Synthesis and in vitro antitumor activity of novel 2-alkyl-5- methoxycarbonyl-11-methyl-6H-pyrido[4,3-b]carbazol-2-ium and 2-alkylellipticin-2-ium chloride derivatives

ACCEPTED MANUSCRIPT

Synthesis and in vitro antitumor activity of novel 2-alkyl-5-methoxycarbonyl-11-

methyl-6H-pyrido[4,3-b]carbazol-2-ium and 2-Alkylellipticin-2-ium chloride

derivatives

a

Ryota Mori, Asako Kato, Kousuke Komenoi, Haruaki Kurasaki, Touru IIjima, Masashi

a

a,b

Kawagoshi, Y. B. Kiran, Sho Takeda, Norio Sakai, and Takeo Konakahara *

a

Department of Pure and Applied Chemistry, Faculty of Science & Technology, Tokyo University of

Science (RIKADAI), Noda, Chiba 278-8510, Japan

b

Center for Technologies against Cancer, Tokyo University of Science (RIKADAI), Noda, Chiba,

2

78-8510, Japan

*konaka@rs.noda.tus.ac.jp

Phone: 81-04-7124-1501 Fax: 81-04-7123-9326

Highlights

1. Twelve novel 2-tethered ellipticine derivatives were synthesized in good yields.

2. Nine novel 2-tethered pyridocarbazole derivatives were synthesized in good yields.

3. Some of these compounds exhibited potent antitumor activity on HeLa S-3 cells.

4. Introduction of a OH group at the 9-position enhanced its antitumor activity.

5. Elongation of the tether had a tendency to suppress the activity of the compounds.

ACCEPTED MANUSCRIPT

Synthesis and in vitro antitumor activity of novel 2-alkyl-5-methoxycarbonyl-11-methyl-6H-

pyrido[4,3-b]carbazol-2-ium and 2-Alkylellipticin-2-ium chloride derivatives

a

Ryota Mori, Asako Kato, Kousuke Komenoi, Haruaki Kurasaki, Touru IIjima, Masashi

a

a,b

Kawagoshi, Y. B. Kiran, Sho Takeda, Norio Sakai, and Takeo Konakahara *

a

Department of Pure and Applied Chemistry, Faculty of Science & Technology, Tokyo University of Science

(

RIKADAI), Noda, Chiba 278-8510, Japan

b

Center for Technologies against Cancer, Tokyo University of Science (RIKADAI), Noda, Chiba, 278-8510,

Japan

*konaka@rs.noda.tus.ac.jp

Phone: 81-04-7124-1501 Fax: 81-04-7123-9326

Abstract

Twenty-one types of novel ellipticine derivatives and pyridocarbazoles (5-methoxycarbonyl-11-methyl-

6

H-pyrido[4,3-b]carbazoles) with a nitrosourea moiety, linked by an oxydiethylene unit at the 2 position,

were synthesized, and their cytotoxicity against HeLa S-3 cells was evaluated. Some of these new

compounds exhibited potent antitumor activity by comparison with that of ellipticine.

Keywords:

Ellipticine, 6H-pyrido[4,3-b]carbazole, nitrosourea, synthesis, antitumor activity, cytotoxicity, HeLa S-3,

SAR

1

. Introduction

A great deal of interest has been generated by indole alkaloids, ellipticines and β-carbolines because

of their intercalation, antitumor properties, and anti-HIV activities. Ellipticine was first identified in 1959 as

1

2

1

g

a compound in the leaves of a small tropical evergreen Ochrosia elliptica Labill (see Fig. 1). Ellipticine

and its derivatives cause selective inhibition of p53 protein phosphorylation in several human cancer cell

3

lines, and this is correlated with their cytotoxic activity. These also uncouple mitochondrial oxidative

4

phosphorylation, thereby disrupting the energy balance of cells. Further, these have been shown to react

1e,f

5

with DNA by an intercalation process, which may account for their cytotoxicity, and the further

1

a

inhibition of DNA topoisomerase-II activity. We have reported the interaction mode of ellipticine and its

6

derivatives with DNA using deflection spectroscopy. Ellipticine is a good intercalator and an antitumor

7

agent like β-carbolines. The unique structural features, limited toxic side effects, and complete lack of

1

ACCEPTED MANUSCRIPT

hematological toxicity of these compounds has prompted chemists to study their synthesis of a number of

8

analogues for pharmacological evaluation.

<

Fig. 1>

9

The syntheses of ellipticine and its derivatives have been reported by many groups. Several simple

1

0

structural modifications to ellipticine derivatives have resulted in compounds with increased toxicity. A

low level of water solubility at physiological pH, as well as systemic toxicity, has prevented the use of

ellipticine as a therapeutic agent. The introduction of a positive charge in the molecule improved water

11

solubility and made a profound difference in the biological activity. For example, the quaternization of the

12

13

endocyclic nitrogen at N5 of 5-aza-ellipticine and the nitrogen at N2 of ellipticine with a methyl group

was found to interact with DNA through intercalation, which resulted in a high affinity for binding with

DNA.

14

This background combined with further work in this area has led us to the development of a novel

15

and efficient method for the synthesis of ellipticine via Suzuki-Miyaura coupling reaction. Nitrosourea

compounds generally are good DNA alkylating agents and are often used in chemotherapy to damage the

1

6

DNA of cancer cells. Herein, we focus on the development of new ellipticine analogues that will have

greater solubility in water by quaternizing the endocyclic nitrogen atom at the 2 position with a tethered

nitrosourea moiety (Fig. 2). In order to prevent a decrease in water solubility with an extension of the

carbon chain, we designed a 3-oxa-pentyl tether (X = O in Fig. 2). In addition, we examined the efficiency

of these new molecules as potent antitumor agents with improvements in the binding ability related to

cytotoxic activity, which is evaluated by MTT assay against HeLa S-3 cells.

<

Fig. 2>

2

. Results and Discussion

.1. Synthesis of N-nitrosoureas linked to 2-alkyl-5-methoxycarbonyl-11-methyl-6H-pyrido[4,3-

2

b]carbazol-2-ium and ellipticin-2-ium chloride derivatives

We selected a (2-aminoethoxy)ethyl (Fig. 2, X = O) and 5-aminopentyl groups (Fig. 2, X = CH

linkers in the target compounds (Fig. 2a,b). Retro synthesis analysis for 2-(2-aminoethoxy)ethyl-5-

methoxycarbonyl-11-methyl-6H-pyrido[4,3-b]carbazol-2-ium chloride derivatives (Fig. 2a;

pyridocarbazole-5-carboxylate derivatives) is briefly shown in Fig. 3 to make the synthetic strategy

2

) as

understandable. We considered that the target compound was obtained from a synthetic equivalent A and a

synthon B, and that the synthetic equivalent A was obtained by successive reactions of methyl 2-(3-(1-

(

pyridin-3-yl)vinyl)-1H-indol-2-yl)acetate D and an aminoalkyl linker E to give the corresponding

precursor compound C (Fig. 3).

2

ACCEPTED MANUSCRIPT

<

Fig. 3>

First, we synthesized various kinds of aminoalkyl linkers to accomplish the target molecules

(

pyridocarbazole-5-carboxylate and ellipticine derivatives), as represented in Fig. 2 and 3 (see Schemes 1

and 2). Treatment of potassium phthalimide with bis(2-chloroethyl) ether (1) in DMF gave the

0

corresponding monoimide 2. The reduction of 2 with NH NH at 60 C for 3 h generated the corresponding

2

monoamine 3 (Scheme 1). The NH group of 3 was protected with Ac O/Et N or 2,5-hexanedione/acetic

2

3

acid to give 4a or 4b in 65 and 45% yields, respectively (Scheme 1; see Supporting Information).

Compounds 6a-d were obtained in a similar manner by protecting the NH group of 2-(2-

2

aminoethoxy)ethanol (5) by treatment with phthalic anhydride, Ac O/Et N, 2,5-hexanedione/acetic acid,

2

3

and Boc O/Et N, respectively (Scheme 2; see Supporting Information). The obtained compounds 6a-d

2

3

were tosylated with TsCl in the presence of Et

3

N at room temperature for 3 h of reaction time, to give the

corresponding O-tosylates 7a-d in high yields.

<

Scheme 1>

Scheme 2>

<

Next, 2-alkyl-5-methoxycarbonyl-11-methyl-6H-pyrido[4,3-b]carbazol-2-ium chloride derivatives,

shown in Fig. 2, were prepared from indole (8), as shown in Scheme 3. First, the NH group of 8 was

protected with benzenesulfonyl group to give N-benzenesulfonylindole (9) in an 85 % yield, and 9 was

oxalylated with dimethyl oxalate on a C-2 atom to give compound 10 in a 61% yield. The benzenesulfonyl

group was easily removed upon treatment with potassium carbonate accompanied by hydrolysis of the ester

group to give the corresponding carboxylic acid 11 in an 85% yield. The carbonyl group of 11 was reduced

with hydrazine leading to the formation of 2-indolylacetic acid (12). The crude product was treated with

trimethylsilyldiazomethane without further purification to give the corresponding methyl ester 13 (75%).

The isolation of carboxylic acid 12 failed because of decarboxylation, which resulted in 2-methylindole.

Finally, a mixture of 13 and 3-acetylpyridine was heated under reflux in methanol for 6.5 h in the presence

of concentrated sulfuric acid leading to methyl 2-(3-(1-(pyridin-3-yl)vinyl)-1H-indol-2-yl)acetate (14) in

6

5% yield.

Furthermore, in order to construct a pyridocarbazole ring, the compound 14 was treated with 2 and 4a

in DMF, and then with sodium methoxide in the presence of 3-ethoxycarbonyl-1-methylpyridinium

chloride (17) (Path A in Scheme 4) leading to 2-alkylpyridocarbazolium derivatives 19a,b in a yield of 10%

and a trace amount, respectively. When conducted in another manner, the 2-alkylpyridocarbazoliums 19a,

3

ACCEPTED MANUSCRIPT

1

7

9b and 19d were prepared from 14 in good yields (55, 60 and 52%) in two steps via treatment of 14 with

a, 7b and 7d, respectively, in the presence of NaOMe and 17 followed by the action of Amberlite IRA-900

(

Path B in Scheme 4). The pyridinium chloride 17 was prepared from the corresponding pyridine

carboxylate 15 and methyl iodide to give the pyridinium iodide 16 followed by treatment with Amberlite

IRA-900 (chloride form). Unexpectedly, the reaction of 4b and 7c with 14 via paths A and B yielded

complex mixtures, and the objective for 19c was not given (Scheme 4). The stability of the quaternary salts

of these molecules increases by converting them into the corresponding tosylate and chloride salts (Scheme

4

). The compound 20 was obtained by the deprotection of the amino groups of 19a, 19b and 19d. The

compound 19b on reflux with 9 M HCl-MeOH in 3 days and compound 19d upon treatment with 12 M

HCl in 5 h gave compound 20 in 50 and 85% yields, respectively (Scheme 4). Contrary to our expectations,

1

9a did not give 20 in methanolic HCl even after reflux for 3 days.

Antitumor activity of 9-hydroxyellipticine (23) and its derivatives has been widely studied, and the

1

7

introduction of a 9-hydroxy group may increase its antitumor activity. We were interested in applying this

substituent to our molecules 35a, 38b and 39a,b tethered at the 2-position. Therefore, we planned to

synthesize the compounds 35b, 38c and 39c together with their 6-methylated derivatives 35c,d, 38d,e, and

3

9d,e and to determine their in vitro antitumor activity. Ellipticine (21), 9-hydroxyellipticine (23), 6-

methylellipticine (27) and 9-hydroxy-6-methylellipticine (29) were prepared via a modification of a

18

previously reported method, as shown in Scheme 5 for 27 and 29. Ellipticine (21) was heated with

hexamethylenetetramine in TFA for 20 min under reflux to give the corresponding 9-formyl derivative

(

92% yield), and was followed by Dakin oxidation leading to the formation of 9-hydroxyellipticine (23) in

18

5

5% yield, according to a reported method. Ellipticine (21), 9-hydroxyellipticine (23), and 9-hydroxy-6-

methylellipticine (29) were treated with 7d to form the corresponding ellipticinium tosylate quaternary salts

3a-c (Scheme 6). In a similar manner, tosylate salt 33d was formed by reacting 29 with 32. The quaternary

3

salts 33a-d were then treated with Amberlite IRA-900 (chloride form) in water at room temperature, and

the reaction mixture was purified by ODS column chromatography to give the corresponding chloride salts

3

4a-d in 52, 52, 52, and 50% yields, respectively. These chloride salts (34a-d) were then refluxed for 5 h in

2 M HCl to give the 2-alkylellipticinim analogues 35a-d in 86, 85, 86, and 82% yields (Scheme 6).

1

<

Scheme 5>

Scheme 6>

<

The obtained 2-(2-aminoethoxy)ethyl-5-methoxycarbonyl-11-methyl-6H-pyrido[4,3-b]carbazol-2-ium

chloride (20) and its analogues 35a-d were treated with p-nitrophenyl N-methylcarbamate 37a and its N-

i

nitroso derivative 37b in the presence of Pt NEt in DMF to get the corresponding urea derivatives 38a-e

2

4

ACCEPTED MANUSCRIPT

(

74, 45, 43, 47, and 63% yields, respectively) and N-nitrosourea derivatives 39a-e (64, 43, 39, 40, and 20%

yields, respectively) (Scheme 7).

Finally, 2-(2-aminoethyl)- and 2-(3-aminopropyl)-pyridocarbazol-2-ium chlorides 40, 41, 42, 43, 44, and

5 were synthesized to verify the effect of linker length on antitumor activity in comparison with those of

8e and 39e, by a method analogous to the synthesis of 38e and 39e in poor to high yields (see Scheme 8).

4

3

Furthermore, 2-methylellipticin-2-ium chloride (46) was synthesized to clarify the effect of the

quaternization of the endocyclic nitrogen at the N2 of ellipticine (21) on antitumor activity. The compound

4

6 can be synthesized by a direct methylation of ellipticine (21) with methyl iodide, but the yield is low and

30

the reaction time is long. Therefore, we decided to synthesize 46 by the similar method for the preparation

of 50 using methyl iodide instead of 49 followed by both reduction with sodium bis(2-

methoxyethoxy)aluminum hydride and the ion-exchange reaction with Amberlite IRA-900 (a chloride

form) in a higher yield as shown in Scheme 9.

<

Scheme 8>

Scheme 9>

<

2

.2. Antitumor activity of ellipticine and pyridocarbazole-5-carboxylate derivatives 20, 21, 35, and 38-46

We used an MTT assay to evaluate the cytotoxicity of ellipticine and pyridocarbazole derivatives on

human cervical cancer cell line HeLa S-3. The IC50 values in the sub-micromolar range of all compounds

are given in Table 1.

2

First, the in vitro antitumor activity of the ellipticine series (Fig. 2; R = CH

3

) is discussed. In

comparison with the antitumor activity of ellipticine (21) itself (IC50 2.1 µM), the quaternization of the

endocyclic nitrogen atom at the 2-position of 21 by a methyl group did not decrease the activity (46, IC50

=

2

.90 µM), as shown in Table 1. Next, the in vitro antitumor activity was compared between the derivatives

with different tether lengths and a different central group X (O and CH ). Prior to discussing the effects of

2

the tether length on activity, it is important to examine the effects of the central X group, the terminal amino

group, and the terminal urea group on antitumor activity. Although the IC₅₀value of 35c (X = O; 17.9 µM)

was near that of 35d (X = CH ; 19.3 µM) in the amine derivatives, the IC₅₀value of 38d (X = O; 6.2 µM)

2

was smaller than that of 38e (X = CH ; 32.5 µM) in the urea derivatives. The effect of the terminal amino

group on antitumor activity was not observed in the MTT assays of either 46, 40, or 41 (IC₅₀= 2.90, 5.8,

and 2.50 µM, respectively). However, the introduction of the terminal urea group in the tether generally

5

ACCEPTED MANUSCRIPT

decreases the antitumor activity in ellipticine derivatives; for example, the IC₅₀values of 35a and 38b were

2

7.8 and 75.3 µM; 35d and 38e were 19.3 and 32.5 µM; and, 41 and 44 were 2.5 and 24 µM, respectively.

An increase in the activity of the 9-hydroxy-6-methylellipticinium derivatives was an exception (35c and

8d: 17.9 and 6.2 µM). In comparing the IC₅₀value of 46 (Entry 2) with those of 35a and 38b, the

3

compounds with the shorter tether showed the highest activity (2.9, 27.8 and 75.3 µM, respectively). As a

result, it was predicted that an elongation of the tether would suppress the expression of the antitumor

activity of ellipticine (21).

In a series of compounds with a (2-aminoethoxy)ethyl tether, replacement of the methyl group at the

2

5

position to the methyl ester group slightly reduced the antitumor activity (Fig. 2; R = COOCH

3

); for

example, the IC50 values of 35a and 20 were 27.8 and 41.3 µM (Entries 3 and 7), respectively.

As expected, the introduction of an OH group at the 9-position increased the antitumor activity of

elliptine as demonstrated by the MTT assay of pairs of compounds, 35a and 35b (IC50 = 27.8 and 17.7 µM),

and 38b and 38c (IC50 = 75.3 and 29.4 µM). The reason for the increased activity by the 9-OH group is

unclear. In addition, introduction of the methyl group at the 6-position also increased the activity, with the

noted exception of 35b,c (IC50 = 17.7 and 17.9 µM). For example, the IC50 values were 29.4 and 6.2 µM for

3

8c and 38d, and were 60 and 18.4 µM for 39c and 39d, respectively. Previously, we reported the synthesis

1

9

of methylnitrosourea derivatives linked to a methidium chloride nucleus. Despite low activity of

methylnitrosourea (MNU) itself (Entry 24, IC50 > 100 µM), the introduction of a methylnitrosourea moiety

in a molecule generally increases its antitumor activity. Although one of the methylnitrosourea derivatives

synthesized in the present study increased the antitumor activity against HeLa S-3 cells, as shown in Table

1

(IC₅₀= 24 and 1.30 µM for 44 and 45), the IC₅₀values of the other methylnitrosourea derivatives 39c and

3 (60 and 15 µM, respectively), were larger than those of the corresponding methylurea derivatives 38c

4

and 42 (29.4 and 6.6 µM, respectively).

The quaternization of the 2-N atom of ellipticine by an alkyl group is useful for increasing solubility

in water of the molecule, although some of quaternized compounds exhibited lower activity than ellipticine.

The highest antitumor activity of all compounds tested was resulted from pyridocarbazole-5-carboxylate

derivative 45, and its IC₅₀value (1.30 µM) almost approximated that of ellipticine (21) (2.1 µM). In

addition, it is remarkable that the activity of 45 (1.3 µM) is higher than that of 43 (15.0 µM) in ten times, in

which only one methylene unit is added in the linker.

3

. Conclusion

We developed a simple and efficient synthetic method of novel 4 ellipticine derivatives, linked by

an oxydiethylene or polymethylene unit at the 2 position with a nitrosourea moiety 39b-e, and that of the

corresponding 8 intermediates 35a-d, 38b-e in good to high yields. In connection with the synthesis of

these compounds, three kinds of novel pyridocarbazole-5-carboxylate derivatives, linked by an

oxydiethylene or polymethylene unit at the 2 position with a nitrosourea moiety 39a, 43, 45, and the

6

ACCEPTED MANUSCRIPT

corresponding 6 intermediates 20, 38a, 40, 41, 42, 44, were also synthesized. These compounds exhibited

higher solubility in water than ellipticine (21) itself. In vitro antitumor activity of these compounds were

evaluated by MTT assay on human cervical cancer cell line HeLa S-3. Some of these compounds exhibited

potent antitumor activity (1.30–19.3 µM). Introduction of an OH group at the 9-position enhanced its

antitumor activity and elongation of the tether at the 2-position had a tendency to suppress the antitumor

activity of ellipticine (21) except in the case of 43 and 45.

4

. Experimental

4

.1. Materials and Methods

All solvents and chemicals were purchased from TCI and used without further purification.

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. Melting points were determined using a Yanaco micro-

1

13

melting-point apparatus and uncorrected values were reported. H and C NMR spectra were recorded

1

using a JNM ECP-500 (500 and 125 MHz) and a JNM ECP 300 (300 and 75 MHz) respectively. The H,

13

C chemical shifts were referenced to TMS (δ = 0.00) using the solvent residual peak as a reference and J

values are reported in Hz. Mass spectra were recorded on a JEOL JMS-MS 700 mass spectrometer using

xenon (6 kV, 10 mA) as the fast atom bombardment (FAB) gas.

4

.2. Synthesis

20

.2.1. 2-[2-(2-Chloroethoxy)ethyl]isoindole-1,3-dione (2)

A mixture of bis(2-chloroethyl)ether (1, 4.6 g, 32.4 mmol) and potassium phthalimide (2.0 g, 10.8

0

mmol) was stirred at 130 C for 20 h in DMF (1 mL). The residue was washed with water and extracted

3 4

with CHCl and dried (MgSO ), and concentrated. The crude residue was purified by flash column

20

0

20

0

1

chromatography (CHCl ) to yield 2. 65%. (lit. 91%): mp 70.5-71.9 C (lit. 71.0-72.0 C). H NMR (500

3

MHz, CDCl ): δ 7.55-7.22 (4H, m, Ar-H), 3.96 (2H, t, J = 6.0 Hz, CH N), 3.82 (2H, t, J = 7.5 Hz CH Cl),

3

2

1

3

.68-3.60 (4H, m, 2 X CH O); C NMR (125 MHz, CDCl ): δ 168.2, 133.9, 132.1, 123.2, 70.6, 67.9, 42.6,

2

3

7.1; MS (FAB) m/z 256 (M+H).

4

.2.2. N-[2-(2-Chloroethoxy)ethyl]acetamide (4a)

0

A mixture of 2 (2g, 7.9 mmol) and N H (1.2g, 23.7 mmol) was stirred in CHCl for 3 h at 60 C,

2

4

3

and then the reaction mixture was slowly cooled to rt to get 3. This crude material 3 was used for further

reactions without purification.

2

To the crude 3, Ac O (0.96 g, 9.4 mmol), triethylamine (9.5 g, 9.4 mmol) and DMAP (97 mg, 0.79

mmol) were added by dissolving in CHCl (10 mL) through a dropping funnel and refluxed for 2 h. The

3

solvent was removed under reduced pressure. The dark brown liquid remained was subjected to fractional

0

1

distillation to give 4a (45%) bp: 84-85 C/ 7~8 mmHg. H NMR (500 MHz, CDCl ): δ 3.69-3.66 (4H, m,

3

7

ACCEPTED MANUSCRIPT

13

CH N, ClCH ), 3.61 (2H, t, J = 5.0 Hz, CH O), 3.46 (2H, t, J = 5.0 Hz, OCH ), 2.09 (3H, s, CH );

C

2

3

NMR (125 MHz, CDCl ): δ 169.0, 66.7, 66.5, 46.5, 41.6, 21.0; MS (FAB) m/z 166 (M+H).

3

4

.2.3. 1-[2-(2-Chloroethoxy)ethyl]-2,5-dimethyl-1H-pyrrole (4b)

To the crude material 3 (prepared in above said lines), 2,5-hexadione (0.45 g, 7.9 mmol) and

AcOH (0.19 g, 3.2 mmol) were added by dissolving in benzene (10 mL) through a dropping funnel and

0

stirring was continued overnight at 50 C. The solvent was removed in a rotary evaporator. The dark brown

0

1

liquid remained was subjected to fractional distillation to get 4b. (55%) bp: 107-109 C / 6~7 mmHg. H

NMR (500 MHz, CDCl ): δ 5.76 (2H, s, pyrrole), 3.96 (2H, t, J = 6.0 Hz, CH N), 3.64 (4H, m, CH O,

ClCH ), 3.57 (2H, t, J = 5.5 Hz, OCH ); C NMR (125 MHz, CDCl ): δ 127.8,

05.3, 71.3, 70.6, 43.3, 42.7, 12.5; MS (FAB) m/z 202 (M+H).

3

2

13

2

), 2.23 (6H, s, 2 X CH

3

1

4

.2.4. 2-[2-(2-Hydroxyethoxy)ethyl]isoindoline-1,3-dione (6a)

-(2-Aminoethoxy)-1-ethanol (5, 2.0 g, 19 mmol), phthalic anhydride (2.8 g, 19 mmol) and CHCl₃

20 mL) were placed in a 100 mL reaction flask. The reaction mixture was refluxed for 4 h. After the

2

(

volatile materials were removed under reduce pressure, the resulting reside was purified by flash column

1

chromatography (CHCl ) to yield 6a (70%). H NMR (500 MHz, CDCl ): δ 7.86-7.85 (2H, m, Ar-H), 7.73-

3

7

.71 (2H, m, Ar-H), 3.91 (2H, t, J = 5.5 Hz, NCH ), 3.75 (2H, t, J = 6.0 Hz, CH OH), 3.69 (2H, t, J = 5.5

2 2

13

Hz, OCH ), 3.61 (2H, t, J = 5.5 Hz, OCH ); C NMR (125 MHz, CDCl ): δ 168.4, 133.9, 131.9, 123.2,

2

3

7

2.1, 68.3, 61.6, 37.5.

4

.2.5. N-[2-(2-Hydroxyethoxy)ethyl]acetamide (6b)

-(2-Aminoethoxy)-1-ethanol (5, 2.0 g, 19 mmol), Ac O (2.1 g, 21 mmol), Et N (2.1 g, 21 mmol),

2

3

DMAP (0.23 g, 1.9 mmol) and CHCl (20 mL) were placed in a 100 mL reaction flask. The reaction

3

mixture was refluxed for 2 h. After the volatile materials were removed under reduced pressure, the

1

resulting reside was purified by flash column chromatography (CHCl ) to yield 6b (78%), H NMR (500

3

MHz, CDCl

3

): δ 6.24 (1H, br, OH), 3.75 (2H, t, J = 4.8 Hz, CH

2

2 2

N), 3.60-3.55 (4H, m, CH OH, OCH ),

13

3

.47 (2H, t, J = 4.8 Hz, OCH

9.2, 23.0.

2

), 1.99 (3H, s, CH

3

); C NMR (125 MHz, CDCl ): δ 170.6, 72.2, 69.7, 61.4,

3

4

.2.6. 2-[2-(2,5-Dimethyl-1H-pyrrol-1-yl)ethoxy]ethan-1-ol (6c)

-(2-Aminoethoxy)-1-ethanol (5, 2.0 g, 19 mmol), 2,5-hexadione (2.2 g, 19 mmol), AcOH (0.46 g,

2

7

.6 mmol), and benzene (20 mL) were placed in a 100 mL reaction flask. The reaction mixture was allowed

0

to stir overnight at 50 C. After the volatile materials were removed under reduced pressure, the resulting

1

reside was purified by flash column chromatography (CHCl ) to get 6c (75%), H NMR (300 MHz,

3

CDCl ): δ 5.77 (2H, s, pyrrole), 4.10 (2H, t, J = 6.0 Hz, CH N), 3.65-3.61 (4H, m, CH O, CH OH), 3.48

3

2

13

(

2H, t, J = 4.5 Hz, OCH ), 2.23 (6H, s, 2 X CH ); C NMR (125 MHz, CDCl ): δ 127.8, 105.3, 72.5, 70.4,

2 3 3

6

1.7, 43.2, 12.5.

8

ACCEPTED MANUSCRIPT

21

4

.2.7. tert-Butyl [2-(2-hydroxyethoxy)ethyl]carbamate (6d)

-(2-Aminoethoxy)-1-ethanol (5, 2.0 g, 19 mmol), Boc O (4.6 g, 21 mmol), Et N (2.1 g, 21 mmol),

2

3

DMAP (0.23 g, 1.9 mmol) and CHCl (20 mL) were placed in a 100 mL reaction flask. The reaction

mixture was allowed to stir for 3 h at rt. After the volatile materials were removed under reduced pressure,

1

the resulting reside on flash column chromatography (CHCl ) gives 6d (85%), H NMR (300 MHz,

3

CDCl ): δ 3.74 (2H. t, J = 4.8 Hz, CH N), 3.59-3.53 (4H, m, CH OH, OCH ), 3.34 (2H, t, J = 5.1 Hz,

3

2

13

OCH ), 1.45 (9H, s, CH ); C NMR (75 MHz, CDCl ): δ 156.0, 79.3, 72.1, 70.2, 61.6, 40.3, 28.3.

2

3

4

.2.8. Preparation of N-protected [2-(2-hydroxyethoxy)ethyl]amine 7a-d

A flame-dried 100 mL round-bottom flask containing a magnetic stirring bar was charged with 6a,

0

Et N (10 mmol), DMAP (0.1 mmol), and CHCl (20 mL). The solution was cooled to 0 C, and TsCl (10

3

mmol) dissolved in CHCl was added to the solution over 10 min. The reaction mixture was stirred at rt for

3

h. After all the volatile materials were removed under reduced pressure, the crude residue was purified by

flash column chromatography (CHCl ) to yield 7a. The 7b, 7c and 7d were prepared in similar lines

3

starting with 6b, 6c and 6d, respectively.

1

7

a: 70 %; H NMR (500 MHz, CDCl ): δ 7.83-7.81 (2H, m, Pht), 7.73-7.69 (4H, m, Pht, Ts), 7.30-7.28 (2H,

3

m, Ts); 4.08 (2H, t, J = 6 Hz, CH N), 3.81 (2H, t, J = 6 Hz, CH O), 3.65-3.63 (4H, m, OCH , CH O), 2.41

2

13

(

3H, s, CH₃); C NMR (75 MHz, CDCl ): δ 168.2, 144.7, 133.9, 132.9, 129.8, 127.9, 123.3, 69.1, 68.1,

3

6

8.0, 37.0, 21.6.

1

7

b: 74 %; H NMR (300 MHz, CDCl

3

): δ 7.78 (2H, d, J = 8.4 Hz, Ts), 7.33 (d, 2H, J = 8.1 Hz, Ts), 4.18

(

2H, t, J = 4.5 Hz, CH OTs), 3.64 (2H, t, J = 4.5 Hz, CH N), 3.49 (2H, t, J = 4.8 Hz, OCH ), 3.39 (2H, t, J

2 2 2

13

=

4.5 Hz, OCH ), 2.43 (3H, s, CH ), 1.97 (3H, s, CH ); C NMR (75 MHz, CDCl ): δ 170.2, 144.9, 133.0,

2 3 3 3

1

29.8, 127.7, 69.8, 69.0, 68.3, 39.0, 23.1, 21.5.

1

7

c: 76 %; H NMR (300 MHz, CDCl

3

): δ 7.78 (2H, d, J = 8.4 Hz, Ts), 7.34 (2H, d, J = 8.1 Hz, Ts), 5.73

), 3.87 (2H, t, J = 4.5 Hz, CH N), 3.56-3.52 (4H, m, OCH

OCH ), 2.45 (3H, s, CH ), 2.16 (6H, s, CH ); C NMR (75 MHz, CDCl ): δ 144.8, 133.0, 129.8, 127.9,

(

2H, s, CH), 4.10 (2H, t, J = 4.5 Hz, OCH

2

,

13

2

3

1

27.7, 105.3, 70.7, 69.1, 68.7, 43.2, 21.6, 12.5.

1

7

d: 78 %; H NMR (300 MHz, CDCl ): δ 7.79 (2H, d, J = 8.4 Hz, Ts), 7.35 (2H, d, J = 8.1 Hz, Ts), 4.16 (2h,

3

t, J = 4.5 Hz, CH

Hz, OCH ), 2.45 (3H, s, CH

27.9, 79.3, 70.3, 69.0, 68.3, 28.4, 27.7, 21.6.

2

OTs), 3.63 (2H, t, J = 4.5 Hz, CH

2

N), 3.44 (2H, t, J = 4.8 Hz, OCH

2

), 3.24 (2H, t, J = 4.5

13

2

3

), 1.45 (9H, s, CH ); C NMR (75 MHz, CDCl ): δ 155.9, 144.9, 133.0, 129.8,

3

1

9

ACCEPTED MANUSCRIPT

22

4

.2.9. 1-(Phenylsulfony1)indole (9)

To a solution of indole (8, 7.25 g, 61.8 mmol) in dry THF (75 mL) under N at -78 C, n-BuLi

0

2

(

1.58 M in hexane; 27.71 g, 64.9 mmol) was added dropwise via syringe over 15 min. The cooling bath was

0

removed and the solution was stirred for 1 h while warming to 0 C. The resulting indole anion precipitated

0

as a very fine white solid in a cloudy colorless solution. After the suspension was recooled to -78 C, neat

benzenesulfonyl chloride (11.99 g, 67.9 mmol) was added via syringe over 20 min, keeping the internal

0

temperature below -60 C. The resulting colorless mixture was allowed to warm slowly to rt overnight,

poured into 2% aq NaHCO (500 mL), and extracted with Et O (4 X 200 mL). The combined extracts were

3

2

washed with 2% aq NaHCO , H O, and brine, dried over K CO , filtered and concentrated. The crude

3

2

3

2

product was washed with chilled MeOH, to get pure 9 as colorless crystals. Yield 85 % (lit. 91%): mp

0

22

0

1

7

7.2-72.9 C. (lit. 76.0-76.5 C). H NMR (500 MHz, CDCl

3

); 7.92 (1H, d, J = 8.0 Hz, Ar-H), 7.79 (2H, d,

J = 7.5 Hz, 2-indolyl-H, Ar-H), 7.48 (1H, d, J = 5.5 Hz, Ar-H), 7.47-7.31 (4H, m, Ar-H), 7.24 (1H, t, J =

13

8

.5 Hz, Ar-H), 7.15 (1H, t, J = 8.5 Hz, Ar-H), 6.57 (1H, d, J = 5.5 Hz, 3-indolyl-H); C NMR (125 MHz,

CDCl ): 138.2, 134.8, 133.7, 130.7, 129.2, 126.6, 126.2, 124.6, 123.3, 121.3, 113.4, 109.2; MS (FAB) m/z

3

+

2

57 (M+H ).

23

4

.2.10. N-Benzenesulfonyl-2-methoxalylindole (10)

A suspension of diisopropylamine (4.44g, 43.9 mmol) in THF (90 mL) was placed into a two-

necked round-bottomed flask (1 L) equipped with a dropping funnel and an air condenser. The mixture was

0

cooled to -78 C and the solution containing n-BuLi (1.58 M in hexane; 17.47 g, 43.9 mmol) in THF was

added into the flask slowly through the dropping funnel stirring the reaction mixture. The stirring was

continued for 30 min under same temperature. Then, 9 (10 g, 38.9 mmol), dissolved in 10 mL of dry THF

0

under N and cooled to 0 C, was added slowly into the reaction mixture and stirred again for 1.5 h at -78

2

0

C then for 1 h at 15 C. After the suspension was cooled to -50 C, dimethyl oxalate (18.91 g, 159.5 mmol)

dissolved in 50 mL of THF was added slowly over 30 min. The resulting reaction mixture was allowed to

warm slowly to rt and stirred continuously for further 2 h. Then about 100 mL of ice-cold water was added

slowly into the reaction mixture and stirred again for 10 min. The organic layer was separated and the

aqueous layer was extracted with Et

2

O (3 X 50 mL). The combined organic layers were washed with brain,

and dried over MgSO . The solvent was removed under reduced pressure, and the residue obtained was

4

23

0

washed with chilled MeOH to get pure 10 as colorless crystals. Yield: 61 % (lit. 70%). mp 108.5-109.8 C.

23

0

1

(

lit. 108.9-109.3 C); H NMR (500 MHz, CDCl ): δ 7.99 (1H, d, J = 7.5 Hz, Ar-H), 7.71 (2H, d, J = 7.5

3

1

3

3 3

Hz, 3-indolyl-H, Ar-H), 7.50-7.18 (7H, m, Ar-H), 3.91 (3H, s, CH ); C NMR (125 MHz, CDCl ): δ 177.2,

1

61.4, 138.6, 136.8, 135.8, 134.1, 129.0, 128.7, 128.6, 127.1, 124.8, 123.4, 122.2, 115.3, 53.3; MS (FAB)

+

m/z 344 (M+H ).

4

.2.11. Synthesis of 2-(1H-indol-2-yl)-2-oxoacetic acid (11)

2 3

A reaction flask was charged with 10 (3.5 g, 10.2 mmol) and K CO (14.2 g, 0.103 mol) followed

2

by MeOH (350 mL) and H O (110 mL). The reaction mixture was refluxed for 3 h and cooled to rt slowly.

1

0

ACCEPTED MANUSCRIPT

Then the reaction mixture was acidified using 10% aq HCl and extracted with EtOAc. The organic fraction

was dried (MgSO ) and the solvent was removed under reduced pressure and the residue obtained was

4

0

24

0

1

washed with chilled MeOH to get pure 11. Yield 85 % mp 162.3-163.8 C (lit. 163-164 C). H NMR (500

1

3

MHz, CDCl ): δ 8.10 (1H, s, NH), 7.78-7.17 (5H, m, 3-indolyl-H, Ar-H); C NMR (125.77 MHz,

CD

3

OD): δ 178.6, 165.0, 140.6, 133.5, 128.8, 128.4, 124.5. 122.0, 116.3, 113.6. MS (FAB) m/z 189

+

(

M+H ).

4

.2.12. Synthesis of methyl 2-(1H-indol-2-yl)acetate (13)

A mixture of 11 (1.2 g, 6.34 mmol) and KOH (6.7, 0.12 mol) taken in a reaction flask was

dissolved in 150 mL of EtOH, to this stirred reaction mixture was slowly added 80% N

2

H

4

(5 mL) at rt.

0

Later the reaction mixture was refluxed for 5 h then the reaction mixture was heated to 150 C and was

0

maintained at the same temperature for 45 min. Then, the reaction mixture was cooled to 0 C and acidified

(

pH = 2-3) with 5% aq HCl. The organic layer was separated and the aqueous layer was extracted with Et O

2

3 X 50 mL). The combined organic layers were washed with brain, and dried over MgSO . The solvent

4

was removed under reduced pressure and the crude product 12 was dissolved in a mixture of MeOH (13

mL) and benzene (48 mL). To this solution, 4 mL of 8 mmol of TMSCHN was added and the resulting

2

reaction mixture was stirred for 30 min at rt. Then all the volatile materials were removed under reduced

pressure, later the solid residue was dissolved in CH Cl and washed with aq Na CO . The organic fraction

2

3

was dried (MgSO ) and the solvent removed under reduced pressure and the crude product was purified by

4

0

25

flash column chromatography (EtOAc : Hexane = 3 : 2) to yield 13 (75 %). mp 70.3-71.4 C (lit. 68.0-

0

1

7

1.0 C); H NMR (500 MHz, CDCl

H), 3.82 (2H, s, CH ), 3.74 (3H, s, CH

21.7, 120.1, 119.7, 110.8, 101.8, 52.3, 33.7. MS (FAB) m/z 189 (M+H).

3

): δ 8.79 (1H, br-s, N-H), 7.54 -7.12 (4H, m, Ar-H), 6.34 (1H, s, Ar-

1

3

2

3 3

); C NMR (125.77 MHz, CDCl ): δ 171.0, 136.3, 130.3, 128.1,

1

2

6

4

.2.13. Synthesis of indolylpyridylethene derivatives (14)

A solution of 0.709 g (3.75 mmol) of 13 and 1.73 g (14.2 mmol) of 3-acetylpyridine, and 4.25 g

43.3 mmol) concentrated H SO in 20 mL of dry MeOH was refluxed for 6.5 h in N atmosphere. The clear

(

2

4

2

red solution was poured onto 500 g of ice. It was made alkaline with Na CO and extracted with ether. The

2

3

ether layer was washed and dried (MgSO ), and concentrated to dryness. The crude residue was purified by

4

0

26

0

1

flash column chromatography to yield 14 (65 %); mp 159-161 C (lit. mp 154-157 C). H NMR (500

MHz, CDCl ): δ 9.02 (1H, br, NH), 8.70 (1H, s, Ar-H), 8.54 (d, 1H, J= 4Hz, Ar-H), 7.61 (d, 1H, J = 8.0 Hz,

Ar-H), 7.30 (d, 1H, J = 8.0 Hz, Ar-H), 7.26-7.12 (m, 3H, Ar-H), 7.00 (dd, 1H, Ar-H), 5.83, 5.57 (s, s, 2H,

3

13

ethenyl CH ), 3.76 (s, 2H, CH ), 3.72 (s, 3H, CH ); C NMR (125 MHz, CDCl ): δ 170.8, 148.7, 148.3,

2

3

1

38.9, 136.9, 135.5, 134.6, 128.5, 127.2, 123.1, 122.2, 120.0, 119.7, 117.1, 114.6, 110.8, 52.3, 32.0; MS

(

FAB) m/z 292 (M+H).

4

.2.14. Synthesis of 4-nitrophenyl N-methylcarbamate (37a)

11

ACCEPTED MANUSCRIPT

A flame-dried 100 mL round-bottom flask was charged with 4-nitrophenylchloroformate (36, 500

mg, 2.54 mmol), MeNH .HCl (170 mg, 2.54 mmol) and dioxane (30 mL). The reaction mixture was

2

refluxed for 12 h under argon atmosphere. After the volatile materials were removed under reduced

pressure the crude residue was dissolved in CHCl

3 3

and washed with aq NaHCO and the organic phase was

dried (Na SO ), filtered, and concentrated under reduced pressure. The crude product was purified by flash

2

4

0

1

column chromatography (AcOEt : hexane = 1 : 1) to give 37a (99 %). mp 137-140 C; H NMR (500 MHz,

CDCl ): δ 8.24 (2H, m, Ar-H), 7.30 (2H, m, Ar-H), 5.16 (1H, s, NH), 2.91 (3H, s, CH ).

3

4

.2.15. Synthesis of 4-nitrophenyl N-nitroso-N-methylcarbamate (37b)

A flame-dried 100 mL round-bottom flask was charged with sodium acetate (1.78 g, 21.7 mmol),

4

2 2

-nitrophenyl N-methylcarbamate (37a, 1.78 g, 7.24 mmol), and CH Cl (30 mL). The reaction mixture

0

was cooled to -40 C and was slowly added N O (21.7 mmol) by dissolving in CH Cl . The reaction

2

4

2

mixture was allowed to stir at the same temperature for 1 h. Later, the reaction mixture was warmed to rt

and washed with aq NaHCO , brine and dried over Na SO . After filtration, the filtrate was concentrated

3

2

4

under reduced pressure. The crude product was purified by flash column chromatography (CHCl /ether) to

3

1

give 37b (56 %). H NMR (500 MHz, CDCl

3

): δ 8.37 (2H, d, J = 9.5 Hz, Ar-H), 7.54 (2H, q, J = 9.5 Hz,

13

Ar-H), 3.27 (3H, s, CH

3

); C NMR (125 MHz CDCl ): δ 154.8, 151.9, 145.9, 125.4, 122.2, 28.1; MS

3

+

(

FAB) m/z 226 (M ).

27

4

.2.15. Synthesis of 3-ethoxycarbonyl-1-methylpyridinium chloride (17)

A reaction flask was charged with ethyl nicotinate 15 (10.87 g, 71.91 mmol), methyl iodide (30.62

g, 215.7 mmol), and benzene (40 mL). The reaction mixture was stirred at rt for 24 h. The volatile

materials were removed under reduced pressure to get 16. After drying, 16 (3.00 g, 10.7 mmol) was

dissolved in 30 mL H O, and treated with Amberlite IRA-900 (Chloride form) 15 g with stirring for 5 h.

2

After the water was removed under reduced pressure, the crude residue was purified by ODS column

0

1

chromatography (H O/MeOH = 8:2) to yield 17 (87 %), mp 101.5-102.5 C; H NMR (500 MHz, CDCl ):

2

3

δ 9.53 (1H, s, Ar-H), 9.18 (1H, d, J = 6.0 Hz, Ar-H), 8.96 (1H, d, J = 8.0 Hz, Ar-H), 8.26 (1H, t, J = 6.5 Hz,

1

3

2 3 3 2 3

Ar-H), 4.46 (2H, q, J = 7.0 Hz, CH CH ), 4.44 (3H, s, CH ), 1.38 (3H, t, J= 7.0 Hz, CH CH ); C NMR

+

-

(

125.77 MHz, CD OD): δ 162.7, 149.7, 148.1, 146.3, 131.9, 129.4, 64.2, 14.5. MS (FAB) m/z 166 (M -Cl ).

3

4

.2.16. Synthesis of N-protected 2-(2-aminoethoxy)ethyl-5-metoxycarbonyl-11-methyl-6H-pyrido[4,3-

b]carbazol-2-ium chloride derivatives (ellipticinium-analogues) 19a-c using N-protected [2-(2-

chloroethoxy)ethyl]amines 2, 4a, and 4b; Path A

A flame-dried 100 mL round-bottom flask containing a magnetic stirring bar was charged with 14

0

(

0.17 mmol), 2, and DMF (1 mL). The reaction mixture was stirred at 110 C for 15 h and cooled to rt, and

then NaOMe (0.51 mmol) in MeOH was added and the reaction mixture was stirred for 1 h at rt. Later, 0.2

mmol of 17 was added by dissolving in MeOH and stirring was continued for 5 h at rt. After the volatile

materials were removed under reduced pressure, the crude residue was purified by ODS column

1

2

ACCEPTED MANUSCRIPT

chromatography (H O/MeOH = 7:3) to yield 19a (10 %). When 4a was similarly used instead of 2, a trace

2

of 19b was obtained. However, 4b did not yield 19c but a complex mixture.

4

.2.17. Synthesis of N-protected 2-(2-aminoethoxy)ethyl-5-metoxycarbonyl-11-methyl-6H-pyrido[4,3-

b]carbazol-2-ium chloride derivatives (ellipticinium-analogues) 19a-d using N-protected 2-(2-

aminoethoxy)ethyl tosylate 7a-d; Path B

A flame-dried 100 mL round-bottom flask containing a magnetic stirring bar was charged with 14

0

(

0.17 mmol), 7a, and DMF (1 mL). The reaction mixture was stirred at 110 C for 15 h. The reaction

mixture was cool to ambient temperature, and then NaOMe (0.51 mmol) in MeOH was added and the

reaction mixture was stirred for 1 h at rt. Later, 0.2 mmol of 17 was added by dissolving in MeOH and

stirring was continued for 5 h at rt. After the volatile materials were removed under reduced pressure, the

crude residue (crude 18a) obtained was dissolved in 30 mL of water. To this solution, Amberlite IRA-900

(

chloride form) (15 g) was added and the reaction mixture was stirred for 5 h at rt. After all the volatile

material was removed under reduced pressure, the crude residue was purified by ODS column

chromatography (H O/MeOH = 7:3) to yield 19a (55 %). Although 19b (60 %), and 19d (52%) were

2

obtained from 7b and 7d, the reaction of 7c gave a complex mixture instead of the expected 19c.

0

1

8

7

9a; decomp. 259 C; H NMR (500 MHz, DMSO-d ): δ 9.90 (1H, s, 1-H), 8.92 (1H, d, J = 7.0 Hz, 7-H),

6

.48 (1H, d, J = 7.5 Hz, 4-H), 8.45 (1H, d, J = 7.5 Hz, 3-H), 7.91 (1H, d, J = 7.0 Hz, 10-H), 7.70 (1H, t, J =

+

.0 Hz, 9-H), 7.45 (1H, t, J = 7.0 Hz, 8-H), 7.31-7.30 (4H, m, Pht-H), 4.86 (2H, t, J = 9.0 Hz, N CH

2

), 4.12

(

3H, s, CH

3

), 3.97 (2H, t, J = 8.5 Hz, CH

2

N), 3.69 (2H, t, J = 9.0 Hz, CH

2

O), 3.63 (2H, t, J = 8.5 Hz,

+

-

+

5

OCH

2

), 3.26 (3H, s, CH

3

); MS (FAB) m/z 508 (M -Cl ); HRMS (FAB): Calcd for C30

H

26

N

3

O

: 508.1872,

found : 508.1857.

0

1

9b; decomp. 229 C; H NMR (500 MHz, D O): δ 8.53 (1H, s, 1-H), 8.15 (1H, d, J = 7.0 Hz, 4-H), 7.69

2

(

1H, d, J = 7.2 Hz, 3-H), 6.88 (1H, d, J = 7.5 Hz, 10-H), 6.73 (1H, t, J = 7.5 Hz, 8-H), 6.52 (1H, t, J = 7.5

Hz, 9-H), 6.35 (1H, d, J = 7.5 Hz, 7-H), 6.52 (1H, t, J = 7.5 Hz, 9-H), 6.35 (1H, d, J = 7.8 Hz, 7-H), 4.31

+

(

2H, t, J = 4.5 Hz, N CH

2

), 3.76 (2H, t, J = 4.8 Hz, CH

2

N), 3.57 (3H, s, CH

3

), 3.45 (2H, t, J = 4.5 Hz,

13

CH O), 3.17 (2H, t, J = 4.8 Hz, OCH ), 1.89 (3H, s, CH ), 1.68 (3H, s, CH ); C NMR (125 MHz,

2

3

CD OD): δ 173.2, 167.3, 147.0, 143.7, 141.4, 136.9, 135.0, 130.5, 129.7, 129.3, 125.4, 123.3, 122.7, 122.6,

3

+

-

1

22.3, 113.5, 102.6, 70.6, 70.0, 61.3, 53.1, 39.9, 22.3, 16.1; MS (FAB) m/z 420 (M -Cl ); HRMS (FAB):

+

Calcd for C24

H

26

N

3

O

4

: 420.1923, found : 420.1930.

0

1

9d; decomp. 210 C; H NMR (300 MHz, D O): δ 8.55 (1H, s, 1-H), 8.13 (1H, d, J = 7.0 Hz, 4-H), 7.69

2

(

1H, d, J = 7.2 Hz, 3-H), 6.83 (1H, d, J = 7.5 Hz, 10-H), 6.74 (1H, t, J = 7.5 Hz, 8-H), 6.50 (1H, t, J = 7.5

+

Hz, 9-H), 6.36 (1H, d, J = 7.8 Hz, 7-H), 4.32 (2H, m, N CH ), 3.78 (2H, m, CH N), 3.57 (3H, s, CH ), 3.40

2

3

1

3

(

2H, t, J = 4.5 Hz, CH O), 3.06 (2H, t, J = 7.5 Hz, OCH ), 1.87 (3H, s, CH ), 1.12 (9H, s, CH ); C NMR

2 2 3 3

3

125 MHz, CD OD): δ 167.2, 158.3, 146.9, 146.7, 143.4, 141.2, 136.7, 134.9, 130.5, 129.0, 125.3, 123.3,

1

3

ACCEPTED MANUSCRIPT

+

-

1

22.7, 122.4, 13.4, 102.4, 80.0, 71.2, 70.2, 61.4, 53.2, 41.1, 28.6, 16.1; MS (FAB) m/z 478 (M -Cl ); HRMS

+

(

FAB): Calcd for C H N O : 478.2342, found : 478.2335.

27

32

3

5

4

.2.18. Deprotetion of NH

2

group in ellipticinium-analogues 19a, 19b, and 19d; synthesis of 20

Path A: A flame-dried 100 mL round-bottom flask was charged with 0.56 mmol of 19a (or 19b)

and 9 M HCl/MeOH (15 mL). The reaction mixture was refluxed for 3 days. Purification of the crude

residue by ODS column chromatography (H O) gives 20 in trace from 19a and 50 % from 19b.

2

Path B: A flame-dried 100 mL round-bottom flask was charged with 19d (0.56 mmol) and 12 M

aq HCl (5 mL). The reaction mixture was stirred for 5 h at rt. Purification of the crude residue by ODS

2

column chromatography (H O) gives 85 % of 20.

0

1

2

0; decomp. 235 C; H NMR (300 MHz, D O): δ 8.71 (1H, s, 1-H), 8.31 (1H, d, J = 7.2 Hz, 4-H), 7.86 (1H,

2

d, J = 7.2 Hz, 3-H), 7.05 (1H, d, J = 7.8 Hz, 10-H), 6.88 (1H, t, J = 7.2 Hz, 8-H), 6.68 (1H, t, J = 7.5 Hz, 9-

+

H), 6.54 (1H, d, J = 7.2 Hz, 7-H), 4.45 (2H, t, J = 4.5 Hz, N CH ), 3.81 (2H, t, J = 4.2 Hz, CH N), 3.65 (5H,

2

13

m, CH

2

O, CH

3

), 3.09 (2H, t, J = 4.5 Hz, OCH

2

), 2.08 (3H, s, CH

3

); C NMR (125 MHz, CD

3

OD): δ 167.5,

1

47.4, 147.3, 143.9, 141.7, 137.2, 135.1, 130.7, 129.6, 125.6, 123.5, 123.0, 122.9, 122.6, 113.6, 102.8, 70.7,

+

-

+

6

8.1, 61.1, 53.2, 40.3, 16.4; MS (FAB) m/z 378 (M -Cl ); HRMS (FAB): Calcd for C H N O : 378.1818,

22 24 3 3

found : 378.1830.

4

.2.19. Synthesis of ellipticinium-analogue 38a linked to a methylurea group

A reaction flask was charged with 20 (24 mg, 0.058 mmol), diisopropylethylamine (22 mg, 0.17

mmol), 4-nitrophenyl N-methylcarbamate (37a, 14 mg, 0.07 mmol), and DMF (1 mL). The reaction

0

mixture was stirred at 0 C for 7 h. After the volatile materials were removed under reduced pressure, the

crude residue purified by ODS column chromatography (H O/MeOH = 8:2) to yield 38a (74 %). Decomp.

2

0

1

2

20 C; H NMR (500 MHz, D O): δ 8.55 (1H, s, 1-H), 8.18 (1H, d, J = 7.0 Hz, 4-H), 7.71 (1H, d, J = 7.2

2

Hz, 3-H), 6.91 (1H, d, J = 7.5 Hz, 10-H), 6.78 (1H, t, J = 7.5 Hz, 8-H), 6.56 (1H, t, J = 7.5 Hz, 9-H), 6.39

+

(

1H, d, J = 7.2 Hz, 7-H), 4.32 (2H, m, N CH

2

), 3.78 (2H, m, CH

), 2.33 (3H, s, CH ), 1.91 (3H, s, CH

CD OD): δ 167.3, 161.7, 147.1, 143.6, 141.4, 136.9, 135.0, 125.4, 123.3, 122.8, 122.6, 113.5, 71.6, 70.1,

2 3

N), 3.60 (3H, s, CH ), 3.42 (2H, t, J = 4.5

1

3

Hz, CH

2

O), 3.10 (2H, t, J = 4.8 Hz, OCH

2

3

); C NMR (500 MHz,

3

+

-

6

1.5, 61.3, 53.1, 40.6, 26.8, 20.8, 16.2, 14.4; MS (FAB) m/z 435 (M -Cl ); HRMS (FAB): Calcd for

+

4

C H N O : 435.2032, found : 435.2045.

2

4

27

4

.2.20. Synthesis of ellipticinium-analogues 39a linked to a methylnitrosourea group

A reaction flask was charged with 20 (40 mg, 0.097 mmol), diisopropylethylamine (37 mg, 0.29

mmol), and 4-nitrophenyl-N-methyl-N-nitrosocarbamate (37b, 25 mg, 0.13 mmol) and DMF (1 mL). The

0

reaction mixture was stirred at 0 C for 7 h. After the volatile materials were removed under reduced

1

4

ACCEPTED MANUSCRIPT

pressure, the crude residue was purified by ODS column chromatography (H O/MeOH = 8:2) to yield 39a

2

0

1

(

64 %). Decomp. 215 C; H NMR (500 MHz, D O): δ 8.67 (1H, s, 1-H), 8.24 (1H, d, J = 7.0 Hz, 4-H),

2

7

3

.80 (1H, d, J = 7.2 Hz, 3-H), 7.00 (1H, d, J = 7.5 Hz, 10-H), 6.87 (1H, t, J = 7.5 Hz, 8-H), 6.65 (1H, t, J =

+

.5 Hz, 9-H), 6.53 (1H, d, J = 7.8 Hz, 7-H), 4.41 (2H, m, N CH

2

), 3.85 (2H, m, CH

2

N), 3.70 (3H, s, CH

3

),

.58 (2H, t, J = 4.5 Hz, CH

2

O), 3.40 (2H, t, J = 4.8 Hz, OCH

2

), 2.60 (3H, s, CH ), 2.05 (3H, s, CH

3

). MS

+

-

+

(

FAB) m/z 464 (M - Cl ); HRMS (FAB): Calcd for C H N O : 464.1934, found: 464.1937.

24 26 5 5

4

.2.21. Synthesis of 2-(N-Boc-2-aminoethoxy)ethylellipticinium chloride (34a)

A reaction flask was charged with 21 (0.17 mmol), 7d (0.51 mmol) and DMF (1 mL). The reaction

0

mixture was stirred at 80 C for 12 h. After the volatile materials were removed under reduced pressure, the

crude residue (crude 33a) was treated with 15 g of Amberlite IRA-900 (Chloride form) in water at rt for 5 h.

The solvent was removed from the reaction mixture and the crude residue was purified by ODS column

0

1

chromatography (H O/MeOH = 7:3) to afford 34a (52 %) as a white solid. Decomp. 219 C; H NMR (500

2

MHz, D O): δ 8.25 (1H, s, 1-H), 7.33 (1H, m, 4-H), 6.94 (1H, m, 3-H), 6.63 (2H, m, 8/ 10-H), 6.34-6.30

2

+

(

2H, m, 7/9-H), 4.16 (2H, m, N CH ), 3.72 (2H, m, CH N), 3.40(2H, t, J = 5.0 Hz, CH O), 3.05 (2H, t, J =

2

1

3

5

1

.0 Hz, OCH

2

), 1.72 (3H, s, CH

3

), 1.55 (3H, s, CH

3

3 3

), 1.09 (9H, s, CH ); C NMR (125 MHz, CD OD): δ

58.3, 146.4, 145.0, 143.2, 133.6, 133.5, 131.7, 129.4, 126.6, 124.6, 122.8, 121.6, 120.9, 120.7, 112.0,

+

-

1

10.7, 80.0, 71.1, 70.3, 61.0, 41.1, 28.6, 15.1, 12.0; MS (FAB) m/z 434 (M -Cl ); HRMS (FAB): Calcd for

+

3

C H N O : 434.2444, found: 434.2469.

2

6

32

3

4

.2.22. Synthesis of 2-(2-aminoethoxy)ethylellipticinium chloride 35a

A reaction flask was charged with 34a (0.20 mmol) and 12 M aq HCl (5 mL). The reaction

mixture was stirred at rt for 5 h. After the volatile materials were removed under reduced pressure, the

crude residue was purified by ODS column chromatography (H O/MeOH = 7:3) to afford 35a (86 %) as a

2

0

1

white solid. Decomp. 260 C; H NMR (500 MHz, D O): δ 8.54 (1H, s, 1-H), 7.52 (1H, m, 4-H), 7.24 (1H,

2

+

m, 3-H), 7.03 (1H, m, 10-H), 6.85 (1H, m, 8-H), 6.60-6.56 (2H, m, 7/9-H), 4.33 (2H, m, N CH ), 3.84 (2H,

2

1

3

m, CH N), 3.64 (2H, m, CH O), 3.07 (2H, m, OCH ), 2.07 (3H, s, CH ), 1.80 (3H, s, CH ); C NMR (125

2

3

MHz, CD OD): δ 164.8, 147.6, 144.3, 134.9, 134.6, 132.1, 130.0, 125.5, 122.3, 122.0, 121.4, 112.7, 111.6,

+

-

7

0.9, 68.1, 60.9, 40.3, 36.9, 31.6, 15.5, 12.1; MS (FAB) m/z 334 (M -Cl ); HRMS (FAB): Calcd for

+

C H N O : 334.1919, found: 334.1916.

2

1

24

3

4

.2.23. Synthesis of ellipticinium chloride derivative 38b linked to a methylnitrosourea group

A reaction flask was charged with 35a (40 mg, 0.11 mmol), diisopropylethylamine (42 mg, 0.33

mmol), 4-nitrophenyl-N-methylcarbamate (37a, 25 mg, 0.13 mmol), and DMF (1 mL). The reaction

0

mixture was stirred at 0 C for 7 h under N

2

atmosphere. After the volatile materials were removed under

reduced pressure, the crude residue was purified by ODS column chromatography (H O/MeOH = 8:2) to

2

0

1

yield 45 % of 38b. Decomp. 226 C; H NMR (500 MHz, D O): δ 8.18 (1H, s, 1-H), 7.27 (1H, d, J = 7.0

2

1

5

ACCEPTED MANUSCRIPT

Hz, 4-H), 6.86 (1H, d, J = 6.0 Hz, 3-H), 6.57 (2H, m, 8/10-H), 6.29-6.23 (2H, m, 7/9-H), 4.11 (2H, m,

+

N CH ), 3.70 (2H, m, CH N), 3.39 (2H, t, J = 5.5 Hz, CH O), 3.07 (2H, t, J = 5.0 Hz, OCH ), 2.35 (3H, s,

2

1

3

NHCH ), 1.66 (3H, s, CH ), 1.50 (3H, s, CH ); C NMR (125 MHz, CD OD): δ 161.7, 146.9, 145.6, 143.7,

3

1

2

3

34.1, 134.0, 131.8, 129.6, 127.3, 125.0, 123.3, 121.9, 121.4, 121.0, 112.3, 111.1, 71.6, 70.3, 61.1, 40.7,

+

-

+

27 4 2

6.9, 15.3, 12.0; MS (FAB) m/z 391 (M -Cl ); HRMS (FAB): Calcd for C23H N O : 391.2134, found:

91.2131.

4

.2.24. Synthesis of ellipticinium chloride derivative 39b linked to a methylnitrosourea group

A reaction flask was charged with 35a (50 mg, 0.14 mmol), diisopropylethylamine (52 mg, 0.4

mmol), 4-nitrophenyl-N-methyl-N-nitrosocarbamate (37b, 36 mg, 0.16 mmol), and DMF (1 mL). The

0

reaction mixture was stirred at 0 C for 7 h. After the volatile materials were removed under reduced

pressure, the crude residue was purified by ODS column chromatography (H O/MeOH = 8:2) to yield 43 %

2

0

1

of 39b. Decomp. 220 C; H NMR (500 MHz, D O): δ 8.66 (1H, s, 1-H), 7.65 (1H, d, J = 7.0 Hz, 4-H),

2

7

.45 (1H, d, J = 7.0 Hz, 3-H), 7.38 (1H, m, 10-H), 7.11 (1H, m, 8-H), 6.85 (2H, d, J = 6.0 Hz, 7/9-H), 4.39

+

(

2H, m, N CH ), 3.83 (2H, m, CH N), 3.54 (2H, t, J = 4.5 Hz, CH O), 3.28 (2H, t, J = 4.5 Hz, OCH ), 2.29

2

+

-

3H, s, CH

3

), 2.12 (3H, s, CH

3

), 2.08 (3H, s, CH ); MS (FAB) m/z 421 (M -Cl ); HRMS (FAB): Calcd for

+

3

C

23

H

26

N

5

O

: 420.2036, found: 420.2045.

4

.2.25. Synthesis of 9-formyl-ellipticine (22)

A reaction flask was charged with 21 (30.0 mg, 0.0122 mmol), hexamethylenetetramine (200 mg,

1

.42 mmol), and TFA (5 mL). The reaction mixture was refluxed for 20 min and cooled to rt. The resulted

reaction mixture was basified with Na

brine and dried over Na SO , and then filtered and evaporated. The crude residue was purified by SiO

column chromatography (CHCl /MeOH = 9:1) to yield 92% of 22 (lit. 97%). Decomp. 360 C (lit

2 3 3

CO , and extracted with CHCl . The organic layer was washed with

2

4

2

1

8

0

18

3

0

1

decomp. > 350 C); H NMR (500 MHz, CD CD): δ 10.6 (1H, s, CHO), 9.66 (1H, s, 1-H), 8.89 (1H, s, 10-

3

H), 8.40 (1H, d, J = 8.4 Hz, 3-H), 8.06 (2H, dd, N-H, 8-H), 7.65 (1H, d, J = 8.4 Hz, 7-H), 2.85 (3H, s,

1

3

CH ); C NMR (125.77 MHz, DMSO-d6): δ 191.9, 149.8, 146.7, 141.0, 140.7, 132.9, 128.8, 128.5, 128.1,

3

+

1

27.2, 123.2, 122.8, 116.0, 115.7, 111.0, 109.4, 14.3, 11.9. MS (FAB) m/z 275 (M+H ).

4

.2.26. Synthesis of 9-hydroxyellipticine (23)

A reaction flask was charged with 22 (100 mg, 0.36 mmol), MeOH (25 mL), 31 % H O (0.2 mL),

2

conc. H SO (2 drops). The reaction mixture was refluxed for 4 h and cooled to rt. The reaction mixture

2

4

was basified with sat. Na CO aq, and extracted with CHCl . The organic layer was washed with brine,

2

3

dried over Na

2

SO

4

, filtered and evaporated. The crude residue was purified by SiO

2

column

18

0

18

chromatography (CHCl

3

/MeOH = 9:1) to yield 23 in 55 % yield (lit 91 %). Decomp. 355 C (lit decomp.

0

1

>

350 C); H NMR (500 MHz, CDMSO-d ): δ 11.04 (1H, s, NH), 9.64 (1H, s, 1-H), 9.09, (1H, s, OH),

6

1

6

ACCEPTED MANUSCRIPT

8

.37 (1H, d, J = 6.0 Hz, 3-H), 7.86 (1H, d, J = 6.0 Hz, 4-H), 7.75 (1H, s, 10-H), 7.36 (1H, d, J = 8.5 Hz, 7-

+

H), 7.00 (1H, d, J = 8.5 Hz, 8-H), 3.19 (3H, S, 11-CH ), 2.73 (3H, s, 5-CH ); MS (FAB) m/z 263 (M+H ).

3

4

.2.27. Synthesis of 2-(N-Boc-2-aminoethoxy)ethyl-9-hydroxyellipticinium chloride (34b)

A reaction flask was charged with 23 (0.17 mmol), 7d (0.51 mmol) and DMF (2 mL). The reaction

0

mixture was stirred at 80 C for 12 h. After the volatile materials were removed under reduced pressure, the

crude residue was treated with 15 g of Amberlite IRA-900 (Chloride form) in water at rt for 5 h. The

solvent was removed from the reaction mixture and the crude residue (crude 33b) was purified by ODS

0

1

column chromatography (H O/MeOH = 7:3) to afford 34b (52 %) as a white solid. Decomp. 250 C; H

2

NMR (500 MHz, D

2

O): δ 9.23 (1H, s, 1-H), 7.94 (1H, m, 4-H), 7.75 (1H, m, 3-H), 7.00-6.96 (2H, m, 8/10-

+

H), 6.73 (1H, m, 7-H), 4.62 (2H, m, N CH

2

), 3.87 (2H, m, CH

2

N), 3.40 (2H, m, CH

2

O), 3.09 (2H, m,

13

OCH ), 2.65 (3H, s, CH ), 2.32 (3H, s, CH ), 1.20 (9H, s, CH ); C NMR (125 MHz, CD OD): δ 158.3,

2

3

1

7

4

53.0, 146.8, 146.1, 137.4, 134.2, 133.7, 131.6, 127.3, 124.0, 121.0, 120.8, 118.2, 112.7, 110.8, 110.4, 80.0,

+

-

+

1.1, 70.3, 61.0, 41.1, 28.6, 15.2, 12.0; MS (FAB) m/z 450 (M -Cl ); HRMS (FAB): Calcd for C H N O :

26

31

4

3

49.2315, found: 434.2317.

4

.2.28. Synthesis of 2-(2-aminoethoxy)ethyl-9-hydroxyellipticinium chloride (35b)

A reaction flask was charged with 34b (0.22 mmol) and 12 M HCl (5 mL). The reaction mixture

0

was stirred at 80 C for 12 h. After the volatile materials were removed under reduced pressure, the crude

residue was purified by ODS column chromatography (H O/MeOH = 9:1) to afford 35b (85 %). Decomp.

2

0

1

2

95 C; H NMR (500 MHz, D O): δ 8.29 (1H, s, 1-H), 7.35 (1H, m, 4-H), 6.83 (1H, m, 3-H), 5.90 (1H, m,

2

+

1

0-H), 5.76 (1H, m, 8-H), 5.50 (1H, m, 7-H), 4.21 (2H, m, N CH

2

), 3.75 (2H, m, CH

2

N), 3.61 (2H, m,

13

CH

2

O), 3.06 (2H, m, OCH

2

), 1.48 (3H, s, CH

3

), 1.36 (3H, s, CH

3

); C NMR (125 MHz, CD OD): δ 152.0,

3

1

46.8, 144.7, 136.0, 133.4, 132.0, 131.1, 125.8, 122.7, 119.6, 119.4, 117.3, 112.0, 109.7, 109.6, 68.9, 66.4,

+

-

+

5

8.6, 38.2, 14.9, 11.9; MS (FAB) m/z 350 (M -Cl ); HRMS (FAB): Calcd for C H N O : 350.1869,

26 31 4 3

found: 350.1866.

4

.2.29. Synthesis of 9-hydroxyellipticinium chloride derivative 38c linked to a methylurea group

A reaction flask was charged with 35b (50 mg, 0.13 mmol), diisopropylethylamine (50 mg, 0.39

mmol), 4-nitrophenyl-N-methylcarbamate (37a, 31 mg, 0.16 mmol), and DMF (1 mL). The reaction

0

mixture was stirred at 0 C for 7 h under N atmosphere. After the volatile materials were removed under

2

reduced pressure, the crude residue was purified by ODS column chromatography (H O/MeOH = 8:2) to

2

0

1

yield 38c (43 %). Decomp. 255 C; H NMR (500 MHz, D O): δ 8.16 (1H, s, 1-H), 7.25 (1H, m, 4-H), 6.74

2

+

(

1H, m, 3-H), 5.86 (1H, m, 10-H), 5.74 (1H, m, 8-H), 5.46 (1H, m, 7-H), 4.12 (2H, m, N CH

2

), 3.69 (2H, m,

), 1.33 (3H, s,

CH ); C NMR (125 MHz, CD OD): δ 161.7, 153.5, 147.4, 138.0, 135.0, 134.1, 131.7, 124.6, 121.4, 121.0,

2 2 2 3 3

CH N), 3.37 (2H, m, CH O), 3.08 (2H, m, OCH ), 2.44 (3H, s, NHCH ), 1.39 (3H, s, CH

1

3

1

7

ACCEPTED MANUSCRIPT

+

-

1

18.6, 113.0, 111.2, 110.9, 71.6, 70.3, 61.0, 40.6, 26.8, 23.6, 15.2, 14.4, 11.9; MS (FAB) m/z 408 (M -Cl );

+

3

HRMS (FAB): Calcd for C H N O : 407.2083, found: 407.2075.

23

27

4

.2.30. Synthesis of 9-hydroxyellipticinium chloride derivative 39c linked to a methylnitrosourea group

A reaction flask was charged with 35b (50 mg, 0.13 mmol), diisopropylethylamine (50 mg, 0.39

mmol), 4-nitrophenyl-N-methyl-N-nitrosocarbamate (37b, 38 mg, 0.17 mmol), and DMF (1 mL). The

0

reaction mixture was stirred at 0 C for 7 h. After the volatile materials were removed under reduced

pressure, the crude residue was purified by ODS column chromatography (H O/MeOH = 8:2) to yield 39c

2

0

1

(

39 %). Decomp. 244 C; H NMR (500 MHz, D O): δ 8.63 (1H, s, 1-H), 7.62 (1H, m, 4-H), 7.35 (1H, m,

2

+

3

-H), 6.54 (1H, m, 10-H), 6.42 (2H, m, 7/8-H), 4.40 (2H, m, N CH

2

), 3.84 (2H, m, CH N), 3.54 (2H, m,

CH

2

O), 3.29 (2H, m, OCH

2

), 2.21 (3H, s, NHCH

3

) 2.07 (3H, s, CH

3

), 1.94 (3H, s, CH ); MS (FAB) m/z 437

3

+

-

+

(

M -Cl ); HRMS (FAB): Calcd for C H N O : 436.1985, found: 436.1966.

2

3

26

5

3

4

.2.31. Synthesis of 1,4,9-trimethylcarbazole-3-carbaldehyde (25)

KOH (3.6 g, 64.1 mmol), 24 (3.6g, 16.1 mmol), and DMSO (20 mL) were placed in a 100 mL

reaction flask and the mixture was stirred at rt for 30 min. To this solution, methyl iodide (4.6g, 32.4 mmol)

was added and stirring was continued for 10 min. The reaction mixture was diluted with water (100 ml) and

extracted with EtOAc. The residue after removal of the solvent was purified by column chromatography to

0

28

0

1

yield 25 (88 %). Decomp. 158-160 C (lit. 157-161 C); H NMR (500 MHz, CDCl ): δ 10.39 (1H, s,

3

CHO), 8.23 (1H, d, J = 7.5 Hz, 5-H), 7.64 (1H, s, 2-H), 7.51 (1H, d, J = 7.5 Hz, 8-H), 7.41 (1H, t, J = 7.5

1

3

Hz, 7-H), 7.30 (1H, t, J = 7.5 Hz, 6-H), 4.07 (3H, s, N-CH ), 3.10 (3H, s, 1-CH ), 2.81 (3H, s, 4-CH );

C

3

NMR (125 MHz, CDCl

3

): δ 191.2, 142.6, 142.0, 136.4, 131.2, 126.0, 125.6, 123.6, 123.0, 122.7, 120.1,

+

1

17.9, 108.9, 32.1, 20.3, 14.9; MS (FAB) m/z 237 (M+H ).

4

.2.32. Synthesis of 26

To a 50 mL round-bottom flask equipped with a magnetic stir bar and condenser, was added 25

0.05g, 0.21 mmol), aminoacetaldehyde diethyl acetal, (0.033g, 0.246 mmol), and benzene (20 mL). This

(

yellow solution was stirred and heated under reflux for 2 h. After completion of the reaction as indicated

by TLC, the solvent was removed under reduced pressure. The residue obtained was evaporated to dryness

to yield brown oil. This brown oil was dissolved in CH OH and treated with NaBH (0.08g, 2.23 mmol)

3

4

and the reaction mixture was stirred at rt for 2 h. Then, the solvent was removed under reduced pressure

and the obtained crude residue was dissolved in 3 mL of dry pyridine and a solution of 2-

nitrobenzenesulfonyl chloride (0.15g, 0.669 mmol) dissolved in 10 mL of dry CH CN was added while the

3

solution was stirring. The resulting solution was allowed to stir at rt for 8 h. The solvent was removed under

2 2

reduced pressure after completion of the reaction, and the residue obtained was extracted with CH Cl and

washed with water, dried over MgSO , filtered, and evaporated to yield orange oil, which is then dissolved

4

1

in chilled CH OH and refrigerated overnight to get yellow solid 26 (54%). H NMR (CDCl , 500 MHz): δ

3

1

8

ACCEPTED MANUSCRIPT

8

3

.17-6.99 (9H, m, Ar-H), 4.86 (2H, s, ArCH N), 4.55 (1H, t, J = 5.5 Hz, CH(OEt) ), 4.05 (3H, s, N-CH ),

2

3

.61 (2H, d, J = 5.5 Hz, CH CH(OEt) ), 3.40 (4H, q, J= 7.0 Hz, O(CH CH ) ), 2.72 (3H, s, 1-CH ), 2.69

2

3 2

3

13

(

3H, s, 4-CH ), 1.14 (6H, t, J=7.0 Hz, O(CH CH ) ); C NMR (CDCl , 125 MHz): δ 147.8, 141.8, 139.3,

3 2 3 2 3

1

34.3, 132.8, 131.1, 131.0, 130.8, 130.6, 124.9, 123.8, 123.3, 123.2, 122.7, 122.4, 118.9, 117.4, 108.3,

+

02.1, 63.3, 50.3, 49.3, 32.2, 20.2, 16.0, 15.2; MS (FAB) m/z 540 (M+H ).

4

.2.33. Synthesis of 6-methylellipticine (27)

To a 100 mL round-bottom flask equipped with a magnetic stirring bar and condenser, was added

6 (100 mg, 0.19 mmol), 6 M HCl/dioxane (1:4) 20 mL. This slurry was stirred and heated to reflux with

2

an oil bath for 3 h. After completion of the reaction as indicated by TLC, pH of the reaction mixture was

adjusted to 8 with Na CO and extracted with 3 X 50 mL of CH Cl . The combined extracts were dried

over MgSO , filtered, and evaporated to yield brownish yellow oil. The residue obtained was purified by

2

3

2

4

0

29

0

1

silica gel chromatography to yield 27 (86 %). mp 206.4-208.1 C (lit. 207-208 C); H NMR (CDCl , 500

3

MHz): δ 9.56 (1H, s, 1-H), 8.45 (1H, d, J = 6.0 Hz, Ar-H), 8.21 (1H, d, J = 8.0 Hz, Ar-H), 7.89 (1H, d, J =

6

.0 Hz, Ar-H), 7.51 (1H, t, J = 8.0 Hz, Ar-H), 7.25-731 (2H, m, Ar-H), 3.93 (3H, s, N-CH ), 3.02 (3H, s,

3

13

1

3 3

1-CH ), 2.86 (3H, s, 5-CH ); C NMR (DMSO, 500 MHz): δ 149.5, 144.9, 141.6, 140.7, 134.2, 128.6,

1

27.0, 124.5, 123.7, 123.3, 122.5, 119.5, 115.8, 108.4, 108.3, 33.7, 14.4, 13.7. MS (FAB) m/z 261 (M+H).

4

.2.34. Synthesis of 9-formyl-6-methylellipticine (28)

To a round-bottom flask equipped with a magnetic stir bar and condenser, was added 27 (30.0 mg,

.11 mmol), hexamethylenetetramine (200 mg, 1.42 mmol), TFA (5 mL). This slurry was stirred and heated

0

to reflux with an oil bath for 20 min. After completion of the reaction as indicated by TLC, the reaction

mixture was basified with Na CO and extracted with 3 X 50 mL of CH Cl . The combined extracts were

dried over MgSO , filtered, and evaporated to dryness. The residue obtained was purified by silica gel

2

3

2

4

18

0

18

0

1

chromatography to yield 28 (84 %) (lit. 96%). mp 222-223 C (lit. 223-226 C); H NMR (CDCl , 500

3

MHz): δ 10.0 (1H, s, CHO), 8.54-8.50 (2H, m, 10/1-H), 7.99 (1H, d, J = 8.5 Hz, 3-H), 7.99 (1H, d, J = 8.5

Hz, 3-H), 7.79 (1H, m, 8-H), 7.20 (1H, d, J = 8.5 Hz, 7-H), 3.96 (3H, s, N-CH ), 3.00 (3H, s, 11-CH ), 2.86

3

13

(

3H, s, 5-CH

3

); C NMR (CDCl

3

, 125 MHz): δ 191.2, 149.6, 148.5, 141.5, 134.6, 129.4, 129.3, 128.8,

1

25.7, 123.6, 123.5, 122.8, 115.9, 109.6, 108.2, 34.0, 14.5, 13.6. MS (FAB) m/z 288 (M+H).

4

.2.35. Synthesis of 9-hydroxy-6-methylellipticine (29)

To a reaction flask equipped with a magnetic stir bar and condenser, was added 28 (100.0 mg, 0.35

mmol), CH OH (25 mL), conc. H SO (2 drops), and 0.2 mL of 31 % H O . This slurry was stirred and

3

2

4

2

heated to reflux with an oil bath for 4 h. The reaction mixture was basified with Na

X 150 mL of CH Cl . The combined extracts were dried over MgSO , filtered, and evaporated to dryness.

The residue obtained was purified by silica gel chromatography to get 29 (53 %) (lit. 93 %). Decomp: 345

2 3

CO and extracted with

3

2

4

18

0

9

0

1

C (it. >300 C); H NMR (CDCl , 500 MHz): δ 9.62 (1H, s, 1-H), 9.18 (1H, s, OH), 8.39 (1H, m, 3-H),

3

1

9

ACCEPTED MANUSCRIPT

7

.94 (1H, d, J = 6.0 Hz, 4-H), 7.76 (1H, s, 10-H), 7.42 (1H, d, J = 8.5 Hz, 7-H), 7.06 (1H, d, J = 8.5 Hz, 8-

13

H), 4.05 (3H, s, N-CH ), 3.14 (3H, s, 11-CH ), 2.97 (3H, s, 5-CH ); C NMR (CDCl , 125 MHz): δ 151.0,

3

1

49.4, 141.7, 140.5, 138.5, 133.4, 128.2, 123.9, 123.1, 121.7, 115.8, 115.6, 109.5, 109.4, 108.5, 33.7, 14.1,

3.3. MS (FAB) m/z 277 (M+H).

4

.2.36. Synthesis of 2-(N-Boc-2-aminoethoxy)ethyl-9-hydroxy-6-methylellipticinium chloride (34c)

A reaction flask was charged with 29 (0.17 mmol), 7d (0.51 mmol) and DMF (2 mL). The reaction

0

mixture was stirred at 80 C for 12 h. After the volatile materials were removed under reduced pressure, the

crude residue was treated with 15 g of Amberlite IRA-900 (Chloride form) in water at rt for 5 h. The

solvent was removed from the reaction mixture and the residue (crude 33c) was purified by ODS column

0

1

2

chromatography (H O/MeOH = 7:3) to afford 34c (52 %) as a white solid. mp 195 C; H NMR (500 MHz,

D O): δ 8.45 (1H, s, 1-H), 7.64 (1H, m, 4-H), 7.33 (1H, m, 3-H), 6.04 (2H, m, 8/10-H), 5.77 (1H, m, 7-H),

2

+

4

1

4

.31 (2H, m, N CH ), 3.37 (2H, m, CH N), 3.00 (2H, m, CH O), 2.67 (2H, m, OCH ), 1.70 (3H, s, CH ),

2

3

1

3

.50 (3H, s, CH ), 1.15 (9H, s, -C(CH ) ); C NMR (125 MHz, CD OD): δ 158.4, 153.4, 146.5, 145.5,

3

3 3

3

39.1, 134.9, 134.0, 132.0, 127.5, 123.1, 121.1, 121.0, 117.9, 111.1, 110.8, 110.3, 80.0, 71.2, 70.3, 61.0,

+

-

+

34 3 4

1.1, 34.0, 28.7, 15.2, 14.0; MS (FAB) m/z 464 (M -Cl ); HRMS (FAB): Calcd for C27H N O : 464.2549,

found: 464.2561.

4

.2.37. Synthesis of 2-(2-aminoethoxy)ethyl-9-hydroxy-6-methylellipticinium chloride (35c)

A reaction flask was charged with 34c (0.25 mmol), and 12 M HCl (5 mL). The reaction mixture

was stirred at rt for 5 h. After the volatile materials were removed under reduced pressure, the crude residue

0

was purified by ODS column chromatography (H

2

O/MeOH = 9:1) to afford 35c (86 %). Decomp. 205 C;

1

H NMR (500 MHz, D

2

O): δ 8.54 (1H, s, 1-H), 7.69 (1H, m, 4-H), 7.40 (1H, m, 3-H), 6.13 (1H, m, 10-H),

+

6

3

.01 (1H, m, 8-H), 5.79 (1H, m, 7-H), 4.37 (2H, m, N CH ), 3.82 (2H, m, CH N), 3.62 (2H, m, CH O),

2

13

.06 (2H, m, OCH ), 1.79 (3H, s, N-CH ), 1.79 (3H, s, 11-CH ), 1.57 (3H, s, 5-CH ); C NMR (125 MHz,

2

3

CD OD): δ 153.8, 147.1, 146.5, 139.9, 135.4, 134.7, 132.1, 128.4, 123.7, 121.6, 121.3, 118.3, 111.7, 111.3,

3

+

-

1

10.9, 70.8, 68.1, 60.7, 40.3, 34.3, 15.6, 14.2; MS (FAB) m/z 365 (M -Cl ); HRMS (FAB): Calcd for

+

22 26 3 2

C H N O : 364.2025, found: 364.2021.

4

.2.38. Synthesis of 9-hydroxy-6-methylellipticine-derivative 38d linked to a methylurea group

A reaction flask was charged with 35c (50 mg, 0.13 mmol), diisopropylethylamine (48 mg, 0.37

mmol) 4-nitrophenyl-N-methylcarbamate (37a, 29 mg, 0.15 mmol) and DMF (1 mL). The reaction mixture

0

was stirred at 0 C for 7 h under N atmosphere. After the volatile materials were removed under reduced

2

pressure, the crude residue purified by ODS column chromatography (H O/MeOH = 8:2) to yield 38d

0

1

(

47 %). Decomp. 203 C; H NMR (500 MHz, D

2

O): δ 8.36 (1H, s, 1-H), 7.58 (1H, m, 4-H), 7.26 (1H, m,

+

3

-H), 5.99 (2H, m, 8/10-H), 5.62 (1H, m, 7-H), 4.26 (2H, m, N CH ), 3.72 (2H, m, CH N), 3.35 (2H, m,

2

CH O), 3.04 (2H, m, OCH ), 2.62 (3H, s, N-CH ), 2.42 (3H, s, NHCH ), 1.64 (3H, s, 11-CH ), 1.35 (3H, s,

2

3

2

0

ACCEPTED MANUSCRIPT

1

3

5

1

4

-CH ); C NMR (125 MHz, CD OD): δ 161.8, 153.4, 146.5, 145.4, 139.0, 134.8, 133.9, 132.1, 127.4,

3 3

23.0, 121.0, 117.9, 111.0, 110.8, 110.3, 71.7, 70.3, 61.0, 40.8, 36.9, 33.9, 27.0, 15.2, 13.9; MS (FAB) m/z

+

-

+

3

22 (M - Cl ); HRMS (FAB): Calcd for C H N O : 421.2240, found: 421.2220.

2

4

29

4

.2.39. Synthesis of 9-hydroxy-6-methylellipticine-derivative 39d linked to a methylnitrosourea group

A reaction flask was charged with 35c (50 mg, 0.13 mmol), diisopropylethylamine (48 mg, 0.38

mmol), 4-nitrophenyl-N-methyl-N-nitrosocarbamate (37b, 37 mg, 0.17 mmol) and DMF (1 mL). The

0

reaction mixture was stirred at 0 C for 7 h. After the volatile materials were removed under reduced

pressure, the crude residue was purified by ODS column chromatography (H O/MeOH = 8:2) to yield 39d

2

0

1

(

40 %). Decomp. 200 C; H NMR (500 MHz, D

2

O): δ 8.52 (1H, s, 1-H), 7.68 (1H, m, 4-H), 7.38 (1H, m,

+

3

-H), 6.16 (2H, m, 8/10-H), 5.94 (1H, m, 7-H), 4.37 (2H, m, N CH

2

), 3.82 (2H, m, CH

2

N), 3.54 (2H, m,

CH O), 3.36 (2H, m, OCH ), 2.85 (3H, s, N-CH ), 2.64 (3H, s, NHCH ), 1.85 (3H, s, 11-CH ), 1.60 (3H, s,

2

3

+

-

+

5

-CH ); MS (FAB) m/z 451 (M -Cl ); HRMS (FAB): Calcd for C H N O : 450.2141, found: 450.2129.

3 24 28 5 4

4

.2.40. Synthesis of tert-butyl 5-hydroxypentylcarbamate (31) and the corresponding O-tosylate 32

-Amino-1-pentanol (30, 2 g, 19 mmol), Boc O (4.5 g, 20 mmol), Et N (2.1 g, 21 mmol), DMAP

0.23 g, 1.9 mmol) and CHCl (20 mL) were placed in a 100 mL reaction flask. The reaction mixture was

5

2

3

(

3

allowed to stir for 3 h at rt. After the volatile materials were removed under reduced pressure, the resulting

reside was purified by flash column chromatography (CHCl ) to yield 31.

3

To a flame-dried 100 mL round-bottom flask containing a magnetic stirring bar were charged 31

0

(

30 mmol), Et N (30 mmol), DMAP (3 mmol), and CHCl (30 mL). The solution was cooled to 0 C, and

3

TsCl (30 mmol) dissolved in CHCl

3

was added to the solution over 10 min. The reaction mixture was

stirred at rt for 3 h. After all the volatile materials were removed under reduced pressure, the crude residue

was purified by flash column chromatography (CHCl ) to get 32.

3

1

3

1: 83%; H NMR (300 MHz, CDCl ): δ 3.63 (2H, m, CH N), 3.11 (2H, m, CH OH), 1.61-1.39 (15H, m, 3

3

2

X CH , -C(CH ) ).

2

3 3

1

3

2: 75 %; H NMR (300 MHz, CDCl

3

): δ 7.75 (2H, d, J = 8.5 Hz, Ts), 7.32 (2H, d, J = 8.5 Hz, Ts), 3.98

(

2H, t, J = 6.0 Hz, CH OTs), 3.02 (2H, m, CH N), 1.63 (2H, m, CH CH CH ), 2.45 (3H, s, Ar-CH ), 1.40

2

3

13

(

11H, m, CH CH CH -C(CH ) ), 1.30 (2H, s, CH CH CH ); C NMR (75 MHz, CDCl ): δ 155.9, 144.6,

2 2 2, 3 3 2 2 2 3

1

33.1, 129.8, 127.8, 70.3, 40.2, 29.4, 28.4, 28.3, 22.6, 21.6, 14.1.

4

.2.41. Synthesis of 2-(N-Boc-5-aminopentyl)-9-hydroxy-6-methylellipticinium chloride (34d)

A reaction flask was charged with 29 (0.17 mmol), 32 (0.51 mmol) and DMF (2 mL). The reaction

0

mixture was stirred at 80 C for 12 h. After the volatile materials were removed under reduced pressure, the

crude residue (crude 33d) was treated with 15 g of Amberlite IRA-900 (Chloride form) in water at rt for 5 h.

2

1

ACCEPTED MANUSCRIPT

The solvent was removed from the reaction mixture and the residue was purified by ODS column

0

1

chromatography (H O/MeOH = 7:3) to afford 34d (50 %) as a white solid. Decomp. 190 C; H NMR (500

2

MHz, D O): δ 8.46 (1H, s, 1-H), 7.62 (1H, m, 4-H), 7.47 (1H, m, 3-H), 6.14 (1H, m, 10-H), 6.05 (1H, m, 8-

2

+

H), 5.87 (1H, m, 7-H), 4.09 (2H, m, N CH

2

), 2.96 (3H, s, NCH

), 1.35 (2H, m, CH

CH CH CH , C(CH ) ); 13C NMR (125 MHz, CD OD): δ 158.6, 153.9, 146.7, 146.5, 140.1, 135.4, 134.7,

3

), 2.89 (2H, m, CH

2

N), 1.98 (3H, s, 11-

CH

3

), 1.74 (2H, m, CH

2

CH

2

CH

2

), 1.61 (3H, s, 5-CH

3

2

CH CH

2

), 1.19 (11H, m,

2

3 3

3

1

31.5, 128.7, 123.9, 121.9, 121.8, 118.4, 111.9, 111.4, 111.0, 79.9, 61.4, 40.8, 34.3, 32.0, 30.5, 28.7, 24.5,

+

-

+

3

5.4, 14.2; MS (FAB) m/z 462 (M -Cl ); HRMS (FAB): Calcd for C H N O : 462.2757, found: 462.2734.

2

8

36

3

4

.2.42. Synthesis of 2-(5-aminopentyl)-9-hydroxy-6-methylellipticinium chloride (35d)

A reaction flask was charged with 34d (0.25 mmol) and 12 M HCl (5 mL). The reaction mixture

was stirred at rt for 5 h. After the volatile materials were removed under reduced pressure, the crude residue

0

was purified by ODS column chromatography (H O/MeOH = 9:1) to afford 35d (86 %). Decomp. 278 C;

2

1

H NMR (500 MHz, D O): δ 8.43 (1H, s, 1-H), 7.59 (1H, m, 4-H), 7.39 (1H, m, 3-H), 6.15 (1H, m, 10-H),

2

+

6

.06 (1H, m, 8-H), 5.80 (1H, m, 7-H), 4.07 (2H, m, N CH ), 2.92 (5H, m, CH N, N-CH ), 1.92 (3H, s, 11-

2

3

CH

3

2

), 1.74 (2H, m, CH

CH CH

2

CH

2

CH

2

), 1.64 (2H, m, CH

2

CH

2

CH

2

3

), 1.55 (3H, s, 5-CH ), 1.36 (2H, m,

+

-

+

CH

2

); MS (FAB) m/z 363 (M -Cl ); HRMS (FAB): Calcd for C23

H

28

N

3

O : 362.2232, found:

3

62.2256.

4

.2.43. Synthesis of 9-hydroxy-6-methylellipticine-derivative 38e linked to a methylurea group

A reaction flask was charged with 35d (50 mg, 0.1 mmol), diisopropylethylamine (39 mg, 0.3

mmol), 4-nitrophenyl-N-methylcarbamate (37a, 24 mg, 0.12 mmol) and DMSO (1 mL). The reaction

mixture was stirred at rt for 3 h under N atmosphere. After the volatile materials were removed under

reduced pressure, the crude residue was purified by ODS column chromatography (H O/MeOH = 8:2) to

2

0

1

yield 38e (63 %). Decomp. 213 C; H NMR (500 MHz, D O): δ 8.34 (1H, s, 1-H), 7.57 (1H, m, 4-H), 7.38

2

+

(

1H, m, 3-H), 6.04 (1H, m, 10-H), 5.97 (1H, m, 8-H), 5.71 (1H, m, 7-H), 4.02 (2H, m, N CH ), 2.95 (2H, m,

2

CH N), 2.85 (3H, s, N-CH ), 2.53 (3H, s, NHCH ), 1.88 (3H, s, 11-CH ), 1.71 (2H, m, CH CH CH ), 1.44-

2

3

2

1

4

.37 (5H, m, CH

2

CH

2

CH

2

, 5-CH

3

2 2 2 3

), 1.18 (2H, m, CH CH CH ); 13C NMR (125 MHz, CD OD): δ 161.9,

53.5, 146.2, 146.0, 139.5, 135.0, 134.2, 131.5, 128.0, 123.4, 121.6, 121.5, 118.1, 111.5, 111.0, 110.6, 61.3,

+

-

0.5, 34.1, 32.0, 30.7, 27.0,24.4, 15.3, 14.1; MS (FAB) m/z 420 (M -Cl ); HRMS (FAB): Calcd for

+

2

C H N O : 419.2447, found: 419.2420.

2

5

31

4

.2.44. Synthesis of 9-hydroxy-6-methylellipticine-derivative 39e linked to a methylnitrosourea group

A reaction flask was charged with 35d (50 mg, 0.13 mmol), diisopropylethylamine (48 mg, 0.38

mmol) 4-nitrophenyl-N-methyl-N-nitrosocarbamate (37b, 37 mg, 0.17 mmol) and DMF (1 mL). The

0

reaction mixture was stirred at 0 C for 7 h. After the volatile materials were removed under reduced

pressure, the crude residue was purified by ODS column chromatography (H O/MeOH = 8:2) to yield 39e

2

ACCEPTED MANUSCRIPT

o

1

(

20 %). Decomp. 241 C; H NMR (500 MHz, D O): δ 8.46 (1H, s, 1-H), 7.62 (1H, m, 4-H), 7.45 (1H, m,

2

+

3

-H), 6.15 (1H, m, 10-H), 6.07 (1H, m, 8-H), 5.90 (1H, m, C-H), 4.09 (2H, m, N CH ), 3.25 (2H, t, J = 6.5

2

Hz, CH N), 2.96 (3H, s, N-CH ),), 2.89 (3H, s, NHCH ), 1.97 (3H, s, 11-CH ), 1.78 (2H, m, CH CH CH ),

2

3

2

+

-

1

.67-1.52 (5H, m, CH

2

CH

2

CH

2

, 5-CH

3

2 2 2

), 1.22 (2H, m, CH CH CH ); MS (FAB) m/z 449 (M -Cl ); HRMS

+

(

FAB): Calcd for C25

H

30

N

5

O

3

: 448.2349, found: 448.2356.

4

.2.45. Synthesis of N-Boc-2-chloroethylamine (49a) and N-Boc-3-chloropropylamine (49b)

A reaction flask was charged with chloroethylamine hydochloride 48a (1.00 g, 8.62 mmol), di-

tert-butyl dicarbonate (1.88 g, 8.62 mmol) and CHCl (50 mL) under argon atmosphere. To a mixture is

3

o

added triethylamine (0.91 g, 9.05 mmol) slowly at 0 C, and the mixture was heated under reflux for 1 h.

After washing the reaction mixture with water, the volatile materials were removed under reduced pressure.

The crude residue was purified by silica gel column chromatography (CHCl /MeOH = 8:2) to yield 49a

3

(

95 %). The 49b was prepared in a 93 % yield by a similar method starting with 48b.

1

4

9a; H NMR (300 MHz, CDCl ): δ 4.99 (1H, brs, NH), 3.60 (2H, t, CH CH Cl), 3.45 (2H, dt, CH CH Cl),

3

2

+

1

.46 (9H, s, C(CH ) ); MS (EI+) m/z : 180 (M ).

3 3

1

4

9b; H NMR (500 MHz, CDCl

3

2 2 2

): δ 4.80 (1H, brs, NH), 3.60 (2H, t, CH CH CH Cl ), 3.45 (2H, dt,

+

CH

2

CH Cl), 1.89 (2H, m, CH CH

2

CH Cl), 1.36 (9H, s, C(CH ); MS (EI+) m/z 193 (M ).

2

3 3

)

4

.2.46. Synthesis of 2-(N-Boc-2-aminoethyl)-5-metoxycarbonyl-11-methyl-6H-pyrido[4,3-b]carbazol-2-ium

chloride (50a) and its 3-proylamino derivative (50b)

A flame-dried round-bottom flask containing a magnetic stirring bar was charged with 14 (0.100 g,

0

.342 mmol), 49a (1.85 g, 10.22 mol), and DMF (5 mL). The reaction mixture was stirred at 120 C for 4 h

and concentrated after cooling to rt, and then NaOMe (0.019 g, 0.352 mmol) in MeOH (5 mL) was added

and the reaction mixture was stirred for 1 h at rt. Later, 0.343 mmol of 17 was added by dissolving in

MeOH and stirring was continued for 5 h at rt. After the volatile materials were removed under reduced

pressure, the crude residue was purified by ODS column chromatography (H O/MeOH = 6:4) to yield 50a

2

(

21 %). When 49b was similarly used instead of 49a and acetonitirile as a solvent, 50b was obtained in a

2

5

8

5 % yield.

0a; decomp. ~230 C; H NMR (300 MHz, CD

.48-8.52 (2H, m, 4, 7-H), 7.85 (1H, d, J = 7.9 Hz, 10-H), 7.74 (1H, dd, J = 7.3, 8.1 Hz, 8-H), 7.53 (1H, dd,

o

1

3

4

OD-d ): δ 9.93 (1H, s, 1-H), 9.51 (1H, d, J = 7.7 Hz, 3-H),

+

J = 7.3, 7.9 Hz, 9-H), 4.82 (2H, t, J = 5.3 Hz, N CH CH ), 3.75 (2H, t, J = 5.3 Hz, N CH CH ), 4.25 (3H, s,

2

+

-

OCH ), 3.39 (3H, s, CH ), 1.19 (9H, s, C(CH ) ). MS (FAB) m/z 434 (M - Cl ); HRMS: Calcd for

3

3 3

+

-

C H N O (M - Cl ): 434.2082, found: 434.2080.

2

5

28

3

4

o

1

5

2

0b: decomp. ~240 C; H NMR (300 MHz, D O): δ 8.54 (1H, s, 1-H), 8.29 (1H, d, J = 7.7 Hz, 3-H), 7.75

(

1H, d, J = 7.7 Hz, 4-H), 7.06 (1H, d, J = 8.1 Hz, 7-H), 6.86 (1H, d, J = 7.9 Hz, 10-H), 6.65 (1H, dd, J = 7.3,

+

8

.1 Hz, 8-H), 6.47 (1H, dd, J = 7.9, 8.1 Hz, 9-H), 4.15 (2H, m, N CH CH CH ), 3.66 (3H, s, OCH ), 3.01

2

3

+

13

(

2H, m, N CH CH CH ), 1.96 (3H, s, CH ), 1.94 (2H, m, N CH CH CH ), 1.23 (9H, s, C(CH ) ); C

2 2 2 3 2 2 2 3 3

2

3

ACCEPTED MANUSCRIPT

NMR (125.77 MHz, DMSO-d6): δ 165.5, 155.7, 146.4, 144.5, 142.2, 140.2, 134.5, 133.6, 128.9, 127.0,

1

24.1, 121.6, 121.0, 120.9, 120.4, 112.7, 101.0, 77.7, 57.6, 52.6, 36.8, 30.9, 28.1, 15.7. MS (FAB) m/z 448

+

-

+

-

(

M - Cl ); HRMS: Calcd for C H N O (M - Cl ): 448.2236, found: 448.2230.

26 30 3 4

4

.2.46. Synthesis of 2-(2-aminoethyl)-5-metoxycarbonyl-11-methyl-6H-pyrido[4,3-b]carbazol-2-ium

chloride (40) and its 3-proylamino derivative (41)

A round-bottom flask was charged with 50a (27.6 g, 0.060 mmol) and 4 M aq HCl (10 mL). The

reaction mixture was stirred for 1 h at rt. Purification of the crude residue by ODS column chromatography

(

H O/MeOH = 9:1) gives 97 % of 40. When 50b was similarly used instead of 50a, the compound 41 was

2

obtained in a 95 % yield.

o

1

4

0: decomp. ~210 C; H NMR (300 MHz, D O): ꢀ 8.70 (1H, s, 1-H), 8.30 (1H, d, J = 7.7 Hz, 3-H), 8.30

2

(

1H, d, J = 7.7 Hz, 4-H), 7.85 (1H, d, J = 8.1 Hz, 7-H), 7.85 (1H, d, J = 7.9 Hz, 10-H), 6.55 (1H, dd, J = 7.4,

+

8

3

.1 Hz, 8-H), 6.39 (1H, dd, J = 7.4, 7.9 Hz, 9-H), 4.54 (2H, t, J = 5.2 Hz, N CH

2

CH

+

2

3

), 3.61 (3H, s, OCH ),

-

+

.50 (2H, t, J = 5.2 Hz, N CH

2

CH

2

), 1.91 (3H, s, CH

3

); MS (FAB+) m/z 334 (M - Cl ); HRMS Calcd for

C H N O : 334.1556, found 334.1546..

2

0

20

3

2

o

1

4

1: decomp. ~240 C; H NMR (300 MHz, D

2

O):

ꢀ

8.99 (1H, s, 1-H), 8.66 (1H, d, J = 7.8 Hz, 3-H), 8.00

(

1H, d, J = 7.8 Hz 4-H), 7.54 (1H, d, J = 8.1 Hz, 7-H), 7.18 (1H, d, J = 7.9 Hz, 10-H), 6.97 (1H, dd, J = 7.4,

+

8

.1 Hz, 8-H), 6.89 (1H, dd, J = 7.4, 7.9 Hz, 9-H), 4.15 (2H, t, N CH CH CH ), 3.66 (3H, s, OCH ), 3.01

2

3

+

-

(

2H, t, N CH CH CH ), 1.96 (3H, s, CH ), 1.94 (2H, m, N CH CH CH ); MS (FAB+) m/z 348 (M - Cl );

2

3

2

HRMS Calcd for C H N O : 348.1712, found 348.1727.

21

22

3

2

4

.2.47. Synthesis of succinimidyl N-methylcarbamate (51a)

A dried round-bottom flask was charged with N-hydroxysuccinimide (5.83 g, 50.7 mmol), ethyl

o

acetate (20 mL) as a solvent and cooled at 0 C. To the mixture were slowly added triethylamine (4.76 g)

and methylisocyanate (6.39 g, 0.112 mmol) at the same temperature. The reaction temperature was slowly

raised to rt, and the mixture was stirred for 24 h at rt. After the volatile materials were removed under

reduced pressure the crude residue was re-crystallized from ethyl acetate/diethyl ether to give 51a (86 %).

0

19

o

1

Colorless needles, mp 148.0-149.0 C (lit. 148.0-152.0 C); H NMR (300 MHz, CDCl

3

): δ 8.15 (1H, br,

2

13

NH), 2.76 (4H, s, CH

2

CH ), 2.67 (3H, s-like, CH

3

); C NMR (22.5 MHz, CDCl ): δ 170.66, 151.98, 27.98,

3

2

5.47; MS (FAB+) m/z 173 (M + H)

1

9

4

.2.48. Synthesis of succinimidyl N-nitroso-N-methylcarbamate (51b)

A dried round-bottom flask was charged with sodium acetate (409 mg, 4.99 mmol), 51a (2.0 g, 8.0

o

mmol), and dichloromethane (25 mL) as a solvent, and a suspension of the mixture was stirred at -40 C. To

the mixture, N

2

O

4

(5.08 g, 55.2 mmol) in dichloromethane (10 mL) was added drop-by-drop with stirring.

o

The mixture was stirred at -40 C for 1h, and then the temperature was slowly raised to the room

temperature followed by an additional 1 h stirring. The reaction mixture was poured into iced water, and an

2

4

ACCEPTED MANUSCRIPT

aqueous layer was extracted with dichloromethane. The combined organic layer was successively washed

with 10 % NaHCO and a brine, and dried over Na SO over-night in a refrigerator. To the concentrated

3

2

4

filtrate (5 mL) was added petroleum ether to precipitate a crude product, which was recrystallized from

3

1

o

1

chloroform/ether to give 51b (73 %). light yellow needles, mp not determined (lit. 148-152 C); H NMR

13

(

300 MHz, CDCl

3

): δ 3.22 (4H, s, CH

2 2 3 3

CH ), 2.95 (3H, s, CH ); C NMR (22.5 MHz, CDCl ): δ 168.53,

1

51.09, 28.44, 25.57.

4

.2.49. Synthesis of ellipticinium-analogues 42 linked to a methylurea group

A reaction flask was charged with 40 (20.5 mg, 0.055 mmol), diisopropylethylamine (9.30 mg,

o

0

1

.072 mmol), and DMF (10 mL), and cooled to 0 C. To the mixture, succinimidyl N-methylcarbamate (51a,

0

0.6 mg, 0.062 mmol) in DMF (3 mL) was slowly added, and the reaction mixture was stirred at 0 C for 2

h. After the volatile materials were removed under reduced pressure, the crude residue, precipitated from

methanol solution with ethyl acetate, was purified by ODS column chromatography (H O/MeOH = 6:4) to

2

0

1

yield 42 (78 %). Decomp. ~250 C; H NMR (500 MHz, D O): δ 8.53 (1H, s, 1-H), 8.19 (1H, d, J = 7.7 Hz,

2

3

-H), 7.72 (1H, d, J = 7.7 Hz, 4-H), 6.88 (1H, d, J = 8.1 Hz, 7-H), 6.79 (1H, d, J = 7.9 Hz, 10-H), 6.55 (1H,

+

dd, J = 7.5, 8.1 Hz, 8-H), 6.38 (1H, dd, J = 7.5, 7.9 Hz, 9-H), 4.02 (2H, t, J = 5.3 Hz, N CH

2

CH

2

), 3.57 (3H,

+

s, OCH

3

), 3.48 (2H, t, J = 5.3 Hz, N CH

2

CH

2

3 3

), 2.47 (3H, s, CH ), 1.83 (3H, s, CH ). MS (FAB) m/z 391

+

-

+

3

(

M - Cl ); HRMS (FAB): Calcd for C H N O : 391.1770, found: 391.1762.

22

23

4

.2.50. Synthesis of ellipticinium-analogues 43 linked to a methylnitrosourea group

A reaction flask was charged with 40 (24.0 mg, 0.064 mmol), diisopropylethylamine (9.29 mg,

o

0

.072 mmol), and DMF (10 mL), and cooled to 0 C. To the mixture, succinimidyl N-methyl-N-

nitrosocarbamate (51b, 13.0 mg, 0.060 mmol) in DMF (3 mL) was slowly added, and the reaction mixture

0

was stirred at 0 C for 2 h. After the volatile materials were removed under reduced pressure, the crude

residue, precipitated from methanol solution with ethyl acetate, was purified by ODS column

0

1

chromatography (H O/MeOH = 6:4) to yield 43 (76 %). Decomp. ~250 C; H NMR (500 MHz, CD OD-

2

3

D ): δ 10.00 (1H, s, 1-H), 9.49 (1H, d, J = 7.7 Hz, 3-H), 8.53 (1H, d, J = 7.7 Hz, 4-H), 8.46 (1H, d, J = 8.1

4

Hz, 7-H), 7.81 (1H, d, J = 7.9 Hz, 10-H), 7.69 (1H, dd, J = 7.5, 8.1 Hz, 8-H), 6.38 (1H, dd, J = 7.5, 7.9 Hz,

+

9

-H), 4.96 (2H, t, J = 5.3 Hz, N CH

2

CH

2

), 4.19 (3H, s, OCH

3

), 4.10 (2H, t, J = 5.3 Hz, N CH

2

CH

2

), 3.48

+

-

+

(

3H, s, CH ), 3.00 (3H, s, CH ). MS (FAB) m/z 420 (M - Cl ); HRMS (FAB): Calcd for C H N O :

3

22 22

5

4

20.1677, found: 420.1671.

4

.2.51. Synthesis of ellipticinium-analogues 44 linked to a methylurea group

A reaction flask was charged with 41 (22.5 mg, 0.066 mmol), diisopropylethylamine (10.23 mg,

o

0

.079 mmol), and DMF (10 mL), and cooled to 0 C. To the mixture, succinimidyl N-methylcarbamate (51a,

0

1

1.7 mg, 0.066 mmol) in DMF (3 mL) was slowly added, and the reaction mixture was stirred at 0 C for 2

h. After the volatile materials were removed under reduced pressure, the crude residue, precipitated from

2

5

ACCEPTED MANUSCRIPT

methanol solution with ethyl acetate, was purified by ODS column chromatography (H O/MeOH = 6:4) to

2

0

1

yield 44 (79 %). Decomp. ~250 C; H NMR (500 MHz, D O): δ 8.53 (1H, s, 1-H), 8.19 (1H, d, J = 7.7 Hz,

2

3

-H), 7.72 (1H, d, J = 7.7 Hz, 4-H), 6.88 (1H, d, J = 8.1 Hz, 7-H), 6.79 (1H, d, J = 7.9 Hz, 10-H), 6.55 (1H,

+

dd, J = 7.5, 8.1 Hz, 8-H), 6.38 (1H, dd, J = 7.5, 7.9 Hz, 9-H), 4.15 (2H, t, N CH

2

CH

2

CH

2

), 3.66 (3H, s,

+

OCH

3

), 3.22 (2H, t, N CH

2

CH

2

CH

2

), 1.96 (3H, s, CH

3

), 2.47 (3H, s, CH

3

), 1.88 (2H, m, N CH

2

CH

2

CH

2

).

+

-

+

3

MS (FAB) m/z 405 (M - Cl ); HRMS (FAB): Calcd for C H N O : 405.1937, found: 405.1941.

23

25

4

.2.52. Synthesis of ellipticinium-analogues 45 linked to a methylnitrosourea group

A reaction flask was charged with 41 (18.0 mg, 0.051 mmol), diisopropylethylamine (8.01 mg,

o

0

.062 mmol), and DMF (10 mL), and cooled to 0 C. To the mixture, succinimidyl N-methyl-N-

nitrosocarbamate (51b, 9.6 mg, 0.056 mmol) in DMF (3 mL) was slowly added, and the reaction mixture

0

was stirred at 0 C for 2 h. After the volatile materials were removed under reduced pressure, the crude

residue, precipitated from methanol solution with ethyl acetate, was purified by ODS column

0

1

chromatography (H O/MeOH = 6:4) to yield 45 (76 %). Decomp. ~240 C; H NMR (500 MHz, D O): δ

2

8

6

.43 (1H, s, 1-H), 8.19 (1H, d, J = 7.7 Hz, 3-H), 7.64 (1H, d, J = 7.7 Hz, 4-H), 6.99 (1H, d, J = 8.1 Hz, 7-H),

.89 (1H, d, J = 7.9 Hz, 10-H), 6.55 (1H, dd, J = 7.5, 8.1 Hz, 8-H), 6.38 (1H, dd, J = 7.5, 7.9 Hz, 9-H), 4.35

+

(

2H, t, N CH

2

CH

2

CH

2

), 3.46 (3H, s, OCH

3

), 3.22 (2H, t, N CH

2

2 2 3

CH CH ), 2.49 (3H, s, CH ), 1.86 (3H, s,

+

-

+

4

CH ), 1.68 (2H, m, N CH CH CH ). MS (FAB) m/z 434 (M - Cl ); HRMS (FAB): Calcd for C H N O :

3

2

23 24

5

4

1

34.1828, found: 434.1819.

.2.53. Synthesis of 2-methyl-5-metoxycarbonyl-11-methyl-6H-pyrido[4,3-b]carbazol-2-ium iodide (52)

A flame-dried round-bottom flask containing a magnetic stirring bar was charged with 14 (0.4 g,

.36 mmol), acetonitirile (15 mL), and methyl iodide (0.85 mL, 13.6 mmol). The reaction mixture was

stirred at rt for 3 h and dissolved in MeOH (2.5 mL) after concentration, and then NaOMe (74 mg, 1.93

mmol) in MeOH (2.5 mL) was added drop-by-drop and the reaction mixture was stirred for 1 h at rt. Later,

3

65 mg (1.42 mmol) of 17 was added by dissolving in MeOH (4 mL) and stirring was continued for 5 h at

3

2

rt. The precipitated crystals were collected by filtration and washed with MeOH to give 52 (75 %; lit.

o

32

o

1

7

8 %) as light yellow crystals. Decomp. ~310 C (lit. 317-321 C); H NMR (300 MHz, DMSO-d

1.98 (1H, s, 6-H), 10.05 (1H, s, 12-H), 9.20 (1H, d, J = 7.4 Hz, 3-H), 8.57 (1H, d, J = 7.4 Hz, 4-H), 8.43

1H, d, J = 8 Hz, 7-H), 7.84 (1H, d, J = 8 Hz, 10-H), 7.76 and 7.46 (1H, dd, J = 8, 7.4 Hz, dd, J = 8, 7.4 Hz,

6

): δ

1

(

+

-

8

/9-H), 4.47 (3H, s, N CH ), 4.12 (3H, s, OCH ), 3.30 (3H, s, CH ). MS (FAB+) m/z 305 (M - I ).

2 3 3

4

.2.53. Synthesis of 2-methyl-ellipticin-2-ium iodide (53) and the corresponding chloride 46

A reaction flask was charged with 52 (74 mg, 0.0171 mmol), Na[Al(OCH2CH2OMe)2] (65%

o

solution in toluene, 86 mg, 0.0685 mmol), and xylene (13 mL), and stirred at 130 C for 6 h. After the

reaction mixture was cooled down to rt, the reaction was quenched with water, and the mixture was

condensed to dryness under reduced pressure. The residue was dissolved in 20 mL of MeOH, and treated

2

6

ACCEPTED MANUSCRIPT

with 17 (50 mg, 0.0171 mmol) at rt for 5 h. After the volatile materials were removed under reduced

pressure, the crude residue was de-salted with Amberlite-XAD2, and purified by silica gel column

32

chromatography (MeOH including 1% acethyl chloride) to yield 53 (84 %; lit. 85 %). The compound 53

100 mg, 0.26 mmol) and Amberlite IRA-900 (chloride form; 2.5 g) were stirred at rt for 5 h in water (30

(

mL), and the mixture was passed through a column of Amberlite IRA-900 (3 g) to give 46 in quantitative

yield.

o

1g

o

1

5

3: decomp. 355 C(lit. decomp. 360 C); H NMR (300 MHz, CD OD): δ 9.56 (1H, s, 1-H), 8.34 (1H, d,

3

J = 6.9 Hz, 3-H), 8.31 (1H, d, J = 6.9 Hz, 4-H), 7.96 (1H, d, J = 6.3 Hz, 10-H), 7.50-7.53 (2H, m, 7/8-H),

+

7

2

4

.26-7.29 (1H, m, 9-H), 4.34 (3H, s, N -CH ), 3.23 (3H, s, 11-CH ), 2.77 (3H, s, 5-CH ). MS (FAB+) m/z

3

+

-

61 (M - I ).

6: decomp. 355 C; H NMR (300 MHz, CD

o

1

3

OD): δ 9.90 (1H, s, 1-H), 8.47 (1H, d, J = 7.5 Hz, 3-H), 8.46

(

1H, d, J = 7.5 Hz, 4-H), 8.29 (1H, d, J = 7.2 Hz, 10-H), 7.67 (2H, m, 7/8-H), 7.40-7.46 (1H, m, 9-H), 4.49

+

-

3H, s, N -CH ), 3.37 (3H, s, 11-CH ), 2.91 (3H, s, 5-CH ). MS (FAB+) m/z 261 (M - Cl ).

3

4

.3. Antitumor activity

.3.1. Cell lines and culture

HeLa S-3 cells were provided by Dr. Okada (Institute for Biological Resources and Functions,

National Institute of Advanced Industrial Science and Technology, Japan). The cells were maintained in

MEM (Nissui) supplemented with 10% FBS (GIBCO), 2% HEPES, 3% NEAA and 2 mM L-glutamine in a

water-saturated atmosphere of 5% CO at 37 °C.

2

4

.3.2. Cell viability assay

Cell viability was determined by a 3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium 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

4

2

× 10 cells/mL and cultured 3 h prior to drug treatment. Cells were exposed at 37 °C for 24-72 h to

ellipticine analogues. The MTT reagent (Nacalai tesuque) was prepared at a concentration of 2 mg/ml in

Dulbecco’s PBS without calcium and magnesium, and stored at 4 °C. After treatment for indicated times,

cells were incubated with MTT reagent for 4 h at 37 °C. The plate was centrifuged at 3,000 rpm for 10 min,

and the medium was removed. To solubilize the resultant MTT-formazan, 200 ꢀL/well of dimethyl

sulfoxide (DMSO) was added to each well followed by thorough mixing with a mechanical plate mixer.

Absorbance at 540 nm was measured on a microplate reader (MTP-500, CORONA), and the percentage of

cell viability was taken as the percentage absorbance at 540 nm of ellipticine-treated cells against control

cells.

Acknowledgements

2

7

Article Doi

Synthesis and in vitro antitumor activity of novel 2-alkyl-5- methoxycarbonyl-11-methyl-6H-pyrido[4,3-b]carbazol-2-ium and 2-alkylellipticin-2-ium chloride derivatives

DOI: 10.1016/j.ejmech.2014.05.032

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2014.05.032

Full text of DOI:10.1016/j.ejmech.2014.05.032

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