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

Article Cite This: J. Med. Chem. 2018, 61, 1499−1518

pubs.acs.org/jmc

Discovery of 4‑((7H‑Pyrrolo[2,3‑d]pyrimidin-4-yl)amino)‑N‑(4-((4methylpiperazin-1-yl)methyl)phenyl)‑1H‑pyrazole-3-carboxamide (FN-1501), an FLT3- and CDK-Kinase Inhibitor with Potentially High Efficiency against Acute Myelocytic Leukemia 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*,†,‡ †

School of Sciences and ‡State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China S Supporting Information *

ABSTRACT: A series of 1-H-pyrazole-3-carboxamide derivatives have been designed and synthesized that exhibit excellent FLT3 and CDK inhibition and antiproliferative activities. A structure−activity-relationship study illustrates that the incorporation of a pyrimidine-fused heterocycle at position 4 of the pyrazole is critical for FLT3 and CDK inhibition. Compound 50 (FN-1501), which possesses potent inhibitory activities against FLT3, CDK2, CDK4, and CDK6 with IC50 values in the nanomolar range, shows antiproliferative activities against MV4-11 cells (IC50: 0.008 μM), which correlates with the suppression of retinoblastoma phosphorylation, FLT3, ERK, AKT, and STAT5 and the onset of apoptosis. 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 a nude-mouse model, 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 a drug applied in acute-myeloidleukemia (AML) therapeutics.

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INTRODUCTION

molecule in hematopoiesis and is overexpressed in most cases of acute leukemia.3 The FLT3 pathway plays an important role in the growth and differentiation of hematopoietic progenitor cells (HPCs).4,5 Many studies have shown that AML and other hematologic cancers are closely related to abnormal FLT3 pathways.6−10 Like other RTKs, the FLT3 receptor dimerizes 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 the cell cycle, cell apoptosis, and cell differentiation. However, mutations of FLT3 lead to the hyperactivation of FLT3 in the absence of ligand binding and the activation of downstream signaling pathways.11 About

Acute myeloid leukemia (AML) is a heterogeneous disease of hematopoietic progenitor cells and is characterized by a block in differentiation and uncontrolled proliferation.1 More than a quarter of a million adults throughout the world are diagnosed with AML annually. Induction chemotherapy with cytarabine (ara-C) and an anthracycline (most often daunorubicin) is usually performed as the first-line treatment of AML.1 Although some improvement has been apparent among younger patients during the last four decades, only approximately 35% of patients entered in clinical trials are cured of the disease, and older people, who are not able to withstand intensive chemotherapy, only survive 5−10 months.2 Thus, there is an unmet need for effective therapeutics. Fms-like receptor tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase (RTK) that acts as an important regulatory © 2018 American Chemical Society

Received: August 24, 2017 Published: January 22, 2018 1499

DOI: 10.1021/acs.jmedchem.7b01261 J. Med. Chem. 2018, 61, 1499−1518

Journal of Medicinal Chemistry

Article

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 a homology model of FLT3.18

signaling pathways and the cell cycle simultaneously, which may exert synergistic effects to increase antileukemic efficiencies.18,28,36 We have reported a series of compounds with moderate CDK2 activities.37−40 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 group has explored the biological properties of variously substituted 1-H-pyrazole-3carboxamide derivatives with the aim to find new active compounds and pharmacophores that are potent FLT3 and CDK inhibitors. Among these compounds, compound 50 was found to be the most active in vitro and in vivo, and it also possesses good pharmacokinetic properties and low toxicity. This report presents the discovery of 50 as a highly efficient multitarget FLT3 and CDK kinase inhibitor suitable for further evaluation.

30% of AML patients harbor some form of FLT3 mutation, and the 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) being marketed or under investigation.12 Although some of these compounds have shown initial therapeutic benefits, responses have been transient and relapses occur within a few weeks, partially because of insufficient target coverage, activation of parallel pathways, and acquisition of resistance mutations.13 Midostaurin (PKC412, N-benzoylstaurosporin) is a multitargeted tyrosine-kinase inhibitor (TKI) used against FLT3, VEGFR2, KIT, PDGFR, and PKC-a. Midostaurin was approved and first launched in 2017 in the United States for the treatment of adult patients with newly diagnosed acute myeloid leukemia in combination with chemotherapy. The success of midostaurin indicates that the inhibition of FLT3 and other antitumor targets can improve antitumor efficiency. Cyclin-dependent kinases (CDKs) are a family of 20 serine and threonine protein kinases that are generally classified as regulators of the cell cycle (CDK1, -2, -4, and -6) or transcription (CDK7, -8, -9, -11, and -20).14 The uncontrolled regulation of CDK activity has been identified as a hallmark of cancer.15−17 Indeed, oncogenic alterations of CDKs, cyclins, and other components of the Rb pathway have been reported in more than 90% of human cancers.18−20 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, and pancreatic neoplasia and sarcoma.21 Cyclin D2 and cyclin D3 belong to D-type cyclins, which bind and activate CDK4 and CDK6 and play important roles in hematologic malignancies. Mutations or overexpression of CDK4 and 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 tumorigenesis.22−24 These studies suggest that the CDK family has been considered as one of the most important targets for therapeutic interventions in cancers, including hematologic malignancies. CDK inhibitors (AT-7519, flavopiridol, AMG-925, and palbociclib)18,25−32 are currently in clinical development in various solid tumors and hematopoietic malignances.33 However, most CDK inhibitors were discontinued during phase II trials because of severe toxicities34 or limited clinical activities as monotherapies.35 In fact, the activation of the FLT3 downstream signaltransduction pathway is an extremely important process in promoting cell proliferation and requires the cooperation of CDKs. Therefore, inhibitors of FLT3 and CDKs can arrest antimitotic

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CHEMISTRY The synthesis of compounds 27−62 was depicted in Scheme 1. In total, 35 pyrazole-carboxamide derivatives were prepared (Tables 1 and 2). Compounds 27−62 were prepared in a similar fashion. 4-Nitrobenzyl bromide was used as the starting material, and compounds 2−7 were prepared through a nucleophilic substitution reaction. The reduction of compounds 2−7 with 80% hydrazine hydrate gave compounds 8−13, and after the coupling of 4-nitropyrazole-3-carboxylic acid with EDC·HCl and HOBt,40 compounds 14−19 were obtained. Compounds 20−25 were obtained via the reduction of the nitro-groups in compounds 14−19. All target compounds (27−62) were prepared by ammonolysis of the appropriate chlorides with 20−25.

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RESULTS AND DISCUSSION In our previous studies,40 a series of pyrazole-3-carboxamide compounds were synthesized. Recently, it has been identified that compound 26 inhibits FLT3 and CDK2 with IC50’s of 42.6 nM and 318 nM, respectively. With the help of computer-aided drug design (CADD), we found that the hydrophobic cavity of CDK2 formed by ASP145, VAL18, PHE80, and LYS33 (corresponding to ASP757, PHE691, PHE627, and LYS650 in FLT3) was wide enough to accommodate a larger hydrophobic group. Guided by our previous studies, we kept the 1-H-pyrazole-3-carboxamide skeleton and the hydrophobic phenyl ring linked to 3-carboxamide (the binding modes were shown in Figure 1). In position 4 of the 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 activities. In the solventaccessible region, different hydrophilic fragments were introduced, leading to a series of 1-H-pyrazole-3-carboxamide derivatives. All derivatives were tested for their inhibitory potencies 1500



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Scheme 1. Synthetic Routes of Compounds 27−62a

Reagents and conditions: (a) relative amine, Et3N, CH2Cl2; (b) FeO(OH)/C, 80% NH2NH2·H2O, 95% EtOH; (c) 4-nitropyrazole-3-carboxylic acid, EDC·HCl, HOBt, DMF, room temperature; (d) FeO(OH)/C, 80% NH2NH2·H2O, 95% EtOH; (e) R2−Cl, AcOH/H2O, 50 °C. a

in C2 position of pyrimidine or pyridine might prevent edge-toface interactions between the aromatic ring and PHE80 (CDK2). However, the 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 the gatekeeper residue PHE675 of FLT3 is far enough to accommodate the chlorine atom. The pyrimidine in the fused heterocycle seems to be optimal for forming an aromatic−aromatic interaction with the gatekeeper PHE80 in CDK2. To further investigate the hydrophobic interactions of aromatic rings with the hydrophobic cavity, the phenyl ring of the fused heterocycle was then replaced with five-membered aromatic heterocycles. Modifications to the phenyl ring resulted in analogues 35−39 and 48−56, among which the thienopyrimidine analogues (35 and 48) retained the same affinities to CDK2 and FLT3 when compared with those of 31 and 47. Even when the position 7 or 8 hydrogen of thieno[2,3d]pyrimidine was substituted with methyl (39, 53, and 54), ethyl (37 and 56), or isopropyl (38 and 55), the strong inhibitory activities against CDK2 and FLT3 were maintained. Moreover, changing the position of the sulfur atom (51) did not affect the kinase-inhibitory activities. In order to obtain additional SAR information, we replaced the thiazole ring with furan (49) and pyrrole (36 and 50) on the basis of the bioisosterism. The pyrrolopyrimidine analogue, 50, exhibited a similar kinaseinhibitory potency to that of 48 for both CDK2 and FLT3. But the furan-substituted compound, 49, showed a decreased inhibitory efficiency against CDK2. Overall, the introduction of pyrrolopyrimidine or thienopyrimidine is important for kinaseinhibitory activities, especially against FLT3. Although having the 1-methylpiperazine or morpholine stretched to hydrophilic region conferred good potency for either CDK2or FLT3-inhibitory activities, there was 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[2,3d]pyrimidine and the 7H-pyrrolo[2,3-d]pyrimidine as the hydrophobic segments. All these compounds are listed in Table 2. It is noteworthy that modifications in the hydrophilic region could maintain the inhibitory activities of the compounds

toward recombinant human CDK2/cyclin A1 and FLT3 and their antiproliferative effects on MV4-11 cells (human-acutemonocytic-leukemia-cell line). Structure−Activity-Relationship (SAR) Studies. The results of the kinase-inhibition assays are listed in Tables 1 and 2 with AT-7519 and sorafenib as the positive controls. Minimizing the diversity of the substituents in the phenyl ring enabled us to investigate how varying the substituents at the C4-position of the pyrazole affected the activity against CDK2 and FLT3. As is shown in Table 1, both CDK2 and FLT3 affinities increased slightly compared with that of the parent compound when the benzoyl in 26 was replaced by quinolone (compounds 27 and 42) or isoquinoline (compounds 28 and 44) . This might be the benefit of the edge-to-face aromatic− aromatic interactions between the pyridine and PHE80 in CDK2 and PHE691 in FLT3 and the hydrophobic interactions formed by the phenyl ring with the ASP145 in CDK2 and ASP698 in FLT3, respectively (Figure 2). 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 with 27 and 42. The introduction of a nitrogen into the pyridine ring of 27 yielded analogue 31, which interestingly showed a significant increase in its inhibitory activities against CDK2 and FLT3 (IC50’s of 5.30 nM and 0.588 nM, respectively). Replacement of the morpholine (31) with N-methylpiperazine (47) also retained excellent activity against CDK2 (IC50: 3.17 nM) and FLT3 (IC50: 8.27 nM). The inhibitory activities were approximately 10 times more potent than those of AT7519 and sorafenib. The addition of a chlorine atom into the 2 position of pyrimidine (32) resulted in a decrease in the affinity to CDK2 when compared with that of 31, supporting the hypothesis that a substituent 1501



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Table 1. Structures and Biological Evaluations of the Target Compounds

against CDK2 and FLT3. However, the 7H-pyrrolo[2,3d]pyrimidine derivatives (61−62, Table 2) led to decreases

in cell-based activities compared with those of the thieno[2,3d]pyrimidine derivatives (57−58, Table 2). These decreases 1502



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Table 1. continued

may be caused by decreases in the lipophilicity of the compounds, which influences the permeability of compounds. Further studies have shown that introducing thieno[2,3-d]pyrimidine led to higher clearance rates in liver microsomes. The introduction of a 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 efficiencies of the compounds. Hence, the introduction of a fused heterocycle that contains a pyrimidine or pyridine structure to pyrazole is suggested to be the most critical factor in determining the potency of pyrazolecarboxamide derivatives toward FLT3 and CDKs. 1503



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Table 1. continued

In the presence of 10 μM ATP and 0.123 μM concentrations of the target compounds. The values are the means ± SD from three independent experiments. bnd: not determined. cn = 3. a

Kinase-Inhibition Profile of Compound 48. 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 results, 28 kinases were selected for further assays (Figure 3B and Table S1). Compound 48 displayed the highest inhibitory activities 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), whose IC50’s are followed by those of CDK9/cyclinK (IC50: 38.9 nM), FLT1/VEGFR1 (IC50: 19.8 nM), KDR/ VEGFR2 (IC50: 35.1 nM), ALK (IC50: 94.1 nM), and CDK1/ cyclinE (IC50: 96 nM). Therefore, compound 48 is identified to be a multiple-kinase inhibitor; inhibition of multiple pathways simultaneously by compound 48 is beneficial in improving its antitumor efficiency and overcoming drug resistance. 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, kinases other than CDK2/cyclinA and FLT3 (Table 3). All the selected compounds showed high inhibitory activities against these kinases with IC50’s lower than 10 nM, suggesting that these compounds might be even more potent CDK and FLT3 inhibitors (i.e., 50: CDK2 IC50: 2.47 nM, CDK4 IC50: 0.85 nM, CDK6 IC50: 1.96 nM, FLT3 IC50: 0.28 nM). In Vitro Cell Assays. All of target compounds (27−62) were evaluated against human-leukemia-cell line MV4-11 (FLT3ITD) using the MTT assay after 72 h incubations (see Tables 1 and 2). Compounds 31, 36−40, 47−48, and 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 certifies the rationality of our design strategy. Compounds 35, 48, and 50 were selected to evaluate their activities against a panel of cell lines that represent different tumor types, including gastric cancer, leukemia, breast cancer, and colorectal cancer. The growth of all the cell lines was inhibited by the

selected compounds, and the 50% growth inhibition (GI50) values ranged from 51 to 620 nM (Table 4). With these findings, we submitted 35, 48, and 50 to the Developmental Therapeutics Program at the National Cancer Institute (NCI, http://dtp.nci.nih.gov) to test their selectivities against the NCI60 cancer-cell lines. In this screen, compounds 35, 48, and 50 exhibited potent inhibitory activities on all 60 cancer cell lines (Figures S1−S3 in the Supporting Information), with GI50 < 0.5 μmol/L (the growth inhibition of 50%, calculated by [(Ti − Tz)/(C − Tz)] × 100 = 50, is the drug concentration resulting in a 50% reduction in the net protein increase, as measured by SRB staining in the control cells during the drug incubation). For the great majority of the lines, the LC50’s (concentrations of the drugs resulting in 50% reductions in the measured proteins at the end of the drug treatments as compared with those at the beginning, which indicates a net loss of cells following treatment and is calculated by [(Ti − Tz)/Tz] × 100 = −50) were >100 μmol/L (mean LC50 > 400 μmol/L). In comparison, the lethal concentration of flavopiridol (mean GI50: 0.074 μmol/L against NCI60 cell lines), a potent inhibitor of CDKs, reached submicromolar concentrations (LC50: 0.904 μmol/L).41 Pharmacokinetics of 35 and 50 in Liver Microsomes of Multiple Species. The preliminary metabolic-stability study of compounds 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 half-lives (t1/2) were moderate in human liver microsomes, rat liver microsomes, mouse liver microsomes, and monkey liver microsomes (ranging between 0.025 and 1.7 h). Compound 50 was metabolically more stable (t1/2 ranging 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 rings are metabolically unstable.42,43 Therefore, this series of compounds has not been considered for further investigation. 1504



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Table 2. Optimization of the Hydrophilic Group in the Solvent-Accessible Area

In the presence of 10 μM ATP and 0.123 μM concentrations of the target compounds. The values are the means ± SD from three independent experiments. bn = 3. a

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 the homology model of FLT3.18

Molecular Modeling of Compound 50 with CDK2 and FLT3. Compound 50 shows optimal FLT3- and CDK-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 (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 and the 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 1505



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Figure 3. (A) Distribution of kinases inhibited by compound 48 within the human kinome. The color code for inhibition is indicated. Residual enzymatic activities were determined in single-dose duplicates at compound concentrations 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 33P-radiolabeled assay. (B) Inhibitory activity of compound 48 against selected kinases using the 33P-radiolabeled assay.

Table 3. Inhibitory Activities of Compounds 35, 48, 49, 50, and 51 toward CDKs and FLT3 Kinases compound IC50 (nM)a kinase

35

CDK2/cyclin A CDK4/cyclin D1 CDK6/cyclin D1 FLT3

3.36 2.40 3.88 0.62

± ± ± ±

48 0.26 0.27 0.17 0.06

5.85 3.33 4.24 0.60

± ± ± ±

49 0.58 0.24 0.23 0.04

16.5 5.67 5.09 2.56

± ± ± ±

50 0.76 0.30 0.44 0.11

2.47 0.85 1.96 0.28

± ± ± ±

51 0.21 0.28 0.08 0.01

9.32 3.10 4.11 0.47

± ± ± ±

0.45 0.34 0.29 0.02

a In the presence of 10 μM ATP and 0.123 μM concentrations of the target compounds. The values are the means ± SD from three independent experiments.

Table 4. In Vitro Growth-Inhibitory Activities of Selected Compounds cell type GI50 (μM)a

a

compound

MGC803

RS4;11

MCF-7

HCT-116

NCI-H82

AT-7519

0.21 ± 0.01

0.20 ± 0.04

0.11 ± 0.02

0.26 ± 0.13

0.27 ± 0.14

35

0.21 ± 0.02

0.17 ± 0.05

0.61 ± 0.07

0.58 ± 0.11

0.29 ± 0.06

48

0.16 ± 0.05

0.13 ± 0.01

0.62 ± 0.09

0.37 ± 0.02

0.17 ± 0.02

50

0.37 ± 0.04

0.05 ± 0.01

2.84 ± 0.25

0.09 ± 0.04

0.11 ± 0.02

n = 3.

Table 5. Pharmacokinetics of Selected Compounds in Liver Microsomes of Multiple Species compounds

HML

RLM

MLM

DLM

CLM

R2 a t1/2 (h)b CLint (mL/min/kg)c R2 t1/2 (h) CLint (mL/min/kg) R2 t1/2 (h) CLint (mL/min/kg) R2 t1/2 (h) CLint (mL/min/kg) R2 t1/2 (h) CLint (mL/min/kg)

35

50

0.9793 0.138 165.1 0.8963 0.118 352.4 0.9056 0.12 765.1 0.9899 0.025 1327.7 0.9929 0.182 170.9

0.3209 1.717 13.3 0.5008 1.032 40.3 0.9465 0.66 138.6 0.9908 0.257 129.3 0.9051 0.855 36.5

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 images of compound 50 (green) and AT7519 (gray) complexed with CDK2. (C) Compound 50 docked into a homology model of FLT3.18 (D) Chemical structure of compound 50.

a 2 R is the correlation coefficient of the linear regression for the determination of the kinetic constant. bt1/2 is the half-life. cCLint is the intrinsic clearance.

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lymphocyte cells. Compound 50 showed little cytotoxicity to normal lymphocyte cells, comparable to those of AC220 (IC50: 0.492 μM) and AT7519 (IC50: 0.554 μM). To further evaluate the safety of 50 in vivo, we conducted additional LD50 tests for AT-7519 and 50; groups of ICR mice (half male and half female) were given single doses of compound 50 or AT7519 by intravenous injections. The survival of the mice was monitored and recorded over 12 days (Table S2). The LD50 values of AT7519 were 32 mg/kg. However, the LD50 values of 50 were 185.67 mg/kg, as shown in Table 6. Compound 50 exhibited significantly reduced toxicity compared with AT7519.

the kinase gatekeeper residue, PHE80. The phenyl ring occupies the small hydrophobic cavity between hinge region and the hydrophilic group. The N-methylpiperazine group sits in a solventaccessible area that is mainly hydrophobic but lined with several polar residues at the edge (LYS89, ASP86, LEU298, and GLN85). 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 and the other is between the N2 of pyrazole and CYS694. The 7H-pyrrolo[2,3-d]pyrimidine substituent of 50 occupies the hydrophobic pocket, and the N-methylpiperazine group extends into the 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 sc 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 mice. In the MV4-11 model, when the tumor grew to a volume of 200 mm3, the mice were grouped and treated intravenously once daily (QD) with 15, 30, or 40 (mg/kg)/d of compound 50 for 21 days. Another two groups were recruited: one group for negative control, which was given only the vehicle, and another group treated intravenously once daily with 50 (mg/kg)/d of cytarabine as a positive control for 21 days. The group injected with 50 (mg/kg)/d of cytarabine only had reduced tumorgrowth rates, and the tumor-growth inhibition was 61.94% on the 21st day, whereas injections of 50 at 15 (mg/kg)/d induced tumor regression, for which the percent-tumor-regression (PTR) rate was −6.11% on the 21st day (Figure 5). Additionally, injections of 50 at 30 and 40 (mg/kg)/d significantly induced tumor regressions, and the PTRs on the 21st day were 78.95% and 81.33%, respectively (Figure 5B−D). Furthermore, 50 was well tolerated with no overt signs of toxicity or changes in body weight (Figure 5A). Experiments in rats demonstrated that 50 does have significant antitumor 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

Table 6. Median-Lethal-Dose (LD50) Values of 50 and AT7519 compounds

LD50(mg/kg)

AT7519

32.00

95% confidence interval (μmol/kg) 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 and propidium iodide (PI) was performed. Inducing apoptosis is considered as one of the major strategies for antitumor-drug development.14 Flow cytometry was used to investigate whether the antiproliferative effect of compound 50 was caused by the activation of cellular apoptosis in MV4-11 cells. Quantitative analysis of early-apoptotic cells and advanced-apoptotic and necrotic cells was determined via an Annexin V-FITC and PI assay. MV4-11 cells were stimulated with 0.2−2 μM concentrations of compound 50 for 24 h. DMSO was used as the negative control, and sorafenib was used as the positive control. As shown in Figure 6, the apoptosis induced by compound 50 was much greater than that of the control, and MV4-11 cells underwent both early apoptosis (Annexin V+ and PI−, lower right) and advanced apoptosis and necrosis (Annexin V+ and PI+, upper right). The results showed that compound 50 could effectively induce cell apoptosis starting from 0.2 μM with enhanced effects 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), and 78.30% (2 μM), and the apoptotic effect increased in a dose-dependent manner. From these results, we

Figure 5. Antitumor efficacy of 50 in the 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, with cytarabine at 50 (mg/kg)/d, or with the vehicle. (A) Body-weight and (B) tumor-size measurements from MV4-11-xenograft mice after 50 or cytarabine administration. Initial body weights and tumor sizes were set as 100%. (C) Representative photographs of tumors in each group after 15, 30, or 40 (mg/kg)/d of 50, 50 (mg/kg)/d of cytarabine, or the vehicle treatment. (D) Comparison of the final tumor weights in each group after the 22 day treatment period. The numbers in columns indicate the mean tumor weight in each group. (*) p < 0.05, (**) p < 0.01. 1507



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

Figure 7. Cancer-cell-growth inhibition and cell-cycle arrest from compound 50 treatments in MV4-11 and HCT116 cells. (A) Effect of compound 50 on MV4-11 cell-cycle kinetics. Compound 50 was added at the concentrations shown to MV4-11 cells 24 h prior to fixation, staining with propidium iodide (PI), and flow-cytometric analysis. (B) Effect of compound 50 on HCT-116 cell cycles.

can conclude that 50 caused effective apoptotic effects in the MV4-11 cell line and showed an efficiency equivalent to that of sorafenib (75.32% at 2 μM). Furthermore, compound 50 was measured in MV4-11 and HCT-116 cell lines to determine if its antiproliferative activity was consistent with FLT3- and CDK2-signaling inhibition. The antiproliferative activity of compound 50 was verified by flowcytometry analysis of the MV4-11 cell line and HCT-116 colon-cell line, through double staining with propidium iodide and 5-bromo-20-deoxyuridine (BrdU). As is shown in Figure 7, compound 50 arrested the cell-cycle progression of the former cell line into the G0 and G1 phases in a dose-dependent manner, which is comparable to action of sorafenib (Figure 7A) in the MV4-11 cell line. Treatment with compound 50 mainly caused G2- or M-phase arrest in the HCT-116 cell lines (Figure 7B), which could be related to the potent inhibition of CDKs. Overall, compound 50 arrested the cell cycle of two humancancer-cell lines as efficiently as AT-7519 and sorafenib, and both cell lines displayed similar patterns of cell-cycle blockades as those with the positive control. We then treated the MV4-11 cell line with increasing doses of compound 50 for 24 h and immunoblotted the lysates with antibodies against the relative proteins involved in the FLT3mediated signaling pathway (Figure 8A). Treatment of MV4-11 cells with compound 50 for 24 h significantly decreased the phosphorylation of FLT3 and that of its downstream mediators STAT5, ERK, and AKT more than treatment with sorafenib did. As shown in Figure 8B, compound 50 inhibited RbSer807/811

phosphorylation in a dose-dependent manner, and its efficiency was equal to that of AT7519 at 1 μM. These findings reveal that compound 50 acts as a highly efficient FLT3 and CDK inhibitor.

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CONCLUSIONS Starting from compound 26, we have discovered a series of N-(4-methylphenyl)-1-H-pyrazole-3-carboxamide derivatives that are potent FLT3 and CDK inhibitors. The structure−activityrelationship analysis involving substitutions with fused heterocycles that contains pyrimidine and pyridine structures in position 4 of the 1-H-pyrazole-3-carboxamide moiety demonstrated that the 1-H-pyrazole-3-carboxamide moiety was a key strategic group in the improvement of CDK2- and FLT3-inhibitory activities and antiproliferative activities. Compound 50 showed nanomolar range CDK and FLT3 inhibition and significant cytotoxic effects in NCI60 cancer-cell lines (IC50 < 1 μM). In addition, 50 exhibited a high metabolic stability in the liver-microsomalmetabolism assay. The in vivo study in the MV4-11-xenograft model showed that 50 had a higher antitumor efficacy at a dosage of 15.0 (mg/kg)/d than cytarabine did at a dosage of 50.0 (mg/kg)/d and furthermore that 50 induced tumor regression when the doses increased to 30.0 (mg/kg)/d or 40.0 (mg/kg)/d. Currently, compound 50 is in phase I clinical trials in the United States and China as a cancer treatment, and it may represent a promising drug with therapeutic potential for the treatment of AML. 1508



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Figure 8. Western blot analyses. (A) Immunoblots showing the phosphorylation of FLT3, STAT5, ERK, and AKT proteins in MV4-11 cells treated with compound 50 or sorafenib for 24 h. (B) Immunoblots showing the phosphorylation of the Rb protein in MV4-11 cells treated for 24 h with compound 50 or AT7519.

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EXPERMENTAL SECTION General Procedures. Unless otherwise specified, reagents were purchased from commercial suppliers and used without further purification. Melting points were determined by an X-4 digital-display micromelting-point apparatus (Beijing Tech Instrument Company, Ltd., Beijing, China). NMR spectra were recorded on a Bruker AVANCE AV-600 spectrometer (800 MHz for 1H and 150 MHz for 13C) or a Bruker AVANCE AV-300 spectrometer (300 MHz for 1H and 75 MHz for 13C; Bruker, Billerica, MA). Mass spectra were obtained on an Agilent 1100 LC/MSD mass spectrometer (Agilent, Santa Clara, CA) and Micromass Q-tofmicro MS (Waters, Milford, MA). All reactions were monitored by TLC (Silica gel GF254, Merck, Kenilworth, NJ), and spots were visualized with UV light or iodine. The purities of the biologically evaluated compounds were >95% as determined by HPLC. General Procedure A for the Synthesis of Compounds 2−7. 4-Nitrobenzyl bromide (46.3 mmol) was dissolved in dichloromethane (100 mL). The solution was added to a mixture of relative amine (47.0 mmol) and triethylamine (70.3 mmol) in dichloromethane (20 mL). The reaction mixture was stirred at room temperature (rt) for 24 h and was extracted with dichloromethane (100 mL × 3). After the removal of the solvent, the residue was crystallized from ethanol, giving a yellow powder. Compounds 2−7 were used for further reactions 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 reflux.40,44,45 When TLC analysis showed the complete conversion of the starting material, the reaction mixture was filtrated through celite, and the filtrate was concentrated in a vacuum. The crude product was purified by silicagel column chromatography (DCM/MeOH) to yield the title compound as a white solid. 4-(Morpholinomethyl)aniline (8). Compound 8 was prepared according to general procedure A on a 36.2 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (6.0 g, 31 mmol, 86% yield). [M + H]+: 193. 1H NMR (DMSO-d6) δ: 2.32 (s, 4 H), 3.24 (s, 4 H), 3.51 (s, 2 H), 4.92 (s, 2 H), 6.53 (d, 2 H, J = 8.4 Hz), 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 A on a 36.2 mmol

scale. Purification by column chromatography (5% MeOH/ DCM) yielded the title compound (7.3 g, 34.2 mmol, 94% yield). [M + H]+: 206. 1H NMR (300 MHz, DMSO-d6) δ: 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 a 36.2 mmol scale. Purification by column chromatography (10% MeOH/DCM) yielded the title compound (8.2 g, 28.1 mmol, 78% yield). [M + H]+: 292. 1H NMR (300 MHz, DMSO-d6) δ: 1.38 (s, 9 H), 2.24−2.26 (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 a 36.2 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (4.9 g, 23.1 mmol, 65% yield). [M + H]+: 220. 1H NMR (DMSO-d6) δ: 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 = 7.05 Hz), 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 a 36.2 mmol scale. Purification by column chromatography (3% MeOH/ DCM) yielded the title compound (2.9 g, 17.1 mmol, 46% yield). [M + H]+: 179. 1H NMR (DMSO-d6) δ: 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 a 36.2 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (3.1 g, 19.8 mmol, 54% yield). [M + H]+: 163. 1H NMR (DMSO-d6) δ: 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 the Synthesis of Compounds 20−25. The mixture of compounds 8−13 (1 equiv, 18.5 mmol), 4-nitro-1H-pyrazole-3-acid (1.1 equiv, 20.4 mmol), EDC (1.2 equiv, 22.2 mmol), and HOBT (1.2 equiv, 22.2 mmol) in DMF (50 mL) was stirred for 24 h. Ice water (100 mL) was added to the reaction mixture. A large amount of yellow, solid precipitation (compounds 3a−f) was acquired. Compounds 1509



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ethyl acetate. After the 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 a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound, (77 mg, 0.18 mmol, 36% yield). Yellow solid; mp 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 = 8.0 Hz, 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, 12 125.24, 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 a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (116 mg, 0.27 mmol, 54% yield). White solid; mp 285−287 °C. HPLC analysis: retention time, 4.109 min; peak area, 100.00%. 1H NMR (300 MHz, DMSOd6) 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); 13C 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 a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (65 mg, 0.13 mmol, 26% yield). Yellow solid; mp 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 a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (49 mg, 0.11 mmol, 21% yield). Yellow solid; mp 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); 13C NMR (75 MHz, DMSO-d6) δ: 21.20, 53.01, 55.27, 61.95, 66.10,

14−19 were used without further purification. Compounds 20−25 were reduced by the same process as compound 8−13; then the resulting compounds 20−25 were purified by column chromatography on silica gel and eluted with the appropriate solvent. 4-Amino-N-(4-(morpholinomethyl)phenyl)-1H-pyrazole-3carboxamide (20). Compound 20 was prepared according to general procedure B on an 18.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (3.6 g, 12.0 mmol, 65% yield). [M + H]+: 302. 1H NMR (300 MHz, DMSO-d6) 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 an 18.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (4.1 g, 15.4 mmol, 76% yield). [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 an 18.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (1.8 g, 4 mmol, 24% yield). [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 an 18.5 mmol scale. Purification by column chromatography (5% MeOH/ DCM) yielded the title compound (1.3 g, 3.9 mmol, 21% yield). [M + H]+: 329.3. 1H NMR (DMSO-d6) δ: 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, 1 H). 4-Amino-N-(4-((diethylamino)methyl)phenyl)-1H-pyrazole3-carboxamide (24). Compound 24 was prepared according to general procedure B on an 18.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (2.0 g, 6.8 mmol, 37% yield). [M + H]+: 288. 1H NMR (300 MHz, DMSO-d6) 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 an 18.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (0.9 g, 3.3 mmol, 18% yield). [M + H]+: 272. 1H NMR (300 MHz, DMSO-d6) 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 the Synthesis of Compounds 27−62. Compounds 20−25 (1 equiv) and the corresponding chorides (1.2 equiv) in AcOH/H2O:1/1 (20 mL) were heated to 50 °C. When TLC analysis showed the complete conversion of the starting material, sodium hydroxide (2 equiv) was added to the mixture. Then, the reaction mixture was extracted with 1510



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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]pyrimidin4-ylamino)-1H-pyrazole-3-carboxamide (35). Compound 35 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (92 mg, 0.22 mmol, 44% yield). White solid; mp 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 a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (138 mg, 0.33 mmol, 66% yield). White solid; mp 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 (37). Compound 37 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (71 mg, 0.15 mmol, 31% yield). White solid; mp 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); 13C NMR (150 MHz, DMSOd6) 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-3-carboxamide (38). Compound 38 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (93 mg, 0.20 mmol, 39% yield). White solid; mp 222−224 °C. HPLC analysis: retention time, 4.433 min; peak area, 97.47%. 1H 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); 13C 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; HRMSEI m/z [M + H]+ calcd for C24H28N7O2S: 478.2025, found: 478.2032.

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 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (120 mg, 0.28 mmol, 56% yield). Yellow solid; mp 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, 2 H), 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 a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (54 mg, 0.12 mmol, 23% yield). Yellow solid; mp 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 a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (46 mg, 0.09 mmol, 19% yield). Yellow solid; mp 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. 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 a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (85 mg, 0.19 mmol, 37% yield). Yellow solid; mp 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, 1511



Article

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 a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (48 mg, 0.11 mmol, 22% yield). Yellow solid; mp 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, 2 H, 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 a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (45 mg, 0.09 mmol, 17% yield). Yellow solid; mp 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, 1 H, 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-3-carboxamide (45). Compound 45 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (39 mg, 0.08 mmol, 16.7% yield). Yellow solid; mp 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-3-carboxamide (46). Compound 46 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (43 mg, 0.09 mmol, 18.2% yield). Yellow solid; mp 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),

4-((5-Methylthieno[2,3-d]pyrimidin-4-yl)amino)-N-(4(morpholinomethyl)phenyl)-1H-pyrazole-3-carboxamide (39). Compound 39 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (92 mg, 0.21 mmol, 41% yield). White solid; mp 205−207 °C. HPLC analysis: retention time, 3.568 min; peak area, 96.37%. 1H 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; HRMSEI m/z [M + H]+ 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 a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (94 mg, 0.21 mmol, 43% yield). White solid; mp 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-3-carboxamide (41). Compound 41 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (89 mg, 0.19 mmol, 38% yield). White solid; mp 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); 13C 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-(quinolin4-ylamino)-1H-pyrazole-3-carboxamide (42). Compound 42 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (44 mg, 0.10 mmol, 19% yield). Yellow solid; mp 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), 7.67−7.66 (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 (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, 1512



Article

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]+ 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-3-carboxamide (51). Compound 51 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (156 mg, 0.35 mmol, 69% yield). White solid; mp 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)-1H-pyrazole-3-carboxamide (52). Compound 52 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (135 mg, 0.28 mmol, 56% yield). White solid; mp 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-((6methylthieno[2,3-d]pyrimidin-4-yl)amino)-1H-pyrazole-3carboxamide (53). Compound 53 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (147 mg, 0.32 mmol, 64% yield). White solid; mp 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 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-((5methylthieno[2,3-d]pyrimidin-4-yl)amino)-1H-pyrazole-3carboxamide (54). Compound 54 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (179 mg, 0.39 mmol, 77% yield). White solid; mp 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,

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); 13C NMR (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-4ylamino)-1H-pyrazole-3-carboxamide (47). Compound 47 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (101 mg, 0.23 mmol, 47% yield). Yellow solid; mp 276−278 °C. HPLC analysis: retention time, 3.558 min; peak area, 100.00%. 1H NMR (300 MHz, DMSOd6) 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. N-(4-((4-Methylpiperazin-1-yl)methyl)phenyl)-4-(thieno[2,3d]pyrimidin-4-ylamino)-1H-pyrazole-3-carboxamide(48). Compound 48 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (152 mg, 0.34 mmol, 68% yield). White solid; mp 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, 121 051, 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-3-carboxamide (49). Compound 49 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (95 mg, 0.22 mmol, 43% yield). White solid; mp 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, 1 H), 8.49− 8.52 (d, 2 H, J = 5.64 Hz), 9.70 (s, 1 H), 10.29 (s, 1 H), 13.52 (s, 1 H); 13C 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-3-carboxamide (50). Compound 50 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (160 mg, 0.37 mmol, 74% yield). White solid; mp 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), 1513



Article

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 a 0.5 mmol scale. Purification by column chromatography (10% MeOH/DCM) yielded the title compound (43 mg, 0.11 mmol, 21% yield). White solid; mp 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; HRMSEI 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-3-carboxamide (60). Compound 60 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (58 mg, 0.13 mmol, 25% yield). White solid; mp 241−244 °C. HPLC analysis: retention time, 3.687 min; peak area, 98.39%. 1 H 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; HRMSEI 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 a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (115 mg, 0.28 mmol, 55% yield). White solid; mp 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), 7.78−7.81 (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-carboxamide (62). Compound 62 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (73 mg, 0.18 mmol, 36% yield). White solid; mp 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); 13C NMR (150 MHz, DMSO-d6)

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)-1H-pyrazole-3-carboxamide (55). Compound 55 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (140 mg, 0.29 mmol, 57% yield). White solid; mp 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); 13C 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-((4methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (56). Compound 56 was prepared according to general procedure C on a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (152 mg, 0.32 mmol, 63% yield). White solid; mp 259−261 °C. HPLC analysis: retention time, 6.032 min; peak area, 96.03%. 1 H 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 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); 13C 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 a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (96 mg, 0.22 mmol, 43% yield). White solid; mp 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 a 0.5 mmol scale. Purification by column chromatography (5% MeOH/DCM) yielded the title compound (72 mg, 0.17 mmol, 33% yield). White solid; mp 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 0.91 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, 1514



Article

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. The activity of the CDKs and FLT3 were determined using a Hot-SpotSM kinase assay, which was performed by Reaction Biology Corporation (Malvern, PA), as described previously.46 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 the indicated doses, were delivered into the kinase reaction mixture by acoustic technology (Echo550, nanoliter range; Labcyte, San Jose, CA) and incubated 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 the filterbinding method. IC50 values and curve fits were obtained by Prism (GraphPad Software, La Jolla, CA). Cell-Growth-Inhibition Assay. The human AML cell line MV4-11 was purchased from the American Type Culture Collection (ATCC, Manassas, VA). MV4-11 was cultured in IMDM media (Corning, Corning, NY) with 10% FBS and supplemented with 2% L-glutamine and 1% penicillin/streptomycin. The MV4-11 cell line was maintained in culture media at 37 °C with 5% CO2. The effects of the target compounds on MV4-11 proliferation were performed by Crown Bioscience Inc. (San Diego, CA). Cells were cultured in 96-well culture plates (10 000 cells/well). The compounds at various concentrations were added to the plates. Cell proliferation was determined after treatment with the compounds for 72 h. Cell viability was measured using the CellTiter-Glo assay (Promega, Madison, WI) according to the manufacturer’s instructions, and luminescence was measured in a multilabel reader (Envision2014, PerkinElmer, Waltham, MA). Data were normalized to control groups (DMSO) and represented as the means of three independent measurements with standard errors of

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

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