Bioorganic & Medicinal Chemistry Letters 24 (2014) 799–807
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Synthesis and biological evaluation of 4-(1,2,3-triazol-1-yl)coumarin derivatives as potential antitumor agents Wenjuan Zhang, Zhi Li, Meng Zhou, Feng Wu, Xueyan Hou, Hao Luo, Hao Liu, Xuan Han, Guoyi Yan, Zhenyu Ding, Rui Li ⇑ State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041 Sichuan, PR China
a r t i c l e
i n f o
Article history: Received 9 October 2013 Revised 21 November 2013 Accepted 23 December 2013 Available online 29 December 2013 Keywords: Coumarin 1,2,3-Triazole Antiproliferative activity G2/M cell-cycle arrest Apoptosis
a b s t r a c t In this research, a series of 4-(1,2,3-triazol-1-yl)coumarin conjugates were synthesized and their anticancer activities were evaluated in vitro against three human cancer cell lines, including human breast carcinoma MCF-7 cell, colon carcinoma SW480 cell and lung carcinoma A549 cell. To increase the biological potency, structural optimization campaign was conducted focusing on the C-4 position of 1,2,3-triazole and the C-6, C-7 positions of coumarin. In addition, to further evaluate the role of 1,2,3-triazole and coumarin for antiproliferative activity, 9 compounds possessing 4-(piperazin-1-yl)coumarin framework and 3 derivatives baring quinoline core were also synthesized. By MTT assay in vitro, most of the compounds display attractive antitumor activities, especially 23. Further flow cytometry assays demonstrate that compound 23 exerts the antiproliferative role through arresting G2/M cell-cycle and inducing apoptosis. Ó 2014 Elsevier Ltd. All rights reserved.
Coumarin derivatives which are widely distributed in the plants1,2 have been extensively investigated as anticoagulation, antiviral,3–5 anti-inflammatory,6,7 antibacterial8 and anticancer9– 17 agents. Another promising group, 1,2,3-triazole, has emerged as one of the most important heterocycles in current medicinal chemistry18–20 and its applications have also been extended to widespread diseases.21–24 In recent years, a library of coumarin derivatives conjugated with 1,2,3-triazole were synthesized and proved to possess different bioactivity. As far as anticancer activity was concerned, H.M. Liu and his coworkers discovered 4-((1,2,3triazol-1-yl)methyl)coumarin derivatives exhibiting obvious anticancer activity through inducing apoptosis.25 Besides, novobiocin analogues with 1,2,3-triazole at the C-3 position of coumarin displayed potent cytotoxic activity against two breast cancer cell lines (SKBr-3 and MCF-7).26 For anti-inflammatory activity, a family of 3-(1,2,3-triazol-1-yl)coumarins, effecting on inducible nitric oxide synthase, were synthesized and proved to reduce neutrophils in the LPS-inflamed subcutaneous tissue,27 while, through inhibiting 5-lipoxygenase, triazole connected to C-7 position of coumarin by a methene can also cause remarkable anti-inflammatory activity.28 In addition, compounds with 1,2,3-triazol-1-yl at C-3 position of coumarin revealed the inhibition of amyloid-b aggregation which plays a pathogenic role in the progression of Alzheimer’s disease.29 Compounds mentioned above are summarized in Figure 1. ⇑ Corresponding author. E-mail address: [email protected] (R. Li). 0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.12.095
While the conjugates of 1,2,3-triazole and coumarin fighting against different diseases have made significant progress, efficient molecules against cancer are still urgently needed. As for 4-(1,2,3triazol-yl)coumarin derivatives, the synthesis methods have been reported,30 however, so far their anticancer activities have rarely been investigated. In our preliminary experiments, we found 6-bromo-4-(4-phenyl-1,2,3-triazol-1-yl)coumarin (compound 1) showed a moderate anticancer activity with the IC50 values of 9.45, 6.66, 11.23 lM against MCF-7, SW480 and A549, respectively, which inspired us to conduct the structural optimization of compound 1 for more potent anticancer agents. Our optimization focused on the following two aspects: (1) in view of the planarity caused by the conjugated system, introducing a series of chemical bridges (–CH2–NH–, –CH2–O– or –CH2–S–) between phenyl and 1,2,3-triazole may be helpful to eliminate the planarity, thereby ameliorate the druggability. Meanwhile, the rotatable bonds may also facilitate the compounds binding to its possible receptor through induced fit. (2) To improve the potency, different substitutions introduced to C-6 and C-7 positions of coumarin were taken into account. Moreover, it should be mentioned that in order to evaluate the contribution of 1,2,3-triazole and coumarin to the antiproliferative activity, respectively, the replacements of 1,2,3-triazole with piperazine and coumarin with quinoline were conducted and their biological activity were also measured. The general procedures for the preparation of the 4-(1,2,3-triazol-1-yl)coumarin derivatives 1–23 and 6e were efficiently synthesized according to the protocol outlined in Schemes 1–3. Different
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W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807
N O
N
O HO F3 C
N N N
O
N
O O
Me
O N N N
F anti-inflammatory agent
amyloid- β aggregation inhibitor
5-lipoxygenase inhibitor
HO
O
O O N
O
N
O
O
NH
N N N
S
N
O
N
N
O
S novobiocin analogue anticancer agent
anticancer agent
Figure 1. Structures of coumarin derivatives conjugated with 1,2,3-triazole.
OH i
R2
R1 R
R2
R2
ii
O O
1
R
OH
R2
iii
1
iv
R2 R
O
O
v
1
R2
O
O
R1 N3 R 2=H R 2=H R 2=OCH3 R 2=H R 2=OH R 2=H
3a R 1 =Br R 2=H 3b R 1=OCH 3 R 2=H 3c R 1 =H R 2=OCH3
vi
R2 R
Cl 4a R 1 =Br 4b R 1=OCH 3 4c R 1 =H 4d R 1=OH 4e R 1 =H 4f R1 =H
OH
2a R 1 =Br R 2=H 2b R 1=OCH 3 R 2=H 2c R 1 =H R 2=OCH3
R 2=H R 2=H R 2=OCH3
R 1 =Br
O
R1 O
1a R 1=Br 1b R1 = OCH3 1c R 1=H
O
5a 5b R 1=OCH 3 5c R 1 =H 5d R 1=OH 5e R 1 =H 5f R1 =H
2
R =H R 2=H R 2=OCH3 R 2=H R 2=OH R 2=H
O
O
R1 N N
N
R 6e, 1- 23
Scheme 1. Reagents and conditions: (i) acetic anhydride, pyridine, 100 °C, 3.5 h, 82–98%; (ii) AlCl3, 150 °C, 3.5 h, 70–75%; (iii) diethyl carbonate, NaH, Toluene, 100 °C, 4 h, 67–86%; (iv) POCl3, Triethylamine, reflux, 1 h, 70–85%; (v) NaN3, NMP, rt, 69–82%; (vi) t-BuOH, Cu, CuSO45H2O, 65 °C, 49–80%.
phenol derivatives were reacted with acetic anhydride at 100 °C to prepare the esters 1a–1c, which were further subjected to the Fries rearrangement reaction with o-hydroxyacetophenone 2a–2c in the presence of aluminum chloride at 150 °C. Compounds 3a–3c, which are crucial to the synthesis of all 4-(1,2,3-triazol-1-yl)coumarin derivatives, were synthesized from o-hydroxyacetophenone derivatives by reacting with diethyl carbonate and sodium hydride at 100 °C. Compounds 4a–4c, with a chlorine group at the C-4 position of the coumarin ring, were synthesized in 60–97% yield by treatment of 3a–3c compounds with phosphorus oxychloride (POCl3) and triethylamine. The product (4b and 4c) in dichloromethane was then hydrolyzed with BBr3 at cooled temperature to obtain the 4-chloro-6-hydroxycoumarin (4d and 4e) (Scheme 2). The treatment of 4-azido-coumarin compounds (5a–5f) which were obtained through 4-chloro-coumarin derivatives in the presence of NaN3 at room temperature, and alkynes (Scheme 3), afforded the 4-(1,2,3-triazol-1-yl) coumarin derivatives in tert-butyl
alcohol at 65 °C with copper sulfate pentahydrate and copper as CuAAC.31 Furthermore, 4-fluorobenzene-1-sulfonyl chloride, methanesulfonyl chloride or morpholine derivative (12a) were reacted with compound 6e or 16 to prepare target compounds 18, 19 and 17 (Scheme 2). To further measure the contribution of 1,2,3-triazole and coumarin to the antiproliferative activity, we next replaced them with piperazine and quinoline, respectively (Scheme 4 and 5). As outlined in Scheme 4, each candidate was obtained starting from 4-chlorocoumarin (4f) and the synthesis began with the substitution of piperazine or 2-(piperazin-1-yl)ethanol to obtain 6a and 6b. The 4-(4-benzylpiperazin-1-yl)coumarin analogues 24–30 were prepared by the reaction of the corresponding benzaldehyde with 6a. On the other hand, analogues 31 and 32 were synthesized from 6b in two steps. In addition, the syntheses of 6-bromoquinolin-4-ol, 2g, was accomplished according to the cyclization of 1g in boiling diphenyl ether, which was achieved with 4-bromoaniline in
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W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807
O
O O
R O
O
O
O
O
22 R=
HO
O
N i
O
ii
HO
Cl
iii
HO
Cl
4b
N3
O
O
O
HO
O
O
O R
N
O 10a
ii
i
iii
N N
iv
4e
N N
O 19 R=
5e
OH H N
18 R=
N3
Cl
4c
O
O N
O
Cl
O
HO
O
16 R=
O
HO O
O
R 16, 22
5d
4d
F N N
O S O
F
O S O
OTs
N
N vi
v
16 vii
O
O
12a
12b
17
Scheme 2. Reagents and conditions: (i) BBr3, DCM, 22 °C (2 h), rt (5 h), 95–97%; (ii) NaN3, NMP, rt, 69–80%; (iii) t-BuOH, Cu, CuSO45H2O, 65 °C, 49–53%; (iv) CH3SO2Cl or 4-fluorobenzene-1-sulfonylchloride, Et3N, THF, 0 °C, 78–80%; (v) K2CO3, MeCN, reflux, 3 h, 78%; (vi) TsCl, Et3N, DMAP, DCM, rt, overnight, 65%; (vii) K2CO3, DMF, 60 °C, 24 h, 57%.
X
X
Br
R5
R5 DMF/K2CO 3 8a-8d,9a-9i,10a-10e X=OH,SH or NH 2
8a R 5=o-COOCH 3, 8b R 5=o-Br, 8c R 5=o-OH,
X=O X=O X=S
10a R 5=H, 10b R 5=o-COOCH3, 10c R 5=H, 10d R 5=H,
X=O X=O X=S X=N
9a R 5=p-F, 9b R 5=p-C2H 5, 9c R 5=p-Br, 9d R 5=p-NH 2, 9e R 5=p-COOCH 3, 9f R 5=p-CF3, 9g R 5=p-NO 2, 9h R 5=p-COOEt,
Br
X=O X=O X=O X=O X=O X=O X=O X=O
N Br Br OH
N O
N
11a
Br O
DMF/K 2CO3 OH
DMF/K 2CO3
N
11b
Scheme 3. Reagents and conditions: 3-bromopropyne, K2CO3, DMF, rt, overnight, 60–98%.
the presence of 2,2-dimethyl-1,3-dioxane-4,6-dione and triethyl orthoformate in ethanol at 90 °C. Nitration occurring at C-3 position of quinoline resulted in 6-bromo-3 -nitroquinolin-4-ol (3g). Compound 4g with a chlorine at C-4 position of quinoline was synthesized by 3g, POCl3 and triethylamine and used to prepare compound 5g in the presence of NaN3 in N-methylpyrrolidone at room temperature. All the 6-bromo-3-nitro-4-(1,2,3-triazol-1-yl)quinoline derivatives 33–35 were obtained from 5g and alkynes via CuAAC (Scheme 5). The general reaction conditions and processing and the Supplementary Data (MS, NMR, yield and purity) are described in Supporting Information. The IC50 values (concentration required to inhibit tumor cell proliferation by 50%) for the synthesized compounds against three human cancer cell lines including human breast carcinoma MCF-7 cell, colon carcinoma SW480 cell and lung carcinoma A549 cell were measured by MTT assay (Tables 1–5). All of our antiproliferation tests were performed three times using each agent. Consideration of the few rotatable bonds of compound 1, which may not bind to the possible target well, a series of chemical
bridges, more specifically, –CH2–NH–, –CH2–O– and –CH2–S– were inserted to phenyl and 1,2,3-triazole. As for the –CH2–NH– and – CH2–S– bridges, compounds 2 and 3 displayed improved inhibitory activity against three tested cancer cell lines with IC50 values of 3.51 lM. To our delight, the introduction of –CH2–O– bridge (compound 4) authorized the potential inhibitory against MCF-7 with IC50 value of 1.72 lM. Subsequently, the replacement of phenyl with heterocycles, including pyridine (5) and quinoline (6), generally resulted in loss in bioactivity on three cancer cell lines. Hence, we use compound 4 for further optimization. Furthermore, we decided to optimize the substituents of the phenyl ring, and the IC50 values were summarized in Table 2. First of all, compounds 7–9 with different substitutions (methoxycarbonyl, bromine and hydroxy, respectively) at the C-2 position showed drastically decreased or equivalent activity against lead compound 4, suggesting that the modification at C-2 position is not allowed. Then we turned our attention to the C-4 position of phenyl. The results in Table 2 show that a lipophilic group, ethyl (10), and polar groups, including amino (11), bromine (12) and
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O
H
O
H N O O
O
O
N H
N
R4
i
O
ii
N
N
Cl R4
N H 4f
24-30
6a HN
iii
N O
O
OH
O
O
O
N
HO N
N
N
v
iv N
O
R3
N O OH
OTs
6b
R3
7b
31,32
Scheme 4. Reagents and conditions: (i) ethanol, rt, 70%; (ii) Na(OAc)3BH, DCM, rt, 70–78%; (iii) ethanol, rt, 70%; (iv) TsCl, Et3N, DMAP, DCM, rt, 31%; (v) 60–71%.
OH
Br
Br
O
i NH 2
Br
ii N
O
iii
NO2
O N O
O
N
O
1g O
OH Br
2g
3g
O R N3
Cl iv
Br
N N
NO2
v
Br
NO2
vi R
N
Br
NO2
N
N
N 4g
5g
33-35
Scheme 5. Reagents and conditions: (i) triethyl orthoformate, 90 °C, 10 min, EtOH, 30 min, 66%; (ii) diphenyl oxide, 220 °C, 5 min, 83.5%; (iii) nitric acid, Propionic acid, 125 °C, 2 h, 50.2%; (iv) POCl3, triethylamine, reflux, 1 h, 68%; (v) NaN3, NMP, rt, 59%; (vi) t-BuOH, Cu, CuSO45H2O, 65 °C, 67–75%.
trifluoromethyl (13), led to the equivalent potent or slightly increased activity. However fluorine and methoxycarbonyl at the C-4 position endowed compounds (14 and 15, respectively) with prominent activity against all the three cancer cell lines as low as 0.66 lM, indicating that the hydrogen acceptor at the C-4 position of phenyl is essential to the activity. Next, we turned our attention to the substituents at the C-6 and C-7 positions of coumarin. Compared with the activity of compound 4, complete loss of antiproliferative activity were observed in Table 3 when the bromine was replaced with hydroxy (16) or 2-morpholinoethoxy (17). Nevertheless, when the bromine was replaced with hydrogen, compound 20, with fluorine at the C-4 position of phenyl, showed more potency than compound 14 in the MCF-7 and A549, indicating that bulky group may be unfavorable. Subsequently, with hydrogen at the C-6 position, the introduction of bulky group, 4-fluorobenzenesulfonate (18), to C-7 position led to total loss of potency, while the introduction of smaller group, methylmethanesulfonate (19), only caused the partial loss of the bioactivity, suggesting that small group at C-7 position may also be advantageous to the bioactivity. Then, methoxy introducing to the C-7 position of coumarin was taken into account. As might
have been expected, compound 23, with fluorine retained at the C-4 position of phenyl, showed higher bioactivity with IC50 values of 5.89, 1.99 and 0.52 lM against MCF-7, SW480 and A549, respectively. It is worth noting that compound 23 is more potent than compound 20, which can be ascribed to the additional hydrogen bond caused by the methoxy at C-7 position. Lastly, with hydrogen at C-7 position of coumarin and fluorine at the C-4 position of phenyl, it was discovered that the replacement of bromine with methoxy (21) or hydroxy (22) group at the C-6 position of coumarin resulted in the approximate activity to compound 14, reconfirming that there may be a steric hindrance at the C-6 region. To further measure the contribution of the triazole and coumarin motifs to the biological activity, a number of 6-bromo-4-(piperazin-1-yl)coumarin and 6-bromo-3-nitro-4-(1,2,3-triazol-1-yl) quinoline derivatives were prepared (Tables 4 and 5). First, the coumarin analogues substituted by piperazine exhibited enormous reduction of potency, especially compounds 24–30. However, the extended chemical bridge (compounds 31, and 32) between phenyl and piperazine could give a slight improvement in inhibitory activity, probably because the rotatable bonds can facilitate the compounds binding to the possible target. On the other hand,
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W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807 Table 1 In Vitro Inhibition of Tumor Cell Growth of compounds 1–6
R N N N Br O Compd
O
IC50a (lM)
R
1
MCF-7
SW480
A549
9.45 ± 0.55
6.66 ± 0.31
11.23 ± 2.0
>50
18.41 ± 3.5
13.2 ± 4.2
2
HN
3
S
36.83 ± 2.3
15.43 ± 0.33
3.51 ± 0.98
4
O
1.72 ± 0.64
4.99 ± 0.42
12.31 ± 0.11
5
O
39.82 ± 1.8
39.77 ± 2.4
24.79 ± 2.3
41.56 ± 1.4
16.95 ± 2.3
13.29 ± 4.9
3.51 ± 0.31
2.43 ± 0.25
1.65 ± 0.09
N N 6
O Br Doxorubicin a
Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard deviation (n = 3).
Table 2 In Vitro Inhibition of Tumor Cell Growth of compounds 7–15
R5 O N N N Br O
O
Compd
R5
IC50a (lM) MCF-7
SW480
A549
7 8 9 10 11 12 13 14 15 Doxorubicin
2-COOCH3 2-Br 2-OH 4-CH2CH3 4-NH2 4-Br 4-CF3 4-F 4-COOCH3
34.14 ± 2.2 28.91 ± 1.3 24.82 ± 0.89 12.67 ± 1.4 2.04 ± 1.1 1.92 ± 0.71 11.41 ± 2.3 7.00 ± 0.25 3.13 ± 0.24 3.51 ± 0.31
20.23 ± 3.4 20.13 ± 1.7 19.14 ± 3.3 10.82 ± 0.60 2.09 ± 0.13 1.31 ± 3.2 3.09 ± 2.0 1.77 ± 0.03 3.62 ± 0.25 2.43 ± 0.25
5.25 ± 0.24 8.43 ± 0.51 7.94 ± 3.4 11.58 ± 0.36 11.85 ± 0.24 24.47 ± 2.5 1.62 ± 1.1 0.85 ± 0.09 0.66 ± 0.03 1.65 ± 0.09
a Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard deviation (n = 3).
compounds 33–35, with coumarin replaced by quinoline, can maintain a part of antiproliferative activity, possibly due to the nitrogen atom of the quinolone which is similar to the oxygen atom of coumarin that can form hydrogen bond with the corresponding acceptor. The fact that the coumarin derivatives are more potent than quinoline can be interpreted by the additional
carbonyl group of coumarin, which may form another hydrogen bond with its possible receptor. Structure activity relationships (SAR) were summarized in Figure 2. Because compound 23 shows the highest antiproliferative activity among all the synthesized compounds, it was selected to assess the effect on cell-cycle arrest of MCF-7 by flow cytometry
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W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807
Table 3 In Vitro Inhibition of Tumor Cell Growth of compounds 16–23
R5 O N N N R1 R2
O
O IC50a (lM)
Compd
R1
R2
R5
MCF-7
SW480
A549
16
OH
H
H
>50
>50
>50
H
H
>50
>50
>50
H
>50
>50
>50
H
45.81 ± 1.4
10.67 ± 0.98
9.14 ± 0.58
4-F 4-F 4-F 4-F
5.84 ± 0.22 8.56 ± 0.23 4.62 ± 0.65 5.89 ± 0.14 3.51 ± 0.31
3.82 ± 0.40 25.87 ± 1.4 2.72 ± 0.30 1.99 ± 0.38 2.43 ± 0.25
0.68 ± 0.08 3.15 ± 0.39 2.01 ± 0.14 0.52 ± 0.21 1.65 ± 0.09
O N
17
O
O 18
O S
H
F
O O 19
O
H
S O
20 21 22 23 Doxorubicin
H OCH3 OH H
H H H OCH3
a Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard deviation (n = 3).
Table 4 In Vitro Inhibition of Tumor Cell Growth of compounds 24–32
R0 N N
O
Compd
R0
24
O
IC50a (lM) MCF-7
SW480
A549
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
F 25
26
HO
27
O
O 28
HO
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W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807 Table 4 (continued) Compd
IC50a (lM)
R0
MCF-7
SW480
A549
>50
>50
>50
>50
>50
>50
24.20 ± 2.5
35.01 ± 5.0
21.58 ± 4.8
>50
24.01
11.92
3.51 ± 0.31
2.43 ± 0.25
1.65 ± 0.09
O 29
O
30
OH
O 31
O O 32
F Doxorubicin a
Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard deviation (n = 3).
Table 5 In Vitro Inhibition of Tumor Cell Growth of compounds 33–35
R5
X N N
N
Br
NO2 N
Compd
X
R5
33 34 35 Doxorubicin
S O O
H H COOEt
IC50a (lM) MCF-7 22.72 ± 3.3 7.69 ± 4.3 13.88 ± 1.3 3.51 ± 0.31
a Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard deviation (n = 3).
Figure 2. Structure-activity relationship revealed by optimization.
assay using propidium iodide (PI) staining. As is seen from Figure 3a, in comparison with the control group, treatment of compound 23 (5 lM) led to the obvious decrease in G1 phase (from 52.82% to 3.06%) and dramatic increase in G2 phase (from 15.78% to 84.21%), indicating that compound 23 is a G2/M cell-cycle arrester. A much stronger accumulation of cell in G2/M phase caused by compound 23 inspired us to test whether it can induce the apoptosis of A549 by flow cytometry analysis using annexin V-FITC and propidium iodide (PI) staining. The percentage of apoptotic cells induced by compound 23 (1.25 lM) is increased by more than 6-fold (Fig. 3b). All these results indicate that the promising antitumor activity of compound 23 may be attributed to the G2/M phase arrest and apoptosis. In summary, a family of 4-(1,2,3-triazol-1-yl)coumarin derivatives were synthesized and their antiproliferative activities were evaluated. Among these compounds, 23, 4-(4-((4-fluorophenoxy) methyl)-1,2,3-triazol-1-yl)-7-methoxycoumarin, exhibited excellent broad spectrum anticancer activity in vitro with IC50 values
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W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807
Figure 3. (a) Flow cytometry analyses of cell cycle distribution of breast cancer cell MCF-7 after treatment of compound 23 (5 lM) and no treatment (Ctrl) as reference control for 48 h. (b) Apoptosis induction in lung carcinoma cell A549 after 48 h treatment with 23 (1.25 lM) and no treatment.
of 5.89, 1.99 and 0.52 lM against MCF-7, SW480 and A549, respectively. The further mechanism study demonstrated that compound 23 could obviously inhibit the proliferation of cancer cells through inducing apoptosis and arresting the cell-cycle at G2/M phase. In addition, structure activity relationship research revealed that – CH2–O– bridge at C-4 position of 1,2,3-triazole core is the best optimal for bioactivity; a hydrogen bond acceptor at C-4 position of phenyl is indispensable for the improvement of potency; the hydrogen bond acceptor at C-7 position of coumarin can make a positive contribution to the activity. All the conclusions have encouraged us to continue the development, and the further validation of mechanism at the molecular level is undergoing. Acknowledgments The authors are grateful for the support from the National Science Foundation of China (30901743), the National Key Program of China (2012ZX09103101-022), the State Key Laboratory of Drug Research and Doctoral Fund of Ministry of Education. Supplementary data Supplementary data (experimental procedures and characterizationdata of compounds) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl. 2013.12.095.
References and notes 1. Murray, R. D. H.; Mendez, J.; Brown, S. A. Chemistry and Biochemistry; John Wiley & Sons Ltd, New York, 1982, 21. 2. Garazd, M. M.; Garadz, Y. L.; Khilya, V. P. Chem. Nat. Comp. 2003, 39, 54. 3. Hwu, J. R.; Singha, R.; Hong, S. C.; Chang, Y. H.; Das, A. R.; Vliegen, I.; Clercq, E. D.; Neyts, J. Antiviral Res. 2008, 77, 157. 4. Neyts, J.; Clercq, E. D.; Singha, R.; Chang, Y. H.; Das, A. R.; Chakraborty, S. K.; Hong, S. C.; Tsay, S. C.; Hsu, M. H.; Hwu, J. R. J. Med. Chem. 2009, 52, 1486. 5. Hwu, J. R.; Lin, S. Y.; Tsay, S. C.; Clercq, E. D.; Leyssen, P.; Neyts, J. J. Med. Chem. 2011, 54, 2114. 6. Li, Z. P.; Hu, J. F.; Sun, M. N.; Ji, H. J.; Chu, S. F.; Liu, G.; Chen, N. H. Int. Immunopharmacol. 2012, 14, 145. 7. Li, Z. P.; Hu, J. F.; Sun, M. N.; Ji, H. J.; Zhao, M.; Wu, D. H.; Li, G. Y.; Liu, G.; Chen, N. H. Eur. J. Pharmacol. 2011, 661, 118. 8. Shi, Y.; Zhou, C. H. Bioorg. Med. Chem. Lett. 2011, 21, 956. 9. Meggio, F.; Pagano, M. A.; Moro, S.; Zagotto, G.; Ruzzene, M.; Sarno, S.; Cozza, G.; Bain, J.; Elliott, M.; Deana, A. D.; Brunati, A. M.; Pinna, L. A. Biochemistry 2004, 43, 12931. 10. Chilin, A.; Battistutta, R.; Bortolato, A.; Cozza, G.; Zanatta, S.; Poletto, G.; Mazzorana, M.; Zagotto, G.; Uriarte, E.; Guiotto, A.; Pinna, L. A.; Meggio, F.; Moro, S. J. Med. Chem. 2008, 51, 752. 11. Bras, G. L.; Radanyi, C.; Peyrat, J. F.; Brion, J. D.; Alami, M.; Marsaud, V.; Stella, B.; Renoir, J. M. J. Med. Chem. 2007, 50, 6189. 12. Zhao, H. P.; Donnelly, A. C.; Kusuma, B. R.; Brandt, G. E. L.; Brown, D.; Rajewski, R. A.; Vielhauer, G.; Holzbeierlein, J.; Cohen, M. S.; Blagg, B. S. J. J. Med. Chem. 2011, 54, 3839. 13. Zhao, H. P.; Yan, B.; Peterson, L. B.; Blagg, B. S. J. ACS Med. Chem. Lett. 2012, 3, 327. 14. Donnelly, A. C.; Mays, J. R.; Burlison, J. A.; Nelson, J. T.; Vielhauer, G.; Holzbeierlein, J.; Blagg, B. S. J. J. Org. Chem. 2008, 73, 8901. 15. Purohit, A.; Woo, L. W. L.; Potter, B. V. L.; Reed, M. J. Cancer Res. 2000, 60, 3394. 16. Woo, L. W. L.; Purohit, A.; Malini, B.; Reed, M. J.; Potter, B. V. L. Chem. Biol. 2000, 7, 773.
W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807 17. Malini, B.; Purohit, A.; Ganeshapillai, D.; Woo, L. W. L.; Potter, B. V. L.; Reed, M. J.; Steroid, J. Biochem Mol. Biol. 2000, 75, 253. 18. Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128. 19. Moorhouse, A. D.; Moses, J. E. Chem. Med. Chem. 2008, 3, 715. 20. Whiting, M.; Muldoon, J.; Lin, Y. C.; Silverman, S. M.; Lindstrom, W.; Olson, A. J.; Kolb, H. C.; Finn, M. G.; Sharp less, K. B.; Elder, J. H.; Fokin, V. V. Chem. Int. Ed. 2006, 45, 1435. 21. Boechat, N.; Ferreira, V. F.; Ferreira, S. B.; Ferreira, M. L. G.; Silva, F. C.; Bastos, M. M.; Costa, M. S.; Lourenco, M. C. S.; Pinto, A. C.; Krettli, A. U.; Aguiar, A. C.; Teixeira, B. M.; Silva, N. V.; Martins, P. R. C.; Bezerra, F. A. F. M.; Camilo, A. L. S.; Silva, G. P.; Costa, C. C. P. J. Med. Chem. 2011, 54, 5988–5999. 22. Hou, J.; Li, Z.; Fang, Q.; Feng, C.; Zhang, H.; Guo, W.; Wang, H.; Gu, G.; Tian, Y.; Liu, P.; Liu, R.; Lin, J.; Shi, Y.; Yin, Z.; Shen, J.; Wang, P. G. J. Med. Chem. 2012, 55, 3066–3075. 23. Glaser, M.; Goggi, J.; Smith, G.; Morrison, M.; Luthra, S. K.; Robins, E.; Aboagye, E. O. Bioorg. Med. Chem. Lett. 2011, 21, 6945–6949.
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 4-(1,2,3-triazol-1-yl)coumarin derivatives as potential antitumor agents Wenjuan Zhang, Zhi Li, Meng Zhou, Feng Wu, Xueyan Hou, Hao Luo, Hao Liu, Xuan Han, Guoyi Yan, Zhenyu Ding, Rui Li ⇑ State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041 Sichuan, PR China
a r t i c l e
i n f o
Article history: Received 9 October 2013 Revised 21 November 2013 Accepted 23 December 2013 Available online 29 December 2013 Keywords: Coumarin 1,2,3-Triazole Antiproliferative activity G2/M cell-cycle arrest Apoptosis
a b s t r a c t In this research, a series of 4-(1,2,3-triazol-1-yl)coumarin conjugates were synthesized and their anticancer activities were evaluated in vitro against three human cancer cell lines, including human breast carcinoma MCF-7 cell, colon carcinoma SW480 cell and lung carcinoma A549 cell. To increase the biological potency, structural optimization campaign was conducted focusing on the C-4 position of 1,2,3-triazole and the C-6, C-7 positions of coumarin. In addition, to further evaluate the role of 1,2,3-triazole and coumarin for antiproliferative activity, 9 compounds possessing 4-(piperazin-1-yl)coumarin framework and 3 derivatives baring quinoline core were also synthesized. By MTT assay in vitro, most of the compounds display attractive antitumor activities, especially 23. Further flow cytometry assays demonstrate that compound 23 exerts the antiproliferative role through arresting G2/M cell-cycle and inducing apoptosis. Ó 2014 Elsevier Ltd. All rights reserved.
Coumarin derivatives which are widely distributed in the plants1,2 have been extensively investigated as anticoagulation, antiviral,3–5 anti-inflammatory,6,7 antibacterial8 and anticancer9– 17 agents. Another promising group, 1,2,3-triazole, has emerged as one of the most important heterocycles in current medicinal chemistry18–20 and its applications have also been extended to widespread diseases.21–24 In recent years, a library of coumarin derivatives conjugated with 1,2,3-triazole were synthesized and proved to possess different bioactivity. As far as anticancer activity was concerned, H.M. Liu and his coworkers discovered 4-((1,2,3triazol-1-yl)methyl)coumarin derivatives exhibiting obvious anticancer activity through inducing apoptosis.25 Besides, novobiocin analogues with 1,2,3-triazole at the C-3 position of coumarin displayed potent cytotoxic activity against two breast cancer cell lines (SKBr-3 and MCF-7).26 For anti-inflammatory activity, a family of 3-(1,2,3-triazol-1-yl)coumarins, effecting on inducible nitric oxide synthase, were synthesized and proved to reduce neutrophils in the LPS-inflamed subcutaneous tissue,27 while, through inhibiting 5-lipoxygenase, triazole connected to C-7 position of coumarin by a methene can also cause remarkable anti-inflammatory activity.28 In addition, compounds with 1,2,3-triazol-1-yl at C-3 position of coumarin revealed the inhibition of amyloid-b aggregation which plays a pathogenic role in the progression of Alzheimer’s disease.29 Compounds mentioned above are summarized in Figure 1. ⇑ Corresponding author. E-mail address: [email protected] (R. Li). 0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.12.095
While the conjugates of 1,2,3-triazole and coumarin fighting against different diseases have made significant progress, efficient molecules against cancer are still urgently needed. As for 4-(1,2,3triazol-yl)coumarin derivatives, the synthesis methods have been reported,30 however, so far their anticancer activities have rarely been investigated. In our preliminary experiments, we found 6-bromo-4-(4-phenyl-1,2,3-triazol-1-yl)coumarin (compound 1) showed a moderate anticancer activity with the IC50 values of 9.45, 6.66, 11.23 lM against MCF-7, SW480 and A549, respectively, which inspired us to conduct the structural optimization of compound 1 for more potent anticancer agents. Our optimization focused on the following two aspects: (1) in view of the planarity caused by the conjugated system, introducing a series of chemical bridges (–CH2–NH–, –CH2–O– or –CH2–S–) between phenyl and 1,2,3-triazole may be helpful to eliminate the planarity, thereby ameliorate the druggability. Meanwhile, the rotatable bonds may also facilitate the compounds binding to its possible receptor through induced fit. (2) To improve the potency, different substitutions introduced to C-6 and C-7 positions of coumarin were taken into account. Moreover, it should be mentioned that in order to evaluate the contribution of 1,2,3-triazole and coumarin to the antiproliferative activity, respectively, the replacements of 1,2,3-triazole with piperazine and coumarin with quinoline were conducted and their biological activity were also measured. The general procedures for the preparation of the 4-(1,2,3-triazol-1-yl)coumarin derivatives 1–23 and 6e were efficiently synthesized according to the protocol outlined in Schemes 1–3. Different
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W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807
N O
N
O HO F3 C
N N N
O
N
O O
Me
O N N N
F anti-inflammatory agent
amyloid- β aggregation inhibitor
5-lipoxygenase inhibitor
HO
O
O O N
O
N
O
O
NH
N N N
S
N
O
N
N
O
S novobiocin analogue anticancer agent
anticancer agent
Figure 1. Structures of coumarin derivatives conjugated with 1,2,3-triazole.
OH i
R2
R1 R
R2
R2
ii
O O
1
R
OH
R2
iii
1
iv
R2 R
O
O
v
1
R2
O
O
R1 N3 R 2=H R 2=H R 2=OCH3 R 2=H R 2=OH R 2=H
3a R 1 =Br R 2=H 3b R 1=OCH 3 R 2=H 3c R 1 =H R 2=OCH3
vi
R2 R
Cl 4a R 1 =Br 4b R 1=OCH 3 4c R 1 =H 4d R 1=OH 4e R 1 =H 4f R1 =H
OH
2a R 1 =Br R 2=H 2b R 1=OCH 3 R 2=H 2c R 1 =H R 2=OCH3
R 2=H R 2=H R 2=OCH3
R 1 =Br
O
R1 O
1a R 1=Br 1b R1 = OCH3 1c R 1=H
O
5a 5b R 1=OCH 3 5c R 1 =H 5d R 1=OH 5e R 1 =H 5f R1 =H
2
R =H R 2=H R 2=OCH3 R 2=H R 2=OH R 2=H
O
O
R1 N N
N
R 6e, 1- 23
Scheme 1. Reagents and conditions: (i) acetic anhydride, pyridine, 100 °C, 3.5 h, 82–98%; (ii) AlCl3, 150 °C, 3.5 h, 70–75%; (iii) diethyl carbonate, NaH, Toluene, 100 °C, 4 h, 67–86%; (iv) POCl3, Triethylamine, reflux, 1 h, 70–85%; (v) NaN3, NMP, rt, 69–82%; (vi) t-BuOH, Cu, CuSO45H2O, 65 °C, 49–80%.
phenol derivatives were reacted with acetic anhydride at 100 °C to prepare the esters 1a–1c, which were further subjected to the Fries rearrangement reaction with o-hydroxyacetophenone 2a–2c in the presence of aluminum chloride at 150 °C. Compounds 3a–3c, which are crucial to the synthesis of all 4-(1,2,3-triazol-1-yl)coumarin derivatives, were synthesized from o-hydroxyacetophenone derivatives by reacting with diethyl carbonate and sodium hydride at 100 °C. Compounds 4a–4c, with a chlorine group at the C-4 position of the coumarin ring, were synthesized in 60–97% yield by treatment of 3a–3c compounds with phosphorus oxychloride (POCl3) and triethylamine. The product (4b and 4c) in dichloromethane was then hydrolyzed with BBr3 at cooled temperature to obtain the 4-chloro-6-hydroxycoumarin (4d and 4e) (Scheme 2). The treatment of 4-azido-coumarin compounds (5a–5f) which were obtained through 4-chloro-coumarin derivatives in the presence of NaN3 at room temperature, and alkynes (Scheme 3), afforded the 4-(1,2,3-triazol-1-yl) coumarin derivatives in tert-butyl
alcohol at 65 °C with copper sulfate pentahydrate and copper as CuAAC.31 Furthermore, 4-fluorobenzene-1-sulfonyl chloride, methanesulfonyl chloride or morpholine derivative (12a) were reacted with compound 6e or 16 to prepare target compounds 18, 19 and 17 (Scheme 2). To further measure the contribution of 1,2,3-triazole and coumarin to the antiproliferative activity, we next replaced them with piperazine and quinoline, respectively (Scheme 4 and 5). As outlined in Scheme 4, each candidate was obtained starting from 4-chlorocoumarin (4f) and the synthesis began with the substitution of piperazine or 2-(piperazin-1-yl)ethanol to obtain 6a and 6b. The 4-(4-benzylpiperazin-1-yl)coumarin analogues 24–30 were prepared by the reaction of the corresponding benzaldehyde with 6a. On the other hand, analogues 31 and 32 were synthesized from 6b in two steps. In addition, the syntheses of 6-bromoquinolin-4-ol, 2g, was accomplished according to the cyclization of 1g in boiling diphenyl ether, which was achieved with 4-bromoaniline in
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O
O O
R O
O
O
O
O
22 R=
HO
O
N i
O
ii
HO
Cl
iii
HO
Cl
4b
N3
O
O
O
HO
O
O
O R
N
O 10a
ii
i
iii
N N
iv
4e
N N
O 19 R=
5e
OH H N
18 R=
N3
Cl
4c
O
O N
O
Cl
O
HO
O
16 R=
O
HO O
O
R 16, 22
5d
4d
F N N
O S O
F
O S O
OTs
N
N vi
v
16 vii
O
O
12a
12b
17
Scheme 2. Reagents and conditions: (i) BBr3, DCM, 22 °C (2 h), rt (5 h), 95–97%; (ii) NaN3, NMP, rt, 69–80%; (iii) t-BuOH, Cu, CuSO45H2O, 65 °C, 49–53%; (iv) CH3SO2Cl or 4-fluorobenzene-1-sulfonylchloride, Et3N, THF, 0 °C, 78–80%; (v) K2CO3, MeCN, reflux, 3 h, 78%; (vi) TsCl, Et3N, DMAP, DCM, rt, overnight, 65%; (vii) K2CO3, DMF, 60 °C, 24 h, 57%.
X
X
Br
R5
R5 DMF/K2CO 3 8a-8d,9a-9i,10a-10e X=OH,SH or NH 2
8a R 5=o-COOCH 3, 8b R 5=o-Br, 8c R 5=o-OH,
X=O X=O X=S
10a R 5=H, 10b R 5=o-COOCH3, 10c R 5=H, 10d R 5=H,
X=O X=O X=S X=N
9a R 5=p-F, 9b R 5=p-C2H 5, 9c R 5=p-Br, 9d R 5=p-NH 2, 9e R 5=p-COOCH 3, 9f R 5=p-CF3, 9g R 5=p-NO 2, 9h R 5=p-COOEt,
Br
X=O X=O X=O X=O X=O X=O X=O X=O
N Br Br OH
N O
N
11a
Br O
DMF/K 2CO3 OH
DMF/K 2CO3
N
11b
Scheme 3. Reagents and conditions: 3-bromopropyne, K2CO3, DMF, rt, overnight, 60–98%.
the presence of 2,2-dimethyl-1,3-dioxane-4,6-dione and triethyl orthoformate in ethanol at 90 °C. Nitration occurring at C-3 position of quinoline resulted in 6-bromo-3 -nitroquinolin-4-ol (3g). Compound 4g with a chlorine at C-4 position of quinoline was synthesized by 3g, POCl3 and triethylamine and used to prepare compound 5g in the presence of NaN3 in N-methylpyrrolidone at room temperature. All the 6-bromo-3-nitro-4-(1,2,3-triazol-1-yl)quinoline derivatives 33–35 were obtained from 5g and alkynes via CuAAC (Scheme 5). The general reaction conditions and processing and the Supplementary Data (MS, NMR, yield and purity) are described in Supporting Information. The IC50 values (concentration required to inhibit tumor cell proliferation by 50%) for the synthesized compounds against three human cancer cell lines including human breast carcinoma MCF-7 cell, colon carcinoma SW480 cell and lung carcinoma A549 cell were measured by MTT assay (Tables 1–5). All of our antiproliferation tests were performed three times using each agent. Consideration of the few rotatable bonds of compound 1, which may not bind to the possible target well, a series of chemical
bridges, more specifically, –CH2–NH–, –CH2–O– and –CH2–S– were inserted to phenyl and 1,2,3-triazole. As for the –CH2–NH– and – CH2–S– bridges, compounds 2 and 3 displayed improved inhibitory activity against three tested cancer cell lines with IC50 values of 3.51 lM. To our delight, the introduction of –CH2–O– bridge (compound 4) authorized the potential inhibitory against MCF-7 with IC50 value of 1.72 lM. Subsequently, the replacement of phenyl with heterocycles, including pyridine (5) and quinoline (6), generally resulted in loss in bioactivity on three cancer cell lines. Hence, we use compound 4 for further optimization. Furthermore, we decided to optimize the substituents of the phenyl ring, and the IC50 values were summarized in Table 2. First of all, compounds 7–9 with different substitutions (methoxycarbonyl, bromine and hydroxy, respectively) at the C-2 position showed drastically decreased or equivalent activity against lead compound 4, suggesting that the modification at C-2 position is not allowed. Then we turned our attention to the C-4 position of phenyl. The results in Table 2 show that a lipophilic group, ethyl (10), and polar groups, including amino (11), bromine (12) and
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O
H
O
H N O O
O
O
N H
N
R4
i
O
ii
N
N
Cl R4
N H 4f
24-30
6a HN
iii
N O
O
OH
O
O
O
N
HO N
N
N
v
iv N
O
R3
N O OH
OTs
6b
R3
7b
31,32
Scheme 4. Reagents and conditions: (i) ethanol, rt, 70%; (ii) Na(OAc)3BH, DCM, rt, 70–78%; (iii) ethanol, rt, 70%; (iv) TsCl, Et3N, DMAP, DCM, rt, 31%; (v) 60–71%.
OH
Br
Br
O
i NH 2
Br
ii N
O
iii
NO2
O N O
O
N
O
1g O
OH Br
2g
3g
O R N3
Cl iv
Br
N N
NO2
v
Br
NO2
vi R
N
Br
NO2
N
N
N 4g
5g
33-35
Scheme 5. Reagents and conditions: (i) triethyl orthoformate, 90 °C, 10 min, EtOH, 30 min, 66%; (ii) diphenyl oxide, 220 °C, 5 min, 83.5%; (iii) nitric acid, Propionic acid, 125 °C, 2 h, 50.2%; (iv) POCl3, triethylamine, reflux, 1 h, 68%; (v) NaN3, NMP, rt, 59%; (vi) t-BuOH, Cu, CuSO45H2O, 65 °C, 67–75%.
trifluoromethyl (13), led to the equivalent potent or slightly increased activity. However fluorine and methoxycarbonyl at the C-4 position endowed compounds (14 and 15, respectively) with prominent activity against all the three cancer cell lines as low as 0.66 lM, indicating that the hydrogen acceptor at the C-4 position of phenyl is essential to the activity. Next, we turned our attention to the substituents at the C-6 and C-7 positions of coumarin. Compared with the activity of compound 4, complete loss of antiproliferative activity were observed in Table 3 when the bromine was replaced with hydroxy (16) or 2-morpholinoethoxy (17). Nevertheless, when the bromine was replaced with hydrogen, compound 20, with fluorine at the C-4 position of phenyl, showed more potency than compound 14 in the MCF-7 and A549, indicating that bulky group may be unfavorable. Subsequently, with hydrogen at the C-6 position, the introduction of bulky group, 4-fluorobenzenesulfonate (18), to C-7 position led to total loss of potency, while the introduction of smaller group, methylmethanesulfonate (19), only caused the partial loss of the bioactivity, suggesting that small group at C-7 position may also be advantageous to the bioactivity. Then, methoxy introducing to the C-7 position of coumarin was taken into account. As might
have been expected, compound 23, with fluorine retained at the C-4 position of phenyl, showed higher bioactivity with IC50 values of 5.89, 1.99 and 0.52 lM against MCF-7, SW480 and A549, respectively. It is worth noting that compound 23 is more potent than compound 20, which can be ascribed to the additional hydrogen bond caused by the methoxy at C-7 position. Lastly, with hydrogen at C-7 position of coumarin and fluorine at the C-4 position of phenyl, it was discovered that the replacement of bromine with methoxy (21) or hydroxy (22) group at the C-6 position of coumarin resulted in the approximate activity to compound 14, reconfirming that there may be a steric hindrance at the C-6 region. To further measure the contribution of the triazole and coumarin motifs to the biological activity, a number of 6-bromo-4-(piperazin-1-yl)coumarin and 6-bromo-3-nitro-4-(1,2,3-triazol-1-yl) quinoline derivatives were prepared (Tables 4 and 5). First, the coumarin analogues substituted by piperazine exhibited enormous reduction of potency, especially compounds 24–30. However, the extended chemical bridge (compounds 31, and 32) between phenyl and piperazine could give a slight improvement in inhibitory activity, probably because the rotatable bonds can facilitate the compounds binding to the possible target. On the other hand,
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W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807 Table 1 In Vitro Inhibition of Tumor Cell Growth of compounds 1–6
R N N N Br O Compd
O
IC50a (lM)
R
1
MCF-7
SW480
A549
9.45 ± 0.55
6.66 ± 0.31
11.23 ± 2.0
>50
18.41 ± 3.5
13.2 ± 4.2
2
HN
3
S
36.83 ± 2.3
15.43 ± 0.33
3.51 ± 0.98
4
O
1.72 ± 0.64
4.99 ± 0.42
12.31 ± 0.11
5
O
39.82 ± 1.8
39.77 ± 2.4
24.79 ± 2.3
41.56 ± 1.4
16.95 ± 2.3
13.29 ± 4.9
3.51 ± 0.31
2.43 ± 0.25
1.65 ± 0.09
N N 6
O Br Doxorubicin a
Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard deviation (n = 3).
Table 2 In Vitro Inhibition of Tumor Cell Growth of compounds 7–15
R5 O N N N Br O
O
Compd
R5
IC50a (lM) MCF-7
SW480
A549
7 8 9 10 11 12 13 14 15 Doxorubicin
2-COOCH3 2-Br 2-OH 4-CH2CH3 4-NH2 4-Br 4-CF3 4-F 4-COOCH3
34.14 ± 2.2 28.91 ± 1.3 24.82 ± 0.89 12.67 ± 1.4 2.04 ± 1.1 1.92 ± 0.71 11.41 ± 2.3 7.00 ± 0.25 3.13 ± 0.24 3.51 ± 0.31
20.23 ± 3.4 20.13 ± 1.7 19.14 ± 3.3 10.82 ± 0.60 2.09 ± 0.13 1.31 ± 3.2 3.09 ± 2.0 1.77 ± 0.03 3.62 ± 0.25 2.43 ± 0.25
5.25 ± 0.24 8.43 ± 0.51 7.94 ± 3.4 11.58 ± 0.36 11.85 ± 0.24 24.47 ± 2.5 1.62 ± 1.1 0.85 ± 0.09 0.66 ± 0.03 1.65 ± 0.09
a Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard deviation (n = 3).
compounds 33–35, with coumarin replaced by quinoline, can maintain a part of antiproliferative activity, possibly due to the nitrogen atom of the quinolone which is similar to the oxygen atom of coumarin that can form hydrogen bond with the corresponding acceptor. The fact that the coumarin derivatives are more potent than quinoline can be interpreted by the additional
carbonyl group of coumarin, which may form another hydrogen bond with its possible receptor. Structure activity relationships (SAR) were summarized in Figure 2. Because compound 23 shows the highest antiproliferative activity among all the synthesized compounds, it was selected to assess the effect on cell-cycle arrest of MCF-7 by flow cytometry
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Table 3 In Vitro Inhibition of Tumor Cell Growth of compounds 16–23
R5 O N N N R1 R2
O
O IC50a (lM)
Compd
R1
R2
R5
MCF-7
SW480
A549
16
OH
H
H
>50
>50
>50
H
H
>50
>50
>50
H
>50
>50
>50
H
45.81 ± 1.4
10.67 ± 0.98
9.14 ± 0.58
4-F 4-F 4-F 4-F
5.84 ± 0.22 8.56 ± 0.23 4.62 ± 0.65 5.89 ± 0.14 3.51 ± 0.31
3.82 ± 0.40 25.87 ± 1.4 2.72 ± 0.30 1.99 ± 0.38 2.43 ± 0.25
0.68 ± 0.08 3.15 ± 0.39 2.01 ± 0.14 0.52 ± 0.21 1.65 ± 0.09
O N
17
O
O 18
O S
H
F
O O 19
O
H
S O
20 21 22 23 Doxorubicin
H OCH3 OH H
H H H OCH3
a Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard deviation (n = 3).
Table 4 In Vitro Inhibition of Tumor Cell Growth of compounds 24–32
R0 N N
O
Compd
R0
24
O
IC50a (lM) MCF-7
SW480
A549
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
F 25
26
HO
27
O
O 28
HO
805
W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807 Table 4 (continued) Compd
IC50a (lM)
R0
MCF-7
SW480
A549
>50
>50
>50
>50
>50
>50
24.20 ± 2.5
35.01 ± 5.0
21.58 ± 4.8
>50
24.01
11.92
3.51 ± 0.31
2.43 ± 0.25
1.65 ± 0.09
O 29
O
30
OH
O 31
O O 32
F Doxorubicin a
Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard deviation (n = 3).
Table 5 In Vitro Inhibition of Tumor Cell Growth of compounds 33–35
R5
X N N
N
Br
NO2 N
Compd
X
R5
33 34 35 Doxorubicin
S O O
H H COOEt
IC50a (lM) MCF-7 22.72 ± 3.3 7.69 ± 4.3 13.88 ± 1.3 3.51 ± 0.31
a Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard deviation (n = 3).
Figure 2. Structure-activity relationship revealed by optimization.
assay using propidium iodide (PI) staining. As is seen from Figure 3a, in comparison with the control group, treatment of compound 23 (5 lM) led to the obvious decrease in G1 phase (from 52.82% to 3.06%) and dramatic increase in G2 phase (from 15.78% to 84.21%), indicating that compound 23 is a G2/M cell-cycle arrester. A much stronger accumulation of cell in G2/M phase caused by compound 23 inspired us to test whether it can induce the apoptosis of A549 by flow cytometry analysis using annexin V-FITC and propidium iodide (PI) staining. The percentage of apoptotic cells induced by compound 23 (1.25 lM) is increased by more than 6-fold (Fig. 3b). All these results indicate that the promising antitumor activity of compound 23 may be attributed to the G2/M phase arrest and apoptosis. In summary, a family of 4-(1,2,3-triazol-1-yl)coumarin derivatives were synthesized and their antiproliferative activities were evaluated. Among these compounds, 23, 4-(4-((4-fluorophenoxy) methyl)-1,2,3-triazol-1-yl)-7-methoxycoumarin, exhibited excellent broad spectrum anticancer activity in vitro with IC50 values
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Figure 3. (a) Flow cytometry analyses of cell cycle distribution of breast cancer cell MCF-7 after treatment of compound 23 (5 lM) and no treatment (Ctrl) as reference control for 48 h. (b) Apoptosis induction in lung carcinoma cell A549 after 48 h treatment with 23 (1.25 lM) and no treatment.
of 5.89, 1.99 and 0.52 lM against MCF-7, SW480 and A549, respectively. The further mechanism study demonstrated that compound 23 could obviously inhibit the proliferation of cancer cells through inducing apoptosis and arresting the cell-cycle at G2/M phase. In addition, structure activity relationship research revealed that – CH2–O– bridge at C-4 position of 1,2,3-triazole core is the best optimal for bioactivity; a hydrogen bond acceptor at C-4 position of phenyl is indispensable for the improvement of potency; the hydrogen bond acceptor at C-7 position of coumarin can make a positive contribution to the activity. All the conclusions have encouraged us to continue the development, and the further validation of mechanism at the molecular level is undergoing. Acknowledgments The authors are grateful for the support from the National Science Foundation of China (30901743), the National Key Program of China (2012ZX09103101-022), the State Key Laboratory of Drug Research and Doctoral Fund of Ministry of Education. Supplementary data Supplementary data (experimental procedures and characterizationdata of compounds) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl. 2013.12.095.
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