Design, Synthesis and Biological Evaluation of Novel Nonsteroidal Progesterone Receptor Antagonists Based on Phenylamino-1,3,5-triazine Scaffold

Article abstract of DOI:10.1248/cpb.c19-00094

We report here the development of phenylamino-1,3,5-triazine derivatives as novel nonsteroidal progesterone receptor (PR) antagonists. PR plays key roles in various physiological systems, including the female reproductive system, and PR antagonists are promising candidates for clinical treatment of multiple diseases. By using the phenylamino-1,3,5-triazine scaffold as a template structure, we designed and synthesized a series of 4-cyanophenylamino-1,3,5-triazine derivatives. The synthesized compounds exhibited PR antagonistic activity, and among them, compound 12n was the most potent (IC₅₀=0.30μM); it also showed significant binding affinity to the PR ligand-binding domain. Docking simulation supported the design rationale of the compounds. Our results suggest that the phenylamino-1,3,5-triazine scaffold is a versatile template for development of nonsteroidal PR antagonists and that the developed compounds are promising lead compounds for further structural development of nonsteroidal PR antagonists.

Full text of DOI:10.1248/cpb.c19-00094

566
Chem. Pharm. Bull. 67, 566–575 (2019)
Vol. 67, No. 6
Regular Article
Highlighted Paper selected by Editor-in-Chief
Design, Synthesis and Biological Evaluation of Novel Nonsteroidal
Progesterone Receptor Antagonists Based on Phenylamino-1,3,5-triazine
Scaffold
Kazuma Kaitoh,a Aki Nakatsu,b Shuichi Mori,b Hiroyuki Kagechika,b Yuichi Hashimoto,a and
,a,b
Shinya Fujii*
a Institute for Quantitative Biosciences, The University of Tokyo; 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–0032,
b
Japan: and Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University; 2–3–10 Kanda-
Surugadai, Chiyoda-ku, Tokyo 101–0062, Japan.
Received February 4, 2019; accepted February 25, 2019
We report here the development of phenylamino-1,3,5-triazine derivatives as novel nonsteroidal proges-
terone receptor (PR) antagonists. PR plays key roles in various physiological systems, including the female
reproductive system, and PR antagonists are promising candidates for clinical treatment of multiple diseases.
By using the phenylamino-1,3,5-triazine scaffold as a template structure, we designed and synthesized a se-
ries of 4-cyanophenylamino-1,3,5-triazine derivatives. The synthesized compounds exhibited PR antagonistic
activity, and among them, compound 12n was the most potent (IC50 =0.30µM); it also showed significant
binding affinity to the PR ligand-binding domain. Docking simulation supported the design rationale of the
compounds. Our results suggest that the phenylamino-1,3,5-triazine scaffold is a versatile template for devel-
opment of nonsteroidal PR antagonists and that the developed compounds are promising lead compounds for
further structural development of nonsteroidal PR antagonists.
Key words progesterone receptor; antagonist; multi-template; triazine
In this paper, we report the design and synthesis of a series of
Introduction
Progesterone receptor (PR) is a member of the nuclear re- 2-(4-cyanophenylamino)-4-phenoxy-1,3,5-triazine derivatives
ceptor superfamily of ligand-dependent transcription factors.1) as novel PR antagonists.
PR is expressed in female tissues including uterus, ovary
and breast, and plays essential roles in female reproductive
systems, being associated with the establishment and main-
Results and Discussion
Chemistry In this work, we applied the multi-template
tenance of pregnancy and with alveolar development in the approach. This is based on the fact that the number of
breast.2,3) Recent studies suggest that PR also has a role in the three-dimensional spatial structures (fold structures) of
central nervous system.4–6) PR agonists, including the endo- human proteins is only approximately 1000, which is much
genous agonist progesterone (1), have been used extensively to smaller than the number of human proteins, estimated to be
treat gynecological disorders, as well as in female contracep- 50000–70000.25,26) Therefore, compounds binding to a par-
tion and hormone replacement therapy.7–9) Among nonsteroidal ticular fold structure are likely to serve as ligands for mul-
PR agonists, the investigational compound tanaproget (2) tiple proteins. We have previously employed multi-template
seems to be the most promising compound for clinical use.10,11) structures such as thalidomide27) and diphenyl-X (Ph-X-Ph)
In addition, PR antagonists are potentially useful for the as represented by diphenylmethane28) to obtain various bio-
treatment of various diseases, including hormone-dependent active compounds. Recently, we also demonstrated that the
cancers,12,13) uterine fibroids,14) and endometriosis,15) and also phenylamino-1,3,5-triazine scaffold is a useful core structure
as abortifacients. The steroidal compound mifepristone (3) for development of nuclear receptor modulators, enabling
is the most intensively investigated PR antagonist,16) but has facile structure–activity relationship (SAR) investigation, and
found only limited clinical use as an abortifacient so far. we developed novel inverse agonists of nuclear retinoic acid
Therefore, to assess the potential clinical utility of this class receptor-related orphan receptor-γ (RORγ).29) In the phenyl-
of compounds, further development, especially of potent non- amino-1,3,5-triazine scaffold, the diarylamine is a diphenyl-X
steroidal PR antagonists, is needed. Based on the structure of homolog and is an established multi-template for nuclear re-
the nonsteroidal PR agonist 2, several nonsteroidal PR antago- ceptor ligands. Further, the 4- and 6-positions of 1,3,5-triazine
nists, such as 4 and 5, have been developed.17–19) In addition to are available as points of systematic structural modification.
these tanaproget derivatives, nonsteroidal PR antagonists with In view of these advantages, we chose this scaffold to develop
novel chemotypes distinct from tanaproget (2) have been also novel PR antagonists.
investigated. For example, 3-aryl indole derivatives including
First, we designed the 2-(4-cyanophenylamino)-4-phenoxy-
6 were developed as PR antagonists to treat uterine fibroids,20) 1,3,5-triazine derivatives shown in Fig. 2. The known non-
and other structures such as 7 and 8 have also been reported steroidal PR antagonists illustrated in Fig. 1 suggested that
in recent years21–24) (Fig. 1). In short, novel types of nonsteroi- the cyanoaryl moiety is an important pharmacophore for
dal PR antagonists are of interest as potential drug candidates. PR-modulating activity, and therefore we chose the 4-cyano
*To whom correspondence should be addressed. e-mail: fujiis@iam.u-tokyo.ac.jp
© 2019 The Pharmaceutical Society of Japan

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567
Fig. 1. Structures of Representative Steroidal and Nonsteroidal PR Ligands
Fig. 2. Design Scheme for Novel PR Antagonists Based on the Phenylamino-1,3,5-triazine Scaffold as a Template
Reagents and conditions: (a) NaHCO3, MeOH, 0°C, 74%; (b) R1-C6H4-OH, i-Pr2EtN, CH2Cl2, r.t., 34–79%; (c) 4-CN-C6H4-NH2, i-Pr2EtN, reflux in THF or 100°C in
DMSO, 8–60%.
Chart 1. Synthesis of the Designed Triazine Derivatives 12a–p
group as the substituent on the phenylamino group. In order to groups and compounds 17a and 17b bearing amino groups at
exert inhibitory activity toward nuclear receptors, antagonists the triazine ring were similarly synthesized by means of se-
must be sufficiently bulky to induce the inactive conforma- quential nucleophilic substitutions (Chart 2).
tion of the receptors.30) Our previous results indicated that the
Biological Activity The PR-agonistic and -antagonistic
phenoxy group is large enough for this purpose.29) Further, activities of synthesized triazine derivatives were evaluated
as substituents on the phenoxy and the triazine moiety, we by means of alkaline phosphatase assay using T47D human
chose a set of substituents present in previously developed PR breast carcinoma cell line.31) None of the triazine derivatives
ligands, such as fluoro, methyl, trifluoromethyl, isopropyl, ace- examined induced alkaline phosphatase activity alone (data
tyl, and dimethylamino groups (Fig. 2).
not shown), i.e., they did not possess PR-agonistic activity.
The designed compounds were synthesized in three steps PR-antagonistic activity was examined in terms of the activity
as illustrated in Charts 1 and 2. Synthesis of methoxytriazine of the test compounds in the presence of 1nM progesterone
derivatives bearing various substituents at the phenoxy group (1) (Table 1).
12a–p is summarized in Chart 1. The methoxy group, phe-
All the synthesized triazine derivatives exhibited PR-
noxy group and 4-cyanophenylamino group were successively antagonistic activity with the exception of 12m. In the case of
connected by means of sequential nucleophilic substitutions tanaproget (2)-based PR ligands such as 4, quite small struc-
(Chart 1). The derivatives 15a–g bearing various alkoxy tural modifications can alter the activity, including agonist/an-

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Reagents and conditions: (a) R2-H, NaHCO3, (for 13a and 13b), or R2-H, i-Pr2EtN, CHCl3, 0°C, 34–64%; (b) phenol, i-Pr2EtN, CH2Cl2, r.t., 62–84%; (c) 4-CN-C6H4-NH2,
i-Pr2EtN, DMSO, 100°C, 23–87%; (d) Me2NH or pyrrolidine, i-Pr2EtN, THF2, r.t., 67% (16a), 86% (16b); (e) 4-CN-C6H4-NH2, i-Pr2EtN, DMSO, 160°C, 33% (17a), 44%
(17b).
Chart 2. Synthesis of the Designed Triazine Derivatives 15a–g and 17a,b
Table 1. PR-Antagonistic Activity of Triazine Derivatives in Alkaline Phosphatase Assay
Compound
R1
R2
IC50 (µM)a)
12a
12b
12c
12d
12e
12f
H
OMe
1.2
0.59
0.44
1.2
2-Me
3-Me
4-Me
2-CF3
3-CF3
4-CF3
2-F
0.58
0.71
2.1
12g
12h
12i
1.1
3-F
2.3
12j
4-F
1.3
12k
12l
2-COMe
3-COMe
4-COMe
2-OMe
3-OMe
4-OMe
H
3.9
1.9
12m
12n
12o
12p
15a
15b
15c
15d
15e
15f
>10
0.30
1.3
1.9
OEt
1.3
O-i-Pr
3.0
O-n-Bu
2.5
OPh
1.9
OCH2CF3
OC2H5OMe
OCH2COMe
NMe2
1.7
4.1
15g
17a
17b
7.7
0.64
1.6
N-Pyrrolidinyl
a) Expression of alkaline phosphatase was induced with 1nM progesterone.
tagonist activity switching.17,18) The results suggested that the compounds, with an IC50 value of 0.30µM. Regarding the
2-(4-cyanophenylamino)-4-phenoxy-1,3,5-triazine structure is substituent at the triazine ring (R2 in Table 1), elongation or
favorable for PR-antagonistic activity. Compound 12a exhib- enlargement of the alkoxy group reduced the PR-antagonistic
ited significant PR-antagonistic activity with an IC50 value of potency (15a–g). Amino-substitution at the triazine ring was
1.2 µM. Introduction of a methyl group at the 2- or 3-position acceptable, and dimethylamino derivative 17a exhibited potent
of the phenoxy moiety (12b and 12c) enhanced the PR-antag- activity with an IC50 value of 0.64µM.
onistic potency, whereas introduction at the 4-position did not
Next the binding affinity of selected compounds was
(12d). The effect of introduction of a trifluoromethyl group examined by competitive binding assay using 3H-labelled
(12e–g) was similar to that of a methyl group, and introduc- progesterone ([3H]-P4) and human PR ligand-binding domain
tion of a fluoro group (12h–j) or an acetyl group (12k–m) re- (LBD) (Fig. 3). Compounds 12n and 17a, which showed po-
duced the potency. The 2-position of the phenoxy group seems tent PR-antagonistic activity in alkaline phosphatase assay,
favorable for substitution, and 2-methoxyphenoxy derivative exhibited dose-dependent binding to PR LBD. Although their
12n exhibited the most potent activity among the examined binding potency was lower than that of steroidal PR antagonist

Vol. 67, No. 6 (2019)
Chem. Pharm. Bull.
569
ID: 2W8Y).32) We first investigated docking of the 2-me-
thoxyphenyl derivative 12n, which exhibited the most potent
PR-antagonistic activity among the synthesized compounds.
Figure 4A shows the docking model of compound 12n with
the PR-LBD, superimposed on the co-crystal structure of 3. In
the calculated structure, the cyano group of 12n interacts with
Gln725 and Arg766; these residues interact with the 3-car-
bonyl group of mifepristone (3). The 2-methoxyphenyl group
occupied the hydrophobic region of the PR LBD, where the
D-ring and 18-methyl group of mifepristone (3) are located.
The methoxytriazine moiety of 12n was placed near helix 12
(H12) of PR, where the 11β-dimethylaminophenyl group of 3
is located in the crystal structure. The ligand-induced folding
of H12 is essential for transcriptional activity of nuclear recep-
tors,30) and the dimethylaminophenyl group plays a key role in
PR-antagonism of mifepristone (3) through steric interference
with Met909 in H12, leading to destabilization of the helix.32)
We also calculated the binding forms of compounds 12a and
17a. Figure 4B shows the calculated structures of triazine
derivatives bound to PR-LBD as a superimposition of three
derivatives including 12n. The calculation suggested that com-
pounds 12a and 17a could bind to PR similarly to 12n. The
dimethylamino group of 17a was located in the same position
as the methoxy group of 12n, suggesting that the dimethyl-
aminotriazine moiety of 17a, as well as the dimethylamino
group of mifepristone (3), could interact with Met909 of PR to
exert PR-antagonistic activity. As for the phenoxy moiety, the
calculation suggested that the unsubstituted phenoxy group of
12a and 17a is located in the hydrophobic cavity of PR-LBD,
and the 2-methoxyphenyl group of 12n is better able to occupy
the cavity effectively. This could be one reason why substitu-
tion of the 2-position of the phenoxy group was favorable for
PR-antagonistic activity of the designed triazine derivatives.
Overall, the simulations suggested that the designed com-
pounds function as PR antagonists in the expected manner,
i.e., the cyanophenyl group serves as a key pharmacophore
to form hydrogen bonds to the PR-LBD, and the bulkiness of
the phenoxytriazine moiety is appropriate to inhibit the tran-
scriptional activity of the receptor. The calculations also sug-
gest avenues for further structural development. The designed
compounds formed hydrogen bonds to Gln725 and Arg766
in a similar manner to mifepristone (3), but mifepristone (3)
forms an additional hydrogen bond; the 17β-hydroxyl on the
D-ring forms a water-mediated hydrogen bond to Asn719.32)
Further structural optimization especially at the phenoxy
moiety that corresponds to the D-ring of mifepristone (3) may
lead to more potent PR-antagonistic potency (Fig. 4).
Fig. 3. Competitive Binding Assay of Compounds 12n and 17a Using
Human PR LBD and [3H]-P4
The concentration of [3H]-P4 was 4.0×10−9 M.
Fig. 4. Docking Models of Triazine Derivatives with the hPR-LBD
(PDB ID: 2W8Y) Obtained with the Docking Program AutoDock33)
(A) Superimposition of PR LBD binding to mifepristone (3) (carbons in gray)
and docking model of 12n (carbons in green). The blue ribbon represents helix
12 of PR in the crystal structure. The protein surface is indicated by a gray mesh.
(B) Superimposition of binding models of compounds 12a (carbon in white), 12n
(carbon in green), and 17a (carbon in pink) at the hPR-LBD. (Color figure can be
accessed in the online version.)
Conclusion We set out to develop a novel type of nonste-
roidal PR antagonists by utilizing the 2-(4-cyanophenylamino)-
4-phenoxy-1,3,5-triazine scaffold as a template structure.
Among the compounds synthesized, 12n exhibited most
mifepristone (3), this seems reasonable, because 3 exhibits potent PR-antagonistic activity, with reasonable binding af-
subnanomolar potency in the alkaline phosphatase assay17,18)
;
finity to the PR-LBD. Docking simulations suggested that the
thus the binding affinity of triazine derivatives was consistent 2-(4-cyanophenylamino)-4-phenoxy-1,3,5-triazine structure is
with the results of alkaline phosphatase assay. The binding a promising scaffold for PR antagonists, i.e., the cyanophenyl
data indicate that the developed compounds function as PR group functions as a key pharmacophore forming hydrogen
antagonists through binding to the PR-LBD.
bonds to the PR-LBD, and the bulkiness of the phenoxytri-
Docking Simulations In order to estimate the binding azine moiety is appropriate to inhibit the transcriptional activ-
mode of the developed triazine derivatives and to investigate ity of the receptor. The docking simulations also suggested
the SAR, we conducted docking simulations, using the co- avenues for further exploration. The compounds developed
crystal structure of the hPR-LBD with mifepristone (3) (PDB in this study should be useful lead compounds for further

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1
structural development of PR antagonists and are expected to tals. H-NMR (500MHz, CDCl3) δ: 7.22 (2H, d, J=8.0Hz),
expand the pharmaceutical potential of PR modulation.
7.05 (2H, td, J=3.2Hz, 5.6Hz), 4.02 (3H, s), 2.37 (3H, s).
2-Chloro-4-methoxy-6-(2-trifluoromethylphenoxy)-1,3,5-
triazine (11e)
Chemistry
General All reagents were purchased from Sigma-
Purification with silica gel column chromatography (elu-
Aldrich Chemicals Co. (U.S.A.), Tokyo Kasei Co. (Japan), ent: n-hexane–ethyl acetate 5:1) gave 11e (63%) as a white
1
Wako Pure Chemical Industries, Ltd. (Japan), and Kanto solid. H-NMR (500MHz, CDCl3) δ: 7.74 (1H, d, J=7.4Hz),
Chemical Co., Inc. (Japan) Flash column chromatography 7.65 (1H, t, J=7.7Hz), 7.43 (1H, t, J=7.7Hz), 7.27 (1H, d,
was performed on silica gel 60 Kanto Kagaku (40–100µm). J=8.0Hz), 4.02 (3H, s).
1H-NMR spectra were recorded on a JEOL JNM-GX 500
2-Chloro-4-methoxy-6-(3-trifluoromethylphenoxy)-1,3,5-
spectrometer at 500MHz. The following abbreviations are triazine (11f)
used for spin multiplicity: s=singlet, d=doublet, t=triplet,
Purification with silica gel column chromatography (eluent:
q=quartet, m=multiplet, br=broad. Melting points (mp) n-hexane–ethyl acetate 10:1) gave 11f (39%) as colorless crys-
1
were determined by using a MP-J3 melting point apparatus tals. H-NMR (500MHz, CDCl3) δ: 7.58 (1H, d, J=4.0Hz),
(Yanaco, Japan). Mass spectral data were obtained with a 7.57 (1H, t, J=6.9Hz), 7.48 (1H, s), 7.39 (1H, m), 4.03 (3H, s).
Bruker microTOF mass spectrometer. Elemental analyses were
2-Chloro-4-methoxy-6-(4-trifluoromethylphenoxy)-1,3,5-
determined by using a MT-6 CHN CORDER analyzer (Ya- triazine (11g)
naco, Japan), and results were within 0.4% of the theoretical
values.
2,4-Dichloro-6-methoxy-1,3,5-triazine
chloride (9.23g, 50.0mmol) was added to a solution of sodium (2H, d, J=8.0Hz), 4.05 (3H, s).
hydrogen carbonate (4.20g, 50.0mmol) in methanol (200mL)
at 0°C. The mixture was stirred for 20min at 0°C, then (11h)
poured into water and extracted with ethyl acetate. The organ-
ic layer was washed with brine, dried over sodium sulfate and (59%) as colorless crystals. H-NMR (500MHz, CDCl3) δ:
concentrated. The residue was purified by recrystallization 7.29 (1H, m), 7.26–7.18 (3H, m), 4.02 (3H, s).
Purification with silica gel column chromatography (eluent:
n-hexane–ethyl acetate 10:1) gave 11g (40%) as a white solid.
(10) Cyanuric 1H-NMR (500MHz, CDCl3) δ: 7.72 (2H, d, J=8.6Hz), 7.32
2-(2-Fluorophenoxy)-4-chloro-6-methoxy-1,3,5-triazine
Recrystallization from n-hexane–chloroform gave 11h
1
from n-hexane to afford 6.67g (74%) of 2 as colorless crystals.
2-(3-Fluorophenoxy)-4-chloro-6-methoxy-1,3,5-triazine
(11i)
Recrystallization from n-hexane–chloroform gave 11i (53%)
1H-NMR (500MHz, CDCl3) δ: 4.14 (3H, s).
General Procedure for the Synthesis of Compounds
1
11a–11p Phenol (ca. 500mg, 1.0eq) in dichloromethane as colorless crystals. H-NMR (500MHz, CDCl3) δ: 7.40 (1H,
(5mL) and N,N-diisopropylethylamine (1.0eq) in dichloro- td, J=6.7, 8.3Hz), 7.03–6.98 (3H, m), 4.04 (3H, s).
methane (5mL) were added dropwise to a solution of 2 (1.0eq)
in dichloromethane (10mL). The mixture was stirred for (11j)
30min at room temperature, washed with aqueous hydrochlo-
ric acid and brine, dried over sodium sulfate and concentrated. as colorless crystals. H-NMR (500MHz, CDCl3) δ: 7.16–7.10
The residue was purified by silica gel column chromatography (4H, m), 7.48 (1H, s), 4.03 (3H, s).
2-(4-Fluorophenoxy)-4-chloro-6-methoxy-1,3,5-triazine
Recrystallization from n-hexane–chloroform gave 11j (68%)
1
or recrystallization.
2-Chloro-4-methoxy-6-phenoxy-1,3,5-triazine (11a)
Purification with silica gel column chromatography (eluent:
2-(2-Acetylphenoxy)-4-chloro-6-methoxy-1,3,5-triazine
(11k)
Purification with silica gel column chromatography (eluent:
n-hexane–ethyl acetate 10:1) gave 11a (69%) as a white solid. n-hexane–ethyl acetate 3:1) gave 11k (34%) as a pale yellow
1
1H-NMR (500MHz, CDCl3) δ: 7.44 (2H, t, J=7.2Hz), 7.30 solid. H-NMR (500MHz, CDCl3) δ: 7.88 (1H, dd, J=1.4Hz,
(1H, t, J=7.4Hz), 7.18 (2H, dq, J=1.5, 8.9Hz), 4.02 (3H, s).
7.7Hz), 7.61 (1H, td, J=1.7Hz, 7.7Hz), 7.42 (1H, t, J=7.7Hz),
2-Chloro-4-methoxy-6-(2-methylphenoxy)-1,3,5-triazine 7.22 (1H, d, J=8.6Hz), 4.02 (3H, s), 2.54 (3H, s).
(11b)
2-(3-Acetylphenoxy)-4-chloro-6-methoxy-1,3,5-triazine
Purification with silica gel column chromatography (elu- (11l)
ent: n-hexane–ethyl acetate 10:1) gave 11b (74%) as a white
solid. H-NMR (500MHz, CDCl3) δ: 7.28 (1H, dd, J=1.7, ent: n-hexane–ethyl acetate 6:4) gave 11l (79%) as a yellow
Hz, 7.4Hz), 7.24 (1H, td, J=1.7Hz, 7.3Hz), 7.21 (1H, td, solid. H-NMR (500MHz, CDCl3) δ: 7.90 (1H, d, J=6.9Hz),
Purification with silica gel column chromatography (elu-
1
1
J=1.3Hz, 7.3Hz), 7.07 (1H, dd, J=1.4Hz, 7.7Hz), 4.00 (3H, 7.78 (1H, t, J=1.7Hz), 7.55 (1H, t, J=7.7Hz), 7.39 (1H, dt,
s), 2.19 (3H, s).
J=1.4Hz, 8.0Hz), 4.03 (3H, s), 2.62 (3H, s).
2-(4-Acetylphenoxy)-4-chloro-6-methoxy-1,3,5-triazine
(11m)
2-Chloro-4-methoxy-6-(3-methylphenoxy)-1,3,5-triazine
(11c)
Purification with silica gel column chromatography (eluent:
Recrystallization from n-hexane–chloroform gave 11 m
1
n-hexane–ethyl acetate 5:1) gave 11c (73%) as a white solid. (53%) as colorless crystals. H-NMR (500MHz, CDCl3) δ:
1H-NMR (500MHz, CDCl3) δ: 7.31 (1H, td, J=1.1Hz, 7.4Hz), 8.06 (2H, dt, J=2.3Hz, 9.4Hz), 7.29 (2H, dt, J=2.3Hz,
7.10 (1H, d, J=7.4Hz), 6.97 (1H, s), 6.96 (1H, d, J=6.9Hz), 9.2Hz), 4.04 (3H, s), 2.63 (3H, s).
4.02 (3H, s), 2.39 (3H, s).
2-Chloro-4-methoxy-6-(2-methoxyphenoxy)-1,3,5-triazine
2-Chloro-4-methoxy-6-(4-methylphenoxy)-1,3,5-triazine (11n)
(11d)
Purification with silica gel column chromatography (eluent:
Purified by silica gel column chromatography (eluent: n- n-hexane–ethyl acetate 5:1) gave 11n (70%) as a white solid.
hexane–ethyl acetate 10:1) gave 11d (68%) as colorless crys- 1H-NMR (500MHz, CDCl3) δ: 7.27 (1H, t, J=7.7Hz), 7.14

Vol. 67, No. 6 (2019)
Chem. Pharm. Bull.
571
(1H, dd, J=1.4Hz, 7.7Hz), 7.01 (1H, t, J=8.0Hz), 6.99 (1H, t,
J=7.7Hz), 4.00 (3H, s), 3.79 (3H, s).
2-Chloro-4-methoxy-6-(3-methoxyphenoxy)-1,3,5-triazine
(11o)
2-(4-Cyanoanilino)-4-methoxy-6-(4-methylphenoxy)-1,3,5-
triazine (12d)
Method B. Recrystallization from methanol gave 12d
(41%) as colorless crystals. mp 189.9–191.2°C; 1H-NMR
Purification with silica gel column chromatography (eluent: (500MHz, CDCl3) δ: 7.52 (4H, br), 7.41 (1H, s), 7.24 (2H, d,
n-hexane–ethyl acetate 3:1) gave 11o (50%) as a yellow solid. J=8.0Hz), 7.08 (2H, td, J=3.4Hz, 5.7Hz), 4.02 (3H, s), 2.41
1H-NMR (500MHz, CDCl3) δ: 7.33 (1H, t, J=8.0Hz), 6.84 (3H, s). HR-MS (ESI-TOF) m/z: 332.1140 ([M−H]− Calcd for
(1H, dd, J=2.6Hz, 8.3Hz), 6.77 (1H, dd, J=2.3Hz, 7.4Hz), C18H15N5O2 332.1142). Anal. Calcd for C18H15N5O2: C, 64.86;
6.72 (1H, t, J=2.3Hz), 4.02 (3H, s), 3.82 (3H, s).
2-Chloro-4-methoxy-6-(4-methoxyphenoxy)-1,3,5-triazine
(11p)
H, 4.54; N, 21.01. Found: C, 64.80; H, 4.67; N, 20.99.
2-(4-Cyanoanilino)-4-methoxy-6-(2-trifluoromethylphenoxy)-
1,3,5-triazine (12e)
Recrystallization from n-hexane–ethyl acetate gave 11p
Method A. Recrystallization from methanol gave 12e (53%)
1
1
(67%) as white crystals. H-NMR (500MHz, CDCl3) δ: 7.09 as colorless crystals. mp 196.0–197.2°C; H-NMR (500MHz,
(2H, td, J=3.8Hz, 6.3Hz), 6.93 (2H, td, J=4.0Hz, 6.4Hz), CDCl3) δ: 7.75 (1H, d, J=8.0Hz), 7.65 (1H, t, J=7.7Hz), 7.53
4.02 (3H, s), 3.83 (3H, s).
(5H, br), 7.44 (1H, t, J=7.4Hz), 7.29 (1H, d, J=8.6Hz), 4.01
General Procedure for the Synthesis of Compounds (3H, s). HR-MS (ESI-TOF) m/z: 386.0862 ([M−H]− Calcd for
12a–12p [Method A] To a solution of a compound (11a, C18H12F3N5O2 386.0859). Anal. Calcd for C18H12F3N5O2: C,
11e–11j, ca. 250mg, 1.0eq) in tetrahydrofuran (THF) (1.0mL) 55.82; H, 3.12; N, 18.08. Found: C, 55.81; H, 3.40; N, 18.09.
were added 4-aminobenzonitrile (2.0eq) and N,N-diisopro-
2-(4-Cyanoanilino)-4-methoxy-6-(3-trifluoromethylphenoxy)-
pylethylamine (2.0eq) at room temperature. The mixture was 1,3,5-triazine (12f)
refluxed for 5h, then poured into dilute hydrochloric acid and
Method A. Recrystallization from methanol gave 12f (16%)
extracted with ethyl acetate. The organic layer washed with as a white solid. mp 192.5–193.8°C; 1H-NMR (500MHz,
brine, dried over sodium sulfate and concentrated. The residue CDCl3) δ: 7.63–7.39 (9H, m), 4.02 (3H, s). HR-MS (ESI-TOF)
was purified by recrystallization. [Method B] To a solution of m/z: 386.0878 ([M−H]− Calcd for C18H12F3N5O2 386.0859).
a compound (11b–11d, 11k) ca. 250mg, 1.0eq) in dimethyl Anal. Calcd for C18H12F3N5O2: C, 55.82; H, 3.12; N, 18.08.
sulfoxide (DMSO) (1.0mL) were added 4-aminobenzonitrile Found: C, 55.91; H, 3.31; N, 18.10.
(2eq) and N,N-diisopropylethylamine (2.0eq) at room tem-
perature. The mixture was stirred for 3h at 100°C, and then 1,3,5-triazine (12g)
poured into chloroform. The organic layer was washed with
Method A. Recrystallization from methanol gave 12g (14%)
brine, dried over sodium sulfate and concentrated. The residue as colorless crystals. mp 200.2–202.0°C; H-NMR (500MHz,
was purified by recrystallization.
CDCl3) δ: 7.73 (2H, d, J=8.6Hz), 7.56 (4H, br), 7.42 (1H, s),
2-(4-Cyanoanilino)-4-methoxy-6-(4trifluoromethylphenoxy)-
1
2-(4-Cyanoanilino)-4-methoxy-6-phenoxy-1,3,5-triazine 7.33 (2H, d, J=8.0Hz), 4.02 (3H, s). HR-MS (ESI-TOF) m/z:
(12a)
386.0871 ([M−H]− Calcd for C18H12F3N5O2 386.0859). Anal.
Method A. Recrystallization from methanol gave 12a (50%) Calcd for C18H12F3N5O2: C, 55.82; H, 3.12; N, 18.08. Found: C,
1
as colorless crystals. mp 197.7–198.5°C; H-NMR (500MHz, 55.74; H, 3.33; N, 18.13.
CDCl3) δ: 7.53 (4H, br), 7.46 (2H, t, J=8.0Hz), 7.33 (1H,
2-(4-Cyanoanilino)-4-(2-fluorophenoxy)-6-methoxy-1,3,5-
t, J=7.2Hz), 7.20 (2H, d, J=7.4Hz), 3.49 (3H, s). High triazine (12h)
resolution (HR)-MS (electrospray ionization-time of flight
Method A. Recrystallization from methanol gave 12h
(ESI-TOF)) m/z: 318.0974 ([M−H]− Calcd for C17H13N5O2: (31%) as colorless crystals. mp 204.5–206.0°C; 1H-NMR
318.0986). Anal. Calcd for C17H13N5O2: C, 63.94; H, 4.10; N, (500MHz, CDCl3) δ: 7.52 (5H, br), 7.32–7.20 (4H, m), 4.02
21.93. Found: C, 63.99; H, 4.38; N, 21.91.
2-(4-Cyanoanilino)-4-methoxy-6-(2-methylphenoxy)-1,3,5- for C17H12FN5O2 336.0891). Anal. Calcd for C17H12FN5O2: C,
(3H, s). HR-MS (ESI-TOF) m/z: 336.0896 ([M−H]− Calcd
triazine (12b)
60.53; H, 3.59; N, 20.76. Found: C, 60.49; H, 3.84; N, 20.71.
2-(4-Cyanoanilino)-4-(3-fluorophenoxy)-6-methoxy-1,3,5-
Method B. Recrystallization from methanol gave 12b (33%)
as colorless crystals. mp 170.3–171.2°C; H-NMR (500MHz, triazine (12i)
1
CDCl3) δ: 7.55 (1H, s), 7.48 (4H, br), 7.29 (1H, t, J=8.9Hz),
Method A. Recrystallization from methanol gave 12i (8%)
7.27 (1H, d, J=8.0Hz), 7.24 (1H, t, J=7.2Hz), 7.10 (1H, d, as a white solid. mp 185.8–187.0°C; 1H-NMR (500MHz,
J=8.0Hz), 4.02 (3H, s), 2.21 (3H, s). HR-MS (ESI-TOF) m/z: CDCl3) δ: 7.56 (4H, br), 7.48 (1H, s), 7.41 (1H, q, J=7.6Hz),
332.1160 ([M−H]− Calcd for C18H15N5O2: 332.1142). Anal. 7.05 (1H, t, J=8.0Hz), 7.01 (1H, d, J=8.0Hz), 6.97 (1H, dt,
Calcd for C18H15N5O2: C, 64.86; H, 4.54; N, 21.01. Found: C, J=2.3, 9.2Hz), 4.02 (3H, s). HR-MS (ESI-TOF) m/z: 336.0887
64.58; H, 4.61; N, 21.03.
2-(4-Cyanoanilino)-4-methoxy-6-(3-methylphenoxy)-1,3,5- C17H12FN5O2: C, 60.53; H, 3.59; N, 20.76. Found: C, 60.51; H,
([M−H]− Calcd for C17H12FN5O2 336.0891). Anal. Calcd for
triazine (12c)
3.93; N, 20.67.
Method B. Recrystallization from methanol gave 12c (18%)
2-(4-Cyanoanilino)-4-(4-fluorophenoxy)-6-methoxy-1,3,5-
as a white solid. mp 182.0–183.5°C; 1H-NMR (500MHz, triazine (12j)
CDCl3) δ: 7.49 (5H, br), 7.33 (1H, t, J=7.7Hz), 7.14 (1H, d,
Method A. Recrystallization from methanol gave 12j (60%)
1
J=7.4Hz), 7.00 (1H, d, J=8.6Hz), 7.00 (1H, s), 4.02 (3H, s), as colorless crystals. mp 210.8–211.6°C; H-NMR (500MHz,
2.40 (3H, s). HR-MS (ESI-TOF) m/z: 332.1140 ([M−H]− Calcd CDCl3) δ: 7.56 (4H, br), 7.40 (1H, s), 7.18–7.11 (4H, m), 4.02
for C18H15N5O2 332.1142). Anal. Calcd for C18H15N5O2: C, (3H, s). HR-MS (ESI-TOF) m/z: 336.0894 ([M−H]− Calcd
64.86; H, 4.54; N, 21.01. Found: C, 65.00; H, 4.75; N, 21.03.
for C17H12FN5O2 336.0891). Anal. Calcd for C17H12FN5O2: C,
60.53; H, 3.59; N, 20.76. Found: C, 60.51; H, 3.88; N, 20.76.

572
Chem. Pharm. Bull.
Vol. 67, No. 6 (2019)
2-(2-Acetylphenoxy)-4-(4-cyanoanilino)-6-methoxy-1,3,5- (40mL) at 0°C. The mixture was stirred for 25min at 0°C,
triazine (12k) then poured into water and extracted with ethyl acetate. The
Method B. Recrystallization from methanol gave 12k (43%) organic layer was washed with brine, dried over sodium sul-
1
as colorless crystals. mp 200.8–202.5°C; H-NMR (500MHz, fate and concentrated. The residue was purified by recrystal-
CDCl3) δ: 7.89 (1H, d, J=7.4Hz), 7.70 (1H, s), 7.61 (1H, t, lization from n-hexane to afford 493mg (2.54mmol, 25%) of
1
J=7.7Hz), 7.51 (4H, br), 7.43 (1H, t, J=7.4Hz), 7.23 (1H, 13a as colorless crystals. H-NMR (500MHz, CDCl3) δ: 4.57
d, J=8.0Hz), 3.99 (3H, s), 2.54 (3H, s). HR-MS (ESI-TOF) (2H, q, J=7.3Hz), 1.47 (3H, t, J=7.2 Hz).
m/z: 360.1091 ([M−H]− Calcd for C19H15N5O3 360.1091). Anal.
2,4-Dichloro-6-isopropoxy-1,3,5-triazine (13b) Cyanu-
Calcd for C19H15N5O3: C, 63.15; H, 4.18; N, 19.38. Found: C, ric chloride (1.85g, 10.0mmol) was added to a solution of
62.97; H, 4.39; N, 19.21. sodium hydrogen carbonate (858mg, 10.2mmol, 1.0eq) in
2-(3-Acetylphenoxy)-4-(4-cyanoanilino)-6-methoxy-1,3,5- 2-propanol (40mL) at room temperature. The mixture was
triazine (12l) stirred for 50min at room temperature, then poured into
Method A. Recrystallization from methanol gave 12l (46%) water and extracted with ethyl acetate. The organic layer was
1
as colorless crystals. mp 205.7–206.4°C; H-NMR (500MHz, washed with brine, dried over sodium sulfate and concen-
CDCl3) δ: 7.90 (1H, d, J=7.4Hz), 7.59 (1H, s), 7.56 (1H, t, trated. The residue was purified by silica gel column chro-
J=7.7Hz), 7.55 (5H, br), 7.41 (1H, dd, J=1.1Hz, 8.0Hz), matography (eluent: n-hexane–ethyl acetate 10:1) to afford
1
4.02 (3H, s), 2.63 (3H, s). HR-MS (ESI-TOF) m/z: 360.1091 1.32g (6.35mmol, 64%) of 13b as a colorless oil. H-NMR
([M−H]− Calcd for C19H15N5O3 360.1091). Anal. Calcd for (500MHz, CDCl3) δ: 4.50 (2H, t, J=6.6Hz), 5.47–5.37 (1H,
C19H15N5O3: C, 63.15; H, 4.18; N, 19.38. Found: C, 63.01; H, m), 1.44 (6H, d, J=6.3 Hz).
4.50; N, 19.15.
2-(4-Acetylphenoxy)-4-(4-cyanoanilino)-6-methoxy-1,3,5- 13c–13g Alcohol or phenol (1.0eq) in chloroform (5mL) and
triazine (12m) N,N-diisopropylethylamine (1eq) in chloroform (5mL) were
General Procedure for the Synthesis of Compounds
Method A. Recrystallization from methanol gave 12m (9%) added dropwise to a solution of cyanuric chloride (ca. 2.0g,
1
as a white solid. mp 229.0°C–229.8°C; H-NMR (500MHz, 1.0eq) in chloroform (10mL) at 0°C. The mixture was stirred
CDCl3) δ: 8.06 (2H, dt, J=2.3Hz, 9.0Hz), 7.57 (4H, br), 7.45 for 20min at 0°C, then washed with dilute sodium hydroxide
(1H, s), 7.30 (2H, dt, J=2.3Hz, 9.4Hz), 4.02 (3H, s), 2.65 and brine, dried over sodium sulfate and concentrated. The
(3H, s). HR-MS (ESI-TOF) m/z: 360.1075 ([M−H]− Calcd for residue was purified by silica gel column chromatography.
C19H15N5O3 360.1091). Anal. Calcd for C19H15N5O3: C, 63.15;
2-n-Butoxy-4,6-dichloro-1,3,5-triazine (13c)
H, 4.18; N, 19.38. Found: C, 63.08; H, 4.45; N, 19.26.
Purification with silica gel column chromatography (elu-
2-(4-Cyanoanilino)-4-methoxy-6-(2-methoxyphenoxy)- ent: n-hexane–ethyl acetate 10:1) gave 13c (55%) as a clear
1
1,3,5-triazine (12n)
oil. H-NMR (500MHz, CDCl3) δ: 4.50 (2H, t, J=6.6Hz),
Method A. Recrystallization from methanol gave 12n (14%) 1.83–1.78 (2H, m), 1.49 (2H, td, J=7.6Hz, 15.2Hz), 0.98 (3H,
1
as colorless crystals. mp 165.2–166.8°C; H-NMR (500MHz, t, J=7.4 Hz).
CDCl3) δ: 7.69 (1H, br), 7.48 (4H, br), 7.30 (1H, t, J=7.7Hz),
7.17 (1H, dd, J=1.7Hz, 8.0Hz), 7.04 (1H, d, J=8.6Hz),
2,4-Dichloro-6-phenoxy-1,3,5-triazine (13d)
Purification with silica gel column chromatography (eluent:
7.02 (1H, t, J=7.7Hz), 4.01 (3H, s), 3.79 (3H, s). HR-MS n-hexane–ethyl acetate 10:1) gave 13d (59%) as a white solid.
(ESI-TOF) m/z: 348.1087 ([M−H]− Calcd for C18H15N5O3 1H-NMR (500MHz, CDCl3) δ: 7.47 (2H, t, J=8.0Hz), 7.35
348.1091)). Anal. Calcd for C18H15N5O3: C, 61.89; H, 4.33; N, (1H, t, J=7.4Hz), 7.18 (2H, d, J=8.0 Hz).
20.05. Found: C, 61.96; H, 4.50; N, 20.15.
2-(4-Cyanoanilino)-4-methoxy-6-(3-methoxyphenoxy)-
1,3,5-triazine (12o)
2,4-Dichloro-6-(2,2,2-trifluoroethoxy)-1,3,5-triazine (13e)
Purification with silica gel column chromatography (eluent:
n-hexane–ethyl acetate 10:1) gave 13e (64%) as a clear oil.
Method A. Recrystallization from methanol gave 12o (44%) 1H-NMR (500MHz, CDCl3) δ: 4.88 (2H, q, J=7.8 Hz).
1
as colorless crystals. mp 163.7–164.5°C; H-NMR (500MHz,
2,4-Dichloro-6-(2-methoxylethoxy)-1,3,5-triazine (13f)
CDCl3) δ: 7.53 (4H, br), 7.42 (1H, s), 7.35 (1H, t, J=8.0Hz),
Purification with silica gel column chromatography (eluent:
6.87 (1H, d, J=8.0Hz), 6.80 (1H, dd, J=2.0Hz, 8.6Hz), 6.76 n-hexane–ethyl acetate 10:1) gave 13f (34%) as a white solid.
(1H, t, J=2.3Hz), 4.03 (3H, s), 3.82 (3H, s). HR-MS (ESI- 1H-NMR (500MHz, CDCl3) δ: 4.65–4.64 (2H, m), 3.77–3.75
TOF) m/z: 348.1101 ([M−H]− Calcd for C18H15N5O3 348.1091). (2H, m), 3.42 (3H, s), 7.35 (1H, t, J=7.4Hz), 7.18 (2H, d,
Anal. Calcd for C18H15N5O3: C, 61.89; H, 4.33; N, 20.05. J=8.0 Hz).
Found: C, 61.96; H, 4.45; N, 20.10.
2-(4-Cyanoanilino)-4-methoxy-6-(4-methoxyphenoxy)-
1,3,5-triazine (12p)
2,4-Dichloro-6-(2-oxopropoxy)-1,3,5-triazine (13g)
Purification with silica gel column chromatography (eluent:
n-hexane–ethyl acetate 6:4) gave 13g (58%) as ocher crystals.
Method A. Recrystallization from methanol gave 12p (28%) 1H-NMR (500MHz, CDCl3) δ: 5.09 (2H, s), 2.27 (3H, s).
1
as colorless crystals. mp 174.2–175.5°C; H-NMR (500MHz,
General Procedure for the Synthesis of Compounds
CDCl3) δ: 7.54 (4H, br), 7.37 (1H, s), 7.11 (2H, td, J=4.0Hz, 14a–14g Phenol (1.0eq) in dichloromethane (5mL) and N,N-
6.4Hz), 6.95 (2H, td, J=3.8Hz, 6.3Hz), 4.02 (3H, s), 3.85 diisopropylethylamine (1 equiv) in dichloromethane (5mL)
(3H, s). HR-MS (ESI-TOF) m/z: 348.1095 ([M−H]− Calcd for were added dropwise to a solution of 13a–g (ca. 700mg,
C18H15N5O3 348.1091). Anal. Calcd for C18H15N5O3: C, 61.89; 1.0eq) in dichloromethane (10mL) at room temperature. The
H, 4.33; N, 20.05. Found: C, 61.94; H, 4.44; N, 19.92.
mixture was stirred for 30min at room temperature. The reac-
2,4-Dichloro-6-ethoxy-1,3,5-triazine (13a) Cyanuric chlo- tion mixture was washed with dilute sodium hydroxide and
ride (1.86g, 10.1mmol) was added to a solution of sodium brine, dried over sodium sulfate and concentrated. The residue
hydrogen carbonate (843mg, 10.0mmol, 0.99eq) in ethanol was purified by silica gel column chromatography.

Vol. 67, No. 6 (2019)
Chem. Pharm. Bull.
573
2-Chloro-4-ethoxy-6-phenoxy-1,3,5-triazine (14a)
2-(4-Cyanoanilino)-4-(1-isopropoxy)-6-phenoxy-1,3,5-
Purification by silica gel column chromatography (eluent: n- triazine (15b)
hexane–ethyl acetate 10:1) gave 14a (84%) as colorless crys- Recrystallization from methanol gave 15b (60%) as color-
1
1
tals. H-NMR (500MHz, CDCl3) δ: 7.43 (2H, tt, J=2.3Hz, less crystals. mp 182.8–184.6°C; H-NMR (500MHz, CDCl3)
8.0Hz), 7.30 (1H, t, J=7.4Hz), 7.17 (2H, dt, J=2.3Hz, δ: 7.59 (2H, br), 7.50 (3H, br), 7.45 (2H, t, J=8.0Hz), 7.32
8.0Hz), 4.44 (2H, q, J=5.9Hz), 1.39 (3H, t, J=7.2 Hz).
2-Chloro-4-(1-isopropoxy)-6-phenoxy-1,3,5-triazine (14b)
(1H, t, J=7.4Hz), 7.19 (2H, d, J=7.4Hz), 5.32–5.24 (2H, m),
1.38 (6H, d, J=6.3Hz). HR-MS (ESI-TOF) m/z: 346.1300
Purification by silica gel column chromatography (eluent: ([M−H]− Calcd for C19H17N5O2 346.1299)). Anal. Calcd for
n-hexane–ethyl acetate 10:1) gave 14b (70%) as a white solid. C19H17N5O2: C, 65.69; H, 4.93; N, 20.16. Found: C, 65.65; H,
1H-NMR (500MHz, CDCl3) δ: 7.43 (2H, t, J=8.0Hz), 7.29 5.22; N, 19.93.
(1H, t, J=7.4Hz), 7.17 (2H, dq, J=1.5, 8.9Hz), 5.26–5.19 (1H,
m), 1.35 (6H, d, J=6.3 Hz).
2-n-Butoxy-4-(4-cyanoanilino)-6-phenoxy-1,3,5-triazine
(15c)
Recrystallization from methanol gave 15c (31%) as colorless
2-n-Butoxy-4-chloro-6-phenoxy-1,3,5-triazine (14c)
1
Purification by silica gel column chromatography (eluent: n- crystals. mp 139.2–139.7°C; H-NMR (500MHz, CDCl3) δ:
hexane–ethyl acetate 10:1) gave 14c (66%) as colorless crys- 7.50 (5H, br), 7.46 (2H, t, J=8.0Hz), 7.33 (1H, t, J=7.4Hz),
1
tals. H-NMR (500MHz, CDCl3) δ: 7.43 (2H, t, J=8.0Hz), 7.20 (2H, d, J=7.4Hz), 4.38 (2H, t, J=6.9Hz), 1.80–1.74 (2H,
7.30 (1H, t, J=7.4Hz), 7.17 (2H, dq, J=1.6Hz, 8.7Hz), 4.36 m), 1.46 (2H, m), 0.96 (3H, t, J=7.4Hz). HR-MS (ESI-TOF)
(2H, t, J=6.6Hz), 1.76–1.70 (2H, m), 1.45–1.39 (2H, m), 0.93 m/z: 360.1455 ([M−H]− Calcd for C20H19N5O2 360.1471). Anal.
(3H, t, J=7.4 Hz).
2-Chloro-4,6-diphenoxy-1,3,5-triazine (14d)
Purification by silica gel column chromatography (eluent:
Calcd for C20H19N5O2: C, 66.47; H, 5.30; N, 19.38. Found: C,
66.31; H, 5.61; N, 19.23.
2-(4-Cyanoanilino)-4,6-diphenoxy-1,3,5-triazine (15d)
n-hexane–ethyl acetate 5:1) gave 14d (68%) as colorless crys-
tals. H-NMR (500MHz, CDCl3) δ: 7.40 (4H, t, J=8.0Hz), less crystals. mp 204.8–205.9°C; H-NMR (500MHz, CDCl3)
Recrystallization from methanol gave 15d (23%) as color-
1
1
7.28 (2H, t, J=7.4Hz), 7.14 (4H, td, J=3.6Hz, 5.3Hz).
δ: 7.61 (1H, s), 7.44 (4H, t, J=7.7Hz), 7.41 (4H, s), 7.32 (2H,
2-Chloro-4-phenoxy-6-(2,2,2-trifluoroethoxy)-1,3,5-triazine br), 7.19 (4H, d, J=8.0Hz). HR-MS (ESI-TOF) m/z: 380.1127
(6e)
([M−H]− Calcd for C22H15N5O2 380.1142). Anal. Calcd for
Purification by silica gel column chromatography (eluent: n- C22H15N5O2: C, 69.28; H, 3.96; N, 18.36. Found: C, 69.45; H,
hexane–ethyl acetate 10:1) gave 14e (70%) as colorless crys- 4.32; N, 18.29.
1
tals. H-NMR (500MHz, CDCl3) δ: 7.46 (2H, t, J=8.0Hz),
2-(4-Cyanoanilino)-4-phenoxy-6-(2,2,2-trifluoroethoxy)-
7.33 (1H, t, J=8.0Hz), 7.17 (2H, dq, J=8.9Hz, 1.7Hz), 4.76 1,3,5-triazine (15e)
(2H, q, J=8.0 Hz).
Purification with silica gel column chromatography (elu-
2-Chloro-4-(2-methoxylethoxy)-6-phenoxy-1,3,5-triazine ent: n-hexane–ethyl acetate 5:1) gave 15e (52%) as colorless
1
(14f)
crystals. mp 161.2–162.0°C; H-NMR (500MHz, CDCl3) δ:
Purification by silica gel column chromatography (eluent: n- 7.61 (1H, s), 7.47 (2H, t, J=8.0Hz), 7.46 (4H, br), 7.35(1H, t,
hexane–ethyl acetate 5:1) gave 14f (76%) as colorless crystals. J=6.9Hz), 7.19 (2H, d, J=8.0Hz), 4.78 (2H, q, J=8.2Hz).
1H-NMR (500MHz, CDCl3) δ: 7.43 (2H, tt, J=2.3Hz, 8.0Hz), HR-MS (ESI-TOF) m/z: 386.0856 ([M−H]− Calcd for
7.29 (1H, t, J=7.4Hz), 7.17 (2H, dq, J=1.6Hz, 8.7Hz), C18H12F3N5O2 386.0859). Anal. Calcd for C18H12F3N5O2: C,
4.52–4.50 (2H, m), 3.70–3.68 (2H, m), 3.38 (3H, s).
2-Chloro-4-(2-oxopropoxy)-6-phenoxy-1,3,5-triazine (14g)
55.82; H, 3.12; N, 18.08. Found: C, 55.81; H, 3.50; N, 17.97.
2-(4-Cyanoanilino)-4-(2-methoxylethoxy)-6-phenoxy-1,3,5-
Purification by silica gel column chromatography (eluent: triazine (15f)
n-hexane–ethyl acetate 3:1) gave 14g (62%) as a white solid.
Purification with silica gel column chromatography (elu-
1H-NMR (500MHz, CDCl3) δ: 7.43 (2H, t, J=7.7Hz), 7.30 ent: n-hexane–ethyl acetate 5:1) gave 15f (88%) as colorless
1
(1H, t, J=7.4Hz), 7.14 (2H, dd, J=1.1Hz, 8.6Hz), 4.88 (2H, crystals. mp 145.8–147.2°C; H-NMR (500MHz, CDCl3) δ:
s), 2.08 (3H, s).
7.90 (1H, br), 7.49 (4H, br). 7.45 (2H, t, J=7.7Hz), 7.32 (1H,
General Procedure for the Synthesis of Compounds t, J=7.2Hz), 7.18 (2H, d, J=7.4Hz), 4.52 (2H, t, J=4.6Hz),
15a–g To a solution of 14a–g (ca. 250mg, 1.0eq) in DMSO 3.71 (2H, t, J=4.9Hz) 3.40 (3H, s). HR-MS (ESI-TOF) m/z:
(1.0mL) were added 4-aminobenzonitrile (2.0eq) and N,N-di- 362.1244 ([M−H]− Calcd for C19H17N5O3 362.1248). Anal.
isopropylethylamine (1.0eq) at room temperature. The mixture Calcd for C19H17N5O3: C, 62.80; H, 4.72; N, 19.27. Found: C,
was stirred for 1h at 100°C, then cooled at room temperature, 62.97; H, 4.94; N, 19.24.
diluted with chloroform, washed with dilute hydrochloric acid
2-(4-Cyanoanilino)-4-(2-oxopropoxy)-6-phenoxy-1,3,5-
and brine, dried over sodium sulfate, and concentrated. The triazine (15g)
residue was purified by silica gel column chromatography or
recrystallization.
Purification with silica gel column chromatography (elu-
ent: n-hexane–ethyl acetate 6:4) gave 15g (27%) as colorless
2-(4-Cyanoanilino)-4-ethoxy-6-phenoxy-1,3,5-triazine (15a) crystals. mp 145.8–147.2°C; H-NMR (500MHz, CDCl3) δ:
Recrystallization from methanol gave 15a (35%) as color- 7.90 (1H, br), 7.49 (4H, br). 7.45 (2H, t, J=7.7Hz), 7.32 (1H,
1
1
less crystals. mp 152.0–152.9°C; H-NMR (500MHz, CDCl3) t, J=7.2Hz), 7.18 (2H, d, J=7.4Hz), 4.52 (2H, t, J=4.6Hz),
δ: 7.50 (5H, br), 7.45 (2H, t, J=8.0Hz), 7.33 (1H, t, J=7.4Hz), 3.71 (2H, t, J=4.9Hz) 3.40 (3H, s). HR-MS (ESI-TOF) m/z:
7.20 (2H, d, J=7.4Hz), 4.45 (2H, q, J=7.1Hz), 1.41 (3H, t, 362.1244 ([M−H]− Calcd for C19H17N5O3 362.1248). Anal.
J=7.1Hz). HR-MS (ESI-TOF) m/z: 332.1142 ([M−H]− Calcd Calcd for C19H17N5O3: C, 62.80; H, 4.72; N, 19.27. Found: C,
for C18H15N5O2 332.1138). Anal. Calcd for C18H12F3N5O2: C, 62.97; H, 4.94; N, 19.24.
64.86; H, 4.54; N, 21.01. Found: C, 64.82; H, 4.93; N, 20.82.
General Procedure for the Synthesis of Compounds 16a

574
Chem. Pharm. Bull.
Vol. 67, No. 6 (2019)
and 16b To a solution of 11d (243mg, 1.00mmol) in THF buffer [1mg/mL p-nitrophenyl phosphate (Thermo, U.S.A.) in
(10mL) were added amine (1.0eq) and N,N-diisopropylethyl- diethanolamine aqueous solution, pH 9.0, 2mM MgCl2] was
amine (1.0eq) at room temperature. The mixture was stirred added. Following incubation at 37°C with shielding from light
for 30min at room temperature, then poured into water and for 3h, the reaction was terminated by the addition of 100µL
extracted with ethyl acetate. The organic layer was washed of 1mol/L NaOH. The absorbance at 405nm was measured
with brine, dried over sodium sulfate and concentrated. The with a Wallac 1420 multilabel counter (ParkinElmer Life Sci-
residue was purified by silica gel column chromatography ences, U.S.A.). All data points were measured in triplicate.
(eluent: n-hexane–ethyl acetate 5:1).
PR Binding Assay PR-binding experiments were per-
2-Chloro-4-dimethylamino-6-phenoxy-1,3,5-triazine (16a)
formed as reported, using recombinant hPR-LBD purchased
Reaction of 11d with dimethylamine 40% in water gave from Invitrogen. hPR-LBD was diluted with buffer (20mM
1
16a (67%) as a white solid. H-NMR (500MHz, CDCl3) δ: Tris–HCl, 300mM NaCl, 1mM ethylenediaminetetraacetic
7.39 (2H, t, J=7.7Hz), 7.24 (1H, t, J=7.4Hz), 7.17 (2H, d, acid (EDTA), 5mM dithiothreitol (DTT), pH 8.0) to 5nM and
J=7.4Hz), 3.19 (3H, s), 3.03 (3H, s).
2-Chloro-4-phenoxy-6-(N-pyrrolidinyl)-1,3,5-triazine (16b)
300µL aliquots were incubated in the dark at 4°C with 4nM
[1,2,6,7-3H]progesterone (PerkinElmer, Inc.) and reference or
Reaction of 11d with pyrrolidine gave 17b (86%) as a white test compounds (dissolved in DMSO; final concentration of
1
solid. H-NMR (500MHz, CDCl3) δ: 7.39 (2H, td, J=1.7Hz, DMSO was 3%). Nonspecific binding was assessed by ad-
7.7Hz), 7.24 (1H, t, J=7.4Hz), 7.18 (2H, td, J=4.3Hz, dition of a 200-fold excess of nonradioactive progesterone.
2.1Hz), 3.61 (2H, t, J=6.6Hz), 3.44 (2H, t, J=6.6Hz), After 24h, 30µL of Dextran T-70/γ-globulin-coated charcoal
1.99–1.90 (4H, m).
suspension was added to the ligand/protein mixtures (1%
General Procedure for the Synthesis of Compounds 17a activated charcoal, 0.05% γ-globulin, 0.05% Dextran 70, final
and 17b To a solution of 17a or 17b (ca. 100mg, 1.0eq) in concentrations) and incubated at 4°C for 5min. The charcoal
DMSO (500µL) were added 4-aminobenzonitrile (2.0eq) and was removed by centrifugation for 5min at 1300×g, and the
N,N-diisopropylethylamine (2.0eq) at room temperature. The radioactivity of the supernatant was measured in Ultima Gold
mixture was stirred for 3h at 160°C, then cooled at room tem- scintillation cocktail (PerkinElmer, Inc.) by using a liquid
perature, and diluted with chloroform. The whole was washed scintillation counter. All experiments were performed in du-
with dilute hydrochloric acid and brine, dried over sodium plicate.
sulfate and concentrated. The residue was purified by recrys-
Docking Simulation The structure of the LBD of hPR
tallization.
was prepared from the Protein Data Bank accession 2W8Y.
2-(4-Cyanoanilino)-4-dimethylamino-6-phenoxy-1,3,5- Polar hydrogens were added, and partial atomic charges were
triazine (17a) assigned using AutoDockTools (ADT). Molecular docking was
Recrystallization from methanol gave 17a (33%) as pale performed using AutoDock 4.2 with the Genetic Algorithm.
yellow crystals. mp 232.0–233.8°C; 1H-NMR (500MHz,
CDCl3) δ: 7.59 (2H, d, J=7.4Hz), 7.50 (2H, d, J=8.0Hz),
Acknowledgments This work was partially supported by
7.42 (2H, td, J=1.7Hz, 7.0Hz), 7.28 (1H, t, J=7.2Hz), 7.20 the Japan Society for the Promotion of Science (JSPS) Grants-
(2H, td, J=1.9Hz, 3.7Hz), 7.13 (1H, s), 3.20 (3H, s), 3.15 in-Aid for Scientific Research No. 17H03887 (S.F.), as well as
(3H, s). HR-MS (ESI-TOF) m/z: 331.1297 ([M−H]− Calcd for by the JSPS Core-to-Core Program, A. Advanced Research
C18H16N6O 331.1302). Anal. Calcd for C18H16N6O: C, 65.05; H, Networks, and Platform Project for Supporting in Drug Dis-
4.85; N, 25.29. Found: C, 65.17; H, 5.16; N, 25.39.
covery and Life Science Research (Platform for Drug Dis-
2-(4-Cyanoanilino)-4-phenoxy-6-(N-pyrrolidinyl)-1,3,5- covery, Informatics, and Structural Life Science) from Japan
triazine (17b)
Agency for Medical Research and Development (AMED).
Recrystallization from methanol gave 17b (44%) as brown
1
crystals. mp 210.0–211.5°C; H-NMR (500MHz, CDCl3) δ:
Conflict of Interest The authors declare no conflict of
7.60 (2H, br), 7.48 (2H, d, J=8.0Hz), 7.42 (2H, t, J=8.0Hz), interest.
7.27 (1H, t, J=7.4Hz), 7.20 (3H, d, J=7.4Hz), 3.62 (2H, t,
J=6.6Hz), 3.58 (2H, t, J=6.6Hz), 2.06–1.90 (4H, m). HR-MS
(ESI-TOF) m/z: 357.1464 ([M−H]− Calcd for C20H18N6O
357.1458). Anal. Calcd for C20H18N6O: C, 67.02; H, 5.09; N,
23.45. Found: C, 67.16; H, 5.35; N, 23.28.
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