Vol. 66, No. 3 Chem. Pharm. Bull. 66, 251–262 (2018) 251 Current Topics Drug Discovery: Recent Progress and the Future Regular Article Discovery of N-{2-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}- N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-triazine-2,4-diamine (ASP3026), a Potent and Selective Anaplastic Lymphoma Kinase (ALK) Inhibitor Kazuhiko Iikubo,* Yutaka Kondoh,† Itsuro Shimada, Takahiro Matsuya, Kenichi Mori, Yoko Ueno, and Minoru Okada‡ Drug Discovery Research, Astellas Pharma Inc.; 21 Miyukigaoka, Tsukuba, Ibaraki 305–8585, Japan. Received September 27, 2017; accepted November 15, 2017 Anaplastic lymphoma kinase (ALK) is a validated therapeutic target for treating echinoderm microtu- bule-associated protein-like 4 (EML4)-ALK positive non-small cell lung cancer (NSCLC). We synthesized a series of 1,3,5-triazine derivatives and identified ASP3026 (14a) as a potent and selective ALK inhibitor. In mice xenografted with NCI-H2228 cells expressing EML4-ALK, once-daily oral administration of 14a demonstrated dose-dependent antitumor activity. Here, syntheses and structure–activity relationship (SAR) studies of 1,3,5-triazine derivatives are described. Key words 1,3,5-triazine; non-small cell lung cancer; anaplastic lymphoma kinase; echinoderm microtubule- associated protein-like 4 The receptor tyrosine kinase anaplastic lymphoma kinase activity of 14a. (ALK) was identified in anaplastic large-cell lymphoma (ALCL) as a fusion gene, comprising portions of the nucleo- Results and Discussion phosmin (NPM) gene and the ALK gene that contains the Chemistry Compounds 5a–5e were synthesized by kinase catalytic domain, which encodes the NPM-ALK fu- reacting 3a–3c, 3d11) and 3e12) with 4-piperidone monohy- sion protein.1) In 2007, the echinoderm microtubule-associated drate monohydrochloride, followed by reductive amination protein-like 4 (EML4)-ALK fusion gene was identified in a with 1-methylpiperazine and sodium triacetoxyborohydride subset of non-small cell lung cancer (NSCLC) patients.2,3) (NaBH(OAc)) and hydrogenation with catalytic palladium 3 EML4-ALK oncogenic fusion kinase plays an essential role on carbon (Chart 1). Compounds 8 and 10 were synthesized in the pathogenesis of NSCLC.4) In addition, EML4-ALK has by introduction of the commercially available corresponding constitutive tyrosine kinase activity.2,5) Furthermore, a number amines into 6 and 3a, followed by hydrogenation of the nitro of ALK inhibitors have been reported to date,6) with crizotinib groups (Charts 2, 3, respectively). having been approved by the U.S. Food and Drug Administra- The synthesis of compounds 14a–14l is shown in Chart 4. tion (FDA) in 20117) (Fig. 1). Given these previous findings, These were prepared by reacting compounds 11a–11c13) with ALK is a validated therapeutic target for treating EML4- corresponding 1,3,5-triazine derivatives 12a, 12b and 12c14) ALK-positive NSCLC. to afford 4-chloro-1,3,5-triazine analogues 13a–13e, followed Several compounds with inhibitory activity against ALK, by the introduction of corresponding aniline derivatives using such as NVP-TAE6848,9) (Fig. 1), had been previously reported methanesulfonic acid (MsOH) in ethanol (EtOH). when we started our project to identify novel ALK inhibitors. The synthesis of 20 is shown in Chart 5. Compound 16 In the course of exploring novel ALK inhibitors, 1,3,5-triazine derivatives were discovered. As a result of detailed structure– activity relationship (SAR) studies on every part of 1,3,5-tri- azine derivatives, 14a (ASP302610)) was identified and selected as a clinical candidate. Here, we describe the syntheses and SAR studies of 1,3,5-triazine derivatives as novel ALK in- hibitors. We also report the kinase selectivity and antitumor † Present address: Research and Development Dept., Omnica Co., Ltd.; TN Koishikawa Bldg. 5F, 1–15–17 Koishikawa, Bunkyo-ku, Tokyo 112–0002, Japan. ‡ Present address: Technology Department, Yonezawa Hamari Chemicals, Ltd.; 2–4300–18 Hachimampara, Yonezawa, Yamagata 992–1128, Japan. Fig. 1. Structures of Crizotinib and NVP-TAE684 * To whom correspondence should be addressed. e-mail: [email protected] © 2018 The Pharmaceutical Society of Japan 252 Chem. Pharm. Bull. Vol. 66, No. 3 (2018) Reagents and conditions: (a) 4-piperidone monohydrate monohydrochloride, KCO, N,N-dimethylformamide (DMF), 70 or 80°C; (b) 1-methylpiperazine, NaBH(OAc), 2 3 3 dichloromethane (DCM) or 1,2-dichloroethane (DCE), rt; (c) H, 10% Pd/C, EtOH or EtOH/tetrahydrofuran (THF), rt. 2 Chart 1. Synthesis of Compounds 5a–5e Reagents and conditions: (a) 1-methyl-4-(piperidin-4-yl)piperazine, KCO, DMF, 80°C; (b) H, 10% Pd/C, EtOH/THF, rt. 2 3 2 Chart 2. Synthesis of Compound 8 14a, showed inhibitory activity against EML4-ALK with IC 50 values of 73 and 360 nM, respectively. Our docking model of 14a with wild type ALK indicates the formation of two hydrogen bonds between one of the nitrogen atoms of the 1,3,5-triazine ring and the NH hydrogen atom of Met1199, and between the hydrogen atom of the 1,3,5-triazine ring and the Reagents and conditions: (a) N-ethyl-2-(4-methylpiperazin-1-yl)ethanamine, carbonyl oxygen atom of Glu1197 in the hinge region (Fig. 2). KCO, DMF, 80°C; (b) H, 10% Pd/C, EtOH, rt. 2 3 2 Therefore, the reduced inhibition of EML4-ALK by 14b and Chart 3. Synthesis of Compound 10 14c compared to that of 14a could be attributed to the attenua- tion of the interaction between these compounds and the hinge was synthesized by reacting commercially available 15 with moiety in ALK. 2-bromopropane using a sodium hydroxymethanesulfinate Table 2 shows SARs of the sulfonyl moiety of 14a. Substi- promoted one-pot reaction.16) Oxidation of 16 with m-chloro- tution of the 2-isopropylsulfonyl group of 14a with a 3-iso- peroxybenzoic acid (mCPBA), followed by reduction of the propylsulfonyl group (20) led to a loss of inhibitory activ- nitro group using iron powder in acetic acid (AcOH), provided ity against EML4-ALK, possibly due to the steric hindrance compound 18, which was converted to 20 in two steps by between 20 and the amino acid residues in ALK, including employing procedures similar to those described for 14a–14l. Gly1125 and Ala1126, as shown in Fig. 3. The methylsulfonyl The synthesis of 25 is shown in Chart 6. Commercially derivative 14d exhibited reduced inhibition of EML4-ALK available 1,4-dioxa-8-azaspiro[4.5] decane was reacted with 3a (IC50=530 nM), whereas the ethylsulfonyl analogue 14e main- to give 21, which was then hydrogenated to give compound tained inhibitory activity against EML4-ALK (IC50=21 nM). 22. The introduction of 22 into 13a successfully proceeded Our docking model suggests that the 2-isopropylsulfonyl moi- using N,N-diisopropylethylamine (DIPEA) in N-methylpyrro- ety plays two important roles in the potent inhibitory activity lidinone (NMP) under microwave conditions at 120°C to give of EML4-ALK (Fig. 3). First, the oxygen atom of the sulfone 23. After acidic hydrolysis of 23, ketone 24 was subjected to interacts with Lys1150 in ALK. Second, one of the two methyl reductive amination conditions with morpholine, then treated groups in the isopropyl moiety extends into and forms a hy- with 4 M HCl in ethyl acetate (EtOAc) to obtain 25 as a trihy- drophobic interaction with the hydrophobic pocket created drochloride salt. by Leu1256 in ALK. Therefore, the decrease in inhibitory Compound 29 was synthesized by reacting 2617) with 13a activity of 14d could be attributed to the loss of the interac- under basic conditions, followed by the removal of the tert- tion between 14d and the hydrophobic pocket. In contrast, 14e butoxycarbonyl group and reductive amination (Chart 7). retained its inhibitory activity, as the ethyl group in 14e can Biological Evaluation The synthesized compounds were interact with the hydrophobic pocket. evaluated via EML4-ALK enzyme and cell growth assays Substituent effects of the methoxy component of 14a were using Ba/F3 expressing EML4-ALK. The SARs of the synthe- then examined, with results shown in Table 3. Replacement sized compounds are summarized in Tables 1–4. of the 2-methoxy group (14a) with a 3-methoxy group (14f) As shown in Table 1, 14a inhibited EML4-ALK with an resulted in a three-fold reduction in inhibition of EML4-ALK. IC50 value of 17 nM.18) Compounds 14b and 14c, with a me- An unsubstituted phenyl ring (14g) resulted in a 19-fold de- thoxy group and a methyl group on the 1,3,5-triazine ring of crease in inhibitory activity against EML4-ALK relative to Vol. 66, No. 3 (2018) Chem. Pharm. Bull. 253 Reagents and conditions: (a) DIPEA, THF, rt or 70°C; (b) 5a–5e, 8, 2-methoxy-4-(4-methylpiperazin-1-yl)aniline,15) or 10, MsOH, EtOH, 80 or 100°C. Chart 4. Synthesis of Compounds 14a–14l Reagents and conditions: (a) 2-bromopropane, sodium hydroxymethanesulfinate, KCO, DMF/HO, rt; (b) mCPBA, CHCl, rt to 50°C; (c) Fe, AcOH, 80°C; (d) 12a, 2 3 2 3 DIPEA, THF, 0°C; (e) 5a, MsOH, EtOH, 100°C. Chart 5. Synthesis of Compound 20 Reagents and conditions: (a) 1,4-dioxa-8-azaspiro[4.5]decane, K2CO3, DMF, 70°C; (b) H2, 10% Pd/C, EtOH/THF, rt; (c) 13a, DIPEA, NMP, microwave, 120°C; (d) 4 M HCl aq, 1,4-dioxane, 80°C; (e) morpholine, NaBH(OAc)3, DCM, rt, then 4 M HCl/EtOAc, THF, rt. Chart 6. Synthesis of Compound 25 254 Chem. Pharm. Bull. Vol. 66, No. 3 (2018) Reagents and conditions: (a) 13a, DIPEA, NMP, microwave, 120°C; (b) 4 M HCl/EtOAc, EtOAc/MeOH, rt; (c) 1-methylpiperidin-4-one, NaBH(OAc)3, DCM, rt. Chart 7. Synthesis of Compound 29 Table 1. Structure–Activity Relationships of Compounds 14a–14c Table 3. Structure–Activity Relationships of Compounds 14a and 14f–14j IC50 (nM) Compound R EML4-ALK (enzyme) Ba/F3 (cell) 14a 2-MeO 17 42 14f 3-MeO 56 107 14g H 330 NTa 14h 2-Me 340 NTa 14i 2-EtO 93 197 14j 2-i-PrO 210 NTa a: Not tested. a: Not tested.; EML4-ALK enzyme IC50 value of compound 2 was 0.63 nM.18) Table 2. Structure–Activity Relationships of Compounds 14a, 14d, 14e Table 4. Structure–Activity Relationships of Compounds 14a, 14k, 14l, and 20 25 and 29 a: 3HCl salt.; b: Not determined. a: Not tested. Vol. 66, No. 3 (2018) Chem. Pharm. Bull. 255 14a. Substitution of the methoxy group with a methyl group consistent with observations in our docking model suggesting (14h) also reduced potency. These results indicate that the that the space between the 2-methoxy group of 14a and ALK oxygen atom at the 2-position is important for the inhibitory is relatively narrow (Fig. 2). activity of 14a, possibly due to the formation of a dipole– Table 4 shows SARs of the amine moiety in 14a, which dipole interaction between the methoxy group of 14a and NH projects into the solvent region located outside the ATP bind- at the 2-position of the 1,3,5-triazine ring, and stabilization of ing pocket and adjacent to Glu1210, as shown in Fig. 2. Com- the desirable conformation, as shown in Fig. 2. Replacing the pound 14k retained inhibitory activity against EML4-ALK methoxy group of 14a with an ethoxy group (14i) or an isop- with an IC50 value of 29 nM.18) Replacement of the piperazine ropoxy group (14j) also resulted in a reduction of inhibitory ring of 14a with a morphorine ring (25) also maintained activity (IC50=93 and 210 nM, respectively). This finding is inhibition of EML4-ALK. Compound 29 potently inhibited Fig. 2. The Docking Mode of 14a with Wild Type ALK 14a and ALK are represented as a ball-and-stick and stick model, respectively. All of the atoms are colored according to element (white: hydrogen, cyan: carbon of 14a, gray: carbon of ALK, blue: nitrogen, red: oxygen, yellow: sulfur). The yellow dotted lines indicate the hydrogen bonds formed between 14a and ALK. For clarity, the non- polar hydrogen atoms of the protein are omitted, and any atoms of ALK closer than the front of the gatekeeper residue are hidden. Fig. 3. The Docking Mode of 14a with Wild Type ALK 14a and ALK are represented as a ball-and-stick and stick model, respectively. The protein surface is colored by the characteristics of the pocket (green: hydrophobic, magenta: polar, red: solvent exposed). The carbon atoms of A1126, G1125, K1150, and L1256 are highlighted in yellow. The other coloring and visualizing schemes are the same as in Fig. 2. 256 Chem. Pharm. Bull. Vol. 66, No. 3 (2018) Table 5. Kinase Selectivity of 1 and 14a10) Compound 1 Compound 14a Kinase Selectivitya Kinase Selectivitya IC50 (nM) IC50 (nM) ROS 0.95 0.63 ALK 3.5 — ALK 1.5 — ALK R1275Q 5.4 1.5 MET 1.5 1.0 ACK 5.8 1.7 ALK F1174L 1.8 1.2 NPM1-ALK 6.8 1.9 LTK 1.8 1.2 ROS 8.9 2.5 NPM1-ALK 2.0 1.3 ALK F1174L 10 2.9 ALK R1275Q 2.0 1.3 TNK1 27 7.7 TRKA 3.2 2.1 FMS 30 8.6 AXL 3.3 2.2 YES 32 9.1 MER 3.4 2.3 DDR1 35 10 TRKC 4.4 2.9 TRKB 4.6 3.1 RON 5.5 3.7 MUSK 9.1 6.1 EPHA1 11 7.3 JAK2 11 7.3 LCK 11 7.3 An inhibitory assay for a panel of 86 tyrosine kinases, including ALK, was conducted. Kinases with IC values 10-fold or less, than that for ALK are shown (minor modifi- 50 cation of ref. 10). a: The ratio of the IC value for each tyrosine kinase relative to ALK. 50 Fig. 4. Antitumor Activity of 14a Subcutaneously xenografted mice with NCI-H2228 cells were treated with once-daily oral administration of 14a at the indicated doses for 14 d. Tumor volume was mea- sured to assess antitumor activity. Each point represents the mean±S.E.M., and the number of animals used is shown in parentheses. The values obtained on day 14 were statistically analyzed and compared. **, p<0.01 compared with the value of the control group on day 14 (Dunnett’s multiple comparison test).10) EML4-ALK with an IC50 value of 7.9 nM, whereas compound tumor growth inhibition at doses of 0.3 mg/kg (4% inhibition) 14l resulted in a five-fold loss of inhibitory activity against and 1 mg/kg (69% inhibition), and tumor regression at doses EML4-ALK. In consideration of the inhibitory activity in of 3 mg/kg (4% regression), 10 mg/kg (45% regression), and cells, in addition to that in enzymes, compound 14a exhibited 30 mg/kg (78% regression) in a dose-dependent manner. Body the most promising inhibitory activity against EML4-ALK. weight was not affected by 14a at the doses used in this ex- The inhibition exerted by 1 and 14a on a panel of 86 ty- periment.10) rosine kinases, including ALK, was examined. Compound 14a inhibited six kinases (ACK, ROS, TNK1, FMS, YES and Conclusion DDR1) with IC values 10-fold or less, than that for ALK We identified 14a (ASP3026) as a potent and selective ALK 50 except ALK R1275Q, NPM1-ALK and ALK F1174L, while inhibitor via SAR studies of 1,3,5-triazine derivatives. Once- compound 1 inhibited 13 kinases (ROS, MET, LTK, TRKA, daily oral administration of 14a to mice bearing NCI-H2228 AXL, MER, TRKC, TRKB, RON, MUSK EPHA1, JAK2 and tumor xenografts demonstrated dose-dependent antitumor LCK) in the same range (Table 5). activity and induced tumor regression. The antitumor activity of 14a was evaluated in mice xe- nografted with NCI-H2228, a human NSCLC tumor cell en- Experimental dogenously expressing EML4-ALK (Fig. 4). Compound 14a Chemistry 1 H-NMR spectra were recorded on JEOL inhibited the growth of NCI-H2228 cells with an IC value of AL400 or Varian 400-MR, and chemical shifts were expressed 50 65 nM.10) Once-daily oral administration of 14a demonstrated in δ (ppm) values with trimethylsilane as an internal reference Vol. 66, No. 3 (2018) Chem. Pharm. Bull. 257 (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, and din-4-yl}piperazine (4e) Compound 4e was prepared with a br=broad peak). MS were recorded on Thermo Electron LCQ yield of 43% from 4-fluoro-1-nitro-2-(propan-2-yloxy) benzene12) Advantage, Waters ultra performance liquid chromatography 3e using a procedure similar to that described for 4a. 1H-NMR (UPLC)/ZQ, Waters UPLC/SQD LC/MS system, JEOL GC- (400 MHz, CDCl) δ: 1.41 (6H, d, J=6.4 Hz), 1.52–1.68 (2H, m), 3 mateII, Thermo Electron TRACE DSQ or Waters Micromass 1.88–2.01 (2H, m), 2.19–2.76 (9H, m), 2.29 (3H, s), 2.87–2.99 LCT Premier Mass Spectrometer. Elemental analyses were (2H, m), 3.82–3.95 (2H, m), 4.53–4.67 (1H, m), 6.35 (1H, d, performed with Yanaco MT-6 (C, H, N) and DIONEX DX-500 J=2.4 Hz), 6.42 (1H, dd, J=2.8, 9.6 Hz), 7.94 (1H, d, J=9.6 Hz). (S, halogen) instruments, and results were within ±0.3% of ESI-MS m/z: 363 [M+H]+. theoretical values. 2-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]- 1-[1-(3-Methoxy-4-nitrophenyl)piperidin-4-yl]-4-methyl- aniline (5a) To a mixture of 1-[1-(3-methoxy-4-nitrophenyl)- piperazine (4a) To a mixture of 4-fluoro-2-methoxy-1-nitro- piperidin-4-yl]-4-methylpiperazine 4a (2.18 g, 6.52 mmol) in benzene 3a (3.00 g, 17.5 mmol) and K CO (6.10 g, 44.1 mmol) EtOH (50 mL) was added 10% palladium on carbon (wet, con- 2 3 in DMF (30 mL) was added 4-piperidone monohydrate mono- tains 53% water; 600 mg). The reaction mixture was stirred at hydrochloride (3.20 g, 20.8 mmol). The reaction mixture was room temperature for 8 h under 1 atm hydrogen atmosphere. stirred at 70°C overnight. Water was added to the mixture, The insoluble material was removed by filtration through and the resulting slurry was extracted with EtOAc. The organ- Celite, and the filtrate was concentrated in vacuo to give ic layer was washed with water and brine, dried over Na SO , 5a (1.96 g, 99%) as a pale purple solid. 1H-NMR (400 MHz, 2 4 and concentrated in vacuo. The residue was washed with Et O CDCl ) δ: 1.62–1.80 (2H, m), 1.86–1.98 (2H, m), 2.22–2.84 2 3 to give an ocher solid (3.85 g). (11H, m), 2.31 (3H, s), 3.23–3.74 (4H, m), 3.83 (3H, s), 6.42 To this product were added 1,2-dichloroethane (40 mL) (1H, dd, J=2.4, 8.0 Hz), 6.52 (1H, d, J=2.4 Hz), 6.63 (1H, d, and 1-methylpiperazine (2 mL, 18.2 mmol). After the mixture J=8.4 Hz). Electron ionization (EI)-MS m/z: 304 [M]+. was stirred for 30 min, sodium triacetoxyborohydride (3.90 g, 4-[4-(4-Methylpiperazin-1-yl)piperidin-1-yl]aniline (5b) 18.4 mmol) was added to the mixture, and the reaction mixture Compound 5b was prepared with a yield of 87% from 4b was stirred at room temperature overnight. Saturated aqueous using a procedure similar to that described for 5a. 1H-NMR NaHCO solution was added to the mixture, and the result- (400 MHz, DMSO-d ) δ: 1.39–1.56 (2H, m), 1.72–1.86 (2H, 3 6 ing slurry was extracted with CHCl . The organic layer was m), 2.07–2.61 (11H, m), 2.13 (3H, s), 3.25–3.46 (2H, m), 4.53 3 dried over Na SO , and concentrated in vacuo. The residue (2H, s), 6.43–6.51 (2H, m), 6.63–6.70 (2H, m). ESI-MS m/z: 2 4 was purified by silica gel column chromatography (CHCl / 275 [M+H]+. 3 MeOH/28% aqueous NH =30 : 1 : 0.1 to 15 : 1 : 0.1). The result- 2-Methyl-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]- 3 ing solid was washed with n-hexane to give 4a (3.30 g, 56%) aniline (5c) Compound 5c was prepared from 4c using a as a yellow solid. 1H-NMR (400 MHz, CDCl ) δ: 1.52–1.69 procedure similar to that described for 5a. ESI-MS m/z: 289 3 (2H, m), 1.90–2.03 (2H, m), 2.20–2.80 (9H, m), 2.31 (3H, s), [M+H]+. 2.90–3.03 (2H, m), 3.89–4.01 (2H, m), 3.95 (3H, s), 6.31 (1H, 2-Ethoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]- d, J=2.0 Hz), 6.42 (1H, dd, J=2.0, 9.2 Hz), 7.96–8.03 (1H, m). aniline (5d) Compound 5d was prepared from 4d using a Electrospray ionization (ESI)-MS m/z: 335 [M+H]+. procedure similar to that described for 5a. ESI-MS m/z: 319 1-Methyl-4-[1-(4-nitrophenyl)piperidin-4-yl]piperazine [M+H]+. (4b) Compound 4b was prepared with a yield of 14% from 4-[4-(4-Methylpiperazin-1-yl)piperidin-1-yl]-2-(propan- 1-fluoro-4-nitrobenzene 3b using a procedure similar to that 2-yloxy)aniline (5e) Compound 5e was prepared from 4e described for 4a. 1H-NMR (400 MHz, CDCl ) δ: 1.52–1.73 using a procedure similar to that described for 5a. ESI-MS 3 (2H, m), 1.88–2.02 (2H, m), 2.18–2.76 (9H, m), 2.29 (3H, s), m/z: 333 [M+H]+. 2.89–3.05 (2H, m), 3.91–4.05 (2H, m), 6.75–6.85 (2H, m), 1-[1-(2-Methoxy-4-nitrophenyl)piperidin-4-yl]-4-methyl- 8.06–8.15 (2H, m). ESI-MS m/z: 305 [M+H]+. piperazine (7) To a solution of 1-fluoro-2-methoxy-4-nitro- 1-Methyl-4-[1-(3-methyl-4-nitrophenyl)piperidin-4-yl]- benzene 6 (4.74 g, 27.7 mmol) in DMF (47 mL) were added piperazine (4c) Compound 4c was prepared with a yield 1-methyl-4-(piperidin-4-yl) piperazine (5.33 g, 29.1 mmol) and of 20% from 4-fluoro-2-methyl-1-nitrobenzene 3c using K CO (4.59 g, 33.2 mmol). The reaction mixture was stirred 2 3 a procedure similar to that described for 4a. 1H-NMR at 80°C for 15 h. Water was added to this reaction mixture (400 MHz, dimethyl sulfoxide (DMSO)-d ) δ: 1.32–1.47 (2H, under cooling in an ice bath, and the resulting precipitate 6 m), 1.75–1.88 (2H, m), 2.12 (3H, s), 2.15–2.63 (8H, m), 2.55 was filtered and washed with water to give 7 (8.79 g, 95%) as (3H, s), 2.85–3.00 (2H, m), 3.36–3.44 (1H, m), 3.95–4.11 (2H, a yellow solid. 1H-NMR (400 MHz, DMSO-d ) δ: 1.44–1.60 6 m), 6.81–6.93 (2H, m), 7.93–8.02 (1H, m). ESI-MS m/z: 319 (2H, m), 1.77–1.89 (2H, m), 2.06–2.61 (9H, m), 2.14 (3H, s), [M+H]+. 2.65–2.80 (2H, m), 3.63–3.76 (2H, m), 3.90 (3H, s), 7.00 (1H, 1-[1-(3-Ethoxy-4-nitrophenyl)piperidin-4-yl]-4-methylpi- d, J=8.8 Hz), 7.67 (1H, d, J=2.4 Hz), 7.82 (1H, dd, J=2.4, perazine (4d) Compound 4d was prepared with a yield of 8.8 Hz). ESI-MS m/z: 335 [M+H]+. 55% from 2-ethoxy-4-fluoro-1-nitrobenzene11) 3d using a pro- 3-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]- cedure similar to that described for 4a. 1H-NMR (400 MHz, aniline (8) Compound 8 was prepared with a yield of 94% CDCl ) δ: 1.50 (3H, t, J=7.0 Hz), 1.53–1.67 (2H, m), 1.88–2.01 from 7 using a procedure similar to that described for 5a. 3 (2H, m), 2.22–2.75 (9H, m), 2.29 (3H, s), 2.86–3.00 (2H, 1H-NMR (400 MHz, CDCl ) δ: 1.72–1.94 (4H, m), 2.29 (3H, s), 3 m), 3.84–3.98 (2H, m), 4.14 (2H, q, J=6.9 Hz), 6.31 (1H, d, 2.32–2.77 (11H, m), 3.32–3.60 (4H, m), 3.81 (3H, s), 6.19–6.29 J=2.8 Hz), 6.41 (1H, dd, J=2.6, 9.4 Hz), 7.97 (1H, d, J=9.2 Hz). (2H, m), 6.77 (1H, d, J=8.4 Hz). ESI-MS m/z: 305 [M+H]+. FAB-MS m/z: 349 [M+H]+. N-Ethyl-3-methoxy-N-[2-(4-methylpiperazin-1-yl)ethyl]- 1-Methyl-4-{1-[4-nitro-3-(propan-2-yloxy)phenyl]piperi- 4-nitroaniline (9) A mixture of 4-fluoro-2-methoxy-1-nitroben- 258 Chem. Pharm. Bull. Vol. 66, No. 3 (2018) zene 3a (900 mg, 5.26 mmol), N-ethyl-2-(4-methylpiperazin-1-yl)- overnight, saturated aqueous NaHCO solution and water were 3 ethanamine (901 mg, 5.26 mmol) and K CO (727 mg, 5.26 mmol) added. The resulting slurry was extracted with EtOAc, and 2 3 in DMF (10 mL) was stirred at 80°C for 6 h. The solvent was the organic layer was washed with water and brine, dried over concentrated, then water was added to the residue. The slurry Na SO , and concentrated in vacuo. The residue was purified 2 4 was extracted with EtOAc, and the organic layer was washed by silica gel column chromatography (n-hexane/EtOAc=3 : 1 with brine, dried over anhydrous MgSO , and concentrated in to 2 : 1) and washed with Et O to give 13d (430 mg, 52%) 4 2 vacuo. The residue was purified by silica gel column chroma- as a white solid. 1H-NMR (400 MHz, CDCl ) δ: 3.10 (3H, 3 tography (CHCl to CHCl /MeOH/28% aqueous NH=10/1/0.1) s), 7.31–7.41 (1H, m), 7.67–7.78 (1H, m), 7.97–8.07 (1H, m), 3 3 3 to give 9 (730 mg, 43%). 1H-NMR (400 MHz, DMSO-d ) δ: 1.14 8.42–8.51 (1H, m), 8.63 (1H, s), 9.48–9.74 (1H, br). FAB-MS 6 (3H, t, J=6.6 Hz), 2.14 (3H, s), 2.17–2.60 (10H, m), 3.42–3.59 (4H, m/z: 285 [M+H]+. m), 3.90 (3H, s), 6.19–6.24 (1H, m), 6.31–6.38 (1H, m), 7.86–7.93 4-Chloro-N-[2-(ethanesulfonyl)phenyl]-1,3,5-triazin- (1H, m). ESI-MS m/z: 323 [M+H]+. 2-amine (13e) Compound 13e was prepared with a yield of N4-Ethyl-2-methoxy-N4-[2-(4-methylpiperazin-1-yl)ethyl]- 47% from 2-(ethanesulfonyl) aniline13) 11c and 2,4-dichloro- benzene-1,4-diamine (10) Compound 10 was prepared 1,3,5-triazine 12a using a procedure similar to that described from 9 using a procedure similar to that described for 5a and for 13d. 1H-NMR (400 MHz, CDCl ) δ: 1.29 (3H, t, J=7.4 Hz), 3 directly used in the next reaction. ESI-MS m/z: 293 [M+H]+. 3.16 (2H, q, J=7.5 Hz), 7.31–7.40 (1H, m), 7.67–7.78 (1H, m), 4-Chloro-N-[2-(propane-2-sulfonyl)phenyl]-1,3,5-triazin- 7.96 (1H, dd, J=1.6, 8.0 Hz), 8.45–8.52 (1H, m), 8.62 (1H, s), 2-amine (13a) To a mixture of 2,4-dichloro-1,3,5-triazine 9.61–9.90 (1H, br). ESI-MS m/z: 299 [M+H]+. 12a (460 mg, 3.07 mmol) and THF (5 mL) was added a mixture N-{2-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin- of 2-(propane-2-sulfonyl) aniline13) 11a (600 mg, 3.01 mmol) 1-yl]phenyl}-N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-tri- and DIPEA (0.58 mL, 3.33 mmol) in THF (10 mL). After the azine-2,4-diamine (14a, ASP3026) A mixture of 5a reaction mixture was stirred at room temperature for three (210 mg, 0.690 mmol) and MsOH (0.13 mL, 2.00 mmol) in days, water (60 mL) and saturated aqueous NaHCO solution EtOH (3 mL) was stirred at room temperature for 15 min. To 3 were added. The resulting slurry was extracted with EtOAc, this mixture was added 13a (170 mg, 0.543 mmol), and the re- and the organic layer was washed with water and brine, dried action mixture was stirred at 100°C for 2 h. After the mixture over Na SO , and concentrated in vacuo. The residue was was cooled to room temperature, water and saturated aque- 2 4 purified by silica gel column chromatography (CHCl ) to ous NaHCO solution were added. The resulting slurry was 3 3 give 13a (340 mg, 36%) as a white solid. 1H-NMR (400 MHz, extracted with CHCl , and the organic layer was dried over 3 CDCl ) δ: 1.32 (6H, d, J=7.2 Hz), 3.15–3.30 (1H, m), 7.30–7.38 Na SO , then concentrated in vacuo. The residue was puri- 3 2 4 (1H, m), 7.67–7.76 (1H, m), 7.92 (1H, dd, J=1.5, 7.8 Hz), fied by silica gel column chromatography (CHCl /MeOH/28% 3 8.47–8.53 (1H, m), 8.61 (1H, s), 9.66–10.13 (1H, br). ESI-MS aqueous NH =50 : 1 : 0.1 to 30 : 1 : 0.1) to give 14a (180 mg, 3 m/z: 313 [M+H]+. 57%). 1H-NMR (400 MHz, CDCl ) δ: 1.31 (6H, d, J=6.8 Hz), 3 4-Chloro-6-methoxy-N-[2-(propane-2-sulfonyl)phenyl]- 1.62–1.79 (2H, m), 1.90–2.02 (2H, m), 2.23–2.81 (11H, m), 1,3,5-triazin-2-amine (13b) To a mixture of 2,4-di- 2.30 (3H, s), 3.18–3.32 (1H, m), 3.63–3.75 (2H, m), 3.88 (3H, s), chloro-6-methoxy-1,3,5-triazine 12b (370 mg, 2.06 mmol) 6.45–6.62 (2H, m), 7.14–7.29 (1H, m), 7.43–7.71 (2H, m), 7.88 and THF (10 mL) was added a mixture of 2-(propane-2- (1H, dd, J=1.6, 8.0 Hz), 8.02–8.17 (1H, m), 8.27–8.61 (2H, m), sulfonyl) aniline13) 11a (400 mg, 2.01 mmol) and DIPEA 9.28 (1H, s). ESI-MS m/z: 581 [M+H]+. High resolution (HR)- (0.72 mL, 4.13 mmol) in THF (5 mL). After the reaction mix- MS (ESI) m/z: 581.3022 [M+H]+ (Calcd for C H NOS: 29 41 8 3 ture was stirred at room temperature overnight and at 70°C for 581.3022). 7 h, water (60 mL) was added under ice-cooling. The resulting 6-Methoxy-N-{2-methoxy-4-[4-(4-methylpiperazin-1- solid was purified by silica gel column chromatography yl)piperidin-1-yl]phenyl}-N′-[2-(propane-2-sulfonyl)phen- (CHCl ) and washed with n-hexane to give 13b (200 mg, 29%) yl]-1,3,5-triazine-2,4-diamine (14b) Compound 14b was 3 as a white solid. 1H-NMR (400 MHz, CDCl ) δ: 1.31 (6H, d, prepared with a yield of 50% from 5a and 13b using a pro- 3 J=6.8 Hz), 3.13–3.28 (1H, m), 4.06 (3H, s), 7.19–7.38 (1H, m), cedure similar to that described for 14a. 1H-NMR (400 MHz, 7.60–7.74 (1H, m), 7.90 (1H, dd, J=1.6, 7.6 Hz), 8.47 (1H, dd, CDCl ) δ: 1.30 (6H, d, J=6.8 Hz), 1.50–1.79 (2H, m), 1.88–2.01 3 J=1.0, 8.2 Hz), 9.69 (1H, s). ESI-MS m/z: 343 [M+H]+. (2H, m), 2.25–2.81 (11H, m), 2.30 (3H, s), 3.19–3.32 (1H, m), 4-Chloro-6-methyl-N-[2-(propane-2-sulfonyl)phenyl]- 3.62–3.74 (2H, m), 3.89 (3H, s), 3.99 (3H, s), 6.44–6.60 (2H, 1,3,5-triazin-2-amine (13c) Compound 13c was prepared m), 7.13–7.24 (1H, m), 7.47 (1H, s), 7.56–7.66 (1H, m), 7.87 with a yield of 18% from 2-(propane-2-sulfonyl)a niline13) 11a (1H, dd, J=1.6, 8.0 Hz), 8.08–8.27 (1H, br), 8.47–8.62 (1H, m), and 2,4-dichloro-6-methyl-1,3,5-triazine14) 12c using a proce- 9.20 (1H, s). ESI-MS m/z: 611 [M+H]+. HR-MS (ESI) m/z: dure similar to that described for 13a. 1H-NMR (400 MHz, 611.3140 [M+H]+ (Calcd for C H NO S: 611.3128). 30 43 8 4 DMSO-d ) δ: 1.14 (6H, d, J=6.8 Hz), 2.40 (3H, s), 3.44–3.57 N-{2-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin- 6 (1H, m), 7.47–7.60 (1H, m), 7.78–7.86 (1H, m), 7.91 (1H, dd, 1-yl]phenyl}-6-methyl-N′-[2-(propane-2-sulfonyl)phenyl]- J=1.6, 8.0 Hz), 7.99–8.10 (1H, m), 10.00 (1H, s). FAB-MS m/z: 1,3,5-triazine-2,4-diamine (14c) Compound 14c was pre- 327 [M+H]+. pared with a yield of 19% from 5a and 13c using a procedure 4-Chloro-N-[2-(methanesulfonyl)phenyl]-1,3,5-triazin- similar to that described for 14a. 1H-NMR (400 MHz, CDCl ) 3 2-amine (13d) To a mixture of 2-(methanesulfonyl) aniline δ: 1.31 (6H, d, J=6.8 Hz), 1.50–1.79 (2H, m), 1.89–2.01 (2H, monohydrochloride 11b (600 mg, 2.89 mmol) and THF (10 mL) m), 2.22–2.79 (11H, m), 2.30 (3H, s), 2.41 (3H, s), 3.19–3.32 were added DIPEA (1.2 mL, 6.89 mmol) and 2,4-dichloro- (1H, m), 3.61–3.74 (2H, m), 3.87 (3H, s), 6.45–6.58 (2H, m), 1,3,5-triazine 12a (880 mg, 5.87 mmol) under ice-cooling. 7.14–7.23 (1H, m), 7.35–7.55 (1H, m), 7.55–7.68 (1H, m), 7.87 After the reaction mixture was stirred at room temperature (1H, dd, J=1.6, 8.0 Hz), 8.04–8.30 (1H, br), 8.53–8.67 (1H, m), Vol. 66, No. 3 (2018) Chem. Pharm. Bull. 259 9.23 (1H, s). ESI-MS m/z: 595 [M+H]+. HR-MS (ESI) m/z: azine-2,4-diamine (14i) Compound 14i was prepared from 595.3171 [M+H]+ (Calcd for C H NOS: 595.3179). 5d, which was obtained from 4d, and 13a using a procedure 30 43 8 3 N-[2-(Methanesulfonyl)phenyl]-N′-{2-methoxy-4-[4- similar to that described for 14a (11% in two steps from (4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}-1,3,5-tri- 4d). 1H-NMR (400 MHz, CDCl ) δ: 1.31 (6H, d, J=6.8 Hz), 3 azine-2,4-diamine (14d) Compound 14d was prepared with 1.46 (3H, t, J=7.0 Hz), 1.53–1.79 (2H, m), 1.88–2.00 (2H, m), a yield of 31% from 5a and 13d using a procedure similar 2.24–2.79 (11H, m), 2.30 (3H, s), 3.18–3.32 (1H, m), 3.60–3.74 to that described for 14a. 1H-NMR (400 MHz, CDCl ) δ: (2H, m), 4.10 (2H, q, J=7.1 Hz), 6.45–6.59 (2H, m), 7.16–7.29 3 1.49–1.79 (2H, m), 1.88–2.01 (2H, m), 2.30 (3H, s), 2.32–2.83 (1H, m), 7.47–7.70 (2H, m), 7.89 (1H, dd, J=1.6, 8.0 Hz), (11H, m), 3.09 (3H, s), 3.62–3.75 (2H, m), 3.88 (3H, s), 8.04–8.20 (1H, m), 8.27–8.48 (1H, br), 8.49–8.60 (1H, m), 6.44–6.61 (2H, m), 7.18–7.29 (1H, m), 7.49–7.71 (2H, m), 7.97 9.22–9.34 (1H, br). ESI-MS m/z: 595 [M+H]+. HR-MS (ESI) (1H, dd, J=1.6, 8.0 Hz) 8.01–8.16 (1H, m), 8.28–8.56 (2H, m), m/z: 595.3180 [M+H]+ (Calcd for C H NOS: 595.3179). 30 43 8 3 9.00 (1H, s). ESI-MS m/z: 553 [M+H]+. HR-MS (ESI) m/z: N-{4-[4-(4-Methylpiperazin-1-yl)piperidin-1-yl]-2-(propan- 553.2701 [M+H]+ (Calcd for C H NOS: 553.2709). 2-yloxy)phenyl}-N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-tri- 27 37 8 3 N-[2-(Ethanesulfonyl)phenyl]-N′-{2-methoxy-4-[4-(4- azine-2,4-diamine (14j) Compound 14j was prepared from methylpiperazin-1-yl)piperidin-1-yl]phenyl}-1,3,5-tri- 5e, which was obtained from 4e, and 13a using a procedure azine-2,4-diamine (14e) Compound 14e was prepared with similar to that described for 14a (9% in two steps from 4e). a yield of 18% from 5a and 13e using a procedure similar 1H-NMR (400 MHz, CDCl) δ: 1.31 (6H, d, J=6.8 Hz), 1.38 3 to that described for 14a. 1H-NMR (400 MHz, CDCl ) δ: (6H, d, J=6.0 Hz), 1.48–1.79 (2H, m), 1.86–2.02 (2H, m), 3 1.27 (3H, t, J=7.4 Hz), 1.49–1.78 (2H, m), 1.89–2.01 (2H, 2.23–2.80 (11H, m), 2.31 (3H, s), 3.18–3.34 (1H, m), 3.59–3.74 m), 2.20–2.80 (11H, m), 2.30 (3H, s), 3.17 (2H, q, J=7.5 Hz), (2H, m), 4.52–4.66 (1H, m), 6.44–6.60 (2H, m), 7.15–7.29 (1H, 3.64–3.75 (2H, m), 3.88 (3H, s), 6.46–6.60 (2H, m), 7.17–7.28 m), 7.50–7.71 (2H, m), 7.89 (1H, dd, J=1.4, 7.8 Hz), 8.05–8.24 (1H, m), 7.43–7.70 (2H, m), 7.92 (1H, dd, J=1.4, 7.8 Hz), (1H, m), 8.30–8.47 (1H, br), 8.49–8.60 (1H, m), 9.19–9.36 (1H, 8.03–8.15 (1H, m), 8.27–8.60 (2H, m), 9.14 (1H, s). ESI-MS br). ESI-MS m/z: 609 [M+H]+. HR-MS (ESI) m/z: 609.3335 m/z: 567 [M+H]+. HR-MS (ESI) m/z: 567.2868 [M+H]+ [M+H]+ (Calcd for C H NOS: 609.3335). 31 45 8 3 (Calcd for C H NOS: 567.2866). N-[2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl]-N′-[2- 28 39 8 3 N-{3-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin- (propane-2-sulfonyl)phenyl]-1,3,5-triazine-2,4-diamine (14k) 1-yl]phenyl}-N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-tri- Compound 14k was prepared with a yield of 39% from azine-2,4-diamine (14f) Compound 14f was prepared with 2-methoxy-4-(4-methylpiperazin-1-yl) aniline15) and 13a using a a yield of 56% from 8 and 13a using a procedure similar to procedure similar to that described for 14a. 1H-NMR (400 MHz, that described for 14a. 1H-NMR (400 MHz, CDCl ) δ: 1.31 CDCl) δ: 1.31 (6H, d, J=6.8 Hz), 2.40 (3H, s), 2.54–2.73 (4H, 3 3 (6H, d, J=6.8 Hz), 1.73–1.97 (4H, m), 2.30 (3H, s), 2.35–2.79 m), 3.16–3.32 (5H, m), 3.89 (3H, s), 6.46–6.60 (2H, m), 7.16–7.29 (11H, m), 3.17–3.32 (1H, m), 3.47–3.58 (2H, m), 3.87 (3H, s), (1H, m), 7.47–7.73 (2H, m), 7.88 (1H, dd, J=1.6, 8.0 Hz), 6.80–7.70 (6H, m), 7.88 (1H, dd, J=1.6, 8.0 Hz), 8.35–8.50 8.04–8.18 (1H, m), 8.26–8.65 (2H, m), 9.29 (1H, s). ESI-MS m/z: (1H, br), 8.53–8.64 (1H, m), 9.26–9.62 (1H, br). ESI-MS m/z: 498 [M+H]+. HR-MS (ESI) m/z: 498.2279 [M+H]+ (Calcd for 581 [M+H]+. HR-MS (ESI) m/z: 581.3020 [M+H]+ (Calcd for C H NOS: 498.2287). 24 32 7 3 C H NOS: 581.3022). N-(4-{Ethyl[2-(4-methylpiperazin-1-yl)ethyl]amino}-2- 29 41 8 3 N-{4-[4-(4-Methylpiperazin-1-yl)piperidin-1-yl]phenyl}- methoxyphenyl)-N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5- N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-triazine-2,4-di- triazine-2,4-diamine (14l) Compound 14l was prepared amine (14g) Compound 14g was prepared with a yield from 10, which was obtained from 9, and 13a using a pro- of 38% from 5b and 13a using a procedure similar to that cedure similar to that described for 14a (16% in two steps described for 14a. 1H-NMR (400 MHz, CDCl ) δ: 1.31 (6H, from 9). 1H-NMR (400 MHz, CDCl ) δ: 1.18 (3H, t, J=7.0 Hz), 3 3 d, J=6.8 Hz), 1.49–1.78 (2H, m), 1.90–2.01 (2H, m), 2.30 (3H, 1.31 (6H, d, J=6.8 Hz), 2.30 (3H, s), 2.34–2.77 (10H, m), s), 2.24–2.86 (11H, m), 3.18–3.31 (1H, m), 3.66–3.79 (2H, 3.18–3.31 (1H, m), 3.32–3.52 (4H, m), 3.87 (3H, s), 6.24–6.38 m), 6.87–7.16 (3H, m), 7.17–7.24 (1H, m), 7.33–7.48 (2H, m), (2H, m), 7.10–7.24 (1H, m), 7.32–7.75 (2H, m), 7.82–8.10 (2H, 7.49–7.71 (1H, br), 7.88 (1H, dd, J=1.6, 8.0 Hz), 8.29–8.47 m), 8.26–8.67 (2H, m), 9.19–9.33 (1H, br). ESI-MS m/z: 569 (1H, br), 8.48–8.61 (1H, m), 9.24–9.43 (1H, br). ESI-MS m/z: [M+H]+. HR-MS (ESI) m/z: 569.3014 [M+H]+ (Calcd for 551 [M+H]+. HR-MS (ESI) m/z: 551.2926 [M+H]+ (Calcd for C H NOS: 569.3022). 28 41 8 3 C H NO S: 551.2917). 1-Nitro-3-(propan-2-ylsulfanyl)benzene (16) A mixture 28 39 8 2 N-{2-Methyl-4-[4-(4-methylpiperazin-1-yl)piperidin- of 1,1′-disulfanediylbis(3-nitrobenzene) 15 (3.00 g, 9.73 mmol) 1-yl]phenyl}-N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-tri- and K CO (2.69 g, 19.5 mmol) in DMF (200 mL) was stirred 2 3 azine-2,4-diamine (14h) Compound 14h was prepared from at room temperature for 2 min. To this mixture were added 5c, which was obtained from 4c, and 13a using a procedure 2-bromopropane (2.01 mL, 21.4 mmol), sodium hydroxymeth- similar to that described for 14a (13% in two steps from anesulfinate (3.45 g, 29.2 mmol) and H O (3 mL), and the reac- 2 4c). 1H-NMR (400 MHz, CDCl ) δ: 1.31 (6H, d, J=6.8 Hz), tion mixture was stirred at room temperature for 2 h. Water 3 1.50–1.80 (2H, m), 1.88–2.02 (2H, m), 2.21–2.87 (11H, m), was added to this mixture, and the resulting slurry was ex- 2.25 (3H, s), 2.30 (3H, s), 3.15–3.33 (1H, m), 3.68–3.80 (2H, tracted with Et O. The organic layer was washed with brine, 2 m), 6.48–7.75 (6H, m), 7.78–7.94 (1H, m), 8.30–8.70 (2H, m), dried over anhydrous MgSO , and concentrated in vacuo to 4 9.24–9.45 (1H, br). ESI-MS m/z: 565 [M+H]+. HR-MS (ESI) give 16 (2.95 g, 77%) as a yellow oil. 1H-NMR (400 MHz, m/z: 565.3077 [M+H]+ (Calcd for C H NO S: 565.3073). CDCl ) δ: 1.32–1.40 (6H, m), 3.45–3.61 (1H, m), 7.41–7.52 (1H, 29 41 8 2 3 N-{2-Ethoxy-4-[4-(4-methylpiperazin-1-yl)piperidin- m), 7.62–7.70 (1H, m), 8.01–8.09 (1H, m), 8.17–8.24 (1H, m). 1-yl]phenyl}-N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-tri- EI-MS m/z: 197 [M]+. 260 Chem. Pharm. Bull. Vol. 66, No. 3 (2018) 1-Nitro-3-(propane-2-sulfonyl)benzene (17) To a solu- 4-(1,4-Dioxa-8-azaspiro[4.5]decan-8-yl)-2-methoxyani- tion of 1-nitro-3-(propan-2-ylsulfanyl) benzene 16 (2.95 g, line (22) Compound 22 was prepared with a yield of 82% 15.0 mmol) in CHCl (60 mL) was added mCPBA (contains from 21 using a procedure similar to that described for 5a. 3 ca. 25% water, 8.60 g, 37.4 mmol), and the reaction mixture 1H-NMR (400 MHz, CDCl ) δ: 1.83–1.94 (4H, m), 3.10–3.22 3 was stirred at room temperature for 2 h, then at 50°C for 12 h. (4H, m), 3.45–3.65 (2H, br), 3.83 (3H, s), 3.99 (4H, s), 6.45 Saturated aqueous NaHCO solution (100 mL) and 5% aque- (1H, dd, J=2.4, 8.4 Hz), 6.55 (1H, d, J=2.4 Hz), 6.63 (1H, d, 3 ous sodium sulfite solution (100 mL) were added to this mix- J=8.4 Hz). ESI-MS m/z: 265 [M+H]+. ture, and the resulting slurry was extracted with CHCl . The N-[4-(1,4-Dioxa-8-azaspiro[4.5]decan-8-yl)-2-methoxy- 3 organic layer was washed with saturated aqueous NaHCO phenyl]-N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-tri- 3 solution and brine, dried over Na SO , and concentrated in azine-2,4-diamine (23) A mixture of 4-chloro-N-[2- 2 4 vacuo to give 17 (3.38 g, 99%) as a white solid. 1H-NMR (propane-2-sulfonyl) phenyl]-1,3,5-triazin-2-amine 13a (1.00 g, (400 MHz, DMSO-d ) δ: 1.19 (6H, d, J=6.8 Hz), 3.57–3.70 3.20 mmol), 4-(1,4-dioxa-8-azaspiro[4.5] decan-8-yl)-2-methoxy- 6 (1H, m), 7.92–8.04 (1H, m), 8.27–8.37 (1H, m), 8.50–8.56 (1H, aniline 22 (845 mg, 3.20 mmol) and DIPEA (0.56 mL, 3.20 mmol) m), 8.56–8.65 (1H, m). Chemical ionization (CI)-MS m/z: 230 in NMP (3.5 mL) was irradiated with microwaves at 120°C [M+H]+. for 40 min. Water was added to this reaction mixture, and the 3-(Propane-2-sulfonyl)aniline (18) To a mixture of resulting solid was filtered and dried to give 23 (1.62 g, 94%). 1-nitro-3-(propane-2-sulfonyl) benzene 17 (3.38 g, 14.3 mmol) 1H-NMR (400 MHz, CDCl) δ: 1.31 (6H, d, J=6.8 Hz), 1.80–1.92 3 and AcOH (35 mL) was added iron powder (2.64 g, 47.2 mmol), (4H, m), 3.18–3.35 (5H, m), 3.88 (3H, s), 4.01 (4H, s), 6.48–6.62 and the reaction mixture was stirred at 80°C for 3 h. To (2H, m), 7.17–7.31 (1H, m), 7.43–7.70 (2H, m), 7.82–7.95 (1H, m), this mixture was added EtOAc; the insoluble material was 8.03–8.18 (1H, m), 8.28–8.64 (2H, m), 9.25–9.32 (1H, br). ESI- removed by filtration, and the filtrate was concentrated in MS m/z: 541 [M+H]+. vacuo. To the residue was added EtOAc, and the insoluble 1-[3-Methoxy-4-({4-[2-(propane-2-sulfonyl)anilino]- material was again removed by filtration. The organic layer 1,3,5-triazin-2-yl}amino)phenyl]piperidin-4-one (24) A was washed with water, saturated aqueous NaHCO solution mixture of N-[4-(1,4-dioxa-8-azaspiro[4.5] decan-8-yl)-2- 3 and brine, dried over Na SO , and concentrated in vacuo. The methoxyphenyl]-N′-[2-(propane-2-sulfonyl) phenyl]-1,3,5- 2 4 residue was purified by silica gel column chromatography triazine-2,4-diamine 23 (2.27 g, 4.20 mmol) and 4 M aqueous (CHCl /MeOH=100 : 0 to 50 : 1) to give 18 (1.79 g, 63%) as a HCl solution (24 mL) in 1,4-dioxane (20 mL) was stirred at 3 pale yellow solid. 1H-NMR (400 MHz, CDCl ) δ: 1.30 (6H, d, 80°C for 2 h. The reaction mixture was concentrated in vacuo, 3 J=6.8 Hz), 3.10–3.26 (1H, m), 3.82–4.08 (2H, br), 6.86–6.96 and saturated aqueous NaHCO solution was added to the 3 (1H, m), 7.13–7.18 (1H, m), 7.19–7.25 (1H, m), 7.25–7.36 (1H, residue. The resulting slurry was extracted with CHCl . The 3 m). EI-MS m/z: 199 [M]+. organic layer was washed with brine, dried over anhydrous 4-Chloro-N-[3-(propane-2-sulfonyl)phenyl]-1,3,5-triazin- MgSO , and concentrated in vacuo. The residue was purified 4 2-amine (19) Compound 19 was prepared with a yield of by silica gel column chromatography (CHCl /MeOH=100 : 0 to 3 92% from 12a and 18 using a procedure similar to that de- 20 : 1) to give 24 (1.49 g, 71%). 1H-NMR (400 MHz, CDCl ) δ: 3 scribed for 13d. 1H-NMR (400 MHz, CDCl ) δ: 1.35 (6H, d, 1.32 (6H, d, J=6.8 Hz), 2.51–2.68 (4H, m), 3.18–3.34 (1H, m), 3 J=6.8 Hz), 3.18–3.33 (1H, m), 7.54–7.65 (1H, m), 7.66–7.74 3.52–3.68 (4H, m), 3.91 (3H, s), 6.51–6.68 (2H, m), 7.17–7.74 (1H, m), 7.76–8.00 (2H, m), 8.01–8.34 (1H, br), 8.49–8.72 (1H, (3H, m), 7.84–7.95 (1H, m), 8.04–8.26 (1H, m), 8.30–8.68 (2H, br). ESI-MS m/z: 313 [M+H]+. m), 9.24–9.38 (1H, m). ESI-MS m/z: 497 [M+H]+. N-{2-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin- N-{2-Methoxy-4-[4-(morpholin-4-yl)piperidin-1-yl]- 1-yl]phenyl}-N′-[3-(propane-2-sulfonyl)phenyl]-1,3,5-tri- phenyl}-N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-tri- azine-2,4-diamine (20) Compound 20 was prepared with a azine-2,4-diamine Trihydrochloride (25) To a solution yield of 36% from 5a and 19 using a procedure similar to that of 1-[3-methoxy-4-({4-[2-(propane-2-sulfonyl) anilino]-1,3,5- described for 14a. 1H-NMR (400 MHz, CDCl ) δ: 1.32 (6H, triazin-2-yl}amino) phenyl] piperidin-4-one 24 (200 mg, 3 d, J=6.8 Hz), 1.51–1.78 (2H, m), 1.88–2.01 (2H, m), 2.30 (3H, 0.403 mmol) in dichloromethane were added morpholine s), 2.33–2.80 (11H, m), 3.12–3.29 (1H, m), 3.63–3.75 (2H, m), (0.14 mL, 1.61 mmol) and sodium triacetoxyborohydride 3.87 (3H, s), 6.48–6.61 (2H, m), 7.16–7.36 (1H, m), 7.45–7.62 (128 mg, 0.604 mmol), and the reaction mixture was stirred (2H, m), 7.85–8.25 (3H, m), 8.30–8.44 (1H, br). ESI-MS m/z: at room temperature for 2 h. Water and saturated aqueous 581 [M+H]+. HR-MS (ESI) m/z: 581.3033 [M+H]+ (Calcd for NaHCO solution were added to the mixture, and the result- 3 C H NOS: 581.3022). ing slurry was extracted with CHCl . The organic layer was 29 41 8 3 3 8-(3-Methoxy-4-nitrophenyl)-1,4-dioxa-8-azaspiro[4.5]- washed with brine, dried over anhydrous MgSO , and concen- 4 decane (21) To a mixture of 4-fluoro-2-methoxy-1-nitro- trated in vacuo. The residue was purified by silica gel column benzene 3a (15 g, 87.7 mmol) and K CO (30.0 g, 217 mmol) chromatography (CHCl /MeOH=100 : 0 to 10 : 1). The obtained 2 3 3 in DMF (150 mL) was added 1,4-dioxa-8-azaspiro[4.5] decane product was dissolved in THF and treated with 4 M HCl in (15 g, 105 mmol), and the reaction mixture was stirred at 70°C EtOAc, and then the mixture was concentrated in vacuo. The overnight. The insoluble material was removed by filtration, residue was treated with acetonitrile, EtOH and water to give and ice water was added to the filtrate. The resulting solid 25 (133 mg, 49%). 1H-NMR (400 MHz, DMSO-d ) δ: 1.15 (6H, 6 was filtered and washed with Et O to give 21 (23.8 g, 92%) d, J=6.8 Hz), 1.82–2.06 (2H, m), 2.18–2.31 (2H, m), 2.78–3.01 2 as a yellow solid. 1H-NMR (400 MHz, CDCl ) δ: 1.78–1.85 (2H, m), 3.01–3.21 (2H, m), 3.29–3.55 (4H, m), 3.80 (3H, s), 3 (4H, m), 3.51–3.59 (4H, m), 3.95 (3H, s), 4.01 (4H, s), 6.33 3.82–4.06 (6H, m), 6.57–6.99 (2H, m), 7.27–7.47 (2H, m), (1H, d, J=2.8 Hz), 6.43 (1H, dd, J=2.4, 9.2 Hz), 8.00 (1H, d, 7.55–7.91 (2H, m), 8.12–8.64 (2H, m), 9.25 (1H, s), 9.51 (1H, s), J=9.2 Hz). ESI-MS m/z: 295 [M+H]+. 11.08–11.35 (1H, br). ESI-MS m/z: 568 [M+H]+. Anal. Calcd