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Oct 14, 2016 - ... Jun-Hong Han†, Guang-Wen Lu†, Yu-Quan Wei†, and Sheng-Yong ... Zhang, Zheng, Li, Lin, Huang, Zhong, Li, Zhang, Lin, Jiao, Wu,...
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From Lead to Drug Candidate: Optimization of 3‑(Phenylethynyl)‑1H‑pyrazolo[3,4‑d]pyrimidin-4-amine Derivatives as Agents for the Treatment of Triple Negative Breast Cancer Chun-Hui Zhang,†,∥ Kai Chen,†,∥ Yan Jiao,†,∥ Lin-Li Li,‡,∥ Ya-Ping Li,† Rong-Jie Zhang,† Ming-Wu Zheng,† Lei Zhong,† Shen-Zhen Huang,† Chun-Li Song,‡ Wan-Ting Lin,† Jiao Yang,† Rong Xiang,§ Bing Peng,† Jun-Hong Han,† Guang-Wen Lu,† Yu-Quan Wei,† and Sheng-Yong Yang*,† †

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and Collaborative Innovation Center for Biotherapy, Sichuan University, Sichuan 610041, China ‡ Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041, China § Department of Clinical Medicine, School of Medicine, Nankai University, Tianjin 300071, China S Supporting Information *

ABSTRACT: Herein we report the sophisticated process of structural optimization toward a previously disclosed Src inhibitor, compound 1, which showed high potency in the treatment of triple negative breast cancer (TNBC) both in vitro and in vivo but had considerable toxicity. A series of 3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine derivatives were synthesized. In vitro cell-based phenotypic screening together with in vivo assays and structure−activity relationship (SAR) studies finally led to the discovery of N-(3-((4-amino-1-(trans-4-hydroxycyclohexyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4methyl-3-(trifluoromethyl)benzamide (13an). 13an is a multikinase inhibitor, which potently inhibited Src (IC50 = 0.003 μM), KDR (IC50 = 0.032 μM), and several kinases involved in the MAPK signal transduction. This compound showed potent antiTNBC activities both in vitro and in vivo, and good pharmacokinetic properties and low toxicity. Mechanisms of action of antiTNBC were also investigated. Collectively, the data obtained in this study indicate that 13an could be a promising drug candidate for the treatment of TNBC and hence merits further studies. treating TNBCs, just showed a limited efficacy in TNBCs.13,14 The fact that dasatinib has subnanomolar inhibitory activity against Src (IC50 = 0.0003 μM)15 implies that it is impractical to further enhance the anti-TNBC activity of a compound by only improving the Src inhibitory potency. A multitarget strategy could be a better choice to enhance the anti-TNBC activity of a Src inhibitor because of the highly heterogeneous property of TNBC.16−20 In a recent study, we developed a multikinase inhibitor, 3-((4-amino-1-ethyl-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methyl-N-(4-((4methylpiperazin-1-yl)methyl)-3-(trifluoromethyl) phenyl)benzamide (1, Figure 1), which potently inhibits Src and RAF kinases with IC50 values of 0.0009 μM for Src, 0.092 μM for B-Raf, and 0.027 μM for C-Raf.21 This compound indeed

1. INTRODUCTION Triple-negative breast cancers (TNBCs), which do not express any of the markers of an estrogen receptor (ER), a progesterone receptor (PR), and a human epidermal growth factor receptor 2 (HER2), constitute 15%−20% of all breast cancers.1,2 A typical characteristic of TNBCs is a lack of specific biomarkers.3,4 Conventional chemotherapy is the only systemic therapy, and the prognosis remains poor.5,6 Recently, molecular profiling efforts have revealed several potential molecular targets of TNBCs.7 Among them, Src is one of the most promising targets.8 Src is a nonreceptor tyrosine kinase. It has been demonstrated to play a role in the proliferation, migration, and invasion of breast cancer cell lines.9−11 Further, the abnormal activation or amplification of Src has also been detected in tumor tissues of TNBC patients.12 Therefore, Src represents a rational molecular target for TNBCs. However, dasatinib, the only Src inhibitor that is in clinical trials for © 2016 American Chemical Society

Received: June 26, 2016 Published: October 14, 2016 9788

DOI: 10.1021/acs.jmedchem.6b00943 J. Med. Chem. 2016, 59, 9788−9805

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Figure 1. Schematic showing the lead optimization process.

Scheme 1. Synthetic Routes for Compounds 8a−ga

Reagents and conditions: (a) (i) SOCl2, 80 °C, (ii) DIPEA, EA, 0 °C to rt; (b) ethynyltrimethylsilane, CuI, PdCl2(PPh3)2, DIPEA, THF, rt, N2; (c) K2CO3, MeOH, rt; (d) NIS, DMF, 80 °C, N2; (e) R1-X(Br, I) or R1-OMs, K2CO3, DMF, 80 °C, N2; (f) 5 N HCl in 1,4-dioxane, MeOH, 0 °C to rt; (g) formaldehyde, acetic acid, sodium cyanoborohydride, 1,2-dichloroethane/methanol, rt; (h) 1 N HCl, acetone, 70 °C to rt; (i) sodium borohydride, MeOH, 0 °C to rt; (j) (PPh3)2PdCl2, CuI, DIPEA, DMF, 80 °C, N2. a

((4-amino-1-(trans-4-hydroxycyclohexyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-methyl-3(trifluoromethyl)benzamide (13an) (Figure 1). This compound not only exhibited potent anti-TNBC activity both in vitro and in vivo but also had a lower toxicity than compound 1 in acute toxicity tests and no hERG activity. In this account, we shall report the sophisticated process of structural optimization from compound 1 to 13an. Evaluations of pharmacodynamic and pharmacokinetic properties and toxicities of 13an, and its mechanisms of action of anti-TNBCs will also be presented.

showed a much higher anti-TNBC activity than dasatinib both in vitro and in vivo. Nevertheless, in preliminary acute toxicity tests with rats, compound 1 caused multiple organ toxicities after a single oral dose of 100 mg/kg (see Figure S1). To reduce the toxicity of compound 1, we subsequently performed structural optimization, which led to the discovery of compound 13b (N-(3-((4-amino-1-(1-methylpiperidin-4-yl)1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3(trifluoromethyl)benzamide) (Figure 1). 13b showed antiTNBC activity very similar to that of 1 but a lower toxicity than that of 1. Unfortunately, 13b displayed considerable ability to block the hERG (human Ether-a-go-go Related Gene) channel, which is a risk factor of cardiotoxicity.22 Thus, further structural optimization was carried out to remove the hERG activity of this compound. Finally, we obtained a new compound, N-(3-

2. CHEMISTRY Synthetic routes of compounds 8a−g with a “forward direction” amide are illustrated in Scheme 1. Briefly, benzoic acids 2a−c were coupled with 3-(trifluoromethyl)aniline to afford amide 9789

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Scheme 3. Synthetic Routes for Compounds 14a−ga

3a−c, followed by a classical Sonogashira reaction and deprotection to produce intermediates 4a−c. Iodination of 1H-pyrazolo[3,4-d]pyrimidin-4-amine (5) yielded compound 6, which was subsequently coupled with commercially available haloalkanes or self-prepared methanesulfonates to afford 7a−h. Intermediates 7i−l containing a tertiary amine were prepared by deprotection of 7a−d and subsequent reductive amination. Stereoisomers 7m and 7n were produced by deprotection of 7e and a succeeding reduction. The target compounds 8a−g were finally obtained by Sonogashira reactions between 4a−c and 7f or 7i−l. Compounds 13a−aq containing a reversed amide linker were prepared according to the routes shown in Scheme 2. First, the Scheme 2. Synthetic Routes for Compounds 1i−ra

Reagents and conditions: (a) triphosgene, DIPEA, EA, 0 °C to rt; (b) 11a−c, EA, 70 °C; (c) (PPh3)2PdCl2, CuI, DIPEA, DMF, 80 °C, N2.

a

structures, a multikinase inhibitor often has potency against many kinases, not just few of the kinases.24 For the toxicity optimization of a multikinase inhibitor, the most ideal strategy is to carry out kinase inhibition profiling against all of the kinase family members for compounds synthesized. However, this is too expensive, and it is often difficult to draw a consistent and meaningful conclusion from the complex data of kinase inhibition profiling. To screen for compounds with potent anti-TNBC activity but low toxicity, we in this investigation adopted a cheaper combination strategy, namely, an in vitro cell-based phenotypic screening together with an in vivo assay. In the cell-based phenotypic screening, three cell lines were selected, including MDA-MB-231, MDA-MB-435, and HepG2. MDA-MB-231 and MDA-MB-435 are typical human TNBC cell lines, which express a high level of Src and are sensitive to Src inhibition.13,21,25,26 HepG2 is a human liver cancer cell line, which expresses a very low level of Src;27 the use of HepG2 is to help determine the toxicity effect due to the inhibition of targets except Src. Compounds with potent activity against MDA-MB-231 and MDA-MB-435 and low cell toxicity against HepG2 were selected to perform in vivo assays. With the aim to optimize compound 1, computational studies were first performed to investigate the binding mode of 1 with Src. The predicted binding mode is shown in Figure 2A. Obviously, compound 1 is a typical type II kinase inhibitor. The moiety 1-ethyl-3-(o-tolylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine suitably resides in the ATP binding site of the kinase domain, and 1-methyl-4-(2-(trifluoromethyl)benzyl)piperazine occupies the allosteric pocket. The two moieties are linked through an amide group. Many studies have shown that, despite a high conservation of the kinase domain, there are still some regions that have a big variation in amino acids among all the kinases, which could be exploited in the structural optimization of ligands to adjust the kinase inhibition profiling, hence achieving the purpose of reducing toxicity.24,28−30 The region where the ethyl group of 1Hpyrazolo[3,4-d]pyrimidin-4-amine resides (the entrance of the ATP binding site, region I) and the allosteric pocket (region II) belong to this type of region. In our previous study that led to the discovery of compound 1, subgroups corresponding to region I and region II have been optimized. However, a retrospective analysis showed that the region I and II subgroups

a

Reagents and conditions: (a) ethynyltrimethylsilane, CuI, PdCl2(PPh3)2, triethylamine, THF, rt, N2; (b) K2CO3, MeOH, rt; (c) Fe, NH4Cl, 70% EtOH, 80 °C; (d) (i) aromatic carboxylic acid, SOCl2, 80 °C, (ii) DIPEA, EA, 0 °C to rt; (e) (PPh3)2PdCl2, CuI, DIPEA, DMF, 80 °C, N2.

starting material iodobenzenes 9a−b underwent a Sonogashira reaction to give 10a−b, followed by reduction to produce 3ethynylaniline derivatives (11a−b). Then, 11a−b together with 3-ethynylaniline (11c), which was purchased from a market, were coupled with commercially available or self-prepared aromatic carboxylic acid under basic conditions to afford intermediate 12a−z. Finally, the target products 13a−aq were obtained through procedures similar to that in Scheme 1. Synthetic routes of compounds 17a−c with a urea linker are outlined in Scheme 3. First, 1-isocyanato-3-(trifluoromethyl)benzene 15 was prepared using the starting material 3(trifluoromethyl)aniline 14, followed by coupling with 11a−c to yield 16a−c. Again, target compounds 17a−c were readily synthesized using procedures similar to that in Scheme 1.

3. RESULTS AND DISCUSSIONS 3.1. Shift of the Lead Compound from Original Compound 1 to 8c. A multikinase inhibitor often inevitably bears some toxicity, and the structural optimization for reducing the toxicity is not an easy task because of the following reasons. Mechanisms responsible for the toxicity of a multikinase inhibitor are often much more complicated; the toxicity might be due to inhibition of kinases that each one could cause toxicity, or inhibition of kinases that each one is safe but that combination of their inhibition could induce toxicity.23 Additionally, due to the high similarity of kinase 9790

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Table 1. Anti-Proliferation Activities of Compounds 1, Dasatinib, and 8a−e against MDA-MB-231, MDA-MB-435, and HepG2 Cells

Figure 2. (A) Predicted binding mode of 1 with Src. The crystal structure of Src was taken from the RCSB Protein Data Bank (PDB entry: 3EL7). (B) The structure of compound 8c. (C) Schematic showing regions that are the focuses of structural modifications.

have not been sufficiently optimized. Therefore, the two regions are still the focus of this investigation. In the first step, we used some new fragments, which have not been employed in our previous study, to replace the ethyl group in region I. Here, the fragments selected are all hydrophilic because region I is actually a solvent accessible region. For simplification, the region II subgroup was temporarily fixed as (trifluoromethyl)benzene because (trifluoromethyl)benzene is structurally simple and also one of the most preferred fragments in this region; of course, structural optimization toward this region would be carried out in the subsequent steps. A total of five compounds (compound 8a−e) were synthesized, and their chemical structures and bioactivities are given in Table 1. From Table 1, we can see that compounds 8b and 8c have antiproliferation activities against TNBC cells comparable to that of compound 1 and that others are obviously weak. Further, compared with 8b, compound 8c showed a relatively poor activity against HepG2, implying low toxicity. We therefore took compound 8c (Figure 2B) as a new starting compound or in other words a new lead to carry out further structural optimization. 3.2. SAR Analyses of 3-(Phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine Derivatives. The structural optimization of compound 8c was focused on the following three regions (see Figure 2C): substituents on the phenyl ring attached to the 1H-pyrazolo[3,4-d]pyrimidine core (Ring A; R2), the aromatic ring at the “head” of the compound and its substituents (ring B; R3), and the linker between the phenyl ring A and aromatic ring B. In this step, the R1 position was fixed as the optimal subgroup 1-methylpiperidine. 3.2.1. Impact of Substituents on Ring A and the Linker Groups. To explore the possible influence of different substituents at the C-4 position (R2) of the phenyl ring and the effects of different linker groups, we varied these two subgroups using different combinations of fragments. R2

a

Each compound was tested in triplicate; the data are presented as the mean ± SD.

substituents include hydrogen, methyl, and chlorine, and the linker groups include forward and reversed amide, and urea. Bioactivities of the resultant compounds are shown in Table 2. From Table 2, we can see that only compound 13b, which contains a methyl group at the R2 position and a reversed amide as the linker, maintained comparable potency against MDA-MB-231 and MDA-MB-435 cells with that of compound 8c. More importantly, the activity against HepG2 was further reduced, implying a decreased toxicity. These data indicate that the presence of the methyl group at the R2 position and a reversed amide in the linker region might be beneficial to reduce the toxicity and maintain the anti-TNBC activity. Here, it is important to mention that in the same case of reversed amide, compound 13b, which contains a methyl group at the R2 position, showed a more potent activity and a lower toxicity compared with that of compound 13a, which has no such a methyl group. A reasonable explanation is that the methyl group in 13b likely constrains conformation, interacts with the gatekeeper side chains, and narrows the kinome profile, making 13b more active and less toxic than 13a. 3.2.2. Influences of Various Substituents on Ring B (R3). In this section, we explore the effects of different substituents on ring B. First, various hydrophobic groups were placed at the meta or para position on ring B. Bioactivities of the synthesized derivatives are shown in Table 3. From Table 3, we can see that bioactivities of compounds with a bulky substituent at the meta position are much higher than corresponding ones substituted at the para position (13b vs 13d; 13e vs 13f). On the contrary, compounds with a small substituent at the para position are more active than those with the same substituent at the meta position (13g vs 13h). Second, various hydrophobic sub9791

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Table 2. Antiviability Activities of Compounds 8f−g, 13a−c, 17a−c, and 8c against MDA-MB-231, MDA-MB-435, and HepG2 Cells

Table 3. Anti-Proliferation Activities of Compounds 13b−v against MDA-MB-231, MDA-MB-435, and HepG2 Cells

cellular inhibition (IC50, μM)a Cpd

R3

13b 13d 13e 13f 13g 13h 13i 13j 13k 13l 13m 13n 13o 13p 13q 13r 13s 13t 13u 13v

3-CF3 4-CF3 3-OMe 4-OMe 3-Cl 4-Cl 3-F 3-OCF3 3-Me 3-tBu 2-F-3-CF3 3-CF3-4-F 3-F-5-CF3 2-F-5-CF3 3-Cl-5-CF3 4-Cl-3-CF3 4-Me-3-CF3 4-OMe-3-CF3 3,5-diCF3 2-F-3-Cl-5-CF3

MDA-MB-231 0.013 0.854 0.101 1.358 0.060 0.033 0.812 0.075 0.133 0.059 0.296 0.523 0.671 0.201 0.085 0.031 0.044 0.024 0.051 1.877

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.002 0.185 0.023 0.333 0.021 0.013 0.162 0.018 0.209 0.012 0.077 0.220 0.121 0.057 0.015 0.012 0.009 0.010 0.020 0.443

MDA-MB-435 0.080 5.135 0.285 8.901 0.733 0.111 2.066 0.093 0.422 0.020 0.273 0.621 1.430 0.117 0.063 0.027 0.040 0.832 0.094 3.377

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.011 1.222 0.138 1.432 0.289 0.031 0.078 0.082 0.172 0.009 0.111 0.180 0.309 0.212 0.017 0.008 0.016 0.177 0.026 1.009

HepG2 >10 >10 >10 >10 9.2 ± 7.6 ± >10 >10 >10 >10 >10 >10 >10 2.7 ± 2.6 ± >10 >10 2.2 ± 7.1 ± >10

2.0 1.8

0.4 0.4

0.2 1.0

a

Each compound was tested in triplicate; the data are presented as the mean ± SD.

a

Each compound was tested in triplicate; the data are presented as the mean ± SD.

3.3. Preliminary in Vivo Assays for Screening of Compounds with Potent Anti-TNBC Activity and Low Toxicity. The SAR analyses above led to the discovery of a number of compounds that exhibited potent antiviability potency against TNBC cell lines MDA-MB-231 and MDAMB-435, and low cytotoxicity in the HepG2 cell assay. To further screen for compounds with potent anti-TNBC activity and tolerable toxicity in vivo, a preliminary in vivo anti-TNBC evaluation in a MDA-MB-231 xenograft mouse model was carried out. Six compounds, namely, 8c, 13b, 13h, 13r, 13s, and 13aa, were selected to do the tests because these compounds showed higher anti-TNBC activity and lower toxicity in in vitro assays. As shown in Table 5, among all the compounds tested, only compound 13b exhibited a high tumor inhibition rate (90.7%) and a low body weight loss of mice (−3.1%) at an oral dose of 40 mg/kg/d. However, in a further safety evaluation, 13b showed a considerable ability to inhibit the hERG channel with an IC50 value of 0.60 μM; substantial evidence has indicated that blockade of the hERG channel has a high risk of cardiotoxicity.22 Therefore, further structural optimization has to be done to rule out the potential cardiac toxicity of 13b. 3.4. Structural Optimization to Remove the Activity against hERG. According to the mapping of the chemical structure of 13b with available pharmacophore models31,32 of

stituents, including fluorine (13i), trifluoromethoxyl (13j), methyl (13k), and tert-butyl (13l), were used to replace the original trifluoromethyl moiety at the meta position, which led to a series of compounds with lower bioactivities. These data indicate that the trifluoromethyl group is the most suitable substituent at the meta position. Third, more hydrophobic substituents were introduced on ring B with the trifluoromethyl group fixed at the meta position (13m−v). As shown in Table 3, compared with 13b, all resulting compounds showed reduced activities against both cell lines, except compound 13r and 13s, which have comparable potencies with 13b. Fourthly, to examine the possible influences of different aromatic rings at the ring B position, a trifluoromethylsubstituted pyridine derivatives (13w−y) and a tert-butylsubstituted five-membered heterocycle (13z and 13aa−ab) were design and synthesized. As shown in Table 4, the bioactivities of the resulting compounds did not exceed that of compound 13b. Therefore, in subsequent structural modifications, ring B and R3 were maintained as a 3-trifluoromethylphenyl group. 9792

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Table 4. Antiviability Activities of Compounds 13w-ab against MDA-MB-231, MDA-MB-435, and HepG2 Cells

Table 6. Antiproliferation Activities of Compounds 13ac-aq and 13b against MDA-MB-231, MDA-MB-435, and HepG2 Cells and Their Inhibitory Potency against hERG

a

Each compound was tested in triplicate; the data are presented as the mean ± SD.

Table 5. Preliminary in Vivo Anti-TNBC Assays of Compounds 8c, 13b, 13h, 13r, 13s, and 13aa Cpd

oral dose (mg/kg)

treatment days

in vivo tumor inhibition rate

body weight change rate

death/ total

8c 13b 13h 13r 13s 13aa

40 40 40 40 40 40

15 21 21 21 21 21

91.2% 90.7% 90.1% 70.5% 69.8% 87.0%

−13.2% −3.1% −16.0% −6.7% −4.5% −5.3%

0/3 0/3 0/3 0/3 0/3 0/3

hERG inhibitors (see Figure S2), the basic nitrogen in the 1methylpiperidin-4-yl moiety might play a critical role, which corresponds to the positive charge feature in the pharmacophore hypothesis. The basic nitrogen (or generally the positive charge feature) could possibly form a good interaction with an aromatic ring of the residue (for example, phenylalanine and tyrosine), or an interaction with a negatively charged residue (for example, aspartic acid and glutamic acid) in the binding pocket of hERG. To test and verify this hypothesis, we used some different hydrophilic groups with and without basic nitrogen to replace the original 1-methylpiperidin-4-yl moiety (the R1 position). A total of 8 compounds were synthesized. Bioactivities and hERG activities of these compounds (13ac− ai) are shown in Table 6. As shown in Table 6, all of the compounds containing a basic nitrogen (13ac−ae) in R1 had considerable activities against hERG. On the contrary, activities of compounds without such a basic nitrogen (13af−ai) were dramatically reduced. This is consistent with the pharmaco-

a

Each compound was tested in triplicate; the data are presented as the mean ± SD. bIC50 values were determined using the service of ChemPartner. The data represent the mean values of three independent experiments. cnt means not tested.

phore hypothesis, for instance, compound 13ai was not mapped with the positive charge feature of the pharmacophore model (see Figure S2). Importantly, antiviability activities of these compounds against MDA-MB-231 and MDA-MB-435 were kept very well. Further, the 3-trifluoromethylphenyl moiety in the head part was also replaced by some other fragments, which was performed for discovering more potent compounds. The fragments include 4-(tert-butyl)thiazole-2-yl, 3-(tert-butyl)phenyl, 4-methyl-3-trifluoromethylphenyl, and 4-chloro-3-trifluoromethylphenyl; these groups have been shown to be beneficial for enhancing the bioactivity (see Tables 3 and 4). 9793

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had better pharmacokinetic properties than 13ai. Collectively, among all the compounds synthesized, 13an is the most promising candidate for the treatment of TNBCs. In-depth studies including in vitro and in vivo anti-TNBC studies and mechanism of action studies were then carried out on 13an. 3.6. Predicted Binding Mode of 13an with Src. Computational studies were performed again to investigate the possible binding mode of 13an with Src. The predicted binding mode is shown in Figure 3A. For comparison, the

We then synthesized a total of 8 compounds. The bioactivities and hERG activities are also presented in Table 6 (13aj−aq). As expected, the hERG activities were all low for the newly synthesized compounds, and the antiviability potencies against TNBC cells were the same or enhanced compared with those of their counterparts. Then, compounds 13af−ai, 13am, 13an, and 13aq were selected to carry out a preliminary in vivo anti-TNBC study in a MDA-MB-231 xenograft mouse model, whose results are summarized in Table 7. Among them, oral dosing of 13ai and Table 7. Preliminary in Vivo Anti-TNBC Assays of Compounds 13af−i, 13am, 13an, and 13aq Cpd

oral dose (mg/kg)

treatment days

in vivo tumor inhibition rate

body weight change rate

death/ total

13af 13ag 13ah 13ai 13ai 13am 13an 13aq

40 40 20 40 20 40 40 40

15 15 15 15 15 15 15 15

78.1% 77.3% 70.2% 89.1% 79.6% 97.1% 96.2% 96.1%

−3.2% −8.2% −2.0% 3.1% 3.5% −11.3% 3.4% −13.4%

0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3

13an at 40 mg/kg/d inhibited the tumor growth with an inhibition rate of 89.1 and 96.2%, respectively. There were no body weight changes or other obvious side effects observed. These data indicate that compound 13ai and 13an are ideal inhibitors, which can potently inhibit the proliferation of TNBC cells both in vitro and in vivo without causing cytotoxicity against HepG2 cells and severe side effects in the treated mice. 3.5. Pharmacokinetic Properties of 13ai and 13an in Rats. Considering the excellent anti-TNBC activities of compounds 13ai and 13an both in vitro and in vivo, pharmacokinetic (PK) properties of these compounds were further evaluated. Key PK parameters are summarized in Table 8. Compared with 13ai, 13an had a longer half-life (T1/2) (4.7 h

Figure 3. (A) Predicted binding mode of 13an with Src. (B) A comparison of the binding modes of 13an (dark slate blue) and compound 1 (cyan) with Src. Hydrogen bonding interactions between the compounds and Src are indicated by dashed yellow lines for compound 1 and dashed green lines for 13an. The crystal structure of Src was taken from the RCSB Protein Data Bank (PDB entry: 3EL7).

Table 8. In Vivo Pharmacokinetic Parameters of 13ai and 13an rat (p.o, 10 mg/kg) parameters a

T1/2 (h) AUC0‑t (h × ng/mL)b AUC0‑∞ (h × ng/mL) Tmax (h)c Cmax (ng/mL)d

13ai

13an

4.7 959.6 975.7 6 128.1

18.9 2861 2946 4 143.0

binding mode of 13an was superimposed on that of 1, which is depicted in Figure 3B. As shown in Figure 3A and B, 13an could tightly bind to the ATP-binding site of Src in a binding mode similar to that of compound 1. The 1H-pyrazolo[3,4d]pyrimidin core occupies the adenine region of Src kinase and forms hydrogen bonds with residues Glu-339 and Met-341 in the hinge region. The trans-4-hydroxycyclohexyl group forms a hydrogen bonding interaction with Asp-348. The amide moiety of 13an also forms hydrogen bonds with Glu-310, which is a common case for type II kinase inhibitors. 28 The 3trifluoromethylphenyl group locates in the allosteric pocket and makes favorable van der Waals interactions with residues Leu-322, Val-402, Val-377, and Leu-317. From Figure 3B, we can see that, compared with compound 1, 13an loses two hydrogen bonding interactions (between 1-methyl-4-(2(trifluoromethyl)benzyl)piperazine and VAL383/HIS-384) but gains another hydrogen bonding interaction (between trans-4-hydroxycyclohexyl and ASP-348), which could be used to interpret the phenomena that 13an showed a slightly weaker activity against the Src kinase than 1.

a

Mean half-life associated with the terminal slope. bMean area under the plasma concentration−time curve. cMean time to reach maximum plasma concentration. dMean peak plasma concentration.

vs 18.99 h) and a larger area under the concentration−time curve (AUC0‑∞, h × ng/mL) (975.7 vs 2946). 13an achieved a maximum plasma concentration (Cmax) of 143.0 ng/mL within approximately 4 h, which is relatively larger and more rapid than 13ai. The microsome stabilities of 13an were also evaluated using liver microsome preparations from mouse, rat, dog, monkey, and human. The results (see Table S1) showed that 13an was very stable; the percentage parent remaining was greater than 90% in all species. These data indicated that 13an 9794

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3.7. Biochemical Activities of 13an against Various Recombinant Human Protein Kinases. To investigate the kinase inhibitory activities and selectivity, kinase inhibition profiling assays with a fixed concentration of 10 μM 13an against a panel of 335 kinases were first carried out through the Eurofins kinase profiling service. The data are presented in Figure 4 and Table S2. To kinases that showed a high

Table 9. Kinase Inhibition Profile of 13an against Selected Protein Kinasesa kinase

IC50 (μM)

kinase

IC50 (μM)

kinase

IC50 (μM)

Src Yes Hck Lyn Fyn Lck Blk PTK5 BRK Abl Abl T315I Arg Flt1 Flt4 KDR DDR1 DDR2

0.003 0.001 0.003 0.004 0.006 0.014 0.016 0.019 0.121 0.002 0.003 0.004 0.015 0.016 0.032 0.015 0.065

Ret Txk Bmx FGFR1 FGFR2 FGFR3 ErbB2 ErbB4 EGFR EGFR T790M, L858R EGFR T790M CSK Tie2 Pyk2 B-Raf B-Raf V600E C-Raf

0.004 0.006 0.021 0.007 0.040 0.120 0.165 0.122 0.651 0.008 0.019 0.009 0.010 0.116 0.110 0.087 0.052

TAK1 ACK1 Mer Rse P38α P38β EphB2 EphA7 EphA4 LIMK1 Rsk1 Rsk2 PDGFRα PDGFRβ ERK2 TrkA TrkB

0.050 0.175 0.052 0.351 0.342 0.084 0.033 0.143 0.227 0.255 0.559 >10 1.287 >10 >10 >10 0.899

a IC50 values were determined using KinaseProfiler by Eurofins. The data represent the mean values of two independent experiments.

Table 10. Antiviability Activities of 13an against Various Cell Linesa

Figure 4. Kinase binding selectivity for 13an shown on the human kinome dendrogram. The inhibition rates were determined using the KinaseProfiler of Eurofins. The figure was generated by using an online Kinome Render program (http://bcb.med.usherbrooke.ca/ kinomerenderLig.php).

inhibitory rate at 10 μM, further half-maximal inhibitory concentrations (IC50s) were examined, and the results are shown in Table 9. 13an potently inhibited Src (IC50 = 0.003 μM), Yes (IC50 = 0.001 μM), Hck (IC50 = 0.003 μM), Lyn (IC50 = 0.004 μM), Fyn (IC50 = 0.006 μM), Lck (IC50 = 0.014 μM), Blk (IC50 = 0.016 μM), and PTK5 (IC50 = 0.019 μM); all of them belong to the Src family kinases (SFKs). 13an also exhibited considerable potency against KDR (IC50 = 0.032 μM) and several kinases involved in the MAPK signal transduction, including members of the P38, RAF, and DDR families of kinases.33−36 13an did not inhibit mitogen-activated protein kinase 1 (ERK2), aurora kinase family members, cyclindependent kinases, or phosphatidylinositide 3-kinases. These data demonstrate that 13an is a multikinase inhibitor with high potencies against Src family kinases, KDR, and several kinases that are involved in the MAPK signaling cascade. 3.8. Antiviability Activities of 13an against Various Cell Lines. Antiviability activities of 13an against various cell lines, including those for TNBCs and a number of other cancer types, as well as two normal cell lines, were examined. As shown in Table 10, 13an potently inhibited the proliferation of

a

cell line

cell type

IC50 (μM)

MDA-MB-231 MDA-MB-435 Hs 578T HCC1937 BT474 MDA-MB-415 ZR-75-1 H1437 PC-9 A375 Miapaca-2 HCT116 HT29 HepG2 plc/prf/5 SMMC7721 RAMOS MV4-11 Hela H4 DU145 L929 LO2

TNBC TNBC TNBC TNBC breast cancer breast cancer breast cancer lung cancer lung cancer melanoma pancreatic cancer colorectal crcinoma colorectal crcinoma hepatocarcinoma hepatocarcinoma hepatocarcinoma lymphoma leukemia cervical cancer neuroglioma prostate carcinoma mouse fibroblast human hepatic cells

0.030 0.008 0.032 0.455 >10 ∼10 ∼10 2.34 0.152 0.031 10 ∼10 >10 ∼10 0.138 6.30 0.670 >10 >10 >10

Each cell line was tested in triplicate.

the TNBC cell lines MDA-MB-231, MDA-MB-435, Hs 578T, and HCC1937, with IC50 values of 0.030 μM, 0.008 μM, 0.032 μM, and 0.455 μM, respectively. It also exhibited considerable potency against several other cell lines, including PC-9 (lung cancer, IC50 = 0.152 μM), A375 (melanoma, IC50 = 0.031 μM), and MV4−11 (leukemia, IC50 = 0.138 μM). Negligible activity was observed for other selected cell lines, including BT474 (breast cancer), H1437 (lung cancer), HCT116 (colorectal carcinoma), HepG2 (hepatocarcinoma), SMMC7721 (hepatocarcinoma), DU145 (prostate carcinoma), L929 (mouse 9795

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Figure 5. (A) 13an inhibited colony formation of MDA-MB-231 cells. MDA-MB-231 cells were seeded in 6-well plates and treated with 13an or dasatinib for 12 days. Colonies were stained with crystal violet, and pictures were taken. (B) 13an induced apoptosis in MDA-MB-231 cells. MDAMB-231 cells were harvested after treatment with various concentrations of 13an and dasatinib for 24 h. Cells were stained using an Annexin VFITC Apoptosis Detection Kit. (C) 13an inhibited MDA-MB-231 cell migration in a wound healing assay. Cells were wounded by a pipet and then treated with various concentrations of compounds for 20 h. Scale bar, 100 μm.

fibroblast), and LO2 (human hepatic cells). These results indicate that 13an has a considerable selectivity for TNBC cells and almost no activity for the normal cells tested. 3.9. Effects of 13an on Colony Formation, Cell Apoptosis, and Migration. Colony-forming assays were then used to examine the antiproliferation activity of 13an. It was found that 13an at concentrations larger than 0.1 μM completely inhibited the colony formation of MDA-MB-231 cells (see Figure 5A). The induction of apoptosis following the treatment of MDA-MB-231 cells by 13an was examined using AnnexinV/PI staining assays. As shown in Figure 5B, 13an treatment led to a concentration-dependent change in the number of apoptotic MDA-MB-231 cells, and approximately 17.35% of the TNBC cells were apoptotic after 24 h of treatment with 0.1 μM 13an. In a wound healing assay, as shown in Figure 5C, after treatment with 13an at a concentration of 0.3 μM for 20 h, the migration of MDAMB-231 cells was significantly inhibited. As a positive control, dasatinib also exhibited the same effects in the three assays but was relatively weaker than 13an in terms of potency. These data demonstrated that 13an could efficiently induce cellular apoptosis and inhibit the migration of tumor cells. 3.10. Inactivation of Key Signaling Proteins in Intact TNBC Cells in Vitro. Western blot analysis was performed to assess the ability of 13an to inhibit the activation of Src and its downstream signaling proteins, as well as MAPK signaling proteins in intact MDA-MB-231 cells. After a 24 h treatment with increasing concentrations of 13an, MDA-MB-231 cells were harvested and lysed for an IP/wt assay. As shown in Figure 6, a dose-dependent reduction in the phosphorylation level of Src was observed following 13an treatment, with an estimated IC50 value of 0.03 μM. Consistent with the inhibition of Src activation, the activation of its downstream signaling proteins FAK and STAT3 was also efficiently inhibited at

Figure 6. 13an inhibited the activation of multiple signaling pathways in intact cells. MDA-MB-231 cells were treated with 13an or dasatinib for 20 h. The cells were lysed, and the proteins were analyzed by Western blot analysis.

concentrations higher than 0.1 μM. Additionally, the phosphorylation of the MAPK signaling proteins MEK and ERK was also strongly inhibited, indicating a blockade of the MAPK signaling pathway. It is also noteworthy that 13an significantly inhibited both the expression and phosphorylation of Fra1 at concentrations larger than 0.03 μM; Fra1 is a key regulator of epithelial-tomesenchymal transition (EMT), which is an important factor responsible for tumor metastasis.37 In addition, 13an increased the level of cleaved-caspase-3 in a dose-dependent manner, indicating the induction of apoptosis.38 In contrast, though dasatinib also efficiently inhibited Src and FAK, it had very 9796

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Figure 7. In vivo antitumor efficacies of 13an against MDA-MB-231 (A) and MDA-MB-435 (B) tumor xenograft models, and average body weights for treated mice in the MDA-MB-231 (C) and MDA-MB-435 (D) xenograft models. Mice implanted with MDA-MB-231 or MDA-MB-435 cells were treated when the tumor grew to about 200 mm3. Animals (6 per group) were treated with solvent control, 13an at oral doses of 10, 20, and 40 mg/kg/d, dasatinib at an oral dose of 40 mg/kg/day, or paclitaxel at a dose of 10 mg/kg/week through a tail vein injection. Points indicate mean tumor volumes (mm3) or mean body weights (g); bars indicate SD.

the formation of intersegmental vessels at all three concentrations (1.25, 2.5, and 5 μM) while preserving fluorescence in the dorsal aorta and major cranial vessels. These results are consistent with the biochemical activity of 13an against KDR (see Table 9); KDR is one of the key regulators of angiogenesis. Taken together, 13an exerted its antitumor effects in vivo by inhibiting both the Src and MAPK signal pathways. Undeniably, the antiangiogenic effects of 13an also played a role in anti-TNBC efficacy. 3.12. Preliminary Toxicity Evaluation of 13an. A single dose toxicity study was carried out to determine the acute toxicity of 13an. After an oral administration of 13an at 200 mg/kg in male rats, the rate of increase of average body weight slowed down in the first 9 days and then recovered to normal (Figure 8C). In female rats, the same dose of 13an did not bring an obvious change in the rate of increase of the body weight. In addition, histological and immunohistochemical techniques were used to detect the pathological changes in the organs of rats after a single dose of 13an at 100 mg/kg. No pathological changes in the major organs (Figure S1) were observed in the treatment groups. All of these indicated that 13an had low toxicity.

weak effects on the MAPK signaling pathway, Fra1, and caspase-3. 3.11. In Vivo Effects and Mechanisms of Action of 13an in TNBC Xenograft Models. The in vivo anti-TNBC efficacies of 13an were assessed using both MDA-MB-231 and MDA-MB-435 xenograft models. Oral dosing of 13an at 10, 20, and 40 mg/kg/d inhibited tumor growth in a dose-dependent manner in both models (Figure 7A and B), with tumor growth completely inhibited at a dose of 40 mg/kg/d. As a positive control, 40 mg/kg/q.d. dasatinib and 10 mg/kg/week (iv) paclitaxel showed even weaker antitumor activities compared with that of the 10 mg/kg/q.d. 13an. During the treatment period, no weight loss (Figure 7C and D) or other side effects were observed in all the treatment groups. To determine the in vivo antitumor mechanisms of action of 13an, immunohistochemical staining assays were carried out on the tumor tissues. Figure 8A shows the immunohistochemical analysis results of the tumor tissues in the MDA-MB-231 xenograft models. Tumor tissues from the 13an treatment groups (40 mg/kg/q.d.) all showed a decrease in the phosphorylation levels of Src, MEK, and ERK and in the tumor mitotic index (Ki67) compared with those from the corresponding control groups. In addition, a significant increase in the level of cleaved caspase-3 was observed in the 13an treatment groups compared with that in the control groups (Figure 8A), indicating the induction of apoptosis. Moreover, compared with the control groups, an obvious reduction of CD31 was observed in the 13an treatment groups, which indicated considerable antiangiogenic effects. The antiangiogenic effects of 13an was further validated by transgenic Tg (flk1: EGFP) zebrafish experiments. As shown in Figure 8B, treatment of live fish embryos with 13an completely blocked

4. CONCLUSION Through a complicated process of structural optimization toward compound 1, we finally discovered a new compound, 13an. 13an is a multikinase inhibitor, which potently inhibited Src and KDR with IC50 values of 0.003 μM and 0.032 μM, respectively. 13an also inhibited several kinases involved in the MAPK signal transduction, including members of the P38, RAF, and DDR families of kinases. Even so, it is not a promiscuous agent and still displayed good selectivity in a 9797

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Figure 8. (A) Mechanisms of action of 13an in human tumor xenograft models. Mice bearing a MDA-MB-231 tumor xenograft treated with 13an at 40 mg/kg/q.d. were humanely euthanized at the end of the experiment, and the tumor tissues were removed for further immunohistochemistry analysis. Scale bar, 100 μm. (B) 30 hpf zebrafish embryos were treated with a blank control or 1.25, 2.5, or 5 μM 13an. ISV budding and outgrowth were completely inhibited by treatment with 1.25 μM 13an. Scale bar, 200 μm. (C) The health status of rats in the acute toxicity evaluation. Average body weights of rats were monitored every 3 days after a single dose of 13an at 200 mg/kg; n = 3; bars indicate SD.

5. EXPERIMENTAL SECTION

kinase profiling assay against 335 kinases. In in vitro antiviability assays, 13an showed potent activities against human TNBC cell lines MDA-MB-231, MDA-MB-435, and Hs 578T with IC50 values of 0.030 μM, 0.008 μM, and 0.032 μM, respectively, but weak activity against other tumor cell lines tested, except A375. In in vivo assays, it completely suppressed tumor growth in MDA-MB-231 and MDA-MB-435 xenograft models at a dose of 40 mg/kg/q.d. Studies of mechanisms of action indicated that 13an down-regulated the activity of Src kinase and the phosphorylation of the MAPK signaling proteins MEK and ERK both in vitro and in vivo. In addition, 13an significantly suppressed the expression of Fra1, which has been demonstrated to play an important role in EMT and hence in tumor metastasis. 13an also inhibited the expression of Ki67 and increased the level of cleaved-caspase-3. It also exhibited very good antiangiogenesis activity in a transgenic zebrafish model [Tg (flk1: EGFP)]. Importantly, 13an did not show an obvious hERG toxicity and had a low acute toxicity at an oral single dose of 200 mg/kg in rats. It also displayed good pharmacokinetic properties. All of the data presented here support 13an as a novel drug candidate for the treatment of TNBCs and deserve further research and development.

5.1. Computational Studies. Molecular docking studies were carried out by GOLD (version 5.0). GOLD adopts the genetic algorithm to dock flexible ligands into protein binding sites. The crystal structure of the Src kinase domain in complex with the pyrazolopyrimidine analogue (PDB entry 3EL7) was used for the docking studies. Hydrogen atoms were added to the protein by using Discovery Studio 3.1 (Accelrys Inc., San Diego, CA, USA). The Charmm force field was assigned. The binding site was defined as a sphere containing the residues that stay within 10 Å from its original ligand, which is large enough to cover the ATP binding region at the active site. GoldScore was selected as the scoring function, and other parameters were set as default. The image was created using PyMOL.39 The 3D pharmacophore model was carried out by Discovery Studio 3.1 (Accelrys Inc., San Diego, CA, USA). The training set is established according to the reference,32 the features of pharmacophore, hydrogen bond acceptor, hydrogen bond donor, hydrophobic, positive ionizable, and negative ionizable were selected, and other parameters were set as default. 5.2. Cell Lines and Cell Culture Conditions. All of the cell lines used were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cell lines were cultured in the designated medium containing 10% fetal bovine serum (FBS) (v/v) at 37 °C in a humidified 5% CO2 incubator according to ATCC guidelines. 5.3. Cell Viability Assays. The viability of cells was determined using the MTT assay method. Cells were seeded (1500−30000 cells per well, depending on the cell type) in 96-well plates. After incubation for 24 h in serum-containing media, the cells were treated with 9798

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13an for 24 h at 37 °C. After incubation, the cells were harvested and washed with ice-cold PBS. Then an Annexin V-FITC Apoptosis Detection Kit (keygentec) was used for apoptosis analysis by flow cytometry as indicated.42 5.11. Wound Healing Assay. MDA-MB-231 cells were cultured to confluence in 24-well plates and wounded using a sterilized pipet tip to make a straight scratch. Cells were rinsed with physiological saline gently, and then PBS was replaced with DMEM medium containing vehicle, dasatinib, or 13an. Pictures were taken by an OLYMPUS digital camera and analyzed by AxioVision Rel 4.8(Carl Zeiss) after 20 h. 5.12. Western Blot Analysis. After treatment with a series of concentrations of 13an for 20 h at 37 °C, MDA-MB-231 cells were harvested, washed with ice-cold physiological saline, and lysed with RIPA lysis buffer (Beyotime) including 1% cocktail (Sigma-Aldrich). Whole-cell protein lysates were prepared and centrifuged for 10 min at 12000 rpm and 4 °C to remove any insoluble material. The total proteins were determined using the Bradford method, and an equivalent quantity of protein was combined with an SDS−PAGE loading buffer (Beyotime) in boiled water for 5 min. Cell lysates were separated by SDS−PAGE and electrotransferred onto PVDF membranes (Millipore). The PVDF membranes were incubated with each antibody and detected according to the immunoblot analysis principle. The antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA), and the dilutions of the antibodies were according to the instructions from CST. 5.13. Immunohistochemistry. SCID mice bearing tumors were treated with control or 13an as described before. At the indicated time points after dosing, individual mice were humanly euthanized. The tumors were fixed with formalin and embedded in paraffin. Then, the immunohistochemistry assays were performed as indicated.43 All of the antibodies were purchased from Cell Signaling Technology or Abcam, except Ki67 (Thermo Fisher Scientific, Fremont, CA) and CD31 (BD Biosciences). Images were captured with a Carl Zeiss digital camera attached to a light microscope. 5.14. Transgenic Zebrafish. The transgenic zebrafish (FLK: EGFP) assay was conducted according to the protocol reported previously.44 Transgenic zebrafish (FLK: EGFP) were maintained normally (temperature, 28 °C; pH 7.2−7.4; 14 h on and 10 h off light cycle). The 30 hpf zebrafish embryos were incubated overnight with 13an. The image was acquired after the zebrafish were anaesthetized by a fluorescence microscope. 5.15. Acute Toxicity. The animal studies were conducted in Sprague−Dawley rats (Chinese Academy of Medical Science, Beijing, China) and under the approval of the Experimental Animal Management Committee of Sichuan University. Compound 1 and 13an were dissolved in sterilization water with 25% (v/v) PEG400 plus 5% DMSO and administered orally at indicated doses. The behavior changes of animals upon drug treatment were monitored, and the side effects were checked every day. The body weights of treated rats were measured every 3 days. Three animals were used in each experiment. 5.16. Chemistry Methods. All reagents and solvents were obtained from commercial suppliers and used without further purification unless otherwise indicated. Anhydrous solvents were dried and purified by conventional methods prior to use. Column chromatography was carried out on silica gel (300−400 mesh). All reactions were monitored by thin-layer chromatography (TLC), and silica gel plates with fluorescence F-254 were used and visualized with UV light. All of the final compounds were purified to >95% purity, as determined by high-performance liquid chromatography (HPLC). HPLC analysis was performed on a Waters 2695 HPLC system with the use of a Kromasil C18 reversed-column (4.6 mm × 250 mm, 5 μm). The binary solvent system (A/B) was chosen between I and II (I, 10 mmol of ammonium acetate in water (pH 9) (A) and acetonitrile (B), A/B = 50/50; II, water (A) and acetonitrile (B), A/B = 40/60) The absorbance was detected at 273 nm, and the flow rate was 1 mL/ min. 1H NMR and 13C NMR spectra were recorded on a Bruker AV400 spectrometer at 400 and 100 MHz, respectively. Coupling constants (J) are expressed in hertz (Hz). Spin multiplicities are

inhibitors (0−10 μg/mL) diluted with culture medium for 72 h at 37 °C under a 5% CO2 atmosphere. Then, 20 μL of the MTT reagent (5 mg/mL) was added to each well, and the plates were incubated for 2− 4 h at 37 °C. For the adherent cells, the media and MTT were carefully aspirated from each well, and the formazan crystals were dissolved in 150 μL of 100% DMSO. For the suspended cells, 50 μL of 20% acidified SDS (w/v) was used to dissolve the oxidative product, and the cells were incubated overnight. Finally, the absorbance at 570 nm was read using a Multiskan MK3 ELISA photometer (Thermo Scientific). All experiments were performed in triplicate. IC50 values were calculated using GraphPad Prism software. 5.4. In Vivo Models. The animal studies were conducted under the approval of the Experimental Animal Management Committee of Sichuan University. MDA-MB-231 and MDA-MB-435 cells were harvested during exponential-growth phase, washed 3 times with serum-free medium, followed by resuspension at a concentration of 5 × 107 per mL. A total of 100 μL of cell suspension was injected into SCID mice (5−6 weeks) subcutaneously. After the tumors grew to volumes of 150−200 mm3, all the mice were randomized into groups (3 or 6 mice for each group) and dosed with indicated compounds, dasatinib (40 mg/kg/q.d.), paclitaxel (10 mg/kg/week (iv)), or vehicle. The compounds were dissolved in sterilization water with 25% (v/v) PEG400 plus 5% DMSO and administered orally. Mice were monitored for side effects every day. Tumor growth and body weight were measured every 3 days. The volume was calculated as follows: tumor size = a × b2/2 (a, long diameter; b, short diameter). The tumor inhibition rate was calculated as (c − d)/c × 100% (c, the tumor volume of the control group; d, the tumor volume of the treatment group). 5.5. Assessments of Pharmacokinetic Properties. The pharmacokinetics analysis of 13ai and 13an was conducted in male Sprague−Dawley rats (Chinese Academy of Medical Science, Beijing, China). Briefly, catheters were surgically placed into the jugular veins of the rats to collect serial blood samples. 13ai was dissolved in saline with 12.5% (v/v) ethanol plus 12.5% kolliphor EL, 13an was dissolved in 20% hydroxypropyl-beta-cyclodextrin saline solution with 12.5% (v/ v) ethanol plus 12.5% kolliphor EL. The animals were administered a single dose of 10 mg/kg 13ai or 13an by oral gavage after fasting overnight. Blood was collected and centrifuged immediately to isolate plasma. The plasma concentrations were determined using high performance liquid chromatography with tandem mass spectrometric detection (3200 QTRAP system, Applied Biosystems). Noncompartmental pharmacokinetic parameters were fitted using DAS software (Enterprise, version 2.0, Mathematical Pharmacology Professional Committee of China). 5.6. Inhibition Evaluation on hERG K+ Channel. The hERG inhibition was measured in hERG-expressing CHO cells using a Qpatch 16× assay as described previously.40 5.7. Microsomal Stability Assay. 13an (1 μM) was incubated with 0.5 mg/mL indicated liver microsomes (liver microsomes of micee, rats, monkeys, and humans were purchased from BD Gentest; liver microsomes of dogs were purchased from GIBCO). NADPH was maintained at 1 mM in 1000 μL of reaction volume. The reaction was then evaluated at 0, 10, 15, 30, 45, and 60 min and was terminated by the addition of acetonitrile. Samples were centrifuged for 5 min at 15000 rpm and the supernatant analyzed using HPLC-MS/MS. Percentage parent remaining was calculated considering percent parent area at 0 min as 100%. 5.8. Kinase Inhibition Assays. Kinase inhibition profiles were determined using KinaseProfiler services provided by Eurofins, and ATP concentrations used are the Km of corresponding kinases. The binding affinities of 13an projected on the human kinome tree were generated using the online Kinome Render program.41 5.9. Colony Formation Assay. Cells were seeded in six-well plates at a density of 5000 per well and treated with vehicle, dasatinib, or 13an the next day. The medium containing vehicle or 13an was replaced every 4 days. Cells were fixed with methanol and stained with crystal violet after treatment for 12 days. 5.10. Apoptosis Assays. A total of 2 × 105 MDA-MB-231 cells were plated in a six-well plate and treated with vehicle, dasatinib, or 9799

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Journal of Medicinal Chemistry

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8.2 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 4.84−4.78 (m, 1H), 3.20 (d, J = 11.6 Hz, 2H), 2.67 (br. s, 2H), 2.49 (s, 3H), 2.31 (dd, J = 22.6, 11.2 Hz, 2H), 2.02 (d, J = 11.4 Hz, 2H). HRMS m/z (ESI) calcd for C27H25F3N7O [M + H]+ 520.2073; found, 520.2088. 5.16.7. 3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4d]pyrimidin-3-yl)ethynyl)-4-chloro-N-(3-(trifluoromethyl)phenyl)benzamide (8g). 1H NMR (400 MHz, DMSO-d6) δ 10.79 (s, 1H), 8.57 (d, J = 1.8 Hz, 1H), 8.30 (s, 1H), 8.27 (s, 1H), 8.19 (br. s, 1H), 8.12−8.07 (m, 2H), 7.84 (d, J = 8.5 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 6.65 (br. s, 1H), 4.93 (t, J = 10.8 Hz, 1H), 3.37 (br. s, 2H), 2.95 (br. s, 2H), 2.64 (s, 3H), 2.40 (dd, J = 22.9, 11.2 Hz, 2H), 2.11 (d, J = 12.0 Hz, 2H). HRMS m/z (ESI) calcd for C27H24ClF3N7O [M + H]+ 554.1683; found, 554.1691. Compound 13a−z and 13aa−aq were prepared in a manner similar to that described for 8c from 12a−z and 7g−n, yield 46.9−67.2%. 5.16.8. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)phenyl)-3-(trifluoromethyl)benzamide (13a). 1H NMR (400 MHz, DMSO-d6) δ 10.63 (s, 1H), 8.33 (s, 1H), 8.29 (d, J = 7.9 Hz, 1H), 8.25 (s, 1H), 8.13 (s, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.82 (t, J = 7.8 Hz, 1H), 7.50 (dt, J = 15.5, 7.7 Hz, 2H), 4.79−4.55 (m, 1H), 2.96 (br. s, 2H), 2.28 (s, 3H), 2.18 (t, J = 8.1 Hz, 4H), 1.92 (d, J = 4.8 Hz, 2H). HRMS m/z (ESI) calcd for C27H25F3N7O [M + H]+ 520.2073; found, 520.2117. 5.16.9. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3-(trifluoromethyl)benzamide (13b). 1H NMR (400 MHz, DMSO-d6) δ 10.56 (s, 1H), 8.32 (s, 1H), 8.29 (d, J = 7.9 Hz, 1H), 8.26 (s, 1H), 8.08 (d, J = 2.2 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.76 (dd, J = 8.3, 2.2 Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H), 4.68−4.61 (m, 1H), 2.94 (d, J = 8.4 Hz, 2H), 2.48 (s, 3H), 2.24 (s, 3H), 2.19 (dd, J = 24.6, 12.0 Hz, 4H), 1.91 (d, J = 9.0 Hz, 2H). HRMS m/z (ESI) calcd for C28H27F3N7O [M + H]+ 534.2229; found, 534.2239. 5.16.10. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-chlorophenyl)-3-(trifluoromethyl)benzamide (13c). 1H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H), 8.32 (s, 1H), 8.29 (d, J = 7.9 Hz, 2H), 8.26 (d, J = 2.4 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.89 (dd, J = 8.9, 2.4 Hz, 1H), 7.83 (t, J = 7.8 Hz, 1H), 7.66 (d, J = 8.9 Hz, 1H), 4.75−4.59 (m, 1H), 2.95 (d, J = 7.5 Hz, 2H), 2.26 (s, 3H), 2.23−2.08 (m, 4H), 1.92 (d, J = 9.3 Hz, 2H). HRMS m/z (ESI) calcd for C27H24ClF3N7O [M + H]+ 554.1683; found, 554.1695. 5.16.11. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-(trifluoromethyl)benzamide (13d). 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 8.27 (s, 1H), 8.18 (d, J = 7.2 Hz, 2H), 8.11 (s, 1H), 7.94 (d, J = 7.2 Hz, 2H), 7.76 (d, J = 7.2 Hz, 1H), 7.37 (d, J = 7.2 Hz, 1H), 4.77−4.68 (m, 1H), 2.87 (br. s, 2H), 2.61 (s, 3H), 2.48 (s, 3H), 2.42−2.23 (m, 4H), 2.09 (d, J = 11.7 Hz, 2H). HRMS m/z (ESI) calcd for C28H27F3N7O [M + H]+ 534.2229; found, 534.2069. 5.16.12. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3-methoxybenzamide (13e). 1H NMR (400 MHz, DMSO-d6) δ 10.31 (s, 1H), 8.29 (s, 1H), 8.12 (s, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 7.4 Hz, 1H), 7.50 (s, 1H), 7.47 (t, J = 7.9 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 5.06−4.87 (m, 1H), 3.85 (s, 3H), 3.58−3.39 (m, 2H), 3.22−2.99 (m, 2H), 2.76 (s, 3H), 2.47 (s, 3H), 2.44−2.28 (m, 2H), 2.22−2.06 (m, 2H). HRMS m/z (ESI) calcd for C28H30N7O2 [M + H]+ 496.2461; found, 496.2453 5.16.13. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-methoxybenzamide (13f). 1H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 8.29 (s, 1H), 8.12 (s, 1H), 7.98 (d, J = 8.6 Hz, 2H), 7.72 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.08 (d, J = 8.6 Hz, 2H), 5.08−4.80 (m, 1H), 3.85 (s, 3H), 3.58−3.44 (m, 2H), 3.21−2.92 (m, 2H), 2.72 (s, 3H), 2.47 (s, 3H), 2.43−2.29 (m, 2H), 2.20−2.08 (m, 2H). HRMS m/z (ESI) calcd for C28H30N7O2 [M + H]+ 496.2461; found, 496.2454 5.16.14. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3-chlorobenzamide (13g). 1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 8.26 (s, 1H), 8.08 (s, 1H), 8.03 (s, 1H), 7.94 (d, J = 7.5 Hz, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.36 (d, J =

described as s (singlet), br. s (broad singlet), t (triplet), q (quartet), and m (multiplet). Chemical shifts (δ) are listed in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. Mass spectral (MS) data were acquired on a Waters Q-TOF Premier mass spectrometer (Micromass, Manchester, U.K.). 5.16.1. 3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4d]pyrimidin-3-yl)ethynyl)-4-methyl-N-(3-(trifluoromethyl)phenyl)benzamide (8c). 3-Ethynyl-4-methyl-N-(3-(trifluoromethyl)phenyl)benzamide 4b (159 mg, 0.53 mmol, 1.05 equiv), 3-iodo-1-(1methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 7i (179.1 mg, 0.5 mmol, 1.0 equiv), CuI (9.5 mg, 0.05 mmol, 0.1 equiv), and (PPh3)2PdCl2 (35.1 mg, 0.05 mmol, 0.1 equiv) were suspended in anhydrous DMF (5 mL). The mixture underwent three cycles of vacuum/filling with N2. Then, DIPEA (0.16 mL, 1 mmol, 2.0 equiv) was added with a syringe. The mixture was stirred at 80 °C for 8 h and then quenched with water. DCM (3× 30 mL) and 5% ammonia (30 mL) were added for extraction. The combined organic layer was concentrated in vacuo, and the crude product was purified using silica gel chromatography with a methanol/dichloromethane gradient to afford the title compound 8c as a white solid (131 mg, yield 49.1%). 1 H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 8.36 (d, J = 1.6 Hz, 1H), 8.26 (s, 2H), 8.09 (d, J = 8.2 Hz, 1H), 7.97 (dd, J = 8.2, 1.6 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.56 (d, J = 8.1 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 4.69−4.56 (m, 1H), 2.92 (d, J = 10.1 Hz, 2H), 2.59 (s, 3H), 2.23 (s, 3H), 2.20−2.02 (m, 4H), 1.90 (d, J = 13.2 Hz, 2H). HRMS m/z (ESI) calcd for C28H27F3N7O [M + H]+ 534.2229; found, 534.2240. Compounds 8a−b and 8d−g were prepared in a manner similar to that described for 8c from 7f, 7i−l, and 4a−c. Yield 43.2−62.3%. 5.16.2. 3-((4-Amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methyl-N-(3-(trifluoromethyl)phenyl)benzamide (8a). 1 H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.35 (d, J = 1.7 Hz, 1H), 8.27 (s, 1H), 8.26 (s, 1H), 8.09 (d, J = 8.2 Hz, 1H), 7.97 (dd, J = 8.0, 1.8 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.55 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H), 4.38 (q, J = 7.2 Hz, 2H), 2.59 (s, 3H), 1.42 (t, J = 7.2 Hz, 3H). HRMS m/z (ESI) calcd for C24H20F3N6O [M + H]+ 465.1651; found, 465.1650. 5.16.3. (R)-3-((4-amino-1-(1-methylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methyl-N-(3-(trifluoromethyl)phenyl)benzamide (8b). 1H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.35 (d, J = 1.9 Hz, 1H), 8.27 (s, 2H), 8.09 (d, J = 8.2 Hz, 1H), 7.97 (dd, J = 8.0, 1.9 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.55 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 5.43−5.36 (m, 1H), 3.05−2.97 (m, 1H), 2.78−2.63 (m, 3H), 2.59 (s, 3H), 2.41−2.36 (m, 1H), 2.32 (s, 3H), 2.29−2.21 (m, 1H). HRMS m/z (ESI) calcd for C27H25F3N7O [M + H]+ 520.2073; found, 520.2073. 5.16.4. 3-((4-Amino-1-((1-methylpyrrolidin-3-yl)methyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methyl-N-(3(trifluoromethyl)phenyl)benzamide (8d). 1H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 8.35 (d, J = 1.7 Hz, 1H), 8.28 (s, 1H), 8.26 (s, 1H), 8.09 (d, J = 8.1 Hz, 1H), 7.97 (dd, J = 8.0, 1.7 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.55 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H), 4.30 (d, J = 7.6 Hz, 2H), 2.82−2.68 (m, 1H), 2.59 (s, 3H), 2.55− 2.52 (m, 1H), 2.42−2.31 (m, 3H), 2.21 (s, 3H), 1.91−1.75 (m, 1H), 1.57−1.49 (m, 1H). HRMS m/z (ESI) calcd for C28H27F3N7O [M + H]+ 534.2229; found, 534.2232. 5.16.5. 3-((4-Amino-1-((1-methylpiperidin-4-yl)methyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methyl-N-(3(trifluoromethyl)phenyl)benzamide hydrochloride salt (8e). 1H NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H), 10.21 (s, 1H), 8.38 (d, J = 1.8 Hz, 1H), 8.28 (s, 2H), 8.12 (d, J = 8.2 Hz, 1H), 8.00 (dd, J = 8.0, 1.8 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.55 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 4.30 (d, J = 6.1 Hz, 2H), 3.29 (s, 2H), 2.85 (s, 2H), 2.66 (s, 3H), 2.58 (s, 3H), 2.20 (s, 1H), 1.69 (d, J = 13.0 Hz, 2H), 1.60−1.52 (m, 2H). HRMS m/z (ESI) calcd for C28H27F3N7O [M + H]+ 548.2386; found, 548.2383. 5.16.6. 3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4d]pyrimidin-3-yl)ethynyl)-N-(3-(trifluoromethyl)phenyl)benzamide (8f). 1H NMR (400 MHz, DMSO-d6) δ 10.74 (s, 1H), 8.41 (s, 1H), 8.28 (s, 1H), 8.26 (s, 1H), 8.10 (d, J = 8.4 Hz, 1H), 8.06 (d, J = 7.9 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.62 (t, J = 9800

DOI: 10.1021/acs.jmedchem.6b00943 J. Med. Chem. 2016, 59, 9788−9805

Journal of Medicinal Chemistry

Article

8.2 Hz, 1H), 4.67−4.60 (m, 1H), 2.94 (d, J = 8.3 Hz, 2H), 2.48 (s, 3H), 2.26 (s, 3H), 2.23−2.05 (m, 4H), 1.91 (d, J = 9.9 Hz, 2H). HRMS m/z (ESI) calcd for C27H27ClN7O [M + H]+ 500.1966; found, 500.1956. 5.16.15. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-chlorobenzamide (13h). 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 8.30 (s, 1H), 8.19 (s, 1H), 8.13 (d, J = 9.0 Hz, 1H), 8.11 (s, 1H), 8.02 (s, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.39 (d, J = 8.5 Hz, 1H), 5.18−4.88 (m, 1H), 3.54 (d, J = 10.8 Hz, 2H), 3.29−3.13 (m, 2H), 2.82 (s, 3H), 2.49 (s, 3H), 2.46−2.29 (m, 2H), 2.19 (d, J = 12.3 Hz, 2H). HRMS m/z (ESI) calcd for C27H27ClN7O [M + H]+ 500.1966; found, 500.1968. 5.16.16. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3-fluorobenzamide (13i). 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.26 (s, 1H), 8.08 (d, J = 1.9 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 9.3 Hz, 1H), 7.75 (dd, J = 8.3, 1.9 Hz, 1H), 7.61 (dd, J = 13.9, 7.9 Hz, 1H), 7.48 (dd, J = 11.7, 5.3 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 4.76−4.52 (m, 1H), 2.95 (d, J = 6.7 Hz, 2H), 2.48 (s, 3H), 2.27 (s, 3H), 2.21− 2.16 (m, 4H), 1.91 (d, J = 7.8 Hz, 2H). HRMS m/z (ESI) calcd for C27H27FN7O [M + H]+ 484.2261; found, 484.2242. 5.16.17. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3(trifluoromethoxy)benzamide (13j). 1H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.29 (s, 1H), 8.11 (d, J = 1.8 Hz, 1H), 8.04 (d, J = 7.7 Hz, 1H), 7.93 (s, 1H), 7.71 (t, J = 7.9 Hz, 2H), 7.63 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 5.03−4.92 (m, 1H), 3.51 (d, J = 11.7 Hz, 2H), 3.18 (t, J = 11.0 Hz, 2H), 2.79 (s, 3H), 2.48 (s, 3H), 2.39 (dd, J = 23.1, 11.4 Hz, 2H), 2.17 (d, J = 12.2 Hz, 2H). HRMS m/z (ESI) calcd for C28H27F3N7O2 [M + H]+ 550.2178; found, 550.2185. 5.16.18. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3-methylbenzamide (13k). 1H NMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H), 8.26 (s, 1H), 8.09 (s, 1H), 7.79 (s, 1H), 7.76 (d, J = 6.9 Hz, 2H), 7.42 (s, 2H), 7.34 (d, J = 8.2 Hz, 1H), 4.73−4.54 (m, 1H), 2.92 (d, J = 9.4 Hz, 2H), 2.47 (s, 3H), 2.41 (s, 3H), 2.24 (s, 3H), 2.22−2.03 (m, 4H), 1.90 (d, J = 10.3 Hz, 2H). HRMS m/z (ESI) calcd for C28H30N7O [M + H]+ 480.2512; found, 480.2500. 5.16.19. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3-(tert-butyl)benzamide (13l). 1H NMR (400 MHz, DMSO-d6) δ 10.30 (s, 1H), 8.29 (s, 1H), 8.11 (d, J = 2.0 Hz, 1H), 7.96 (s, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.75 (dd, J = 8.3, 2.0 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.47 (t, J = 7.8 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 5.04−4.98 (m, 1H), 3.56 (br. s, 2H), 3.28 (br. s, 2H), 2.84 (s, 3H), 2.48 (s, 3H), 2.43 (d, J = 12.9 Hz, 2H), 2.20 (d, J = 11.9 Hz, 2H), 1.35 (s, 9H). HRMS m/z (ESI) calcd for C31H36N7O [M + H]+ 522.2981; found, 522.2980. 5.16.20. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-2-fluoro-3(trifluoromethyl)benzamide (13m). 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.29 (s, 1H), 8.11 (d, J = 2.0 Hz, 1H), 8.00 (d, J = 8.5 Hz, 2H), 7.72 (dd, J = 8.4, 2.0 Hz, 1H), 7.63 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 8.4 Hz, 1H), 5.04−4.88 (m, 1H), 3.47 (d, J = 11.4 Hz, 2H), 3.11 (t, J = 11.0 Hz, 2H), 2.76 (s, 3H), 2.47 (s, 3H), 2.38 (t, J = 12.0 Hz, 2H), 2.16 (d, J = 12.0 Hz, 2H). HRMS m/z (ESI) calcd for C28H26F4N7O [M + H]+ 552.2135; found, 552.2135 5.16.21. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-fluoro-3(trifluoromethyl)benzamide (13n). 1H NMR (400 MHz, DMSO-d6) δ 10.56 (s, 1H), 8.39−8.35 (m, 2H), 8.29 (s, 1H), 8.09 (d, J = 2.0 Hz, 1H), 7.80−7.67 (m, 2H), 7.38 (d, J = 8.4 Hz, 1H), 5.00−4.84 (m, 1H), 3.39 (br. s, 2H), 3.00 (br. s, 2H), 2.69 (s, 3H), 2.48 (s, 3H), 2.35 (d, J = 12.0 Hz, 2H), 2.13 (d, J = 11.7 Hz, 2H). HRMS m/z (ESI) calcd for C28H26F4N7O [M + H]+ 552.2135; found, 552.2145. 5.16.22. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3-fluoro-5(trifluoromethyl)benzamide (13o). 1H NMR (400 MHz, DMSO-d6) δ 10.73 (s, 1H), 8.29 (s, 1H), 8.06 (d, J = 2.0 Hz, 1H), 8.05−7.94 (m, 2H), 7.64 (dd, J = 8.3, 2.0 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H),5.03−4.87 (m, 1H), 3.49 (d, J = 11.6 Hz, 2H), 3.15 (t, J

= 11.0 Hz, 2H), 2.78 (s, 3H), 2.48 (s, 3H), 2.38 (dd, J = 23.1, 11.3 Hz, 2H), 2.16 (d, J = 12.0 Hz, 2H). HRMS m/z (ESI) calcd for C28H26F4N7O [M + H]+ 552.2135; found, 552.2127. 5.16.23. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-2-fluoro-5(trifluoromethyl)benzamide (13p). 1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 8.26 (s, 1H), 8.09 (d, J = 4.6 Hz, 1H), 8.02 (s, 2H), 7.79−7.57 (m, 2H), 7.37 (d, J = 4.0 Hz, 1H), 4.67−4.60 (m, 1H), 2.93 (d, J = 8.3 Hz, 2H), 2.48 (s, 3H), 2.25 (s, 3H), 2.21−2.05 (m, 4H), 1.90 (d, J = 10.7 Hz, 2H). HRMS m/z (ESI) calcd for C28H26F4N7O [M + H]+ 552.2135; found, 552.2173. 5.16.24. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3-chloro-5(trifluoromethyl)benzamide (13q). 1H NMR (400 MHz, DMSO-d6) δ 10.62 (s, 1H), 8.34 (s, 1H), 8.31 (s, 1H), 8.27 (s, 1H), 8.15 (s, 1H), 8.10 (d, J = 1.8 Hz, 1H), 7.72 (dd, J = 8.3, 1.8 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 5.03−4.98 (m, 1H), 3.56 (d, J = 9.5 Hz, 2H), 3.26 (d, J = 11.0 Hz, 2H), 2.85 (s, 3H), 2.49 (s, 3H), 2.40 (dd, J = 23.4, 11.6 Hz, 2H), 2.20 (d, J = 11.9 Hz, 2H). HRMS m/z (ESI) calcd for C28H26ClF3N7O [M + H]+ 568.1839; found, 568.1842. 5.16.25. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-chloro-3(trifluoromethyl)benzamide (13r). 1H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 8.41 (s, 1H), 8.28 (d, J = 10.8 Hz, 2H), 8.11 (s, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 5.03−4.97 (m, 1H), 3.55 (d, J = 10.6 Hz, 2H), 3.24 (d, J = 11.2 Hz, 2H), 2.83 (s, 3H), 2.48 (s, 3H), 2.39 (dd, J = 21.0, 9.5 Hz, 2H), 2.20 (d, J = 12.6 Hz, 2H). HRMS m/z (ESI) calcd for C28H26ClF3N7O [M + H]+ 568.1839; found, 568.1836. 5.16.26. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-methyl-3(trifluoromethyl)benzamide (13s). 1H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.28 (s, 1H), 8.27 (s, 1H), 8.19 (d, J = 7.5 Hz, 1H), 8.11 (s, 1H), 7.75 (d, J = 7.5 Hz, 1H), 7.65 (d, J = 8.3 Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 5.06−4.80 (m, 1H), 3.38 (br. s, 2H), 3.00 (br. s, 2H), 2.68 (s, 3H), 2.54 (s, 3H), 2.48 (s, 3H), 2.39 (d, J = 12.9 Hz, 2H), 2.12 (d, J = 12.8 Hz, 2H). HRMS m/z (ESI) calcd for C29H29F3N7O [M + H]+ 548.2386; found, 548.2392. 5.16.27. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-methoxy-3(trifluoromethyl)benzamide (13t). 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.49 (br. s, 1H), 8.30 (s, 2H), 8.27 (s, 1H), 8.11 (s, 1H), 7.72 (d, J = 7.9 Hz, 1H), 7.45 (d, J = 8.7 Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 6.70 (br. s, 1H), 5.02−4.97 (m, 1H), 4.00 (s, 3H), 3.54 (d, J = 9.9 Hz, 2H), 3.25 (d, J = 11.4 Hz, 2H), 2.83 (s, 3H), 2.48 (s, 3H), 2.40 (dd, J = 11.4, 12.9 Hz, 2H), 2.19 (d, J = 12.4 Hz, 2H). HRMS m/ z (ESI) calcd for C29H29F3N7O2 [M + H]+ 564.2335; found, 564.2333. 5.16.28. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3,5-bis(trifluoromethyl)benzamide (13u). 1H NMR (400 MHz, DMSO-d6) δ 10.71 (s, 1H), 8.57 (s, 2H), 8.33 (s, 1H), 8.22 (s, 1H), 8.00 (d, J = 1.5 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 4.64− 4.58 (m, 1H), 2.90 (d, J = 7.9 Hz, 2H), 2.44 (s, 3H), 2.21 (s, 3H), 2.11 (q, J = 13.2 Hz, 4H), 1.87 (d, J = 9.0 Hz, 2H). HRMS m/z (ESI) calcd for C29H26F6N7O [M + H]+ 602.2103; found, 602.2123. 5.16.29. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3-chloro-2-fluoro-5(trifluoromethyl)benzamide (13v). 1H NMR (400 MHz, DMSO-d6) δ 10.79 (s, 1H), 8.33 (d, J = 4.5 Hz, 1H), 8.30 (s, 1H), 8.09 (d, J = 3.4 Hz, 1H), 8.04 (d, J = 2.0 Hz, 1H), 7.63 (dd, J = 8.4, 2.0 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 4.94 (m, 1H), 3.42 (s, 2H), 3.04 (s, 2H), 2.73 (s, 3H), 2.48 (s, 3H), 2.35 (dd, J = 22.9, 11.0 Hz, 2H), 2.14 (d, J = 12.5 Hz, 2H). HRMS m/z (ESI) calcd for C28H25ClF4N7O [M + H]+ 586.1745; found, 586.1752. 5.16.30. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-6-(trifluoromethyl)picolinamide (13w). 1H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 8.44−8.33 (m, 2H), 8.29 (s, 1H), 8.23−8.16 (m, 2H), 7.87 (dd, J = 8.4, 1.8 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H),5.06−4.78 (m, 1H), 3.44 (d, J = 11.5 Hz, 2H), 3.06 (t, J = 11.1 Hz, 2H), 2.73 (s, 3H), 2.49 (s, 3H), 2.37 (dd, J = 22.7, 11.1 Hz, 2H), 2.15 (d, J = 11.8 Hz, 2H). 9801

DOI: 10.1021/acs.jmedchem.6b00943 J. Med. Chem. 2016, 59, 9788−9805

Journal of Medicinal Chemistry

Article

HRMS m/z (ESI) calcd for C27H26F3N8O [M + H]+ 535.2182; found, 535.2178. 5.16.31. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-5-(trifluoromethyl)nicotinamide (13x). 1H NMR (400 MHz, DMSO-d6) δ 10.71 (s, 1H), 9.40 (s, 1H), 9.21 (s, 1H), 8.71 (s, 1H), 8.27 (s, 1H), 8.08 (s, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 4.74−4.59 (m, 1H), 2.97 (s, 2H), 2.49 (s, 3H), 2.29 (s, 3H), 2.20 (br. s, 4H), 1.92 (s, 2H). HRMS m/z (ESI) calcd for C27H26F3N8O [M + H]+ 535.2182; found, 535.2174. 5.16.32. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide (13y). 1H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H), 8.99 (d, J = 4.7 Hz, 1H), 8.41 (s, 1H), 8.26 (s, 2H), 8.11 (s, 1H), 7.79 (d, J = 8.1 Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 4.87 (t, J = 10.8 Hz, 1H), 3.36 (d, J = 7.3 Hz, 2H), 2.90 (br. s, 2H), 2.60 (s, 3H), 2.47 (s, 3H), 2.38 (d, J = 11.5 Hz, 2H), 2.07 (d, J = 11.3 Hz, 2H). HRMS m/z (ESI) calcd for C27H26F3N8O [M + H]+ 535.2182; found, 535.2186. 5.16.33. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-5-(tert-butyl)isoxazole-3-carboxamide (13z). 1H NMR (400 MHz, DMSO-d6) δ 10.71 (s, 1H), 8.27 (s, 1H), 8.06 (d, J = 2.1 Hz, 1H), 7.70 (dd, J = 8.4, 2.1 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H),6.67 (s, 1H), 4.97 (t, J = 11.5 Hz, 1H), 3.51 (br. s, 2H), 3.22 (br. s, 2H), 2.80 (s, 3H), 2.45 (s, 3H), 2.33 (dd, J = 25.3, 10.0 Hz, 2H), 2.16 (d, J = 12.2 Hz, 2H), 1.34 (s, 9H). HRMS m/z (ESI) calcd for C28H33N8O2 [M + H]+ 513.2726; found, 513.2726. 5.16.34. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-2-(tert-butyl)thiazole-5-carboxamide (13aa). 1H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.30 (d, J = 7.6 Hz, 2H), 8.14 (s, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.36 (d, J = 7.9 Hz, 1H), 5.08−4.92 (m, 1H), 3.54 (d, J = 7.9 Hz, 2H), 3.30−3.15 (m, 2H), 2.82 (s, 3H), 2.47 (s, 3H), 2.43 (d, J = 11.2 Hz, 2H), 2.19 (d, J = 11.5 Hz, 2H), 1.47 (s, 9H). HRMS m/z (ESI) calcd for C28H33N8OS [M + H]+ 529.2498; found, 529.2495. 5.16.35. N-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3-(tert-butyl)-1methyl-1H-pyrazole-5-carboxamide (13ab). 1H NMR (400 MHz, DMSO-d6) δ 10.19 (s, 1H), 8.26 (s, 1H), 8.04 (d, J = 2.0 Hz, 1H), 7.70 (dd, J = 8.3, 2.0 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 6.97 (s, 1H), 4.74−4.54 (m, 1H), 4.04 (s, 3H), 2.93 (d, J = 9.9 Hz, 2H), 2.47 (s, 3H), 2.24 (s, 3H), 2.22−2.02 (m, 4H), 1.90 (d, J = 10.9 Hz, 2H), 1.29 (s, 9H). HRMS m/z (ESI) calcd for C29H36N9O [M + H]+ 526.3043; found, 526.3041. 5.16.36. N-(3-((4-Amino-1-((1-methylpiperidin-4-yl)methyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3(trifluoromethyl)benzamide hydrochloride salt (13ac). 1H NMR (400 MHz, DMSO-d6) δ 10.63 (s, 1H), 10.13 (s, 1H), 8.33 (s, 1H), 8.31 (d, J = 8.2 Hz, 1H), 8.28 (s, 1H), 8.13 (d, J = 2.2 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.75 (dd, J = 8.3, 2.2 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 4.30 (s, 2H), 3.32−3.25 (m, 2H), 2.87 (s, 2H), 2.67 (s, 3H), 2.48 (s, 3H), 2.20 (s, 1H), 1.70 (d, J = 12.9 Hz, 2H), 1.60−1.52 (m, 2H). HRMS m/z (ESI) calcd for C29H29F3N7O [M + H]+ 548.2386; found, 548.2384. 5.16.37. N-(3-((4-Amino-1-((1-methylpyrrolidin-3-yl)methyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3(trifluoromethyl)benzamide (13ad). 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 8.33 (s, 1H), 8.31 (s, 1H), 8.28 (s, 1H), 8.11 (d, J = 2.2 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.76 (dd, J = 8.3, 2.2 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 4.44−4.30 (m, 2H), 2.92−2.71 (m, 3H), 2.71−2.58 (m, 2H), 2.48 (s, 3H), 2.42 (s, 3H), 2.00−1.85 (m, 1H), 1.70−1.56 (m, 1H). HRMS m/z (ESI) calcd for C28H27F3N7O [M + H]+ 534.2229; found, 534.2231. 5.16.38. (R)-N-(3-((4-amino-1-(1-methylpyrrolidin-3-yl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3(trifluoromethyl)benzamide (13ae). 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 8.32 (s, 1H), 8.29 (s, 1H), 8.27 (s, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.75 (dd, J = 8.3, 2.2 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 5.47−5.34 (m, 1H), 3.08 (t, J = 8.6 Hz, 1H), 2.85−2.69 (m, 3H), 2.49 (s, 3H), 2.36 (s,

3H), 2.34−2.22 (m, 2H). HRMS m/z (ESI) calcd for C27H25F3N7O [M + H]+ 520.2073; found, 520.2071. 5.16.39. (R)-N-(3-((4-Amino-1-(3-hydroxy-2-methylpropyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3(trifluoromethyl)benzamide (13af). 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 8.32 (s, 1H), 8.29 (s, 1H), 8.27 (s, 1H), 8.08 (d, J = 2.2 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.76 (dd, J = 8.3, 2.2 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 4.67 (s, 1H), 4.36 (dd, J = 13.7, 6.1 Hz, 1H), 4.18 (dd, J = 13.7, 8.1 Hz, 1H), 3.34 (s, 2H), 2.48 (s, 3H), 2.28−2.20 (m, 1H), 0.79 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 164.00, 157.90, 156.47, 153.51, 136.67, 135.64, 135.54, 131.85, 130.02, 129.79, 129.22 (d, J = 32.0 Hz), 128.24 (d, J = 3.0 Hz), 125.32, 124.22 (d, J = 3.8 Hz), 123.87, 122.62, 121.84, 121.32, 100.44, 91.90, 84.65, 63.67, 49.92, 36.37, 19.86, 14.48. HRMS m/z (ESI) calcd for C26H24F3N6O2 [M + H]+ 509.1913; found, 509.1912. 5.16.40. (S)-N-(3-((4-Amino-1-(3-hydroxy-2-methylpropyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3(trifluoromethyl)benzamide (13ag). 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 8.32 (s, 1H), 8.29 (s, 1H), 8.27 (s, 1H), 8.08 (d, J = 2.2 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.76 (dd, J = 8.3, 2.2 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 4.67 (t, J = 5.3 Hz, 1H), 4.36 (dd, J = 13.7, 6.1 Hz, 1H), 4.18 (dd, J = 13.7, 8.1 Hz, 1H), 3.35 (d, J = 5.6 Hz, 2H), 2.48 (s, 3H), 2.28−2.20 (m, 1H), 0.79 (d, J = 6.8 Hz, 3H). HRMS m/z (ESI) calcd for C26H24F3N6O2 [M + H]+ 509.1913; found, 509.1906. 5.16.41. N-(3-((4-Amino-1-(cis-4-hydroxycyclohexyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3(trifluoromethyl)benzamide (13ah). 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 8.36−8.22 (m, 3H), 8.08 (d, J = 1.9 Hz, 1H), 7.99 (d, J = 7.7 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.76 (dd, J = 8.3, 1.9 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 4.75−4.61 (m, 1H), 4.51 (d, J = 2.5 Hz, 1H), 3.90 (s, 1H), 2.42−2.26 (m, 2H), 1.82 (d, J = 11.7 Hz, 2H), 1.64 (t, J = 12.8 Hz, 4H). HRMS m/z (ESI) calcd for C28H26F3N6O2 [M + H]+ 535.2069; found, 535.2070. 5.16.42. N-(3-((4-Amino-1-(trans-4-hydroxycyclohexyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3(trifluoromethyl)benzamide (13ai). 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 8.32−8.26 (M, 3H), 8.08 (s, 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 4.73 (d, J = 4.0 Hz, 1H), 4.64 (t, J = 11.1 Hz, 1H), 3.61−3.47 (m, 1H), 2.47 (s, 3H), 2.09−1.83 (m, 6H), 1.49−1.31 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 164.01, 157.87, 156.24, 152.51, 136.67, 135.61, 135.54, 131.86, 130.02, 129.80, 129.21 (d, J = 32.0 Hz), 128.25 (d, J = 4.0 Hz), 125.33, 125.03, 124.22 (d, J = 3.9 Hz), 123.86, 122.62, 121.81, 121.32, 100.74, 91.79, 84.70, 67.87, 55.42, 34.08, 29.81, 19.88. HRMS m/z (ESI) calcd for C28H26F3N6O2 [M + H]+ 535.2069; found, 535.2067. 5.16.43. (R)-N-(3-((4-Amino-1-(3-hydroxy-2-methylpropyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-2-(tertbutyl)thiazole-5-carboxamide (13aj). 1H NMR (400 MHz, DMSOd6) δ 10.01 (s, 1H), 8.32 (s, 1H), 8.27 (s, 1H), 8.12 (d, J = 1.5 Hz, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 4.69 (s, 1H), 4.36 (dd, J = 13.6, 5.9 Hz, 1H), 4.18 (dd, J = 13.6, 8.2 Hz, 1H), 3.49− 3.39 (m, 2H), 2.48 (s, 3H), 2.28−2.20 (m, 1H), 1.47 (s, 9H), 0.79 (d, J = 6.8 Hz, 3H). HRMS m/z (ESI) calcd for C26H30N7O2S [M + H]+ 504.2182; found, 504.2184. 5.16.44. N-(3-((4-Amino-1-(trans-4-hydroxycyclohexyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3-(tertbutyl)benzamide (13ak). 1H NMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H), 8.26 (s, 1H), 8.07 (d, J = 2.1 Hz, 1H), 7.95 (s, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.75 (dd, J = 8.4, 2.1 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.34 (d, J = 8.5 Hz, 1H), 4.80−4.56 (m, 2H), 3.55 (t, J = 10.4 Hz, 1H), 2.47 (s, 3H), 2.11−1.83 (m, 6H), 1.51−1.37 (m, 2H), 1.35 (s, 9H). HRMS m/z (ESI) calcd for C31H35N6O2 [M + H]+ 523.2821; found, 523.2844. 5.16.45. (R)-N-(3-((4-Amino-1-(3-hydroxy-2-methylpropyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-methyl3-(trifluoromethyl)benzamide (13al). 1H NMR (400 MHz, DMSOd6) δ 10.48 (s, 1H), 8.27 (s, 2H), 8.18 (d, J = 7.9 Hz, 1H), 8.08 (d, J = 2.2 Hz, 1H), 7.75 (dd, J = 8.3, 2.2 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 9802

DOI: 10.1021/acs.jmedchem.6b00943 J. Med. Chem. 2016, 59, 9788−9805

Journal of Medicinal Chemistry

Article

7.36 (d, J = 8.5 Hz, 1H), 4.68 (s, 1H), 4.36 (dd, J = 13.7, 6.1 Hz, 1H), 4.18 (dd, J = 13.7, 8.1 Hz, 1H), 3.34 (s, 2H), 2.54 (s, 3H), 2.48 (s, 3H), 2.28−2.20 (m, 1H), 0.79 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.95, 157.91, 156.47, 153.51, 139.97, 136.74, 135.50, 132.61, 132.50, 131.54, 129.99, 127.48 (d, J = 30.0 Hz), 125.48 (d, J = 30.0 Hz), 124.91 (q, J = 5.5 Hz), 123.84, 122.92, 121.82, 121.28, 100.46, 91.92, 84.62, 63.67, 49.92, 36.37, 19.85, 18.82, 14.47. HRMS m/z (ESI) calcd for C27H26F3N6O2 [M + H]+ 523.2069; found, 523.2066. 5.16.46. (S)-N-(3-((4-Amino-1-(3-hydroxy-2-methylpropyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-methyl3-(trifluoromethyl)benzamide (13am). 1H NMR (400 MHz, DMSOd6) δ 10.48 (s, 1H), 8.27 (s, 2H), 8.18 (d, J = 7.9 Hz, 1H), 8.07 (d, J = 2.2 Hz, 1H), 7.75 (dd, J = 8.3, 2.2 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 4.67 (t, J = 5.3 Hz, 1H), 4.36 (dd, J = 13.7, 6.0 Hz, 1H), 4.18 (dd, J = 13.7, 8.1 Hz, 1H), 3.35 (d, J = 5.6 Hz, 2H), 2.54 (s, 3H), 2.48 (s, 3H), 2.28−2.20 (m, 1H), 0.79 (d, J = 6.8 Hz, 3H). HRMS m/z (ESI) calcd for C27H26F3N6O2 [M + H]+ 523.2069; found, 523.2076. 5.16.47. N-(3-((4-Amino-1-(trans-4-hydroxycyclohexyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-methyl3-(trifluoromethyl)benzamide (13an). 1H NMR (400 MHz, DMSOd6) δ 10.48 (s, 1H), 8.27 (s, 1H), 8.26 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 2.2 Hz, 1H), 7.74 (dd, J = 8.3, 2.2 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 4.71 (s, 1H), 4.69−4.56 (m, 1H), 3.63−3.47 (m, 1H), 2.54 (s, 3H), 2.47 (s, 3H), 2.10−1.83 (m, 6H), 1.46−1.36 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 163.95, 157.67, 155.96, 152.43, 139.98, 136.76, 135.47, 132.61, 132.49, 131.54, 129.97, 127.48 (d, J = 30.0 Hz), 125.39 (d, J = 50.0 Hz), 124.92 (q, J = 5.7 Hz), 123.83, 122.92, 121.80, 121.27, 100.73, 91.90, 84.61, 67.87, 55.45, 34.08, 29.82, 19.86, 18.80. HRMS m/z (ESI) calcd for C29H28F3N6O2 [M + H]+ 549.2226; found, 549.2229. 5.16.48. (R)-N-(3-((4-Amino-1-(3-hydroxy-2-methylpropyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-chloro-3(trifluoromethyl)benzamide (13ao). 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 8.41 (s, 1H), 8.31 (s, 1H), 8.28 (d, J = 8.4 Hz, 1H), 8.06 (d, J = 1.8 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.74 (dd, J = 8.4, 1.8 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 4.67 (t, J = 5.3 Hz, 1H), 4.36 (dd, J = 13.7, 6.0 Hz, 1H), 4.18 (dd, J = 13.6, 8.1 Hz, 1H), 3.35 (d, J = 5.6 Hz, 2H), 2.48 (s, 3H), 2.24 (dq, J = 12.9, 6.4 Hz, 1H), 0.79 (d, J = 6.8 Hz, 3H). HRMS m/z (ESI) calcd for C26H23ClF3N6O2 [M + H]+ 543.1523; found, 543.1522. 5.16.49. (S)-N-(3-((4-Amino-1-(3-hydroxy-2-methylpropyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-chloro-3(trifluoromethyl)benzamide (13ap). 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 8.41 (d, J = 1.5 Hz, 1H), 8.28 (d, J = 8.4 Hz, 2H), 8.06 (d, J = 2.0 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.74 (dd, J = 8.4, 2.0 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 4.67 (t, J = 5.3 Hz, 1H), 4.36 (dd, J = 13.7, 6.1 Hz, 1H), 4.18 (dd, J = 13.7, 8.1 Hz, 1H), 3.35 (d, J = 5.8 Hz, 2H), 2.48 (s, 3H), 2.24 (dq, J = 13.1, 6.4 Hz, 1H), 0.79 (d, J = 6.8 Hz, 3H). HRMS m/z (ESI) calcd for C26H23ClF3N6O2 [M + H]+ 543.1523; found, 543.1524. 5.16.50. N-(3-((4-Amino-1-(trans-4-hydroxycyclohexyl)-1Hpyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-4-chloro-3(trifluoromethyl)benzamide (13aq). 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 8.41 (s, 1H), 8.28 (d, J = 5.9 Hz, 2H), 8.06 (s, 1H), 7.94 (d, J = 7.9 Hz, 1H), 7.73 (d, J = 7.7 Hz, 1H), 7.37 (d, J = 7.9 Hz, 1H),4.70 (s, 1H), 4.64 (d, J = 9.9 Hz, 1H), 3.56 (s, 1H), 2.14−1.81 (m, 6H), 1.53−1.31 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 163.09, 157.85, 156.21, 152.50, 136.52, 135.73, 134.01, 133.89, 133.38, 132.00, 130.03, 127.03 (d, J = 5.0 Hz), 126.69 (d, J = 31.0 Hz), 125.02, 123.96 (d, J = 17.0 Hz), 121.79, 121.32 (d, J = 6.0 Hz), 100.74, 91.74, 84.74, 67.87, 55.42, 34.08, 29.81, 19.88. HRMS m/z (ESI) calcd for C28H25ClF3N6O2 [M + H]+ 569.1680; found, 569.1679. Compounds 17a−c were prepared in a manner similar to that described for 8c from 7i and 16a−c; yield 47.9−55.0%. 5.16.51. 1-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)phenyl)-3-(3-(trifluoromethyl)phenyl)urea (17a). 1H NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1H), 9.25 (s, 1H), 8.26 (s, 1H), 8.04 (s, 1H), 7.87 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.58−7.47 (m, 2H), 7.38 (d, J = 7.1 Hz, 2H), 7.33 (d, J = 7.5 Hz, 1H),

4.89−4.65 (m, 1H), 3.42 (br. s, 2H), 3.12 (d, J = 7.1 Hz, 2H), 2.43 (s, 3H), 2.25 (dd, J = 22.2, 11.0 Hz, 2H), 1.99 (d, J = 10.7 Hz, 2H). HRMS m/z (ESI) calcd for C27H26F3N8O [M + H]+ 535.2182; found, 535.2200. 5.16.52. 1-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3-(3(trifluoromethyl)phenyl)urea (17b). 1H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 8.93 (s, 1H), 8.26 (s, 1H), 8.03 (s, 1H), 7.81 (d, J = 2.2 Hz, 1H), 7.59 (d, J = 8.3 Hz, 1H), 7.52 (t, J = 7.9 Hz, 1H), 7.43 (dd, J = 8.3, 2.2 Hz, 1H), 7.32 (d, J = 7.5 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 4.79−4.49 (m, 1H), 2.99 (br. s, 2H), 2.44 (s, 3H), 2.31 (s, 3H), 2.20 (dd, J = 18.5, 7.4 Hz, 4H), 1.93 (d, J = 11.4 Hz, 2H). HRMS m/z (ESI) calcd for C28H28F3N8O [M + H]+ 549.2338; found, 549.2357. 5.16.53. 1-(3-((4-Amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-chlorophenyl)-3-(3(trifluoromethyl)phenyl)urea (17c). 1H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 9.51 (s, 1H), 8.29 (s, 1H), 8.02 (s, 1H), 7.98 (t, J = 1.4 Hz, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.55 (d, J = 1.4 Hz, 2H), 7.52 (d, J = 8.1 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H), 4.83 (t, J = 11.2 Hz, 1H), 3.24 (d, J = 10.6 Hz, 2H), 2.70 (br. s, 2H), 2.52 (s, 3H), 2.31 (dd, J = 22.7, 10.7 Hz, 2H), 2.06 (d, J = 12.1 Hz, 2H). HRMS m/z (ESI) calcd for C27H25ClF3N8O [M + H]+ 569.1792; found, 569.1805.



ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00943. Chemical data of key intermediates, acute toxicity evaluation of compound 1 and 13an, mappings of 13b and 13ai with the pharmacophore hypothesis of hERG blockers, and kinase profiling results of 13an (PDF) SMILES data (CSV)



AUTHOR INFORMATION

Corresponding Author

*Tel: +86-28-85164063. Fax: +86-28-85164060. E-mail: [email protected]. Author Contributions ∥

C.-H.Z., K.C., Y.J., and L.-L.L. contributed equally to this work. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the 973 Program (2013CB967204), the National Natural Science Foundation of China (81325021, 81473140, and 81573349), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT13031).



ABBREVIATIONS USED TNBC, triple-negative breast cancer; hERG, human Ether-a-gogo Related Gene; FAK, focal adhesion kinase; ERK, extracellular regulated protein kinases; MAPK, mitogenactivated protein kinase; Fra1, fos-related antigen 1; SFKs, Src family kinases; SAR, structure−activity relationship; Ar, aromatic; PK, pharmacokinetic; EA, ethyl acetate



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