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

Article abstract of DOI:10.1248/cpb.c17-00784

Anaplastic lymphoma kinase (ALK) is a validated therapeutic target for treating echinoderm microtubule- 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.

Full text of DOI:10.1248/cpb.c17-00784

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}-
Nꢀ-[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-
phosmin (NPM) gene and the ALK gene that contains the
Results and Discussion
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)3) and hydrogenation with catalytic palladium
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.
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
The synthesis of 20 is shown in Chart 5. Compound 16
† 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: kazuhiko.iikubo@astellas.com
© 2018 The Pharmaceutical Society of Japan

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Reagents and conditions: (a) 4-piperidone monohydrate monohydrochloride, K2CO3, N,N-dimethylformamide (DMF), 70 or 80°C; (b) 1-methylpiperazine, NaBH(OAc)3,
dichloromethane (DCM) or 1,2-dichloroethane (DCE), rt; (c) H2, 10% Pd/C, EtOH or EtOH/tetrahydrofuran (THF), rt.
Chart 1. Synthesis of Compounds 5a–5e
Reagents and conditions: (a) 1-methyl-4-(piperidin-4-yl)piperazine, K2CO3, DMF, 80°C; (b) H2, 10% Pd/C, EtOH/THF, rt.
Chart 2. Synthesis of Compound 8
14a, showed inhibitory activity against EML4-ALK with IC50
values of 73 and 360nM, 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
carbonyl oxygen atom of Glu1197 in the hinge region (Fig. 2).
Reagents and conditions: (a) N-ethyl-2-(4-methylpiperazin-1-yl)ethanamine,
K2CO3, DMF, 80°C; (b) H2, 10% Pd/C, EtOH, rt.
Therefore, the reduced inhibition of EML4-ALK by 14b and
14c compared to that of 14a could be attributed to the attenua-
tion of the interaction between these compounds and the hinge
Chart 3. Synthesis of Compound 10
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 4M 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 17nM.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

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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, K2CO3, DMF/H2O, rt; (b) mCPBA, CHCl3, rt to 50°C; (c) Fe, AcOH, 80°C; (d) 12a,
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) 4M
HCl aq, 1,4-dioxane, 80°C; (e) morpholine, NaBH(OAc)3, DCM, rt, then 4M HCl/EtOAc, THF, rt.
Chart 6. Synthesis of Compound 25

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Reagents and conditions: (a) 13a, DIPEA, NMP, microwave, 120°C; (b) 4M 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
14f
14g
14h
14i
2-MeO
3-MeO
H
17
56
42
107
NTa
NTa
197
NTa
330
340
93
2-Me
2-EtO
2-i-PrO
14j
210
a: Not tested.
a: Not tested.; EML4-ALK enzyme IC50 value of compound 2 was 0.63nM.18)
Table 2. Structure–Activity Relationships of Compounds 14a, 14d, 14e
and 20
Table 4. Structure–Activity Relationships of Compounds 14a, 14k, 14l,
25 and 29
a: 3HCl salt.; b: Not determined.
a: Not tested.

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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 29nM.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 210nM, 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.

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Selectivitya
Table 5. Kinase Selectivity of 1 and 14a10)
Compound 1
IC50 (nM)
Compound 14a
Kinase
Selectivitya
Kinase
IC50 (nM)
ROS
0.95
1.5
1.5
1.8
1.8
2.0
2.0
3.2
3.3
3.4
4.4
4.6
5.5
9.1
0.63
—
ALK
3.5
5.4
5.8
6.8
8.9
10
—
1.5
1.7
1.9
2.5
2.9
7.7
8.6
9.1
10
ALK
ALK R1275Q
ACK
MET
1.0
1.2
1.2
1.3
1.3
2.1
2.2
2.3
2.9
3.1
3.7
6.1
7.3
7.3
7.3
ALK F1174L
LTK
NPM1-ALK
ROS
NPM1-ALK
ALK R1275Q
TRKA
AXL
ALK F1174L
TNK1
27
FMS
30
YES
32
MER
DDR1
35
TRKC
TRKB
RON
MUSK
EPHA1
JAK2
11
11
11
LCK
An inhibitory assay for a panel of 86 tyrosine kinases, including ALK, was conducted. Kinases with IC50 values 10-fold or less, than that for ALK are shown (minor modifi-
cation of ref. 10). a: The ratio of the IC50 value for each tyrosine kinase relative to ALK.
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 14d. 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 7.9nM, whereas compound tumor growth inhibition at doses of 0.3mg/kg (4% inhibition)
14l resulted in a five-fold loss of inhibitory activity against and 1mg/kg (69% inhibition), and tumor regression at doses
EML4-ALK. In consideration of the inhibitory activity in of 3mg/kg (4% regression), 10mg/kg (45% regression), and
cells, in addition to that in enzymes, compound 14a exhibited 30mg/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 IC50 values 10-fold or less, than that for ALK
We identified 14a (ASP3026) as a potent and selective ALK
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-
dogenously expressing EML4-ALK (Fig. 4). Compound 14a
Experimental
Chemistry 1H-NMR spectra were recorded on JEOL
inhibited the growth of NCI-H2228 cells with an IC50 value of AL400 or Varian 400-MR, and chemical shifts were expressed
65nM.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)
1
Advantage, Waters ultra performance liquid chromatography 3e using a procedure similar to that described for 4a. H-NMR
(UPLC)/ZQ, Waters UPLC/SQD LC/MS system, JEOL GC- (400MHz, CDCl3) δ: 1.41 (6H, d, J=6.4Hz), 1.52–1.68 (2H, m),
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=2.4Hz), 6.42 (1H, dd, J=2.8, 9.6Hz), 7.94 (1H, d, J=9.6Hz).
(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 (2.18g, 6.52mmol) in
benzene 3a (3.00g, 17.5mmol) and K2CO3 (6.10g, 44.1mmol) EtOH (50mL) was added 10% palladium on carbon (wet, con-
in DMF (30mL) was added 4-piperidone monohydrate mono- tains 53% water; 600mg). The reaction mixture was stirred at
hydrochloride (3.20g, 20.8mmol). The reaction mixture was room temperature for 8h 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
1
ic layer was washed with water and brine, dried over Na2SO4, 5a (1.96g, 99%) as a pale purple solid. H-NMR (400MHz,
and concentrated in vacuo. The residue was washed with Et2O CDCl3) δ: 1.62–1.80 (2H, m), 1.86–1.98 (2H, m), 2.22–2.84
to give an ocher solid (3.85g).
(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 (40mL) (1H, dd, J=2.4, 8.0Hz), 6.52 (1H, d, J=2.4Hz), 6.63 (1H, d,
and 1-methylpiperazine (2mL, 18.2mmol). After the mixture J=8.4Hz). Electron ionization (EI)-MS m/z: 304 [M]+.
was stirred for 30min, sodium triacetoxyborohydride (3.90g,
4-[4-(4-Methylpiperazin-1-yl)piperidin-1-yl]aniline (5b)
18.4mmol) was added to the mixture, and the reaction mixture Compound 5b was prepared with a yield of 87% from 4b
1
was stirred at room temperature overnight. Saturated aqueous using a procedure similar to that described for 5a. H-NMR
NaHCO3 solution was added to the mixture, and the result- (400MHz, DMSO-d6) δ: 1.39–1.56 (2H, m), 1.72–1.86 (2H,
ing slurry was extracted with CHCl3. The organic layer was m), 2.07–2.61 (11H, m), 2.13 (3H, s), 3.25–3.46 (2H, m), 4.53
dried over Na2SO4, and concentrated in vacuo. The residue (2H, s), 6.43–6.51 (2H, m), 6.63–6.70 (2H, m). ESI-MS m/z:
was purified by silica gel column chromatography (CHCl3/ 275 [M+H]+.
MeOH/28% aqueous NH3=30:1:0.1 to 15:1:0.1). The result-
2-Methyl-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]-
ing solid was washed with n-hexane to give 4a (3.30g, 56%) aniline (5c) Compound 5c was prepared from 4c using a
1
as a yellow solid. H-NMR (400MHz, CDCl3) δ: 1.52–1.69 procedure similar to that described for 5a. ESI-MS m/z: 289
(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,
d, J=2.0Hz), 6.42 (1H, dd, J=2.0, 9.2Hz), 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
2-Ethoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]-
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 (400MHz, CDCl3) δ: 1.52–1.73 using a procedure similar to that described for 5a. ESI-MS
(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-
piperazine (7) To a solution of 1-fluoro-2-methoxy-4-nitro-
8.06–8.15 (2H, m). ESI-MS m/z: 305 [M+H]+.
1-Methyl-4-[1-(3-methyl-4-nitrophenyl)piperidin-4-yl]- benzene 6 (4.74g, 27.7mmol) in DMF (47mL) were added
piperazine (4c) Compound 4c was prepared with a yield 1-methyl-4-(piperidin-4-yl)piperazine (5.33g, 29.1mmol) and
of 20% from 4-fluoro-2-methyl-1-nitrobenzene 3c using K2CO3 (4.59g, 33.2mmol). The reaction mixture was stirred
a procedure similar to that described for 4a. 1H-NMR at 80°C for 15h. Water was added to this reaction mixture
(400MHz, dimethyl sulfoxide (DMSO)-d6) δ: 1.32–1.47 (2H, under cooling in an ice bath, and the resulting precipitate
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 (8.79g, 95%) as
1
(3H, s), 2.85–3.00 (2H, m), 3.36–3.44 (1H, m), 3.95–4.11 (2H, a yellow solid. H-NMR (400MHz, DMSO-d6) δ: 1.44–1.60
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=8.8Hz), 7.67 (1H, d, J=2.4Hz), 7.82 (1H, dd, J=2.4,
perazine (4d) Compound 4d was prepared with a yield of 8.8Hz). 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]-
1
cedure similar to that described for 4a. H-NMR (400MHz, aniline (8) Compound 8 was prepared with a yield of 94%
CDCl3) δ: 1.50 (3H, t, J=7.0Hz), 1.53–1.67 (2H, m), 1.88–2.01 from 7 using a procedure similar to that described for 5a.
(2H, m), 2.22–2.75 (9H, m), 2.29 (3H, s), 2.86–3.00 (2H, 1H-NMR (400MHz, CDCl3) δ: 1.72–1.94 (4H, m), 2.29 (3H, s),
m), 3.84–3.98 (2H, m), 4.14 (2H, q, J=6.9Hz), 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=2.8Hz), 6.41 (1H, dd, J=2.6, 9.4Hz), 7.97 (1H, d, J=9.2Hz). (2H, m), 6.77 (1H, d, J=8.4Hz). 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-

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zene 3a (900mg, 5.26mmol), N-ethyl-2-(4-methylpiperazin-1-yl)- overnight, saturated aqueous NaHCO3 solution and water were
ethanamine (901mg, 5.26mmol) and K2CO3 (727mg, 5.26mmol) added. The resulting slurry was extracted with EtOAc, and
in DMF (10mL) was stirred at 80°C for 6h. The solvent was the organic layer was washed with water and brine, dried over
concentrated, then water was added to the residue. The slurry Na2SO4, and concentrated in vacuo. The residue was purified
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 MgSO4, and concentrated in to 2:1) and washed with Et2O to give 13d (430mg, 52%)
vacuo. The residue was purified by silica gel column chroma- as a white solid. 1H-NMR (400MHz, CDCl3) δ: 3.10 (3H,
tography (CHCl3 to CHCl3/MeOH/28% aqueous NH3=10/1/0.1) s), 7.31–7.41 (1H, m), 7.67–7.78 (1H, m), 7.97–8.07 (1H, m),
1
to give 9 (730mg, 43%). H-NMR (400MHz, DMSO-d6) δ: 1.14 8.42–8.51 (1H, m), 8.63 (1H, s), 9.48–9.74 (1H, br). FAB-MS
(3H, t, J=6.6Hz), 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-
2-amine (13e) Compound 13e was prepared with a yield of
(1H, m). ESI-MS m/z: 323 [M+H]+.
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
1
from 9 using a procedure similar to that described for 5a and for 13d. H-NMR (400MHz, CDCl3) δ: 1.29 (3H, t, J=7.4Hz),
directly used in the next reaction. ESI-MS m/z: 293 [M+H]+.
3.16 (2H, q, J=7.5Hz), 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, 8.0Hz), 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 (460mg, 3.07mmol) and THF (5mL) was added a mixture
of 2-(propane-2-sulfonyl)aniline13) 11a (600mg, 3.01mmol) 1-yl]phenyl}-N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-tri-
and DIPEA (0.58mL, 3.33mmol) in THF (10mL). After the azine-2,4-diamine (14a, ASP3026) mixture of 5a
N-{2-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-
A
reaction mixture was stirred at room temperature for three (210mg, 0.690mmol) and MsOH (0.13mL, 2.00mmol) in
days, water (60mL) and saturated aqueous NaHCO3 solution EtOH (3mL) was stirred at room temperature for 15min. To
were added. The resulting slurry was extracted with EtOAc, this mixture was added 13a (170mg, 0.543mmol), and the re-
and the organic layer was washed with water and brine, dried action mixture was stirred at 100°C for 2h. After the mixture
over Na2SO4, and concentrated in vacuo. The residue was was cooled to room temperature, water and saturated aque-
purified by silica gel column chromatography (CHCl3) to ous NaHCO3 solution were added. The resulting slurry was
1
give 13a (340mg, 36%) as a white solid. H-NMR (400MHz, extracted with CHCl3, and the organic layer was dried over
CDCl3) δ: 1.32 (6H, d, J=7.2Hz), 3.15–3.30 (1H, m), 7.30–7.38 Na2SO4, then concentrated in vacuo. The residue was puri-
(1H, m), 7.67–7.76 (1H, m), 7.92 (1H, dd, J=1.5, 7.8Hz), fied by silica gel column chromatography (CHCl3/MeOH/28%
8.47–8.53 (1H, m), 8.61 (1H, s), 9.66–10.13 (1H, br). ESI-MS aqueous NH3=50:1:0.1 to 30:1:0.1) to give 14a (180mg,
1
m/z: 313 [M+H]+.
57%). H-NMR (400MHz, CDCl3) δ: 1.31 (6H, d, J=6.8Hz),
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 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),
a
chloro-6-methoxy-1,3,5-triazine 12b (370mg, 2.06mmol) 6.45–6.62 (2H, m), 7.14–7.29 (1H, m), 7.43–7.71 (2H, m), 7.88
and THF (10mL) was added a mixture of 2-(propane-2- (1H, dd, J=1.6, 8.0Hz), 8.02–8.17 (1H, m), 8.27–8.61 (2H, m),
sulfonyl)aniline13) 11a (400mg, 2.01mmol) and DIPEA 9.28 (1H, s). ESI-MS m/z: 581 [M+H]+. High resolution (HR)-
(0.72mL, 4.13mmol) in THF (5mL). After the reaction mix- MS (ESI) m/z: 581.3022 [M+H]+ (Calcd for C29H41N8O3S:
ture was stirred at room temperature overnight and at 70°C for 581.3022).
7h, water (60mL) 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-
(CHCl3) and washed with n-hexane to give 13b (200mg, 29%) yl]-1,3,5-triazine-2,4-diamine (14b) Compound 14b was
1
as a white solid. H-NMR (400MHz, CDCl3) δ: 1.31 (6H, d, prepared with a yield of 50% from 5a and 13b using a pro-
1
J=6.8Hz), 3.13–3.28 (1H, m), 4.06 (3H, s), 7.19–7.38 (1H, m), cedure similar to that described for 14a. H-NMR (400MHz,
7.60–7.74 (1H, m), 7.90 (1H, dd, J=1.6, 7.6Hz), 8.47 (1H, dd, CDCl3) δ: 1.30 (6H, d, J=6.8Hz), 1.50–1.79 (2H, m), 1.88–2.01
J=1.0, 8.2Hz), 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)aniline13) 11a (1H, dd, J=1.6, 8.0Hz), 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:
1
dure similar to that described for 13a. H-NMR (400MHz, 611.3140 [M+H]+ (Calcd for C30H43N8O4S: 611.3128).
DMSO-d6) δ: 1.14 (6H, d, J=6.8Hz), 2.40 (3H, s), 3.44–3.57
N-{2-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-
(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, 8.0Hz), 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
1
4-Chloro-N-[2-(methanesulfonyl)phenyl]-1,3,5-triazin- similar to that described for 14a. H-NMR (400MHz, CDCl3)
2-amine (13d) To a mixture of 2-(methanesulfonyl)aniline δ: 1.31 (6H, d, J=6.8Hz), 1.50–1.79 (2H, m), 1.89–2.01 (2H,
monohydrochloride 11b (600mg, 2.89mmol) and THF (10mL) m), 2.22–2.79 (11H, m), 2.30 (3H, s), 2.41 (3H, s), 3.19–3.32
were added DIPEA (1.2mL, 6.89mmol) 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 (880mg, 5.87mmol) 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, 8.0Hz), 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 C30H43N8O3S: 595.3179).
5d, which was obtained from 4d, and 13a using a procedure
N-[2-(Methanesulfonyl)phenyl]-N′-{2-methoxy-4-[4- similar to that described for 14a (11% in two steps from
1
(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}-1,3,5-tri- 4d). H-NMR (400MHz, CDCl3) δ: 1.31 (6H, d, J=6.8Hz),
azine-2,4-diamine (14d) Compound 14d was prepared with 1.46 (3H, t, J=7.0Hz), 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 (400MHz, CDCl3) δ: (2H, m), 4.10 (2H, q, J=7.1Hz), 6.45–6.59 (2H, m), 7.16–7.29
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, 8.0Hz),
(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, 8.0Hz) 8.01–8.16 (1H, m), 8.28–8.56 (2H, m), m/z: 595.3180 [M+H]+ (Calcd for C30H43N8O3S: 595.3179).
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-
2-yloxy)phenyl}-N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-tri-
553.2701 [M+H]+ (Calcd for C27H37N8O3S: 553.2709).
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 (400MHz, CDCl3) δ: 1.31 (6H, d, J=6.8Hz), 1.38
to that described for 14a. 1H-NMR (400MHz, CDCl3) δ: (6H, d, J=6.0Hz), 1.48–1.79 (2H, m), 1.86–2.02 (2H, m),
1.27 (3H, t, J=7.4Hz), 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=7.5Hz), (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, 7.8Hz), 8.05–8.24
(1H, m), 7.43–7.70 (2H, m), 7.92 (1H, dd, J=1.4, 7.8Hz), (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 C31H45N8O3S: 609.3335).
(Calcd for C28H39N8O3S: 567.2866).
N-[2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl]-N′-[2-
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
1
a yield of 56% from 8 and 13a using a procedure similar to procedure similar to that described for 14a. H-NMR (400MHz,
1
that described for 14a. H-NMR (400MHz, CDCl3) δ: 1.31 CDCl3) δ: 1.31 (6H, d, J=6.8Hz), 2.40 (3H, s), 2.54–2.73 (4H,
(6H, d, J=6.8Hz), 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, 8.0Hz),
6.80–7.70 (6H, m), 7.88 (1H, dd, J=1.6, 8.0Hz), 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 C24H32N7O3S: 498.2287).
C29H41N8O3S: 581.3022).
N-(4-{Ethyl[2-(4-methylpiperazin-1-yl)ethyl]amino}-2-
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
1
1
described for 14a. H-NMR (400MHz, CDCl3) δ: 1.31 (6H, from 9). H-NMR (400MHz, CDCl3) δ: 1.18 (3H, t, J=7.0Hz),
d, J=6.8Hz), 1.49–1.78 (2H, m), 1.90–2.01 (2H, m), 2.30 (3H, 1.31 (6H, d, J=6.8Hz), 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, 8.0Hz), 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 C28H41N8O3S: 569.3022).
C28H39N8O2S: 551.2917).
1-Nitro-3-(propan-2-ylsulfanyl)benzene (16) A mixture
N-{2-Methyl-4-[4-(4-methylpiperazin-1-yl)piperidin- of 1,1′-disulfanediylbis(3-nitrobenzene) 15 (3.00g, 9.73mmol)
1-yl]phenyl}-N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-tri- and K2CO3 (2.69g, 19.5mmol) in DMF (200mL) was stirred
azine-2,4-diamine (14h) Compound 14h was prepared from at room temperature for 2min. To this mixture were added
5c, which was obtained from 4c, and 13a using a procedure 2-bromopropane (2.01mL, 21.4mmol), sodium hydroxymeth-
similar to that described for 14a (13% in two steps from anesulfinate (3.45g, 29.2mmol) and H2O (3mL), and the reac-
1
4c). H-NMR (400MHz, CDCl3) δ: 1.31 (6H, d, J=6.8Hz), tion mixture was stirred at room temperature for 2h. Water
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 Et2O. The organic layer was washed with brine,
m), 6.48–7.75 (6H, m), 7.78–7.94 (1H, m), 8.30–8.70 (2H, m), dried over anhydrous MgSO4, and concentrated in vacuo to
9.24–9.45 (1H, br). ESI-MS m/z: 565 [M+H]+. HR-MS (ESI) give 16 (2.95g, 77%) as a yellow oil. 1H-NMR (400MHz,
m/z: 565.3077 [M+H]+ (Calcd for C29H41N8O2S: 565.3073).
CDCl3) δ: 1.32–1.40 (6H, m), 3.45–3.61 (1H, m), 7.41–7.52 (1H,
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]+.

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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 (2.95g, line (22) Compound 22 was prepared with a yield of 82%
15.0mmol) in CHCl3 (60mL) was added mCPBA (contains from 21 using a procedure similar to that described for 5a.
ca. 25% water, 8.60g, 37.4mmol), and the reaction mixture 1H-NMR (400MHz, CDCl3) δ: 1.83–1.94 (4H, m), 3.10–3.22
was stirred at room temperature for 2h, then at 50°C for 12h. (4H, m), 3.45–3.65 (2H, br), 3.83 (3H, s), 3.99 (4H, s), 6.45
Saturated aqueous NaHCO3 solution (100mL) and 5% aque- (1H, dd, J=2.4, 8.4Hz), 6.55 (1H, d, J=2.4Hz), 6.63 (1H, d,
ous sodium sulfite solution (100mL) were added to this mix- J=8.4Hz). ESI-MS m/z: 265 [M+H]+.
ture, and the resulting slurry was extracted with CHCl3. The
N-[4-(1,4-Dioxa-8-azaspiro[4.5]decan-8-yl)-2-methoxy-
organic layer was washed with saturated aqueous NaHCO3 phenyl]-N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-tri-
solution and brine, dried over Na2SO4, and concentrated in azine-2,4-diamine (23)
A
mixture of 4-chloro-N-[2-
vacuo to give 17 (3.38g, 99%) as a white solid. 1H-NMR (propane-2-sulfonyl)phenyl]-1,3,5-triazin-2-amine 13a (1.00g,
(400MHz, DMSO-d6) δ: 1.19 (6H, d, J=6.8Hz), 3.57–3.70 3.20mmol), 4-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)-2-methoxy-
(1H, m), 7.92–8.04 (1H, m), 8.27–8.37 (1H, m), 8.50–8.56 (1H, aniline 22 (845mg, 3.20mmol) and DIPEA (0.56mL, 3.20mmol)
m), 8.56–8.65 (1H, m). Chemical ionization (CI)-MS m/z: 230 in NMP (3.5mL) was irradiated with microwaves at 120°C
[M+H]+.
3-(Propane-2-sulfonyl)aniline (18) To
for 40min. Water was added to this reaction mixture, and the
mixture of resulting solid was filtered and dried to give 23 (1.62g, 94%).
a
1-nitro-3-(propane-2-sulfonyl)benzene 17 (3.38g, 14.3mmol) 1H-NMR (400MHz, CDCl3) δ: 1.31 (6H, d, J=6.8Hz), 1.80–1.92
and AcOH (35mL) was added iron powder (2.64g, 47.2mmol), (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 3h. 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
material was again removed by filtration. The organic layer 1,3,5-triazin-2-yl}amino)phenyl]piperidin-4-one
was washed with water, saturated aqueous NaHCO3 solution mixture
of N-[4-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)-2-
1-[3-Methoxy-4-({4-[2-(propane-2-sulfonyl)anilino]-
(24)
A
and brine, dried over Na2SO4, and concentrated in vacuo. The methoxyphenyl]-N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-
residue was purified by silica gel column chromatography triazine-2,4-diamine 23 (2.27g, 4.20mmol) and 4M aqueous
(CHCl3/MeOH=100:0 to 50:1) to give 18 (1.79g, 63%) as a HCl solution (24mL) in 1,4-dioxane (20mL) was stirred at
1
pale yellow solid. H-NMR (400MHz, CDCl3) δ: 1.30 (6H, d, 80°C for 2h. The reaction mixture was concentrated in vacuo,
J=6.8Hz), 3.10–3.26 (1H, m), 3.82–4.08 (2H, br), 6.86–6.96 and saturated aqueous NaHCO3 solution was added to the
(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 CHCl3. The
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- MgSO4, and concentrated in vacuo. The residue was purified
2-amine (19) Compound 19 was prepared with a yield of by silica gel column chromatography (CHCl3/MeOH=100:0 to
1
92% from 12a and 18 using a procedure similar to that de- 20:1) to give 24 (1.49g, 71%). H-NMR (400MHz, CDCl3) δ:
1
scribed for 13d. H-NMR (400MHz, CDCl3) δ: 1.35 (6H, d, 1.32 (6H, d, J=6.8Hz), 2.51–2.68 (4H, m), 3.18–3.34 (1H, m),
J=6.8Hz), 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]+.
N-{2-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-
m), 9.24–9.38 (1H, m). ESI-MS m/z: 497 [M+H]+.
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-
1
described for 14a. H-NMR (400MHz, CDCl3) δ: 1.32 (6H, triazin-2-yl}amino)phenyl]piperidin-4-one
24
(200mg,
d, J=6.8Hz), 1.51–1.78 (2H, m), 1.88–2.01 (2H, m), 2.30 (3H, 0.403mmol) in dichloromethane were added morpholine
s), 2.33–2.80 (11H, m), 3.12–3.29 (1H, m), 3.63–3.75 (2H, m), (0.14mL, 1.61mmol) and sodium triacetoxyborohydride
3.87 (3H, s), 6.48–6.61 (2H, m), 7.16–7.36 (1H, m), 7.45–7.62 (128mg, 0.604mmol), 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 2h. Water and saturated aqueous
581 [M+H]+. HR-MS (ESI) m/z: 581.3033 [M+H]+ (Calcd for NaHCO3 solution were added to the mixture, and the result-
C29H41N8O3S: 581.3022).
ing slurry was extracted with CHCl3. The organic layer was
8-(3-Methoxy-4-nitrophenyl)-1,4-dioxa-8-azaspiro[4.5]- washed with brine, dried over anhydrous MgSO4, and concen-
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 (15g, 87.7mmol) and K2CO3 (30.0g, 217mmol) chromatography (CHCl3/MeOH=100:0 to 10:1). The obtained
in DMF (150mL) was added 1,4-dioxa-8-azaspiro[4.5]decane product was dissolved in THF and treated with 4M HCl in
(15g, 105mmol), 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
1
and ice water was added to the filtrate. The resulting solid 25 (133mg, 49%). H-NMR (400MHz, DMSO-d6) δ: 1.15 (6H,
was filtered and washed with Et2O to give 21 (23.8g, 92%) d, J=6.8Hz), 1.82–2.06 (2H, m), 2.18–2.31 (2H, m), 2.78–3.01
1
as a yellow solid. H-NMR (400MHz, CDCl3) δ: 1.78–1.85 (2H, m), 3.01–3.21 (2H, m), 3.29–3.55 (4H, m), 3.80 (3H, s),
(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=2.8Hz), 6.43 (1H, dd, J=2.4, 9.2Hz), 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=9.2Hz). ESI-MS m/z: 295 [M+H]+.
11.08–11.35 (1H, br). ESI-MS m/z: 568 [M+H]+. Anal. Calcd

Vol. 66, No. 3 (2018)
Chem. Pharm. Bull.
261
for C28H37N7O4S.2.8HCl.H2O: C, 48.89; H, 6.13; N, 14.25; S, 8.47–8.60 (1H, m), 9.31 (1H, s). ESI-MS m/z: 580 [M+H]+.
4.66; Cl, 14.43. Found: C, 48.62; H, 6.05; N, 14.14; S, 4.73; Cl, HR-MS (ESI) m/z: 580.3077 [M+H]+ (Calcd for C30H42N7O3S:
14.43.
tert-Butyl
580.3069).
4-[3-Methoxy-4-({4-[2-(propane-2-sulfonyl)-
In Vitro Kinase Inhibitory Assay EML4-ALK variant
anilino]-1,3,5-triazin-2-yl}amino)phenyl]piperidine-1-car- 1 protein was isolated from Ba/F3 cells transformed with
boxylate (27)
mixture of 4-chloro-N-[2-(propane-2- EML4-ALK. Kinase activity was measured using HTRF®
sulfonyl)phenyl]-1,3,5-triazin-2-amine 13a
(700mg, KinEASE™-TK (Cisbio Bioassays, Codolet, France). The final
2.24mmol), tert-butyl 4-(4-amino-3-methoxyphenyl)- concentration of ATP for EML4-ALK variant 1 protein was
piperidine-1-carboxylate17) 26 (686mg, 2.24mmol) and DIPEA 100 µM.
A
(0.47mL, 2.69mmol) in NMP (4mL) was irradiated with
An inhibitory assay for a panel of 86 tyrosine kinases was
microwaves at 120°C for 20min. Water was added to this re- conducted using ATP concentrations that were approximately
action mixture, and the resulting solid was filtered and dried equal to the Km value for each kinase using a TK-enzyme-
in vacuo. This solid was dissolved in EtOAc, and the organic linked immunosorbent assay (ELISA) or Off-chip Mobility
layer was dried over anhydrous MgSO4, then concentrated in Shift Assay (MSA) at Carna Biosciences, Inc. (Kobe, Japan).
vacuo. The residue was purified by silica gel column chro- The percent inhibition of 1 and 14a against kinase activity
matography (n-hexane/EtOAc=100:0 to 50:50) to give 27 was first determined at concentrations of 100 and 1000nM in
1
(960mg, 74%). H-NMR (400MHz, CDCl3) δ: 1.31 (6H, d, a single experiment. IC50 values of 1 and 14a were then deter-
J=6.8Hz), 1.49 (9H, s), 1.53–1.71 (2H, m), 1.77–1.90 (2H, m), mined in the presence of various concentrations of each com-
2.58–2.70 (1H, m), 2.73–2.91 (2H, m), 3.18–3.32 (1H, m), 3.91 pound in three individual experiments. The 86 tyrosine ki-
(3H, s), 4.16–4.37 (2H, m), 6.74–6.78 (1H, m), 6.80–6.86 (1H, nases were as follows: ABL, ACK, ALK, ALK F1174L, ALK
m), 7.21–7.30 (1H, m), 7.53–7.79 (2H, m), 7.86–7.94 (1H, m), R1275Q, ARG, AXL, BLK, BMX, BRK, BTK, CSK, CTK,
8.26 (1H, d, J=8.4Hz), 8.35–8.60 (2H, m), 9.33 (1H, s). ESI- DDR1, DDR2, EGFR, EGFR L858R, EGFR L861Q, EGFR
MS m/z: 583 [M+H]+.
T790M, EGFR T790M/L858R, EPHA1, EPHA2, EPHA3,
N-[2-Methoxy-4-(piperidin-4-yl)phenyl]-N′-[2-(propane-2- EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2,
sulfonyl)phenyl]-1,3,5-triazine-2,4-diamine (28) To
EPHB3, EPHB4, FAK, FER, FES, FGFR1, FGFR2, FGFR3,
a
solution of tert-butyl 4-[3-methoxy-4-({4-[2-(propane-2- FGFR4, FGR, FLT1, FLT3, FLT4, FMS, FRK, FYN, HCK,
sulfonyl)anilino]-1,3,5-triazin-2-yl}amino)phenyl]piperidine-1- HER2, HER4, IGF1R, INSR, IRR, ITK, JAK1, JAK2, JAK3,
carboxylate 27 (980mg, 1.68mmol) in EtOAc (10mL) and KDR, KIT, LCK, LTK, LYNa, LYNb, MER, MET, MUSK,
MeOH (10mL) was added 4M HCl in EtOAc (20mL). After NPM1-ALK, PDGFRα, PDGFRβ, PYK2, RET, RON, ROS,
stirring at room temperature for 1h, the mixture was con- SRC, SRM, SYK, TEC, TIE2, TNK1, TRKA, TRKB, TRKC,
centrated in vacuo. Saturated aqueous NaHCO3 solution was TXK, TYK2, TYRO3, YES, and ZAP70.10)
added to the residue, and the resulting slurry was extracted
In Vitro Cell Growth Assay Ba/F3 cells transformed
with CHCl3. The organic layer was washed with brine, dried with EML4-ALK variant 1 were seeded in 96-well plates at
over anhydrous MgSO4, and concentrated in vacuo to give 2×103 cells per well and treated with various concentrations
28 (840mg, quantitative). 1H-NMR (400MHz, CDCl3) δ: of compounds (14a, 14b and 14k) for 3d, then cell viability
1.31 (6H, d, J=6.8Hz), 1.68–2.30 (4H, m), 2.58–2.71 (1H, m), was measured using the CellTiter-Glo™ Luminescent Cell Vi-
2.74–2.90 (2H, m), 3.17–3.37 (3H, m), 3.91 (3H, s), 6.77–6.82 ability Assay (Promega Corporation, Madison, WI, U.S.A.) or
(1H, m), 6.82–6.90 (1H, m), 7.20–7.29 (1H, m), 7.49–7.83 seeded in 384-well plates at 2.5×102 or 5×102 cells per well,
(2H, m), 7.89 (1H, dd, J=1.6, 8.0Hz), 8.25 (1H, d, J=8.0Hz), and treated with various concentrations of compounds (14e,
8.33–8.48 (1H, br), 8.48–8.62 (1H, m), 8.90–9.70 (1H, br). ESI- 14f, 14i, 25 and 29) for 2d, then cell viability was measured
MS m/z: 483 [M+H]+.
N-[2-Methoxy-4-(1′-methyl[1,4′-bipiperidin]-4-yl)- entific Inc., Waltham, MA, U.S.A.).
phenyl]-N′-[2-(propane-2-sulfonyl)phenyl]-1,3,5-tri- NCI-H2228 cells were seeded in 96-well spheroid plates
azine-2,4-diamine (29)
mixture of N-[2-methoxy-4- (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) at 2×103 cells per
with alamarBlue® Cell Viability Reagent (Thermo Fisher Sci-
A
(piperidin-4-yl)phenyl]-N′-[2-(propane-2-sulfonyl)- well and incubated overnight. Cells were then exposed to 14a
phenyl]-1,3,5-triazine-2,4-diamine 28 (300mg, 0.622mmol), for 5d. Cell viability was measured using the CellTiter-Glo™
1-methylpiperidin-4-one (87µL, 0.746mmol) and sodium Luminescent Cell Viability Assay (Promega Corporation).10)
triacetoxyborohydride (158mg, 0.746mmol) in dichlorometh-
In Vivo NCI-H2228 Xenograft Model10) All experi-
ane (11mL) was stirred at room temperature overnight. Water ments were performed in accordance with the regulation
and saturated aqueous NaHCO3 solution were added to the of the Animal Ethics Committee of Astellas Pharma Inc.
reaction mixture, and the resulting slurry was extracted with NCI-H2228 cells were subcutaneously inoculated into the
CHCl3. The organic layer was washed with brine, dried over flank of male non-obese diabetic-severe combined immuno-
anhydrous MgSO4, and concentrated in vacuo. The residue deficiency (NOD-SCID) mice (NOD.CB17-Prkdcscid/J, Charles
was purified by silica gel column chromatography (CHCl3/ River Laboratories Japan, Inc., Kanagawa, Japan) at 5×106
MeOH/28% aqueous NH3=100:0:0 to 10:1:0.1) to give 29 cells/0.1mL/mouse. 14a was suspended in 0.5% methylcel-
(140mg, 39%). 1H-NMR (400MHz, CDCl3) δ: 1.31 (6H, d, lulose solution and orally administered once daily. Tumor di-
J=6.8Hz), 1.49–2.04 (10H, m), 2.22–2.40 (3H, m), 2.28 (3H, ameter was measured using a caliper, and tumor volume was
s), 2.42–2.55 (1H, m), 2.88–2.99 (2H, m), 3.00–3.12 (2H, m), determined by calculating the volume of an ellipsoid using the
3.18–3.33 (1H, m), 3.89 (3H, s), 6.77–6.83 (1H, m), 6.83–6.92 following formula: length×width2×0.5.
(1H, m), 7.19–7.29 (1H, m), 7.53–7.82 (2H, m), 7.89 (1H, dd,
Computational Modeling of ALK Inhibitors Docking
J=1.6, 8.0Hz), 8.22 (1H, d, J=8.4Hz), 8.34–8.47 (1H, br), simulation of 14a with wild-type ALK was performed using

262
Chem. Pharm. Bull.
Vol. 66, No. 3 (2018)
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the docking software GLIDE (Schrödinger, LLC, New York,
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pound 2 (PDB ID: 2XB719)) was used as a template for the
docking, and hydrogen atoms were added using the modeling
software MOE with the function Protonate3D (Chemical Com-
puting Group Inc., Montreal, Quebec, Canada). The docking
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Acknowledgments The authors thank Tatsuya Niimi and
Masamichi Mori for their helpful support in the preparation
of this manuscript, and Kazuo Kurosawa and Akio Kamikawa
for their support. The authors thank Sadao Kuromitsu, Satoshi
Konagai, Nobuaki Shindoh, Takatoshi Soga, Takashi Furu-
tani and Hideki Sakagami for their contributions to biological
studies. The authors thank the staff of the Division of Analyti-
cal Science Laboratories and Astellas Research Technologies
Co., Ltd. for conducting spectral measurements and elemental
analysis.
Conflict of Interest Kazuhiko Iikubo, Yutaka Kondoh,
Itsuro Shimada and Takahiro Matsuya are inventors of the
following patents: WO 2009/008371 (A1), US 8318702 (B2)
and JP 5233996 (B2). Kazuhiko Iikubo, Itsuro Shimada, Taka-
hiro Matsuya, Kenichi Mori, and Yoko Ueno are employees of
Astellas Pharma Inc. as of the date of submitting this article.
Minoru Okada has no conflict of interest.
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