Antimalarial activity and docking studies of novel bi-functional hybrids derived from 4-aminoquinoline and 1,3,5-triazine against wild and mutant malaria parasites as pf-DHFR inhibitor

Article abstract of DOI:10.1039/c2ra21915h

Bi-functional conjugates comprised of 4-aminoquinoline and 1,3,5-triazine were synthesized through facile synthetic routes. These compounds were rigorously screened for determination of their antimalarial activity against wild and mutant cultured Plasmodium falciparum. The results disclosed that the conjugates have considerable antimalarial activity against both wild and mutant parasites with marked variation on changing the pattern of substitutions. The observed activity profiles were additionally substantiated by docking studies on both wild and quadruple mutant P. falciparum dihydrofolate reductase thymidylate synthase (pf-DHFR-TS).

Full text of DOI:10.1039/c2ra21915h

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Antimalarial activity and docking studies of novel bi-
functional hybrids derived from 4-aminoquinoline and
1,3,5-triazine against wild and mutant malaria parasites
as pf-DHFR inhibitor
Cite this: DOI: 10.1039/c2ra21915h
Hans Raj Bhat,*a Udaya Pratap Singh,3ae Prashant Gahtori,b Surajit Kumar Ghosh,c
Kabita Gogoi,d Anil Prakashd and Ramendra K. Singhe
Bi-functional conjugates comprised of 4-aminoquinoline and 1,3,5-triazine were synthesized through facile
synthetic routes. These compounds were rigorously screened for determination of their antimalarial activity
against wild and mutant cultured Plasmodium falciparum. The results disclosed that the conjugates have
considerable antimalarial activity against both wild and mutant parasites with marked variation on changing
the pattern of substitutions. The observed activity profiles were additionally substantiated by docking studies
on both wild and quadruple mutant P. falciparum dihydrofolate reductase thymidylate synthase (pf-DHFR-TS).
Received 24th August 2012,
Accepted 19th December 2012
DOI: 10.1039/c2ra21915h
www.rsc.org/advances
The 4-amino-quinoline-type antimalarial drugs are supposed to
exert their action through a p–p stacking interaction with the
Introduction
porphyrin ring system.7–9 In this regard, researchers try to
integrate this moiety with prevailing groups, for example 1,4-
bis(3-aminopropyl)piperazine, istatin, b-carboline etc. to advanta-
geously improve activity against the CQ-resistant strain.10–14 1,3,5-
triazine, which is another core scaffold in clinically used
antimalarial drugs, for instance in cycloguanil, chlorcycloguanil,
clociguanil, and WR99210, has gained attraction due to its
potential to deliver potent molecules.14–16 Recently, conjugates
of 4-aminoquinoline with 1,3,5-triazine have been employed
widely as novel DHFR inhibitors.16,18 Our research endeavour to
discover newer hybrid molecules of 1,3,5-triazine as antibacter-
ial,19 antimalarial,17 antifungal20 agents continues. We herein
disclose a new series of hybrid conjugates of N-(7-chloroquinolin-
4-yl)piperazine-1-carbothioamide and 1,3,5-triazine derivatives as
core scaffolds. These molecules were tested for plausible
antimalarial activity against 3D-7 (chloroquine sensitive) and
RKL-2 (chloroquine resistant) strains of P. falciparum. Additionally,
a molecular docking study was performed to elucidate the
necessary key structural features of the potential antimalarial
agents, see Fig. 1.
Malaria is a complex disease causing one of the major public
health problems in developing countries. Recent statistics indicate
that it accounted for about 219 million cases in 2010 (with an
uncertainty range of 154 million to 289 million) and an estimated
660 000 deaths (with an uncertainty range of 610 000 to 971 000)
and most of deaths prevail among children living in Africa, where
a child dies every minute from malaria.1 Plasmodium falciparum is
the most virulent parasite of the malaria species, which is
responsible for more than 95% of malaria related morbidity and
mortality.2 Modern medicinal chemistry explores the bi-functional
enzyme dihydrofolate reductase-thymidylate synthase of the
parasite Plasmodium falciparum (Pf-DHFR-TS) as an active binding
site for 1,3,5-triazine derivatives.3,4 The amino acids, Ala16, Asn51,
Cys59, Ser108, and Ile164 are considered as active site residues for
Pf-DHFR-TS.5 Over the past few years, several computational
techniques have been applied to understand better the Pf-DHFR-
TS ligand interactions and intense research activity has been
undertaken by both private companies and academic institutions,
aimed at the discovery of new, effective DHFR inhibitors.6
aDepartment 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
bFaculty of Pharmacy, Uttarakhand Technical University, Dehradun, 248007 India
cDepartment of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, 786004
India
Results and discussion
Chemistry
dRegional Medical Research Centre, ICMR, Dibrugarh, 786005 India
eNucleic Acids Research Laboratory, Department of Chemistry, University of
Allahabad, Allahabad, 211002 India
The chemistry of this group of compounds has been studied
intensively and has been the subject of many reviews. The
various 2,4,6-mono, di- or tri-substituted, symmetrical and
nonsymmetrical 1,3,5-triazines can be prepared with a high
Present address: Archimedes DoRa5 Visiting Fellow, Institute of Chemistry,
3
University of Tartu, Estonia.
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Fig. 1 Orientation of the most active hybrid compounds (7c, 7e, 7h and 7i) in the active site of pf-DHFR-TS (wild and quadruple mutant).
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Scheme 1 Reagents and conditions: (a) Reflux with stirring for 18 h at 90–120 uC. (b) Dry acetone, 0–5 uC, 1 h, then 40–45 uC for a further 3 h, NaHCO3. (c) Piperazine,
1,4-dioxane, 120–130 uC, 5–6 h, K2CO3. (d) Dry acetone, 40–45 uC, 18 h.
level of sophistication by the nucleophilic substitution of
chlorine atoms in 2,4,6-trichloro-1,3,5-triazine. Indeed, 2,4,6-
trichloro-1,3,5-triazine is a cheap and commercially available
reagent, which becomes the most intriguing starting approach
for its wide application. This reaction therefore offers a
convenient route for the preparation of substituted amino-
1,3,5-triazines.
Antimalarial activity and structure–activity relationship
The level of parasitemia in terms of % dead rings along with
schizonts was determined for each compound 7(a–j) along
with the use of Chloroquine as the standard control against
3D-7 (Chloroquine sensitive) and RKL-2 (Chloroquine resis-
tant) strains of P. falciparum (Table 1). Overall, the 3D-7 strain
(avg% inhibition ¡ std dev = 13.44 ¡ 3.96) was found to be
more sensitive as compared to the RKL-2 strain (avg%
inhibition ¡ std dev = 7.50 ¡ 2.44) for the tested set of
compounds. The % dead rings values for this series of
compounds showed moderate activity with a wide degree of
variation. It was found that the compounds 7c and 7(h–j)
displayed substantial activity against both the strains and the
remaining compounds demonstrated weak to moderate
activities. Compound 7d exhibited the weakest activity for
the tested 3D-7 and RKL-2 strains.
It is imperative to determine the important structural
features of these putative triazine hybrid compounds for
antimalarial activity. The initial exploration performed was
aimed at substituting the triazine core with the corresponding
4-aminoquinoline triazine template analogue. To this end,
various 4-nitro, 4-chloro, 4-bromo, 4-methoxy, 4-methyl,
2-methyl, and 4-hydroxy anilines, morpholine, piperidine
and aminopropyl amine substituted hybrid compounds were
prepared as described above and characterized in terms of
their in vitro antimalarial activity as well as their activity at the
pf-DHFR receptor binding site through molecular docking. As
shown in Table 1, these compounds exhibited good anti-
malarial activity, among them one compound, 7c, has
demonstrated the optimal level of paracitemia of 19.3 in
terms of % dead rings along with schizonts, whereas the
compound 7j showed the level of paracetemia as 17.6 for the
The synthesis of compound (3) was achieved by the
nucleophilic substitution of silver thiocyanate (2) with the
4-chloro of 4,7-dichloroquinoline (1) in anhydrous toluene at
90–120 uC for 18 h. A controlled temperature of 40–45 uC, for
3–4 h in dry acetone drove the ease of replacement of the
chlorine atoms in 2,4,6-trichloro-1,3,5-triazine to afford
various di-substituted 1,3,5-triazine derivatives 5(a–j). The tri-
substituted 6(a–j) 1,3,5-triazine derivatives were obtained on
refluxing 5(a–j) in 1,4-dioxane for 6–7 h (Scheme 1). At the end,
the desired compounds 7(a–j) were furnished on stirring 6(a–j)
with 7-chloro-4-isothiocyanatoquinoline in dry acetone at 40–
45 uC for 8–9 h. The structures of all the newly synthesized
1
compounds were ascertained on the basis of FT-IR, H-NMR,
13C-NMR, mass and elemental analysis.
Bands in the FT-IR spectra of the products in the range
1548.28–1387.38 cm21 are attributable to the aromatic CLN
group of 1,3,5-triazine, whereas the CLC aromatic group
appears nearer to 1475 cm21. Many strong absorption bands
at 850–670 cm21 confirm the existence of the aromatic ring.
1
The H-NMR spectra reveal a signal corresponding to quino-
line at 7.27–8.85 ppm. For the tri-substituted 1,3,5-triazine,
shielding for the bridged NH was usually observed, for
instance the chemical shift of the –NH bridge was lowered
approximately by 0.55 ppm in the case of 6(a–j). Moreover, all
mass spectra and elemental analyses are found to be in
agreement with the proposed structures.
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Table 1 In vitro antimalarial activity of the hybrid derivatives 7(a–j)
Strains
3D-7a
3D-7a
RKL-2b
RKL-2b
Compounds
5 mg ml21 (dose)
50 mg ml21 (dose)
5 mg ml21 (dose)
50 mg ml21 (dose)
7a
7b
7c
7d
7e
7f
7g
7h
11.6
12.3
19.3
8.3
12.3
9
10
17
17
17.6
51
22.6
25
4
4.5
11
11.5
10
16
16.5
18
16
16.5
17.5
17.5
14
34.6
13.3
28.6
15.3
18.6
27.3
29
5.5
7.5
7.5
6.5
8.5
10.5
9.5
–
7i
7j
26.7
Chloroquine(CQ) (0.7 mg ml21
)
)
Chloroquine(CQ) (1.2 mg ml21
–
50
50
50
Proguanil (200 mg ml21
)
a
b
Wild malaria parasite (Chloroquine sensitive). Mutant malaria parasite (Chloroquine resistant).
same strain. Further to this encouraging result, a careful
selection of the substituents of the pendent triazine ring was
made to clarify the effects on biological activity.
site were illustrated in Table 2 and Fig. 1, respectively. These
results showed that the targeted molecules were snugly fitted
into the active site with considerable and diverse binding
affinities towards the wild (263.3 to 299.3) and quadruple
mutant (43.6 to 280.0) pf-DHFR-TS along with the formation
of numerous hydrogen bonds and p–p, p–+, and p–s
interactions. The results disclosed that compound 7c has
high antimalarial activity and very low binding energies for
wild (264.1) and mutant (273.3) pf-DHFR-TS. It showed the
generation of two hydrogen bonds with Ser108 and Ser167
through the involvement of 1,3,5-triazine and thioamide,
respectively. The stability of these complexes (post docked
ligand–receptor) and their low binding energies are attributed
to the creation of greater intermolecular hydrogen bonding
and non-bonding (p–p and covalent, +– p, and s–p) forces
amid the amino acid residues of both enzymes, as depicted in
Fig. 1. The thioamide bridge used to join 4-aminoquinoline
with 1,3,5-triazines was predicted to be predominantly
engaged in hydrogen bonding with key amino acids (Ser167,
Gly44) and minor bonding was disclosed through the
participation of 1,3,5-triazine, the substituted phenyl and its
amine linkage with Ser108, Arg106, Asp54 and Thr107,
suggesting a role of biological activity for these compounds
in wild malaria parasites. The formation of hydrophobic
interactions was also shown by quinoline and the substituted
phenyl ring with Phe58, and other non-bonded forces (s–p)
through the 1,3,5-triazine and quinoline with Leu46. On the
contrary, for quadruple mutant pf-DHFR-TS, from the entire
set of ligands, thioamide was again prevalent for the
generation of H-bonds with Ser111, Asn108 and Leu164 and
minor interactions through Arg59, Leu46, Lys49 and Arg122.
Whereas, a hydrophobic interaction was reported for 1,3,5-
triazine and the phenyl ring with Phe116 and Arg59,
respectively. Additionally, the phenyl ring is also engaged in
the formation of +–p interactions with Arg59.
Notably, the presence of an appropriate ortho substituent
seems to escalate the in vitro potency; in fact, the correspond-
ing methyl substituted compound 7f was found to be less
active than 7g. Moreover, where the para substitution of the
pendant phenyl ring is concerned, most of the substituents
introduced were well tolerated, with the exception of the
4-methoxy, 4-methyl and 2-methyl groups, respectively, of
compounds 7d, 7f and 7g, whose lower activity is considered to
be due to the high steric bulk hindrance of the methyl moiety.
The replacement of the phenyl ring by a 3-aminopropyl or
morpholine was tolerated. In particular, as shown in Table 1,
the 3-aminopropyl 7i or morpholine 7e derivative exhibited
similar in vitro activity compared to the corresponding
4-bromo or 7-chloro derivatives. Whereas, the piperidino
analogue was still active with a level of paracitemia (7h).
The SARs clearly postulate that 4-Br, 4-OH substitution play
an essential role in escalating activity. Whereas, the similar
effect was observed in the case of amino propyl and piperidino
substitution. On the other hand, decline in antimalarial
activity was observed for 7f, 7g and 7d with 4-methyl, 2-methyl
and 4-methoxy substitutions, respectively. It is imperative
from this studies that electron withdrawing substituent such
as bromo, hydroxy are endowed with high activity, whereas,
electron donating substituent such as methyl and methoxy
degraded antimalarial activity. Furthermore, amino propyl and
piperidino substitution was well tolerated.
Molecular docking study
In order to gain insight into the key structural requirements
and the basis of the distinct activity profile of the test
compounds in both wild and mutant P. falciparum parasites, a
molecular docking study was undertaken. The docking studies
of the target compounds were performed into the binding
pocket of both the wild type (1J3I.pdb) and quadruple mutant
(N51I, C59R, S108N, I164L, 3QG2.pdb) pf-DHFR. The docking
results and docked conformations of the ligands in the active
Compound 7a was revealed to make two hydrogen bonds
with Arg106 and Thr107 with the involvement of the oxygen of
the nitro present on the phenyl of 1,3,5-triazine. Additionally,
it also created one s–p bond through Leu46 with the quinoline
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Table 2 Docking interaction of hybrid derivatives 7(a–j) in wild and quadruple mutant pf-DHFR-TS
Wild type pf-DHFR-TS
Quadruple mutant pf-DHFR-TS
Donor–acceptor
Compound Hydrogen bond
Binding energy Donor–acceptor
Binding energy
Non-bonded forces
(Kcal mol21
hydrogen bond
)
Non-bonded forces
(Kcal mol21
)
7a
1,3,5-triazine-phenyl
NO ARG106:HH21
p–s Quinoline- LEU 46 278.1
1,3,5-triazine-Phenyl- NO
253.0
…
…
O
HE-ARG59
Triazine-phenyl
…
NO THR 107:HG1
…
…
7b
7c
Quinoline-S HN-SER167 p–p Quinoline-PHE58
263.3
264.1
Quinoline-S
HD-ASN108
p–+ Benzene—ARG59 43.6
p–p Benzene—ARG59 273.3
…
…
…
…
Triazine-N HG-SER-108 NO
Quinoline-S HG-SER167
Triazine-N HG-SER-108 p–s Quinoline—THR107 282.8
7d
7e
7f
NO
NO
2107.2
288.9
Quinoline-S HN-SER167 NO
270.4
…
…
…
NH HO-SER111
p–p Quinoline—PHE58 270.2
Quinoline-S
HG-SER111
p–+ Benzene—ARG59 2162.5
…
7g
7h
7i
Quinoline-S HN-GLY44 NO
271.4
270.9
299.2
NO
…
NH HO-SER110
…
Quinoline-S HN-GLY44 p–s Triazine—LEU46
Quinoline-S
HO-LEU164
NO
NO
261.1
280.0
…
…
Quinoline-S HN-GLY44 NO
Amino-N
HH-ARG122
Amino-N HN-LEU46
…
…
NH HO-SER110
…
…
7j
Quinoline-S HG-SER167 p–p Phenyl—PHE58
293.3
Phenyl-O
p–p Triazine—PHE116 248.36
HE-ARG59
…
Phenyl-O
HH-LYS49
1,3,5-triazine-phenyl-
OH HO-APS54
…
ring. In the quadruple mutant, 7a shows no hydrophobic
interaction, and formed only one hydrogen bond, with Arg59,
along with having lower binding energy (253.0). On the
contrary, the introduction of a halogen atom (4-Cl) on the
phenyl ring of 1,3,5-triazine, 7b, causes the formation of new
H-bonds to Ser167 with the S of the thioamide linkage and a p–
p non-bonded interaction through the involvement of quino-
line with Phe58 in the wild pf-DHFR-TS. The same ligand led to
the creation of a p–p interaction with Arg59 in the mutant,
with lower binding energy (243.6). On replacing p-Cl with
another halogen (p-Br), 7c, a minor variation in the binding
energy and interactions was observed with an additional
H-bond inbetween 1,3,5-triazine and Ser108, in the wild type,
while no hydrogen bonds were reported with the same ligand
in the mutant. It was surprising to see that no interactions
were reported for 7d and 7e in the mutant, but again 1,3,5-
triazine and the thioamide were involved in the generation of
H-bonds with an additional s–p interaction of Thr107 with
quinoline in the wild type. In the case of compounds 7f and 7g,
where a methyl group was present at the fourth and second
positions of the phenyl, respectively, these compounds showed
a diverse pattern of interaction with the binding site. In the
wild type, compound 7f formed a hydrogen bond with Ser111
with the additional creation of p–p interactions through
quinoline with Phe58. Whereas, in the case of the mutant, 7f
caused the formation of a H-bond with Ser111 through
involvement of the thioamide linkage with +–p interactions
through Arg59. Thioamide was again disclosed as a common
structural feature among the rest of the compounds (7h,7i and
7j) for the creation of H-bonds through Gly44, Ser167 in the
case of the wild type and Leu164 (for 7h) for the quadruple
mutant. The hydroxyl present in 7j causes the formation of
H-bonds with Arg59 and Lys49 in the mutant and Asp54 in the
wild type with an additional H-bond with Ser167 through the
thioamide.
These results corroborate the idea that the creation of
H-bonds is the main predictor for the activity of the ligands.
Nevertheless, the formation of other non-bonded forces (p–p,
p–+, and p–s) appeared to stabilize the post-docked conforma-
tion of the ligands in the inner groove of the binding site.
Finally, the native ligands were allowed to dock into the active
site of proteins (WR99210 and NDP 610, for wild type and
quadruple pf-DHFR, respectively) for the validation of the
docking protocol. The docked result was then compared with
the crystal structure of the bound ligand–protein complex. It
was revealed that the title ligands were bound in a similar
fashion to the reference ligands with the root mean square
deviation (RMSD) of .1.5 Å. It has been confirmed that the
accuracy and performance of the LigandFit/Discovery Studio
2.5 was highly satisfactory.
Experimental
All commercially available solvents and reagents were of
analytical grade and used without further purification. Melting
points were determined on a Veego, MPI melting point
apparatus and FT-IR (2.0 cm21, flat, smooth, abex) were
recorded on Perkin Elmer RX-I Spectrophotometer. 1H-NMR
spectra were recorded on a Bruker Avance II 400 NMR and 13C-
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Rf: 0.55; FT-IR (nmax; cm21 KBr): 3289.56 (N–H secondary),
3055.70 (C–H broad), 1548.28–1446.06 (aromatic CLN); 1H-
NMR (400 MHz, CDCl3-d6, TMS) d (ppm): 7.40–7.38 (m, 4H,
4xCH, Ar–H), 7.32–7.28(m, 4H, 4xCH, Ar–H), 3.62 (br, s, 2H,2x
NH); 13C-NMR (100MHz, CDCl3) d (ppm): 173.56, 168.85,
148.26, 143.16, 131.36, 126.23; Mass: 388.10 (M + H)+;
Elemental analysis for C15H10ClN7O4: calculated: C, 46.46; H,
2.60; N, 25.29. Found: C, 46.48; H, 2.65; N, 25.26.
6-Chloro-N2,N4-bis(4-chlorophenyl)-1,3,5-triazine-2,4-dia-
mine (5b). White crystals, yield: 75%; M.p: 135–137 uC; MW:
366.63; Rf: 0.48; FT-IR (nmax; cm21 KBr): 3243.26 (N–H
secondary), 2965.46 (C–H broad), 1387.38 (aromatic 2CLN);
1H-NMR (400 MHz, CDCl3-d6, TMS) d (ppm): 7.32–7.30 (m, 4H,
4xCH, Ar–H), 7.08–7.04 (m, 4H, 4xCH, Ar–H), 4.82 (br, s, 2H,
2xNH); 13C-NMR (100 MHz, CDCl3) d (ppm): 168.40, 167.85,
137.10, 129.60, 127.70, 122.25,; Mass: 366 (M + H)+; Elemental
analysis for C15H10Cl3N5: calculated: C, 49.14; H, 2.75; N,
19.10. Found: C, 49.17; H, 2.77; N, 19.15.
N2,N4-Bis(4-bromophenyl)-6-chloro-1,3,5-triazine-2,4-dia-
mine (5c). Brownish black crystals, yield: 81%; M.p: 169–170
uC; MW: 455.53; Rf: 0.35; FT-IR (nmax; cm21 KBr): 3350.62 (N–H
secondary), 3015.43 (C–H broad), 1656.15 (CLC stretch); 1H-
NMR (400 MHz, CDCl3-d6, TMS) d (ppm): 7.26–7.21 (m, 4H,
4xCH, Ar–H), 7.06–7.02 (m, 4H, 4xCH, Ar–H), 4.81 (br, s, 2H, 2x
NH); 13C-NMR (100 MHz, CDCl3) d (ppm): 168.35, 167.85,
137.85, 132.45, 118.50, 116.80; Mass: 455.85 (M + H)+;
Elemental analysis for C15H10Br2ClN5: calculated: C, 39.55;
H, 2.21; N, 15.37. Found: C, 39.53; H, 2.20; N, 15.34.
6-Chloro-N2,N4-bis(4-methoxyphenyl)-1,3,5-triazine-2,4-dia-
mine (5d). Yellow crystals, yield: 88%; M.p: 235 uC; MW:
357.79; Rf: 0.69; FT-IR (nmax; cm21 KBr): 3300 (N–H secondary),
3015 (C–H), 1670–1685 (CLN), 1630–1640 (CLC), 1585 (CLC
aromatic ring), 1460 (CLC aromatic ring), 1100–1230 (C–N);
1H-NMR (400 MHz, CDCl3-d6, TMS) d (ppm): 7.52–7.49 (m, 4H,
4xCH, Ar–H), 7.43–7.38 (m, 4H,4xCH, Ar–H), 5.49 (br, s, 2H,
2xNH), 3.65 (s, 6H, 2xOCH3); 13C-NMR (100 MHz, CDCl3) d
(ppm): 168.35, 167.85, 153.40, 131.10, 121.80, 115.10, 55.90;
Mass: 359 (M + H)+; Elemental analysis for C17H16ClN5O2:
calculated: C, 57.07; H, 4.51; N, 19.57. Found: C, 57.02; H, 4.45,
N, 19.58.
NMR spectra on a Bruker Avance II 100 NMR spectrometer in
DMSO-d6 using TMS as an internal standard. Mass spectra
were obtained on a VG-AUTOSPEC spectrometer equipped
with electrospray ionization (ESI) sources. Elemental analysis
was carried out on a Vario EL-III CHNOS elemental analyzer.
The desired compounds 3, 5(a–j), 6(a–j) and 7(a–j) were
synthesised by the synthetic protocols as outlined in Scheme 1.
The synthesis of compound (3) was achieved by the nucleo-
philic substitution of silver thiocynate (2) with the 4-chloro of
4,7-dichloroquinoline (1). The synthesis of di-substituted 1,3,5-
triazines 5(a–j) were accomplished by the nucleophilic
substitution of the Cl atom of the 2,4,6-trichloro-1,3,5-triazine
(4) with distinguished primary and secondary amines (a–j) as
shown in Scheme 1. Whereas, the trisubstituted 1,3,5-triazines
6(a–j) were acheived by the nucleophilic substitution (SNAr) of
the Cl atom of the disubstituted s-triazine 5(a–j) with
piperazine. Finally, new antimalarial compounds 7(a–j) were
synthesized by incorporating the tri-substituted 1,3,5-triazine
moieties 6(a–j) in the thiocynate attached to 4-aminoquinoline
pharmacophore (3).
7-Chloro-4-isothiocyanatoquinoline (3)
A solution of 4,7-dichloroquinoline (1) (0.01 mol) and silver
thiocynate (2) (0.02 mol) in anhydrous toluene was refluxed at
90–120 uC for 18 h. The completion of the reaction was
monitored by TLC using ethanol : acetone (1 : 1) as the mobile
phase. The reaction mixture was filtered and concentrated
under reduced pressure. The resulting residue was dissolved
in dichloromethane and washed with brine and dried over
Na2SO4. The dried solution was concentrated under reduced
pressure to obtain the title compound (3).
Brown crystals, yield: 68%; M.p: 197–198 uC; MW: 220.68; Rf:
0.48; FT-IR (nmax; cm21 KBr): 3000 (C–H), 1690–1640 (CLN),
1630 (CLC), 1600 (CLC, aromatic ring), 1470 (CLC, aromatic
ring), 1275 (C–N); 1H-NMR (400 MHz, CDCl3-d6, TMS) d (ppm):
8.85 (d, 1H J = 4.97 Hz, quinoline ring), 8.40 (d, 1H J = 8.60 Hz,
quinoline ring), 8.01 (d, 1H J = 1.96 Hz, quinoline ring), 7.76
(d, 1H J = 8.60 Hz, quinoline ring), 7.27 (d, 1H J = 4.97 Hz,
quinoline ring); 13C-NMR (100 MHz, CDCl3); d (ppm): 152.30,
138.40, 137.20, 135.20, 129.80, 129.10, 128.60, 124.10, 118.30;
Mass: 221.60 (M + H)+; Elemental analysis for C10H5ClN2S:
Calculated: C, 54.43; H, 2.28; N, 12.69. Found: C, 54.41; H,
2.23; N, 12.58.
6-Chloro-2,4-dimorpholino-1,3,5-triazine (5e). Buff white,
yield: 73.32%, M.p: 132–135 uC; MW: 285.73; Rf: 0.62; FT-IR
(nmax; cm21 KBr): 2966 (C–H stretch), 1574–1451, 1362 (C–N
stretch), 1116(C–O stretch); 1H-NMR (400 MHz, CDCl3) d
(ppm): 3.78–3.74 (m, 8H, 4xCH2-O, Morpholine), 3.70–3.67 (m,
8H, 4xCH2-N, Morpholine); 13C-NMR (100 MHz, CDCl3) d
(ppm): 169.69, 164.48, 66.56, 43.86; Mass: 286.10 (M + H)+;
Elemental analysis for C11H16ClN5O2: C, 46.24; H, 5.64; N,
24.51. Found: C, 46.28; H, 5.58; N, 24.56.
General procedure for the synthesis of di-substituted 1,3,5-
triazine derivatives 5(a–j)
Various distinguished anilines (a–j) (0.2 mol) were added into
100 mL of acetone, maintaining the temperature at 40–45 uC. A
solution of 2,4,6-trichloro-1,3,5-triazine (4) (0.1 mol) in 25 mL
of acetone was added, and stirred for 3 h followed by the drop-
wise addition of NaHCO3 solution (0.1 mol) taking care that
the reaction mixture does not become acidic. The completion
of the reaction was monitored by TLC using benzene : ethyl
acetate (9 : 1) as the mobile phase. The product was filtered
and washed with cold water and recrystallized with ethanol to
afford pure products 5(a–j).
6-Chloro-N2,N4-di-p-tolyl-1,3,5-triazine-2,4-diamine (5f). Pale
yellowish crystals, yield: 78%; M.p: 212–214 uC; MW: 332.80;
Rf: 0.72; FT-IR (nmax; cm21 KBr): 3310 (N–H secondary), 3000
(C–H), 1620–1650 (CLC), 1605 (CLC aromatic ring), 1475 (CLC
aromatic ring); 1H-NMR (400 MHz, CDCl3-d6, TMS) d (ppm):
7.31–7.26 (m,4H, 4xCH, Ar–H), 7.06–7.02 (m, 4H, 4xCH, Ar–H),
5.24 (br, s, 2H, 2xNH), 2.53 (s, 6H, 2xCH3); 13C-NMR (100 MHz,
CDCl3) d (ppm): 168.35, 167.85, 135.85, 131, 129.85, 120.40,
6-Chloro-N2,N4-bis(4-nitrophenyl)-1,3,5-triazine-2,4-diamine
(5a). Yellow crystals, yield: 86%; M.p: 143–145 uC; MW: 387.74;
21.5; Mass: 326.20 (M
+
H)+; Elemental analysis for
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C17H16ClN5: calculated: C, 62.67; H, 4.95; N, 21.50. Found: C,
62.63; H, 4.98; N, 21.55.
MW: 437.20; Rf: 0.69; FT-IR (nmax; cm21 KBr): 3289.56 (N–H
secondary), 3055.70 (C–H broad), 1675 (CLN), 1548.28–1446.06
(aromatic CLN), 1525 (NO2 aromatic), 1250 (C–N); 1H-NMR
(400 MHz, CDCl3-d6, TMS) d (ppm): 7.40–7.37 (m, 4H, 4xCH,
Ar–H), 7.29–7.25 (m, 4H, 4xCH, Ar–H), 3.62 (br, s, 2H, 2xNH),
3.25–3.19 (m, 4H, 2xCH2), 2.79–2.74(m, 4H, 2xCH2), 1.95(s, 1H,
NH); 13C-NMR (100 MHz, CDCl3) d (ppm): 173.56, 168.85,
148.26, 143.16, 131.36, 126.23, 48.25, 45.80; Mass: 438.20 (M +
H)+; Elemental analysis for C19H19N9O4: calculated: C, 52.17;
H, 4.38; N, 28.82. Found: C, 52.10; H, 4.39; N, 28.85.
N2,N4-Bis(4-chlorophenyl)-6-(piperazin-1-yl)-1,3,5-triazine-
2,4-diamine (6b). White crystals, yield: 76%; M.p: 234–236 uC;
MW: 416.31; Rf: 0.52; FT-IR (nmax; cm21 KBr): 3243.26 (N–H
secondary), 3000 (C–H aromatic ring), 2965.46 (C–H broad),
1475 (CLC aromatic ring), 1387.38 (aromatic –CLN), 1250 (C–
N); 1H-NMR (400 MHz, CDCl3-d6, TMS) d (ppm): 7.32 (d, 2H J =
8.52 Hz, 2xCH, Ar–H), 7.29 (d, 2H J = 8.02 Hz, 2xCH, Ar–H),
7.08 (d, 2H J = 5.52 Hz, 2xCH, Ar–H), 7.02 (d, 2H J = 5.12 Hz,
2xCH, Ar–H), 4.82 (br, s, 2H, 2xNH), 3.25–3.15 (m, 4H, 2xCH2),
2.79–7.72 (m, 4H, 2xCH2), 1.95 (s, 1H, NH); 13C-NMR (100
MHz, CDCl3) d (ppm): 168.40, 167.85, 137.10, 129.50, 127.70,
122.25, 48.30, 45.50; Mass: 438.20 (M + H)+; Elemental analysis
for C19H19N9O4: Calculated: C, 52.17; H, 4.38; N, 28.82. Found:
C, 51.90; H, 4.36; N, 28.81.
N2,N4-Bis(4-bromophenyl)-6-(piperazin-1-yl)-1,3,5-triazine-
2,4-diamine (6c). Brown crystals, yield: 63%; M.p: 258–259 uC;
MW: 505; Rf: 0.48; FT-IR (nmax; cm21 KBr): 3350.62 (N–H
secondary), 3015.43 (C–H broad), 1656.15 (CLC stretch), 1650
(CLN), 1475(CLC), 1250 (C–N); 1H-NMR (400 MHz, CDCl3-d6,
TMS) d (ppm): 7.26 (d, 2H J = 8.80 Hz, 2xCH, Ar–H), 7.21 (d, 2H
J = 8.02 Hz, 2xCH, Ar–H), 7.06 (d, 2H J = 5.50 Hz, 2xCH, Ar–H),
6.95 (d, 2H J = 5.03 Hz, 2xCH, Ar–H), 4.81 (br, s, 2H, 2xNH),
3,28 (d, 4H J = 13.03 Hz, 2xCH2), 2.91 (d,4H J = 13.29 Hz,
2xCH2), 1.95 (s,1H, NH); 13C-NMR (100 MHz, CDCl3) d (ppm):
168.35, 167.85, 137.85, 132.45, 118.50, 116.80, 48.20, 45.50;
Mass: 506.20(M + H)+; Elemental analysis for C19H19Br2N7:
calculated: C, 45.17; H, 3.79; N, 19.41. Found: C, 45.10; H, 3.82;
N, 19.40.
6-Chloro-N2,N4-di-o-tolyl-1,3,5-triazine-2,4-diamine (5g).
Yellowish crystals, yield: 72%; M.p: 225–226 uC; MW: 332.80;
Rf: 0.67; FT-IR (nmax; cm21 KBr): 3310 (N–H secondary), 3000
(C–H), 1620–1650 (CLC), 1605 (CLC aromatic ring), 1475 (CLC
1
aromatic ring); H- NMR (400 MHz, CDCl3-d6, TMS) d (ppm):
7.10–7.07 (m, 4H, 4xCH, Ar–H), 7.05–7.02 (m, 4H, 4xCH, Ar–H),
5.24 (br, s, 2H, 2xNH), 2.20 (s, 6H, 2xCH3); 13C-NMR (100 MHz,
CDCl3) d (ppm): 168.35, 167.85, 141.50, 131.20, 129, 126.40,
123.85, 123.60, 17.45; Mass: 326.20 (M + H)+; Elemental
analysis for C17H16ClN5: calculated: C, 62.67; H, 4.95; N, 21.50.
Found: C, 62.63; H, 4.98; N, 21.55.
2-Chloro-4,6-di(piperidin-1-yl)-1,3,5-triazine
(5h).
Light
brown, yield: 64%; M.p: 256–258 uC; MW: 281.78; Rf: 0.73;
FT-IR (nmax; cm21 KBr): 3000 (C–H), 1675 (CLN), 1475 (CLC
1
aromatic ring), 1250 (C–N aromatic ring); H-NMR (400 MHz,
CDCl3-d6, TMS) d (ppm): 3.71–3.67 (m, 8H, 4xCH2, Piperidine),
1.63–1.60 (m, 8H, 4xCH2, Piperidine), 1.57–1.54 (m, 4H, 2xCH2,
Piperidine); 13C-NMR (100 MHz, CDCl3) d (ppm): 164.20,
160.50, 52.10, 26.50, 24.50; Mass: 282.10 (M + H)+; Elemental
analysis for C13H20ClN5: calculated: C, 55.41; H, 7.15; N, 24.85.
Found: C, 55.48; H, 7.13; N, 24.87.
N1,N19-(6-Chloro-1,3,5-triazine-2,4-diyl)dipropane-1,3-dia-
mine (5i). Brown crystals, yield: 57%; M.p: 234–236 uC; MW:
259.74; Rf: 0.57; FT-IR (nmax; cm21 KBr): 3390 (N–H primary),
3300 (N–H secondary), 3000 (C–H), 1675 (CLN), 1475 (CLC
1
aromatic ring), 1250 (C–N aromatic ring); H-NMR (400 MHz,
CDCl3-d6, TMS) d (ppm): 5.10–5.08 (m, 4H, 2xNH2), 4.13 (br, s,
2H, 2xNH), 3.20–3.18 (m, 4H, 2xCH2), 2.63–2.61 (m, 4H,
2xCH2), 1.68–1.65 (m, 4H, 2x CH2); 13C-NMR (100 MHz, CDCl3)
d (ppm): 164.20, 160.50, 50.10, 29.60; Mass: 260.23 (M + H)+;
Elemental analysis for C9H18ClN7: calculated: C, 41.62; H, 6.99;
N, 37.75. Found: C, 41.60; H, 7.03; N, 37.78.
4,49-(6-Chloro-1,3,5-triazine-2,4-diyl)bis(azanediyl)diphenol
(5j). Black crystals, yield: 67%; M.p: 251–252 uC; MW: 329.74;
Rf: 0.47; FT-IR (nmax; cm21 KBr): 3400 (OH aromatic), 3300 (N–
H secondary), 3000 (C–H aromatic ring), 1675 (CLN), 1475
N2,N4-Bis(4-methoxyphenyl)-6-(piperazin-1-yl)-1,3,5-triazine-
2,4-diamine (6d). Light yellow crystals, yield: 79%; M.p: 261–
263 uC; MW: 407.47; Rf: 0.58; FT-IR (nmax; cm21 KBr): 3300 (N–
H secondary), 3015 (C–H), 1670–1685 (CLN), 1630–1640 (CLC),
1585 (CLC aromatic ring), 1460 (CLC aromatic ring), 1100–
1
(CLC aromatic ring), 1250 (C–N aromatic ring); H-NMR (400
MHz, CDCl3-d6, TMS) d (ppm): 7.26–7.21 (m, 4H, 4xCH, Ar–H),
6.93–6.84 (m,4H, 4xCH, Ar–H), 5.25 (s, 2H, Ar–OH), 4.13 (br, s,
2H, 2xNH); 13C-NMR (100 MHz, CDCl3) d (ppm): 164.20,
160.50, 153.60, 131.50, 122.10, 116.70; Mass: 330.20 (M + H)+;
Elemental analysis for C15H12ClN5O2: calculated: C, 54.64; H,
3.67; N, 21.24. Found: C, 54.63; H, 3.69; N, 21.23.
1
1230 (C–N). H-NMR (400 MHz, CDCl3-d6, TMS) d (ppm): 7.52
(d, 2H J = 5.49 Hz, 2xCH, Ar–H), 7.48 (d, 2H J = 5.01 Hz, 2xCH,
Ar–H), 7.43 (d, 2H J = 8.68 Hz, 2xCH, Ar–H), 7.39 (d, 2H J = 8.76
Hz, 2xCH, Ar–H), 5.49 (br, s, 2H, 2xNH), 3.65 (s, 6H, 2x OCH3),
3.26 (d, 4H J = 13.03 Hz, 2xCH2), 2.92 (d, 4H J = 13.29 Hz,
2xCH2), 1.94 (s,1H, NH); 13C-NMR (100 MHz, CDCl3) d (ppm):
168.35, 167.85, 153.40, 131.10, 121.80, 115.10, 55.90, 47.70,
General procedure for the synthesis of tri-substituted 1,3,5-
triazine derivatives. 6(a-j)
A solution of the di-substituted 1,3,5-triazine compound, 5(a–
j), (0.01 mol), piperazine (0.01 mol) and K2CO3 (0.01 mol) in
1,4-dioxane was refluxed for 5–6 h. The completion of the
reaction was monitored by TLC using benzene : ethyl acetate
(9 : 1) as the mobile phase. The reaction mixture was filtered
and concentrated under reduced pressure. The resulting
residue was recrystallised by ethanol to afford the desired
product 6(a–j)
45.10; Mass: 408.50 (M
+
H)+; Elemental analysis for
C21H25N7O2: calculated: C, 61.90; H, 6.18; N, 24.06. Found:
C, 61.94; H, 6.19, N, 24.05.
4,49-(6-(Piperazin-1-yl)-1,3,5-triazine-2,4-diyl)dimorpholine
(6e). White crystals, yield: 79%: M.p: 281–283 uC; MW: 335.40;
Rf: 0.72; FT-IR (nmax; cm21 KBr): 2966 (C–H aromatic), 1574–
1451 (CLC aromatic ring), 1362 (C–N), 1116; 1H-NMR (400
MHz, CDCl3) d 3.78–3.73 (m, 8H, 4xCH2-O), 3.70–3.64 (m, 8H,
N2,N4-Bis(4-nitrophenyl)-6-(piperazin-1-yl)-1,3,5-triazine-2,4-
diamine (6a). Yellow crystals, yield: 74%; M.p: 261–263 uC;
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4xCH2-N), 3.26 (d, 4H, J = 13.03 Hz, 2xCH2), 2.92 (d, 4H, J =
13.29 Hz, 2xCH2), 1.94 (s,1H NH); 13C-NMR (100 MHz, CDCl3)
d (ppm): 169.69, 164.48, 66.56, 47.60, 45.20, 43.86; Mass:
336.50 (M + H)+; Elemental analysis for C15H25N7O2: calcu-
lated: C, 53.71; H, 7.51; N, 29.23. Found: C, 53.64; H, 7.57; N,
29.26.
6-(Piperazin-1-yl)-N2,N4-dip-tolyl-1,3,5-triazine-2,4-diamine
(6f). Dark yellowish crystals, yield: 65%; M.p: 284–286 uC; MW:
375.47; Rf: 0.63; FT-IR (nmax; cm21 KBr): 3310 (N–H secondary),
3000 (C–H), 1605 (CLC aromatic ring), 1620–1650 (CLC), 1475
(CLC aromatic ring); 1H-NMR (400 MHz, CDCl3-d6, TMS) d
(ppm): 7.31 (d, 2H J = 8.21 Hz, 2xCH, Ar–H), 7.28 (d, 2H J = 8.02
Hz, 2xCH, Ar–H), 7.23 (d, 2H J = 6.21 Hz, 2xCH, Ar–H), 7.06 (d,
2H J = 5.43 Hz, 2xCH, Ar–H), 5.24 (br, s, 2H, 2xNH), 3.26 (d, 4H
J = 13.03 Hz, 2xCH2), 2.92 (d, 4H J = 13.29 Hz, 2xCH2), 2.53 (s,
6H, 2xCH3), 1.94 (s,1H, NH); 13C-NMR (100 MHz, CDCl3) d
(ppm):168.35, 167.85, 135.85, 131, 129.85, 120.40, 47.80, 45.30,
21.5; Mass: 376.45 (M + H)+; Elemental analysis for C21H25N7:
calculated: C, 67.18; H, 6.71; N, 26.11. Found: C, 66.97; H, 6.73;
N, 26.10.
6-(Piperazin-1-yl)-N2,N4-dio-tolyl-1,3,5-triazine-2,4-diamine
(6g). Brown crystals, yield: 82%; M.p: 291–293 uC; MW:375.47;
Rf: 0.46; FT-IR (nmax; cm21 KBr): 3310 (N–H secondary), 3000
(C–H), 1620–1650 (CLC), 1605 (CLC aromatic ring), 1475 (CLC
aromatic ring); 1H-NMR (400 MHz, CDCl3-d6, TMS) d (ppm):
7.12 (d, 2H J = 8.68 Hz, 2xCH, Ar–H), 7.10 (d, 2H J = 8.05 Hz,
2xCH, Ar–H), 7.08 (d, 2H J = 7.89 Hz, 2xCH, Ar–H), 7.05 (d, 2H J
= 7.50 Hz, 2xCH, Ar–H), 5.24 (br, s, 2H, 2xNH), 3.26 (d, 4H J =
13.03 Hz, 2xCH2), 2.92 (d, 4H J = 13.29 Hz, 2xCH2), 2.20 (s, 6H,
2xCH3), 1.94 (s,1H, NH); 13C-NMR (100 MHz, CDCl3) d (ppm):
168.35, 167.85, 141.50, 131.20, 129.20, 126.40, 123.85, 123.60,
47.5, 44.97, 17.30; Mass: 376.30 (M + H)+; Elemental analysis
for C21H25N7: calculated: C, 62.67; H, 4.95; N, 21.50. Found: C,
62.69; H, 4.93; N, 21.51.
4,49-(6-(Piperazin-1-yl)-1,3,5-triazine-2,4diyl)bis(azanediyl)
diphenol (6j). Dark black crystals, yield: 82%; M.p: 287–289 uC;
MW: 379.42; Rf: 0.56; FT-IR (nmax; cm21 KBr): 3400 (OH
aromatic), 3300 (N–H secondary), 3000 (C–H aromatic ring),
1675 (CLN), 1475 (CLC aromatic ring), 1250 (C–N aromatic
ring); 1H-NMR (400 MHz, CDCl3-d6, TMS) d (ppm): 7.32 (d, 2H J
= 8.72 Hz, 2xCH, Ar–H), 7.26 (d, 2H J = 8.53 Hz, 2xCH, Ar–H),
6.93 (d, 2H J = 3.72 Hz, 2xCH, Ar–H), 6.84 (d, 2H J = 3.64 Hz,
2xCH, Ar–H), 5.25 (s, 2H, 2x Ar-OH), 4.13 (br, s, 2H, 2xNH),
3.26 (d, 4H J = 13.03 Hz, 2xCH2), 2.92 (d, 4H, J = 13.29 Hz,
2xCH2), 1.94 (s, 1H, NH); 13C-NMR (100 MHz, CDCl3) d (ppm):
164.20, 160.50, 153.60, 131.50, 122.10, 116.70, 48.80, 45.30;
Mass: 380.40 (M + H)+; Elemental analysis for C19H21N7O2:
calculated: C, 60.15; H, 5.58; N, 25.84. Found: C, 60.13; H, 5.59;
N, 25.84.
General procedure for the synthesis of title compounds. 7(a–j)
A solution of compound (3) (0.01 mol) and the tri-substituted
1,3,5-triazine compounds 6(a–j) (0.01 mol) in dry acetone was
stirred at 40–45 uC for 18 h. The completion of the reaction was
monitored by TLC using ethanol : acetone (1 : 1) as the mobile
phase. The reaction mixture was filtered and concentrated
under reduced pressure. The resulting residue was dissolved
in dichloromethane, washed with brine and dried over
anhydrous Na2SO4. The dried solution was concentrated under
reduced pressure to obtain the titled compounds 7(a–j).
4-(4,6-Bis(4-nitrophenylamino)-1,3,5-triazin-2-yl)-N-(7-chlor-
oquinolin-4-yl)piperazine-1-carbothioamide (7a). Yellow crys-
tals, yield: 58%; M.p: 230–232 uC; MW: 658.09; Rf: 0.81; FT-IR
(nmax; cm21 KBr): 3300 (N-H secondary), 3000 (C–H aromatic
ring), 1675 (CLN), 1640 (CLC), 1525 (NO2 aromatic), 1475 (CLC
aromatic ring), 1235 (C–N); 1H-NMR (400 MHz, CDCl3-d6, TMS)
d (ppm): 8.82 (d, 1H J = 6.60 Hz, quinoline ring), 8.10 (d, 1H J =
8.43 Hz, quinoline ring), 8.03–7.98 (m, 4H, 4xC-H, Ar–H), 7.94
(d, 1H J = 8.35 Hz, quinoline ring), 7.35 (d, 1H J = 6.65 Hz,
quinoline ring), 7.02 (d, 1H J = 6.25 Hz, quinoline ring), 6.90–
6.87 (m, 4H 4xC-H, Ar–H), 4.13 (br, s, 1H, NH), 4.07–4.02 (m,
4H, 2xCH2), 4.01 (s, 2H, 2xNH), 3.18–3.12 (m, 4H, 2xCH2); 13C-
NMR (100 MHz, CDCl3); d (ppm): 181.40, 176.10, 167, 152.70,
150.70, 149.60, 145.35, 136.80, 134.90, 129.40, 126.70, 124.70,
121.60, 119.60, 118.20, 112.70, 56.70, 52.70; Mass: 659.09 (M +
H)+; Elemental analysis for C29H24ClN11S: calculated: C, 51.90;
H, 5.50; N, 21.68. Found: C, 51.85; H, 5.53; N, 21.70.
2-(Piperazin-1-yl)-4,6-di(piperidin-1-yl)-1,3,5-triazine (6h).
Brown crystals, yield: 72%; M.p: 305–306 uC; MW: 323–325;
Rf: 0.71; FT-IR (nmax; cm21 KBr): 3000 (C–H), 1675 (CLN), 1475
1
(CLC aromatic ring), 1250 (C–N aromatic ring); H-NMR (400
MHz, CDCl3-d6, TMS) d (ppm): 3.71–3.67 (m, 8H, 4xCH2), 3.26
(d, 4H J = 13.03 Hz, 2xCH2), 2.92 (d, 4H J = 13.29 Hz, 2xCH2),
1.94 (s,1H, NH), 1.63–1.59 (m, 8H, 4xCH2), 1.51–1.49 (m, 4H,
2xCH2); 13C-NMR (100 MHz, CDCl3) d (ppm):164.20, 160.50,
52.10, 48.93, 45.65, 26.50, 24.50; Mass: 332.40 (M + H)+;
Elemental analysis for C17H29N7: calculated: C, 61.60; H, 8.82;
N, 29.58. Found: C, 61.56; H, 8.83; N, 29.56.
4-(4,6-Bis(4-chlorophenylamino)-1,3,5-triazin-2-yl)-N-(7-
chloroquinolin-4-yl)piperazine-1-carbothioamide (7b). Whitish
yellow crystals, yield: 68%; M.p: 213–215 uC; MW: 636.99; Rf:
0.75; FT-IR (nmax; cm21 KBr): 3420 (N–H secondary), 3010 (C–H
aromatic ring), 1675 (CLN), 1645 (CLC), 1470 (CLC aromatic
N1,N19-(6-(Piperazin-1-yl)-1,3,5-triazine-2,4-diyl)dipropane-
1,3-diamine (6i). Yellow brown crystals, yield: 69%; M.p: 324–
325 uC;MW: 309.41; Rf: 0.52; FT-IR (nmax; cm21 KBr): 3390 (N–H
primary), 3300 (N–H secondary), 3000 (C–H), 1675 (CLN), 1475
1
ring), 1232 (C–N), 1007, 671 (Cl aromatic); H-NMR (400 MHz,
1
CDCl3-d6, TMS) d (ppm): 8.76 (d, 1H J = 6.53 Hz, quinoline
ring), 8.12 (d, 1H J = 8.43 Hz, quinoline ring),7.83 (d, 1H J =
6.58 Hz, quinoline ring), 7.68–7.63 (m, 4H, 4xC-H, Ar–H),7.13–
7.09 (m, 4H, 4xC-H, Ar–H), 7.33 (d, 1H J = 6.42 Hz, quinoline
ring), 6.87 (d, 1H J = 5.43 Hz, quinoline ring), 4.20–4.17 (m, 4H,
2xCH2), 3.34–3.30 (m, 4H, 2xCH2), 4.13 (br, s, 1H, NH), 3.98 (s,
2H, 2xNH); 13C-NMR (100 MHz, CDCl3); d (ppm): 181.80, 176,
167.30, 152.70, 149.50, 149.30, 137.40, 134.9, 129.70, 129.20,
127.60, 124.80, 122, 121.60, 119.70, 113, 56.70, 52.20; Mass:
(CLC aromatic ring), 1250 (C–N aromatic ring); H-NMR (400
MHz, CDCl3-d6, TMS) d (ppm): 5.10 (d, 4H J = 6.75 Hz, 2xNH2),
4.13 (br, s, 2H, 2xNH)), 3.26 (d, 4H J = 13.03 Hz, 2xCH2), 3.20
(d, 4H J = 6.74 Hz, 2xCH2), 2.92 (d,4H J = 13.29 Hz, 2xCH2), 2.63
(d, 4H J = 7.23 Hz, 2xCH2), 1.94 (s,1H, NH), 1.68 (d, 4H J = 7.26
Hz, 2xCH2); 13C-NMR (100 MHz, CDCl3) d (ppm): 164.20,
160.50, 50.10, 48.20, 45.60, 29.60; Mass: 310.40(M + H)+;
Elemental analysis for C13H27N9: calculated: C, 50.46; H, 8.80;
N, 40.74. Found: C, 50.43; H, 8.82; N, 40.76.
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+
H)+; Elemental analysis for C29H24Cl3N9S:
cm21 KBr): 3405 (N–H secondary), 3020 (C–H aromatic ring),
1693 (CLN), 1556 (CLC), 1421 (CLC aromatic ring), 1261 (C–N),
1220, 1000.6, 671; 1H-NMR (400 MHz, CDCl3-d6, TMS) d (ppm):
8.86 (d, 1H J = 7.44 Hz, quinoline ring), 8.23 (d, 1H J = 8.54 Hz,
quinoline ring), 7.75 (d, 1H J = 6.30 Hz, quinoline ring), 7.45–
7.35 (m, 4H, 4xCH, Ar–H), 7.13 (d, 1H J = 8.14 Hz, quinoline
ring), 7.06–7.04 (m, 4H, 4xCH, Ar–H), 6.93 (d, 1H J = 5.38 Hz,
quinoline ring), 4.10 (br, s, 1H, NH), 4.05–4.01 (m, 4H, 2xCH2),
3.92 (s, 2H, 2xNH), 3.17–3.07 (m, 4H, 2xCH2), 2.43 (s, 6H,
2xCH3); 13C-NMR (100 MHz, CDCl3); d (ppm): 179.30, 176,
160.50, 153.30, 149.40, 146.90, 142.50, 140.40, 135.90, 131.70,
130.50, 129.80, 125.30, 121.20, 120.40, 113.70, 56.70, 52, 21.30;
Mass: 597.20 (M + H)+; Elemental analysis for C31H30ClN9S:
calculated: C, 61.96; H, 6.94; N, 20.65. Found: C, 61.98; H, 7.01;
N, 20.64.
calculated: C, 55.35; H, 5.13; N, 17.23. Found: C, 54.68; H,
4.97; N, 19.79.
4-(4,6-Bis(4-bromophenylamino)-1,3,5-triazin-2-yl)-N-(7-
chloroquinolin-4-yl)piperazine-1-carbothioamide (7c).
Yellowish brown crystals, yield: 71%; M.p: 315–317 uC; MW:
725.89; Rf: 0.76; FT-IR (nmax; cm21 KBr): 3435 (N–H secondary),
3025 (C–H aromatic ring), 1671 (CLN), 1647 (CLC), 1475 (CLC
aromatic ring), 1236 (C–N), 1072, 769, 672; 1H-NMR (400 MHz,
CDCl3-d6, TMS) d (ppm): 8.72 (d, 1H J = 6.57 Hz, quinoline
ring), 8.23 (d, 1H J = 8.48 Hz, quinoline ring), 7.87 (d, 1H J =
6.65 Hz, quinoline ring), 7.53–7.46 (m, 4H, 4xC-H, Ar–H), 7.31
(d, 1H J = 6.52 Hz, quinoline ring), 7.01–6.95 (m, 4H, 4xC-H,
Ar–H), 6.93 (d, 1H J = 5.37 Hz, quinoline ring), 3.87–3.83 (m,
4H, 2xCH2), 3.33–3.29 (m, 4H, 2xCH2), 4.10 (br, s, 1H, NH),
4.02 (s, 2H, 2xNH); 13C-NMR (100 MHz, CDCl3); d,ppm: 180.40,
178.30, 167.20, 155.60, 149.20, 147.30, 138.40, 135.90, 132.80,
129.80, 125.30, 121.60, 119.50, 118.70, 115.70, 113.20, 56.40,
4-(4,6-Bis(o-tolylamino)-1,3,5-triazin-2-yl)-N-(7-chloroquino-
lin-4-yl)piperazine-1-carbothioamide (7g). Yellowish crystals,
yield: 71%; M.p: 396–398 uC; MW: 596.15; Rf: 0.78; FT-IR (nmax
;
50.20; Mass: 726.90 (M
+
H)+; Elemental analysis for
cm21 KBr): 3404 (N–H secondary), 3020 (C–H aromatic ring),
C29H24Br2ClN9S: calculated: C, 46.67; H, 3.09; N, 19.40.
Found: C, 46.60; H, 3.10; N, 19.56.
1693 (CLN), 1556(CLC), 1421 (CLC aromatic ring), 1261 (C–N),
1225, 1020, 672; H-NMR (400 MHz, CDCl3-d6, TMS) d (ppm):
1
8.85 (d, 1H J = 7.44 Hz, quinoline ring), 8.35 (d, 1H J = 8.49 Hz,
quinoline ring), 7.73 (d, 1H J = 6.32 Hz, quinoline ring), 7.28
(d, 1H J = 8.62 Hz, quinoline ring), 7.12–7.04 (m, 4H, 4xCH, Ar–
H), 6.95 (d, 1H J = 5.37 Hz, quinoline ring), 6.69–6.56 (m, 4H,
4xCH, Ar–H), 4.06–3.98 (m, 4H, 2xCH2), 3.17–3.12 (m, 4H,
2xCH2), 2.15 (t, 6H, 2xCH3), 4.05 (br, s, 1H, NH) 3.93 (s, 2H,
2xNH); 13C-NMR (100 MHz, CDCl3); d (ppm): 179.30, 176,
167.50, 153.40, 149.20, 148.30, 142.50, 135.90, 130.50, 129.80,
129.40, 128.30, 126.50, 124.70, 123.70, 121.10, 119.70, 113.20,
56.80, 51.90, 17.70; Mass: 597.20 (M + H)+; Elemental analysis
for C31H30ClN9S: calculated: C, 61.96; H, 6.94; N, 20.65. Found:
C, 61.92; H, 7.05; N, 20.58.
4-(4,6-Bis(4-methoxyphenylamino)-1,3,5-triazin-2-yl)-N-(7-
chloroquinolin-4-yl)piperazine-1-carbothioamide (7d).
Yellowish crystals, yield: 66%; M.p: 320–322 uC; MW: 625.15;
Rf: 0.73; FT-IR (nmax; cm21 KBr): 3410 (N–H secondary), 3018
(C–H aromatic ring), 1678 (CLN), 1650 (CLC), 1570, 1463 (CLC
aromatic ring), 1236 (C–N), 1176, 1033, 671; 1H-NMR (400
MHz, CDCl3-d6, TMS) d (ppm): 8.79 (d, 1H J = 7.6 Hz, quinoline
ring), 8.35 (d, 1H J = 8.49 Hz, quinoline ring), 7.61 (d, 1H J =
6.20 Hz, quinoline ring), 7.55–7.48 (m, 4H, 4xC-H, Ar–H), 7.28
(d, 1H J = 6.59 Hz, quinoline ring), 7.10–7.03 (m, 4H, 4xC-H,
Ar–H), 6.85 (d, 1H J = 5.28 Hz, quinoline ring), 3.98–3.93 (m,
4H, 2xCH2), 3.34–3.29 (m, 4H, 2xCH2), 3.83 (t, 6H, 2xOCH3),
4.20 (br, s, 1H, NH) 4.03 (s 2H, 2xNH); 13C-NMR (100 MHz,
CDCl3); d,ppm: 181.20, 178.30, 166.70, 155.20, 152.60, 149.20,
148.90, 139.40, 133.50, 131.20, 128.70, 127.60, 121.30, 119.60,
115.40, 113.70, 56.20, 54.90, 51.20; Mass: 626.10 (M + H)+;
Elemental analysis for C31H30ClN9O2S: calculated: C, 59.22; H,
6.57; N, 21.42. Found: C, 59.24; H, 6.56; N, 21.47.
N-(7-Chloroquinolin-4-yl)-4-(4,6-di(piperidin-1-yl)-1,3,5-tria-
zin-2-yl)piperazine-1-carbothioamide (7h). Brown crystals,
yield: 66%; M.p: 270–272 uC; MW: 552.14; Rf: 0.73; FT-IR (nmax
;
cm21 KBr): 3415 (N–H secondary), 3032 (C–H aromatic ring),
1685 (CLN), 1565(CLC), 1475 (CLC aromatic ring), 1262 (C–N),
1
1228, 1027, 670; H-NMR (400 MHz, CDCl3-d6, TMS) d (ppm):
N-(7-Chloroquinolin-4-yl)-4-(4,6-dimorpholino-1,3,5-triazin-
2-yl)piperazine-1-carbothioamide (7e). Brown crystals, yield:
77%; M.p: 333–334 uC; MW: 557.08; Rf: 0.82; FT-IR (nmax; cm21
KBr): 3347 (N–H secondary), 3042 (C–H aromatic ring), 1689
(CLN), 1579 (CLC), 1444 (CLC aromatic ring), 1301, 1243 (C–
N), 1218, 699; 1H-NMR (400 MHz, CDCl3-d6, TMS) d (ppm):
8.82 (d, 1H J = 7.40 Hz, quinoline ring), 8.28 (d, 1H J = 8.43 Hz,
quinoline ring), 7.68 (d, 1H J = 6.40 Hz, quinoline ring), 7.23
(d, 1H J = 8.73 Hz, quinoline ring), 6.94 (d, 1H J = 5.38 Hz,
quinoline ring), 4.25 (br, s, 1H, NH), 4.10–4.04 (m, 4H, 2xCH2),
3.78–3.73 (m,8H, 4x CH2), 3.58–3.51 (m, 8H, 4xCH2), 3.08–3.02
(m, 4H, 2xCH2); 13C-NMR (100 MHz, CDCl3); d (ppm): 181.30,
179.40, 177.60, 160.50, 146.90, 142.40, 134.40, 130.50, 129.80,
125.30, 121.60, 113.60, 66.30, 56.20, 50.10, 48.70; Mass: 558.10
(M + H)+; Elemental analysis for C25H30ClN9O2S: calculated: C,
46.12; H, 6.38; N, 24.33. Found: C, 46.10; H, 6.32; N, 24.30.
4-(4,6-Bis(p-tolylamino)-1,3,5-triazin-2-yl)-N-(7-chloroquino-
lin-4-yl)piperazine-1-carbothioamide (7f). Yellowish crystals,
8.85 (d, 1H J = 7.44 Hz, quinoline ring), 8.12 (d, 1H J = 8.14 Hz,
quinoline ring), 7.70 (d, 1H J = 6.32 Hz, quinoline ring), 7.43
(d, 1H J = 8.75 Hz, quinoline ring), 7.02 (d, 1H J = 5.63 Hz,
quinoline ring), 4.03–4.01 (m, 4H, 2xCH2), 3.75–3.71 (m, 8H,
4xCH2), 3.18–3.15 (m, 4H, 2xCH2), 1.63–1.61 (m, 4H 2xCH2),
1.58–1.53 (m, 8H, 4xCH2), 4.08 (br, s, 1H, NH); 13C-NMR (100
MHz, CDCl3); d (ppm): 181.80, 180.50, 176.50, 152.30, 149.60,
148.30, 134.90, 129.40, 124.80, 121.60, 119.70, 114.10, 56.70,
54.80, 52.10, 25.50, 24.50; Mass: 553.10 (M + H)+; Elemental
analysis for C27H34ClN9S: calculated: C, 56.86; H, 8.57; N,
22.63. Found: C, 57.01; H, 8.54; N, 22.64.
4-(4,6-Bis(3-aminopropylamino)-1,3,5-triazin-2-yl)-N-(7-
chloroquinolin-4-yl)piperazine-1-carbothioamide (7i). Yellow
brown crystals, yield: 69%; M.p: 288–290 uC; MW: 530.09; Rf:
0.68; FT-IR (nmax; cm21 KBr): 3415 (N–H secondary), 3390 (N–H
primary), 3030 (C–H aromatic ring), 1670 (CLN), 1555 (CLC),
1475 (CLC aromatic ring), 1269 (C–N), 1220, 1020, 670; 1H-
NMR (400 MHz, CDCl3-d6, TMS) d (ppm): 8.78 (d, 1H J = 7.40
Hz, quinoline ring), 8.43 (d, 1H J = 8.31 Hz, quinoline ring),
yield: 64%; M.p: 342–343 uC; MW: 596.15; Rf: 0.83; FT-IR (nmax
;
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7.78 (d, 1H J = 6.35 Hz, quinoline ring), 7.24 (d, 1H J = 8.74 Hz,
quinoline ring), 6.94 (d, 1H J = 5.38 Hz, quinoline ring), 5.20
(d, 4H J = 2.34 Hz, 2xNH2), 4.08 (br, s, 1H, NH), 4.03–3.98 (m,
4H, 2xCH2), 3.95 (s, 2H, 2xNH), 3.18–3.12 (m, 4H, 2xCH2), 3.35
(m, 4H, 2xCH2), 2.68 (m, 4H, 2xCH2), 1.83 (m, 4H, 2xCH2); 13C-
NMR (100 MHz, CDCl3); d (ppm): 181.80, 176.50, 164.30,
152.70, 149.30, 148.70, 134.90, 129.60, 124.80, 121.60, 119.70,
112.90, 56.70, 52.50, 40.30, 31.50; Mass: 531.05 (M + H)+;
Elemental analysis for C23H32ClN11S: calculated: C, 47.67; H,
6.64; N, 30.61. Found: C, 47.65; H, 6.67; N, 30.62.
4-(4,6-Bis(4-hydroxyphenylamino)-1,3,5-triazin-2-yl)-N-(7-
chloroquinolin-4-yl)piperazine-1-carbothioamide (7j). Black
crystals, yield: 74%; M.p: 278–280 uC MW: 600.09; Rf: 0.64;
FT-IR (nmax; cm21 KBr): 3420 (N–H secondary), 3395 (OH
aromatic ring), 3035 (C–H aromatic ring), 1676 (CLN), 1557
(CLC), 1477 (CLC aromatic ring), 1269 (C–N), 1210, 1003, 678;
1H-NMR (400 MHz, CDCl3-d6, TMS) d (ppm): 8.90 (d, 1H J =
7.50 Hz, quinoline ring), 8.28 (d, 1H J = 8.43 Hz, quinoline
ring), 7.76 (d, 1H J = 6.33 Hz, quinoline ring), 7.53–7.48 (m, 4H,
2xCH2, Ar–H), 7.23 (d, 1H J = 8.73 Hz, quinoline ring), 6.78 (d,
1H J = 5.32 Hz, quinoline ring), 6.75–6.68 (m, 4H, 2xCH2, Ar–
H), 5.40 (s, 2H, 2x Ar–OH), 4.05 (br, s, 1H, NH), 4.02–3.96 (m,
4H, 2xCH2), 3.87 (s, 2H, 2xNH), 3.16–3.09 (m, 4H, 2xCH2); 13C-
NMR (100 MHz, CDCl3); d (ppm): 181.80, 176.20, 167.30,
152.70, 149.50, 149.20, 148.50, 134.80, 131.50, 129.40, 124.80,
122.20, 121.60, 119.70, 115.90, 113.10, 56.70, 52.50; Mass:
601.01 (M + H)+; Elemental analysis for C29H26ClN9O2S:
calculated: C, 58.04; H, 4.37; N, 21.01. Found: C, 58.06; H,
4.39; N, 20.95.
counting a total of 100 asexual parasites (both live and dead)
microscopically using Chloroquine and Proguanil as reference
drugs.
Molecular docking studies
The 3D X-ray crystal structure of wild type (1J3I.pdb) and
quadruple mutant (N51I, C59R, S108 N, I164L, 3QG2.pdb) pf-
DHFR were used as the starting model for this study. The
protein was prepared, docked and the molecular dynamics
simulation carried out. Computational analysis was carried
out using Discovery Studio 2.5 (Accelrys Software Inc., San
Diego; http://www.accelrys.com).
Preparation of receptor
The target wild and quadruple mutant pf-DHFR proteins with
the removal of the co-crytallised ligands were taken and the
bond order was corrected. The hydrogen atoms were added,
and their positions were optimized using the all-atom
CHARMm (version c32b1) forcefield with Adopted Basis set
Newton Raphson (ABNR) minimization algorithm until the
root mean square (r.m.s) gradient for potential energy was less
than 0.05 kcal mol21 Å21 22,23
Using the ’Binding Site’ tool
.
panel available in DS 2.5, the minimized protein structure was
defined as the receptor, the binding site was defined as the
volume occupied by the ligand in the receptor, and an input
site sphere was defined over the binding site with a radius of
5 Å. The center of the sphere was taken to be the center of the
binding site, and the side chains of the residues in the binding
site within the radius of the sphere were assumed to be flexible
during the refinement of postdocking poses. The receptor with
a defined binding site was used for the docking studies.
Ligand setup
Antimalarial activity
Using the built-and-edit module of DS 2.5, various ligands
were built, all-atom CHARMm forcefield parameterization was
assigned and then minimized using the ABNR method. A
conformational search of the ligand was carried out using a
stimulated annealing molecular dynamics (MD) approach. The
ligand was heated to a temperature of 700 K and then
annealed to 200 K. Thirty such cycles were carried out. The
transformation obtained at the end of each cycle was further
subjected to local energy minimization, using the ABNR
method. The 30 energy-minimized structures were then
superimposed and the lowest energy conformation occurring
in the major cluster was taken to be the most probable
conformation.
Preparation of parasites
The chloroquine sensitive 3D-7 and chloroquine resistant RKL-
2 strain (Raurkela, Orissa, India) of P. falciparum were
routinely maintained in stock cultures in the medium RPMI-
1640 supplemented with 25 mmol of HEPES, 1% D-glucose,
0.23% sodium bicarbonate and 10% heat inactivated human
serum. The asynchronous parasites of P. falciparum were
synchronized after 5% D-sorbitol treatment to obtain only the
ring stage parasitized cells. For carrying out the assay, the
initial ring stage parasitaemia of 0.8–1.5% at 3% haematocrit
in a total volume of 200 mL of medium RPMI-1640 was
uniformly maintained.
In vitro antimalarial efficacy test
Docking
The in vitro antimalarial assay was carried out according to
microassay of Reickmann et al. (1978) in 96 well-microtitre
plates,21 with minor modifications. A stock solution of 5 mg
mL21 of each of the test samples was prepared in DMSO and
subsequent dilutions were prepared with culture medium. The
test compounds in 20 mL volume concentration at 5 mg mL21
and 50 mg mL21 in duplicate wells were incubated with the
parasitized cell preparation at 37 uC in a candle jar. After 36–40
h of incubation, the blood smears were prepared from each
well and stained with Giemsa stain. The level of parasitemia in
terms of % dead rings along with Schizonts was determined by
Docking, a significant computational method used to foretell
the binding of the ligand to the receptor binding site by
varying the position and conformation of the ligand keeping
the receptor rigid. LigandFit23 protocol of DS 2.5 was used for
the docking of ligands with wild and quadruple mutant pf-
DHFR proteins.24 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.25
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6 E. F. F. D. Cunha, T. C. Ramalho, E. R. Maia and R. B.
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The receptor protein conformation was kept fixed during
docking, and the docked poses were further minimized using
the all-atom CHARMm (version c32b1) forcefield and smart
minimization method (steepest descent followed by conjugate
gradient) until the r.m.s gradient for potential energy was less
than 0.05 kcal mol21 Å21. The atoms of the ligand and the side
chains of the residues of the receptor within 5 Å from the
center of the binding site were kept flexible during minimiza-
tion.
Conclusions
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Fontaine, R. Vandaele, E. Mouray, P. Grellier and
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In conclusion, the present study describes the facile synthetic
routes to give rise to bi-functional hybrid derivatives com-
prised of 4-aminoquinoline and 1,3,5-triazine and their
evaluation as antimalarial agents against chloroquine sensi-
tive (3D7) and chloroquine resistant (RKL-2) strain of P.
falciparum. The observed activity profiles were further sub-
stantiated and illustrated using docking simulations per-
formed using the Ligandfit module within Discovery Studio
2.5. Additional studies to develop newer analogues of this core
scaffold are in progress and will be reported subsequently in
the future.
Acknowledgements
The financial support from the Department of Science and
Technology (DST), New Delhi, India is gratefully acknowledged
(grant no. SR/SO/HS-125/2010) and the authors also are
thankful to S.A.I.F., Punjab University, Chandigarh, India for
providing spectroscopic data.
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Notes and references
1 World Health Organistion, Malaria, Fact sheet Nu94,
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