Synthesis and biological evaluation of 4-(pyridin-4-oxy)-3-(3,3-difluorocyclobutyl)- pyrazole derivatives as novel potent transforming growth factor-β type 1 receptor inhibitors
Guofeng Xu, Yan Zhang, Hai Wang, Zhuang Guo, Xiaowei Wang, Xue Li, Shaohua Chang, Tianwen Sun, Zhuangzhuang Yu, Tianwei Xu, Liwen Zhao, Yazhou Wang, Wenying Yu
PII: S0223-5234(20)30324-X
DOI: https://doi.org/10.1016/j.ejmech.2020.112354
Reference: EJMECH 112354
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 December 2019 Revised Date: 13 April 2020 Accepted Date: 16 April 2020
Please cite this article as: G. Xu, Y. Zhang, H. Wang, Z. Guo, X. Wang, X. Li, S. Chang, T. Sun, Z. Yu, T. Xu, L. Zhao, Y. Wang, W. Yu, Synthesis and biological evaluation of 4-(pyridin-4-oxy)-3- (3,3-difluorocyclobutyl)-pyrazole derivatives as novel potent transforming growth factor-β type 1 receptor inhibitors, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112354.
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Graphical Abstract
Synthesis and Biological Evaluation of
4-(Pyridin-4-oxy)-3-(3,3-difluorocyclobutyl)-pyrazole Derivatives as Novel Potent
Transforming Growth Factor-β Type 1 Receptor Inhibitors
Guofeng Xua,b,#, Yan Zhangb,#, Hai Wangb, Zhuang Guob, Xiaowei Wangb, Xue Lib, Shaohua Changb, Tianwen Sunb, Zhuangzhuang Yub, Tianwei Xub, Liwen Zhaob,*,Yazhou Wangb,*and Wenying Yua,*
Synthesis and Biological Evaluation of
4-(Pyridin-4-oxy)-3-(3,3-difluorocyclobutyl)-pyrazole Derivatives as Novel Potent
Transforming Growth Factor-β Type 1 Receptor Inhibitors
Guofeng Xua,b,#, Yan Zhangb,#, Hai Wangb, Zhuang Guob, Xiaowei Wangb, Xue Lib, Shaohua Changb, Tianwen Sunb, Zhuangzhuang Yub, Tianwei Xub, Liwen Zhaob,*,Yazhou Wangb,*and Wenying Yua,*
aState Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China.
bNanjing Sanhome Pharmaceutical Co. Ltd., No. 99, West Yunlianghe Road, Jiangning District, Nanjing, 210049, People’s Republic of China.
*Corresponding Authors. Tel/Fax: +86-25-83271405; E-mail: [email protected] (W. Yu), [email protected] (Y. Wang), [email protected] (L. Zhao)
#These authors contributed equally.
Abstract: Inhibition of transforming growth factor β (TGF-β) type 1 receptor (ALK5) provides a feasible approach for the treatment of fibrotic diseases and malignant tumors. In this study, we designed and synthesized a new series of
4-(pyridin-4-oxy)-3-(3,3-difluorocyclobutyl)-pyrazole derivatives, and evaluated biologically as TGF-β type 1 receptor inhibitors. The most potent compound 15r inhibited the ALK5 enzyme and NIH3T3 cell viability with IC50 values of 44 and 42.5 nM, respectively. Compound 15r also displayed better oral plasma exposure and excellent bioavailability than LY-3200882, and in vivo inhibited 65.7% of the tumor growth in a CT26 xenograft mouse model.
Keywords: ALK5; TGF-β inhibitor; Antitumor activity.
Introduction
The transforming growth factor β (TGF-β) superfamily of cytokines is evolutionarily conserved and actively involved in numerous cellular processes, including cell proliferation, differentiation, self-renewal and apoptosis [1]. TGF-β1 is a prototypic cytokine of TGF-β family which transduces signals through two highly conserved single transmembrane serine/threonine kinase receptors termed, the type 1 receptors (also known as activin-like kinase 5 or ALK5) and type 2 receptors. When the signaling cascade is initiated, TGF-β1 dimers bind to type 2 receptor and induce
the phosphorylation of GS domain of type 1 receptor, ALK5. The ALK5 receptor subsequently recruits and phosphorylates Smad2 or Smad3 at carboxy-terminal serines. The phosphorylated Smad proteins form heteromeric complexes with the common mediator, Smad 4 and translocates into the nucleus to regulate the expression of specific genes, thereby participating in the process of angiogenesis, epithelial-mesenchymal transition and extracellular matrix remodeling [2-4]. Moreover, TGF-β balances the generation and effector functions of many immune cell types in adaptive immunity and innate immune system [5]. Latest research revealed that TGF-β played a central role in tumor immune evasion and poor responses to cancer immunotherapy. Combination of TGF-β signaling blockade and checkpoint therapies, primary tumors and metastases rendered susceptible to anti-PD-1/L1 therapy and immune response was enhanced therefore [6]. Unlike its tumor suppressor function in normal tissue, deregulation of TGF-β signaling has been implicated in various diseases. Current studies showed that consecutively activation of TGF-β1 is closely related to pulmonary fibrosis[7], liver fibrosis[8] and hepatocellular cancer[9]. Thus, inhibition of TGF-β signaling pathway is considered to present a feasible approach to alleviate progression of these diseases.
Fig. 1. Representative small-molecular ALK5 inhibitors.
Along with the attempt to target TGF-β signaling pathway, several small-molecular ATP-competitive ALK5 inhibitors have come to light. Compound 1 (SB-505124) [10], 2 (SD-208) [11], 3 (GFH018) [12], 4 (AZ12601011) [13], 5 (TEW-7197) [14], 6 (Galunisertib) [15] and 7 (LY-3200882) [16] have emerged over the past two decades (Fig. 1). Among them, Lilly’s Galunisertib, which has entered advanced clinical trials, can notably inhibit the invasion and metastasis of tumor cells
in vivo through blocking TGF-β-mediated signal [15]. It also inhibited p38α due to the fact that p38α MAP kinase domain was highly homologous to that of ALK5 [17]. However, there are still concerns about the long-term tolerability of p38α blockade. In p38α knockout mice, increased lung/liver tumorigenesis was observed. In clinical trials, inhibition of p38α might increase the risk of potentially serious side effects and several inhibitors failed therefore [18, 19].
As a new follow-up generation of ALK5 inhibitor, LY-3200882 has demonstrated strong ALK5 inhibitory activity with high kinase selectivity improvement against p38α, following with remarkable anti-cancer and anti-fibrotic efficacy in various models. Nevertheless, it is to be further improved due to some flaw such as moderate oral bioavailability. LY-3200882 was selected as a lead compound. A series of 4-pyrazolyl- oxy-2-aminopyridine derivatives were designed and synthesized to provide an opportunity for further optimization of pharmacokinetics properties and in vivo efficacy.
Initially, we noted that the tetrahydro-2H-pyran moiety in LY-3200882 was readily underwent oxidative metabolism (see Supplementary Material for details). Thus the effect of various substitutions in R1 position was first evaluated (Fig. 2). This resulted in the identification of 1,1-difluorocyclobutanyl group as optimal substituent according to the ALK5 inhibitory activity and NIH3T3 cellular potency. Further structural modifications at the R2 position of solvent channel were executed. Ultimately, compound 15r was discovered with better performance than LY-3200882 in comprehensive consideration of in vitro potency, p38α selectivity, pharmacokinetics profiles and in vivo efficacy.
Fig. 2. Current design of new TGF-βR1 inhibitors
Chemistry
A series of derivatives were prepared as shown in Scheme 1. Commercially available carboxylic acid 8 were treated with N,O-dimethylhydroxylamine hydrochloride to afford Weinreb’s amide 9. Compound 9 reacted with methyl magnesium bromide in anhydrous tetrahydrofuran to yield ketone 10. Bromination of compound 10 led to α-bromoketone 11, which was then treated with 2-chloropyridin-4-ol hydrochloride under base conditions to give compound 12.
Condensation of compound 12 with N,N-dimethylformamide dimethyl acetal (DMF-DMA) gave the crude enamine intermediates, which were cyclized directly to produce pyrazole derivatives 13. Chan-Lam coupling of 13 with cyclopropylboronic acid in the presence of Cu(OAc)2 and 2,2′-bipyridine afforded the key intermediate 14. Divergent Buchwald coupling [20] of compound 14 with a range of amino fragments under palladium-catalyzed conditions led to target compounds 15a-r and 16a-j.
Scheme 1. Reagents and conditions: (i) N,O-dimethylhydroxylamine hydrochloride, HATU, DIPEA, THF, 81-93%; (ii) methyl magnesium bromide, THF, 0 °C, 81-93% (iii) Br2, H2SO4, MeOH; (iv) 2-chloropyridin-4-ol, K2CO3, DMF, 28-99%; (v) DMF-DMA, DMF, reflux; (vi) hydrazine monohydrate, DMF; (vii) cyclopropylboronic acid, Cu(OAc)2, 2,2′-bipyridine, O2, Na2CO3, DCE, 80 °C, 47-94%; (viii) Pd(OAc)2, Cs2CO3, Xantphos, 1,4-dioxane, 100 °C, 4 h, 3-70%; (ix) Pd2(dba)3, sodium tert-butoxide, Xphos, Tert-butanol, 100 °C, overnight, 20-65%; (x) Pd2(dba)3, sodium benzenolate, Xantphos, 1,4-dioxane, 100 °C, overnight, for 16b only, 19%.
Results and Discussion
Binding mode hypothesis. The published literature was reviewed [14, 21-27] and the binding modes of ALK5 inhibitors shared several conserved interactions. There was a hydrogen bond acceptor in the skeleton of ATP-competitive inhibitors, usually from a pyridine or quinoline moiety in the inhibitor, which binds to the kinase hinge region (His283 in ALK5). There also existed a water-mediated hydrogen bond between the nitrogen-containing heterocycles and “selectivity pocket” [24, 26]. In
addition, substitutions at the ortho-position of the pyridine ring extended to “solvent channel”. Thus, we designed and synthesized a variety of compounds to investigate the effects of different side chains in R1 and R2 positions.
Structure-activity relationship (SAR) studies. Guided by the binding mode hypothesis, we examined the structure-activity relationship of the series. LY-3200882 was used as starting point and a reference compound for calibration of assay results. The result of optimization in R1 position was shown in Table 1. It was found that introduction of phenyl groups led to slightly enhanced ALK5 inhibitory activity (15a – c, 15f, 15g). Electron-withdrawing or electron-donating groups had almost the same performance. However, derivatives 15d and 15e bearing amide and carboxyl brought out a major decrease in inhibitory activity, which might arise from strong polarity or hydrogen donors in these groups. N-heterocycle substituent derivatives were well tolerant with an exception of 15h. The two ortho-methyl groups on the isoxazole of compound 15h form steric hindrance and accordingly perpendicular conformation, which might account for the significant decrease in enzymatic activity. In addition, aliphatic substitutions were explored and exhibited a similar pattern compared with aromatic groups. Compounds 15o and 15p abolished the binding affinity, which might be similar to that of 15h. To our delight, compounds 15l and 15q reached single digit nanomolar-level ALK5 inhibitory activity. Besides, cellular antiproliferation assay of several active compounds were performed. Among this series, compound 15r bearing 1,1-difluorocyclobutyl exhibited 2-fold enhanced NIH3T3 cellular potency relative to LY-3200882. As a result, 1,1-difluorocyclobutyl was selected to fix at the R1 position for further modification.
Table 1
Optimization of R1 position
Compound R1 ALK5 IC50 (nM)a NIH3T3 IC50 (nM) a
15a phenyl 26.2 28.9
15b 4-fluorophenyl 19.7 119
15c 4-cyanophenyl 31.4 162
15d 4-carbamoylphenyl 282 2036
15e 4-carboxyphenyl 814 >10000
15f 21.7 91.9
15g 44.2 225
15h 6736 >10000
15i 30.2 188
15j pyridin-4-yl 84.0 326
15k 4,4-difluoro-cyclohexyl 13.2 86.5
15l 4-hydroxy-4-methylcyclohexyl 8.2 136
15m O 47.2 116
15o 528 6689
15p 308 748
15q 8.2 102
15r 44.0 42.5
LY-3200882 38.2 82.9
a Values are means of two independent experiments, and standard deviation values were ignored for clarity; /
means not tested.
It is reported that modifications in the solvent channel position might offer the potential to improve the cellular potency and physicochemical properties [23, 25]. Thus several hydrophilic groups were introduced to replace the tertiary alcohol in R2 position. As shown in Table 2, most of small substituent were well tolerant. Compounds 16a-g exhibited increased ALK5 inhibitory potency than that of 15r and LY-3200882, with IC50 varied from 11.4 to 34.6 nM. The two analogs 16h and 16i bearing larger size of tertiary alcohol showed comparable activity. However, compound 16j displayed significantly decreased potency, perhaps due to the much bigger 3,5-dimethyl morpholine. Meanwhile, it seems that cellular potency were associated with ClogP values closely. The much hydrophobic trifluoromethyl of compound 16f (ClogP = 3.92) and 2-azaspiro[3.3]heptane of compound 16i (ClogP = 5.25) might result in their lower potency, which was consistent with the hydrophilic environment in the solvent channel.
Table 2
Optimization of R2 position
Compound
R2
ALK5 IC50
(nM)a
NIH3T3 IC50 (nM) a
CLogP b
15r 44.0 42.5 2.64
16a 34.6 90.2 3.29
16b 13.2 28.4 2.94
16c 26.0 45.3 2.84
16d 11.4 103 2.26
16e 27.5 57.1 3.08
16f 24.3 127 3.92
16g 31.4 134 1.59
16h 69.2 116 3.41
16i 61.3 283 5.25
16j 416 972 3.07
LY-3200882 38.2 82.9 2.59
a Values are means of two independent experiments, and standard deviation values were ignored for clarity; /
means not tested. b ClogP Calculated using ACD/LogP DB (ACD/Labs 10).
To evaluate the druggability of these new ALK5 inhibitors, the most potent compounds were further investigated by pharmacokinetics (PK) profiles (Table 3). After administration at an intravenous dose of 1 mg/kg and oral dose of 10 mg/kg in mice, all the area-under-curve (AUC) of compounds 15r, 16a, 16b, 16c and 16d reached 1223.4 to 4831.3 h*ng/mL, larger than that of LY-3200882 (943.9 h*ng/mL).
Such more plasma exposure is speculated to be contributed to better metabolic stability of 1,1-difluorocyclobutyl than tetrahydro-2H-pyran moiety, as expected. Particularly, compound 16b had better pharmacokinetic profiles of clearance rates, maximum concentration, oral plasma exposure than that of 15r. However, compound 16b was temporary inferior due to the chiral center, which may result in difficulties in CMC development subsequently. Compounds 16f and 16h bearing trifluoromethyl showed poor PK characteristics both by iv and po administration. Noted that 16f had possessed much higher lipophilicity (ClogP = 3.92), such lower plasma exposure might be caused by the larger volume of distribution (2.8 L/kg) and higher systemic clearance (6.2 L/h/kg). This might also explain for unsatisfactory oral bioavailability of compound 16h (CLogP = 3.41). Overall, compound 15r displayed good oral plasma exposure and excellent bioavailability with AUC > 2000 h*ng/mL and F > 80%, respectively.
Table 3
Pharmacokinetic profiles of selected compounds a
IV (1 mg/kg) PO (10 mg/kg)
Compd.
T1/2b (h)
CLc (L/h/kg)
Vssd (L/kg)
AUC(0-24h)e (h*ng/mL)
T1/2 (h)
Cmaxf (ng/mL)
AUC(0-24h) (h*ng/mL)
F g (%)
15r 0.5 4.9 3.0 195.2 1.8 926.0 2351.2 120.5
16a 1.8 4.6 6.9 205.7 1.8 549.0 1621.7 78.8
16b 0.6 2.2 1.5 408.7 1.3 1552.0 4831.3 118.2
16c 0.6 3.1 2.2 293.0 0.9 736.4 2059.1 70.3
16d 0.4 4.6 2.3 220.0 0.9 676.5 1223.4 55.6
16f 0.4 6.2 2.8 158.8 0.7 83.0 107.5 6.8
16h 0.2 5.3 1.3 180.4 0.9 450.7 692.3 38.4
LY-3200882 0.4 5.2 2.3 211.7 1.1 747.0 943.9 45.6
a Values are means of three independent experiments, and standard deviation values were ignored for clarity. b Half-life. c Clearance. d Volume of distribution at steady state. e AUC from 0 to 24 h. f Maximum concentration observed. g Oral bioavailability.
In addition, compound 15r also showed kinase selectivity against p38α inhibition with an IC50 >10000 nM. Considering its superior potency, remarkable oral exposure and improved oral bioavailability, we further evaluated in vivo antitumor efficacy of compound 15r.
Evaluation of in vivo efficacy was then performed in a BALB/C mouse model, which bears CT26 mouse colon carcinoma (Fig. 3). LY-3200882 was used as a
reference and both compounds were administrated 60 mg/kg BID via oral gavage starting on day 7 post-implantation and continued for 21 days. Compared with the positive control LY-3200882, a statistically significant tumor growth delay in CT26 model was observed. Oral administration of compound 15r efficiently suppressed tumor growth (tumor growth inhibition = 65.7%) and tumor weight. Dose at 60 mg/kg of 15r was well tolerated, with no mortality or obvious body weight loss observed during the study.
Fig. 3. In vivo CT26 xenograft model study of compound 15r and LY-3200882. (A) The tumor growth curve of three groups, including vehicle control, 15r (60mg/kg, bid) and LY-3200882 (60 mg/kg, bid). (B) The tumor weight of three groups. (C) The body weights of the group mice over time. *P < 0.05 vs control, **P < 0.01 vs control. To rationalize the observed activities of these 4-(pyridin-4-oxy)-3-(3,3-difluorocyclobutyl)-pyrazole derivatives against TGF-β R1, molecular modeling was performed. Due to the complex crystal of LY-3200882 is undisclosed, a structure bound the closest 4-pyridinoxy-2-anilinopyridine-based ALK5 inhibitors was used for docking studies [23] (Fig. 4). It was found that compound 15r overlaid well with LY-3200882 on the hinge region, selectivity pocket, and solvent channel, respectively. The typically conserved interactions in the ATP-competitive kinase inhibitors were observed, namely two hydrogen bonds formed by 2-aminopyridine moiety with the hinge residue His-283. Tertiary alcohol formed another two hydrogen bonds with the residues Glu-284 and Arg-294. Besides, the pyrazole of 15r formed a critically strong hydrogen bond network through a water bridge with Glu-245, Tyr-249 and Asp351 in the selectivity pocket. The 1,1-difluorocyclobutanyl of 15r superimposed with the chair-configurational tetrahydro-2H-pyran in the relatively tight but variable space. In accordance with the SAR of table 1, small to moderate sized substituents of tetrahydro-2H-pyran were well tolerant. Fig. 4. Predicted binding mode of compound 15r (green) overlaid with the proposed binding mode of LY-3200882 (magentas) in the TGF-βR1 ATP binding site (PDB id: 2wou). Conclusion In conclusion, a new series of TGF-βR1 inhibitors based on 4-(pyridin-4-oxy)-3-(3,3-difluorocyclobutyl)-pyrazole scaffold have been designed and synthesized. Through SAR study, quite a few inhibitors were found to display stronger potency than LY-3200882. Among them, the most potent compound 15r also exhibited excellent selectivity against p38α, which might reduce the undesired side effects. The substructural 1,1-difluorocyclobutyl at the R1 position was of critical importance for the observed cellular potency and particularly for favorable PK properties. The in vivo investigation indicated that the most potent compound 15r possessed favorable PK properties and displayed better anti-tumor efficacy. This study provides a potential candidate for further comprehensive safety evaluation. 4.Experimental Section 4.1Chemistry General. All starting materials were commercially available and used without further purification. All reactions were carried out without purification unless otherwise stated. All reactions were monitored by thin-layer chromatography (TLC) carried out on silica gel plates (Yantai Jiangyou silica gel development Co. Ltd., HSGF254) and components were visualized using UV light or iodine vapor. Solvent removal refers to rotary evaporation under reduced pressure at 40-45 °C. All HRMS data were obtained using a Thermo Q Exacetive HPLC/MS system. The mass spectra were obtained on an Agilent 1290 LC-MS system. The 1H NMR and 13C NMR spectra were collected on a 400 MHz Bruker spectrometer. Flash column chromatography was performed using silica gel (Qingdao Haiyang Chemical Co. Ltd, ZCX-II, 200-300 mesh) on an EZ Plus 100D preparative liquid chromatography system. Purity was ascertained from AUC by HPLC using a Waters e2695 system (254 nM). Molecular docking was carried out using Glide 5.9 in Schrödinger 2013 suite, with OPLS_2005 as force field and Extra Precision (XP) as algorithm. 4.1.1General procedure for synthesis of compounds 15a-c, 15f-m, 15q, 15r, 16a, 16d, 16g-j. A schlenk tube was charged with the previous intermediate, divergent amines, palladium (II) acetate (0.1 eq.), 4,5-bis(diphenylphosphino)-9,9-dimethyl xanthene (0.2 eq.) and cesium carbonate (2 eq.). The vessel was evacuated and backfilled with argon. 1,4-dioxane was added. Then the schlenk tube was heated to 100 °C for 4 hours. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate, filtered and the filtrate was concentrated in vacuo. The residue was purified by a silica gel column chromatography to give target compounds. 4.1.2General procedure for synthesis of compounds 15n, 15q, 16c, 16e, 16f. A schlenk tube was charged with the previous intermediate, divergent amines, tris (dibenzylideneacetone)dipalladium (0.1 eq.), X-phos (0.2 eq.) and sodium tert-butoxide (3 eq.). The vessel was evacuated and backfilled with argon. Tert-butanol was added. Then the schlenk tube was heated to 100 °C overnight. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate, filtered and the filtrate was concentrated in vacuo. The residue was purified by a silica gel column chromatography to give target compounds. 4.1.32-(4-((4-((1-cyclopropyl-3-phenyl-1H-pyrazol-4-yl)oxy)pyridin-2-yl)amino) pyridine-2-yl)propan-2-ol (15a). Following the general procedure, compound 15a was obtained in 30% yield. 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 5.6 Hz, 1H), 8.15 (d, J = 5.6 Hz, 1H), 7.76 (d, J = 7.2 Hz, 2H), 7.44 (s, 1H), 7.36 – 7.27 (m, 4H), 7.14 (d, J = 3.6 Hz, 1H), 6.91 (s, 1H), 6.62 (d, J = 3.6 Hz, 1H), 6.43 (s, 1H), 5.05 (s, 1H), 3.66 – 3.58 (m, 1H), 1.49 (s, 6H), 1.18 (s, 2H), 1.05 (d, J = 6.2 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 167.11, 166.69, 155.61, 149.87, 148.30, 148.21, 141.93, 134.40, 131.18, 128.63, 128.02, 126.08, 122.89, 110.49, 106.45, 106.01, 96.85, 71.62, 33.65, 30.60, 6.62. HRMS (ESI): m/z calcd for C25H25N5O2, 428.2081, found 428.2069 [M + H]+. 4.1.42-(4-((4-((1-cyclopropyl-3-(4-fluorophenyl)-1H-pyrazol-4-yl)oxy)pyridin-2-yl) amino)pyridin-2-yl)propan-2-ol (15b). Following the general procedure, compound 15b was obtained in 24% yield. 1H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 5.6 Hz, 1H), 8.15 (d, J = 5.6 Hz, 1H), 7.78 – 7.70 (m, 2H), 7.45 (s, 1H), 7.34 (s, 1H), 7.20 (d, J = 4.0 Hz, 1H), 7.01 (m, 3H), 6.59 (d, J = 4.0 Hz, 1H), 6.46 (s, 1H), 3.67 – 3.57 (m, 1H), 1.50 (s, 6H), 1.19 (s, 2H), 1.06 (d, J = 6.2 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 166.92, 166.57, 162.50 (d, J = 248.5 Hz), 155.61, 149.91, 148.50, 147.87, 141.08, 134.16, 127.88 (d, J = 8.1 Hz), 127.41 (d, J = 3.0 Hz), 122.92, 115.60 (d, J = 21.2 Hz), 110.59, 106.55, 105.90, 96.97, 71.65, 33.69, 30.58, 6.62. HRMS (ESI): m/z calcd for C25H24FN5O2, 446.1987, found 446.1974 [M + H]+. 4.1.54-(1-cyclopropyl-4-((2-((2-(2-hydroxypropan-2-yl)pyridin-4-yl)amino)pyridin-4 -yl)oxy)-1H-pyrazol-3-yl)benzonitrile (15c). Following the general procedure, compound 15c was obtained in 52% yield. 1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 8.16 (d, J = 4.4 Hz, 1H), 7.89 (d, J = 8.3 Hz, 2H), 7.61 (d, J = 8.3 Hz, 2H), 7.49 (s, 1H), 7.32 (s, 1H), 7.20 (s, 1H), 7.11 (s, 1H), 6.59 (d, J = 4.4 Hz, 1H), 6.47 (s, 1H), 3.71 – 3.62 (m, 1H), 1.50 (s, 6H), 1.22 (s, 2H), 1.10 (d, J = 6.1 Hz, 2H).13C NMR (101 MHz, CDCl3) δ 167.00, 166.33, 155.80, 149.94, 148.31, 139.72, 135.68, 135.24, 132.44, 127.68, 126.32, 123.24, 118.81, 111.16, 110.75, 106.57, 105.59, 96.87, 71.70, 34.05, 29.66, 6.70. HRMS (ESI): m/z calcd for C26H24N6O2, 453.2034, found 453.2027 [M + H]+. 4.1.6 4-(1-cyclopropyl-4-((2-((2-(2-hydroxypropan-2-yl)pyridin-4-yl)amino)pyridin-4-yl)o xy)-1H-pyrazol-3-yl)benzamide (15d). To a stirred solution of 4-(1-cyclopropyl-4-((2-((2-(2-hydroxypropan-2-yl)pyridin-4-yl)amino)pyridin-4-yl)o xy)-1H-pyrazol-3-yl)benzonitrile (15c, 200 mg, 0.44 mmol) in DMSO (1 mL) in an ice bath was added 30% H2O2 (150 mg, 1.32 mmol) and K2CO3 (182 mg, 1.32 mmol). The reaction was allowed to warm up to room temperature and stirred for 30 min. The residue was then purified by a silica gel column chromatography to give compound 15d (135 mg, 65%). 1H NMR (400 MHz, CDCl3) δ 8.27 (s, 1H), 8.15 (s, 1H), 7.85 (d, J = 7.6 Hz, 2H), 7.76 (d, J = 7.6 Hz, 2H), 7.48 (s, 1H), 7.31 (s, 1H), 7.19 (s, 1H), 7.01 (s, 1H), 6.60 (s, 1H), 6.43 (s, 1H), 3.65 (s, 1H), 1.48 (s, 6H), 1.21 (s, 2H), 1.09 (d, J = 6.0 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 168.82, 167.15, 166.44, 155.76, 149.99, 148.22, 140.70, 135.02, 134.85, 132.32, 127.76, 126.10, 123.12, 110.62, 106.55, 105.75, 96.87, 71.70, 33.90, 30.60, 6.68. HRMS (ESI): m/z calcd for C26H26N6O3, 471.2139, found 471.2130 [M + H]+. 4.1.7 4-(1-cyclopropyl-4-((2-((2-(2-hydroxypropan-2-yl)pyridin-4-yl)amino)pyridin-4-yl)o xy)-1H-pyrazol-3-yl)benzoic acid (15e). 4-(1-cyclopropyl-4-((2-((2-(2-hydroxy propan-2-yl)pyridin-4-yl)amino)pyridin-4-yl)oxy)-1H-pyrazol-3-yl) benzonitrile (15c, 100mg, 0.22 mol) was dissolved in 3 M NaOH solution (1 mL) and stirred at 60 °C overnight. The residue was purified by a silica gel column chromatography to give compound 15e (56 mg, 54%). 1H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 1H), 8.26 – 8.12 (m, 3H), 7.92 (d, J = 7.8 Hz, 2H), 7.81 (d, J = 7.8 Hz, 2H), 7.67 (d, J = 15.2 Hz, 2H), 6.67 (d, J = 4.3 Hz, 1H), 6.45 (s, 1H), 5.32 (s, 1H), 5.10 (s, 1H), 1.99 (m, 1H), 1.39 (s, 6H), 1.04 (d, J = 5.8 Hz, 2H), 0.85 (m, 2H). LC-MS (ESI) m/z 472.2 [M+H]+. 4.1.8 2-(4-((4-((3-(benzo[d][1,3]dioxol-5-yl)-1-cyclopropyl-1H-pyrazol-4-yl)oxy) pyridine-2-yl)amino)pyridin-2-yl)propan-2-ol (15f). Following the general procedure, compound 15f was obtained in 38% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1H), 8.19 (d, J = 5.6 Hz, 1H), 8.15 (d, J = 5.6 Hz, 1H), 8.07 (s, 1H), 7.70 (d, J = 2.0 Hz, 1H), 7.66 (dd, J = 5.6, 2.0 Hz, 1H), 7.20 – 7.15 (m, 2H), 6.90 (d, J = 8.4 Hz, 1H), 6.64 (dd, J = 5.6, 2.0 Hz, 1H), 6.40 (d, J = 2.4 Hz, 1H), 6.00 (s, 2H), 5.09 (s, 1H), 3.81 – 3.76 (m, 1H), 1.40 (s, 6H), 1.17 – 1.11 (m, 2H), 1.04 - 1.00 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 167.04, 166.59, 155.74, 149.79, 148.39, 148.14, 147.85, 147.38, 141.74, 133.89, 125.23, 122.87, 120.00, 110.52, 108.49, 106.65, 106.45, 105.91, 101.00, 96.83, 71.65, 33.51, 30.58, 6.56. HRMS (ESI): m/z calcd for C26H25N5O4, 472.1979, found 472.1969 [M + H]+. 4.1.9 2-(4-((4-((1-cyclopropyl-3-(2,3-dihydrobenzo[b][1,4]dioxin-5-yl)-1H-pyrazol-4-yl)ox y)pyridin-2-yl)amino)pyridin-2-yl)propan-2-ol (15g). Following the general procedure, compound 15g was obtained in 8% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H), 8.18 (d, J = 5.6 Hz, 1H), 8.07 (d, J = 5.6 Hz, 1H), 8.04 (s, 1H), 7.70 (d, J = 2.0 Hz, 1H), 7.65 (dd, J = 5.6, 2.0 Hz, 1H), 6.88 (dd, J = 6.0, 3.2 Hz, 1H), 6.83 – 6.78 (m, 2H), 6.54 (dd, J = 5.6, 2.0 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 5.09 (s, 1H), 4.12 – 4.05 (m, 2H), 3.87 – 3.81 (m, 2H), 3.78 (ddd, J = 11.2, 7.4, 3.8 Hz, 1H), 1.39 (s, 6H), 1.16 – 1.10 (m, 2H), 1.00 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 167.10, 166.98, 155.39, 149.34, 148.43, 148.16, 143.60, 141.37, 140.08, 134.83, 122.67, 122.06, 121.00, 120.37, 117.65, 110.47, 106.38, 106.01, 96.78 , 71.66, 63.98, 63.93, 33.63, 30.59, 6.67. HRMS (ESI): m/z calcd for C27H27N5O4, 486.2136, found 486.2124 [M + H]+. 4.1.10 2-(4-((4-((1-cyclopropyl-3-(3,5-dimethylisoxazol-4-yl)-1H-pyrazol-4-yl)oxy) pyridin-2-yl)amino)pyridin-2-yl)propan-2-ol (15h). Following the general procedure, compound 15h was obtained in 61% yield. 1H NMR (400 MHz, CDCl3) δ 8.30 (d, J = 5.6 Hz, 1H), 8.11 (d, J = 5.6 Hz, 1H), 7.50 (s, 1H), 7.32 (s, 1H), 7.21 (d, J = 5.2 Hz, 1H), 6.83 (s, 1H), 6.45 (d, J = 4.4 Hz, 1H), 6.40 (s, 1H), 5.00 (s, 1H), 3.68 – 3.61 (m, 1H), 2.38 (s, 3H), 2.27 (s, 3H), 1.52 (s, 6H), 1.20 (s, 2H), 1.08 (d, J = 6.8 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 167.20, 166.25, 159.10, 155.63, 150.00, 148.30, 148.19, 135.06, 133.87, 121.96, 118.76, 110.67, 107.20, 106.69, 105.04, 96.66, 71.71, 33.91, 30.61, 11.95, 10.96, 6.59. HRMS (ESI): m/z calcd for C24H26N6O3, 447.2139, found 447.2127 [M + H]+. 4.1.11 2-(4-((4-((1-cyclopropyl-1'-methyl-1H,1'H-[3,4'-bipyrazol]-4-yl)oxy)pyridin-2-yl)ami no)pyridin-2-yl)propan-2-ol (15i). Following the general procedure, compound 15i was obtained in 30% yield. 1H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 8.08 (d, J = 5.6 Hz, 1H), 7.91 (s, 1H), 7.71 (s, 1H), 7.66 (s, 1H), 7.56 (s, 1H), 7.42 (s, 1H), 6.85 (s, 1H), 6.55 (d, J = 3.6 Hz, 1H), 3.80 (s, 3H), 3.56 (s, 1H), 1.52 (s, 6H), 1.14 (s, 2H), 0.99 (d, J = 6.0 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 166.36, 163.35, 155.44, 152.38, 149.22, 142.64, 137.01, 136.63, 133.13, 127.72, 122.48, 112.88, 111.01, 107.32, 106.37, 99.19, 71.48, 38.92, 33.42, 30.14, 6.59. HRMS (ESI): m/z calcd for C23H25N7O2, 432.2142, found 432.2129 [M + H]+. 4.1.122-(4-((4-((1-cyclopropyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl)oxy)pyridin-2-yl) amino)pyridin-2-yl)propan-2-ol (15j). Following the general procedure, compound 15j was obtained in 69% yield. 1H NMR (400 MHz, CDCl3) δ 8.50 (s, 2H), 8.19 (s, 1H), 8.13 (s, 1H), 7.73 (s, 1H), 7.65 (s, 2H), 7.57 (s, 1H), 7.52 (s, 1H), 6.77 (s, 1H), 6.58 (s, 1H), 3.66 (s, 1H), 1.51 (s, 6H), 1.25 (s, 2H), 1.07 (d, J = 6.0 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 166.06, 164.66, 155.65, 150.97, 149.88, 149.63, 144.51, 138.92, 138.76, 135.66, 123.55, 120.24, 110.93, 107.17, 106.11, 98.56, 71.57, 34.12, 30.33, 6.72. LC-MS m/z: 429.0 [M+H]+. 4.1.132-(4-((4-((1-cyclopropyl-3-(4,4-difluorocyclohexyl)-1H-pyrazol-4-yl)oxy) pyridin-2-yl)amino)pyridin-2-yl)propan-2-ol (15k). Following the general procedure, compound 15k was obtained in 61% yield. 1H NMR (400 MHz, CDCl3) δ 8.31 (d, J = 5.6 Hz, 1H), 8.16 (d, J = 5.6 Hz, 1H), 7.36 (s, 1H), 7.31 (s, 1H), 7.28 (s, 1H), 6.86 (s, 1H), 6.52 (d, J = 4.0 Hz, 1H), 6.42 (s, 1H), 5.04 (s, 1H), 3.53 (s, 1H), 2.64 (s, 1H), 2.12 (d, J = 9.7 Hz, 2H), 1.90 (d, J = 5.4 Hz, 4H), 1.72 (d, J = 12.9 Hz, 2H), 1.52 (s, 6H), 1.10 (s, 2H), 1.02 (d, J = 6.5 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 167.18, 167.10, 155.66, 149.91, 148.26, 146.10, 134.10, 125.40, 123.01, 121.82, 110.66, 106.54, 105.43, 96.71, 71.68, 33.61, 33.29 (t , J = 24.2 Hz), 30.63, 27.66, 27.57, 6.51. HRMS (ESI): m/z calcd for C25H29F2N5O2, 470.2362, found 470.2351 [M + H]+. 4.1.14 4-(1-cyclopropyl-4-((2-((2-(2-hydroxypropan-2-yl)pyridin-4-yl)amino)pyridin-4-yl)o xy)-1H-pyrazol-3-yl)-1-methylcyclohexan-1-ol (15l). Following the general procedure, compound 15l was obtained in 61% yield. 1H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 5.6 Hz, 1H), 8.14 (d, J = 5.6 Hz, 1H), 7.49 (s, 1H), 7.40 (s, 1H), 7.30 (s, 1H), 6.55 (s, 1H), 6.53 (s, 1H), 3.56 – 3.49 (m, 1H), 2.58 – 2.53 (m, 1H), 1.82 (d, J = 12.8 Hz, 2H), 1.72 (d, J = 12.8 Hz, 2H), 1.69 – 1.55 (m, 4H), 1.53 (s, 6H), 1.22 (s, 3H), 1.09 (s, 2H), 1.00 (d, J = 6.1 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 166.21, 165.00, 154.66, 148.62, 148.42, 146.45, 145.55, 133.00, 120.79, 109.76, 105.80, 104.75, 96.36, 70.68, 69.45, 38.83, 34.03, 32.15, 29.47, 27.88, 24.80, 5.52. HRMS (ESI): m/z calcd for C26H33N5O3, 464.2656, found 464.2649 [M + H]+. 4.1.152-(4-((4-((3-(3-oxabicyclo[3.1.0]hexan-6-yl)-1-cyclopropyl-1H-pyrazol-4-yl) oxy)pyridin-2-yl)amino)pyridin-2-yl)propan-2-ol (15m). Following the general procedure, compound 15m was obtained in 25% yield. 1H NMR (400 MHz, CDCl3) δ 8.30 (d, J = 5.6 Hz, 1H), 8.15 (d, J = 5.6 Hz, 1H), 7.36 (s, 1H), 7.30 (s, 1H), 7.26 (s, 1H), 7.16 (s, 1H), 6.53 (d, J = 3.9 Hz, 1H), 6.45 (s, 1H), 3.83 (d, J = 8.4 Hz, 2H), 3.69 (d, J = 8.4 Hz, 2H), 3.53 – 3.43 (m, 1H), 3.33 (dd, J = 14.4, 7.2 Hz, 1H), 2.01 (s, 2H), 1.51 (s, 6H), 1.06 (s, 2H), 0.99 (d, J = 6.9 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 166.97, 155.66, 149.70, 148.40, 148.20, 142.98, 134.65, 130.55, 121.74, 110.68, 106.54, 105.55, 96.78, 71.69, 69.56, 33.19, 30.61, 25.72, 15.59, 6.48. HRMS (ESI): m/z calcd for C24H27N5O3, 434.2187, found 434.2177 [M + H]+. 4.1.162-(4-((4-((3-(azetidin-3-yl)-1-cyclopropyl-1H-pyrazol-4-yl)oxy)pyridin-2-yl) amino)pyridin-2-yl)propan-2-ol (15n). Following the general procedure, intermediate 15n was obtained in 58% yield. 4.1.17Cyclopropyl(3-(1-cyclopropyl-4-((2-((2-(2-hydroxypropan-2-yl)pyridin-4-yl) amino)pyridin-4-yl)oxy)-1H-pyrazol-3-yl)azetidin-1-yl)methanone (15o). To a suspension of 2-(4-((4-((3-(azetidin-3-yl)-1-cyclopropyl-1H-pyrazol-4-yl)oxy)pyridin-2-yl)amino)p yridin-2-yl)propan-2-ol (15n, 43 mg, 0.1 mmol) and Na2CO3 (43 mg, 0.42 mmol) in THF-MeOH(2 mL, 1:1) was added cyclopropanecarbonyl chloride (14 mg, 12 μL, 0.128 mmol) dropwise. The mixture was stirred at room temperature for 1 h. After completion, the reaction mixture was diluted with ethyl acetate, filtered and the filtrate was concentrated in vacuo. The residue was purified by a silica gel column chromatography to give compound 15o (30 mg, 60%). 1H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 5.8 Hz, 1H), 8.21 (s, 1H), 8.13 (d, J = 5.8 Hz, 1H), 7.57 (s, 1H), 7.46 (s, 1H), 7.40 (s, 1H), 6.56 (s, 1H), 6.47 (d, J = 5.7 Hz, 1H), 5.29 (s, 1H), 4.49 – 4.40 (m, 2H), 4.21 – 4.12 (m, 2H), 3.74 (dt, J = 15.1, 7.6 Hz, 1H), 3.58 – 3.53 (m, 1H), 2.01 (dd, J = 12.2, 6.3 Hz, 1H), 1.53 (s, 6H), 1.13 (d, J = 3.1 Hz, 2H), 1.02 (d, J = 6.0 Hz, 2H), 0.83 (d, J = 6.0 Hz, 2H), 0.70 – 0.65 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 173.90, 166.50, 155.99, 149.78, 149.52, 146.34, 142.99, 134.66, 122.48, 110.84, 106.90, 105.27, 97.81, 71.69, 54.56, 53.25, 52.93, 33.47, 29.66, 14.08, 9.88, 6.52. HRMS (ESI): m/z calcd for C26H30N6O3, 475.2452, found 475.2444 [M + H]+. 4.1.18 2-(4-((4-((1-cyclopropyl-3-(1-(2,2,2-trifluoroethyl)azetidin-3-yl)-1H-pyrazol-4-yl)oxy )pyridin-2-yl)amino)pyridin-2-yl)propan-2-ol (15p). To a solution of 2-(4-((4-((3-(azetidin-3-yl)-1-cyclopropyl-1H-pyrazol-4-yl)oxy)pyridin-2-yl)amino)p yridin-2-yl)propan-2-ol (15n, 30 mg, 0.072 mmol) in THF (1 mL) was added triethylamine (40 μL, 0.29 mmol) and CF3CH2OSO2CF3 (21 mg, 0.09 mmol). The reaction mixture was heated at 70 °C for 1 h. Then the mixture was poured into water and extracted with ethyl acetate. The organic layers were combined and then dried with anhydrous sodium sulfate. After removal of the solvent, the residue was purified by a silica gel column chromatography to give compound 15p (20 mg, 60%). 1H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 5.2 Hz, 1H), 8.13 (d, J = 5.2 Hz, 1H), 7.39 (s, 2H), 7.34 (s, 1H), 7.29 (s, 1H), 6.47 (s, 1H), 6.46 (s, 1H), 3.74 (s, 1H), 3.69 – 3.63 (m, 1H), 3.54 – 3.52 (m, 2H), 3.45 – 3.42 (m, 2H), 2.98 (dd, J = 18.7, 9.3 Hz, 2H), 1.52 (s, 6H), 1.11 (s, 2H), 1.02 (d, J = 6.5 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 166.87, 155.77, 149.71, 148.59, 147.90, 143.06, 134.51, 124.69 (dd, J = 556.5 Hz, 277.8 Hz), 122.00, 110.77, 106.66, 105.33, 96.85, 71.73, 59.18 (dd, J = 61.6 Hz, 31.3 Hz) , 33.37, 30.57, 29.67, 27.73, 6.54. HRMS (ESI): m/z calcd for C24H27F3N6O2, 489.2220, found 489.2212 [M + H]+. 4.1.19 3-(1-cyclopropyl-4-((2-((2-(2-hydroxypropan-2-yl)pyridin-4-yl)amino)pyridin-4-yl)o xy)-1H-pyrazol-3-yl)-1-(trifluoromethyl)cyclobutan-1-ol (15q). Following the general procedure, compound 15q was obtained in 20% yield. 1H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 6.0 Hz, 1H), 8.16 (d, J = 6.0 Hz, 1H), 7.43 (s, 1H), 7.35 (s, 1H), 7.32 (s, 1H), 7.18 (s, 1H), 6.50 (d, J = 4.0 Hz, 1H), 6.45 (s, 1H), 3.56 (s, 1H), 3.23 – 3.13 (m, 1H), 2.89 – 2.81 (m, 2H), 2.43 – 2.35 (m, 2H), 1.53 (s, 6H), 1.13 (s, 2H), 1.04 (d, J = 6.0 Hz, 2H). HRMS (ESI): m/z calcd for C24H26F3N5O3, 490.2061, found 490.2052 [M + H]+. 4.1.202-(4-((4-((1-cyclopropyl-3-(3,3-difluorocyclobutyl)-1H-pyrazol-4-yl)oxy) pyridin-2-yl)amino)pyridin-2-yl)propan-2-ol (15r). Following the general procedure, compound 15r was obtained in 70% yield. 1H NMR (400 MHz, CDCl3) δ 8.31 (d, J = 5.6 Hz, 1H), 8.16 (d, J = 5.6 Hz, 1H), 7.36 (s, 1H), 7.35 (s, 1H), 7.27 (s, 1H), 7.01 (s, 1H), 6.49 (d, J = 4.0 Hz, 1H), 6.41 (s, 1H), 3.60 – 3.51 (m, 1H), 3.21 – 3.13 (m, 1H), 2.91 – 2.73 (m, 4H), 1.52 (s, 6H), 1.11 (s, 2H), 1.03 (d, J = 6.5 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 166.93, 166.73, 155.89, 149.78, 148.49, 148.09, 144.20, 134.37, 122.14, 119.62 (dd, J = 278.8 Hz, 8.1 Hz), 110.73, 106.58, 105.19, 96.73, 71.78, 40.66 (t, J = 23.2 Hz), 33.34, 30.56, 19.45 (dd, J = 17.2 Hz, 4.0 Hz), 6.47. HRMS (ESI): m/z calcd for C25H27F2N3O2, 442.2049, found 442.2039 [M + H]+. 4.1.212-(4-((4-((1-cyclopropyl-3-(3,3-difluorocyclobutyl)-1H-pyrazol-4-yl)oxy) pyridin-2-yl)amino)pyridin-2-yl)-1,1,1-trifluoropropan-2-ol (16a). Following the general procedure, compound 15r was obtained in 61% yield. 1H NMR (400 MHz, CDCl3) δ 8.36 (d, J = 4.8 Hz, 1H), 8.18 (d, J = 4.8 Hz, 1H), 7.50 (s, 2H), 7.35 (s, 1H), 6.75 (s, 1H), 6.53 (d, J = 5.6 Hz, 1H), 6.37 (s, 1H), 3.56 (s, 1H), 3.23 – 3.12 (m, 1H), 2.92 – 2.74 (m, 4H), 1.69 (s, 3H), 1.12 (s, 2H), 1.04 (d, J = 5.7 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 173.77, 166.93, 155.35, 153.96, 144.17, 134.42, 122.49, 122.14, 119.66, 116.99, 113.42, 108.20, 105.25, 97.91, 72.23, 47.27, 47.13, 40.75 (t, J = 23.2 Hz), 33.37, 30.83 (t, J = 4.0 Hz), 29.68, 19.47 (dd, J = 17.2 Hz, 2.0 Hz), 10.02, 7.26, 6.53. HRMS (ESI): m/z calcd for C23H22F5N5O2, 496.1766, found 496.1753 [M + H]+. 4.1.221-(4-((4-((1-cyclopropyl-3-(3,3-difluorocyclobutyl)-1H-pyrazol-4-yl)oxy) pyridin-2-yl)amino)pyridin-2-yl)-2,2,2-trifluoroethan-1-ol (16b). A two-necked flask was charged with 2-chloro-4-((1-cyclopropyl-3-(3,3-difluorocyclobutyl)-1H-pyrazol-4-yl)oxy)pyridine (293 mg, 0.90 mmol), 1-(4-aminopyridin-2-yl)-2,2,2-trifluoroethan-1-ol hydrochloride (287 mg, 1.08 mmol), tris(dibenzylideneacetone) dipalladium (41 mg, 0.05 mmol), Xantphos (52 mg, 0.09 mmol) and sodium benzenolate (418 mg, 3.60 mmol). The vessel was evacuated and backfilled with argon. 1,4-dioxane (20 mL) was added under argon. Then the schlenk tube was heated to 100 °C and was stirred overnight. After completing the reaction, the reaction mixture was diluted with ethyl acetate, filtered and the filtrate was concentrated in vacuo. The residue was purified by a silica gel column chromatography to give compound 16b (80 mg, 19%). 1H NMR (400 MHz, CDCl3) δ 8.36 (d, J = 5.6 Hz, 1H), 8.18 (d, J = 5.6 Hz, 1H), 7.51 (s, 1H), 7.44 (d, J = 4.8 Hz, 1H), 7.35 (s, 1H), 6.53 (d, J = 4.8 Hz, 1H), 6.39 (s, 1H), 4.94 (d, J = 6.6 Hz, 1H), 3.54 (s, 1H), 3.17 (t, 1H), 2.90 – 2.70 (m, 4H), 1.11 (s, 2H), 1.02 (d, J = 6.4 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 166.85, 155.41, 152.00, 149.82, 148.70, 148.54, 144.25, 134.33, 124.16 (dd, J = 576.6 Hz, 283.8 Hz), 122.19, 119.62 (dd, J = 567.6 Hz, 283.8 Hz), 112.72, 110.20, 105.70, 97.15, 70.74 (dd, J = 53.5 Hz, 31.3 Hz) , 40.69 (t, J = 23.2 Hz), 33.37, 19.47 (dd, J = 17.2 Hz, 4.0 Hz), 6.49. HRMS (ESI): m/z calcd for C22H20F5N5O2, 482.1610, found 482.1596 [M + H]+. 4.1.23 4-((1-cyclopropyl-3-(3,3-difluorocyclobutyl)-1H-pyrazol-4-yl)oxy)-N-(2-morpholino pyridin-4-yl)pyridin-2-amine (16c). Following the general procedure, compound 16c was obtained in 65% yield. 1H NMR (400 MHz, CDCl3) δ 8.12 (d, J = 6.0 Hz, 1H), 8.02 (d, J = 6.0 Hz, 1H), 7.34 (s, 1H), 6.89 (s, 1H), 6.83 (s, 1H), 6.56 (d, J = 5.2 Hz, 1H), 6.45 (s, 2H), 3.83 (d, J = 4.0 Hz, 4H), 3.60 – 3.53 (m, 1H), 3.48 (d, J = 4.0 Hz, 4H), 3.17 (dt, J = 17.3, 8.8 Hz, 1H), 2.92 – 2.74 (m, 4H), 1.12 (s, 2H), 1.03 (d, J = 6.5 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 166.76, 160.91, 156.08, 149.83, 1498.89, 144.20, 134.48, 122.13, 104.74, 104.32, 96.50, 94.58, 66.78, 45.80, 40.71 (t, J = 22.2 Hz), 33.40, 19.51 (dd, J = 16.2 Hz, 3.0 Hz), 6.52. HRMS (ESI): m/z calcd for C24H26F2N6O2, 469.2158, found 469.2145 [M + H]+. 4.1.244-(4-((4-((1-cyclopropyl-3-(3,3-difluorocyclobutyl)-1H-pyrazol-4-yl)oxy) pyridine-2-yl)amino)pyridin-2-yl)tetrahydro-2H-pyran-4-ol (16d). Following the general procedure, compound 16d was obtained in 57% yield. 1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 8.06 (s, 1H), 7.82 (s, 1H), 7.55 (s, 1H), 7.34 (s, 1H), 7.03 (s, 1H), 6.69 (d, J = 4.3 Hz, 1H), 5.05 (s, 1H), 3.88 (t, J = 11.2 Hz, 2H), 3.76 (d, J = 8.8 Hz, 2H), 3.57 (s, 1H), 3.17 (dt, J = 16.7, 8.4 Hz, 1H), 2.90 – 2.73 (m, 4H), 1.95 (t, J = 11.2 Hz, 2H), 1.71 (d, J = 13.2 Hz, 2H), 1.20 (s, 2H), 0.98 (d, J = 6.4 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 167.81, 163.92, 153.96, 152.46, 143.54, 142.34, 140.19, 133.45, 122.29, 114.95, 110.94, 106.92, 98.98, 70.25, 63.37, 40.78 (t, J = 23.2 Hz), 37.21, 33.57, 19.36 (dd, J = 17.2 Hz, 4.0 Hz), 6.54. HRMS (ESI): m/z calcd for C25H27F2N5O3, 484.2155, found 484.2142 [M + H]+. 4.1.251-(4-((4-((1-cyclopropyl-3-(3,3-difluorocyclobutyl)-1H-pyrazol-4-yl)oxy) pyridin-2-yl)amino)pyridin-2-yl)-4,4-difluorocyclohexan-1-ol (16e). Following the general procedure, compound 16e was obtained in 46% yield. 1H NMR (400 MHz, CDCl3) δ 8.11 (s, 2H), 7.53 (s, 1H), 7.39 (s, 1H), 7.33 (s, 1H), 6.92 (s, 1H), 6.53 (s, 1H), 3.57 (s, 1H), 3.24 – 3.15 (m, 1H), 2.90 – 2.74 (m, 4H), 2.35 – 2.21 (m, 2H), 2.06 (s, 4H), 1.84 (d, J = 11.7 Hz, 2H), 1.14 (s, 2H), 1.03 (d, J = 6.4 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 169.33, 166.18, 155.14, 155.04, 153.61, 144.07, 135.26, 133.78, 122.84, 122.17, 100.45, 99.91, 71.48, 40.77 (t, J = 23.2 Hz), 34.47, 33.44, 29.69, 19.44 (dd, J = 20.2 Hz, 4.0 Hz), 6.53. HRMS (ESI): m/z calcd for C26H27F4N5O2, 518.2174, found 518.2164 [M + H]+. 4.1.261-(4-((4-((1-cyclopropyl-3-(3,3-difluorocyclobutyl)-1H-pyrazol-4-yl)oxy) pyridin-2-yl)amino)pyridin-2-yl)-4-(trifluoromethyl)piperidin-4-ol (16f). Following the general procedure, compound 16f was obtained in 21% yield. 1H NMR (400 MHz, CDCl3) δ 8.13 (d, J = 5.6 Hz, 1H), 7.86 (d, J = 5.6 Hz, 1H), 7.36 (s, 1H), 7.31 (s, 1H), 6.90 (s, 1H), 6.66 (s, 1H), 6.48 (d, J = 3.9 Hz, 1H), 4.15 (d, J = 12.2 Hz, 2H), 3.59 – 3.51 (m, 1H), 3.29 (t, J = 12.4 Hz, 2H), 3.21 – 3.13 (m, 1H), 2.88 – 2.73 (m, 4H), 1.97 – 1.81 (m, 4H), 1.13 (s, 2H), 1.01 (d, J = 6.2 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 166.60, 159.98, 155.86, 151.25, 149.44, 144.13, 142.95, 134.37, 122.96 (dd, J = 570.6 Hz, 285.8 Hz), 122.26, 119.69 (dd, J = 285.8 Hz, 269.7 Hz), 105.49, 104.57, 98.13, 94.64, 71.00 (dd, J = 58.6 Hz, 29.3 Hz), 41.06, 40.68 (t, J = 22.2 Hz), 33.38, 29.30, 19.47 (dd, J = 17.2 Hz, 4.0 Hz), 6.52. HRMS (ESI): m/z calcd for C26H27F5N6O2, 551.2188, found 551.2178 [M + H]+. 4.1.273-(4-((4-((1-cyclopropyl-3-(3,3-difluorocyclobutyl)-1H-pyrazol-4-yl)oxy) pyridine-2-yl)amino)pyridin-2-yl)oxetan-3-ol (16g). Following the general procedure, compound 16g was obtained in 3% yield. 1H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 5.6 Hz, 1H), 8.20 (d, J = 5.6 Hz, 1H), 7.87 (s, 1H), 7.59 (d, J = 5.2 Hz, 1H), 7.38 (s, 1H), 7.20 (s, 1H), 6.53 (d, J = 5.2 Hz, 1H), 6.41 (s, 1H), 5.07 (d, J = 6.4 Hz, 2H), 4.71 (d, J = 6.4 Hz, 2H), 3.61 – 3.53 (m, 1H), 3.24 – 3.11 (m, 1H), 2.91 – 2.73 (m, 4H), 1.13 (s, 2H), 1.03 (d, J = 6.4 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 166.83, 161.29, 155.58, 149.94, 149.43, 147.54, 144.28, 134.34, 122.22, 111.80, 106.10, 105.60, 96.96, 85.93, 73.95, 40.72 (t, J = 23.2 Hz), 33.42, 19.50 (dd, J = 17.2 Hz, 4.0 Hz), 6.53. HRMS (ESI): m/z calcd for C23H23F2N5O3, 456.1842, found 456.1831 [M + H]+. 4.1.283-(4-((4-((1-cyclopropyl-3-(3,3-difluorocyclobutyl)-1H-pyrazol-4-yl)oxy) pyridine-2-yl)amino)pyridin-2-yl)-1-(2,2,2-trifluoroethyl)azetidin-3-ol (16h). Following the general procedure, compound 16h was obtained in 52% yield. 1H NMR (400 MHz, CDCl3) δ 8.16 (d, J = 5.2 Hz, 1H), 8.04 (d, J = 5.2 Hz, 1H), 7.51 (s, 1H), 7.50 (s, 1H), 7.37 (s, 1H), 7.19 (d, J = 4.0 Hz, 1H), 6.45 (d, J = 4.0 Hz, 1H), 3.78 (d, J = 7.6 Hz, 2H), 3.60 (d, J = 7.6 Hz, 2H), 3.58 – 3.54 (m, 1H), 3.24 – 3.07 (m, 3H), 2.95 – 2.71 (m, 4H), 1.12 (s, 2H), 1.03 (d, J = 6.4 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 166.83, 161.29, 155.58, 149.94, 149.43, 147.54, 144.28, 134.34, 122.22, 119.66 (dd, J = 277.8 Hz, 8.1 Hz), 111.80, 106.10, 105.60, 96.96, 85.93, 73.95, 40.72 (t, J = 23.2 Hz), 33.42, 19.50 (dd, J = 17.2 Hz, 4.0 Hz), 6.53. HRMS (ESI): m/z calcd for C25H25F5N6O2, 537.2032, found 537.2024 [M + H]+. 4.1.29 Cyclopropyl(6-(4-((4-((1-cyclopropyl-3-(3,3-difluorocyclobutyl)-1H-pyrazol-4-yl)ox y)pyridin-2-yl)amino)pyridin-2-yl)-6-hydroxy-2-azaspiro[3.3]heptan-2-yl) methanone (16i). Following the general procedure, compound 16i was obtained in 65% yield. 1H NMR (400 MHz, CDCl3) δ 8.15 (s, 2H), 8.10 (s, 1H), 7.54 (d, J = 18.4 Hz, 1H), 7.39 (s, 1H), 7.21 (s, 1H), 7.16 (s, 1H), 6.90 (s, 1H), 6.50 (s, 1H), 4.42 (s, 1H), 4.24 (s, 1H), 4.16 (s, 1H), 4.01 (s, 1H), 3.57 (s, 1H), 3.25 – 3.14 (m, 1H), 2.95 – 2.66 (m, 7H), 2.62 (s, 1H), 2.59 (s, 1H), 1.14 (s, 2H), 1.03 (d, J = 5.6 Hz, 2H), 0.94 (s, 2H), 0.74 (s, 2H). HRMS (ESI): m/z calcd for C30H32F2N6O3, 563.2577, found 563.2568 [M + H]+. 4.1.30 4-((1-cyclopropyl-3-(3,3-difluorocyclobutyl)-1H-pyrazol-4-yl)oxy)-N-(2-((3,5-dimeth ylmorpholino)methyl)pyridin-4-yl)pyridin-2-amine (16j). Following the general procedure, compound 16j was obtained in 61% yield. 1H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 8.17 (s, 1H), 7.47 – 7.41 (m, 2H), 7.29 (d, J = 7.6 Hz, 1H), 6.59 (d, J = 9.2 Hz, 2H), 3.89 – 3.65 (m, 4H), 3.61 – 3.55 (m, 2H), 3.21 – 3.17 (m, 1H), 2.92 – 2.78 (m, 7H), 1.16 (s, 6H), 1.04 – 1.02 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 165.93, 155.92, 152.31, 148.71, 143.61, 134.29, 122.37, 122.00, 119.53, 116.86, 110.75, 105.45, 99.69, 59.20, 45.78, 40.49 (t, J = 23.2 Hz), 33.18, 29.44, 19.26 (dd, J = 17.2 Hz, 3.0 Hz), 8.51, 6.32. HRMS (ESI): m/z calcd for C26H27F5N6O2, 511.2628, found 511.2620 [M + H]+. 4.2ALK5 inhibitory activity. A luminescent ADP detection assay was used to assess the ALK5 binding capacity of compounds (ADP-Glo™ Kinase Assay, Promega). Serially dilute the stock solution of 10 mM 3-fold in DMSO to obtain a ten-point dilution curve with final compound concentrations ranging from 3.333 μM to 0.5 nM. Assay measurements were performed in 1X kinase reaction buffer containing 40 mM Tris pH 7.5, 20 mM MgCl2, 0.1% BSA, 1 mM DTT in a final assay volume of 5 μL. Briefly 2.5 μL of ALK5 protein (Carna Biosciences), final concentration of 3 μg/mL, was added to each well of a 384 well assay plate containing 100 nL of each concentration of test compound dissolved in DMSO. 2.5 μL of TGF-βR1 peptide (SignalChem), final concentration 3 μg/mL, and ATP, final concentration 1 mM. Following incubation for 120 minutes at 28 °C, add 5 μL ADP-Glo™ Reagent to terminate the kinase reaction and deplete the remaining ATP . After incubation for 120 minutes at 28 °C, add 10 μL of Kinase Detection Reagent to convert ADP to ATP and record luminescence. 4.3Cell-Based luciferase reporter assay for TGF-β type 1 receptor activity. The aim of this experiment is to identify compounds which interfere with SMAD 2,3-dependent gene expression selectively in cell-based assays demonstrating that they inhibit ALK5 at cellular level. Plate the Luc-Smad2/3-NIH3T3 cells (lab of assistant professor Xiao-Jun Xu, China Pharmaceutical University) from assay-ready frozen stocks at 4000 cells per well in 96-well plates in DMEM medium (Invitrogen). After overnight attachment of the cells, media was changed to 2% FBS. Prepare test compounds in DMSO to make 4 mM stock solutions. Serially dilute the stock solutions 4-fold in DMSO to obtain an eight-point dilution curve with final compound concentrations ranging from 20 μM to 1.22 nM and test compounds are added. After 24 hours, add Glo Lysis Buffer (Progema) and Bright-Glo Luciferase assay system (Promega) to each well to double the well volume. Transfer aliquots (180 μL) to white solid bottom plates for reading luminescence on a plate reader (1 second read). 4.4In vitro p38α enzymatic activity assay. All of the enzymatic reactions were conducted at 28 °C for 40 min. The 25 μL reaction mixture contains 50 mM HEPES, pH 7.5, 0.0015% Brij-35, 25 ng kinase, 10 μM ATP, and the FAM-labled peptide. The compounds were tested from 100 µM, 3-fold dilution, 10 concentration. The assay was performed by ChemPartner. It measures kinase activity by quantitating the amount of ATP remaining in solution following a kinase reaction. The luminescent signal from the assay is correlated with the amount of ATP present and is inversely correlated with the amount of kinase activity. The IC50 values were calculated in XLFit excel add-in version 5.4.0.8 to obtain IC50 values using the equation: Y=Bottom + (Top－Bottom)/(1＋(IC50/X) ^Hill Slope) where X is Compound Concentration and Y is Inhibition Rate(%). 4.5Pharmacokinetics procedures. This study was conducted in BALB/C male subjects to investigate the pharmacokinetic profiles of test compounds. The mice were randomized and divided into two groups consisting of 3 mice/group. Prepare test compounds in PEG200-EtOH-solutol-physiological saline (4:1:1:14) to make 0.5 mg/mL solutions for oral gavage and to make 0.1 mg/mL solutions for intravenous injection. Sample collection was performed as follows: 1) single oral administration (PO) group: 5 mg/kg, 0.2 mL/10g; 2) single tail vein injection (IV) group: 1 mg/kg, 0.1 mL/10g. Blood was collected from orbital venous plexus in heparinized EP tube at 5, 15, 30 minutes, 1, 2, 6, 10, 24 hours after intravenous or oral administration, and the contents of the blood were analyzed by LC-MS/MS(API 4500 ). 4.6In vivo tumor xenograft model. A well-established tumorigenesis assay was used to evaluate the antitumor effect of compound 15r in BALB/C female mice model. All mice were housed under standard specific-pathogen-free (SPF) conditions and the animal experiments strictly complied with protocols approved by the Animal Welfare and Ethics Committee (AWEC). 1×106 cells/mouse of CT26 cells were injected subcutaneously into the 5-to-8-week-old BALB/C female mice. All compounds were administrated by oral gavage. Mice were examined thrice a week for the development of tumors by palpation, and tumor volumes calculated using the formula V=0.5× length×width2. The investigators were not blinded to allocation during experiments and outcome assessment. Mice were randomly allocated to three groups consisting of 6 mice/group by an independent person in the laboratory. No statistical method was used to predetermine sample size. The antitumor effect of the compound was assessed by tumor growth inhibition (TGI) or relative tumor proliferation rate (T/C): TGI(%)=[1－(Vt1－Vt0)/(Vc1－Vc0)]×100%, where Vc1 and Vt1 are the mean volumes of control and treated groups at time of tumor extraction, while Vc0 and Vt0 are the same groups at the start of dosages; T/C (%)＝TRTV / CRTV×100%, where TRTV is the relative tumor volume (RTV) of treated groups, while CRTV is the RTV of control groups. (RTV= Vt/V0, Vt is the mean volumes of treated groups at time of tumor extraction, V0 is the mean volumes of the same groups at the start of dosages). Acknowledgments This work was supported by the National Major Science and Technology Project of China (Innovation and Development of New Drugs, No. 2018ZX09301014-006), and the National Natural Science Foundation of China (Grant No. 81973180 and No. 81673298). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/ References [1]R.J. 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Highlights: Synthesis and Biological Evaluation of 4-(Pyridin-4-oxy)-3-(3,3-difluorocyclobutyl)-pyrazole Derivatives as Novel Potent Transforming Growth Factor-β Type 1 Receptor Inhibitors ti Novel 4-(pyridin-4-oxy)-3-(3,3-difluorocyclobutyl)-pyrazole derivatives were designed, synthesized and evaluated as TGF-βR1 inhibitors. ti The key moiety of 1,1-difluorocyclobutyl was proven to improve the potency and metabolic stability. ti Compound 15r exhibited potent antitumor activity in vitro and in vivo. LY 3200882
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.