Novel antitumor indolizino[6,7-b]indoles with multiple modes of action: DNA cross-linking and topoisomerase i and II inhibition

Article

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Novel Antitumor Indolizino[6,7‑b]indoles with Multiple Modes of

Action: DNA Cross-Linking and Topoisomerase I and II Inhibition

Ravi Chaniyara,^†,⊥,∞Satishkumar Tala,^†,∞Chi-Wei Chen,^†,§,∞Xiuguo Zang,^‡Rajesh Kakadiya,^†

Li-Fang Lin,^†Ching-Huang Chen,^†Shin-I Chien,^∥Ting-Chao Chou,^‡Tung-Hu Tsai,^∥Te-Chang Lee,*^,†

Anamik Shah,^⊥and Tsann-Long Su*^,†,#

^†Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan

^‡Preclinical Pharmacology Core Laboratory, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer

Center, New York, New York 10021, United States

^§Institute of Biopharmaceutical Science and ^∥Institute of Traditional Medicine, National Yang-Ming University, Taipei 11221, Taiwan

^⊥Department of Chemistry, Saurashtra University, Rajkot, Gujarat, India

^#Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan

S

* Supporting Information

ABSTRACT: A series of bis(hydroxymethyl)indolizino[6,7-b]indoles and their bis-

(alkylcarbamates) were synthesized for antitumor studies. These agents were designed as hybrid

molecules of β-carboline (topoisomerase inhibition moiety) and bis(hydroxymethyl)pyrrole (DNA

cross-linking moiety). The preliminary antitumor studies indicated that these agents exhibited

signiﬁcant cytotoxicity against a variety of human tumor cells in vitro. Treatment of human breast

carcinoma MX-1 xenograft-bearing nude mice with compounds 18b and 28c achieved more than

99% tumor remission. We also observed that 18a displayed potent therapeutic eﬃcacy against

human lung adenocarcinoma A549 and colon cancer HT-29 xenografts. These results revealed that

compound 18a was more potent than irinotecan against HT-29 cells and was as potent as irinotecan against A549 cells in

xenograft models. Furthermore, we demonstrated that these derivatives possess multiple modes of action, such as induction of

DNA cross-linking, inhibition of topoisomerase I and II, and cell-cycle arrest at the S-phase.

derivatives of β-carboline preserve a unique pharmacophore

and exhibit a wide spectrum of biological activities,¹¹⁻¹⁷such as

antimalarial, antithrombotic, sedative, anti-HIV, and anticancer

activities. Of these derivatives, naturally occurring β-carboline

alkaloids, such as callophycin A (2)¹⁸and fascaplysin (3),¹⁹

displayed antiproliferative eﬀects in vitro against a variety of

human cancer cell lines. Many synthetic β-carboline deriva-

tives,^20,21including 1,⁹were also observed to have moderate to

good cytotoxicity against tumor cell growth in vitro. These

reports indicated that β-carboline derivatives exhibit antitumor

activity through DNA intercalation²¹and the inhibition of

topoisomerase I (topo I) and II,²⁰⁻²²cyclin-dependent kinase

(CDK),²³and IkK kinase complex (IkK).²⁴

Another pharmacophoric group in the target compounds is

bis(hydroxymethyl)pyrrole, which was observed to induce

DNA cross-linking. Previous reports revealed that bis-

(hydroxymethyl)pyrroles (4 and 5)²⁵or pyrrolizines (6 and

7)²⁶displayed signiﬁcant antitumor activity. The bis-

(hydroxymethyl) functionality located at the adjuvant position

of these agents can form two electrophilic centers, leading to

DNA double-strand cross-linking. The proposed mechanism of

action for DNA interstrand cross-linking induced by these

derivatives occurs most likely via an S_N1 electrophilic

INTRODUCTION

■

A hybrid molecule comprising two pharmacophoric groups is

often used for the design of new drugs.¹The hybrid may have

increased potencies and/or modiﬁed selectivity proﬁles as

receptor ligands compared to the corresponding single drug.

Accordingly, several hybrid compounds in which the known

antitumor compounds have been tethered to distamycin and

netropsin frames have been designed, synthesized, and

subjected to biological studies.²⁻⁴We have also designed and

synthesized various DNA-directed alkylating agents, which were

prepared by linking various DNA-aﬃnic molecules, such as 9-

anilinoacridines, acridines, or quinolines, with phenyl N-

mustard, a DNA alkylator.^5,6Our studies demonstrated that

these conjugates were generally more cytotoxic than the

individual intercalator or alkylating agent alone. Azatoxin (1,

Figure 1), a hybrid of etoposide, which is a topoisomerase II

(topo II) inhibitor,⁷and ellipticine, a DNA intercalator, topo II

inhibitor, and DNA binding agent,⁸was reported to have a dual

mechanism of action, including the inhibition of topo II and

tubulin polymerization.^9,10As a result, we designed and

synthesized novel hybrid indolizino[6,7-b]indole derivatives,

which contain biologically active β-carboline and bis-

(hydroxymethyl)pyrrole pharmacophores, as shown in Figure

2.

β-Carboline alkaloids were originally identiﬁed in nature.

Naturally occurring β-carboline alkaloids and synthetic

Received: September 19, 2012

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Figure 1. Structures of several anticancer β-carboline derivatives (1−3), bis(hydroxymethyl)pyrrole and their carbamate derivatives (4−12), and the

newly synthesized novel tetracyclic indolizino[6,7-b]indole derivatives (13).

synergistic antitumor eﬀects when coadministered with DNA

repair inhibitors, such as arsenic trioxide (ATO), in nude mice

transplanted with cisplatin-resistant lung and bladder cancer

cells.³¹Recently, we further synthesized a set of bis-

(hydroxymethyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline ana-

logues (11)³²and bis(hydroxymethyl)benzo[d]pyrrolo[2,1-

b]thiazoles (12),³³which can be considered “bioisoteres” of

3a-azacyclopenta[a]indene (10). We demonstrated that these

analogues also exhibited a broad spectrum of antitumor activity

against the growth of various human solid tumor cells both in

vitro and in xenograft models.

Figure 2. Novel indolizino[6,7-b]indole derivatives comprising β-

carboline and bis(hydroxymethyl)pyrroles moieties.

reaction.²⁷The potential electrophilic reactivity of these agents,

in which the carbamate moieties act as leaving groups in an

alkyl−oxygen cleavage mechanism, would be modulated by the

degree of electronic perturbation in the participating pyrrole.²⁷

Of these analogues, compound 6 was found to have signiﬁcant

antitumor activity against a broad range of human tumor

xenografts.²⁸After further structural modiﬁcation, pyrrolo[2,1-

a]isoquinolines (8) and pyrrolo[1,2-a]quinolines (9) were

synthesized and found to have a broad spectrum of activity

against a wide range of tumors.²⁹

We have previously designed and synthesized a series of

bis(hydroxymethyl)-8H-3a-azacyclopenta[a]indene-1-yl and

their bis(methylcarbamate) derivatives (10).³⁰These analogues

exhibited signiﬁcant cytotoxicity against human lymphoblastic

leukemia CCRF-CEM and a variety of human tumors in vitro

and showed potent therapeutic eﬃcacy in xenograft models.

We also found that these derivatives possessed signiﬁcant

To continue the investigation of bis(hydroxymethyl)pyrrole

analogues as potential antitumor agents, we synthesized novel

tetracyclic indolizino[6,7-b]indole derivatives (13), which were

designed as a hybrid molecule of β-carboline, a DNA

intercalating agent and topoisomerase inhibitor, and bis-

(hydroxymethyl)pyrroles, a DNA double-strand cross-linking

agent. We anticipate that the hybrid may possess an interesting

mechanism of action and may display potent antitumor activity.

Here, we report the chemical modiﬁcation of β-carboline

derivatives for a better understanding of the structure−activity

relationships (SAR), antitumor activity, and mechanism of

action of these newly synthesized compounds.

RESULTS AND DISCUSSION

■

Chemistry. The synthesis of 1,2-bis(hydroxymethyl)-

indolizino[6,7-b]indole derivatives (18a−c) and their bis-

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a

Scheme 1. 6-Substituted Indolizino[6,7-b]indole Derivatives

a

Reagents and conditions: (a) formaldehyde, sulfuric acid, rt; (b) DMAD, Ac₂O, 70 °C; (c) NaH, DMF, R¹-I or R¹-Br; (d) LAH, ether, DCM, 0 °C;

(e) R²NCO, TEA.

a

Scheme 2. 3-Substituted Indolizino[6,7-b]indole Derivatives

a

Reagents and conditions: (a) formaldehyde, MeOH; (b) R³COCl, TEA; (c) NaH, MeI, THF; (d) DMAD, Ac₂O, 60−75 °C; (e) LAH, ether,

DCM, 0 °C; (f) R²NCO, TEA.

(alkylcarbamate) derivatives (19a−c and 20a−c) is depicted in

Scheme 1. Compound 15 was prepared by reacting

commercially available ^L-tryptophan (14) with 37% form-

aldehyde in the presence of sulfuric acid according to the

procedure described in the literature.³⁴The reaction of 15 with

dimethyl acetylenedicarboxylate (DMAD) in acetic anhydride

(Ac₂O) yielded the diester 16,³⁵which was N-alkylated by

treatment with sodium hydride (NaH) and either an alkyl or

benzyl halide, such as iodomethane (MeI), iodoethane (EtI), or

benzyl bromide, in dimethylformamide (DMF) at ambient

temperature to yield 17a−c. The ester functions of 17a−c were

reduced to the corresponding bis(hydroxymethyl) derivatives

(18a−c) by reaction with lithium aluminum hydride (LAH) in

a mixture of ether/dichloromethane (DCM) in an ice bath.

Treatment of 18a−c with alkyl isocyanates, such as ethyl

isocyanate or isopropyl isocyanate, in the presence of

triethylamine (TEA) resulted in a good yield of the desired

bis(alkylcarbamate) derivatives 19a−c and 20a−c.

Pictet−Spengler cyclization according to a previously described

method.³⁶Acylation of 22 with various acid chlorides in the

presence of TEA produced the corresponding N-acyl

derivatives 23a−i.³⁷Similarly, compounds 23a−i were treated

with NaH and MeI in tetrahydrofuran (THF) at ambient

temperature to yield the carboxylic acids 24a−i, which were

further converted into the diesters 25a−i by treatment with

DMAD in Ac₂O at 60−75 °C. The diesters 25a−i were

reduced to the bis(hydroxymethyl) derivatives 26a−i by

reaction with LAH in a mixture of ether/DCM in an ice

bath. Similarly, treatment of 26a−i with alkyl isocyanates

resulted in the desired bis(alkylcarbamate) derivatives 27a−i

and 28a−i.

In Vitro Cytotoxicity. In our anticancer-agent-screening

program, we used the CCRF-CEM cell line for evaluating the

primary antiproliferative activity of the tested compounds for

SAR studies. Compounds that exhibited signiﬁcant in vitro

cytotoxicity against this cancer cell line were selected for further

evaluation of their antiproliferative activity against the growth

of a variety of human solid tumor cells in culture. The

antiproliferative activities of the newly synthesized bis-

(hydroxymethyl)indolizino[6,7-b]indole (18a−c, 26a−i), bis-

( e t h y l c a r b a m a t e ) ( 1 9 a − c , 2 7 a − i) , a n d b i s -

(isopropylcarbamate) derivatives (20a−c, 28a−i) against

Because the solubility of compound 15 is very poor and is

not suitable for N-acylation with other alkyl acid chlorides or

benzoyl chlorides, an alternative method for synthesizing the

target compounds was developed. We used ^L-tryptophan

methyl ester hydrochloride (21) as the starting material

(Scheme 2). Compound 22 was synthesized from 21 by

C

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Table 2. Cytotoxicity of Selected 2,3-Bis(hydroxymethyl)-5H-indolizino[6,7-b]indoles and Their Bis(alkylcarbamate)

a

Derivatives against KB, KBvin10, MES-SA, and MES-SA/dx5 As Measured by Cell Growth in Vitro

compd

b

c

b

18a

18b

19a

26a

28c

vincristine

doxorubicin

ND

KB

2.51 1.14

1.31 0.20

1.47 0.40

0.90 0.51

2.75 0.46

2.60 0.50

1.97 0.39

1.56 0.51

14.22 6.57

15.60 2.61

2.11 0.45

KBvin10

303.2 34.12

d

[0.52×]

[0.62×]

[0.94×]

[0.79×]

[1.1×]

[143.7×]

MES-SA

0.24 0.04

0.12 0.02

0.40 0.13

0.24 0.04

0.78 0.14

0.28 0.06

0.77 0.08

0.47 0.12

ND

0.038 0.0028

2.30 0.98

MES-SA/dx5

d

[0.52×]

[0.60×]

[0.36×]

[0.61×]

[60.52×]

a

b

The data represent the mean STDEV of each compound from three to six independent experiments. ND: not determined. IC₅₀values in μM.

c

d

IC₅₀values in nM. Resistance factor.

CCRF-CEM cells are shown in Table 1. We synthesized

compounds with a methyl, ethyl, and benzyl substituent at the

hetero nitrogen atom (N⁶) at the indole ring because we

observed that the N-Me derivatives, such as compounds 18a,

19a, and 20a, are generally more cytotoxic than the

corresponding N-Et (18b, 19b, and 20b) and N-Bn derivatives

(18c, 19c, and 20c) Therefore, we focused primarily on the N-

Me derivatives for our antitumor studies. In the series of 6-

methyl-1,2-bis(hydroxymethyl) derivatives, 18a and 26a−i, the

order of potency in this series is 18a (C3-Me) > 26a (C3-Et) >

26b−i (C3-Ph), indicating that the size of the substituent may

aﬀect the antiproliferative activity. The C3-Me derivative (18a)

is the most cytotoxic, with an IC₅₀of 0.04 μM against the

CCRF-CEM cell line in vitro. Among the C3-Ph substituted

derivatives 26b−i, the C3-4′-F-Ph (26c) is more potent than

the chloro-substituted derivatives (26b−f), and the C3-4′-OMe

and C3-3′,4′-di-OMe derivatives 26g and 26h, respectively, are

more cytotoxic than the C3-3′,4′,5′-tri-OMe derivative (26i).

However, the OMe substituted derivatives are generally more

cytotoxic than the halogen-substituted compounds.

For the bis(alkylcarbamate) derivatives, we found that the

bis(ethylcarbamate) derivatives, 27a−i, are more potent than

the corresponding bis(isopropylcarbamate) compounds, 28a−i,

for the inhibition of cancer cell growth. Unlike the 1,2-

bis(hydroxymethyl) derivatives, the substituent(s) on the

phenyl ring of the bis(alkylcarbamate) derivatives do not

have a large inﬂuence on potency. However, compounds with a

3′-Cl,4′-F-Ph substituent at C3, such as 26f, 27f, and 28f, are

less cytotoxic than compounds with other substituent(s). For

the compounds with a OMe group on the C3-substituted

phenyl derivatives, the cytotoxicity is decreased when the

number of OMe group is increased (i.e., 4′-MeO-Ph > 3′,4′-di-

MeO-Ph > 3′,4′,5′-tri-MeO-Ph). In general, the bis-

(alkylcarbamate) derivatives 27b−i and 28b−i are more potent

than the corresponding bis(hydroxymethyl) congeners, 26b−i,

with the exception of the C3-Me- (18a, 19a, and 20a) and C3-

Et-substituted derivatives (18b, 19b, and 20b).

(hydroxymethyl) derivatives against all the tumor cells tested.

The most cytotoxic compound of this series is compound 18a,

which has signiﬁcant cytotoxicity against the tested tumor cells

in vitro. We also examined the cytotoxic eﬀects of several newly

synthesized compounds on human embryonic lung ﬁbroblast

HEL299 cells. As shown in Table 1, compounds 19a and 28c

were much less toxic to the HEL299 cell line than to the other

tested tumor cell lines, whereas compounds 18a, 18b, and 26a

were slightly less or similarly cytotoxic to the HEL299 cells.

Notably, the HEL299 cell line is relatively more susceptible to

cisplatin than the other tested tumor cells.

Overcoming the multidrug resistance (MDR) of resistant

tumor cells is a major concern for new drug discovery and

development. To determine whether tumor cells are resistant to

the newly synthesized derivatives in a manner similar to

currently used anticancer drugs, we selected ﬁve compounds

(18a, 18b, 19a, 26a, and 28c) and evaluated their in vitro

cytotoxicity against human drug-sensitive cancer cell lines and

their resistant subcell lines, including a human oral cancer cell

line KB, the subcell line resistant to vincristine KBvin10,³⁸

a

human uterine sarcoma cell line MES-SA, and the subcell line

resistant to doxorubicin MES-SA/dx5. As shown in Table 2,

almost all the tested compounds were relatively more potent

against the MDR cell lines than the parental lines. This ﬁnding

indicates that these cell lines, which are resistant to vincristine

or doxorubicin, are not cross-resistant to the tested compounds.

In Vivo Antitumor Activity. On the basis of the in vitro

antiproliferative eﬀects of the newly synthesized derivatives, we

selected compounds 18a, 18b, and 28c and evaluated their

therapeutic eﬃcacy against a variety of solid tumors in

xenograft models. In all experiments, the tumors were

subcutaneously implanted in nude mice, and the maximum

tolerated dose (MTD) of the tested compound was

administered via intravenous injection (iv inj). The MX-1

xenograft-bearing mice were treated with compound 18b at 25

mg/kg once per day for 3 consecutive days (QD×3) or

compound 28c at 35 mg/kg once per day for 4 consecutive

days (QD×4). In all cases, greater than 99% tumor suppression

and no relapses were observed on day 20 (D20) (Figure 3A).

The average body weight of all the drug-treated mice recovered

after the cessation of treatment (Figure 3B). In another study,

we treated HT-29 xenograft-bearing nude mice with either

compound 18a or irinotecan, which is used as a standard

chemotherapeutic for advanced colorectal cancer. As shown in

Figures 4, compound 18a signiﬁcantly suppressed tumor

growth at the same treatment regime (25 mg/kg, QD×4, iv

inj). Treatment with irinotecan also signiﬁcantly suppressed the

growth of HT-29 cells in nude mice; however, the tumor began

growing on D22 after the tumor cells were implanted. These

To further investigate the antiproliferative activity of the

newly synthesized derivatives, the cytotoxicity of the selected

compounds was evaluated with regard to the inhibition of cell

growth in vitro of other human solid tumors, such as human

prostate cancer (PC3), human oral epidermoid carcinoma

(OECM1), human breast cancer (MX-1), human colon cancer

(HCT-116), human colon adenocarcinoma (HT-29), and

human lung adenocarcinoma (CL141T, A549, and H460). As

summarized in Table 1, all of the compounds tested displayed a

broad spectrum of antitumor activity against the solid tumor

cell lines tested in culture. In general, the bis(alkylcarbamate)

derivatives are more cytotoxic than the corresponding bis-

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Figure 4. Therapeutic eﬀects of 18a and irinotecan in HT-29

xenograft-bearing nude mice: (A) average tumor size changes; (B)

average body weight changes.

Figure 3. Therapeutic eﬀect of 28c and 18b in MX-1 xenograft-

bearing nude mice (iv inj, n = 4): (A) average tumor size changes; (B)

average body weight changes.

results indicated that compound 18a is more potent than

irinotecan against HT-29 cells. We further evaluated the

antitumor activity of compound 18a against A549 cells in a

xenograft model and compared the activity of compound 18a

to that of irinotecan (Figure 5). Under the same treatment

conditions (25 mg/kg, QD×4 via iv inj), compound 18a was as

potent as irinotecan (60 mg/kg, every 2 days for three times

(Q2D×3), iv inj). Both compounds suppressed tumor growth

by approximately 50%. For the changes in body weight (Figures

4B and 5B) in the drug-treated mice, we found that compound

18a was less toxic than irinotecan. Our current studies

demonstrate that compound 18a is most likely more potent

than irinotecan against the human colon and lung tumor

xenograft models and is also less toxic to the host based on the

average body weight loss.

Cell-Cycle Inhibition. Guan et al. synthesized a series of β-

carboline derivatives, which could induce DNA damage and

G2/M arrest in human epithelial carcinoma (HeLa) cells.²¹

Additionally, a distinct S phase arrest was observed, which may

be caused by the inhibition of topo I and II at the beginning of

the DNA synthesis. Recently, we demonstrated that treatment

of human lung adenocarcinoma (H1299) cells with bis-

(hydroxymethyl) derivatives of benzo[d]pyrrolo[2,1-b]thiazole

at higher concentrations for 24 h induced the accumulation of

the G1 population. This treatment also resulted in accumu-

lation at the G1 and G2/M phases and the appearance of a sub-

G1 population at 48 h.³³In the present studies, we investigated

the eﬀects of compounds 18a, 18b, and 28c on cell-cycle

progression in CL141T cells. We treated CL141T cells with

compounds 18a, 18b, and 28c at 0.5-, 1-, and 2-fold their IC₅₀

values for 24, 48, and 72 h and analyzed the cell-cycle

distributions by ﬂow cytometry. The IC₅₀values of 18a, 18b,

and 28c were 0.5, 0.8, and 7.5 μM, respectively. As shown in

Figure 6, the distribution of S phase of the CL141T cells was

increased by treatment with the bis(hydroxymethyl) derivatives,

18a and 18b, at 0.5-fold IC₅₀for 24 h. Increasing the

concentrations to 1- and 2-fold IC₅₀, we observed an

accumulation of the G1 and S phases. However, treatment of

CL141T cells with bis(isopropylcarbamate) 28c caused an S

phase delay after 24 h at various concentrations. The previous

S-phase delayed cells were progressed to the G2/M phase after

48 and 72 h of treatment, when the cells were treated with 0.5-

and 1-fold IC₅₀. However, these cells did not progress to the

G2/M phase when the cells were treated with 2-fold

concentration of IC₅₀. In general, all three compounds, 18a,

18b, and 28c, induced S-phase delay initially, followed by G2

arrest. Compounds 18a and 18b, but not 28c, treated cells at 1-

and 2-fold IC₅₀resulted in the delay of G1 to S progression,

suggesting that 18a and 18b produced more severe DNA

damage than 28c. In addition to cell cycle disruption, we

observed an increase of a sub-G1 population in CL141T cells

treated with various doses of 18a, 18b, and 28c for 72 h,

suggesting that these compounds can trigger apoptotic cell

death.

Induction of Apoptosis. Our cell-cycle progression studies

suggested that newly synthesized compounds could cause

apoptosis of cancer cells. Therefore, we determined the

induction of apoptosis by compounds 18a and 18b with an

annexin V binding assay³⁹in H460 cells. As shown in Figure

7A, after a 24 h treatment with compounds 18a and 18b,

apoptotic cells increased from 4.10% to 15.09% and 12.92%

and was signiﬁcantly increased to 61.35% and 43.52%,

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cytotoxicity and is the main mechanism of inhibiting cancer cell

growth.

Inhibition of Topoisomerase I. As noted previously, the

novel tetracyclic indolizino[6,7-b]indole derivative is designed

as a hybrid molecule of β-carboline and a bis(hydroxymethyl)-

pyrrole. We demonstrated that the newly synthesized

derivatives with a bis(hydroxymethyl)pyrrole moiety can

induce DNA cross-linking. Because these agents bear a β-

carboline moiety, we also investigated whether the synthesized

derivatives act as inhibitors of topo I. A supercoiled plasmid

DNA relaxation assay was used to determine whether topo I

activity was inhibited by the newly synthesized compounds.

Irinotecan was used as a positive control. As shown in Figure 9,

irinotecan, 18a, and 19a interfered with the ability of topo I to

relax DNA supercoils. These results demonstrate that the newly

synthesized compounds exhibit inhibitory eﬀects against topo I.

However, 18a and 19a are less potent than irinotecan in

inhibiting topo I.

Inhibition of Topoisomerase II. Previous studies have

shown that β-carboline analogues also exhibit inhibitory eﬀects

against topo II.^7,8To determine whether the newly synthesized

compounds also inhibit topo II, an ATP-dependent topo II

mediated relaxation assay was performed. Etoposide was used

as a positive control. The results for the representative

compounds (18a and 19a) are shown in Figure 10, where

etoposide, 18a, and 19a inhibit DNA topo II activation in a

concentration-dependent manner (25−200 μM). These results

demonstrate that the newly synthesized derivatives exhibit

inhibitory activity against topo II but are less potent than

etoposide.

Stability of Compound 18a in Rat Plasma. A

bioanalytical system was used to evaluate the plasma stability

of compound 18a in rat plasma. The analytical validations were

performed according to the U.S. Food and Drug Admin-

istration (U.S. FDA) guidelines.⁴²The stability tests of

compound 18a in rat plasma samples were performed for

short-term stability at concentrations of 1 and 10 μg/mL

incubated for 1, 2, 3, and 4 h at room temperature. The

degradation of compound 18a was detected by high-perform-

ance liquid chromatography (HPLC). HPLC indicated that

Figure 5. Therapeutic eﬀects of 18a and irinotecan in A549 xenograft-

bearing nude mice: (A) average tumor size changes; (B) average body

weight changes.

respectively, after 72 h. These results conﬁrmed that the

damage induced by the tested compounds can trigger apoptotic

cell death. We also investigated the expression of caspase-3,

caspase-7, and poly ADP-ribose polymerase (PARP), indicator

proteins of apoptosis,^40,41in H460 cells treated with

compounds 18a and 18b using Western blot analysis. As

shown in Figure 7B, we found that cleaved caspase-3 (c-

caspase-3), cleaved caspase-7 (c-caspase-7), and cleaved PARP

(c-PARP) were signiﬁcantly increased after treatment with

compounds 18a and 18b at the IC₅₀dose after 24 and 72 h.

This ﬁnding indicates that these compounds cause cancer cell

apoptosis via reduction of the membrane integrity and increase

of the levels of c-caspase-3, c-caspase-7, and c-PARP.

DNA Cross-Linking Study. Our previous studies revealed

that bis(hydroxymethyl)pyrrole analogues^30,32,33are capable of

forming DNA interstrand cross-links. To determine whether

the newly synthesized compounds are also able to produce

DNA cross-links, we used an alkaline agarose gel shift assay in

which linearized pEGFP-N1 plasmid DNA was treated with

bis(hydroxymethyl) derivatives (18a and 18b) and bis-

(alkylcarbamate) derivatives (19b, 20b, and 28c) at various

concentrations (1, 5, and 10 μM). The results show that

compounds 18a, 18b, 19b, and 20b formed DNA interstrand

cross-links eﬃciently, with the exception of compound 28c,

which produced less DNA cross-linking (Figure 8). This

ﬁnding suggests that DNA cross-linking correlates with in vitro

compound 18a is stable in rat plasma with a long half-life (t_1/2

=

12.4 1.25 h, n = 3). These results demonstrate that the newly

prepared bis(hydroxymethyl)indolizino[6,7-b]indole deriva-

tives are chemically and metabolically stable.

CONCLUSIONS

■

In this study, we synthesized a series of new, substituted

bis(hydroxymethyl)indolizino[6,7-b]indole derivatives and

their bis(alkylcarbamate) derivatives for antitumor studies.

These agents were designed as hybrid molecules of β-carboline,

a topo I and II inhibitory moiety, and bis(hydroxymethyl)-

pyrrole, a DNA cross-linking moiety. The preliminary

antitumor studies revealed that the newly synthesized

compounds exhibited a broad spectrum of signiﬁcant antitumor

activity by inhibiting the growth of various human tumor cell

lines with no cross-resistance to vinblastine and doxorubicin.

Among the newly synthesized derivatives, compounds 18a,

18b, and 28c were selected for further evaluation of their

therapeutic eﬃcacy against human solid tumors in xenograft

models. We observed that the synthesized compounds

displayed multiple modes of action. These compounds are

capable of inducing DNA cross-linking and exhibit inhibitory

eﬀects against topo I and II. To the best of our knowledge, this

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Figure 6. Eﬀects of various compounds on cell-cycle progression in CL141T cells. The cells were treated with 18a (0.25, 0.5, 1 μM), 18b (0.4, 0.8,

1.6 μM), and 28c (3.25, 7.5, 15 μM) for 24, 48, and 72 h. The cells were then harvested and analyzed for cell-cycle progression by ﬂow cytometry.

analysis was performed on a Heraeus CHN-O Rapid instrument.

HPLC was performed on an Elite instrument with a Mightysil RP-18

(250 mm × 4.6 mm) column. Compounds were detected by UV at

260 nm. The mobile phase was acetonitrile/THF (80:20 v/v) with a

ﬂow rate of 1 mL/min. The purity of all tested compounds was ≥95%

is the ﬁrst time bis(hydroxymethyl)indolizino[6,7-b]indole

derivatives have been synthesized as DNA cross-linkers and

topo I and II inhibitors. Flow cytometry studies of cell-cycle

distribution indicated that the bis(hydroxymethyl) derivatives

may have diﬀerent modes of action from the bis-

(alkylcarbamate) derivatives. Further studies on the levels of

the apoptotic proteins, caspase-3, caspase-7, and PARP suggest

that the studied compounds are able to cleave these proteins

and able to induce apoptosis. Of these agents, compound 18a

was found to have superior antitumor activity to irinotecan in

A549 and HT-29 xenograft-bearing nude mice and with less

toxicity. This derivative may potentially be selected as a

candidate for preclinical studies.

1

based on analytical HPLC. H NMR spectra and ¹³C NMR spectra

were recorded on a Bruker AVANCE 600 DRX and/or 400 MHz

Bruker Top-Spin spectrometer in the solvents indicated. The proton

chemical shifts were reported in parts per million (δ ppm) relative to

(CH₃)₄Si (TMS), and coupling constants (J) were reported in hertz

(Hz). Abbreviations used are the following: s, singlet; d, doublet; t,

triplet; m, multiplet; br s, broad peak.

1,2,3,4-Tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic Acid

(15). Formalin [37% (80 mL)] was added to a mixture of ^L-tryptophan

(14, 50 g, 245 mmol) and 0.1 N H₂SO₄(150 mL). The resulting

mixture was stirred at room temperature for 4 h. A white solid

separated out and was ﬁltered, washed with water, and dried. The

resulting powder weighed 14.41 g (78%); mp 275−276 °C (lit.³⁴mp

280−282 °C). ¹H NMR (DMSO-d₆) δ 2.84 (1H, m, CH₂), 3.16 (1H,

m, CH₂), 3.66 (1H, m, CH₂), 4.23 (1H, m, CH₂), 4.38 (1H, m, CH),

6.98−7.02 (1H, m, ArH), 7.13−7.16 (1H, m, ArH), 7.32−7.34 (1H,

m, ArH), 7.45 −7.48 (1H, m, ArH), 11.03 (1H, brs, exchangeable,

NH).

EXPERIMENTAL SECTION

■

General Chemistry. All commercial chemicals and solvents were

reagent grade and were used without further puriﬁcation unless

otherwise speciﬁed. Melting points were determined in open

capillaries on a Fargo melting point apparatus and are uncorrected.

Thin-layer chromatography (TLC) was performed on silica gel G60

F254 (Merck, Merck KGaA, Darmstadt, Germany), and short-

wavelength ultraviolet (UV) light was used for visualization. Elemental

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Figure 7. (A) Eﬀects of compounds 18a and 18b on the induction of apoptosis in human non-small-cell lung adenocarcinoma H460 cells. The cells

were harvested and analyzed for apoptosis by ﬂow cytometric analysis of phosphatidylserine externalization (annexin V binding) and cell membrane

integrity (PI staining) after treatment with 18a (0.4 μM) and 18b (0.8 μM) for 24 and 72 h. (B) Eﬀect of compounds 18a and 18b on the levels of

the apoptotic proteins, caspase-3, caspase-7, and PARP and their cleaved forms using Western blot analysis.

Figure 8. Representative DNA cross-linking gel shift assay for bis(hydroxymethyl) derivatives (18a and 18b) and bis(alkylcarbamate) derivatives

(19b, 20b, and 28c) at 1, 5, and 10 μM: XL, cross-linking DNA; SS, single-strand DNA.

Dimethyl 3-Methyl-6,11-dihydro-5H-indolizino[6,7-b]indole-

1,2-dicarboxylate (16). DMAD (8.4 g, 59.5 mmol) was added to a

solution of 15 (10 g, 39.6 mmol) in Ac₂O (70 mL), and the reaction

mixture was heated at 70 °C for 2 h with stirring. The reaction mixture

was evaporated to dryness in vacuo, and the residue was recrystallized

from MeOH to give 16, 11.5 g (86%); mp 252−253 °C (lit.³⁵mp

255−260 °C). ¹H NMR (DMSO-d₆) δ 2.43 (3H, s, Me), 3.73 (3H, s,

COOMe), 3.75 (3H, s, COOMe), 4.16 (2H, s, CH₂), 5.21 (2H, s,

CH₂), 7.02−7.06 (1H, m, ArH), 7.11−7.14 (1H, m, ArH), 7.39 (1H,

d, J = 8.0 Hz, ArH), 7.52 (1H, d, J = 7.8 Hz, ArH), 11.12 (1H, brs,

exchangeable, NH).

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Figure 9. Inhibition of topo I activity by 18a and 19a: lane 1, marker for relaxed pHOT1 DNA; lane 2, supercoiled pHOT DNA, no enzyme; lane 3,

DNA plus 1 unit of topo I; lanes 4−7, DNA plus 1 unit of topo I in the presence of 25, 50, 100, and 200 μM 18a; lanes 8−11, DNA plus 1 unit of

topo I in the presence of 25, 50, 100, and 200 μM 19a; lanes 12−14, DNA plus 1 unit of topo I in the presence of 25, 50, and 100 μM irinotecan. All

reaction samples were separated by electrophoresis on a 1% agarose gel, stained with ethidium bromide, and photographed under UV light as

described in the topo I mediated DNA relaxation assay.

Figure 10. Inhibition of topo II catalytic activity by 18a and 19a: lane 1, marker for linear pHOT1 DNA; lane 2, supercoiled pHOT DNA, no

enzyme; lane 3, DNA plus 5 units of topo II; lanes 4−7, DNA plus 5 units of topo II in the presence of 10, 50, 100, and 200 μM 18a; lanes 8−11,

DNA plus 5 units of topo II in the presence of 10, 50, 100, and 200 μM 19a; lanes 12−14, DNA plus 5 units of topo II plus 50, 100, and 200 μM

etoposide. All reaction samples were electrophoresed in 1% agarose gels as described in the topo I mediated DNA relaxation assay.

Dimethyl 3,6-Dimethyl-6,11-dihydro-5H-indolizino[6,7-b]-

indole-1,2-dicarboxylate (17a). Compound 16 (6 g, 17.7 mmol)

was dissolved in DMF (150 mL) and cooled to 0−5 °C. NaH (0.63 g,

26.5 mmol) was then added. After the mixture was stirred for 15 min,

MeI (2.5 g, 17.7 mmol) was added, and the reaction mixture was

stirred for 1 h at 0−5 °C and then at room temperature for 9 h. The

excess hydride was decomposed with MeOH, and the reaction mixture

was evaporated to dryness in vacuo. The residue was crystallized from

MeOH to give 17a, 5.8 g (94%); mp 240−241 °C. ¹H NMR (DMSO-

d₆) δ 2.45 (3H, s, Me), 3.73 (6H, s, COOMe and Me), 3.74 (3H, s,

COOMe), 4.16 (2H, s, CH₂), 5.27 (2H, s, CH₂), 7.05−7.09 (1H, m,

ArH), 7.17−7.21 (1H, m, ArH), 7.47−7.49 (1H, m, ArH), 7.52−7.54

(1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 10.3, 20.8, 29.4, 51.0, 51.3,

102.3, 109.2, 109.4, 112.8, 117.9, 118.9, 121.4, 125.2, 128.4, 132.4,

132.5, 137.4, 164.5, 165.5. Anal. Calcd for C₂₀H₂₀N₂O₄: C, 68.17; H,

5.72; N, 7.95. Found: C, 67.79; H, 5.73; N, 7.92.

Dimethyl 6-Benzyl-3-methyl-6,11-dihydro-5H-indolizino-

[6,7-b]indole-1,2-dicarboxylate (17c). Compound 17c was pre-

pared from 16 (5.1 g, 15 mmol), NaH (0.54 g, 22.5 mmol), and benzyl

1

bromide (2.65 g, 15 mmol). Yield 6.0 g (90%); mp 195−196 °C. H

NMR (DMSO-d₆) δ 2.39 (3H, s, Me), 3.72 (3H, s, COOMe), 3.74

(3H, s, COOMe), 4.21 (2H, s, CH₂), 5.23 (2H, s, CH₂), 5.46 (2H, s,

NCH₂), 7.06−7.14 (4H, m, 4 × ArH), 7.21−7.23 (1H, m, ArH),

7.24−7.29 (2H, m, 2 × ArH), 7.40−7.42 (1H, m, ArH), 7.56−7.58

(1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 10.3, 20.9, 46.2, 51.0, 51.2,

103.3, 109.3, 110.1, 112.9, 118.2, 119.4, 121.8, 125.6, 126.6, 127.3,

128.3, 128.7, 132.3, 132.6, 137.1, 137.9, 164.5, 165.4. Anal. Calcd for

C₂₆H₂₄N₂O₄: C, 72.88; H, 5.65; N, 6.54. Found: C, 72.65; H, 5.64; N,

6.46.

[3,6-Dimethyl-6,11-dihydro-5H-indolizino[6,7-b]indole-1,2-

diyl]dimethanol (18a). A solution of 17a (3.5 g, 10.0 mmol) in

anhydrous DCM (35 mL) was added dropwise to a stirred suspension

of LAH (0.9 g, 25.0 mmol) in anhydrous diethyl ether (20 mL) at 0 to

−5 °C. The reaction mixture was further stirred for 15 min after the

addition was completed. The excess hydride was destroyed by the

sequential addition of water (1 mL), 15% aqueous (aq) NaOH (1

mL), and water (1 mL) at 0 °C. The mixture was ﬁltered through a

pad of Celite and washed with DCM. The combined ﬁltrate and

washings were evaporated to dryness in vacuo. The residue was

crystallized from ether to give 18a. Yield 2.3 g. ¹H NMR (DMSO-d₆) δ

2.29 (3H, s, Me), 3.73 (3H, s, NMe), 3.99 (2H, s, CH₂), 4.38 (4H, br

s, CH₂and exchangeable, OH), 4.44 (2H, s, CH₂), 5.13 (2H, s, CH₂),

7.05 (1H, t, J = 7.4 Hz, ArH), 7.16 (1H, t, J = 7.4 Hz, ArH), 7.46 (1H,

d, J = 7.8 Hz, ArH), 7.53 (1H, d, J = 7.8 Hz, ArH). ¹³C NMR (DMSO-

d₆) δ 9.4, 18.6, 29.3, 54.1, 54.2, 103.5, 109.2, 116.8, 117.9, 118.7, 119.2,

121.1, 121.2, 123.6, 125.6, 130.0, 137.3. Anal. Calcd for

Following the same synthetic procedure as for 17a, the following

compounds were prepared.

Dimethyl 6-Ethyl-3-methyl-6,11-dihydro-5H-indolizino[6,7-

b]indole-1,2-dicarboxylate (17b). Compound 17b was prepared

from 16 (5.1 g, 15 mmol), NaH (0.54 g, 22.5 mmol), and EtI (2.3 g,

15 mmol). Yield 5.1 g (92%); mp 202−203 °C. ¹H NMR (DMSO-d₆)

δ 1.29 (3H, t, J = 6.9 Hz, Me), 2.46 (3H, s, Me), 3.73 (3H, s,

COOMe), 3.74 (3H, s, COOMe), 4.17 (2H, s, CH₂), 4.20 (2H, q, J =

6.9 Hz, CH₂), 5.29 (2H, s, CH₂), 7.05−7.09 (1H, m, ArH), 7.16−7.20

(1H, m, ArH), 7.49−7.51 (1H, m, ArH), 7.53−7.55 (1H, m, ArH).

¹³C NMR (DMSO-d₆) δ 10.4, 15.3, 20.8, 37.6, 51.0, 51.2, 102.6, 109.2,

109.5, 112.9, 118.1, 119.0, 121.5, 125.5, 127.7, 132.4, 132.6, 136.4,

164.5, 165.5. Anal. Calcd for C₂₁H₂₂N₂O₄: C, 68.84; H, 6.05; N, 7.65.

Found: C, 68.46; H, 6.06; N, 7.59.

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C₁₈H₂₀N₂O₂·0.5H₂O: C, 70.80; H, 6.93; N, 9.17. Found: C, 70.85; H,

6.65; N, 9.02.

Following the same synthetic procedure as for 18a, the following

(2H, s, CH₂), 5.09 (2H, s, CH₂), 5.47 (2H, s, NCH₂), 6.89 (2H, br s,

exchangeable, NH), 7.06−7.09 (3H, m, 3 × ArH), 7.11−7.14 (1H, m,

ArH), 7.20−7.22 (1H, m, ArH), 7.27−7.30 (2H, m, 2 × ArH), 7.40−

7.42 (1H, m, ArH), 7.57−7.59 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ

9.3, 15.1, 18.7, 34.9, 46.1, 56.8, 56.9, 104.2, 109.9, 112.5, 114.7, 118.1,

119.1, 121.6, 124.4, 125.9, 126.4, 127.2, 128.6, 129.3, 137.1, 138.0,

156.3, 156.4. Anal. Calcd for C₃₀H₃₄N₄O₄: C, 70.02; H, 6.66; N, 10.89.

Found: C, 69.76; H, 6.64; N, 10.52.

compounds were prepared.

[6-Ethyl-3-methyl-6,11-dihydro-5H-indolizino[6,7-b]indole-

1,2-diyl]dimethanol (18b). Compound 18b was prepared from 17b

1

(3.6 g, 10 mmol) and LAH (0.92 g, 25 mmol). Yield 2.5 g. H NMR

(DMSO-d₆) δ 1.29 (3H, t, J = 7.1 Hz, Me), 2.30 (3H, s, Me), 4.00

(2H, s, CH₂), 4.21 (2H, q, J = 7.1 Hz, CH₂), 4.38 (3H, br s, CH₂and

exchangeable, OH), 4.44 (3H, br s, CH₂and exchangeable, OH), 5.13

(2H, s, CH₂), 7.04−7.07 (1H, m, ArH), 7.14−7.18 (1H, m, ArH),

7.47−7.49 (1H, m, ArH), 7.53−7.55 (1H, m, ArH). ¹³C NMR

(DMSO-d₆) δ 9.4, 15.3, 18.6, 37.5, 54.2, 54.3, 103.8, 109.3, 116.8,

118.0, 118.7, 119.2, 121.1, 121.2, 123.7, 125.9, 129.1, 136.3. Anal.

Calcd for C₁₉H₂₂N₂O₂: C, 73.52; H, 7.14; N, 9.03. Found: C, 73.68;

H, 6.93; N, 8.66.

[6-Benzyl-3-methyl-6,11-dihydro-5H-indolizino[6,7-b]-

indole-1,2-diyl]dimethanol (18c). Compound 18c was prepared

from 17c (5.3 g, 12 mmol) and LAH (1.10 g, 30 mmol). Yield 3.6 g.

¹H NMR (DMSO-d₆) δ 2.21 (3H, s, Me), 4.04 (2H, s, CH₂), 4.36

(3H, br s, CH₂and exchangeable, OH), 4.44 (3H, br s, CH₂and

exchangeable, OH), 5.04 (2H, s, CH₂), 5.47 (2H, s, NCH₂), 7.04−

7.07 (3H, m, 3 × ArH), 7.08−7.10 (1H, m, ArH), 7.20−7.22 (1H, m,

ArH), 7.26−7.30 (2H, m, 2 × ArH), 7.41−7.43 (1H, m, ArH), 7.57−

7.59 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 9.4, 18.7, 46.1, 54.1, 54.2,

104.6, 109.9, 116.9, 118.2, 119.1, 119.4, 121.5, 121.9, 123.6, 125.9,

126.3, 127.2, 128.7, 129.7, 137.1, 138.1. Anal. Calcd for C₂₄H₂₄N₂O₂:

C, 77.39; H, 6.49; N, 7.52. Found: C, 77.19; H, 6.28; N, 7.16.

General Procedure for the Preparation of Bis-

(alkylcarbamate) Derivatives (19a−c and 20a−c). Alkyl iso-

cyanate (5 equiv) was added to a solution of bis(hydroxymethyl)

derivative (17, 1.0 equiv) and TEA (2−3 equiv) in anhydrous DMF.

The reaction mixture was stirred at ambient temperature (for 8−12 h)

under argon. After completion of the reaction, the reaction mixture

was evaporated to dryness in vacuo. The residue was triturated with

ether, and the separated solid was collected by ﬁltration. The desired

product was obtained by either recrystallization or column

chromatography.

[3,6-Dimethyl-6,11-dihydro-5H-indolizino[6,7-b]indole-1,2-

diyl]bis(methylene) Bis(isopropylcarbamate) (20a). Compound

20a was prepared from 18a (0.29 g, 1 mmol), TEA (0.3 mL), and i-Pr

1

isocyanate (0.34 g, 4 mmol). Yield 0.32 g. H NMR (DMSO-d₆) δ

1.02 (12H, d, J = 6.4 Hz, 4 × Me), 2.31 (3H, s, Me), 3.56 (2H, m,

CH), 3.73 (3H, s, NMe), 4.03 (2H, s, CH₂), 4.94 (2H, s, CH₂), 4.98

(2H, s, CH₂), 5.16 (2H, s, CH₂), 6.82 (2H, br s, exchangeable, NH),

7.04−7.07 (1H, m, ArH), 7.15−7.18 (1H, m, ArH), 7.45−7.47 (1H,

m, ArH), 7.52−7.54 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 9.4, 18.6,

22.6, 23.3, 29.3, 42.1, 56.6, 56.8, 103.2, 109.3, 112.4, 114.6, 117.9,

118.8, 121.2, 124.4, 125.5, 126.1, 129.7, 137.4, 155.6. Anal. Calcd for

C₂₆H₃₄N₄O₄: C, 66.93; H, 7.35; N, 12.01. Found: C, 66.79; H, 7.15;

N, 11.73.

[6-Ethyl-3-methyl-6,11-dihydro-5H-indolizino[6,7-b]indole-

1,2-diyl]bis(methylene) Bis(isopropylcarbamate) (20b). Com-

pound 20b was prepared from 18b (0.62 g, 2 mmol), TEA (0.6 mL),

and i-Pr isocyanate (0.68 g, 8 mmol). Yield 0.61 g. ¹H NMR (DMSO-

d₆) δ 1.01 (12H, d, J = 6.4 Hz, 4 × Me), 1.29 (3H, t, J = 7 Hz, Me),

2.33 (3H, s, Me), 3.57 (2H, m, CH), 4.04 (2H, s, CH₂), 4.21 (2H, q, J

= 7 Hz, CH₂), 4.94 (2H, s, CH₂), 4.99 (2H, s, CH₂), 5.17 (2H, s,

CH₂), 6.82 (2H, br s, exchangeable, NH), 7.04−7.08 (1H, m, ArH),

7.15−7.18 (1H, m, ArH), 7.45−7.49 (1H, m, ArH), 7.52−7.54 (1H,

m, ArH). ¹³C NMR (DMSO-d₆) δ 9.4, 15.2, 18.6, 22.6, 37.6, 42.1,

56.6, 56.8, 103.4, 109.3, 112.4, 114.7, 118.0, 118.8, 121.2, 124.4, 125.8,

126.2, 128.8, 136.3, 155.6, 155.7. Anal. Calcd for C₂₇H₃₆N₄O₄: C,

67.48; H, 7.55; N, 11.66. Found: C, 67.26; H, 7.59; N, 11.54.

[6-Benzyl-3-methyl-6,11-dihydro-5H-indolizino[6,7-b]-

indole-1,2-diyl]bis(methylene) Bis(isopropylcarbamate) (20c).

Compound 20c was prepared from 18c (0.18 g, 0.5 mmol), TEA (0.2

1

mL), and i-Pr isocyanate (0.17 g, 2 mmol). Yield 0.18 g. H NMR

(DMSO-d₆) δ 1.01 (12H, d, J = 6.4 Hz, 4 × Me), 2.25 (3H, s, Me),

3.57 (2H, m, CH), 4.09 (2H, s, CH₂), 4.92 (2H, s, CH₂), 4.99 (2H, s,

CH₂), 5.10 (2H, s, CH₂), 5.47 (2H, s, NCH₂), 6.81 (2H, br s,

exchangeable, NH), 7.06−7.09 (3H, m, 3 × ArH), 7.11−7.14 (1H, m,

ArH), 7.20−7.24 (1H, m, ArH), 7.27−7.30 (2H, m, 2 × ArH), 7.41−

7.43 (1H, m, ArH), 7.56−7.58 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ

9.3, 18.7, 22.6, 42.1, 46.1, 56.6, 56.8, 104.2, 109.9, 112.6, 114.8, 118.1,

119.2, 121.6, 124.3, 125.9, 126.1, 127.2, 128.6, 129.3, 137.1, 138.0,

155.6. Anal. Calcd for C₃₂H₃₈N₄O₄: C, 70.82; H, 7.06; N, 10.32.

Found: C, 70.55; H, 6.93; N, 10.03.

Methyl 1,2,3,4-Tetrahydro-9H-pyrido[3,4-b]indole-3-carbox-

ylate (22). Formalin [37% (12.5 mL)] was added to ^L-tryptophan

methyl ester hydrochloride (21, 25.4 g, 100 mmol) in aqueous MeOH

(170 mL; ratio 10:1). The resulting mixture was stirred at room

temperature for 5 h. The reaction mixture was evaporated to dryness

in vacuo. The residue was treated with sodium bicarbonate. A white

solid separated out and was ﬁltered, washed with water, and dried. The

resulting powder weighed 14.4 g (63%); mp 161−162 °C (lit.³⁵mp

164−165 °C). ¹H NMR (DMSO-d₆) δ 2.78 (1H, m, CH₂), 2.98 (1H,

m, CH₂), 3.66 (3H, s, COOMe), 3.85 (1H, m, CH), 4.02 (2H, q, J =

15.8 Hz, NCH₂), 6.92−6.96 (1H, m, ArH), 7.00−7.04 (1H, m, ArH),

7.27−7.29 (1H, m, ArH), 7.37−7.39 (1H, m, ArH), 10.78 (1H, br s,

exchangeable NH).

[3,6-Dimethyl-6,11-dihydro-5H-indolizino[6,7-b]indole-1,2-

diyl]bis(methylene) Bis(ethylcarbamate) (19a). Compound 19a

was prepared from 18a (0.29 g, 1 mmol), TEA (0.3 mL), and ethyl

isocyanate (0.28 g, 4 mmol). Yield 0.3 g. ¹H NMR (DMSO-d₆) δ 0.98

(6H, t, J = 6.9 Hz, 2 × Me), 2.32 (3H, s, Me), 2.98 (4H, q, J = 6.9 Hz,

CH₂), 3.73 (3H, s, NMe), 4.04 (2H, s, CH₂), 4.94 (2H, s, CH₂), 4.98

(2H, s, CH₂), 5.17 (2H, s, CH₂), 6.89 (2H, br s, exchangeable, NH),

7.04−7.08 (1H, m, ArH), 7.15−7.19 (1H, m, ArH), 7.46−7.48 (1H,

m, ArH), 7.52−7.54 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 9.4, 15.1,

18.6, 29.4, 34.9, 56.8, 56.9, 103.2, 109.3, 112.4, 114.6, 117.9, 118.8,

121.2, 124.5, 125.5, 126.2, 129.7, 137.4, 156.3. Anal. Calcd for

C₂₄H₃₀N₄O₄: C, 65.73; H, 6.90; N, 12.78. Found: C, 65.53; H, 6.75;

N, 12.44.

[6-Ethyl-3-methyl-6,11-dihydro-5H-indolizino[6,7-b]indole-

1,2-diyl]bis(methylene) Bis(ethylcarbamate) (19b). Compound

19b was prepared from 18b (0.62 g, 2 mmol), TEA (0.6 mL), and

ethyl isocyanate (0.56 g, 8 mmol). Yield 0.52 g. ¹H NMR (DMSO-d₆)

δ 0.98 (6H, t, J = 7.1 Hz, 2 × Me), 1.29 (3H, t, J = 7 Hz, Me), 2.33

(3H, s, Me), 2.97 (4H, q, J = 7.1 Hz, CH₂), 4.04 (2H, s, CH₂), 4.21

(2H, q, J = 7 Hz, CH₂), 4.94 (2H, s, CH₂), 4.99 (2H, s, CH₂), 5.17

(2H, s, CH₂), 6.89 (2H, br s, exchangeable, NH), 7.04−7.08 (1H, m,

ArH), 7.15−7.18 (1H, m, ArH), 7.47−7.49 (1H, m, ArH), 7.52−7.54

(1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 9.4, 15.1, 15.2, 18.6, 34.9,

37.5, 56.8, 56.9, 103.4, 109.4, 112.4, 114.6, 118.0, 118.8, 121.2, 124.4,

125.8, 128.8, 136.3, 156.3. Anal. Calcd for C₂₅H₃₂N₄O₄·0.5H₂O: C,

65.06; H, 7.21; N, 12.14. Found: C, 64.71; H, 7.09; N, 12.03.

[6-Benzyl-3-methyl-6,11-dihydro-5H-indolizino[6,7-b]-

indole-1,2-diyl]bis(methylene) Bis(ethylcarbamate) (19c).

Compound 19c was prepared from 18c (0.37 g, 1 mmol), TEA (0.3

Methyl 2-Propionyl-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]-

indole-3-carboxylate (23a). A mixture of 22 (6.9 g, 30 mmol),

propionyl chloride (2.7 g, 30 mmol), and TEA (4.8 mL) in anhydrous

THF (150 mL) was reﬂuxed for 9 h. The reaction mixture was

concentrated under vacuo and then diluted with water. The solid

separated out and was ﬁltered and washed with water and hexane to

1

mL), and ethyl isocyanate (0.28 g, 4 mmol). Yield 0.3 g. H NMR

give 23a. Yield 5.1 g (60%); mp 73−74 °C. H NMR (DMSO-d₆) δ

(DMSO-d₆) δ 0.98 (6H, t, J = 7.1 Hz, 2 × Me), 2.24 (3H, s, Me), 2.97

1.05 (3H, m, Me), 2.50 (2H, m, CH₂), 3.02 (1H, m, CH₂), 3.34 (1H,

(4H, q, J = 7.1 Hz, CH₂), 4.09 (2H, s, CH₂), 4.92 (2H, s, CH₂), 4.99

m, CH₂), 3.55 and 3.57 (3H, s, COOMe), 4.14 and 5.06 (1H, d, J = 17

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Hz, NCH₂), 4.60 and 4.90 (1H, d, J = 15.8 Hz, NCH₂), 5.29 and 5.72

(1H, d, J = 5.2 Hz, CH), 6.94−6.98 (1H, m, ArH), 7.03−7.07 (1H, m,

ArH), 7.27−7.31 (1H, m, ArH), 7.41−7.43 (1H, m, ArH), 10.89 and

10.90 (1H, br s, exchangeable NH). ¹³C NMR (DMSO-d₆) δ 9.0, 22.6,

23.3, 38.6, 52.2, 67.0, 104.2, 111.0, 117.6, 118.6, 118.7, 120.9, 126.2,

129.8, 136.2, 171.4, 173.7. Anal. Calcd for C₁₆H₁₈N₂O₃·0.55H₂O: C,

64.87; H, 6.50; N, 9.46. Found: C, 65.03; H, 6.75; N, 9.06.

Following the same synthetic procedure as for 23a, the following

compounds were prepared.

ArH), 7.25−7.31 (1H, m, ArH), 7.48−7.55 (3H, m, 3 × ArH), 7.68−

7.72 (1H, m, ArH), 10.67 and 10.95 (1H, br s, exchangeable NH). ¹³C

NMR (DMSO-d₆) δ 22.6, 43.8, 52.6, 56.9, 104.6, 111.1, 117.5, 118.7,

120.2, 121.2, 126.3, 127.7, 129.4, 130.4, 131.5, 133.4, 136.2, 158.3,

164.1, 168.9, 170.8. Anal. Calcd for C₂₀H₁₆ClFN₂O₃: C, 62.10; H,

4.17; N, 7.24. Found: C, 61.89; H, 4.03; N, 6.97.

Methyl 2-(4-Methoxybenzoyl)-1,2,3,4-tetrahydro-9H-

pyrido[3,4-b]indole-3-carboxylate (23g). Compound 23g was

prepared from 22 (6.9 g, 30 mmol), 4-methoxybenzoyl chloride (5.1 g,

30 mmol), and TEA (4.8 mL). Yield 7.3 g (67%); mp 159−160 °C. ¹H

NMR (DMSO-d₆) δ 3.11 (1H, m, CH₂), 3.36 (1H, m, CH₂), 3.54 and

3.64 (3H, s, COOMe), 3.82 (3H, s, OMe), 4.37 and 5.11 (1H, d, J =

17.6 Hz, NCH₂), 4.66 (1H, m, NCH₂), 4.97 and 5.76 (1H, m, CH),

6.96−6.99 (1H, m, ArH), 7.03−7.06 (3H, m, 3 × ArH), 7.25−7.28

(1H, m, ArH), 7.43−7.46 (3H, m, 3 × ArH), 10.67 and 10.94 (1H, br

s, exchangeable NH). ¹³C NMR (DMSO-d₆) δ 22.6, 44.1, 52.6, 55.2,

57.1, 104.6, 111.1, 114.1, 117.7, 118.7, 121.1, 126.2, 127.6, 128.7,

129.6, 130.0, 136.2, 160.5, 170.9, 171.4. Anal. Calcd for C₂₁H₂₀N₂O₄:

C, 69.22; H, 5.53; N, 7.69. Found: C, 69.07; H, 5.58; N, 7.57.

Methyl 2-(3,4-Dimethoxybenzoyl)-1,2,3,4-tetrahydro-9H-

pyrido[3,4-b]indole-3-carboxylate (23h). Compound 23h was

prepared from 22 (6.9 g, 30 mmol), 3,4-dimethoxybenzoyl chloride (6

g, 30 mmol), and TEA (4.8 mL). Yield 7.2 g (61%); mp 195−196 °C.

¹H NMR (DMSO-d₆) δ 3.12 (1H, m, CH₂), 3.34 (1H, m, CH₂), 3.55

and 3.64 (3H, s, COOMe), 3.78 (3H, s, OMe), 3.81 (3H, s, OMe),

4.37 and 5.13 (1H, d, J = 17.8 Hz, NCH₂), 4.67 (1H, m, NCH₂), 5.02

and 5.74 (1H, m, CH), 6.96−6.99 (1H, m, ArH), 7.02−7.08 (4H, m, 4

× ArH), 7.26−7.31 (1H, m, ArH), 7.44−7.46 (1H, m, ArH), 10.66

and 10.94 (1H, br s, exchangeable NH). ¹³C NMR (DMSO-d₆) δ 22.7,

44.2, 51.3, 52.6, 57.1, 104.6, 110.3, 111.3, 117.7, 118.7, 121.1, 123.2,

126.3, 127.7, 129.7, 136.2, 149.4, 170.1, 171.4. Anal. Calcd for

C₂₂H₂₂N₂O₅: C, 66.99; H, 5.62; N, 7.10. Found: C, 66.63; H, 5.62; N,

6.89.

Methyl 2-(Benzoyl)-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]-

indole-3-carboxylate (23b). Compound 23b was prepared from

22 (6.9 g, 30 mmol), benzoyl chloride (4.2 g, 30 mmol), and TEA (4.8

mL). Yield 7.5 g (75%); mp 193−194 °C (lit.⁴³218−219 °C); H

1

NMR (DMSO-d₆) δ 3.09 (1H, m, CH₂), 3.33 (1H, m, CH₂), 3.54 and

3.65 (3H, s, COOMe), 4.39 and 5.18 (1H, d, J = 17.1 Hz, NCH₂),

4.52 and 4.64 (1H, d, J = 16.6 Hz, NCH₂), 4.86 and 5.81 (1H, m,

CH), 6.95−6.99 (1H, m, ArH), 7.04−7.06 (1H, m, ArH), 7.24−7.31

(1H, m, ArH), 7.43−7.45 (2H, m, 2 × ArH), 7.46−7.51 (4H, m, 4 ×

ArH), 10.66 and 10.95 (1H, br s, exchangeable NH). ¹³C NMR

(DMSO-d₆) δ 22.4, 43.8, 52.5, 64.9, 104.5, 111.1, 117.7, 118.7, 121.2,

126.2, 126.5, 128.7, 129.4, 130.0, 135.7, 136.1, 170.8, 171.1. Anal.

Calcd for C₂₀H₁₈N₂O₃: C, 71.84; H, 5.43; N, 8.38. Found: C, 71.80;

H, 5.42; N, 8.46.

Methyl 2-(4-Fluorobenzoyl)-1,2,3,4-tetrahydro-9H-pyrido-

[3,4-b]indole-3-carboxylate (23c). Compound 23c was prepared

from 22 (6.9 g, 30 mmol), 4-ﬂuorobenzoyl chloride (4.7 g, 30 mmol),

1

and TEA (4.8 mL). Yield 9.2 g (88%); mp 191−192 °C. H NMR

(DMSO-d₆) δ 3.10 (1H, m, CH₂), 3.33 (1H, m, CH₂), 3.55 and 3.65

(3H, s, COOMe), 4.38 and 5.16 (1H, d, J = 17.1 Hz, NCH₂), 4.54 and

4.66 (1H, d, J = 16.2 Hz, NCH₂), 4.89 and 5.80 (1H, m, CH), 6.96−

7.00 (1H, m, ArH), 7.05−7.07 (1H, m, ArH), 7.26−7.33 (3H, m, 3 ×

ArH), 7.43−7.45 (1H, m, ArH), 7.53−7.57 (2H, m, 2 × ArH), 10.68

and 10.95 (1H, br s, exchangeable NH). ¹³C NMR (DMSO-d₆) δ 22.4,

43.9, 52.5, 57.0, 104.6, 111.1, 115.8, 117.7, 118.7, 121.2, 126.3, 129.3,

132.1, 136.2, 161.9, 170.3, 171.0. Anal. Calcd for C₂₀H₁₇FN₂O₃: C,

68.17; H, 4.86; N, 7.95. Found: C, 68.43; H, 4.85; N, 7.77.

Methyl 2-(3,4,5-Trimethoxybenzoyl)-1,2,3,4-tetrahydro-9H-

pyrido[3,4-b]indole-3-carboxylate (23i). Compound 23i was

prepared from 22 (4.6 g, 20 mmol), 3,4,5-trimethoxybenzoyl chloride

(4.6 g, 20 mmol), and TEA (3.2 mL). Yield 4.9 g (58%); mp 207−208

Methyl 2-(4-Chlorobenzoyl)-1,2,3,4-tetrahydro-9H-pyrido-

[3,4-b]indole-3-carboxylate (23d). Compound 23d was prepared

from 22 (6.9 g, 30 mmol), 4-chlorobenzoylchloride (5.2 g, 30 mmol),

and TEA (4.8 mL). Yield 9 g (82%); mp 243−244 °C (lit.^35,37245−

1

°C. H NMR (DMSO-d₆) δ 3.11 (1H, m, CH₂), 3.34 (1H, m, CH₂),

3.57 and 3.65 (3H, s, COOMe), 3.71 (3H, s, OMe), 3.80 (6H, s, 2 ×

OMe), 4.36 and 5.16 (1H, d, J = 16.9 Hz, NCH₂), 4.64 (1H, m,

NCH₂), 4.99 and 5.76 (1H, m, CH), 6.72 (2H, s, 2 × ArH), 6.96−6.99

(1H, m, ArH), 7.04−7.07 (1H, m, ArH), 7.31−7.33 (1H, m, ArH),

7.42−7.44 (1H, m, ArH), 10.66 and 10.94 (1H, br s, exchangeable

NH). ¹³C NMR (DMSO-d₆) δ 22.6, 43.9, 52.5, 56.1, 60.1, 104.1,

111.1, 117.7, 118.1, 121.1, 126.3, 129.5, 130.9, 136.2, 138.6, 153.1,

170.5, 171.1. Anal. Calcd for C₂₃H₂₄N₂O₆: C, 65.08; H, 5.70; N, 6.60.

Found: C, 64.79; H, 5.75; N, 6.45.

1

246 °C). H NMR (DMSO-d₆) δ 3.10 (1H, m, CH₂), 3.35 (1H, m,

CH₂), 3.55 and 3.65 (3H, s, COOMe), 4.41 and 5.18 (1H, d, J = 17.4

Hz, NCH₂), 4.51 and 4.64 (1H, d, J = 16.4 Hz, NCH₂), 4.88 and 5.82

(1H, d, J = 5.4 Hz, CH), 6.96−6.99 (1H, m, ArH), 7.03−7.08 (1H, m,

ArH), 7.25−7.32 (1H, m, ArH), 7.42−7.60 (5H, m, 5 × ArH), 10.65

and 10.95 (1H, br s, exchangeable NH). ¹³C NMR (DMSO-d₆) δ 22.5,

43.8, 52.5, 67.0, 104.6, 111.1, 117.7, 118.7, 121.1, 126.1, 128.6, 128.8,

129.2, 134.5, 136.1, 169.4, 170.8.

Methyl 2-(3,4-Diﬂuorobenzoyl)-1,2,3,4-tetrahydro-9H-

pyrido[3,4-b]indole-3-carboxylate (23e). Compound 23e was

prepared from 22 (4.6 g, 20 mmol), 3,4-diﬂuorobenzoylchloride (3.5

g, 20 mmol), and TEA (3.2 mL). Yield 5.8 g (78%); mp 81−82 °C. ¹H

NMR (DMSO-d₆) δ 3.10 (1H, m, CH₂), 3.35 (1H, m, CH₂), 3.55 and

3.65 (3H, s, COOMe), 4.36 and 5.14 (1H, d, J = 16.6 Hz, NCH₂),

4.55 and 4.67 (1H, d, J = 15.5 Hz, NCH₂), 4.92 and 5.78 (1H, d, J =

5.2 Hz, CH), 6.96−6.99 (1H, m, ArH), 7.04−7.06 (1H, m, ArH),

7.27−7.33 (2H, m, 2 × ArH), 7.43−7.45 (1H, m, ArH), 7.52−7.57

(2H, m, 2 × ArH), 10.67 and 10.94 (1H, br s, exchangeable NH). ¹³C

NMR (DMSO-d₆) δ 22.5, 43.8, 52.6, 56.9, 104.6, 111.1, 116.5, 117.7,

118.2, 118.7, 121.1, 124.1, 126.2, 129.2, 132.9, 136.2, 149.4, 169.4,

170.7. Anal. Calcd for C₂₀H₁₆F₂N₂O₃: C, 64.86; H, 4.35; N, 7.56.

Found: C, 64.63; H, 4.23; N, 7.18.

2-Propionyl-9-methyl-1,2,3,4-tetrahydropyrido[3,4-b]-

indole-3-carboxylic Acid (24a). Compound 23a (4.2 g, 15 mmol)

was dissolved in THF (150 mL), and NaH (0.72 g, 30 mmol) was

added to the reaction mixture at 0−5 °C. After the mixture was stirred

for 15 min, MeI (2.1 g, 15 mmol) was added and the reaction mixture

was stirred for 1 h at 0−5 °C and then at room temperature for 8 h.

The excess of hydride was decomposed by the treatment of MeOH.

The reaction mixture was concentrated in vacuo and diluted with

water. The pH was adjusted to 6 with acetic acid to give 2.8 g (67%);

1

mp 246−247 °C. H NMR (acetic acid-d₄) δ 1.20 (3H, m, Me), 2.67

(2H, m, CH₂), 3.09 (1H, m, CH₂), 3.52 (1H, m, CH₂), 3.62 and 3.64

(3H, s, NMe), 4.45 and 5.28 (1H, d, J = 17.5 Hz, NCH₂), 4.83 and

4.92 (1H, d, J = 15.2 Hz, NCH₂), 5.23 and 6.02 (1H, d, J = 5.2 Hz,

CH), 7.02−7.07 (1H, m, ArH), 7.12−7.17 (1H, m, ArH), 7.28−7.30

(1H, m, ArH), 7.44−7.46 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 9.1,

22.6, 26.2, 29.1, 37.9, 67.0, 104.3, 109.2, 117.7, 118.7, 120.9, 125.8,

131.1, 136.9, 172.4, 173.8. Anal. Calcd for C₁₆H₁₈N₂O₃: C, 67.12; H,

6.34; N, 9.78. Found: C, 66.97; H, 6.72; N, 9.43.

Methyl 2-(3-Chloro-4-ﬂuorobenzoyl)-1,2,3,4-tetrahydro-9H-

pyrido[3,4-b]indole-3-carboxylate (23f). Compound 23f was

prepared from 22 (4.6 g, 20 mmol), 3-chloro-4-ﬂuorobenzoyl chloride

(3.8 g, 20 mmol), and TEA (3.2 mL). Yield 6.1 g (79%); mp 97−98

Following the same synthetic procedure as for 24a, the following

compounds were prepared.

1

°C. H NMR (DMSO-d₆) δ 3.09 (1H, m, CH₂), 3.32 (1H, m, CH₂),

3.56 and 3.65 (3H, s, COOMe), 4.37 and 5.15 (1H, d, J = 17.4 Hz,

NCH₂), 4.55 and 4.67 (1H, d, J = 16.2 Hz, NCH₂), 4.93 and 5.79 (1H,

d, J = 4.9 Hz, CH), 6.96−6.99 (1H, m, ArH), 7.05−7.08 (1H, m,

2-(Benzoyl)-9-methyl-1,2,3,4-tetrahydropyrido[3,4-b]indole-

3-carboxylic Acid (24b). Compound 24b was prepared from 23b

(6.7 g, 20 mmol), NaH (0.96 g, 40 mmol), and MeI (2.8 g, 20 mmol).

L

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Yield 4.8 g (73%); mp 164−165 °C (lit.⁴⁴175−176 °C). H NMR

(acetic acid-d₄) δ 3.18 (1H, m, CH₂), 3.52 (1H, m, CH₂), 3.43 and

3.68 (3H, s, NMe), 4.64 and 5.40 (1H, m, NCH₂), 4.71 and 4.91 (1H,

m, NCH₂), 5.03 and 6.14 (1H, m, CH), 7.03−7.06 (1H, m, ArH),

7.14−7.17 (1H, m, ArH), 7.30−7.33 (1H, m, ArH), 7.50−7.56 (4H,

m, 4 × ArH), 7.57−7.59 (2H, m, 2 × ArH). ¹³C NMR (DMSO-d₆) δ

22.5, 29.1, 38.3, 56.8, 104.4, 109.3, 117.8, 118.7, 120.9, 125.8, 126.4,

128.8, 129.8, 130.9, 136.1, 136.9, 171.5, 171.9. Anal. Calcd for

C₂₀H₁₈N₂O₃: C, 71.84; H, 5.43; N, 8.38. Found: C, 71.68; H, 5.37; N,

8.36.

NMR (DMSO-d₆) δ 23.2, 29.2, 50.8, 55.3, 56.9, 104.4, 109.3, 114.1,

117.8, 118.8, 120.9, 125.8, 128.0, 128.5, 129.1, 131.0, 136.9, 160.4,

170.3, 171.9. Anal. Calcd for C₂₁H₂₀N₂O₄: C, 69.22; H, 5.53; N, 7.69.

Found: C, 69.11; H, 5.77; N, 7.96.

1

2 - ( 3 , 4 - D i m e t h o x y b e n z o y l ) - 9 - m e t h y l - 1 , 2 , 3 , 4 -

tetrahydropyrido[3,4-b]indole-3-carboxylic Acid (24h). Com-

pound 24h was prepared from 23h (5.9 g, 15 mmol), NaH (0.72 g, 30

mmol), and MeI (2.1 g, 15 mmol). Yield 4.7 g (79%); mp 197−198

1

°C. H NMR (acetic acid-d₄) δ 3.19 (1H, m, CH₂), 3.56 (1H, m,

CH₂), 3.47 and 3.68 (3H, s, NMe), 3.86 (3H, s, OMe), 3.88 (3H, s,

OMe), 4.66 and 5.38 (1H, d, J = 17.4 Hz, NCH₂), 4.86 and 4.98 (1H,

d, J = 15.6 Hz, NCH₂), 5.22 and 6.09 (1H, d, J = 5.2 Hz, CH), 6.99−

7.02 (1H, m, ArH), 7.04−7.07 (1H, m, ArH), 7.14−7.18 (2H, m, 2 ×

ArH), 7.21−7.25 (1H, m, ArH), 7.31−7.33 (1H, m, ArH), 7.46−7.48

(1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 23.4, 29.2, 50.8, 55.5, 57.4,

104.8, 109.2, 110.4, 111.5, 117.7, 118.6, 119.4, 120.8, 131.3, 136.9,

148.5, 170.9, 172.3. Anal. Calcd for C₂₂H₂₂N₂O₅: C, 66.99; H, 5.62; N,

7.10. Found: C, 66.73; H, 5.85; N, 6.81.

2-(4-Fluorobenzoyl)-9-methyl-1,2,3,4-tetrahydropyrido[3,4-

b]indole-3-carboxylic Acid (24c). Compound 24c was prepared

from 23c (3.5 g, 10 mmol), NaH (0.46g, 20 mmol), and MeI (1.4 g,

10 mmol). Yield 2.6 g (74%); mp 266−267 °C. ¹H NMR (acetic acid-

d₄) δ 3.18 (1H, m, CH₂), 3.56 (1H, m, CH₂), 3.46 and 3.68 (3H, s,

NMe), 4.64 and 5.40 (1H, d, J = 17.1 Hz, NCH₂), 4.70 and 4.93 (1H,

d, J = 15.7 Hz, NCH₂), 5.04 and 6.11 (1H, d, J = 4.7 Hz, CH), 7.03−

7.07 (1H, m, ArH), 7.14−7.18 (1H, m, ArH), 7.21−7.25 (2H, m, 2 ×

ArH), 7.31−7.33 (1H, m, ArH), 7.45−7.49 (1H, m, ArH), 7.60−7.66

(2H, m, 2 × ArH). ¹³C NMR (DMSO-d₆) δ 23.2, 43.9, 50.9, 57.1,

104.8, 111.1, 115.8, 117.6, 118.6, 121.1, 126.3, 129.2, 132.5, 136.2,

161.2, 170.6, 172.0. Anal. Calcd for C₂₀H₁₇FN₂O₃: C, 68.17; H, 4.86;

N, 7.95. Found: C, 68.21; H, 5.21; N, 7.88.

2-(3,4,5-Trimethoxybenzoyl)-9-methyl-1,2,3,4-

tetrahydropyrido[3,4-b]indole-3-carboxylic Acid (24i). Com-

pound 24i was prepared from 23i (4.2 g, 10 mmol), NaH (0.48 g,

20 mmol), and MeI (1.4 g, 10 mmol). Yield 3.2 g (76%); mp 280−282

1

°C. H NMR (acetic acid-d₄) δ 3.21 (1H, m, CH₂), 3.56 (1H, m,

CH₂), 3.47 and 3.69 (3H, s, NMe), 3.84 (3H, s, OMe), 3.87 (6H, s, 2

× OMe), 4.66 and 5.39 (1H, d, J = 16.9 Hz, NCH₂), 4.83 and 4.95

(1H, d, J = 16.1 Hz, NCH₂), 5.18 and 6.08 (1H, d, J = 5.2 Hz, CH),

6.86 (2H, s, 2 × ArH), 7.04−7.07 (1H, m, ArH), 7.15−7.18 (1H, m,

ArH), 7.32−7.34 (1H, m, ArH), 7.45−7.47 (1H, m, ArH). ¹³C NMR

(DMSO-d₆) δ 24.0, 29.2, 56.1, 59.0, 60.2, 104.3, 109.1, 117.8, 118.5,

120.6, 126.5, 131.8, 132.9, 136.9, 138.1, 152.9, 170.8, 172.5. Anal.

Calcd for C₂₃H₂₄N₂O₆: C, 65.08; H, 5.70; N, 6.60. Found: C, 64.89;

H, 5.31; N, 6.87.

Dimethyl 3-(Ethyl)-6-methyl-6,11-dihydro-5H-indolizino-

[6,7-b]indole-1,2-dicarboxylate (25a). DMAD (1.7 g, 12 mmol)

was added to a solution of 24a (2.2 g, 8 mmol) in Ac₂O (30 mL), and

the reaction mixture was heated at 70 °C for 2 h with stirring. The

reaction mixture was evaporated to dryness in vacuo, and the residue

was recrystallized from MeOH to give 1.8 g (64%); mp 231−232 °C.

¹H NMR (DMSO-d₆) δ 1.19 (3H, t, J = 7.4 Hz, Me), 2.87 (2H, q, J =

7.4 Hz, CH₂), 3.73 (3H, s, COOMe), 3.74 (3H, s, COOMe), 3.75

(3H, s, NMe), 4.17 (2H, s, CH₂), 5.34 (2H, s, CH₂), 7.06−7.09 (1H,

m, ArH), 7.17−7.21 (1H, m, ArH), 7.47−7.49 (1H, m, ArH), 7.53−

7.55 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 14.3, 17.7, 20.9, 29.5,

51.1, 51.3, 102.3, 109.4, 112.6, 118.0, 119.0, 121.5, 125.2, 128.7, 132.2,

137.5, 137.8, 164.6, 165.5. Anal. Calcd for C₂₁H₂₂N₂O₄: C, 68.84; H,

6.05; N, 7.65. Found: C, 68.73; H, 5.96; N, 7.64.

2-(4-Chlorobenzoyl)-9-methyl-1,2,3,4-tetrahydropyrido[3,4-

b]indole-3-carboxylic Acid (24d). Compound 24d was prepared

from 23d (7.3 g, 20.0 mmol), NaH (0.96 g, 40.0 mmol), and MeI (2.8

g, 20.0 mmol). Yield 5.4 g (74%); mp 262−263 °C. H NMR (acetic

1

acid-d₄) δ 3.18 (1H, m, CH₂), 3.56 (1H, m, CH₂), 3.45 and 3.68 (3H,

s, NMe), 4.64 and 5.40 (1H, d, J = 15.8 Hz, NCH₂), 4.70 and 4.93

(1H, d, J = 17.1 Hz, NCH₂), 5.02 and 6.12 (1H, d, J = 5.4 Hz, CH),

7.04−7.07 (1H, m, ArH), 7.15−7.18 (1H, m, ArH), 7.31−7.33 (1H,

m, ArH), 7.45−7.47 (1H, m, ArH), 7.50−7.55 (3H, m, 3 × ArH),

7.59−7.61 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 23.1, 29.2, 50.8,

56.8, 104.4, 109.3, 117.8, 118.8, 121.0, 125.8, 128.7, 130.7, 134.6,

136.9, 170.6, 171.7. Anal. Calcd for C₂₀H₁₇ClN₂O₃: C, 65.13; H, 4.65;

N, 7.60. Found: C, 64.77; H, 4.79; N, 7.68.

2-(3,4-Diﬂuorobenzoyl)-9-methyl-1,2,3,4-tetrahydropyrido-

[3,4-b]indole-3-carboxylic Acid (24e). Compound 24e was

prepared from 23e (5.5 g, 15 mmol), NaH (0.72 g, 30 mmol), and

MeI (2.1 g, 15 mmol). Yield 4.2 g (76%); mp 231−232 °C. ¹H NMR

(acetic acid-d₄) δ 3.17 (1H, m, CH₂), 3.55 (1H, m, CH₂), 3.46 and

3.68 (3H, s, NMe), 4.64 and 5.37 (1H, m, NCH₂), 4.69 and 4.92 (1H,

m, NCH₂), 5.02 and 6.08 (1H, m, CH), 7.03−7.08 (1H, m, ArH),

7.13−7.17 (1H, m, ArH), 7.31−7.38 (3H, m, 3 × ArH), 7.47−7.51

(2H, m, 2 × ArH). ¹³C NMR (DMSO-d₆) δ 23.0, 29.2, 50.9, 56.9,

104.6, 109.3, 111.1, 116.3, 117.7, 118.3, 121.0, 123.9, 130.6, 136.2,

136.9, 170.6, 171.8. Anal. Calcd for C₂₀H₁₆F₂N₂O₃: C, 64.86; H, 4.35;

N, 7.56. Found: C, 64.63; H, 4.47; N, 7.57.

Following the same synthetic procedure as for 25a, the following

compounds were prepared:

2-(3-Chloro-4-ﬂuorobenzoyl)-9-methyl-1,2,3,4-

tetrahydropyrido[3,4-b]indole-3-carboxylic Acid (24f). Com-

pound 24f was prepared from 23f (6 g, 15 mmol), NaH (0.72 g, 30

mmol), and MeI (2.1 g, 15 mmol). Yield 5.1 g (85%); mp 183−184

Dimethyl 3-(Phenyl)-6-methyl-6,11-dihydro-5H-indolizino-

[6,7-b]indole-1,2-dicarboxylate (25b). Compound 25b was

prepared from 24b (3.3 g, 10 mmol) and DMAD (2.1 g, 15 mmol)

in AC₂O (40 mL). Yield 3.0 g, (74%); mp 249−250 °C. H NMR

(DMSO-d₆) δ 3.58 (6H, s, 2 × COOMe), 3.77 (3H, s, NMe), 4.30

(2H, s, CH₂), 5.17 (2H, s, CH₂), 7.06−7.9 (1H, m, ArH), 7.16−7.19

(1H, m, ArH), 7.44−7.46 (1H, m, ArH), 7.47−7.53 (5H, m, 5 ×

ArH), 7.55−7.57 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 21.3, 29.4,

41.9, 51.2, 51.7, 102.2, 109.2, 116.4, 118.0, 119.1, 121.5, 125.1, 128.5,

129.7, 130.3, 133.0, 133.9, 137.4, 164.1, 165.8. Anal. Calcd for

C₂₅H₂₂N₂O₄: C, 72.45; H, 5.35; N, 6.76. Found: C, 72.34; H, 5.33; N,

6.72.

1

°C. H NMR (acetic acid-d₄) δ 3.18 (1H, m, CH₂), 3.57 (1H, m,

CH₂), 3.47 and 3.69 (3H, s, NMe), 4.65 and 5.37 (1H, d, J = 17.2 Hz,

NCH₂), 4.71 and 4.94 (1H, d, J = 15.8 Hz, NCH₂), 5.03 and 6.09 (1H,

d, J = 4 Hz, CH), 7.04−7.08 (1H, m, ArH), 7.15−7.19 (1H, m, ArH),

7.32−7.37 (2H, m, 2 × ArH), 7.45−7.51 (2H, m, 2 × ArH), 7.68−

7.70 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 23.1, 29.1, 50.6, 57.4,

104.3, 109.2, 117.3, 118.7, 120.1, 129.0, 130.7, 136.9, 170.8, 171.5.

Anal. Calcd for C₂₀H₁₆ClFN₂O₃: C, 62.10; H, 4.17; N, 7.24. Found: C,

61.88; H, 4.37; N, 7.31.

2-(4-Methoxybenzoyl)-9-methyl-1,2,3,4-tetrahydropyrido-

[3,4-b]indole-3-carboxylic Acid (24g). Compound 24g was

prepared from 23g (3.6 g, 10 mmol), NaH (0.48 g, 20 mmol), and

MeI (1.4 g, 10 mmol). Yield 2.3 g (64%); mp 222−223 °C. ¹H NMR

(acetic acid-d₄) δ 3.19 (1H, m, CH₂), 3.56 (1H, m, CH₂), 3.47 and

3.68 (3H, s, NMe), 3.85 (3H, s, OMe), 4.66 and 5.37 (1H, d, J = 17.2

Hz, NCH₂), 4.81 and 4.97 (1H, d, J = 15.2 Hz, NCH₂), 5.16 and 6.10

(1H, d, J = 5 Hz, CH), 7.01−7.05 (3H, m, 3 × ArH), 7.14−7.17 (1H,

m, ArH), 7.31−7.33 (1H, m, ArH), 7.46−7.52 (3H, m, 3 × ArH). ¹³C

Dimethyl 3-(4-Fluorophenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-dicarboxylate (25c). Compound 25c

was prepared from 24c (1.7 g, 4.8 mmol) and DMAD (1.0 g, 7.2

1

mmol) in AC₂O (15 mL). Yield 1.45 g (73%); mp 253−254 °C. H

NMR (DMSO-d₆) δ 3.59 (6H, s, 2 × COOMe), 3.78 (3H, s, NMe),

4.31 (2H, s, CH₂), 5.16 (2H, s, CH₂), 7.07−7.10 (1H, m, ArH), 7.17−

7.21 (1H, m, ArH), 7.33−7.37 (2H, m, 2 × ArH), 7.45−7.47 (1H, m,

ArH), 7.56−7.59 (3H, m, 3 × ArH). ¹³C NMR (DMSO-d₆) δ 21.3,

29.4, 41.8, 51.2, 51.6, 102.2, 109.3, 115.4, 116.3, 117.9, 119.0, 121.5,

125.1, 126.0, 128.7, 132.2, 133.8, 137.5, 164.0, 165.5. Anal. Calcd for

M

dx.doi.org/10.1021/jm301788a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

C₂₅H₂₁FN₂O₄: C, 69.44; H, 4.89; N, 6.48. Found: C, 69.21; H, 4.93;

N, 6.47.

Dimethyl 3-(4-Chlorophenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-dicarboxylate (25d). Compound

25d was prepared from 24d (4 g, 10.8 mmol) and DMAD (2.3 g,

16.3 mmol) in AC₂O (30 mL). Yield 3.5 g, (72%); mp 264−265 °C.

¹H NMR (DMSO-d₆) δ 3.60 (6H, s, 2 × COOMe), 3.78 (3H, s,

NMe), 4.30 (2H, s, CH₂), 5.18 (2H, s, CH₂), 7.06−7.10 (1H, m,

ArH), 7.17−7.20 (1H, m, ArH), 7.45−7.47 (1H, m, ArH), 7.54−7.59

(5H, m, 5 × ArH). ¹³C NMR (DMSO-d₆) δ 21.3, 29.5, 41.9, 51.3,

51.7, 102.1, 109.3, 116.5, 118.0, 119.1, 121.6, 125.1, 128.6, 131.9,

132.3, 133.7, 134.1, 137.5, 164.0, 165.5. Anal. Calcd for

C₂₅H₂₁ClN₂O₄: C, 66.89; H, 4.72; N, 6.24. Found: C, 66.82; H,

4.63; N, 6.24.

m, ArH), 7.55−7.57 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 21.4,

29.5, 51.1, 51.7, 56.1, 60.0, 102.2, 107.8, 116.4, 118.0, 121.5, 125.1,

128.9, 132.7, 133.0, 133.7, 152.7, 164.0, 165.9. Anal. Calcd for

C₂₈H₂₈N₂O₇: C, 66.66; H, 5.59; N, 5.55. Found: C, 66.31; H, 5.63; N,

5.45.

[3-Ethyl-6-methyl-6,11-dihydro-5H-indolizino[6,7-b]indole-

1,2-diyl]dimethanol (26a). A solution of 25a (1.46 g, 4 mmol) in

anhydrous DCM (30 mL) was added dropwise to a stirred suspension

of LAH (0.37 g, 10 mmol) in anhydrous diethyl ether (10 mL) at 0 to

−5 °C. The reaction mixture was further stirred for 30 min after the

addition was completed. The excess hydride was decomposed by the

sequential addition of water (1 mL), 15% aqueous NaOH (1 mL), and

water (1 mL) at 0 °C. The mixture was ﬁltered through a pad of

Celite, and the solid residue was washed with DCM. The ﬁltrate was

washed with water and brine solution. The organic layer was dried

over anhydrous Na₂SO₄and concentrated to dryness in vacuo. The

oily residue was crystallized from ether to give 0.81 g. ¹H NMR

(DMSO-d₆) δ 1.17 (3H, t, J = 7.4 Hz, Me), 2.74 (2H, q, J = 7.4 Hz,

CH₂), 3.75 (3H, s, NMe), 4.00 (2H, s, CH₂), 4.34 (1H, br s,

exchangeable, OH), 4.36 (2H, s, CH₂), 4.42 (3H, br s, CH₂and

exchangeable, OH), 5.19 (2H, s, CH₂), 7.04−7.07 (1H, m, ArH),

7.15−7.19 (1H, m, ArH), 7.45−7.47 (1H, m, ArH), 7.53−7.55 (1H,

m, ArH). ¹³C NMR (DMSO-d₆) δ 15.7, 16.8, 18.6, 19.4, 54.1, 54.2,

103.5, 109.2, 116.9, 117.9, 118.7, 118.9, 121.1, 121.9, 125.6, 129.8,

130.0, 137.4. Anal. Calcd for C₁₉H₂₂N₂O₂: C, 73.52; H, 7.14; N, 9.03.

Found: C, 73.16; H, 7.18; N, 8.92.

Dimethyl 3-(3,4-Diﬂuorophenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-dicarboxylate (25e). Compound 25e

was prepared from 24e (3.7 g, 10 mmol) and DMAD (2.1 g, 15

1

mmol) in AC₂O (50 mL). Yield 3.2 g (71%); mp 253−254 °C. H

NMR (DMSO-d₆) δ 3.61 (6H, s, 2 × COOMe), 3.78 (3H, s, NMe),

4.29 (2H, s, CH₂), 5.19 (2H, s, CH₂), 7.06−7.08 (1H, m, ArH), 7.17−

7.20 (1H, m, ArH), 7.36−7.38 (1H, m, ArH), 7.45−7.47 (1H, m,

ArH), 7.55−7.59 (2H, m, 2 × ArH), 7.63−7.64 (1H, m, ArH). ¹³C

NMR (DMSO-d₆) δ 21.2, 29.4, 41.8, 51.2, 51.6, 102.1, 109.4, 116.5,

118.0, 119.1, 121.5, 125.1, 128.7, 131.2, 133.9, 137.5, 164.0, 165.2.

Anal. Calcd for C₂₅H₂₀F₂N₂O₄: C, 66.66; H, 4.48; N, 6.22. Found: C,

66.63; H, 4.47; N, 6.22.

Dimethyl 3-(3-Chloro-4-ﬂuorophenyl)-6-methyl-6,11-dihy-

dro-5H-indolizino[6,7-b]indole-1,2-dicarboxylate (25f). Com-

pound 25f was prepared from 24f (4.6 g, 12 mmol) and DMAD

(2.5 g, 18 mmol) in AC₂O (90 mL). Yield 4.1 g (73%); mp 250−251

Following the same synthetic procedure as for 26a, the following

compounds were prepared.

[3-Phenyl-6-methyl-6,11-dihydro-5H-indolizino[6,7-b]-

indole-1,2-diyl]dimethanol (26b). Compound 26b was prepared

from 25b (2.1 g, 5.0 mmol) and LAH (0.46 g, 12.5 mmol). Yield 1.48

g. ¹H NMR (DMSO-d₆) δ 3.60 (3H, s, NMe), 4.10 (2H, s, CH₂), 4.30

(2H, m, CH₂), 4.57 (4H, br s, CH₂and 2 × OH), 5.15 (2H, s, CH₂),

7.04−7.08 (1H, m, ArH), 7.14−7.18 (1H, m, ArH), 7.38−7.42 (2H,

m, 2 × ArH), 7.45−7.48 (2H, m, 2 × ArH), 7.51−7.58 (3H, m, 3 ×

ArH). ¹³C NMR (DMSO-d₆) δ 18.9, 29.3, 41.2, 54.2, 54.4, 103.3,

109.3, 117.9, 118.3, 118.8, 121.2, 121.7, 124.6, 125.5, 127.1, 128.3,

129.5, 130.2, 130.3, 131.8, 137.4. Anal. Calcd for C₂₃H₂₂N₂O₂: C,

77.07; H, 6.19; N, 7.82. Found: C, 76.83; H, 5.94; N, 7.50.

[3-(4-Fluorophenyl)-6-methyl-6,11-dihydro-5H-indolizino-

[6,7-b]indole-1,2-diyl]dimethanol (26c). Compound 26c was

prepared from 25c (1.3 g, 3 mmol) and LAH (0.27 g, 7.5 mmol).

Yield 0.86 g. ¹H NMR (DMSO-d₆) δ 3.61 (3H, s, NMe), 4.10 (2H, s,

CH₂), 4.27 (2H, s, CH₂), 4.56 (4H, br s, CH₂and exchangeable, 2 ×

OH), 5.13 (2H, s, CH₂), 7.04−7.08 (1H, m, ArH), 7.14−7.18 (1H, m,

ArH), 7.31−7.35 (2H, m, 2 × ArH), 7.44−7.46 (1H, m, ArH), 7.56−

7.60 (3H, m, 3 × ArH). ¹³C NMR (DMSO-d₆) δ 18.9, 29.3, 41.1, 54.2,

54.3, 103.3, 109.3, 115.1, 115.3, 117.9, 118.2, 118.8, 121.2, 121.8,

124.6, 125.5, 128.1, 128.5, 130.1, 132.3, 132.4, 137.4. Anal. Calcd for

C₂₃H₂₁FN₂O₂: C, 73.39; H, 5.62; N, 7.44. Found: C, 73.16; H, 5.33;

N, 7.09.

1

°C. H NMR (DMSO-d₆) δ 3.60 (6H, s, 2 × COOMe), 3.78 (3H, s,

NMe), 4.28 (2H, s, CH₂), 5.17 (2H, s, CH₂), 7.05−7.09 (1H, m,

ArH), 7.16−7.19 (1H, m, ArH), 7.44−7.46 (1H, m, ArH), 7.54−7.58

(3H, m, 3 × ArH), 7.75−7.78 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ

21.2, 29.5, 41.8, 51.2, 51.6, 102.1, 109.5, 116.5, 118.0, 119.1, 121.6,

125.1, 128.7, 131.3, 132.7, 133.9, 137.5, 164.0, 165.2. Anal. Calcd for

C₂₅H₂₀ClFN₂O₄: C, 64.31; H, 4.32; N, 6.00. Found: C, 63.92; H, 4.09;

N, 5.92.

Dimethyl 3-(4-Methoxyphenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-dicarboxylate (25g). Compound

25g was prepared from 24g (2 g, 5.4 mmol) and DMAD (1.2 g, 8.2

1

mmol) in AC₂O (15 mL). Yield 1.5 g (61%); mp 211−212 °C. H

NMR (DMSO-d₆) δ 3.59 (6H, s, 2 × COOMe), 3.77 (3H, s, NMe),

3.83 (3H, s, OMe), 4.29 (2H, s, CH₂), 5.14 (2H, s, CH₂), 7.04−7.09

(3H, m, 3 × ArH), 7.16−7.20 (1H, m, ArH), 7.43−7.47 (3H, m, 3 ×

ArH), 7.56−7.58 (1H, m, ArH). 13C NMR (DMSO-d₆) δ 21.4, 29.4,

41.8, 51.2, 51.6, 55.2, 102.3, 109.0, 113.9, 116.0, 118.0, 119.1, 121.5,

125.1, 128.8, 131.8, 132.9, 133.6, 133.9, 137.5, 159.5, 164.1, 165.8.

Anal. Calcd for C₂₆H₂₄N₂O₅: C, 70.26; H, 5.44; N, 6.30. Found: C,

69.94; H, 5.43; N, 6.21.

Dimethyl 3-(3,4-Dimethoxyphenyl)-6-methyl-6,11-dihydro-

5H-indolizino[6,7-b]indole-1,2-dicarboxylate (25h). Compound

25h was prepared from 24h (4.3 g, 11 mmol) and DMAD (2.3 g, 16.5

[3-(4-Chlorophenyl)-6-methyl-6,11-dihydro-5H-indolizino-

[6,7-b]indole-1,2-diyl]dimethanol (26d). Compound 26d was

prepared from 25d (3.2 g, 7 mmol) and LAH (0.6g, 17.8 mmol).

1

mmol) in AC₂O (60 mL). Yield 3.7 g (72%); mp 241−242 °C. H

1

NMR (DMSO-d₆) δ 3.60 (3H, s, COOMe), 3.61 (3H, s, COOMe),

3.78 (3H, s, NMe), 3.79 (3H, s, OMe), 3.83 (3H, s, OMe), 4.30 (2H,

s, CH₂), 5.19 (2H, s, CH₂), 7.05−7.10 (4H, m, 4 × ArH), 7.17−7.20

(1H, m, ArH), 7.45−7.48 (1H, m, ArH), 7.56−7.58 (1H, m, ArH).

¹³C NMR (DMSO-d₆) δ 21.3, 29.4, 41.9, 51.1, 51.6, 55.5, 102.3, 109.4,

Yield 2.2 g. H NMR (DMSO-d₆) δ 3.62 (3H, s, NMe), 4.10 (2H, s,

CH₂), 4.29 (2H, m, CH₂), 4.55 (2H, s, CH₂), 4.59 (2H, br s,

exchangeable, 2 × OH), 5.16 (2H, s, CH₂), 7.04−7.08 (1H, m, ArH),

7.14−7.18 (1H, m, ArH), 7.43−7.45 (1H, m, ArH), 7.53−7.59 (5H,

m, 5 × ArH). ¹³C NMR (DMSO-d₆) δ 18.9, 29.3, 41.2, 54.1, 54.2,

103.3, 109.3, 117.9, 118.4, 118.8, 121.2, 122.1, 125.1, 125.4, 128.3,

128.4, 130.1, 130.6, 131.9, 137.4. Anal. Calcd for

C₂₃H₂₁ClN₂O₂·0.5H₂O: C, 68.74; H, 5.52; N, 6.97. Found: C,

68.72; H, 5.16; N, 6.94.

111.5, 113.9, 116.1, 118.0, 119.0, 121.5, 125.1, 128.9, 133.3, 137.5,

148.3, 164.1, 165.9. Anal. Calcd for C₂₇H₂₆N₂O₆: C, 68.34; H, 5.52; N,

5.90. Found: C, 68.60; H, 5.90; N, 5.61.

Dimethyl 3-(3,4,5-Trimethoxyphenyl)-6-methyl-6,11-dihy-

dro-5H-indolizino[6,7-b]indole-1,2-dicarboxylate (25i). Com-

pound 25i was prepared from 24i (3 g, 7 mmol) and DMAD (1.5

g, 10.5 mmol) in AC₂O (40 mL). Yield 3.0 g (84%); mp 253−254 °C.

¹H NMR (DMSO-d₆) δ 3.62 (3H, s, COOMe), 3.63 (3H, s,

[3-(3,4-Diﬂuorophenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-diyl]dimethanol (26e). Compound

26e was prepared from 25e (2.7 g, 6 mmol) and LAH (0.55 g, 15

1

mmol). Yield 1.8 g. H NMR (DMSO-d₆) δ 3.63 (3H, s, NMe), 4.10

COOMe), 3.73 (3H, s, OMe), 3.77 (3H, s, NMe), 3.80 (6H, s, 2 ×

OMe), 4.30 (2H, s, CH₂), 5.26 (2H, s, CH₂), 6.82 (2H, s, 2 × ArH),

7.05−7.09 (1H, m, ArH), 7.15−7.19 (1H, m, ArH), 7.45−7.47 (1H,

(2H, s, CH₂), 4.28 (2H, m, CH₂), 4.55 (2H, s, CH₂), 4.62 (2H, br s,

exchangeable, 2 × OH), 5.19 (2H, s, CH₂), 7.05−7.08 (1H, m, ArH),

7.14−7.16 (1H, m, ArH), 7.39−7.41 (1H, m, ArH), 7.44−7.46 (1H,

N

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m, ArH), 7.54−7.59 (2H, m, 2 × ArH), 7.64−7.68 (1H, m, ArH). ¹³

C

(2H, s, CH₂), 6.92 (2H, br s, exchangeable, NH), 7.05−7.08 (1H, m,

ArH), 7.16−7.20 (1H, m, ArH), 7.46−7.48 (1H, m, ArH), 7.53−7.55

(1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 15.1, 15.4, 16.8, 18.6, 29.4,

34.9, 56.7, 57.0, 103.1, 109.3, 112.4, 114.3, 117.9, 118.8, 121.2, 122.6,

124.4, 125.5, 129.7, 132.1, 137.4, 156.3, 156.4. Anal. Calcd for

C₂₅H₃₂N₄O₄: C, 66.35; H, 7.13; N, 12.38. Found: C, 66.19; H, 7.23;

N, 12.28.

NMR (DMSO-d₆) δ 18.9, 29.3, 411, 54.1, 54.2, 103.2, 109.3, 117.3,

117.4, 117.9, 118.4, 118.8, 119.0, 119.1, 121.2, 122.3, 125.2, 125.5,

127.3, 127.5, 129.3, 130.1, 137.4. Anal. Calcd for C₂₃H₂₀F₂N₂O₂: C,

70.04; H, 5.11; N, 7.10. Found: C, 69.86; H, 4.95; N, 7.24.

[3-(3-Chloro-4-ﬂuorophenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-diyl]dimethanol (26f). Compound

26f was prepared from 25f (3.7 g, 8 mmol) and LAH (0.74 g, 20

[3-Phenyl-6-methyl-6,11-dihydro-5H-indolizino[6,7-b]-

indole-1,2-diyl]bis(methylene) Bis(ethylcarbamate) (27b).

Compound 27b was prepared from 26b (0.36 g, 1 mmol), TEA

1

mmol). Yield 2.6 g. H NMR (DMSO-d₆) δ 3.61 (3H, s, NMe), 4.08

(2H, s, CH₂), 4.26 (2H, m, CH₂), 4.54 (2H, s, CH₂), 4.62 (2H, br s,

exchangeable, 2 × OH), 5.17 (2H, s, CH₂), 7.03−7.07 (1H, m, ArH),

7.13−7.17 (1H, m, ArH), 7.42−7.44 (1H, m, ArH), 7.52−7.56 (3H,

m, 3 × ArH), 7.76−7.78 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 18.9,

29.3, 41.1, 54.1, 54.2, 103.2, 109.3, 116.7, 116.8, 117.9, 118.3, 118.8,

119.3, 119.5, 121.2, 122.3, 125.2, 125.4, 127.2, 129.6, 129.7, 130.1,

130.9, 131.0, 137.4. Anal. Calcd for C₂₃H₂₀ClFN₂O₂: C, 67.23; H,

4.91; N, 6.82. Found: C, 66.86; H, 5.00; N, 6.57.

1

(0.3 mL), and ethyl isocyanate (0.28 g, 4 mmol). Yield 0.32 g. H

NMR (DMSO-d₆) δ 1.00 (6H, t, J = 6.8 Hz, 2 × Me), 2.99 (4H, q, J =

6.8 Hz, CH₂), 3.60 (3H, s, NMe), 4.15 (2H, s, CH₂), 4.81 (2H, s,

CH₂), 5.08 (2H, s, CH₂), 5.16 (2H, s, CH₂), 6.98 (2H, br s,

exchangeable, NH), 7.05−7.09 (1H, m, ArH), 7.15−7.19 (1H, m,

ArH), 7.45−7.47 (2H, m, 2 × ArH), 7.51−7.55 (4H, m, 4 × ArH),

7.56−7.58 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 15.1, 18.6, 29.3,

35.0, 41.3, 56.9, 57.3, 103.0, 109.3, 113.6, 116.7, 117.9, 118.8, 121.2,

125.3, 126.8, 127.7, 128.5, 129.8, 130.4, 130.9, 131.5, 137.4, 156.1,

156.4. Anal. Calcd for C₂₉H₃₂N₄O₄: C, 69.58; H, 6.44; N, 11.19.

Found: C, 69.26; H, 6.25; N, 10.83.

[3-(4-Methoxyphenyl)-6-methyl-6,11-dihydro-5H-indolizino-

[6,7-b]indole-1,2-diyl]dimethanol (26g). Compound 26g was

prepared from 25g (1.3 g, 3 mmol) and LAH (0.27 g, 7.5 mmol).

1

Yield 0.9 g. H NMR (DMSO-d₆) δ 3.61 (3H, s, NMe), 3.83 (3H, s,

[3-(4-Fluorophenyl)-6-methyl-6,11-dihydro-5H-indolizino-

[6,7-b]indole-1,2-diyl]bis(methylene) Bis(ethylcarbamate)

(27c). Compound 27c was prepared from 26c (0.2 g, 0.5 mmol),

TEA (0.2 mL), and ethyl isocyanate (0.14 g, 2 mmol). Yield 0.2 g. ¹H

NMR (DMSO-d₆) δ 0.99 (6H, t, J = 7.1 Hz, 2 × Me), 2.99 (4H, q, J =

7.1 Hz, CH₂), 3.60 (3H, s, NMe), 4.14 (2H, s, CH₂), 4.79 (2H, s,

CH₂), 5.07 (2H, s, CH₂), 5.12 (2H, s, CH₂), 6.97 (2H, br s,

exchangeable, NH), 7.05−7.08 (1H, m, ArH), 7.15−7.19 (1H, m,

ArH), 7.32−7.36 (2H, m, 2 × ArH), 7.44−7.46 (1H, m, ArH), 7.54−

7.57 (3H, m, 3 × ArH). ¹³C NMR (DMSO-d₆) δ 15.1, 18.9, 29.3, 35.0,

56.9, 57.2, 102.9, 109.3, 113.5, 115.3, 115.5, 117.9, 118.8, 121.3, 125.4,

126.8, 127.4, 129.8, 132.5, 132.6, 137.4, 156.1, 156.4, 160.9. Anal.

Calcd for C₂₉H₃₁FN₄O₄: C, 67.17; H, 6.03; N, 10.80. Found: C, 67.48;

H, 5.96; N, 10.63.

[3-(4-Chlorophenyl)-6-methyl-6,11-dihydro-5H-indolizino-

[6,7-b]indole-1,2-diyl]bis(methylene) Bis(ethylcarbamate)

(27d). Compound 27d was prepared from 26d (0.4 g, 1 mmol),

TEA (0.4 mL), and ethyl isocyanate (0.28 g, 4 mmol). Yield 0.31 g. ¹H

NMR (DMSO-d₆) δ 0.98 (6H, t, J = 6.4 Hz, 2 × Me), 2.98 (4H, q, J =

6.4 Hz, CH₂), 3.60 (3H, s, NMe), 4.13 (2H, s, CH₂), 4.80 (2H, s,

CH₂), 5.07 (2H, s, CH₂), 5.15 (2H, s, CH₂), 6.97 (2H, br s,

exchangeable, NH), 7.04−7.08 (1H, m, ArH), 7.14−7.18 (1H, m,

ArH), 7.43−7.45 (1H, m, ArH), 7.54−7.60 (5H, m, 5 × ArH). ¹³C

NMR (DMSO-d₆) δ 15.1, 18.9, 29.3, 35.0, 41.3, 56.8, 57.1, 102.9,

109.3, 113.7, 117.1, 117.9, 118.8, 121.3, 125.3, 127.1, 128.5, 129.8,

130.1, 132.2, 132.7, 137.4, 156.1, 156.4. Anal. Calcd for

C₂₉H₃₁ClN₄O₄·0.5H₂O: C, 64.02; H, 5.93; N, 10.30. Found: C,

63.97; H, 6.01; N, 10.29.

[3-(3,4-Diﬂuorophenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-diyl]bis(methylene) Bis-

(ethylcarbamate) (27e). Compound 27e was prepared from 26e

(0.2 g, 0.5 mmol), TEA (0.2 mL), and ethyl isocyanate (0.14 g, 2

mmol). Yield 0.15 g. ¹H NMR (DMSO-d₆) δ 0.99 (6H, t, J = 7 Hz, 2

× Me), 2.98 (4H, q, J = 7 Hz, CH₂), 3.62 (3H, s, NMe), 4.14 (2H, s,

CH₂), 4.82 (2H, s, CH₂), 5.07 (2H, s, CH₂), 5.17 (2H, s, CH₂), 6.97

(2H, br s, exchangeable, NH), 7.05−7.09 (1H, m, ArH), 7.15−7.19

(1H, m, ArH), 7.35−7.38 (1H, m, ArH), 7.44−7.46 (1H, m, ArH),

7.53−7.57 (2H, m, 2 × ArH), 7.63−7.65 (1H, m, ArH). ¹³C NMR

(DMSO-d₆) δ 15.1, 15.7, 18.9, 29.4, 34.0, 35.0, 41.2, 56.8, 57.0, 102.8,

109.3, 113.7, 117.4, 117.6, 118.0, 118.9, 119.4, 119.5, 121.3, 125.3,

127.1, 127.6, 128.5, 129.2, 129.8, 137.4, 156.0, 156.3. Anal. Calcd for

C₂₉H₃₀F₂N₄O₄: C, 64.91; H, 5.64; N, 10.44. Found: C, 65.57; H, 6.01;

N, 10.07.

[3-(3-Chloro-4-ﬂuorophenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-diyl]bis(methylene) Bis-

(ethylcarbamate) (27f). Compound 27f was prepared from 26f

(0.4 g, 1 mmol), TEA (0.4 mL), and ethyl isocyanate (0.28 g, 4

mmol). Yield 0.32 g. ¹H NMR (DMSO-d₆) δ 0.97 (6H, t, J = 7.1 Hz, 2

× Me), 2.97 (4H, q, J = 7.1 Hz, CH₂), 3.61 (3H, s, NMe), 4.13 (2H, s,

CH₂), 4.80 (2H, s, CH₂), 5.07 (2H, s, CH₂), 5.16 (2H, s, CH₂), 6.98

OMe), 4.09 (2H, s, CH₂), 4.27 (2H, s, CH₂), 4.55 (4H, br s, CH₂and

exchangeable, 2 × OH), 5.10 (2H, s, CH₂), 7.04−7.08 (3H, m, 3 ×

ArH), 7.14−7.18 (1H, m, ArH), 7.44−7.47 (3H, m, 3 × ArH), 7.56−

7.58 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 15.1, 18.9, 29.3, 41.1,

54.5, 55.1, 103.4, 109.3, 113.8, 117.8, 117.9, 118.8, 121.1, 121.2, 123.9,

124.0, 125.5, 129.3, 130.2, 131.6, 137.4, 158.5. Anal. Calcd for

C₂₄H₂₄N₂O₃: C, 74.21; H, 6.23; N, 7.21. Found: C, 73.94; H, 6.11; N,

7.48.

[3-(3,4-Dimethoxyphenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-diyl]dimethanol (26h). Compound

26h was prepared from 25h (3.3 g, 7 mmol) and LAH (0.64 g, 17.5

1

mmol). Yield 2.3 g. H NMR (DMSO-d₆) δ 3.62 (3H, s, NMe), 3.80

(3H, s, OMe), 3.82 (3H, s, OMe), 4.09 (2H, s, CH₂), 4.29 (2H, s,

CH₂), 4.52 (1H, br s, exchangeable, OH) 4.55 (3H, br s, CH₂and

exchangeable, OH), 5.14 (2H, s, CH₂), 7.04−7.08 (3H, m, 3 × ArH),

7.12 (1H, s, ArH), 7.14−7.18 (1H, m, ArH), 7.44−7.46 (1H, m, ArH),

7.56−7.58 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 18.9, 29.3, 41.1,

54.3, 54.5, 55.5, 55.6, 103.4, 109.3, 111.7, 114.3, 117.9, 118.0, 118.7,

121.1, 121.2, 122.6, 124.0, 124.2, 125.5, 129.6, 130.3, 137.4, 148.1,

148.4. Anal. Calcd for C₂₅H₂₆N₂O₄: C, 71.75; H, 6.26; N, 6.69. Found:

C, 71.66; H, 6.29; N, 6.53.

[3-(3,4,5-Trimethoxyphenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-diyl]dimethanol (26i). Compound

26i was prepared from 25i (2.5 g, 5 mmol) and LAH (0.46 g, 12.5

1

mmol). Yield 1.7 g. H NMR (DMSO-d₆) δ 3.64 (3H, s, NMe), 3.73

(3H, s, OMe), 3.82 (6H, s, 2 × OMe), 4.10 (2H, s, CH₂), 4.31 (2H, s,

CH₂), 4.55 (4H, br s, CH₂and exchangeable, 2 × OH), 5.20 (2H, s,

CH₂), 6.83 (2H, s, 2 × ArH), 7.04−7.08 (1H, m, ArH), 7.14−7.18

(1H, m, ArH), 7.44−7.46 (1H, m, ArH), 7.56−7.58 (1H, m, ArH).

¹³C NMR (DMSO-d₆) δ 18.9, 29.4, 41.3, 54.2, 54.5, 56.1, 60.0, 103.3,

107.9, 109.3, 117.9, 118.2, 118.8, 121.2, 121.5, 124.4, 125.5, 127.2,

129.8, 130.3, 136.7, 137.4, 152.7. Anal. Calcd for C₂₆H₂₈N₂O₅: C,

69.63; H, 6.29; N, 6.25. Found: C, 69.25; H, 6.30; N, 6.08.

General Procedure for Preparing Bis(alkylcarbamate) De-

rivatives (27a−i and 28a−i). Alkyl isocyanate (5 equiv) was added

to a solution of bis(hydroxymethyl) derivatives (26a−i, 1.0 equiv) and

TEA (2−3 equiv) in anhydrous DMF or THF. The reaction mixture

was stirred at ambient temperature (for 8−12 h) under argon. After

completion of the reaction, the reaction mixture was evaporated to

dryness in vacuo. The residue was triturated with ether, and the

separated solid was collected by ﬁltration. The desired product was

obtained by either recrystallization or column chromatography.

[3-Ethyl-6-methyl-6,11-dihydro-5H-indolizino[6,7-b]indole-

1,2-diyl]bis(methylene) Bis(ethylcarbamate) (27a). Compound

27a was prepared from 26a (0.15 g, 0.5 mmol), TEA (0.2 mL), and

ethyl isocyanate (0.14 g, 2 mmol). Yield 0.18 g. ¹H NMR (DMSO-d₆)

δ 0.99 (6H, t, J = 7.1 Hz, 2 × Me), 1.17 (3H, t, J = 7.4 Hz, Me), 2.77

(2H, q, J = 7.4 Hz, CH₂), 2.99 (4H, q, J = 7.1 Hz, CH₂), 3.75 (3H, s,

NMe), 4.04 (2H, s, CH₂), 4.95 (2H, s, CH₂), 4.99 (2H, s, CH₂), 5.23

O

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(2H, br s, exchangeable, NH), 7.04−7.08 (1H, m, ArH), 7.14−7.18

(1H, m, ArH), 7.43−7.45 (1H, m, ArH), 7.53−7.57 (3H, m, 3 ×

ArH), 7.72−7.74 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 15.1, 15.7,

18.9, 29.4, 34.0, 35.0, 41.2, 56.7, 57.0, 102.8, 109.3, 113.7, 116.8, 116.9,

117.4, 117.9, 118.8, 119.5, 121.3, 125.3, 127.2, 128.8, 129.0, 129.7,

131.2, 132.3, 137.4, 155.9, 156.3, 157.9. Anal. Calcd for

C₂₉H₃₀ClFN₄O₄: C, 62.98; H, 5.47; N, 10.13. Found: C, 62.64; H,

5.60; N, 10.34.

[3-(4-Methoxyphenyl)-6-methyl-6,11-dihydro-5H-indolizino-

[6,7-b]indole-1,2-diyl]bis(methylene) Bis(ethylcarbamate)

(27g). Compound 27g was prepared from 26g (0.2 g, 0.5 mmol),

TEA (0.2 mL), and ethyl isocyanate (0.14 g, 2 mmol). Yield 0.2 g. ¹H

NMR (DMSO-d₆) δ 1.00 (6H, t, J = 6.9 Hz, 2 × Me), 2.99 (4H, q, J =

6.9 Hz, CH₂), 3.61 (3H, s, NMe), 3.84 (3H, s, OMe), 4.14 (2H, s,

CH₂), 4.79 (2H, s, CH₂), 5.07 (2H, s, CH₂), 5.11 (2H, s, CH₂), 6.97

(2H, br s, exchangeable, NH), 7.06−7.09 (3H, m, 3 × ArH), 7.15−

7.19 (1H, m, ArH), 7.43−7.47 (3H, m, 3 × ArH), 7.56−7.58 (1H, m,

ArH). ¹³C NMR (DMSO-d₆) δ 15.1, 18.8, 29.3, 35.0, 41.3, 55.1, 56.9,

57.0, 103.0, 109.3, 113.9, 118.0, 118.8, 121.2, 123.1, 125.4, 131.7,

137.4, 158.9. Anal. Calcd for C₃₀H₃₄N₄O₅: C, 67.91; H, 6.46; N, 10.56.

Found: C, 67.60; H, 6.35; N, 10.25.

[3-(3,4-Dimethoxyphenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-diyl]bis(methylene) Bis-

(ethylcarbamate) (27h). Compound 27h was prepared from 26h

(0.21 g, 0.5 mmol), TEA (0.2 mL), and ethyl isocyanate (0.14 g, 2

mmol). Yield 0.15 g. ¹H NMR (DMSO-d₆) δ 0.98 (6H, t, J = 7 Hz, 2

× Me), 2.98 (4H, q, J = 7 Hz, CH₂), 3.61 (3H, s, NMe), 3.78 (3H, s,

OMe), 3.82 (3H, s, OMe), 4.13 (2H, s, CH₂), 4.79 (2H, s, CH₂), 5.06

(2H, s, CH₂), 5.15 (2H, s, CH₂), 6.97 (2H, br s, exchangeable, NH),

7.04−7.10 (4H, m, 4 × ArH), 7.14−7.18 (1H, m, ArH), 7.44−7.46

(1H, m, ArH), 7.55−7.57 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ

15.1, 18.8, 25.1, 29.4, 34.9, 35.0, 41.3, 55.5, 57.5, 67.0, 103.0, 109.3,

111.7, 113.4, 114.0, 116.2, 118.0, 118.8, 121.2, 122.7, 123.3, 125.4,

130.0, 137.4, 148.5, 156.2, 156.4. Anal. Calcd for C₃₁H₃₆N₄O₆: C,

66.41; H, 6.47; N, 9.99. Found: C, 66.02; H, 6.18; N, 9.63.

[3-(3,4,5-Trimethoxyphenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-diyl]bis(methylene) Bis-

(ethylcarbamate) (27i). Compound 27i was prepared from 26i

(0.45 g, 1 mmol), TEA (0.4 mL), and ethyl isocyanate (0.28 g, 4

mmol). Yield 0.35 g. ¹H NMR (DMSO-d₆) δ 0.98 (6H, t, J = 7.1 Hz, 2

× Me), 2.98 (4H, q, J = 7.1 Hz, CH₂), 3.64 (3H, s, NMe), 3.74 (3H, s,

OMe), 3.81 (6H, s, 2 × OMe), 4.14 (2H, s, CH₂), 4.81 (2H, s, CH₂),

5.07 (2H, s, CH₂), 5.22 (2H, s, CH₂), 6.79 (2H, m, 2 × ArH), 6.96

(2H, br s, exchangeable, NH), 7.05−7.08 (1H, m, ArH), 7.15−7.18

(1H, m, ArH), 7.45−7.47 (1H, m, ArH), 7.56−7.58 (1H, m, ArH).

¹³C NMR (DMSO-d₆) δ 15.1, 18.9, 29.4, 34.9, 35.0, 41.4, 55.9, 56.8,

ArH), 7.48−7.52 (4H, m, 4 × ArH), 7.55−7.57 (1H, m, ArH). ¹³C

NMR (DMSO-d₆) δ 18.9, 22.6, 23.3, 29.3, 40.6, 41.3, 42.2, 56.8, 57.2,

102.9, 109.3, 113.6, 116.7, 117.9, 118.8, 121.2, 125.3, 126.7, 127.7,

128.5, 129.8, 130.4, 130.9, 137.4, 155.4, 155.7. Anal. Calcd for

C₃₁H₃₆N₄O₄: C, 70.43; H, 6.86; N, 10.60. Found: C, 70.25; H, 7.15;

N, 10.75.

[3-(4-Fluorophenyl)-6-methyl-6,11-dihydro-5H-indolizino-

[6,7-b]indole-1,2-diyl]bis(methylene) Bis(isopropylcarbamate)

(28c). Compound 28c was prepared from 26c (0.2 g, 0.5 mmol), TEA

(0.2 mL), and i-Pr isocyanate (0.18 g, 2 mmol). Yield 0.19 g. ¹H NMR

(DMSO-d₆) δ 1.02 (12H, d, J = 6.4 Hz, 4 × Me), 3.55 (2H, m, CH),

3.60 (3H, s, NMe), 4.13 (2H, s, CH₂), 4.79 (2H, s, CH₂), 5.07 (2H, s,

CH₂), 5.13 (2H, s, CH₂), 6.88 (2H, br s, exchangeable, NH), 7.05−

7.08 (1H, m, ArH), 7.15−7.18 (1H, m, ArH), 7.31−7.35 (2H, m, 2 ×

ArH), 7.44−7.46 (1H, m, ArH), 7.54−7.57 (3H, m, 3 × ArH). ¹³C

NMR (DMSO-d₆) δ 18.9, 22.6, 29.3, 41.2, 42.2, 56.7, 57.0, 102.9,

109.2, 113.5, 115.3, 115.5, 116.9, 117.9, 118.8, 121.2, 125.3, 126.7,

127.4, 129.8, 130.3, 132.5, 132.6, 137.4, 155.4, 155.7, 160.9, 162.8.

Anal. Calcd for C₃₁H₃₅FN₄O₄: C, 68.11; H, 6.45; N, 10.25. Found: C,

68.41; H, 6.33; N, 9.96.

[3-(4-Chlorophenyl)-6-methyl-6,11-dihydro-5H-indolizino-

[6,7-b]indole-1,2-diyl]bis(methylene) Bis(isopropylcarbamate)

(28d). Compound 28d was prepared from 26d (0.4 g, 1 mmol), TEA

(0.4 mL), and i-Pr isocyanate (0.34 g, 4 mmol). Yield 0.34 g. ¹H NMR

(DMSO-d₆) δ 1.02 (12H, d, J = 6.4 Hz, 4 × Me), 3.57 (2H, m, CH),

3.61 (3H, s, NMe), 4.14 (2H, s, CH₂), 4.81 (2H, s, CH₂), 5.08 (2H, s,

CH₂), 5.16 (2H, s, CH₂), 6.88 (2H, br s, exchangeable, NH), 7.05−

7.09 (1H, m, ArH), 7.15−7.19 (1H, m, ArH), 7.44−7.46 (1H, m,

ArH), 7.54−7.58 (5H, m, 5 × ArH). ¹³C NMR (DMSO-d₆) δ 18.9,

22.6, 23.3, 29.3, 41.3, 42.2, 56.7, 56.9, 102.9, 109.3, 113.7, 117.2, 117.9,

118.8, 121.3, 123.1, 125.3, 127.1, 128.5, 129.7, 129.8, 130.0, 132.1,

132.6, 137.4, 155.4, 155.7. Anal. Calcd for C₃₁H₃₅ClN₄O₄·0.5H₂O: C,

65.08; H, 6.34; N, 9.79. Found: C, 64.94; H, 6.21; N, 9.73.

[3-(3,4-Diﬂuorophenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-diyl]bis(methylene) Bis-

(isopropylcarbamate) (28e). Compound 28e was prepared from

26e (0.2 g, 0.5 mmol), TEA (0.2 mL), and i-Pr isocyanate (0.18 g, 2

mmol). Yield 0.22 g. ¹H NMR (DMSO-d₆) δ 1.03 (12H, d, J = 6.4 Hz,

4 × Me), 3.57 (2H, m, CH), 3.63 (3H, s, NMe), 4.14 (2H, s, CH₂),

4.82 (2H, s, CH₂), 5.08 (2H, s, CH₂), 5.18 (2H, s, CH₂), 6.92 (2H, br

s, exchangeable, NH), 7.06−7.10 (1H, m, ArH), 7.16−7.20 (1H, m,

ArH), 7.36−7.39 (1H, m, ArH), 7.45−7.47 (1H, m, ArH), 7.55−7.62

(3H, m, 3 × ArH). ¹³C NMR (DMSO-d₆) δ 18.9, 22.6, 23.3, 29.4,

41.2, 42.2, 56.6, 56.8, 102.8, 109.2, 113.7, 117.4, 117.6, 117.9, 118.8,

119.4, 119.5, 121.3, 125.3, 127.1, 127.5, 129.2, 129.7, 137.4, 148.2,

155.3, 155.7. Anal. Calcd for C₃₁H₃₄F₂N₄O₄: C, 65.94; H, 6.07; N,

9.92. Found: C, 65.67; H, 6.07; N, 9.71.

[3-(3-Chloro-4-ﬂuorophenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-diyl]bis(methylene) Bis-

(isopropylcarbamate) (28f). Compound 28f was prepared from

26f (0.2 g, 0.5 mmol), TEA (0.2 mL), and i-Pr isocyanate (0.17 g, 2

mmol). Yield 0.2 g. ¹H NMR (DMSO-d₆) δ 1.02 (12H, d, J = 6.5 Hz,

4 × Me), 3.58 (2H, m, CH), 3.62 (3H, s, NMe), 4.14 (2H, s, CH₂),

4.80 (2H, s, CH₂), 5.07 (2H, s, CH₂), 5.17 (2H, s, CH₂), 6.91 (2H, br

s, exchangeable, NH), 7.05−7.09 (1H, m, ArH), 7.15−7.19 (1H, m,

ArH), 7.44−7.46 (1H, m, ArH), 7.54−7.58 (3H, m, 3 × ArH), 7.71−

7.73 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 18.9, 22.6, 25.1, 29.4,

41.2, 42.2, 56.6, 56.8, 66.99, 102.8, 109.3, 113.7, 116.8, 117.0, 117.5,

117.9, 118.8, 119.5, 119.6, 121.3, 125.3, 127.2, 128.8, 128.9, 129.7,

131.2, 132.3, 137.4, 155.3, 155.6, 155.9, 157.9. Anal. Calcd for

C₃₁H₃₄ClFN₄O₄: C, 64.08; H, 5.90; N, 9.64. Found: C, 63.89; H, 5.76;

N, 9.39.

[3-(4-Methoxyphenyl)-6-methyl-6,11-dihydro-5H-indolizino-

[6,7-b]indole-1,2-diyl]bis(methylene) Bis(isopropylcarbamate)

(28g). Compound 28g was prepared from 26g (0.2 g, 0.5 mmol),

TEA (0.2 mL), and i-Pr isocyanate (0.17 g, 2 mmol). Yield 0.21 g. ¹H

NMR (DMSO-d₆) δ 1.03 (12H, d, J = 6.3 Hz, 4 × Me), 3.57 (2H, m,

CH), 3.60 (3H, s, NMe), 3.83 (3H, s, OMe), 4.13 (2H, s, CH₂), 4.78

(2H, s, CH₂), 5.07 (2H, s, CH₂), 5.10 (2H, s, CH₂), 6.88 (2H, br s,

exchangeable, NH), 7.05−7.08 (3H, m, 3 × ArH), 7.15−7.18 (1H, m,

ArH), 7.42−7.46 (3H, m, 3 × ArH), 7.55−7.57 (1H, m, ArH). ¹³C

57.5, 60.0, 102.9, 107.7, 109.3, 113.7, 116.4, 117.9, 118.8, 121.3, 125.4,

126.3, 126.6, 130.0, 131.7, 137.0, 137.4, 152.8, 156.2, 156.3. Anal.

Calcd for C₃₂H₃₈N₄O₇: C, 65.07; H, 6.48; N, 9.49. Found: C, 64.69;

H, 6.52; N, 9.26.

[3-Ethyl-6-methyl-6,11-dihydro-5H-indolizino[6,7-b]indole-

1,2-diyl]bis(methylene) Bis(isopropylcarbamate) (28a). Com-

pound 28a was prepared from 26a (0.31 g, 1 mmol), TEA (0.3 mL),

and i-Pr isocyanate (0.36 g, 4 mmol). Yield 0.32 g. ¹H NMR (DMSO-

d₆) δ 1.00 (12H, d, J = 6.4 Hz, 4 × Me), 1.17 (3H, t, J = 7.4 Hz, Me),

2.77 (2H, q, J = 7.4 Hz, CH₂), 3.59 (2H, m, CH), 3.75 (3H, s, NMe),

4.03 (2H, s, CH₂), 4.94 (2H, s, CH₂), 4.99 (2H, s, CH₂), 5.22 (2H, s,

CH₂), 6.83 (2H, br s, exchangeable, NH), 7.04−7.08 (1H, m, ArH),

7.15−7.19 (1H, m, ArH), 7.46−7.48 (1H, m, ArH), 7.52−7.54 (1H,

m, ArH). ¹³C NMR (DMSO-d₆) δ 15.4, 16.8, 18.6, 22.6, 23.3, 29.4,

42.2, 45.6, 56.5, 56.8, 103.1, 109.3, 112.5, 117.9, 118.8, 121.2, 124.4,

125.5, 129.7, 132.1, 137.4, 155.6, 155.7. Anal. Calcd for C₂₇H₃₆N₄O₄:

C, 67.48; H, 7.55; N, 11.66. Found: C, 67.27; H, 7.77; N, 12.01.

[3-Phenyl-6-methyl-6,11-dihydro-5H-indolizino[6,7-b]-

indole-1,2-diyl]bis(methylene) Bis(isopropylcarbamate) (28b).

Compound 28b was prepared from 26b (0.36 g, 1 mmol), TEA (0.3

1

mL), and i-Pr isocyanate (0.34 g, 4 mmol). Yield 0.34 g. H NMR

(DMSO-d₆) δ 1.01 (12H, d, J = 7 Hz, 4 × Me), 3.58 (2H, m, CH),

3.60 (3H, s, NMe), 4.14 (2H, s, CH₂), 4.80 (2H, s, CH₂), 5.07 (2H, s,

CH₂), 5.15 (2H, s, CH₂), 6.89 (2H, br s, exchangeable, NH), 7.04−

7.08 (1H, m, ArH), 7.14−7.18 (1H, m, ArH), 7.44−7.46 (2H, m, 2 ×

P

dx.doi.org/10.1021/jm301788a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

NMR (DMSO-d₆) δ 18.9, 22.6, 23.3, 29.3, 41.2, 42.2, 56.8, 57.2, 103.1,

109.2, 113.3, 113.9, 116.3, 117.9, 118.8, 121.3, 123.1, 125.4, 126.2,

129.9, 131.3, 137.4, 155.5, 155.7, 158.9. Anal. Calcd for C₃₃H₃₈N₄O₅:

C, 66.80; H, 6.86; N, 10.03. Found: C, 66.57; H, 6.55; N, 9.81.

[3-(3,4-Dimethoxyphenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-diyl]bis(methylene) Bis-

(isopropylcarbamate) (28h). Compound 28h was prepared from

26h (0.21 g, 0.5 mmol), TEA (0.2 mL), and i-Pr isocyanate (0.17 g, 2

mmol). Yield 0.18 g. ¹H NMR (DMSO-d₆) δ 1.02 (12H, d, J = 6.3 Hz,

4 × Me), 3.58 (2H, m, CH), 3.61 (3H, s, NMe), 3.78 (3H, s, OMe),

3.82 (3H, s, OMe), 4.13 (2H, s, CH₂), 4.78 (2H, s, CH₂), 5.06 (2H, s,

CH₂), 5.15 (2H, s, CH₂), 6.89 (2H, br s, exchangeable, NH), 7.04−

7.10 (4H, m, 4 × ArH), 7.14−7.18 (1H, m, ArH), 7.44−7.46 (1H, m,

ArH), 7.55−7.57 (1H, m, ArH). ¹³C NMR (DMSO-d₆) δ 18.9, 22.0,

22.6, 23.3, 29.3, 41.3, 42.2, 55.5, 56.8, 57.4, 103.0, 109.3, 111.7, 113.4,

114.1, 116.2, 117.9, 118.8, 121.2, 122.7, 123.3, 125.4, 126.2, 129.9,

131.6, 137.4, 148.5, 155.5, 155.7. Anal. Calcd for C₃₃H₄₀N₄O₆: C,

67.33; H, 6.85; N, 9.52. Found: C, 67.04; H, 7.14; N, 9.16.

volume was assessed by measuring the length × width × height (or

width) using a caliper. The vehicle used was 50 μL of DMSO and 40

μL of Tween 80 in 160 μL of saline. The MTD of the tested

compound was determined and used for the in vivo therapeutic

eﬃcacy assay. For tumor-bearing nude mice during the course of the

experiment, the body weight refers to the total weight minus the

weight of the tumor. All animal studies were conducted in accordance

with the guidelines for the National Institutes of Health Guide for the

Care and Use of Animals, and the protocol was approved by the

Institutional Animal Care and Use Committee.

Alkaline Agarose Gel Shift Assay. Formation of DNA cross-links

was analyzed by alkaline agarose gel electrophoresis as previously

described.³³Brieﬂy, puriﬁed pEGFP-N1 plasmid DNA (1500 ng) was

mixed with various concentrations (1, 5, and 10 μM) of 18a, 18b, 19b,

20b, and 28c in 40 μL of binding buﬀer (3 mM sodium chloride/1

mM sodium phosphate, pH 7.4, and 1 mM EDTA). The reaction

mixture was incubated at 37 °C for 2 h. At the end of the reaction, the

plasmid DNA was linearized by digestion with BamHI followed by

precipitation with EtOH. The DNA pellets were dissolved and

denatured in alkaline buﬀer (0.5 N NaOH and 10 mM EDTA). An

aliquot of 20 μL of DNA solution (1500 ng) was mixed with 4 μL of

6× alkaline loading dye and then electrophoretically resolved on a

0.8% alkaline agarose gel with NaOH−EDTA buﬀer at 4 °C. The

electrophoresis was performed at 18 V for 22 h. After the gels were

stained with ethidium bromide solution, the DNA was visualized under

UV light.

Cell-Cycle Analysis. The eﬀects of compounds 18a, 18b, and 28c

on cell-cycle progression were analyzed with a ﬂow cytometer as

described previously.⁵²Brieﬂy, CL141T cells were treated with the

compounds at 0.5-, 1- and 2-fold IC₅₀for 24, 48, and 72 h before the

cells were harvested and ﬁxed with ice-cold 70% EtOH. The cells were

then stained with 4 μg/mL propidium iodide (PI) in phosphate

buﬀered saline (PBS) containing 1% Triton X-100 and 0.1 mg/mL

RNase A. The cells were then subjected to ﬂow cytometric analysis

(FACScan ﬂow cytometer; Becton Dickinson, San Jose, CA). The cell-

cycle phase distribution was analyzed using the ModFit LT 3.0

software (Verity Software House, Topsham, ME) and was based on

the DNA histograms.

Annexin V-FITC Apoptosis Detection. An annexin V-FITC

apoptosis detection kit was purchased from eBioscience Inc. (San

Diego, CA) and was used to study apoptosis induced by the

synthesized compounds. This study was performed according to the

instructions provided by the manufacturer. H460 cells were treated

with compounds 18a and 18b at the IC₅₀dose for 24 and 72 h prior to

harvesting. The cells were resuspended in annexin V-FITC containing

binding buﬀer. After incubation at room temperature for 10 min, the

cells were washed and resuspended with PI and subjected to ﬂow

cytometric analysis (FACScan ﬂow cytometer; Becton Dickinson, San

Jose, CA).

Western Blot Analysis. For electrophoresis, the proteins were

separated by sodium dodecyl sulfate−polyacrylamide gel electro-

phoresis (SDS−PAGE). The proteins were then transferred to a

nitrocellulose membrane, which was blocked with 5% skimmed milk in

phosphate buﬀered saline Tween-20 (PBST). A speciﬁc primary

antibody was added to bind the target proteins for either 1 h at room

temperature or overnight at 4 °C. A horseradish peroxidase (HRP)

conjugated secondary antibody was added to the membrane after the

primary antibody was washed oﬀ. All signals were detected after the

HRP was activated by enhanced chemiluminescence. The antibodies

anti-caspase-3, anti-cascase-7, and anti-PARP (no. 9662, no. 9492, and

no. 9542, Cell Signaling, Danvers, MA) and anti-β-actin (GTX100313,

GeneTex, San Antonio, TX) were used for analyzing the protein levels

of caspase-3, cascase-7, and PARP, as well as their cleaved forms by

diﬀerent exposure periods and β-actin.

Topoisomerase I Mediated DNA Relaxation Assay. The topo I kit

from TopoGEN Inc. (Port Orange, FL, U.S.) was used to determine

whether the synthesized compounds inhibited the topo I mediated

relaxation of supercoiled DNA. The assay was performed according to

the manufacturer’s instructions. The reaction mixture (20 μL)

contained 200 ng of plasmid pHOT DNA and drug in an incubation

[3-(3,4,5-Trimethoxyphenyl)-6-methyl-6,11-dihydro-5H-

indolizino[6,7-b]indole-1,2-diyl]bis(methylene) Bis-

(isopropylcarbamate) (28i). Compound 28i was prepared from

26i (0.22 g, 0.5 mmol), TEA (0.2 mL), and i-Pr isocyanate (0.17 g, 2

mmol). Yield 0.21 g. ¹H NMR (DMSO-d₆) δ 1.02 (12H, d, J = 6.3 Hz,

4 × Me), 3.59 (2H, m, CH), 3.64 (3H, s, NMe), 3.74 (3H, s, OMe),

3.82 (6H, s, 2 × OMe), 4.15 (2H, s, CH₂), 4.81 (2H, s, CH₂), 5.07

(2H, s, CH₂), 5.23 (2H, s, CH₂), 6.79 (2H, m, 2 × ArH), 6.92 (2H, br

s, exchangeable, NH), 7.05−7.09 (1H, m, ArH), 7.15−7.19 (1H, m,

ArH), 7.45−7.47 (1H, m, ArH), 7.56−7.58 (1H, m, ArH). ¹³C NMR

(DMSO-d₆) δ 18.9, 22.6, 23.3, 29.4, 41.4, 42.1, 55.9, 57.4, 60.0, 102.9,

107.7, 109.3, 116.3, 117.9, 118.8, 121.2, 125.4, 126.3, 126.6, 129.9,

131.7, 137.0, 137.4, 152.8, 155.5, 155.7. Anal. Calcd for C₃₄H₄₂N₄O₇:

C, 66.00; H, 6.84; N, 9.06. Found: C, 65.70; H, 6.79; N, 8.86.

Biological Assays. Cytotoxicity Assays. The cytotoxic eﬀects of

the newly synthesized compounds were determined in the T-cell acute

lymphocytic leukemia cell line CCRF-CEM by the XTT assay⁴⁵and in

human solid tumor cell lines such as MX-1 cell line by the SRB assay.⁴⁶

The cells were incubated for 72 h, and the assays were read using a

microplate spectrophotometer as previously described.⁴⁷After the

addition of phenazine methosulfate-XTT solution, the cells were

incubated at 37 °C for 6 h. The absorbance at 450 and 630 nm was

detected on a microplate reader (EL 340). The cytotoxicity of the

newly synthesized compounds against CL141T (the parental cell,

CL141, was isolated from a lung cancer patient and was kindly

provided by Dr. Pan-Chyr Yang, Academia Sinica, Taiwan, and the

CL141T cell line was isolated from a xenograft tumor grown in a

SCID mouse), the lung adenocarcinoma cell lines A549 and H460, the

prostate cancer cell line PC3, the oral cancer cell line OECM1, the oral

carcinoma cell line KB, the vincristine-resistant oral carcinoma cell line

KBvin10, and the colorectal cancer cell lines HCT-116 and HT-29 was

determined by Alamar blue assay⁴⁸in a 72 h incubation using a

microplate spectrophotometer as previously described. After addition

of the Alamar blue solution, the cells were incubated at 37 °C for 10−

12 h. PrestoBlue (Invitrogen, U.S.) assay was used to evaluate the

cytotoxicity of the newly synthesized compounds against the MES-SA

and MES-SA/dx5 cell lines. The MES-SA and MES-SA/dx5 cells were

kindly provided by Dr. Hong-Lin Chan, College of Life Science,

National Tsing-Hua University. In detail, cells were incubated for 72 h

using a microplate spectrophotometer. After addition of the

PrestoBlue reagent, the cells were incubated at 37 °C for 3 h. The

absorbance at 570 and 600 nm was detected on a microplate reader.

The IC₅₀values were determined from the dose−eﬀect relationship

with six or seven concentrations of each drug using the CompuSyn

software by Chou and Martin,⁴⁹which is based on the median-eﬀect

principle and plot.^50,51The ranges given for the IC₅₀values were the

mean STDEV (n = 3).

In Vivo Studies. Athymic nude mice bearing the nu/nu gene were

obtained from National Cancer Institute (NCI), Frederick, MD, and

used for all human tumor xenografts. Male nude mice 6 weeks old or

older weighing 20−24 g or more were used. The compounds were

administered via tail vein iv inj as described previously.⁴⁷Tumor

Q

dx.doi.org/10.1021/jm301788a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

buﬀer (10 mM Tris-HCl, pH 7.9, and 1 mM EDTA). The mixture was

incubated at 37 °C and was initiated by the addition of 1 unit of topo I.

After reaction for 30−45 min, the reaction was stopped by the addition

of 2 μL of 10% SDS. The samples were then loaded onto a 1% agarose

gel and electrophoresed at 50 V for 90 min. The gels were stained with

ethidium bromide, destained with water, and photographed.

Topoisomerase II Mediated DNA Relaxation Assay. The topo II

kit from TopoGEN Inc. (Port Orange, FL, U.S.) was used to evaluate

the inhibitory activity of the synthetic compounds against the topo II

mediated relaxation of supercoiled DNA. The assay was performed

according to the manufacturer’s instructions. The reaction mixture (20

μL) contained 125 ng of plasmid pHOT DNA and drug in an assay

incubation buﬀer (0.5 M Tris-HCl, pH 8.0, 1.5 M NaCl, 100 mM

MgCl₂, 5 mM dithiothreitol, 300 μg/mL BSA, and 20 mM ATP). The

mixture was warmed to 37 °C. The reaction was initiated by the

addition of 6 units of topo II. The mixture was incubated for 30−45

min, and the reaction was stopped by the addition of 2 μL of 10%

SDS. The samples were then loaded onto a 1% agarose gel and

electrophoretically separated at 50 V for 90 min. The gels were stained

with ethidium bromide, destained with water, and photographed.

Stability Assay of Compound 18a in Rat Plasma. HPLC

Conditions. A reversed-phase C18 column (4.6 mm × 150 mm, 5 μm,

Extend-C18, Agilent, Palo Alto, CA, U.S.) was used as a stationary

phase for the separation of compound 18a. The mobile phase

comprised acetonitrile/10 mM monosodium phosphate (pH 4.5)

(32:68, v/v) with at ﬂow rate of 1.1 mL/min. The identiﬁcation and

quantiﬁcation of compound 18a were performed on an HPLC system

equipped with a chromatographic pump (LC-20AT, Shimadzu, Kyoto,

Japan), diode array detector (SPD-M20A, Shimadzu), autosampler

(SIL-20AT, Shimadzu), and degasser (DG-2410). The UV wavelength

of detection was set at 228 nm, and the injection volume of all samples

was 20 μL.

Method Validation. The stock solution of compound 18a in

MeOH (0.5 mg/mL) was diluted with 50% acetonitrile to yield serial

working standard solutions (1.25, 2.5, 5, 25, 50, and 250 μg/mL).

Propyl paraben (10 μg/mL) was dissolved in acetonitrile as the

internal standard. The sample was extracted by acetonitrile protein

precipitation as described in the sample preparation section. The

calibration curves were constructed as follows: compound 18a peak

area/IS peak area for the y-axis and compound 18a concentration for

the x-axis.

Short-Term Stability. The stability analysis followed the U.S. FDA

guidelines. Short-term temperature stability for the spiked plasma

samples was assessed by incubation for 1, 2, 3, and 4 h at room

temperature. The samples were then vortex-mixed with 140 μL of

internal standard solution containing 10 μg/mL propyl paraben

dissolved in acetonitrile for protein precipitation. The stability was

represented as the mean relative error (%) of at least three replicates

per concentration of the freshly prepared samples and the samples

prepared for stability analysis as the peak area ratio of compound 18a

and propyl paraben (internal standard). The sample stability was

deﬁned as the stability ([(C_obs− C_pre)/C_pre] × 100%) within 15%

deviation of the freshly prepared samples and the samples under a

short-term cycle.

(ethylcarbamates) (19a−c, 27a−i), and bis-

(isopropylcarbamate) derivatives (20a−c, 28a−i). This materi-

al is available free of charge via the Internet at http://pubs.acs.

org.

AUTHOR INFORMATION

Corresponding Author

■

*For T.-C.L.: phone, +886-2-27899045; fax, +886-2-27829142;

e-mail, bmtcl@ibms.sinica.edu.tw. For T.-L.S.: phone, +886-2-

27899145; fax, +886-2-27829142; e-mail, tlsu@ibms.sinica.edu.

tw.

Author Contributions

^∞These authors contributed equally.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We are grateful for support from the National Science Council,

Taiwan, Republic of China (Grant NSC 99-2320-B-001-014 to

T.-L.S. and Grant NSC 100-2325-B-001-003 to T.-C.L. and T.-

L.S.), and Academia Sinica, Taipei, Taiwan (Grant AS-100-TP-

B13 to T.-C.L. and T.-L.S.). T.-C.C. was supported by the

Sloan-Kettering Institute General Fund. The NMR spectra of

the synthesized compounds were obtained at the High-Field

Biomolecular NMR Core Facility supported by the National

Research Program for Genomic Medicine (Taiwan). The NMR

spectra were acquired on a Bruker 400 MHz spectrometer at

the Institute of Biological Chemistry, Academia Sinica, and the

National Center for High-Performance Computing is acknowl-

edged for our use of computer time and facilities.

ABBREVIATIONS USED

■

MX-1, human breast carcinoma; A549, human lung adeno-

carcinoma; CL141T, human lung adenocarcinoma; HT-29,

human colon cancer; HCT-116, human colon cancer; topo I,

topoisomerase I; topo II, topoisomerase II; IkK, IkK kinase

complex; ATO, arsenic trioxide; DMAD, dimethyl acetylene-

dicarboxylate; NaH, sodium hydride; MeI, iodomethane; EtI,

iodoethane; TEA, triethylamine; Ac₂O, acetic anhydride;

CCRF-CEM, human lymphoblastic leukemia; PC3, human

prostate cancer; OECM1, human oral epidermoid carcinoma;

HEL299, human embryonic lung ﬁbroblast; KB, human oral

cancer; KBvin10, human oral cancer resistant to vincristine;

MES-SA, human uterine sarcoma cell line; MES-SA/dx5,

human uterine sarcoma cell line resistant to doxorubicin; iv

inj, intravenous injection; QD×3, once per day for 3 days;

QD×4, once per day for 4 days; Q2D×3, every 2 days for three

times; HeLa, human epithelial carcinoma; H1299, human lung

adenocarcinoma; PARP, poly ADP-ribose polymerase; c-

caspase-3, cleaved caspase-3; c-caspase-7, cleaved caspase-7; c-

PARP, cleaved poly ADP-ribose polymerase; U.S. FDA, U.S.

Food and Drug Administration; PI, propidium iodide; SDS−

PAGE, sodium dodecyl sulfate−polyacrylamide gel electro-

phoresis; PBST, phosphate buﬀered saline Tween-20; HRP,

horseradish peroxidase; Topo, topoisomerases

Sample Preparation. The plasma was prepared by centrifugation of

the blood samples at 3000g for 10 min at 4 °C. The plasma samples

were stored at −20 °C until analysis. For each sample, 14 μL of

compound 18a was spiked into 56 μL of plasma and was vortex-mixed

with 140 μL of IS solution containing 10 μg/mL propyl paraben

dissolved in acetonitrile for protein precipitation. The sample was then

centrifuged at 16000g for 10 min at 4 °C. The supernatant was ﬁltered

through a 0.22 μm ﬁlter, and 50 μL of ﬁltrate was transferred to

autosampler vials. A ﬁxed sample loop volume (20 μL) was injected

into the HPLC−photodiode array system.

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ASSOCIATED CONTENT

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* Supporting Information

Physical data of the newly synthesized bis(hydroxymethyl)-

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Novel antitumor indolizino[6,7-b]indoles with multiple modes of action: DNA cross-linking and topoisomerase i and II inhibition

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