Identification and Optimization of Novel Cathepsin C Inhibitors Derived

Article Cite This: J. Med. Chem. 2019, 62, 5901−5919

pubs.acs.org/jmc

Identification and Optimization of Novel Cathepsin C Inhibitors Derived from EGFR Inhibitors Weijie Hou,† Huan Sun,† Yongfen Ma, Chunyan Liu, and Zhiyuan Zhang* National Institute of Biological Sciences (NIBS), 7 Science Park Road, ZGC Life Science Park, Beijing 102206, China

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S Supporting Information *

ABSTRACT: In the course of developing the biochemistry to chemistry activity-based protein profiling (BTC-ABPP) method, we herein unexpectedly discovered that the epidermal growth factor receptor irreversible inhibitor WZ4002 also functioned as a low micromolar inhibitor of cathepsin C (CatC), a promising target for the treatment of numerous inflammatory and autoimmune diseases. Building on from this discovery, and following structure−activity relationship investigations guided by computational modeling, a novel series of pyridine scaffold compounds were developed as irreversible CatC inhibitors, further culminated in identifying a highly potent and selective inhibitor 22, which displays good metabolic stability and oral bioavailability. In vivo studies revealed that compound 22 clearly displays the ability to inhibit CatC, consequently leading to efficient inhibition of downstream neutrophil serine proteases in both bone marrow and blood. The overall excellent profile of compound 22 made it an interesting candidate for further preclinical investigation.

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INTRODUCTION Cathepsin C (CatC), also known as dipeptidyl peptidase 1, is an important lysosomal cysteine protease1 that can activate granule-associated proinflammatory serine proteases, including neutrophil elastase (NE), cathepsin G (CatG), and proteinase 3 (PR3). Upon such activation, these neutrophil serine proteases (NSPs) are secreted into the extracellular space, where their hydrolysis activity protects host cells from invading pathogens.2,3 However, in certain cases, excessive recruitment of neutrophils can increase the secretion of NSPs to such an extent that they become destructive even after the clearance of pathogens have been completed, thereby driving the pathogenesis of several disorders. Specifically, NSPs participate in the progression of a variety of chronic inflammatory diseases, such as rheumatoid arthritis,4,5 sepsis,6 and antineutrophil cytoplasmic antibody-associated necrotizing crescentic glomerulonephritis.7 Further, chronic respiratory disorders, including chronic obstructive pulmonary disease8 (COPD; the fourth leading cause of death globally in 2010 and rose to the third position in 20169), bronchiectasis, and cystic fibrosis,10 are also now understood to be NSPs related. Owing to its function in activating NSPs, CatC is now viewed as a promising therapeutic target for a variety of NSPs-related diseases.11 Biological studies have shown that CatC knock-out mice and CatC inhibitor-treated wild-type (WT) mice, demonstrate protection from smoke-induced pulmonary inflammation and associated alveolar destruction.12 These observations support the hypothesis that CatC is viewed as a therapeutic target against COPD. Human CatC is known to occur as a homotetrameric complex comprising four identical subunits, each of which © 2019 American Chemical Society

contains a light chain, a heavy chain, and an exclusion domain (unique to CatC amongst the cathepsin family proteases).1 As a peptidase, CatC removes dipeptide moieties from the Nterminal of the substrates.13 Studies have shown that the aspartic acid at position 1 of the N-terminal exclusion domain keeps the exopeptidase activity of CatC via its interaction with α-amino groups of peptide substrates; moreover, Cys234 has been demonstrated as a key catalytic residue in the active site.14 A number of CatC inhibitors have been developed in recent decades; most of these are peptide mimics derived from known preferred dipeptide substrates of CatC, including irreversible inhibitors such as diazoketones,15 vinyl sulfone,16,17 O-acylhydroxamates,17 as well as reversible covalent inhibitors with nitrile groups18−21 as warheads. In the development of CatC inhibitors, metabolic stability given by their peptidic and electrophilic nature may be a serious issue.14,22 To date, only two drug candidates entered clinical trials: GSK-279366023−25 (terminated in phase I) and INS1007/AZD798626,27 (currently in phase II) (Figure 1), and their chemotypes are dipeptide derived. In the course of a biochemistry to chemistry activity-based protein profiling (BTC-ABPP) study using an activity-based probe derived from an irreversible epidermal growth factor receptor (EGFR) inhibitor (canertinib) to study off-target effects, we identified a probe-modified peptide from CatC, thus indicating binding to this protease potentially via the catalytic cysteine.28 Subsequently, results from both enzymatic assay and Received: April 13, 2019 Published: May 30, 2019 5901

DOI: 10.1021/acs.jmedchem.9b00631 J. Med. Chem. 2019, 62, 5901−5919

Journal of Medicinal Chemistry

Article

obtained in a similar manner, using an appropriate substituted pyridine in step (a) of Scheme 2. The synthetic routes of compounds 14, 17−20, and 21−26 are illustrated in Schemes 3 and 4 and start from 3-bromo-2,6dichloropyridine. For preparation of compounds 14 and 17−20, appropriate phenols were regioselectively introduced to the C-2 position of 3-bromo-2,6-dichloropyridine to afford the intermediate 47a−e. Amination of C-3 with pyrrolidine and subsequent amination of C-6 with 2-methoxy-4-(4-methylpiperazin-1-yl) aniline was accomplished through palladiumcatalyzed Buchwald−Hartwig coupling. The nitro group in 49a−e was reduced by PtO2/H2. Acylation of 50a−e by acryloyl chloride gave rise to compounds 14 and 17−20. The analogues of 21−26 and 27−31 were obtained in a similar manner, using the appropriate amines in step (a) of Scheme 4 and appropriate substituted anilines in step (a) of Scheme 5, respectively. Compounds 32 and 33 were synthesized as shown in Scheme 6. Hit Compound Development. In our previous work, CatC was identified as one of the off-targets of canertinib; the CatC active site residue Cys234, known to be essential for its peptidase, was modified by our probe.28 Most irreversible CatC inhibitors reported to date covalently bind to Cys234 via their electrophilic warheads.14,22 We therefore asked whether other known irreversible inhibitors of EGFR could likewise inhibit CatC activity. As shown in Figure 2, canertinib and WZ4002 displayed better inhibition of CatC (IC50 = 2.7 and 2.1 μM, respectively) than the other three inhibitors (IC50 > 10 μM). Both canertinib and WZ4002 were selected as the initial hits for further optimization; the rest of the paper reports our SAR development of WZ4002. Understanding the possible binding mode of WZ4002 in the active site of CatC could provide useful guidance for designing analogues to conduct structure−activity relationship (SAR) studies. Docking studies with WZ4002 based on the CatC/ AZD5248 complex crystal structure (Protein Data Bank (PDB) ID: 4CDE) identified two inhibitor−acceptor interactions as

Figure 1. Chemical structures of GSK-2793660 and INS1007/ AZD7986.

cell assays confirmed that canertinib was indeed a CatC inhibitor, and a small screen of other irreversible EGFR inhibitors identified WZ4002 as an additional CatC inhibitor. Docking studies informed our proposal of a plausible binding mode for WZ4002 within the CatC active site, and subsequent rational design efforts based on this mode, alongside structure− activity relationship (SAR) studies, ultimately enabled the identification of a low nanomolar inhibitor. Extensive in vitro/in vivo assays demonstrate that the candidate compound 22 from our study is highly metabolically stable, exhibits potent CatC inhibitory effects, and results in the anticipated inhibition on the activation of downstream NSPs in mice.

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RESULTS AND DISCUSSION Chemistry. The preparation of this series compounds was achieved using the synthetic routes illustrated in scheme 1−6. The synthetic routes of compounds 1−8 started from 3nitrophenol. 3-Nitrophenol was regioselectively introduced to the C-2 position of 2,3,6-trichloropyridine to afford the intermediate 36. Amination of C-6 with different substituted anilines was accomplished through a palladium-catalyzed Buchwald−Hartwig coupling. The nitro group in 38a−f was reduced by iron power/ammonium chloride. Acylation of 39a−f by acryloyl chloride gave rise to compounds 1, 2, 4, and 40d−f. The subsequent step of tert-butoxycarbonyl (Boc) deprotection afforded compounds 3−6. The analogues of 10 and 11 were Scheme 1. Synthesis of Compounds 1−8a

Reagents and conditions: (a) Cs2CO3, dimethylformamide (DMF), 110 °C; (b) Cs2CO3, (±)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP), Pd(OAc)2, dioxane, microwave, 130 °C, 1 h; (c) Fe, NH4Cl, EtOH/tetrahydrofuran (THF)/H2O, 80 °C; (d) acryloyl chloride, triethylamine (TEA), dichloromethane (DCM), 0 °C, 1 h; (e) trifluoroacetic acid (TFA), DCM, room temperature (RT), 2 h; (f) (bromomethyl)cyclopropane or (bromomethyl)cyclopentane, K2CO3, 70 °C, 4 h.

a

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Scheme 2. Synthesis of Compounds 10 and 11a

a Reagents and conditions: (a) Cs2CO3, DMF, 110 °C; (b) 2-methoxy-4-(4-methylpiperazin-1-yl)aniline, Cs2CO3, BINAP, Pd(OAc)2, dioxane, microwave, 130 °C, 1 h; (c) Fe, NH4Cl, EtOH/THF/H2O, 80 °C; (d) acryloyl chloride, TEA, DCM, 0 °C, 1 h.

Scheme 3. Synthesis of Compounds 14 and 17−20a

Reagents and conditions: (a) Cs2CO3, DMF, 110 °C; (b) pyrrolidine, Cs2CO3, BINAP, Pd2(dba)3, toluene, 110 °C; (c) 2-methoxy-4-(4methylpiperazin-1-yl)aniline, Cs2CO3, BINAP, Pd(OAc)2, dioxane, microwave, 130 °C, 1 h; (d) PtO2, H2, MeOH/THF, RT, overnight; (e) acryloyl chloride, TEA, DCM, 0 °C, 1 h.

a

Scheme 4. Synthesis of Compounds 21−26a

Reagents and conditions: (a) Cs2CO3, BINAP, Pd2(dba)3, toluene, 110 °C or Pd(PPh3)4, Na2CO3 (2 M), 1,2-dimethoxyethane, 80 °C; (b) 2methoxy-4-(4-methylpiperazin-1-yl)aniline, Cs2CO3, BINAP, Pd(OAc)2, dioxane, microwave, 130 °C, 1 h; (c) PtO2, H2, MeOH/THF, RT, overnight; (d) acryloyl chloride, TEA, DCM, 0 °C, 1 h; (e) tetra-n-butylammonium fluoride (TBAF), THF, 0 °C to RT, 2 h. a

potentially related to our observed effects: a potential covalent attachment between the acrylamide fragment of WZ4002 and Cys234 in the CatC active site and salt-bridge interaction

between the piperazine N-4 of WZ4002 and Asp1 in the exclusion domain (Figure 3). Its predicted binding mode suggested that improvements in potency could perhaps be 5903



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a Reagents and conditions: (a) Cs2CO3, BINAP, Pd(OAc)2, dioxane, microwave, 130 °C, 1 h; (b) PtO2, H2, MeOH/THF, RT, overnight; (c) acryloyl chloride, TEA, DCM, 0 °C, 1 h.


Reagents and conditions: (a) Cs2CO3, DMF, 110 °C; (b) Fe, NH4Cl, EtOH/H2O, 80 °C; (c) (Boc)2O, 4-dimethylaminopyridine (DMAP), TEA, THF, RT, overnight; (d) pyrrolidine or piperidine, Cs2CO3, BINAP, tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3), toluene, 110 °C; (e) 2-methoxy-4-(4-methylpiperazin-1-yl)aniline, Cs2CO3, BINAP, Pd(OAc)2, dioxane, microwave, 130 °C, 1 h; (f) TFA, DCM, RT; (g) acryloyl chloride, TEA, DCM, 0 °C, 1 h.

a

Figure 2. Chemical structures and CatC enzymatic inhibitory activity of selected irreversible inhibitors of EGFR and AZD524829 (a known CatC inhibitor as a tool compound).

Using the docking study data as a guide, we aimed to develop a highly potent CatC inhibitor, while depleting the activity against EGFR. We first explored the possibility of eliminating WZ4002’s

achieved via modification of the pyrimidine at C-5 position and/ or the phenyl at the C-4 position. 5904



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with the piperazine analogue 2. Piperazine compound 3 maintained its inhibitory activity, while the salt-bridge interaction between piperazine and Asp1 was undisrupted (according to the proposed binding mode); however, morpholine compound 4 showed a significant loss in potency (IC50 > 10 μM), which could be explained by the absence of salt-bridge interaction. Compounds 5 and 6, with flexible amine chains at different positions of the phenyl ring, were synthesized and tested, and 4-fold enzymatic activity reduction was observed with both compounds as compared with compound 3. On adding a larger alkyl group on N-4 with the hope of improving potency, cyclopropylmethyl compound 7 maintained the potency, whereas the cyclopentylmethyl group of compound 8 was apparently too bulky to be tolerated by the surrounding environment of CatC, leading to a more than 10-fold loss in potency. Although compound 2 was slightly more potent than 1, its liver microsome stability in mouse and rat was poorer (Table S1, Supporting Information). Thus, we decided to continue to use the C-6 2-methoxy-4-(4-methylpiperazin-1-yl)-aniline moiety in further SAR investigation. Reviewing our proposed docking mode, the unfilled space near the C-3 position at pyridine of compound 1 appeared attractive as a potential site for generating additional desired binding interactions. We therefore conducted SAR studies for the modification of the C-3 position: the results are shown in Table 2. Replacing the −Cl with −H (compound 9) or cyano (compound 11) at pyridine C-3 position had little impact on IC50 values. Trifluoromethyl compound 10 and dimethylamino compound 13 conferred a slight loss (2-fold) of potency. However, replacement with a larger pyrrolidine group as in compound 14 resulted in a 5-fold increase in potency. Another two cyclic amine compounds were also synthesized: 15 with a piperdinyl moiety and 16 with an azepanyl moiety. These two compounds showed similar activity to compound 14. Compounds 14−16 strongly suggested that the increased potency likely results from additional hydrophobic interaction between the cyclic amines and the nearby CatC Trp405 residue. These encouraging results further supported our proposed binding mode and led us to suspect that fine tuning of the phenyl ring bearing the acrylamide could confer yet additional improvements in potency. As per results shown in Table 3, when a methyl group was introduced to the C-2 position, compound 17 lost almost all CatC inhibitory activity (IC50 > 10 μM). However, a 4-fold improvement in potency was observed when a methyl group (19) was incorporated into C-4 position of 14. These results implied that the orientation of acrylamide relative to Cys234 made a significant influence on potency. In addition, the methyl group of compound 19 established hydrophobic interaction with nearby residues. Compound 18, bearing a methyl group at the C-6 position, was 3-fold less potent than 14, probably due to the unfavorable angle between phenyl-O-pyridine. Having successfully improved the potency from a low micromolar initial hit WZ4002 to low nanomolar compounds in enzymatic and cellular assays, we next turned our attention to the metabolic stability issue. As a starting point, compound 19 suffered from poor stability in liver microsomes (Table 4a) and in plasma (data not shown). The liver microsome stability data (Table S1, Supporting Information) suggested that pyridine C-3 substituents apparently strongly influenced metabolic stability. To address this issue, we conducted optimization of substitutions at pyridine C-3 position.

Figure 3. Putative binding mode of WZ4002 (orange) within the active site of CatC (PDB ID: 4CDE).

kinase binding capability by focusing on the known “hinge binding” regions of the inhibitor: a hydrogen bond between pyrimidine N1 and EGFR Met793 facilitates the crucial hinge binding.30 Notably, according to our proposed binding mode, however, WZ4002’s pyrimidine N1 does not contribute to ligand interactions with CatC, suggesting that replacing N1 with a carbon may eliminate its EGFR inhibitory effects without affecting its binding affinity for the CatC active site (Figure 3). We synthesized compound 1 to test this hypothesis and observed that the EGFR IC50 of compound 1 was reduced by more than 5000-fold (IC50 > 10 μM) compared to that of WZ4002 (IC50 = 1.7 nM) (Figure 4) whereas its inhibitory effect

Figure 4. Hit validation.

against CatC was slightly increased compared to that of WZ4002 (IC50 of 810 nM vs 2.1 μM in the enzymatic assay and IC50 = 538 nM in the U937 cell). We also tested the metabolic stability of compound 1: their CLint (μL/(min mg protein)) in liver microsomes (human, rat, and mouse) were 5.4, 39.7, and 26.3, respectively (Table S1, Supporting Information), which strongly encouraged our additional efforts to further develop this novel series of compounds. Hit To Lead. First, 4-methyl-piperazine compound 2, removal of the methoxy group from the phenyl ring, showed slightly better potency than 1 (Table 1). In general, a salt-bridge provides strong interactions between a ligand and its receptor so we made efforts to identify potentially superior salt-bridge interaction groups. Several analogues with various aniline substitutions at pyridine C-6 position were prepared to compare 5905



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Table 1. SAR of Substitutions at the Pyridine Core C-6 Position

a

Human recombinant CatC isolated enzyme assay. Mean values from a minimum of two experiments.

Table 2. SAR of Substitutions at the Pyridine Core C-3 Position

a

Human recombinant CatC isolated enzyme assay. Mean values from a minimum of two experiments.

First, we synthesized compound 21, in which an additional hydroxyl was introduced to the meta position of compound 19. Compound 21 was rapidly metabolized in all three tested liver microsomes systems; note that it had slightly increased potency over compound 19. We then replaced the pyrrolidine with a piperidine to obtain compound 22: excitingly, compound 22 exhibited an improvement in liver microsome stability (human/ rat/mouse liver microsomes CLint = 1.8, 6.3, and 10.8 μL/(min mg), Table 4a) and was more potent than compound 19 in both enzymatic and cellular assays. Next, adding a hydroxyl group to the meta position of the piperidine as in compound 23 led to a slight improvement in metabolic stability in mouse liver

microsomes but reduced stability in both human and rat liver microsomes. When the piperidine was replaced by morpholine as in compound 24 or 4-methylpiperazine as in compound 25, the enzymatic activity decreased about 3-fold compared with compound 22; further, although the stability of compound 25 was maintained, compound 24 showed was less stable in rat and mouse liver microsomes. A planar structure phenyl group was also introduced to the pyridine C-3 position (compound 26); however, this led to a 9-fold loss in inhibitory activity. The effect of pyridine C-6 substituents on the metabolic stability was also evaluated in all three liver microsome systems (Table 4b). Removing the methoxy group from the phenyl 5906



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Table 3. Phenyl Ring Substituent Modifications

compd 14 17 18 19 20

R3 =

CatC Enz (IC50, nM)a

H 2-Me 6-Me 4-Me 4-OMe

175 ± 77 >10 000 505 ± 35 44 ± 3.8 139.8 ± 41.6

THP1 cell (IC50, nM)b d

NT NT NT 12.3 ± 7.3 21.3 ± 5.9

U937 cell (IC50, nM)c NT NT NT 40.8 ± 16.9 70 ± 3.0

a Human recombinant CatC isolated enzyme assay. Mean values from a minimum of two experiments. bCatC cell assay using THP1 cell line. Mean values from a minimum of two experiments. cCatC cell assay using U937 cell line. Mean values from a minimum of two experiments. dNT = not test.

tested compounds, no inhibition of EGFR WT kinase was observed (when tested up to 10 μM). To evaluate the cathepsin selectivity for our compounds, assays with compounds 19, 22, and 24 were conducted against human recombinant enzymes CatB, CatL, and CatS. These three compounds exhibited generally high cathepsin selectivity (Table 5). We also tested the inhibitory activity against cytochrome P450s using five recombinant human cytochrome P450 isoforms (1A2, 2D6, 3A4, 2C9, and 2C19) (Table S3, Supporting Information). For compounds 22 and 24, the IC50 values were higher than 10 μM, suggesting a low potential involved in drug−drug interactions. In contrast, compound 19 showed weak inhibition against 1A2 and 2C19, with IC50 values of 8.75 and 9.91 μM, respectively; the other three isoforms had IC50 values over 10 μM. Compound 22 was then processed into mouse in vivo pharmacokinetic (PK) studies, which demonstrated desirable results upon oral (PO) delivery, with a half-life of 3.06 h and bioavailability of 22.3% (Table 6). Tissue distribution studies showed that lung had the highest compound concentration and kidney had the second highest compound concentration (Figure S2, Supporting Information). On the basis of its overall favorable profiles, compound 22 was thusly selected as a potential drug candidate for further in vivo profiling. In Vivo Efficacy of Compound 22. In chronic inflammatory diseases, CatC has been linked with activation of neutrophil elastase (NE), cathepsin G (CatG) and proteinase 3 (PR3).11 These three serine proteases are major components of neutrophil granules, which are secreted and which cause both inflammation and destruction of extracellular matrices. Specifically, it has been found that CatC functions to peptidolytically remove N-terminal dipeptides from pro-NSPs during the promyelocyte stage of neutrophil maturation in bone marrow.31,32 Then, the activated NSPs (packaged in granules) in mature neutrophils are eventually released from bone marrow into the circulating blood. It has been reported that a continuous very high fractional inhibition of the CatC activity is required to effectively prevent the activation of NSPs in vivo.33,34 In vitro, 22 showed concentration-dependent inhibition of CatC in both THP1 and U937 cell-based assays, with IC50 values of 2 and 3 nM, respectively. Subsequently, we tested whether the treatment of 22 could inhibit the activity of CatC and NSPs in vivo. First, the effect of oral administration of 22 on CatC inhibition was examined in mice. Within 15 min of oral

(compound 27) or replacing the methoxyphenyl with a methoxypyridine (compound 28) caused poorer stability than 22 in liver microsomes of all three species. Compound 29, with an acetyl group at its N4-position that disrupts the salt-bridge interaction with CatC, exhibited only a moderate decrease in potency (about 8-fold less potent than 22). The change in potency might be explained by a likely hydrogen bond occurring between the carbonyl of 29 and CatC Asp1 that was predicted in docking simulations. Replacement of piperazine (compound 22) with N,N-dimethylpiperidin (compound 30) had little effect on potency and liver microsome metabolic stability. Compound 27 implied that the modification of ortho position at phenyl probably improved the metabolic stability, so an N-atom substitution analogue was prepared (compound 31), for which a dramatic loss (about 20-fold) in potency was observed. Although introduction of trifluoromethyl to the 4-position of phenyl at pyridine C-2 increased the potencies (compounds 32 and 33), these modifications made the compounds metabolically unstable in liver microsomes. Overall, compound 22 exhibited the best combination of strong potency and liver microsome stability so it was selected as a potential candidate for further in vitro evaluation. Molecular Docking. To better understand the SAR results observed for our compounds, we performed molecular docking of compound 22 within the active site of CatC based on the CatC/AZD5248 complex crystal structure (PDB ID: 4CDE). The acrylamide formed two hydrogen bond donor−acceptor motifs, respectively, with Asn380 and Gln228, and these two hydrogen bonds enabled the β-carbon atoms of acrylamide to readily reach the sulfhydryl group of Cys234 (within 4.0 Å), thereby facilitating a Michael addition to form the covalent bond (Figure 5 and Table S2, Supporting Information). Salt-bridge interaction was also observed between the piperazine N-4 of compound 22 and the Asp1 residue of the CatC exclusion domain, and hydrophobic interaction was predicted between the piperidine and Trp405 (with a 4−5 Å distance between the two rings). The methyl group of the phenyl ring generated slightly hydrophobic interaction with nearby residues and also forced the acrylamide to retain its proper orientation relative to Cys234 for Michael addition to form the covalent bond. In Vitro/in Vivo Profiling. As our compounds are derived from an EGFR inhibitor, several compounds with favorable CatC activity were selected for evaluation of their EGFR inhibitory activity (Figure S1, Supporting Information). For all 5907



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Table 4. Further Optimization at the Pyridine C-3 Position (a) and C-6 Position (b)

a Human recombinant CatC isolated enzyme assay. Mean values from a minimum of two experiments. bCatC cell assay using THP1 cell line. Mean values from a minimum of two experiments. cCatC cell assay using U937 cell line. Mean values from a minimum of two experiments. dCLint: μL/ (min mg protein), determined in human/rat/mouse microsomes. eIt was racemic. fND = not detected at 0 min. gNT = not test.

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Table 6. Pharmacokinetic Parameters for Intravenous (IV), Intraperitoneal (IP), and Oral (PO) Administration of 22 in C57BL/6 Mice route

AUCinf (h ng/mL)

t1/2 (h)

tmax (h)

Cmax (ng/mL)

F%

IVa IPb POc

1225 1243 1364

1.62 1.19 3.06

0 0.5

766 184

20.3 22.3

a IV dose: 2 mg/kg. bIP dose: 10 mg/kg. cPO dose: 10 mg/kg. All administrated with 0.5% Methocel and 0.1% Tween-80 in citrate buffer, pH 3. Values were the mean values of male for mouse (n = 3).

injections of caerulein. Compound 22 and AZD5248 both caused significant inhibition of CatC enzymatic activity in the pancreas and displayed a considerable pancreas-protecting effect based on assessment of photomicrographs of hematoxylin- and eosin (H&E)-stained pancreas sections (Figure 7). On the basis of its attractive performance, both in vitro and in vivo, we have initiated further druggability evaluations of compound 22 that are currently underway.

Figure 5. Putative binding mode of compound 22 (orange) within the active site of CatC (PDB ID: 4CDE).

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administration of 22 at a dose of 20 mg/kg, the blood concentration of 22 was 200 ng/mL and CatC activity in blood was inhibited by 75%, and time-course activity studies showed statistically significant inhibition (80%) of CatC by 22 in blood lasting for 8 h post administration (Figure S3, Supporting Information). Next, the effect of compound 22 on the activation of NE, PR3, and CatG was assessed in vivo by oral administration of 22 twice daily to mice (6.7, 20, and 60 mg/kg) over a 6 day time course. In Figure 6, dose-dependent decreases of CatC activity were observed in both bone marrow and blood. We also found that the activation of downstream NE, PR3, and CatG was also inhibited, in a dose-dependent manner, both in bone marrow and in blood (Figure 6). Oral administration of compound 22 (60 mg/kg; twice daily) was tolerated, and we detected no difference in the white blood cell count (Figure S4, Supporting Information) between the vehicle-treated and 60 mg/kg treatment groups, thus suggesting that compound 22 might not exert direct effects on the maturation or release of neutrophil into the blood circulation. Collectively, these in vivo studies robustly confirmed that compound 22 can block CatC enzymatic activity and can, further, inhibit the activation of downstream NSPs (both in bone marrow and in blood samples). These results demonstrated that compound 22 could potentially be a therapeutic compound for the treatment of neutrophilassociated inflammatory diseases. Recently, Aghdassi et al. reported that deficiency of cathepsin C ameliorated severity of acute pancreatitis in mice.35 Next, we examined the potential protective effect of compound 22 on the pancreas using a caerulein-induced acute pancreatitis mouse model. In this model, mice were orally administrated 22 or AZD5248 twice daily at 20 mg/kg for 6 days. On the 6th day, acute pancreatitis was induced via hourly intraperitoneal

CONCLUSIONS Starting from hit compound WZ4002 (a low micromolar CatC inhibitor), structure−activity relationship (SAR) investigations were performed guided by a plausible binding mode of WZ4002 within the CatC active site. These efforts ultimately led to the discovery of a novel series of CatC inhibitors with significant improvement in enzymatic/cellular potency, culminating finally in the identification of compound 22 as a highly potent and selective irreversible inhibitor of CatC. In vitro and in vivo profiling assays demonstrated that compound 22 is an extremely promising drug candidate, and further evaluations of compound 22 as a preclinical candidate are currently in progress.

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EXPERIMENTAL SECTION

Biology. Recombinant human cathepsin C (1071-CY-010), cathepsin L (952-CY-010), Mca-RPKPVE-Nval-WRK (Dnp)-NH2 (ES002), and Z-Leu-Arg-AMC (ES008) were purchased from R&D (Akron, OH). Recombinant human cathepsin B (10483-H08H) and cathepsin S (10487-H08H) were purchased from Sino Biological Inc. (Beijing, CN). Human THP1 cells and U937 cells were purchased from ATCC (Manassas, VA) and maintained in Roswell Park Memorial Institute (RPMI) 1640 culture medium. The medium was supplemented with 10% fetal bovine serum (12483-020) and 100 unit/mL penicillin−streptomycin (15140-122), which were bought from Life Technology (Beijing, CN). Human and mouse liver microsomes were purchased from BD Biosciences (Beijing, CN). Gly-Phe-AFC (SMAFC041) was bought from SM Biochemicals (Anaheim, CA). Cisbio HTRF KinEASE-TK kit (62TK0PEC) was purchased from Cisbio (Shanghai, CN). All other chemicals were purchased from Sigma-Aldrich (Shanghai, CN). The assay and cell culture plates were purchased from Corning (Shanghai, CN). C57BL/6 mice matched for gender and age were purchased from Vitalriver (Beijing, CN). All experiments on mice were conducted according to institutional and

Table 5. Cathepsin Selectivity of Compound 19, 22, and 24 compd

CatC Enz (IC50, nM)a

CatB Enz (IC50, μM)a

CatL Enz (IC50, μM)a

CatS Enz (IC50, μM)a

19 22 24

44 17 59

3−10 3−10 3−10

3−10 3−10 >10

>10 >10 >10

a

Human recombinant CatC/CatB/CatL/CatS isolated enzyme assay. 5909



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Figure 6. In vivo profiling data (mice) of compound 22. Compound 22 inhibited CatC and the activation of NE, PR3, and CatG in bone marrow and blood, and did so in a dose-dependent manner. Compound 22 was administered orally twice daily to naive mice (6.7, 20, and 60 mg/kg) for 6 days. The activity inhibition data of compound-treating groups were showed as percentage activity of the vehicle mean (set to 100% activity).

Figure 7. Compound 22 reduced the severity of acute pancreatitis: (a) CatC activity in homogenates prepared from mice pancreas samples. Compound 22 and AZD5248 were administered orally twice daily at 20 mg/kg. The activity inhibition data of the compound-treated groups are shown as the percentage activity of a sham mean (set to 100% activity). Values are means ± standard error of the mean. Statistical significance was considered: ## p < 0.05 vs sham; ** p < 0.05 vs caelurein + vehicle group. (b) Pancreas histology representative photomicrographs. Hematoxylin and eosin staining of formalin-fixed sections of pancreas tissue. Scale bar: 50 μm. pH 6.0) at RT for 2 h and then activated rhCatC was diluted to 4.4 nM in assay buffer (25 mM MES, 50 mM NaCl, 5 mM DTT, 0.01% (v/v) Triton X-100, pH 6.0). Fourteen microliters of activated rhCatC and 1 μL of compound in 10% dimethyl sulfoxide (DMSO) or 10% DMSO were incubated at RT for 3 h, and 5 μL of the substrate (Gly-Phe-AFC) was added to the final concentration of 100 μM. After 60 min of reaction, the product of the reaction was measured in the EnSpire plate

national animal regulations. Animal protocols were approved by the ethics committee of the National Institute of Biological Sciences. Mice were used at the age of 6−8 weeks. In Vitro CatC Enzyme Assay. The CatC enzyme assay was performed on a black 384-well plate. First, rhCatC (440 nM) was activated by rhCatL (300 nM) in activation buffer (25 mM 2-(Nmorpholino)ethanesulfonic acid (MES), 5 mM dithiothreitol (DTT), 5910



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reader using Ex λ 400 nm and Em λ 505 nm for AFC. A standard irreversible CatC inhibitor AZD5248 by AstraZeneca was used as an inhibitor control in the assay. CatC Cell Assay. The cell assay was carried out in a 96-well clear black plate. Thirty microliters of 1.0 × 106/mL THP1 or U937 cells in medium were added to all wells. Then, 10 μL of 5% (v/v) DMSO positive controls or 10 μL of threefold serial diluted compound were dispensed into the appropriate well. The plate was preincubated at 37 °C for 4 h in a cell culture incubator, and then 10 μL of H-Gly-Phe-AFC (100 μM) was added to start the assay reaction. The plate was further incubated for 60 min at 37 °C and measured same as the above CatC enzyme assay. EGFR Kinase Assay. The EGFR kinase assays were performed on a black 384-well plate using Cisbio HTRF KinEASE-TK kit. The assay buffer contained 50 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (pH 7.0), 0.02% NaN3, 0.01% bovine serum albumin, 0.1 mM orthovanadate, 10 mM MgCl2, 2.5 mM DTT, and 6.25 nM SEB. EGFR kinases was first incubated with compounds for 2 h, respectively; then, adenosine 5′-triphosphate (ATP)/peptide substrate mixture was added to initiate the reaction. The final concentration EGFR WT was 0.1 nM. The final concentration of the biotin-labeled peptide substrate was 1.4 μM, and ATP was 20 μM for EGFR WT. After 30 min of reaction at RT, the detection reagents were added. The time-resolved fluorescence energy transfer signal was measured on PerkinElmer Envision using Ex λ 320 nm and Em λ 615/665 nm. Data Analysis. Data were analyzed using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). The curves were fitted using a nonlinear regression model with a sigmoidal dose response. Selectivity Profile of Cathepsin B/L/S. Selectivity assay was conducted on a black 384-well plate. Briefly, compounds and recombinant human cathepsin enzymes were incubated at RT for 30 min followed by reaction with substrates for further 30 min, respectively. Z-Leu-Arg-AMC (as a substrate of cathepsin B and L) was measured by PerkinElmer Envision using Ex λ 380 nm and Em λ 460 nm. MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)NH2 (as a substrate of cathepsin S) was measured at Ex λ 320 nm and Em λ 405 nm using Enspire plate reader. E-64, a natural inhibitor of cysteine proteases, was used as an inhibitor control in three assays. Compounds Stability Test in Liver Microsomes Assays. Compounds were incubated with human, rat or mouse liver microsomes and reduced nicotinamide adenine dinucleotide phosphate in 0.05 M phosphate buffer (pH = 7.4) at 37 °C for 0−60 min. The reaction was quenched, and the amount of the remaining compound was analyzed using liquid chromatography tandem mass spectrometry (LC−MS/MS). Pharmacokinetics Study in Mice. Following intravenous (IV), intraperitoneal (IP), or oral administration (PO) of 22 to C57BL/6 mice (n = 3), blood was sampled through eye puncture at various time points. Compound concentrations in the blood samples were analyzed by LC−MS/MS. Pharmacokinetic parameters were determined from individual animal data using noncompartmental analysis in phoenix 64 (winNonlin 6.3). Mice in Vivo Study. C57BL/6 mice (20−25 g) were randomly divided into four groups (n = 10) and kept at five animals per cage. Compound 22 was administered orally twice daily at 6.7, 20, and 60 mg/kg for 6 days. The matched vehicle controls (0.5% Methocel, 0.1% Tween-80 in 100 mM citrate buffer, pH 3) were also administered twice daily for 6 days. Mice were weighed and the dose adjusted accordingly on each day. Blood was sampled through eye puncture at 14 h intervals after the end of the administration. Then, mice were terminated and bone marrow cells were immediately extracted from femurs and tibias using ice cold RPMI 1640 medium. Blood cells and bone marrow cells were lysed according to the protocols previously described8 with some modifications. Briefly, cell lysis buffer contained 50 mM Tris (pH 7.4), 750 mM NaCl, and 1% (v/v) Triton X-100, and cell lysates were mildly treated by ultrasonic treatment in ice. Following cell lysis, the supernatants were kept at −80 °C until NSPs’ activity analyses were carried out.

Analysis of Downstream NSPs Activities. The analysis was performed as previously described. Briefly, cell lysates were diluted according to protein concentration using different assay buffers. The assay buffers used were 25 mM MES, 50 mM NaCl, 5 mM DTT, pH 6.0 for CatC, 50 mM Tris, 750 mM NaCl, pH 7.4 for NE and PR3 and 50 mM Tris, 750 mM NaCl, 5 mM ethylenediaminetetraacetic acid, pH 7.4 for CatG. Blood lysates were diluted 5-fold higher than bone marrow lysates. Lysates were added to 384-well plates together with DMSO control or protease inhibitors (inhibitor AZD5248 by AstraZeneca for CatC, avelestat for NE, sivelestat for PR3, cathepsin G inhibitor 1 for CatG). Synthetic peptide substrates (Gly-Phe-AFC (SM Biochemicals) for CatC, methoxysuccinyl-Ala-Ala-Pro-Val-AMC (Sigma) for NE, aminobenzoyl-Val-Ala-Asp-Cys-Ala-Asp-Gln-ethylenediamine 2,4-dinitrophenyl (Peptide Synthetics) for PR3, and Nsuccinyl-Ala-Ala-Pro-Phe-pNA (Sigma) for CatG) were added before plates were read at fluorescence or absorbance. Assay kinetics was monitored for up to 90 min. Data were analyzed using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). Induction of Pancreatitis. The acute pancreatitis was induced by hourly intraperitoneal injections of caerulein (50 μg/(kg body weight)) for up to 8 h.36 C57BL/6 mice (20−25 g) were randomly divided into four groups (n = 5). Compound 22 and AZD5248 were administered orally twice daily at 20 mg/kg for 6 days. The matched vehicle controls (0.5% Methocel, 0.1% Tween-80 in 100 mM citrate buffer, pH 3) were also administered twice daily for 6 days. On the 6th day, mice were induced pancreatitis after compound administration, and after the last caerulein injection, mice were administered the compound. The shamtreated group was treated the same as vehicle controls except that saline was administered instead of caerulein. Mice were killed 1 h after the last caerulein injection. Tissue was harvested immediately after sacrificing the animals. A portion of pancreas samples was frozen in liquid nitrogen for preparation of homogenate. For histology, pancreas were fixed in 4.5% formalin for paraffin embedding and stained with hematoxylin and eosin. Serum samples were prepared and stored at −20 °C. Molecule Docking. Molecular docking was performed using MOE 2015.10 software. The cocrystal structure of CatC in complex with AZD5248 (PDB ID: 4CDE) was used for docking studies. The optimized structure of 22 was generated by MOE 2015.10. Before docking simulation, ligands and protein were prepared with the standard protocol using MOE 2015.10. 22 was inserted into the substrate-binding pocket of CatC to replace AZD5248. The complex of 22-CatC was used for geometrical optimization. All docking calculations were performed using default settings. Chemistry. The reagents and solvents for reactions were reagentgrade and used without further purification. Glass apparatus was used for reaction dewater, and deoxygenization was carried out under a nitrogen atmosphere at high temperature. Reactions sensitive to water and air rigorously followed water-free and oxygen-free reaction procedures, wherein reagents were added with an injection syringe. LC−mass spectrometry (MS) system: Waters 2545 (XBridge C18)/ 3100 mass detector. 1H NMR spectra were recorded on a Varian 400 MHz spectrometer (Varian, Inc., Palo Alto, CA) at ambient temperature with tetramethylsilane as an internal standard. 13C NMR spectra were recorded on a Varian 100 MHz spectrometer (with complete proton decoupling) at ambient temperature. Chemical shifts are reported in ppm. Data for 1H NMR are reported as follows: chemical shift (δ) in ppm, multiplicity (s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constants and integration. High-resolution mass spectra were obtained by Agilent Technologies 6540 UHD Accurate-Mass Q-TOF LC/MS (Santa Clara, CA). The samples were analyzed by high-performance liquid chromatography (HPLC)/MS on a Waters Auto Purification LC/MS system (3100 mass detector, 2545 binary gradient module, 2767 sample manager, and 2998 photodiode array detector), which was purchased from Waters (Milford, MA). The system was equipped with a Waters C18 5 μm SunFire separation column (150 × 4.6 mm2), equilibrated with HPLC grade water (solvent A) and HPLC grade acetonitrile (solvent B) with a flow rate of 0.3 mL/min. Thin-layer chromatography was carried out using HS-GF 254 silica plates; chromatographic purification was carried out using silica gel (Yan tai mou ping yuan bo 5911



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jing xi silica gel plant). The purity of all final compounds was confirmed to be >95% by HPLC. N-(5-((3-Chloro-6-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-2-yl)oxy)-2-methylphenyl)acrylamide (1). Step a: 3,6-Dichloro-2-(3-nitrophenoxy)pyridine (36). Potassium carbonate (1.52 g, 10.96 mmol) and 2,3,6-trichloropyridine (1.0 g, 5.48 mmol) were added to the solution of 3-nitrophenol (763 mg, 5.48 mmol) in anhydrous DMF (20 mL). The reaction mixture was heated to 100 °C for 4 h. The reaction mixture was filtered, and the filtrate was diluted with ethyl acetate and washed with brine three times. The organic layer was dried over anhydrous sodium sulfate and concentrated; the residue product was purified by silica gel chromatography to afford 1.02 g of a white solid (yield: 65%). 1H NMR 400 MHz (CDCl3) δ 8.14−8.11 (m, 1H), 8.06 (t, J = 2.4 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H), 7.55−7.52 (m, 1H), 7.08 (d, J = 8.0 Hz, 1H). LC−MS m/z: 285.02 (M + H)+. Step b: 5-Chloro-N-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)-6-(3-nitrophenoxy)pyridin-2-amine (38a). An anhydrous dioxane solution of 3,6-dichloro-2-(3-nitrophenoxy)pyridine (300 mg, 1.05 mmol), Pd(OAc)2 (24 mg, 0.11 mmol), BINAP (98 mg, 0.16 mmol), cesium carbonate (515 mg, 1.58 mmol), and 2-methoxy-4(4-methylpiperazin-1-yl)aniline (256 mg, 1.16 mmol) was added into a 20 mL microwave reaction vessel and capped. The reaction mixture was stirred for 1 h at 130 °C using a Biotage optimizer reactor. Water and ethyl acetate were added to the reaction solution and separated, and then the aqueous phase was extracted with ethyl acetate three times. The combined organic phase was washed with saturated brine, then dried over anhydrous sodium sulfate. The organic phase was filtered off and concentrated under reduced pressure. The obtained residue was purified by silica gel chromatography to afford 309 mg of a brown solid (yield: 62.6%). 1H NMR 400 MHz (DMSO-d6) δ 8.23 (s, 1H), 8.15− 8.12 (m, 1H), 8.00 (t, J = 2.4 Hz, 1H), 7.73 (t, J = 8.4 Hz, 1H), 7.68− 7.65 (m, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.07 (d, J = 8.8 Hz, 1H), 6.59 (d, J = 8.4 Hz, 1H), 6.50 (d, J = 2.4 Hz, 1H), 5.97 (dd, J = 2.4, 8.8 Hz, 1H), 3.74 (s, 3H), 3.00 (t, J = 4.8 Hz, 4H), 2.43 (t, J = 4.8 Hz, 4H), 2.21 (s, 3H). LC−MS m/z: 470.36 (M + H)+. Step c: 6-(3-Aminophenoxy)-5-chloro-N-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)pyridin-2-amine (39a). 5-Chloro-N-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)-6-(3-nitrophenoxy)pyridin2-amine (305 mg, 0.65 mmol) was dissolved in ethanol (2 mL) and THF (2 mL), and water (2 mL) was added. Iron powder (363 mg, 6.49 mmol) and ammonium chloride (344 mg, 6.49 mmol) were then added, and the resulting mixture was heated to 75 °C for 4 h The reaction mixture was cooled to RT and filtered through celite. The solvent was removed in vacuo, and the resulting residue was basified with saturated sodium bicarbonate and extracted with ethyl acetate three times. The combined organic phase was dried with anhydrous sodium sulfate, concentrated, and purified by silica gel chromatography with DCM−MeOH to afford 240 mg of a brown solid (yield: 83.9%). 1 H NMR 400 MHz (DMSO-d6) δ 8.07 (s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H), 7.03 (t, J = 8.0 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H), 6.52 (d, J = 8.4 Hz, 1H), 6.42 (dd, J = 2.4, 8.0 Hz, 1H), 6.27 (t, J = 2.4 Hz, 1H), 6.23−6.17 (m, 1H), 5.21 (s, 2H), 3.77 (s, 3H), 3.04 (t, J = 4.8 Hz, 4H), 2.44 (t, J = 4.8 Hz, 4H), 2.22 (s, 3H). LC−MS m/z: 440.33 (M + H)+. Step d: N-(5-((3-Chloro-6-((2-methoxy-4-(4-methylpiperazin-1yl)phenyl)amino)pyridin-2-yl)oxy)-2-methylphenyl)acrylamide (1). Acryloyl chloride (14 mg, 0.16 mmol) was added dropwise to a solution of 6-(3-aminophenoxy)-5-chloro-N-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)pyridin-2-amine (63 mg, 0.14 mmol) and triethylamine (30 μL, 0.21 mmol) in anhydrous DCM (2 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h. Water and DCM were added to the reaction solution, separated and then the aqueous phase was extracted with DCM twice. The combined organic phase was washed with brine, then dried over anhydrous sodium sulfate. The organic phase was filtered off and concentrated under reduced pressure. The obtained residue was dissolved in DCM (2 mL) and TFA (0.25 mL) was added and stirred at RT for 30 min to afford the TFA salt of the title compound. DCM and excess TFA were removed in vacuo; then, the residue was dissolved in MeOH and purified by reverse-phase

HPLC eluting with acetonitrile/water (containing 0.15‰ TFA). Fractions containing the product were concentrated in vacuo to remove the solvent to give the title compound (TFA salt, 20 mg, 19.3%) as a gray solid. 1H NMR (400 MHz, DMSO-d6) δ 10.30 (s, 1H), 8.23 (s, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.44 (t, J = 2.4 Hz, 1H), 7.40−7.35 (m, 2H), 6.86 (dd, J = 8.4, 2.4 Hz, 1H), 6.64 (d, J = 8.4 Hz, 1H), 6.59 (d, J = 2.4 Hz, 1H), 6.42 (dd, J = 17.2, 10.0 Hz, 1H), 6.26 (dd, J = 17.2, 2.0 Hz, 1H), 6.07 (dd, J = 8.8, 2.8 Hz, 1H), 5.77 (dd, J = 10.0, 2.0 Hz, 1H), 3.78 (s, 3H), 3.68 (d, J = 13.6 Hz, 2H), 3.50 (d, J = 12.0 Hz, 2H), 3.17−3.08 (m, 2H), 2.86−2.80 (m, 5H). 13C NMR (100 MHz, DMSO-d6) δ 163.29, 156.25, 154.13, 153.35, 150.03, 145.18, 140.24, 140.12, 131.72, 129.79, 127.28, 122.38, 120.87, 116.36, 115.50, 112.40, 106.91, 106.14, 103.18, 100.71, 55.67, 52.33, 46.31, 42.10. High-resolution mass spectrometry (HRMS) C26H28ClN5O3 (M + H)+ calcd mass, 494.1959; found, 494.1972. N-(3-((3-Chloro-6-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-2-yl)oxy)phenyl)acrylamide (2). This compound was prepared according to the preparation of compound 1 using 4-(4methylpiperazin-1-yl)aniline to replace 2-methoxy-4-(4-methylpiperazin-1-yl)aniline in step b to afford the title compound (TFA salt, 8 mg, 7%) as a brown solid. The title compound was purified by reverse-phase HPLC eluting with acetonitrile/water (containing 0.15‰ TFA). 1H NMR (400 MHz, DMSO-d6) δ 10.36 (s, 1H), 9.11 (s, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.65−7.63 (m, 1H), 7.49 (t, J = 2.4 Hz, 1H), 7.41 (t, J = 8.4 Hz, 1H), 7.14 (d, J = 8.8 Hz, 2H), 6.91−6.88 (m, 1H), 6.64 (d, J = 9.2 Hz, 2H), 6.47 (d, J = 8.4 Hz, 1H), 6.43 (dd, J = 16.8, 10.0 Hz, 1H), 6.26 (dd, J = 16.8, 2.0 Hz, 1H), 5.77 (dd, J = 10.0, 2.0 Hz, 1H), 3.57 (bs, 2H), 3.47 (bs, 2H), 3.12 (bs, 2H), 2.85 (s, 3H), 2.80 (bs, 2H). 13C NMR (100 MHz, DMSO-d6) δ 163.30, 156.59, 154.02, 152.78, 143.77, 140.30, 140.24, 134.19, 131.71, 129.86, 127.34, 118.94, 116.70, 116.52, 115.74, 112.80, 105.69, 102.96, 52.36, 46.46, 42.11. HRMS C25H27ClN5O2 (M + H)+ calcd mass, 464.1853; found, 464.1866. N-(3-((3-Chloro-6-((4-(piperazin-1-yl)phenyl)amino)pyridin-2yl)oxy)phenyl)acrylamide (3). tert-Butyl 4-(4-((6-(3-acrylamidophenoxy)-5-chloropyridin-2-yl)amino)phenyl)piperazine-1-carboxylate (40d) was prepared according to the preparation method of compound 1 using tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate to replace 2-methoxy-4-(4-methylpiperazin-1-yl) aniline in step b. tert-Butyl 4-(4-((6-(3-acrylamidophenoxy)-5-chloropyridin-2-yl)amino)phenyl)piperazine-1-carboxylate (40d) was dissolved in DCM/TFA (2:1), and the mixture was stirred at RT for 2 h. The mixture was evaporated to dryness, dissolved in MeOH, and purified by reverse-phase HPLC eluting with acetonitrile/water (containing 0.15‰ TFA) to afford the title compound (TFA salt, 10 mg, 14.6% over the last two steps) as a gray solid. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 9.09 (s, 1H), 8.77 (bs, 1H), 7.66 (t, J = 8.8 Hz, 1H), 7.64 (t, J = 8.4 Hz, 1H), 7.50 (t, J = 2.4 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 8.4 Hz, 2H), 6.89 (dd, J = 8.4, 2.4 Hz, 1H), 6.63 (d, J = 8.8 Hz, 2H), 6.47 (d, J = 8.8 Hz, 1H), 6.43 (dd, J = 16.8, 10.0 Hz, 1H), 6.26 (dd, J = 16.8, 2.0 Hz, 1H), 5.77 (dd, J = 10.0, 2.0 Hz, 1H), 3.21 (bs, 4H), 3.14 (d, J = 4.4 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 163.32, 156.60, 154.02, 152.80, 144.32, 140.27, 134.20, 131.73, 129.81, 127.26, 118.96, 116.69, 116.59, 115.71, 112.83, 105.68, 102.94, 46.42, 42.79. HRMS C24H25ClN5O2 (M + H)+ calcd mass, 450.1697; found, 450.1704. N-(3-((3-Chloro-6-((4-morpholinophenyl)amino)pyridin-2-yl)oxy)phenyl)acrylamide (4). This compound was prepared according to the preparation of compound 1 using 4-morpholinoaniline to replace 2-methoxy-4-(4-methylpiperazin-1-yl)aniline in step b to afford the title compound (TFA salt, 20 mg, 16.7%) as a brown solid. The title compound was purified by reverse-phase HPLC eluting with acetonitrile/water (containing 0.15‰ TFA). 1H NMR (400 MHz, DMSO-d6) δ 10.31 (s, 1H), 9.09 (s, 1H), 7.67−7.61 (m, 2H), 7.54 (t, J = 2.4 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.13 (bs, 2H), 6.89 (dd, J = 2.4, 8.0 Hz, 1H), 6.67 (bs, 2H), 6.47 (s, 1H), 6.42 (dd, J = 16.8, 10.0 Hz, 1H), 6.25 (dd, J = 16.8, 2.0 Hz, 1H), 5.77 (dd, J = 10.0, 2.0 Hz, 1H), 3.73 (bs, 4H), 2.98 (bs, 4H). 13C NMR (100 MHz, DMSO-d6) δ 163.26, 153.94, 140.25, 131.69, 129.85, 127.27, 118.89, 116.73, 115.69, 112.87, 65.84, 49.92. HRMS C24H24ClN4O3 (M + H)+ calcd mass, 451.1537; found, 451.1521. 5912



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N-(3-((6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-2-yl)oxy)phenyl)acrylamide (9). The title compound was obtained in the preparation of compound 14 as a brown solid (TFA salt, 16 mg). 1H NMR (400 MHz, DMSO-d6) δ 10.27 (s, 1H), 8.00 (s, 1H), 7.57−7.55 (m, 1H), 7.54−7.49 (m, 2H), 7.46−7.44 (m, 1H), 7.36 (t, J = 8.0 Hz, 1H), 6.84−6.81 (m, 1H), 6.62 (d, J = 2.8 Hz, 1H), 6.56 (d, J = 8.0 Hz, 1H), 6.42 (dd, J = 16.8, 10.0 Hz, 1H), 6.25 (dd, J = 16.8, 2.0 Hz, 1H), 6.21 (d, J = 8.0 Hz, 1H), 6.18 (dd, J = 8.8, 2.4 Hz, 1H), 5.76 (dd, J = 10.0, 2.0 Hz, 1H), 3.79 (s, 3H), 3.70 (d, J = 13.2 Hz, 2H), 3.50 (d, J = 12.0 Hz, 2H), 3.18−3.09 (m, 2H), 2.87−2.82 (m, 5H). HRMS C26H30N5O3 (M + H)+ calcd mass, 460.2349; found, 460.2361. N-(3-((6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)3-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)acrylamide (10). This compound was prepared according to the preparation method of compound 1 using 2,6-dichloro-3-(trifluoromethyl)pyridine and 3nitrophenol as the starting material. The product was purified by silica gel column chromatography eluting with DCM/MeOH as a yellow solid (20 mg, 35.9%). 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.66−7.64 (m, 1H), 7.55 (t, J = 2.4 Hz, 1H), 7.46 (d, J = 2.0 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.33 (d, J = 8.8 Hz, 1H), 6.91−6.89 (m, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.46 (d, J = 8.8 Hz, 1H), 6.41 (dd, J = 16.8, 10.0 Hz, 1H), 6.25 (dd, J = 16.8, 2.0 Hz, 1H), 6.00 (dd, J = 8.8, 2.4 Hz, 1H), 5.77 (dd, J = 10.0, 2.0 Hz, 1H), 3.80 (s, 3H), 3.01 (t, J = 4.8 Hz, 4H), 2.42 (t, J = 4.8 Hz, 4H), 2.21 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 164.65, 163.27, 153.34, 150.65, 149.14, 147.33, 140.44, 139.63, 131.68, 130.05, 127.28, 126.02, 123.33, 120.51, 120.22, 116.92, 116.05, 113.09, 106.29, 99.67, 98.42, 56.09, 54.65, 48.60, 45.82. HRMS C27H29F3N5O3 (M + H)+ calcd mass, 528.2222; found, 528.2232. N-(3-((3-Cyano-6-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-2-yl)oxy)phenyl)acrylamide (11). This compound was prepared according to the preparation method of compound 1 using 2,6-dichloronicotinonitrile and 3-nitrophenol as the starting material. The product was purified by silica gel column chromatography eluting with DCM/MeOH as a yellow solid (19 mg, 42.2%). 1H NMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H), 8.31 (s, 1H), 7.59−7.57 (m, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 2.4 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 6.85−6.82 (m, 1H), 6.54 (d, J = 8.8 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 6.42 (dd, J = 16.8, 10.0 Hz, 1H), 6.25 (dd, J = 16.8, 2.0 Hz, 1H), 6.02 (dd, J = 8.8, 2.4 Hz, 1H), 5.76 (dd, J = 10.0, 2.0 Hz, 1H), 3.75 (s, 3H), 3.00 (t, J = 4.8 Hz, 4H), 2.41 (t, J = 4.8 Hz, 4H), 2.21 (s, 3H). HRMS C27H29N6O3 (M + H)+ calcd mass, 485.2301; found, 485.2312. N-(3-((3-Ethynyl-6-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-2-yl)oxy)phenyl)acrylamide (12). Step a: 3Bromo-6-chloro-2-(3-nitrophenoxy)pyridine was prepared using 3nitrophenol and 3-bromo-2,6-dichloropyridine as starting materials according to the method of step a mentioned in preparation of compound 1. Step b: A flask containing 3-bromo-6-chloro-2-(3-nitrophenoxy)pyridine (275 mg, 0.83 mmol), Pd(PPh3)2Cl2 (59 mg, 0.083 mmol) and CuI (16 mg, 0.083 mmol) was evacuated and back-filled with nitrogen three times. Then, sequentially, with stirring were added anhydrous dioxane (4 mL), triethylamine (126 mg, 1.25 mmol), and ethynyltrimethylsilane (98 mg, 1.00 mmol). The resulting solution was stirred at 70 °C for 4 h, before quenching with sat. ammonium chloride (aq). The mixture was diluted with ethyl acetate and washed with brine. The organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo to afford the crude, which was purified by silica gel chromatography to afford the title product (224 mg, 77%). LC−MS m/z: 347.12 (M + H)+. Steps c and d: Same as the method in steps b and c mentioned in preparation of compound 1. Step e: To a solution of intermediate 6-(3-aminophenoxy)-N-(2methoxy-4-(4-methylpiperazin-1-yl)phenyl)-5-((trimethylsilyl)ethynyl)pyridin-2-amine in THF was added a 1 M solution of TBAF in THF (10 equiv). The mixture was stirred at RT for 2 h. Water and ethyl acetate were added to the reaction solution and separated, and then the aqueous phase was extracted with ethyl acetate. The combined organic phase was washed with saturated brine, then dried over anhydrous

N-(3-((6-((3-(Aminomethyl)phenyl)amino)-3-chloropyridin-2-yl)oxy)phenyl)acrylamide (5). This compound was prepared according to the preparation of compound 3 using tert-butyl (3-aminobenzyl)carbamate to replace the tert-butyl 4-(4-aminophenyl)piperazine-1carboxylate in step b to afford a brown solid (TFA salt, 13 mg, 18.4% over the last two steps). The title compound was purified by reversephase flash chromatography eluting with MeOH/water (containing 0.15‰ TFA). 1H NMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H), 9.39 (s, 1H), 8.03 (s, 2H), 7.76 (d, J = 8.8 Hz, 1H), 7.58−7.54 (m, 2H), 7.43 (t, J = 8.4 Hz, 1H), 7.30−7.27 (m, 1H), 7.20 (t, J = 2.0 Hz, 1H), 7.00 (t, J = 8.0 Hz, 1H), 6.97−6.94 (m, 1H), 6.87 (d, J = 8.0 Hz, 1H), 6.60 (d, J = 8.4 Hz, 1H), 6.40 (dd, J = 16.8, 10.0 Hz, 1H), 6.24 (dd, J = 17.2, 2.0 Hz, 1H), 5.76 (dd, J = 10.0, 2.0 Hz, 1H), 3.74 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 163.33, 156.57, 153.96, 152.45, 140.95, 140.60, 140.32, 134.13, 131.68, 129.83, 128.74, 127.31, 120.67, 118.07, 117.91, 116.88, 115.81, 112.68, 106.28, 104.20, 42.48. HRMS C21H20ClN4O2 (M + H)+ calcd mass, 395.1275; found, 395.1284. N-(3-((6-((4-(2-Aminoethyl)phenyl)amino)-3-chloropyridin-2-yl)oxy)phenyl)acrylamide (6). This compound was prepared according to the preparation of compound 3 using tert-butyl (4-aminophenethyl)carbamate to replace tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate in step b to afford the title compound (TFA salt, 11 mg, 19.6% over the last two steps) as a brown solid. The product was purified by reverse-phase HPLC eluting with acetonitrile/water (containing 0.15‰ TFA). 1H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H), 9.26 (s, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.72−7.70 (m, 2H), 7.64−7.61 (m, 1H), 7.51−7.50 (m, 1H), 7.42 (t, J = 8.4 Hz, 1H), 7.20 (d, J = 8.8 Hz, 1H), 7.21−7.18 (m, 1H), 6.93−6.90 (m, 1H), 6.85 (d, J = 8.4 Hz, 1H), 6.87−6.84 (m, 1H), 6.54 (d, J = 8.4 Hz, 1H), 6.41 (dd, J = 16.8, 10.0 Hz, 1H), 6.26 (dd, J = 17.2, 2.0 Hz, 1H), 5.77 (dd, J = 10.0, 2.0 Hz, 1H), 2.91 (bs, 2H), 2.67 (t, J = 8.0 Hz, 2H). HRMS C22H22ClN4O2 (M + H)+ calcd mass, 409.1431; found, 409.1437. N-(3-((3-Chloro-6-((4-(4-(cyclopropylmethyl)piperazin-1-yl)phenyl)amino)pyridin-2-yl)oxy)phenyl)acrylamide (7). Compound 3 (30 mg, 0.067 mmol) was dissolved in anhydrous DMF (2 mL); then, potassium carbonate (18 mg, 0.13 mmol) and (bromomethyl)cyclopropane (9 mg, 0.067 mmol) were added. The mixture was stirred at 70 °C for 4 h. Water and ethyl acetate were added to the reaction mixture, separated, and then the aqueous phase was extracted with ethyl acetate three times. The combined organic phase was washed with saturated brine, then dried over anhydrous sodium sulfate. The organic phase was filtered off and then concentrated under reduced pressure. The obtained residue was purified by reverse-phase HPLC eluting with acetonitrile/water (containing 0.15‰ TFA) to afford the title compound (TFA salt, 5 mg, 10.2%) as a gray solid. 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 9.09 (s, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.67−7.64 (m, 1H), 7.47 (t, J = 2.0 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 9.2 Hz, 2H), 6.91−6.88 (m, 1H), 6.65 (d, J = 9.2 Hz, 2H), 6.47 (d, J = 8.8 Hz, 1H), 6.42 (dd, J = 17.2, 10.0 Hz, 1H), 6.26 (dd, J = 17.2, 2.0 Hz, 1H), 5.77 (dd, J = 10.0, 2.0 Hz, 1H), 3.60 (d, J = 11.6 Hz, 2H), 3.15−3.12 (m, 2H), 3.09−3.06 (m, 2H), 2.86 (t, J = 12.0 Hz, 2H), 1.12−1.06 (m, 1H), 0.70−0.65 (m, 2H), 0.41−0.37 (m, 2H). HRMS C28H31ClN5O2 (M + H)+ calcd mass, 504.2166; found, 504.2181. N-(3-((3-Chloro-6-((4-(4-(cyclopentylmethyl)piperazin-1-yl)phenyl)amino)pyridin-2-yl)oxy)phenyl)acrylamide (8). This compound was prepared according to the preparation of compound 7 using (bromomethyl)cyclopentane to replace the (bromomethyl)cyclopropane in step f to afford the title compound (TFA salt, 7 mg, 12.7% over the last two steps) as a gray solid. The product was purified by reverse-phase HPLC eluting with acetonitrile/water (containing 0.15‰ TFA). 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 9.09 (s, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.67−7.64 (m, 1H), 7.47 (t, J = 2.0 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 8.8 Hz, 2H), 6.91−6.88 (m, 1H), 6.64 (d, J = 8.8 Hz, 2H), 6.47 (d, J = 8.8 Hz, 1H), 6.43 (dd, J = 17.2, 10.4 Hz, 1H), 6.26 (dd, J = 17.2, 2.0 Hz, 1H), 5.77 (dd, J = 10.0, 2.0 Hz, 1H), 3.56 (d, J = 11.6 Hz, 2H), 3.15−3.07 (m, 4H), 2.91 (t, J = 11.6 Hz, 2H), 1.87−1.81 (m, 2H), 1.65−1.52 (m, 4H), 1.28−1.16 (m, 5H). HRMS C30H35ClN5O2 (M + H)+ calcd mass, 532.2479; found, 532.2522. 5913



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sodium sulfate. The organic phase was filtered off and then concentrated under reduced pressure. The obtained residue was purified by silica column chromatography to afford 6-(3-aminophenoxy)-5-ethynyl-N-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)pyridin-2-amine. LC−MS m/z: 430.20 (M + H)+. Step f: This method is the same as the method in step d mentioned in preparation of compound 1. The product was purified by silica gel column chromatography eluting with DCM/MeOH as pale yellow solid (29 mg, 65.3%). 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.89 (s, 1H), 7.61−7.58 (m, 1H), 7.56 (t, J = 2.0 Hz, 1H), 7.40 (t, J = 8.4 Hz, 1H), 7.26 (d, J = 8.8 Hz, 1H), 6.90− 6.87 (m, 1H), 6.55 (d, J = 2.8 Hz, 1H), 6.42 (d, J = 8.4 Hz, 1H), 6.42 (dd, J = 16.8, 10.0 Hz, 1H), 6.25 (dd, J = 16.8, 2.0 Hz, 1H), 6.07 (dd, J = 8.8, 2.4 Hz, 1H), 5.77 (dd, J = 10.0, 2.0 Hz, 1H), 3.79 (s, 3H), 3.03 (t, J = 4.8 Hz, 4H), 2.42 (t, J = 4.8 Hz, 4H), 2.22 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 164.78, 163.28, 155.56, 152.95, 150.36, 148.21, 145.59, 140.36, 131.68, 129.96, 127.29, 121.87, 119.37, 116.81, 116.76, 116.13, 112.89, 106.23, 99.61, 84.83, 55.85, 54.64, 48.48, 45.81. HRMS C28H30N5O3 (M + H)+ calcd mass, 484.2349; found, 484.2358. N-(3-((3-(Dimethylamino)-6-((2-methoxy-4-(4-methylpiperazin1-yl)phenyl)amino)pyridin-2-yl)oxy)phenyl)acrylamide (13). This compound was prepared according to the preparation of compound 22 using 3-bromo-2,6-dichloropyridine and 3-nitrophenol as starting materials, and in step b, dimethylamine was used to replace piperidine. The title compound was purified by silica gel column chromatography eluting with DCM/MeOH as a gray solid (14 mg, 46%). 1H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 7.57−7.54 (m, 1H), 7.50 (s, 1H), 7.46 (t, J = 2.4 Hz, 1H), 7.37−7.33 (m, 2H), 7.28 (d, J = 8.4 Hz, 1H), 6.82−6.79 (m, 1H), 6.56 (d, J = 8.4 Hz, 1H), 6.50 (d, J = 2.8 Hz, 1H), 6.41 (dd, J = 16.8, 10.0 Hz, 1H), 6.24 (dd, J = 16.8, 2.0 Hz, 1H), 6.02 (dd, J = 8.8, 2.4 Hz, 1H), 6.75 (dd, J = 10.0, 2.0 Hz, 1H), 3.75 (s, 3H), 2.97 (t, J = 5.2 Hz, 4H), 2.69 (s, 6H), 2.43 (t, J = 5.2 Hz, 4H), 2.22 (s, 3H). HRMS C28H35N6O3 (M + H)+ calcd mass, 503.2771; found, 503.2770. N-(3-((6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)3-(pyrrolidin-1-yl)pyridin-2-yl)oxy)phenyl)acrylamide (14). This compound was prepared according to the preparation of 22 using 3bromo-2,6-dichloropyridine and 3-nitrophenol as starting materials, and in step b, pyrrolidine was used to replace piperidine. In step b, we obtained a mixture of 6-chloro-2-(3-nitrophenoxy)-3-(pyrrolidin-1yl)pyridine and 2-chloro-6-(3-nitrophenoxy)pyridine, and in the next three steps, we used the mixture for reactions. In step e, the crude was purified by reverse-phase HPLC eluting with acetonitrile/water (containing 0.15‰ TFA) to afford 14 (TFA salt, 14 mg) as a brown solid and 9 (TFA salt, 16 mg) as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 9.88 (s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.55−7.52 (m, 1H), 7.42−7.36 (m, 2H), 6.91−6.87 (m, 1H), 6.68− 6.55 (m, 2H), 6.43 (dd, J = 16.8, 10.0 Hz, 1H), 6.25 (dd, J = 16.8, 2.0 Hz, 1H), 6.11−6.03 (m, 1H), 5.76 (dd, J = 10.0, 2.0 Hz, 1H), 3.79 (s, 3H), 3.66 (bs, 2H), 3.50 (d, J = 12.0 Hz, 2H), 3.12 (bs, 2H), 2.86 (s, 3H), 2.85 (bs, 2H), 2.05−1.97 (m, 4H). HRMS C30H37N6O3 (M + H)+ calcd mass, 529.2927; found, 529.2923. N-(3-((6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)3-(piperidin-1-yl)pyridin-2-yl)oxy)phenyl)acrylamide (15). This compound was prepared according to the preparation of 22 using 3bromo-2,6-dichloropyridine and 3-nitrophenol as starting materials. In step e, the crude was purified by reverse-phase HPLC eluting with acetonitrile/water (containing 0.15‰ TFA) to afford 15 (TFA salt, 18 mg, 16.4%) as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.91 (s, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.40 (t, J = 8.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.63 (d, J = 8.4 Hz, 1H), 6.59 (s, 1H), 6.44 (dd, J = 16.8, 10.0 Hz, 1H), 6.26 (dd, J = 16.8, 2.0 Hz, 1H), 6.05 (d, J = 8.8 Hz, 1H), 5.77 (dd, J = 10.0, 2.0 Hz, 1H), 3.78 (s, 3H), 3.67 (d, J = 13.2 Hz, 2H), 3.51 (d, J = 12.0 Hz, 2H), 3.16− 3.09 (m, 2H), 2.86 (s, 3H), 2.84−2.81 (m, 2H), 1.83 (bs, 4H), 1.58 (bs, 2H). HRMS C31H38N6O3 (M + H)+ calcd mass, 543.3084; found, 543.3133. N-(3-((3-(Azepan-1-yl)-6-((2-methoxy-4-(4-methylpiperazin-1yl)phenyl)amino)pyridin -2-yl)oxy)phenyl)acrylamide (16). This compound was prepared according to Scheme S1, Supporting

Information. In step d, the crude was purified by reverse-phase HPLC eluting with acetonitrile/water (containing 0.15‰ TFA) to afford 16 (TFA salt, 16 mg, 15.0%) as a brown solid. 1H NMR (400 MHz, MeOD) δ 7.84 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.40 (t, J = 8.4 Hz, 1H), 7.60 (s, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H), 6.60 (bs, 1H), 8.44 (dd, J = 16.8, 9.6 Hz, 1H), 6.37 (dd, J = 16.8, 2.4 Hz, 1H), 6.13 (bs, 1H), 5.80 (dd, J = 9.6, 2.4 Hz, 1H), 3.83 (bs, 4H), 3.59 (d, J = 12.0 Hz, 2H), 3.25−3.19 (m, 2H), 2.97 (s, 3H), 2.13 (bs, 4H), 1.87 (bs, 4H). HRMS C32H41N6O3 (M + H)+ calcd mass, 557.3240; found, 557.3273. N-(3-((6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)3-(pyrrolidin-1-yl)pyridin-2-yl)oxy)-2-methylphenyl)acrylamide (17). This compound was prepared according to the preparation of compound 22 using 3-bromo-2,6-dichloropyridine and 2-methyl-3nitrophenol as starting materials, and in step b, pyrrolidine was used to replace piperidine to afford the title compound as a brown solid (TFA salt, 12 mg, 21.7%). The obtained residue was purified by reverse-phase flash chromatography eluting with MeOH/water (containing 0.15‰ TFA). 1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 9.62 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.30−7.17 (m, 2H), 7.00−6.91 (m, 2H), 6.62 (dd, J = 16.8, 10.0 Hz, 1H), 6.28 (dd, J = 17.2, 2.0 Hz, 1H), 5.78 (dd, J = 10.0, 2.0 Hz, 1H), 3.78 (s, 3H), 3.51 (d, J = 11.6 Hz, 2H), 3.17−3.06 (m, 2H), 2.86 (s, 3H), 2.06 (s, 3H), 1.98 (bs, 4H). HRMS C31H39N6O3 (M + H)+ calcd mass, 543.3084; found, 543.3085. N-(3-((6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)3-(pyrrolidin-1-yl)pyridin-2-yl)oxy)-4-methylphenyl)acrylamide (18). This compound was prepared according to the preparation method of compound 22 using 3-bromo-2,6-dichloropyridine and 2methyl-5-nitrophenol as starting materials; in step b, pyrrolidine was used to replace piperidine to afford the title compound as a brown solid (TFA salt, 13 mg, 20.6%). The product was purified by reverse-phase HPLC eluting with acetonitrile/water (containing 0.15‰ TFA). 1H NMR (400 MHz, DMSO-d6) δ 10.19 (s, 1H), 9.64 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.39−7.22 (m, 3H), 6.67−6.54 (m, 2H), 6.39 (dd, J = 16.8, 10.0 Hz, 1H), 6.23 (dd, J = 16.8, 2.0 Hz, 1H), 6.04−5.94 (m, 1H), 5.74 (dd, J = 10.0, 2.0 Hz, 1H), 3.77 (s, 3H), 3.51−3.48 (m, 4H), 3.11 (bs, 2H), 2.87−2.79 (m, 5H), 2.09 (s, 3H), 1.97 (bs, 4H). HRMS C31H38N6O3 (M + H)+ calcd mass, 543.3084; found, 543.3091. N-(5-((6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)3-(pyrrolidin-1-yl)pyridin-2-yl)oxy)-2-methylphenyl)acrylamide (19). This compound was prepared according to the preparation of compound 22 using pyrrolidine to replace piperidine in step b to afford the title compound as a gray solid (25 mg, 50.4%). The title compound was purified by reverse-phase flash chromatography eluting with acetonitrile/water. 1H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 7.37−7.33 (m, 2H), 7.22 (d, J = 8.4 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H), 6.80 (dd, J = 8.0, 2.4 Hz, 1H), 6.57 (dd, J = 16.8, 10.0 Hz, 1H), 6.54 (d, J = 8.8 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 6.22 (dd, J = 17.2, 2.0 Hz, 1H), 6.05 (dd, J = 8.8, 2.4 Hz, 1H), 5.73 (dd, J = 10.0, 2.0 Hz, 1H), 3.76 (s, 3H), 3.17 (t, J = 6.4 Hz, 4H), 2.99 (t, J = 5.2 Hz, 4H), 2.43 (t, J = 5.2 Hz, 4H), 2.25 (s, 3H), 2.21 (s, 3H), 1.86−1.83 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 163.16, 153.10, 150.71, 149.04, 147.18, 145.64, 136.92, 131.75, 130.79, 127.01, 126.79, 125.96, 125.66, 123.41, 118.81, 117.32, 116.02, 106.90, 140.51, 100.43, 55.49, 54.81, 50.09, 49.19, 45.82, 24.18, 17.49. HRMS C31H39N6O3 (M + H)+ calcd mass, 543.3084; found, 543.3088. N-(2-Methoxy-5-((6-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-3-(pyrrolidin-1-yl)pyridin-2-yl)oxy)phenyl)acrylamide (20). This compound was prepared according to the preparation of compound 22 using 3-bromo-2,6-dichloropyridine and 4-methoxy-3-nitrophenol as the starting material; in step b, pyrrolidine was used to replace the piperidine to afford the title compound as a gray solid (TFA salt, 8 mg, 11.4%). 1H NMR (400 MHz, DMSO-d6) δ 9.64 (s, 1H), 9.48 (s, 1H), 8.08−8.03 (m, 1H), 7.45−7.38 (m, 1H), 7.12− 7.08 (m, 1H), 6.90−6.85 (m, 1H), 6.75 (dd, J = 17.2, 10.0 Hz, 1H), 6.61−6.55 (m, 2H), 6.20 (dd, J = 17.2, 2.0 Hz, 1H), 6.08−6.03 (m, 1H), 5.72 (dd, J = 10.0, 2.0 Hz, 1H), 3.89 (s, 3H), 3.77 (s, 3H), 3.66 (bs, 4H), 3.49 (bs, 4H), 3.17−3.12 (m, 2H), 2.80−2.87 (m, 5H), 1.96 (bs, 4H). HRMS C31H39N6O4 (M + H)+ calcd mass, 559.3033; found, 559.3047. 5914



Article

N-(5-((3-(3-Hydroxypyrrolidin-1-yl)-6-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-2-yl)oxy)-2-methylphenyl)acrylamide (21). (±)-3-(tert-Butyldimethylsilyloxy)pyrrolidine hydrochloride was prepared following the reference: Tetrahedron, 2012, 68, 7295−7301. N-(5-((3-(3-((tert-Butyldimethylsilyl)oxy)pyrrolidin-1-yl)-6-((2methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-2-yl)oxy)2-methylphenyl)acrylamide (54a) was prepared according to the preparation method of compound 22 using (±)-3-(tertbutyldimethylsilyloxy)pyrrolidine hydrochloride to replace piperidine in step b. To a solution of N-(5-((3-(3-((tert-butyldimethylsilyl)oxy)pyrrolidin-1-yl)-6-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-2-yl)oxy)-2-methylphenyl)acrylamide (54a) (1 equiv) in THF was added TBAF (1 M THF solution, 5 equiv) on an ice bath, which was stirred for 4 h at RT. Water was added to the reaction solution, which was then extracted with ethyl acetate three times. The extract was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by reverse-phase flash chromatography eluting with acetonitrile/water to give the title compound (68 mg, yield: 41.8% over the last two steps) as a brown solid. 1H NMR (400 MHz, CDCl3) δ 7.72 (s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.20−7.18 (m, 2H), 7.04 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.49−6.48 (m, 2H), 6.42 (d, J = 8.4 Hz, 1H), 6.41−6.36 (m, 2H), 6.29−6.22 (m, 2H), 5.75−5.73 (m, 1H), 4.48−4.45 (m, 1H), 3.80 (s, 3H), 3.59−3.53 (m, 1H), 3.49−3.45 (m, 1H), 3.29−3.25 (m, 1H), 3.16−3.13 (m, 1H), 3.10 (t, J = 5.6 Hz, 4H), 2.59 (t, J = 5.2 Hz, 4H), 2.36 (s, 3H), 2.28 (s, 3H), 2.23−2.14 (m, 2H), 1.96−1.89 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 163.80, 155.59, 153.58, 152.01, 149.20, 147.30, 146.00, 136.13, 131.11, 130.90, 127.78, 127.07, 126.83, 124.74, 118.46, 118.13, 116.03, 108.37, 103.59, 101.23, 71.05, 59.66, 55.63, 55.24, 50.48, 48.64, 46.11, 34.29, 17.46. HRMS C31H38N6O4 (M + H)+ calcd mass, 559.3033; found, 559.3017. N-(5-((6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)3-(piperidin-1-yl) pyridin-2-yl)oxy)-2-methylphenyl)acrylamide (22). Step a: 3-Bromo-6-chloro-2-(4-methyl-3-nitrophenoxy)pyridine (47d). Cesium carbonate (14.4 g, 44.07 mmol) and 3bromo-2,6-dichloropyridine (5.0 g, 22.04 mmol) were added to the solution of 4-methyl-3-nitrophenol (3.4 g, 22.04 mmol) in anhydrous DMF (50 mL). The reaction mixture was heated to 90 °C for 8 h. The reaction mixture was filtered, and the filtrate was diluted with ethyl acetate and washed with brine three times. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was purified by silica gel chromatography to afford 5.31 g of a white solid (yield: 70%). 1H NMR 400 MHz (DMSO-d6) δ 8.26 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 2.4 Hz, 1H), 7.60−7.53 (m, 2H), 7.27 (d, J = 8.0 Hz, 1H), 2.53 (s, 3H); LC−MS m/z: 343.03 (M + H)+. Step b: 6-Chloro-2-(4-methyl-3-nitrophenoxy)-3-(piperidin-1-yl)pyridine (51b). Piperidine (94 mg, 1.10 mmol) in anhydrous toluene (5 mL) was treated with 3-bromo-6-chloro-2-(4-methyl-3-nitrophenoxy)pyridine (345 mg, 1.00 mmol), Pd2(dba)3 (95 mg, 0.10 mmol), BINAP (65 mg, 0.10 mmol), and cesium carbonate (491 mg, 1.51 mmol). The reaction mixture was heated to 100 °C overnight in the absence of light. The mixture was allowed to cool to ambient temperature, diluted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to provide the title compound (110 mg, 31%) as a brown oil. 1H NMR 400 MHz (CDCl3) δ 7.79 (d, J = 2.0 Hz, 1H), 7.34−7.33 (m, 2H), 7.24−7.22 (m, 1H), 7.02 (d, J = 8.0 Hz, 1H), 3.05 (t, J = 4.2 Hz, 4H), 2.61 (s, 3H), 1.72−1.69 (m, 4H), 1.61−1.56 (m, 2H). LC−MS m/z: 348.28 (M + H)+. Step c: N-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)-6-(4methyl-3-nitrophenoxy)-5-(piperidin-1-yl)pyridin-2-amine (52b). An anhydrous dioxane solution (2 mL) of 6-chloro-2-(4-methyl-3nitrophenoxy)-3-(piperidin-1-yl)pyridine (100 mg, 0.29 mmol), Pd(OAc)2 (6.5 mg, 0.029 mmol), BINAP (27 mg, 0.043 mmol), cesium carbonate (141 mg, 0.43 mmol), and 2-methoxy-4-(4-methylpiperazin1-yl)aniline (70 mg, 0.32 mmol) was added into a 5 mL microwave reaction vessel and capped. The reaction mixture was reacted at 130 °C for 1 h using a Biotage optimizer reactor. Water and ethyl acetate were

added to the reaction solution and separated, and then the aqueous phase was extracted with ethyl acetate two times. The combined organic phase was washed with saturated brine, then dried over anhydrous sodium sulfate. The organic phase was filtered off and then concentrated under reduced pressure. The obtained residue was purified by silica gel chromatography to afford a brown solid (69 mg, 45%). 1H NMR 400 MHz (DMSO-d6) δ 7.73 (d, J = 2.4 Hz, 1H), 7.61 (s, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.40 (dd, J = 8.4, 4.2 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 8.8 Hz, 1H), 6.56 (d, J = 8.4 Hz, 1H), 6.51 (d, J = 2.8 Hz, 1H), 5.98 (dd, J = 8.8, 2.4 Hz, 1H), 3.75 (s, 3H), 3.01 (t, J = 5.2 Hz, 4H), 2.90 (t, J = 5.2 Hz, 4H), 2.55 (s, 3H), 2.44 (t, J = 4.8 Hz, 4H), 2.22 (s, 3H), 1.62−1.56 (m, 4H), 1.50−1.45 (m, 2H). LC−MS m/z: 533.42 (M + H)+. Step d: 6-(3-Amino-4-methylphenoxy)-N-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)-5(piperidin-1-yl)pyridin-2-amine (53b). Platinum(IV) oxide (6 mg) was added to the solution of N-(2methoxy-4-(4-methylpiperazin-1-yl)phenyl)-6-(4-methyl-3-nitrophenoxy)-5-(piperidin-1-yl)pyridin-2-amine (60 mg, 0.11 mmol) in MeOH/THF (2/2 mL). The reaction mixture was stirred at RT overnight under a hydrogen atmosphere. The reaction mixture was filtrated through celite and concentrated to afford the crude product, which was used without further purification. 1H NMR (400 MHz, DMSO-d6) δ 7.42 (s, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H), 6.51 (d, J = 2.8 Hz, 1H), 6.46 (d, J = 8.4 Hz, 1H), 6.33 (d, J = 2.4 Hz, 1H), 6.17 (dd, J = 2.4, 8.0 Hz, 1H), 6.04 (dd, J = 2.8, 8.8 Hz, 1H), 4.92 (s, 2H), 3.76 (s, 3H), 3.02 (t, J = 4.8 Hz, 4H), 2.90 (t, J = 4.8 Hz, 4H), 2.26 (s, 3H), 2.08 (s, 3H), 1.64−1.59 (m, 4H), 1.51−1.46 (m, 2H). LC−MS m/z: 503.32 (M + H)+. Step e: N-(5-((6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-3-(piperidin-1-yl)pyridin-2-yl)oxy)-2-methylphenyl)acrylamide (22). Acryloyl chloride (8 mg, 0.088 mmol) was added dropwise to a solution of 6-(3-amino-4-methylphenoxy)-N-(2methoxy-4-(4-methylpiperazin-1-yl)phenyl)-5(piperidin-1-yl)pyridin2-amine (40 mg, 0.08 mmol) and TEA (17 μL, 0.12 mmol) in anhydrous DCM (2 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h. Water and DCM were added to the reaction solution and separated, and the aqueous phase was extracted with DCM twice. The combined organic phase was washed with saturated brine, then dried over anhydrous sodium sulfate. The organic phase was filtered off and then concentrated under reduced pressure. The obtained residue was dissolved in DCM (2 mL); TFA (0.25 mL) was added and stirred at RT for 30 min to afford the TFA salt of the title compound. DCM and excess TFA were removed in vacuo; then, the residue was dissolved in MeOH and purified by reverse-phase HPLC eluting with acetonitrile/ water (containing 0.15‰ TFA). Fractions containing the product were concentrated in vacuo to remove the solvent to give the title compound (TFA salt, 15 mg, 24%) as a gray solid. 1H NMR (400 MHz, CDCl3) δ 7.71 (s, 1H), 7.39 (d, J = 8.8 Hz, 1H), 7.33−7.31 (m, 1H), 7.18 (d, J = 8.8 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.45 (d, J = 2.8 Hz, 1H), 6.43 (s, 1H), 6.38−6.34 (m, 2H), 6.25 (dd, J = 16.8, 10.0 Hz, 1H), 6.19 (dd, J = 8.8, 2.4 Hz, 1H), 5.69 (d, J = 10.0 Hz, 1H), 3.76 (s, 3H), 3.09 (t, J = 5.2 Hz, 4H), 2.96 (t, J = 5.2 Hz, 4H), 2.58 (t, J = 4.8 Hz, 4H), 2.34 (s, 3H), 2.27 (s, 3H), 1.72−1.68 (m, 4H), 1.54−1.48 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 163.46, 155.18, 153.47, 149.17, 148.86, 146.05, 136.11, 131.19, 130.73, 130.06, 129.13, 127.65, 124.68, 124.43, 119.07, 118.82, 116.63, 108.18, 102.66, 101.08, 55.59, 55.25, 52.58, 50.44, 46.11, 26.32, 24.32, 17.51. HRMS C32H40N6O3 (M + H)+ calcd mass, 557.3240; found, 557.3245. N-(5-((3-(3-Hydroxypiperidin-1-yl)-6-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-2-yl)oxy)-2-methylphenyl)acrylamide (23). (±)3-((tert-Butyldimethylsilyl)oxy)piperidine was prepared according to methods mentioned in preparation of (±)-3(tert-butyldimethylsilyloxy)pyrrolidine hydrochloride in compound 21. Compound 23 was prepared according to the method of preparation of compound 21 using (±)3-((tert-butyldimethylsilyl)oxy)piperidine to replace (±)-3-(tert-butyldimethylsilyloxy)pyrrolidine in step b and purified by reverse-phase flash chromatography eluting with acetonitrile/water as a white solid (49 mg, 36.1% over the last two steps). 1H NMR (400 MHz, CDCl3) δ 7.79 (s, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.21−7.18 (m, 2H), 7.11 (s, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.49−6.47 5915



Article

52.41, 46.44, 42.11, 17.62. HRMS C33H35N5O3 (M + H)+ calcd mass, 550.2818; found, 550.2834. N-(2-Methyl-5-((6-((4-(4-methylpiperazin-1-yl)phenyl)amino)-3(piperidin-1-yl)pyridin-2-yl)oxy)phenyl)acrylamide (27). This compound was prepared according to the preparation method of compound 22 using 4-(4-methylpiperazin-1-yl)aniline in step c. The product was purified by reverse-phase HPLC eluting with acetonitrile/water (containing 0.15‰ TFA) as a gray solid (TFA salt, 13 mg, 11.6%). 1 H NMR (400 MHz, DMSO-d6) δ 9.85 (s, 1H), 9.58 (s, 1H), 7.49 (s, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.12 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 8.8 Hz, 2H), 6.58 (dd, J = 16.8, 10.0 Hz, 1H), 6.46 (d, J = 8.4 Hz, 1H), 6.22 (dd, J = 16.8, 2.0 Hz, 1H), 5.75 (dd, J = 10.0, 2.0 Hz, 1H), 3.61 (d, J = 12.8 Hz, 2H), 3.51 (d, J = 12.0 Hz, 2H), 3.18− 3.10 (m, 2H), 2.86−2.80 (m, 5H), 2.30 (s, 3H), 1.81 (bs, 4H), 1.57 (bs, 2H). HRMS C31H39N6O2 (M + H)+ calcd mass, 527.3134; found, 527.3146. N-(5-((6-((2-Methoxy-6-(4-methylpiperazin-1-yl)pyridin-3-yl)amino)-3-(piperidin-1-yl)pyridin-2-yl)oxy)-2-methylphenyl)acrylamide (28). 2-Methoxy-6-(4-methylpiperazin-1-yl)pyridin-3amine (55b) was prepared following the patent: CN105218561, 2016. Compound 28 was prepared according to the preparation method of 22 using 2-methoxy-6-(4-methylpiperazin-1-yl)pyridin-3-amine in step c. The product was purified by reverse-phase flash chromatography eluting with MeOH/water (containing 0.15‰ TFA) as a gray solid (TFA salt, 19 mg, 18.7%). 1H NMR (400 MHz, CD3OD) δ 7.81 (d, J = 8.8 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 2.4 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.02 (dd, J = 8.0, 2.4 Hz, 1H), 6.57 (d, J = 8.0 Hz, 1H), 6.54 (dd, J = 16.8, 10.0 Hz, 1H), 6.36 (dd, J = 16.8, 1.6 Hz, 1H), 6.00 (d, J = 8.8 Hz, 1H), 5.80 (dd, J = 10.0, 1.6 Hz, 1H), 4.33 (bs, 2H), 3.90 (s, 3H), 3.72 (bs, 4H), 3.58 (bs, 2H), 3.13 (bs, 4H), 2.95 (s, 3H), 2.36 (s, 3H), 2.06 (bs, 4H), 1.79 (bs, 2H). HRMS C31H40N7O3 (M + H)+ calcd mass, 558.3193; found, 558.3222. N-(5-((6-((4-(4-Acetylpiperazin-1-yl)-2-methoxyphenyl)amino)3-(piperidin-1-yl)pyridin-2-yl)oxy)-2-methylphenyl)acrylamide (29). This compound was prepared according to the preparation method of 22 using 1-(4-(4-amino-3-methoxyphenyl)piperazin-1-yl)ethan-1-one in step c. The product was purified by reverse-phase flash chromatography eluting MeOH/water (containing 0.15‰ TFA) as a gray solid (TFA salt, 25 mg, 31.6%). 1H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H), 7.58 (s, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.26−7.23 (m, 1H), 6.96 (s, 1H), 6.64−6.57 (m, 2H), 6.61 (dd, J = 16.8, 10.0 Hz, 1H), 6.23 (dd, J = 17.2, 2.0 Hz, 1H), 6.06 (d, J = 8.0 Hz, 1H), 5.76 (dd, J = 10.0, 2.0 Hz, 1H), 3.77 (s, 3H), 3.57−3.55 (m, 4H), 3.06−3.01 (m, 4H), 2.30 (s, 3H), 2.05 (s, 3H), 1.88 (bs, 4H), 1.53 (bs, 2H). HRMS C33H41N6O4 (M + H)+ calcd mass, 585.3189; found, 585.3220. N-(5-((6-((4-(4-(Dimethylamino)piperidin-1-yl)-2methoxyphenyl)amino)-3-(piperidin-1-yl)pyridin-2-yl)oxy)-2methylphenyl)acrylamide (30). 1-(4-Amino-3-methoxyphenyl)-N,Ndimethylpiperidin-4-amine (55d) was prepared following the patent: CN106905245, 2017. Compound 30 was prepared according to the preparation method of 22 using 1-(4-amino-3-methoxyphenyl)-N,N-dimethylpiperidin-4amine (55d) in step c to afford the title compound (TFA salt, 22 mg, 20.5%) as a gray solid. The product was purified by reverse-phase flash chromatography eluting with MeOH/water (containing 0.15‰ TFA). 1 H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 9.56 (s, 1H), 7.52 (s, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.26−7.24 (m, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.61 (dd, J = 17.2, 10.0 Hz, 1H), 6.61−6.57 (m, 2H), 6.23 (dd, J = 17.2, 2.0 Hz, 1H), 6.10 (d, J = 8.4 Hz, 1H), 5.76 (dd, J = 10.0, 2.0 Hz, 1H), 3.77 (s, 3H), 3.72−3.69 (m, 4H), 3.30−3.24 (m, 3H), 2.78 (s, 3H), 2.77 (s, 3H), 2.66 (bs, 2H), 2.30 (s, 3H), 2.09−2.06 (m, 2H), 1.86−1.73 (m, 6H), 1.55 (bs, 2H). HRMS C34H45N6O3 (M + H)+ calcd mass, 585.3553; found, 585.3608. N-(5-((6-((5-(4-(Dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-3-(piperidin-1-yl)pyridin-2-yl)oxy)-2-methylphenyl)acrylamide (31). 5-(4-(Dimethylamino)piperidin-1-yl)pyridin-2amine (55e) was prepared following the reference: J. Med. Chem., 2017, 60, 1892−1915. Compound 31 was prepared according to the preparation method of 22 using 5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-amine (55e)

(m, 2H), 6.42−6.37 (m, 2H), 6.30−6.23 (m, 2H), 5.75 (d, J = 10.0, 1H), 3.89−3.87 (m, 1H), 3.81 (s, 3H), 3.12 (bs, 4H), 3.04−3.01 (m, 3H), 2.87 (t, J = 10.4 Hz, 1H), 2.61 (t, J = 5.2 Hz, 4H), 2.37 (s, 3H), 2.31 (s, 3H), 1.93−1.87 (m, 1H), 1.72−1.58 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 163.59, 155.34, 153.55, 149.87, 149.60, 146.52, 136.17, 131.48, 131.16, 130.86, 128.28, 127.88, 124.43, 124.06, 119.48, 118.06, 115.81, 108.28, 102.99, 101.11, 66.18, 58.74, 55.67, 55.27, 52.11, 50.42, 46.15, 31.47, 21.97, 17.47. HRMS C32H40N6O4 (M + H)+ calcd mass, 573.3189; found, 573.3195. N-(5-((6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)3-morpholinopyridin-2-yl)oxy)-2-methylphenyl)acrylamide (24). This compound was prepared according to the preparation method of compound 22 using morpholine to replace piperidine in step b to afford the title compound as a gray solid (23 mg, 59.4%). The product was purified by silica gel column chromatography eluting with DCM/ MeOH. 1H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 7.53 (s, 1H), 7.39 (s, 1H), 7.32−7.23 (m, 3H), 6.83 (dd, J = 8.4, 2.8 Hz, 1H), 6.58 (dd, J = 17.6, 10.8 Hz, 1H), 6.53−6.51 (m, 2H), 6.21 (dd, J = 17.2, 2.0 Hz, 1H), 6.02 (dd, J = 8.8, 2.4 Hz, 1H), 5.73 (dd, J = 10.4, 2.0 Hz, 1H), 3.75 (s, 3H), 3.70 (t, J = 4.8 Hz, 4H), 3.02 (bs, 4H), 2.95 (t, J = 4.8 Hz, 4H), 2.46 (bs, 4H), 2.27 (s, 3H), 2.24 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.19, 154.21, 152.47, 149.70, 149.41, 146.02, 136.93, 131.74, 130.78, 130.27, 126.83, 126.08, 122.71, 119.77, 118.20, 116.77, 106.74, 103.80, 100.32, 66.41, 55.50, 54.72, 50.98, 48.99, 45.71, 17.52. HRMS C31H38N6O4 (M + H)+ calcd mass, 559.3033; found, 559.3063. N-(5-((6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)3-(4-methylpiperazin-1-yl)pyridin-2-yl)oxy)-2-methylphenyl)acrylamide (25). This compound was prepared according to the preparation method of compound 22 using 1-methylpiperazine to replace piperidine in step b to afford the title compound as a brown solid (TFA salt, 18 mg, 19.4%). The product was purified by reversephase HPLC eluting with acetonitrile/water (containing 0.15‰ TFA). 1 H NMR (400 MHz, DMSO-d6) δ 9.53 (s, 1H), 7.73 (s, 1H), 7.41− 7.34 (m, 3H), 7.26 (d, J = 8.4 Hz, 1H), 6.86 (dd, J = 8.4, 2.8 Hz, 1H), 6.59−6.57 (m, 2H), 6.58 (dd, J = 17.2, 10.0 Hz, 1H), 6.22 (dd, J = 17.2, 2.0 Hz, 1H), 6.06 (dd, J = 8.8, 2.4 Hz, 1H), 5.74 (dd, J = 10.4, 2.4 Hz, 1H), 3.77 (s, 3H), 3.67 (bs, 2H), 3.51−3.48 (m, 4H), 3.19−3.14 (m, 4H), 3.03−2.97 (m, 4H), 2.90−2.81 (m, 2H), 2.86 (s, 3H), 2.85 (s, 3H), 2.27 (s, 3H). HRMS C32H42N7O3 (M + H)+ calcd mass, 572.3349; found, 572.3361. N-(5-((6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)3-phenylpyridin-2-yl)oxy)-2-methylphenyl)acrylamide (26). Step a: Tetrakistriphenylphosphine palladium (116 mg, 0.1 mmol), phenylboronic acid (146 mg, 1.2 mmol), and a 2 M sodium carbonate solution (1 mL) were sequentially added to a solution of 3-bromo-6-chloro-2(4-methyl-3-nitrophenoxy)pyridine (47d) (344 mg, 1 mmol) in 1,2dimethoxyethane (5 mL) in a nitrogen atmosphere, and the mixture was stirred at 80 °C for 8 h. Water was added to the reaction solution at RT, followed by extraction with ethyl acetate three times. The combined organic phase was washed with brine, dried over anhydrous sodium sulfate, and filtered. The solvent was then evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give the title product (304 mg, yield: 89%). 6-Chloro-2-(4-methyl-3-nitrophenoxy)-3-phenylpyridine (51f). LC− MS m/z: 341.23 (M + H)+. Steps b−d are the same as those in the method mentioned in preparation of compound 22. The product was purified by silica gel column chromatography eluting with DCM/MeOH as a gray solid (22 mg, 58.4%). 1H NMR (400 MHz, DMSO-d6) δ 9.54 (s, 1H), 8.10 (s, 1H), 7.67−7.60 (m, 3H), 7.45−7.38 (m, 4H), 7.30−7.24 (m, 2H), 6.88 (dd, J = 8.4, 2.4 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 6.62 (d, J = 2.4 Hz, 1H), 6.57 (dd, J = 16.8, 10.0 Hz, 1H), 6.22 (dd, J = 16.8, 2.0 Hz, 1H), 6.10 (dd, J = 8.8, 2.4 Hz, 1H), 5.74 (dd, J = 10.0, 2.0 Hz, 1H), 3.80 (s, 3H), 3.72 (d, J = 12.8 Hz, 2H), 3.52 (d, J = 12.0 Hz, 2H), 3.19−3.11 (m, 2H), 2.90 (d, J = 12.0 Hz, 2H), 2.86 (s, 3H), 2.26 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.15, 158.30, 158.15, 157.98, 154.01, 152.53, 149.94, 144.89, 140.60, 137.00, 136.67, 131.72, 130.88, 128.45, 128.28, 126.87, 126.58, 126.38, 122.85, 120.87, 118.43, 116.97, 111.96, 107.14, 104.96, 100.89, 55.70, 5916



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in step c to afford the title compound (TFA salt, 32 mg, 14%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.79 (s, 1H), 9.76 (s, 1H), 7.72−7.64 (m, 2H), 7.46−7.45 (m, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 9.6 Hz, 1H), 7.04 (dd, J = 8.0, 2.4 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 6.55 (dd, J = 17.2, 10.0 Hz, 1H), 6.22 (dd, J = 17.2, 2.0 Hz, 1H), 5.77 (dd, J = 10.0, 2.0 Hz, 1H), 3.69 (d, J = 8.8 Hz, 2H), 3.34−3.27 (m, 1H), 3.14 (bs, 4H), 2.80 (s, 3H), 2.78 (s, 3H), 2.66 (t, J = 12.0 Hz, 2H), 2.31 (s, 3H), 2.07 (d, J = 12.0 Hz, 2H), 1.75−1.64 (m, 6H), 1.58 (bs, 2H). HRMS C34H45N6O3 (M + H)+ calcd mass, 556.3400; found, 556.3466. N-(5-((6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)3-(pyrrolidin-1-yl)pyridin-2-yl)oxy)-2-(trifluoromethyl)phenyl)acrylamide (32). Step a: This is according to the method of step a mentioned in Scheme 1. 3-Bromo-6-chloro-2-(3-nitro-4(trifluoromethyl)phenoxy)pyridine (59), LC−MS m/z: 396.90 (M + H)+. Step b: This is according to the method of step c mentioned in Scheme 1. 5-((3-Bromo-6-chloropyridin-2-yl)oxy)-2(trifluoromethyl)aniline (60), LC−MS m/z: 367.08 (M + H)+. Step c: 5-((3-Bromo-6-chloropyridin-2-yl)oxy)-2-(trifluoromethyl)aniline (60) (284 mg, 0.77 mmol) was dissolved in THF (4 mL), (Boc)2O (387 mg, 1.78 mmol), and DMAP (9 mg, 0.077 mmol), and TEA (195 mg, 1.93 mmol) was added; the reaction mixture was stirred at RT overnight. The solvent was removed under reduced pressure, and the residue was partitioned between DCM and saturated aqueous sodium bicarbonate solution. The aqueous phase was extracted with DCM three times. The combined organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by silica gel chromatography to afford the solid product (235 mg, yield: 65%). tert-Butyl (5-((3-bromo-6-chloropyridin-2-yl)oxy)-2(trifluoromethyl)phenyl)carbamate (61), LC−MS m/z: 467.35 (M + H)+. Steps d and e: This is according to the method of steps b and c mentioned in Scheme 3. 6-(3-Aminophenoxy)-N-(2-methoxy-4-(4methylpiperazin-1-yl)phenyl)-5-(pyrrolidin-1-yl)pyridin-2-amine (63a). LC−MS m/z: 643.02 (M + H)+. Step f: tert-Butyl(3-((6-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-3-(pyrrolidin-1-yl)pyridin-2-yl)oxy)phenyl)carbamate (63a) was dissolved in DCM/TFA (2:1); the reaction mixture was stirred at RT for 2 h. The solvent and excess TFA were removed in vacuo. The crude product was used for next transformation without further purification. 6-(3-Amino-4-(trifluoromethyl)phenoxy)-N-(2methoxy-4-(4-methylpiperazin-1-yl)phenyl)-5-(pyrrolidin-1-yl)pyridin-2-amine (64a). LC−MS m/z: 542.58 (M + H)+. Step g: This is according to the method of step e mentioned in Scheme 3. The crude product was purified by silica gel column chromatography eluting with DCM/MeOH to afford the product as a gray solid (16 mg, 29%). 1H NMR (400 MHz, DMSO-d6) δ 9.76 (s, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.47 (s, 1H), 7.32 (d, J = 8.8 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 7.10 (dd, J = 8.8, 2.4 Hz, 1H), 6.62 (d, J = 8.4 Hz, 1H), 6.53 (d, J = 2.4 Hz, 1H), 6.54 (dd, J = 17.2, 10.4 Hz, 1H), 6.23 (dd, J = 17.2, 2.0 Hz, 1H), 6.10 (dd, J = 8.8, 2.8 Hz, 1H), 5.76 (dd, J = 10.4, 2.0 Hz, 1H), 3.76 (s, 3H), 3.16 (t, J = 6.4 Hz, 4H), 3.00 (t, J = 4.8 Hz, 4H), 2.42 (t, J = 4.8 Hz, 4H), 2.21 (s, 3H), 1.85−1.82 (m, 4H). HRMS C31H35F3N6O3 (M + H)+ calcd mass, 597.2801; found, 597.2819. N-(5-((6-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)3-(piperidin-1-yl) pyridin-2-yl)oxy)-2-(trifluoromethyl)phenyl)acrylamide (33). This compound was prepared according to the preparation method of 32 using piperidine instead of pyrrolidine to afford the title compound. The product was purified by reverse-phase HPLC eluting with acetonitrile/water (containing 0.15‰ TFA) as a gray solid (TFA salt, 30 mg, 18.2% over the last two steps). 1H NMR (400 MHz, DMSO-d6) δ 9.82 (s, 1H), 9.64 (s, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.38−7.30 (m, 2H), 7.22 (s, 1H), 6.65 (d, J = 8.4 Hz, 1H), 6.62 (d, J = 2.4 Hz, 1H), 6.54 (dd, J = 17.2, 10.0 Hz, 1H), 6.23 (dd, J = 17.2, 2.0 Hz, 1H), 6.12 (d, J = 9.2 Hz, 1H), 5.78 (dd, J = 10.4, 2.0 Hz, 1H), 3.78 (s, 3H), 3.68 (d, J = 13.2 Hz, 2H), 3.52 (d, J = 12.0 Hz, 2H), 3.16−3.08 (m, 2H), 2.89−2.82 (m, 5H), 1.67 (bs, 4H), 1.51 (bs, 2H). HRMS C32H38F3N6O3 (M + H)+ calcd mass, 611.2957; found, 611.2964.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.9b00631. Summary of liver microsome stability data of the selected compounds; exploration of electrophilic warheads; cytochromes P450 (CYPs) inhibition and plasma protein binding study of the selected compounds; EGFR inhibitory activity of the selected compounds; tissue distribution of compound 22 in mouse (PO); some in vivo data of compound 22; preparation and NMR data of compounds 65−72; 1H and 13C NMR spectra of compounds (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86-10-80726688. Ext 8575. ORCID

Zhiyuan Zhang: 0000-0002-2910-1986 Author Contributions †

W.H. and H.S. contributed equally to this work.

Author Contributions

All authors have given approval to the final version of the manuscript. Z.Z. conceived the project; Z.Z. and W.H. designed, W.H. synthesized all of the compounds; H.S. designed and conducted the biochemical experiments in vitro and in vivo; H.S. tested liver microsomes stability and prepared compound formulations, Y.M. tested CYP inhibition, PPB, and pharmacokinetic parameters; H.S., C.L., and W.H. conducted mice experiments; W.H. and H.S. drafted the manuscript. Funding

This work was supported by the National Major Scientific and Technological Special Project for “Significant New Drugs Development” during the 12th 5 year plan period 2013ZX0950910 from the Chinese Ministry of Science and Technology. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Kang Wang for the help with mice assays. We thank Li Li for the help with liver microsome stability test. We thank the metabolomics center and proteomics center for mass analysis.

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ABBREVIATIONS CatC, cathepsin C; NSPs, neutrophil serine proteases; EGFR, epidermal growth factor receptor; SAR, structure−activity relationship; Compd, compound; Enz, enzyme; COPD, chronic obstructive pulmonary disease; PK, pharmacokinetic; WT, wild type; RT, room temperature; t1/2, half-life; Pd2(dba)3, tris(dibenzylideneacetone)dipalladium(0); BINAP, (±)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl

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REFERENCES

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Identification and Optimization of Novel Cathepsin C Inhibitors Derived

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