4-Anilinoquinoline triazines: A novel class of hybrid antimalarial agents

Article abstract of DOI:10.1016/j.ejmech.2010.12.003

A novel class of hybrid 4-anilinoquinoline triazines have been synthesized and evaluated in vitro for their antimalarial activity against CQ-sensitive 3D7 strain of P. falciparum as well as for their cytotoxicity toward VERO cell line. Five compounds (19,

Full text of DOI:10.1016/j.ejmech.2010.12.003

European Journal of Medicinal Chemistry 46 (2011) 676e690
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
4-Anilinoquinoline triazines: A novel class of hybrid antimalarial agents
Ashok Kumar a, Kumkum Srivastava b, S. Raja Kumar b, M.I. Siddiqi c, Sunil K. Puri b, Jitendra K. Sexana d,
,
Prem M.S. Chauhan a
*
a Division of Medicinal & Process Chemistry, Central Drug Research Institute, Lucknow 226001, India
b Division of Parasitology, Central Drug Research Institute, Lucknow 226001, India
c Molecular and Structural Biology Division, Central Drug Research Institute, Lucknow 226001, India
d Division of Biochemistry, Central Drug Research Institute, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of hybrid 4-anilinoquinoline triazines have been synthesized and evaluated in vitro for their
antimalarial activity against CQ-sensitive 3D7 strain of P. falciparum as well as for their cytotoxicity
toward VERO cell line. Five compounds (19, 20, 23, 41 and 45) exhibited the antimalarial potency
superior to CQ. Compounds 14 and 16 were found to be orally active at a dose of 100 mg/kg ꢀ 4 days
Received 9 August 2010
Received in revised form
26 November 2010
Accepted 3 December 2010
Available online 9 December 2010
against CQ-resistant strain of P. yoelii. Inhibition of b-hematin formation assay and molecular docking
study has been conducted in order to gain insight into the mechanism of action of proposed targets for
the 4-anilinoquinoline and triazine moiety of the hybrid compounds.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
4-anilinoquinoline triazine
Antimalarial
P. falciparum
P. yoelii
1. Introduction
semi-synthetic derivatives (artemether, arteether and artesunate)
are the most potent and fast acting antimalarials effective against
Malaria, a deadly parasitic disease, is a major cause of concern to
human health and economies of poor countries. The gravity of
malaria reflects by the facts that 40% of the global population is at
risk of infection, 1.5e2.5 million people die every year especially
children under the age of five years [1]. There are four major species
of the malaria parasite of which Plasmodium falciparum causes the
most virulent form of malaria and is responsible for more than 95%
of malaria-related morbidity and mortality [2]. Due to unavail-
ability of effective vaccine, chemotherapy remains the only option
for treatment of malaria [3]. Since the discovery of natural product
quinine, structural modifications of its quinoline pharmacophore
led to the development of most effective antimalarial agents
namely chloroquine (CQ) mefloquine and amodiaquine (AQ) (Fig. 1)
[4e6]. CQ has been the mainstay of malaria therapy for decades
because of its efficacy, safety and low cost until the emergence and
spread of CQ-resistance. Pyrimethamine-sulfadoxine (fansidar)
was another best therapeutic option after CQ but rendered inef-
fective in most of malaria endemic regions due to spread of resis-
tance [7,8]. Currently, natural endoperoxide artemisinin and its
resistant strains of P. falciparum [9]. In view of prevailing danger of
resistance; combination therapy has been introduced as a measure
to safeguard the few available antimalarial drugs. WHO recom-
mended the use of artemisinin and its analogue in combination
with 4-aminoquinoline antimalarials like lumefantrine and
mefloquine [10,11]. However, the world-wide deployment of arte-
misinin based combination therapy is limited due to some serious
issues like higher cost of treatment, safety in pregnancy and
imbalance in demand than supply [12,13]. In addition, resistance to
artemisinin derivatives was also emerged in Southeast Asian
countries which will continue to evolve and spread, subsequently
making malaria chemotherapy more complicated [14]. To keep
pace with the continuously evolving resistant parasite, there is
challenge and urgency to develop cost-effective and efficacious
antimalarials with low potential of inducing resistance.
Despite the emergence of resistance to heme-targeted anti-
malarials mainly quinoline-based drugs, drug target heme is still
being exploited extensively for designing new antimalarial agents
because biochemical drug target heme cannot either be mutated or
expressed by the parasite [15]. During the intraerythrocytic stage of
life cycle, parasite invades the red blood cells of human host for
feeding the hemoglobin which is a major source of nutrition for its
growth and development. In the acidic food vacuole of the parasite,
a toxic by-product free heme is generated as a result of hemoglobin
* Corresponding author. Tel.: þ91 522 221 2411x4332; fax: þ91 522 262 3405.
E-mail addresses: premsc58@hotmail.com, prem_chauhan_2000@yahoo.com
(P.M.S. Chauhan).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.12.003

A. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 676e690
677
parasite, hybrid drug approach is gaining attention as aviable option
for effective long term strategy of antimalarial chemotherapy [36].
Hybrid drug approach involves the incorporation of two drug
pharmacophore in one single molecule with attention of dual drug
action. Given the unique pharmacological effect of quinoline based
antimalarials targeting the heme, quinoline moiety has been the
integral component of designing hybrid antimalarials. It was
exemplified by several potential antimalarials that include triox-
aferroquines [37], trioxaquines [38], artemisinin-quinine hybrid
[39], 4-aminoquinoline based tetraoxanes [40], clotrimazole-based
4-aminoquinolines [41], ferrocene-chloroquine analogues [42]
inhibitors of glutathione reductase conjugated to a 4-aminoquino-
line [43] and 4-aminoquinolines based on natural product scaffold
isatin [44]. In addition, a chloroquine reversal agent hybrid of CQ-
like moiety and imipramine was identified as potential antimalarial
agent [45]. More recently, the dual function acridones as new anti-
malarial chemotype were discovered that combined the heme tar-
geting character of acridones, together with a chemosensitizing
component that counteracts resistance to quinoline antimalarial
drugs [46].
The dihydrofolate reductase (DHFR) is one of the well-defined
and successfully exploited targets in malarial chemotherapy. Pyri-
methamine and cycloguanil (Fig. 1), the two important therapeutic
drugs commonly employed for the prophylaxis and treatment of
malaria target the DHFR. However, in the recent years, rapid spread
of antifolate resistant P. falciparum seriously compromised the
clinical utilities of these drugs and consequently necessitates the
need to search for new potent antifolate antimalarials [8,47,48].
Toward this goal, additional pyrimethamine and cycloguanil
analogues were identified as potential inhibitors of resistant DHFR
[49e51]. Apart from this, structurally similar to cycloguanil,
triazines have been reported to possess promising antimalarial
activity [52]. As part of our research programme devoted to
synthesis of various class of heterocycles as antimalarial agents, our
group identified trisubstituted triazines [53], pyrazole based
triazines [54], hybrid 4-aminoquinoline triazines [55], 9-anilinoa-
OH
HO
N
H
N
N
HN
HN
N
CF3
Cl
N
CF3
Cl
N
CQ
AQ
Mefloquine
R1
N
N
N
Cl
H
N
HN
N
N
R2
H2N
N
NH2
Cl
N
Cycloguanil,
13-45 (Table 1)
Fig. 1. Structures of CQ, AQ, mefloquine, cycloguanil and title compounds (13e45).
degradation into amino acids by enzymes aspartic, cysteine, and
metallo proteases [16,17]. As a defense mechanism for its survival,
parasite converts the free heme into non-toxic inert crystalline
pigment called hemozoin [18]. CQ and other aminoquinoline drugs
disrupt the conversion of toxic heme into hemozoin, consequently
substantial accumulation of toxic heme lead to the death of the
parasite [19e21]. Despite the persistent heavy drug pressure of CQ
for several decades, the delayed emergence of resistance to CQ is
considered due to the complexity of digestive vacuole environment
and the immutable nature of heme target [22]. Multiple point
mutations in P. falciparum chloroquine resistance transporter
protein (pfcrt) conferred resistance to CQ characterized by the
substantially reduced accumulation of CQ level in food vacuole.
Interaction of CQ with pfcrt induces resistance very slowly to
P. falciparum owing to the complexity in amino acid substitutions in
pfcrt [23e25].
The comprehensive structure-activity relationship studies on
CQeHematin binding have been explored to identify the optimal
structural requirements for designing the new CQ-based antima-
larial agents. It was established that the 7-chloro-4-aminoquino-
line is critical for the antimalarial activity and basicity of the side
chain nitrogen is also equally important for accumulation of drug
within acidic food vacuole of the parasite [26,27]. Modification in
the side chain of CQ led to the new CQ analogues with improved
activity effective against CQ-resistant strain. Altering the chain
length of CQ had the little effect on CQ-sensitive strain but
produced the remarkable effect on the CQ-resistant strain sug-
gesting that CQ-resistance is compound specific [28e30]. In
addition to the side chain modified new CQ analogues; several
functionalized 4-anilinoquinolines were identified as potential
antimalarials. Amodiaquine is still clinically useful for low degree
CQ-resistant parasites but it has some serious side effects like
heptatotoxicity and agranulocytosis [31,32]. To prevent the
formation of toxic metabolite, amodiaquine quinoneimine, a direct
regioisomer of amodiaquine, isoquine was developed having
potent in vitro activity against CQ-resistant parasites, however the
unacceptable high first pass metabolism of isoquine to dealkylated
metabolites did not allow further drug development process [33].
Modification of isoquine guided the discovery of drug candidate
N-tert-butyl isoquine [34]. In addition, 40-Fluoro-N-tert-butyla-
modiaquine was identified as a back-up compound for N-tert-
butyl isoquine based on potent activity against CQ-sensitive
and CQ-resistant parasites [35]. Thus, these findings manifest
considerable scope for developing new antimalarial with quino-
line nucleus.
cridinetriazines [56] and 4-aminoquinoline-based
b-carbolines
[57] as potential antimalarials. The 4-anilinoquinoline moiety has
been shown to be responsible for CQ-sensitive and CQ-resistant
activity in P. falciparum. We envisaged that combining two intrin-
sically active antimalarial moiety 4-anilinoquinoline and triazine
would lead to develop more potent antimalarials. The present
paper describes the synthesis and in vitro and in vivo antimalarial
evaluation of 4-anilinoquinoline triazines. Inhibition of b-hematin
formation assay and molecular docking study has been conducted
in order to gain insight into the mechanism of action of proposed
targets for the 4-anilinoquinoline and triazine moiety of the hybrid
compounds.
2. Chemistry
Since malaria severely affects the health and economies of the
poorest countries, low-cost of antimalarials are equally important
alongside efficacy and safety. An easy access to the title compounds
using readily available and inexpensive starting materials is a signifi-
cant feature of our simple and efficient synthetic approach. The
synthesis of title compounds 13e53 were achieved by a synthetic
protocol as shown in Scheme 1.To begin with 4,6-dichloro-6-
substituted-[1,3,5]triazine 1e4 were synthesized by nucleophilic
substitutionofcyanuricchloridewithamines. Commerciallyavailable
4,7-dichloroquinoline was condensed with p-phenylenediamine/
m-phenylenediamine in presence of catalyst p-TSA to afford the N-(7-
chloro-quinolinyl-4-yl)-benzene-1,4-diamine 5 or N-(7-chloro-qui-
nolinyl-4-yl)-benzene-1,3-diamine 6 [58]. The compound 5 or 6 was
refluxed with 4,6-dichloro-6-substituted-[1,3,5]triazines in THF to
Along with improvement of efficacy, preventing and delaying the
emergence of drug resistance is an essential goal of antimalarial
drug development. To combat the increasingly becoming resistant

678
A. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 676e690
Cl
N
R1
Cl
N
Cl
a
N
N
N
N
1; R1 = piperidino
2; R1 = anilino
Cl
Cl
3; R1 = o-toluidino
4; R1 = p-toluidino
Cl
N
Cl
b
b
HN
NH2
HN
N
NH2
Cl
N
c
Cl
5
6
c
R1
N
R1
N
H
N
N
N
HN
N
N
N
HN
N
N
H
Cl
Cl
11; R1 = anilino
12; R1 = p-toluidino
Cl
7; R1 = piperidino
8; R1 = anilino
Cl
9; R1 = o-toluidino
10; R1 = p-toluidino
d
d
R1
R1
N
N
N
H
HN
N
N
N
HN
N
N
N
R2
N
H
R2
Cl
Cl
13 - 45
46 - 53
Scheme 1. Reagents and conditions: (a) anilines/piperidine, 0 ꢁC, THF, 2 h; (b) p-phenylenediamine/m-phenylenediamine, p-TSA, EtOH, 3 h; (c) monosubstituted triazines,
THF, reflux, 8 h; (d) various amines, THF, reflux, 5 h.
yield the corresponding 6-chloro-N-[4-(7-chloro-quinolinyl-4-yl-
amino)-phenyl]- N0-substituted-[1,3,5]triazine-2,4-diamine 7e12.
The compounds (7e12) were subjected to nucleophilic substitution
with various amines to furnish the title compounds 13e53 (Table 1).
P. falciparum using a standardized inexpensive assay based on
SYBER Green I [59]. The IC50 values were calculated from experi-
ments carried out in triplicate. The cytotoxicity of all the
compounds was determined against VERO cell line using MTT assay
[60]. The in vivo drug responses of selected compounds were
evaluated in swiss mice infected with N-67 strain of P. yoelii which
is innately resistant to CQ [62]. Effects of selected compounds upon
3. Pharmacology
All the synthesized title compounds were evaluated in vitro
for their antimalarial activity against CQ-sensitive strain 3D7 of
the inhibition of
b-hematin formation were investigated by using
the -hematin inhibitory (BHIA) assay [62].
b

A. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 676e690
679
Table 1
In vitro antimalarial activity of compounds against 3D7 strain of P. falciparum and their cytotoxicity on VERO cell lines.
R1
N
R1
N
N
N
N
H
N
HN
N
HN
N
H
R2
R2
Cl
N
Cl
N
13 - 45
46 - 53
Compound No.
R1
R2
IC50 (nM)a
SIb
106.35
124.85
59.71
170.18
189.52
98.42
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
CQ
piperidino
piperidino
piperidino
piperidino
piperidino
piperidino
anilino
anilino
anilino
anilino
anilino
N-methyl piperazino
N-ethyl piperazino
N,N-diethylethylenediamino
4-(3-aminopropyl)morpholino
cyclohexylamino
34.77
29.74
38.77
23.13
313
234.63
5.85
5.92
18.53
60.41
3.01
64.84
33.65
41.5
19.03
18.85
102.43
31.52
26.05
13.42
162.99
27.78
29.65
37.63
12.44
11.90
44.49
82.85
7.03
73.95
63.50
11.88
6.41
47.17
60.07
26.30
291.73
110.67
43.17
40.95
34.56
8.15
benzylamino
N-methyl piperazino
N-ethyl piperazino
n-butylamino
N,N-diethylethylenediamino
4-(3-aminopropyl)morpholino
cyclohexylamino
benzylamino
amino
methylamino
N-methyl piperazino
N-ethyl piperazino
n-butylamino
N,N-diethylethylenediamino
4-(3-aminopropyl)morpholino
cyclohexylamino
benzylamino
amino
methylamino
N-methyl piperazino
N-ethyl piperazino
n-butylamino
N,N-diethylethylenediamino
4-(3-aminopropyl)morpholino
cyclohexylamino
benzylamino
amino
methylamino
N-methyl piperazino
N-ethyl piperazino
n-butylamino
N,N-diethylethylenediamino
4-(3-aminopropyl)morpholino
N-methyl piperazino
N-ethyl piperazino
4-(3-aminopropyl)morpholino
92.06
192.66
287.22
79.47
154.29
76.97
151.04
132.63
290.68
16.33
anilino
anilino
anilino
anilino
o-toluidino
o-toluidino
o-toluidino
o-toluidino
o-toluidino
o-toluidino
o-toluidino
o-toluidino
o-toluidino
p-toluidino
p-toluidino
p-toluidino
p-toluidino
p-toluidino
p-toluidino
p-toluidino
p-toluidino
p-toluidino
anilino
2.58
821.15
136.49
418.75
449.80
2129.68
163.54
240.35
36.39
35.61
101.03
64.38
610.98
137.42
79.44
134.89
491.91
223.40
90.51
anilino
anilino
anilino
anilino
p-toluidino
p-toluidino
p-toluidino
156.25
135.37
90.51
226.18
229.50
321.48
8983
a
IC50 (nM): concentration corresponding to 50% growth inhibition of the parasite.
SI: Selectivity index (IC50 values of toxic activity/IC50 values of antimalarial activity).
b
4. Results and discussion
exhibited the IC50 values ranging from 11.88 nM to 19.03 nM, 16
compounds displayed the IC50 values ranging from 23.13 nM to
44.49 nM, 7 compounds showed the IC50 values ranging from
47.17 nM to 82.85 nM and the rest six compounds showed the IC50
values ranging from 102.43 nM to 313 nM. As can be seen from
Table 1, compounds having anilino unit as substituent at triazine
nucleus have shown the superior potency among the screened
compounds. While keeping N-methylpiperizino unit fixed, replacing
the piperidino (13) with anilino (19) led to the six times increase in
antimalarial potency (13, IC50 ¼ 18.85 nM; 19, IC50 ¼ 3.15 nM).
Further, introducing the methyl group at ortho (28) and para (37)
position to the phenyl ring (19) resulted in three times and two times
4.1. In vitro antimalarial activity
The antimalarial activity of all the synthesized hybrid 4-anilino-
quinoline triazine derivatives was determined in vitro against the
CQ-sensitive 3D7 strain of P. falciparum. In vitro activity results are
summarized in Table 1. Considerable variations around the triazine
nucleus of the hybrid compounds have been done in order to
establish structure-activity relationship among the screened
compounds. Among the 41 compounds evaluated, five compounds
expressedthesuperiorantimalarialpotencytoCQ, sevencompounds

680
A. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 676e690
decrease in antimalarial potency, respectively (28, IC50 ¼ 10.41 nM;
37, IC50 ¼ 6.87 nM). In case of compounds having piperidino, anilino,
and p-toluidino unit, replacement of N-mehtylpiperazino with N-
ethyl piperizino had slight effect on the antimalarial activity pattern.
Surprisingly, compounds having o-toluidinoassubstituent, replacing
the N-methylpiperizino (28) with N-ethyl piperizino (29) produced
dramatic decrease in the antimalarial activity (28, IC50 ¼ 18.85 nM;
29, IC50 ¼ 102.43 nM). The IC50 values for compounds substituted
with n-butylamino and N,N-diethylethylenediamino were relatively
high. These results suggest that n-butylamino and N,N-dieth-
ylethylenediamino substituents were not favorable for the antima-
larial activity. Compound 23 having anilino and aminopropyl
morpholino substituents was the most potent among the evaluated
compounds being the almost three times more active than CQ (23,
IC50 ¼ 3.01 nM). Aminopropyl morpholino in combination with
piperidino (16), o-toluidino (32) and p-toluidino (41) produced good
in vitro antimalarial activity with IC50 values of 23.13 nM, 13.42 nM
and 7.03 nM, respectively. This observation indicates that amino-
propyl morpholino substituent was highly favorable for antimalarial
effect. Introducing the cyclohexylamino and benzylamino units to
the triazine nucleus displayed the poor antimalarial activity. It might
be attributed to the non-basic nature of these substituents.
Compounds 26, 35, and 44 with amino group exhibited modest to
good antimalarial activity (26, IC50 ¼ 41.51 nM; 35, IC50 ¼ 29.65 nM;
44, IC50 ¼ 11.86 nM). Replacing the amino group with methylamino
group, the corresponding compounds having anilino (27) and
p-toluidino (45) led to the two times improvement of the antima-
larial potency while the activity was considerably reduced for the
compound substituted with o-toluidino group. A small set of mole-
cules (46e53) with m-phenylenediamine linker were synthesized in
order to investigate the effect of position of linkage on the antima-
larial activity. Comparison of the antimalarial activity of compounds
with the corresponding p-phenylenediamine derived compounds
reflects that m-phenylenediamine linker significantly reduced the
antimalarial potency. Compounds 46 and 47 were 8 and 10 times less
active than compounds 19 and 20, respectively. Similarly,
Compounds 51 and 52 were almost 3 times less potent than
compounds 37 and 38. Compound 49 was virtually inactive with an
IC50 value of 291.73 nM. Surprisingly, compound 50 with amino-
propyl morpholine was considerably less active with an IC50 value of
110.67 nM given the highly favorable nature of aminopropyl mor-
pholine on the antimalarial activity as exemplified by compounds
with p-phenylenediamine linker. However, introducing the methyl
group to the para position (53) of phenyl ring (50) resulted in 3 times
improvement of the antimalarial activity.
with an IC50 value of 27.78 nM exhibited the highest selectivity
2129.68 while compound 29 having an IC50 value of 102.43 nM was
the most toxic with selectivity index 2.58. In general, most of the
compounds having good in vitro activity endowed with fairly high
selectivity index.
4.3. In vivo antimalarial activity
Compounds characterized by good in vitro antimalarial activity
were selected for in vivo study. Compounds were tested in swiss
mice infected with CQ-resistant N-67 strain of P. yoelii parasite
(Table 2) [61]. Initially; in vivo activity of compounds was evalu-
ated by intraperitoneal route at the dose 50 mg/kg ꢀ 4 days once
daily. Compounds 14, 16, and 23 provided the 100% protection to
the treated mice at 50 mg/kg ꢀ 4 days while the rest of
compounds only managed to exert suppressive effect. Compounds
14, 16, and 23 when further screened at 25 mg/kg by i.p. route
exhibited the complete clearance of parasitemia on day 4 but none
of the mice survived beyond day 28. It is interesting that
compound 23 substituted with anilino unit was active by i.p route
while introducing the methyl group (41) to the para position of
phenyl ring (23) abolished the in vivo antimalarial activity.
Compounds 14, 16 and 23 active by i.p route were further assessed
for oral activity. Compounds 14 and 16 having piperidino
substituent provided the 100% protection to the treated mice at
the dose 100 mg/kg for four days whereas compound 23 with
anilino substituent was inactive. This observation suggests that
oral activity of compounds 14 and 16 having piperidino substit-
uent might be attributed to their more metabolic stability than
compound 23 with anilino substituent. Though the efficacy of
orally active compounds 14 and 16 was found to be modest,
considering the hybrid nature of compounds able to overcome
CQ-resistance, these molecules represent promising leads for the
development of novel class of potent antimalarials.
4.4.
b-Hematin inhibitory activity
The selected compounds were tested for their ability to inhibit
the -hematin formation by using the -hematin inhibitory activity
b
b
(BHIA) assay (Table 3) [62]. All the tested compounds showed
a dose dependent inhibition in the BHIA assay and exhibited the
stronger b-hematin inhibitory activity than CQ. Compounds 14, 16,
Table 2
The structure-activity relationship emerged from the activity
results revealed that combination of aminopropyl morpholino with
piperidino, anilino, o-toluidino and p-toluidino, combination of
N-methylpiperizino with anilino, o-toluidino and p-toluidino,
combination of amino with p-toluidino and combination of methy-
lamino with anilino and p-toluidino at the 4- and 6-positions of the
triazine nucleus were well tolerated for the in vitro antimalarial
activity.
In vivo antimalarial activity of selected compounds against CQ-resistant N-67 strain
of P. yoelii in swiss mice.
Compound
Dose
(mg/kg ꢀ 4 days)
% Suppression
on day 4
Mice alive
on day 28
14
50 (i.p.)
25 (i.p.)
100 (oral)
50 (oral)
50 (i.p.)
25 (i.p.)
100 (oral)
50 (oral)
50 (i.p.)
50 (i.p.)
50 (i.p.)
25 (i.p.)
100 (oral)
50 (i.p.)
50 (i.p.)
50 (i.p.)
50 (i.p.)
50 (i.p.)
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
5/5
0/5
5/5
0/5
5/5
0/5
5/5
0/5
0/5
0/5
5/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
16
4.2. In vitro cytotoxicity
The cytotoxicity of all the synthesized hybrid 4-anilinoquinoline
triazines was determined against VERO cells using MTT assay
(Table 1) [60]. Compounds 19, 20, 23, 41 and 45 which have shown
in vitro antimalarial activity (IC50 range ¼ 3.01e7.03 nM) superior
to CQ possessed the selective index ranging from 92.06 to 610.98.
Thus these compounds demonstrated the promising safe activity
profile. Compounds 32, 37, 38, and 44 which were slightly less
active (IC50 range ¼ 11.90e13.42 nM) than CQ displayed the fairly
safe selectivity index range from 35.61 to 418.75. Compound 34
20
21
23
30
41
45
48
53

A. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 676e690
681
Table 3
-hematin inhibitory activity of selected
b
compounds.
a
Compound
IC50
14
16
20
21
23
30
41
45
48
53
CQ
2.79
2.71
2.69
3.08
2.98
2.96
2.85
3.16
2.55
2.70
3.65
Data are the mean of three different experi-
ments in triplicate.
a
The IC50 represents the concentration of
compound that inhibit
50%.
b-hematin formation by
21 and 30 exerted the similar inhibitory effect on the b-hematin
formation like compounds 20, 23, 41 and 45 but exhibited the
decreased in vitro antimalarial activity (Table 1). These results
suggested that apart from 4-anilinoquinoline triazine derivatives
targeting heme, the possibility of other mechanism of action for the
antimalarial activity cannot be ruled out.
Fig. 3. Structure of WR99210 and its docked conformation in yellow. Crystal structure
conformation of WR99210 (blue) in the pfDHFR. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article).
4.5. Molecular docking studies
Molecular docking studies of 4-anilinoquinoline triazine
derivatives were performed on the binding model based on the
P. falciparum Dihydrofolate Reductase- Thymidylate Synthase
(pfDHFR-TS) complexed With Wr99210 (Fig. 2) to identify a plau-
sible binding mode (Fig. 3). LigandFit, a modern docking program
within Cerius2 version 4.10 (Cerius2 Version 4.10 (2005), Accelrys
Inc., San Diego, USA), was used for docking runs [63]. The repre-
sentative compounds 14e16, 18, 20, 21, 23, 30, 33, 40, 41, 45, 48, 53
were subjected to molecular docking studies. In this model, most of
the derivatives bind in more or less similar fashion with its phenyl
ring occupying the interior of the deep hydrophobic cleft, while the
quinoline moiety containing lateral chain toward the solvent. The
NADP. This edge to face or T shaped aromatic stacking interaction
seems very important for pfDHFR inhibition and is responsible for
the proteineligand complex. Due to the non-aromatic and
nonplanar nature of piperidine derivatives, compounds 14, 15 and
16 were not able to accomodate inside the same binding pocket
suggests that non-aromatic bulky substituents at this position
may cause steric clashes with active site residues. As revealed from
the docked conformation, the phenyl moiety attached with the
aminoquinoline subunit make lipophilic interactions with Pro113
and Phe116 and it extends toward the solvent. It is hypothesized
that these
pep stacking interactions are responsible for optimal
orientation of 4-p-toluidino-6-methylamino triazine part of the
hybrid compound into the binding pocket. These results, along with
in vitro antimalarial activity of compounds against 3D7 strain of
P. falciparum suggestthattraizinemoietytargetingthepfDHFRseems
to account for in part the antimalarial activity of hybrid compounds.
hydrophobic and
pep stacking interactions seem to be mainly
responsible for orientation of these analogues in pfDHFR binding
pocket. In order to compare the binding pattern of these molecules,
the compound 45 that produced the best result in docking analysis
was analyzed, along with the active site of the crystallographic
structure of pfDHFR. All the amino acid residues which had inter-
actions with P. falciparum DHFR were showed in Fig. 4. In the
binding model of compound 45 and P. falciparum DHFR, carbonyl
oxygen of Ser111 forms hydrogen bond with hydrogen of methyl-
amino group substituent attached with triazine ring of compound
45 which is found to be conserved in other analogues also. More-
over, hydrophobic interactions probably existed between
compound 45 and Ile14, Ala16, Leu56, Val45, Phe58 and Ile164
residues of active site while benzene ring act as possible basic and
5. Conclusions
Despite the extensive research efforts directed toward the
development of new drugs, malaria continues to devastate the
poorest countries due to the lack of affordable and efficacious drugs
after the resistance to the most widely employed and successful
drugs, CQ and fansidar. Therefore, there is challenge and urgency to
develop the affordable, safe and effective drugs with low potential
of inducing resistance. In search of new antimalarials, a new class of
hybrid 4-anilinoquinoline triazine derivatives was synthesized
using readily available and inexpensive chemicals. The assessment
of compounds for in vitro antimalarial activity against 3D7 strain of
P. falciparum led to the identification of five potent compounds
superior to CQ. Most of the compounds have shown the fairly high
selectivity index. Compounds 14 and 16 were found to be orally
active at a dose of 100 mg/kg ꢀ 4 days in swiss mice infected with
CQ-resistance N-67 strain of P. yoelii. Though the efficacy of orally
active compounds 14 and 16 was modest, considering the hybrid
nature of compounds able to overcome CQ-resistance, these
p-donor site and is involved in edge to face strong pep stacking
interactions with the residues of Phe58, and nicotinamide ring of
Cl
NH2
O
N
N
Cl
Cl
H2N
N
Fig. 2. Structure of WR99210.

682
A. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 676e690
Fig. 4. Docked conformation of 45 in the pfDHFR.
molecules represent promising leads for the development of novel
class of potent antimalarials.
temperature. The reaction mixture was filtered and concentrated
under reduced pressure. The resulting residue was purified by
column chromatography using ethylacetateehexane (10:100) as
eluents to afford the desired products 1e4.
6. Experimetal
6.2.1. 2,4-Dichloro-6-piperidin-1-yl-[1,3,5]triazine (1)
6.1. General
Yield: 78%; mp 72e74 ꢁC; MS: 233 (M þ 1); 1H NMR (300 MHz,
CDCl3):
d
3.83 (t, 4H, J ¼ 5.7 Hz, NeCH2). Anal. Calc. for C8H10Cl2N4:
IR spectra were recorded on Beckman Aculab-10, Perkin Elmer
881 and FTIR 8210 PC, Schimadzu spectrophotometers either on KBr
discs or in neat. Nuclear magnetic resonance (NMR) spectra were
recorded on either Bruker Avance DRX-300 MHz or Bruker DPX 200
FT spectrometers using TMS as an internal reference. The samples
(dissolved in suitable solvents such as methanol/acetonitrile/water)
were introduced into the ESI source through a syringe pump at the
CalculatedC:41.22, H:4.32, N:24.04. FoundC: 41.03, H:4.21, N:24.33.
6.2.2. (4,6-Dichloro-[1,3,5]triazin-2-yl)-phenyl-amine (2)
Yield: 92%; mp 136e138 ꢁC; MS: 241 (M þ 1); 1H NMR
(300 MHz, CDCl3):
d
7.61 (bs,1H, NH), 7.74 (d, 2H, J ¼ 7.74 Hz, AreH),
7.41 (t, 2H, J ¼ 7.59 Hz, AreH), 7.21 (t, 1H, J ¼ 7.35 Hz, AreH). Anal.
Calc. for C9H6Cl2N4: Calculated C: 44.84, H: 2.51, N: 23.24. Found C:
44.69, H: 2.37, N: 23.42.
rate of 5 ml per min. The ESI capillary was set at 3.5 kV and the cone
voltage was 40 V. The spectra were collected in 6 s scans and the
printouts are averagedspectra of 6e8 scans. In case of multiplets the
signals are reported as intervals. Signals were abbreviated as
s, singlet; d, doublet; t, triplet; m, multiplet. The electron spray mass
spectra were recorded on triple quadrupole mass spectrometer. EI
mass spectra were recorded on JEOL JMS-D-300 spectrometer with
the ionization potential of 70 eV and ES mass on Quantro-II, micro
mass. Purity of all tested compounds was ascertained on the basis of
their elemental analysis and was carried out on Carlo-Erba-1108
instrument. The melting points were recorded on an electrically
heated melting point apparatus and are uncorrected.
6.2.3. (4,6-Dichloro-[1,3,5]triazin-2-yl)-o-tolyl-amine (3)
Yield: 90%; mp 132e134 ꢁC; MS: 255 (M þ 1); 1H NMR
(300 MHz, CDCl3):
7.29e7.21 (m. 3H, AreH). Anal. Calc. for C10H8Cl2N4: Calculated C:
d
7.69 (d, 1H, J ¼ 7.86 Hz, AreH), 7.3 (bs, 1H, NH),
47.08, H: 3.16, N: 21.96. Found C: 46.87, H: 3.26, N: 22.11.
6.2.4. (4,6-Dichloro-[1,3,5]triazin-2-yl)-p-tolyl-amine (4)
Yield: 94%; mp 142e144 ꢁC; MS: 255 (M þ 1); 1H NMR (300 MHz,
CDCl3):
d
7.51 (bs, 1H, NH), 7.42 (d, 2H, J ¼ 8.37 Hz, AreH), 7.22 (d,
2H, J ¼ 8.37 Hz, AreH). Anal. Calc. for C10H8Cl2N4: Calculated C:
47.08, H: 3.16, N: 21.96. Found C: 47.29, H: 3.21, N: 21.78.
6.2. General procedure for the synthesis of monosubstituted triazine
derivatives (1e4)
6.3. Generalprocedureforthesynthesisof4-anilinoquinolines(5 & 6)
To an ice cold solution of cyanuric chloride (1.0 equiv.) and
K2CO3 (2 equiv.) in dry THF was added dropwise the solution of
desired amine in THF for 20 min and stirred for 2 h at room
A solution of 4,7-dichloroquinoline (1.0 equiv.) and p-phenyl-
enediamine or m-phenylenediamine (2.0 equiv.) in absolute

A. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 676e690
683
ethanol was refluxed in presence of p-TSA as a catalyst for 3 h.
During the refluxing, precipitation of product occurred. The
precipitate was collected through filtration, washed with ethanol
and dried under vacuum to get the desired 4-anilinoquinoline 5 & 6
with excellent yields.
CH3). Anal. Calc. for C25H19Cl2N7: Calculated: C: 61.48, H: 3.92; N:
20.08. Found C: 61.35, H: 4.05; N: 20.23.
6.4.4. 6-Chloro-N-[4-(7-chloro-quinolin-4-ylamino)-phenyl]-N0-p-
tolyl-[1,3,5]triazine-2,4-diamine (10)
Yield: 72%; mp 172e174 ꢁC; MS: 488 (M þ 1); 1H NMR
6.3.1. N-(7-Chloro-quinolin-4-yl)-benzene-1,4-diamine (5)
(300 MHz, CDCl3 þ DMSO-d6):
d 9.56 (bs, 1H, NH), 9.29 (bs, 1H, NH),
Yield: 95%; mp >210 ꢁC; MS: 269 (M þ 1); 1H NMR (300 MHz,
8.35 (d, 1H, J ¼ 5.28 Hz, AreH), 8.25 (d, 1H, J ¼ 9.03 Hz, AreH), 7.88
(d, 1H, J ¼ 1.65 Hz, AreH), 7.68 (d, 2H, J ¼ 7.65 Hz, AreH), 7.48 (d,
2H, J ¼ 7.52 Hz, AreH), 7.35 (dd, 1H, J ¼ 1.65 Hz, J ¼ 9.03 Hz, AreH),
7.21 (d, 2H, J ¼ 7.65 Hz, AreH), 7.04 (d, 2H, J ¼ 7.52 Hz, AreH), 6.75
(d, 1H, J ¼ 5.28 Hz, AreH), 2.35 (s, 3H, CH3). Anal. Calc. for
C25H19Cl2N7: Calculated C: 61.48, H: 3.92, N: 20.08. Found C: 61.67,
H: 3.85, N: 20.11.
DMSO-d6):
d
8.54 (d, 1H, J ¼ 5.31 Hz, AreH), 8.04 (d, 1H,
J ¼ 2.04 Hz, AreH), 8.85 (d, 1H, J ¼ 8.97 Hz, AreH), 7.54 (d, 2H,
J ¼ 8.74 Hz, AreH), 7.49 (dd, 1H, J ¼ 2.04, 8.97 Hz, AreH), 7.10 (d,
2H, J ¼ 8.74 Hz, AreH), 6.83 (d, 1H, J ¼ 5.31, AreH). Anal. Calc. for
C15H12ClN3: Calculated C: 66.79, H: 4.48, N: 15.58. Found C: 66.87,
H: 4.29, N: 15.83.
6.3.2. N-(7-Chloro-quinolin-4-yl)-benzene-1,3-diamine (6)
6.4.5. 6-Chloro-N-[3-(7-chloro-quinolin-4-ylamino)-phenyl]-N0-
phenyl-[1,3,5]triazine-2,4-diamine (11)
Yield: 93%; mp 205e207 ꢁC; MS: 270 (M þ 1); 1H NMR
(300 MHz, DMSO-d6):
d
8.56 (d, 1H, J ¼ 5.32 Hz, AreH), 8.06 (d, 1H,
Yield: 65%; mp 171e173 ꢁC; MS: 474 (M þ 1); 1H NMR (300 MHz,
J ¼ 1.98 Hz, AreH), 7.94 (s, 1H, AreH), 7.87 (d, 1H, J ¼ 8.94 Hz,
AreH), 7.44 (dd, 1H, J ¼ 1.98, 8.94 Hz, AreH), 7.34 (t, 1H,
J ¼ 7.98 Hz, AreH), 7.18 (d, 1H, J ¼ 7.21 Hz, AreH), 6.96 (d, 1H,
J ¼ 7.22 Hz, AreH), 6.89 (d, 1H, J ¼ 5.32 Hz, AreH). Anal. Calc. for
C15H12ClN3: Calculated C: 66.79, H: 4.48, N, 15.58. Found C: 67.06,
H: 4.61, N, 15.49.
CDCl3):
d
8.59 (d, 1H, J ¼ 5.28 Hz, AreH), 8.07 (d, 1H, J ¼ 2.04 Hz,
AreH), 7.92 (s,1H, AreH), 7.86 (d,1H, J ¼ 7.93 Hz, AreH), 7.56 (d, 2H,
J ¼ 7.86 Hz, AreH), 7.51 (dd, 1H, J ¼ 2.04, 8.93 Hz, AreH), 7.34 (t, 1H,
J ¼ 7.86 Hz, AreH), 7.31e7.25 (m, 3H, AreH), 7.11 (bs, 1H, NH),
7.04e6.93 (m, 4H, AreH). Anal. Calc. for C24H17Cl2N7: Calculated C:
60.77, H: 3.61, N: 20.67. Found C: 60.45, H: 3.74, N: 20.89.
6.4. General procedure for the synthesis of compounds (7e12)
6.4.6. 6-Chloro-N-[3-(7-chloro-quinolin-4-ylamino)-phenyl]-N0-p-
tolyl-[1,3,5]triazine-2,4-diamine (12)
A reaction mixture of 4-anilinoquinoline (1.0 equiv.), mono-
substituted triazine (1e4, 1.5 equiv.) and K2CO3 (2.0 equiv.) in dry
THF was refluxed for 8 h. The reaction mixture was filtered and
concentrated under reduced pressure. The resulting residue was
dissolved in CHCl3, washed with water and dried over Na2SO4. The
solution was concentrated under reduced pressure and purified by
column chromatography using MeOH-CHCl3 (1:99) as eluents to
afford the pure compounds (7e12).
Yield: 67%; mp 165e167 ꢁC; MS: 488 (M þ 1); 1H NMR
(300 MHz, CDCl3):
d
8.59 (d, 1H, J ¼ 5.32 Hz, AreH), 8.07 (d, 1H,
J ¼ 2.01 Hz, AreH), 7.93 (s, 1H, AreH).7.87 (d, 1H, J ¼ 8.82 Hz,
AreH), 7.49 (dd, 1H, J ¼ 2.01, 8.82 Hz, AreH). 7.46 (d, 2H,
J ¼ 8.45 Hz, AreH), 7.35 (t, 1H, J ¼ 8.1 Hz, AreH). 7.18 (d, 1H,
J ¼ 7.32 Hz, AreH), 7.11e7.07 (m, 3H, AreH), 6.97 (d, 1H, J ¼ 5.32 Hz,
AreH), 6.91 (bs, 1H, NH), 6.78 (bs, 1H, NH), 6.63 (bs, 1H, NH). Anal.
Calc. for C25H19Cl2N7: Calculated C: 61.48, H: 3.92, N: 20.08. Found
C: 61.27, H: 4.03, N: 20.21.
6.4.1. N-(4-Chloro-6-piperidin-1-yl-[1,3,5]triazin-2-yl)-N0-(7-
chloro-quinolin-4-yl)-benzene-1,4-diamine (7)
Yield: 72%; mp 135e138 ꢁC; MS: 466 (M þ 1); 1H NMR
6.5. General procedure for the synthesis of titled compounds
(13e53)
(300 MHz, CDCl3 þ DMSO-d6):
d
9.56 (bs, 1H, NH), 9.29 (bs, 1H, NH),
8.35 (d, 1H, J ¼ 5.28 Hz, AreH), 8.25 (d, 1H, J ¼ 9.03 Hz, AreH), 7.88
(d, 1H, J ¼ 1.65 Hz, AreH), 7.68 (d, 2H, J ¼ 7.65 Hz, AreH), 7.48 (d,
2H, J ¼ 7.52 Hz, AreH), 7.35 (dd, 1H, J ¼ 1.65 Hz, J ¼ 9.03 Hz, AreH),
7.21 (d, 2H, J ¼ 7.65 Hz, AreH), 7.04 (d, 2H, J ¼ 7.52 Hz, AreH), 6.75
(d, 1H, J ¼ 5.28 Hz, AreH), 2.35 (s, 3H, CH3). Anal. Calc. for
C23H21Cl2N7: Calculated C: 59.23, H: 4.54, N: 21.02. Found C: 59.41,
H: 4.39, N: 21.18.
A solution of compound (7e12, 1.0 equiv.), desired amine (2.0
equiv.) and K2CO3 (2.0 equiv.) in dry THF was refluxed for 5 h. The
reaction mixture was filtered and concentrated under reduced
pressure. The resulting residue was dissolved in CHCl3, washed
with water and dried over Na2SO4. The dried solution was
concentrated under reduced pressure. The resulting crude mass
was purified by column chromatography using 2% MeOH/CHCl3 as
eluents to obtain the titled compounds (13e53).
6.4.2. 6-Chloro-N-[4-(7-chloro-quinolin-4-ylamino)-phenyl]-N0-
phenyl-[1,3,5]triazine-2,4-diamine (8)
Yield: 75%; mp 161e163 ꢁC; MS: 474 (M þ 1); 1H NMR (300 MHz,
6.5.1. N-(7-Chloro-quinolin-4-yl)-N0-[4-(4-methyl-piperazin-1-yl)-
6-piperidin-1-yl-[1,3,5]triazin-2-yl]-benzene-1,4-diamine (13)
CDCl3 þ DMSO-d6):
d
9.91 (bs, 2H, NH), 8.40 (d, 1H, J ¼ 5.49 Hz,
AreH), 8.37 (d,1H, J ¼ 9.09 Hz, AreH), 7.87 (d,1H, J ¼ 2.07 Hz, AreH),
7.74e7.65 (m, 4H, AreH), 7.41 (dd,1H, J ¼ 2.07 Hz, J ¼ 9.09 Hz, AreH),
7.30e7.25 (m, 4H, AreH), 7.03 (t, 1H, J ¼ 7.32 Hz, AreH), 6.81 (d,
1H, J ¼ 5.49 Hz, AreH). Anal. Calc. for C24H17Cl2N7: Calculated C:
60.77, H, 3.61, N: 20.67. Found C: 60.94, H, 3.52, N: 20.77.
Yield: 92%; mp >230 ꢁC; MS: 530 (M
þ
1); 1H NMR
(300 MHz, CDCl3):
d
8.54 (d, 1H, J ¼ 5.31 Hz, AreH)), 8.04 (d, 1H,
J ¼ 2.07 Hz, AreH), 7.89 (d, 1H, J ¼ 9.00 Hz, AreH), 7.68 (d, 2H,
J ¼ 8.76 Hz, AreH), 7.48 (dd, 1H, J ¼ 2.07 Hz, J ¼ 9.00 Hz, AreH),
7.27 (d, 2H, J ¼ 8.76 Hz, AreH), 6.84 (d, 1H, J ¼ 5.31 Hz, AreH),
6.79 (bs, 1H, NH), 6.60 (bs, 1H, NH), 3.86 (t, 4H, J ¼ 4.62 Hz, CH2),
3.78 (t, 4H, J ¼ 5.37 Hz, NeCH2), 2.47 (t, 4H, J ¼ 5.37 Hz, NeCH2),
2.36 (s, 3H, CH3), 1.68e1.61 (m, 6H, CH2). 13C NMR (75 MHz,
6.4.3. 6-Chloro-N-[4-(7-chloro-quinolin-4-ylamino)-phenyl]-N0-o-
tolyl-[1,3,5]triazine-2,4-diamine (9)
Yield: 71%; mp 167e169 ꢁC; MS: 488 (M þ 1); 1H NMR
CDCl3 þ DMSO-d6):
d 167.90, 167.56, 167.15, 154.13, 151.89, 140.61,
(300 MHz, CDCl3 þ DMSO-d6):
d
9.94 (bs, 1H, NH), 9.23 (bs, 1H, NH),
137.71, 135.92, 129.92, 127.95, 127.31, 126.65, 123.15, 120.86,
103.82, 57.54, 48.69, 46.94, 45.59, 28.54, 27.58. Anal. Calc. for
C28H32ClN9: Calculated C: 63.44, H: 6.08, N: 23.78. Found C:
63.26, H: 6.13, N: 23.95.
8.40e8.35 (m, 2H, AreH), 7.90 (d, 1H, J ¼ 1.95 Hz, AreH), 7.76e7.63
(m, 3H, AreH), 7.43 (dd, 1H, J ¼ 1.98 Hz, J ¼ 8.92 Hz, AreH),
7.25e7.12 (m, 4H, AreH), 6.74 (d, 1H, J ¼ 5.32 Hz, AreH), 2.31 (s, 3H,

684
A. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 676e690
6.5.2. N-(7-Chloro-quinolin-4-yl)-N0-[4-(4-ethyl-piperazin-1-yl)-6-
piperidin-1-yl-[1,3,5]triazin-2-yl]-benzene-1,4-diamine (14)
Yield: 92%; mp 215e218 ꢁC; MS: 544 (M þ 1); 1H NMR
6.5.6. N-Benzyl-N0-[4-(7-chloro-quinolin-4-ylamino)-phenyl]-6-
piperidin-1-yl-[1,3,5]triazine-2,4-diamine (18)
Yield: 89%; mp 119e121 ꢁC; MS: 537 (M þ 1); 1H NMR
(300 MHz, CDCl3):
d
8.55 (d, 1H, J ¼ 5.34 Hz, AreH), 8.05 (d, 1H,
(300 MHz, CDCl3):
d
8.54 (d, 1H, J ¼ 5.31 Hz, AreH), 8.05 (d,
J ¼ 2.1 Hz, AreH), 7.88 (d, 1H, J ¼ 9.03 Hz, AreH), 7.68 (d, 2H,
J ¼ 8.76 Hz, AreH), 7.49 (dd, 1H, J ¼ 2.1 Hz, J ¼ 9.03 Hz, AreH), 7.26
(d, 2H, J ¼ 8.76 Hz, AreH), 6.84 (d, 1H, J ¼ 5.34 Hz, AreH), 6.76 (bs,
1H, NH), 6.57 (bs, 1H, NH), 3.86 (t, 4H, J ¼ 4.86 Hz, NeCH2), 3.78 (t,
4H, J ¼ 5.43 Hz, NeCH2), 2.52e2.45 (m, 6H, NeCH2), 1.68e1.61 (m,
6H, CH2), 1.15 (t, 3H, J ¼ 7.14 Hz, CH3). 13C NMR (75 MHz, CDCl3):
1H, J ¼ 2.04 Hz, AreH), 7.88 (d, 1H, J ¼ 8.97 Hz, AreH), 7.66 (d, 2H,
J ¼ 8.7 Hz, AreH), 7.48 (dd, 1H, J ¼ 2.04 Hz, J ¼ 8.97 Hz, AreH),
7.39e7.33 (m, 5H, AreH), 7.24 (d, 2H, J ¼ 8.7 Hz, AreH), 6.83 (d,
1H, J ¼ 5.31 Hz, AreH), 6.72 (bs, 1H, NH), 6.58 (bs, 1H, NH), 5.25
(bs, 1H, NH), 4.66 (s, 2H. CH2), 3.78 (t, 4H, J ¼ 4.92 Hz, NeCH2),
1.65e1.61 (m, 6H, CH2). 13C NMR (75 MHz, CDCl3):
d 164.97,
d
163.84, 163.62, 163.11, 150.59, 148.26, 147.42, 136.34, 133.89,
163.52, 163.12, 150.55, 148.23, 147.42, 138.14, 136.07, 133.91,
131.93, 127.47, 127.23, 126.24, 125.87, 124.51, 123.22, 120.08,
119.53, 116.47, 100.52, 43.55, 43.05, 24.53, 23.58. Anal. Calc. for
C30H29ClN8: Calculated C: 67.09, H: 5.44, N: 20.86. Found C: 67.28,
H: 5.29, N: 20.73.
131.73, 127.51, 124.51, 123.34, 119.99, 119.22, 116.44, 100.53, 51.45,
51.16, 43.01, 41.87, 24.53, 23.61, 10.63. Anal. Calc. for C29H34ClN9:
Calculated C: 64.02, H: 6.30, N: 23.17. Found C: 64.27, H: 6.08,
N: 23.42.
6.5.7. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-6-(4-methyl-
piperazin-1-yl)-N0-phenyl-[1,3,5]triazine-2,4-diamine (19)
Yield: 82%; mp >220 ꢁC; MS: 538 (M þ 1); 1H NMR (300 MHz,
6.5.3. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-(2-
diethylamino-ethyl)-6-piperidin-1-yl-[1,3,5]triazine-2,4-diamine (15)
Yield: 85%; mp 150e151 ꢁC; MS: 546 (M þ 1); 1H NMR
CDCl3):
d
8.47 (d, 1H, J ¼ 5.4 Hz, AreH), 8.00 (d, 1H, J ¼ 1.98 Hz,
(300 MHz, CDCl3):
d
8.54 (d, 1H, J ¼ 5.34 Hz, AreH), 8.04 (d, 1H,
AreH), 7.92 (d, 1H, J ¼ 8.94 Hz, AreH), 7.65 (d, 2H, J ¼ 8.73 Hz,
AreH), 7.58 (d, 2H, J ¼ 7.77 Hz, AreH), 7.46 (dd, 1H, J ¼ 1.98 Hz,
J ¼ 8.94 Hz, AreH), 7.32 (t, 2H, J ¼ 7.65 Hz, AreH), 7.26 (d, 2H,
J ¼ 8.73 Hz, AreH), 7.15 (bs, 1H, NH), 7.06 (t, 1H, J ¼ 7.65 Hz, AreH),
6.83 (d, 1H, J ¼ 5.4 Hz, AreH), 6.63 (bs, 1H, NH), 3.87 (t, 4H,
J ¼ 4.77 Hz, NeCH2), 2.48 (t, 4H, J ¼ 4.77 Hz, NeCH2), 2.35 (s, 3H,
J ¼ 2.04 Hz, AreH), 7.90 (d, 1H, J ¼ 9.00 Hz, AreH), 7.69 (d, 2H,
J ¼ 7.92 Hz, AreH), 7.48 (dd, 1H, J ¼ 2.04, J ¼ 9.00 Hz, AreH), 7.26 (d,
2H, J ¼ 7.92 Hz, AreH), 6.83 (d, 1H, J ¼ 5.34 Hz, AreH), 6.8 (bs, 1H,
NH), 6.67 (bs, 1H, NH), 5.42 (bs, 1H, NH), 3.78 (t, 4H, J ¼ 5.21 Hz,
NeCH2), 3.48 (t, 2H, J ¼ 5.52 Hz, CH2), 2.70e2.55 ( m, 6H, NeCH2),
1.68e1.61 (m, 6H, CH2), 1.07 (t, 6H, J ¼ 4.65 Hz, CH3). 13C NMR
NeCH3). 13C NMR (50 MHz, DMSO-d6):
d 165.44, 165.01, 164.96,
(75 MHz, CDCl3):
d 163.51, 163.01, 150.48, 148.16, 147.57, 136.15,
152.78, 150.42, 149.69, 141.04, 137.91, 134.70, 134.53, 129.24, 128.49,
125.58, 125.24, 124.78, 122.63, 121.83, 120.90, 118.89, 101.90, 55.31,
46.68, 43.67. Anal. Calc. for C29H28ClN9: Calculated C: 64.74, H: 5.25,
N: 23.43. Found C: 64.92, H: 5.13, N: 23.67.
133.92, 131.89, 127.31, 124.48, 123.30, 120.30, 119.42, 116.49, 100.44,
50.54, 45.49, 45.32, 43.01, 37.02, 24.55, 23.59, 10.16. Anal. Calc. for
C29H36ClN9: Calculated C: 63.78, H: 6.64, N: 23.08. Found C: 63.56,
H: 6.92, N: 23.19.
6.5.8. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-6-(4-ethyl-
piperazin-1-yl)-N0-phenyl-[1,3,5]triazine-2,4-diamine (20)
Yield: 90%; mp 190e192 ꢁC; MS: 552 (M þ 1); 1H NMR
6.5.4. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-(3-
morpholin-4-yl-propyl)-6-piperidin-1-yl-[1,3,5]triazine-2,4-
diamine (16)
(300 MHz, CDCl3):
d
8.4 (d, 1H, J ¼ 5.4 Hz, AreH), 8.00 (d, 1H,
Yield: 87; mp 116e118 ꢁC; MS: 574 (M þ 1); 1H NMR (300 MHz,
J ¼ 2.04 Hz, AreH), 7.93 (d, 1H, J ¼ 8.97 Hz, AreH), 7.66 (d,
2H, J ¼ 8.76 Hz, AreH), 7.58 (d, 2H, J ¼ 7.83 Hz, AreH), 7.47 (dd,
1H, J ¼ 2.04 Hz, J ¼ 8.97 Hz, AreH), 7.32 (t, 2H, J ¼ 7.75 Hz, AreH),
7.27 (d, 2H, J ¼ 8.97 Hz, AreH), 7.16 (bs, 1H, NH), 7.06 (t, 1H,
J ¼ 7.41 Hz, AreH), 6.84 (d, 1H, J ¼ 5.4 Hz, AreH), 6.64 (bs,
1H, NH), 3.88 (t, 4H, J ¼ 4.29 Hz, NeCH2), 2.54e2.42 (m, 6H,
NeCH2), 1.14 (t, 3H, J ¼ 7.17 Hz, CH3). 13C NMR (50 MHz, DMSO-
CDCl3):
d
8.54 (d, 1H, J ¼ 5.31 Hz, AreH), 8.04 (d, 1H, J ¼ 1.8 Hz,
AreH), 7.89 (d, 1H, J ¼ 9.0 Hz, AreH), 7.70 (d, 2H, J ¼ 8.04 Hz, AreH),
7.47 (dd, 1H, J ¼ 1.8 Hz, J ¼ 9.0 Hz, AreH), 7.26 (d, 2H, J ¼ 8.04 Hz,
AreH), 6.84 (d, 1H, J ¼ 5.31 Hz, AreH), 6.63 (bs, 1H, NH), 5.74 (bs,
1H, NH), 5.33 (bs, 1H, NH), 3.79e3.76 (m, 8H, NeCH2 & OeCH2),
3.53 (t, 2H, J ¼ 6.03 Hz, NeCH2), 2.52e2.48 (m, 6H, NeCH2), 1.79
(quint, 2H, J ¼ 6.03 Hz, CH2), 1.68e1.61 (m, 6H, CH2). 13C NMR
d6):
d 165.37, 165.01, 164.98, 152.75, 150.41, 149.71, 141.05, 134.72,
(75 MHz, CDCl3):
d 163.50, 163.02, 150.57, 148.22, 147.48, 136.19,
129.23, 128.48, 125.57, 125.24, 124.77, 121.82, 120.89, 118.75,
102.32, 53.11, 52.55, 43.76, 12.80. Anal. Calc. for C30H30ClN9:
Calculated C: 65.27, H: 5.48, N: 22.83. Found: C: 65.05, H: 5.32,
N: 22.98.
133.90, 131.87, 127.43, 124.50, 123.27, 120.16, 119.42, 116.47, 100.46,
65.74, 55.59, 52.43, 42.99, 38.62, 24.70, 24.56, 23.60. Anal. Calc. for
C30H36ClN9O: Calculated C: 62.76, H: 6.32, N: 21.96. Found C: 62.95,
H: 6.45, N: 22.15.
6.5.9. N-Butyl-N0-[4-(7-chloro-quinolin-4-ylamino)-phenyl]-N00-
phenyl-[1,3,5]triazine-2,4,6-triamine (21)
6.5.5. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-cyclohexyl-
6-piperidin-1-yl-[1,3,5]triazine-2,4-diamine (17)
Yield: 87%; mp 125e127 ꢁC; MS: 511 (M þ 1); 1H NMR
Yield: 85%, mp 156e158 ꢁC; MS: 529 (M þ 1); 1H NMR
(300 MHz, CDCl3):
d
8.51 (d, 1H, J ¼ 5.34 Hz, AreH), 8.01 (d, 1H,
(300 MHz, CDCl3):
d
8.51(d, 1H, J ¼ 5.31 Hz, AreH), 8.02 (d, 1H,
J ¼ 2.01 Hz, AreH), 7.85 (d, 1H, J ¼ 8.97 Hz, AreH), 7.63e7.58 (m,
4H, J ¼ AreH), 7.45 (dd, 1H, J ¼ 2.01 Hz, J ¼ 8.97 Hz, AreH), 7.32 (t,
2H, J ¼ 7.62 Hz, AreH), 7.23 (d, 2H, J ¼ 8.85 Hz, AreH), 7.03 (t,
1H, J ¼ 7.62 Hz, AreH), 6.92 (bs, 2H, NH), 6.81 (d, 1H, J ¼ 5.34 Hz,
AreH), 6.56 (bs, 1H, NH), 5.06 (bs, 1H, NH), 3.46 (t, 2H, J ¼ 6.36 Hz,
CH2), 1.59 (quint, 2H, J ¼ 7.62 Hz, CH2), 1.42 (sext, 2H, J ¼ 7.5
Hz, CH2), 0.94 (t, 3H, J ¼ 7.29 Hz, CH3). 13C NMR (50 MHz,
J ¼ 1.68 Hz, AreH), 7.86 (d, 1H, J ¼ 8.94 Hz, AreH), 7.65 (d, 2H,
J ¼ 8.64 Hz, AreH), 7.46 (dd, 1H, J ¼ 1.68, J ¼ 8.94 Hz, AreH), 7.23
(d, 2H, J ¼ 8.64 Hz, AreH), 6.87 (bs, 1H, NH), 6.81 (d, 1H, J ¼ 5.31 Hz,
AreH), 6.59 (bs, 1H, NH), 4.83 (bs, 1H, NH), 3.81e3.74 (m, 5H, CH &
CH2), 2.05e2.02 (m, 2H, CH2), 1.71e1.60 (m, 6H, CH2), 1.45e1.33 (m,
2H, CH2), 1.25e1.14 (m, 4H, CH2). 13C NMR (75 MHz, CDCl3):
d
165.38, 164.48, 164.46, 152.22, 149.87, 149.28, 138.23, 134.13,
CDCl3 þ DMSO-d6):
d 171.35, 169.55, 156.92, 154.85, 154.58, 144.77,
133.25, 127.95, 124.99, 124.68, 124.35, 120.54, 118.30, 101.22, 49.30,
43.87, 33.09, 25.82, 25.43, 24.83. Anal. Calc. for C29H33ClN8:
Calculated C: 65.83, H: 6.29, N: 21.18. Found C: 65.68, H: 6.47,
N: 21.32.
142.07, 139.94, 139.27, 133.29, 130.31, 129.59, 128.57, 127.59, 126.42,
125.55, 123.37, 106.53, 45.79, 36.97, 25.30, 19.08. Anal. Calc. for
C28H27ClN8: Calculated C: 65.81, H: 5.33, N: 21.93. Found C: 65.59,
H: 5.54, N: 22.15.

A. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 676e690
685
6.5.10. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-(2-
diethylamino-ethyl)-N00-phenyl-[1,3,5]triazine-2,4,6-triamine (22)
Yield: 78%; mp 130e132 ꢁC; MS: 554 (M þ 1); 1H NMR
C31H25ClN8: Calculated C: 68.31, H: 4.62, N: 20.56. Found C: 68.38,
H: 4.49, N: 20.81.
(300 MHz, CDCl3):
d
8.55 (d, 1H, J ¼ 5.34 Hz, AreH), 8.05 (d, 1H,
6.5.14. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-phenyl-
[1,3,5]triazine-2,4,6-triamine (26)
J ¼ 2.04 Hz, AreH), 7.90 (d, 1H, J ¼ 9.00 Hz, AreH), 7.68e7.61 (m,
4H, AreH), 7.49 (dd, 1H, J ¼ 2.04 Hz, J ¼ 9.00 Hz, AreH), 7.34 (t, 2H,
J ¼ 8.22 Hz, AreH), 7.24 (d, 2H, J ¼ 8.82 Hz, AreH), 7.07 (t, 1H,
J ¼ 8.22 Hz, AreH), 6.99 (bs, 2H, NH), 6.86 (d, 1H, J ¼ 5.34 Hz,
AreH), 6.60 (bs, 1H, NH), 3.54 (t, 2H, J ¼ 6.09 Hz, NHeCH2), 2.70 (t,
2H, J ¼ 6.09 Hz, NeCH2), 2.66 (quart, 4H, J ¼ 6.99 Hz, NeCH2), 1.07
(t, 6H, J ¼ 6.99 Hz, CH3). 13C NMR (50 MHz, CDCl3 þ DMSO-d6):
Yield: 76%; mp 178e180 ꢁC; MS: 455 (M þ 1); 1H NMR (300 MHz,
CDCl3 þ CD3OD):
d
8.57 (d, 1H, J ¼ 9.09 Hz, AreH), 8.22 (d, 1H,
J ¼ 7.08 Hz, AreH), 8.00 (d, 1H, J ¼ 1.89 Hz, AreH), 7.83 (d,
2H, J ¼ 8.73 Hz, AreH), 7.71 (dd,1H, J ¼ 1.89, 9.09 Hz, AreH), 7.58 (d,
2H, J ¼ 7.71 Hz, AreH), 7.42 (d, 2H, J ¼ 8.73 Hz, AreH), 7.36e7.30 (m,
3H, AreH), 7.12 (t, 1H, J ¼ 7.35 Hz, AreH), 6.83 (d, 1H, J ¼ 7.08 Hz,
d
166.39, 164.75, 151.99, 149.87, 149.65, 139.69, 137.01, 135.29,
AreH).13C NMR (50 MHz, DMSO-d6):
d 167.13,165.45,155.99,143.74,
134.55, 128.96, 128.47, 125.66, 124.73, 123.37, 123.00, 121.74,
120.79, 118.47, 101.82, 52.13, 47.01, 38.76, 11.89. Anal. Calc. for
C30H32ClN9: Calculated C: 65.03, H: 5.82, N: 22.75. Found C: 64.78,
H: 5.65, N: 22.97.
139.88,139.46,132.02,129.23,128.05,127.00,126.42,123.91,122.50,
121.97, 120.01, 116.71, 100.91. Anal. Calc. for C24H19ClN8: Calculated
C: 63.37, H: 4.21, N: 24.63. Found C: 63.29, H: 4.16, N: 24.48.
6.5.15. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-methyl-N00-
phenyl-[1,3,5]triazine-2,4,6-triamine (27)
6.5.11. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-(3-
Yield: 78%; mp 158e161 ꢁC; MS: 469 (M þ 1); 1H NMR (300 MHz,
morpholin-4-yl-propyl)-N00-phenyl-[1,3,5]triazine-2,4,6-triamine(23)
Yield: 89%; mp 151e153 ꢁC; MS: 582 (M þ 1); 1H NMR
CDCl3):
d
8.56 (d, 1H, J ¼ 5.31 Hz, AreH), 8.22 (d, 1H, J ¼ 7.08 Hz,
AreH), 8.01 (d,1H, J ¼ 1.98 Hz, AreH), 7.84 (d, 2H, J ¼ 8.73 Hz, AreH),
7.70 (dd, 1H, J ¼ 1.98, 9.08 Hz, AreH), 7.58 (d, 2H, J ¼ 7.71 Hz, AreH),
7.43 (d, 2H, J ¼ 8.73 Hz, AreH), 7.36e7.29 (m, 3H, AreH), 7.11 (t,
1H, J ¼ 7.43 Hz, AreH), 6.84 (d, 1H, J ¼ 5.31 Hz, AreH). 13C NMR
(300 MHz, CDCl3):
d
8.54 (d, 1H, J ¼ 5.31 Hz, AreH), 8.05 (d, 1H,
J ¼ 2.04 Hz, AreH), 7.90 (d, 1H, J ¼ 8.9 Hz, AreH), 7.70 (d, 2H,
J ¼ 8.22 Hz, AreH), 7.63 (d, 2H, J ¼ 7.89 Hz, AreH), 7.49 (dd, 1H,
J ¼ 2.04 Hz, J ¼ 8.94 Hz, AreH), 7.33 (d, 2H, J ¼ 7.62 Hz, AreH), 7.24
(d, 2H, J ¼ 8.22 Hz, AreH), 7.07 (t, 1H, J ¼ 7.38 Hz, AreH), 6.93 (bs,
2H, NH), 6.85 (d, 1H, J ¼ 5.31 Hz, AreH), 6.64 (bs, 1H, NH), 6.62 (bs,
1H, NH), 3.78 (t, 4H, J ¼ 4.47 Hz, OeCH2), 3.59 (t, 2H, J ¼ 5.88 Hz,
NHeCH2), 2.55e2.51 (m, 6H, NeCH2), 1.82 (quint, 2H, J ¼ 6.42 Hz,
(50 MHz, DMSO-d6): d 167.08, 165.31, 156.01, 143.65, 139.73, 139.48,
132.13, 129.13, 127.92, 127.05, 126.39, 123.96, 122.48, 121.98, 119.96,
116.69, 101.13, 19.03. Anal. Calc. for C25H21ClN8: Calculated C: 64.03,
H: 4.51, N: 23.90. Found C: 64.25, H: 4.36, N: 24.12.
CH2). 13C NMR (50 MHz, CDCl3):
d 166.42, 164.79, 151.98, 149.68,
6.5.16. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-6-(4-methyl-
piperazin-1-yl)-N0-o-tolyl-[1,3,5]triazine-2,4-diamine (28)
Yield: 81%; mp 212e215 ꢁC; MS: 552 (M þ 1); 1H NMR (300 MHz,
149.29, 139.48, 136.99, 135.68, 134.41, 129.13, 128.75, 126.18, 124.71,
123.39, 122.27, 122.06, 120.78, 118.24, 102.13, 67.38, 57.76, 54.09,
40.75, 25.82. Anal. Calc. for C31H32ClN9O: Calculated C: 63.89, H:
5.48, N: 21.78.
CDCl3):
d
8.56 (d, 1H, J ¼ 5.34 Hz, AreH), 8.05 (d, 1H, J ¼ 2.07 Hz,
AreH), 7.95 (d,1H, J ¼ 8.04 Hz, AreH), 7.89 (d,1H, J ¼ 9.00 Hz, AreH),
7.66 (d, 2H, J ¼ 8.73 Hz, AreH), 7.50 (dd, 1H, J ¼ 2.07 Hz, J ¼ 9.00 Hz,
AreH), 7.26e7.22 (m, 4H, AreH), 7.08 (t,1H, J ¼ 7.29 Hz, AreH), 6.87
(d,1H, J ¼ 5.34 Hz, AreH), 6.83 (bs,1H, NH), 6.56 (bs, 2H, NH), 3.88 (t,
4H, J ¼ 4.68 Hz, NeCH2), 2.49 (t, 4H, J ¼ 4.68 Hz, NeCH2), 2.37 (s, 3H,
6.5.12. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-cyclohexyl-
N00-phenyl-[1,3,5]triazine-2,4,6-triamine (24)
Yield: 83%; mp 165e167 ꢁC; MS: 537 (M þ 1); 1H NMR
(300 MHz, CDCl3):
d
8.55 (d, 1H, J ¼ 5.31 Hz, AreH), 8.05 (d, 1H,
CH3), 2.35 (s, 3H, NeCH3).13C NMR (75 MHz, CDCl3):
d 163.68,163.41,
J ¼ 2.01 Hz, AreH), 7.89 (d, 1H, J ¼ 8.97 Hz, AreH), 7.64e7.58 (m,
4H, AreH), 7.49 (dd, 1H, J ¼ 2.01 Hz, J ¼ 8.97 Hz, AreH), 7.33 (t, 2H,
J ¼ 7.65 Hz, AreH), 7.27 (d, 2H, J ¼ 8.85 Hz, AreH), 7.07 (t, 1H,
J ¼ 7.32 Hz, AreH), 6.93 (bs, 2H, NH), 6.85 (d, 1H, J ¼ 5.31 Hz,
AreH), 6.60 (bs, 1H, NH), 5.04 (bs, 1H, NH), 3.90e3.87 (m, 1H,
NHeCH), 2.10e2.07 (m, 2H, CH2), 1.82e1.78 (m, 2H, CH2),
1.44e1.37 (m, 2H, CH2), 1.30e1.23 (m, 4H, CH2). 13C NMR (50 MHz,
162.93, 150.03, 147.94, 147.71 135.35, 134.15, 132.50, 129.14, 128.92,
126.51, 124.91, 124.98, 123.10, 122.55, 120.64, 119.82, 116.49, 100.21,
53.44, 47.35, 41.70, 16.69. Anal. Calc. for C30H30ClN9; Calculated C:
65.27, H: 5.48, N: 2.83. Found C: 65.42, H: 5.62, N: 3.05.
6.5.17. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-6-(4-ethyl-
piperazin-1-yl)-N0-o-tolyl-[1,3,5]triazine-2,4-diamine (29)
Yield: 83%; mp 200e202 ꢁC; MS: 566 (M þ 1); 1H NMR
CDCl3 þ DMSO-d6):
d 170.52, 169.64, 156.92, 154.91, 154.71, 145.04,
142.33, 139.84, 139.16, 133.68, 133.19, 130.19, 129.59, 128.91, 127.39,
126.40, 125.67, 123.44, 106.44, 54.76, 38.35, 30.82, 30.37. Anal. Calc.
for C30H29ClN8: Calculated C: 67.09, H: 5.44, N: 20.86. Found C:
67.29, H: 5.63, N: 21.07.
(300 MHz, CDCl3):
d
8.55 (d, 1H, J ¼ 5.34 Hz, AreH), 8.05 (d, 1H,
J ¼ 2.07 Hz, AreH), 7.94 (d, 1H, J ¼ 7.98 Hz, AreH), 7.89 (d, 1H,
J ¼ 8.97 Hz, AreH), 7.66 (d, 2H, J ¼ 8.76 Hz, AreH), 7.49 (dd, 1H,
J ¼ 2.07 Hz, J ¼ 8.97 Hz, AreH), 7.27e7.22 (m, 4H, AreH), 7.08 (t, 1H,
J ¼ 7.38 Hz, AreH), 6.97 (bs, 1H, NH), 6.85 (d, 1H, J ¼ 5.34 Hz, AreH),
6.62 (bs, 2H, NH), 3.89 (t, 4H, J ¼ 4.74 Hz, NeCH2), 2.54e2.46 (m,
6H, NeCH2), 2.35 (s, 3H, CH3), 1.16 (t, 3H, J ¼ 7.11 Hz, CH3). 13C NMR
6.5.13. N-Benzyl-N0-[4-(7-chloro-quinolin-4-ylamino)-phenyl]-N00-
phenyl-[1,3,5]triazine-2,4,6-triamine (25)
Yield: 86%; mp 155e157 ꢁC; MS: 545 (M þ 1); 1H NMR
(75 MHz, CDCl3):
d 163.73, 163.56, 163.09, 150.62, 148.28, 147.25,
(300 MHz, CDCl3):
d
8.54 (d, 1H, J ¼ 5.34 Hz, AreH), 8.05 (d, 1H,
135.63, 133.98, 132.31, 129.18, 128.43, 127.59, 125.00, 124.63, 123.17,
122.90, 122.29, 119.99, 119.73, 116.49, 100.61, 51.41, 51.16, 42.01,
16.89, 10.69. Anal. Calc. for C31H32ClN9: Calculated C: 65.77, H: 5.70,
N: 22.27. Found C: 65.58, H: 5.93, N: 22.43.
J ¼ 2.07 Hz, AreH), 7.88 (d, 1H, J ¼ 8.94 Hz, AreH), 7.64 (d, 2H,
J ¼ 8.41 Hz, AreH), 7.58 (d, 2H, J ¼ 7.71 Hz, AreH), 7.48 (dd, 1J,
J ¼ 2.07 Hz, J ¼ 8.94 Hz, AreH), 7.38e7.34 (m, 5H, AreH), 7.31 (t, 2H,
J ¼ 7.70 Hz, AreH), 7.24 (d, 2H, J ¼ 8.41 Hz, AreH), 7.07 (t, 1H,
J ¼ 7.38 Hz, AreH), 7.01 (bs, 2H, NH), 6.84 (d, 1H, J ¼ 5.34 Hz, AreH),
6.61 (bs, 1H, NH), 5.49 (bs, 1H, NH), 4.68 (s, 2H, CH2). 13C NMR
6.5.18. N-Butyl-N0-[4-(7-chloro-quinolin-4-ylamino)-phenyl]-N00-
o-tolyl-[1,3,5]triazine-2,4,6-triamine (30)
Yield: 87%; mp 110e112 ꢁC; MS: 525 (M þ 1); 1H NMR (300 MHz,
(50 MHz, CDCl3):
d 166.61, 164.76, 152.18, 149.85, 149.00, 139.22,
136.77, 135.67, 134.49, 129.16, 129.05, 127.76, 126.25, 124.63, 123.59,
121.94, 121.10, 118.22, 109.98, 102.26, 45.28. Anal. Calc. for
CDCl3):
d
8.55 (d, 1H, J ¼ 5.31 Hz, AreH), 8.05 (d, 1H, J ¼ 2.01 Hz,
AreH), 7.98e7.86 (m, 2H, AreH), 7.67 (d, 2H, J ¼ 8.78 Hz, AreH),

686
A. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 676e690
7.49 (dd, 1H, J ¼ 2.01 Hz, J ¼ 8.91 Hz, AreH), 7.27e7.22 (m, 4H,
AreH), 7.09 (t, 1H, J ¼ 7.17 Hz, AreH), 6.98 (bs, 1H, NH), 6.82 (d, 1H,
J ¼ 5.31 Hz, AreH), 6.60 (bs, 1H, NH), 5.07 (bs, 1H, NH), 3.48 (t,
2H, J ¼ 6.42 Hz, NHeCH2), 2.34 (s, 3H, CH3), 1.62 (quint, 2H,
J ¼ 6.81 Hz, CH2), 1.42 (sext, 2H, J ¼ 7.21 Hz, CH2), 0.98 (t, 3H,
AreH), 7.89e7.86 (m, 2H, AreH), 7.64 (d, 2H, J ¼ 8.28 Hz, AreH),
7.50 (dd, 1H, J ¼ 2.04 Hz, J ¼ 8.94 Hz, AreH), 7.38e7.32 (m, 4H,
AreH), 7.25e7.20 (m, 5H, AreH), 7.09 (t, 1H, J ¼ 7.53 Hz, AreH), 6.93
(bs, 1H, NH), 6.84 (d, 1H, J ¼ 5.31 Hz, AreH), 6.58 (bs, 2H, NH), 5.41
(bs, 1H, NH), 4.68 (s, 2H, CH2), 2.34 (s, 3H, CH3). 13C NMR (75 MHz,
J ¼ 7.21 Hz, CH3). 13C NMR (75 MHz, CDCl3 þ DMSO-d6):
d
164.61,
CDCl3): d 164.97, 163.66, 163.10, 150.51, 148.19, 147.31, 137.57, 135.26,
163.49, 162.86, 150.11, 147.76, 135.30, 134.13, 132.52, 129.21, 126.63,
124.97, 124.51, 123.71, 123.07, 120.55, 116.47, 100.22, 39.36, 30.38,
18.77, 16.70, 12.48. Anal. Calc. for C29H29ClN8: Calculated C: 66.34,
H: 5.57, N: 21.34. found C: 66.25, H: 5.43, N: 21.49.
133.97, 132.52, 129.20, 127.33, 126.03, 125.01, 124.57, 122.99, 120.18,
116.51, 100.54, 43.52, 16.80. Anal. Calc. for C32H27ClN8: Calculated C:
68.75, H; 4.87, N: 20.04. Found C: 68.69, H; 5.04, N: 19.83.
6.5.23. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-o-tolyl-
[1,3,5]triazine-2,4,6-triamine (35)
6.5.19. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-(2-
diethylamino-ethyl)-N00-o-tolyl-[1,3,5]triazine-2,4,6-triamine (31)
Yield: 72%; mp 150e152 ꢁC; MS: 68 (M þ 1); 1H NMR (300 MHz,
Yield: 76%; mp 183e185 ꢁC; MS: 469(M þ 1); 1H NMR (300 MHz,
CDCl3):
d
8.41 (d, 1H, J ¼ 5.13 Hz, AreH), 8.27 (d, 1H, J ¼ 9.00 Hz,
AreH), 7.99 (d, 1H, J ¼ 2.04 Hz, AreH), 7.90 (bs, 1H, NH), 7.78 (d, 1H,
J ¼ 7.71 Hz, AreH), 7.70 (d, 2H, J ¼ 8.55 Hz, AreH), 7.44 (dd,
1H, J ¼ 2.04 Hz, J ¼ 9.00 Hz, AreH), 7.41 (bs, 1H, NH), 7.23e7.20 (m,
4H, AreH), 7.06 (t, 1H, J ¼ 7.71 Hz, AreH), 7.00 (bs, 1H, NH), 6.77
(d, 1H, J ¼ 5.13 Hz, AreH), 6.64 (bs, 1H, NH), 5.42 (bs, 2H, NH), 2.31
CDCl3):
d
8.54 (d, 1H, J ¼ 5.34 Hz, AreH), 8.05 (d, 1H, J ¼ 2.1 Hz,
AreH), 7.90e7.87 (m, 2H, AreH), 7.66 (d, 2H, J ¼ 8.88 Hz, AreH),
7.49 (dd, 1H, J ¼ 2.1 Hz, J ¼ 8.88 Hz, AreH), 7.24e7.22 (m, 4H,
J ¼ AreH), 7.11 (t, 1H, J ¼ 6.57 Hz, AreH), 6.92 (bs, IH, NH), 6.84 (d,
1H, J ¼ 5.34 Hz, AreH). 6.62 (bs, 2H, NH), 5.72 (bs, 1H, NH), 3.54 (t,
2H, J ¼ 5.46 Hz, NHeCH2), 2.71e2.57 (m, 6H, NeCH2), 2.34 (s, 3H,
CH3), 1.03 (t, 6H, J ¼ 3.57 Hz, CH3). 13C NMR (75 MHz, CDCl3):
(s, 3H, CH3). 13C NMR (50 MHz, DMSO-d6):
d 167.93, 166.29, 165.37,
151.17, 150.57, 149.14, 138.46, 135.22, 134.00, 133.53, 130.98, 127.49,
126.88, 125.82, 125.63, 125.43, 124.98, 121.25, 118.57, 101.66, 18.99.
Anal. Calc. for C25H21ClN8: Calculated C: 64.03, H: 4.51, N: 23.90.
Found C: 63.86, H: 4.72, N: 23.79.
d
164.84, 163.67, 163.17, 159.98, 150.50, 148.22, 147.38, 135.44,
133.94, 132.48, 129.15, 127.40, 124.98, 123.08, 120.24, 119.91, 116.53,
100.52, 50.44, 45.44, 37.11, 34.14, 16.80, 10.28. Anal. Calc. for
C31H34ClN9: Calculated C: 65.54, H: 6.03, N: 22.19. Found C: 65.48,
H: 6.17, N: 22.34.
6.5.24. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-methyl-
N00-o-tolyl-[1,3,5]triazine-2,4,6-triamine (36)
Yield: 79%; mp 147e149 ꢁC; MS: 483(M þ 1); 1H NMR (300 MHz,
6.5.20. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-(3-
morpholin-4-yl-propyl)-N00-o-tolyl-[1,3,5]triazine-2,4,6-triamine (32)
Yield: 87%; mp 140e142 ꢁC; MS: 596 (M þ 1); 1H NMR
CDCl3):
d
8.54 (d, 1H, J ¼ 5.34 Hz, AreH), 8.05 (d, 1H, J ¼ 2.01 Hz,
AreH), 7.88e7.85 (m, 2H, AreH), 7.64e7.67 (m, 2H, AreH), 7.49 (dd,
1H, J ¼ 2.01 Hz, J ¼ 8.97 Hz, AreH), 7.24e7.21 (m, 4H, AreH), 7.08 (t,
1H, J ¼ 7.86 Hz, AreH), 6.95 (bs, 1H, NH), 6.83 (d, 1H, J ¼ 5.34 Hz,
AreH), 6.60 (bs, 2H, NH), 5.06 (bs, 1H, NH), 3.03 (s, 3H, NHeCH3),
(300 MHz, CDCl3):
d
8.55 (d, 1H, J ¼ 5.34 Hz, AreH), 8.06 (d, 1H,
J ¼ 2.07 Hz, AreH), 7.96e7.87 (m, 2H, AreH), 7.69 (d, 2H, J ¼ 8.4 Hz,
AreH), 7.50 (dd, 1H, J ¼ 2.07 Hz, J ¼ 8.94 Hz, AreH), 7.25e7.22 (m,
4H, AreH), 7.08 (t, 1H, J ¼ 7.41 Hz, AreH), 6.93 (bs, 1H, NH), 6.83 (d,
1H, J ¼ 5.34 Hz, AreH), 6.61 (bs, 2H, NH), 6.23 (bs, 1H, NH), 3.78 (t,
4H, J ¼ 5.49 Hz, OeCH2), 3.55 (t, 2H, J ¼ 6.06 Hz, NHeCH2),
2.55e2.50 (m, 6H, NeCH2), 2.35 (s, 3H, CH3), 1.81 (quint, 2H,
2.34 (s, 3H, CH3). 13C NMR (50 MHz, CDCl3):
d 167.14, 165.24, 164.73,
152.03, 149.71, 149.23, 137.11, 137.02, 135.70, 134.21, 130.88, 128.83,
126.67, 126.22, 124.74, 122.07, 121.67, 118.20, 102.16, 28.14, 18.48.
Anal. Calc. for C26H23ClN8: Calculated C: 64.66, H: 4.80, N: 23.20.
Found C: 64.73, H: 4.63, N: 23.41.
J ¼ 6.42 Hz, CH2). 13C NMR (75 MHz, CDCl3):
d 164.84, 163.32,
163.00, 150.50, 148.18, 147.34, 135.50, 133.97, 132.42, 129.17, 127.43,
124.97, 124.58, 123.07, 122.43, 120.14, 119.79, 116.49, 100.51, 65.72,
56.06, 52.43, 38.97, 24.32, 16.83. Anal. Calc. for C32H34ClN9O:
Calculated C: 64.47, H: 5.75, N: 21.15. Found C: 64.56, H: 5.89, N:
21.23.
6.5.25. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-6-(4-methyl-
piperazin-1-yl)-N0-p-tolyl-[1,3,5]triazine-2,4-diamine (37)
Yield: 88%; mp 201e203 ꢁC; MS: 552 (M þ 1); 1H NMR
(300 MHz, CDCl3):
d
8.56 (d, 1H, J ¼ 5.28 Hz, AreH), 8.05 (d, 1H,
J ¼ 2.01 Hz, AreH), 7.89 (d, 1H, J ¼ 9.00 Hz, AreH), 7.68 (d, 2H,
J ¼ 8.7 Hz, AreH), 7.49e7.45 (m, 3H, AreH), 7.25 (d, 2H, J ¼ 8.7
Hz, AreH), 7.16 (d, 2H, J 8.19 Hz, AreH), 6.86 (d, 1H, J ¼ 5.28 Hz,
AreH), 6.84 (bs, 1H, NH), 6.77 (bs, 1H, NH), 6.57 (bs, 1H, NH), 3.88 (t,
4H, J ¼ 5.56 Hz, NeCH2), 2.48 (t, 4H, J ¼ 4.56 Hz, NeCH2), 2.36 (s,
3H, NeCH3), 2.34 (s, 3H, CH3). 13C NMR (50 MHz, CDCl3 þ DMSO-
6.5.21. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-cyclohexyl-
N00-o-tolyl-[1,3,5]triazine-2,4,6-triamine (33)
Yield: 81%; mp 160e162 ꢁC; MS: 551(M þ 1); 1H NMR (300 MHz,
CDCl3):
d
8.55 (d, 1H, J ¼ 5.34 Hz, AreH), 8.05 (d, 1H, J ¼ 2.07 Hz,
AreH), 7.89 (d, 1H, J ¼ 9.00 Hz, AreH), 7.68 (d, 2H, J ¼ 7.98
Hz, AreH), 7.49 (dd, 1H, J ¼ 2.07 Hz, J ¼ 9.00 Hz, AreH), 7.27 (bs, 1H,
NH), 7.25e7.22 (m, 4H, AreH), 7.08 (t, 1H, J ¼ 7.2 Hz, AreH),
6.97e6.93 (m, 1H, AreH), 6.84 (d, 1H, J ¼ 5.34 Hz, AreH), 6.60 (bs,
2H, NH), 5.04 (bs, 1H, NH), 3.89e3.86 (m, 1H, NHeCH), 2.34 (s, 3H,
CH3), 2.09e2.06 (m, 2H, CH2), 1.82e1.76 (m, 2H, CH2), 1.45e1.36 (m,
2H, CH2), 1.26e1.18 (m, 4H, CH2). 13C NMR (75 MHz, CDCl3):
d6):
d 170.24, 169.63, 156.96, 154.97, 154.70, 142.64, 142.54, 139.74,
136.47, 134.19, 133.14, 130.10, 129.54, 129.24, 126.25, 125.65, 123.51,
106.44, 60.08, 51.43, 48.38, 25.99. Anal. Calc. for C30H30ClN9:
Calculated C: 65.27, H: 5.48, N: 22.83. Found C: 65.05, H: 5.37, N:
22.63.
6.5.26. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-6-(4-ethyl-
piperazin-1-yl)-N0-p-tolyl-[1,3,5]triazine-2,4-diamine (38)
Yield: 90%; mp 192e194 ꢁC; MS: 566 (M þ 1); 1H NMR
d
164.02, 163.52, 163.13, 150.64, 148.27, 147.24, 135.31, 132.34,
129.28, 127.59, 125.03, 124.67, 123.15, 120.09, 119.62, 116.58, 100.53,
98.72, 31.95, 24.32, 23.73, 16.84. Anal. Calc. for C31H31ClN8: Calcu-
lated C: 67.56, H: 5.67, N: 20.33. Found C: 67.67, H: 5.53, N: 20.43.
(300 MHz, CDCl3):
d
8.54 (d, 1H, J ¼ 5.34 Hz, AreH), 8.05 (d, 1H,
J ¼ 2.04 Hz, AreH), 7.89 (d, 1H, J ¼ 9.00 Hz, AreH), 7.67 (d, 2H,
J ¼ 8.73 Hz, AreH), 7.49e7.44 (m, 3H, AreH), 7.27 (d, 2H, J ¼ 8.73 Hz,
AreH), 7.16 (d, 2H, J ¼ 8.22 Hz, AreH), 6.90 (bs, 1H, NH), 6.85 (d,
1H, J ¼ 5.34 Hz, AreH), 6.81 (bs, 1H, NH), 6.59 (bs, 1H, NH), 3.89 (t,
4H, J ¼ 4.53 Hz, NeCH2), 2.54e2.45 (m, 6H, NeCH2), 2.34 (s, 3H,
CH3), 1.15 (t, 3H, J ¼ 7.14 Hz, CH3). 13C NMR (50 MHz, CDCl3):
6.5.22. N-Benzyl-N0-[4-(7-chloro-quinolin-4-ylamino)-phenyl]-N00-
o-tolyl-[1,3,5]triazine-2,4,6-triamine (34)
Yield: 86%; mp 145e147 ꢁC; MS: 559(M þ 1); 1H NMR (300 MHz,
CDCl3):
d
8.55 (d, 1H, J ¼ 5.31 Hz, AreH), 8.06 (d, 1H, J ¼ 2.04 Hz,

A. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 676e690
687
d
165.36, 164.78, 164.72, 152.22, 149.89, 149.08, 137.18, 136.81,
CH2), 1.82e1.77 (m, 2H, CH2), 1.44e1.30 (m, 2H, CH2), 1.27e1.19 (m,
4H, CH2). 13C NMR (50 MHz, CDCl3):
165.68,164.83,152.22,149.90,
149.05, 136.88, 135.65, 134.29, 132.92, 129.69, 129.09, 126.24,
124.75, 122.16, 121.89, 121.55, 120.75, 118.20, 102.22, 50.16, 33.59,
26.06, 25.43, 21.23. Anal. Calc. for C31H31ClN8: Calculated C: 67.56,
H: 5.67, N: 20.33. Found C: 67.79, H: 5.59, N: 20.18.
135.63, 134.18, 132.93, 129.69, 129.05, 126.21, 124.81, 121.92, 121.72,
120.96, 118.21, 102.23, 53.05, 52.82, 43.75, 21.23, 12.35. Anal. Calc.
for C31H32ClN9: Calculated C: 65.77, H: 5.70, N: 22.27. Found C:
65.98, H: 5.56, N: 22.13.
d
6.5.27. N-Butyl-N0-[4-(7-chloro-quinolin-4-ylamino)-phenyl]-N00-
p-tolyl-[1,3,5]triazine-2,4,6-triamine (39)
6.5.31. N-Benzyl-N0-[4-(7-chloro-quinolin-4-ylamino)-phenyl]-N00-
p-tolyl-[1,3,5]triazine-2,4,6-triamine (43)
Yield: 84%; mp 141e143 ꢁC; MS: 525 (M þ 1); 1H NMR
Yield: 86%; mp 138e140 ꢁC; MS: 559 (M þ 1); 1H NMR
(300 MHz, CDCl3):
d
8.55 (d, 1H, J ¼ 5.34 Hz, AreH), 8.05 (d, 1H,
(300 MHz, CDCl3):
d
8.55 (d, 1H, J ¼ 5.28 Hz, AreH), 8.05 (d, 1H,
J ¼ 1.98 Hz, AreH), 7.89 (d, 1H, J ¼ 9.00 Hz, AreH), 7.68 (d,
2H, J ¼ 8.94 Hz, AreH), 7.49e7.46 (m, 3H, AreH), 7.27 (d, 2H,
J ¼ 8.94 Hz, AreH), 7.15 (d, 2H, J ¼ 8.25 Hz, AreH), 7.02 (bs, 1H, NH),
6.85 (d, 1H, J ¼ 5.34 Hz, AreH), 6.73 (bs, 1H, NH), 6.58 (bs, 1H, NH),
5.06 (bs, 1H, NH), 3.46 (t, 2H, J ¼ 6.33 Hz, NHeCH2), 2.34 (s,
3H, CH3), 1.60 (quint, 2H, J ¼ 7.59 Hz, CH2), 1.45 (sext, 2H,
J ¼ 7.59 Hz, CH2), 0.98 (t, 3H, J ¼ 7.29 Hz, CH3). 13C NMR (50 MHz,
J ¼ 2.01 Hz, AreH), 7.88 (d, 1H, J ¼ 9.00 Hz, AreH), 7.65 (d, 2H,
J ¼ 8.4 Hz, AreH), 7.49 (dd. 1H, J ¼ 2.01 Hz, J ¼ 9.00 Hz, AreH), 7.46
(d, 2H, J ¼ 8.16 Hz, AreH), 7.38e7.30 (m, 5H, AreH), 7.24 (d, 2H,
J ¼ 8.4 Hz, AreH), 7.14 (d, 2H, J ¼ 8.16 Hz, AreH), 6.93 (bs, 2H, NH),
6.84 (d, 1H, J ¼ 5.28 Hz, AreH), 6.57 (bs, 1H, NH), 5.43 (bs, 1H, NH),
4.68 (s, 2H, CH2), 2.33 (s, 3H, CH3). 13C NMR (50 MHz, CDCl3):
d
166.48, 164.68, 151.97, 149.93, 149.65, 139.92, 137.19, 134.88,
CDCl3):
d 166.55, 164.78, 152.24, 149.91, 149.02, 136.67, 135.64,
134.20, 131.94, 129.23, 128.63, 128.27, 127.45, 125.24, 124.58, 123.82,
121.43, 120.82, 118.44, 101.48, 44.62, 21.00. Anal. Calc. for
C32H27ClN8: Calculated C: 68.75, H: 4.87, N: 20.04. Found C: 68.98,
H: 5.09, N: 20.13.
134.30, 129.69, 129.11, 126.24, 124.73, 121.87, 120.90, 118.21, 102.23,
41.13, 32.19, 21.24, 20.54, 14.26. Anal. Calc. for C29H29ClN8: Calcu-
lated C: 66.34, H: 5.57, N: 21.34. Found C: 66.52, H: 5.45, N: 21.38.
6.5.28. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-(2-
diethylamino-ethyl)-N00-p-tolyl-[1,3,5]triazine-2,4,6-triamine (40)
Yield: 71%; mp 128e130 ꢁC; MS: 568 (M þ 1); 1H NMR
6.5.32. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-p-tolyl-
[1,3,5]triazine-2,4,6-triamine (44)
Yield: 76%; mp 188e190 ꢁC; MS: 469 (M þ 1); 1H NMR
(300 MHz, CDCl3):
d
8.55 (d, 1H, J ¼ 5.34 Hz, AreH), 8.05 (d, 1H,
(300 MHz, CDCl3 þ CD3OD):
d
8.37 (d, 1H, J ¼ 5.58 Hz, AreH), 8.01
J ¼ 2.04 Hz, AreH), 7.90 (d, 1H, J ¼ 9.00 Hz, AreH), 7.49 (d, 2H,
J ¼ 8.7 Hz, AreH), 7.49e7.45 (m, 3H, AreH), 7.26 (d, 2H, J ¼ 8.7
Hz, AreH), 7.15 (d, 2H, J ¼ 8.25 Hz, AreH), 7.05 (bs, 1H, NH), 6.85 (d,
1H, J ¼ 5.34 Hz, AreH), 6.64 (bs, 1H, NH), 5.67 (bs, 1H, NH), 3.53 (t,
2H, J ¼ 5.58 Hz, NHeCH2), 2.70e2.57 (m, 6H, NeCH2), 2.34 (s, 3H,
CH3), 1.05 (t, 6H, J ¼ 4.11 Hz, CH3). 13C NMR (75 MHz, CDCl3):
(d,1H, J ¼ 1.74 Hz, AreH), 7.98 (d,1H, J ¼ 8.88 Hz, AreH), 7.61 (d, 2H,
J ¼ 8.64 Hz, AreH), 7.44 (dd, 1H, J ¼ 1.74 Hz, 8.88 Hz, AreH), 7.39 (d,
2H, J ¼ 8.91 Hz, AreH), 7.23 (d, 2H, J ¼ 8.64 Hz, AreH), 7.11 (d, 2H,
J ¼ 8.19 Hz, AreH), 6.79 (d, 1H, J ¼ 5.58 Hz, AreH), 2.29 (s, 3H, CH3).
13C NMR (50 MHz, DMSO-d6):
d 167.71, 165.34, 165.28, 152.33,
149.16, 146.14, 139.29, 138.54, 136.52, 132.89, 131.38, 129.60, 126.54,
125.50, 125.03, 121.61, 121.08, 117.98, 101.48, 21.28. Anal. Calc. for
C25H21ClN8: Calculated C: 64.03, H: 4.51, N: 23.90. Found C: 64.21,
H: 4.34, N: 24.16.
d
164.76, 163.19, 150.55, 148.26, 147.30, 135.37, 134.99, 133.94,
132.59, 131.44, 127.97, 127.47, 124.55, 123.06, 120.14, 119.69, 116.52,
100.56, 50.46, 45.42, 37.22, 19.52, 10.26. Anal. Calc. for C31H34ClN9:
Calculated C: 65.54, H; 6.03, N: 22.19. Found C: 65.36, H; 6.21, N:
22.04.
6.5.33. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-methyl-
N00-p-tolyl-[1,3,5]triazine-2,4,6-triamine (45)
6.5.29. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-(3-
morpholin-4-yl-propyl)-N00-p-tolyl-[1,3,5]triazine-2,4,6-triamine (41)
Yield: 87%; mp 158e160 ꢁC; MS: 596 (M þ 1); 1H NMR
Yield: 78%; mp 165e167 ꢁC; MS: 483 (M þ 1); 1H NMR
(300 MHz, CDCl3):
d
8.55 (d, 1H, J ¼ 6.30 Hz, AreH), 8.05 (d, 1H,
J ¼ 2.07 Hz, AreH), 7.89 (d, 1H, J ¼ 9.00 Hz, AreH), 7.68 (d,
2H, J ¼ 8.83 Hz, AreH), 7.49e7.46 (m, 3H, AreH), 7.24 (d, 2H,
J ¼ 8.83 Hz, AreH), 7.16 (d, 2H, J ¼ 8.25 Hz, AreH), 6.94 (bs, 2H, NH),
6.85 (d, 1H, J ¼ 6.30 Hz, AreH), 6.60 (bs, 1H, NH), 5.05 (bs, 1H, NH),
3.04 (s, 3H, CH3), 2.34 (s, 3H, CH3). 13C NMR (50 MHz, DMSO-d6):
(300 MHz, CDCl3):
J ¼ 2.07 Hz, AreH), 7.89 (d, 1H, J ¼ 9.00 Hz, AreH), 7.70 (d, 2H,
d
8.55 (d, 1H, J ¼ 5.31 Hz, AreH), 8.05 (d, 1H,
J
¼
8.34 Hz, AreH), 7.50e7.45 (m, 3H, AreH), 7.26 (d,
2H, J ¼ 8.34 Hz, AreH), 7.15 (d, 2H, J ¼ 8.28 Hz, AreH), 6.93 (bs,
2H, NH), 6.84 (d, 1H, J ¼ 5.31 Hz, AreH), 6.61 (bs, 1H, NH), 6.21
(bs, 1H, NH), 5.12 (bs, 1H, NH), 3.78 (t, 4H, J ¼ 4.5 Hz, OeCH2),
3.57 (t, 2H, J ¼ 5.97 Hz, NHeCH2), 2.54e2.50 (m, 6H, NeCH2), 2.34
(s, 3H, CH3), 1.81 (quint, 2H, J ¼ 5.49 Hz, CH2). 13C NMR (50 MHz,
d
167.75,165.98,165.35,152.31,149.21,146.11,139.25,138.51,136.45,
132.95, 131.29, 129.63, 126.43, 125.62, 124.98, 121.63, 121.15, 118.05,
101.42, 21.23, 18.73. Anal. Calc. for C26H23ClN8: Calculated C: 64.66,
H: 4.80, N: 23.20. Found C: 64.89, H: 4.73, N: 23.47.
CDCl3):
d 166.42, 164.68, 152.21, 149.88, 149.06, 137.06, 136.76,
135.64, 134.30, 133.07, 129.06, 126.22, 124.72, 121.95, 121.08,
118.21, 102.19, 67.42, 57.79, 54.11, 40.73, 25.95, 21.24. Anal. Calc.
for C32H34ClN9O: Calculated C: 64.47, H: 5.75, N: 21.15. Found C:
64.67, H: 5.83, N: 21.27.
6.5.34. N-[3-(7-Chloro-quinolin-4-ylamino)-phenyl]-6-(4-methyl-
piperazin-1-yl)-N0-phenyl-[1,3,5]triazine-2,4-diamine (46)
Yield: 82%; mp 201e203 ꢁC; MS: 538 (M þ 1); 1H NMR
(300 MHz, CDCl3):
d
8.58 (d, 1H, J ¼ 5.28 Hz, AreH), 8.07 (d, 1H,
J ¼ 2.04 Hz, AreH), 7.92 (s, 1H, AreH), 7.87 (d, 1H, J ¼ 8.97
Hz, AreH), 7.55 (d, 2H, J ¼ 7.86 Hz, AreH), 7.50 (dd, 1H,
J ¼ 2.04 Hz, 8.97 Hz, AreH), 7.34 (t, 1H, J ¼ 8.07 Hz, AreH),
7.30e7.24 (m, 3H, AreH), 7.18 (bs, 1H, NH), 7.09 (d, 1H, J ¼ 5.28 Hz,
AreH), 6.97e6.93 (m, 3H, AreH), 6.87 (bs, 1H, NH), 6.58 (bs,
1H, NH), 3.82 (t, 4H, J ¼ 4.75 Hz, NeCH2), 2.40 (t, 4H, J ¼ 4.75 Hz,
6.5.30. N-[4-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-
cyclohexyl-N00-p-tolyl-[1,3,5]triazine-2,4,6-triamine (42)
Yield: 81%; mp 171e173 ꢁC; MS: 551 (M þ 1); 1H NMR (300 MHz,
CDCl3):
d
8.55 (d, 1H, J ¼ 5.34 Hz, AreH), 8.05 (d, 1H, J ¼ 2.04 Hz,
AreH), 7.89 (d, 1H, J ¼ 8.97 Hz, AreH), 7.68 (d, 2H, J ¼ 8.82
Hz, AreH), 7.49e7.45 (m, 3H, AreH), 7.27 (d, 2H, J ¼ 8.82 Hz, AreH),
7.15 (d, 2H, J ¼ 8.22 Hz, AreH), 6.93 (bs, 1H, NH), 6.85 (d, 1H,
J ¼ 5.34 Hz, AreH), 6.75 (bs, 1H, NH), 6.59 (bs, 1H, NH), 5.00 (bs,
1H, NH), 3.89e3.87 (m, 1H, NH), 2.34 (s, 3H, CH3), 2.10e2.06 (m, 2H,
NeCH2), 2.33 (s, 3H, NeCH3). 13C NMR (75 MHz, CD3OD):
d 165.34,
164.85, 151.39, 150.21, 149.20, 141.50, 140.38, 139.91, 135.76, 129.69,
128.85, 126.89, 125.74, 123.69, 122.54, 120.57, 118.62, 117.54, 116.78,
115.25, 102.09, 54.64, 45.28, 42.96. Anal. Calc. for C29H28ClN9:

688
A. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 676e690
Calculated C: 64.74, H: 5.25, N: 23.43. Found C: 64.91, H: 5.39, N:
23.26.
109.99, 102.93, 67.34, 57.63, 54.01, 40.66, 25.88. Anal. Calc. for
C31H32ClN9O: Calculated C: 63.96, H: 5.54, N: 21.66. Found C: 64.17,
H: 5.37, N: 21.89.
6.5.35. N-[3-(7-Chloro-quinolin-4-ylamino)-phenyl]-6-(4-ethyl-
piperazin-1-yl)-N0-phenyl-[1,3,5]triazine-2,4-diamine (47)
Yield: 85%; mp 191e193 ꢁC; MS: 552 (M þ 1); 1H NMR
6.5.39. N-[3-(7-Chloro-quinolin-4-ylamino)-phenyl]-6-(4-methyl-
piperazin-1-yl)-N0-p-tolyl-[1,3,5]triazine-2,4-diamine (51)
(300 MHz, CDCl3):
d
8.52 (d, 1H, J ¼ 5.25 Hz, AreH), 8.04 (d, 1H,
Yield: 82%; mp 187e189 ꢁC; MS: 552 (M þ 1); 1H NMR
(300 MHz, CDCl3): 8.58 (d, 1H, J ¼ 5.28 Hz, AreH), 8.07 (d, 1H,
J ¼ 2.04 Hz, AreH), 7.93 (s, 1H, AreH), 7.87 (d, 1H, J ¼ 8.82
Hz, AreH), 7.49 (dd, 1H, J ¼ 2.04, 8.82 Hz, AreH), 7.43 (d, 2H,
J ¼ 8.34 Hz, AreH), 7.34 (t, 1H, J ¼ 8.1 Hz, AreH), 7.18 (d, 1H,
J ¼ 7.32 Hz, AreH), 7.09e7.03 (m, 3H, AreH), 6.97 (d,1H, J ¼ 5.28 Hz,
AreH), 6.90 (bs, 1H, NH), 6.79 (bs, 1H, NH), 6.60 (bs, 1H, NH), 3.80 (t,
4H, J ¼ 4.72 Hz, NeCH2), 2.39 (t, 4H, J ¼ 4.72 Hz, NeCH2), 2.32 (s, 3H,
J ¼ 1.89 Hz, AreH), 8.00 (s, 1H, AreH), 7.91 (d, 1H, J ¼ 8.94 Hz,
AreH), 7.53 (d, 2H, J ¼ 7.83 Hz, AreH), 7.46 (dd,1H, J ¼ 1.89, 8.94 Hz,
AreH), 7.33e7.21 (m, 5H, AreH & NH), 7.11 (s, 1H, NH), 7.04e7.02
(m, 2H, AreH & NH), 6.95e6.93 (m, 2H, AreH & NH), 3.81 (t, 4H,
J ¼ 4.73 Hz, NeCH2), 2.48e2.41(m, 6H, NeCH2), 1.12 (t, 3H,
J ¼ 7.17 Hz, CH3). 13C NMR (50 MHz, CD3OD):
d 169.16, 168.57,
155.44, 153.51, 153.25, 144.99, 144.41, 143.30, 139.92, 133.96, 132.92,
131.59, 130.14, 127.18, 127.02, 124.80, 122.59, 121.40, 120.68, 118.92,
106.51, 56.67, 47.21, 15.59. Anal. Calc. for C30H30ClN9: Calculated C:
65.27, H: 5.48, N: 22.83. Found C: 65.49, H: 5.31, N: 22.69.
CH3). 13C NMR (50 MHz, CDCl3):
d 165.27, 164.45, 152.18, 149.88,
148.13, 141.41, 136.73, 135.70, 132.92, 130.18, 129.62, 129.08, 126.43,
121.87, 120.86, 118.55, 116.86, 114.39, 103.14, 55.16, 46.54, 43.65,
21.08. Anal. Calc. for C30H30ClN9: Calculated C: 65.27, H: 5.48, N:
22.83. Found C: 65.46, H: 5.69, N: 23.04.
6.5.36. N-Butyl-N0-[3-(7-chloro-quinolin-4-ylamino)-phenyl]-N00-
phenyl-[1,3,5]triazine-2,4,6-triamine (48)
Yield: 87%; mp 128e131 ꢁC; MS: 511 (M þ 1); 1H NMR
6.5.40. N-[3-(7-Chloro-quinolin-4-ylamino)-phenyl]-6-(4-ethyl-
piperazin-1-yl)-N0-p-tolyl-[1,3,5]triazine-2,4-diamine (52)
(300 MHz, CDCl3):
d
8.58 (d, 1H, J ¼ 5.31 Hz, AreH), 8.06 (d, 1H,
J ¼ 2.07 Hz, AreH), 7.99 (s, 1H, AreH), 7.85 (d, 1H, J ¼ 8.97
Hz, AreH), 7.55 (d, 2H, J ¼ 7.78 Hz, AreH), 7.49 (dd, 1H, J ¼ 2.07,
8.97 Hz, AreH), 7.32 (t, 1H, J ¼ 7.98 Hz, AreH), 7.26e7.23 (m, 3H,
AreH), 7.11 (bs, 1H, NH), 7.07 (d, 1H, J ¼ 5.31 Hz, AreH), 6.97e6.93
(m, 3H, AreH & NH), 6.60 (bs, 1H, NH), 5.10 (bs, 1H, NH), 3.40 (q, 2H,
J ¼ 5.92 Hz, NHeCH2), 1.58 (quint, 2H, J ¼ 7.18 Hz, CH2), 1.44 (sext,
2H, J ¼ 7.45 Hz, CH2), 0.92 (t, 3H, J ¼ 7.35 Hz, CH3). 13C NMR
Yield: 86%; mp 163e165 ꢁC; MS: 566 (M þ 1); 1H NMR
(300 MHz, CDCl3): 8.59 (d, 1H, J ¼ 5.28 Hz, AreH), 8.07 (d, 1H,
J ¼ 2.04 Hz, AreH), 7.95 (s,1H, AreH), 7.87 (d,1H, J ¼ 8.94 Hz, AR-h),
7.49 (dd, 1H,J ¼ 2.04 8.94 Hz, AreH), 7.43 (d, 2H, J ¼ 8.31 Hz, AreH),
7.34 (t, 1H, J ¼ 7.98 Hz, AR-h), 7.18 (d, 1H, J ¼ 8.24 Hz, AreH),
7.09e7.03 (m, 3H, AreH), 6.97 (d, 1H, J ¼ 5.28 Hz, AreH), 6.89 (bs,
1H, NH), 6.78 (bs, 1H, NH), 6.59 (bs, 1H, NH), 3.81 (t, 4H, J ¼ 4.83 Hz,
NeCH2), 2.48e2.41 (m, 6H, NeCH2), 2.20 (s, 3H, CH3), 1.12 (t, 3H,
(50 MHz, CDCl3):
d 166.17, 164.42, 151.71, 149.44, 148.86, 140.84,
140.39, 139.08, 135.87, 130.01, 129.04, 128.16, 126.27, 123.57, 122.43,
121.34, 118.51, 117.30, 116.74, 114.98, 102.59, 41.08, 31.96, 20.41,
14.06. Anal. Calc. for C28H27ClN8: Calculated C: 65.81, H: 5.33, N:
21.93. Found C: 65.67, H: 5.51, N: 22.14.
J ¼ 7.2 Hz, NeCH3). 13C NMR (50 MHz, CDCl3):
d 165.19, 164.65,
152.23, 149.93, 148.11, 140.43, 136.75, 135.67, 132.89, 130.17, 129.62,
126.41, 121.86, 120.85, 118.58, 116.83, 114.35, 103.59, 52.93, 52.75,
43.70, 21.08, 12.29. Anal. Calc. for C31H32ClN9: Calculated C: 65.77,
H: 5.70, N: 22.27. Found C: 65.46, H: 5.91, N: 22.45.
6.5.37. N-[3-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-(2-
diethylamino-ethyl)-N00-phenyl-[1,3,5]triazine-2,4,6-triamine (49)
Yield: 72%; mp 156e158 ꢁC; MS: 554 (M þ 1); 1H NMR
(300 MHz, CDCl3): 8.58 (d, 1H, J ¼ 5.28 Hz, AreH), 8.06 (d,
1H, J ¼ 2.04 Hz, AreH), 7.98 (s, 1H, NH), 7.90 (d, 1H, J ¼ 8.7 Hz,
AreH), 7.62 (d, 2H, J ¼ 7.86 Hz, AreH), 7.50 (dd, 1H, J ¼ 2.04, 8.7 Hz,
AreH), 7.33 (t, 1H, J ¼ 7.92 Hz, AreH), 7.29e7.23 (m, 3H, AreH), 7.13
(bs, 1H, NH), 7.11 (d, 1H, J ¼ 5.28 Hz, AreH), 6.99e6.96 (m, 3H,
AreH), 6.93 (bs, 1H, NH), 6.67 (bs, 1H, NH), 5.68 (bs, 1H, NH), 3.48
(q, 2H, J ¼ 6.52 Hz, NHeCH2), 2.59e2.55 (m, 6H, NeCH2), 1.04 (t, 6H,
6.5.41. N-[3-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-(3-
morpholin-4-yl-propyl)-N00-p-tolyl-[1,3,5]triazine-2,4,6-triamine(53)
Yield: 89%; mp 145e147 ꢁC; MS: 596 (M þ 1); 1H NMR
(300 MHz, CDCl3): 8.58 (d, 1H, J ¼ 5.28 Hz, AreH), 8.06 (d, 1H,
J ¼ 2.04 Hz, AreH), 7.90 (d, 1H, J ¼ 8.73 Hz, AreH), 7.49e7.43 (m,
3H, AreH), 7.33 (t, 1H, J ¼ 7.95 Hz, AreH), 7.11e7.05 (m, 5H, AreH),
6.98 (d, 1H, J 5.28 Hz, AreH), 6.81 (bs, 1H, NH), 6.68 (bs, 1H, NH),
6.58 (bs, 1H, NH), 3.73 (t, 4H, J ¼ 4.47 Hz, OeCH2), 3.51 (q, 2H,
J ¼ 6.33 Hz, NHeCH2), 2.44e2.34 (m, 6H, NeCH2), 2.22 (s, 3H,
CH3), 1.77 (quint, 2H, J ¼ 7.60 Hz, CH2). 13C NMR (50 MHz, CDCl3):
166.29, 164.64, 152.25, 149.96, 148.16, 141.16, 140.42, 136.71, 135.64,
133.00, 130.09, 129.61, 126.36, 121.90, 120.90, 118.57, 116.32, 103.06,
67.38, 57.67, 54.03, 40.68, 25.84, 21.09. Anal. Calc. for C32H34ClN9O:
Calculated C: 64.47, H: 5.75, N: 21.15. Found C: 64.72, H: 5.61,
N: 21.38.
J ¼ 6.85 Hz, CH3). 13C NMR (50 MHz, DMSO-d6):
d 166.56, 164.95,
161.99, 152.79, 150.47, 149.09, 142.23, 141.09, 134.72, 130.03, 129.11,
128.49, 125.65, 125.42, 122.40, 120.78, 119.20, 117.23, 116.80, 115.25,
102.77, 52.51, 52.36, 47.38, 36.37, 12.54. Anal. Calc. for C30H32ClN9:
Calculated C: 65.03, H: 5.82, N: 22.75. Found C: 64.86, H: 5.88, N:
22.91.
6.5.38. N-[3-(7-Chloro-quinolin-4-ylamino)-phenyl]-N0-(3-
morpholin-4-yl-propyl)-N00-phenyl-[1,3,5]triazine-2,4,6-triamine (50)
Yield: 89%; mp 167e169 ꢁC; MS: 582 (M þ 1); 1H NMR
7. Materials and methods
7.1. In vitro antimalarial activity assay
(300 MHz, CDCl3):
d
8.56 (d, 1H, J ¼ 5.04 Hz, AreH), 8.05 (d,
1H, J ¼ 1.89 Hz, AreH), 7.98 (s, 1H, AreH), 7.91 (d, 1H, J ¼ 9.42 Hz,
AreH), 7.56 (d, 2H, J ¼ 7.88 Hz, AreH), 7.49 (dd, 1H, J ¼ 1.89,
9.42 Hz, AreH), 7.33 (t, 1H, J ¼ 8.07 Hz, AreH), 7.30e7.27 (m, 3H,
AreH), 7.13 (bs, 1H, NH), 6.98e6.96 (m, 4H, J ¼ AreH & NH), 6.72
(bs, 1H, NH), 3.74 (t, 4H, J ¼ 4.41 Hz, OeCH2), 3.51 (q, 2H,
J ¼ 6.24 Hz, NHeCH2), 2.63e2.46 (m, 6H, NeCH2), 1.71 (quint, 2H,
The in vitro antimalarial activity of compounds was determined
by using a standardized inexpensive assay based on Syber Green I
[59]. The compounds were dissolved in DMSO at 5 mg/ml. For the
assays, fresh dilutions of all compounds in screening medium were
prepared and 50 ml of highest starting concentration (500 ng/ml)
was dispensed in duplicate wells in row B of 96 well tissue culture
plate. The highest concentration for chloroquine was 25 ng/ml.
Subsequently two fold serial dilutions were prepared up to row
J ¼ 6.52 Hz, CH2). 13C NMR (50 MHz, CDCl3):
d 166.25, 164.67,
151.88, 149.61, 148.42, 141.14, 140.35, 139.43, 135.75, 130.02, 129.08,
128.68, 126.33, 123.37, 122.18, 120.76, 118.51, 117.01, 116.39, 114.77,
H (seven concentrations). Finally 50
ml of 2.5% parasitized cell

A. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 676e690
689
suspension containing 0.5% parasitemia was added to each well
7.5. Molecular docking study
except 4 wells in row A which received non infected cell suspen-
sion. These wells containing non infected erythrocytes in the
absence of drugs served as negative controls, while parasitized
erythrocytes in the presence of CQ served as positive control. After
LigandFit, a modern docking program within Cerius2 version
4.10 (Cerius2 Version 4.10 (2005), Accelrys Inc., San Diego, USA),
was used for docking runs [63]. Reference protein coordinates for
docking were taken from the X-ray structure of Plasmodium falci-
parum TS-DHFR in complex with the pfDHFR inhibitor WR99210.
The site search was performed in the shape-based mode based on
the crystal structure of the compound WR99210. All calculations
were performed with the CFF 1.01 force field. Conformations were
generated with Monte Carlo simulations (10,000 trials) and Flexible
fit was selected. A grid resolution was set to 0.5 Å. Electrostatic
energy was included in the calculation of the ligand internal
energy. In order to avoid identical conformations, root mean square
deviation threshold of 1.5 Å and a score threshold of 20 kcal/mol
were used while saving the final conformations. The top 10
conformations were saved after rigid body minimizations of 1000
steps. Scoring was performed for each of the 10 saved ligand
conformations using a set of scoring functions as implemented in
Cerius2, including LigScore1, LigScore2,13 PLP1, PLP2,14 JAIN,15
PMF16 and LUDI.17 Consensus scoring function was also used to
evaluate and rank the ligand binding affinities.
72 h of incubation, 100 ml of lysis buffer [20 mM tris (Ph 7.5), 5 mM
EDTA, 0.008% (wt/vol) saponin, and 0.08% (vol/vol) Triton X e 100]
containing 1 ꢀ concentration of SYBER Green I (Invitrogen) was
added to each cell. The plates were re-incubated for 1 h at room
temperature and examined for the relative fluoroscence units
(RFUs) per well using the FLUOstar, BMG lab technologies. The 50%
inhibitory concentration (IC50) was determined using non-linear
regression analysis dose-response curves.
7.2. In vitro cytotoxicity assay
Cytotoxicity of the compounds was determined against VERO
cell lines (C-1008; Monkey kidney fibroblast cells) using MTT assay
[60]. A total of 1 ꢀ 104 cells/well were incubated with varying
concentrations of compound for 72 h. The highest concentration of
compound was 100 mg/ml. The 50% inhibitory concentration (IC50)
was determined using non-linear regression analysis dose-
response curves and represented the concentration of compound
required to kill 50% of the fibroblast cells.
Acknowledgments
A. Kumar thanks the Council of Scientific and Industrial
Research, India, for the award of Senior Research Fellowship. We
are also thankful to S.A.I.F. Division, CDRI, Lucknow, for providing
spectroscopic data. CDRI Communication No.7995.
7.3. In vivo activity assay
The in vivo drug response was evaluated in Swiss mice infected
with P. yoelii (N-67 strain) which is innately resistant to CQ [61].
The mice (22ꢂ2g) were inoculated with 1 ꢀ106 parasitized RBC on
day 0 and treatment was administered to a group of five mice from
day 0e3, once daily. The aqueous suspensions of compounds were
prepared with a few drops of Tween 80. Initially, the efficacy of test
compounds was evaluated at 50.0 mg/kg/day and required daily
dose was administered in 0.2 mL volume via intraperitoneal route.
The efficacy of test compounds was evaluated at 100 mg/kg/day
and required daily dose was administered in 0.1 mL volume via oral
route. Parasitemia levels were recorded from thin blood smears
between days 4 and 6. The mean value determined for a group of 5
mice was used to calculate the percent suppression of parasitemia
with respect to the untreated control group. Mice treated with CQ
served as reference controls.
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