Novel indolizino[8,7-b]indole hybrids as anti-small cell lung cancer agents: Regioselective modulation of topoisomerase II inhibitory and DNA crosslinking activities

Article abstract of DOI:10.1016/j.ejmech.2016.12.046

A novel series of bis(hydroxymethyl)indolizino[8,7-b]indole hybrids composed of β-carboline (topoisomerase I/II inhibition) and bis(hydroxymethyl)pyrrole (DNA cross-linking) are synthesized for antitumor evaluation. Of tumor cell lines tested, small cell

Full text of DOI:10.1016/j.ejmech.2016.12.046

Accepted Manuscript
Novel indolizino[8,7-b]indole hybrids as anti-small cell lung cancer agents:
Regioselective modulation of topoisomerase II inhibitory and DNA crosslinking
activities
Sue-Ming Chang, Wilson Christian, Ming-Hsi Wu, Tai-Lin Chen, Yi-wen Lin, Ching-
Shu Suen, Hima Bindu Pidugu, Dilip Detroja, Anamik Shah, Ming-Jing Hwang, Tsann-
Long Su, Te-Chang Lee
PII:
S0223-5234(16)31052-2
10.1016/j.ejmech.2016.12.046
EJMECH 9137
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 November 2016
Revised Date: 20 December 2016
Accepted Date: 23 December 2016
Please cite this article as: S.-M. Chang, W. Christian, M.-H. Wu, T.-L. Chen, Y.-w. Lin, C.-S. Suen,
H.B. Pidugu, D. Detroja, A. Shah, M.-J. Hwang, T.-L. Su, T.-C. Lee, Novel indolizino[8,7-b]indole
hybrids as anti-small cell lung cancer agents: Regioselective modulation of topoisomerase II inhibitory
and DNA crosslinking activities, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2016.12.046.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Novel indolizino[8,7-b]indole hybrids as anti-small cell lung cancer agents:
Regioselective modulation of topoisomerase II inhibitory and DNA crosslinking
activities
a
a,b
a
a,c
a
Sue-Ming Chang , Wilson Christian , Ming-Hsi Wu , Tai-Lin Chen , Yi-wen Lin ,
a
a
a,b
b
Ching-Shu Suen , Hima Bindu Pidugu , Dilip Detroja , Anamik Shah , Ming-Jing
a
a**
a*
Hwang , Tsann-Long Su , Te-Chang Lee
Graphical Abstract
1

ACCEPTED MANUSCRIPT
Novel indolizino[8,7-b]indole hybrids as anti-small cell lung cancer agents:
Regioselective modulation of topoisomerase II inhibitory and DNA crosslinking
activities
a
a,b
a
a,c
a
Sue-Ming Chang , Wilson Christian , Ming-Hsi Wu , Tai-Lin Chen , Yi-Wen Lin ,
a
a
a,b
b
Ching-Shu Suen , Hima Bindu Pidugu , Dilip Detroja , Anamik Shah , Ming-Jing
a
a**
a*
Hwang , Tsann-Long Su , Te-Chang Lee
a
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
b
Department of Chemistry, Saurashtra University, Rajkot, Gujarat, India
c
Institute of Biochemistry and Molecular Biology, National Yang-Ming University,
Taipei, Taiwan
*
*
Corresponding author. Tel.: +886-2-27899145, Fax: +886-2-27829142
*Corresponding author. Tel.: +886-2-27899045, Fax: +886-2-27829142
E-mail: tlsu@ibms.sinica.edu.tw (T.L. Su), bmtcl@ibms.sinica.edu.tw (T.C. Lee).
Abstract
A novel series of bis(hydroxymethyl)indolizino[8,7-b]indole hybrids composed of
β-carboline (topoisomerase I/II inhibition) and bis(hydroxymethyl)pyrrole (DNA
cross-linking) are synthesized for antitumor evaluation. Of tumor cell lines tested,
small cell lung cancer (SCLC) cell lines are the most sensitive to the newly
synthesized compounds. These hybrids induce cell cycle arrest at the G2/M phase,
trigger tumor cell apoptotic death, and display diverse mechanisms of action
1

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involving topoisomerase II (Topo II) inhibition and induction of DNA cross-linking.
1
1
Intriguingly, the substituent at N (H or Me) plays a critical role in modulating Topo
1
1
II inhibition and DNA cross-linking activities. N -Me derivatives predispose to
1
1
induce DNA crosslinks, whereas N -H derivatives potently inhibit Topo II.
1
1
Computational analysis implicates that N -Me restrict the torsion angles of the two
adjacent OH on pyrrole resulting in a favorable of DNA cross-linking. Among these
1
1
hybrids, compound 17a with N -H is more effective than cisplatin and etoposide, but
as potent as irinotecan, against the growth of SCLC H526 cells in xenograft model.
Keywords:
anti-SCLC
hybrids;
β-carboline;
bis(hydroxymethyl)pyrrole;
indolizino[8,7-b]indole; DNA cross-linking; topoisomerase inhibition
Abbreviations
Ac O, acetic anhydride; ACN, acetonitrile; CCRF-CEM, human lymphoblastic
2
leukemia; CCRF-CEM/VBL, vinblastine-resistant CCRF-CEM subline; CL,
cross-linking; DCM, dichloromethane; DMAD, dimethyl acetylenedicarboxylate;
DMF, dimethylformamide; HCT-116, human colon cancer; IC , half maximal
5
0
inhibitory concentration; LiAlH , lithium aluminium hydride; MDR, multidrug
4
resistance; MeI, iodomethabe; MTD, maximum tolerated dose; NaH, sodium hydride;
NSCLC, non-small cell lung cancer; PC3, human prostate cancer; PI, propidium
iodide; QD, once per day; SAR, structure-activity relationship; SCLC, small cell lung
cancer; THF, tetrahydrofuran; Topo, topoisomerases.
2

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1
Introduction
DNA or the enzymes participating in DNA replication and RNA transcription
are common targets for anticancer agents. Among these therapeutic agents,
DNA-alkylating drugs and topoisomerase I and II (Topo I and Topo II) inhibitors are
widely used for the treatment of patients with various cancers [1,2]. Several
experimental trials of simultaneous or sequential administration of etoposide (Topo II
inhibitor) and topotecan or irinotecan (Topo I inhibitor) to patients have revealed not
only severe to life-threatening neutropenia and anemia [3-6] but also antagonistic
rather than synergistic effects. However, compounds, such as saintopin (1, Fig. 1) [7],
intoplicin (2) [8], fagaronine (3) [9], and batracylin (4) [10], which consist of
tetra-heterocyclic rings, are of interest because they display dual or multiple modes of
action and directly target DNA, Topo I, and Topo II. Consequently, these
compounds may have a broader spectrum and more favorable antitumor activity.
Several single agents with dual inhibitory activities against both Topo I and Topo II
display more promising anti-tumor activity than those with solo mechanism of action
[
11-13].
Small cell lung cancer (SCLC) is an aggressive type of lung cancer and
spreads much more quickly than non-small cell lung cancer (NSCLC). SCLC
accounts for 10% to 15% of all lung cancer cases [14]. The 5-year survival rates for
people with SCLC have not significantly improved over the last 40 years and have
currently plateaued [15]. Platinum-based agents alone or in combination with a
second chemotherapeutic drug (such as etoposide, docetaxel, or vinorelbine) are
currently used as a standard of therapy for SCLC patients [16]. However, the crucial
defects of platinum based drugs for anticancer therapy are side effects such as
3

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nephrotoxicity, neurotoxicity, and occurrence of inherent and acquired resistance to
the drug [17,18]. The development of drug-resistant tumor cells remains one of the
major obstacles underlying treatment failure in clinical oncology [19,20].
Because cancer is a complex disease with multiple alterations, treatments
using a single-target approach are usually unsuccessful [21,22]. Hybrid molecules
consisting of two pharmacophores have been designed to develop new anticancer
drugs with multiple modes of action [23]. The designs of hybrid drugs in general aims
to amplify their effects, reduce side effects, lower the risk of drug-drug interactions,
and overcome drug resistance. However, to maintain the functions of the original
pharmacophores after covalent linkages, the fusion of the two templates must be
carefully designed [24,25].
To discover new agent for treating lung cancer, we recently synthesized a
series of bis(hydroxymethyl)indolizino[6,7-b]indoles and their bis(alkylcarbamate)
derivatives 5 (Fig. 1) for antitumor studies [26] and demonstrated that these
derivatives exhibit potent inhibitory activity against NSCLC [35]. These agents were
designed as hybrid molecules by fusing two pharmacophores, β-carboline (6) and
bis(hydroxymethyl)pyrrole (7). Many natural and synthetic β-carboline derivatives,
such as compounds 8, 9 and 10 (Fig. 1), exhibit antiproliferative effects against a
variety of human cancer cell lines in vitro [10,26-29]. The anti-proliferative activity of
β-carboline derivatives is mediated by DNA intercalation [29] as well as the
inhibition of Topo I and Topo II [28-30], cyclin-dependent kinase, and IkK kinase
complex [31]. The bis(hydroxymethyl)pyrrole pharmacophore induces DNA
cross-linking and acts as potent antitumor agents [32,33]. Mechanistically, the
adjuvant bis(hydroxymethyl) groups on the pyrrole ring form two electrophilic centers,
4

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leading to DNA double-strand cross-linking via an S 1 electrophilic reaction [34].
N
The potential electrophilic reactivity of these agents, in which the two hydroxyl
groups act as a leaving group, is determined by the degree of participation of the
lone-pair
bis(hydroxymethyl)indolizino[6,7-b]indole hybrids, such as compounds 5a, b (5a: R
Me, 5b: R = Et), exhibit significant cytotoxicity against various human cancer cell
electron
in
the
pyrrole
nitrogen
[34].
As
expected,
=
lines in culture and potent antitumor activity in mouse xenograft models and display
multiple modes of action (DNA cross-linking and Topo I/II inhibition) [26]. Our
recent study further demonstrated that 5b (BO-1978) significantly suppresses the
growth of EGFR wild-type and mutant NSCLC tumors in tumor xenograft and
orthotopic models [35]. The combination of BO-1978 with gefitinib further
suppressed EGFR mutant NSCLC cell growth in xenograft tumor and orthotopic lung
tumor models.
<
Insert Figure 1 here>
To continue the investigation of new hybrids bearing potent inhibitory activity
against lung cancer with multiple modes of action, we further designed and
synthesized a new series of tetracyclic bis(hydroxymethyl)indolizino[8,7-b]indole
derivatives (11) (Fig. 1) bearing β-carboline and bis(hydroxymethyl)pyrrole moieties,
as shown in Fig. 2. These new hybrids were subjected to antitumor evaluation and the
preliminary results demonstrated that the new derivatives possess significant
cytotoxicity against various human tumor cell lines in vitro. Of the tumor cell lines
tested, human SCLC H526 cell line was the most sensitive to the newly synthesized
compounds. Intriguingly, the structure-activity relationship (SAR) study demonstrated
1
1
that the substituent at N (H or Me) influences the conformation of the adjuvant
5

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bis(hydroxymethyl) groups on the pyrrole ring, thus affecting DNA cross-linking and
inhibition of Topo II. We report herein the chemical synthesis, antitumor evaluation
and mechanism of action of the newly synthesized compounds.
<
Insert Figure 2 here>
2
Results and Discussion
2
.1 Chemistry
The
synthetic
route
for
the
preparation
of
bis(hydroxymethyl)indolizino[8,7-b]indole derivatives (17a-d, 18a-d) is shown in
Scheme 1 and was modified from that used for the synthesis of
indolizino[6,7-b]indoles [26]. The commercially available tryptamine hydrochloride
(12) was reacted with glyoxalic acid in aqueous solution at pH 3 (adjusted with
aqueous KOH solution) to obtain pyrido[3,4-b]indole-1-carboxylic acid (13),
according to a previously reported procedure [36]. Acylation of 13 with various acid
chlorides in the presence of NaOH produced the corresponding N-acyl derivatives
1
4c-d, which were further reacted with dimethyl acetylenedicarboxylate (DMAD) to
1
yield the diesters 15c-d. Compounds 15a and 15b (where R is Me or Et, respectively)
were prepared directly from 13 in a one-pot reaction by treatment with acetic
anhydride (Ac O)/DMAD or propionic anhydride/DMAD, respectively. The diesters
2
1
5a-d were then reduced to the corresponding diols 17a-d via reaction with LiAlH in
4
a mixture of ether and dichloromethane (DCM). Alternatively, the N-atom on the
indole ring of compounds 15a-d was N-methylated by treatment with sodium hydride
and methyl iodide in DMF to give compounds 16a-d, which were then reduced to the
corresponding diols 18a-d by the same procedure (LiAlH /DCM). We also attempted
4
6

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to convert diols 17a-d and 18a-d into their corresponding bis(alkylcarbamates)
derivatives. However, many by-products were obtained under the reaction conditions,
either by following the reaction conditions previously established in our laboratory or
with modification, and we failed to obtain the desired products by liquid column
chromatography (silica gel column eluted with a variety of eluents, such as CHCl3,
CH Cl or hexane/ EtOAc, EtOH, or MeOH). In the current study, we merely
2
2
1
1
11
synthesized N -H derivatives (17a-d) and their N -Me derivatives (18a-d) for
®
anti-proliferative studies. HPLC analysis (Column: PrincetonSPHER -C18, 250 X 4.6
mm; condition: ACN/THF/H O = 50/25/25; flow rate = 0.5 mL/min; and UV detector)
2
showed the purity of these compounds ranged from 90.2 to 98.8%.
<Insert Scheme 1 here>
2
.2 In vitro cytotoxicity
The synthesized hybrid compounds (17a-d and 18a-d) were subjected to in
vitro anti-proliferative evaluation against a panel of human tumor cell lines, including
lymphoblastic leukemia (CCRF-CEM) cells and the vinblastine-resistant subline
CCRF-CEM/VBL, colon cancer cells (HCT-116), prostate cancer cells (PC3),
NSCLC (H460), and SCLC (H526 and H211) (Table 1). The SAR study of the newly
synthesized compounds was mainly determined based on their anti-proliferative
activities against human lymphoblastic leukemia CCRF-CEM cells in culture.
Compounds with an alkyl substituent at C3 (Me or Et), such as 17a, 18a, 17b, and
1
8b, were equally cytotoxic to CCRF-CEM cells but were more potent than the
C3-aryl-substituted compounds. The IC (the half maximal inhibitory concentration)
5
0
values of the C3-Me- or Et-substituted derivatives (17a, 18a, 17b, and 18b) were
approximately 2- to 12 fold lower than those of the C3-aryl-substituted compounds.
1
1
Among the C3-aryl-substituted compounds, the N -H derivatives of
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bis(hydroxymethyl)indolizino[8,7-b]indole (17c-d) were more cytotoxic than their
1
1
corresponding N -Me derivatives (18c-d). These results indicate that the substituent
1
1
at the indole N -atom (H vs. Me) and the C3 position (alkyl vs. aryl) affects the
cytotoxicity of the new target compounds.
The cytotoxicity of the synthesized derivatives was also assessed against a
variety of human solid tumor cell lines (Table 1). Similarly, the C3-alkyl derivatives
(17a, b and 18a, b) were generally more cytotoxic than the C3-aryl-substituted
derivatives and cisplatin against the solid tumor cell lines, including HCT-116, PC3,
H460, H526, and H211 cells. Of the human solid tumor cell lines examined, SCLC
H526 and H211 cells were the most sensitive to the C3-alkyl derivatives and cisplatin.
SCLC is a very chemosensitive tumor, and a variety of chemotherapeutic agents are
active against SCLC. Platinum-based treatment and irradiation have been the standard
of care since the early 1980s [37]. Unfortunately, monodrug-based therapy is
associated with low complete response rates. Because SCLC has a greater tendency to
be widely disseminated by the time of diagnosis and to develop early resistance to
conventional treatments, a cure is difficult to achieve [38]. Thus, the poor prognosis
of SCLC is likely due to rapid growth, early tumor spread, and inevitable drug
resistance [39]. Only 5% of patients remain alive 2 years after diagnosis. Combination
therapy using multiple drugs has become a common strategy for SCLC treatment [40].
In general, compared to single-drug-based treatments, multidrug therapy based on
alkylating agents improves overall survival of SCLC [41,42]. However, nearly all
patients eventually relapse with refractory disease. Therefore, a hybrid molecule with
dual functions is likely an alternative approach for the treatment of patients with
SCLC.
To determine whether the newly synthesized derivatives were active against
multi-drug-resistant (MDR) cell lines, we evaluated the cytotoxicity of several tested
compounds against CCRF-CEM/VBL cells, which are approximately 880-fold more
resistant to vincristine than the parent cell line, CCRF-CEM. As shown in Table 1, the
resistance factors (RF) of all tested compounds were less than 1 (0.35-0.95), revealing
8

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that the newly synthesized hybrid molecules were not affected by P-glycoprotein, a
multidrug resistance protein overexpressed in vincristine-resistant CCRF-CEM/VBL
cells.
<Insert Table 1 here>
9

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2
.3 Cell cycle interference and apoptosis induction
Because these hybrid molecules were designed to fuse pharmacophores with Topo
I- and II-inhibitory and DNA-crosslinking activities [43], we examined their effects on
cell cycle progression and triggering apoptosis. Because hybrids 17a and 18a were the
most cytotoxic agents, we investigated their effects on cell cycle progression by treating
H460 lung cancer cells at concentrations of 1, 2, 4, and 8 µM for 24, 48, and 72 h. The
cell cycle distribution was analyzed by flow cytometry. As shown in Fig. 3, treatment
with compounds 17a and 18a interfered with cell cycle progression. Compound 17a
mainly resulted in the accumulation of cells at the G2/M phase at doses of 1 and 2 µM.
Intriguingly, as the concentration of compound 17a was increasing, we observed the
delayed progression of the S phase (4 µM treatment) and the G1 arrest (8 µM treatment).
However, at the similar toxic dose range, compound 18a significantly induced the typical
G2/M accumulation in a dose- and time-dependent manner. These results imply that
1
1
11
N -modifications, N -H (17a) vs. -Me (18a), may affect the modes of action of these
two compounds. The representative flow histograms were shown in Supplementary
Information S1.
In addition to the disturbance of the cell cycle, we also observed increased sub-G1
population, an indicator of apoptotic cells, in H460 cells treated with compounds 17a or
1
8a following a dose- and time-dependent manner (Fig. 3). To confirm the appearance of
apoptotic cells, we performed Annexin V staining assay to determine if compounds 17a
and 18a trigger apoptotic death in H460 cells. After treatment with increasing
concentrations (4, 8 and 16 µM) of compounds 17a or 18a for 48 h, the H460 cells were
harvested, stained with Annexin V-FITC and propidium iodide, and analyzed with a flow
cytometer. As shown in Fig. 4, both 17a and 18a significantly increased the proportion of
+
Annexin V cells in a dose-dependent manner. The present results confirm that these
DNA-targeting agents typically interfere with cell cycle progression by activating cell
cycle checkpoints [44]. Unrepaired DNA is cytotoxic and triggers cell death via apoptosis,
necrosis, and several other death mechanisms [45].
1
0

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<
Insert Figure 3 here>
Insert Figure 4 here>
<
2
.4 Inhibition of Topoisomerases I and II
Because β-carboline, one pharmacophore of the newly synthesized hybrid
derivatives (9), can inhibit Topo I and Topo II [28-30], a Topo I- and II-mediated DNA
relaxation assay using supercoiled pHOT plasmid DNA was performed to ensure that the
inhibitory activity was maintained in these new hybrid molecules. Camptothecin and
etoposide, well-known Topo I and II inhibitors, respectively, were included as positive
controls. The potency of the inhibitory activities of all synthesized hybrids against Topo I
and II are depicted in Fig. 5 and 6. None of the tested compounds inhibited on Topo I at
the concentration of 20 µM (data not shown). At the concentration of 100 µM, only
C3-4’-F-Ph-substituted hybrids (18d) exhibited weak inhibition of Topo I (Fig. 5).
<Insert Figure 5 here>
1
1
Alternatively, the N -H compounds (17a-d) possessed significant Topo II
1
1
inhibitory activity at 20 and 100 µM (Fig. 6). However, most of the N -Me derivatives
(18a-d) displayed a very weak inhibitory effect against Topo II. By increasing the
1
1
concentration to 100 µM, only 18a and 18c in the series of N -Me derivatives displayed
weak to moderate inhibition of Topo II activity. The inhibitory activity of Topo II by
compounds 17a-d and 18a-d was summarized in Supplementary Information S2.
<Insert Figure 6 here>
1
1
11
To further confirm that the N -H derivatives are more potent than their N -Me
counterparts against Topo II, we evaluated the inhibitory effect of 17a, 17c, 18a, and 18c
on Topo II at various concentrations. As shown in Fig. 7, compounds 17a and 17c
inhibited Topo II activity in a dose-dependent manner at the dose range from 12.5 µM to
1
00 µM, whereas compounds 18a and 18c displayed a weak effect on Topo II-mediated
1
1
DNA relaxation at the concentrations used. These results demonstrate that the N -H
1
1

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1
1
derivatives are generally more effective than their corresponding N -Me derivatives in
inhibiting Topo II.
<Insert Figure 7 here>
The cytotoxicity, DNA intercalation, and/or Topo I- and II-inhibitory activities of
diverse natural and synthetic β-carbolines have been attributed, at least in part, to the
9
planar and rigid aromatic β-carboline moiety [46]. Substitutions at N of β-carboline (6,
Fig. 1) have been shown to be critical for cytotoxicity and DNA binding activity [47,48].
9
Furthermore, the N -substituent of β-carboline was shown to boost cytotoxicity by
enhancing the DNA-intercalating ability [49]. However, there was no clear correlation
9
between cytotoxic activity and Topo I and II inhibition among N -arylated alkly
substituted
β-carbolines
[31,50].
In
addition,
we
observed
that
bis(hydroxymethyl)indolizino[8,7-b]indoles hybrids (11) exhibited little or limited
inhibition of Topo I compared to bis(hydroxymethyl)indolizino[6,7-b]indoles (Fig. 1, 5a,
b) [26]. These results might also explain why 17a induced cell cycle disturbance
similar that of the Topo II inhibitor etoposide [51]. Whether the conjugation position of
these newly synthesized compounds interferes with their binding to the interfacial region
of DNA and Topo I warrants further investigation [52].
1
1
Intriguingly, our present results demonstrated that the N -substituent of the
1
1
hybrids modulate Topo II inhibitory activity. Compared to N -H (17a and 17c), Me
1
1
substitution at N (18a and 18c) drastically reduced Topo II inhibitory activity. Because
several natural β-carboline alkaloids [53] and synthetic β-carboline hybrids [54] with no
9
9
N substitution (i.e., N -H) have been reported to inhibit Topo I and/or Topo II activity,
1
1
our results confirm that N -Me of the tetra-cyclic angular hybrid molecules of
β-carboline and bis(hydroxymethyl)pyrrole plays a crucial to interfere with Topo II
11
inhibitory activity of newly synthesized hybrids. Atom N of our hybrid molecules is the
9
same as N of β-carboline alkaloids.
1
2

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2
.5 DNA cross-linking study
Pyrroles have been widely used as scaffolds for the development of a variety of
therapeutic agents [55,56]. Because the adjuvant two hydroxymethyl groups can serve as
a leaving group, leading to the formation of electrophilic centers, the pyrrole derivatives
induce DNA CLs [34]. In early studies, bis(hydroxymethyl)pyrrole or pyrrolizine
analogues mainly alkylated DNA at the N2 amino group of guanine residues via a
substitution reaction on the exterior of the helix [57-59]. While we demonstrated that
compounds 17a, 17b, 18a, and 18b were equally cytotoxic to all cell lines tested, we
thought that in addition to Topo II inhibitory activity, DNA-crosslinking activity also
played certain roles in killing the cancer cells. To demonstrate that the
bis(hydroxymethyl)pyrrole moiety of the synthesized hybrid molecules maintains its
DNA cross-linking (CL) activity, we performed alkaline agarose gel shift assay by
treating plasmid pEGFP-N1with compounds 17a, 18a, 17c, and 18c at various
concentrations (1, 2.5, 5 and 10 µM). As shown in Figure 8, compounds 18a and 18c
1
1
(
N -Me derivatives) induced interstrand CLs in a dose-dependent manner. However, the
C3-aryl-substitued 18a was apparently more potent than the C3-4’-Ph-substituted 18c for
the induction of DNA interstrand CLs. Intriguingly, the DNA interstrand CL activity of
1
1
compounds 17a and 17c (N -H compound) was drastically reduced compared to the
1
1
corresponding N -Me derivatives (Fig. 8).
<Insert Figure 8 here>
1
1
It is unclear how N -Me substitution enhances the interaction with DNA. We therefore
analyzed the structural conformations of hybrids 17a and 18a with the aid of
computational calculation [60]. Energy minimization and molecular dynamics
simulations were used to calculate the torsion angles of the two terminal hydroxyl groups
of 17a and 18a. As shown in Fig. 9, the range of torsion angles accessed by the two
A
D
C
B
A
A
B
C
D
D
hydroxyl groups (O H and O H, i.e. C -C -C -O and C -C -C -O torsions) was
1
1
A
D
affected by the substituent (H or Me) at N . Apparently, the torsion angle of C and C
1
1
A
is not altered by the substituent (H or Me) at N , but that of two OH groups (O H vs
D
11
O H) is apparently more restricted in 18a than in 17a, implying that N -Me does
1
3

ACCEPTED MANUSCRIPT
influence the torsion angle of these two adjuvant OH groups on pyrrole by limiting the
D
11
rotation of O H. We may infer that the steric hindrance caused by the Me group at N
A
D
plays certain role on limiting the rotations of O H and O H in 18a. Consequently, the
localized orientation of the adjuvant hydroxymethyl groups in 18a may easily
accommodate the parallel bond at 5’-CG and favor their interaction with the N2-amino
group of guanine residues to form interstrand CL products [61]. However, further
11
structural analysis is required to confirm how N -Me affects the configuration of the two
adjacent OH groups on the pyrrole moiety.
<Insert Figure 9 here>
2
.6 In vivo antitumor activity against small cell lung cancer (SCLC)
Based on the in vitro anti-proliferative effects of the newly synthesized
derivatives, the therapeutic efficacy of compounds 17a, 17b, and 18a was preliminarily
evaluated in nude mice bearing human SCLC H526 in xenografts. According to the
modes of action of our compounds, cisplatin, etoposide, and irinotecan, which are
currently used in clinics for the treatment of SCLC patients, were included as the positive
controls. In all experiments, the tumor cells were subcutaneously implanted in nude mice,
and the maximum tolerated dose (MTD) of the tested compound was intravenously
administered via the tail vein. The H526 xenograft-bearing mice were treated with 17a,
1
7b, and 18a at doses of 30, 40, and 20 mg/kg, respectively, every day for five times
QD×5). Among them, compound 17a was the most potent agent against H526 cells (Fig.
0). To compare its antitumor potency with other therapeutic agents, the H526
(
1
xenograft-bearing mice were treated with 17a (30 mg/kg, QD×5), cisplatin (4 mg/kg,
Q4D×3) and etoposide (10 mg/kg, Q2D×3), respectively. As shown in Fig. 11A, 17a,
cisplatin, and etoposide suppressed the tumor volumes of H526 xenografts by
approximately 93, 74, and 60%, respectively, on day32 (Fig. 11A). Notably, 17a was
significantly more effective than cisplatin and etoposide against SCLC H526 tumor
xenografts. In addition, the therapeutic efficacy of 17a is as potent as that of irinotecan
(Fig. 11B). Furthermore, there was no significant change of body weight of mice treated
1
4

ACCEPTED MANUSCRIPT
with 17a (Fig. 10 and Supplementary Information S3), suggesting that 17a at the dose
used did not cause severe systematic toxicity to mice.
<
Insert Figure 10 here>
Insert Figure 11 here>
<
Conclusion
In the present study, we synthesized a series of new hybrids, namely
bis(hydroxymethyl)indolizino[8,7-b]indole derivatives (11), containing β-carboline
(topoisomerase inhibition moiety) and bis(hydroxymethyl)pyrrole (DNA cross-linking
moiety) fragments, for antitumor studies. Preliminary antitumor studies revealed that the
newly synthesized compounds exhibit a broad spectrum of antitumor activity against the
cell growth of various human tumor cell lines in vitro. The in vivo study further
demonstrated that compound 17a, with potent Topo II inhibitory activity but moderate
DNA cross-linking activity, significantly suppressed the tumor growth rate in nude mice
bearing SCLC H526 xenografts and was more potent than cisplatin and etoposide, but
was as potent as irinotecan. Our results suggest that 17a may be further developed as a
potential agent for the treatment of SCLC.
In contrast to bis(hydroxymethyl)indolizino[6,7-b]indole hybrids (5), the new
iso-steric hybrids (11) mainly targeted on DNA and Topo II, but not Topo I. Furthermore,
1
1
the N -Me derivatives enhanced DNA crosslinking activity, but attenuate Topo II
11
inhibition. The computational analysis suggested that the N -Me affects the torsion
angles of two adjacent OH groups on the pyrrole moiety and hence favors the formation
of DNA interstrand CLs. The SAR profile clearly revealed that the substituents (H and
1
1
Me) at the indole N -atom in these hybrid molecules play a regioselective role in
modulating Topo II inhibition and DNA crosslinking activity.
1
5

ACCEPTED MANUSCRIPT
3
Experimental
3
.1 Materials and methods
All commercial chemicals and solvents were reagent grade and used without
further purification unless otherwise specified. Melting points were determined in open
capillaries on a Fargo MP-2D melting point apparatus and are uncorrected. Thin-layer
chromatography (TLC) was performed on silica gel 60 F254 (Merck KGaA, Darmstadt,
Germany) with short-wavelength ultraviolet (UV) light for visualization.
High-performance liquid chromatography (HPLC) was performed on an Elite Lachrom
instrument (HITACHI) with a Mightysil RP-18 (250×4.6 mm) column. Compounds were
detected by UV light at 260 nm. The mobile phase was acetonitrile/THF (80:20 v/v) with
a flow rate of 1 mL/min. The purity of all tested compounds was ≥95% based on
1
13
analytical HPLC. H NMR spectra and C NMR spectra were recorded on Bruker
AVANCE Top-Spin spectrometers (400 or 500 MHz) in the solvents indicated. Proton
chemical shifts are reported in parts per million (δ ppm) relative to (CH ) Si (TMS), and
3
4
coupling constants (J) are reported in Hertz (Hz). NMR peak splittings are given by the
following abbreviations: s, singlet; d, doublet; t, triplet; m, multiplet; and brs, broad
singlet. High-resolution mass spectra (HRMS) were recorded on a Waters HDMS G1
+
/-
instrument with ESI in centroid mode, and the samples were dissolved in MeOH.
3
.2 Synthesis
derivatives
of
6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-diyl)dimethanol
3
.2.1 2,3,4,9-Tetrahydro-1H-pyrido[3,4-b]indole-1-carboxylic acid (13)
An aqueous solution of 50% glyoxylic acid (1.7 g, 20 mmol) was added dropwise
to a stirred solution of tryptamine hydrochloride (2.0 g, 10 mmol) in water (200 mL). The
pH of the reaction mixture was maintained at 3 by adjusting with KOH (0.5 g, 90 mmol)
in water (10 mL). The reaction mixture was stirred at room temperature for 1 h. A white
solid product was collected by filtration, washed with water and dried to give compound
1
1
1. Yield 1.6 g (75%); mp 239–240 °C (mp 205–208 °C [62]); H NMR (DMSO-d ) δ
6
2
.81–2.85 (m, 1H, CH ), 2.89–2.95 (m, 1H, CH ), 3.26–3.44 (m, 2H, CH ), 4.67 (s, 1H,
2
2
2
CH), 6.96–7.02 (m, 2H, ArH), 7.38–7.45 (m, 2H, ArH), 8.90 (brs, 1H, NH), 10.68 (s, 1H,
1
6

ACCEPTED MANUSCRIPT
1
3
COOH); C NMR (DMSO-d ) δ 18.5, 41.0, 55.8, 104.6, 112.3, 117.9, 118.9, 121.3,
6
+
+
1
2
26.3,129.1, 136.5, 165.9; HRMS (ESI ): calcd for [C12
H
12
N
2
O
2
+ H] , 217.0972, found
17.0985.
3.2.2 2-Benzoyl-2,3,4,9,-tetrahydro-1H-pyrido[3,4-b]indole-1-carboxylic acid (14c)
Benzoyl chloride (2.3 mL, 20 mmol) was added dropwise to compound 13 (4.3 g,
0 mmol) in a mixture of 50% aqueous acetone containing 10% NaOH(aq) solution. The
2
pH of the reaction mixture was maintained at PH >10 by the addition of 10% NaOH(aq)
solution. The reaction mixture was stirred at room temperature for 10 h, followed by
evaporation to dryness in vacuo. The residue was triturated with diluted acetic acid, and
the separated solid was collected by filtration, washed with water, and dried to afford
1
compound 14c. Yield 2.3 g (71%); mp 151–152 °C; H NMR (DMSO-d ) δ 2.82–2.87
6
(
m, 2H, CH
2
), 3.90–3.92 (m, 2H, CH
2
), 5.97 (s, 1H, CH), 7.00–7.10 (m, 2H, ArH), 7.31–
7
1
1
.35 (m, 2H, ArH), 7.41–7.44 (m, 2H, ArH), 7.50–7.52 (m, 4H, ArH + NH), 11.00 (s,
1
3
H, COOH); C NMR (DMSO-d ) δ 21.1, 43.9, 52.7, 108.0, 111.4, 111.6, 117.9, 118.7,
6
21.5, 126.2, 126.8, 127.1, 127.4, 128.6, 128.7, 128.8, 130.0, 135.7, 136.5, 170.0, 170.6;
-
-
HRMS (ESI ): calcd for [ C H N O - H] 319.1083, found 319.1072.
19
16
2
3
Following the same synthetic procedure as that used for 13c, the following
compounds were prepared:
3.2.3 2-(4-Fluorobenzoyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-1-carboxylic
acid (14d)
Compound 14d was prepared from compound 13 (2.2 g, 10 mmol) and
1
4
-florobenzoyl chloride (1.2 mL, 10 mmol). Yield 3.0 g (85%); mp 248–249 °C; H
NMR (DMSO-d ) δ 2.62–2.77 (m, 2H, CH ), 3.73–3.75 (m, 1H, CH ), 4.82 (s, 1H, CH),
6
2
2
6
.91–7.0 (m, 2H, ArH), 7.17-7.19 (m, 1H, ArH), 7.27–7.35 (m, 3H, ArH), 7.50–7.53 (m,
1
3
2
H, ArH), 10.44 (s, 1H, NH), 10.65 (s, 1H, COOH); C NMR (DMSO-d
6
) δ 21.1, 44.2,
5
6.0, 61.0, 105.7, 106.2, 111.6, 111.7, 115.4, 117.5, 120.6, 126.6, 129.5, 133.3, 134.2,
+
+
1
61.8, 163.7, 169.0, 170.3; HRMS (ESI ): calcd for [C H FN O + H] 339.1139,
19 15 2 3
found 339.1182.
1
7

ACCEPTED MANUSCRIPT
3
.2.4 Dimethyl
15a)
3-methyl-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-dicarboxylate
(
DMAD (1.4 g, 10 mmol) was added to a solution of compound 13 (2.2 g, 10
mmol) in Ac O (20 mL) and the reaction mixture was heated at 70 °C for 2 h with
2
stirring. The reaction mixture was evaporated to dryness in vacuo and the residue was
1
recrystallized from MeOH to give 15a, Yield 2.4 g (72%); mp 160–161 °C; H NMR
(
DMSO-d ) δ 2.39 (s, 3H, CH ), 3.12 (t, 2H, J = 7.2 Hz, CH ), 3.76 (3H, s, COOCH ),
6 3 2 3
3
.83 (3H, s, COOCH ), 4.11 (t, 2H, J = 7.2 Hz, CH ), 7.05–7.07 (m, 1H, ArH), 7.12–7.14
3 2
1
3
(
m, 1H, ArH), 7.52–7.55 (m, 1H, ArH), 7.60–7.62 (m, 1H, ArH), 10.6 (s, 1H, NH); C
NMR (DMSO-d ) δ 11.8, 21.3, 45.3, 51.5, 111.1, 113.8, 115.3, 115.5, 118.8, 119.8,
6
+
1
21.7, 124.8, 134.3, 131.9, 132.7, 136.5, 162.7, 163.7; HRMS (ESI ): calcd for
+
[
C H N O + H] 339.1393, found 339.1410.
1
9
18
2
4
Following the same synthetic procedure as that used for 15a, the following
compounds 15b were prepared:
3
.2.5 Dimethyl
15b)
3-ethyl-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-dicarboxylate
(
Compound 15b was prepared from compound 13 (2.2 g, 10 mmol) and DMAD
1
(
1.4 g, 10 mmol) in propionic anhydride (20 mL). Yield 2.5 g, 71%; mp 170–171 °C; H
NMR (DMSO-d ) δ 1.12–1.16 (t, 3H, J = 7.4 Hz, CH ), 2.81–2.86 (t, 2H, J = 7.3 Hz,
6
3
CH
2
), 3.11–3.15 (t, 2H, J = 7.0 Hz, CH
2
3 3
), 3.76 (s, 3H, COOCH ), 3.83 (s, 3H, COOCH ),
4
7
1
1
3
.16 (t, 2H, J = 7.0 Hz, CH ), 7.02–7.05 (m, 1H, ArH), 7.12–7.16 (m, 1H, ArH), 7.53–
2
1
3
.56 (m, 1H, ArH), 7.60–7.61 (m, 1H, ArH), 10.6 (s, 1H, NH); C NMR (DMSO-d ) δ
6
4.2, 18.1, 20.3, 42.2, 51.8, 52.3, 108.1, 109.2, 112.5, 112.9, 118.8, 120.0, 122.8, 125.7,
-
-
25.9, 128.0, 136.8, 140.4, 165.7, 166.1; HRMS (ESI ): calcd for [C20
H
20
N
2
O
4
- H]
51.1350, found 351.1384.
3
.2.6 Dimethyl
15c)
3-phenyl-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-dicarboxylate
(
DMAD (1.8 g, 12 mmol) was added to a mixture of compound 14c (4.0 g, 12
o
mmol) in Ac O (20 mL), and the reaction mixture was heated at 70 C for 2 h with
2
1
8

ACCEPTED MANUSCRIPT
stirring. The mixture was then evaporated to dryness in vacuo, and the residue was
1
recrystallized from MeOH to give 15c, 3.5 g, (70%); mp 167–168 °C; H NMR
(
DMSO-d ) δ 3.11 (t, 2H, J = 6.9 Hz, CH ), 3.63 (3H, s, COOCH ), 3.86 (3H, s,
6
2
3
COOCH
3
2
), 4.05 (t, 2H, J = 6.9 Hz, CH ), 7.04–7.07 (m, 1H, ArH), 7.14–7.18 (m, 1H,
1
3
ArH), 7.50–7.56 (m, 6H, ArH), 7.64–7.65 (m, 1H, ArH), 10.81 (s, 1H, NH); C NMR
(
DMSO-d ) δ 20.3, 43.9, 52.1, 52.5, 107.9, 110.0, 113.1, 115.9, 118.9, 120.1, 123.1,
6
+
1
25.6, 125.8, 128.9, 129.1, 129.1, 130.5, 135.4, 136.8, 165.5, 165.9; HRMS (ESI ): calcd
+
for [C H N O + H] 401.1496, found 401.1508.
24
20
2
4
Following the same synthetic procedure as that used for 15c, the following
compounds were prepared:
3
.2.7 Dimethyl
-(4-fluorophenyl)-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-dicarboxylate
15d)
3
(
Compound 15d was prepared from compound 14d (4.0 g, 12 mmol) and DMAD
1
(
1.8 g, 12 mmol) in Ac O (20 mL). Yield 3.8 g, (75%); mp 181–182 °C; H NMR
2
6 2 3
(DMSO-d ) δ 3.09 (t, 2H, J = 6.9 Hz, CH ), 3.63 (s, 3H, COOCH ), 3.86 (s, 3H,
COOCH ), 4.02 (t, 2H, J = 6.9 Hz, CH ), 7.04–7.06 (m, 1H, ArH), 7.14–7.17 (m, 1H,
3
2
ArH), 7.32–7.35 (m, 2H, ArH), 7.52–7.55 (m, 3H, ArH), 7.64–7.66 (d, 1H, ArH), 10.80
1
3
(
brs, 1H, NH); C NMR (DMSO-d
6
) δ 20.3, 43.8, 52.1, 52.5, 108.0, 110.0, 113.1, 115.8,
1
1
4
15.9, 118.9, 120.1, 123.1, 125.6, 125.7, 126.1, 129.1, 132.9, 133.0, 134.7, 136.8, 161.8,
-
-
63.8, 165.6, 165.7; HRMS (ESI ): calcd for [C H FN O - H] 417.1256, found
24
19
2
4
17.1260.
3
.2.8 Dimethyl
,11-dimethyl-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-dicarboxylate (16a)
3
Compound 16a (3.4 g, 10 mmol) was dissolved in DMF (30 ml) and sodium
hydride (NaH) (0.4 g, 15 mmol) was added into reaction mixture at 0–5 °C. After stirring
for 15 min, iodomethane (MeI) (1.4 g, 10 mmol) was added and the reaction mixture was
stirred for 1 h at 0–5 °C and then at rt for 8 h. The excess of NaH was decomposed by the
treatment of MeOH and the reaction mixture was concentrated in vacuo. The residue was
1
9

ACCEPTED MANUSCRIPT
1
crystallized from MeOH to give 16a. Yield 2.3 g, (65%); mp 162–163 °C; H NMR
(
DMSO-d ) δ 2.52 (s, 3H, CH ), 3.03 (t, 2H, J = 6.5 Hz, CH ), 3.59 (s, 3H, N-CH ), 3.72
6 3 2 3
(
s, 3H, COOCH ), 3.78 (s, 3H, COOCH ), 4.04 (t, 2H, J = 6.4 Hz, CH ), 7.08–7.11 (m,
3 3 2
1
H, ArH), 7.18–7.21 (m, 1H, ArH), 7.43–7.45 (m, 1H, ArH), 7.56–7.58 (m, 1H, ArH);
1
3
C NMR (DMSO-d ) δ 12.8, 21.3, 33.6, 46.3, 52.6, 106.7, 109.6, 113.4, 115.7, 117.8,
6
+
1
19.8, 124.6, 132.7, 134.3, 138.6, 161.6, 164.5; HRMS (ESI ): calcd for [C H N O +
2
0
20
2
4
+
H] 353.1496, found 353.1499.
Following the same synthetic procedure as that used for 16a, the following
compounds were prepared:
3
.2.9 Dimethyl
-ethyl-11-methyl-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-dicarboxylate
16b)
3
(
Compound 16b was prepared from compound 15b (3.5 g, 10 mmol), NaH (0.4 g,
1
1
5 mmol) and MeI (1.4 g, 10 mmol). Yield 2.5 g (70%); mp 178–179 °C; H NMR
(
DMSO-d ) δ 1.15 (t, 3H, J = 7.3 Hz, CH ), 2.95-3.01 (m, 2H, J = 7.5 Hz, CH ), 3.04 (t,
6
3
2
2
H, J = 6.5 Hz, CH
2
), 3.59 (s, 3H, N-CH
3
), 3.73 (s, 3H, COOCH
3
), 3.82 (s, 3H,
COOCH ), 4.08 (t, 2H, J = 6.4 Hz, CH ), 7.08–7.11 (m, 1H, ArH), 7.18–7.21 (m, 1H,
3
2
1
3
ArH), 7.43–7.45 (m, 1H, ArH), 7.57–7.58 (m, 1H, ArH); C NMR (DMSO-d ) δ 14.1,
6
1
1
3
8.5, 21.0, 32.4, 42.0, 51.6, 52.6, 109.8, 110.3, 110.6, 111.7, 118.9, 120.2, 122.3, 122.6,
+
+
25.6, 129.3, 138.9, 141.7, 164.4, 167.4; HRMS (ESI ): calcd for [C21
H
22
N
2
O
4
+ H]
67.1652, found 367.1684.
3
.2.10 Dimethyl
1-methyl-3-phenyl-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-dicarboxylate
16c)
1
(
Compound 16c was prepared from compound 15c (4.0 g, 10 mmol), NaH (0.4 g,
1
1
5 mmol) and MeI (1.4 g, 10 mmol). Yield 2.3 g (76%); mp 182–183 °C; H NMR
6 2 3 3
(DMSO-d ) δ 2.94–2.97 (m, 2H, CH ), 3.56 (s, 3H, N-CH ), 3.82 (s, 3H, COOCH ), 3.90
(
s, 3H, COOCH ), 4.03–4.07 (m, 2H, CH ), 7.06–7.08 (m, 1H, ArH), 7.14–7.18 (m, 1H,
3
2
1
3
ArH), 7.49-7.55 (m, 8H, ArH); C NMR (DMSO-d ) δ 14.9, 21.0, 43.5, 51.7, 52.7,
6
2
0

ACCEPTED MANUSCRIPT
1
11.4, 111.5, 112.0, 113.3, 119.2, 120.3, 122.8, 123.4, 126.3, 128.5, 128.6, 129.2, 130.3,
-
-
1
30.8, 137.1, 138.4, 164.5, 166.6; HRMS (ESI ): calcd for [C H N O - H] 413.1507,
25 22 2 4
found 413.1530.
3
.2.11 Dimethyl
-(4-fluorophenyl)-11-methyl-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-dicar
3
boxylate (16d)
Compound 16d was prepared from compound 15d (4.0 g, 10 mmol), NaH (0.4 g,
1
1
5.0 mmol) and MeI (1.4 g, 10 mmol) to give 2.6 g (65%); mp 188–189 °C; H NMR
(
DMSO-d
6
) δ 2.97–2.99 (m, 2H, m, CH
2 3 3
), 3.58 (s, 3H, N-CH ), 3.64 (s, 3H, COOCH ),
3
.84 (s, 3H, COOCH ), 3.87–3.89 (m, 2H, CH ), 7.09–7.13 (m, 1H, ArH), 7.20–7.24 (m,
3
2
1
3
1
H, ArH), 7.32–7.37 (m, 1H, ArH), 7.47–7.58 (m, 4H, ArH); C NMR (DMSO-d ) δ
6
2
1.4, 34.7, 45,3, 50.2, 106.0, 108.9, 112.8, 115.8, 117.6, 124.3, 127.0, 128.6, 130.5,
+
+
1
34.4, 130.0, 138.3, 162.7,162.9; HRMS (ESI ): calcd for [C H FN O + H] 433.1558,
25 21 2 4
found 433.1577.
3
.2.12 (3-Methyl-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-diyl)dimethanol (17a)
A solution of compound 15a (0.5 g, 1.5 mmol) in anhydrous dichloromethane (10
mL) was added dropwise to a stirred suspension of lithium aluminum hydride (LAH)
0.17 g, 4.5 mmol) in anhydrous diethyl ether (10 mL) at 0–5 °C. The reaction mixture
(
was stirred for further 30 min after the addition was completed. The excess hydride was
decomposed by the sequential addition of 15% aqueous NaOH (0.5 mL) and water (0.5
L) at 0 °C. The mixture was filtered through a pad of Celite and washed with DCM. The
combined filtrate was washed with water and brine solution. The organic layer was dried
over anhydrous Na
2
SO
4
and concentrated to dryness in vacuo. The oily residue was
1
crystallizes from ether to give 17a. Yield 0.37 g, (90%); mp 99–100 °C; H NMR
6 3 2 2
(DMSO-d ) δ 2.2 (s, 3H, CH ), 2.99 (t, 2H, J = 6.6 Hz, CH ), 3.90–3.91 (m, 5H, CH ),
4
.42–4.43 (m, 2H, CH ), 4.70 (brs, 2H, OH), 7.03–7.12 (m, 2H, ArH), 7.39–7.48 (m, 2H,
2
1
3
ArH); C NMR (DMSO-d ) δ 10.1, 20.9, 41.8, 54.2, 55.1, 104.0, 111.8, 117.6, 118.1,
6
-
1
19.4, 119.6, 120.7, 120.8, 126.9, 127.6, 129.5, 137.0; HRMS (ESI ): calcd for
-
[
C
17
H
18
N
2
O
2
- H] 281.1296, found 281.1286; HPLC, t
r
. 5.67 min (95.4% at 254 nm).
2
1

ACCEPTED MANUSCRIPT
3
.2.13 (3-Ethyl-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-diyl)dimethanol (17b)
Compound 17b was prepared from compound 15b (1.0 g, 3.0 mmol) and LAH
1
(
0.35 g, 9.0 mmol) in DCM. Yield 0.85 g (88%); mp 117–118 °C; H NMR (DMSO-d
6
) δ
1
3
2
.13 (t, 3H, J = 6.8 Hz, CH ), 2.64–2.70 (m, 2H, CH ), 3.00 (t, 2H, J = 6.7 Hz, CH ),
3
2
2
.99 (t, 2H, J = 6.7 Hz, CH
2
), 4.35 (d, 2H, J = 4.9 Hz, CH
2
), 4.42 (s, 1H, OH), 4.69 (d,
H, J = 5.0 Hz, CH ), 5.03 (brs, 1H, OH), 6.97–7.00 (m, 2H, ArH), 7.39–7.43 (m, 2H,
2
1
3
ArH), 10.59 (s, 1H, NH); C NMR (DMSO-d ) δ 15.5, 17.4, 21.1, 41.8, 54.1, 55.1,
6
1
04.2, 111.8, 117.7, 118.1, 119.1, 119.4, 120.7, 120.8, 126.8, 129.5, 133.5; HRMS
+
+
(
ESI ): calcd for [C H N O + H] 297.1598, found 297.1603; HPLC, t . 5.90 min
18 20 2 2 r
(96.2% at 254 nm).
3
.2.14 (3-Phenyl-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-diyl)dimethanol (17c)
Compound 17c was prepared from compound 15c (2.0 g, 5.0 mmol) and LAH
1
(
0.6 g, 15 mmol) in DCM. Yield 1.5 g (92%); mp 145–146 °C; H NMR (DMSO-d ) δ
6
2
.98–3.00 (m, 2H, CH
2 2 2
), 4.00–4.02 (m, 2H, CH ), 4.28–4.30 (m, 2H, CH ), 4.69–4.79
(
m, 3H, CH + OH), 7.00–7.05 (m, 2H, ArH), 7.45–7.49 (m, 7H, ArH), 10.58 (brs, 1H,
2
1
3
NH); C NMR (DMSO-d ) δ 21.2, 43.6, 54.5, 55.1,105.6, 112.0, 117.9, 119.0, 119.5,
6
1
19.8, 121.1, 121.5, 122.7, 126.7, 127.8, 128.8, 129.0, 129.1, 130.4, 131.6, 132.4, 137.2;
-
-
HRMS (ESI ): calcd for [C H N O - H] 343.1447, found 343.1418; HPLC, t . 6.27 min
22
20
2
2
r
(92.4% at 254 nm).
3
.2.15 (3-(4-Fluorophenyl)-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-diyl)dimethano
l (17d).
Compound 17d was prepared from compound 14d (1.5 g, 3.5 mmol) and LAH
1
(
0.4 g, 10.5 mmol) in DCM to give 1.0 g (90%); mp 113–114 °C; H NMR (DMSO-d
6
) δ
2
4
7
.99 (t, 2H, J = 6.3 Hz, CH ), 4.00 (t, 2H, J = 6.3 Hz, CH ), 4.26–4.28 (m, 2H, CH ),
2
2
2
2
.70 (s, 1H, OH), 4.78–4.80 (m, 2H, CH ), 5.23 (s, 1H, OH), 6.98–7.07 (m, 2H, ArH),
.31–7.35 (m, 2H, ArH), 7.43–7.47 (m, 2H, ArH), 7.51–7.54 (m, 2H, ArH), 10.71 (s, 1H,
1
3
NH); C NMR (DMSO-d ) δ 21.2, 43.5, 54.4, 55.1, 105.6, 112.0, 115.8, 117.9, 118.9,
6
1
19.5, 121.1, 121.6, 122.7, 126.7, 128.0, 129.0, 131.4, 132.4, 132.5, 137.2, 163.0; HRMS
2
2

ACCEPTED MANUSCRIPT
+
+
(
ESI ): calcd for [C22
H19FN
2
O
2
+ H] 363.1503, found 363.1504; HPLC, t
r
. 6.23 min
(90.4% at 254 nm).
3
.2.16 (3,11-Dimethyl-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-diyl)dimethanol
18a)
(
Compound 18a was prepared from compound 16a (0.7 g, 2.0 mmol) and LAH
1
(
0.25 g, 6.0 mmol) in DCM. Yield 0.52 g (90%); mp 155–157 °C; H NMR (DMSO-d ) δ
6
2
4
.24 (s, 3H, CH
3 2 2 3
), 2.91 (t, 2H, J = 5.1 Hz, CH ), 3.89–3.92 (m, 5H, CH + N-CH ), 4.42–
.43 (m, 2H, CH ), 4.54–4.75 (m, 2H, OH), 7.03–7.06 (m, 1H, ArH), 7.09–7.11 (m, 1H,
2
1
3
ArH), 7.39–7.41 (m, 1H, ArH), 7.47–7.48 (m, 1H, ArH); C NMR (DMSO-d ) δ 10.3,
6
2
1
2
1.6, 33.0, 41.6, 54.4, 55.0, 106.9, 110.5, 117.7, 117.9, 119.6, 119.9, 120.5, 121.1, 126.6,
+
+
28.1, 132.7, 139.0; HRMS (ESI ): calcd for [C H N O + H] 297.1598, found
18
20
2
2
r
97.1603; HPLC, t . 5.84 min (98.8% at 254 nm).
3
.2.17 (3-Ethyl-11-methyl-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-diyl)dimethanol
18b)
(
Compound 18b was prepared from compound 16b (1.1 g, 3.0 mmol) and LAH
1
(
0.35 g, 9.0 mmol) in DCM. Yield 0.9 g, (90%); mp 118–120 °C; H NMR (DMSO-d ) δ
6
1
.11 (t, 3H, J = 7.2 Hz, CH ), 2.37–2.72 (m, 2H, J = 6.4 Hz, CH ), 2.90–2.92 (m, 2H,
3
2
CH
2
), 3.92 (s, 3H, N-CH
3
), 4.08 (t, 2H, J = 4.9 Hz, CH
2
2
), 4.43–4.44 (m, 2H, CH ), 4.51
(
brs, 1H, OH), 4.58–4.60 (m, 2H, CH ), 4.75 (brs, 1H, OH), 7.03–7.06 (m, 1H, ArH),
2
13
7
.09–7.12 (m, 1H, ArH), 7.39–7.41 (m, 1H, ArH), 7.47–7.48 (m, 1H, ArH);
C NMR
(
DMSO-d
6
) δ 15.4, 17.6, 21.5, 21.8, 30.8, 33.0, 54.33, 55.8, 67.4, 107.1, 110.5,117.7,
-
1
18.0, 119.6, 119.9, 121.1, 126.5, 132.6, 134.0, 139.0; HRMS (ESI ): calcd for
-
[
C H N O - H] 309.1609, found 309.1618; HPLC, t . 6.11 min (95.5% at 254 nm).
1
9
22
2
2
r
3
.2.18 (11-Methyl-3-phenyl-6,11-dihydro-5H-indolizino[8,7-b]indole-1,2-diyl)
dimethanol (18c)
Compound 18c was prepared from compound 16c (2.0 g, 5.0 mmol) and LAH
1
(
0.6 g, 15 mmol) in DCM. Yield 1.6 g (94%); mp 182–184 °C; H NMR (DMSO-d
6
) δ
2
.84–2.86 (m, 2H, CH ), 3.97 (s, 3H, N-CH ), 4.38–4.39 (m, 2H, CH ), 4.54–4.55 (m,
2 3 2
2
3

ACCEPTED MANUSCRIPT
2
2
1
1
H, CH
H, ArH), 7.42–7.52 (m, 7H, ArH); C NMR (DMSO-d
09.5, 111.4, 118.4, 118.5, 120.0, 121.5, 122.1, 127.2, 127.8, 128.7, 130.5, 131.4, 131.7,
2 2
), 4.65–4.66 (m, 2H, CH ), 4.79 (s, 1H, OH), 4.90 (s, 1H, OH), 7.06–7.14 (m,
1
3
6
) δ 15.3, 21.8, 43.1, 54.6, 54.9,
+
+
33.1, 138.3; HRMS (ESI ): calcd for [C H N O + H] 359.1754, found 359.1760;
2
3
22
2
2
HPLC, t . 6.83 min (92.9% at 254 nm).
r
3.2.19 (3-(4-Fluorophenyl)-11-methyl-6,
11-dihydro-5H-indolizino[8,7-b]indole-1,2-diyl) dimethanol (18d)
Compound 18d was prepared from compound 16d (1.0 g, 2.5 mmol) and LAH
1
(
0.3 g, 0.75 mmol) in DCM. Yield 0.77 g (90%); mp 205–206 °C; H NMR (DMSO-d ) δ
6
2
2 3
.49–2.50 (m, 2H, CH ), 3.98 (s, 3H, N-CH ), 4.36–4.37 (m, 2H, ArH), 4.53–4.55 (m,
2
H, CH ), 4.69–4.70 (m, 2H, CH ), 4.80 (s, 1H, OH), 4.91 (s, 1H, OH), 7.06–7.07 (m,
2
2
1
H, ArH), 7.12–7.14 (m, 1H, s, ArH) , 7.34–7.35 (m, 1H, ArH), 7.44–7.48 (m, 2H, ArH),
1
3
7
.52–7.55 (m, 2H, ArH); C NMR (DMSO-d
6
) δ 21.8, 33.0, 43.1, 54.5, 55.0, 55.3,
1
08.4, 110.7, 115.6, 115.7, 118.2, 118.6, 120.0, 121.3, 121.5, 122.2, 126.4, 127.9, 132.2,
-
-
1
32.5, 132.6, 139.2, 161.1, 163.0; HRMS (ESI ): calcd for [C H FN O - H] 375.1509,
23 21 2 2
r
found 375.1566; HPLC, t . 6.60 min (96.4% at 254 nm).
3
.3 Biological assays
.3.1 Cytotoxicity assays
The antiproliferative activities of the newly synthesized compounds against the
3
human lymphoblastic leukemia cell line CCRF-CEM, its vincristine-resistant sub-cell
line CCRF-CEM/VBL, and human solid tumor cell lines (H460 lung cancer cells,
HCT-116 colon cancer cells, and PC3 prostate cancer cells) were determined after a 72-h
®
incubation using the Alamar Blue (AbD Serotec, UK) assay with a microplate
spectrophotometer, as previously described [63]. After the addition of the Alamar Blue
solution, the cells were incubated at 37°C for 10-12 h. The absorbance at 570 and 600 nm
was measured with a microplate reader. The IC50 values were determined from the
dose-effect relationship at six or seven concentrations of each drug using CompuSyn
software (version 1.0.1; CompuSyn, Inc., Paramus, NJ) by Chou and Martin, which is
based on the median-effect principle and plot [64,65].
2
4

ACCEPTED MANUSCRIPT
3
.3.2 Cell cycle analysis
The effects of the compounds on cell cycle progression were analyzed by flow
5
cytometry as previously described [26]. Briefly, 1.0×10 H460 cells were seeded in each
well of a 6-well plate and incubated at 37°C in a 5% CO humidified atmosphere
2
overnight. The cells were then incubated with the test compounds for different time
periods. At the end of treatment, the attached cells were trypsinized, fixed in ice-cold
7
0% EtOH, and stored at -20°C overnight. The cells were then stained with 4 µg/mL
propidium iodide (PI) in phosphate-buffered saline containing 0.1 mg/mL RNase A and
% Triton X-100 and subjected to flow cytometry analysis (FACScan flow cytometer;
1
Becton Dickinson, San Jose, CA). The cell cycle phase distribution was analyzed with
ModFit LT 3.0 software (Verity Software House, Topsham, ME) based on the DNA
histograms.
3
.3.3 Apoptosis assay
H460 cells were treated with the test compounds or cisplatin, and apoptosis was
detected by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit
eBiosciences, San Diego, CA, USA) according to the manufacturer's instructions. Cells
(
positive for Annexin V staining only (bottom right quadrant) and positive for both
Annexin V and PI (propidium iodide) staining (top right quadrant) represent the early and
late apoptotic populations, respectively, whereas cells positive for PI staining only (top
left quadrant) represent the necrotic population.
3
.3.4 Topoisomerase I-mediated DNA relaxation assay
The topo I kit from TopoGEN, Inc. (Port Orange, FL, U.S.), was used to
determine whether the synthesized compounds inhibited the topo I-mediated relaxation of
supercoiled DNA. The assay was performed according to the manufacturer’s instructions.
The reaction mixture (20 µL) contained 200 ng of plasmid DNA and the test compound
in incubation buffer (10 mM Tris-HCl, pH 7.9, and 1 mM EDTA). The reaction was
incubated at 37 °C and was initiated by the addition of 1.5 units of topo I. After 30-45
min, the reaction was stopped by the addition of 2 µL of 10% SDS. The samples were
then loaded onto a 1% agarose gel and electrophoresed at 50 V for 90 min. The gels were
2
5

ACCEPTED MANUSCRIPT
stained with ethidium bromide, destained with water, and photographed.
3
.3.5 Topoisomerase II-mediated DNA relaxation assay
The topo II kit from TopoGEN, Inc. (Port Orange, FL, U.S.), was used to evaluate
the inhibitory activity of the synthetic compounds against topo II-mediated relaxation of
supercoiled DNA. The assay was performed according to the manufacturer’s instructions.
Briefly, the reaction mixture (20 µL) contained 200 ng of pHOT plasmid DNA and the
test compound in assay incubation buffer (0.5 M Tris-HCl, pH 8.0, 1.5 M NaCl, 100 mM
2
MgCl , 5 mM dithiothreitol, 300 µg/mL BSA, and 20 mM ATP). The mixture was
warmed to 37 °C, and the reaction was initiated by the addition of 4 units of topo II. The
reaction was incubated for 30-45 min, followed by termination by the addition of 2 µL of
1
0% SDS. The samples were then loaded onto a 1% agarose gel and separated by
electrophoresis at 50 V for 90 min. The gels were stained with ethidium bromide,
destained with water, and photographed.
3
.3.6 Alkaline agarose gel shift assay
The formation of DNA cross-links was analyzed by alkaline agarose gel
electrophoresis as previously described [29]. In brief, purified pEGFP-N1 plasmid DNA
1,500 ng) was reacted with various concentrations (1, 2.5, 5, and 10 µM) of the tested
(
compounds in 40 µL of binding buffer (30 mM sodium chloride/10 mM sodium
phosphate, pH 7.4/10 mM EDTA) at 37°C for 2 h. The plasmid DNA was linearized by
the BamH1 restriction enzyme (New England Biolabs) for 3 h and separated by
electrophoresis. The DNA bands were visualized under UV light after staining the gels
with ethidium bromide.
3
.3.7 In Vivo Studies
Athymic nude mice bearing the nu/nu gene were obtained from the National
Laboratory Animal Center (Taipei, Taiwan) and housed for 1 week before performing
experiments. Male nude mice 6 weeks old or older weighing 20−24 g or more were used.
The compounds were administered via tail vein as described previously [66]. Tumor
volume was assessed by measuring the length × width × height (or width) using a caliper.
2
6

ACCEPTED MANUSCRIPT
The vehicle used was 50 µL of DMSO and 40 μL of Tween 80 in 160 µL of saline.
®
®
Cisplatin and irinotecan were obtained from Across and Cayman Medical , and
dissolved in a 0.9% NaCl solution, respectively. The MTD of the tested compound was
determined and used for the in vivo therapeutic efficacy assay. For tumor-bearing nude
mice during the course of the experiment, the body weight refers to the total weight
minus the weight of the tumor. All animal studies were conducted in accordance with the
guidelines for the National Institutes of Health Guide for the Care and Use of Animals,
and the protocol was approved by the Institutional Animal Care and Use Committee.
3
.4 Evaluation of the conformational behavior
The molecular structures of 17a and 18a were constructed by modifying the DNA
binding agent Ellipticine (PDB code 1Z3F), and CHARMM forcefiled (Brooks et al.,
983)[60] was used for their energy calculations. Each of the two chemicals contains four
1
rotatable bonds as indicated in Fig. 9. These four rotatable bonds were subjected to
conformational search by using the Boltzmann Jump method implemented in the
molecular modeling package of BIOVIA Discovery Studio (Accelrys Software Inc., San
Diego, USA). The five lowest energy conformers (out of randomly sampled 100) were
then selected to monitor the rotational behavior in molecular dynamic simulations, which
were carried out in three stages, Heating (20 ns), Equilibration (10 ns) and Production
(20ns), at 300 K target temperature with a time step of 1fs. Two hundred trajectory
frames were captured during the last 20ns of the production stage in an NVT statistical
C
B
A
A
B
C
D
D
ensemble to measure the angles of the C -C -C -O and C -C -C -O torsions. BIOVIA
Discovery Studio was used for all these computations.
4
Acknowledgment
We are grateful for support from the Ministry of Science and Technology, Taiwan
(MOST103-2325-B-001-018 and MOST103-3111-Y-001-031) and Academia Sinica,
Taipei, Taiwan. We thank Mr. Ching_Huang Chen (Chemical Lab of IBMS, Academia
Sinica) for technical help on synthesis of hybrid molecules. The NMR spectra of the
synthesized compounds were obtained at the Chemical Lab and High-Field Biomolecular
2
7

ACCEPTED MANUSCRIPT
NMR Core Facility supported by the National Research Program for Genomic Medicine
(Taiwan) and the Core Facility for Protein Structural Analysis supported by the National
Core Facility Program for Biotechnology (Taiwan). The NMR spectra were acquired on a
Bruker 400 MHz spectrometer at the Institute of Biological Chemistry, a Bruker 500 MHz
spectrometer at the Institute of Biomedical Sciences and at the Core Facility for Protein
Structural Analysis supported by National Core facility Program for Biotechnology.
5
References
[
[
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[
[
[
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2
8