Synthesis and antitumor activity of novel: N -substituted tetrahydro-β-carboline-imidazolium salt derivatives

Article abstract of DOI:10.1039/c6ob01495j

The synthesis of a series of novel N-substituted tetrahydro-β-carboline-imidazolium salt derivatives is presented. The biological properties of the compounds were evaluated in vitro against a panel of human tumor cell lines. The results suggest that the benzimidazole ring and 1-(naphthalen-2-yl)ethan-1-one or 2-naphthylmethyl substituent at the imidazolyl-3-position were vital for modulating cytotoxic activity. Compound 41 was observed as a potent derivative with IC₅₀ values of 3.24-8.78 μM and exhibited cytotoxic activity selectively against HL-60, A-549 and MCF-7 cell lines. Meanwhile, high inhibitory activities selectively against HL-60 and MCF-7 cell lines were observed for compound 51. Moreover, compound 51 was able to induce G1 phase cell cycle arrest and apoptosis in MCF-7 cells. The cytotoxicity of compound 51 against human normal lung epithelial cell line BEAS-2B was further evaluated.

Full text of DOI:10.1039/c6ob01495j

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1
2
Synthesis and antitumor activity of novel Nꢀsubstituted tetrahydroꢀβꢀ
carboline–imidazolium salt derivatives
3
4
Bei Zhoua, ZhengꢀFen Liua, GuoꢀGang Denga, Wen Chena, MinꢀYan Lic, LiꢀJuan Yangd, Yan Lib,*, Xiaoꢀ
Dong Yanga,*, HongꢀBin Zhanga,*
5
6
7
8
Received Xth XXXXXXXXX 2016, Accepted Xth XXXXXXXXX 2016
First published on the web Xth XXXXXXXXX 2016
DOI: 10.1039/c1ob00000x
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17
18
aKey Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical
Science and Technology, Yunnan University, Kunming, 650091, P. R. China. Tel.: +86-871-65031119; Fax.:
+86-871-65035538. E-mail: xdyang@ynu.edu.cn, zhanghb@ynu.edu.cn
bState Key Laboratory for Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,
Chinese Academy of Science, Kunming, 650204, P. R. China. E-mail: liyanb@mail.kib.ac.cn
cDepartment of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, 19104-6323, United States
dSchool of Chemistry & Environment, Yunnan Minzu University, Kunming, 650500, P. R. China.
† Electronic supplementary information (ESI) available: Details of experimental procedure, spectral data and
copies of all novel compounds. For ESI or other electronic format see DOI: 10.1039/c1ob00000x.
19
20
21
22
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DOI: 10.1039/C6OB01495J
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24
25
26
27
28
29
30
31
The synthesis of a series of novel Nꢀsubstituted tetrahydroꢀβꢀcarboline–imidazolium salt derivatives is
presented. Biological property of such compounds was further evaluated in vitro against a panel of human tumor
cell lines. The results suggest that benzimidazole ring and 1ꢀ(naphthalenꢀ2ꢀyl)ethanꢀ1ꢀone or 2ꢀnaphthylmethyl
substituent at imidazolylꢀ3ꢀposition were vital for modulating cytotoxic activity. Compound 41 was observed as
potent derivative with IC50 values of 3.24ꢀ8.78 ꢁM and exhibited cytotoxic activity selectively against HLꢀ60,
Aꢀ549 and MCFꢀ7 cell lines, additionally. Meanwhile, high inhibitory activities selectively against HLꢀ60 and
MCFꢀ7 cell lines were observed in compound 51. Moreover, compound 51 was able to induce the G1 phase cell
cycle arrest and apoptosis in MCFꢀ7 cells. The cytotoxicity of compound 51 against human normal lung
epithelial cell line (BEASꢀ2B) was further evaluated.
32
33 Introduction
34
35
36
37
38
39
40
41
42
43
44
45
46
47
βꢀcarboline alkaloids are widely present as anticancer natural products and are widely distributed in plants,
animals and humans.1ꢀ3 They have attracted significant attention due to their biological effects such as antitumor,
antiꢀleishmanial, antiꢀtrypanosomal, antiꢀHIV and antiꢀinflammatory, etc.4 Specifically, tetrahydroꢀβꢀcarbolines5,
such as eleagnine, harmicine and Nꢀsubstituted 2ꢀbenzoylꢀ1,3,4,9ꢀtetrahydroꢀβꢀcarboline, are one of the
important analogs in carboline alkaloid (Fig. 1), which are reported possessing antinociceptive, antiꢀ
inflammatory and antioxidant, antileishmanial and antinociceptive activities.6
Imidazolium salts are important building block in drug discovery with pharmacological activities7 and
antitumor activity.8 For example, two new imidazolium chlorides, Lepidiline A and B, illustrated in Figure 1,
displayed potent cytotoxic activity against human cancer cell lines (UMUC3, PACA2, MDA231, and
FDIGROV).9 Considering the value of imidazolium salts, we recently introduced the synthesis of a novel
imidazolium salt NMIB (Fig. 1) and studied their potential antitumor activity.10 Further study of molecular
mechanisms showed that the imidazolium salt hybrids can induce the cell cycle arrest and apoptosis in tumor
cells.10i
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49
Fig. 1 Representative structures of tetrahydroꢀβꢀcarboline and its Nꢀsubstituted derivative and imidazolium salts.
50
51
52
53
54
55
56
Molecular hybridization is a useful tool in new drug design and development during the past two decades.11
We were curious if any pharmacological activities would be identified in the hybridizing compounds of Nꢀ
substituted tetrahydroꢀβꢀcarboline together with imidazole moieties. To the best of our knowledge, antitumor
activity hasn’t been observed in any Nꢀsubstituted tetrahydroꢀβꢀcarboline–imidazole hybrids in the literature.
Herein, we have designed and synthesized a series of novel imidazole scaffoldꢀbased Nꢀsubstituted tetrahydroꢀβꢀ
carboline and investigated their antitumor activity.
57 Results and discussion
58
Chemistry
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OH
OTBS
OTBS
NaH, PhSO2Cl, THF
82%
TBSCl, Imidazole,
DCM, 99%
N
H
N
H
N
SO2Ph
3
1
2
O
S
H2N
OTBS
O
OTBS
(R)
n-BuLi, DMF
1,2-dimethoxyethane, 72%
5
O
N
S
Ti(EtO)4, THF
92%
N
N
SO2Ph
SO2Ph
6
4
OH
OMs
OTBS
TBAF
(1M in THF)
THF, 90%
NaBH4,
MeOH
99%
O
O
O
MsCl, Et3N
DCM, 96%
H
H
H
N S
N S
N S
N
N
N
SO2Ph
SO2Ph
SO2Ph
8
9
7
Mg, MeOH, NH4Cl
Ultrasonic, 91%
NH
N
H
11a
O
Cs2CO3
DMF, 99%
N
S
N
SO2Ph
HCl (4M in dioxane)
MeOH, 86%
NH
10
N
SO2Ph
11b
O
Et3N, DCM
0oC, 0.5h
Cl
NH
N
Cl
Cl
N
N
O
then rt, 1h
R1
R1
1
12
a
: 89%
1
13a
: R =H
11a
11b
: R =H
b: 93%
13b: R1=PhSO2
1
: R =PhSO2
59
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60
61
Scheme 1 Synthesis of hybrid compounds 16ꢀ21.
62
63
64
65
66
67
68
69
70
71
72
73
74
75
For the synthesis of Nꢀsubstituted tetrahydroꢀβꢀcarboline–imidazole derivatives (Compounds 16ꢀ21), we started
with commercial tryptophol 1, after OH and NH groups were protected with tertꢀbutyl(dimethyl)silyl with
phenylsulfonyl groups, and an aldehyde group was installed at the 3ꢀposition of indole motif to afford
compound 4. The condensation of 4 with (R)ꢀ(+)ꢀ2ꢀmethylꢀ2ꢀpropanesulfinamide 5 led to imine 6. Started
form compound 6, tetrahydroꢀβꢀcarboline 10 was formed through sequential three steps including reduction of
imine, deprotection of OH group and ring closure. Next, key intermediate 10 was converted to 11a and 11b
under two deportation conditions. The acylation of 11a and 11b led to amides 13a and 13b. Subsequently, Nꢀ
substituted tetrahydroꢀβꢀcarboline–imidazole hybrids 16–21 were formed from 13a and 13b with various
substituted imidazole (imidazole, 2ꢀethylꢀimidazole, benzimidazole or 5,6ꢀdimethylꢀbenzimidazole) at 72–88%
yields (Scheme 1).
Finally, imidazolium salts 22–51 were prepared from coupling of imidazole hybrids 16–21 with various alkyl
and phenacyl bromides at 72–92% yields. The structures and yields of derivatives are shown in Table 1.
Table 1 Synthesis of Nꢀsubstituted tetrahydroꢀβꢀcarboline imidazolium salt derivatives 22ꢀ54 from 16ꢀ21
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Entry Compound R1
No.
Imidazole ring
R3
Molecular
formula
mp (oC)
Yields (%)
1
2
3
4
5
6
7
8
H
H
imidazole
–
C16H16N4O
85ꢀ86
85
88
82
88
72
76
78
92
16
17
18
19
20
21
22
23
benzimidazole
–
C20H18N4O
275ꢀ276
77ꢀ79
PhSO2 imidazole
–
C22H20N4O3S
C24H24N4O3S
C26H22N4O3S
C28H26N4O3S
C28H25BrN4O2
C28H25BrN4O2
PhSO2 2ꢀethylꢀimidazole
PhSO2 benzimidazole
–
147ꢀ148
139ꢀ140
278ꢀ279
201ꢀ202
249ꢀ251
–
PhSO2 5,6ꢀdimethylꢀbenzimidazole
–
H
H
imidazole
imidazole
phenacyl
1ꢀ(naphthalenꢀ2ꢀ
yl)ethanꢀ1ꢀone
phenacyl
9
H
H
H
H
benzimidazole
benzimidazole
benzimidazole
benzimidazole
C28H25BrN4O2
C29H27BrN4O3
C28H24Br2N4O2
C32H27BrN4O2
236ꢀ238
258ꢀ260
240ꢀ242
270ꢀ271
92
88
78
83
24
25
26
27
10
11
12
4ꢀmethoxyphenacyl
4ꢀbromophenacyl
1ꢀ(naphthalenꢀ2ꢀ
yl)ethanꢀ1ꢀone
13
14
15
17
18
H
H
H
benzimidazole
benzimidazole
benzimidazole
4ꢀbromobenzyl
4ꢀnitrobenzyl
3ꢀnaphthylmethyl
phenacyl
C27H24Br2N4O
C27H24BrN5O3
C31H27BrN4O
272ꢀ274
279ꢀ280
272ꢀ273
88
82
76
89
86
28
29
30
31
32
PhSO2 imidazole
PhSO2 imidazole
C30H27BrN4O4S 222ꢀ223
C34H29BrN4O4S 195ꢀ196
1ꢀ(naphthalenꢀ2ꢀ
yl)ethanꢀ1ꢀone
29
20
21
PhSO2 2ꢀethylꢀimidazole
PhSO2 2ꢀethylꢀimidazole
PhSO2 2ꢀethylꢀimidazole
phenacyl
C32H31BrN4O4S 272ꢀ273
C32H30Br2N4O4S 279ꢀ280
C36H33BrN4O4S 273ꢀ274
86
89
78
33
34
35
4ꢀbromophenacyl
1ꢀ(naphthalenꢀ2ꢀ
yl)ethanꢀ1ꢀone
22
23
24
25
26
27
PhSO2 2ꢀethylꢀimidazole
PhSO2 2ꢀethylꢀimidazole
PhSO2 benzimidazole
PhSO2 benzimidazole
PhSO2 benzimidazole
PhSO2 benzimidazole
4ꢀmethylbenzyl
3ꢀnaphthylmethyl
phenacyl
C32H33BrN4O3S 244ꢀ245
C35H33BrN4O3S 253ꢀ254
C34H29BrN4O4S 200ꢀ201
C34H28Br2N4O4S 197ꢀ198
C35H31BrN4O5S 209ꢀ210
C38H31BrN4O4S 214ꢀ215
81
87
86
75
89
77
36
37
38
39
40
41
4ꢀbromophenacyl
4ꢀmethoxyphenacyl
1ꢀ(naphthalenꢀ2ꢀ
yl)ethanꢀ1ꢀone
4ꢀmethylbenzyl
28
29
30
31
32
33
34
PhSO2 benzimidazole
PhSO2 benzimidazole
PhSO2 benzimidazole
C34H31BrN4O3S 290ꢀ291
C33H28BrN5O5S 272ꢀ273
C37H31BrN4O3S 136ꢀ137
C36H33BrN4O4S 239ꢀ240
C36H32Br2N4O4S 250ꢀ252
C37H35BrN4O5S 244ꢀ245
C40H35BrN4O4S 234ꢀ235
84
86
72
91
88
90
91
42
43
44
45
46
47
48
4ꢀnitrobenzyl
3ꢀnaphthylmethyl
PhSO2 5,6ꢀdimethylꢀbenzimidazole phenacyl
PhSO2 5,6ꢀdimethylꢀbenzimidazole 4ꢀbromophenacyl
PhSO2 5,6ꢀdimethylꢀbenzimidazole 4ꢀmethoxyphenacyl
PhSO2 5,6ꢀdimethylꢀbenzimidazole 1ꢀ(naphthalenꢀ2ꢀ
yl)ethanꢀ1ꢀone
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PhSO2 5,6ꢀdimethylꢀbenzimidazole 4ꢀmethylbenzyl
PhSO2 5,6ꢀdimethylꢀbenzimidazole 4ꢀnitrobenzyl
PhSO2 5,6ꢀdimethylꢀbenzimidazole 3ꢀnaphthylmethyl
C36H35BrN4O3S 270ꢀ271
C35H32BrN5O5S 227ꢀ228
C39H35BrN4O3S 252ꢀ254
79
49
50
51
36
37
89
82
77
78
79
80
81
82
83
84
Biological evaluation and structureꢀactivity relationship analysis
The potential cytotoxicity of all newly synthesized hybrid compounds was assessed in vitro against a panel of
human tumor cell lines, on the basis of the procedures in the literature12. The panel comprised with myeloid
leukaemia (HLꢀ60), liver carcinoma (SMMCꢀ7721), lung carcinoma (Aꢀ549), breast carcinoma (MCFꢀ7) and
colon carcinoma (SW480). Cisplatin (DDP) were used as reference drug (See Table 2 to for complete results).
Table 2 Cytotoxic activities of hybrid compounds 16ꢀ51 in vitrob (IC50, µMa)
Entry
Compound No.
HLꢀ60
SMMCꢀ7721
Aꢀ549
MCFꢀ7
SW480
1
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
>40
>40
>40
>40
>40
2
>40
>40
>40
>40
>40
3
17.81
36.73
>40
>40
28.16
>40
21.00
>40
29.80
>40
4
>40
5
>40
>40
>40
>40
6
>40
>40
>40
>40
>40
7
>40
>40
>40
>40
>40
8
>40
>40
>40
>40
>40
9
>40
>40
>40
>40
>40
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
21.81
11.09
10.68
>40
27.59
19.80
30.39
>40
>40
20.47
17.56
19.51
>40
32.35
16.69
>40
>40
>40
>40
>40
3.54
>40
13.23
>40
18.02
>40
12.24
>40
17.46
>40
3.32
>40
12.11
>40
14.21
>40
3.74
>40
11.80
>40
11.87
2.47
2.56
2.77
14.39
10.61
3.97
3.24
3.04
16.77
10.67
12.48
12.81
24.60
17.28
14.95
15.03
14.78
>40
8.28
10.44
3.37
2.61
16.44
16.59
11.34
8.05
7.68
35.62
10.14
11.84
12.81
13.60
11.81
13.58
11.01
11.70
13.39
22.13
14.46
21.41
31.23
18.27
8.78
17.01
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27
28
29
30
31
32
33
34
35
36
43
44
45
46
47
48
49
50
51
13.58
4.34
10.18
3.75
3.39
3.08
4.30
12.81
2.61
23.35
14.74
14.50
15.30
13.18
14.77
15.13
33.19
14.15
9.98
35.36
17.28
22.75
>40
17.42
10.33
11.26
4.97
12.44
11.76
13.19
10.47
16.37
16.17
14.20
>40
23.70
>40
8.23
3.90
29.52
>40
10.17
10.81
2.79
17.13
8.25
9.46
DDP (MW 300) 2.27
14.69
15.11
a Cytotoxicity as IC50 for each cell line, is the concentration of compound which reduced by 50%
the optical density of treated cells with respect to untreated cells using the MTT assay.
b Data represent the mean values of three independent determinations.
85
86
As shown in Table 2, at the concentration of 40 µM, imidazole derivatives 17, 18 and 21 were inactive at
concentrations lower than 40 µM, and 19 and 20 were observed processing weak activities against all tumor cell
lines, while their imidazolium salts anallogs 26, 27, 28, 30, 32, 34ꢀ51 exhibited cytotoxic activities. Such
difference in cytotoxicity between neutral compounds and imidazolium salts may be attributed to the differences
in molecular structure, charge distribution and water solubility.13
87
88
89
90
91
In the case of the Nꢀsubstituents of indole moiety, Nꢀbenzenesulfonylated imidazolium salts 32, 34ꢀ51 showed
higher cytotoxic activities than NꢀH imidazolium salts 22ꢀ29. All of Nꢀbenzenesulfonylated salts (except 31 and
33) exhibited moderated or high inhibitory activities. Thus, we will mainly focus on the Nꢀbenzenesulfonylated
indole moiety in the following discussion.
92
93
94
95
Substituted imidazole part of imidazolium salts plays an important role in bioactivity, for example, only
compounds 32 and 37 which have 1ꢀ(naphthalenꢀ2ꢀyl)ethanꢀ1ꢀone and 3ꢀnaphthylmethyl at positionꢀ3 of the
imidazole and 2ꢀethylꢀimidazole rings, showed higher cytotoxic activity with IC50 values of 2.61–14.46 ꢁM.
Meanwhile, compounds 38ꢀ44 with benzimidazole ring exhibited medium inhibitory activities. Different
substituents at positionꢀ3 of the benzimidazole ring such as 4ꢀmethoxyphenacyl (40), 1ꢀ(naphthalenꢀ2ꢀyl)ethanꢀ
1ꢀone (41) and 4ꢀmethylbenzyl (42) substituent displayed better inhibitory activity toward MCFꢀ7 and SW480
compared with DDP. Notably, compounds 45ꢀ51 with 5,6ꢀdimethylꢀbenzimidazole rings showed significant
inhibitory activities. Among them, compounds 46, 49 and 51, with a 4ꢀbromophenacyl, 4ꢀmethylbenzyl or 3ꢀ
96
97
98
99
100
101
102
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103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
naphthylmethyl, displayed potent higher inhibitory activity in vitro than DDP with IC50 values of 2.79ꢀ14.20 ꢁM
against MCFꢀ7 and SW480 tumor cell lines.
In the case of the substituent at positionꢀ3 of imidazole ring, imidazolium salt 31, 33, 38 and 45 with phenacyl
substituent, as well as derivatives 43 and 50 with 4ꢀnitrobenzyl substituent showed either negative or weak
activities against five tumor cell lines. Compounds 34, 36, 39, 40, 42, 46, 47 and 49 with, 4ꢀbromophenacyl, 4ꢀ
methylbenzyl or 4ꢀmethoxyphenacyl substituent at positionꢀ3 of benzimidazole ring exhibited medium cytotoxic
activities (IC50 = 2.56–35.62 ꢁM). However, compared with above phenacyl or substituted phenacyl substituent
derivatives, compounds 30, 37, 44 and 51 with 3ꢀnaphthylmethyl substituent, as well as compounds 32, 35, 41
and 48 with 1ꢀ(naphthalenꢀ2ꢀyl)ethanꢀ1ꢀone substituent at positionꢀ3 of the imidazole ring displayed much
higher cytotoxic activity in vitro. Interestingly, compound 51, bearing a 3ꢀnaphthylmethyl substituent at
positionꢀ3 of 5,6ꢀdimethylꢀbenzimidazole, was found to be one of the most potent derivatives with IC50 values
of 2.61–17.13 ꢁM against the five human tumor cell lines investigated. Notably, compound 51 exhibited
cytotoxic activity selectively against MCFꢀ7 and SW480 cell lines with IC50 values 5.3ꢀfold and 1.6ꢀfold more
sensitive to DDP. This finding shows that steric and electronic effects have an important role in the cytotoxic
activity of imidazolium salts.
The results suggest that the existence of substituted 5,6ꢀdimethylꢀbenzimidazoless ring and substitution of the
imidazolylꢀ3ꢀposition with a 1ꢀ(naphthalenꢀ2ꢀyl)ethanꢀ1ꢀone or 2ꢀnaphthylmethyl group could enhance the
cytotoxic activity of imidazolium salts. In addition, the structureꢀactivity relationship (SAR) results were
illustrated in Scheme 2.
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R2
R2
N
N
N
N
N
N
R3
N
R1
N
O
O
R1
Br
(A)
(B)
N-substituted tetrahydro- -carboline derivatives:
imidazolium salts (B) > imidazole hybrids (A)
Better
imidazole ring:
5,6-dimethyl-benzimidazole > 2-ethyl-imidazole > benzimidazole > imidazole
Best
O
O
R3
=
>
>
O
Best
O
O
>
>
>
>
Br
Br
NO2
Best Potent Compounds
Cytotoxic activity
IC50,
M
2.61
HL-60
A549
SMMC-7721
MCF-7
14.15
17.13
2.79
N
N
Br
N
N
O
9.46
SW480
SO2Ph
51
122
123
Scheme 2 Structureꢀactivity relationship of Nꢀsubstituted tetrahydroꢀβꢀcarboline imidazolium salts.
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125
126
127
Furthermore, the cytotoxicity of the representative compound 51 against human normal lung epithelial cell
line (BEASꢀ2B) was also evaluated. The results were showed in Table 2. By comparing the IC50 values of the
tested compounds towards cancer cell lines with those towards the normal lung epithelial cells BEASꢀ2B,
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compound 51 exhibited selective cytotoxicity between cancer and normal cells, with an IC50 value above 40 ꢁM
against normal BEASꢀ2B cells, and less toxic than that against lung carcinoma A549 cancer cells. Contrarily,
DDP had not obvious selectivity between cancer and normal cells.
Table 2 Cytotoxicity of compound 15 against A549 and BEASꢀ2B cells in vitro (IC50, µM)
Entry
Compound no. BEASꢀ2B
A549
17.13
8.25
1
2
>40
51
DDP
9.12
133
134
135
136
137
Compound 51 induces G1 phase arrest and apoptosis in cancer cells
MCFꢀ7 cells were exposed to increasing concentrations of compound 51 and cell apoptosis was determined with
Annexin VꢀFITC/PI doubleꢀlabeled cell cytometry. As shown in Fig. 2, after treatment of cells with compound
51 at 1, 3, 9 ꢁM for 48 h, the apoptotic cell rate was 15.1±0.9 %, 25.5±1.7 % and 59.8±2.6 %, respectively,
138
which were statistically different from the control (6.5±0.9%).
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140
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Fig. 2 Compound 51 caused significant apoptosis of MCFꢀ7 cells. (A) Cells were treated with 1, 3 and 9 ꢁM
compound 51 for 48 h. Cell apoptosis was determined by Annexin VꢀFITC/PI doubleꢀstaining assay. (B) The
quantification of cell apoptosis. Data represents the mean ± S.D. of three independent experiments.
The results of cell cycle analysis on MCFꢀ7 cells treated with compound 51 were summarized in Fig. 3.
Compared with the control cells, the percentage of cells of G1 phase increased during the cells incubated with
compound 51 with a dose dependent manner. In the meanwhile, the fraction of cells in S phase decreased
slightly accordingly, while the proportion of G2 phase cells showed no obvious change. Our data suggest that
compound 51 may induce G1 phase arrest in the cell cycle.
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Fig. 3 Compound 51 induces G1 phase arrest in MCFꢀ7 cells. (A) Cells were treated with 1, 3 and 9 ꢁM of
compound 51 for 24 h. Cell cycle was determined by PI staining and cell cytometry. (B) The percentages of
cells in different phases were quantified. At least three independent experiments were performed and data of one
representative experiment is shown.
Disruption or malfunction of cell cycle control within the G1 phase has been recognized as the most
important biochemical phenomenon for tumor progression and tumorigenesis. The ability of certain small
molecules to control cell cycle machinery within the G1 phase has provided exciting new opportunities with
hopes of developing new types of drugs efficacious against refractory cancers.14
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162 Conclusion
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In summary, a series of novel Nꢀsubstituted tetrahydroꢀβꢀcarboline imidazolium salt derivatives prepared in
this research proved to be potent antitumor agents. The imidazolium salt derivatives 37, 41, 44 and 51, bearing
2ꢀethylꢀimidazole, benzimidazole or 5,6ꢀdimethylꢀbenzimidazole ring and a 3ꢀnaphthylmethyl or 1ꢀ(naphthalenꢀ
2ꢀyl)ethanꢀ1ꢀone at positionꢀ3 of the imidazole ring, were found to be the most potent compounds. Compound
41, bearing a 1ꢀ(naphthalenꢀ2ꢀyl)ethanꢀ1ꢀone substituent at positionꢀ3 of benzimidazole, was found to be the
most potent derivatives with IC50 values of 3.24–15.03 ꢁM against five human tumor cell lines. Notably,
compound 51 exhibited cytotoxic activity selectively against MCFꢀ7 and SW480 cell lines with IC50 values 5.3ꢀ
fold and 1.6ꢀfold more sensitive to DDP. Compound 51 can induce the G1 phase cell cycle arrest and apoptosis
in MCFꢀ7 cells. The tetrahydroꢀβꢀcarbolineꢀbased imidazolium salts 32, 35, 37, 41, 44 and 51 can be considered
promising leads for further structural modifications guided by the valuable information derivable from our
detailed SARs.
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175 Experimental Section
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General procedures
Melting points were obtained on a XTꢀ4 meltingꢀpoint apparatus and were uncorrected. Proton nuclear magnetic
resonance (1HꢀNMR) spectra were recorded on a Bruker Avance 400 (600) spectrometer at 400 (400) MHz.
Carbonꢀ13 nuclear magnetic resonance (13CꢀNMR) was recorded on Bruker Avance 400 (600) spectrometer at
100 (120) MHz. Chemical shifts are reported as δ values in parts per million (ppm) relative to tetramethylsilane
(TMS) for all recorded NMR spectra. Lowꢀresolution Mass spectra were recorded on a VG Auto Specꢀ3000
magnetic sector MS spectrometer. High Resolution Mass spectra were taken on AB QSTAR Pulsar mass
183
spectrometer. Elemental analysis (%CHN) was conducted on a Vario EL Ⅲ spectrometer. Silica gel
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(200–300 mesh) for column chromatography and silica GF254 for TLC were produced by Qingdao Marine
Chemical Company (China). All airꢀ or moistureꢀsensitive reactions were conducted under an argon
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atmosphere. Starting materials and reagents used in reactions were obtained commercially from Acros, Aldrich,
Fluka and were used without purification, unless otherwise indicated.
Synthesis of compounds 2ꢀ4, 6ꢀ11 and 13. See ESI file for characterization data.†
Synthesis of hybrid compounds 16ꢀ21. A mixture of compound 13 (2 mmol) and imidazole or substituted
imidazole (6mmol) and K2CO3 (3 mmol) was stirred in DMF (20 ml) at 50 ℃ for 1 h (monitored by TLC).
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200
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205
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211
After cooling to room temperature, the solvent was concentrated, and the residue was diluted with EtOAc (20
mL). The organic layer was washed with water (20 mL) and brine (20 mL), dried over anhydrous Na2SO4 and
concentrated. The residue was purified by column chromatography (silica gel, petroleum ether : EtOAc : Et3N =
1:1:0.1) to afford 16ꢀ21 in 62ꢀ82% yield as yellow powder. See ESI file for characterization data.†
Synthesis of compounds 22ꢀ51. A mixture of substituted imidazole 16ꢀ21 (0.25 mmol) and
phenacylbromides or phenacyl or benzyl or 1ꢀ(naphthalenꢀ2ꢀyl)ethanꢀ1ꢀone or naphthylmethyl (0.75 mmol) was
stirred in acetone (10 ml) at reflux 8ꢀ36 h. An insoluble substance was formed. After completion of the reaction
as indicated by TLC, the precipitate was filteredand washed with acetone (3 × 10 ml), then dried to afford
imidazolium salts 22ꢀ51 in 64–95% yields. See ESI file for characterization data.†
Cytotoxicity assay. The assay was in five kinds of cell lines (HLꢀ60, SMMCꢀ7721, A549, MCFꢀ7 and
o
SW480). Cells were cultured at 37 C under a humidified atmosphere of 5% CO2 in RPMI 1640 medium
supplemented with 10% fetal serum and dispersed in replicate 96ꢀwell plates. Compounds were then added.
After 48 h exposure to the compounds, cells viability were determined by the [3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide] (MTT) cytotoxicity assay by measuring the absorbance at 570 nm with a
microplate spectrophotometer. Each test was performed in triplicate.
Cell apoptosis analysis. Cell apoptosis was analyzed using the Annexin VꢀFITC/PI Apoptosis kit (BD
Biosciences, Franklin Lakes, NJ) according to the manufacturer’s protocols. Cells were seeded in 6ꢀwell plates
at a density of 1.2 × 106 cells/well. After 48 h of compound treatment at the indicated concentrations, cells were
collected and then washed twice with cold PBS, and then resuspended in a binding buffer containing Annexin
VꢀFITC and propidium iodine (PI). After incubation for 15 min at room temperature in the dark, the fluorescent
intensity was measured using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ).
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Cell cycle analysis. To analyze the DNA content by flow cytometry, cells were collected and washed twice
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with PBS. Cells were fixed with 70% ethanol overnight. Fixed cells were washed with PBS, and then stained
with a 50 ꢁg/ml propidium iodide (PI) solution containing 50 ꢁg/ml RNase A for 30 min at room temperature.
Fluorescence intensity was analyzed by FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).
The percentages of the cells distributed in different phases of the cell cycle were determined using ModFIT LT
2.0.
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Acknowledgments
This work was supported by grants from the Program for Changjiang Scholars and Innovative Research Team in
University (IRT13095), Natural Science Foundation of China (21462049, 21332007, 21572197 and U1402227),
Yunnan Province (2013FA028), Education Department of Yunnan Province (ZD2014010), and Program for
China Scholarship Council (201408535034) and excellent young talents of Yunnan University.
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FIGURE TITLES
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Fig. 1 Representative structures of tetrahydroꢀβꢀcarboline and its Nꢀsubstituted derivative and imidazolium
salts.
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Fig. 2 Compound 51 caused significant apoptosis of MCFꢀ7 cells. (A) Cells were treated with 1, 3 and 9 ꢁM
compound 51 for 48 h. Cell apoptosis was determined by Annexin VꢀFITC/PI doubleꢀstaining assay. (B) The
quantification of cell apoptosis. Data represents the mean ± S.D. of three independent experiments.
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Fig. 3 Compound 51 induces G1 phase arrest in MCFꢀ7 cells. (A) Cells were treated with 1, 3 and 9 ꢁM of
compound 51 for 24 h. Cell cycle was determined by PI staining and cell cytometry. (B) The percentages of
cells in different phases were quantified. At least three independent experiments were performed and data of one
representative experiment is shown.
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SCHEME TITLES
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306
Scheme 1 Synthesis of hybrid compounds 16ꢀ21.
Scheme 2 Structureꢀactivity relationship of Nꢀsubstituted tetrahydroꢀβꢀcarboline imidazolium salts.
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TABLE TITLES
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Table 1 Synthesis of Nꢀsubstituted tetrahydroꢀβꢀcarboline imidazolium salt derivatives 22ꢀ51 from 16ꢀ21
Table 2 Cytotoxic activities of hybrid compounds 17ꢀ51 in vitrob (IC50, µMa)
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