Indazole-Based Potent and Cell-Active Mps1 Kinase Inhibitors

Article pubs.acs.org/jmc

Indazole-Based Potent and Cell-Active Mps1 Kinase Inhibitors: Rational Design from Pan-Kinase Inhibitor Anthrapyrazolone (SP600125) Ken-ichi Kusakabe,*,† Nobuyuki Ide,† Yataro Daigo,⊥,∥ Yuki Tachibana,† Takeshi, Itoh,† Takahiko Yamamoto,§ Hiroshi Hashizume,† Yoshio Hato,† Kenichi Higashino,§ Yousuke Okano,§ Yuji Sato,† Makiko Inoue,† Motofumi Iguchi,† Takayuki Kanazawa,† Yukichi Ishioka,† Keiji Dohi,† Yasuto Kido,‡ Shingo Sakamoto,‡ Kazuya Yasuo,§ Masahiro Maeda,§ Masayo Higaki,§ Kazuo Ueda,† Hidenori Yoshizawa,† Yoshiyasu Baba,† Takeshi Shiota,† Hitoshi Murai,† and Yusuke Nakamura∥ †

Medicinal Research Laboratories, ‡Drug Developmental Research Laboratories, and §Innovative Drug Discovery Research Laboratories, Shionogi Pharmaceutical Research Center, 1-1 Futaba-cho 3-chome, Toyonaka, Osaka 561-0825, Japan ∥ Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan ⊥ Department of Medical Oncology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan S Supporting Information *

ABSTRACT: Monopolar spindle 1 (Mps1) is essential for centrosome duplication, the spindle assembly check point, and the maintenance of chromosomal instability. Mps1 is highly expressed in cancer cells, and its expression levels correlate with the histological grades of cancers. Thus, selective Mps1 inhibitors offer an attractive opportunity for the development of novel cancer therapies. To design novel Mps1 inhibitors, we utilized the pan-kinase inhibitor anthrapyrazolone (4, SP600125) and its crystal structure bound to JNK1. Our design efforts led to the identification of indazole-based lead 6 with an Mps1 IC50 value of 498 nM. Optimization of the 3- and 6-positions on the indazole core of 6 resulted in 23c with improved Mps1 activity (IC50 = 3.06 nM). Finally, application of structurebased design using the X-ray structure of 23d bound to Mps1 culminated in the discovery of 32a and 32b with improved potency for cellular Mps1 and A549 lung cancer cells. Moreover, 32a and 32b exhibited reasonable selectivities over 120 and 166 kinases, respectively.

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INTRODUCTION Most human cancer cells accumulate genetic changes at an aberrantly rapid rate, which is referred to as genetic instability.1−3 This is a hallmark of cancer and can arise through one of two different pathways, microsatellite instability (MIN) and chromosomal instability (CIN).4 Some cancer cells cannot repair certain types of DNA damage or correct errors in replication. These cells tend to accumulate more point mutations or DNA sequence changes. This creates the genetic condition known as MIN.5,6 Other cancer cells have defects in maintaining either the number or the integrity of their chromosomes. Consequently, they become aneuploid, having an abnormal number of chromosomes, and this is linked to CIN.2,3,7 Duesberg et al. have demonstrated that aneuploidy is a sufficient cause of genetic instability, that is, aneuploidy (or CIN) is a common cause of cancer and genetic instability.8 Therefore, strategies that contribute to the suppression of aneuploidy or the selective death of aneuploid cells could be therapeutically beneficial.9,10 © 2013 American Chemical Society

Monopolar spindle 1 (Mps1), also known as TTK, is a dual specificity protein kinase that phosphorylates tyrosine, serine, or threonine residues.11 Mps1 is activated during mitosis and is essential for centrosome duplication, mitotic checkpoint signaling, and the maintenance of CIN.12−15 Increased expression of Mps1 is observed in cancer cells,16−19 and its expression levels correlate with the histological grades of breast cancers.19 Interestingly, high levels of Mps1 provide stability and protection for aneuploid cancer cells during mitosis, while reduction of Mps1 levels by RNAi in aneuploid cells increases the frequency of aberrant and catastrophic mitoses, which results in decreased survival and induction of apoptosis.19 In contrast, there is no significant increase in apoptosis in Mps1depleted nonmalignant cells.19 These findings suggest that inhibition of Mps1 should lead to the selective death of cancer cells with aneuploidy. Therefore, Mps1 is a promising target in the development of cancer therapeutics. Received: January 4, 2013 Published: May 1, 2013 4343

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cells were moderate, and the variety of their chemotypes was limited. Anthrapyrazolone (SP600125), a c-Jun N-terminal kinase (JNK) inhibitor, is also known as a potent Mps1 inhibitor (Figure 2).15,26−29,32,33 Although 4 (SP600125) has good inhibitory activity against Mps1 (IC50 = 98 nM),28 it shows poor selectivity over other kinases, which limits further development and its use as a research tool.28,29 However, we felt that pan-kinase inhibitor 4 would be a good template for the design of novel Mps1 inhibitors due to its good lead profiles: low molecular weight (220), low topological polar surface area (TPSA)30 (42 Å), and high ligand efficiency (LE)31 (0.57). Furthermore, to design advanced Mps1 inhibitors, the X-ray structure of 4 bound to JNK132 was available in the Protein Data Bank (PDB) at the start of this study.29,33 Traditionally, kinase inhibitors have been discovered by highthroughput screening; the identified hits are selected on the basis of their inhibitory activity and their selectivity profiles.34 Despite recent progress in structure-based drug discovery, the design of highly selective inhibitors remains a major challenge.34−40 Herein, we describe our efforts to identify novel indazole-based Mps1 inhibitors that were designed from pan-kinase inhibitor 4. Using the X-ray structure of 4 bound to JNK1, we successfully designed and synthesized indazole-based lead 6. Further optimization led to the discovery of selective Mps1 inhibitors 32a and 32b with improved cellular activities. Moreover, 32a and 32b exhibited reasonable selectivities over 120 and 166 kinases, respectively.

A few potent and selective Mps1 inhibitors have been disclosed (Figure 1).20−25 For example, Nerviano Medical

Figure 1. Structures of potent and selective Mps1 inhibitors.

Sciences and Myrexis (Myriad) have reported on 1 (NMSP715) and 2 (MPI-0479605), respectively. Both inhibitors have pyrimidine-based scaffolds and inhibited tumor growth in preclinical cancer models. In our efforts to discover Mps1 inhibitors, we identified diaminopyridine-based compound 3 as a selective inhibitor with in vivo activity.25 These Mps1 inhibitors exhibit single-digit nanomolar potency on biochemical assay; however, their antiproliferative activities in tumor

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CHEMISTRY The initial set of indazole derivatives including 6 and 9a−d was synthesized according to the procedures outlined in Scheme 1.

Figure 2. (A) Design of indazoles 5 and 6 from 4 (SP600125). (B) X-ray structure of 4 bound to JNK1 (1UKI). Key residues are shown as sticks with green carbon. Hydrogen bonds are shown as dotted lines in red. (C) Docking model of 5 bound to JNK1 (1UKI). Residues with green carbon are identical in Mps1, while residues with yellow carbon are different in Mps1. Upper residue labels are JNK1, and lower residue labels are Mps1. (D) Docking model of 5 bound to JNK1 (1UKI) with pocket surface colored in orange. The binding pockets and solvent region are indicated. HBA is the approximate position of the hinge carbonyl (JNK1, Met111; Mps1, Gly605). Figures were generated with PyMOL.41 4344

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Scheme 1. Synthesis of Indazoles 6 and 9a−da

prepared in a manner similar to that for compounds 23a−c and 23e. 5-Amino-indazole analogues 32a and 32b were prepared according to the procedures in Scheme 4, in order to improve the cellular activity of the alkoxy analogue 23c. Nitration of 5 followed by cyclization of 24 with hydrazine hydrate gave 25, which was protected with the 2-(trimethylsilyl)ethoxymethyl (SEM) group and subsequent Suzuki coupling reaction followed by SEM deprotection to afford 26. Iodination of 26 followed by SEM protection gave 28. Suzuki reactions of 28 with 3-sulfamoylphenylboronic acid and 3-acetamidophenylboronic acid afforded 29a and 29b, respectively. Nitro groups of 29a and 29b were reduced to give 30a and 30b, which were then reductive-alkylated with cyclohexanone followed by deprotection of the SEM groups to afford the final compounds 32a and 32b, respectively.

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a

Reagents and conditions: (a) (i) I2, KOH, DMF, rt; (ii) Boc2O, DMAP, Et3N, MeCN, rt (36%); (b) R-B(OH)2, Pd(PPh3)4, aq. Na2CO3, THF, reflux; (ii) TFA, DCM, rt (50−78%).

RESULTS AND DISCUSSION Design of the Indazole Scaffold and Identification of Initial Lead 6. At the start of this study, the crystal structure of 4 bound to JNK1 was available (PDB ID: 1UKI).29,32 Therefore, we used this crystal structure as a guide to predict the orientation of key substituents on 4 at the ATP binding site (Figure 2B). As shown in Figure 2A, indazole 5 was designed by truncating the carbonyl group of 4 for the following reasons: (1) there are no specific interactions of the carbonyl group in 4 (Figure 2B) with amino acid residues; (2) introduction of various substituents on the fused ring of 4 is limited because of the synthetic inaccessibility of the fused ring system. We evaluated the newly designed indazole 5 and found that it showed moderate activity with an Mps1 IC50 value of 2.1 μM (Figure 2A). Although the Mps1 inhibitory activity of 5 was lower than that of 4, this compound was expected to provide a good starting point for the design of indazole-based Mps1 inhibitors. To obtain its inhibitory activity and a selectivity profile, we considered reasonable positions for introducing substituents to the indazole ring using the docking model of 5 bound to JNK1 (Figure 2C and D). Position 7 comes into close contact with the gatekeeper residue Met, which is conserved in Mps1, and was therefore left unsubstituted. However, a substituent at the 6-position can provide a suitable vector toward the back pocket. In addition, introduction of substituents at the 5-position offered opportunities for contact with the ribose binding pocket. In general, these two regions are not conserved and can be used to gain affinity as well as selectivity.35,38 Unlike the 5and 6-positions, the vector of the 4-position is directed toward the solvent region, which means that this site is less important for achieving high affinity for ligand binding. Therefore, we selected the 5- and 6-positions to explore the structure−activity relationships. To test our hypothesis, the ethoxy group was introduced at the 5-position of indazole 5, which gave rise to compound 6 (Figure 2A). As expected, 5-ethoxy indazole 6 showed improved potency with an Mps1 IC50 value of 498 nM as compared with 5, supporting our design hypothesis. Initial SAR around Compound 6. Once we identified initial lead 6, our attention was focused on the phenyl ring of 6. Given the docking model of 5 bound to JNK1, the phenyl ring was expected to lie in the front pocket. This pocket is frequently used to gain affinity and selectivity, similar to the back pocket and the ribose binding pocket.35,38 Furthermore, introduction of substituents on the phenyl ring appeared to

Iodination of indazole 7 followed by tert-butoxycarbonyl (Boc) protection provided 8. A variety of aryl groups was introduced at the 3-positon via Suzuki coupling reaction followed by Bocdeprotection to afford 6 and 9a−d. For the syntheses of 6-cyanoindazoles 17a and 17b, we utilized the commercially available 11 (Scheme 2). Compound Scheme 2. Synthesis of Indazole 17a

a

Reagents and conditions: (a) NaOEt, EtOH, rt (99%); (b) CuCN, DMF, 150 °C (quant.); (c) Pd/C, H2, THF, rt; (d) NaNO2, AcOH, rt (82% in 2 steps); (e) (i) I2, KOH, DMF, rt; (ii) Boc2O, DMAP, MeCN, rt (78%); (f) 3-sulfamoylphenylboronic acid, PdCl2(dppf)·DCM, aq. Na2CO3, THF, reflux; (ii) TFA, DCM, rt (12−68%).

11 was reacted with sodium ethoxide to afford 12, which was then treated with copper cyanide to give 13. Reduction of the nitro group, diazotization with sodium nitrite, iodination, and subsequent Boc protection provided 16. Finally, 17a was prepared by Suzuki coupling reaction of 16 with 3sulfamoylpheyl boronic acid and subsequent deprotection of the Boc group. Compound 17b was prepared in a manner similar to that for 17a. To explore the 6-position of the indazole scaffold, a variety of 6-aryl indazole analogues 23a−f were prepared as shown in Scheme 3. Reduction of 12 with sodium hydrosulfite followed by cyclization provided 6-bromo-indazole 20a. Iodination of 20a and subsequent Boc-protection gave compound 21a, which was then made to react with 3-sulfamoylphenylboronic acid to afford the key intermediate 22a. Suzuki reactions of 22a with a variety of arylboronic acids followed by Boc-deprotection afforded 23a−c and 23e. Compounds 23d and 23f were 4345

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Scheme 3. Synthesis of Indazoles 23a−ea

Reagents and conditions: (a) NaOEt, EtOH, rt or cyclohexanol, KOH, MeCN, rt (36−99%); (b) Na2S2O4, EtOH/H2O, 100 °C (85−87%); (c) NaNO2, AcOH, rt (75−88%); (d) (i) I2, KOH, DMF, rt; (ii) Boc2O, DMAP, MeCN, rt (91−95%); (e) R-B(OH)2, PdCl2(dppf)·DCM, aq. Na2CO3, THF, reflux (59−67%); (f) (i) R-B(OH)2, S-Phos, aq. K2CO3, THF, reflux; (ii) TFA, DCM, rt (11−100%). a

Scheme 4. Synthesis of Indazoles 32a and 32ba

Reagents and conditions: (a) KNO3, H2SO4, 0 °C (94%); (b) hydrazine hydrate, DMF, 150 °C (93%); (c) (i) SEMCl, NaH, DMF, 0 °C; (ii) 1methyl-1H-pyrazole boronic acid pinacol ester, PdCl2(dtbpf), aq. K2CO3, THF, reflux; (iii) TBAF, ethylenediamine, THF, reflux (79%); (d) I2, KOH, DMF, rt (98%); (e) SEMCl, NaH, DMF, rt (88%); (f) R-B(OH)2, PdCl2(dppf)·CH2Cl2, aq. Na2CO3, THF, reflux (89%); (g) Fe, NH4Cl, EtOH/H2O, reflux (89−97%); (h) cyclohexanone, NaBH(OAc)3, AcOH, DCM, rt (87−100%); (i) TBAF, ethylenediamine, THF, reflux (55− 79%). a

when a sulfonamide was introduced at the meta-position (9d). This compound showed an IC50 value of 28.0 nM, which was an 18-fold increase over lead compound 6 and was more potent than 4. Furthermore, 9d showed improved selectivity for JNK1 when compared with the analogues described in Table 1. Next, we turned our attention to the 6-position of the indazole, pointing toward the back pocket. As a general trend, introduction of substituents bearing hydrogen bond acceptors and their sizes was important for binding to the back pocket.35,39,43 For example, 6-cyano indazole 17a was more

furnish an access point to the hinge carbonyl group as shown in Figure 2D, which might be expected to gain additional potency. To form a hydrogen bond with the hinge carbonyl of Gly605, hydrogen bond donating groups were investigated (Table 1). Introduction of the acetamide group at the para-position of the phenyl ring led to 9a, which showed equal potency against Mps1 (IC50 = 438 nM). The meta-substituted acetamide 9b was more potent than 9a with an IC50 value of 116 nM. A “reversed” amide analogue such as 9c had potency similar to that of 9a. Finally, a significant increase in activity was observed 4346

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Table 1. SAR of the Indazole 3-Positiona

hypothesis, small-sized heterocycles with hydrogen bond acceptor capability such as pyrazole analogues were prepared (23b−e). As expected, the N-H-pyrazole 23b was nearly equipotent to the cyano analogue 17. Importantly, the Nmethyl analogue of 23c showed improved Mps1 activity (IC50 = 3.06 nM), while a larger group of N-isobutyl pyrazole 23e resulted in a 38-fold decrease in activity (IC50 = 119 nM). Furthermore, 23c provided superior selectivity for JNK1 (31fold). Introduction of a methyl group at the 4-position of the phenyl ring of 23c led to 23d, which retained its inhibitory activity. Having identified the compounds with biochemical potency, the cellular potencies for the compounds described in Table 2 were evaluated. To investigate the cellular inhibition of Mps1, we used an autophosphorylation assay on a cell line that stably expresses FLAG-tagged Mps1 under the control of a tetracycline-suppressible promoter.25 The antiproliferative activity was measured in A549 lung carcinoma cell lines. As a general trend, there was a significant correlation between biochemical and cellular IC50 values as shown in Figure 4A (R2 = 0.737 for the compounds in Table 2). Indeed, the promising compound 23c exhibited the highest cellular Mps1 potency in Table 2 (85.8 nM), consistent with its biochemical potency. Unfortunately, these analogues with cellular Mps1 potency such

biochemical IC50 (nM)

a

compd

R1

R2

Mps1

JNK1

Mps1/JNK1

9a 9b 9c 9d

H NHAc CONH2 SO2NH2

NHAc H H H

438 116 189 28.0

169 79.5 42.8 150

0.39 0.69 0.23 5.4

Assay protocols are described in ref 25.

potent than 6-unsubstituted analogue 9d, while installation of the phenyl group diminished activity (23a). Although 17a showed improved Mps1 activity, the effect on selectivity for JNK1 was not observed when compared with the unsubstituted analogue 9d. We hypothesized that the cyano group would form a hydrogen bond with the conserved Lys553, and the large gatekeeper Met would limit the size of the back pocket as reported for other kinase inhibitors.35,39,40 To test this Table 2. SAR of the Indazole 6-Positiona

a

Assay protocols are described in ref 25. bInhibition of autophosphorylation in RERF cells. cCell viability in A549 cells after 72 h. dLipophilicity index determined by RP-HPLC. 4347

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Figure 3. X-ray structure of 23d bound to Mps1 (PDB ID: 3W1F). (A) 23d and key residues within the kinase active site. 23d is shown as sticks with green carbons, and the key residues are shown as lines with cyan carbons. (B) Binding site surface of 23d in Mps1. The surface is colored orange. (C) 23d and the hinge residues. Hydrogen bonds are shown as red dotted lines. (D) The key residues around the sulfonamide group of 23d. Hydrogen bonds with the sulfonamide are shown as red dotted lines. Figures were generated with PyMOL.41

Table 3. SAR of the Indazole 5-Positiona

a

Assay protocols are described in ref 25; nd = not determined. bInhibition of autophosphorylation in RERF cells. cCell viability in A549 cells after 72 h. dLipophilicity index determined by RP-HPLC. eApparent permeability in MDCK cells. This assay was conducted at Absorption Systems.42

hydrogen bonds with the hinge backbone carbonyl of Glu603 and NH of Gly605 (Figure 3C), respectively, which is similar to that observed in the crystal structures of JNK1 and Mps1 in complex with 4.29,32 Consistent with the hypothesis as shown in Figure 2D, the sulfonamide NH2 on the phenyl ring forms a hydrogen bond with the carbonyl of Gly605 (Figure 3C). Interestingly, this NH2 of sulfonamide also forms a hydrogen

as 23c and 23d were limited by their moderate antiproliferative IC50 values in A549 cell lines. Crystal Structure of 23d Bound to the Mps1 Kinase Domain. To confirm our hypothesis, as well as to gain insight into the binding mode of these indazoles, the crystal structure of 23d bound to Mps1 was solved. The key residues are shown in Figure 3A. The indazole nitrogens of N1 and N2 form 4348

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Figure 4. Logarithmic scale is used for both x- and y-axes. The compounds in Table 2 are colored magenta, and the compounds in Table 3 are colored cyan. (A) Cellular Mps1 IC50 versus biochemical IC50. R2 = 0.737 (for the compounds in Table 2). R2 = 0.503 (for all compounds in the figure). The linear regression shown is generated based on all compounds. (B) A549 IC50 versus cellular Mps1 IC50. R2 = 0.862. The scatter plots were prepared using TIBCO Spotfire, version 4.5.0.

provided a 4-fold increase in antiproliferative activity (A549 IC50 = 152 nM), as compared with alkoxy analogues 23f. Also, the acetamide analogue 32b was roughly equipotent with 32a. Unfortunately, both 32a and 32b showed lower selectivity for JNK1 than 23c (3.6-fold and 1.0-fold, respectively) possibly because the residues around the 5-position between Mps1 and JNK1 are conserved (Ile531, Gly532, and Val539), which led to increases in both Mps1 and JNK1 activities. To gain insight into the improvement in the cellular activity of Mps1, we analyzed the relationship between biochemical and cellular Mps1 IC50 values (Figure 4A). As a general trend, the compounds in Table 2 (including 23c) exhibited log k′ values of less than 0.90, while the compounds in Table 3 (other than 23c), which showed improved cellular Mps1 activity but a similar level of biochemical activity to 23c, had log k′ values of more than 0.90, indicating that increased lipophilicity could contribute to improvement in the cellular activity of Mps1. Furthermore, as shown in Table 3, log k′ correlated roughly with Papp, suggesting the importance of permeability in cellular potency. The relationship between cellular Mps1 and A549 antiproliferative activity was also investigated. As shown in Figure 4B, we observed a strong correlation between cellular Mps1 and A549 IC50 values (R2 = 0.862), which indicated that increased antiproliferative activity appeared to be responsible for the increased inhibitory activity of Mps1. The optimized Mps1 inhibitors 32a and 32b showed more potent antiproliferative activities in A549 lung cancer cells than the known potent Mps1 inhibitors 1, 2, and 3. For example, 1 and 2 had biochemical Mps1 IC50 values of 8 nM and 1.8 nM, respectively,20,22 which were comparable to our compounds described here; however, despite these single digit nanomolar IC50 values, these antiproliferative activities in A549 cells (after 72 h) were only 1.25 μM and >10 μM.20,22 Our previously reported Mps1 inhibitor 3 was equipotent with 1 and 2 in the biochemical assay (6.4 nM) but showed moderate antiproliferative activity (A549 IC50 = 840 nM).25 Kinase Selectivity. To investigate kinase selectivity, compounds 32a and 32b, which were very potent in both enzyme and cellular assays within our indazole series, were evaluated at 1 μM against panels of 120 and 166 human

bond with the carbonyl of Asn606 (Figure 3C). Furthermore, four additional hydrogen bonds are formed among the sulfonamide oxygen atoms and the side chains of Lys529, Gln541, and Cys604 (Figure 3D). These observations provide an explanation for the importance of the sulfonamide at this position, as shown in Table 1. As expected, the N-methyl pyrazole ring lies in the back pocket (Figure 3B) and leads to favorable van der Waals interactions with the gatekeeper Met602, Met600, and Ile663. This observation explains why the larger N-isobutyl pyrazole 23e is significantly less potent. The N2 pyrazole nitrogen forms a weak hydrogen bond or ionic interaction with NH3+ of Lys553 (Figure 3A), with a distance of 3.20 Å, which explains the increase in potency observed when the phenyl ring was replaced with the pyrazole ring (23a and 23c) or the cyano group (17a). The phenyl ring on the 3position of the indazole scaffold lies in the front pocket and causes hydrophobic interactions with Ile531 and Leu654. Finally, the ethoxy group at the 5-position of indazole occupies the ribose binding pocket (Figure 3B), which was defined by the hydrophobic residues Gly532, Val539, Met671, and Pro673. Improvement in Cellular Activity: Optimization of the 5-Position. To aid further design of compounds with improved cellular activity, lipophilicity was experimentally determined as log k′ by a method using RP-HPLC on the basis of the compound retention time (a higher value means greater lipophilicity) as shown in Tables 2 and 3. For selected compounds, the apparent permeability coefficient (Papp) was also measured in MDCK cells. Consistent with its moderate cellular activity, 23c showed low permeability (Papp = 0.60 × 10−6 cm/s) and a log k′ value of 0.79 (Table 3). To increase the lipophilicity as well as inhibitory activity of 23c, we explored substituents at the 5-position of the indazole scaffold. The X-ray structure of 23d revealed that this position lies in the ribose binding pocket of Mps1 defined by hydrophobic residues. Therefore, we selected a small set of hydrophobic residues. Replacement of the ethoxy group with a cyclohexyloxy group led to 23f, which retained the cellular activity of Mps1 and showed a 2-fold improvement in A549 activity (IC50 = 646 nM). Finally, we were pleased to observe that a simple change to the amino analogue 32a exhibited a cellular Mps1 IC50 value of 20.5 nM with only a 2-fold cell-shift. Furthermore, 32a 4349

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Table 4. Pharmacokinetic Properties of 32a, 32b, and 32c iv (rat, 0.5 mpk)

po (rat, 1 mpk)

compd

rat Hep (%)a

Papp (× 10−6 cm/s)b

CL (mL/min/kg)c

Vdss (L/kg)d

AUC (ng·h/mL)e

Cmax (ng/mL)f

F (%)g

32a 32b

69 64

2.69 20.5

33 19

2.8 1.0

11.5 149

1.4 18

2.3 17

a

Percent remaining in rat hepatocytes after 1 h. bApparent permeability in MDCK cells. This assay was conducted at Absorption Systems.42 cPlasma clearance. dVolume of distribution at steady state. ePlasma area under the curve. fMaximal plasma concentration. gOral bioavailability.

appeared to be limited because 32a with a larger substituent, cyclohexylamino group, showed a level of kinase selectivity similar to that of 23c with a smaller substituent ethoxy group; both compounds inhibited 2 kinases (AURKA and FLT3) when using 48 kinases that were used for both 17b and 23c. As demonstrated above, all compounds tested for kinase selectivity showed more than 50% inhibition of AURKA, regardless of the size of the substituents at the 5- and 6positions. To better understand the selectivity for AURKA, we examined the residue difference between Mps1 and AURKA in the vicinity of 23d. For 21 residues within 4.5 Å from 23d, AURKA showed 43% identity and 87% similarity, which explains why AURKA was subject to more than 50% inhibition by our indazoles. Pharmacokinetic Profiles of 32a and 32b. On the basis of these favorable in vitro profiles, compounds 32a and 32b were further characterized through pharmacokinetic studies in rats (Table 4). Compound 32a showed a moderate clearance of 33 mL/min/kg but lacked oral bioavailability at a dose of 1 mg/ kg (2.3%). In contrast, 32b improved the pharmacokinetic profiles, that is, it exhibited a lower clearance (19 mL/min/kg) and a higher oral bioavailability (17%). In particular, 32b showed a 12-fold increase in oral plasma exposure (AUC, po) over that of 32a. The difference in oral bioavailability between these compounds could be explained by their passive permeability; 32b displayed a higher permeability in MDCK cells than 32a (32a, Papp = 2.69 × 10−6 cm/s; 32b, Papp = 20.5 × 10−6 cm/s),42 while they had similar metabolic stabilities for rat hepatocytes as shown in Table 4. Clearly, a lower TPSA of 32b seems to contribute to an increase in permeability (32a, TPSA = 112; 32b, TPSA = 81).30 Finally, both compounds demonstrated detectable oral absorption but limited bioavailability. Future studies will be aimed at improving the oral bioavailability of this indazole series as well as exploring other scaffolds.

kinases, respectively (Supporting Information, Table S1). Both compounds showed reasonable selectivity against these kinases. Of the 120 kinases, six were inhibited by more than 50% by 32a: AURKA (Aurora kinase A), AURKB, AURKC, FGFR2 (N549H), FLT3, and FLT4. Compound 32b also inhibited six kinases out of 166 by more than 50%: AURKA, AURKB, AURKC, FLT3(D835Y), RPS6KA1, and MELK. In the past decade, a number of mitotic kinases such as AURKA, AURKB, and PLK1 (polo-like kinase 1) were identified as promising cancer therapeutic targets; the recently identified Mps1 kinase is another novel target, whose inhibition is expected to lead to the selective death of cancer cells.22,44 Unlike other Mps1 kinase inhibitors 1, 2, and 3, indazoles 32a and 32b showed inhibitory activity for AURKA and AURKB. To investigate the inhibitory activity of another mitotic kinase PLK1, IC50 values of PLK1 for 32a and 32b were measured.45 As a result, both compounds showed IC50 values of >5 μM, indicating that the contribution of PLK1 to the improved antiproliferative activity in A549 cells would be minimal. As mentioned above, 32a and 32b were found to have potent inhibitory activity against JNK1 and the Aurora kinase family, which are an important factor in the regulation of cell proliferation. Although the strong correlation between cellular Mps1 and A549 activity was observed as shown in Figure 4B, determination of cellular activity of these targets for 32a and 32b would be required to properly understand the increased antiproliferative activity in A549 cells. Our indazoles were designed from a pan-kinase inhibitor 4 that inhibits 21 kinases out of 119 with IC50 values of less than 1 μM.28 We compared our kinase inhibition data for 32b with the kinase inhibition data for 4 in ref 28 (Table S1, Supporting Information). Of the 119 kinases in the reference, 57 kinases were identical to the kinase panel used for 32b. Compound 4 inhibited 6 kinases (STK3, CLK2, DAPK3, PIM2, PHKG2, and AURKB) out of the 57 with affinities of less than 1 μM, while 32b showed more than 50% inhibition at 1 μM against only 2 kinases (AURKB and AURKC), indicating that 32b seems to have higher selectivity than 4. However, for strict comparison, kinase selectivity data obtained under the same conditions would be needed. To gain insight into the effects of the use of the back and the ribose binding pockets on kinase selectivity, the kinase selectivity profiles of 17b and 23c were also evaluated against a smaller panel of 47 human kinases (Table S2, Supporting Information). The major difference between 17b and 23c was the size of substituents at the 6-position of the indazole ring that could occupy the back pocket: 17b (cyano group) and 23c (1-methyl-pyrazole-4-yl ring). When treated with 1 μM of 17b, 17 kinases out of 48 showed more than 50% inhibition, while 23c inhibited only 2 kinases (AURKA and FLT3) with more than 50% inhibition, indicating that the size of substituents in the back pocket is important for gaining kinase selectivity. In contrast, the effect of kinase selectivity on substituents at the 5position, which probably occupy the ribose binding pocket,

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CONCLUSIONS Starting with a pan-kinase inhibitor 4, we developed indazolebased selective and cell-active inhibitors of Mps1. The crystal structure of 4 bound to JNK1 guided the design of indazolebased lead 6. Analysis of the crystal structure suggested that introduction of substituents on the phenyl ring, the 5-, and 6positions of the indazole scaffold, would lead to an increase in both Mps1 activity and selectivity because these can provide suitable vectors toward the hinge region, the ribose binding pocket, and the back pocket, respectively. Indeed, introduction of hydrogen donating groups, such as a sulfonamide and an acetamide, to the phenyl ring of 6 and N-methyl pyrazole at the 6-position of 9d led to an increase in Mps1 inhibitory activity. As expected, the X-ray structure of 23d bound to Mps1 agrees well with the above-described design hypothesis. Finally, replacement of the ethoxy group at the 5-position with the cyclohexyl amino group was crucial for improvement in cellular activity, which led to the discovery of 32a and 32b. 4350

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7.63 (m, 6H), 7.63−7.94 (m, 2H), 13.08 (s, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C15H15N2O, 239; found, 239. N-(4-(5-Ethoxy-1H-indazol-3-yl)phenyl)acetamide (9a). Compound 9a was prepared in a manner similar to that for 6 by substituting 4-acetamidophenylboronic acid pinacol ester for phenylboronic acid in 78% yield (60 mg). 1H NMR (300 MHz, DMSO-d6) δ 1.37 (t, J = 6.9 Hz, 3H), 2.08 (s, 3H), 4.10 (q, J = 6.9 Hz, 2H), 7.04 (dd, J = 8.7, 2.7 Hz, 1H), 7.37 (d, 1H), 7.47 (d, J = 9.0 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.88 (d, 2H), 10.04 (s, 1H), 12.99 (s, 1H). LRMSESI (m/z): [M + H]+ calcd for C17H18N3O2, 296; found, 296. N-(3-(5-Ethoxy-1H-indazol-3-yl)phenyl)acetamide (9b). Compound 9b was prepared in a manner similar to that for 6 by substituting 3-acetamidophenylboronic acid for phenylboronic acid in 73% yield (56 mg). 1H NMR (400 MHz, DMSO-d6) δ 1.38 (br s, 3H), 2.10 (s, 3H), 4.10−4.11 (br m, 2H), 7.08 (d, J = 8.3 Hz, 1H), 7.43− 7.64 (m, 5H), 8.30 (s, 1H), 10.09 (s, 1H), 13.12 (s, 1H). HRMS-ESI (m/z): [M + H]+ calcd for C17H18N3O2, 296.1394; found, 296.1392. 3-(5-Ethoxy-1H-indazol-3-yl)benzamide (9c). Compound 9c was prepared in a manner similar to that for 6 by substituting 3carbamoylphenylboronic acid for phenylboronic acid in 66% yield (91 mg). 1H NMR (300 MHz, DMSO-d6) δ 1.38 (t, J = 7.2 Hz, 3H), 4.11 (q, J = 7.2 Hz, 2H), 7.08 (dd, J = 9.0, 2.4 Hz, 1H), 7.39−7.61 (m, 4H), 7.86 (m, J = 7.8 Hz, 1H), 8.09 (m, 1H), 8.14 (br, 1H), 8.41 (s, 1H), 13.19 (s, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C16H16N3O2, 282; found, 282. 3-(5-Ethoxy-1H-indazol-3-yl)benzenesulfonamide (9d). A mixture of compound 8 (150 mg, 0.386 mmol), 3-sulfamoylphenylboronic acid (93 mg, 0.464 mmol), PdCl2(dtbpf) (25 mg, 0.039 mmol), and 2 M aqueous K2CO3 (386 μL, 0.773 mmol) in DMF (2 mL) was stirred at 60 °C for 1 h. The mixture was cooled and diluted with EtOAc and H2O. The aqueous layer was separated and extracted with EtOAc. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated to give tert-butyl 5-ethoxy-3-(3-sulfamoylphenyl)-1H-indazole-1-carboxylate (140 mg, 0.336 mmol) as a solid. 1H NMR (300 MHz, DMSO-d6) δ 1.38 (t, J = 6.9 Hz, 3H), 1.68 (s, 9H), 4.16 (q, J = 6.9 Hz, 2H), 5.76 (s, 2H), 7.34 (dd, J = 9.0, 2.3 Hz, 1H), 7.45 (d, J = 2.3 Hz, 1H), 7.55 (s, 2H), 7.80 (t, J = 7.9 Hz, 1H), 7.97 (d, J = 7.9 Hz, 1H), 8.08 (d, J = 9.0 Hz, 1H), 8.23 (d, J = 7.9 Hz, 1H), 8.40 (s, 1H). LCMS-ESI (m/z): [M + H]+ calcd for C20H24N3O5S, 418, found, 418. A solution of this compound (126 mg, 0.302 mmol) and TFA (1.0 mL) in DCM (1.0 mL) was stirred at room temperature for 1 h. The mixture was diluted with DCM and 10% aqueous K2CO3 solution. The aqueous layer was separated and extracted with DCM. The combined organic extracts were dried over MgSO4, filtered, and evaporated. The residue was purified by flash chromatography (silica gel; hexane/EtOAc, gradient, 30−50% EtOAc) to give compound 9d (48.3 mg, 50%) as a solid. 1H NMR (400 MHz, DMSO-d6) δ 1.38 (t, J = 6.8 Hz, 3H), 4.12 (q, J = 6.8 Hz, 2H), 7.11 (d, J = 9.0 Hz, 1H), 7.43 (s, 1H), 7.51 (s, 2H), 7.54 (d, J = 9.0 Hz, 1H), 7.72 (t, J = 7.8 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 8.20 (d, J = 7.8 Hz, 1H), 8.42 (s, 1H), 13.32 (s, 1H). LRMS-ESI (m/ z): [M + H]+ calcd for C15H16N3O3S, 318; found, 318. 1-Bromo-2-ethoxy-4-methyl-5-nitrobenzene (12). To a solution of 1-bromo-2-fluoro-4-methyl-5-nitrobenzene (1.0 g, 4.27 mmol) in ethanol (5 mL) was added 20% sodium ethoxide in ethanol (1.84 mL, 4.70 mmol). The mixture was stirred at room temperature for 1 h and then acidified with aqueous 2 M HCl solution. The resulting solid was collected on a glass filter to give compound 9d as a yellow solid (1.1 g, 99%). 1H NMR (300 MHz, DMSO-d6) δ 1.39 (t, J = 6.9 Hz, 3H), 2.55 (s, 3H), 4.25 (q, J = 6.9 Hz, 2H), 7.22 (s, 1H), 8.27 (s, 1H). 2-Ethoxy-4-methyl-5-nitrobenzonitrile (13). A mixture of compound 9d (500 mg, 1.92 mmol) and CuCN (207 mg, 2.31 mmol) in DMF (2 mL) was stirred at 150 °C for 5 h. The mixture was cooled and diluted with H2O and EtOAc. The aqueous layer was separated and extracted with EtOAc. The combined extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated to give compound 13 (471 mg, quant.) as a solid. 1H NMR (300 MHz, DMSO-d6) δ 1.39 (t, J = 6.9 Hz, 3H), 2.62 (s, 3H), 4.25 (q, J = 6.9 Hz, 2H), 7.36 (s, 1H), 8.51 (s, 1H).

Importantly, both compounds showed better antiproliferative activity in A549 lung cancer cells than the known Mps1 inhibitors 1, 2, and 3, and had a reasonable selectivity over other kinases. Both 32a and 32b were found to be excellent lead compounds as well as favorable biological tools to further evaluate the therapeutic potential of Mps1 inhibitors in the treatment of cancer.

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EXPERIMENTAL SECTION

General. All commercial reagents and solvents were used as received unless otherwise noted. Thin layer chromatography (TLC) analysis was performed using Merck silica gel 60 F254 thin layer plates (250 μm thickness). Flash column chromatography was carried out with an automated purification system using Yamazen prepacked silica gel columns. Microwave reactions were performed with a Biotage Initiator 60. 1H NMR spectra were recorded on a Varian Gemini 300 MHz or a Bruker Advance 400 MHz spectrometer. Spectral data are reported as follows: chemical shift (as ppm referenced to tetramethylsilane), multiplicity (s = singlet, d = doublet, dd = double doublets, t = triplet, q = quartet, m = multiplet, br = broad peak), coupling constant, and integration value. Analytical LC/MS was performed on a Waters X-Bridge (C18, 5 μm, 4.6 mm × 50 mm, a linear gradient from 10% to 100% B over 3 min and then 100% B for 1 min (A = H2O + 0.1% formic acid; B = MeCN + 0.1% formic acid), flow rate 3.0 mL/min) using a Waters system equipped with a ZQ2000 mass spectrometer, 2525 binary gradient module, 2996 photodiode array detector (detection at 254 nm), and 2777 sample manager. The purity of all compounds used in bioassays was determined by this method to be >95%. High resolution mass spectra were recorded on a Thermo Fisher Scientific LTQ Orbitrap using electrospray positive ionization. tert-Butyl 5-ethoxy-3-iodo-1H-indazole-1-carboxylate (8). To a solution of 7 (4.23 g, 26.1 mmol) and I2 (5.96 g, 47.0 mol) in DMF (20 mL) was added KOH (5.13 g, 91.4 mol). The mixture was stirred at room temperature for 4 h and diluted with Et2O and 10% aqueous citric acid solution. The aqueous layer was separated and extracted with Et2O. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated to give 5ethoxy-3-iodo-1H-indazole. This compound, triethylamine (4.73 mL, 33.9 mmol), and DMAP (319 mg, 2.61 mol) were dissolved in MeCN (30 mL). Boc2O (7.80 mL, 33.9 mmol) was added to this solution, and the reaction mixture was stirred overnight at room temperature. The mixture was diluted with EtOAc and H2O. The aqueous layer was separated and then extracted with EtOAc. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The residue was purified by flash column chromatography (silica gel; hexane/EtOAc = 10:1) to give 8 (3.60 g, 36%) as a solid. 1H NMR (400 MHz, CDCl3) δ 1.47 (t, J = 6.9 Hz, 3H), 1.71 (s, 9H), 4.12 (q, J = 6.9 Hz, 2H), 6.81 (d, J = 2.3 Hz, 1H), 7.20 (dd, J = 9.0, 2.3 Hz, 1H), 7.99 (d, J = 9.0 Hz, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C14H18IN2O3, 389; found, 389. 5-Ethoxy-3-phenyl-1H-indazole (6). A suspension of 8 (100 mg, 0.258 mmol), Pd(PPh3)4 (15 mg, 0.013 mmol), phenylboronic acid (38 mg, 0.31 mmol), and aqueous Na2CO3 solution (2 M, 0.52 mL, 1.03 mmol) in THF (1.0 mL) was heated to reflux for 3 h. The mixture was allowed to cool to room temperature and diluted with EtOAc and H2O. The aqueous layer was separated and extracted with EtOAc. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The residue was dissolved in DCM (0.5 mL), and then TFA (0.5 mL) was added to this solution. The mixture was stirred at room temperature for 45 min and then neutralized with 10% aqueous K2CO3 solution. The aqueous layer was separated and extracted with DCM. The combined organic extracts were dried over MgSO4, filtered, and evaporated. The residue was purified by flash column chromatography (silica gel; EtOAc/ hexane, gradient, 10−50% EtOAc) to give compound 6 (39 mg, 63%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 1.34 (t, J = 7.2 Hz, 3H), 4.07 (q, J = 7.2 Hz, 2H), 7.04 (dd, J = 9.0, 2.4 Hz, 1H), 7.33− 4351

dx.doi.org/10.1021/jm4000215 | J. Med. Chem. 2013, 56, 4343−4356

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5-Ethoxy-1H-indazole-6-carbonitrile (15). A mixture of compound 13 (250 mg, 1.21 mmol) and 10% Pd/C (50 mg) in THF (3 mL) was stirred under an atmosphere of hydrogen at room temperature for 2 h. The mixture was filtered though Celite and evaporated to give compound 14. LRMS-ESI (m/z): [M + H]+ calcd for C10H13N2O, 177; found, 177. To a mixture of 14 in acetic acid (10 mL) was added NaNO2 (83.5 mg, 1.21 mmol). The mixture was stirred at room temperature overnight. The resulting solid was collected on a glass filter and recrystallized from EtOAc/hexane to give compound 15 (186 mg, 82%) as a solid. 1H NMR (300 MHz, DMSOd6) δ 1.40 (t, J = 7.2 Hz, 3H), 4.16 (q, J = 7.2 Hz, 2H), 7.44 (s, 1H), 8.08 (d, J = 9.0 Hz, 2H), 13.39 (br, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C10H10N3O, 188; found, 188. tert-Butyl 6-cyano-5-ethoxy-3-iodo-1H-indazole-1-carboxylate (16). To a mixture of compound 15 (150 mg, 0.80 mmol) and KOH (157 mg, 2.80 mmol) in DMF (2 mL) was added I2 (178 mg, 1.4 mmol). The mixture was stirred at room temperature for 3 h. The mixture was acidified with aqueous 2 M HCl solution and then extracted with EtOAc. The combined extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated to give 5-ethoxy3-iodo-1H-indazole-6-carbonitrile. LRMS-ESI: [M + H]+ calcd for C10H9IN3O, 314; found, 314. To a solution of this compound, Et3N (0.145 mL, 1.04 mmol), and DMAP (4.9 mg, 0.04 mmol) in MeCN (2 mL) was added Boc2O (0.239 mL, 1.04 mmol). The mixture was stirred at room temperature for 50 min. The mixture was diluted with EtOAc and H2O. The aqueous layer was separated and extracted with EtOAc. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The resulting solid was crystallized from EtOAc/hexane to give compound 16 (257 mg, 78%) as a solid. 1H NMR (300 MHz, DMSO-d6) δ 1.42 (t, J = 6.9 Hz, 3H), 1.65 (s, 9H), 4.30 (q, J = 6.9 Hz, 2H), 7.14 (s, 1H), 8.33 (s, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C15H17IN3O3, 414; found, 414. 3-(6-Cyano-5-ethoxy-1H-indazol-3-yl)benzenesulfonamide (17a). A mixture of compound 16 (30 mg, 0.073 mmol), aqueous 2 M Na2CO3 (0.15 mL), 3-sulfamoylphenylboronic acid (17.7 mg, 0.088 mmol), and PdCl2(dppf)·DCM (3.3 mg, 0.004 mmol) in THF (1 mL) was heated to reflux for 6 h under an atmosphere of nitrogen. The mixture was diluted with EtOAc and H2O. The aqueous layer was separated and extracted with EtOAc. The combined extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. To a solution of the resulting residue in DCM (1 mL) was added TFA (1 mL). The mixture was stirred at room temperature for 30 min. The mixture was diluted with saturated aqueous NaHCO3 solution and EtOAc. The aqueous layer was separated and extracted with EtOAc. The combined extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The residue was purified by flash chromatography (silica gel; hexane/EtOAc, gradient, 10−50% EtOAc) to give compound 17a (3.1 mg, 12%) as a solid. 1H NMR (300 MHz, DMSO-d6) δ 1.43 (t, J = 7.2 Hz, 3H), 4.27 (q, J = 7.2 Hz, 2H), 7.49 (s, 2H), 7.60 (s, 1H), 7.74 (t, J = 7.5 Hz, 1H), 7.86 (m, J = 8.1 Hz, 1H), 8.19−8.23 (m, 2H), 8.41 (br, 1H), 13.81 (br s, 1H). HRMS-ESI (m/z): [M + H]+ calcd for C16H15N4O3S, 343.0859; found, 343.0850. N-(3-(6-Cyano-5-ethoxy-1H-indazol-3-yl)phenyl)acetamide (17b). Compound 17b was prepared in a manner similar to that for 17a by substituting 3-acetamidophenylboronic acid for 3-sulfamoylphenylboronic acid in 68% yield (37 mg). 1H NMR (300 MHz, DMSO-d6) δ 1.43 (t, J = 6.9 Hz, 3H), 2.09 (s, 3H), 4.24 (q, J = 6.9 Hz, 2H), 7.38−7.49 (m, 1H), 7.49−7.67 (m, 3H), 8.13 (s, 1H), 8.32 (s, 1H), 10.08 (s, 1H), 13.60 (br s, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C18H17N4O2, 321; found, 321. 5-Bromo-4-ethoxy-2-methylaniline (19a). To a mixture of compound 12 (1.0 g, 3.84 mmol) in ethanol/H2O (10 mL/2 mL) was added Na2S2O4 (3.4 g, 19.5 mmol). The mixture was heated to 100 °C for 5 h and allowed to cool to room temperature. The mixture was then neutralized with concentrated HCl and diluted with EtOAc and H2O. The aqueous layer was separated and extracted with EtOAc. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated to give compound 19a (766 mg, 87%) as a solid. 1H NMR (300 MHz, DMSO-d6) δ 1.10 (t, J = 6.9 Hz,

3H), 1.84 (s, 3H), 3.74 (q, J = 6.9 Hz, 2H), 4.47 (br, 2H), 6.57 (s, 1H), 6.64 (s, 1H). 6-Bromo-5-ethoxy-1H-indazole (20a). To a solution of compound 19a (2.5 g, 10.9 mmol) in acetic acid (120 mL) was added NaNO2 (750 mg, 10.9 mmol) in H2O. The mixture was stirred at room temperature overnight and then diluted with saturated aqueous NaHCO3 solution. The mixture was extracted with EtOAc, and the combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The residue was purified by flash chromatography (silica gel; hexane/EtOAc, gradient, 0−50% EtOAc) to give compound 20a (2.3 g, 88%) as a solid. 1H NMR (400 MHz, DMSO-d6) δ 1.42 (t, J = 6.8 Hz, 3H), 1.64 (s, 9H), 4.21 (q, J = 6.8 Hz, 2H), 6.97 (s, 1H), 8.26 (s, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C9H10BrN2O, 241; found, 241. tert-Butyl 6-bromo-5-ethoxy-3-iodo-1H-indazole-1-carboxylate (21a). To a solution of compound 20a (1.16 g, 4.56 mmol) and I2 (1.16 g, 4.56 mmol) in DMF (8 mL) was added KOH (931 mg, 16.6 mmol). The mixture was stirred at room temperature for 4 h and acidified with aqueous 2 M HCl solution. The resulting solid was collected on a glass filter to give 6-bromo-5-ethoxy-3-iodo-1Hindazole. To a solution of this compound and Et3N (747 μL, 5.39 mmol) in MeCN (3 mL) was added Boc2O (1.25 mL, 5.39 mmol). The mixture was stirred at room temperature for 50 min and diluted with H2O. The resulting solid was collected on a glass filter to give compound 21a (1.85 g, 95%) as a solid. 1H NMR (400 MHz, DMSOd6) δ 1.42 (t, J = 6.8 Hz, 3H), 1.65 (s, 9H), 4.21 (dd, J = 6.8 Hz, 2H), 6.95 (s, 1H), 8.25 (s, 1H). tert-Butyl 6-bromo-5-ethoxy-3-(3-sulfamoylphenyl)-1H-indazole-1-carboxylate (22a). A mixture of compound 21a (500 mg, 1.07 mmol), 3-sulfamoylphenylboronic acid (258 mg, 1.29 mmol), aqueous 2 M Na2CO3 (2.14 mL, 4.28 mmol), and PdCl2(dppf)·DCM (87 mg, 0.107 mmol) in THF (5 mL) was heated to reflux for 6 h under an atmosphere of nitrogen. The mixture was cooled to room temperature and diluted with H2O and EtOAc. The aqueous layer was separated and extracted with EtOAc. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The residue was purified by flash chromatography (silica gel; hexane/EtOAc, gradient, 0−70% EtOAc) to give compound 22a (358 mg, 67%) as a solid. 1H NMR (400 MHz, DMSO-d6) δ 1.42 (t, J = 6.8 Hz, 3H), 1.68 (s, 9H), 4.23 (q, J = 6.8 Hz, 2H), 7.52−7.56 (m, 3H), 7.81 (t, J = 7.6 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.38 (s, 1H), 8.39 (s, 1H). 3-(5-Ethoxy-6-(1-methyl-1H-pyrazol-4-yl)-1H-indazol-3-yl)benzenesulfonamide (23c). A mixture of compound 22a (30 mg, 0.060 mmol), 1-methylpyrazole-4-boronic acid pinacol ester (18.9 mg, 0.091 mmol), Pd(OAc)2 (1.40 mg, 6.2 μmol), S-Phos (4.96 mg, 0.12 mmol), and aqueous 2 M K2CO3 (121 μL, 0.242 mmol) in THF (1 mL) was heated to reflux for 5 h. The mixture was allowed to cool to room temperature and diluted with H2O and EtOAc. The aqueous layer was separated and extracted with EtOAc. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated to give Boc protected 23c. To a solution of this compound in DCM (0.5 mL) was added TFA (0.5 mL). The mixture was stirred at room temperature and diluted with DCM and 10% aqueous K2CO3 solution. The aqueous layer was separated and extracted with EtOAc. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The residue was purified by flash chromatography (silica gel; CHCl3/ MeOH, gradient, 3−10% MeOH) to give compound 23c (16 mg, 67%) as a solid. 1H NMR (400 MHz, DMSO-d6) δ 1.48 (t, J = 6.8 Hz, 3H), 3.90 (s, 3H), 4.19 (q, J = 6.8 Hz, 2H), 7.46 (s, 1H), 7.49 (s, 2H), 7.70−7.83 (m, 3H), 7.99 (s, 1H), 8.19−8.22 (m, 2H), 8.43 (brs, 1H), 13.25 (brs, 1H). HRMS-ESI (m/z): [M + H]+ calcd for C19H20N5O3S, 398.1281; found, 398.1269. 3-(5-Ethoxy-6-phenyl-1H-indazol-3-yl)benzenesulfonamide (23a). Compound 23a was prepared in a manner similar to that for 23c by substituting phenylboronic acid for 1-methylpyrazole-4-boronic acid pinacol ester in 11% yield (2.5 mg). 1H NMR (400 MHz, DMSOd6) δ 1.28 (t, J = 6.8 Hz, 3H), 4.11 (q, J = 6.8 Hz, 2H), 7.37−7.59 (m, 8H), 7.74 (t, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.0 4352

dx.doi.org/10.1021/jm4000215 | J. Med. Chem. 2013, 56, 4343−4356

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Hz, 1H), 8.45 (brs, 1H), 13.36 (s, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C21H20N3O3S, 394; found, 394. 3 - ( 5- E t h ox y - 6 - (1 H - py r az o l - 4 - yl ) - 1H - i n d a z o l - 3 - y l ) benzenesulfonamide (23b). Compound 23b was prepared in a manner similar to that for 23c by substituting 1H-pyrazole-4-boronic acid pinacol ester for 1-methylpyrazole-4-boronic acid pinacol ester in 100% yield (24.2 mg). 1H NMR (400 MHz, DMSO-d6) δ 1.48 (t, J = 6.8 Hz, 3H), 4.20 (q, J = 6.8 Hz, 2H), 7.47−7.48 (m, 3H), 7.70−7.74 (m, 1H), 7.78 (s, 1H), 7.81−7.83 (m, 1H), 8.21 (d, J = 8.6 Hz, 1H), 8.44 (s, 1H), 13.25 (s, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C18H18N5O3S, 384; found, 384. 5-(5-Ethoxy-6-(1-methyl-1H-pyrazol-4-yl)-1H-indazol-3-yl)2-methylbenzenesulfonamide (23d). Compound 23d was prepared in 2 steps from 21a in a manner similar to that for 22a and 23c by substituting 4-methyl-3-sulfamoylphenylboronic acid for 3sulfamoylphenylboronic acid in 68% yield (119 mg). 1H NMR (400 MHz, DMSO-d6) δ 1.48 (t, J = 6.9 Hz, 3H), 2.65 (s, 3H), 3.90 (s, 3H), 4.18 (q, J = 6.9 Hz, 2H), 7.45 (s, 1H), 7.50 (d, J = 8.1 Hz, 1H), 7.54 (brs, 2H,), 7.71 (s, 1H), 7.99 (s, 1H), 8.08 (dd, J = 8.1, 1.8 Hz, 1H), 8.18 (s, 1H), 8.48 (d, J = 1.8 Hz, 1H), 13.16 (brs, 1H). HRMS-ESI (m/z): [M + H]+ calcd for C20H22N5O3S, 412.1438; found, 412.1425. 3-(5-Ethoxy-6-(1-isobutyl-1H-pyrazol-4-yl)-1H-indazol-3-yl)benzenesulfonamide (23e). Compound 23e was prepared in a manner similar to that for 23c by substituting 1-isobutyl-1H-pyrazole4-boronic acid pinacol ester for 1-methylpyrazole-4-boronic acid pinacol ester in 64% yield (11.4 mg). 1H NMR (400 MHz, DMSO-d6) δ 0.89 (d, J = 6.8 Hz, 6H), 1.48 (t, J = 6.8 Hz, 1H), 2.15 (m, 1H), 3.98 (d, J = 6.8 Hz, 1H), 4.20 (q, J = 6.8 Hz, 2H), 7.48 (m, 3H), 7.70−7.83 (m, 3H), 8.02 (s, 1H), 8.21 (m, 2H), 8.44 (s, 1H), 13.25 (s, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C22H26N5O3S, 440; found, 440. 1-Bromo-2-(cyclohexyloxy)-4-methyl-5-nitrobenzene (18). To a solution of compound 11 (26 g, 111 mmol) and cyclohexanol (20.0 mL, 189 mmol) in MeCN (107 mL) was added KOH (9.35 g,) at 0 °C, and the reaction mixture was then stirred at room temperature for 22 h. The mixture was diluted with H2O and EtOAc. The aqueous layer was separated and extracted with EtOAc. The combined extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The residue was purified by flash chromatography (silica gel; hexane/EtOAc, gradient, 5−10% EtOAc) to give compound 18 (12.5 g, 36%) as a solid. 1H NMR (300 MHz, DMSO-d6) δ 1.37−1.62 (m, 6H), 1.70−1.76 (m, 2H), 1.85−1.91 (m, 2H), 2.54 (s, 3H), 4.68− 4.75 (m, 1H), 7.27 (s, 1H), 8.26 (d, J = 1.0 Hz, 1H). 5-Bromo-4-(cyclohexyloxy)-2-methylaniline (19b). Compound 19b was prepared in a manner similar to that for 19a in 85% yield (9.6 g). 1H NMR (300 MHz, DMSO-d6) δ 1.24−1.35 (m, 3H), 1.42−1.48 (m, 3H), 1.67−1.86 (m, 4H), 2.00 (s, 3H), 3.99−4.08 (m, 1H), 4.68 (s, 2H), 6.75 (s, 1H), 6.79 (s, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C13H19BrNO, 284; found, 284. 6-Bromo-5-(cyclohexyloxy)-1H-indazole (20b). Compound 20b was prepared in a manner similar to that for 20a in 75% yield (7.5 g). 1H NMR (300 MHz, DMSO-d6) δ 1.32−1.43 (m, 3H), 1.51− 1.58 (m, 3H), 1.71−1.77 (m, 2H), 1.85−1.93 (m, 2H), 4.36−4.43 (m, 1H), 7.40 (s, 1H), 7.78 (s, 1H), 7.95 (s, 1H), 12.96 (s, 1H). LRMSESI (m/z): [M + H]+ calcd for C13H16BrN2O, 295; found, 295. tert-Butyl 6-bromo-5-(cyclohexyloxy)-3-iodo-1H-indazole-1carboxylate (21b). Compound 21b was prepared in a manner similar to that for 21a in 91% yield (13 g). 1H NMR (300 MHz, DMSO-d6) δ 1.53−1.77 (m, 10H), 1.63 (s, 9H), 4.66−4.71 (m, 1H), 7.08 (s, 1H), 8.27 (s, 1H). tert-Butyl 6-bromo-5-(cyclohexyloxy)-3-(3-sulfamoylphenyl)-1H-indazole-1-carboxylate (22b). Compound 22b was prepared in a manner similar to that for 22a in 59% yield (1.2 g). 1H NMR (300 MHz, DMSO-d6) δ 1.35−1.92 (m, 19H), 1.68 (s, 9H), 4.65−4.72 (m, 1H), 7.54 (s, 2H), 7.61 (s, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.98 (dt, J = 7.8, 1.5 Hz, 1H), 8.21 (dt, J = 7.8, 1.5 Hz, 1H), 8.37−8.38 (m, 2H). 3-(5-(Cyclohexyloxy)-6-(1-methyl-1H-pyrazol-4-yl)-1H-indazol-3-yl)benzenesulfonamide (23f). Compound 23f was prepared in a manner similar to that for 23c in 56% yield (37.8 mg). 1H NMR (300 MHz, DMSO-d6) δ 1.28−1.75 (m, 8H), 1.99−2.06 (m, 2H), 3.90

(s, 3H), 4.50−4.58 (m, 1H), 7.47 (s, 2H), 7.50 (s, 1H), 7.69−7.74 (m, 2H), 7.81 (d, J = 7.1 Hz, 1H), 7.97 (s, 1H), 8.16−8.19 (m, 2H), 8.42 (s, 1H), 13.22 (s, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C23H26N5O3S, 452; found, 452. 4-Bromo-2-fluoro-5-nitrobenzaldehyde (24). To a solution of KNO3 (38 g, 379 mmol) in H2SO4 (288 mL) was added compound 5 (70 g, 345 mmol) at 0 °C, and the mixture was stirred at room temperature for 1 h. The mixture was poured into ice water, and the resulting solid was collected on a glass filter and washed with H2O and saturated aqueous NaHCO3 solution to give compound 24 (80 g, 94%) as a solid. 1H NMR (300 MHz, DMSO-d6) δ 8.22 (d, J = 9.9 Hz, 1H), 8.48 (d, J = 6.6 Hz, 1H), 10.12 (s, 1H). 6-Bromo-5-nitro-1H-indazole (25). To a solution of compound 24 (80.2 g, 323 mmol) in DMF (1280 mL) was added hydrazine monohydrate (17.3 mL, 356 mmol) at room temperature. The mixture was stirred at 150 °C for 1.5 h and then cooled to room temperature. The mixture was evaporated and diluted with EtOAc and H2O. The aqueous layer was separated and extracted with EtOAc. The combined extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The resulting solid was crystallized from EtOAc/ hexane to give compound 25 (72.4 g, 93%) as a solid. 1H NMR (300 MHz, DMSO-d6) δ 8.07 (t, J = 0.6 Hz, 1H), 8.35 (d, J = 0.9 Hz, 1H), 8.62 (s, 1H), 13.71 (brs, 1H). 6-(1-Methyl-1H-pyrazol-4-yl)-5-nitro-1H-indazole (26). To a mixture of compound 25 (5.0 g, 20.7 mmol) and NaH (60%; 909 mg, 22.7 mg) in DMF (40 mL) was added 2-(trimethylsilyl)ethoxymethyl chloride (3.83 mL, 21.7 mmol) at 0 °C. The mixture was stirred at room temperature for 40 min and then poured into ice water. The mixture was extracted with EtOAc, and the combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The residue was purified by flash chromatography (silica gel; hexane/EtOAc, gradient, 5−20% EtOAc) to give SEM-protected 26 (5.82 g, 76%). A mixture of this compound, aqueous 2 M K2CO3 (31.9 mL, 63.8 mmol), 1-methylpyrazole-4-boronic acid pinacol ester (3.98 g, 19.1 mmol), and PdCl2(dtbpf) (520 mg, 0.797 mmol) in THF was heated to reflux for 3.5 h under an atmosphere of nitrogen. The mixture was diluted with EtOAc and H2O. The aqueous layer was separated and extracted with EtOAc. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The residue was dissolved in THF (32 mL), and ethylenediamine (5.54 mL, 82 mmol) and TBAF (1 M solution in THF; 82 mL, 82 mmol) were added to the solution. The reaction mixture was heated to reflux for 8 h and allowed to cool to room temperature. The mixture was then diluted with EtOAc and H2O, and the aqueous layer was separated. The aqueous layer was extracted with EtOAc, and the combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The residue was purified by flash chromatography (silica gel; hexane/EtOAc, gradient, 30−100% EtOAc) to give compound 26 (3.11 g, 79%) as a solid. 1H NMR (300 MHz, DMSO-d6) δ 3.88 (s, 3H), 7.57 (d, J = 0.9 Hz, 1H), 7.59 (dd, J = 0.9, 0.6 Hz, 1H), 7.93 (d, J = 0.6 Hz, 1H), 8.29 (d, J = 0.9 Hz, 1H), 8.43 (d, J = 0.6 Hz, 1H), 13.56 (brs, 1H). 3-Iodo-6-(1-methyl-1H-pyrazol-4-yl)-5-nitro-1H-indazole (27). To a solution of compound 26 (3.10 g, 12.8 mmol) and I2 (4.69 g, 18.5 mmol) in DMF (34 mL) was added KOH (3.58 g, 63.7 mmol). The mixture was stirred at room temperature for 2 h and then diluted with aqueous 1 M HCl solution and aqueous 5% Na2S2O3 solution. The mixture was stirred for 20 min, and the resulting solid was collected. This solid was dissolved in EtOAc, and the insoluble residue was filtered off. The filtrate was evaporated to give compound 27 (4.62 g, 98%) as a solid. 1H NMR (300 MHz, DMSO-d6) δ 3.88 (s, 3H), 7.59 (d, J = 0.9 Hz, 1H), 7.64 (d, J = 0.6 Hz, 1H), 7.95 (d, J = 0.3 Hz, 1H), 8.05 (d, J = 0.6 Hz, 1H), 13.97 (s, 1H). 3-Iodo-6-(1-methyl-1H-pyrazol-4-yl)-5-nitro-1-((2(trimethylsilyl)ethoxy)methyl)-1H-indazole (28). To a mixture of compound 27 (4.62 g, 12.5 mmol) and NaH (60%; 651 mg, 16.3 mmol) in DMF (40 mL) was added 2-(trimethylsilyl)ethoxymethyl chloride (2.76 mL, 15.7 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 40 min and then diluted with EtOAc and H2O. The aqueous layer was separated and extracted with EtOAc. 4353

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Journal of Medicinal Chemistry

Article

evaporated. The residue was purified by flash chromatography (silica gel; hexane/EtOAc, gradient, 30−60% EtOAc) to give compound 32a (104 mg, 79%) as a solid. 1H NMR (300 MHz, DMSO-d6) δ 1.13− 1.29 (m, 3H), 1.31−1.48 (m, 2H), 1.54−1.72 (m, 3H), 1.95−2.06 (m, 2H), 3.37 (m, 1H), 3.92 (s, 3H), 4.15 (d, J = 7.8 Hz, 1H), 7.07 (s, 1H), 7.32 (s, 1H), 7.46 (brs, 2H), 7.70 (t, J = 7.8 Hz, 1H), 7.72 (d, J = 0.6 Hz, 1H), 7.79 (dt, J = 7.8, 1.5 Hz, 1H), 8.03 (s, 1H), 7.14 (m, 1H), 8.42 (t, J = 1.5 Hz, 1H), 13.04 (s, 1H). HRMS-ESI (m/z): [M + H]+ calcd for C23H27N6O2S, 451.1911; found, 451.1897. N-(3-(6-(1-Methyl-1H-pyrazol-4-yl)-5-nitro-1-((2(trimethylsilyl)ethoxy)methyl)-1H-indazol-3-yl)phenyl)acetamide (29b). Compound 29b was prepared in a manner similar to that for 29a by substituting 3-acetamidophenylboronic acid for 3sulfamoylphenylboronic acid in 89% yield (543 mg). 1H NMR (300 MHz, DMSO-d6) δ −0.11 (s, 9H), 0.83 (t, J = 7.9 Hz, 2H), 2.09 (s, 3H), 3.61 (t, J = 7.9 Hz, 2H), 3.91 (s, 3H), 5.89 (s, 2H), 7.47 (t, J = 7.9 Hz, 1H), 7.64 (s, 1H), 7.70 (t, J = 9.0 Hz, 2H), 7.97 (s, 1H), 8.03 (s, 1H), 8.25 (s, 1H), 8.60 (s, 1H), 10.13 (s, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C25H31N6O4Si, 507; found, 507. N-(3-(5-Amino-6-(1-methyl-1H-pyrazol-4-yl)-1-((2(trimethylsilyl)ethoxy)methyl)-1H-indazol-3-yl)phenyl)acetamide (30b). Compound 30b was prepared in a manner similar to that for 30a in 97% yield (487 mg). 1H NMR (300 MHz, DMSOd6) δ −0.11 (s, 9H), 0.81 (t, J = 7.9 Hz, 2H), 2.08 (s, 3H), 3.56 (t, J = 7.9 Hz, 2H), 3.91 (s, 3H), 4.76 (s, 2H), 5.71 (s, 2H), 7.34 (s, 1H), 7.42 (t, J = 7.8 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.59 (s, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.78 (s, 1H), 8.08 (s, 1H), 8.15 (s, 1H), 10.08 (s, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C25H33N6O2Si, 477; found, 477. N-(3-(5-(Cyclohexylamino)-6-(1-methyl-1H-pyrazol-4-yl)-1((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-3-yl)phenyl)acetamide (31b). Compound 31b was prepared in a manner similar to that for 31a in 87% yield (132 mg). 1H NMR (300 MHz, DMSOd6) δ −0.10 (s, 9H), 0.81 (t, J = 7.9 Hz, 2H), 1.15−1.27 (m, 3H), 1.36−1.48 (m, 2H), 1.57−1.69 (m, 3H), 1.99−2.03 (m, 2H), 2.09 (s, 3H), 3.55 (t, J = 7.9 Hz, 2H), 3.93 (s, 3H), 4.18 (d, J = 7.9 Hz, 1H), 5.70 (s, 2H), 7.14 (s, 1H), 7.41−7.43 (m, 2H), 7.54 (s, 1H), 7.58− 7.59 (m, 1H), 7.72 (s, 1H), 8.02 (s, 1H), 8.46 (s, 1H), 10.08 (s, 1H). LRMS-ESI (m/z): [M + H]+ calcd for C31H43N6O2Si, 559; found, 559. N-(3-(5-(Cyclohexylamino)-6-(1-methyl-1H-pyrazol-4-yl)-1Hindazol-3-yl)phenyl)acetamide (32b). Compound 32b was prepared in a similar manner to that for 32a in 55% yield (54 mg). 1 H NMR (300 MHz, DMSO-d6) δ 1.12−1.31 (m, 3H), 1.32−1.52 (m, 2H), 1.54−1.74 (m, 3H), 1.96−2.10 (m, 2H), 2.09 (s, 3H), 3.35 (m, 1H), 3.92 (s, 3H), 4.09 (d, J = 6.6 Hz, 1H), 7.14 (s, 1H), 7.29 (s, 1H), 7.36−7.45 (m, 2H), 7.60 (m, 1H), 7.71 (s, 1H), 8.02 (s, 1H), 8.47 (s, 1H), 10.05 (s, 1H), 12.83 (s, 1H). HRMS-ESI (m/z): [M + H]+ calcd for C25H29N6O, 429.2397; found, 429.2385. Biological Assays. Enzymatic assays of Mps1 and JNK1, Mps1 autophosphorylation assay, and A549 antiproliferative assay were performed as described previously.25 Reported values are means of n ≥ 2 determinations, standard deviation ≤15%. Pharmacokinetic Studies. Male Sprague−Dawley rats (8 weeks) were purchased from Charles River Laboratories. Compounds were formulated as suspensions in 0.5% methylcellulose (0.2 mg/mL) and dosed orally at 1 mg/kg (n = 2) in nonfasted condition. Blood samples (0.2 mL) were collected with 1-mL syringes containing anticoagulants (EDTA-2K and heparin) at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after dosing. Compounds were formulated as solutions in DMA/propylene glycol (1:1, 0.5 mg/mL) and dosed intravenously from the tail vein at 0.5 mg/kg (n = 2) under isoflurane anesthesia under the nonfasted condition. Blood samples (0.2 mL) were collected with 1-mL syringes containing anticoagulants (EDTA-2K and heparin) at 2, 5, 15, 30, 60, 120, 240, and 360 min after dosing. Blood samples were centrifuged for 10 min at 3500 rpm at 4 °C to obtain plasma samples, which were transferred to each tube and stored in a freezer until analysis. Plasma concentrations were determined by LC/MS/MS following protein precipitation with acetonitrile. Pharmacokinetic parameters were calculated using WinNonlin based on a noncompartment model.

The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The residue was purified by flash chromatography (silica gel; hexane/EtOAc, gradient, 10−50% EtOAc) to give compound 28 (5.51 g, 88%) as a solid, which contained 2-SEM protected isomer (3-iodo-6-(1-methyl-1H-pyrazol-4yl)-5-nitro-2-((2-(trimethylsilyl)ethoxy)methyl)-2H-indazole) (28:2SEM isomer = 1:0.2). 1H NMR (300 MHz, DMSO-d6) δ −0.11 (s, 9H), 0.82 (t, J = 7.8 Hz, 2H), 3.62 (t, J = 7.8 Hz, 2H), 3.83 (s, 3H), 5.78 (s, 2H), 7.52 (d, J = 0.6 Hz, 1H), 7.78 (d, J = 0.3 Hz, 1H), 7.85 (s, 1H), 8.09 (d, J = 0.3 Hz, 1H). 3-(6-(1-Methyl-1H-pyrazol-4-yl)-5-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-3-yl)benzenesulfonamide (29a). A mixture of compound 28 (5.49 g, 11 mmol), aqueous 2 M Na2CO3 (22 mL, 44 mmol), PdCl2(dppf)·DCM (898 mg, 1.1 mmol), and 3sulfamoylphenylboronic acid (3.74 g, 13.2 mmol) in THF (52 mL) was heated to reflux for 3 h under an atmosphere of nitrogen. The mixture was allowed to cool to room temperature and diluted with EtOAc and H2O. The aqueous layer was separated and extracted with EtOAc. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The residue was purified by flash chromatography (silica gel; hexane/EtOAc, gradient, 20−100% EtOAc) to give compound 29a (5.16 g, 89%) as a solid. 1H NMR (300 MHz, DMSO-d6) δ −0.11 (s, 9H), 0.84 (t, J = 7.8 Hz, 2H), 3.63 (t, J = 7.8 Hz, 2H), 3.91 (s, 3H), 5.92 (s, 2H), 7.52 (brs, 2H), 7.65 (d, J = 0.6 Hz, 1H), 7.76 (t, J = 7.8 Hz, 1H), 7.91 (m, 1H), 7.97 (s, 1H), 8.07 (s, 1H), 8.27 (m, 1H), 8.42 (t, J = 1.5 Hz, 1H), 8.69 (s, 1H). 3-(5-Amino-6-(1-methyl-1H-pyrazol-4-yl)-1-((2(trimethylsilyl)ethoxy)methyl)-1H-indazol-3-yl)benzenesulfonamide (30a). A mixture of 29a (5.09 g, 9.63 mmol), NH4Cl (558 mg, 10.4 mmol), and iron powder (2.33 g, 41.7 mmol) in ethanol/H2O (40 mL/44 mL) was heated to reflux for 45 min. The mixture was cooled and filtered though Celite. The filtrate was evaporated, and the resulting residue was diluted with EtOAc and H2O. The aqueous layer was separated and extracted with EtOAc. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated. The residue was purified by flash chromatography (silica gel; hexane/EtOAc, gradient, 30−100% EtOAc) to give compound 30a (4.27 g, 89%) as a solid. 1H NMR (300 MHz, DMSO-d6) δ −0.11 (s, 9H), 0.82 (t, J = 7.8 Hz, 2H), 3.57 (t, J = 7.8 Hz, 2H), 3.91 (s, 3H), 4.81 (s, 2H), 5.74 (s, 2H), 7.39 (s, 1H), 7.46 (brs, 2H), 7.63 (s, 1H), 7.72 (t, J = 7.8 Hz, 1H), 7.77−7.83 (m, 2H), 8.07−8.14 (m, 2H), 8.39 (s, 1H). LRMS-ESI (m/z): [M + H]+ C23H31N6O3SSi, 499; found, 499. 3-(5-(Cyclohexylamino)-6-(1-methyl-1H-pyrazol-4-yl)-1-((2(trimethylsilyl)ethoxy)methyl)-1H-indazol-3-yl)benzenesulfonamide (31a). To a solution of compound 30a (150 mg, 0.301 mmol), cyclohexanone (37 μL, 0.361 mmol), and acetic acid (34 μL, 0.602 mmol) in DCM (3.0 mL) was added NaBH(OAc)3 (128 mg, 0.602 mmol). The mixture was stirred at room temperature for 3 h. The mixture was diluted with saturated aqueous NaHCO3 solution and EtOAc. The aqueous layer was separated and extracted with EtOAc. The combined organic extracts were washed with H2O and brine, dried over MgSO4, filtered, and evaporated to give compound 31a (175 mg, 100%) as a solid. 1H NMR (300 MHz, DMSO-d6) δ −0.10 (s, 9H), 0.82 (t, J = 7.9 Hz, 2H), 1.16−1.48 (m, 5H), 1.55−1.77 (m, 3H), 1.96−2.06 (m, 2H), 3.57 (t, J = 7.9 Hz, 2H), 3.93 (s, 3H), 4.25 (d, J = 7.8 Hz, 1H), 5.74 (s, 2H), 7.07 (s, 1H), 7.47 (s, 2H), 7.59 (s, 1H), 7.70−7.77 (m, 3H), 7.82−7.85 (m, 1H), 8.04 (s, 1H), 8.11−8.17 (m, 1H), 8.40−8.43 (m, 1H). LRMS-ESI (m/z): [M + H]+ C29H41N6O3SSi, 581; found, 581. 3-(5-(Cyclohexylamino)-6-(1-methyl-1H-pyrazol-4-yl)-1H-indazol-3-yl)benzenesulfonamide (32a). To a solution of compound 31a (169 mg, 0.291 mmol) and ethylenediamine (138 μL, 2.04 mmol) in THF (2 mL) was added TBAF (1 M solution in THF, 2.04 mL, 2.04 mmol). The mixture was heated to reflux for 24 h and then allowed to cool to room temperature. The mixture was diluted with EtOAc and H2O. The aqueous layer was separated and extracted with EtOAc. The combined organic extracts were washed with saturated aqueous NH4Cl, H2O, and brine, dried over MgSO4, filtered, and 4354

dx.doi.org/10.1021/jm4000215 | J. Med. Chem. 2013, 56, 4343−4356

Journal of Medicinal Chemistry Hepatocyte Metabolic Stability Studies. Rat fresh hepatocytes were prepared from male Sprague−Dawley rats (8 weeks). The compounds (2 μM) were incubated with approximately 1.0 × 106 cells/mL in suspension in Williams’ medium E at 37 °C in an atmosphere of 5% CO2. Hepatocyte incubations were initiated by the addition of 100-fold concentrated solutions of compounds. Incubations were terminated by the addition of 4-fold organic solvent (acetonitrile/methanol = 1/1) after 0, 60, and 120 min of incubation at 37 °C in an atmosphere of 5% CO2 and centrifuged. The supernatants were analyzed by LC/MS/MS. All incubations were conducted in duplicate, and the percentage of compounds remaining at the end of the incubation was determined from the LC/MS/MS peak area ratio. log k′. The lipophilicity index log k′ was determined from the RPHPLC retention time. Compounds were dissolved in DMSO (10 mM) and then diluted with 1% formamide in 50 mM ammonium bicarbonate/acetic acid buffer (pH 6.8) and acetonitrile (1/1, 50 μM). The RP-HPLC method conditions were as follows: Waters Alliance2795 HPLC system, XTerra MS C18 column, 2.1 × 100 mm, 5 μm, column temperature 30 °C; solvent A consisting of water (50 mM ammonium bicarbonate/acetic acid buffer, pH 6.8); solvent B consisting of acetonitrile; 5% B (1 min), gradient of 5−95% B (20 min), 95% B (1 min), 5% B (7 min); flow rate of 0.25 mL/min; injection volume of 4 μL. The log k′ of the compounds was calculated according to the following equation:

ASSOCIATED CONTENT

S Supporting Information *

Kinase selectivity data for compounds 17b, 23c, 32a, and 32b. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

PDB ID is 3W1F (Figure 3, crystal structure).

AUTHOR INFORMATION

Corresponding Author

*Phone: +81 6 6331 6190. Fax: +81 6 6332 6385. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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where tR and t0 are the retention times of the test compounds and formamide, respectively. Cocrystallization of Mps1 with 23d. Protein expression and purification were performed as described previously.25 Cocrystals of Mps1 in complex with 23d were prepared using the sitting-drop vapor diffusion method. Equal volumes of protein solution (8.7 mg/mL containing 0.5 mM of 23d) and the mother liquor were mixed in a single droplet and equilibrated against 0.1 mL of mother liquor at 293 K. The mother liquor conditions were 11.5% w/v PEG 4000 and 0.15 M magnesium formate. Data collection and structure solution were performed as described previously.25

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ABBREVIATIONS USED

AURK, aurora kinase; CIN, chromosomal instability; CL, plasma clearance; Cmax, maximum plasma concentration; dppf, 1,1′-bis(diphenylphosphino)ferrocene; dtbpf, 1,1′-bis(ditertbutylphosphino)]ferrocene; FGFR, fibroblast growth factor receptor kinase; FLT, Fms-like tyrosine kinase; JNK, c-Jun Nterminal kinase; LE, ligand efficiency; MDCK, Madin-Darby canine kidney; MELK, maternal embryonic leucine zipper kinase; MIN, microsatellite instability; Mps1, monopolar spindle 1; Papp, apparent permeability coefficient; RNAi, RNA interference; RPS6KA1, ribosomal protein S6 kinase alpha1; RP-HPLC, reverse-phase high-performance liquid chromatography; SEM, 2-(trimethylsilyl)ethoxymethyl; TPSA, topological polar surface area; Vdss, volume of distribution at steady state

log k′ = log{(t R − t0)/t0}

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ACKNOWLEDGMENTS

We thank Yoshiharu Hiramatsu, Yasuhiko Fujii, Masayoshi Miyagawa, Yo Adachi, and Masahiko Fujioka for their support with the syntheses, and Naoko Umesako for her HRMS analysis. K.K. also acknowledges Robert Hall for his assistance in preparing this manuscript. We gratefully thank Kenji Abe, Yoshiyuki Matsuo, Akira Kato, Norihiko Tanimoto, Takuji Nakatani, Hirosato Kondo, and Kohji Hanasaki for their helpful advice and suggestions during the course of this research. 4355

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