Bioorganic & Medicinal Chemistry Letters 25 (2015) 431–434
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Identification of indole-3-carboxylic acids as non-ATP-competitive Polo-like kinase 1 (Plk1) inhibitors Meng Liu a,b, Jie Huang c, Dong-Xing Chen a,b, Cheng Jiang a,b,⇑ a
Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China Department of Medicinal Chemistry, School of Pharmacy,China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China c Gansu Institute for Food and Drug Control, Yinan road 7, Lanzhou 730070, China b
a r t i c l e
i n f o
Article history: Received 28 September 2014 Revised 2 December 2014 Accepted 18 December 2014 Available online 24 December 2014 Keywords: Polo-like kinase 1 Non-ATP-competitive Inhibitors Structure–activity relationship
a b s t r a c t A series of indole-3-carboxylic acids were designed as novel small molecular non-ATP-competitive Plk1 inhibitors. The designed compounds were synthesized and evaluated. Most of the targeted compounds showed potent Plk1 inhibitory activities and anti-proliferative characters. Particularly, 4f and 4g showed Plk1 inhibitory activity with IC50 values of 0.41 and 0.13 lM, which were about 5 and 17 times more potent compared to thymoquinone, respectively. Compound 4g also showed inhibitory activity to HeLa and MCF-7 cell lines with IC50 values of 0.72 and 1.15 lM, which was almost 3 and 4 times more potent than thymoquinone. Study of mechanism of action suggested that 4g was an ATP-independent and substrate-dependent Plk1 inhibitor. Moreover, 4g showed excellent Plk1 inhibitory selectivity against Plk2 and Plk3. Fluorescein isothiocyanate Annexin V/propidium iodide (PI) double-staining assay and western-blot results indicate that induction of apoptosis by 4g is involved in its anti-tumor activity. This study may provide a support for further optimization of non-ATP-competitive Plk1 inhibitors. Ó 2014 Elsevier Ltd. All rights reserved.
The serine/threonine kinase Polo-like kinase 1 (Plk1) is a member of the polo-like kinases family which is overexpressed in many types of human cancers. Plk1 acts as a regulator in multiple stages of mitotic progression and is considered an attractive anti-cancer drug target due to its ability to promote tumorigenesis.1 For a long period, several potent ATP-competitive Plk1 inhibitors targeting the ATP-binding catalytic domain have been reported.1 However, the ATP-binding sites of the catalytic domains of protein kinases are closely related, these efforts suffered from a lack of target specificity. Thus discovery of non-ATP-competitive Plk1 inhibitors targeting other binding pockets rather than the ATP-binding domain is more suited to the exploration of the feasibility of inhibiting this kinase. Several series of peptides have been designed and synthesized as non-ATP-competitive Plk1 inhibitors with high affinity and specificity.2–8 However, these peptides suffered from less drugablity. A lot of small molecular non-ATP-competitive Plk1 inhibitors were also reported. Some of the small molecular non-ATP-competitive Plk1 inhibitors were thymoquinone and its derivatives such as poloxin and poloxime (Fig. 1) with moderate Plk1 inhibitory activity.9–11 ON01910 and I2 were disclosed as potent non-ATP-competitive Plk1 inhibitors by screen, which showed potent antitumor ⇑ Corresponding author. E-mail address: [email protected] (C. Jiang). http://dx.doi.org/10.1016/j.bmcl.2014.12.060 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
O
O N
N
OH
O
O
O O
Thymoquinone
Poloxin
Poloxime(PXE)
Figure 1. Chemical structures of thymoquinone and its derivatives.
activity both in vitro and in vivo (Fig. 2).12,13 A series of benzimidazole and indole derivatives (e.g., 5i) were identified by our group as potent non-ATP-competitive Plk1 inhibitors (Fig. 2).14 To our knowledge, only a limited number of small molecular nonATP-competitive Plk1 inhibitors have been reported. Herein, we would like to report the discovery of a series of small molecular non-ATP-competitive Plk1 inhibitors with a new chemical scaffold. Compound CJ-054 was obtained from the screening of a series of in-house compounds. The IC50 value of CJ-054 was 1.82 lM in inhibition of plk1. In order to obtain potent non-ATP-competitive Plk1 inhibitors, we modified CJ-054 based on the structure of reported non-ATP-competitive Plk1 inhibitors as displayed in Figure 2.
432
M. Liu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 431–434 N S
H N O
O N O
O I2 N
OH N
O N
N H
O
CJ-054 N
N
OH
O
5i
COOH O
O
H S H O O
O
O
HN COOH ON010910
Figure 2. Design of new small molecular Plk1 inhibitors by modification of CJ-054.
Analysis of several classes of reported non-ATP-competitive small molecular Plk1 inhibitors showed that their structures contain three parts: the head group (in red), a proper linker (in green) and the tail (in blue) (Fig. 2). The head group and tail were usually hydrophobic groups and the length of the linker was 3 to 4 atoms. In the chemical structure of CJ-054, the indole core could be looked as head group while the phenyl and the piperidine could both act as the tail. Thus one of the phenyl and the piperidine could be removed. Among the reported non-ATP-competitive small molecular Plk1 inhibitors, ON01910 was the most potent (Plk1 IC50 = 9 nM).12 There is a residue with terminal carboxylic acid attached to the phenyl ring as the head group (Fig. 2). Based on that, a side chain with terminal carboxylic acid could be introduced to the indole core of CJ-054. In this report, the piperidine group was kept as the tail part and another side chain containing phenyl group was replaced by an additional side chain with terminal carboxylic acid.
Thus a series of indole-3-carboxylic acids were designed as shown in Table 1. The synthesis of designed compounds is provided in Scheme 1. Commercially available methyl indole-3-carboxic acid, methyl indole-3-acetate or methyl indole-3-propanoate (1) was reacted with 1,2-epoxy-3-chloropropane in DMSO to give epoxide 2. Epoxide 2 was reacted with proper second-amine in methanol to give 3. Then target compound 4 (Table 1) was obtained after hydrolysis of 3 under the condition of sodium methoxide in methanol. Target compounds were all synthesized as racemic mixtures. Designed compounds were synthesized and evaluated using Plk1 inhibitory assays and in vitro cell lines proliferation inhibition assays.12 Thymoquinone, a reported natural product as non-ATPcompetitive Plk1 inhibitor was used as control. As shown in Table 1, compounds with acetic acid residue at 3-position of indole core show better Plk1 inhibitory characters than compounds with
Table 1 In vitro Plk1 and cancer cell lines growth inhibition of 4 COOH n N N
R1 R2
OH
a
Compound
n
-NR1R2
Plk1 inhibition, IC50 ± SDa (lM)
4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k Thymoquinone
0 0 0 0 1 1 1 1 1 2 2 —
DiethylaminoPiperdin-1Pyrrolidin-14-CH3-piperazin-1DiethylaminoPiperdin-1Pyrrolidin-14-CH3-piperazin-1Morphilin-4Piperdin-1Pyrrolidin-1—
1.91 ± 0.66 2.10 ± 0.27 4.84 ± 1.04 3.47 ± 0.82 2.64 ± 0.19 0.41 ± 0.09 0.13 ± 0.02 1.88 ± 0.71 0.96 ± 0.14 5.71 ± 0.89 2.90 ± 0.51 2.18 ± 0.31
Values are the average of three independent experiments.
Cell growth inhibition, IC50 ± SDa (lM) HeLa
MCF-7
6.72 ± 1.25 4.92 ± 1.19 7.34 ± 2.46 >10 6.63 ± 2.07 0.97 ± 0.21 0.72 ± 0.13 3.97 ± 0.92 2.13 ± 0.49 >10 4.95 ± 1.78 2.06 ± 0.27
4.37 ± 0.93 5.33 ± 0.98 >10 >10 3.16 ± 0.84 1.89 ± 0.44 1.15 ± 0.37 2.44 ± 0.83 >10 8.79 ± 1.60 >10 6.19 ± 0.97
433
M. Liu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 431–434
O n O
n a N
N H
O
O
n
O b
N
O
OH
c, d
N
OH R1 N R2
1
O
R
N R2
1
2
3
OH
n
O
4
Scheme 1. Synthetic route of the designed compounds. Reagents and conditions: (a) 1,2-epoxy-3-chloropropane, NaH, DMSO, rt, 12 h, 19–34%; (b) HN(R1R2), MeOH, reflux, 6 h, 34–92%; (c) NaOMe, MeOH, reflux, 15 h; (d) 36% HCl, 4–56% yield for c and d steps.
Table 2 In vitro Plks kinase inhibitory profile of 4g
a
Compound
Plk1 inhibition, IC50 ± SDa (lM)
Plk2 inhibition, IC50 ± SDa (lM)
Plk3 inhibition, IC50 ± SDa (lM)
4g Thymoquinone
0.13 ± 0.02 2.18 ± 0.31
>50 14.72 ± 4.07
>50 >50
Values are the average of three independent experiments.
formic acid or propanoic acid residues. This result confirms that the distance between the carboxylic acid and the indole core may be important for Plk1 inhibitory activity. Among the indole3-acetic acid compounds, compounds with 4-methyl piperazine (4h) and morphiline (4i) at the side chain showed similar Plk1 inhibitory activities comparing to thymoquinone and the hit compound CJ-054. While compounds with piperdine (4f) and pyrrolidine (4g) at the side chain exhibit very potent Plk1 inhibitory activities. Compound 4f and 4g showed Plk1 inhibitory activity with an IC50 value of 0.41 and 0.13 lM, about 5 and 17 times more potent than thymoquinone. In particular, compound 4g showed inhibitory activity to HeLa and MCF-7 with an IC50 value of 0.72
and 1.15 lM, which was about 3 and 4 times more potent compared to thymoquinone. A selectivity test for 4g against two closely related kinases Plk2 and 3 was then carried out using the protocol described in support information. These two kinases were available as recombinant proteins purchased from commercial sources (Abnova Inc.). As shown in Table 2, thymoquinone showed excellent Plk1 inhibitory selectivity against Plk3 (IC50 >50 lM) and moderate inhibitory selectivity against Plk2 (IC50 = 14.72 ± 4.07 lM, about 7-fold higher than Plk1 IC50). Compound 4g showed excellent Plk1 inhibitory selectivity against Plk2 (IC50 >50 lM) and Plk3 (IC50 >50 lM). This result proved that the 4g was a highly selective inhibitor of Plk1. The effects of increasing concentrations of ATP or the substrate Cdc25C on the inhibitory activity of the compound were examined using the method reported by Gumireddy et al.12 Ten nanograms of recombinant Plk1 was mixed with different concentrations of 4g, followed by the addition of a reaction mixture containing c32P-ATP and varying concentrations of ATP (20, 60 and 120 lM). The values from individual samples were analyzed and plotted as a function of inhibitor concentration using Prism 4 Graphpad software. The results were displayed as Figure 3. These analyses
Figure 3. Character of Plk1 inhibition by 4g, (A) IC50 curves for Plk1 with different concentrations of 4g in the presence of varying concentrations of ATP. (B) IC50 curves for Plk1 with different concentrations of 4g in the presence of varying concentrations of Cdc25C substrate.
Figure 4. The apoptotic effects of 4g in HeLa cells. (A) Induction of apoptosis, measured by Annexin V/PI assay following treatment with 4g (1 lM) for 24 h. Control cells were treated with DMSO alone. (B) Effect on PARP and Caspase-3 in HeLa cells after treatment with 4g (1 lM) for 24 h and continued to 48 h.
434
M. Liu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 431–434
showed that the IC50 values for the inhibitor were similar in the presence of increasing concentrations of ATP, suggesting that 4g is not an ATP-competitive inhibitor (Fig. 3A). Then the effects of increasing concentrations of substrate Cdc25C (100, 200, 400 and 800 ng) on the inhibitory activity of the compound in the presence of a constant amount of ATP (100 lM) were examined. It was shown that increasing the concentration of the substrate resulted in increased IC50 values of the inhibitor (Fig. 3B, IC50 curves shift right). Thus 4g demonstrates the substrate-dependent and ATPindependent nature of inhibition. This result may also confirm that 4g binds to the binding pocket of Cdc25C rather than the ATP binding pocket. To further confirm the apoptosis induced by 4g, flow cytometry with the fluorescein isothiocyanate Annexin V/propidium iodide (PI) double-staining assay was carried out to evaluate whether 4g-induced cell death was due to apoptosis or necrosis. As shown in Fig. 4A, after treated with1 lM 4g for 24 h, the early and median apoptotic cells (right low section of fluorocytogram) represented 12.7% of the total cells compared to the DMSO group with only 4.4%. To examine the apoptotic events associated with 4g-induced apoptosis, we analyzed the expression of the apoptotic proteins. Results showed that the apoptotic protein PARP cleavage were activated significantly after the treatment of 4g (1 lM) for 24 h and continued to 48 h (Fig. 4B). The Caspase protein is a member of the cysteine-aspartic acid protease (Caspase) family. Sequential activation of Caspase-3 plays a central role in the phase of cell apoptosis. In our assays we found that the expression of Caspase-3 protein did not change much, however the active form of Caspase-3, cleaved Caspase-3 were significant increased from 24 h to 48 h after the treatment of 4g (1 lM). These results indicate that induction of apoptosis by 4g is involved in its anti-tumor activity. In this study, a series of indole-3-carboxylic acids were designed as new Plk1 inhibitors based on the structure of reported non-ATPcompetitive Plk1 inhibitors. Two indole-3-acetic acids (4f and 4g) showed more potent Plk1 inhibitory activity together with potent anti-proliferative activity comparing to thymoquinone. The mechanism of action of the optimal compound 4g were also examined, which suggested that 4g was an ATP-independent (non-ATPcompetitive) and substrate-dependent Plk1 inhibitor. In the
specificity test, 4g showed excellent selectivity to Plk1 against Plk2 and 3. Fluorescein isothiocyanate Annexin V/propidium iodide (PI) double-staining assay and western-blot results indicate that 4g could be an effective apoptosis inducer in HeLa cells. Our study may be a good starting point for further discovery of highly selective non-ATP-competitive Plk1 inhibitors. Acknowledgments This work was financially supported by Jiangsu Province Natural Science Foundation (No. BK20110623) and ‘Qinglan project’ of Jiangsu province. References and notes 1. Strebhardt, K.; Ullrich, A. Nat. Rev. Cancer 2006, 6, 321. 2. Kang, Y. H.; Park, J. E.; Yu, L. R.; Soung, K. K.; Yun, S. M.; Bang, J. K.; Seong, Y. S.; Yu, H.; Garfield, S.; Veenstra, T. D.; Lee, K. S. Mol. Cell 2006, 24, 409. 3. Yun, S.-M.; Moulaei, T.; Lim, D.; Bang, J. K.; Park, J.-E.; Shenoy Shilpa, R.; Liu, F.; Kang, Y. H.; Liao, C.; Soung, N.-K.; Lee, S.; Yoon, D.-Y.; Lim, Y.; Lee, D.-H.; Otaka, A.; Appella, E.; McMahon, J. B.; Nicklaus, M. C.; Burke, T. R., Jr.; Yaffe, M. B.; Wlodawer, A.; Lee, K. S. Nat. Struct. Mol. Biol. 2009, 16, 876. 4. Liu, F.; Park, J.-E.; Qian, W.-J.; Lim, D.; Garber, M.; Berg, T.; Yaffe, M. B.; Lee, K. S., ; Burke, T. R., Jr. Nat. Chem. Biol. 2011, 7, 595. 5. Liu, F.; Park, J.-E.; Qian, W.-J.; Lim, D.; Scharow, A.; Berg, T.; Yaffe, M. B.; Lee, K. S., ; Burke, T. R., Jr. ACS Chem. Biol. 2012, 7, 805. 6. Liu, F.; Park, J.-E.; Qian, W.-J.; Lim, D.; Scharow, A.; Berg, T.; Yaffe, M. B.; Lee, K. S., ; Burke, T. R., Jr. ChemBioChem 2012, 13, 1291. 7. Qian, W. J.; Park, J. E.; Lee, K. S.; Burke, T. R., Jr. Bioorg. Med. Chem. Lett. 2012, 15, 7306. 8. Murugan, R. N.; Park, J.; Lim, D.; Ahn, M.; Cheong, C.; Kwon, T.; Nam, K. Y.; Choi, S. H.; Kim, B. Y.; Yoon, D. Y.; Yaffe, M. B.; Yu, D. Y.; Lee, K. S.; Bang, J. K. Bioorg. Med. Chem. 2013, 21, 2623. 9. Reindl, W.; Yuan, J.; Kramer, A.; Strebhardt, K.; Berg, T. Chem. Biol. 2008, 15, 459. 10. Yin, Z.; Song, Y.; Rehse, P. H. ACS Chem. Biol. 2013, 8, 303. 11. Yuan, J.; Sanhaji, M.; Kramer, A.; Reindl, W.; Hofmann, M.; Kreis, N. N.; Zimmer, B.; Berg, T.; Strebhardt, K. Am. J. Pathol. 2011, 179, 2091. 12. Gumireddy, K.; Reddy, M. V. R.; Cosenza, S. C.; Boominathan, R.; Baker, S. J.; Papathi, N.; Jiang, J.; Holland, J.; Reddy, E. P. Cancer Cell 2005, 7, 275. 13. Lu, J.; Xin, S.; Meng, H.; Veldman, M.; Schoenfeld, D.; Che, C.; Yan, R.; Zhong, H.; Li, S.; Lin, S. PLoS ONE 2013, 8, e53317. 14. Chen, D.; Huang, J.; Liu, M.; Xu, Y.; Jiang, C. Arch. Pharm. 2014. http://dx.doi.org/ 10.1002/ardp.201400294.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Identification of indole-3-carboxylic acids as non-ATP-competitive Polo-like kinase 1 (Plk1) inhibitors Meng Liu a,b, Jie Huang c, Dong-Xing Chen a,b, Cheng Jiang a,b,⇑ a
Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China Department of Medicinal Chemistry, School of Pharmacy,China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China c Gansu Institute for Food and Drug Control, Yinan road 7, Lanzhou 730070, China b
a r t i c l e
i n f o
Article history: Received 28 September 2014 Revised 2 December 2014 Accepted 18 December 2014 Available online 24 December 2014 Keywords: Polo-like kinase 1 Non-ATP-competitive Inhibitors Structure–activity relationship
a b s t r a c t A series of indole-3-carboxylic acids were designed as novel small molecular non-ATP-competitive Plk1 inhibitors. The designed compounds were synthesized and evaluated. Most of the targeted compounds showed potent Plk1 inhibitory activities and anti-proliferative characters. Particularly, 4f and 4g showed Plk1 inhibitory activity with IC50 values of 0.41 and 0.13 lM, which were about 5 and 17 times more potent compared to thymoquinone, respectively. Compound 4g also showed inhibitory activity to HeLa and MCF-7 cell lines with IC50 values of 0.72 and 1.15 lM, which was almost 3 and 4 times more potent than thymoquinone. Study of mechanism of action suggested that 4g was an ATP-independent and substrate-dependent Plk1 inhibitor. Moreover, 4g showed excellent Plk1 inhibitory selectivity against Plk2 and Plk3. Fluorescein isothiocyanate Annexin V/propidium iodide (PI) double-staining assay and western-blot results indicate that induction of apoptosis by 4g is involved in its anti-tumor activity. This study may provide a support for further optimization of non-ATP-competitive Plk1 inhibitors. Ó 2014 Elsevier Ltd. All rights reserved.
The serine/threonine kinase Polo-like kinase 1 (Plk1) is a member of the polo-like kinases family which is overexpressed in many types of human cancers. Plk1 acts as a regulator in multiple stages of mitotic progression and is considered an attractive anti-cancer drug target due to its ability to promote tumorigenesis.1 For a long period, several potent ATP-competitive Plk1 inhibitors targeting the ATP-binding catalytic domain have been reported.1 However, the ATP-binding sites of the catalytic domains of protein kinases are closely related, these efforts suffered from a lack of target specificity. Thus discovery of non-ATP-competitive Plk1 inhibitors targeting other binding pockets rather than the ATP-binding domain is more suited to the exploration of the feasibility of inhibiting this kinase. Several series of peptides have been designed and synthesized as non-ATP-competitive Plk1 inhibitors with high affinity and specificity.2–8 However, these peptides suffered from less drugablity. A lot of small molecular non-ATP-competitive Plk1 inhibitors were also reported. Some of the small molecular non-ATP-competitive Plk1 inhibitors were thymoquinone and its derivatives such as poloxin and poloxime (Fig. 1) with moderate Plk1 inhibitory activity.9–11 ON01910 and I2 were disclosed as potent non-ATP-competitive Plk1 inhibitors by screen, which showed potent antitumor ⇑ Corresponding author. E-mail address: [email protected] (C. Jiang). http://dx.doi.org/10.1016/j.bmcl.2014.12.060 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
O
O N
N
OH
O
O
O O
Thymoquinone
Poloxin
Poloxime(PXE)
Figure 1. Chemical structures of thymoquinone and its derivatives.
activity both in vitro and in vivo (Fig. 2).12,13 A series of benzimidazole and indole derivatives (e.g., 5i) were identified by our group as potent non-ATP-competitive Plk1 inhibitors (Fig. 2).14 To our knowledge, only a limited number of small molecular nonATP-competitive Plk1 inhibitors have been reported. Herein, we would like to report the discovery of a series of small molecular non-ATP-competitive Plk1 inhibitors with a new chemical scaffold. Compound CJ-054 was obtained from the screening of a series of in-house compounds. The IC50 value of CJ-054 was 1.82 lM in inhibition of plk1. In order to obtain potent non-ATP-competitive Plk1 inhibitors, we modified CJ-054 based on the structure of reported non-ATP-competitive Plk1 inhibitors as displayed in Figure 2.
432
M. Liu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 431–434 N S
H N O
O N O
O I2 N
OH N
O N
N H
O
CJ-054 N
N
OH
O
5i
COOH O
O
H S H O O
O
O
HN COOH ON010910
Figure 2. Design of new small molecular Plk1 inhibitors by modification of CJ-054.
Analysis of several classes of reported non-ATP-competitive small molecular Plk1 inhibitors showed that their structures contain three parts: the head group (in red), a proper linker (in green) and the tail (in blue) (Fig. 2). The head group and tail were usually hydrophobic groups and the length of the linker was 3 to 4 atoms. In the chemical structure of CJ-054, the indole core could be looked as head group while the phenyl and the piperidine could both act as the tail. Thus one of the phenyl and the piperidine could be removed. Among the reported non-ATP-competitive small molecular Plk1 inhibitors, ON01910 was the most potent (Plk1 IC50 = 9 nM).12 There is a residue with terminal carboxylic acid attached to the phenyl ring as the head group (Fig. 2). Based on that, a side chain with terminal carboxylic acid could be introduced to the indole core of CJ-054. In this report, the piperidine group was kept as the tail part and another side chain containing phenyl group was replaced by an additional side chain with terminal carboxylic acid.
Thus a series of indole-3-carboxylic acids were designed as shown in Table 1. The synthesis of designed compounds is provided in Scheme 1. Commercially available methyl indole-3-carboxic acid, methyl indole-3-acetate or methyl indole-3-propanoate (1) was reacted with 1,2-epoxy-3-chloropropane in DMSO to give epoxide 2. Epoxide 2 was reacted with proper second-amine in methanol to give 3. Then target compound 4 (Table 1) was obtained after hydrolysis of 3 under the condition of sodium methoxide in methanol. Target compounds were all synthesized as racemic mixtures. Designed compounds were synthesized and evaluated using Plk1 inhibitory assays and in vitro cell lines proliferation inhibition assays.12 Thymoquinone, a reported natural product as non-ATPcompetitive Plk1 inhibitor was used as control. As shown in Table 1, compounds with acetic acid residue at 3-position of indole core show better Plk1 inhibitory characters than compounds with
Table 1 In vitro Plk1 and cancer cell lines growth inhibition of 4 COOH n N N
R1 R2
OH
a
Compound
n
-NR1R2
Plk1 inhibition, IC50 ± SDa (lM)
4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k Thymoquinone
0 0 0 0 1 1 1 1 1 2 2 —
DiethylaminoPiperdin-1Pyrrolidin-14-CH3-piperazin-1DiethylaminoPiperdin-1Pyrrolidin-14-CH3-piperazin-1Morphilin-4Piperdin-1Pyrrolidin-1—
1.91 ± 0.66 2.10 ± 0.27 4.84 ± 1.04 3.47 ± 0.82 2.64 ± 0.19 0.41 ± 0.09 0.13 ± 0.02 1.88 ± 0.71 0.96 ± 0.14 5.71 ± 0.89 2.90 ± 0.51 2.18 ± 0.31
Values are the average of three independent experiments.
Cell growth inhibition, IC50 ± SDa (lM) HeLa
MCF-7
6.72 ± 1.25 4.92 ± 1.19 7.34 ± 2.46 >10 6.63 ± 2.07 0.97 ± 0.21 0.72 ± 0.13 3.97 ± 0.92 2.13 ± 0.49 >10 4.95 ± 1.78 2.06 ± 0.27
4.37 ± 0.93 5.33 ± 0.98 >10 >10 3.16 ± 0.84 1.89 ± 0.44 1.15 ± 0.37 2.44 ± 0.83 >10 8.79 ± 1.60 >10 6.19 ± 0.97
433
M. Liu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 431–434
O n O
n a N
N H
O
O
n
O b
N
O
OH
c, d
N
OH R1 N R2
1
O
R
N R2
1
2
3
OH
n
O
4
Scheme 1. Synthetic route of the designed compounds. Reagents and conditions: (a) 1,2-epoxy-3-chloropropane, NaH, DMSO, rt, 12 h, 19–34%; (b) HN(R1R2), MeOH, reflux, 6 h, 34–92%; (c) NaOMe, MeOH, reflux, 15 h; (d) 36% HCl, 4–56% yield for c and d steps.
Table 2 In vitro Plks kinase inhibitory profile of 4g
a
Compound
Plk1 inhibition, IC50 ± SDa (lM)
Plk2 inhibition, IC50 ± SDa (lM)
Plk3 inhibition, IC50 ± SDa (lM)
4g Thymoquinone
0.13 ± 0.02 2.18 ± 0.31
>50 14.72 ± 4.07
>50 >50
Values are the average of three independent experiments.
formic acid or propanoic acid residues. This result confirms that the distance between the carboxylic acid and the indole core may be important for Plk1 inhibitory activity. Among the indole3-acetic acid compounds, compounds with 4-methyl piperazine (4h) and morphiline (4i) at the side chain showed similar Plk1 inhibitory activities comparing to thymoquinone and the hit compound CJ-054. While compounds with piperdine (4f) and pyrrolidine (4g) at the side chain exhibit very potent Plk1 inhibitory activities. Compound 4f and 4g showed Plk1 inhibitory activity with an IC50 value of 0.41 and 0.13 lM, about 5 and 17 times more potent than thymoquinone. In particular, compound 4g showed inhibitory activity to HeLa and MCF-7 with an IC50 value of 0.72
and 1.15 lM, which was about 3 and 4 times more potent compared to thymoquinone. A selectivity test for 4g against two closely related kinases Plk2 and 3 was then carried out using the protocol described in support information. These two kinases were available as recombinant proteins purchased from commercial sources (Abnova Inc.). As shown in Table 2, thymoquinone showed excellent Plk1 inhibitory selectivity against Plk3 (IC50 >50 lM) and moderate inhibitory selectivity against Plk2 (IC50 = 14.72 ± 4.07 lM, about 7-fold higher than Plk1 IC50). Compound 4g showed excellent Plk1 inhibitory selectivity against Plk2 (IC50 >50 lM) and Plk3 (IC50 >50 lM). This result proved that the 4g was a highly selective inhibitor of Plk1. The effects of increasing concentrations of ATP or the substrate Cdc25C on the inhibitory activity of the compound were examined using the method reported by Gumireddy et al.12 Ten nanograms of recombinant Plk1 was mixed with different concentrations of 4g, followed by the addition of a reaction mixture containing c32P-ATP and varying concentrations of ATP (20, 60 and 120 lM). The values from individual samples were analyzed and plotted as a function of inhibitor concentration using Prism 4 Graphpad software. The results were displayed as Figure 3. These analyses
Figure 3. Character of Plk1 inhibition by 4g, (A) IC50 curves for Plk1 with different concentrations of 4g in the presence of varying concentrations of ATP. (B) IC50 curves for Plk1 with different concentrations of 4g in the presence of varying concentrations of Cdc25C substrate.
Figure 4. The apoptotic effects of 4g in HeLa cells. (A) Induction of apoptosis, measured by Annexin V/PI assay following treatment with 4g (1 lM) for 24 h. Control cells were treated with DMSO alone. (B) Effect on PARP and Caspase-3 in HeLa cells after treatment with 4g (1 lM) for 24 h and continued to 48 h.
434
M. Liu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 431–434
showed that the IC50 values for the inhibitor were similar in the presence of increasing concentrations of ATP, suggesting that 4g is not an ATP-competitive inhibitor (Fig. 3A). Then the effects of increasing concentrations of substrate Cdc25C (100, 200, 400 and 800 ng) on the inhibitory activity of the compound in the presence of a constant amount of ATP (100 lM) were examined. It was shown that increasing the concentration of the substrate resulted in increased IC50 values of the inhibitor (Fig. 3B, IC50 curves shift right). Thus 4g demonstrates the substrate-dependent and ATPindependent nature of inhibition. This result may also confirm that 4g binds to the binding pocket of Cdc25C rather than the ATP binding pocket. To further confirm the apoptosis induced by 4g, flow cytometry with the fluorescein isothiocyanate Annexin V/propidium iodide (PI) double-staining assay was carried out to evaluate whether 4g-induced cell death was due to apoptosis or necrosis. As shown in Fig. 4A, after treated with1 lM 4g for 24 h, the early and median apoptotic cells (right low section of fluorocytogram) represented 12.7% of the total cells compared to the DMSO group with only 4.4%. To examine the apoptotic events associated with 4g-induced apoptosis, we analyzed the expression of the apoptotic proteins. Results showed that the apoptotic protein PARP cleavage were activated significantly after the treatment of 4g (1 lM) for 24 h and continued to 48 h (Fig. 4B). The Caspase protein is a member of the cysteine-aspartic acid protease (Caspase) family. Sequential activation of Caspase-3 plays a central role in the phase of cell apoptosis. In our assays we found that the expression of Caspase-3 protein did not change much, however the active form of Caspase-3, cleaved Caspase-3 were significant increased from 24 h to 48 h after the treatment of 4g (1 lM). These results indicate that induction of apoptosis by 4g is involved in its anti-tumor activity. In this study, a series of indole-3-carboxylic acids were designed as new Plk1 inhibitors based on the structure of reported non-ATPcompetitive Plk1 inhibitors. Two indole-3-acetic acids (4f and 4g) showed more potent Plk1 inhibitory activity together with potent anti-proliferative activity comparing to thymoquinone. The mechanism of action of the optimal compound 4g were also examined, which suggested that 4g was an ATP-independent (non-ATPcompetitive) and substrate-dependent Plk1 inhibitor. In the
specificity test, 4g showed excellent selectivity to Plk1 against Plk2 and 3. Fluorescein isothiocyanate Annexin V/propidium iodide (PI) double-staining assay and western-blot results indicate that 4g could be an effective apoptosis inducer in HeLa cells. Our study may be a good starting point for further discovery of highly selective non-ATP-competitive Plk1 inhibitors. Acknowledgments This work was financially supported by Jiangsu Province Natural Science Foundation (No. BK20110623) and ‘Qinglan project’ of Jiangsu province. References and notes 1. Strebhardt, K.; Ullrich, A. Nat. Rev. Cancer 2006, 6, 321. 2. Kang, Y. H.; Park, J. E.; Yu, L. R.; Soung, K. K.; Yun, S. M.; Bang, J. K.; Seong, Y. S.; Yu, H.; Garfield, S.; Veenstra, T. D.; Lee, K. S. Mol. Cell 2006, 24, 409. 3. Yun, S.-M.; Moulaei, T.; Lim, D.; Bang, J. K.; Park, J.-E.; Shenoy Shilpa, R.; Liu, F.; Kang, Y. H.; Liao, C.; Soung, N.-K.; Lee, S.; Yoon, D.-Y.; Lim, Y.; Lee, D.-H.; Otaka, A.; Appella, E.; McMahon, J. B.; Nicklaus, M. C.; Burke, T. R., Jr.; Yaffe, M. B.; Wlodawer, A.; Lee, K. S. Nat. Struct. Mol. Biol. 2009, 16, 876. 4. Liu, F.; Park, J.-E.; Qian, W.-J.; Lim, D.; Garber, M.; Berg, T.; Yaffe, M. B.; Lee, K. S., ; Burke, T. R., Jr. Nat. Chem. Biol. 2011, 7, 595. 5. Liu, F.; Park, J.-E.; Qian, W.-J.; Lim, D.; Scharow, A.; Berg, T.; Yaffe, M. B.; Lee, K. S., ; Burke, T. R., Jr. ACS Chem. Biol. 2012, 7, 805. 6. Liu, F.; Park, J.-E.; Qian, W.-J.; Lim, D.; Scharow, A.; Berg, T.; Yaffe, M. B.; Lee, K. S., ; Burke, T. R., Jr. ChemBioChem 2012, 13, 1291. 7. Qian, W. J.; Park, J. E.; Lee, K. S.; Burke, T. R., Jr. Bioorg. Med. Chem. Lett. 2012, 15, 7306. 8. Murugan, R. N.; Park, J.; Lim, D.; Ahn, M.; Cheong, C.; Kwon, T.; Nam, K. Y.; Choi, S. H.; Kim, B. Y.; Yoon, D. Y.; Yaffe, M. B.; Yu, D. Y.; Lee, K. S.; Bang, J. K. Bioorg. Med. Chem. 2013, 21, 2623. 9. Reindl, W.; Yuan, J.; Kramer, A.; Strebhardt, K.; Berg, T. Chem. Biol. 2008, 15, 459. 10. Yin, Z.; Song, Y.; Rehse, P. H. ACS Chem. Biol. 2013, 8, 303. 11. Yuan, J.; Sanhaji, M.; Kramer, A.; Reindl, W.; Hofmann, M.; Kreis, N. N.; Zimmer, B.; Berg, T.; Strebhardt, K. Am. J. Pathol. 2011, 179, 2091. 12. Gumireddy, K.; Reddy, M. V. R.; Cosenza, S. C.; Boominathan, R.; Baker, S. J.; Papathi, N.; Jiang, J.; Holland, J.; Reddy, E. P. Cancer Cell 2005, 7, 275. 13. Lu, J.; Xin, S.; Meng, H.; Veldman, M.; Schoenfeld, D.; Che, C.; Yan, R.; Zhong, H.; Li, S.; Lin, S. PLoS ONE 2013, 8, e53317. 14. Chen, D.; Huang, J.; Liu, M.; Xu, Y.; Jiang, C. Arch. Pharm. 2014. http://dx.doi.org/ 10.1002/ardp.201400294.
