Bioorganic & Medicinal Chemistry Letters 25 (2015) 2749–2752

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

Antiproliferative activity of O4-benzo[c]phenanthridine alkaloids

against HCT-116 and HL-60 tumor cells
Noriyuki Hatae a,⇑, Erina Fujita a, Saori Shigenobu a, Sayumi Shimoyama a, Yuhsuke Ishihara b,
Yuhki Kurata b, Tominari Choshi b, Takashi Nishiyama b, Chiaki Okada a, Satoshi Hibino b
a
School of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
b
Graduate School of Pharmacy & Pharmaceutical Sciences, and Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan

a r t i c l e i n f o a b s t r a c t

Article history: The O4-benzo[c]phenanthridine alkaloids exhibit potent antiproliferative activity against cancer cells,
Received 14 April 2015 which is derived from their ability to inhibit of topoisomerase I and II. It has been reported that in the
Revised 12 May 2015 alkaloids a cationic quaternary ammonium atom, which results in resonance effects between ring A
Accepted 14 May 2015
and B, is necessary for increased antiproliferative activity. These findings indicate the role of their sub-
Available online 19 May 2015
stituents at ring A on inhibition of tumor cell proliferation. In the present study, we systematically
assessed the cytotoxic activities of naturally occurring alkaloids and their derivatives containing various
Keywords:
ring A substituents against two tumor cell lines, HCT-116 colon tumor cells and HL-60 promyelocytic leu-
Benzo[c]phenanthridine alkaloid
Antiproliferative activity
kemia cells. Among the cationic iminium alkaloids, which displayed more potent activity than the corre-
DFT calculation sponding neutral derivatives, and the 7,8-oxygenated benzo[c]phenanthridine alkaloids, chelerythrine
Antitumor and NK109, exhibited stronger antiproliferative activity than the 8,9- and 9,10-oxygenated alkaloids.
Iminium ion The activity of cationic iminium alkaloids could be correlated with the bond lengths of their ring A sub-
stituents and the electrostatic potentials of their ammonium molecules by DFT calculation.
Ó 2015 Elsevier Ltd. All rights reserved.

Benzo[c]phenanthridine alkaloids are commonly isolated from
Rutaceae, Papaveraceae, and Fumariaceae plants,1,2 and exhibit
various biological activities, like as antitumor and antituberculosis
activity.3,4 Some naturally occurring O4-alkaloids, including niti-
dine (1c), fagaronine, and chelerythrine (1a), demonstrated signif-
icant antitumor activity in preclinical studies conducted at
National Cancer Institute in 1970s,5,6 and the novel synthetic
benzo[c]phenanthridine derivatives, NK109 (1b) and NK314 (1h),
were developed.7–10 However NK109 has to be administered at
high doses in vivo because it is converted to an inactive metabolite
under biological conditions. The NK314 is currently being exam-
ined in clinical trials in Japan. The antitumor activities of O4-
benzo[c]phenanthridine alkaloids are based on their ability to inhi-
bit topoisomerases; that is, nitidine (1c) and fagaronine cause
tumor cell death by inhibiting topoisomerases I and II,11–13 and
NK109 (1b) and NK314 (1h) also induce drug-resistant tumor cells
Abbreviations: IC50, half maximal inhibitory concentration; MTT, thiazolyl blue
tetrazolium bromide; WST-1, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disul-
fophenyl)-2H-tetrazolium monosodium salt; 1-methoxy PMS, 1-methoxy-5-
methylphenazinium methylsulfate; DFT, density functional theory.
⇑ Corresponding author.
E-mail address: nhatae@hoku-iryo-u.ac.jp (N. Hatae).
death by suppressing topoisomerase II activity.14–17 Furthermore,
the chelerythrine (1a) induces tumor cell toxicity and delays tumor
cell growth by inhibiting protein kinase C.18
Previous studies have reported that the cationic iminium
benzo[c]phenanthridines, sanguinarine and nitidine (1c), exhibited
strong in vitro antiproliferative activity against KB tumor cells.19,20
Stermitz and co-workers suggested that the activity of these mole-
cules can be explained by the fact that they contain a cationic
quaternary nitrogen atom in a heteroaromatic system.19,20
Furthermore, Zee-Cheng et al. proposed that molecular planarity
might be a prerequisite for desired activity.21 As for NK109 (1b),
Nakanishi et al. considered that the presence of a resonance hybrid
involving the cationic nitrogen atom and molecular planarity are
essential for its antiproliferative activity.22 Quantitative struc-
ture–activity relationship modeling of benzo[c]phenanthridine
analogs indicated that the substituents at the 8,9-dimethoxy and
2-methoxy positions of their skeletons are necessary for increasing
their inhibitory activity against topoisomerase I and their anti-can-
cer effects.23
These findings suggest that ring A substituents could play
an important role in the antiproliferative activity of
O4-benzo[c]phenanthridines and their derivatives against tumor
cells. It has been reported that 7,8-oxygenated alkaloids,

http://dx.doi.org/10.1016/j.bmcl.2015.05.031
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
2750 N. Hatae et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2749–2752
sanguinarine and chelerythrine (1a), demonstrated strong in vitro
antiproliferative activity against KB tumor cells,20 while the 8,9-
oxygenated alkaloids, nitidine (1c) and fagaronine, exhibited
potent activity against HeLa S3 cervical tumor cells.22 It was con-
sidered that the strength of the antiproliferative activity of O4-
benzo[c]phenanthridine alkaloids might depend on the character-
istics of the target tumor cells; therefore, the effects of such alka-
loids should be compared using the same tumor cells. In the
present study, we systematically assessed the cytotoxic activities
of naturally occurring 7,8-; 8,9-; and 9,10-oxygenated alkaloids
(1a–g in Fig. 1) against human colon tumor-derived HCT-116 cells
and human leukemia-derived HL-60 cells. Furthermore, the struc-
tural characteristics of the O4-benzo[c]phenanthridines that dis-
played potent antiproliferative activity were examined by
performing DFT calculations in Spartan 14.
The cationic iminium benzo[c]phenanthridine alkaloids have
been demonstrated to be potent antitumor agents in vitro.20 In
addition, some 5,6-dihydrobenzo[c]phenanthridines, such as dihy-
drofagaridine and dihydronitidine (2c), have been reported to dis-
play in vitro antitumor activity against tumor cells;24,25 however,
their activities were a little bit weaker than those of the corre-
sponding cationic iminium alkaloids. In the current study, antipro-
liferative activities of naturally occurring benzo[c]phenanthridine
alkaloids and their 5,6-dihydro derivatives with various ring A sub-
stituents against two tumor cell lines, HCT-116 and HL-60 cells,
were systematically assessed (Table 1). Assay of HCT-116 and
HL-60 cell viability were conducted using the MTT and WST-1
methods, respectively.26 All of the alkaloids were synthesized via
our microwave-assisted electrocyclic reaction of the 6p-elec-
tron,27–29 although we were not able to isolate dihydro terihanine
because of its instability.
All of the tested neutral alkaloids which did not possess N-Me
substituents, 3a–g, exhibited weak or no antiproliferative activity.
Specifically, they demonstrated half-maximal inhibitory concen-
trations (IC50) of over 10 lM toward both HCT-116 and HL-60 cells.
These findings were in good agreement with the previous consid-
eration that the presence of a cationic quaternary nitrogen atom
in a heteroaromatic system is quite important for antiproliferative
activity in O4-benzo[c]phenanthridine alkaloids.21 All of the
iminium alkaloids except 1d and 1g, displayed potent antiprolifer-
ative activity against both tumor cell lines, and NK109 (1b)
demonstrated the strongest activity among the tested compounds.
In a comparison of the effects of three types of alkaloids; that
is, 7,8-, 8,9-, and 9,10-oxygenated alkaloids, 7,8-oxygenated
benzo[c]phenanthridines displayed the strongest antiproliferative
Figure 1. Structures of O4-benzo[c]phenanthridine alkaloids.
activity against both tumor cell lines. It was reported that the
8,9-oxygenated alkaloid nitidine exhibited the IC50 value of
0.13 lM toward tumor cells and demonstrated greater activity that
the 7,8-oxygenated chelerythrine.22 However, another group found
that nitidine’s IC50 value against HepG2 tumor cells was 3.52 lM;30
therefore, nitidine’s activity could depend on the characteristics of
the target tumor cells. In addition, our data suggested that the con-
version of a dimethoxy group into a hydroxyl group could influ-
ence the antiproliferative activity of O4-benzo[c]phenanthridine
alkaloids; for example, compared with the corresponding mono-
methoxy molecules terihanine (1d) and N-methylzanthoxyline
(1g) exhibited decreased or no activities, but NK109 (1b) and iso-
terihanine (1e) displayed increased activity. These findings indi-
cate that the position of the substituted hydroxyl group could
also have important effects on the activities of O4-benzo[c]phenan-
thridine alkaloids. Among the 5,6-dihydro derivatives, three mole-
cules, 2b–c and 2e, exhibited antiproliferative activities, but their
IC50 values were increased compared to those of the corresponding
cationic iminium derivatives, 1b–c and 2e, respectively.
A previous study examining the correlation between the struc-
tures of benzo[c]phenanthridine alkaloids and antiproliferative
activity reported that molecular planarity and a cationic character
of the quaternary nitrogen atom are required for antiproliferative
activity;21 while the effects of ring A substituents were not
described in detail. In the present study, the structural characteris-
tics of the iminium alkaloids 1a–g were examined by performing
DFT calculations at the B3LYP/6-31+G(d) level in Spartan 14,31,32
and the resultant data are shown in Table 2. The calculated data
indicated that the molecules all have planar framework (see
Supplementary information). To analyze the resonance between
the C7(9)-carbon to N5-cationic nitrogen atom of iminium alka-
loids, the lengths of the bonds between each ring A substituent
and backbone were investigated. It has been reported that an
increasing electron withdrawing effect of substituted groups leads
to a shortened the other substituents bond distance on disubsti-
tuted benzene.33 In all of the iminium alkaloids except for teriha-
nine (1d), the O–C7(9) bond was shorter than the O–C8(10)
bond, which makes sense as C7 is able to form a resonance hybrid
with iminium ions, but C8 is not. In terihanine (1d), the fact that
the O–C9 bond is same length as the O–C8 bond could lead to a
reduction in antiproliferative activity. These findings could agree
with those of a previous report that examined the correlation
between bioactivity and the resonance effects between the C7(9)
atom and the cationic N5 atom.22 The effects of the electrostatic
potential of the cationic quaternary nitrogen atom were also
 N. Hatae et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2749–2752 2751

Table 1
Antiproliferative activities of O4-benzo[c]phenanthridine alkaloids against HCT-116 and HL-60 cells

The upper and lower rows were indicated the alkaloids’ IC50 values for HCT-116 and HL-60 cells, respectively.

Table 2
Acknowledgments
Ring A substituent bond lengths and ammonium ion charges of benzo[c]phenan-
thridine alkaloids This study was supported in part by a Grant-in-Aid for Scientific
Compound IC50 (lM) Bond length (Å) Electrostatic
Research from the Japan Society for the Promotion of Science (No.
potential at 14572026).
HCT-116 HL-60 C7(9)-O C8(10)-O
ammonium
cells cells
atom
Supplementary data
7,8-Oxygenated type
1a 2.13 ± 0.02 2.99 ± 0.05 1.351 1.355 +0.277
Supplementary data associated with this article can be found, in
1b 1.31 ± 0.23 0.79 ± 0.14 1.350 1.359 +0.242
8,9-Oxygenated type the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.05.
1c 4.42 ± 0.93 3.41 ± 0.21 1.351 1.362 +0.320 031.
1d >10 >10 1.347 1.347 +0.307
1e 3.35 ± 0.80 3.28 ± 0.20 1.340 1.353 +0.312 References and notes
9,10-Oxygenated type
1f 3.23 ± 0.63 7.82 ± 0.45 1.341 1.363 +0.330
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