Optimization and biological evaluation of 2-aminobenzothiazole derivatives as Aurora B kinase inhibitors

Accepted Manuscript Optimization and Biological Evaluation of 2-Aminobenzothiazole Derivatives as Aurora B Kinase Inhibitors Eun Lee, Ying An, Junhee Kwon, Keun Il Kim, Raok Jeon PII: DOI: Reference:

S0968-0896(16)31511-5 http://dx.doi.org/10.1016/j.bmc.2017.04.004 BMC 13673

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

28 December 2016 1 April 2017 4 April 2017

Please cite this article as: Lee, E., An, Y., Kwon, J., Kim, K.I., Jeon, R., Optimization and Biological Evaluation of 2-Aminobenzothiazole Derivatives as Aurora B Kinase Inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.04.004

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Bioorganic & Medicinal Chemistry j o ur n al h o m e p a g e : w w w . e l s e v i e r . c o m

Optimization and Biological Evaluation of 2-Aminobenzothiazole Derivatives as Aurora B Kinase Inhibitors Eun Leea, Ying Ana, Junhee Kwonb, Keun Il Kimb, and Raok Jeon a, * a b

Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women's University, Cheongpa-ro 47-gil 100, Yongsan-gu, Seoul, 04310, Korea Department of Biological Sciences, Sookmyung Women's University, Cheongpa-ro 47-gil 100, Yongsan-gu, Seoul, 04310, Korea

AR T IC LE IN F O

A B S TR A C T

Article history: Received Received in revised form Accepted Available online

A strong relationship between abnormal functions of Aurora kinases and tumorigenesis has been reported for decades. Consequently, Aurora kinases serve as potential targets for anticancer agents. Here, we identified aminobenzothiazole derivatives as novel inhibitors of Aurora B kinase through bioisosteric replacement of the previous inhibitors, aminobenzoxazole derivatives. Most of the urea-linked aminobenzothiazole derivatives showed potent and selective inhibitory activity against Aurora B kinase over Aurora A kinase. Molecular modeling indicated that compound 15g bound well to the active site of Aurora B kinase and formed the essential hydrogen bonds. The potent compounds, 15g and 15k, were selected, and their biological effects were evaluated using HeLa cell lines. It was found that these compounds inhibited the phosphorylation of histone H3 at Ser10 and induced G2 /M cell cycle arrest. We suggest that the reported compounds have the potential to be further developed as anticancer therapeutics.

Keywords: Aminobenzothiazole Aurora B kinase Antiproliferation Keyword_4 Keyword_5

2016 Elsevier Ltd. All rights reserved.

——— *

Corresponding author. Tel.: +82-2-710 9571; fax: +82-2-715-9571; E-mail address: [email protected]

1. Introduction The Aurora family of serine/threonine kinases plays important roles during mitosis and meiosis. This kinase family consists of three paralogues, designated as Aurora A, B, and C, which show quite different features as regards to their expression, cellular localization, and function. Aurora A is localized in the centrosome and is important for mitotic processes1. It is involved in the mitotic entry, centrosome maturation, and spindle dynamics2. Aurora B adjusts the chromosomal bi-orientation, regulates the association between kinetochores and microtubules, and controls cytokinesis. Aurora B phosphorylates histone H3 at Ser10, which then regulates chromatin condensation and separation3,4. Aurora C exhibits functions similar to those of Aurora B during meiosis5. Aurora kinases are over-expressed in various tumor cell-lines, suggesting that these kinases might play a crucial role in cancer progression6. There is a correlation reported between the overexpression of Aurora kinases and various tumors such as breast cancer, colorectal cancer, and acute myeloid leukemia7, leading to considerable interest in Aurora kinases as possible anticancer targets. A number of Aurora kinase inhibitors have been identified as chemotherapeutic candidates8, and several compounds are undergoing preclinical and clinical trials9.

heterocycle of the inhibitor can increase its affinity to Aurora B kinase. Hence, the benzoxazole scaffold was altered to a benzothiazole one, which is derived by replacing the oxygen atom of the benzoxazole core with sulfur (Figure 1C). This could increase the lipophilicity of the molecule without any significant change in shape. In this study, we described the SAR of a novel series of aminobenzothiazole derivatives as Aurora B kinase inhibitors and their inhibitory effect on the phosphorylation of Histone H3 and cell cycle progression.

2. Results and discussion 2.1. Synthesis The synthetic routes of the targeted molecules are depicted in scheme 1-4. Our previous approach for the synthesis of aminobenzoxazole derivatives 12 was revised by changing the site of disconnection to improve the overall yield and overcome the limited structure variation. At first, the solvent-accessible moiety was introduced to the benzothiazole core. Demethylation of methoxy substituted 2-aminobenzothiazole was followed by alkylation of the resulting alcohol with R1-substituted ethyl chloride to give the ether compounds, 3a-d (Scheme 1). 4

N O 6

N

a NH2

N

b

HO

NH2

S 1a: 4-OCH3 1b: 6-OCH3

O

R1

S

NH2 S

3a: 4, R1 = Morphorinyl 3b: 6, R1 = Morphorinyl 3c: 6, R1 = N,N-dimethylamino 3d: 6, R1 = Piperidinyl

2a: 4-OH 2b: 6-OH

Figure 1. Modification of the hinge binding heterocyclic scaffold. (A) Lipophilic potential property map of the ATP binding site in human Aurora B kinase (PDB ID: 4AF3) calculated by MOLCAD. (B) Docked pose of benzothiazole compound 15g in the ATP binding site of Aurora B kinase (C) Bioisosteric replacement of the previously reported benzoxazole scaffold with benzothiazole. Ar, Aromatic ring; HA, Hydrogen bond acceptor; HD, Hydrogen bond donor.

Aurora A and B share most of their primary protein structures, 71% of the whole protein6. Regardless of the structural similarity between Aurora A and B, they perform distinct roles in the mitotic process. The consequences of Aurora A and B inhibition are quite different, and the proliferating cells are more sensitive to the inhibition of Aurora B than that of Aurora A10. The important differences between the structures of Aurora A and B kinases exist at the residues Arg175, Glu177, and Lys180 of Aurora B kinase vs Lue215, Thr217, and Arg220 of Aurora A kinase3. At the active site of the Aurora kinases, Glu177 in Aurora B kinase corresponds to a smaller amino acid, Thr, in Aurora A kinase11. This results in a difference in the size, shape, and charge of the binding cavity at the active site. By targeting this residue, some inhibitors such as barasertib as a representative selective Aurora B kinase inhibitor, can acquire subtype selectivity. In a former study, we identified aminobenzoxazole derivatives as lead compounds of Aurora B kinase inhibitors, and the most potent compound showed a half maximal inhibitory concentration (IC50) value of 0.6 µM12. In our endeavor to improve the potency of the lead compound, detailed molecular modeling was performed. The results brought us to become aware of a lipophilic cavity which aromatic heterocycle of hinge binder positioned in and is composed of hydrophobic amino acid residues such as Val, Lue, and Ala (Figure 1A, 1B)13. It is implied that enhancing the lipophilic property of the aromatic

O a

N

O

NH

b

N

HO

N

O

NH 6

NH 7

O c

d

N

HO

NH 8

N

H

NH 9

Scheme 1. Synthesis of the benzothiazole derivatives. Reagents and conditions: a) catechol, HBr (aq), reflux; b) R1-substituted ethyl chloride HCl, Cs2 CO3, DMF, 80°C.

Two types of linkers, benzyl and methyl pyrazole, were introduced between the benzothiazole core and the terminal AspPhe-Gly (DFG) interacting group. The aromatic linker groups were prepared as their respective aldehydes 5 and 9. Bocprotected aminobenzyl alcohol 4 was oxidized to give benzaldehyde 5 (Scheme 2). 3-Methylpyrazole was oxidized to produce the corresponding carboxylic acid 6. Esterification and subsequent reduction of the carboxylic acid 6 gave the O

corresponding alcohol 8, which was then oxidized to aldehyde 9 (Scheme 3).

The IC50 values of the active compounds, which showed more than 50 % inhibition relative to the maximum value of the positive control, staurosporine, were determined. Most of the benzothiazole derivatives showed improved enzyme inhibitory activities when compared with the previous benzoxazole derivatives. For instance, compound 15j, which is the benzothiazole equivalent of the most potent benzoxazole compound12, was more potent than its counterpart with an IC50 value of 0.27 µM. The clogP value of 15j were calculated 7.0804, which was higher than those of benzoxazole counterpart 6.3517. These results support our hypothesis that replacing the heteroatom oxygen with sulfur could enhance the potency. Overall tendency of the structure activity relationship corresponded with the former case of benzoxazole and was more explicit.

Scheme 2. Preparation of hydrophobic substituents. Reagents and conditions: a) (Boc)2 O, THF, RT, 6 h; b) MnO2 , THF, 50°C.

The aldehyde linkers 5 or 9 underwent reductive amination reactions with aminobenzothiazole derivatives 3a-d to provide the corresponding amines 10 or 12a-e. The target compound 11 was obtained via the reaction of 10 with aryl isocyanate. Hydrolysis of the Boc-protected amine 12 followed by reaction with acyl chloride, sulfonyl chloride, isocyanate, or isothiocyanate produced the corresponding compounds 14a-j, 14k-m, 15a-k, or 15l-m, respectively (Scheme 4).

The linker moiety between the aminobenzothiazole core and the DFG interacting group was examined. Replacing the phenyl ring (14g and 14i) with a 5-membered heterocyclic pyrazole (11a-c) led to decreased activity. The hydrogen bond acceptor/donor moiety, which is an essential interacting group in the DFG pocket, was thoroughly investigated. Compared with carboxamide (14g and 14i), the sulfonamide-linked compounds (14k-m) showed lower inhibitory activities. In addition, the replacement of the amide linkage with urea mostly increased the activity. Regarding the position of the linker group on the phenyl ring, the para-substituted 14h, 14i, 15d, 15f, and 15g compounds showed a better activity than the ortho-substituted 14a and the meta-substituted 14c-e, and 15a-c compounds. Compounds which have thiourea linkage, 15m and 15n, showed no significant differences compared with its urea counterpart, 15g and 15l, respectively. The effect of the linker position and the property of the solvent-accessible moiety were also examined. The 6-Alkoxybenzothiazole compounds showed a better activity than the 4-alkoxy substituted ones (data not shown no activities at 10 µM). Changing the morpholinyl (15g) group to either a dimethylamino (15h) or a piperidinyl (15i) group resulted in a lower inhibitory activity.

Scheme 3. Preparation of hydrophobic substituents. Reagents and conditions: a) KMnO4 , H2O, reflux; b) SOCl2, MeOH, 45°C, 3 h; c) LiAlH4 , THF, RT, 2 h; d) MnO2 , DMF, 70°C.

2.2. In vitro kinase inhibitory activity The enzyme inhibitory activities of the synthesized compounds were determined quantitatively by the ADP-Glo kinase assay, which detects the ATP consumption during a kinase reaction. The inhibitory activity of compounds, 11 and 14 (10 µM) was evaluated using the same concentration of staurosporine as a positive control (Table 1). Several compounds that completely suppressed the enzymatic activity of Aurora B kinase at a concentration of 10 µM were further subjected to evaluation of their inhibitory activity at a concentration of 1 µM (Table 2).

Table 1. In vitro kinase inhibitory µM N

O

Type A

N

N N O

NH2

S

O

N H

c

N NH

S

O

N H

NHBoc

N H

NH2

N S

O

X Y

N

O O

S

O

N N

N H

HN X

11d: X = 4-Br 11e: X = 3,4-diCl

12a: R1 = Morpholinyl, ortho 12b: R1 = Morpholinyl, meta 12c: R1 = Morpholinyl, para 12d: R1 = N,N-dimethylamino, para 12e: R1 = Piperidinyl, para

b

R1

N

pH

11a: X = H 11b: X = 4-F 11c: X = 4-Cl

N R1

Y

N

S

3a: 4, R1 = Morpholinyl 3b: 6, R1 = Morpholinyl 3c: 6, R1 = N,N-dimethylamino 3d: 6, R1 = Piperidinyl

H N

m

N H

S

O

10

a

N X

N N

N

O

N

N H

S

O

O

Type B

R1

activities of compound 11 and 14 at 10

R

c

N

O N

1

13a: R = Morpholinyl, ortho 13b: R1 = Morpholinyl, meta 13c: R1 = Morpholinyl, para 13d: R1 = N,N-dimethylamino, para 13e: R1 = Piperidinyl, para

O

S

N H

14a: ortho, X = CO, Y = H 14b: meta, X = CO, Y = H 14c: meta, X = CO, Y = 4-F 14d: meta, X = CO, Y = 3-Cl 14e: meta, X = CO, Y = 4-Cl 14f: meta, X = CO, Y = 4-NO2

c

N H

O

S

15a: R1 = Morpholinyl, meta, X = O, Y = H

Y

14g: para, X = CO, Y = H 14h: para, X = CO, Y = 3-Cl 14i: para, X = CO, Y = 4-Cl 14j: para, X = CO, Y = 4-OMe 14k: para, X = SO2, Y = H 14l: para, X = SO2, Y = 4-F 14m: para, X = SO2, Y = 4-Cl X

N R1

X

N H

N H

Y N H

15h: R1 = N,N-Dimethylamino, para, X = O, Y = 4-Cl

R Inhibitory activity (%)a AURB IC50 (µM) X Y AURA AURB A CO H ND b 12 ND 11a A CO 4-F ND 5 ND 11b A CO 4-Cl ND 31 ND 11c A CO 4-Br ND 31 ND 11d A CO 3,4-diCl ND 46 ND 11e 14a o B CO H NAc NA ND B m CO H NA 24 ND 14b B m CO 4-F NA 56 9.0 14c B m CO 3-Cl NA 30 ND 14d B m CO 4-Cl NA 42 6.2 14e 14f B m CO 4-NO 2 NA 17 ND B p CO H NA 25 ND 14g B p CO 3-Cl NA 91 1.4 14h B p CO 4-Cl NA 59 3.0 14i B p CO 4-OMe NA 85 0.7 14j B p SO2 H NA 16 ND 14k B p SO2 4-F NA 30 ND 14l B p SO2 4-Cl NA 28 ND 14m a Relative percent inhibition against staurosporine at a concentration of 10 µM. Compounds were tested at a concentration of 10 µM. b Not determined. c Not active at 10 µM. Comp.

Type

position

Table 2. In vitro kinase inhibitory activities of compounds 15 at 1 µM R1 X

N 2

R O

N H

Y N H

R1 Inhibitory activity (%)a AURB IC50 (µM) position X Y AURA AURB 2-Morpholin-4-ylethyl m O H NAb 37 3.1 15a 2-Morpholin-4-ylethyl m O 3-Cl NA 33 1.0 15b 2-Morpholin-4-ylethyl m O 4-Cl NA 31 1.2 15c 2-Morpholin-4-ylethyl p O H NA 91 0.27 15d 2-Morpholin-4-ylethyl p O 4-F NA 44 0.70 15e 2-Morpholin-4-ylethyl p O 3-Cl NA 67 0.46 15f 2-Morpholin-4-ylethyl p O 4-Cl NA 100 0.12 15g N,N-Dimethylaminoethyl p O 4-Cl NA 65 0.32 15h 2-Piperidinylethyl p O 4-Cl NA 5 2.3 15i 15j 2-Morpholin-4-ylethyl p O 3,4-diCl 2 77 0.27 2-Morpholin-4-ylethyl p O 4-Br 9 83 0.09 15k 2-Morpholin-4-ylethyl p O 4-OMe NA 79 0.25 15l 2-Morpholin-4-ylethyl p S 4-Cl 3 94 0.11 15m 2-Morpholin-4-ylethyl p S 4-OMe NA 72 0.36 15n a Relative percent inhibition against staurosporine at a concentration of 10 µM. Compounds were tested at a concentration of 1 µM. b Not active at 1 µM. Comp.

R2

S

N H

2.3. Cellular inhibitory activities

Regarding the in vitro kinase inhibitory activity, compounds 15g and 15k were chosen for further evaluation of their cellular effect via suppressing the enzymatic activity of Aurora B kinase. The selected compounds were tested using cell cyclesynchronized HeLa cells. The phosphorylation level of the 10th serine residue on histone H3, which represents the known substrate of Aurora B kinase, was decreased in a dose-dependent manner. Moreover, flow cytometry was performed to determine the cell cycle distribution in HeLa cells treated with 15g, 15k (10 µM, 50 µM), or DMSO for 24 h. Treatment of the cells with the benzothiazole compounds arrested the majority of the cell population in the G2/M phase. The G2/M phase percentage of 15k-treated HeLa cells was more than 2-fold higher than that of the DMSO-treated group.

Figure 2. Cellular effect of compounds 15g and 15k. (A) Inhibition of histone H3S10 phosphorylation. HeLa cells (4 × 105 cells) were seeded in 6well plates, and the cell cycle was synchronized at mitosis using Monastrol block. Cells were released from the block after 16 h by washing and changing with media containing no Monastrol. At the time of release, cells were treated with the indicated concentrations of the inhibitors. Cells were harvested 1 h after their release and used for immunoblotting. (B) Cell cycle analysis of HeLa cells after treatment with 15g and 15k. G2/M phase percentage values of treated compound were marked on the graph. HeLa cells (5x105 cells) were seeded in 60 mm dish on day before treatment of inhibitors and cell were treated with the indicated concentrations of the inhibitors for 24 hrs. The cells were analyzed for cell cycle using flow cytometry. (C) G2/M phase percentage of benzothiazole compound-treated HeLa cells. D, DMSO; H, Hesperadin (5 µM), pH3S10, phosphorylated histone H3 on the 10th serine residue; AUKB, Aurora B kinase.

2.4. Molecular docking analysis Molecular docking analysis was performed to predict the binding mode of the aminobenzothiazole derivatives using Surflex Dock interfaced with Sybyl-X version 2.1.1. The crystal structure of human Aurora B kinase complexed with its potent inhibitor, VX-680 (PDB ID: 4AF3), was chosen for the docking study. Compound 15g fits well in the active site of Aurora B kinase, as shown in Figure 3. The aminobenzothiazole core of 15g occupies the adenine-binding region and forms the essential hydrogen bonds with Ala157 and Glu155 in the hinge backbone (Figure 3A). The urea carbonyl oxygen forms a hydrogen bonding interaction with the catalytic Lys106, which is critical for the enzyme activity. The oxygen linking with solventaccessible moiety also makes a water-mediated interaction with Glu161 (Thr217 in Aurora A), which is known to serve for subtype selectivity11. In contrast, there was no meaningful result when our benzothiazole compounds were docked with Aurora A kinase. To compare the structure of Aurora A to B, Aurora A was merged with compound 15g docked Aurora B. The side chain of DFG motif at Aurora A occupied the space where the terminal lipophilic group of 15g was positioned. It reflected narrower positional options for Aurora A than Aurora B in the region of DFG, which was caused by the differences of arrangement of the DFG motif. It can also support our selectivity data. In addition, the terminal phenyl ring, located at a distance of 2.95 Å from the carbonyl group of Phe219, possibly forms an electrostatic interaction with the carbonyl oxygen (Figure 3B)14. In the case of compound 15g, chlorine substitution could enhance the chargetransfer interactions at this position because of the relative lack of electrons at the center of the terminal phenyl ring.

Figure 3. The predicted molecular docking results of compound 15g. (A) The active site of human Aurora B-INCENP complex colored purple (PDB ID: 4AF3), with compound 15g. (B) Electrophilic potential property map with MMFF charge calculated by MOLCAD.

3. Conclusion A series of aminobenzothiazole derivatives were investigated as possible Aurora B kinase inhibitors. Bioisosteric replacement of the heterocyclic core of the lead compound was performed regarding the docking analysis. Replacement of the oxygen atom of the bicyclic heterocycle with sulfur enhanced the inhibitory activity and the selectivity for Aurora B kinase. The SAR results implied the importance of the phenylureido moiety as a linker and the docking analysis provided an insight into their possible interaction with Aurora B kinase. The potent compounds were selected and their intrinsic enzyme inhibitory activity was evaluated in HeLa cells. Compounds 15g and 15k decreased the cellular phosphorylation level and arrested the mitotic progression at the G2/M phase. These results suggest that our compounds have a potential for further development of chemotherapeutic agent.

4. Experimental 4.1. Chemistry The majority of the reagents and solvents were purchased from Sigma-Aldrich chemicals and were used without purification. Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck) with the indicated solvents. Thin-layer chromatography (TLC) was performed using Kieselgel 60 F254 plates (Merck). IR spectra were recorded using a JASCO FT/IR 430 spectrophotometer. NMR spectra were recorded using a Varian YH 400 spectrometer (1H 400 MHz and 13C 100 MHz). Chemical shifts are expressed in parts per million (ppm, δ) relative to the internal standard, tetramethylsilane. Mass spectra (ESI) were obtained using an Agilent single Quadrople LC/MS high-resolution mass spectrometer.

4.2. Synthesis of compounds 4.2.1. General procedure for the preparation of compounds 11, 14 and 15 To a solution of 10 or 13 in MeCN (0.1 M) was added pyridine (1.2 equiv.) under nitrogen atmosphere. The mixture was stirred at room temperature for 30 min. Then, substituted acyl chloride, aryl sulfonyl chloride, aryl isocyanate, or aryl isothiocyanate (1.2 equiv.) was added. The reaction mixture was stirred for 15 min to 1 h and diluted with water. The resulting mixture was extracted with EtOAc. The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (nhexane:EtOAc:MeOH = 1:2:0.3; CHCl3:EtOAc:MeOH = 1:1:0.1). 4.2.1.1. 3-((6-(2-Morpholin-4-ylethoxy)benzothiazol-2ylamino)methyl)pyrazole-1-carboxylic acid phenylamide (11a) A mixture of 10 and phenyl isocyanate in MeCN afforded 11a (44% yield): 1H NMR (400 MHz, CDCl3) δ 8.98 (1H, br s), 8.26 (1H, d, J = 2.4 Hz), 7.60 (2H, d, J = 8.0 Hz), 7.47 (1H, d, J = 8.8 Hz), 7.39 (2H, dd, J = 7.6, 8.0 Hz), 7.17 (1H, t, J = 7.6 Hz), 7.15 (1H, d, J = 2.8 Hz), 6.92 (1H, dd, J = 2.8, 8.8 Hz), 6.46 (1H, d, J = 2.4 Hz), 4.71 (2H, s), 4.11 (2H, t, J = 5.6 Hz), 3.74 (4H, t, J = 4.8 Hz), 2.80 (2H, t, J = 5.6 Hz), 2.58 (4H, t, J = 4.8 Hz); 13C NMR (100 MHz, CDCl3 ) δ 165.1, 154.5, 152.4, 146.5, 136.4,

131.6, 130.0, 129.2, 124.8, 119.8, 119.5, 114.3, 108.0, 106.5, 66.9, 66.6, 57.7, 54.1, 42.7. 4.2.1.2. 3-((6-(2-Morpholin-4-ylethoxy)benzothiazol-2ylamino)methyl)pyrazole-1-carboxylic acid (4fluorophenyl)amide (11b) A mixture of 10 and 4-fluorophenyl isocyanate in MeCN afforded 11b (18% yield): 1 H NMR (400 MHz, CDCl3 ) δ 9.00 (1H, br s), 8.25 (1H, d, J = 2.8 Hz), 7.56 (2H, dd, J = 4.4, 8.8 Hz), 7.45 (1H, d, J = 8.8 Hz), 7.15 (1H, d, J = 2.8 Hz), 7.07 (2H, dd, J = 8.4, 8.8 Hz), 6.91 (1H, dd, J = 2.8, 8.8 Hz), 6.46 (1H, d, J = 2.8 Hz), 4.69 (2H, s), 4.11 (2H, t, J = 5.6 Hz), 3.74 (4H, t, J = 4.8 Hz), 2.81 (2H, t, J = 5.6 Hz), 2.59 (4H, t, J = 4.8 Hz). 4.2.1.3. 3-((6-(2-Morpholin-4-ylethoxy)benzothiazol-2ylamino)methyl)pyrazole-1-carboxylic acid (4chlorophenyl)amide (11c) A mixture of 10 and 4-chlorophenyl isocyanate in MeCN afforded 11c (40% yield): 1H NMR (400 MHz, CDCl3) δ 9.07 (1H, br s), 8.24 (1H, d, J = 2.8 Hz), 7.56 (2H, d, J = 8.8 Hz), 7.45 (1H, d, J = 8.4 Hz), 7.34 (2H, d, J = 8.8 Hz), 7.15 (1H, d, J = 2.4 Hz), 6.92 (1H, dd, J = 2.4, 8.4 Hz), 6.46 (1H, d, J = 2.8 Hz), 4.67 (2H, s), 4.12 (2H, t, J = 5.6 Hz), 3.74 (4H, t, J = 4.4 Hz), 2.81 (2H, t, J = 5.6 Hz), 2.60 (4H, t, J = 4.4 Hz). 4.2.1.4. 3-((6-(2-Morpholin-4-ylethoxy)benzothiazol-2ylamino)methyl)pyrazole-1-carboxylic acid (4bromophenyl)amide (11d) A mixture of 10 and 4-bromophenyl isocyanate in MeCN afforded 11d (48% yield): 1H NMR (400 MHz, CDCl3) δ 9.00 (1H, br s), 8.22 (1H, d, J = 2.8 Hz), 7.47 (4H, s), 7.44 (1H, d, J = 8.8 Hz), 7.14 (1H, d, J = 2.4 Hz), 6.90 (1H, dd, J = 2.4, 8.8 Hz), 6.45 (1H, d, J = 2.8 Hz), 4.68 (2H, s), 4.10 (2H, t, J = 5.6 Hz), 3.74 (4H, t, J = 4.8 Hz), 2.80 (2H, t, J = 5.6 Hz), 2.58 (4H, t, J = 4.8 Hz); 13C NMR (100 MHz, CDCl 3) δ 165.2, 154.5, 152.6, 146.4, 135.6, 132.2, 131.5, 130.0, 121.4, 121.2, 119.5, 117.5, 114.3, 108.2, 106.5, 66.9, 66.6, 57.7, 54.1, 42.7. 4.2.1.5. 3-((6-(2-Morpholin-4-ylethoxy)benzothiazol-2ylamino)methyl)pyrazole-1-carboxylic acid (3,4dichlorophenyl)amide (11e) A mixture of 10 and 3,4-dichlorophenyl isocyanate in MeCN afforded 11e (46% yield): 1H NMR (400 MHz, CDCl3) δ 9.02 (1H, br s), 8.21 (1H, d, J = 2.8 Hz), 7.76 (1H, s), 7.44-7.41 (3H, m), 7.13 (1H, d, J = 2.8 Hz), 6.90 (1H, dd, J = 2.8, 8.8 Hz), 6.46 (1H, d, J = 2.8 Hz), 4.69 (2H, s), 4.10 (2H, t, J = 6.0 Hz), 3.74 (4H, t, J = 4.8 Hz), 2.80 (2H, t, J = 6.0 Hz), 2.58 (4H, t, J = 4.8 Hz); 13C NMR (100 MHz, CDCl3) δ 165.2, 154.5, 152.8, 146.4, 146.3, 136.0, 133.0, 131.5, 130.7, 130.0, 128.0, 121.3, 119.5, 118.9, 114.3, 108.4, 106.4, 66.9, 66.6, 57.7, 54.1, 42.6. 4.2.1.6. N-(2-((6-(2-Morpholin-4-yl-ethoxy)benzothiazol-2ylamino)methyl)phenyl)benzamide (14a) A mixture of 13a and benzoyl chloride in MeCN afforded 14a: 1H NMR (400 MHz, CDCl3 ) δ 11.09 (1H, s), 8.16 (2H, d, J = 7.2 Hz), 8.02 (1H, d, J = 8.0 Hz), 7.55 (1H, dd, J = 7.2, 7.6 Hz), 7.46 (2H, dd, J = 7.2, 7.6 Hz), 7.37 (1H, dd, J = 7.2, 8.0 Hz), 7.34 (1H, t, J = 7.6 Hz), 7.18 (1H, dd, J = 7.2, 7.6 Hz), 7.05 (1H, d, J = 2.8 Hz), 6.84 (1H, d, J = 8.8 Hz), 6.72 (1H, dd, J = 2.8, 8.8 Hz), 5.86 (1H, br s), 4.64 (2H, s), 4.06 (2H, t, J = 5.6 Hz), 3.73 (4H, t, J = 4.4 Hz), 2.77 (2H, t, J = 5.6 Hz), 2.56 (4H, t, J = 4.4 Hz). 4.2.1.7. N-(3-((6-(2-Morpholin-4-yl-ethoxy)-benzothiazol-2ylamino)methyl)phenyl)benzamide (14b)

A mixture of 13b and benzoyl chloride in MeCN afforded 14b: 1H NMR (400 MHz, CDCl 3) δ 8.07 (1H, s), 7.82 (2H, d, J = 7.2 Hz), 7.64 (1H, s), 7.58 (1H, d, J = 8.0 Hz), 7.51 (1H, t, J = 7.2 Hz), 7.43 (2H, t, J = 7.2 Hz), 7.35 (1H, d, J = 8.8 Hz), 7.30 (1H, dd, J = 7.6, 8.0 Hz), 7.12 (1H, d, J = 7.6 Hz), 7.09 (1H, d, J = 2.4 Hz), 6.86 (1H, dd, J = 2.4, 8.8 Hz), 6.11 (1H, br s), 4.56 (2H, s), 4.08 (2H, t, J = 5.6 Hz), 3.72 (4H, t, J = 4.4 Hz), 2.78 (2H, t, J = 5.6 Hz), 2.56 (4H, t, J = 4.4 Hz); 13C NMR (100 MHz, CDCl3) δ 154.2, 146.6, 138.8, 138.4, 134.7, 131.9, 131.4, 129.4, 128.7, 127.0, 123.7, 119.6, 119.2, 114.2, 106.5, 66.9, 66.6, 57.7, 54.1, 48.9; HRMS (ESI) m/z calcd for C27H28N4 O3S, 488.1882; found: 489.1950 [M+H]+.

A mixture of 13c and benzoyl chloride in MeCN afforded 14g: 1H NMR (400 MHz, CDCl3) δ 7.87 (2H, dd, J = 1.6, 7.2 Hz), 7.81 (1H, br s), 7.64 (2H, d, J = 8.4 Hz), 7.56 (1H, tt, J = 1.6, 7.2 Hz), 7.50 (2H, t, J = 7.2 Hz), 7.45 (1H, d, J = 8.8 Hz), 7.40 (2H, d, J = 8.4 Hz), 7.14 (1H, d, J = 2.8 Hz), 6.91 (1H, dd, J = 2.8, 8.8 Hz), 5.37 (1H, br s), 4.61 (2H, s), 4.12 (2H, t, J = 5.6 Hz), 3.74 (4H, t, J = 4.8 Hz), 2.80 (2H, t, J = 5.6 Hz), 2.58 (4H, t, J = 4.8 Hz); 13C NMR (100 MHz, CD3OD + CDCl3) δ 188.7, 166.6, 166.4, 154.0, 146.2, 137.5, 134.7, 133.7, 131.8, 131.0, 128.5, 128.2, 127.2, 120.8, 118.8, 114.1, 106.4, 66.7, 66.2, 57.6, 53.9; HRMS (ESI) m/z calcd for C27H28N4O3S, 488.1882; found: 489.1950 [M+H]+.

4.2.1.8. 4-Fluoro-N-(3-((6-(2-morpholin-4-ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)benzamide (14c)

4.2.1.13. 3-Chloro-N-(4-((6-(2-morpholin-4-ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)benzamide (14h)

A mixture of 13b and 4-fluorobenzoyl chloride in MeCN afforded 14c (14% yield): 1H NMR (400 MHz, CDCl 3) δ 8.20 (1H, br s), 7.81 (2H, dd, J = 5.2, 8.4 Hz), 7.61 (1H, s), 7.56 (1H, d, J = 8.0 Hz), 7.32 (1H, d, J = 8.8 Hz), 7.28 (1H, dd, J = 7.6, 8.0 Hz), 7.11-7.05 (4H, m), 6.84 (1H, dd, J = 2.4, 8.8 Hz), 4.53 (2H, s), 4.07 (2H, t, J = 5.6 Hz), 3.72 (4H, t, J = 4.4 Hz), 2.78 (2H, t, J = 5.6 Hz), 2.57 (4H, t, J = 4.4 Hz); HRMS (ESI) m/z calcd for C27H27FN4O3S, 506.1788; found: 507.1849 [M+H]+.

A mixture of 13c and 3-chlorobenzoyl chloride in MeCN afforded 14h (14% yield): 1 H NMR (400 MHz, CDCl3 ) δ 7.85 (1H, t, J = 1.2 Hz), 7.81 (1H, br s), 7.74 (1H, d, J = 7.6 Hz), 7.62 (2H, d, J =8.4 Hz), 7.53 (1H, dd, J = 1.2, 8.0 Hz), 7.45 (1H, d, J = 8.8 Hz), 7.44 (1H, dd, J = 7.6, 8.0 Hz), 7.40 (2H, d, J = 8.4 Hz), 7.14 (1H, d, J = 2.4 Hz), 6.91 (1H, dd, J = 2.4, 8.8 Hz), 5.47 (1H, br s), 4.61 (2H, s), 4.12 (2H, t, J = 5.6 Hz), 3.74 (4H, t, J = 4.4 Hz), 2.80 (2H, t, J = 5.6 Hz), 2.58 (4H, t, J = 4.4 Hz); 13C NMR (100 MHz, CDCl 3 + CD3OD) δ 166.5, 165.4, 154.0, 146.1, 137.3, 136.5, 134.5, 133.9, 131.6, 130.8, 129.8, 128.1, 127.5, 125.5, 121.0, 118.6, 114.0, 106.3, 66.5, 66.1, 57.5, 53.8; HRMS (ESI) m/z calcd for C27H27ClN4O3S, 522.1492; found: 523.1557 [M+H]+.

4.2.1.9. 3-Chloro-N-(3-((6-(2-morpholin-4-ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)benzamide (14d) A mixture of 13b and 3-chlorobenzoyl chloride in MeCN afforded 14d: 1H NMR (400 MHz, CDCl3) δ 8.03 (1H, s), 7.80 (1H, t, J = 1.6 Hz), 7.68 (1H, d, J = 8.0 Hz), 7.64 (1H, s), 7.57 (1H, d, J = 8.0 Hz), 7.49 (1H, td, J = 1.2, 7.6 Hz), 7.40-7.30 (4H, m), 7.15 (1H, d, J = 7.6 Hz), 7.10 (1H, d, J = 2.4 Hz), 6.87 (1H, dd, J = 2.4, 8.8 Hz), 5.97 (1H, br s), 4.59 (2H, s), 4.09 (2H, t, J = 5.6 Hz), 3.73 (4H, t, J = 4.4 Hz), 2.79 (2H, t, J = 5.6 Hz), 2.57 (4H, t, J = 4.4 Hz); 13C NMR (100 MHz, CDCl3 + CD3OD) δ 154.0, 146.0, 138.5, 138.2, 136.4, 134.4, 131.6, 129.7, 129.1, 127.5, 126.4, 125.5, 123.8, 120.1, 119.9, 118.5, 113.9, 106.3, 66.5, 66.0, 57.5, 53.7; HRMS (ESI) m/z calcd for C27H27ClN4O3S, 522.1492; found: 523.1558 [M+H]+. 4.2.1.10. 4-Chloro-N-(3-((6-(2-morpholin-4-ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)benzamide (14e) A mixture of 13b and 4-chlorobenzoyl chloride in MeCN afforded 14e (26% yield): 1H NMR (400 MHz, CDCl 3) δ 8.33 (1H, br s), 7.72 (2H, d, J = 8.4 Hz), 7.61 (1H, s), 7.56 (1H, d, J = 8.4 Hz), 7.34 (2H, d, J = 8.4 Hz), 7.30-7.25 (2H, m), 7.09 (1H, d, J = 7.6 Hz), 7.06 (1H, d, J = 2.4 Hz), 6.83 (1H, dd, J = 2.4, 8.8 Hz), 4.52 (2H, s), 4.07 (2H, t, J = 5.6 Hz), 3.72 (4H, t, J = 4.4 Hz), 2.78 (2H, t, J = 5.6 Hz), 2.57 (4H, t, J = 4.4 Hz); HRMS (ESI) m/z calcd for C27H27ClN4O3S, 522.1492; found: 523.1559 [M+H]+. 4.2.1.11. N-(3-((6-(2-Morpholin-4-yl-ethoxy)benzothiazol-2ylamino)methyl)phenyl)-4-nitrobenzamide (14f) A mixture of 13b and 4-nitrobenzoyl chloride in MeCN afforded 14f: 1H NMR (400 MHz, CDCl3) δ 8.28 (2H, d, J = 8.8 Hz), 8.12 (1H, br s), 7.99 (2H, d, J = 8.8 Hz), 7.65 (1H, s), 7.61 (1H, d, J = 8.0 Hz), 7.39 (1H, d, J = 8.8 Hz), 7.36 (1H, dd, J = 7.6, 8.0 Hz), 7.18 (1H, d, J = 7.6 Hz), 7.10 (1H, d, J = 2.4 Hz), 6.88 (1H, dd, J = 2.4, 8.8 Hz), 4.61 (2H, s), 4.10 (2H, t, J = 5.6 Hz), 3.74 (4H, t, J = 4.4 Hz), 2.80 (2H, t, J = 5.6 Hz), 2.59 (4H, t, J = 4.4 Hz); HRMS (ESI) m/z calcd for C27H27N5O5S, 533.1733; found: 534.1799 [M+H]+. 4.2.1.12. N-(4-((6-(2-Morpholin-4-yl-ethoxy)benzothiazol-2ylamino)methyl)phenyl)benzamide (14g)

4.2.1.14. 4-Chloro-N-(4-((6-(2-morpholin-4-yl-ethoxy)benzothiazol-2-ylamino)methyl)phenyl)benzamide (14i) A mixture of 13c and 4-chlorobenzoyl chloride in MeCN afforded 14i (39% yield): 1H NMR (400 MHz, CDCl3) δ 7.96 (1H, s), 7.79 (2H, d, J = 8.8 Hz), 7.58 (2H, d, J = 8.4 Hz), 7.43 (2H, d, J = 8.8 Hz), 7.41 (1H, d, J = 8.8 Hz), 7.35 (2H, d, J = 8.4 Hz), 7.13 (1H, d, J = 2.4 Hz), 6.90 (1H, dd, J = 2.4, 8.8 Hz), 5.85 (1H, br s), 4.58 (2H, s), 4.11 (2H, t, J = 5.6 Hz), 3.73 (4H, t, J = 4.4 Hz), 2.80 (2H, t, J = 5.6 Hz), 2.58 (4H, t, J = 4.4 Hz) ); 13C NMR (100 MHz, CDCl 3 + CD3OD) δ 166.5, 165.7, 154.0, 146.1, 137.8, 137.4, 133.8, 133.1, 130.8, 128.8, 128.6, 128.1, 121.0, 118.6, 114.0, 106.3, 66.5, 66.1, 57.5, 53.8; HRMS (ESI) m/z calcd for C27H27ClN4O3S, 522.1492; found: 523.1559 [M+H]+. 4.2.1.15. 4-Methoxy-N-(4-((6-(2-morpholin-4ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)benzamide (14j) A mixture of 13c and 4-methoxybenzoyl chloride in MeCN afforded 14j: 1H NMR (400 MHz, CDCl3) δ 7.94 (1H, br s), 7.83 (2H, d, J = 8.4 Hz), 7.58 (2H, d, J = 8.4 Hz), 7.40 (1H, d, J = 8.8 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.13 (1H, d, J = 2.4 Hz), 6.94 (2H, d, J = 8.4 Hz), 6.89 (1H, dd, J = 2.4, 8.8 Hz), 5.94 (1H, br s), 4.56 (2H, s), 4.10 (2H, t, J = 5.6 Hz), 3.86 (3H, s), 3.73 (4H, t, J = 4.4 Hz), 2.79 (2H, t, J = 5.6 Hz), 2.57 (4H, t, J = 4.4 Hz); HRMS (ESI) m/z calcd for C28H30N4O4S, 518.1988; found: 519.2053 [M+H]+. 4.2.1.16. N-(4-((6-(2-Morpholin-4-ylethoxy)benzothiazol-2ylamino)methyl)phenyl)benzenesulfonamide (14k) A mixture of 13c and phenyl sulfonyl chloride in MeCN afforded 14k: 1H NMR (400 MHz, CDCl3) δ 7.76 (2H, d, J = 8.4 Hz), 7.50 (1H, t, J = 7.6 Hz), 7.40 (2H, dd, J = 7.6, 8.4 Hz), 7.32 (1H, d, J = 8.8 Hz), 7.22 (2H, d, J = 8.4 Hz), 7.11 (1H, d, J = 2.4 Hz), 7.03 (2H, d, J = 8.4 Hz), 6.86 (1H, dd, J = 2.4, 8.8 Hz), 4.49 (2H, s), 4.10 (2H, t, J = 5.6 Hz), 3.73 (4H, t, J = 4.4 Hz), 2.80 (2H, t, J = 5.6 Hz), 2.58 (4H, t, J = 4.4 Hz); 13C NMR (100 MHz, CDCl 3) δ 166.0, 154.3, 146.3, 139.1, 136.1, 134.6, 133.0, 131.2, 129.0, 128.6, 127.1, 121.9, 119.2, 114.3, 106.5, 66.9, 66.5, 57.7,

54.0, 48.6; HRMS (ESI) m/z calcd for C26H28N4O4S2, 524.1552; found: 525.1619 [M+H]+.

4.2.1.22. 4-((6-(2-Morpholin-4-ylethoxy)benzothiazol-2ylamino)methyl)phenyl-3-phenylurea (15d)

4.2.1.17. 4-Fluoro-N-(4-((6-(2-morpholin-4ylethoxy)benzothiazol-2ylamino)methyl)phenyl)benzenesulfonamide (14l)

A mixture of 13c and phenyl isocyanate in MeCN afforded 15d: 1H NMR (400 MHz, CD3OD + CDCl3) δ 7.41-7.39 (4H, m), 7.34-7.31 (3H, m), 7.27 (2H, dd, J = 7.2, 8.8 Hz), 7.20 (1H, d, J = 2.8 Hz), 7.00 (1H, dd, J = 7.2, 7.6 Hz), 6.88 (1H, dd, J = 2.8, 8.8 Hz), 4.53 (2H, s), 4.12 (2H, t, J = 5.6 Hz), 3.71 (4H, t, J = 4.4 Hz), 2.79 (2H, t, J = 5.6 Hz), 2.59 (4H, t, J = 4.4 Hz); HRMS (ESI) m/z calcd for C27H29N5O3S, 503.1991; found: 504.2056 [M+H]+.

A mixture of 13c and phenyl 4-fluorosulfonyl chloride in MeCN afforded 14l: 1 H NMR (400 MHz, CD3OD + CDCl3) δ 7.73 (2H, dd, J = 4.8, 8.8 Hz), 7.31 (1H, d, J = 8.8 Hz), 7.23 (2H, d, J = 8.4 Hz), 7.12 (1H, d, J = 2.4 Hz), 7.10-7.03 (4H, m), 6.86 (1H, dd, J = 2.4, 8.8 Hz), 4.48 (2H, s), 4.10 (2H, t, J = 5.2 Hz), 3.72 (4H, t, J = 4.0 Hz), 2.80 (2H, t, J = 5.2 Hz), 2.60 (4H, t, J = 4.0 Hz); 13C NMR (100 MHz, CD3OD + CDCl3) δ 167.3, 167.1, 164.5, 154.9, 146.9, 137.2, 136.3, 135.6, 131.7, 130.6, 130.5, 129.0, 122.2, 119.1, 116.7, 116.5, 114.6, 107.0, 78.0, 67.2, 66.7, 58.3, 54.5; HRMS (ESI) m/z calcd for C26H27FN4O4 S2, 542.1458; found: 543.1531 [M+H]+. 4.2.1.18. 4-Chloro-N-(4-((6-(2-morpholin-4ylethoxy)benzothiazol-2ylamino)methyl)phenyl)benzenesulfonamide (14m) A mixture of 13c and phenyl 4-chlorosulfonyl chloride in MeCN afforded 14m: 1H NMR (400 MHz, CD3OD) δ 7.68 (2H, d, J = 8.8 Hz), 7.44 (2H, d, J = 8.8 Hz), 7.31 (1H, d, J = 8.8 Hz), 7.26 (2H, d, J = 8.8 Hz), 7.21 (1H, d, J = 2.4 Hz), 7.07 (2H, d, J = 8.8 Hz), 6.89 (1H, dd, J = 2.4, 8.8 Hz), 4.50 (2H, s), 4.13 (2H, t, J = 5.2 Hz), 3.72 (4H, t, J = 4.4 Hz), 2.82 (2H, t, J = 5.2 Hz), 2.62 (4H, t, J = 4.4 Hz); 13C NMR (100 MHz, CD3 OD) δ 167.8, 155.7, 147.4, 140.1, 139.7, 137.9, 136.6, 132.3, 130.2, 130.0, 129.5, 122.7, 119.4, 115.1, 107.4, 67.5, 67.0, 58.8, 55.1, 48.4; HRMS (ESI) m/z calcd for C26H27 ClN4O4S2, 558.1162; found: 559.1234 [M+H]+. 4.2.1.19. 3-((6-(2-Morpholin-4-yl-ethoxy)benzothiazol-2ylamino)methyl)phenyl-3-phenylurea (15a) A mixture of 13b and phenyl isocyanate in MeCN afforded 15a (42% yield): 1H NMR (400 MHz, CDCl3) δ 7.67 (1H, br s), 7.55 (1H, br s), 7.33 (1H, d, J = 8.8 Hz), 7.27-7.25 (2H, m), 7.21 (2H, dd, J = 7.6, 8.4 Hz), 7.15 (1H, d, J = 7.6 Hz), 7.10-7.07 (3H, m), 7.01 (1H, t, J = 7.2 Hz), 6.89 (1H, d, J = 8.0 Hz), 6.82 (1H, dd, J = 2.0, 8.8 Hz), 4.38 (2H, s), 4.06 (2H, t, J = 5.6 Hz), 3.72 (4H, t, J = 4.4 Hz), 2.78 (2H, t, J = 5.6 Hz), 2.57 (4H, t, J = 4.4 Hz); HRMS (ESI) m/z calcd for C27H29N5 O3S, 503.1991; found: 538.1692 [M+Cl]-. 4.2.1.20. (3-Chlorophenyl)-3-(3-((6-(2-morpholin-4ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)urea (15b) A mixture of 13b and 3-chlorophenyl isocyanate in MeCN afforded 15b: 1H NMR (400 MHz, CDCl3 + CD3 OD) δ 7.52 (1H, s), 7.94-7.29 (3H, m), 7.25-7.15 (3H, m), 7.11 (1H, d, J = 2.4 Hz), 7.02 (1H, d, J = 7.6 Hz), 6.97 (1H, d, J = 8.4 Hz), 6.88 (1H, dd, J = 2.4, 8.8 Hz), 4.51 (2H, s), 4.10 (2H, t, J = 6.4 Hz), 3.75 (4H, t, J = 4.8 Hz), 2.81 (2H, t, J = 6.4 Hz), 2.60 (4H, t, J = 4.8 Hz); HRMS (ESI) m/z calcd for C27H28ClN5O3S, 537.1601; found: 538.1664 [M+H]+. 4.2.1.21. (4-Chlorophenyl)-3-(3-((6-(2-morpholin-4ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)urea (15c) A mixture of 13b and 3-chlorophenyl isocyanate in MeCN afforded 15c: 1H NMR (400 MHz, CD3OD) δ 7.43-7.35 (2H, m), 7.39 (2H, d, J = 8.8 Hz), 7.32 (1H, d, J = 8.8 Hz), 7.28-7.23 (3H, m), 7.20 (1H, d, J = 2.8 Hz), 7.05 (1H, d, J = 7.6 Hz), 6.88 (1H, dd, J = 2.8, 8.8 Hz), 4.57 (2H, s), 4.11 (2H, t, J = 5.6 Hz), 3.70 (4H, t, J = 4.4 Hz), 2.80 (2H, t, J = 5.6 Hz), 2.60 (4H, t, J = 4.4 Hz); HRMS (ESI) m/z calcd for C27H28ClN5O3S, 537.1601; found: 538.1666 [M+H]+.

4.2.1.23. (4-Fluorophenyl)-3-(4-((6-(2-morpholin-4ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)urea (15e) A mixture of 13c and 4-fluorophenyl isocyanate in MeCN afforded 15e: 1H NMR (400 MHz, CDCl3 + CD3OD) δ 7.41-7.35 (5H, m), 7.29 (2H, d, J = 8.4 Hz), 7.13 (1H, d, J = 2.4 Hz), 6.99 (2H, dd, J = 8.4, 9.2 Hz), 6.90 (1H, dd, J = 2.4, 8.8 Hz), 4.50 (2H, s), 4.12 (2H, t, J = 5.6 Hz), 3.75 (4H, t, J = 4.8 Hz), 2.82 (2H, t, J = 5.6 Hz), 2.61 (4H, t, J = 4.8 Hz); HRMS (ESI) m/z calcd for C27H28FN5O3S, 521.1897; found: 522.1961 [M+H]+. 4.2.1.24. (3-Chlorophenyl)-3-(4-((6-(2-morpholin-4ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)urea (15f) A mixture of 13c and 3-chlorophenyl isocyanate in MeCN afforded 15f: 1H NMR (400 MHz, CD3OD) δ 7.62-7.61 (1H, m), 7.41 (2H, d, J = 8.8 Hz), 7.34 (1H, s), 7.32 (2H, d, J = 8.8 Hz), 7.24-7.22 (2H, m), 7.21 (1H, d, J = 2.8 Hz), 7.00-6.97 (1H, m), 6.88 (1H, dd, J = 2.8, 8.8 Hz), 4.54 (2H, s), 4.12 (2H, t, J = 5.6 Hz), 3.71 (4H, t, J = 4.4 Hz), 2.79 (2H, t, J = 5.6 Hz), 2.59 (4H, t, J = 4.4 Hz); 13C NMR (100 MHz, CD3OD + CDCl3) δ 166.5, 154.2, 153.5, 146.1, 140.6, 138.1, 134.1, 132.7, 130.9, 129.6, 127.9, 122.1, 119.2, 118.5, 118.0, 116.7, 113.7, 106.0, 66.2, 65.7, 57.4, 53.7; HRMS (ESI) m/z calcd for C27H28ClN5O3S, 537.1601; found: 538.1662 [M+H]+. 4.2.1.25. (4-Chlorophenyl)-3-(4-((6-(2-morpholin-4ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)urea (15g) A mixture of 13c and 4-chlorophenyl isocyanate in MeCN afforded 15g: 1 H NMR (400 MHz, CD3OD) δ 7.41 (2H, d, J = 8.8 Hz), 7.40 (2H, d, J = 8.8 Hz), 7.33 (3H, m), 7.25 (2H, d, J = 8.8 Hz), 7.21 (1H, d, J = 2.8 Hz), 6.88 (1H, dd, J = 2.8, 8.8 Hz), 4.54 (2H, s), 4.12 (2H, t, J = 5.2 Hz), 3.71 (4H, t, J = 4.4 Hz), 2.80 (2H, t, J = 5.2 Hz), 2.60 (4H, t, J = 4.4 Hz); 13C NMR (100 MHz, CD3 OD + CDCl3) δ 166.5, 154.2, 154.0, 146.0, 138.2, 137.9, 132.7, 130.8, 128.4, 127.9, 127.2, 120.1, 119.2, 118.0, 113.7, 106.0, 66.2, 65.7, 57.4, 53.7; HRMS (ESI) m/z calcd for C27H28ClN5O3S, 537.1601; found: 538.1665 [M+H]+. 4.2.1.26. (4-Chlorophenyl)-3-(4-((6-(2dimethylaminoethoxy)benzothiazol-2ylamino)methyl)phenyl)urea (15h) A mixture of 13d and 4-chlorophenyl isocyanate in MeCN afforded 15h: 1H NMR (400 MHz, CD3OD + CDCl3) δ 7.39-7.30 (7H, m), 7.22 (2H, d, J = 9.2 Hz), 7.19 (1H, d, J = 2.4 Hz), 6.88 (1H, dd, J = 2.4, 8.8 Hz), 4.53 (2H, s), 4.35 (2H, t, J = 5.2 Hz), 3.33 (2H, t, J = 5.2 Hz), 2.79 (6H, s); HRMS (ESI) m/z calcd for C25H26ClN5O2S, 495.1496; found: 494.1418 [M-H]-. 4.2.1.27. (4-Chlorophenyl)-3-(4-((6-(2piperidinylethoxy)benzothiazol-2-ylamino)methyl)phenyl)urea (15i) A mixture of 13e and 4-chlorophenyl isocyanate in MeCN afforded 15i: 1H NMR (400 MHz, CDCl3 + CD3 OD) δ 7.70 (1H, d, J = 9.2 Hz), 7.47 (2H, d, J = 8.4 Hz), 7.40 (2H, d, J = 8.8 Hz), 7.33 (2H, d, J = 8.4 Hz), 7.25 (2H, d, J = 8.8 Hz), 7.16 (1H, d, J

= 2.4 Hz), 7.07 (1H, dd, J = 2.4, 9.2 Hz), 4.58 (2H, s ), 4.15 (2H, t, J = 5.6 Hz), 2.86 (2H, t, J = 5.6 Hz), 2,61 (4H, t, J = 5.6 Hz), 1.66 (4H, quin, J = 5.6 Hz), 1.50 (2H, quin, J = 5.6 Hz). 4.2.1.28. (3,4-Dichlorophenyl)-3-(4-((6-(2-morpholin-4ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)urea (15j) A mixture of 13c and 3,4-dichlorophenyl isocyanate in MeCN afforded 15j: 1H NMR (400 MHz, CD3OD + CDCl3) δ 7.69 (1H, d, J = 2.4 Hz), 7.38-7.29 (6H, m), 7.21 (1H, dd, J = 2.4, 8.8 Hz), 7.12 (1H, d, J = 2.0 Hz), 6.86 (1H, dd, J = 2.0 Hz, 8.4 Hz), 4.51 (2H, s), 4.10 (2H, t, J = 5.6 Hz), 3.72 (4H, t, J = 4.4 Hz), 2.80 (2H, t, J = 5.6 Hz), 2.60 (4H, t, J = 4.4 Hz); 13C NMR (100 MHz, CD3OD + CDCl3) δ 167.3, 154.7, 154.0, 146.8, 139.5, 138.6, 133.2, 132.9, 131.6, 130.8, 128.8, 125.9, 120.9, 120.0, 118.9, 118.7, 114.5, 107.0, 67.1, 66.6, 58.2, 54.4, 48.5; HRMS (ESI) m/z calcd for C27H27Cl2N5O3 S, 571.1212; found: 572.1276 [M+H]+. 4.2.1.29. (4-Bromophenyl)-3-(4-((6-(2-morpholin-4ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)urea (15k) A mixture of 13c and 4-bromophenyl isocyanate in MeCN afforded 15k: 1H NMR (400 MHz, CD3OD + CDCl3) δ 7.42-7.32 (9H, m), 7.16 (1H, d, J = 2.8 Hz), 6.90 (1H, dd, J = 2.4, 8.8 Hz), 4.55 (2H, s), 4.14 (2H, t, J = 5.6 Hz), 3.76 (4H, t, J = 4.8 Hz), 2.83 (2H, t, J = 5.6 Hz), 2.63 (4H, t, J = 4.8 Hz); 13 C NMR (100 MHz, CD3 OD + CDCl3) δ 167.4, 154.8, 154.4, 146.9, 139.0, 138.8, 133.1, 132.3, 131.6, 128.9, 121.3, 120.0, 119.0, 115.5, 114.6, 107.0, 67.1, 66.6, 58.3, 54.5; HRMS (ESI) m/z calcd for C27H28BrN5O3S, 581.1096; found: 584.1142 [M+H]+. 4.2.1.30. (4-Methoxyphenyl)-3-(4-((6-(2-morpholin-4ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)urea (15l) A mixture of 13c and 4-methoxyhenyl isocyanate in MeCN afforded 15l: 1 H NMR (400 MHz, CDCl3 + CD3 OD) δ 7.38 (1H, d, J = 8.8 Hz), 7.36 (2H, d, J = 8.4 Hz), 7.29 (2H, d, J = 8.8 Hz), 7.28 (2H, d, J = 8.4 Hz), 7.14 (1H, d, J = 2.4 Hz), 6.89 (1H, dd, J = 2.4, 8.8 Hz), 6.84 (2H, d, J = 8.4 Hz), 4.50 (2H, s), 4.12 (2H, t, J = 5.6 Hz), 3.77 (3H, s), 3.76 (4H, t, J = 4.4 Hz), 2.82 (2H, t, J = 5.6 Hz), 2.62 (4H, t, J = 4.4 Hz); 13C NMR (100 MHz, CDCl3 + CD3OD) δ 166.4, 155.3, 153.9, 153.7, 145.8, 138.1, 131.5, 131.4, 130.6, 127.9, 121.3, 119.0, 118.1, 113.8, 113.7, 106.1, 66.2, 65.7, 57.2, 55.0, 53.5, 47.8; HRMS (ESI) m/z calcd for C28H31N5O4S, 533.2097; found: 534.2164 [M+H]+. 4.2.1.31. (4-Chlorophenyl)-3-(4-((6-(2-morpholin-4ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)thiourea (15m) A mixture of 13c and 4-chlorophenyl isothiocyanate in MeCN afforded 15m: 1H NMR (400 MHz, CD3OD + CDCl3) δ 7.39 (2H, d, J = 8.8 Hz), 7.35 (2H, d, J = 9.2 Hz), 7.34 (1H, d, J = 8.8 Hz), 7.29 (2H, d, J = 8.8 Hz), 7.21 (2H, d, J = 9.2 Hz), 7.12 (1H, d, J = 2.8 Hz), 6.86 (1H, dd, J = 2.8, 8.8 Hz), 4.51 (2H, s), 4.12 (2H, t, J = 5.2 Hz), 3.74 (4H, t, J = 4.8 Hz), 2.84 (2H, t, J = 5.2 Hz), 2.64 (4H, t, J = 4.8 Hz). 4.2.1.32. (4-Methoxyphenyl)-3-(4-((6-(2-morpholin-4ylethoxy)benzothiazol-2-ylamino)methyl)phenyl)thiourea (15n) A mixture of 13c and 4-methoxyphenyl isothiocyanate in MeCN afforded 15n: 1H NMR (400 MHz, CD3OD + CDCl3) δ 7.33 (2H, d, J = 8.8 Hz), 7.31 (1H, d, J = 8.8 Hz), 7.26-7.23 (4H, m), 7.09 (1H, d, J = 2.4 Hz), 6.83 (1H, dd, J = 2.4, 8.8 Hz), 6.80 (2H, d, J = 8.8 Hz), 4.47 (2H, s), 4.07 (2H, t, J = 5.6 Hz), 3.73 (3H, s), 3.70 (4H, t, J = 4.8 Hz), 2.77 (2H, t, J = 5.6 Hz), 2.57 (4H, t, J = 4.8 Hz).

4.3 in vitro assay 4.3.1. In vitro kinase activities The compounds were dissolved completely in DMSO. For the determination of primary efficacy, a 10 µM concentration of each compound was screened using purified active Aurora B or Aurora A kinase as an enzyme and myelin basic protein as a substrate with 10 µM of ATP. 10 µM staurosporine (multiple kinase inhibitor) was used as a positive control. The activity and inhibition were determined using the luciferase activity mediated by ATP (ADP–Glo assay kit, Promega, USA). After kinase reaction for 60 minutes at room temperature, the kinase reaction solution and ADP-Glo reagent were mixed at a 1:1 ratio (total 20 µl), and incubated at room temperature for 30 minutes. Kinase detection reagent (20 µl) was then added, and the mixture was further incubated for 30 minutes before the plate was read on a Glo-Max discover (Promega, USA) to measure the luminescence intensity. IC50 values were calculated using the Sigmaplot software ver. 12.0. 4.3.2. Western blot (WB) analysis and antibodies HeLa cells (4 × 105 cells) were seeded in 6-well plates, and the cell cycle was synchronized at mitosis using Monastrol block. Cells were released from the block after 16 h by washing and changing with media containing no Monastrol. At the time of release, cells were treated with the indicated concentrations of the inhibitors. Cells were harvested 1 h after their release and used for immunoblotting. Cells were lysed with RIPA buffer (20 Mm Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% NP-40) in the presence of Xpert protease inhibitor and Xpert phosphatase inhibitor (GenDEPOT, USA) followed by sonication. Then, 20 µg of protein extracts in 2x SDS-sample buffer were run on a 8~15% gradient SDS-PAGE, transferred to nitrocellulose membrane (GE Healthcare, UK) and probed with the appropriate antibodies. Antibodies used in this blot were antiphospho-H3S10 (Abcam, UK), anti-Histone H3 (Cell Signaling Technology, USA), anti-α-Tubulin (Sigma, USA), and antiAurora kinase B (Cell Signaling Technology, USA). 4.3.3. Cell cycle analysis HeLa cells (5x105 cells) were seeded in 60 mm dish on day before treatment of inhibitors and cell were treated with the indicated concentrations of the inhibitors for 24 hrs. Cells treated with inhibitors were harvested with Non-enzymatic Cell Dissociation Solution (Sigma, USA) and washed twice by centrifugation at 15,000 rpm for 5 min in cold PBS. Cells then were resuspended in 1 ml cold PBS and 9 ml of cold 70% EtOH was added slowly to the cell suspension dropwise with gentle vortex. It was stored at 4 °C for 2 h and cells were washed again by centrifugation at 15,000 rpm for 5 min in cold PBS. For staining with Propidium Iodide (PI), cells were resuspended in 300~500 µl PI/Triton X-100 staining solution (0.1% Triton X100, 0.1% EDTA, 50 µg/ml RNase A, 50 µg/ml PI in PBS) and incubated at 25 oC for 20 min in dark. Fluorescence-activated cell sorting (FACS) analysis was measured using the FACSCanto II (BD Biosciences, USA) and data were analyzed by Flow Jo software (FlowJo LLC, USA).

4.4. Molecular modeling All molecular modeling calculations were carried out using the Surflex Dock and MOLCAD interfaced with SYBYL-X, ver. 2.1.1. The 3D coordinates of the active site were taken from the reported crystal structure of the human Aurora B kinase in complex with the Aurora kinase inhibitor VX-680 (PDB ID:

4AF3). In this automated docking program, the flexibility was considered as rigid, the ligand was built in an incremental fashion where each new fragment was added in all possible positions and conformations to a pre-placed base fragment inside the active site. All the molecules used for docking were sketched in the SYBYL, and energy minimizations were performed using Tripos Force Field and Gasteiger-Huckel charged with 100,000 iterations of the conjugate gradient method with convergence criterion of 0.05 kcal/mol.

11. Sessa, F.; Villa, F. Acta Crystallogr. F 2014, 70, 294. 12. An, Y.; Lee, E.; Yu, Y.; Yun, J.; Lee, M. Y.; Kang, J. S.; Kim, W.; Jeon, R. Bioorg. Med. Chem. Lett. 2016, 26, 3067. 13. Elkins, J. M.; Santaguida, S.; Musacchio, A.; Knapp, S. J. Med. Chem. 2012, 55, 7841.

Acknowledgments We dedicate this article to Professor Young-Ger Suh on the occasion of his retirement. This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2011-0030074 and 2014R1A2A1A11052761).

14. Li, Y.; Snyder, L. B.; Langley, D. R. Bioorg. Med. Chem. Lett. 2003, 13, 3261.

Supplementary Material References and notes

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