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Optimization of Ligand and Lipophilic Efficiency To Identify an in Vivo Active Furano-Pyrimidine Aurora Kinase Inhibitor Hui-Yi Shiao,†,∥ Mohane Selvaraj Coumar,‡,∥ Chun-Wei Chang,†,∥ Yi-Yu Ke,† Ya-Hui Chi,§ Chang-Ying Chu,† Hsu-Yi Sun,† Chun-Hwa Chen,† Wen-Hsing Lin,† Ka-Shu Fung,† Po-Chu Kuo,† Chin-Ting Huang,† Kai-Yen Chang,† Cheng-Tai Lu,† John T. A. Hsu,† Chiung-Tong Chen,† Weir-Torn Jiaang,† Yu-Sheng Chao,† and Hsing-Pang Hsieh*,† †

Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 350, Taiwan, ROC ‡ Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Kalapet, Puducherry 605014, India § Institute of Cellular and System Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 350, Taiwan, ROC S Supporting Information *

ABSTRACT: Ligand efficiency (LE) and lipophilic efficiency (LipE) are two important indicators of “drug-likeness”, which are dependent on the molecule’s activity and physicochemical properties. We recently reported a furano-pyrimidine Aurora kinase inhibitor 4 (LE = 0.25; LipE = 1.75), with potent activity in vitro; however, 4 was inactive in vivo. On the basis of insights obtained from the X-ray co-crystal structure of the lead 4, various solubilizing functional groups were introduced to optimize both the activity and physicochemical properties. Emphasis was placed on identifying potential leads with improved activity as well as better LE and LipE by exercising tight control over the molecular weight and lipophilicity of the molecules. Rational optimization has led to the identification of Aurora kinase inhibitor 27 (IBPR001; LE = 0.26; LipE = 4.78), with improved in vitro potency and physicochemical properties, resulting in an in vivo active (HCT-116 colon cancer xenograft mouse model) anticancer agent.

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therapy” for the treatment of cancer.1 In particular, the 2001 approval of imatinib, which inhibits the activity of Bcr-Abl kinase, for the treatment of chronic myelogenous leukemia (CML), has opened a new field of “kinase targeted therapy”.2 Currently a total of 17 small molecule kinase inhibitors are approved by U.S. Food and Drug Administration (FDA) for the treatment of various cancers.3 A subclass of protein kinases is known as Aurora kinases, which phosphorylate serine/threonine amino acid residues and are involved in the regulation of various stages of mitosis.4,5 Because the mitotic phase of the cell cycle is a key step in ensuring the genetic integrity of daughter cells, any aberration in the regulation of the mitotic phase could lead to genetic instability and ultimately cancer. Therefore, Aurora kinases are an important regulator in maintaining normal cell cycle process. Three Aurora kinase isoforms, A, B, and C, have a conserved ATP binding pocket but different amino acid sequences at the

INTRODUCTION Cancer is caused by abnormalities in the genetic material of the transformed cells. Cancer-promoting oncogenes are activated and tumor suppressor genes are often inactivated in cancer cells, leading to abnormal protein expression which result in an aberrant cell cycle. Traditional approaches to cancer chemotherapy using cytotoxic drugs such as antimitotic agents are hindered by a narrow therapeutic window, severe toxicity, and development of drug resistance. These limitations have fueled interest in developing “targeted therapies” to minimize toxicity to healthy tissues. Targeted therapy constitutes the use of agents specific for the deregulated proteins of cancer cells, with an aim to avoid unwanted toxicity to normal tissues. Protein kinases, the enzymes that catalyze phosphorylation of serine, threonine, or tyrosine residues of predetermined target proteins, are deregulated in a number of diseases, including cancer. Because protein phosphorylation is an important step in the cellular signal transduction pathway regulating cell differentiation, growth, and migration, targeting abnormal protein kinase activity has become an attractive “targeted © XXXX American Chemical Society

Received: November 3, 2012

A

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Figure 1. Aurora kinase inhibitors. Compound 17 was developed by Vertex Pharmaceuticals and has undergone testing in phase II clinical trials. Compound 2 was developed by Sunesis Pharmaceuticals and has undergone testing in phase I clinical trials. Compounds 3,12 4,13 and 2714 were developed in our laboratory.

Scheme 1a

a

(a) R-NH2, ethanol, 8 h, reflux, 86−50%; (b) method A, R-NCO, CH2Cl2, rt, 8 h, 86−73%; (c) method B, CDI, R-NH2, CH2Cl2, rt, 8 h, 78−70%.

N-terminal domain.6 Aurora A is expressed from the late S phase, peaking at G2/M phase and declining at the G1 phase and is involved in centrosome maturation and separation, bipolar spindle assembly, and mitotic entry. Aurora B, known as the chromosomal passenger protein, is essential for accurate chromosome segregation and cytokinesis, and Aurora C complements the function of Aurora B. It is expressed in the testes. Aurora kinases are known to be overexpressed in solid tumors and also may be inappropriately activated in certain cell types.4 Inhibition of Aurora activity by inhibitors such as 17 and 28 (Figure 1) is known to slow cell growth and division and encouraged us to initiate a kinase-based drug discovery program,9−11 which has led to the identification of novel furano-pyrimidine Aurora kinase inhibitor 3 from our in-house compound library.12 Further modification of hit 3 through construction of a small molecule library and structure-based drug design concepts has led to the development of potent Aurora kinase inhibitor lead 4.13 Importantly, lead 4 exhibited antiproliferative activity when tested against the HCT-116 colon cancer cell line and showed pan Aurora kinase inhibition (Aurora A, IC50 = 43 nM; Aurora B, IC50 = 97 nM). However, 4 did not demonstrate significant in vivo antitumor activity up to 100 mg/kg, perhaps due to poor drug physicochemical properties. Hence we started a lead optimization program to modify lead 4, with an aim to identify potent compounds with an improved in vitro activity profile as well as good in vivo efficacy in a HCT-116 tumor xenograft mouse model.

Structure-based lead optimization of 4 by introduction of various amino functionality, along with tight control over drug properties such as molecular weight and lipophilicity, resulted in the identification of 27 (IBPR001)14 as a potent Aurora kinase inhibitor with improved physicochemical properties (Figure 1). Compound 27 showed superior tumor growth inhibition in an in vivo HCT-116 xenograft mouse model compared to the known Aurora kinase inhibitor 17 and has potential for further development as an anticancer agent. The results leading to the identification of 27 are reported here.

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CHEMISTRY Synthesis of compounds 15−24 bearing modifications on the urea side chain were carried out starting from the previously reported chloropyrimidine 512 (Scheme 1). Substitution reactions of 5 with appropriate amines/anilines (6a−e) in ethanol gave the desired intermediate anilines 7a−e, which were converted to the corresponding urea compounds 15−24, either by reacting with appropriate isocyanates (method A) or by first reacting with CDI and then treating with desired substituted anilines (method B). Compound 25 was synthesized from the chloro compound 9a12 by reacting first with 4-(2-amino-ethyl)-phenylamine in ethanol, followed by reaction with phenyl isocyanate. Compounds 25a,b were prepared from 9b,c in a manner similar to 25. The chloro compounds 9b,c were synthesized from substituted 2-bromo-1-phenyl-ethanones 8b,c in a manner similar to 9a (Scheme 2). B

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Scheme 2a

(a) (i) Malononitrile, DMF, Et2NH, rt, 16 h, (ii) HCO2H, Ac2O, 0 °C → reflux, 8 h, (iii) POCl3, neat, 70 °C, 3 h, 40−55% for three steps; (b) (i) 4-(2-amino-ethyl)-phenylamine, Et3N, EtOH, reflux, 5 h, (ii) PhNCO, CH2Cl2, rt, 8 h, 88−79% for two steps. a

Scheme 3a

(a) BBr3, CH2Cl2, 0 °C, 96−89%; (b) method C, R2N-(CH2)n-CH2Cl, K2CO3, KI, acetone/DMF, reflux 6 h, 52−29%; (c) method D, R2N(CH2)n-CH2OMs, K2CO3, DMF, reflux overnight, 41−27%; (d) method E, Br-(CH2)n-Cl, K2CO3, KI, acetone/DMF, reflux 6 h, then R2-NH, DMF, reflux, 8 h, 40−35%. a

Scheme 4a

(a) (i) Malononitrile, DMF, Et2NH, rt, 16 h, 71%, (ii) HCO2H, Ac2O, 0 °C → reflux, 8 h, (iii) POCl3, neat, 70 °C, 3 h, 37% for two steps; (b) (i) 4-(2-amino-ethyl)-phenylamine, Et3N, EtOH, reflux, 5 h, (ii) PhNCO, CH2Cl2, rt, 8 h, 92% for two steps.

a

Figure 2. X-ray co-crystal structure of Aurora kinase A in complex with inhibitor 4 (PDB ID: 3M11). Hydrogen bonding interactions between the protein and the ligand are shown as yellow lines. Interacting amino acid residues shown in stick form. The 6-position phenyl ring (attached to the furano-pyrimidine core) projecting toward the solvent exposed section of the Aurora A protein could be an appropriate place for further chemical modification. Figure was created using UCSF Chimera package.15

Demethylation of 25a,b with BBr3 at low temperature gave the corresponding phenolic compounds 10a,b. Alkylation of

10a,b using three different methods C−E gave the desired compounds 27−37. Method C: Alkylation was carried out C

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compared to lead 4 (Table 2). Moreover, removal of the phenyl ring and replacement with a hydrogen atom (20), methyl group

using chloro compound R2N-(CH2)n-CH2Cl under basic condition in acetone/DMF mixture. Method D: Alkylation was carried out using mesityl ester R2N-(CH2)n-CH2OMs under basic condition in DMF. Method E: Alkylation was carried out using chlorobromo compound Br-(CH2)n-Cl under basic condition in acetone/DMF mixture, followed by reaction with the amine R2-NH in DMF (Scheme 3). For compound 26, 2-hydroxy-1-phenyl-ethanone (11) was converted to 12 in a manner similar to that reported earlier,12 which was later reacted with 4-(2-amino-ethyl)-phenylamine and phenyl isocyanate, in a manner similar to 25 (Scheme 4).

Table 2. SAR Investigation of the Urea Functional Group of Lead 4

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RESULTS AND DISCUSSION Before we set out to optimize the lead 4, we solved the X-ray co-crystal structure of 4−Aurora A complex structure (PDB ID: 3M11) to determine the important structural features essential for activity and to identify possible modification sites.13 The Xray structure revealed that 4 binds to the Aurora A ATP binding pocket with the furano-pyrimidine core forming two essential hydrogen bonds with the hinge region Ala213 (Figure 2). In addition to these hydrogen bonds, the urea carbonyl group forms a hydrogen bond interaction with Lys162 of Aurora A. Disruption of this hydrogen bond interaction, by replacement of the urea function of 4 with either an amide (13)13 or sulphonamide (14)13 linkage, led to loss of potency at enzyme as well as cellular level (Table 1). Moreover, moving

n

R

Aurora kinase A IC50 (nM)a

HCT-116 EC50 (nM)a

413 1313 1413 15 16 17 18

2 2 2 2 0 1 3

p-NH(CO)NHPh p-NH(CO)Ph p-NHSO2Ph m-NH(CO)NHPh p-NH(CO)NHPh p-NH(CO)NHPh p-NH(CO)NHPh

43 304 5831 >10000b 201 852 >10000b

400 >10000 >10000 >10000 2747 >10000 >10000

R

Aurora kinase A IC50 (nM)a

HCT-116 EC50 (nM)a

LigandFit scoreb

413 19 20 21 22 2317 2417

Ph CH2Ph H CH3 cC6H11 p-OMe-Ph p-F-Ph

43 160 929 203 141 367 123

400 822 >10000 1786 756 2201 770

155.858 148.505 127.233 135.981 147.692 140.318 153.602

a

Values are expressed as the mean of at least two independent determinations and are within ±15%. bLigandFit score calculated by docking of compounds to Aurora kinase A X-ray crystal structure (PDB ID: 3M11) using DS/LigandFit program (Discovery studio 2.1).

Table 1. SAR Investigation of the Side Chain of Lead 4

compd

compd

(21), or cyclohexyl group (22) all led to decreased activity, with 20 being completely inactive in the cellular assay. These results indicate the importance of aromatic hydrophobic interactions in the back pocket for cellular inhibition. Introduction of either an electron donating (23)17 or withdrawing group (24)17 in the terminal phenyl ring was found to be detrimental to activity, suggesting a strict steric requirement to exist in the back pocket region for Aurora kinase inhibition. To better understand the SAR trend for Aurora kinase inhibition of compounds 19−24, they were docked to the Aurora kinase A X-ray crystal structure (PDB ID: 3M11) using the DS/LigandFit program (Discovery studio 2.1). The LigandFit scores are shown in Table 2 and further emphasize the importance of aromatic hydrophobic interaction (4 vs 20−22) and limited steric bulk accommodation (4 vs 23) in the back pocket region of the enzyme. Having found that the modifications in the urea side chain of 4 could not improve the cellular activity, we turned our attention toward the two phenyl groups appended to the furan ring of the furano-pyrimidine core. The X-ray co-crystal study showed both to form hydrophobic interactions with the surrounding amino acid residues. More importantly, the 6position phenyl group in the furano-pyrimdine ring is directed toward the solvent exposed part of the enzyme (Figure 2). On the basis of this information, we envisioned introduction of solubilizing functional groups bearing amino functionality into this phenyl ring. It has been shown previously that the presence of basic amino functionality can improve aqueous solubility, decrease lipophilic character, and enhance activity by improving interactions between the ligand and the enzyme.18−20 Before attempting the introduction of solubilizing functionality, we were interested to know the importance of phenyl rings (attached to the furano-pyrimidine ring) for maintaining the activity. In particular, we were concerned about the increase in molecular weight that would result by the introduction of solubilizing functionality to the lead 4 (Ml. Wt, 526). With the

a

Values are expressed as the mean of at least two independent determinations and are within ±15%. bInhibition 500 nM of 27 delayed mitotic progression for approximately 4 h (Figure 4B), suggesting Aurora kinase A inhibition.26,27 These cells that were delayed in mitotic progression were finally flattened with decondensed chromosomes and reformed nuclear envelope without cell division/cytokinesis (Figure 4C). Consistently, accumulated

Figure 4. Functional study of 27 on mitotic progression. (A) Western blot analysis for phosphorylation inhibition of Aurora substrates in HCT-116 cells treated with 27 for 2 h. Compound 27 inhibited phospho Aurora A (Thr288) and phospho histone H3 (Ser10) formation in HCT-116 cells in a dose dependent manner, suggesting inhibition of kinase activity for both Aurora A and B. (B) Mitotic index from HeLa cells treated with mock (DMSO) or with various concentrations of 27 following release from the double thymidine block. Mitotic index was scored every 2 h between 10 and 18 h after the second thymidine release by quantifying cells that harbor condensed chromosomes using the MetaMorph software (Molecular Devices). Errors in SEM were obtained from triplicate experiments. (C) HeLa cells released from double thymidine block were treated with the control DMSO (Mock) or 1.0 μM of 27. After 18 h of treatment, cells were fixed and subjected for immunofluorescence staining. Nuclear membrane was marked with lamin B1 (green) staining. DNA was stained with Hoechst33342 (red). Bars: 10 μm. (D) Flow cytometry analysis for DNA content in HCT-116 cells treated with 27 at various concentrations for 48 h. G

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the aim of improving in vitro activity and identifying in vivo active compounds. SAR exploration of the urea side chain revealed that modification in this part is detrimental to activity. On the basis of the X-ray co-crystal structure of 4 in complex with Aurora A, as well as a docking study, various solubilizing functional groups were introduced to the 6-position phenyl group attached to the furano-pyrimidine core to optimize both the activity (Aurora kinase A/B inhibition and HCT-116 antiproliferation) and properties (mol wt and lipophilicity). Several secondary amino groups with varying size and basicity (pKa) were introduced through a linker chain to the phenyl group attached to the furano-pyrimidine core. All the compounds bearing the solubilizing functional group showed improved antiproliferative activity compared to the initial lead 4. Overall, smaller amino groups are well tolerated when placed at para-position of the phenyl ring through a two-carbon side chain linker. In particular, compound 27 bearing an N,Ndimethyl amino group showed a 2-fold improvement in Aurora kinase A and B activity compared to the lead 4. Most importantly, these improvements in enzyme inhibition were reflected at the cellular level by 7-fold improvement in antiproliferative activity in HCT-116 cells for 27 compared to the lead 4. Furthermore, compound 27 possessed the best ligand efficiency (LE = 0.26) and lipophilicity efficiency (LipE = 4.78) among the significantly active compounds, and was therefore selected for further in vitro and in vivo profiling. Compound 27 inhibited the kinase activity of both Aurora A and B, interfered with the cell cycle progression, and affected HURP (hepatoma up-regulated protein) phosphorylation for the assembly of mitotic spindles.14 Moreover, preliminary in vivo evaluation of 27 in a HCT-116 xenograft mouse model suggested it holds great promise as an anticancer agent. Further preclinical investigations are underway to determine the full potential of this compound as an anticancer therapy.

multinucleated cells with 4N or 8N DNA content were observed in a flow cytometry analysis of HCT-116 cells treated with compound 27 for 48 h, an indicator of mitotic checkpoint override by the inhibition of Aurora kinase B (Figure 4D).28,29 This effect could be observed at much lower concentrations of 27 than 1, emphasizing the superior potency of 27 compared with 1 and consistent with the improved antiproliferative activity observed for 27 compared to 1. These experiments suggest potent in vitro efficacy of 27 to both Aurora kinase A and B inhibition. Further to determine the selectivity, 27 was tested against a panel of 31 therapeutically important kinases (Table S2, Supporting Information). At a dose of 1 μM, 27 was found to inhibit seven kinases with >50% inhibition, including Auroras A, B, and C. Except for CHK2, the other three kinases (Flt3, NTRK1, RAF1) showed inhibition levels lower than Aurora kinases, suggesting 27 to be a potent and selective Aurora kinase inhibitor. Once 27 had been confirmed as a suitable candidate for further development, it was tested in an in vivo mouse acute tolerability assay. The hydrochloride salt of 27 (free form has poor solubility in the dosing formulation) of 27 was injected intravenously into three mice at a dose of 50 mg/kg QD for every day for five days. The mice were observed for another 5 days; no notable changes in activity were observed, and none of the mice died. Building on this preliminary investigation, HCT116 tumor bearing nude mice were administered 27 at a dose of 50 and 10 mg/kg iv QD, and 20 mg/kg iv BID for 5 days in two cycles, with a two-day break between the cycles. Similarly, 1 was administered at 50 mg/kg iv using the same schedule and treatment cycle. Significant tumor growth suppression was observed for 27 when administered at 50 mg/kg QD as well as at 20 mg/kg BID, similar to that of 1 at 50 mg/kg QD (Figure 5).

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CONCLUSION Our studies toward targeted anticancer agents have included the disclosure of the novel furano-pyrimdine 4 as an Aurora kinase inhibitor. Modification of the lead 4 was initiated with

EXPERIMENTAL SECTION GENERAL METHODS

All commercial chemicals and solvents are of reagent grade and were used without further purification unless otherwise stated. All reactions were carried out under dry nitrogen atmosphere and were monitored for completion by TLC using Merck 60 F254 silica gel glass backed plates (5 cm × 10 cm); zones were detected visually under UV irradiation (254 nm) or by spraying with phosphomolybdic acid reagent (Aldrich) followed by heating at 80 °C. Flash column chromatography was carried out using silica gel (Merck Kieselgel 60, no. 9385, 230−400 mesh ASTM). 1H and 13C NMR spectra were obtained with a Varian Mercury-300 or Varian Mercury-400 spectrometers, and the chemical shifts were recorded in parts per million (ppm, δ) and reported relative to TMS or the solvent peak. High-resolution mass spectra (HRMS) were measured with a Finnigan (MAT95XL) electron impact (EI) or by using Finnigan/Thermo Quest MAT 95XL FAB mass spectrometer. Low-resolution mass spectra (LRMS) data were measured on an Agilent MSD1100 ESI-MS/MS system. Purity of the final compounds were determined with an Hitachi 2000 series HPLC system using C18 column (Agilent ZORBAX Eclipse XDB-C18 5 μm, 4.6 mm × 150 mm) operating at 25 °C. Elution was carried out using acetonitrile as mobile phase A and water containing 0.1% formic acid +10 mmol NH4OAc as mobile phase B. Elution condition: at 0 min, phase A 10% + phase B 90%; at 45 min,

Figure 5. In vivo antitumor effect of 27 in human colon cancer (HCT116) xenograft nude mice model. The growth of HCT-116 tumor xenograft is inhibited by 27 (50 mg/kg, iv, QD; 20 mg/kg, iv, BID) and 1 (50 mg/kg, iv, QD) with *p < 0.05. Drug treatments on days 1− 5 and 8−12. For all groups except for treatment with 27 at 10 mg/kg, n = 8 mice; n = 7 mice for the treatment group 27 at 10 mg/kg. H

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7.01−6.94 (m, 2H), 6.72 (d, J = 7.6 Hz, 1H), 4.68 (bt, J = 5.6 Hz, 1H), 3.67 (q, J = 6.4 Hz, 2H), 2.72 (t, J = 6.4 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 164.5 (C), 157.4 (C), 153.7 (C), 146.7 (C), 139.5 (C), 138.8 (C), 138.4 (C), 131.8 (C), 129.6 (CH), 129.4 (CH), 129.2 (CH), 129.1 (C), 129.0 (CH), 128.9 (CH), 128.4 (CH), 126.2 (CH), 123.4 (CH), 120.2 (CH), 119.9 (CH), 118.0 (CH), 114.9 (C), 103.1 (C), 41.7 (CH2), 34.9 (CH2). HRMS (EI) calcd for C33H27N5O2 [M]+, 525.2165; found, 525.2155. 1-{4-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]phenyl}-3-phenylurea 16. Method A, using 7c and phenylisocyanate to give 16. Purification: column chromatography over silica gel column using MeOH:CH2Cl2 (1:20). Yield, 73%. 1 H NMR (400 MHz, DMSO-d6) δ 8.64 (s, 1H), 8.62 (s, 1H), 8.48 (s, 1H), 7.68−7.63 (m, 6H), 7.51−7.48 (m, 2H), 7.43− 7.36 (m, 8H), 7.28−7.24 (m, 3H), 6.71 (s, 1H). LRMS (ESI) m/z: 498 [M + H]+. 1-(4-{[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]methyl}phenyl)-3-phenylurea 17. Method A, using 7d and phenylisocyanate to give 17. Purification: column chromatography over silica gel column using MeOH:CH2Cl2 (1:20). Yield, 82%. 1H NMR (300 MHz, CDCl3) δ 8.44 (s, 1H), 7.86− 7.83 (m, 3H), 7.59−7.45 (m, 13H), 7.29−7.24 (m, 4H), 7.14 (d, J = 7.2 Hz, 2H), 4.94 (t, J = 5.7 Hz, 1H), 4.64 (d, J = 5.7 Hz, 1H). LRMS (ESI) m/z: 512 [M + H]+. 1-(4-{3-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]propyl}phenyl)-3-phenylurea 18. Method A, using 7e and phenylisocyanate to give 18. Purification: column chromatography over silica gel column using MeOH:CH2Cl2 (1:20). Yield, 78%. 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 7.59− 7.50 (m, 8H), 7.35−7.23 (m, 6H), 7.14−7.11 (m, 1H), 7.01 (d, J = 8.4 Hz, 2H), 6.56 (d, J = 8.4 Hz, 2H), 4.67 (bt, J = 5.6 Hz, 1H), 3.43 (q, J = 6.8 Hz, 2H), 2.47 (t, J = 6.8 Hz, 2H), 1.73 (q, J = 6.8 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 164.5 (C), 157.6 (C), 153.9 (C), 153.9 (CH), 146.6 (C), 138.4 (C), 136.3 (C), 136.1 (C), 132.3 (C), 129.7 (CH), 129.6 (CH), 129.3 (C), 129.0 (CH), 128.9 (CH), 128.8 (CH), 128.5 (CH), 128.4 (CH), 126.2 (CH), 123.3 (CH), 120.2 (CH), 114.8 (C), 103.2 (C), 49.9 (CH2), 32.0 (CH2), 30.7 (CH2). HRMS (EI) calcd for C34H29N5O2 [M]+, 539.2321; found, 539.2327. HPLC purity: 92%. 1-Benzyl-3-(4-{2-[(5,6-diphenylfuro[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)urea 19. Method A, using 7a and benzylisocyanate to give 19. Purification: column chromatography over silica gel column using MeOH:CH2Cl2 (1:30). Yield, 86%. 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 7.48− 7.37 (m, 6H), 7.32−7.23 (m, 9H), 7.17 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 6.37 (brs, 1H), 5.05 (bt, J = 5.6 Hz, 1H), 4.64 (bt, J = 5.2 Hz, 1H), 4.46 (d, J = 5.6 Hz, 2H), 3.67 (q, J = 6.4 Hz, 2H), 2.73 (t, J = 6.4 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 164.6 (C), 157.4 (C), 156.2 (C), 153.9 (CH), 146.6 (C), 138.9 (C), 137.2 (C), 133.3 (C), 132.0 (C), 129.6 (CH), 129.5 (CH), 129.3 (C), 129.1 (CH), 128.8 (CH), 128.5 (CH), 128.4 (CH), 128.4 (CH), 127.2 (CH), 126.3 (CH), 120.5 (CH), 114.8 (C), 103.1 (C), 44.0 (CH2), 41.9 (CH2), 34.3 (CH2). HRMS (EI) calcd for C34H29N5O2 [M]+, 539.2321; found, 539.2320. 1-(4-{2-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)urea 20. Method B, using 7a and aqueous ammonia to give 20. Purification: column chromatography over silica gel column using MeOH:CH2Cl2:NH4OH (5:100:1). Yield, 78%. 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 7.47− 7.38 (m, 5H), 7.32−7.28 (m, 2H), 7.25−7.18 (m, 5H), 6.97 (d,

phase A 90% + phase B 10%; at 50 min, phase A 10% + phase B 90%; at 60 min, phase A 10% + phase B 90%. The flow-rate of the mobile phase was 0.5 mL/min, and the injection volume of the sample was 5 μL. Peaks were detected at 210 nm. Purity of all the tested compounds were found to be >95% unless otherwise stated. N-[2-(4-Aminophenyl)ethyl]-5,6-diphenylfuro[2,3-d]pyrimidin-4-amine 7a. 4-Chloro-5,6-diphenylfuro[2,3-d]pyrimidine (5) (0.200 g, 65.2 mmol) and 4-(2-aminoethyl)aniline (6a) (0.107 g, 70.8 mmol) in ethanol (5 mL) were heated at reflux for 8 h. The reaction mixture was concentrated under vacuum, and the residue partitioned between water and ethyl acetate. The organic layer separated, dried over Na2SO4, concentrated, and the crude compound was purified by silica gel column chromatography using a mixture of MeOH:CH2Cl2 (1:40), to give 7a (0.195 g, 74%). 1H NMR (300 MHz, CDCl3): δ 8.43 (s, 1H), 7.50−7.23 (m, 10H), 6.79 (d, J = 8.4 Hz, 2H), 6.59 (d, J = 8.4 Hz, 2H), 4.68 (t, J = 5.2 Hz, 1H), 3.65 (q, J = 6.4 Hz, 2H), 3.62 (bs, 2H), 2.67 (t, J = 6.4 Hz, 2H). HRMS (EI) calcd for C26H22N4O [M]+, 406.1794; found, 406.1789. Compounds 7b−e were prepared from 5 and appropriate amines (6b−e), in a manner similar to 7a. N-[2-(3-Aminophenyl)ethyl]-5,6-diphenylfuro[2,3-d]pyrimidin-4-amine 7b. Yield, 86%. 1H NMR (300 MHz, CDCl3) δ 8.43 (s, 1H), 7.50−7.21 (m, 12H), 7.00 (t, J = 8.1 Hz, 1H), 6.55 (ddd, J = 8.1, 1.2, 0.9 Hz, 1H), 6.38−6.31 (m, 2H), 4.72 (t, J = 5.7 Hz, 1H), 3.68 (q, J = 6.3 Hz, 2H), 2.70 (t, J = 6.3 Hz, 2H). LRMS (ESI) m/z: 407 [M + H]+. N-(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)-benzene-1,4-diamine 7c. Yield, 80%. 1H NMR (300 MHz, DMSO-d6) δ 8.39 (s, 1H), 7.67−7.61 (m, 6H), 7.50−7.46 (m, 2H), 7.40−7.34 (m, 3H), 6.99 (d, J = 6.9 Hz, 2H), 6.49 (d, J = 6.9 Hz, 2H), 6.44 (s, 2H). LRMS (ESI) m/z: 379 [M + H]+. N-(4-Aminobenzyl)-5,6-diphenylfuro[2,3-d]pyrimidin-4amine 7d. Yield, 85%. 1H NMR (300 MHz, CDCl3) δ 8.44 (s, 1H), 7.54−7.44 (m, 8H), 7.27−7.25 (m, 2H), 6.93 (d, J = 6.9 Hz, 2H), 6.10 (d, J = 6.9 Hz, 2H), 4.87 (t, J = 5.7 Hz, 1H), 4.51 (d, J = 5.7 Hz, 2H), 3.65 (brs, 2H). LRMS (ESI) m/z: 393 [M + H]+. N-[3-(4-Aminophenyl)propyl]-5,6-diphenylfuro[2,3-d]pyrimidin-4-amine 7e. Yield, 50%. 1H NMR (400 MHz, CDCl3) δ 8.41 (s, 1H), 7.58−7.49 (m, 8H), 7.29−7.25 (m, 2H), 6.83 (d, J = 8.0 Hz, 2H), 6.60 (d, J = 8.0 Hz, 2H), 4.66 (t, J = 5.2 Hz, 1H), 3.57 (bs, 2H), 3.41 (q, J = 6.8 Hz, 2H), 2.38 (t, J = 6.8 Hz, 2H), 1.69 (quin, J = 6.8 Hz, 2H). HRMS (EI) calcd. for C27H24N4O [M]+, 420.1950; found, 420.1949. Urea Side Chain Installation in Compounds 15−24. Method A. The urea side chain was installed by reaction between 7b−e and appropriate phenylisocyanates to give the final products in a manner similar to that for 4 reported by us.13 Method B. A mixture of 7a−e (1.0 mmol) and carbonyldiimdazole (CDI, 2.0 mmol) in THF (10 mL) was stirred at ambient temperature for 4 h, followed by addition of the required amine (2.0 mmol) and heating to reflux for another 16 h. After cooling the reaction, the mixture was evaporated and purified by silica gel column chromatography. 1-(3-{2-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea 15. Method A, using 7b and phenylisocyanate to give 15. Purification: column chromatography over silica gel column using MeOH:CH2Cl2 (1:20). Yield, 82%. 1H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 7.45− 7.27 (m, 11H), 7.24−7.16 (m, 4H), 7.09 (t, J = 7.2 Hz, 1H), I

dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1H), 7.08 (s, 1H), 7.10 (ddd, J = 9.0, 2.1, 1.2 Hz, 1H), 3.90 (s, 3H). LCMS (ESI) m/z: 261 [M + H]+. 1-Phenyl-3-(4-{2-[(6-phenylfuro[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)urea 25. The mixture of 9a (2.0 g, 8.69 mmol), 2-(4-aminophenyl)-ethyl amine (1.3 mL, 10.43 mmol), and triethyl amine (3.56 mL, 26.07 mmol) was heated to reflux in EtOH (40 mL) for 5 h. The precipitate formed after cooling to room temperature was filtered, washed with EtOH, and then dried under vacuum. The solid product obtained was dissolved in CH2Cl2 (45 mL) and phenyl isocyanate (1.38 mL, 12.76 mmol) was added slowly. After stirring at room temperature for 8 h, the precipitate formed was filtered and washed with CH2Cl2 to obtain 25 as a white solid. Yield, 88% (two steps). 1 H NMR (300 MHz, DMSO-d6) δ 8.62 (s, 1H), 8.59 (s, 1H), 8.28 (s, 1H), 8.10 (br, 1H), 7.78 (d, J = 7.2 Hz, 2H), 7.52−7.37 (m, 8H), 7.28−7.17 (m, 4H), 6.94 (t, J = 7.2 Hz, 1H), 3.70 (q, J = 7.2 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 165.7 (C), 157.0 (C), 153.9 (CH), 152.6 (C), 150.2 (C), 139.8 (C), 137.9 (C), 132.8 (C), 129.2 (CH), 129.0 (CH), 128.8 (CH × 2), 124.1 (CH), 121.7 (CH), 118.3 (CH), 118.1 (CH), 102.6 (C), 98.2 (CH), 42.1 (CH2), 34.4 (CH2). HRMS (FAB) m/z: calcd for C27H24N5O2 ([M + H]+), 450.1930; found, 450.1936. 1-[4-(2-{[6-(4-Methoxyphenyl)furo[2,3-d]pyrimidin-4-yl]amino}ethyl)phenyl]-3-phenylurea 25a. Synthesized from 9b in a manner similar to 25. Yield, 85%. 1H NMR (300 MHz, DMSO-d6) δ 8.62 (s, 1H), 8.59 (s, 1H), 8.25 (s, 1H), 8.01 (bs, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.43 (d, J = 8.1 Hz, 2H), 7.37 (d, J = 8.1 Hz, 2H), 7.27 (t, J = 8.4 Hz, 2H), 7.20−7.16 (m, 3H), 7.07 (d, J = 9.0 Hz, 2H), 6.95 (t, J = 7.2 Hz, 1H), 3.81 (s, 3H), 3.69 (q, J = 7.2 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H). LCMS (FAB) m/z: 480 [M + H]+. 1-[4-(2-{[6-(3-Methoxyphenyl)furo[2,3-d]pyrimidin-4-yl]amino}ethyl)phenyl]-3-phenylurea 25b. Synthesized from 9c in a manner similar to 25. Yield, 79%. 1H NMR (300 MHz, CDCl3+CD3OD): δ 8.30 (s, 1H), 7.44−7.30 (m, 9H), 7.19 (d, J = 6.3 Hz, 2H), 7.09−7.01 (m, 2H), 7.00 (s, 1H), 6.93−6.89 (m, 1H), 3.88 (s, 3H), 3.82 (t, J = 6.9 Hz, 2H), 3.37 (brs, 2H), 2.96 (t, J = 6.9 Hz, 2H). LCMS (ESI) m/z: 480 [M + H]+. 1-[4-(2-{[6-(4-Hydroxyphenyl)furo[2,3-d]pyrimidin-4-yl]amino}ethyl)phenyl]-3-phenylurea 10a. Treated 25a (1.0 mmol) with BBr3 (9 mL, 1.0 M in CH2Cl2) in CH2Cl2 (10 mL) at 0 °C for 1 h, then H2O (10 mL) was added to quench the reaction and neutralized by the addition of NaHCO3(aq). The precipitate formed was filtered and purified by silica gel column chromatography using MeOH:CH2Cl2 (1:20). Yield, 96%. 1H NMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H), 8.66 (s, 1H), 8.63 (s, 1H), 8.23 (s, 1H), 8.01 (bs, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 7.2 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 7.27 (t, J = 7.6 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H), 7.11 (s, 1H), 6.95 (t, J = 7.6 Hz, 1H), 6.88 (d, J = 8.4 Hz, 2H), 3.68 (q, J = 7.6 Hz, 2H), 2.86 (t, J = 7.6 Hz, 2H). LCMS (ESI) m/z: 466 [M + H]+. 1-[4-(2-{[6-(3-Hydroxyphenyl)furo[2,3-d]pyrimidin-4-yl]amino}ethyl)phenyl]-3-phenylurea 10b. Synthesis carried out from 25b in a manner similar to 10a. Purification: column chromatography over silica gel column using MeOH:CH2Cl2 (1:20). Yield, 89%. 1H NMR (400 MHz, CDCl3 + CD3OD) δ 8.23 (s, 1H), 7.43−7.30 (m, 9H), 7.18 (d, J = 8.4 Hz, 2H), 7.06−6.99 (m, 3H), 6.93−6.89 (m, 1H), 3.82 (q, J = 7.6 Hz, 2H), 2.96 (t, J = 7.6 Hz, 2H). LCMS (ESI) m/z: 466 [M + H]+.

J = 8.4 Hz, 2H), 4.79 (brs, 2H), 4.65 (bt, J = 5.6 Hz, 1H), 3.69 (q, J = 6.4 Hz, 2H), 2.75 (t, J = 6.4 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 164.7 (C), 157.4 (C), 157.2 (C), 154.0 (C), 146.6 (C), 136.8 (C), 134.2 (C), 132.0 (C), 129.6 (CH), 129.5 (CH), 129.3 (C), 129.2 (CH), 128.8 (CH), 128.4 (CH), 126.3 (CH), 121.2 (CH), 114.8 (C), 103.1 (C), 41.9 (CH2), 34.4 (CH2). HRMS (FAB) calcd for C27H24N5O2 [M + H]+, 450.1930; found, 450.1933. HPLC purity: 86%. 1-(4-{2-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-methylurea 21. Method B, using 7a and methylamine to give 21. Purification: column chromatography over silica gel column using MeOH:CH 2 Cl 2 :NH 4 OH (5:100:1). Yield, 72%. 1H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 7.49−7.39 (m, 6H), 7.33−7.30 (m, 2H), 7.27−7.23 (m, 2H), 7.18 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 8.0 Hz, 2H), 6.31 (brs, 1H), 4.67 (bq, J = 4.8 Hz, 1H), 4.65 (bt, J = 5.6 Hz, 1H), 3.69 (q, J = 6.4 Hz, 2H), 2.86 (d, J = 4.8 Hz, 3H), 2.75 (t, J = 6.4 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 164.7 (C), 157.4 (C), 156.9 (C), 154.0 (CH), 146.6 (C), 137.3 (C), 133.5 (C), 132.0 (C), 129.6 (CH), 129.5 (CH), 129.4 (C), 129.2 (CH), 128.8 (CH), 128.5 (CH), 128.4 (CH), 126.3 (CH), 120.8 (CH), 114.9 (C), 103.1 (C), 41.9 (CH2), 34.4 (CH2), 26.9 (CH3). HRMS (FAB) m/z: calcd for C28H26N5O2 ([M + H]+), 464.2087; found, 464.2096. 1-Cyclohexyl-3-(4-{2-[(5,6-diphenylfuro[2,3-d]pyrimidin-4yl)amino]ethyl}phenyl)urea 22. Method B, using 7a and cyclohexylamine to give 22. Purification: column chromatography over silica gel column using MeOH:CH2Cl2 (1:20). Yield, 75%. 1H NMR (300 MHz, CDCl3): δ 8.56 (s, 1H), 7.44−7.38 (m, 4H), 7.30−7.17 (m, 10H), 4.66 (t, J = 5.4 Hz, NH), 3.68−3.60 (m, 2H), 2.67 (t, J = 6.6 Hz, 2H), 2.17−1.93 (m, 1H), 1.70−1.56 (m, 4H), 1.41−1.26 (m, 4H), 1.17−1.06 (m, 2H). LCMS (ESI) m/z: 532 [M + H]+. 1-(4-{2-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-(4-methoxyphenyl)urea 23.17 Method B, using 7a and p-amino-anisole to give 23. Purification: column chromatography over silica gel column using MeOH:CH2Cl2 (1:20). Yield, 70%. 1H NMR (300 MHz, CDCl3): δ 8.42 (s, 1H) 7.46−7.43 (m, 4H), 7.41−7.22 (m, 10H), 6.90 (q, J = 6.8 Hz, 4H), 4.49 (t, J = 5.4 Hz, 1H), 3.80 (s, 3H), 3.68 (q, J = 6.0 Hz, 2H), 2.73 (t, J = 6.8 Hz, 2H). LCMS (ESI) m/z: 556 [M + H]+. 1-(4-{2-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-(4-fluorophenyl)urea 24.17 Method B, using 7a and p-fluoro-aniline to give 24. Purification: column chromatography over silica gel column using MeOH:CH2Cl2 (1:20). Yield, 78%. 1H NMR (300 MHz, CDCl3) δ 8.43 (s, 1H), 7.47−7.39 (m, 4H), 7.34−7.29 (m, 4H), 7.25−7.22 (m, 5H), 7.03−6.93 (m, 4H), 6.88 (s, 1H), 4.67 (t, J = 5.4 Hz, 1H), 3.69 (q, J = 6.0 Hz, 2H), 2.74 (t, J = 6.0 Hz, 2H). LCMS (ESI) m/z: 544 [M + H]+. 4-Chloro-6-(4-methoxyphenyl)furo[2,3-d]pyrimidine 9b. Synthesized in a manner similar to 9a12 starting from 2bromo-1-(4-methoxy-phenyl)-ethanone (8b) in 28% yield. 1H NMR (300 MHz, CDCl3) δ 8.71 (s, 1H) 7.85 (d, J = 9.0 Hz, 2H), 7.02 (d, J = 9.0 Hz, 2H), 6.93 (s, 1H), 3.89 (s, 3H). LCMS (ESI) m/z: 261 [M + H]+. 4-Chloro-6-(3-methoxyphenyl)furo[2,3-d]pyrimidine 9c. Synthesized in a manner similar to 9a12 starting from 2bromo-1-(3-methoxy-phenyl)-ethanone (8c) in 24% yield. 1H NMR (300 MHz, CDCl3) δ 8.75 (s, 1H) 7.50 (ddd, J = 9.0, 1.5, 1.2 Hz, 1H), 7.44 (dd, J = 2.1, 1.5 Hz, 1H), 7.39 (t, J = 9.0 Hz, J

dx.doi.org/10.1021/jm4006059 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1-Phenyl-3-(4-{2-[(5-phenylfuro[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)urea 26. Compound 26 was prepared from 1212 using 4-(2-amino-ethyl)-phenylamine and PhNCO in a manner similar to 25. Yield, 92% (two steps). 1H NMR (400 MHz, CDCl3) δ 8.44 (s, 1 H), 7.50 (brs, 1H), 7.49 (brs, 1H), 7.36−7.29 (m, 6H), 7.25−7.21 (m, 4H), 7.19 (d, J = 8.4 Hz, 2H), 7.04−7.00 (m, 1H), 6.93 (d, J = 8.4 Hz, 2H), 5.00 (t, J = 6.4 Hz, 1H), 3.72 (q, J = 6.4 Hz, 2H), 2.77 (t, J = 6.4 Hz, 2H). HRMS (FAB) calcd for C27H24N5O2 [M + H]+, 450.1930; found, 450.1937. HPLC purity: 86%. Solubilizing Group Installation in Compounds 27−37. Method C. A mixture of 10a,b (1.0 mmol), potassium carbonate (3.0 mmol), potassium iodide (1.0 mmol), and R2N(CH2)2Cl (1.5 mmol) in 10 mL of DMF:acetone (1:1) was heated to reflux for 6 h. Then the mixture was concentrated, water was added, and then extracted with ethyl acetate. The organics were separated, concentrated under vacuum, and the solid obtained was purified by silica gel column chromatography using MeOH:CH2Cl2:NH4OH (10:200:1) to get the desired product. Method D. A mixture of 10a (0.50 mmol) and potassium carbonate (1.60 mmol) in 5 mL of DMF was heated to 75 °C, then R2N(CH2)2OMs (0.75 mmol) in 4 mL of DMF was slowly added. After stirring overnight, the resulting mixture was filtered, then water was added and extracted with ethyl acetate. The organics were separated and concentrated under vacuum, and the solid obtained was purified by silica gel column chromatography using MeOH:CH2Cl2:NH4OH (10:200:1) to get the desired product. Method E. A mixture of 10a (1.0 mmol), potassium carbonate (3.0 mmol), potassium iodide (1.5 mmol), and Br(CH2)nCl (1.5 mmol) in 10 mL of DMF:acetone (1:1) was heated to reflux for 6 h. Then added R2NH (2.0 mmol) and refluxed for another 8 h. After cooling, the mixture was concentrated, then H2O (20 mL) was added and then extracted with ethyl acetate. The organics were separated, concentrated under vacuum, and the solid obtained was purified by silica gel column chromatography using MeOH:CH 2 Cl 2 :NH 4 OH (10:200:1) to get the desired product. 1-(4-{2-[(6-{4-[2-(Dimethylamino)ethoxy]phenyl}furo[2,3d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea 27.14 Method C using 10a and 2-(chloroethyl)dimethyl amine to give 27. Yield, 52%. 1H NMR (300 MHz, CD3OD) δ 8.20 s, 1H), 8.01 (brs, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H), 7.26 (t, J = 7.8 Hz, 2H), 7.19 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 9.0 Hz, 2H), 6.99 (s, 1H), 6.98−6.96 (m, 1H), 4.15 (t, J = 5.4 Hz, 2H), 3.78 (t, J = 7.2 Hz, 2H), 2.91 (t, J = 7.2 Hz, 2H), 2.78 (t, J = 5.4 Hz, 2H), 2.35 (s, 6H). LCMS (ESI) m/z: 537 [M + H] +. The HCl salt of 27 was prepared by dissolving 27 in CH2Cl2/MeOH mixture and then passing HCl gas (prepared by reacting concentrated H2SO4 with NaCl) into the mixture for 2 h with stirring at room temperature. Solvent was evaporated under reduced pressure to afford HCl salt of 27. 1-(4-{2-[(6-{4-[2-(Diethylamino)ethoxy]phenyl}furo[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea 28. Method D using 10a and methanesulfonic acid 2-diethylamino-ethyl ester to give 28. Yield, 41%. 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.60 (s, 1H), 8.25 (s, 1H), 8.01 (brs, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.27 (t, J = 7.6 Hz, 2H), 7.18 (d, J = 8 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.95 (t, J = 7.2 Hz, 2H), 4.07 (t, J = 6.0 Hz, 2H), 3.69 (q, J = 6.8 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H), 2.79 (t, J =

6.0 Hz, 2H), 2.56 (q, J = 7.2 Hz, 4H), 0.98 (t, J = 7.2 Hz, 6H). LCMS (ESI) m/z: 565 [M + H]+. 1-(4-{2-[(6-{4-[2-(Dibutylamino)ethoxy]phenyl}furo[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea 29. Method D using 10a and methanesulfonic acid 2-dibutylamino-ethyl ester to give 29. Yield, 30%. 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.60 (s, 1H), 8.25 (s, 1H), 8.01 (brs, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.27 (t, J = 8.0 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 9.2 Hz, 2H), 6.95 (t, J = 7.2 Hz, 2H), 4.07 (2H), 3.69 (q, J = 6.8 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H), 2.79 (brs, 2H), 2.47 (brs, 4H), 1.39 (t, J = 6.4 Hz, 4H), 1.28 (m, J = 7.2 Hz, 4H), 0.87 (t, J = 7.2 Hz, 6H). LCMS (ESI) m/z: 621 [M + H]+. 1-Phenyl-3-(4-{2-[(6-{4-[2-(pyrrolidin-1-yl)ethoxy]phenyl}furo[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)urea 30. Method D using 10a and methanesulfonic acid 2-pyrrolidin-1-ylethyl ester to give 30. Yield, 32%. 1H NMR (400 MHz, DMSOd6) δ 8.62 (s, 1H), 8.59 (s, 1H), 8.25 (s, 1H), 8.01 (brs, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.27 (t, J = 8.0 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 6.95 (t, J = 7.2 Hz, 2H), 4.13 (t, J = 6.0 Hz, 2H), 3.69 (q, J = 6.0 Hz, 2H), 2.89−2.82 (m, 4H), 2.54 (brs, 4H), 1.79 (m, 4H). LCMS (ESI) m/z: 563 [M + H]+. HPLC purity: 94% . 1-Phenyl-3-(4-{2-[(6-{4-[2-(piperidin-1-yl)ethoxy]phenyl}furo[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)urea 31. Method D using 10a and methanesulfonic acid 2-piperidin-1-yl-ethyl ester to give 31. Yield, 27%. 1H NMR (300 MHz, DMSO-d6) δ 8.62 (s, 1H), 8.59 (s, 1H), 8.25 (s, 1H), 8.00 (brs, 1H), 7.71 (d, J = 9.0 Hz, 2H), 7.45−7.37 (m, 4H), 7.27 (t, J = 7.8 Hz, 2H), 7.18 (d, J = 8.1 Hz, 2H), 7.07 (d, J = 8.7 Hz, 2H), 6.95 (t, J = 7.2 Hz, 2H), 4.13 (t, J = 5.7 Hz, 2H), 3.67 (q, J = 6.3 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H), 2.54 (brs, 2H), 2.27 (brs, 4H), 1.50 (m, 4H), 1.39 (m, 2 H). LCMS (ESI) m/z: 577 [M + H]+. 1-(4-{2-[(6-{4-[2-(4-Methylpiperazin-1-yl)ethoxy]phenyl}furo[2,3-d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea 32. Method E using 10a, bromochloroethane, and 4methylpiperazine to give 32. Yield, 40%. 1H NMR (300 MHz, DMSO-d6) δ 8.72 (s, 1H), 8.70 (s, 1H), 8.25 (s, 1H), 8.00 (brs, 1H), 7.71 (d, J = 9.0 Hz, 2H), 7.44 (d, J = 7.8 Hz, 2H), 7.38 (d, J = 8.7 Hz, 2H), 7.26 (t, J = 7.6 Hz, 2H), 7.21− 7.16 (m, 3H), 7.07 (d, J = 8.7 Hz, 2H), 6.95 (t, J = 6.6 Hz, 1H), 4.13 (t, J = 5.7 Hz, 2H), 3.68 (q, J = 6.6 Hz, 2H), 2.87 (t, J = 6.6 Hz, 2H), 2.70 (t, J = 6.0 Hz, 2H), 2.35−2.25 (m, 8H), 2.14 (s, 3H). LCMS (ESI) m/z: 592 [M + H]+. 1-(4-{2-[(6-{4-[(2-Morpholin-4-yl)ethoxy]phenyl}furo[2,3d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea 33. Method C using 10a and 4-(2-chloro-ethyl)-morpholine to give 33. Yield, 29%. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 8.59 (s, 1H), 8.25 (s, 1H), 8.01 (brs, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.27 (t, J = 8.0 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 6.95 (t, J = 7.2 Hz, 2H), 4.15 (t, J = 6.0 Hz, 2H), 3.68 (q, J = 6.0 Hz, 2H), 3.58 (t, J = 4.8 Hz, 4H), 2.87 (t, J = 7.2 Hz, 2H), 2.71 (t, J = 6.0 Hz, 2H), 2.48 (brs, 4H). LCMS (ESI) m/z: 579 [M + H]+. 1-[4-(2-{[6-(4-{2-[Ethyl(2-hydroxyethyl)amino]ethoxy}phenyl)furo[2,3-d]pyrimidin-4-yl]amino}ethyl)phenyl]-3-phenylurea 34. Method E using 10a, bromochloroethane, and 2hydroxyethyl-ethylamine to give 34. Yield, 38%. 1H NMR (400 MHz, CD3OD) δ 8.22 (s, 1 H), 7.75 (s, 1 H), 7.74 (s, 1 H), 7.41 (d, J = 8.4 Hz, 2 H), 7.35 (d, J = 8.4 Hz, 2 H), 7.28 (m, 2 H), 7.21 (d, J = 8.4 Hz, 2 H), 7.02 (m, 4H), 4.15 (t, J = 5.6 Hz, K

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2 H), 3.78 (t, J = 7.2 Hz, 2 H), 3.67 (t, J = 6.4 Hz, 2 H), 2.97 (m, 4 H), 2.75 (m, 4H), 1.11 (t, J = 7.2 Hz, 2 H). LCMS (ESI) m/z: 581 [M + H]+. 1-[4-(2-{(6-{4-[2-(4-Hydroxypiperidin-1-yl)ethoxy]phenyl}furo[2,3-d]pyrimidin-4-yl)amino}ethyl)phenyl]-3-phenylurea 35.14 Method E using 10a, bromochloroethane, and 2hydroxypiperidine to give 35. Yield, 40%. 1H NMR (300 MHz, CD3OD) δ 8.29 (s, 1H), 7.70 (d, J = 9.3 Hz, 2H), 7.44− 7.26 (m, 7H), 7.13 (d, J = 8.4 Hz, 2H), 7.05−7.00 (m, 1H), 6.94 (d, J = 8.7 Hz, 2H), 6.77 (s, 1H), 4.14 (t, J = 5.7 Hz, 2H), 3.81−3.78 (m, 4H), 3.41−3.39 (m, 1H), 3.23−3.17 (m, 2H), 2.95−2.92 (m, 2H), 2.85 (t, J = 5.7 Hz, 2H), 1.99−1.90 (m, 2H), 1.44−1.37 (m, 2H). LCMS (ESI) m/z: 593 [M + H]+. 1-(4-{2-[(6-{4-[3-(Dimethylamino)prooxy]phenyl}furo[2,3d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea 36. Method E using 10a, bromochloropropane, and dimethylamine to give 36. Yield, 35%. 1H NMR (300 MHz, CD3OD) δ 8.22 (s, 1H), 8.03 (s, 1H), 7.75 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.7 Hz, 2H), 7.27 (t, J = 7.8 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 9.0 Hz, 2H), 7.00 (s, 1H), 6.98− 6.96 (m, 1H), 4.15 (t, J = 5.4 Hz, 2H), 3.78 (t, J = 7.2 Hz, 2H), 2.91 (t, J = 7.2 Hz, 2H), 2.78 (t, J = 5.4 Hz, 2H), 2.35 (s, 6H), 1.83 (m, 2H). LCMS (ESI) m/z: 551 [M + H]+. HPLC purity: 93%. 1-(4-{2-[(6-{3-[2-(Dimethylamino)ethoxy]phenyl}furo[2,3d]pyrimidin-4-yl)amino]ethyl}phenyl)-3-phenylurea 37. Method C using 10b and 2-(chloroethyl)dimethylamine to give 37. Yield, 38%. 1H NMR (300 MHz, CD3OD) δ 8.31 (s, 1H), 8.01 (d, J = 5.7 Hz, 2H), 7.39−7.18 (m, 9H), 7.00−6.93 (m, 2H), 6.95 (s, 1H), 6.86−6.85 (m, 1H), 4.04 (t, J = 5.7 Hz, 2H), 3.66 (t, J = 6.3 Hz, 2H), 2.80 (t, J = 6.3 Hz, 2H), 2.72 (t, J = 5.7 Hz, 2H), 2.34 (s, 6H). LCMS (ESI) m/z: 537 [M + H] +. HPLC purity: 90%. Aqueous Solubility Determination of 4 and 27. Solubility tests were carried out by suspending 10 mg of test compound in double distilled water (1 mL), followed by stirring the mixture at rt for 24 h. The mixture was filtered to remove undissolved test compound. The amount of test compound dissolved in the filtrate was determined by HPLC. Calibration curves were prepared by dissolving known concentration of the test compounds in DMSO and determining the AUC of the peaks obtained from HPLC. HPLC conditions were same as that used for the purity determination of the compounds. Aurora Kinase A Protein Purification. The GST-tAurora A (123−401aa) fusion protein was produced by baculovirus expression system. The Aurora A catalytic domain with an Nterminal GST tag was constructed in pBacPAK8 plasmid and expressed in sf9 cells. Recombinant baculovirus infected sf9 cells were harvested by centrifugation, and the pellets were resuspended in PBS buffer (PBS, pH 7.3, 0.2 mM PMSF, 0.5 mM Na3VO4, 0.5 mM EDTA, 2 mM DTT, Complete Protease Inhibitor Cocktail table (1125700, Roche). Cells were lysed by sonication, and lysates were cleared by centrifugation at 15000 rpm for 30 min. The supernatants were loaded into 1 mL of GST Sepharose 4 Fast Flow (17-5132-01, GE healthcare) column previously washed with PBS buffer. The column were washed with 30 volumes of PBS buffer and then eluted by elution buffer (50 mM Tris (pH 8.0), 10 mM glutathione). To concentrate GST-tAurora A, buffer was replaced with Tris buffer (100 mM Tris (pH 7.5), 300 mM NaCl, 1 mM EDTA, 4 mM DTT) using Amicon ultra-15 (MWCO:30K, Millipore) to 2.4 mg/mL. After the addition of equal volume of glycerol and

0.04% Triton X-100, the proteins were aliquoted and stored at −80 °C. Aurora Kinase A Enzyme Inhibition Assay.30 Test compounds, enzyme, substrate-tetra(LRRWSLG), DTT, and ATP were dissolved in Aur buffer (50 mM Tris-HCl pH 7.4, 10 mM NaCl, 10 mM MgCl2, 100 μg/mL BSA) individually before the assay. Compounds were series diluted from 10 mM stock (for single dose, compounds were diluted from 10 mM stock to 100 and 20 μM; for IC50, 5× serial dilution was made from 100 to 0.16 μM) in Aur buffer. Diluted compounds (25 μL) were preincubated with purified 105 ng (10 μL) of GSTtAurora A (123−401aa) fusion protein at 25 °C for 15 min into 96-well U-bottomed plates (268152, NUNC). Then 5 μM ATP (5 μL), 1 mM DTT (5 μL), and 0.1 mM tetra(LRRWSLG) peptide substrate (5 μL) were added into the reactions of test compounds and GST-tAurora A. The reactions were incubated at 37 °C for 90 min. Then 50 μL of Kinase-Glo Plus reagent (V3771, Promega) was added into the reactions, followed by the incubation at 25 °C for 20 min. Finally, 70 μL of reactions were transferred to 96-well black plates (237108, NUNC) to quantify the ATP remaining in the solution. The luminescence was recorded by vector2 V (1420 multilable HTS counter, Perkin-Elmer). Aurora Kinase B Enzyme Inhibition Assay.30 Test compounds, recombinant human Aurora kinase B (PV3970, purchased from Invitrogen, Carlsbad, CA, USA), peptide substrate tetra(LRRASLG), DTT, and ATP were dissolved in Aur buffer (50 mM Tris-HCl pH 7.4, 10 mM NaCl, 10 mM MgCl2, 100 μg/mL BSA) individually before the assay. Compounds were series diluted from 10 mM stock (for single dose, compounds were diluted from 10 mM stock to 10 μM and 1 μM; for IC50, 4× serial dilution was made from 10 μM to 0.16 nM) in Aur buffer. Diluted compounds were preincubated with 40 ng Aurora B proteins at 25 °C for 15 min into 96 well U-bottomed plate (268152, NUNC), 5 μM ATP (5 μL), 1 mM DTT (5 μL), and 0.1 mM tetra(LRRASLG) peptide substrate (5 μL) were added into the reactions of test compound and Aurora B mixture. The reactions were incubated at 37 °C for 90 min. Then 50 μL of Kinase-Glo Plus reagent (V3771, Promega, Madison, WI, USA) was added into the reactions, followed by the incubation at 25 °C for 20 min. Then 70 μL of reactions were transferred to 96-well black plates (237108, NUNC) to quantify the ATP remaining in the solution. The luminescence was recorded by vector2 V (1420 multilable HTS counter, Perkin-Elmer). HCT-116 Antiproliferative Assay, Flow Cytometry Analysis, and Western Blot Analysis. HCT-116 antiproliferative assay of newly synthesized compounds and flow cytometry analysis of HCT-116 cells treated with compound 27 was carried out as reported in our previous publication.13 Western blot analysis of HCT-116 cells treated with compound 27 was performed as reported in our previous publication.10 Mitotic Progression Assay. To determine mitotic progression, HeLa cells were first synchronized in S phase with double thymidine block. Accordingly, cells were treated with 2.5 mM thymidine for 16 h, washed with 1× phosphate buffered saline (PBS), trypsinized, and reseeded in culture dish. After 12 h, cells were treated again with 2.5 mM thymidine for another 12 h. Then cells were released from the second thymidine block by washing twice with culture medium. To determine the mitotic index, cells were stained with Hoechst33342, and the cell-cycle content was scored from cells harvested every 2 h between 10 and 18 h after the second L

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thymidine release by the cell cycle analysis using MetaMorph software (Molecular Devices). Immunofluorescence Staining and Confocal Microscopy. The immunofluorescence staining and confocal microscopy were performed as described previously.14 In brief, cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.1% TritonX-100, and immunostained with a goat antilamin B1 antibody (Santa Cruz). After three washes with PBS, cells were probed with Alexa-488-conjugated secondary antibody (Invitrogen). Cell nuclei were counterstained with Hoechst33342 (Invitrogen). Cells were mounted onto glass slides with ProLong Gold antifade reagent (Invitrogen) and were visualized using a Leica TCS SP5 confocal microscope. Images were processed by the Imaris 7.2.1 (Bitplane) software. Docking of Ligands to Aurora Kinase A Co-crystal Structure. X-ray co-crystal structure of Aurora A protein in complex with inhibitor 4 (PDB ID: 3M11) was used for docking utilizing Discovery Studio 2.1.31 The docking of the ligands was done by DS/LigandFit program with CHARMm forcefield.32 To bring the docked ligand−protein complex to equilibrium, the DS/Simulation program was used to run molecular dynamics simulation. The Minimization convergent was used in two step methods: Steepest Descent with RMS gradient convergent to 0.1, and the final step was Conjugate Gradient with RMS Gradient convergent to 0.0001. Default set of parameters were used for the simulation, except for the Production parameter, which was set as 100000 steps. All the minimization process was in water environment by the Distance-Dependent Dielectrics methods. After molecular dynamics simulation, the binding energy was calculated by DS/Calculate Binding Energies protocol. In Vivo Pharmacological Evaluation of 27 in HCT-116 Subcutaneous Xenograft Nude Mice Model. Adapted from the previous report,33 adult male athymic nu/nu nude mice (BioLASCO, Ilan, Taiwan) were sc implanted with human colorectal HCT 116 (1 × 106) cancer cells per mouse. Human colorectal HCT 116 cancer cells were propagated in McCoy’s 5A Medium containing 10% fetal bovine serum (Gibco, Gaitherburg, MD). Tumor sizes were measured using an electronic caliper and calculated in length × (width)2/2. Tumor size and animal body weight were monitored twice a week. The animals were treated with the HCl salt of compound 27 (10 and 50 mg kg−1, QD; 20 mg kg−1, BID) and compound 1 (50 mg kg−1, QD) when the tumor size reached 100−200 mm3. The compounds were dissolved in a mixture of DMSO/ Cremophor EL/saline (10/20/70% in v/v/v) and administered iv into the tumor-bearing nude mice once or twice daily for 5 days per week for 2 weeks. Animal body weight and tumor size were measured twice a week. For all groups except for treatment with 27 at 10 mg/kg, n = 8 mice; n = 7 mice for the treatment group 27 at 10 mg/kg. Data were subjected to ANOVA followed by Student−Newman−Keuls test using SPSS software (SPSS, Chicago, IL, USA). A p value