Synthesis, antimalarial activity, heme binding and docking studies of 4-aminoquinoline-pyrimidine based molecular hybrids

Article abstract of DOI:10.1039/c4ra09768h

A series of novel 4-aminoquinoline-pyrimidine hybrids was synthesized and evaluated for their antimalarial activity. Several compounds showed potent antimalarial activity against both CQ-sensitive and CQ-resistant strains of P. falciparum with no cytotoxi

Full text of DOI:10.1039/c4ra09768h

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DOI: 10.1039/C4RA09768H
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ARTICLE TYPE
Synthesis, antimalarial activity, heme binding and docking studies of 4ꢀ
aminoquinolineꢀpyrimidine based molecular hybrids
Deepak Kumar,1 Shabana I. Khan,2,3,4 Prija Ponnan,1 Diwan S. Rawat1*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
A series of novel 4ꢀaminoquinolineꢀpyrimidine hybrids was synthesized and evaluated for their
antimalarial activity. Several compounds showed potent antimalarial activity against both CQꢀsensitive
and CQꢀresistant strains of P. falciparum with no cytotoxicity against Vero cell lines. Selected compound
7f, when evaluated for in vivo activity showed mild suppression of parasite in P. bergheiꢀmouse malaria
10 model. The heme binding studies were conducted to determine the probable mode of action of these
hybrids. Compound 8d formed a stable 1:1 complex with hematin suggesting that these hybrids act on a
heme polymerization target. The binding of most active hybrids were studied by molecular docking
analysis in the active site of PfꢀDHFRꢀTS. The top scored compounds having low binding energy interact
in the active site of PfꢀDHFRꢀTS similar to the protein natural substrate dihydrofolate. The
15 pharmacokinetics properties of best active compounds were also assessed using ADMET prediction.
dihydroartemisinin, artemether, artesunate along with a partner
Introduction
drug such as lumefantrine, mefloquine, amodiaquine,
50 piperaquine, sulfadoxine, pyrimethamine, dapsone etc.2 Although
ACTs are fast acting, highly effective and reduce the chances of
resistance development,8 few cases of artemisinin resistance have
recently been reported in SouthꢀEast Asia.9,10 Hence the
development of new antimalarial agents is urgently needed to
55 counter the everꢀincreasing spread of drug resistant malaria.
Despite the long efforts in eradicating the malaria, the disease
continues to present a major public health challenge especially in
tropical and subtropical areas affecting millions of people.1
20 According to 2013 WHO report, there were 207 million cases and
an estimated 627000 deaths due to malaria worldwide.2 The
African subcontinent has the highest disease burden with more
than 75% related deaths in pregnant women and children under
the age of five.3 The disease is transmitted by female mosquitoes
25 and is caused by five different species of the protozoan
Plasmodium parasite, namely, falciparum, vivax, malariae, ovale
and knowlesi.4 Amongst these P. falciparum is the most prevalent
species and accounts for more than 95% of clinical cases and
deaths due to malaria. Malaria in most instances is a curable
30 disease if it is diagnosed in time and treated with proper
medicines. However, the rapid development of drug resistance
has compromised the use of conventional antimalarial drugs such
as chloroquine, amodiaquine, pamaquine, mefloquine and
pyrimethamine (Fig. 1).5 Moreover, there is no commercially
35 available malaria vaccine developed despite the intensive efforts
of scientist working in this field. Though, recently a vaccine
candidate, RTS, S/AS01, developed by GlaxoSmithKline
pharmaceuticals is being evaluated in Phase III clinical trials
providing only modest protection against both clinical and severe
40 malaria in young infant.6,7 Therefore chemotherapy remains the
mainstay for dealing with this enormous problem. The growing
resistance and the lack of an effective antimalarial vaccine
Fig. 1 Antimalarial drugs
In order to search a new drug, various approaches are being
60 engaged of which the molecular hybridization is quite an
attractive strategy which involves designing of new chemical
entities by covalent linking of two pharmacophore units derived
from the known biologically active molecules with complimentary
activities and multiple pharmacological targets.11ꢀ13 Various
65 research groups across the world have successfully employed this
emphasize the need to develop
a novel, safe, affordable
antimalarial drug effective against multi drugꢀresistant malaria.
45 Currently, the best option for the treatment of uncomplicated
malaria is artemisininꢀbased combination therapies (ACTs) which
includes rapidly acting artemisinin or its analogues such as
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hybridization approach towards the synthesis of novel hybrids
novel hybrid molecules was synthesized in which 4ꢀ
which have shown potent antimalarial activity against sensitive 25 aminoquinoline moiety was covalently attached to pyrimidine
and resistant strain of P. falciparum.1,14ꢀ22 Heme and Plasmodium
falciparum dihydrofolate reductase (PfꢀDHFR) are among the
most important targets for antimalarial drug discovery. Quinoline
based drugs such as chloroquine and its derivatives are known to
ring present in antifolate class of antimalarial drugs such as
cycloguanil/pyrimethamine.39,40 All these compounds displayed
potent in-vitro antimalarial activity (IC50 = 0.005ꢀ0.44 µM)
against both CQꢀsensitive (D6 clone) and CQꢀresistant (W2
5
affect the parasite metabolism and causes death of the parasite by 30 clone) of P. falciparum with no cytotoxicity against VERO cells
blocking the polymerization of toxic heme into an insoluble and
nonꢀtoxic pigment, hemozoin, resulting in cell lysis.23 On the
(Fig. 2). The hybrids also possessed excellent in-vivo antimalarial
activity without any apparent toxicity when tested in P. berghei
infected mouse malaria model. It was found that the activity of
these hybrids was dependent upon the substituents on the
10 other hand, triazine and pyrimidineꢀbased compounds such as
cycloguanil and pyrimethamine exhibit the antimalarial activity
due to their ability to inhibit dihydrofolate reductase enzyme.24 35 pyrimidine nucleus as well as the linker connecting the two
Thus, linking of the quinoline unit with triazine or pyrimidine
nucleus can deliver hybrids that might show potent antimalarial
15 activity than each of the parent molecules. Several research
groups have synthesized such kind of hybrid molecules25ꢀ33 and
pharmacophores.
In continuation of our efforts towards the development of novel
antimalarial agents and in order to gain complete structural
information to establish structure activity relationship, herein we
many of these derivatives have shown excellent in vitro and in 40 present the synthesis and antimalarial activity of a new series of
vivo activity against both chloroquineꢀsensitive and chloroquineꢀ
resistant strains of P. falciparum.
20 Our lab has also successfully adopted the hybrid concept to
generate new antimalarial agents in which 4ꢀaminoquinoline
4ꢀaminoquinolineꢀpyrimidine (7aꢀ7g, 8aꢀ8g, 9aꢀ9g) hybrids in
which the secondary cyclic amines were replaced by substituted
anilines at the pyrimidine nucleus (Fig. 2). The docking studies
were also performed in the binding site of P. falciparum
moiety was covalently attached to different pharmacophores 45 dihydrofolate reductase (PfꢀDHFR) to investigate the interaction
present in other antimalarial agents.34ꢀ38 Recently, a series of
of these hybrids in PfꢀDHFR protein.
Fig. 2 Design strategy for the synthesis of novel 4ꢀaminoquinolineꢀpyrimidine hybrids
o
Chemistry
60 different substituted anilines at 0 C to room temperature in the
presence of triethylamine using THF as a solvent (scheme 2).42,43
The reaction of pyrimidine with substituted anilines yielded two
regioꢀisomers 5aꢀ5g as a major and 6aꢀ6g as minor isomers. The
major products were separated by column chromatography.
50 Synthesis of aminoquinolineꢀpyrimidine conjugates was carried
out as outlined in schemes 1ꢀ3. Firstly, N1ꢀ(7ꢀchloroquinolinꢀ4ꢀ
yl)ethaneꢀ1,2ꢀdiamine (2a), N1ꢀ(7ꢀchloroquinolinꢀ4ꢀyl)propaneꢀ
1,3ꢀdiamine (2b) and N1ꢀ(7ꢀchloroquinolinꢀ4ꢀyl)butaneꢀ1,4ꢀ
diamine (2c) were synthesized by the reaction of 4,7ꢀ 65 Finally the 4ꢀaminoquinolines (2aꢀ2c) with free NH2 group were
55 dichloroquinoline (1) with the excess of ethaneꢀ1,2ꢀdiamine,
propaneꢀ1,3ꢀdiamine and butaneꢀ1,4ꢀdiamine, respectively under
neat condition at 120 oC (scheme 1).41 The substituted
pyrimidines were synthesized by the reaction between
commercially available 2,4ꢀdichloroꢀ6ꢀmethylpyrimidine (3) with
coupled with aniline substituted pyrimidines (5aꢀ5g) in the
presence of K2CO3 and using Nꢀmethyl pyrrolidone (NMP) as
solvent at reflux condition to give 4ꢀaminoquinolineꢀpyrimidine
hybrids (7ꢀ9) (scheme 3).
70
Scheme 1
2
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Scheme 2
5
Scheme 3
Biological activity
mammalian cells was also determined. The assay was performed
in 96ꢀwell tissue cultureꢀtreated plates as described earlier.46 Vero
cells (monkey kidney fibroblasts) were seeded to the wells of 96ꢀ
well plate at a density of 25,000 cells/well and incubated for 24 h.
35 Compounds at different concentrations were added and plates
were again incubated for 48 h. The number of viable cells was
determined by Neutral Red assay. The IC50 values were obtained
from dose response curves.
In Vitro antimalarial activity
The antimalarial activity was determined by measuring
10 plasmodial LDH activity as described in the literature.44
A
suspension of red blood cells infected with D6 or W2 strain of P.
falciparum (200 µL, with 2% parasitemia and 2% hematocrit in
RPMI 1640 medium supplemented with 10% human serum and
60 µg/mL amikacin) was added to the wells of a 96ꢀwell plate
15 containing 10 µL of serially diluted test samples. The plate was
flushed with a gas mixture of 90% N2, 5% O2, and 5% CO2 and
incubated at 37 ºC, for 72 h in a modular incubation chamber
(BillupsꢀRothenberg, CA). Parasitic LDH activity was
determined according to the procedure of Makler and Hinrichs.45
20 Briefly, 20 µL of the incubation mixture was mixed with 100 µL
of the MalstatTM reagent (Flow Inc., Portland, OR) and incubated
at room temperature for 30 min. Twenty microliters of a 1:1
mixture of NBT/PES (Sigma, St. Louis, MO) was then added and
the plate is further incubated in the dark for 1 h. The reaction was
25 then stopped by the addition of 100 µL of a 5% acetic acid
solution. The plate was read at 650 nm. Chloroquine and
pyrimethamine were included in each assay as antimalarial drug
controls. IC50 values were computed from the dose response
curves. To determine the selectivity index of antimalarial activity
30 of compounds, in vitro cytotoxicity of these compounds against
Heme binding studies
40 Chloroquine and other 4ꢀaminoquinoline are believed to exert
their antimalarial activity by binding with heme and thereby
interfering with the formation of hemozoin through the π−π
stacking interaction of the quinoline ring with the porphyrin
ring.47,48 This can be demonstrated in in vitro by showing the
45 capability of chloroquine to inhibit the formation of βꢀhematin, a
process that is similar to the hemozoin synthesis within the
parasite food vacuole.49 Cohen et al were the first to show that
CQ forms a complex with ferriprotoporphyrin IX (FPIX) in the
aqueous solution, which was demonstrated on the basis of red
50 shifts observed in the heme absorbance data in the presence of a
drug.50 Since then several studies have explained the quinolineꢀ
FPIX complex formation by computational methods as well as
the spectroscopic methods.51ꢀ53 The FPIX exists either as a
monomer, µꢀoxoꢀdimer or an aggregate of µꢀoxoꢀdimers, and the
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relative proportions of these species are pHꢀdependent.54,55 55 Results and Discussion
Recently it has been determined that CQ forms complexes with
The aminoquinolineꢀpyrimidine hybrids were evaluated for their
in vitro antimalarial activity against both CQꢀsensitive (D6 clone)
and CQꢀresistant (W2 clone) strains of P. falciparum using
chloroquine and pyrimethamine as reference drugs (Table 1).
60 Cytotoxicity was also determined against Vero cell lines (Table
1). Most of the compounds showed potent antimalarial activity.
Eight compounds (7a, 7f, 7g, 8d, 8f, 8g, 9d and 9b) exhibited
antimalarial activity with IC50 < 0.05 µM, whereas one compound
8f was found to be equally potent to chloroquine against CQꢀ
65 sensitive strain. All the compounds except compounds 9c and 7e
showed better activity than chloroquine against CQꢀresistant
strain (W2 clone). Compounds which showed better activity
against CQꢀsensitive strain also found to possess good activity
against CQꢀresistant strain.
both monomeric and ꢁꢀoxoꢀdimeric FPIX.29,56,57 Therefore, we
decided to evaluate the binding of the most potent compound 8d
from the series with heme.
5
Docking Studies
The PfꢀDHFR has been considered as an important target for the
antimalarial drug discovery. Several antimalarial drugs such as
pyrimethamine and cycloguanil exhibit antimalarial activity due
10 to their ability to inhibit dihydrofolate reductase enzyme. In the
present manuscript, we have reported molecular docking studies
of the most active hybrids using wild type and quadruple mutant
PfꢀDHFRꢀTS (N51I, C59R, S108N, I164L) to study the inhibitory
effect of these compounds on wild and mutant type PfꢀDHFR.
15 Clinical isolates of P. falciparum resistant to antifolates such as
pyrimethamine and cycloguanil is caused by various combinations
of four point mutations in the active site of DHFR domain of Pfꢀ
DHFRꢀTS. In the quadruple mutant protein, the first mutation
occur at codon 108 (S108N), followed by codon 59 (C59R) and
20 codon 51 (N51I), finally at codon 164 (I164L). The effect of
quadruple mutations (N51I, C59R, S108 N, I164L) is ascribed to
the movement of residues in the active site of DHFR and
interferes in the inhibitor binding.58 For docking, molecular
structures of all the compounds were drawn using ChemBioDraw
25 Ultra 12.0 (www.cambridgesoft.com). These structures were then
imported into Maestro implemented in Schrödinger and Ligprep
module was used to generate energy minimized 3D structures.
The possible Lewis structure, tautomers and ionization states (pH
7.0 +/− 2.0) for each of these compounds were generated and
30 optimized with default settings provided in the LigPrep module
(Ligprep 2.5, Schrödinger, LLC, New York, NY, 2012). Partial
atomic charges were computed using the OPLS_2005 force field.
The crystal structures of wild type PfꢀDHFRꢀTS (PDB ID: 3QGT;
resolution 2.30 Å) and qradruple mutant (N51I+C59R+S108N+
35 I164L) PfDHFRꢀTS (PDB ID: 3QG2; resolution: 2.30 Å)
complexed with pyrimethamine was extracted from protein data
bank (www.rcsb.org). Protein Preparation Wizard (Maestro 10.0
Schrödinger, LLC, New York, NY, 2012) was used to prepare
proteins for docking. Water molecules within 5 Å of the protein
40 structures were considered. Bond order and formal charges were
assigned and hydrogen atoms were added to the crystal structure.
Further to refine the structure OPLSꢀ2005 force field parameter
was used to remove the steric clashes and the minimization was
terminated when RMSD reached maximum cut off value of 0.30 Å.
70 Table 1: In-vitro antimalarial activity and cytotoxicity of aminoquinolineꢀ
pyrimidine conjugates
P. falciparum
P. falciparum
Cytotoxicity
(Vero cells)
(D6 Clone)
(W2 Clone)
Com
p
IC50
IC50
SI
SI
IC50 (µM)
(µM)
0.049
0.052
0.050
0.056
0.360
0.041
0.045
0.051
0.058
0.060
0.043
0.350
0.034
0.046
0.074
0.047
4.589
0.045
0.328
0.054
0.076
0.035
0.01
(µM)
0.142
0.046
0.056
0.183
0.403
0.051
0.050
0.075
0.107
0.094
0.048
0.349
0.060
0.079
0.070
0.047
6.502
0.126
0.267
0.063
0.082
0.367
NA
7a
7b
7c
7d
7e
7f
>239.79
>216.34
>216.60
>175.53
>31.55
>266.82
>227.33
>222.74
>187.75
>174.83
>222.32
29.85
>82.74
>244.56
>193.39
>53.71
>28.18
>214.50
>204.60
>151.46
>101.77
>111.59
>199.16
29.94
>11.75
>11.25
>10.83
>9.83
>11.36
>10.94
>10.23
>11.36
>10.89
>10.49
>9.56
10.45
7g
8a
8b
8c
8d
8e
8f
>311.76
209.13
>148.51
>224.46
>2.22
>176.66
121.77
>157.0
>224.46
>1.56
>10.60
9.62
8g
9a
9b
9c
9d
9e
9f
>10.99
>10.55
>10.18
>9.29
9.09
>206.44
27.71
>73.73
34.04
45 In silico ADMET prediction
The pharmacokinetic profile of the test compounds showing good
antimalarial activity were predicted by using programs Qikprop
v3.5 (Schrödinger, Inc., New York, NY, 2012). All the
compounds prepared by LigPrep were used for the calculation of
50 pharmacokinetic properties by QikProp. The program QikProp,
utilizes the method of Jorgensen59 to compute pharmacokinetic
properties and descriptors such as octanol/water partitioning
coefficient, aqueous solubility, brain/blood partition coefficient,
intestinal wall permeability, plasma protein binding and others.
>190.37
110.78
>212.57
–
>163.17
102.68
>20.27
–
>10.28
8.42
9g
CQ
Pyr
>7.440
NT
a IC50 the concentration that causes 50% growth inhibition; S.I. selectivity
index (IC50 for cytotoxicity to Vero cells/IC50 for antimalarial activity);
NA: Not active up to 19 µM; NT: Not tested; Pyrꢀpyrimethamine.
4
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For particular aniline substituted 4ꢀaminoquinoineꢀpyrimidine 45 days for chloroquine treated animals.
conjugates (7aꢀ9a or 7bꢀ9b or 7cꢀ9c or 7dꢀ9d or 7eꢀ9e or 7fꢀ9f
Table 2: In Vivo antimalarial activity of compound 7f in P. bergheiꢀ
or 7gꢀ9g), no obvious trend of activity was observed with
increasing or decreasing the length of carbon chain linker.
Compound having phenyl ring at pyrimidine nucleus (7a, 8a, 9a)
showed antimalarial activity in the range of 0.049ꢀ0.074 µM (D6)
and 0.075ꢀ0.142 µM (W2) with high selectivity index and
compound with ethylene linker (7a) was found to be most active
against CQꢀsensitive strain while compound with butylene linker
mouse malaria model
Treatment Dose (mg/kg × % Parasitemia
MSTb
Toxicity
5
(PO)
no. of days
postꢀinfection)
NA × 3
suppressiona
Day 5
Day 7
ꢀ
Vehicle
CQ
ꢀ
7.6
26.2
7.6
7
NA
NC
NC
NC
NC
100 × 3
100
100
10 (9a) was the most active against CQꢀresistant strain. When
phenyl ring was substituted with halogen groups (7bꢀ7d, 8bꢀ8d,
9bꢀ9d), most of the compounds showed potent antimalarial
activity against both the strains with exception of compound 9c in
which chloro group is present at para position of the phenylene
15 ring attached to pyrimidine ring and spacer is butylene but same
compound with ethylene linker (7c) showed improved
antimalarial activity against both the strains. Compounds with
methyl group at para position of the phenyl ring (7e, 8e, 9e) led
to partial decrease in the antimalarial activity against both the
20 starins, while methoxy substitution at 4ꢀposition (7f, 8f, 9f) and
3,5ꢀpositions (7g, 8g, 9g) of the phenyl ring led to increase in the
antimalarial activity with all the compounds more active against
CQꢀsensitive strain of P. falciparum. Compound 8f having 4ꢀ
OCH3 group at phenyl ring with propylene linker was found to be
25 the most potent against CQꢀsensitive strain with IC50 value of
0.034 µM. While compound 7b with 4ꢀF group and ethylene
linker displayed the most potent activity against CQꢀresistant
strain with IC50 value of 0.046 µM. Cytotoxicity was also
determined against Vero cell lines (Table 1). All the compounds
30 showed toxicity at very high concentration as compared to their
concentrations (IC50) responsible for their antimalarial activity.
(Table 1).
Comp 7f
11.1 × 3
17.85
37.62
96.42
11.07
12.79
2.68
33.3 × 3
100 × 3
12.2
a
% suppression in parasitemia is calculated by considering the mean
parasitemia in the vehicle control as 100%; bMST ꢀ mean survival time (days);
50 NAꢀNot active; NCꢀ No cytotoxicity up to highest concentration tested
In order to understand the primary mode of action of these
hybrids, heme binding studies were performed using compound
8d. A solution of hematin in 40% DMSO/water shows an
absorption band at 402 nm, indicating the presence of monomeric
55 heme, Fe(III) PPIX, under the conditions used (0.02 M HEPES
buffer, pH 7.4 and 0.02 M MES buffer, pH 5.6). When compound
8d was added into a constant concentration of monomeric heme
(5.0 ꢁM), a substantial decrease in the intensity of the absorption
band at 402 nm was observed with no shift in the absorption
60 maxima. This indicates the association of compounds with
hematin. The stoichiometry ratio of the most stable complexes of
compound 8d with monomeric heme at pH 7.4 and 5.6 was
inferred from the Job’s plot. The absorbance at 402 nm got
maximum value when mole fraction of compound was
65 approximately 0.5, which confirms the association of compound
with heme in 1:1 ratio at both the pH values (Figure 4).
Compound 7f with significant activity in vitro was selected for
further in vivo evaluation. In vivo antimalarial activity was
35 determined through oral route of administration in P. bergheiꢀ
mouse malaria model. The compounds were administered to the
P. berghei infected mice, through oral gavage, once daily on days
0, 1 and 2 post infection and monitored for apparent signs of
toxicity, parasitemia and survival till day 28 post infection (Table
40 2). It was found that the compound 7f causes 17.85, 37.62 and
96.42% parasite suppression at 11.1, 33.3 and 100 mg/kg doses
on day 5 as compared to 100% suppression displayed by CQ. On
day 7, the effect almost disappeared at the highest dose level and
the mean survival time was only 12.2 days as compared to 26.2
As discussed earlier that CQ and its derivatives also bind to heme
dimer (µꢀoxoꢀheme). Therefore, the binding of compound 8d was
also studied with ꢁꢀoxo dimer of heme at pH 5.8. In aqueous
70 NaOH solution heme exists as ꢁꢀoxoꢀdimer and shows an
absorption band at 362 nm. Addition of compound 8d (0−20 ꢁM)
to a solution of ꢁꢀoxo dimer (10 ꢁM) in 20 mM phosphate buffer
at pH 5.8 resulted in a decrease in intensity of absorbance at 362
nm (Figure 5), which shows the interaction between heme and
75 compound 8d. The Job’s plot indicated a 1:1 stoichiometry for
the most stable complex formed between ꢁꢀoxo heme and
compound 8d (Figure 5).
1
1
A
B
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
350
370
390
410
430
450
350
370
390
410
430
450
Wavelength
Wavelength
Fig. 3 A) Titration of compound 8d with monomeric heme at pH 7.4; B) Titration of compound 8d with monomeric heme at pH 5.6
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0.1
0.08
0.06
0.04
0.02
0
0.014
0.012
0.01
A
B
0.008
0.006
0.004
0.002
0
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
X
X
Fig. 4 Job’s plot of monomeric heme complex formation with compound 8d; A) at pH 7.4; B) at pH 5.6; X (mole fraction of compound 8d) = [compd
8d]/[compd 8d]+[ heme]; A0 is the absorbance, when x = 1 and A is the absorbance at respective values of x
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0.05
0.04
0.03
0.02
0.01
0
A
B
300
350
400
450
500
0
0.2
0.4
0.6
0.8
1
Wavelength
X
5
Fig. 5 A) Titration of compound 8d with ꢁꢀoxodimeric heme at pH 5.8; B) Job’s plot of ꢁꢀoxodimeric heme complex formation with compound 8d at pH 5.8
The association constants for the complexes formed between
most active compounds in the study exhibited significant binding
monomeric Fe(III) PPIX and compound 8d at pH 7.4 and 5.6 35 affinities towards the wild (Glide energy range ꢀ65.11 kcalmolꢀ1
were calculated by the analysis of titration data and the results are
to ꢀ37.00 kcalmolꢀ1) and quadruple mutant (Glide energy range ꢀ
72.10 kcalmolꢀ1 to ꢀ36.43 kcalmolꢀ1) PfꢀDHFRꢀTS structures and
the energy ranges are comparable to that of reference compounds
(pyrimethamine, cycloguanil and WR99210) and the native
10 shown in Table 3. The association constant at pH 7.4 (log K
5.048) was comparable to that of the standard antimalarial drug
CQ (log K 5.15). Furthermore, decreasing the pH from 7.4 to 5.6
(food vacuole pH), compound displayed improved binding 40 substrate of DHFR dihydrofolate (Table 4).
constant (log K 5.272) indicating that binding is stronger even at
Table 3 Binding constants for compound 8d and chloroquine with heme
15 acidic pH of food vacuole. Interestingly, at pH 5.6, compound
showed large value of binding constant than the standard drug
chloroquine. The association constants for the binding with µꢀ
oxoꢀheme at pH 5.8 (log K 5.171) was found to be even greater
than that of monomeric heme complexes at pH 7.4. From the data
20 shown in table 3, it is clear that the compound 8d binds strongly
with monomeric heme (log K 5.272) as well as ꢁꢀoxoꢀheme (log
K 5.171) and the observed results are comparable to the standard
CQ (log K 5.58). Thus the formation of complex between heme
and compound 8d suggests the inhibition of formation of βꢀ
25 hematin, which could be correlated to the observed antimalarial
activity of these compounds.
The mode of action of these 4ꢀaminoquinolineꢀpyrimidine
hybrids were further substantiated by molecular docking studies
into the binding pocket of wild type PfꢀDHFRꢀTS and quadruple
30 mutant PfꢀDHFRꢀTS (N51I, C59R, S108 N, I164L). The results
of molecular docking of active compounds in the binding site of
both wild type and mutant PfꢀDHFRꢀTS are shown in Table 4.
The Glide XP Gscores and glide energies clearly indicate that the
Comp
Monomeric heme Monomeric heme µꢀoxoꢀheme
log K (pH = 5.6) log K (pH = 7.4) log K (pH = 5.8)
8d
5.272
4.65a
5.048
5.15a
1:1
5.171
5.58a
1:1
CQ
Stoichiometry 1:1
a See text reference 29
Figure 6 and 7 represents the binding mode of the two best
selected active compounds 7f and 8d having high Glide XP
45 scores and low Glide binging energies. These compounds occupy
similar binding pocket as the native PfꢀDHFR substrate
dihyrofolate. Compound 7f showed lowest binding energy (ꢀ
65.11 kcalmolꢀ1) and considerably high Glide XP score (ꢀ8.91
kcalmolꢀ1) for wild type PfꢀDHFR and ꢀ72.10 and ꢀ8.59
50 respectively for mutant type PfꢀDHFR. A hydrogen bond
interaction was observed between NH group of ethylene linker
6
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attached to pyrimidine nucleus and oxygen side chain of Asp54
of both wild and mutant PfꢀDHFR. An additional Hꢀbond
interaction was also observed between the NH group attached
type, one between the aromatic ring of Phe116 and pyridine ring
of quinoline nucleus and the other between the aromatic ring of
Phe58 and the pyrimidine aromatic ring. In mutant type Pfꢀ
between pyrimidine and methoxy substituted phenyl ring, and 10 DHFR, a πꢀπ interaction was observed between the aromatic ring
5
main chain oxygen of Ile164 in wild type PfDHFR. (Fig. 6).
of Phe58 and the methoxy substituted phenyl ring.
Further, compound 7f showed two πꢀπ interactions in case of wild
Table 4 Glide docking scores (kcal molꢀ1) and docking energies of best active molecules along with the reference compounds (pyrimethamine,
cycloguanil and WR99210) and dihydrofolate bound to wild and mutant PfDHFRꢀTS binding site
Compounds
Docking results with wild PfDHFR
XP GScore Van der waals Coulumbic Glide Energy XP GScore Van der waals
energy
ꢀ48.64
Docking results with mutant PfDHFR
Coulumbic
Energy
ꢀ16.87
ꢀ21.86
ꢀ10.44
ꢀ8.44
Glide Energy
Energy
ꢀ16.47
ꢀ12.83
ꢀ15.60
ꢀ11.92
ꢀ10.18
ꢀ14.57
ꢀ4.45
energy
ꢀ51.62
ꢀ50.23
ꢀ32.68
ꢀ27.99
ꢀ28.46
ꢀ46.82
ꢀ46.99
ꢀ37.44
ꢀ48.20
ꢀ25.06
ꢀ43.68
ꢀ33.65
ꢀ34.30
ꢀ27.37
7f
8f
9f
7g
8g
7c
8d
9d
ꢀ8.91
ꢀ6.94
ꢀ6.89
ꢀ6.82
ꢀ5.88
ꢀ4.28
ꢀ7.61
ꢀ6.21
ꢀ5.82
ꢀ5.61
ꢀ9.33
ꢀ65.11
ꢀ56.90
ꢀ44.85
ꢀ37.60
ꢀ37.97
ꢀ51.59
ꢀ62.37
ꢀ37.00
ꢀ41.48
ꢀ37.77
ꢀ64.84
ꢀ44.91
ꢀ38.55
ꢀ37.03
ꢀ8.59
ꢀ7.13
ꢀ6.89
ꢀ6.60
ꢀ5.80
ꢀ4.20
ꢀ8.07
ꢀ4.77
ꢀ5.32
ꢀ5.32
ꢀ11.00
ꢀ9.39
ꢀ8.95
ꢀ5.48
ꢀ72.10
ꢀ61.18
ꢀ43.12
ꢀ36.43
ꢀ37.60
ꢀ51.51
ꢀ64.28
ꢀ45.13
ꢀ68.49
ꢀ37.99
ꢀ61.30
ꢀ43.55
ꢀ46.60
ꢀ34.30
ꢀ44.06
ꢀ29.25
ꢀ25.68
ꢀ27.78
ꢀ47.79
ꢀ47.13
ꢀ23.47
ꢀ34.43
ꢀ26.43
ꢀ52.14
ꢀ31.70
ꢀ30.12
ꢀ51.18
ꢀ9.13
ꢀ4.69
ꢀ17.28
ꢀ7.69
ꢀ13.52
ꢀ7.05
7b
9b
ꢀ12.98
ꢀ12.92
ꢀ17.61
ꢀ12.06
ꢀ8.60
ꢀ11.34
ꢀ14.19
ꢀ15.51
ꢀ10.74
ꢀ6.91
Dihydrofolate
Pyrimethamine ꢀ9.04
Cycloguanil
WR99210
ꢀ8.94
ꢀ4.84
ꢀ8.07
15
Fig. 6 2D and 3D docking pose showing interaction for compounds 7f in the binding site of (A) mutant type PfꢀDHFRꢀTS (PDB ID: 3QG2) and (B) wild
type PfꢀDHFRꢀTS (PDB ID: 3QGT).
20
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Fig. 7 2D and 3D docking pose showing interaction for compounds 8d in the binding site of (A) mutant type PfꢀDHFRꢀTS (PDB ID: 3QG2) and (B) wild
type PfꢀDHFRꢀTS (PDB ID: 3QGT).
Another compound predicted to have low binding energy (ꢀ62.37
Table 5 Prediction of Lipinski’s ‘Rule of 5’ for the active test
5
kcalmolꢀ1) and high glide score (ꢀ7.61) was 8d, showing similar 30 compoundsa
Comp mol_MW donor
accpt
HB
(<10)
QPlogPo/w
(<5)
‘N’ of
violations
(<2)
0
Hꢀbond pattern between NH group of propylene linker of
compound and oxygen side chain of Asp54. The πꢀπ interactions
were also observed between the two aromatic rings (4ꢀ
bromophenyl and pyrimidine ring) of compound 8d and the
10 aromatic ring of Phe58 of mutant PfꢀDHFR. In wild type Pfꢀ
DHFR, a πꢀπ interaction was observed between the aromatic ring
of Phe116 and 4ꢀbromophenyl ring attached to pyrimidine
nucleus. Also, the compound 8d forms Hꢀbond interaction
through its NH group of propylene linker attached to the
15 aminoquinoline ring with the main chain oxygen atom of Ile164
in the wild type protein binding site.
Pharmacokinetic parameters of best active compounds were
calculated using ADMET predictions by Qikprop v3.5. The most
important of these parameters together with its permissible ranges
20 are listed in the Table 5 and Table S1. Qikprop results for the
Lipinski’s rule of 5 parameters, a preliminary test of the drugꢀ
likeness of the compounds is presented in Table 5. An orally
active compound should not have more than 2 violations of these
rules.60 In the present study, all the active test compounds showed
25 values for Lipinski’s rule of 5 violations less than the maximum
permissible value of 2, indicating that these active test
compounds are having good drug likeness properties.
(<500
amu)
HB
(<5)
3
3
3
3
3
3
3
3
3
3
4
7f
8f
9f
7g
8g
434.92
448.95
462.98
464.95
478.98
425.32
497.82
511.85
422.89
450.94
248.71
253.73
6
6
6
6
6
5
5
5
5
5
3
3
4.316
5.288
5.217
4.475
5.445
4.365
5.366
5.436
4.763
5.503
1.809
0.888
1
1
0
1
0
1
2
0
7c
8d
9d
7b
9b
Pyr
Cyg
1
0
0
5
a
All values calculated by QikProp v 3.5 and the explanations of
the descriptors are given in the text.
The optimum values of descriptors such as number of rotable
bonds (<15) and polar surface area (7 Å2 – 200 Å2) can also have
35 significant influence on oral bioavailability of compounds.61 In
the present study, all the test compounds possess a number of
rotatable bonds <15 and polar surface area in the permissible
range (7 Å – 200 Å). Similarly, the test molecules were checked
for their intestinal absorption or permeation property, which is
40 confirmed by the predicted Cacoꢀ2 cells permeability (QPPCaco),
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used as a model for the gut–blood barrier.62 QPPCaco predictions 55 71%; mp 242ꢀ244 oC; IR (Film, cmꢀ1): 3274, 3100, 2921, 1636,
1
for all the test compounds showed values >500. Further,
QPlogKhsa, the prediction for human serum albumin binding
were calculated and all inhibitors were predicted to lie within the
expected range for 95% of known drugs (–1.5 to 1.5). The
aqueous solubility (QPlogS) parameters for the test compounds
were assessed and all the compounds were predicted to have
QPlogS values in the permissible range (–6.5 to 0.5).
Furthermore, QPlogHERG descriptor for the prediction of IC50
1559, 1525, 1495, 1458, 1236, 1045, 828; H NMR (400 MHz,
CDCl3): δ 2.34 (s, 3H, CH3), 6.45 (s, 1H, ArH), 6.98 (brs, 1H,
NH), 7.28ꢀ7.30 (m, 3H, ArH), 7.39ꢀ7.43 (m, 2H, ArH); ESIꢀMS
(m/z) calculated for C11H10ClN3: 219.05, found: 220.16 (M + H)+.
5
60 2ꢀChloroꢀNꢀ(4ꢀfluorophenyl)ꢀ6ꢀmethylpyrimidinꢀ4ꢀamine (5b):
Yield 68%; mp 147ꢀ150 oC; IR (Film, cmꢀ1): 3229, 3081, 3003,
1
1591, 1507, 1418, 1370, 1276, 1212, 1156, 1033, 974, 914; H
NMR (400 MHz, CDCl3): δ 2.32 (s, 3H, CH3), 6.29 (s, 1H, ArH),
7.09ꢀ7.13 (m, 2H), 7.17 (brs, 1H, NH), 7.27ꢀ7.30 (m, 2H, ArH);
65 13C NMR (100 MHz, CDCl3): δ 23.90, 100.52, 116.39, 126.13,
133.04, 160.18, 160.89, 163.34, 168.87; ESIꢀMS (m/z) calculated
for C11H9ClFN3: 237.04, found: 238.12 (M + H)+.
10 value of HERG K+ channel blockage was predicted for the test
compounds. Compounds 7f and 7g have been predicted to
possess values for in QPlogHERG less than the value of concern
(–5), comparable to reference compounds pyrimethamine and
cycloguanil (Table S1).
2ꢀChloroꢀNꢀ(4ꢀchlorophenyl)ꢀ6ꢀmethylpyrimidinꢀ4ꢀamine (5c):
Yield 75%; mp 129ꢀ131 oC; IR (Film, cmꢀ1): 3290, 3208, 3161,
70 3075, 1606, 1584, 1492, 1412, 1369, 1274, 1240, 1217, 1176,
15 Experimental Section
All the chemicals were purchased from SigmaꢀAldrich. Solvents
used for the chemical synthesis were of analytical grade and used
without further purification. Thin layer chromatography (Merck
Kiesel 60 F254, 0.2 mm thickness) was used to monitor the
20 progress of the reactions and the compounds were purified by
silica gel (60ꢀ120 mesh) column chromatography. IR spectra
were recorded on Perkinꢀelmer FTꢀIR spectrophotometer using
KBr pellets or as film in chloroform and the values were
1
1090, 1031, 974; H NMR (400 MHz, CDCl3): δ 2.34 (s, 3H,
CH3), 6.38 (s, 1H, ArH), 7.22 (brs, 1H, NH), 7.27 (d, J = 8.79 Hz,
2H, ArH), 7.37 (d, J = 8.79 Hz, 2H, ArH); 13C NMR (100 MHz,
CDCl3): δ 23.91, 101.04, 124.56, 129.66, 131.05, 135.86, 160.22,
75 162.70, 168.98; ESIꢀMS (m/z) calculated for C11H9Cl2N3: 253.01,
found: 254.13 (M + H)+.
Nꢀ(4ꢀBromophenyl)ꢀ2ꢀchloroꢀ6ꢀmethylpyrimidinꢀ4ꢀamine (5d):
Yield 78%; mp 141ꢀ144 oC; IR (Film, cmꢀ1): 3291, 3204, 3158,
3078, 2924, 1603, 1580, 1488, 1409, 1368, 1274, 1238, 1216,
80 1177, 1074, 1031, 974, 915; 1H NMR (400 MHz, CDCl3): δ 2.35
(s, 3H, CH3), 6.39 (s, 1H, ArH), 7.19 (brs, 1H, NH), 7.22 (d, J =
8.79 Hz, 2H, ArH), 7.52 (d, J = 8.79 Hz, 2H, ArH); 13C NMR
(100 MHz, CDCl3): δ 24.03, 95.83, 116.79, 123.54, 132.11,
138.87, 159.37, 161.23, 167.25; ESIꢀMS (m/z) calculated for
85 C11H9BrClN3: 296.96, found: 298.04 (M + H)+.
1
expressed in cmꢀ1. The H NMR (400 MHz) and 13C NMR (100
25 MHz) spectra were recorded on Jeol ECX spectrospin instrument
using CDCl3 or DMSOꢀd6 as solvent and TMS as internal
reference. The chemical shift values were expressed on δ scale
and the coupling constant (J) in Hz. Melting points were recorded
on EZꢀMelt automated melting point apparatus, Stanford
30 Research Systems and are uncorrected. Mass data were recorded
in JeolꢀAccu TOF JMSꢀT100LC mass spectrometer.
2ꢀChloroꢀ6ꢀmethylꢀNꢀpꢀtolylpyrimidinꢀ4ꢀamine (5e): Yield 76%;
Typical procedure for the synthesis of N1ꢀ(7ꢀchloroquinolinꢀ4ꢀ
yl)ethaneꢀ1,2ꢀdiamine (2a) and related compounds (2b and
2c): A mixture of 4,7ꢀdichloroquinoline (1, 5.0 g, 0.025 mol) and
35 1,2ꢀethylene diamine (5.8 g, 0.125 mol) was heated slowly from
RT to 120 oC and the reaction mixture was stirred at this
temperature for 6 h (scheme 1). Reaction mixture was cooled
down to room temperature and ice cold water added to it. The
solid thus obtained was filtered and washed with excess of water.
40 The crude product was crystallized by using ethanol and the data
corresponds to that reported in the literature.
mp 152ꢀ154 oC; IR (Film, cmꢀ1): 3289, 3176, 2921, 1653, 1559,
1
1439, 1358, 1298, 1237, 1175, 1096, 1031, 974; H NMR (400
MHz, CDCl3): δ 2.31 (s, 3H, CH3), 2.37 (s, 3H), 6.36 (s, 1H,
90 ArH), 7.05 (brs, 1H, NH), 7.15 (d, J = 8.05 Hz, 2H, ArH), 7.21
(d, J = 8.05 Hz, 2H, ArH); ESIꢀMS (m/z) calculated for
C12H12ClN3: 233.07, found: 234.21 (M + H)+.
2ꢀChloroꢀNꢀ(4ꢀmethoxyphenyl)ꢀ6ꢀmethylpyrimidinꢀ4ꢀamine
o
(5f): Yield 75%; mp 170ꢀ172 C; IR (Film, cmꢀ1): 3214, 3087,
95 2924, 1591, 1510, 1420, 1394, 1368, 1276, 1241, 1223, 1173,
1034, 972, 912; 1H NMR (400 MHz, CDCl3): δ 2.29 (s, 3H,
CH3), 3.83 (s, 3H, 2×OCH3), 6.23 (brs, 1H, NH), 6.94 (d, J = 8.79
Hz, 2H, ArH), 7.19 (d, J = 8.79 Hz, 3H, ArH); 13C NMR (100
MHz, CDCl3): δ 23.78, 55.42, 100.20, 114.73, 126.45, 129.67,
100 157.98, 159.99, 163.97, 168.38; ESIꢀMS (m/z) calculated for
C12H12ClN3O: 249.06, found: 250.18 (M + H)+.
Typical procedure for the synthesis of 2ꢀchloroꢀNꢀ
phenylpyrimidinꢀ4ꢀamine (5a) and related compounds (5bꢀ
5g): To a solution of 2,4ꢀdichloropyrimidine (3, 2.0 g, 0.013 mol)
o
45 and triethylamine (1.63 g, 0.016 mol) in ethanol at 0 C, aniline
(4a, 1.2 g, 0.013 mol) was added (scheme 2). The reaction
mixture was stirred overnight at room temperature. After
completion of the reaction as observed by TLC, excess ethanol
was evaporated and the reaction mixture was diluted with water.
50 The solid thus obtained was filtered and washed with excess
water. The crude product was purified by column
chromatography using EtOAc/Hexane as eluent to afford pure
compound 5a.
2ꢀChloroꢀNꢀ(3,5ꢀdimethoxyphenyl)ꢀ6ꢀmethylpyrimidinꢀ4ꢀ
amine (5g): Yield 70%; mp 185ꢀ188 oC; IR (Film, cmꢀ1): 3293,
1
3092, 2925, 1599, 1526, 1474, 1339, 1205, 1154, 1063, 829; H
105 NMR (400 MHz, CDCl3): δ 2.34 (s, 3H, CH3), 3.80 (s, 6H,
2OCH3), 6.33 (brs, 1H, NH), 6.46 (d, J = 1.46 Hz, 2H), 6.51 (s,
1H), 7.12 (s, 1H); ESIꢀMS (m/z) calculated for C13H14ClN3O2:
279.07, found: 280.23 (M + H)+.
2ꢀChloroꢀ6ꢀmethylꢀNꢀphenylpyrimidinꢀ4ꢀamine (5a): Yield
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Typical procedure for the synthesis of N2ꢀ(2ꢀ((7ꢀ
chloroquinolinꢀ4ꢀyl)amino)ethyl)ꢀN4ꢀphenylpyrimidineꢀ2,4ꢀ
diamine (7a) and related compounds (7bꢀ7g, 8aꢀ8g and 9aꢀ
238ꢀ242 oC; IR (KBr, cmꢀ1): 3245, 3189, 3066, 2974, 2935, 1610,
1583, 1519, 1488, 1459, 1398, 1341, 1329, 1247, 1074, 1006,
1
906, 870, 804; H NMR (400 MHz, DMSOꢀd6): δ 2.15 (s, 3H,
9g): To a stirred solution of compound 2a (400 mg, 1.8 mmol) 60 CH3), 3.43ꢀ3.44 (m, 2H, NCH2), 3.55 (brs, 2H, NCH2), 5.90 (s,
5
and compound 5a (372 mg, 1.8 mmol) in Nꢀmethyl pyrrolidinone
(NMP), K2CO3 (750 mg, 5.4 mmol) was added. Reaction mixture
was stirred at 140 oC for 12 h (scheme 3). After completion of the
reaction, water was added to the reaction mixture and the product
1H, ArH), 6.79 (brs, 1H, NH), 6.94 (s, 1H, ArH), 7.37 (d, J = 8.7
Hz, 2H, ArH), 7.42 (dd, J = 2.2, 8.7 Hz, 2H, ArH), 7.68 (d, J =
6.5 Hz, 2H, ArH), 7.77 (d, J = 2.2 Hz, 1H, ArH), 8.17 (d, J = 8.7
Hz, 1H, ArH), 8.36 (brs, 1H, NH), 9.21 (brs, 1H, NH); 13C NMR
was extracted with chloroform (3 × 20 mL). The combined 65 (100 MHz, DMSOꢀd6): δ 23.72, 42.61, 98.85, 112.60, 117.98,
10 organic layer was dried over sodium sulphate and excess solvent
was evaporated under reduced pressure. The crude product was
purified by column chromatography using MeOH/CHCl3 as
eluent to afford pure compound 7a in quantitative yield.
121.04, 123.89, 124.06, 127.47, 131.25, 133.41, 140.16, 148.96,
150.18, 151.81, 160.89, 161.90; ESIꢀHRMS (m/z) calculated for
C22H20BrClN6: 482.0621, found: 482.9981 (M + H)+, 484.9908
(M + 2)+; Anal. calcd. for C22H20BrClN6: C, 54.62; H, 4.17; Br,
70 16.52; Cl, 7.33; N, 17.37, found: C, 54.71; H, 4.12; Br, 16.49; Cl,
7.19; N, 17.40.
N2ꢀ(2ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)ethyl)ꢀ6ꢀmethylꢀN4ꢀphenyl
15 pyrimidineꢀ2,4ꢀdiamine (7a): Yield 75%; mp 214ꢀ215o C; IR
(KBr, cmꢀ1): 3254, 3061, 2936, 2858, 1578, 1553, 1497, 1443,
N2ꢀ(2ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)ethyl)ꢀ6ꢀmethylꢀN4ꢀpꢀtolyl
1
o
1355, 1326, 1248, 1228, 1141, 908, 870; H NMR (400 MHz,
pyrimidineꢀ2,4ꢀdiamine (7e): Yield 80%; mp 214ꢀ215 C; IR
DMSOꢀd6): δ 2.15 (s, 3H, CH3), 3.42ꢀ3.44 (m, 2H, NCH2), 3.56
(KBr, cmꢀ1): 3253, 3062, 2936, 2859, 1644, 1576, 1508, 1447,
(brs, 2H, NCH2), 5.91 (s, 1H, ArH), 6.54 (brs, 1H, NH), 6.88ꢀ6.92 75 1407, 1355, 1326, 1291, 1247, 1233, 1139, 1084, 1033, 907, 870,
1
20 (m, 2H, ArH), 7.22 (t, J = 7.3 Hz, 2H, ArH), 7.41 (d, J = 8.0 Hz,
2H, ArH), 7.68 (d, J = 8.0 Hz, 2H, ArH), 7.77 (d, J = 2.2 Hz, 1H,
ArH), 8.16 (d, J = 9.5 Hz, 1H, ArH), 8.35 (brs, 1H, NH), 9.06 (s,
1H, NH); ESIꢀHRMS (m/z) calculated for C22H21ClN6: 404.1516,
814; H NMR (400 MHz, DMSOꢀd6): δ 2.13 (s, 3H, CH3), 2.20
(s, 3H, CH3), 3.40ꢀ3.44 (m, 2H, NCH2), 3.54 (brs, 2H, NCH2),
5.87 (s, 1H, ArH), 6.54 (brs, 1H, NH), 6.83 (s, 1H, ArH), 7.02 (d,
J = 7.3 Hz, 2H, ArH), 7.41 (d, J = 8.0 Hz, 2H, ArH), 7.53 (d, J =
found: 405.1381 (M + H)+, 407.1700 (M + 2)+; Anal. calcd. for 80 8.0 Hz, 2H, ArH), 7.77 (d, J = 2.2 Hz, 1H, ArH), 8.15 (d, J = 8.7
25 C22H21ClN6: C, 65.26; H, 5.23; Cl, 8.76; N, 20.76, found: C,
65.40; H, 5.20; Cl, 8.65; N, 20.82.
Hz, 1H, ArH), 8.34 (brs, 1H, NH), 8.94 (s, 1H, NH); 13C NMR
(100 MHz, DMSOꢀd6): δ 20.34, 23.72, 42.80, 98.82, 117.38,
119.63, 123.85, 123.98, 127.52, 129.0, 130.30, 133.34, 138.04,
149.02, 150.12, 151.89, 161.23, 162.04; ESIꢀHRMS (m/z)
85 calculated for C23H23ClN6: 418.1673, found: 419.1968 (M + H)+,
421.2253 (M + 2)+; Anal. calcd. for C23H23ClN6: C, 65.94; H,
5.53; Cl, 8.46; N, 20.06, found: C, 66.14; H, 5.70; Cl, 8.41; N,
19.82.
N2ꢀ(2ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)ethyl)ꢀN4ꢀ(4ꢀfluoro
phenyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine (7b): Yield 55%; mp
216ꢀ218 oC; IR (KBr, cmꢀ1): 3299, 3186, 3061, 3006, 1642, 1583,
30 1504, 1458, 1408, 1368, 1330, 1244, 1216, 1152, 1079, 1013,
1
906, 872, 821; H NMR (400 MHz, DMSOꢀd6): δ 2.14 (s, 3H,
CH3), 3.40ꢀ3.44 (m, 2H, NCH2), 3.54 (brs, 2H, NCH2), 5.87 (s,
1H, ArH), 6.53 (brs, 1H, NH), 6.88 (s, 1H, ArH), 7.05 (d, J = 8.7
N2ꢀ(2ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)ethyl)ꢀN4ꢀ(4ꢀmethoxy
Hz, 2H, ArH), 7.41 (d, J = 8.7 Hz, 2H, ArH), 7.68 (brs, 2H, 90 phenyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine (7f): Yield 65%; mp
35 ArH), 7.77 (d, J = 2.2 Hz, 1H, ArH), 8.16 (d, J = 9.5 Hz, 1H,
ArH), 8.35 (brs, 1H, NH), 9.08 (brs, 1H, NH); Anal. calcd. for
C22H20ClFN6: C, 62.48; H, 4.77; Cl, 8.38; F, 4.49; N, 19.87,
found: C, 62.43; H, 4.65; Cl, 8.44; F, 4.51; N, 19.90.
200ꢀ201oC; IR (KBr, cmꢀ1): 3248, 3057, 2929, 1582, 1509, 1409,
1354, 1245, 1166, 1139, 1033, 922, 874, 828, 795; 1H NMR (400
MHz, DMSOꢀd6): δ 2.11 (s, 3H, CH3), 3.37ꢀ3.43 (m, 2H, NCH2),
3.53ꢀ3.56 (m, 2H, NCH2), 3.65 (s, 3H, OCH3), 5.81 (s, 1H, ArH),
95 6.53 (brs, 1H, NH), 6.79ꢀ6.81 (m, 3H, ArH), 7.41 (d, J = 7.3 Hz,
2H, ArH), 7.52 (d, J = 8.7 Hz, 2H, ArH), 7.76 (d, J = 2.2 Hz, 1H,
ArH), 8.15 (d, J = 9.5 Hz, 1H, ArH), 8.34 (brs, 1H, NH), 8.85
(brs, 1H, NH); ESIꢀHRMS (m/z) calculated for C23H23ClN6O:
434.1681, found: 435.1748 (M + H)+, 437.1731 (M + 2)+; Anal.
100 calcd. for C23H23ClN6O: C, 63.52; H, 5.33; Cl, 8.15; N, 19.32; O,
3.68, found: C, 63.41; H, 5.46; Cl, 8.22; N, 19.10; O, 3.73.
N4ꢀ(4ꢀChlorophenyl)ꢀN2ꢀ(2ꢀ((7ꢀchloroquinolinꢀ4ꢀyl)amino)
40 ethyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine (7c): Yield 70%; mp
238ꢀ242 oC; IR (KBr, cmꢀ1): 3250, 3005, 2881, 1610, 1586, 1521,
1491, 1459, 1402, 1342, 1330, 1248, 1133, 1087, 1010, 906, 870,
1
818, 804; H NMR (400 MHz, DMSOꢀd6): δ 2.15 (s, 3H, CH3),
3.42ꢀ3.45 (m, 2H, NCH2), 3.55 (brs, 2H, NCH2), 5.89 (s, 1H,
45 ArH), 6.81 (brs, 1H, NH), 6.93 (s, 1H, ArH), 7.25 (d, J = 9.2 Hz,
2H, ArH), 7.42 (dd, J = 2.2, 8.7 Hz, 2H, ArH), 7.73 (d, J = 8.0
Hz, 2H, ArH), 7.77 (d, J = 2.2 Hz, 1H, ArH), 8.16 (d, J = 9.5 Hz,
1H, ArH), 8.35 (brs, 1H, NH), 9.20 (brs, 1H, NH); ESIꢀHRMS
N2ꢀ(2ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)ethyl)ꢀN4ꢀ(3,5ꢀdimethoxy
phenyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine (7g): Yield 50%; mp
229ꢀ231 oC;1H NMR (400 MHz, DMSOꢀd6): δ 2.13 (s, 3H, CH3),
(m/z) calculated for C22H20Cl2N6: 438.1127, found: 439.0452 (M 105 3.35 (brs, 2H, NCH2), 3.57 (brs, 2H, NCH2), 3.66 (s, 6H,
50 + H)+, 441.0577 (M + 2)+; 13C NMR (100 MHz, DMSOꢀd6): δ
23.72, 42.63, 98.83, 117.39, 120.63, 123.87, 124.03, 124.73,
127.51, 128.36, 133.39, 139.74, 149.0, 150.16, 151.83, 160.92,
161.92; Anal. calcd. for C22H20Cl2N6: C, 60.14; H, 4.59; Cl,
16.14; N, 19.13, found: C, 60.28; H, 4.64; Cl, 16.30; N, 19.33.
2×OCH3), 5.89 (s, 1H, ArH), 6.06 (s, 1H, ArH), 6.49 (brs, 1H,
NH), 6.88 (s, 1H, ArH), 6.96 (d, J = 2.2 Hz, 2H, ArH), 7.41 (d, J
= 8.7 Hz, 2H, ArH), 7.77 (d, J = 2.2 Hz, 1H, ArH), 8.14 (d, J =
8.0 Hz, 1H, ArH), 8.34 (brs, 1H, NH), 9.05 (brs, 1H, NH); Anal.
110 calcd. for C24H25ClN6O2: C, 62.00; H, 5.42; Cl, 7.63; N, 18.08;
O, 6.88, found: C, 62.15; H, 5.38; Cl, 7.50; N, 17.87; O, 7.04.
55 N4ꢀ(4ꢀBromophenyl)ꢀN2ꢀ(2ꢀ((7ꢀchloroquinolinꢀ4ꢀyl)amino)
ethyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine (7d): Yield 70%; mp
N2ꢀ(3ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)propyl)ꢀ6ꢀmethylꢀN4ꢀ
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1
phenylpyrimidineꢀ2,4ꢀdiamine (8a): Yield 65%; mp 199ꢀ200o C;
IR (KBr, cmꢀ1): 3231, 3052, 2954, 1583, 1526, 1496, 1401, 1365,
855, 812, 795; H NMR (400 MHz, DMSOꢀd6): δ 1.88ꢀ1.95 (m,
2H, NCH2CH2CH2N), 2.08 (s, 3H, CH3), 3.32ꢀ3.39 (m, 4H,
1284, 1266, 1130, 877, 855, 801, 754; 1H NMR (400 MHz, 60 2×NCH2), 5.83 (s, 1H, ArH), 6.44 (d, J = 5.8 Hz, 1H, ArH), 6.84
DMSOꢀd6): δ 1.88ꢀ1.95 (m, 2H, NCH2CH2CH2N), 2.08 (s, 3H,
CH3), 3.37ꢀ3.40 (m, 4H, 2×NCH2), 5.85 (s, 1H, ArH), 6.44 (d, J =
5.8 Hz, 1H, ArH), 6.78 (brs, 1H, NH), 6.88 (t, J = 7.3 Hz, 1H,
ArH), 7.21 (t, J = 7.3 Hz, 2H, ArH), 7.30 (t, J = 5.1 Hz, 1H, NH),
(brs, 1H, NH), 7.30 (t, J = 5.1 Hz, 1H, NH), 7.34 (d, J = 8.7 Hz,
2H, ArH), 7.41 (dd, J = 2.2, 8.7 Hz, 1H, ArH), 7.68 (d, J = 8.7
Hz, 2H, ArH), 7.75 (d, J = 2.2 Hz, 1H, ArH), 8.24 (d, J = 9.5 Hz,
1H, ArH), 8.33 (d, J = 5.1 Hz, 1H, ArH), 9.14 (brs, 1H, NH); 13C
5
7.41 (dd, J = 2.2, 9.5 Hz, 1H, ArH), 7.69 (d, J = 7.3 Hz, 2H, 65 NMR (100 MHz, DMSOꢀd6): δ 24.52, 28.68, 40.99, 41.14, 99.48,
ArH), 7.76 (d, J = 2.2 Hz, 1H, ArH), 8.24 (d, J = 8.7 Hz, 1H,
113.25, 118.33, 121.74, 124.85, 128.38, 132.03, 134.23, 141.15,
149.94, 150.90, 152.70, 161.68, 162.83, 166.10; ESIꢀHRMS
(m/z) calculated for C23H22BrClN6: 496.0788, found: 497.0862
(M + H)+, 499.0841 (M + 2)+; Anal. calcd. for C23H22BrClN6: C,
10 ArH), 8.33 (d, J = 5.1 Hz, 1H, ArH), 8.99 (brs, 1H, NH); 13C
NMR (100 MHz, DMSOꢀd6): δ 24.57, 28.74, 39.50, 41.22, 99.53,
118.39, 120.10, 122.07, 124.89, 124.95, 128.40, 129.42, 134.26,
141.76, 150.0, 150.94, 152.78, 162.02, 162.96, 165.88; ESIꢀ 70 55.49; H, 4.45; Br, 16.05; Cl, 7.12; N, 16.88, found: C, 55.53; H,
HRMS (m/z) calculated for C23H23ClN6: 418.1724, found:
15 419.1797 (M + H)+, 421.1778 (M + 2)+; Anal. calcd. for
C23H23ClN6: C, 65.94; H, 5.53; Cl, 8.46; N, 20.06, found: C,
66.04; H, 5.59; Cl, 8.53; N, 19.88.
4.39; Br, 16.12; Cl, 7.23; N, 16.76.
N2ꢀ(3ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)propyl)ꢀ6ꢀmethylꢀN4ꢀpꢀ
o
tolylpyrimidineꢀ2,4ꢀdiamine (8e): Yield 70%; mp 238ꢀ241 C;
IR (KBr, cmꢀ1): 3229, 3041, 2955, 1584, 1510, 1450, 1406, 1364,
N2ꢀ(3ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)propyl)ꢀN4ꢀ(4ꢀfluoro
phenyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine (8b): Yield 65%; mp
20 230ꢀ232 oC; IR (KBr, cmꢀ1): 3232, 3049, 2954, 2922, 1613, 1583,
75 1286, 1265, 1134, 878, 855; H NMR (400 MHz, DMSOꢀd6): δ
1
1.87ꢀ1.94 (m, 2H, NCH2CH2CH2N), 2.05 (s, 3H, CH3), 2.17 (s,
3H, CH3), 3.30ꢀ3.32 (m, 2H, NCH2), 3.35ꢀ3.38 (m, 2H, NCH2),
5.80 (s, 1H, ArH), 6.43 (d, J = 5.8 Hz, 1H, ArH), 6.72 (brs, 1H,
NH), 6.97 (d, J = 7.3 Hz, 2H, ArH), 7.30 (t, J = 5.8 Hz, 1H, NH),
1
1504, 1449, 1407, 1365, 1284, 1207, 1130, 1079, 876; H NMR
(400 MHz, DMSOꢀd6): δ 1.87ꢀ1.94 (m, 2H, NCH2CH2CH2N),
2.07 (s, 3H, CH3), 3.30ꢀ3.33 (m, 4H, 2×NCH2), 5.81 (s, 1H, 80 7.40 (dd, J = 2.2, 8.7 Hz, 1H, ArH), 7.54 (d, J = 8.0 Hz, 2H,
ArH), 6.43 (d, J = 5.8 Hz, 1H, ArH), 6.78 (brs, 1H, NH), 7.03 (t,
25 J = 8.7 Hz, 2H, ArH), 7.30 (t, J = 5.1 Hz, 1H, NH), 7.40 (dd, J =
2.2, 8.7 Hz, 1H, ArH), 7.67ꢀ7.70 (m, 2H, ArH), 7.76 (d, J = 2.2
Hz, 1H, ArH), 8.23 (d, J = 9.5 Hz, 1H, ArH), 8.33 (d, J = 5.8 Hz,
ArH), 7.75 (d, J = 2.2 Hz, 1H, ArH), 8.23 (d, J = 9.5 Hz, 1H,
ArH), 8.33 (d, J = 5.1 Hz, 1H, ArH), 8.87 (brs, 1H, NH); 13C
NMR (100 MHz, DMSOꢀd6): δ 21.20, 24.54, 28.77, 39.50, 41.22,
99.52, 118.37, 120.25, 124.88, 124.95, 128.37, 129.81, 130.90,
1H, ArH), 9.02 (brs, 1H, NH); 13C NMR (100 MHz, DMSOꢀd6): 85 134.25, 139.13, 149.97, 150.95, 152.76, 162.03, 162.96, 165.66;
δ 24.57, 28.75, 39.50, 39.81, 99.55, 115.77, 115.99, 118.40,
30 121.65, 121.72, 124.89, 124.94, 128.42, 134.28, 138.13, 150.02,
150.96, 152.77, 156.69, 159.06, 161.91, 162.93, 165.92; ESIꢀ
HRMS (m/z) calculated for C23H22ClFN6: 436.1579, found:
437.1331 (M + H)+, 439.1596 (M + 2)+; Anal. calcd. for
C23H22ClFN6: C, 63.23; H, 5.08; Cl, 8.11; F, 4.35; N, 19.24,
35 found: C, 63.40; H, 5.02; Cl, 8.14; F, 4.55; N, 19.38.
ESIꢀMS (m/z) calculated for C24H25ClN6: 432.1829, found:
433.1929 (M + H)+, 435.2094 (M + 2)+; Anal. calcd. for
C24H25ClN6: C, 66.58; H, 5.82; Cl, 8.19; N, 19.41, found: C,
66.52; H, 5.94; Cl, 8.30; N, 19.21.
90 N2ꢀ(3ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)propyl)ꢀN4ꢀ(4ꢀmethoxy
phenyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine (8f): Yield 65%; mp
216ꢀ219 oC; IR (KBr, cmꢀ1): 3230, 3052, 2931, 1585, 1530, 1508,
1451, 1409, 1368, 1265, 1246, 1208, 1174, 1133, 1040, 877, 854,
797; 1H NMR (400 MHz, DMSOꢀd6): δ 1.87ꢀ1.94 (m, 2H,
N4ꢀ(4ꢀChlorophenyl)ꢀN2ꢀ(3ꢀ(7ꢀchloroquinolinꢀ4ꢀylamino)
propyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine (8c): Yield 68%; mp
235ꢀ238 oC; IR (KBr, cmꢀ1): 3234, 3064, 2956, 1610, 1584, 1526, 95 NCH2CH2CH2N), 2.05 (s, 3H, CH3), 3.30ꢀ3.38 (m, 4H, 2×NCH2
1
1490, 1406, 1362, 1305, 1287, 1131, 1089, 878, 855; H NMR
40 (400 MHz, DMSOꢀd6): δ 1.88ꢀ1.95 (m, 2H, NCH2CH2CH2N),
2.08 (s, 3H, CH3), 3.29ꢀ3.31 (m, 2H, NCH2), 3.36ꢀ3.39 (m, 2H,
NCH2), 5.84 (s, 1H, ArH), 6.44 (d, J = 5.1 Hz, 1H, ArH), 6.84
3.66 (s, 3H, OCH3), 5.76 (s, 1H, ArH), 6.44 (d, J = 5.1 Hz, 1H,
ArH), 6.69 (brs, 1H, NH), 6.80 (d, J = 9.5 Hz, 2H, ArH), 7.29 (t,
J = 5.1 Hz, 1H, NH), 7.41 (dd, J = 2.2, 8.7 Hz, 1H, ArH), 7.54 (d,
J = 8.7 Hz, 2H, ArH), 7.75 (d, J = 2.2 Hz, 1H, ArH), 8.24 (d, J =
(brs, 1H, NH), 7.22 (d, J = 8.7 Hz, 2H, ArH), 7.31 (t, J = 5.1 Hz, 100 8.7 Hz, 1H, ArH), 8.34 (d, J = 5.1 Hz, 1H, ArH), 8.79 (s, 1H,
1H, NH), 7.41 (dd, J = 2.2, 9.5 Hz, 1H, ArH), 7.73 (d, J = 8.7 Hz,
45 2H, ArH), 7.76 (d, J = 2.2 Hz, 1H, ArH), 8.24 (d, J = 8.7 Hz, 1H,
ArH), 8.33 (d, J = 5.8 Hz, 1H, ArH), 9.15 (brs, 1H, NH); 13C
NMR (100 MHz, DMSOꢀd6): δ 24.51, 28.69, 39.50, 41.15, 99.47,
NH); 13C NMR (100 MHz, DMSOꢀd6): δ 24.59, 28.81, 39.50,
41.28, 56.03, 99.59, 114.71, 118.42, 122.14, 124.93, 124.99,
128.42, 134.32, 134.79, 150.02, 151.01, 152.80, 155.13, 162.19,
163.04, 165.59; ESIꢀMS (m/z) calculated for C24H25ClN6O:
118.34, 121.33, 124.83, 124.87, 125.41, 128.38, 129.14, 134.24, 105 448.1778, found: 449.1529 (M + H)+, 451.1860 (M + 2)+; Anal.
140.73, 149.95, 150.90, 152.68, 161.72, 162.85, 166.08; ESIꢀ
50 HRMS (m/z) calculated for C23H22Cl2N6: 452.1283, found:
453.0463 (M + H)+, 455.0528 (M + 2)+; Anal. calcd. for
C23H22Cl2N6: C, 60.93; H, 4.89; Cl, 15.64; N, 18.54, found: C,
61.07; H, 5.00; Cl, 15.51; N, 18.66.
calcd. for C24H25ClN6O: C, 64.21; H, 5.61; Cl, 7.90; N, 18.72; O,
3.56, found: C, 64.29; H, 5.56; Cl, 7.88; N, 18.70; O, 3.64.
N2ꢀ(3ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)propyl)ꢀN4ꢀ(3,5ꢀ
dimethoxyphenyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine
(8g):
o
110 Yield 55%; mp 168ꢀ170 C; IR (KBr, cmꢀ1): 3258, 2944, 1583,
N4ꢀ(4ꢀBromophenyl)ꢀN2ꢀ(3ꢀ((7ꢀchloroquinolinꢀ4ꢀyl)amino)
55 propyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine (8d): Yield 75%; mp
240ꢀ243 oC; IR (KBr, cmꢀ1): 3235, 3163, 3062, 2957, 1612, 1583,
1527, 1487, 1405, 1361, 1304, 1285, 1263, 1135, 1074, 903, 878,
1521, 1450, 1400, 1356, 1230, 1203, 1151, 1060, 869, 823; H
1
NMR (400 MHz, DMSOꢀd6): δ 1.96 (quintet, J = 6.5 Hz, 2H,
NCH2CH2CH2N), 2.08 (s, 3H, CH3), 3.40ꢀ3.43 (m, 4H, 2×NCH2),
3.68 (s, 6H, 2×OCH3), 5.85 (s, 1H, ArH), 6.06 (s, 1H, ArH), 6.56
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(d, J = 5.1 Hz, 1H, ArH), 6.91 (brs, 1H, NH), 6.98 (s, 2H, ArH),
7.49ꢀ7.52 (m, 1H, ArH), 7.81 (d, J = 1.4 Hz, 1H, ArH), 8.02 (brs,
1H, NH), 8.33ꢀ8.39 (m, 2H, ArH), 9.08 (s, 1H, NH); Anal. calcd.
192ꢀ194 oC; IR (KBr, cmꢀ1): 3246, 3059, 2957, 2914, 1641, 1583,
1551, 1515, 1488, 1397, 1368, 1237, 1138, 1072, 996, 908, 856,
1
821, 801; H NMR (400 MHz, DMSOꢀd6): δ 1.64ꢀ1.71 (m, 4H,
for C25H27ClN6O2: C, 62.69; H, 5.68; Cl, 7.40; N, 17.55; O, 6.68, 60 NCH2CH2CH2CH2N), 2.08 (s, 3H, CH3), 3.25ꢀ3.33 (m, 4H,
5
found: C, 62.74; H, 5.88; Cl, 7.57; N, 17.50; O, 6.55.
2×NCH2), 5.83 (s, 1H, ArH), 6.43 (d, J = 5.8 Hz, 1H, ArH), 6.80
(brs, 1H, NH), 7.29 (t, J = 5.1 Hz, 1H, NH), 7.37 (d, J = 8.7 Hz,
2H, ArH),7.41 (dd, J = 2.2, 8.7 Hz, 1H, ArH), 7.70 (d, J = 8.7 Hz,
2H, ArH), 7.76 (d, J = 2.2 Hz, 1H, ArH), 8.25 (d, J = 9.5 Hz, 1H,
65 ArH), 8.34 (d, J = 5.1 Hz, 1H, ArH), 9.14 (s, 1H, NH); 13C NMR
(100 MHz, DMSOꢀd6): δ 23.66, 25.42, 26.91, 40.42, 42.28,
98.61, 112.35, 117.44, 120.85, 123.96, 124.10, 127.42, 131.17,
133.36, 140.35, 149.03, 150.12, 151.81, 160.81, 161.97; ESIꢀ
HRMS (m/z) calculated for C24H24BrClN6: 510.0934, found:
70 511.0450 (M + H)+, 513.0376 (M + 2)+; Anal. calcd. for
C24H24BrClN6: C, 56.32; H, 4.73; Br, 15.61; Cl, 6.93; N, 16.42,
found: C, 56.37; H, 4.84; Br, 15.58; Cl, 7.13; N, 16.31.
N2ꢀ(4ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)butyl)ꢀ6ꢀmethylꢀN4ꢀ
phenylpyrimidineꢀ2,4ꢀdiamine (9a): Yield 65%; mp 210ꢀ212oC;
IR (KBr, cmꢀ1): 3249, 3060, 2957, 2934, 2865, 1626, 1581, 1551,
1512, 1436, 1365, 1227, 1204, 1146, 1081, 894, 864, 848, 790,
10 753; 1H NMR (400 MHz, DMSOꢀd6): δ 1.64ꢀ1.70 (m, 4H,
NCH2CH2CH2CH2N), 2.06 (s, 3H, CH3), 3.24ꢀ3.31 (m, 4H,
2×NCH2), 5.83 (s, 1H, ArH), 6.42 (d, J = 5.8 Hz, 1H, ArH), 6.71
(brs, 1H, NH), 6.87 (t, J = 7.3 Hz, 1H, ArH), 7.21 (t, J = 7.3 Hz,
2H, ArH), 7.29 (t, J = 5.1 Hz, 1H, NH), 7.40 (dd, J = 2.2, 8.7 Hz,
15 1H, ArH), 7.69 (d, J = 8.0 Hz, 2H, ArH), 7.74 (d, J = 2.2 Hz, 1H,
ArH), 8.24 (d, J = 8.7 Hz, 1H, ArH), 8.33 (d, J = 5.8 Hz, 1H,
ArH), 8.98 (brs, 1H, NH); 13C NMR (100 MHz, DMSOꢀd6): δ
23.68, 25.43, 26.96, 40.44, 42.29, 98.63, 117.46, 119.13, 121.13,
N2ꢀ(4ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)butyl)ꢀ6ꢀmethylꢀN4ꢀpꢀ
o
tolylpyrimidineꢀ2,4ꢀdiamine (9e): Yield 70%; mp 175ꢀ178 C;
123.96, 124.11, 127.46, 128.50, 133.36, 140.92, 149.09, 150.10, 75 IR (KBr, cmꢀ1): 3250, 3059, 2924, 2856, 1627, 1612, 1581, 1514,
20 151.87, 161.12, 162.06; ESIꢀHRMS (m/z) calculated for
C24H25ClN6: 432.1829, found: 433.2361 (M + H)+, 435.2553 (M
+ 2)+; Anal. calcd. for C24H25ClN6: C, 66.58; H, 5.82; Cl, 8.19; N,
19.41, found: C, 66.64; H, 5.79; Cl, 8.06; N, 19.11.
1450, 1422, 1366, 1336, 1315, 1229, 1204, 1134, 1081, 846, 805;
1H NMR (400 MHz, DMSOꢀd6):
1.64ꢀ1.70 (m, 4H,
δ
NCH2CH2CH2CH2N), 2.05 (s, 3H, CH3), 2.18 (s, 3H, CH3), 3.25ꢀ
3.30 (m, 4H, 2×NCH2), 5.79 (s, 1H, ArH), 6.43 (d, J = 5.8 Hz,
80 1H, ArH), 6.68 (brs, 1H, NH), 7.01 (d, J = 8.0 Hz, 2H, ArH), 7.30
(t, J = 5.1 Hz, 1H, NH), 7.41 (dd, J = 2.2, 8.7 Hz, 1H, ArH), 7.55
(d, J = 8.0 Hz, 2H, ArH), 7.75 (d, J = 2.2 Hz, 1H, ArH), 8.25 (d, J
= 9.5 Hz, 1H, ArH), 8.34 (d, J = 5.1 Hz, 1H, ArH), 8.87 (brs, 1H,
NH); 13C NMR (100 MHz, DMSOꢀd6): δ 20.31, 23.63, 25.45,
85 27.02, 40.43, 42.31, 98.61, 117.47, 119.36, 123.95, 124.11,
127.44, 128.91, 130.01, 133.38, 138.30, 149.06, 150.12, 151.82,
161.17, 162.09, 164.75; ESIꢀMS (m/z) calculated for C25H27ClN6:
446.2049, found: 447.2117 (M + H)+, 449.2099 (M + 2)+; Anal.
calcd. for C25H27ClN6: C, 67.18; H, 6.09; Cl, 7.93; N, 18.80,
90 found: C, 67.30; H, 6.14; Cl, 8.12; N, 18.74.
N2ꢀ(4ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)butyl)ꢀN4ꢀ(4ꢀfluoro
25 phenyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine (9b): Yield 52%; mp
188ꢀ200 oC; IR (KBr, cmꢀ1): 3229, 2955, 2868, 1613, 1580, 1505,
1450, 1412, 1366, 1335, 1206, 1137, 1079, 847, 827, 804; 1H NMR
(400 MHz, DMSOꢀd6): δ 1.63ꢀ1.69 (m, 4H, NCH2CH2CH2CH2N),
2.07 (s, 3H, CH3), 3.29ꢀ3.30 (m, 4H, 2×NCH2), 5.79 (s, 1H,
30 ArH), 6.44 (d, J = 5.1 Hz, 1H, ArH), 6.72 (brs, 1H, NH), 7.05 (d,
J = 8.7 Hz, 2H, ArH), 7.33 (brs, 1H, NH), 7.42 (d, J = 8.7 Hz,
1H, ArH), 7.68ꢀ7.69 (m, 2H, ArH), 7.75 (d, J = 2.2 Hz, 1H, ArH),
8.25 (d, J = 9.5 Hz, 1H, ArH), 8.34 (d, J = 5.1 Hz, 1H, ArH), 9.01
(brs, 1H, NH); ESIꢀHRMS (m/z) calculated for C24H24ClFN6:
35 450.1735, found: 451.3680 (M + H)+,453.3787 (M + 2)+; Anal.
calcd. for C24H24ClFN6: C, 63.92; H, 5.36; Cl, 7.86; F, 4.21; N,
18.64, found: C, 64.05; H, 5.29; Cl, 8.00; F, 4.27; N, 18.88.
N2ꢀ(4ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)butyl)ꢀN4ꢀ(4ꢀmethoxy
phenyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine (9f): Yield 62%; mp
134ꢀ138 oC; IR (KBr, cmꢀ1): 3281, 3181, 3067, 2945, 1582, 1508,
1
1445, 1369, 1241, 1138, 1029, 906, 856; H NMR (400 MHz,
N4ꢀ(4ꢀChlorophenyl)ꢀN2ꢀ(4ꢀ(7ꢀchloroquinolinꢀ4ꢀylamino)
butyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine (9c): Yield 70%; mp
40 197ꢀ198 oC; IR (KBr, cmꢀ1): 3251, 3070, 2956, 2915, 1582, 1552,
1514, 1491, 1401, 1368, 1332, 1238, 1138, 1081, 908, 856, 823,
801; 1H NMR (400 MHz, DMSOꢀd6): δ 1.64ꢀ1.71 (m, 4H,
NCH2CH2CH2CH2N), 2.08 (s, 3H, CH3), 3.24ꢀ3.33 (m, 4H,
2×NCH2), 5.83 (s, 1H, ArH), 6.42 (d, J = 5.8 Hz, 1H, ArH), 6.79
45 (brs, 1H, NH), 7.25 (d, J = 8.7 Hz, 2H, ArH), 7.28 (t, J = 5.1 Hz,
1H, NH), 7.40 (dd, J = 2.2, 8.7 Hz, 1H, ArH), 7.73 (s, 1H, ArH),
7.76 (d, J = 2.2 Hz, 2H, ArH), 8.24 (d, J = 8.7 Hz, 1H, ArH), 8.34
(d, J = 5.1 Hz, 1H, ArH), 9.14 (brs, 1H, NH); 13C NMR (100
MHz, DMSOꢀd6): δ 23.66, 25.44, 26.93, 42.29, 98.61, 117.46,
50 120.43, 123.94, 124.10, 124.51, 127.47, 128.28, 133.36, 139.94,
149.09, 150.10, 151.84, 160.85, 162.0; ESIꢀHRMS (m/z) calculated
for C24H24Cl2N6: 466.1497, found: 467.1569 (M + H)+, 469.1546
(M + 2)+; Anal. calcd. for C24H24Cl2N6: C, 61.67; H, 5.18; Cl,
15.17; N, 17.98, found: C, 61.65; H, 5.23; Cl, 15.20; N, 18.14.
95 DMSOꢀd6): δ 1.63ꢀ1.70 (m, 4H, NCH2CH2CH2CH2N), 2.04 (s,
3H, CH3), 3.24ꢀ3.32 (m, 4H, 2×NCH2), 3.66 (s, 3H, OCH3), 5.75
(s, 1H, ArH), 6.43 (d, J = 5.1 Hz, 1H, ArH), 6.62 (brs, 1H, NH),
6.81 (d, J = 8.7 Hz, 2H, ArH), 7.29 (t, J = 5.1 Hz, 1H, NH), 7.41
(dd, J = 2.2, 8.7 Hz, 1H, ArH), 7.55 (d, J = 8.7 Hz, 2H, ArH),
100 7.75 (d, J = 2.2 Hz, 1H, ArH), 8.25 (d, J = 8.7 Hz, 1H, ArH), 8.34
(d, J = 5.8 Hz, 1H, ArH), 8.79 (brs, 1H, NH); ESIꢀHRMS (m/z)
calculated for C25H27ClN6O: 462.1935, found: 463.2074 (M +
H)+, 465.2463 (M + 2)+; Anal. calcd. for C25H27ClN6O: C, 64.86;
H, 5.88; Cl, 7.66; N, 18.15; O, 3.46, found: C, 64.77; H, 5.92; Cl,
105 7.71; N, 18.28; O, 3.61.
N2ꢀ(4ꢀ(7ꢀChloroquinolinꢀ4ꢀylamino)butyl)ꢀN4ꢀ(3,5ꢀdimethoxy
phenyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine (9g): Yield 55%; mp
188ꢀ190 oC; IR (KBr, cmꢀ1): 3259, 3142, 2932, 1581, 1476, 1452,
1
1354, 1333, 1248, 1233, 1204, 1155, 1073, 970, 846; H NMR
110 (400 MHz, DMSOꢀd6): δ 1.64ꢀ1.70 (m, 4H, NCH2CH2CH2CH2N),
2.06 (s, 3H, CH3), 3.27ꢀ3.35 (m, 4H, 2×NCH2), 3.67 (s, 6H,
2×OCH3), 5.81 (s, 1H, ArH), 6.05 (t, J = 2.2 Hz, 1H, ArH), 6.45
(d, J = 5.8 Hz, 1H, ArH), 6.77 (brs, 1H, NH), 6.98 (s, 2H, ArH),
55 N4ꢀ(4ꢀBromophenyl)ꢀN2ꢀ(4ꢀ((7ꢀchloroquinolinꢀ4ꢀyl)amino)
butyl)ꢀ6ꢀmethylpyrimidineꢀ2,4ꢀdiamine (9d): Yield 70%; mp
12 | Journal Name, [year], [vol], 00–00
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7.42ꢀ7.45 (m, 2H, NH, ArH), 7.76 (d, J = 2.2 Hz, 1H, ArH), 8.26
(d, J = 8.7 Hz, 1H, ArH), 8.34 (d, J = 5.8 Hz, 1H, ArH), 8.99
(brs, 1H, NH); 13C NMR (100 MHz, DMSOꢀd6): δ 23.53, 25.33,
26.86, 40.48, 42.37, 54.93, 93.45, 97.34, 98.60, 117.24, 124.19,
126.66, 133.77, 142.55, 148.07, 150.57, 150.98, 160.42, 161.14,
161.94, 164.84; ESIꢀHRMS (m/z) calculated for C26H29ClN6O2:
492.2110, found: 493.2181 (M + H)+; Anal. calcd. for C26H29ClN6O2:
C, 63.34; H, 5.93; Cl, 7.19; N, 17.05; O, 6.49, found: C, 63.44; H,
5.85; Cl, 7.22; N, 17.16; O, 6.53.
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10 Conclusions
In summary, we reported the synthesis and antimalarial activity
evaluation of a series of 4ꢀaminoquinolineꢀpyrimidine hybrids.
The in vitro evaluation of these hybrids against D6 and W2
strains of P. falciparum depicted activity in the micromolar
15 range. Also, these hybrids exhibited high selectivity indices and
low toxicity against the tested cell lines. Further, the binding
capability of compound 8d was evaluated with heme to find out
the probable mode of action of these hybrids. The best active
compounds showed good interaction with PfꢀDHFRꢀTS similar to
20 the protein native substrate dihydrofolate. The compounds with
best antimalarial activity also showed good ADMET properties.
The promising antimalarial activity exhibited by the novel 4ꢀ
aminoquinolineꢀpyrimidine conjugates and their good safety
profile described in the present study emphasizes their potential
25 for further development as antimalarial drugs.
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Acknowledgements
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D.S.R. thanks Council of Scientific and Industrial Research [No.
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D.K. is thankful to CSIR for the award of junior and senior
30 research fellowship. P.P. is thankful to CSIR for Research
Associate. SIK is thankful to United States Department of
Agriculture (USDA), Agricultural Research Service Specific
Cooperative Agreement No. 58ꢀ6408ꢀ2ꢀ0009 for partial support
of this work. The Authors are also thankful to CIFꢀUSIC,
35 University of Delhi, Delhi for NMR spectral data and RSIC,
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DOI: 10.1039/C4RA09768H
Synthesis, antimalarial activity, heme binding and docking studies of 4-
aminoquinoline-pyrimidine based molecular hybrids
Deepak Kumar,1 Shabana I. Khan,2,3,4 Prija Ponnan,1 Diwan S. Rawat1*
1Department of Chemistry, University of Delhi, Delhi-110007, India
2National Center for Natural Products Research, 3Department of Biomolecular Sciences,
University of Mississippi, MS-38677, USA
4Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, KSA
Corresponding author: dsrawat@chemistry.du.ac.in
Phone No: 91-11-27667465, Fax No: 91-11-27667501
Graphical Abstract