Synthesis and antitumor activity of aza-brazilan derivatives containing imidazolium salt pharmacophores

Article abstract of DOI:10.1039/c9md00112c

The synthesis of a series of novel aza-brazilan derivatives containing imidazolium salt pharmacophores is presented. The biological activity of such imidazolium salts was further evaluated in vitro against a panel of human tumor cell lines. The results suggest that the electron-withdrawing group on the aza-brazilan moiety, substituted 5,6-dimethyl-benzimidazole ring and substitution of the imidazolyl-3-position with a 4-methylbenzyl group were essential for modulating the cytotoxic activity. Compounds 55 and 39, bearing a 4-methylbenzyl substituent at position-3 of 5,6-dimethyl-benzimidazole, were found to be the most potent compounds with IC₅₀ values of 0.52-1.30 μM and 0.56-1.51 μM against four human tumor cell lines investigated. Particularly, compound 57 exhibited inhibitory activity against the MCF-7 cell line with an IC₅₀ value of 0.35 μM and was 56-fold more sensitive than DDP. Moreover, compound 55 inhibited cell proliferation through inducing G0/G1 cell cycle arrest and apoptosis in SMMC-7721 cells.

Full text of DOI:10.1039/c9md00112c

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Synthesis and antitumor activity of aza-brazilan derivatives containing
imidazolium salt pharmacophore
Mingqin Huang,a Shengzu Duan,a Xueqiong Ma,a Bicheng Cai,a,b Dongmei Wu,a,b Yan Li,*b Liang Li,*a
Hongbin Zhanga and Xiaodong Yang*a
Received Xth XXXXXXXXX 2019, Accepted Xth XXXXXXXXX 2019
First published on the web Xth XXXXXXXXX 2019
DOI: 10.1039/c1ob00000x
aKey Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education and Yunnan Province,
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, liliang5758@sina.com
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
† 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.
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The synthesis of a series of novel aza-brazilan derivatives containing imidazolium salt pharmacophores is
presented. Biological activity of such imidazolium salts was further evaluated in vitro against a panel of human
tumor cell lines. The results suggest that the electron-withdrawing group on the aza-brazilan moiety, substituted
5,6-dimethyl-benzimidazoless ring and substitution of the imidazolyl-3-position with a 4-methylbenzyl group
were essential for modulating cytotoxic activity. Compounds 55 and 39, bearing a 4-methylbenzyl substituent at
position-3 of 5,6-dimethyl-benzimidazole, were found to be the most potent compounds with IC50 values of
0.52-1.30 μM and 0.56-1.51 μM against four human tumor cell lines investigated. Particularly, compound 57
exhibited inhibitory activity against MCF-7 cell line with IC50 value of 0.35 μM and 56-fold more sensitive to
DDP. Moreover, compound 55 inhibited cell proliferation through inducing the G0/G1 cell cycle arrest and
apoptosis in SMMC-7721 cells.
Introduction
Caesalpinia sappan L. (Chinese name Su-Mu) has long been used as a traditional folk medicine in Southeast
and East Asia.1 Scientific studies have confirmed that the heartwood of C. Sappan is rich homoisoflavones.
Among them, brazilin (Fig. 1) is considered one of the most significant bioactive constituents and attracts the
attention of researchers owing to its many pharmacological activities2. Previous studies have reported that
brazilin possesses widely pharmacological activities such as anticancer, antioxidant, antibacterial, anti-
inflammatory, hypoglycemic, vasorelaxant and hepatoprotective properties.3-9 We initiated the research towards
antitumor activity of aza-brazilin derivatives in 2011 and a number of cytotoxic aza-brazilin derivatives were
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designed and synthesized. We found that aza-brazilin derivatives (such as DMAB, Fig. 1) showed potent
cytotoxic activity against a panel of human cancer cell lines.10
On the other hand, N-heterocycles are of great interest to medicinal chemists because of their widespread
occurrence in active natural products and drug molecules.11-15 Of them, imidazolium salts have obtained
significant concerns due to their significant and widespread biological and pharmacological activities,16-20
especially antitumor activity.21-24 For instance, two novel imidazolium salts, Lepidiline A and B (Fig. 1), showed
effective cytotoxic activity against human tumor cell lines (UMUC3, PACA2, MDA231, and FDIGROV).25 In
this respect, our group has long been devoted to the synthesis of novel imidazolium salt derivatives and their
potential antitumor activity, aiming to discover new antitumor agents.26-31 One representative example is the
33
imidazolium salt NMIB (Fig. 1) which exerts potent in vitro and in vivo antitumor activity.32,
Further
mechanism research demonstrated that these imidazolium salt derivatives could induce the cell cycle arrest and
apoptosis in tumor cells.34-36 Recently, we report that imidazolium salt, as a novel specific pan-PI3K inhibitor
with potent inhibitory activity against class I PI3K isoforms, showed effective inhibition of PI3K/mTOR
signaling pathway. Recently, we report that imidazolium salt, as a novel specific pan-PI3K inhibitor with potent
inhibitory activity against class I PI3K isoforms, showed effective inhibition of PI3K/mTOR signaling
pathway.37
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HO
HO
OH
N
N
O
H
Cl
R
OMe
Lepidiline A R = H
Lepidiline B R = Me
Brazilin
MeO
MeO
OH
NH
OMe
O
H
N
N
Br
DMAB
NMIB
Fig. 1 Representative structures of brazilin, aza-brazilin derivatives and imidazolium salts.
Molecular hybridization has played an important role in drug discovery during the past two decades. Thus, it
is clear that new pharmacologically interesting hybrid compounds would directly benefit the access to
novel drug for the patients.38-41 In view of the potential anticancer activity of aza-brazilan derivatives and
imidazolium salts, we launched the synthesis of hybridized compounds bearing aza-brazilan derivatives and
imidazolium salts. Although some hybrid compounds between aza-brazilan (desoxo-brazilin) and imidazole
were synthesized and found to display moderate antitumor activity in our previous study42, further design,
synthesis and structural modification of new aza-brazilan-imidazolium salt derivatives guided by these
structure-activity relationship (SAR) results are needed. With this in mind, we turned our attention to synthesis
and antitumor activity of a series of novel aza-brazilan derivatives containing imidazolium salt pharmacophore
and report our results herein.
Results and discussion
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Chemistry
O
O
R1
a) (COCl)2, DMF
CH2Cl2, rt, 12 h
R2
R1
R2
R1
O
NaH, DMC
b) AlCl3, CH2Cl2
rt, 2 h
reflux, 1 - 3 h
90-92%
R2
HO
R1 = R2 = H
R1 = R2 = OMe
R1 = H, R2 = Br
O
OMe
95-97%
1a
xylene
reflux
2-4
NH2
5-7
1b
1c
3 - 12 h
74-80%
OMe
OH
OH
O
R2
R1
R2
R1
R2
R1
O
O
NaBH4
DCM/MeOH
LiAlH4
NH
OMe
NH
NH
rt, 2 - 4 h
80-92%
reflux, 12 - 24 h
50-75%
OMe
OMe
11-13
8-10
14-16
PPTS, toluene
reflux, 5 - 12 h
50-63%
R1
R1
O
a)
H
H
Cl
Cl
b) BH3Me2S
R2
R2
NH
OMe
N
Cl
H
H
THF
reflux, 12 h
84-86% for two steps
K2CO3 (aq.)
CH2Cl2
OMe
rt, 30 min
17-19
20-22
R
O
a)
Cl
b)
HN
R
Cl
K2CO3, DMF
120 oC, 12 h
53-74%
N
K2CO3 (aq.)
CH2Cl2
K2CO3, DMF
120 oC, 12 h
HN
N
rt, 30 min
61-82% for two steps
R1
R2
R1
R2
R
R
H
H
O
N
N
N
N
R1 = R2 = H
R1 = R2 = OMe
R1 = H, R2 = Br
N
N
H
H
OMe
23-28
OMe
2-methylbenzimidazole
5,6-dimethylbenzimidazole
29-34
Scheme 1 Synthesis of aza-brazilan-imidazole hybrid compounds 23-34
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For the synthesis of hybrid molecules containing aza-brazilan derivatives and imidazolium salts, we utilized
commercially available imidazole derivatives that were alkylated with N-chloropropyl substituted aza-brazilan
derivatives, which was prepared from readily obtainable starting materials as illustrated in Scheme 1.
Commercial substituted phenylpropanoic acid 1a-1c was selected as the starting material for the synthesis of a
series of aza-brazilan -imidazole hybrids (compounds 23-28). Phenylpropanoic acids 1a-1c were treated with
oxalyl chloride, and then under Friedel-Crafts alkylation conditions yielded the corresponding indanones 2-4 in
95-97% yields. The acylation of compounds 2-4 with dimethyl carbonate led to methyl carboxylate 5-7 in 90-
92% yields. Compounds 5-7 reacted with o-methoxyaniline to give amides 8-10 in refluxing xylene (74-80%
yields). Subsequently, the ketone groups of compounds 8-10 were reduced by NaBH4 yielding the hydroxy
groups (11-13, 74-80% yields), and amides 11-13 were reduced via LiAlH4 leading to amines (14-16, 50-75%
yields). Next, the intramolecular Friedel-Crafts alkylation of compounds 14-16 synthesized aza-brazilan
derivatives 17-19 in refluxing toluene and pyridinium p-toluenesulfonate (PPTS) in 50-63% yields. Key
intermediates 17-19 were treated with chloropropanoyl chloride, and then reduced via borane giving to N-
chloropropyl substituted aza-brazilan derivatives (20-22, 84-86% yields for two steps). Compounds 20-22 were
transformed to the respective aza-brazilan-imidazole hybrids 23-28 with various substituted benzimidazole (2-
methylbenzimidazole and 5,6-dimethylbenzimidazole) by heat under DMF with 53-74% yields. At the same
time, key intermediates 17-19 were also treated with chloropropanoyl chloride, and then coupled directly with
various substituted benzimidazole to give respective aza-brazilan-imidazole hybrids 29-34 under the same
conditions with 61-82% yields.
Finally, thirty-six aza-brazilan-imidazolium salt derivatives 35-58 and 59-70 were synthesized by reaction of
aza-brazilan-imidazole hybrids 23-28 and 29-34 with the corresponding alkyl and phenacyl bromides in the
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refluxing mixed solvents of toluene and acetone (49-87% and 13-45% yields). The structures and yields of aza-
brazilan-imidazolium salt derivatives were listed in Table 1.
Table 1 Synthesis of aza-brazilan-imidazolium salt derivatives 35-70 from hybrids 23-34
R1
R2
R1
R2
R
R
R
H
H
N
N
N
N
N
N
N
R3
N
N
H
H
H
H
Br
OMe
OMe
R3 Br
23-28
O
35-58
R1
R2
R1
R2
toluene/acetone
reflux, 24 - 48 h
R
H
H
O
N
R3
13-87%
N
N
Br
OMe
29-34
OMe
59-70
R1 = R2 = H
R1 = R2 = OMe
R1 = H, R2 = Br
2-methylbenzimidazole
5,6-dimethylbenzimidazole
R3 = alkyl, phenacyl
Entry Compound R1
No.
R2
Imidazole ring
R3
Molecular
formula
Yields (%)
1
2
3
4
5
6
7
8
9
23
24
25
26
27
28
29
30
31
H
H
H
H
2-Methylbenzimidazole
5,6-Dimethylbenzimidazole
2-Methylbenzimidazole
5,6-Dimethylbenzimidazole
2-Methylbenzimidazole
5,6-Dimethylbenzimidazole
2-Methylbenzimidazole
5,6-Dimethylbenzimidazole
2-Methylbenzimidazole
-
-
-
-
C28H29N3O
C29H31N3O
C30H33N3O3
C31H35N3O3
74
66
74
53
69
71
64
61
82
OMe
OMe
H
OMe
OMe
Br
-
-
-
-
-
C28H28BrN3O
C29H30BrN3O
C28H27N3O2
C29H29N3O2
C30H31N3O4
H
Br
H
H
H
H
OMe
OMe
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10
11
12
13
14
15
16
17
18
29
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
32
OMe
H
OMe
Br
5,6-Dimethylbenzimidazole
-
C31H33N3O4
C28H26BrN3O2
C29H28BrN3O2
C36H38BrN3O
C39H38BrN3O
C37H38BrN3O3
C40H38BrN3O2
C36H40BrN3O
C40H40BrN3O
C38H40BrN3O3
C41H40BrN3O2
C38H42BrN3O4
C42H42BrN3O3
C39H42BrN3O5
C42H42BrN3O4
C39H44BrN3O3
C42H44BrN3O3
C40H44BrN3O5
C43H44BrN3O4
C36H37Br2N3O
C39H37Br2N3O
C37H37Br2N3O3
C40H37Br2N3O2
C37H39Br2N3O
C40H39Br2N3O
C38H39Br2N3O3
C41H39Br2N3O2
81
69
71
66
81
84
58
63
87
80
71
65
76
68
86
74
52
63
71
53
49
67
80
69
56
69
68
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
2-Methylbenzimidazole
5,6-Dimethylbenzimidazole
2-Methylbenzimidazole
2-Methylbenzimidazole
2-Methylbenzimidazole
2-Methylbenzimidazole
5,6-Dimethylbenzimidazole
5,6-Dimethylbenzimidazole
5,6-Dimethylbenzimidazole
5,6-Dimethylbenzimidazole
2-Methylbenzimidazole
2-Methylbenzimidazole
2-Methylbenzimidazole
2-Methylbenzimidazole
5,6-Dimethylbenzimidazole
5,6-Dimethylbenzimidazole
5,6-Dimethylbenzimidazole
5,6-Dimethylbenzimidazole
2-Methylbenzimidazole
2-Methylbenzimidazole
2-Methylbenzimidazole
2-Methylbenzimidazole
5,6-Dimethylbenzimidazole
5,6-Dimethylbenzimidazole
5,6-Dimethylbenzimidazole
5,6-Dimethylbenzimidazole
-
H
Br
-
H
H
4-Methylbenzyl
2-Naphthylmethyl
4-Methoxyphenacyl
2-Naphthylacyl
4-Methylbenzyl
2-Naphthylmethyl
4-Methoxyphenacyl
2-Naphthylacyl
4-Methylbenzyl
2-Naphthylmethyl
4-Methoxyphenacyl
2-Naphthylacyl
4-Methylbenzyl
2-Naphthylmethyl
4-Methoxyphenacyl
2-Naphthylacyl
4-Methylbenzyl
2-Naphthylmethyl
4-Methoxyphenacyl
2-Naphthylacyl
4-Methylbenzyl
2-Naphthylmethyl
4-Methoxyphenacyl
2-Naphthylacyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
Br
H
Br
H
Br
H
Br
H
Br
H
Br
H
Br
H
Br
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37
38
39
40
41
42
43
44
45
46
47
48
59
H
H
H
H
2-Methylbenzimidazole
4-Methylbenzyl
2-Naphthylmethyl
2-Naphthylacyl
C36H36BrN3O2
C39H36BrN3O2
C37H36BrN3O4
C40H36BrN3O3
C40H38BrN3O2
C38H38BrN3O4
C41H38BrN3O3
C39H40BrN3O6
C42H40BrN3O5
C40H35Br2N3O3
C40H37Br2N3O2
C41H37Br2N3O3
28
27
33
34
16
17
13
21
20
45
27
24
60
61
62
63
64
65
66
67
68
69
70
2-Methylbenzimidazole
2-Methylbenzimidazole
2-Methylbenzimidazole
5,6-Dimethylbenzimidazole
5,6-Dimethylbenzimidazole
5,6-Dimethylbenzimidazole
2-Methylbenzimidazole
2-Methylbenzimidazole
2-Methylbenzimidazole
5,6-Dimethylbenzimidazole
5,6-Dimethylbenzimidazole
H
H
H
H
4-Methylbenzyl
H
H
2-Naphthylmethyl
4-Methoxyphenacyl
2-Naphthylacyl
H
H
H
H
OMe
OMe
H
OMe
OMe
Br
Br
Br
4-Methoxyphenacyl
2-Naphthylacyl
2-Naphthylmethyl
4-Methoxyphenacyl
2-Naphthylacyl
H
H
Biological evaluation and structure-activity relationship analysis
The potential cytotoxicity of all prepared aza-brazilan-imidazolium salts was evaluated in vitro against a panel
of human tumor cell lines. The panel comprised with liver carcinoma (SMMC-7721), lung carcinoma (A-549),
breast carcinoma (MCF-7) and colon carcinoma (SW480). Cisplatin (DDP) was served as reference drug. The
results were listed in Table 2.
Table 2 Cytotoxic activities of aza-brazilan-imidazole hybrids 23-34 and aza-brazilan-imidazolium salts 35-
70 in vitrob (IC50, Ma)
Entry
Compound No.
SMMC-7721
>20
A-549
MCF-7
>20
SW480
>20
1
2
23-34
35
>20
1.80 ± 0.24
4.25 ± 0.32
1.47 ± 0.17
1.66 ± 0.18
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3
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
2.06 ± 0.37
1.32 ± 0.14
2.01 ± 0.19
0.66 ± 0.11
0.85 ± 0.17
1.42 ± 0.14
1.09 ± 0.11
6.49 ± 0.76
1.13 ± 0.08
5.36 ± 1.12
5.46 ± 0.50
1.71 ± 0.35
1.82 ± 0.41
4.50 ± 0.50
1.71 ± 0.04
0.98 ± 0.29
1.08 ± 0.26
0.93 ± 0.05
1.12 ± 0.09
0.52 ± 0.06
2.16 ± 0.74
0.89 ± 0.02
1.35 ± 0.16
2.04 ± 0.57
1.45 ± 0.32
4.62 ± 0.73
1.51 ± 0.11
5.74 ± 0.39
1.02 ± 0.03
0.40 ± 0.25
1.36 ± 0.17
0.67 ± 0.10
0.91 ± 0.27
1.26 ± 0.13
0.91 ± 0.15
5.54 ± 1.06
1.07 ± 0.06
3.36 ± 0.45
2.16 ± 0.21
2.00 ± 0.01
1.49 ± 0.07
2.14 ± 0.19
1.12 ± 0.14
1.08 ± 0.28
0.92 ± 0.01
1.01 ± 0.12
0.95 ± 0.10
0.68 ± 0.14
0.84 ± 0.78
0.35 ± 0.13
1.58 ± 0.15
1.57 ± 0.38
0.96 ± 0.20
3.12 ± 0.71
1.46 ± 0.06
2.16 ± 0.51
1.27 ± 0.17
1.80 ± 0.16
0.56 ± 0.08
0.93 ± 0.32
1.73 ± 0.06
1.46 ± 0.19
3.51 ± 0.59
1.50 ± 0.03
2.34 ± 0.61
2.06 ± 0.25
2.00 ± 0.09
1.62 ± 0.16
1.80 ± 0.29
1.38 ± 0.12
1.62 ± 0.43
1.28 ± 0.05
1.24 ± 0.06
1.26 ± 0.06
0.75± 0.01
3.92 ± 0.64
1.40 ± 0.18
4.26 ± 0.67
1.57 ± 0.24
0.77 ± 0.19
2.65 ± 0.38
1.44 ± 0.03
4
3.65 ± 1.56
5.32 ± 0.15
1.51 ± 0.07
1.51 ± 0.18
1.40 ± 0.10
1.47 ± 0.06
14.40 ± 2.16
1.53 ± 0.15
14.42 ± 0.37
8.26 ± 0.84
4.83 ± 0.35
5.83 ± 0.78
8.27 ± 0.17
4.91 ± 0.51
1.42 ± 0.45
1.89 ± 0.47
1.22 ± 0.08
1.61 ± 0.23
1.30 ± 0.01
5.62 ± 0.76
1.53 ± 0.11
3.48 ± 1.00
6.27 ± 0.42
3.68 ± 0.62
10.61 ± 0.91
4.71 ± 0.26
5
6
7
8
9
10
11
12
13
14
15
16
17
18
29
20
21
22
23
24
25
26
27
28
29
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30
31
32
33
34
35
36
37
63
64
65
66
67
68
69
70
1.03 ± 0.15
3.16 ± 1.72
1.12 ± 0.23
>20
2.58 ± 0.24
0.93 ± 0.14
2.54 ± 0.81
1.01 ± 0.25
>20
0.97 ± 0.21
0.80 ± 0.32
1.35 ± 0.28
15.40 ± 2.36
3.79 ± 1.38
1.75 ± 0.19
1.05 ± 0.17
1.83 ± 0.10
7.53 ± 0.98
1.99 ± 0.14
>20
6.53 ± 0.72
1.58 ± 0.20
1.09 ± 0.01
1.00 ± 0.08
9.25 ± 0.39
9.29 ± 1.12
2.63 ± 0.49
1.45 ± 0.07
1.11 ± 0.05
5.55 ± 0.30
4.67 ± 0.20
1.43 ± 0.20
1.05 ± 0.10
1.22 ± 0.16
38
DDP
19.56 ± 0.49 6.84 ± 0.10
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 MTS assay.
b Data represent the mean values of three independent determinations.
As shown in Table 2, the structures of aza-brazilan derivatives have an essential control on the cytotoxic
potential. The corresponding imidazoles 23-34, as controls, lacked activity against all tumor cell lines
investigated at the concentration of 20 μM (Entry 1). For the substituents of aza-brazilan moiety, aza-
brazilan-imidazolium salts 35-42 and 59-65 with no substituents on aza-brazilan moiety (R1 = R2 = H) showed
moderate cytotoxic activities with IC50 values of 0.56–10.61 μM. When the substituents were replaced with
electron-donating groups (R1 = R2 = OMe), cytotoxic activities of aza-brazilan-imidazolium salts 43-50 and 66-
67 were decreased remarkably (IC50 values of 1.07->20 μM). On the contrary, when the substituents were
changed with electron-withdrawing group (R2 = Br), aza-brazilan-imidazolium salts 51-58 and 68-70 tend
towards higher inhibitory activities with IC50 values of 0.35-5.62 μM than the others.
For the chain between aza-brazilan and imidazole ring, the cytotoxic activities of aza-brazilan-imidazolium
salt derivatives (35-58) with alkyl chain displayed higher cytotoxic activities against four tumor cell lines. When
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the chain was replaced with acyl chain (59-70), cytotoxic activities of aza-brazilan-imidazolium salts were
dropped off slightly, and some compounds kept the same activity.
In the case of imidazole ring, aza-brazilan-imidazolium salt derivatives (35-38/43-46/51-54/59-62/66-68)
with 2-methyl-benzimidazole ring exhibited medium cytotoxic activities. However, aza-brazilan-imidazolium
salt derivatives (39-42/47-50/55-58/63-65/69-70) with 5,6-dimethyl-benzimidazole ring displayed significant
inhibitory activities. Interestingly, aza-brazilan-imidazolium salt derivatives 39-42/55/57/69/70, with 5,6-
dimethyl-benzimidazole ring, showed powerful inhibitory activity with IC50 values of below 2.00 μM (0.35-1.83
μM) against four tumor cell lines.
For the substituent at position-3 of imidazole ring, aza-brazilan-imidazolium salt derivatives with 4-
methoxyphenacyl substituent such as 37, 41, 45, 49, 53, 57, 61, 64 and 69, showed relative weak inhibitory
activities against four tumor cell lines with IC50 values of 0.35-14.42 μM. Compound 66 lacked activities
against three tumor cell lines tested at the concentration of 20 M. Imidazolium salts with 2-naphthylmethyl
substituent such as 36, 40, 44, 48, 52, 56, 60, 63 and 68, as well as imidazolium salts with 2-naphthylacyl
substituent such as 38 42, 46, 50, 54, 58, 62, 65, 67 and 70 had moderate cytotoxic activities (with IC50 values of
0.85-5.83 μM and 0.91-8.26 μM). Notably, imidazolium salts with 4-methylbenzyl substituent such as 35, 39,
47, 51, 55 and 59 exhibited higher inhibitory activity (IC50 = 0.52-6.27 μM). Among them, compounds 55 and
39, bearing a 4-methylbenzyl substituent at position-3 of 5,6-dimethyl-benzimidazole, were found to be the
most potent compounds with IC50 values of 0.52-1.30 μM and 0.56-1.51 μM against four human tumor cell lines
investigated. Particularly, compound 57, with a 2-bromobenzyl substituent at position-3 of 5,6-dimethyl-
benzimidazole, exhibited inhibitory activity against MCF-7 cell line with IC50 value of 0.35 μM and 56-fold
more sensitive to DDP.
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The results implied that the existence of electron-withdrawing group on aza-brazilan moiety, substituted 5,6-
dimethyl-benzimidazoless ring and substitution of the imidazolyl-3-position with a 4-methylbenzyl group could
be important for improving cytotoxic activity. The SAR results were summarized in Scheme 2.
R1
R2
R1
R2
R
R
O
N
N
R3
N
N
R3
N
N
Br
Br
OMe
OMe
A
B
aza-brazilin-imidazolium salt derivatives
alkyl chain (A) ≈ acyl chain (B)
Better
R1 = Br, R2 = H > R1 = R2 = H > R1 = R2 = OMe
Best
imidazole ring:
5,6-dimethyl-benzimidazole > 2-methyl-benzimidazole
Better
O
O
R2 =
≈
>
>
OMe
Best
Best Potent Compound
Br
N
N
N
Br
OMe
55
Scheme 2 Structure-activity relationship of aza-brazilan-imidazolium salt derivatives.
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Imidazolium salt 55 induced G0/G1 phase arrest and apoptosis in cancer cells
To determine the proliferation selectivity of compound 55, the IC50 of compound 55 on three normal cell lines
(normal human liver cell line L02, normal human lung cell line Beas-2B and normal human colon cell line
NCM-460) were detected. As shown in Fig 2, compound 55 inhibited the growth of human cancer cells with
moderate selectivity, compared with the corresponding normal human cell lines. All compounds used as
chemotherapeutic agents do have an effect on normal cells as do the present compounds. However, their effect
is more on cancer cells. This is the reason the present drugs are having a potential. To determine whether the
proliferation inhibitory effect of aza-brazilan-imidazolium salt 55 caused by cell cycle arrest, propidium iodide
(PI) staining and flow cytometry analysis of cells was performed in SMMC-7721 cells treated with indicated
concentrations of imidazolium salt 55 (1, 2, 4 μM). As shown in Fig. 3, the results suggested that imidazolium
salt 55 may induce G0/G1 phase arrest in the cell cycle, and a sub-G1 peak (apoptotic peak) appeared when the
concentration was at 4 μM.
Fig. 2 The proliferation selectivity of imidazolium salt 55 against human cancer cells and normal human cell
lines.
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A
1 μM
2 μM
4 μM
DMSO
DNAContent
B
Cells (%)
Treatment
DMSO
sub-G1
G0/G1
S
G2/M
4.03 ± 0.47
1.32 ± 0.14
1.21 ± 0.18
18.65 ± 0.43
72.17 ± 1.0
80.32 ± 0.25
80.22 ± 0.89
62.54 ± 0.53
14.733 ± 1.29
10.18 ± 1.67
9.27 ± 1.69
8.21 ± 0.38
7.64 ± 1.56
9.16 ± 0.93
5.98 ± 1.11
Compound 55 (1 μM)
Compound 55 (2 μM)
Compound 55 (4 μM)
12.76 ± 1.69
Fig. 3 Imidazolium salt 55 induced G0/G1 phase arrest in SMMC-7721 cells. (A) Cells were treated with 1, 2
and 4 μM of compound 55 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 was shown.
Aza-brazilan-imidazolium salt 55 induced cell apoptosis was determined with Annexin V-FITC/PI double-
labeled cell cytometry. As shown in Fig. 4, after treatment of cells with imidazolium salt 55 at 0.5, 1, 2, 4 and 8
μM for 48 h, cell apoptosis in SMMC-7721 cells remarkably elevated to 8.97%, 15.13%, 16.22%, 77.72% and
81.56%, respectively. The data suggested that illustrated that steroidal imidazolium salt 55 inhibited cell
proliferation through induction of G0/G1 cell cycle arrest and apoptosis of the SMMC-7721 cells.
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Fig. 4 Imidazolium salt 55 caused apoptosis of SMMC-7721 cells. (A) Cells were treated with 0.5, 1, 2, 4 and
8 μM imidazolium salt 55 for 48 h. Cell apoptosis was determined by Annexin V-FITC/PI double-staining
assay. (B) The quantification of cell apoptosis. Four independent experiments were performed and data of one
representative experiment was shown. The significance was determined by Student’s test (**p < 0.01 and ***p <
0.001 vs. DMSO).
Conclusion
In sum, a series of novel aza-brazilan derivatives containing imidazolium salt pharmacophore prepared in
this research has been verified to be potential antitumor agents. The aza-brazilan-imidazolium salt derivatives
39, 40, 51, 52, 53, 55 and 57, with electron-withdrawing group on aza-brazilan moiety, or 5,6-dimethyl-
benzimidazole ring or a 4-methylbenzyl at position-3 of the imidazole ring, were found to be the most potent
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compounds. Notably, compounds 55 and 39, bearing a 4-methylbenzyl substituent at position-3 of 5,6-dimethyl-
benzimidazole, were found to be the most potent compounds with IC50 values of 0.52-1.30 μM and 0.56-1.51
μM against four human tumor cell lines investigated. Particularly, compound 57 exhibited inhibitory activity
against MCF-7 cell line with IC50 value of 0.35 μM and 56-fold more sensitive to DDP. Compound 55 can
induce the G0/G1 phase cell cycle arrest and apoptosis in SMMC-7721 cells. This kind of aza-brazilan-
imidazolium salt derivatives could thus serve as a novel starting point to investigate better lead compounds for
further structural modifications.
Experimental Section
General procedures
Melting points were obtained on a XT-4 melting-point apparatus and were uncorrected. Proton nuclear magnetic
resonance (1H-NMR) spectra were recorded on a Bruker Avance 300 or Bruker DRX 400 spectrometer at 300 or
400 MHz. Carbon-13 nuclear magnetic resonance (13C-NMR) was recorded on Bruker DRX 400spectrometer at
100 MHz. Chemical shifts are reported as δ values in parts per million (ppm) relative to tetramethylsilane
(TMS) for all recorded NMR spectra. Low-resolution Mass spectra were recorded on a VG Auto Spec-3000
magnetic sector MS spectrometer. High Resolution Mass spectra were taken on AB QSTAR Pulsar mass
spectrometer. Silica gel (200-300 mesh) for column chromatography and silica GF254 for TLC were produced by
Qingdao Marine Chemical Company (China). All air- or moisture- sensitive reactions were conducted under an
argon atmosphere. Starting materials and reagents used in reactions were obtained commercially from Acros,
Aldrich, Fluka and were used without purification, unless otherwise indicated.
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Synthesis of compounds 2-4, 5-7, 8-10, 11-13, 14-16 17-19 and 20-22.
See ESI file for characterization data.†
Synthesis of aza-brazilan-imidazole hybrid compounds 23-28.
To a mixture of compound 20-22 (0.44 mmol) and 2-methylbenzimidazole or 5,6-dimethylbenzimidazole (0.53
mmol) and K2CO3 (365 mg, 2.64 mmol) was stirred in DMF (20 ml) at 120 oC under nitrogen for 12 h. The
reaction progress was monitored by TLC. After cooling to room temperature, the solvent was evaporated under
reduced pressure and the crude product was purified by flash column chromatography on silica gel (eluted with
CH2Cl2:MeOH:Et3N = 500:1:5→300:1:3) to give 23-28 as white powder or colorless oil in 53-74% yields. See
ESI file for characterization data.†
Synthesis of aza-brazilan-imidazole hybrid compounds 29-34.
To a solution of compound 17-19 (0.96 mmol) and K2CO3 aqueous solution (0.5 M, 6 ml, 3.00 mmol) in CH2Cl2
(10 mL) was added slowly 3-chloropropanoyl chloride (367 mg, 2.89 mmol) at an ice bath. The solution was
warmed to room temperature and stirred for 30 min. After cooling at 0 °C, it was quenched with H2O. The
resulting mixture was extracted with CH2Cl2, and the organic layer was washed with brine, dried over Na2SO4,
and concentrated. The obtained product was used without any further purification. A mixture of the previous
product (0.44 mmol) and 2-methylbenzimidazole or 5,6-dimethylbenzimidazole (0.53 mmol) and K2CO3 (365
mg, 2.64 mmol) in dry DMF (5 ml) was stirred in DMF (20 ml) at 120 oC under nitrogen for 12 h. The reaction
progress was monitored by TLC. The solvent was evaporated under reduced pressure and the crude product was
purified by flash column chromatography on silica gel (eluted with CH2Cl2:MeOH:Et3N = 500:1:5→300:1:3) to
give 29-34 as white powder or colorless oil in 61-82% yields. See ESI file for characterization data.†
Synthesis of aza-brazilan-imidazolium salt compounds 38-58 and 59-70.
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A mixture of compound 23-28 or 29-34 (0.19mmol) and the corresponding alkyl and phenacyl bromides (0.38
mmol) was stirred in acetone/toluene (1:1, 6 ml) at reflux for 24-48 h. An insoluble substance was formed. After
completion of the reaction as indicated by TLC, the precipitate was filtered through a small pad of Celite, and
washed with ethyl acetate (3 × 30 ml), then dried to afford imidazolium salts 35-58 and 59-70 in 49-87% and
13-45% yields. See ESI file for characterization data.†
Cytotoxicity assay.
Cytotoxicity of compounds was determined by MTS method. All cell lines used in this study were obtained
from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China).
Briefly, 5×103 cells were plated in 96-well plates 12 h before treatment and continuously exposed to test
compounds for 48 h. Then MTS (Promega, Madison, WI, USA) was added to each well. The samples were
incubated at 37 °C for 1~4 h and the optical density (OD) was measured at 490 nm using a microplate reader
(Bio-Rad Laboratories, Hercules, CA, USA). The IC50 values are calculated from appropriate dose-response
curves.
Cell cycle analysis.
To analyze the DNA content by flow cytometry, cells were collected and washed twice with PBS. Cells were
fixed with 70% ethanol overnight. Fixed cells were washed with PBS, and then stained with a 50 μg/ml
propidium iodide (PI) solution (Sigma-Aldrich, Saint-Louis, MO, USA) containing 50 μg/ml RNase A (Sigma-
Aldrich) 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 FlowJo V 7.6.1 software.
Cell apoptosis analysis.
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Cell apoptosis was analyzed using the Annexin V-FITC/PI Apoptosis kit (BD Biosciences, Franklin Lakes, NJ)
according to the manufacturer’s protocols. Cells were seeded in 6-well plates at a density of 3 × 105 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 V-FITC 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).
Acknowledgments
This work was supported by grants from the Natural Science Foundation of China (21662043, U1402227 and
U1702286), Program for Changjiang Scholars and Innovative Research Team in University (IRT17R94) and
IRTSTYN, Yun-Ling Scholar of Yunnan Province and Donglu Scholar & Excellent Young Talents of YNU,
State Key Laboratory of Phytochemistry and Plant Resources in West China (P2017-KF12).
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FIGURE TITLES
Fig. 1 Representative structures of brazilin, aza-brazilin derivatives and imidazolium salts.
Fig. 2 The proliferation selectivity of imidazolium salt 55 against human cancer cells and normal human cell
lines.
Fig. 3 Imidazolium salt 55 induced G0/G1 phase arrest in SMMC-7721 cells. (A) Cells were treated with 1, 2
and 4 μM of compound 55 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 was shown.
Fig. 4 Imidazolium salt 55 caused apoptosis of SMMC-7721 cells. (A) Cells were treated with 0.5, 1, 2, 4 and
8 μM imidazolium salt 55 for 48 h. Cell apoptosis was determined by Annexin V-FITC/PI double-staining
assay. (B) The quantification of cell apoptosis. Four independent experiments were performed and data of one
representative experiment was shown. The significance was determined by Student’s test (**p < 0.01 and ***p <
0.001 vs. DMSO).
SCHEME TITLES
Scheme 1 Synthesis of aza-brazilan-imidazole hybrid compounds 23-34.
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DOI: 10.1039/C9MD00112C
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Scheme 2 Structure-activity relationship of aza-brazilan-imidazolium salt derivatives.
TABLE TITLES
Table 1 Synthesis of aza-brazilan-imidazolium salt derivatives 35-70 from hybrids 23-34 from 16-21
Table 2 Cytotoxic activities of aza-brazilan-imidazole hybrids 23-34 and aza-brazilan-imidazolium salts 35-
70 in vitrob (IC50, Ma)
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DOI: 10.1039/C9MD00112C
Graphical Abstracts
R1
R2
R1
R2
R
H
H
R1
N
N
R3
NH
OMe
N
H
H
Br
R2
HO
O
OMe
35-70
17-19
1
Br
H
cytotoxic activity
IC50, μM
N
N
N
H
Br
SMMC-7721
A549
0.52
1.30
0.68
0.75
OMe
MCF-7
SW480
55
A series of novel aza-brazilan derivatives containing imidazolium salt pharmacophore were
synthesized and their antitumor structure-activity relationship studies were reported.