Discovery of New Benzothiazole-Based Inhibitors of Breakpoint

Article pubs.acs.org/jmc

Discovery of New Benzothiazole-Based Inhibitors of Breakpoint Cluster Region-Abelson Kinase Including the T315I Mutant Seunghee Hong,† Jinhee Kim,† Sun-Mi Yun,‡ Hyunseung Lee,‡ Yoonsu Park,† Soon-Sun Hong,*,‡ and Sungwoo Hong*,† †

Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea Department of Biomedical Sciences, College of Medicine, Inha University, Incheon 400-712, Korea

‡

S Supporting Information *

ABSTRACT: The existence of drug resistance caused by mutations in the break-point cluster region-Abelson tyrosine kinase (Bcr-Abl) kinase domain remains a clinical challenge due to limited effective treatment options for chronic myeloid leukemia (CML). Herein we report a novel series of benzothiazole-based inhibitors that are effective against wild-type and T315I mutant Bcr-Abl kinases. The original hit compound, nocodazole, was extensively modified through a structure-based drug design strategy, especially by varying the groups at the C2 and C6 positions of the scaffold. In addition, the introduction of water-solubilizing groups at the terminal ethyl group resulted in enhanced physicochemical properties and potency in cellular inhibition. Several compounds inhibited the kinase activity of both wild-type Bcr-Abl and the T315I mutant with IC50 values in the picomolar range and exhibited good antiproliferative effects on Ba/F3 cell lines transformed with either wild-type or T315I mutant Bcr-Abl.

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INTRODUCTION The appearance of Philadelphia (Ph) chromosome is characteristic of chronic myeloid leukemia (CML) and is a result of the reciprocal translocation between the Abelson (ABL) gene on chromosome 9 and the break-point cluster region (Bcr) gene on chromosome 22. This chimeric Bcr-Abl gene generated by the recombination of two genes leads to the production of BcrAbl fusion protein, a constitutively active tyrosine kinase that drives clonal malignancy characterized by unchecked myeloid proliferation.1 Therefore, the fusion protein Bcr-Abl kinase has become a well-validated target for the development of therapeutics to treat CML as demonstrated by the clinical success of drug imatinib.2 Imatinib is a potent first-generation Bcr-Abl kinase inhibitor approved for clinical use and effective for most patients diagnosed with chronic phase disease.3 Despite the clear benefits of imatinib, many patients with advanced phases of CML frequently develop resistance to imatinib therapy that is often associated with the emergence of a point mutation in the Bcr-Abl kinase domain that disturbs effective inhibitor binding.4 To date, at least 100 different point mutations have been identified in CML patients with resistance to this drug.4b Several second-generation Bcr-Abl kinase inhibitors (e.g., nilotinib, bafetinib, dasatinib, and bosutinib) have been developed to overcome the imatinib resistance in CML.5 These currently approved drugs are effective against the majority of imatinib-resistant forms of Bcr-Abl, but none of the compounds are capable of inhibiting cases with the gatekeeper T315I mutation in Bcr-Abl that accounts for about 15−20% of all clinically relevant CML mutations.6 The © 2013 American Chemical Society

T315I mutation is a major obstacle for drug design because mutation at the gatekeeper site can alter the geometry of the ATP-binding pocket to interrupt several critical protein−drug interaction sites and eliminate a critical hydrogen bond required for tight binding of the inhibitors.7 In this regard, the identification of potent Bcr-Abl inhibitors that target the T315I mutation would have important therapeutic implications. Recently, Bcr-Abl inhibitors capable of inhibiting the T315I mutant were disclosed to inhibit the full range of Bcr-Abl kinase domain mutations as well as the wild-type kinase.8 Several compounds, such as ponatinib9a−c and rebastinib,9d are currently under clinical investigation in CML patients harboring the Bcr-Abl T315I mutation. Recently, we have disclosed that nocodazole may serve as a promising starting point for the development of new inhibitors targeting T315I mutant Bcr-Abl to treat CML (Figure 1).10

Figure 1. Chemical structure of nocodazole. Received: December 22, 2012 Published: April 19, 2013 3531

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Figure 2. (a) Predicted binding mode of nocodazole with Abl. (b) Predicted binding mode of nocodazole with AblT315I. Carbon atoms of nocodazole are shown in pink. (c) Predicted binding mode of imatinib with AblT315I. Each dotted line represents a hydrogen bond.

code 2GQG)12 and that of the T315I mutant bound to PPY-A (PDB code 2QOH).13 Structural analysis demonstrated that currently approved drugs (e.g., imatinib, nilotinib, and dasatinib) establish a key hydrogen bond with the OH group at the Thr315 residue. However, a mutation of threonine to isoleucine prevented the formation of this critical hydrogen bond. Furthermore, the increased bulk of the Ile315 side chain caused steric repulsion, thereby blocking the access of the inhibitor to the hydrophobic pocket near the gatekeeper residue (Figure 2c).14 In an effort to identify potent Bcr-Abl inhibitors, our structural design plan was initiated based on the docking and structural analysis of wild-type and T315I mutant of Abl bound to nocodazole (Figure 2a,b). The AutoDock program was used while accounting for the motional flexibility of the amino acid side chains in the ATP-binding site. The docking simulation was initiated by establishing 3D grids of interaction energy for all possible atom types. It was noted that two nitrogen atoms in the aminobenzimidazole group of nocodazole formed two hydrogen bonds with the backbone amidic nitrogen and the aminocarbonyl oxygen of Met318 in the Abl kinase. Interestingly, a larger gatekeeper isoleucine residue was found at the interface formed by van der Waals contacts between nocodazole and the T315I mutant of Abl (Figure 2b), indicating that favorable hydrophobic interactions were formed in the T315I mutant. For these reasons, nocodazole was selected as an initial template for further optimization by indepth structural modification. In an initial effort to enhance binding affinity, we explored the substituent space on nocodazole at two positions (C2 and C6) and compared the calculated binding free energies of the derivatives with respect to the wild-type and T315I mutant Abl kinase. First, we hypothesized that the overall activity of nocodazole derivatives might be improved by introducing a

Nocodazole has been used as a standard antimicrotubule agent that interferes with the polymerization of microtubules.11 Our enzymatic kinase assays and docking simulation studies have shown that nocodazole is a common inhibitor of various cancer-related kinases including Abl, c-Kit, MEK, and Braf. In particular, nocodazole has submicromolar activities against both phosphorylated wild-type and T315I mutant forms of Bcr-Abl kinase (Ablphos IC50 = 0.21 μM; AblT315I IC50 = 0.64 μM). We extensively modified the structure of nocodazole using a structure-based drug design strategy and discovered a new class of potent benzothiazole-based inhibitors. Herein, we report our studies on the design and synthesis of a new series of Bcr-Abl inhibitors effective against the wild-type and the T315I mutant of Bcr-Abl kinase and demonstrate the biological activities of these compounds.

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RESULTS AND DISCUSSION Design of the Lead Compound from Nocodazole. The availability of the 3D structure data for therapeutic targets has enhanced opportunities for the rapid identification of biologically active compounds utilizing structure-based drug design strategies. Conformation of the Asp381-Phe382-Gly383 (DFG) motif in the Abl activation loop is very important for kinase activity because this motif is positioned adjacent to the ATPbinding site. The “DFG-in” conformation is referred to as the side chain of Asp381 is directed toward the ATP-binding site in the active conformation to coordinate an ATP-bound Mg2+ ion. Biochemical assay results showed that nocodazole derivatives target the active form of the phosphorylated Abl protein. Therefore, the active conformations (DFG-in conformation) of the wild-type and T315I mutant of Abl were selected as the target proteins in the present study. We prepared the receptor models in the DFG-in conformation based on the X-ray crystal structure of the wild-type Abl complexed with dasatinib (PDB 3532

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Figure 3. (a) The overall process for deriving the lead from nocodazole. (b) Predicted binding mode of compound 2 with AblT315I. Carbon atoms of compound 2 are indicated in pink. (c) The simplified binding mode of compound 2 with the ATP binding site of AblT315I.

slight loss of potency (2, AblT315I IC50 = 43.7 nM). Figure 3b shows the lowest energy conformation of compound 2 in the ATP binding site as calculated with the modified AutoDock program.15 The overall structural features derived from the docking simulations confirmed that the inhibitory activities of compound 2 were attributed to the multiple hydrogen bonds and hydrophobic interactions established simultaneously in the ATP-binding site of Abl. Considering the enzymatic activity within the active site of the T315I mutant and the synthetic flexibility via standard Suzuki coupling chemistry, compound 2 was selected as a lead compound from which more potent inhibitors could be derived. To identify improved enzyme interactions and physicochemical properties of compound 2, a systematic evaluation of the structural features necessary to impart activity was performed. We initiated our investigation by modifying the aryl group at the C6 position. The docking simulation predicted that the C6 phenyl group is directed toward the region near the gatekeeper and catalytic lysine region (Figure 3c). In this region, space was available for substituents on the phenyl ring that may participate in hydrogen bonding with Lys271 or Asp381. Based on our structural analysis, we planned to install a variety of substituents as the phenyl group at the C6 position, while the ethyl urea moiety at the C2 position remained fixed. Scheme 1 illustrates the general synthetic route for the preparation of benzimidazole derivatives with C6-aryl substitution. A variety of aryl groups were attached to the 6-

hydrogen-donating moiety into the molecule to establish an additional hydrogen bond with the hinge region. Thus, the terminal methoxy group was replaced with an ethylamine group to afford the compound 1 containing an ethyl urea group at the C2 position (Figure 3a). To our delight, this modification led to about a 30-fold increase in inhibitory activity against both Abl wild-type and T315I mutant (Abl IC50 = 8.67 nM; AblT315I IC50 = 23.1 nM). This significant enhancement of potency most likely stemmed from the strengthening of hydrogen bonds between the inhibitor and protein groups because the docking study showed that the terminal ethyl urea group in compound 1 was found to establish multiple hydrogen bonds with Met318 of Abl. The importance of the hydrogen bond network was further demonstrated by removal of the terminal ethyl urea group in other related derivatives (e.g., amide and carbamate). Therefore, the ethyl urea moiety at the C2 position was fixed to catch two pairs of hydrogen bonds when designing the new derivatives. Next, a survey of the C6 group was conducted to evaluate the structural differences affecting the binding pockets of wild-type and T315I mutant Abl kinase. To identify a scaffold that can be easily diversified for our structure−activity relationship (SAR) study, we planned to modify the thiophene-2-one group at the C6 position. Of particular significance was the observation that the benzene ring could form favorable van der Waals interactions with the gatekeeper Ile315 residue of Bcr-Abl without causing steric clash, although this substitution led to a 3533

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Scheme 1a

arylboronic acids. Diaminobenzene 4 was then reacted with N,N′-bis[(ethylamino)carbonyl] carbamimidothioic acid methyl ester to allow for the facile conversions of o-phenylenediamines directly into the desired product 5 with high yields (Scheme 1). Aryl ether derivatives (5v and 5w) were obtained from 4-phenoxybenzene-1,2-diamines, which were prepared by a two-step sequence: (1) SNAr reaction between 5-chloro-2nitroaniline and substituted phenol under basic conditions, followed by (2) reduction of the nitro group with tin chloride. Similarly, amide derivatives (5x and 5y) were obtained from 3,4-diaminobenzamides. Thus, 3,4-dinitrobenzoyl chloride was reacted with primary or secondary amines to produce 3,4-

a (a) Arylboronic acid, Pd(dppf)Cl2·CH2Cl2, K2CO3, 1,4-dioxane/H2O = 3:1, 100 °C, microwave, 90 min; (b) N,N′-bis[(ethylamino)carbonyl] carbamimidothioic acid methyl ester, H2O/AcOH = 10:3, 110 °C, 5 h.

position of the benzimidazole core using palladium-catalyzed Suzuki couplings of diaminobenzene 3 with corresponding Table 1. Exploration of the Groups at C6

3534

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Scheme 2a

dinitrobenzamides, followed by the reduction of the nitro groups by tin chloride. The IC50 values of the resulting compounds were determined for both wild-type and T315I Abl (Table 1). The orientation and pattern of the substitutions on the phenyl ring are critical for enzyme potency. In general, substitution at the ortho position of the phenyl ring enhanced its potency (5e, Abl IC50 = 7.8 nM; AblT315I IC50 = 16.9 nM), whereas substitution at the meta or para position led to reduced activity (5a and 5c). This prompted us to investigate in detail the potential effect of various substitutions at the ortho position. Substituents at the ortho position of the phenyl ring included ethyl and chloro groups. These derivatives were tolerated for both wild-type and T315I Abl kinase (5f, 5g). It was noteworthy that the methoxy derivative 5h exerted potent effects on wild-type and T315I mutant of Abl (Abl IC50 = 18.8 nM; AblT315I IC50 = 6.2 nM). The oxygen in the 2-methoxy group appeared to establish an additional hydrogen bond with the Asp381 backbone, and such a strengthening of hydrogen bonds would be responsible for the higher inhibitory activity. The optimal compound in this series was obtained by introducing an ethoxy group as exemplified by compound 5j (Abl IC50 = 1.6 nM; AblT315I IC50 = 2.3 nM). We also examined the effect of size of the groups at the C6 position and observed that inhibitory activity was reduced when the ethoxy group was replaced with more sterically hindered isopropoxy (5k) or phenoxy groups (5l). The next round of analogues had an additional substituent incorporated into the phenyl ring. Attempts to further increase potency through disubstitution at the two ortho positions (5m, 5n, 5o, and 5p) were unremarkable, and no compound offered any advantage relative to the corresponding derivatives with a single substitution. In order to determine whether the methoxyor ethoxy-phenyl groups at the C6 position were optimal, we next evaluated that the effect of a heteroaryl group, such as pyridyl and pyrazole, at this position and found that these groups decreased the inhibitory activity (5q, 5r, 5s, 5t, and 5u). We further attempted to explore the SAR of the side chain linked to the C6 position. Replacement of the aryl group with aryl ether (5v and 5w) had a negative effect on potency. In particular, replacement of the aryl group with the amide groups (5x and 5y) resulted in a complete loss of activity, thus revealing the indispensable nature of the aryl group at this position for maintaining activity in this series. Based on these findings, we determined that o-methoxy- or ethoxy-phenyl moieties at the C6 position were most effective and planned to explore the potential influence of the imidazole core for further structural modification to investigate SAR. Identification of Benzothiazole Scaffold. Based on the structures of compounds 5h and 5j, the enhanced potency due to the ethyl urea group at the C2 position and the 2-methoxyor 2-ethoxyphenyl moieties at the C6 position suggested a need to more broadly explore analogs incorporating these key functional groups. We therefore turned to our attention to core modification with these groups fixed in position. An investigation was thus conducted to evaluate the impact of core modification on inhibitory activities of the compounds, and a series of derivatives with benzothiazole and benzoxazole cores were synthesized (Scheme 2). In all cases, an aryl group at C6 was introduced prior to the urea group at C2. Compound 6 was efficiently converted into the aryl intermediate 7 by Suzuki coupling with appropriate aryl boronic acid. Finally, the treatment of 7 with alkyl-isocyanate or carbodiimidazole yielded the desired target compounds 8 and 9.

(a) Arylboronic acid, Pd(dppf)Cl2·CH2Cl2, K2CO3, 1,4-dioxane/H2O = 3:1, 120 °C, microwave, 90 min; (b) alkyl-isocyanate, 1,4-dioxane, 80 °C, 5 h; (c) carbodiimidazole, R-NH2, DMF, rt, 10 h. a

IC50 values of the synthesized derivatives were then determined as summarized in Table 2. Notably, the conversion Table 2. Comparison of Core on Enzyme Potency

compd

X

Abl wt IC50 (nM)

Abl T315I IC50 (nM)

5h 8a 9a

NH O S

18.8 40.8 0.06

6.22 6.12 0.11

of benzimidazole into benzothiazole increased in the inhibitory activity compared with the parent compounds. Collectively, the combination of 2-methoxybenzene, the benzothiazole core, and the urea group at the C2 position yielded exceptionally potent inhibitors 9a and 9b with picomolar activities (9a, Abl IC50 = 0.06 nM, AblT315I IC50 = 0.11 nM; 9b, Abl IC50 = 0.03 nM, AblT315I IC50 = 0.064 nM). Further investigation of alternate scaffolds revealed that the benzothiazole was optimal for this series (Table 2 and Table S2 in Supporting Information). To the best of our knowledge, the benzothiazole scaffold is not present in any of the Abl inhibitors reported to date. We next prepared more benzothiazole derivatives to investigate the SAR profiles based on our previous docking study. The enzyme potency of the derivatives was decreased when a trifluoromethoxy or propoxy group was introduced (9c and 9d), indicating that the ethoxy group is maximum size suitable for this position on the benzothiazole core. We also introduced polar groups at the para position of the C6 aryl ring to establish additional hydrogen bonds with the Lys271 or Asp381 side chains. However, the 4-pyridyl (9h and 9l), 4methoxyphenyl (9i and 9k), and 4-aminophenyl derivatives (9j) did not provide any advantages compared with the compounds 9a or 9b (Table 3). To obtain structural insight into the mechanisms underlying the inhibitory activities of the newly identified benzothiazole derivatives, binding modes in the ATP-binding sites were investigated using the modified AutoDock scoring function.15 The calculated binding modes of compounds 9a and 5h with the Abl T315I mutant are presented in Figure 4. The results of the docking simulations were internally consistent in that compounds 9a and 5h were positioned in similar configurations and had comparable interactions with the amino acid residues in the ATP binding site. The docking study revealed that 9a appeared to bind tightly at the ATP binding site by forming multiple hydrogen bonds and hydrophobic interactions with the pocket created by Ile313, Met290, Glu286, Val299, Lys271, and Phe382. One explanation for the differences in potency between compounds 9a and 5h is that the ethyl urea group of 9a can participate in more favorable hydrogen bonding. 3535

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Compound 9a was further subjected to kinase selectivity profiling with a panel of 96 cancer-related kinases at 1 μM concentrations in a high-throughput binding assay (Table 4, KINOMEscan, Ambit Biosciences).16 Remarkably, compound 9a displayed a high degree of selectivity over other kinases including Src, Aurora, and c-Kit, which are known to have high structural similarity to Abl. The only kinase against which 9a demonstrated meaningful off-target activity was PDGFRB (POC = 16). Optimization for Improving Cell Activity. With the impressive enzyme activity profile, the ability of these inhibitors to suppress the growth of Ba/F3 cells ectopically expressing wild-type or T315I mutant Bcr-Abl was assessed. For this study, cell viability was measured using a 2,3-bis(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboanilide inner salt (XTT) assay. Despite their excellent enzymatic inhibition profiles, these derivatives exerted modest effects on cell growth (e.g., 9a WT cell IC50 = 3.57 μM, T315I cell IC50 = 2.14 μM), which might have been due to poor solubility of the compounds (e.g., 9a 0.0227 μg/mL, Table 6). Our docking study suggested that the terminal ethylamine group appeared to orient to the solvent-exposed region of the enzyme and could provide a good location for modulating the physicochemical properties of the compounds (Figures 3 and 4). Therefore, we hypothesized that the cell activities might be improved by introducing an additional solubilizing group into the terminal ethylamine group without significant influencing the enzyme inhibitory activities. To this end, the water-soluble morpholine moiety was employed to enhance the physicochemical properties. Consistent with our docking analysis, replacement of the terminal ethyl group with morpholinoethane, 10a, increased the potency in cellular inhibition in a dose-dependent manner (WT cell IC50 = 0.10 μM, T315I cell IC50 = 0.29 μM) while retaining an excellent enzymatic potency. Encouraged by these results, a series of new derivatives were further designed and synthesized by modifying the terminal ethyl group (Table 5). In general, derivatives incorporating a 2-methoxyphenyl group at the C6 position (10a, 10b, 10c, and 10d) were slightly superior to the corresponding 2-ethoxyphenyl (10e, 10f, 10g, and 10h) and 2ethylphenyl (10i and 10j) derivatives in terms of both enzyme and cell growth inhibition. The best result was obtained with 10d, in which a widely utilized solubilizing group, methyl piperazine was incorporated. The IC50 values for 10d against wild-type and T315I Ba/F3 cells were 0.046 and 0.078 μM, respectively. On the other hand, imatinib strongly inhibited proliferation of wild-type Ba/F3 cells but not T315I Abl mutant transformed Ba/F3 cells, indicating that the T315I mutation conferred resistance to imatinib. Compound 10d also exhibited desirable pharmaceutical properties, such as aqueous solubility, stability, and lipophilicity, and deserves consideration for further development (Table 6). The lead compounds 10b and 10d were further compared with imatinib and ponatinib in both kinase assay and cellular assay including parental Ba/F3 cells. Although 10d is about 4fold less potent in the antiproliferative effects on T315I cells when compared with ponatinib, the ratio for selectivity relative to untransformed Ba/F3 cells of 10d is more desirable than that of ponatinib (ponatinib, parental Ba/F3 IC50 = 1.9 μM) as summarized in Table 7. Effect on Cell Proliferation and Downstream Cell Signaling. As the most potent compounds shown in Table 5, 10b and 10d were selected for evaluation with additional biological assays to better understand the antiproliferative

Table 3. Structure and Activity Relationship of Benzothiazole Derivatives

a

5-Methoxy benzothiazole. b7-N-Benzothiazole.

Although the overall binding mode of benzothiazole 9a was similar to the corresponding benzimidazole 5h, the urea group of 9a was more closely directed toward the pocket around Met318 due to the increased size of the sulfur atom in benzothiazole. The lengths of the two hydrogen bonds between the ethyl urea group in 9a and Met318 were calculated to be 1.90 and 2.03 Å, respectively, indicating that these were stronger hydrogen bonds than those formed by 5h (2.06 and 3.59 Å). Moreover, the intramolecular hydrogen bond between the NH in benzimidazole and CO in ethyl urea of 5h appeared to fix the geometry in the same plane. This spatial restriction prevented tight hydrogen bond interactions with Met318 by distorting the angle of the urea group (Figure 4b). Unlike the benzimidazole core, the urea group of the benzothiazole derivatives had sufficient flexibility and rotation required for tight hydrogen bonding. We also turned to the energetic features associated with binding of 9a and 5h in the ATP-binding sites of T315I mutant of ABL. The calculated binding free energies of 9a and 5h compare reasonably well with their inhibitory activities found in docking simulations. The predicted binding free energy of 9a is lower than that of 5h by 2.24 kcal/mol (9a −9.78 kcal/mol; 5h = −7.54 kcal/mol). 3536

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Figure 4. (a) Predicted binding mode of 9a with the ATP-binding site of AblT315I. Carbon atoms of the protein and the ligand are indicated in gray and cyan, respectively. (b) Predicted binding mode of 5h with the ATP-binding site of AblT315I. Carbon atoms of the protein and the ligand are indicated in gray and green, respectively. Each dotted line represents a hydrogen bond.

Table 4. KINOMEscan Profile of Compound 9aa kinase phos

ABL ABLT315I ABLE255K AKT1 AURKA AURKB BRAF CDK2 CDK11 EGFR ERK1 FAK FGFR2

POC

b

5 4.5 4.5 67 100 100 100 100 100 100 100 100 100

kinase

POC

FLT3 GSK3b IGF1R IKKα JAK2 JNK1 KIT MEK1 MEK2 MET P38α PAK1 PDGFRA

94 100 100 100 100 100 79 100 100 100 100 93 79

b

kinase

POC

PDGFRB PDPK1 PKAC-a PLK1 PI3KCA PIM1 PLK1 PLK3 ROCK2 RSK2 SRC TIE2 VEGFR2

16 67 100 100 100 100 100 87 100 58 72 100 98

inhibitors that were effective against the wild-type and T315I mutant of Bcr-Abl kinase. Through the systematic exploration of the functional groups in the C2 and C6 positions along with modulating the core template of the original hit compound, nocodazole, we successfully developed a SAR profile for the series. Our results showed that the urea group at the C2 position and a selection of moieties (e.g., 2-methoxy and 2ethoxy group on the phenyl ring) at the C6 position of the benzothiazole scaffold resulted in optimal activity of the compounds. This profoundly influenced the enzymatic inhibitory activities and antiproliferative effects on corresponding Ba/F3 cell lines transformed with wild-type or T315I mutant of Bcr-Abl kinase. In addition, the introduction of water-solubilizing groups at the terminal ethyl group resulted in the enhanced physicochemical properties and enhanced potency in cellular inhibition. These new inhibitors may serve as promising lead compounds for further development of new therapeutic agents to treat drug-resistant cases of CML with the T315I mutation.

b

a A panel of 96 kinases were tested at 1 μM concentrations in a highthroughput binding assay (Ambit Bioscience). Only representative kinases are shown; see Supporting Information for complete panel. b Lower numbers of POC (percent of control) values indicate stronger hits. Values shown are the average of duplicate measurements.

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EXPERIMENTAL SECTION

General Chemistry. Unless stated otherwise, reactions were performed in flame-dried glassware under a positive pressure of nitrogen using freshly distilled solvents. Analytical thin layer chromatography (TLC) was performed on precoated silica gel 60 F254 plates and visualization on TLC was achieved by UV light (254 and 354 nm). Flash column chromatography was undertaken on silica gel (400−630 mesh). 1H NMR was recorded at 400 or 300 MHz, and chemical shifts were quoted in parts per million (ppm) referenced to the appropriate solvent peak or 0.0 ppm for tetramethylsilane. The following abbreviations were used to describe peak splitting patterns when appropriate: br = broad, s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, dd = doublet of doublet, td = triplet of doublet, qd = quartet of doublet, ddd = doublet of doublet of doublet. Coupling constants, J, were reported in hertz. 13C NMR was recorded at 100 MHz, and chemical shifts were reported in ppm referenced to the center line of a triplet at 77.0 ppm of chloroform-d. Mass spectral data were obtained from the KAIST Basic Science Institute using the EI method. High-performance liquid chromatog-

activities against leukemia cells. To determine whether the antiproliferative effects of 10b and 10d were dependent on the inhibition of Bcr-Abl activity, we measured the phosphorylation of Bcr-Abl and the downstream signals, STAT5 and CrkL.17 As shown in Figure 5, 10b and 10d strongly inhibited the phosphorylation of Bcr-Abl in Ba/F3 cells expressing either wild-type or T315I mutant. Likewise, the phosphorylation levels of STAT5 and CrkL were effectively suppressed. On the other hand, both imatinib and dasatinib were significantly less able to reduce the phosphorylation levels of Bcr-Abl, STAT5, and CrkL in Ba/F3 cells expressing the T315I mutant (Figure 5).

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CONCLUSION Utilizing a structure-based design strategy, we designed and synthesized a new class of potent benzothiazole-based 3537

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Table 5. Potency Profiles for Selected Benzothiazole Derivatives on Antiproliferative Effects on Bcr-Abl Transformed Ba/F3 Cells

maintained for 23 min; the solvent ratio was changed to MeOH (0.1% TFA) 10% and H2O (0.1% TFA) 90% (25 min). All final compounds were found to have >95% purity. Commercial grade reagents and solvents were used without further purification except as indicated below. Dichloromethane was distilled from calcium hydride. THF was distilled from sodium. Unless otherwise stated, all commercial reagents and solvents were used without additional purification. General Procedure (GP I) for Suzuki Coupling. A solution of 4bromo-1,2-diaminobenzene 3 (100 mg, 0.535 mmol), phenylboronic acid (72 mg, 0.59 mmol), K2CO3 (221 mg, 1.60 mmol), and Pd(dppf)Cl2·CH2Cl2 (87 mg, 0.11 mmol) in 1,4-dioxane/H2O = 3:1 (4 mL) was heated to 100 °C for 4 h. The resulting solution was concentrated in vacuo and filtered on silica−Celite using EtOAc and hexane. The filtrate was concentrated and purified with flash column chromatography (EtOAc/hexane) to give a biphenyl-3,4-diamine (30 mg, 30%). 1H NMR (300 MHz, MeOH-d4) δ 6.69 (d, J = 8.0 Hz, 1H), 6.81 (dd, J = 2.0, 8.0 Hz, 1H), 6.93 (d, J = 2.0 Hz, 1H), 7.13 (t, J = 7.3 Hz, 1H), 7.23−7.29 (m, 2H), 7.42−7.45 (m, 2H). General Procedure (GP II) for Introducing 2-Urea. 1-Ethyl-3(6-phenyl-1H-benzo[d]imidazol-2-yl)urea (2). Biphenyl-3,4-diamine (15 mg, 0.081 mmol) and N,N′-bis[(ethylamino)carbonyl] carbamimidothioic acid methyl ester (23 mg, 0.098 mmol) was dissolved in 0.5 mL of AcOH/H2O = 3:10 and stirred for 5 h at 100 °C. The reaction mixture was cooled to room temperature and then neutralized using sat. NaHCO3. It was concentrated in vacuo and purified with flash column chromatography to give the product 2 as a white solid (18 mg, 78% yield). 1H NMR (400 MHz, DMSO-d6) δ 1.11 (t, J = 7.2 Hz, 3H), 3.14−3.27 (m, 2H), 7.27−7.33 (m, 3H), 7.40−7.44 (m, 3H), 7.60−7.63 (m, 3H), 9.89 (s, 1H), 11.54 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.3, 34.1, 119.7, 126.4, 126.6, 128.8, 133.1, 141.5, 148.9, 154.1. HRMS (EI+) m/z calcd for C16H16N4O [M + H]+, 281.1402; found, 281.1401.

Table 6. Physicochemical Properties of Lead Compounds compd

solubilitya (μg/ mL)

stabilityb (%)

log Pe (cm/s)c

clogPd

MW

9a 10b 10d

0.0227 7.49 2240

100 100 99.7

−4.11 −4.29 −4.93

3.5 2.5 3.1

327.4 343.4 425.6

Aqueous solubility determined at 25 °C (pH 6.7). bMonitored by UPLC for 24 h at 37 °C (pH 6.7). cPermeability coefficient (log Pe) obtained in PAMPA test. dPredicted by Pipeline Pilot.

a

Table 7. Inhibitory Activities and Potency Profiles for Selected Benzothiazole Derivatives, Imatinib, and Ponatinib IC50 (μM) compd 10b 10d imatinib ponatinib

Abl T315I kinase IC50 (nM)

WT Ba/ F3

T315I Ba/F3

K562

parental Ba/F3

0.064 0.015

0.089 0.046 0.092 0.0023

0.14 0.078 4.79 0.018

0.12 0.42 0.17 0.023

>10 >10 >10 1.9

0.58

raphy analyses for checking purity (>95% area) of synthesized compounds were performed on a Waters HPLC equipped with an Agilent Prep-C18 reverse phase column (21.2 mm × 150 mm, 10 μm) and by HRMS. The mobile phase was a mixture of MeOH (0.1% TFA) and H2O (0.1% TFA). Compound purity was determined by integrating peak areas of the liquid chromatogram, monitored at 254 nm. Flow rate (10 mL/min). Gradient system: from MeOH (0.1% TFA) 10% and H2O (0.1% TFA) 90% (0 min) to MeOH (0.1% TFA) 90% and H2O (0.1% TFA) 10% (12 min), the solvent ratio was 3538

dx.doi.org/10.1021/jm301891t | J. Med. Chem. 2013, 56, 3531−3545

Journal of Medicinal Chemistry

Article

Figure 5. Western blot showing the inhibition of Bcr-Abl signaling in Ba/F3 cells expressing wild-type Bcr-Abl (left panel) or Bcr-Abl T315I (right panel). Cells were treated with 1 μM of imatinib, dasatinib, 10b, and 10d, incubated 37 °C for 1 h. 1-Ethyl-3-(6-(thiophene-2-carbonyl)-1H-benzo[d]imidazol-2-yl)urea (1). The solution of 3,4-dinitrobenzoic acid (300 mg, 1.414 mmol) in thionyl chloride (5 mL) was stirred for 5 h at 60 °C and cooled to room temperature. The mixture was concentrated and 3,4dinitrobenzoyl chloride was obtained as yellow solid. The yellow solid (326 mg, 1.41 mmol), AlCl3 (566 mg, 4.25 mmol), and thiophene (113 μL, 1.41 mmol) were dissolved in anhydrous CH2Cl2 and stirred 5 h at 60 °C. The reaction was quenched with H2O at 0 °C and filtered to remove solid. The filtrate was extracted 3 times with CH2Cl2 and dried over MgSO4. The organic layer was concentrated in vacuo and purified with flash column chromatography (hexane/EtOAc = 5:1) to give 3,4-(dinitrophenyl)(thiophen-2-yl)methanone (59 mg, 15% yield). The 3,4-(dinitrophenyl)(thiophen-2-yl)methanone (42 mg, 0.151 mmol) and SnCl2·2H2O (206 mg, 0.910 mmol) in EtOH/ H2O = 1:1 were stirred for 12 h at 80 °C. After the mixture cooled to room temperature, sat. NaHCO3 was added to the reaction mixture. The precipitate was removed by filtration, and the filtrate was extracted 3 times with CH2Cl2, dried over MgSO4, and concentrated in vacuo. The residue was purified with flash column chromatography (hexane/ EtOAc = 1:2) to give (3,4-diaminophenyl)(thiophen-2-yl)methanone (33 mg, 99.7% yield). Finally, 1 was prepared (15 mg, 32% yield) according to GP II from (3,4-diaminophenyl)(thiophen-2-yl)methanone (33 mg, 0.15 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.10 (t, J = 7.2 Hz, 3H), 2.95−4.06 (m, 2H), 7.11 (s, 1H), 7.20−7.35 (m, 1H), 7.47 (d, J = 8.3 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.73 (d, J = 3.8 Hz, 1H), 7.94 (s, 1H), 8.04 (d, J = 5.0 Hz, 1H), 10.17 (s, 1H), 11.82 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.2, 34.1, 114.6, 122.7, 128.4, 129.8, 134.3, 134.5, 143.5, 150.5, 153.9, 186.8. HRMS (EI+) m/z calcd for C15H14N4O2S [M + H]+, 315.0916; found, 315.0923. 1-Ethyl-3-(6-p-tolyl-1H-benzo[d]imidazol-2-yl)urea (5a). Compound 5a was prepared (12 mg, 55% yield) according to GP II from 4′-methylbiphenyl-3,4-diamine (15 mg, 0.075 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.11 (t, J = 7.1 Hz, 3H), 2.32 (s, 3H), 3.16− 3.28 (m, 2H), 7.23 (d, J = 7.9 Hz, 2H), 7.26−7.34 (m, 2H), 7.39 (d, J = 8.2 Hz, 1H), 7.50 (d, J = 7.8 Hz, 2H), 7.58 (s, 1H), 9.89 (s, 1H), 11.48 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.2, 20.6, 34.1, 119.5, 126.4, 129.4, 133.0, 135.5, 138.6, 148.9, 154.1. HRMS (EI+) m/ z calcd for C17H18N4O [M + H]+, 295.1559; found, 295.1537. 1-Ethyl-3-(6-(3-fluorophenyl)-1H-benzo[d]imidazol-2-yl)urea (5b). Compound 5b was prepared (7 mg, 22% yield) according to GP II from 3′-fluorobiphenyl-3,4-diamine (20 mg, 0.098 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.11 (t, J = 7.2 Hz, 3H), 2.91−3.73 (m, 2H), 7.10 (td, J = 2.9, 6.6 Hz, 1H), 7.27 (s, 1H), 7.35 (dd, J = 1.8, 8.3 Hz, 1H), 7.39−7.50 (m, 4H), 7.66 (s, 1H), 9.98 (s, 1H), 11.51 (s, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 15.2, 34.1, 112.8, 113.0, 113.0, 113.2, 119.7, 122.5, 122.6, 130.5, 130.6, 131.6, 144.0, 144.0, 149.1, 154.0, 161.4, 163.9. HRMS (EI+) m/z calcd for C16H15FN4O [M + H]+, 299.1308; found, 299.1317. 1-Ethyl-3-(6-m-tolyl-1H-benzo[d]imidazol-2-yl)urea (5c). Compound 5c was prepared (4 mg, 19% yield) according to GP II from 3′-methylbiphenyl-3,4-diamine (15 mg, 0.075 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.11 (t, J = 7.1 Hz, 3H), 2.36 (s, 3H), 3.18−3.25

(m, 2H), 7.10 (d, J = 7.4 Hz, 1H), 7.28−7.32 (m, 3H), 7.33−7.48 (m, 3H), 7.59 (s, 1H), 9.98 (s, 1H), 11.54 (s, 1H). 13C NMR (100 MHz DMSO-d6) δ 15.3, 21.2, 34.1, 113.1, 119.7, 123.8, 127.1, 127.4, 128.7, 133.2, 137.8, 141.5, 149.0, 154.1. HRMS (EI+) m/z calcd for C17H18N4O [M + H]+, 295.1559; found, 295.1562. 1-(6-(3-Cyanophenyl)-1H-benzo[d]imidazol-2-yl)-3-ethylurea (5d). Compound 5d was prepared (10 mg, 47% yield) according to GP II from 3′,4′-diaminobiphenyl-3-carbonitrile (15 mg, 0.071 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 1.11 (t, J = 7.2 Hz, 3H), 3.14−3.27 (m, 2H), 7.25 (s, 1H), 7.39 (dd, J = 1.8, 8.2 Hz, 1H), 7.44 (d, J = 8.3 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.69 (d J = 1.7 Hz, 1H), 7.74 (d, J = 7.7 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 8.08 (s, 1H), 9.93 (s, 1H), 11.64 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.2, 34.1, 111.9, 118.9, 119.8, 130.0, 130.8, 131.4, 142.6, 149.2, 154.0. HRMS (EI+) m/z calcd for C17H15N5O [M + H]+, 306.1355; found, 306.1351. 1-Ethyl-3-(6-o-tolyl-1H-benzo[d]imidazol-2-yl)urea (5e). Compound 5e was prepared (10 mg, 34% yield) according to GP II from 2′-methylbiphenyl-3,4-diamine (20 mg, 0.10 mmol). 1H NMR (300 MHz, DMSO-d6) δ 1.10 (t, J = 7.2 Hz, 3H), 2.22 (s, 3H), 3.16− 3.25 (m, 2H), 6.95 (dd, J = 1.5, 8.1 Hz, 1H), 7.20−7.27 (m, 6H), 7.37 (d, J = 8.1 Hz, 1H), 9.85 (s, 1H), 11.52 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.2, 20.3, 34.0, 121.7, 125.7, 126.6, 129.8, 130.1, 133.7, 134.8, 142.4, 148.7, 154.1. HRMS (EI+) m/z calcd for C17H18N4O [M + H]+, 295.1559; found, 295.1557. 1-(6-(2-Chlorophenyl)-1H-benzo[d]imidazol-2-yl)-3-ethylurea (5f). Compound 5f was prepared (5 mg, 38% yield) according to GP II from 2′-chlorobiphenyl-3,4-diamine (15 mg, 0.068 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.11 (t, J = 7.2 Hz, 3H), 3.05−3.29 (m, 2H), 7.07 (dd, J = 1.9, 8.1 Hz, 1H), 7.25 (s, 1H), 7.31−7.45 (m, 5H), 7.53 (dd, J = 1.7, 7.4 Hz, 1H) 10.19 (s, 1H), 11.36 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.2, 34.1, 113.1, 114.1, 122.1, 127.3, 128.5, 129.7, 131.2, 131.5, 131.8, 136.7, 140.8, 148.9, 154.0. HRMS (EI+) m/z calcd for C16H15ClN4O [M + H]+, 315.1013; found, 315.1001. 1-Ethyl-3-(6-(2-ethylphenyl)-1H-benzo[d]imidazol-2-yl)urea (5g). Compound 5g was prepared (18 mg, 56% yield) according to GP II from 2′-ethylbiphenyl-3,4-diamine (23 mg, 0.10 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.01 (t, J = 7.5 Hz, 3H), 1.10 (t, J = 7.2 Hz, 3H), 2.55 (q, J = 7.5 Hz, 2H), 3.19−3.23 (m, 2H), 6.93 (dd, J = 1.7, 8.1 Hz, 1H), 7.15 (dd, J = 1.6, 7.5 Hz, 1H), 7.20 (td, J = 1.9, 7.1 Hz, 1H), 7.23−7.32 (m, 4H), 7.37 (d, J = 8.1 Hz, 1H), 9.87 (s, 1H), 11.49 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.2, 15.7, 25.6, 34.1, 121.7, 125.5, 126.9, 128.4, 130.0, 133.8, 141.1, 142.1, 148.7, 154.1. HRMS (EI+) m/z calcd for C18H20N4O [M + H]+, 309.1715; found, 309.1690. Ethyl-3-(6-(2-methoxyphenyl)-1H-benzo[d]imidazol-2-yl)urea (5h). Compound 5h was prepared (31 mg, 71% yield) according to GP II from 2′-methoxybiphenyl-3,4-diamine (30 mg, 0.14 mmol). 1H NMR (300 MHz, DMSO-d6) δ 1.11 (t, J = 7.2 Hz, 3H), 3.17−3.26 (m, 2H), 3.75 (s, 3H), 7.00 (t, J = 7.3 Hz, 1H), 7.07 (d, J = 8.2 Hz, 1H), 7.11 (dd, J = 1.7, 8.1 Hz, 1H) 7.26−7.30 (m, 2H), 7.31−7.37 (m, 2H), 7.46 (d, J = 1.6 Hz, 1H), 9.90 (s, 1H), 11.36 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.2, 34.1, 55.4, 111.7, 120.6, 122.2, 127.9, 3539

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6.83 (dd, J = 1.5, 8.1 Hz, 1H), 7.15 (s, 1H), 7.20−7.38 (m, 2H), 7.33 (s, 1H), 9.79 (s, 1H), 11.37 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.3, 34.1, 55.6, 104.4, 119.8, 123.5, 126.2, 128.3, 148.3, 154.2, 157.4. HRMS (EI+) m/z calcd for C18H20N4O3 [M + H]+, 341.1614; found, 341.1605. 1-Ethyl-3-(6-(pyridin-4-yl)-1H-benzo[d]imidazol-2-yl)urea (5q). Compound 5q was prepared (9 mg, 30% yield) according to GP II from 4-(pyridin-4-yl)benzene-1,2-diamine (20 mg, 0.10 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.11 (t, J = 7.2 Hz, 3H), 3.19−3.26 (m, 2H), 7.23 (s, 1H), 7.47 (s, 2H), 7.66 (d, J = 6.1 Hz, 2H), 7.78 (s, 1H), 8.53−8.60 (m, 2H), 9.97 (s, 1H), 11.69 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.2, 34.1, 119.6, 121.0, 129.6, 148.1, 149.4, 150.0, 154.0. HRMS (EI+) m/z calcd for C15H15N5O [M + H]+, 282.1355; found, 282.1360. 1-Ethyl-3-(6-(2-methylpyridin-4-yl)-1H-benzo[d]imidazol-2-yl)urea (5r). Compound 5r was prepared (14 mg, 48% yield) according to GP II from 4-(2-methylpyridin-4-yl)benzene-1,2-diamine (20 mg, 0.10 mmol). 1H NMR (300 MHz, DMSO-d6) δ 1.08 (t, J = 7.1, 3H), 2.48 (s, 3H), 3.14−3.23 (m, 2H), 7.31 (s, 1H), 7.42 (s, 3H), 7.50 (s, 1H), 7.71 (s, 1H), 9.93 (d, J = 5.2 Hz, 1H). HRMS (EI+) m/z calcd for C16H17N5O [M + H]+, 296.1511; found, 296.1498. 1-Ethyl-3-(6-(3-methylpyridin-4-yl)-1H-benzo[d]imidazol-2-yl)urea (5s). Compound 5s was prepared (6 mg, 20% yield) according to GP II from 4-(3-methylpyridin-4-yl)benzene-1,2-diamine (20 mg, 0.10 mmol). 1H NMR (300 MHz, CDCl3) δ 1.13−1.18 (m, 3H), 2.27 (s, 3H), 3.04−3.10 (m, 2H), 7.08−7.11 (m, 2H), 7.30 (s, 1H), 7.40 (d, J = 8.2 Hz, 1H), 8.44 (d, J = 4.8 Hz, 1H), 8.49 (s, 1H). HRMS (EI+) m/z calcd for C16H17N5O [M + H]+, 296.1511; found, 296.1497. 1-Ethyl-3-(6-(pyridin-3-yl)-1H-benzo[d]imidazol-2-yl)urea (5t). Compound 5t was prepared (10 mg, 46% yield) according to GP II from 4-(pyridin-3-yl)benzene-1,2-diamine (15 mg, 0.081 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.11 (t, J = 7.2 Hz, 3H), 3.17−3.26 (m, 2H), 7.26 (s, 1H), 7.36 (dd, J = 1.8, 8.3 Hz, 1H), 7.41−7.47 (m, 2H), 7.67 (d, J = 1.7 Hz, 1H), 8.02 (d, J = 7.9 Hz, 1H), 8.50 (dd, J = 1.6, 4.7 Hz, 1H), 8.85 (d, J = 2.6 Hz, 1H), 9.92 (s, 1H), 11.64 (s, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 15.2, 34.1, 119.8, 123.7, 129.8, 133.9, 136.8, 147.4, 147.6, 149.1, 154.0. HRMS (EI+) m/z calcd for C15H15N5O [M + H]+, 282.1355; found, 282.1343. 1-Ethyl-3-(6-(1-ethyl-1H-pyrazol-5-yl)-1H-benzo[d]imidazol-2-yl)urea (5u). Compound 5u was prepared (15 mg, 50% yield) according to GP II from 4-(1-ethyl-1H-pyrazol-5-yl)benzene-1,2-diamine (20 mg, 0.099 mmol). 1H NMR (300 MHz, DMSO-d6) δ 1.12 (t, J = 7.2 Hz, 3H), 1.32 (t, J = 7.2 Hz, 3H), 3.17−3.26 (m, 2H), 4.11 (q, J = 7.2 Hz, 2H), 6.27 (d, J = 1.8 Hz, 1H), 7.09 (dd, J = 1.7, 8.1 Hz, 1H), 7.20 (s, 1H), 7.40−45 (m, 2H), 7.47 (d, J = 1.8 Hz, 1H), 9.96 (s, 1H), 11.69 (s, 1H). HRMS (EI+) m/z calcd for C15H18N6O [M + H]+, 299.1620; found, 299.1613. 1-(6-(2-Chlorophenoxy)-1H-benzo[d]imidazol-2-yl)-3-ethylurea (5v). 2-Chlorophenol (125 μL, 1.23 mmol) and 60% NaH (70 mg, 1.75 mmol) in anhydrous DMF were stirred for 10 min at 90 °C, and 5-chloro-2-nitroaniline (200 mg, 1.16 mmol) was added to reaction mixture. It was additionally stirred for 24 h and cooled to room temperature. To the reaction mixture was added sat. NH4Cl, and the generated precipitate was filtered. The solid was purified with flash column chromatography to give the 5-(2-chlorophenoxy)-2-nitroaniline (31 mg, 11% yield). The 5-(2-chlorophenoxy)-2-nitroaniline (31 mg, 0.12 mmol) and SnCl2·2H2O (133 mg, 0.59 mmol) in EtOH/ H2O = 4:1 were stirred for 12 h at 80 °C. After the mixture cooled to room temperature, sat. NaHCO3 was added to the reaction mixture. The precipitate was removed by filtration, and the filtrate was extracted 3 times with CH2Cl2, dried over MgSO4, and concentrated in vacuo. The residue was purified with flash column chromatography (hexane/ EtOAc) to give 4-(2-chlorophenoxy)-benzene-1,2-diamine (14 mg, 48% yield). Compound 5v was prepared (10 mg, 52% yield) according to GP II from 4-(2-chlorophenoxy)-benzene-1,2-diamine (14 mg, 0.058 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.09 (t, J = 7.2 Hz, 3H), 3.19 (qd, J = 5.5, 7.2 Hz, 2H), 6.73 (dd, J = 2.4, 8.5 Hz, 1H), 6.89 (dd, J = 1.5, 8.2 Hz, 1H), 6.97 (s, 1H), 7.10 (td, J = 1.5, 7.7 Hz, 1H), 7.21 (s, 1H), 7.27 (ddd, J = 1.6, 7.4, 8.2 Hz, 1H), 7.35 (d, J = 8.5 Hz, 1H), 7.54 (dd, J = 1.6, 8.0 Hz, 1H), 9.90 (s, 1H), 11.52 (s, 1H). 13C

130.6, 131.0, 148.7, 154.1, 156.1. HRMS (EI+) m/z calcd for C17H18N4O2 [M + H]+, 311.1508; found, 311.1511. 1-Ethyl-3-(6-(2-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol2-yl)urea (5i). Compound 5i was prepared (47 mg, 71% yield) according to GP II from 2′-(trifluoromethoxy)biphenyl-3,4-diamine (49 mg, 0.18 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.10 (t, J = 7.2 Hz, 3H), 3.18−3.25 (m, 2H), 7.11 (dd, J = 1.8, 8.2 Hz, 1H), 7.24 (s, 1H), 7.42−7.46 (m, 5H), 7.49−7.53 (m, 1H), 9.94 (s, 1H), 11.61 (s, 1H). HRMS (EI+) m/z calcd for C17H15F3N4O2 [M + H]+, 365.1225; found, 365.1203. 1-(6-(2-Ethoxyphenyl)-1H-benzo[d]imidazol-2-yl)-3-ethylurea (5j). Compound 5j was prepared (15 mg, 62% yield) according to GP II from 2′-ethoxybiphenyl-3,4-diamine (17 mg, 0.072 mmol). 1H NMR (300 MHz, DMSO-d6) δ 1.10 (t, J = 7.2 Hz, 3H), 1.25 (t, J = 6.9 Hz, 3H), 3.16−3.25 (m, 2H), 4.02 (q, J = 6.8 Hz, 2H), 6.99 (t, J = 7.5 Hz, 1H), 7.06 (d, J = 7.9 Hz, 1H), 7.15 (dd, J = 8.2, 1.7 Hz, 1H), 7.23−7.35 (m, 4H), 7.49 (s, 1H), 9.81 (s, 1H), 11.50(s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 14.6, 15.2, 34.0, 63.5, 112.9, 120.7, 122.2, 127.9, 130.6, 131.1, 148.6, 154.1, 155.3. HRMS (EI+) m/z calcd for C18H20N4O2 [M + H]+, 325.1665; found, 325.1653. 1-Ethyl-3-(6-(2-isopropoxyphenyl)-1H-benzo[d]imidazol-2-yl)urea (5k). Compound 5k was prepared (10 mg, 55% yield) according to GP II from 2′-isopropoxybiphenyl-3,4-diamine (12 mg, 0.051 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.11 (t, J = 7.2 Hz, 3H), 1.18 (d, J = 6.1 Hz, 6H), 3.15−3.27 (m, 2H), 4.50 (p, J = 6. 0 Hz, 1H), 6.98 (td, J = 1.1, 7.4 Hz, 1H), 7.06 (d, J = 7.4 Hz, 1H), 7.15 (dd, J = 1.7, 8.3 Hz, 1H), 7.20−7.30 (m, 3H), 7.33 (d, J = 8.2 Hz, 1H), 7.50(s, 1H), 9.85 (s, 1H), 11.52 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.3, 21.8, 34.1, 69.8, 115.0, 120.8, 122.2, 127.7, 130.9, 132.1, 148.6, 153.9, 154.2. HRMS (EI+) m/z calcd for C19H22N4O2 [M + H]+, 339.1821; found, 339.1798. 1-Ethyl-3-(6-(2-phenoxyphenyl)-1H-benzo[d]imidazol-2-yl)urea (5l). Compound 5l was prepared (13 mg, 62% yield) according to GP II from 2′-phenoxybiphenyl-3,4-diamine (15 mg, 0.054 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.09 (t, J = 7.2 Hz, 3H), 3.18−3.22 (m, 2H), 6.87 (d, J = 7.6 Hz, 2H), 7.01 (t, J = 7.2 Hz, 2H), 7.18 (dd, J = 1.7, 8.3 Hz, 1H), 7.21−7.36 (m, 6H), 7.45−7.55 (m, 2H), 9.82 (s, 1H), 11.51 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.2, 34.0, 117.4, 120.4, 121.8, 122.5, 124.5, 128.3, 129.8, 131.4, 134.3, 148.8, 152.6, 154.0, 157.4. HRMS (EI+) m/z calcd for C22H20N4O2 [M + H]+, 373.1665; found, 373.1646. 1-(6-(2,6-Dimethylphenyl)-1H-benzo[d]imidazol-2-yl)-3-ethylurea (5m). Compound 5m was prepared (10 mg, 44% yield) according to GP II from 2′,6′-dimethylbiphenyl-3,4-diamine (15 mg, 0.070 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.10 (t, J = 7.2 Hz, 3H), 1.95 (s, 6H), 3.18−3.23 (m, 2H), 6.73 (dd, J = 1.6, 8.0 Hz, 1H), 7.03− 7.15 (m, 4H), 7.32 (s, 1H), 7.39 (d, J = 8.0 Hz, 1H), 9.85 (s, 1H), 11.51 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.2, 20.6, 34.0, 121.2, 126.5, 127.1, 132.6, 135.6, 142.4, 148.6, 154.1. HRMS (EI+) m/ z calcd for C18H20N4O [M + H]+, 309.1715; found, 309.1716. 1-(6-(2-chloro-6-methylphenyl)-1H-benzo[d]imidazol-2-yl)-3-ethylurea (5n). Compound 5n was prepared (5 mg, 41% yield) according to GP II from 2′-chloro-6′-methylbiphenyl-3,4-diamine (8 mg, 0.035 mmol). 1H NMR (300 MHz, DMSO-d6) δ 1.11 (t, J = 7.3 Hz, 3H), 2.02 (s, 3H), 3.18−3.23 (m, 2H), 6.79 (d, J = 8.2 Hz, 1H), 7.12 (s, 1H), 7.26−7.42 (m, 5H), 9.88 (s, 1H), 11.58 (s, 1H). HRMS (EI+) m/z calcd for C17H17ClN4O [M + Na]+, 351.0989; found, 351.0983. 1-(6-(2,6-dichlorophenyl)-1H-benzo[d]imidazol-2-yl)-3-ethylurea (5o). Compound 5o was prepared (9 mg, 45% yield) according to GP II from 2′,6′-dichlorobiphenyl-3,4-diamine (15 mg, 0.059 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.11 (t, J = 7.2 Hz, 3H), 3.15−3.26 (m, 2H), 6.85 (dd, J = 1.6, 8.1 Hz, 1H), 7.19 (s, 1H), 7.24 (s, 1H), 7.36−7.43 (m, 2H), 7.55 (d, J = 8.0 Hz, 2H), 9.91 (s, 1H), 11.64 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.2, 34.1, 121.8, 128.2, 129.6, 134.5, 139.8, 149.0, 154.0. HRMS (EI+) m/z calcd for C16H14Cl2N4O [M + H]+, 349.0623; found, 349.0607. 1-(6-(2,6-Dimethoxyphenyl)-1H-benzo[d]imidazol-2-yl)-3-ethylurea (5p). Compound 5p was prepared (16 mg, 26% yield) according to GP II from 2′,6′-dimethoxybiphenyl-3,4-diamine (40 mg, 0.17 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.11 (t, J = 7.2 Hz, 3H), 3.21 (qd, J = 5.4, 7.1 Hz, 2H), 3.62 (s, 6H), 6.71 (d, J = 8.4 Hz, 2H), 3540

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Journal of Medicinal Chemistry

Article

NMR (100 MHz, DMSO-d6) δ 15.3, 34.1, 112.1, 119.1, 123.3, 124.0, 128.5, 130.5, 149.2, 150.3, 153.5, 154.0. HRMS (EI+) m/z calcd for C16H15ClN4O2 [M + H]+, 331.0962; found, 331.0952. 1-Ethyl-3-(6-(3-methoxyphenoxy)-1H-benzo[d]imidazol-2-yl)urea (5w). Compound 5v was prepared (17 mg, 43% yield) according to GP II from 4-(3-methoxyphenoxy)-benzene-1,2-diamine (28 mg, 0.12 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.09 (t, J = 7.2 Hz, 3H), 3.19 (qd, J = 5.4, 7.1 Hz, 2H), 3.69 (s, 3H), 6.44 (ddd, J = 0.9, 2.4, 8.2 Hz, 1H), 6.48 (t, J = 2.3 Hz, 1H), 6.55−6.66 (m, 1H), 6.75 (dd, J = 2.3, 8.5 Hz, 1H), 7.02 (s, 1H), 7.20 (t, J = 8.2 Hz, 2H), 7.34 (d, J = 8.5 Hz, 1H), 9.87 (s, 1H), 11.52 (s, 1H). 13C NMR (100 MHz, DMSOd6) δ 15.2, 34.1, 55.1, 103.3, 107.8, 109.2, 113.0, 130.2, 149.0, 150.0, 154.0, 159.8, 160.6. HRMS (EI+) m/z calcd for C17H18N4O3 [M + H]+, 327.1457; found, 327.1453. 1-Ethyl-3-(6-(4-methylpiperazine-1-carbonyl)-1H-benzo[d]imidazol-2-yl)urea (5x). Compound 5x was prepared (17 mg, 35% yield) according to GP II from (3,4-diaminophenyl)(4-methylpiperazin-1-yl)methanone (40 mg, 0.17 mmol). 1H NMR (400 MHz, DMSO-d6, 55 °C) δ 1.09 (t, J = 7.2 Hz, 3H), 2.17 (s, 3H), 2.24−2.36 (m, 4H), 3.19 (qd, J = 7.2, 5.6 Hz, 2H), 3.47 (t, J = 5.0 Hz, 4H), 7.03 (dd, J = 8.1, 1.6 Hz, 1H), 7.18 (t, J = 5.4 Hz, 1H), 7.34 (d, J = 8.2 Hz, 1H), 7.36 (s, 1H), 10.58 (s, 2H). 13C NMR (100 MHz, DMSO-d6, 55 °C) δ 14.8, 33.8, 44.4, 45.3, 54.4, 112.4, 119.7, 127.9, 149.3, 153.8, 169.8. HRMS (EI+) m/z calcd for C16H21N6O2 [M + H]+, 331.1882; found, 331.1866. 2-(3-Ethylureido)-N-(pyridin-4-yl)-1H-benzo[d]imidazole-6-carboxamide (5y). Compound 5y was prepared (10 mg, 21% yield) according to GP II from 3,4-diamino-N-(pyridin-4-yl)benzamide (32 mg, 0.14 mmol). 1H NMR (300 MHz, DMSO-d6) δ 1.11 (t, J = 7.2 Hz, 3H), 3.17−3.26 (m, 2H), 7.30 (s, 1H), 7.44 (d, J = 8.3 Hz, 1H), 7.70 (dd, J = 1.6, 8.3 Hz, 1H), 7.80 (d, J = 6.4 Hz, 2H), 8.01 (d, J = 1.3 Hz, 1H), 8.44 (d, J = 6.3 Hz, 2H), 10.13 (s, 1H), 10.45 (s, 1H), 12.0 (s, 1H).HRMS (EI+) m/z calcd for C16H16N6O2 [M + H]+, 325.1413; found, 325.1397. General Procedure (GP III) for Preparation of 6-Aryl 2Aminobenzothiazole. A solution of 2-amino-6-bromobenzothiazole (300 mg, 1.31 mmol), 2-methoxyphenylboronic acid (299 mg, 1.96 mmol), K2CO3 (543 mg, 3.93 mmol), and Pd(dppf)Cl2·CH2Cl2 (214 mg, 0.26 mmol) in 1,4-dioxane/H2O = 3:1 (4 mL) was heated to 120 °C for 6 h. The resulting solution was concentrated in vacuo and filtered on silica−Celite using EtOAc and hexane. The filtrate was concentrated and purified with flash column chromatography (EtOAc/ hexane) to give the 6-(2-methoxyphenyl)benzo[d]thiazol-2-amine (188 mg, 56%). 1H NMR (400 MHz, CDCl3) δ 3.82 (s, 3H), 5.27 (m, 2H), 6.99 (dd, J = 1.0, 8.2 Hz, 1H), 7.03 (td, J = 1.1, 7.5 Hz, 1H), 7.28−7.38 (m, 2H), 7.48 (dd, J = 1.8, 8.3 Hz, 1H), 7.58 (dd, J = 0.6, 8.3 Hz, 1H), 7.77 (dd, J = 0.5, 1.8 Hz, 1H). General Procedure (GP IV) for Introducing 2-Urea. 1-Ethyl-3(6-(2-methoxyphenyl)benzo[d]thiazol-2-yl)urea (9a). To a solution of 6-(2-methoxyphenyl)benzo[d]thiazol-2-amine (26 mg, 0.10 mmol) in 1,4-dioxane was added ethyl isocyanate (40 μL, 0.50 mmol), and the mixture was stirred for 15 h at 90 °C. After the reaction finished, the resulting solution was concentrated in vacuo, and then, H2O was added. It was heated to 100 °C for 3 h. The resulting solid was filtered and washed with H2O and additionally purified with flash column chromatography to give compound 9a (18 mg, 54% yield). 1H NMR (300 MHz, CDCl3) δ 1.25 (t, J = 7.2 Hz, 3H), 3.37−3.46 (m, 2H), 3.81 (s, 3H), 6.75−7.12 (m, 2H), 7.29−7.35 (m, 2H), 7.56 (dd, J = 1.8, 8.4 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 1.7 Hz, 1H). 13 C NMR (100 MHz, CDCl3) δ 15.1, 35.1, 55.5, 111.2, 119.1, 120.9, 122.0, 127.9, 128.6, 130.0, 130.9, 130.9, 133.9, 148.0, 154.7, 156.4, 161.9. HRMS (EI+) m/z calcd for C17H17N3O2S [M + H]+, 328.1120; found, 328.1117. 1-(6-(2-Ethoxyphenyl)benzo[d]thiazol-2-yl)-3-ethylurea (9b). Compound 9b was prepared (27 mg, 64% yield) according to GP IV from 6-(2-ethoxyphenyl)benzo[d]thiazol-2-amine (33 mg, 0.12 mmol). 1H NMR (300 MHz, CDCl3) δ 1.26 (t, J = 7.2 Hz, 3H), 1.33 (t, J = 7.0 Hz, 3H), 3.44 (p, J = 6.9 Hz, 2H), 4.04 (q, J = 6.9 Hz, 2H), 6.85−7.09 (m, 2H), 7.25−7.33 (m, 1H), 7.36 (dd, J = 1.7, 7.5 Hz, 1H), 7.55−7.67 (m, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 1.7 Hz,

1H), 10.95 (s, 1H).13C NMR (100 MHz, CDCl3) δ 14.8, 15.1, 35.2, 64.0, 112.7, 119.1, 120.9, 122.0, 128.0, 128.6, 130.2, 130.7, 130.9, 134.2, 148.0, 154.7, 155.8, 161.9. HRMS (EI+) m/z calcd for C18H19N3O2S [M + Na]+, 364.1096; found, 364.1073. 1-Ethyl-3-(6-(2-(trifluoromethoxy)phenyl)benzo[d]thiazol-2-yl)urea (9c). Compound 9c was prepared (51 mg, 69% yield) according to GP IV from 6-(2-(trifluoromethoxy)phenyl)benzo[d]thiazol-2amine (60 mg, 0.19 mmol). 1H NMR (400 MHz, CDCl3) δ 1.27 (t, J = 7.2 Hz, 1H), 3.45 (qd, J = 5.3, 7.1 Hz, 1H), 7.28−7.40 (m, 3H), 7.44−7.48 (m, 1H), 7.50 (dd, J = 1.8, 8.4 Hz, 1H), 7.79 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 1.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 15.5, 34.7, 116.6, 119.1, 119.7, 121.7, 122.0, 122.2, 124.3, 127.3, 128.3, 129.5, 130.9, 132.2, 135.1, 145.8, 149.2, 154.1, 161.1. HRMS (EI+) m/ z calcd for C17H14F3N3O2S [M + H]+, 382.0837; found, 382.0814. 1-Ethyl-3-(6-(2-propoxyphenyl)benzo[d]thiazol-2-yl)urea (9d). Compound 9d was prepared (50 mg, 68% yield) according to GP IV from 6-(2-propoxyphenyl)benzo[d]thiazol-2-amine (58 mg, 0.20 mmol). 1H NMR (400 MHz, CDCl3) δ 0.94 (t, J = 7.4 Hz, 3H), 1.27 (t, J = 7.2 Hz, 3H), 1.72 (h, J = 7.1 Hz, 2H), 3.19−3.54 (m, 2H), 3.92 (t, J = 6.4 Hz, 2H), 6.90−7.10 (m, 2H), 7.28 (t, J = 7.9 Hz, 1H), 7.36 (dd, J = 1.8, 7.3 Hz, 1H), 7.49 (s, 1H), 7.56−7.65 (m, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.91 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 10.7, 15.2, 22.6, 35.2, 70.0, 112.5, 119.1, 120.8, 122.1, 128.1, 128.6, 130.2, 130.6, 130.8, 134.2, 147.9, 154.7, 156.0, 161.9. HRMS (EI+) m/z calcd for C19H21N3O2S [M + H]+, 356.1433; found, 356.1413. 1-Ethyl-3-(5-methoxy-6-(2-methoxyphenyl)benzo[d]thiazol-2-yl)urea (9e). Compound 9e was prepared (51 mg, 69% yield) according to GP IV from 5-methoxy-6-(2-methoxyphenyl)benzo[d]thiazol-2amine (60 mg, 0.19 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.06 (t, J = 7.2 Hz, 3H), 3.15 (qd, J = 5.4, 7.1 Hz, 2H), 3.65 (s, 3H), 3.69 (s, 3H), 6.72 (t, J = 5.7 Hz, 1H), 6.93 (td, J = 1.1, 7.4 Hz, 1H), 7.01 (dd, J = 1.0, 8.3 Hz, 1H), 7.11 (dd, J = 1.8, 7.4 Hz, 1H), 7.22 (s, 1H), 7.28 (ddd, J = 1.8, 7.4, 8.2 Hz, 1H), 7.51 (s, 1H), 10.62 (s, 1H).13C NMR (100 MHz, DMSO-d6) δ 15.2, 34.3, 55.4, 55.7, 102.2, 111.2, 120.0, 122.5, 122.8, 123.6, 127.5, 128.5, 131.3, 149.9, 153.6, 156.0, 156.9, 160.8. HRMS (EI+) m/z calcd for C18H19N3O3S [M + Na]+, 380.1045; found, 380.1031. 1-Ethyl-3-(5-(2-methoxyphenyl)thiazolo[5,4-b]pyridin-2-yl)urea (9f). Compound 9f was prepared (16 mg, 83% yield) according to GP IV from 5-(2-methoxyphenyl)thiazolo[5,4-b]pyridin-2-amine (15 mg, 0.05 mmol). 1H NMR (300 MHz, CDCl3) δ 1.32 (t, J = 7.2 Hz, 3H), 3.37−3.59 (m, 2H), 3.91 (s, 3H), 7.05 (d, J = 8.3 Hz, 1H), 7.12 (td, J = 1.1, 7.5 Hz, 1H), 7.41 (ddd, J = 1.8, 7.4, 8.9 Hz, 1H), 7.57 (s, 1H), 7.84 (dd, J = 1.8, 7.6 Hz, 1H), 7.92 (d, J = 8.5 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H), 10.90 (s, 1H).13C NMR (100 MHz, CDCl3) δ 15.2, 35.2, 55.6, 111.5, 121.1, 123.2, 126.0, 128.5, 130.0, 131.2, 141.4, 152.0, 154.4, 154.4, 156.9, 161.4. HRMS (EI+) m/z calcd for C16H16N4O2S [M + H]+, 329.1072; found, 329.1061. 1-Ethyl-3-(6-phenylbenzo[d]thiazol-2-yl)urea (9g). Compound 9g was prepared (24 mg, 48% yield) according to GP IV from 6phenylbenzo[d]thiazol-2-amine (38 mg, 0.17 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.06 (t, J = 7.2 Hz, 3H), 3.04−3.22 (m, 2H), 6.72 (t, J = 5.7 Hz, 1H), 7.30 (t, J = 7.3 Hz, 1H), 7.41 (t, J = 7.6 Hz, 2H), 7.55−7.74 (m, 4H), 8.16 (s, 1H), 10.72 (s, 1H).13C NMR (100 MHz, DMSO-d6) δ 15.2, 34.3, 119.4, 119.8, 124.7, 126.7, 127.1, 128.9, 132.4, 134.8, 140.1, 148.6, 153.7, 160.3. HRMS (EI+) m/z calcd for C16H15N3OS [M + Na]+, 320.0834; found, 320.0817. 1-Ethyl-3-(6-(pyridin-4-yl)benzo[d]thiazol-2-yl)urea (9h). Compound 9h was prepared (10 mg, 25% yield) according to GP IV from 6-(pyridin-4-yl)benzo[d]thiazol-2-amine (41.2 mg, 0.137 mmol). 1 H NMR (400 MHz, CDCl3) δ 1.26 (t, J = 7.2 Hz, 3H), 3.43 (qd, J = 5.4, 7.2 Hz, 2H), 7.41−7.56 (m, 2H), 7.62 (dd, J = 1.8, 8.5 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 1.8 Hz, 1H), 8.47−8.80 (m, 2H). 13 C NMR (100 MHz, CDCl3) δ 15.1, 35.2, 104.1, 119.7, 120.4, 121.5, 125.2, 132.1, 133.3, 147.9, 150.1, 154.5, 162.6. HRMS (EI+) m/z calcd for C15H14N4OS [M + H]+, 299.0967; found, 299.0960. 1-Ethyl-3-(6-(4-methoxyphenyl)benzo[d]thiazol-2-yl)urea (9i). Compound 9i was prepared (12 mg, 47% yield) according to GP IV from 6-(4-methoxyphenyl)benzo[d]thiazol-2-amine (20 mg, 0.07 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.06 (t, J = 7.2 Hz, 3H), 3541

dx.doi.org/10.1021/jm301891t | J. Med. Chem. 2013, 56, 3531−3545

Journal of Medicinal Chemistry

Article

161.6. HRMS (EI+) m/z calcd for C18H19N3O3S [M + H]+, 358.1225; found, 358.1212. 1-(6-(2-Methoxyphenyl)benzo[d]thiazol-2-yl)-3-(2-(4-methylpiperazin-1-yl)ethyl)urea (10d). Compound 10d was prepared (53 mg, 40% yield) according to GP V from 6-(2-methoxyphenyl)benzo[d]thiazol-2-amine (79 mg, 0.308 mmol). 1H NMR (400 MHz, CDCl3) δ 2.26 (s, 3H), 2.43 (s, 4H), 2.53 (s, 6H), 3.41 (s, 2H), 3.78 (s, 3H), 6.87−7.14 (m, 2H), 7.25−7.32 (m, 2H), 7.39 (s, 1H), 7.50 (dd, J = 1.7, 8.4 Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.86 (dd, J = 0.6, 1.8 Hz, 1H), 10.89 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 37.6, 45.8, 52.9, 55.0, 55.7, 57.2, 111.7, 119.2, 121.0, 122.0, 127.8, 128.6, 130.5, 130.9, 131.4, 134.1, 148.3, 154.9, 156.7, 161.7. HRMS (EI+) m/z calcd for C22H27N5O2S [M + H]+, 426.1964; found, 426.1955. 1-(6-(2-Ethoxyphenyl)benzo[d]thiazol-2-yl)-3-(2morpholinoethyl)urea (10e). Compound 10e was prepared (39 mg, 83% yield) according to GP V from 6-(2-ethoxyphenyl)benzo[d]thiazol-2-amine (30 mg, 0.11 mmol). 1H NMR (400 MHz, CDCl3, 55 °C) δ 1.34 (t, J = 7.0 Hz, 3H), 2.54 (d, J = 4.6 Hz, 4H), 2.62 (t, J = 6.0 Hz, 2H), 3.52 (q, J = 5.7 Hz, 2H), 3.75 (t, J = 4.4 Hz, 4H), 4.06 (q, J = 7.0 Hz, 2H), 6.96−7.07 (m, 2H), 7.29 (ddd, J = 1.8, 7.4, 8.2 Hz, 1H), 7.36 (dd, J = 1.8, 7.5 Hz, 1H), 7.59 (dd, J = 1.8, 8.4 Hz, 1H), 7.62 (s, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.93 (d, J = 1.6 Hz, 1H) 10.84 (br, 1H). 13 C NMR (100 MHz, CDCl3, 55 °C) δ 14.8, 37.4, 53.6, 57.8, 64.4, 67.0, 113.3, 119.2, 121.1, 122.0, 128.0, 128.6, 130.7, 130.9, 131.1, 134.4, 148.4, 155.0, 156.1, 161.6. HRMS (EI+) m/z calcd for C22H26N4O3S [M + H]+, 427.1804; found, 427.1792. 1-(6-(2-Ethoxyphenyl)benzo[d]thiazol-2-yl)-3-(2-hydroxyethyl)urea (10f). Compound 10f was prepared (28 mg, 67% yield) according to GP V from 6-(2-ethoxyphenyl)benzo[d]thiazol-2-amine (32 mg, 0.11 mmol). 1H NMR (400 MHz, MeOH-d4) δ 1.29 (t, J = 7.0 Hz, 3H), 3.41 (t, J = 5.5 Hz, 2H), 3.69 (t, J = 5.5 Hz, 2H), 4.02 (q, J = 7.0 Hz, 2H), 6.95−7.05 (m, 2H), 7.23−7.33 (m, 2H), 7.52 (dd, J = 1.8, 8.4 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 1.7 Hz, 1H). 13 C NMR (100 MHz, MeOH-d4) δ 15.1, 43.4, 61.9, 65.2, 114.0, 119.9, 122.0, 122.8, 129.0, 129.7, 131.7, 131.8, 132.4, 135.6, 149.0, 156.3, 157.2, 162.2. HRMS (EI+) m/z calcd for C18H19N3O3S [M + Na]+, 380.1045; found, 380.1027. 1-(6-(2-Ethoxyphenyl)benzo[d]thiazol-2-yl)-3-(3-hydroxypropyl)urea (10g). Compound 10g was prepared (26 mg, 61% yield) according to GP V from 6-(2-ethoxyphenyl)benzo[d]thiazol-2-amine (31 mg, 0.11 mmol). 1H NMR (400 MHz, CDCl3) δ 1.34 (t, J = 6.9 Hz, 3H), 1.81−1.90 (m, 2H), 3.60 (q, J = 6.1 Hz, 2H), 3.80 (t, J = 5.6 Hz, 2H), 4.05 (q, J = 7.0 Hz, 2H), 6.95−7.06 (m, 2H), 7.27−7.33 (m, 1H), 7.35 (dd, J = 1.8, 7.6 Hz, 1H), 7.60 (dd, J = 1.7, 8.4 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 1.7 Hz, 1H) 8.08 (br, 1H). 13C NMR (100 MHz, CDCl3) δ 14.7, 32.4, 37.1, 59.5, 64.0, 112.7, 119.2, 120.9, 122.0, 128.0, 128.6, 130.1, 130.6, 130.9, 134.3, 147.8, 155.6, 155.8, 161.5. HRMS (EI+) m/z calcd for C19H21N3O3S [M + Na]+, 394.1201; found, 394.1191. 1-(6-(2-Ethoxyphenyl)benzo[d]thiazol-2-yl)-3-(2-(piperazin-1-yl)ethyl)urea (10h). Compound 10h (TFA salt) was prepared (11 mg, 23% yield) according to GP V from 6-(2-ethoxyphenyl)benzo[d]thiazol-2-amine (30 mg, 0.11 mmol). 1H NMR (300 MHz, MeOH-d4) δ 1.30 (t, J = 7.0 Hz, 3H), 2.57−2.64 (m, 6H), 3.06 (br, 4H), 3.43 (t, J = 6.1 Hz, 2H), 4.03 (q, J = 6.9 Hz, 2H), 6.95−7.08 (m, 2H), 7.22− 7.35 (m, 2H), 7.53 (dd, J = 1.7, 8.4 Hz, 1H) 7.64 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 1.7 Hz, 1H). 1C NMR (100 MHz, MeOH-d4) δ 15.1, 37.8, 45.9, 53.7, 58.6, 65.2, 114.0, 119.9, 122.0, 122.8, 129.0, 129.7, 131.7, 131.8, 132.4, 135.5, 149.0, 157.2, 162.3. HRMS (EI+) m/z calcd for C22H27N5O2S [M + H]+, 426.1964; found, 426.1940. 1-(6-(2-Ethylphenyl)benzo[d]thiazol-2-yl)-3-(2-morpholinoethyl)urea (10i). Compound 10i was prepared (31 mg, 64% yield) according to GP V from 6-(2-ethylphenyl)benzo[d]thiazol-2-amine (30 mg, 0.12 mmol). 1H NMR (400 MHz, CDCl3, 55 °C) δ 1.09 (t, J = 7.5 Hz, 3H), 2.54−2.59 (m, 4H), 2.59−2.69 (m, 4H), 3.52 (q, J = 5.6 Hz, 2H), 3.62−4.04 (m, 4H), 7.16−7.22 (m, 2H), 7.27−7.37 (m, 3H), 7.64 (dd, J = 0.5, 1.7 Hz, 1H), 7.72 (dd, J = 0.6, 8.2 Hz, 1H). 13C NMR (100 MHz, CDCl3, 55 °C) δ 15.5, 26.2, 37.4, 53.7, 57.9, 66.9, 119.5, 121.6, 125.6, 127.7, 127.7, 128.6, 130.2, 131.2, 137.8, 141.1,

2.31 (s, 3H), 3.16 (qd, J = 5.5, 7.2 Hz, 2H), 6.72 (t, J = 5.8 Hz, 1H), 7.23 (d, J = 8.0 Hz, 2H), 7.43−7.74 (m, 4H), 7.82−8.61 (m, 1H), 10.69 (s, 1H).13C NMR (100 MHz, DMSO-d6) δ 15.6, 21.1, 34.7, 119.4, 120.2, 124.9, 126.9, 129.9, 132.8, 135.2, 136.7, 137.6, 148.9, 154.1, 160.6. HRMS (EI+) m/z calcd for C17H19N3O2S [M + H]+, 328.1120; found, 328.1118. 1-(6-(4-Amino-2-methoxyphenyl)benzo[d]thiazol-2-yl)-3-ethylurea (9j). Compound 9j was prepared (4.9 mg, 7.1% yield) according to GP III from 1-(6-bromobenzo[d]thiazol-2-yl)-3-ethylurea (60 mg, 0.2 mmol) and 3-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (74 mg, 0.30 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.26 (t, J = 7.1 Hz, 3H), 3.18 (s, 2H), 3.32−3.53 (m, 2H), 3.77 (s, 3H), 6.33 (d, J = 2.1 Hz, 1H), 6.36 (dd, J = 2.2, 8.1 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.48−7.54 (m, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 1.6 Hz, 1H). HRMS (EI+) m/z calcd for C17H17N4O2S [M + Na]+, 380.1045; found, 380.1021. 1-(6-(2,4-Dimethoxyphenyl)benzo[d]thiazol-2-yl)-3-ethylurea (9k). Compound 9k was prepared (8 mg, 35% yield) according to GP IV from 6-(2,4-dimethoxyphenyl)benzo[d]thiazol-2-amine (28 mg, 0.098 mmol). 1H NMR (400 MHz, DMSO-d6) δ 1.08 (t, J = 7.2 Hz, 3H), 3.11−3.26 (m, 2H), 3.75 (s, 3H), 3.79 (s, 3H), 6.60 (dd, J = 2.4, 8.4 Hz, 1H), 6.65 (d, J = 2.5 Hz, 1H), 6.73 (t, J = 5.9 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.39 (dd, J = 1.8, 8.3 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.87 (s, 1H), 10.64 (s, 1H). HRMS (EI+) m/z calcd for C18H19N3O3S [M + Na]+, 380.1045; found, 380.1021. 1-Ethyl-3-(6-(3-methoxypyridin-4-yl)benzo[d]thiazol-2-yl)urea (9l). Compound 9l was prepared (5.2 mg, 27% yield) according to GP IV from 6-(3-methoxypyridin-4-yl)benzo[d]thiazol-2-amine (15 mg, 0.058 mmol). 1H NMR (300 MHz, CDCl3) δ 1.31 (t, J = 7.3 Hz, 3H), 3.35−3.54 (m, 2H), 3.97 (s, 3H), 7.32 (d, J = 4.9 Hz, 1H), 7.64 (dd, J = 1.8, 8.4 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 8.00 (d, J = 1.7 Hz, 1H), 8.36 (d, J = 4.8 Hz, 1H), 8.42 (s, 1H), 10.99 (s, 1H). HRMS (EI+) m/ z calcd for C16H16N4O2S [M + H]+, 329.1072; found, 329.1059. General Procedure (GP V) for Introducing 2-Urea. 1-(6-(2Methoxyphenyl)benzo[d]thiazol-2-yl)-3-(2-morpholinoethyl)urea (10a). 6-(2-Methoxyphenyl)benzo[d]thiazol-2-amine (58 mg, 0.23 mmol) and carbonyldiimidazole (112 mg, 0.679 mmol) was dissolved into anhydrous DMF and stirred for 8 h at room temperature. 2Morpholinoethanamine (150 μL, 1.13 mmol) was added. After the reaction finished, the resulting solution was concentrated in vacuo. Then the crude mixture was purified with flash column chromatography to give compound 10a (74 mg, 79% yield). 1H NMR (300 MHz, CDCl3, 55 °C) δ 2.46−2.56 (m, 4H), 2.61 (t, J = 5.9 Hz, 3H), 3.50 (q, J = 5.6 Hz, 2H), 3.68−3.77 (m, 4H), 3.80 (s, 3H), 6.87−7.10 (m, 2H), 7.26−7.40 (m, 2H), 7.53 (dd, J = 1.7, 8.4 Hz, 1H), 7.60 (s, 1H), 7.71 (dd, J = 0.6, 8.4 Hz, 1H), 7.87 (dd, J = 0.5, 1.8 Hz, 1H).13C NMR (100 MHz, CDCl3, 55 °C) δ 37.3, 53.6, 55.7, 57.8, 66.9, 111.8, 119.3, 121.1, 122.0, 128.0, 128.7, 130.5, 131.0, 131.2, 134.3, 148.4, 154.9, 156.7, 161.6. HRMS (EI+) m/z calcd for C21H24N4O3S [M + H]+, 413.1647; found, 413.1635. 1-(2-Hydroxyethyl)-3-(6-(2-methoxyphenyl)benzo[d]thiazol-2-yl)urea (10b). Compound 10b was prepared (13 mg, 66% yield) according to GP V from 6-(2-methoxyphenyl)benzo[d]thiazol-2amine (15 mg, 0.058 mmol). 1H NMR (400 MHz, CDCl3) δ 3.59 (q, J = 5.3 Hz, 2H), 3.82 (s, 3H), 3.89 (t, J = 5.1 Hz, 2H), 4.63 (br, 1H), 6.98−7.04 (m, 2H), 7.29−7.33 (m, 2H), 7.52 (dd, J = 8.4 Hz, 1.7 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 1.4 Hz, 1H), 8.05 (s, 1H), 10.26 (br, 1H). 13C NMR (100 MHz, CDCl3) δ 42.7, 55.5, 61.9, 104.1, 111.2, 119.2, 120.9, 121.9, 128.0, 128.7, 130.0, 130.7, 131.0, 134.1, 155.3, 156.4, 161.3. HRMS (EI+) m/z calcd for C17H17N3O3S [M + Na]+, 366.0888; found, 366.0878. 1-(3-Hydroxypropyl)-3-(6-(2-methoxyphenyl)benzo[d]thiazol-2yl)urea (10c). Compound 10c was prepared (12 mg, 57% yield) according to GP V from 6-(2-methoxyphenyl)benzo[d]thiazol-2amine (15 mg, 0.059 mmol). 1H NMR (300 MHz, CDCl3) δ 1.86 (p, J = 6.0 Hz, 2H), 3.62 (q, J = 6.2 Hz, 2H), 3.79 (t, J = 5.6 Hz, 2H), 3.83 (s, 3H), 6.97−7.09 (m, 2H), 7.31−7.36 (m, 2H), 7.57 (dd, J = 1.7, 8.4 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 1.7 Hz, 1H). 13 C NMR (100 MHz, CDCl3) δ 32.4, 37.0, 55.6, 59.5, 111.3, 119.4, 120.9, 122.1, 128.0, 128.7, 130.0, 130.7, 131.0, 134.2, 155.6, 156.4, 3542

dx.doi.org/10.1021/jm301891t | J. Med. Chem. 2013, 56, 3531−3545

Journal of Medicinal Chemistry

Article

Western Blot Analysis. After cells were treated with 1 μM imatinib, dasatinib, 10b, or 10d and incubated 37 °C for 1 h, cells were collected and washed with ice-cold phosphate-buffered saline (PBS). Cells were lysed with buffer containing 1% Triton X-100, 1% Nonidet P-40 (NP40), followed by protease and phosphatase inhibitor cocktails (GenDEPOT, Barker, TX). Cell lysates were collected by dissolving cells in 1× SDS sample lysis buffer then boiling. Equal amounts of cell lysates were loaded to 12% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and separated by electrophoresis. Separated proteins were then electrically transferred onto poly(vinylidene fluoride) (PVDF) membranes (Millipore, Bedford, MA). The film was blocked with 1× TBS containing 0.1% Tween-20 and 5% bovine serum albumin (BSA) and then incubated with primary antibody followed by horseradish peroxidase (HRP)-conjugated secondary antibody. The protein lanes were visualized using an enhanced chemiluminescence (ECL) plus system (Amersham Biosciences, Piscataway, NJ). Primary monoclonal antibodies against the following factors were used: pBcrAbl (Tyr245), pSTAT5 (Tyr694), pCrkL (Tyr207), and elF4E (Cell Signaling Technology Inc., Danvers, MA). The secondary antibodies were purchased from Amersham Biosciences (Piscataway, NJ).

141.8, 148.5, 155.0, 161.4. HRMS (EI+) m/z calcd for C22H26N4O2S [M + H]+, 411.1855; found, 411.1849. 1-(2-(Dimethylamino)ethyl)-3-(6-(2-ethylphenyl)benzo[d]thiazol2-yl)urea (10j). Compound 10j was prepared (18 mg, 41% yield) according to GP V from 6-(2-ethylphenyl)benzo[d]thiazol-2-amine (30 mg, 0.12 mmol). 1H NMR (400 MHz, CDCl3) δ 1.07 (t, J = 7.5 Hz, 3H), 2.38 (s, 6H), 2.59 (q, J = 7.5 Hz, 2H), 2.65 (t, J = 5.8 Hz, 2H), 3.52 (d, J = 6.1 Hz, 2H), 7.14−7.23 (m, 2H), 7.25−7.33 (m, 3H), 7.60 (d, J = 1.7 Hz, 1H), 7.64 (d, J = 8.2 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 15.6, 26.1, 45.1, 119.2, 121.4, 125.5, 127.4, 127.5, 128.5, 130.2, 137.2, 141.1, 141.7, 148.0, 161.3. HRMS (EI+) m/z calcd for C20H24N4OS [M + H]+, 369.1749; found, 369.1741. Docking Study. The active conformations of the wild-type and T315I mutant of ABL were selected as the target proteins in this study. We prepared the two receptor models in the “DFG-in” conformation from the X-ray crystal structure of the wild-type Abl in complex with dasatinib (PDB code 2GQG) and that of T315I mutant in complex with PPY-A (PDB code 2QOH). We used the AutoDock program because the outperformance of its scoring function over those of the others had been shown in several target proteins. To obtain the allatom models for the two receptors, hydrogen atoms were added to each protein atom in the wild-type and mutant enzymes. A special attention was paid to assign the protonation states of the ionizable aspartate, glutamate, histidine, and lysine residues in the X-ray crystal structures of the wild-type and T315I mutant. The side chains of aspartate and glutamate residues were assumed to be neutral if one of their carboxylate oxygens pointed toward a hydrogen bond accepting group including the backbone aminocarbonyl oxygen at a distance within 3.5 Å, a generally accepted distance limit for a hydrogen bond of moderate strength. Similarly, the lysine side chains were assumed to be protonated unless the NZ atom was in proximity to a hydrogen bond donating group. The same procedure was also applied to determine the protonation states of ND and NE atoms in histidine residues. Biological Assays and Methods. Enzyme Assay. The IC50 determinations were performed using radiometric kinase assays ([γ-33P]-ATP) with 1 μM ATP at the Reaction Biology Corp. (Malvern, PA, U.S.A.). Compounds were tested in a 10-dose IC50 mode with 3-fold serial dilution starting at 1 μM. POC and Kd determinateions were performed at the Ambit Bioscience Corp. (San Diego, CA, USA). Compound 9a was profiled at 1 μM against a panel of 96 kinases in a high-throughput binding assay (Ambit Bioscience). Cells and Reagents. Ba/F3 cells were kindly provided by Dr. Deininger (Huntsman Cancer Institute, Salt Lake City, UT). Ba/F3WT and Ba/F3T315I cells, which inducibly express Bcr-Abl with wild-type and Bcr-Abl with T315I mutation, respectively, were derived as described previously by retroviral transfection using the mammalian expression vector pSRα.18 Ba/F3WT and Ba/F3T315I cells were grown in Roswell Park Memorial Institute media 1640 (RPMI 1640) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin. RPMI 1640, FBS, and penicillin/streptomycin were purchased from Gibco (Grand Island, NY). Imatinib and dasatinib were purchased from LC laboratories (Woburn, MA). XTT was purchased from WELGENE Inc. (Daegu, Korea). Antibodies against p-Bcr-Abl, p-STAT5, p-CrkL, and elF4E were all purchased from Cell Signaling Technology Inc. (Danvers, MA). Cellular Antiproliferation Assay Using XTT Assay. Cellular antiproliferation effect of corresponding compounds was determined by sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium5-carboxanilide inner salt (XTT) assay. Cells were plated at a density of 2.5 × 103 cells/well in 96-well culture dishes and incubated in 37 °C for 24 h. Cells were treated with the corresponding compounds or control at the indicated concentration (0.001−50 μM) for 48 h. Then 10 μL of XTT labeling mixture (1 mL of XTT/20 μL of PMS (phenazine methosulfate)) was added into each well and incubated for 2 h. Optical density (OD) was measured by a microplate reader at OD540 and OD620. Absorbance rate for each well was calculated as OD540 − OD620. The data were further analyzed using Graphpad Prism5 (Graphpad Software, Inc.)

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ASSOCIATED CONTENT

S Supporting Information *

Spectra, more chemical information, and biological data. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*S. Hong: tel (+82) 42-350-2811; fax (+82) 42-350-2810; email [email protected]. S.-S. Hong: tel (+82) 32-890-3683; fax (+82) 32-890-2462; e-mail: [email protected]. Author Contributions

Seunghee Hong, Jinhee Kim, and Sun-Mi Yun contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dedicated to Professor Deukjoon Kim on the occasion of his retirement. This research was supported by National Research Foundation of Korea (NRF) through General Research Grants NRF-2010-0022179, 2011-0016436, and 2011-0020322.

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ABBREVIATIONS USED Bcr-Abl, break-point cluster region−Abelson tyrosine kinase; CML, chronic myeloid leukemia; c-Kit, receptor tyrosine kinase for the cytokine stem cell factor; WT, wild-type; DFG, AspPhe-Gly; MEK1, MAP kinase kinase 1; Braf, v-Raf murine sarcoma viral oncogene homolog B1; ATP, adenosine-5′triphosphate; SAR, structure−activity relationship; DCM, dichloromethane; DMF, N,N-dimethylformamide; DIPE, diisopropyl ether; DPPF(dppf), 1,1′-bis(diphenylphosphino)ferrocene; CDI, carbondiimidazole; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; XTT, 2,3-bis(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboanilid inner salt

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REFERENCES

(1) (a) Quintas-Cardama, A.; Cortes, J. Molecular biology of bcrabl1-positive chronic myeloid leukemia. Blood 2009, 113, 1619−1630. (b) Faderl, S.; Kantarjian, H. M.; Talpaz, M. Chronic myelogenous leukemia: Update on biology and treatment. Oncology (Williston Park, N.Y.) 1999, 13, 169−184.

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