Article pubs.acs.org/jmc
Discovery of [5-Amino-1-(2-methyl‑3H‑benzimidazol-5-yl)pyrazol-4yl]-(1H‑indol-2-yl)methanone (CH5183284/Debio 1347), An Orally Available and Selective Fibroblast Growth Factor Receptor (FGFR) Inhibitor Hirosato Ebiike,*,† Naoki Taka,‡ Masayuki Matsushita,† Masayuki Ohmori,† Kyoko Takami,‡ Ikumi Hyohdoh,† Masami Kohchi,† Tadakatsu Hayase,† Hiroki Nishii,† Kenji Morikami,‡ Yoshito Nakanishi,† Nukinori Akiyama,† Hidetoshi Shindoh,† Nobuya Ishii,† Takehito Isobe,‡ and Hiroharu Matsuoka‡ †
Research Division, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
‡
S Supporting Information *
ABSTRACT: The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases regulates multiple biological processes, such as cell proliferation, migration, apoptosis, and differentiation. Various genetic alterations that drive activation of the receptors and the pathway are associated with tumor growth and survival; therefore, the FGFR family represents an attractive therapeutic target for treating cancer. Here, we report the discovery and the pharmacological profiles of 8 (CH5183284/Debio 1347), an orally available and selective inhibitor of FGFR1, FGFR2, and FGFR3. The chemical modifications, which were guided by 3D-modeling analyses of the inhibitor and FGFRs, led to identifying an inhibitor that is selective to FGFR1, FGFR2, and FGFR3. In in vitro studies and xenograft models in mice, 8 shows antitumor activity against cancer cell lines that harbor genetically altered FGFRs. These results support the potential therapeutic use of 8 as a new anticancer agent.
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INTRODUCTION Tyrosine phosphorylation is a critical event in the signal transduction that is mediated by growth factor, and the protein tyrosine kinases are key components of the phosphorylation process. Several molecular targeted kinase inhibitors have been launched, such as epidermal growth factor receptor (EGFR) inhibitors (erlotinib and gefitinib),1 erb-b2 receptor tyrosine kinase 2 (HER2) inhibitor (lapatinib),1 breakpoint cluster region abelson tyrosine kinase (BCR-ABL) inhibitors (imatinib and dasatinib),1 B-RAF inhibitors (vemurafenib and dabrafenib),1 and anaplastic lymphoma kinase (ALK) inhibitors (crizotinib and alectinib).1 While these kinase inhibitors that can treat patients according to their genetic status have contributed remarkable progress to cancer therapy, these drugs cannot cover all tumors, and the need to treat tumors harboring other genetic alterations still remains.2 Thus, the potential therapeutic strategy using a molecular targeted drug to satisfy the unmet medical © 2016 American Chemical Society
needs of a variety of cancers harboring novel genetic alterations has attracted attention over the years.3 In cancer, genetic alterations that constitutively activate FGFR, such as amplification and mutation, drive activation of the receptors and the pathway in several tumor types and are known to be associated with cell growth, angiogenesis, cell migration, invasion, and metastasis (Table 1).4,5 Several FGFR inhibitors targeting multiple kinases have been reported and are currently under clinical trial. While these drugs show some clinical benefits in patients that show positive for FGFR genetic alterations, some of the drugs, such as dovitinib (TKI258,6a Figure 1) and cediranib (AZD2171,6b Figure 1), also target kinase insert domain receptor (KDR), which causes grade 3/4 hypertension, and dose limiting toxicity has been observed in a clinical trial.7 Received: August 1, 2016 Published: November 7, 2016 10586
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Table 1. Genetic Alteration and Amplification of FGFR’s and Related Cancers4,5 gene
alteration
prevalence
FGFR1
amplification
FGFR2
amplification mutation mutation
squamous NSCLC (20%), breast (10%), ovarian (∼5%), bladder (3%) gastric (10%), breast (4% of triple-negative cases) endometrial (12%), squamous NSLC (5%) bladder (50−60% nonmuscle invasive type, 10− 15% invasive type) myeloma (15−20%)
FGFR3
translocation
with dimethylformamide dimethyl acetal (DMF-DMA) and yielded enamines (intermediate d). The aminopyrazoles were formed by treating the intermediate d with aryl hydrazine hydrochlorides at 100 °C. The synthesis of indole derivatives is outlined in Scheme 2. Because a protective group at 1-position of indole gave more selective cyclization to aminopyrazole, a benzenesulfonyl (Bs) group or a tosyl (Ts) group was introduced in the synthesis of indole derivatives (Scheme 2). The functional groups at 5-position or 6-position of indoles were introduced in advance to form the aminopyrazole ring, except for the sulfone compound (16, Scheme 3). The methanesulfonyl group in 16 was introduced to the aminopyrazole derivative (16c) by Cucatalyzed reaction with methanesulfinic acid sodium salt in DMSO.15 Aryl hydrazines were obtained by reducing diazonium salts with SnCl2 (procedure C, Scheme 4) or introducing the hydrazine moiety to heteroaryl bromide derivatives by Pdcatalyzed coupling reaction of di-tert-butyl hydrazodiformate (BocNHNHBoc) as a hydrazine source (procedure D, Scheme 4).16 The aryl hydrazines, especially heteroaryl hydrazines, were unstable in air. The aryl hydrazines were each isolated as a salt of hydrochloric acid and subjected to the aminopyrazole formation steps using in situ neutralization protocols with a neutralizing reagent (see Supporting Information).
Since compounds that are selective to one target kinase can potentially claim a more favorable toxicity profile than multitargeted compounds, selective and potent FGFR inhibitors are required. Currently, several FGFR-selective inhibitors, such as 21 (AZD4547)8 and 22 (NVP-BGJ398)9 (Figure 1), are under clinical trials targeting patients who have FGFR genetic alterations. These inhibitors, as well as the FGFR-selective inhibitor 20 (PD173074),10 share a common 3,5-dimethoxyphenyl ring that interacts with the backpocket region of the FGFR, according to X-ray structures of the binary complex of FGFR with each inhibitor.8,9,11 Since it is possible that a common single mutation, such as the gatekeeper mutation, is causing resistance to these inhibitors,12 a potential strategy to overcome this issue is the rational design of a novel inhibitor with a different chemical scaffold. This report describes the optimization program that led to identifying the novel inhibitor (8, (CH5183284/Debio 1347)), which potently and selectively inhibits the FGFR family of receptor tyrosine kinases and which is currently in Phase I clinical trial.13
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MOLECULAR DESIGN In order to obtain an FGFR-selective inhibitor, we performed a high-throughput screening against our chemical library and identified a screening hit (1) (Table 3) that inhibited a relatively broad range of kinases but had a different chemical scaffold from the other known FGFR inhibitors (Figure 1). The chemical modifications strategy guided by 3D modeling of inhibitors and FGFR1 is outlined in Figure 2. A 3D model structure of 1 and FGFR1 suggested that compound 1 was located in the ATPbinding site of FGFR1 (Figure 2).17 Analysis of the 3D modeling structure also suggested an interaction of the amino group and the carbonyl group of 1 with the backbone of the hinge region (E562 and A564) of FGFR1. Additionally, the phenolic hydroxyl group of 1 was supposed to interact with D641 through a hydrogen bond in the backpocket region of FGFR1. In addition to these interactions, we assumed that a further functional group
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CHEMISTRY The synthesis of aminopyrazole derivatives 1−3 is outlined in Scheme 1.14 Cyanomethylation of commercially available esters (intermediate b) with acetonitrile anion, which had been generated by treating acetonitrile (MeCN) with lithium hexamethyldisilazide (LHMDS) in THF at −78 °C, provided cyanomethyl compounds (intermediate c). The intermediate c that was available commercially or obtained above was treated
Figure 1. FGFR inhibitors. 10587
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Scheme 1. Syntheses of Aminopyrazole Derivativesa
a
Reagents and conditions: (a) LHMDS, MeCN, THF −78 °C; (b) DMF-DMA; (c) Ar2NHNH2 HCl, 100 °C.
Scheme 2. Syntheses of Indole Aminopyrazole Derivatives (4−15 and 17−19)a
a
Reagents and conditions: (a) NaH, ArylSO2Cl, THF; (b) LHMDS, MeCN, THF −78 °C; (c) DMF-DMA; (d) Ar2NHNH2 HCl, 100 °C.
Scheme 3. Synthesis of 16a
a Reagents and conditions: (a) LHMDS, MeCN, THF −78 °C; (b) DMF-DMA, THF; (c) ArNHNH2 HCl, 100 °C; (d) sodium methanesulfinate, CuI, N1,N2-dimethylcyclohexane-1,2-diamine, DMSO, 120 °C.
Scheme 4. Syntheses of aryl Hydrazines (7a, 9c, 10c, 11c, and 12a)a
Reagents and conditions: (a) H2, Pd-C, H2O-EtOH; (b) NaNO2, dil. HCl then SnCl2, 0 °C; (c) Pd2dba3, t-Bu XPhos, BocNHNHBoc, Cs2CO3, toluene, 100 °C; (d) 4 M HCl-AcOEt.
a
residue (Y563) in the hinge region, and replacing the m-tolyl moiety was expected to strengthen the π−π interaction between
could be introduced to 1 to gain additional interaction with FGFRs. In 1, an m-tolyl group was located near the tyrosine 10588
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antiproliferative activity against HCT116 (colorectal cancer, WT FGFR) was evaluated as a negative control cell lines. As an entry to the lead optimization process, we prepared a series of compounds bearing an aminopyrazole scaffold to explore their structure−activity relationships (SARs) by replacing the m-tolyl group in the solvent-exposed region of 1.
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RESULTS AND DISCUSSION Hit-to-Lead. The m-tolyl group was replaced into bicyclic aromatics in order to direct the aromatic ring of inhibitors in the solvent-exposed region near Y563 in FGFR1. As we expected, the bicyclic compounds 2 and 3 showed better kinase inhibitory activity than the monocyclic compound (1) because the π−π interaction between Y563 of FGFR1 and inhibitors was enhanced (Table 3). Interestingly, compound 4 showed the most potent kinase inhibitory activity out of the bicyclic compounds. The 3D modeling analysis of 4 with FGFR1 suggested that the phenyl moiety of the indole was forced into parallel geometry with Y563, and the hydrogen of the NH in the indole additionally interacted with the backbone of A564. We concluded that the indole was the optimal bicyclic heteroaromatic and explored modification of the backpocket region and the solvent-exposed region in next step, using 4 as the lead compound. Compared with the lead compound (4), a drop in inhibitory activity of 1 order of magnitude was observed in 5 by removing the phenolic hydroxyl group in 4. The hydroxyl group in 4 probably interacted with the carboxylic acid at D641 in FGFR1 by a hydrogen bond. This interaction was supposed to be essential to keep the inhibitory activity against FGFRs, because the aspartic acids corresponding to D641 in FGFR1 were well conserved in FGFRs. On the other hand, the aspartic acids corresponding to D641 in FGFR1 were also conserved in several kinases including KDR and SRC (Table 2). As described earlier, the inhibition of KDR is known to induce hypertension. We were also concerned about inhibition of SRC which is reported to lead to osteopetrosis in animal study.18 In addition to the interaction between inhibitors and kinases, a phenolic hydroxyl group was generally known to form a conjugate metabolite. To avoid both a broad kinase inhibition spectrum and drug metabolism, it was necessary to replace the phenol to its isostere. We analyzed the 3D structure of the backpocket region and the hinge region and hypothesized that compounds in FGFR1 interacting with M535, V559, and V561, which are the commonly conserved amino acid residues in FGFRs, could improve the specificity to FGFRs over SRC and KDR (Table 2 and Figure 3A and Figure 3B). The discrimination between FGFR1 and SRC would be considered to be due to the different characteristic of V561 (in FGFR1) versus T341 (in SRC) and of V559 (in FGFR1) versus I339 (in
Figure 2. Structure analysis of 3D modeling of 1 (magenta) and FGFR1. The side chains of amino acid residues important for ligand recognition of FGFR1 are highlighted as sticks (green). Hydrogen bonds at hinge region and backpocket region are indicated in blue dotted lines.
Y563 and the inhibitors. Addition of a hydrogen donor to 1 in the direction of the carbonyl group of A564 was predicted to enhance the interaction at the hinge region of FGFRs. Replacement of the phenol moiety by an isostere of phenol was considered to be essential to avoid metabolic instability. We envisioned that we could improve the specificity to FGFRs by implementing either a favorable interaction between the inhibitor and amino acid residues common in FGFRs or an unfavorable interaction between the inhibitor and amino acid residues in off-target kinases. Interaction of inhibitors with both the methionine residue (M535 in FGFR1) and the valine residue (V561 in FGFR1) could improve both kinase inhibitory activity and specificity to FGFRs (Table 2). Introducing a solubilizing group at the m-tolyl moiety was considered to be beneficial if the solubility of the inhibitors was insufficient. To confirm the selective antitumor efficacy of the inhibitors, we tested them in a kinase inhibitory assay and an in vitro antiproliferative activity assay. Because compound 1 inhibited a relatively broad range of kinases such as KDR and protooncogene (SRC) which were considered to potentially be offtarget kinases, the inhibitory activity against FGFRs, KDR, and SRC was measured in a cell-free system to evaluate selectivity of kinase inhibition.18 The sequence identity between FGFR1 and FGFR2 at the kinase domain was 87.6%, and the pivotal interactions between 1 and the amino acid residues of FGFR1 were expected to agree with those of FGFR2. Because the inhibitors had the same binding mode with FGFR1 and FGFR2, the inhibitory activity against FGFR1 correlated well with that against FGFR2. We concluded that the antiproliferative activity of the inhibitors in SNU-16 (gastric cancer, FGFR2 amplification) was appropriate for the in vitro assay, even though the 3D modeling of the inhibitors was performed in FGFR1. The Table 2. Amino Acid Sequences of FGFR1, KDR, and SRC
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Table 3. SAR of Hit-to-Lead
potential risk of drug related toxicity from having a broad spectrum of kinase inhibition. We explored the 2-position of the benzimidazole of 7, and it was revealed that 2-methyl-benzimidazole derivative (8) was optimal in terms of kinase selectivity. In addition to kinase selectivity, the profiles of 8 were well balanced in terms of in vitro cellular antiproliferative activity against SNU-16 and stability in human liver microsome. While the 2-ethyl benzimidazole derivative (9) showed equivalent kinase inhibition and cellular growth inhibition profiles as 8, its stability in human liver microsome did not meet our criteria (Table 5). The low kinase inhibitory activity of the benzimidazolinone derivative (12) against all the tested kinases suggested the importance of both the hydrogen acceptor and the hydrogen donor of the benzimidazole group. We speculated that the nitrogen of the benzimidazole group of 8 formed a hydrogen bond with the backbone of A640, and the NH group of the benzimidazole formed another hydrogen bond with the side chain of E531. In addition to these hydrogen bonds, the benzimidazole ring was located in the hydrophobic pocket formed by M535, V561, and F642, according to an X-ray structure of the binary complex of 8 and FGFR1 (Figure 5A). Furthermore, an S−π interaction between the sulfur atom of M535 and the benzimidazole ring was suggested.19 The selectivity of 8 to inhibit FGFR over KDR was suggested to be caused by the difference in the interaction with M535 in FGFR1 and L889 in KDR, because the leucine residue in KDR would disrupt the formation of the S−π interaction with the
SRC)(Figure 3B). Though the only amino acid residues that were different at the ATP-binding site in FGFR1 and KDR were M535 (FGFR1) and L889 (KDR), a steric repulsion between the inhibitors and L889 (KDR) could be expected at this site (Figure 3A). When a solubilizing group was introduced in the solventexposed indole moiety of 4, the kinase inhibitory activity of 6 was improved (Table 3). On the other hand, the selectivity to FGFRs over SRC and KDR was insufficient due to the high sequence similarity of FGFRs, SCR, and KDR at the solvent-exposed regions. Thus, we tried to modify the backpocket region to improve the specificity to FGFRs. Modification of the Backpocket Region. By analyzing a 3D structure of the backpocket region of FGFR1, we designed several bicyclic heteroaromatics with a hydrogen donor that could form a hydrogen bond to D641 to fit the cavity formed by E531, M535, V561, A640, and D641 in the binding pocket (Figure 2 and Table 4). A screening of the heteroaromatics revealed that the benzimidazole derivatives (7, 8, and 9) showed greater specificity to FGFRs than the low specificity to FGFRs of indole derivatives (10 and 11, Table 4). According to 3D modeling of 10 and 11 with FGFR1, the indole derivatives (10 and 11) still showed inhibitory activity against FGFRs probably due to the hydrogen bond between the NH group of indole and E531 in FGFRs. Interestingly, compound 11 showed better inhibitory activity against FGFRs than 10 probably because of CH−π interaction between the 2-methyl group of the indole in 11 and F642 in FGFR1, but the specificity of these indole derivatives to FGFRs needed to be improved to avoid the 10590
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Figure 3. (A, B) Neighboring amino acid residues of FGFR1 (green), KDR (magenta), and SRC (cyan) around 4 (orange). The binding models were created by overlapping X-ray structures of FGFR1 (2FGI), KDR (3WZD), and SRC (3G5D). (C, D) Superimposed structures of 4 (purple) and 8 (green) in FGFR1. The hydrogen bonds between 8 and FGFR1 (E531 and D641) are indicated in blue dotted lines. The benzimidazole in 8 interacts better with V561, I545, and F641 by CH−π interaction (orange dotted lines) than phenoxy group in 4.
was reported to be the dose-limiting toxicity of KDR inhibitors in clinical trials. We hypothesized that modifying the solventexposed region of 8 might improve the hERG inhibition without losing the inhibitory activity against FGFRs. Both lipophilic and hydrophilic functionalities were introduced to 8 to reduce hERG inhibition by controlling log P.22 First, amino groups with low pKa, such as morpholine or 4difluoropiperidine, were introduced at the 5- or 6-position of indole in 8. The inhibitory activities against FGFRs and SNU-16 were maintained by introducing amino groups (Table 6). Because introducing an electronegative oxygen at β-position or an electron-withdrawing fluorine atom at γ-position of basic nitrogen reduced the pKa value of amino groups, the reduction of the hERG binding affinity by lowering the basicity of nitrogen in amino groups was expected.23 The pKa value of nitrogen of morpholine in 13 and 14 was reduced from 9.23 (piperidine) to 7.11 (13) and 7.24 (14) by replacing piperidine to morpholine, and the pKa value of nitrogen in 4-difluoropiperidine derivative (15) was also reduced to 6.39 (Figure 4). The strategy to reduce inhibition of hERG by polar groups worked well in 13 and 14, but not in 15 (Table 7). The solubility of these amino derivatives (13, 14, and 15) was improved by introducing hydrophilic groups, but the permeability in a Caco2 study was reduced, compared to 8 (Table 7). The low permeability of these compounds resulted in low oral bioavailability (BA) in a monkey pharmacokinetic (PK) study (Table 7). Next, lipophilic groups were introduced in the 6-position of indole, and the in vitro profiles are summarized in Table 6. The 6-bromo-indole derivative (17) and the 6-methyl-indole derivative (18) showed better permeability in the Caco2 study among lipophilic derivatives, and compound 17 exhibited 20% BA in the monkey PK study (Table 7). Unfortunately, a weaker inhibition of hERG
benzimidazole moiety of 8 (Figure 5B). The selectivity to FGFR over SRC was also suggested to be the result of a disruption of a crucial interaction between 8 and the lipophilic binding pocket formed by V561 and V559 in FGFR1, which were T314 and I339 in SRC (Figure 5B). On the other hand, compound 4 showed a broad kinase inhibitory activity against the tested kinases because the chloro-phenyl moiety of 4 had effective interactions with amino acid residues other than those in FGFR1. The 3D modeling of 4 and 8 (Figure 3C and Figure 3D) suggested that the chloro-phenyl ring of 4 and the benzimidazole ring of 8 had different geometry, and the chloro-phenyl moiety of 4 had no repulsive interaction against either FGFR1, KDR, or SRC (Figure 3A,B). Furthermore, the benzimidazole derivatives were much soluble in fasted state simulated intestinal fluid (FaSSIF) than indole derivatives (Table 5). As described above, we concluded that the 2-methyl-benzimidazole group was optimal in the backpocket region of an inhibitor and planned to modify the solvent-exposed region of compounds that had 2-methylbenzimidazole group in 1-position of the aminopyrazole. Modification of the Solvent-Exposed Region to Reduce Cardiovascular Effects. In spite of 8’s high specificity to FGFRs, an inhibition of cardiac K+ channel encoded by the human ether-a-go-go-related gene (hERG) (IC50 6.9 μM, 69% inhibition at 10 μM, Table 7) was observed in an early safety study.20 In addition to hERG inhibition, the inhibitory activity of 8 on KDR (IC50 2.1 μM) was also suggested to cause hypertension. Cardiovascular side effects, especially QTc prolongation, are associated with inhibition of the hERG channel, which has resulted in several drugs being withdrawn from the market and which is still one of the potential risks in the R&D process of new molecular entities.21 As we mentioned above, hypertension (grades 3 and 4) caused by inhibiting KDR 10591
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Table 4. SAR in Modification of Backpocket Regiona
a
N.T.: Not tested.
blood pressure compared with vehicle-dosed rats and monkeys (data not shown). The effect of 8 on blood pressure in telemetryinstrumented rats at a dose of 30 mg/kg did not lead to significant changes in blood pressure compared with vehicledosed animals.24 Of note, the plasma exposure of 8 in rats (AUC: 102 μg·h·mL−1) at a dose of 30 mg/kg was 4-fold higher than the exposure inducing a >100% tumor growth inhibition in the SNU16 mouse xenograft model (AUC: 26 μg·h·mL−1 at a dose of 30 mg/kg).24 Therefore, compound 8 was predicted to have no biologically significant effect on blood pressure at multiples of the efficacious dose. Efficacy in Tumor Xenograft Studies. The in vivo efficacy was evaluated in mice bearing an SNU-16 xenograft. Compound 8 was orally administered once daily for 11 days, and the body weight of mice and the volume of the tumors were measured twice a week. A dose-dependent tumor regression (tumor growth inhibition (TGI) = 106% at 30 mg/kg and 147% at 100 mg/kg) was observed without apparent body weight loss.24 Compound 8 also showed significant in vivo efficacy in xenograft mice models with FGFR genetic alterations, such as KG1 (leukemia, FGFR1OP-FGFR1 fusion), MFE280 (endometrial cancer, FGFR2 S252W mutation), UM-UC-14 (bladder cancer, FGFR3 S249C mutation), and RT112/84 (bladder cancer, FGFR3-TACC3 fusion).24 These results suggested 8’s therapeutic potential of cancers harboring FGFR genetic alterations.
Table 5. Solubility in FaSSIF and Stability (CLint) in Human Liver Microsome cmpd
solubility (μg/mL)
CLint (μL/min/mg)
4 6 7 8 9 10 11
20 40 37 29 42 4.0 10
15 20 4.2 13 23 4.4 10
was observed in 17 than 8, but another cardiovascular side-effect was suggested by a safety study (data not shown). The 6-phenylindole derivative (19) showed the same in vitro properties as 8 and a weak hERG inhibition, but the BA of 19 was low (3% in monkey). We concluded that oral BA could be predicted by the permeability in Caco2 and that hERG inhibition and oral BA could not be improved at the same time by modifying the solvent-exposed region. Assessment of Cardiovascular Effect of 8. We tried to evaluate the profiles of 8 in detail, because there was some concern around possible cardiovascular risk due to inhibitory activity against KDR and hERG inhibition as described earlier. We examined a safety assessment study to evaluate a potential risk of cardiovascular effect of 8. Compound 8 did not lead to either QTc prolongation in Langendorff’s isolated heart perfusion model in guinea pig or to significant changes in 10592
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Table 6. SAR in Optimization of Solvent Exposed Region
Figure 4. pKa values of amines in the solvent exposed region.
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CONCLUSION
at the backpocket region of FGFR1 according to the X-ray crystal structure of binary complex of FGFR1 and 8 (Figure 5A). The recognition of the different amino acid residues in FGFRs, KDR, and SRC was achieved by introducing 2-methyl-benzimidazole in the backpocket region of 8 (Figure 5B). The benzimidazole moiety was positioned in the same region of FGFR1 as the 3,5dimethoxy-phenyl groups in 21 and 22 (Figure 5C). It may be
We identified 8 as a potent and selective FGFR inhibitor with a novel chemical scaffold by exploiting crystallographic structure information on FGFRs with their inhibitors. Compound 8 only bound to five kinases, including FGFR1, FGFR2, and FGFR3, out of 442 WT and mutant kinases at 100 nM in KINOMEscan panel (DiscoveRx).24 The benzimidazole moiety of 8 was located 10593
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Table 7. Physicochemical Properties, Suppression of hERG, and BA in Monkey
a
cmpd
CLint (μL/min/mg) Human
Caco2 (10−6 cm/s)
hERG IC50 (μM)
hERG % inhibition @10 μM
monkey BA (%)
8 13 14 15 16 17 18 19
13 15 9.6 14 5.4 4.2 140 3.2
28 0.35 1.2 19 0.075 2.2a 32 0.070
6.9 >10 >10 8.6 >10 >10 6.9 >10
69 7.0 15 58 4.0 31 64 15
30 N.T. 0 5.0 N.T. 20 N.T. 3.0
Caco2 assay at 100 μM except for 17 (10 μM). N.T.: Not tested. 1 H NMR (400 MHz, DMSO-d6) δ: 10.13 (1H, s), 7.76 (1H, s), 7.56− 7.54 (2H, m), 7.46 (1H, d, J = 8.8 Hz), 7.41−7.39 (2H, m), 7.04 (2H, s), 6.96 (1H, dd, J = 8.8, 2.7 Hz), 6.87 (1H, d, J = 2.7 Hz), 2.40 (3H, s); 13C NMR (100 MHz, DMSO-d6) δ: 187.74, 157.14, 152.15, 141.40, 139.71, 137.89, 134.79, 131.84, 130.90, 128.42, 128.31, 125.07, 120.75, 118.36, 116.64, 102.50, 20.97. HRMS (ESI): m/z calculated for C17H14ClN3O2 + H+ [M + H]+: 328.0853, found: 328.0852. [5-Amino-1-(2-chloro-5-hydroxy-phenyl)pyrazol-4-yl]-(benzofuran-2-yl)methanone (2). Prepared by following the general procedure A. 1 H NMR (400 MHz, DMSO-d6) δ: 10.20 (1H, s), 8.47 (1H, s), 7.83 (1H, dd, J = 2.7, 1.1 Hz), 7.81 (1H, dd, J = 3.3, 1.1 Hz), 7.77 (1H, d, J = 1.1 Hz), 7.55−7.51 (1H, m), 7.49 (1H, d, J = 8.8 Hz), 7.38 (1H, td, J = 7.7, 0.9 Hz), 7.21 (2H, s), 6.99 (1H, dd, J = 8.8, 2.7 Hz), 6.92 (1H, d, J = 2.7 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 174.34, 157.12, 154.75, 153.52, 152.64, 140.87, 134.63, 130.92, 127.43, 126.94, 123.86, 123.03, 120.84, 118.43, 116.69, 112.34, 111.38, 101.95. HRMS (ESI): m/z calculated for C18H12ClN3O3 + H+ [M + H]+: 354.0645, found: 354.0645. [5-Amino-1-(2-chloro-5-hydroxy-phenyl)pyrazol-4-yl]-(6quinolyl)methanone (3). Prepared by following the general procedure A. 1 H NMR (400 MHz, DMSO-d6) δ: 11.93 (1H, s), 8.47 (1H, dd, J = 4.4, 1.6 Hz), 8.39 (1H, s), 7.86 (1H, d, J = 8.2 Hz), 7.63 (1H, br s), 7.55 (1H, s), 7.30 (1H, dd, J = 8.2, 1.6 Hz), 7.26 (1H, dd, J = 8.2, 4.4 Hz), 7.09 (2H, s), 2.54 (3H, s); 13C NMR (100 MHz, DMSO-d6) δ: 186.73, 157.12, 152.31, 152.10, 148.68, 141.62, 137.54, 137.13, 134.77, 130.93, 129.36, 128.72, 128.33, 127.29, 122.13, 120.82, 118.40, 116.65, 102.64. HRMS (ESI): m/z calculated for C19H13ClN4O2 + H+ [M + H]+: 365.0805, found: 365.0795. General Procedure B for Synthesis of the Aminopyrazole Compounds 4−7, 9−15, and 17−19 (Scheme 2). [5-Amino-1-(2chloro-5-hydroxy-phenyl)pyrazol-4-yl]-(1H-indol-2-yl)methanone (4). (E)-3-(Dimethylamino)-2-[1-(p-tolylsulfonyl)indole-2-carbonyl]prop-2-enenitrile (4c, 100 mg, 0.254 mmol) and 4-chloro-3hydrazino-phenol hydrochloride (74.4 mg, 0.381 mmol) were placed in a screw-capped vial, and then NMP (1.0 mL) was added to the vial. 4Methylmorpholine (0.042 mL, 0.381 mmol) was added to the solution, and then nitrogen gas was bubbled into the reaction mixture. The mixture was stirred at 100 °C for 1 h under nitrogen atmosphere. Five M NaOH (0.250 mL) was added to the solution, and then the mixture was stirred at 100 °C for 2 h under nitrogen atmosphere. The reaction mixture was subjected to a C18 MPLC column, and then the column was eluted with 0.1% formic acid-H2O-MeCN by a linear gradient. The fractions containing the target were collected. The eluent was passed through MP-CO3 cartridges (Biotage, one cartridge (3 g) per 36 mL of fraction) to remove formic acid. The resulting solution was concentrated under reduced pressure to give the titled compound as a light yellow amorphous (47 mg, 52%). 1 H NMR (600 MHz, DMSO-d6) δ: 11.67 (1H, s), 10.17 (1H, s), 8.30 (1H, s),7.69 (1H, d, J = 8.1 Hz), 7.49−7.48 (2H, m), 7.44 (1H, d, J = 1.5 Hz), 7.26−7.23 (1H, m), 7.09−7.06 (1H, m), 7.02 (2H, s), 6.98 (1H, dd, J = 8.8, 2.9 Hz), 6.90 (1H, d, J = 2.9 Hz); 13C NMR (150 MHz, DMSO-d6) δ: 177.75, 156.99, 152.09, 140.16, 136.73, 135.41, 134.73, 130.79, 127.39, 124.23, 122.14, 120.70, 119.81, 118.23, 116.57, 112.37,
possible to overcome acquired resistance, such as a gatekeeper mutation, because the binding mode of 8 is different from that of 21 and 22 at both the backpocket region and the hinge region of FGFR1.24 A preclinical PK evaluation in mice, rats, and monkeys revealed that the compound was orally bioavailable in both rodents and nonrodents. When the anticancer efficacy was evaluated, compound 8 potently inhibited the proliferation of gastric cancer cell lines with amplified FGFR2 and exhibited dose-dependent antitumor efficacy in SNU-16 xenograft models. Compound 8 will provide therapeutic opportunities for patients who have FGFR genetic alterations and patients with acquired resistance to existing FGFR-selective inhibitors.
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EXPERIMENTAL SECTION
General Synthetic and Analytical Chemistry Methods. All reagents and solvents were purchased from commercial sources and used as received. 1H and 13C NMR spectra were recorded on a JEOL JNM-ECA400 (400 MHz), a Varian MR400 (400 MHz), a Bruker AVANCE III HD600 (600 MHz), or a Bruker AVANCE III 600 CryoTCI (600 MHz). The chemical shifts are expressed as δ units using tetramethylsilane as an internal standard. The following abbreviations are used: singlet (s), doublet (d), double doublets (dd), triplet (t), quartet (q), multiplet (m), broad peak (br), and broad singlet (brs). LCMS analyses were performed on a Waters Micromass SQD instrument coupled to a Waters Acuity UPLC and PDA instruments. Analytical reverse-phase ultra-high-performance liquid chromatography (UPLC) experiment was carried out on an Ascentis Express C18 column (2.1 × 50 mm, 2.7 μm) or Acquity UPLC BEH Phenyl column (2.1 × 50 mm, 1.7 μm). Eluents in UPLC used were solvent A (H2O with 0.1% formic acid) and solvent B (MeCN with 0.1% formic acid) or solvent C (H2O with 0.03% TFA) and solvent D (MeCN with 0.03% TFA) and eluted by a linear gradient at 1.0 mL/min. High-resolution mass spectra (HRMS) were measured with a Xevo G2-SL (Waters) MS spectrometer using an ESI source coupled to a Waters HPLC system operating in reversed phase with an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm). Normal phase and reverse-phase flash column chromatography were performed with Biotage SNAP cartridges equipped with SP1 or Isolera One (Biotage). The purities of all tested compounds are >95%. The purity was determined by LC-MS analysis. General Procedure A for Synthesis of the Aminopyrazole Compounds 1−3 (Scheme 1). [5-Amino-1-(2-chloro-5-hydroxyphenyl)pyrazol-4-yl]-(m-tolyl)methanone (1). (E)-3-(Dimethylamino)-2-(3-methylbenzoyl)prop-2-enenitrile (1a, 107 mg, 0.500 mmol) and 4-chloro-3-hydrazino-phenol hydrochloride (146 mg, 0.750 mmol) were placed in a screw-capped vial, and then EtOH (1.0 mL) was added to the vial. Nitrogen gas was bubbled into the reaction mixture, and then the mixture was stirred at 78 °C for 10 h under nitrogen atmosphere. The solution was diluted with DMSO (2 mL) and then subjected to a C18 medium-pressure liquid chromatography (MPLC) column. The column was eluted with 0.1% formic acid-H2O-MeCN by a linear gradient. The fractions containing the target were collected and then concentrated under reduced pressure to give the titled compound as a light yellow powder (26 mg, 15%). 10594
DOI: 10.1021/acs.jmedchem.6b01156 J. Med. Chem. 2016, 59, 10586−10600
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[5-Amino-1-(2-chloro-5-hydroxy-phenyl)pyrazol-4-yl]-[5-(morpholinomethyl)-1H-indol-2-yl]methanone (6). Prepared by following the general procedure B. 1 H NMR (400 MHz, DMSO-d6) δ: 11.66 (1H, s), 8.29 (1H, s), 7.57 (1H, s), 7.50−7.46 (2H, m), 7.41 (2H, d, J = 2.2 Hz), 7.22 (1H, dd, J = 8.4, 1.3 Hz), 7.03 (2H, s), 6.98 (1H, dd, J = 8.8, 3.1 Hz), 6.90 (1H, d, J = 3.1 Hz), 3.57 (4H, t, J = 4.4 Hz), 3.52 (2H, s), 2.37 (4H, br s); 13C NMR (100 MHz, DMSO-d6) δ: 177.72, 157.07, 152.06, 140.18, 136.11, 135.62, 134.71, 130.78, 129.12, 129.04, 128.33, 127.27, 122.26, 120.63, 116.59, 112.12, 106.19, 102.03, 66.12 (2C), 62.92, 53.09 (2C). HRMS (ESI): m/z calculated for C23H22ClN5O3 + H+ [M + H]+: 452.1489, found: 452.1492. 3H-Benzimidazol-5-ylhydrazine dihydrochloride (7a). Prepared by following the general procedure C. 1 H NMR (400 MHz, DMSO-d6) δ: 10.63 (3H, br s), 9.47 (1H, s), 8.73 (1H, s), 7.77 (1H, d, J = 8.8 Hz), 7.39 (1H, s), 7.23 (1H, d, J = 8.8 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 144.96, 140.10, 131.68, 125.81, 115.73, 115.51, 98.09. MS (ESI): m/z 149 [M + H]+. [5-Amino-1-(3H-benzimidazol-5-yl)pyrazol-4-yl]-(1H-indol-2-yl)methanone (7). Prepared by following the general procedure B. 1 H NMR (400 MHz, DMSO-d6) δ: 11.74 (1H, s), 8.46 (1H, s), 8.36 (1H, s), 7.82−7.81 (1H, m), 7.79 (1H, s), 7.71 (1H, d, J = 7.7 Hz), 7.53−7.50 (1H, m), 7.48 (1H, d, J = 1.1 Hz), 7.44 (1H, dd, J = 8.2, 2.2 Hz), 7.27 (1H, td, J = 7.7, 1.1 Hz), 7.13−7.09 (3H, m); 13C NMR (100 MHz, DMSO-d6) δ: 178.15, 151.32, 143.71, 140.08, 136.89, 135.58, 131.91, 128.12, 127.52, 125.53, 124.42, 122.30, 119.98, 119.25, 115.97, 112.51, 111.69, 106.55, 103.18. HRMS (ESI): m/z calculated for C19H14N6O + H+ [M + H]+: 343.1307, found: 343.1311. [5-Amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-(1Hindol-2-yl)methanone (8). (E)-3-(Dimethylamino)-2-[1-(ptolylsulfonyl)indole-2-carbonyl]prop-2-enenitrile (4c, 79 mg, 0.200 mmol) and (2-methyl-3H-benzimidazol-5-yl)hydrazine dihydrochloride (56 mg, 0.240 mmol) were placed in a round-bottomed flask, and then EtOH (2.0 mL) was added thereto. The mixture was stirred at 80 °C for 2 h under nitrogen atmosphere. The mixture was cooled to 25 °C, and then the precipitate was collected by filtration. The cake was washed with H2O (2 mL). The powder was dried under reduced pressure. The powder was suspended in EtOH (17 mL), and then 4 M NaOH (1.7 mL) was added to the suspension. The mixture was stirred at 25 °C for 4 h under nitrogen atmosphere. The volatile was removed under reduced pressure to 5 mL. H2O (5 mL), and 4 M HCl (1.7 mL) was added to the mixture at 0 °C. The precipitate was collected by filtration. The cake was washed with H2O (2 mL). The powder was dried under reduced pressure to give the titled compound as a light yellow powder (58 mg, 81%). 1 H NMR (400 MHz, DMSO-d6) δ: 11.73 (1H, s), 8.36 (1H, s), 7.80 (1H, d, J = 1.6 Hz), 7.77 (1H, d, J = 8.8 Hz), 7.70 (1H, d, J = 8.2 Hz), 7.49 (3H, td, J = 8.7, 1.6 Hz), 7.26 (1H, t, J = 7.7 Hz), 7.14 (2H, s), 7.09 (1H, t, J = 7.7 Hz), 2.69 (3H, s); 13C NMR (100 MHz, DMSO-d6) δ: 178.11, 153.06, 151.32, 140.27, 136.88, 135.62, 135.51, 134.78, 132.58, 127.48, 124.43, 122.28, 119.97, 119.84, 114.62, 112.50, 110.31, 106.57, 103.19, 13.74. HRMS (ESI): m/z calculated for C20H16N6O + H+ [M + H]+: 357.1464, found: 357.1475. General Procedure C for Synthesis of the Aryl Hydrazines 9c, 7a, and 12a (Scheme 4). 2-Ethyl-6-nitro-1H-benzimidazole (9a). 4Nitrobenzene-1,2-diamine (766 mg, 5.00 mmol) was suspended in MeCN (7.6 mL), and then N,N-diisopropylethylamine (DIPEA) (1.31 mL, 7.50 mmol) was added to the suspension. Propionyl chloride (0.524 mL, 6.00 mmol) was added to the suspension with ice-cooling keeping the inner temperature of the reaction mixture below 25 °C. The mixture was stirred at 25 °C for 1 h under nitrogen atmosphere. H2O (15 mL) was added to the solution at 25 °C, and then the precipitate was collected by filtration. The cake was washed with H2O/methanol (MeOH) (2/1, 5.0 mL). The powder was dried under reduced pressure. The powder was suspended in MeOH (7.0 mL), and then concentrated HCl (2.1 mL) was added to the suspension. The mixture was stirred at 70 °C for 1 h. Five M NaOH (5.0 mL) was added to the suspension, and then the precipitate was collected by filtration. The powder was dissolved in DMSO (2.0 mL), and then the solution was subjected to a C18 MPLC column. The column was eluted with 0.1% formic acid-H2O-
Figure 5. 3D structures of 8, 21, and 22. (A) The X-ray crystal structure (PDB code: 5B7V) of 8 (purple) and FGFR1. The side chains of the amino acid residues important for ligand recognition of FGFR1 are highlighted as sticks (green). Hydrogen bonds are indicated in blue dotted lines. The CH−π and S−π interactions are indicated in orange dotted lines. (B) The overlapped structure of 8 (green), FGFR1 (green), KDR (magenta), and SRC (light blue). The side chains of the amino acid residues around 8 in the backpocket regions are reported as sticks. (C) The overlapped structure of 8 (magenta), 21 (light blue), and 22 (green) in FGFR1. The side chains of the amino acid residues around 8, 21, and 22 in the backpocket regions are reported as sticks. 106.28, 102.04. HRMS (ESI): m/z calculated for C18H13ClN4O2 + H+ [M + H]+: 353.0805, found: 353.0810. [5-Amino-1-(2-chlorophenyl)pyrazol-4-yl]-(1H-indol-2-yl)methanone (5). Prepared by following the general procedure B. 1 H NMR (400 MHz, DMSO-d6) δ: 11.70 (1H, s), 8.34 (1H, s), 7.74− 7.73 (1H, m), 7.69 (1H, d, J = 7.5 Hz), 7.63−7.53 (3H, m), 7.49 (1H, dd, J = 8.4, 0.9 Hz), 7.46 (1H, d, J = 1.3 Hz), 7.27−7.23 (1H, m), 7.09−7.07 (3H, m); 13C NMR (100 MHz, DMSO-d6) δ: 177.76, 152.31, 140.29, 136.73, 135.39, 134.40, 131.67, 131.27, 130.36, 130.31, 128.35, 127.39, 124.25, 122.15, 119.82, 112.37, 106.30, 102.07. HRMS (ESI): m/z calculated for C18H13ClN4O + H+ [M + H]+: 337.0856, found: 337.0852. 10595
DOI: 10.1021/acs.jmedchem.6b01156 J. Med. Chem. 2016, 59, 10586−10600
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Article
MeCN by a linear gradient. The fractions containing the target were collected and concentrated under reduced pressure to give the titled compound as a light brown powder (343 mg, 35%). 1 H NMR (400 MHz, DMSO-d6) δ: 12.90 (1H, s), 8.38 (1H, d, J = 1.6 Hz), 8.07 (1H, dd, J = 8.8, 2.2 Hz), 7.65 (1H, d, J = 8.8 Hz), 2.91 (2H, q, J = 7.6 Hz), 1.35 (3H, t, J = 7.6 Hz). HRMS (ESI): m/z calculated for C9H9N3O2 + H+ [M + H]+: 192.0773, found: 192.0772. 2-Ethyl-3H-benzimidazol-5-amine hydrochloride (9b). 2-Ethyl-6nitro-1H-benzimidazole (9a, 200 mg, 1.05 mmol) was dissolved in EtOH (2.0 mL), and then 5 M HCl (0.50 mL) and Pd−C (50.0 mg) suspended in H2O (0.50 mL) were added to the solution. The mixture was stirred at 25 °C for 3 h under hydrogen atmosphere. The catalyst was removed by filtration, and then the filter paper was washed with EtOH (4 mL). The filtrate was concentrated under reduced pressure to give the titled compound as a light yellow powder (241 mg, 100%). 1 H NMR (400 MHz, DMSO-d6) δ: 12.21 (4H, br s), 7.81 (1H, d, J = 8.8 Hz), 7.73 (1H, s), 7.43−7.42 (1H, m), 3.16 (2H, q, J = 7.5 Hz), 1.44 (3H, t, J = 7.7 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 156.08, 132.10, 131.05, 128.71, 119.72, 114.58, 106.50, 19.85, 10.68. HRMS (ESI): m/z calculated for C9H11N3 + H+ [M + H]+: 162.1031, found: 162.1033. (2-Ethyl-3H-benzimidazol-5-yl)hydrazine dihydrochloride (9c). NaNO2 (101 mg, 1.46 mmol) in H2O (0.25 mL) was added to the suspension of 2-ethyl-3H-benzimidazol-5-amine hydrochloride (9b, 241 mg, 1.22 mmol) in diluted HCl (concentrated HCl/H2O: 1 mL/0.25 mL) at 0 °C over 5 min. SnCl2·2H2O (605 mg, 2.68 mmol) in concentrated HCl (2.23 mL) was added to the resulting solution at 0 °C over 5 min. The resulting mixture was stirred at 0 °C for 30 min. The solution was charged onto SCX cartridges (5 g × 3), and then the cartridges were eluted with MeOH (10 mL each). The cartridges were eluted with 2 M NH3 MeOH (10 mL each), and then the eluent was evaporated off with 5 M HCl (15 mL). The residue was dissolved in H2O (5 mL), and then the solution was subjected to a C18 MPLC column. The column was eluted with 0.1% formic acid-H2O-MeCN by a linear gradient. The fractions containing the target were collected and concentrated with 5 M HCl (2 mL) under reduced pressure to give the titled compound as a colorless powder (183 mg, 60%). 1 H NMR (400 MHz, DMSO-d6) δ: 7.66 (1H, d, J = 8.8 Hz), 7.40 (1H, d, J = 1.8 Hz), 7.18 (1H, dd, J = 9.0, 2.0 Hz), 3.12 (2H, q, J = 7.6 Hz), 1.42 (3H, t, J = 7.7 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 154.49, 143.97, 131.24, 125.38, 114.29, 114.04, 97.54, 19.68, 10.83. MS (ESI): m/z 177 [M + H]+. [5-Amino-1-(2-ethyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-(1Hindol-2-yl)methanone (9). Prepared by following the general procedure B. 1 H NMR (400 MHz, DMSO-d6) δ: 11.72 (1H, s), 8.33 (1H, s), 8.15 (1H, s), 7.70 (1H, d, J = 8.2 Hz), 7.64 (1H, s), 7.62 (1H, s), 7.50 (1H, d, J = 8.2 Hz), 7.46 (1H, d, J = 2.2 Hz), 7.31 (1H, dd, J = 8.2, 1.6 Hz), 7.28− 7.24 (1H, m), 7.09 (1H, td, J = 7.4, 1.1 Hz), 7.02 (2H, s), 2.89 (2H, q, J = 7.5 Hz), 1.35 (3H, t, J = 7.4 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 178.00, 162.99, 158.02, 151.11, 139.79, 136.75, 135.47, 130.96, 127.96, 127.39, 125.40, 124.27, 122.16, 119.85, 118.24, 117.01, 112.38, 106.37, 103.00, 21.93, 12.11. HRMS (ESI): m/z calculated for C21H18N6O + H+ [M + H]+: 371.1620, found: 371.1625. General Procedure D for Synthesis of Aryl Hydrazines 10c and 11c (Scheme 4). 5-Bromo-1-(p-tolylsulfonyl)indole (10a). 5Bromo-1H-indole (1.96 g, 10.0 mmol) was dissolved in THF (20 mL), and then NaH (60% in mineral oil, 520 mg, 13.0 mmol) was added to the mixture at 0 °C. The mixture was stirred at 25 °C for 15 min under nitrogen atmosphere. TsCl (tosyl chloride) (2.29 g, 12.0 mmol) was added to the mixture at 0 °C, and then the mixture was stirred at 25 °C for 18 h under nitrogen atmosphere. The mixture was diluted with H2O (20 mL) with ice-cooling. The mixture was extracted with AcOEt (20 mL × 2), and then the combined organic layers were washed with brine (20 mL). The organic layer was dried on Na2SO4, and then the desiccant was removed by filtration. The filtrate was concentrated under reduced pressure, and then the residue was diluted with dichloromethane (20 mL). The mixture was absorbed in ISOLUTE HM-N (Biotage, 10 g), and then the volatile was removed under reduced pressure. The powder was subjected to a silica gel column cartridge (100 g), and then the column was eluted with n-hexane-AcOEt by a linear gradient. The
fractions containing the target were collected and concentrated under reduced pressure to give the titled compound as a colorless powder (3.03 g, 87%). 1 H NMR (400 MHz, DMSO-d6) δ: 7.88−7.85 (5H, m), 7.48 (1H, dd, J = 8.8, 2.2 Hz), 7.39 (2H, d, J = 8.2 Hz), 6.81 (1H, d, J = 3.3 Hz), 2.32 (3H, s); 13C NMR (100 MHz, DMSO-d6) δ: 145.67, 133.78, 132.83, 132.34, 130.23 (2C), 128.35, 127.12 (2C), 126.63, 123.96, 116.01, 114.89, 108.69, 20.93. HRMS (ESI): m/z calculated for C15H12BrNO2S - H+ [M - H]−: 347.9694, found: 347.9693. tert-Butyl N-(tert-Butoxycarbonylamino)-N-[1-(p-tolylsulfonyl)indol-5-yl]carbamate (10b). 5-Bromo-1-(p-tolylsulfonyl)indole (10a, 350 mg, 1.00 mmol), tert-butyl N-(tert-butoxycarbonylamino)carbamate (279 mg, 1.20 mmol), and Cs2CO3 (489 mg, 1.50 mmol) were placed in a screw-capped vial, and then toluene (2.0 mL) was added thereto. Nitrogen gas was bubbled into the suspension for 30 s. Tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3) (45.8 mg, 0.0500 mmol) and 2-di-tert-butylphosphino-2′,4′,6′-triiso-propyl-1,1′biphenyl (t-Bu XPhos, 63.7 mg, 0.150 mmol) were added to the vial, and then nitrogen gas was bubbled into the suspension for 30 s. The mixture was stirred at 100 °C for 6 h under nitrogen atmosphere. The mixture was diluted with AcOEt (5 mL), and then the suspension was washed with H2O (5 mL) and brine (5 mL). The organic layer was concentrated under reduced pressure, and then the residue was diluted with DMSO (2 mL). The solution was subjected to a C18 MPLC column, and then the column was eluted with 0.1% formic acid-H2O-MeCN by a linear gradient. The fractions containing the target were collected and concentrated under reduced pressure to give the titled compound as a light yellow amorphous (359 mg, 72%). 1 H NMR (400 MHz, DMSO-d6) δ: 9.64 (1H, s), 7.87−7.85 (3H, m), 7.78 (1H, d, J = 3.5 Hz), 7.51 (1H, s), 7.37 (2H, d, J = 7.9 Hz), 7.28 (1H, d, J = 9.3 Hz), 6.85 (1H, d, J = 3.5 Hz), 2.31 (3H, s), 1.42 (18H, s). HRMS (ESI): m/z calculated for C25H31N3O6S - H+ [M - H]−: 500.1855, found: 500.1846. [1-(p-Tolylsulfonyl)indol-5-yl]hydrazine hydrochloride (10c). tertButyl N-(tert-butoxycarbonylamino)-N-[1-(p-tolylsulfonyl)indol-5-yl]carbamate (10b, 300 mg, 0.598 mmol) was suspended in AcOEt (3.0 mL), and then 4 M HCl AcOEt (3.0 mL) was added to the suspension at 25 °C. The mixture was stirred at 25 °C for 4 h under nitrogen atmosphere. The precipitate was collected by filtration, and then the cake was washed with AcOEt (2 mL). The powder was dried under reduced pressure to give the titled compound as a colorless powder (156 mg, 77%). 1 H NMR (400 MHz, DMSO-d6) δ: 10.08 (3H, br s), 8.19 (1H, s), 7.85−7.82 (3H, m), 7.75 (1H, d, J = 3.5 Hz), 7.37 (2H, d, J = 7.9 Hz), 7.14 (1H, d, J = 1.8 Hz), 7.01 (1H, dd, J = 9.0, 2.4 Hz), 6.79 (1H, d, J = 3.1 Hz), 2.31 (3H, s); 13C NMR (100 MHz, DMSO-d6) δ: 145.30, 142.00, 133.92, 130.95, 130.06 (2C), 129.56, 127.78, 126.54 (2C), 113.64, 113.57, 109.36, 106.20, 20.91. MS (ESI): m/z 302 [M + H]+. [5-Amino-1-(1H-indol-5-yl)pyrazol-4-yl]-(1H-indol-2-yl)methanone (10). Prepared by following the general procedure B. 1 H NMR (400 MHz, DMSO-d6) δ: 11.70 (1H, s), 11.40 (1H, s), 8.30 (1H, s), 7.71−7.69 (2H, m), 7.57 (1H, d, J = 8.8 Hz), 7.50−7.46 (3H, m), 7.25−7.23 (2H, m), 7.08 (1H, t, J = 7.1 Hz), 6.93 (2H, s), 6.56 (1H, s); 13C NMR (100 MHz, DMSO-d6) δ: 177.96, 151.06, 139.49, 136.72, 135.51, 135.07, 129.01, 127.52, 127.39, 127.03, 124.22, 122.14, 119.82, 118.11, 116.24, 112.36, 112.04, 106.28, 102.92, 101.73. HRMS (ESI): m/z calculated for C20H15N5O + H+ [M + H]+: 342.1355, found: 342.136. [2-Methyl-1-(p-tolylsulfonyl)indol-5-yl]hydrazine hydrochloride (11c). Prepared by following the general procedure D. 1 H NMR (400 MHz, DMSO-d6) δ: 10.06 (3H, s), 8.17 (1H, s), 7.92 (1H, d, J = 8.8 Hz), 7.71 (2H, d, J = 8.4 Hz), 7.36 (2H, d, J = 8.8 Hz), 7.02 (1H, d, J = 2.2 Hz), 6.94 (1H, dd, J = 9.0, 2.4 Hz), 6.53 (1H, d, J = 0.9 Hz), 2.57 (3H, d, J = 0.9 Hz), 2.32 (3H, s).; 13C NMR (100 MHz, DMSO-d6) δ: 145.15, 141.99, 138.05, 134.73, 131.37, 130.14 (2C), 129.87 (2C), 126.07, 114.38, 112.41, 109.78, 105.12, 20.90, 15.28. MS (ESI): m/z 316 [M + H]+. [5-Amino-1-(2-methyl-1H-indol-5-yl)pyrazol-4-yl]-(1H-indol-2-yl)methanone (11). Prepared by following the general procedure B. 10596
DOI: 10.1021/acs.jmedchem.6b01156 J. Med. Chem. 2016, 59, 10586−10600
Journal of Medicinal Chemistry
Article
H NMR (400 MHz, DMSO-d6) δ: 11.70 (1H, s), 11.22 (1H, s), 8.28 (1H, s), 7.70 (1H, d, J = 7.7 Hz), 7.55 (1H, s), 7.49−7.43 (3H, m), 7.25 (1H, t, J = 7.1 Hz), 7.15−7.06 (2H, m), 6.89 (2H, s), 6.25 (1H, s), 2.43 (3H, s); 13C NMR (100 MHz, DMSO-d6) δ: 177.94, 150.97, 139.40, 137.47, 136.71, 135.52, 135.28, 128.82, 128.57, 127.39, 124.22, 122.13, 119.81, 116.95, 115.13, 112.36, 110.97, 106.27, 102.92, 99.73, 13.35. HRMS (ESI): m/z calculated for C21H17N5O + H+ [M + H]+: 356.1511, found: 356.1518. 5-Hydrazino-1,3-dihydrobenzimidazol-2-one hydrochloride (12a). Prepared by following the general procedure C. 1 H NMR (400 MHz, DMSO-d6) δ: 10.66 (1H, s), 10.49 (1H, s), 10.06 (4H, s), 6.84 (1H, d, J = 8.4 Hz), 6.75 (1H, d, J = 1.3 Hz), 6.66 (1H, dd, J = 8.4, 1.8 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 155.93, 140.00, 130.64, 125.65, 109.10, 108.98, 98.05. MS (ESI): m/z 165 [M + H]+. 5-[5-Amino-4-(1H-indole-2-carbonyl)pyrazol-1-yl]-1,3-dihydrobenzimidazol-2-one (12). Prepared by following the general procedure B. 1 H NMR (400 MHz, DMSO-d6) δ: 11.71 (1H, s), 10.97 (2H, br s), 8.30 (1H, s), 7.70 (1H, d, J = 7.7 Hz), 7.49 (1H, d, J = 8.2 Hz), 7.45 (1H, d, J = 1.1 Hz), 7.25 (1H, td, J = 7.7, 1.1 Hz), 7.12−7.09 (4H, m), 7.01 (2H, s); 13C NMR (100 MHz, DMSO-d6) δ: 177.96, 155.43, 151.02, 139.79, 136.74, 135.43, 130.29, 130.07, 129.35, 127.37, 124.27, 122.15, 119.84, 116.97, 112.38, 108.59, 106.38, 105.20, 102.95. HRMS (ESI): m/z calculated for C19H14N6O2 + H+ [M + H]+: 359.1257, found: 359.1251. [5-Amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-[5(morpholinomethyl)-1H-indol-2-yl]methanone (13). Prepared by following the general procedure B. 1 H NMR (400 MHz, DMSO-d6) δ: 11.68 (1H, s), 8.31 (1H, s), 7.61− 7.59 (3H, m), 7.44−7.42 (2H, m), 7.30 (1H, dd, J = 8.4, 2.2 Hz), 7.22 (1H, dd, J = 8.4, 1.3 Hz), 7.01 (2H, s), 3.58 (4H, t, J = 4.4 Hz), 3.53 (2H, s), 2.54 (3H, s), 2.37 (4H, s); 13C NMR (100 MHz, DMSO-d6) δ: 177.96, 163.72, 153.17, 151.07 (2C), 139.79, 136.12, 135.67, 132.46, 130.94, 129.04, 128.37, 127.26, 126.01, 122.27, 118.09, 112.12, 106.29, 102.99, 66.12 (2C), 62.91, 53.08 (2C), 14.64. HRMS (ESI): m/z calculated for C25H25N7O2 + H+ [M + H]+: 456.2148, found: 456.2141. [5-Amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-[6(morpholinomethyl)-1H-indol-2-yl]methanone (14). Prepared by following the general procedure B. 1 H NMR (400 MHz, DMSO-d6) δ: 12.48 (1H, s), 11.66 (1H, s), 8.31 (1H, s), 7.63−7.61 (3H, m), 7.43 (1H, d, J = 1.6 Hz), 7.42 (1H, s), 7.30 (1H, dd, J = 8.2, 1.6 Hz), 7.06 (1H, dd, J = 8.2, 1.6 Hz), 7.00 (2H, s), 3.58 (4H, t, J = 4.4 Hz), 3.35 (2H, s), 2.54 (3H, s), 2.38 (4H, br s); 13C NMR (100 MHz, DMSO-d6) δ: 178.04, 153.25, 151.17, 143.60, 139.85, 136.98, 135.63, 134.35, 134.04, 131.14, 126.70, 121.91, 121.65, 118.55, 117.93, 112.51, 111.18, 106.47, 103.08, 66.24 (2C), 63.16, 53.27 (2C), 14.72. HRMS (ESI): m/z calculated for C25H25N7O2 + H+ [M + H]+: 456.2148, found: 456.2138. [5-Amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-[6[(4,4-difluoro-1-piperidyl)methyl]-1H-indol-2-yl]methanone (15). Prepared by following the general procedure B. 1 H NMR (400 MHz, DMSO-d6) δ: 12.48 (1H, s), 11.67 (1H, s), 8.31 (1H, s), 7.63−7.60 (3H, m), 7.43 (1H, d, J = 2.2 Hz), 7.42 (1H, s), 7.30 (1H, d, J = 8.2 Hz), 7.06 (1H, dd, J = 8.2, 1.1 Hz), 6.99 (2H, s), 3.63 (2H, s), 2.54 (3H, s), 2.51−2.49 (4H, m), 2.01−1.91 (4H, m); 13C NMR (100 MHz, DMSO-d6) δ: 178.04, 153.25, 151.17, 143.59, 139.86, 136.98, 135.68, 134.65, 134.41, 130.97, 126.74, 122.88 (t, J = 240.7 Hz), 121.96, 121.53, 118.54, 117.86, 112.40, 111.20, 106.47, 103.09, 61.61, 49.33 (2C, t, J = 5.0 Hz), 33.50 (2C, t, J = 22.4 Hz), 14.72. HRMS (ESI): m/z calculated for C26H25F2N7O + H+ [M + H]+: 490.2167, found: 490.2158. 3-[6-Iodo-1-(p-tolylsulfonyl)indol-2-yl]-3-oxo-propanenitrile (16a). Ethyl 6-iodo-1-(p-tolylsulfonyl)indole-2-carboxylate (14a, 469 mg, 1.00 mmol) was dissolved in THF (4.7 mL), and then MeCN (0.157 mL, 3.00 mmol) was added to the solution. LHMDS (1.3 M in THF, 3.86 mL, 5.00 mmol) was added to the mixture at −78 °C over 10 min, and then the mixture was stirred at −78 °C for 1 h under nitrogen atmosphere. 10% AcOH (13 mL) was added at −78 °C, and then the mixture was warmed up to 25 °C. The volatile was removed under 1
reduced pressure to dryness, and then MeOH (5 mL) was added to the suspension. The suspension was stirred at 25 °C for 1 h, and then H2O (5 mL) was added to the suspension at 25 °C. The precipitate was collected by filtration, and then the cake was washed with MeOH/H2O (1/2, 2 mL). The powder was dried under reduced pressure. The powder was dissolved in DMSO (5 mL), and then the solution was subjected to a C18 MPLC column. The column was eluted with 0.1% formic acid-H2O-MeCN by a linear gradient. The fractions containing the target were collected and concentrated under reduced pressure to give the titled compound as a light brown powder (397 mg, 86 mg). 1 H NMR (400 MHz, CDCl3) δ: 8.52 (1H, s), 7.67 (2H, d, J = 8.8 Hz), 7.62 (1H, dd, J = 8.2, 1.6 Hz), 7.29 (1H, d, J = 8.2 Hz), 7.24 (2H, d, J = 8.2 Hz), 7.13 (1H, s), 4.13 (2H, s), 2.36 (3H, s); 13C NMR (100 MHz, CDCl3) δ: 181.94, 146.04, 139.49, 136.94, 134.34, 133.10, 129.83 (2C), 127.81, 127.43 (2C), 124.95, 124.41, 119.30, 113.34, 93.78, 32.41, 21.69. HRMS (ESI): m/z calculated for C18H13IN2O3S − H+ [M − H]−: 462.9613, found: 462.9608. (E)-3-(Dimethylamino)-2-[6-iodo-1-(p-tolylsulfonyl)indole-2carbonyl]prop-2-enenitrile (16b). 3-[6-Iodo-1-(p-tolylsulfonyl)indol2-yl]-3-oxo-propanenitrile (16a, 338 mg, 0.728 mmol) was dissolved in THF (3.3 mL), and then DMF-DMA (0.107 mL, 0.801 mmol) was added to the solution at 25 °C. The mixture was stirred at 25 °C for 1 h under nitrogen atmosphere, and then n-hexane (2 mL) was added to the solution. The precipitate was collected by filtration, and then the cake was washed with n-hexane (1 mL). The powder was dried under reduced pressure to give the titled compound as a yellow amorphous (359 mg, 95%). 1 H NMR (400 MHz, DMSO-d6) δ: 8.25 (1H, s), 7.97−7.95 (3H, m), 7.63 (1H, dd, J = 8.5, 1.4 Hz), 7.48 (1H, d, J = 8.2 Hz), 7.45 (2H, d, J = 8.2 Hz), 7.03 (1H, s), 3.40 (3H, s), 3.31 (3H, s), 2.36 (3H, s); 13C NMR (100 MHz, DMSO-d6) δ: 181.24, 158.63, 145.74, 137.37, 136.67, 134.28, 132.67, 130.05 (2C), 128.01, 127.22 (2C), 127.08, 124.29, 122.35, 118.89, 112.14, 91.00, 47.67, 38.92, 21.08. HRMS (ESI): m/z calculated for C21H18IN3O3S + H+ [M + H]+: 520.0192, found: 520.0197. [5-Amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-[6iodo-1-(p-tolylsulfonyl)indol-2-yl]methanone (16c). (E)-3-(Dimethylamino)-2-[6-iodo-1-(p-tolylsulfonyl)indole-2-carbonyl]prop-2-enenitrile (16b, 331 mg, 0.637 mmol) was dissolved in NMP (3.3 mL), and then 4-methylmorpholine (0.210 mL, 1.91 mmol) was added to the solution. Nitrogen gas was bubbled into the solution, and then (2methyl-3H-benzimidazol-5-yl)hydrazine dihydrochloride (300 mg, 1.28 mmol) was added to the mixture. The mixture was stirred at 100 °C for 30 min under nitrogen atmosphere. The mixture was dissolved in DMSO (3 mL), and then the solution was subjected to a C18 MPLC column. The column was eluted with 0.1% formic acid-H2O-MeCN by a linear gradient. The fractions containing the target was collected and concentrated under reduced pressure to give the titled compound as a light brown amorphous (331 mg, 82%). 1 H NMR (400 MHz, DMSO-d6) δ: 12.61 (1H, s), 8.31 (1H, s), 8.01 (2H, d, J = 8.2 Hz), 7.77 (1H, s), 7.67 (1H, dd, J = 8.2, 1.6 Hz), 7.63− 7.62 (2H, m), 7.52 (1H, d, J = 8.2 Hz), 7.48 (2H, d, J = 8.2 Hz), 7.30 (1H, dd, J = 8.2, 2.2 Hz), 7.25 (1H, s), 7.06 (2H, s), 2.54 (3H, s), 2.38 (3H, s); 13C NMR (100 MHz, DMSO-d6) δ: 178.10, 163.05, 158.83, 153.31, 150.65, 145.64, 141.18, 138.22, 137.44, 134.52, 132.83, 130.82, 130.06 (2C), 129.18, 127.99, 127.26 (2C), 126.40, 124.58, 122.77, 118.18, 113.82, 105.07, 91.50, 21.12, 14.69. HRMS (ESI): m/z calculated for C27H21IN6O3S + H+ [M + H]+: 637.0519, found: 637.0517. [5-Amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-(6methylsulfonyl-1H-indol-2-yl)methanone (16). [5-Amino-1-(2-methyl-3H-benzimidazol-5-yl)pyrazol-4-yl]-[6-iodo-1-(p-tolylsulfonyl)indol-2-yl]methanone (16c, 63.6 mg, 0.100 mmol), sodium methansulfinate (20.4 mg, 0.22 mmol), (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (2.84 mg, 0.0200 mmol), and CuI (1.91 mg, 0.0010 mmol) were placed in a round-bottomed flask, and then DMSO (0.4 mL) was added to the mixture. The flask was evacuated and then backfilled with nitrogen. The mixture was stirred at 120 °C for 18 h under nitrogen atmosphere. Five M NaOH (0.400 mL) was added to the mixture, and then the mixture was stirred at 80 °C for 1 h under nitrogen 10597
DOI: 10.1021/acs.jmedchem.6b01156 J. Med. Chem. 2016, 59, 10586−10600
Journal of Medicinal Chemistry
Article
Calculation of pKa Values. The pKa values were calculated in a prediction software (Moka 2.0) developed by Molecular Discovery. http://www.moldiscovery.com/. Caco2 Assay. Caco-2 cells were grown on culture flasks in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10%FBS, 1% nonessential amino acids, 1% L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cells were harvested with trypsinEDTA and seeded onto polyester filters (1.0 μm pores, 0.14 cm2 growth area) inside Transwell cell culture chambers (Costar, Cambridge, MA) at a density of 2.8 × 104 cells/filter. The culture medium was changed every other day. The cells were cultured for 2 weeks, and the Caco-2 monolayers were utilized for the following assay. Apical to basolateral permeability assays were carried out with apical and basolateral Hanks’ Balanced Salt Solution (HBSS) adjusted at 6.0 and 7.4, respectively (HBSS with 4% bovine serum albumin (BSA) in basolateral compartment). The donor concentration of test compound was set at 10 μmol/L, and basolateral compartment was sampled at 120 min. The compound concentration in basolateral compartment was measured by LC-MS/MS. The apparent permeability coefficient (Papp) value was calculated from the following equation: Papp (cm/s) = Q/t/A/C0 where Q, t, A, and C0 represent permeation amount of the compound (nmol), incubation time (s), surface area of cell monolayer (cm2) and initial concentration of test compound (μmol/L). Intrinsic Clearance (CLint) Determination in Microsomes. Each compound (1 μM) was incubated with human (or mouse) liver microsome (0.5 mg protein/mL) in 50 mM phosphate buffer (pH 7.4) containing 1 mM NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate) at 37 °C for 30 min. After the enzyme reaction was terminated with the addition of a 3-fold volume of acetonitrile, the reaction mixture was centrifuged at 1500 rpm for 10 min. The resultant supernatant was used as a test sample to measure the stability in human (or mouse) liver microsome by quantitating the compound in the sample using LC/MS. Solubility Assay. An aliquot of 50 μL of 4 mM or 1 mM sample in DMSO was freeze-dried to remove DMSO. To the resulting residue was added 50 μL of FaSSIF (pH = 6.5), which was then irradiated ultrasonically for 10 min, shaken for 2 h, centrifuged for 10 min (3000 rpm), and filtered by Whatman Unifilter. Concentration of the filtrate was analyzed by HPLC-UV based on the calibration curve of each sample. Composition of FaSSIF: Sodium taurocholate (1.61 g), lecithin (0.59 g), KH2PO4 (3.9 g), KCl (7.7 g), and NaOH (pH 6.5) per 1 L. Protein Kinase Enzyme Assay. The measurement of inhibitory activities against kinases and cell proliferation was described previouly.24 Cell Proliferation Assay. Cell lines were obtained from American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Health Science Research Resources Bank (HSRRB), Asterand Inc., and Health Protection Agency Culture Collections (HPACC). All cell lines were authenticated by the cell banks with cytogenic analysis, DNA profiling, or growth properties and were propagated for
