Synthesis and antitumor evaluation of a novel series of triaminotriazine derivatives

Article abstract of DOI:10.1016/j.bmc.2006.11.028

A series of triaminotriazine derivatives (compounds 5a-f, 6a-x, and 7a-g) was designed, synthesized, and evaluated for their inhibition activities to colorectal cancer (CRC) cell lines (HCT-116 and HT-29). Most of the synthesized compounds demonstrated moderate anti-proliferatory effects on both HCT-116 and HT-29 cell lines at the concentration of 10 μM. The inhibitory activities against HCT-116 and HT-29 cell lines were discussed to develop the structure-activity relationships of this new series. Compounds 6l and 6o exhibited prominent inhibition activities toward HCT-116, with IC₅₀s of 0.76 and 0.92 μM, respectively. The in vivo antitumor studies and pharmacokinetics of compound 6l showed that it might be a promising new hit for further development of antitumor agents.

Full text of DOI:10.1016/j.bmc.2006.11.028

Bioorganic & Medicinal Chemistry 15 (2007) 1815–1827
Synthesis and antitumor evaluation of a novel series of
triaminotriazine derivatives
Mingfang Zheng,a Chenghui Xu,b Jianwei Ma,a Yan Sun,c Feifei Du,c Hong Liu,a,*
Liping Lin,b,* Chuan Li,c Jian Ding,b Kaixian Chena and Hualiang Jianga,d,
*
aDrug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China
bDivision of Anti-tumor Pharmacology, Shanghai institute of Materia Medica, Shanghai, PR China
cCenter for DMPK Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China
dSchool of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
Received 4 September 2006; revised 16 November 2006; accepted 17 November 2006
Available online 19 November 2006
Abstract—A series of triaminotriazine derivatives (compounds 5a–f, 6a–x, and 7a–g) was designed, synthesized, and evaluated for
their inhibition activities to colorectal cancer (CRC) cell lines (HCT-116 and HT-29). Most of the synthesized compounds demon-
strated moderate anti-proliferatory effects on both HCT-116 and HT-29 cell lines at the concentration of 10 lM. The inhibitory
activities against HCT-116 and HT-29 cell lines were discussed to develop the structure–activity relationships of this new series.
Compounds 6l and 6o exhibited prominent inhibition activities toward HCT-116, with IC50s of 0.76 and 0.92 lM, respectively.
The in vivo antitumor studies and pharmacokinetics of compound 6l showed that it might be a promising new hit for further devel-
opment of antitumor agents.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Triazines have been widely studied due to its broad
range of biological activities, such as anti-microbial ef-
fects,1 Erm (erythromycin-resistance methylase) methyl-
transferase inhibition,2 anti-trypanosomal activity,3
VLA-4 (integrin very late antigen-4) antagonism,4 estro-
gen receptor modulation,5 and cytotoxic activity.6 Hex-
amethylmelamine (HMM) 1 (Fig. 1) was discovered as
an effective agent against breast, lung, and ovarian
cancers, but unfortunately it causes many severe adverse
effects, including nausea, vomiting, abdominal cramps,
and anorexia.7 Recently, more studies based on the
triazine scaffold toward antitumor activity have been
carried out.8–10 Compound 2 (Fig. 1) was reported
by Moon et al.8 as a microtubule destabilizing entity
with potent growth inhibition against U937 cells
OMe
OMe
Me
N
Me
N
HN
N
N
N
N
Me
Me
N
H
N
NH
N
N
Me
Me
MeO
1
2
N
H
N
O
N
N
OH
S
N
O
HN
N
N
N
N
N
3
N
N
N
H
Keywords: Antitumor; Triazine derivatives; Inhibitor; Colorectal
cancer.
4
*
Corresponding authors. Tel.: +86 21 50807042; fax: +86 21
50807088; e-mail addresses: hliu@mail.shcnc.ac.cn; lplin@jding.
dhs.org; hljiang@mail.shcnc.ac.cn
Figure 1. Reported triaminotriazine compounds with antitumor
activity.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.11.028

1816
M. Zheng et al. / Bioorg. Med. Chem. 15 (2007) 1815–1827
(GI50 = 1 lM). Leftheris et al.9 found compound 3 as a
potent inhibitor of p38 MAP kinase with oral activities.
Compound 4 was recently investigated by Baindur
et al.10 as potent VEGF-R2 (KDR) tyrosine kinase
inhibitor. From the reported structures, we notice that
it is a common feature for compounds to exhibit antitu-
mor activities by introducing structural units of various
arylamino groups into the triazine scaffold.
mor cell lines. One of the exciting screening results is
that compound (4,6-bis(N-morpholino)-[1,3,5]triazin-2-
yl)-phenylamine (5a, Table 1) exhibited moderate
inhibition activity toward HT-29 (one of the cell lines
of colorectal cancer), with 80.5% inhibition at the con-
centration of 10 lM. Nowadays, colorectal cancer
(CRC) has become one of the major cancers that threat-
en people’s lives. The American Cancer Society esti-
mates that there will be about 106,680 new cases of
colon cancer and 41,930 new cases of rectal cancer in
2006 in the United States, and they will cause about
55,170 deaths.11 Though the death rate from colorectal
cancer has been going down for the past 15 years, there
Based on the above findings and the availability of
abundant tri-substituted 1,3,5-triazine compounds in
our in-house collection, we were intrigued to screening
our compounds on selected targets, especially some tu-
Table 1. Chemical structures of compounds 5a–f and 6a–g, and their inhibitory effects on the growth of tumor cell lines
R3
N
N
R2
R2
N
R1
Compound
R1
R3
%Inhibition at 10 lMa
HT-29 HCT-116
5a
80.5
44.9
NH
O
O
O
O
O
O
N
N
N
N
N
N
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
N
N
N
N
N
N
N
5b
5c
5d
5e
5f
NIb
74.2
80.2
5.2
NIb
6.9
H2NO2S
H3CO
F
NH
NH
NIb
NIb
NIb
48.7
NIb
20.1
10.1
NIb
15.7
81.3
NH
NH
NIb
90.5
68.6
88.4
87.1
50.0
75.3
85.0
O
N
NH
NH
NH
NH
NH
6a
6b
6c
6d
6e
6f
NH
H2NO2S
H3CO
F
NH
NH
NH
NH
NH
NH
NH
6g
NH
a Values are means of three determinations and deviation from the mean is <10% of the mean value.
b NI, no inhibition.

M. Zheng et al. / Bioorg. Med. Chem. 15 (2007) 1815–1827
1817
Table 2. Chemical structures of compounds 6h–x and their inhibitory effects on the growth of tumor cell lines
R3
N
N
R2
N
R1
Compound
R1
R2
R3
%Inhibition at 10 lMa
HT-29 HCT-116
6h
87.1
51.7
NH
NH
NH
NH
NH
NH
Cl
Br
F
NH
NH
O
O
O
O
O
O
N
N
N
N
N
N
6i
89.7
90.0
87.3
77.7
60.1
63.4
81.4
78.3
6j
NH
6k
6l
F3CO
H3CO
NH
100
NH
NH
OCH3
6m
89.3
87.4
NH
6n
82.6
NH
O
N
H3CO
H3CO
H3CO
NH
6o
6p
6q
6r
76.4
87.5
80.4
87.8
80.6
82.3
85.5
54.1
81.4
83.2
84.7
64.1
96.2
85.8
53.3
NH
NH
NH
NH
O
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
N
N
N
N
C2H5O
H2NO2S
H2NOC
H3CO
NH
NH
NH
6s
H3CO
NH
NH
6t
NIb
F
F
NH
NH
H CO
3
NH
NH
6u
6v
6w
6x
62.0
71.1
79.2
69.0
F CO
3
H C
3
NH
H3CS
H3CO
NH
NH
H3CS
NH
H3CO
H3CO
H2NO2S
NH
NH
a Values are means of three determinations and deviation from the mean is <10% of the mean value.
b NI, no inhibition.

1818
M. Zheng et al. / Bioorg. Med. Chem. 15 (2007) 1815–1827
is a continuing urgent need to develop new potent
chemical agents.11,12 The finding of compound 5a
prompted us to undertake a study of the in vitro inhibi-
tion activities of triazine derivatives substituted by sub-
units of morpholino and arylamino toward CRC cell
lines, HCT-116 and HT-29. Rita Menicagli et al.6 had
reported 2,4,6-tris(N-morpholino)-1,3,5-triazine and
several 2-alkyl-4,6-bis(N-morpholino)-[1,3,5]triazines,
with negligible cytotoxic activities against leukemia cell
lines, L1210 and HL60, and glioma cell line C6, but
no (4,6-bis(N-morpholino)-[1,3,5]triazin-2-yl)-arylam-
ines and 6-morpholino-N,N0-diaryl-[1,3,5]triazine-2,4-
diamines have been considered.
2. Materials and methods
2.1. Chemistry
2.1.1. Design of analogues of triazine compounds. Based
on the structural feature of the screening hit 5a, 12 com-
pounds (5b–f and 6a–g, Table 1) were designed and syn-
thesized for the first round. Keeping the two morpholino
groups of 5a, we obtained compounds 5b–e by introduc-
ing different electronic substituents to the para position
of the phenyl ring of 5a, or substituting the phenyl ring
of 5a with benzyl. Substitution of the anilino group of 5a
with morpholino gave the tris(N-morpholino)-1,3,5-tri-
azine (5f). Replacing one of the morpholino units of
compounds 5a–e with benzylamino, p-methylbenzylami-
no or anilino unit, the corresponding mono-N-morpho-
lino substituted triazine derivatives (6a–g) were
obtained. According to the bioassay results of the first
round, we further designed and synthesized compounds
6h–x (Table 2), using 6-morpholino-N,N0-diphenyl-
[1,3,5]triazine-2,4-diamine (6g) as the benchmark com-
pound. Compounds 6h–r were obtained by introducing
various steric, electronic, and hydrophobic groups to
one of the phenyl rings of 6g. Compounds 6s–x were
prepared by introducing various substituents to both
of the phenyl rings of 6g. Displacing the morpholino
unit of the potent compounds 6 with the desired amines,
we synthesized compounds 7a–g (Table 3).
Herein, the design, synthesis, and biological activities of
a series of novel N-morpholino triaminotriazine deriva-
tives (5a–f, 6a–x, and 7a–g) are reported. The cellular
activities in relative colorectal tumor cell lines (HCT-
116 and HT-29) were examined and discussed to develop
the preliminary structure–activity relationships (SAR) of
this series. Most of the compounds exhibited moderate
inhibitory potency against the HCT-116 and HT-29 cell
lines at the concentration of 10 lM. Significantly, four
compounds (6g, 6l, 6o, and 6q) showed higher inhibition
activities against the proliferation of the HCT-116 cell
lines. The in vivo antitumor studies and pharmacokinet-
ics of compound 6l showed that it might be a promising
new hit for further development of antitumor agents.
Table 3. Chemical structures of compounds 7a–g and their inhibitory effects on the growth of tumor cell lines
R3
N
N
R2
N
R1
Compound
R1
R2
R3
%Inhibition at 10 lMa
HT-29
HCT-116
30.1
7a
23.4
O
O
N
N
NH
NH
NH
NH
NH
NH
NH
NH
NH
7b
7c
7d
7e
7f
74.6
33.9
13.7
76.1
77.3
49.3
NIb
NIb
NIb
22.0
19.0
NIb
H3CO
NH
NH
NH
NH
NH
NH
HO
H3CO
HO
HO
N
N
N
N
HO
HO
H3CO
H3CO
NH
NH
N
N
7g
H3CO
NH
a Values are means of three determinations and deviation from the mean is <10% of the mean value.
b NI, no inhibition.

M. Zheng et al. / Bioorg. Med. Chem. 15 (2007) 1815–1827
1819
d (R1 = R2)
Cl
N
Cl
R3
N
Cl
N
N
N
N
N
N
N
N
a
b
c
R2
R1
Cl
N
R1
R2
R1
Cl
N
8
Cl
–
–
–
5(a f)
9(a g)
10(a x)
–
6(a x)
–
7(a g)
f (R2 = R3 = morpholino)
e (R1 = R2 = R3 = morpholino)
Scheme 1. Synthetic procedures of target compounds 5–7. Reagents and conditions: (a) R1H, Na2CO3, acetone, 0 ꢁC, 3 h; (b) R2H, K2CO3 or
NaOH, acetone, rt, 5–12 h; (c) morpholine or 4-(2-aminoethyl)morpholine or ethanolamine or N-(2-hydroxyethyl)piperazine, K2CO3, DMF, rt; (d) 2
equiv R1H, Na2CO3, acetone, 0 ꢁC–rt, 3–8 h; (e) 6 equiv morpholine, acetone; (f) 4 equiv morpholine, acetone.
2.1.2. Synthetic methods. Scheme 1 depicts the sequence
of reactions that led to the preparation of compounds
5a–f, 6a–x, and 7a–g, using cyanuric chloride (8) as
the starting material. In general, treatment of commer-
cially available arylamine with 8 through chloride dis-
placement formed derivatives 9, which were then
reacted with 4 equiv morpholine to give compounds
5a–e. 8 was reacted with 6 equiv morpholine to produce
the tris(N-morpholino)-1,3,5-triazine (5f). Cyanuric
chloride (8) reacted with 2 equiv arylamine in the pres-
ence of Na2CO3 in acetone to afford compounds 10e,
10g, and10s. Compounds 9 were substituted by various
arylamines to produce derivatives 10a–d, 10f, 10h–r,
and10t–x. Subsequent coupling of 10 with the desired
amines under basic conditions formed the correspond-
ing triaminotriazines 6a–x and7a–g.
tion of 10 lM, respectively. Introducing substituents to
the para position of the phenyl ring of 5a (compounds
5b–d) did not improve the inhibition activities. Substitu-
tion of the anilino unit of 5a with benzylamino (5e) or
morpholino (5f) resulted in a complete loss of inhibitory
activities to both HCT-116 and HT-29. These findings
indicate that the anilino group of compound 5a plays
an important role in the potent inhibition activities of
the CRC cell lines. The mono-N-morpholino substituted
triazine derivatives (6a–e) presented more potent inhibi-
tory effects against HT-29 than their corresponding 4,6-
bis(N-morpholino)-[1,3,5]triazine derivatives (5a–e).
Among them, compounds 6a, 6c, and 6d were a little
more active than the initial compound 5a, with 90.5%,
88.4%, and 87.1% inhibition against HT-29 at 10 lM,
respectively. Dianilino derivative (6g) showed more po-
tent activity against HT-29 and a large improved inhib-
itory activity against HCT-116 in comparison with 5a,
with 85% and 81.3% inhibition at 10 lM, respectively.
Thus, compound 6g was chosen as the benchmark com-
pound for subsequent optimization studies.
3. Results and discussion
3.1. Analogue synthesis
In total, 36 compounds (5b–f, 6a–x, and 7a–g) were de-
signed and synthesized based on the structural feature of
the screening hit 5a, and their chemical structures are
shown in Tables 1–3. These compounds were synthe-
sized through the routes outlined in Scheme 1, and the
details of synthetic procedures and structural character-
izations are described in Section 5.
The inhibitory activities of the second round of com-
pounds 6h–x against HCT-116 and HT-29 were tested
and the results are summarized in Table 2. Compounds
6h–r, which retained the morpholino group and one of
the anilino units of 6g, were first investigated. Halogen
(F, Cl, and Br) substituted derivatives (6h–j) demon-
strated improved activities toward HT-29. However,
their inhibition activities toward HCT-116 were de-
creased. Electron-donating groups substituted on the
phenyl ring of 6g produced excellent to good anti-pro-
liferatory potencies against HT-29. For instance, com-
pounds 6l (4-OCH3), 6m (2-OCH3), 6n (3-OCH3), and
6p (4-OCH2CH3) exhibited high inhibition activity to-
ward HT-29, with 100%, 89.3%, 87.4%, and 87.5% inhi-
bition at 10 lM, respectively. The similar potency of
compounds 6l–n indicates that a methoxy substituent
walking on the phenyl ring had little impact on the inhi-
bition potency to both HCT-116 and HT-29. Synergistic
increase in activity was not found for the two methoxy-
substituted derivative (6o) toward HT-29, but was
found in its activity against HCT-116, with 84.7% inhi-
bition at 10 lM. The sulfanilamide derivative (6q) and
3.2. Biological activities
Compounds 5a–f, 6a–x, and 7a–g were evaluated and
analyzed by the sulforhodamine B (SRB, Sigma) assay13
for their inhibitory activities toward CRC cell lines
(HCT-116 and HT-29). For the primary assay, the per-
cent inhibitions of the compounds at the concentration
of 10 lM against HCT-116 and HT-29 were measured.
The results are summarized in Tables 1–3. The details
for bioassay procedures are described in Section 5.
As shown in Table 1, the initial compound 5a exhibited
potential inhibition activities toward HT-29 and HCT-
116, with 80.5% and 44.9% inhibition at the concentra-

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M. Zheng et al. / Bioorg. Med. Chem. 15 (2007) 1815–1827
p-aminobenzamide derivative (6r) exhibited the high
inhibitory activities against HCT-116, with 96.2% and
85.8% inhibition at 10 lM, respectively. Among the sec-
ond round of compounds, four compounds (6l, 6n, 6q,
and 6r) exhibited significant inhibitory potency against
both HT-29 and HCT-116.
for 50% growth inhibition of tumor cells) against HT-
29. Most of these compounds showed moderate growth
inhibition activities with IC50s ranging from 8.1 to
39 lM. Compound 6r (IC50 = 8.1 lM), which was most
prominent in this series of compounds against HT-29,
was nearly four times more active than the benchmark
compound 6g (IC50 = 31 lM). Compounds 6g, 6l, 6o,
6q–r, and 6v were tested for their IC50s against HCT-
116. Most of them proved to be potent inhibitors with
IC50 values below 5 lM, except compounds 6r and 6v.
Among them, compounds 6l (IC50 = 0.76 lM) and 6o
(IC50 = 0.92 lM) were the optimal ones, which were five
times more active relative to the benchmark compound
6g (IC50 = 4.7 lM). Compound 6l, which was the most
potent one in vitro against HCT-116, was chosen as
the representative one of this class of triaminotriazines
to undertake a study of its pharmacokinetic properties
and in vivo antitumor activities.
Compounds (6s–x), with substituents on both of the two
phenyl rings of 6g, showed similar inhibition potency
with the benchmark compound 6g. However, they were
all less active than the p-methoxy analogue (6l). De-
creased potency toward HCT-116 was observed
throughout this subseries of compounds, and the p-fluo-
ro derivative (6t) proved to be virtually inactive against
HCT-116 at 10 lM.
Table 3 lists the biological results of derivatives 7a–g,
which were designed based on the potent inhibitors of
6g and 6l. All these compounds showed decreased inhib-
itory activities toward both HCT-116 and HT-29, and a
few of them proved to have completely lost inhibitory
activity. These results suggest the morpholino subunit
directly introduced to the 1,3,5-triazine nuclear is an
important determinant of inhibitory activity toward
both HCT-116 and HT-29.
3.3. Pharmacokinetic properties and in vivo antitumor
activity of 6l
The pharmacokinetic (PK) properties of 6l were assessed
in Sprague–Dawley rats. The details of procedures are
described in Section 5. The orally administered 6l was
found to be rapidly absorbed from the gastrointestinal
To determine the exact potency of the compounds that
exhibited significant inhibition toward HCT-116 or
HT-29 at 10 lM, ten compounds (6g–i, 6l, 6n, 6o, 6q–
s, and 6v) were further investigated in concentration–
response studies, and the results are summarized in
Table 4. Compounds 6g–i, 6l, 6n, and 6r–s were tested
for their IC50s (the compound concentration required
tract. The mean peak concentration (Cmax) was
1.29 lg/mL achieved at 15 min after oral administration.
By comparing with intravenous data, the oral bioavail-
ability (F) of 6l was 30.9%. The elimination half-lives
(T1/2) of 6l by oral and intravenous administration were
1.16 and 1.09 h, respectively, while the mean resident
times (MRT) were 1.88 and 1.06 h, respectively. The dis-
tribution volume (Vd) and clearance (CL) of intravenous
6l were 2.11 and 1.36 L/h/kg, respectively.
Table 4. Determination of IC50 values of selected compounds of 6 on
the growth of CRC cell line
When evaluated for antitumor efficacy in a sarcoma 180
mice model, compound 6l demonstrated modest tumor
inhibitory activity with 40.7% inhibition at a dose of
200 mg/kg/day. The detailed experimental data are
shown in Table 5.
a
Compound
IC50 (lM)
HT-29
HCT-116
6g
6h
6i
31
37
4.7
NDb
NDb
0.76
NDb
0.92
2.0
25
30
6l
4. Conclusions
6n
6o
6q
6r
6s
6v
39
NDb
NDb
8.1
100
NDb
A series of triaminotriazine derivatives (5–7) was de-
signed and synthesized, based on the screening hit 5a.
All the compounds were evaluated for their growth inhi-
bition activities to colorectal tumor cell lines, HCT-116
and HT-29. 6-Morpholino-N,N0-diphenyl-[1,3,5]tri-
azine-2,4-diamine (6g) was discovered from the first
subseries of triazine derivatives with potent anti-prolif-
9.6
NDb
120
a Values are means of three determinations and deviation from the
mean is <10% of the mean value.
b ND, not determined.
Table 5. In vivo experimental data of compound 6la
Group
Dose (mg/kg)
Mice (n) Initial/end
Body weight (g) Initial/end
Tumor weight (g) X SD
Inhibition rate (%)
P
Control
—
100
200
20/20
10/10
10/10
18.6/29.4
18.5/29.3
18.6/27.3
1.50 0.52
1.65 0.42
0.89 0.44
—
—
6l
6l
ncb
40.7
ncb
<0.05
a The in vivo experiment was carried out in the mice sarcoma 180 model, using intraperitoneal (ip) treatment. For other detailed procedures, please
see Section 5.1.2.
b nc, not calculated.

M. Zheng et al. / Bioorg. Med. Chem. 15 (2007) 1815–1827
1821
erative activities against both HCT-116 and HT-29.
Using 6g as the benchmark compound, structural
modification led to a series of compounds (6h–x), which
demonstrated significant anti-proliferatory effects at the
concentration of 10 lM. Among them, compounds 6l
and 6o exhibited prominent activities to HCT-116, with
IC50s of 0.76 and 0.92 lM, respectively, which were five
times more active than the benchmark compound 6g.
Compound 6r was the most prominent derivative
against HT-29, which was nearly four times more active
than the benchmark compound 6g. The preliminary
in vivo antitumor studies and pharmacokinetics studies
on compound 6l showed that it might be promising
for the development of new antitumor agents. Further
investigations are in progress for understanding the
mechanism of action of this novel class of triaminotri-
azine compounds.
axilla of mice on Day 0, and mice were randomly
grouped (20 mice/group for control, and 10 mice/group
for drug-treated group) on Day 1. Intraperitoneal (ip)
treatment of the drug-treated groups was then adminis-
tered once daily for 7 days, with 200 mg/kg of 6l, which
was suspended in 0.5% CMC. Animals were sacrificed
24 h after the last administration, and mice and tumor
weights were measured. The rate of inhibition of tumor
growth in vivo was calculated using the following for-
mula: growth inhibition (%) = (average tumor weight
of control group ꢀ average tumor weight of test
group)/average tumor weight of control group · 100%.
5.2. Pharmacokinetics
Pharmacokinetic animal experiments were conducted
according to protocols approved by the Review Com-
mittee of Animal Care and Use at the Shanghai Institute
of Materia Medica (Shanghai, China).15 Six male Spra-
gue–Dawley rats (ꢃ250 g; Shanghai Laboratory Ani-
mals Co., Shanghai, China) were housed in two rat
cages (48 · 29 · 18 cm3) and maintained in an air-condi-
tioned rat room. Animals were allowed to acclimatize
for one week prior to experiments. Rats were given a
single oral dose (by gavage) of 6l at 10 mg/kg (suspended
in 0.5% CMC-Na); or a single intravenous dose at 2 mg/
kg [dissolved in a solvent containing PEG400 and
cremophor (100:15, v/v)], and serial blood samples
(0.22–0.25 mL; 0, 5, 15, 30 min, 1, 2, 4, 6, and 8 h) were
collected in heparinized tubes from the orbital sinus under
light ether anesthesia. The blood samples were centri-
fuged, and the plasma was frozen at ꢀ70 ꢁC until use.
5. Experimental
5.1. Biology
5.1.1. Cell growth inhibition assay. Two human colorectal
cell lines, HCT-116 and HT-29, obtained from American
Type Culture Collection (Rockville, MD) were used for
the cell proliferation assay. Cells were maintained in
RPMI-1640 medium supplemented with 10% (v/v) fetal
bovine serum, 2 mmol/L glutamine, 100 U/mL penicillin,
and 100 lg/mL streptomycin (Gibco, Grand Island, NY,
USA) in a highly humidified atmosphere of 95% air with
5% CO2 at 37 ꢁC. The growth inhibition was analyzed by
the sulforhodamine B (SRB, Sigma) assay.13 Briefly, the
cells were seeded at 6000 cells/well in 96-well plates (Fal-
con, CA, USA) and allowed to attach overnight. The cells
were treated in triplicate with grade concentrations of
compounds at 37 ꢁC for 72 h. Then, they were fixed with
10% trichloroacetic acid and incubated for 60 min at 4 ꢁC.
Then, the plates were washed and dried. SRB solution
(0.4% w/v in 1% acetic acid) was added and the culture
was incubated for an additional 15 min. After the plates
were washed and dried, bound stain was solubilized with
Tris buffer, and the optical densities were read on the plate
reader (model VERSA Max, Molecular Devices) at
515 nm (A515). The growth inhibitory rate (GIR) of treat-
ed cells was calculated by the following Eq. (1),
To determine the plasma concentration of 6l, 50 lL of
the thawed rat plasma sample were extracted with
1 mL EtOAc by vortex-mixing for 5 min and then cen-
trifuging at 16,060g for 5 min. The upper organic phase
(850 lL) was removed and dried at 35 ꢁC under a stream
of nitrogen. The residue was reconstituted in 50 lL of an
LC mobile phase and centrifuged at 13,000 rpm for
5 min. The supernatant (10 lL) was used for LC–MS/
MS analysis. The LC–MS/MS system consisted of a
Thermo Finnigan TSQ Quantum triple-quadrupole
mass spectrometer (Thermo Finnigan, San Jose, CA,
USA) interfaced via an electrospray ionization probe
with a Surveyor series liquid chromatography system,
an MS pump, and an autosampler (Thermo Finnigan).
The Xcalibur software (Version 1.3) was used to control
the LC–MS/MS system, as well as for data acquisition
and processing (Thermo Finnigan, San Jose, CA,
USA). Chromatographic separations were achieved on
a 5-lm Zorbax SB-C18 column (50 · 2.1 mm id; Agilent
Technologies, Chadds Ford, PA, USA) maintained at
25 ꢁC. A 0.2-lm filter (Upchurch Scientific, Oak Har-
bor, WA, USA) was used before the analytical column.
The LC mobile phase was CH3CN/H2O (636:364 v/v,
containing a mass fraction of 0.02% HCOONH4) at a
flow rate of 0.2 mL/min for an isocratic elution. The
mass spectrometer was operated in the positive ion elec-
trospray ionization and multiple-reaction monitoring
modes for 6l. The instrument parameters were optimized
for the analyte to maximize generation of the protonat-
ed molecules and to efficiently produce the characteristic
GIRð%Þ ¼ ½1 ꢀ ðA515 treated=A515ðcontrolÞÞꢁ
ꢂ 100%
ð1Þ
The results were also expressed as IC50 (the compound
concentration required for 50% growth inhibition of tu-
mor cells), which was calculated by the Logit method.
The mean IC50 was determined from the results of three
independent tests.
5.1.2. In vivo tumor growth inhibition assay. Female KM
mice (6–8 weeks of age) were used to study the inhibition
of tumor growth in vivo.14 The use of lab animals was in
accordance with guidelines of Experimental Animal
Association of China (certificate number: SYXK
[Shanghai] 2003-0029). Well-grown sarcoma 180 cells
(1–2 · 106/mL) were subcutaneously implanted into the

1822
M. Zheng et al. / Bioorg. Med. Chem. 15 (2007) 1815–1827
fragment ions. The precursor-to-product ion transitions
m/z 379 ! 250 for 6l were monitored with a scan time of
0.2 s per transition. The peak widths of the precursor
and product ions were maintained at 0.7 U.
5.3.2. General procedures for the preparations of 5b–e are
described as those for 5b.
5.3.2.1. 4-(4,6-Bis(N-morpholino)-[1,3,5]triazin-2-yla-
mino)-benzenesulfonamide (5b). To a solution of 4-(4,6-
dichloro-1,3,5-triazin-2-ylamino)benzenesulfonamide
(9b, 250 mg, 0.78 mmol) in acetone (10 mL) was added
morpholine (0.27 mL, 3.12 mmol). The reaction mixture
was stirred at room temperature for 5 h. The solvent
was removed under reduced pressure and ice water
(20 mL) was added. The solid was collected by vacuum fil-
tration, washed with water (3· 20 mL), dried in vacuo,
and purified by flash chromatography on silica gel to af-
To determine the pharmacokinetics of 6l, concentration-
time data were analyzed by noncompartmental methods
using KineticaTM 2000 software package (version 3.0,
InnaPhase Corp. Philadelphia, PA, USA). The peak
concentration (Cmax) and the time taken to achieve peak
concentration (Tpeak) were obtained directly from the
data without interpolation. The terminal elimination
half-life (t1/2) was calculated using the relationship
0.693/k, where k is the elimination rate constant. The
area unde r concentration-time curve up to the last mea-
surable time point (AUC0!t) was calculated by the trap-
ezoidal rule. The oral bioavailability (F) of orally
administered 6l was calculated by the following
equation:
ford 5b as
a white solid (300 mg, 92% yield).
1
Mp > 300 ꢁC. H NMR (300 MHz, DMSO-d6): d 7.84
(d, J = 9.0 Hz, 2H), 7.71 (t, J = 8.7 Hz, 2H), 3.71 (m,
8H), 3.64 (m, 8H). LRMS (EI) m/z 421 (M+), 391
(100%); HRMS (EI) m/z calcd for C17H23N7O4S (M+)
421.1532, found 421.1529.
5.3.2.2. (4,6-Bis(N-morpholino)-[1,3,5]triazin-2-yl)-(4-
methoxyphenyl)-amine (5c). Compound 5c was prepared
from 4,6-dichloro-N-(4-methoxyphenyl)-1,3,5-triazin-2-
amine (9c) and morpholine using a procedure similar
to that described for the preparation of 5b as a white sol-
AUCoral ꢂ Doseintravenous
F ¼
ꢂ 100%
ð2Þ
AUCintravenous ꢂ Doseoral
All results were expressed as arithmetic mean stan-
dard deviation (SD).
1
id. Yield: 96%; mp 206–207 ꢁC. H NMR (300 MHz,
DMSO-d6):
d 7.55 (d, J = 9.0 Hz, 2H), 6.84 (t,
5.3. Chemistry
J = 8.7 Hz, 2H), 3.60–3.70 (m, 19H). LRMS (EI) m/z
372 (M+, 100%); HRMS (EI) m/z calcd for
C18H24N6O3 (M+) 372.1910, found 372.1906.
The reagents (chemicals) were purchased from Shanghai
Chemical Reagent Company, Lancaster, and Acros, and
used without further purification. The type of analytical
thin-layer chromatography (TLC) was HSGF 254 (0.15–
0.2 mm thickness, Yantai Huiyou Company, China).
Yields were not optimized. Melting points were mea-
sured in capillary tube on a SGW X-4 melting point
apparatus without correction. Nuclear magnetic reso-
nance (NMR) spectra were recorded on a Brucker
AMX-300 NMR (TMS as IS). Chemical shifts were
reported in parts per million (ppm, d) downfield from tet-
ramethylsilane. Proton coupling patterns were described
as singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), and broad (br). Low- and high-resolution mass
spectra (LRMS and HRMS) were given with electric
(EI) produced by Finnigan MAT-95 mass spectrometer.
5.3.2.3. (4,6-Bis(N-morpholino)-[1,3,5]triazin-2-yl)-(4-
fluorophenyl)-amine (5d). Compound 5d was prepared
from
4,6-dichloro-N-(4-fluorophenyl)-1,3,5-triazin-2-
amine (9d) and morpholine using a procedure similar
to that described for the preparation of 5b as a white sol-
1
id. Yield: 95%; mp 199–200 ꢁC. H NMR (300 MHz,
CDCl3): d 7.48 (m, 2H), 7.01 (t, J = 8.4 Hz, 2H), 3.71–
3.81 (m, 16H). LRMS (EI) m/z 360 (M+, 100%); HRMS
(EI) m/z calcd for C17H21FN6O2 (M+) 360.1710, found
360.1709.
5.3.2.4. Benzyl-(4,6-bis(N-morpholino)-[1,3,5]triazin-
2-yl)-amine (5e). Compound 5e was prepared from N-
benzyl-4,6-dichloro-1,3,5-triazin-2-amine (9e) and mor-
pholine using a procedure similar to that described for
the preparation of 5b as a white solid. Yield: 92%; mp
160–161 ꢁC. 1H NMR (300 MHz, DMSO-d6): d 7.36
(t, J = 6.6 Hz, 1H), 7.17–7.29 (m, 5H), 4.40 (d,
J = 6.3 Hz, 2H), 3.55–3.61 (m, 16H). LRMS (EI) m/z
356 (M+, 100%); HRMS (EI) m/z calcd for
C18H24N6O2 (M+) 356.1961, found 356.1950.
5.3.1. General procedures for the preparations of inter-
mediates 9 are described as those for (4,6-dichloro-
[1,3,5]triazin-2-yl)-phenyl-amine (9a).
5.3.1.1. (4,6-Dichloro-[1,3,5]triazin-2-yl)phenylamine
(9a). To a solution of cyanuric chloride (8, 1.0 g,
5.4 mmol) in acetone (15 mL) at 0 ꢁC were added
Na2CO3 (5.4 mmol) and aniline (0.49 mL, 5.4 mmol).
The resulting mixture was stirred at 0 ꢁC for 2 h and then
for another 2 h at room temperature. The solvent was re-
moved under reduced pressure and ice water (20 mL) was
added. The solid was collected by vacuum filtration,
washed with water (3· 20 mL), dried in vacuo, and puri-
fied by flash chromatography on silica gel (EtOAc/petro-
leum ether 1:10) to afford the title compound as a white
solid (1.22 g, 93% yield). Mp 136–138 ꢁC. 1H NMR
(300 MHz, CDCl3): d 7.61 (s, 1H), 7.57 (d, J = 8.1 Hz,
2H), 7.43 (t, J = 8.1 Hz, 2H), 7.25 (t, J = 7.8 Hz, 2H).
LRMS (EI) m/z 241 (M+), 239 (100%).
5.3.3. Procedures for 2,4,6-tris(N-morpholino)-1,3,5-tri-
azine (5f). An ice-cooled solution of cyanuric chloride (8,
300 mg, 1.63 mmol) in acetone (8 mL) was treated with
morpholine (0.85 mL, 9.76 mmol). The reaction temper-
ature was gradually raised to 40 ꢁC until the complete
disappearance of the cyanuric chloride. The solvent
was removed under reduced pressure and 30 mL water
was added to the residue. The precipitate was filtered,
washed with water (3· 20 mL), and dried to afford com-
pound 5f as a white solid (525 mg, 96%). Mp 267 ꢁC. 1H
NMR (300 M, CDCl3): d 3.69–3.78 (m, 24H). LRMS

M. Zheng et al. / Bioorg. Med. Chem. 15 (2007) 1815–1827
1823
(EI) m/z 336 (M+, 100%); HRMS (EI) m/z calcd for
7.75 (d, J = 8.7 Hz, 1H), 7.63 (m, 2H), 7.17–7.31 (m, 5H),
4.46 (d, J = 12 Hz, 2H), 3.59–3.66 (m, 8H). LRMS (EI)
m/z 441 (M+, 100%); HRMS (EI) m/z calcd for
C20H23N7O3S (M+) 441.1583, found 441.1591.
C15H24N6O3 (M+) 336.1910, found 336.1910.
5.3.4. General procedures for the preparations of inter-
mediates 10, except for 10e, 10g, and 10s, are described as
those for 10a.
5.3.6.3. N-Benzyl-N0-(4-methoxyphenyl)-6-morpholi-
no-[1,3,5]triazine-2,4-diamine (6c). Compound 6c was
prepared from N-benzyl-6-chloro-N0-(4-methoxyphe-
nyl)-1,3,5-triazine-2,4-diamine (10c) and morpholine
using a procedure similar to that described for the prep-
aration of 6a as a white solid. Yield: 95%; mp 152–
153 ꢁC. 1H NMR (300 MHz, CDCl3): d 7.40 (d,
J = 9.0 Hz, 2H), 7.26–7.33 (m, 5H), 6.83 (d,
J = 9.0 Hz, 2H), 6.75 (br s, 1H), 5.27 (br s, 1H), 4.59
(d, J = 5.7 Hz, 2H), 3.68–3.78 (m, 11H). LRMS (EI)
m/z 392 (M+, 100%); HRMS (EI) m/z calcd for
C21H24N6O2 (M+) 392.1961, found 392.1962.
5.3.4.1. N-Benzyl-6-chloro-N0-phenyl-[1,3,5]triazine-
2,4-diamine (10a). To a solution of 9a (500 mg,
2.07 mmol) in acetone (10 mL) were added K2CO3
(286 mg, 2.07 mmol) and benzylamine (144 lL,
2.07 mmol). The resulting mixture was stirred at room
temperature for 5 h. The solvent was removed under re-
duced pressure and ice water (20 mL) was added. The sol-
id was collected by vacuum filtration, washed with water
(3· 20 mL), dried in vacuo, and purified by flash chroma-
tography on silica gel (EtOAc/petroleum ether 1:5) to give
10a as a white solid (530 mg, 82% yield). Mp 173–175 ꢁC.
1H NMR (300 MHz, DMSO-d6): d 7.55 (d, J = 8.1 Hz,
2H), 7.21–7.36 (m, 7H), 7.00 (t, J = 8.1 Hz, 1H), 4.49(s,
1H). LRMS (EI) m/z 311 (M+, 100%).
5.3.6.4. N-Benzyl-N0-(4-fluorophenyl)-6-morpholino-
[1,3,5]triazine-2,4-diamine (6d). Compound 6d was
prepared from N-benzyl-6-chloro-N0-(4-fluorophenyl)-
1,3,5-triazine-2,4-diamine (10d) and morpholine using a
procedure similar to that described for the preparation
5.3.5. General procedures for the preparations of inter-
mediates 10e, 10g, and 10s are described as those for 10g.
5.3.5.1. 6-Chloro-N,N0-diphenyl-[1,3,5]triazine-2,4-di-
amine (10g). To a solution of cyanuric chloride (8,
500 mg, 2.7 mmol) in acetone (10 mL) at 0 ꢁC were added
Na2CO3 (575 mg, 5.4 mmol) and aniline (494 lL,
5.4 mmol). The resulting mixture was stirred at 0 ꢁC for
2 h and then at room temperature for an additional 5 h.
The solvent was removed under reduced pressure and ice
water (20 mL) was added. The solid was collected by vacu-
um filtration, washed with water (3· 20 mL), dried in vac-
uo, and purified by flash chromatography on silica gel
(EtOAc/petroleum ether 1:5) to afford 10g as a white solid
1
of 6a as a white solid. Yield: 95%; mp 143–144 ꢁC. H
NMR (300 MHz, CDCl3): d 7.45 (m, 2H), 7.25–7.33
(m, 5H), 6.96 (t, J = 8.7 Hz, 2H), 6.78 (s, 1H), 5.28 (t,
J = 5.7 Hz, 1H), 4.59 (d, J = 6.0 Hz, 2H), 3.69–3.76 (m,
8H). LRMS (EI) m/z 380 (M+, 100%); HRMS (EI) m/z
calcd for C20H21FN6O (M+) 380.1761, found 380.1753.
5.3.6.5. N,N0-Dibenzyl-6-morpholino-[1,3,5]triazine-
2,4-diamine (6e). Compound 6e was prepared from
N,N0-dibenzyl-6-chloro-1,3,5-triazine-2,4-diamine (10e)
and morpholine using a procedure similar to that de-
scribed for the preparation of 6a as a white solid. Yield:
1
(694 mg, 86% yield). Mp 197 ꢁC. H NMR (300 MHz,
1
DMSO-d6): d 7.76 (s, 4H), 7.35 (t, J = 7.5 Hz, 4H), 7.10
92%; mp 135–136 ꢁC. H NMR (300 MHz, CDCl3): d
(t, J = 7.5 Hz, 2H). LRMS (EI) m/z 297 (M+, 100%).
7.23–7.31 (m, 10H), 5.17 (br s, 2H), 4.57 (d,
J = 5.7 Hz, 4H), 3.66–3.73 (m, 8H). LRMS (EI) m/z
376 (M+), 73 (100%); HRMS (EI) m/z calcd for
C21H24N6O (M+) 376.2012, found 376.2016.
5.3.6. General procedures for the preparations of target
compounds 6 are described as those for 6a.
5.3.6.1. N-Benzyl-6-morpholino-N0-phenyl-[1,3,5]tri-
azine-2,4-diamine (6a). To a solution of 10a (250 mg,
0.80 mmol) in acetone (10 mL) at room temperature
was added morpholine (140 lL, 1.6 mmol). The reaction
mixture was stirred at room temperature for 3 h. The sol-
vent was removed under reduced pressure and ice water
(20 mL) was added. The solid was collected by vacuum fil-
tration, washed with water (3· 20 mL), dried in vacuo,
and purified by flash chromatography on silica gel to give
6a as a white solid in 93% yield. Mp 121–123 ꢁC. 1H NMR
(300 MHz, CDCl3): d 7.53 (d, J = 8.7 Hz, 2H), 7.25–7.34
(m, 7H), 7.01 (t, J = 7.2 Hz, 1H), 6.87 (s, 1H), 5.31 (br s,
1H), 4.61 (d, J = 5.7 Hz, 2H), 3.69–3.77 (m, 8H). LRMS
(EI) m/z 362 (M+, 100%); HRMS (EI) m/z calcd for
C20H22N6O (M+) 362.1855, found 362.1867.
5.3.6.6. N-(4-Methylbenzyl)-6-morpholino-N0-phenyl-
[1,3,5]triazine-2,4-diamine (6f). Compound 6f was
prepared from N-benzyl-6-chloro-N0-p-tolyl-1,3,5-
triazine-2,4-diamine (10f) and morpholine using a pro-
cedure similar to that described for the preparation of
6a as a white solid. Yield: 91%; mp 80–85 ꢁC. 1H
NMR (300 MHz, DMSO-d6): d 7.73 (d, J = 7.8 Hz,
1H), 7.63 (d, J = 8.1 Hz, 1H), 7.22 (m, 4H), 7.11 (d,
J = 8.1 Hz, 2H), 6.89 (t, J = 7.2 Hz, 1H), 4.42 (d,
J = 13.2 Hz, 2H), 3.60–3.67 (m, 8H), 2.25 (s, 3H).
LRMS (EI) m/z 376 (M+, 100%); HRMS (EI) m/z
calcd for C21H24N6O (M+) 376.2012, found 376.2010.
5.3.6.7. 6-Morpholino-N,N0-diphenyl-[1,3,5]triazine-
2,4-diamine (6g). Compound 6g was prepared from 6-
chloro-N,N0-diphenyl-1,3,5-triazine-2,4-diamine (10g)
and morpholine using a procedure similar to that de-
scribed for the preparation of 6a as a white solid. Yield:
5.3.6.2. 4-(4-Benzylamino-6-morpholino-[1,3,5]triazin-
2-ylamino)-benzenesulfonamide (6b). Compound 6b was
prepared from 4-(4-(benzylamino)-6-chloro-1,3,5-triazin-
2-ylamino)benzenesulfonamide (10b) and morpholine
using a procedure similar to that described for the prepara-
tion of 6a as a white solid. Yield: 92%; mp 205–206 ꢁC. 1H
NMR (300 MHz, DMSO-d6): d 7.89 (d, J = 8.7 Hz, 1H),
1
96%; mp 195–197 ꢁC. H NMR (300 MHz, CDCl3): d
7.55 (d, J = 7.8 Hz, 4H), 7.32 (t, J = 6.8 Hz, 4H), 7.05
(t, J = 7.8 Hz, 2H), 6.88 (s, 2H), 3.82 (t, J = 4.8 Hz,
4H), 3.75 (t, J = 4.8 Hz, 4H). LRMS (EI) m/z 348

1824
M. Zheng et al. / Bioorg. Med. Chem. 15 (2007) 1815–1827
(M+, 100%); HRMS (EI) m/z calcd for C19H20N6O (M+)
348.1699, found 348.1699.
(t, J = 7.6 Hz, 1H), 6.91 (s, 1H), 6.88 (d, J = 6.8 Hz,
2H), 6.81 (s, 1H), 3.81 (m, 7H), 3.75 (t, J = 4.8 Hz,
4H). LRMS (EI) m/z 378 (M+, 100%); HRMS (EI) m/
z calcd for C20H22N6O2 (M+) 378.1804, found 378.1875.
5.3.6.8. N-(4-Chlorophenyl)-6-morpholino-N0-phenyl-
[1,3,5]triazine-2,4-diamine (6h). Compound 6h was
prepared from 6-chloro-N-(4-chlorophenyl)-N0-phen-
yl-1,3,5-triazine-2,4-diamine (10h) and morpholine
using a procedure similar to that described for the
preparation of 6a as a white solid. Yield: 96%; mp
226 ꢁC. 1H NMR (300 MHz, CDCl3): d 7.55 (d,
J = 7.8 Hz, 4H), 7.50 (d, J = 6.6 Hz, 2H), 7.33 (t,
J = 7.8 Hz, 2H), 7.27 (dd, J = 7.2 and 1.5 Hz, 2H),
7.08 (t, J = 7.2 Hz, 1H), 7.02 (br s, 2H), 3.74–3.84 (m,
8H). LRMS (EI) m/z 382 (M+, 100%); HRMS (EI)
m/z calcd for C19H19ClN6O (M+) 382.1309, found
382.1304.
5.3.6.13. N-(2-Methoxyphenyl)-6-morpholino-N0-phen-
yl-[1,3,5]triazine-2,4-diamine (6m). Compound 6m was
prepared from 6-chloro-N-(2-methoxyphenyl)-N0-phen-
yl-1,3,5-triazine-2,4-diamine (10m) and morpholine
using a procedure similar to that described for the prep-
aration of 6a as a white solid. Yield: 90%; mp 162–
163 ꢁC. 1H NMR (300 MHz, CDCl3): d 8.13 (br s,
1H), 7.74 (br s, 1H), 7.70 (d, J = 8.1 Hz, 2H), 7.26 (t,
J = 8.1 Hz, 2H), 7.05 (d, J = 3.6 Hz, 2H), 6.92–6.98
(m, 2H), 3.86 (s, 3H), 3.73 (m, 4H), 3.66 (m, 4H). LRMS
(EI) m/z 378 (M+), 347 (100%); HRMS (EI) m/z calcd
for C20H22N6O2 (M+) 378.1804, found 378.1800.
5.3.6.9. N-(4-Bromophenyl)-6-morpholino-N0-phenyl-
[1,3,5]triazine-2,4-diamine (6i). Compound 6i was
prepared from N-(4-bromophenyl)-6-chloro-N0-phen-
yl-1,3,5-triazine-2,4-diamine (10i) and morpholine
using a procedure similar to that described for the
preparation of 6a as a white solid. Yield: 97%; mp
5.3.6.14. N-(3-Methoxyphenyl)-6-morpholino-N0-phen-
yl-[1,3,5]triazine-2,4-diamine (6n). Compound 6n was pre-
pared from 6-chloro-N-(3-methoxyphenyl)-N0-phenyl-
1,3,5-triazine-2,4-diamine (10n) and morpholine using
a procedure similar to that described for the preparation
of 6a as a white solid. Yield: 94%; mp 164 ꢁC. 1H NMR
(300 MHz, DMSO-d6): d 7.75 (d, J = 7.5 Hz, 2H), 7.43
(s, 1H), 7.27 (m, 3H), 7.17 (t, J = 8.1 Hz, 1H), 6.96 (t,
J = 7.5 Hz, 1H), 6.55 (d, J = 7.5 Hz, 1H), 3.65–3.75
(m, 11H). LRMS (EI) m/z 378 (M+, 100%); HRMS
(EI) m/z calcd for C20H22N6O2 (M+) 378.1804, found
378.1792.
1
227–228 ꢁC. H NMR (300 MHz, DMSO-d6): d 7.73
(m, 4H), 7.43 (d, J = 9.0 Hz, 2H), 7.29 (t, J = 7.8 Hz,
2H), 6.98 (t, J = 7.5 Hz, 1H), 3.65–3.74 (m, 8H).
LRMS (EI) m/z 426 (M+, 100%); HRMS (EI) m/z calcd
for C19H19BrN6O (M+) 426.0804, found 426.0790.
5.3.6.10. N-(4-Fluorophenyl)-6-morpholino-N0-phenyl-
[1,3,5]triazine-2,4-diamine (6j). Compound 6j was pre-
pared from 6-chloro-N-(4-fluorophenyl)-N0-phenyl-
1,3,5-triazine-2,4-diamine (10j) and morpholine using a
procedure similar to that described for the preparation
5.3.6.15.
N-(3,4-Dimethoxyphenyl)-6-morpholino-N0-
phenyl-[1,3,5]triazine-2,4-diamine (6o). Compound 6o was
prepared from 6-chloro-N-(3,4-dimethoxyphenyl)-N0-
phenyl-1,3,5-triazine-2,4-diamine (10o) and morpholine
using a procedure similar to that described for the prepa-
ration of 6a as a white solid. Yield: 96%; mp 132–135 ꢁC.
1H NMR (300 MHz, CDCl3): d 7.53 (d, J = 7.6 Hz, 2H),
7.30 (t, J = 7.6 Hz, 2H), 7.23 (s, 1H), 7.04 (t, J = 7.2 Hz,
1H), 6.95 (dd, J = 8.4 and 2.4 Hz, 2H), 6.91 (s, 1H),
6.82 (d, J = 8.4 Hz, 1H), 3.87 (s, 3H), 3.82 (m, 7H), 3.73
(t, J = 5.2 Hz, 4H). LRMS (EI) m/z 408 (M+, 100%);
HRMS (EI) m/z calcd for C21H24N6O3 (M+) 408.1910,
found 408.1917.
1
of 6a as a white solid. Yield: 96%; mp 210–211 ꢁC. H
NMR (300 MHz, CDCl3): d 7.54 (dd, J = 8.7 &
1.2 Hz, 2H), 7.48 (m, 2H), 7.31 (t, J = 8.4 Hz, 2H),
6.97–7.08 (m, 3H), 6.96 (s, 1H), 6.91 (s, 1H), 3.72–3.83
(m, 8H). LRMS (EI) m/z 366 (M+, 100%); HRMS (EI)
m/z calcd for C19H19FN6O (M+) 366.1604, found
366.1602.
5.3.6.11. 6-Morpholino-N-phenyl-N0-(4-trifluorometh-
oxyphenyl)-[1,3,5]triazine-2,4-diamine (6k). Compound
6k was prepared from 6-chloro-N-phenyl-N0-(4-(triflu-
oromethoxy)phenyl)-1,3,5-triazine-2,4-diamine (10k)
and morpholine using a procedure similar to that de-
scribed for the preparation of 6a as a white solid. Yield:
93%; mp 178–180 ꢁC. H NMR (300 MHz, DMSO-d6):
d 7.85 (d, J = 9.0 Hz, 2H), 7.72 (d, J = 7.8 Hz, 2H),
7.28 (m, 4H), 6.97 (t, J = 7.5 Hz, 1H), 3.64–3.85 (m,
8H). LRMS (EI) m/z 432 (M+, 100%); HRMS (EI) m/
z calcd for C20H19F3N6O2 (M+) 432.1522, found
432.1521.
5.3.6.16. N-(4-Ethoxyphenyl)-6-morpholino-N0-phen-
yl-[1,3,5]triazine-2,4-diamine (6p). Compound 6p was
prepared from 6-chloro-N-(3-ethoxyphenyl)-N0-phenyl-
1,3,5-triazine-2,4-diamine (10p) and morpholine using
a procedure similar to that described for the preparation
1
1
of 6a as a white solid. Yield: 95%; mp 174–175 ꢁC. H
NMR (300 MHz, DMSO-d6): d 7.74 (d, J = 7.8 Hz,
2H), 7.59 (d, J = 9.0 Hz, 2H), 7.27 (t, J = 7.8 Hz, 2H),
6.95 (t, J = 7.2 Hz, 1H), 6.85 (d, J = 9.0 Hz, 2H), 3.98
(q, J = 6.9 Hz, 2H), 3.64–3.73 (m, 8H), 1.31 (t,
J = 6.9 Hz, 3H). LRMS (EI) m/z 392 (M+, 100%);
HRMS (EI) m/z calcd for C21H24N6O2 (M+) 392.1961,
found 392.1958.
5.3.6.12. N-(4-Methoxyphenyl)-6-morpholino-N0-phen-
yl-[1,3,5]triazine-2,4-diamine (6l). Compound 6l was
prepared from 6-chloro-N-(4-methoxyphenyl)-N0-phen-
yl-1,3,5-triazine-2,4-diamine (10l) and morpholine using
a procedure similar to that described for the preparation
5.3.6.17. 4-(4-Morpholino-6-phenylamino-[1,3,5]tria-
zin-2-ylamino)-benzenesulfonamide (6q). Compound 6q
was prepared from 4-(4-chloro-6-(phenylamino)-1,3,5-
triazin-2-ylamino)benzenesulfonamide (10q) and mor-
1
of 6a as a white solid. Yield: 94%; mp 198–200 ꢁC. H
NMR (300 MHz, CDCl3): d 7.55 (d, J = 7.6 Hz, 2H),
7.45 (d, J = 7.2 Hz, 2H), 7.30 (t, J = 7.6 Hz, 2H), 7.05

M. Zheng et al. / Bioorg. Med. Chem. 15 (2007) 1815–1827
1825
pholine using a procedure similar to that described for
the preparation of 6a as a white solid. Yield: 90%; mp
245–248 ꢁC. 1H NMR (300 MHz, DMSO-d6): d 7.95
(d, J = 8.7 Hz, 2H), 7.78 (d, J = 8.7 Hz, 2H), 7.74 (d,
J = 8.7 Hz, 2H), 7.29 (t, J = 7.6 Hz, 2H), 7.00 (t,
J = 7.6 Hz, 1H), 3.79 (t, J = 4.8 Hz, 4H), 3.68 (t,
J = 4.8 Hz, 4H). LRMS (EI) m/z 427 (M+, 100%);
HRMS (EI) m/z calcd for C19H21N7O3S (M+)
427.1427, found 427.1411.
6v was prepared from 6-chloro-N-(4-methylsulfanylphe-
nyl)-N0-p-tolyl-1,3,5-triazine-2,4-diamine (10v) and mor-
pholine using a procedure similar to that described for
the preparation of 6a as a white solid. Yield: 97%; mp
230–231 ꢁC. 1H NMR (300 MHz, DMSO-d6): d 7.70
(d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.1 Hz, 2H), 7.20 (d,
J = 8.7 Hz, 2H), 7.07 (t, J = 8.4 Hz, 2H), 3.63–3.71 (m,
8H), 2.43 (s, 3H), 2.24 (s, 3H). LRMS (EI) m/z 408
(M+, 100%); HRMS (EI) m/z calcd for C21H24N6OS
(M+) 408.1732, found 408.1735.
5.3.6.18. 4-(4-Morpholino-6-phenylamino-[1,3,5]tria-
zin-2-ylamino)-benzamide (6r). Compound 6r was
prepared from N-(4-(aminooxycarbonyl)phenyl)-6-
chloro-N-phenyl-1,3,5-triazine-2,4-diamine (10r) and
morpholine using a procedure similar to that described
for the preparation of 6a as a white solid. Yield: 87%;
mp 222–223 ꢁC. 1H NMR (300 MHz, DMSO-d6): d
7.78–7.85 (m, 5H), 7.73 (d, J = 8.1 Hz, 2H), 7.30 (t,
J = 8.1 Hz, 2H), 7.18 (s, 1H), 6.99 (t, J = 7.5 Hz, 1H),
3.66–3.76 (m, 8H). LRMS (EI) m/z 391 (M+, 100%);
HRMS (EI) m/z calcd for C20H21N7O2 (M+)
391.1757, found 391.1741.
5.3.6.23. N-(3,4-Dimethoxyphenyl)-N0-(4-methylsulfa-
nylphenyl)-6-morpholino-[1,3,5]triazine-2,4-diamine (6w).
Compound 6w was prepared from 6-chloro-N-(3,4-
dimethoxyphenyl)-N0-(4-methylsulfanylphenyl)-1,3,5-
triazine-2,4-diamine (10w) and morpholine using a
procedure similar to that described for the preparation
1
of 6a as a white solid. Yield: 95%; mp 124–126 ꢁC. H
NMR (300 MHz, DMSO-d6): d 7.70 (d, J = 6.3 Hz,
2H), 7.41 (s, 1H), 7.16–7.20 (m, 3H), 6.86 (d, J =
9.0 Hz, 2H), 3.63–3.71 (m, 14H), 2.43 (s, 3H). LRMS
(EI) m/z 454 (M+, 100%); HRMS (EI) m/z calcd for
C22H26N6O3S (M+) 454.1787, found 454.1790.
5.3.6.19. N,N0-Bis(4-methoxyphenyl)-6-morpholino-
[1,3,5]triazine-2,4-diamine (6s). Compound 6s was
prepared from 6-chloro-N,N0-bis(4-methoxyphenyl)-
1,3,5-triazine-2,4-diamine (10s) and morpholine using
a procedure similar to that described for the prepara-
tion of 6a as a white solid. Yield: 96%; mp 192–
193 ꢁC. 1H NMR (300 MHz, CDCl3): d 7.42 (d,
J = 8.7 Hz, 4H), 6.86 (d, J = 8.7 Hz, 4H), 6.78 (s,
2H), 3.72–3.80 (m, 14H). LRMS (EI) m/z 408 (M+,
100%); HRMS (EI) m/z calcd for C21H24N6O3 (M+)
408.1910, found 408.1904.
5.3.6.24. 4-(4-(4-Methoxyphenylamino)-6-morpholino-
1,3,5-triazin-2-ylamino)benzenesulfonamide (6x). Com-
pound 6x was prepared from 4-(4-chloro-6-(4-methoxy-
phenylamino)-1,3,5-triazin-2-ylamino)benzenesulfona-
mide (10x) and morpholine using a procedure similar to
that described for the preparation of 6a as a white solid.
Yield: 94%; mp 257–258 ꢁC. 1H NMR (300 MHz,
DMSO-d6): d 7.91 (d, J = 7.2 Hz, 2H), 7.68 (d,
J = 8.7 Hz, 2H), 7.57 (d, J = 8.7 Hz, 2H), 6.88 (d,
J = 7.2 Hz, 2H), 3.73 (m, 7H), 3.65 (m, 4H). LRMS
(EI) m/z 457 (M+, 100%); HRMS (EI) m/z calcd for
C20H23N7O4S (M+) 457.1532, found 457.1522.
5.3.6.20. N-(4-Fluorophenyl)-N0-(4-methoxyphenyl)-6-
morpholino-[1,3,5]triazine-2,4-diamine (6t). Compound
6t was prepared from 6-chloro-N-(4-fluorophenyl)-N0-
(4-methoxyphenyl)-1,3,5-triazine-2,4-diamine (10t) and
morpholine using a procedure similar to that described
for the preparation of 6a as a white solid. Yield: 93%;
5.3.7. General procedures for the preparations of com-
pounds 7 are described as those for 7a.
5.3.7.1. N-(2-Morpholinoethyl)-N0,N00-diphenyl-[1,3,5]
triazine-2,4,6-triamine (7a). To a solution of 6-chloro-
N,N0-diphenyl-1,3,5-triazine-2,4-diamine (10g, 250 mg,
0.84 mmol) in acetone (10 mL) at room temperature were
added 2-(4-morpholino)ethylamine (110 lL, 0.84 mmol)
and K2CO3 (116 mg, 0.84 mmol). The reaction mixture
was stirred at room temperature for 5 h. The solvent
was removed under reduced pressure and ice water
(20 mL) was added. The resulting mixture was extracted
with ethyl acetate (3· 15 mL). The combined extracts
were washed with brine (3· 10 mL), dried over anhydrous
sodium sulfate, filtered, concentrated, and purified by
flash chromatography on silica gel to afford 7a as a white
1
mp 200–201 ꢁC. H NMR (300 MHz, CDCl3): d 7.47
(m, 2H), 7.41 (d, J = 9.0 Hz, 2H), 6.70 (t, J = 8.7 Hz,
2H), 6.87 (d, J = 9.3 Hz, 2H), 6.82 (s, 1H), 6.77 (s,
1H), 3.71–3.81 (m, 11H). LRMS (EI) m/z 396 (M+,
100%); HRMS (EI) m/z calcd for C20H21FN6O2 (M+)
396.1710, found 396.1710.
5.3.6.21. N-(4-Fluorophenyl)-6-morpholino-N0-(4-tri-
fluoromethoxyphenyl)-[1,3,5]triazine-2,4-diamine (6u).
Compound 6u was prepared from 6-chloro-N-(4-fluor-
ophenyl)-N0-(4-(trifluoromethoxy)phenyl)-1,3,5-triazine-
2,4-diamine (10u) and morpholine using a procedure
similar to that described for the preparation of 6a as a
white solid. Yield: 89%; mp 198–199 ꢁC. 1H NMR
(300 MHz, CDCl3): d 7.55 (d, J = 9.3 Hz, 2H), 7.47
(m, 2H), 7.15 (d, J = 8.7 Hz, 2H), 7.01 (t, J = 9.0 Hz,
2H), 6.88 (s, 1H), 6.80 (s, 1H), 3.73–3.82 (m, 8H).
LRMS (EI) m/z 450 (M+, 100%); HRMS (EI) m/z calcd
for C20H18F4N6O2 (M+) 450.1427, found 450.1441.
1
solid (270 mg, 82% yield). Mp 167–168 ꢁC. H NMR
(300 MHz, CDCl3): d 7.59 (m, 4H), 7.34 (t, J = 7.5 Hz,
4H), 7.07 (t, J = 7.2 Hz, 2H), 6.96 (s, 2H), 5.58 (t,
J = 6.0 Hz, 1H), 3.74 (t, J = 4.8 Hz, 4H), 3.55 (q,
J = 5.7 Hz, 2H), 2.59 (t, J = 6.0 Hz, 2H), 2.50 (t,
J = 4.8 Hz, 4H). LRMS (EI) m/z 391 (M+), 279 (100%);
HRMS (EI) m/z calcd for C21H25N7O (M+) 391.2121,
found 391.2120.
5.3.6.22. N-(4-Methylsulfanylphenyl)-6-morpholino-
N0-p-tolyl-[1,3,5]triazine-2,4-diamine (6v). Compound
5.3.7.2. N-(4-Methoxyphenyl)-N0-(2-morpholinoethyl)-
N00-phenyl-[1,3,5]triazine-2,4,6-triamine (7b). Compound

1826
M. Zheng et al. / Bioorg. Med. Chem. 15 (2007) 1815–1827
7b was prepared from 6-chloro-N-(4-methoxyphenyl)-
N0-phenyl-1,3,5-triazine-2,4-diamine (10l) and 2-(4-mor-
pholino)ethylamine using a procedure similar to that de-
scribed for the preparation of 7a as a white solid. Yield:
3.80 (s, 3H), 3.69 (t, J = 5.7 Hz, 2H), 3.00 (s, 2H), 2.58–
2.64 (m, 6H). LRMS (EI) m/z 421 (M+), 321 (100%);
HRMS (EI) m/z calcd for C22H27N7O2 (M+) 421.2226,
found 421.2201.
1
88%; mp 147–148 ꢁC. H NMR (300 MHz, CDCl3): d
7.57 (d, J = 7.5 Hz, 2H), 7.45 (d, J = 7.5 Hz, 2H), 7.30
(t, J = 8.7 Hz, 2H), 7.04 (t, J = 7.2 Hz, 2H), 6.89 (br s,
1H), 6.87 (d, J = 9.0 Hz, 2H), 6.85 (br s, 1H), 5.54 (t,
J = 6.0 Hz, 1H), 3.81 (s, 3H), 3.71 (t, J = 4.8 Hz, 4H),
3.51 (q, J = 5.7 Hz, 2H), 2.56 (t, J = 5.4 Hz, 2H), 2.47
(t, J = 4.5 Hz, 4H). LRMS (EI) m/z 421 (M+), 309
(100%); HRMS (EI) m/z calcd for C22H27N7O2 (M+)
421.2226, found 421.2211.
5.3.7.7. 2-(4-(4,6-Bis(4-methoxyphenylamino)-1,3,5-
triazin-2-yl)piperazin-1-yl)ethanol (7g). Compound 7g
was prepared from 6-chloro-N,N0-bis(4-methoxyphe-
nyl)-1,3,5-triazine-2,4-diamine (10s) and 2-(piperazin-1-
yl)ethanol using a procedure similar to that described
for the preparation of 7a as a white solid. Yield: 75%;
1
mp 149–151 ꢁC. H NMR (300 MHz, CDCl3): d 7.44
(d, J = 9.3 Hz, 4H), 6.86 (d, J = 9.0 Hz, 4H), 3.84 (m,
10H), 3.68 (t, J = 6.8 Hz, 2H), 2.54–2.61 (m, 6H).
LRMS (EI) m/z 451 (M+), 352 (100%); HRMS (EI) m/
z calcd for C23H29N7O3 (M+) 451.2332, found 451.2305.
5.3.7.3. 2-(4,6-Bis(phenylamino)-[1,3,5]triazin-2-ylami-
no)ethanol (7c). Compound 7c was prepared from 6-
chloro-N,N0-diphenyl-1,3,5-triazine-2,4-diamine (10g)
and 2-aminoethanol using a procedure similar to that
described for the preparation of 7a as a white sol-
id.Yield: 85%; mp 182–183 ꢁC. 1H NMR (300 MHz,
Acknowledgments
DMSO-d6):
d
7.80 (d, J = 8.1 Hz, 4H), 7.25 (t,
We gratefully acknowledge financial support from the
National Natural Science Foundation of China (Grants
20372069, 29725203, 20472094, and 20102007), the Basic
Research Project for Talent Research Group from the
Shanghai Science and Technology Commission, the
Key Project from the Shanghai Science and Technology
Commission (Grant 02DJ14006), the Key Project for
New Drug Research from CAS, and the 863 Hi-Tech
Programm (Grants 2002AA233061 and 2003AA235030).
J = 7.8 Hz, 4H), 6.94 (t, J = 7.5 Hz, 2H), 3.54 (t,
J = 6.0 Hz, 2H), 3.38 (q, J = 5.7 Hz, 2H). LRMS (EI)
m/z 322 (M+), 304 (100%); HRMS (EI) m/z calcd for
C17H18N6O (M+) 322.1542, found 322.1540.
5.3.7.4. 2-(4-(4-Methoxyphenylamino)-6-(phenylamino)-
1,3,5-triazin-2-ylamino)ethanol (7d). Compound 7d was
prepared from 6-chloro-N-(4-methoxyphenyl)-N0-phenyl-
1,3,5-triazine-2,4-diamine (10l) and 2-aminoethanol using
a procedure similar to that described for the preparation
1
of 7a as a white solid. Yield: 84%; mp 173–174 ꢁC. H
References and notes
NMR (300 MHz, CDCl3): d 7.55 (d, J = 8.7 Hz, 4H),
7.42 (d, J = 9.0 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 7.06
(t, J = 7.5 Hz, 1H), 6.86 (t, J = 8.7 Hz, 2H), 6.79 (s,
1H), 6.70 (s, 1H), 5.45 (t, J = 5.4 Hz, 1H), 3.78–3.82 (m,
5H), 3.58 (q, J = 5.1 Hz, 2H). LRMS (EI) m/z 352 (M+,
100%); HRMS (EI) m/z calcd for C18H20N6O2 (M+)
352.1648, found 352.1631.
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3.88 (t, J = 5.1 Hz, 4H), 3.69 (t, J = 5.7 Hz, 2H), 2.90
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