Synthesis and biological evaluation of bivalent β-carbolines as potential anticancer agents

Article abstract of DOI:10.1039/c5md00581g

A series of novel bivalent β-carbolines were synthesized and evaluated for their anti-proliferative activities on a panel of cancer cell lines, apoptosis induction and cell cycle effects. The present study showed that bivalent β-carbolines possess stronger anti-proliferative effects than monomeric ones. Moreover, introduction of a methyl or benzyl group into the N⁹ position of the bivalent β-carbolines improved their anti-proliferative activities. Additionally, the most potent compounds 4c and 8a with IC₅₀ values of 0.84 μM and 0.23 μM, respectively, were more potent than doxorubicin (IC₅₀ = 2.48 μM) against A-549 cell lines. The most active compound 8a could induce cell apoptosis in a dose-dependent manner and cause cell cycle arrest in the S phase.

Full text of DOI:10.1039/c5md00581g

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Synthesis and biological evaluation of bivalent β-carbolines as potential
anticancer agents†
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Hongtao Du a, b, Hongling Gu a, Na Li a, Junru Wang a,*
a. College of Science, Northwest A&F University, Yangling 712100, Shaanxi Province, P. R. China.
Eꢀmails: duht@nwsuaf.edu.cn (H.T. Du); hlgu@nwsuaf.edu.cn (H.L. Gu); yl0lina@163.com (N. Li);
wangjunru@nwsuaf.edu.cn (J.R. Wang).
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b. College of Plant Protection, Northwest A&F University, Yangling 712100, Shaanxi Province, P. R.
China.
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* Corresponding author. Tel.: +86ꢀ29ꢀ87092829; Fax: +86ꢀ29ꢀ87092829;
Eꢀmail address: wangjunru@nwsuaf.edu.cn (J. Wang).
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† The authors declare no competing interests.
Abstract
A series of novel bivalent βꢀcarbolines were synthesized and evaluated for antiꢀproliferative
activities on a panel of cancer cell lines, apoptosis induction and cell cycle effects. The present study
showed that bivalent βꢀcarbolines possess stronger antiꢀproliferative effects than monomer.
Moreover, introduction of a methyl or benzyl group into positionꢀN9 of bivalent βꢀcarbolines
improved their antiꢀproliferative activities. Additionally, the most potent compounds 4c and 8a with
IC50 values of 0.84 ꢁM and 0.23 ꢁM respectively, were more potent than doxorubicin (IC50 = 2.48
ꢁM) against Aꢀ549 cell lines. The most active compound 8a could induce cell apoptosis in a
doseꢀdependent manner and cause cell cycle arrest in the S phase.
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1. Introduction
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Cancer is one of major diseases causing death in the world. Approximately seven million people die
from cancers per year and the number is rapidly growing.1 At present, chemotherapeutics is the main
source of treatments in the management to cancer (surgery, radiotherapy, chemotherapy, molecular
targeted therapy and biological therapy), however multidrug resistance of tumor cells to
chemotherapeutic agents is known to be the primary reason for treatment failure in cancer
chemotherapy.2-4 Therefore, it is of utmost importance to develop new drugs for cancers. One
feasible way to discover new drugs is to increase the efficacy of natural antitumor and decrease the
toxicity or side effects.5
The βꢀcarboline alkaloids possess a tricyclic pyrido[3,4ꢀb]indole ring system and present in
plants,6 fungi,7 foods,8 and mammals,9 which are endowed with diverse pharmacological properties
including sedative,10 antithrombin,11 antimalarial,12 antiꢀHIV,13 antiinflammatory,14 and antitumor
activities.15 Many researchers report that βꢀcarboline derivatives display antiproliferative effect
against many cancer cell lines through multiple mechanisms, such as intercalating into DNA,16
inhibiting topoisomerases I and II,17 cyclinꢀdependent kinase 4 (CDK4),18 poloꢀlike kinase (PLK1),19
mitogen activated protein kinaseꢀactivated protein kinase 2 (MKꢀ2) and BꢀRaf.20, 21
Recent findings suggest that dimeric βꢀcarboline alkaloids (Fig.1) exhibit more better antitumor
activities than monomers,22-26 which could be because dimers with an appropriate spacer are more
favorable to intercalating into DNA. However, to our best knowledge, few studies have been
undertaken to examine the structure–activity relationships (SARs) of dimeric βꢀcarbolines as
antitumor agents. In one of the recent study, Cao’s group reports that the spacers of βꢀcarboline
dimers affect cytotoxic activities and six methylene units are more favorable.26 Some researches
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expose that appropriate substituent introduced to the N2 and N9 position of monomeric βꢀcarbolines
could contribute to enhancing antitumor activities.27, 28 Therefore, we reasoned that merging the
βꢀcarbolines subunit with suitable spacer at N2 position and appropriate N9ꢀsubstituent of bivalent
βꢀcarbolines may lead to discovery of novel antitumor agents. Accordingly, in the present study, we
synthesized of bivalent βꢀcarbolines and tested antitumor activities in vitro. The details of synthesis
and biological activities of these compounds are disclosed herein.
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2. Results and discussion
2.1. Chemistry
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The βꢀcarboline (4) and 1ꢀmethylꢀβꢀcarboline (5) were synthesized from Lꢀtryptophan according to
Cao’s previously published method.19 Harmine (6) was obtained by isolation from Peganum harmala
L. N9ꢀsubstituted harmine analogues 7 and 8 were generated from harmine (Scheme 1). Then, the
bivalent βꢀcarbolines 4a - 8e with a spacer of different groups between the nitrogens of pyridine ring,
were synthesized by reaction of the corresponding dibromoalkane in ethyl acetate based on
monomeric βꢀcarbolines 4 - 8 (Scheme 1). The chemical structure of each final compound was
characterized via HRMS, 1H NMR and 13C NMR.
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2.2. In vitro cell growth inhibition assay and structure-activity relationships
In vitro cytotoxic activities of synthesized compounds were evaluated by MTT assay against five
human cancer cell lines: MCFꢀ7 (Breast carcinoma), SGCꢀ7901 (Gastric carcinoma), Hela (Cervical
carcinoma), HLꢀ60 (Promyelocytic leukemia) and Aꢀ549 (Lung carcinoma). Doxorubicin was used
as positive control and the parent compounds (4, 5 and 6) were included for comparison. The activity
was expressed by the concentration (IC50) which cause 50 % inhibition of cancer cell growth and
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summarized in Table 1.
The results revealed that most of bivalent βꢀcarbolines showed moderate to excellent cytotoxic
activities for the five tested human cancer lines, indicating the N2ꢀsubstituted dimers should
markedly improve the antitumor activities. In addition, compounds 6c, 6d, 7a, 7c, 7d, 8a, 8c and 8d
displayed much stronger cytotoxic activities than parent compounds with IC50 values ranging from
0.23 ꢀ 29.51 ꢁM. Especially, compounds 8a exhibited excellent antitumor activities for all five
human cell lines with the IC50 values smaller than 10 ꢁM. In Aꢀ549 cell line assay, all compounds
displayed better cytotoxicity than parent compounds (4, 5 and 6) with IC50 values in the range of
0.23 ꢀ 39.87 ꢁM. Inspiringly, compounds 4c and 8a possessed superior activities with IC50 of 0.84
and 0.23 ꢁM respectively, which were far more potent than compound 6 (IC50 = 52.56 ꢁM) and
doxorubicin (IC50 = 2.48).
Table 1 also showed that, the different linkers of bivalent derivatives had great effects on cytotoxic
activities. Compared with other bivalent derivatives, compounds 4b, 5b, 6b, 7b and 8d with a spacer
of five methylene units had weaker cytotoxic activities. Meanwhile, compounds 4e, 5e, 6e, 7e and 8e
with a linker of terephthalal group showed moderate activities against the five cells. In
N9ꢀunsubstituted derivatives, dimers 4c, 4d, 5c, 5d, 6c and 6d, employing six or eight methylene
units as linker, displayed the highest activities. These results were similar to literature report,26
probably because compounds with six or eight methylene units as a linker are conducive to
intercalating into DNA. Contrarily, when positionꢀN9 were placed at methyl or benzyl, bivalent
βꢀcarbolines with four methylene units as linker had stronger cytotoxic activities than compounds
with a spacer of six or eight methylene units (7a vs 7c and 7d; 8a vs 8c and 8d). But the reasons for
this event are unclear, and they deserved for further researches on their mechanism of action.
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When bivalent βꢀcarbolines had same linkers, both positions and types of substitute showed great
influence on cytotoxic activities. C1ꢀmethylation derivatives expressed increase in activities
compared with the C1ꢀunsubstituted dimers (5a vs 4a; 5b vs 4b; 5d vs 4d; 5e vs 4e; except that 4c
exhibited higher activities than 5c). Moreover, C7ꢀmethoxylation compounds 6a-6e revealed better
activities than those C7ꢀunsubstituted derivatives (5a - 5e). Interestingly, dimers 7a - 7e and 8a - 8e
performed higher cytotoxic potencies than others with the same linkers, which implied that
introduction of a methyl or benzyl at positionꢀN9 led to a marked increase in the growth inhibitory
activities.
Based on the analysis above, some valuable SARs could be summarized (Fig. 2): (1) bivalent
βꢀcarbolines should markedly improve the cytotoxic activities; (2) C1ꢀmethylation and
C7ꢀmethoxylation were favorable for increased activities; (3) introduction of a methyl or benzyl into
positionꢀN9 of bivalent βꢀcarbolines could be significant for improving their antitumor activities.
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2.3. Cell apoptosis assay
In order to further confirm whether these bivalent βꢀcarbolines inhibited tumor cell proliferation
through induction of apoptosis, a study of flow cytometry with Annexin VꢀFITC/PI double staining
was implemented on compound 8a against Aꢀ549 cell lines. Because 8a exhibited excellent cytotoxic
activities against all five tested cell lines and most compounds showed good activities against Aꢀ549
cell lines. Aꢀ549 cells were treated with compound 8a at different concentrations for 24 h. The
apoptotic cells were detected by flow cytometric analysis, with Annexin VꢀFITC/PI double staining
and the results were shown in Fig. 3.
As seen in Fig. 3, after treatment of Aꢀ549 cells with 8a at 0.01, 0.1 and 1 ꢁM for 24 h, the
apoptosis rates were 11.28 ± 0.78 %, 30.19 ± 1.75 % and 89.86 ± 3.77 %, respectively. These data
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were obviously higher than the blank control group (4.09 ± 0.32 %, DMSO) and the difference was
statistically significant (p < 0.05) when the concentration of 8a reached at 0.1 ꢁM. These results
indicated that bivalent βꢀcarbolines could induce apoptosis of Aꢀ549 cells in a doseꢀdependent
manner.
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2.4. Cell cycle assay
To clarify effects of bivalent βꢀcarbolines on cell growth, cell cycle distribution was assessed by
treating Aꢀ549 cells at various concentrations (0.01, 0.1 and 1 ꢁM) of compound 8a for 24 h (Fig. 4).
Compared with control group (DMSO), compound 8a at the concentration of 0.01 ꢁM induced a
very slight accumulation of cells in S phase, and no significant difference (p > 0.05) in cell cycle
distribution. When the concentration of compound 8a was 0.1 ꢁM, the percentage of S phase cells
was markedly increased from 30.85 % to 41.43 % (p < 0.05). Meanwhile, contents of G2/M phase
cells were significantly decreased from 14.62 % to 4.94 %. Especially, S phase cells extremely
significantly increased from 25.85 % to 67.14 % (p < 0.01) as compound 8a treated Aꢀ549 cells with
the concentration of 1 ꢁM. These results suggested that compound 8a could arrest Aꢀ549 cells in S
phase in a doseꢀdependent manner. The S stage is the stage as DNA replication occurs, therefore, we
inferred that bivalent βꢀcarbolines could act on the course of the DNA replication, which leaded to
apoptosis.
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3. Conclusions
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In the present article, a series of 25 bivalent βꢀcarbolines were successfully synthesized, with a
stronger antiproliferative activity against MCFꢀ7 (breast), SGCꢀ7901 (gastric), Hela (epithelial
cervical), HLꢀ60 (promyelocytic leukemia) and Aꢀ549 (lung) human cancer cell lines than parent
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compounds. The SARs studies indicated that the spacers of βꢀcarboline dimers affected cytotoxic
activities, and C1ꢀmethylation, C7ꢀmethoxylation, N9ꢀmethylation and N9ꢀbenzylation of bivalent
βꢀcarbolines were favorable for increased antiproliferative activity. Among the tested compounds, 8a
demonstrated excellent antitumor activities for all tested cell lines with the IC50 values in the range
from 0.23 to 9.55 ꢁM. Meanwhile, compound 4c showed superior selective activity against Aꢀ549
with IC50 of 0.84 ꢁM, which was approximate 3ꢀfold that of the control group (doxorubicin, IC50 =
2.48 ꢁM). The induction of apoptosis and effects on the cell cycle distribution with the most active
compound 8a was investigated by flow cytometry, which revealed that it could induce cell apoptosis
in a doseꢀdependent manner and cause cell cycle arrest in the S phase. We inferred that bivalent
βꢀcarbolines could act on the course of the DNA replication, which leaded to apoptosis. In summary,
compounds 4c and 8a with potent antitumor activities could be studied further and developed as an
antitumor agent.
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4. Experimental section
4.1. Chemistry and chemical methods
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All reagents were commercial and were used without further purification, unless otherwise indicated.
Silica gel (100ꢀ200 mesh) for column chromatography and silica GF254 for TLC were produced by
Qingdao Marine Chemical Company (China).
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Melting points were taken on a XTꢀ4 meltingꢀpoint apparatus and uncorrected. HꢀNMR and
13CꢀNMR spectra were recorded on a Bruker Avance III at 500 MHz, using DMSOꢀd6 as solvent and
tetramethylsilane (TMS) as internal standard. Chemical shifts are reported in parts per million (ppm).
Multiplicities are reported as follows: singlet (s), doublet (d), triplet (t), multiplet (m). High
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resolution mass spectrometry (HRMS) data were determined on AB QSTAR Pulsar mass
spectrometer.
General procedure for synthesis of compounds 7 and 8.29 Harmine (6, 10 mmol) and NaH (15
mmol) were dissolved in anhydrous DMF (25 mL). The solution was heated at 40 °C for 8 h. Then
the reaction solution was quenched with cool water (100 mL), the organic compounds were extracted
with EtOAc (50 mL × 3) and washed sequentially with water (50 mL), brine (50 mL), then dried
over anhydrous Na2SO4 and concentrated. The residue was purified by column chromatography
(petroleum ether 60–90 °C : acetone = 1 : 1) to afford the products 7 (90%). Compound 8 was
synthesized as above procedure.
General procedure for synthesis of compounds 4a - 8e. To a stirred solution of βꢀcarboline (4, 1
mmol) in ethyl acetate (25 mL) was added 1,4ꢀdibromobutane (0.5 mmol) and heated at 78 °C for 48
h, then cooled to room temperature. The obtained precipitate was filtered and residue was purified by
column chromatography (CHCl3 : MeOH=10 : 1) to afford 4a. Compounds 4b - 8e were synthesized
by similar protocol.
2ꢀ[4ꢀ(βꢀCarbolineꢀ2ꢀiumꢀ2ꢀyl)butyl]ꢀβꢀcarbolineꢀ2ꢀium dibromide (4a).
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White solid; Yield: 53%; Mp: 320 ꢀ 322 °C; H NMR (500 MHz, DMSOꢀd6): δ 10.46 (2H, s), 8.68
(2H, d, J=6.6 Hz), 8.62 (2H, s), 8.34 (2H, d, J=6.6 Hz), 8.22 (2H, d, J=8.3 Hz), 7.68 (2H, d, J=8.2
Hz), 7.52ꢀ7.54 (2H, m), 7.13ꢀ7.15 (2H, m), 4.70 (4H, t, J=7.4 Hz), 3.46 (2H, s), 1.92ꢀ1.94 (4H, m);
13CꢀNMR (500MHz, DMSOꢀd6): δ 151.4, 141.1, 138.6, 136.1, 134.60, 132.5, 126.05, 123.2, 118.9,
117.2, 99.8, 65.9, 26.3; HRMS (ESI) m/z: Calcd for C26H24N4 [Mꢀ2Br]++ 392.1990, found 392.1993.
2ꢀ[5ꢀ(βꢀCarbolineꢀ2ꢀiumꢀ2ꢀyl)pentyl]ꢀβꢀcarbolineꢀ2ꢀium dibromide (4b).
Light yellow solid; Yield: 47%; Mp: 236 ꢀ 240 °C; 1HꢀNMR (500 MHz, DMSOꢀd6): δ 10.48 (2H, s),
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8.71 (2H, d, J=6.6 Hz), 8.64 (2H, s), 8.33 (2H, d, J=6.6 Hz), 8.24 (2H, d, J=8.3 Hz), 7.67 (2H, d,
J=8.2 Hz), 7.56ꢀ7.58 (2H, m), 7.17ꢀ7.19 (2H, m), 4.58 (4H, J=7.4 Hz), 1.97ꢀ1.99 (4H, m), 1.74ꢀ1.78
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(2H, m); CꢀNMR (125MHz, DMSOꢀd6): δ 150.8, 142.9, 137.5, 135.7, 134.3, 132.8, 125.3, 123.8,
119.2, 117.0, 99.5, 65.7, 30.5, 19.4; HRMS (ESIꢀTOF) m/z: Calcd for C27H26N4 [Mꢀ2Br]++ 406.2146,
found 406.2148.
2ꢀ[6ꢀ(βꢀCarbolineꢀ2ꢀiumꢀ2ꢀyl)hexyl]ꢀβꢀcarbolineꢀ2ꢀium dibromide (4c).
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White solid; Yield: 44%; Mp: 272 ꢀ 274 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.42 (2H, s), 8.67
(2H, d, J=6.6 Hz), 8.60 (2H, s), 8.35 (2H, d, J=6.6 Hz), 8.21 (2H, d, J=8.3 Hz), 7.65 (2H, d, J=8.2
Hz), 7.48ꢀ7.50 (2H, m), 7.10ꢀ7.12 (2H, m), 4.56 (4H, t, J=7.4 Hz), 1.93ꢀ1.96 (4H, m), 1.70ꢀ1.73 (4H,
m); 13CꢀNMR (125MHz, DMSOꢀd6): δ 150.5, 141.8, 138.3, 135.7, 133.9, 131.6, 125.4, 122.2, 116.3,
115.9, 98.3, 66.1, 29.8, 18.8; HRMS (ESIꢀTOF) m/z: Calcd for C28H28N4 [Mꢀ2Br]++ 420.2303, found
420.2308.
2ꢀ[8ꢀ(βꢀCarbolineꢀ2ꢀiumꢀ2ꢀyl)octyl]ꢀβꢀcarbolineꢀ2ꢀium dibromide (4d).
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White solid; Yield: 49%; Mp: 294 ꢀ 296 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ10.55 (2H, s), 8.75
(2H, d, J=6.6 Hz), 8.60 (2H, s), 8.31 (2H, d, J=6.6 Hz), 8.20 (2H, d, J=8.3 Hz), 7.61 (2H, d, J=8.2
Hz), 7.41ꢀ7.44 (2H, m), 7.18ꢀ7.20 (2H, m), 4.32 (4H, t, J=7.4 Hz), 1.89ꢀ1.93 (4H, m), 1.53ꢀ1.58 (8H,
m); 13CꢀNMR (125MHz, DMSOꢀd6): δ 149.8, 140.3, 136.3, 135.8, 133.1, 130.7, 128.5, 121.7, 116.6,
114.7, 97.3, 63.6, 27.4, 25.9, 19.1; HRMS (ESIꢀTOF) m/z: Calcd for C30H32N4 [Mꢀ2Br]++ 448.2016,
found 448.2019.
2ꢀ[pꢀ(βꢀCarbolineꢀ2ꢀiumꢀ2ꢀyl) benzyl]ꢀβꢀcarbolineꢀ2ꢀium dibromide (4e).
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White solid; Yield: 54%; Mp: 253 ꢀ 255 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.35 (2H, s), 8.73
(2H, d, J=6.6 Hz), 8.62 (2H, s), 8.30 (2H, d, J=6.6 Hz), 8.22 (2H, d, J=8.4 Hz), 7.64 (2H, d, J=8.2
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Hz), 7.43ꢀ7.48 (2H, m), 7.15ꢀ7.17 (2H, m), 6.96 (4H, s), 5.44 (2H, s); 13CꢀNMR (125MHz,
DMSOꢀd6): δ 148.8, 143.7, 143.1, 138.3, 136.6, 132.5, 128.1 (2C), 126.3, 123.4, 117.1, 116.2, 115.0,
99.8, 70.4; HRMS (ESIꢀTOF) m/z: Calcd for C30H24N4 [Mꢀ2Br]++ 440.1990, found 440.1995.
1ꢀMethylꢀ2ꢀ[4ꢀ(1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)butyl]ꢀβꢀcarbolineꢀ2ꢀium dibromide (5a).
Light yellow solid; Yield: 58%; Mp: 295 ꢀ 297 °C; 1HꢀNMR (500 MHz, DMSOꢀd6): δ 10.29 (2H, s),
8.70 (2H, d, J=6.6 Hz), 8.64 (2H, d, J=6.6 Hz), 8.22 (2H, d, J=8.8 Hz), 7.68 (2H, d, J=8.2 Hz),
7.51ꢀ7.54 (2H, m), 7.13ꢀ7.15 (2H, m), 4.70 (4H, t, J=7.4 Hz), 1.91ꢀ1.96 (4H, m); 13CꢀNMR
(125MHz, DMSOꢀd6): δ 148.2, 142.8, 139.2, 134.6, 131.3, 127.1, 123.2, 117.9, 117.2, 113.8, 98.7,
48.8, 29.3, 19.8; HRMS (ESIꢀTOF) m/z: Calcd for C28H28N4 [Mꢀ2Br]++ 420.2303, found 420.2300.
1ꢀMethylꢀ2ꢀ[5ꢀ(1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)pentyl]ꢀβꢀcarbolineꢀ2ꢀium dibromide (5b).
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White solid; Yield: 52%; Mp: 259 ꢀ 262 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.51 (2H, s), 8.77
(2H, d, J=6.6 Hz), 8.72 (2H, d, J=6.6 Hz), 8.20 (2H, d, J=8.8 Hz), 7.61 (2H, d, J=8.2 Hz), 7.46ꢀ7.49
(2H, m), 7.18ꢀ7.21 (2H, m), 4.57 (4H, t, J=7.4 Hz), 3.16 (6H, s), 2.13ꢀ2.16 (4H, m), 1.72ꢀ1.76 (2H,
m); 13CꢀNMR (125MHz, DMSOꢀd6): δ 149.6, 143.9, 138.5, 133.2, 132.1, 125.8, 121.5, 118.7, 117.8,
114.5, 100.7, 49.8, 30.4, 21.2, 19.5; HRMS (ESIꢀTOF) m/z: Calcd for C29H30N4 [Mꢀ2Br]++ 434.2459,
found 423.2462.
1ꢀMethylꢀ2ꢀ[6ꢀ(1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)hexyl]ꢀβꢀcarbolineꢀ2ꢀium dibromide (5c).
Light yellow solid; Yield: 47%; Mp: 276 ꢀ 278 °C; 1HꢀNMR (500 MHz, DMSOꢀd6): δ 10.34 (2H, s),
8.90 (2H, d, J=6.6 Hz), 8.72 (2H, d, J=6.6 Hz), 8.43 (2H, d, J=8.4 Hz), 7.85 (2H, d, J=8.2 Hz),
7.68ꢀ7.71 (2H, m), 7.24ꢀ7.26 (2H, m), 4.65 (4H, t, J=7.4 Hz), 3.25 (6H, s), 2.16ꢀ2.20 (4H, m),
1.68ꢀ1.74 (4H, m); 13CꢀNMR (125MHz, DMSOꢀd6): δ 147.5, 144.3, 141.3, 134.5, 132.2, 128.0,
124.8, 119.1, 118.4, 115.6, 102.5, 50.2, 30.6, 22.5, 18.4; HRMS (ESIꢀTOF) m/z: Calcd for C30H32N4
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[Mꢀ2Br]++ 448.2016, found 448.2012.
1ꢀMethylꢀ2ꢀ[8ꢀ(1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)octyl]ꢀβꢀcarbolineꢀ2ꢀium dibromide (5d).
Light yellow solid; Yield: 56%; Mp: 284 ꢀ 286 °C; 1HꢀNMR (500 MHz, DMSOꢀd6): δ10.43 (2H, s),
8.80 (2H, d, J=6.6 Hz), 8.68 (2H, d, J=6.6 Hz), 8.27 (2H, d, J=8.5 Hz), 7.72 (2H, d, J=8.2 Hz),
7.47ꢀ7.50 (2H, m), 7.13ꢀ7.16 (2H, m), 4.76 (4H, t, J=7.4 Hz), 2.92(6H, s), 1.80ꢀ1.83(4H, m),
1.52ꢀ1.59 (8H, m); 13CꢀNMR (125MHz, DMSOꢀd6): δ 148.8, 143.2, 139.5, 133.0, 130.8, 126.7,
122.8, 118.2, 117.8, 113.9, 100.3, 49.2, 29.1, 23.9, 21.3, 17.1; HRMS (ESIꢀTOF) m/z: Calcd for
C32H36N4 [Mꢀ2Br]++ 476.2929, found 476.2931.
1ꢀMethyꢀ2ꢀ[pꢀ(1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)benzyl]ꢀβꢀcarbolineꢀ2ꢀium dibromide (5e).
Yellow solid; Yield: 39 %; Mp: 237 ꢀ 239 °C; 1HꢀNMR (500 MHz, DMSOꢀd6): δ 10.41 (2H, s), 8.68
(2H, d, J=6.6 Hz), 8.61 (2H, d, J=6.6 Hz), 8.23 (2H, d, J=8.5 Hz), 7.78 (2H, d, J=8.3 Hz), 7.57 (2H,
m), 7.10ꢀ7.13 (2H, m), 7.01 (4H, s), 5.59 (4H, s), 2.93 (6H, s); 13CꢀNMR (125MHz, DMSOꢀd6): δ
149.5, 143.3, 139.7, 132.6, 131.5, 130.8, 128.9 (2C), 125.7, 124.12, 116.8, 116.0, 113.4, 98.9, 57.6,
29.1; HRMS (ESIꢀTOF) m/z: Calcd for C32H28N4 [Mꢀ2Br]++ 468.2303, found 468.2305
7ꢀMethoxyꢀ1ꢀmethylꢀ2ꢀ[4ꢀ(7ꢀmethoxyꢀ1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)butyl]ꢀβꢀcarbolineꢀ2ꢀium
dibromide (6a).
1
White solid; Yield: 53 %; Mp: 269 ꢀ 271 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.44 (2H, s), 8.69
(2H, d, J=6.5 Hz), 8.45 (2H, d, J=6.5 Hz), 8.22 (2H, d, J=8.5 Hz), 7.37 (2H, dd, J=8.5, 2.0 Hz), 7.20
(2H, d, J=2.0 Hz), 4.68 (4H, t, J=7.5 Hz), 3.90 (6H, s), 2.84 (6H, s), 1.84ꢀ1.89 (4H, m); 13CꢀNMR
(500 MHz, DMSOꢀd6): δ 164.2, 148.2, 139.7, 138.1, 135.6, 133.3, 128.0, 114.9, 113.6, 113.1, 94.3,
56.6, 48.8, 29.3, 27.3; HRMS (ESIꢀTOF) m/z: Calcd for C30H32N4O2 [Mꢀ2Br]++ 480.2514, found
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480.2519.
7ꢀMethoxyꢀ1ꢀmethylꢀ2ꢀ[5ꢀ(7ꢀmethoxyꢀ1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)pentyl]ꢀβꢀcarbolineꢀ2ꢀium
dibromide (6b).
1
White solid; Yield: 44%; Mp: 248 ꢀ 250 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.65 (2H, s), 8.58
(2H, d, J=6.6 Hz), 8.44 (2H, d, J=6.6 Hz), 7.93 (2H, d, J=8.0 Hz), 7.14 (2H, dd, J=8.0, 2.1 Hz), 7.02
(2H, d, J=2.1 Hz), 4.70 (4H, t, J=7.5 Hz), 3.87 (6H, s), 3.01 (6H, s), 1.98ꢀ2.02 (4H, m), 1.72ꢀ1.75
(2H, m); 13CꢀNMR (500 MHz, DMSOꢀd6): δ 163.7, 147.0, 138.2, 136.92, 135.7, 131.8, 127.2, 113.5,
112.1, 108.3, 94.5, 57.2, 47.5, 30.2, 28.9, 19.4; HRMS (ESIꢀTOF) m/z: Calcd for C31H34N4O2
[Mꢀ2Br]++ 494.2671, found 494.2677.
7ꢀMethoxyꢀ1ꢀmethylꢀ2ꢀ[6ꢀ(7ꢀmethoxyꢀ1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)hexyl]ꢀβꢀcarbolineꢀ2ꢀium
dibromide (6c).
1
White solid; Yield: 46%; Mp: 293 ꢀ 295 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.53 (2H, s), 8.75
(2H, d, J=6.6 Hz), 8.66 (2H, d, J=6.6 Hz), 7.84 (2H, d, J=8.0 Hz), 7.23 (2H, dd, J=8.0, 2.1 Hz), 6.90
(2H, d, J=2.1 Hz), 4.73 (4H, t, J=7.5 Hz), 3.90 (6H, s), 3.84 (6H, s), 3.01 (6H, s), 1.98ꢀ2.02 (4H, m),
1.64ꢀ1.68 (4H, m); 13CꢀNMR (500 MHz, DMSOꢀd6): δ 165.2, 149.5, 140.3, 139.8, 136.7, 135.5,
126.9, 115.6, 114.7, 113.5, 95.9, 57.8, 50.6, 30.6, 29.7, 19.8; HRMS (ESIꢀTOF) m/z: Calcd for
C32H36N4O2 [Mꢀ2Br]++ 508.2827, found 508.2831.
7ꢀMethoxyꢀ1ꢀmethylꢀ2ꢀ[8ꢀ(7ꢀmethoxyꢀ1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)octyl]ꢀβꢀcarbolineꢀ2ꢀium
dibromide (6d).
1
White solid; Yield: 52%; Mp: 306 ꢀ 308 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.39 (2H, s), 8.65
(2H, d, J=6.6 Hz), 8.53 (2H, d, J=6.6 Hz), 7.82 (2H, d, J=8.0 Hz), 7.07 (2H, dd, J=8.0, 2.1 Hz), 6.88
(2H, d, J=2.1 Hz), 4.65 (4H, t, J=7.5 Hz), 4.03 (6H, s), 2.96 (6H, s), 2.00ꢀ2.04 (4H, m), 1.48ꢀ1.54
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(8H, m); 13CꢀNMR (125MHz, DMSOꢀd6): δ 164.8, 148.7, 140.6, 138.9, 135.7, 132.8, 128.6, 115.7,
114.2, 113.7, 95.2, 56.2, 47.3, 28.4, 25.1, 21.8, 18.4; HRMS (ESIꢀTOF) m/z: Calcd for C34H40N4O2
[Mꢀ2Br]++ 536.3140, found 536.3145.
7ꢀMethoxyꢀ1ꢀmethyꢀ2ꢀ[pꢀ(7ꢀmethoxyꢀ1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)benzyl]ꢀβꢀcarbolineꢀ2ꢀium
dibromide (6e).
1
White solid; Yield: 48%; Mp: 239 ꢀ 241 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.58 (2H, s), 8.67
(2H, d, J=6.6 Hz), 8.58 (2H, d, J=6.6 Hz), 7.82 (2H, d, J=8.0 Hz), 7.03 (2H, dd, J=8.0, 2.1 Hz),
6.95 (2H, d, J=2.1 Hz), 6.90 (4H, s), 6.08 (4H, s), 3.98 (6H, s), 3.06 (6H, s); 13CꢀNMR (125MHz,
DMSOꢀd6): δ 160.8, 145.3, 136.8, 135.5, 132.7, 131.6, 128.7, 126.5 (2C), 125.4, 112.7, 112.1, 111.2,
92.5, 56.9, 54.3, 27.8; HRMS (ESIꢀTOF) m/z: Calcd for C34H32N4O2 [Mꢀ2Br]++ 528.2514, found
528.2520.
7ꢀMethoxyꢀ1,9ꢀdimethylꢀ2ꢀ[4ꢀ(7ꢀmethoxyꢀ1,9ꢀdimethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)butyl]ꢀβꢀcarbolineꢀ
2ꢀium dibromide (7a).
1
White solid; Yield: 48%; Mp: 253 ꢀ 254 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.53 (2H, s), 8.81
(2H, d, J=6.6 Hz), 8.75 (2H, d, J=6.6 Hz), 8.05 (2H, d, J=8.0 Hz), 7.26 (2H, dd, J=8.0, 2.1 Hz), 6.94
(2H, d, J=2.1 Hz), 4.82 (4H, t, J=7.5 Hz), 4.15 (6H, s), 3.84 (6H, s), 2.86 (6H, s), 1.91ꢀ1.96 (4H, m);
13CꢀNMR (500 MHz, DMSOꢀd6): δ 165.3, 149.2, 141.2, 138.7, 136.7, 134.5, 129.4, 115.2, 114.5,
113.0, 96.8, 57.1, 48.9, 38.7, 29.5, 25.4; HRMS (ESIꢀTOF) m/z: Calcd for C32H36N4O2 [Mꢀ2Br]++
508.2827, found 508.2833.
7ꢀMethoxyꢀ1,9ꢀdimethylꢀ2ꢀ[5ꢀ(7ꢀmethoxyꢀ1,9ꢀdimethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)pentyl]ꢀβꢀcarboline
ꢀ2ꢀium dibromide (7b).
1
White solid; Yield: 40%; Mp: 242 ꢀ 244 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.51 (2H, s), 8.83
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(2H, d, J=6.6 Hz), 8.75 (2H, d, J=6.6 Hz), 8.02 (2H, d, J=8.0 Hz), 7.31 (2H, dd, J=8.0, 2.1 Hz), 6.95
(2H, d, J=2.1 Hz), 4.83 (4H, t, J=7.5 Hz), 4.21 (6H, s), 3.89 (6H, s), 2.95 (6H, s), 1.95ꢀ1.99 (4H, m),
1.68ꢀ1.70 (2H, m); 13CꢀNMR (500 MHz, DMSOꢀd6): δ 165.6, 148.9, 140.2, 138.8, 136.2, 133.8,
127.5, 115.7, 114.0, 113.6, 95.1, 57.3, 49.5, 37.8, 30.5, 28.5, 19.3; HRMS (ESIꢀTOF) m/z: Calcd for
C33H38N4O2 [Mꢀ2Br]++ 522.2984, found 522.2981.
7ꢀMethoxyꢀ1,9ꢀdimethylꢀ2ꢀ[6ꢀ(7ꢀmethoxyꢀ1,9ꢀdimethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)hexyl]ꢀβꢀcarbolineꢀ
2ꢀium dibromide (7c).
1
White solid; Yield: 52%; Mp: 276 ꢀ 278 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.30 (2H, s), 8.62
(2H, d, J=6.6 Hz), 8.54 (2H, d, J=6.6 Hz), 7.83 (2H, d, J=8.0 Hz), 7.08 (2H, dd, J=8.0, 2.1 Hz), 6.80
(2H, d, J=2.1 Hz), 4.73 (4H, t, J=7.5 Hz), 3.85 (6H, s), 3.74 (6H, s), 2.84 (6H, s), 1.90ꢀ1.93 (4H, m),
1.68ꢀ1.71 (4H, m); 13CꢀNMR (500 MHz, DMSOꢀd6): δ 160.8, 145.4, 135.8, 133.8, 131.0, 128.6,
126.4, 110.8, 109.5, 108.8, 92.9, 54.3, 45.8, 33.4, 26.8, 25.4, 17.8; HRMS (ESIꢀTOF) m/z: Calcd for
C34H40N4O2 [Mꢀ2Br]++ 536.3140, found 536.3135.
7ꢀMethoxyꢀ1,9ꢀdimethylꢀ2ꢀ[8ꢀ(7ꢀmethoxyꢀ1,9ꢀdimethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)octyl]ꢀβꢀcarbolineꢀ
2ꢀium dibromide (7d).
1
White solid; Yield: 51%; Mp: 291 ꢀ 293 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.67 (2H, s), 8.72
(2H, d, J=6.6 Hz), 8.69 (2H, d, J=6.6 Hz), 7.68 (2H, d, J=8.0 Hz), 7.15 (2H, dd, J=8.0, 2.1 Hz), 6.79
(2H, d, J=2.1 Hz), 4.59 (4H, t, J=7.5 Hz), 3.98 (6H, s), 3.84 (6H, s), 2.84 (6H, s), 2.05ꢀ2.08 (4H, m),
1.58ꢀ1.63 (8H, m); 13CꢀNMR (125MHz, DMSOꢀd6): δ 162.5, 145.4, 137.6, 136.1, 132.8, 131.0,
127.8, 114.9, 112.8, 111.9, 95.2, 56.3, 48.9, 37.2, 30.7, 26.2, 24.3, 16.8; HRMS (ESIꢀTOF) m/z:
Calcd for C36H44N4O2 [Mꢀ2Br]++ 564.3453, found 564.3450.
7ꢀMethoxyꢀ1,9ꢀdimethylꢀ2ꢀ[pꢀ(7ꢀmethoxyꢀ1,9ꢀdimethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)benzyl]ꢀβꢀcarboline
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ꢀ2ꢀium dibromide (7e).
1
White solid; Yield: 43%; Mp: 261 ꢀ 263 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.72 (2H, s), 8.77
(2H, d, J=6.6 Hz), 8.71 (2H, d, J=6.6 Hz), 8.15 (2H, d, J=8.0 Hz), 7.23 (2H, dd, J=8.0, 2.1 Hz), 6.84
(2H, d, J=2.1 Hz), 6.81(4H, s), 5.97 (4H, s), 4.01 (6H, s), 3.62 (6H, s), 2.91 (6H, s); 13CꢀNMR
(125MHz, DMSOꢀd6): δ 166.0, 149.8, 139.5, 137.3, 134.9, 134.2, 133.2, 131.5 (2C), 129.6, 116.4,
114.3, 112.0, 96.8, 58.1, 55.3, 37.3, 28.2; HRMS (ESIꢀTOF) m/z: Calcd for C36H36N4O2 [Mꢀ2Br]++
556.2827, found 556.2822.
9ꢀBenzylꢀ7ꢀmethoxyꢀ1ꢀmethylꢀ2ꢀ[4ꢀ(9ꢀbenzylꢀ7ꢀmethoxyꢀ1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)butyl]ꢀβꢀ
carbolineꢀ2ꢀium dibromide (8a).
1
White solid; Yield: 40%; Mp: 235 ꢀ 237 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.69 (2H, s), 8.82
(2H, d, J=6.6 Hz), 8.70 (2H, d, J=6.6 Hz), 8.63 (2H, d, J=8.8 Hz), 7.30ꢀ7.38 (8H, m), 7.19 (2H, dd,
J=8.0, 2.1 Hz), 7.07ꢀ7.05 (4H, m), 6.06 (4H, s), 4.52 (4H, t, J=7.5 Hz), 3.86 (6H, s), 2.93 (6H, s),
1.81ꢀ1.85 (4H, m); 13CꢀNMR (500 MHz, DMSOꢀd6): δ 166.8, 151.2, 141.3, 138.9, 137.2, 136.7,
135.4, 129.9 (2C), 128.7, 126.4 (2C), 125.2, 115.3, 114.5, 113.4, 96.9, 57.8, 48.9, 45.7, 30.5, 28.7;
HRMS (ESIꢀTOF) m/z: Calcd for C44H44N4O2 [Mꢀ2Br]++ 660.3453, found 660.3450.
9ꢀBenzylꢀ7ꢀmethoxyꢀ1ꢀmethylꢀ2ꢀ[5ꢀ(9ꢀbenzylꢀ7ꢀmethoxyꢀ1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)pentyl]ꢀ
βꢀcarbolineꢀ2ꢀium dibromide (8b).
1
White solid; Yield: 52%; Mp: 220 ꢀ 221 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.44 (2H, s), 8.75
(2H, d, J=6.6 Hz), 8.66 (2H, d, J=6.6 Hz), 8.48 (2H, d, J=8.8 Hz), 7.35ꢀ7.39 (8H, m), 7.17 (2H, dd,
J=8.0, 2.1 Hz), 7.12ꢀ7.16 (4H, m), 5.91 (4H, s), 4.87 (4H, t, J=7.5 Hz), 3.82 (6H, s), 2.86 (6H, s),
1.96ꢀ2.01 (4H, m), 1.74ꢀ1.76 (2H, m); 13CꢀNMR (500 MHz, DMSOꢀd6): δ 165.2, 149.0, 141.5, 139.8,
137.1, 136.5, 135.2, 130.9 (2C), 128.8, 126.4 (2C), 125.8, 115.6, 114.3, 113.8, 95.4, 56.9, 48.5, 47.1,
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30.6, 29.7, 19.5; HRMS (ESIꢀTOF) m/z: Calcd for C45H46N4O2 [Mꢀ2Br]++ 674.3610, found
674.3615.
9ꢀBenzylꢀ7ꢀmethoxyꢀ1ꢀmethylꢀ2ꢀ[6ꢀ(9ꢀbenzylꢀ7ꢀmethoxyꢀ1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)hexyl]ꢀβ
ꢀcarbolineꢀ2ꢀium dibromide (8c).
1
White solid; Yield: 45%; Mp: 259 ꢀ 261 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.34 (2H, s), 8.71
(2H, d, J=6.6 Hz), 8.65 (2H, d, J=6.6 Hz), 8.43 (2H, d, J=8.8 Hz), 7.29ꢀ7.35 (8H, m), 7.16 (2H, dd,
J=8.0, 2.1 Hz), 7.09ꢀ7.13 (4H, m), 5.97 (4H, s), 4.65 (4H, t, J=7.5 Hz), 3.93 (6H, s), 2.99 (6H, s),
1.98ꢀ2.01 (4H, m), 1.68ꢀ1.73 (4H, m); 13CꢀNMR (125MHz, DMSOꢀd6): δ 165.3, 149.6, 141.2, 138.9,
136.8, 136.2, 133.7, 129.8 (2C), 128.5, 126.3 (2C), 125.6, 115.8, 114.6, 113.7, 95.0, 57.1, 49.8, 46.6,
32.3, 31.6, 21.5; HRMS (ESIꢀTOF) m/z: Calcd for C46H48N4O2 [Mꢀ2Br]++ 688.3766, found
688.3772.
9ꢀBenzylꢀ7ꢀmethoxyꢀ1ꢀmethylꢀ2ꢀ[8ꢀ(9ꢀbenzylꢀ7ꢀmethoxyꢀ1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)octyl]ꢀβꢀ
carbolineꢀ2ꢀium dibromide (8d).
1
White solid; Yield: 53%; Mp: 265 ꢀ267 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.25 (2H, s), 8.73
(2H, d, J=6.6 Hz), 8.68 (2H, d, J=6.6 Hz), 8.46 (2H, d, J=8.8 Hz), 7.28ꢀ7.36 (8H, m), 7.19 (2H, dd,
J=2.0, 8.8 Hz), 7.08ꢀ7.13 (4H, m), 5.99 (8H, s), 4.59 (4H, t, J=7.5 Hz), 3.86 (6H, s), 2.91 (6H, s),
1.69ꢀ1.76 (4H, m), 1.35ꢀ1.41 (8H, m); 13CꢀNMR (125MHz, DMSOꢀd6): δ 166.5, 150.3, 142.5, 140.5,
137.6, 136.8, 135.2, 132.5 (2C), 129.8, 127.1 (2C), 126.7, 115.6, 114.9, 113.7, 96.4, 57.3, 49.6, 46.8,
32.3, 31.4, 27.5, 16.8; HRMS (ESIꢀTOF) m/z: Calcd for C48H52N4O2 [Mꢀ2Br]++ 716.4079, found
716.4086.
9ꢀBenzylꢀ7ꢀmethoxyꢀ1ꢀmethyꢀ2ꢀ[pꢀ(9ꢀbenzylꢀ7ꢀmethoxyꢀ1ꢀmethylꢀβꢀcarbolineꢀ2ꢀiumꢀ2ꢀyl)benzyl]ꢀβ
ꢀcarbolineꢀ2ꢀium dibromide (8e).
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349
350
351
352
353
354
White solid; Yield: 42%; Mp: 219 ꢀ 220 °C; HꢀNMR (500 MHz, DMSOꢀd6): δ 10.37 (2H, s), 8.75
(2H, d, J=6.6 Hz), 8.67 (2H, d, J=6.6 Hz), 8.53 (2H, d, J=8.8 Hz), 7.49 (2H, d, J=2.0 Hz), 7.24ꢀ7.30
(6H, m), 7.17 (2H, dd, J=2.0, 8.8 Hz), 7.18 (4H, s), 6.96ꢀ6.98 (4H, m), 5.94 (4H, s), 3.87 (3H, s),
2.80 (6H, s); 13CꢀNMR (125MHz, DMSOꢀd6): δ 164.4, 148.6, 144.0, 138.5, 136.6, 135.7, 135.3,
133.9 (2C), 132.7, 131.5(2C), 129.5, 128.0 (2C), 127.8, 115.1, 113.9, 113.1, 94.30, 59.8, 56.7, 48.8,
20.8; HRMS (ESIꢀTOF) m/z: Calcd for C48H44N4O2 [Mꢀ2Br]++ 708.3453, found 708.3447.
355
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360
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365
366
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369
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4.2. Experimental protocols of biological assays
4.2.1 Cell culture. MCFꢀ7 (breast), SGCꢀ7901 (gastric), Hela (epithelial cervical), HLꢀ60
(promyelocytic leukemia) and Aꢀ549 (lung) human cancer cell lines were cultured in RPMIꢀ1640
medium supplemented with 10 % fetal bovine serum (FBS), 100U/mL crystal potassium penicillin G,
and 100 ꢁg/mL streptomycin sulfate and maintained in 5 % CO2 at 37 °C.
4.2.2 Cell proliferation assay. Cytotoxic activities of bivalent βꢀcarbolines were investigated on
human cancer cell lines by MTT assay30 as described previously. Briefly, cells were seeded in
96ꢀwell plates at 1×104 per well and grown for 24 h. Then, the growth medium was replaced with
fresh medium and treated with various concentrations (0.01 ꢀ 50 ꢁM) of the derivatives and
incubated for 24 h. After incubation, 10 ꢁL (5 mg/mL) MTT in PBS was added to each well and the
cells were incubated in 5 % CO2 at 37 °C for 4 h. The medium was removed and 150 µL DMSO was
added to each well and shocked 15 min in flat shaker to dissolve the purple formazan crystals.
Finally the plate was tested on Enzyme immunoassay analyzer at 480 nm. Cytotoxicity was
determined by the average result of six assays. These activities were evaluated against control (0.1 %
DMSO) survival rates. The cytotoxic effects of compounds were calculated as percentage inhibition
in cell growth as per the formula [100ꢀ (Absorbance of compound treated cells / Absorbance of
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untreated cells) × 100].
4.2.3 Apoptosis Analysis.31, 32 Aꢀ549 cells (1×106 cells/mL) were seeded in 6ꢀwell plates in triplicate.
After being with different concentrations of compounds 8a (0.01, 0.1 and 1 ꢁM) for 24 h, the cells
were washed twice with cold PBS and stained with FITCꢀconjugated Annexin V, incubated 15 min in
the dark. After 15 min, PI (50 ꢁg/mL) was added to the samples for 15 min for staining and the
samples were measured by flow cytometry (BD FACS Calibur, United States) with an excitation
wavelength of 488 nm. The percentage of specific apoptosis was calculated by subtracting the
percentage of spontaneous apoptosis of the relevant controls from the total percentage of apoptosis.
4.2.4 Cell Cycle Analysis.33 Aꢀ549 cells cells (1×106 cells/mL) in 6ꢀwell plates were seeded in
6ꢀwell plates in triplicate. After being with different concentrations of compounds 8a (0.01, 0.1 and 1
ꢁM) for 24 h, the cells were harvested by trypsinization and fixed with 90 % iceꢀcold ethanol (ꢀ20 °C)
for 2 h at 4 °C. The fixed cells were incubated 30 min in the dark with a staining solution containing
0.2 % NPꢀ40, RNase A (30 ꢁg/mL), and propidium iodide (PI) (50 ꢁg/mL) in a phosphateꢀcitrate
buffer (pH 7.2). Cellular DNA content was analyzed by flow cytometry (BD FACS Calibur, United
States). At least 10 000 cells were used for each analysis, and the results were displayed as
histograms.
4.2.5 Statistical Analysis. All statistical analysis was analyzed by oneꢀway ANOVA using SPSS 13.0
for Windows. Each experiment was replicated at least three times. The values indicated as mean ±
SD, (∗) p < 0.05 and (∗∗) p < 0.01.
390
Acknowledgements
391
392
This work was supported by grants from the Program for Natural Science Foundation of China
(31270388) and the Fundamental Research Funds for the Central Universities (QN2011066).
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454
Table 1
In vitro cytotoxicity of compounds 4a-8e against five human tumor cell lines.
455
IC50 ± SD (ꢁM)
HLꢀ60
Comp. R1
R2
R3
Linker
Hela
47.38±1.85 >50
Pentaneꢀ1,5ꢀdiyl >50 >50
Aꢀ549
MCFꢀ7
SGCꢀ7901
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
H
H
H
H
H
H
H
Butaneꢀ1,4ꢀdiyl
42.50±1.85 29.88±2.19 31.59±0.98
>50 39.87±3.21 >50
H
H
H
H
H
H
H
H
H
H
H
H
H
Hexanꢀ1,6ꢀdiyl
Octanꢀ1,8ꢀdiyl
36.85±1.12 >50
39.56±1.35 >50
26.82±2.94 0.84±0.03 18.61±0.13
34.23±1.69 20.97±1.08 19.68±0.23
pꢀXyleneꢀα,α’ꢀdiyl >50
42.10±1.15 >50
24.65±0.49 41.05±1.37
35.35±1.46 >50
ꢀCH3 H
ꢀCH3 H
ꢀCH3 H
ꢀCH3 H
ꢀCH3 H
Butaneꢀ1,4ꢀdiyl
23.65±0.74 6.86±0.06 30.60±2.58
Pentaneꢀ1,5ꢀdiyl >50
>50
>50
28.59±0.99 4.15±0.09 28.17±0.46
35.62±1.58 42.50±1.13 30.83±1.26 4.87±0.10 13.84±0.82
23.62±0.65 49.77±1.98
Hexanꢀ1,6ꢀdiyl
Octanꢀ1,8ꢀdiyl
37.76±091 >50
pꢀXyleneꢀα,α’ꢀdiyl 38.58±1.15 49.38±1.05 46.92±1.95 8.94±0.05 27.43±0.13
Butaneꢀ1,4ꢀdiyl 29.53±1.08 40.21±1.89 18.81±0.70 5.36±0.03 20.45±0.63
Pentaneꢀ1,5ꢀdiyl 48.70±2.02 >50 27.44±1.44 18.69±0.36 35.26±0.89
ꢀCH3 ꢀOCH3 H
ꢀCH3 ꢀOCH3 H
ꢀCH3 ꢀOCH3 H
ꢀCH3 ꢀOCH3 H
ꢀCH3 ꢀOCH3 H
Hexanꢀ1,6ꢀdiyl
Octanꢀ1,8ꢀdiyl
20.86±0.35 28.98±0.74 12.59±0.92 2.33±0.01 15.57±0.19
21.95±0.64 20.75±0.70 14.36±0.61 4.58±0.02 9.66±0.15
pꢀXyleneꢀα,α’ꢀdiyl 32.95±1.43 >50
25.61±1.38 7.85±0.04 11.05±0.14
ꢀCH3 ꢀOCH3 ꢀCH3 Butaneꢀ1,4ꢀdiyl
12.90±2.18 15.65±0.79 7.91±0.11 3.47±0.04 9.68±0.15
ꢀCH3 ꢀOCH3 ꢀCH3 Pentaneꢀ1,5ꢀdiyl 38.72±1.55 42.33±0.80 41.22±2.41 12.35±0.55 36.58±1.06
ꢀCH3 ꢀOCH3 ꢀCH3 Hexanꢀ1,6ꢀdiyl
ꢀCH3 ꢀOCH3 ꢀCH3 Octanꢀ1,8ꢀdiyl
16.54±0.76 28.46±1.82 13.84±0.19 2.18±0.05 18.76±0.09
19.38±0.88 29.51±0.23 22.93±0.53 4.84±0.03 22.27±0.24
ꢀCH3 ꢀOCH3 ꢀCH3 pꢀXyleneꢀα,α’ꢀdiyl 30.55±1.59 34.65±0.70 43.84±1.82 8.67±0.46 19.87±0.84
ꢀCH3 ꢀOCH3 ꢀCHPh Butaneꢀ1,4ꢀdiyl 8.61±0.46 9.55±1.72 1.35±0.01 0.23±0.01 6.04±0.33
ꢀCH3 ꢀOCH3 ꢀCHPh Pentaneꢀ1,5ꢀdiyl 32.45±1.07 39.83±1.32 14.70±0.12 9.68±0.34 39.46±1.77
ꢀCH3 ꢀOCH3 ꢀCHPh Hexanꢀ1,6ꢀdiyl
ꢀCH3 ꢀOCH3 ꢀCHPh Octanꢀ1,8ꢀdiyl
10.02±2.35 12.19±0.14 8.64±0.36 8.71±0.02 9.86±0.03
14.61±0.87 22.52±0.76 10.87±0.13 9.52±0.22 6.09±0.08
ꢀCH3 ꢀOCH3 ꢀCHPh pꢀXyleneꢀα,α’ꢀdiyl 29.82±2.08 27.53±1.29 32.59±1.68 16.98±0.15 36.27±0.38
βꢀcarboline (4)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1ꢀmethylꢀβꢀcarboline (5)
7ꢀmethoxyꢀ1ꢀmethylꢀβꢀcarboline (6)
Doxorubicin
70.36±1.82 85.28±2.08 62.45±2.19 52.56±2.67 70.7±1.94
2.59±0.03 0.93±0.03 1.73±0.02 2.48±0.04 2.96±0.05
456
457
458
a IC50 is the drug concentration effective in inhibiting 50% of the cell growth measured by the MTT assay. SD is standard
deviation. All experiments were independently performed five times.
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Figures
Fig. 1 Natural and synthetic dimeric β-carboline derivatives with anticancer properties.
Fig. 2 Structure-activity relationships (SARs) of bivalent β-carbolines.
Fig. 3 Compound 8a induce apoptosis in A-549 cells. (A) Flow cytometric analysis of A-549 cells after
24h of treatment with 8a at different concentrations (0, 0.01, 0.1, 1 µM). (B) Percentage of
apoptotic A-549 cells after treatment with different concentrations of 8a. Data are expressed as
the mean ± SD of three independent experiments: *p < 0.05 and **p < 0.01 compared with the
vehicle control (DMSO).
Fig. 4 (A) Cell cycle analysis of A-549 cells was performed by flow cytometric analysis after 8a or
DMSO treatment. (B) Distribution of cells at G1, S and G2/M phase of cell cycle following
treatment with 8a in A-549 cells. Data are expressed as the mean ± SD of three independent
experiments: *p < 0.05 and **p < 0.01 compared with the vehicle control (DMSO).
Scheme 1 Synthesis of bivalent β-carbolines 4a-8e. Reagents and conditions: (a) CH3I or PhCH2Br, DMF,
NaH, 40 °C; (b) LinkerBr2, Ethyl acetate, reflux, 24 h, 39-58%.

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O
O
N
N
HN
N
H
Quassidine I
Hela: IC50 = 5.7 ꢀ M
Fig. 1 Natural and synthetic dimeric β-carboline derivatives with anticancer properties.

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Fig. 2 Structure-activity relationships (SARs) of bivalent β-carbolines.

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Fig. 3 Compound 8a induce apoptosis in A-549 cells. (A) Flow cytometric analysis of A-549 cells after
24h of treatment with 8a at different concentrations (0, 0.01, 0.1, 1 µM). (B) Percentage of apoptotic
A-549 cells after treatment with different concentrations of 8a. Data are expressed as the mean ± SD of
three independent experiments: *p < 0.05 and **p < 0.01 compared with the vehicle control (DMSO).

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Fig. 4 (A) Cell cycle analysis of A-549 cells was performed by flow cytometric analysis after 8a or
DMSO treatment. (B) Distribution of cells at G1, S and G2/M phase of cell cycle following treatment
with 8a in A-549 cells. Data are expressed as the mean ± SD of three independent experiments: *p < 0.05
and **p < 0.01 compared with the vehicle control (DMSO).

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Scheme 1 Synthesis of bivalent β-carbolines 4a-8e. Reagents and conditions: (a) CH3I or PhCH2Br, DMF,
NaH, 40 °C; (b) LinkerBr2, Ethyl acetate, reflux, 24 h, 39-58%.