CDC2-Like Kinase (CLK2

Article Cite This: J. Med. Chem. 2017, 60, 8989-9002

pubs.acs.org/jmc

The Discovery of a Dual TTK Protein Kinase/CDC2-Like Kinase (CLK2) Inhibitor for the Treatment of Triple Negative Breast Cancer Initiated from a Phenotypic Screen Jennifer R. Riggs,* Mark Nagy, Jan Elsner, Paul Erdman, Dan Cashion, Dale Robinson, Roy Harris, Dehua Huang, Lida Tehrani, Gordafaried Deyanat-Yazdi, Rama Krishna Narla, Xiaohui Peng, Tam Tran, Leo Barnes, Terra Miller, Jason Katz, Yang Tang, Ming Chen, Mehran F. Moghaddam, Sogole Bahmanyar, Barbra Pagarigan, Silvia Delker, Laurie LeBrun, Philip P. Chamberlain, Andrew Calabrese, Stacie S. Canan, Katerina Leftheris, Dan Zhu, and John F. Boylan Celgene Corporation, 10300 Campus Pointe Drive, Suite 100, San Diego, California 92121, United States S Supporting Information *

ABSTRACT: Triple negative breast cancer (TNBC) remains a serious unmet medical need with discouragingly high relapse rates. We report here the synthesis and structure−activity relationship (SAR) of a novel series of 2,4,5-trisubstituted-7Hpyrrolo[2,3-d]pyrimidines with potent activity against TNBC tumor cell lines. These compounds were discovered from a TNBC phenotypic screen and possess a unique dual inhibition profile targeting TTK (mitotic exit) and CLK2 (mRNA splicing). Design and optimization, driven with a TNBC tumor cell assay, identified potent and selective compounds with favorable in vitro and in vivo activity profiles and good iv PK properties. This cell-based driven SAR produced compounds with strong single agent in vivo efficacy in multiple TNBC xenograft models without significant body weight loss. These data supported the nomination of CC671 into IND-enabling studies as a single agent TNBC therapy.

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INTRODUCTION

kinase, and CDC2-like kinase (CLK2). TTK is a dual serine/ threonine kinase that regulates the spindle assembly checkpoint (SAC), thereby controlling the progression of cells through mitosis.7,8 TTK siRNA knockout accelerates mitotic progression and induces cell death in TNBC cell lines.9,10 TTK also phosphorylates key oncogenic proteins such as p53, CHK2, and MDM2, critical to DNA damage repair.11,12 TTK has been shown in the literature to be a promising TNBC targeting opportunity. TTK mRNA and protein are overexpressed in a TNBC cohort of breast cancer primary tumors, and high TTK expression in TNBC patients is linked to a poor patient prognosis.10 These data provided sufficient rationale to gain the interest of a number of pharmaceutical companies and academic institutions leading to the publication of multiple, potent TTK inhibitors.9,13−23 Thus far, four TTK inhibitors have reportedly entered phase I clinical trials: BAY 1161909

Phenotypic-based drug discovery has proven successful in the generation of novel, first-in-class drugs as compared to targetbased approaches.1−3 Although triple negative breast cancer (TNBC) patients have a reasonable response rate to current standard of care chemotherapy, the relapse rates are high and treatment options remain limited. Because of the heterogeneous nature of TNBC, attempts have been made to further categorize TNBC into as many as 10 different subtypes.4 Because of this heterogeneity and lack of well-defined clinical targets, we used a phenotypic approach to identify a novel treatment for TNBC. By targeting preferential induction of apoptosis in TNBC cell lines over luminal breast cancer cell lines, we identified a series of 2,4,5-trisubstituted-7H-pyrrolo[2,3-d]pyrimidines with the desired phenotypic profile.5,6 During the phenotypic SAR chemistry campaign, we established two kinases that were selectively inhibited by our molecules and likely responsible for the cellular activity: monopolar spindle 1 (Mps1), also known as TTK protein © 2017 American Chemical Society

Received: August 18, 2017 Published: October 9, 2017 8989

DOI: 10.1021/acs.jmedchem.7b01223 J. Med. Chem. 2017, 60, 8989−9002

Journal of Medicinal Chemistry

Article

Figure 1. Structures of TTK inhibitors reportedly in phase I clinical trials. The structure of S 81694 has not been published.

(clinical trials.gov ID: NCT02138812), BAY 1217389 (clinical trials.gov ID: NCT02366949), S 81694 (EudraCT no: 2014002023-10), and CFI-402257 (clinicaltrials.gov ID: NCT02792465) (Figure 1). While all are potent TTK inhibitors, they differ in several aspects including kinase selectivity profiles, route of administration, preclinical efficacy model type, ability to produce single agent efficacy with tolerated doses, dosing paradigm, PK, and likely mean resonance time on the target protein. The novel kinase profile, the long time on target which allows for intermittent dosing, and strong single agent efficacy provide possible competitive advantages for our series of TTK inhibitors. SR specific kinases (SRPKs) and CLK2 phosphorylate serine- and arginine-rich proteins (SR) within the spliceosome and modulate mRNA splicing activity and consequently protein synthesis.24,25 The high level of cancer associated mutations or overexpression across the splicing process highlights the growth and survival advantage obtained when tumor cells co-opt this pathway. Downregulation of CLK2 alters splicing patterns and inhibits breast cancer growth in vitro and in vivo.26 Consistent with these data, small molecule CLK2 inhibition alters expression of SR proteins by changing the splicing pattern in mRNAs involved in growth and survival leading to cancer cell apoptosis.27 A dual TTK/CLK2 inhibitor would produce mitotic acceleration and RNA splicing modification. Given the rapid adaptation and growth requirements of a tumor cell, this unique kinase combination profile should prove impactful in a number of cancer subtypes including high unmet medical need indications such as TNBC. Here we describe the optimization of a phenotypic hit (1, Figure 2) and SAR exploration, leading to favorable in vivo iv PK profiles and the selection of 4-((4(cyclopentyloxy)-5-(2-methylbenzo[d]oxazol-6-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (CC-671) for IND-enabling studies.5

Figure 2. Structure of phenotypic hit 1.

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CHEMISTRY The synthesis of 2,4,5-trisubstituted-7H-pyrrolo[2,3-d]pyrimidines is outlined in Schemes 1 and 2. Commercially available 2,4-dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine was converted to the SEM-protected intermediate 2 using 2(trimethylsilyl)ethoxymethyl chloride (Scheme 1). The C-4 substituent was introduced by displacement of the chloride with appropriate alcohols to give 3a−3c. Suzuki−Mukiyama cross coupling reaction of iodide 3 with aryl boronic acids or esters resulted in disubstituted pyrrolopyrimidines 4a−4u. Alternatively, the C-5 substituent could be installed first from intermediate 2. Suzuki−Mukiyama coupling with appropriate aryl boronic esters or acids affords 5. Chloride displacement at C-4 with alkyl and cycloalkyl alcohols provides disubstituted pyrrolopyrimidines 6a−6g. The C-2 substituent was installed using Pd-catalyzed Buchwald coupling to afford 7−44 SEM (Scheme 2). Final analogues (7−44) were obtained after deprotection using either TBAF or a two-step process of TFA followed by NH4OH-assisted cleavage of the hemiaminal intermediate.

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RESULTS AND DISCUSSION Our desired profile for TNBC therapy from this phenotypic approach was to retain TNBC sensitivity while sparing luminal cancer cell lines to ensure a lack of general cytotoxicity. With this in mind, two cellular assays, Cal-51, a TNBC cell line, and BT-474, a luminal breast cancer cell line, were utilized to evaluate analogues. Potency in the Cal-51 assay was primarily 8990



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Scheme 1. Synthesis of Disubstituted Pyrimidine Intermediates 4a−4u and 6a−6ga

Reagents and conditions: (a) SEM-Cl, NaH, DMF, 0−22 °C; (b) R2-alcohol, 1, 4-dioxane, NaOtBu, 70−90 °C; (c) R1-boronate ester or acid, sodium carbonate in water, PdCl2(dppf)-CH2Cl2 or Pd(PPh3)2Cl2, 1, 4-dioxane, 70−90 °C.

a

In an effort to replace the carboxamide, substituting the aryl ring at the para position with five-membered heterocycles was explored. Again, the positioning of the H-bond acceptor in the pyrazole, imidazole, and triazole compounds (17−19) impacted the potency. Fused 6,5-heterocycles 20−23 gave mixed results, as expected from the H-bond acceptor SAR presented thus far. Depending on the position of the nitrogen H-bond acceptor, analogues either maintained potency (20, 23), or lost potency (21, 22). On the basis of the overall profile of potency in Cal-51, selectivity over the luminal line BT-474, and high S9 stability, the 2-methyl benzoxazole 23 was held constant at C-5 in the exploration of C-4 and C-2. Our exploration at C-4 revealed a general tolerance for a variety of cycloalkyl and oxygen-containing functional groups for Cal-51 potency (Table 2). We first explored contraction of the cyclopentyl ring, resulting in equipotent cyclobutyl 24 and cyclopropyl 25. Small alkyl substituents such as isopropyl (26), hydroxy-ethyl (27), or methoxy-ethyl (28) also maintained Cal51 potency although a general erosion of selectivity was seen in BT-474 with the smaller cyclopropyl and alkyl substituents. The free OH at C-4, 29, caused a complete loss in potency. Introduction of heteroatoms into the cyclic C-4 substituent generally led to a slight improvement in Cal-51 potency (30 vs 24 and 31 vs 23). The six-membered THP and cyclohexanols 32−34 generally led to an increase in potency as compared to the cyclopentyl 23. Overall, the C-4 position was tolerant of various groups with respect to potency and reasonable rat S9 stability was maintained (data not shown). We felt this position could be used to improve properties, such as lowering lipophilicity. Considering the potency, selectivity against BT474, and synthetic ease, we chose the cyclopentyl at C-4 to hold constant for SAR studies at C-2. On the C-2 aryl group, we extensively explored the para substituents (R4) while holding the ortho-methoxy constant (R5). A subset of these analogues are shown in Table 3. Removal of the R4 substituent (35) resulted in a loss of potency as compared to the methyl carboxamide 23. The dimethyl tertiary carboxamide 36 maintained potency. Cyclic tertiary amides such as piperazine 37 and morpholine 38 were explored with mixed results. Piperazine 37 maintained good

Scheme 2. Synthesis of 2,4,5-Trisubstituted-7H-pyrrolo[2,3d]pyrimidines 7−44a

a

Reagents and conditions: (a) R3-amine, Pd2(dba)3, Xanphos, Cs2CO3, 1,4-dioxane, MW 150 °C; (b) TFA, DCM; (c) NH4OH, MeOH; (d) TBAF, THF, 50 °C.

used to drive the SAR. While the phenotypic activity of hit 1 exhibited the desired phenotypic profile, it was metabolically unstable in liver S9 fractions. Glucuronidation and sulfation of the phenol were the major metabolites. Our first goal was to replace the metabolically labile phenol at C-5 of 1. The contribution of the phenol moiety to the metabolic instability of 1 is further illustrated by the significant improvement in S9 stability of the unsubstituted phenyl 7 (Table 1). The dramatic loss in potency of 7 suggests the phenol in 1 is making a key interaction, contributing to the observed cellular effect. We further examined the hydrogen bonding requirements and the positioning of the phenol. The m-phenol 8 suffered from a slight reduction in potency as compared to the p-phenol 1. Replacement of the phenol with an unsubstituted 4- or 3pyridyl (9 and 10) afforded analogues with similar potency to their respective phenol counterparts (9 vs 1 and 10 vs 8), suggesting the hydrogen bond donor is not necessary, but a hydrogen bond acceptor is contributing significantly to potency. Five-membered heterocycles were tolerated as in pyrazole 12, but isomeric pyrazole 11 resulted in a loss of potency, suggesting the correct orientation of the H-bond acceptor is critical. Interestingly, capping the phenol to give the p-methoxy 13 resulted in a loss in potency. Extending the alcohol by adding a methylene as in 14 and 15 was tolerated, and the proper positioning of the H-bond acceptor (15) resulted in improved potency. The p-carboxamide, 16, was equipotent to the original hit 1. 8991



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Table 1. C-5 Position Potency and Metabolic Stability SAR

a

Mean ± SEM. bPercent remaining at 60 min. cNot determined. dn = 2. 8992



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Table 2. C-4 Position Potency SAR

a

Table 3. C-2 Position Potency SAR

Mean ± SEM. a

Cal-51 potency, while the corresponding morpholine 38 was ∼6-fold less potent. Secondary amides (oxetane 39 and alcohols 40−42), explored to decrease lipophilicity and increase solubility, generally maintained potency. SAR at R5 of the C-2 aryl group was also explored. Hydrogen substitution at this position led to an erosion of kinase selectivity (data not shown), therefore small changes were assessed. While replacement of the methoxy with a methyl (43) was not well tolerated, with a ∼6-fold loss in potency, the corresponding chloro analogue (44) provided an equipotent analogue to 23. Extending the alkyl chain, such as ethoxy at R5, resulted in a loss of potency (data not shown). With multiple analogues in hand that met the desired TNBC potency, selectivity against BT-474, and metabolic stability, the iv PK properties of a number of compounds were assessed. The results of both rat and mouse iv PK are provided in Table 4. Comparing analogue 23 to analogue 16, the clearance in both rat and mouse are lower for the C-5 methyl benzoxazole (23) vs methyl amide (16), leading to a longer MRT. Compounds containing C-4 alkyl and cycloalkyl substituents 23, 25, 26, 31, 32, and 34 have slow to moderate clearance in rat, but THP 32 and methylcyclohexanol 34 suffer from rapid clearance in mouse. Substitution of the amide at C-2 with the solubilizing

Mean ± SEM.

hydroxy-ethyl group (40) showed increased clearance as compared to 23 in both rat and mouse. The oxetane 39 displayed low clearance in both rat and mouse, resulting in a longer MRT. Although the project was initiated on a phenotypic premise and TNBC Cal-51/Luminal BT-474 cell assays drove the SAR exploration, target identification was critical in supporting potential clinical advancement. In particular, a target biomarker would be required to guide dose and schedule during development. The pyrrolo[2,3-d]pyrimidine core along with the C-2 amine arranged a network of hydrogen bonds in a donor−acceptor−donor pattern resembling the hinge-binding motif of known kinase inhibitors. We therefore profiled project compounds in a commercial panel of >250 kinases.28 On the basis of the comparison of results for TNBC selective and nonselective compounds, a small set of candidate targets emerged. For example, when 23 was assessed in a panel of 255 kinases, only seven were inhibited by more than 80% at 3 μM (Supporting Information, Table 1). IC50 values for the seven kinases are shown in Table 5, with only TTK and CLK2 having single-digit nanomolar potency. 8993



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Table 4. PK Properties of Selected Analogues rat IV PKb,c a

analogue

rat S9 met. stability 87 98 63 69 62 75 76 91 80

16 23 25 26 31 32 34 39 40

± ± ± ± ± ± ± ± ±

a

f

human S9 met. stability

CL

± ± ± ± ± ± ± ± ±

61.3i 9.30 ± 1.31 11.4i 33.9 ± 5.4 27.4i 22.6i 17.6 ± 3.4 13.0 ± 1.8 50.8 ± 9.8

10 6 8 6 1 7 2 8 1

79 91 75 78 62 70 65 79 90

13 11 9 4 3 4 1 7 3

g

mouse IV PKb,d,e h

Vss

MRT

4.0i 2.3 ± 0.22 1.5i 7.2 ± 1.2 1.6i 3.8i 2.9 ± 1.2 3.7 ± 0.8 3.9 ± 0.6

1.1i 4.1 ± 0.3 2.5i 3.5 ± 0.3 1.0i 2.9i 2.9 ± 1.5 4.7 ± 0.8 1.3 ± 0.1

CLf

MRTh

63.1 8.48j 10.4 7.71 19.1 67.6 52.2 14.3 31.6

0.36 5.2j 3.1 4.8 0.73 1.8 1.0 1.4 0.59

a Percent remaining at 60 min. bFormulated as a solution in 15% DMA, 50% PEG, 35% D5W. cDose 2 mg/kg. dDose 10 mg/kg in SCID mice. eSD not available with composite PK. fmL/min/kg. gL/kg. hHours. iSD not available n = 2. jDose 5 mg/kg.

Table 5. IC50 (μM) of the Seven Kinases That Were >80% Inhibition at 3 μM of 23 TTK

CLK2

DYRK3

DYRK1A

PHKG

DYRK1B

CLK1

0.005

0.006

0.099

0.104

0.136

0.157

0.30

HCT-116 cell lysates were treated with compound 23 at 3 μM for 1 h and tested in the ActivX KiNative profiling assay. Only four kinases showed cellular binding of 75% or more including CLK2, CAMKK2, PIP4K22, and JNK (TTK not offered, full data in Supporting Information, Table 2). The IC50 values for the four kinases were determined in the HCT-116 cell line (Table 6). CLK2 was determined to be the most Table 6. Kinase Binding in the HCT-116 Cell Line kinase

IC50 (μM)

CLK2 JNK1,2,3 CAMKK2 PIP4K2C

0.015 0.68 1.10 1.30

potent with an IC50 value of 15 nM. From the seven kinases that were identified in the biochemical kinase screen, only one additional kinase (PHKG-1) was monitored in the HCT-116 KiNative kinase panel, and PhKG-1 only showed 26% binding in the 3 μM screen. Inhibition of TTK and CLK2 phosphorylation substrates was monitored to confirm the cellular inhibitory mechanism of action. Cal-51 cells were treated with compound 23 for 1 h and analyzed by Western blot using antibodies against the autophosphorylation of TTK at T686 or phosphorylation of SR protein 75, a substrate of CLK2. Quantification of the Western blots enabled IC50 determinations. The p-TTK and pSRp75 potency of 23 was shown to be 0.057 and 0.549 μM, respectively (Figure 3a). To assess the scope of the TNBC sensitivity versus Luminal BC resistance to compound 23, we chose two additional cell lines in each category. As seen in Figure 3b, the three TNBC lines are highly sensitive to compound 23 while the three Luminal BC lines are resistant. The identification of the key targets responsible for TNBC cellular activity, namely TTK and CLK2, allowed us to initiate crystallography efforts to enable SBDD. We obtained a crystal structure of compound 23 with TTK at 2.99 Å resolution (Figure 4). A detailed view of 23 bound in the TTK binding pocket is shown in Figure 4a. When compared to the apo structure of TTK, an induced fit of the glycine-rich loop (G-

Figure 3. (a) Biochemical and cellular biomarker potency of compound 23, confirming inhibition of TTK and CLK2 and phosphorylation of downstream substrates. (b) Compound 23 demonstrated preferential antiproliferative activities in TNBC lines.

loop) is observed (see Supporting Information, Figure 1a,1b). As anticipated, the pyrrolopyrimidine core of 23 forms three hydrogen bonds to the hinge region of the TTK ATP binding pocket. The pyrrole NH (N7) forms a hydrogen bond to backbone carbonyl of Glu603. The amino-pyrimidine moiety of the core forms two additional hydrogen bond interactions with the hinge residue Gly605. The C5 methyl benzoxazole binds deep in the ATP binding pocket and forms favorable van der Waals interactions with the gatekeeper residue Met602, the side chain carbons of catalytic residue Lys553, and the side chain of activation loop residue Met671. In addition, N3 of the methyl benzoxazole forms a direct hydrogen bond with the side chain amine of Lys553. The C4 cyclopentyl substituent is enclosed in the ribose pocket by a portion of the activation loop, where residues Met671, Gln672, Pro673, and Asp674 are ordered in the crystal structure (Figure 4b). This conformation of the activation loop has been observed in other crystal structures of TTK.21 The ordered portion of the activation loop and the G-loop appear to interact though a network of hydrogen bonds (see Supporting 8994



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Figure 4. (a) Crystal structure of 23 bound in the TTK ATP pocket, and interactions with residues within 4.0 Å of the ligand are shown (PDB 6B4W). (b) TTK ATP binding pocket with the ordered portion of the activation loop shown in green.

Information, Figure 1a,1b for additional views). This arrangement of the activation loop residues encapsulates the cyclopentyl ring in a hydrophobic pocket. The C2 aniline binds toward the “solvent” pocket. The ortho-methoxy substituent binds in a pocket formed by hinge residue Cys604 and G-loop residue Gln541. Cys604 is a small and fairly unique residue at the hinge important for obtaining kinase selectivity in this series.29 The para amide substituent of the C2 aniline is within 3.3 Å of Asp608 but does not appear to form specific hydrogen bonds. On the basis of modeling and docking studies, compound 23 binds similarly in the CLK2 binding pocket (Supporting Information, Figure 2a). The overall residue homology of CLK2 and TTK is low, however, there are similarities in the binding pocket that allows both kinases to accommodate compound 23 (Supporting Information, Figure 2b).

With numerous potent and selective compounds demonstrating good rat and mouse iv PK in hand, we set out to assess the time and exposure necessary to illicit an antiproliferative effect. Cal-51 cells were treated in cellular wash-out experiments with compound 23 for 6, 12, 24, 48, and 72 h followed by a wash out. Antiproliferative activity was measured at 3 days post washout. From the data represented in Figure 5, at time points >12 h, a considerable left shift in the potency is observed. This in vitro data suggests that >12 h coverage by the compound is necessary to give maximal antiproliferative activity. Compound 23 was chosen for in vivo efficacy assessment based on potency, selectivity, slow clearance, and long MRT in both rat and mouse species. We sought to investigate the optimal in vivo dosing schedule and finalize the target product profile including route of administration. On the basis of the 8995



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Figure 5. In vitro wash-out data in Cal-51 cell line of compound 23.

washout data presented in Figure 5, it is likely that daily dosing would be required. It is plausible that with the TTK mechanism, 33 μM. Full biological characterization and additional in vivo TNBC patient derived xenograft (PDX) data where compound 23 causes regression at well tolerated doses will be published in due course.

Figure 6. Cal-51 tumor xenograft studies with q3d (a) and q7d (b) dosing schedules of 23 (*p < 0.001, one way ANNOVA).

compound 23 for IND-enabling studies. Compound 23 has excellent kinase selectivity, favorable iv pharmacokinetic properties in all species tested, superior in vivo efficacy and tolerability as compared to Taxotere, and an acceptable in vitro and in vivo toxicology profile. Additional biological characterization of compound 23 will be disclosed elsewhere.

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MATERIALS AND METHODS

Chemistry. All materials were obtained from commercial sources and used without further purification unless otherwise noted. Chromatography solvents were HPLC grade and used as purchased. All air-sensitive reactions were carried out under a positive pressure of an inert nitrogen atmosphere. 1H NMR spectra were obtained on 300, 400, or 500 MHz spectrometers. Chemical shifts are relative to tetramethylsilane (TMS) as the internal standard or relative to a residual solvent peak. Chemical shifts (δ) are reported in ppm, and coupling constants (J) are given in Hz. Thin layer chromatography (TLC) analysis was performed on Whatman thin layer plates. LCMS analysis was performed on a PE Sciex ESI MS or Agilent 1100 MS. Semipreparative reverse phase HPLC was performed on a Shimadzu system equipped with a Phenomenex 15 μm C18 column (250 mm ×

CONCLUSION In summary, we have described the SAR and optimization of the 2,4,5-trisubstituted-7H-pyrrolo[2,3-d]pyrimidine series emanating from a TNBC phenotypic screen. Interrogation of the biology of these compounds identified two inhibitory targets, TTK and CLK2. This unique dual kinase profile of accelerated mitotic progression and mRNA splicing has been shown to be efficacious in TNBC cell lines in vitro and in xenograft models in vivo. Multiple compounds in this series were generated with the desired in vitro profile and good iv PK properties. This work ultimately led to the identification and nomination of 8996



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4-((4-(Cyclopentyloxy)-5-(pyridin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (10). Yield 0.033 g, 0.071 mmol, 19%, HPLC purity >93%. MS (ESI) m/z 459 [M + 1]+. 4-((4-(Cyclopentyloxy)-5-(1H-pyrazol-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (11). Yield 0.018 g, 0.040 mmol, 16%, HPLC purity >91%. 1H NMR (400 MHz, DMSO-d6) δ 12.71−12.76 (m, 1H), 11.87−11.94 (m, 1H), 8.58−8.67 (m, 1H), 8.34−8.44 (m, 1H), 7.66−7.75 (m, 1H), 7.44−7.57 (m, 3H), 6.63−6.74 (m, 1H), 5.65−5.73 (m, 1H), 4.08−4.17 (m, 1H), 3.92− 4.03 (m, 3H), 2.74−2.84 (m, 3H), 1.98−2.09 (m, 2H), 1.82−1.93 (m, 2H), 1.58−1.82 (m, 4H). MS (ESI) m/z 448 [M + 1]+. 4-((4-(Cyclopentyloxy)-5-(1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (12). Yield 0.012 g, 0.027 mmol, 8%, HPLC purity >97%. 1H NMR (400 MHz, DMSO-d6) δ 12.67−12.77 (m, 1H), 11.53−11.65 (m, 1H), 8.61−8.69 (m, 1H), 8.28−8.37 (m, 1H), 7.94−8.02 (m, 1H), 7.83−7.91 (m, 1H), 7.64−7.69 (m, 1H), 7.49−7.58 (m, 2H), 7.25−7.33 (m, 1H), 5.64− 5.76 (m, 1H), 3.97 (s, 3H), 2.77−2.85 (m, 3H), 2.00−2.14 (m, 2H), 1.84−1.94 (m, 2H), 1.64−1.81 (m, 4H). MS (ESI) m/z 448 [M + 1]+. 4-((4-(Cyclopentyloxy)-5-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (13). Yield 0.033 g, 0.067 mmol, 17%, HPLC purity >95%. 1H NMR (400 MHz, DMSO-d6) δ 11.66−11.70 (m, 1H), 8.61−8.67 (m, 1H), 8.27−8.33 (m, 1H), 7.66−7.70 (m, 1H), 7.57−7.63 (m, 2H), 7.48−7.53 (m, 2H), 7.18−7.22 (m, 1H), 6.88−6.95 (m, 2H), 5.63−5.69 (m, 1H), 3.96 (s, 3H), 3.77 (s, 3H), 2.76−2.84 (m, 3H), 1.92−2.03 (m, 2H), 1.76−1.86 (m, 2H), 1.61−1.76 (m, 4H). MS (ESI) m/z 488 [M + 1]+. 4-((4-(Cyclopentyloxy)-5-(4-(hydroxymethyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (14). Yield 0.020 g, 0.041 mmol, 60%, HPLC purity >97%. 1H NMR (400 MHz, DMSO-d6) δ 11.73−11.79 (m, 1H), 8.62−8.68 (m, 1H), 8.26− 8.36 (m, 1H), 7.69−7.71 (m, 1H), 7.62−7.68 (m, 2H), 7.49−7.54 (m, 2H), 7.27−7.32 (m, 3H), 5.65−5.71 (m, 1H), 5.16 (t, J = 5.75 Hz, 1H), 4.52 (d, J = 5.87 Hz, 2H), 3.97 (s, 3H), 2.78−2.83 (m, 3H), 1.94−2.06 (m, 2H), 1.78−1.87 (m, 2H), 1.62−1.77 (m, 4H). MS (ESI) m/z 488.3 [M + 1]+. 4-[4-Cyclopentyloxy-5-(3-hydroxymethyl-phenyl)-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-ylamino]-3methoxy-N-methyl-benzamide (15). Yield 0.008 g, 0. 016 mmol, 5%, HPLC purity >95%. 1H NMR (400 MHz, DMSO-d6) δ 8.70 (d, J = 8.4 Hz, 1H), 7.58 (s, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.41 (d, J = 7.6 Hz, 2H), 7.27 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 7.6 Hz, 1H), 7.03 (s, 1H), 5.62 (s, 1H), 4.60 (s, 2H), 3.95 (s, 3H), 2.87 (s, 3H), 2.01−1.91 (m, 2H), 1.86−1.83 (m, 2H), 1.69−1.60 (m, 4H). MS (ESI) m/z 488.2 [M + H]+. 4-((4-(Cyclopentyloxy)-5-(4-(methylcarbamoyl)phenyl)-7Hpyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (16). Yield 0.335 g, 0.651 mmol, 56%, HPLC purity >99%. 1H NMR (400 MHz, DMSO-d6) δ 11.91 (d, J = 1.95 Hz, 1 H), 8.63 (d, J = 8.98 Hz, 1 H), 8.43 (q, J = 4.43 Hz, 1 H), 8.29−8.35 (m, 1 H), 7.76−7.88 (m, 4 H), 7.74 (s, 1 H), 7.49−7.55 (m, 2 H), 7.46 (d, J = 2.34 Hz, 1 H), 5.64−5.75 (m, 1 H), 3.97 (s, 3 H), 2.74−2.85 (m, 6 H), 1.93−2.07 (m, 2 H), 1.78−1.90 (m, 2 H), 1.60−1.78 (m, 4 H). MS (ESI) m/z 515.0 [M + 1]+. Elemental Analysis calculated (%) for (C28H30N6O4·0.35H2O): C 64.52, H 5.94, N 16.12. Found C 64.21, H 5.85, N 15.90. KF = 1.26%. 4-(5-(4-(1H-Pyrazol-5-yl)phenyl)-4-(cyclopentyloxy)-7H-pyrrolo[2,3-d]pyrimidin-2-ylamino)-3-methoxy-N-methylbenzamide (17). Yield 0.046 g, 0.088 mmol, 12% over two steps, HPLC purity >96%). 1H NMR (300 MHz, DMSO-d6) δ 8.65 (m, 1H), 8.35 (m, 1H), 7.80−7.72 (m, 6H), 7.53 (m, 2H), 7.37 (s, 1H), 6.73 (s, 1H), 5.70 (m, 1H), 3.97 (s, 3H), 2.80 (s, 3H), 2.00−1.64 (m, 8H). MS (ESI) m/z 524.2 [M + 1]+. 4-(5-(4-(1H-Imidazol-2-yl)phenyl)-4-(cyclopentyloxy)-7H-pyrrolo[2,3-d] pyrimidin-2-ylamino)-3-methoxy-N-methylbenzamide (18). Yield 0.076 g, 0.15 mmol, 28%, HPLC purity >96%. 1H NMR (300 MHz, CD3OD) δ 8.79−8.78 (m, 1H), 7.89−7.80 (m, 4H), 7.51−7.48 (m, 2H), 7.22 (s, 1H), 5.75−5.73 (m, 1H), 4.03 (s, 3H), 2.94 (s, 3H), 2.04−1.72 (m, 8H). MS (ESI) m/z 524.2 [M + H]+.

10 mm). Preparative reverse phase HPLC was performed on a Shimadzu system equipped with a Phenomenex 15 μm C18 column (250 mm × 50 mm). Compounds were analyzed for purity by one of two methods: (A) gradient (0−75% acetonitrile + 0.1% formic acid in water + 0.1% formic, over 7 min, followed by 75% acetonitrile + 0.1% formic acid for 2 min); flow Rate 1 mL/min, column Phenomenex Gemini-NX 5 μ C18 110A (50 mm × 4.60 mm); (B) gradient (0− 75% acetonitrile + 0.1% formic acid in water + 0.1% formic, over 20 min, followed by 75% acetonitrile + 0.1% formic acid for 5 min); flow Rate 1 mL/min, column Phenomenex Gemini-NX 5 μ C18 110A (250 mm × 4.60 mm). The purity of final tested compounds was determined to be ≥95% by HPLC conducted on an Agilent 1100 system using a reverse phase C18 column and diode array detector (compounds 8, 9, 10, 11, 20, 21, and 44 are >90% pure). Elemental analysis was performed at Robertson Microlit Laboratories, Ledgewood, New Jersey. Reported yields are unoptimized. Compounds were named using ChemDraw Ultra. Synthetic procedures for all intermediates 2, 3a−3u, 4a−4u, 6a−6g, and 7−44 SEM are included in the Supporting Information. General Procedure for the Synthesis of Compounds 1, 7− 23, 25−29, and 31−44. The appropriate SEM protected intermediate (1 equiv) was diluted with dichloromethane (0.2 M) and treated with trifluoroacetic acid (25−200 equiv). The solution was stirred at rt for 1−18 h. The mixture was concentrated under reduced pressure to afford an oil. The residue was treated with 2 M ammonia in methanol (50−500 equiv) for 3−18 h. The mixture was concentrated under reduced pressure. The residue was diluted with either acetonitrile, dioxane, or methanol (0.1 M), treated with a concentrated aqueous solution of ammonium hydroxide (50−500 equiv), and stirred for 2−24 h at temperatures of rt to 50 °C. The mixture was concentrated under reduced pressure and purified by reverse phase preparative HPLC. Fractions containing pure product were loaded onto a Phenomenex Strata-XC strong cation ion exchange column. The column was washed successively with water and methanol, and the product was eluted with 2 M ammonia in methanol. The combined product fractions were concentrated under reduced pressure to afford the title compound. 4-((4-(Cyclopentyloxy)-5-(4-hydroxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (1). Yield 0.120 g, 0.253 mmol, 12% over 2 steps, HPLC purity >99%. 1H NMR (400 MHz, DMSO-d6) δ 11.62 (br s, 1H), 9.27 (s, 1H), 8.65− 8.63 (d, 1 H, J = 9 Hz), 8.30−3.28 (m, 1 H), 7.66 (s, 1H), 7.52−7.46 (m, 4 H), 7.12 (s, 1 H), 6.76−6.73 (d, 2 H, J = 9 Hz), 5.67−5.65 (br m., 1 H), 3.97 (s, 3 H), 2.80−2.79 (d, J = 4.4 Hz, 3 H), 1.99−1.94 (m, 2 H), 1.82−1.62 (m, 6 H). MS (ESI) m/z 474.2 [M + 1]+. 4-((4-(Cyclopentyloxy)-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-2yl)amino)-3-methoxy-N-methylbenzamide (7). Yield 0.155 g, 0.339 mmol, 64%, HPLC purity >99%. 1H NMR (499 MHz, DMSO-d6) δ 11.78 (s, 1 H) 8.63 (d, J = 8.86 Hz, 1 H) 8.25−8.34 (m, 1 H) 7.63− 7.75 (m, 3 H) 7.46−7.55 (m, 2 H) 7.29 (d, J = 2.46 Hz, 1 H) 7.18 (t, J = 8.86 Hz, 2 H) 5.58−5.70 (m, J = 5.78, 5.78, 2.71, 2.46 Hz, 1 H) 3.96 (s, 3 H) 2.79 (d, J = 4.43 Hz, 3 H) 1.90−2.03 (m, 2 H) 1.74−1.85 (m, 2 H) 1.56−1.73 (m, 4 H). MS (ESI) m/z 458.1 [M + 1]+. 4-((4-(Cyclopentyloxy)-5-(3-hydroxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (8). Yield 0.014 g, 0.030 mmol, 9%, HPLC purity >93%. 1H NMR (400 MHz, DMSO-d6) δ 11.69−11.75 (m, 1H), 9.17−9.25 (m, 1H), 8.59−8.68 (m, 1H), 8.27−8.33 (m, 1H), 7.66−7.71 (m, 1H), 7.47−7.56 (m, 2H), 7.20−7.24 (m, 1H), 7.05−7.16 (m, 3H), 6.62−6.67 (m, 1H), 5.62− 5.69 (m, 1H), 3.91−4.07 (m, 3H), 2.74−2.84 (m, 3H), 1.92−2.02 (m, 2H), 1.55−1.87 (m, 6H). MS (ESI) m/z 474 [M + 1]+. 4-((4-(Cyclopentyloxy)-5-(pyridin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (9). Yield 0.067 g, 0.15 mmol, 28%, HPLC purity >93%. 1H NMR (400 MHz, DMSO-d6) δ 12.03−12.08 (m, 1H), 8.58−8.62 (m, 1H), 8.48−8.52 (m, 2H), 8.28−8.34 (m, 1H), 7.76−7.78 (m, 1H), 7.70−7.74 (m, 2H), 7.64 (d, J = 2.45 Hz, 1H), 7.49−7.54 (m, 2H), 5.68−5.74 (m, 1H), 3.96 (s, 3H), 2.79 (d, J = 4.65 Hz, 3H), 1.96−2.06 (m, 2H), 1.81−1.90 (m, 2H), 1.63−1.79 (m, 4H). MS (ESI) m/z 459 [M + 1]+. 8997



Article

4-((5-(4-(4H-1,2,4-Triazol-3-yl)phenyl)-4-(cyclopentyloxy)-7Hpyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (19). Yield 0.049 g, 0.093 mmol, 27%, HPLC purity >97%. 1H NMR (400 MHz, DMSO-d6) δ 11.83−11.92 (m, 1H), 8.61−8.66 (m, 1H), 8.28−8.34 (m, 1H), 7.97−8.02 (m, 2H), 7.80−7.87 (m, 2H), 7.71−7.74 (m, 1H), 7.49−7.54 (m, 2H), 6.97−7.27 (m, 3H), 5.68− 5.72 (m, 1H), 3.97 (s, 3H), 2.76−2.82 (m, 3H), 1.95−2.05 (m, 2H), 1.81−1.89 (m, 2H), 1.63−1.79 (m, 4H). MS (ESI) m/z 525.3 [M + 1]+. 4-(5-(1H-Benzo[d]imidazol-6-yl)-4-(cyclopentyloxy)-7H-pyrrolo[2,3-d]pyrimidin-2-ylamino)-3-methoxy-N-methylbenzamide (20). Yield 0.014 g, 0.02 mmol, 5% over two steps, HPLC purity >94%. 1 H NMR (400 MHz, CD3OD) δ 8.70−8.68 (d, J = 8.8 Hz, 1H), 8.11 (br s, 1H), 7.79 (br s, 1H), 7.49 (br s, 2H), 7.41−7.38 (m, 2H), 7.00 (s, 1H), 5.61 (m, 1H), 3.93 (s, 3H), 2.83 (s, 3H), 1.91−1.51 (m, 8H). MS (ESI) m/z 498.2 [M + 1]+. 4-[4-Cyclopentyloxy-5-(1H-indazol-6-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-ylamino]-3-methoxy-N-methylbenzamide (21). Yield 0.023 g, 0. 046 mmol, 21%, HPLC purity >90%. 1H NMR (400 MHz, DMSO-d6) δ 13.21−12.86 (m, 1H), 12.04−11.65 (m, 1H), 8.82−8.55 (m, 1H), 8.47−8.23 (m, 1H), 8.14−8.01 (m, 1H), 7.72 (s, 3H), 7.51 (s, 4H), 5.82−5.62 (m, 1H), 3.97 (s, 3H), 2.80 (s, 3H), 2.07−1.90 (m, 2H), 1.88−1.73 (m, 2H), 1.63−1.52 (m, 5H). MS (ESI) m/z 498.2 [M + 1]+. 4-((4-(Cyclopentyloxy)-5-(2-methylbenzo[d]oxazol-5-yl)-7Hpyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (22). Yield 0.045 g, 0.088 mmol, 26%, HPLC purity >97%. 1H NMR (400 MHz, CD3OD) δ 8.78 (d, J = 8.9 Hz, 1H), 7.88 (s, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.56−7.41 (m, 3H), 7.11 (s, 1H), 5.71 (m, 1H), 4.03 (s, 3H), 2.93 (s, 3H), 2.66 (s, 3H), 2.09−1.84 (m, 4H), 1.80−1.58 (m, 4H). MS (ESI) m/z 513 [M + 1]+. 4-((4-(Cyclopentyloxy)-5-(2-methylbenzo[d]oxazol-6-yl)-7Hpyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (23). Yield 0.310 g, 0.60 mmol, 72%, HPLC purity >99%. 1H NMR (400 MHz, DMSO-d6) δ 8.66 (d, J = 8.5 Hz, 1H), 8.31 (s, 1H), 7.93 (s, 1H), 7.79−7.29 (m, 6H), 5.69 (s, 1H), 3.97 (s, 3H), 2.80 (s, 3H), 2.61 (s, 3H), 2.16−1.43 (m, 8H). MS (ESI) m/z 513 [M + 1]+. Elemental Analysis calculated (%) for (C28H28N6O4): C 65.61, H 5.51, N 16.40. Found: C 65.79, H 5.48, N 16.37. 4-((4-Cyclobutoxy-5-(2-methylbenzo[d]oxazol-6-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (24). Tetrabutylammonium fluoride (5.57 mL, 5.57 mmol) was added to a stirred solution of 4-((4-cyclobutoxy-5-(2-methylbenzo[d]oxazol-6yl)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin2-yl)amino)-3-methoxy-N-methylbenzamide (0.350 g, 0.557 mmol) in tetrahydrofuran (20 mL). The resulting mixture was capped and stirred at 50 °C for 2 h. The solvent was removed on a rotary evaporator. The reaction mixture was purified using silica gel chromatography (0−25% methanol in dichloromethane) to afford the title compound (0.110 g, 0.221 mmol, 40% yield, HPLC purity >98%). 1H NMR (400 MHz, DMSO-d6) δ 11.88 (d, J = 1.95 Hz, 1 H) 8.61 (d, J = 8.59 Hz, 1 H) 8.33 (d, J = 4.69 Hz, 1 H) 8.00 (s, 1 H) 7.66−7.75 (m, 2 H) 7.63 (d, 1 H) 7.49−7.56 (m, 2 H) 7.42 (d, J = 2.34 Hz, 1 H) 5.39 (t, J = 7.22 Hz, 1 H) 3.97 (s, 3 H) 2.80 (d, J = 4.69 Hz, 3 H) 2.63 (s, 3 H) 2.45−2.49 (m, 2 H) 2.06−2.20 (m, 2 H) 1.79− 1.91 (m, 1 H) 1.74 (t, 1 H). MS (ESI) m/z 499.5 [M + l]+. 4-(4-Cyclopropoxy-5-(2-methylbenzo[d]oxazol-6-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-ylamino)-3-methoxy-N-methylbenzamide (25). Yield 1.3 g, 2.7 mmol, 68%, HPLC purity >98%). 1H NMR (500 MHz, DMSO-d6) δ 11.90 (s, 1 H) 8.73 (d, J = 8.83 Hz, 1 H) 8.31 (d, J = 4.73 Hz, 1 H) 7.85 (s, 1 H) 7.77 (s, 1 H) 7.57−7.64 (m, 2 H) 7.50− 7.55 (m, 2 H) 7.41 (s, 1 H) 4.50−4.58 (m, J = 6.19, 6.19, 3.07, 2.84 Hz, 1 H) 3.98 (s, 3 H) 2.80 (d, J = 4.41 Hz, 3 H) 2.63 (s, 3 H) 0.88 (d, J = 6.94 Hz, 2 H) 0.76−0.82 (m, 2 H). MS (ESI) m/z 485.2 [M + 1]+. Elemental Analysis calculated (%) for (C26H24N6O4): C 64.45, H 4.99, N 17.35. Found: C 64.28, H 4.72, N 17.25. 4-(4-Isopropoxy-5-(2-methylbenzo[d]oxazol-6-yl)-7H-pyrrolo[2,3d]pyrimidin-2-ylamino)-3-methoxy-N-methylbenzamide (26). Yield 0.459 g, 0.943 mmol, 86%, HPLC purity >99%. 1H NMR (400 MHz, DMSO-d6) δ 11.77−11.95 (m, 1 H), 8.53−8.69 (m, 1 H), 8.27−8.37 (m, 1 H), 7.94−8.00 (m, 1 H), 7.70−7.75 (m, 1 H), 7.65−7.70 (m, 1

H), 7.57−7.64 (m, 1 H), 7.47−7.55 (m, 2 H), 7.34−7.43 (m, 1 H), 5.46−5.58 (m, 1 H), 3.97 (s, 3 H), 2.75−2.83 (m, 3 H), 2.62 (s, 3 H), 1.38 (d, J = 6.25 Hz, 6 H). MS (ESI) m/z 487.4 [M + 1]+. Elemental Analysis calculated (%) for (C26H26N6O4·0.19H2O): C 63.74, H 5.43, N 17.15. Found: C 64.07, H 5.30, N 17.14. KF = 0.70%. 4-((4-(2-Hydroxyethoxy)-5-(2-methylbenzo[d]oxazol-6-yl)-7Hpyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (27). Yield 0.057 g, 0.12 mmol, 11%, HPLC purity >98%. 1H NMR (500 MHz, DMSO-d6) δ 8.54−8.69 (m, 1 H), 8.22−8.36 (m, 1 H), 7.93−8.04 (m, 1 H), 7.68−7.76 (m, 2 H), 7.55−7.61 (m, 1 H), 7.47−7.54 (m, 2 H), 7.36−7.44 (m, 1 H), 4.48−4.56 (m, 2 H), 3.92− 4.00 (m, 3 H), 3.74−3.83 (m, 2 H), 2.77−2.83 (m, 3 H), 2.58−2.66 (m, 3 H). MS (ESI) m/z 488.9 [M + l]+. 3-Methoxy-4-((4-(2-methoxyethoxy)-5-(2-methylbenzo[d]oxazol6-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-N-methylbenzamide (28). Yield 0.031 g, 0.062 mmol, 19%, HPLC purity >98%). 1H NMR (400 MHz, DMSO-d6) δ 11.9 (s, 1H), 8.61 (d, J = 8.8 Hz, 1H), 8.30 (m, 1H), 8.03 (d, J = 1.6 Hz, 1H), 7.76 (s, 1H), 7.70 (dd, J = 8.3, 1.7 Hz, 1H), 7.59 (d, J = 8.3 Hz, 1H), 7.51 (m, 2H), 7.42 (d, J = 2.5 Hz, 1H), 4.60 (m, 2H), 3.97 (s, 3H), 3.72 (m, 2H), 3.33 (s, 3H), 2.80 (d, J = 4.5 Hz, 3H), 2.62 (s, 3H). MS (ESI) m/z 503 [M + 1]+. 4-((4-Hydroxy-5-(2-methylbenzo[d]oxazol-6-yl)-7H-pyrrolo[2,3d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (29). Compound 6e was treated under the standard SEM deprotection conditions. The title compound was isolated as a byproduct (0.017 g, 0.038 mmol, 16% yield, HPLC purity >95%). 1H NMR (400 MHz, DMSO-d6) δ 11.66 (s, 1 H) 10.95 (br s, 1 H) 8.78 (s, 1 H) 8.34 (s, 1 H) 8.29 (s, 1 H) 8.22 (s, 1 H) 7.91 (d, 1 H) 7.55 (d, J = 8.20 Hz, 1 H) 7.49 (d, 1 H) 7.19 (s, 1 H) 7.10 (d, J = 8.20 Hz, 1 H) 3.94 (s, 3 H) 2.78 (d, J = 3.90 Hz, 3 H) 2.60 (s, 3 H). MS (ESI) m/z 445.5 [M + 1]+. 3-Methoxy-N-methyl-4-((5-(2-methylbenzo[d]oxazol-6-yl)-4-(oxetan-3-yloxy)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)benzamide (30). Tetrabutylammonium fluoride (7.93 mL, 7.93 mmol) was added to a stirred solution of 3-methoxy-N-methyl-4-((5-(2-methylbenzo[d]oxazol-6-yl)-4-(oxetan-3-yloxy)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)benzamide (0.5 g, 0.793 mmol) in tetrahydrofuran (20 mL). The resulting mixture was capped and stirred at 50 °C for 2 h. The solvent was removed on a rotary evaporator. The reaction mixture was purified using silica gel chromatography (0−25% methanol in dichloromethane) to afford the title compound (0.190 g, 0.380 mmol, 48% yield, HPLC purity >99%). 1 H NMR (500 MHz, DMSO-d6) δ 11.94 (d, J = 1.89 Hz, 1 H) 8.48 (d, J = 8.20 Hz, 1 H) 8.33 (d, J = 4.41 Hz, 1 H) 8.02 (s, 1 H) 7.79 (s, 1 H) 7.70−7.75 (m, 1 H) 7.66 (d, 1 H) 7.54 (d, J = 8.51 Hz, 1 H) 7.52 (s, 1 H) 7.45 (d, J = 2.52 Hz, 1 H) 5.80 (q, 1 H) 4.96 (t, J = 6.94 Hz, 2 H) 4.62 (td, J = 7.41, 5.20 Hz, 2 H) 3.96 (s, 3 H) 2.81 (d, J = 4.41 Hz, 3 H) 2.64 (s, 3 H). MS (ESI) m/z 500.1 [M + l]+. (S)-3-Methoxy-N-methyl-4-((5-(2-methylbenzo[d]oxazol-6-yl)-4((tetrahydrofuran-3-yl)oxy)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)benzamide (31). Yield 0.130 g, 0.253 mmol, 96%, HPLC purity >98%. 1H NMR (400 MHz, DMSO-d6) δ 11.90 (s, 1 H) 8.58 (d, J = 8.59 Hz, 1 H) 8.32 (d, J = 5.08 Hz, 1 H) 7.93 (s, 1 H) 7.78 (s, 1 H) 7.57−7.71 (m, 2 H) 7.46−7.56 (m, 2 H) 7.41 (d, J = 1.95 Hz, 1 H) 5.71−5.86 (m, 1 H) 4.00 (dd, J = 10.35, 4.88 Hz, 1 H) 3.96 (s, 3 H) 3.87 (s, 1 H) 3.75−3.86 (m, 3 H) 2.80 (d, J = 4.29 Hz, 3 H) 2.62 (s, 3 H) 2.29 (d, 1 H). MS (ESI) m/z 515.0 [M + 1]+. 3-Methoxy-N-methyl-4-((5-(2-methylbenzo[d]oxazol-6-yl)-4-((tetrahydro-2H-pyran-4-yl)oxy)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)benzamide (32). Yield 0.148 g, 0.280 mmol, 14% over 2 steps, HPLC purity >96%. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.96 (s, 1 H), 8.67 (d, J = 8.93 Hz, 1 H), 8.41 (br d, J = 4.52 Hz, 1 H), 8.06 (d, J = 1.10 Hz, 1 H), 7.85 (s, 1 H), 7.69−7.79 (m, 2 H), 7.58−7.64 (m, 2 H), 7.49 (d, J = 2.08 Hz, 1 H), 5.62 (tt, J = 7.90, 4.02 Hz, 1 H), 4.06 (s, 3 H), 3.80−3.93 (m, 2 H), 3.66 (ddd, J = 11.46, 8.22, 3.18 Hz, 2 H), 2.90 (d, J = 4.52 Hz, 3 H), 2.72 (s, 3 H), 2.20 (br dd, J = 8.86, 4.95 Hz, 2 H), 1.76−1.86 (m, 2 H). MS (ESI) m/z 529.2 [M + 1]+. 4-((4-(((1r,4r)-4-Hydroxy-4-methylcyclohexyl)oxy)-5-(2methylbenzo[d]oxazol-6-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (33). Compound 33 SEM was deprotected by the standard conditions and purified by prep 8998



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(m, 1H), 7.91 (s, 1H), 7.71 (s, 1H), 7.52−7.64 (m, 4H), 7.37 (s, 1H), 5.67 (s, 1H), 4.74 (s, 1H), 3.95 (s, 3H), 3.49−3.51 (m, 2H), 3.32 (m, 2H), 2.60 (s, 3H), 1.95−1.96 (m, 2H), 1.82 (m, 2H), 1.64−1.67 (m, 4H). MS (ESI) m/z 543.2 [M + 1]+. Elemental Analysis calculated (%) for (C29H30N6O5·0.17H2O): C 63.83, H 5.60, N 15.40. Found: C 63.81, H 5.49, N 15.31. KF = 0.57%. 4-((4-(Cyclopentyloxy)-5-(2-methylbenzo[d]oxazol-6-yl)-7Hpyrrolo[2,3-d]pyrimidin-2-yl)amino)-N-(2-hydroxy-2-methylpropyl)3-methoxybenzamide (41). Yield 0.061 g, 0.11 mmol, 21% over two steps, HPLC purity >97%. 1H NMR (300 MHz, CD3OD) δ 8.79 (d, J = 8.3 Hz, 1H), 7.88 (s, 1H), 7.73−7.60 (m, 1H), 7.54 (dd, J = 8.5, 3.3 Hz, 3H), 7.18 (s, 1H), 5.73 (s, 1H), 4.04 (s, 3H), 3.44 (s, 2H), 2.66 (s, 3H), 2.00−1.94 (m, 4H), 1.77−1.69 (m, 4H), 1.27 (s, 6H). MS (ESI) m/z 571.3 [M + 1]+. 4-((4-(Cyclopentyloxy)-5-(2-methylbenzo[d]oxazol-6-yl)-7Hpyrrolo[2,3-d]pyrimidin-2-yl)amino)-N-(1-hydroxy-2-methylpropan-2-yl)-3-methoxybenzamide (42). Yield 0.025 g, 0.044 mmol, 23%, HPLC purity >96%. 1H NMR (400 MHz, DMSO-d6) δ 11.83 (d, 1 H), 8.62 (d, 1 H), 7.93 (d, 1 H), 7.73 (s, 1 H), 7.68−7.57 (m, 2 H), 7.53−7.42 (m, 3 H), 7.39 (d, 1 H), 5.72−5.65 (m, 1 H), 5.00−4.93 (m, 1 H), 3.97 (s, 3 H), 3.53 (d, 2 H), 2.62 (s, 3 H), 2.04−1.92 (m, 2 H), 1.88−1.78 (m, 2 H), 1.67 (dd, 4 H), 1.33 (s, 6 H). MS (ESI) m/z 571.6 [M + l]+. 4-((4-(Cyclopentyloxy)-5-(2-methylbenzo[d]oxazol-6-yl)-7Hpyrrolo[2,3-d]pyrimidin-2-yl)amino)-N,3-dimethylbenzamide (43). Yield 0.045 g, 0.091 mmol, 13% over 2 steps, HPLC purity >98%. 1 H NMR (400 MHz, CD3OD) δ 8.33 (d, J = 8.59 Hz, 1 H), 7.85 (s, 1 H), 7.57−7.69 (m, 3 H), 7.46−7.52 (m, 1 H), 7.06 (s, 1 H), 5.64 (br s, 1 H), 2.89 (s, 3 H), 2.63 (s, 3 H), 2.38 (s, 3 H), 1.54−1.99 (m, 8 H). MS (ESI) m/z 497.0 [M + 1]+. 3-Chloro-4-((4-(cyclopentyloxy)-5-(2-methylbenzo[d]oxazol-6yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-N-methylbenzamide (44). Yield 0.051 g, 0.099 mmol, 64%, HPLC purity >94%. 1H NMR (400 MHz, DMSO-d6) δ 11.87 (s, 1 H), 8.43−8.50 (m, 2 H), 8.15 (s, 1 H), 7.97 (d, J = 1.95 Hz, 1 H), 7.94 (d, J = 1.17 Hz, 1 H), 7.83 (dd, J = 8.59, 1.95 Hz, 1 H), 7.64−7.67 (m, 1 H), 7.58−7.62 (m, 1 H), 7.41 (d, J = 2.34 Hz, 1 H), 5.66 (tt, J = 5.52, 2.69 Hz, 1 H), 2.79 (d, J = 4.30 Hz, 3 H), 2.62 (s, 3 H), 1.90−2.00 (m, 2 H), 1.77−1.86 (m, 2 H), 1.58−1.74 (m, 4 H). MS (ESI) m/z 517.6 [M + 1]+. Cellular Proliferation Assays. Cal-51 was purchased from DSMZ. MDA-MB-231, MDA-MB-468, BT-474, MDA-MB-361, and ZR-75-30 cells were purchased from the American Tissue Culture Collection. All cell lines were maintained in growth media as recommended by the vendors. The growth inhibitory effect was determined using the CellTiter-Glo luminescent cell viability assay. The cells were incubated with compound for 3 days. At the end of the assay period, CellTiter-Glo reagent was added to each well and the luminescence was measured on the Envision Multilabel (PerkinElmer) plate reader. The percent inhibition at each compound concentration was determined by normalizing data to the DMSO control values for each set of triplicate wells. All data were analyzed using XLfit from IDBS. The formula used for determining IC50 in XLfit was model number 205, which utilizes a four-parameter logistic model or sigmoidal dose−response model to calculate IC50 values. Full experimental details have been previously described.5 In Vitro Kinase Selectivity Profiling. The kinase selectivity profile of compound 23 was assessed using the Invitrogen panel of 255 kinases using one of the following Invitrogen protocols: Z′-Lyte protocol, ADAPTA, or the Lantha Binding Assay. The screen was conducted with the concentration of compound 23 held constant at 3 μM. The TTK binding affinity was measured at Invitrogen using the LanthaScreen Eu kinase binding assays. The LanthaScreen Eu kinase binding assays are based on the binding and displacement of a proprietary, Alexa Fluor 647-labeled, ATP-competitive kinase inhibitor scaffold (kinase tracer). ActivX Cellular Kinase Profiling. The HCT-116 cell line (CRL1619) was acquired from American Type Culture Collection (ATCC) and cultured in RPMI-1640 media with 10% fetal bovine serum. Upon confluence, compound was added to form a 1000× DMSO stock solution so that the final compound concentration was 3 μM. For

HPLC (10−95% acetonitrile in water) to give the racemic product (280 mg, 0.50 mmol, 23% yield over two steps) as a yellow solid. This material was further purified by chiral HPLC (30% ethanol in hexane) to obtain the title compound (0.041 g, 0.07 mmol, 3% yield, HPLC purity >97%). 1H NMR (400 MHz, DMSO-d6) δ 11.84 (s, 1H), 8.59 (d, J = 8.8 Hz, 1H), 8.32 (d, J = 4.8 Hz, 1H), 7.90 (s, 1H), 7.72 (s, 1H), 7.63−7.58 (m, 2H), 7.51 (d, J = 5.2 Hz, 2H), 7.34 (s, 1H), 5.56 (br s, 1H), 4.12 (s, 1H), 3.97 (s, 3H), 2.79 (d, J = 4.4 Hz, 3H), 2.61 (s, 3H), 2.01−1.94 (m, 2H), 1.71−1.68 (m, 2H), 1.48−1.42 (m, 2H), 1.37−1.34 (m, 2H), 1.01 (s, 3H). MS (ESI) m/z 557.2 [M + 1]+. 4-((4-(((1s,4s)-4-Hydroxy-4-methylcyclohexyl)oxy)-5-(2methylbenzo[d]oxazol-6-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (34). Compound 33 SEM was deprotected by the standard conditions and purified by prep HPLC (10−95% acetonitrile in water) to give the racemic product (280 mg, 0.50 mmol, 23% yield over two steps) as a yellow solid. This material was further purified by chiral HPLC (30% ethanol in hexane) to obtain the title compound (0.163 g, 0.29 mmol, 13% yield, HPLC purity >99%). 1H NMR (400 MHz, DMSO-d6) δ 11.83 (s, 1H), 8.60 (d, J = 8.0 Hz, 1H), 8.31 (d, J = 4.8 Hz, 1H), 7.97 (s, 1H), 7.71−7.68 (m, 2H), 7.69 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 9.2 Hz, 2H), 7.39 (d, J = 2.4 Hz, 1H), 5.25−5.20 (m, 1H), 4.19 (s, 1H), 3.97 (s, 3H), 2.80 (d, J = 4.4 Hz, 3H), 2.62 (s, 3H), 1.96−1.92 (m, 2H), 1.87−1.78 (m, 2H), 1.66−1.63 (m, 2H), 1.52−1.45 (m, 2H), 1.17 (s, 3H). MS (ESI) m/z 557.3 [M + 1]+. 4-(Cyclopentyloxy)-N-(2-methoxyphenyl)-5-(2-methylbenzo[d]oxazol-6-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (35). Yield 0.058 g, 0.13 mmol, 34%, HPLC purity >95%. 1H NMR (400 MHz, CDCl3) δ 9.05 (s, 1H), 8.60−8.58 (dd, J = 2.0 Hz, 1H), 7.82 (s, 1H), 7.59−7.59 (t, J = 1.6 Hz, 2H), 7.51 (s, 1H), 7.01−6.90 (m, 4H), 5.74−5.71 (m, 1H), 3.90 (s, 3H), 2.66 (s, 3H), 1.99−1.92 (m, 4H), 1.77−1.73 (m, 2H), 1.66−1.63 (d, J = 12 Hz, 2H). MS (ESI) m/z 456.2 [M + 1]+. 4-((4-(Cyclopentyloxy)-5-(2-methylbenzo[d]oxazol-6-yl)-7Hpyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N,N-dimethylbenzamide (36). Yield 0.073 g, 0.139 mmol, 87%, HPLC purity >96%. 1H NMR (400 MHz, DMSO-d6) δ 11.74 (s, 1 H) 8.55 (d, J = 8.20 Hz, 1 H) 7.89 (s, 1 H) 7.48−7.71 (m, 3 H) 7.28 (s, 1 H) 6.84−7.13 (m, 2 H) 5.43−5.75 (m, 1 H) 3.89 (s, 3 H) 2.95 (s, 6 H) 2.58 (s, 3 H) 1.49− 2.00 (m, 8 H). MS (ESI) m/z 527.2 [M + 1]+. (4-((4-(Cyclopentyloxy)-5-(2-methylbenzo[d]oxazol-6-yl)-7Hpyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxyphenyl)(4-methylpiperazin-1-yl)methanone (37). Yield 0.250 g, 0.420 mmol, 78%, HPLC purity >97%. 1H NMR (500 MHz, DMSO-d6) δ 1.59−1.76 (m, 4 H) 1.83 (br s, 2 H) 1.94−2.02 (m, 3 H) 2.22 (s, 3 H) 2.34 (br s, 4 H) 2.63 (s, 3 H) 3.54 (br s, 3 H) 3.95 (s, 3 H) 5.69 (br s, 1 H) 7.01− 7.09 (m, 2 H) 7.35−7.40 (m, 1 H) 7.58−7.73 (m, 3 H) 7.94 (s, 1 H) 8.60 (d, J = 8.20 Hz, 1 H) 11.82 (s, 1 H). MS (ESI) m/z 582.0 [M + l]+. (4-((4-(Cyclopentyloxy)-5-(2-methylbenzo[d]oxazol-6-yl)-7Hpyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxyphenyl)(morpholino)methanone (38). Yield 0.110 g, 0.193 mmol, 68%, HPLC purity >98%. 1H NMR (400 MHz, DMSO-d6) δ 11.74 (br s, 1 H) 8.58 (d, J = 8.20 Hz, 1 H) 7.89 (s, 1 H) 7.64 (s, 1 H) 7.50−7.62 (m, 2 H) 7.28 (s, 1 H) 6.95−7.09 (m, 2 H) 5.55−5.72 (m, 1 H) 3.90 (s, 3 H) 3.44−3.66 (m, 8 H) 2.58 (s, 3 H) 1.84−2.00 (m, 2 H) 1.71− 1.81 (m, 2 H) 1.63−1.70 (m, 2 H) 1.57 (br s, 2 H). MS (ESI) m/z 569.3 [M + 1]+. 4-((4-(Cyclopentyloxy)-5-(2-methylbenzo[d]oxazol-6-yl)-7Hpyrrolo[2,3-d]pyrimidine-2-yl)amino)-3-methoxy-N-(oxetan-3-yl)benzamide (39). Yield 0.127 g, 0.23 mmol, 45% over two steps, HPLC purity >97%. 1H NMR (400 MHz, DMSO-d6) δ 11.88 (s, 1H), 8.69−8.67 (d, J = 8.0 Hz, 1H), 7.93 (s, 1H), 7.77 (s, 1H), 7.66−7.58 (m, 2H), 7.51−7.45 (m, 2H), 7.39 (s, 1H), 5.70−5.67 (m, 1H), 4.86− 4.83 (m, 1H), 4.44−4.41 (m, 1H), 4.29−4.22 (m, 2H), 3.96 (s, 3H), 3.62−3.58 (m, 1H), 3.48−3.42 (m, 1H), 2.62 (s, 3H), 1.98−1.95 (m, 2H), 1.83−1.78 (m, 2H), 1.75−1.63 (m, 4H). MS (ESI) m/z 555.3 [M + 1]+. 4-((4-(Cyclopentyloxy)-5-(2-methylbenzo[d]oxazol-6-yl)-7Hpyrrolo[2,3-d]pyrimidin-2-yl)amino)-N-(2-hydroxyethyl)-3-methoxybenzamide (40). Yield 6 g, 11.0 mmol, 68%, HPLC purity >99%. 1H NMR (400 MHz DMSO-d6) δ 11.83 (s, 1H), 8.61−8.63 (m, 1H), 8.34 8999



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control cells (no compound), an equivalent volume of DMSO was added. After 1 h, cells were washed once with ice-cold 1× phosphate buffered saline (PBS), scrapped, and collected by centrifugation. The cell pellets were stored at −80 °C until ready for processing. The samples were analyzed using the KiNativ approach (ActivX, San Diego). The KiNativ (ActivX, San Diego) approach allows for selective enrichment of native kinases based on covalent modification of the active site with ATP and adenosinediphosphate (ADP) acylphosphate probes. Quantitation of enriched kinases is achieved through liquid chromatography (LC)-mass spectrometry (MS)/mass spectrometry. Procedure describe in Patricelli et al.36 Immunoblot Analyses. Cells were lysed in RIPA buffer (Thermos Scientific, catalogue no. 89900). The protein concentration of the lysates was determined by the Bio-Rad protein assay (Bio-Rad, catalogue no. 500-0001). Then 30 μg of protein from each lysate were loaded onto NuPAGE Novex 4−12% Bis-Tris gels (Invitrogen catalogue no. EA0375) and run in MES SDS running buffer. Protein was then transferred and blotted with primary antibodies. Primary antibodys used were as follows: Phospho-TTK T686 (custom-made rabbit monoclonal antibody), Phospho-SR (mouse 1H4G7) (Invitrogen catalogue no. 339400). Goat antirabbit AlexaFluor680 (Invitrogen catalogue no. A21076) and goat antimouse IRDye800 (Rockland Immunochemicals catalogue 610-132-121) were then used to detect the protein bands. The membranes were then scanned using the Odyssey infrared imaging system (LI-COR Biosciences). Wash-out Studies. Cal-51 cells were plated at a density of 3000 cells per well in a 96-well plate (Costar catalogue no. 33595) in 100 μL of growth media. The following day, compound dilutions were prepared with a final DMSO concentration of 0.2% in each well. All concentrations were assessed in triplicate within each 96-well assay plate. The cells were incubated with compound in 5% CO2 at 37 °C for 6, 12, 24, 48, and 72 h. At the end of each time point, wells were washed three times with fresh cell culture medium. The cells were cultured in 100 μL growth medium for up to 72 h post compound treatment. At the end of the assay, 100 μL of CellTiter-Glo reagent was added to each well. The luminescence was measured on the Envision Multilabel (2104 PerkinElmer) plate reader. Crystallization. The TTK kinase domain with a thrombincleavable, N-terminal 6XHis tag was expressed in E. coli strain BL21(DE3) (Life Technologies). Cells were grown in LB media, and induced at 20 °C for 4 h. Initial capture was performed via nickel affinity chromatography using nickel-NTA resin. The 6XHis tag was subsequently cleaved by addition of thrombin, and the protein passed back over nickel-NTA resin. Cleaved TTK was further purified by S-75 size exclusion chromatography (GE Healthcare), and concentrated to 11 mg/mL. TTK was crystallized by sitting drop vapor diffusion at 4 °C in the presence of 1 mM of compound 23. The TTK:23 complex was mixed 1:1 with, and subsequently equilibrated against, a solution of 100 mM sodium cacodylate pH 7, 200 mM potassium thiocyanate, 15% PEG 4K. Crystals were cryo-protected by addition of 20% ethylene gycol, and flash cooled under liquid nitrogen. Diffraction data was collected at the Canadian Light Source, beamline CMCF-08ID. Data were indexed, integrated and scaled using HKL2000.30 The structure was solved using molecular replacement using PHASER.31 Manual model building was performed using COOT, with subsequently rounds of refinement using CCP4.32,33 Coordinates and structure factors have been deposited in the Protein Data Bank with the accession code 6B4W. Computational Methods and Modeling. The Schrodinger Small-Molecule Drug Discovery Suite was used for all crystal structure and small molecule-protein visualization and analysis, including SiteMap generation (Maestro, Schrödinger Release 2017-1; MS Jaguar, Schrödinger, LLC, New York, NY, 2017; SiteMap (version 4201).34,35 CLK2 crystal structure (PDB 3NR9) was obtained from http://www.rcsb.org. Docking experiments were conducted with Glide as implemented in Schrodinger Small-molecule Suite (Schrödinger Release 2017-1, Glide; Schrödinger, LLC, New York, NY, 2017). Alignments and sequence comparisons were done with Clustal Omega Program ((http://www.ebi.ac.uk/Tools/msa/clustalw2/) and with the Schrodinger multiple sequence alignment tool PrimeX (Schrödinger

Release 2017-1: PrimeX; Schrödinger, LLC, New York, NY, 2017). Some portions of the modeling studies are included in the Supporting Information. In Vivo Studies. All animal studies were performed under protocols approved by Institutional Animal Care and Use Committees. Female SCID mice were inoculated subcutaneously with 5 × 106 Cal51 cells. Mice with tumors of approximately 125 mm3 were randomized and treated intravenously at various doses and schedules of compound 23 (n = 8−10/group). Tumors were measured twice a week for the duration of the study. The long and short axes of each tumor were measured using a digital caliper in millimeters and the tumor volumes were calculated using the formula: width 2 x_ length/ 2. The tumor volumes were expressed in cubic millimeters (mm3).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01223. In vitro and cellular kinase selectivity data for compound 23, additional TTK crystal structure views, docked structure of 23 in CLK2, experimental procedures for intermediates (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 858-795-4854. E-mail: [email protected]. ORCID

Jennifer R. Riggs: 0000-0001-9012-1428 Philip P. Chamberlain: 0000-0002-6407-7344 Present Address

All authors are employees of Celgene, except P. Erdman, G. Deyanat-Yazdi, M. Moghaddam, S. Delker, A. Calabrese, K. Leftheris, who were Celgene employees at the time of their contribution to this work. Current address information for P. Erdman, G. Deyanat-Yazdi, M. Moghaddam, S. Delker, A. Calabrese, and K. Leftheris is available from the corresponding author upon request. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Celgene San Diego DMPK department for analysis, the Celgene San Diego CLMD and analytical groups for project support, and Deborah Mortensen for helpful discussions and editorial comments. Crystal structure determination described in this paper was performed using beamline 08ID-1 at the Canadian Light Source, which is supported by the Canada Foundation for Innovation, Natural Sciences and Engineering Research Council of Canada, the University of Saskatchewan, the Government of Saskatchewan, Western Economic Diversification Canada, the National Research Council Canada, and the Canadian Institutes of Health Research.

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ABBREVIATIONS USED Mps1, monopolar spindle 1; CLK2, CDC2-like kinase; DYRK1A, 1B, 3, dual specificity tyrosine-phosphorylationregulated kinase 1A, 1B, 3; PHKG1, phosphorylase kinase catalytic subunit gamma 1; CAMKK2, calcium/calmodulin dependent protein kinase kinase 2; PIP4K22, phosphatidylino9000



Article

on N-(3-(3-sulfamoylphenyl)-1H-indazol-5-yl)-acetamides and carboxamides. Bioorg. Med. Chem. 2014, 22, 4968−4997. (15) Liu, Y.; Lang, Y.; Patel, N. K.; Ng, G.; Laufer, R.; Li, S. W.; Edwards, L.; Forrest, B.; Sampson, P. B.; Feher, M.; Ban, F.; Awrey, D. E.; Beletskaya, I.; Mao, G.; Hodgson, R.; Plotnikova, O.; Qiu, W.; Chirgadze, N. Y.; Mason, J. M.; Wei, X.; Lin, D. C.; Che, Y.; Kiarash, R.; Madeira, B.; Fletcher, G. C.; Mak, T. W.; Bray, M. R.; Pauls, H. W. The discovery of orally bioavailable tyrosine threonine kinase (TTK) inhibitors: 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides as anticancer agents. J. Med. Chem. 2015, 58, 3366−3392. (16) Liu, Y.; Laufer, R.; Patel, N. K.; Ng, G.; Sampson, P. B.; Li, S. W.; Lang, Y.; Feher, M.; Brokx, R.; Beletskaya, I.; Hodgson, R.; Plotnikova, O.; Awrey, D. E.; Qiu, W.; Chirgadze, N. Y.; Mason, J. M.; Wei, X.; Lin, D. C.; Che, Y.; Kiarash, R.; Fletcher, G. C.; Mak, T. W.; Bray, M. R.; Pauls, H. W. Discovery of pyrazolo[1,5-a]pyrimidine TTK inhibitors: CFI-402257 is a potent, selective, bioavailable anticancer agent. ACS Med. Chem. Lett. 2016, 7, 671−675. (17) Mason, J. M.; Wei, X.; Fletcher, G. C.; Kiarash, R.; Brokx, R.; Hodgson, R.; Beletskaya, I.; Bray, M. R.; Mak, T. W. Functional characterization of CFI-402257, a potent and selective Mps1/TTK kinase inhibitor, for the treatment of cancer. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 3127−3132. (18) Faisal, A.; Mak, G. W. Y.; Gurden, M. D.; Xavier, C. P. R.; Anderhub, S. J.; Innocenti, P.; Westwood, I. M.; Naud, S.; Hayes, A.; Box, G.; Valenti, M. R.; De Haven Brandon, A. K.; O’Fee, L.; Schmitt, J.; Woodward, H. L.; Burke, R.; vanMontfort, R. L. M.; Blagg, J.; Raynaud, F. I.; Eccles, S. A.; Hoelder, S.; Linardopoulos, S. Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy. Br. J. Cancer 2017, 116, 1166−1176. (19) Innocenti, P.; Woodward, H. L.; Solanki, S.; Naud, S.; Westwood, I. M.; Cronin, N.; Hayes, A.; Roberts, J.; Henley, A. T.; Baker, R.; Faisal, A.; Mak, G. W.; Box, G.; Valenti, M.; De Haven Brandon, A.; O’Fee, L.; Saville, H.; Schmitt, J.; Matijssen, B.; Burke, R.; van Montfort, R. L.; Raynaud, F. I.; Eccles, S. A.; Linardopoulos, S.; Blagg, J.; Hoelder, S. Rapid discovery of pyrido[3,4-d]pyrimidine inhibitors of monopolar spindle kinase 1 (MPS1) using a structurebased hybridization approach. J. Med. Chem. 2016, 59, 3671−3688. (20) Kusakabe, K.; Ide, N.; Daigo, Y.; Itoh, T.; Yamamoto, T.; Hashizume, H.; Nozu, K.; Yoshida, H.; Tadano, G.; Tagashira, S.; Higashino, K.; Okano, Y.; Sato, Y.; Inoue, M.; Iguchi, M.; Kanazawa, T.; Ishioka, Y.; Dohi, K.; Kido, Y.; Sakamoto, S.; Ando, S.; Maeda, M.; Higaki, M.; Baba, Y.; Nakamura, Y. Discovery of imidazo[1,2b]pyridazine derivatives: selective and orally available Mps1 (TTK) kinase inhibitors exhibiting remarkable antiproliferative activity. J. Med. Chem. 2015, 58, 1760−1775. (21) Uitdehaag, J. C. M.; de Man, J.; Willemsen-Seegers, N.; Prinsen, M. B. W.; Libouban, M. A. A.; Sterrenburg, J. G.; de Wit, J. J. P.; de Vetter, J. R. F.; de Roos, J.; Buijsman, R. C.; Zaman, G. J. R. Target residence time-guided optimization on TTK kinase results in inhibitors with potent anti-proliferative activity. J. Mol. Biol. 2017, 429, 2211−2230. (22) Jemaa, M.; Galluzzi, L.; Kepp, O.; Senovilla, L.; Brands, M.; Boemer, U.; Koppitz, M.; Lienau, P.; Prechtl, S.; Schulze, V.; Siemeister, G.; Wengner, A. M.; Mumberg, D.; Ziegelbauer, K.; Abrieu, A.; Castedo, M.; Vitale, I.; Kroemer, G. Characterization of novel MPS1 inhibitors with preclinical anticancer activity. Cell Death Differ. 2013, 20, 1532−1545. (23) Wengner, A. M.; Siemeister, G.; Koppitz, M.; Schulze, V.; Kosemund, D.; Klar, U.; Stoeckigt, D.; Neuhaus, R.; Lienau, P.; Bader, B.; Prechtl, S.; Raschke, M.; Frisk, A. L.; von Ahsen, O.; Michels, M.; Kreft, B.; von Nussbaum, F.; Brands, M.; Mumberg, D.; Ziegelbauer, K. Novel Mps1 kinase inhibitors with potent antitumor activity. Mol. Cancer Ther. 2016, 15, 583−592. (24) Yomoda, J.; Muraki, M.; Kataoka, N.; Hosoya, T.; Suzuki, M.; Hagiwara, M.; Kimura, H. Combination of Clk family kinase and SRp75 modulates alternative splicing of adenovirus E1A. Genes Cells 2008, 13, 233−244.

sitol 5-phosphate 4-kinase; JNK, c-Jun N-terminal kinase; SAC, spindle assembly checkpoint; TNBC, triple negative breast cancer; SR proteins, serine- and arginine-rich proteins; SEM, standard error of the mean; TGI, tumor growth inhibition; BWL, body weight loss

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