Highly active 4-aminoquinoline-pyrimidine based molecular hybrids as potential next generation antimalarial agents

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Highly active 4-aminoquinoline-pyrimidine based molecular

hybrids as potential next generation antimalarial agents

Sunny Manohar, V. Satya Pavan, Dale Taylor, Deepak Kumar, Prija Ponnan, Lubbe Wiesner and Diwan S.

Rawat

Novel 4-aminoquinoline-pyrimidine based antimalarial hybrids were discovered to show

potent activity against NF54 and Dd2 strains of P. falciparum.

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ARTICLE TYPE

Highly active 4ꢀaminoquinolineꢀpyrimidine based molecular hybrids as

potential next generation antimalarial agents

a#

a

b

a

b

Sunny Manohar, V. Satya Pavan, Dale Taylor, Deepak Kumar, Prija Ponnan, Lubbe Wiesner and Diwan S.

Rawat*

a

5

Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX

DOI: 10.1039/b000000x

In order to overcome the problem of emerging drug resistance in malarial chemotherapy, a series of

highly active 4ꢀaminoquinolineꢀpyrimidine hybrids were synthesized and evaluated for their antimalarial

activity against CQꢀsensitive (NF54) and CQꢀresistant (Dd2) strains of P. falciparum in an in-vitro assay.

The most active hybrid 19f exhibited 74ꢀfold better potency than chloroquine and 4ꢀfold better potency

than artesunate against drugꢀresistant strain of P. falciparum. Compound 19e, when evaluated for in vivo

activity in P. bergheiꢀmouse malaria model showed 93.9% parasite suppression at 30 mg/kg dose on day

1

0

4

with mean survival time of 11 days. To gain insights towards mechanism of action of these hybrids,

heme binding and molecular modelling studies were performed on the most active hybrids. It was

observed that inhibition of formation of βꢀhematin and dihydrofolate reductaseꢀthymidylate synthase

1

5

(PfDHFRꢀTS) enzyme could be associated with the observed antimalarial activity of these compounds.

Introduction

broad spectrum of antimalarial activity and such compounds

should be robust enough to reduce the evolution of new resistance

for a longer period of time. The multiꢀtargeting approaches based

on combination therapies such as ACTs suffers from several

In recent times, the emergence of drug resistance is one of the

worst nightmares to tackle in the present scenario of malaria

related public health programs. The severity of this problem is

indicated in the WHO recent report, which revealed an estimated

5

0

2

3

4

0

5

0

5

0

5

4

chemicals, therapeutic and treatment related limitations. In

addition, a greater precision is required for the fineꢀtuning of the

formulation in combination therapies as the chemical species

involved have different solubility’s and pharmacokinetic

properties.

2

6

07 million cases of malaria in 2012 with an approximately

27,000 deaths. Out of these deaths, 482,000 deaths were of

55

1

children under the age of five. The statistics of this level are

highly alarming and a message for the entire world to devote

considerable efforts towards this problem. The P. falciparum, P.

vivax, P. ovale and P. malariae P. knowlesi are the main

causative species of malaria but P. falciparum is the most virulent

species of the malaria parasite causing most of the malaria related

a

OH

H

O

N

O

HO

N

HN

N

HN

O

H

O

H3CO

O

Cl

N

2

N

deaths in humans. The traditional first line antimalarial drugs

such as quinine (1), chloroquine (CQ, 2) and amodiaquine (3)

Artemisinin (4)

Quinine (1)

Chloroquine (CQ, 2)

Cl

Amodiaquine (3)

H2N

N

NH2

H

O

Cl

N

(figure 1) considered as a mainstay for antimalarial chemotherapy

N

O

for a long period of time have lost their efficacy and their usage

as antimalarial drugs has been limited due to the ever increasing

H

N

Cl

O

Piperaquine (8)

OH

Pyrimethamine (9)

3

1

OR

P. falciparum drug resistance. According to the WHO, currently

the only best available treatment, particularly for P. falciparum

related uncomplicated and severe malarial infection is

artemisininꢀbased doubleꢀ or tripleꢀcombination therapies (ACTs)

where the core compound artemisinin (4) or its derivatives

[dihydroartemisinin (5)/artemether (6)/arteether (7)] (figure 1) is

administered in combination with locally active partner drug

R = H, Dihydroartemisinin (5)

R = Me, Artemether (6)

R = Et, Arteether (7)

N

HN

HO

Cl

OCH3

a

H3CO

O

Cl

N

CF3

H2N

S

NH

CF3

O

Cl

Lumefantrine (11)

Sulfadoxine (10)

Mefloquine (12)

Fig. 1: Antimalarial drugs

[piperaquine

(8)/pyrimethamine

(9)/

sulfadoxine

To overcome these challenges, a better and rational alternative

is to gain therapeutic benefits by developing single multiꢀtargeted

hybrid by covalent hybridization of two or more differential

(

10)/lumefantrine (11)/ mefloquine (12)] (figure 1). However,

60

recent reports on the development of resistance towards

artemisinin in some southꢀeast Asian countries including

Cambodia, Myanmar, Thailand and Vietnam has again propelled

5

1

targetꢀselective antimalarial entities. These dualꢀdrug kinds of

hybrids have the potential to increase bioꢀpharmaceutical

the dire need of novel next generation antimalarials having a

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efficacy, reduce cost, decreases risk of drugꢀdrug interactions and

interested to generate

a

new series of 4ꢀaminoquinolineꢀ

overcome rapid development of resistance problems. Various 60 pyrimidine hybrids, so as to present their elaborated structure

research groups across the world have successfully employed this

hybridization approach towards the synthesis of interesting

hybrids which have the potential of eliminating drug resistance.

activity relationship (SAR) and to recognize novel molecular

leads for finding alternative next generation antimalarial drugs. In

the present investigation, we report herein new pyrimidine

tethered 4ꢀaminoquinoline based molecular hybrids synthesized

6

5

0

5

0

5

0

5

0

5

0

5

Our lab has successfully adopted this covalent biꢀtherapy

strategy to generate hybrid antimalarials in which 4ꢀ 65 by systematic chemical modification of previous lead molecules

aminoquinoline entity derived from CQ was covalently attached

to pharmacophoric framework present in antifolate class of

and to evaluate their antimalarial activity against both CQꢀ

sensitive and resistant strains of P. falciparum in vitro and against

P. berghei mouse malaria model in-vivo.

7ꢀ9

1

2

3

4

5

antimalarial drugs such as cycloguanil/pyrimethamine.

The

hybrids were designed in such a way that quinoline part of CQ is

retained, in order to retain its hemozoin inhibition ability while

the side chain of CQ was modified by conjugating it with

triazine/pyrimidine moieties which can inhibit plasmodial DHFR

and at the same time not be recognized by drug effluxing

proteins. The rationality behind choosing 4ꢀaminoquinoline

skeleton, despite growing evidences of the skeleton becoming

ineffective due to resistance, is based on its brilliant features like

excellent clinical efficacy, ease of administration, low toxicity

and cheap synthesis which makes the pharmacophore very

difficult to abandon. Moreover, the hybridization strategy has the

potential to restore the efficacy of traditional 4ꢀaminoquinoline

drugs such as CQ. So, the conceptual idea was to develop 4ꢀ

aminoquinoline based multiꢀactionable architectures having dual

functionality of inhibiting two conventional targets (hemozoin

and plasmodial DHFR) simultaneously and inꢀturn could bring an

upsurge of antimalarial activity particularly against resistant

8

,9

strains of P. falciparum. In our previous report, we have

identified two highly active 4ꢀaminoquinolineꢀpyrimidine hybrids

(13 and 14; figure 2a) as lead compounds having potent in-vitro

antimalarial activity against both CQꢀsensitive (D6 clone) and

CQꢀresistant (W2 clone) of P. falciparum with no cytotoxicity

against mammalian cells. The hybrids also possessed excellent

in-vivo antimalarial activity when tested in P. berghei infected

mouse malaria model. This level of activity warrants their

selection as a potential drug candidate for preclinical trials. An

inꢀdepth analysis of antimalarial activity pattern reported for this

series of 4ꢀaminoquinolineꢀpyrimidine hybrids revealed three

important points which are kept in mind while synthesizing new

hybrid molecules (figure 2b). These are: 1) lengthening and

shortening of carbon spacer linker does not play an important role

in influencing antimalarial activity when Cl is substituted with

amino functionalities; 2) The activity increases manifold when Cl

is replaced with amino functionalities (morpholine, piperidine, Nꢀ

methyl piperidine and Nꢀethyl piperazine); 3) The comparison of

antimalarial activity of two groups of regioisomers clearly

indicated that both the regioisomers displayed more or less

similar potency against both the strains of P. falciparum. This

shows that the point of attachment of the spacer to the pyrimidine

nucleus may not have a great impact on antimalarial activity

7

0

Fig. 2: a) 4ꢀAminoquinolineꢀpyrimidine hybrids (13 and 14) identified as

8,9

lead candidates from our earlier studies. b) Useful structural features as

determined by SAR studies on our previously reported 4ꢀaminoquinolineꢀ

pyrimidine hybrids.

75

Results and discussion

Chemistry

In order to gain insights towards the determination of structural

features required for displaying potent antimalarial activity and

keeping the above mentioned SAR studies in mind, a series of

thirty two 4ꢀaminoquinolineꢀpyrimidine hybrids (17aꢀ17d, 18aꢀ

8

9

0

5

0

5

1

8d, 19aꢀ19l, 20aꢀ20l) were prepared by taking same amino

functionalities, but altering the carbon spacer from C2 to C3 only.

Additionally, the methyl group attached to pyrimidine nucleus is

replaced by H and Cl, so as to see its effect on antimalarial

activity pattern. The synthesis of these hybrids took place

8

,9

following a three step procedure reported by our lab previously.

In short, commercially available starting material 4,7ꢀ

dichloroquinoline (15) was taken and converted to substituted 4ꢀ

aminoquinolines (16a and 16b) by treatment with an excess of

aliphatic linear chain diaminoalkanes having 2ꢀ3 carbon spacer

10

profile. During the progress of this work, Singh et al also

reported antimalarial activity of 4ꢀaminoquinolineꢀpyrimidine

hybrids against Dd2 and D10 strains of P. falciparum with high

selectivity indices and inferior toxicity when tested in-vitro.

However, they did not carry out in vivo antimalarial activity of

these hybrids.

via a S Ar reaction under neat conditions. The free terminal

N

amino groups of the substituted 4ꢀaminoquinolines (16a and 16b)

were then reacted with substituted pyrimidines to yield a set of

two regioisomeric intermediates (17aꢀ17d and 18aꢀ18d) differing

in the position of attachment of the pyrimidine nucleus to the

Encouraged by these results and as a part of our onꢀgoing work

7

ꢀ9,11

towards the synthesis of novel antimalarial agents,

we got

2

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Scheme 1: a) Diaminoalkanes (n = 1ꢀ2), neat, 120ꢀ130 °C, 6ꢀ8 h, 80ꢀ90%; b) 2,4ꢀdichloropyrimidine or 2,4,6ꢀtrichloropyrimidine, TEA, EtOH, RT, 10ꢀ12

h; c) secondary amines, DMF, 100ꢀ120 °C, 10ꢀ12 h, 50ꢀ85%.

alkyl chain linker. The intermediates (17aꢀ17d and 18aꢀ18d)

were finally converted to the targeted 4ꢀaminoquinolineꢀ

pyrimidine hybrids (19aꢀ19l and 20aꢀ20l) in moderate to

linker). Additionally, the presence of a second Cl atom at the

pyrimidine appears to be critical for determining antimalarial

activity against CQꢀresistant strains (hybrid 17a/18a vs 17b/18b

5

excellent yield by treatment with various cyclic secondary amines 40 and hybrid 17d/18d vs 17c/18c). The noticeable lack of activity

(

scheme 1).

with hybrid 17a/18a may well be a combination of the shortened

linking chain and the single Cl in the pyrimidine ring. The two

sets of regioꢀisomers (hybrids 17aꢀ17d vs 18aꢀ18d and hybrids

Antimalarial Activity and Cytotoxicity

1

9aꢀ19l vs 20aꢀ20l) were mostly potent to same extent which is

1

2

3

0

5

0

5

0

5

In the present investigation, we also determined antimalarial 45 in accordance with our earlier observation that point of

8

activity of two previously identified lead molecules (13 and 14)

for the validation and comparison purpose and to check their

potency against CQꢀsensitive (NF54) and CQꢀresistant (Dd2)

strains of P. falciparum. Most of the hybrids showed potent

attachment of pyrimidine nucleus to 4ꢀaminoquinoline scaffold

via carbon spacer linker doesn’t have any major impact on

determining antimalarial activity pattern. Ethyl/methyl

piperazines as amino functionalities in the pyrimidine nucleus

antimalarial activity against both the strains of P. falciparum. Out 50 generally bring an upsurge of antimalarial activity when

of thirty two synthesized hybrids, nineteen hybrids (18d, 19aꢀ19f,

9i, 19k, 19l, 20a, 20b, 20dꢀ20f, and 20iꢀ20l) with IC₅₀values

ranging from 0.003ꢀ0.198 µM, displayed superior activity than

compared to morpholine/piperidine counterparts. The most active

hybrid of the present series was found to be hybrid 19f (IC50

value 0.003 µM against CQꢀresistant and IC50 value 0.028 µM

against CQꢀsensitive strain of P. falciparum) with C2 carbon

1

CQ (IC₅₀= 0.222 µM), while four hybrids (19f, 19l, 20e and 20f)

with IC₅₀values ranging from 0.003ꢀ0.011 µM) exhibited 55 spacer linker attached at one of the position while Cl and ethyl

superior activity than artesunate (IC₅₀= 0.013 µM) when tested

against CQꢀresistant (Dd2 clone) strain of P. falciparum.

Moreover, six hybrids (19a, 19b, 20b, 20f, 20k and 20l) with IC₅₀

values ranging from 0.009ꢀ0.025 µM also exhibited enhanced

piperazine as the other two groups attached to pyrimidine

nucleus. SAR studies further revealed that, when a methyl group

attached to pyrimidine nucleus was replaced with H or Cl,

activity was retained against drugꢀresistant P. falciparum strains,

activity than CQ (IC₅₀= 0.027 µM) when tested against CQꢀ 60 but dropped slightly against drugꢀsensitive P. falciparum strains.

sensitive (NF54 clone) strain of P. falciparum. The lead

molecules (13 and 14) from our previously published results also

showed an equal level of potency against both the strains in the

present investigation. Increasing the length of the linker that

This observation showed that methyl group is essential for

imparting improved antimalarial activity against CQꢀsensitive

strain of P. falciparum.

One of the highly active hybrids, 19e was further evaluated for

connects 4ꢀaminoquinoline to pyrimidine by one carbon seems to 65 the in-vivo antimalarial activity against P. berghei infected mice

significantly increase activity (hybrid 17a vs 17c, 17b vs 17d, 18a

vs 18c and 18b vs 18d); however, this does not appear to be an

important factor if the secondary cyclic amine is attached to

pyrimidine (for example if we move from hybrid 19c to 19i or

(table 2 and figure 3) and it was found out that it causes 93.9 %

parasite suppression at 30 mg/kg dose on day 4 with mean

survival time being 11 days postꢀinfection. In comparison, CQ

showed 90.3 % suppression at 15 mg/kg dose. The parasite

20c to 20i, activity increases whereas moving from hybrid 19e to 70 suppression data was calculated by the formula given below.

1

9k or 19f to 19l activity drops with increase in carbon chain

Parasite suppression% = [1ꢀ(group parasitemia/placebo parasitemia)]×100

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Table 1: In-vitro antimalarial activity of synthesized 4ꢀaminoquinolineꢀpyrimidine hybrids

d

Com

N

R

1

R

2

Antimalarial Activity

IC50 ± SD (µM)

CQꢀsensitive Strain

Cytotoxicity (CHO

SI

a

b

c

.

No.

cells)_a

b

CQꢀresistant Strain

(Dd2 Clone)

5.044 ± 0.392

1.846 ± 0.030

0.362 ± 0.056

0.104 ± 0.008

3.096 ± 0.222

1.001 ± 0.044

1.736 ± 0.108

0.096 ± 0.006

0.016 ± 0.003

0.090 ± 0.012

0.157 ± 0.014

0.074 ± 0.004

0.014 ± 0.002

0.003 ± 0.0009

2.384 ± 0.248

0.747 ± 0.055

0.067 ± 0.009

0.861 ± 0.020

0.024 ± 0.006

0.010 ± 0.004

0.112 ± 0.015

0.022 ± 0.002

3.710 ± 0.152

0.051 ± 0.007

0.011 ± 0.002

0.007 ± 0.001

0.373 ± 0.041

0.227 ± 0.021

0.097 ± 0.017

0.051 ± 0.008

0.198 ± 0.015

0.182 ± 0.024

0.010 ± 0.0007

0.008 ± 0.002

0.222 ± 0.009

0.013 ± 0.002

IC50 ± SD (µM)

(

NF54 Clone)

1

2

1

7a

7b

7c

7d

8a

8b

8c

8d

9a

9b

9c

9d

9e

9f

9g

9h

9i

9j

9k

9l

0a

0b

0c

0d

0e

0f

0g

0h

0i

0j

0k

0l

3

4

1

2

1

2

1

2

1

2

1

2

H

Cl

H

Cl

H

Cl

H

Cl

H

Cl

H

Cl

1.761 ± 0.072

1.838 ± 0.071

0.092 ± 0.013

0.144 ± 0.021

0.413 ± 0.057

1.218 ± 0.105

0.300 ± 0.060

0.121 ± 0.020

0.013 ± 0.002

0.023 ± 0.004

0.234 ± 0.031

0.109 ± 0.019

0.027 ± 0.004

0.028 ± 0.004

0.088 ± 0.014

0.352 ± 0.023

0.102 ± 0.011

0.880 ± 0.046

0.052 ± 0.008

0.031 ± 0.005

0.035 ± 0.006

0.025 ± 0.002

2.735 ± 0.170

0.039 ± 0.007

0.046 ± 0.009

0.022 ± 0.005

0.027 ± 0.005

0.262 ± 0.039

0.097 ± 0.012

0.082 ± 0.007

0.009 ± 0.004

0.022 ± 0.006

0.004 ± 0.002

0.005 ± 0.0009

0.027 ± 0.006

0.010 ± 0.005

*

1396

*

145.19 ± 10.82

*

144.17 ± 4.32

78.99 ± 5.45

132.86 ± 10.71

*

31.15 ± 2.13

15.84 ± 2.10

21.79 ± 2.41

*

1501

4936

1476

*

421

1131

7263

*

Morpholine

Piperidine

Morpholine

Piperidine

NꢀMethylpiperazine

NꢀEthylpiperazine

NꢀMethylpiperazine

NꢀEthylpiperazine

Morpholine

Piperidine

NꢀMethylpiperazine

NꢀEthylpiperazine

Morpholine

Piperidine

H

*

1485

*

Cl

H

99.54 ± 7.26

*

126.19 ± 4.51

107.58 ± 4.26

19.61 ± 1.08

44.46 ± 2.70

*

17.82 ± 3.14

22.70 ± 3.10

28.56 ± 5.41

*

5257

10758

175

2020

*

350

2063

4080

*

H

Cl

H

Morpholine

Piperidine

NꢀMethylpiperazine

NꢀEthylpiperazine

NꢀMethylpiperazine

NꢀEthylpiperazine

Morpholine

Piperidine

NꢀMethylpiperazine

NꢀEthylpiperazine

NꢀMethylpiperazine

NꢀEthylpiperazine

H

*

Cl

CH

230.77 ± 0.00

11.09 ± 2.55

*

19.93 ± 2.31

20.78 ± 3.06

*

2379

217

*

3

1993

2597

*

CQ

Artesunate

*

a

b

c

d

IC50, the concentration that causes 50% growth inhibition; SD, Standard Deviation; CHO cells, Chinese Hamster Ovary cells; SI, Selectivity index (IC50

for cytotoxicity to CHO cells/IC50 for antimalarial activity against CQꢀresistant strains); *Not Determined

#

5

Table 2: In-vivo antimalarial activity of hybrid 19e against P. berghei infected mice

Day

Controls

Compound 19e

Oral 30 mg/kg

Placebo

Mean

CQ 15 mg/kg

Intravenous 5 mg/kg

Oral 10 mg/kg

Mean SEM

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

4

7

9

0

ꢀ

0

ꢀ

0

ꢀ

2.05

ꢀ

0

1.28

2.08

14.94

26.95°

ꢀ

0

ꢀ

21.10

ND

54.70

68.05

2.75

ꢀ

2.74

1.89

2.06

2.52

6.42

8.22

0.46

0.33

1.66

0.66

18.43*

ND

0.23

0.45

5.15

6.39

14.54

18.95

38.22

58.06

1.59

2.05

5.76

4.68

ꢀ

1

62.40**

5.50

#

Mean parasitemia levels in the treatment groups after dosing with 19e for 4 days. ND = not determined. *n=4; one animal excluded owing to a technical

error. **n=3; one animal euthanized on Day 7. n=4; one animal euthanized on Day 10.

Although hybrid 19e is able to suppress parasitemia for the

The cytotoxicity of the selected active hybrids (13, 14, 17d,

duration of the dosing period, it was unable to completely clear

the parasites from infected mice.

18d, 19a, 19b, 19dꢀf, 19i, 19k, 19l, 20a, 20b, 20dꢀf, 20i and 20j)

was assessed against CHO (Chinese Hamster Ovary) cells (table

10

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1

). For the tested hybrids, cytotoxicity appeared at much higher

50

A solution of hematin in 40% DMSO showed a Soret band at

402 nm, indicating the presence of monomeric heme under the

conditions used (0.02 M HEPES buffer, pH 7.4 and 0.02 M MES

buffer, pH 5.6). The stepwise addition of small increments of

compound 19f into a constant concentration of monomeric heme

concentrations than the concentrations responsible for their

antimalarial activity indicating compounds safe toxicity profile.

The most toxic of the hybrids was found to be compound 20j

with IC₅₀value 11.09 µM while the least toxic is hybrid 20i (IC₅₀

5

0

5

value is 230.77 µM). The high selectivity index (the ratio of IC₅₀55 (5.0 ꢁM) resulted in a substantial decrease in the intensity of the

for cytotoxicity to CHO cells and IC₅₀for antimalarial activity

against CQꢀresistant strains revealing an estimate of a therapeutic

window) for most of the hybrids further revealed their potency

against the tested Plasmodium isolates. Three hybrids (19f, 19k

Fe(III) PPIX Soret band at 402 nm with no shift in the absorption

maximum. This indicates the association of compounds with

hematin (figure 4). Solvent (DMSO) did not affect the binding of

compound 19f with heme at the pH values used in this

1

and 19l) showed selectivity index > 5000 whereas other displayed 60 experiment. The stoichiometry ratio of the most stable complexes

in the range of 220 to 4347. Five hybrids (19a, 19f, 19k, 19l and

of compound 19f with monomeric heme at pH 7.5 and 5.6 was

deduced from the Job’s plot. The absorbance at 402 nm got to

maximum when the mole fraction of the compound was

approximately 0.5. Thus 1:1 ratio was established for the

2

4

0f) were found to have greater selectivity index (in the range of

070ꢀ10758) than the previously identified lead molecules 13 and

1

14. To know the probable mode of action of these hybrids, heme

binding and molecular modelling studies were further performed 65 association of compound heme at both the pH values (figure 5).

on the most active hybrid.

As discussed earlier that CQ and its derivatives also bind to

heme dimer (µꢀoxo heme). Therefore, the binding of compound

1

9f was also studied with ꢁꢀoxo dimers of heme at pH 5.8. A

1

9e

8

6

4

2

0

solution of heme in aqueous NaOH showed a peak at 362 nm.

Addition of compound 19f (0ꢀ20 ꢁ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 6A), which

shows the interaction between heme and the compound 19f. The

Job’s plot indicated a 1:1 stoichiometry for the most stable ꢁꢀoxo

75 heme:compound 19f complex (figure 6B). The association

constants for the complexes formed between monomeric Fe (III)

PPIX and compound 19f at pH 7.4 and 5.6 were calculated by the

analysis of titration data and are presented in Table 3.

Placebo

70

CQ 15mg/kg

IV 5mg/kg

Oral 30mg/kg

Oral 10mg/kg

0

2

4

6

8

10

12

Day post-infection

2

0

Fig. 3: Parasite levels in mice infected with Plasmodium berghei ANKA

and treated orally or intravenously with compound 19e for 4 consecutive

days. The data represent mean ± SEM.

80

Table 3: Binding constants for compound 19f and chloroquine with

heme.

Heme Binding Studies

Chloroquine and other 4ꢀaminoquinoline derivatives are believed

to show their antimalarial action by interfering with the parasite

feeding process during which heme ferriprotoporphyrinꢀIX is

generated which is very toxic to the parasite but through a unique

mechanism, parasite detoxifies it within the food vacuole by

crystallizing it to insoluble granules of nonꢀtoxic material called

hemozoin. Hemozoin is an insoluble ionic polymer in which

adjacent hematin units are linked via a carboxylate iron (III)

coordinate bond. Hemozoin was originally considered to be

formed by the polymerization of heme, but it has now been

2

3

4

5

0

5

0

5

Compound

Monomeric

heme log K

Monomeric

heme log K

(pH = 5.6)

5.81

4.65

1:1

µꢀoxoꢀheme

log K

(pH = 5.8)

(pH = 7.4)

1

9f

5.19

5.15

1:1

5.83

5.58

10

CQ

Stoichiometry

1:1

The association constants for the complexes formed between

monomeric heme and compound 19f at pH 7.5 (log K 5.19) were

demonstrated to be

ferriprotoporphyrinꢀIX.

a

crystalline cyclic dimer of 85 found to be almost equivalent to the standard antimalarial drug

It is widely accepted that CQ

1

2

10

CQ (log K 5.15). Furthermore, decreasing the pH from 7.4 to

5.6 (food vacuole pH), compound has shown improved binding

constant (log K 5.81) indicating that binding is stronger even at

acidic pH of food vacuole. Interestingly, compound 19f showed

accumulates in the plasmodium food vacuole and inhibit the

process of formation of hemozoin, by forming a complex with

heme/hematin which in turn leaves heme in an uncrystallised

form which has a toxic effect and eventually kills the parasite. 90 the larger value of the binding constant than the standard drug

Cohen et al were the first to show that CQ forms a complex with

ferriprotoporphyrinꢀIX (FPIX) in aqueous solution, which was

proved by the changes in the UVꢀspectrum of aqueous hematin in

chloroquine at pH 5.6. The association constants for the binding

with µꢀoxo heme at pH 5.8 (log K 5.83) was found to be even

greater than the monomeric heme complexes. From the data

shown in table 3, it is clear that the compound 19f binds strongly

1

3

the presence of drug. Later on, several studies explained the

formation of CQꢀhematin complex by computational methods as 95 with monomeric heme (log K 4.63) as well as ꢁꢀoxoꢀheme (log K

1

4

well as the spectroscopic methods. More recently it has been

determined that CQ forms complexes with both monomeric and

5.83) and the observed results are comparable to the standard CQ

(log K 5.58). Thus the formation of a complex between heme and

compound 19f suggests the inhibition of formation of βꢀhematin,

which could be correlated to the observed antimalarial activity of

100 these compounds in a fashion similar to that of CQ.

1

5

ꢁ

ꢀoxo dimeric FPIX. Therefore, we decided to evaluate the

binding of the one of the most active compound 19f from the

series with heme.

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1

.9

.8

.7

.6

.5

.4

.3

.2

.1

0

1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

A

B

0

3

60

380

400

420

440

360

380

400

420

440

Wavelength (nm)

Fig. 4: A) Titration of compound 19f with monomeric heme at pH 7.5; B) Titration of compound 19f with monomeric heme at pH 5.6

0

.05

.045

.04

.035

.03

.025

.02

.015

.01

0.007

.006

0.005

0

A

B

0

.004

.003

0

0.002

0

.001

0

.005

0

0.2

0.4

0.6

0.8

1

0

0.2

0.4

0.6

0.8

1

X

5

Fig. 5: Job’s plot of monomeric heme complex formation with compound 19f; A) at pH 7.4; B) at pH 5.6; X (mole fraction of the

compound 19f) = [compound 19f]/[ compound 19f] + [ 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

0.014

0.012

0.01

A

B

0.008

0.006

0.004

0.002

0

3

00

350

400

450

500

550

0

0.2

0.4

0.6

0.8

1

Wavelength

X

Fig. 6: A) Titration of compound 19f with ꢁꢀoxo dimeric heme at pH 5.8; B) Job’s plot of ꢁꢀoxo dimeric heme complex formation with

compound 19f at pH 5.8.

1

0

16

the cause of dihyrofolate resistance. In the present work we

have attempted to study the interactions of novel 4ꢀ

25 aminoquinolineꢀpyrimidine based hybrid compounds with Pfꢀ

DHFRꢀTS.

For this purpose molecular docking studies of best active

compounds (19a, 19e, 19f, 19l, 20b, 20e, 20f) were performed in

the binding pocket of both the wild type PfꢀDHFRꢀTS (PDB

Molecular Modelling studies

a) Binding mode analysis

With the emergence of resistance to most of the antimalarial

drugs especially quinoline and antifolate class of drugs, the quest

for novel drug candidate for malaria is needed. Antifolates act by

inhibiting dihydrofolate reductase activity of Plasmodium

falciparum bifunctional enzyme dihydrofolate reductaseꢀ

thymidylate synthase (PfꢀDHFRꢀTS). Occurrence of four point

mutations in codons 51, 59, 108, and 164 (N51I, C59R, S108N

and I164L) in the DHFR domain of PfꢀDHFRꢀLS gene from the

clinical isolates of dihydrofolate resistant parasite shed light on

15

3

0

ID:3QGT) and quadruple mutant PfꢀDHFRꢀTS (N51I, C59R,

S108 N, I164L, PDB ID:3QG2) structures. The results of docking

studies and the docked conformations of best scored ligands (19a

and 20e) in the active site of wild and mutant PfꢀDHFRꢀTS are

illustrated in table 4 and figures 6 and 7. These docking results

clearly indicate that the most active compounds in the study

exhibited significant binding affinities towards the wild (Glide

20

35

ꢀ1

energy range ꢀ51.64 kcalmol to ꢀ40.52 kcalmol ) and quadruple

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ꢀ

1

ꢀ1

mutant (Glide energy range ꢀ54.09 kcalmol to ꢀ42.81 kcalmol )

PfꢀDHFRꢀTS structures and the energy ranges are comparable to

that of reference compounds (pyrimethamine, cycloguanil and

compound predicted to have low binding energy (ꢀ42.55

Kcalmol ) and high glide score (ꢀ7.09) was compound 19a,

showing similar Hꢀbond pattern between linker NH group of

ꢀ

1

WR99210) and the native substrate of DHFR dihydrofolate 20 compound and oxygen side chain of Asp54. The πꢀπ interactions

5

0

5

(Table 4).

The compounds showed hydrogen bond interactions along

with πꢀπ interactions in the predicted binding poses. Compound

between aromatic ring of Phe58 and 4ꢀchloropyrimidine ring of

compound were also observed (figure 8). No Hꢀbond was

observed between the compound and Ser111 of the proteins. The

influence of quadruple mutations in DHFR (N51I, C59R, S108 N,

ꢀ

1

2

0e showing lowest binding energy (ꢀ54.09 kcalmol ) and

ꢀ

1

considerable high Glide score (ꢀ8.18 kcalmol ) for mutant Pfꢀ 25 I164L) is attributed to the movement of the active site residues

1

DHFR with formation of hydrogen bonds between a linker NH

group of compound 20e and oxygen side chain of Asp54 of both

wild and mutant PfꢀDHFR. Further πꢀπ interactions between

aromatic ring of Phe58 and 4ꢀchloropyrimidine ring of compound

are observed (figure 7). An additional Hꢀbond between main 30 which has been reported crucial for inhibitors and substrate

chain oxygen atom of Ser111 and quinolone nitrogen was

and interferes in the inhibitor binding. N51I causes movement in

main chain atoms of residues 48ꢀ51 and C59R mutation does not

cause any significant changes in the protein structure. Residues

51 and 59 lie in the proximity of the active site residue Asp54,

1

binding.

observed in the docking mode of wild PfꢀDHFR. Another

ꢀ1

Table 4: Glide docking scores (kcal mol ) 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

Docking results with wild PfDHFR

Glide

Docking results with mutant PfDHFR

Glide

Comp

Glide

Score

Glide lipo

Glide Energy

Glide Score

Glide lipo

Glide Energy

Hꢀbond

ꢀ0.19

ꢀ0.18

ꢀ0.26

ꢀ0.22

0.25

Hꢀbond

ꢀ0.35

ꢀ0.17

ꢀ0.21

ꢀ0.46

ꢀ0.32

ꢀ0.42

ꢀ0.45

ꢀ0.74

ꢀ0.82

ꢀ0.73

ꢀ0.30

1

9a

9e

ꢀ7.09

ꢀ5.63

ꢀ5.88

ꢀ6.40

ꢀ6.97

ꢀ8.21

ꢀ6.44

ꢀ9.34

ꢀ8.82

ꢀ8.77

ꢀ5.61

ꢀ3.05

ꢀ2.82

ꢀ2.87

ꢀ3.27

ꢀ3.14

ꢀ2.83

ꢀ2.29

ꢀ2.27

ꢀ2.55

ꢀ1.89

ꢀ2.0

ꢀ42.55

ꢀ40.52

ꢀ43.55

ꢀ44.23

ꢀ51.64

ꢀ51.20

ꢀ41.80

ꢀ44.85

ꢀ65.11

ꢀ57.97

ꢀ39.04

ꢀ7.37

ꢀ7.17

ꢀ5.36

ꢀ6.28

ꢀ6.31

ꢀ8.18

ꢀ6.56

ꢀ8.88

ꢀ9.39

ꢀ9.28

ꢀ7.61

ꢀ3.29

ꢀ1.95

ꢀ1.78

ꢀ3.73

ꢀ3.51

ꢀ3.20

ꢀ4.47

ꢀ2.37

ꢀ3.01

ꢀ2.43

ꢀ2.51

ꢀ51.07

ꢀ44.48

ꢀ42.81

ꢀ46.77

ꢀ48.93

ꢀ54.09

ꢀ43.8

1

9f

9l

1

2

0b

0e

0f

2

ꢀ0.32

ꢀ0.27

ꢀ0.72

ꢀ0.85

ꢀ0.88

0.52

2

Dihydrofolate

Pyrimethamine

Cycloguanil

WR99210

ꢀ59.48

ꢀ32.48

ꢀ39.05

ꢀ40.08

Comp 19a

A

Comp 20e

A

NADH

Asp54

Phe58

Asp54

Phe58

B

Phe58

Asp54

Ser111

NADH

3

5

40

Fig. 8: 2D and 3D docking pose showing interaction for compound 19a

in the binding site of (A) mutant PfDHFRꢀTS (PDB ID: 3QG2); (B) wild

PfꢀDHFRꢀTS (PDB ID: 3QGT).

Fig. 7: 2D and 3D docking pose showing interaction for compound 20e in

the binding site of (A) mutant PfDHFRꢀTS (PDB ID: 3QG2); (B) wild Pfꢀ

DHFRꢀTS (PDB ID: 3QGT)

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In the present docking study, we have observed Hꢀbond 40 sticks),Cycloguanil (red sticks), WR99210 (blue sticks) and the PfꢀDHFR

substrate dihydrofolate (yellow ball and stick) bound to the binding site of

interactions of test compound with Asp54 similar to the DHFR

native substrate dihydrofolate. Moreover, the function of Asp54

residue is preserved in the mutant protein and not affected by the

quadruple mutant PfꢀDHFR (PDB ID: 3QG2) showing the fitting of the

test compounds and the PfꢀDHFR inhibitors in the surface area occupied

by the PfꢀDHFR substrate (dihydrofolate).

1

7

5

0

5

0

5

0

5

two proximal mutations N51I and C59R. Further, I164L

mutation causes shifts in the residues 164ꢀ167 and affects the gap

in the active site causing steric interactions of Phe58 with small

inhibitors such as pyrimethamine and cycloguanil. The test

compounds form a long distance interaction with Phe58 forming

πꢀπ interaction, thus avoiding steric clash with the aromatic side

chain of Phe58. In PfꢀDHFR S108N mutation, the pꢀchlorophenyl

moiety in pyrimethamine and cycloguanil cause steric

interference with the side chain of Asn108 in the modified active

site. Thus, it becomes appropriate to explore the binding pattern

of novel lead compound in the preliminary stages of drug design

against mutant proteins. Figure 9 shows the binding mode of test

compounds used in the study in comparison with the PfDHFR

inhibitors (Pyrimethamine, Cycloguanil and WR99210) in the

active site of the mutant PfꢀDHFR structure. In S108N mutation,

the pꢀchlorophenyl moiety in pyrimethamine and cycloguanil

cause steric interference with the side chain of Asn108 in the

modified active site mutant PfꢀDHFR. Whereas, WR99210

having flexible linker does not show steric hindrance with

Asn108. It was observed that WR99210 occupies similar surface

volume as the PfꢀDHFR native substrate (dihydrofolate). Several

observations have shown that drug molecules designed to occupy

the surface volume of the native substrate of the protein will be

less susceptible to resistance occurring due to steric clashes in the

4

5

b) Prediction of Pharmacokinetic Properties

Different pharmacokinetic parameters of compounds in the study,

showing good inhibitory potential in malarial parasites were

calculated using ADMET predictions by Qikprop. The most

19

1

2

3

important of these parameters together with its permissible ranges

are listed in the table 5 and 6. As a preliminary test of the drugꢀ

likeness of the compounds, we calculated Lipinski’s rule of 5

using QikProp (table 5). An orally active compound should not

have more than four violations of these rules. In the present

study, all the active compounds showed “0” value for Lipinski’s

rule of 5 violations, indicating that these active test compounds

have good drug likeness properties similar to the reference

molecules pyrimethamine and cycloguanil.

Prediction of oral drug absorption (Percent Human Oral

Absorption) was highly satisfactory for all the test compounds.

Studies have suggested that oral bioavailability is influenced by

compound’s flexibility and can be measured by the number of

rotable bonds (<15) and polar surface area (70 Å ꢀ200 Å ). In

the present study, all the test compounds have a number of

rotable bonds <15 and polar surface area falls satisfactorily in the

permissible range (table 6). Similarly, molecules following

Lipinski’s rule of 5 are more likely to have good intestinal

absorption or permeation which is confirmed by the predicted

5

6

7

8

0

5

0

5

0

5

0

2

2 20

1

8

mutated protein binding site. Hence, we have performed

molecular overlay of docking poses of active test compounds

along PfꢀDHFR inhibitors (Pyrimethamine, Cycloguanil and

WR99210) on the dihydrofolate surface envelope and it is clear

from the figure 9 that the test compounds occupy the similar

volume as that of the protein substrate unlike Pyrimethamine and

Cycloguanil to avoid the steric clash with the side chain of

Asn108.

Cacoꢀ2 cells permeability (QPPCaco), used as a model for the

21

gutꢀblood barrier.

QPPCaco predictions for all the test

compounds showed very good values with the exception for

compound 19e having slightly good values for Cacoꢀ2 cells

permeability and is comparable to the value predicted for the drug

pyremethamine. Further, QPlogKhsa, the prediction for human

serum albumin binding and all inhibitors were predicted to lie

within the expected range for 95% of known drugs (ꢀ1.5 to 1.5).

Also, the QikProp descriptor for brain/blood partition coefficient

(QPlogBB) and the bloodꢀbrain barrier mimic MDCK cell

permeability (QPPMDCK) show satisfactory predictions for all

the test compounds and the reference compounds.

Table 5: Prediction of Lipinski’s ‘Rule of 5’ for the active test

a

compounds

Rule

of

Five

mol_M dono

accpt

HB

(<10)

QPlog

Po/w

(<5)

Comp

W

> 500)

rHB

(<5)

(

(<4)

1

9a

9e

9f

9l

384.86

432.35

446.38

460.40

382.89

432.35

446.38

248.7

2

4

5

7

5

7

3

3.74

4.11

4.61

4.9

4.72

4.1

4.47

1.81

0.89

0

2

0b

0e

0f

Pyremethamine

Cycloguanil

253.73

a

All values calculated by QikProp v 3.5 and the explanations of the

Fig. 9: Molecular overlay of most active docked test compounds

descriptors are given in the text

(represented as wireframe format with atom colours), Pyrimethamine (red

8

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Table 6: Calculated ADMET properties

ꢀ

1

Compound

Percent Human Oral

Absorption (>80%

high,<25% poor)

QPPCaco nms (<25 QPlogBB_a

QPPMDCK

QPlogKhsa_a

(ꢀ1.5 to 1.5)

PSA (7.0ꢀ

200.0)

#rotor_a

(0ꢀ15)

a

poor, >500 great)

(ꢀ3.0ꢀ1.2)

(<25 poor; >500

a

great)

1

2

9a

9e

9f

9l

0b

0e

0f

100

84.34

68.8

2159.46

497.61

564.96

596.79

2432.89

584.59

632.19

412.28

111.854

ꢀ0.26

0.22

0.21

0.14

ꢀ0.23

0.27

0.23

ꢀ0.78

ꢀ0.17

2084.40

1479.7

0.24

68.4

5

6

7

5

6

4

2

0.529

0.611

0.75

0.65

0.51

0.611

ꢀ0.24

ꢀ0.30

64.08

68.75

63.43

56.91

61.93

61.42

73.73

76.36

1714.28

1967.53

3190.13

1556.19

1693.53

468.85

Pyrimethamine

Cycloguanil

126.64

a

Calculated using QikProp v 3.5. Range/recommended values calculated for 95% known drugs

Conclusions

Experimental Section

In conclusion,

a series of thirty two highly active 4ꢀ

Instrumentation and chemicals

5

0

5

0

5

0

aminoquinolineꢀpyrimidine hybrids were synthesised using a

three step procedure and evaluated for their antimalarial activity

against both CQꢀsensitive and CQꢀresistant strains of P.

falciparum. 19 hybrids (18d, 19aꢀ19f, 19i, 19k, 19l, 20a, 20b,

4

5

6

5

0

5

0

All of the chemicals used in the synthesis were purchased from

SigmaꢀAldrich and were used as such. Thin layer

chromatography was used to monitor the progress of the reactions

and checked by preꢀcoated TLC plates (E. Merck Kieselgel 60

F₂₅₄) with spots being visualized by iodine vapors. Compounds

were purified over silica gel (60ꢀ120 mesh) column or

recrystallized with suitable solvents. Solvents were distilled

before using for purification purposes. Meting points were

recorded on an ERS automated melting point apparatus and are

uncorrected. IR spectra were recorded using PerkinꢀElmer and

2

0dꢀ20f, and 20iꢀ20l) displayed superior antimalarial activity

1

2

3

than CQ while 4 hybrids (19f, 19l, 20e and 20f) exhibited better

antimalarial activity than artesunate against drugꢀresistant P.

falciparum strain. The most active hybrid 19f showed 74ꢀfold

better activity than CQ and around 4ꢀfold better activity than

artesunate in the assay. Inhibition of formation of βꢀhematin and

dihydrofolate reductaseꢀthymidylate synthase (PfꢀDHFRꢀTS)

enzyme could be associated to the observed antimalarial activity

of these compounds as observed by heme binding and molecular

modelling studies. Compounds 19a and 20e were predicted to

show good binding with wild type and mutant PfꢀDHFR proteins

with interaction pattern comparable to that of DHFR inhibitors

and native DHFR substrate. Moreover, the test compounds

efficiently bind with the mutant protein avoiding steric clashes

resulting from the amino acid mutations. The calculated in silico

ADMET parameters for all the active test compounds indicated

good drug likeness character and pharmacokinetic properties,

making them important candidates in the antiꢀmalarial drug

discovery process. This level of activity coupled with their

possible mechanism of action of inhibiting two targets

simultaneously within a system permits their further investigation

as a lead candidate in malarial chemotherapy for the production

of next generation antimalarials.

ꢀ1

Bruker FTꢀIR and the values are expressed as λ_m_a_xcm . HRMS

data were recorded on Agilent G6530 AA LCꢀHRMSQꢀTOF

1

13

instrument. The H NMR and C NMR spectra were recorded on

Jeol Spectrospin spectrometer at 400 MHz and 100 MHz

respectively. The referencing was done using TMS as an internal

standard in case of CDCl solvent and with residual DMSO peak

3

in case of DMSOꢀd6 solvent. The chemical shift values are

recorded on δ scale and the coupling constants (J) are in Hz.

General procedure for the synthesis of compounds 16a-b

A mixture of 4,7ꢀdichloroquinoline (1 eq.) and diaminoalkane

o

6

5

(5 eq.) was heated at 120ꢀ130 C under nitrogen atmosphere for

6

ꢀ8 h. Ice was added to the reaction mixture and reaction mixture

o

was stirred at 0 C for 1 h. The precipitate thus formed was

filtered, washed with cold water (100 mL) and dried. The crude

product was dissolved in 50 ml CHCl and washed with water (3

3

70

× 250 mL) and finally with brine. Organic layer was dried over

Na SO and excess of solvent was removed under vacuum. The

Acknowledgements

2

4

crude product thus obtained was recrystallized by CHCl /hexane

system to get pale yellow solid of compound 16a and 16b.

3

DSR thank University of Delhi and DUꢀDST PURSE for

financial support. SM and DK are thankful to CSIR for the award

of junior and senior research fellowship. SPV is thankful to UGC

for junior research fellowship [File No: 41ꢀ20282012(SR)]. PP is

thankful to CSIR for Research Associate. DT and LW are

thankful to South African National Research Foundation for

funding support. The authors are also thankful to CIFꢀUSIC,

University of Delhi, Delhi for NMR & HRMS data.

3

5

22

N1-(7-Chloroquinolin-4-yl)ethane-1,2-diamine (16a) : Yield:

o

1

75

90%; mp 139ꢀ141 C; H NMR (400 MHz, DMSOꢀd ) 2.86 (t, J

6

=

6.0 Hz, 2 H, CH ); 3.27ꢀ3.29 (m, 2 H, CH ); 4.53 (brs, 2 H,

2 2

NH ); 6.47 (d, J = 5.3 Hz, 1 H, ArH); 7.37ꢀ7.42 (m, 2 H, NH and

2

4

0

ArH); 7.75 (d, J = 2.2 Hz, 1 H); 8.26ꢀ8.29 (m, 1 H, ArH); 8.37 (d,

J = 5.3 Hz, 1 H, ArH); HRMS (ESI, m/z) calcd for C H ClN :

1

13

3

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+

2

22.0793 (MH) ; Found: 222.0796.

ArH); 7.32 (brs, 1 H, NH); 7.43 (dd, J = 8.7 Hz, 2.2 Hz, 1 H,

ArH); 7.75ꢀ7.77 (m, 2 H, NH and ArH); 8.19ꢀ8.25 (m, 2 H, ArH);

2

13

N1-(7-Chloroquinolin-4-yl)propane-1,3-diamine (16b) : Yield: 55 8.36 (d, J = 5.1 Hz, 1 H, ArH); C NMR (100 MHz, DMSOꢀd

):

6

o

1

8

(

0%; mp 95ꢀ97 C; H NMR (400 MHz, DMSOꢀd ) 1.73ꢀ1.76

m, 2 H, CH ); 2.71 (t, J = 6.0 Hz, 2 H, CH ); 3.29ꢀ3.32 (m, 2 H,

CH ); 4.04 (brs, 2 H, NH ); 6.45 (d, J = 5.3 Hz, 1 H, ArH); 7.40ꢀ

.42 (m, 2 H, NH and ArH); 7.75 (d, J = 2.2 Hz, 1 H); 8.22ꢀ8.25

28.1, 39.5, 99.5, 109.6, 118.3, 124.9, 128.1, 134.2, 149.7, 150.9,

152.5, 160.7, 163.1; HRMS (ESI, m/z) calcd for C16H16Cl N :

2 5

6

2

+

5

348.0777 (MH) ; Found: 348.0782; ESIꢀMS (m/z): 348.13,

2

+

7

350.13 (MH + 2) ; Anal. Calcd for C16H15Cl N : C, 55.19; H,

2 5

(

m, 1 H, ArH); 8.35ꢀ8.37 (m, 1 H, ArH); HRMS (ESI, m/z) calcd 60 4.34; N, 20.11; Found: C, 55.25; H, 4.49; N, 20.01.

+

for C H ClN : 236.0949 (MH) ; Found: 236.0946.

12

15

3

1

3

N -(7-chloroquinolin-4-yl)-N -(2,6-dichloropyrimidin-4-

o

General procedure for the synthesis of compounds 17a-d

and 18a-d

yl)propane-1,3-diamine (17d): Yield: 55%; mp 205ꢀ207 C; IR

ꢀ

1

10

(cm , KBr): 3259, 3116, 2937, 1609, 1590, 1457, 1141, 1092,

61, 794; H NMR (400 MHz, DMSOꢀd ): 1.87ꢀ1.93 (m, 2 H,

6

1

8

To a well stirred solution of 2,4ꢀdichloropyrimidine or 2,4,6ꢀ 65 CH

trichloropyrimidine (1 eq.) and triethylamine (2 eq.) in ethanol

50 ml) at room temperature was added diamines (1 eq.). The

); 3.31ꢀ3.37 (m, 4 H, CH ), 6.44ꢀ6.45 (m, 1 H, ArH); 6.81ꢀ

2

6.84 (m, 1 H, ArH); 7.28 (brs, 1 H, NH); 7.42 (dd, J = 8.7 Hz, 2.2

Hz, 1 H, ArH); 7.76 (d, J = 2.2 Hz, 1 H, ArH); 8.22ꢀ8.24 (m, 2 H,

(

13

reaction mixture was allowed to stir overnight at room

temperature. After completion of reaction as evident by TLC,

NH and ArH), 8.36 (d, J = 5.1 Hz, 1 H, ArH); C NMR (100

15

MHz, DMSOꢀd ): 27.5, 39.4, 99.3, 107.9, 118.0, 124.65, 124.68,

6

reaction mixture was poured into ice cold water (250 ml) and 70 128.1, 133.9, 149.6, 150.6, 152.4, 161.3, 161.7, 162.0; HRMS

+

precipitate thus formed was filtered and washed with excess of

water at vaccum pump. The crude precipitate was dissolved in

(ESI, m/z) calcd for C16

382.0384; Anal. Calcd for C16

18.30; Found: C, 50.12; H, 3.60; N, 18.43.

H

15Cl

H

3

N

5

: 382.0387 (MH) ; Found:

14Cl N : C, 50.22; H, 3.69; N,

3 5

1

00 ml of CHCl and extracted with water (2 × 500 ml) and

3

20

finally with brine. Excess of solvent was evaporated to dryness

under vaccum and the crude product thus obtained was purified

1

2

N -(4-chloropyrimidin-2-yl)-N -(7-chloroquinolin-4-yl)ethane-

o

ꢀ1

by SiO column using MeOH/CHCl as eluent to yield respective 75 1,2-diamine (18a): Yield: 30%; mp 216ꢀ218 C; IR (cm , KBr):

2

3

1

compounds 17aꢀd and 18aꢀd.

2427, 1600, 1431, 1384, 1242, 978, 763; H NMR (400 MHz,

DMSOꢀd ): 3.50ꢀ3.52 (m, 4 H, CH ); 6.44 (d, J = 5.8 Hz, 1 H,

ArH); 6.79 (d, J = 5.8 Hz, 1 H, ArH); 7.48 (dd, J = 8.7 Hz, 2.2

Hz, 1 H, ArH); 7.82 (d, J = 2.2 Hz, 1 H, ArH); 7.86 (d, J = 5.8

6

2

1

2

N -(2-chloropyrimidin-4-yl)-N -(7-chloroquinolin-4-yl)ethane-

o

ꢀ1

25

30

35

1,2-diamine (17a): Yield: 65%; mp 234ꢀ236 C; IR (cm , KBr):

1

3

253, 2967, 1608, 1585, 1427, 1237, 1142, 901, 792; H NMR 80 Hz, 1 H, ArH); 8.19 (brs, 1 H, NH); 8.30 (brs, 1 H, NH); 8.34 (d,

1

3

(

400 MHz, DMSOꢀd ): 3.42ꢀ3.46 (m, 4 H, CH ); 6.62ꢀ6.75 (m, 2

H, ArH); 7.45 (dd, J = 8.7 Hz, 2.2 Hz, 1 H, ArH); 7.53 (brs, 1 H,

NH); 7.78ꢀ7.79 (m, 2 H); 8.20 (d, J = 8.7 Hz, 1 H, ArH); 8.28

J = 8.7 Hz, 1 H, ArH); 8.40 (d, J = 5.8 Hz, 1 H, ArH); C NMR

(100 MHz, DMSOꢀd ): 38.2, 41.3, 98.8, 105.2, 116.8, 124.7,

124.9, 134.8, 145.6, 148.9, 151.9, 155.6, 159.8, 163.5; HRMS

6

2

6

1

3

+

(brs, 1 H, NH); 8.40 (d, J = 5.1 Hz, 1 H, ArH); C NMR (100

MHz, DMSOꢀd ): 41.5, 98.8, 109.2, 117.3, 124.1, 124.3, 126.9, 85 334.0625; Anal. Calcd for C15

(ESI, m/z) calcd for C15

H

14Cl

H

2

N

5

: 334.0621 (MH) ; Found:

: C, 53.91; H, 3.92; N,

20.96; Found: C, 53.99; H, 3.76; N, 20.88.

13Cl N

2 5

6

1

33.7, 148.3, 150.5, 151.3, 159.9, 160.1, 162.3; HRMS (ESI,

m/z) calcd for C H Cl N : 334.0621 (MH) ; Found: 334. 0627;

Anal. Calcd for C H Cl N : C, 53.91; H, 3.92; N, 20.96; Found:

C, 53.73; H, 3.98; N, 21.03.

+

15

14

2

5

1

2

N -(7-chloroquinolin-4-yl)-N -(4,6-dichloropyrimidin-2-

15

13

2

5

o

yl)ethane-1,2-diamine (18b): Yield: 28%; mp 235ꢀ237 C; IR

ꢀ

1

(

cm , KBr): 3352, 2944, 2860, 1612, 1568, 1451, 1366, 1272,

1

2

1

N -(7-chloroquinolin-4-yl)-N -(2,6-dichloropyrimidin-4-

90 1173, 985, 829, 764; H NMR (400 MHz, DMSOꢀd

): 3.47ꢀ3.57

6

o

yl)ethane-1,2-diamine (17b): Yield: 68%; mp 237ꢀ239 C; IR

cm , KBr): 3266, 3113, 1610, 1572, 1449, 1239, 1099, 811,

94; H NMR (400 MHz, DMSOꢀd ): 3.44ꢀ3.50 (m, 4 H, CH );

6 2

(m, 4 H, CH ); 6.53 (s, 1 H, ArH); 6.71 (d, J = 5.1 Hz, 1 H, ArH);

2

ꢀ

1

(

7.46 (d, J = 8.8 Hz, 1 H, ArH); 7.58 (brs, 1 H, NH); 7.78 (d, J =

2.2 Hz, 1 H, ArH); 8.21 (d, J = 8.8 Hz, 1 H, ArH); 8.41ꢀ8.42 (m,

1

7

13

40

6.69 (d, J = 5.1 Hz, 1 H, ArH); 6.88 (s, 1 H); 7.42ꢀ7.45 (m, 2 H,

NH and ArH); 7.77 (d, J = 2.2 Hz, 1 H, ArH); 8.18 (d, J = 9.5 Hz, 95 124.7, 124.9, 127.4, 134.4, 148.8, 151.0, 151.8, 157.5, 159.6,

2 H); C NMR (100 MHz, DMSOꢀd ): 41.7, 99.5, 103.5, 117.9,

6

1

H, ArH); 8.27 (brs, 1 H, NH); 8.40 (d, J = 5.1 Hz, 1 H, ArH);

164.9; HRMS (ESI, m/z) calcd for C15

(MH) ; Found: 368.0228; Anal. Calcd for C15

H

13Cl

12Cl

3

N

5

: 368.0231

N : C, 48.87;

3 5

1

3

+

C NMR (100 MHz, DMSOꢀd ): 41.1, 98.7, 107.7, 117.4, 123.9,

6

1

24.1, 127.3, 133.4, 148.8, 150.1, 151.6, 160.8, 161.1, 161.5;

H, 3.28; N, 19.00; Found: C, 48.95; H, 3.44; N, 19.13.

+

45

HRMS (ESI, m/z) calcd for C H Cl N : 368.0231 (MH) ;

Found: 368.0235; Anal. Calcd for C H Cl N : C, 48.87; H,

3

1

5

13

3

5

1

3

N -(4-chloropyrimidin-2-yl)-N -(7-chloroquinolin-4-yl)propane-

1

5

12

3

5

o

ꢀ1

.28; N, 19.00; Found: C, 48.96; H, 3.45; N, 18.82.

100 1,3-diamine (18c): Yield: 35%; mp 234ꢀ236 C; IR (cm , KBr):

1

3

255, 2964, 2920, 1597, 1463, 1431, 1343, 1083, 977, 765; H

NMR (400 MHz, DMSOꢀd ): 1.90ꢀ1.94 (m, 2 H, CH ); 3.30ꢀ3.35

(m, 4 H, CH ); 6.44ꢀ6.48 (m, 2 H, ArH); 7.35 (brs, 1 H, NH);

7.44 (dd, J = 8.7 Hz, 2.2 Hz, 1 H, ArH); 7.78 (d, J = 2.2 Hz, 1 H,

MHz, DMSOꢀd ): 1.87ꢀ1.92 (m, 2 H, CH ); 3.30ꢀ3.35 (m, 4 H, 105 ArH); 7.87 (d, J = 5.8 Hz, 1 H, ArH); 8.00 (brs, 1 H, NH); 8.27

1

3

N -(2-chloropyrimidin-4-yl)-N -(7-chloroquinolin-4-yl)propane-

1

3264, 2956, 1596, 1430, 1364, 1165, 1093, 787; H NMR (400

6

2

o

ꢀ1

,3-diamine (17c): Yield: 60%; mp 185ꢀ187 C; IR (cm , KBr):

2

1

50

6

2

1

3

CH ); 6.46 (d, J = 5.8 Hz, 1 H, ArH); 6.63 (d, J = 5.1 Hz, 1 H,

(d, J = 8.7 Hz, 1 H, ArH); 8.38 (d, J = 5.1 Hz, 1 H, ArH);

C

2

1

0

| Journal Name, [year], [vol], 00–00

This journal is © The Royal Society of Chemistry [year]

RSC Advances

Page 12 of 17

View Article Online

DOI: 10.1039/C4RA16032K

+

NMR (100 MHz, DMSOꢀd ): 27.0, 37.9, 98.7, 105.1, 117.4,

(ESI, m/z) calcd for C H ClN : 383.1745 (MH) ; Found:

6

20 24

6

1

24.1, 127.3, 133.5, 148.8, 150.1, 151.7, 155.3, 159.9, 163.4; 55 383.1745; Anal. Calcd for C H ClN : C, 62.74; H, 6.05; N,

20 23 6

+

HRMS (ESI, m/z) calcd for C H Cl N : 348.0777 (MH) ;

21.95; Found: C, 62.88; H, 6.15; N, 22.10.

1

6

16

2

5

Found: 348.0783; Anal. Calcd for C H Cl N : C, 55.19; H,

4.34; N, 20.11; Found: C, 55.13; H, 4.41; N, 20.24.

1

6

15

2

5

1

2

5

N -(6-chloro-2-morpholinopyrimidin-4-yl)-N -(7-

chloroquinolin-4-yl)ethane-1,2-diamine (19c): Yield: 80%; mp

1

3

o

ꢀ1

N -(7-chloroquinolin-4-yl)-N -(4,6-dichloropyrimidin-2-

148ꢀ150 C; IR (cm , KBr): 3378, 2971, 2853, 1580, 1516, 1369,

o

1

yl)propane-1,3-diamine (18d): Yield: 35%; mp 195ꢀ198 C; IR 60 1331, 1248, 1121, 974, 784; H NMR (400 MHz, CDCl ): 3.44ꢀ

3

ꢀ

1

(

cm , KBr): 3266, 3141, 2870, 1578, 1450, 1118, 973, 826, 796;

H NMR (400 MHz, DMSOꢀd ): 1.89ꢀ1.93 (m, 2 H, CH ); 3.28ꢀ

3.48 (m, 2 H, CH ); 3.54ꢀ3.56 (m, 4 H, CH ), 3.70ꢀ3.73 (m, 4 H,

2 2

CH ), 3.82ꢀ3.88 (m, 2 H, CH ), 5.47 (brs, 1 H, NH); 5.99 (s, 1 H,

2 2

1

6

2

1

0

3.32 (m, 4 H, CH ), 6.45ꢀ6.48 (m, 2 H, ArH); 7.29 (brs, 1 H,

NH); 7.42 (dd, J = 8.8 Hz, 2.2 Hz, 1 H, ArH); 7.77 (d, J = 2.2 Hz,

ArH); 6.33 (d, J = 5.1 Hz, 1 H, ArH); 6.87 (brs, 1 H, NH); 7.31

(d, J = 8.0 Hz, 1 H, ArH); 7.80ꢀ7.92 (m, 2 H, ArH); 8.48 (d, J =

2

13

1

H, ArH); 8.23ꢀ8.25 (m, 2 H, NH and ArH), 8.37 (d, J = 5.8 Hz, 65 5.1 Hz, 1 H, ArH); C NMR (100 MHz, DMSOꢀd ): 42.1, 43.9,

6

13

H, ArH); C NMR (100 MHz, DMSOꢀd ): 26.9, 38.4, 98.7,

65.7, 90.3, 98.6, 117.4, 123.9, 124.0, 127.5, 133.3, 149.0, 150.0,

6

02.7, 117.4, 124.0, 127.3, 133.4, 148.9, 150.0, 151.7, 156.6,

151.8, 159.3, 161.3, 164.3; HRMS (ESI, m/z) calcd for

+

1

5

159.0, 164.1; HRMS (ESI, m/z) calcd for C H Cl N : 382.0388

C H Cl N O: 419.1148 (MH) ; Found: 419.1154; Anal. Calcd

1

6

15

3

5

19 21

2

6

+

(

MH) ; Found: 382.0385; Anal. Calcd for C H Cl N : C, 50.22;

for C H Cl N O: C, 54.42; H, 4.81; N, 20.04; Found: C, 54.56;

19 20 2 6

1

6

14

3

5

H, 3.69; N, 18.30; Found: C, 50.27; H, 3.75; N, 18.40.

70 H, 4.98; N, 19.92.

1

2

General procedure for the synthesis of compounds 19a-l

N -(6-chloro-2-(piperidin-1-yl)pyrimidin-4-yl)-N -(7-

chloroquinolin-4-yl)ethane-1,2-diamine (19d): Yield: 75%; mp

o

ꢀ1

In a 100 mL round bottom flask, compound 17aꢀd (1 eq.) was

taken and dissolved in 10 mL of DMF. To this, a solution of

139ꢀ141 C; IR (cm , KBr): 3372, 2938, 2854, 1579, 1517, 1447,

1

2

0

1331, 1130, 973, 784; H NMR (400 MHz, DMSOꢀd

); 1.57ꢀ1.58 (m, 2 H, CH ), 3.36ꢀ3.52 (m, 8 H, CH

2

6

): 1.45ꢀ1.46

respective amine (3 eq.) in DMF (5 ml) was added dropwise. 75 (m, 4 H, CH

),

2

o

Reaction mixture was allowed to stir at 100ꢀ120 C for 10 h

monitored by TLC (scheme 1.5). After completion, water (50 ml)

was added to reaction mixture and it was extracted with EtOAc (2

× 25 mL). Organic layer was then collected, washed with water

6.05ꢀ6.15 (m, 1 H, ArH); 6.52 (d, J = 5.1 Hz, 1 H, ArH); 7.14

(brs, 1 H, NH); 7.38 (brs, 1 H, NH); 7.43 (dd, J = 8.8 Hz, 2.2 Hz,

1 H, ArH); 7.77 (d, J = 2.2 Hz, 1 H, ArH); 8.18 (d, J = 8.0 Hz, 1

H, ArH), 8.35ꢀ8.38 (m, 1 H, ArH); C NMR (100 MHz, DMSOꢀ

): 24.4, 25.1, 43.4, 44.6, 90.0, 98.5, 117.4, 123.9, 124.0, 127.5,

133.3, 149.0, 150.0, 151.8, 159.2, 161.5, 163.9; HRMS (ESI,

13

2

5

(

2 × 100 mL) and brine, dried over Na SO and finally excess of

80

d

6

2

4

solvent was evaporated under vaccum. The crude residue thus

obtained was purified by SiO₂column using MeOH/CHCl₃as

eluent to afford compounds 19aꢀl.

+

m/z) calcd for C20

Anal. Calcd for C20

H23Cl

N : 417.1355 [MH] ; Found 417.1350;

2 6

H

22Cl

N

6

: C, 57.56; H, 5.31; N, 20.14; Found:

2

C, 57.64; H, 5.28; N, 20.29.

1

2

3

4

0

5

0

N -(7-chloroquinolin-4-yl)-N -(2-morpholinopyrimidin-4-

o

1

2

yl)ethane-1,2-diamine (19a): Yield: 66%; mp 128ꢀ130 C; IR 85 N -(6-chloro-2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)-N -(7-

ꢀ

1

(

cm , KBr): 3248, 2969, 1585, 1481, 1240, 1116, 976, 793; H

NMR (400 MHz, DMSOꢀd ): 3.37ꢀ3.51 (m, 8 H, CH ); 3.56ꢀ3.58

m, 4 H); 6.00 (d, J = 5.1 Hz, 1 H, ArH); 6.53 (d, J = 5.1 Hz, 1 H,

ArH); 6.72 (brs, 1 H, NH); 7.40ꢀ7.42 (m, 2 H, NH and ArH); 7.74

chloroquinolin-4-yl)ethane-1,2-diamine (19e): Yield: 66%; mp

o

ꢀ1

118ꢀ120 C; IR (cm , KBr): 3266, 2941, 2850, 1584, 1514, 1376,

6

2

1

(

1228, 1139, 971, 782; H NMR (400 MHz, DMSOꢀd

(m, 7 H, CH and CH ); 3.48ꢀ3.54 (m, 8 H, CH ), 6.05ꢀ6.15 (m, 1

d, J = 2.2 Hz, 1 H, ArH); 7.84 (s, 1 H, ArH); 8.13 (d, J = 8.7 Hz, 90 H, ArH); 6.51ꢀ6.75 (m, 1 H, ArH); 7.17 (brs, 1 H, NH); 7.37 (brs,

6

): 2.00ꢀ2.25

2

3

2

(

1

3

H, ArH); 8.34 (d, J = 5.8 Hz, 1 H, ArH); C NMR (100 MHz,

DMSOꢀd ): 42.7, 43.6, 65.8, 93.1, 98.6, 117.4, 123.8, 124.1,

27.5, 133.3, 149.0, 150.1, 151.9, 156.7, 161.9, 162.4; HRMS

(ESI, m/z) calcd for C H ClN O: 385.1537 (MH) ; Found:

1 H, NH); 7.42 (dd, J = 8.8 Hz, 2.2 Hz, 1 H, ArH); 7.77 (d, J =

2.2 Hz, 1 H, ArH); 8.17 (d, J = 8.0 Hz, 1 H, ArH), 8.37 (d, J = 5.0

6

13

1

Hz, 1 H, ArH); C NMR (100 MHz, DMSOꢀd

54.1, 66.3, 90.3, 98.5, 117.4, 123.9, 124.1, 127.5, 133.4, 149.0,

85.1542; Anal. Calcd for C H ClN O: C, 59.29; H, 5.50; N, 95 150.1, 151.8, 159.4, 161.4, 163.0; HRMS (ESI, m/z) calcd for

6

): 42.1, 43.5, 45.6,

+

1

9

22

6

3

2

1

9

21

6

+

1.84; Found: C, 59.18; H, 5.35; N, 21.92.

C

20

H24Cl

2

N

7

: 432.1465 (MH) ; Found: 432.1466; Anal. Calcd for

C H Cl N : C, 55.56; H, 5.36; N, 22.68; Found: C, 55.71; H,

5.26; N, 22.77.

20

23

2

7

1

2

N -(7-chloroquinolin-4-yl)-N -(2-(piperidin-1-yl)pyrimidin-4-

o

yl)ethane-1,2-diamine (19b): Yield: 72%; mp 147ꢀ149 C; IR

(cm , KBr): 3386, 2940, 2853, 1565, 1584, 1365, 1236, 1128,

ꢀ

1

2

4

5

N -(6-chloro-2-(4-ethylpiperazin-1-yl)pyrimidin-4-yl)-N -(7-

1

9

78, 792; H NMR (400 MHz, DMSOꢀd ): 1.42ꢀ1.55 (m, 6 H, 100 chloroquinolin-4-yl)ethane-1,2-diamine (19f): Yield: 72%; mp

6

o

ꢀ1

CH ); 3.43ꢀ3.48 (m, 8 H, CH ); 5.99 (d, J = 5.1 Hz, 1 H, ArH);

.52 (d, J = 5.8 Hz, 1 H, ArH); 6.62 (brs, 1 H, NH); 7.40 (dd, J =

.7 Hz, 2.2 Hz, 1 H, ArH); 7.46 (brs, 1 H, NH); 7.74 (d, J = 2.2

Hz, 1 H, ArH); 7.78 (s, 1 H, ArH); 8.12 (d, J = 8.7 Hz, 1 H,

97ꢀ99 C; IR (cm , KBr): 3265, 2932, 2816, 1582, 1449, 1245,

2

1

6

8

970, 782; H NMR (400 MHz, DMSOꢀd

3 H, CH ); 2.29ꢀ2.30 (m, 6 H, CH ); 3.44ꢀ3.49 (m, 8 H, CH

6.05ꢀ6.17 (m, 1 H, ArH); 6.52ꢀ6.76 (m, 1 H, ArH); 7.19 (brs, 1 H,

ArH); 8.34 (d, J = 5.8 Hz, 1 H, ArH); C NMR (100 MHz, 105 NH); 7.37 (brs, 1 H, NH); 7.42 (dd, J = 8.8 Hz, 2.2 Hz, 1 H,

6

): ): 0.98 (t, J = 7.3 Hz,

),

3

2

5

0

1

3

1

DMSOꢀd ): 24.2, 25.1, 44.2, 93.2, 98.6, 117.3, 123.8, 124.1,

ArH); 7.77 (d, J = 2.2 Hz, 1 H, ArH); 8.17 (d, J = 8.0 Hz, 1 H,

ArH), 8.37 (d, J = 5.0 Hz, 1 H, ArH); HRMS (ESI, m/z) calcd for

6

1

27.4, 133.3, 149.0, 150.1, 151.8, 156.3, 161.8, 162.0; HRMS

Journal Name, [year], [vol], 00–00 | 11

Page 13 of 17

RSC Advances

View Article Online

DOI: 10.1039/C4RA16032K

+

1

3

C H Cl N : 446.1621 (MH) ; Found: 446.1619; Anal. Calcd for

N -(6-chloro-2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)-N -(7-

2

1

26

2

7

C H Cl N : C, 56.50; H, 5.65; N, 21.97; Found: C, 56.62; H, 55 chloroquinolin-4-yl)propane-1,3-diamine (19k): Yield: 66%; mp

2

1

25

2

7

o

ꢀ1

5

.76; N, 21.81.

230ꢀ232 C; IR (cm , KBr): 3314, 2938, 2850, 1579, 1515, 1449,

1

373, 1279, 1143, 1001, 967, 779; H NMR (400 MHz, DMSOꢀ

): 1.83ꢀ1.86 (m, 2 H, CH ); 2.08ꢀ2.12 (m, 7 H, CH and CH );

), 6.00 (s, 1 H, ArH); 6.44 (d, J = 5.1 Hz,

1

3

N -(7-chloroquinolin-4-yl)-N -(2-(4-methylpiperazin-1-

d

6

2

3

5

yl)pyrimidin-4-yl)propane-1,3-diamine (19g): Yield: 61%; mp

72ꢀ175 C; IR (cm , KBr): 3343, 2928, 1582, 1367, 1138, 1001, 60 1 H, ArH); 7.16 (brs, 1 H, NH); 7.32 (brs, 1 H, NH); 7.43 (dd, J =

92; H NMR (400 MHz, DMSOꢀd ): 1.85ꢀ2.25 (m, 9 H, CH and

3.29ꢀ3.31 (m, 8 H, CH

2

o

ꢀ1

1

7

1

8.8 Hz, 2.2 Hz, 1 H, ArH); 7.76 (d, J = 2.2 Hz, 1 H, ArH); 8.24

6

2

1

3

CH ); 3.43ꢀ3.59 (m, 8 H, CH ), 5.96 (s, 1 H, ArH); 6.45ꢀ6.50 (m,

(d, J = 8.7 Hz, 1 H, ArH), 8.35 (d, J = 5.8 Hz, 1 H, ArH);

NMR (100 MHz, DMSOꢀd ): 27.5, 38.5, 43.3, 45.5, 54.0, 89.9,

98.5, 117.4, 123.9, 127.4, 133.3, 149.0, 149.9, 151.8, 159.3,

ArH); HRMS (ESI, m/z) calcd for C H ClN : 412.2010 (MH) ; 65 161.3, 162.9; HRMS (ESI, m/z) calcd for C21 : 446.1621;

C

3

2

1

H, ArH); 7.35ꢀ7.42 (m, 3 H, ArH and NH); 7.75ꢀ7.77 (m, 2 H,

6

10

ArH); 8.23 (d, J = 8.7 Hz, 1 H, ArH), 8.35 (d, J = 5.1 Hz, 1 H,

+

H

26Cl

25Cl

N

2

1

27

7

2

7

+

Found: 412.2007; Anal. Calcd for C H ClN : C, 61.23; H, 6.36;

(MH) ; Found: 446.1617; Anal. Calcd for C21

2

N

7

: C, 56.50;

2

1

26

7

N, 23.80; Found: C, 61.33; H, 6.38; N, 23.70.

H, 5.65; N, 21.97; Found: C, 56.51; H, 5.76; N, 21.76.

1

3

1

3

N -(7-chloroquinolin-4-yl)-N -(2-(4-ethylpiperazin-1-

N -(6-chloro-2-(4-ethylpiperazin-1-yl)pyrimidin-4-yl)-N -(7-

15

20

25

yl)pyrimidin-4-yl)propane-1,3-diamine (19h): Yield: 55%; mp

chloroquinolin-4-yl)propane-1,3-diamine (19l): Yield: 58%; mp

o

ꢀ1

o

ꢀ1

1

78ꢀ180 C; IR (cm , KBr): 3245, 2932, 1581, 1438, 1370, 1244, 70 203ꢀ205 C; IR (cm , KBr): 3292, 2931, 2816, 1581, 1449, 1368,

1

140, 974, 790; H NMR (400 MHz, DMSOꢀd ): 0.96 (t, J = 7.3

1247, 1139, 970, 853, 781; H NMR (400 MHz, DMSOꢀd

(t, J = 7.3 Hz, 3 H, CH ); 1.83ꢀ1.86 (m, 2 H, CH ); 2.09ꢀ2.23 (m,

6 H, CH ); 3.29ꢀ3.37 (m, 8 H, CH ), 5.98 (s, 1 H, ArH); 6.44 (d, J

= 5.1 Hz, 1 H, ArH); 7.15 (brs, 1 H, NH); 7.32 (brs, 1 H, NH);

H, NH); 7.43 (dd, J = 8.7 Hz, 2.2 Hz, 1 H, ArH); 7.74ꢀ7.76 (m, 2 75 7.42 (dd, J = 8.8 Hz, 2.2 Hz, 1 H, ArH); 7.75 (d, J = 2.2 Hz, 1 H,

6

): 0.95

6

Hz, 3 H, CH ); 1.84ꢀ1.86 (m, 2 H, CH ); 2.19ꢀ2.25 (m, 6 H,

3

2

3

2

CH ); 3.31ꢀ3.38 (m, 8 H, CH ); 5.95 (d, J = 5.8 Hz, 1 H, ArH);

2

6.43(d, J = 5.8 Hz, 1 H, ArH); 6.60 (brs, 1 H, NH); 7.35 (brs, 1

H, ArH); 8.23 (d, J = 8.7 Hz, 1 H, ArH); 8.34 (d, J = 5.1 Hz, 1 H,

ArH); 8.24 (d, J = 8.7 Hz, 1 H, ArH), 8.34 (d, J = 5.8 Hz, 1 H,

+

13

ArH); HRMS (ESI, m/z) calcd for C H ClN : 426.2167 (MH) ;

ArH); C NMR (100 MHz, DMSOꢀd ): 11.8, 27.6, 38.5, 43.4,

6

2

29

7

Found: 421.2160; Anal. Calcd for C H ClN : C, 62.03; H, 6.63;

51.4, 51.8, 89.8, 98.5, 117.4, 123.9, 124.0, 127.4, 133.3, 149.0,

149.9, 151.8, 159.2, 161.3, 162.9; HRMS (ESI, m/z) calcd for

2

28

7

N, 23.02; Found: C, 62.13; H, 6.55; N, 23.16.

+

80

C H Cl N : 406.1777 (MH) ; Found: 460.1783; Anal. Calcd for

2

28

2

7

1

3

N -(6-chloro-2-morpholinopyrimidin-4-yl)-N -(7-

C

22

H27Cl

2

N

7

: C, 57.39; H, 5.91; N, 21.30; Found: C, 57.46; H,

chloroquinolin-4-yl)propane-1,3-diamine (19i): Yield: 78%; mp

5.96; N, 21.48.

o

ꢀ1

1

78ꢀ180 C; IR (cm , KBr): 3338, 2924, 2855, 1581, 1448, 1368,

1

245, 1115, 972, 784; H NMR (400 MHz, DMSOꢀd ): 1.83ꢀ1.88

General procedure for the synthesis of compounds 20a-l

In a 100 mL round bottom flask, compound 18aꢀd (1 eq.) was

6

30

(m, 2 H, CH ); 3.28ꢀ3.50 (m, 12 H, CH ), 6.00 (s, 1 H, ArH);

2 2

6

.44 (d, J = 5.1 Hz, 1 H, ArH); 7.21 (brs, 1 H, NH); 7.31 (brs, 1

H, NH); 7.42 (dd, J = 8.8 Hz, 2.2 Hz, 1 H, ArH); 7.75 (d, J = 2.2 85 taken and dissolved in 10 mL of DMF. To this, a solution of

Hz, 1 H, ArH); 8.23 (d, J = 8.0 Hz, 1 H, ArH), 8.35 (d, J = 5.0

respective amine (3 eq.) in DMF (5 ml) was added dropꢀwise.

Reaction mixture was allowed to stir at 100ꢀ120 C for 10 h

1

3

o

Hz, 1 H, ArH); C NMR (100 MHz, DMSOꢀd ): 27.5, 38.5, 43.8,

6

35

44.2, 65.6, 65.9, 89.9, 98.6, 117.4, 124.0, 127.4, 133.3, 149.0,

monitored by TLC. After completion, water (50 ml) was added to

reaction mixture and it was extracted with EtOAc (2 × 25 mL).

1

50.0, 151.8, 159.3, 161.2, 163.2; HRMS (ESI, m/z) calcd for

+

C H Cl N O: 433.1305 (MH) ; Found: 433.1312; Anal. Calcd 90 Organic layer was then collected, washed with water (2 × 100

2

0

23

2

6

for C H Cl N O: C, 55.43; H, 5.12; N, 19.39; Found: C, 55.54;

mL) and brine, dried over Na SO and finally excess of solvent

2 4

20

22

2

6

H, 5.21; N, 19.53.

was evaporated under vaccum. The crude residue thus obtained

was purified by SiO column using MeOH/CHCl as eluent to

afford compounds 20aꢀl.

2

3

1

3

40

45

50

N -(6-chloro-2-(piperidin-1-yl)pyrimidin-4-yl)-N -(7-

chloroquinolin-4-yl)propane-1,3-diamine (19j): Yield: 82%; mp

o

ꢀ1

1

2

1

35ꢀ238 C; IR (cm , KBr): 3245, 2930, 2852, 1610, 1588, 1522, 95 N -(7-chloroquinolin-4-yl)-N -(4-morpholinopyrimidin-2-

1

o

368, 1125, 973, 774; H NMR (400 MHz, DMSOꢀd ): 1.34ꢀ1.50

yl)ethane-1,2-diamine (20a): Yield: 85%; mp 258ꢀ260 C; IR

(cm , KBr): 3216, 2925, 1579, 1369, 1243, 1067, 977; H NMR

(400 MHz, DMSOꢀd ): 3.25ꢀ3.31 (m, 8 H, CH ); 3.52ꢀ3.69 (m, 4

H, CH ); 6.42 (d, J = 5.1 Hz, 1 H, ArH); 6.64 (d, J = 5.1 Hz, 1 H,

2

6

ꢀ

1

(

m, 6 H); 1.80ꢀ1.85 (m, 2 H, CH ); 3.26ꢀ3.28 (m, 4 H); 3.36ꢀ3.40

2

(m, 4 H, CH ), 5.97 (s, 1 H, ArH); 6.42 (d, J = 5.1 Hz, 1 H, ArH);

2

6

2

7

.06 (brs, 1 H, NH); 7.27 (brs, 1 H, NH); 7.39 (dd, J = 8.8 Hz, 2.2

Hz, 1 H, ArH); 7.73 (d, J = 2.2 Hz, 1 H, ArH); 8.20 (d, J = 8.0 100 ArH); 7.16 (brs, 1 H, NH); 7.34 (dd, J = 8.7 Hz, 2.2 Hz, 1 H,

1

3

Hz, 1 H, ArH), 8.32 (d, J = 5.0 Hz, 1 H, ArH); C NMR (100

ArH); 7.80 (d, J = 2.2 Hz, 1 H, ArH); 7.87ꢀ7.93 (m, 2 H, ArH);

8.14 (d, J = 8.7 Hz, 1 H, ArH); 8.44 (d, J = 5.1 Hz, 1 H, ArH);

HRMS (ESI, m/z) calcd for C H ClN O: 385.1537 (MH) ;

MHz, DMSOꢀd ): 24.1, 25.0, 27.5, 38.4, 44.5, 89.6, 98.6, 117.4,

6

+

124.0, 127.4, 133.3, 149.0, 150.0, 151.7, 159.2, 161.3, 162.6;

19

22

6

+

HRMS (ESI, m/z) calcd for C H Cl N : 431.1512 (MH) ;

Found: 385.1534; Anal. Calcd for C H ClN O: C, 59.29; H,

19 21 6

2

1

25

2

6

Found: 431.1518; Anal. Calcd for C H Cl N : C, 58.47; H, 105 5.50; N, 21.84; Found: C, 59.42; H, 5.61; N, 21.92.

2

1

24

2

6

5

.61; N, 19.48; Found: C, 58.35; H, 5.54; N, 19.32.

1

2

N -(7-chloroquinolin-4-yl)-N -(4-(piperidin-1-yl)pyrimidin-2-

1

2

| Journal Name, [year], [vol], 00–00

RSC Advances

Page 14 of 17

View Article Online

DOI: 10.1039/C4RA16032K

o

yl)ethane-1,2-diamine (20b): Yield: 82%; mp 198ꢀ200 C; IR 55 chloroquinolin-4-yl)ethane-1,2-diamine (20f): Yield: 60%; mp

ꢀ

1

o

ꢀ1

(

cm , KBr): 3322, 2931, 2855, 1585, 1499, 1341, 1243, 796; H

116ꢀ118 C; IR (cm , KBr): 3310, 2969, 2853, 1576, 1489, 1447,

1

NMR (400 MHz, DMSOꢀd ): 1.45ꢀ1.58 (m, 6 H, CH ); 3.42ꢀ3.52

1140, 968, 805; H NMR (400 MHz, DMSOꢀd ): 0.95 (t, J = 7.3

6

2

6

(

m, 4 H, CH ); 3.64ꢀ3.67 (m, 4 H); 5.70 (d, J = 5.1 Hz, 1 H,

Hz, 3 H, CH ); 2.24ꢀ2.30 (m, 6 H, CH ); 3.42ꢀ3.54 (m, 8 H, CH ),

2

3

2

5

ArH); 6.54 (d, J = 5.1 Hz, 1 H, ArH); 7.11 (brs, 1 H, NH); 7.42ꢀ

5.73 (s, 1 H, ArH); 6.49 (d, J = 5.1 Hz, 1 H, ArH); 7.38ꢀ7.42 (m,

7

.44 (m, 2 H, NH and ArH); 7.69 (d, J = 5.1 Hz, 1 H, ArH); 7.77 60 3 H, NH and ArH); 7.74 (d, J = 2.2 Hz, 1 H, ArH); 8.12 (d, J =

(

d, J = 2.2 Hz, 1 H, ArH); 8.17 (d, J = 8.7 Hz, 1 H, ArH); 8.37 (d,

8.7 Hz, 1 H, ArH), 8.34 (d, J = 5.4 Hz, 1 H, ArH); HRMS (ESI,

1

3

+

J = 5.1 Hz, 1 H, ArH); C NMR (100 MHz, DMSOꢀd ): 24.4,

m/z) calcd for C H Cl N : 446.1621 (MH) ; Found: 446.1623;

6

21 26

2

7

2

5.2, 42.1, 44.1, 95.0, 98.5, 117.4, 123.8, 123.9, 127.4, 133.3,

Anal. Calcd for C H Cl N : C, 56.50; H, 5.65; N, 21.97; Found:

21 25 2 7

1

0

149.0, 150.0, 151.7, 154.9, 161.1, 162.5; HRMS (ESI, m/z) calcd

C, 56.58; H, 5.81; N, 21.79.

+

for C H ClN : 383.1745 (MH) ; Found: 383.1743; Anal. Calcd

20

24

6

1

3

for C H ClN : C, 62.74; H, 6.05; N, 21.95; Found: C, 62.81; H, 65 N -(7-chloroquinolin-4-yl)-N -(4-(4-methylpiperazin-1-

20

23

6

.11; N, 21.96.

yl)pyrimidin-2-yl)propane-1,3-diamine (20g): Yield: 54%; mp

o

ꢀ1

7

6ꢀ78 C; IR (cm , KBr): 3276, 2936, 2851, 1581, 1490, 1343,

1

2

1

N -(4-chloro-6-morpholinopyrimidin-2-yl)-N -(7-

1244, 1138, 1003, 974, 794; H NMR (400 MHz, CDCl ): 2.05ꢀ

3

1

2

5

0

5

chloroquinolin-4-yl)ethane-1,2-diamine (20c): Yield: 80%; mp

2.08 (m, 2 H, CH ); 2.30ꢀ2.39 (m, 7 H, CH and CH ); 3.44ꢀ3.55

2

3

o

ꢀ1

1

15ꢀ117 C; IR (cm , KBr): 3369, 2967, 2857, 1576, 1478, 1451, 70 (m, 4 H, CH ), 3.74ꢀ3.78 (m, 4 H, CH ); 4.86 (brs, 1 H, NH);

2 2

1

247, 1105, 967, 783; H NMR (400 MHz, DMSOꢀd ): 3.40ꢀ3.52

5.29 (brs, 1 H, NH); 5.68 (d, J = 5.1 Hz, 1 H, ArH); 6.38 (d, J =

4.4 Hz, 1 H, ArH); 7.32 (dd, J = 8.7 Hz, 2.2 Hz, 1 H, ArH); 7.55

(d, J = 8.7 Hz, 1 H, ArH); 7.88 (d, J = 5.1 Hz, 1 H, ArH); 7.94 (d,

6

(

m, 12 H, CH ), 5.77 (s, 1 H, ArH); 6.49 (d, J = 5.8 Hz, 1 H,

2

ArH); 7.36 (brs, 1 H, NH); 7.40 (dd, J = 8.8 Hz, 2.2 Hz, 1 H,

ArH); 7.48 (brs, 1 H, NH); 7.75 (d, J = 2.2 Hz, 1 H, ArH); 8.12

1

3

J = 2.2 Hz, 1 H, ArH), 8.49 (d, J = 5.1 Hz, 1 H, ArH); C NMR

13

(d, J = 8.7 Hz, 1 H, ArH), 8.34 (d, J = 5.4 Hz, 1 H, ArH);

C

75 (100 MHz, DMSOꢀd ): 27.6, 36.2, 37.6, 43.0, 45.8, 54.3, 98.6,

6

NMR (100 MHz, DMSOꢀd ): 38.1, 41.8, 43.8, 65.9, 93.2, 98.6,

117.4, 124.00, 124.09, 127.4, 133.3, 149.0, 150.0, 151.8, 154.5,

161.2, 162.4; HRMS (ESI, m/z) calcd for C H ClN : 412.2010

6

1

17.4, 124.0, 124.1, 127.5, 133.4, 149.1, 150.0, 151.8, 157.3,

60.8, 163.6; HRMS (ESI, m/z) calcd for C H Cl N O:

2

1

27

7

+

(MH) ; Found: 412.2005; Anal. Calcd for C H ClN : C, 61.23;

1

9

21

2

6

21 26

7

+

419.1148 (MH) ; Found: 419.1144; Anal. Calcd for

C H Cl N O: C, 54.42; H, 4.81; N, 20.04; Found: C, 54.35; H,

H, 6.36; N, 23.80; Found: C, 61.29; H, 6.19; N, 23.62.

1

9

20

2

6

1

3

4

.90; N, 20.12.

80 N -(7-chloroquinolin-4-yl)-N -(4-(4-ethylpiperazin-1-

yl)pyrimidin-2-yl)propane-1,3-diamine (20h): Yield: 50%; mp

1

2

o

ꢀ1

N -(4-chloro-6-(piperidin-1-yl)pyrimidin-2-yl)-N -(7-

160ꢀ163 C; IR (cm , KBr): 246, 2926, 2850, 1578, 1443, 1327,

1

chloroquinolin-4-yl)ethane-1,2-diamine (20d): Yield: 85%; mp

201ꢀ203 C; IR (cm , KBr): 3327, 2932, 2850, 1578, 1486, 1329,

1236, 1137, 791; H NMR (400 MHz, CDCl ): 1.10 (t, J = 7.3

3

o

ꢀ1

3

4

0

5

0

Hz, 3 H, CH ); 2.04ꢀ2.09 (m, 4 H, CH ); 2.39ꢀ2.43 (m, 4 H,

3

2

1

286, 1142, 1064, 797; H NMR (400 MHz, DMSOꢀd ): 1.40ꢀ 85 CH ); 3.41ꢀ3.46 (m, 2 H); 3.52ꢀ3.57 (m, 2 H, CH ), 3.75ꢀ3.77 (m,

6

2

.42 (m, 4 H, CH ); 1.52ꢀ1.53 (m, 2 H, CH ); 3.45ꢀ3.57 (m, 8 H,

4 H, CH ); 4.88 (brs, 1 H, NH); 5.26 (brs, 1 H, NH); 5.67 (d, J =

2

CH ), 5.69 (s, 1 H, ArH); 6.49 (d, J = 5.1 Hz, 1 H, ArH); 7.38ꢀ

5.8 Hz, 1 H, ArH); 6.38 (d, J = 5.1 Hz, 1 H, ArH); 7.31 (dd, J =

9.5 Hz, 2.2 Hz, 1 H, ArH); 7.55 (d, J = 9.5 Hz, 1 H, ArH); 7.88

(d, J = 5.1 Hz, 1 H, ArH); 7.94 (d, J = 2.2 Hz, 1 H, ArH), 8.50 (d,

2

7

.40 (m, 3 H, NH and ArH); 7.74 (d, J = 2.2 Hz, 1 H, ArH); 8.12

13

(d, J = 8.7 Hz, 1 H, ArH), 8.33 (d, J = 5.4 Hz, 1 H, ArH);

C

13

NMR (100 MHz, DMSOꢀd ): 24.3, 25.3, 41.9, 44.2, 66.4, 92.3, 90 J = 5.1 Hz, 1 H, ArH); C NMR (100 MHz, DMSOꢀd ): 11.8,

6

9

1

4

8.6, 117.5, 124.0, 124.2, 127.5, 133.5, 149.1, 150.1, 151.8,

27.2, 38.8, 43.0, 51.6, 52.1, 95.7, 98.6, 117.4, 123.9, 124.0,

57.5, 160.6, 163.7; HRMS (ESI, m/z) calcd for C H Cl N :

127.4, 133.3, 149.0, 150.0, 151.8, 154.6, 161.1, 162.3; HRMS

2

0

23

2

6

+

17.1356 (MH) ; Found: 417.1363; Anal. Calcd for C H Cl N :

(ESI, m/z) calcd for C H ClN : 426.2167 (MH) ; Found:

20

22

2

6

22 29

7

C, 57.56; H, 5.31; N, 20.14; Found: C, 57.72; H, 5.48; N, 20.02.

426.2169; Anal. Calcd for C H ClN : C, 62.03; H, 6.63; N,

22 28 7

95

23.02; Found: C, 62.10; H, 6.71; N, 23.11.

1

2

N -(4-chloro-6-(4-methylpiperazin-1-yl)pyrimidin-2-yl)-N -(7-

chloroquinolin-4-yl)ethane-1,2-diamine (20e): Yield: 62%; mp

1

3

N -(4-chloro-6-morpholinopyrimidin-2-yl)-N -(7-

o

ꢀ1

1

19ꢀ121 C; IR (cm , KBr): 3309, 2937, 2851, 1575, 1489, 1447,

138, 1003, 943, 785; H NMR (400 MHz, DMSOꢀd ): 2.15ꢀ2.23

chloroquinolin-4-yl)propane-1,3-diamine (20i): Yield: 79%; mp

1

o

ꢀ1

230ꢀ232 C; IR (cm , KBr): 3397, 3326, 2927, 2859, 1579, 1491,

6

1

4

5

(m, 7 H, CH and CH ); 3.34ꢀ3.58 (m, 8 H, CH ), 5.76 (s, 1 H,

1447, 1247, 1111, 1073, 787; H NMR (400 MHz, DMSOꢀd ):

2

3

2

6

ArH); 6.51 (d, J = 5.8 Hz, 1 H, ArH); 7.41ꢀ7.44 (m, 2 H, NH and 100 1.84ꢀ1.89 (m, 2 H, CH ); 3.28ꢀ3.45 (m, 12 H, CH ), 5.78 (s, 1 H,

2

ArH); 7.47 (brs, 1 H, NH); 7.77 (d, J = 2.2 Hz, 1 H, ArH); 8.16

ArH); 6.45 (d, J = 5.1 Hz, 1 H, ArH); 7.32 (brs, 1 H, NH); 7.40

(brs, 1 H, NH); 7.44 (dd, J = 8.8 Hz, 2.2 Hz, 1 H, ArH); 7.76 (d, J

= 2.2 Hz, 1 H, ArH); 8.25 (d, J = 8.7 Hz, 1 H, ArH), 8.36 (d, J =

13

(d, J = 8.7 Hz, 1 H, ArH), 8.37 (d, J = 5.4 Hz, 1 H, ArH);

C

NMR (100 MHz, DMSOꢀd ): 38.1, 41.8, 43.2, 45.7, 54.3, 92.8,

6

13

5

0

98.5, 117.4, 124.0, 124.1, 127.5, 133.4, 149.0, 150.0, 151.7,

5.8 Hz, 1 H, ArH); C NMR (100 MHz, DMSOꢀd ): 27.5, 37.9,

6

1

4

57.3, 160.6, 163.6; HRMS (ESI, m/z) calcd for C H Cl N : 105 43.6, 65.7, 92.9, 98.6, 117.4, 123.96, 123.99, 127.4, 133.3, 149.0,

20 24 2 7

+

32.1464 (MH) ; Found: 432.1473; Anal. Calcd for C H Cl N :

149.9, 151.8, 157.1, 160.6, 163.5; HRMS (ESI, m/z) calcd for

20

23

2

7

+

C, 55.56; H, 5.36; N, 22.68; Found: C, 55.68; H, 5.42; N, 22.73.

C H Cl N O: 433.1304 (MH) ; Found: 433.1308; Anal. Calcd

20

23

2

6

for C H Cl N O: C, 55.43; H, 5.12; N, 19.39; Found: C, 55.24;

H, 5.31; N, 19.44.

20

22

2

6

1

2

N -(4-chloro-6-(4-ethylpiperazin-1-yl)pyrimidin-2-yl)-N -(7-

Journal Name, [year], [vol], 00–00 | 13

Page 15 of 17

RSC Advances

View Article Online

DOI: 10.1039/C4RA16032K

1

3

N -(4-chloro-6-(piperidin-1-yl)pyrimidin-2-yl)-N -(7-

55 tested as a suspension. Stock solutions were stored at ꢀ20°C until

use. For the in vitro evaluation, dilutions to the desired starting

concentration of each compound were prepared in complete

medium immediately prior to use on each occasion.

chloroquinolin-4-yl)propane-1,3-diamine (20j): Yield: 72%; mp

5ꢀ87 C; IR (cm , KBr): 3310, 2933, 2853, 1581, 1489, 1447,

369, 1247, 1136, 967, 786; H NMR (400 MHz, DMSOꢀd ):

o

ꢀ1

8

1

6

5

1.33ꢀ1.50 (m, 6 H, CH ); 1.85ꢀ1.88 (m, 2 H, CH ); 3.27ꢀ3.51 (m,

Doseꢀresponse experiments were carried out in both isolates in

2

8

7

8

H, CH ), 5.69 (s, 1 H, ArH); 6.44 (d, J = 5.1 Hz, 1 H, ArH); 60 order to determine the IC₅₀value of each compound. The

2

.29ꢀ7.31 (brs, 2 H, NH); 7.42 (dd, J = 8.8 Hz, 2.2 Hz, 1 H, ArH);

.75 (d, J = 2.2 Hz, 1 H, ArH); 8.24 (d, J = 8.7 Hz, 1 H, ArH),

experiment was conducted using 2% parasitemia and 1%

haematocrit in the plate. Compounds were prepared to double the

desired highest starting concentration in a 96ꢀwell plate and then

serially diluted 2ꢀfold in complete medium to produce a wide

1

3

.35 (d, J = 5.4 Hz, 1 H, ArH); C NMR (100 MHz, DMSOꢀd₆):

10

24.2, 25.1, 27.5, 37.9, 44.0, 91.9, 98.6, 117.4, 123.9, 124.0,

1

27.4, 133.3, 149.0, 149.9, 151.7, 160.4, 163.4; HRMS (ESI, 65 range of different concentrations, to which an equivalent volume

+

m/z) calcd for C H Cl N : 431.1512 (MH) ; Found: 431.1514;

Anal. Calcd for C H Cl N : C, 58.47; H, 5.61; N, 19.48; Found:

C, 58.51; H, 5.50; N, 19.49.

of prepared parasite stock was added, yielding the desired

concentration of each compound. An erythrocyte control and a

drugꢀfree parasite control were included for each row,

representing 0% and 100% parasite survival respectively. Plates

70 were housed in airtight chambers containing 4% CO2 and 3% O2

in nitrogen and left for 48 hours at 37 °C.

2

1

25

2

6

2

1

24

2

6

1

3

1

2

5

0

5

N -(4-chloro-6-(4-methylpiperazin-1-yl)pyrimidin-2-yl)-N -(7-

chloroquinolin-4-yl)propane-1,3-diamine (20k): Yield: 63%; mp

1

CH ); 2.06ꢀ2.16 (m, 7 H, CH and CH ); 3.41ꢀ3.47 (m, 8 H,

parasite lactate dehydrogenase assay described by Makler et al.

CH ), 5.74 (s, 1 H, ArH); 6.44 (d, J = 5.1 Hz, 1 H, ArH); 7.28ꢀ 75 The IC50ꢀvalues were obtained using a nonꢀlinear doseꢀresponse

o

ꢀ1

06ꢀ108 C; IR (cm , KBr): 3327, 2929, 1578, 1449, 1248, 1138,

001, 785; H NMR (400 MHz, DMSOꢀd ): 1.85ꢀ1.90 (m, 2 H,

Quantitative assessment of antimalarial activity was

1

determined from the doseꢀresponse experiments using the

6

2

4

2

3

2

7

(

.32 (brs, 2 H, NH); 7.43 (dd, J = 8.8 Hz, 2.2 Hz, 1 H, ArH); 7.76

d, J = 2.2 Hz, 1 H, ArH); 8.25 (d, J = 8.7 Hz, 1 H, ArH), 8.36 (d,

J = 5.4 Hz, 1 H, ArH); HRMS (ESI, m/z) calcd for C H Cl N :

curve fitting analysis via Graphpad Prism v.4.0 software.

Assay for in-vivo antimalarial activity

2

1

26

2

7

+

4

46.1620 (MH) ; Found: 446.1621; Anal. Calcd for C H Cl N :

21 25 2 7

C, 56.50; H, 5.65; N, 21.97; Found: C, 56.48; H, 5.52; N, 21.83.

The compound was tested using the Peters 4ꢀDay test. The

parasite strain used was Plasmodium berghei ANKA transfected

80 with the green fluorescent protein construct to enable detection of

parasite using a flow cytometer. Mice were male Balb C (albino)

mice, randomly divided into groups of five animals. Approval for

the study was obtained following submission of the protocol for

review by the Animal Ethics Committee at the University of Cape

1

3

N -(4-chloro-6-(4-ethylpiperazin-1-yl)pyrimidin-2-yl)-N -(7-

chloroquinolin-4-yl)propane-1,3-diamine (20l): Yield: 61%; mp

7

1

o

ꢀ1

9ꢀ81 C; IR (cm , KBr): 3278, 2928, 1576, 1442, 1240, 1137,

1

005, 785; H NMR (400 MHz, DMSOꢀd ): 0.92 (t, J = 7.3 Hz, 3

6

30

H, CH ); 1.81ꢀ1.86 (m, 2 H, CH ); 2.07ꢀ2.20 (m, 6 H, CH ); 3.25ꢀ

3 2 2

3

.39 (m, 8 H, CH ), 5.70 (s, 1 H, ArH); 6.41 (d, J = 5.1 Hz, 1 H, 85 Town. The experiment continued until it became apparent that

2

ArH); 7.30ꢀ7.32 (brs, 2 H, NH); 7.40 (dd, J = 8.8 Hz, 2.2 Hz, 1 H,

ArH); 7.73 (d, J = 2.2 Hz, 1 H, ArH); 8.22 (d, J = 8.7 Hz, 1 H,

ArH), 8.32 (d, J = 5.8 Hz, 1 H, ArH); C NMR (100 MHz,

parasite levels were no longer suppressed and were increasing.

Several days prior to the evaluation, donor mice were infected

with parasites which were left to multiply to sufficiently high

numbers. On the first day (Day 0) of the experiment, donor mice

1

3

35

DMSOꢀd ): 11.8, 27.6, 37.9, 43.1, 51.5, 51.9, 92.5, 98.6, 117.4,

6

1

24.01, 124.07, 127.4, 133.3, 149.0, 150.0, 151.8, 157.2, 160.5, 90 were killed by inhalation of excess isoflurane and their blood

63.4; HRMS (ESI, m/z) calcd for C H Cl N : 460.1777

collected into heparinised tubes to prevent clotting. Parasitemia

was determined via microscopic analysis, as was cell density.

2

28

2

7

+

(MH) ; Found: 460.1766; Anal. Calcd for C H Cl N : C, 57.39;

22 27 2 7

H, 5.91; N, 21.30; Found: C, 57.25; H, 5.95; N, 21.41.

From this, blood was diluted into phosphateꢀbuffered saline

8

(PBS) such that each mL of the stock preparation contained 2x10

40

Assay for in-vitro antimalarial activity

95 parasitised erythrocytes. Mice were anaesthetised by

intramuscular injection of a fixedꢀdose preparation of ketamine

and xylizine (3:2 v/v), diluted 1:4 in phosphateꢀbuffered saline.

Each mouse was then infected with 100µL (2x10 ) of the parasite

stock intravenously. Mice were left for two hours to recover from

Activity was determined against both chloroquineꢀsensitive

isolate of the human malaria parasite (Plasmodium falciparum

Nf54) and chloroquineꢀresistant isolate (P. falciparum Dd2).

7

Parasites were maintained in continuous culture using the method 100 the anaesthesia. After that, each group was dosed with either the

2

3

4

5

of Trager and Jensen with modifications. Growth medium was

supplemented with Albumax II (Gibco), a bovine serum albumin

preparation, instead of human serum. Cultures did not exceed 4%

haematocrit and parasitemia was diluted to 1% when the cultures

were in the trophozoite stage.

appropriate compound or the placebo.

Compounds were dosed on Day 0 and then 24, 48 and 72 hours

later. Compounds were prepared immediately prior to dosing.

Chloroquine was prepared to a dose of 15mg/kg in phosphateꢀ

105 buffered saline and 200µL administered by gavage. The placebo

group received 200µL of 0.5% (w/v) hydroxypropyl

methylcellulose (HPMC) prepared in water, given by gavage.

Two groups of mice received compound 30 orally, at 10mg/kg

and 30mg/kg respectively. The compound was weighed out and

50

The compounds were tested in triplicate on at least three

occasions in vitro against the human malaria parasite.

Compounds were prepared to 20mg/mL stock solutions in

dimethyl sulfoxide and sonicated for 10 minutes to enhance

solubility. Compounds which did not dissolve completely were 110 dissolved in dimethyl sulphoxide (DMSO; the volume used was

1

4

| Journal Name, [year], [vol], 00–00

RSC Advances

Page 16 of 17

View Article Online

DOI: 10.1039/C4RA16032K

1

5% of the total final volume calculated), then made up to the

55

The pharmacokinetic profile of compounds showing good antiꢀ

malarial activity was predicted by using programs Qikprop v3.5

(Schrödinger, Inc., New York, NY, 2012). All the compounds

were prepared in neutralized form for the calculation of

pharmacokinetic properties by QikProp using Schrodinger’s

final volume in 0.5% HPMC as above, and sonicated for ten

minutes. A final volume of 200µL was dosed to each animal by

gavage on each occasion. For intravenous dosing the compound

was prepared to 5mg/kg and dissolved in 15% of the final

5

0

5

calculated volume in DMSO and sonicated for ten minutes. A 60 Maestro Build module and LigPrep, saved in SD format. The

25

solution of propylene glycol and ethanol (4:1 v/v) was added,

comprising 40% of the total calculated volume. The balance of

the volume was made up with polyethylene glycol. A total of

50µL was administered to each animal intravenously each day

after the animals were anaesthetised as described above.

programs QikProp utilizes the method of Jorgensen to compute

pharmacokinetic properties and descriptors such as octanol/water

partitioning coefficient, aqueous solubility, brain/blood partition

coefficient, intestinal wall permeability, plasma protein binding

65 and others.

1

Parasite counts were determined by flow cytometry after 96

hours (Day 4) and then again on Days 7, 9 and 11, using a

BectonꢀDickinson FACSCalibur. On those days, a small incision

was made on the tail of each animal and a small volume of blood

collected into a heparinised capillary tube. The blood was

Notes and references

a

Department of Chemistry, University of Delhi, Delhi-110007, India.

1

Fax: 91-11-27667501; Tel: 91-11-27662683; E-mail:

dsrawat@chemistry.du.ac.in

#

expelled from the capillary into a separate tube containing 0.5mL 70 Current address: Chemistry Department, Deen Dayal Upadhyaya

PBS and transferred to the flow cytometer for analysis.

College (University of Delhi), Shivaji Marg, Karampura, New Delhi-

1

10015.

b

Division of Clinical Pharmacology, Department of Medicine, University

Molecular docking studies

of Cape Town, Rondebosch 7700, South Africa

1

13

7

8

9

5

0

5

0

5

† Electronic Supplementary Information (ESI) available: [ H NMR,

NMR and HPLC data of selected compounds]. See

DOI: 10.1039/b000000x/

C

2

3

4

5

0

5

0

5

0

5

0

The 2D structures of all the compounds were generated by

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| Journal Name, [year], [vol], 00–00

Article Doi

Highly active 4-aminoquinoline-pyrimidine based molecular hybrids as potential next generation antimalarial agents

DOI: 10.1039/c4ra16032k

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Article abstract of DOI:10.1039/c4ra16032k

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