Design and Synthesis of 4-(Heterocyclic Substituted Amino)-1H-Pyrazole-3-Carboxamide Derivatives and Their Potent Activity against Acute Myeloid Leukemia (AML)

Fms-like receptor tyrosine kinase 3 (FLT3) has been emerging as an attractive target for the treatment of acute myeloid leukemia (AML). By modifying the structure of FN-1501, a potent FLT3 inhibitor, 24 novel 1H-pyrazole-3-carboxamide derivatives were designed and synthesized. Compound 8t showed strong activity against FLT3 (IC50: 0.089 nM) and CDK2/4 (IC50: 0.719/0.770 nM), which is more efficient than FN-1501(FLT3, IC50: 2.33 nM; CDK2/4, IC50: 1.02/0.39 nM). Compound 8t also showed excellent inhibitory activity against a variety of FLT3 mutants (IC50 < 5 nM), and potent anti-proliferative effect within the nanomolar range on acute myeloid leukemia (MV4-11, IC50: 1.22 nM). In addition, compound 8t significantly inhibited the proliferation of most human cell lines of NCI60 (GI50 < 1 μM for most cell lines). Taken together, these results demonstrated the potential of 8t as a novel compound for further development into a kinase inhibitor applied in cancer therapeutics.


Introduction
Acute myeloid leukemia (AML) is a malignant hematopoietic disease, characterized by uncontrolled proliferation of hematopoietic progenitor cells of the myeloid lineage within the bone marrow [1]. Fms-like receptor tyrosine kinase 3 (FLT3) represents a promising target for treatment of AML [2,3]. High expression of FLT3 is a common feature of AML and acute lymphoblastic leukemia (ALL) [4]. Furthermore, FLT3 mutations occur in approximately 30% of new diagnosed AML patients and tend to have a negative prognostic effect [5,6]. Two major classes of activating FLT3 mutations have been identified in AML patients, internal-tandem duplications (ITDs) and tyrosine kinase domain (TKD) point mutations [7]. Mutation of FLT3 causes ligand-independent autophosphorylation and constitutive activation of downstream pathways, including RAS/MEK, PI3K/AKT/mTOR, and JAK/STAT. Excessive activation of these pathways always results in uncontrolled cell proliferation [8,9]. PI3K/AKT/mTOR, and JAK/STAT. Excessive activation of these pathways always results in uncontrolled cell proliferation [8,9].
As shown in Figure 1, various small molecular FLT3 inhibitors have been developed [7, [10][11][12]. As a first-generation FLT3 inhibitor, midostaurin was first approved by FDA (in April 2017) for the treatment of newly diagnosed FLT3-mutant AML in combination with chemotherapy [13]. However, other first-generation FLT3 inhibitors were characterized by low clinical efficacy and significant toxicity. Despite initial optimism, response rates and response duration of those inhibitors were limited in patients with relapsed or refractory AML, due to the FLT3 mutation and lack of sustained FLT3 inhibition in vivo [14][15][16]. Subsequently, a series of second-generation FLT3 inhibitors were developed and achieved significant therapeutic effects. Up to now, the second-generation FLT3 inhibitors gilteritinib (approved by FDA) and quizartinib (approved in Japan) have been used for the treatment of relapsed or refractory AML [17]. Crenolanib also entered Phase III clinical research in 2017 (For treatment of Newly Diagnosed FLT3 Mutated AML). As shown in Table 1, although high selectivity against FLT3 was achieved for these second-generation FLT3 inhibitors, they still inhibit other targets that are also associated with the tumorigenesis [14,18,19]. Therefore, new agents with potent and sustained inhibition of FLT3 and the related signal pathways were noted to be beneficial to the suppression of tumor cell proliferation and overcoming drug resistance.

Chemistry
Coupling 4-nitropyrazole-3-carboxylic acid with a series of amine, followed by the reduction reaction, generated the intermediates 2a-2c. Compounds 3a-3c were then obtained by the substitution of 2a-2c with 4-chloro-7H-pyrrolo [2,3-d]pyrimidine (Scheme 1). Boc group was removed from 3c to produce 3d in the last step. Cyclin-dependent kinases (CDKs) are a family of serine/threonine protein kinases that are known to play a vital role in cell cycle regulation and modulating the transcription activity [20]. Cell cycle dysregulation, resulting from aberrant mitogenic signaling and leading to uncontrolled proliferation, is one of the hallmarks of cancer [21]. Thus, inhibitors that simultaneously block FLT3 and CDKs could synergistically improve the response rate and duration in the treatment of AML. For instance, FN-1501 ( Figure 1) is a FLT3 and CDKs inhibitor that we have reported, showing significant anti-AML activity [22]. In this paper, we further modified the structure of FN-1501 by optimizing the moieties that bind to the hydrophobic zone and hydrophilic regions in FLT3, and then a series of compounds with better FLT3/CDKs inhibitory activities were discovered.
Compounds 8a-8t were prepared as shown in Scheme 2. Intermediates 4a and 4b were prepared by coupling p-nitrobenzoic acid with the corresponding amines. Nucleophilic substitution of the corresponding amines with 5-fluoro-2-nitropyridine or 1-fluoro-4-nitrobenzene afforded the intermediates 4c-4h. Then hydrogenation of the nitro group of 4a-4h yielded intermediates 5a-5h. Intermediates 5a-5h were reacted with 4-nitro-1H-pyrazole-3-carbonyl to give 6a-6h, followed by the reduction reaction, to yield the intermediate products 7a-7h. Intermediates 7a-7b and 7e-7h were reacted with the appropriate chlorides to yield the desired products 8a-8g and 8r. Intermediates 7c and 7d were reacted with the appropriate chlorides under the appropriate temperature (initially 50 • C), and the Boc group were then removed by increasing the temperature to 70 • C when 7c and 7d vanished (TLC detection), to yield the desired products 8h-8q, 8s, and 8t (Scheme 2).

Structure-Activity Relationship Study
The enzymatic inhibitory activities of the target compounds were evaluated by CDK2, CDK4, and FLT3 kinase activity assays, and the cell-growth inhibitory potency against AML cell line MV4-11 were further evaluated for selected compounds (8a-8t). The results were summarized in Tables 2-4.

Structure-Activity Relationship Study
The enzymatic inhibitory activities of the target compounds were evaluated by CDK2, CDK4, and FLT3 kinase activity assays, and the cell-growth inhibitory potency against AML cell line MV4-11 were further evaluated for selected compounds (8a-8t). The results were summarized in Table 2

Structure-Activity Relationship Study
The enzymatic inhibitory activities of the target compounds were evaluated by CDK2, CDK4, and FLT3 kinase activity assays, and the cell-growth inhibitory potency against AML cell line MV4-11 were further evaluated for selected compounds (8a-8t). The results were summarized in Table 2

Structure-Activity Relationship Study
The enzymatic inhibitory activities of the target compounds were evaluated by CDK2, CDK4, and FLT3 kinase activity assays, and the cell-growth inhibitory potency against AML cell line MV4-11 were further evaluated for selected compounds (8a-8t). The results were summarized in Table 2-4.

Structure-Activity Relationship Study
The enzymatic inhibitory activities of the target compounds were evaluated by CDK2, CDK4, and FLT3 kinase activity assays, and the cell-growth inhibitory potency against AML cell line MV4-11 were further evaluated for selected compounds (8a-8t). The results were summarized in Table 2-4.

Structure-Activity Relationship Study
The enzymatic inhibitory activities of the target compounds were evaluated by CDK2, CDK4, and FLT3 kinase activity assays, and the cell-growth inhibitory potency against AML cell line MV4-11 were further evaluated for selected compounds (8a-8t). The results were summarized in Table 2    In the presence of 10 μM ATP, the values are the mean ± SD from three independent experiments.  In the presence of 10 μM ATP, the values are the mean ± SD from three independent experiments. In the presence of 10 μM ATP, the values are the mean ± SD from three independent experiments.      In the presence of 10 μM ATP, the highest test concentration is 1 μM. The values are the mean ± SD from three independent experiments. 2 The highest test concentration is 1 μM, the values are the mean ± SD from three independent experiments. In the presence of 10 μM ATP, the highest test concentration is 1 μM. The values are the mean ± SD from three independent experiments. 2 The highest test concentration is 1 μM, the values are the mean ± SD from three independent experiments. In the presence of 10 μM ATP, the highest test concentration is 1 μM. The values are the mean ± SD from three independent experiments. 2 The highest test concentration is 1 μM, the values are the mean ± SD from three independent experiments. In the presence of 10 μM ATP, the highest test concentration is 1 μM. The values are the mean ± SD from three independent experiments. 2 The highest test concentration is 1 μM, the values are the mean ± SD from three independent experiments. In the presence of 10 μM ATP, the highest test concentration is 1 μM. The values are the mean ± SD from three independent experiments. 2 The highest test concentration is 1 μM, the values are the mean ± SD from three independent experiments. In the presence of 10 μM ATP, the highest test concentration is 1 μM. The values are the mean ± SD from three independent experiments. 2 The highest test concentration is 1 μM, the values are the mean ± SD from three independent experiments. In the presence of 10 μM ATP, the highest test concentration is 1 μM. The values are the mean ± SD from three independent experiments. 2 The highest test concentration is 1 μM, the values are the mean ± SD from three independent experiments. In the presence of 10 μM ATP, the highest test concentration is 1 μM. The values are the mean ± SD from three independent experiments. 2 The highest test concentration is 1 μM, the values are the mean ± SD from three independent experiments. In the presence of 10 μM ATP, the highest test concentration is 1 μM. The values are the mean ± SD from three independent experiments. 2 The highest test concentration is 1 μM, the values are the mean ± SD from three independent experiments. As shown in Table 2, compounds 3a and 3b were synthesized to evaluate if the hydrophilic groups were necessary for the inhibitory activity against CDK2/4 and FLT3. Their activities against CDK4 largely decreased compared with FN-1501, which confirmed that the hydrophilic group was important for binding to CDK4. Furthermore, replacement of benzene and pyridine rings (3a and 3b) 5 As shown in Table 2, compounds 3a and 3b were synthesized to evaluate if the hydrophilic groups were necessary for the inhibitory activity against CDK2/4 and FLT3. Their activities against CDK4 largely decreased compared with FN-1501, which confirmed that the hydrophilic group was important for binding to CDK4. Furthermore, replacement of benzene and pyridine rings (3a and 3b) with piperidine (3c, 3d)  As shown in Table 2, compounds 3a and 3b were synthesized to evaluate if the hydrophilic groups were necessary for the inhibitory activity against CDK2/4 and FLT3. Their activities against CDK4 largely decreased compared with FN-1501, which confirmed that the hydrophilic group was important for binding to CDK4. Furthermore, replacement of benzene and pyridine rings (3a and 3b) with piperidine (3c, 3d) caused reduction of their activity against CDK2/4 and FLT3. This suggested 0.719 ± 0.064 0.770 ± 0.007 0.089 ± 0.013 1.22 ± 0.06 As shown in Table 2, compounds 3a and 3b were synthesized to evaluate if the hydrophilic groups were necessary for the inhibitory activity against CDK2/4 and FLT3. Their activities against CDK4 largely decreased compared with FN-1501, which confirmed that the hydrophilic group was important for binding to CDK4. Furthermore, replacement of benzene and pyridine rings (3a and 3b) with piperidine (3c, 3d) caused reduction of their activity against CDK2/4 and FLT3. This suggested that both hydrophilic group and aromatic-ring structure were necessary for the compounds to inhibit CDK2/4 and FLT3.
In order to find out the optimal groups in the hydrophilic region of ATP-binding site, a series of substitutions were introduced to the benzene ring (8a-8j, as shown in Table 3). A decrease of kinase inhibitory activity was observed when fixing the N-methylpiperazine or morpholine to benzene by carbonyl group (8a and 8b) compared with FN-1501. Bulkier groups (such as homopiperazine), when directly connected to the benzene ring, generally had no obvious influence on the activities of compunds 8c and 8d against CDK2/CDK4 and FLT3. Changing the N-methylhomopiperazine (8c and 8d) to meta-position (8e and 8f) in the benzene ring caused an obvious decrease in the inhibitory activities against CDK4 and the antiproliferative activity against MV4-11. Similarly, replacement of homopiperazine (8d) with morpholine (8g) caused a 10-fold decrease of IC 50 value against FLT3 compared with FN-1501. However, replacement of NH by S in the hydrophobic ring structure has little effect on their kinase inhibitory activity. Moreover, compound 8h with a single piperazine group showed improved inhibitory effects against CDK2 and FLT3, probably benefiting from the extra intermolecular interaction between the secondary amine of piperazine with CDK2 (GLU8)/FLT3 (ASN701) (Figure 2A,B). Compound 8h also exhibited high inhibitory activity against MV4-11 (IC 50 : 7.3 nM). However, the derivatives 8i and 8j consisted of the bridged-piperazine rings displayed weaker inhibitory effects against CDK2/4 and FLT3, when compared with compound 8h. Overall, a piperazine ring that directly attaches to the para position of the benzene ring is beneficial in elevating the inhibitory activity against CDK2/4 and FLT3. compunds 8c and 8d against CDK2/CDK4 and FLT3. Changing the N-methylhomopiperazine (8c and 8d) to meta-position (8e and 8f) in the benzene ring caused an obvious decrease in the inhibitory activities against CDK4 and the antiproliferative activity against MV4-11. Similarly, replacement of homopiperazine (8d) with morpholine (8g) caused a 10-fold decrease of IC50 value against FLT3 compared with FN-1501. However, replacement of NH by S in the hydrophobic ring structure has little effect on their kinase inhibitory activity. Moreover, compound 8h with a single piperazine group showed improved inhibitory effects against CDK2 and FLT3, probably benefiting from the extra intermolecular interaction between the secondary amine of piperazine with CDK2 (GLU8)/FLT3 (ASN701) (Figure 2A,B). Compound 8h also exhibited high inhibitory activity against MV4-11 (IC50: 7.3 nM). However, the derivatives 8i and 8j consisted of the bridged-piperazine rings displayed weaker inhibitory effects against CDK2/4 and FLT3, when compared with compound 8h. Overall, a piperazine ring that directly attaches to the para position of the benzene ring is beneficial in elevating the inhibitory activity against CDK2/4 and FLT3. We next turned our attention to the aromatic linker that connected the piperazine ring and the structure bound in the hinge area. As shown in Table 4, replacement of the benzene ring with 2pyridine (8k, 8l) significantly decreased their inhibitory activities against CDK2, and attenuated the inhibitory activities against CDK4 and FLT3 moderately. Molecular modeling showed that an unoccupied space existed in the deep hydrophobic pocket ( Figure 2C,D), suggesting that the additional hydrophobic substitution was beneficial to the inhibitory activity against CDK2/4 and FLT3. Therefore, we retained the pyridine ring as aromatic linker and introduced different groups in the deep hydrophobic pocket. When the five-member ring in the bicycle system was replaced by benzene ring (8m), the kinase inhibitory activities were basically maintained compared with 8l. Further alteration with the saturated five-member ring (8q), led to overall reduction in kinase and cell inhibitory activity . However, compounds 8n, 8o, and 8p, which contained the additional bulkier We next turned our attention to the aromatic linker that connected the piperazine ring and the structure bound in the hinge area. As shown in Table 4, replacement of the benzene ring with 2-pyridine (8k, 8l) significantly decreased their inhibitory activities against CDK2, and attenuated the inhibitory activities against CDK4 and FLT3 moderately. Molecular modeling showed that an unoccupied space existed in the deep hydrophobic pocket ( Figure 2C,D), suggesting that the additional hydrophobic substitution was beneficial to the inhibitory activity against CDK2/4 and FLT3. Therefore, we retained the pyridine ring as aromatic linker and introduced different groups in the deep hydrophobic pocket. When the five-member ring in the bicycle system was replaced by benzene ring (8m), the kinase inhibitory activities were basically maintained compared with 8l. Further alteration with the saturated five-member ring (8q), led to overall reduction in kinase and cell inhibitory activity. However, compounds 8n, 8o, and 8p, which contained the additional bulkier hydrophobic groups in different size in the deep hydrophobic pocket, showed improved inhibitory activities against CDK4 and FLT3 compared with 8k. Unfortunately, these compounds exhibited weaker anti-proliferative effects on MV4-11 cells (IC 50 value: 0.045-0.35 µM). It was presumed that the introduction of pyridine ring may reduce the permeability of the entire molecule, and thus decrease their activity to MV4-11. Therefore, benzene ring was reserved as the aromatic linker.Combining the bulkier hydrophobic substituents in deep pocket and the benzene as aromatic linker, compounds 8r-8t were designed (as shown in Table 4). Generally, compounds 8r and 8s showed enhanced CDK2/4 and FLT3 inhibitory activities as well as the antiproliferative potency against MV4-11 cells, compared with compound 8n. As the optimal compound, 8t exhibited the sub-nanomolar IC 50 values against CDK2 (0.719 nM), CDK4 (0.770 nM), and FLT3 (0.0890 nM), and consistently strong anti-proliferative activity in MV4-11 cells (IC 50 : 1.22 nM). Thus, the step-by-step structural optimization demonstrated that the combination of piperazine in the hydrophilic pocket, benzene ring as the aromatic linker, and the bulkier fused ring in the deep hydrophobic pocket were beneficial for kinase inhibitory activity and anti-proliferative activity to MV4-11.

Molecular Modeling of Compound 8t with CDK2 and FLT3
Compound 8t showed optimal FLT3/CDK2/CDK4 inhibitory activities. Hence, the binding mode of compound 8t with CDK2 and FLT3 were elucidated using a docking model. Since 8t is a type I FLT3 inhibitor, we used our homology model structure of "DFG-in" FLT3 [22]. As shown in Figure 3, compound 8t binded to the ATP-binding site of CDK2 and FLT3 in an orientation similar to FN-1501 [22]. The pyrazole-3-carboxamide skeleton of compound 8t formed three conserved hydrogen bonds with the hinge region of CDK2 and FLT3 respectively (Figure 3). The aromatic heterocycle moiety occupied the hydrophobic pocket and the piperazine group extended to solvent accessible area. The difference was that the NH of piperazine formed a hydrogen bond with GLU85 in CDK2 and it also formed a hydrogen bond with ASN701 in FLT3. The cyclopentane extended to ribose zones which were not occupied by FN-1501. This binding mode was beneficial to improving the inhibitory activity of 8t against CDK2 and FLT3. In general, the docking results further confirmed the rationality of our design strategy.  1.22 nM). Thus, the step-by-step structural optimization demonstrated that the combination of piperazine in the hydrophilic pocket, benzene ring as the aromatic linker, and the bulkier fused ring in the deep hydrophobic pocket were beneficial for kinase inhibitory activity and anti-proliferative activity to MV4-11.

Molecular Modeling of Compound 8t with CDK2 and FLT3
Compound 8t showed optimal FLT3/CDK2/CDK4 inhibitory activities. Hence, the binding mode of compound 8t with CDK2 and FLT3 were elucidated using a docking model. Since 8t is a type I FLT3 inhibitor, we used our homology model structure of "DFG-in" FLT3 [22]. As shown in Figure  3, compound 8t binded to the ATP-binding site of CDK2 and FLT3 in an orientation similar to FN-1501 [22]. The pyrazole-3-carboxamide skeleton of compound 8t formed three conserved hydrogen bonds with the hinge region of CDK2 and FLT3 respectively (Figure 3). The aromatic heterocycle moiety occupied the hydrophobic pocket and the piperazine group extended to solvent accessible area. The difference was that the NH of piperazine formed a hydrogen bond with GLU85 in CDK2 and it also formed a hydrogen bond with ASN701 in FLT3. The cyclopentane extended to ribose zones which were not occupied by FN-1501. This binding mode was beneficial to improving the inhibitory activity of 8t against CDK2 and FLT3. In general, the docking results further confirmed the rationality of our design strategy.

Kinase Profiling
In order to investigate the kinase profile vulnerable to compound 8t, its enzymatic inhibitory effects against 32 kinases were tested (Table S1), which were the representative kinase drug targets. Among those kinases (Table 5), compound 8t showed significant inhibitory activities against CDKs and FLT3, except for CDK1 that is considered not reasonable as an anti-tumor target [20]. In addition,

Kinase Profiling
In order to investigate the kinase profile vulnerable to compound 8t, its enzymatic inhibitory effects against 32 kinases were tested (Table S1), which were the representative kinase drug targets. Among those kinases (Table 5), compound 8t showed significant inhibitory activities against CDKs and FLT3, except for CDK1 that is considered not reasonable as an anti-tumor target [20]. In addition, compound 8t exhibited inhibitory activities against KDR/VEGFR2, ERK7, FLT1, FLT4, and GSK3β (Table 5), which were related to the tumorigenesis. These data indicated that compound 8t is a highly potent pan-kinase inhibitor with the prominent inhibitory potency against CDKs and FLT3. Furthermore, compound 8t potently inhibited eight FLT3 mutants (IC 50 values less than 5 nM) that are correlated to the drug resistance [23,24]. Compound 8t also showed significant inhibitory activity against the FLT3 (ITD)-F691L mutation (IC 50 : 0.6 nM), which led to the drug resistance to FLT3 inhibitors, such as quizartinib. Accordingly, in the BaF3 cells that are transformed with FLT3-ITD-F691L, compound 8t showed the improved antiproliferative activity over quizartinib (Table S2).

In vitro Cell Assays
With these findings, we submitted compound 8t to National Cancer Institute (NCI) to evaluate their antitumor efficacy against 60 human cancer cell lines. As shown in Table 6, compound 8t exhibited anti-proliferative activities against a variety of cancer cell lines, which was consistent with its multi-kinase inhibition potency, indicating that 8t has the potential of further development as a powerful anti-tumor agent for various human cancers, including AML.

Cellular Mode of Action
To characterize the mode of cellular effects induced by compound 8t, flow cytometry was performed in MV4-11 cell line. Since compound 8t had strong inhibitory activity against FLT3 and CDK, sorafenib with potent inhibitory activity against FLT3 and pan-CDK inhibitor AT-7519 were selected as the positive controls [25,26]. As detected by annexin V staining, not only was the apoptosis triggered, but also a dose-dependent increase in the percentage of apoptotic and dead cells was seen ( Figure 4). In the presence of vehicle alone for 24 h, only 4.7% of the cells underwent apoptosis, while treatment with compound 8t at 2 µM for 24 h led to an apoptosis rate up to 51.36%.
To further investigate whether the antitumor activities were relevant to the inhibition of FLT3 and CDK2, we then examined the relative signaling proteins in MV4-11 cells treated with compound 8t by western blot. According to the MV4-11 cell-based western blot assays, the phosphorylation of the FLT3 was weakened by compound 8t in a dose-dependent manner, and eliminated when the compound concentration was increased to 1 µM ( Figure 5), which exhibited more potent inhibition effects than sorafenib. As downstream signal pathways of FLT3, the phosphorylation of STAT5/AKT/ERK were also completely blocked at 1 µM. Compound 8t also inhibited the phosphorylation of the retinoblastoma protein (Rb), a key downstream factor of CDK2/4, in a dose-dependent manner. At the concentration of 1 µM, compound 8t exhibited comparable inhibitory effects against the phosphorylation of Rb with AT-7519. In all, the anti-proliferative activity of compound 8t was associated with the inhibition of FLT3 and CDK2/4.
To characterize the mode of cellular effects induced by compound 8t, flow cytometry was performed in MV4-11 cell line. Since compound 8t had strong inhibitory activity against FLT3 and CDK, sorafenib with potent inhibitory activity against FLT3 and pan-CDK inhibitor AT-7519 were selected as the positive controls [25,26]. As detected by annexin V staining, not only was the apoptosis triggered, but also a dose-dependent increase in the percentage of apoptotic and dead cells was seen ( Figure 4). In the presence of vehicle alone for 24 h, only 4.7% of the cells underwent apoptosis, while treatment with compound 8t at 2 μM for 24 h led to an apoptosis rate up to 51.36%. To further investigate whether the antitumor activities were relevant to the inhibition of FLT3 and CDK2, we then examined the relative signaling proteins in MV4-11 cells treated with compound 8t by western blot. According to the MV4-11 cell-based western blot assays, the phosphorylation of the FLT3 was weakened by compound 8t in a dose-dependent manner, and eliminated when the compound concentration was increased to 1 μM ( Figure 5), which exhibited more potent inhibition effects than sorafenib. As downstream signal pathways of FLT3, the phosphorylation of STAT5/AKT/ERK were also completely blocked at 1 μM. Compound 8t also inhibited the phosphorylation of the retinoblastoma protein (Rb), a key downstream factor of CDK2/4, in a dosedependent manner. At the concentration of 1 μM, compound 8t exhibited comparable inhibitory effects against the phosphorylation of Rb with AT-7519. In all, the anti-proliferative activity of compound 8t was associated with the inhibition of FLT3 and CDK2/4.

Materials and Methods
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 (Tech Instrument Co., Ltd., Beijing, China); NMR spectra were recorded on Bruker AVANCE AV-600 spectrometer (600 MHz for 1 H, 150 MHz for 13 C) or Bruker AVANCE AV-300 spectrometer (300 MHz for 1 H, 75 MHz for 13 C); Mass spectra were obtained on the Agilent 1100 LC/MSD mass spectrometer (Agilent, Santa Clara, CA, USA). All reactions were monitored by TLC (Merck Kieselgel GF254, Merck, Kenilworth, NJ, China) and spots were visualized with UV light or iodine. The purity of biologically evaluated compounds was >95% as determined by HPLC.

Procedure A For the Synthesis of Compounds 4a and 4b
The mixture of appropriate amine (18.5 mmol), p-nitrobenzoic acid (20.4 mmol), EDC (22.2 mmol), HOBT (22.2 mmol) in DMF (30 mL) was stirred for 24 h. The ice water (100 mL) was added to the reaction mixture. A large amount of yellow solid precipitation (compounds 4a and 4b) was acquired. Compounds 4a and 4b were used without further purification.

Materials and Methods
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 (Tech Instrument Co., Ltd., Beijing, China); NMR spectra were recorded on Bruker AVANCE AV-600 spectrometer (600 MHz for 1 H, 150 MHz for 13 C) or Bruker AVANCE AV-300 spectrometer (300 MHz for 1 H, 75 MHz for 13 C); Mass spectra were obtained on the Agilent 1100 LC/MSD mass spectrometer (Agilent, Santa Clara, CA, USA). All reactions were monitored by TLC (Merck Kieselgel GF254, Merck, Kenilworth, NJ, China) and spots were visualized with UV light or iodine. The purity of biologically evaluated compounds was >95% as determined by HPLC.

Procedure A For the Synthesis of Compounds 4a and 4b
The mixture of appropriate amine (18.5 mmol), p-nitrobenzoic acid (20.4 mmol), EDC (22.2 mmol), HOBT (22.2 mmol) in DMF (30 mL) was stirred for 24 h. The ice water (100 mL) was added to the reaction mixture. A large amount of yellow solid precipitation (compounds 4a and 4b) was acquired. Compounds 4a and 4b were used without further purification.

Procedure B For the Synthesis of Compounds 4c-4h
Fluorobenzene or fluoropyridine (46.3 mmol) and K 2 CO 3 (69.5 mmol) were dissolved in DMSO (50 mL). The reaction mixture was stirred at r.t. for 30 min and then amine (69.5 mmol) was added. The reaction mixture was stirred at 70 • C for 5 h. The ice water (500 mL) was added to the reaction mixture. A large amount of yellow solid precipitation (compounds 4c-4h) was acquired. Compounds 4c-4h were used for further reaction without purification. 2a-2c, 5a-5h, and 7a-7h To a suspension of compounds 1a-1c, 4a-4h, 6c-6h, or 4a-4h (26.2 mmol) in 95% ethanol (100 mL), 85% NH 2 NH 2 . H 2 O (262 mmol), 95% ethanol (100 mL), and iron (III) oxide hydroxide (FeO(OH)/C, 0.5 g) were added and heated to reflux. When TLC analysis showed complete conversion of the starting material, the reaction mixture was filtrate through Celite ® 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.

Procedure D for the Synthesis of Compounds 1a-1c and 6a-6h
4-nitro-1H-pyrazole-3-carboxylic acid (4.19 g, 13.94 mmol) was dissolved in 20 mL THF, DMF (0.5 mL) and oxalyl chloride (1.78 mL, 20.91 mmol) were added at 0 • C, the resultant mixture was stirred at room temperature for 60 min. After the mixture was concentrated in vacuo, the residue was dissolved in pyridine (20 mL), and the solution was added dropwise into the solution of 7 (dissolved in 20 mL pyridine) at 0 • C. The solution was stirred for 6 h at 25 • C. Upon completion of the reaction, the solvent was removed on a rotary evaporator. Then water (100 mL) was added, and the mixture was basified using 10% NaOH until pH 8~9. The solid precipitation was filtered to give the crude product, which was used for next step without further purification.
Detailed synthetic process and structural characterization were provided in the Supplementary Materials. pocket can significantly increase the inhibitory activity of these kinds of compounds against CDK2/4 and FLT3. Among these compounds, compound 8t showed significant potency against CDKs and FLT3, almost 10 times more powerful than FN-1501. Compound 8t also exhibited significant inhibitory activity against various FLT3 mutations, especially against FLT3 (ITD)-F691L, indicating its potential to overcome drug resistance caused by FLT3 mutation. Compound 8t showed potent anti-proliferative activity against a variety of cancer cell lines including MV4-11 cells, and inhibited phosphorylation of CDK and FLT3 pathways in a dose-dependent manner. These results demonstrated the potential of this compound (8t) for further development as a promising agent for treatment of AML as well as other cancers.