Synthesis and biological evaluation of novel 6-hydrazinyl-2,4-bismorpholino pyrimidine and 1,3,5-triazine derivatives as potential antitumor agents

Article abstract of DOI:10.1002/ardp.201200074

A series of 6-hydrazinyl-2,4-bismorpholino pyrimidine and 1,3,5-triazine derivatives (5a-5l and 8a-8o) were synthesized and their chemical structures as well as the relative stereochemistry were confirmed. All the synthesized compounds were evaluated for antiproliferative activity against three cancer cell lines (H460, HT-29, and MDA-MB-231). Several potent compounds were further evaluated against two other cell lines (U87MG, H1975). Most of the prepared compounds, particularly compounds 5c and 5j with IC₅₀ values (0.07 and 0.05 μM, respectively) in the nM range, exhibited moderate to excellent antiproliferative activity and high selectivity against the H460 cancer cell line as compared with compound 1. The most promising compound 5j, possessing a cyano group at the 3-position of the benzene ring, showed strong antiproliferative activity against H460, HT-29, and MDA-MB-231 cell lines with IC₅₀ values of 0.05, 6.31, and 6.50 μM, which were 4.6- to 190.4-fold more active than compound 1 (9.52, 29.24, and 36.21 μM), respectively. Copyright

Full text of DOI:10.1002/ardp.201200074

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
1
Full Paper
Synthesis and Biological Evaluation of Novel 6-Hydrazinyl-
2,4-bismorpholino pyrimidine and 1,3,5-Triazine Derivatives
as Potential Antitumor Agents
Wufu Zhu, Yajing Liu, Yanfang Zhao, Haiyan Wang, Li Tan, Weijie Fan, and Ping Gong
Key Laboratory of Original New Drugs Design and Discovery of Ministry of Education, Shenyang
Pharmaceutical University, Shenhe District, P. R. China
A series of 6-hydrazinyl-2,4-bismorpholino pyrimidine and 1,3,5-triazine derivatives (5a–5l and 8a–8o)
were synthesized and their chemical structures as well as the relative stereochemistry were
confirmed. All the synthesized compounds were evaluated for antiproliferative activity against
three cancer cell lines (H460, HT-29, and MDA-MB-231). Several potent compounds were further
evaluated against two other cell lines (U87MG, H1975). Most of the prepared compounds,
particularly compounds 5c and 5j with IC50 values (0.07 and 0.05 mM, respectively) in the nM
range, exhibited moderate to excellent antiproliferative activity and high selectivity against the
H460 cancer cell line as compared with compound 1. The most promising compound 5j, possessing a
cyano group at the 3-position of the benzene ring, showed strong antiproliferative activity against
H460, HT-29, and MDA-MB-231 cell lines with IC50 values of 0.05, 6.31, and 6.50 mM, which were 4.6- to
190.4-fold more active than compound 1 (9.52, 29.24, and 36.21 mM), respectively.
Keywords: Antiproliferative activity / Bismorpholino pyrimidines and 1,3,5-triazines / Synthesis
Received: February 27, 2012; Revised: May 4, 2012; Accepted: May 11, 2012
DOI 10.1002/ardp.201200074
Supporting information available online
:
Introduction
pound 1 is a triazine-hydrazone derivative which exhibited
potent antitumor activity with an mTOR IC50 of 0.27 mM [24].
To our knowledge, few studies on pyrimidine- or triazine-
hydrazone have been reported to date. Importantly, the
hydrazinyl group and its mimics are key pharmacophores
in the design of different potential anticancer agents [24–26]
and some of these compounds are undergoing clinical trials,
such as PAC-1 [25]. Therefore, the researches on this aspect
seemed to be an attractive task.
Pyrimidine- and triazine-containing compounds, particularly
1,3,5-triazines, have received considerable attention owing to
their broad biological activities, such as antitumor activity
involving different targets [1–4], antimalarial [5–7], antileish-
manial [8, 9], and antiinflammatory activity [10]. Especially in
recent years, more studies based on pyrimidine and 1,3,5-
triazine scaffolds toward antitumor activity have been car-
ried out [11–24]. According to these studies, we notice that
the pyrimidine and 1,3,5-triazine cores occur usually in con-
junction with bis-morpholine groups, such as NVP-BKM120
[16–18], ZSTK474 [19–21], PKI-587 [22, 23], and compound 1
[24] (Fig. 1). Interestingly, nearly all of these compounds
showed potent antitumor activity, and NVP-BKM120 is cur-
rently undergoing phase II clinical trials. Among them, com-
Indole nucleus is frequently found in medicinal chemistry
and is considered as a privileged scaffold [27–30]. In the past
few years, many of the indole-based small molecules were
identified as potent anticancer agents [31–37]. It is note-
worthy that indole derivatives oncrasin-1 and bis(indolyl)
hydrazide-hydrazones (Fig. 1) showed potent antiprolifera-
tive activity against several cancer cell lines [34–36].
In continuation of our previous research toward the identi-
fication of hydrazones as potent antitumor agents [26], our
group focused on the synthesis of novel hydrazinyl group
derivatives based on pyrimidine and triazine scaffolds.
Design of novel compounds was carried out using 1 as
Correspondence: Ping Gong, 103 Wenhua Road, Shenhe District,
110016 Shenyang, Liaoning, P. R. China.
E-mail: gongpinggp@126.com
Fax: þ86 24 23986429
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W. Zhu et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Figure 1. Structures of pyrimidine and 1,3,5-triazine derivatives, hydrazones, substituted benzyl-1H-indoles and target compounds.
reference molecule, incorporating the two active pharmaco- Chemistry
phores, bis-morpholine pyrimidine or 1,3,5-triazine and
N-benzyl indolyl-methylene group in a single molecular
framework with the hydrazinyl group as the linker. As a
result, a series of compounds (5a–5l and 8a–8o, Fig. 1) were
achieved, expected to possess excellent antiproliferative
activity and mTOR potency with improved pharmacokinetic
properties.
The 6-hydrazinyl-2,4-bismorpholino pyrimidine and 1,3,5-tri-
azine derivatives 5a–5l and 8a–8o, which are exemplified in
the present article, were prepared starting from the commer-
cially available cyanuric chloride or 2,4,6-trichloropyri-
midine, as outlined in Scheme 1. Regioselective nucleophilic
displacement of the 2- and 4-chloride of cyanuric chloride
with morpholine, followed by substitution with
NH2NH2 ꢀ H2O, gave access to key intermediate 3 as a pale
yellow solid. Subsequently, intermediate 3 was condensed
Herein, we report the newly synthesized target compounds
and their biological activity against human cancer cell lines
and mTOR kinase.
Scheme 1. Synthesis of target compounds.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
6-Hydrazinyl-2,4-bismorpholino pyrimidines and 1,3,5-Triazines
3
with the corresponding benzyl-1H-indole-3-carbaldehydes
4a–4l in EtOH with two drops of glacial acetic acid as catalyst
to afford the target compounds 5a–5l, respectively. The target
compounds 8a–8o were prepared in a similar procedure as
described for compounds 5a–5l starting from 2,4,6-trichloro-
pyrimidine. The substituted benzyl-1H-indole-3-carbalde-
hydes 4a–4o were prepared by nucleophilic substitution of
the various benzyl chlorides with 1H-indole-3-carbaldehyde
in the presence of K2CO3 in N,N-dimethylformamide (DMF).
The chemical structures of the target compounds were con-
with compound 1 and PAC-1 as the controls by MTT assay [40].
Furthermore, several potent compounds were further eval-
uated against U87MG (human glioblastoma cell line) and
H1975 (human lung cancer) cell lines. To investigate the
mechanism of this series of compounds, the most promising
compound 5j was evaluated for mTOR activity by LANCE1
Ultra kinase assay, using mTOR inhibitors PI103 and 1
as positive controls [41]. The results expressed as IC50 are
summarized in Tables 1 and 2. The IC50 values are the average
of at least two independent experiments.
1
firmed by IR, H NMR, 2D NOESY NMR, and mass spectrom-
As illustrated in Table 1, most of the synthesized compounds
showed moderate to excellent antiproliferative activity against
one or more cancer cell lines. Among them, compounds 8a–8c,
8e, and 8m–8n were superior to compound 1 against all the
tested cancer cell lines. Furthermore, compounds 5a, 5c, 5f, 5h,
5j–5k, 8a–8c, 8h, 8j–8k, and 8m–8n, especially 5c and 5j (with
IC50 values of 0.07 and 0.05 mM, respectively), exhibited signifi-
cant antiproliferative activity and high selectivity against the
H460 cancer cell line. The most promising compound 5j,
possessing a cyano group substituted at the C-3 position of
the benzene ring, showed strong antiproliferative activity
against the H460, HT-29, and MDA-MB-231 cell lines with
IC50 values of 0.05, 6.31, and 6.50 mM, which were 190.4,
4.6, and 5.6 times more active than compound 1 (9.52,
29.24, and 36.21 mM), respectively.
etry (MS) spectra.
All the target compounds can exist in the E or Z isomeric
form due to the imine bonds formed in condensation reac-
tions. The presence/absence of bulky groups and the differ-
ences in stability and energy are generally accepted as the
main reasons for the preferential formation of one isomer
over the other. It is interesting to note that the experimental
procedure shown in Scheme 1 led selectively to the E isomer,
which was confirmed by 2D NOESY NMR spectroscopic
approach [38, 39] (Fig. 2). For the representative compound
5j, the NOESY effect was observed between the proton of N–H
(10.58), the proton in C-2 position of indole ring (7.88) and the
proton in imine (8.27) in the E isomer (E-5j) (see Supporting
Information), whereas the putative Z isomer (Z-5j) should not
show this effect due to the larger intra-molecular H–H dis-
tances (Fig. 2). Moreover, variable-temperature experiments
performed on E-5j in the range of 220–300 K did not produce
any changes in the NMR spectra, revealing that the Z and E
isomers are not easily interconverted. On the basis of the
above analysis, the relative stereochemistry of the target
compounds was established unambiguously.
Based on the pharmacological data shown in Table 1, the
following conclusions could be drawn: (a) The 1-benzyl-1H-
indol-3-yl-methylene moiety is essential for the compound to
possess potent antiproliferative activity, as all the target com-
pounds (5a–5l and 8a–8o) showed greater antiproliferative
effects than the parent intermediates 3 and 7 against all
the cancer cell lines. (b) In most cases, 1,3,5-triazine derivatives
were more potent than the pyrimidine derivatives against the
H460 cancer cell line (5a vs. 8a, 5c vs. 8c, 5f vs. 8f, 5h vs. 8h, 5j–
5l vs. 8j–8l), but less effective against the other cancer cell lines
(5a–5c vs. 8a–8c, 5e–5f vs. 8e–8f, 5h–5j vs. 8h–8j, 5l vs. 8l). (c) A
fluorine atom at the 2-position of the benzene ring was more
favorable, as compound 8m was more potent than com-
pounds 8e, 8i, 8o against all the tested cancer cell lines.
Biological evaluation and discussion
Compounds 5a–5l, 8a–8o, and their precursors 3, 7 were
evaluated for their antiproliferative activity against H460
(human lung cancer), HT-29 (human colon cancer) and
MDA-MB-231 (human breast cancer) cancer cell lines together
Figure 2. Key NOESY of the representative
compound 5j.
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4
W. Zhu et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Table 1. Structures and antiproliferative activities of compounds against H460, HT-29, MDA-MB-231, U87MG, and H1975 cancer cell lines
in vitro
O
N
N
X
N
N
N
H
N
R
O
_
N
a
=
5
5l X
N
C
,
_
a
o
X
,
=
8
8
Compound
X
R
IC50 (mM)a)
H460
HT-29
MDA-MB-231
U87MG
H1975
3
7
–
–
–
–
35.6
>100
0.39
6.25
0.07
23.45
7.89
0.68
10.13
0.18
8.12
0.05
0.54
2.30
0.45
0.92
0.92
4.88
3.20
1.35
3.44
0.25
3.53
0.54
0.86
6.01
0.98
0.78
2.29
3.57
9.52
>100
59.22
45.03
2.65
28.26
3.1
10.9
11.44
4.6
6.42
28.3
6.31
>100
>100
ND
>100
>100
>100
>100
>100
>100
9.03
68.88
6.50
78.92
67.11
12.41
6.45
9.53
4.88
11.84
16.08
21.05
2.52
30.01
24.68
10.34
10.61
16.10
9.31
ND
ND
ND
ND
>100
ND
ND
>100
ND
ND
ND
ND
ND
ND
ND
ND
>100
ND
5a
5b
5c
5d
5e
5f
5g
5h
5i
N
N
N
N
N
N
N
N
N
N
N
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
–
3-Chloro
4-Methyl
3,4-Dichloro
4-tert-butyl
2-Chloro
H
4-Cyano
3-Fluoro
2,4-Dichloro
3-Cyano
4-Chloro
3-Trifluoromethyl
3-Chloro
4-Methyl
3,4-Dichloro
4-tert-butyl
2-Chloro
H
>100
ND
>100
ND
5j
5k
5l
>100
>100
88.30
11.28
29.32
5.47
ND
22.56
>100
>100
ND
ND
ND
44.24
ND
56.13
5.82
ND
ND
>100
>100
>100
>100
45.11
29.32
26.48
ND
52.63
>100
>100
ND
ND
ND
33.13
ND
47.06
25.21
ND
ND
>100
7.52
13.42
3.01
5.08
11.83
>100
7.71
16.08
40.18
1.28
8.47
3.64
24.44
17.50
11.64
1.86
13.77
0.97
29.24
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
PAC-1b)
1b)
4-Cyano
3-Fluoro
2,4-Dichloro
3-Cyano
4-Chloro
3-Trifluoromethyl
2-Fluoro
4-Fluoro
2,6-Dichloro
–
8.65
6.11
36.21
–
–
ND, not determined.
Bold values show the IC50 of the most promising compound in each series or the IC50 of compounds that exhibited potent
antiproliferative activity against the H460 cell line.
a)
IC50: concentration of the compound (mM) producing 50% cell growth inhibition after 72 h of drug exposure, as determined by the
MTT assay. Each experiment was run at least two times.
b)
Used as a positive control.
(d) The substituent in the 3-position of the benzene ring
appears to play an important role in activity. It could clearly
be seen that chloro, fluoro, and cyano groups (5a, 5c, 5h, 5j, 8a,
8c, 8h, 8j) in the 3-position of the benzene ring were favorable
for a good antiproliferative potency, whereas a drop of potency
could be observed for compounds with bulky trifluoromethyl
group (5l, 8l), confirming that the bulky groups may be
unfavorable. (e) Introduction of smaller groups (F, Cl) at
the 4-position of the benzene ring were more favorable for
increasing the antiproliferative activity against all five cancer
cell lines, while introduction of bulkier groups (tert-butyl,
methyl group, etc.) reduced their activity significantly (5c, 5k,
8k, 8n vs. 5b, 5d, 5g, 8d, 8g). (f) The positions of substituents
in the benzene ring appear to have significant effects on the
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
6-Hydrazinyl-2,4-bismorpholino pyrimidines and 1,3,5-Triazines
5
Table 2. mTOR kinase activity of selected compound 5j, positive
controls 1 and PI103
Experimental
Chemistry
Compound
IC50 on mTOR (mM)a)
All melting points were obtained on a Bu¨chi Melting Point B-540
apparatus (Bu¨chi Labortechnik, Flawil, Switzerland) and were
uncorrected. The IR spectra were recorded by means of the KBr
pellet technique on a Bruker FTS 135 spectrometer. NMR spectra
were performed using Bruker 300/600 MHz spectrometers (Bruker
Bioscience, Billerica, MA, USA) with TMS as internal standard. MS
were taken in ESI mode on an Agilent 1100 LC–MS (Agilent, Palo
Alto, CA, USA). Elemental analysis was determined on a Carlo-Erba
1106 Elemental analysis instrument (Carlo Erba, Milan, Italy). All
the materials were obtained from commercial suppliers and used
without purification, unless otherwise specified. Yields were not
optimized. TLC analysis was carried out on silica gel plates GF254
(Qindao Haiyang Chemical, China).
5j
16.624
1.366
0.0053
1b)
PI103b)
a)
The values are an average of two separate determinations.
Used as a positive control.
b)
antiproliferative activity. We can easily find that compounds
with a cyano substituent in the C-3 position (5j, 8j) were
13.8- to 202.6-fold more active than those in the C-4 position
(5g, 8g).
General procedure for the synthesis of 4,40-(6-chloro-
From these preliminary results we tentatively conclude
that the substituents in the benzene ring had a significant
effect on the antiproliferative activity and smaller groups in
the benzene ring were favorable for a good potency.
Furthermore, the results of the mTOR kinase assay
in Table 2 showed that compound 5j was less active
than the mTOR inhibitors 1 and PI103, suggesting that
this series of compounds may act through some other
mechanism rather than by inhibiting mTOR kinase.
Further studies on the mechanism of action of these
compounds are in progress.
1,3,5-triazine-2,4-diyl)dimorpholine 2 and 4,40-
(6-chloropyrimidine-2,4-diyl)dimorpholine 6 [22]
To a stirred solution of cyanuric chloride (18.4 g, 10 mmol) or
2,4,6-trichloropyrimidine (18.4 g, 10 mmol) in acetone (100 mL)
and crushed ice (500 g), a mixture of triethylamine (3.03 g,
30 mmol) and morpholine (17.4 g, 20 mmol) was added at
ꢁ108C. After the addition, the reaction mixture was stirred at
room temperature for 1 h and diluted with 50 mL of water.
Separated white solid was filtered and washed with water. The
white solid was dried. The crude products 2 and 6 were found to
be pure and taken to the next step without purification.
General procedure for the synthesis of 4,40-(6-hydrazinyl-
1,3,5-triazine-2,4-diyl)dimorpholine 3 and 4,40-
(6-hydrazinylpyrimidine-2,4-diyl)dimorpholine 7
Conclusions
In summary, a series of 6-hydrazinyl-2,4-bismorpholino
pyrimidine and 1,3,5-triazine derivatives were synthesized
and evaluated for their biological activity. Most of the
prepared compounds exhibited moderate to excellent anti-
proliferative activity and high selectivity against the H460
cancer cell line as compared with compound 1. In particular,
compounds 5c and 5j displayed excellent antiproliferative
activity against the H460 cell line with IC50 values (0.07 and
0.05 mM, respectively) in the nM range. The most promising
compound 5j (0.05, 6.31, and 6.50 mM) was 4.6- to 190.4-fold
more active than compound 1 (9.52, 29.24, and 36.21 mM)
against the H460, HT-29, and MDA-MB-231 cell lines,
respectively. The initial structure-activity relationships
showed that the 1-benzyl-1H-indol-3-yl-methylene moiety is
necessary for the compound to possess potent antiprolifer-
ative activity. Moreover, the substituents in the benzene
ring had significant effects on the antiproliferative activity
and a smaller atom or group in the benzene ring was favor-
able for a good activity. The results of the mTOR kinase
assay showed that this series of compounds act not through
inhibiting mTOR kinase. Further studies on the mechanism
of action are currently underway and will be reported in the
future.
A solution of 80% hydrazine hydrate (NH2NH2 ꢀ H2O, 170 mL,
4.9 mol) and 4,40-(6-chloro-1,3,5-triazine-2,4-diyl)dimorpholine
(2, 22.2 g, 0.078 mol) or 4,40-(6-chloropyrimidine-2,4-diyl)dimor-
pholine (6, 22.2 g, 0.078 mol) was stirred at 808C for 4–6 h. The
precipitate was collected by filtration and washed with water,
and dried to give 3 or 7, respectively.
4,40-(6-Hydrazinyl-1,3,5-triazine-2,4-diyl)dimorpholine 3
This compound was obtained as white solid in 77% yield; m.p.:
1
171–1738C. ESI–MS m/z: 282.1 [MþH]þ. H NMR (300 MHz, CDCl3)
d: 3.73–3.65 (m, 8H), 3.75 (d, J ¼ 4.6 Hz, 8H), 3.90 (s, 2H), 6.16 (s, 1H).
4,40-(6-Hydrazinylpyrimidine-2,4-diyl)dimorpholine 7
This compound was obtained as white solid in 67% yield and
should be taken to the next step quickly for its instability. ESI–MS
m/z: 281.2 [MþH]þ.
General procedure for the preparation of compounds
4a–4o [32, 34]
1H-Indole-3-carbaldehyde (14.5 g, 0.1 mol), together with
the respective substituted benzyl chloride (0.11 mol) and
anhydrous K2CO3 (27.6 g, 0.2 mol), was dissolved in DMF
(30 mL) and heated at 808C for 5–8 h. Upon cooling to room
temperature, the mixture was poured into ice-water and then
the precipitate was collected by filtration and dried. The crude
product was recrystallized from ethanol to give solids 4a–4o.
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6
W. Zhu et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
(d, J ¼ 8.1 Hz, 1H), 7.82 (s, 1H), 8.26 (s, 1H), 8.45 (d, J ¼ 7.6 Hz,
1H), 10.57 (s, 1H). Anal. Calcd. for C31H38N8O2 (%): C, 67.12; H, 6.91;
N, 20.20. Found (%): C, 67.05; H, 6.90; N, 20.26.
General procedure for the preparation of compounds 5a–5l
and 8a–8o
A mixture of 4,40-(6-hydrazinyl-1,3,5-triazine-2,4-diyl)dimorpho-
line (3, 0.13 g, 0.46 mmol) or 4,40-(6-hydrazinylpyrimidine-2,4-
diyl)dimorpholine (7, 0.13 g, 0.46 mmol), substitued benzyl-1H-
indole-3-carbaldehydes 4a–4o (0.46 mmol) and two drops of gla-
cial acetic acid in absolute ethanol (8 mL) was refluxed for 4–8 h.
The mixture was cooled, separated by filtration and washed with
EtOH to give off a white to light yellow solid. The crude product
was recrystallized from ethanol or purified by chromatography
on silica gel using MeOH/CH2Cl2 to afford solids 5a–5l and 8a–8o.
(E)-4,40-(6-(2-((1-(2-Chlorobenzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)-1,3,5-triazine-2,4-diyl)-
dimorpholine 5e
This compound was obtained as white solid in 81% yield; m.p.:
128–1308C. ESI–MS m/z: 533.2 (Cl ¼ 35), 535.2 (Cl ¼ 37) [MþH]þ,
555.0 [MþNa]þ. IR (KBr) cmꢁ1: 3444.1, 2963.0, 2853.1, 1522.5,
1
1258.5, 1114.9, 745.6. H NMR (300 MHz, DMSO-d6) d: 3.65 (br s,
8H), 3.74 (br s, 8H), 5.53 (s, 2H), 6.76 (d, J ¼ 7.5 Hz, 1H), 7.10–7.22
(m, 3H), 7.32 (t, J ¼ 7.0 Hz, 1H), 7.41 (d, J ¼ 7.9 Hz, 1H), 7.52
(d, J ¼ 7.8 Hz, 1H), 7.76 (s, 1H), 8.26 (s, 1H), 8.49 (d, J ¼ 7.5 Hz,
1H), 10.62 (s, 1H). Anal. Calcd. for C27H29ClN8O2 (%): C, 60.84;
H, 5.48; N, 21.02. Found (%): C, 60.75; H, 6.64; N, 21.09.
(E)-4,40-(6-(2-((1-(3-Chlorobenzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)-1,3,5-triazine-2,4-diyl)-
dimorpholine 5a
This compound was obtained as white solid in 87% yield; m.p.:
129–1308C. ESI–MS m/z: 533.3 [MþH]þ. IR (KBr) cmꢁ1: 3433.9,
2962.3, 2853.4, 1568.6, 1516.7, 1258.1, 1115.6, 858.9, 804.7,
741.1. 1H NMR (300 MHz, DMSO-d6) d: 3.64 (br s, 8H), 3.73 (br s,
8H), 5.45 (s, 2H), 7.10–7.22 (m, 3H), 7.31–7.37 (m, 3H), 7.49
(d, J ¼ 8.1 Hz, 1H), 7.87 (s, 1H), 8.27 (s, 1H), 8.46 (d, J ¼ 7.7 Hz,
1H), 10.59 (s, 1H). Anal. Calcd. for C27H29ClN8O2 (%): C, 60.84;
H, 5.48; N, 21.02. Found (%): C, 60.76; H, 5.51; N, 21.12.
(E)-4,40-(6-(2-((1-Benzyl-1H-indol-3-yl)methylene)-
hydrazinyl)-1,3,5-triazine-2,4-diyl)dimorpholine 5f
This compound was obtained as white solid in 75% yield;
m.p.: 129–1318C. ESI–MS m/z: 499.2 [MþH]þ, 521.2 [MþNa]þ.
IR (KBr) cmꢁ1: 3432.6, 2853.0, 1567.6, 1520.1, 1388.5, 1258.8,
1114.2, 740.4. 1H NMR (300 MHz, DMSO-d6) d: 3.64 (br s, 8H),
3.74 (br s, 8H), 5.42 (s, 2H), 7.02–7.39 (m, 7H), 7.47 (d, J ¼ 8.0 Hz,
1H), 7.83 (s, 1H), 8.27 (s, 1H), 8.46 (d, J ¼ 7.7 Hz, 1H), 10.55 (s, 1H).
Anal. Calcd. for C27H30N8O2 (%): C, 65.04; H, 6.06; N, 22.47. Found
(%): C, 64.91; H, 6.02; N, 22.59.
(E)-4,40-(6-(2-((1-(4-Methylbenzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)-1,3,5-triazine-2,4-diyl)-
dimorpholine 5b
This compound was obtained as yellow solid in 78% yield; m.p.:
137–1398C. ESI–MS m/z: 533.1 [MþH]þ. IR (KBr) cmꢁ1: 3428.2,
2864.0, 2852.3, 1568.8, 1518.5, 1257.7, 1116.0, 804.0, 742.4.
1H NMR (300 MHz, DMSO-d6) d: 1.06 (s, 3H), 3.64 (br s, 8H),
3.73 (br s, 8H), 5.43 (s, 2H), 7.02–7.32 (m, 4H), 7.38
(d, J ¼ 8.3 Hz, 2H), 7.46 (d, J ¼ 8.0 Hz, 1H), 7.84 (s, 1H), 8.26
(s, 1H), 8.45 (d, J ¼ 7.4 Hz, 1H), 10.58 (s, 1H). Anal. Calcd.
for C28H32N8O2 (%): C, 65.61; H, 6.29; N, 21.86. Found (%): C,
65.56; H, 6.31; N, 21.90.
(E)-4-((3-((2-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-
hydrazono)methyl)-1H-indol-1-yl)methyl)benzonitrile 5g
This compound was obtained as white solid in 79% yield;
m.p.: 225–2268C. ESI–MS m/z: 524.2 [MþH]þ, 546.2 [MþNa]þ.
IR (KBr) cmꢁ1: 3436.5, 3253.8, 2222.5, 1529.6, 1257.3, 1169.2,
1114.4, 803.8. 750.1. 1H NMR (300 MHz, DMSO-d6) d: 3.65 (br s,
8H), 3.74 (br s, 8H), 5.68 (s, 2H), 6.94 (d, J ¼ 7.8 Hz, 1H), 7.12–7.21
(m, 2H), 7.47 (dd, J1 ¼ 14.5 Hz, J2 ¼ 7.6 Hz, 2H), 7.60
(t, J ¼ 7.7 Hz, 1H), 7.79 (s, 1H), 7.91 (d, J ¼ 7.5 Hz, 1H), 8.27
(s, 1H), 8.50 (d, J ¼ 7.4 Hz, 1H), 10.61 (s, 1H). Anal. Calcd.
for C28H29N9O2 (%): C, 64.23; H, 5.58; N, 24.08. Found (%): C,
64.12; H, 5.60; N, 24.14.
(E)-4,40-(6-(2-((1-(3,4-Dichlorobenzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)-1,3,5-triazine-2,4-diyl)-
dimorpholine 5c
This compound was obtained as carnation solid in 82%
yield; m.p.: 135–1378C. ESI–MS m/z: 566.9 (Cl ¼ 35), 568.2
(E)-4,40-(6-(2-((1-(3-Fluorobenzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)-1,3,5-triazine-2,4-diyl)-
dimorpholine 5h
(Cl ¼ 37) [MþH]þ. IR (KBr) cmꢁ1
: 3426.1, 2853.2, 1526.8,
1387.4, 1257.3, 1114.9, 805.4, 741.4. 1H NMR (300 MHz, DMSO-
d6) d: 3.65 (br s, 8H), 3.74 (br s, 8H), 5.45 (s, 2H), 7.04–7.30 (m, 3H),
7.49 (d, J ¼ 8.1 Hz, 1H), 7.56 (dd, J1 ¼ 9.4 Hz, J2 ¼ 5.0 Hz, 2H),
7.87 (s, 1H), 8.27 (s, 1H), 8.47 (d, J ¼ 7.7 Hz, 1H), 10.60 (s, 1H). Anal.
Calcd. for C27H28Cl2N8O2 (%): C, 57.15; H, 4.97; N, 19.75. Found
(%): C, 57.09; H, 4.99; N, 19.83.
This compound was obtained as white solid in 76%
:
yield; m.p.: 128–1288C. ESI–MS m/z: 517.2 [MþH]þ. IR (KBr) cmꢁ1
3439.9, 2854.0, 1520.6, 1388.5, 1258.9, 1112.9, 805.4, 742.5.
1H NMR (300 MHz, DMSO-d6) d: 3.64 (br s, 8H), 3.73 (br s, 8H),
5.49 (s, 2H), 7.05–7.28 (m, 5H), 7.29–7.40 (m, 1H), 7.49
(d, J ¼ 8.1 Hz, 1H), 7.78 (s, 1H), 8.25 (s, 1H), 8.46 (d, J ¼ 7.7 Hz,
1H), 10.58 (s, 1H). Anal. Calcd. for C27H29FN8O2 (%): C, 62.78; H, 5.66;
N, 21.69. Found (%): C, 62.70; H, 5.63; N, 21.75.
(E)-4,40-(6-(2-((1-(4-tert-Butylbenzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)-1,3,5-triazine-2,4-diyl)-
dimorpholine 5d
This compound was obtained as carnation solid in 71% yield; m.p.:
128–1308C. ESI–MS m/z: 555.2 [MþH]þ. IR (KBr) cmꢁ1: 3439.4,
2959.4, 2854.6, 1525.2, 1258.3, 1115.1, 806.3, 744.0. 1H NMR
(300 MHz, DMSO-d6) d: 1.22 (s, 9H), 3.64 (br s, 8H), 3.73 (br s, 8H),
5.37 (s, 2H), 7.03–7.26 (m, 4H), 7.32 (d, J ¼ 8.2 Hz, 2H), 7.50
(E)-4,40-(6-(2-((1-(2,4-Dichlorobenzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)-1,3,5-triazine-2,4-diyl)-
dimorpholine 5i
This compound was obtained as white solid in 85% yield; m.p.:
206–2078C. ESI–MS m/z: 567.1 (Cl ¼ 35), 569.1 (Cl ¼ 37) [MþH]þ,
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
6-Hydrazinyl-2,4-bismorpholino pyrimidines and 1,3,5-Triazines
7
589.0 [MþNa]þ. IR (KBr) cmꢁ1: 3442.7, 2853.4, 1529.3, 1386.6,
1257.4, 1113.8, 805.5, 743.1. 1H NMR (300 MHz, DMSO-d6) d: 3.65
(br s, 8H), 3.74 (br s, 8H), 5.52 (s, 2H), 6.74 (d, J ¼ 8.4 Hz, 1H), 7.06–
7.28 (m, 2H), 7.34 (dd, J1 ¼ 8.4 Hz, J2 ¼ 2.1 Hz, 1H), 7.41
(d, J ¼ 8.1 Hz, 1H), 7.70 (d, J ¼ 2.1 Hz, 1H), 7.75 (s, 1H), 8.25
(s, 1H), 8.49 (d, J ¼ 7.5 Hz, 1H), 10.60 (s, 1H). Anal. Calcd.
for C27H28Cl2N8O2 (%): C, 57.15; H, 4.97; N, 19.75. Found (%): C,
57.06; H, 4.99; N, 19.79.
(E)-4,40-(6-(2-((1-(4-Methylbenzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)pyrimidine-2,4-diyl)dimorpholine 8b
This compound was obtained as white solid in 72% yield;
:
m.p.: 226–2278C. ESI–MS m/z: 512.5 [MþH]þ. IR (KBr) cmꢁ1
3444.6, 2961.8, 2851.3, 1559.4, 1435.0, 1258.8, 1169.0, 1116.1,
789.3, 742.3. 1H NMR (300 MHz, DMSO-d6) d: 2.24 (s, 3H), 3.49
(s, 4H), 3.62 (s, 8H), 3.68 (d, J ¼ 4.4 Hz, 4H), 5.38 (s, 2H), 5.81
(s, 1H), 7.08–7.22 (m, 6H), 7.43–7.53 (m, 1H), 7.84 (s, 1H), 8.13–
8.21 (m, 1H), 8.23 (s, 1H), 10.29 (s, 1H). Anal. Calcd. for C29H33N7O2
(%): C, 68.08; H, 6.50; N, 19.16. Found (%): C, 68.02; H, 6.53;
N, 19.19.
(E)-3-((3-((2-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-
hydrazono)methyl)-1H-indol-1-yl)methyl)benzonitrile 5j
This compound was obtained as white solid in 81% yield;
m.p.: 226–2278C. ESI–MS m/z: 524.2 [MþH]þ, 546.0 [MþNa]þ.
IR (KBr) cmꢁ1: 3434.3, 2853.3, 2228.4, 1518.6, 1258.3, 1115.5,
1004.9, 742.4. 1H NMR (600 MHz, DMSO-d6) d: 3.64 (br s, 8H),
3.72 (br s, 8H), 5.50 (s, 2H), 7.13 (t, J ¼ 7.4 Hz, 1H), 7.20
(t, J ¼ 7.6 Hz, 1H), 7.52 (dd, J1 ¼ 15.7, J2 ¼ 6.4 Hz, 3H), 7.75
(d, J ¼ 10.7 Hz, 2H), 7.88 (s, 1H), 8.27 (s, 1H), 8.46 (d, J ¼ 7.9 Hz,
1H), 10.58 (s, 1H). Anal. Calcd. for C28H29N9O2 (%): C, 64.23; H, 5.58;
N, 24.08. Found (%): C, 64.15; H, 5.53; N, 24.17.
(E)-4,40-(6-(2-((1-(3,4-Dichlorobenzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)pyrimidine-2,4-diyl)dimorpholine 8c
This compound was obtained as white solid in 87% yield; m.p.:
235–2388C. ESI–MS m/z: 565.9 (Cl ¼ 35), 567.9 (Cl ¼ 37) [MþH]þ.
IR (KBr) cmꢁ1: 3439.6, 2951.2, 2851.5, 1586.2, 1559.6, 1465.7,
1435.8, 1435.8, 1258.4, 1172.9, 1115.0, 1030.3, 1006.4, 745.2.
1H NMR (300 MHz, DMSO-d6) d: 3.49 (s, 4H), 3.62 (s, 8H), 3.69
(s, 4H), 5.45 (s, 2H), 5.82 (s, 1H), 7.20 (s, 4H), 7.51 (d, J ¼ 9.0 Hz,
2H), 7.89 (s, 1H), 8.15–8.27 (m, 2H), 10.31 (s, 1H). Anal. Calcd.
for C28H29Cl2N7O2 (%): C, 59.37; H, 5.16; N, 17.31. Found (%): C,
59.33; H, 5.14; N, 17.36.
(E)-4,40-(6-(2-((1-(4-Chlorobenzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)-1,3,5-triazine-2,4-diyl)-
dimorpholine 5k
This compound was obtained as yellow solid in 89% yield;
m.p.: 138–1398C. ESI–MS m/z: 533.2 [MþH]þ, 555.1 [MþNa]þ.
IR (KBr) cmꢁ1: 3431.6, 2964.2, 2852.2, 1518.7, 1257.7, 1116.6,
804.1, 742.4. 1H NMR (300 MHz, DMSO-d6) d: 3.64 (br s, 8H),
3.74 (br s, 8H), 5.43 (s, 2H), 7.09–7.21 (m, 4H), 7.37
(d, J ¼ 8.2 Hz, 2H), 7.46 (d, J ¼ 8.0 Hz, 1H), 7.83 (s, 1H), 8.26
(s, 1H), 8.46 (d, J ¼ 7.6 Hz, 1H), 10.56 (s, 1H). Anal. Calcd.
for C27H29ClN8O2 (%): C, 60.84; H, 5.48; N, 21.02. Found (%): C,
60.78; H, 5.47; N, 21.09.
(E)-4,40-(6-(2-((1-(4-tert-Butylbenzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)pyrimidine-2,4-diyl)dimorpholine 8d
This compound was obtained as pale yellow solid in 73%
yield; m.p.: 199–2018C. ESI–MS m/z: 554.3 [MþH]þ. IR
(KBr) cmꢁ1: 3447.2, 2963.2, 1555.7, 1431.4, 1259.6, 1118.5,
914.9, 752.1. 1H NMR (300 MHz, DMSO-d6) d: 1.22 (s, 9H), 3.49
(s, 4H), 3.62 (s, 8H), 3.69 (s, 4H), 5.38 (s, 2H), 5.82 (s, 1H), 7.19 (s, 4H),
7.32 (d, J ¼ 7.9 Hz, 2H), 7.52 (d, J ¼ 6.5 Hz, 1H), 7.85 (s, 1H), 8.19
(d, J ¼ 6.0 Hz, 1H), 8.24 (s, 1H), 10.27 (s, 1H). Anal. Calcd.
for C32H39N7O2 (%): C, 69.41; H, 7.10; N, 17.71. Found (%): C,
69.36; H, 7.13; N, 17.77.
(E)-4,40-(6-(2-((1-(3-(Trifluoromethyl)benzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)-1,3,5-triazine-2,4-diyl)-
dimorpholine 5l
(E)-4,40-(6-(2-((1-(2-Chlorobenzyl)-1H-indol-3-yl)-
This compound was obtained as white solid in 74% yield;
m.p.: 183–1848C. ESI–MS m/z: 567.3 [MþH]þ, 589.3 [MþNa]þ.
IR (KBr) cmꢁ1: 3437.4, 2853.3, 1566.6, 1526.1, 1259.1, 1168.8,
1116.4, 805.4, 748.6. 1H NMR (300 MHz, DMSO-d6) d: 3.65 (br s,
8H), 3.74 (br s, 8H), 5.55 (s, 2H), 7.06–7.22 (m, 2H), 7.43–7.59
(m, 3H), 7.64 (d, J ¼ 9.5 Hz, 2H), 7.89 (s, 1H), 8.27 (s, 1H),
8.47 (d, J ¼ 7.7 Hz, 1H), 10.60 (s, 1H). Anal. Calcd. for
C28H29F3N8O2 (%): C, 59.36; H, 5.16; N, 19.78. Found (%): C,
59.26; H, 5.19; N, 19.86.
methylene)hydrazinyl)pyrimidine-2,4-diyl)dimorpholine 8e
This compound was obtained as purple solid in 79% yield; m.p.:
216–2178C. ESI–MS m/z: 532.1 (Cl ¼ 35), 534.1 (Cl ¼ 37) [MþH]þ.
IR (KBr) cmꢁ1: 3438.6, 1582.3, 1557.1, 1259.9, 1174.2, 1113.6,
790.4, 747.5. 1H NMR (300 MHz, DMSO-d6) d: 3.49 (s, 4H), 3.62
(s, 8H), 3.69 (s, 4H), 5.54 (s, 2H), 5.83 (s, 1H), 6.75 (d, J ¼ 7.4 Hz,
1H), 7.14–7.37 (m, 4H), 7.42 (d, J ¼ 3.1 Hz, 1H), 7.52 (d, J ¼ 7.9 Hz,
1H), 7.79 (s, 1H), 8.23 (s, 2H), 10.32 (s, 1H). Anal. Calcd.
for C28H30ClN7O2 (%): C, 63.21; H, 5.68; N, 18.43. Found (%): C,
63.14; H, 5.70; N, 18.46.
(E)-4,40-(6-(2-((1-(3-Chlorobenzyl)-1H-indol-3-yl)-
(E)-4,40-(6-(2-((1-Benzyl-1H-indol-3-yl)methylene)-
hydrazinyl)pyrimidine-2,4-diyl)dimorpholine 8f
methylene)hydrazinyl)pyrimidine-2,4-diyl)dimorpholine 8a
This compound was obtained as gray solid in 80% yield; m.p.:
118–1208C. ESI–MS m/z: 532.1 (Cl ¼ 35), 534.2 (Cl ¼ 37) [MþH]þ,
546.7 [MþNa]þ. IR (KBr) cmꢁ1: 3434.3, 2955.9, 2851.5, 1558.9,
1464.6, 1432.7, 1258.8, 1170.7, 1116.4, 968.2, 788.9, 744.7.
1H NMR (300 MHz, DMSO-d6) d: 3.49 (s, 4H), 3.62 (s, 8H),
3.69 (s, 4H), 5.46 (s, 2H), 5.82 (s, 1H), 7.20–7.22 (m, 3H),
7.33–7.35 (m, 3H), 7.45–7.60 (m, 1H), 7.90 (s, 1H), 8.20
(d, J ¼ 5.3 Hz, 1H), 8.24 (s, 1H), 10.32 (s, 1H). Anal. Calcd.
for C28H30ClN7O2 (%): C, 63.21; H, 5.68; N, 18.43. Found (%): C,
63.14; H, 5.65; N, 18.54.
This compound was obtained as white solid in 77% yield;
m.p.: 226–2278C. ESI–MS m/z: 498.4 [MþH]þ, 520.1 [MþNa]þ.
IR (KBr) cmꢁ1: 3439.6, 2852.9, 1582.8, 1557.0, 1440.2, 1174.0,
1113.0, 742.9. 1H NMR (300 MHz, DMSO-d6) d: 3.49 (s, 4H),
3.62 (s, 8H), 3.69 (s, 4H), 5.44 (s, 2H), 5.82 (s, 1H), 7.12–
7.29 (m, 7H), 7.48–7.51 (m, 1H), 7.76 (s, 1H), 8.09–8.18
(m, 1H), 8.24 (s, 1H), 10.29 (s, 1H). Anal. Calcd. for
C28H31ClN7O2 (%): C, 67.59; H, 6.28; N, 19.70. Found (%): C,
67.51; H, 6.30; N, 19.74.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
W. Zhu et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
(E)-4-((3-((2-(2,6-Dimorpholinopyrimidin-4-yl)-
(E)-4,40-(6-(2-((1-(3-(Trifluoromethyl)benzyl)-1H-indol-3-yl)-
hydrazono)methyl)-1H-indol-1-yl)methyl)benzonitrile 8g
This compound was obtained as off-white solid in 78% yield;
m.p.: 236–2388C. ESI–MS m/z: 523.2 [MþH]þ, 546.1 [MþNa]þ.
IR (KBr) cmꢁ1: 3448.2, 2954.5, 2853.1, 2226.2, 1560.1, 1434.5,
1258.5, 1115.4, 743.9. 1H NMR (300 MHz, DMSO-d6) d: 3.55–
3.46 (m, 4H), 3.62 (s, 8H), 3.69 (s, 4H), 5.69 (s, 2H), 5.83 (s, 1H),
6.89–7.03 (m, 1H), 7.19–7.29 (m, 2H), 7.48 (dd, J1 ¼ 12.4 Hz,
J2 ¼ 5.5 Hz, 2H), 7.61 (t, J ¼ 7.8 Hz, 1H), 7.82 (s, 1H), 7.92
(t, J ¼ 7.0 Hz, 1H), 8.23 (d, J ¼ 8.0 Hz, 2H), 10.33 (s, 1H). Anal.
Calcd. for C29H30N8O2 (%): C, 66.65; H, 5.79; N, 21.44. Found (%): C,
66.54; H, 5.82; N, 21.52.
methylene)hydrazinyl)pyrimidine-2,4-diyl)dimorpholine 8l
This compound was obtained as gray solid in 70% yield; m.p.:
240–2428C. ESI–MS m/z: 566.3 [MþH]þ. IR (KBr) cmꢁ1: 3441.5,
2958.6, 2854.2, 1557.6, 1437.8, 1331.0, 1170.8, 1117.4, 790.9,
747.0. 1H NMR (300 MHz, DMSO-d6) d: 3.47 (s, 4H), 3.61 (s, 8H),
3.66 (s, 4H), 5.42 (s, 2H), 5.80 (s, 1H), 7.06–7.17 (m, 2H), 7.23–
7.46 (m, 3H), 7.49 (s, 1H), 7.84 (s, 1H), 8.07(s,1H), 8.14–8.21
(m, 1H), 8.23 (s, 1H), 10.29 (s, 1H). Anal. Calcd. for
C29H30F3N7O2 (%): C, 61.58; H, 5.35; N, 17.34. Found (%): C,
61.50; H, 5.26; N, 17.42.
(E)-4,40-(6-(2-((1-(2-Fluorobenzyl)-1H-indol-3-yl)-
(E)-4,40-(6-(2-((1-(3-Fluorobenzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)pyrimidine-2,4-diyl)dimorpholine 8m
This compound was obtained as lilac solid in 82% yield; m.p.:
217–2198C. ESI–MS m/z: 516.3 [MþH]þ. IR (KBr) cmꢁ1: 3438.5,
3298.3, 1557.0, 1462.3, 1435.9, 1112.9, 1006.1, 758.4, 741.4.
1H NMR (300 MHz, DMSO-d6) d: 3.49 (s, 4H), 3.62 (s, 8H), 3.69
(s, 4H), 5.50 (s, 2H), 5.81 (s, 1H), 7.05–7.29 (m, 5H), 7.34 (s, 1H), 7.51
(d, J ¼ 8.0 Hz, 1H), 7.80 (s, 1H), 8.07–8.35 (m, 2H), 10.30 (s, 1H).
Anal. Calcd. for C28H30FN7O2 (%): C, 65.23; H, 5.86; N, 19.02.
Found (%): C, 65.14; H, 5.85; N, 19.11.
methylene)hydrazinyl)pyrimidine-2,4-diyl)dimorpholine 8h
This compound was obtained as white solid in 65% yield; m.p.:
202–2048C. ESI–MS m/z: 516.5 [MþH]þ. IR (KBr) cmꢁ1: 3444.2,
2853.2, 1558.4, 1437.6, 1259.6, 1114.0, 789.9, 742.5. 1H NMR
(300 MHz, DMSO-d6) d: 3.49 (s, 4H), 3.62 (s, 8H), 3.69 (s, 4H),
5.42 (s, 2H), 5.81 (s, 1H), 7.18–7.21 (m, 3H), 7.35–7.39 (m, 3H),
7.41–7.56 (m, 1H), 7.92 (s, 1H), 8.20 (d, J ¼ 5.1 Hz, 1H), 8.24 (s, 1H),
10.32 (s, 1H). Anal. Calcd. for C28H30FN7O2 (%): C, 65.23; H, 5.86;
N, 19.02. Found (%): C, 65.14; H, 5.83; N, 19.12.
(E)-4,40-(6-(2-((1-(4-Fluorobenzyl)-1H-indol-3-yl)-
(E)-4,40-(6-(2-((1-(2,4-Dichlorobenzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)pyrimidine-2,4-diyl)dimorpholine 8n
This compound was obtained as yellow solid in 76% yield; m.p.:
193–1958C. ESI–MS m/z: 516.2 [MþH]þ. IR (KBr) cmꢁ1: 3438.3,
2960.2, 2854.1, 1583.3, 1558.5, 1439.4, 1260.0, 1176.1, 1113.9,
790.4, 743.1. 1H NMR (300 MHz, DMSO-d6) d: 3.49 (s, 4H), 3.62
(s, 8H), 3.69 (s, 4H), 5.42 (s, 2H), 5.81 (s, 1H), 7.12–7.23 (m, 4H),
7.29–7.31 (m, 2H), 7.52 (d, J ¼ 9 Hz, 1H), 7.87 (s, 1H), 8.18
(d, J ¼ 9 Hz, 1H), 8.23 (s, 1H), 10.29 (s, 1H). Anal. Calcd.
for C28H30FN7O2 (%): C, 65.23; H, 5.86; N, 19.02. Found (%): C,
65.11; H, 5.89; N, 19.14.
methylene)hydrazinyl)pyrimidine-2,4-diyl)dimorpholine 8i
This compound was obtained as off-white solid in 81% yield; m.p.:
226–2288C. ESI–MS m/z: 566.5 (Cl ¼ 35), 568.5 (Cl ¼ 37) [MþH]þ.
IR (KBr) cmꢁ1: 3439.8, 2852.3, 2851.9, 1586.5, 1560.2, 1434.7,
1258.1, 1115.2, 741.6. 1H NMR (300 MHz, DMSO-d6) d: 3.49
(s, 4H), 3.62 (s, 8H), 3.69 (d, J ¼ 4.5 Hz, 4H), 5.52 (s, 2H), 5.83
(s, 1H), 6.73 (d, J ¼ 6.8 Hz, 1H), 7.20–7.26 (m, 2H), 7.32–7.51
(m, 2H), 7.71 (dd, J1 ¼ 6.9 Hz, J2 ¼ 2.1 Hz, 1H), 7.78 (s, 1H), 8.22
(d, J ¼ 5.7 Hz, 2H), 10.33 (s, 1H). Anal. Calcd. for C28H29Cl2N7O2 (%):
C, 59.37; H, 5.16; N, 17.31. Found (%): C, 59.24; H, 5.18; N, 17.39.
(E)-4,40-(6-(2-((1-(2,6-Dichlorobenzyl)-1H-indol-3-yl)-
(E)-3-((3-((2-(4,6-Dimorpholinopyrimidin-2-yl)hydrazono)-
methyl)-1H-indol-1-yl)methyl)benzonitrile 8j
methylene)hydrazinyl)pyrimidine-2,4-diyl)dimorpholine 8o
This compound was obtained as white solid in 86% yield; m.p.:
268–2698C. ESI–MS m/z: 566.2 (Cl ¼ 35), 568.3 (Cl ¼ 37) [MþH]þ.
IR (KBr) cmꢁ1: 3444.2, 2955.6, 1560.8, 1435.6, 1118.1, 787.3,
740.5. 1H NMR (300 MHz, DMSO-d6) d: 3.49 (s, 4H), 3.61 (s, 8H),
3.69 (s, 4H), 5.58 (s, 2H), 5.80 (s, 1H), 7.09–7.37 (m, 3H), 7.42–7.54
(m, 1H), 7.61 (d, J ¼ 8.2 Hz, 3H), 8.13 (s, 1H), 8.22 (d, J ¼ 7.6 Hz,
1H), 10.23 (s, 1H). Anal. Calcd. for C28H29Cl2N7O2 (%): C, 59.37;
H, 5.16; N, 17.31. Found (%): C, 59.28; H, 5.15; N, 17.42.
This compound was obtained as white solid in 80% yield; m.p.: 221–
:
2238C. ESI–MS m/z: 523.5 [MþH]þ, 545.5 [MþNa]þ. IR (KBr) cmꢁ1
3448.1, 2850.1, 2229.5, 1555.5, 1434.0, 1259.6, 1117.3, 1007.7,
748.9. 1H NMR (300 MHz, DMSO-d6) d: 3.49 (s, 4H), 3.63 (s, 8H),
3.70 (s, 4H), 5.51 (s, 2H), 5.83 (s, 1H), 7.12–7.28 (m, 2H), 7.54 (s, 3H),
7.68–7.82 (m, 2H), 7.91 (s, 1H), 8.13–8.24 (m, 1H), 8.25 (s, 1H), 10.31
(s, 1H). Anal. Calcd. for C29H30N8O2 (%): C, 66.65; H, 5.79; N, 21.44.
Found (%): C, 66.56; H, 5.80; N, 21.51.
Pharmacology
MTT assay in vitro [40]
(E)-4,40-(6-(2-((1-(4-Chlorobenzyl)-1H-indol-3-yl)-
methylene)hydrazinyl)pyrimidine-2,4-diyl)dimorpholine 8k
This compound was obtained as off-white solid in 79% yield; m.p.:
236–2378C. ESI–MS m/z: 531.6 (Cl ¼ 35), 533.5 (Cl ¼ 37) [MþH]þ.
IR (KBr) cmꢁ1: 3440.4, 2964.2, 2851.4, 1620.9, 1559.5, 1464.3,
1437.4, 1258.7, 1168.9, 1114.8, 1011.5, 807.5, 744.6. 1H NMR
(300 MHz, DMSO-d6) d: 3.49 (s, 4H), 3.62 (s, 8H), 3.69
(d, J ¼ 4.7 Hz, 4H), 5.44 (s, 2H), 5.82 (s, 1H), 7.21–7.25 (m, 2H),
7.26–7.29 (m, 2H), 7.44–7.59 (m, 2H), 7.87 (s, 1H), 8.09 (s, 1H),
8.14–8.22 (m, 1H), 8.24 (s, 1H), 10.29 (s, 1H). Anal. Calcd.
for C28H30ClN7O2 (%): C, 63.21; H, 5.68; N, 18.43. Found (%): C,
63.12; H, 5.65; N, 18.51.
The antiproliferative activities of compounds (5a–5l, 8a–8o) were
evaluated with the H460, HT-29, MDA-MB-231, U87MG, and
H1975 cell lines by the standard MTT assay in vitro, with com-
pound 1 and PAC-1 as positive controls. The cancer cell lines were
cultured in minimum essential medium (MEM) supplement with
10% fetal bovine serum (FBS). Approximately 4 ꢂ 103 cells, sus-
pended in MEM medium, were plated onto each well of a 96-well
plate and incubated in 5% CO2 at 378C for 24 h. The test com-
pounds at the indicated final concentrations were added to the
culture medium and the cell cultures were continued for 72 h.
Fresh MTT was added to each well at a terminal concentration of
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
6-Hydrazinyl-2,4-bismorpholino pyrimidines and 1,3,5-Triazines
9
5 mg/mL and incubated with cells at 378C for 4 h. The formazan
crystals were dissolved in 100 mL DMSO each well, and the
absorbency at 492 nm (for absorbance of MTT formazan) and
630 nm (for the reference wavelength) was measured with an
ELISA reader. All of the compounds were tested two times in each
of the cell lines. The results expressed as IC50 (inhibitory con-
centration 50%) were the averages of two determinations and
calculated by using the Bacus Laboratories Incorporated Slide
Scanner (Bliss) software.
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the LANCE1 Ultra time-resolved fluorescence resonance energy
transfer assay following the manufacturer’s instructions, with
compound 1 and PI103 as positive controls. Briefly, mTOR
enzyme (10 nM), ATP (21.6 mM), ULight-4E-BP1 peptide (100 nM)
and test compounds were diluted in kinase buffer (50 mM
HEPES pH 7.5, 1 mM EGTA, 3 mM MnCl2, 10 mM MgCl2,
2 mM DTT, and 0.01% Tween-20). The reactions were performed
in white 384-well Optiplates (PerkinElmer, MA, USA) at room
temperature for 1 h and stopped by adding EDTA to 16 mM. Eu-
anti-phospho-4E-BP1 (Thr37/46) antibody (PerkinElmer, MA, USA)
was then added to each well to a final concentration of 2 nM. The
intensity of the light emission was measured with an EnVision1
Multilabel Reader (PerkinElmer, MA, USA) in TR-FRET mode
(excitation at 320 nm and emission at 665 nm). All of the
compounds were tested two times. The results expressed as
IC50 (inhibitory concentration 50%) were the averages of two
determinations.
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