4-Aminoquinoline-1,3,5-triazine: Design, synthesis, in vitro antimalarial activity and docking studies

Article abstract of DOI:10.1039/c3nj00317e

A series of hybrid 4-aminoquinoline 1,3,5-triazine derivatives was synthesized and their chemical structure were confirmed by ¹H-NMR, ¹³C-NMR, FT-IR and mass spectrometric analyses. In vitro antimalarial activity of these compounds was evaluated against chloroquine-sensitive (3D-7) and chloroquine resistant (RKL-2) strains of P. falciparum. Results showed that all compounds had considerable antimalarial activity against both the strains and further docking studies were performed on both wild type (1J3I.pdb) and quadruple mutant (N51I, C59R, S108 N, I164L, 3QG2.pdb) pf-DHFR-TS to quantify the structural parameter necessary for the activity.

Full text of DOI:10.1039/c3nj00317e

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4-Aminoquinoline-1,3,5-triazine: Design, synthesis,
in vitro antimalarial activity and docking studies
Cite this: DOI: 10.1039/c3nj00317e
Hans Raj Bhat,*a Udaya Pratap Singh,ab Prashant Gahtori,c Surajit Kumar Ghosh,d
Kabita Gogoi,e Anil Prakashe and Ramendra K. Singhb
A series of hybrid 4-aminoquinoline 1,3,5-triazine derivatives was synthesized and their chemical
structure were confirmed by 1H-NMR, 13C-NMR, FT-IR and mass spectrometric analyses. In vitro
antimalarial activity of these compounds was evaluated against chloroquine-sensitive (3D-7) and
chloroquine resistant (RKL-2) strains of P. falciparum. Results showed that all compounds had
considerable antimalarial activity against both the strains and further docking studies were performed
on both wild type (1J3I.pdb) and quadruple mutant (N51I, C59R, S108 N, I164L, 3QG2.pdb) pf-DHFR-TS
to quantify the structural parameter necessary for the activity.
Received (in Montpellier, France)
26th March 2013,
Accepted 4th June 2013
DOI: 10.1039/c3nj00317e
www.rsc.org/njc
molecules using 2-(piperazin-1-yl) ethylamine as a linker to
connect covalently 1,3,5-triazine and 4-aminoquinoline. These
Introduction
Growing levels of antibiotic resistance have resulted in significant
morbidity and mortality worldwide and significant evidence has
emerged of the resistance of Plasmodium falciparum (malignant
tertian malaria) to almost all antimalarial agents.1–3 Unfortunately,
despite the indisputable need for newer therapeutic agents, drug
discovery for malaria is very challenging for several reasons, such
as (i) low levels of medical care as malaria is prevalent in poor
resource countries, (ii) non-availability of oral administered
drugs and (iii) unaffordable therapy etc.4 One of the possible
drug discovery approaches for such an infectious disease is by
combining two pharmacophoric groups that may act as a dual
drug targeting more than one site.5 As a consequence, a few
aminoquinoline- and trioxane-based hybrid molecules have
already been developed6,7 and some are in clinical trials as
antimalarial agents.8 To revalidate an earlier concept and in
continuation of our research program to develop novel hybrid
molecules, we have already reported 4-aminoquinoline-triazine,9,10
thiazole-1,3,5-triazines,11–13 1,3-thiazine-1,3,5-triazine,14 1,3,4-
thiadiazole-1,3,5-triazine,15 phenyl thiazolyl-1,3,5-triazine deri-
vatives.16 We herein devised a new series of hybrid target
identified hybrid molecules were screened for their antimalarial
activity against laboratory adapted 3D-7 (chloroquine sensitive)
and RKL-2 (chloroquine resistant) strains of Plasmodium
falciparum. Additionally, molecular docking studies were also
performed to get insight of the essential key structural require-
ment for antimalarial activity.
Result and discussion
Chemistry
Cynauric chloride (2,4,6-trichloro 1,3,5-triazine), due to
presence of three chloro groups offers numerous possible
modifications to be carried out via nucleophilic substitution
reactions. This structural parameter is quite important in terms
of medicinal chemistry to explore the SAR and thus, the
chemistry of 1,3,5-triazine has been exploited experimentally
and has been the subject of many research and review articles.
The synthesis of compound 3 was achieved by the nucleophilic
substitution of 2-(piperazin-1-yl)ethanamine (2) with 4,7-
dichloroquinoline (1) at 120–130 1C for 6–8 h. Whereas, 2,4,6-
trichloro 1,3,5-triazine (4) was allowed to react with different
aromatic amines (a–j) in the presence of a saturated solution of
NaHCO3 to afford monochloro di-substituted 1,3,5-triazine
5(a–j) derivatives. Later, the desired compounds 6(a–j) were
synthesized by refluxing the monochloro di-substituted 1,3,5-
triazine 5(a–j) with 7-chloro-N-(2-(piperazin-1-yl)ethyl)quinolin-
4-amine (3) in 1,4-dioxane for 6–7 h as shown in Scheme 1. The
completion of reaction was ascertained on the basis of TLC
using appropriate solvent systems. FT-IR spectra of compounds
a Department of Pharmaceutical Sciences, Sam Higginbottom Institute of
Agriculture, Technology & Science, Deemed University, Allahabad, 211007 India.
E-mail: pharmahans@gmail.com; Fax: +91-532-2684394; Tel: +91-9616574197
b Nucleic Acids and Antiviral Research Laboratory, Department of Chemistry,
University of Allahabad, Allahabad, 211002 India
c Faculty of Pharmacy, Uttarakhand Technical University, Dehradun, 248007 India
d Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh,
786004 India
e Regional Medical Research Centre, ICMR, Dibrugarh, 786005 India
c
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Scheme 1 Reagents and conditions: R–H (a–j) various amines, (i) reflux 1 h at 80 1C followed by 6–8 h at 120–130 1C (ii) 1,4-dioxane 0–5 1C 1 h, 40–45 1C, 3 h,
NaHCO3 (iii) 1,4-dioxane 120–130 1C, 6–7 h, K2Co3.
Table 1 In vitro antimalarial activity of hybrid derivatives 6(a–j)
6(a–j) are presented in the experimental section. The CQN
group of 1,3,5-triazine was observed at 1675–1250 cmꢀ1
.
% Dead asexual parasites
3D-7a 3D-7a
A strong band at 1475 cmꢀ1 is characteristic of the stretching
frequencies of the Ar CQC group. The FT-IR spectra show a
band at 850–670 cmꢀ1 corresponding to the aromatic ring.
The Ar C–H group absorption is observed as a distinct band at
2965 cmꢀ1, whereas the NH linkage present on 1,3,5-triazine
RKL-2b
RKL-2b
5 mg mLꢀ1 50 mg mLꢀ1 5 mg mLꢀ1 50 mg mLꢀ1
Compound
(Dose)
(Dose)
(Dose)
(Dose)
6a
6b
6c
6d
6e
6f
6g
6h
6i
13.5
11
16.5
15
20.5
15.5
18
26.5
15.5
6
25.5
23.5
29.5
24
30.5
36
25
36
31
18
13.5
7.5
11
9.5
10
16
15.5
11
29
15.5
22
17
17
26.5
30.5
24
15.5
16
ring is observed at 3450–3100 cmꢀ1
.
Hybrid derivatives show a characteristic strong band at
1650–1683 cmꢀ1, which is attributed to the CQN stretching
vibrations. The 1H-NMR spectra shows a doublet at 8.65–7.57 ppm
corresponding to the presence of the quinoline ring. A distinct
resonance at 2.14 ppm is due to the presence of the NH group
situated on the carbon atom of the s-triazine ring. The resonance
due to Ar–H is observed at 6.83 to 8.30 ppm for the disubstituted
1,3,5-triazine derivatives 5(a–j). This is further supported by the
resonance at 3.56 and 2.53 ppm for the piperazine protons and
3.45–2.78 ppm for methylene protons. Finally structures of all
compounds were confirmed on the basis of mass spectra and
elemental analysis.
10
8
6j
Chloroquine (CQ) 51
—
(0.7 mg mLꢀ1
)
Chloroquine (CQ)
—
50
46
50
(1.2 mg mLꢀ1
)
Proguanil
(4200 mg mLꢀ1
)
a
Wild malaria parasite (chloroquine sensitive). b Mutant malaria parasite
(chloroquine resistant).
Antimalarial activity and structure–activity relationship
as 1,3-diamino propane containing 1,3,5-triazine (6h) showed
The antimalarial screening results of compounds 6(a–j), against the highest activity against chloroquine-sensitive (3D-7) but
chloroquine-sensitive (3D-7) and chloroquine resistant (RKL-2) low activity against chloroquine resistant (RKL-2) strains. The
strains at 5 mg mLꢀ1 and 50 mg mLꢀ1 dosages are presented in replacement of aliphatic with aromatic groups 6(a–g) and 6(i–j),
Table 1 using chloroquine and proguanil as standard drugs. displayed mid to moderate activity against both the strains of P.
It was observed that the entire set of analogues 6a–j displayed falciparum. Compound 6a having a p-Cl substituent at the
killing of parasitemia ranging from 18–36% at 50 mg mLꢀ1 and phenyl ring connected to 1,3,5-triazine showed 13.5% parasite-
6–26.5% at 5 mg mLꢀ1 against 3D-7, whereas, 16–30.5% at mia kill against both the strains of P. falciparum at 5 mg mLꢀ1
,
50 mg mLꢀ1 and 7.5–15.5% at 5 mg mLꢀ1 were reported against while incorporation of p-Br at the phenyl ring (6g) displayed 18
RKL-2 strains. Results revealed that aliphatic analogous, such and 15.5% parasitemia kill against chloroquine-sensitive and
c
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chloroquine resistant strains at 5 mg mLꢀ1 and compound 6f while, one s–p interaction was revealed between the quinoline
having a substituent (p-OCH3) showed similar antimalarial ring and Arg59 and Arg122 have been reported. Compound 6a
activity with 15.5–16% parasitemia kill against both strains. was reported to exhibit one hydrogen bond with Ser111 with the
The replacement of the para methoxy group with p-OH (6b) and involvement of the amine on the phenyl ring of 1,3,5-triazine
p-NO2 (6i) showed mild to moderate activity against both the and p–p, +–p bonds through Phe58 and Lys49 with the phenyl
strains. Compounds 6c and 6d containing electron donating ring of 1,3,5-triazine as well. In the quadruple mutant, it
substituents (p-CH3 and o-CH3) at the phenyl ring connected to showed no hydrogen bond except the formation of +–p bonds
1,3,5-triazine showed similar activity against a chloroquine- through Arg59 with the phenyl ring of 1,3,5-triazine along with
sensitive strain with 15–16.5% of parasitemia kill but with the lower BE (ꢀ80.4). Introduction of a hydroxyl group (4-OH) on
loss of the antimalarial activity against a chloroquine resistant the phenyl ring of 1,3,5-triazine (6b) caused formation of new
strain at 5 mg mLꢀ1 with 9.5–11% of parasitemia kill only. The H-bonds between Asp54 and the hydroxyl group of the phenyl
morpholine substituted derivative 6j showed marginally less linked to 1,3,5-triazine and p–p, s–p non-bonded interactions
activity against both the strains with 6–8% of parasitemia kill. through the involvement of phenyl with Phe58 and 1,3,5-
This was further subtantiated by the study carried out by triazine with Leu46 in wild pf-DHFR-TS. The same ligand led
Sunduru et al.23 Introducing piperidine at 1,3,5-triazine 6e to the creation of two hydrogen bonds between Asp54 and
showed improved activity with 20.5% parasitemia kill against Ser111 and the hydroxyl group of the phenyl and the amine
a chloroquine sensitive strain but with loss of activity to 10% of group of the 1,3,5-triazine as well as a p–p interaction with
parasitemia kill against a chloroquine resistant strain at 5 mg mLꢀ1
Among all derivatives, compounds 6h and 6g showed good activity cing p-hydroxy aniline with o-toluidine, 6c, led to major varia-
against both strains at 5 mg mLꢀ1 concentration.
tion in the binding energy and the absence of hydrogen bonds
.
Phe58 in the mutant with lower binding energy (ꢀ17.2). Repla-
An SAR study indicated that aromatic derivatives, such as were reported by in both wild and mutant type, however, one
6(a–j) except 6h showed mild to moderate activity but an p–p stacking interaction was observed between Phe58 and the
aliphatic derivative 6h showed good activity against a chloro- phenyl of 1,3,5-triazine in wild type. Surprisingly, no H-bond
quine sensitive strain. The compound with an electron with- was reported in compounds 6d in the wild and mutant protein.
drawing substituent at the phenyl ring, such as 6g showed good Further, compound 6d showed one p–p interaction between
activity against a chloroquine resistant strain at 5 mg mLꢀ1 Phe58 and phenyl 1,3,5-triazine in wild type and p–s, +–p of
concentration. However, introduction of basic moieties, like Phe116 andArg59 with quinoline in the mutant. Compound 6h
piperidine, morpholine, and 1,3-diaminopropane 6e, 6h & 6g at showed one hydrogen bond between Asp54 and Cys15 and the
the 1,3,5-triazine enhances the activity confirming that the NH group of propane linked to 1,3,5-triazine in wild type; in
presence of basic moieties at 1,3,5-triazine is imperative for mutant type formation of two hydrogen bonds between Asn108
antimalarial activity. Lastly, compounds 6c and 6d having and Lys49 with the nitrogen atom of the quinoline ring and the
electron donating groups at the ortho and para position on amine of the propane group linked to 1,3,5-triazine ring. In
the phenyl ring displayed no antimalarial activity. Our results wild type, compound 6e showed the formation of two hydrogen
are in good agreement with the similar study carried out by bonds with Ser167 and Ser108 and in addition to the p–s
Manoher et al., which reported that the length of the covalent interactions through the 1,3,5-triazine and piperidine linkage
linker between piperazine and 4,7-dichloroquinoline moieties with Leu46 and Phe58. However, in the case of the mutant, no
should be 2–3 carbon units for better activity.22
hydrogen bond was reported. The presence of a bromo group in
compound 6g exhibited H-bonds with Ser111 in wild and
mutant type and additional p–p, p–+ interactions with Phe58,
Molecular docking study
The docking studies of target compounds were performed Lys49 and the phenyl of 1,3,5-triazine in wild type; it also
using the binding pocket of both the wild type (1J3I.pdb) and showed p–+ interactions of Arg59 with the benzene ring in
quadruple mutant (N51I, C59R, S108 N, I164L, 3QG2.pdb) mutant type. Compound 6i showed two hydrogen bond with
pf-DHFR. The docking results and docked conformations of Ser167 and Arg122 through involvement of the nitrogen atom
ligands in the active site are illustrated in Table 2 and Fig. 1. of the quinoline ring and the nitro group of the phenyl linked
These results disclosed that targeted molecules exhibited con- to 1,3,5-triazine. It also exhibited two p–+ bonds with Arg122
siderable and diverse binding affinities towards the wild (153.8 and Phe116 utilising the phenyl ring and nitro group linked to
to ꢀ84.2) and quadruple mutant (ꢀ11.7 to ꢀ80.4) pf-DHFR-TS the 1,3,5-triazine ring. In the case of the mutant, no hydrogen
along with the formation of numerous hydrogen bonds and bond was reported. On the other hand, the introduction of
p–p, p–+, p–s interactions. Further, compound 6f exhibited higher morpholine on 1,3,5-triazine, 6j, showed formation of the p–p
antimalarial activity and considerably low binding energies (BE) interaction through involvement of the 1,3,5-triazine ring with
for wild (ꢀ33.9) and mutant (ꢀ70.0) pf-DHFR-TS with the the Phe116 in mutant pf-DHFR-TS.
formation of one hydrogen bond with Ser111 through the
In conclusion, we can say that most of the ligands exhibited
involvement of the NH of the phenyl linkage to 1,3,5-triazine a hydrophobic interaction between the phenyl ring on the
ring, while the s–p bond with Ser167 through the involvement ligand and the Phe58 residue as well as a s–p interaction
of the quinoline ring in wild type. The one hydrogen bond was between the 1,3,5-triazine ring and the Leu46 residue in the
observed between Leu164 and the phenyl ring of 1,3,5-triazine wild type strain. Similarly in the case of the quadruple mutant
c
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Table 2 Docking interaction of hybrid derivatives 6(a–j) in wild type and quadruple mutant pf-DHFR-TS
Wild type pf-DHFR-TS
Quadruple mutant pf-DHFR-TS
Donor/acceptor
Compound hydrogen bond
Non-bonded
forces
Binding energy Donor/acceptor
Binding energy
(kcal molꢀ1
)
hydrogen bond
Non-bonded forces (kcal molꢀ1
)
6a
1,3,5-triazine-
NHꢁ ꢁ ꢁSER 111
p–p
—
p–+, p–s
ꢀ80.4
Benzene–PHE58
p–+
Benzene–ARG59
Benzene–LYS49
p–p
6b
1,3,5-triazine-phenyl
ꢀ31.7
1,3,5-triazine-phenyl-
OHꢁ ꢁ ꢁOD-ASP54
1,3,5-triazine-NHꢁ ꢁ ꢁOG-
SER111
p–p
ꢀ17.2
OHꢁ ꢁ ꢁASP54
Benzene–PHE58
Benzene–PHE58
p–s
Triazine-LEU46
6c
6d
—
—
p–p Benzene–PHE58 ꢀ84.2
—
—
—
p–s
ꢀ18.0
ꢀ52.7
p–p Benzene–PHE58 153.8
Quinoline–PHE116
p–+
Quinoline–ARG59
—
6e
Quinoline-NHꢁ ꢁ ꢁSER167 p–s
Piperazine-NHꢁ ꢁ ꢁSER108 Triazine–LEU46
p–s
ꢀ14.2
—
ꢀ19.1
Piperidine–PHE58
p–s
Quinoline–SER167
6f
1,3,5-triazine-phenyl
NHꢁ ꢁ ꢁSER111
ꢀ33.9
1,3,5-triazine-phenyl
NHꢁ ꢁ ꢁLEU164
p–+
ꢀ70.0
ꢀ42.4
Quinoline–ARG122,
ARg-159
p–+
6g
1,3,5-triazine-phenyl
NHꢁ ꢁ ꢁSER111
p–p
52.2
1,3,5-triazine-phenyl
NHꢁ ꢁ ꢁSER111
Benzene–PHE58
p–+
Benzene–ARG59
Benzene–LYS49
—
6h
6i
1,3,5-triazine-1,3-diamino
propane
NHꢁ ꢁ ꢁASP54, CYS15
28.3
Quinoline Nꢁ ꢁ ꢁASP108
—
—
ꢀ46.5
1,3,5-triazine-1,3-diamino
propane
NHꢁ ꢁ ꢁLYS49
Quinoline
p–+
Nꢁ ꢁ ꢁSER167
Benzene–ARG122
p–+
ꢀ53.3
ꢀ24.8
—
ꢀ11.7
ꢀ59.7
1,3,5-triazine-phenyl
NOꢁ ꢁ ꢁARG122
Benzene NO–PHE116
—
6j
—
—
p–p
Triazine–PHE116
of pf-DHFR-TS, a hydrophobic interaction was observed between Synthesis of compound 3 was achieved by the nucleophilic
the phenyl ring and Phe58 in addition to H-bond formation substitution of N-aminoethyl piperazine (2) at the fourth chloro of
between the 1,3,5-triazine ring and Ser111. Additionally, the 4,7-dichloroquinoline (1). The syntheses of disubstituted s-triazines
quinoline ring was engaged in the formation of a +–p inter- 5(a–j) were accomplished by the nucleophilic substitution of the Cl
action with Arg59.
atom of the 2,4,6-trichloro-1,3,5-triazine (cyanuric chloride) (4) with
different primary and secondary amines (a–j). Finally, compounds
6(a–j) were synthesized by incorporating a di-substituted 1,3,5-
Experimental
All commercially available solvents and reagents of analytical triazine moiety 5(a–j) in the piperazine attached to 4-amino-
grade were used without further purification. Melting points quinoline pharmacophore (3).
were determined on a Veego, MPI melting point apparatus and
7-Chloro-N-(2-(piperazin-1-yl)ethyl)quinolin-4-amine (3). A
FT-IR (2.0 cmꢀ1, flat, smooth, abex) were recorded on a Perkin mixture of 4,7-dichloroquinoline (1 eq.) and N-aminoethyl
1
Elmer RX-I Spectrophotometer. H-NMR spectra were recorded piperazine (5 eq.) was heated slowly and raised to 80 1C over 1 h
on a Bruker Avance II 400 NMR and 13C-NMR spectra on Bruker with stirring and subsequently at 120–130 1C for 6–8 h with
Avance II 100 NMR spectrometer in DMSO-d6 using TMS as continued stirring to drive the reaction to completion. The reaction
internal standard. Mass spectra were obtained on VG-AUTO- mixture was cooled to room temperature and taken up in dichloro-
SPEC spectrometer equipped with electrospray ionization (ESI) methane. The organic layer was successively washed with 5%
sources. Elemental analysis was carried out on Vario EL-III aqueous NaHCO3 followed by a water wash and finally with brine.
CHNOS elemental analyzer.
The organic layer was dried over anhydrous Na2SO4 and solvent was
The desired compounds 3, 5(a–j) and 6(a–j) were obtained removed under reduced pressure. The residue was precipitated by
through the synthetic protocol as outlined in Scheme 1. the addition of (80 : 20) hexane–chloroform to obtain compound 3.
c
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Fig. 1 Docking image of hybrid 4-aminoquinoline 1,3,5-triazine derivatives (6h, 6f, 6i and 6e) in wild and (6h, 6f, 6g and 6a) in mutant pf-DHFR-TS.
Brown-crystals; yield: 78%; M.p: 139–140 1C; MW: 290.79; monitored by TLC using benzene : ethyl acetate (9 : 1) as mobile
Rf : 0.73; FT-IR (nmax; cmꢀ1 KBr): 3450 (N–H stretch), 3022, 2950 phase. The product was filtered and washed with cold water and
(C–H stretch), 1652 (CQN stretch), 1217 (C–N stretch), recrystallized with ethanol to afford pure products 5(a–j).
762 cmꢀ1
;
1H-NMR (400 MHz, CDCl3-d6, TMS) d ppm: 8.65
6-Chloro-N2,N4-bis(4-chlorophenyl)-1,3,5-triazine-2,4-diamine
(d, 1H, J = 5.02 Hz, quinoline), 8.12–7.91 (m, 3H, quinoline), (5a). White-crystals; yield: 75%; M.p: 135–137 1C; MW: 366.63;
7.57 (dd, 1H, J = 2.10, 8.62 Hz, quinoline), 3.48–3.32 (m, 4H, Rf : 0.48; FT-IR (nmax; cmꢀ1 KBr): 3243 (N–H secondary), 2965
methylene), 2.73–2.47 (m, 4H, 2CH2, piperazine), 2.82–2.76 (m, (C–H broad), 1387 (aromatic –CQN); 1H-NMR (400 MHz, CDCl3-
4H, 2CH2, piperazine), 2.14 (br–s, 1H, Quinoline, NH), 1.89 (s, 1H, d6, TMS) d ppm: 7.32 (d, 4H J = 8.72 Hz, Ar–H), 7.08 (d, 4H J =
piperazine NH); 13C-NMR (100 MHz, CDCl3) d, ppm: 158.5, 152.1, 8.52 Hz Ar–H), 4.82 (s, 2H, 2NH); 13C-NMR (100 MHz, CDCl3) d,
151.1, 136.2, 129.4, 129.2, 124.8, 121.6, 116.8, 61.2, 57.2, 55.5, 42.2; ppm: 168.4, 167.8, 137.1, 129.6, 127.7, 122.2; mass: 366 (M + H)+;
mass: 291 (M + 1); elemental analysis for C15H19ClN4: calculated: C, elemental analysis for C15H10Cl3N5: calculated: C, 49.14; H, 2.75;
61.96; H, 6.59; N, 19.27; found: C, 60.84; H, 6.63; N, 19.26.
N, 19.10. Found: C, 49.17; H, 2.77; N, 19.15.
General procedure for synthesis of disubstituted 1,3,5-triazine
4,40-(6-Chloro-1,3,5-triazine-2,4-diyl)bis(azanediyl)diphenol (5b).
derivatives 5(a–j). To a stirred solution of ice cold 2,4,6-trichloro- Black crystals; yield%: 67; M.p: 251–252 1C; MW: 329.74; Rf: 0.47;
1,3,5-triazine (4) (0.1 mol) in acetone (25 mL), different anilines (a–j) FT-IR (nmax; cmꢀ1 KBr): 3400 (OH aromatic), 3300 (N–H secondary),
(0.2 mol) were added drop wise at 0–5 1C. The resulting reaction 3000 (C–H aromatic ring), 1675 (CQN), 1475 (CQC aromatic ring),
mixture was stirred at 40–45 1C for 3 h followed by drop-wise 1250 (C–N aromatic ring); 1H-NMR (400 MHz, CDCl3-d6, TMS)
addition of NaHCO3 solution (0.1 mol) taking care that the reaction d ppm: 7.26–7.21 (m, 4H, 4CH, Ar–H), 6.93–6.84 (m, 4H, 4CH,
mixture did not become acidic. The completion of reaction was Ar–H), 5.25 (s, 2H, Ar–OH), 4.13 (br, s, 2H, 2NH); 13C-NMR
c
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(100 MHz, CDCl3) d, ppm: 164.2, 160.5, 153.6, 131.5, 122.1, 116.7; 3300 (N–H secondary), 3000 (C–H), 1675 (CQN), 1475 (CQC
mass 330.20 (M + H)+; elemental analysis for C15H12ClN5O2: calcu- aromatic ring), 1250 (C–N aromatic ring); 1H-NMR (400 MHz,
lated: C, 54.64; H, 3.67; N, 21.24. Found: C, 54.63; H, 3.69; N, 21.23. CDCl3-d6, TMS) d ppm: 5.10–5.08 (m, 4H, 2NH2), 4.13 (br, s, 2H,
6-Chloro-N2,N4-di-o-tolyl-1,3,5-triazine-2,4-diamine (5c). Yellowish 2NH), 3.20–3.18 (m, 4H, 2CH2), 2.63–2.61 (m, 4H, 2CH2),
crystals; yield: 72%; M.p: 225–226 1C; MW: 332.80; Rf : 0.67; FT-IR 1.68–1.65 (m, 4H, 2CH2); 13C-NMR (100 MHz, CDCl3) d, ppm:
(nmax; cmꢀ1 KBr): 3310 (N–H secondary), 3000 (C–H), 1620–1650 164.2, 160.5, 50.1, 29.6; mass 260.23 (M + H)+; elemental analysis
(CQC), 1605 (CQC aromatic ring), 1475 (CQC aromatic ring); for C9H18ClN7: calculated: C, 41.62; H, 6.99; N, 37.75. Found: C,
1H-NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.10–7.07 (m, 4H, 41.60; H, 7.03; N, 37.78.
4CH, Ar–H), 7.05–7.02 (m, 4H, 4CH, Ar–H), 5.24 (br, s, 2H, 2NH),
6-Chloro-N2,N4-bis(4-nitrophenyl)-1,3,5-triazine-2,4-diamine
2.20 (s, 6H, 2CH3); 13C-NMR (100 MHz, CDCl3) d, ppm: 168.3, 167.8, (5i). Yellow-crystals; yield: 86%; M.p: 143–145 1C; MW: 387.74;
141.5, 131.2, 129, 126.4, 123.8, 123.6, 17.2; mass 326.20 (M + H)+; Rf: 0.55; FT-IR (nmax; cmꢀ1 KBr): 3289 (N–H secondary), 3055
elemental analysis for C17H16ClN5: calculated: C, 62.67; H, 4.95; N, (C–H broad), 1548–1446 (aromatic CQN); H-NMR (400 MHz,
1
21.50. Found: C, 62.63; H, 4.98; N, 21.55.
CDCl3-d6, TMS) d ppm: 7.40–7.38 (m, 4H, 4CH, Ar–H), 7.32–7.30
6-Chloro-N2,N4-di-p-tolyl-1,3,5-triazine-2,4-diamine (5d). Pale- (m, 4H, 4CH, Ar–H), 3.62 (br, s, 2H, 2NH); 13C-NMR (100 MHz,
yellowish crystals; yield: 78%; M.p: 212–214 1C; MW: 332.80; Rf : CDCl3) d, ppm: 173.5, 168.8, 148.2, 143.1, 131.3, 126.2;
0.72; FT-IR (nmax; cmꢀ1 KBr): 3310 (N–H secondary), 3000 (C–H), mass 388.10 (M + H)+; elemental analysis for C15H10ClN7O4:
1650 (CQC), 1620–1475 (CQC aromatic ring); 1H-NMR (400 MHz, calculated: C, 46.46; H, 2.60; N, 25.29. Found: C, 46.48; H, 2.65;
CDCl3-d6, TMS) d ppm: 7.31–7.26 (m, 4H, 4CH, Ar–H), 7.06–7.02 N, 25.26.
(m, 4H, 4CH, Ar–H), 5.24 (br, s, 2H, 2NH), 2.53 (s, 6H, 2CH3);
6-Chloro-2,4-dimorpholino-1,3,5-triazine (5j). White crystals;
13C-NMR (100 MHz, CDCl3) d, ppm: 168.3, 167.8, 135.8, 131, yield: 73.32%; M.p: 132–135 1C; FTIR (KBr) cmꢀ1 2966 (C–H
129.8, 21.5, 20.4; mass 326.20 (M + H)+; elemental analysis for stretch), 1574 (CQN stretch), 1451 (CQC stretch), 1362 (C–N
C
17H16ClN5: calculated: C, 62.67; H, 4.95; N, 21.50. Found: C, stretch), 1116; 1H-NMR (400 MHz, CDCl3) d ppm: 3.78–3.74 (m,
62.63; H, 4.98; N, 21.55.
8H, 4CH2–O, morpholine), 3.70–3.67 (m, 8H, 4CH2–N, morpho-
2-Chloro-4,6-di(piperidin-1-yl)-1,3,5-triazine (5e). Light brown line); 13C-NMR (100 MHz, CDCl3) d, ppm: 169.6, 164.4, 66.5,
crystals; yield: 64%; M.p: 256–258 1C; MW: 281.78; Rf : 0.73; FT-IR 43.8; mass 286.10 (M
+
H)+; elemental analysis for
C11H16ClN5O2: C, 46.24; H, 5.64; N, 24.51. Found: C, 46.28; H,
(nmax; cmꢀ1 KBr): 3000 (C–H), 1675 (CQN), 1475 (CQC aromatic
1
ring), 1250 (C–N aromatic ring); H-NMR (400 MHz, CDCl3-d6, 5.58; N, 24.56.
TMS) d ppm: 3.71–3.67 (m, 8H, 4CH2, piperidin), 1.63–1.60
General procedure for synthesis of compounds 6(a–j). A
(m, 8H, 4CH2, piperidin), 1.57–1.54 (m, 4H, 2CH2, piperidin); solution of disubstituted 1,3,5-triazines 5(a–j) (0.01 eq.), piper-
13C-NMR (100 MHz, CDCl3) d, ppm: 164.2, 160.5, 52.1, 26.5, 24.5; azine attached to 4-aminoquinoline pharmacophore 3 (0.01 eq.)
mass 282.10 (M + H)+; elemental analysis for C13H20ClN5: and K2CO3 (0.01 eq.) in 1,4-dioxane was refluxed for 6–7 h. The
calculated: C, 55.41; H, 7.15; N, 24.85. Found: C, 55.48; H, completion of reaction was monitored by TLC using ethanol :
7.13; N, 24.87.
acetone (1 : 1) as mobile phase. The reaction mixture was
6-Chloro-N2,N4-bis(4-methoxyphenyl)-1,3,5-triazine-2,4-diamine filtered and concentrated under reduced pressure. The result-
(5f). Yellow crystals; yield: 88%; M.p: 235 1C; MW: 357.79; Rf: 0.69; ing residue was recrystallised from ethanol to afford the desired
FT-IR (nmax; cmꢀ1 KBr): 3300 (N–H secondary), 3015 (C–H), 1670– products 6(a–j).
1685 (CQN), 1630–1640 (CQC), 1585–1460 (CQC aromatic ring),
N2,N4-Bis(4-chlorophenyl)-6-(4-(2-((7-chloroquinolin-4-yl)amino)-
1100–1230 (C–N); 1H-NMR (400 MHz, CDCl3-d6, TMS) d ppm: ethyl)piperazin-1-yl)-1,3,5-triazine-2,4-diamine (6a). White crystals;
7.52–7.49 (m, 4H, 4CH, Ar–H), 7.43–7.38 (m, 4H, 4CH, Ar–H), 5.49 yield: 67%; M.P: 289–290 1C; MW: 620.96; Rf: 0.69; FT-IR (nmax
;
(br, s, 2H, 2NH), 3.65–3.63 (s, 6H, 2OCH3); 13C-NMR (100 MHz, cmꢀ1 KBr): 3415 (N–H stretch secondary amine), 2937 (C–H
CDCl3) d, ppm: 168.3, 167.8, 153.4, 131.1, 121.8, 115.1, 55.9; mass: stretch), 1583 (CQC stretch), 1368 (C–N stretch), 1012, 857, 637;
359 (M + H)+; elemental analysis for C17H16ClN5O2: calculated: C, 1H-NMR (400 MHz, CDCl3-d6, TMS) d ppm: 8.72 (d, 1H J = 6.63 Hz,
57.07; H, 4.51; N, 19.57. Found: C, 57.02; H, 4.45, N, 19.58.
quinoline), 8.03 (d, 1H J = 8.58 Hz, quinoline), 7.92 (dd, 1H J = 8.31,
N2,N4-Bis(4-bromophenyl)-6-chloro-1,3,5-triazine-2,4-diamine 1.52 Hz, Ar–H), 7.52 (d, 1H J = 6.34 Hz, quinoline), 7.48–7.42
(5g). Brownish-black crystals; yield: 81%; M.p: 169–170 1C; MW: (m, 4H, 4CH, Ar–H), 7.46 (dd, 1H J = 8.32, 1.90 Hz, Ar–H), 7.15–7.09
455.53; Rf: 0.35; FT-IR (nmax; cmꢀ1 KBr): 3350 (N–H secondary), (m, 4H, 4CH, Ar–H), 3.82 (s, 2H, 2NH), 3.45 (d, 2H J = 5.03 Hz,
3015 (C–H broad), 1656 (CQC stretch); 1H-NMR (400 MHz, methylene), 3.25–3.19 (m, 4H, piperazine), 2.78 (d, 2H J = 2.63 Hz,
CDCl3-d6, TMS) d ppm: 7.26–7.21 (m, 4H, 4CH, Ar–H), 7.06– methylene), 2.53–2.47 (m, 4H, piperazine), 2.16 (br–s, 1H, quino-
7.02 (m, 4H, 4CH, Ar–H), 4.81 (br, s, 2H, 2NH); 13C-NMR line, NH); 13C-NMR (100 MHz, CDCl3) d, ppm: 178.3, 164.3, 158.5,
(100 MHz, CDCl3) d, ppm: 168.3, 167.8, 137.8, 132.4, 118.5, 152.2, 151.1, 137.5, 136.3, 129.4, 128.2, 127.7, 124.7, 122.8, 121.6,
116.8; mass: 455.85 (M
C15H10Br2ClN5: calculated: C, 39.55; H, 2.21; N, 15.37. Found: analysis for C30H28Cl3N9: calculated: C, 58.03; H, 4.54; N, 20.30.
+
H)+; elemental analysis for 116.5, 94.4, 61.2, 56.2, 55.5, 54.2; mass: 622 (M + 1)+; elemental
C, 39.53; H, 2.20; N, 15.34.
Found: C, 58.01; H, 4.58; N, 20.42.
1
1
0
N ,N -(6-Chloro-1,3,5-triazine-2,4-diyl)dipropane-1,3-diamine
4,40-((6-(4-(2-((7-Chloroquinolin-4-yl)amino)ethyl)piperazin-
(5h). Brown crystals; yield: 57%; M.p: 234–236 1C; MW: 1-yl)-1,3,5-triazine-2,4 diyl)bis(azanediyl))diphenol (6b). Brown-
259.74; Rf: 0.57; FT-IR (nmax; cmꢀ1 KBr): 3390 (N–H primary), crystals; yield: 72%; M.p: 292–294 1C; MW: 584.07; Rf: 0.62; FT-IR
c
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(nmax; cmꢀ1 KBr): 3487 (O–H stretch), 3389 (N–H stretch second- stretch), 1021, 848, 764; 1H-NMR (400 MHz, CDCl3-d6, TMS) d ppm:
ary amine), 1589 (CQC stretch), 1359 (C–N stretch), 1016, 853, 8.54 (d, 1H, J = 5.35 Hz, quinoline), 8.06 (d, 1H, J = 2.03 Hz,
1
734; H-NMR (400 MHz, CDCl3-d6, TMS) d ppm: 8.62 (d, 1H J = quinoline), 7.47 (dd, 1H, J = 2.05, 8.98 Hz, quinoline), 6.48 (d, 1H, J =
6.52 Hz, quinoline), 8.05 (dd, 1H J = 8.53, 1.98 Hz, quinoline), 5.34 Hz, quinoline), 4.75 (br s, 1H, NH), 3.85 (t, 4H, J = 4.83 Hz,
7.54 (dd, 1H J = 8.67, 2.40 Hz, quinoline), 7.52–7.48 (m, 4H, 4CH, piperazine), 3.74 (t, 4H, J = 5.52 Hz, piperazine), 2.02–1.98 (m, 2H,
Ar–H), 7.45 (dd, 1H J = 6.53, 1.90 Hz, quinoline), 6.84 (d, 1H J = CH2), 1.76–1.72 (m, 2H, CH2); 13C-NMR (100 MHz, CDCl3) d, ppm:
4.84, Hz, quinoline), 6.43–6.41 (m, 4H, 4CH, Ar–H), 5.42 (s, 2H, 186.84, 178.63, 161.21, 152.75, 151.36, 137.96, 131.27, 128.86,
2OH, Ar–OH), 3.87 (s, 2H, 2NH), 3.60–3.56 (m, 4H, 2CH2, 124.62, 118.53, 101.28, 57.83, 55.29, 53.86, 52.19, 47.63, 26.75,
piperazine), 3.32 (d, 2H J = 2.67 Hz, methylene), 3.08–3.02 (m, 24.62; mass: 537.16 (M + H)+; elemental analysis for C28H38ClN9:
4H, 2CH2, piperazine), 2.48 (d, 2H J = 1.93 Hz, methylene), 2.15 calculated: C, 62.73; H, 7.14; N, 23.51. Found: C, 62.76; H, 7.13;
(br–s, 1H, quinoline, NH); 13C-NMR (100 MHz, CDCl3) d, ppm: N, 23.54.
178.3, 164.3, 158.5, 152.2, 151.1, 148.5, 136.3, 131.3, 128.2, 125.3,
6-(4-(2-((7-Chloroquinolin-4-yl)amino)ethyl)piperazin-1-yl)-N2,N4-
122.1, 121.6, 119.8, 116.7, 96.7, 60.2, 55.2, 51.3, 47.2; mass: 585 bis(4-methoxyphenyl)-1,3,5-triazine-2,4-diamine (6f). Light brown
(M + H)+; elemental analysis for C30H30ClN9O2: calculated: C, crystals; yield: 76%; M.p: 302–303 1C; MW: 612.12; Rf: 0.58; FT-IR
61.69; H, 5.18; N, 21.58. Found: C, 60.98; H, 5.28; N, 21.59.
(nmax; cmꢀ1 KBr): 3408 (N–H stretch), 2958 (C–H stretch), 1650
6-(4-(2-((7-Chloroquinolin-4-yl)amino)ethyl)piperazin-1-yl)-N2,N4- (CQN stretch), 1589 (CQC stretch), 1358 (C–N stretch), 1009, 852,
1
di-o-tolyl-1,3,5-triazine-2,4-diamine (6c). Light yellow crystals; 768; H-NMR (400 MHz, CDCl3-d6, TMS) d ppm: 8.65 (d, 1H, J =
yield: 68%; M.p: 287–288 1C; MW: 580.13; Rf: 0.47; FT-IR (nmax
;
6.56 Hz, quinoline), 8.03 (d, 1H, J = 2.01 Hz, quinoline), 7.49
cmꢀ1 KBr): 3426 (N–H stretch secondary amine), 2948 (C–H (dd, 1H, J = 2.03, 8.69 Hz, Ar–H), 7.02 (d, 1H, J = 5.36 Hz, Ar–H), 4.38
stretch), 1587 (CQC stretch), 1365 (C–N stretch), 1023, 862, (br s, 1H, NH), 3.83 (t, 4H, J = 4.76 Hz, piperazine), 3.64 (t, 4H,
773; 1H-NMR (400 MHz, CDCl3-d6, TMS) d ppm: 8.36 (d, 1H J = J = 5.47 Hz, piperazine), 3.45 (m, 2H, CH2), 2.53 (m, 2H, CH2), 3.87
5.49 Hz, quinoline), 8.03 (dd, 1H J = 8.51, 1.95 Hz, quinoline), (m, 3H, OCH3); 13C-NMR (100 MHz, CDCl3) d, ppm: 184.24, 174.27,
7.74–7.69 (m, 2H, 2CH, Ar–H), 7.48 (dd, 1H J = 6.48, 1.88 Hz, 157.83, 156.39, 154.58, 149.63, 132.43, 126.51, 123.58, 118.93,
quinoline), 7.36 (dd, 1H J = 7.38, 2.46 Hz, quinoline), 7.01–6.95 115.75, 83.48, 55.86, 53.27, 52.43, 47.83; mass: 613.08 (M + H)+;
(m, 2H, 2CH, Ar–H), 6.87 (d, 1H J = 3.42 Hz, quinoline), 6.65– elemental analysis for C32H34ClN9O2: calculated: C, 62.79; H, 5.60;
6.53 (m, 4H, 4CH, Ar–H), 3.82 (s, 2H, 2NH), 3.54–3.68 (m, 4H, N, 20.59. Found: C, 62.81; H, 5.62; N, 20.57.
piperazine), 3.32 (d, 2H J = 2.63 Hz, methylene), 3,09–3.04 (m,
N2,N4-Bis(4-bromophenyl)-6-(4-(2-((7-chloroquinolin-4-yl)amino)-
4H, piperazine), 2.51–2.45 (d, 2H J = 1.97 Hz, methylene), 2.18 ethyl)piperazin-1-yl)-1,3,5-triazine-2,4-diamine (6g). Brown crystals;
(br–s, 1H, quinoline NH), 2.15–2.12 (m, 6H, 2CH3); 13C-NMR yield: 64%; M.p: 276–277 1C; MW: 709.86; Rf: 0.46; FT-IR (nmax; cmꢀ1
(100 MHz, CDCl3) d, ppm: 183.2, 178.6, 163.4, 156.7, 151.4, KBr): 3415 (N–H stretch), 2978 (C–H stretch), 1658 (CQN stretch),
1
143.3, 138.9, 134.6, 132.3, 129.4, 128.9, 127.8, 125.6, 124.5, 1596 (CQC stretch), 1367 (C–N stretch), 1014, 858, 798; H-NMR
123.4, 121.6, 118.2, 96.7, 58.9, 53.4, 48.3, 21.2; mass: 581.18 (400 MHz, CDCl3-d6, TMS) d ppm: 8.78 (d, 1H, J = 6.63 Hz,
(M + H)+; elemental analysis for C32H34ClN9: calculated: C, quinoline), 7.53 (d, 1H, J = 3.23 Hz, quinoline), 7.49 (dd, 1H, J =
66.25; H, 5.91; N, 21.73. Found: C, 66.27; H, 5.87; N, 21.76.
5.30, 8.79 Hz, quinoline), 7.32 (d, 1H, J = 8.79 Hz, Ar–H), 7.15 (d, 1H,
J = 5.32 Hz, Ar–H), 4.32 (br s, 1H, NH), 3.63 (t, 4H, J = 4.76 Hz,
6-(4-(2-((7-Chloroquinolin-4-yl)amino)ethyl)piperazin-1-yl)-
N2,N4-di-p-tolyl-1,3,5-triazine-2,4-diamine (6d). Yellow crystals; piperazine), 3.56 (t, 4H, J = 5.47 Hz, piperazine), 3.38 (m, 2H, CH2),
yield: 76%; M.p: 293–294 1C; MW: 580.13; Rf : 0.43; FT-IR 2.79 (m, 2H, CH2); 13C-NMR (100 MHz, CDCl3) d, ppm: 182.35,
(nmax; cmꢀ1 KBr): 3438 (N–H stretch secondary amine), 2942 173.38, 156.73, 152.74, 149.38, 137.94, 134.82, 132.48, 128.41,
(C–H stretch), 1585 (CQC stretch), 1369 (C–N stretch), 1027, 124.85, 121.62, 118.57, 117.52, 114.73, 55.52, 52.56, 51.13, 47.61;
858, 782; 1H-NMR (400 MHz, CDCl3-d6, TMS) d ppm: 8.42 (d, 1H mass: 710.98 (M + H)+; elemental analysis for C30H28Br2ClN9:
J = 5.38 Hz, quinoline), 7.49 (dd, 1H J = 6.42, 1.85 Hz, quino- calculated: C, 50.76; H, 3.98; N, 17.76. Found: C, 50.80; H, 3.96;
line), 7.32 (dd, 1H J = 7.36, 2.43 Hz, quinoline), 8.08 (dd, 1H J = N, 17.79.
1
1
0
8.57, 1.92 Hz, quinoline), 6.87 (d, 1H J = 3.53 Hz, quinoline),
N ,N -(6-(4-(2-((7-Chloroquinolin-4-yl)amino)ethyl)piperazin-
7.76–7.69 (m, 4H, Ar–H), 6.57–6.52 (m, 4H, Ar–H), 3.38 (d, 2H 1-yl)-1,3,5-triazine-2,4-diyl)bis(propane-1,3-diamine) (6h). Light
J = 2.68 Hz, methylene), 2.46 (d, 2H J = 1.96 Hz, methylene), brown crystals; yield: 67%; M.p: 246–248 1C; MW: 514.07; Rf :
3.57–3.62 (m, 4H, piperazine), 3.07–3.02 (m, 4H, piperazine), 0.42; FT-IR (nmax; cmꢀ1 KBr): 3458 (N–H stretch primary amine),
2.18 (m, 3H, CH3) 2.27 (br–s, 1H, quinoline NH); 13C-NMR 3312 (N–H stretch secondary amine) 2986 (C–H stretch), 1667
(100 MHz, CDCl3) d, ppm: 183.42, 172.64, 163.49, 156.72, (CQN stretch), 1573 (CQC stretch), 1359 (C–N stretch), 1018,
151.29, 138.92, 136.94, 132.31, 129.85, 127.32, 123.47, 121.42, 863, 792; 1H-NMR (400 MHz, CDCl3-d6, TMS) d ppm: 8.68 (d, 1H,
58.72, 51.38, 48.47, 23.51; mass: 581.10 (M + H)+; elemental J = 5.45 Hz, quinoline), 7.81 (d, 1H, J = 2.55 Hz, quinoline), 7.38
analysis for C32H34ClN9: calculated: C, 66.25; H, 5.91; N, 21.73. (dd, 1H, J = 5.45, 8.51 Hz, quinoline), 7.10 (d, 1H, J = 8.51 Hz,
Found: C, 66.24; H, 5.89; N, 21.74.
Ar–H), 6.83 (d, 1H, J = 5.46 Hz, quinoline), 4.36 (br s, 1H, NH),
7-Chloro-N-(2-(4-(4,6-di(piperidin-1-yl)-1,3,5-triazin-2-yl)piperazin- 3.58 (t, 4H, J = 3.11 Hz, piperazine), 3.28 (t, 4H, J = 7.01 Hz,
1-yl)ethyl)quinolin-4-amine (6e). Light yellow crystals; yield: 63%; piperazine), 3.38 (t, 2H J = 6.48 Hz, CH2), 2.56 (d, 1H J = 10.24 Hz,
M.p: 278–279 1C; MW: 536.11; Rf: 0.41; FT-IR (nmax; cmꢀ1 KBr): 3417 CH2), 2.65 (d, 1H J = 3.11 Hz, CH2), 1.74 (t, 2H J = 7.01 Hz, CH2);
(N–H stretch), 2942 (C–H stretch), 1583 (CQC stretch), 1363 (C–N 13C-NMR (100 MHz, CDCl3) d, ppm: 179.21, 164.25, 156.72,
c
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152.13, 149.42, 134.94, 129.42, 124.53, 121.61, 117.58, 103.42, plates,17 with minor modifications. A stock solution of 5 mg mLꢀ1
55.27, 52.54, 51.10, 48.68, 39.42, 31.54; mass: 515.12 (M + H)+; of each test compound was prepared in DMSO and subsequent
elemental analysis for C24H36ClN11: calculated: C, 56.07; H, 7.06; dilutions were prepared with culture medium. The test compounds
N, 29.97. Found: C, 56.05; H, 7.04; N, 29.98.
in 20 mL volume at 5 mg mLꢀ1 and 50 mg mLꢀ1 concentrations in
6-(4-(2-((7-Chloroquinolin-4-yl)amino)ethyl)piperazin-1-yl)-N2,N4- triplicate wells were incubated with parasitized cell preparation at
bis(4-nitrophenyl)-1,3,5-triazine-2,4-diamine (6i). Yellow crystals; 37 1C in a CO2 incubator set at 37 1C and 5% CO2 level. After 40 h of
yield: 72%; M.p: 296–297 1C; MW: 709.86; Rf: 0.46; FT-IR (nmax
;
incubation, the blood smears were prepared from each well and
cmꢀ1 KBr): 3443 (N–H stretch secondary amine), 2963 (C–H stained with Giemsa stain. The level of parasitemia in terms of %
stretch), 1683 (CQN stretch), 1528 (N–O stretch), 1475 (CQC dead rings along and trophozoites was determined by counting a
1
stretch), 1245 (C–N stretch), 1009, 853, 784; H-NMR (400 MHz, total of 100 asexual parasites (both live and dead) microscopically
CDCl3-d6, TMS) d ppm: 8.65 (d, 1H, J = 6.58 Hz, quinoline), 7.97 using chloroquine and proguanil as reference drugs.
(d, 1H, J = 2.12 Hz, quinoline), 7.61 (dd, 1H, J = 1.93, 8.52 Hz,
quinoline), 8.06 (d, 1H, J = 8.45 Hz, Ar–H), 6.93 (d, 1H, J = 1.76 Hz,
Ar–H), 4.27 (br s, 1H, NH), 3.61 (t, 4H, J = 3.69 Hz, piperazine), 3.36
Molecular docking studies
(t, 4H, J = 7.01 Hz, piperazine), 3.32 (t, 2H J = 2.67 Hz, CH2), 2.79 The 3D X-ray crystal structure of wild type (1J3I.pdb) and
(t, 2H J = 8.26 Hz, CH2); 13C-NMR (100 MHz, CDCl3) d, ppm: 182.36, quadruple mutant (N51I, C59R, S108 N, I164L, 3QG2.pdb)
167.35, 154.53, 152.75, 151.78, 149.36, 146.24, 137.95, 134.84, pf-DHFR-TS were used as starting models for this study. The
129.52, 123.49, 121.65, 119.23, 118.54, 99.45, 57.83, 55.52, 53.42, protein was prepared, docked and the molecular dynamics
51.63, 48.68; mass: 643.13 (M + H)+; elemental analysis for
simulation carried out. All computational analyses were carried
C30H28ClN11O4: calculated: C, 56.12; H, 4.40; N, 24.00. Found: C, out using Discovery Studio 2.5 (Accelrys Software Inc., San
56.14; H, 4.36; N, 23.97.
Diego; http://www.accelrys.com).
7-Chloro-N-(2-(4-(4,6-dimorpholino-1,3,5-triazin-2-yl)piperazin-
1-yl)ethyl)quinolin-4-amine (6j). White-crystals; yield: 65%, M.p:
Preparation of receptor
306–307 1C; MW: 540.06; Rf: 0.41; FT-IR (nmax; cmꢀ1 KBr): 3352 The target wild and quadruple mutant pf-DHFR proteins with
(N–H stretch secondary amine) 2967 (C–H stretch), 1682 (CQN removal of the co-crytallised ligands were taken and the bond
stretch), 1578 (CQC stretch), 1362 (C–N stretch), 1058, 876, 797; order was corrected. The hydrogen atoms were added, and their
1H-NMR (400 MHz, CDCl3-d6, TMS) d ppm: 8.59 (d, 1H, J = 5.42 Hz, positions were optimized using the all-atom CHARMm (version
quinoline), 8.10 (d, 1H, J = 1.17 Hz, quinoline), 7.83 (dd, 1H, J = c32b1) forcefield with Adopted Basis set Newton Raphson (ABNR)
2.83, 1.91 Hz, quinoline), 7.06 (d, 1H, J = 8.49 Hz, quinoline), minimization algorithm until the root mean square (r.m.s.) gradient
6.80 (d, 1H, J = 2.82 Hz, quinoline), 4.39 (br s, 1H, NH), 3.69 for potential energy was less than 0.05 kcal molꢀ1 Å
.
ꢀ1 18,19 Using the
(t, 4H, J = 3.86 Hz, piperazine), 2.70 (t, 4H, J = 6.12 Hz, ‘Binding Site’ tool panel available in DS 2.5, the minimized protein
piperazine), 3.38 (t, 2H J = 2.67 Hz, CH2), 2.79 (t, 2H J = structures were defined as the receptor, the binding site was
2.67 Hz, CH2), 3.18 (d, 1H J = 10.24 Hz, Ar–H), 3.52 (d, 1H defined as the volume occupied by the ligand in the receptor,
J = 2.51 Hz, Ar–H); 13C-NMR (100 MHz, CDCl3) d, ppm: 183.26, and an input site sphere was defined over the binding site with
177.63, 154.52, 152.75, 149.38, 134.92, 129.42, 124.83, 121,72, a radius of 5 Å. The center of the sphere was taken to be the
117.58, 66.36, 55.21, 54.96, 51.26, 48.73, 46.53; mass: 541.12 center of the binding site, and side chains of the residues in the
(M + H)+; elemental analysis for C26H34ClN9O2: calculated: C, binding site within the radius of the sphere were assumed to be
57.82; H, 6.35; N, 23.34. Found: C, 57.87; H, 6.34; N, 23.41.
flexible during refinement of postdocking poses. The receptor
having a defined binding site was used for the docking studies.
Antimalarial activity
Ligand setup
Preparation of parasites
Using the built-and-edit module of DS 2.5, various ligands were
The chloroquine sensitive 3D7 and chloroquine resistant RKL-2 built, all-atom CHARMm forcefield parameterization was assigned
strain (Raurkela, Orissa, India) of P. falciparum were routinely and then minimized using the ABNR method. A conformational
maintained in stock cultures in a RPMI-1640 medium supple- search of the ligand was carried out using a stimulated annealing
mented with 25 mmol HEPES, 1% D-glucose, 0.23% sodium molecular dynamics (MD) approach. The ligand was heated to a
bicarbonate and 10% heat inactivated human serum. The temperature of 700 K and then annealed to 200 K. Thirty such cycles
asynchronous parasites of P. falciparum were synchronized after were carried out. The transformation obtained at the end of each
5% D-sorbitol treatment to obtain only the ring stage parasitized cycle was further subjected to local energy minimization, using the
cells. For carrying out the assay, the initial ring stage para- ABNR method. The 30 energy-minimized structures were then
sitaemia of 1.05% at 3% haematocrit in a total volume of 200 mL superimposed and the lowest energy conformation occurring in
of medium RPMI-1640 was uniformly maintained.
the major cluster was taken to be the most probable conformation.
In vitro antimalarial efficacy test
Docking
The in vitro antimalarial assay was carried out according to Docking, a significant computational method was used to
microassay of Reickmann and co-workers in 96 well microtitre foretell the binding of the ligand to the receptor binding site
c
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Paper
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by varying the position and conformation of the ligand keeping
the receptor rigid. The LigandFit19 protocol of DS 2.5 was used
for the docking of ligands with wild and quadruple mutant
pf-DHFR proteins.20 The LigandFit docking algorithm combines a
shape comparison filter with a Monte Carlo conformational search
to generate docked poses consistent with the binding site shape.
These initial poses are further refined by rigid body minimization
of the ligand with respect to the grid based calculated interaction
energy using the Dreiding forcefield.21 The receptor protein con-
formation was kept fixed during docking, and the docked poses
were further minimized using an all-atom CHARMm (version
c32b1) forcefield and smart minimization method (steepest des-
cent followed by conjugate gradient) until the r.m.s. gradient for
the potential energy was less than 0.05 kcal molꢀ1 Åꢀ1. The atoms
of the ligand and the side chains of the residues of the receptor
within 5 Å of the center of the binding site were kept flexible
during minimization.
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Conclusion
The present study describes the synthesis of hybrid com-
pounds, 4-aminoquinoline and 1,3,5-triazine and their antima-
larial evaluation against chloroquine sensitive (3D7) and
chloroquine resistance (RKL-2) strains of P. falciparum. It was
observed on the basis of results that compounds 6h and 6e
exhibited considerable activity against wild type, while, the
compounds 6g, 6a and 6f exhibited enhanced activity in case
of mutant strains. Thus, the present study provides a new
approach for the ongoing malarial drug discovery initiatives.
Conflict of interest
Authors declare no conflict of interest.
Acknowledgements
Financial support from the Department of Science and Tech-
nology (DST), New Delhi, India is gratefully acknowledged
(Grant no. SR/SO/HS-125/2010) and the authors also are thank-
ful to S. A. I. F., Punjab University, Chandigarh, India for
providing spectroscopic data.
21 S. L. Mayo, B. D. Olafson and W. A. III Goddard, J. Phys.
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Notes and references
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New J. Chem.