Bioorganic & Medicinal Chemistry Letters 24 (2014) 4659–4663

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Discovery of (E)-5-(benzylideneamino)-1H-benzo[d]imidazol-2(3H)-one derivatives as inhibitors for PTK6

http://dx.doi.org/10.1016/j.bmcl.2014.08.0360960-894X/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Tel.: +82 31 290 7711; fax: +82 31 290 7773 (Y.H.J.);tel.: +82 2 2123 2703; fax: +82 2 362 9897 (S.-T.L.).

E-mail addresses: yhjung@skku.edu (Y.H. Jung), stlee@yonsei.ac.kr (S.-T. Lee).� Present address: Department of Life Science and Biotechnology, Shingyeong

University, Hwaseong, Gyeonggi-do, 445-741, Republic of Korea.

Hyun Jae Shim a, Hye Ran Yang b, Han Ie Kim a,�, Shin-Ae Kang a, Kyoung Tai No c,d, Young Hoon Jung b,⇑,Seung-Taek Lee a,⇑a Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Koreab School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Koreac Bioinformatics and Molecular Design Research Center, 120-749 Seoul, Republic of Koread Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 June 2014Revised 12 August 2014Accepted 14 August 2014Available online 23 August 2014

Keywords:Catalytic activityInhibitorPTK6Structure–activity relationship

A lead compound 1, which inhibits the catalytic activity of PTK6, was selected from a chemical library.Derivatives of compound 1 were synthesized and analyzed for inhibitory activity against PTK6 in vitroand at the cellular level. Selected compounds were analyzed for cytotoxicity in human foreskin fibro-blasts using MTT assays and for selectivity towards PTK members in HEK 293 cells. Compounds 20(in vitro IC50 = 0.12 lM) and 21 (in vitro IC50 = 0.52 lM) showed little cytotoxicity, excellent inhibitionof PTK6 in vitro and at the cellular level, and selectivity for PTK6. Compounds 20 and 21 inhibited phos-phorylation of specific PTK6 substrates in HEK293 cells. Thus, we have identified novel PTK6 inhibitorsthat may be used as treatments for PTK6-positive carcinomas, including breast cancer.

� 2014 Elsevier Ltd. All rights reserved.

Protein tyrosine kinase 6 (PTK6), also known as Brk, is an intra-cellular tyrosine kinase containing an SH3 domain, an SH2 domain,and a catalytic tyrosine kinase domain.1,2 PTK6 is expressed inmore than 60% of breast carcinoma tissue samples and breast can-cer cell lines, but not in normal mammary tissue or benign lesions.3

PTK6 is expressed in approximately 86% of invasive ductal breasttumors; this is a significantly higher incidence than that of othermolecular alterations, such as p53 mutation (around 20%) or c-erbB-2 overexpression (25–30%).4,5 In addition to its involvementin breast cancer, PTK6 is also increased in colon,6 head, neck,7

and ovarian cancers.8

Expression of PTK6 renders mammary epithelial cells more sen-sitive to the mitogenic effects of epidermal growth factor (EGF) andpromotes breast cancer cell proliferation.9 PTK6 is also capable ofpromoting cell migration and invasion.10 Various substrates andinteracting proteins, such as signal transducing adaptor protein-2(STAP-2), paxillin, AKT, p130 CRK-associated substrate (p130CAS),and p190RhoGAP-A, contribute to the oncogenic roles of PTK6.11–14 Recently we reported that Arf-GAP, Rho-GAP, ankyrin repeat,and pleckstrin homology (PH) domain-containing protein 1 (ARAP1)

is a novel interacting protein and substrate of PTK6. EGFR signaling isenhanced due to reduced down-regulation of EGFR subsequent toPTK6-induced phosphorylation of ARAP1 in breast cancer cells.15

In view of its oncogenic activity and its presence in various carcino-mas, including breast cancer, PTK6 is a potentially valuable thera-peutic target for slowing or arresting tumor growth.16

We have shown that the intra-molecular interaction betweenthe SH2-kinase linker (Linker) region and tyrosine kinase (Kinase)domain of PTK6 is essential for positive regulation of PTK6 activ-ity.17 Our recombinant PTK6 variant, PTK6-Linker(DN)-Kinase,18

retains the catalytic activity but is free from auto-inhibition.PTK6-Linker(DN)-Kinase was used in an in vitro non-isotopicenzyme-linked immunosorbent assay (ELISA) for PTK6 catalyticactivity. This assay was based on the detection of phosphorylatedtyrosine residues with a monoclonal antibody against phosphoty-rosine.19 We used the PTK6 catalytic activity assay system (definedas an in vitro assay)20 to screen a chemical library for PTK6 inhib-itors. Several compounds inhibited the catalytic activity of PTK6.Compound 1 (IC50 = 1.90 lM in vitro) was selected as a lead com-pound based on Lipinski’s rule21 and the novelty of its chemicalscaffold (Fig. 1). Nevertheless, assays for PTK6 inhibitory activityin HEK 293 cells (defined as a cellular assay)22,23 showed that com-pound 1 did not inhibit PTK6-dependent phosphorylation of cellu-lar proteins at concentrations up to 100 lM.

To analyze whether the hydroxyl and bromine groups in the ben-zylidene of compound 1 were important for PTK6 inhibition, deriv-

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Table 2PTK6 inhibitory activities of derivatives of compound 1 optimized at the side chains ofthe benzylidene ring

Compound R1 R2 R3 R4 R5 IC50 (lM)

In vitro At cellular level

1 OH H H Br H 1.90 ± 0.11 >10019 OH Br H Br Br 0.41 ± 0.02 11.75 ± 1.5020 OH OCH3 H Br Br 0.12 ± 0.004 1.46 ± 0.5321 OH OCH2CH3 H Br Br 0.52 ± 0.08 1.60 ± 0.5222 OH OCH2CH3 H Cl Cl 1.29 ± 0.29 3.48 ± 0.33

Figure 1. Structure of compound 1, a lead compound for PTK6 inhibitors. To findcompounds that inhibit PTK6 activity, a chemical library was screened by an ELISA-based method as described.20 Compound 1 was selected as a lead compound.

4660 H. J. Shim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4659–4663

atives of compound 1 were synthesized (Table 1).24,25 Compound 2,which lacks a side chain in benzylidene, did not inhibit PTK6 in vitro.Compound 3, which has a hydroxyl group at the R1 position, showedPTK6 inhibitory activity (IC50 = 2.00 lM in vitro). Replacement ofthe hydroxy group at the R1 position with a methoxy groupdecreased the inhibitory activity against PTK6; 1 versus 4 and 5 ver-sus 6. These results indicate that the hydroxyl group of compound 1was essential for the PTK6-inhibitory activity.

Removal of the bromine group from the R4 position of com-pound 1 did not change the PTK6 inhibitory activity (3 vs 1). How-ever, the bromine groups in other compounds are beneficial for thePTK6 inhibitory activities (5 vs 8 and 11 vs 12). Chemicals thathave chloride or iodine instead of bromine at the R4 position arealso able to inhibit PTK6 (compounds 7, 9, 10, and 13). Thus, thepresence of bromine, chloride, or iodine at the R4 position tendsto improve PTK6 inhibition in vitro.

We then analyzed the effects of a side chain at the R2 position ofcompound 1. The addition of bromine, methoxy, or ethoxy groupsto the R2 position reduced PTK6 inhibitory activity in vitro butenhanced PTK6 inhibition at the cellular level (14, 5, and 11).

Table 1PTK6 inhibitory activities of lead compound 1 and its derivatives, which were modified at

Compound R1 R2 R3

1 OH H H2 H H H3 OH H H4 OCH3 H H5 OH OCH3 H6 OCH3 OCH3 H7 OH H H8 OH OCH3 H9 OH OCH3 H10 OH OCH3 H11 OH OCH2CH3 H12 OH OCH2CH3 H13 OH OCH2CH3 H14 OH Br H15 OH H OH16 OH H OCH317 OH H H18 OH OCH3 H

AG808b

a IC50 values for inhibition of PTK6 catalytic activity in vitro and at the cellular level werof at least three independent experiments performed in duplicate.

b Tyrphostin AG808 (Sigma–Aldrich), which is reported to be a PTK6 inhibitor, was u

Next, we analyzed the effects of side chains at the R3 or R5 posi-tion. Compounds 15 and 16 with a hydroxyl or methoxy group atthe R3 position of compound 3 did not inhibit PTK6 catalytic activ-ity. A bromine group at the R5 position improved PTK6 inhibitoryactivity in vitro (3 vs 17 and 8 vs 18) and achieved stronger inhibi-tion at the cellular level (3 vs 17).

Based on the effects of side chains in the benzylidene group ofcompound 1, we synthesized compounds that are likely to showimproved inhibition of PTK6 in vitro and at the cellular level(Table 2). Among them, compounds 20 and 21 showed significantlyimproved PTK6 inhibition in vitro and at the cellular level

the side chains of the benzylidene ring

R4 R5 IC50 (lM)a

In vitro At cellular level

Br H 1.90 ± 0.11 >100H H >10 NDH H 2.00 ± 0.48 >100Br H 17.11 ± 1.99 >100Br H 4.73 ± 0.50 52.44 ± 6.34Br H >10 NDCl H 1.41 ± 0.25 >100H H >10 NDCl H 1.68 ± 0.14 74.04 ± 2.32I H 4.98 ± 0.81 58.10 ± 8.01Br H 5.83 ± 0.46 23.51 ± 3.64H H >10 NDCl H 1.99 ± 0.29 51.25 ± 3.78Br H 5.60 ± 0.69 48.96 ± 8.38H H >10 NDH H >10 NDH Br 1.35 ± 0.29 24.49 ± 1.17H Br 3.90 ± 0.41 16.63 ± 1.07

2.32 ± 0.07 53.24 ± 3.31

e determined as described.20,23 Each value represents the mean ± standard deviation

sed as a positive control.

Table 3PTK6 inhibitory activities of derivatives on the linkage between benzylidene and benzimidazoline rings

Compound R1 R2 R3 R4 R5 R6 IC50 (lM)

In vitro At cellular level

1 OH H H Br HNH

HN

ON

1.90 ± 0.11 >100

20 OH OCH3 H Br BrNH

HN

ON

0.12 ± 0.004 1.46 ± 0.53

21 OH OCH2CH3 H Br BrNH

HN

ON

0.52 ± 0.08 1.60 ± 0.52

23 OH OCH3 H Br Br NH

HN

ONH

>10 ND

24 OH OCH2CH3 H Br Br NH

HN

ONH

4.79 ± 0.63 —

Table 4PTK6 inhibitory activities of derivatives of compound 1 derivatives modified at benzimidazoline ring

Compound R1 R2 R3 R4 R5 R7 IC50 (lM)

In vitro At cellular level

1 OH H H Br HNH

HN

O 1.90 ± 0.11 >100

20 OH OCH3 H Br BrNH

HN

O 0.12 ± 0.004 1.46 ± 0.53

21 OH OCH2CH3 H Br BrNH

HN

O 0.52 ± 0.08 1.60 ± 0.52

25 OH OCH3 H Br Br

HN

0.27 ± 0.01 3.49 ± 0.48

26 OH OCH3 H Br BrN

HN

0.20 ± 0.03 5.52 ± 0.78

27 OH OCH3 H Br Br N

S1.30 ± 0.13 4.61 ± 0.87

28 OH OCH2CH3 H Br Br

HN

0.36 ± 0.05 7.58 ± 0.61

29 OH OCH2CH3 H Br BrN

HN

0.13 ± 0.02 4.71 ± 0.45

30 OH OCH2CH3 H Br Br N

S1.17 ± 0.13 2.54 ± 0.36

H. J. Shim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4659–4663 4661

Figure 2. Effect of PTK6 inhibitors on phosphorylation of specific PTK6 substrates.HEK293 cells stably expressing PTK6 were incubated with indicated concentrationsof compounds 20 or 21 for 48 h. Cell lysates were analyzed by Western blottingusing anti-phosphotyrosine (4G10) antibody (A) or anti-phospho-STAT3 antibody(B). Cell lysates were immunoprecipitated with anti-paxillin antibody and analyzedby Western blotting using anti-phosphotyrosine antibody (C).

Figure 3. Effect of PTK6 inhibitors on cytotoxicity in human foreskin fibroblasts.Human foreskin fibroblasts were incubated in complete media supplemented with5% fetal bovine serum in the presence of various concentrations of compounds 20 or21 for 24 h. Media were removed, and fresh media containing 0.5 mg/ml 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were added to eachwell. After incubation for 4 h, media were replaced with DMSO to dissolve the dye.The absorbance was measured on a microplate reader at wavelength 565 nm. Theviability of the no-chemical control (DMSO) was set as 100%. All data wereexpressed as mean ± standard deviation of three independent experiments per-formed in duplicate. ⁄p < 0.05; ⁄⁄⁄p < 0.001 versus no compound.

4662 H. J. Shim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4659–4663

compared to that of lead compound 1. Tyrphostin AG808, whichwas reported to be a PTK6 inhibitor and then as an inhibitor ofPDGFR and EGFR,26 was used as a positive control for PTK6 inhibi-tion. In our assay, AG808 inhibited PTK6 in vitro (IC50 = 2.33 lM)and at the cellular level (IC50 = 53.24 lM), but its inhibitory activitywas much weaker than those of compounds 20 and 21.

We then analyzed the effect of reduction on the linkagebetween the benzylidene and benzimidazoline rings. Compounds23 and 24 showed low inhibitory activities compared to those ofcompounds 20 and 21, respectively (Table 3).

To analyze whether the benzimidazoline ring structure isimportant for PTK6 inhibition, derivatives with modified benzimi-dazoline were tested for PTK6 inhibition. Compounds 25, 26, 28,and 29 had similar in vitro inhibitory activities as those of com-pounds 20 and 21 but show decreased inhibition at the cellularlevel (Table 4). Compounds 30 and 27, in which an NH in benzim-idazoline was changed to S, showed decreased PTK6 inhibitioneven in vitro (Table 4).

Table 5Selectivity of 20 and 21 against PTK family members

Compound PTK6 Src

20 1.46 ± 0.53 >10021 1.60 ± 0.52 >100Genistein 43.46 ± 4.51 >100

Selectivity of inhibitors against various PTK members was analyzed in HEK 293 cells expIC50 values (cellular inhibition activity) were determined by quantifying phosphorylatio

Based on the structure–activity relationships (SARs) of leadcompound 1 and its derivatives, we obtained compounds 20(IC50 = 0.12 lM in vitro and IC50 = 1.46 lM at cellular level) and21 (IC50 = 0.52 lM in vitro and IC50 = 1.60 lM at cellular level) aspotent PTK6 inhibitors.

We further analyzed whether compounds 20 and 21 could inhi-bit phosphorylation of PTK6 substrates in HEK293 cells expressingPTK6. Tyrosine phosphorylation of cellular proteins was increasedby expression of PTK6. Both PTK6 inhibitors decreased tyrosine-phosphorylation levels in a dose-dependent manner (Fig. 2a).Moreover, phosphorylation levels of paxillin and STAT3, whichare PTK6 substrates, were also decreased in the presence of PTK6inhibitors (Fig. 2b and c).

We also analyzed cytotoxicities of the selected PTK6 inhibitors,compounds 20 and 21, in human foreskin fibroblasts (Fig. 3).22,27

Compound 21 did not exhibit cytotoxicity up to 100 lM. Com-pound 20 showed dose-dependent cytotoxicity; however, theIC50 (1.46 lM) of compound 20 at the cellular level was muchlower than the concentration (10 lM) that showed cytotoxicity.

Compounds 20 and 21 were evaluated for selectivity towardsother tyrosine kinases at the cellular level.23 Both chemicals inhib-ited the non-receptor type tyrosine kinases, Src, Fyn, and Bmx, anda receptor-type tyrosine kinase, EGFR, much less efficiently thanPTK6 (Table 5). Even Src family tyrosine kinases (Src and Fyn),which are evolutionarily very close to PTK6,28 were poorly inhib-ited. These results demonstrated that compounds 20 and 21 werequite selective for PTK6.

Fyn Bmx EGFR

38.67 ± 10.86 38.57 ± 7.69 88.67 ± 3.6868.54 ± 7.04 42.29 ± 7.33 76.70 ± 11.5558.18 ± 8.58 67.99 ± 3.70 25.12 ± 4.31

ressing hyperactive PTK6 (PTK6-3PA/Y447F), Src, Fyn, Bmx, or EGFR, as described.23

n levels.

H. J. Shim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4659–4663 4663

In summary, we screened a lead compound that inhibited PTK6catalytic activity. Through the synthesis of derivatives of com-pound 1 and SAR analysis, compounds 20 and 21 were selectedas PTK6 inhibitors. These compounds showed improved PTK6inhibitory activity in vitro and at the cellular level with low cyto-toxicity and selectivity for PTK6. Therefore, compounds 20 and21 may be valuable therapies for controlling PTK6-positive malig-nant diseases, such as breast cancer.

Acknowledgments

This work was supported by a National Research FoundationGrant funded by the Government of Republic of Korea (MSIP)(No. 2013R1A2A2A01013884).

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2014.08.036.

References and notes

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4280.13. Zheng, Y.; Asara, J. M.; Tyner, A. L. J. Biol. Chem. 2012, 287, 148.14. Shen, C. H.; Chen, H. Y.; Lin, M. S.; Li, F. Y.; Chang, C. C.; Kuo, M. L.; Settleman, J.;

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285, 26013.16. Harvey, A. J.; Crompton, M. R. Anticancer Drugs 2004, 15, 107.17. Kim, H.; Lee, S. T. J. Biol. Chem. 2005, 280, 28973.18. GST-PTK6-Linker(DN)-Kinase was prepared as described previously.17

19. Farley, K.; Mett, H.; McGlynn, E.; Murray, B.; Lydon, N. B. Anal. Biochem. 1992,203, 151.

20. ELISA plates (96-well; GREINER BIO-ONE) were incubated with 100 ll of0.1 mg/ml poly(Glu and Tyr) (Glu/Tyr = 4:1, Sigma) in PBS for 16 h at 37 �C,washed three times with PBS, blocked in 1% BSA in PBS for 1 h at 37 �C, andwashed three times with PBS. Phosphorylation of tyrosine residues wasinitiated by the addition of 10 nM GST-PTK6-Linker(DN)-Kinase and 300 lMATP in kinase buffer (20 mM Tris–HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2,50 lM Na3VO4). After incubation for 20 min at room temperature, the platewas washed five times with PBS. For quantification of phosphorylatedtyrosines, the plate was incubated with anti-phosphotyrosine antibody(4G10; Millipore Corp.) for 1 h at room temperature, washed five times withPBS (0.05% Tween-20), and incubated with anti-mouse peroxidase-conjugatedantibody for 1 h at room temperature. Detection was carried out using a colorreagent, TMB solution (Thermo Scientific). After incubation of TMB for 5 min,the OD at 450 nm was measured.

21. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.2001, 46, 3.

22. HEK293 cells and human foreskin fibroblasts were maintained in Dulbecco’sModified Eagle Medium (DMEM) containing 5% fetal bovine serum (FBS;HyClone).

23. HEK293 cells overexpressing Flag-PTK6-3PA/Y447F, wild type Src, Fyn, Bmx, orEGFR were treated with the indicated concentrations of compounds for 2 days.Western blot analysis and immunoprecipitations were performed aspreviously described.15 Immunoreactive proteins were visualized with anti-phosphotyrosine, anti-PTK6 (Santa Cruz Biotechnologies), anti-paxillin (SantaCruz Biotechnologies), anti-phospho-STAT2 (Santa Cruz Biotechnologies), anti-STAT3 (Santa Cruz Biotechnologies), and anti-GAPDH (AbClon) primaryantibodies, horseradish peroxidase-conjugated secondary antibody, and anenhanced chemiluminescent detection kit (Millipore Corp.) For quantificationof phosphorylation levels of cell lysates, chemiluminescence was detected byLAS-3000 (Fujifilm) and quantified by Multi Gauge V2.2 software (Fujifilm).

24. General procedure for the preparation of imine compounds: Benzaldehyde(0.30 mmol), 5-amino-2-benzimidazolinone (0.20 mmol), and isopropylalcohol (2 ml) were stirred at room temperature under N2. Acetic acid(0.07 ml) was added drop-wise, and the mixture was refluxed overnight. Thereaction mixture was filtered, washed with CH2Cl2, MeOH, and dried to givethe corresponding imine compounds. Procedures for individual compounds aredescribed in Supplementary data.

25. The physical and spectral data of compound 1 and its derivatives are shown inSupplementary data. Data for representative compounds are as follows.Compound 20: Yield 952%; dark orange powder; 1H NMR (DMSO, 300 MHz) d15.49 (s, 1H), 10.83 (s, 1H), 10.80 (s, 1H), 9.09 (s, 1H), 7.36 (s, 1H), 7.09 (s, 1H),7.18–6.98 (m, 2H), 3.84 (s, 3H); 13C NMR (DMSO, 500 MHz) d 161.20, 156.17,154.65, 149.60, 139.41, 131.45, 130.71, 119.00, 117.88, 117.56, 115.92, 113.08,109.71, 101.56, 57.00. Rf = 0.52 (CH2Cl2/MeOH 9:1).Compound 21: Yield 900%; orange powder; 1H NMR (DMSO, 300 MHz) d 15.51(s, 1H), 10.83 (s, 1H), 10.81 (s, 1H), 9.09 (s, 1H), 7.34 (s, 1H), 7.09 (s, 1H), 7.15–6.92 (m, 2H), 4.08 (q, 2H, J = 6.6 Hz), 1.34 (t, 3H, J = 6.6 Hz); 13C NMR (DMSO,700 MHz) d 159.87, 154.87, 153.39, 147.46, 138.11, 130.13, 129.39, 118.57,116.65, 116.25, 114.66, 111.80, 108.42, 100.25, 63.91, 13.75. Rf = 0.70 (CH2Cl2/MeOH 9:1).

26. Gazit, A.; App, H.; McMahon, G.; Chen, J.; Levitzki, A.; Bohmer, F. D. J. Med.Chem. 1996, 39, 2170.

27. Subconfluent cells of human foreskin fibroblasts were incubated in DMEM-5%FBS containing various concentrations of the chemicals for 24 h. Viable cellswere measured with an MTT assay as previously described.17 The viability ofthe no-chemical control (DMSO) was set as 100%. All data were expressed asmean ± SD of three independent experiments performed in duplicate.

28. Lee, H.; Kim, M.; Lee, K. H.; Kang, K. N.; Lee, S. T. Mol. Cells 1998, 8, 401.

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Discovery of (E)-5-(benzylideneamino)-1H-benzo[d]imidazol-2 (3H)-one derivatives as inhibitors for PTK6AcknowledgmentsSupplementary dataReferences and notes