Synthesis and in vitro biological evaluation of hybrids from tetrahydro-β-carboline and hydroxylcinnamic acid as antitumor carcinoma agents

Article abstract of DOI:10.1248/cpb.c13-00902

Novel hybrids 8a-j and 9a-j were designed and synthesized by coupling the carboxyl group of hydroxylcinnamic acids with tetrahydro-β-carboline alkaloids which were linked with different substituted nitrogen-containing heterocycles at the positions-N9, and

Full text of DOI:10.1248/cpb.c13-00902

April 2014
Regular Article
Chem. Pharm. Bull. 62(4) 343–349 (2014)
343
Synthesis and in Vitro Biological Evaluation of Hybrids from
Tetrahydro-β-carboline and Hydroxylcinnamic Acid as Antitumor
Carcinoma Agents
,a
Ying Lin,* Xuanping Xia,b Rongxin Yao,a Li Ni,c Jie Hu,d Wenjian Guo,a and Baoling Zhua
aꢀDepartmentꢀofꢀHematologyꢀandꢀOncology,ꢀTheꢀSecondꢀAffiliatedꢀHospital,ꢀWenzhouꢀMedicalꢀUniversity;ꢀ
bꢀDepartmentꢀofꢀGastroenterology,ꢀTheꢀSecondꢀAffiliatedꢀHospital,ꢀWenzhouꢀMedicalꢀUniversity; c Department of
ClinicalꢀLaboratory,ꢀTheꢀSecondꢀAffiliatedꢀHospital,ꢀWenzhouꢀMedicalꢀUniversity;ꢀandꢀd Department of Pharmacy,
WenzhouꢀMedicalꢀUniversity;ꢀWenzhou,ꢀZhejiangꢀ325035,ꢀP.ꢀR.ꢀChina.
Received November 18, 2013; accepted December 26, 2013
Novel hybrids 8a–j and 9a–j were designed and synthesized by coupling the carboxyl group of hy-
droxylcinnamic acids with tetrahydro-β-carboline alkaloids which were linked with different substituted
nitrogen-containing heterocycles at the positions-N9, and their in vitro biological activities were evaluated.
It was found that most hybrids showed good to moderate anti-tumor activities. Especially, compound 9j had
a great potency superior to 5-fluorouracil (5-FU) and comparable to adriamycin in human cancer cells, and
could selectively inhibit tumor cells, but not inhibit non-tumor cell proliferation in vitro. More importantly,
apoptosis assay indicated that 9j could significantly induce tumor cell apoptosis in a dose-dependent manner.
Therefore, our novel findings may provide a new framework for the design of new hybrids for the interven-
tion of human cancers.
Key words synthesis; tetrahydro-β-carboline; hydroxylcinnamic acid; anti-tumor agent; inhibitory activity;
cell apoptosis
Cancer is a major health problem worldwide and is the There is an increased interest in the use of hybrid molecules
leading cause of human mortality exceeded only by cardio- for drug discovery against a multitude of disease indica-
vascular diseases.1) Therefore, development of new anticancer tions.20,21) Many tetrahydro-β-carboline hybrids bearing cin-
drugs and more effective treatment strategies for cancer are namoyl or aroyl group at the N2 position, reported in recent
of utmost importance when traditionally prescribed chemo- literatures, exhibit excellent biological acitivities.22,23) With
therapeutic agents have problems with toxicity and drug re- these ideas in mind, we conjugated tetrahydro-β-carboline
sistance.2)
alkaloid with hydroxylcinnamic acid and different nitrogen-
The tetrahydro-β-carboline alkaloids, a large group of syn- containing heterocycle was introduced to positions-N9 of
thetic and naturally occurring polycyclic indolic compounds, tetrahydro-β-carboline through carbonchain with the purpose
have attracted a great deal of interest amongst medicinal for the improvement of water-solubility. Thus, we hypoth-
chemists and pharmacologists over the years, partially due to esized that the novel types of tetrahydro-β-carboline/hydroxyl-
their widespread potent biological and pharmaceutical proper- cinnamic acid hybrids will be more efficacious and selectively
ties including antimalarial, antitumor, anti-human immuno- inhibit tumor cell proliferation. Therefore, a total of twenty
deficiency virus (HIV), anti-thrombotic, and antimicrobial target compounds (8a–j, 9a–j) were designed and synthesized,
activities.3–8) Recently, more attention has been focused on and their inꢀvitro antitumor effects were investigated. Herein,
the study of the tetrahydro-β-carboline derivatives about their the synthesis and preliminary biological evaluation of these
potential antitumor activities.4,5,9,10) Current issues in cancer compounds were reported.
research show that many tricyclic ring carboline compounds
which display potent cytotoxicity against numerous cancer
Results and Discussion
Chemistry The synthesis of 8a–j and 9a–j was described
cell lines are harman and norharman derivatives,11) and more
complex structures such as manzanine,12) azatoxin,13) eudis- in Chart 1. The 1,2,3,4-tetrahydro-β-carboline-3-carboxylic
tomine K,14) fascaplysine,15) and picrasidine L.16) Therefore, acid 2 was prepared by the condensation of L-tryptophan 1
the structural fragment of tetrahydro-β-carboline seems to be with aqueous 37% formaldehyde according to the Pictet–Spen-
essential for many biologically important indole alkaloids and gler reaction. Then the carboxyl group of compound 2 was
represents an important lead structure for the development of subsequently methyl-esterified through SOCl2 in MeOH solu-
novel anticancer agents.
tion to afford 3. In addition, the phenolic hydroxyl of hydrox-
In an ongoing study on discovering new anti-tumor agents ylcinnamic acid 4a or 4b was protected by acetic anhydride to
for human use, we were inspired by the fact that hydroxylcin- form acetylated product 5a or 5b, which was further reacted
namic acids, such as ferulic acid and p-hydroxy-cinnamic with intermediate 3 to obtain compound 6 in the presence of
acid, are known as phenolic compounds occurring in natural N-methylmorpholine and ethyl chloroformate. The aromatic
plant product, and their derivatives displayed selective anti- secondary amine of compound 6 was substituted with dibro-
proliferative activity against some types of cancer cells,17–19) mopropane to yield compound 7, which was then transformed
which attracts considerable attention from medicinal chemists. to 8a–j after treatment with nitrogen-containing heterocycles.
Finally, compounds 8a–j were hydrolyzed to gain the target
The authors declare no conflict of interest.
compounds 9a–j. The products 8a–j and 9a–j were puri-
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: wzly_onco@163.com

344
Vol. 62, No. 4
Reaction conditions and reagents: a) 1N NaOH, HCHO, r.t. to 38°C, 3h, 82%; b) SOCl2, MeOH, 0°C-reflux, 6 h, 94%; c) Ac2O, NaOH, 0°C, 1h, 88%; d) ethyl chlorofor-
mate, N-methylmorpholine, THF, 0°C to r.t., 1h; e) 3, TEA, THF, 0°C, 1h, 56–75%; f) NaH, Br(CH2)3Br, DMF, 0°C, 1h; g) KI, K2CO3, nitrogen-containing heterocycles,
CH3CN, 50°C, 3–6h, 52–69%; h) 2N NaOH, MeOH, 65°C, 2–6h, 81–90%.
Chart 1
Table 1. The IC50 Values of Synthetic Compounds 8a–j, 9a–j against Six Human Cancer Cell Lines
Inꢀvitro inhibition of human cancer cells proliferation (IC50a), µM)
Compound
SMMC-7721
SGC-7901
HCT116
Hep G2
MCF-7
SKOV-3
5-FU
39.03
1.92
41.35
3.39
32.84
4.72
43.52
3.48
19.36
2.78
41.65
4.63
Adriamycin
8a
8b
8c
8d
8e
8f
>50
>50
>50
32.53
15.27
18.09
41.34
9.53
>50
32.46
37.01
26.65
30.98
28.00
25.38
38.98
8.72
>50
43.11
27.82
30.02
15.21
18.62
33.77
10.03
13.98
11.35
9.74
29.12
23.71
23.26
26.00
>50
10.12
14.56
7.22
25.28
35.13
17.88
21.14
>50
>50
26.58
23.22
>50
8g
8h
8i
9.18
9.24
11.50
9.26
18.66
11.89
10.75
28.36
25.09
18.45
15.23
14.85
>50
11.83
13.95
10.07
9.32
19.56
8.36
20.10
9.01
8j
7.02
8.44
7.71
7.93
9a
9b
9c
9d
9e
9f
>50
32.09
21.87
29.91
17.55
18.42
22.29
8.25
30.08
22.90
24.34
15.23
12.58
26.32
9.78
>50
35.91
29.38
22.38
13.35
11.33
25.99
9.18
16.73
26.15
21.16
18.25
31.69
12.28
11.09
7.98
19.25
20.62
15.22
16.75
24.37
9.86
9g
9h
9i
9.46
10.16
6.68
12.11
9.17
11.26
8.21
7.25
9j
6.45
6.63
5.93
7.76
9.14
a) The inhibitory effects of individual compounds on the proliferation of cancer cell lines were determined by the MTT assay. The data are the mean values of IC50 from at
least three independent experiments.

April 2014
345
fied by column chromatography, and their structures were ties, which were significantly superior to 5-FU. Among these
1
characterized by IR, H-NMR, 13C-NMR, MS, and elemental compounds, compound 9j (IC50=6.93–9.32 µM) exhibited high-
analyses.
er antiproliferative activities than the other target compounds
Biological Evaluation The inhibitory activities of syn- against six tumor cells, and its IC50 values were especially
thetic compounds 8a–j, 9a–j against six human cancer cells 3–6-fold less than those of 5-FU (IC50=19.36–43.52 µM), and
SMMC-7721, Hep G2 (human hepatoma cells), SGC7901 comparable to or slightly weaker than those of adriamycin
(human gastric cancer cells), HCT-116 (human colon carci- (IC50=1.92–4.72 µM).
noma cells), MCF-7 (human breast adenocarcinoma cells),
Given that most of tetrahydro-β-carboline/hydroxylcinnam-
SKOV-3 (human ovarian cancer cells) were evaluated by ic acid hybrids show strong anticancer activities, we wonder to
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide know whether those compounds were toxic to the normal cells
(MTT) assays inꢀvitro, and 5-fluorouracil (5-FU) and adriamy- or not. So compound 9j with the highest anticancer activities
cin were used as positive controls. The results (shown in Table was further determined to compare the inhibitory activities
1) illustrated the IC50 values of target compounds against each of hepatocellular carcinoma cells (SMMC-7721, Hep G2) with
tumor cell line. The antitumor activities of intermediates 8a–j those of human normal cell (LO2) by MTT assay. As shown in
were slightly weaker than target compounds 9a–j. Compounds Fig. 1, it was found that compound 9j (1.5–12.5µM) inhibited
8g, 8i–j, and 9g–j showed good to moderate cytotoxic activi- hepatocellular carcinoma cell proliferation in a concentration-
dependent manner, and treatment with 12.5µM 9j promoted
nearly 90% of the inhibition in tumor cells, but the same treat-
ment had no significant inhibitory effect on non-tumor liver
LO2 cell proliferation (only 20% of inhibition). Apparently,
these results indicated that compound 9j had strong cytotoxic-
ity selectively to human cancer cells inꢀvitro.
To determine whether the inhibitory activities on tumor
cells of 9j were due to cell apoptosis, apoptosis assay was fur-
ther assessed employing Hep G2 cells which were incubated
with vehicle alone or with different concentrations of 9j (3.0,
6.0 or 12µM) for 48h. The percentages of the apoptotic Hep
G2 cells, presented in Fig. 2, were determined by fluorescein
isothiocyanate (FITC)-Annexin V/propidium iodide (PI) stain-
ing and flow cytometry analysis. In the untreated group, the
frequency of Hep G2 cell apoptosis was unobvious. However,
the percentage of apoptotic Hep G2 cells was gradually in-
creased for those cells exposed to increasing concentrations of
Fig. 1. Inhibitory Effects of 9j on the Proliferation of SMMC-7721, Hep
G2, and LO2 Cells
9j (23.4% for 3.0µM; 45.8% for 6.0µM; and 84.5% for 12µM),
SMMC-7721, Hep G2, and LO2 cells were incubated with the indicated concen-
which demonstrated that incubation with 9j could induce
trations of 9j for 48h. Cell proliferation was assessed using the MTT assay. Data
are means S.E.M. of the inhibition (%) from three independent experiments.
tumor cell apoptosis in a dose-dependent manner.
Fig. 2. Compound 9j Induced Hep G2 Cell Apoptosis inꢀVitro
Hep G2 cells were incubated with the indicated concentrations of 9j for 48h, and the cells were stained with FITC-Annexin V and PI, followed by flow cytometry
analysis. (A) Flow cytometry analysis. (B) Quantitative analysis of apoptotic cells. Data are expressed as means S.E.M. of the percentages of apoptotic cells from three
independent experiments.

346
Vol. 62, No. 4
Structure–Activity Relationship (SAR) Analysis of SAR were concentrated using a rotary evaporator operating at a
revealed that the antiproliferative activities of tetrahydro-β- reduced pressure of ca. 20Torr. Organic solutions were dried
carboline/ferulic acid hybrids 8f–j and 9f–j were more potent over anhydrous sodium sulfate.
than that of tetrahydro-β-carboline/p-hydroxy-cinnamic acid
General Procedure for the Synthesis of 6a,b To a
hybrids 8a–d and 9a–d, which suggested that the electron- solution of 5 (12mmol) in 50mL anhydrous tetrahydro-
donating substitutions (OCH3) on the ferulic acid derivatives furan (THF) at 0°C were added N-methylmorpholine (1.57g,
were able to confer antitumor activities to these molecules. 18mmol) and ethyl carbonochloridate (1.30g, 12mmol), and
Secondary, different substituted nitrogen-containing hetero- the mixture was stirred at room temperature for 1–2h, which
cycles connected with tetrahydro-β-carboline were also crucial was then added dropwise to a solution of 3 (2.76g, 12mmol)
for their anticancer activities inꢀvitro. These hybrid molecules in 20mL anhydrous THF. After the reaction was completed,
linked with piperazine ring, especially for N-methylpipera- the resulting mixture was allowed to pour into ice-water, and
zine, exibited the outstanding antiprofication effects while the extracted with ethyl acetate (30mL×3). The organic phase
hybrids linked with tetrahydropyrrole group showed extremely was washed with water and brine, then dried over anhydrous
weak activities. The relative antitumor activities of tetrahydro- sodiumsulfate, filtered and evaporated to afford the crude
β-carboline/hydroxylcinnamic acid hybrids with varying het- product. The crude was purified by column chromatography
erocycles were as following: N-methylpiperazine>piperazine on silica gel to give compound 6.
>piperide>morpholine>tetrahydropyrrole. Finally, the anti-
(S,E)-Methyl 2-(3-(4-Acetoxyphenyl)acryloyl)-2,3,4,9-tetra-
tumor activities of intermediates 8a–j containing ester groups hydro-1H-pyrido[3,4-b]indole-3-carboxylate (6a): The title
were slightly weaker than their hydrolyzates 9a–j containing compound was obtained starting from 5a and 3. White solid,
carboxyl and phenolic hydroxyl groups. However, further mp 191–194°C; yield: 73%. MS (ESI) m/z=419 [M+H]+.
investigation about the precise SAR of these compounds is
ongoing.
(S,E)-Methyl 2-(3-(4-Acetoxy-3-methoxyphenyl)acryloyl)-
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (6b):
The title compound was obtained starting from 5b and 3.
White solid, mp 176–179°C; yield: 70%. MS (ESI) m/z=449
Conclusion
In conclusion, a novel series of hybrids from tetrahydro-β- [M+H]+.
carboline and hydroxylcinnamic acid were designed and syn-
General Procedure for the Synthesis of 8a–j Compound
thesized, and their inꢀvitro antitumor effects were investigated. 6 (2.0mmol) was dissolved in 25mL anhydrous N,N-dimeth-
It was found that most hybrids displayed strong antitumor ylformamide (DMF) until clear, and then 60% NaH (0.083g,
activities against six human cancer cells inꢀ vitro. The hybrid 2.1mmol) and 1,3-dibromopropane (0.43g, 2.0mmol) were
9j containing ferulic acid and N-methylpiperazine ring was slowly added in sequence at 0°C. The mixture was stirred
the most active, with IC50s of 5.93–9.14µM against all tested at room temperature for 1h, and then nitrogen-containing
cancer cells, which were even more potent than 5-FU and heterocycles (2.0mmol), KI (0.033g, 0.2mmol), and K2CO3
comparable to adriamycin. Furthermore, compound 9j could (0.42g, 3.0mmol) were added to the reaction solution and
selectively inhibit tumor cells, but not inhibit non-tumor cell continuously stirred at 50°C for 3–6h. After the reaction was
proliferation. More importantly, apoptosis assay indicated completed, the resulting mixture was filtered and evaporated
that 9j could significantly induce cell apoptosis in a dose- to obtain the crude product. The obtained was purified by
dependent manner. Our findings suggest that the tetrahydro- silica column chromatography (CH2Cl2–MeOH=10:1–5:1, v/v
β-carboline/hydroxylcinnamic acid hybrids may hold greater as the eluent) to gain compound 8a–j.
promise as therapeutic agents for the intervention of human
cancers.
(S,E)-Methyl
2-(3-(4-Acetoxyphenyl)acryloyl)-9-(3-(pyr-
rolidin-1-yl)propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-
3-carboxylate (8a): The title compound was obtained starting
from 6a, 1,3-dibromopropane, and pyrrolidine. White solid,
Experimental
Melting points were determined on Yanaco MP-J3 micro- mp 118–121°C; yield: 62%; MS (ESI) m/z=530 [M+H]+.
scope melting point apparatus. Infrared (IR) spectra (KBr)
(S,E)-Methyl 2-(3-(4-Acetoxyphenyl)acryloyl)-9-(3-(piper-
were recorded on a Nicolet Impact 410 instrument (KBr idin-1-yl)propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-
pellet). Proton and carbon nuclear magnetic resonance (1H- carboxylate (8b): The title compound was obtained starting
and 13C-NMR) spectra were recorded with a Bruker Avance from 6a, 1,3-dibromopropane, and piperidine. Pale yellow
300MHz spectrometer at 300K, with chemical shift in parts solid, mp 97–100°C; yield: 69%; MS (ESI) m/z=544 [M+H]+.
per million (ppm, δ) downfield from TMS as an internal
(S,E)-Methyl 2-(3-(4-Acetoxyphenyl)acryloyl)-9-(3-morpho-
standard. MS spectra were recorded on a Mariner Mass Spec- linopropyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-car-
trum (electrospray ionization (ESI)). Element analysis was boxylate (8c): The title compound was obtained starting from
performed on an Eager 300 instrument. All compounds were 6a, 1,3-dibromopropane, and morpholine. Pale yellow solid,
1
routinely checked by TLC and H-NMR. TLCs and prepara- mp 106–109°C; yield: 58%; MS (ESI) m/z=546 [M+H]+.
tive thin-layer chromatography were performed on silica gel
(S,E)-Methyl 2-(3-(4-Acetoxyphenyl)acryloyl)-9-(3-(piper-
GF/UV 254, and the chromatograms were conducted on silica azin-1-yl)propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-
gel (200–300 mesh, Merck) and visualized under UV light at carboxylate (8d): The title compound was obtained starting
254 and 365nm. L-Tryptophan 1 and compounds 4a,b were from 6a, 1,3-dibromopropane, and piperazine. Pale yellow solid,
commercially available, and compounds 2, 3, and 5a,b were mp 123–125°C; yield: 53%; MS (ESI) m/z=545 [M+H]+.
prepared according the literatures.24,25) All solvents were re-
(S,E)-Methyl 2-(3-(4-Acetoxyphenyl)acryloyl)-9-(3-(4-meth-
agent grade and, when necessary, were purified and dried by ylpiperazin-1-yl)propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]-
standards methods. Solutions after reactions and extractions indole-3-carboxylate (8e): The title compound was obtained

April 2014
347
starting from 6a, 1,3-dibromopropane, and N-methylpipera- Acid (9b): The title compound was obtained starting from 8b.
zine. Pale yellow solid, mp 127–130°C; yield: 53%; MS (ESI) Pale yellow solid, mp 128–131°C; yield: 88%. Analytical data
m/z=559 [M+H]+.
for 9b: IR (KBr, cm−1): 3446, 2951, 1723, 1618, 1436, 1219,
1
(S,E)-Methyl 2-(3-(4-Acetoxy-3-methoxyphenyl)acryloyl)-9- 1028; H-NMR (DMSO-d6, 300MHz, δ ppm): 7.65–7.69 (m,
(3-(pyrrolidin-1-yl)propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4- 2H, Ar-H), 7.62 (d, 1H, J=16.2Hz, CH), 7.33 (m, 1H, Ar-H),
b]indole-3-carboxylate (8f): The title compound was obtained 7.15–7.22 (m, 3H, Ar-H), 7.03 (d, J=7.5Hz, 1H, Ar-H), 6.90
starting from 6b, 1,3-dibromopropane, and pyrrolidine. Pale (m, 1H, Ar-H), 6.45 (d, 1H, J=16.2Hz, CH), 4.48–4.56 (m,
yellow solid, mp 110–112°C; yield: 65%; MS (ESI) m/z=560 2H, NCH, NCH2Ar), 4.28 (m, 1H, NCH2Ar), 4.07 (t, J=6.9Hz,
[M+H]+.
2H, NCH2), 2.95 (m, 1H, ArCH2), 2.59 (d, J=7.5Hz, 1H,
(S,E)-Methyl 2-(3-(4-Acetoxy-3-methoxyphenyl)acryloyl)-9- ArCH2), 2.23–2.38 (m, 6H, 3×NCH2), 1.83–1.92 (m, 2H,
(3-(piperidin-1-yl)propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]- NCH2CH2), 1.57–1.63 (m, 4H, 2×NCH2CH2), 1.38–1.48 (m,
indole-3-carboxylate (8g): The title compound was obtained 2H, CH2); 13C-NMR (DMSO-d6, 75MHz, δ ppm): 174.2, 161.9,
starting from 6b, 1,3-dibromopropane, and piperidine. Pale 153.5, 138.3, 136.5, 130.4, 128.5, 127.4, 125.5, 121.2, 118.8,
yellow solid, mp 102–105°C; yield: 61%; MS (ESI) m/z=574 117.7, 115.9, 111.7, 105.2, 61.1, 56.8, 54.4, 51.5, 45.7, 28.8,
[M+H]+.
26.3, 24.3, 22.9; MS (ESI) m/z=488 [M+H]+; Anal. Calcd for
(S,E)-Methyl 2-(3-(4-Acetoxy-3-methoxyphenyl)acryloyl)-9- C29H33N3O4: C, 71.44; H, 6.82; N, 8.62; Found: C, 71.52; H,
(3-morpholinopropyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]- 6.93; N, 8.51.
indole-3-carboxylate (8h): The title compound was obtained
(S,E)-2-(3-(4-Hydroxyphenyl)acryloyl)-9-(3-morpho-
starting from 6b, 1,3-dibromopropane, and morpholine. Yel- linopropyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-car-
low solid, mp 104–106°C; yield: 56%; MS (ESI) m/z=576 boxylic Acid (9c): The title compound was obtained starting
[M+H]+.
from 8c. Pale yellow solid, mp 141–144°C; yield: 85%. Ana-
(S,E)-Methyl 2-(3-(4-Acetoxy-3-methoxyphenyl)acryloyl)-9- lytical data for 9c: IR (KBr, cm−1): 3440, 2943, 1710, 1609,
1
(3-(piperazin-1-yl)propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]- 1425, 1207, 1020; H-NMR (DMSO-d6, 300MHz, δ ppm): 7.67
indole-3-carboxylate (8i): The title compound was obtained (m, 2H, Ar-H), 7.58 (d, 1H, J=16.2Hz, CH), 7.17–7.29 (m, 4H,
starting from 6b, 1,3-dibromopropane, and piperazine. Pale Ar-H), 7.06 (d, J=7.5Hz, 1H, Ar-H), 6.91 (m, 1H, Ar-H), 6.41
yellow solid, mp 119–122°C; yield: 52%; MS (ESI) m/z=575 (d, 1H, J=16.2Hz, CH), 4.45–4.57 (m, 2H, NCH, NCH2Ar),
[M+H]+.
4.24 (m, 1H, NCH2Ar), 4.03 (t, J=6.9Hz, 2H, NCH2), 3.72
(S,E)-Methyl 2-(3-(4-Acetoxy-3-methoxyphenyl)acryloyl)-9- (t, J=4.2Hz, 4H, 2×OCH2), 2.92 (m, 1H, ArCH2), 2.55 (m,
(3-(4-methylpiperazin-1-yl)propyl)-2,3,4,9-tetrahydro-1H-pyr- 1H, ArCH2), 2.26–2.41 (m, 6H, 3×NCH2), 1.85–1.93 (m, 2H,
ido[3,4-b]indole-3-carboxylate (8j): The title compound was NCH2CH2); 13C-NMR (DMSO-d6, 75MHz, δ ppm): 174.7,
obtained starting from 6b, 1,3-dibromopropane, and N-methyl- 164.7, 153.8, 138.1, 135.9, 130.9, 128.6, 127.7, 125.8, 121.6,
piperazine. Pale yellow solid, mp 127–130°C; yield: 54%; MS 118.7, 115.7, 111.3, 105.9, 66.1, 61.2, 59.2, 56.5, 55.1, 51.6,
(ESI) m/z=589 [M+H]+.
46.0, 29.2, 26.9; MS (ESI) m/z=490 [M+H]+; Anal. Calcd for
General Procedure for the Synthesis of 9a–j A suspen- C28H31N3O5: C, 68.69; H, 6.38; N, 8.58; Found: C, 68.82; H,
sion of 8 (1mmol) in 5mL methanol containing 1.5mL 2N 6.54; N, 8.37.
NaOH was stirred and refluxed for 2–6h, and then cooled.
(S,E)-2-(3-(4-Hydroxyphenyl)acryloyl)-9-(3-(piperazin-1-
The solvent was evaporated and the residue was dissolved yl)propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-
in 1M HCl solution. The solvent was evaporated, the residue carboxylic Acid (9d): The title compound was obtained start-
was neutralized to pH=5 with 1M HCl. The precipitate was ing from 8d. Pale yellow solid, mp 175–177°C; yield: 81%.
filtered, washed with water, and dried in vacuum to afford the Analytical data for 9d: IR (KBr, cm−1): 3465, 2954, 1733,
1
target compound 9a–j.
1626, 1434, 1223, 1117, 1032; H-NMR (DMSO-d6, 300MHz,
(S,E)-2-(3-(4-Hydroxyphenyl)acryloyl)-9-(3-(pyrrolidin-1- δ ppm): 7.66–7.71 (m, 2H, Ar-H), 7.60 (d, 1H, J=16.2Hz, CH),
yl)propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3- 7.33 (m, 1H), 7.15–7.26 (m, 3H, Ar-H), 7.02 (d, J=7.5Hz, 1H,
carboxylic Acid (9a): The title compound was obtained start- Ar-H), 6.89 (d, J=7.5Hz, 1H, Ar-H), 6.37 (d, 1H, J=16.2Hz,
ing from 8a. Pale yellow solid, mp 136–139°C; yield: 85%. CH), 4.59 (m, 1H, NCH), 4.47 (m, 1H, NCH2Ar), 4.27 (m,
Analytical data for 9a: IR (KBr, cm−1): 3432, 2944, 1719, 1H, NCH2Ar), 4.08 (t, J=6.9Hz, 2H, NCH2), 3.05 (m, 4H,
1610, 1432, 1215, 1021; 1H-NMR (DMSO-d6, 300MHz, δ 2×NCH2), 2.92 (m, 1H, ArCH2), 2.31–2.67 (m, 7H, ArCH2,
ppm): 7.64 (m, 2H, Ar-H), 7.59 (d, 1H, J=16.2Hz, CH), 7.30 3×NCH2), 1.83–1.94 (m, 2H, NCH2CH2); 13C-NMR (DMSO-
(m, 1H, Ar-H), 7.18–7.24 (m, 3H, Ar-H), 7.00 (d, J=7.5Hz, 1H, d6, 75MHz, δ ppm): 173.5, 161.8, 153.2, 137.7, 135.6, 130.1,
Ar-H), 6.88 (d, J=7.5Hz, 1H, Ar-H), 6.41 (d, 1H, J=16.2Hz, 127.9, 126.8, 125.3, 120.2, 118.3, 117.4, 115.3, 111.8, 106.2,
CH), 4.57 (m, 1H, NCH), 4.46 (m, 1H, NCH2Ar), 4.28 (m, 1H, 60.8, 56.1, 54.2, 51.0, 47.8, 45.5, 28.8, 27.3; MS (ESI) m/z=489
NCH2Ar), 4.10 (t, J=6.9Hz, 2H, NCH2), 2.97 (m, 1H, ArCH2), [M+H]+; Anal. Calcd for C28H32N4O4: C, 68.83; H, 6.60; N,
2.62 (d, J=7.5Hz, 1H, ArCH2), 2.22–2.35 (m, 6H, 3×NCH2), 11.47; Found: C, 68.76; H, 6.73; N, 11.59.
1.80–1.88 (m, 2H, NCH2CH2), 1.60–1.65 (m, 4H, CH2CH2);
(S,E)-2-(3-(4-Hydroxyphenyl)acryloyl)-9-(3-(4-methyl-
13C-NMR (DMSO-d6, 75MHz, δ ppm): 174.6, 163.2, 153.7, piperazin-1-yl)propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]in-
138.1, 136.2, 130.3, 128.5, 127.4, 125.3, 120.9, 118.5, 117.8, dole-3-carboxylic Acid (9e): The title compound was obtained
115.6, 111.5, 105.4, 60.5, 56.3, 54.6, 51.2, 45.8, 28.5, 26.2, 23.5; starting from 8e. Pale yellow solid, mp 157–160°C; yield:
MS (ESI) m/z=474 [M+H]+; Anal. Calcd for C28H31N3O4: C, 83%. Analytical data for 9e: IR (KBr, cm−1): 3443, 2940,
71.01; H, 6.60; N, 8.87; Found: C, 70.83; H, 6.72; N, 8.98.
1
1726, 1618, 1423, 1170, 1025; H-NMR (DMSO-d6, 300MHz,
(S,E)-2-(3-(4-Hydroxyphenyl)acryloyl)-9-(3-(piperidin-1-yl)- δ ppm): 7.69 (m, 2H, Ar-H), 7.57 (d, 1H, J=16.2Hz, CH),
propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic 7.17–7.30 (m, 4H, Ar-H), 6.98 (d, J=7.5Hz, 1H, Ar-H), 6.85

348
Vol. 62, No. 4
(m, 1H, Ar-H), 6.33 (d, 1H, J=16.2Hz, CH), 4.41–4.55 (m, 2H, 118.1, 117.4, 115.8, 111.8, 106.7, 66.5, 61.7, 58.8, 56.9, 55.5,
NCH, NCH2Ar), 4.22 (m, 1H, NCH2Ar), 4.01 (t, J=6.9Hz, 2H, 52.0, 47.2, 29.9, 27.5; MS (ESI) m/z=520 [M+H]+; Anal. Calcd
NCH2), 2.99–3.08 (m, 4H, 2×NCH2), 2.92 (m, 1H, ArCH2), for C29H33N3O6: C, 67.04; H, 6.40; N, 8.09; Found: C, 67.11; H,
2.59 (m, 1H, ArCH2), 2.55 (s, 3H, NCH3), 2.27–2.50 (m, 6H, 6.53; N, 7.96.
3×NCH2), 1.80–1.91 (m, 2H, NCH2CH2); 13C-NMR (DMSO-
(S,E)-2-(3-(4-Hydroxy-3-methoxyphenyl)acryloyl)-9-(3-
d6, 75MHz, δ ppm): 174.2, 163.5, 153.6, 138.6, 136.8, 131.1, (piperazin-1-yl)propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]-
128.7, 127.7, 125.3, 121.5, 118.8, 117.9, 116.0, 112.1, 106.0, 62.4, indole-3-carboxylic Acid (9i): The title compound was
57.7, 56.1, 54.9, 49.6, 47.1, 29.5, 26.5; MS (ESI) m/z=503 [M+ obtained starting from 8i. Pale yellow solid, mp 161–163°C;
H]+; Anal. Calcd for C29H34N4O4: C, 69.30; H, 6.82; N, 11.15; yield: 82%. Analytical data for 9i: IR (KBr, cm−1): 3467,
1
Found: C, 69.44; H, 6.71; N, 11.32.
2953, 1742, 1630, 1425, 1217, 1129, 1021; H-NMR (DMSO-d6,
(S,E)-2-(3-(4-Hydroxy-3-methoxyphenyl)acryloyl)-9-(3- 300MHz, δ ppm): 7.66 (m, 1H, Ar-H), 7.55 (d, 1H, J=16.2Hz,
(pyrrolidin-1-yl)propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]- CH), 7.32 (m, 1H, Ar-H), 7.20 (m, 1H, Ar-H), 7.03 (m, 1H,
indole-3-carboxylic Acid (9f): The title compound was Ar-H), 6.72–6.91 (m, 3H, Ar-H), 6.41 (d, 1H, J=16.2Hz, CH),
obtained starting from 8f. Pale yellow solid, mp 130–132°C; 4.47–4.61 (m, 2H, NCH, NCH2Ar), 4.28 (m, 1H, NCH2Ar),
yield: 87%. Analytical data for 9f: IR (KBr, cm−1): 3416, 4.08 (t, J=6.9Hz, 2H, NCH2), 2.97–3.09 (m, 5H, 2×NCH2,
2933, 1709, 1611, 1405, 1131, 1017; 1H-NMR (DMSO-d6, ArCH2), 2.63 (m, 1H, ArCH2), 2.30–2.45 (m, 6H, 3×NCH2),
300MHz, δ ppm): 7.69 (m, 1H, Ar-H), 7.61 (d, 1H, J=16.2Hz, 1.85–1.92 (m, 2H, NCH2CH2); 13C-NMR (DMSO-d6, 75MHz,
CH), 7.27 (m, 1H, Ar-H), 7.18 (m, 1H, Ar-H), 6.99 (m, 1H, δ ppm): 174.1, 164.0, 153.9, 139.1, 136.3, 130.7, 128.5, 127.6,
Ar-H), 6.71–6.88 (m, 3H, Ar-H), 6.36 (d, 1H, J=16.2Hz, CH), 125.8, 121.1, 118.7, 117.8, 115.6, 111.9, 106.6, 62.1, 57.6, 54.7,
4.44–4.58 (m, 2H), 4.25 (m, 1H), 4.05 (t, J=6.9Hz, 2H), 2.94 51.8, 48.3, 45.7, 29.4, 27.6; MS (ESI) m/z=519 [M+H]+; Anal.
(m, 1H), 2.59 (d, J=7.5Hz, 1H), 2.20–2.33 (m, 6H, 3×NCH2), Calcd for C29H34N4O5: C, 67.16; H, 6.61; N, 10.80; Found: C,
1.85–1.94 (m, 2H, NCH2CH2), 1.57–1.66 (m, 4H, CH2CH2); 66.98; H, 6.74; N, 10.68.
13C-NMR (DMSO-d6, 75MHz, δ ppm): 173.7, 164.5, 153.1,
(S,E)-2-(3-(4-Hydroxy-3-methoxyphenyl)acryloyl)-9-(3-(4-
149.2, 137.4, 135.6, 130.2, 128.4, 127.1, 125.6, 121.3, 118.4, methylpiperazin-1-yl)propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-
117.3, 116.3, 112.3, 105.8, 60.8, 56.3, 54.8, 51.6, 46.5, 28.7, b]indole-3-carboxylic Acid (9j): The title compound was
26.5, 23.2; MS (ESI) m/z=504 [M+H]+; Anal. Calcd for obtained starting from 8j. Pale yellow solid, mp 156–158°C;
C29H33N3O5: C, 69.17; H, 6.60; N, 8.34; Found: C, 69.06; H, yield: 83%. Analytical data for 9j: IR (KBr, cm−1): 3451,
6.75; N, 8.23.
2940, 1727, 1614, 1410, 1136, 1012; 1H-NMR (DMSO-d6,
(S,E)-2-(3-(4-Hydroxy-3-methoxyphenyl)acryloyl)-9-(3- 300MHz, δ ppm): 7.73 (m, 2H, Ar-H), 7.62 (d, 1H, J=16.2Hz,
(piperidin-1-yl)propyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]- CH), 7.23–7.30 (m, 2H, Ar-H), 7.05 (d, J=7.5Hz, 1H, Ar-H),
indole-3-carboxylic Acid (9g): The title compound was 6.76–6.93 (m, 3H, Ar-H), 6.40 (d, 1H, J=16.2Hz, CH),
obtained starting from 8g. Pale yellow solid, mp 123–125°C; 4.43–4.65 (m, 2H, NCH, NCH2Ar), 4.27 (m, 1H, NCH2Ar),
yield: 90%. Analytical data for 9g: IR (KBr, cm−1): 3445, 4.11 (t, J=6.9Hz, 2H, NCH2), 3.11 (m, 4H, 2×NCH2), 2.96
2952, 1731, 1617, 1422, 1231, 1024; 1H-NMR (DMSO-d6, (m, 1H, ArCH2), 2.63 (m, 1H, ArCH2), 2.59 (s, 3H, NCH3),
300MHz, δ ppm): 7.67–7.71 (m, 1H, Ar-H), 7.58 (d, 1H, 2.31–2.54 (m, 6H, 3×NCH2), 1.84–1.97 (m, 2H, NCH2CH2);
J=16.2Hz, CH), 7.30 (m, 1H, Ar-H), 7.19 (m, 1H, Ar-H), 7.02 13C-NMR (DMSO-d6, 75MHz, δ ppm): 173.6, 162.7, 151.8,
(d, J=7.5Hz, 1H, Ar-H), 6.66–6.87 (m, 3H, Ar-H), 6.35 (d, 1H, 138.0, 137.2, 130.3, 127.9, 127.0, 125.1, 120.2, 118.4, 117.3,
J=16.2Hz, CH), 4.59 (m, 1H, NCH), 4.47 (m, 1H, NCH2Ar), 115.3, 110.8, 105.2, 62.8, 56.8, 55.9, 54.1, 49.0, 46.6, 28.5, 26.1;
4.29 (m, 1H, NCH2Ar), 4.07 (t, J=6.9Hz, 2H, NCH2), 2.97 MS (ESI) m/z=533 [M+H]+; Anal. Calcd for C30H36N4O5: C,
(m, 1H, ArCH2), 2.60 (m, 1H, ArCH2), 2.21–2.38 (m, 6H, 67.65; H, 6.81; N, 10.52; Found: C, 67.82; H, 6.93; N, 10.41.
3×NCH2), 1.87–1.95 (m, 2H, NCH2CH2), 1.59–1.65 (m, 4H,
Cell Culture SMMC-7721, HepG2, SGC7901, HCT-116,
2×NCH2CH2), 1.41–1.49 (m, 2H, CH2); 13C-NMR (DMSO-d6, MCF-7, SKOV-3, or human normal liver cell line LO2 cells
75MHz, δ ppm): 174.3, 163.5, 152.6, 149.1, 138.1, 136.3, 130.7, were maintained in 10% fetal bovine serum (FBS) Dulbecco’s
128.4, 127.1, 124.2, 120.9, 118.3, 116.9, 112.7, 106.1, 61.5, 57.0, modified Eagle’s medium (DMEM) (Gibco, Invitrogen), which
54.3, 50.8, 45.9, 27.8, 26.6, 22.7; MS (ESI) m/z=518 [M+H]+; were supplemented with 10% fetal calf serum (PAA, Austria)
Anal. Calcd for C30H35N3O5: C, 69.61; H, 6.82; N, 8.12; Found: and antibiotics [100IU/mL penicillin and 100IU/mL strepto-
C, 69.74; H, 6.69; N, 8.18.
mycin (Amresco)]. All of the cell lines were purchased from
(S,E)-2-(3-(4-Hydroxy-3-methoxyphenyl)acryloyl)-9-(3- the Shanghai Institute of Cell Biology (Shanghai, China) and
morpholinopropyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]- were grown at 37°C in a 5% CO2 atmosphere.
indole-3-carboxylic Acid (9h): The title compound was ob-
MTT Assay The inhibitory effects on cell proliferation
tained starting from 8h. White solid, mp 139–141°C; yield: of test compounds were investigated by the MTT method.
85%. Analytical data for 9h: IR (KBr, cm−1): 3432, 2943, SMMC-7721, HepG2, SGC7901, HCT-116, MCF-7, SKOV-3, or
1
1728, 1610, 1413, 1146, 1009; H-NMR (DMSO-d6, 300MHz, LO2 cells at a final density of 1.0×104 cells/well were placed
δ ppm): 7.70 (m, 1H, Ar-H), 7.61 (d, 1H, J=16.2Hz, CH), in 96-well cell plates overnight and treated with or without
7.19–7.27 (m, 2H, Ar-H), 7.01 (m, 1H, Ar-H), 6.69–6.90 (m, different concentrations of test compounds for various periods
3H, Ar-H), 6.35 (d, 1H, J=16.2Hz, CH), 4.42–4.55 (m, 2H, of time. During the last 4h culture, the cells were exposed to
NCH, NCH2Ar), 4.23 (m, 1H, NCH2Ar), 4.02 (m, 2H, NCH2), MTT (5mg/mL), and the resulting formazan crystals were dis-
3.75 (t, J=4.2Hz, 4H, 2×OCH2), 2.89 (m, 1H, ArCH2), 2.52 solved in 150µL of dimethyl sulfoxide (DMSO) and measured
(m, 1H, ArCH2), 2.25–2.42 (m, 6H, 3×NCH2), 1.83–1.95 (m, using a spectrophotometer (Tecan) at a test wavelength of
2H, NCH2CH2); 13C-NMR (DMSO-d6, 75MHz, δ ppm): 172.8, 570nm. Experiments were conducted in triplicate. Inhibition
162.5, 151.8, 137.5, 135.2, 130.1, 128.8, 127.5, 125.3, 120.5, rate (%)=[(Acontrol−Atreated)/Acontrol]×100%.

April 2014
349
10) Shen L., Park E. J., Kondratyuk T. P., Guendisch D., Marler L.,
Pezzuto J. M., Wright A. D., Sun D., Bioorg.ꢀ Med.ꢀ Chem., 19,
6182–6195 (2011).
11) Herraiz T., Guillén H., Arán V. J., Chem.ꢀ Res.ꢀ Toxicol., 21, 2172–
2180 (2008).
12) Kallifatidis G., Hoepfner D., Jaeg T., Guzmán E. A., Wright A. E.,
Mar.ꢀDrugs, 11, 3500–3516 (2013).
13) Cline S. D., Macdonald T. L., Osheroff N., Biochemistry, 36,
13095–13101 (1997).
14) Kobayashi J., Harbour G. C., Gilmore J., Rinehart Jr. K. L., J. Am.
Chem. Soc., 106, 1526–1528 (1984).
15) Bharate S. B., Manda S., Mupparapu N., Battini N., Vishwakarma
R. A., MiniꢀRev.ꢀMed.ꢀChem., 12, 650–664 (2012).
Apoptotic Cell Analysis HepG2 cells were cultured
overnight and incubated in triplicate with or without various
concentraions of 9j (3.0, 6.0, or 12µM) for 48h. To quantify
apoptosis, prepared cells were washed twice with cold PBS
and then resuspended in 500µL of binding buffer at a concen-
tration of 1×106 cells/mL. Five microliters of annexin-V-FITC
and 5µL of PI were then added to these cells, which were kept
in the dark at 25°C for 10min. Data acquisition and analysis
were performed in a FACS calibur flow cytometer (Becton
Dickinson) and values calculated with Cell Quest (BD Biosci-
ences, Franklin Lakes, NJ, U.S.A.).
16) Mitsunaga K., Koike K., Tanaka T., Ohkawa Y., Kobayashi Y.,
Sawaguchi T., Ohmoto T., Phytochemistry, 35, 799–802 (1994).
17) Murakami A., Nakamura Y., Koshimizu K., Takahashi D., Matsu-
moto K., Hagihara K., Taniguchi H., Nomura E., Hosoda A., Tsuno
T., Maruta Y., Kim H. W., Kawabata K., Ohigashi H., CancerꢀLett.,
180, 121–129 (2002).
18) Huang W. Y., Cai Y. Z., Zhang Y., Nutr. Cancer, 62, 1–20 (2010).
19) Balakrishnan S., Menon V. P., Manoharan S., J.ꢀ Med.ꢀ Food, 11,
693–700 (2008).
20) Getlik M., Grutter C., Simard J. R., Kluter S., Rabiller M., Rode H.
B., Robubi A., Rauh D., J.ꢀMed.ꢀChem., 52, 3915–3926 (2009).
21) Morphy R., Kay C., Rankovic Z., DrugꢀDiscov.ꢀToday, 9, 641–651
(2004).
22) Miller J. F., Turner E. M., Sherrill R. G., Gudmundsson K., Spalten-
stein A., Sethna P., Brown K. W., Harvey R., Romines K. R.,
Golden P., Bioorg.ꢀMed.ꢀChem.ꢀLett., 20, 256–259 (2010).
23) Bombrun A., US Patent 6306870 (2001).
References
1) Varmus H., Science, 312, 1162–1165 (2006).
2) Alexander-Bryant A. A., Vanden Berg-Foels W. S., Wen X., Adv.ꢀ
CancerꢀRes., 118, 1–59 (2013).
3) Valdez R. H., Tonin L. T., Ueda-Nakamura T., Silva S. O., Dias-Fil-
ho B. P., Kaneshima E. N., Yamada-Ogatta S. F., Yamauchi L. M.,
Sarragiotto M. H., Nakamura C. V., Antimicrob.ꢀAgentsꢀChemother.,
56, 507–512 (2012).
4) Shen Y. C., Chang Y. T., Lin C. L., Liaw C. C., Kuo Y. H., Tu L. C.,
Yeh S. F., Chern J. W., Mar.ꢀDrugs, 9, 256–277 (2011).
5) Guan H., Chen H., Peng W., Ma Y., Cao R., Liu X., Xu A., Eur. J.
Med.ꢀChem., 41, 1167–1179 (2006).
6) Tang J. G., Wang Y. H., Wang R. R., Dong Z. J., Yang L. M., Zheng
Y. T., Liu J. K., Chem.ꢀBiodivers., 5, 447–460 (2008).
7) Bi W., Cai J., Liu S., Baudy-Floc’h M., Bi L., Bioorg.ꢀMed.ꢀChem.,
15, 6909–6919 (2007).
8) Miller J. F., Turner E. M., Sherrill R. G., Gudmundsson K., Spalten-
stein A., Sethna P., Brown K. W., Harvey R., Romines K. R.,
Golden P., Bioorg.ꢀMed.ꢀChem.ꢀLett., 20, 256–259 (2010).
9) Wang F. X., Deng A. J., Li M., Wei J. F., Qin H. L., Wang A. P.,
Molecules, 18, 418–429 (2012).
24) Liu J., Jiang X., Zhao M., Zhang X., Zheng M., Peng L., Peng S., J.
Med.ꢀChem., 53, 3106–3116 (2010).
25) Lu M. D., Zhou X., Yu Y. J., Li P. H., Sun W. J., Zhao C. G., Zheng
Z. Q., You T., Wang F. H., Chin.ꢀChem.ꢀLett., 24, 415–418 (2013).