Pyrimidine-chloroquinoline hybrids: Synthesis and antiplasmodial activity

Article abstract of DOI:10.1016/j.ejmech.2018.02.021

Triazole tethered 7-chloroquinoline-pyrimidine-5-carboxylate hybrids were synthesized and evaluated for antiplasmodial activity against chloroquine sensitive (CQ^S) NF54 strain of Plasmodium falciparum. The most active hybrids of the series were

Full text of DOI:10.1016/j.ejmech.2018.02.021

Accepted Manuscript
Pyrimidine-chloroquinoline hybrids: Synthesis and antiplasmodial activity
Rakesh Chopra, Kelly Chibale, Kamaljit Singh
PII:
S0223-5234(18)30143-0
10.1016/j.ejmech.2018.02.021
EJMECH 10198
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 December 2017
Revised Date: 27 January 2018
Accepted Date: 6 February 2018
Please cite this article as: R. Chopra, K. Chibale, K. Singh, Pyrimidine-chloroquinoline hybrids:
Synthesis and antiplasmodial activity, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.02.021.
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ACCEPTED MANUSCRIPT
Pyrimidine-chloroquinoline hybrids: Synthesis and antiplasmodial activity
Rakesh Chopra,♦ Kelly Chibale♠ and Kamaljit Singh♦ ,*
Heme
375 nm
402 nm
IC (µµM) = 0.048 (NF54),
µµ
50
4.59 (Dd2)
♦ Department of Chemistry, UGC-Centre for Advanced Studies-II, Guru Nanak Dev
University, Amritsar 143005, India. Email: kamaljit.chem@gndu.ac.in
♠ Department of Chemistry, South African Medical Research Council Drug Discovery and
Development Research Unit, Institute of Infectious Disease and Molecular Medicine,
University of Cape Town, Rondebosch 7701, South Africa
1

ACCEPTED MANUSCRIPT
Pyrimidine-chloroquinoline hybrids: Synthesis and antiplasmodial activity
Rakesh Chopra,♦ Kelly Chibale♠ and Kamaljit Singh♦ ,*
Abstract: Triazole tethered 7-chloroquinoline-pyrimidine-5-carboxylate hybrids were
synthesized and evaluated for antiplasmodial activity against chloroquine sensitive (CQS)
NF54 strain of Plasmodium falciparum. The most active hybrids of the series were further
screened against the chloroquine resistant (CQR) Dd2 strain of the parasite and for in vitro
cytotoxicity against mammalian Vero cell lines. Further, their physico-chemical properties,
binding studies with hemin (monomeric & µ-oxo dimeric) and DNA [pUC-18, calf thymus
(CT)] led us to plausible proposed binding mode of the most active member of the present
series.
Keywords: Pyrimidine hybrids; 7-chloroquinoline; triazole; Huisgen 1,3-dipolar
cycloaddition; antiplasmodial studies; lipophilicity; binding with DNA.
1. Introduction
Malaria continues to be a lead killer disease. As an estimate, a total of 216 million
cases of malaria were reported by World Health Organization (WHO) in the World
Malaria Report 2017, recording an increase of about 5 million cases compared to
2015. Further, nearly 445,000 deaths were attributed to malaria [1]. However, these
figures represented a nearly 17.5% reduction in comparison to the malaria incidences
recorded in 2010 (262 million) and a 53% decline in the number of deaths (839,000)
reported in that year. At least 80% of the cases and deaths (90%) occurred in sub-
Saharan Africa [1]. The disease is estimated to claim life of a child every 2 minutes in
♦ Department of Chemistry, UGC-Centre for Advanced Studies-II, Guru Nanak Dev
University, Amritsar 143005, India. Email: kamaljit.chem@gndu.ac.in
♠ Department of Chemistry, South African Medical Research Council Drug Discovery and
Development Research Unit, Institute of Infectious Disease and Molecular Medicine,
University of Cape Town, Rondebosch 7701, South Africa
2

ACCEPTED MANUSCRIPT
sub-Saharan Africa [1-4]. Malaria case incidences are highest among pregnant women
and children under five years of age, owing to their weak immune system.
Out of more than 100 species of Plasmodium that have been identified to cause
malaria, only five: P. falciparum, P. malariae, P. vivax, P. ovale and P. knowlesi [5]
are known to cause human malaria. P. falciparum is responsible for the most severe
form of the disease [6], and contributes most significantly to morbidity and mortality
numbers associated with the disease. On the other hand, although severity of the
disease caused by P. vivax and P. ovale is mild owing to persistence of dormant
hypnozoite forms in the liver [7-9], they are responsible for relapses. Likewise, P.
malariae is typically linked with chronic infections and can persist in blood
asymptomatically for long periods of time [10]. The zoonotic parasite P. knowlesi was
previously known to cause malaria only in Macaque monkeys (Macaca fascicularis),
but has now also been reported to infect humans [11-13]. Further, various species of
Anopheles mosquitoes are responsible for human transmission of these parasites [14].
Among the front-line drugs, artemisinin combination therapy (ACT), involving a fast
acting derivative of artemisinin
1 [15,16] along with a longer-acting blood
schizonticide is the treatment of choice recommended by the WHO for adults,
pregnant women as well as children diagnosed with uncomplicated falciparum malaria
[1]. The latter class of blood schizonticidal drugs are intended to minimize chances of
recrudescence after clearance of the initial blood parasite load [17,18]. The most
commonly implicated ACT combinations are: artemether
2
, lumefantrine
, dihydroartemisinin , piperaquine
9 and pyrimethamine 10 (Figure 1). On the other hand, primaquine 11 is
3
, artesunate
4
(ASN), amodiaquine
5
, 4-mefloquine
6
7
8
and 4-
sulfadoxine
the only antimalarial known to be active [1,19] against the hypnozoite stage of
Plasmodia and also possesses activity against P. falciparum gametocytes and is
consequently capable of preventing transmission of the parasite [20]. However,
hemolysis in glucose-6-phosphate (G6PD)-deficient individuals constitutes the major
drawback [21]. The development of resistance to the one-time front-line antimalarial
agents such as quinine 12, chloroquine 13 (CQ), sulfadoxine-pyrimethamine [22,23],
emerging resistance to artemisinin [24,25] and non-availability of a vaccine casts a
grim picture of malaria treatment. Intense efforts are currently underway to develop
antimalarials, which are active against drug-resistant mutants of Plasmodium, and are
3

ACCEPTED MANUSCRIPT
both, safe, effective with minimal side effects, orally active and above all possess
novel mechanisms of action.
Cl
CH3
CH3
OH
N
H
CH3
H
H
O
O
O
O
Cl
O
O
H3C
H3C
HN
N
H3C
O
O
O
H
CH3
H
CH3
C2H5
C2H5
O
H
CH3
O
O
N
Cl
OMe
O
Na+
HO
OCOCH2CH2COO
Cl
1
2
3
4
5
O
CH3
N
N
HO
H
H
O
N
H
N
N
N
O
S
O
O
H3C
O
N
N
N
N
O
H
H2N
N
CF3
O
CH3
CF3
Cl
Cl
OH
6
7
8
9
CH3
C2H5
N
Cl
CH3
NH2
N
HO
NH2
C2H5
HN
HN
N
MeO
N
2H3PO4
CH3
H2N
N
N
Cl
N
OCH3
10
11
12
13
Figure 1. Chemical structures of representative ACT and chloroquine based antimalarials.
In this context, hybrid drugs are endowed with many advantages over single drug
and/or multicomponent combination therapy [26-29]. These are capable of exerting
dual mode of action and address issues related to drug resistance, solubility,
formulation/delivery etc. In addition to structural diversity, a distinctive feature of
hybrid drugs is the presence of two different antimalarial pharmacophores joined
covalently, endowing them the ability to hit multiple targets. Consequently, hybrid
drugs have been theme of several reviews [30-37]. Recently, synthesis and activity of a
number of antimalarial hybrids based upon the pyrimidine core have been reported
[38,39].
Amongst the pyrimidine hybrids, pyrimidine-4-aminoquinoline hybrids 14 containing
a rigid p-phenylene linker exhibited moderate antipalsmodial activity [40], while the
hybrids 15 (Figure 2) bearing diaminoalkane substituents on the pyrimidine core, were
active in the nM range [41]. Using a 2,6-diaminopyrimidine core, Pretorius et. al.
synthesized series of quinoline-pyrimidine hybrids 16 bearing different linkers (Figure
2), and these were active in µM range [42].
Recently, our group reported [43] pyrimidine-4-aminoquinoline hybrids 17 with potent
antiplasmodial activity in the nM range. One of these hybrids 17 [R1 = CH(CH3)2, R2
4

ACCEPTED MANUSCRIPT
=
o-NO2C6H5, R3 = (CH2)4] proved to be the most potent of this series. Hemin and
DNA binding studies indicated that these hybrids acted on multiple targets. Likewise,
hybrids 18 of pyrimidine-5-carboxylates and primaquine 11 showed, both blood stage
and liver stage activity against NF54 strain and P. berghei, respectively [43]. Further,
these hybrids exhibited a higher (upto 6.7 times) liver stage activity compared to 11
.
Likewise, hybrids 19 23 lacking 5-carboxylate units [44] depicted nM range in vitro
-
antiplasmodial activity against the chloroquine sensitive (CQS) D10 and chloroquine
resistant (CQR) Dd2 strains of P. falciparum. The activity was 3.2 times more than CQ
[IC50 = 140 nM] and comparable to artesunate [IC50 = 31.2 nM] against the Dd2 strain.
Figure 2. Pyrimidine based antimalarial hybrids.
Further, the in vitro antiplasmodial testing of pyrimidine-quinoline hybrids 24 (Figure
2) bearing a nitrile functionality at the C-5 position of pyrimidines [45] against two
strains (NF54 and Dd2) of P. falciparum revealed that most of the hybrids displayed
activity in nM range and seemed to inhibit hemozoin formation [45].
5

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The quest for developing new hybrids led us to employ heterocyclic moieties such as
1,2,3-triazoles in view of their medicinal potential [46-48]. Further, 1,2,3-triazoles are
stable as well as readily available through efficacious synthesis. In the present
investigation, we report synthesis and antiplasmodial activity of 1,2,3-triazoles
tethered 7-chloroquinoline-pyrimidine-5-carboxylates hybrids 30a-l and 32a-f. These
were initially tested against the NF54 strain. Hybrids 30a-f and 30h-l, were found to
be the most active hybrids in this series against the NF54 strain and were also tested
against the Dd2 strain. Further, hybrids 30a-f and 30h-l were further screened for in
vitro cytotoxicity against the mammalian Vero cell line. Physicochemical properties
such as aqueous solubility (Sw), pKa, log
D were also determined. We also conducted
hemin (monomeric & -oxo dimeric) and DNA [pUC-18, calf thymus (CT)] binding
µ
studies in order to explore the mode of action of these hybrids.
2. Results and discussion
2.1 Synthesis and characterization
Two series of 1,2,3-triazole tethered pyrimidine-chloroquinoline hybrids 30 and 32
were prepared as outlined in Scheme 1.
Scheme 1. Synthesis of pyrimidine-chlororoquinolinetriazole tethered hybrids 30 and
pyrimidine-chloroquinolinetriazole tethered hybrids 32
.
6

ACCEPTED MANUSCRIPT
For preparing 28 (Scheme 1), 25 was treated with the appropriate amino alcohol to
give 4-hydroxyethyl/propyllamino-7-chloroquinoline 26. Treatment of 26 with
methane sulphonyl chloride in dry THF and triethylamine (Et3N) furnished mesylated
intermediate 27, which upon treatment with sodium azide in dry DMF yielded 28. The
C-2 propargylated pyrimidines 29 were synthesized from the appropriate
dihydropyrimidin-2-(1H)-one (obtained via conventional Biginelli condensation) [49]
through pyridinium chlorochromate (PCC) mediated dehydrogenation to furnish the
corresponding oxidized intermediate [50]. Subsequent treatment in refluxing POCl3
furnished chloropyrimidine [51], which upon nucleophilic substitution reaction with
propargyl alcohol under basic conditions yielded 29. In the first series of hybrids
comprising of 30, the 1,2,3-triazole ring was assembled using the click reaction of
appropriate azide 28 with an alkyne 29 using sodium ascorbate and copper sulphate.
For the second series of hybrids, the azide 31 was obtained from 4,7-dichloroquinoline
25 and sodium azide in dimethylformamide. Click reaction of 29 with 4-azido-7-
chloroquinoline 31 using standard reaction conditions (Scheme 1) afforded the second
series of triazole tethered hybrids in excellent yields (Table 1). All intermediates and
products were unambiguously characterized (Fig. S1-S48) using spectroscopic and
microanalytical data (vide experimental).
2.2 In vitro antiplasmodial activity of triazole tethered pyrimidine chloroquinoline
hybrids and structure-activity relationships (SARs)
The in vitro antiplasmodial activity (Table 1) of the hybrids 30 and 32 was determined
against the NF54 strain using CQ and ASN as reference drugs. The compounds 30a-f
and 30h-l, which turned out to be the most potent against NF54 were also evaluated
for their in vitro antiplasmodial activity against the Dd2 strain. Hybrids 30a-l (with a
spacer between the triazole and chloroquinoline rings) were more potent (Table 1)
than the hybrids 32a-f with the triazole ring directly attached to chloroquinoline ring,
against both NF54 and Dd2 strains. Further, on comparison of hybrids differing only
in the length of the methylene spacer between the triazole and chloroquinoline rings, it
is evident that the hybrid 30a with a three carbon methylene spacer were more potent
than 30g with two carbon methylene spacer against both the plasmodium strains.
Similarly, 30b was more active than 30h [52]. Hybrid 30d, which was most active of
all the hybrids against the CQS strain proved to be less active against the CQR strain,
whereas, 30j turned out to be more potent than 30d against the CQR strain (Table 1).
7

ACCEPTED MANUSCRIPT
Replacement of the methyl ester in 30a with ethyl and isopropyl esters to form 30b
and 30c, respectively, led to an increase in clog
P value as well as antiplasmodial
activity in that order against NF54 and Dd2 strains (Table 1).
Likewise, among the hybrids 32a-f, lacking a spacer between triazole and
chloroquinoline unit, the ethyl ester substituted 32b was more active compared to 32a
against the CQS NF54 strain. However, 32c bearing an isopropyl ester, despite being
more lipophilic than 32b (ethyl ester), was less active than 32b. The observed
variation in the activity of these hybrids could possibly arise due to the variable
accumulation of hybrids in the food vacuole (FV) of appropriate strain influencing the
antiplasmodial activity. Keeping all other structural features intact, introducing a polar
nitro group at ortho-position of the C-6 phenyl substituent on the pyrimidine ring of
30c to produce 30e led to a decrease in the antiplasmodial activity (Table 1).
Interestingly, introduction of a polar nitro group in the para position of C-6 phenyl
group in hybrid 30c resulted in enhancement of antiplasmodial activity of the resultant
compound 30d compared to 30c. This is in contrast to the above (30c vs. 30e) trend as
well as our earlier findings, where the hybrid bearing C-6 o-NO2 phenyl group was
more active compared to its p-NO2 phenyl analogue [53]. However, the observed
lower activity of 30d (Table 1) compared to 30c against Dd2 might be attributed to
factors such as mutations in CQR strain leading to lower accumulation of 30d in FV of
CQR strain. Finally, replacing the C-6 phenyl group in 30h with a methyl group in 30l
resulted in a decrease in antiplasmodial activity of 30l (Table 1).
2.3 Cytotoxicity
The most potent hybrids 30a-f and 32h-l were also screened for cytotoxicity against
the mammalian Vero cell line (Table 1) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide (MTT) assay. The IC50 values (Table 1) suggested that
most of the hybrids were devoid of any significant cytotoxicity. Hybrid 30d, which
was the most active against the NF54 strain exhibited high selectivity index (SI =
317.50).
8

ACCEPTED MANUSCRIPT
Table 1. Yield, antiplasmodial activity, and cytotoxicity of the 1,2,3-triazole tethered
pyrimidine chloroquinoline hybrids 30a-l/32a-f
.
R1
R2
Yield
(%)a
86
84
85
92
89
87
89
85
88
81
86
82
82
88
83
86
87
85
-
NF54b
IC50 (µM)d,e
1.01±0.10
Dd2c
ClogPf
Cytotoxicity
IC50 (µM)g,h
155.65±1.55
152.82±0.90
28.52±3.40
15.24±0.20
23.35±1.1
>201± n.d
n.d
SIi
CH3
C2H5
i-C3H7
i-C3H7
i-C3H7
C2H5
CH3
C6H5
C6H5
1.65±0.22
1.37±0.17
0.61±0.01
4.59±0.13
0.86±0.06
1.11±0.14
n.dj
5.555
6.084
6.393
6.139
6.139
4.485
5.278
5.807
6.116
5.862
5.862
4.208
5.485
6.014
6.323
6.069
6.069
4.415
n.d
154.10
171.70
86.42
317.50
38.91
>287
n.d
30a
30b
30c
0.89±0.04
0.33±0.03
0.048±0.01
0.60±0.04
0.70±0.05
10.38±0.30
1.49±0.10
0.64±0.10
0.18±0.02
0.53±0.01
2.49±0.20
15.82±0.50
12.24±2.40
13.99±4.10
7.86±0.10
16.08±0.10
84.15±14.2
0.018±0.002
C6H5
p-NO2C6H4
o-NO2C6H4
CH3
30d
30e
30f
C6H5
30g
30h
30i
C2H5
i-C3H7
i-C3H7
i-C3H7
C2H5
CH3
C6H5
2.52±0.29
2.00±0.26
0.99±0.06
1.08±0.07
8.14±0.17
n.d
149.40±2.50
148.52±1.0
149.87±2.8
145.39±4.0
>207± n.d
n.d
100.26
232.06
832.61
274.32
>83
C6H5
p-NO2C6H4
o-NO2C6H4
CH3
30j
30k
30l
C6H5
n.d
32a
32b
32c
C2H5
i-C3H7
i-C3H7
i-C3H7
C2H5
-
C6H5
n.d
n.d
n.d
C6H5
n.d
n.d
n.d
p-NO2C6H4
o-NO2C6H4
CH3
n.d
n.d
n.d
32d
32e
n.d
n.d
n.d
n.d
n.d
n.d
32f
CQk
ASNl
EMEm
-
0.275±0.02
n.d
n.d
-
-
-
<5.202±ND 0.015±0.0001
n.d
n.d
n.d
-
-
-
n.d n.d
n.d
0.176±0.02
n.d
b
c
d
e
aIsolated yield, Against CQS NF54 strain, Against CQR Dd2 strain, activity in µM, mean of three independent
f
g
experiments, ClogP calculated from ChemDraw Ultra 11.0 plus, Cytotoxicity determined on mammalian Vero
cells, 50% inhibitory concentration, as determined by measuring the cell viability with MTT assay, Selectivity
Index (SI) is calculated as IC50 (Vero cell line)/IC50 (NF54), not determined, Standard references: chloroquine,
lartesunate, memetine.
h
i
j
k
2.4 Physicochemical parameters
Aqueous solubility (SW) and lipophilicity represent important indicators of the
pharmacokinetics of a drug in a biological system. An oral drug formulation must
possess critical balance of the lipophilic and hydrophilic properties so as to pass
through biological membranes and barriers to eventually enter the systemic circulation
[54,55]. Various physicochemical parameters such as aqueous solubility SW,
distribution coefficient (log D) and pKa of the hybrids of this series were evaluated and
results are summarised in Table 2. The pKa of quinoline-based hybrids is one of the
9

ACCEPTED MANUSCRIPT
factors that governs accumulation of the drug in the acidic FV of the parasite [56]. The
solubility in n-octanol (SOC) was calculated from the experimentally determined SW
and log
D
data using the equation: log SOC = log
D
+ log SW [57]. The aqueous
in an -octanol/PBS bu er
solubility (SW) in PBS (phosphate buffer saline) and log
D
n
ff
mixture at the cytosolic pH 7.4 of the parasite was evaluated using HPLC (Fig. S49-
S66) [58].
Table 2.a Acid dissociation constants (pKa), aqueous solubility (Sw), distribution
coefficient (log
D) and solubility in
n
-octanol (SOC) of 30 and 32
.
b
b
Hybrid
30a
30b
30c
30d
30e
30f
pKa1
7.38
7.49
7.50
7.15
7.55
7.25
6.55
7.25
7.20
7.40
7.45
7.10
9.78
9.65
9.20
7.70
7.50
8.30
pKa2
Sw (µM)
5.64±0.43
3.11±0.89
1.21±0.43
0.55±0.14
0.58±0.07
8.01±0.09
log D
Soc (µM)c
34.19
48.3
10.78
10.90
-
0.78±0.27
1.19±0.01
1.72±0.05
1.43±0.02
1.31±0.31
0.42±0.17
63.53
14.87
12.14
21.24
44.67
21.72
53.08
54.38
16.72
35.63
16.34
66.22
52.48
60.53
37.41
17.78
10.65
9.75
-
8.65
10.60
9.55
9.15
10.60
10.75
-
11.30±0.89 0.58±0.23
30g
30h
30i
3.30±0.82
3.21±0.37
7.03±0.25
2.30±0.11
0.81±0.50
1.21±0.11
0.88±0.28
0.86±0.33
30j
30k
30l
19.10±0.03 0.27±0.08
3.31±0.05
5.47±0.87
1.78±0.17
4.54±0.41
2.93±0.33
7.18±0.89
0.69±0.24
1.08±0.37
1.46±0.07
1.12±0.49
1.10±0.23
0.39±0.12
32a
32b
32c
32d
32e
32f
-
-
-
aThe data represents mean of three experiments conducted at 298.1
b
b
K. pKa of triazole nitrogen atom; pKa of N atom quinolone;
cSolubility in n-octanol at pH 7.4, calculated from experimental
aqueous solubility (Sw) and distribution coefficient log D (n-
octanol/PBS buffer) using equation: log SOC = log D + log Sw.
Reasonable correlations could be drawn between the calculated distribution
coefficients (Table 2) and the observed trends in the antiplasmodial activity of the
hybrids. Most of the potent hybrids 30a-f (n = 3) group have higher clog
P and log D
values compared to the analogues 30g-l (n = 2). The more lipophilic 30a [log
D =
10

ACCEPTED MANUSCRIPT
0.78, clog
than 30g [log
= 1.19, clog
P
= 5.555, IC50= 1.01 µM (NF54) and 1.65 µM (Dd2)] is also more potent
D
= 0.58, clog
P = 5.278, IC50= 10.38 µM (NF54)]. Similarly, 30b [log
D
P = 6.084, IC50 = 0.89 µM (NF54) and 1.37 µM (Dd2)] being more
lipophilic is more active than 30h [log
(NF54) and 2.52 µM (Dd2)].
D = 0.81, clog P = 5.807, IC50 = 1.49 µM
Since quinoline-based hybrids are expected to accumulate in acidic FV of the parasite
in much the same way as CQ and inhibit hemazoin formation, pKa’s of the synthesized
hybrids 30 and 32 were determined [59] using UV-visible absorption
spectrophotometric titrations (Fig. S67-S102) and the data is summarized in Table 2.
Hybrids 30 and 32 showed insignificant variation in the pKa values. The higher
magnitude of the pKa values (Table 2) suggest significant basic character of these
hybrids and are thus expected to accumulate in the FV of the parasite. However, the
antiplasmodial activity of these hybrids bears no regular trend with the experimentally
determined pKa values, which was not unexpected as other factors such as lipophilicity
etc. also important parameters influencing activity.
2.5 Mode of action studies
2.5.1 Interaction with hemin chloride
4-Aminoquinoline base antimalarials are known to exert their antiplasmodial effect by
complexation with Fe3+PPIX thus inhibiting the polymerization of toxic heme to
hemozoin [60,61]. In order to rationalize the binding mode of this series of hybrids,
interaction of the most potent member 30d of the current series with hemin chloride
was studied by performing spectrophotometric titrations at pH 7.4 and 5.6
(approximate pH of parasitic FV) [62]. Hemin is expected to be in monomeric state in
40% aqueous DMSO solution [56]. Coupled with this, the higher clog P (6.13) of 30d
compared to CQ (5.10) suggested the use of a mixture of aqueous buffer/DMSO as
solvent. The UV-visible absorption spectrum (Figure 3) recorded a gradual decrease in
the intensity of the Soret band (Fe3+PPIX) at 402 nm, upon incremental addition (upto
40 µM) of 30d in hemin chloride solution, both at pH 7.4 and 5.6. Further increase in
concentration of 30d did not lead to any significant change in absorbance of the Soret
band.
As CQ is known to interact with dimeric
µ-oxoheme, the interaction between µ-
oxoheme and 30d (Figure S105) was also determined at pH 5.8 [63,64]. The UV-
visible absorption spectrum (Figure S105A) showed a gradual decrease in the intensity
11

ACCEPTED MANUSCRIPT
of the Soret band (Fe3+PPIX) at 360 nm, upon incremental addition of 30d (upto 26.7
µM) to hemin chloride solution (10 µM) in 20 mM phosphate buffer saline (pH 5.8).
Employing Job’s method of continuous variation, a 1:1 stoichiometry of the most
stable complex between 30d and monomeric hemin chloride and
established (Figure S103 and S105B). The binding constants (log
for complex of 30d with momomeric heme and -oxoheme, were calculated using
Hyp-Spec, a non-linear square fitting programme [65], which suggested stronger
binding of 30d with -oxoheme. For comparison purposes, similar titrations (at pH 7.4
µ
-oxoheme was
K
= 5.434, Table 3)
µ
µ
and 5.6) of monomeric hemin chloride were also performed with CQ (Figure S104).
The binding constants of 30d were found to be less than standard CQ consistent with
the observed trend of the antiplasmodial activity (Table 1). The decrease in pH from
7.4 to 5.6 resulted in only an insignificant change in the binding constant, which
suggests strong binding of 30d with hemin chloride at both the experimental pH
values.
0.6
0.4
0.2
0
0.4
0.2
(B)
(A)
0 µM
0 µM
0
0.000025 0.00005
Conc/M
0
0.00E+00 2.50E-05 5.00E-05
Conc/M
40 µM
40 µM
Figure 3. (A) Changes in absorption of monomeric hemin chloride (402 nm) (2.4 µM) at pH
7.4 (0.02 M HEPES buffer) and (B) at pH 5.6 (0.02 M MES buffer), upon incremental
addition of 30d (up to 40 µM). (Inset: Plot of A402 nm vs. conc.)
Thus, we propose that hybrid 30d and other hybrids of this series in analogy could
potentially inhibit hemozoin formation by blocking the growing face of heme. Further,
to provide evidence for inhibition of
with 30d, polymerization of hemin chloride into
sodium acetate buffer (4.5 µM) [66]. FT-IR for
β
-hematin formation by complexation of heme
-hematin was carried out at 60 C in
-hematin exhibited peaks at 1209 and
β
°
β
1662 cm-1, characteristic of iron carboxylate bonds (Figure S106A). However, the FT-
12

ACCEPTED MANUSCRIPT
IR of the complex prepared according to a literature report [67] upon incubation of 1.2
equiv of 30d with hemin chloride showed absence of these peaks, which further
corroborated complexation of hemin chloride with 30d (Figure S106B). Also, the FT-
IR spectrum of the complex prepared as above differ significantly from that of both
30d (Figure S107A) as well as hemin chloride (Figure S107B).
Table 3. Binding constant (log K) of 30d and CQ with hemic chloride (pH 7.4), µ-oxo
dimeric heme (pH 5.8), CT DNA and pUC18 DNA.
log K
hemina
CT DNA
pUC18 DNA
µ-oxo dimeric
heme
4.679± 0.0104 (4.274± 0.0284)
5.16±0.0230(4.67±0.6546)
5.434 ±0.3162
n.d
3.74
n.d
3.92
n.d
30d
CQ
aValues in parenthesis correspond to the titration at pH 5.6; n.d: not determined.
To obtain further insight into the binding interaction of 30d with monomeric hemin
1
chloride, H NMR titration of a solution (40% DMSO-d6 in D2O) of 30d was
performed with increasing concentration of hemin chloride (Figure 4). Upon addition
of 10 mol% of hemin chloride, the spectra depicted significant shift (Figure 4) in the
relevant signals, indicative of an interaction between hemin chloride and hybrid 30d
.
Subsequent addition of hemin chloride (20 mol%) led to considerable broadening of
1
the H NMR peaks possibly due to increased concentration of the paramagnetic
Fe3+PPIX.
NO2
i
i
o
CH3
d
d
O
o
H3
e
C
O
N
j
a
k
n
m
N
O
H3C
l
N
NH
N
N
N
c
b
g
h
Cl
f
(C)
(B)
(A)
e d c
b
gf
a
i,h
1
Figure 4. H NMR spectral changes of 30d [40% DMSO:D2O\D2SO4 (10 µl)] upon
incremental [0, 10 and 20 mol% (A-C, respectively)] addition of hemin chloride.
13

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Further, HRMS mass spectrum (Figure 5A) of equimolar mixture of 30d and hemin
chloride in (40% DMSO+H2O) exhibited a peak at m/z 1268.3775 corresponding to
the formula C64H61N12Cl2O9Fe, which further corroborated a 1:1 stoichiometry of the
complex formed between 30d and hemin chloride. Thus, on the basis of results
obtained above as well as according to literature precedence [68], we propose an
interaction between the iron atom of hemin chloride with the quinoline nitrogen as
shown in Figure 5B.
Intensity.
+MS, 0.0-0.2min #2-10
1269.3972
(A)
12.5
10.0
30d: Hemin
1268.3775
1270.3946
7.5
5.0
2.5
0.0
1264
1266
1268
1270
1272
1274
1276
m/z
(B)
N
N
N
N H
N
Fe
N
N
C l
N
N
H
O
H
O
O
O
N
O
N
N O2
H 3
C
O
O
C H 3
H 3
C
Figure 5. (A) The solution phase mass spectra of 30d (5 µmol) upon addition of hemin
chloride (5 µmol) in 40% aq. DMSO solution. (B) Proposed binding model of 30d with
hemin chloride.
2.5.2 DNA binding studies
Ionic interactions between protonated amine residues of the drugs and phosphate
groups of DNA [69,70] and/or interaction between the nucleotide bases of DNA and
the aromatic nucleus of a drug may result in the enhanced stability of DNA helical
configuration towards thermal denaturation. The buffering action of CQ improves
gene transfection efficiency by inhibiting lysosomal enzyme degradation or by
facilitating the release of DNA from endocytic pathways [71]. The DNA of P.
falciparum has poor GC content (17-19%) [72] compared to the human DNA (43%).
However, CQ is known to bind predominantly to GC rich DNA. Thus a decisive
relationship between antimalarial activity and DNA intercalation ability of CQ is still
ambiguous. Thus, complexation studies of the most active hybrid 30d of the current
14

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series were undertaken with both GC rich CT DNA and AT rich pUC 18 DNA using
absorption as well as fluorescence spectrophotometry.
Incremental addition of CT DNA (upto 260.42 µM) to a buffered methanolic solution
(66 µM) of 30d, showed a gradual decrease (Figure S108) in absorption at 298 nm,
and characteristic quinoline bands at 330 nm. An isosbestic point was observed at 280
nm, which suggested the intercalative nature of the interaction between DNA and 30d
.
The association constant (log = 2.29) was calculated from the Benesi-Hildebrand
K
equation [73]. The increase in thermal stability of DNA (Figure S109) marked by an
increase in melting temperature (Table S1) of CT DNA in the presence of 30d, further
corroborated the intercalative nature of interactions [74,75].
Likewise, the incremental addition of CT DNA (upto 260.42 µM) to a solution (17.1
µM) of hybrid 30d in buffered methanol led to a gradual decrease in emission intensity
at 375 nm (Figure 6A), which eventually got completely quenched. Similar changes
were observed upon incremental addition of pUC 18 DNA (upto 148 µM) to a solution
(17.1 µM) of 30d (Figure 6B). The binding constants for CT DNA (log
pUC 18 (log = 3.92) suggested that 30d might also interact with parasitic DNA
besides causing inhibition of heme polymerization to exert its antiplasmodial effect.
K = 3.74) and
K
(B)
375 nm
375 nm
(A)
1000
900
800
700
600
500
400
300
200
100
0
0 µM
0 µM
1000
900
800
700
600
500
400
300
200
100
0
148 µM
260.42 µM
350
375
400
425
450
475
350
375
400
425
450
475
Wavelength (nm)
Wavelength (nm)
Figure 6. Changes in fluorescence emission spectra of 30d (17.1 µM, in buffered
CH3OH, λex = 330 nm, λem = 375 nm) upon incremental addition of (A) CT DNA
(upto 260.42 µM); (B) pUC18 DNA (upto 148 µM).
3. Conclusions
A new series of 1,2,3-triazole tethered pyrimidine chloroquinoline hybrids 30a-l and
32a-f was initially screened for in vitro antiplasmodial activity against the drug
15

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sensitive P. falciparum NF54 strain. The most active hybrids against NF54 were
further tested against the drug resistant Dd2 strain. Hybrids 30d and 30c were the most
potent against both strains. Cytotoxicity studies were performed against the
mammalian vero cell line suggested that most of the hybrids were devoid of any
significant cytotoxicity at sub-micromolar concentrations. The most potent hybrid
30d, exhibited high selectivity index (SI = 317.50). Structure activity relationships
delineated the effect of both the nature of the substituent on the pyrimidine ring and
the length of the linker on the antiplasmodial activity of the hybrids. Hemin and DNA
binding studies were performed for 30d to shed light on mode of action of synthesized
hybrids. The binding (DNA and hemin) studies suggest multiple site of action for
these hybrids, which could also account for the observed antiplasmodial activity.
4. Experimental
4.1.1 Materials and reagents
Most reactions were performed under inert atmosphere created by using purified
(oxygen free) dry nitrogen gas. Thin layer chromatography (TLC) was performed on
pre-coated aluminium sheets (Merck 60F254, 0.2 mm) and the chromatograms were
visualized under UV light (254 nm). For column chromatography silica gel (60–120
mesh) was employed and ethyl acetate/or mixtures of ethyl acetate/hexanes/or ethyl
acetate/methanol were used as eluents. Liquid reagents used for the synthesis were
purified/dried following standard drying agents and/or distilled over 4 Å molecular
sieves: DCM (CaCl2), CH3CN (P2O5), CH3OH/C2H5OH (Mg treatment), DMF (4 Å
molecular sieves), tetrahydrofuran/ benzene and toluene (Na-benzophenone ketyl).
K2CO3 was dried overnight in furnace (300°C) before use. The reagents were used as
purchased.
HPLC
grade
solvents
were
utilized
for
spectrophotometric/spectrofluorimetric studies. Recrystallization of the solid products
was achieved using appropriate mixtures of DCM and hexane.
4.1.2 Instrumentation
13
1H NMR (500 MHz) and C NMR (125 MHz) spectra were recorded on Bruker
Biospin Avance III HD (500 MHz), instrument using tetramethylsilane (TMS) as
internal standard and CDCl3 and/or DMSO-d6 as deuterated solvents. The purity of the
products was checked by elemental analysis performed on a Thermoelectron FLASH
EA1112 CHNS analyzer and was within ± 0.4% of the calculated values. High
16

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resolution mass spectrum (HRMS) was recorded using Bruker HRMS MICROTOF-Q
II mass spectrometer. IR spectra were recorded using Perkin-Elmer FTIR-C92035
spectrometer in the range 400–4000 cm-1 using KBr pellets or in DCM solutions.
Fluorescence measurements were performed on a Perkin Elmer LS-50 instrument.
UV-visible absorption studies were carried out using UV-1800 SHIMADZU UV-
Spectrophotometer using matched quartz cuvettes (path length = 1 cm). The cell
holder was maintained at 25 ± 1 °C using a Peltier temperature controller. The pH
titrations were performed on Equip-Tronics Digital pH meter (model–EQ 610) The
HPLC chromatograms were recorded using a Waters 2489 HPLC separations module
equipped with Waters 2489 photodiode array detector, Waters 515 HPLC pump with
20 µL standard sample injector, Symmetry (4.6 mm × 75 mm), 3.5 µm C-18 reverse
phase column, flow rate of 0.5 mL/min., Empower software (Waters Corporation,
Milford, MA, USA). A calibration plot of the peak area versus compound
concentration for each compound showed excellent linearity (0.99 < r2< 1) over the
concentration range (0−10 µM) employed for analysis. The purity of the products was
also checked by HPLC and was ≥ 96%. All reported yields are isolated yields.
4.1.3 Determination of aqueous solubility
The aqueous solubility was determined by preparing saturated solutions in PBS
(phosphate buffer saline) buffer (10 mM) at pH 7.4. The compounds were stirred in a
water bath at 32 °C for 24 h. The saturated solution was filtered and the filtrate was
analyzed using HPLC. Further solutions of known concentrations of test compounds
were prepared in DMSO and HPLC chromatograms recorded. Plotting concentration
(1 x 10-3 to 1 x 10-7 M) vs area under the peak furnished calibration curve. The exact
concentration of the test compounds in saturated aqueous filtrate could be deduced
from the calibration curve, which corresponded to the limit of solubility of the
compound under the experimental conditions used.
4.1.4 Determination of partition coefficient (log D)
n
-Octanol and PBS buffer solutions (pH 7.4) were mixed in equal volumes and were
saturated with each other by stirring for 24 h and then separated. Accurately weighed
30 and 32 (0.002 g) were dissolved in 0.75 mL of pre-saturated -octanol in graduated
n
(2 mL) test tube and sonicated for 10 min. To the above sonicated solutions 0.75 mL
pre-saturated PBS buffer solution was added and solutions were again sonicated for 1h
17

ACCEPTED MANUSCRIPT
and centrifuged for 30 min at 4000 rpm. The ratio of final volume of the PBS buffer
and
n-octanol was close to 1. Aqueous and organic phases were separated and
concentration of 30 and 32 in both the phases was measured by HPLC using the
calibration (concentration vs area) curve prepared above. The log D values were
calculated as logarithmic ratios of concentrations in the n-octanol phase compared to
the concentrations in the buffer. The experiments were performed in triplicate.
4.1.5 Determination of pKa
Stock solutions (10 mM) of compounds 30 and 32 were prepared in DMSO. The
working solutions (30 µM) were obtained by diluting stock solution of a compound
with distilled water so that overall DMSO content in the solution corresponded to 30%
v/v. The pH of the solutions was adjusted to 3 by adding HCl solution (0.1 M) and in
the range 3-12 using NaOH (0.1 M). The UV-visible absorption spectra were recorded
in the order of increasing pH (3-12). All measurements were carried out in triplicate at
298.1 K. Plots of absorption vs apparent pH values at 330 nm of sample solution
furnished sigmoid curves, from where pKa values were determined as the center point
in the respective titration curve.
4.2 Heme binding studies
4.2.1 Binding with monomeric heme
The stock solution (1.2 mM) of hemin chloride was prepared by dissolving hemin
chloride (7.8 mg) in DMSO (10 mL). Working solution (2.4 µM) of hemin chloride
was prepared by diluting (20 µL) hemin stock solution with 1 mL 0.2 M HEPES
buffer (pH 7.4), 4 mL DMSO and making final volume up to 10 mL with double
distilled deionized water. Likewise, the stock solution (2 mM) of 30d was prepared in
DMSO. Hemin chloride solution (2.4 µM, 3mL) was titrated with increasing
concentrations (upto 40 µM) of 30d. Subsequent to each addition of aliquot of the
compound in the solution of hemic chloride, absorbance was recorded at 402 nm.
Likewise, solution of hemin chloride and 30d were titrated at pH 5.6 (2-[N-
morpholino]ethanesulphonate (MES, pH 5.4) buffer was used).
4.2.2 Binding with dimeric
µ
-oxoheme
-oxoheme was prepared by dissolving hemin chloride
The stock solution (1 mM) of
µ
in NaOH (0.1 M). To ensure complete dissolution of hemin chloride, solution was
sonicated for 30 min. The resulting solution was subsequently diluted to achieve
18

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working concentration (10 µM) by adding phosphate buffer (20 mM, pH 5.8). On the
other hand, stock solution (2 mM) of 30d was prepared in DMSO. Titration was
performed by successive addition of aliquots of stock solution of 30d (upto 26.7 µM)
to the solution of
µ-oxoheme (10 µM, 3 mL) prepared above and the absorbance was
recorded at 364 nm.
4.3.1 Complexation of hemin chloride and 30d
The complex was prepared by dissolving equimolar solution of hemin chloride (15
mg) and 30d (9.14 mg) in NaOH (0.1 N, 3mL). A solution containing 1.74 mL of
aqueous sodium acetate (12.9 M, pH 5.00) and 0.3 mL of HCl (1.0 M) pre-warmed at
60 °C, was then added. After 1 h incubation at 60 °C, the reaction mixture was filtered
over millipore filters (0.45 µm). The solid obtained was thoroughly washed with
distilled water (3 x 30 mL) and was vacuum dried over P2O5. Then, the solid
precipitate was characterized by FT-IR spectroscopy.
4.4 DNA binding studies
A stock solution (2 mM) of 30d was prepared in methanol. The DNA binding
experiments were carried out by diluting the stock solution of 30d with 1:3 Tris-
EDTA (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) (TE) buffer/methanol. Likewise, a
stock solution of DNA (pUC 18 and CT DNA) was prepared by dissolving DNA pellet
in the TE buffer. The DNA concentration was estimated by comparing the absorbance
intensity (260 nm) with a known molar absorption coefficient value 6600 dm3 mol-1
cm-1. The purity of DNA was established from ratio of absorbance intensity at 260 and
280 nm, which in the present study is 1.88, suggesting that DNA is free from protein.
The titration experiments were performed by varying the concentration of DNA and
keeping the 30d concentration constant (66.6 µM). All the UV-visible absorption
spectra were recorded after equilibration of solution for 5 min. Similarly using
fluorimetry, the titration was performed by varying the concentration of DNA [30d
:
CT DNA (upto 260.42 µM) and pUC 18 DNA (upto 148 µM)] and keeping the
compound concentration constant (30d: 17.1 µM).
4.5 Thermal denaturation of DNA
DNA melting experiment was performed by monitoring the absorbance of CT DNA
(151 µM NP) at 260 nm at various temperatures in absence and presence 30d/CQ in
5:1 ratio of the DNA and 30d/CQ with ramp rate of 0.5 °C/min in a 40% DMSO/TE
19

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buffer (pH 7.4) with NaCl (0.5 mM). The thermal melting temperature was calculated
by plotting dA/dT vs temperature.
4.6.1 Synthesis of alkyl 4-methyl-6-aryl-2-(prop-2-yn-1-yloxy) pyrimidine-5-
carboxylates
To a solution of appropriate 2-chloropyrimidine (3.80 mmol) in dry CH3CN (30 mL),
dry K2CO3 (1.57 g, 11.40 mmol) and propargyl alcohol (0.32 g, 5.70 mmol) were
added in that sequence and the reaction mixture was refluxed overnight. After
completion (TLC), CH3CN was removed under vacuum. Water (30 mL) was then
added to the residue and the product extracted with DCM (2 x 20 mL).
Dichloromethane extract was dried over anhydrous sodium sulphate and the solvent
was removed under vacuum. The crude product was purified by column
chromatography using ethyl acetate/hexane mixtures as eluent to obtain pure 29. The
characteristic data of compounds prepared as above is given below.
4.6.1.1 Methyl 4-methyl-6-phenyl-2-(prop-2-yn-1-yloxy)pyrimidine-5-carboxylate
29a
White solid. Yield: 94%. Rf: 0.5 (30% ethyl acetate/ hexane). Mp 69-73 °C. IR (KBr):
νmax 1531, 1527, 1710, 2987, 3252 cm-1.1H NMR (500 MHz, CDCl3, 25 °C): δ 2.48 (t,
J
= 2.5 Hz, 1H, CCH), 2.58 (s, 3H, C-6 CH3), 3.69 (s, C-5 OCH3), 5.09 (d, J = 5.0, 2H,
13
C-2 OCH2), 7.43-7.50 (m, 3H, ArH), 7.64-7.69 (m, 2H, ArH). C NMR (125 MHz,
CDCl3, 25 °C): 22.7, 52.4, 55.1, 74.9, 78.2, 120.0, 128.3, 128.4, 130.3, 137.6, 163.0,
166.2, 168.5 and 168.9. Anal. calcd. for C16H14N2O3: C, 68.07; H, 5.00; N, 9.92;
found: C, 68.26; H, 4.85, N, 9.10. HRMS calcd. for C16H14N2O3: m/z 282.1004; found:
m/z 282.1008 (M+).
4.6.1.2 Ethyl 4-methyl-6-phenyl-2-(prop-2-yn-1-yloxy)pyrimidine-5-carboxylate
29b
White solid. Yield: 94%. Rf: 0.5 (30% ethyl acetate/ hexane). Mp 65-67 °C. IR (KBr):
νmax 1533, 1552, 1708, 2985, 3250 cm-1.1H NMR (500 MHz, CDCl3, 25 °C): δ 1.07 (t,
J
= 5.0 Hz, 3H, C-5 OCH2CH3), 2.50 (t,
J
= 2.5 Hz, 1H, CCH), 2.61 (s, 3H, C-6 CH3),
= 2.5 Hz, C-2 OCH2), 7.44-7.69
4.18 (q, = 10.0 Hz, 2H, C-5 OCH2CH3), 5.11 (d,
J
J
13
(m, 5H, ArH). C NMR (125 MHz, CDCl3, 25 °C): δ 13.6, 22.7, 55.1, 61.7, 74.7,
78.2, 120.5, 128.3, 128.4, 130.2, 137.5, 163.0, 166.3, 168.1 and 168.9. Anal. calcd. for
C17H16N2O3: C, 68.91; H, 5.44; N, 9.45; found: C, 68.95; H, 5.51, N, 9.41. HRMS
calcd. for C17H16N2O3: m/z 296.1161; found: m/z 296.1169 (M+).
20

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4.6.1.3
Isopropyl
4-methyl-6-phenyl-2-(prop-2-yn-1-yloxy)pyrimidine-5-
carboxylate 29c
Colourless oil. Yield: 94%. Rf: 0.5 (30% ethyl acetate/hexane). IR (dichloromethane):
νmax 1556, 1585, 1713, 2939, 2981, 3047, 3255 cm-1.1H NMR (500 MHz, CDCl3, 25
°C): δ 1.09 (d,
J = 10.0 Hz, 6H, C-5 OCH(CH3)2), 2.49 (t, J = 2.5 Hz, 1H, CCH), 2.59
(s, 3H, C-6 CH3), 5.05-5.10 (m, 1H, C-5 OCH(CH3)2 and 2 H C-2 OCH2), 7.43-7.49
13
(m, 3H, ArH), 7.67-7.69 (m, 2H, ArH). C NMR (125 MHz, CDCl3, 25 °C): δ 21.2,
22.6, 55.1, 69.6, 74.7, 78.3, 121.0, 128.4, 130.1, 137.5, 162.9, 166.1, 167.6 and 168.6.
Anal. calcd. for C18H18N2O3: C, 69.66; H, 5.85; N, 9.03; found: C, 69.71; H, 5.71, N,
9.08. HRMS calcd. for C18H18N2O3: m/z 310.1314; found: m/z 310.1314 (M+).
4.6.1.4 Isopropyl 4-methyl-6-(4-nitrophenyl)-2-(prop-2-yn-1-yloxy)pyrimidine-5-
carboxylate 29d
White solid. Yield: 94%. Rf: 0.5 (30% ethyl acetate/ hexane). Mp 96-98 °C. IR (KBr):
νmax 1520, 1554, 1606, 1721, 2937, 2985, 3084, 3109, 3279 cm-1.1H NMR (500 MHz,
CDCl3, 25 °C): δ 1.12 (d,
J = 5.0 Hz, 6H, C-5 OCH(CH3)2), 2.50 (t, J = 2.5 Hz, 1H,
CCH), 2.62 (s, 3H, C-6 CH3), 5.08-5.12 (m, 1H, C-5 OCH(CH3)2 and 2 H C-2 OCH2),
13
7.83 (d,
J = 5.0 Hz, 2H, ArH), 8.31 (d, J = 10.0 Hz, 2H, ArH). C NMR (125 MHz,
CDCl3, 25 °C): δ 21.3, 22.9, 55.4, 70.1, 75.0, 77.9, 121.1, 123.57, 129.5, 143.5, 148.7,
163.0, 163.9, 166.7 and 169.6. Anal. calcd. for C18H17N3O5: C, 60.84; H, 4.82; N,
11.83; found: C, 60.96; H, 4.61, N, 11.76. HRMS calcd. for C18H17N3O5: m/z
355.1168; found: m/z 355.1172 (M+).
4.6.1.5 Isopropyl 4-methyl-6-(2-nitrophenyl)-2-(prop-2-yn-1-yloxy)pyrimidine-5-
carboxylate 29e
White solid. Yield: 94%. Rf: 0.5 (30% ethyl acetate/ hexane). Mp 98-100 °C. IR
(KBr): νmax 1532, 1552, 1708, 2938, 3041, 3250 cm-1.1H NMR (500 MHz, CDCl3, 25
°C): δ 0.94 (d,
J
= 5.0 Hz, 6H, C-5 OCH(CH3)2), 2.47 (t,
J
= 2.5 Hz, 1H, CCH), 2.70
= 2.5 Hz, 2H, C-2
= 5.0 Hz, 1H, ArH), 7.61-7.70 (m, 2H, ArH), 8.20 (d, = 10.0 Hz,
(s, 3H, C-6 CH3), 4.91-4.96 (m, 1H, C-5 OCH(CH3)2), 5.02 (d,
J
OCH2), 7.34 (d,
J
J
13
1H, ArH). C NMR (125 MHz, CDCl3, 25 °C): δ 21.3, 24.1, 55.7, 69.5, 75.2, 77.9,
119.9, 124.7, 130.0, 130.3, 133.4, 134.5, 147.5, 162.9, 165.4, 166.7 and 171.0. Anal.
calcd. for C18H17N3O5: C, 60.84; H, 4.82; N, 11.83; found: C, 60.71; H, 4.67, N, 11.97.
HRMS calcd. for C18H17N3O5: m/z 355.1168; found: m/z 355.1160 (M+).
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4.6.1.6 Ethyl 4,6-dimethyl-2-(prop-2-yn-1-yloxy)pyrimidine-5-carboxylate 29f
Colorless oil. Yield: 94%. Rf: 0.5 (30% ethyl acetate/hexane). IR (dichloromethane):
νmax 1510, 1577, 1715, 2989, 3257 cm-1. 1H NMR (500 MHz, CDCl3, 25 °C): δ 1.40 (t,
J
= 7.5 Hz, 3H, C-5 OCH2CH3), 2.48 (t,
J
= 2.5 Hz, 1H, CCH), 2.52 (s, 3H, C-4 CH3
and 3H, C-6 CH3), 4.41 (q,
J
= 5.0 Hz, 2H, C-5 OCH2CH3), 5.03 (d, = 5.0 Hz, 2H,
J
13
C-2 OCH2). C NMR (125 MHz, CDCl3, 25 °C): δ 14.1, 23.1, 52.6, 54.9, 74.6, 75.6,
120.9, 162.7, 167.3 and 168.2. Anal. calcd. for C12H14N2O3: C, 61.53; H, 6.02; N,
11.96; found: C, 61.64; H, 6.25, N, 11.78. HRMS calcd. forC12H14N2O3: m/z
234.1004; found: m/z 234.1006 (M+).
4.6.2 Synthesis of triazole tethered pyrimidine-chloroquinoline hybrids
A solution of appropriate 29 (0.35 mmol) and 28/31 (0.35 mmol) in EtOH: H2O (8:2,
v/v) (10 mL), CuSO4.5H2O (0.02 mmol) and sodium ascorbate (0.1 mmol) was stirred
at ambient temperature. After completion (TLC), solvent was removed under vacuum.
Water (30 mL) was then added to the residue and the product was extracted with DCM
(2 x 20 mL). The organic phase was dried over anhydrous sodium sulphate. After
removing DCM under reduced pressure, the crude product was purified by column
chromatography using ethyl acetate/hexane mixtures as eluent to obtain pure 30a-l and
32a-f, respectively. The characteristic data of compounds prepared as above is given
below.
4.6.2.1 Methyl 2-[(1-(3-(7-chloroquinolin-4-ylamino)propyl)-1H-1,2,3-triazol-4-
yl)methoxy]-4-methyl-6-phenylpyrimidine-5-carboxylate 30a
White solid. Yield: 86%. Rf: 0.4 (10% ethyl acetate/MeOH). Mp 113-115 °C. IR
(KBr): νmax 1550, 1582, 1611, 1726, 2853, 2924, 3369 cm-1.1H NMR (500 MHz,
CDCl3, 25 °C): δ 2.29 (m, 2H, NHCH2CH2CH2N-), 2.56 (s, 3H, C-6 CH3), 3.38 (m,
2H, NHCH2CH2CH2N-), 3.69 (s, 3H, C-5 OCH3), 4.49 (t,
NHCH2CH2CH2N-), 5.62 (s, 2H, C-2 OCH2), 5.86 (br, 1H, NH, D2O exchangeable),
6.34 (d, = 5.0 Hz, 1H, CQ), 7.33-7.79 (m, 5H, ArH and 3H, CQ), 7.92 (s, 1H,
J = 7.5Hz, 2H,
J
triazolyl H), 8.48 (s, 1H, CQ). 13C NMR (125 MHz, CDCl3, 25 °C): δ 22.8, 28.4, 39.9,
47.9, 52.5, 61.1, 98.8, 117.2, 120.1, 121.5, 123.6, 125.5, 128.1, 128.3, 128.5, 130.4,
135.0, 137.3, 143.8, 148.8, 149.6, 151.6, 163.4, 166.3, 168.6 and 169.0. HPLC
[Methanol/H2O, 25:75, 0.5 mL/min, 330 nm] tR= 1.019 min. Anal. calcd. for
C28H26N7ClO3: C, 61.82; H, 4.82; N, 18.02; found: C, 61.71; H, 4.80, N, 18.07. HRMS
calcd. for C28H26N7ClO3: m/z 543.1786; found: m/z 544.1978 (M++1).
22

ACCEPTED MANUSCRIPT
4.6.2.2 Ethyl 2-[(1-(3-(7-chloroquinolin-4-ylamino)propyl)-1H-1,2,3-triazol-4-
yl)methoxy]-4-methyl-6-phenylpyrimidine-5-carboxylate 30b
White solid. Yield: 84%. Rf: 0.4 (10% ethyl acetate/MeOH). Mp 93-95 °C. IR (KBr):
νmax 1552, 1583, 1719, 2956, 2982, 3326 cm-1. 1H NMR (500 MHz, CDCl3, 25 °C): δ
1.04 (t,
(s, 3H, C-6 CH3), 3.34 (m, 2H, NHCH2CH2CH2N-), 4.16 (q,
OCH2CH3), 4.51 (t, = 7.5 Hz, 2H, NHCH2CH2CH2N-) , 5.62 (s, 2H, C-2 OCH2),
= 5.0 Hz, 1H, CQ), 7.22 (d, = 10.0 Hz, 1H, CQ), 7.41-7.47 (m, 3H, ArH),
= 10.0 Hz, 2H, ArH), 7.74 (s, 1H, CQ), 7.81 (s, 1H, triazolyl H), 7.96 (d,
= 5.0 Hz, 1H, CQ). 13C NMR (125 MHz, CDCl3, 25 °C):
J
= 7.5 Hz, 3H, C-5 OCH2CH3), 2.32-2.34 (m, 2H, NHCH2CH2CH2N-), 2.57
J
= 5.0 Hz, 2H, C-5
J
6.12 (d,
7.62 (d,
J
J
J
J
=
10.0 Hz, 1H, CQ), 8.15 (d,
J
δ 13.6, 22.7, 28.2, 40.3, 47.8, 61.0, 61.7, 97.7, 116.0, 120.4, 123.1, 123.6, 124.0,
126.2, 128.2, 128.4, 130.2, 137.1, 137.4, 142.9, 143.5, 146.2, 152.7, 163.4, 166.5,
168.0 and 168.9. HPLC [Methanol/H2O, 25:75, 0.5 mL/min, 330 nm] tR= 1.280 min.
Anal. calcd. for C29H28N7ClO3: C, 62.42; H, 5.06; N, 17.57; found: C, 62.37; H, 4.80,
N, 17.61. HRMS calcd. for C29H28N7ClO3: m/z 557.1942; found: m/z 558.2265
(M++1).
4.6.2.3 Isopropyl 2-[(1-(3-(7-chloroquinolin-4-ylamino)propyl)-1
yl)methoxy]-4-methyl-6-phenylpyrimidine-5-carboxylate 30c
H-1,2,3-triazol-4-
White solid. Yield: 85%. Rf: 0.3 (10% ethyl acetate/MeOH). Mp 108-111 °C. IR
(KBr): νmax 1554, 1584, 1714, 2853, 2924, 2980, 3316 cm-1.1H NMR (500 MHz,
CDCl3, 25 °C): δ 1.07 (d,
NHCH2CH2CH2N-) 2.56 (s, 3H, C-6 CH3), 3.34 (m, 2H, NHCH2CH2CH2N-), 4.50 (t,
= 7.5 Hz, 2H, NHCH2CH2CH2N-), 5.03-5.08 (m, 1H, C-5 OCH(CH3)2), 5.60 (s, 2H,
C-2 OCH2), 6.18 (d, = 5.0 Hz, 1H, CQ), 7.24 (d, = 5.0 Hz, 1H, CQ), 7.40-7.47 (m,
3H, ArH), 7.62 (d, = 10.0 Hz, 2H, ArH), 7.77 (s, 1H, CQ), 7.80 (s, 1H, triazolyl H),
7.91 (d,
= 5.0 Hz, 1H, CQ), 8.25 (s, 1H, CQ). 13C NMR (125 MHz, CDCl3, 25 °C): δ
J = 5.0 Hz, 6H, C-5 OCH(CH3)2), 2.31 (m, 2H,
J
J
J
J
J
21.2, 22.6, 29.1, 40.2, 47.9, 61.0, 69.7, 98.1, 114.0, 116.5, 120.9, 123.9, 125.9, 128.3,
128.4, 130.2, 136.3, 137.4, 143.6, 145.0, 151.7, 163.3, 166.3, 167.5, 168.6 and 177.2.
HPLC [Methanol/H2O, 25:75, 0.5 mL/min, 330 nm] tR= 1.390 min. Anal. calcd. for
C30H30N7ClO3: C, 62.99; H, 5.29; N, 17.14; found: C, 62.71; H, 5.37, N, 17.33. HRMS
calcd. for C30H30N7ClO3: m/z 571.2099; found: m/z 572.2127 (M++1).
23

ACCEPTED MANUSCRIPT
4.6.2.4 Isopropyl 2-[(1-(3-(7-chloroquinolin-4-ylamino)propyl)-1H-1,2,3-triazol-4-
yl)methoxy]-4-methyl-6-(4-nitrophenyl)pyrimidine-5-carboxylate 30d
Pale yellow solid. Yield: 92%. Rf: 0.3 (10% ethyl acetate/MeOH). Mp 145-147 °C. IR
(KBr): νmax 1551, 1610, 1717, 2929, 2981, 3154, 3327 cm-1.1H NMR (500 MHz,
CDCl3, 25 °C): δ 1.11 (d,
NHCH2CH2CH2N-), 2.62 (s, 3H, C-6 CH3), 3.42-3.46 (m, 2H, NHCH2CH2CH2N-),
4.55 (t, = 7.5 Hz, 2H, NHCH2CH2CH2N-), 5.07-5.12 (m, 1H, C-5 OCH(CH3)2), 5.40
(br, 1H, NH, D2O exchangeable), 5.63 (s, 2H, C-2 OCH2), 6.38 (d, = 5.0 Hz, 1H,
CQ), 7.40 (d, = 5.0 Hz, 1H , CQ), 7.67 (s, 1H, CQ), 7.69 (d, = 5.0 Hz, 1H, CQ),
7.81 (d, = 5.0 Hz, 2H, ArH), 7.96 (s, 1H, triazolyl H), 8.31 (d, = 10.0 Hz, 2H,
ArH), 8.54 (d,
= 5.0 Hz, 1H, CQ). 13C NMR (125 MHz, CDCl3, 25 °C): δ 21.3, 22.9,
J = 10.0 Hz, 6H, C-5 OCH(CH3)2), 2.32-2.37 (m, 2H,
J
J
J
J
J
J
J
28.4, 40.0, 47.9, 61.4, 70.1, 98.8, 117.1, 121.1, 123.5, 123.6, 125.6, 128.7, 128.7,
129.5, 135.1, 143.5, 143.7, 148.7, 151.8, 163.4, 164.2, 166.7 and 169.5. HPLC
[Methanol/H2O, 25:75, 0.5 mL/min, 330 nm] tR= 1.381 min. Anal. calcd. for
C30H29N8ClO5: C, 58.39; H, 4.74; N, 18.16; found: C, 58.46; H, 4.84, N, 18.31. HRMS
calcd. for C30H29N8ClO5: m/z 616.1949; found: m/z 617.2198 (M++1).
4.6.2.5 Isopropyl 2-[(1-(3-(7-chloroquinolin-4-ylamino)propyl)-1H-1,2,3-triazol-4-
yl)methoxy]-4-methyl-6-(2-nitrophenyl)pyrimidine-5-carboxylate 30e
Pale yellow solid. Yield: 89%. Rf: 0.3 (10% ethyl acetate/MeOH). Mp 110-112 °C. IR
(KBr): νmax 1581, 1611, 1717, 2980, 3089, 3146, 3342 cm-1.1H NMR (500 MHz,
CDCl3, 25 °C): δ 0.94 (d,
NHCH2CH2CH2N-), 2.69 (s, 3H, C-6 CH3), 3.38-3.40 (m, 2H, NHCH2CH2CH2N-),
4.50 (t, = 5.0 Hz, 2H, NHCH2CH2CH2N-), 4.91-4.96 (m, 1H, C-5 OCH(CH3)2), 5.57
(s, 2H, C-2 OCH2), 5.64 (br, 1H, NH, D2O exchangeable), 6.37 (d, = 5.0 Hz, 1H,
J = 5.0 Hz, 6H, C-5 OCH(CH3)2), 2.29-2.33 (m, 2H,
J
J
CQ), 7.32-7.70 (m, 4H, ArH and 1H, CQ), 7.74 (d,
1H, triazolyl H), 8.16 (d,
J
= 10.0 Hz, 1H, CQ), 7.94 (s,
13
J
= 10.0 Hz, 1H, CQ), 8.51 (d, J = 10.0 Hz, 1H, CQ). C
NMR (125 MHz, CDCl3, 25 °C): 21.1, 23.9, 28.3, 39.8, 47.8, 61.3, 69.4, 98.9, 117.2,
119.6, 121.3, 123.5, 124.6, 125.5, 128.6, 129.9, 130.1, 133.4, 134.3, 135.0, 143.6,
147.1, 149.1, 149.4, 151.8, 163.1, 165.2, 166.3 and 171.0. HPLC [Methanol/H2O,
25:75, 0.5 mL/min, 330 nm] tR= 1.360 min. Anal. calcd. for C30H29N8ClO5: C, 58.39;
H, 4.74; N, 18.16; found: C, 58.41; H, 4.80, N, 18.17. HRMS calcd. for
C30H29N8ClO5: m/z 616.1949; found: m/z 617.2198 (M++1).
24

ACCEPTED MANUSCRIPT
4.6.2.6 Ethyl 2-[(1-(3-(7-chloroquinolin-4-ylamino)propyl)-1H-1,2,3-triazol-4-
yl)methoxy]-4,6-dimethylpyrimidine-5-carboxylate 30f
White soild. Yield: 87%. Rf: 0.4 (10% ethyl acetate/MeOH). Mp 98-100 °C. IR (KBr):
νmax 1591, 1611, 1729, 2850, 2918, 2960, 3429 cm-1.1H NMR (500 MHz, CDCl3, 25
°C): δ 1.40 (t,
2.50 (s, 3H, C-4 CH3 and 3H, C-6 CH3), 3.37-3.41 (m, 2H, NHCH2CH2CH2N-), 4.40
(q, = 10.0 Hz, 2H, C-5 OCH2CH3), 4.50 (t, = 5.0 Hz, 2H, NHCH2CH2CH2N-) ,
5.53 (s, 2H, C-2 OCH2), 5.83 (br, 1H, NH, D2O exchangeable), 6.33 (d, = 5.0 Hz,
1H, CQ), 7.31-7.33 ( m, 1H, CQ), 7.70 (s, 1H, triazolyl H), 7.81 (d, = 10.0, 1H, CQ),
7.91 (s, 1H, CQ), 8.47 (d,
= 5.0 Hz, 1H, CQ). 13C NMR (125 MHz, CDCl3, 25 °C): δ
J = 5.0 Hz, 3H, C-5 OCH2CH3), 2.28-2.33 (m, 2H, NHCH2CH2CH2N-),
J
J
J
J
J
14.1, 23.2, 28.4, 39.9, 47.9, 60.9, 61.6, 98.8, 117.3, 120.8, 121.7, 123.6, 125.4, 128.2,
135.0, 143.8, 148.8, 149.7, 151.6, 163.1, 167.3 and 168.3. HPLC [Methanol/H2O,
25:75, 0.5 mL/min, 330 nm] tR= 1.341 min. Anal. calcd. for C24H26N7ClO3: C, 58.12;
H, 5.28; N, 19.77; found: C, 58.34; H, 5.33, N, 19.81. HRMS calcd. for
C24H26N7ClO3: m/z 495.1786; found: m/z 496.1920 (M++1).
4.6.2.7 Methyl 2-[(1-(2-(7-chloroquinolin-4-ylamino)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy]-4-methyl-6-phenylpyrimidine-5-carboxylate 30g
White solid. Yield: 89%. Rf: 0.4 (10% ethyl acetate/MeOH). Mp 163-165 °C. IR
(KBr): νmax 1551, 1579, 1610, 1724, 2950, 3075, 3290 cm-1.1H NMR (500 MHz,
CDCl3, 25 °C): δ 2.54 (s, 3H, C-6 CH3), 3.68 (s, 3H, C-5 OCH3), 3.83-3.90 (m , 2H,
NHCH2CH2N-), 4.70 ( t,
J = 7.5 Hz, 2H, NHCH2CH2N-), 5.63 (s, 2H, C-2 OCH2),
5.86 (br, 1H, NH, D2O exchangeable), 6.41 (d,
J
= 5.0 Hz, 1H, CQ), 7.36 (d,
J
= 10.0
Hz, 1H, CQ), 7.41-7.48 (m, 3H, ArH), 7.60 (d,
J
= 10.0 Hz, 2H, ArH), 7.68 (d,
J = 5.0
Hz, 2H, CQ), 7.95 (s, 1H, triazolyl H), 8.55 (s, 1H, CQ). 13C NMR (125 MHz, CDCl3,
25 °C): δ 22.8, 42.7, 48.5, 52.5, 60.9, 98.7, 120.0, 121.4, 124.4, 125.8, 128.1, 128.5,
130.4, 135.2, 137.3, 143.6, 149.0, 151.8, 163.4, 166.3, 168.5 and 169.0. HPLC
[Methanol/H2O, 25:75, 0.5 mL/min, 330 nm] tR= 1.306 min. Anal. calcd. for
C27H24N7ClO3: C, 61.19; H, 4.56; N, 18.50; found: C, 61.34; H, 4.67, N, 18.54. HRMS
calcd. for C27H24N7ClO3: m/z 529.1629; found: m/z 530.1751 (M++1).
4.6.2.8
Ethyl
2-[(1-(2-(7-chloroquinolin-4-ylamino)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy]-4-methyl-6-phenylpyrimidine-5-carboxylate 30h
White solid. Yield: 85%. Rf: 0.4 (10% ethyl acetate/MeOH). Mp 148-151 °C. IR
(KBr): νmax 1553, 1578, 1610, 1718, 2928, 2983, 3079, 3280 cm-1.1H NMR (500 MHz,
25

ACCEPTED MANUSCRIPT
CDCl3, 25 °C): δ 1.04 (t,
J
= 7.5 Hz, 3H, C-5 OCH2CH3), 2.55 (s, 3H, C-6 CH3), 3.86-
3.88 (m, 2H, NHCH2CH2N-), 4.16 (q,
=5Hz, 2H, NHCH2CH2N-), 5.62 (s, 2H, C-2 OCH2), 6.14 (br, 1H, NH, D2O
J = 5.0 Hz, 2H, C-5 OCH2CH3), 4.71 (t, J
exchangeable), 6.39 (d,
(m, 3H, ArH), 7.59 (d,
J
J
= 5.0 Hz, 1H, CQ), 7.35 (d,
= 5.0 Hz, 2H, ArH), 7.72 (s, 1H, CQ), 7.74 (d,
= 5.0 Hz, 1H, CQ). 13C NMR (125 MHz,
J
= 10.0 Hz, 1H, CQ), 7.40-7.46
J
= 10.0 Hz,
1H, CQ), 7.93 (s, 1H, triazolyl H), 8.50 (d,
J
CDCl3, 25 °C): δ 13.6, 22.7, 42.8, 48.6, 60.9, 61.8, 98.7, 117.1, 120.4, 121.5, 124.3,
126.0, 128.0, 128.2, 128.4, 130.3, 135.5, 137.4, 143.8, 149.4, 151.2, 163.3, 166.5,
168.0 and 168.9. HPLC [Methanol/H2O, 25:75, 0.5 mL/min, 330 nm] tR= 1.275 min.
Anal. calcd. for C28H26N7ClO3: C, 61.82; H, 4.82; N, 18.02; found: C, 61.68; H, 4.80,
N, 18.16. HRMS calcd. for C28H26N7ClO3: m/z 543.1786; found: m/z 544.1853
(M++1).
4.6.2.9 Isopropyl 2-[(1-(2-(7-chloroquinolin-4-ylamino)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy]-4-methyl-6-phenylpyrimidine-5-carboxylate 30i
White solid. Yield: 88%. Rf: 0.4 (10% ethyl acetate/MeOH). Mp 158-160 °C. IR
(KBr): νmax 1555, 1578, 1721, 2930, 2979, 3081, 3285 cm-1.1H NMR (500 MHz,
CDCl3, 25 °C): δ 1.07 (d,
3.85-3.87 (m, 2H, NHCH2CH2N-), 4.71 (t,
(m, 1H, C-5 OCH(CH3)2), 5.62 (s, 2H, C-2 OCH2), 6.06 (br, 1H, NH, D2O
exchangeable), 6.41 (s, 1H, CQ), 7.36 (d, = 5.0 Hz, 1H, CQ), 7.40-7.47 (m, 3H,
J
= 5.0 Hz, 6H, C-5 OCH(CH3)2), 2.55 (s, 3H, C-6 CH3),
J
= 5.0 Hz, 2H, NHCH2CH2N-), 5.03-5.07
J
ArH), 7.60 (d,
J
= 5.0 Hz, 2H, CQ), 7.71-7.74 (m, 2H, ArH), 7.94 (s, 1H, triazolyl H),
13
8.52 (s, 1H, CQ). C NMR (125 MHz, CDCl3, 25 °C): δ 21.3, 22.7, 42.8, 48.6, 61.0,
69.7, 120.9, 121.4, 124.3, 126.0, 128.3, 128.4, 130.2, 135.5, 137.4, 143.9, 163.3,
166.3, 167.5 and 168.6. HPLC [Methanol/H2O, 25:75, 0.5 mL/min, 330 nm] tR= 1.036
min. Anal. calcd. for C29H28N7ClO3: C, 62.42; H, 5.06; N, 17.57; found: C, 62.57; H,
5.15, N, 17.69. HRMS calcd. for C29H28N7ClO3: m/z 557.1942; found: m/z 558.2824
(M++1).
4.6.2.10 Isopropyl 2-[(1-(2-(7-chloroquinolin-4-ylamino)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy]-4-methyl-6-(4-nitrophenyl)pyrimidine-5-carboxylate 30j
Pale yellow solid. Yield: 81%. Rf: 0.3 (10% ethyl acetate/MeOH). Mp 177-179 °C. IR
(KBr): νmax 1547, 1609, 1737, 2931, 2981, 3083, 3286 cm-1.1H NMR (500 MHz,
CDCl3, 25 °C): δ 1.11 (d,
J
= 5.0 Hz, 6H, C-5 OCH(CH3)2), 2.62 (s, 3H, C-6 CH3),
= 5.0 Hz, 2H, NHCH2CH2N-), 5.08-5.12
3.88-3.91 (m, 2H, NHCH2CH2N-), 4.73 (t,
J
26

ACCEPTED MANUSCRIPT
(m, 1H, C-5 OCH(CH3)2), 5.63 (s, 2H, C-2 OCH2), 5.76 (br, 1H, NH, D2O
exchangeable), 6.41 (d,
= 10.0 Hz, 1H, CQ), 7.70 (s, 1H, CQ), 7.78 (d,
triazolyl H), 8.29 (d,
J = 5.0 Hz, 1H, CQ), 7.39 (d, J = 10.0 Hz, 1H , CQ), 7.67 (d, J
J
= 10.0 Hz, 2H, ArH), 7.97 (s, 1H,
13
J
= 10.0 Hz, 2H, ArH), 8.55 (d, J = 5.0 Hz, 1H CQ). C NMR
(125 MHz, CDCl3, 25 °C): 21.3, 22.9, 42.8, 48.6, 61.1, 70.1, 98.8, 117.2, 121.0, 121.2,
123.5, 124.3, 125.8, 128.7, 129.4, 135.2, 143.4, 143.5, 148.7, 148.9, 151.8, 163.3,
164.1, 166.6 and 169.5. HPLC [Methanol/H2O, 25:75, 0.5 mL/min, 330 nm] tR= 1.049
min. Anal.calcd. for C29H27N8ClO5: C, 57.76; H, 4.51; N, 18.58; found: C, 57.71; H,
4.76, N, 18.63. HRMS calcd. for C29H27N8ClO5: m/z 602.1793; found: m/z 603.1927
(M++1).
4.6.2.11 Isopropyl 2-[(1-(2-(7-chloroquinolin-4-ylamino)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy]-4-methyl-6-(2-nitrophenyl)pyrimidine-5-carboxylate 30k
Pale yellow solid. Yield: 86%. Rf: 0.3 (10% ethyl acetate/MeOH). Mp 175-177 °C. IR
(KBr): νmax 1581, 1611, 1718, 2980, 3108, 3148, 3322 cm-1.1H NMR (500 MHz,
CDCl3, 25 °C): δ 0.94 (d,
3.86-3.90 (m, 2H, NHCH2CH2N-), 4.69 (t,
(m, 1H, C-5 OCH(CH3)2), 5.56 (s, 2H, C-2 OCH2), 5.92 (br, 1H, NH, D2O
exchangeable), 6.42 (d, = 5Hz, 1H, CQ), 7.29-7.68 (m, 3H, ArH and 3H, CQ), 7.95
(s, 1H , triazolyl H), 8.13 (d, = 10.0 Hz, 1H, ArH), 8.54 (d,
= 5.0 Hz, 1H, CQ). 13C
J
= 5.0 Hz, 6H, C-5 OCH(CH3)2), 2.66 (s, 3H, C-6 CH3),
J
= 7.5 Hz, 2H, NHCH2CH2N-), 4.92-4.97
J
J
J
NMR (125 MHz, CDCl3, 25 °C): δ 21.1, 23.8, 42.7, 48.6, 61.2, 69.4, 98.8, 117.2,
119.7, 121.3, 124.0, 124.6, 125.8, 128.6, 129.9, 130.0, 133.3, 134.2, 135.2, 143.6,
147.2, 149.0, 151.8, 163.0, 165.2, 166.2 and 171.0. HPLC [Methanol/H2O, 25:75, 0.5
mL/min, 330 nm] tR= 1.066 min. Anal. calcd. for C29H27N8ClO5: C, 57.76; H, 4.51; N,
18.58; found: C, 57.81; H, 4.71, N, 18.43. HRMS calcd. for C29H27N8ClO5: m/z
602.1793; found: m/z 603.1927 (M++1).
4.6.2.12 Ethyl 2-[(1-(2-(7-chloroquinolin-4-ylamino)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy]-4,6-dimethylpyrimidine-5-carboxylate 30l
White solid. Yield: 82%. Rf: 0.4 (10% ethyl acetate/MeOH). Mp 130-132 °C. IR
1
(KBr): νmax 1557, 1585, 1613, 1721, 2979, 3142, 3274 cm-1. H NMR (500 MHz,
CDCl3, 25 °C): δ 1.40 (t,
3H, C-6 CH3), 3.87-3.89 (m, 2H, NHCH2CH2N-), 4.41 (q,
OCH2CH3), 4.72 (t, = 5.0 Hz, 2H, NHCH2CH2N-), 5.56 (s, 2H, C-2 OCH2), 5.89 (br,
1H, NH, D2O exchangeable), 6.41 (d, = 5.0 Hz, 1H, CQ), 7.38 (d, J = 10.0 Hz, 1H,
J
= 7.5 Hz, 3H, C-5 OCH2CH3), 2.49 (s, 3H, C-4 CH3 and
J
= 10.0 Hz, 2H, C-5
J
J
27

ACCEPTED MANUSCRIPT
CQ), 7.70 (d,
J
= 5.0 Hz, 2H, CQ), 7.96 (s, 1H, triazolyl H), 8.54 (d, J = 5.0 Hz, 1H,
13
CQ). C NMR (125 MHz, CDCl3, 25 °C): δ 14.1, 23.2, 42.8, 48.6, 60.8, 61.6, 98.8,
117.2, 120.8, 121.3, 124.2, 125.9, 128.7, 135.2, 143.9, 148.9, 149.0, 151.8, 163.1,
167.3 and 168.3. HPLC [Methanol/H2O, 25:75, 0.5 mL/min, 330 nm] tR= 1.084 min.
Anal. calcd. for C23H24N7ClO3: C, 57.32; H, 5.02; N, 20.34; found: C, 57.21; H, 4.89,
N, 20.41. HRMS calcd. for C23H24N7ClO3: m/z 481.1629; found: m/z 482.1731
(M++1).
4.6.2.13 Methyl 2-[(1-(7-chloroquinolin-4-yl)-1
methyl-6-phenylpyrimidine-5-carboxylate 32a
H-1,2,3-triazol-4-yl)methoxy]-4-
White soild. Yield: 82%. Rf: 0.3 (70% ethyl acetate/hexane). Mp 118-120 °C. IR
(KBr): νmax 1556, 1612, 1726, 2854, 2927, 2951, 3091, 3131 cm-1.1H NMR (500 MHz,
CDCl3, 25 °C): δ 2.61 (s, 3H, C-6 CH3), 3.70 (s, 3H, C-5 OCH3), 5.83 (s, 2H, C-2
OCH2), 7.45- 7.50 (m, 3H, ArH and 1H, CQ), 7.59 (d , J = 10.0 Hz, 1H, CQ), 7.68 (d,
J
= 10.0 Hz, 2H, ArH), 7.97 (d,
J
J
= 10.0 Hz, 1H, CQ), 8.17 (s, 1H, triazolyl H), 8.25
= 5.0 Hz, 1H, CQ). 13C NMR (125 MHz, CDCl3, 25
(d, = 1.5 Hz, 1H, CQ), 9.06 (d,
J
°C): δ 22.8, 52.6, 61.0, 116.0, 120.3, 120.5, 124.5, 125.0, 128.2, 128.6, 129.0, 129.5,
130.5, 136.9, 137.3, 140.9, 144.4, 150.2, 151.4, 163.5, 166.4, 168.6 and 169.2. HPLC
[Methanol/H2O, 25:75, 0.5 mL/min, 330 nm] tR= 1.054 min. Anal. calcd. for
C25H19N6ClO3: C, 61.67; H, 3.93; N, 17.26; found: C, 61.54; H, 4.05, N, 17.45. HRMS
calcd. for C25H19N6ClO3: m/z 486.1207; found: m/z 487.1464 (M++1).
4.6.2.14 Ethyl 2-[(1-(7-chloroquinolin-4-yl)-1
methyl-6-phenylpyrimidine-5-carboxylate 32b
H-1,2,3-triazol-4-yl)methoxy]-4-
White solid. Yield: 88%. Rf: 0.3 (70% ethyl acetate/hexane). Mp 103-105 °C. IR
(KBr): νmax 1556, 1611, 1722, 2928, 2979, 3090, 3131 cm-1.1H NMR (500 MHz,
CDCl3, 25 °C): δ 1.06 (t,
(q, = 7.5 Hz, 2H, C-5 OCH2CH3), 5.83 (s, 2H, C-2 OCH2), 7.44-7.49 (m, 3H, ArH
and 1H, CQ), 7.58-7.60 (m, 1H, CQ), 7.67 (d, = 5.0 Hz, 2H, ArH), 7.97 (d, = 10.0
= 1.5 Hz, 1H, CQ), 9.06 (d, = 5.0
J = 7.5 Hz, 3H, C-5 OCH2CH3), 2.62 (s, 3H, C-6 CH3), 4.18
J
J
J
Hz, 1H, CQ), 8.17 (s, 1H, triazolyl H), 8.25 (d,
J
J
13
Hz, 1H, CQ). C NMR (125 MHz, CDCl3, 25 °C): δ 13.6, 22.8, 61.0, 61.8, 116.0,
120.5, 120.6, 124.5, 125.0, 128.2, 128.5, 129.0, 129.5, 130.3, 136.9, 137.4, 140.9,
144.4, 150.2, 151.3, 163.4, 166.6, 168.0 and 169.1. HPLC [Methanol/H2O, 25:75, 0.5
mL/min, 330 nm] tR= 1.408 min. Anal. calcd. for C26H21N6ClO3: C, 62.34; H, 4.23; N,
28

ACCEPTED MANUSCRIPT
16.78; found: C, 62.31; H, 4.07, N, 16.89. HRMS calcd. for C26H21N6ClO3: m/z
500.1364; found: m/z 523.1345 (M++23).
4.6.2.15 Isopropyl 2-[(1-(7-chloroquinolin-4-yl)-1
methyl-6-phenylpyrimidine-5-carboxylate 32c
H-1,2,3-triazol-4-yl)methoxy]-4-
White solid. Yield: 83%. Rf: 0.4 (70% ethyl acetate/hexane). Mp 123-125 °C. IR
(KBr): νmax 1558, 1592, 1611, 1719, 2936, 2979, 3089, 3130 cm-1.1H NMR (500 MHz,
CDCl3, 25 °C): δ 1.09 (d,
5.06-5.11 (m, 1H, C-5 OCH(CH3)2), 5.82 (s, 2H, C-2 OCH2), 7.44-7.51 (m, 3H, ArH
and 1H, CQ), 7.59-7.61 (m, 1H, CQ), 7.67-7.69 (m, 2H, ArH), 7.98 (d, = 10.0 Hz,
= 4.5 Hz,
J = 5.0 Hz, 6H, C-5 OCH(CH3)2), 2.61 (s, 3H, C-6 CH3),
J
1H, CQ), 8.18 (s, 1H, triazolyl H), 8.25 (d,
J = 5.0 Hz, 1H, CQ), 9.07 (d, J
13
1H, CQ). C NMR (125 MHz, CDCl3, 25 °C): δ 21.3, 22.7, 61.0, 69.7, 116.0, 120.5,
121.1, 124.5, 125.0, 128.3, 128.5, 129.0, 129.5, 130.3, 136.9, 137.4, 140.9, 144.5,
150.2, 151.4, 163.3, 166.4, 167.5 and 168.7. HPLC [Methanol/H2O, 25:75, 0.5
mL/min, 330 nm] tR= 1.402 min. Anal. calcd. for C27H23N6ClO3: C, 62.97; H, 4.50; N,
16.32; found: C, 62.71; H, 4.24, N, 16.47. HRMS calcd. for C27H23N6ClO3: m/z
514.1520; found: m/z 515.1683 (M++1).
4.6.2.16 Isopropyl 2-[(1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methoxy]-4-
methyl-6-(4-nitrophenyl)pyrimidine-5-carboxylate32d
Pale yellow solid. Yield: 86%. Rf: 0.3 (70% ethyl acetate/hexane). Mp 97-100 °C. IR
(KBr): νmax 1584, 1610, 2853, 2925, 3088, 3126 cm-1.1H NMR (500 MHz, CDCl3, 25
°C): δ 1.12 (d,
1H, C-5 OCH(CH3)2), 5.82 (s, 2H, C-2 OCH2), 7.49 (d,
= 10.0 Hz, 1H, CQ), 7.84 (d, = 5.0 Hz, 2H, ArH), 7.98 (d, = 10.0 Hz, 1H, CQ),
= 2.0 Hz, 1H, CQ), 8.32 (d, J = 10.0 Hz, 2H, ArH),
J
= 5.0 Hz, 6H, C-5 OCH(CH3)2), 2.65 (s, 3H, C-6 CH3), 5.08-5.13 (m,
J
= 5.0 Hz, 1H , CQ), 7.61 (d,
J
J
J
8.18 (s, 1H, triazolyl H), 8.26 (d,
J
13
9.07 (d,
J = 5.0 Hz, 1H, CQ). C NMR (125 MHz, CDCl3, 25 °C): 21.3, 23.0, 61.2,
70.2, 116.0, 120.5, 121.3, 123.6, 124.4, 125.0, 129.0, 129.5, 137.0, 143.4, 144.1,
148.7, 150.2, 151.4, 163.4, 164.2, 166.6 and 169.6. HPLC [Methanol/H2O, 25:75, 0.5
mL/min, 330 nm] tR= 1.381 min. Anal. calcd. for C27H22N7ClO5: C, 57.91; H, 3.96; N,
17.51; found: C, 57.83; H, 3.80, N, 17.55. HRMS calcd. for C27H22N7ClO5: m/z
559.1371; found: m/z 582.1384 (M++23).
29