pyrimidine Derivatives Leading to the D - American Chemical Society

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Structure−Activity Relationship Studies of Pyrazolo[3,4‑d]pyrimidine Derivatives Leading to the Discovery of a Novel Multikinase Inhibitor That Potently Inhibits FLT3 and VEGFR2 and Evaluation of Its Activity against Acute Myeloid Leukemia in Vitro and in Vivo Ling-Ling Yang,†,‡,§ Guo-Bo Li,†,§ Shuang Ma,† Chan Zou,† Shu Zhou,† Qi-Zheng Sun,† Chuan Cheng,† Xin Chen,† Li-Jiao Wang,† Shan Feng,† Lin-Li Li,† and Sheng-Yong Yang*,† †

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, and ‡College of Chemical Engineering, Sichuan University, Sichuan 610041, China S Supporting Information *

ABSTRACT: We describe the structural optimization of a hit compound, 1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)phenyl)-3-(3-methoxyphenyl)urea (1), which exhibits inhibitory activity but low potency against FLT3 and VEGFR2. A series of pyrazolo[3,4-d]pyrimidine derivatives were synthesized, and structure−activity relationship analysis using cell- and transgenic-zebrafish-based assays led to the discovery of a number of compounds that exhibited both high potency against FLT3driven human acute myeloid leukemia (AML) MV4-11 cells and a considerable antiangiogenic effect in transgenic-zebrafish-based assays. The compound 1-(4-(1H-pyrazolo[3,4-d]pyrimidin −4yloxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (33), which exhibited the highest activity in preliminary in vivo antiAML assays, was chosen for further anti-AML studies. The results demonstrated that compound 33 is a multikinase inhibitor that potently inhibits FLT3 and VEGFR2. In an MV4-11 xenograft mouse model, a once-daily dose of compound 33 at 10 mg/kg for 18 days led to complete tumor regression without obvious toxicity. Western blot and immunohistochemical analyses were performed to determine the mechanism of action of compound 33.

1. INTRODUCTION Acute myeloid leukemia (AML) is a malignant disorder of hematopoietic cells that is characterized by the rapid growth of abnormal white blood cells, which accumulate in the bone marrow and hinder the development of normal blood cells.1 AML has become one of the leading causes of cancer-related mortality worldwide. Recent studies regarding the pathogenesis of AML have revealed that mutations and/or aberrant expression of specific protein tyrosine kinases (PTKs) are frequently responsible for the development of AML.2−5 Of special note is the FMS-like tyrosine kinase 3 (FLT3), which is a class III receptor tyrosine kinase. Activating mutations in FLT3 kinase are found in up to one-third of AML cases; the most prevalent activating mutations are “internal tandem duplications” (ITDs) in the juxtamembrane domain that lead to constitutive, ligand-independent activation of the kinase.6−8 Numerous studies have demonstrated that FLT3-ITD mutations represent a driving mutation for the development of AML and are associated with a poor prognosis for overall survival.9−11 Therefore, FLT3 has been considered a valid therapeutic target for AML treatment, and many pharmaceutical companies and research institutes have been involved in the discovery of FLT3 inhibitors. © XXXX American Chemical Society

A number of small-molecule FLT3 inhibitors have been reported, and several, such as SU-11248, CHIR-258, PKC412, CEP-701, MLN518, and AC220, have been investigated in clinical trials.12−18 However, with few exceptions, such as AC220, the clinical efficacy of these FLT3 inhibitors in patients with AML has been unimpressive, with responses characterized by transient clearance of leukemia blast cells, followed by progressive disease or resistance to the treatment.9 The causes of this unimpressive efficacy are complex, but a critical factor may be aberrant VEGF signaling in the bone marrow of AML patients, which promotes autocrine AML blast cell proliferation, survival, and chemotherapy resistance and mediates paracrine vascular endothelial cell-controlled angiogenesis in AML.19−24 More recently, researchers focusing on bone marrow stem cell niches have demonstrated a role for VEGF signaling in the preservation of several cell types within these niches.23,25−28 Bone marrow niches have been proposed to be a protective microenvironment for AML cells that could be responsible for relapses in AML patients.19 These studies have indicated that therapeutics targeting VEGF signaling could Received: October 20, 2012

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dx.doi.org/10.1021/jm301537p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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chloride with commercially available 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (34) with a yield of 56% (Scheme 1).31 All the target compounds can be classified into two categories: 4-N-pyrazolopyrimidine derivatives 1−5 and 4-Opyrazolopyrimidine derivatives 6−33. Scheme 2 depicts the general synthetic route for the 4-N-pyrazolopyrimidine derivatives 1−5. First, intermediates 38−42 were prepared by a synthetic method similar to that described previously.32 Target compounds 1−5 were then obtained by a classic nucleophilic substitution reaction between 38−42 and the previously synthesized intermediate 35 in 1-butanol in the presence of concentrated HCl in 74−80% yield. The 4-O-pyrazolopyrimidine derivatives 6−9, 20, and 25−33 were obtained via the two routes outlined in Scheme 3.31,33 In route 1, condensation of 4-aminophenol with the corresponding isocyanates in THF provided the desired urea derivative intermediates 62−75 in high yields with short reaction times. Subsequent nucleophilic substitution of intermediate 35 with the phenolic hydroxyl group of the urea derivatives 62−75 in the presence of sodium hydroxide produced the target compounds 6−9, 20, and 25−33 in low yield (see Scheme 3). In route 2, 4-aminophenol initially forms 4-aminophenolate in a sodium hydroxide solution. Selective nucleophilic substitution reaction of phenolate anion (bearing an oxygen anion) with the chloride of compound 35 resulted in intermediate 76 in a yield of 32% since the nucleophilicity of the oxygen anion is relatively stronger than that of nitrogen in 4-aminophenol.33 Then target compounds 6−9, 20, and 25−33 were produced in excellent yield by the reaction of intermediate 76 with the corresponding isocyanates under reflux in acetonitrile (see Scheme 3). The second route gave high yields and thus was adopted here. In addition, intermediate 76 was reacted with commercially available 4-bromobenzoic acid and 2-(4-bromophenyl)acetic acid to give compounds 18 and 19, respectively, which contain an amide or acetamide moiety (Scheme 3). Compounds 10 and 11, which contain a methyl substituent at the 1-NH of pyrazolopyrimidine, were prepared from the corresponding analogues 6 and 9 (Scheme 4). In this reaction, sodium hydroxide was used to deprotonate the NH group in DMF at ambient temperature. Iodomethane was then added, and the mixture continued to react overnight. The 4-O-pyrazolopyrimidine derivatives 12−17 were synthesized as outlined in Scheme 5. Urea compounds 12 and 13 were obtained via the second route (see Scheme 3), as described above. Compounds 14−17 were obtained following the condensation of commercially available carboxylic acids and intermediate 77 in a mixture of 1.0 equiv of HOBT, 1.0 equiv of EDCI, and 1.5 equiv of DIEA. Compounds 12−17 were all obtained in high yields. Scheme 6 depicts the general reaction route of compounds 21−24. Intermediate 76, which was obtained via route 2 in Scheme 3, was reacted with various isocyanates to give the final products with yields ranging from 44% to quantitative.

potentially prevent relapses of AML. Because VEGFR2 is the key receptor of VEGF, we hypothesized that a dual FLT3/ VEGFR2 tyrosine kinase inhibitor could function in both clearing leukemia blast cells and preventing relapses of AML. To identify potent dual FLT3/VEGFR2 inhibitors, we recently performed virtual screening against several commercial chemical databases and an in-house chemical library, followed by an in vitro kinase inhibition assay. From the hit compounds obtained, we chose the compound 1-(4-(1H-pyrazolo[3,4d]pyrimidin-4-ylamino)phenyl)-3-(3-methoxyphenyl)urea (1; Figure 1a) for further structural optimization. Compound 1

Figure 1. (a) Structure of compound 1. (b) Schematic showing subgroups or atoms that were the focus of structural modifications.

was chosen because the 1-(4-(1H-pyrazolo[3,4-d]pyrimidin-4ylamino)phenyl)-3-phenylurea scaffold is novel for FLT3 and VEGFR2 inhibitors (no FLT3 or VEGFR2 inhibitors possess this scaffold) and has a relatively low molecular weight (375 Da).29,30 Compound 1 inhibits both FLT3 and VEGFR2 with a half-maximal inhibitory concentration (IC50) of 1.278 μM for FLT3 and 0.305 μM for VEGFR2. Thus, the potency of compound 1 against FLT3 and VEGFR2 is poor and must be optimized further. The purpose of this study was to perform structural modifications to optimize the potency of compound 1 against both FLT3 and VEGFR2. A series of novel pyrazolo[3,4-d]pyrimidine derivatives were synthesized and tested for their in vitro anti-AML activity and antiangiogenic effect. The most potent derivatives were then subjected to further in vitro and in vivo assays. In this paper, we report the chemical synthesis and structure−activity relationship (SAR) studies of this novel series of compounds, as well as the in vitro and in vivo anti-AML activities of the most active compounds.

2. CHEMISTRY To explore the SAR of the pyrazolo[3,4-d]pyrimidine compound class, a series of derivatives were synthesized according to the synthetic routes outlined in Schemes 1−6. All of the target compounds were prepared from the general intermediate 4-chloro-1H-pyrazolo[3,4-d]pyrimidine (35), which was obtained through reaction of phosphorus oxyScheme 1a

3. RESULTS AND DISCUSSION 3.1. SAR of Pyrazolo[3,4-d]pyrimidine Derivatives Using Cell- and Transgenic-Zebrafish-Based Assays. For SAR analysis, cell- and transgenic-zebrafish-based assays were used. To screen FLT3 kinase inhibitors, two tumor cell lines, MV4-11 and HeLa, were chosen. MV4-11 is a human AML cell line whose viability depends on the activation of mutated FLT3 kinase (FLT3-ITD).34−37 HeLa is a human

a

Reagents and conditions: (a) phosphorus oxychloride, DIEA, toluene, reflux, 56%. B



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Scheme 2a

a Reagents and conditions: (b) triphosgene, Et3N, THF, rt; (c) RNH2, THF, 45 °C, 72−96% for two steps; (d) Fe, NH4Cl, EtOH, H2O, 91−98%; (e) HCl, 1-BuOH, 100 °C, 74−80%.

Scheme 3a

Reagents and conditions: (f) triphosgene, THF, Et3N, rt; 4-aminophenol, THF, 45 °C, 64−99%; (g) sodium hydroxide, H2O, THF, 60 °C, 21− 34%; (h) 4-aminophenol, sodium hydroxide, H2O, THF, 60 °C, 32%; (i) substituted anilines, triphosgene, THF, Et3N, rt; acetonitrile, reflux, 61− 84%; (j) HOBT, EDCI, DIEA, THF, rt to reflux, 66−73%. a

utilized as a less costly and more rapid in vivo method for the screening of agents with antiangiogenic activity.38−41 The combination of cell- and transgenic-zebrafish-based assays is an effective method for identifying dual inhibitors of FLT3 and VEGFR2. There are some advantages of performing SAR analysis with cell- and transgenic-zebrafish-based assays rather than enzymatic assays for hit/lead optimization. For example, SAR analysis based on cellular assays may permit the evaluation of both the intrinsic activity of the compounds against the

cancerous cervical tumor cell line whose viability is independent of FLT3; Hela cells were used to rule out activity due to non-FLT3-mediated effects (“off-target” or cellular toxic effects). Compounds are likely better FLT3 inhibitors if they have high potency against MV4-11 cells but low or no activity against HeLa cells. To screen VEGFR2 kinase inhibitors, an in vivo live fluorescent transgenic zebrafish (FLK-1:EGFP) assay was adopted; VEGFR2 is one of the key regulators of angiogenesis, and the transgenic zebrafish assay has been C



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Scheme 4a

Scheme 6a

a

Reagents and conditions: (k) iodomethane (MeI), sodium hydroxide, DMF, rt, 65−70%.

a Reagents and conditions: (n) amine, triphosgene, THF, Et3N, rt; acetonitrile, reflux, 44−77%.

protein target and the ability of the compounds to permeate the cell wall. The transgenic zebrafish assay combines the physiological complexity of an in vivo vertebrate model with the speed of high-throughput screening. Thus, SAR studies based on both cell (in vitro) and zebrafish (in vivo) assays can potentially accelerate the hit/lead optimization process to rapidly obtain an agent with bioactivity both in vitro and in vivo. Certainly, there may exist some possibility of off-target effects contributing to cell growth inhibitory potency or antiangiogenic activity, although some strategies, such as the use of Hela cells, have been adopted to rule out this possibility. Thus, enzymatic assays were also performed but only on compounds with higher potency in cell and zebrafish assays. The most active compounds in the enzyme, cell, and zebrafish assays were selected for further in vivo experiments to evaluate their anti-AML activity. The SAR analysis below focuses on four positions: the 4position linker atom (Y), the N-1 position of pyrazolo[3,4d]pyrimidine (R1), the bridge group that connects rings A and B, and the ring B moiety (see Figure 1b). 3.1.1. Impact of the 4-Position Linker Atom (Y) of Pyrazolo[3,4-d]pyrimidine. Table 1 shows the bioactivities of two series of 1H-pyrazolo[3,4-d]pyrimidine derivatives, namely, 4-anilino-1H-pyrazolo[3,4-d]pyrimidine and 4-phenoxy-1Hpyrazolo[3,4-d]pyrimidine derivatives, which contain a nitrogen (−NH) and oxygen (−O) atom at the 4-position linker site, respectively. The cell growth inhibitory activities (IC50) against MV4-11 of the 4-phenoxy-1H-pyrazolo[3,4-d]pyrimidine derivatives 6, 7, 8, 9, and 22 were 0.020, 0.083, 0.037, 0.005, and

1.077 μM, respectively, corresponding to a higher potency than that of the corresponding 4-anilino-1H-pyrazolo[3,4-d]pyrimidine derivatives 1, 2, 3, 4, and 5 (IC50 = 1.65, 1.24, 1.69, 0.26, and 2.05 μM, respectively). Here one may argue that the lower MV4-11 cell growth inhibitory potency observed with compounds 1−5 might be due to poorer cell permeability. To clarify this issue, we tested the enzymatic inhibitory potency of compounds 1−5 against the FLT3 kinase, and the results showed that compounds 1−5 had a poor FLT3 kinase inhibitory potency (1 μM < IC50 < 10 μM). These results together with the fact that many of their analogues displayed a higher MV4-11 cell growth inhibitory potency (see below) demonstrated that the lower MV4-11 cell growth inhibitory potency observed with compounds 1−5 should not be, at least not mainly, due to poorer cell permeability. All of the 1Hpyrazolo[3,4-d]pyrimidine derivatives had IC50 values greater than 10 μM against HeLa cells (the same phenomenon will be seen below), indicating that the cell growth inhibitory activities of these compounds against the MV4-11 cells were not due to cellular toxicity. By contrast, all of the 4-phenoxy-1H-pyrazolo[3,4-d] pyrimidine derivatives also exhibited slightly more potent antiangiogenic activity in zebrafish assays than did the corresponding 4-anilino-1H-pyrazolo[3,4-d]pyrimidine derivatives. Thus, we can conclude that the 4-phenoxy-1H-pyrazolo[3,4-d]pyrimidine derivatives are more potent than the 4anilino-1H-pyrazolo[3,4-d]pyrimidine derivatives, in terms of their bioactivities in both cell and zebrafish assays. 3.1.2. Substitution Effect of the N-1 Position (R1) of Pyrazolo[3,4-d]pyrimidine. The possible influence of the

Scheme 5a

Reagents and conditions: (h) 3-aminophenol, sodium hydroxide, THF, H2O, 60 °C, 38%; (i) for 12 and 13, substituted aniline, triphosgene, THF, Et3N, rt; acetonitrile, reflux, 87−89%; for 14 and 15, HOBT, EDCI, DIEA, THF, rt to reflux, 71−74%; (m) HOBT, EDCI, DIEA, THF, rt to reflux, 79−86%. a

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Table 1. Cell Growth Inhibitory Activities of Pyrazolo[3,4-d]pyrimidine Derivatives with Different 4-Position Linker Atoms (Y) and Different Substituents at the N-1 Position (R1) of Pyrazolo[3,4-d]pyrimidine against FLT3-ITD-Dependent MV4-11 Cells and FLT3-Independent HeLa Cells, as Well as Their Antiangiogenic Activities in Transgenic-Zebrafish-Based Assays

a

Each compound was tested in triplicate; the data are presented as the mean ± SD.

substitution of the N-1 position (R1) of pyrazolo[3,4d]pyrimidine was examined next. The N-1 hydrogen of the pyrazolo[3,4-d]pyrimidine moiety of the two most potent 4phenoxy-1H-pyrazolo[3,4-d]pyrimidine derivatives (6 and 9) was replaced by a methyl group, yielding compounds 10 and 11 (Table 1), respectively. Compounds 10 and 11 exhibited much lower antiproliferative and antiangiogenic activities than their unsubstituted counterparts 6 and 9, implying that substitution at the N-1 position of pyrazolo[3,4-d]pyrimidine is not beneficial for activity. 3.1.3. Influence of Different Bridge Groups Connecting Ring A and Ring B. The bioactivities of a series of 4-phenoxy1H-pyrazolo[3,4-d]pyrimidine derivatives with different bridge

groups connecting ring A and ring B, including p-urea, m-urea, p-amide, m-amide, p-acetamide, and m-acetamide, are presented in Table 2.42 Replacement of the urea group by an amide or acetamide led to a decreased bioactivity (12 vs 14 and 16; 13 vs 15 and 17; 7 vs 18 and 19) in the cellular assay. Furthermore, a comparison of the bioactivities of the compound pairs [12 (4.53 μM), 9 (0.005 μM)] and [13 (7.82 μM), 7 (0.083 μM)], in which the urea group is attached to the para- or metaposition, respectively, indicated that substitution at the paraposition was preferred to substitution at the meta-position to increase bioactivity in the cellular assay. 3.1.4. Influence of Variation of the Ring B Moiety. Table 3 presents the bioactivities of a series of 4-phenoxy-1HE



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based assays. The three most potent compounds, 9, 27, and 33, displayed nanomolar potencies at the cellular level and low micromolar potencies in the zebrafish assay. The kinase inhibitory potencies (IC50) against FLT3 and VEGFR2, as well as the binding affinity (Kd, dissociation constant) for FLT3-ITD of the three compounds were then measured, and the results are presented in Table 4. The three compounds are all potent FLT3 and VEGFR2 inhibitors, and their kinase inhibitory activities are at least as potent as that of sorafenib, which is a multikinase inhibitor and can potently inhibit VEGFR2 and FLT3; sorafenib is currently in clinical studies for the treatment of AML.43,44 Of note is that compounds 27 and 33 showed lower nanomolar binding affinity (Kd) against the FLT3-ITD mutant, which is at least 10 times more potent compared with sorafenib. We subsequently performed a preliminary in vivo anti-AML study in a xenograft MV4-11 mouse model. Compound 33 was the most active in this study (see Figure 2). This increased activity could be due to the potentially increased bioavailability of compound 33 compared with the other compounds. Further in-depth in vitro (including enzymatic and cellular assays) and in vivo anti-AML studies were subsequently performed with compound 33. 3.2. Enzymatic Activity of Compound 33 against Various Kinases. The kinase inhibition profile of compound 33 against a panel of selected recombinant human protein kinases is presented in Table 5. Compound 33 potently inhibited FLT3 and VEGFR2 kinases (IC50 = 0.039 μM for FLT3 and 0.012 μM for VEGFR2). Compound 33 also exhibits considerable potency against several other kinases, including cRAF (0.072 μM), PDGFRα (0.223 μM), PDGFRβ (0.408 μM), c-Kit (0.507 μM), and FGFR2 (1.805 μM). Compound 33 displayed almost no inhibitory activity against 23 other tested protein kinases. These data demonstrate that compound 33 is a multikinase inhibitor that potently inhibits FLT3 and VEGFR2 kinases. Furthermore, a primary kinase selectivity assay was performed through measuring the binding affinities of compound 33 with a large set of representative kinases. A total of 131 kinases were selected; these kinases cover all of the kinase subfamilies, and most of them are common kinases. Compound 33 was screened against the kinase set at a fixed concentration of 10 μM. The results, shown in Figure S1 and Table S1 (see the Supporting Information), indicate that compound 33 also has a considerable binding affinity for a few of the kinases in addition to FLT3 and VEGFR2. The calculated selectivity scores, S(1), S(10), and S(35), whose definitions are given in the Experimental Section, are 0.076, 0.122, and 0.206, respectively. 3.3. In Vitro Cell Growth Inhibitory Activity of Compound 33. The cell growth inhibitory potency of compound 33 against various leukemia and solid tumor cell lines was examined, and the results are presented in Table 6. As noted previously, compound 33 potently inhibits the viability of FLT3-driven AML MV4-11 cells, with an IC50 value of 0.004 μM (also see Figure 3a). It exhibited weak inhibitory activity against several other cell lines, including Jurkat (leukemia, IC50 = 8.72 μM), MKN45 (gastric cancer, IC50 = 8.23 μM), MDAMB-468 (breast cancer, IC50 = 6.68 μM), and TT (thyroid cancer, IC50 = 3.42 μM). Negligible activity against the remaining 16 human cancer cell lines was observed. These results indicate that compound 33 is relatively selective for the human AML cell line MV4-11.

Table 2. 4-Phenoxy-1H-pyrazolo[3,4-d]pyrimidine Derivatives with Different Bridge Groups Connecting Rings A and B, Together with Their Cell Growth Inhibitory Activities against FLT3-ITD-Dependent MV4-11 Cells and FLT3-Independent HeLa Cells and Their Zebrafish-Based Antiangiogenic Activities

a

Each compound was tested in triplicate; the data are presented as the mean ± SD.

pyrazolo[3,4-d] pyrimidine derivatives containing a variety of moieties at the ring B position. From Table 3, we can see that, compared with other moieties at the ring B position, including a six-membered heteroaromatic ring (21) and other sixmembered cyclic groups (22−24), a phenyl group at the ring B position yielded a more potent compound (20) in both the cell and zebrafish assays. We then explored the influence of different substituents on the phenyl ring (ring B). It is clear that, compared with compound 20, which has no substituent on ring B, substitution at the meta- and/or para-position of ring B considerably increased the potency of cell growth inhibition against MV4-11. Substitution on ring B also increased the antiangiogenic potency, with the exception of compound 26, which contains a morpholine ring. Furthermore, for both the cell growth inhibitory activity against MV4-11 and antiangiogenic activity, compounds with substituents at the metaposition were identically potent or slightly more potent compared with compounds with substituents at the paraposition (see 27 vs 9, 29 vs 25). Collectively, the structural optimization and SAR studies led to the discovery of a number of compounds that exhibited both high potency against FLT3-driven human AML MV4-11 cells and a considerable antiangiogenic effect in transgenic-zebrafishF



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Table 3. 4-Phenoxy-1H-pyrazolo[3,4-d]pyrimidine Derivatives Containing Various Moieties at the Ring B Position Together with Their Cell Growth Inhibitory Activities against FLT3-ITD-Dependent MV4-11 Cells and FLT3-Independent Hela Cells and Zebrafish-Based Antiangiogenic Activities

a

Each compound was tested in triplicate; the data are presented as the mean ± SD. G



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concentration of 0.10 μM, an apoptosis rate of 35.8% was observed. The ability of compound 33 to inhibit the activation of FLT3 and downstream signaling proteins in intact cells was assessed by Western blot analysis. After a 5 h treatment with increasing concentrations of compound 33, MV4-11 cells were harvested and lysed for an IP/wt assay. As shown in Figure 3c, compound 33 inhibited FLT3 phosphorylation in a dose-dependent manner. Consistent with the downregulation of the phosphorylation of FLT3, the phosphorylation of the downstream signaling proteins STAT5 and ERK1/2 was also significantly inhibited at concentrations of compound 33 >0.003 μM (Figure 3c). The observed IC50 value was approximately 0.003 μM for phosphorylation of FLT3, 0.003 μM for STAT5, and 0.001 μM for Erk1/2. Here we have to point out that inhibition of VEGF signaling in intact MV4-11 cells was not measured because MV-411 cells express very low levels of VEGFR2, below that which can be detected by Western blot. VEGFR2 inhibition should not significantly contribute to the cell viability of MV4-11 cells. As mentioned before, aberrant VEGF signaling often exists in the bone marrow of AML patients, which has been demonstrated to be associated with drug resistance and relapses of AML after treatment with FLT3 inhibitors. Blockade of VEGF signaling is expected to play important roles in preventing drug resistance and relapses of AML. Thus, we shall in the next section examine the suppressive ability of compound 33 against the VEGF signaling using transgenic-zebrafish-based assays. 3.5. Antiangiogenic Activity of Compound 33. The effect of compound 33 on embryonic angiogenesis in zebrafish was examined. Figure 4a shows 33hpf zebrafish embryos treated with 1.25, 2.5, and 5 μM 33, as well as a blank control. Treatment of live fish embryos with compound 33 completely blocked the formation of intersegmental vessels (ISVs) at a concentration of 5 μM while preserving fluorescence in the doral aorta and major cranial vessels. At 1.25 or 2.5 μM 33, the formation of intersegmental vessels was considerably inhibited compared with that of the vehicle control group, indicating a dose-dependent inhibition pattern. The IC50 value was approximately 2.5 μM (Figure 4b). 3.6. In Vivo Effects of 33 against sc MV4-11 Tumor Xenografts. The in vivo anti-AML activity of compound 33 was evaluated using the FLT3-ITD-positive MV4-11 xenograft model. When the tumor grew to a volume of 300−500 mm3, the mice were grouped and treated orally once daily with 1, 3, or 10 mg/kg/d of compound 33 for 18 days. The tumor

Table 4. Kinase Inhibitory Potency against FLT3 and VEGFR2 for the Most Active Compounds in Cell and Zebrafish Assays compd

FLT3 IC50, μM

VEGFR2 IC50, μM

FLT3-ITD Kd, μM

9 27 33 sorafenib

0.032 0.009 0.039 0.033

0.038 0.011 0.012 0.090

0.91 0.0066 0.0043 0.079

Figure 2. Preliminary in vivo anti-AML assays. Daily oral administration of compounds 9, 27, and 33 at concentrations of 5, 7.5, and 5 mg/kg/d, respectively, was initiated when the MV4-11 tumors reached approximately 400 mm3 in volume (three mice per group).

Table 5. Kinase Inhibition Profile for Compound 33 against Human FLT3/VEGFR2 and a Panel of Other Selected Protein Kinases kinase

IC50, μM

kinase

IC50, μM

kinase

IC50, μM

Flt3 VEGFR2 c-RAF PDGFRα PDGFRβ c-Kit FGFR2 EGFR ErBB2 ErBB4

0.039 0.012 0.072 0.223 0.408 0.507 1.805 >10 >10 >10

Pim-1 Syk aurora A aurora B PLK1 CDK2 CHEK1 CAMK4 CTK DLK

>10 >10 >10 >10 >10 >10 >10 >10 >10 >10

DMPK ERN1 IGF1R MLK1 MEK1 PAK1 PAK2 PAK4 ERK Lck

>10 >10 >10 >10 >10 >10 >10 >10 >10 >10

3.4. Apoptosis Assays and Signaling Inhibition in MV4-11 Cells. A flow cytometry assay was performed to examine cell apoptosis upon treatment with compound 33. Compound 33 induced apoptosis in MV4-11 cells in a concentration-dependent manner (see Figure 3b). At a

Table 6. Cell Growth Inhibitory Potency of Compound 33 against Various Leukemia and Solid Tumor Cell Lines

a

tumor type

cell line

IC50,a μM

tumor type

cell line

IC50,a μM

leukemia, AML leukemia, ALL leukemia, APL leukemia, CML cervical cancer colon cancer gastric cancer breast cancer breast cancer thyroid cancer

MV4-11 Jurkat HL60 K562 Hela HCT116 MKN45 MDA-MB-468 MCF-7 TT

0.004 ± 0.0004 8.72 ± 0.440 >10 >10 >10 >10 8.23 ± 0.485 6.68 ± 0.652 >10 3.42

liver cancer liver cancer lung cancer lymphoma lymphoma lymphoma colorectal carcinoma prostate cancer multiple myeloma ovarian cancer ovarian cancer

Bel7402 SMMC7721 A549 Raji U937 Karpass299 LoVo DU145 U266 SKOV3 SK

>10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10

Each compound was tested in triplicate; the data are presented as the mean ± SD. H



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Figure 3. (a) Growth inhibitory profile of compound 33 against MV4-11 cells using the MTT assay method. (b) A flow cytometry assay was performed to examine cell apoptosis. MV4-11 cells (1 × 105) were harvested after treatment with various drug concentrations for 24 h. Cells were stained with an Annexin V & PI Kit and analyzed by flow cytometry. Percentages in the bottom right quadrants are the ratio of cells in early apoptosis (annexin V positive and PI negative). Percentages in the upper right quadrants are the ratio of cells in late apoptosis (Annexin V positive and PI positive). (c) Western blot analysis was used to examine the phosphorylation of FLT3 and downstream signals in MV4-11 cells treated with different doses of compound 33 for 20 h.

volumes were measured every 3 d. Treatment with compound 33 at 10 mg/kg/d resulted in rapid and complete tumor regression in all mice in this group. Compound 33 treatment at 3 mg/kg/d stopped tumor growth and resulted in a slight regression of tumor growth (Figure 5a). Moreover, throughout the experiment, no significant weight loss or any other obvious signs of toxicity were observed for any of the compound 33treated mice (Figure 5b). Compound 33 was also evaluated for its effects on the tumor mitotic index (Ki67) and apoptosis, as well as the FLT3 signaling, using histological and immunohistochemical techniques. Similar to the tumor xenograft models, a dose of 10 mg/ kg/d of compound 33 was administered by oral gavage for the MV4-11 model. After treatment for 3 or 4 d, tumors were collected and analyzed. Tumor tissues from the vehicle group stained strongly with Ki67, indicating a large number of highly proliferative cells. Conversely, the tumor tissues from the compound 33-treated groups exhibited significantly fewer Ki67positive cells (Figure 6a). Furthermore, the TUNEL data

demonstrated an obvious increase in the percentage of apoptotic cells in a time-dependent manner (Figure 6b). In addition, the phosphorylation of STAT5, which is an important downstream signaling protein of FLT3 signaling, was also significantly reduced in MV4-11 tumor tissues of the compound 33-treated group compared with the vehicle group (Figure 6c), indicating that the observed efficacy in vivo was consistent with FLT3 signaling inhibition. It is important to mention again that the MV4-11 cell line expresses very low levels of VEGFR2. VEGFR2 inhibition should not have a significant effect on the AML cell viability, but is expected to contribute to blockade of aberrant VEGF signaling that often exists in the bone marrow of AML patients, which may be helpful in preventing relapses of AML. We acknowledged that the in vivo MV4-11 xenograft model used here is not suitable for examining the effects of compound 33 on preventing relapses of AML. Other more sophisticated models such as orthotopic AML models should be adopted for this purpose; the related studies are still under way. I



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Figure 5. (a) Daily oral administration of compound 33 at concentrations of 1, 3, and 10 mg/kg/d and sorafenib at a dose of 3 mg/kg/d was initiated when the MV4-11 tumors reached approximately 500 mm3 in volume (six mice per group). (b) Average body weights for 33-treated mouse groups in the MV4-11 xenograft model. (c) Pharmacokinetics of compound 33 in SD rats (five rats per group). The rats were treated orally with 10 mg/kg/d of compound 33, and plasma was collected via jugular vein cannulation. Finally, compound concentrations in the plasma were detected by liquid−liquid extraction followed by LC−MS detection.

Figure 4. (a) 33hpf zebrafish embryos treated with blank control or 1.25, 2.5, or 5 μM 33. ISV budding and outgrowth were almost completely inhibited by treatment with 5 μM 33. (b) Statistics of ISV length in embryos treated with blank control and compound 33. ISVs above the yolk extension were counted. Key: columns, mean; bars, SD (n = 11; ANOVA; **, P < 0.01 vs the control).

once-daily dose of compound 33 at 10 mg/kg for 18 days led to complete tumor regression in the MV4-11 xenograft model. Preliminary pharmacokinetic studies indicated that compound 33 possesses good pharmacokinetic properties. By the way, since the AML MV4-11 cell line used in this study expresses very low levels of VEGFR2, inhibition of VEGFR2 should not significantly contribute to the suppression of tumor cell growth, but might play some important roles in preventing relapses of AML due to the fact that aberrant VEGF signaling often exists in the bone marrow of AML patients. Issues related to the VEGFR2 inhibition and its roles in preventing the relapses of AML still need further intense studies.

3.7. Pharmacokinetic Characteristics of Compound 33. The preliminary pharmacokinetic characteristics of compound 33 following po administration to rats were also analyzed. As shown in Figure 5c, the compound was absorbed well, achieving a maximum plasma level (Cmax) of 15.12 μg/mL within 7 h after oral administration at a dose of 10 mg/kg/d. The measured plasma protein binding value was 87.96%. Furthermore, the apparent plasma half-life was approximately 12.6 h, the AUC0−24h was approximately 318817.4 μg/(L·h), and the oral bioavailability was about 66.7%. All of these indicated that compound 33 has good pharmacokinetic properties.

5. EXPERIMENTAL SECTION 5.1. Chemistry Methods. All reagents were purchased and used without further purification unless otherwise indicated. Reactions were monitored by thin-layer chromatography (TLC) on Merck silica gel 60 F-254 thin-layer plates. All of the final compounds were purified to >95% purity, as determined by high-performance liquid chromatography (HPLC). HPLC analysis was performed on a Waters 2695 HPLC system with the use of a Kromasil C18 column (4.6 mm × 250 mm, 5 um). 1H NMR and 13C NMR spectra were recorded on a Bruker AV-400 spectrometer at 400 and 100 MHz, respectively. Chemical shifts (δ) are listed in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. Multiplicities are given as s (singlet), d (doublet), dd (double−doublet), t (triplet), q (quadruplet), m (multiplet), and br s (broad signal). Low-resolution and high-resolution mass spectral (MS) data were acquired on an Agilent 1100 series LC−MS instrument with UV detection at 254 nm in low-resonance electrospray mode (ESI). 5.1.1. 4-Chloro-1H-pyrazolo[3,4-d]pyrimidine (35). To a cooled solution of 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (34; 5.0 g, 36.7 mmol) and N,N-diisopropylethylamine (3.0 mL, 18.4 mmol) in

4. CONCLUSIONS In this paper, a series of pyrazolo[3,4-d]pyrimidine derivatives was synthesized, and an SAR analysis of these compounds based on cell and zebrafish assays led to the identification of three pyrazolo[3,4-d]pyrimidine derivatives with considerable potency. Further in-depth in vitro and in vivo assays were performed with compound 33, which exhibited the highest in vivo anti-AML activity in a preliminary in vivo assay. The kinase inhibition profile revealed that compound 33 was a multikinase inhibitor that potently inhibits FLT3 and VEGFR2 and showed good kinase spectrum selectivity. Western blot analysis demonstrated that compound 33 significantly inhibited FLT3 phosphorylation and the activation of downstream signaling proteins. In vivo anti-AML activity assays showed that an oral, J



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Figure 6. Ki67, TUNEL, and p-STAT5 detection of MV4-11 tumor tissues after treatment with compound 33 for 72 or 96 h. At 24 h after the final dose of compound 33, the tumor samples were collected for analysis. toluene was slowly added phosphorus oxychloride (16.8 mL, 183.5 mmol). The reaction mixture was warmed to room temperature and refluxed for 5 h. After evaporation of the organic solvent, the residue was cooled to room temperature and treated with excess ice−water. At this time, a buff solid was formed and collected by filtration. The crude product was dried in a vacuum oven to give the title compound 35 (3.2 g, 56%). This product was used in the next step without purification. 1H NMR (400 MHz, DMSO-d6): δ 14.12 (s, 1H), 9.32 (s, 1H), 7.55 (s, 1H). LC−MS: m/z 155.0 [M + H]+. 5.1.2. 1-(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-ylamino)phenyl)-3-(3methoxyphenyl)urea (1). To a mixture of 1-(4-aminophenyl)-3-(3methoxyphenyl)urea (180 mg, 0.7 mmol) in 1-butanol (10 mL) was added 6 N HCl (0.2 mL). The reaction mixture was stirred at room temperature for 20 min. Then a solution of 35 (108 mg, 0.7 mmol) in 1-butanol (10 mL) was added to the reaction mixture, followed by heating to 100 °C for 3.5 h. The solvent was removed by rotary evaporation. The crude mixture was added to 100 mL of water, and the pH was adjusted to 7−8 with K2CO3. The aqueous layer was extracted with ethyl acetate (2 × 120 mL). Then the combined organic layers were dried over MgSO4 and concentrated. The residue was purified by column chromatography (eluent gradient CH2Cl2:MeOH = 35:1 to 20:1) and recrystallized from EtOAc and petroleum to afford the product 1 (205 mg, 78% yield, 98% HPLC purity). 1H NMR (400 MHz, DMSO-d6): δ 13.59 (s, 1H), 9.93 (s, 1H), 8.69 (d, J = 5.6 Hz, 2H), 8.35 (s, 1H), 8.15 (br s, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 8.8 Hz, 2H), 7.21−7.16 (m, 2H), 6.93 (d, J = 8.0 Hz, 1H), 6.55 (dd, J = 2.4 Hz, J = 5.6 Hz, 1H), 3.74 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 160.2, 155.8, 155.0, 153.0, 141.5, 133.8, 132.9, 130.0, 122.8, 119.1, 110.9, 107.6, 104.3, 100.8, 55.4. LC−MS: m/z 376.1 [M + H]+. Compounds 2−5 were synthesized using the same methodology. Experimental data for compound 2−5 are given in the Supporting Information. 5.1.3. General Procedure for the Synthesis of Ureas 62−75. A solution of substituted anilines (10.0 mmol) dissolved in THF (80 mL) was slowly dripped into a stirred solution of triphosgene (2.98 g, 10.0 mmol) in THF (10 mL) by using a constant-pressure dropping funnel. NEt3 (3 mL, 21.0 mmol) was then added slowly to the reaction mixture after the anilines were added. After evaporation of the solvent, the residue was taken up in THF (80 mL), and 4-aminophenol (0.93 g, 8.5 mmol) was added directly to the residue. The reaction mixture was stirred at 45 °C for 2 h, and the solvent was subsequently removed

in vacuo. The residue obtained was dissolved in acetone (4 mL), and upon addition of water, a solid precipitated. This solid was collected, washed with water and Et2O, and dried under vacuum to give the products 62−75 (64−99%). 5.1.4. 4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yloxy)aniline (76). To a solution of 4-aminophenol (3.53 g, 32.4 mmol) in H2O (40 mL) was added a sodium hydroxide solution (1.30 g, 32.4 mmol in 40 mL of water). The mixture was stirred at room temperature for 30 min. Then the intermediate 35 (5.00 g, 32.4 mmol) in THF (40 mL) was slowly added, and the reaction mixture was heated to 60 °C for 1.5 h. The solvent was then partially evaporated on a rotary evaporator. The crude mixture was extracted with ethyl acetate (2 × 120 mL) and water. The combined organic layers were dried over MgSO4 and concentrated. The obtained residue was purified by column chromatography (eluent gradient CH2Cl2:MeOH = 100:1 to 60:1) without final recrystallization to give the product 76 (2.35 g, 32%) as a brown solid. 1H NMR (400 MHz, DMSO-d6): δ 14.07 (s, 1H), 8.50 (s, 1H), 7.67 (s, 1H), 6.96 (d, J = 8.8 Hz, 2H), 6.64 (d, J = 8.8 Hz, 2H), 5.20 (s, 2H). LC−MS: m/z 228.1 [M + H]+. 5.1.5. 1-(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(3methoxyphenyl)urea (6). Two methods were used to obtain the final product. Method A: A suspension of 1-(4-hydroxyphenyl)-3-(3methoxyphenyl)urea (0.35 g, 1.35 mmol) and sodium hydroxide (0.065 g, 1.62 mmol) in THF (10 mL) and water (2 mL) was stirred at room temperature for 30 min. Then the intermediate 35 (0.31 g, 2.0 mol) was added, and the resulting mixture was heated to 60 °C for 6 h. The solvent was concentrated in vacuo, treated with water (100 mL), and extracted with EtOAc (3 × 60 mL). The combined organic layers were removed in vacuo. The residue obtained was purified by column chromatography (eluent gradient CH2Cl2:MeOH = 70:1) and recrystallized from EtOAc and petroleum ether to provide 6 (0.13 g, 26.3% yield, 98% HPLC purity). Method B: A solution of 3methoxyaniline (0.62 g, 5.0 mmol) dissolved in THF (40 mL) was slowly dripped into a stirred solution of triphosgene (1.49g, 5.0 mmol) in THF (10 mL) by using a constant-pressure dropping funnel. NEt3 (1.5 mL, 10.5 mmol) was then added slowly to the reaction mixture after the aniline was added. After evaporation of the solvent, the residue was taken up in acetonitrile (80 mL), and compound 76 (1.02 g, 4.5 mmol) was added directly to the residue. Next the reaction mixture was stirred at 90 °C for 6 h, and the solvent was removed in vacuo. The residue obtained was purified by column chromatography (eluent gradient CH2Cl2:MeOH = 70:1) and recrystallized from K



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purity: 98%. 1H NMR (400 MHz, DMSO-d6): δ 14.14 (s, 1H), 9.10 (s, 1H), 8.93 (s, 1H), 8.51 (s, 1H), 8.04 (s, 2H), 7.61−7.51 (m, 4H), 7.32 (d, J = 7.6 Hz, 1H), 7.26 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 163.2, 156.7, 154.9, 152.6, 146.6, 140.5, 137.2, 131.8, 129.9, 122.3, 121.8, 119.7, 101.3. HRMS: m/z calcd for C19H13F3N6O2 [M + H]+ 415.1052, found 415.1062. 5.1.11. 1-(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(4chloro-3-(trifluoromethyl)phenyl)urea (33). The title compound was prepared from 76 and 4-chloro-3-(trifluoromethyl)aniline using method B, purified by column chromatography (eluent gradient CH2Cl2:MeOH = 75:1), and recrystallized from EtOAc and petroleum ether. Yield: 82%. HPLC purity: 99%. 1H NMR (400 MHz, DMSOd6): δ 14.14 (s, 1H), 9.22 (s, 1H), 8.99 (s, 1H), 8.51 (s, 1H), 8.13 (s, 1H), 8.05 (s, 1H), 7.64 (q, J = 6.8 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 163.2, 156.7, 154.9, 152.5, 146.7, 139.3, 137.1, 131.9, 131.8, 123.0, 122.3, 119.9, 101.3. HRMS: m/z calcd for C19H12ClF3N6O2 [M + H]+ 449.0662, found 449.0680. Experimental data for compound 7, 8, 11, 13−17, 19−26, and 28− 32 are given in the Supporting Information. 5.2. Kinase Inhibition, Binding Affinity Assays, and Kinase Selectivity. The kinase inhibition assays were performed according to the KinaseProfiler assay protocols of Upstate Biotechnology (Millipore). The Ambit in vitro KINOMEscan kinase binding assays (Ambit Biosciences) were employed to determine the binding affinity of the compounds. The kinase selectivity is described by the selectivity score, S-score, which is calculated by dividing the number of kinases that compounds bind to by the total number of distinct kinases tested, excluding mutant variants. Here S-score is defined as follows: S(X) = (number of nonmutant kinases with %Ctrl < X)/(number of nonmutant kinases tested). 5.3. Cell Lines and Cell Culture. All cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA). MV411 was maintained in IMDM culture medium according to ATCC guidelines. The other cell lines were grown in RPMI 1640 or DMEM culture medium containing 10% fetal bovine serum (v/v) in 5% CO2 at 37 °C. 5.4. Cell Viability Assays. The MTT assay was used to detect the viability of cells. Leukemia cells were collected and seeded in a 96-well plate at (1−4) × 104 cells per well. Different concentrations of inhibitors were added to the cells and incubated at 37 °C for 72 h. Then 20 μL of 5 mg/mL MTT reagent was added to each well and incubated for 2−4 h, and 50 μL of 20% acidified SDS was used to lyse the oxidative product. The other cell lines were seeded in 96-well plates at a density of (2−5) × 103 cells per well, and after 24 h, the medium in the plates was replaced by medium containing serial dilutions of inhibitors. Following a 72 h incubation, the MTT reagent was added for a 2−4 h incubation, and 100% DMSO was used to dissolve the oxidative product. Finally, the light absorption (OD) of the dissolved cells was measured at 570 nm using a SpectraMAX M5 microplate spectrophotometer (Molecular Devices). All experiments were performed in triplicate. The IC50 values were calculated using GraphPad Prism software. 5.5. Western Blot Analysis. After treatment with a series of concentrations of compound 33 for 20 h at 37 °C, MV4-11 cells were harvested, washed with ice-cold physiological saline, and lysed with RIPA lysis buffer (Beyotime) including 1% cocktail (Sigma-Aldrich). Cell lysates were separated by SDS−PAGE and electrotransferred onto PVDF membranes (Millipore). The PVDF membranes were incubated with each antibody and detected according to the immunoblot analysis principle. The antibodies were purchased from Cell Signaling Technology, with the exception of the anti-FLT3 antibody, which was obtained from Abcam. 5.6. Apoptosis Analysis in MV4-11 Cells. A total of 4 × 105 MV4-11 cells were plated in a six-well plate and treated with compound 33 for 24 h at 37 °C. After incubation, the cells were harvested and washed with PBS. The apoptosis ratio was analyzed using an Annexin V-FITC Apoptosis Analysis Kit (Tianjin Suangene Biotech Co.) and BD FACSCalibur.

EtOAc and petroleum ether to provide 6 (1.37 g, yield 81%, 98% HPLC purity). 1H NMR (400 MHz, DMSO-d6): δ 14.14 (s, 1H), 8.79 (s, 1H), 8.73 (s, 1H), 8.51 (s, 1H), 8.03 (s, 1H), 7.60 (d, J = 8.8 Hz 2H), 7.26−7.21 (m, 4H), 6.96 (d, J = 8.0 Hz, 1H), 6.57 (dd, J = 2.0 Hz, J = 6.0 Hz, 1H), 3.74 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 163.2, 159.6, 156.7, 154.9, 152.5, 146.4, 140.8, 137.5, 131.8, 129.5, 122.2, 119.4, 110.5, 107.2, 104.0, 101.3, 54.9. LC−MS: m/z 377.1 [M + H]+. 5.1.6. 1-(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(4(trifluoromethyl)phenyl)urea (9). The title compound was prepared from 76 and 4-(trifluoromethyl)aniline using method B, purified by column chromatography (eluent gradient CH2Cl2:MeOH = 70:1), and recrystallized from EtOAc and petroleum ether. Yield: 83%. HPLC purity: 98%. 1H NMR (400 MHz, DMSO-d6): δ 14.15 (s, 1H), 9.13 (s, 1H), 8.97 (s, 1H), 8.51 (s, 1H), 8.04 (s, 1H), 7.60−7.51 (m, 5H), 7.32 (d, J = 7.2 Hz, 1H), 7.26 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 163.2, 155.8, 155.0, 152.6, 146.6, 140.5, 137.3, 131.8, 130.0, 129.6, 122.3, 121.8, 119.7, 118.1, 114.1, 101.3. HRMS: m/z calcd for C19H13F3N6O2 [M + H]+ 415.1052, found 415.1064. 5.1.7. 1-(3-Methoxyphenyl)-3-(4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)urea (10). A suspension of compound 6 (752 mg, 2.0 mmol) and sodium hydroxide (80 mg, 2.0 mmol) in dry DMF (10 mL) was stirred at room temperature for 30 min. Iodomethane (0.15 mL, 2.4 mmol) was added, and the resulting mixture was stirred overnight. The reaction mixture was treated with water (100 mL) and extracted with EtOAc (3 × 60 mL). The combined organic layers were removed in vacuo. The residue obtained was purified by column chromatography (eluent gradient CH2Cl2: MeOH = 90:1) and recrystallized from EtOAc and petroleum ether to provide 10 (546 mg, 70%, 98% HPLC purity). 1H NMR (400 MHz, DMSO-d6): δ 8.82 (s, 1H), 8.75 (s, 1H), 8.55 (s, 1H), 8.05 (s, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.26−7.17 (m, 4H), 6.95 (d, J = 8.4 Hz, 1H), 6.57 (d, J = 8.4 Hz, 1H), 4.04(s, 3H), 3.74(s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 163.3, 159.7, 154.9, 154.8, 152.5, 146.4, 140.8, 137.6, 130.9, 129.5, 122.1, 119.4, 110.5, 107.2, 104.0, 101.7, 54.9, 34.0. LC−MS: m/z 391.4 [M + H]+. 5.1.8. 1-(3-(1H-Pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(4(trifluoromethyl)phenyl)urea (12). The title compound was prepared from 77 and 4-(trifluoromethyl)aniline using method B, purified by column chromatography (eluent gradient CH2Cl2:MeOH = 75:1), and recrystallized from EtOAc and petroleum ether. Yield: 89%. PHLC purity: 97%. 1H NMR (400 MHz, DMSO-d6): δ 14.16 (s, 1H), 9.17 (s, 1H), 9.03 (s, 1H), 8.53 (s, 1H), 8.10 (s, 1H), 7.66−7.57 (m, 5H), 7.42 (t, J = 8.0 Hz, 1H), 7.33 (d, J = 8.8 Hz, 1H), 6.97 (d, J = 7.2 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6): δ 162.8, 156.7, 154.9, 152.3, 152.1, 143.2, 140.8, 131.9, 130.0, 126.1, 123.1, 122.0, 121.7, 117.9, 115.9, 115.5, 111.7, 101.4. LC−MS: m/z 415.1 [M + H]+. 5.1.9. N-(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-4-bromobenzamide (18). A mixture of 4-bromobenzoic acid (265 mg, 1.32 mmol), 1-hydroxybenzotriazole (HOBT; 178.8 mg, 1.32 mmol), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDCI; 254.6 mg, 1.32 mmol), and N,N-diisopropylethylamine (DIEA; 0.33 mL, 2.0 mmol) in THF (25 mL) was stirred at room temperature for 0.5 h. Next compound 76 (300 mg, 1.32 mmol) was added to the mixture, which was heated to reflux for 12 h. Then the mixture was cooled to room temperature, and water (5 mL) was added. The precipitate was filtered and washed with ice−water and THF to obtain compound 18 (390 mg, 73%). The white precipitate was purified by recrystallization from EtOAc and petroleum ether (97% HPLC purity). 1H NMR (400 MHz, DMSO-d6): δ 14.18 (s, 1H), 10.49 (s, 1H), 8.53 (s, 1H), 8.11 (br s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 8.8 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 9.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ164.7, 162.8, 156.8, 154.9, 152.2, 141.0, 133.7, 131.7, 131.4, 129.8, 125.5, 122.3, 117.7, 117.2, 113.7, 101.4. LC−MS: m/z 411.2 [M + H]+. 5.1.10. 1-(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yloxy)phenyl)-3-(3(trifluoromethyl)phenyl)urea (27). The title compound was prepared from 76 and 3-(trifluoromethyl)aniline using method B, purified by column chromatography (eluent gradient CH2Cl2:MeOH = 70:1), and recrystallized from EtOAc and petroleum ether. Yield: 84%. HPLC L



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5.7. In Vivo Models. For the subcutaneous xenograft model, MV411 cells were harvested and washed three times with serum-free medium, followed by resuspension at a concentration of 1 × 108 per mL. A total of 100 μL of cell suspension was injected into NOD-SCID mice (5−6 weeks) subcutaneously. When the tumors had grown to 300−500 mm3, all the mice were grouped and administered different doses of compound or vehicle. The compounds were dissolved in 12.5% (v/v) castor oil (Sigma-Aldrich), 12.5% ethanol, and 75% distilled water. Tumor growth was measured every 3, and the volume was calculated as follows: tumor size = long diameter × (short diameter)2/2. 5.8. Histopathology. NOD-SCID mice bearing tumors were administered compound 33 at a dose of 10 mg/kg/d orally. Two or three days later, the mice were killed, and the tumors were fixed in formalin. Proliferation was assessed by immunostaining with Ki67 (Thermo Fisher Scientific, Fremont, CA). Apoptosis was determined using transferase-mediated dUTP nick-end labeling (TUNEL) staining (Roche Applied Science). The p-STAT5 staining was performed using the p-STAT5 antibody from Cell Signaling Technology. Images were acquired on an Olympus digital camera attached to a light microscope. 5.9. In Vivo Live Fluorescent Zebrafish Assay. Transgenic zebrafish (FLK-1:EGFP) embryos were grown and maintained according to the same protocols as given in ref 41. The screen was carried out in 24-well plates. Each compound was prepared initially as 10 mmol/L stock solution in dimethyl sulfoxide (DMSO). Stock solution was diluted in the relevant assay concentration with fish water, and 0.3% DMSO served as a vehicle control. The embryos were distributed to 24-well plates with 10 embryos placed in each well. Then a diluted solution of each compound was added. The embryos were exposed to compound solution and incubated at 28.5 °C from 15 h postfertilization (hpf) until 31 hpf. At 31 hpf, zebrafish were anesthetized with 0.01% tricaine and imaged under a fluorescence microscope (Carl Zeiss Microimaging Inc.) equipped with an AxioCam MRc5 digital CCD camera (Carl Zeiss Microimaging Inc.).

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layer chromatography; STAT5, signal transducer and activator of transcription 5; HOBT, 1-hydroxybenzotriazole; EDCI, 1-(3(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride; DIEA, N,N-diisopropylethylamine

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ASSOCIATED CONTENT

* Supporting Information S

Table S1 listing the binding affinities of compound 33 with various kinases, Figure S1 showing the TREEspot interaction maps for compound 33, experimental details of the synthesis and analytical characterization of compounds 2−5, 7, 8, 11, 13−17, 19−26, and 28−32 described in this paper, and purity data for the final compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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*Phone: +86-28-85164063. Fax: +86-28-85164060. E-mail: [email protected]. Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant 81172987), the 973 Program (Grant 2013CB967204), and the 863 Hi-Tech Program (Grants 2012AA020301 and 2012AA0203).

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ABBREVIATIONS USED FLT3, FMS-like tyrosine kinase 3; VEGFR2, vascular endothelial growth factor receptor 2; AML, acute myeloid leukemia; PTK, protein tyrosine kinase; SAR, structure−activity relationship; ITDs, internal tandem duplications; TLC, thinM



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pyrimidine Derivatives Leading to the D - American Chemical Society

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