Synthesis and structure-activity relationship study of triazine-based inhibitors of the DNA binding of NF-κB

Article abstract of DOI:10.1248/cpb.c14-00218

Nuclear transcription factor nuclear factor-kappa B (NF-κB) has diverse pathophysiological functions, and NF-κ B inhibitors are considered to be candidates for multiple therapeutic applications. We previously reported a novel triazine-based NF-κB inhibitor, 2-anilino-4,6-dichloro-1,3,5- triazine (NI241), that directly inhibits DNA binding of NF-κB. Here, we report synthesis of a series of triazine derivatives and evaluation of their structure-activity relationships for NF-κB inhibition. We found that 2-amino-4,6-dichloro-1,3,5-triazine substructure is essential for the inhibitory activity of the lead compound NI241, and modification of NI241 by introduction of an m-methoxy substituent on the phenyl ring afforded the more potent derivative 28. The structure-activity relationships identified in this study suggested a possible mechanism of irreversible NF-κB inhibition by NI241, and should be helpful in the design of other NF-κB inhibitors.

Full text of DOI:10.1248/cpb.c14-00218

700
Regular Article
Chem. Pharm. Bull. 62(7) 700–708 (2014)
Vol. 62, No. 7
Synthesis and Structure–Activity Relationship Study of Triazine-Based
Inhibitors of the DNA Binding of NF-κB
Shinya Fujii,a,b,# Takanobu Kobayashi,c,# Aki Nakatsu,a Hiroshi Miyazawa,c and
,a
Hiroyuki Kagechika*
a Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University; 2–3–10 Kanda-Surugadai,
b
Chiyoda-ku, Tokyo 101–0062, Japan: Institute of Molecular and Cellular Biosciences, The University of Tokyo;
c
1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–0032, Japan: and Kagawa School of Pharmaceutical Sciences, Tokushima
Bunri University; 1314–1 Shido, Sanuki, Kagawa 769–2193, Japan
Received March 9, 2014; accepted April 15, 2014
Nuclear transcription factor nuclear factor-kappa B (NF-κB) has diverse pathophysiological functions,
and NF-κB inhibitors are considered to be candidates for multiple therapeutic applications. We previously
reported a novel triazine-based NF-κB inhibitor, 2-anilino-4,6-dichloro-1,3,5-triazine (NI241), that directly
inhibits DNA binding of NF-κB. Here, we report synthesis of a series of triazine derivatives and evaluation
of their structure–activity relationships for NF-κB inhibition. We found that 2-amino-4,6-dichloro-1,3,5-
triazine substructure is essential for the inhibitory activity of the lead compound NI241, and modification of
NI241 by introduction of an m-methoxy substituent on the phenyl ring afforded the more potent derivative
28. The structure–activity relationships identified in this study suggested a possible mechanism of irrevers-
ible NF-κB inhibition by NI241, and should be helpful in the design of other NF-κB inhibitors.
Key words nuclear factor-kappa B (NF-κB); DNA binding; inhibitor; triazine
Nuclear factor-kappa B (NF-κB) is a transcriptional factor
Results and Discussion
that regulates many crucial physiological and pathological pro-
Chemistry NI241 consists of three parts: dichlorotri-
cesses, such as immune response, inflammation, infection, cell azine, a phenyl ring, and the linking secondary amino group.
differentiation, and cancer development.1–5) NF-κB is activated C-Chlorinated triazine is chemically reactive toward nu-
by various factors, such as cytokines, toxins, and other cellu- cleophiles. Here, we focused on pyrimidine and triazine de-
lar stimuli, through multiple pathways including the canonical rivatives bearing dichloro groups or other functional group(s)
pathway and non-canonical pathway.6–11) Inhibitors of NF-κB such as alkoxy or amino groups (Fig. 1). Since our previous
are expected to be candidates for multiple therapeutic applica- studies on docking simulation of NI241 to NF-κB suggested
tions, and various compounds that inhibit NF-κB signaling the importance of both the molecular shape of NI241 and a
pathways have been developed in recent decades.12–14) Though secondary amino group that interacts with amino acid resi-
activation of NF-κB can be blocked at a variety of steps, com- dues of NF-κB, we also examined various N-substituents on
pounds that block upstream and midstream steps of the NF-κB 2-amino-4,6-dichloro-1,3,5-triazine.
pathway might affect multiple targets and pathways. On the
Synthesis of pyrimidine derivatives is illustrated in Chart
other hand, compounds that directly inhibit DNA binding of 1. Aromatic nucleophilic substitution reactions using aniline
NF-κB might be very effective in regulating the expression and 2,4,6-trichloropyrimidine gave anilinopyrimidines 2 and
of target genes, because NF-κB signaling pathways converge 3. Further substitution of 2 or 3 using alkoxides afforded
at the level of DNA binding. On the basis of these consider- compounds 4–8. The use of excess alkoxide afforded di-
ations, we previously screened NF-κB DNA-binding inhibi- alkoxylated compounds 5 and 8. Amination of 2 or 3 using
tors and identified the 2,4-dichloro-1,3,5-triazine derivative 4-methoxybenzylamine gave compounds 9 or 11, respectively.
NI241.15) Structurally, NI241 has a quite simple scaffold, and Cleavage of the 4-methoxybenzyl group of 9 and 11 under
in the present work we planned to investigate its structure– acidic conditions gave compounds 10 and 12, respectively.
activity relationship (SAR) with the aim of developing more Methylpyrimidine derivative 14 was prepared from 4,6-dichlo-
potent compounds.
ro-2-methylpyrimidine (13) and aniline (Chart 1).
Synthesis of 1,3,5-triazine derivatives is illustrated in
Charts 2 and 3. Nucleophilic substitution reaction of 2,4,6-tri-
Fig. 1. Structures of NF-κB-DNA Binding Inhibitor NI241 and Its Derivatives Synthesized for SAR Study
The authors declare no conflict of interest.
#These authors contributed equally to this work.
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: kage.chem@tmd.ac.jp

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Chart 1. Synthesis of Pyrimidine Derivatives
chloro-1,3,5-triazine (15) by aniline gave mono-substituted the synthesized compounds. These results suggest that 2,4-di-
compound 16 (NI241) and disubstituted compound 17. Further chloro-1,3,5-triazine substructure is the essential pharmaco-
substitution of 16 with various nucleophiles afforded mono- phore for NF-κB inhibitory activity of NI241, and aromatic
chlorotriazine derivatives 18–23. Compounds 24 and 25 were nucleophilic substitution of NI241 decreased the activity.
prepared from compound 18 by amination using methylam-
Thus, we next investigated the inhibitory activity of various
monium chloride or dimethylammonium chloride, respectively 2,4-dichloro-1,3,5-triazine derivatives toward DNA binding
(Chart 2). Various 2,4-dichloro-1,3,5-triazine derivatives were of NF-κB. Table 4 summarizes the results of EMSA evalua-
synthesized in one step by substitution of 15 using corre- tion. Most of the synthesized compounds exhibited inhibitory
sponding amines (Chart 3).
activity, supporting the hypothesis that the 2,4-dichloro-1,3,5-
Biological Evaluation The inhibitory activity of syn- triazine substructure is the key pharmacophore for this activ-
thesized compounds toward DNA binding of NF-κB was ity. N-Phenyl derivatives including naphthyl derivative 37 and
evaluated by means of electrophoretic mobility shift assays tetrahydroquinoline derivative 39 exhibited significant activ-
(EMSA) using Alexa680-labeled NF-κB probe and His-tagged ity, whereas N-alkyl derivatives exhibited moderate activity.
p50 recombinants (Tables 1, 2). All 4-phenylaminopyrimidine N-Methylation of secondary amines NI241 and 30, yielding 26
derivatives exhibited only weak activities, significantly lower and 31, respectively, increased the inhibitory activity. The re-
than that of NI241 (Table 1). As for the 2-phenylaminopyrimi- sults suggested that aromatic and bulky amines are favorable
dines, 4-ethoxy derivative 7 exhibited moderate activity, and for the inhibitory activity. Finally we examined the IC50 values
p-methoxybenzylamino derivative 11 exhibited weak activity. of the most potent compounds. Table 5 summarized the IC50
However, the activity of these compounds was lower than values of the selected compounds. m-Methoxy derivative 28
that of NI241. These results suggested that the 1,3,5-triazine exhibited the most potent inhibitory activity, showing an ap-
substructure is essential for the inhibitory activity of NI241. proximately 50% increase of potency over the lead compound
Therefore, we investigated structural development based on NI241.
1,3,5-triazine structure.
In our previous report, the binding mode of NI241 to NF-κB
Table 3 summarizes the inhibitory activity of 2-phenyl- was simulated in silico and it was suggested that the N–H hy-
amino-1,3,5-triazine derivatives. Compounds bearing a bulky drogen of the aminophenyl group and two nitrogen atoms of
substituent, such as n-butoxy (19), phenoxy (20) or pyrrolidi- triazine adjacent to the amino group form hydrogen bonds to
nyl (23), exhibited quite low activity. Though methoxy de- NF-κB.15) However, the present structure–activity relationship
rivative 18 exhibited moderate activity, the lead compound 16 data indicate that the N–H hydrogen is not important, and that
(NI241) exhibited the most potent inhibitory activity among the whole 2,4-dichloro-1,3,5-triazine substructure is required

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Vol. 62, No. 7
Chart 2. Synthesis of 1,3,5-Triazine Derivatives
Chart 3. Synthesis of 2,4-Dichloro-1,3,5-triazine Derivatives
for inhibitory activity of NI241. Dichloro-1,3,5-triazine is an
electrophilic species that could react with nucleophilic tar-
Conclusion
We investigated the SAR of the NF-κB DNA-binding in-
gets in proteins or water molecules in assay media. Thus, we hibitor NI241. Its 2,4-dichloro-1,3,5-triazine substructure was
speculate that NI241 and its derivatives function as irrevers- identified as the essential pharmacophore for this activity.
ible inhibitors by reacting with NF-κB protein, or activated by Based on the structure of NI241, we developed the more po-
conversion into hydrated form. These are also possible reasons tent inhibitor 28. Since NF-κB has diverse pathophysiologi-
of low biological activity of 2 and 3, the dichloropyrimidine cal functions, the SAR information obtained in this study is
derivatives with lower activity, corresponding to NI241. Fur- expected to contribute to the development of novel NF-κB
ther binding simulation studies based on the present results inhibitors as candidates for multiple therapeutic applications.
are in progress.

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Table 1. Inhibitory Activity of 4-Phenylaminopyrimidine Derivatives Determined by EMSA
Compound
R1
R2
Inhibition (%)a)
NI241
—
—
Cl
40
<5
<5
11
10
7
2
4
Cl
MeO
Cl
5
i-PrO
Oi-Pr
Cl
9
NH–CH2C6H4–p-OMe
10
14
NH2
Me
Cl
Cl
12
a) Inhibition ratio was determined from the decrease of Alexa680-labeled NF-κB probe at the concentration of 100µM bound to His-tagged p50 recombinants.
Table 2. Inhibitory Activity of 2-Phenylaminopyrimidine Derivatives Determined by EMSA
Compound
R1
R2
Inhibition (%)a)
NI241
—
—
Cl
40
<5
<5
31
3
6
Cl
MeO
Cl
7
EtO
i-PrO
Cl
8
Oi-Pr
Cl
<5
22
11
12
NH–CH2C6H4–p-OMe
NH2
Cl
14
a) Inhibition ratio was determined from the decrease of Alexa680-labeled NF-κB probe at the concentration of 100µM bound to His-tagged p50 recombinants.
Table 3. Inhibitory Activity of 2-Phenylamino-1,3,5-triazine Derivatives Determined by EMSA
Compound
R1
R2
Inhibition (%)a)
16 (NI241)
Cl
NHPh
Cl
Cl
51
15
31
5
17
18
19
20
21
22
23
24
25
MeO
Cl
n-BuO
PhO
On-Bu
Cl
9
MeNH
Me2N
Cl
14
18
11
<5
22
Cl
Pyrrolidin-1-yl
MeO
Oi-Pr
NHMe
NMe2
MeO
a) Inhibition ratio was determined from the decrease of Alexa680-labeled NF-κB probe at the concentration of 100µM bound to His-tagged p50 recombinants.
Table 4. Inhibitory Activity of 2-Amino-4,6-dichloro-1,3,5-triazine Derivatives Determined by EMSA
Compound
R1
R2
Inhibition (%)a)
16 (NI241)
Ph
Ph
H
Me
H
51
57
52
63
51
31
49
<5
51
29
7
26
27
28
29
30
31
32
33
34
35
36
37
38
39
o-OMe–C6H4
m-OMe–C6H4
p-OMe–C6H4
Bn
H
H
H
Bn
Me
Et
H
Et
t-Bu
n-Hex
H
c-Hex
H
Furfuryl
1-Naphthyl
H
59
62
47
55
H
–(CH2)5–
–C6H4–o-(CH2)3–
a) Inhibition ratio was determined from the decrease of Alexa680-labeled NF-κB probe at the concentration of 100µM bound to His-tagged p50 recombinants.

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Table 5. IC50 Values of Selected Triazine Derivatives
J=7.4, 0.9Hz), 7.32 (dd, 2H, J=8.6, 1.7Hz), 7.22 (tt, 1H,
J=7.3, 1.2Hz), 6.97 (brs, 1H), 6.35 (s, 1H), 3.96 (s, 3H);
13C-NMR (CDCl3, 125MHz) δ: 165.2, 163.6, 161.2, 137.4,
129.5, 125.6, 123.2, 96.8, 55.0. Anal. Calcd for C11H10ClN3O:
C, 56.06; H, 4.28; N, 17.83. Found: C, 56.07; H, 4.34; N, 17.88.
2,4-Diisopropoxy-6-phenylaminopyrimidine (5) Sodium
hydride (60% dispersion in mineral oil, 66.6mg, 1.67mmol)
Compound
16 (NI241)
26
28
29
37
IC50 (µM)
97
77
60
97
68
Experimental
General Methods All reagents were purchased from in 2-propanol (6mL) was added to 2 (100mg, 0.42mmol)
Sigma-Aldrich Chemicals Co., Tokyo Kasei Co., Wako under Ar. The mixture was stirred at room temperature for
Pure Chemical Industries, Ltd., and Kanto Chemical Co., 1h and at 100°C for 24h, then sodium hydride (60% disper-
Inc. Silica gel column chromatography was purchased from sion in mineral oil, 16.66mg) suspended in 2-propanol (3mL)
Kanto Chemical Co., Inc. 1H-NMR spectra were recorded was further added in one portion. The resulting mixture was
at 400MHz on a Brucker Avance 400 spectrometer or stirred at 100°C for 24h, then poured into water and extracted
500MHz on a Brucker Avance 500 spectrometer. 13C-NMR with ethyl acetate. The organic layer was washed with brine,
spectra were recorded on at 125MHz on a Brucker Avance dried over sodium sulfate and concentrated. Purification of the
500 spectrometer. Chemical shifts are reported as parts per residue by silica gel flash chromatography (eluent: hexane–
1
million (ppm) relative to chloroform (7.26ppm for H-NMR ethyl acetate 5:1) gave 5 (67%) as colorless needles (hexane);
1
and 77.16ppm for 13C-NMR) or dimethylsulfoxide (DMSO) mp 86.1–88.8°C; H-NMR (CDCl3, 400MHz) δ: 7.57 (dd, 2H,
1
(2.50ppm for H-NMR and 39.52ppm for 13C-NMR). Data are J=8.7, 1.1Hz), 7.25 (dd, 2H, J=7.1, 0.8Hz), 7.12 (tt, 1H, J=7.4,
reported as follows; chemical shift, multiplicity (s, singlet; d, 1.2Hz) 6.54 (brs, 1H), 5.32 (sep, 1H, J=6.2Hz), 5.21 (sep,
doublet; t, triplet; q, quartet; quint, quintet; sex, sextet; sep, 1H, J=6.3Hz), 1.38 (d, 6H, J=6.4Hz), 1.30 (d, 6H, J=6.0Hz);
septet; br, broad; m, multiplet) coupling constants (Hz), in- 13C-NMR (CDCl3, 125MHz) δ: 171.7, 164.5, 163.6, 138.8,
tegration. Melting points were taken on a Yanagimoto micro 129.3, 124.3, 122.4, 80.9, 69.5, 68.7, 22.1, 22.0. Anal. Calcd for
melting point apparatus. Mass spectral data were obtained C15H18N3O2: C, 66.88; H, 7.37; N, 14.62. Found: C, 66.82; H,
with a JEOL AX-505 spectrometer. Elemental analyses were 7.19; N, 14.72.
carried out on a Yanaco MT-6 CHN CORDER.
4-Chloro-6-methoxy-2-phenylaminopyrimidine (6) So-
2,6-Dichloro-4-phenylaminopyrimidine (2) and 4,6-Di- dium methoxide (58.50mg, 1.08mmol) was added to 3
chloro-2-phenylaminopyrimidine (3) Aniline (1.0mL, (200mg, 0.83mmol) in methanol (4mL) at 0°C. The mixture
10.90mmol) was added dropwise to 2,4,6-trichloropyrimidine was stirred at room temperature for 23h, then poured into
(2.00g, 10.90mmol) in tetrahydrofuran (30mL). The mixture saturated aqueous ammonium chloride and extracted with
was stirred at room temperature for 3h, then Et3N (2.27mL, ethyl acetate. The organic layer was washed with brine,
16.35mmol) and aniline (0.50mL, 5.45mmol) were further dried over sodium sulfate, and evaporated. Recrystallization
added in one portion. The mixture was stirred at room temper- of the residue gave 6 (quant.) as colorless plates (hexane);
1
ature for 18h, then poured into water and extracted with ethyl mp 105.0–105.9°C; H-NMR (CDCl3, 400MHz) δ: 7.59 (dd,
acetate. The organic layer was washed with brine, dried over 2H, J=8.7, 1.1Hz), 7.34 (td, 2H, J=7.1, 1.0Hz), 7.22 (brs,
sodium sulfate and concentrated. Purification of the residue by 1 H), 7.07 (tt, 1H, J=7.4, 1.1Hz), 6.22 (s, 1H), 3.97 (s, 3H);
silica gel chromatography (eluent: hexane–ethyl acetate 10:1 13C-NMR (CDCl3, 125MHz) δ: 171.2, 160.6, 159.0, 138.8,
to 4:1) gave 2 (70%) and 3 (25%). 2: colorless crystals (di- 128.9, 123.1, 119.4, 97.9, 54.3. Anal. Calcd for C11H10ClN3O:
1
chloromethane–hexane); mp 143.2–144.6°C; H-NMR (CDCl3, C, 56.06; H, 4.28; N, 17.83. Found: C, 56.26; H, 4.31; N, 17.82.
400MHz) δ: 7.45 (td, 2H, J=7.0, 0.6Hz), 7.30 (tt, 1H, J=7.5,
4-Chloro-6-ethoxy-2-phenylaminopyrimidine (7) Sodi-
1.0Hz), 7.28 (dd, 2H, J=7.4, 1.2Hz), 7.15 (brs, 1H), 6.57 (s, um hydride (60% dispersion in mineral oil) (33.6mg,
1H); 13C-NMR (CDCl3, 125MHz) δ: 163.3, 161.2, 160.1, 136.2, 0.84mmol) in ethanol (6mL) was added to 3 (70mg,
129.9, 126.8, 123.8, 100.7. Anal. Calcd for C10H7Cl2N3: C, 0.29mmol) at 0°C under Ar. The mixture was stirred at room
50.03; H, 2.94; N, 17.50. Found: C, 50.00; H, 2.96; N, 17.39. temperature for 5h, then poured into water and extracted
3: colorless needles (hexane); mp 115.1–116.9°C; 1H-NMR with ethyl acetate. The organic layer was washed with brine,
(CDCl3, 400MHz) δ: 7.57 (dd, 2H, J=8.7, 1.1Hz), 7.36 (td, 2H, dried over sodium sulfate and concentrated. Purification of
J=7.1, 1.0Hz), 7.23 (brs, 1 H), 7.11 (tt, 1H, J=7.4, 1.1Hz), 6.79 the residue by silica gel chromatography (eluent: hexane–ethyl
(s, 1H); 13C-NMR (CDCl3, 125MHz) δ: 161.8, 158.9, 137.7, acetate 5:1) gave 7 (quant.) as pale-yellow needles (hexane);
1
129.1, 124.0, 119.7, 111.2. Anal. Calcd for C10H7Cl2N3: C, 50.03; mp 109.1–111.2°C; H-NMR (CDCl3, 400MHz) δ: 7.57 (dd,
H, 2.94; N, 17.50. Found: C, 50.16; H, 3.08; N, 17.46.
2H, J=8.8, 1.2Hz), 7.33 (td, 2H, J=7.2, 2.0Hz), 7.06 (tt, 1H,
4-Chloro-2-methoxy-6-phenylaminopyrimidine (4) So- J=7.6, 0.8Hz), 7.08–7.04 (brs, 1H), 6.20 (s, 1H), 4.41 (q, 2H,
dium methoxide (58.5mg, 1.08mmol) was added to 2 (200mg, J=7.2Hz), 1.40 (t, 3H, J=7.2Hz); 13C-NMR (CDCl3, 125MHz)
0.83mmol) in methanol (4.2mL) at 0°C. The mixture was δ: 170.9, 159.6, 158.5, 138.5, 129.0, 123.2, 119.5, 98.2, 63.4,
stirred at room temperature for 4h. The mixture was stirred 14.3. Anal. Calcd for C12H12ClN3O: C, 57.72; H, 4.84; N, 16.83.
at 50°C for 2h. The contents were poured into saturated aque- Found: C, 57.65; H, 4.80; N, 16.93.
ous ammonium chloride and extracted with ethyl acetate.
4,6-Diisopropoxy-2-phenylaminopyrimidine (8) Sodium
The organic layer was washed with brine, dried over sodium hydride (60% dispersion in mineral oil, 45.5mg, 1.14mmol)
sulfate and concentrated. Purification of the residue by silica in 2-propanol (3mL) was added to 3 (136.4mg, 0.57mmol)
gel chromatography (eluent: hexane–ethyl acetate 5:1) gave at 0°C under Ar. The mixture was stirred at room tempera-
4 (32%) as colorless needles (dichloromethane–hexane); mp ture for 1h and at 100°C for 24h, then sodium hydride (60%
1
174.2–176.8°C; H-NMR (CDCl3, 400MHz) δ: 7.40 (td, 2H, dispersion in mineral oil, 68.17mg, 1.70mmol) suspended

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in 2-propanol (3mL) was further added in one portion. The 159.2, 139.2, 128.8, 112.5, 119.3, 114.2, 55.3, 53.4, 45.2, 45.1.
resulting mixture was stirred at 100°C for 24h, then poured 4-Amino-6-chloro-2-phenylaminopyrimidine (12) Tri-
into water and extracted with ethyl acetate. The organic layer fluoroacetic acid (2.0mL) was added to a solution of 11
was washed with brine, dried over sodium sulfate and con- (92.0mg, 0.42mmol). The mixture was stirred at room tem-
centrated. Purification of the residue by silica gel flash chro- perature for 17h, then poured into saturated aqueous sodium
matography (eluent: hexane–ethyl acetate 20:1) gave 8 (56%) hydrogen carbonate and extracted with ethyl acetate. The
1
as a colorless oil; H-NMR (CDCl3, 400MHz) δ: 7.59 (dd, 2H, organic layer was washed with brine, dried over sodium sul-
J=8.8, 1.2Hz), 7.30 (td, 2H, J=8.0, 1.2Hz), 7.00 (tt, 1H, J=7.4, fate and concentrated. Purification of the residue by silica gel
1.2Hz), 6.83 (brs, 1H), 5.48 (s, 1H), 5.23 (sep, 2H, J=7.4Hz), chromatography (eluent: hexane–ethyl acetate 1:1) gave 2–13
1.35 (d, 12 H J=6.4Hz); 13C-NMR (CDCl3, 125MHz) δ: 171.1, (quant.) as colorless plates (dichloromethane–hexane); mp
1
158.8, 139.8, 128.7, 122.0, 118.8, 82.4, 69.0, 22.0.
139.2–141.0°C; H-NMR (CDCl3, 400MHz) δ: 7.55 (dd, 2H,
4-Chloro-2-(4-methoxybenzyl)amino-6-phenylaminopy- J=8.8, 1.2Hz), 7.31 (td, 2H, J=7.6, 2.0Hz), 7.04 (tt, 1H, J=7.2,
rimidine (9) 4-Methoxybenzylamine (68.6mg, 0.50mmol) 1.2Hz), 6.91 (brs, 1H), 5.97 (s, 1H), 4.81 (brs, 2H); 13C-NMR
and Et3N (63.3mg, 0.63mmol) were added to 2 (100mg, (CDCl3, 125MHz) δ: 164.3, 160.0, 159.6, 139.0, 128.8, 122.8,
0.42mmol) in THF (4mL) at 0°C. The mixture was stirred 119.6, 95.0. Anal. Calcd for C10H9ClN4: C, 54.43; H, 4.11; N,
at room temperature for 18h and at 50°C for 4h, then 4- 25.39. Found: C, 54.29; H, 4.26; N, 25.52.
methoxybenzylamine (28.6mg, 0.21mmol) and Et3N (21.1mg,
4-Chloro-2-methyl-6-phenylaminopyrimidine (14) Ani-
0.21mmol) were further added in one portion. The result- line (560µL, 6.13mmol) was added dropwise to 4,6-dichloro-
ing mixture was stirred at 50°C for 3h, then poured into 2-methylpyrimidine (1.0g, 6.13mmol) in ethanol (30mL) at
water and extracted with ethyl acetate. The organic layer 0°C. The mixture was stirred at room temperature for 19h,
was washed with brine, dried over sodium sulfate and then then poured into water and extracted with ethyl acetate. The
concentrated. Purification of the residue by silica gel chro- organic layer was washed with brine, dried over sodium sul-
matography (eluent: hexane–ethyl acetate 5:1) gave 9 (88%). fate and concentrated. Purification of the residue by silica gel
9: colorless crystals (hexane); mp 143.8–146.3°C; 1H-NMR flash chromatography (eluent: hexane–ethyl acetate 5:1) gave
(CDCl3, 400MHz) δ: 7.35 (td, 2H, J=7.0, 2.3Hz), 7.30 (dd, 14 (53%) as colorless needles (hexane); mp 109.1–111.2°C;
2H, J=8.6, 1.5Hz), 7.26 (dd, 2H, J=6.1, 2.0Hz), 7.15 (tt, 1H, 1H-NMR (CDCl3, 400MHz) δ: 7.42 (td, 2H, J=7.6, 0.8Hz),
J=7.0, 1.5Hz), 6.87 (dd, 2H, J=6.6, 2.1Hz), 6.57 (brs, 1 H), 7.24 (dd, 2H, J=8.8, 1.2Hz), 7.24 (tt, 1H, J=7.6, 0.8Hz),
6.04 (s, 1H), 5.27 (s, 1H), 4.53 (d, 2H, J=5.8Hz), 3.80 (s, 3H); 6.87 (brs, 1H), 6.54 (s, 1H), 2.54 (s, 3H); 13C-NMR (CDCl3,
13C-NMR (CDCl3, 125MHz) δ: 162.4, 161.8, 160.4, 158.8, 125MHz) δ: 167.43, 161.1, 160.9, 136.5, 129.9, 129.6, 129.2,
138.0, 130.9, 129.3, 128.7, 124.7, 122.4, 114.0 93.1, 55.3, 44.9. 126.3, 123.4, 99.9, 25.2. Anal. Calcd for C11H10ClN3: C, 60.14;
Anal. Calcd for C18H17ClN4O: C, 63.44; H, 5.03; N, 16.44. H, 4.59; N, 19.13. Found: C, 60.36; H, 4.79; N, 19.15.
Found: C, 63.28; H, 5.03; N, 16.45.
2,4-Dichloro-6-phenylamino-1,3,5-triazine (16) and 2-
2-Amino-4-chloro-6-phenylaminopyrimidine (10) Tri- Chloro-4,6-bis(phenylamino)-1,3,5-triazine (17) Aniline
fluoroacetic acid (1.0mL) was added to a solution of 9 (0.98 µL, 10.85mmol) was added dropwise to cyanuric chlo-
(34.6mg, 0.10mmol). The mixture was stirred at room temper- ride (2.0g, 10.85mmol) in acetone (60mL) at 0°C. Et3N
ature for 25h, then poured into saturated aqueous sodium hy- (1.5mL, 10.85mmol) was added in one portion. The mixture
drogen carbonate and extracted with ethyl acetate. The organ- was stirred at room temperature for 7h, then poured into
ic layer was washed with brine, dried over sodium sulfate and water and extracted with ethyl acetate. The organic layer was
concentrated. Purification of the residue by silica gel chroma- washed with brine, dried over sodium sulfate and concen-
tography (eluent: hexane–ethyl acetate 2:1) gave 10 (quant.) trated. Purification of the residue by silica gel chromatography
as colorless powder (ethyl acetate–hexane); mp 209.6–210.4°C; (eluent: hexane–ethyl acetate 8:1 to 6:1) gave 16 (53%) and
1H-NMR (CDCl3, 400MHz) δ: 7.38 (td, 2H, J=8.0, 0.8Hz), 17 (10%). 16: colorless crystals (ethyl acetate–hexane); mp
1
7.30 (dd, 2H, J=8.8, 1.6Hz), 7.19 (tt, 1H, J=7.4, 1.2Hz), 6.59 134.1–140.6°C; H-NMR (CDCl3, 400MHz) δ: 7.54 (dd, 2H,
(brs, 1 H), 6.11 (s, 1 H), 4.90 (brs, 2H); 13C-NMR (CDCl3, J=8.8, 1.2Hz), 7.49 (brs, 1H), 7.42 (td, 2H, J=7.2, 5.6Hz),
125MHz) δ: 162.8, 162.4, 160.7, 137.6, 129.5, 125.2, 122.9, 7.23 (tt, 1H, J=7.6, 1.2Hz). 13C-NMR (CDCl3, 125MHz) δ:
93.8. Anal. Calcd for C10H9ClN4: C, 54.43; H, 4.11; N, 25.39. 171.4, 170.2, 164.1, 135.7, 129.3, 125.9, 121.2. Anal. Calcd for
Found: C, 54.38; H, 4.22; N, 25.15.
C9H6Cl2N4: C, 44.84; H, 3.51; N, 23.24. Found: C, 45.13; H,
4-Chloro-6-(4-methoxybenzylamino)-2-phenylaminopy- 2.69; N, 23.43. 17: colorless crystals (ethyl acetate–hexane);
1
rimidine (11) 4-Methoxybenzylamine (57.1mg, 0.42mmol) mp 201.8–204.0°C; H-NMR (CDCl3, 400MHz) δ: 7.54 (d, 4H,
and Et3N (31.6mg, 0.31mmol) were added to 3 (50mg, J=8.0Hz), 7.36 (t, 4H, J=7.6Hz), 7.29 (brs, 2H), 7.16 (t, 2H,
0.20mmol) in THF (2mL). The mixture was stirred at room J=7.4Hz); 13C-NMR (CDCl3, 125MHz) δ: 168.6, 164.0, 137.0,
temperature for 1h and at 50°C for 14h, then poured into 129.1, 121.4, 121.4. Anal. Calcd for C15H12ClN5: C, 60.51; H,
water and extracted with ethyl acetate. The organic layer was 4.06; N, 23.52. Found: C, 60.75; H, 4.18; N, 23.65.
washed with brine, dried over sodium sulfate and concen-
2-Chloro-4-methoxy-6-phenylamino-1,3,5-triazine (18)
trated. Purification of the residue by silica gel chromatography Sodium methoxide (22.1mg, 0.41mmol) was added to 16
(eluent: hexane–ethyl acetate 2:1) gave 11 (81%) as a color- (100mg, 0.41mmol) in methanol (2.0mL) at 0°C. The mixture
1
less oil; H-NMR (CDCl3, 400MHz) δ: 7.53 (dd, 2H, J=8.4, was stirred at room temperature for 18h. Sodium methox-
0.8Hz), 7.27 (td, 2H, J=7.2, 2.0Hz), 7.24 (dd, 2H, J=6.8, ide (11.1mg, 0.21mmol) was further added in one portion
2.0Hz), 7.00 (tt, 1H, J=8.0, 1.2Hz), 7.02–6.98 (brs, 1H), 6.89 and stirring was continued at room temperature for 3h, then
(dd, 2H, J=6.8, 2.0Hz), 5.88 (s, 1H), 5.18 (brs, 1H), 4.46 the mixture was poured into water and extracted with ethyl
(brs, 2H), 3.80 (s, 3 H); 13C-NMR (CDCl3, 125MHz) δ: 163.7, acetate. The organic layer was washed with brine, dried over

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Vol. 62, No. 7
sodium sulfate and concentrated. Purification of the residue 29.72. Found: C, 51.17; H, 4.45; N, 29.74.
by silica gel chromatography (eluent: hexane–ethyl acetate
2-Chloro-4-dimethylamino-6-phenylamino-1,3,5-tri-
7:1) gave 18 (78%) as colorless needles (ethyl acetate–hex- azine (22) Dimethylammonium chloride (33.0mg, 0.40mmol)
1
ane); mp 174.2–176.8°C; H-NMR (CDCl3, 400MHz) δ: 7.56 and Et3N (57µL, 0.41mmol) were added to 16 (100.2mg,
(dd, 2H, J=8.8, 1.2Hz), 7.38 (td, 2H, J=7.6, 2.0Hz), 7.41–7.36 0.41mmol) in 2-propanol (2mL) at 0°C. The mixture was
(brs, 1H), 7.17 (tt, 1H, J=7.6, 0.8Hz), 4.04 (s, 3H); 13C-NMR stirred at room temperature for 4h, then poured into water and
(CDCl3, 125MHz) δ: 171.6, 170.7, 165.2, 136.7, 129.2, 124.9, extracted with ethyl acetate. The organic layer was washed
120.8, 55.8. Anal. Calcd for C10H9ClN4O: C, 50.75; H, 3.83; N, with brine, dried over sodium sulfate and concentrated. Pu-
23.67. Found: C, 50.74; H, 3.94; N, 23.88.
rification of the residue by silica gel chromatography (eluent:
2,4-Di-n-butoxy-6-phenylamino-1,3,5-triazine (19) So- hexane–ethyl acetate 10:1) gave 22 (85%) as colorless needles
1
dium hydride (60% dispersion in mineral oil, 18.0mg, (ethyl acetate–hexane); mp 162.2–163.4°C; H-NMR (CDCl3,
0.45mmol) in 1-butanol (2mL) was added to 16 (101mg, 400MHz) δ: 7.57 (dd, 2H, J=8.8, 1.2Hz), 7.34 (td, 2H, J=7.2,
0.42mmol) at 0°C under Ar. The mixture was stirred at room 2.0Hz), 7.09 (tt, 1H, J=7.4, 1.2Hz), 7.00 (brs, 1H), 3.20 (s,
temperature for 1h and at 40°C for 1h, then sodium hydride 6H); 13C-NMR (CDCl3, 125MHz) δ: 169.0, 165.2, 163.4, 138.0,
(60% dispersion in mineral oil, 33.4mg, 0.84mmol) suspended 128.9, 123.7, 120.1, 36.8, 36.7. Anal. Calcd for C11H12ClN5: C,
in 1-butanol (2mL) was further added in one portion. The 52.91; H, 4.82; N, 28.05. Found: C, 52.80; H, 4.99; N, 28.10.
resulting mixture was stirred at 40°C for 4h, then poured into
2-Chloro-4-phenylamino-6-(pyrrolidin-1-yl)-1,3,5-tri-
water and extracted with ethyl acetate. The organic layer was azine (23) Pyrrolidine (45µL, 0.54mmol) was added to
washed with brine, dried over sodium sulfate and concen- a solution of 16 (101mg, 0.42mmol) in dichloromethane
trated. Purification of the residue by silica gel chromatography (2mL) at 0°C. The mixture was stirred at room temperature
(eluent: hexane–ethyl acetate 10:1) gave 19 (86%) as colorless for 15min, then poured into water and extracted with ethyl
1
plates (hexane); mp 58.4–59.4°C; H-NMR (CDCl3, 400MHz) acetate. The organic layer was washed with brine, dried over
δ: 7.58 (dd, 2H, J=8.8, 1.2Hz), 7.34 (td, 2H, J=7.2, 1.2Hz), sodium sulfate and concentrated. Purification of the residue
7.10 (tt, 1H, J=7.4, 1.6Hz), 7.12–7.08 (brs, 1H), 4.38 (t, 4H), by silica gel chromatography (eluent: hexane–ethyl acetate
1.78 (quint, 4H, J=7.2Hz), 1.48 (sex, 4H, J=7.4Hz), 0.96 (t, 7:1) gave 23 (34%) as colorless needles (ethyl acetate–hex-
1
4H, J=7.4Hz); 13C-NMR (CDCl3, 125MHz) δ: 172.0, 166.32, ane); mp 191.0–192.0°C; H-NMR (CDCl3, 400MHz) δ: 7.40
137.9, 128.9, 123.8, 120.4, 67.7, 30.7, 19.1, 13.8. Anal. Calcd for (td, 2H, J=7.4, 0.9Hz), 7.33 (dd, 2H, J=7.2, 2.0Hz), 7.08 (tt,
C17H24N4O2: C, 64.53; H, 7.65; N, 17.71. Found: C, 64.45; H, 1H, J=7.6, 1.2Hz), 7.00 (brs, 1H), 3.63 (t, 4H, J=6.8Hz),
7.37; N, 17.80.
1.99 (tt, 4H, J=3.4, 3.2Hz); 13C-NMR (CDCl3, 125MHz) δ:
2-Chloro-4-phenylamino-6-phenoxy-1,3,5-triazine (20) 168.7, 163.2, 1629, 138.2, 128.9, 123.5, 119.8, 46.8, 46.6, 25.2,
Sodium hydride (60% dispersion in mineral oil, 20.1mg, 25.0. Anal. Calcd for C11H12ClN5: C, 56.63; H, 5.12; N, 25.40.
0.49mmol) suspended in N,N-dimethylformamide (DMF) Found: C, 56.62; H, 5.17; N, 25.43.
(1mL) was added to phenol (67.3mg, 0.72mmol) and 16
2-Methylamino-4-methoxy-6-phenylamino-1,3,5-triazine
(101mg, 0.42mmol) in DMF at 0°C under Ar. The mixture (24) Methylammonium chloride (36.1mg, 0.42mmol) and
was stirred at 0°C for 2h, then poured into water and extracted Et3N (70µL, 0.50mmol) were added to 18 (99.9mg, 0.42mmol)
with ethyl acetate. The organic layer was washed with brine, in methanol (2mL) at 0°C. The mixture was stirred at room
dried over sodium sulfate and concentrated. Purification of temperature for 2h and at 40°C for 4h, then methylammoni-
the residue by silica gel chromatography (eluent: hexane–ethyl um chloride (34.5mg, 0.51mmol) and Et3N (70µL, 0.50mmol)
acetate 2:1) gave 20 (73%) as colorless crystals (hexane); mp were further added. The resulting mixture was refluxed for
1
137.1–138.8°C; H-NMR (DMSO-d6, 373K, 400MHz) δ: 10.36 1h, then poured into water and extracted with ethyl acetate.
(brs, 1H) 7.50–7.45 (m, 4H), 7.32 (tt, 1H, J=7.4, 1.2Hz), 7.28 The organic layer was washed with brine, dried over sodium
(dd, 2H, J=9.2, 1.6Hz), 7.23 (t, 1H, J=7.6Hz), 7.07 (t, 2H, sulfate and concentrated. Purification of the residue by silica
J=7.4Hz); Anal. Calcd for C15H11ClN4O: C, 60.31; H, 3.71; N, gel chromatography (eluent: hexane–ethyl acetate 5:1) gave
18.76. Found: C, 60.53; H, 3.85; N, 18.87.
24 (88%) as colorless crystals (ethyl acetate–hexane); mp
1
2-Chloro-4-methylamino-6-phenylamino-1,3,5-triazine 163.1–164.5°C; H-NMR (DMSO-d6, 373K, 400MHz) δ: 9.06
(21) Methylammonium chloride (70mg, 0.49mmol) and Et3N (brs, 1H), 7.73 (d, 2H, J=8.0Hz), 7.25 (t, 2H, J=7.8Hz), 7.01
(70µL, 0.50mmol) were added to 16 (101mg, 0.42mmol) in (brs, 1H), 6.95 (t, 1H, J=7.4Hz), 3.83 (s, 3H), 2.84 (d, 3 H
2-propanol (2mL) at 0°C. The mixture was stirred at room J=4.8Hz); Anal. Calcd for C11H13N5O: C, 57.13; H, 5.67; N,
temperature for 4h, and then methylammonium chloride 30.28. Found: C, 57.31; H, 5.65; N, 30.28.
(4mg, 0.06mmol) was further added in one portion. The
2-Dimethylamino-4-methoxy-6-phenylamino-1,3,5-
resulting mixture was stirred at room temperature for 1h, triazine (25) Dimethylammonium chloride (69.0mg,
then poured into water and extracted with ethyl acetate. The 0.85mmol) and Et3N (120µL, 0.80mmol) were added to a
organic layer was washed with brine, dried over sodium sul- solution of 18 (101mg, 0.43mmol) in methanol (2mL) at 0°C.
fate and concentrated. Purification of the residue by silica gel The mixture was stirred at room temperature for 2h and
chromatography (eluent: hexane–ethyl acetate 2:1) gave 21 at 50°C for 2h, then methylammonium chloride (38.2mg,
(53%) as a colorless powder (ethyl acetate); mp 243.9–245.3°C; 0.47mmol) and Et3N (60.0µL 0.43mmol) were added in one
1H-NMR (DMSO-d6, 373K, 400MHz) δ: 9.57 (brs, 1H), 7.69 portion. The resulting mixture was refluxed for 1h, then
(d, 2H, J=8.0Hz), 7.60 (brs, 1H), 7.29 (t, 2H, J=8.0Hz), 7.02 poured into water and extracted with ethyl acetate. The organ-
(t, 1H, J=7.2Hz), 2.84 (s, 3H); 13C-NMR (CDCl3, 125MHz) ic layer was washed with brine, dried over sodium sulfate and
δ: 168.9, 168.1, 166.2, 164.1, 163.6, 139.4, 129.1, 123.3, 120.5, concentrated. Purification of the residue by silica gel chroma-
17.9, 27.8. Anal. Calcd for C10H10ClN5: C, 50.96; H, 4.28; N, tography (eluent: hexane–ethyl acetate 5:1) gave 25 (78%) as

July 2014
707
colorless needles (ethyl acetate–hexane); mp 163.1–164.5°C;
2,4-Dichloro-6-benzylamino-1,3,5-triazine (30): Yield: 34%;
1H-NMR (CDCl3, 400MHz) δ: 7.61 (dd, 2H, J=8.8, 1.2Hz), colorless needles (dichloromethane–hexane); mp 120.1–121.3°C;
7.32 (td, 2H, J=7.2, 2.0Hz), 7.04 (tt, 1H, J=7.2, 1.2Hz), 1H-NMR (CDCl3, 400MHz) δ: 7.40 (m, 5H), 6.15 (brs, 1H),
6.85 (brs, 1H), 3.94 (s, 3H), 3.19 (s, 6H). 13C-NMR (CDCl3, 4.68 (d, 2H, J=6.0Hz); 13C-NMR (CDCl3, 125MHz) δ: 171.3,
125MHz) δ: 170.9, 166.6, 165.0, 139.0, 128.8, 122.8, 119.8, 170.3, 166.0, 136.3, 129.2, 128.4, 127.9, 45.6. Anal. Calcd for
54.0, 36.6, 36.3. Anal. Calcd for C12H15N5O: C, 58.76; H, 6.16; C10H8Cl2N4: C, 47.08; H, 3.16; N, 21.96. Found: C, 47.05; H,
N, 28.55. Found: C, 58.90; H, 6.04; N, 28.66.
3.22; N, 21.99.
2,4-Dichloro-6-methyl(phenyl)amino-1,3,5-triazine (26)
2,4-Dichloro-6-benzyl(methyl)amino-1,3,5-triazine
(31):
N-Methylaniline (58µL, 0.54mmol) and Et3N (75µL, Yield: 48%; colorless needles (hexane); mp 99.4–101.6°C;
0.54mmol) were added dropwise to a solution of cyanuric 1H-NMR (CDCl3, 400MHz) δ: 7.38–7.30 (m, 3H), 7.26–7.23
chloride (101mg, 0.55mmol) in acetone (2mL) at 0°C under (m, 2H), 4.87 (s, 2H), 3.15 (s, 3H); 13C-NMR (CDCl3,
Ar. The mixture was stirred at room temperature for 2h, 125MHz) δ: 170.5, 170.2, 165.3, 135.5, 129.1, 128.3, 128.0,
and then poured into water. The precipitate was collected 52.8, 35.0. Anal. Calcd for C11H10Cl2N4: C, 49.09; H, 3.75; N,
by filtration, washed with water, dried, and recrystallized 20.82. Found: C, 49.19; H, 3.64; N, 20.92.
to gave 26 (91%) as colorless prisms (CH2Cl2–hexane); mp
2,4-Dichloro-6-diethylamino-1,3,5-triazine (32): Yield: 54%;
1
1
134.0–132.7°C; H-NMR (CDCl3, 400MHz) δ: 7.46 (td, 2H, colorless crystals (hexane); mp 78.0–78.8°C; H-NMR (CDCl3,
J=7.8, 2.0Hz), 7.36 (tt, 1H, J=7.4, 2.0Hz), 7.24 (dd, 2H, 400MHz) δ: 3.63 (q, 4H, J=7.2Hz), 1.22 (t, 6H, J=7.2Hz);
J=7.2, 1.2Hz), 3.55 (s, 3H); 13C-NMR (CDCl3, 125MHz) δ: 13C-NMR (CDCl3, 125MHz) δ: 169.0, 163.0, 41.6, 11.7. Anal.
170.3, 170.0, 165.1, 142.0, 129.5, 127.8, 126.1, 39.2. Anal. Calcd Calcd for C7H10Cl2N4: C, 38.03; H, 4.56; N, 25.34. Found: C,
for C10H8Cl2N4: C, 47.12; H, 3.33; N, 21.85. Found: C, 47.08; 38.30; H, 4.56; N, 25.52.
H, 3.16; N, 21.96.
2,4-Dichloro-6-t-butylamino-1,3,5-triazine (33): Yield: 65%;
General Procedure for the Synthesis of Compounds colorless plates (hexane); mp 129.4–132.0°C; 1H-NMR
27–29 Anisidine (1eq) was added dropwise to a solution of (CDCl3, 400MHz) δ: 5.78 (brs, 1H), 1.46 (s, 9H).
cyanuric chloride (1eq) in acetone (2mL) at 0°C. The mixture
2,4-Dichloro-6-n-hexylamino-1,3,5-triazine (34): Yield: 56%;
was stirred at room temperature for 2h. After the completion colorless needles (hexane); mp 54.8–55.7°C; 1H-NMR
of the reaction, the mixture was evaporated. The crude prod- (CDCl3, 400MHz) δ: 3.47 (q, 2H, J=6.8Hz), 1.60 (quint, 2H,
uct was purified by silica gel chromatography (eluent: hexane– J=7.2Hz), 1.40–1.26 (m, 6H), 0.90 (t, 3H, J=6.8Hz).
ethyl acetate 5:1) and then recrystallized.
2,4-Dichloro-6-cyclohexylamino-1,3,5-triazine (35): Yield:
1
2,4-Dichloro-6-(2-methoxyphenyl)amino-1,3,5-triazine (27): 40%; colorless oil; H-NMR (CDCl3, 400MHz) δ: 5.76 (brs,
Yield: 86%; pale-yellow plates (ethyl acetate–hexane); mp 1H), 3.93–3.88 (m, 1H), 2.01–1.96 (m, 2H), 1.77–1.70 (m, 2H),
1
179.4–181.2°C; H-NMR (CDCl3, 400MHz) δ: 8.31 (dd, 1H, 1.67–1.61 (m, 1H), 1.47–1.36 (m, 2H), 1.29–1.20 (m, 3H);
J=8.0, 1.6Hz), 8.20 (brs, 1H), 7.15 (td, 1H, J=7.8, 1.2Hz), 13C-NMR (CDCl3, 125MHz) δ: 171.1, 170.0, 165.1, 50.3, 32.6,
7.04 (td, 1H, J=7.8, 1.2Hz), 6.94 (dd, 1H, J=8.0, 1.2Hz), 25.4, 24.5. Anal. Calcd for C9H12Cl2N4: C, 47.08; H, 3.16; N,
3.91 (s, 3H); 13C-NMR (CDCl3, 125MHz) δ: 171.4, 170.1, 21.96. Found: C, 47.05; H, 3.22; N, 21.99.
148.9, 125.7, 125.5, 121.2, 120.9, 110.5, 56.0. Anal. Calcd for
2,4-Dichloro-6-(piperidin-1-yl)-1,3,5-triazine (38): Yield:
C10H8Cl2N4O: C, 44.30; H, 2.97; N, 20.67. Found: C, 44.24; H, 40%; colorless plates (hexane); mp 88.9–89.4°C; 1H-NMR
3.09; N, 20.71.
(CDCl3, 400MHz) δ: 3.81 (t, 4H, J=5.6Hz), 1.75–1.69 (m, 2H),
2,4-Dichloro-6-(3-methoxyphenyl)amino-1,3,5-triazine (28): 1.67–1.61 (m, 4H); 13C-NMR (CDCl3, 125MHz) δ: 170.3, 163.7,
Yield: 72%; colorless needles (dichloromethane–hexane); mp 45.5, 25.8, 24.4. Anal. Calcd for C8H10Cl2N4: C, 41.22; H, 4.32;
1
116.7–117.8°C; H-NMR (CDCl3, 400MHz) δ: 7.50 (brs, 1H), N, 23.98. Found: C, 41.19; H, 4.68; N, 24.04.
7.30 (t, 1H, J=8.2Hz), 7.26 (t, 1H, J=2.4Hz), 7.05 (ddd, 1H,
General Procedure for the Synthesis of Compounds
J=8.0, 2.0, 0.8Hz), 6.77 (ddd, 1H, J=8.4, 2.4. 0.8Hz), 3.84 36, 37 and 39 Amine (1eq) in tetrahydrofuran was added
(s, 3H); 13C-NMR (CDCl3, 125MHz) δ: 171.5, 170.4, 164.1, dropwise to cyanuric chloride (1.2eq) and K2CO3 (1eq) in
160.4, 137.0, 130.2, 113.4, 111.3, 107.4, 55.6. Anal. Calcd for tetrahydrofuran at 0°C. The mixture was stirred at room tem-
C10H8Cl2N4O: C, 44.30; H, 2.97; N, 20.67. Found: C, 44.68; H, perature for 3–4h then poured into water and extracted with
3.25; N, 20.32.
ethyl acetate. The organic layer was washed with brine, dried
2,4-Dichloro-6-(4-methoxyphenyl)amino-1,3,5-triazine (29): over sodium sulfate and concentrated. The crude product was
Yield: 67%; pale-yellow plates (ethyl acetate–hexane); mp purified by silica gel chromatography (eluent: hexane–ethyl
1
170.0–173.4°C; H-NMR (CDCl3, 400MHz) δ: 7.43–7.40 (brs, acetate) and then recrystallized.
1H), 7.41 (dd, 2H, J=6.8, 2.4Hz), 6.93 (dd, 2H, J=6.8, 2.4Hz),
2,4-Dichloro-6-(furfurylamino)-1,3,5-triazine (36): Yield:
3.82 (s, 3H); 13C-NMR (CDCl3, 125MHz) δ: 171.5, 170.2, 92%; colorless needles (hexane–ethyl acetate); mp 104.7–
1
164.4, 128.5, 123.6, 114.6, 55.7. Anal. Calcd for C10H8Cl2N4O: 105.4°C; H-NMR (CDCl3, 400MHz) δ: 7.38 (dd, 1H, J=1.6,
C, 44.30; H, 2.97; N, 20.67. Found: C, 47.43; H, 3.26; N, 20.66. 0.4Hz), 6.35 (dd, 1H, J=3.2, 2.0Hz), 6.32 (dd, 1H, J=3.2,
General Procedure for the Synthesis of Compounds 0.8Hz), 6.20 (brs, 1H), 4.67 (d, 2H, J=6.0Hz); 13C-NMR
30–35 and 38 Amine (1eq) was added dropwise to cyanuric (CDCl3, 125MHz) δ: 171.2, 170.3, 165.8, 149.3, 143.0, 110.1,
chloride (1.2eq) in acetone at 0°C. The mixture was stirred at 108.7, 38.5. Anal. Calcd for C8H6Cl2N4O: C, 39.21; H, 2.47; N,
room temperature for 1h then poured into water and extracted 22.86. Found: C, 39.28; H, 2.49; N, 22.98.
with ethyl acetate. The organic layer was washed with brine,
2,4-Dichloro-6-(1-naphthylamino)-1,3,5-triazine (37): Yield:
dried over sodium sulfate and concentrated. The crude prod- 93%; colorless needles (hexane–ethyl acetate); mp 156.7–
1
uct was purified by silica gel chromatography (eluent: hexane– 157.8°C; H-NMR (CDCl3, 400MHz) δ: 7.92–7.80 (m, 5H),
ethyl acetate) and then recrystallized.
7.60–7.52 (m, 3H); 13C-NMR (CDCl3, 125MHz) δ: 171.8,

708
Vol. 62, No. 7
170.7, 165.9, 134.4, 130.5, 129.0, 128.2, 128.0, 127.3, 126.7, labeled NF-κB probe was added and incubation was continued
125.7, 122.9, 120.9. Anal. Calcd for C13H8Cl2N4: C, 53.63; H, at room temperature for 30min. The samples in a volume of
2.77; N, 19.24. Found: C, 53.58; H, 2.98; N, 19.26.
10 µL were loaded on native 5% polyacrylamide gel prepared
2,4-Dichloro-6-(1,2,3,4-tetrahydroquinolin-1-yl)-1,3,5-tri- in 0.5×TBE and electrophoresed at 120CV for 90min. The
azine (39): Yield: 93%; orange needles (hexane–ethyl acetate); gels were scanned and analyzed with an Odyssey Infrared
1
mp 159.2–160.6°C; H-NMR (CDCl3, 400MHz) δ: 7.72 (d, 1H, Imaging System (LI-COR).
J=8.0Hz), 7.27–7.16 (m, 3H),4.07 (t, 2H, J=6.4Hz), 2.83 (t,
2H, J=6.8Hz), 2.05 (quint, 2H, J=6.5Hz); 13C-NMR (CDCl3,
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125MHz) δ: 170.5, 170.4, 164.1, 136.7, 132.4, 129.0, 126.2,
126.0, 125.1, 45.7, 26.8, 23.6. Anal. Calcd for C12H10Cl2N4: C,
51.27; H, 5.59; N, 19.93. Found: C, 51.38; H, 3.60; N, 20.08.
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Alexa680-labeled NF-κB probe was prepared by annealing
with Alexa680-labeled single-stranded oligonucleotide and
unlabeled complementary single-stranded oligonucleotide:
5′-Alexa680-AGTTGAGGGGACTTTCCCAGGC-3′
(sense)
and 5′-GCCTGGGAAAGTCCCCTCAACT-3′ (antisense). The
underlines show the sequence of the κB site. His-tagged
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containing binding buffer (15mM Tris–HCl (pH 7.5), 75mM
NaCl, 1.5mM ethylenediamine tetraacetic acid (EDTA),
1.5 mM dithiothreitol, 7.52% glycerol, and 0.3% Nonidet P-40),
0.5µg of poly(dI-dC)·(dI-dC) and 62.5ng of His-tagged p50
recombinants were left to stand on ice for 10min, and then
the candidate inhibitors were added and the mixtures were
incubated at room temperature. After 30min, 20nM Alexa680
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