performed in silico docking
simulations using single X-ray crystallographic data of the
topotecan (2)–DNA–topoisomerase I ternary complex.26 The
interfacial inhibitor 2 was replaced by docking with BBPI 22a
and the resulting complex was minimized by the Molecular
Operating Environment (MOE) program.27 The two most
plausible models (poses A and B) are shown in Fig. 5. In the case
of pose A, the pentacyclic core (ABCDE-ring) of BBPI 22a
intercalated at the DNA cleavage site in such a way that the
methyl group at N13 was directed toward the DNA major groove
cavity. The A-ring pointed in toward the backbone of the DNA
non-scissile strand whereas the E-ring was oriented toward the
DNA cleaved strand. The pentacyclic core was stabilized by both
+1 (C·G) and −1 (A·T) base pairs by forming stacking
interactions. The hydroxy groups at C3 and C10 and the carbonyl
oxygen of the lactone ring were hydrogen-bonded with Asn722,
Glu356, and Arg364, respectively. The docking score of pose A
was estimated to be −9.58 kcal/mol. In the case of pose B, as
compared to pose A, the pentacyclic core was turned around and
the orientations of the A- and E-rings were reversed. Similar
hydrogen bonding interactions were observed between the
hydroxy group at C10 and Asn722 and between the carbonyl
oxygen and Arg364 while the hydroxy group at C3 was not
hydrogen-bonded to Glu356 owing to the longer distance
between O3 and the carboxylate oxygen of Glu356 (ca. 4.0 Å).
Despite the lack of the hydrogen bonding interaction, the docking
score of pose B was scarcely affected (−9.47 kcal/mol) because
the additional stacking interactions stabilized this orientation.
The small difference of the docking scores (0.11 kcal/mol) may
indicate that both poses A and B are possible.
Fig. 3. ORTEP drawing of 21a·AcOEt. Thermal ellipsoids are drawn at 50%
probability.
2.2. Topoisomerase I inhibitory activity
Topoisomerase I inhibitory activities of the selected BBPIs 20,
22a, 22e, and 24 were evaluated in DNA relaxation assay24 using
supercoiled pBR322 plasmid DNA and topoisomerase I isolated
from calf thymus (Fig. 4). Camptothecin (1) and lamellarin D (5)
were used for the positive controls. The experiments were
performed at 10 µM concentration of the inhibitors. The
supercoiled DNA changed to the relaxed DNA in the absence of
an inhibitor, which appeared as multiple bands corresponding to
the topoisomers of the different linking numbers in the agarose
gel electrophoresis, while the treatment with topoisomerase I
2.4. In vitro antiproliferative activity
The antiproliferative activities of all synthesized BBPIs, i.e.,
20, 22a–e, and 24, were evaluated on a panel of JFCR39.17 The
50% growth-inhibitory concentration (GI50) values of the selected
nine cell lines and the mean-graph midpoint (MG-MID) of the
average GI50 values across the entire panel of JFCR39 are shown
in Table 1. For comparison, the activities of SN-38 (4) and
lamellarin D (5) have also been included. All the N-substituted
BBPIs 22a–e and 24 exhibited potent activities at low nanomolar
concentrations (MG-MID = 22.9–89.1 nM). Among them, the
activities of 22a (R = Me), 22b (R = Et), 22e (R =
CH2CH2NMe2), and 24 (valine ester) were shown to be higher
than that of SN-38 (4) by comparison of their MG-MID values.
The BBPIs 22c and 22d, which had an unsaturated double or
triple bond in the N-substituent, were somewhat less potent. In
Fig. 4. DNA relaxation assay of the BBPIs 20, 22a, 22e, and 24. All
inhibitors including camptothecin (1) and lamellarin D (5) were used at
10 µM. Agarose gel electrophoresis was performed in the absence or
presence of ethidium bromide.
inhibitors resulted in accumulation of the nicked DNA. The

Fig. 5. Plausible docking models of the BBPI 22a–DNA–topoisomerase I ternary complex. (A) View along the axis of the DNA double helix; (B) ligand–
receptor interaction diagram.
Table 1
In vitro antiproliferative activities of BBPIs 20, 22a–e, and 24 against selected human cancer cell lines.
Antiproliferative activity (GI50 in nM)a
Human tumor
20e
68
22af
9.6
22be
<10
<10
<10
<10
<10
<10
<10
<10
<10
24.5
0.39
3.11
22cg
14.0
7.6
22de
18
22eg
2.6
4.0
9.7
3.9
2.7
26
24g
6.1
3.4
15
SN-38 (4)f
3.0
lamellarin D (5)e
cell line
Breast
CNS
MCF-7
U251
<10
<10
<10
<10
<10
38
<10
88
1.3
<10
34
2.8
Colon
HCT-116
NCI-H522
LOX-IMVI
SK-OV-3
ACHN
8.8
25
23
Lung
<10
21
1.8
9.5
<10
<10
56
3.4
2.5
20
3.7
Melanoma
Ovarian
Renal
2.6
6.4
5.3
330
110
1600
51
9.4
29
23
<1.0
33
4.2
<10
66
1.9
67
2.9
41
5.9
<10
50
Stomach
Prostate
MKN28
82
110
4.3
DU-145
MG-MIDb
Deltac
Ranged
2.5
6.5
27
2.0
30.9
1.26
2.77
3.2
27.5
1.04
2.60
<10
41.7
0.62
2.30
660.7
1.82
4.00
22.9
1.36
4.00
58.9
1.15
2.37
89.1
0.95
3.11
40.7
1.26
2.91
a Concentration for 50% inhibition of cell growth relative to control. Cell growth was determined according to sulforhodamine B assay.
b Mean GI50 value in all cell lines tested.
c Difference in log GI50 value between the most sensitive cells and the MG-MID value.
d Difference in log GI50 value between the most and least sensitive cells.
e The GI50 value was obtained from the dose-response curve in the test range between 10−4–10−8 M.
f The GI50 value was obtained from the dose-response curve in the test range between 10−5–10−9 M.
g The GI50 value was obtained from the dose-response curve in the test range between 10−6–10−10 M.
contrast to the high activities of the N-substituted BBPIs, the
activity of N-unsubstituted BBPI 20 was much lower (MG-MID
= 660.7 nM). The diminished activity may be accounted for by
considering the factors unique to living cells such as cell
membrane permeability, since 20 showed good topoisomerase I
inhibitory activity like the N-substituted BBPIs in the DNA
relaxation assay.
cellular target of these compounds was topoisomerase I. For
typical examples, the mean graph patterns, also called
fingerprints, of 22a and 24 were derived from their log GI50
values against JFCR39 and are shown in Fig. 6. The patterns of
these compounds showed good correlation to those of the known
topoisomerase I inhibitors such as SN-38 (4) and TAS-10328 (r =
0.726–0.793).
The COMPARE analyses17 of the chemosensitivity patterns of
the BBPIs against a panel of JFCR39 suggested that the major
2.5. In vivo antitumor activity

Fig. 6. Chemosensitivity patterns of BBPIs 22a and 24 against a panel of JFCR39 and the results of COMPARE analyses. The mean graph shows the deviation
of log GI50 value of each cell line from MG-MID. The correlation coefficient r (r = 0–1) shows the similarity of the chemosensitivity pattern of the tested
compound to that of the known antitumor agent in the database.
was administered intravenously every two days seven times and
the tumor volumes were recorded daily until day 20. As shown in
Fig. 7, 24 showed antitumor activity comparable to that of 3.
Body weight loss in the treated mice was compared with that of
controls and found to be at an acceptable level, suggesting that 24
1,400
control
irinotecan (3)
1,200
24
P < 0.01
1,000
caused no serious damage to the mice at doses less than 50 mg/kg
(Supplementary data).
800
P < 0.01
600
3. Conclusion
400
In this study, a new class of topoisomerase I inhibitors with
the benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one (BBPI)
200
0
scaffold were designed and synthesized by structural
0
2
4
6
8
10 12 14 16 18 20
modification of the marine natural product lamellarin D (5). The
topoisomerase I inhibitory activities of the selected BBPIs were
found to be higher than those of the reference compounds,
camptothecin (1) and the parental 5, in DNA relaxation assay.
Although the antiproliferative activity of the N-unsubstituted
BBPI 20 was modest, those of the N-substituted BBPIs 22a, b, e,
and 24 were higher than that of SN-38 (4) against the JFCR39
panel. Furthermore, the water-soluble BBPI 24 exhibited
antitumor activity against colon 26. The activity was comparable
to the approved anticancer agent irinotecan (3). Unlike
Days after the first drug administration
Fig. 7. In vivo antitumor activity of 24 in comparison with irinotecan (3)
toward murine colon carcinoma colon 26 implanted in BALB/c mice.
Finally, we evaluated the in vivo antitumor activity of the
water-soluble BBPI 24 against colon 26 (murine colon carcinoma
cell line) implanted on the BALB/c mice (Fig. 7).29 For
comparison, the antitumor activity of irinotecan (3) was also
estimated. One day after implantation, 50 mg/kg dose of 24 or 3

lamellarins, a variety of BBPIs can be produced by introducing
diverse substituents on the ring nitrogen. This provides an
excellent opportunity to discover more effective topoisomerase I
inhibitors having improved antitumor activity and such
investigations are currently in progress in our laboratories.
ethereal extracts were combined, washed with brine, dried
(Na2SO4), and concentrated in vacuo. The residual solid was
triturated with hexane and filtered to give 9 (15.6 g, 75%) as
1
white powder. H NMR (500 MHz, CDCl3):  1.62 (s, 9H), 6.26
(t, J = 3.2 Hz, 1H), 7.10 (dd, J = 1.6 and 3.2 Hz, 1H), 7.18 (br s,
2H), 7.45 (dd, J = 1.6 and 3.2 Hz, 1H). 13C NMR (126 MHz,
CDCl3):  28.0, 85.6, 112.0, 127.1, 128.7, 152.2. These physical
and spectroscopic data are in good agreement with those
previously reported.19
4. Experimental section
4.1. Synthesis–general
Melting points were determined using the Yanagimoto micro
melting point apparatus and are uncorrected. IR spectra were
obtained with a Thermo Nicolet Nexus 670 NT FT-IR instrument
and are reported in terms of absorption frequency (cm−1). NMR
spectra were recorded on a JEOL JNM-AL400 instrument
(400 MHz for 1H and 100 MHz for 13C) or a Varian NMR
System 500PS SN instrument (500 MHz for 1H and 126 MHz for
4.2.3. 2-Bromo-5-isopropoxy-4-methoxybenzaldehyde (10). A
mixture of isovanillin (25.0 g, 164 mmol), K2CO3 (45.1 g,
329 mmol), 2-bromopropane (23.1 mL, 247 mmol) in DMSO
(300 mL) was heated at 55 °C for 2 h. After cooling, the mixture
was poured into water and extracted 3 times with ether. The
ethereal extracts were combined, washed successively with
water, 10% NaOH solution and brine, dried (Na2SO4), and
concentrated in vacuo. The oily residue was distilled to give 3-
isopropoxy-4-methoxybenzaldehyde (10′) (29.0 g, 91%) as a
colorless oil, bp 85–87 °C/0.2 mmHg. 1H NMR (500 MHz,
CDCl3): δ 1.40 (d, J = 6.1 Hz, 6H), 3.94 (s, 3H), 4.65 (sep, J =
6.1 Hz, 1H), 6.98 (d, J = 8.2 Hz, 1H), 7.42 (d, J = 1.7 Hz, 1H),
7.45 (dd, J = 1.7 and 8.2 Hz, 1H), 9.84 (s, 1H). 13C NMR
(126 MHz, CDCl3):  21.9, 56.2, 71.3, 110.9, 112.7, 126.5,
130.1, 147.9, 155.7, 191.0. These physical and spectroscopic data
are in good agreement with those previously reported.31
1
13C). Chemical shifts for H NMR are expressed in parts per
million (ppm) relative to the following internal standards: CDCl3
(tetramethylsilane,  0.0 ppm); acetone-d6 (tetramethylsilane, 
0.0 ppm); DMSO-d6 (DMSO,

2.50 ppm); methanol-d4
1
(tetramethylsilane,  0.0 ppm). Data for H NMR spectra are
reported as follows: chemical shift (δ ppm), multiplicity (s =
singlet, d = doublet, dd = double doublet, t = triplet, sext = sextet,
sep = septet, m = multiplet, br s = broad singlet), coupling
constant (Hz), and integration. Chemical shifts for 13C NMR are
expressed in ppm relative to the following internal standards:
CDCl3
(tetramethylsilane,
(tetramethylsilane,

0.0 ppm);
acetone-d6
To a solution of 10′ (19.4 g, 99.9 mmol) in DMF (50 mL), was
added dropwise a solution of NBS (35.6 g, 199 mmol) in DMF
(100 mL) at room temperature. After 19.5 h, the mixture was
diluted with water, extracted with ether, washed successively
with Na2SO3 solution, water and brine, dried (Na2SO4), and
concentrated in vacuo. The crude product was purified by
recrystallization from ether–hexane to give 10 (17.0 g, 62%) as
pale brown needles. The mother liquor was evaporated and the
residue was chromatographed on silica gel using hexane–EtOAc
(10:1) as an eluent to give an additional 10 (3.36 g, 12%) as pale
brown solid. IR (KBr): 1681, 1588, 1508, 1269, 1217, 1158,

0.0 ppm); DMSO-d6 (DMSO-d6,

39.52 ppm). High-resolution mass spectra (HRMS) were
recorded on a JEOL JMS-T100TD (direct analysis in real-time
mass spectrometry, DARTMS) instrument or a JEOL JMS-700N
(fast atom bombardment mass spectrometry, FABMS or electron
ionized mass spectrometry, EIMS) instrument. Column
chromatography was conducted using silica gel 60N, 63–210 µm
(Kanto Chemical Co., Inc.) or Chromatorex NH-DM1020 (Fuji
Silysia Chemical Ltd.). Flash chromatography was conducted
using silica gel 60N, 40–50 µm (Kanto Chemical Co., Inc.).
1
1021 cm−1. H NMR (500 MHz, CDCl3):  1.38 (d, J = 6.1 Hz,
4.2. Synthesis of N-unsubstituted BBPI 20
6H), 3.94 (s, 3H), 4.63 (sep, J = 6.1 Hz, 1H), 7.05 (s, 1H), 7.43
(s, 1H), 10.18 (s, 1H). 13C NMR (126 MHz, CDCl3):  21.8, 56.5,
71.5, 113.6, 115.9, 120.1, 126.5, 147.2, 155.7, 190.9. Anal. Calcd
for C11H13BrO3: C, 48.37; H, 4.80. Found: C, 48.23; H, 4.72.
These physical and spectroscopic data are in good agreement
with those previously reported.32
4.2.1. 1-(tert-Butoxycarbonyl)-1H-pyrrole (8). To a solution of
pyrrole (30.9 g, 460 mmol), Et3N (31.9 mL, 229 mmol) and
DMAP (2.81 g, 23.0 mmol) in THF (250 mL), was added
dropwise via syringe a solution of Boc2O (100 g, 458 mmol) in
THF (30 mL) at 0 °C under Ar atmosphere. After the mixture had
been stirred at rt for 18 h, the solvent was distilled off in vacuo.
Distillation of the crude product yielded 8 (58.5 g, 76%) as a
colorless oil, bp 80 °C/47 mmHg. 1H NMR (500 MHz, CDCl3): 
4.2.4. tert-Butyl 2-(2-formyl-4-isopropoxy-5-methoxyphenyl)-1H-
pyrrole-1-carboxylate (11). Under Ar atmosphere, a mixture of
10 (5.49 g, 20.1 mmol), 9 (5.06 g, 24.0 mmol), Pd(PPh3)4 (2.42 g,
2.10 mmol), and Na2CO3 (12.9 g, 126 mmol), THF (420 mL),
and degassed water (38 mL) was refluxed for 18 h. After cooling
to room temperature, the solvent was removed in vacuo and the
residue was diluted with water and extracted 3 times with DCM.
The organic extracts were combined, washed successively with
water (twice) and brine, dried (Na2SO4), and concentrated in
vacuo. The oily residue was chromatographed on silica gel eluted
with hexane–EtOAc (10:1) to give 11 (6.78 g, 94%) as a reddish
viscous oil. IR (KBr): 1743, 1683, 1511, 1332, 1155, 1124 cm−1.
1H NMR (400 MHz, CDCl3): δ 1.29 (s, 9H), 1.41 (d, J = 6.1 Hz,
6H), 3.92 (s, 3H), 4.70 (sep, J = 6.1 Hz, 1H), 6.24 (dd, J = 1.8
and 3.3 Hz, 1H), 6.29 (t, J = 3.3 Hz, 1H), 6.82 (s, 1H), 7.44 (dd, J
= 1.8 and 3.3 Hz, 1H), 7.43 (s, 1H), 9.74 (s, 1H). 13C NMR
(100 MHz, CDCl3): δ 21.9, 27.6, 56.2, 71.2, 84.0, 110.6, 111.2,
113.9, 116.9, 122.6, 128.7, 129.3, 132.8, 147.4, 148.9, 154.0,
190.7. HRFABMS m/z. Calcd for C20H25NO5 (M+): 359.1733.
Found: 359.1738.
1.59 (s, 9H), 6.21 (t, J = 2.3 Hz, 2H), 7.23 (t, J = 2.3 Hz, 2H). 13
NMR (126 MHz, CDCl3):  28.0, 83.6, 111.8, 120.0, 148.9.
These physical and spectroscopic data are in good agreement
with those previously reported.30
C
4.2.2. [1-(tert-Butoxycarbonyl)-1H-pyrrol-2-yl]boronic acid (9).
To a solution of diisopropylamine (18.2 mL, 130 mmol) in THF
(450 mL), was added dropwise a hexane solution of BuLi
(1.61 M, 74.8 mL, 120 mmol) at −78 °C under Ar atmosphere.
The mixture was stirred at −78 °C for 5 min, gradually warmed
up to 0 °C, and kept at the same temperature for 10 min. The
whole was again cooled to −78 °C and a solution of 8 (16.7 g,
100 mmol) in THF (30 mL) was added dropwise. After 1 h at
−78 °C, trimethyl borate (16.7 mL, 150 mmol) was added
dropwise. After 1 h, the mixture was gradually warmed up to
room temperature and stirred for 15 h. The reaction was then
quenched by adding saturated aqueous NH4Cl and the THF was
removed in vacuo. The pH of the residual liquid was made 3 with
AcOH and the whole was extracted 3 times with ether. The

4.2.5.
methoxyphenyl]-1H-pyrrole-1-carboxylate (12). To a mixture of
(methoxymethyl)triphenylphosphonium chloride (2.68 g,
tert-Butyl
2-[2-(2-methoxyethenyl)-4-isopropoxy-5-
(s, 1H), 7.38 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 8.5 Hz, 1H), 7.49
(s, 1H). 13C NMR (100 MHz, CDCl3): δ 22.0, 27.9, 55.8, 71.0,
85.7, 102.5, 107.4, 111.3, 111.9, 116.9, 117.2, 123.1, 125.6,
127.4, 131.0, 146.1, 149.8, 151.0. HRFABMS m/z Calcd for
C21H24BrNO4 (M+): 433.0889. Found: 433.0903.
7.82 mmol) in THF (39 mL) cooled to 0 °C under Ar atmosphere,
was added dropwise a suspension of t-BuOK (1.05 g, 9.36 mmol)
in THF (9.4 ml). After 10 min at 0 °C, a solution of 11 (2.31 g,
6.26 mmol) in THF (27 mL) was added dropwise. After 3 h at
0 °C, the reaction was quenched by adding water (100 mL). The
THF was removed in vacuo and the residue was extracted 3 times
with ether. The combined ethereal layers were washed
successively with water and brine, dried (Na2SO4), and
concentrated in vacuo. The oily residue was chromatographed on
silica gel using hexane–EtOAc (10:1) as an eluent to give 12
(2.26 g, 93%) as an E/Z mixture (E:Z = 64:36, based on NMR) as
a reddish viscous oil. IR (KBr): 1736, 1509, 1336, 1158,
4.2.8. tert-Butyl 7-isopropoxy-2-[4-isopropoxy-5-methoxy-2-
(methoxymethoxy)phenyl]-8-methoxy-1H-benzo[g]indole-1-
carboxylate (16). In a Schlenk tube, 14 (454 mg, 1.05 mmol),
arylboronic acid 1522 (421 mg, 1.56 mmol), Na2CO3 (727 mg,
6.86 mmol) and Pd(PPh3)4 (116 mg, 0.100 mmol) were placed
and the whole purged with Ar. To this mixture, DME (15 mL)
and degassed (Ar) water (2.0 mL) were added and the whole was
heated under reflux under Ar atmosphere for 23 h. After cooling
to room temperature, the solvent was removed in vacuo and the
residue was diluted with water and extracted 3 times with DCM.
The combined organic layers were washed successively with
water and brine, dried (Na2SO4), and concentrated in vacuo. The
oily residue was chromatographed on silica gel using hexane–
EtOAc (5:1) as an eluent to give 16 (589 mg, 97%) as white
crystals, mp 102.5–103.0 °C (DCM/hexane). IR (KBr): 1738,
1494, 1391, 1308, 1150, 1022 cm−1. 1H NMR (400 MHz,
CDCl3): δ 1.33 (s, 9H), 1.42 (d, J = 6.1 Hz, 6H), 1.46 (d, J =
6.1 Hz, 6H), 3.29 (s, 3H), 3.84 (s, 3H), 3.97 (s, 3H), 4.59 (sep, J
= 6.1 Hz, 1H), 4.72 (sep, J = 6.1 Hz, 1H), 4.94 (s, 2H), 6.59 (s,
1H), 6.82 (s, 1H), 6.93 (s, 1H), 7.27 (s, 1H), 7.49 (s, 2H), 7.95 (s,
1H). 13C NMR (100 MHz, CDCl3): δ 22.0, 22.0, 27.3, 55.7, 56.3,
56.6, 71.0, 71.9, 83.6, 96.1, 103.6, 106.2, 108.9, 112.2, 115.2,
117.8, 118.1, 118.1, 122.8, 125.7, 127.7, 130.5, 135.5, 145.5,
145.7, 147.9, 149.3, 149.5, 152.0. Anal. Calcd for C33H41NO8: C,
68.37; H, 7.13; N, 2.42. Found: C, 68.36; H, 7.28; N, 2.26.
1
1125 cm−1. H NMR (400 MHz, CDCl3): δ 1.22 (s, 3.28H), 1.26
(s, 5.72H), 1.38 (d, J = 6.1 Hz, 3.82H), 1.40 (d, J = 6.2 Hz,
2.18H), 3.50 (s, 1.91H), 3.71 (s, 1.09H), 3.82 (s, 1.91H), 3.82 (s,
1.09H), 4.50–4.61 (m, 1H), 4.84 (d, J = 7.2 Hz, 0.364H), 5.47 (d,
J = 12.9 Hz, 0.636H), 5.95 (d, J = 7.2 Hz, 0.364H), 6.11 (dd, J =
1.8 and 3.4 Hz, 0.364H), 6.12 (dd, J = 1.9 and 3.3 Hz, 0.636H),
6.24 (t, J = 3.4 Hz, 0.364H), 6.24 (t, J = 3.3 Hz, 0.636H), 6.72 (d,
J = 12.9 Hz, 0.636H), 6.74 (s, 0.364H), 6.76 (s, 0.636H), 6.87 (s,
0.636H), 7.37 (dd, J = 1.9 and 3.3 Hz, 0.636H), 7.38 (dd, J = 1.8
and 3.4 Hz, 0.364H), 7.70 (s, 0.364H). 13C NMR (100 MHz,
CDCl3): δ 22.2, 27.3, 27.5, 56.0, 56.1, 56.3, 60.4, 71.4, 71.7,
76.7, 77.0, 77.4, 83.1, 83.1, 103.3, 103.4, 110.3, 110.4, 112.1,
114.0, 114.2, 114.3, 114.6, 116.1, 121.3, 121.4, 125.8, 128.8,
129.0, 132.9, 133.1, 146.4, 146.5, 147.0, 147.7, 148.1, 148.3,
149.4, 149.6. HRFABMS m/z Calcd for C22H29NO5 (M+):
387.2046. Found: 387.2053.
4.2.9.
tert-Butyl
3-bromo-7-isopropoxy-2-(4-isopropoxy-5-
4.2.6. tert-Butyl 7-isopropoxy-8-methoxy-1H-benzo[g]indole-1-
carboxylate (13). A solution of 12 (1.49 g, 3.85 mmol) in DCM
(25 mL) was cooled to 0 °C and methanesulfonic acid (0.025 mL,
0.365 mmol) was added. After 23 h at 0 °C, Na2CO3 (103.7 mg,
0.948 mmol) and MgSO4 (101.8 mg, 0.846 mmol) were added as
solids and the mixture was stirred for a while and filtered. The
filtrate was concentrated and the residue was purified by a
column chromatography on silica gel using hexane–EtOAc
(10:1) as an eluent to give 13 (1.26 g, 92%) as white crystals, mp
102.5–103 °C (DCM/hexane). IR (KBr): 1740, 1491, 1389, 1308,
1250, 1165, 1112 cm−1. 1H NMR (400 MHz, CDCl3): δ 1.45 (d, J
= 6.1 Hz, 6H), 1.68 (s, 9H), 4.02 (s, 3H), 4.72 (sep, J = 6.1 Hz,
1H), 6.61 (d, J = 3.7 Hz, 1H), 7.25 (s, 1H), 7.46 (d, J = 8.4 Hz,
1H), 7.52 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 3.7 Hz, 1H), 8.43 (s,
1H). 13C NMR (100 MHz, CDCl3): δ 22.0, 28.1, 55.8, 70.9, 83.6,
105.8, 108.0, 111.6, 111.6, 118.0, 118.8, 123.8, 126.9, 127.8,
128.2, 130.5, 146.0, 149.2, 150.5. Anal. Calcd for C21H25NO4: C,
70.96; H, 7.09; N, 3.94. Found: C, 71.06; H, 7.26; N, 3.76.
methoxy-2-methoxymethoxyphenyl)-8-methoxy-1H-
benzo[g]indole-1-carboxylate (17). To a cooled (0 °C) solution
of 16 (2.21 g, 3.81 mmol) in DMF (40 mL), was added dropwise
a solution of NBS (746 mg, 4.19 mmol) in DMF (5.0 mL). After
6.5 h at 0 °C, the reaction was quenched by adding water. The
mixture was extracted 3 times with ether and the combined
extracts were washed successively with water and brine, dried
(Na2SO4), and concentrated in vacuo. The crude product was
purified by a column chromatography on silica gel using hexane–
EtOAc (3:1) as an eluent to give 17 (2.50 g, 99%) as pale yellow
crystals, mp 64.5–66.0 °C. IR (KBr): 1743, 1494, 1304, 1250,
1219, 1152 cm−1. H NMR (400 MHz, CDCl3): δ 1.32 (s, 9H),
1
1.42 (d, J = 6.1 Hz, 3H), 1.44 (d, J = 6.1 Hz, 3H), 1.46 (d, J =
6.1 Hz, 6H), 3.30 (s, 3H), 3.84 (s, 3H), 3.96 (s, 3H), 4.62 (sep, J
= 6.1 Hz, 1H), 4.74 (sep, J = 6.1 Hz, 1H), 4.94 (d, J = 6.7 Hz,
1H), 5.00 (d, J = 6.7 Hz, 1H), 6.87 (s, 1H), 6.90 (s, 1H), 7.28 (s,
1H), 7.50 (d, J = 8.6 Hz, 1H), 7.57 (d, J = 8.6 Hz, 1H), 7.79 (s,
1H). 13C NMR (100 MHz, CDCl3): δ 21.8, 22.0, 22.0, 22.2, 27.2,
55.7, 56.1, 56.6, 71.0, 71.7, 84.1, 96.0, 99.7, 103.4, 105.3, 111.9,
115.0, 115.7, 116.3, 117.6, 123.5, 124.9, 128.3, 129.7, 132.6,
145.3, 146.2, 148.6, 149.7, 150.0, 151.3. HRFABMS m/z Calcd
for C33H40BrNO8 (M+): 657.1937. Found: 657.1925.
4.2.7.
tert-Butyl
2-bromo-7-isopropoxy-8-methoxy-1H-
cooled (−78 °C)
benzo[g]indole-1-carboxylate (14). To
a
solution of 12 (491 mg, 1.38 mmol) in THF (50 mL), was added
dropwise a pentane solution of t-BuLi (1.46 M, 1.14 mL,
1.66 mmol) under Ar atmosphere. After 1 h at this temperature,
1,2-dibromo-1,1,2,2-tetrafluoroethane (0.246 mL, 2.07 mmol)
was added and the mixture was stirred for 1 h. The reaction was
quenched by adding water and the THF was removed in vacuo.
The residue was extracted 3 times with DCM and the combined
organic layers were washed successively with water and brine,
dried (Na2SO4), and concentrated in vacuo. The oily residue was
chromatographed on silica gel using hexane–EtOAc (10:1) as an
eluent to give 14 (567 mg, 95%) as white solid, mp 108.5–
109.0 °C. IR (KBr): 1749, 1493, 1299, 1247, 1168, 1106 cm−1.
1H NMR (400 MHz, CDCl3): δ 1.45 (d, J = 6.1 Hz, 6H), 1.75 (s,
9H), 3.96 (s, 3H), 4.70 (sep, J = 6.1 Hz, 1H), 6.70 (s, 1H), 7.23
4.2.10. 1-tert-Butyl 3-methyl 7-isopropoxy-2-(4-isopropoxy-5-
methoxy-2-methoxymethoxyphenyl)-8-methoxy-1H-
benzo[g]indole-1,3-dicarboxylate (18). To a cooled (−78 °C)
solution of 17 (2.50 g, 3.80 mmol) in THF (100 mL), was added
dropwise a 1.59 M solution of t-BuLi in pentane (1.43 M,
5.85 mL, 8.36 mmol) under Ar atmosphere. After 1 h at −78 °C,
a solution of methyl chloroformate (0.883 mL, 11.4 mmol) in
THF (8 mL) was dropwise added. After 1 h at −78 °C, the
reaction was quenched by adding saturated aqueous NH4Cl. The
THF was removed in vacuo and the residue was extracted 3 times
with ether. The ethereal extracts were combined, washed

successively with water and brine, dried (Na2SO4), and
concentrated in vacuo. The crude product was purified by a
column chromatography on silica gel using with hexane–EtOAc
(3:1) as an eluent to give 18 (1.95 g, 81%) as reddish crystals, mp
147–149 °C (DCM/hexane). IR (KBr): 1754, 1700, 1503, 1251,
added dropwise a solution of 19 (32.0 mg, 69.3 µmol) in DMF
(1.0 mL) under Ar atmosphere. After 40 min, 18-crown-6
(32.8 mg, 0.124 mmol) dissolved in DMF (1.0 mL) was added.
After 40 min, iodomethane (18.0 µL, 0.289 mmol) was added
and the mixture was stirred for 3 h. The reaction was then
quenched by adding saturated aqueous NH4Cl. The mixture was
diluted with EtOAc and the two layers were separated. The
organic layer was washed with water and brine, dried (Na2SO4),
and concentrated in vacuo. The residue was chromatographed on
silica gel using hexane–EtOAc (2:1–0:1) as an eluent to give 21a
(29.0 mg, 88%) as pale yellow solid, mp 231.5–232 °C (EtOAc).
1
1217, 1149 cm−1. H NMR (400 MHz, CDCl3): δ 1.33 (s, 9H),
1.42 (d, J = 6.1 Hz, 3H), 1.44 (d, J = 6.1 Hz, 3H), 1.47 (d, J =
6.1 Hz, 6H), 3.32 (s, 3H), 3.75 (s, 3H), 3.81 (s, 3H), 3.94 (s, 3H),
4.63 (sep, J = 6.1 Hz, 1H), 4.74 (sep, J = 6.1 Hz, 1H), 4.94 (d, J
= 6.8 Hz, 1H), 4.99 (d, J = 6.8 Hz, 1H), 6.86 (s, 1H), 6.90 (s,
1H), 7.31 (s, 1H), 7.56 (s, 1H), 7.60 (d, J = 8.7 Hz, 1H), 8.17 (d,
J = 8.7 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 21.8, 22.0, 22.0,
22.2, 27.1, 51.0, 55.7, 55.9, 56.5, 71.0, 71.7, 85.1, 95.9, 102.3,
104.9, 110.2, 111.9, 114.6, 115.3, 116.8, 118.4, 123.6, 123.6,
127.7, 129.4, 139.8, 144.9, 146.2, 148.6, 149.8, 150.3, 151.6,
165.1. Anal. Calcd for C35H43NO10: C, 65.92; H, 6.80; N, 2.20.
Found: C, 65.92; H, 6.89; N, 2.02.
1
IR (KBr): 1709, 1519, 1427, 1266, 1227, 1110 cm−1. H NMR
(500 MHz, CDCl3): δ 1.45 (d, J = 6.1 Hz, 6H), 1.48 (d, J =
6.1 Hz, 6H), 3.99 (s, 3H), 4.04 (s, 3H), 4.48 (s, 3H), 4.60 (sep, J
= 6.1 Hz, 1H), 4.72 (sep, J = 6.1 Hz, 1H), 6.94 (s, 1H), 7.27 (s,
1H), 7.41 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.66 (s, 1H), 8.20 (d,
J = 8.4 Hz, 1H). 13C NMR (126 MHz, CDCl3): δ 21.8, 22.0, 37.7,
56.0, 56.9, 71.1, 71.5, 101.6, 101.8, 103.8, 105.0, 105.8, 112.3,
116.8, 117.9, 121.0, 123.1, 128.3, 134.4, 141.5, 146.3, 146.4,
148.7, 149.3, 149.9, 159.2. HREIMS m/z Calcd for C28H29NO6
(M+): 475.1995. Found: 475.1991.
4.2.11.
3,10-Diisopropoxy-2,11-
dimethoxybenzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one (19).
A solution of 18 (2.02 g, 3.17 mmol) in a mixture of CHCl3
(40 mL) and MeOH (40 mL) containing conc. HCl (8.0 mL) was
stirred at 50 °C for 24.5 h. After cooling to room temperature, the
solvents were removed in vacuo, and the residue was diluted with
water and extracted 3 times with DCM. The combined organic
extracts was washed successively with saturated aqueous
NaHCO3, water, and brine, dried (Na2SO4), and concentrated in
4.3.2.
13-Ethyl-3,10-diisopropoxy-2,11-
dimethoxybenzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one
(21b). To a cooled (0 °C) and stirred slurry of NaH (31.3 mg,
0.783 mmol of 60% NaH in mineral oil which had been freed
from mineral oil by washing with hexane) in DMF (2.0 mL), was
added dropwise a solution of 19 (82.6 mg, 0.179 mmol) in DMF
(3.0 mL) under Ar atmosphere. After 30 min, iodoethane
(56.0 µL, 0.716 mmol) was added. After 30 min, the mixture was
allowed to warm to room temperature and stirred for 23.5 h. The
reaction was then quenched by adding saturated aqueous NH4Cl
and 28% aqueous ammonia. The mixture was diluted with EtOAc
and the two layers were separated. The organic layer was washed
with water and brine, dried (Na2SO4), and concentrated in vacuo.
The residue was chromatographed on silica gel using hexane–
EtOAc (1:2) as an eluent to give 21b (50.9 mg, 58%) as colorless
powder, mp 219.2–220.0 °C (EtOAc). IR (KBr): 1711, 1520,
vacuo. The crude product was purified by
a column
chromatography on silica gel using CHCl3–EtOAc (1:1) as an
eluent to give 19 (1.45 g, 99%) as white crystals, mp 186–187 °C
(EtOAc). IR (KBr): 1680, 1515, 1470, 1257, 1216, 1108 cm−1. 1H
NMR (400 MHz, acetone-d6) δ 1.40 (d, J = 6.0 Hz, 12H), 3.89 (s,
3H), 3.94 (s, 3H), 4.76 (sep, J = 6.0 Hz, 1H), 4.77 (sep, J =
6.0 Hz, 1H), 7.05 (s, 1H), 7.48 (s, 1H), 7.51 (s, 1H), 7.62 (d, J =
8.5 Hz, 1H), 7.66 (s, 1H), 8.04 (d, J = 8.5 Hz, 1H), 12.03 (br s,
1H). 13C NMR (100 MHz, acetone-d6): δ 22.2, 22.3, 56.1, 56.7,
71.4, 72.0, 101.9, 104.4, 104.7, 106.5, 113.0, 117.6, 118.1, 121.3,
122.8, 128.0, 133.7, 141.3, 148.0, 148.3, 149.2, 150.7, 152.0,
159.5. HREIMS m/z Calcd for C27H27NO6 (M+): 461.1838.
Found: 461.1833.
1
1438, 1256, 1228, 1112 cm−1. H NMR (400 MHz, CDCl3): δ
1.46 (d, J = 6.1 Hz, 6H), 1.49 (d, J = 6.1 Hz, 6H), 1.98 (t, J =
7.2 Hz, 3H), 4.00 (s, 3H), 4.06 (s, 3H), 4.61 (sep, J = 6.1 Hz,
1H), 4.74 (sep, J = 6.1 Hz, 1H), 4.94 (q, J = 7.2 Hz, 2H), 6.97 (s,
1H), 7.30 (s, 1H), 7.45 (s, 1H), 7.54 (d, J = 8.5 Hz, 1H), 7.69 (s,
1H), 8.26 (d, J = 8.5 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ
15.9, 21.8, 22.0, 42.9, 55.9, 56.8, 71.0, 71.5, 101.4, 101.4, 104.0,
104.6, 105.7, 112.6, 116.4, 118.1, 121.3, 123.1, 128.5, 132.7,
139.6, 146.2, 146.6, 148.7, 149.2, 150.2, 159.2. Anal. Calcd for
C29H31NO6: C, 71.15; H, 6.38; N, 2.86. Found: C, 71.06; H, 6.45;
N, 2.79.
4.2.12.
3,10-Dihydroxy-2,11-
dimethoxybenzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one (20).
To a cooled (−78 °C) solution of 19 (99.1 mg, 0.215 mmol) in
DCM (37 mL), was added dropwise a solution of BCl3 in heptane
(1.0 M, 2.58 mL, 2.58 mmol) under Ar atmosphere. The mixture
was stirred at −78 °C for 30 min, 0 °C for 1 h and room
temperature for 22 h. The reaction was quenched by adding
saturated aqueous NaHCO3 and the DCM was removed in vacuo.
The precipitated solid was filtered, washed successively washed
with water and 2 M HCl solution, and dried overnight in vacuo to
give 1a (71.3 mg, 88%) as white crystals, mp 254–255 °C
(decomp). IR (KBr): 3503, 3237, 1653, 1500, 1288, 1209,
4.3.3.
13-Allyl-3,10-diisopropoxy-2,11-
dimethoxybenzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one
(21c). To a cooled (0 °C) and stirred slurry of NaH (12.1 mg,
0.303 mmol of 60% NaH in mineral oil which had been freed
from mineral oil by washing with hexane) in DMF (2.0 mL), was
added dropwise a solution of 19 (43.4 mg, 94.0 µmol) in DMF
(2.0 mL) under Ar atmosphere. After 30 min, allyl bromide
(32.0 µL, 0.370 mmol) was added. After 30 min, the mixture was
allowed to warm to room temperature and stirred for 22 h. The
reaction was then quenched by adding saturated aqueous NH4Cl
and 28% aqueous ammonia. The mixture was diluted with EtOAc
and the two layers were separated. The organic layer was washed
with water and brine, dried (Na2SO4), and concentrated in vacuo.
The residue was chromatographed on silica gel using hexane–
EtOAc (1:2) as an eluent to give 21c (35.1 mg, 74%) as pale
yellow solid, mp 219.2–220.0 °C (ether/hexane). IR (KBr): 1704,
1516, 1436, 1258, 1226, 1039 cm−1. 1H NMR (400 MHz,
1
1145 cm−1. H NMR (400 MHz, DMSO-d6): δ 3.97 (s, 3H), 4.04
(s, 3H), 6.94 (s, 1H), 7.30 (s, 1H), 7.48 (d, J = 8.6 Hz, 1H), 7.85
(d, J = 8.6 Hz, 1H), 8.32 (s, 1H), 8.36 (s, 1H), 13.70 (s, 1H). 13
C
NMR (100 MHz, DMSO-d6) δ 56.2, 56.6, 99.4, 102.6, 103.7,
104.9, 105.6, 111.4, 116.2, 116.7, 118.9, 121.0, 126.7, 133.1,
140.9, 145.3, 146.2, 147.7, 149.1, 149.2, 158.8. HRFABMS m/z
Calcd for C21H15NO6 (M+): 377.0899. Found: 377.0892.
4.3. Synthesis of N-substituted BBPIs 22a–e
4.3.1.
3,10-Diisopropoxy-2,11-dimethoxy-13-
methylbenzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one (21a).
To a cooled (0 °C) and stirred slurry of NaH (22.4 mg,
0.560 mmol of 60% NaH in mineral oil which had been freed
from mineral oil by washing with hexane) in DMF (0.5 mL), was

CDCl3): δ 1.45 (d, J = 6.1 Hz, 6H), 1.48 (d, J = 6.1 Hz, 6H), 3.92
(s, 3H), 3.97 (s, 3H), 4.61 (sep, J = 6.1 Hz, 1H), 4.74 (sep, J =
6.1 Hz, 1H), 5.45 (br s, 1H), 5.55–5.63 (m, 1H), 5.71–5.77 (m,
1H), 6.56–6.67 (m, 1H), 6.98 (s, 1H), 7.32 (s, 1H), 7.43 (s, 1H),
7.57 (d, J = 8.5 Hz, 1H), 7.67 (s, 1H), 8.24 (d, J = 8.5 Hz, 1H).
13C NMR (100 MHz, CDCl3): δ 21.8, 22.0, 51.4, 56.0, 56.6, 71.1,
71.6, 101.8, 102.1, 103.8, 104.6, 105.5, 112.3, 116.4, 117.9,
119.8, 121.3, 123.2, 128.3, 133.0, 134.5, 142.0, 146.4, 146.6,
148.7, 149.3, 150.2, 159.3. HRFABMS m/z Calcd for C30H31NO6
(M+): 501.2151. Found: 501.2139.
4.60–4.72 (m, 2H), 4.71 (sep, J = 6.1 Hz, 1H), 6.92 (s, 1H), 7.24
(s, 1H), 7.45 (s, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.61 (s, 1H), 8.22
(d, J = 8.5 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 21.8, 22.0,
46.0, 47.4, 56.1, 56.6, 58.8, 71.0, 71.5, 100.9, 101.3, 103.7,
103.8, 105.4, 112.4, 116.4, 117.9, 121.2, 123.2, 128.3, 132.4,
139.6, 146.1, 146.8, 148.4, 148.9, 150.4, 159.1. Anal. Calcd for
C31H36N2O6: C, 69.91; H, 6.81; N, 5.26. Found: C, 69.62; H,
6.97; N, 5.16.
4.3.6.
3,10-Dihydroxy-2,11-dimethoxy-13-
methylbenzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one (22a).
The title compound was obtained in a similar manner as
described for the synthesis of 20. Yellow crystals, mp 188–
192 °C (decomp). Yield, 94%. IR (KBr): 3227, 1670, 1502,
1424, 1271, 1212, 1159 cm−1. 1H NMR (400 MHz, DMSO-d6): δ
3.96 (s, 3H), 4.01 (s, 3H), 4.56 (s, 3H), 6.95 (s,1H), 7.34 (s, 1H),
7.53 (d, J = 8.5 Hz, 1H), 7.61 (s, 1H), 7.81 (s, 1H), 7.94 (d, J =
8.5 Hz, 1H), 9.69 (br s, 2H). 13C NMR (100 MHz, DMSO-d6): δ
37.9, 55.4, 56.1, 99.8, 101.9, 104.3, 104.6, 105.3, 112.2, 116.1,
116.6, 119.7, 122.3, 128.0, 134.2, 141.5, 144.8, 145.9, 148.1,
148.5, 149.2, 158.2. HRFABMS m/z Calcd for C22H17NO6 (M+):
391.1056. Found: 391.1062.
4.3.4.
3,10-Diisopropoxy-2,11-dimethoxy-13-
propargylbenzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one
(21d). To a cooled (0 °C) and stirred slurry of NaH (45.3 mg,
1.13 mmol of 60% NaH in mineral oil which had been freed from
mineral oil by washing with hexane) in DMF (2.0 mL), was
added dropwise a solution of 19 (96.7 mg, 0.210 mmol) in DMF
(2.0 mL) under Ar atmosphere. After 30 min, propargyl bromide
(63.0 µL, 0.836 mmol) was added. After 30 min, the mixture was
allowed to warm to room temperature and stirred for 3.5 h. The
reaction was then quenched by adding saturated aqueous NH4Cl
and 28% aqueous ammonia. The mixture was diluted with EtOAc
and the two layers were separated. The organic layer was washed
with water and brine, dried (Na2SO4), and concentrated in vacuo.
The residue was chromatographed on silica gel using hexane–
EtOAc (1:1) as an eluent to give 21d (68.6 mg, 65%) as pale
yellow solid, mp 196–197 °C (ether/hexane). IR (KBr): 1711,
1514, 1434, 1257, 1227, 1163 cm−1. 1H NMR (400 MHz,
CDCl3): δ 1.45 (d, J = 6.0 Hz, 6H), 1.48 (d, J = 6.0 Hz, 6H), 3.96
(s, 3H), 4.01 (s, 3H), 4.59 (sep, J = 6.0 Hz, 1H), 4.70 (sep, J =
6.0 Hz, 1H), 5.35 (d, J = 6.2 Hz, 2H), 6.89 (s, 1H), 7.20 (s, 1H),
7.32 (t, J = 6.2 Hz, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.75 (s, 1H),
8.04 (s, 1H), 8.20 (d, J = 8.5 Hz, 1H). 13C NMR (100 MHz,
CDCl3): δ 21.8, 22.0, 55.8, 56.5, 71.0, 71.5, 83.6, 98.1, 101.4,
101.8, 103.3, 104.3, 105.8, 111.8, 116.9, 117.6, 120.7, 123.2,
128.2, 132.8, 139.7, 146.2, 146.3, 148.6, 149.2, 149.9, 159.3,
209.7. HRFABMS m/z Calcd for C30H30NO6 [(M+H)+]:
500.2073. Found: 500.2076.
4.3.7.
13-Ethyl-3,10-dihydroxy-2,11-
dimethoxybenzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one
(22b). The title compound was obtained in a similar manner as
described for the synthesis of 20. Deep green crystals, mp 185–
186 °C (decomp). Yield, 94%. IR (KBr): 3370, 1683, 1504,
1440, 1296, 1208, 1159 cm−1. 1H NMR (400 MHz, DMSO-d6): δ
1.93 (t, J = 7.1 Hz, 3H), 3.96 (s, 3H), 4.02 (s, 3H), 5.07 (q, J =
7.1 Hz, 2H), 6.99 (s, 1H), 7.37 (s, 1H), 7.57 (d, J = 8.5 Hz, 1H),
7.59 (s, 1H), 7.79 (s, 1H), 8.02 (d, J = 8.5 Hz, 1H), 9.73 (br s,
1H), 10.36 (br s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 15.7,
43.1, 55.5, 56.2, 100.0, 101.5, 104.6, 104.7, 105.0, 112.7, 115.9,
117.0, 120.1, 122.8, 128.4, 132.7, 139.8, 145.3, 145.9, 148.2,
149.0, 149.3, 158.5. HRFABMS m/z Calcd for C23H19NO6 (M+):
405.1212. Found: 405.1237.
4.3.8.
13-Allyl-3,10-dihydroxy-2,11-
dimethoxybenzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one
(22c). The title compound was obtained in a similar manner as
described for the synthesis of 20. Yellow crystals, mp 170–
172 °C (decomp). Yield, 50%. IR (KBr): 3453, 1677, 1504,
1438, 1296, 1244, 1169 cm−1. 1H NMR (400 MHz, DMSO-d6): δ
3.88 (s, 3H), 3.94 (s, 3H), 5.38 (d, J = 17.3 Hz, 1H), 5.61 (br s,
2H), 5.66 (d, J = 11.0 Hz, 1H), 6.70–6.82 (m, 1H), 7.01 (s, 1H),
7.37 (s, 1H), 7.50 (s, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.70 (s, 1H),
8.02 (d, J = 8.5 Hz, 1H), 9.65 (s, 1H), 10.30 (s, 1H). 13C NMR
(100 MHz, DMSO-d6): δ 51.4, 55.6, 56.2, 100.5, 102.0, 104.4,
104.5, 105.2, 112.3, 115.8, 116.9, 118.3, 120.0, 122.8, 128.2,
134.2, 134.2, 141.8, 145.1, 146.1, 148.3, 148.8, 149.4, 158.6.
HRFABMS m/z Calcd for C24H19NO6 (M+): 417.1212. Found:
417.1227.
4.3.5.
13-[2-(Dimethylamino)ethyl]-3,10-diisopropoxy-2,11-
dimethoxybenzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one
(21e). To a cooled (0 °C) and stirred slurry of NaH (8.6 mg,
0.215 mmol of 60% NaH in mineral oil which had been freed
from mineral oil by washing with hexane) in DMF (2.0 mL), was
added dropwise a solution of 19 (32.0 mg, 69.3 µmol) in DMF
(2.0 mL) under Ar atmosphere. After 20 min, 2-
(dimethylamino)ethyl
chloride
hydrochloride
(23.2 mg,
0.161 mmol) was added and the mixture was warmed to 75 °C.
After 8 h, the mixture was cooled to 0 °C. Sodium hydride (60%
dispersion in mineral oil, 3.8 mg, ca 95 µmol) and 2-
(dimethylamino)ethyl
chloride
hydrochloride
(23.2 mg,
0.161 mmol) was added to the mixture, and then warmed again to
75 °C. After 13 h, the mixture was cooled to 0 °C. Sodium
hydride (60% dispersion in mineral oil, 3.9 mg, ca 98 µmol) and
2-(dimethylamino)ethyl chloride hydrochloride (23.2 mg,
0.161 mmol) was added to the mixture, and then warmed again to
75 °C. After 4 h, the mixture was cooled to room temperature.
The reaction was quenched by adding saturated aqueous NH4Cl.
The mixture was diluted with EtOAc and the two layers were
separated. The organic layer was washed with water and brine,
dried (Na2SO4), and concentrated in vacuo. The residue was
chromatographed on silica gel using hexane–EtOAc (1:1) as an
eluent to give 21e (13.4 mg, 47%) as pale yellow solid, mp
226.5–227.5 °C (EtOAc). IR (KBr): 1727, 1521, 1437, 1245,
1228, 1110, 1035 cm−1. 1H NMR (400 MHz, CDCl3): δ 1.46 (d, J
= 6.1 Hz, 6H), 1.49 (d, J = 6.1 Hz, 6H), 2.38 (s, 6H), 2.94–3.14
(m, 2H), 3.99 (s, 3H), 4.02 (s, 3H), 4.60 (sep, J = 6.1 Hz, 1H),
4.3.9.
3,10-Dihydroxy-2,11-dimethoxy-13-
propargylbenzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one
(22d). The title compound was obtained in a similar manner as
described for the synthesis of 20. Yellow crystals, mp 247–
250 °C (decomp). Yield, 63%. IR (KBr): 3216, 1708, 1437,
1
1245, 1210, 1162 cm−1. H NMR (400 MHz, DMSO-d6) δ 3.92
(s, 3H), 3.97 (s, 3H), 5.70 (d, J = 6.3 Hz, 2H), 6.95 (s, 1H), 7.33
(s, 1H), 7.57 (d, J = 8.6 Hz, 1H), 7.95 (d, J = 8.6 Hz, 1H), 7.96
(s, 1H), 7.97 (t, J = 6.3 Hz, 1H), 8.23 (s, 1H). 13C NMR
(100 MHz, DMSO-d6): δ 55.5, 56.1, 84.4, 97.9, 100.3, 101.5,
104.3, 104.6, 104.7, 112.1, 116.4, 116.8, 119.6, 122.8, 128.2,
132.8, 139.8, 145.0, 146.2, 148.3, 148.7, 149.4, 158.5, 209.0.
HRFABMS m/z Calcd for C24H18NO6 [(M+H)+]: 416.1134.
Found: 416.1113.

4.3.10. Trifluoroacetic acid salt of 13-[2-(Dimethylamino)ethyl]-
3,10-dihydroxy-2,11-dimethoxybenzo[g][1]benzopyrano[4,3-
b]indol-6(13H)-one (22e). To a solution of 21e (100 mg,
0.188 mmol) in DCM (6.0 mL), was added dropwise a 1.0 M
solution of AlCl3 in nitrobenzene (1.01 mL, 1.01 mmol) under Ar
atmosphere. The mixture was stirred at room temperature for
48 h. The reaction was quenched by adding a solution of
NaHCO3 (255 mg, 3.04 mmol) and Rochelle salt (857 mg,
3.04 mmol) in water (6.0 mL). The mixture was stirred for 1 h
and the DCM was removed in vacuo. The nitrobenzene was
removed azeotropically with water under reduced pressure. The
precipitated solid was filtered, washed successively washed with
water, and dried overnight in vacuo to give 13-[2-
(dimethylamino)ethyl]-3,10-dihydroxy-2,11-
dimethoxybenzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one
(22e′) (78.9 mg, 94%) as pale yellow solid, mp >300 °C. IR
(KBr): 3535, 3207, 1682, 1440, 1298, 1207, 1154 cm−1. 1H NMR
(400 MHz, DMSO-d6): δ 2.29 (s, 6H), 2.93 (br s, 2H), 3.91 (s,
3H), 3.92 (s, 3H), 4.57 (br s, 2H), 6.88 (s, 1H), 7.30 (s, 1H), 7.42
(s, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.57 (s, 1H), 7.93 (d, J =
8.5 Hz, 1H), 9.57 (br s, 1H), 10.18 (br s, 1H). 13C NMR
(100 MHz, DMSO-d6): δ 45.6, 47.0, 55.3, 55.9, 58.1, 99.8, 100.8,
104.0, 104.0, 104.4, 112.4, 115.5, 116.6, 119.8, 122.5, 128.1,
132.2, 139.4, 145.0, 145.7, 147.9, 148.8, 148.9, 158.0.
HRFABMS m/z Calcd for C25H25N2O6 [(M+H)+]: 449.1713.
Found: 449.1734.
140.1, 140.6, 146.9, 147.5, 149.8, 155.7, 155.7, 158.3, 170.2,
170.8. HRFABMS m/z. Calcd for C42H51N3O12 (M+): 789.3473.
Found: 789.3456.
4.4.2. Trifluoroacetic acid salt of 2,11-dimethoxy-13-methyl-6-
oxo-6,13-dihydrobenzo[g][1]benzopyrano[4,3-b]indole-3,10-diyl
bis[(2S)-2-amino-3-methylbutanoate] (24). To the compound 23
(20.5 mg, 26.0 µmol) was added trifluoroacetic acid (1.0 mL) at
room temperature. After stirring for 1 h at room temperature, the
mixture was evaporated. The residue was chromatographed on
Sephadex LH-20 using methanol as an eluent to give 24
(25.4 mg, quant) as a pale yellow powder, mp 158.5–160 °C. IR
(KBr): 3435, 1771, 1678, 1520, 1428, 1260, 1204 cm−1. 1H NMR
(400 MHz, methanol-d4):  1.25–1.32 (m, 12H), 2.52–2.62 (m,
2H), 3.95 (s, 3H), 4.00 (s, 3H), 4.30 (s, 3H), 4.34 (d, J = 4.3 Hz,
2H), 7.18 (s, 1H), 7.48 (d, J = 8.6 Hz, 1H), 7.55 (s, 1H), 7.64 (s,
1H), 7.67 (s, 1H), 7.89 (d, J = 8.6 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6):  17.4, 17.4, 17.9, 17.9, 29.0, 29.7, 38.2, 55.8, 56.3,
57.3, 101.8, 103.2, 106.4, 112.2, 112.4, 117.5, 120.8, 121.7,
122.0, 123.8, 126.7, 134.1, 137.7, 139.4, 140.5, 146.5, 147.1,
149.3, 157.5, 167.0, 167.5. HRFABMS (m/z) Calcd for
C32H36N3O8 [(M−2CF3COO−H)+]: 590.2502. Found: 590.2527.
4.5. X-ray crystallographic analysis of 21a
Crystallographic data for 21a can be summarized as follows:
compound formula C32H37NO8, Mw = 563.63, Triclinic, P–1, a =
9.221(5) Å, b = 10.578(4) Å, c = 16.355(7) Å,  = 74.248(13)°, 
= 79.774(18)°,  = 69.424(17)°, V = 1431.5(11) Å3, Z = 2, Dcalc
=
To a solution of 22e′ (100 mg, 0.223 mmol) in DCM (2.0 mL)
was added trifluoroacetic acid (2.0 mL) at room temperature.
After stirring for 5 min, the mixture was evaporated. The residue
was purified by column chromatography over Sephadex LH-20
(MeOH) to give 22e (115 mg, 91%) as a brown powder, mp 263–
264 °C (decomp) (sealed capillary). IR (KBr): 3265, 1684, 1519,
1.308 g/cm3, monochromatized radiation λ(Mo Kα) = 0.71075 Å,
µ = 0.094 mm−1, F(000) = 600, T = 123(2) K. Data were collected
on an Rigaku FR-E SuperBright diffractometer. There were 6500
unique reflections with 3657 being observed at the 2σ level. The
structure was solved by direct methods (SHELXS-97)33 and the
non-hydrogen atoms were refined anisotropically by full-matrix
least-squares procedures on F2 using SHELXL Version 2018/3
software.33 Hydrogen atoms were refined using the riding model.
All calculations were performed using the CrystalStructure34
crystallographic software package except for refinement, which
was performed using SHELXL Version 2018/3. The final model
was refined using 382 parameters and data from all 6500
reflections. The final agreement statistics are as follows: R =
0.0767 (based on 3657 reflections with I > 2σ(I)), wR = 0.2505, S
= 1.040. The maximum peak height in the final difference
Fourier map was 1.005 eÅ3, which is without chemical
significance. The program ORTEP-3 for Windows was used to
generate the X-ray structural diagrams.35 CCDC 1857984
contains the supplementary crystallographic data for this paper.
The data can be obtained free of charge from The Cambridge
1
1439, 1298, 1205 cm−1. H NMR (400 MHz, DMSO-d6):  2.78
(s, 6H), 3.46 (t, J = 6.9 Hz, 2H), 4.01 (s, 3H), 4.08 (s, 3H), 5.55
(br s, 2H), 7.02 (s, 1H), 7.40 (s, 1H), 7.60 (s, 1H), 7.63 (d, J =
8.6 Hz, 1H), 7.69 (s, 1H), 8.04 (d, J = 8.6 Hz, 1H), 9.70 (br s,
1H), 10.41 (br s, 2H). 13C NMR (100 MHz, DMSO-d6):  42.9,
44.1, 54.8, 55.9, 56.6, 101.6, 102.1, 104.4, 104.6, 105.6, 112.5,
116.0, 116.7, 121.0, 123.4, 128.4, 133.0, 141.4, 145.2, 146.2,
148.3, 149.1, 149.7, 158.0. HRFABMS m/z. Calcd for
C25H25N2O6 [(M−CF3COO)+]: 449.1713. Found: 449.1721.
4.4. Synthesis of the water-soluble valine ester derivative 24
4.4.1.
dihydrobenzo[g][1]benzopyrano[4,3-b]indole-3,10-diyl bis{(2S)-
2-[(tert-butoxycarbonyl)amino]-3-methylbutanoate} (23).
solution of N-(tert-butoxycarbonyl)-L-valine (150 mg,
2,11-Dimethoxy-13-methyl-6-oxo-6,13-
A
0.690 mmol), EDCI·HCl (132 mg, 0.690 mmol) and DMAP
(6.2 mg, 51 µmol) in DMF (7.0 mL) was stirred at room
temperature for 10 min under Ar atmosphere. To the mixture,
22a (50.0 mg, 0.128 mmol) was added and stirred for 24 h. The
reaction was quenched by adding water. The mixture was diluted
with DCM and the two layers were separated. The organic layer
was washed with water and brine, dried (Na2SO4), and
concentrated in vacuo. The residue was chromatographed on
silica gel using hexane–EtOAc (1:1) as an eluent to give 23
(66.9 mg, 66%) as white crystals, mp 127–128 °C (ether/hexane).
Crystallographic
www.ccdc.cam.ac.uk/data_request/cif.
Data
Centre
via
4.6. Biological assays–general
The purity of all tested compounds [5, 20, 22a–e, and 24] was
determined to be >95% using HPLC with a GL Science Inertsil
diol column (4.6 mm i.d. × 250 mm). The detection wavelength
was fixed at 305 nm. The HPLC profiles of 5, 20, 22a–e, and 24
are shown in Supplementary data (Figure S53).
1
4.7. Topoisomerase I inhibitory assay
IR (KBr): 1766, 1717, 1497, 1367, 1258, 1160 cm−1. H NMR
(400 MHz, CDCl3):  1.06–1.20 (m, 12H), 1.51 (s, 18H), 2.30–
2.56 (m, 2H), 3.89 (s, 3H), 3.92 (s, 3H), 4.18 (s, 3H), 4.53–4.66
(m, 2H), 5.22–5.32 (m, 2H), 7.02 (s, 1H), 7.27 (s, 1H), 7.35 (d, J
= 8.5 Hz, 1H), 7.43 (s, 2H), 8.01 (d, J = 8.4 Hz, 1H). 13C NMR
(100 MHz, CDCl3):  17.3, 17.3, 19.2, 19.2, 28.4, 31.3, 31.4,
37.5, 55.6, 56.3, 58.6, 79.9, 80.0, 101.8, 102.5, 104.7, 111.5,
112.9, 117.8, 120.3, 121.9, 122.2, 123.8, 127.3, 133.8, 138.6,
The topoisomerase
I relaxation assay was performed
according to previous reports.24,25 In brief, 2U of DNA
topoisomerase I from calf thymus (TaKaRa Bio) was mixed with
250 ng of supercoiled DNA pBR322 (TaKaRa Bio) in 20 µL of a
reaction buffer (35 mM Tris–HCl, pH 8, 72 mM KCl, 5 mM
MgCl2, 5 mM DTT, 5 mM spermidine, 0.01% BSA) in the
presence or absence of the test drugs previously dissolved in

DMSO. The mixture was incubated at 37 °C for 30 min and then
the reaction was terminated by adding 2 µL of 10% SDS
solution. After digestion of the enzyme by adding 2 µL of
2 mg/mL of proteinase K and incubating at 37 °C for 30 min,
excess compounds were removed by extraction with
CHCl3/isoamyl alcohol (24:1). The reaction product was
subjected to 1% agarose gel electrophoresis in the absence or
presence of ethidium bromide (0.5 µg/mL). When the agarose gel
electrophoresis was performed in the absence of ethidium
bromide, the gel was stained with 0.5 µg/mL ethidium bromide
after electrophoresis.
cage, under standardized 12 h light–dark cycle conditions in a
specific pathogen-free room for three days. During the
acclimatization period, food and water were freely available.
4.10.2. Preparation of dosing drugs
Irinotecan hydrochloride trihydrate (3) was dissolved in
distilled water (final concentration of 6.25 mg/mL). Compound
24 was dissolved in 5% glucose solution (final concentration of
6.25 mg/mL). The drugs (50 mg/kg) were administered by tail
vein injection. Controls were given the vehicle (saline solution).
4.10.3. Cell culture
4.8. Docking simulation
Colon 26 cell line was purchased from Cell Bank Riken
BioResource Center. Cells were maintained in RPMI 1640
supplemented with 10% fetal bovine serum (FBS) and 0.5%
Docking studies were performed using MOE 2014.0901.27
Crystal structure of the topotecan (2)–DNA–topoisomerase I
ternary complex (PDB ID: 1K4T)26 was obtained from the
Protein Data Bank. The protein structures for the docking process
were prepared according to the following sequence: (i) The
topoisomerase I–DNA–topotecan ternary complex was loaded.
(ii) Hydrogen atoms and electric charge were added to the
complex by Protonate 3D (default settings). (iii) The hydrogen
atoms were optimized by MMFF94x force field (heavy atoms
were fixed during the optimization). (iv) The dummy atoms were
disposed in the docking site by Site Finder (default settings). On
the other hand, the conformers of 22a were obtained by
Conformational Search using the LowModeMD search method
with default parameters except for the following: The check box
for the hydrogens was selected in order to include both hydrogen
and heavy atoms in the RMSD calculation for duplication
detection and the value of energy window was set to 10 kcal/mol.
Finally, the ligands were docked into the binding site of the
kinases by using the Dock docking program according to the
following sequence: (i) Initial poses were obtained using the
Triangle Matcher placement (timeout: 3000 s; No. of return
poses: 10000), London dG rescoring 1 (maximum number of
poses: 500), GridMin refinement (default settings), and
GBVI/WSA dG rescoring 2 (maximum number of poses: 100).
(ii) The poses obtained in (i) were refined by Forcefield
refinement (default settings) and rescored by GBVI/WSA dG
scoring function (maximum number of poses: 100). (iii) The
poses obtained in (ii) were refined by Forcefield refinement
(sidechain: tether, the tether value was set to 10) and rescored by
GBVI/WSA dG scoring function (maximum number of poses:
100). (iv) The poses obtained in (iii) were refined by Forcefield
refinement (sidechain: free) and rescored by GBVI/WSA dG
scoring function (maximum number of poses: 100). The obtained
poses were evaluated using the GBVI/WSA dG scoring function
and the two most plausible models (pose A and B) are shown in
Fig. 5.
penicillin
G/streptomycin
(penicillin
G:
25 units/mL,
streptomycin: 25 µg/mL) at 37 °C in a humidified 5% CO2
atmosphere. Before use, the concentration of colon 26 was
adjusted to 2  107 cells/mL.
4.10.4. Determination of antitumor effect
Colon 26 cells (5  105 cells) were subcutaneously injected
into the footpad of each mouse. One day after the injection of
colon 26 cells, the drug was administered to the animal at two
day intervals. A total of seven doses were administered and the
mice were weighted daily. Tumor dimensions was measured
using a Vernier caliper and the tumor volume was calculated
daily according to the following equation: tumor volume (mm3) =
4/3  3.14  (A/2)  (B/2)  (C/2), where A is the longer
dimension, B is the shorter dimension, and C is the thickness
(mm).
4.10.5. Statistical analysis
Data are shown as the mean  s.d. Groups were compared by
one-way analysis of variance (ANOVA) and repeated ANOVA,
and the differences between the groups were determined by
Scheffe’s test. All P values less than 0.05 were considered to be
significant.
Acknowledgments
We are grateful to the Japan Science and Technology Agency
(JST) for financial support through the Adaptable & Seamless
Technology Transfer Program through Target-driven R&D (A-
STEP) (Grant No. AS231Z02077G) and Intellectual Property
Highway Program (Grant No. HWY2012-1-144). We also thank
the Screening Committee of Anticancer Drugs supported by a
Grant-in-Aid for Scientific Research on Innovative Areas,
Scientific Support Programs for Cancer Research, from The
Ministry of Education, Culture, Sports, Science and Technology-
Japan for the evaluation of the antiproliferative activities of the
compounds described in this paper. A part of this work (X-ray
crystallographic analysis) was performed under the Cooperative
Research Program of "Network Joint Research Center for
Materials and Devices". In addition, we would like to thank Dr.
Hiroshi Furuno, Green Asia Education Center, Kyushu
University, and Dr. Atsushi Tahara, Institute for Materials
Chemistry and Engineering (IMCE), Kyushu University, for X-
ray crystallographic analysis of compound 21a. We are grateful
to Dr. Gen Onodera, Graduate School of Engineering, Nagasaki
University, for helpful discussions.
4.9. Antiproliferative activity against 39 human cancer cell lines
(JFCR39)
This experiment was carried out at the Screening Committee
of Anticancer Drugs according to the standard protocol used by
the Committee. Inhibition of cell growth was assessed by
measuring the changes in the total cellular protein levels
following 48 h treatment with a given test compound, using the
sulforhodamine B colorimetric assay. The molar concentration of
a test compound required for 50% growth inhibition (GI50) of
cells was calculated as reported previously.17
4.10. Evaluation of in vivo antitumor activity
4.10.1. Animals
Six-week-old male BALB/c mice were purchased from Japan
SLC Ltd, Sizuoka, Japan. The mice were maintained, six per

17. Kong D, Yamori T. JCFR39, a panel of 39 human cancer cell lines, and
its application in the discovery and development of anticancer drugs.
Bioorg Med Chem. 2012;20:1947–1951.
18. Sun H, Tawa G, Wallqvist A. Classification of scaffold-hopping
approaches. Drug Discovery Today. 2011;17:310–324.
19. Martina S, Enkelmann V, Wegner G, Schlülter A-D. N-Protected
pyrrole derivatives substituted for metal-catalyzed cross-coupling
reactions. Synthesis. 1991;613–615.
Supplementary Material
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2018.00.000.
References and notes
20. Hessian KO, Flynn BL. Iodine-induced reaction cascades for the rapid
construction of variously substituted benzothiophenes. Org Lett.
2003;5:4377–4380.
21. Harvey RG, Dai Q, Ran C, Penning TM. Synthesis of the o-quinones
and other metabolites of polyaromatic hydrocarbons implicated in
carcinogenesis. J Org Chem. 2004;69:2024–2032.
22. Fujikawa N, Ohta T, Yamaguchi T, Fukuda T, Ishibashi F, Iwao M.
Total synthesis of lamellarins D, L, and N. Tetrahedron. 2006;62:594–
604.
23. Tardy C, Facompré M, Laine W, Baldeyrou B, García-Galvalos D,
Francesch A, Mateo C, Pastor A, Jiménez JA, Manzanares I, Cuevas C,
Bailly C. Topoisomerase I-mediated DNA cleavage as a guide to the
development of antitumor agents derived from the marine alkaloid
lamellarin D: Triester derivatives incorporating amino acid residues.
Bioorg Med Chem. 2004;12:1607–1712.
24. Bailly C. DNA relaxation and cleavage assays to study topoisomerase I
inhibitors. In: Chaires JB, Waring MJ, eds. Methods in Enzymology. San
Diego, California, USA: Academic Press; 2001;340:610–623.
25. Bailly C. Topoisomerase I poisons and suppressors as anticancer drugs.
Curr Med Chem. 2000;7:39–58.
26. Staker BL, Hjerrild K, Feese MD, Benke CA, Burgin AB, Stewart L.
The mechanism of topoisomerase I poisoning by a camptothecin analog.
Proc Natl Acad Sci USA. 2002;99:15387–15392.
27. Molecular Operating Environment (MOE), version 2014.0901;
Chemical Computing Group Inc.: Montreal, Quebec, Canada, 2014;
https://www.chemcomp.com.
28. Utsugi T, Aoyagi K, Asao T, Okazaki S, Aoyagi Y, Sano M, Wierzba
K, Yamada Y. Antitumor activity of a novel quinolone derivative,
TAS-103, with inhibitory effects of topoisomerases I and II. Jpn J
Cancer Res. 1997;88:992–1002.
29. Okazaki F, Matsunaga N, Hamamura K, Suzuki K, Nakao T, Okazaki
H, Kutsukake M, Fukumori S, Tsuji Y, To H. Administering xCT
inhibitors based on circadian clock improves antitumor effects. Cancer
Res. 2017;77:6603–6613.
30. Taylor JE, Jones MD, Williams JMJ, Bull SD. Friedel–Crafts acylation
of pyrroles and indoles using 1,5-diazabicyclo[4.3.0]non-5-ene (DBN)
as a nucleophilic catalyst. Org Lett. 2010;12:5740–5743.
31. Beugelmans R, Roussi G, González Zamora E, Carbonnelle A-C.
Synthetic studies towards western and eastern macropolypeptide
subunits of kistamycin. Tetrahedron. 1999;55:5089–5112.
32. Hessian KO, Flynn BL. Iodine-induced reaction cascades for the rapid
construction of variously substituted benzothiophenes. Org Lett.
2003;5:4377–4380.
1.
2.
3.
4.
Campoux JJ. DNA topoisomerases: Structure, function, and mechanism.
Annu Rev Biochem. 2001;70:369–413.
Pommier Y. Topoisomerase I inhibitors: camptothecins and beyond.
Nat Rev Cancer. 2006;6:789–802.
Pommier Y. DNA topoisomerase I inhibitors: chemistry, biology, and
interfacial inhibition. Chem Rev. 2009;109:2894–2902.
Rahier NJ, Thomas CJ, Hecht SM. Camptothecin and its analogues. In:
Cragg GM, Kingston DGI, Newman DJ, eds. Anticancer Agents from
Natural Products. Boca Rto, FL, USA: Taylor & Francis; 2005:5–21.
Beretta GL, Zuco V, Perego P, Zaffaroni N. Targeting DNA
topoisomerase I with non-camptothecin poisons. Curr Med Chem.
2012;19:1238–1257.
Fukuda T, Ishibashi F, Iwao M. Synthesis and biological activity of
lamellarin alkaloids: an overview. Heterocycles. 2011;83:491–529.
Bailly C. Lamellarins: a tribe of bioactive marine natural products. In:
La Barre S, Kornprobs J-M, eds. Outstanding Marine Molecules:
Chemistry, Biology, Analysis. Weinheim, Germany: Wiley-Blackwell;
2014:377–386.
Bailly C. Anticancer properties of lamellarins. Mar Drugs.
2015;13:1105–1123.
Quesada AR, Grávalos MDG, Puentes JLF. Polyaromatic alkaloids
from marine invertebrates as cytotoxic compounds and inhibitors of
multidrug resistance caused by P-glycoprotein. Br J Cancer.
1996;74:677–782.
5.
6.
7.
8.
9.
10. Ishibashi F, Miyazaki Y, Iwao M. Total syntheses of lamellarin D and
H. the first synthesis of lamellarin-class marine alkaloids. Tetrahedron.
1997;53:5951–5962.
11. Ishibashi F, Tanabe S, Oda T, Iwao M. Synthesis and structure–activity
relationship study of lamellarin derivatives. J Nat Prod. 2002;65:500–
504.
12. Chittchang M, Batsomboon P, Ruchirawat S, Ploypradith P.
Cytotoxicities and structure–activity relationships of natural and
unnatural lamellarins toward cancer cell lines. ChemMedChem.
2009;4:457–465.
13. Facompré M, Tardy C, Bal-Mahieu C, Colson P, Perez C, Manzanares
I, Cuevas C, Bailly C. Lamellarin D. a novel potent inhibitor of
topoisomerase I. Cancer Res. 2003;63:7392–7399.
14. Marco E, Laine W, Tardy C, Lansiaux A, Iwao M, Ishibashi F, Bailly
C, Gago F. Molecular determinants of topoisomerase I poisoning by
lamellarins: comparison with camptothecin and structure–activity
relationships. J Med Chem. 2005;48:3796–3807.
15. Ohta, T, Fukuda T, Ishibashi F, Iwao M. Design and synthesis of
lamellarin D analogues targeting topoisomerase I. J Org Chem. 2009;
74:8143–8153.
33. Sheldrick GM. A short history of SHELX. Acta Crystallogr, Sect A:
Found Crystallogr. 2008;64:112–122.
34. CrystalStructure 4.3, Crystal Structure Analysis Package; Rigaku
Corporation (2000-2018): Tokyo, 196-8666, Japan.
16. Antiproliferative activities of 6 in the panel of JFCR 39 human cancer
cell lines are shown in Supplementary data (Table S1).
35. Farrugia LJ. WinGX and ORTEP for Windows: an update. J Appl Cryst
2012;45:849–854.