Synthesis and cytotoxicity of pyrido[4,3-b]carbazole alkaloids against HCT-116 and HL-60 cells

Article abstract of DOI:10.1007/s00044-017-2068-6

Ellipticine, olivacine, and their five reduced natural variants were synthesized via a palladium-catalyzed tandem cyclization/cross-coupling reaction as the key step. In addition, a previously unknown conformer of janetine was obtained through conformational inversion of the D ring in janetine. Because there are few synthetic approaches for reduced natural variants, little is known about the biological activities of these compounds. Six synthetic natural alkaloids and five of their derivatives were evaluated for their antiproliferative activity against HCT-116 and HL-60 cells. The activities of variants with the D-reduced ring or without the C(11)-Me group were lower than those of ellipticine. The conformer of guatambuine showed higher activities than guatambuine.

Full text of DOI:10.1007/s00044-017-2068-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2068-6
ORIGINAL RESEARCH
Synthesis and cytotoxicity of pyrido[4,3-b]carbazole alkaloids
against HCT-116 and HL-60 cells
1 ●
1 ●
2 ●
2 ●
Tomoki Itoh Noriyuki Hatae Takashi Nishiyama Tominari Choshi
2 ●
1 ●
1
Satoshi Hibino Teruki Yoshimura Minoru Ishikura
Received: 3 July 2017 / Accepted: 5 September 2017
©
Springer Science+Business Media, LLC 2017
Abstract Ellipticine, olivacine, and their five reduced nat-
ural variants were synthesized via a palladium-catalyzed
tandem cyclization/cross-coupling reaction as the key step.
In addition, a previously unknown conformer of janetine
was obtained through conformational inversion of the D
ring in janetine. Because there are few synthetic approaches
for reduced natural variants, little is known about the bio-
logical activities of these compounds. Six synthetic natural
alkaloids and five of their derivatives were evaluated for
their antiproliferative activity against HCT-116 and HL-60
cells. The activities of variants with the D-reduced ring or
without the C(11)-Me group were lower than those of
ellipticine. The conformer of guatambuine showed higher
activities than guatambuine.
Introduction
Ellipticine (1) and olivacine (5), which are typical pyrido
[4,3-b]carbazole alkaloids, were first isolated from the
leaves of Ochrosia elliptica Labill (Goodwin et al. 1959)
and from the bark and stems of Aspidosperma olivaceum
Müll. Arg., respectively (Schmutz and Hunzicker 1958).
Several alkaloids of this class have been isolated from
various plants (Fig. 1). Ellipticine and olivacine exhibit
significant antitumor activity based on topoisomerase II
inhibition and/or DNA intercalation (Kohn et al. 1975;
Monnot et al. 1991) as well as antimalarial activity (Mon-
toia et al. 2014). To investigate the mode of interaction of
ellipticine with DNA, the contribution of DNA adduct
formation with biologically oxidized ellipticine to cyto-
toxicity has been evaluated (Stiborová et al. 2004).
Structure–activity relationship analysis of ellipticine
derivatives has clarified the interactions of some of their
structural features with biomolecules (O’Sullivan et al.
2013). The pyrido[4,3-b]carbazole ring plays an important
role in DNA intercalation because of its structural resem-
blance to a purine–pyrimidine complementary base pair
●
●
Keywords Pyrido[4,3-b]carbazole alkaloids Cytotoxicity
●
HCT-116 cell HL-60 cell
Abbreviations
IC50
MTT
WST-1
Half maximal inhibitory concentration
Thiazolyl blue tetrazolium bromide
Water-soluble tetrazolium salt-1
(Auclair 1987), and the crystal structure of ellipticine
complexed to a 6 bp oligonucleotide has been published
(
(
Canals et al. 2005). Introducing a hydroxyl group at the C
9) position of ellipticine may lead to enhanced anti-
Electronic supplementary material The online version of this article
https://doi.org/10.1007/s00044-017-2068-6) contains supplementary
material, which is available to authorized users.
(
proliferative activity. The C(9)-hydroxyl group is hypothe-
sized to facilitate isomerization to the quinone–imine form
and to form a hydrogen bond with biomolecules. Ellipti-
cium salts have been found to improve the uptake and
cytosol localization of ellipticine derivatives (Charcosset
et al. 1983). A number of following analogs of ellipticine
and olivacine have been synthesized, and the increase in
their cytotoxicity was evaluated. Success in a clinical trial of
9-hydroxy-N-methylellipticinium acetate (Celiptium®)
*
Minoru Ishikura
ishikura@hoku-iryo-u.ac.jp
1
2
School of Pharmaceutical Sciences, Health Sciences University of
Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
Graduate School of Pharmacy & Pharmaceutical Sciences, and
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama
University, Fukuyama, Hiroshima 729-0292, Japan

Med Chem Res
(
Ohashi and Oki 1996; Treat et al. 1989) spurred the further
HCT-116 human colon tumor cells and HL-60 human
promyelocytic leukemia cells.
development of other analogs such as datelliptium (Khayat
et al. 1992), S16020 (Vassal et al. 2003), and S30972
(
Kraus-Berthier et al. 2002) (Fig. 2).
The diverse biological activities and intriguing structures
of pyridocarbazole alkaloids have prompted a large number
of synthetic studies (Sainsbury 1977; Hewlins et al. 1984;
Gribble and Saulnier 1985; Kansal and Potier 1986; Alvarez
and Joule 2001; Knölker and Reddy 2002; Miller and
McCarthy 2012). We have developed a concise synthetic
approach to pyrido[4,3-b]carbazole alkaloids 1–8 via a
palladium-catalyzed tandem cross-coupling reaction of
indolylborate (Itoh et al. 2016). Compounds 3, 4, 6, 7, and 8
are reduced natural variants of 1 and 5 with partially
reduced D rings, and there are few reports of the synthesis
of these derivatives. Moreover, little is known about the
biological activities of 3, 4, 6, 7, and 8, whereas 1 and 5
have been extensively studied. In this paper, the anti-
proliferative activities of the synthetic natural products 3, 4,
Experimental
Chemistry
General methods
Melting points were measured with a micro melting point
apparatus (Yanagimoto) and are uncorrected. High-
resolution mass spectrometry (HRMS) was performed
(JMS-T100LP, JEOL), and infrared (IR) spectra were
recorded (IRAffinity-1, Shimadzu). The nuclear magnetic
resonance (NMR) experiments were performed with a
500 MHz spectrometer (JNM-ECA500, JEOL), and the
chemical shifts are expressed in ppm (δ). Flash column
chromatography was performed on silica gel (Silica Gel 60
N, Kanto Chemical Co., Ltd.). Compounds 16–20 were
6
, 7, and 8 and their analogs 16, 17, 18, 19 (Ishikura et al.
008), and 20 (Ishikura et al. 2000) were assessed against
2
Fig. 1 Pyrido[4,3-b]carbazole
alkaloids
Fig. 2 Ellipticine (1) and
olivacine (5) analogs used in
clinical trials

Med Chem Res
synthesized according to our previous reports (Ishikura
et al. 2000, 2008).
indicated concentrations for a further 48 h, and then 10 μL
of MTT (5 mg/mL in phosphate-buffered saline) per well
was added. After 4 h incubation to allow the MTT to be
metabolized by cellular mitochondrial dehydrogenase, the
excess MTT was aspirated and the formazan was dissolved
in 100 μL of dimethyl sulfoxide. The absorbance of for-
mazan at 570 nm was measured using a microplate reader.
The results were calculated as a percentage relative to
untreated controls.
Synthesis of conformational isomer of janetine (7a)
A solution of 11 (100 mg, 0.2 mmol) was stirred in the
presence of 20% Pd(OH) /C (100 mg) in THF under
2
atmospheric pressure of hydrogen for 1 h, and then the
catalyst and the solvent were removed. A solution of the
residue (15) was heated with Cs CO (200 mg) in MeOH/
The HL-60 cell viability assay used the WST-1 method,
which was based on Ishiyama’s method (Ishiyama et al.
1993). The cells were seeded in 96-well flat-bottomed clear
2
3
THF (20 mL, 1:1) under reflux for 3 h. The solvent was
removed, and the residue was diluted with CH Cl
2
2
4
(
100 mL), washed with brine, and dried over Na SO . The
tissue culture plates with 2.0 × 10 cells/well in 100 μL of
2
4
solvent was removed, and the residue was separated by thin
layer chromatography (TLC) (aluminum oxide 60 F254
basic, Merck) with hexane/AcOEt (1:1) to give 7a (30 mg,
culture medium. The cells were treated with the compounds
for 48 h, and then 10 μL of WST-1/1-methoxy phenazine
methoxysulfate (1-methoxy PMS) solution containing
5 mM WST-1 and 0.2 mM 1-methoxy PMS in 20 mM
HEPES-NaOH (pH 7.4) per well was added. After 3 h
incubation to allow the WST-1/1-methoxy PMS to be
metabolized by cellular mitochondrial dehydrogenase, the
formazan absorbance was measured at 415 nm by using a
microplate reader. The results were calculated as a percen-
tage relative to untreated controls.
6
1
0%) as colorless solid; mp > 300 °C; IR(KBr) v
3645,
max
−1 1
610 cm ; H NMR (CD OD, 500 MHz): δ = 1.68 (3H, d,
3
J = 6.8 Hz, 1-CH ), 2.74 (3H, s, 5-CH ), 3.00–3.13 (2H, m,
3
3
4
4
8
-H ), 3.26–3.34 (1H, m, 3-H), 3.48–3.54 (1H, m, 3-H),
.54 (1H, q, J = 6.8 Hz, 1-H), 7.13 (1H, dt, J = 1.2, 7.5 Hz,
-H), 7.18 (1H, s, 11-H), 7.34 (1H, dt, J = 1.1, 7.5 Hz, 9-
2
H), 7.41 (1H, d, J = 8.0 Hz, 7-H), 8.16 (1H, d, J = 8.0 Hz,
1
3
1
0-H); C NMR (CD OD, 125 MHz): δ = 14.7 (CH , C5-
3
3
CH ), 19.8 (CH , C1-CH ), 24.0 (CH , C4), 40.2 (CH ,
3
3
3
2
2
Statistical analysis
C3), 52.1 (CH, C1), 105.2 (CH, C11), 110.2 (CH, C7),
18.3 (CH, C9), 120.4 (C, C5), 121.4 (C, C10b), 122.1
CH, C10), 123.1 (C, C10a), 124.9 (CH, C8), 130.8 (C,
1
(
Compound concentration–cell viability curves were fitted
with a four-parameter logistic equation using a nonlinear
curve-fitting program, and the IC50 values were obtained
using Kaleida-graph (Synergy Software). Wherever appro-
priate, the results were expressed as mean ± standard error
of the mean, with n = 3 or higher in at least one out of three
similar experiments.
C4a), 133.1 (C, C11a), 138.9 (C, C5a), 140.9 (C, C6a);
HRESIMS m/z (pos): 251.1549 C H N (calcd.
1
7 20 2
2
51.1548).
Cell lines and cell cultures
The antiproliferative activities of the compounds were tes-
ted in HCT-116 human colon tumor cells and HL-60 human
promyelocytic leukemia cells, which were purchased from
the American Type Culture Collection. The HCT-116 cells
were maintained in McCoy’s 5a medium containing L-glu-
tamine and 10% heat-inactivated (55 °C for 30 min) fetal
bovine serum (hiFBS). The HL-60 cells were maintained in
RPMI-1640 medium containing L-glutamine and 10%
hiFBS.
Results and discussion
Syntheses of pyrido[4,3-b]carbazole alkaloids
In our ongoing studies on the use of indolylborate 10 as a
synthetic intermediate, we reported the syntheses of pyr-
idocarbazole alkaloids 1–8 by the one-pot tandem cycliza-
tion/cross-coupling reaction of bromide 11 with
indolylborate 10, which was generated in situ from indole 9,
to produce hexatriene intermediate 12. This was followed
by 6π-electrocyclization of 12 to produce pyridocarbazole
core 13. Carbazole intermediates 13a were transformed
into ellipticine (1), 9-methoxyellipticine (2), 3,4-dihy-
droellipticine (3), and 1,2,3,4-tetrahydroellipticine (4).
Olivacine (5), (±)-guatambuine (6), (±-janetine (7), and
3,4-dihydroolivacine (8) were synthesized from common
intermediate 14 derived from 13b (Scheme 1).
Cell viability assays
The HCT-116 cell viability assay used the MTT method,
which was based on Mosmann’s method (Mosmann 1983)
The cells were seeded in 96-well flat-bottomed clear tissue
3
culture plates with 2.0 × 10 cells/well in 100 μL of culture
medium. The cells were incubated at 37 °C in an atmo-
sphere of 5% CO for 24 h to allow the cells to attach to the
2
wells. The cells were treated with the compounds at the

Med Chem Res
Scheme 1 Synthetic scheme for pyrido[4,3-b]carbazole alkaloids
(
±)-Guatambuine (6) and (±-janetine (7) were obtained
values of ellipticine (1) against HL-60 cells have been
reported in previous studies as 0.66 μM (Dalla via et al.
2002), 0.9 μM (Gopal and Shahabuddin 2004), and 1.6 μM
(Shenoy et al. 2007). Our IC50 value for the corresponding
cells was slightly higher than, but less than five-fold, the
previously reported values. Ellipticine’s cytotoxicity against
HCT-116 cells has not been reported previously, and was
similar to or slightly higher than the activity against HL-60
cells. The IC50 values against HCT-116 cells of compounds
2, 6, 7, 7a, and 16–20 were lower than those against HL-60
cells. Because HL-60 cells lacking p53 protein (Gorczyca
et al. 1993) are highly sensitive to ellipticine (Shenoy et al.
2007), HCT-116 cells may also be sensitive to the current
compounds.
by treating 15a and 15b with BBr at −78 °C, respectively
Scheme 2). Cleavage of the N-Boc group in 15a with
3
(
Cs CO in MeOH/THF under reflux gave previously
2
3
unknown conformer 6a (Itoh et al. 2016). The structure of
natural (+)-guatambuine (6) was unequivocally confirmed
by X-ray crystal structure analysis, which showed that the D
ring is in a half chair conformation and the C(1)-Me group
is axial (De Simone et al. 2006). NOE experiments for 6a
showed that the C(1)-Me group switched from axial in 6 to
equatorial in 6a through a conformational inversion of the D
ring accompanied by nitrogen inversion at the N(2) position.
Moreover, conformer 7a was obtained by heating 15b with
Cs CO in MeOH/THF. The structure of 7a was confirmed
2
3
based on NOE experiments, similarly to 6a (Scheme 2).
Irradiation of C(1)-H in 7a produced NOE correlations
We identified the D ring’s aromaticity and the C(11)-Me
group as key structural features that affect the cytotoxicity
of ellipticine. Among natural carbazoles 1–8, the alkaloids
with an aromatic D ring, such as ellipticine (1) and 9-
methoxyellipticine (2), exhibited potent cytotoxicity against
both tumor cell lines. In contrast, the compounds with a
reduced D ring, such as tetrahydroellipticine (4), gua-
tambuine (6), and janetine (7), showed low or no activity.
The cytotoxicities of dihydroellipticine (3) and tetra-
hydroellipticine (4) decreased with the decrease in D ring
aromaticity. A similar trend was observed for dihy-
droolivacine (8) and janetine (7). In a previous study by
Guillonneau et al. (2005) on D ring-modified olivacine
analogs, the calculated global properties, such as molecular
electrostatic potential maps and partition coefficients (log
P), were reported to play a pivotal role in the biological
b
between the C(1)-Me, C(3)-H , and C(11)-H peaks, and
a
between the C(1)-Me and C(3)-H peaks in 7.
Cytotoxicities of pyrido[4,3-b]carbazole alkaloids
against HCT-116 and HL-60 cells
The HCT-116 and HL-60 cell viabilities after treatment
with synthetric pyrido[4,3-b]carbazole alkaloids and their
analogs were assessed by the MTT (Mosmann 1983) and
WST-1 (Ishiyama et al. 1993) methods, respectively. All the
compounds exhibited concentration-dependent cytotoxic
activities, and their IC50 values are shown in Table 1. The
IC50 values of the reference ellipticine against HCT-116 and
HL-60 cells were 2.58 and 3.10 μM, respectively. The IC50

Med Chem Res
Scheme 2 Conformations of 6a and 7a
activities of these compounds (Guillonneau et al. 2005).
Our results may also be explained by these properties.
Furthermore, the correlation between the degree of reduc-
tion of the D ring and cytotoxicity was observed in N(6)-
methylated analogs 16–18. The C(11)-Me group also
affected the cytotoxicity; C(11)-demethylated analogs 19
and 20 had lower activities against both tumor cell lines
compared with the corresponding C(11)-methylated com-
pounds 1 and 16. Because of the decrease or loss of cyto-
toxicity in all of the C(11)-demethylated analogs (6, 6a, 7,
This is the first report of the cytotoxicities of gua-
tambuine (6) and janetine (7), including their conforma-
tional isomers 6a and 7a, against tumor cells. These
compounds showed decreased or no activity against HCT-
116 and HL-60 cells. The decrease in the activities was
related to D ring reduction and C(11) demethylation. The
isomerization of naturally isolated 6 and 7 provided ther-
modynamically more stable conformational isomers 6a and
7a (Scheme 2). The conformational isomer of guatambuine
(6a) showed higher cytotoxicity than that of its natural form
(6), with IC50 values of 18.87 μM against HCT-116 cells
and 13.37 μM against HL-60 cells for 6a, compared with
36.85 and 44.61 μM, respectively, for 6. Guatambuine may
not be isomerized in the cells.
7
a, 8, 19, and 20), the methyl group at C(11) was identified
as important for their activity.
The N(6)-methylated
analogs,
except
6-
methyldihydroellipticine (17) against HL-60 cells, exhib-
ited higher cytotoxicities against both tumor cell lines than
did the corresponding demethylated analogs, and the IC50
values of 6-methylellipticine (16) of 1.46 and 0.26 μM
against HCT-116 and HL-60 cells, respectively, were the
lowest among the tested compounds. These results were in
good agreement with a previous study that found that N(6)-
alkylation of ellipticine increased growth inhibition against
Conclusions
The cytotoxicities of pyrido[4,3-b]carbazole alkaloids were
determined. Alkaloids 1–8 and conformational isomers 6a
and 7a were synthesized with our synthetic method
using indolylborate 10 as a pivotal synthetic intermediate.
1
2 human cancer cell lines (Deane et al. 2013).

Med Chem Res
Table 1 Cytotoxicity of ellipticine analogs against HCT-116 and HL-60 cells
1
2
3
Compounds
R
R
R
D ring
IC50 value (μM)
HCT-116 cells
HL-60 cells
Ellipticine (1)
H
H
Me
Me
Me
Me
Me
Me
Me
H
2.58 ± 0.75
1.46 ± 0.86
7.81 ± 0.89
3.53 ± 0.24
16.45 ± 1.41
5.85 ± 0.03
2.33 ± 1.13
11.29 ± 0.79
8.74 ± 1.30
3.10 ± 0.12
6
-Me ellipticine (16)
H
Me
H
2.26 ± 0.29
3.11 ± 0.08
3.70 ± 0.45
12.80 ± 1.37
6.57 ± 0.21
6.51 ± 0.44
12.21 ± 1.64
9.47 ± 1.50
Dihydroellipticine (3)
H
6
-Me dihydroellipticine (17)
H
Me
H
Tetrahydroellipticine (4)
H
6
9
1
6
-Me tetrahydroellipticine (18)
H
Me
H
-Methoxyellipticine (2)
OMe
H
1-Demethylellipticine (19)
-Me 11-demethyl-ellipticine (20)
H
H
Me
H

Med Chem Res
Table 1 continued
1
2
3
Compounds
R
R
R
D ring
IC50 value (μM)
HCT-116 cells
HL-60 cells
Dihydroolivacine (8)
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
8.57 ± 0.23
5.91 ± 1.36
Janetine (natural type) (7)
>100
>100
Janetine (conformational isomer) (7a)
Guatambuine (natural type) (6)
>100
>100
36.85 ± 1.10
18.87 ± 1.60
44.61 ± 1.58
13.37 ± 1.41
Guatambuine (conformational isomer) (6a)
Compliance with ethical standards
The cytotoxicities of the synthetic alkaloids and their deri-
vatives (16–20) against HCT-116 and HL-60 cells were
determined. Structural properties, such as the aromaticity of
the D ring and the presence of a Me group at the N(6)- and
C(11)-positions, affected the activities of the compounds.
The cytotoxicities of guatambuine and janetine were eval-
uated, and conformer 6a of guatambuine exhibited higher
activities than synthetic guatambuine (6).
Conflict of interest The authors declare that they have no competing
interests.
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