1H-pyrazole-3-carboxamide (FN-1501), an FLT3 ... - ACS Publications

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Discovery of 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3carboxamide (FN-1501), as a FLT3/CDKs kinase inhibitor with potential high efficiency against acute myelocytic leukemia (AML) Yue Wang, Yanle Zhi, Qiaomei Jin, Shuai Lu, Guowu Lin, Haoliang Yuan, Taotao Yang, Zhanwei Wang, Chao Yao, Jun Ling, Hao Guo, Tonghui Li, Jianlin Jin, Baoquan Li, Li Zhang, Yadong Chen, and Tao Lu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01261 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Discovery of 4-((7H-pyrrolo[2,3-d]pyrimidin-4yl)amino)-N-(4-((4-methylpiperazin-1yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (FN-1501), as a FLT3/CDKs kinase inhibitor with potential high efficiency against acute myelocytic leukemia (AML) ⊥

⊥

Yue Wang†,⋚, , Yanle zhi†,⋚, , Qiaomei Jin†, Shuai Lu†, Guowu Lin†,‡, Haoliang Yuan†, Taotao Yang†, Zhanwei Wang†, Chao Yao†, Jun Ling†, Hao Guo†, Tonghui Li†, Jianlin Jin†, Baoquan Li†, Li Zhang†, Yadong Chen*,†, Tao Lu *,†,⋚ . †

⋚

School of Sciences, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China.

State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China.

‡

Department of Structural Biology, University of Pittsburgh, School of medicine, Pittsburgh, PA 1526, U.S.A., current address.

ACS Paragon Plus Environment

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ABSTRACT A series of 1-H-pyrazole-3-carboxamide derivatives have been designed and synthesized, exhibiting excellent FLT3/CDKs inhibition and anti-proliferative activity. Structure-activity relationship study illustrates that the incorporation of a pyrimidine fused heterocycle at position 4 of the pyrazole is critical for FLT3/CDKs inhibition. Compound 50 (FN-1501), possessing potent inhibitory activities against FLT3, CDK2, CDK4 and CDK6 with IC50 values in the nanomolar range, shows anti-proliferative activity against MV4-11 cells (IC50: 0.008 μM), which correlates with the suppression of retinoblastoma phosphorylation, FLT3, ERK, AKT, STAT5 and the onset of apoptosis. The acute toxicity studies in mice show that compound 50 (LD50: 186 mg/kg) is safer than AT7519 (32 mg/kg). In MV4-11 xenografts in nude mice mode, compound 50 can induce tumor regression at the dose of 15 mg/kg, which is more efficient than Cytarabine (50 mg/kg). Taken together, these results demonstrate the potential of this unique compound for further development into drug applied in acute myeloid leukemia (AML) therapeutics.


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INTRODUCTION AML is a heterogeneous disease of the hematopoietic progenitor cell, characterized by a block in differentiation and uncontrolled proliferation1. More than one quarter of a million adults throughout the world

are

diagnosed

with

AML

annually.

Induction

chemotherapy with Cytarabine (ara-C)

and Anthracycline (most often Daunorubicin) are usually performed as the first-line treatment of AML1. Although some improvement has been apparent among younger patients during the last four decades, only approximately 35% of those patients entered on clinical trials are cured of their disease, but older people who are not able to withstand intensive chemotherapy can only survive 5-10 months2. Thus there is an unmet need for effective therapeutics. Fms-like receptor tyrosine kinase 3 (FLT3) is a Class III receptor tyrosine kinase (RTK), which acts as an important regulatory molecule in hematopoiesis and is overexpressed in most cases of acute leukemia3. FLT3 pathway plays an important role in the growth and differentiation of hematopoietic progenitor cells (Hematopoietic progenitor cells, HPCs)4, 5. Many studies have shown that AML and other hematologic cancers are closely related to abnormal FLT3 pathway6-10. As other RTKs, FLT3 receptor dimerized when binding to the FLT3 ligand, resulting in autophosphorylation and activation of downstream signaling pathways, such as RAS/MEK, PI3K/AKT/mTOR and JAK/STAT. These pathways play important roles in regulating cell cycle, cell apoptosis and cell differentiation. However, mutations of FLT3 lead to the hyperactivation of FLT3 in the absence of ligand binding and activation of downstream signaling pathways11. About 30% of AML patients harbor some form of FLT3 mutation, constitutively activating internal tandem duplication (ITD) mutations (of 1–100 amino acids) in the juxtamembrane domain (in approximately 25% of AML patients) invariably present the greatest clinical challenge. FLT3 has always been an important target for the treatment of AML, with a lot of small molecule FLT3 inhibitors (Quizartinib , Sorafenib , Midostaurin , Lestaurtinib , Crenolanib) marketed or under investigation12. Although some of these compounds have shown initial therapeutic benefits, responses have been transient and relapse occurs within a few weeks, partially due to insufficient target coverage, activation of parallel pathways, and acquisition of resistance mutations13. Midostaurin (PKC412; N-


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benzoylstaurosporin) is a multi-targeted tyrosine kinase inhibitor (TKI) against FLT3, VEGFR2, KIT, PDGFR and PKC-a. Midostaurin was approved and first launched in 2017 in the U.S. for the treatment of adult patients with newly diagnosed acute myeloid leukemia, in combination with chemotherapy. The success of Midostaurin indicates that inhibition of FLT3 and other antitumor target can improve the antitumor efficiency. Cyclin-Dependent Kinases (CDKs) are a family of 20 serine/threonine protein kinases that are generally classified as regulators of the cell cycle (CDK1, 2, 4, 6) or transcription (CDK7, 8, 9, 11, 20)14. The uncontrolled regulation of CDK activity has been identified as a hallmark of cancer15-17. Indeed, oncogenic alterations of CDKs, cyclins and other components of the Rb pathway have been reported in more than 90% of human cancers18-20. For example, overexpression of cyclin E or cyclin A results in CDK2 hyperactivation in several human malignancies, including AML, lung cell carcinoma, melanoma, osteosarcoma, ovarian carcinoma, pancreatic neoplasia and sarcomas21. Cyclin D2 and cyclin D3 belong to D-type cyclins, binding and activating CDK4/CDK6, play important roles in hematologic malignancies. Mutations or overexpression of CDK4, cyclin D, as well as reduced levels of p27Kip1 have also been found in various tumors, indicating that deregulation at checkpoints throughout the cell cycle contributes to the process of tumorigenesis22-24. These studies suggest that the CDK family has been considered as one of the most important targets for therapeutic intervention in cancer, including hematologic malignancies. CDK inhibitors (AT-7519, Flavopiridol, AMG-925, Palbociclib)18, 2532

are currently in clinical development in various solid tumors and hematopoietic malignances33. However,

most of CDKs inhibitors were discontinued during phase II trials because of severe toxicity34 or limited clinical activity as monotherapy35. In fact, activation of FLT3 downstream signal transduction pathway is an extremely important process in promoting cell proliferation, which requires the cooperation of CDKs. Therefore, inhibitor of FLT3 and CDKs can arrest anti-mitotic signaling pathways and cell cycle simultaneously, which may exert synergistic effect to increase the anti-leukemic efficiency

18, 28, 36

. We have reported a series of compounds with moderate CDK2

activity37-40. Among these compounds, 26 (Figure 1) has a certain inhibitory activity against CDK2 (IC50: 318.21 nM) and FLT3 (IC50: 42.60 nM) and shows moderate activity in MV4-11 (IC50: 0.45 μM). Starting from 26, our


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group has explored the biological properties of variously substituted 1-H-pyrazole-3-carboxamide derivatives with the aim to find new active compounds and pharmacophores as the potent FLT3/CDKs inhibitors. Among these compounds, compound 50 was found to be the most active in vitro and in vivo, which also possesses good pharmacokinetic properties and low toxicity. The report presents the discovery of 50 as a highly efficient multi-target FLT3/CDKs kinase inhibitor suitable for further evaluation.

Figure 1. Molecular docking analysis of compound 26 to CDK2 kinase and FLT3 kinase. (A) Chemical structure of 26. (B) Compound 26 docked into CDK2 kinase (PDB code 2VU3). (C) Compound 26 docked into homology model of FLT318.

CHEMISTRY Scheme 1. Synthetic routes of compounds 27-62 R1

a

Br NO2

R1

O N H R1

70 - 95 %

NO2

d

80 - 95 %

NO 2

c NH 2

2: R 1 = morpholine 3: R 1 = 1-methylpiperazine 4: R 1 = piperazine 5: R 1 = 1-methyl-1,4-diazepane 6: R 1 = diethylamine

8: R 1 = morpholine 9: R 1 = 1-methylpiperazine 10: R 1 = piperazine 11: R 1 = 1-methyl-1,4-diazepane 12: R 1 = diethylamine

7: R 1 = cyclopropan amine

13: R 1 = cyclopropan amine

R1

O N H

N NH

14: = morpholine 15: R 1 = 1-methylpiperazine 16: R 1 = piperazine 17: R 1 = 1-methyl-1,4-diazepane 18: R 1 = diethylamine 19: R 1 = cyclopropan amine

R1

b

NH 2

e

N NH

R1

20: = morpholine 21: R 1 = 1-methylpiperazine 22: R 1 = piperazine 23: R 1 = 1-methyl-1,4-diazepane 24: R 1 = diethylamine 25: R 1 = cyclopropan amine

R1

O N H

2 HN R

N NH

27- 57 as in Table 1 57-62 as in Table 2

Reagents and conditions: (a) relative amine, Et3N, CH2Cl2; (b) FeO(OH)/C, 80% NH2NH2.H2O, 95% EtOH; (c) 4-


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Nitropyrazole-3-carboxylic acid, EDC.HCl, HOBt, DMF, r.t; (d) FeO(OH)/C, 80% NH2NH2.H2O, 95% EtOH; (e) R2Cl, AcOH/H2O, 50 °C The synthesis of compound 27-62 was depicted in Scheme 1. In total, 35 pyrazole-carboxamide derivatives were prepared (Table 1 and Table 2). Compounds 27-62 were prepared in a similar fashion. 4Nitrobenzyl bromide was used as the starting material and compound 2-7 were prepared through a nucleophilic substitution reaction. Reduction of compound 2-7 with 80% hydrazine hydrate gave compound 813 and after coupling of 4-nitropyrazole-3-carboxylic acid with EDC.HCl and HOBt40, compound 14-19 were obtained. Compound 20-25 were obtained via the reduction of the nitro-group in compound 14-19. All target compounds (27-62) were prepared by ammonolysis of appropriate chlorides with 20-25.

RESULTS AND DISCUSSION In our previous studies40, a series of pyrazole-3-carboxamide compounds were synthesized. Recently, it has been identified that compound 26 inhibits FLT3 and CDK2 with IC50 of 42.6 nM and 318 nM, respectively. With the aid of CADD (Computer Aided Drug Design), we found that the hydrophobic cavity of CDK2 formed by ASP145, VAL18, PHE80 and LYS33 (corresponding to ASP757, PHE691, PHE627 and LYS650 in FLT3) are wide enough to accommodate a larger hydrophobic group. Guided by our previous studies, we kept the 1-Hpyrazole-3-carboxamide skeleton and the hydrophobic phenyl ring linked to 3-carboxamide (The binding modes were shown in Figure 1). In position 4 of pyrazole, the benzoyl group was cyclized to give a pyrimidobenzene ring structure, and then various binary aromatic heterocycles were incorporated to study the effects of the hydrophobic group on kinase inhibitory activity. In solvent accessible region, different hydrophilic fragments were introduced, leading to a series of 1-H-pyrazole-3-carboxamide derivatives. All derivatives were tested for their inhibitory potency toward recombinant human CDK2/Cyclin A1 and FLT3 and their antiproliferative effects on MV4-11 cells (human acute monocytic leukemia cell line).

Structure-Activity Relationship (SAR) Studies The results of the kinase inhibition assays are listed in Table 1 and Table 2 with AT-7519 and Sorafenib as positive controls. Minimizing the diversity of the substituents in the phenyl ring enabled us to investigate how


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varying the substituents at the C4-position of the pyrazole will affect the activity against CDK2 and FLT3. As is shown in Table 1, both CDK2 and FLT3 affinity were increased slightly compared with the parent compound when the benzoyl in 26 were replaced by quinolone (compound 27, 42) or isoquinoline (compound 28, 44) .This may benefit from the edge-to-face aromatic-aromatic interaction between the pyridine and PHE80 in CDK2 and PHE691 in FLT3 and the hydrophobic interaction formed by the phenyl ring with the ASP145 in CDK2 and ASP698 in FLT3, respectively (Figure 2).

Figure 2. Binding mode analysis of compound 42 to CDK2 kinase and FLT3 kinase. (A) Compound 42 docked into CDK2 kinase (PDB code 2VU3). (B) Compound 42 docked into homology model of FLT318. CDK2 and FLT3 inhibitory activities were lost when the hydrogen at position 2 of quinoline was replaced by trifluoromethyl (33), probably because trifluoromethyl prevented the edge-to-face interaction between the aromatic ring and PHE80. Further introduction of different substituents to isoquinoline or quinoline yield analogues 29-30 and 44-46. Electron withdrawing groups such as bromine in 29 and 46 can improve the affinity to FLT3. However, compounds 30, 44 and 46, substituted with electron-donating groups such as methoxyl were slightly less active against both CDK2 and FLT3 when compared to 27 and 42. Interestingly, introduction of a nitrogen to the pyridine ring of 27 yielded analogues 31, which showed significant increase of inhibitory activities against CDK2 and FLT3 (IC50: 5.30 nM and 0.588 nM, respectively). Replacement of the morpholine (31) to N-methylpiperazine (47) also retained excellent activity against CDK2 (IC50: 3.17 nM) and FLT3 (IC50: 8.27 nM). The inhibitory activity was approximately 10 times more potent than that of AT7519 and Sorafenib. Addition of the chlorine atom into 2 position of pyrimidine (32) resulted in decrease of affinity to CDK2 when compared with 31, supporting the hypothesis that substituent in C2 position of pyrimidine or pyridine might prevent edge-to-face interaction between the aromatic ring and


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PHE80 (CDK2). However, introduction of a chlorine atom has no impact on the inhibitory activity of 32 against FLT3. We speculate that the distance between the pyrimidine ring and gatekeeper residues PHE675 of FLT3 are far enough to accommodate the chlorine atom. Table 1. Structures and biological evaluation of target compounds

( % cell growth (% enzyme activity at 0.123 µM)

Compounds

A

inhibition at 1µM)

IC50(nM)a

R2

IC50 (µM)c

CDK2

FLT3

MV4-11

AT7519

57.20 ± 1.31

nd b

0.232 ± 0.003

Sorafenib

nd

15.48

0.004 ± 0.0002

42.60 ± 1.6

0.45 ± 0.009

(57.67%)

(40.75%)

(41.97%)

nd

nd

nd

287.19 ± 11.4

42.38 ± 4.3

26

27

O

318.21 ± 13.5

O

(50.40%) 28

O

nd (93.66%) 29

O

24.15 ± 1.8

16.90 ± 0.5 nd


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(74.99%) 30

O

(22.20%) 97.4 ± 2.1

nd

nd

31

O

5.30 ± 0.32

0.588 ± 0.019

32

O

58.63 ± 1.6

0.958 ± 0.023

0.023 ± 0.003

(91.10%) nd

33

(99.7%)

(83.22%)

(4.7%)

nd

nd

nd

O

（53.11%） 34

O

29.8 ± 2.4

3.78 ± 0.3 nd (98.49%)

35

O

3.36 ± 0.6

0.623 ± 0.04 nd

36

O

7.25 ± 0.28

2.06 ± 0.05

0.021 ± 0.003

37

O

3.30 ± 0.01

1.56 ± 0.02

0.034 ± 0.004

38

O

9.85 ± 0.91

7.63 ± 0.65

0.015 ± 0.002

39

O

1.08 ± 0.04

1.37 ± 0.06

0.011 ± 0.003


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40

O

11.64 ± 1.56

6.15 ± 0.87

0.035 ± 0.008

41

O

7.34 ± 0.24

3.52 ± 0.15

0.030 ± 0.005

(26.51%)

(27.72%)

(98.79%)

42

NCH3 nd

nd

nd

(60.47%)

(24.49%)

(66.46%)

nd

nd

nd

5.74 ± 0.73

4.84 ± 0.21

43

NCH3

(97.24%) 44

NCH3

nd (22.60%) 45

NCH3

697 ± 23.05

197 ± 24.71 nd

46

(32.45%)

(10.45%)

(64.40%)

nd

nd

nd

NCH3

47

NCH3

8.27 ± 1.32

3.17 ± 0.57

0.017 ± 0.004

48

NCH3

5.85 ± 0.61

0.60 ± 0.05

0.019 ± 0.004

49

NCH3

16.5 ± 0.85

2.65 ± 0.24

(94.69%) nd


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50

NCH3

2.47 ± 0.06

0.27 ± 0.01

0.008 ± 0.001

51

NCH3

9.32 ± 0.27

0.47 ± 0.01

0.023 ± 0.003

52

NCH3

1.90 ± 0.03

0.576 ± 0.01

>0.00015

53

NCH3

1.74 ± 0.04

0.384 ± 0.012

0.017 ± 0.002

54

NCH3

4.49 ± 0.19

0.167 ± 0.003

0.021 ± 0.004

55

NCH3

3.89 ± 0.24

2.31 ± 0.32

0.027 ± 0.001

NCH3

1.87 ± 0.16

0.101 ± 0.02

0.029 ± 0.001

56

a

In the presence of 10 μM ATP with 0.123 μM compound, the values are the mean ± SD from three

independent experiments. b nd: not determined. c n = 3. The pyrimidine in the fused heterocycle seems to be optimal to form aromatic-aromatic interaction with the gatekeeper PHE80 in CDK2. To further investigate the hydrophobic interaction of aromatic rings with the hydrophobic cavity, the phenyl ring of the fused heterocycle was then replaced by a 5-membered aromatic heterocycles. Modifications to the phenyl ring resulted in analogies 35-39 and 48-56, among which the thienopyrimidine analogues (35, 48) retained the same affinity with CDK2 and FLT3 when compared to (31, 47). Even, substituting 7 or/and 8 hydrogen of thieno[2,3-d]pyrimidine with methyl (39, 53, 54), ethyl (37, 56)


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or isopropyl(38, 55) also maintained strong inhibitory activities against CDK2 and FLT3. Besides, changing the position of the sulfur atom (51) did not affect kinase activities. In order to obtain additional SAR information, we replaced the thiazole ring with the furan (49) and pyrrole (36, 50) based on the bioisosterism. The pyrrolopyrimidine analogue 50 exhibited similar kinase inhibitory potency to that of 48 for both CDK2 and FLT3. But the furan substituted compound 49 showed a decreasing inhibitory efficiency against CDK2. Overall， introduction of pyrrolopyrimidine or thienopyrimidine is important for kinase inhibit activity, especially for FLT3. Although the 1-methylpiperazine or morpholine stretched to hydrophilic region conferred good potency for either CDK2 or FLT3 inhibitory activities, there had been no evidence to clarify which hydrophilic group would be optimal in consideration of potency and physical properties. Thus, a series of piperazine replacements was investigated using both the thieno[2,3-d]pyrimidine and the 7H-pyrrolo[2,3-d]pyrimidine as the hydrophobic segments. All these compounds are listed in Table 2. Table 2. Optimization of the hydrophilic group in the solvent accessible area

( % cell ( % Enzyme growth activity at inhibition at

Compounds

R1

B

0.123µM) IC50 (nM)a

1µM)b

LogP

CLogP

TPSA

2.65

2.80

105

IC50 (µM)

57

CDK2

FTL3

2.08 ±

0.34 ±

0.18

0.03

MV4-11

0.019 ± 0.004

S


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58

59

1.59 ±

5.36 ±

0.02

0.98

2.05 ±

1.05 ±

0.42

0.11

S

S

0.007 ± 0.001

3.95

3.97

93

0.013 ± 0.004

3.19

3.02

102

0.047 ± 0.006

3.14

3.31

98

0.053 ± 0.006

1.22

2.32

118

0.012 ± 0.004

2.51

3.48

105

3.03 ± 60

0.32 ±

S 0.11

0.03

61

4.49 ±

1.03 ±

0.72

0.05

NH

0.13 ± 62

NH

13.6 ± 0.03 1.12

a


independent experiments. b n = 3. It is noteworthy that modifications in the hydrophilic region can maintain the inhibitory activity of the compounds against CDK2 and FLT3. However, 7H-pyrrolo[2,3-d]pyrimidine derivatives (Table 2, 61-62) led to a decrease in cell-based activity compared to thieno[2,3-d]pyrimidine derivatives (Table 2, 57-58). These may be caused by the decrease of compounds’ lipophilicity which influences the permeability of compounds. Further studies have shown that introducing thieno[2,3-d]pyrimidine led to high clearance rates in liver microsomes. Introduction of diethylamine group (62, Table 2) maintained cell potency with acceptable lipophilicity, but showed poor CDK2 activity. Taken together, optimizing the hydrophilic region did not significantly improve the efficiency of the compounds. Hence, introducing of the fused heterocycle that contains pyrimidine/pyridine structure to pyrazole is suggested to be the most critical factor determining potency of pyrazole-carboxamide derivatives towards FLT3 and CDKs.


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Kinase Inhibition Profile of compound 48

Figure 3. (A) Distribution of inhibited kinases of compound 48 within the human kinome. The color code for inhibition is indicated. Residual enzymatic activity was determined in single dose duplicate at a compound concentration of 10 μM. The ATP concentration was 10 μM. Staurosporine served as a positive control. The experiment was performed by Reaction Biology Corp. using a P33-radiolabel assay. (B) Inhibitory activity of compound 48 against selected kinases using the P33-Radiolabeled assay. Among the series of compounds mentioned above, one promising compound 48 was tested against a panel of 339 kinases by Reaction Biology Corporation at a single concentration of 10 μM (Figure 3A). On the basis of kinase activity result, 28 kinases were selected for further assays (Figure 3B, Table S1). Compound 48 displayed highest inhibitory activity against CDK2/CyclinA1 (IC50: 9.47 nM) and FLT3 (IC50: 0.438 nM). It should be noted that 48 also has inhibitory activities against CDK4/cyclinD1 (IC50: 5.57 nM) and CDK6/cyclinD1 (IC50: 4.5 nM), followed by CDK9/cyclinK (IC50: 38.9 nM), FLT1/VEGFR1 (IC50: 19.8 nM), KDR/VEGFR2 (IC50: 35.1 nM), ALK (IC50: 94.1nM) and CDK1/cyclinE (IC50: 96 nM). Therefore, compound 48 is identified to be a multiple kinases inhibitor, inhibition of multiple pathways simultaneously by compound 48 is beneficial to improve its antitumor efficiency and overcome drug resistant. Inspired by the kinase profiling result, five representative CDK2 and FLT3 inhibitors 35, 48, 49, 50 and 51 were further evaluated for inhibitory activities against CDK4/cyclin D1, CDK6/cyclin D1 other than CDK2/cyclinA and FLT3 (Table 3). All selected compounds showed high inhibitory activities against these


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kinases with IC50 lower than 10 nM, suggesting that these compounds may be even more potent CDKs and FLT3 inhibitors. (ie. 50: CDK2, IC50: 2.47 nM; CDK4, IC50: 0.85 nM; CDK6, IC50: 1.96 nM; FLT3, IC50: 0.28 nM). Table 3. Inhibitory activities of compound 35, 48, 49, 50 and 51 toward CDKs and FLT3 kinases

Compounds IC50 (nM) Kinases

a

35

48

49

50

51

CDK2/cyclin A

3.36±0.26

5.85±0.58

16.5±0.76

2.47±0.21

9.32±0.45

CDK4/cyclin D1

2.40±0.27

3.33±0.24

5.67±0.30

0.85±0.28

3.10±0.34

CDK6/cyclin D1

3.88±0.17

4.24±0.23

5.09±0.44

1.96±0.08

4.11±0.29

FLT3

0.62±0.06

0.60±0.04

2.56±0.11

0.28±0.01

0.47±0.02


independent experiments.

In Vitro Cell Assays Table 4. In vitro growth inhibitory activities of selected compounds

Cell type GI50 (µM)a compounds

a

MGC803

RS4;11

AT-7519

0.21±0.01

0.20±0.04

35

0.21±0.02

48 50

MCF-7

HCT-116

NCI-H82

0.11±0.02

0.26±0.13

0.27±0.14

0.17±0.05

0.61±0.07

0.58±0.11

0.29±0.06

0.16±0.05

0.13±0.01

0.62±0.09

0.37±0.02

0.17±0.02

0.37±0.04

0.05±0.01

2.84±0.25

0.09±0.04

0.11±0.02

n = 3. All of target compounds (27-62) were evaluated against human leukemia cell line MV4-11 (FLT3ITD) using

the MTT assay after 72 h incubation (see Table 1 and Table 2). Compounds 31, 36-40, 47-48, 50-62 showed significant antiproliferative activities against MV4-11, with IC50 values ranging from 0.0015 to 0.035 μM. Almost all of these derivatives proved to be more antiproliferative than 26, which further certify the


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rationality of our design strategy. Compounds 35, 48 and 50 were selected to evaluate their activity against a panel of cell lines that represent different tumor types, including gastric, leukemia, breast cancer, colorectal cancer. The growth of all cell lines were inhibited by selected compounds and the 50% growth inhibition (GI50) values range from 51 to 620 nM (Table 4). With these findings, we submitted 35, 48, 50 to the Developmental Therapeutics Program at NCI (http://dtp.nci.nih.gov) to test their selectivity against the NCI60 cancer cell lines. In this screen, compounds 35, 48 and 50 exhibit potent inhibit activity on all 60 cancer cell lines (Figure S1, S2, S3 in Supporting Information), with GI50100 μmol/L (mean LC50>400 μmol/L). In comparison, the lethal concentration of flavopiridol (mean GI50: 0.074 μmol/L against NCI60 cell lines), one of a potent inhibitor of CDKs, reached submicromolar concentrations (LC50: 0.904 μmol/L) 42. Table 5. Pharmacokinetics of selected compounds in liver microsomes of multiple species

Compounds

HML

RLM

35

50

R2a

0.9793

0.3209

T1/2 (h)b

0.138

1.717

CLint(mL/min/kg)c

165.1

13.3

R2

0.8963

0.5008

T1/2 (h)

0.118

1.032

CLint(mL/min/kg)

352.4

40.3


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MLM

DLM

CLM

R2

0.9056

0.9465

T1/2 (h)

0.12

0.66

CLint(mL/min/kg)

765.1

138.6

R2

0.9899

0.9908

T1/2 (h)

0.025

0.257

CLint(mL/min/kg)

1327.7

129.3

R2

0.9929

0.9051

T1/2 (h)

0.182

0.855

CLint(mL/min/kg)

170.9

36.5

a

R2 is the correlation coefficient of the linear regression for the determination of kinetic constant

b

T1/2 is half life, c CLint is the intrinsic clearance

Pharmacokinetics of 35 and 50 in Liver Microsomes of Multiple Species The preliminary metabolic stability study of the compound 35 and 50 were performed by using human liver microsomes (HML), rat liver microsomes (RLM), mouse liver microsomes (MLM), dog liver microsomes (DLM) and monkey liver microsomes(CLM) (Table 5). The terminal t1/2 was moderate in human liver microsomes, rat liver microsomes, mouse liver microsomes and monkey liver microsomes (ranged between 0.025 and 1.7 h). Compound 50 was metabolically more stable (t1/2 ranged between 0.25 and 1.7 h) than 35 (t1/2 shorter than 0.18 h). Given the drastic metabolic instability in the liver microsomes experiment, compound 35 was not considered for further study. Some other studies have demonstrated that compounds with pyrimidothiophene ring are metabolically unstable43, 44. Therefore, this series of compounds had not been considered for further investigation.

Molecular Modeling of Compound 50 with CDK2 and FLT3 Compound 50 showed optimal FLT3/CDKs inhibitory activities, and importantly, it is metabolically stable. Hence, the binding mode of compound 50 within CDK2 and FLT3 were elucidated using a docking model


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(Figure 4). As is shown in Figure 4A, compound 50 binds to the ATP-binding site of CDK2 in an orientation similar to that of AT-7519 (Figure 4B). The pyrazole-3-carboxamide skeleton of compound 50 forms three conserved hydrogen bonds with the hinge region of CDK2, one between the N1 of pyrazole and GLU81, and the other two between the NH of formamide, N2 of pyrazole and LEU83 (Figure 4A). The fused heterocycle substituent has complementary packing and hydrophobic interactions with LYS33, VAL18, ASP145, ALA144 and the hydrophobic residue of the ILE10 side chain. The pyrrole NH forms additional polar interactions with the side chains of ASP145. A face-to-face interaction with the adjacent aromatic planes is observed between the pyrimidine and the kinase gatekeeper residue, PHE80. Phenyl ring occupied the small hydrophobic cavity between hinge region and the hydrophilic group. The N-methylpiperazine group sits in a solvent accessible area which is mainly hydrophobic but lined with several polar residues at the edge (LYS89, ASP86, LEU298, GLN85).

Figure 4. Binding mode analysis of compound 50 to CDK2 kinase and FLT3 kinase. (A) Compound 50 docked into CDK2 kinase (PDB code 2VU3). (B) Overlapping of compound 50 (green) and AT7519 (gray) complexed with CDK2. (C) Compound 50 docked into homology model of FLT318. (D) Chemic structure of compound 50. In Figure 4B, we found that compound 50 binds to FLT3 in a similar fashion to its binding mode to CDK2, with the core pyrazole-3-carboxamide skeleton forming two conserved hydrogen bonds in the hinge region of FLT3. One is between the N1 of pyrazole and GLU692, the other one is between the N2 of pyrazole and


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CYS694. The 7H-pyrrolo[2,3-d]pyrimidine substituent of 50 occupies the hydrophobic pocket and the Nmethylpiperazine group extends into solvent accessible area (Figure 4C). In general, the docking results further confirm the rationality of our design strategy.

In Vivo Effects of Compound 50 In s.c. MV4-11 Tumor Xenografts On the basis of a desirable set of in vitro properties, compound 50 was finally selected for further in vivo evaluation. We proceeded to test the antitumor efficacy of compound 50 in MV4-11 cell inoculated xenograft mouse.

Figure 5. The antitumor efficacy of 50 in MV4-11 xenograft model. Female nu/nu mice bearing established MV4-11 tumor xenografts were treated by intravenous injection (iv, QD) with 50 at 15, 30, or 40 (mg/kg)/d or Cytarabine at 50 (mg/kg)/d or vehicle. (A) Body weight and (B) tumor size measurements from MV4-11 xenograft mice after 50 or Cytarabine administration. Initial body weight and tumor size was set as 100%. (C) Representative photographs of tumors in each group after 15, 30, or 40 (mg/kg)/d of 50 or 50 (mg/kg)/d of Cytarabine or vehicle treatment. (D) Comparison of the final tumor weight in each group after 22-day treatment period. Numbers in columns indicate the mean tumor weight in each group. ns, p > 0.05, (∗) p < 0.05, (∗∗) p < 0.01. In the MV4-11 model, when the tumor grew to a volume of 200 mm3, the mice were grouped and treated intravenous once daily with 15, 30, or 40 mg/kg/d compound 50 for 21 days. Another two groups were recruited, one group for negative control (only vehicle) and another group treated intravenous once daily with


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50 mg/kg/d Cytarabine as a positive control for 21 days. The group injected with 50 mg/kg/d Cytarabine only reduced the tumor growth rate, and the tumor growth inhibition was 61.94% on the 21th day. While injected with 50 by 15 mg/kg/d induced tumor regression and the PTR (percent tumor regression) rate was -6.11% on the 21th day (Figure 5). Meanwhile, groups injected with 50 by 30 and 40 mg/kg/d significantly induce tumor regression and the PTR on the 21th day were 78.95% and 81.33% (Figure5B, 5C and 5D), respectively. Furthermore, it was well tolerated with no overt signs of toxicity or changes in body weight (Figure 5A). Experiments in rats demonstrated that 50 did have significant anti-tumor activity in vivo compared with Cytarabine which is used as a first-line chemotherapy drug against AML.

Toxicity Studies Inspired by the excellent antitumor activity of compound 50, we assessed its cytotoxicity on normal lymphocytes cells. Compound 50 shows little cytotoxicity to normal lymphocytes cells, which are comparable with AC220 (IC50:0.492 μM) or AT7519 (IC50:0.554 μM). To further evaluate the safety of 50 in vivo, we conducted additional LD50 tests for AT-7519 and 50, and groups of ICR mice (half male and half female) were administered with compound 50 or AT7519 for a single dose by intravenous injection. The survival of the mice was monitored and recorded over 12 days (Table S2). The LD50 values of AT7519 are 32 mg/kg. However, the LD50 values of 50 were 185.67 mg/kg as is shown in Table 6. Compound 50 exhibited significantly reduced toxicity compared with AT7519. Table 6. Median Lethal Dose (LD50) Values of 50 and AT7519

Compounds

LD50(mg/kg)

95% confidence interval (μ μmol/kg)

AT7519

32.00

28.20-36.32

50

185.67

158.57-208.12

Cellular Mode of Action To characterize the mode of cell death induced by compound 50, biparametric flow cytometric analysis with annexin-V/propidium iodide (PI) was performed. Inducing apoptosis is considered as one of the major


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strategies for antitumor drug development14. Flow cytometry was used to investigate whether the antiproliferative effect of compound 50 was caused by activation of cellular apoptosis in MV4-11 cells. Quantitative analysis of early apoptotic cells and advanced apoptotic/necrotic cells was determined via an Annexin V-FITC/ PI assay. MV4-11 cells were stimulated with either 0.2-2 μM of compound 50 for 24 h, and DMSO was used as a negative control. Sorafenib was used as positive control. As shown in Figure 6, apoptosis induced by compound 50 was much greater than the control, and MV4-11 cells underwent both early apoptosis (Annexin V+/PI-, lower right) and advanced apoptosis/necrosis (Annexin V+/PI+, upper right). The results showed that compound 50 could effectively induce cell apoptosis starting from 0.2 μM with an enhanced effect at higher concentrations. The apoptosis ratios of compound 50 measured at different concentrations were 24.35% (0.2 μM), 41.98% (0.5 μM), 46.34% (1 μM), 78.30% (2 μM) and the apoptotic effect increases in a dose-dependent manner. From these results, we can conclude that 50 caused effective apoptotic effect in MV4-11 cell line, which shows equivalent efficiency as Sorafenib (75.32% at 2 μM).

Figure 6. MV4-11 cells were treated with compound 50 for 24 h and analysed by Annexin V/PI staining. The percentage of cells undergoing apoptosis was defined as the sum of early apoptosis, advanced apoptosis and necrotic cells. UR (upper right): advanced apoptotic cells and necrotic cells labeled with PI and Annexin, LL (lower left): fully viable cells, LR (lower right): early apoptotic cells labeled with Annexin V but not with PI. (A) DMSO control; (B) 0.2 µM compound 50; (C) 0.5 µM compound 50; (D) 1 µM compound 50; (E) 2 µM compound 50; (F) 2 µM Sorafenib; (G) Percentage of apoptosis cells.


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Figure 7. Compound 50 treatment of MV4-11 and HCT116 cells lead to cancer cell growth inhibition, cell cycle arrest. (A) Effect of compound 50 on MV4-11 cell cycle kinetics. Compound 50 at the concentrations shown was added to MV4-11 cells for 24 h prior to fixation, staining with propidium iodide (PI), and flow cytometric analysis. (B) Effect of compound 50 on HCT-116 cell cycle. Furthermore, compound 50 was measured in MV4-11 and HCT-116 cell lines to consider if the antiproliferative activity is consistent with FLT3 and CDK2 signaling inhibition. The antiproliferative activity of compound 50 was verified by flow cytometry analysis of MV4-11 and the colon HCT-116 cell line, through double staining with propidium iodide and 5-bromo-20-deoxyuridine (BrdU). As is shown in Figure 7, compound 50 arrested cell cycle progression of this cell line into the G0/G1 phase in a dose-dependent manner, which is consisted with Sorafenib (Figure 7A) in MV4-11 cell line. Treatment with compound 50 mainly caused G2/M phase arrest in the HCT-116 cell lines (Figure 7B), which would be related to the potent inhibition of CDKs. Overall, compound 50 arrested the cell cycle of two human cancer cell lines as efficiency as AT-7519 and Sorafenib, and both cell lines displayed similar patterns as the positive control against cell-cycle blockade. We then treated MV4-11 cell line with increasing doses of compound 50 for 24 h and immune blotted the lysates with antibodies against relative proteins involved in FLT3 mediated signaling pathway (Figure 8A). Treatment of MV4-11 cells with compound 50 for 24h significantly decrease the phosphorylation of FLT3 and its downstream mediators STAT5, ERK and AKT’s phosphorylation than Sorafenib. As shown in Figure 8B, compound 50 inhibited RbSer807/811 phosphorylation in a dose-dependent manner and its efficiency was equal


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to AT7519 at 1μM. These findings reveal that compound 50 acts as a highly efficient FLT3 and CDKs inhibitor.

Figure 8. . Western blot analysis. (A) MV4-11 cells were treated with compound 50 or Sorafenib for 24 h and phosphorylation of FLT3, STAT5, ERK and AKT protein were analyzed by immune blotting. (B) MV4-11 cells were treated for 24 h with compound 50 or AT7519 and phosphorylation of Rb protein was analyzed by immune blotting.

CONCLUSIONS Starting from compound 26, we have discovered a series of N-(4-methylphenyl)-1-H-pyrazole-3carboxamide derivatives as potent FLT3/CDKs inhibitors. The structure-activity relationship analysis demonstrated that substitution with fused heterocycle that contains pyrimidine/pyridine structure in position 4 of the 1-H-pyrazole-3-carboxamide moiety was considered to be key strategic group to improve CDK2 and FLT3 inhibitory activity and antiproliferative activity. Compound 50 showed nanomolar range CDKs and FLT3 inhibition and significant cytotoxic effect on NCI60 cancer cell lines (IC50 < 1μM). In addition, 50 exhibited higher metabolic stability in liver microsomal metabolism essay. In vivo study of 50 in MV4-11 xenograft model showed higher tumor efficacy at a dosage of 15.0 (mg/kg)/d than Cytarabine (50.0 (mg/kg)/d), and furthermore, it induces tumor regression when increasing the doses to 30.0 (mg/kg)/d or 40.0 (mg/kg)/d. Currently compound 50 is in phase I clinical trials for cancer (In the United States and China) and it may represent a promising drug with therapeutic potential for the AML.

EXPERMENTAL SECTION


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General Procedures Unless otherwise specified, reagents were purchased from commercial suppliers and used without further purification. Melting points were determined by X-4 digital display micro-melting point apparatus (Beijing Tech Instrument Co., Ltd.); NMR spectra were recorded on Bruker AVANCE AV-600 spectrometer (800 MHz for 1H, 150 MHz for 13C) or Bruker AVANCE AV-300 spectrometer (300 MHz for 1H, 75 MHz for 13C); Mass spectra were obtained on the Agilent 1100 LC / MSD mass spectrometer (Agilent, USA) and Q-tofmicro MS (micromass company). All reactions were monitored by TLC (Merck Kieselgel GF254) and spots were visualized with UV light or iodine. The purity of biologically evaluated compounds was >95% as determined by HPLC.

General Procedure A for Synthesis of Compound 2-7 4-Nitrobenzyl bromide (46.3 mmol) was dissolved in dichloromethane (100 mL). The solution was added to the mixture of relative amine (47.0 mmol) and triethylamine (70.3 mmol) in dichloromethane (20 ml). The reaction mixture was stirred at r.t. for 24 h and was extracted with dichloromethane (100 ml × 3). After removal of the solvent, the residue was crystallized from ethanol, giving yellow powder. Compounds 2-7 were used for further reaction without purification. To a suspension of compounds 2-7 (36.2 mmol) in 95% ethanol (100 ml), 85% NH2NH2 .H2O (362 mmol), 95% ethanol (100 ml) and iron (III) oxide hydroxide (FeO(OH)/C, 2.0 g) were added and heated to reflux40, 45, 46

. When TLC analysis showed complete conversion of the starting material, the reaction mixture was filtrated

through Cellit and the filtrate was concentrated in vacuum. The crude product was purified by silica gel column chromatography (DCM/MeOH) to yield the title compound as white solid. 4-(morpholinomethyl)aniline (8). Compound 8 was prepared according to general procedure A on 36.2 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound (6.0 g, 31 mmol, yield 86%). [M+H]+: 193. 1H-NMR (DMSO-d6): 2.32 (s, 4 H), 3.24 (s, 4 H), 3.51 (s, 2 H), 4.92 (s, 2 H), 6.53 (d, J = 8.4 Hz, 2 H), 6.90 (d, 2 H, J = 8.4 Hz). 4-((4-methylpiperazin-1-yl)methyl)aniline (9). Compound 9 was prepared according to general procedure


24

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A on 36.2 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 7.3 g, 34.2 mmol Yield 94%). [M+H]+: 206. 1H-NMR (300 MHz, DMSO-d6): 2.11 (s, 3 H), 2.37 (br, 8 H), 3.50 (s, 2 H), 4.01 (s, 2 H), 7.54 (d, 2 H, J = 8.7 Hz), 8.23 (d, 2 H, J = 8.7 Hz). Tert-butyl-4-(4-aminobenzyl)piperazine-1-carboxylate (10). Compound 10 was prepared according to general procedure A on 36.2 mmol scale. Purification by column chromatography (10% MeOH/DCM) yield the title compound (8.2 g, 28.1 mmol Yield 78%). [M+H]+: 292. 1H-NMR (300 MHz, DMSO-d6): 1.38 (s, 9 H), 2.242.26 (m, 4 H), 3.26 (s, 4 H), 3.41 (s, 2 H), 4.92 (s, 2 H), 6.48-6.51 (d, 2 H, J = 8.7 Hz), 6.89-6.92 (d, 2 H, J = 8.7 Hz). 4-((4-methyl-1,4-diazepan-1-yl)methyl)aniline (11). Compound 11 was prepared according to general procedure A on 36.2 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound (4.9 g, 23.1 mmol Yield 65%). [M+H]+: 220. 1H-NMR (DMSO-d6): 1.64−1.69 (m, 2 H), 1.81−1.96 (m, 4 H), 2.09−2.15 (m, 2 H), 4.59 (d, 2 H, J = 6.72 Hz), 4.77 (m, 1 H, J = 7.05Hz), 7.28(d, 2 H, J = 8.22 Hz), 7.49 (d, 2 H, J = 8.22 Hz), 8.26 (s, 1 H),8.83 (t, 1 H, J = 6.72 Hz). 4-((diethylamino)methyl)aniline (12). Compound 12 was prepared according to general procedure A on 36.2 mmol scale. Purification by column chromatography (3 % MeOH/DCM) yield the title compound (2.9 g, 17.1 mmol Yield 46%). [M+H]+: 179. 1H-NMR (DMSO-d6): 1.04−1.09 (t, 6 H), 2.61-2.69 (q, 4 H, J = 6.72 Hz), 3.65 (s, 2 H), 5.10 (br, 2 H), 6.52-6.55 (d, 2 H, J = 8.31 Hz), 7.03-7.07 (d, 2 H, J = 8.22 Hz). 4-((cyclopropylamino)methyl)aniline (13). Compound 13 was prepared according to general procedure A on 36.2 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound (3.1 g, 19.8 mmol Yield 54%). [M+H]+: 163. 1H-NMR (DMSO-d6): 0.05-0.14 (m, 4 H), 1.76−1.83 (m, 2 H), 3.00 (s, 1 H), 4.64 (d, 2 H, J = 6.72 Hz), 6.26-6.29 (d, J = 8.22 Hz, 2 H), 6.71-6.75 (d, 2 H, J = 8.22 Hz).

General Procedure B for Synthesis of Compound 20-25 The mixture of compound 8-13 (1 eq., 18.5 mmol), 4-Nitro-1H-pyrazole-3-acid (1.1equiv, 20.4mmol), EDC (1.2equiv, 22.2mmol), HOBT (1.2equiv, 22.2mmol) in DMF (50 ml) was stirred for 24 hours. The ice water (100 ml) added to the reaction mixture. A large amount of yellow solid precipitation (compound 3a-f) was


25


Page 26 of 54

acquired. 14-19 were used without further purification. Compounds 20-25 were reduced by the same process of compound 8-13, then the resulting compound 20-25 were purified by column chromatography on silica gel, eluted with the appropriate solvent. 4-amino-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3-carboxamide (20). Compound 20 was prepared according to general procedure B on 18.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound (3.6 g, 12.0 mmol Yield 65%). [M+H]+: 302. 1H-NMR (300 MHz, DMSOd6) δ: 2.51 (s, 4 H), 3.42 (s, 2 H), 3.61 (s, 4 H), 4.71 (s, 2 H), 7.21-7.26 (m, 3 H), 7.72-7.58 (d, 2 H, J = 8.4 Hz), 9.7 (s, 1 H), 12.7 (s, 1 H); 4-amino-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (21). Compound 21 was prepared according to general procedure B on 18.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound (4.1 g, 15.4 mmol Yield 76%). [M+H]+: 315. 1H-NMR (300 MHz, DMSO-d6) δ: 2.13 (s, 3 H), 2.38 (s, 8 H), 3.43 (s, 2 H), 4.71 (s, 2 H), 7.11-7.24 (m, 3 H), 7.71-7.81 (d, 2 H, J = 8.4 Hz), 9.72 (s, 1 H), 12.71 (s, 1 H). Tert-butyl-4-(4-(4-amino-1H-pyrazole-3-carboxamido)benzyl)piperazine-1-carboxylate (22). Compound 22 was prepared according to general procedure B on 18.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound (1.8 g, 4 mmol Yield 24%). [M+H]+: 401. 1H-NMR (300 MHz, DMSO-d6) δ: 1.39 (s, 9 H), 2.31 (s, 4 H), 3.43 (s, 2 H), 4.68 (s, 2 H), 7.17-7.20(d, 2 H, J = 7.71 Hz), 7.22 (s, 1 H), 7.72-7.74 (d, 2 H, J = 7.95 Hz), 9.70 (s, 1 H), 12.75 (s, 1 H). 4-amino-N-(4-((4-methyl-1,4-diazepan-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (23). Compound 23 was prepared according to general procedure B on 18.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound (1.3 g, 3.9 mmol Yield 21%). [M+H]+: 329.3. 1HNMR (DMSO-d6): 1.66−1.73 (m, 2 H), 2.24 (s, 2 H), 2.51−2.64 (m, 8 H), 3.38 (s, 2 H), 4.73 (br, 2 H), 7.14 (s, 1 H), 7.21-7.24 (d, 2 H, J = 8.37 Hz), 7.68-7.71 (d, 2 H, J = 8.40 Hz), 9.94 (s, 1H). 4-amino-N-(4-((diethylamino)methyl)phenyl)-1H-pyrazole-3-carboxamide

(24).

Compound

24

was

prepared according to general procedure B on 18.5 mmol scale. Purification by column chromatography (5%


26

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MeOH/DCM) yield the title compound (2.0 g, 6.8 mmol Yield 37%). [M+H]+: 288. 1H-NMR (300 MHz, DMSOd6) δ: 1.06 (s, 6 H), 2.52 (s, 2 H), 2.61-2.69 (m, 4 H), 4.70 (d, 2 H, J = 6.72 Hz), 7.18 (s, 1 H, J = 7.05 Hz), 7.32 (d, 2 H, J = 8.22 Hz), 7.76 (d , 2 H, J = 8.22 Hz), 9.76 (s, 1 H ), 12.79 (s, 1 H). 4-amino-N-(4-((cyclopropylamino)methyl)phenyl)-1H-pyrazole-3-carboxamide (25). Compound 25 was prepared according to general procedure B on 18.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound (0.9 g, 3.3 mmol Yield 18%). [M+H]+: 272. 1H-NMR (300 MHz, DMSOd6) δ: 0.34-0.36 (m, 4 H), 2.05 (s, 2 H), 3.69 (s, 2 H), 4.70 (s, 2 H), 7.17-7.26 (m, 3 H), 7.69-7.70 (d, 2 H, J = 4.5 Hz), 9.61 (s, 1 H), 12.74 (s, 1 H).

General Procedure C for Synthesis of Compound 27-62 Compound 20-25 (1 equiv.) and corresponding chorides (1.2 equiv.) in AcOH/H2O:1/1 (20 ml) was heated to 50 °C. When TLC analysis showed complete conversion of the starting material, sodium hydroxide (2 equiv.) was added to the mixture. Then, the reaction mixture was extracted with ethyl acetate. After removal of the solvent, the residue was purified by column chromatography and the corresponding compound was obtained. N-(4-(morpholinomethyl)phenyl)-4-(quinolin-4-ylamino)-1H-pyrazole-3-carboxamide (27). Compound 27 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound (77 mg, 0.18 mmol Yield 36%). yellow solid; m.p 175-279 °C. HPLC analysis: retention time =5.168 min; peak area, 98.43%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.35 (m, 4 H,), 3.42 (s, 2 H), 3.57 (m, 4 H) , 7.00 (d, 1 H, J = 4.8 Hz), 7.25-7.28 (d, J = 7.9 Hz, 2 H), 7.62 (t, J = 8.0Hz, 1 H), 7.69-7.72 (d, 2 H, J = 7.9 Hz), 7.76 (d, 2 H, J = 8.0 Hz), 7.90 (d, 1 H, J = 8.1 Hz), 8.13 (d, 1 H, J = 8.0 Hz), 8.23 (s ,1 H), 8.6 (d, 1 H, J = 4.8 Hz), 9.9 (br, 1 H); 13C-NMR (75 MHz, DMSO-d6) δ: 25.08, 53.10, 62.03, 66.18, 100.71; 118.90, 12125.24, 125.87, 129.12, 129.31, 132.05, 132.95, 137.33, 145.56, 148.21, 150.78, 161.97, 175.20; HRMS-EI m/z [M+H] + calcd for C24H25N6O2: 429.2039, found: 429.2049. 4-(isoquinolin-1-ylamino)-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3-carboxamide (28). Compound 28 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 116 mg, 0.27 mmol Yield 54%). white solid; m.p 285-287 °C. HPLC


27


Page 28 of 54

analysis: retention time =4.109 min; peak area,100.00%. 1H NMR (300 MHz, DMSO-d6) δ: 2.38 (s, 4 H), 3.46 (s, 2 H), 3.59 (s, 4 H) , 7.19 (d, 1 H, J = 5.8 Hz) , 7.31 (d, 2 H, J = 8.1 Hz) , 7.75 (dd, 2 H, J = 9.1 Hz) , 7.85 (d, 3 H, J = 8.1 Hz) , 8.03 (d, 1 H, J = 6.7 Hz) , 8.13 (d, 1 H, J = 5.7 Hz) , 8.66 (s, 1 H) , 10.30 (d, 2 H, J = 6.5 Hz) , 13.42 (s, 1 H);

13

C-NMR (150 MHz, DMSO-d6) δ: 53.09, 62.01, 66.15, 112.04, 117.67, 119.39, 120.40, 120.87, 121.18,

125.84, 127.27, 127.36, 129.47, 130.38, 132.01, 136.68, 137.34, 141.24, 150.48, 163.39; HRMS-EI m/z [M+H] +

calcd for C24H24N6O2: 429.2039, found: 429.2049.

4-((6-bromoquinolin-4-yl)amino)-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3-carboxamide (29). Compound 29 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 65 mg, 0.13 mmol Yield 26%). yellow solid; m.p 231-235 °C. HPLC analysis: retention time =4.360 min; peak area, 99.49%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.35 (s, 4 H), 3.43 (s, 2 H), 3.57 (s, 4 H), 6.83-6.86 (d, 1 H, J=5.13 Hz), 7.24-7.27 (d, 2 H, J= 8.25 Hz), 7.73-7.76 (d, 2 H, J =8.16 Hz), 7.84 (s, 1 H), 8. 28 (s, 1 H), 8.47 (s, 1 H), 8.52 (s, 1 H), 9.38 (s, 1 H), 10.23 (s, 1 H), 13.65 (s, 1 H); 13C-NMR (150 MHz, DMSO-d6) δ: 53.16, 62.05, 66.22, 101.52, 118.03, 120.27, 120.48, 122.84, 123.51, 123.89, 129.34, 131.51, 132.44, 132.98, 136.30, 137.36, 146.50, 146.91, 151.30, 161.65; HRMS-EI m/z [M+H] + calcd for C24H24BrN6O2: 507.1144, found: 507.1143. 4-((7-Methoxyquinolin-4-yl)amino)-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3-carboxamide (30). Compound 30 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 49 mg, 0.11 mmol Yield 21%). yellow solid; m.p 185-187 °C. HPLC analysis: retention time = 3.516 min; peak area, 100.00%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.34 (s, 4 H), 3.42 (s, 2 H), 3.57 (s, 4 H), 3.91 (s, 3 H), 6.88 (d, 1 H, J = 4.2 Hz), 7.28 (s, 4 H), 7.75 (d, 1 H, J =7.5 Hz), 8.02 (d, 1 H, J =8.7 Hz), 8.35 (s, 1 H), 8.49 (d, 1 H, J = 4.2 Hz), 9.64 (s, 1 H), 10.31 (s, 1 H), 13.6 (s, 1 H); 13CNMR (75 MHz, DMSO-d6) δ: 21.20, 53.01, 55.27, 61.95, 66.10, 99.63, 107.64, 113.18, 117.31, 120.20, 120.51, 121.66, 125.31, 129.17, 133.08, 133.79, 137.00, 145.94, 149.60, 150.74, 160.00, 162.04; HRMS-EI m/z [M+H] + calcd for C25H27N6O3: 459.2145, found: 459.2134. N-(4-(Morpholinomethyl)phenyl)-4-(quinazolin-4-ylamino)-1H-pyrazole-3-carboxamide (31). Compound 31


28

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was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 120 mg, 0.28 mmol Yield 56%). yellow solid; m.p 215-217 °C. HPLC analysis: retention time = 4.234 min; peak area, 99.18%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.33 (s, 4 H), 3.17 (s, 2 H), 3.41 (s, 2H), 3.56 (s, 4 H), 7.27-7.29 (d, 2 H, J = 7.86 Hz), 7.71-7.73 (t, 1 H), 7.79-7.88 (m, 4 H), 7.98-8.00 (1 H, d, J = 7.98 Hz), 8.64 (s, 1 H), 8.73 (s, 1 H), 10.45 (s, 1 H), 10.63 (s, 1 H); 13C-NMR (150 MHz, DMSO-d6) δ: 53.23, 62.15, 66.30, 114.65, 120.95, 121.04, 123.94, 127.27, 128.25, 129.29, 132.87, 133.29, 133.44, 137.04, 149.21, 154.91, 155.29, 163.00; HRMS-EI m/z [M+H] + calcd for C23H24N7O2: 430.1991, found: 430.2006. 4-((2-chloroquinazolin-4-yl)amino)-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3-carboxamide (32). Compound 32 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 54 mg, 0.12 mmol Yield 23%). yellow solid; m.p 235-237 °C. HPLC analysis: retention time = 3.758 min; peak area, 99.00%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.36 (s, 4 H), 3.45(s, 2 H), 3.60 (s, 4 H), 7.28-7.31(d, 2 H, J = 8.28 Hz), 7.72-7.81(m, 4 H), 7.90-7.95 (s, 1 H, J = 7.35 Hz), 8.0-8.1(d, 1 H, J = 8.01 Hz), 8.47 (s, 1 H), 10.42 (s, 1 H), 10.87(s, 1 H), 13.62 (s, 1 H); 13C-NMR (75 MHz, DMSO-d6) δ: 162.86, 156.82, 156.23, 150.36, 136.82, 134.21, 133.42, 129.13, 127.45, 127.18, 122.83, 121.58, 120.87, 113.19, 66.17, 62.00, 53.10; HRMS-EI m/z [M+H] + calcd for C23H23ClN7O2: 464.1602, found: 464.1602. N-(4-(morpholinomethyl)phenyl)-4-((2-(trifluoromethyl)quinolin-4-yl)amino)-1H-pyrazole-3-carboxamide (33). Compound 33 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound (46 mg, 0.09 mmol Yield 19%). yellow solid; m.p 215-218 °C. HPLC analysis: retention time = 4.386 min; peak area, 100.00%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.45 (s, 4 H), 3.42 (s, 2 H), 3.63 (s, 4 H), 7.18-7.20 (d, 1 H, J = 5.73 Hz), 7.34-7.36 (d, 2 H, J = 6.63 Hz), 7.73-7.76 (m, 2 H), 8.01-8.04 (d, 1 H, J = 6.57 Hz), 8.12-8.14 (d, 1 H, J = 5.73 Hz), 8.66 (s, 1 H), 10.28 (s, 1 H), 10.33 (s, 1 H), 13.48 (s, 1 H); 13C-NMR (75 MHz, DMSO-d6) δ: 51.99, 52.82, 65.86, 111.89, 117.55, 119.26, 120.71, 121.05, 125.70, 127.10, 127.20, 129.23, 129.49, 130.20, 136.56, 137.33, 141.09, 150.37,163.26; HRMS-EI m/z [M+H] + calcd for C25H24F3N6O2: 497.1913, found: 497.1921.


29


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4-((6-methoxyquinazolin-4-yl)amino)-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3-carboxamide (34). Compound 34 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 85 mg, 0.19 mmol Yield 37%). yellow solid; m.p 251-252 °C. HPLC analysis: retention time = 3.255 min; peak area, 100.00%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.55 (s, 4 H), 3.66 (s, 6 H), 3.97 (s, 3 H), 7.33-7.38 (m, 3 H ), 7.55-7.57 (d, 2 H, J = 7.53 Hz), 7.78-7.88 (m, 3 H), 8.60 (s, 1 H ), 8.63 (s, 1 H ), 10.38 (s, 1 H), 10.42 (s, 1 H), 13.62 (s, 1 H); 13C-NMR (75 MHz, DMSO-d6) δ: 52.44, 55.59, 61.20, 65.39, 100.92, 115.07, 120.61, 121.11, 123.41, 123.76, 129.76, 133.08, 137.60, 144.36, 152.63, 154.60, 157.41, 162.77; HRMS-EI m/z [M+H]+ calcd for C24H26N7O3: 460.2097, found: 460.2096. N-(4-(morpholinomethyl)phenyl)-4-(thieno[2,3-d]pyrimidin-4-ylamino)-1H-pyrazole-3-carboxamide (35). Compound 35 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 92 mg, 0.22 mmol Yield 44%). white solid; m.p 262-265 °C. HPLC analysis: retention time = 6.105 min; peak area, 98.44%. 1H-NMR (300 MHz, DMSO-d6): δ 2.35 (s, 4 H), 3.43 (s, 2 H), 3.58 (s, 4 H), 7.27-7.30 (d, 2 H, J = 8.28 Hz), 7.48-7.50 (d, 1 H, J = 5.97 Hz), 7.77-7.82 (m, 3 H), 8.55 (s, 1 H), 8.62 (s, 1 H), 9.99 (s, 1 H), 10.29 (s, 1 H), 13.51 (s, 1 H); 13C-NMR (75 MHz, DMSO-d6) δ: 53.10, 62.47, 66.18, 116.49, 117.53, 120.62, 121.10, 123.44, 124.96, 129.10, 133.21, 137.00, 152.96, 153.54, 162.45, 165.97; HRMS-EI m/z [M+H] + calcd for C21H22N7O2S: 436.1556, found: .436.1560. 4-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3-carboxamide (36). Compound 36 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 138 mg, 0.33 mmol Yield 66%).White solid; m.p 213-214 °C. HPLC analysis: retention time = 6.541 min; peak area, 95.47%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.38 (s, 4 H), 3.35 (s, 2 H), 3.60 (s, 4 H), 6.50 (s, 1 H), 7.31 (s, 3 H), 7.79-7.82 (d, 2 H, J = 6.75 Hz), 8.40 (s, 1 H ), 8.58 (s, 1 H), 9.53 (s, 1 H), 10.25 (s, 1 H), 11.89 (s, 1 H), 13.45 (s, 1 H); 13C-NMR (75 MHz, DMSO-d6) δ: 53.10, 61.94, 66.07, 96.69, 103.07, 120.05, 120.58, 122.98, 124.58, 129.20, 132.26, 137.20, 150.56, 151.16, 151.65, 162.78; HRMS-EI m/z [M+H] + calcd for C21H23N8O2: 419.1944, found: . 419.1953. 4-((6-ethylthieno[2,3-d]pyrimidin-4-yl)amino)-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3-carboxamide


30

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(37). Compound 37 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 71 mg, 0.15 mmol Yield 31%). White solid; m.p 220-222 °C. HPLC analysis: retention time = 5.701 min; peak area, 97.42%. 1H-NMR (300 MHz, DMSO-d6) δ: 1.30-1.53 (t, 3 H, J = 7.47 Hz), 2.36 (s, 4 H), 2.95-3.03 (q, 2 H, J = 7.38 Hz), 3.44 (s, 2 H), 3.58 (s, 4 H), 7.17 (s, 1 H), 7.26-7.30 (d, 2 H, J = 8.28 Hz), 7.77-7.80 (d, 2 H, J = 8.10 Hz), 8.55 (s, 1 H), 9.81 (s, 1 H), 10.28 (s, 1 H), 13.49 (s, 1 H);

13

C-NMR (150 MHz, DMSO-d6) δ: 15.99, 23.05, 53.53, 62.44, 66.59, 113.73, 117.30, 121.25,

121.38, 124.09, 129.71, 131.04, 133.41, 137.56, 146.11, 152.46, 153.43, 163.09, 165.55; HRMS-EI m/z [M+H] +

calcd for C23H26N7O2S: 464.1869, found: 464.1871.

4-((6-isopropylthieno[2,3-d]pyrimidin-4-yl)amino)-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3carboxamide (38). Compound 38 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 93 mg, 0.20 mmol Yield 39%).white solid; m.p 222-224 °C. HPLC analysis: retention time = 4.433 min; peak area, 97.47%.

1

H-NMR

(300 MHz, DMSO-d6) δ: 1.36-1.38 (d, 2 H, J = 6.78 Hz), 2.35 (s, 1 H), 3.35-3.40 (s, 1 H), 3.44 (s, 2 H), 3.58 (s, 4 H), 7.18 (s, 1 H), 7.26-7.29 (d, 2 H, J = 7.35 Hz),7.76-7.79 (d, 2 H, J = 8.10 Hz), 8.54 (s, 1 H), 9.79 (s, 1 H), 10.28 (s, 1 H), 13.51 (s, 1 H);

13

C-NMR (75 MHz, DMSO-d6) δ: 13.07, 13.93, 53.09, 62.00, 66.17, 72.31, 116.95,

120.60, 123.81, 129.13, 129.91, 132.39, 133.16, 136.99, 152.32, 152.42, 164.25; HRMS-EI m/z [M+H] + calcd for C24H28N7O2S: 478.2025, found: 478.2032. 4-((5-methylthieno[2,3-d]pyrimidin-4-yl)amino)-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3carboxamide (39). Compound 39 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 92 mg, 0.21 mmol Yield 41%).white solid; (41% Yield); m.p 205-207 °C. HPLC analysis: retention time = 3.568 min; peak area, 96.37%. 1

H-NMR (300 MHz, DMSO-d6) δ: 2.35 (s, 4 H), 2.81 (s, 3 H), 3.43 (s, 2 H), 3.58 (s, 4 H) , 7.26-7.29 (d, 2 H, J =

8.37 Hz), 7.34 (s, 1 H), 7.76-7.79 (d, 2 H, J = 8.34 Hz), 8.58 (s, 1 H), 8.67 (s, 1 H), 10.14 (s, 1 H), 10.26 (s, 1 H),13.48 (s, 1 H); 13C-NMR (150 MHz, DMSO-d6) δ: 17.75, 53.57, 62.47, 66.64, 116.53, 119.90, 120.73, 121.18, 121.28, 124.22, 129.46, 129.66, 132.94, 133.65, 137.49, 153.71, 153.84, 163.12, 167.64; HRMS-EI m/z [M+H]


31


+

Page 32 of 54

calcd for C22H24N7O2S: 450.1712, found: 450.1723.

N-(4-(morpholinomethyl)phenyl)-4-(thieno[3,2-d]pyrimidin-4-ylamino)-1H-pyrazole-3-carboxamide

(40).

Compound 40 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 94 mg, 0.21 mmol Yield 43%).white solid; m.p 156-158 °C. HPLC analysis: retention time = 3.138 min; peak area, 95.66%.1H-NMR (300 MHz, DMSO-d6) δ: 2.35 (s, 4 H), 3.43 (s, 2 H), 3.58 (s, 4 H), 7.27-7.30 (d, 2 H, J = 8.28 Hz), 7.52-7.54 (d, 1 H, J = 5.28 Hz), 7.77-7.81 (d, 2 H, J = 8.28 Hz), 8.25-8.27 (d, 1 H, J = 5.31 Hz), 8.57 (s, 1 H), 8.73 (s, 1 H), 9.74 (s, 1 H), 10.30 (s, 1 H), 13.54 (s, 1 H); 13C-NMR (75 MHz, DMSO-d6) δ: 53.11, 62.02, 66.18, 101.35, 108.49, 115.23, 120.63, 123.45, 124.83, 129.13, 133.12, 133.26, 133.61, 136.94, 153.27, 154.55, 159.99, 162.58; HRMS-EI m/z [M+H] + calcd for C21H22N7O2S: 436.1556, found: 436.1556. 4-((5,6-dimethylthieno[2,3-d]pyrimidin-4-yl)amino)-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3carboxamide (41). Compound 41 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 89 mg, 0.19 mmol Yield 38%). white solid; m.p 254-256 °C. HPLC analysis: retention time = 6.625 min; peak area, 95.14%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.35 (s, 4 H), 2.44 (s, 3 H), 2.66 (s, 3 H), 3.43 (s, 2 H), 3.56-3.59 (s, 4 H), 7.27-7.30 (d, 2 H, J = 8.37 Hz), 7.76-7.79 (d, 2 H, J = 8.34 Hz), 8.50 (s, 1 H), 8.63 (s, 1 H), 10.12 (s, 1 H), 10.25 (s, 1 H), 13.46 (s, 1 H);

13

C-NMR (150 MHz, DMSO-d6) δ: 13.59, 14.46, 53.57, 62.47, 66.63, 117.44, 120.65, 121.10, 124.33,

129.64, 130.38, 131.31, 132.86, 133.58, 137.53, 152.84, 152.89, 163.12, 164.72; HRMS-EI m/z [M+H] + calcd for C23H26N7O2S: 464.1869, found: 464.1874. N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-4-(quinolin-4-ylamino)-1H-pyrazole-3-carboxamide (42). Compound 42 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 44 mg, 0.10 mmol Yield 19%). yellow solid; m.p 224-226 °C. HPLC analysis: retention time = 5.358 min; peak area, 99.76%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.14 (s, 3 H), 2.34 (br, 8 H), 3.41 (s, 2 H), 7.01-7.03 (d, 1 H, J = 5.22 Hz), 7.24-7.27 (d, 2 H, J = 8.25 Hz), 7.677.66 (t, 1 H, J = 7.40 Hz), 7.71-7.79 (m, 4 H), 7.92-7.94 (d, 1 H, J = 8.13 Hz), 8.15-8.18 (d, 1 H, J = 8.04 Hz), 8.32


32

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(s, 1 H), 8.56-8.58 (d, 1 H, J = 5.1 Hz), 9.86-10.27 (br, 2 H); 13C-NMR (75 MHz, DMSO-d6) δ: 45.69, 52.47, 54.71, 64.65, 100.81, 118.88, 120.19, 120.53, 121.23, 125.32, 125.65, 129.00, 129.15, 129.31, 133.01, 133.67, 137.01, 145.51, 148.16, 150.74, 162.03; HRMS-EI m/z [M+H]

+

calcd for C25H28N7O: 442.2355, found:

442.2359. 4-(isoquinolin-1-ylamino)-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (43). Compound 43 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 48 mg, 0.11 mmol Yield 22%). yellow solid; (65.1% Yield); m.p 224-225 °C. HPLC analysis: retention time = 5.134 min; peak area, 96.60%. 1H-NMR (300 MHz, DMSO-d6) δ: 1.99 (s, 3 H), 2.15 (br, 8 H), 3.43 (s, 2 H), 7.17-7.20 (d, 1 H, J = 5.73 Hz), 7.26-7.30 (d, 2H, J = 8.25 Hz), 7.73-7.75 (t, 2 H, J = 3.55 Hz), 7.79-7.83 (d, 2 H, J = 8.28 Hz), 7.86-7.88 (d, 1 H, J = 6.72 Hz), 7.99-8.02 (d, 1 H, J = 8.10 Hz), 8.11-8.13 (d, 1 H, J = 5.73 Hz), 8.64 (s, 1 H), 10.26 (s, 2 H), 13.38 (s,1 H); 13C-NMR (75 MHz, DMSO-d6) δ: 45.71, 52.48, 54.72, 61.67, 111.87, 117.56, 120.72, 121.05, 125.73, 127.11, 127.20, 129.02, 130.21, 133.72, 136.55, 136.93, 141.09, 150.39, 163.12; HRMS-EI m/z [M+H] + calcd for C25H28N7O: 442.2355, found: 442.2360. 4-((6-bromoquinolin-4-yl)amino)-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (44). Compound 44 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 45 mg, 0.09 mmol Yield 17%). yellow solid; m.p 231-235 °C. HPLC analysis: retention time = 4.526 min; peak area, 98.25%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.13 (s, 3 H), 2.33 (br, 8 H), 3.40 (s, 2 H), 6.83-6.84 (d, 1H, J =5.13 Hz), 7.21-7.24 (d, 2 H, J = 8.28 Hz), 7.69-7.73 (d, 2 H, J = 8.28 Hz), 7.83 (s, 2 H), 8.27 (s, 1 H), 8.44 (s, 1 H), 8.52 (s, 1 H), 9.31 (s, 1 H), 10.17 (s, 1 H), 13.60 (s, 1 H); 13C-NMR (150 MHz, DMSO-d6) δ: 46.15, 52.90, 55.15, 62.10, 101.90, 118.33, 120. 567, 120.84, 123.78, 124.35, 129.51, 132.14, 134.05, 136.22, 137.56, 146.58, 147.57, 151.90, 161.87, 170.80, 172.62; HRMS-EI m/z [M+H] + calcd for C25H27BrN7O: 520.1460, found: 520.1461. 4-((7-methoxyquinolin-4-yl)amino)-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3carboxamide (45). Compound 45 was prepared according to general procedure C on 0.5 mmol scale.


33


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Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 39 mg, 0.08 mmol Yield 16.7%). Yellow solid; m.p 226-228 °C. HPLC analysis: retention time = 6.589 min; peak area, 99.61%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.1 (s, 3 H), 2.35 (br, 8 H), 3.41 (s, 2 H), 3.92 (s, 3 H), 6.88-6.89 (d, 1 H, J = 4.71 Hz), 7.24-7.30 (m, 4 H), 7.74-7.77 (d, 2 H, J = 7.71 Hz), 8.03-8.06 (d, 1 H, J = 8.4 Hz), 8.32 (s, 1 H), 8.50 (s, 1 H), 9.63 (br, 1 H), 10.29 (s, 1 H); 13C-NMR (150 MHz, DMSO-d6) δ: 46.10, 52.85, 55.11, 55.81, 62.10, 100.15, 108.57, 113.70, 117.85, 121.05, 122.04, 125.82, 129.55, 134.12, 134.33, 137.46, 145.97, 150.58, 151.71, 160.42, 162.68, 172.62; HRMS-EI m/z [M+H] + calcd for C26H30N7O2: 472.2461, found: 472.2446. 4-((6-methoxyquinolin-4-yl)amino)-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3carboxamide (46). Compound 46 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 43 mg, 0.09 mmol Yield 18.2%). yellow solid; m.p 272-274 °C. HPLC analysis: retention time = 3.607 min; peak area, 97.10%.1H-NMR (300 MHz, DMSO-d6) δ: 2.16 (s, 3 H), 2.35 (br, 8 H), 3.40 (s, 2 H), 3.97 (s, 3 H), 6.87-6.89 (d, 1 H, J = 3.31 Hz), 7.22-7.25 (d, 2 H, J = 8.28 Hz), 7.39-7.43 (m, 1 H), 7.53-7.54 (d, 1 H, J = 2.04 Hz), 7.74-7.77 (d, 2 H, J = 8.28 Hz), 7.84-7.87 (d, 1 H, J = 9.15 Hz), 8.30 (s, 1 H), 8.41-8.43 (d, 1 H, J = 5.16 Hz), 9.34 (br, 1 H), 10.24 (s, 1 H); 13CNMR (150 MHz, DMSO-d6) δ: 46.16, 52.92, 55.16, 56.00, 62.11, 100.47, 101.46, 119.87, 120.78, 120.86, 125.46, 129.54, 131.54, 134.04, 135.27, 137.61, 144.49, 145.72, 149.00, 157.11, 162.34, 162.78, 172.62; HRMS-EI m/z [M+H] + calcd for C26H30N7O2: 472.2461, found: 472.2446. N-(4-((4-Methylpiperazin-1-yl)methyl)phenyl)-4-(quinazolin-4-ylamino)-1H-pyrazole-3-carboxamide (47). Compound 47 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 101 mg, 0.23 mmol Yield 47%). yellow solid; m.p 276-278 °C. HPLC analysis: retention time = 3.558 min; peak area, 100.00%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.22 (s, 3 H), 2.42 (br, 8 H), 3.45 (s, 2 H), 7.28-7.30 (d, 2 H, J = 6.0 Hz), 7.75-7.87 (m, 6 H), 8.65 (s, 1 H), 8.76 (s, 1 H), 10.40 (s, 1 H), 10.64 (s, 1 H), 13.60 (s, 1 H); 13C-NMR (150 MHz, DMSO-d6) δ: 45.99, 52.75, 55.05, 62.05, 115.00, 121.31, 121.41, 124.28, 127.66, 128.61, 129.55, 133.18, 133.67, 134.26, 137.31, 149.57, 155.27, 155.66, 163.30; HRMS-EI m/z [M+H] + calcd for C24H27N8O: 443.2308, found: 443.2319.


34

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N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-4-(thieno[2,3-d]pyrimidin-4-ylamino)-1H-pyrazole-3carboxamide(48). Compound 48 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 152 mg, 0.34 mmol Yield 68%).white solid; m.p 262-265 °C. HPLC analysis: retention time = 3.904 min; peak area, 98.63%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.15 (s, 1 H), 2.34 (br, 8 H), 3.56 (s, 2 H), 7.24-7.27 (m, 2 H), 7.48-7.51 (d, 1 H, J = 5.88 Hz), 8.54 (s, 1 H), 8.61 (s, 1 H), 9.98 (s, 1 H), 10.28 (s, 1 H), 13.60 (s, 1 H); 13C-NMR (75 MHz, DMSO-d6) δ: 45.67, 52.44, 54.68, 61.63, 116.48, 117.56, 120.61, 121051, 123.41, 124.99, 129.00, 129.19, 132.94, 133.73, 136.90, 152.97, 153.55, 162.36, 165.95; HRMS-EI m/z [M+H] + calcd for C22H25N8OS: 449.1872, found: 449.1880. 4-(Furo[2,3-d]pyrimidin-4-ylamino)-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3carboxamide (49). Compound 49 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 95 mg, 0.22 mmol Yield 43%). white solid; m.p 255-257 °C. HPLC analysis: retention time = 4.625 min; peak area, 100.00%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.14 (s, 3 H), 2.3 (br, 8 H), 3.41 (s, 2 H), 7.10 (s, 1 H, ArH), 7.23-7.26 (d, 2 H, J = 8.3 Hz), 7.75-7.79 (d, 2 H, J = 8.3 Hz), 8.24 (s, 1H), 8.49-8.52 (d, 2 H, J = 5.64Hz), 9.70 (s, 1 H), 10.29 (s, 1 H), 13.52 (s, 1 H);

13

C-NMR (75 MHz, DMSO-d6) δ: 45.54, 52.31, 54.57, 61.57, 101.59, 103.10, 120.24, 120.49, 122.10,

123.28, 129.00, 133.55, 136.99, 142.65, 153.49, 153.71, 162.02, 166.14; HRMS-EI m/z [M+H]

+

calcd for

C22H25N8O2: 433.2100, found: 433.2093. 4-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3carboxamide (50). Compound 50 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 160 mg, 0.37 mmol Yield 74%). white solid; m.p 229-230 °C. HPLC analysis: retention time = 3.696 min; peak area, 98.54%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.15 (s, 3 H), 2.35 (br, 8 H), 3.42 (s, 2 H), 6.48 (s, 1 H), 7.24-7.27 (d, 2 H, J = 8.07 Hz), 7.31 (s, 1 H), 7.76-7.79 (d, 2 H, J = 8.22 Hz), 8.38 (s, 1 H), 8.57 (s, 1 H), 9.52 (s, 1 H), 10.28 (s, 1 H), 11.90 (s, 1 H), 13.44 (s, 1 H); 13C-NMR (75 MHz, DMSO-d6) δ: 44.65, 51.45, 53.98, 61.25, 96.71, 103.08, 120.57, 121.94, 123.00, 124.70, 129.13, 131.92, 133.06, 137.14, 150.53, 151.18, 151.67, 162.56, 169.17; HRMS-EI m/z [M+H]


35


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+ calcd for C22H26N9O: 432.2260, found: 432.2271. N-(4-((4-Methylpiperazin-1-yl)methyl)phenyl)-4-(thieno[3,2-d]pyrimidin-4-ylamino)-1H-pyrazole-3carboxamide (51). Compound 51 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 156 mg, 0.35 mmol Yield 69%). white solid; m.p 278-280 °C. HPLC analysis: retention time = 4.505 min; peak area, 99.28%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.17 (s, 3 H), 2.36 (br, 8 H), 3.42 (s, 2 H), 7.25-7.28 (d, 2 H, J = 8.46 Hz), 7.52-7.54 (d, 1 H, J = 5.34 Hz), 7.75-7.79 (d, 2 H, J = 8.43 Hz), 8.26-8.28 (d, 1 H, J = 5.37 Hz), 8.56 (s, 1 H), 8.72 (s, 1 H), 9.74 (s, 1 H), 10.35 (s, 1 H), 13.57 (s, 1 H); 13C-NMR (150 MHz, DMSO-d6) δ: 46.00, 52.77, 55.05, 62.07, 115.00, 121.11, 122.06, 123.93, 125.32, 129.55, 134.14, 134.20, 137.38, 153.73, 155.06, 160.46, 162.90; HRMS-EI m/z [M+H] +

calcd for C22H25N8OS: 449.1872, found: 449.1884.

4-((5,6-dimethylthieno[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1Hpyrazole-3-carboxamide (52). Compound 52 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 135 mg, 0.28 mmol Yield 56%). white solid; m.p 264-267 °C. HPLC analysis: retention time = 6.249 min; peak area, 96.23%. 1

H-NMR (300 MHz, DMSO-d6) δ: 2.23 (s, 3 H), 2.43 (br, 8 H), 2.65 (s, 3 H), 3.44 (s, 2 H), 3.68 (br, 3 H), 7.27 (s, 2

H), 7.79 (s, 2 H), 8.25 (s, 1 H), 8.62 (s, 1 H), 10.11 (s, 1 H), 10.29 (s, 1 H), 13.52 (s, 1 H); 13C-NMR (75 MHz, DMSO-d6) δ: 13.02, 13.90, 45.28, 52.06, 54.43, 61.48, 116.90, 120.56, 120.89, 123.76, 123.91, 129.04, 129.80, 132.04, 133.42, 136.99, 152.26, 152.36, 162.50, 164.22; HRMS-EI m/z [M+H]

+

calcd for C24H29N8OS:

477.2185, found: 477.2193. N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-4-((6-methylthieno[2,3-d]pyrimidin-4-yl)amino)-1H-pyrazole3-carboxamide (53). Compound 53 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 147 mg, 0.32 mmol Yield 64%). white solid; m.p 254-256°C. HPLC analysis: retention time = 5.101 min; peak area, 96.47%. 1H-NMR (300 MHz, DMSO-d6) δ: 1.98 (s, 3 H), 2.35 (br, 8 H), 2.62 (s, 3 H), 3.42 (s, 2 H), 7.16 (s, 1 H), 7.24-7.28 (d, 2 H, J = 8.07 Hz), 7.76-7.79 (d, 2 H, J = 8.19 Hz), 8.54 (s, 2 H), 9.83 (s, 1 H), 10.27 (s, 1 H), 13.49 (s, 1 H); 13C-NMR (75


36

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MHz, DMSO-d6) δ: 15.81, 45.70, 52.47, 54.72, 61.64, 114.80, 117.08, 120.74, 123.72, 128.98, 132.56, 133.80, 136.84, 138.56, 151.78, 152.87, 162.52, 165.40; HRMS-EI m/z [M+H] + calcd for C23H27N8OS: 463.2029, found: 463.2035. N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-4-((5-methylthieno[2,3-d]pyrimidin-4-yl)amino)-1H-pyrazole3-carboxamide (54). Compound 54 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 179 mg, 0.39 mmol Yield 77%). white solid; m.p 247-249 °C. HPLC analysis: retention time = 4.625 min; peak area, 100.00%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.17 (s, 3 H), 2.36 (br, 8 H), 2.82 (s, 3 H), 3.42 (s, 2 H), 7.28-7.33 (m, 3 H), 7.79(s, 2 H), 8.59 (s, 1 H), 8.68 (s, 1 H), 10.17 (s, 1 H), 10.30 (s, 1 H), 13.06 (s, 1 H); 13C-NMR (75 MHz, DMSO-d6) δ: 17.74, 46.07, 52.85, 55.12, 62.11, 116.52, 119.81, 121.14, 121.49, 124.28, 129.44, 129.51, 132.74, 134.15, 137.39, 153.67, 153.84, 163.02, 167.65; HRMS-EI m/z [M+H] + calcd for C23H27N8OS: 463.2029, found: 463.2035. 4-((6-isopropylthieno[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1Hpyrazole-3-carboxamide (55). Compound 55 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 140 mg, 0.29 mmol Yield 57%). white solid; (39.0% Yield); m.p 220-222 °C. HPLC analysis: retention time = 12.72 min; peak area, 97.99%. 1H-NMR (300 MHz, DMSO-d6) δ: 1.35-1.38 (d, J =6.78 Hz, 6 H), 2.31 (s, 3 H), 2.46 (s, 4 H), 2.55 (s, 4 H), 3.29-3.39 (m, 1 H), 3.47 (s, 2 H), 7.18 (s, 1 H), 7.26-7.29 (d, 2 H, J = 8.3 Hz), 7.78-7.82 (d, 2 H, J =8.3 Hz), 8.54-8.55 (m, 2 H), 9.82 (s, 1 H), 10.36 (s, 1 H), 13.61 (s, 1 H);

13

C-NMR (75 MHz, DMSO-d6) δ: 24.11,

30.08, 44.67, 51.46, 54.01, 64.25, 111.83, 116.58, 120.69, 123.59, 129.12, 133.19, 137.06, 150.98, 152.14, 152.92, 162.34, 164.74; HRMS-EI m/z [M+H] + calcd for C25H31N8OS: 491.2342, found: 491.2359. 4-((6-ethylthieno[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3carboxamide (56). Compound 56 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 152 mg, 0.32 mmol Yield 63%). white solid; m.p 259-261 °C. HPLC analysis: retention time = 6.032 min; peak area, 96.03%. 1H-NMR (300 MHz, DMSO-d6) δ: 1.30-1.35 (t, 3 H, J = 7.47 Hz), 2.15 (s, 3 H), 2.34 (br, 8 H), 2.94-3.02 (q, 2 H, J = 5.83


37


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Hz), 3.42 (s, 2 H), 7.16 (s, 1 H), 7.25-7.28 (d, 2 H, J = 8.31 Hz), 7.77-7.80 (d, 2 H, J = 8.32 Hz), 8.55 (s, 2 H), 10.14 (s, 1 H), 10.28 (s, 1 H), 13.50 (s, 1 H);

13

C-NMR (150 MHz, DMSO-d6) δ: 15.95, 24.03, 46.20, 52.95, 55.19,

62.13, 113.65, 117.28, 121.20, 121.51, 124.13, 129.47, 133.14, 134.22, 137.36, 146.05, 152.40, 153.38, 162.97, 165.52; HRMS-EI m/z [M+H] + calcd for C24H29N8OS: 477.2185, found: 477.2194. N-(4-(piperazin-1-ylmethyl)phenyl)-4-(thieno[2,3-d]pyrimidin-4-ylamino)-1H-pyrazole-3-carboxamide (57). Compound 57 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 96 mg, 0.22 mmol Yield 43%). white solid; m.p 243-245 °C. HPLC analysis: retention time = 3.858 min; peak area, 98.43%. 1H-NMR (300 MHz, DMSO-d6) δ: 2.34 (br, 4 H), 2.94 (br, 4 H), 3.18 (s, 1 H), 3.48 (s, 2 H), 7.27-7.30 (d, J = 7.62 Hz, 2 H), 7.50-7.53 (d, J = 5.52 Hz, 1 H), 7.81-7.84 (m, 3 H), 8.54 (s, 1 H), 8.62 (s, 1 H), 10.02 (s, 1 H), 10.43 (s, 1 H) ; 13C NMR (300 MHz, DMSO-d6) δ: 43.69, 50.54, 61.50, 116.47, 117.61, 120.24, 120.58, 121.92, 123.41, 125.00, 129.16, 132.66, 132.85, 137.15, 153.00, 153.56, 158.08, 162.24, 165.93; HRMS-EI m/z [M+H] + calcd for C21H23N8OS: 435.1716, found: 435.1722. N-(4-((diethylamino)methyl)phenyl)-4-(thieno[2,3-d]pyrimidin-4-ylamino)-1H-pyrazole-3-carboxamide (58). Compound 58 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 72 mg, 0.17 mmol Yield 33%). white solid; m.p 233-235 °C. HPLC analysis: retention time = 5.505 min; peak area, 96.03%. 1H-NMR (300 MHz, DMSO-d6): δ 1.18-1.29 (m, 6 H), 2.88-2.90 (d, 4 H, J = 7.17 Hz), 4.05 (s, 2 H), 7.52-7.55 (d, 2 H, J = 5.61 Hz), 7.62-7.65 (d, 2 H, J = 8.25 Hz), 7.78-7.80 (d, 1 H, J = 5 .91 Hz), 7.94-7.97 (d, 2 H, J = 8.28 Hz), 8.54 (s, 1 H), 8.60 (s, 1 H), 9.95 (s, 1 H), 10.51 (s, 1 H), 13.73 (s, 1 H); 13C-NMR (150 MHz, DMSO-d6) δ: 8.75, 45.94, 54.72, 116.99, 118.16, 121.11, 121.89, 123.88, 125.52, 125.74, 132.00, 133.72, 139.67, 153.53, 154.06, 163.20, 166.46; HRMS-EI m/z [M+H] +

calcd for C21H24N7OS: 422.1763, found: 422.1774.

N-(4-((cyclopropylamino)methyl)phenyl)-4-(thieno[2,3-d]pyrimidin-4-ylamino)-1H-pyrazole-3-carboxamide (59). Compound 59 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (10% MeOH/DCM) yield the title compound ( 43 mg, 0.11 mmol Yield 21%). white solid;


38

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(56.1% Yield); m.p 210-213 °C. HPLC analysis: retention time = 3.846 min; peak area, 97.49%. 1H-NMR (300 MHz, DMSO-d6) δ 0.28 (br, 2 H), 0.35-0.38 (m, 2 H), 2.1 (m, 1 H), 3.72 (s , 2 H), 7.28-7.31 (d, 2 H, J = 8.13 Hz), 7.49-7.51 (d, 2 H, J = 8.25 Hz), 7.76-7.79 (d, 2 H, J = 8.25 Hz), 8.54 (s, 1 H), 8.60 (s, 1 H), 9.98 (s, 1 H), 10.28 (s, 1 H), 13.52 (s, 1 H); 13C-NMR (75 MHz, DMSO-d6) δ: 5.97, 29.79, 52.32, 96.76, 103.07, 120.32, 120.44, 122.98, 124.61, 126.29, 127.71, 128.22, 130.28, 131.03, 134.37, 136.13, 136.68, 140.04, 150.55, 151.16, 151.69, 157.66, 162.54; HRMS-EI m/z [M+H] + calcd for C20H20N7OS: 406.1450, found: 406.1437. N-(4-((4-methyl-1,4-diazepan-1-yl)methyl)phenyl)-4-(thieno[2,3-d]pyrimidin-4-ylamino)-1H-pyrazole-3carboxamide (60). Compound 60 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 58 mg, 0.13 mmol Yield 25%). white solid; m.p 241-244 °C. HPLC analysis: retention time = 3.687 min; peak area, 98.39%. 1H NMR (300 MHz, DMSO-d6): δ 1.71-1.77 (m, 2 H), 2.27 (s, 3 H), 2.53-2.67 (m, 8 H), 3.58 (s, 2 H), 7.29-7.31 (d, 2 H, J = 8.4 Hz), 7.51-7.53 (d, 2 H, J = 6.0 Hz), 7.78-7.82 (t, 3 H), 8.56 (s, 1 H), 8.63 (s, 1 H), 10.00 (s, 1 H), 10.29 (s, 1 H), 13.53 (s, 1 H); 13C NMR (75 MHz, DMSO-d6): δ 26.99, 46.51, 53.71, 54.12, 56.13, 57.42, 61.47, 94.95, 100.43, 117.83, 120.47, 128.66, 131.03, 134.87, 137.04, 148.05, 153.67, 161.81; HRMS-EI m/z [M+H]

+

calcd for

C23H27N8OS: 463.2029, found: 463.2041. N-(4-(piperazin-1-ylmethyl)phenyl)-4-(thieno[2,3-d]pyrimidin-4-ylamino)-1H-pyrazole-3-carboxamide (61). Compound 61 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 115 mg, 0.28 mmol Yield 55%). white solid; m.p 239-242 °C. HPLC analysis: retention time = 5.505 min; peak area, 96.03%. 1H NMR (300 MHz, DMSO-d6): δ 1.86 (s, 1 H), 2.34 (S, 4 H), 2.77 (s, 4 H), 3.42 (s, 2 H), 6.49-6.51 (d, 1 H, J = 3.45 Hz), 7.26-7.32 (m, 3 H), 7.787.81 (d, 2 H, J = 8.40 Hz), 8.40 (s, 1 H), 8.58 (s, 1 H), 9.54 (s, 1 H), 10.30 (s, 1 H), 11.92 (s, 1 H); 13C NMR (75 MHz, DMSO-d6): δ 44.88, 52.98, 62.15, 96.69, 103.07, 111.08, 120.57, 120.75, 122.97, 124.67, 129.08, 131.79, 133.37, 137.01, 150.55, 151.16, 151.69, 161.03, 162.55; HRMS-EI m/z [M+H] + calcd for C21H24N9O: 418.2104, found: 418.2113 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((diethylamino)methyl)phenyl)-1H-pyrazole-3-


39


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carboxamide (62). Compound 62 was prepared according to general procedure C on 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yield the title compound ( 73 mg, 0.18 mmol Yield 36%). white solid; m.p 217-220 °C. HPLC analysis: retention time = 3.334 min; peak area, 99.79%. 1H-NMR (300 MHz, DMSO-d6): δ 0.95-1.00 (m, 6 H), 2.43-2.49 (q, 4 H, J = 7.98 Hz), 3.50 (s, 2 H), 6.49 (s, 1 H), 7.26-7.29 (d, 2 H, J = 8.64 Hz), 7.29 (s, 1 H), 7.75-7.84 (d, 2 H, J = 8.31 Hz), 8.39 (s, 1 H), 8.57 (s, 1 H), 9.52 (s, 1 H), 10.19 (s, 1 H), 11.87 (s, 1 H), 13.39 (s, 1 H);

13

C-NMR (150 MHz, DMSO-d6)δ: 11.74, 46.09, 56.55, 98.82, 103.17,

120.27, 120.72, 123.14, 124.70, 128.78, 135.38, 136.84, 150.65, 151.29, 151.76, 162.83; HRMS-EI m/z [M+H] +

calcd for C21H25N8O: 405.2151, found: 405.2165.

Kinase Inhibition Assay Activity of CDKs and FLT3 were determined using Hot-SpotSM kinase assay which was performed by Reaction Biology Corp. (Malvern PA) as described previously47. Kinase activities were assayed in reaction buffer (20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO) at room temperature, at a final ATP concentration of 10 mM. Then compounds dissolved in 100% DMSO at indicated doses were delivered into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), incubate for 20 min at room temperature. After 10 μM [γ-33P] ATP (specific activity 10 Ci/μL) was added to initiate the reaction, the reactions were carried out at 25°C for 120 min. The kinase activities were detected by filterbinding method. IC50 values and curve fits were obtained by Prism (GraphPad Software).

Cell Growth Inhibition Assay The human AML cell line MV4-11 was purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). MV4-11 was cultured in IMDM media (Corning, USA) with 10% FBS and supplemented with 2% L-glutamine and 1% pen/strep. The MV4-11 cell line was maintained in culture media at 37 °C with 5% CO2. The effects of target compounds on MV4-11 proliferation were performed by Crown Bioscience Inc. Cells were cultured in 96-well culture plates (10000/well). The compounds of various concentrations were added into the plates. Cell proliferation was determined after treatment with compounds for 72 h. Cell


40

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viability was measured using the CellTiter-Glo assay (Promega, USA) according to the manufacturer’s instructions, and luminescence was measured in a multilabel reader (Envision2014, PerkinElmer, USA). Data were normalized to control groups (DMSO) and represented by the mean of three independent measurements with standard error of

1H-pyrazole-3-carboxamide (FN-1501), an FLT3 ... - ACS Publications

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