(2-(trifluoromethyl)phenyl)acetamide - ACS Publications

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Discovery of N-(4-(6-acetamidopyrimidin-4-yloxy)phenyl)-2-(2(trifluoromethyl)phenyl)acetamide (CHMFL-FLT3-335) as a Potent Fms-like Tyrosine Kinase 3 Internal Tandem Duplications (FLT3ITD) Mutant Selective Inhibitor for Acute Myeloid Leukemia Xiaofei Liang, Beilei Wang, Cheng Chen, Aoli Wang, Chen Hu, Fengming Zou, Kailin Yu, Qingwang Liu, Feng Li, Zhenquan Hu, Tingting Lu, Junjie Wang, Li Wang, Ellen Weisberg, Lili Li, Ruixiang Xia, Wenchao Wang, Tao Ren, Jian Ge, Jing Liu, and Qingsong Liu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01594 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 19, 2018

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Journal of Medicinal Chemistry

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Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Discovery

of

N-(4-(6-acetamidopyrimidin-4-

yloxy)phenyl)-2-(2(trifluoromethyl)phenyl)acetamide (CHMFL-FLT3335) as a Potent Fms-like Tyrosine Kinase 3 Internal Tandem Duplications (FLT3-ITD) Mutant Selective Inhibitor for Acute Myeloid Leukemia Xiaofei Liang1,2,8, Beilei Wang1,3.8, Cheng Chen1,3,8, Aoli Wang1,2,8, Chen Hu1,3, Fengming Zou1,2, Kailin Yu1,2, Qingwang Liu2,4, Feng Li1,3, Zhenquan Hu1,2, Tingting Lu1,3, Junjie Wang1,3, Li Wang1,3, Ellen L. Weisberg5, Lili Li6, Ruixiang Xia6, Wenchao Wang1,2, Tao Ren2,4, Jian Ge6*, Jing Liu1,2,4*, Qingsong Liu1,2,3,4,7* 1.

High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, 230031, P. R. China

2.

Precision Medicine Research Laboratory of Anhui Province, Hefei, Anhui 230088, P. R. China

3.

University of Science and Technology of China, Hefei, Anhui 230036, P. R. China


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4.

Precision Targeted Therapy Discovery Center, Institute of Technology Innovation, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230088, P. R. China

5.

Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School, 450 Brookline Ave., Boston, MA 02115, USA

6.

Department of Hematology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, P. R. China

7.

Institute of Physical Science and Information Technology, Anhui University, Hefei, Anhui 230601, P. R. China

8.

These authors contribute equally

ABSTRACT Most of the current FLT3 kinase inhibitors lack selectivity between FLT3 kinase and cKIT kinase as well as the FLT3-wt and ITD mutants. We report a new compound 27 which displays GI50 values of 30-80 nM against different ITD mutants and achieves selectivity over both FLT3wt (8-fold) and cKIT kinase in the transformed BaF3 cells (>300-fold). 27 potently inhibits the proliferation of the FLT3-ITD positive AML cancer lines through suppression of the phosphorylation of FLT3 kinase and downstream signaling pathways, induction of apoptosis and arresting the cell cycle into G0/G1 phase. 27 also displays potent antiproliferative effect against FLT3-ITD positive patient primary cells while it does not apparently affect FLT3-wt primary cells. In addition, it also exhibits good therapeutic window to PBMC compared to PKC412. In the in vivo studies, 27 demonstrates favorable PK profiles and suppresses the tumor growth in MV4-11 cell inoculated mouse xenograft model.



INTRODUCTION FMS-like tyrosine kinase 3 (FLT3), a trans-membrane receptor tyrosine kinase belongs to the type III receptor tyrosine kinase including PDGFRα, PDGFRβ, CSF1R, cKIT and FLT3 kinase13,

is one of the most attractive targets in acute myeloid leukemia (AML)4. Upon binding to FLT3

ligand (FL), FLT3 kinase will be autophosphorylated and subsequently activates multiple downstream signaling pathways including signal transducer and activator of transcription 5 (STAT5), Ras/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K)/AKT pathways, resulting in survival and proliferation of the leukemic cell5-7. Two main types of FLT3 mutations including internal tandem duplications (ITD) located in both juxtamembrane domain (JM)8 and tyrosine kinase domain 1 (TKD1)9 and point mutations in the tyrosine kinase domain (TKD) can lead to ligand-independent dimerization, autophosphorylation and constitutively activate FLT3 signaling pathways10. The clinical data showed that among different mutants FLT3-ITD accounts for 20~30%9, 11 and FLT3-TKD accounts for approximately 7%10,

12.

FLT3-ITD has been strongly associated with leukocytosis, high blast

counts, increased risk of relapse and adversely impacts overall prognosis13-14. Therefore, FLT3ITD has been considered as an important molecular target for the treatment of acute myeloid leukemia (AML). A number of potent small molecule FLT3 kinase inhibitors such as midostaurin (PKC412, approved for AML by FDA)15, quizartinib (AC220)16, lestaurtinib (CEP-701)17, sunitinib (SU11248)18, tandutinib (MLN-518)19, crenolanib(CP-868596)20-21, gilteritinib (ASP2215)22 and sorafenib (BAY 43-9006)23 have been investigated in the clinic for the treatment of FLT3-ITD+ AML. Recently there are also several inhibitors against FLT3 kinase24-30 that were developed and investigated in preclinical stages and they bear different selectivity profiles. However, most of


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the current inhibitors lack proper selectivity among other protein kinases, especially over the structurally similar cKIT kinase which may induce synthetic lethal myelosuppreion31-33. In addition, in present there is no inhibitor could achieve the selectivity between the FLT3 wt and FLT3-ITD mutants, while FLT3 wt is essential for the proliferation of normal primitive hematopoietic cells34. Thus, development of novel FLT3-ITD inhibitors with higher selectivity is important for both the physiological and pathological mechanistic study points of view. Based on the binding modes, kinase inhibitors can be classified as type I (binding to ATP binding site with “DFG-in” kinase active conformation), type II (binding to ATP binding site with “DFG-out” kinase inactive conformation) and type III (binding to non-ATP binding site in an allosteric binding mode)35-38. The lipophilic interactions with “DFG-out” shifting generated pocket sometimes can provide type II inhibitor a higher selectivity profile39. Sorafenib has been reported as a type II multitargeted tyrosine kinase inhibitor, which was originally developed as an inhibitor of Raf-1 and subsequently found to also bear activity against B-RAF, VEGFR-2, PDGFR, FLT3 and cKIT40-41. AZD2932 (1) has been reported as a type II VEGFR-2 and PDGFR tyrosine kinases inhibitor. It also potently inhibits both FLT3 and cKIT kinases42. Starting from sorafenib and compound 1, via a hybrid binding elements approach, the focused medicinal chemistry effort led us to discover a novel phenoxypyrimidine scaffold-based type II FLT3-ITD mutant selective inhibitor 27, which achieves the selectivity over both FLT3 wt and cKIT kinase (Figure 1).



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Tail

Linker H N

N N

O

O

Hydrogen bonds

Hydrogen bonds

O

N

O

N Head

CF3

1 (AZD2932)

H N

H N O

O

N CF3 Cl

H N

N

N H

CF3

O N

N H

O

O

Tail

Linker

Head

N H

O

O N

H N

O N

Hydrogen bonds Hinge binding

H N

Hydrogen bonds Hinge binding 2

27 (CHMFL-FLT3-335)

O Sorafenib

Figure 1. Schematic illustration of the discovery of 27 (CHMFL-FLT3-335). RESULTS AND DISCUSSION Structure-Activity Relationships (SAR) Exploration. Based on the canonical type II kinase inhibitor’s chemical structural elements, we divided both Sorafenib and AZD2932 into three parts (Figure 1), i.e., the “head” part which occupies the hinge binding area, the “linker” part which occupies the “gatekeeper” residue adjacent area, and the “tail” part which occupies the DFG shifting created hydrophobic area (Figure 2A). We redesigned the “head” part as shown in 2 based on Sorafenib and AZD2932 in order to form one more hydrogen bond with the backbone residue of Cys694 in FLT3, which presumably would increase the binding affinity (Figure 2B). We kept the “linker” moiety in 2 as in Sorafenib and the “tail” moiety was slightly modified by switching the chlorine substituent to methyl substituent (Figure 1). A preliminary docking study suggested that 2 might bind to the inactive conformation of FLT3 (PDB: 4XUF) with a typical type II binding mode. The pyrimidineacetamide moiety of 2 forms bidentate hydrogen bonds with Cys694 in the hinge


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region (Figure 2B). The urea moiety of 2 forms three hydrogen bonds with conserved residues Glu661 and Asp829 (Figure 2C). The methyl-trifluoromethyaniline moiety of 2 occupies the hydrophobic pocket formed by shift of hydrophobic amino acids from the DFG motif in the DFG-out form (Met664, Met665, Leu668, Leu802 and Ile827) (Figure 2C). 2 also displayed a similar binding mode with cKIT kinase to FLT3 kinase (Figure 3D and 3E, PDB:1T46). After careful analysis of DFG-out shifting created hydrophobic area in FLT3 and cKIT, we found that the residues Met664 and Met665 in FLT3 were replaced with Val643 and Leu644 in cKIT, which might provide the selectivity for inhibitor between FLT3 and cKIT (Figure 2F). Since the FLT3-ITD mutations are located outside the ATP binding domain, the selectivity between FLT3wt and FLT3-ITD could not be previewed through the binding model.



Figure 2. Molecular modeling of sorafenib, 1 and 2 with FLT3 (PDB: 4XUF) and 2 with cKIT (PDB:1T46). (A) The basis of the design rationale is illustrated by the superposition of the structure of FLT3 bound to 1 (green) and sorafenib (yellow). (B) The pyrimidineamide moiety of 2 forms two hydrogen bonds in hinge region with FLT3. (C) The urea moiety of 2 forms three hydrogen bonds with conserved residues and the methyl-trifluoromethyaniline moiety of 2 occupies hydrophobic pocket formed by shift of hydrophobic amino acids from the DFG motif in the DFG-out form with FLT3. (D) The pyrimidineamide moiety of 2 forms two hydrogen bonds in hinge region with cKIT. (E) The urea moiety of 2 forms two hydrogen bonds with conserved residues and the methyl-trifluoromethyaniline moiety of 2 occupies hydrophobic pocket formed by shift of hydrophobic amino acids from the DFG motif in the DFG-out form with cKIT. (F) Superposition of the structure of FLT3 (white) bound to 2 (yellow) with the structure of cKIT (cyan) bound to 2 (orange). The structure-activity relationships investigation were conducted with cell-based assays using FLT3, FLT3-ITD (sequence from MV4-11) transfused isogenic BaF3 cells as the preliminary readout and compound 1 as positive control (Table 1). The results showed that 2 displayed increased potencies against FLT3 wt, FLT3-ITD and cKIT kinases in comparison with 1. Since the acetyl group directed to the solvent exposed area, we then tried to make this moiety more feasibly ionized by removing the acetyl group (3) or replacing it with a methyl group (4). The data showed that this did not improve the selectivity over both FLT3 and cKIT, although it exhibited better selectivity over parental BaF3 cells compared to 2. This indicated that both 3 and 4 had a potent on-target antiproliferative effect in the TEL transfused isogenic BaF3 cells whose growth were dependent on the constitutively activated FLT3 and cKIT kinases. Introduction of larger groups such as benzene derivatives (5 and 6) in R1 which might bind to the area near the


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hinge binding area did not improve the selectivity between FLT3 and cKIT either. Increasing the size of the acetyl group in 2 to a larger group such as propionyl group (7) still exhibited potent activity against cKIT kinase. Interestingly, a conformationally more restricted acryloyl group (8) helped to achieve 30-fold selectivity over FLT3 and cKIT. Replacement of the acetyl amide of 2 with more ionizable moiety N, N-dimethylformimidamide (9) did not improve the selectivity between FLT3 and cKIT either. Introduction of more hydrophilic PEG-like moieties at R1 of 2 (10-12) retained the activities against both FLT3 and cKIT but did not achieve the selectivity. Masking the nitrogen atom with methyl group (13-15) which presumably would form a hydrogen bond in the hinge binding area resulted in great activity loss. Introduction of chiral methyl substituted piperazine in R1 of 2 (16-18) lost activity against those targets as well but gained selectivity between FLT3 and cKIT kinases, indicating that there might be some inhibitor induced conformational differences between the FLT3 and cKIT near the hinge binding area although their amino acid sequences are identical. Replacement of the methyl group by larger groups such as (4-methylpiperazin-1-yl)methyl (19) and (4-ethylpiperazin-1-yl)methyl (20) in R2 of 2 led to toxicity against parental BaF3 cells. However, attachment of larger hydrophobic moiety 4-methyl-1H-imidazol to the meta-position of the CF3 group and removal of the orthomethyl group (21) displayed strong activities against FLT3, FLT3-ITD and cKIT kinases although it still showed no selectivity. Table 1. SAR Exploration of R1 and R2 Moietiesa H N

H N O

O N

O N

O

O N

N H

H N

H N

CF3

N 2


R1

R2


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Compd.

R1

R2

BaF3 (GI50: M)

BaF3-TELFLT3 (GI50: nM)

BaF3-FLT3ITD (GI50: nM)

BaF3TELcKIT (GI50: nM)

1

-

-

>10000

3.1±0.3

2±0.2

45±1

3230±520

0.6±0.06

10000

10000

3±0.2

2±0.1

28±2

3010±380

7±0.5

4±0.1

51±2

1000±50

0.9±0.1

0.3±0.1

58±2

990±30

1±0.1

1±0.1

13±0.2

1940±18

8±0.5

2±0.1

240±10

6610±420

3±0.8

4±0.6

12±0.8

0.2±0.6

15±0.4

O

2

CF3

N H

CF3

3

NH2

4

NH

5

6

CF3

CF3

HN

CF3

HN O

O

7

CF3

N H O

8

N H CF3

N

9

N

10

N H

11

N H

12

CF3

N H

NH2

CF3

OH

CF3

O

CF3

>10000

4±0.4

>10000

18±0.9

9±0.8

39±1

7950±450

9±0.8

4±0.4

15±0.5


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13

N

14

N

15

OH

CF3

H N

CF3

CF3

N

N

N

16

CF3

7210±320

169±6

58±3

354±8

>10000

3121±412

8582±142

8341±215

9410±180

2943±213

3227±254

3415±251

9670±120

200±51

86±12

993±32

8120±310

340±22

152±14

2853±451

8920±810

460±40

300±20

1732±200

50±14

10000

97±5

37±2

242±12

>10000

249±22

32±1

>10000

CF3

25

NH

CH2 CF3

26

NH

CH2

CF3

CF3

27

NH

CH2



28

NH

CH2 F3C

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>10000

325±22

109±14

1421±18

>10000

744±21

158±13

6932±56

>10000

663±17

63±4

>10000

>10000

30±3

38±5

264±64

>10000

17±1

4±0.1

584±13

CF3 Cl

29

NH

CH2 CF3 Cl

30

NH

CH2 Cl

31

NH

Cl

CH2

Cl Cl

32

NH

CH2 Cl

33

NH

CH2

>10000

25±2

11±0.2

824±50

34

NH

CH2

>10000

113±11

19±1

313±14

35

NH

CH2

>10000

>10000

746±38

>10000

aAll

GI50 values were obtained by triplet testings.

In order to understand the selectivity of compound 27 between FLT3 and cKIT, we then tried to dock it into both of these two kinases (Figure 3). The model illustrated that it did prefer to adopt a type II binding mode as designed. In FLT3 kinase, the pyrimidineacetamide moiety of 27 forms bidentate hydrogen bonds with Cys694 in the hinge region (Figure 3A). The urea moiety of 27 forms two hydrogen bonds with conserved residues Glu661 and Asp829. The benzene ring of the “linker” moiety in 27 forms edge to face - stacking interactions with both Phe691 and


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Phe830 (Figure 3A). The trifluoromethylaniline moiety of 27 occupies the hydrophobic pocket formed by shift of hydrophobic amino acids from the DFG motif in the DFG-out form (formed by residues Met664, Met665, Leu668, Leu802 and Ile827) (Figure 3B). In this pocket, the trifluoromethylaniline moiety of 27 forms more favorable interactions in FLT3 (Figure 3C) than in cKIT (Figure 3D) because the residues Met664 and Met665 in FLT3 are replaced with Val643 and Leu644 in cKIT which makes the size of the hydrophobic pocket in FLT3 more suitable for 27 than in cKIT. This also explained the reason why compound 28 which bears a relatively larger size of “tail” (2,4- trifluoromethylaniline) gained activity to cKIT.

Figure 3. Molecular modeling of compound 27 with FLT3 (PDB:4XUF) and cKIT (PDB:1T46). Hydrogen bonds are indicated by red dashed lines to key amino acid residues. (A) and (B) Cartoon view of the binding mode of 27 with FLT3 kinase. (C) Solid surface view of the hydrophobic pocket formed by residues Met664, Met665, Leu668, Leu802 and Ile827 in FLT3.



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(D) Solid surface view of the hydrophobic pocket formed by residues Val643, Leu644, Leu668, Leu802 and Ile827 in cKIT. Since compound 27 displayed the best potency against FLT3-ITD mutant and best selectivity over FLT3 wt and cKIT kinase in the primary test, we then further examined its activity against a panel of FLT3 mutants in the transformed BaF3 cells. Since different amino acid sequences of ITD mutants were observed in the AML patients, we first tested whether 27 was potent to different ITD mutants (Figure 4). Interestingly, it was potent to ITD insertion extracted from MOLM13 cells (GI50: 0.082 M). It was also potent to the randomly inserted 22 amino acids sequences (GI50: 0.032 M), 33 amino acids sequences (GI50: 0.082 M) and even a FLAG insertion (GI50: 0.085 M). These data indicated that the activity of 27 was not ITD sequence and location dependent. In addition, 27 was not potent to any of the FLT3 mutations located in the TKD including K663Q, G697R, D815H/N, D835V, Y842R/H as well as ITD/F691L, ITD/D835I/N (Table 3). FLT3

ITD hotspot

Transmembrane . . . TGSSDNEYFYVDFREYEYDLKWEFPREN Kinase domain domain

DFREYE DYKDDDDK HVDFREYEYD GLVQVTGSSDNEYFYVDFREYE LKWEFPRENLEFEVTGSSDNEYFYVDFREYEYD

ITD length (aa)

Source

Compd. 27 (GI50: M)

6 8 10 22 33

MOLM-13 cell line FLAG tag MV4-11 cell line Patient derived Patient derived

0.082 0.085 0.032 0.032 0.082

Figure 4. Antiproliferative effects of compound 27 against FLT3-ITD mutants isogenic cell lines. Table 3. Antiproliferative Effects of Compound 27 against a Panel of FLT3 Mutants Transformed BaF3 Cellsa


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Cell line

Compd. 27 (GI50: μM)

Cell line


BaF3-TEL-FLT3-K663Q

9.4±1.1

BaF3-TEL-FLT3-Y842H

7.5±0.75

BaF3-TEL-FLT3-G697R

0.54±0.03

BaF3-FLT3-ITD-F691L

>10

BaF3-TEL-FLT3-D815H

>10

BaF3-FLT3-ITD-D835I

>10

BaF3-TEL-FLT3-D815N

>10

BaF3-FLT3-ITD-D835N

>10

BaF3-TEL-FLT3-D835V

>10

BaF3-FLT3-ITD-D835del

>10

BaF3-TEL-FLT3-Y842R

>10

aAll

GI50 values were obtained by triplet testings.

We then examined the selectivity profile of compound 27 in the kinome with KINOMEscan technology. The results showed that 27 displayed a good selectivity (S score(35)=0.022) in a panel of 468 kinases and mutants at 1 μM concentration (Figure 5A). Besides FLT3-ITD, 27 also displayed strong binding against KIT, PDGFRβ, HPK1, CSF1R, MEK5, PDGFRα and FLT3 kinases (percent activity less than 15 % at 1 μM) (Figure 5B). Given the fact that KINOMEscan is a binding assay and may not fully reflect the inhibitory activities, these potential targets were then tested with Invitrogen’s SelectScreen technology based biochemical activity assay. The results demonstrated that 27 inhibited KIT, PDGFRβ, HPK1, CSF1R, PDGFRα, FLT3 and FLT3-ITD with IC50 values of 681.1, 207.1, >10000, 212, 83.8, 90.8 and 23.9 nM, respectively. The biochemical assays indicated that 27 had 28-fold selectivity over KIT (23.9 nM versus 681.1 nM) and also had 4-fold selectivity over FLT3 wt (23.9 nM versus 90.8 nM) (Figure 5B). The on-target activity of 27 and its selectivity against other potential off-targets revealed by KINOMEscan binding assay was further examined using the TEL transformed BaF3 system (Figure 5C). The data indicated that 27 could moderately inhibit CSF1R (GI50: 0.83 μM), and potently inhibit PDGFRα (GI50: 0.007 μM) as well as PDGFRβ (GI50: 0.040 μM) kinase ACS Paragon Plus Environment


transformed BaF3 cells. It is not surprising because FLT3 and PDGFR kinases belong to the type III receptor tyrosine kinase family and the ATP binding pocket of these kinases are highly conserved. The above encouraging results indicated that 27 is a highly potent and selective FLT3-ITD kinase inhibitor.

Figure 5. Kinase selectivity profiling of compound 27. (A) KINOMEscan profiling of 27 at 1 μM against 468 kinases and mutants. (B) Kinases exhibited strong binding to 27 (less than 15% activity remaining compared to DMSO control in the assay format at 1 μM of 27) and the


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biochemical IC50 values of 27 measured with Invitrogen SelectScreen technology. (C) Antiproliferative effects of 27 against a panel of kinase transformed BaF3 cells. In Cell Evaluation of Compound 27 Compound 27 was also tested against a panel of established AML cancer cell lines (Table 4). The results showed that it could selectively and potently inhibit the proliferation of FLT3-ITD positive AML cell lines such as MV4-11(GI50: 0.284 μM), MOLM13 (GI50: 0.466 μM) and MOLM14 (GI50: 0.343μM) meanwhile exhibited excellent selectivity over FLT3 wt expressing cell lines such as HL-60, OCI-AML-2 and U937 (GI50: >10 μM). In addition, 27 did not inhibit the proliferation of normal cells CHL and CHO indicating that it has no general toxicity. In the FLT3-ITD positive cells, compound 27 completely suppressed FLT3 autophosphorylation of the Tyr589/591 site in MOLM13 and MOLM14 cells at a concentration of 1 μM, and in MV4-11 cells at a concentration of 0.3 μM (Figure 6A). This difference may due to the homozygous expression of FLT3-ITD in the MV4-11 cells and heterozygous expression of FLT3 wt/ITD in the MOLM13/14 cells. Downstream signaling pathway mediators including pSTAT5, pAKT, pERK and expression of c-Myc were also dose-dependently inhibited. Flow cytometry analysis demonstrated that 27 strongly arrested cell cycle progression in the G0/G1 phase starting from the concentration of 0.1 μM (Figure 6B). Dose-dependent apoptosis induction by 27 was also observed as evidenced by an increase in PARP and Caspase-3 cleavage (Figure 6C). Table 4. Antiproliferative Effects of Compound 27 against a Panel of AML Cancer Cell lines

Cell line MV4-11 (FLT3-ITD)


Cell line


0.284±0.018

OCI-AML-2(FLT3 wt)

>10



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MOLM13 (FLT3-ITD)

0.466±0.026

U937 (FLT3 wt)

>10

MOLM14 (FLT3-ITD)

0.343±0.025

CHL

>10

>10

CHO

>10

HL-60 (FLT3 wt)

Figure 6. Cellular effects of compound 27 in FLT3-ITD positive AML cell lines. (A) Effects of 27 on the FLT3 kinase mediated signaling pathway in MOLM13, MOLM14 and MV4-114 cell lines. Cells were treated with 27 at the indicated concentrations for 4 h, and whole cell lysates


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were then subjected to Western blot analyses. (B) 27 arrested the cell cycle progression. (C) 27 induced apoptosis in MOLM13, MOLM14 and MV4-11 cell lines. Inhibition of the proliferation of primary AML patient cells We then investigated the effects of 27 in patient derived primary cells (Figure 7). The results indicated that 27 effectively suppressed the proliferation of FLT3-ITD positive AML patient cells (GI50: 25.8 nM) but did not affect FLT3 wt primary cells (Figure 7A and 7B). In addition, 27 did not affect the human PBMC even at 5 M and multi-target FLT3 kinase inhibitor PKC412 exhibited none-dose dependent antiproliferative effects (Figure 7C).

Figure 7. (A) Antiproliferative effects of 27 against AML patient primary cells (GI50: nM). (B) Antiproliferative effects of 27 on patient FLT3-ITD positive AML cell and none FLT3-ITD positive AML cell. (C) Antiproliferative effects of 27 on normal PBMC.



Page 22 of 57

Pharmacokinetic properties in rats of compound 27 The pharmacokinetic properties of compound 27 were evaluated in Sprague-Dawley rats following intravenous and oral administration (Table 5). With the oral gavage administration, 27 has a half-life of 2.8 h and a volume distribution 1.46 L/kg. In addition, 27 exhibited good systemic exposure (AUC) of 26693 ng/mL·h and Cmax of 3625 ng/mL, which indicated that it would be suitable for the oral administration. Table 5. Pharmacokinetic Properties of Compound 27 in Sprague-Dawley Ratsa

AUC(0-t) ng/mL·h

AUC(0-∞) ng/mL·h

MRT(0-t) h

T1/2 h

Tmax h

Vz L/kg

CLz L/h/kg

Cmax ng/mL

Mean

1559.0

1591.2

1.9

1.3

0.02

1.2

0.7

1032.7

SD

329.1

362.2

0.3

0.3

0.00

0.2

0.1

103.0

Mean

26692.7

28026.8

4.5

2.8

2.00

1.5

0.4

3625.4

F%

IV 1 mg/kg

PO 10 mg/kg

171% SD

aAll

5638.0

3623.0

1.1

0.7

0.00

0.4

0.05

490.9

testing data were obtained from three independent mice (±SD).

In Vivo Effects of compound 27 against MV4-11 Tumor Xenografts The in vivo antitumor effects of compound 27 was evaluated in MV4-11 cell inoculated mouse xenografts. When the tumor grew to a mean volume of around 200 mm3, the mice were treated orally with vehicle, 25, 50, and 100 mg/kg/day of 27 and 50 mg/kg/day of PKC412 for 28 days (Figure 8). The results showed that all dosages did not significantly affect the mice body weight (Figure 6A). Compound 27 showed a dose-dependent in vivo antitumor efficacy with TGIs of 48%, 68%, 80% at 25, 50 and 100 mg/kg/day, respectively (Figure 8B, 8C). Immunohistochemical (IHC) analyses demonstrated that 27 dose-dependently suppressed cell


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proliferation (Ki-67 stain) and induced apoptosis (TUNEL stain) in the tumor tissues compared with vehicles (Figure 8D).

Figure 8. In vivo effects of compound 27 against MV4-11 tumor xenografts via oral administration. (A) Body weight change in mice for each daily dosing group of 27 and PKC412. (B) Tumor size measurements of xenograft mice after 27 and PKC412 treatment. Initial body weight and tumor size were set as 100%. (C) Comparison of the final tumor weight in each group. Numbers in columns indicate the mean tumor weight in each group. (D) Representative micrographs of hematoxylin and eosin (HE), Ki-67 and TUNEL staining of tumor tissues with 27 treatment groups in comparison with the vehicle treatment group. Note the specific nuclear staining of cells with morphology consistent with proliferative and apoptosis (D, blue arrows).



CONCLUSION In summary, starting from the nonspecific but potent FLT3 receptor tyrosine kinase inhibitors compound 1 and sorafenib, we have discovered a novel type II FLT3-ITD kinase inhibitor compound 27, which exhibited potent inhibitory activities against a panel of different lengths of inserted ITD mutants. Furthermore, 27 was selective for FLT3-ITD mutants compared to FLT wt which has not been observed in current FLT3 kinase inhibitors. This might be of clinical value because FLT3 wt kinase plays essential roles in the proliferation of normal primitive hematopoietic cells34. ITD insertions are located in the JM domain of the FLT3 kinase and away from the kinase domain. Therefore, how the selectivity is achieved is not feasibly explained by the typical inhibitor-kinase binding models. However, constitutively active, ligand-independently autophosphorylated FLT3-ITD homodimers probably differ in their conformation of the ATP site (DFG-in) from that of the wild type (DFG-out), favoring binding of 27 at FLT3-ITD compared to FLT3 wt. 27 achieved good selectivity over cKIT kinase (over 300-fold) which might potentially avoid the myelosuppression toxicity observed from the dual FLT3/cKIT kinase inhibitor. Besides FLT3-ITD kinase, 27 also inhibited structurally similar PDGFRα and PDGFRβ kinases, which might also contribute positive antileukemic effect of 27. In addition, 27 exhibited good selectivity over other protein kinases which are reflected in the KINOMEscan profiling. Combined the good in vivo PK/PD efficacy, we believed that compound 27 would be a valuable research tool for the FLT3-ITD mediated physiology and pathology studies as well as a potential drug candidate for the FLT3-ITD positive AML. EXPERIMENTAL SECTION Chemistry.


Page 24 of 57

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As depicted in Scheme 1, the syntheses of compounds 2-10 started from the reaction of 4,6dichloropyrimidine and 4-aminophenol in the presence of CuBr2, which provided the intermediate 36. Substitution of the chloride in 36 with different amines afforded the intermediates 37a-d, and then 37a-c were converted to ureas 4-6 in the presence of 4-methyl-3(trifluoromethyl)aniline and CDI. Urea 10 was prepared from 37d in a similar way after deprotection of the N-Boc group using HCl in methanol. The amino protecting group in 6 was removed with trifluoroacetic acid to provide 3, which was further acylated with corresponding acyl chloride to offer products 2 and 7-8. Compound 9 was obtained from 3 in the presence of N, N-dimethylformamide under reflux condition. To

obtain

compounds

11-18,

the

intermediate

36

reacted

with

4-methyl-3-

(trifluoromethyl)aniline in the presence of triphosgene and DIPEA to afford the urea 38, followed by substitution reaction of 38 with different amines under acidic condition (Scheme 2). The N-Boc group was cleanly deprotected by HCl in methanol. The syntheses of compounds 19-21 and 25-35 were accomplished in a three-step process as outlined in Scheme 3. Commercially available 6-chloropyrimidin-4-amine reacted with acetic anhydride at 120 °C under argon to afford the intermediate 39, which was then alkylated with 4aminophenol in the presence of CuBr2 to provide the intermediate 40. The ureas 19-21 were obtained from the reaction of 40 with corresponding amines in the presence of triphosgene and DIPEA. Amidation reactions of 40 with varieties of carboxylic acids were carried out in the presence of HATU and DIPEA to provide 25-35. Compounds 22-24 were prepared from intermediate 39 in three steps (Scheme 4). Firstly, 39 was alkylated with methyl 2-(4-hydroxyphenyl)acetate in the presence of Cs2CO3 to provide



Page 26 of 57

ester 41, which was then cleanly hydrolyzed under basic condition to give carboxylic acid 42. Finally the amidation reaction of 42 and amines afforded the desired products 22-24. Scheme 1. Synthetic Route to Compounds 2-10a.

Cl N

a

O

b

O

Cl

c

N

N

Cl 36

N H 37

6

d

H N

N

H N e

N

4: R2=NHCH3 5: R2=NHCH2Ph 6: R2=NHCH2PhOCH3 10: R2=NHCH2CH2NH2

H N

CF3 2: R3=NHCOCH3 7: R3=NHCOCH2CH3 8: R3=NHCOCHCH2 9: R3=NCHN(CH3)2

O

O N

N

NH2

N

R3

3

aReagents

R2 4-6, 10

CF3

O

O

CF3

N

R1

37a: R1=CH3 37b: R1=CH2Ph 37c: R1=CH2PhOCH3 37d: R1=CH2CH2NHBoc

H N

H N O

O

N

N

N

H N

NH2

NH2

2, 7-9

and conditions: (a) 4-aminophenol, CuBr2, K2CO3, CH3CN, rt, 14 h; (b) R1-NH2, THF

or dioxane, 120 °C, 24 h; (c) for 4-6, 4-methyl-3-(trifluoromethyl)aniline, CDI, DCM, rt, 14 h; for 10, (i) 4-methyl-3-(trifluoromethyl)aniline, CDI, DCM, rt, 14 h; (ii) 4 M HCl in MeOH, MeOH, rt, 1 h; (d) TFA, 80 °C, 14 h; (e) for 2, 7-8, acyl chloride, DIPEA, THF, 0 °C, 30 min; for 9, DMF, DIPEA, reflux, 14 h. Scheme 2. Synthetic Route to Compounds 11-18a. H N

NH2 a

O N

H N O

O

Cl 36

b

H N O

O N

N N

H N

CF3

N

N

Cl 38

R 11-18


CF3

11: R=NHCH2CH2OH 12: R=NHCH2CH2OCH3 13: R=CH3NCH2CH2OH 14: R=CH3NCH2CH2NHCH3 15: R=CH3NCH2CH2N(CH3)2 16: R=(R)-2-methylpiperazine 17: R=(S)-2-methylpiperazine 18: R=(R)-3-methylpiperazine

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aReagents

and conditions: (a) 4-methyl-3-(trifluoromethyl)aniline, triphosgene, DIPEA, THF, 0

°C to rt, 4 h; (b) for 11-15, amines, PTSA, t-butanol, 60 °C, 14 h; for 16-18, (i) amines, PTSA, tButanol, 60 °C, 14 h; (ii) 4 M HCl in MeOH, MeOH, rt, 1 h. Scheme 3. Synthetic Route to Compounds 19-21 and 25-35 a. H N Cl

Cl N

N

a

N

b

N

N

O

N

O HN 40

R

O N

39

X O

O

c

N

HN

NH2

NH2

O

N H 19-21, 25-35

19: X=NH, R=4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)aniline 20: X=NH, R=4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)aniline 21: X=NH, R=3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)aniline 25: X=CH2, R=4-CF3Ph 26: X=CH2, R=3-CF3Ph 27: X=CH2, R=2-CF3Ph 28: X=CH2, R=2-CF3-4-CF3Ph 29: X=CH2, R=2-Cl-4-CF3Ph 30: X=CH2, R=2,6-dichloroPh 31: X=CH2, R=3,4-dichloroPh 32: X=CH2, R=2,4-dichloroPh 33: X=CH2, R=2-(naphthalen-1-yl) 34: X=CH2, R=2-(naphthalen-2-yl) 35: X=CH2, R=4-biphenyl

aReagents

and conditions: (a) Ac2O, 120 °C, 5 h; (b) 4-aminophenol, CuBr2, K2CO3, CH3CN, rt,

14 h; (c) for 19-21, amine, triphosgene, DIPEA, THF, 0 °C to rt, 3 h; for 25-35, carboxylic acid, HATU, DIPEA, THF, rt, 14 h. Scheme 4. Synthetic Route to Compounds 22-24a.

a

N

O HN 39

O

b

N N

O

O

c

N N

O HN

HN 41

NHR

OH

O

N

O

O

O Cl

42

O N N

O HN 22-24 22: R=4-CH3-3-CF3Ph 23: R=Ph 24: R=4-CF3Ph



aReagents

and conditions: (a) methyl 2-(4-hydroxyphenyl)acetate, Cs2CO3, DMF, rt, 14 h; (b)

NaOH, MeOH/H2O, 80 °C, 2 h (c) R-NH2, HATU, DIPEA, THF, 14 h. All reagents and solvents were purchased from commercial sources and were used as received unless specified otherwise, or prepared as described in the literature. All moisture sensitive reactions were carried out using dry solvents under ultrapure argon protection. Glassware was dried in an oven at 140 °C for at least 12 h prior to use and then assembled quickly while hot, sealed with rubber septa, and allowed to cool under a stream of argon. Reactions were stirred magnetically using Teflon-coated magnetic stirring bars. Commercially available disposable syringes were used for transferring the reagents and solvents. LC/MS were performed on an Agilent 6224 TOF using an ESI source coupled to an Agilent 1260 Infinity HPLC system operating in reverse mode with an Agilent XDB-C18 column (4.6 × 50 mm, 1.8 μm) using a water/acetonitrile (each with 0.2% (v/v) formic acid) gradient at a flow rate at 0.5 mL/min. 1H and

13C

spectra were recorded with a Bruker 400 MHz or 850 MHz NMR spectrometer.

Chemical shifts are expressed in ppm. In the NMR tabulation, s indicates singlet; d, doublet; t, triplet; q, quartet and m, multiplet. Flash column chromatography was conducted using silica gel (Silicycle40−64 μm). The purities of all final compounds were determined to be above 95% by HPLC. N-(6-(4-(3-(4-Methyl-3-(trifluoromethyl)phenyl)ureido)phenoxy)pyrimidin-4yl)acetamide (2). To a solution of 3 (50 mg, 0.12 mmol) in anhydrous THF (3 mL) was added acetyl chloride (11 mg, 0.136 mmol) and DIPEA (0.05 mL, 0.36 mmol) at 0 C under argon. Then the reaction mixture was stirred at 0 C for 30 min. The resulting mixture was concentrated to dryness, and the residue was diluted with water and extracted with EtOAc. The combined organic layers were washed with water, brine and dried over anhydrous Na2SO4. Evaporation of


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the solvent provided the crude product, which was purified by flash chromatography (eluting with 0-5% MeOH in DCM) to afford 2 as a white solid (42 mg, 76%). 1H NMR (850 MHz, DMSO-d6) δ 10.93 (s, 1H), 8.96 (s, 1H), 8.82 (s, 1H), 8.49 (s, 1H), 7.94 (s, 1H), 7.57 – 7.49 (m, 4H), 7.34 – 7.29 (m, 1H), 7.13 (d, J = 7.6 Hz, 2H), 2.37 (s, 3H), 2.12 (s, 3H).

13C

NMR (214

MHz, DMSO-d6) δ 171.16, 171.08, 160.05, 158.32, 153.00, 147.36, 138.46, 137.47, 133.06, 129.09, 128.06, 127.92, 125.51, 124.27, 122.36, 122.16, 120.18, 94.30, 24.34, 18.49. LC-MS (ESI, m/z): 446.1419 [M+H]+. 1-(4-((6-Aminopyrimidin-4-yl)oxy)phenyl)-3-(4-methyl-3-(trifluoromethyl)phenyl)urea (3). To a solution of TFA (5 mL) was added 6 (1.4 g, 2.67 mmol) at room temperature under argon. The reaction mixture was heated to 80 C and stirred for 14 h. The resulting mixture was concentrated to dryness. The residue was diluted with saturated Na2CO3, and extracted with EtOAc. The combined organic layers were washed with water, brine and dried over anhydrous Na2SO4. Evaporation of the solvent provided the crude product, which was purified by flash chromatography (eluting with 0-10 % MeOH in DCM) to afford 3 as a yellow solid (800 mg, 74%). 1H NMR (400 MHz, CDCl3) δ 8.97 (s, 1H), 8.85 (s, 1H), 8.10 (s, 1H), 7.96 (s, 1H), 7.53 (s, 3H), 7.32 (s, 1H), 7.09 (s, 2H), 6.85 (s, 2H), 5.72 (s, 1H), 2.37 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 170.22, 166.46, 158.69, 153.12, 147.81, 138.48, 137.13, 133.06, 129.08, 122.38, 122.13, 120.30, 115.57, 115.07, 86.14, 18.13. LC-MS (ESI, m/z): 404.1349 [M+H]+. 1-(4-Methyl-3-(trifluoromethyl)phenyl)-3-(4-((6-(methylamino)pyrimidin-4yl)oxy)phenyl)urea (4). To a solution of 37a (100 mg, 0.46 mmol) in anhydrous DCM (10 mL) was added 4-methyl-3-(trifluoromethyl)aniline (162 mg, 0.93 mmol) and CDI (150 mg,0.93 mmol) at room temperature under argon. Then the reaction mixture was stirred at room temperature for 14 h. The resulting mixture was concentrated to dryness. The residue was diluted



with water, and extracted with EtOAc. The combined organic layers were washed with water, brine and dried over anhydrous Na2SO4. Evaporation of the solvent provided the crude product, which was purified by flash chromatography (eluting with 0-5% MeOH in DCM) to afford 4 as a yellow solid (129 mg, 67%). 1H NMR (850 MHz, DMSO-d6) δ 8.93 (s, 1H), 8.80 (s, 1H), 8.11 (s, 1H), 7.94 (s, 1H), 7.51 (s, 3H), 7.32 (s, 1H), 7.27 (s, 1H), 7.07 (s, 2H),, 5.74 (s, 1H), 2.78 (s, 3H), 2.36 (s, 3H).

13C

NMR (214 MHz, DMSO-d6) δ 169.41, 165.60, 158.31, 153.11, 147.91,

138.50, 137.00, 133.05, 128.90, 128.07, 127.93, 125.62, 124.34, 122.28, 122.12, 120.22, 115.62, 86.92, 27.59, 18.47. LC-MS (ESI, m/z): 418.1473 [M+H]+. Compounds 5 and 6 were prepared following the synthetic procedure of 4. 1-(4-((6-(Benzylamino)pyrimidin-4-yl)oxy)phenyl)-3-(4-methyl-3(trifluoromethyl)phenyl)urea (5). Yield = 71%. 1H NMR (850 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.86 (s, 1H), 8.17 (s, 1H), 7.96 (s, 1H), 7.86 (s, 1H), 7.52 (s, 3H), 7.32 (s, 5H), 7.24 (s, 1H), 7.08 (s, 2H), 5.83 (s, 1H), 4.54 (s, 2H), 2.37 (s, 3H).

13C

NMR (214 MHz, DMSO-d6) δ 169.85,

165.07, 158.49, 153.13, 147.81, 138.61, 136.99, 132.87, 129.06, 128.79, 128.08, 127.96, 127.81, 127.27, 125.63, 124.35, 122.32, 122.12, 120.21, 115.62, 87.24, 43.81. LC-MS (ESI, m/z): 494.1851[M+H]+. 1-(4-((6-((4-Methoxybenzyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-(4-methyl-3(trifluoromethyl)phenyl)urea (6). Yield = 63%. 1H NMR (400 MHz, CDCl3) δ 9.00 (s, 1H), 8.87 (s, 1H), 8.16 (s, 1H), 7.96 (s, 1H), 7.81 (s, 1H), 7.51 (s, 3H), 7.33 (s, 1H), 7.22 (s, 2H), 7.07 (s, 2H), 6.89 (, 2H), 5.79 (s, 1H), 4.44 (s, 2H), 3.73 (s, 3H), 2.37 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 169.77, 164.79, 158.72, 153.00, 147.70, 143.61, 138.51, 137.01, 132.96, 129.03, 128.12, 127.83, 126.35, 123.63, 122.43, 122.33, 122.12, 120.18, 115.4, 114.21, 55.48, 43.66, 18.52. LC-MS (ESI, m/z): 524.1882 [M+H]+.


Page 30 of 57

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Compounds 7 and 8 were prepared following the synthetic procedure of 2. N-(6-(4-(3-(4-Methyl-3-(trifluoromethyl)phenyl)ureido)phenoxy)pyrimidin-4yl)propionamide (7). Yield = 59%. 1H NMR (400 MHz, DMSO-d6) δ 10.89 (s, 1H), 9.00 (s, 1H), 8.89 (s, 1H), 8.50 (s, 1H), 7.95 (s, 1H), 7.54 (d, J = 6.3 Hz, 4H), 7.34 (d, J = 8.0 Hz, 1H), 7.13 (d, J = 8.6 Hz, 2H), 2.46 – 2.41 (m, 2H), 2.38 (s, 3H), 1.04 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 174.70, 171.16, 160.13, 158.27, 152.94, 147.15, 138.63, 137.52, 133.10, 129.10, 128.12, 127.83, 126.35, 123.63, 122.37, 122.20, 120.20, 115.41, 94.05, 29.67, 18.12, 9.11. LC-MS (ESI, m/z): 460.1589 [M+H]+. N-(6-(4-(3-(4-Methyl-3-(trifluoromethyl)phenyl)ureido)phenoxy)pyrimidin-4yl)acrylamide (8). Yield = 31%. 1H NMR (400 MHz, DMSO-d6) δ 11.17 (s, 1H), 9.06 (s, 1H), 8.95 (s, 1H), 8.54 (s, 1H), 7.95 (s, 1H), 7.64 (s, 1H), 7.54 (d, J = 7.8 Hz, 3H), 7.35 (d, J = 8.0 Hz, 1H), 7.15 (d, J = 8.2 Hz, 2H), 6.60 (dd, J = 17.2, 9.9 Hz, 1H), 6.35 (d, J = 16.9 Hz, 1H), 5.87 (d, J = 10.0 Hz, 1H), 2.39 (s, 3H).

13C

NMR (214 MHz, DMSO-d6) δ 171.20, 165.30, 160.14,

158.46, 153.14, 147.26, 138.52, 137.60, 133.11, 131.20, 129.85, 129.05, 128.05, 122.39, 122.09, 120.10, 115.56, 94.75, 18.20. LC-MS (ESI, m/z): 458.1467 [M+H]+. (N,N-Dimethyl-N'-(6-(4-(3-(4-methyl-3(trifluoromethyl)phenyl)ureido)phenoxy)pyrimidin-4-yl)formimidamide (9). To a solution of 3 (50 mg, 0.12 mmol) in anhydrous DMF (1 mL) was added DIPEA (0.1 mL, 0.72 mmol) at room temperature under argon. Then the reaction mixture was heated to reflux for 14 h. The resulting mixture was concentrated to dryness. The residue was diluted with water and extracted with EtOAc. The combined organic layers were washed with water, brine and dried over anhydrous Na2SO4. Evaporation of the solvent provided the crude product, which was purified by flash chromatography (eluting with 0-5% MeOH in DCM) to afford 9 as a white solid (46 mg,



81%). 1H NMR (400 MHz, CDCl3) δ 8.52 (s, 1H), 8.44 (s, 3H), 7.55 (s, 1H), 7.35 (s, 3H), 7.07 (s, 1H), 6.99 (s, 2H), 6.21 (s, 1H), 3.11 (s, 3H), 3.04 (s, 3H), 2.33 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 171.28, 169.70, 157.68, 156.92, 153.83, 147.88, 136.60, 136.34, 132.41, 130.81, 129.10, 128.59, 122.82, 121.98, 121.32, 117.20, 97.05, 35.06, 18.53. LC-MS (ESI, m/z): 459.1733 [M+H]+. 1-(4-((6-((2-Aminoethyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-(4-methyl-3(trifluoromethyl)phenyl)urea (10). To a solution of 37d (200 mg, 0.58 mmol) in anhydrous DCM (10 mL) was added 4-methyl-3-(trifluoromethyl)aniline (203 mg, 1.16 mmol) and CDI (187 mg,1.16 mmol) at room temperature under argon. Then the reaction mixture was stirred at room temperature for 14 h. The resulting mixture was concentrated to dryness. The residue was diluted with water and extracted with EtOAc. The combined organic layers were washed with water, brine and dried over anhydrous Na2SO4. Evaporation of the solvent provided the crude product, which was directly dissolved in MeOH (1 mL) and 4 M HCl in MeOH (1 mL) was added at room temperature. The reaction mixture was stirred at room temperature for 1 h and then concentrated. The residue was purified by flash chromatography (eluting with 0-10 % MeOH in DCM) to afford 10 as a yellow solid (140 mg, 54%). 1H NMR (850 MHz, DMSO-d6) δ 9.77 (s, 1H), 9.64 (s, 1H), 8.43 (s, 1H), 8.35 (s, 1H), 8.12 (s, 3H), 7.93 (s, 1H), 7.54 (d, J = 6.2 Hz, 1H), 7.51 (d, J = 6.9 Hz, 1H), 7.38 – 7.30 (m, 1H), 7.12 (d, J = 6.7 Hz, 1H), 4.32 (s, 2H), 3.57 (s, 2H), 2.96 (s, 2H), 2.37 (s, 3H).

13C

NMR (214 MHz, DMSO-d6) δ 168.14, 164.28,

156.56, 153.28, 146.95, 138.66, 137.96, 133.14, 128.82, 128.05, 127.83, 125.63, 124.35, 122.23, 121.75, 119.90, 115.18, 87.47, 50.33, 36.82, 18.34. LC-MS (ESI, m/z): 447.1784 [M+H]+. 1-(4-((6-((2-Hydroxyethyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-(4-methyl-3(trifluoromethyl)phenyl)urea (11). To a solution of 38 (50 mg, 0.12 mmol) in t-butanol (1 mL)


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was added 2-aminoethan-1-ol (7 mg, 0.13 mmol) and PTSA (2 mg, 0.012 mmol) at room temperature under argon. The reaction mixture was heated to 60 C and stirred for 14 h. The resulting mixture was concentrated to provide the crude product, which was purified by flash chromatography (eluting with 0-10% MeOH in DCM) to afford 11 as an off-white solid (43 mg, 81%). 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.86 (s, 1H), 8.13 (s, 1H), 7.95 (s, 1H), 7.51 (s, 3H), 7.34 (s, 2H), 7.08 (s, 2H), 5.81 (s, 1H), 4.75 (s, 1H), 3.50 (s, 2H), 3.41 (s, 2H), 2.37 (s, 3H).

13C

NMR (101 MHz, DMSO-d6) δ 169.75, 165.03, 158.42, 152.93, 147.88, 138.43,

137.02, 133.07, 129.07, 128.13, 127.84, 126.34, 123.62, 122.29, 122.06, 120.02, 115.57, 87.22, 59.89, 43.60, 18.49. LC-MS (ESI, m/z): 448.1585 [M+H]+. Compounds 12-15 were prepared following the synthetic procedure of 11. 1-(4-((6-((2-Methoxyethyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-(4-methyl-3(trifluoromethyl)phenyl)urea (12). Yield = 79%. 1H NMR (400 MHz, DMSO-d6) δ 8.94 (s, 1H), 8.81 (s, 1H), 8.15 (s, 1H), 7.95 (s, 1H), 7.52 (s, 3H), 7.37 (s, 1H), 7.33 (s, 1H), 7.08 (s, 2H), 5.82 (s, 1H), 3.43 (s, 4H), 3.26 (s, 3H), 2.37 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 169.63, 165.09, 158.41, 153.09, 147.87, 138.50, 136.93, 133.07, 129.00, 127.79, 122.30, 122.14, 120.22, 115.56, 95.45, 70.85, 57.96, 40.60, 18.48. LC-MS (ESI, m/z): 462.1781 [M+H]+. 1-(4-((6-((2-Hydroxyethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-(4-methyl-3(trifluoromethyl)phenyl)urea (13). Yield = 72%. 1H NMR (400 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.82 (s, 1H), 8.17 (s, 1H), 7.96 (s, 1H), 7.51 (s, 3H), 7.32 (s, 1H), 7.06 (s, 2H), 6.03 (s, 1H), 4.68 (s, 1H), 3.58 (s, 4H), 3.04 (s, 3H), 2.37 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 170.00, 164.08, 157.66, 153.12, 148.00, 138.52, 136.82, 133.05, 128.96, 128.13, 127.84, 126.35 , 123.52, 122.21, 122.11, 120.06, 115.64, 85.87, 58.85, 51.59, 36.65, 18.12. LC-MS (ESI, m/z): 462.1736 [M+H]+.



1-(4-((6-(Methyl(2-(methylamino)ethyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-(4-methyl-3(trifluoromethyl)phenyl)urea (14). Yield = 61%. 1H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H), 9.37 (s, 1H), 8.22 (s, 1H), 7.98 (s, 1H), 7.59 (s, 3H), 7.31 (s, 1H), 7.05 (s, 2H), 6.10 (s, 1H), 3.85 (s, 1H), 3.18 (s, 2H), 3.00 (s, 2H), 2.59 (s, 3H), 2.36 (s, 3H), 2.30 (s, 3H). 13C NMR (214 MHz, DMSO-d6) δ 170.13, 164.22, 157.70, 153.23, 147.83, 138.65, 137.04, 133.05, 128.87, 128.06, 127.92, 125.64, 124.36, 122.22, 121.88, 119.84, 115.36, 85.96, 48.25, 36.25, 35.36, 18.49. LC-MS(ESI, m/z): 475.2077[M+H]+. 1-(4-((6-((2-(Dimethylamino)ethyl)(methyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-(4methyl-3-(trifluoromethyl)phenyl)urea (15). Yield = 65%. 1H NMR (400 MHz, DMSO-d6) δ 9.42 (s, 1H), 9.28 (s, 1H), 8.21 (s, 1H), 7.97 (s, 1H), 7.54 (s, 3H), 7.32 (s, 1H), 7.06 (s, 2H), 6.06 (s, 1H), 3.84 (s, 2H), 2.98 (s, 5H), 2.63 (s, 6H), 2.36 (s, 3H). 13C NMR (214 MHz, DMSO-d6) δ 170.22, 164.11, 157.73, 157.14, 153.20, 147.81, 138.62, 137.04, 133.05, 128.89, 128.06, 125.63, 124.35, 122.25, 121.89, 119.86, 115.38, 85.83, 55.76, 46.26, 45.03, 35.94, 18.49. LC-MS (ESI, m/z): 489.2253 [M+H]+ (R)-1-(4-Methyl-3-(trifluoromethyl)phenyl)-3-(4-((6-(2-methylpiperazin-1-yl)pyrimidin4-yl)oxy)phenyl)urea (16). To a solution of 38 (50 mg, 0.12 mmol) in t-butanol (1 mL) was added tert-butyl (R)-3-methylpiperazine-1-carboxylate (50 mg, 0.24 mmol) and PTSA (2 mg, 0.012 mmol) at room temperature under argon. The reaction mixture was heated to 120 C and stirred for 5 h. The resulting mixture was concentrated to provide the crude product, which was directly dissolved in MeOH (1 mL) and 4 M HCl in MeOH (1 mL) was added at room temperature. The reaction mixture was stirred at room temperature for 1 h and then concentrated. The residue was purified by flash chromatography (eluting with 0-10 % MeOH in DCM) to afford 16 as a yellow solid (24 mg, 41%). 1H NMR (850 MHz, DMSO-d6) δ 8.27 (s, 1H), 7.92 (s,


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1H), 7.49 (s, 3H), 7.30 (s, 2H), 7.25 (s, 1H), 7.04 (s, 2H), 6.25 (s, 1H), 4.81 (s, 1H), 4.33 (s, 1H), 3.41 (s, 1H), 3.26 (s, 3H), 3.09 (s, 1H), 2.91 (s, 1H), 2.35 (s, 3H), 1.21 (s, 3H). 13C NMR (214 MHz, DMSO-d6) δ 170.59, 163.39,157.87, 153.12, 147.15, 138.55, 137.12, 133.06, 128.81, 127.85, 125.68, 124.31, 122.33, 121.72, 119.66, 115.35, 87.36, 50.50, 46.98, 45.51, 42.50, 18.50, 15.89. LC-MS (ESI, m/z): 487.2039 [M+H]+. Compounds 17-18 were prepared following the synthetic procedure of 16. (S)-1-(4-Methyl-3-(trifluoromethyl)phenyl)-3-(4-((6-(2-methylpiperazin-1-yl)pyrimidin-4yl)oxy)phenyl)urea (17). Yield = 40%. 1H NMR (850 MHz, DMSO-d6) δ 8.25 (s, 1H), 7.91 (s, 1H), 7.48 (s, 3H), 7.31 (s, 2H), 7.23 (s, 1H), 7.04 (s, 2H), 6.25 (s, 1H), 4.75 (s, 1H), 4.31 (s, 1H), 3.42 (s, 1H), 3.23 (s, 3H), 3.11 (s, 1H), 2.93 (s, 1H), 2.35 (s, 3H), 1.21 (s, 3H). 13C NMR (214 MHz, DMSO-d6) δ 170.55, 163.44, 157.94, 153.02, 147.49, 138.53, 137.16, 133.18, 128.70, 127.89, 125.62, 124.28, 122.29, 121.74, 119.68, 115.16, 86.94, 48.33, 46.36, 44.46, 42.62, 18.60, 15.82. LC-MS (ESI, m/z): 487.2092 [M+H]+. (R)-1-(4-Methyl-3-(trifluoromethyl)phenyl)-3-(4-((6-(3-methylpiperazin-1-yl)pyrimidin4-yl)oxy)phenyl)urea (18). Yield = 65%. 1H NMR (850 MHz, DMSO-d6) δ 8.35 (s, 1H), 7.92 (s, 3H), 7.52 (s, 3H), 7.31 (s, 1H), 7.10 (s, 2H), 6.38 (s, 1H), 4.41 (s, 2H), 3.41 (m, 1H), 3.33 – 3.27 (m, 2H), 3.21 – 3.17 (m, 1H), 3.01 (d, J = 8.7 Hz, 1H), 2.35 (s, 3H), 1.30 (s, 3H). 13C NMR (214 MHz, DMSO-d6) δ 168.98, 163.40, 156.19, 153.28, 147.07, 138.66, 137.68, 133.10, 128.80, 128.04, 127.91, 125.61, 124.33, 122.06, 121.66, 119.68, 115.11, 87.12, 50.38, 47.24, 42.22, 41.01, 18.50, 15.74. LC-MS (ESI, m/z): 487.2081 [M+H]+. N-(6-(4-(3-(4-((4-Methylpiperazin-1-yl)methyl)-3(trifluoromethyl)phenyl)ureido)phenoxy)pyrimidin-4-yl)acetamide (19). To a solution of 40 (30 mg, 0.12 mmol) in anhydrous THF (3 mL) was added 4-((4-methylpiperazin-1-yl)methyl)-3-



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(trifluoromethyl)aniline (100 mg, 0.37 mmol) and DIPEA (0.11 mL, 0.61 mmol) at 0 C under argon. The reaction mixture was stirred at 0 C for 10 min, and then triphosgene (25 mg, 0.09 mmol) was added. The reaction mixture was stirred at 0 C for 1 h, then it was allowed to come to room temperature and stirred for 3 h. The resulting mixture was concentrated to dryness. The residue was diluted with water and extracted with EtOAc. The combined organic layers were washed with water, brine and dried over anhydrous Na2SO4. Evaporation of the solvent provided the crude product, which was purified by flash chromatography (eluting with 0-5% MeOH in DCM) to afford 19 as a yellow solid (38 mg, 57%). 1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 9.08 (s, 1H), 8.91 (s, 1H), 8.49 (s, 1H), 7.97 (s, 1H), 7.61 (s, 1H), 7.59 (s, 1H), 7.52 (s, 3H), 7.13 (d, J = 8.3 Hz, 2H), 3.53 (s, 2H), 2.37 (s, 8H), 2.16 (s, 3H), 2.11 (s, 3H).

13C

NMR (101

MHz, CDCl3) δ 171.13, 171.08, 160.04, 158.36, 153.06, 147.34, 139.31, 137.43, 131.81, 130.46, 127.83, 126.17, 123.26, 122.38, 122.07, 120.16 , 115.51, 94.27, 57.85, 55.17, 53.08, 46.12, 24.54. LC-MS (ESI, m/z): 544.2251 [M+H]+. Compounds 20-21 were prepared following the synthetic procedure of 19. N-(6-(4-(3-(4-((4-Ethylpiperazin-1-yl)methyl)-3(trifluoromethyl)phenyl)ureido)phenoxy)pyrimidin-4-yl)acetamide (20). Yield = 39%. 1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 9.14 (s, 1H), 8.99 (s, 1H), 8.49 (s, 1H), 7.97 (s, 1H), 7.61 (s, 2H), 7.52 (s, 3H), 7.17 (s, 2H), 3.54 (s, 2H), 2.42 (s, 5H), 2.11 (s, 3H), 1.02 (s, 3H). 13C

NMR (214 MHz, DMSO-d6) δ 171.18, 171.10, 160.06, 158.36, 153.13, 147.32, 139.47,

137.50, 131.88, 128.07, 125.42, 122.38, 121.95, 121.07, 120.04, 115.66, 94.28, 57.71, 52.45, 51.82, 24.55, 11.74. LC-MS (ESI, m/z): 558.2427 [M+H]+. N-(6-(4-(3-(3-(4-Methyl-1H-imidazol-1-yl)-5(trifluoromethyl)phenyl)ureido)phenoxy)pyrimidin-4-yl)acetamide (21). Yield = 43%. 1H


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NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 9.28 (s, 1H), 9.13 (s, 1H), 8.50 (s, 1H), 8.20 (s, 1H), 7.86 (s, 2H), 7.53 (s, 4H), 7.49 (s, 1H), 7.15 (s, 2H), 2.18 (s, 3H), 2.11 (s, 3H). 13C NMR (214 MHz, DMSO-d6) δ 171.20, 171.08, 160.07, 158.39, 153.05, 147.55, 142.57, 139.27, 138.55, 137.23, 135.47, 131.38, 124.82, 122.42, 120.35, 114.72, 113.34, 112.67, 110.22, 94.29, 24.55, 14.02. LC-MS (ESI, m/z): 512.1623 [M+H]+. 2-(4-((6-Acetamidopyrimidin-4-yl)oxy)phenyl)-N-(4-methyl-3(trifluoromethyl)phenyl)acetamide (22). To a solution of 42 (30 mg, 0.1 mmol) in anhydrous THF (1 mL) was added 4-methyl-3-(trifluoromethyl)aniline (30 mg, 0.16 mmol), HATU (60 mg, 0.16 mmol) and DIPEA (0.05 mL, 0.5 mmol) at room temperature under argon. The reaction mixture was stirred at room temperature for 14 h. The resulting mixture was concentrated to give the crude product, which was purified by flash chromatography (eluting with 0-10 % MeOH in DCM) to afford 22 as a white solid (38 mg, 86%). 1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 10.44 (s, 1H), 8.48 (s, 1H), 8.04 (s, 1H), 7.73 (s, 1H), 7.57 (s, 1H), 7.40 (s, 3H), 7.16 (s, 2H), 3.69 (s, 2H), 2.38 (s, 3H), 2.12 (s, 3H). 13C NMR (214 MHz, DMSO-d6) δ 171.19, 170.76, 169.85, 160.09, 158.30, 151.61, 137.87, 133.45, 133.13, 130.98, 130.71, 128.00, 127.86, 122.90, 121.98, 116.48, 94.55, 43.01, 24.53, 18.61. LC-MS (ESI, m/z): 445.1472 [M+H]+. Compounds 23-24 were prepared following the synthetic procedure of 22. 2-(4-((6-Acetamidopyrimidin-4-yl)oxy)phenyl)-N-phenylacetamide (23). Yield = 79%.1H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 1H), 10.22 (s, 1H), 8.48 (s, 1H), 7.58 (m, 3H), 7.41 (s, 2H), 7.30 (s, 2H), 7.16 (s, 2H), 7.04 (s, 1H), 3.67 (s, 2H), 2.11 (s, 3H).

13C

NMR (214 MHz,

DMSO-d6) δ 171.19, 170.77, 169.47, 160.08, 158.31, 151.55, 139.66, 133.79, 130.92, 129.18, 123.71, 121.96, 119.60, 119.45, 94.55, 42.77, 24.30. LC-MS (ESI, m/z): 363.1433 [M+H]+.



2-(4-((6-Acetamidopyrimidin-4-yl)oxy)phenyl)-N-(4-(trifluoromethyl)phenyl)acetamide (24). Yield = 81%.1H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H), 10.59 (s, 1H), 8.48 (s, 1H), 7.82 (s, 2H), 7.69 (s, 2H), 7.56 (s, 1H), 7.41 (s, 2H), 7.17 (s, 2H), 3.73 (s, 2H), 2.11 (s, 3H). 13C NMR (214 MHz, DMSO-d6) δ 171.19, 170.74, 170.16, 160.09, 158.29, 151.64, 143.19, 133.33, 131.00 , 126.51, 125.45, 123.90, 121.98, 119.48, 94.57, 43.06, 24.52. LC-MS (ESI, m/z): 431.1365 [M+H]+. N-(4-((6-Acetamidopyrimidin-4-yl)oxy)phenyl)-2-(4-(trifluoromethyl)phenyl)acetamide (25). To a solution of 40 (50 mg, 0.2 mmol) in anhydrous THF (1 mL) was added 2-(4(trifluoromethyl)phenyl)acetic acid (82 mg, 0.4 mmol), HATU (152 mg, 0.4 mmol) and DIPEA (0.2 mL, 1.0 mmol) at room temperature under argon. The reaction mixture was stirred at room temperature for 14 h. The resulting mixture was concentrated to give the crude product, which was purified by flash chromatography (eluting with 0-10 % MeOH in DCM) to afford 25 as a white solid (75 mg, 88%). 1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 10.37 (s, 1H), 8.49 (s, 1H), 7.70 (s, 2H), 7.65 (s, 2H), 7.58 (s, 2H), 7.51 (s, 1H), 7.15 (s, 2H), 3.79 (s, 2H), 2.10 (s, 3H). 13C NMR (214 MHz, DMSO-d6) δ 171.17, 170.99, 168.82, 160.00, 158.32, 148.22, 141.20, 137.06, 130.41, 127.62, 125.57, 124.02, 122.35, 120.79, 94.03, 43.32, 24.26. LC-MS (ESI, m/z): 431.1315 [M+H]+. Compounds 26-35 were prepared following the synthetic procedure of 25. N-(4-((6-Acetamidopyrimidin-4-yl)oxy)phenyl)-2-(3(trifluoromethyl)phenyl)acetamide (26). Yield = 84%.1H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H), 10.34 (s, 1H), 8.48 (s, 1H), 7.72 (s, 1H), 7.65 (s, 5H), 7.51 (s, 1H), 7.15 (s, 2H), 3.81 (s, 2H), 2.11 (s, 3H).

13C

NMR (214 MHz, DMSO-d6) δ 171.19, 170.97, 168.96, 160.09, 158.34,


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148.21, 137.65, 136.87, 129.76, 126.27, 125.35, 124.11, 123.79, 122.16, 120.83, 94.34, 42.31, 24.64. LC-MS (ESI, m/z): 431.1349[M+H]+. N-(4-((6-Acetamidopyrimidin-4-yl)oxy)phenyl)-2-(2(trifluoromethyl)phenyl)acetamide (27). Yield = 80%. 1H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 10.33 (s, 1H), 8.48 (s, 1H), 7.71 (s, 1H), 7.65 (s, 3H), 7.52 (s, 3H), 7.15 (s, 2H), 3.94 (s, 2H), 2.11 (s, 3H).

13C

NMR (214 MHz, DMSO-d6) δ 171.18, 170.90, 168.44, 159.96, 158.23,

148.11, 137.15, 134.36, 133.86, 132.58, 127.75, 126.09, 125.51, 124.36, 122.36, 120.81, 93.67, 39.68, 24.00. LC-MS (ESI, m/z): 431.1308 [M+H]+. N-(4-((6-Acetamidopyrimidin-4-yl)oxy)phenyl)-2-(2,4bis(trifluoromethyl)phenyl)acetamide (28). Yield = 76%. 1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 10.39 (s, 1H), 8.49 (s, 1H), 8.06 (s, 2H), 7.83 (s, 1H), 7.64 (s, 2H), 7.52 (s, 1H), 7.16 (s, 2H), 4.08 (s, 2H), 2.11 (s, 3H).

13C

NMR (214 MHz, DMSO-d6) δ 171.20, 171.06,

167.72, 159.96, 158.30, 148.23, 139.38, 136.84, 135.19, 129.02, 128.45, 125.87, 124.58, 123.41, 122.96, 122.37, 120.85, 94.24, 42.25, 24.49. LC-MS (ESI, m/z): 499.1179 [M+H]+. N-(4-((6-Acetamidopyrimidin-4-yl)oxy)phenyl)-2-(2-chloro-4(trifluoromethyl)phenyl)acetamide (29). Yield = 79%.1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 10.33 (s, 1H), 8.48 (s, 1H), 7.75 (s, 1H), 7.68 (s, 1H), 7.62 (s, 3H), 7.52 (s, 1H), 7.16 (s, 2H), 3.97 (s, 2H), 2.11 (s, 3H).

13C

NMR (214 MHz, DMSO-d6) δ 171.18, 170.96, 167.92,

159.98, 158.25, 148.19, 137.35, 137.01, 136.77, 133.87, 128.13, 127.83, 126.97, 123.80, 122.36, 120.73, 94.36, 42.31, 24.52. LC-MS(ESI, m/z): 465.0922[M+H]+. N-(4-((6-Acetamidopyrimidin-4-yl)oxy)phenyl)-2-(2,6-dichlorophenyl)acetamide

(30).

Yield = 75%. 1H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 10.44 (s, 1H), 8.48 (s, 1H), 7.63 (s, 2H), 7.51 (s, 3H), 7.35 (s, 1H), 7.16 (s, 2H), 4.07 (s, 2H), 2.11 (s, 3H). 13C NMR (214 MHz,



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DMSO-d6) δ 171.21, 171.01, 166.99, 159.96, 158.22, 147.98, 137.14, 136.03, 132.66, 129.90, 128.59, 122.35, 120.72, 94.32, 42.20, 24.45. LC-MS (ESI, m/z): 431.0691 [M+H]+. N-(4-((6-Acetamidopyrimidin-4-yl)oxy)phenyl)-2-(3,4-dichlorophenyl)acetamide

(31).

Yield = 86%. 1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 10.31 (s, 1H), 8.48 (s, 1H), 7.63 (s, 4H), 7.51 (s, 1H), 7.35 (s, 1H), 7.15 (s, 2H), 3.71 (s, 2H), 2.11 (s, 3H). 13C NMR (214 MHz, DMSO-d6) δ 171.18, 170.97, 168.70, 160.08, 148.21, 137.43, 137.01, 131.77, 131.22, 130.82, 130.16, 129.80, 122.35, 120.91, 94.34, 42.34, 24.63. LC-MS (ESI, m/z): 431.0683 [M+H]+. N-(4-((6-Acetamidopyrimidin-4-yl)oxy)phenyl)-2-(2,4-dichlorophenyl)acetamide

(32).

Yield = 78%. 1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 10.36 (s, 1H), 8.48 (s, 1H), 7.64 (s, 3H), 7.51 (s, 1H), 7.46 (s, 1H), 7.44 (s, 1H), 7.16 (s, 2H), 3.86 (s, 2H), 2.10 (s, 3H). 13C NMR (214 MHz, DMSO-d6) δ 171.19, 170.99, 167.99, 160.07, 158.22, 148.00, 137.11, 135.16, 133.97, 133.59, 132.70, 128.91, 127.66, 122.21, 120.81, 94.19, 42.29, 24.54. LC-MS (ESI, m/z): 431.0642 [M+H]+. N-(4-((6-Acetamidopyrimidin-4-yl)oxy)phenyl)-2-(naphthalen-1-yl)acetamide (33). Yield = 78%. 1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 10.44 (s, 1H), 8.48 (s, 1H), 8.13 (s, 1H), 7.92 (s, 1H), 7.86 (s, 1H), 7.67 (s, 2H), 7.51 (s, 5H), 7.14 (s, 2H), 4.17 (s, 2H), 2.11 (s, 3H). 13C NMR (214 MHz, DMSO-d6) δ 171.20, 170.96, 169.49, 160.03, 158.35, 148.05, 137.19, 133.85, 132.86, 132.49, 128.91, 128.30, 127.72, 126.61, 126.16, 126.01, 124.62, 122.35, 120.83, 94.32, 38.70, 24.55. LC-MS (ESI, m/z): 413.1635 [M+H]+. N-(4-((6-Acetamidopyrimidin-4-yl)oxy)phenyl)-2-(naphthalen-2-yl)acetamide (34). Yield = 74%. 1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 10.36 (s, 1H), 8.49 (s, 1H), 7.89 (s, 4H), 7.67 (s, 2H), 7.51 (s, 4H), 7.14 (s, 2H), 3.85 (s, 2H), 2.11 (s, 3H). 13C NMR (214 MHz, DMSOd6) δ 171.19, 171.00, 169.53, 159.99, 158.36, 147.99, 134.10, 133.42, 132.34, 128.22, 128.14,


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127.97, 127.92, 126.60, 126.09, 122.34, 120.89, 94.33, 43.86, 24.55. LC-MS (ESI, m/z): 413.1602 [M+H]+. 2-([1,1'-Biphenyl]-4-yl)-N-(4-((6-acetamidopyrimidin-4-yl)oxy)phenyl)acetamide

(35).

Yield = 82%. 1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 10.32 (s, 1H), 8.49 (s, 1H), 7.66 (s, 6H), 7.46 (s, 5H), 7.36 (s, 1H), 7.15 (s, 2H), 3.71 (s, 2H), 2.11 (s, 3H).

13C

NMR (214 MHz,

DMSO-d6) δ 171.19, 171.00, 169.51, 160.07, 158.35, 140.32, 139.01, 137.23, 135.67, 130.16, 129.37, 127.56, 127.13, 127.04, 122.34, 120.71, 94.21, 43.39, 24.46. LC-MS (ESI, m/z): 439.1792 [M+H]+. 4-((6-Chloropyrimidin-4-yl)oxy)aniline (36). To a solution of 4,6-dichloropyrimidine (10.0 g, 67.1 mmol) in anhydrous CH3CN (120 mL) was added 4-aminophenol (7.32 g, 67.1 mmol), K2CO3 (18.55 g, 134.2 mmol) and CuBr2 (10 mg, 0.045 mmol) at room temperature under argon. Then the reaction mixture was stirred at room temperature for 14 h. The resulting mixture was concentrated to dryness. The residue was diluted with water, and extracted with EtOAc. The combined organic layers were washed with water, brine and dried over anhydrous Na2SO4. Evaporation of the solvent provided the crude product, which was purified by flash chromatography (eluting with 0-50% EtOAc in hexane) to afford 36 as a brown solid (11.6 g, 78%). 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 7.12 (s, 1H), 6.88 (s, 1H), 6.86 (s, 1H), 6.63 (s, 1H), 6.61 (s, 1H), 5.15 (s, 2H).

13C

NMR (101 MHz, DMSO-d6) δ 171.38, 161.17,

159.12, 147.37, 142.27, 122.16, 114.91, 107.72. LC-MS (ESI, m/z): 222.0451 [M+H]+. 6-(4-Aminophenoxy)-N-methylpyrimidin-4-amine (37a). To a solution of 36 (3.43 g, 15.5 mmol) in anhydrous THF (5 mL) was added MeNH2 (16.2 mL, 32.4 mmol, 2.0 M in THF) at room temperature under argon. Then the reaction mixture was heated to 120 C and stirred for 24 h. The resulting mixture was concentrated to dryness. The residue was diluted with water, and



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extracted with EtOAc. The combined organic layers were washed with water, brine and dried over anhydrous Na2SO4. Evaporation of the solvent provided the crude product, which was purified by flash chromatography (eluting with 0-5% MeOH in DCM) to afford 37a as a brown solid (2.8 g, 83%). 1H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H), 7.21 (s, 1H), 6.84 (s, 2H), 6.66 (s, 2H), 5.63 (s, 1H), 5.06 (s, 2H), 2.76 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 170.43, 165.52, 158.41, 145.52, 143.79, 122.41, 115.67, 105.35, 27.90. LC-MS (ESI, m/z): 217.1065 [M+H]+. Compounds 37b-d were prepared following the synthetic procedure of 37a. 6-(4-Aminophenoxy)-N-benzylpyrimidin-4-amine (37b). Yield = 81%. 1H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 7.83 (s, 1H), 7.30 (s, 4H), 7.25 (s, 1H), 6.81 (s, 2H), 6.63 (s, 2H), 5.73 (s, 1H), 5.19 (s, 2H), 4.51 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 170.69, 164.99, 158.59, 146.57, 143.18, 128.79, 127.67, 127.25, 122.40, 115.14, 44.20. LC-MS (ESI, m/z): 293.1386 [M+H]+. 6-(4-Aminophenoxy)-N-(4-methoxybenzyl)pyrimidin-4-amine (37c). Yield = 77%.

1H

NMR (400 MHz, DMSO-d6) δ 8.15 (s, 1H), 7.74 (s, 1H), 7.22 (s, 1H), 7.20 (s, 1H), 6.89 (s, 1H), 6.87 (s, 1H), 6.81 (s, 1H), 6.80 (s, 1H), 6.62 (s, 1H), 6.60 (s, 1H), 5.69 (s, 1H), 5.09 (s, 2H), 4.34 (s, 2H), 3.72 (s, 3H).

13C

NMR (101 MHz, CDCl3) δ 170.67, 164.91, 158.69, 146.74, 143.07,

129.02, 122.40, 115.01, 114.19, 55.48, 43.74. LC-MS (ESI, m/z): 323.1525 [M+H]+. tert-Butyl (2-((6-(4-aminophenoxy)pyrimidin-4-yl)amino)ethyl)carbamate (37d). Yield = 74%. 1H NMR (400 MHz, CDCl3) δ 8.04 (s, 1H), 6.74 (d, J = 7.2 Hz, 2H), 6.55 (d, J = 7.0 Hz, 2H), 6.28 (s, 1H), 5.71 (s, 1H), 5.51 (s, 1H), 3.87 (s, 2H), 3.20 (s, 2H), 3.18 (s, 2H), 1.30 (s, 9H). 13C

NMR (101 MHz, CDCl3) δ 170.48, 164.70, 158.02, 156.59, 144.21, 122.03, 115.97, 41.64,

28.31. LC-MS (ESI, m/z): 346.1891 [M+H]+. 1-(4-((6-Chloropyrimidin-4-yl)oxy)phenyl)-3-(4-methyl-3-(trifluoromethyl)phenyl)urea (38). To a solution of 36 (1.5 g, 6.77 mmol) in anhydrous THF (30 mL) was added 4-methyl-3-


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(trifluoromethyl)aniline (2.37 g, 13.54 mmol) and DIPEA (3.6 mL, 20.31 mmol) at 0 C under argon. The reaction mixture was stirred at 0 C for 10 min, and then triphosgene (0.8 g, 2.71 mmol) was added. The reaction mixture was stirred at 0 C for 1 h. Then the reaction mixture was stirred at room temperature for 3 h. The resulting mixture was concentrated to dryness. The residue was diluted with water and extracted with EtOAc. The combined organic layers were washed with water, brine and dried over anhydrous Na2SO4. Evaporation of the solvent provided the crude product, which was purified by flash chromatography (eluting with 0-5% MeOH in DCM) to afford 38 as a yellow solid (1.4 g, 49%). 1H NMR (400 MHz, CDCl3) δ 8.97 (s, 1H), 8.86 (s, 1H), 8.65 (d, J = 2.7 Hz, 1H), 7.95 (s, 1H), 7.55 (d, J = 3.2 Hz, 3H), 7.31 (d, J = 6.1 Hz, 2H), 7.16 (s, 2H), 2.37 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 170.71, 161.38, 159.01, 152.85, 146.75, 138.24, 137.85, 133.04, 128.96, 127.96, 122.25, 122.13, 120.04, 115.45, 108.30, 18.12. LC-MS (ESI, m/z): 423.0844 [M+H]+. N-(6-Chloropyrimidin-4-yl)acetamide (39). To a solution of Ac2O (50 mL) was added 6chloropyrimidin-4-amine (5.0 g, 38.6 mmol) at room temperature under argon. The reaction mixture was heated to 140 C and stirred for 5 h. The resulting mixture was concentrated to dryness. The residue was washed with methanol to afford 39 as a yellow solid (5.0 g, 76%). 1H NMR (400 MHz, CDCl3) δ 11.20 (s, 1H), 8.70 (s, 1H), 8.05 (s, 1H), 2.14 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 171.46, 161.04, 159.42, 158.89, 108.99, 24.55. LC-MS (ESI, m/z): 172.0245 [M+H]+. N-(6-(4-Aminophenoxy)pyrimidin-4-yl)acetamide (40). To a solution of 39 (2.0 g, 11.7 mmol,) in CH3CN (50 mL) was added 4-aminophenol (1.27 g, 11.7 mmol), K2CO3 (1.94 g, 14.04 mmol,) and CuBr2 (10 mg, 0.05 mmol) at room temperature under argon. The reaction mixture was stirred at room temperature for 14 h. The resulting mixture was concentrated to dryness. The



residue was diluted with water and extracted with EtOAc. The combined organic layers were washed with water, brine and dried over anhydrous Na2SO4. Evaporation of the solvent provided the crude product, which was purified by flash chromatography (eluting with 0-10% MeOH in DCM) to afford 40 as a yellow solid (800 mg, 28%). 1H NMR (400 MHz, DMSO-d6) δ 10.87 (s, 1H), 8.48 (s, 1H), 7.44 (s, 1H), 6.61 (d, J = 6.1 Hz, 2H), 6.48 (s, 2H), 5.09 (s, 2H), 2.10 (s, 3H). 13C

NMR (101 MHz, DMSO-d6) δ 171.68, 170.90, 159.94, 158.38, 147.15, 142.62, 122.15,

115.86, 93.82, 24.04. LC-MS (ESI, m/z): 245.1004 [M+H]+. Methyl 2-(4-((6-acetamidopyrimidin-4-yl)oxy)phenyl)acetate (41). To a solution of 39 (1.0 g, 5.83 mmol) in anhydrous DMF (10 mL) was added methyl 2-(4-hydroxyphenyl)acetate (1.45 g, 8.24 mmol) and Cs2CO3 (3.8 g, 11.66 mmol) at room temperature under argon. The reaction mixture was stirred at room temperature for 14 h. The resulting mixture was concentrated to dryness. The residue was diluted with water and extracted with EtOAc. The combined organic layers were washed with water, brine and dried over anhydrous Na2SO4. Evaporation of the solvent provided the crude product, which was purified by flash chromatography (eluting with 010 % MeOH in DCM) to afford 41 as a white solid (1.4 g, 79%). 1H NMR (400 MHz, CDCl3) δ 10.99 (s, 1H), 8.50 (s, 1H), 7.61 (s, 1H), 7.05 (s, 2H), 6.73 (d, J = 1.9 Hz, 2H), 3.72 (s, 2H), 3.64 (s, 3H), 2.14 (s, 3H).

13C

NMR (101 MHz, CDCl3) δ 172.49, 171.20, 170.68, 160.13, 158.25,

151.72, 131.97, 131.12, 124.87, 94.58, 51.97, 39.79, 24.37. LC-MS (ESI, m/z): 302.1128 [M+H]+. 2-(4-((6-Acetamidopyrimidin-4-yl)oxy)phenyl)acetic acid (42). To a solution of 41 (1.3 g, 4.3 mmol) in MeOH (10 mL) was added 1 M NaOH (5 mL) at room temperature under argon. The reaction mixture was heated to 80 C and stirred for 2 h. The resulting mixture was concentrated to dryness. The residue was diluted with water and 1 M HCl was added to adjust


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the pH to ~3. The yellow precipitate was filtered and dried to afford 42 as a yellow solid (430 mg, 35%). 1H NMR (400 MHz, CDCl3) δ 12.41 (s, 1H), 10.97 (s, 1H), 8.49 (s, 1H), 7.58 (s, 1H), 7.33 (s, 2H), 7.15 (d, J = 2.9 Hz, 2H), 3.62 (s, 2H), 2.13 (s, 3H).

13C

NMR (101 MHz, CDCl3) δ

173.17, 171.20, 170.73, 160.07, 158.28, 151.52, 132.84, 131.23, 121.86, 94.56, 40.43, 24.39. LC-MS (ESI, m/z): 288.0969 [M+H]+. Biochemical Kinase Assay. The inhibitory activities of compound 27 against KIT, PDGFRα, PDGFRβ, FLT3 and FLT3-ITD kinases were determined by ADP-GloTM assay. The optimized enzyme concentrations were chosen as follows: KIT 20 ng/µL, PDGFRα 2.5 ng/µL, PDGFRβ 1.25 ng/µL, FLT3 10 ng/µL, FLT3-ITD 10 ng/µL (Promega, USA). In all cases, 2.5 µL samples of kinase was incubated with compound 27 for 60 min at room temperature in reaction buffer followed by addition of 2.5 µL ATP/substrate mixture. The ATP concentration was chosen as follows: 50 µM ATP for KIT, 200 µM ATP for FLT3, 100 µM ATP for FLT3-ITD, 25 µM ATP for PDGFRα and PDGFRβ. The assay was conducted for 1 h at 37 C before addition of 5 µL ADP-Glo reagent and incubation for 40 min at room temperature. 10 µL Kinase detection reagent was added and incubated for 30 min at room temperature before the luminescence signal was read with an envision Perkin Elmer plate reader (Envision, PE, USA), and the dose– response curve was fitted using Prism 5.0 (GraphPad Software Inc., San Diego, CA). Cell Culture and Proliferation Study. The human AML cell lines MV4-11 and U937 were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Other cell lines OCI-AML-2 and HL-60 were purchased from Cobioer Biosciences CO., Ltd. (Nanjing, China). MOLM13 and MOLM14 cell lines were provided by Dr. Scott Armstrong, Dana Farber Cancer Institute (DFCI), Boston, MA, USA. MV4-11cells was cultured in IMDM media (Corning, USA) with 10% FBS and supplemented with 2% L-glutamine and 1% pen/strep.



MOLM13, MOLM14, U937, HL-60 and kinase isogenic BaF3 cells were cultured in RPMI 1640 media (Corning, USA) with 10% fetal bovine serum (FBS) and supplemented with 2% Lglutamine 1% penicillin/streptomycin. OCI-AML-2 was cultured in α-MEM media (Corning, USA) with 20% FBS and supplemented with 2% L-glutamine and 1% pen/strep. Parental BaF3 cells were cultured RPMI 1640 media (Corning, USA) with 10% fetal bovine serum (FBS), supplemented with 2% L-glutamine, 1% penicillin/streptomycin and 100 ng/ml IL-3. All cell lines were maintained in culture media at 37 °C with 5% CO2. Cells were grown in 96-well culture plates (1500-3000/well). The compounds of various concentrations were added into the plates. Cell proliferation was determined after treatment with compounds for 72 h. Cell viability was measured using the CCK8 assay (MedChemExpress, China) according to the manufacturer’s instructions, and absorbance was measured using a iMark microplate reader (Bio-Red, USA) at 450 nm. Data were normalized to control groups (DMSO) and represented by the mean of three independent measurements with standard error of

(2-(trifluoromethyl)phenyl)acetamide - ACS Publications

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