4-Oxo-1,4-dihydroquinoline-3-carboxamide Derivatives as New Axl

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4-Oxo-1, 4-Dihydroquinoline-3-Carboxamide Derivatives as New Axl Kinase Inhibitors Li Tan, Zhang Zhang, Donglin Gao, Jinfeng Luo, ZhengChao Tu, Zhengqiu Li, Lijie Peng, Xiaomei Ren, and Ke Ding J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00608 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 5, 2016

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Journal of Medicinal Chemistry

4-Oxo-1, 4-Dihydroquinoline-3-Carboxamide Derivatives as New Axl Kinase Inhibitors Li Tan, α, δ, # Zhang Zhang, α, β, # Donglin Gao,α, δ Jinfeng Luo,α Zheng-Chao Tu,α Zhengqiu Li, β Lijie Peng, β Xiaomei Ren,α , β, *and Ke Ding α, β, * α

State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and

Health, Chinese Academy of Sciences, # 190 Kaiyuan Avenue, Guangzhou 510530, China β

School of Pharmacy, Jinan University, # 601 Huangpu Avenue West, Guangzhou 510632,

China. δ

University of Chinese Academy of Sciences, #19 Yuquan Road, Beijing 100049, China

Key Words: Axl, EMT, inhibitor, cancer metastasis

ABSTRACT: Axl is a new potential target for anticancer drug discovery. A series of 4-oxo-1, 4dihydroquinoline-3-carboxamides were designed and synthesized as highly potent Axl kinase inhibitors. One of the most promising compounds 9im tightly bound with Axl protein and potently inhibited its kinase function with a Kd value of 2.7 nM and an IC50 value of 4.0 nM, respectively, while was obviously less potent against most of the 403 wild-type kinases evaluated at a relatively high concentration. The compound dose-dependently inhibited the TGF-β1 induced epithelial-mesenchymal transition (EMT) and suppressed the migration and invasion of

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MDA-MB-231 breast cancer cells. In addition, 9im also demonstrated reasonable pharmacokinetics properties in rats and exhibited in vivo therapeutic effect on hepatic metastasis in a xenograft model of highly metastatic 4T1 murine breast cancer cells. Compound 9im may serve as a lead compound for new anticancer drug discovery and a valuable research probe for further biological investigation on Axl.

INTRODUCTION Axl is a member of receptor tyrosine kinase TAM (i.e. Tyro3, Axl and c-mer proto-oncogene tyrosine kinase (Mer)) subfamily which was originally identified as a transforming gene in cells from chronic myeloid leukemia (CML) patients. 1 Upon activation by the ligand growth arrestspecific 6 (Gas6),

2

Axl demonstrates pivotal roles in many fundamental cellular processes,

including survival, 3 proliferation, 4 differentiation, 5 migration, invasion and angiogenesis etc.

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Dysregulation of Axl signaling has been implicated in various types of human cancer as well as inflammation, autoimmune vascular and kidney diseases. 7 Over-expression of Axl is frequently detected and closely correlated to the poor prognosis in lung cancer, 8 breast cancer, 9 hepatic cellular cancer (HCC), carcinoma, (RCC),

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pancreatic cancer,

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squamous-cell carcinoma (SCC),

gastrointestinal stromal tumors (GIST),

leukemia,

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colorectal carcinoma (CRC),

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melanomas,

ovarian cancer

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prostate

renal cell carcinoma

and others.

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Collective

studies have suggested that abnormal activation of Axl signaling is one prominent mechanism by which tumor cells undergo epithelial-mesenchymal transition (EMT) resistance to both targeted therapies and chemotherapy.

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and develop drug

For instance, high level of Axl is an

important hallmark in invasive estrogen receptor positive (ER+) breast cancer cells, while it is generally expressed at low concentration in healthy breast tissue. 9a Axl was also identified as an


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essential EMT-induced regulator of metastasis to mediate the clinical resistance against lapatinib and traztuzumab in human epidermal growth factor receptor 2 positive (Her2+) and/or ER+ breast cancer patients.

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Thus, Axl becomes a new promising molecular target for anticancer drug

discovery.

Figure 1. Chemical structures of reported selective and non-selective Axl inhibitors. Several well-characterized receptor tyrosine kinase inhibitors were discovered to nonselectively inhibit Axl with low nM IC50 values, among which 1 (Cabozantinib),

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(R)-3-(1-(2,

6-dichloro-3-fluorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine (Crizotinib),

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N-(4-bromo-2-fluorophenyl)-6-methoxy-7-((1-methylpiperidin-4-yl)methoxy)

quinazolin-4-amine (Bosutinib),

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and (Z)-N-(2-(diethylamino)ethyl)-5-(5-fluoro-2-oxo-2, 3-

dihydro-1H-indol-3-ylidenemethyl)-2, 4-dimethyl-1H-pyrrole-3-carboxamide (Sunitinib)

27

have

been approved by US Food and Drug Administration (FDA) for clinical management of different


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human cancers. Other molecules, such as 2 (Foretinib),

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3 (Merestinib),

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6-ethyl-3-((3-

methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)-5-(tetrahydro-2H-pyran-4ylamino) pyrazine-2-carboxamide (Gilteritinib), (Z)-3-((3-((4-(morpholinomethyl)-1H-pyrrol-2yl) methylene)-2-oxoindolin-5-yl)methyl)thiazolidine-2, 4-dione (S49076), 31 4 (BMS777607), 32 5 (MGCD265)

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and 6 (NPS-1034)

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are also in different stages of clinical investigation.

However, none of these inhibitors were developed by using Axl as the primary target. Only recently, 7 (R428, BGB324)

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was disclosed as the first selective Axl inhibitor to potently

inhibit the kinase with an IC50 value of 14.0 nM and demonstrated promising therapeutic efficacy against various human cancer models. The molecule has been advanced into phase I clinical trial and was granted as “orphan-drug” designation for treatment of acute myeloid leukemia (AML). Compound 8 (TP-0903),

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another selective Axl inhibitor, also displayed promising therapeutic

potential in preclinical evaluations. However, given the fact that there is no selective Axl inhibitor has been approved for clinical application, it is highly desirable to discover new Axl inhibitors with distinct chemical scaffolds and improved target selectivity. In this paper, we report a series of 4-oxo-1, 4-dihydroquinoline-3-carboxamides as new Axl inhibitors.


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Figure 2. Design strategy of the new potential selective Axl inhibitors. MOLECULAR DESIGN There is no X-ray crystal structure of Axl kinase domain reported to date. Structural feature analysis of the reported selective/non-selective Axl inhibitors (Figure 1) revealed that most of the compounds consist of several key structural elements which might form crucial interactions with the protein: 37 1) a heterocyclic moiety (so called “head region”) possibly binding to the adenine pocket and forming hydrogen bond network with residues in the hinge region of the kinase; 2) a dual hydrogen bond acceptor (DHBA) moiety (e.g. 1, 3-diketone) which might form additional hydrogen bond networks with back pocket of the protein (“DHBA group”); 3) a 1, 4disubstituted phenyl group as a potential linker between the “head region” and the “DHBA group”. Based on this observation, a series of 4-oxo-1, 4- dihydroquinoline-3-carboxamide derivatives were designed as new potential selective Axl inhibitors in which a privileged quinolone scaffold, which has been widely applied in a number of FDA approved antibacterial drugs (e.g. ciprofloxacin 38 and levofloxacin 39), was utilized as the “DHBA group”.


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CHEMISTRY Synthesis of the designed compounds was outlined in Scheme 1. Briefly, commercially available 4-chloro-7H-pyrrolo[2, 3-d]pyrimidine (10) was reacted with substituted 4nitrophenols (11) under microwave to yield compounds 12 which were further iodinated by Iodine and subsequently protected with (2-(chloromethoxy)ethyl)trimethylsilane (SEMCl) to produce intermediates 14. Suzuki coupling

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of compounds 14 with different substituted

phenylboronic acids (15) under air-free condition produced the corresponding products 16. Reduction of 16 yielded the key intermediates 17 which were condensed with quinolonic acids 29 to afford compounds 18. Deprotection of 18 under standard conditions produced the final products 9 in good or moderate yields. The key intermediates 29 were synthesized via two different methods (Scheme 2) by starting from 2-aminobenzoic acids (20) or anilines 25, respectively. Substituted 2-aminobenzoic acids (20) reacted with triphosgene to produce compounds 21 which was subsequently N-alkalized and condensed with alkyl 3-oxobutanoate 22 to yield intermediates 24. Compounds 24 were hydrolyzed under base conditions to obtain the intermediates 29. Alternatively, substituted anilines 25 reacted with diethyl 2-acetylmalonate or diethyl 2-(ethoxymethylene)malonate to produce intermediates 27. Compounds 27 were alkylated to yield esters 28 which were hydrolyzed to produce quinolonic acids 29 in good yields. Compound 9im was prepared by utilizing commercially available 4-chloro-6, 7dimethoxyquinazoline as the starting material and following the procedures of the other designed molecules. Scheme 1. Synthesis of compounds 9 a


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a

Reagents and conditions: (a) ethyl diisopropylamine (DIPEA), 1-methyl-2-pyrrolidinone

(NMP), Microwave, 200 °C, 1.0 hr, 30-85%; (b) iodine, KOH, N,N-dimethylformamide (DMF), 0 °C, 4.0 hrs, 75-80%; (c) SEMCl, NaOH, DMF, room temperature (rt), overnight, 70-78%; (d) Pd(PPh3)4, Na2CO3, toluene, 90 °C, overnight, 65-75%; (e) NiCl2·6H2O, NaBH4, tetrahydrofuran (THF), MeOH, rt, 2.0 hrs, 60-72%; (f) 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl uronium hexafluoro phosphate (HATU), DIPEA, DMF, rt, overnight, 68-85%; (g) trifluoroacetic acid (F3CCOOH), dichloromethane (DCM), rt, overnight, 85-90%; (h) NaOH, THF-H2O, rt, overnight, 80-90%. Scheme 2. Synthesis of intermediates 29 a


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a

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Reagents and conditions: (i) triphosgene, pyridine, acetonitrile (MeCN), 0-55 °C, 2.0 hrs, 82-

94%; (j) R2I, DIPEA, DMF, 40 °C, overnight, 75-82%; (k) 22, NaH, DMF, 120 °C, overnight, 70-85%; (l) diethyl 2-acetylmalonate or diethyl 2-(ethoxymethylene) malonate, p-toluenesulfonic acid, H2O, pentane, reflux, overnight, 75-90%; (m) diphenyl oxide, 200 °C, 2.0 hrs, or polyphosphoric acid (PPA), 100 °C, overnight, 75-90%; (n) MeI, K2CO3, DMF, 50 °C, overnight, 78-88%; (o) NaOH, THF-H2O, reflux, overnight, 84-93%. RESULTS AND DISCUSSION Axl inhibitory activities of the designed compounds (9) were preliminarily evaluated via a well-established fluorescence resonance energy transfer (FRET)-based Z’-Lyte assays according to the manufactory’s instructions (Invitrogen, Carsbad, USA).

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Two well characterized Axl

inhibitors 4 and 7 were included as the positive controls to validate the screening conditions. Under the experimental conditions, both of the compounds displayed strong inhibition against Axl with IC50 values of 6.0 and 5.0 nM, respectively, which are similar to the previously reported data. 32, 35


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We initially investigated if the quinolone moiety is suitable to serve as a “DHBA group” to demonstrate Axl inhibition. Therefore, a bivalent 5-phenyl-7H-pyrrolo-[2, 3-d]pyrimidinyl group was applied as the potential hinge binding “head region” based on the chemical structure of compound 6 in the newly designed compounds 9. A generally utilized 2-fluoro-1, 4-substituted phenyl linker was also adopted from the newly reported inhibitors. Although the R1unsubstituted dihydroquinoline derivative 9a did not show obvious inhibition against Axl at 10.0 µM, a 4-chloro substitution at the quinolone scaffold (9c) significantly improved the kinase inhibitory potency. Compound 9c displayed an IC50 value of 1.89 µM. Though this compound is approximately 500 times less potent than the reference molecules 4 and 7, it could provide a starting point for further structural optimization. Further investigation also suggested that the 4position of the quinolone is optimal for substitution. When the R1-chloro group was introduced at 3- or 5- position, the resulting compounds (9b and 9d, respectively) were totally inactive. Thus, a variety of substituted groups were introduced in the 4- position with the aim of improving the Axl inhibitory activity. It was shown that 4- substituent had significant and diversified impact on the Axl kinase inhibitory activity. Although the 4-bromo compound 9f displayed comparable Axl inhibitory effect to that of compound 9c, the 4-fluoro derivative (9e) totally abolished its potency. Encouragingly, when the 4-chloro group in 9c was replaced by 4-methyl (9h) or 4trifluoromethyl (9g), the Axl inhibitory activity was obviously improved by 12-19 folds. The IC50 values of the corresponding compounds were 0.10 and 0.15 µM, respectively. Further investigation also revealed that the 4-choro group in 9c could also be replaced by 4-cyclopropyl (9j), 4-isopropyl (9k), 4-prop-1-en-2-yl (9l), 4-n-propyl (9m), 4-tert-butyl (9n), 4-cyclopentyl (9o), 4-methoxyl (9s) or 4-trifluoromethoxyl (9t) substituent to obviously improve the potency. The compounds exhibited IC50 values ranged from 0.23 to 0.026 µM. Significantly, 4-ethyl


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compound (9i) demonstrated the strongest potency with an IC50 value of 6.0 nM against Axl kinase, which is equally potent to the positive controls 7 and 4. However, when a phenyl (9q) or methoxy carbonyl (9u) was introduced at the 4-position, the resulting compounds totally abolished their Axl inhibitory activities. A cyclopent-1-en-1-yl substitution at the 4-position also caused a 2-fold potency loss. A 1-oxo-1, 4-dihydrobenzo[f]quinoline-2-carboxamide derivative (9r) was also designed and synthesized to display no inhibition against Axl at 10.0 µM. Table 1. In Vitro Axl Kinase Inhibitory Activities of Inhibitors 9a-9ic. a

Axl inhibition Compds

R1

R2

R3

R4 (IC50, µM)

9a

H

Me

Me

2’-F

>10

9b

3-Cl

Me

Me

2’-F

>10

9c

4-Cl

Me

Me

2’-F

1.89

9d

5-Cl

Me

Me

2’-F

>10

9e

4-F

Me

Me

2’-F

>10

9f

4-Br

Me

Me

2’-F

3.12


10

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9g

4-CF3

Me

Me

2’-F

0.15

9h

4-Me

Me

Me

2’-F

0.10

9i

4-Et

Me

Me

2’-F

0.006

9j

4-cyclopropyl

Me

Me

2’-F

0.23

9k

4-iso-Pr

Me

Me

2’-F

0.026

Me

Me

2’-F

0.17

9l

(4)

9m

4- n-Pr

Me

Me

2’-F

0.061

9n

4- t-Bu

Me

Me

2’-F

0.042

9o

4-cyclopentyl

Me

Me

2’-F

0.20

Me

Me

2’-F

3.32

Me

Me

2’-F

>10

Me

Me

2’-F

>10

9p

9q

(4) 4-phenyl

9r

9s

4-OMe

Me

Me

2’-F

0.099

9t

4-OCF3

Me

Me

2’-F

0.074


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9u

a

(4)

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Me

Me

2’-F

>10

9ha

4-Me

H

Me

2’-F

0.56

9hb

4-Me

Et

Me

2’-F

0.15

9hc

4-Me

n-Pr

Me

2’-F

0.53

9hd

4-Me

n-Bu

Me

2’-F

0.48

9he

4-Me

Ph

Me

2’-F

0.62

9hf

4-Me

Me

H

2’-F

0.15

9hg

4-Me

Me

Et

2’-F

0.078

9hh

4-Me

Me

Ph

2’-F

0.078

9ia

4-Et

Me

Me

3’-F

0.030

9ib

4-Et

Me

Me

H

0.105

9ic

4-Et

Me

Me

2’-Me

1.12

4

0.006

7

0.005

Axl kinase inhibition was determined by using an FRET-based Z’-Lyte assay according to the

manufactory’s instructions (Invitrogen, Carsbad, USA).

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The compounds were incubated with

the kinase reaction mixture for 1-1.5 hrs before measurement. Reported data are means from 2 independent experiments in which the variation is less than 20%.


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Potential impact of R2 or R3 substituent on the Axl inhibition was also investigated. It was shown that the N-methyl (R2) group in 9h was able to be replaced by an ethyl group (9hb) without obviously affecting the Axl inhibitory potency. However, when the R2 position was introduced with a large hydrophobic group such as an n-propyl (9hc), n-butyl (9hd) or phenyl (9he) moiety, the potency was decreased by about 4-5 folds. The investigation also revealed that R3 position is well tolerated to either small or large hydrophobic group without obviously affecting the kinase inhibitory activity. For instance, compounds 9hf, 9hg and 9hh displayed IC50 values of 0.15, 0.078 and 0.078 µM, respectively, which are almost identical to that of 9h. Table 2. In Vitro Axl Kinase Inhibitory Activities of Inhibitors 9id-9im.a

Axl inhibition

Axl inhibition Compds

Compds

Het (IC50, µM)

Het (IC50, µM)

9i

0.006

9ii

0.888

9id

0.157

9ij

4.377


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9ie

0.034

9ik

0.072

9if

0.030

9il

>10

9ig

0.192 9im

0.004

9ih

a

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>10

Axl kinase inhibition was determined by uing an FRET-based Z’-Lyte assay according to the

manufactory’s instructions (Invitrogen, Carsbad, USA).

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The compounds were incubated with

the kinase reaction mixture for 1-1.5 hrs before measurement. Reported data are means from 2 independent experiments in which the variation is less than 20%. Further investigation also suggested that the 2’-fluoro substituent was critical to the strong Axl inhibition of compound 9i. When the 2’-fluoro group was removed (9ib) or merged to 3’ position (9ia), the resulting compounds displayed 5-16 fold potency loss. It was noteworthy that the 2’methyl compound 9ic also showed significantly decreased Axl inhibitory activity with an IC50 value of 1.12 µM, which is approximately 170 folds less potent than the parental compound 9i. We further investigated contribution of the potential hinge binding 7H-pyrrolo[2, 3-d] pyrimidine group to the Axl kinase inhibition by replacing it with a variety of heterocyclic groups by using 9i as the template molecule. It was found that the 7’’-phenyl group is important


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for the strong Axl kinase inhibition of 9i. When the 7’’-phenyl group was removed (9id) or replaced with a 1-methyl-1H-pyrazol-4-yl (9ie) group, the resulting compounds exhibited 6-26 folds less potency. The replacement of 5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl hinge binding moiety with a 3-phenyl-1H-pyrrolo[2, 3-b]pyridin-4-yl group (9if) also caused a 5-time potency loss. The IC50 value of 9if was 0.03 µM against Axl. More significantly, the 3-phenyl benzofuran-4-yl derivative displayed an IC50 value of 0.19 µM, which is 32 times less potent than the parental compound 9i. The potential hinge binding group was further changed to various monocyclic pyridinyl moieties, such as 2-chloropyridinyl (9ih), 2-aminopyridinyl (9ii), 2aminocarbonatepyridinyl (9ij), 2-amino-3-choloropyridinyl (9ik) or 2-aminocarbonate-3choloropyridinyl (9il). All of the resulting compounds displayed significantly less potencies than the original molecule 9i. Particularly, compounds 9ih and 9il totally abolished the Axl inhibitory potencies. Interestingly, when a 6, 7-dimethoxyquinazolin-4-yl group was utilized as the potential hinge binding moiety, the resulting compound 9im displayed an IC50 value of 4.0 nM against Axl, which was equally potent to that of compound 9i. Thus, compounds 9i and 9im represented the most potent Axl inhibitors for further biological investigation. The binding of compounds 9i and 9im with Axl protein was further determined by using an active-site-dependent competition binding assay (conducted by DiscoveRx Corporation, San Diego, USA). 42 It was found that the compounds tightly bound to ATP-binding site of the kinase with a binding constant (Kd) value of 1.2 and 2.7 nM, respectively, which validated their strong kinase inhibition against Axl. To demonstrate the target selectivity of the compounds, a kinase selectivity profiling study was conducted against a panel of 468 kinases (including 403 nonmutated kinases) at 1.0 µM by using the DiscovRx screening platform. It was shown that both 9i and 9im displayed good target selectivity (Supporting Information). For instance, 9im exhibited


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S(1), S(10) and S(35) scores of 0.005, 0.037 and 0.082, respectively, while the corresponding S(1), S(10) and S(35) scores of the clinical investigated inhibitor 7 were 0.032, 0.117 and 0.243, respectively, at the same concentration (Supporting Information).

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These data implied that

compound 9im might achieve a better target selectivity than the positive control 7. In addition to its primary target Axl, compound 9im also displayed obvious binding with several tyrosine or serine/threonine kinases including abelson murine leukemia viral oncogene (Abl), Aurora A, Aurora B, B lymphoid tyrosine kinase (BLK), cyclin-dependent-kinase 11 (CDK 11), CDK 8, discoidin domain receptor 1 (DDR1), DDR2, death-associated protein kinase related 1 (DRAK1), FMS-like tyrosine kinase 3 (Flt3), homeodomain interacting protein kinase 4 (HIPK4), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (Kit), serine/threonine kinase 10 (LOK), mitogen-activated protein kinase kinase 5 (MEK1), Ste-like kinase (SLK), nerve growth factor receptor A (TrkA) and TrkB etc. The binding affinities (Kd) or kinase inhibitory activities (IC50) of compound 9im against these “off targets” were further determined by using DiscoveRx’s platform or our in-house kinase assays (Table 3). It was shown that compound 9im displayed approximately 25-430 fold higher potency against Axl than most of the “off target” kinases. However, it also exhibited strong potencies against several human cancer related kinase such as DDR1, Flt3, LOK, MEK5, Met, Trk A and Trk B with Kd or IC50 values below 100 nM.

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It is noteworthy that some of these kinases (e.g. Flt3, Met and MEK5)

have been well validated as molecular targets for new anticancer drug discovery. 43b, c, d However, strong inhibition against the “off targets” may also raise some potential toxicity concern about the further development of this compound. The binding affinities of 9im with the other TAM tyrosine kinase members (Tyro3 and Mer) were also determined. It was shown that 9im


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exhibited a similar potency to Mer with a Kd value of 1.4 nM, while it was less potent to Tyro3 with a Kd value of 58 nM. Table 3. Binding affinities or kinase inhibition of compound 9im against a panel of “off target” kinases.

a

Kinase

Kd value or IC50 (nM)

Kinase

Kd value or IC50(nM)

ALK

>1000 a

HIPK4

11.0 a

Axl

2.7 a (4.0 b)

Kit

1673 a

BLK

200 a

LOK

10 a

Aurora A

192.4 b

MEK5

38 a

Aurora B

114.4 b

Mer

1.4 a

CDK11

470 a

Met

79.0 a

CDK8

>1000 a

SLK

240 a

DDR1

22.2 b

TrkA

50 a

DDR2

349.6 b

TrkB

54 a

Flt3

4.0 a (300.0 °C. The other designed inhibitors were prepared by following a similar procedure as that of 9h. 6-Ethyl-N-(3-fluoro-4-((5-phenyl-7H-pyrrolo[2,

3-d]pyrimidin-4-yl)oxy)phenyl)-1,

2-

dimethyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9i). White solid (225 mg, 72%; Rf: 0.44, DCM: Methonal = 10: 1 ). 1H NMR (500 MHz, d6-DMSO) δ 11.02 (s, 1 H), 8.33 (s, 1 H), 8.09 (s, 1 H), 7.93 (d, J = 1.5 Hz, 1 H), 7.91-7.76 (m, 4 H), 7.65 (m, 1 H), 7.46 (d, J = 9.5 Hz, 1 H), 7.41 (m, 3 H), 7.26 (m, 1 H), 3.82 (s, 3 H), 2.75 (q, J = 7.5 Hz, 2 H), 2.65 (s, 3 H), 1.24 (t, J = 7.5 Hz, 3 H). 13C NMR (125 MHz, d6-DMSO) δ 174.0, 166.1, 161.7, 154.8, 154.1 (d, J = 242.6 Hz, 1 C), 152.4, 150.5, 139.9, 139.6, 138.5 (d, J = 9.8 Hz, 1 C), 135.0 (d, J = 13.1 Hz, 1 C), 133.3, 128.8, 128.6, 126.6, 126.2, 124.9, 124.6, 124.1, 119.0, 117.5, 115.9, 107.9 (d, J = 23.8 Hz, 1 C), 102.3, 35.6, 27.9, 19.4, 15.9. HRMS (ESI) for C32H26FN5O3 [M+H]+, calcd: 548.2092, found: 548.2089. HPLC analysis: MeOH-H2O (85:15), 5.61 min, 97.02% purity. Melting point: 283.8-285.1 °C. 6-Cyclopropyl-N-(3-fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-1, 2-dimethyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9j). White solid (65 mg, 58%; Rf:


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0.24, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 11.02 (s, 1 H), 8.29 (s, 1 H), 7.95 (d, J = 2.0 Hz, 1 H), 7.90 (dd, J1 = 2.0 Hz, J2 = 12.5 Hz, 1 H), 7.79-7.77 (m, 3 H), 7.73 (s, 1 H), 7.51 (dd, J1 = 2.0 Hz, J2 = 8.5 Hz, 1 H), 7.45 (dd, J1 = 2.0 Hz, J2 = 9.5 Hz, 1 H), 7.41-7.37 (m, 3 H), 7.24 (t, J = 7.0 Hz, 1 H), 3.82 (s, 3 H), 2.64 (s, 3 H), 2.14-2.09 (m, 1 H), 1.04 (m, 2 H), 0.76 (m, 2 H). 13C NMR (125 MHz, d6-DMSO) δ 173.8, 166.1, 161.6, 154.2 (d, J = 242.8 Hz, 1 C), 152.4, 150.1, 140.1, 139.4, 138.4 (d, J = 9.9 Hz, 1 C), 135.2 (d, J = 13.1 Hz, 1 C), 135.0, 130.9, 128.8, 128.7, 126.4, 126.3, 125.0, 121.8, 119.0, 117.6, 115.9, 115.6, 108.0 (d, J = 23.4 Hz, 1 C), 102.4, 35.7, 19.5, 15.0, 10.2. HRMS (ESI) for C33H26FN5O3 [M+H]+, calcd: 560.2092, found: 560.2081. HPLC analysis: MeOH-H2O (75:25), 5.63 min, 95.14% purity. Melting point: 270.4-272.6 °C. N-(3-Fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-6-isopropyl-1, 2dimethyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9k). White solid (106 mg, 50%; Rf: 0.32, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 11.02 (s, 1 H), 8.31 (s, 1 H), 8.12 (s, 1 H), 7.90 (d, J = 12.5 Hz, 1 H), 7.84-7.72 (m, 5 H), 7.46-7.27 (m, 4 H), 7.27 (d, J = 6.5 Hz, 1 H), 3.84 (s, 3 H), 3.07 (m, 1 H), 2.66 (s, 3 H), 1.27 (m, 6 H).

13

C NMR (125 MHz, d6-

DMSO) δ 174.1, 166.1, 161.7, 154.2 (d, J = 246.0 Hz, 1 C), 152.5, 150.4, 144.5, 139.8, 138.5 (d, J = 9.1 Hz, 1 C), 135.1 (d, J = 12.6 Hz, 1 C), 134.8, 132.1, 128.8, 128.7, 126.6, 126.2, 125.1, 125.0, 122.6, 119.0, 117.6, 115.9, 115.8, 108.0 (d, J = 23.3 Hz, 1 C), 102.4, 35.7, 33.2, 24.2, 19.5. HRMS (ESI) for C33H28FN5O3 [M+H]+, calcd: 562.2249, found: 562.2253. HPLC analysis: MeOH-H2O (85:15), 6.28min, 98.65% purity. Melting point: 268.9-270.2 °C. N-(3-Fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-1, 2-dimethyl-4oxo-6-(prop-1-en-2-yl)-1, 4-dihydroquinoline-3-carboxamide (9l). White solid (34 mg, 55%; Rf: 0.25, DCM: Methonal = 20: 1). 1H NMR (400 MHz, d6-DMSO) δ 12.53 (s, 1 H), 10.93 (s, 1


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H), 8.32 (d, J = 11.2 Hz, 1 H), 8.01 (d, J = 8.4 Hz, 1 H), 7.92-7.87 (m, 2 H), 7.79-7.76 (m, 3 H), 7.45-7.41 (m, 4 H), 7.27 (t, J = 6.8 Hz, 1 H), 5.62 (s, 1 H), 5.23 (s, 1 H), 3.86 (s, 3 H), 2.65 (s, 3 H), 2.20 (s, 3 H).

13

C NMR (125 MHz, d6-DMSO) δ 174.0, 166.0, 161.8, 154.6, 154.1 (d, J =

242.3 Hz, 1 C), 152.5, 150.7, 141.6, 140.7, 138.5 (d, J = 9.6 Hz, 1 C), 135.9, 135.1 (d, J = 12.8 Hz, 1 C), 134.6, 130.2, 128.9, 128.7, 126.8, 126.0, 125.0, 124.3, 122.1, 119.7, 117.7, 116.0, 113.9, 108.0 (d, J = 21.9 Hz, 1 C), 102.3, 35.7, 21.8, 19.5. HRMS (ESI) for C33H26FN5O3 [M+H]+, calcd: 560.2092, found: 560.2081. HPLC analysis: MeOH-H2O (90:10), 8.13 min, 97.79% purity. Melting point: 233.3-235.8 °C. N-(3-Fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-1, 2-dimethyl-4oxo-6-propyl-1, 4-dihydroquinoline-3-carboxamide (9m). White solid (88 mg, 77%; Rf: 0.23, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 11.00 (s, 1 H), 8.30 (s, 1 H), 8.06 (d, J = 2.0 Hz, 1 H), 7.91 (dd, J1 =2.0 Hz, J2 = 12.5 Hz, 1 H), 7.81-7.77 (m, 3 H), 7.74 (s, 1 H), 7.64 (dd, J1 = 2.0 Hz, J2 = 9.0 Hz, 1 H), 7.45 (dd, J1 = 1.5 Hz, J2 = 9.0 Hz, 1 H), 7.41-7.38 (m, 3 H), 7.25 (m, 1 H), 3.83 (s, 3 H), 2.71 (t, J = 7.2 Hz, 2 H), 2.64 (s, 3 H), 1.69-1.61 (q, J = 7.1 Hz, 2 H), 0.89 (t, J = 7.0 Hz, 3 H). 13C NMR (125 MHz, d6-DMSO) δ 174.0, 166.1, 161.6, 155.4, 154.2 (d, J = 242.8 Hz, 1 C), 152.4, 150.3, 139.7, 138.5 (d, J = 9.8 Hz, 1 C), 138.3, 135.2 (d, J = 12.8 Hz, 1 C), 134.9, 133.8, 128.8, 128.7, 126.5, 126.2, 125.5, 125.0, 119.1, 117.5, 115.9 (d, J = 2.6 Hz, 1 C), 115.7, 108.0 (d, J = 23.1 Hz, 1 C), 102.4, 36.9, 35.7, 24.4, 19.5, 13.9. HRMS (ESI) for C33H28FN5O3 [M+H]+, calcd: 562.2249, found: 562.2249. HPLC analysis: MeOH- H2O (85:15), 6.60 min, 95.83% purity. Melting point: 271.5-273.2 °C. 6-(Tert-butyl)-N-(3-fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-1, 2-dimethyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9n). White solid (55 mg, 75%; Rf: 0.24, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 11.03 (s, 1 H), 8.32 (s, 1 H),


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8.25 (d, J = 2.0 Hz, 1 H), 7.92-7.87 (m, 2 H), 7.83 (m, 1 H), 7.78 (d, J = 7.5 Hz, 2 H), 7.75 (s,1 H), 7.45 (dd, J1 = 2.0 Hz, J2 = 8.5 Hz, 1 H), 7.42-7.38 (m, 3 H), 7.26 (t, J = 7.5 Hz, 1 H), 3.84 (s, 3 H), 2.66 (s, 3 H), 1.36 (s, 9 H). 13C NMR (125 MHz, d6-DMSO) δ 174.2, 166.1, 161.7, 154.8, 154.1 (d, J = 242.6 Hz, 1 C), 152.7, 150.6 146.8, 139.4, 138.5 (d, J = 9.8 Hz, 1 C), 135.0 (d, J = 13.0 Hz, 1 C), 134.7, 131.1, 128.9, 128.7, 126.7, 125.8, 125.0, 124.6, 121.5, 118.9, 117.5, 115.9, 108.0 (d, J = 23.4 Hz, 1 C), 102.3, 35.6, 34.8, 31.5, 19.5. HRMS (ESI) for C34H30FN5O3 [M+H]+, calcd: 576.2405, found: 576.2411. HPLC analysis: MeOH-H2O (85:15), 7.12 min, 97.03% purity. Melting point: 272.7-274.3 °C. 6-Cyclopentyl-N-(3-fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-1, 2 -dimethyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9o). White solid (25 mg, 59%; Rf: 0.36, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 11.03 (s, 1 H), 8.33 (d, J = 2.0 Hz, 1 H), 8.12 (d, J = 1.5 Hz, 1 H), 7.91 (dd, J1 = 2.0 Hz, J2 = 12.5 Hz, 1 H), 7.82-7.75 (m, 4 H), 7.69 (dd, J1 = 2.0 Hz, J2 = 9.0 Hz, 1 H), 7.45 (dd, J1 = 1.5 Hz, J2 = 9.0 Hz, 1 H), 7.42-7.38 (m, 3 H), 7.27 (m, 1 H), 3.83 (s, 3 H), 3.15 (m, 1 H), 2.65 (s, 3 H), 2.10-2.06 (m, 2 H), 1.82-1.78 (m, 2 H), 1.71-1.68 (m, 2 H), 1.62-1.56 (m, 2 H).

13

C NMR (125 MHz, d6-DMSO) δ 174.0, 166.1,

161.7, 154.7, 154.1 (d, J = 242.5 Hz, 1 C), 152.6, 150.6, 142.2, 139.7, 138.5 (d, J = 9.9 Hz, 1 C), 135.0 (d, J = 12.8 Hz, 1 C), 134.6, 132.6, 128.9, 128.8, 128.7, 126.7, 126.1, 125.4 124.9, 124.4, 123.3, 118.9, 117.5, 116.0, 115.9 (d, J = 2.8 Hz, 1 C), 108.0 (d, J = 23.0 Hz, 1 C), 102.3, 45.0, 35.7, 34.6, 30.9, 25.5, 19.5. HRMS (ESI) for C35H30FN5O3 [M+H]+, calcd: 588.2405, found: 588.2394. HPLC analysis: MeOH-H2O (85:15), 8.40 min, 96.84% purity. Melting point: 286.8288.1 °C. 6-(Cyclopent-1-en-1-yl)-N-(3-fluoro-4-((5-phenyl-7H-pyrrolo[2,

3-d]pyrimidin-4-yl)oxy)

phenyl)-1, 2-dimethyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9p). Beige solid (64 mg,


33


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52%; Rf: 0.21, DCM: Methonal = 20: 1). 1H NMR (400 MHz, d6-DMSO) δ 10.95 (s, 1 H), 8.32 (s, 1 H), 8.16 (s, 1 H), 8.00 (d, J = 8.4 Hz, 1 H), 7.92-7.84 (m, 2 H), 7.78-7.75 (m, 3 H), 7.477.39 (m, 4 H), 7.27 (t, J = 7.2 Hz, 1 H), 6.45 (s, 1 H), 3.85 (s, 3 H), 2.75 (m, 2 H), 2.64 (s, 3 H), 2.54 (m, 2 H), 2.01 (m, 2 H).

13

C NMR (125 MHz, d6-DMSO) δ 174.0, 166.0, 161.8, 154.6,

154.1 (d, J = 243.0 Hz, 1 C), 152.4, 151.9, 150.7, 141.2, 140.3, 139.6, 138.5 (d, J = 10.0 Hz, 1 C), 135.1 (d, J = 12.6 Hz, 1 C), 134.6, 132.1, 130.7, 128.9, 128.7, 127.5, 127.8, 126.8, 126.2, 125.4, 124.9, 124.2, 122.0, 119.5, 117.7, 116.0 (d, J = 2.1 Hz, 1 C), 108.0 (d, J = 23.3 Hz, 1 C), 102.3, 67.5, 35.7, 34.8, 33.6, 33.2, 30.9, 25.6, 23.3, 21.5, 19.5. HRMS (ESI) for C35H28FN5O3 [M+H]+, calcd: 586.2249, found: 586.2243. HPLC analysis: MeOH-H2O (85:15), 11.32 min, 97.72% purity. Melting point: 279.2-281.8 °C. N-(3-Fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-1, 2-dimethyl-4oxo-6-phenyl-1, 4-dihydroquinoline-3-carboxamide (9q). White solid (55 mg, 62%; Rf: 0.26, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 10.94 (s, 1 H), 8.51 (d, J = 2.0 Hz, 1 H), 8.31 (s, 1 H), 8.13 (dd, J1 = 2.5 Hz, J2 = 9.0 Hz, 1 H), 7.99 (d, J = 9.0 Hz, 1 H), 7.91 (dd, J1 = 2.0 Hz, J2 = 13.0 Hz, 1 H), 7.78 (m, 4 H), 7.75 (s, 1 H), 7.52 (t, J = 7.5 Hz, 2 H), 7.47 (dd, J1 = 2 Hz, J2 = 9 Hz, 1 H), 7.43-7.39 (m, 4 H), 7.26 (t, J = 7.5 Hz, 1 H), 3.88 (s, 3 H), 2.65 (s, 3 H). 13C NMR (125 MHz, d6-DMSO) δ 174.0, 166.0, 161.7, 155.2, 154.2 (d, J = 242.9 Hz, 1 C), 152.5, 150.4, 140.8, 139.2, 138.4 (d, J = 9.8 Hz, 1 C), 135.9, 135.2 (d, J = 12.9 Hz, 1 C), 134.8, 131.5, 129.7, 128.8, 128.7, 128.3, 127.1, 126.6, 125.0, 123.3, 119.9, 118.4, 116.0 (d, J = 2.9 Hz, 1 C), 115.8, 108.0 (d, J = 23.0 Hz, 1 C), 102.4, 35.8, 19.5. HRMS (ESI) for C36H26FN5O3 [M+H]+, calcd: 596.2092, found: 596.2089. HPLC analysis: MeOH-H2O (85:15), 6.78 min, 95.15% purity. Melting point: 255.4-257.7 °C. N-(3-Fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-1, 2-dimethyl-4-


34

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oxo-1, 4-dihydrobenzo[g]quinoline-3-carboxamide (9r). Yellow solid (46 mg, 68%; Rf: 0.23, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 10.91 (s, 1 H), 8.91 (s, 1 H), 8.40 (s, 1 H), 8.34 (s, 1 H), 8.2 (d, J = 8.0 Hz, 1 H), 8.12 (d, J = 8.0 Hz, 1 H), 7.93 (d, J = 12.0 Hz, 1 H), 7.79-7.76 (m, 3 H), 7.68-7.65 (t, J = 7.5 Hz, 1 H), 7.55 (d, J = 7.5 Hz, 1 H), 7.48 (d, J = 8.5 Hz, 1 H), 7.43-7.40 (m, 3 H), 7.27 (t, J = 7.5 Hz, 1 H), 3.92 (s, 3 H), 2.69 (s, 3 H). 13C NMR (125 MHz, d6-DMSO) δ 174.9, 166.1, 161.8, 154.6, 154.2, 154.1 (d, J = 242.3 Hz, 1 C), 150.7, 138.6 (d, J = 8.5 Hz, 1 C), 135.4, 135.0 (d, J = 12.6 Hz, 1 C), 134.6, 129.5, 129.4, 128.9, 128.8, 128.7, 128.2, 126.8, 126.7, 126.3, 125.3, 125.0, 124.3, 117.6, 116.0, 115.9 (d, J = 2.8 Hz, 1 C), 114.5, 107.9 (d, J = 22.6 Hz, 1 C), 102.3, 35.8, 19.9. HRMS (ESI) for C34H24FN5O3 [M+H]+, calcd: 570.1936, found: 570.1931. HPLC analysis: MeOH-H2O (85:15), 6.37 min, 95.08% purity. Melting point: 278.3-280.5 °C. N-(3-Fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-6-methoxy-1, 2dimethyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9s). Beige solid (43 mg, 56%; Rf: 0.28, DCM: Methonal = 20: 1). 1H NMR (400 MHz, d6-DMSO) δ 11.10 (s, 1 H), 8.30 (s, 1 H), 7.92 (dd, J1 = 2.0 Hz, J2 = 12.8 Hz, 1 H), 7.85 (d, J = 9.6 Hz, 1 H), 7.75-7.78 (m, 3 H), 7.67 (d, J = 3.2 Hz, 1 H), 7.46 (dd, J1 = 1.6 Hz, J2 = 8.8 Hz, 1 H), 7.42-7.38 (m, 4 H), 7.25 (m, 1 H), 3.87 (s, 3 H), 3.83 (s, 3 H), 2.65 (s, 3 H). 13C NMR (125 MHz, d6-DMSO) δ 173.3, 166.1, 161.6, 156.3, 154.2 (d, J = 242.3 Hz, 1 C), 151.9, 150.2, 138.4 (d, J = 9.4 Hz, 1 C), 135.9, 135.1 (d, J = 13.6 Hz, 1 C), 134.8, 128.7, 128.6, 127.5, 126.4, 125.4, 124.9, 122.6, 119.4, 118.2, 115.9, 115.7, 107.9 (d, J = 22.6 Hz, 1 C), 105.9, 102.3, 55.9, 35.8, 19.4. HRMS (ESI) for C31H24FN5O4 [M+H]+, calcd: 550.1885, found: 550.1881. HPLC analysis: MeOH-H2O (85:15), 4.69 min, 95.04% purity. Melting point: 271.4-273.5 °C. N-(3-Fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-1, 2-dimethyl-4-


35


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oxo-6-(trifluoromethoxy)-1, 4-dihydroquinoline-3-Carboxamide (9t). White solid (67 mg, 67%; Rf: 0.30, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 12.53 (s, 1 H), 10.76 (s,1 H), 8.33 (s, 1 H), 8.09 (m, 2 H), 7.89 (d, J = 12.5 Hz, 1 H), 7.82-7.76 (m, 4 H), 7.46-7.40 (m, 4 H), 7.27 (m, 1 H), 3.87 (s, 3 H), 2.63 (s, 3 H). 13C NMR (125 MHz, d6-DMSO) δ 172.9, 165.6, 161.7, 154.6, 154.1 (d, J = 242.8 Hz, 1 C), 152.8, 150.7, 144.9, 140.1, 138.4 (d, J = 9.8 Hz, 1C), 135.2 (d, J = 12.5 Hz, 1 C), 134.6, 128.9, 128.7, 126.8, 126.2, 125.0, 124.3, 121.7, 120.7, 120.3, 119.6, 116.8, 116.0, 115.9, 108.0 (d, J = 23.4 Hz, 1 C), 102.3, 36.0, 19.6. HRMS (ESI) for C31H21F4N5O4 [M+H]+, calcd: 604.1602, found: 604.1597. HPLC analysis: MeOH-H2O (85:15), 5.31 min, 97.59% purity. Melting point: 284.1-286.8 °C. Methyl-3-((3-fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)carbamoy l)-1, 2-dimethyl-4-oxo-1, 4-dihydroquinoline-6-carboxylate (9u). A mixture of 9f (200 mg, 0.3 mmol), PdCl2(dppf) (24 mg, 0.033 mmol) and triethylamine in methanol (15 mL) and DMF (50 µL) was stirred at 95 °C overnight under the pressure of CO and then cooled to rt. The mixture was filtratead, the filtrate was concentrated in vacuo and further purified by flash chromatography on silica gel to get the product as a yellow solid (60 mg, 33%; Rf: 0.29, DCM: Methonal = 10: 1). 1H NMR (500 MHz, d6-DMSO) δ 12.52 (s, 1 H), 10.76 (s, 1 H), 8.82 (d, J = 2.0 Hz, 1 H), 8.33 (s, 1 H), 8.25 (dd, J1 = 2.0 Hz, J2 = 9.0 Hz, 1 H), 8.00 (d, J = 9.0 Hz, 1 H), 7.91-7.88 (dd, J1 = 2.0 Hz, J2 = 12.5 Hz, 1 H), 7.77 (m, 3 H), 7.46 (dd, J1 = 1.0 Hz, J2 = 9.0 Hz, 1 H), 7.41 (m, 3 H), 7.28 (t, J = 7.0 Hz, 1 H), 3.92 (s, 3 H), 3.86 (s, 3 H), 2.62 (s, 3 H). 13C NMR (125 MHz, d6-DMSO) δ 173.7, 166.0, 165.5, 161.7, 154.6, 154.1 (d, J = 242.8 Hz, 1 C), 152.8, 150.7, 144.2, 138.3 (d, J = 10.0 Hz, 1 C), 135.2 (d, J = 12.8 Hz, 1 C), 134.7, 132.7, 128.9, 128.7, 128.1, 126.8, 125.8, 125.0, 124.9, 124.3, 121.4, 118.3, 116.0, 115.9 (d, J = 2.5 Hz, 1 C), 108.0 (d, J = 23.0 Hz, 1 C), 102.3, 52.8, 35.9, 19.6. HRMS (ESI) for C32H24FN5O5 [M+H]+, calcd:


36

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578.1834, found: 578.1833. HPLC analysis: MeOH-H2O (85:15), 4.26 min, 96.31% purity. Melting point: >300.0 °C. N-(3-Fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-2, 6-dimethyl-4oxo-1, 4-dihydroquinoline-3-carboxamide (9ha). White solid (65 mg, 73%; Rf: 0.26, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 12.96 (s, 1 H), 12.52 (s, 1 H), 8.34 (s, 1 H), 8.03 (s, 1 H), 7.97 (dd, J1 = 2.0 Hz, J2 = 14.0 Hz, 1 H), 7.78 (dd, J1 = 1.0 Hz, J2 = 8.0 Hz, 2 H), 7.75 (s, 1 H), 7.59-7.54 (m, 2 H), 7.43-7.38 (m, 4 H), 7.27 (t, J = 7.5 Hz, 1 H), 2.83 (s, 3 H), 2.44 (s 3 H). 13C NMR (125 MHz, d6-DMSO) δ 176.6, 165.2, 161.8, 156.2, 154.6, 154.2 (d, J = 242.4 Hz, 1 C), 150.7, 138.4 (d, J = 9.4 Hz, 1 C), 136.9, 134.8 (d, J = 12.4 Hz, 1 C), 134.6, 128.9, 128.7, 126.8, 125.1, 125.0, 124.2, 118.8, 116.2 (d, J = 2.6 Hz, 1 C), 116.0, 110.7, 108.3 (d, J = 23.3 Hz, 1 C), 102.3, 22.0, 21.3. HRMS (ESI) for C30H22FN5O3 [M+H]+, calcd: 520.1779, found: 520.1784. HPLC analysis: MeOH-H2O (85:15), 8.86 min, 98.25% purity. Melting point: 292.3293.5 °C. 1-Ethyl-N-(3-fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-2, 6-dime thyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9hb). White solid (87 mg, 66%; Rf: 0.29, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 10.87 (s, 1 H), 8.32 (s, 1 H), 8.07 (s, 1 H), 7.92 (d, J = 2.0 Hz, 1 H), 7.89-7.76 (m, 4 H), 7.62 (dd, J1 = 1.0 Hz, J2 = 8.5 Hz, 1 H), 7.45 (m, 1 H), 7.40 (m, 3 H), 7.26 (m, 1 H), 4.38 (q, J = 6.5 Hz, 2 H), 2.62 (s, 3 H), 2.45 (s, 3 H), 1.35 (t, J = 6.5 Hz, 3 H). 13C NMR (125 MHz, d6-DMSO) δ 173.7, 166.2, 161.7, 154.9, 154.1 (d, J = 242.5 Hz, 1 C), 150.8, 150.5, 138.5 (d, J = 9.6 Hz, 1 C), 138.3, 135.1 (d, J = 12.6 Hz, 1 C), 134.7, 134.6, 133.7, 128.9, 128.7, 126.7, 126.5, 125.7, 125.0, 124.8, 119.9, 117.3, 115.9 (d, J = 3.8 Hz, 1 C), 107.9 (d, J = 23.0 Hz, 1 C), 102.3, 42.3, 20.9, 18.6, 14.1. HRMS (ESI) for C32H26FN5O3 [M+H]+, calcd: 548.2092, found: 548.2085. HPLC analysis: MeOH- H2O (85:15), 5.32 min,


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98.32% purity. Melting point: 273.7-274.8 °C. N-(3-Fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-2, 6-dimethyl-4oxo-1-propyl-1, 4-dihydroquinoline-3-carboxamide (9hc). White solid (70 mg, 73%; Rf: 0.37, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 10.85 (s, 1 H), 8.29 (s, 1 H), 8.06 (s, 1 H), 7.91 (d, J = 2.0 Hz, 1 H), 7.89-7.75 (m, 4 H), 7.61 (dd, J1 = 2.0 Hz, J2 = 9.0 Hz, 1 H), 7.45 (m, 1 H), 7.44-7.38 (m, 3 H), 7.25 (m, 1 H), 4.25 (t, J = 7.5 Hz, 2 H), 2.61 (s, 3 H), 2.45 (s, 3 H), 1.77-1.73 (m, 2 H), 1.02 (t, J = 7.5 Hz, 3 H).

13

C NMR (125 MHz, d6-DMSO) δ 173.6, 166.2,

161.7, 155.3, 154.1 (d, J = 242.4 Hz, 1 C), 150.8, 150.3, 138.5 (d, J = 14.3 Hz, 1 C), 135.1 (d, J = 12.6 Hz, 1 C), 134.9, 134.5, 133.7, 128.8, 128.7, 126.5, 126.5, 125.6, 125.3, 125.0, 120.0, 117.5, 115.9 (d, J = 2.5 Hz,1 C), 115.8, 107.9 (d, J = 22.9 Hz, 1 C), 102.3, 48.5, 22.0, 20.9, 18.7, 11.1. HRMS (ESI) for C33H28FN5O3 [M+H]+, calcd: 562.2249, found: 562.2245. HPLC analysis: MeOH-H2O (85:15), 6.12 min, 98.11% purity. Melting point: 267.9-269.2 °C. 1-Butyl-N-(3-fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-2, 6-dime thyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9hd). White solid (87 mg, 75%; Rf: 0.27, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 10.87 (s, 1 H), 8.28 (s, 1 H), 8.06 (s, 1 H), 7.92 (dd, J1 = 2.0 Hz, J2 = 13.0 Hz, 1 H), 7.78-7.74 (m, 4 H), 7.61 (dd, J1 = 1.5 Hz, J2 = 8.5 Hz, 1 H), 7.45 (dd, J1 = 1.0 Hz, J2 = 9.0 Hz, 1 H), 7.41-7.38 (m, 3 H), 7.24 (t, J = 7.5 Hz, 1 H), 4.31-4.27 (t, J = 8.0 Hz, 2 H), 2.61 (s, 3 H), 2.45 (s, 3 H), 1.69 (m, 2 H), 1.50-1.45 (q, J = 7.5 Hz, 2 H), 0.97 (t, J = 7.5 Hz, 3 H).

13

C NMR (125 MHz, d6-DMSO) δ 173.6, 166.2, 161.6, 155.8,

154.2 (d, J = 242.6 Hz, 1 C), 150.7, 150.0, 138.5, 138.4 (d, J = 10.0 Hz, 1 C), 135.3 (d, J = 12.9 Hz, 1 C), 135.1, 134.5, 133.6, 128.7, 128.6, 126.5, 126.3, 126.1, 125.6, 125.0, 120.0, 117.4, 115.8 (d, J = 2.3 Hz, 1 C), 115.5, 107.9 (d, J = 23.1 Hz, 1 C), 102.4, 46.9, 30.7, 26.0, 20.9, 19.7, 18.7, 14.1. HRMS (ESI) for C34H30FN5O3 [M+H]+, calcd: 576.2405, found: 576.2407. HPLC


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analysis: MeOH-H2O (85:15), 7.46 min, 98.19% purity. Melting point: 260.4-261.6 °C. N-(3-Fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-2, 6-dimethyl-4oxo-1-phenyl-1, 4-dihydroquinoline-3-carboxamide (9he). White solid (96 mg, 71%; Rf: 0.37, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 11.25 (s, 1 H), 8.29 (s, 1 H), 8.11 (s, 1 H), 7.90-7.93 (dd, J1 = 2.0 Hz, J2 = 12.5 Hz, 1 H), 7.78-7.68 (m, 6 H), 7.47 (m, 3 H), 7.38-7.44 (m, 4 H), 7.25 (t, J = 7.5 Hz, 1 H), 6.57 (d, J = 9.0 Hz, 1 H), 2.42 (s, 3 H), 2.22 (s, 3 H).

13

C

NMR (125 MHz, d6-DMSO) δ 175.0, 165.9, 162.0, 156.0, 154.6 (d, J = 242.8 Hz, 1 C), 152.2, 150.6, 140.7, 139.4, 138.7 (d, J = 9.6 Hz, 1 C), 135.7 (d, J = 12.8 Hz, 1 C), 135.4, 134.7, 134.6, 131.6, 130.8, 129.8, 129.2, 129.1, 126.8, 126.1, 125.9, 125.6, 125.4, 118.9, 118.8, 116.4, 116.0, 108.5 (d, J = 23.5 Hz, 1 H), 102.8, 21.3, 20.8. HRMS (ESI) for C36H26FN5O3 [M+H]+, calcd: 596.2092, found: 596.2098. HPLC analysis: MeOH-H2O (85:15), 8.93 min, 97.96% purity. Melting point: 278.8-280.3 °C. N-(3-Fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-1, 6-dimethyl-4oxo-1, 4-dihydroquinoline-3-carboxamide (9hf). White solid (55 mg, 66%; Rf: 0.20, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 12.67 (s, 1 H), 8.97 (s, 1 H), 8.30 (s, 1 H), 8.20 (s, 1 H), 7.99-7.97 (d, J = 12.5 Hz, 1 H), 7.80-7.74 (m, 5 H), 7.42-7.38 (m, 4 H), 7.25 (m,1 H), 4.06 (s, 3 H), 2.49 (s, 3 H).

13

C NMR (125 MHz, d6-DMSO) δ 176.0, 163.5, 161.6, 155.3,

154.3 (d, J = 242.5 Hz, 1 C), 150.3, 149.4, 138.6, 137.7 (d, J = 9.6 Hz, 1 C), 135.8, 135.3 (d, J = 12.9 Hz, 1 C), 135.1, 134.9, 128.8, 128.7, 127.1, 126.5, 125.8, 125.2, 118.2, 116.3 (d, J = 2.9 Hz, 1C), 115.8, 110.2, 108.5 (d, J = 23.3 Hz, 1 C), 102.4, 41.9, 21.1. HRMS (ESI) for C31H24FN5O3 [M+H]+, calcd: 534.1936, found: 534.19324. HPLC analysis: MeOH-H2O (85:15), 3.78 min, 97.54% purity. Melting point: 280.1-281.7 °C. 2-Ethyl-N-(3-fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-1, 6-dime


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thyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9hg). White solid (76 mg, 70%; Rf: 0.19, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 10.84 (s, 1 H), 8.29 (s, 1 H), 8.05 (s, 1 H), 7.90 (dd, J1 = 2.5 Hz, J2 = 13.0 Hz, 1 H), 7.79-7.76 (m, 3 H), 7.74 (s, 1 H), 7.62 (dd, J1 = 2.0 Hz, J2 = 9.0 Hz, 1 H), 7.45 (dd, J1 = 2.0 Hz, J2 = 9.0 Hz, 1 H), 7.41-7.37 (m, 3 H), 7.24 (t, J = 7.0 Hz, 1 H), 3.84 (s, 3 H), 2.96 (d, J = 7.0 Hz, 2 H), 2.46 (s, 3 H), 1.30 (t, J = 7.0 Hz, 3 H). 13C NMR (125 MHz, d6-DMSO) δ 174.0, 166.2, 161.6, 155.7, 155.5, 154.2 (d, J = 242.0 Hz, 1 C), 150.1, 139.8, 138.4 (d, J = 9.9 Hz, 1 C), 135.2 (d, J = 13.6 Hz, 1 C), 135.0, 134.4, 133.8, 128.8, 128.7, 126.4, 126.3, 125.9, 125.3, 125.0, 119.6, 117.7, 115.9 (d, J = 2.5 Hz, 1 C), 115.6, 107.9 (d, J = 22.5 Hz, 1 C), 102.4, 35.3, 25.2, 20.9, 13.7. HRMS (ESI) for C32H26FN5O3 [M+H]+, calcd: 548.2092, found: 548.2080. HPLC analysis: MeOH-H2O (85:15), 5.08 min, 95.14% purity. Melting point: 292.2-294.5 °C. N-(3-Fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-1, 6-dimethyl-4oxo-2-phenyl-1, 4-dihydroquinoline-3-carboxamide (9hh). White solid (58 mg, 60%; Rf: 0.36, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 10.60 (s, 1 H), 8.27 (s, 1 H), 8.14 (s, 1 H), 7.78 (d, J = 9.0 Hz, 1 H), 7.74 (d, J = 8.5 Hz, 3 H), 7.71-7.69 (dd, J1 = 2.0 Hz, J2 = 9.0 Hz, 1 H), 7.55-7.50 (m, 6 H), 7.39-7.36 (m, 2 H), 7.30-7.27 (m, 1 H), 7.23 (t, J = 7.5 Hz, 1 H), 7.19 (dd, J1 = 1.5 Hz, J2 = 8.5 Hz, 1 H), 3.50 (s, 3 H), 2.51 (s, 3 H). 13C NMR (125 MHz, d6-DMSO) δ 173.8, 164.7, 161.5, 155.6, 154.0 (d, J = 242.4 Hz, 1 C), 152.6, 150.1, 139.5, 138.1 (d, J = 9.8 Hz, 1 C), 135.0 (d, J = 12.8 Hz, 1 C), 134.7, 134.2, 134.1, 130.0, 129.1, 128.9, 128.7, 128.6, 126.7, 126.4, 125.3, 124.8, 120.8, 118.0, 115.6 (d, J = 2.5 Hz, 1 C), 115.5, 107.6 (d, J = 22.9 Hz, 1 C), 102.3, 37.6, 21.0. HRMS (ESI) for C36H26FN5O3 [M+H]+, calcd: 596.2092, found: 596.2096. HPLC analysis: MeOH-H2O (85:15), 5.14 min, 98.3% purity. Melting point: 281.2282.6 °C.


40

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6-Ethyl-N-(2-fluoro-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl)-1, 2-dime thyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9ia). White solid (180 mg, 77%; Rf: 0.35, DCM: Methonal = 20: 1). 1H NMR (400 MHz, d6-DMSO) δ 12.13 (s, 1 H), 8.29 (s, 1 H), 8.24 (t, J = 8.8 Hz, 1 H), 8.17 (s, 1 H), 7.88 (d, J = 8.8 Hz, 1 H), 7.76 (d, J = 7.6 Hz, 2 H), 7.71-7.69 (m, 2 H), 7.40-7.34 (m, 3 H), 7.22 (t, J = 8.0 Hz, 1 H), 7.10 (d, J = 7.2 Hz, 1 H), 3.90 (s, 3 H), 2.91 (s, 3 H), 2.79 (q, J = 7.6 Hz, 2 H), 1.26 (t, J = 7.6 Hz, 3 H).

13

C NMR (125 MHz, d6-DMSO) δ

175.6, 165.9, 162.4, 157.4, 156.2, 153.9 (d, J = 244.0 Hz, 1 C), 150.5, 149.3 (d, J = 10.5 Hz, 1 C), 141.1, 139.7, 135.6, 134.1, 129.3, 129.0, 126.7, 126.5, 126.4, 125.0 (d, J = 11.1 Hz, 1 C), 124.8, 124.1, 118.7, 118.2, 116.0, 115.2, 110.9 (d, J = 22.3 Hz, 1 C), 103.5, 36.6, 28.4, 20.3, 16.3. HRMS (ESI) for C32H26FN5O3 [M+H]+, calcd: 548.2092, found: 548.2098. HPLC analysis: MeOH-H2O (85:15), 7.37 min, 95.08% purity. Melting point: 241.1-243.7 °C. 6-Ethyl-1,2-dimethyl-4-oxo-N-(4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phenyl) -1, 4-dihydroquinoline-3-carboxamide (9ib). White solid (66 mg, 73%; Rf: 0.32, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 10.81 (s, 1 H), 8.29 (s, 1 H), 8.09 (s, 1 H), 7.81 (d, J = 9.0 Hz, 1 H), 7.78-7.75 (m, 4 H), 7.70 (s, 1 H), 7.65 (dd, J1 = 1.5 Hz, J2 = 8.5 Hz, 1 H), 7.38 (m, 2 H), 7.26-7.21 (m, 3 H), 3.83 (s, 3 H), 2.79-274 (q, J = 7.5 Hz, 2 H), 2.64 (s, 3 H), 1.24 (t, J = 7.5 Hz, 3 H).

13

C NMR (125 MHz, d6-DMSO) δ 174.9, 174.0, 165.8, 162.5, 152.2,

150.4, 148.5, 139.9, 139.7, 137.0, 135.1, 133.3, 128.9, 128.6, 126.4, 126.3, 124.2, 122.6, 120.9, 119.5, 117.5, 115.8, 103.0, 35.6, 27.9, 19.5, 16.0. HRMS (ESI) for C32H27N5O3 [M+H]+, calcd: 530.2187, found: 530.2182. HPLC analysis: MeOH-H2O (85:15), 5.25 min, 97.83% purity. Melting point: 271.8-273.2 °C. 6-Ethyl-1,2-dimethyl-N-(3-methyl-4-((5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)ph enyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9ic). White solid (45 mg, 56%; Rf: 0.19,


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DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 10.79 (s, 1 H), 8.28 (s, 1 H), 8.09 (s, 1 H), 7.80 (m, 3 H), 7.71-.65 (m, 3 H), 7.53 (d, J = 7.5 Hz, 1 H), 7.40 (t, J = 7.5 Hz, 2 H), 7.26 (t, J = 7.5 Hz, 1 H), 7.11 (d, J = 8.5 Hz, 1 H), 3.83 (s, 3 H), 2.76 (q, J = 7.5 Hz, 2 H), 2.65 (s, 3 H), 2.09 (s, 3 H), 1.24 (t, J = 7.5 Hz, 3 H). 13C NMR (125 MHz, d6-DMSO) δ 174.0, 165.7, 162.2, 154.7, 152.3, 150.7, 147.0, 139.9, 139.7, 137.2, 134.8, 133.3, 130.7, 128.9, 128.6, 126.6, 126.3, 124.2, 123.0, 122.2, 119.3, 118.6, 117.5, 116.1, 102.6, 35.6, 27.9, 19.5, 16.9, 15.9. HRMS (ESI) for C33H29N5O3 [M+H]+, calcd: 544.2343, found: 544.2348. HPLC analysis: MeOH-H2O (85:15), 5.74 min, 96.04% purity. Melting point: 263.3-265.2 °C. N-(4-((7H-Pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)-3-fluorophenyl)-6-ethyl-1, 2-dimethyl-4oxo-1, 4-dihydroquinoline-3-carboxamide (9id). Yellow solid (65 mg, 72%; Rf: 0.18, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 12.28 (s, 1 H), 10.98 (s, 1 H), 8.30 (d, J = 2.0 Hz, 1 H), 7.92 (dd, J1 = 2.0 Hz, J2 = 12.5 Hz, 1 H), 7.81 (d, J = 9.0 Hz, 1 H), 7.66 (dd, J1 = 2.0 Hz, J2 = 9.0 Hz, 1 H), 7.51 (m, 1 H), 7.87 (dd, J1 = 2.0 Hz, J2 = 9.0 Hz, 1 H), 7.39 (m, 1 H), 6.59 (m, 1 H), 3.83 (s, 3 H), 2.79-2.75 (q, J = 7.5 Hz, 2 H), 2.64 (s, 3 H), 1.24 (t, J = 7.5 Hz, 3 H). 13

C NMR (125 MHz, d6-DMSO) δ 174.0, 166.2, 161.5, 154.2 (d, J = 242.9 Hz, 1 C), 154.1,

152.3, 150.5, 140.0, 138.6 (d, J = 9.9 Hz, 1 C), 135.1 (d, J = 12.9 Hz, 1 C), 133.4, 126.3, 126.0, 124.9, 124.2, 119.2, 117.6, 115.9 (d, J = 2.6 Hz, 1 C), 108.0 (d, J = 23.2 Hz, 1 C), 104.5, 98.3, 35.7, 27.9, 19.5, 16.0. HRMS (ESI) for C26H22FN5O3 [M+H]+, calcd: 472.1779, found: 472.1776. HPLC analysis: MeOH-H2O (85:15), 3.99 min, 96.56% purity. Melting point: 253.9-255.6 °C. 6-Ethyl-N-(3-fluoro-4-((5-(1-methyl-1H-pyrazol-4-yl)-7H-pyrrolo[2, 3-d]pyrimidin-4-yl) oxy)phenyl)-1, 2-dimethyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9ie). White solid (180 mg, 73%; Rf: 0.19, DCM: Methonal = 20: 1). 1H NMR (400 MHz, d6-DMSO) δ 12.30 (s, 1 H), 11.00 (s, 1 H), 8.28 (s, 1 H), 8.09 (s, 1 H), 8.00 (s, 1 H), 7.90 (m, 1 H), 7.83 (m, 2 H), 7.66


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(m, 2 H), 7.49-7.42 (m, 2 H), 3.85 (s, 3 H), 3.84 (s, 3 H), 2.78 (q, J = 7.5 Hz, 2 H), 2.65 (s, 3 H), 1.2 (t, J = 7.5 Hz, 3 H).

13

C NMR (125 MHz, d6-DMSO) δ 174.4, 166.6, 162.1, 154.7 (d. J =

242.5 Hz, 1 C), 154.5, 152.8, 151.0, 140.4, 140.1, 139.0 (d, J = 10.0 Hz, 1 C), 138.4, 135.4 (d, J = 12.5 Hz, 1 C), 133.8, 129.4, 126.7, 125.4, 124.6, 122.5, 119.5, 118.0, 116.4 (d, J = 1.3 Hz, 1 C), 115.6, 108.4 (d, J = 22.5 Hz, 1 C), 107.4, 102.6, 40.0, 39.5, 36.1, 28.2, 19.9, 16.4. HRMS (ESI) for C30H26FN7O3 [M+H]+, calcd: 552.2154, found: 552.2156. HPLC analysis: MeOH-H2O (75:25), 5.34 min, 99.20% purity. Melting point: >300.0 °C. 6-Ethyl-N-(3-fluoro-4-((3-phenyl-1H-pyrrolo[2, 3-b]pyridin-4-yl)oxy)phenyl)-1, 2-dimeth yl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9if). White solid (62 mg, 68%; Rf: 0.25, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 12.03 (s, 1 H), 11.02 (s, 1 H), 8.08 (m, 2 H), 7.98-7.95 (d, J = 13.0 Hz, 2 H), 7.81 (d, J = 8.5 Hz, 1 H), 7.70 (d, J = 7.5 Hz, 2 H), 7.66 (d, J = 8.5 Hz, 1 H), 7.60 (s, 1 H), 7.47 (d, J = 9.0 Hz, 1 H), 7.54 (m, 3 H), 7.21 (t, J = 7 Hz, 1 H), 6.31 (d, J = 5.0 Hz, 1 H), 3.83 (s, 3 H), 2.79-2.74 (q, J = 7.5 Hz, 2 H), 2.64 (s, 3 H), 1.24 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, d6-DMSO) δ 174.0, 166.2, 158.8, 153.9 (d, J = 243.8 Hz, 1 C), 152.4, 152.0, 145.1, 140.0, 139.7, 138.5 (d, J = 9.4 Hz, 1 C), 136.1 (d, J = 11.9 Hz, 1 C), 135.8, 133.4, 129.1, 128.4, 126.3, 126.2, 124.2, 119.1, 117.6, 116.4 (d, J = 2.6 Hz, 1 C), 115.6, 108.5 (d, J = 22.0 Hz, 1 C), 107.6, 101.1, 35.7, 27.9, 19.5, 16.0. HRMS (ESI) for C33H27FN4O3 [M+H]+, calcd: 547.214, found: 547.2145. HPLC analysis: MeOH-H2O (85:15), 6.31 min, 95.08% purity. Melting point: 274.7-276.7 °C. 6-Ethyl-N-(3-fluoro-4-((3-phenylfuro[2,

3-b]pyridin-4-yl)oxy)phenyl)-1,

2-dimethyl-4-

oxo-1, 4-dihydroquinoline-3-carboxamide (9ig). Beige solid (220 mg, 64%; Rf: 0.58, DCM: Methonal = 20: 1).1H NMR (400 MHz, d6-DMSO) δ 11.04 (s, 1 H), 8.58 (s, 1 H), 8.51 (s, 1 H), 8.09 (s, 1 H), 7.94 (d, J = 13.2 Hz, 1 H), 7.83 (m, 3 H), 7.66 (dd, J1 = 1.6 Hz, J2 = 8.8 Hz, 1 H),


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7.52-7.48 (m, 4 H), 7.41 (t, J = 7.2 Hz, 1 H), 3.84 (s, 3 H), 2.80-.74 (q, J = 7.6 Hz, 2 H), 2.65 (s, 3 H), 1.24 (t, J = 7.6 Hz, 3 H).

13

C NMR (125 MHz, d6-DMSO) δ 174.0, 169.2, 166.2, 163.2,

153.9 (d, J = 243.8 Hz, 1 C), 153.4, 152.5, 142.3, 140.0, 139.7, 139.0 (d, J = 10.0 Hz, 1 C), 134.5 (d, J = 12.5 Hz, 1 C), 133.4, 130.2, 129.1, 128.9, 128.6, 126.3, 124.7, 124.1, 120.8, 119.0, 117.6, 116.0, 108.0 (d, J = 22.5 Hz, 1 C), 103.3, 35.7, 27.3, 19.49, 15.9. HRMS (ESI) for C32H25FN4O4 [M+H]+, calcd: 549.1933, found: 549.1936. HPLC analysis: MeOH-H2O (85:15), 6.91 min, 97.17% purity. Melting point: 257.8-259.1 °C. N-(4-((2-Chloropyridin-4-yl)oxy)-3-fluorophenyl)-6-ethyl-1,

2-dimethyl-4-oxo-1,

4-

dihydro quinoline-3-carboxamide (9ih). White solid (50 mg, 70%; Rf: 0.58, DCM: Methonal = 20: 1). 1H NMR (500 MHz, d6-DMSO) δ 11.06 (s, 1 H), 8.31 (d, J = 6.0 Hz, 1 H), 8.06 (d, J = 2.0 Hz, 1 H), 8.00 (dd, J1 = 2.0 Hz, J2 = 13.0 Hz, 1 H), 7.79 (d, J = 9.0 Hz, 1 H), 7.65 (dd, J1 = 2.0 Hz, J2 = 9.0 Hz, 1 H), 7.52 (dd, J1 = 1.0 Hz, J2 = 9.0 Hz, 1 H), 7.39 (t, J = 9.0 Hz, 1 H), 7.09 (d, J = 2.0 Hz, 1 H), 7.00 (dd, J1 = 2.0 Hz, J2 = 6.0 Hz, 1 H), 3.81 (s, 3 H), 2.78-2.74 (q, J = 7.5 Hz, 2 H), 2.63 (s, 3 H), 1.24 (t, J = 7.5 Hz, 3 H). 13C NMR (125 MHz, d6-DMSO) δ 174.2, 166.3, 166.1, 153.7 (d, J = 244.1 Hz, 1 C), 153.1, 152.1, 151.8, 140.1, 139.6, 139.2 (d, J = 9.8 Hz, 1 C), 135.2 (d, J = 12.3 Hz, 1 C), 133.3, 126.3, 124.2, 124.0, 118.3, 117.5, 116.6, 111.4, 111.2, 108.6 (d, J = 22.8 Hz, 1 C), 35.7, 27.9, 19.4, 15.7. HRMS (ESI) for C25H21ClFN3O3 [M+H]+, calcd: 466.1328, found: 466.1325. HPLC analysis: MeOH-H2O (85:15), 5.44 min, 98.91% purity. Melting point: 193.6-194.7 °C. N-(4-((2-Aminopyridin-4-yl)oxy)-3-fluorophenyl)-6-ethyl-1,

2-dimethyl-4-oxo-1,

4-

dihydro quinoline-3-carboxamide (9ii). To a solution of 9ij (106 mg, 0.2 mmol) and pyridine (70 mg, 0.9 mmol) in 2 mL DMF and H2O (11 mg, 0.6 mmol) was added (bis(trifluoro acetoxy)iodo)benzene (135 mg, 0.3 mmol) and the reaction was stirred at rt for 4.0 hrs. Then


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water was added into the mixture and the resulting solution was stirred for another 30 min. The mixture was filtrated and the filter residue was diluted with EA (3×50 mL). The organic solution was washed with brine (2×50 mL), dried over Na2SO4 and concentrated in vacuo. The crude material was purified by flash chromatography on silica gel to get the product as a light yellow solid (38 mg, 38%; Rf: 0.35, DCM: Methanol = 20: 1). 1H NMR (400 MHz, d6-DMSO) δ 10.94 (s, 1 H), 8.08 (s, 1 H), 7.95 (d, J = 13.2 Hz, 1 H), 7.81 (m, 2 H), 7.67 (m, 1 H), 7.48 (d, J = 8.8 Hz, 1 H), 7.30 (t, J = 8.8 Hz, 1 H), 6.19 (dd, J1 = 2.0 Hz, J2 = 5.6 Hz, 1 H), 5.95 (s, 2 H), 5.80 (s, 1 H), 3.83 (s, 3 H), 2.79-.74 (q, J = 7.6 Hz, 2 H), 2.62 (s, 3 H), 1.24 (t, J = 7.6 Hz, 3 H).

13

C

NMR (125 MHz, d6-DMSO) δ 174.4, 166.6, 166.2, 154.5 (d, J = 243.6 Hz, 1 C), 152.6, 150.3, 140.4, 140.1, 139.0 (d, J = 9.8 Hz, 1 C), 136.3 (d, J = 12.1 Hz, 1 C), 133.8, 126.7, 124.9, 124.6, 119.7, 118.0, 116.7, 108.7 (d, J = 23.1 Hz, 1 C), 102.1, 93.3, 28.4, 19.9, 16.4. HRMS (ESI) for C25H23FN4O3 [M+H]+, calcd: 447.1827, found: 447.1822. HPLC analysis: MeOH-H2O (75:25), 4.92 min, 95.41% purity. Melting point: 204.5-206.8 °C. N-(4-((2-Carbamoylpyridin-4-yl)oxy)-3-fluorophenyl)-6-ethyl-1, 2-dimethyl-4-oxo-1, 4-di hydroquinoline-3-carboxamide (9ij). Brown solid (260 mg, 60%; Rf: 0.39, DCM: Methonal = 20: 1). 1H NMR (400 MHz, d6-DMSO) δ 11.06 (s, 1 H), 8.54 (d, J = 5.6 Hz, 1 H), 8.12 (s, 1 H), 8.08 (s, 1 H), 8.00 (d, J = 13.2 Hz, 1 H), 7.82 (d, J = 8.8 Hz, 1 H), 7.72 (s, 1 H), 7.67 (d, J = 8.0 Hz, 1 H), 7.53 (d, J = 8.8 Hz, 1 H), 7.41 (m, 2 H), 7.22 (m, 1 H), 3.84 (s, 3 H), 2.80-2.74 (q, J = 7.6 Hz, 2 H), 2.64 (s, 3 H), 1.24 (t, J = 7.6 Hz, 3 H). 13C NMR (125 MHz, d6-DMSO) δ 174.0, 166.3, 165.8, 165.7, 153.9 (d, J = 243.8 Hz, 1 C), 153.3, 152.4, 151.1, 140.0, 139.6, 139.2 (d, J = 10.0 Hz, 1 C), 135.2 (d, J = 12.5 Hz, 1 C), 133.4, 126.2, 124.4, 124.1, 119.0, 117.6, 116.6, 113.9, 108.7, 108.4 (d, J = 22.5 Hz, 1 C), 35.7, 27.9, 19.5, 15.9. HRMS (ESI) for C26H23FN4O4 [M+H]+, calcd: 475.1776, found: 475.1772. HPLC analysis: MeOH-H2O (75:25), 4.92 min, 98.23% purity.


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Melting point: 250.4-251.8 °C. N-(4-((2-Amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)-6-ethyl-1, 2-dimethyl-4-oxo-1, 4 -dihydroquinoline-3-carboxamide (9ik). To a mixture of 9il (100 mg, 0.2 mmol) in ethyl acetate (2.0 mL), CH3CN (2.0 mL) and H2O (1.0 mL) was added PhI(OCCH3)2 (80 mg, 0.3 mmol) and the resulting solution was stirred at 0 °C overnight. The mixture was concentrated in vacuo and further purified by flash chromatography on silica gel to get the product as a beige solid (50 mg, 53%; Rf: 0.39, DCM: Methonal = 20: 1). 1H NMR (400 MHz, d6-DMSO) δ 10.99 (s, 1 H), 8.08 (s, 1 H), 7.98-7.94 (dd, J1 = 2.4 Hz, J2 = 13.6 Hz,1 H), 7.82 (d, J = 8.8 Hz, 1 H), 7.75 (d, J = 5.6 Hz, 1 H), 7.68-7.65 (dd, J1 = 2.0 Hz, J2 = 8.8 Hz, 1 H), 7.48 (d, J = 9.6 Hz, 1 H), 7.42 (t, J = 8.8 Hz, 1 H), 6.40 (s, 2 H), 5.95 (d, J = 5.6 Hz, 1 H), 3.83 (s, 3 H), 2.80-2.74 (q, J = 7.2 Hz, 2 H), 2.63 (s, 3 H), 1.25 (t, J =7.2 Hz, 3 H).

13

C NMR (125 MHz, d6-DMSO) δ 174.4,

166.6, 160.7, 158.3, 154.1 (d, J = 243.8 Hz, 1 C), 152.8, 148.1, 140.4, 140.1 139.2 (d, J = 9.9 Hz, 1 C), 136.4 (d, J = 12.1 Hz, 1 C), 133.8, 126.6, 124.5, 124.3, 119.5, 118.0, 116.7, 108.7 (d, J = 22.8 Hz, 1 C), 101.1, 101.0, 36.1, 28.3, 19.9, 16.3. HRMS (ESI) for C25H22ClFN4O3 [M+H]+, calcd: 481.1437, found: 481.1435. HPLC analysis: MeOH-H2O (80:20), 5.43 min, 99.59% purity. Melting point: 230.6-231.7 °C. N-(4-((2-Carbamoyl-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)-6-ethyl-1,

2-dimethyl-4-

oxo-1, 4-dihydroquinoline-3-carboxamide (9il). Brown solid (162 mg, 62%; Rf: 0.31, DCM: Methonal = 20: 1). 1H NMR (400 MHz, d6-DMSO) δ 11.05 (s, 1 H), 8.33 (d, J = 5.6 Hz, 1 H), 8.08-8.00 (m, 3 H), 7.83 (d, J = 8.8 Hz, 1 H), 7.74 (s, 1 H), 7.67 (m, 1 H), 7.53 (d, J = 8.8 Hz, 1 H), 7.42 (t, J = 8.8 Hz, 1 H), 6.87 (d, J = 5.2 Hz, 1 H), 3.84 (s, 3 H), 2.80-2.74 (q, J = 7.2 Hz, 2 H), 2.64 (s, 3 H), 1.25 (t, J = 7.2 Hz, 3 H).

13

C NMR (125 MHz, d6-DMSO) δ 174.4, 167.4,

166.7, 161.0, 155.1, 153.9 (d, J = 244.3 Hz, 1 C), 152.8, 149.6, 140.5, 140.1, 139.8 (d, J = 9.5


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Hz, 1 C), 135.6 (d, J = 12.4 Hz, 1 C), 133.8, 126.7, 124.6, 124.5, 119.5, 118.0, 117.3, 117.0, 111.6, 108.9 (d, J = 22.8 Hz, 1 C), 36.1, 28.4, 19.9, 16.4. HRMS (ESI) for C26H22ClFN4O4 [M+H]+, calcd: 509.1386, found: 509.1380. HPLC analysis: MeOH-H2O (70:30), 6.43 min, 99.81% purity. Melting point: 256.8-257.7 °C. N-(4-((6, 7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-6-ethyl-1, 2-dimethyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (9im). White solid (220 mg, 78%; Rf: 0.23, DCM: Methonal = 20: 1). 1H NMR (500 MHz, CDCl3) δ 12.69 (s, 1 H), 8.64 (s, 1 H), 8.34 (s, 1 H), 7.99 (dd, J1= 2.0 Hz, J2 = 12.5 Hz, 1 H), 7.60 (m, 2 H), 7.53 (d, J = 8.5 Hz, 1 H), 7.45 (d, J = 8.5 Hz, 1 H), 7.36 (s, 1 H), 7.29-7.26 (m, 1 H), 4.09 (s, 3 H), 4.07 (s,3 H), 3.91 (s, 3 H), 3.08 (s, 3 H), 2.84-2.80 (q, J = 7.5 Hz, 2 H), 1.35-1.32(t, J = 7.5 Hz, 3 H).

13

C NMR (125 MHz, CDCl3) δ

176.6, 165.0, 164.9, 158.2, 156.0, 154.3 (d, J = 245.6 Hz, 1 C), 152.9, 150.3, 149.4, 141.5, 138.7, 138.3 (d, J = 9.6 Hz, 1 C), 135.1 (d, J = 12.9 Hz, 1 C), 133.6, 126.4, 125.4, 125.3, 123.7, 123.7, 116.2 (d, J = 2.8 Hz, 1 C), 115.8, 113.6, 110.3, 109.4 (d, J = 22.9 Hz, 1 C), 106.8, 101.1, 56.4, 35.7, 28.3, 20.4, 15.3. HRMS (ESI) for C26H22ClFN4O4 [M+H]+, calcd: 543.2038, found: 543.2035. HPLC analysis: MeOH-H2O (85:15), 5.03 min, 99.75 %. Melting point: 260.7261.6 °C. 4-(2-Fluoro-4-nitrophenoxy)-7H-pyrrolo[2, 3-d]pyrimidine (12h). To a mixture of 4chloro-7H-pyrrolo[2, 3-d] pyrimidine (7.7 g, 50.0 mmol) and 2-fluoro-4-nitrophenol (11.0 g, 70.0 mmol) in 50 mL 1-methyl-2-pyrrolidinone (NMP) was added N,N-diisopropylethyl amine (12.0 mL, 70.0 mmol). The reaction was heated to 200 °C under microwave irradiation and stirred for 1.0 hr. The reaction mixture was cooled to room temperature and poured into ice water. The resulting mixture was filtrated, the filter residue was rinsed with additional water (3×100 mL) and then dried in a vacuum oven to afford 12h as a brown solid (12.0 g, 85%). 1H NMR (400


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MHz, d6-DMSO) δ 12.42 (s, 1 H), 8.38 (dd, J1 = 2.8 Hz, J2 = 10.4 Hz, 1 H), 8.33 (s, 1 H), 8.21 (m, 1 H), 7.81-7.77 (m, 1 H), 7.58 (t, J = 2.8 Hz, 1 H), 6.68 (dd, J1 = 1.6 Hz, J2 = 3.6 Hz, 1 H). 4-(2-Fluoro-4-nitrophenoxy)-5-iodo-7H-pyrrolo[2, 3-d]pyrimidine (13h). To a solution of compound 12h (11.2 g, 40.0 mmol) and KOH (6.7 g, 120.0 mmol) in 200 mL of DMF was added iodides (15.2 g, 60.0 mmol) over 10 minutes at 0 °C. The reaction mixture was stirred for 4.0 hrs at 0 °C and then poured into ice water. The precipitate was filtered, the filter cake was rinsed with additional cool water and then dried in a vacuum oven to afford 13h as a yellow solid (14.0 g, 88%). 1H NMR (400 MHz, d6-DMSO) δ 12.74 (s, 1 H), 8.38 (dd, J1 = 2.4 Hz, J2 = 10.0 Hz, 1 H), 8.34 (s, 1 H), 8.21 (m, 1 H), 7.80 (m, 2 H). 4-(2-Fluoro-4-nitrophenoxy)-5-iodo-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2, 3d]pyrimidine (14h). To a solution of 13h (12.2 g, 30.0 mmol) in DMF (150 mL) was added NaH (60%, 1.3 g, 33.0 mmol) under ice bath. The reaction was stirred at 0 °C for 15 min, then (2-(chloromethoxy)ethyl)trimethylsilane (5.8 mL, 33.0 mmol) was added to the mixture. The reaction solution was stirred at room temperature overnight and quenched with ice water. The mixture was extracted with DCM (4×300mL). The combined organic layer was washed with brine (2×400 mL), dried over Na2SO4, concentrated in vacuo and further purified by flash chromatography on silica gel to get the product 14h as a white solid (13.2 g, 83%). 1H NMR (400 MHz, d6-DMSO) δ 8.43 (s, 1 H), 8.39 (dd, J1 = 2.8 Hz, J2 = 10.4 Hz, 1 H), 8.22 (m, 1 H), 8.00 (s, 1 H), 7.82 (t, J = 8.4 Hz, 1 H), 5.61 (s, 2 H), 3.54 (t, J = 8.0 Hz, 2 H), 0.84 (t, J = 8.0 Hz, 2 H), -0.08 (s, 9 H). 4-(2-fluoro-4-nitrophenoxy)-5-phenyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo [2, 3-d]pyrimidine (16h). To a solution of 14h (7, 12.0 g, 22.7 mmol) and phenyl boronic acid (15h, 3.3 g, 27.2 mmol) in 200 mL PhMe were added Pd(PPh3)4 (1.3 g, 1.1 mmol) and Na2CO3 (7.2 g,


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68.1 mmol). The mixture was stirred at 90 °C under argon overnight and cooled to room temperature. The reaction mixture was filtrated, and the filtrate was extracted with DCM (3×400 mL). The combined organic layer was washed with brine (2×400 mL), dried over Na2SO4 and concentrated in vacuo. The crude material was purified by flash chromatography on silica gel to get the product as a yellow solid (16h, 8.3 g, 76%). 1H NMR (400 MHz, d6-DMSO) δ 8.45 (s, 1 H), 8.37 (dd, J1 = 2.8 Hz, J2 = 10.0 Hz, 1 H), 8.21 (m, 1 H), 8.01 (s, 1 H), 7.86-7.83 (t, J = 8.0 Hz, 1 H), 7.76 (m, 2 H), 7.43 (t, J = 8.0 Hz, 1 H), 7.31 (m, 1 H), 5.70 (s, 2 H), 3.61 (t, J = 8.0 Hz, 2 H), 0.86 (t, J = 8.0 Hz, 2 H), -0.08 (s, 9 H). 3-Fuoro-4-((5-phenyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-Pyrrolo[2, 3-d]pyrimidin4-yl)oxy)aniline (17h). To a solution of 16h (8.0 g, 16.6 mmol) in 90 mL of THF and 30 mL of methanol was added NaBH4 (3.8 g, 99.6 mmol) and NiCl2·6H2O (394 mg, 1.7 mmol). The mixture was stirred at room temperature for 2.0 hrs. The reaction mixture was quenched with water and filtrated, the filtrate was concentrated in vacuo and further purified by flash chromatography on silica gel to get the product 17h as a solid (5.3 g, 71%). 1H NMR (400 MHz, d6-DMSO) δ 8.39 (s, 1 H), 7.89 (s, 1 H), 7.74 (m, 2 H), 7.41 (t, J = 7.6 Hz, 2 H), 7.29 (t, J = 7.6 Hz, 1 H), 7.01 (t, J = 8.8 Hz, 1 H), 6.47 (dd, J1 = 2.4 Hz, J2 = 12.8 Hz, 1 H), 6.39 (dd, J1 = 2.0 Hz, J2 = 8.8 Hz, 1 H), 5.66 (s, 2 H), 3.59 (t, J = 8.0 Hz, 2 H), 0.85 (t, J = 8.0 Hz, 2 H), -0.08 (s, 9 H). N-(3-Fluoro-4-((5-phenyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2, 3-d]pyrimid in-4-yl)oxy)phenyl)-1, 2, 6-trimethyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (18h). A mixture of 17h (450 mg, 1.0 mmol), 29h (277 mg, 1.2 mmol), HATU (570 mg, 1.5 mmol) and DIEA (0.5 mL, 3.0 mmol) in DMF (30 mL) was stirred at room temperature overnight. Then water was added to the mixture and the precipitate was filtrated. The filter cake was washed with water, diluted with DCM and concentrated in vacuo. The resulting residue was purified by flash


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chromatography on silica gel to get the product as a white solid (18h, 478 mg, 72%). 1H NMR (400 MHz, d6-DMSO) δ 11.01 (s, 1 H), 8.43 (s, 1 H), 8.07 (s, 1 H), 7.95 (s, 1 H), 7.94-7.90 (dd, J1 = 2.0 Hz, J2 = 12.8 Hz, 1 H), 7.82-7.77 (m, 3 H), 7.63 (dd, J1 = 2.0 Hz, J2 = 8.8 Hz, 1 H), 7.487.40 (m, 4 H), 7.34 (m, 1 H), 5.70 (s, 2H), 3.84 (s, 3 H), 3.62 (t, J = 8.0 Hz, 2 H), 2.74 (s, 3 H), 2.46 (s, 3 H), 0.88 (t, J = 8.0 Hz, 2 H), -0.06 (s, 9 H). N-(3-Fluoro-4-((7-(hydroxymethyl)-5-phenyl-7H-pyrrolo[2, 3-d]pyrimidin-4-yl)oxy)phen yl)-1, 2, 6-trimethyl-4-oxo-1, 4-dihydroquinoline-3-carboxamide (19h). To a solution of compound 18h (400 mg, 0.6 mmol ) in DCM (20 mL) was added trifluoroacetic acid (3 mL). The resulting mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and water was added to the resulting residue. The precipitate was filtrated, the filter cake was washed with methanol and dried in a vacuum oven to obtain the title product as a white solid (300 mg, 89 %). 1H NMR (400 MHz, d6-DMSO) δ 11.00 (s, 1 H), 8.41 (s, 1 H), 8.07 (s, 1 H), 7.89 (d, J = 12.0 Hz, 1 H), 7.85 (s, 1 H), 7.81-7.76 (m, 3 H), 7.63 (d, J = 8.0 Hz, 1 H), 7.47-7.39 (m, 4 H), 7.31 (t, J = 8.0 Hz, 1 H), 5.68 (s, 2 H), 3.83 (s, 3 H), 2.64 (s, 3 H), 2.46 (s, 3 H). 6-Methyl-1H-benzo[d][1, 3]oxazine-2, 4-dione (21h). To a solution of 2-amino-5-methyl benzoic acid (20h, 3.6 g, 23.8 mmol) and pyridine (3.8 mL, 47.6 mmol) in acetonitrile (50 mL) was added triphosgene (2.4 g, 8.0 mmol) under ice bath. The resulting solution was stirred at 55 °C for 2.0 hrs. After cooling to room temperature, the reaction was quenched with concentrated NaHCO3. The precipitate was filtrated, the filter cake was washed with water and petroleum ether (PE), then dried under vacuum to afford the title product (21h, 3.9 g , 94%). 1H NMR (400 MHz, d6-DMSO) δ 11.63 (s, 1 H), 7.72 (s, 1 H), 7.58-7.55 (dd, J1 = 4.0 Hz, J2 = 8.0 Hz, 1 H), 7.06 (d, J = 8.0 Hz, 1 H), 2.33 (s, 3 H).


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1, 6-Dimethyl-1H-benzo[d][1, 3]oxazine-2,4-dione (23h). To a solution of 21h (3.0 g, 16.9 mmol) and DIEA (5.6 mL, 33.8 mmol) in DMF (50 mL) was added MeI (2.1 mL, 33.8 mmol) dropwise. The mixture was stirred at 40 °C overnight. After cooling to room temperature, the reaction was quenched with water. The precipitate was filtrated, the filter cake was washed with additional water. The crude material was purified by flash chromatography on silica gel to yield the product as a yellow solid (23h, 2.8 g, 88%). 1H NMR (400 MHz, d6-DMSO) δ 7.8 (s, 1 H), 7.68 (dd, J1 = 4.0 Hz, J2 = 8.0 Hz, 1 H), 7.34 (d, J = 8.0 Hz, 1 H), 3.44 (s, 3 H), 2.36 (s, 3 H). Methyl 1, 2, 6-trimethyl-4-oxo-1, 4-dihydroquinoline-3-carboxylate (24h). To a solution of 22h (1.9 mL, 18.0 mmol) in DMF (50 mL) was added NaH (60%, 720 mg, 18.0 mmol) under ice bath. After stirring at room temperature for 30 min, 23h (2.9 g, 15.0 mmol) was added to the reaction solution. The mixture was warmed to 120 °C and strried overnight. The reaction mixture was cooled to room temperature and quenched with ice water. The resulting residue was extracted with DCM (3×150 mL). The combined organic layer was washed with brine (2 × 150 mL), dried over Na2SO4, concentrated in vacuo, and futher purified by flash chromatography on silica gel to yield the title product (24h, 2.8 g, 60%). 1H NMR (400 MHz, d6-DMSO) δ 7.95 (s, 1 H), 7.74 (d, J = 8.0 Hz, 1 H), 7.58 (dd, J1 = 4.0 Hz, J2 = 8.0 Hz, 1 H), 3.77 (s, 3 H), 3.75 (s, 3 H), 2.44 (s, 3 H), 2.42 (s, 3 H). 1, 2, 6-Trimethyl-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (29h). To a solution of 24h (3.0 g, 12.2 mmol) in 40 mL of THF and 20 mL of water was added NaOH (2.0 g, 48.8 mmol). The mixture was refluxed overnight and then cooled to room temperature. The solvent was concentrated in vacuo and the residue was diluted with water. The resulting solution was acidified to pH 6-7 with 1N aqueous HCl. The precipitate was filtrated and the filter cake was dried under vacuum to afford 29h as a white solid (2.6 g, 92%). 1HNMR (400 MHz, d6-DMSO)


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δ 8.16 (s, 1 H), 7.99 (d, J = 8.0 Hz, 1 H), 7.77 (d, J = 8.0 Hz, 1 H), 3.96 (s, 3 H), 3.11 (s, 3 H), 2.49 (s, 3 H). Cell Culture. MDA-MB-231 cells were purchased from American type culture collection (ATCC). MDA-MB-231 were maintained in Roswell Park Memorial Institute (RPMI)-1640 supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 50 mg/mL streptomycin, and 2 mmol/L glutamine in a humidified CO2 incubator at 37 °C. All cells were passaged for less than 3 months before renewal from frozen, early-passage stocks obtained from the indicated sources. Reagents and Antibodies. The compound was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) at a concentration of 10 mmol/L and stored at -20 °C. Primary antibodies against Axl

(8661),

phosphor-Axl

(Tyr702,

5724),

E-cadherin

(3195),

N-cadherin

(13116),

glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 2118) and anti-rabbit or anti-mouse IgG horseradish peroxidase (HRP)-linked secondary antibodies were purchased from Cell Signaling Technology (CST, Boston, MA, USA). Primary antibodies against v-akt murine thymoma viral oncogene homolog (AKT, SC8312), phosphor-AKT (SC16646R, SC7985R) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). In Vitro Enzymatic Activity Assay. Axl and the Z′- Lyte Kinase Assay Kit were purchased from Invitrogen. The experiments were performed according to the instructions of the manufacturer. The concentrations of kinases were determined by optimization experiments and the respective concentration was: Axl (PV3971, Invitrogen) 0.22 µg/ µL. Western blot analysis. The western blot analysis was carried out by following the protocol described before. Briefly, after the indicated treatment, cell lysates were collected dissolving cells in 1×SDS sample lysis buffer (CST recommended). After being sonicated and boiled, the


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supernatant of cell lysate were used for western blot analysis. Cell lysates were loaded to 8-12% SDS-PAGE and separated by electrophoresis. Separated proteins were then electrically transferred to a PVDF film. After being blocked with 1×TBS containing 0.5% Tween-20 and 5% non-fat milk, the film was incubated with corresponding primary antibody followed by HRPconjugated secondary antibody. And the protein lanes were visualized using ECL Western Blotting Detection Kit (Thermo Scientific, USA). Cell Proliferation Assay. Tumor cells were plated into 96-well plates (3000 cell/well) in complete medium. After incubation overnight, cells were exposed to various concentrations (0.016~50 µM) of the test compounds for 24, 48 or 72 hrs. Cell proliferation was evaluated by 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay or Cell Counting Kit 8 (CCK8, CK04, Dojindo laboratories, Japan). IC50 values were calculated by concentrationresponse cure fitting using GraphPad Prism 5.0 software. Each IC50 value was expressed as mean ± SD. Wound Healing Assay. Cells were seeded in a 6-well plate and allowed to grow to nearly 100% confluence in culture medium. Subsequently, a cell-free line was manually created by scratching the confluent cell monolayers with a 200-µl pipette tip. The wounded cell monolayers were washed three times with PBS and incubated in RPMI-1640 (containing 2% FBS) with or without TGF-β1 of 10 ng/ml and different concentration of test compounds for 24 h. Three scratched fields were randomly chosen and the images were captured by bright-field microscope (CKX41; Olympus). The percentage of wound closure was measured using Adobe Photoshop 7.0.1 (Adobe Systems Inc., San Jose, CA). The experiment was performed three times and in triplicate. Cell Migration and Invasion Assay. Cell migration assays were evaluated in Transwell chambers (Corning Costar). Cell invasion assays were evaluated in Matrigel invasion chambers


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(Corning Costar). 0.2~1 x 105 tumor cells were plated in the top chamber with medium without FBS. RPMI-1640 medium containing 2% FBS with or without TGF- β1 of 10 ng/ml and test compound (0.04~5 µmol/L) was added to the bottom chamber. After incubation for 24 hrs at 37 °C, the cells were fixed in 100% methanol and stained with 0.25% crystal violet; and the cells that had not migrated from the top surface of the filters were removed with cotton. Migrated cells were quantitated by counting cells in six randomly selected fields on each filter under a microscope at 200 magnification and graphed as the mean of three independent experiments. Immunofluorescence. The cells were seeded on coverslips, which were placed in the 6-well plate in advance. Following treatment with or without TGF-β1 (10 ng/mL) and test compounds (0.04, 0.2, 1, 5 µM) for 96-144 hours, the cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, then blocked with 3% normal goat serum albumin for 1.0 hour at room temperature and incubated with anti-E-cadherin and anti-N-cadherin antibody at room temperature for 1.5 hrs. Subsequently, the cells were rinsed thoroughly with PBST, and were then incubated with Alexa Fluor 555-conjugated goat anti-rabbit IgG (CST, USA) or Alexa Fluor 488-conjugated goat anti-mouse IgG (CST, USA) for 1.0 hour at room temperature in the dark. ProLong Gold antifade reagent (Invitrogen, USA) was used to stain the nuclei for 5 min at room temperature. The cells were visualized with Laser scanning confocal microscope (Zeiss710; Germany). Determination of Pharmacokinetic Parameters in Rats. Male Sprague−Dawley rats (190−230 g) were dosed with the test compounds intravenously (iv) at 2.5 or 5.0 mg/kg and by oral gavage (po) at 25 mg/kg. The compounds was dissolved in mixed solvents (5% EtOH, 3% Tween and a bit of 0.1 mol/L HCl). Blood samples (0.5 mL) were then obtained via orbital sinus puncture at 0.25 h, 0.75 h, 1.5 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h and 96 h time


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points and collected into heparinized tubes. Samples were centrifuged for cell removal, and the plasma supernatant was then transferred to a clean vial and subsequently stored in -80 °C prior to analysis. Test sample concentrations were determined by LC/MS and pharmacokinetic parameters were calculated using DAS 2.0 software. 4T1 Xenograft Model. 0.5×106 4T1 cells were implanted in the right flank of female BALB/c mice. Twenty-four hours after inoculation, mice were randomized into different treatment groups (n = 10). 9im (30, 90 mg/kg, qd) or vehicle were administrated orally through 21 days. Body weight and tumor size were measured every two days. After the mice death or being sacrificed at the end point, mice livers were collected by resection and fixed in 10% buffered formalin, After being paraffin embedded and sectioned, the liver tissue section slides were subjected for hematoxylin and eosin (H&E) staining. Two H&E-stained liver sections per animal were examined by microscope for micrometastases in at least three view fields. The number of liver micrometastases events were counted. ASSOCIATED CONTENT Supporting Information. Biological data, and assay details, 1H NMR, and

13

C NMR for final compounds and the

intermediates. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors *Tel: +86-20-85228025. Fax: +86-20-85224766. E-mail: [email protected] (K.D.); [email protected] (R.M.).


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Author Contributions Li Tan and Zhang Zhang contribute equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors thank the financial support from National Natural Science Foundation of China (81425021 and 21572230), Guangzhou Science Technology and innovation commission (201508030036)

and

the

Natural

Science

Foundation

of

Guangdong

Province

(2015A030312014). ABBREVIATIONS Mer, c-mer proto-oncogene tyrosine kinase; CML, chronic myeloid leukemia; Gas6, growth arrest-specific 6; HCC, hepatic cellular cancer; SCC, squamous-cell carcinoma; GIST, gastrointestinal stromal tumors; RCC, renal cell carcinoma; CRC, colorectal carcinoma; EMT, epithelial-mesenchymal transition; ER+, estrogen receptor positive; Her2+, Human epidermal growth factor receptor 2 positive; FDA, Food and Drug Administration; AML, acute myeloid leukemia;

DHBA,

dual

hydrogen

bond

acceptor;

SEMCl,

(2-(chloromethoxy)ethyl)

trimethylsilane; rt, room temperature; DIPEA, N,N-diisopropylethylamine; NMP, 1-methyl-2pyrrolidinone;

DMF,

N,N-dimethyl

formamide;

[bis(dimethylamino)methylene]-1H-1,2,3-triazolo

THF,

tetrahydrofuran;

[4,5-b]pyridinium

HATU,

1-

3-oxid

hexafluorophosphate; F3CCOOH, trifluoroacetic acid; DCM, dichloromethane; MeCN, acetonitrile; PPA, polyphosphoric acid; FRET, fluorescence resonance energy transfer; IC50, the


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half maximal (50%) inhibitory concentration (IC) of a substance; Kd, binding constant; Abl, abelson murine leukemia viral oncogene; BLK, B lymphoid tyrosine kinase; CDK, cyclindependent-kinase; DDR, discoidin domain receptor; DRAK1, death-associated protein kinase related 1; Flt3, FMS-like tyrosine kinase 3; HIPK, homeodomain interacting protein kinase; Kit, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog; LOK, serine/threonine kinase 10; MEK, mitogen-activated protein kinase kinase; Slk, Ste-like kinase; Trk, nerve growth factor receptor; PK, pharmacokinetic; TGFβ, transforming growth factor β; H&E, hematoxylin-eosin; ATCC, American type culture collection; RPMI, Roswell Park Memorial Institute; FBS, fetal bovine serum. DMSO, dimethyl sulfoxide; ERK, mitogen-activated protein kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; AKT, v-akt murine thymoma viral oncogene homolog; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CCK, Cell Counting Kit; REFERENCES (1) (a) O’Bryan, J. P.; Frye, R. A.; Cogswell, P. C.; Neubauer, A.; Kitch, B.; Prokop, C.; Espinosa, R.; Le Beau, M. M.; Earp, H. S.; Liu, E. T. Axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase. Mol. Cell. Biol. 1991, 11, 5016−5031. (b) Myers, S. H.; Brunton, V. G.; Unciti-Broceta, A., Axl inhibitors in cancer: a medicinal chemistry perspective. J. Med. Chem. 2016, 59, 3593-3608. (2) (a) Varnum, B. C.; Young, C.; Elliott, G.; Garcia, A.; Bartley, T. D.; Fridell, Y. W.; Hunt, R. W.; Trail, G.; Clogston, C.; Toso, R. J.; Yanagihara, Donna.; Bennett, L.; Sylber, M.; Merewether, L. A.; Tseng, A.; Escobar, E.; Liu, E. T.; Yamane, H. K. Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrestspecific gene 6. Nature 1995, 373, 623-626. (b) Nagata, K.; Ohashi, K.; Nakano, T.; Arita,


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H.; Choi, Y. H.; Kwon, T. K. Axl is a novel target of withaferin A in the induction of apoptosis and the suppression of invasion. Biochem. Biophys. Res. Commun. 2014, 451, 455-460. (c) Abu-Thuraia, A.; Gauthier, R.; Chidiac, R.; Fukui, Y.; Screaton, R. A.; Gratton, J. P.; Côté, J. F. Axl phosphorylates Elmo scaffold proteins to promote Rac activation and cell invasion. Mol. Cell Biol. 2015, 35, 76-87. (7) (a) Seitz, H. M.; Camenisch, T. D.; Lemke, G.; Earp, H. S.; Matsushima, G. K. Macrophages and dendritic cells use different Axl/Mertk/Tyro3 receptors in clearance of apoptotic cells. J. Immunol. 2007, 178, 5635-5642. (b) Rothlin, C. V.; Ghosh, S.; Zuniga, E. I.; Oldstone, M. B.; Lemke, G. TAM receptors are pleiotropic inhibitors of the innate immune response. Cell 2007, 131, 1124-1136. (c) Lemke, G.; Rothlin, C. V. Immunobiology of the TAM receptors. Nat. Rev. Immunol. 2008, 8, 327–336. (d) Rothlin, C. V.; Lemke, G. TAM receptor signaling and autoimmune disease. Curr. Opin. Immunol. 2010, 22, 740–746. (e) Shibata, T.; Habiel, D. M.; Coelho, A. L.; Kunkel, S. L.; Lukacs, N. W.; Hogaboam, C. M. Axl receptor blockade ameliorates pulmonary pathology resulting from primary viral infection and viral exacerbation of asthma. J. Immunol. 2014, 192, 35693581. (8) (a) Wimmel, A.; Glitz, D.; Kraus, A.; Roeder, J.; Schuermann, M. Axl receptor tyrosine kinase expression in human lung cancer cell lines correlates with cellular adhesion. Eur. J. Cancer 2001, 37, 2264–2274. (b) Shinh, Y. S.; Lai, C. Y.; Kao, Y. R.; Shiah, S. G.; Chu, Y. W.; Lee, H. S.; Wu, C. W. Expression of Axl in lung adenocarcinoma and correlation with tumor progression. Neoplasia 2005, 7, 1058-1064. (c) Ishikawa, M.; Sonobe, M.; Nakayama, E.; Kobayashi, M.; Kikuchi, R.; Kitamura, J.; Imamura, N.; Date, H. Higher expression of receptor tyrosine kinase Axl, and differential expression of its ligand, Gas6,


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predict poor survival in lung adenocarcinoma patients. Ann. Surg. Oncol. 2013, 20 467-476. (d) Linger, R. M.; Cohen, R. A.; Cummings, C. T.; Sather, S.; Migdall-Wilson, J.; Middleton, D. H.; Lu, X.; Baron, A. E.; Franklin, W. A.; Merrick, D. T.; Jedlicka, P.; DeRyckere, D.; Heasley, L. E.; Graham, D. K. Mer or Axl receptor tyrosine kinase inhibition promotes apoptosis, blocks growth and enhances chemosensitivity of human nonsmall cell lung cancer. Oncogene 2013, 32, 3420-3431. (e) Bivona, T.; Okimoto, R. AXL receptor tyrosine kinase as a therapeutic target in NSCLC. Lung Cancer: Targets and Ther. 2015, 6, 27–34. (9) (a) Berclaz, G.; Altermatt, H. J.; Rohrbach, V.; Kieffer, I.; Dreher, E.; Andres, A. C. Estrogen dependent expression of the receptor tyrosine kinase axl in normaland malignant human breast. Ann. Oncol. 2001, 12, 819-824. (b) Zhang, Y. X.; Knyazev, P. G.; Cheburkin, Y. V.; Sharma, K.; Knyazev, Y. P.; Orfi, L.; Szabadkai, I.; Daub, H.; Keri, G.; Ullrich, A. AXL is a potential target for therapeutic intervention in breast cancer progression. Cancer Res. 2008, 68, 1905-1915. (c) Gjerdrum, C.; Tiron, C.; Hoiby, T.; Stefansson, I.; Haugen, H.; Sandal, T.; Collett, K.; Li, S.; McCormack, E.; Gjertsen, B. T.; Micklem, D. R.; Akslen, L. A.; Glackin, C.; Lorens, J. B. Axl is an essential epithelial-to-mesenchymal transitioninduced regulator of breast cancer metastasis and patient survival. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 1124-1129. (d) Asiedu, M. K.; Beauchamp-Perez, F. D.; Ingle, J. N.; Behrens, M. D.; Radisky, D. C.; Knutson, K. L. AXL induces epithelial-to-mesenchymal transition and regulates the function of breast cancer stem cells. Oncogene 2014, 33, 13161324. (e) D’Alfonso, T. M.; Hannah, J.; Chen. Z. M.; Liu, Y. F.; Zhou, P. B.; Shin, S. J. Axl receptor tyrosine kinase expression in breast cancer. J. Clin. Pathol. 2014, 67, 690–696.


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(10) (a) Xu, M. Z.; Chan, S. W.; Liu, A. M.; Wong, K. F.; Fan, S. T.; Chen, J.; Poon, R. T.; Zender, L.; Lowe, S. W.; Hong, W.; Luk, J. M. AXL receptor kinase is a mediator of YAPdependent oncogenic functions in hepatocellular carcinoma. Oncogene. 2011, 30, 1229– 1240. (b) Lee, H. J.; Jeng, Y. M.; Chen, Y. L.; Chung, L.; Yuan, R. H. Gas6/Axl pathway promotes tumor invasion through the transcriptional activation of Slug in hepatocellular carcinoma. Carcinogenesis 2014, 35, 769-775. (c) Reichl, P.; Dengler, M.; van Zijl, F.; Huber, H.; Fuhrlinger, G.; Reichel, C.; Sieghart, W.; Peck-Radosavljevic, M.; Grubinger, M.; Mikulits, W. Axl activates autocrine transforming growth factor-beta signaling in hepatocellular carcinoma. Hepatol. 2015, 61, 930-941. (11) (a) Koorstra, J. B.; Karikari, C. A.; Feldmann, G.; Bisht, S.; Rojas, P. L.; Offerhaus, G. J.; Alvarez, H.; Maitra, A. The Axl receptor tyrosine kinase confers an adverse prognostic influence in pancreatic cancer and represents a new therapeutic target. Cancer Biol. Ther. 2009, 8, 618–626. (b) Leconet, W.; Larbouret, C.; Chardes, T.; Thomas, G.; Neiveyans, M.; Busson, M.; Jarlier, M.; Radosevic-Robin, N.; Pugniere, M.; Bernex, F.; Penault-Llorca, F.; Pasquet, J. M.; Pelegrin, A.; Robert, B. Preclinical validation of AXL receptor as a target for antibody-based pancreatic cancer immunotherapy. Oncogene 2014, 33, 5405–5414. (12) (a) Green, J.; Ikram, M.; Vyas, J.; Patel, N.; Proby, C. M.; Ghali, L.; Leigh, I. M.; O'Toole, E. A.; Storey, A. Overexpression of the Axl tyrosine kinase receptor in cutaneous SCC-derived cell lines and tumours. Br. J. Cancer. 2006, 94, 1446-1451. (b) Papadakis, E. S.; Cichon, M. A.; Vyas, J. J.; Patel, N.; Ghali, L.; Cerio, R.; Storey, A.; O'Toole, E. A. Axl promotes cutaneous squamous cell carcinoma survival through negative regulation of proapoptotic Bcl-2 family members. J. Invest. Dermatol. 2011, 131, 509-517. (c) Lee, C. H.; Yen, C. Y.; Liu, S. Y.; Chen, C. K.; Chiang, C. F.; Shiah, S. G.; Chen, P. H.; Shieh, Y. S.


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Axl is a prognostic marker in oral squamous cell carcinoma. Ann. Surg. Oncol. 2012, 19, 500-508. (13) Sainaghi, P. P.; Castello, L.; Bergamasco, L.; Galletti, M.; Bellosta, P.; Avanzi, G. C. Gas6 induces proliferation in prostate carcinoma cell lines expressing the Axl receptor. J. Cell Physiol. 2005, 204, 36-44. (14) Mahadevan, D.; Cooke, L.; Riley, C.; Swart, R.; Simons, B.; Della Croce, K.; Wisner, L.; Iorio, M.; Shakalya, K.; Garewal, H.; Nagle, R.; Bearss, D. A novel tyrosine kinase switch is a mechanism of imatinib resistance in gastrointestinal stromal tumors. Oncogene 2007, 26, 3909-3919. (15) (a) Sensi, M.; Catani, M.; Castellano, G.; Nicolini, G.; Alciato, F.; Tragni, G.; De Santis, G.; Bersani, I.; Avanzi, G.; Tomassetti, A.; Canevari, S.; Anichini, A. Human cutaneous melanomas lacking MITF and melanocyte differentiation antigens express a functional Axl receptor kinase. J. Invest. Dermatol. 2011, 131, 2448-2457. (b) Muller, J.; Krijgsman, O.; Tsoi, J.; Robert, L.; Hugo, W.; Song, C.; Kong, X.; Possik, P. A.; Cornelissen-Steijger, P. D.; Foppen, M. H.; Kemper, K.; Goding, C. R.; McDermott, U.; Blank, C.; Haanen, J.; Graeber, T. G.; Ribas, A.; Lo, R. S.; Peeper, D. S. Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma. Nat. Commun. 2014, 5, 5712. (16) (a) Yu, H.; Liu, R.; Ma, B.; Li, X.; Yen, H. Y.; Zhou, Y.; Krasnoperov, V.; Xia, Z.; Zhang, X.; Bove, A. M.; Buscarini, M.; Parekh, D.; Gill, I. S.; Liao, Q.; Tretiakova, M.; Quinn, D.; Zhao, J.; Gill, P. S. Axl receptor tyrosine kinase is a potential therapeutic target in renal cell carcinoma. Br. J. Cancer. 2015, 113, 616-625. (b) Rankin, E. B.; Fuh, K. C.;


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4-Oxo-1,4-dihydroquinoline-3-carboxamide Derivatives as New Axl

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