Nek2

Article pubs.acs.org/jmc

Discovery of 4‑Aryl‑N‑arylcarbonyl-2-aminothiazoles as Hec1/Nek2 Inhibitors. Part I: Optimization of in Vitro Potencies and Pharmacokinetic Properties Ying-Shuan E. Lee,† Shih-Hsien Chuang,† Lynn Y. L. Huang,‡ Chun-Liang Lai,† Yu-Hsiang Lin,† Ju-Ying Yang,† Chia-Wei Liu,† Sheng-chuan Yang,† Her-Sheng Lin,† Chia-chi Chang,‡ Jun-Yu Lai,§ Pei-Shiou Jian,§ King Lam,† Jia-Ming Chang,† Johnson Y. N. Lau,‡ and Jiann-Jyh Huang*,†,§ †

Development Center for Biotechnology, No. 101, Lane 169, Kangning Street, Xizhi District, New Taipei City 22180, Taiwan Taivex Therapeutics Corporation, 17th Floor, No. 3, Yuanqu Street, Nangang District, Taipei City 115, Taiwan § Department of Applied Chemistry, National Chiayi University, No. 300, Syuefu Road, Chiayi City 60004, Taiwan ‡

S Supporting Information *

ABSTRACT: A series of 4-aryl-N-arylcarbonyl-2-aminothiazoles of scaffold 4 was designed and synthesized as Hec1/ Nek2 inhibitors. Structural optimization of 4 led to compound 32 bearing C-4′ 4-methoxyphenoxy and 4-(o-fluoropyridyl)carbonyl groups that showed low nanomolar in vitro antiproliferative activity (IC50: 16.3−42.7 nM), high intravenous AUC (64.9 μM·h, 2.0 mg/kg) in SD rats, and significant in vivo antitumor activity (T/C = 32%, 20 mg/kg, IV) in mice bearing human MDA-MB-231 xenografts. Cell responses resulting from Hec1/Nek2 inhibition were observed in cells treated with 32, including a reduced level of Hec1 coimmunoprecipitated with Nek2, degradation of Nek2, mitotic abnormalities, and apoptosis. Compound 32 showed selectivity toward cancer cells over normal phenotype cells and was inactive in a [3H]astemizole competitive binding assay for hERG liability screening. Therefore, 32 is as a good lead toward the discovery of a preclinical candidate targeting Hec1/Nek2 interaction.

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INTRODUCTION Highly expressed in cancer 1 (Hec1) is a key component of the kinetochore that regulates the spindle check point and plays an essential role in mitosis.1 With Nuf2, Spc24, and Spc25, it forms a dumbbell-like heterotetramer called the Ndc80 complex, in which Hec1 and Nuf2 dimerize as a subcomplex, with their globular domains heading toward the microtubulebinding interface.2−4 Hec1 has microtubule-binding activity at its N-terminal region (aa 1−196) and is responsible for proper kinetochore−microtubule attachment.2,5,6 Hec1 possesses oncogenic properties: its hyperactivation in transgenic mice leads to tumor formation.7 Overexpression of Hec1 is found in various cancers8 and correlates with poor prognosis for cancer patients.9,10 RNA interference of Hec1 enhances the sensitivity of human ovarian cancer cells to paclitaxel11 and reduces the size of induced adenocarcinomas in nude mice.12 Because phosphorylation of Hec1 by Nek2 kinase is essential for its mitotic function,13−15 disruption of Hec1/ Nek2 protein−protein interaction by small molecules shows anticancer activities16,17 and may have potential for cancer treatment. 4-Aryl-N-phenylcarbonyl-2-aminothiazole 1 (INH1, Figure 1) is the prototype Hec1 inhibitor discovered from a chemical genetic screening.17 It specifically disrupts Hec1/Nek2 interaction via direct Hec1 binding and shows in vitro © 2014 American Chemical Society

Figure 1. Structures of 4-aryl-N-arylcarbonyl-2-aminothiazoles 1−3 and scaffold 4.

antiproliferative activity (IC50: 8.6−11.7 μM) as well as in vivo antitumor activity in MDA-MB-468 xenografts. Subsequent lead optimizations of the N-phenylcarbonyl moiety and C-6′ position of 1 afforded compounds 2 and 3 that demonstrated improved in vitro potency (IC50: 1.1−3.4 μM).16 Compounds 1−3 trigger the cellular responses resulting from Hec1 inhibition, including the reduction of Nek2 protein Received: December 28, 2013 Published: April 28, 2014 4098

dx.doi.org/10.1021/jm401990s | J. Med. Chem. 2014, 57, 4098−4110

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level and mitotic abnormalities.16,17 Although they are the only reported Hec1/Nek2 inhibitors to date, none of them, 1−3, have been moved into clinical trials. When the structure−activity relationship of 1−3, shown in Figure 1, was examined, compound 2, bearing C-2′ and C-6′ dimethyls, was found to be ∼5-fold more potent than compound 1, bearing a sole C-2′ methyl (IC50: 1.7−2.5 μM for 2 and 8.6−11.7 μM for 1). Compound 3, with a 4pyridylcarbonyl group, also showed ∼5-fold improved potency (IC50: 1.1−3.4 μM) compared to 1, which has a phenylcarbonyl group at the same position. As for the C-4′ position, no substituent other than a methyl group has been reported on 1− 3 and their analogues in the literature.16 This information suggested that the scaffold of 1−3 possessing C-2′ and C-6′ dimethyl and 4-pyridylcarbonyl groups could show improved potency and that further structural optimization could be performed on the less explored C-4′ position. Herein, we report the design and synthesis of 4-aryl-Narylcarbonyl-2-aminothiazoles of scaffold 4 as Hec1 inhibitors (Figure 1). Scaffold 4 possesses 2′,6′-dimethyl and 4pyridylcarbonyl groups as the core, various C-4′ alkoxy or aryloxyl groups, and o-halo at the pyridyl group for optimization. After evaluation, we obtained compound 32 with low nanomolar in vitro IC50 values, good pharmacokinetic properties, and significant in vivo antitumor activity. Compound 32 induced cellular responses resulting from Hec1 inhibition and was less active on normal cells, kinases, and hERG. In our attempt to discover a preclinical candidate that disrupts Hec1/Nek2 interaction, compound 32 serves as a good lead for future development.

For the synthesis of 4-aryl-N-arylcarbonyl-2-aminothiazoles of scaffold 4 bearing various C-4′ alkoxy substituents (R = alkyl in 4, Figure 1), acetophenones 10a−f were brominated by TBABr3 in CH3CN and then reacted with thiourea in EtOH to provide corresponding 4-aryl-2-aminothiazoles 11a−f in 57− 82% yields (Scheme 2). Acylation of 11a−f with isonicotinoyl chloride, 2-fluoroisonicotinoyl chloride, or 2-chloroisonicotinoyl chloride in CH2Cl2 using DMAP as the base afforded desired 4-aryl-N-arylcarbonyl-2-aminothiazoles 12−20 possessing various C-4′ alkoxy groups in 49−99% yields. The synthesis of scaffold 4 bearing various C-4′ aryloxy substitutents (R = aryl in 4, Figure 1) was carried out by the use of 1-(4-amino-2,6-dimethylphenyl)ethanone 21 as the starting material (Scheme 3). The amino group in 21 was transformed into a chloro group by Sandermeyer-type reaction using tBuONO and CuCl219 in CH3CN to provide 1-(4-chloro-2,6dimethylphenyl)ethanone 22 in 76% yield. O-Arylation of various aryl alcohols by 22 using the Pd(OAc)2/t-BuXPhos catalytic system20 afforded 4-aryloxy-2,6-dimethylacetophenones 23a−i in 19−86% yields. After reacting with TBABr3 and thiourea, 23a−i were converted to corresponding 4-aryl-2aminothiazoles 24a−i in 53−96% yields. Acylation of 24a−i with isonicotinoyl chloride or 2-fluoroisonicotinoyl choride afforded desired 4-aryl-N-arylcarbonyl-2-aminothiazoles 25−34 bearing various C-4′ aryloxy groups in 17−95% yields.

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RESULTS AND DISCUSSION In Vitro Potency of Scaffold 4. The in vitro potency of the 4-aryl-N-arylcarbonyl-2-aminothiazoles of scaffold 4 (Figure 1) was evaluated against the proliferation of HeLa (cervical cancer), K562 (leukemia), MDA-MB-468 (breast cancer), and MDA-MB-231 (breast cancer) cancer cells. Reference compound 2 was tested first. As presented in Table 1, compound 2 showed similar potency (IC50: 2.2−3.6 μM) to that reported in the literature (IC50: 1.7−2.5 μM).16 Compound 9a, bearing 2′,6′-dimethyl and 4-pyridylcarbonyl groups, was 5.4−10.7-fold more potent than 2. The results verified the design of scaffold 4 to improve in vitro potency. We then substituted the 4pyridylcarbonyl in 9a with various acyl groups to establish the structure−activity relationship. Compound 9b with a methylene elongation to 9a showed similar potency (IC50: 0.30−0.62 μM) to that of 9a. Bioisosteric replacement of the 4-pyridyl in 9a with a 4-cyanophenyl group generated 9c with ∼2-fold reduced potency on K562, MDA-MB-468, and MDA-MB-231 cells. Conversion of the pyridyl group in 9a to pyridyl N-oxide afforded compound 9d with >4-fold reduced potency (IC50: 2.7 to >10 μM). Introduction of an o-chloro group to the 4-pyridyl group in 9a provided compound 9e with slightly reduced potency (IC50: 0.42−0.77 μM). Considering the structural complexities and possible physicochemical properties of 9a, 9b, and 9e, which possessed similar in vitro potencies, we selected the 4-pyridylcarbonyl group for the following optimization. Turning to the C-4′ position of scaffold 4 (Figure 1), we found that the introduction of an alkoxy group showed enhanced antiproliferative activity (Table 2). Compounds 12, 16, 17, 19, and 20, with the corresponding C-4′ methoxy, ethoxy, i-propoxy, ibutoxy, and c-pentoxy groups, were more potent than 9a, which possesses a C-4′ methyl group, and displayed IC50 values ranging from 42.1−392 nM (cf. Table 1, IC50: 300−760 nM for 9a). The increasing size of the C-4′ substituents from the methoxy to the c-pentoxy groups did not reduce the potency,

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CHEMISTRY Scheme 1 presents the synthesis of 4-mesityl-N-acyl-2-aminothiazoles 2 and 9a−e to verify the design of scaffold 4. Scheme 1. Synthesis of 4-Mesityl-N-acyl-2-aminothiazoles 2 and 9a−ea

a Reagents and conditions: (i) BiCl3, acetyl chloride, 70 °C, 83%; (ii) CuBr2, EtOAc, reflux, 95%; (iii) thiourea, EtOH, reflux, 90%; (iv) RCOCl, DMAP, CH2Cl2, 65−87%.

Mesitylene (5) was acylated with acetyl chloride using BiCl3 as the catalyst to provide acetyl mesitylene 6 in 83% yield.18 Compound 6 was monobrominated at the α carbon of the acetyl group by CuBr2 in EtOAc to afford compound 7 in 95% yield. After reacting with thiourea, 7 was converted to 4mesityl-2-aminothiazole 8 in 90% yield. Acylation of 8 with various acyl chlorides gave corresponding compounds 2 and 9a−e in 65−87% yields. 4099

dx.doi.org/10.1021/jm401990s | J. Med. Chem. 2014, 57, 4098−4110

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Scheme 2. Synthesis of 4-Aryl-N-(4-pyridylcarbonyl)-2-aminothiazole 12−20 Bearing C-4′ Alkoxy, C-2′ Methyl or Fluoro, and o-Hydro, Fluoro, or Chloro at the Pyridyl Groupa

a

Reagents and conditions: (i) (a) TBABr3, CH3CN, rt, (b) thiourea, EtOH, reflux; 57−82% (two steps); (ii) isonicotinoyl chloride, 2fluoroisonicotinoyl chloride, or 2-chloroisonicotinoyl chloride, DMAP, CH2Cl2, 49−99%.

Scheme 3. Synthesis of 4-Aryl-N-(4-pyridylcarbonyl)-2-aminothiazole 25−34 Bearing C-4′ Aryloxy and/or Fluoro at the Pyridyl Groupa

Reagents and conditions: (i) t-BuONO, CuCl2, CH3CN, 76%; (ii) aryl alcohol, Pd(OAc)2, t-BuXPhos, toluene, 100 °C, 19−86%; (iii) (a) TBABr3, CH3CN, rt, (b) thiourea, EtOH, reflux; 53−96% (two steps); (iv) isonicotinoyl chloride or 2-fluoroisonicotinoyl chloride, DMAP, CH2Cl2, 17−95%.

a

comparison with reference compound 2, 31 showed ∼100-fold improvement (e.g., 32.0 vs 3200 nM for MDA-MB-231). Movement of the C-4 methoxy group in 31 to the C-3 position reduced the antiproliferative activity of 33 (IC50: 102−253 nM). Compound 34, with a 3,4-methylenedioxy moiety on the C-4′ phenoxy group, was not as potent as 31 (IC50: 45.7−128 nM). For further improvement of the antiproliferative activity of scaffold 4, compounds 13−15, 18, and 32, bearing 2′,6′difluoro and/or o-halo atoms at the 4-pyridyl group, were evaluated (Table 3). C-4′ methoxy-substituted compound 13, bearing a 2′,6′-difluoro group, showed reduced in vitro activity (IC50: 170−450 nM) in comparison with its 2′,6′-dimethyl analogue, 12 (IC50: 99.5−172 nM, Table 2). Introduction of an o-fluoro or o-chloro atom to the pyridyl group of 12 generated compounds 14 and 15 that possessed slightly reduced potencies, with IC50 values of 75.4−220 and 160−280 nM, respectively. Compound 18, the o-fluoropyridyl analogue of 17, showed similar potency (IC50: 42.7−167 nM) to that of 17 (IC50: 52.0−162 nM, Table 2). Introduction of a fluoro atom to

indicating that the binding pocket of the Hec1 protein left a space headed by the C-4′ substituents of scaffold 4 (Figure 1). Accordingly, we replaced the alkoxy groups in scaffold 4 with aryloxy groups (R = aryl in 4, Figure 1). C-4′ phenoxy replacement of the c-pentoxy group in compound 20 generated compound 25 with a similar potency (IC50: 94.4−236 nM) to that of 20 (IC50: 96.0−215 nM). The introduction of a methyl or ethyl group to the C-4 position of the phenoxy group in 25 provided compounds 26 and 27 with similar potency (IC50: 134−237 nM). However, the presence of 3,5-dimethyls at the phenoxy group deactivated compound 28 (IC50: 727 to >1000 nM). Compound 29, with a C-4′ 4-fluorophenoxy group, displayed similar potency (IC50: 239−280 nM) to that of 25, 26, and 27. Compound 30, with a C-4′ 4-(trifluoromethyl)phenoxy group, showed a ∼2-fold improvement in antiproliferative activity (IC50: 58.2−132 nM) compared to 29. The most potent analogues of scaffold 4 in Table 2 was compound 31, which possessed a C-4′ 4-methoxyphenoxy functionality. Regarding the four cancer cell lines, 31 displayed low nanomolar antiproliferative activity (32.0−48.6 nM). In 4100

dx.doi.org/10.1021/jm401990s | J. Med. Chem. 2014, 57, 4098−4110

Journal of Medicinal Chemistry

Article

Table 1. In Vitro Antiproliferative Activity of N-Acyl-4-mesityl-2-aminothiazoles 2 and 9a−e toward HeLa, K562, MDA-MB468, and MDA-MB-231 Cancer Cells

a

The IC50 values were averaged from two independent dose−response curves; variation was generally 9.0 μM >9.0 μM >9.0 μM >9.0 μM

The GI50 values were averaged from two independent dose−response curves; variation was generally