Synthesis and in vitro antitumor activity of 9-hydroxyellipticine derivatives with glucose conjugation via triazolylmethyl succinate linker

Article abstract of DOI:10.3987/COM-16-13404

Three types of novel 9-hydroxyellipticine derivatives 7a-c with glucose conjugation, linked by a triazolylmethyl succinic ester bond, were synthesized, and their cytotoxicity against HeLa S-3 and 293T cells was evaluated together with ellipticine (1) and 9-hydroxyellipticine (3). These new compounds 7a-c exhibited potent antitumor activity against HeLa S-3 and 293T, and water solubility of 7a was greatly higher than those of 1 and 3. The effects of an -OCH2CH2-spacer and the amino sugar moiety on the antitumor activity were examined using 7b,c. Furthermore, one of these glucose-conjugates 7a was applied to hydrolysis, catalyzed by carboxyesterase, leading to the formation of 3 under physiological conditions.

Full text of DOI:10.3987/COM-16-13404

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HETEROCYCLES, Vol. 92, No. 4, 2016
HETEROCYCLES, Vol. 92, No. 4, 2016, pp. 664 - 679. © 2016 The Japan Institute of Heterocyclic Chemistry
Received, 8th January, 2016, Accepted, 3rd February, 2016, Published online, 10th February, 2016
DOI: 10.3987/COM-16-13404
SYNTHESIS AND IN VITRO ANTITUMOR ACTIVITY OF
9-HYDROXYELLIPTICINE DERIVATIVES WITH GLUCOSE
CONJUGATION VIA TRIAZOLYLMETHYL SUCCINATE LINKER
Naoya Sato, Yu Kawai, Yosuke Akaba, Shoji Honma, Norio Sakai, and
Takeo Konakahara*
Department of Pure and Applied Chemistry, Faculty of Science and Technology,
Tokyo University of Science (RIKADAI), Noda, Chiba 278-8510, Japan; E-Mail:
konaka@rs.noda.tus.ac.jp
Abstract – Three types of novel 9-hydroxyellipticine derivatives 7a-c with
glucose conjugation, linked by a triazolylmethyl succinic ester bond, were
synthesized, and their cytotoxicity against HeLa S-3 and 293T cells was evaluated
together with ellipticine (1) and 9-hydroxyellipticine (3). These new compounds
7a-c exhibited potent antitumor activity against HeLa S-3 and 293T, and water
solubility of 7a was greatly higher than those of 1 and 3. The effects of an
-OCH2CH2- spacer and the amino sugar moiety on the antitumor activity were
examined using 7b,c. Furthermore, one of these glucose-conjugates 7a was
applied to hydrolysis, catalyzed by carboxyesterase, leading to the formation of 3
under physiological conditions.
INTRODUCTION
A great deal of interest has been generated by indole alkaloids like ellipticines1 and β-carbolines2 because
of their intercalation, antitumor properties, anti-HIV activities and inhibition of RNA polymerase I
transcription.1b Ellipticine (1) was first identified in 1959 as a compound in the leaves of a small tropical
evergreen Ochrosia sandwicensis A. DC. and Ochrosia elliptica Labill3 (see Figure 1). Ellipticine and its
derivatives cause the selective inhibition of p53 protein phosphorylation in several human cancer cell
lines,1 and it correlates with their cytotoxic activity. These compounds also uncouple mitochondrial
oxidative phosphorylation, thereby disrupting the energy balance of cells. Further, they are known to react
with DNA by an intercalation process,4a which may account for their cytotoxicity, and the inhibition of
DNA topoisomerase II activity.4b We have reported the interaction mode of ellipticine and its derivatives

HETEROCYCLES, Vol. 92, No. 4, 2016
665
with DNA using deflection spectroscopy.5 Ellipticine (1) is a good intercalator and an antitumor agent
like derivatives of β-carboline (2).6 The unique structural features, limited toxic side effects, and complete
lack of hematological toxicity of these compounds have prompted chemists to study their synthesis of a
number of analogues for pharmacological evaluation.
10
7
1
11
2
9
N
6
N
H
3
N
8
N
H
5
4
1
2
Figure 1. Chemical structures of ellipticine (1) and β-carboline (2)
The total synthesis of ellipticine itself was first achieved by Woodward et al. in 1959,7 and since then
ellipticine and its derivatives have been synthesized by many groups. Previously, we have developed a
novel and efficient method for the synthesis of ellipticine (1) via Suzuki-Miyaura coupling reaction.8
Several simple structural modifications to ellipticine derivatives have resulted in compounds with
increased antitumor activity. A low level of water solubility at physiological pH, as well as systemic
toxicity, has prevented the use of ellipticine (1) as a therapeutic agent. The introduction of a positive
charge in the molecule improved water solubility and made a profound difference in the biological
activity. Recently, we have reported the syntheses of ellipticin-2-ium and 9-hydroxyellipticin-2-ium salts
along with their 5-ethoxycarbonyl analogs and their antitumor activity.9 These compounds exhibited
potent antitumor activity against the human cervical cancer cell line HeLa S-3 and higher solubility in
water than ellipticine itself. Because of their positive charge, the cellular uptake of these compounds,
however, is affected by the membrane potential,10,11 and is expected to influence their membrane
permeability. Another problem was noted when these compounds showed low selectivity against tumor
cells. An alternative method to resolve this discrepancy is to introduce hydrophilic substituents like a
glucose or polyethylene glycol moiety into the molecule. Chen et al. have improved water-solubility,
cell-selectivity, and membrane permeability of paclitaxel prodrugs via the introduction of a glycan moiety.
This modification has promoted the targeted delivery of this drug to cancer cells via glucose transporters
(GLUTs).12 In medicinal chemistry, the water solubility and lipophilicity of a drug is considered to be
important molecular parameters determining the absorption, the bioavailability and, sometimes, the
bioactivity,13,14 and it has been reported that a therapeutic agent must be relatively soluble in water in
order to pass through biological membranes.14 There were few reports on the glucose-conjugates of
ellipticine (1) although many analogous reports on the acceleration of drug activity by glycan-conjugation
have been published.

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Herein, we focus on the development of new uncharged ellipticine derivatives that will have greater
solubility in water via modification of the carbon atom at the 9-position with glucose conjugation (Figure
2). In addition, we examined the efficiency of these new drugs as potent antitumor agents with
improvements in the binding ability that was related to cytotoxic activity, with evaluation by an MTT
assay against HeLa S-3 and 293T cells.
Figure 2. 9-Hydroxyellipticine derivatives with glucose conjugation
RESULTS AND DISCUSSION
In a previous paper, we noted that the introduction of a 9-OH group enhanced the antitumor activity of
ellipticine, and that the elongation of the tether had a tendency to suppress antitumor activity.9 For this
reason, we designed 9-hydroxyellipticine derivatives with glucose conjugation at the 9-OH group (Figure
2). An ellipticine nucleus was linked with a glucose moiety by both a succinic ester bond and a triazole
ring-spacer. We considered that this target compound was obtained in a high yield from the
corresponding glucose azide and the ellipticin-9-yl propargyl succinate by a click chemistry (Huisgen
reaction) in the presence of a Cu(I) catalyst. The compound is expected intravitally to release
9-hydroxyellipticine (3) through the action of a carboxylic ester hydrolase in cells.15
9-Hydroxyellipticine (3) was synthesized by a previously reported method,9 and then treated with
monopropargyl succinate (4)16 leading to the formation of ellipticin-9-yl propargyl succinate (5) in a 78%
yield under the DCC-DMAP-MS 4A conditions in a mixed solvent of dichloromethane (DCM) and DMF
at room temperature (rt) for 24 h, as shown in Scheme 1.
Me
O
O
Me
Me
HO
DCC (1.5 equiv)
DMAP (0.3 equiv), MS 4A
OH
N
O
O
+
O
N
O
N
H
O
CH2Cl2 / DMF, rt, 24 h
N
H
Me
4
3
5, 78%
Scheme 1. Synthesis of ellipticin-9-yl propargyl succinate (5)

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667
Table 1. Huisgen reaction of 2-azidoethyl β-D-glucopyranoside (6a) and ellipticin-9-yl propargyl
succinate (5)
O
Me
OH
O
O
O
N
HO
HO
O
O
N
OH
N3
H
Me
6a (1.0 equiv)
5 (1.0 equiv)
OH
O
O
Me
Me
O
catalyst (0.3 equiv), additive
HO
N
O
N
N
O
HO
DMSO/H2O (10:1), temperautre, time
O
N
OH
N
H
7a
Entry
Catalyst
Additive (equiv)
sodium ascorbate (1.2)
sodium ascorbate (1.2)
none
Temperature Time (h)
Yield (%)
15a,b
•
CuSO4 5H2O
1
2
3
4
5
rt
60 oC
rt
72
72
tracec
trace
56a,d
•
CuSO4 5H2O
CuI
CuI
CuI
72
DIPEA (1.0)
rt
< 1
< 1
DIPEA (1.0)
rt
92e
a
The product was purified by silica gel column chromatography after the reaction solvent was
o
b
c
removed in vacuo at 60 C. A great deal of unreacted starting materials was accompanied. The
reaction gave a complex mixture. d The starting materials were completely consumed. e The reaction
mixture was directly loaded on the top of the silica gel column to remove the reaction solvent and to
purify by column chromatography
First, a reaction of ellipticin-9-yl propargyl succinate (5) and 2-azidoethyl β-D-glucopyranoside (6a) was
performed (Table 1). When a mixture of 5 and 6a was stirred in the presence of copper(II) sulfate (0.3
equiv) and sodium ascorbate (1.2 equiv) in a mixture of DMSO and H2O (10:1) at rt for 72 h, a small
amount of the objective product 7a was obtained (a 15% yield) accompanied by a great deal of unreacted
o
starting materials (entry 1). Therefore, the reaction temperature was elevated to 60 C. However, only a
trace of the product was obtained from the complex reaction mixture (entry 2). Next, although copper(I)
iodide was used instead of copper(II) sulfate/sodium ascorbate at rt, the yield remained low (entry 3). The
addition of a stoichiometric amount of an amine base such as Et3N and diisopropylethylamine (DIPEA)
reportedly accelerates the Huisgen reaction.17 When an equimolar amount of DIPEA was added to the
reaction mixture (entry 4), the starting material was completely consumed in less than 1 h of the reaction

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time leading to the formation of 7a with a drastically improved yield (56%). The yield, however, was by
no means satisfactory. This moderate yield may have been caused by decomposition of the product, while
DMSO as a solvent was removed in vacuo at about 60 oC. Indeed, 7a was obtained in a quantitative yield
(92%) if the reaction mixture was directly loaded on the top of the silica gel column to remove DMSO
(entry 5). As a result, we decided to use entry 5 as the optimal conditions.
It has been reported that the length of an ethylene glycol-linker changed the antitumor activity of
podphyllotoxin derivatives in a subtle way,18 and that the β-D-glucosamine derivatives showed good
antitumor activity.19 Therefore, in order to clarify the effects of the -OCH2CH2- spacer and the amino
sugar moiety on the antitumor activity in our ellipticine system, we planed to synthesize two types of
sugar-conjugated 9-hydroxyellipticine derivatives 7b,c under the optimal conditions. The reaction of 5
with β-D-glucopyranosyl azide (6b) and 2-acetamido-2-deoxy-β-D-gluco- pyranosyl azide (6c) gave the
corresponding objective products 7b,c in 94 and 90% yields, respectively (Scheme 2).
O
Me
Me
OH
O
O
X
O
N
HO
HO
N3
O
N
H
6b, X = OH
6c, X = NHAc
5 (1.0 equiv)
OH
O
Me
Me
CuI (0.3 equiv)
DIPEA (1.0 equiv)
O
O
HO
HO
O
N
N
N
O
N
DMSO/H2O (10:1), rt, 2 h
X
N
H
7b, X = OH, 94%
7c, X = NHAc, 90%
Scheme 2. Huisgen reaction of β-D-glucopyranosyl azide 6b,c and ellipticin-9-yl propargyl succinate (5)
Subsequently, we evaluated the cytotoxicity of the glucose-conjugates of 9-hydroxyellipticine 7a-c,
ellipticine (1) and 9-hydroxyellipticine (3) on the human cervical cancer cell line HeLa S-3 and on the
human embryonic kidneys (HEK) cell line 293T. We used an MTT assay to evaluate the cytotoxicity. The
IC50 values of all compounds are given in Table 2.
First, the in vitro antitumor activity of the glucose-conjugated 9-hydroxyellipticine derivative 7a against
HeLa S-3 was compared with those of ellipticine (1) and 9-hydroxyellipticine (3). The IC50 value of 7a
was 0.50 µM (entry 1), which was smaller than any of the mother compounds, ellipticine (1; IC50 = 2.1
µM) and 9-hydroxyellipticine (3; IC50 = 1.6 µM); that is to say, the antitumor activity of 7a is enhanced 3-
to 4-fold that of 3 and 1 in spite of the elongated tether. As previously reported, the elongation of the

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669
tether had a tendency to suppress antitumor activity of ellipticine derivatives.9 Therefore, this opposite
effect may be explained by the accelerated delivery of 7a into the cells by the glucose-conjugation.
Although the power of enhancement (3- to 4-fold) in the antitumor activity of 7a is not always high, the
IC50 value of 0.50 µM for 7a is noteworthy if we take account of the suppression in the activity caused by
the elongated tether. As reported for pacritaxel,12 introduction of a glucose moiety is useful to increase the
uptake of glucose derivatives with bulky parent structures.
Table 2. In vitro antitumor activity (IC50) of the glucose-conjugated 9-hydroxyellipticine derivatives 7a-c,
ellipticine (1) and 9-hydroxyellipticine (3) against HeLa S-3 and 293T cells
IC50 (µM)a
Entry
1
Compound
HeLa S-3
293T
OH
O
Me
Me
O
O
HO
HO
O
N
N
O
7a
7b
0.50 ± 0.02b
0.28 ± 0.01
O
N
OH
N
N
H
OH
O
Me
O
O
O
HO
HO
O
N
N
2
3
0.42 ± 0.06
0.74 ± 0.03
0.298 ± 0.001
N/Ac
N
OH
N
N
H
Me
OH
O
Me
O
O
HO
HO
O
N
N
N
7c
O
N
AcNH
N
H
Me
Me
N
4
5
1
3
2.1 ± 0.5
1.6 ± 0.1
1.5 ± 0.5
1.2 ± 0.2
N
H
Me
Me
HO
N
N
H
Me
a Values are reported as the means of three experiments; the standard deviations are given together. b Mean
of IC50 values of 7a before treatment with Amberlite XAD2 resin (0.43 ± 0.02 µM) and that after
treatment (0.571 ± 0.004 µM). c N/A: Not assayed.
Second, the effect of the length of the linker (entry 2, 7b) on the antitumor activity was evaluated. In
contrast to the antitumor activity of podphyllotoxin derivatives,18 the antitumor activity of 7b against
HeLa S-3 remained strong (IC50 = 0.42 µM) in spite of a lack of the -OCH2CH2- spacer (Figure 2, n = 0).
Furthermore, the 2-acetamido group (entry 3, 7c) did not affect the corresponding antitumor activity (IC50
= 0.74 µM).19 T. Honda et al. reported that the L-arabinopyranoside was the most promising antitumor
agent of a series of 9-hydroxyellipticin-2-ium glycosides.20 The compounds, however, regrettably have a
positive charge, caused by glycosidation on the N2 atom of ellipticine (1), in spite of its attractive

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HETEROCYCLES, Vol. 92, No. 4, 2016
property. Although the glucose moiety and the positive charge accelerates the water solubility of the
compound, the latter induced both the decay of the antitumor activity10 and the cell membrane
permeability.11 We have reported antitumor activity of many kinds of novel 2-alkyl-5-methoxy-
carbonyl-11-methyl-6H-pyrido[4,3-b]carbazol-2-ium and 2-alkylellipticin-2-ium chloride derivatives.
Their IC50 values against HeLa S-3 cells varied from 2.90 µM to 75.3 µM with the structure of the
substituent on the N2 atom.9 The in vitro antitumor activity of the present 9-hydroxyellipticine glycosides
are higher than those of the ellipticin-2-ium derivatives by ten-fold to two hundred-fold. On the other
hand, IC50 values of compounds 7a, 7b, 1 and 3 for 293T (IC50 = 0.28, 0.298, 1.5 and 1.2 µM,
respectively) were almost same as those against HeLa S-3 (IC50 = 0.50, 0.42, 2.1 and 1.6 µM,
respectively).21
Next, in order to clarify whether the glucose-conjugated elliticine derivative 7a is hydrolyzed by the
action of an enzyme or not, 7a was treated with carboxyesterase (from a porcine liver) in a phosphate
o
buffer (PBS, pH 7.4), including 1% DMSO, at 37 C. The time course of the absorbance is shown in
Figure 3. The absorbance of 7a at 296 nm decreased gradually with the reaction time. If the phenolic ester
bond of 7a was hydrolyzed, the increasing absorption bands of 9-hydroxyellipticine (3) should be
appeared at 445-450 nm.22 Any increasing absorption band of the hydrolysates of 7a, however, was
unable to find in the spectra of the reaction mixture (Figure 3). Alternatively, a colorless precipitate was
appeared in the reaction mixture during the reaction. The precipitate, collected by filtration after the
reaction completion, was composed from two components by HPLC/ESI-MS analysis. The major product
was identified as 3 by high resolution ESI-MS. An observed exact mass of the molecular ion [M+ H]+ of
3 was 263.11849 [error for C17H15N2O: +0.05 millimass unit (mmu)] and that of the minor product was
523.21496 (see Experimental). The yield of 3 was 18.5% (by HPLC/ESI-MS), and another possible
hydrolysate ellipticin-9-yl succinate (8) was not detected in the reaction mixture by HPLC/ESI-MS
analysis. When 3 was treated under the same conditions, the corresponding oxidative dimer was formed
as reported previously.23,24 In this control reaction, an observed exact mass of the molecular ion [M+ H]+
of the dimer product was 523.21324 (error for C34H27N4O2: -0.16 mmu), which was in agreement with that
of the minor product from 7a within the margin of error. Therefore, we concluded that the minor product
was the dimer of 9-hydroxyellipticine (3).25 In the absence of the
O
Me
esterase, 7a was not reacted at all even after 3.5 h in the reaction for
comparison. These facts strongly suggest that 7a works as a prodrug
leading to the formation of 3 by enzymatic hydrolysis under
physiological conditions in accordance with our expectation.
O
HO
N
O
N
H
Me
8
Finally, we determined the water solubility of 7a, as a typical example. The concentration of a saturated
solution of the compound 7a in PBS was 0.77 mg/mL (see Experimental), while ellipticine (1) and

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671
9-hydroxyellipticine (3) were only minutely soluble in water. This significant increase of the water
solubility of 7a is noteworthy. It is not far behind from those of paclitaxels with glycan-conjugation,12
although we cannot simply compare the solubility of two kinds of compounds. The water solubility of
drugs without a positive charge is generally improved by introduction of a hydrophilic substituent like a
glucose or polyethylene glycol moiety as described above. In relation to the present study, synthesis and
evaluation of antitumor activity of ellipticine derivatives possessing an oligoethylene glycol tether are in
progress.
Figure 3. Time-course for hydrolysis of 7a catalyzed by carboxyesterase in phosphate buffer (PBS, pH
7.4) at 37 oC. Reaction times, 1: 7 min, 2: 12 min, 3: 22 min, 4: 32 min, 5: 42 min, 6: 52 min, 7: 62 min,
8: 82 min, 9: 102 min, 10: 142 min, 11: 182 min, respectively.
CONCLUSION
We succeeded in developing the simple and efficient synthesis of three novel 9-hydroxyellipticine
derivatives 7a-c, linked with a glucose moiety by a triazole ring-succinate tether at the 9-position of an
ellipticine nucleus. The glucose moiety was introduced via the Huisgen reaction. The in vitro antitumor
activity of these compounds was evaluated by an MTT assay on human cervical cancer cell line HeLa S-3
and on human embryonic kidney (HEK) cell line 293T. These compounds exhibited potent antitumor

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activity for both cell lines. The introduction of glucose conjugation at the 9-position enhanced its
solubility in water compared with those of ellipticine alone. Furthermore, 7a was hydrolyzed by esterase
to generate 9-hydroxyellipticine (3) under physiological conditions. The results shown in this paper
strongly suggest that the 9-hydroxyellipticine derivatives 7a-c must work as prodrugs. This is the first
report of the synthesis and evaluation of the antitumor activity of the uncharged glucose-conjugates of
9-hydroxyellipticine (3) with increased water solubility.
EXPERIMENTAL
Materials and methods
All chemicals including glucose-azides 6a-c were purchased from Tokyo Chemical Industry and used
without further purification, unless otherwise noted. Monopropargyl succinate (4) was synthesized from
succinic anhydride and propargyl alcohol by the reported methods.16 3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) was purchased from Nacalai Tesuque. Solvents were basically
dried and distilled before use. Reaction progress and compound purity were monitored using thin-layer
chromatography (TLC) with hexane-ethyl acetate as the irrigating system and UV light at shorter
wavelengths as the visualizer. Flash chromatography was performed using Silica gel 60 (Merck).
Carboxy esterase (EC 3.1.1.1; E2884; containing 6100 units of enzyme per mL) was obtained from Sigma
as a suspension in a 3.2 M (NH4)2SO4 solution (pH 8). Melting points were determined using a Yanaco
1
13
micro- melting-point apparatus, and the uncorrected values were reported. The H and C NMR spectra
were recorded using a JEOL JNM ECP-500 (500 and 125 MHz) and a JEOL JNM ECP-300 (300 and 75
MHz) spectrometer, respectively. The 1H and 13C chemical shifts were referenced to TMS (δ 0.00) using
the solvent residual peak as a reference, and the J values are reported in Hz. The low-resolution (MS) and
high-resolution mass spectra (HRMS) were recorded on a JEOL JMS-MS 700 (FAB) mass spectrometer,
using xenon (6 kV, 10 mA) as the fast atom bombardment gas, and a JEOL JMS-T100CS (ESI) mass
spectrometer. A quantitative and qualitative HPLC/MS analysis were determined on the JEOL
JMS-T100CS equipped with a Agilent 1200 HPLC apparatus with a ZORBAX Eclipse XDB-C18 column
o
(1 x 30 mm, 3.5 mm) eluting with MeOH (0.05 mL/min) at 40 C. Absorbance of MTT formazan was
measured on a CORONA MTP-500 microplate reader.
Synthesis of ellipticine (1)
The method for the synthesis of ellipticine (1) was principally same as a previously reported method for
6-methylellipticine.9 A usual Pomeranz-Fritsch reaction was modified to give higher yield.
Pomeranz-Fritsch type reaction (a modified step-wise method): A solution of 1,4-dimethyl-3-
formylcarbazole (500 mg, 2.2 mmol) in benzene (15 mL) and 2,2-diethoxyethylamine (330 mg, 2.5
mmol) was placed in a flask. The mixture was refluxed for 2 h. After the reaction, the solvent was

HETEROCYCLES, Vol. 92, No. 4, 2016
673
evaporated under the reduced pressure. MeOH (15 mL) was added to the residue. NaBH4 (800 mg, 22
mmol) was then added slowly, and the reaction mixture was stirred at rt for 2 h. The solvent was
evaporated under the reduced pressure. The residue was extracted with CHCl3 and dried over Na2SO4, and
concentrated under reduced pressure. A solution of the residue in a mixture of MeCN (10 mL) and
pyridine (1 mL) was placed in a flask. After the solution was stirred at rt for 0.5 h, 2-nitrobenzenesulfonyl
chloride (1.0 g, 4.5 mmol) was added, and the reaction mixture was stirred at rt for 24 h. After the
reaction, the solvent was evaporated under reduced pressure. The crude product was recrystallized from
MeOH, affording N-(2,2-diethoxyethyl)-N-((1,4-dimethyl-9H-carbazol-3-yl)methyl)-2-nitrobenzene-
sulfonamide (85%, 1.0 g); yellow solid; mp 175.0-176.0 ºC; 1H NMR (500 MHz, CDCl3) δ 1.46 (6H, t, J
= 7 Hz), 2.41 (3H, s), 2.73 (3H, s), 3.41-3.46 (4H, m), 3.61-3.64 (2H, m), 4.56 (1H, t, J = 5.5 Hz), 4.88
(2H, s), 7.07 (1H, s), 7.21-7.26 (1H, m), 7.36-7.42 (2H, m), 7.47 (1H, d, J = 8 Hz), 7.52 (1H, d, J = 8 Hz),
7.60 (1H, d, J = 8 Hz), 7.77 (1H, d, J = 8 Hz), 8.03 (1H, brs), 8.15 (1H, d, J = 8 Hz); 13C NMR (CDCl3,
125 MHz) δ 15.3, 15.9, 16.5, 49.6, 50.6, 63.4, 102.2, 110.6, 116.8, 119.5, 121.9, 122.8, 123.7, 123.8,
124.3, 125.1, 128.7, 130.4, 130.8, 131.1, 132.8, 134.4, 138.4, 139.6, 147.8; MS (FAB+) m/z 525 [M+];
HRMS (FAB+): calcd for C27H31N3O6S (M+) 525.1933, found 525.1921.
A suspension of N-(2,2-diethoxyethyl)-N-((1,4-dimethyl-9H-carbazol-3-yl)methyl)-2-nitrobenzenesulfon-
amide (500 mg, 0.95 mmol) in 6 M HCl/1,4-dioxane (1:4, 20 mL) was placed in a flask. The mixture was
refluxed for 3 h. After the reaction, the reaction mixture was cooled to rt. The mixture was neutralized by
the addition of aqueous sodium carbonate. The solution was extracted with CHCl3 (40 mL × 4), and dried
over Na2SO4, which was removed by filtration. The filtrate wasconcentrated under the reduced pressure.
The crude product was purified by column chromatography on silica gel [CHCl3/MeOH = 9:1 (v/v)],
affording ellipticine (1) (91%, 213 mg; lit.26 40%); yellow solid; mp 309.0-310.0 ºC (lit.26 313-315 oC); 1H
NMR (500 MHz, DMSO-d6), δ 2.78 (3H, s), 3.25 (3H, s), 7.25 (1H, t, J = 7 Hz), 7.51 (1H, t, J = 7 Hz),
7.56 (1H, d, J = 7 Hz), 7.91 (1H, d, J = 6 Hz), 8.37 (1H, d, 7 Hz), 8.42 (1H, d, 6 Hz), 9.69 (1H, s), 11.37
13
(1H, s); C NMR, (125 MHz, DMSO-d6) δ 11.9, 14.3, 108.0, 110.6, 115.8, 119.1, 122.0, 123.1, 123.3,
123.7, 127.0, 128.0, 132.4, 140.4, 140.5, 142.7, 149.6; MS (FAB+) m/z 247 [M+ H]+.
Pomeranz-Fritsch reaction: A solution of 1,4-dimethyl-3-formylcarbazole (50 mg, 0.22 mmol) and
2,2-diethoxyethylamine (33 mg, 0.25 mmol) in benzene (20 mL) was refluxed for 2 h in a flask equipped
with a Dean-Stark trap. After the reaction, the solvent was evaporated under the reduced pressure, and the
crude product was recrystallized from benzene just one time to give the corresponding crude imino
compound. A solution of the crude imiono compound in MeOH was added drop-by-drop to a mixture (5
o
mL) of H3PO4 and H2O (91:1) with stirring at 160 C, and then stirring was continued for 10 min. After
cooling to rt, H2O was added to the reaction mixture, which was then filtered. The filtrate was extracted
with CHCl3 after neutralization with NH3 aq., and dried over MgSO4, which was removed by filtration.

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The filtrate was concentrated under the reduced pressure. The crude product was purified by column
chromatography on silica gel (CHCl3), affording ellipticine (1) (28%, 15 mg; lit.26 28%).
Synthesis of 9-hydroxyellipticine (3)
The method for the synthesis of 9-hydroxyellipticine (3) was principally same as a previously reported
method for 9-hydroxy-6-methylellipticine.9
A suspension of ellipticine (1) (150 mg, 0.61 mmol) and hexamethylenetetramine (1.0 g, 7.1 mmol) in
TFA (25 mL) was placed in a flask. The mixture was refluxed for 30 min. After the reaction, the reaction
mixture was cooled to rt. The mixture was neutralized by the addition of aqueous sodium carbonate. The
mixture was extracted with CHCl3/MeOH (9:1, 50 mL × 4). The organic layers were washed with H2O.
The organic layer was dried over Na2SO4 and concentrated. The solid was triturated in H2O and filtered,
affording 9-formylellipticine. This product was used for the next reaction without further purification.
Yield: 92% (147 mg) (lit.27 97%); yellow solid; mp 360.0 ºC (lit.27 decomp. >350 oC); 1H NMR (500 MHz,
DMSO-d6), δ 2.76 (3H, s), 3.25 (3H, s), 7.64 (1H, d, J = 8.5 Hz), 7.91 (1H, d, J = 6 Hz), 8.03 (1H, d, J =
13
8.5 Hz), 8.46 (1H, d, J = 6 Hz), 8.83 (1H, s), 9.70 (1H, s), 10.08 (1H, s), 11.91 (1H, s); C NMR (125
MHz, DMSO-d6), δ 11.9, 14.3, 109.4, 111.0, 116.0, 122.3, 122.7, 123.2, 127.1, 128.1, 128.4, 128.8, 132.9,
140.7, 140.9, 146.7, 149.8, 191.9; MS (FAB+) m/z 275 [M+ H]+.
A suspension of 9-formylellipticine (100 mg, 0.36 mmol) in MeOH (25 mL) was placed in a flask. The
mixture was cooled to 0 ºC, and concentrated H2SO4 (100 μL) and 30% H2O2 aq were added. The mixture
was refluxed for 30 min. After the reaction, the reaction mixture was cooled to 0 ºC. The mixture was
neutralized by the addition of aqueous sodium carbonate. The mixture was extracted with CHCl3/MeOH
(9:1, 50 mL × 2), and dried over Na2SO4, which was removed by filtration. The filtrate wasconcentrated
under the reduced pressure. The crude product was purified by column chromatography on silica gel
[CHCl3/MeOH = 9:1 to 85:15 (v/v)], affording compound 3 (69%, 66 mg; lit.27 91%); orange solid; mp
355.0 oC (lit.27 decomp. >350 oC); 1H NMR (500 MHz, DMSO-d6), δ 2.74 (3H, s), 3.20 (3H, s), 7.02 (1H,
dd, J = 9, 2 Hz), 7.37 (1H, d, J = 9 Hz), 7.76 (1H, d, J = 2 Hz), 7.87 (1H, d, J = 6 Hz), 8.38 (1H, d, J = 6
Hz), 9.09 (1H, s), 9.64 (1H, s), 11.04 (1H, s); MS (FAB+) m/z 263 [M+ H]+
Synthesis of ellypticin-9-yl propargyl succinate (5)
A solution of DCC (142 mg, 0.69 mmol) in CH2Cl2 (2 mL) was added to a stirred mixture of MS 4A,
monopropargyl succinate (4) (107 mg, 0.69 mmol) and DMAP (17 mg, 0.14 mmol) in CH2Cl2 (8 mL) at
rt. After 1 h, 9-hydroxyellipticine (3) (120 mg, 0.46 mmol) in DMF (6 mL) were added to this mixture.
After 24 h, the mixture was evaporated under the reduced pressure, and extracted with CHCl3/MeOH (9:1,
100 mL × 2), washed with H2O, and dried over Na2SO4, which was removed by filtration. The filtrate was
concentrated under the reduced pressure. The crude product was purified by column chromatography on
silica gel [CHCl3/MeOH = 95:5 (v/v)], affording compound 5 (78%, 143 mg); yellow solid. 1H NMR (300

HETEROCYCLES, Vol. 92, No. 4, 2016
675
MHz, DMSO-d6), δ 2.78 (3H, s), 2.84 (2H, t, J = 6.5 Hz), 2.99 (2H, t, J = 6.5 Hz), 3.20 (3H, s), 3.63 (1H,
t, J = 2.5 Hz), 4.82 (2H, d, J = 2.5 Hz), 7.31 (1H, d, J = 8.5 Hz), 7.60 (1H, d, J = 8.5 Hz), 7.91 (1H, d, J =
6 Hz), 8.09 (1H, s), 8.46 (1H, d, J = 6 Hz), 9.70 (1H, s), 11.46 (1H, s); 13C NMR (75 MHz, DMSO-d6), δ
11.9, 14.2, 28.6, 28.8, 52.0, 77.8, 78.4, 108.3, 110.8, 115.8, 116.4, 120.9, 121.8, 123.0, 123.2, 128.4,
132.5, 140.3, 140.5, 141.1, 143.3, 149.7, 171.4, 171.6; HRMS (FAB+), calcd for C24H21N2O4 [M+ H]+:
401.1501, found 401.1488.
General procedure for the synthesis of glucose-conjugated ellipticines 7a-c (Huisgen reaction)
To a solution of CuI (17 mg, 0.089 mmol) in DMSO (0.5 mL), compound 3 (120 mg, 0.30 mmol) in
DMSO (2 mL) and azide 6a-c (0.3 mmol) in a mixed solvent of DMSO (1 mL) and H2O (0.3 mL) were
added. The mixture was stirred and N,N-diisopropylethylamine (DIPEA) (50 μL, 0.3 mmol) was added.
After the reaction, the mixture was directly loaded onto silica gel and purified by column chromatography
(CHCl3 : MeOH = 9:1 to 8:2), then the solid of the compound 7 was re-precipitated by addition of an
appropriate solvent.
1
7a: Yield 92%; yellow solid; mp 136.0-137.0 ºC; H NMR (500 MHz, DMSO-d6), δ 2.78 (3H, t, J = 6
Hz), 2.80 (3H, s), 2.93 (3H, m), 3.05 (1H, m), 3.13 (2H, m), 3.21 (2H, s), 3.67 (1H, d, J = 10 Hz), 3.89
(1H, m), 4.07 (1H, m), 4.22 (1H, d, J = 10 Hz), 4.57 (3H, t, J = 5 Hz), 4.98 (1H, m), 5.02 (1H, s), 5.13
(1H, d, J = 4 Hz), 5.19 (2H, s), 7.26 (1H, dd, J = 8, 2 Hz), 7.58 (1H, d, J = 8.5 Hz), 8.09 (1H, d, J = 2 Hz),
8.25 (1H, s), 11.55 (1H, s);13C NMR (125 MHz, DMSO-d6), δ 12.01, 14.30, 28.69, 28.89, 49.73, 57.52,
61.06, 64.86, 67.28, 70.01, 73.32, 76.60, 77.00, 79.39, 102.90, 108.54, 110.90, 116.52, 121.13, 123.22,
125.70, 128.27, 128.73, 128.93, 123.60, 140.35, 141.28, 141.51, 143.36, 149.60, 171.71, 171.85; HRMS
(ESI+), calcd for C32H35N5O10Na [M+ Na]+: 672.2282, found 672.2275; calcd for C32H36N5O10 [M + H]+:
650.24622, found 650.24515; UV, λmax(PBS) 296 nm (ε = 2.98 × 104 cm-1 L mol-1).
1
7b: Yield 94%; yellow solid; mp 145.9-146.6 ºC; H NMR (500 MHz, DMSO-d6), δ 2.79 (2H, t, J = 7
Hz), 2.80 (3H, s), 2.94 (2H, t, J = 7 Hz), 3.22 (3H, s), 3.44 (1H, d, J = 7.5 Hz), 3.68 (1 H, m), 3.77 (1H,
m), 4.68 (1H, s), 5.22 (3H, s), 5.35 (1H, s), 5.45 (1H, d, J = 6 Hz), 5.56 (1H, d, J = 9.5 Hz), 7.29 (1H, dd,
J = 9, 2.5 Hz), 7.59 (1H, d, J = 9.5 Hz), 7.97 (1H, d, J = 6 Hz), 8.11 (1H, d, J = 2.5 Hz), 8.41 (1H, s), 8.43
13
(1H,d, J = 6 Hz), 9.72 (1H, s), 11.62 (1H, s); C NMR (125 MHz, DMSO-d6), δ 12.02, 14.33, 28.64,
28.86, 60.71, 64.94, 69.53, 72.06, 76.94, 79.98, 87.49, 108.64, 110.97, 116.29, 116.55, 121.21, 121.67,
123.15, 123.28, 124.10, 128.99, 132.72, 139.40, 140.35, 141.49, 141.67, 143.41, 149.29, 171.69, 171.84;
HRMS (ESI+), calcd for C30H31N5O9Na [M+ Na]+: 628.2019, found 628.2013.
7c: Yield 90%; yellow solid; decomp. 178.3 ºC; 1H NMR (500 MHz, DMSO-d6), δ 1.63 (3H, s), 2.77 (2H,
t, J = 6.5 Hz), 2.82 (3H, s), 2.94 (2H, t, J = 6.5 Hz), 3.23 (3H, s), 3.59 (1H, t, J = 9 Hz), 3.69 (1H, d, J =
10 Hz), 4.07 (1H, q, J = 10 Hz), 4.73 (1H, s), 5.19 (2H, s), 5.38 (2H, s), 5.73 (1H, d, J = 10 Hz), 7.31 (1H,
d, J = 8.5 Hz), 7.64 (1H, d, J = 8.5 Hz), 8.01 (1H, brs), 8.11 (2H, s), 8.23 (1H, s), 8.42 (1H, brs), 9.79 (1H,

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HETEROCYCLES, Vol. 92, No. 4, 2016
13
s), 12.06 (1H, s); C NMR (125 MHz, DMSO-d6), δ 12.15, 14.53, 22.70, 28.67, 28.86, 54.41, 57.37,
60.61, 69.93, 73.85, 80.10, 86.12, 109.28, 111.34, 117.41, 121.03, 121.64, 122.85, 123.51, 123.99, 130.59,
133.10, 135.39, 140.41, 141.58, 142.60, 143.70, 147.43, 149.53, 169.23, 171.66, 171.78; HRMS (ESI+),
calcd for C32H34N6O9Na [M+ Na]+: 669.2285, found 669.2289.
Hydrolysis of 7a by carboxyesterase from porcine liver
Determination of time course of the hydrolysis: A carboxyesterase suspension (2 μL) was diluted with
9.9 mL of phosphate buffer (PBS, pH 7.4), which contained NaCl (8.00 g), KCl (0.20 g),
Na2HPO4·12H2O (2.88 g), and KH2PO4 (0.20 g) in 1.0 L of the buffer solution. Then, 0.1 mL of the 3.52
mM stock solution of compound 7a (0.352 µmol) in DMSO was then combined with the
above-mentioned buffer-diluted esterase solution. It was shaken for 30 s, and then kept in a water bath at
37 ºC with stirring. Aliquots (0.2 mL) were withdrawn from the reaction mixture after 7, 12, 22, 32, 42,
52, 62, 82, 102, 142 and 182 min and each of these samples was diluted to 3 mL with PBS and then
frozen with acetone/liquid-nitrogen bath. They were analyzed by UV-Vis spectrophotometer, later. The
time course for hydrolysis by the absorbance changes is shown in Figure 3.
Determination of hydrolysis product: A carboxyesterase suspension (2 μL) was diluted with 9.9 mL of
PBS (pH 7.4). Aliquots (0.1 mL) of the 35.4 mM stock solution of compound 7a (3.54 µmol) in DMSO
were then combined with the above-mentioned buffer-diluted esterase solution. It was shaken for 30 s,
and then kept in a water bath at 37 ºC for 3 h with stirring. The reaction mixture was stood overnight at
o
room temperature (< 10 C) leading to the formation of precipitates, which were filtrated with a
membrane-filter and collected with 3.3 mL of MeOH. The solution was applied to HPLC/ESI-MS, which
showed that the precipitates were composed of two products. The major product was 9-hydroxyellipticine
(3) and the minor product was a dimer of 3 as shown bellow.ꢀ
12
9-Hydroxyellipticine (3): yield 18.5% (by HPLC/ESI-MS); HRMS (ESI+), calcd for C17H15N2O [M +
H]+: 263.11844, found 263.11849.
The minor product; HRMS (ESI+), calcd for 12C34H27N4O2 [M+ H]+: 523.21340, found 523.21496; HRMS
(ESI-), calcd for 12C34H25N4O2 [M- H]-: 521.19775, found 521.19656.
Dimerization of 9-hydroxyellipticine (3): A solution of 3 (1.85 mg, 7.05 mmol) in 0.2 mL DMSO was
o
combined with 20 mL PBS and the thus-obtained turbid solution was incubated at 37 C for 3 h in the
absence of the esterase and stood overnight at room temperature in the same way as the hydrolysis
reaction of 7a mentioned above. After the reaction, the appeared precipitates were collected by filtration,
and dissolved in 5 mL of a mixed solvent of CH2Cl2 and MeOH (3:2). The solution was applied to
HPLC/ESI-MS. The reaction mixture contains only the dimer of 3 except for the starting material 3.
12
9-Hydroxyellipticine (3): recovery 43% (by HPLC/ESI-MS); HRMS (ESI+), calcd for C17H15N2O [M +
H]+: 263.11844, found 263.11839.

HETEROCYCLES, Vol. 92, No. 4, 2016
677
The dimer of 9-hydroxyellipticine: yield 57%; HRMS (ESI+), calcd for 12C34H27N4O2 [M+H]+: 523.21340,
found 523.21324.
The reaction of 3 in the presence of the esterase also afforded the same dimer. Recovery and the yield
were not determined. HRMS (ESI+), calcd for 12C34H27N4O2 [M+ H]+: 523.21340, found 523.21359.
Evaluation of the water solubility of 7a
A stock solution of 7a (1.5 mg/4 mL H2O) was diluted to 1/10 concentration. The obtained solution was
additionally diluted to 1/2, 1/4, and 1/8 concentrations. The absorbance of each solution was determined
at 296 nm to make a calibration curve. Subsequently, compound 7a (3.5 mg) was suspended in H2O (4.0
mL) and sonicated at rt to prepare a saturated solution, which was filtered to remove the undissolved 7a.
An aliquot of the filtrate was diluted to the appropriate concentration and the absorbance of the solution
was measured at 296 nm leading to the solubility of 7a (0.77 mg/mL).
Evaluation of Antitumor Activity
Cell lines and culture: HeLa S-3 cells were maintained in MEM (Nissui) supplemented with 10% FBS
(GIBCO), 2% HEPES, 3% NEAA, 1.5% NaHCO3 (7.5 w/v% solution) and 2 mM L-glutamine in a
o
water-saturated atmosphere of 5% CO2 at 37 C. The 293T cells were maintained in DMEM (Nissui)
supplemented with 10% FBS and 1% penicillin/streptomycin in a water-saturated atmosphere of 5% CO2
at 37 oC.
Cell viability assay: Cell viability was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) assay, which is a method for determining cell viability by measuring the
mitochondrial dehydrogenase action. Cells were seeded in a 96-well cell culture cluster (Becton
Dickinson) at a density of 2 × 104 cells/mL and cultured 3 h prior to drug treatment. Cells were exposed at
37 oC for 24-72 h to ellipticine derivatives. The MTT reagent was prepared at a concentration of 2 mg/mL
in Dulbecco’s PBS without calcium and magnesium, and stored at 4 oC. After treatment for the indicated
times, the cells were incubated with MTT reagent for 4 h at 37 oC. The plate was centrifuged at 3000 rpm
for 10 min, and the medium was removed. To solubilize the resultant MTT formazan, 200 µL/well of
dimethyl sulfoxide (DMSO) was added to each well followed by thorough mixing using a mechanical
plate mixer. Absorbance at 540 nm was measured on a microplate reader, and the percentage of cell
viability was taken as the percentage absorbance at 540 nm of ellipticine derivative-treated cells against
control cells. IC50 values are reported as the means of three experiments and the standard deviations are
given together.
ACKNOWLEDGMENTS
This work was partially supported by a Grant-in-Aid for Scientific Research from MEXT, a matching
fund subsidy from MEXT 2004-2006 (No.16550148), 2009-2011 (No. 21590025) and 20012–2014 (No.
24590025); a Grant from the Japan Private School Promotion Foundation (2008-2009); and a grant for

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HETEROCYCLES, Vol. 92, No. 4, 2016
the Development of strategic Research Center in Private Universities supported by MEXT, Center for
Technologies against Cancer (CTC), 2009-2013. We would like to thank Dr. Tomoko Okada (Institute for
Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology)
for kindly providing the HeLa S-3 cells and Mrs. Hitomi Isoda (Research Equipment Center, Tokyo
University of Science) for HPLC/ESI-MS analysis.
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1
The succinate was synthesized in a 69% yield as yellow solid by the same method as 5; H NMR
(500 MHz, DMSO-d6), δ 2.67 (3H, s), 2.74 (2H, t, J = 6.5 Hz), 2.92 (2H, t, J = 6.5 Hz), 3.09 (3H, s),
3.66 (3H, s), 7.25 (1H, dd, J = 8.5, 2 Hz), 7.53 (1H, d, J = 9 Hz), 7.80 (1H, d, J = 6 Hz), 8.00 (1H, s),
13
8.38 (1H, d, J = 6 Hz), 9.61 (1H, s), 11.36 (1H, s); C NMR (125 MHz, DMSO-d6), δ 11.8, 14.0,
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