In vitro antimalarial activity, β-haematin inhibition and structure-activity relationships in a series of quinoline triazoles

Article abstract of DOI:10.1016/j.ejmech.2013.08.046

A novel series of quinoline triazole amide analogues (38-51) has been synthesized. Analogues 38-44 had a Cl substituent at the 7-position of the quinoline ring, while 45-51 had a CN substituent at this position. Compounds 40, 45 and 49 were found to be the most active in the series against the Plasmodium falciparum chloroquine-sensitive D10 strain, with IC₅₀ values in the range of 349-1247 nM, with 40 and 45, but not 49 also exhibiting similar activity against the chloroquine-resistant K1 strain of parasite. Quinoline triazoles 40 and 44 were the most active β-haematin inhibitors, with 50% inhibitory concentrations of 14.7 and 8.9 μM respectively. In vitro antimalarial activity of the 7-Cl bearing analogues 38-44 exhibited a strong linear dependence of log(1/IC₅₀) on log P. Thus, the more lipophilic, the more active it was found be. The 7-CN series 45-51 showed no such dependence. The resistance index (IC₅₀ K1/IC₅₀ D10) also exhibited a linear dependence on log P, with a substantially steeper slope in the case of the 7-Cl series. The findings demonstrate the feasibility of producing hydrophilic analogues with strong activity and low cross-resistance with chloroquine.

Full text of DOI:10.1016/j.ejmech.2013.08.046

European Journal of Medicinal Chemistry 69 (2013) 338e347
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
In vitro antimalarial activity, b-haematin inhibition and structuree
activity relationships in a series of quinoline triazoles
Mukesh C. Joshi a, Kathryn J. Wicht a, Dale Taylor b, Roger Hunter a, Peter J. Smith b,
,
Timothy J. Egan a
*
a Department of Chemistry, University of Cape Town, Private Bag, Rondebosch 7701, South Africa
b Division of Pharmacology, Department of Medicine, University of Cape Town, Observatory 7925, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of quinoline triazole amide analogues (38e51) has been synthesized. Analogues 38e44
had a Cl substituent at the 7-position of the quinoline ring, while 45e51 had a CN substituent at this
position. Compounds 40, 45 and 49 were found to be the most active in the series against the Plasmodium
falciparum chloroquine-sensitive D10 strain, with IC50 values in the range of 349e1247 nM, with 40 and
45, but not 49 also exhibiting similar activity against the chloroquine-resistant K1 strain of parasite.
Received 25 May 2013
Received in revised form
19 August 2013
Accepted 25 August 2013
Available online 12 September 2013
Quinoline triazoles 40 and 44 were the most active
b-haematin inhibitors, with 50% inhibitory con-
centrations of 14.7 and 8.9 M respectively. In vitro antimalarial activity of the 7-Cl bearing analogues 38
m
Keywords:
Antimalarial
Plasmodium falciparum
e44 exhibited a strong linear dependence of log(1/IC50) on log P. Thus, the more lipophilic, the more
active it was found be. The 7-CN series 45e51 showed no such dependence. The resistance index (IC50
K1/IC50 D10) also exhibited a linear dependence on log P, with a substantially steeper slope in the case of
the 7-Cl series. The findings demonstrate the feasibility of producing hydrophilic analogues with strong
activity and low cross-resistance with chloroquine.
b-Haematin
Structureeactivity relationships
Resistance
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
the recent observation that 4-amino-7-cyanoquinolines retain
good b-haematin inhibition activity and probably also retain anti-
Quinolines have formed a historically important class of anti-
malarial drugs. Although compromised by resistance, they remain
of interest (e.g. Ref. [1]). The 4-aminoquinolines, such as chloro-
malarial activity we have proposed that less lipophilic analogues of
the 4-amino-7-chloroquinoline antimalarials can be made [10] and
might show improved toxicity profiles. Lately, 4-aminoquinolines
covalently linked to triazine groups were also synthesized in the
expectation of improving activity against drug resistant parasites
[11e14] owing to the altered side-chain, suggesting that other ni-
trogen heterocycles may also be advantageous. Triazoles are an
important class of compounds known to exhibit a variety of bio-
logical activities including antifungal, antimicrobial [15e17] and
antitubercular activity [18] that might also be expected to show
improved antimalarial activity. Herein we report the synthesis,
quine are known to inhibit the crystallisation of Fe(III)haem to b-
haematin, the synthetic counterpart of haemozoin [2], an insoluble
product formed in the parasite digestive vacuole following diges-
tion of human haemoglobin [3]. Inhibition of haemozoin formation
leads to intraparasitic accumulation of free Fe(III)haem [4], which is
highly toxic to the parasite. Several studies have shown that side
chain modification of 4-aminoquinolines can lead to increased
potency of these compounds against drug-resistant Plasmodium
falciparum strains [5,6]. Recently, hybrid 4-aminoquinolines with
suitable side chains have been designed to act as chloroquine
chemosensitizers with strong activity against resistant strains [5e
8]. However, many of these compounds were very lipophilic, an
undesirable trait from the point of view of adverse drug effects such
as hERG inhibition and cytochrome P450 inhibition [9]. Based on
antimalarial activity and b-haematin inhibition activity of a series
of 7-chloro- and 7-cyano-4-aminoquinolines with side chains
containing a triazole as a linker, readily prepared using click
chemistry [19e23]. We further report on their structureeactivity
relationships.
2. Chemistry
The synthesis sequence for producing the triazolyl-amines 29e
35 for coupling is shown in Scheme 1. According to an established
method [24e28] N-substituted amines (1e7, groups R1 and R2 with
* Corresponding author. Tel.: þ27 21 650 2528; fax: þ27 21 650 5195.
E-mail addresses: Timothy.Egan@uct.ac.za, timothy.john.egan@gmail.com
(T.J. Egan).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.08.046

M.C. Joshi et al. / European Journal of Medicinal Chemistry 69 (2013) 338e347
339
Cl
2
2
Cl
R
N
R
N
1
A
R
A
NaN3
A
O
1
1
NH
1
R
N
3
R
Cl
R
DCM
0 - 10 oC
30 min - 1 h
8 - 12 h, RT
O
O
1 - 7
8 - 14
15 - 21
t-BuOH:H2O (1:1)
60 - 80 oC
NHBoc
sodium ascorbate
4 - 8 h
CuSO4.5H2O
1
1
R
R
N
1. TCA, DCM/MeOH, 2 - 4 h
2. MeOH, dioxane.HCl, 1 - 2 h
A
A
N
2
2
R
N
R
N
N
N
N
O
NH
2
O
NHBoc
N
29 - 35
22 - 28
Scheme 1. Three-step sequence for synthesis of the amine coupling partners 29e35.
or without a bridging group A, as specified in Table 1) were reacted
with chloroacetyl chloride (1.1 equiv.) in dichloromethane at 0e
10 ꢀC for 30 min to 1 h to form amides 8e14. Compounds 8e14
were further reacted [25,27,29] with NaN3 (3.0 equiv.) in dime-
thylformamide at room temperature for 8e12 h to give azides 15e
21, which were then reacted at 60e80 ꢀC for 4e8 h in a click re-
action with N-Boc-propargylamine (1.6 equiv.) in t-BuOH and H2O
(1:1) in the presence of sodium ascorbate (0.4 equiv.) and
CuSO4$5H2O (0.22 equiv.) as catalyst. This furnished compounds
22e28, which were then deprotected by trifluoroacetic acid
(5.0 equiv.) in dichloromethane/methanol (2:1). Isolation involved
converting the residue to its hydrochloride salt using dioxane HCl
to yield triazolyl amines 29e35 as hydrochloride salts in a purity of
>90% as judged by 1H NMR spectroscopy. The only chromato-
graphic purification utilized in the three-step sequence was for the
N-Boc derivatives 22e28 (Scheme 1).
(Scheme 2). 4-Chloro-7-cyanoquinoline needed to be synthesized
by a literature procedure [30], while 4,7-dichloroquinoline was
used as received from a commercial supplier. The adducts were
quite difficult to isolate as single spots on TLC in view of their high
polarity, and it was found that column chromatographic purifica-
tion using ethyl acetate/methanol/triethylamine as eluent needed
to be carried out twice to obtain material of better than 85% purity
by HPLC. Final purification to obtain material pure enough for
biological testing was achieved using preparative-plate chroma-
tography, which afforded the quinoline triazole analogues (38e51)
in >95% purity by HPLC, except 39 and 43 which were more than
90% pure. A full analysis using 1H and 13C NMR spectroscopy, as well
as high resolution mass spectrometry, confirmed the correct
structures for the library.
3. Biological and physico-chemical testing
The final quinoline-triazole analogues 38e51 were synthesized
by SNAr reaction of triazolyl-amines 29e35 with 4,7-dichlor-
oquinoline (36) or 4-chloro-7-cyanoquinoline (37, 1.0e2.5 equiv.)
using triethylamine (3.0e5.0 equiv.) as base in ethanol at 130e
135 ꢀC for 8 h under a nitrogen atmosphere in a sealed tube
3.1. In vitro antimalarial activity
Quinolone-triazole amides (QTA) 38e51 were tested for their
in vitro antimalarial activity against the P. falciparum chloroquine-
sensitive (CQS) D10 and chloroquine-resistant K1 strains
(Table 2), and compared to chloroquine (CQ) as a standard drug.
Compounds 40, 45 and 49 were the most active analogues in the
series against the P. falciparum D10 strain. With the exception of 45
and 49, compounds having a 7-CN group in the quinoline core were
less effective than those with a 7-Cl group. Resistance indices varied
from low values in 40 and 47 (1.5 and 2.5 respectively) to high in 41
and 49 (10.8 and 12.1 respectively). Compounds 45 and 49 were
also tested for cytotoxicity in mammalian (Chinese Hamster
Ovarian, CHO) cells. Both compounds were cytotoxic at much
higher concentrations than their antimalarial activity (Table 3),
with selectivity indices versus the D10 strain of P. falciparum
(IC50(CHO)/IC50(D10)) of 31 and 125 respectively and versus the K1
strain of 20 and 31 respectively.
Table 1
Side chains of amines 1e7 in Scheme 1.
eR1
eR2
A
1
2
eCH2CH3
eCH(CH3)2
eCH2CH3
eCH(CH3)2
e
e
3
e
4
5
eH
e
e
3.2. Inhibition of
b-haematin formation
eCH3
Synthesised QTA compounds 38e51 were tested for
inhibiting properties using an NP-40 detergent mediated assay, the
details of which have been reported elsewhere [31e33]. Standard
b
-haematin
6
7
CH2
CH2
CH2
CH2
CH2OCH2
CH2CH2CH2

340
M.C. Joshi et al. / European Journal of Medicinal Chemistry 69 (2013) 338e347
2
R
A
1
N
R
2
N
HN
R
Cl
N
N
A
N
O
N
NH
N
2
N
N
1
R
O
TEA, EtOH
130 - 135 oC
sealed tube
X
X
N
36 X = Cl
37 X = CN
38 - 51
Scheme 2. SNAr coupling to the final adducts 38e51.
drugs such as CQ and amodiaquine were assayed prior to testing
and were found to have IC50 values of 18.2 M and 6.8 mM
piperazinyl linkers were described by these authors, with the
former showing good activity. By contrast with the 7-Cl series, no
correlation of in vitro antimalarial activity with lipophilicity was
found for compounds 45e51 with a 7-CN group (Fig. 1b).
m
respectively. The assay results are reported in Table 2. The most
active QTA compounds in the group with the 7-Cl substituent are
40 and 44, which contain dicyclohexyl and piperidinyl substituents
on the amides respectively. These results differ from the 7-CN
substituent case where the most active compounds (48 and 49)
contained cyclopentyl and benzyl substituents. There is no statis-
The resistance index was found to decrease with increasing log P
in both the 7-Cl and 7-CN series. The gradient is much larger in the
former series (Fig. 1c). As a result, the most hydrophobic analogues
show almost no cross-resistance with chloroquine, while the least
hydrophobic compounds show strong cross-resistance. Thus com-
pound 40 is the most active against both the CQS D10 and CQR K1
parasite strains, and exhibits little evidence of cross-resistance with
CQ. Interestingly, the linear dependence of resistance index on log P
suggests that the lack of cross-resistance in this analogue may be
related to its physico-chemical properties, rather than its resem-
blance to a CQ chemosensitizing pharmacophore.
tically significant difference in average IC50 values for b-haematin
inhibition between the compounds with a 7-Cl substituent and
those with a 7-CN substituent on the quinoline ring (P ¼ 0.25).
Furthermore, differences are generally relatively small between the
various compounds, with the most active only about five-fold more
active than the least active.
The dependence of resistance index on log P is much less steep
in the case of the 7-CN series (Fig. 1d). This means that these
compounds have comparable resistance index values to the 7-Cl
compounds of similar polarity at the hydrophobic end of the
scale, but lower resistance index values at the hydrophilic end.
4. Discussion
Most highly active 4-aminoquinoline antimalarials contain side
chains with a basic amino group thought to play a crucial role in pH
trapping [34,35]. The QTA series contains an amide group which is
unable to assist in pH trapping. It is therefore notable that com-
pounds 40 and 45 have parasite IC50 values below 1
mM in the CQS
5. Conclusion
D10 strain of parasite. The relatively strong activity of 45 is also
noteworthy given that the 7-Cl group was replaced with a more
hydrophilic 7-CN group in this analogue. Activity is not a mere
result of cytotoxicity, because both 40 and 45 exhibit selective ac-
tivity against malaria parasites.
Previous studies on 4-amino-7-chloroquinolines have shown
that biological activity is often correlated with b-haematin inhibi-
tion, albeit only after taking into account varying levels of vacuolar
accumulation via pH trapping [5,35]. In the case of the analogues
studied here, there is a relatively narrow range of b-haematin in-
hibition IC50 values and the compounds possess only one at best
moderately basic nitrogen atom, that in the 4-aminoquinoline
group (with a conjugate acid pKa estimated at 7.16 for the 7-Cl
series and 5.71 for the 7-CN series, while the triazole is only very
weakly basic, with a conjugate acid pKa below 2) [36]. As a result,
pH trapping is expected to occur to a much smaller extent for the Cl
series and is probably negligible for the 7-CN series, possibly ac-
counting for the generally weaker activity of the latter series rela-
tive to the former. On the other hand, the biological activity
(expressed as log (1/IC50) or ꢁlog IC50) was found to be strongly
linearly correlated with calculated log of the partition coefficient
(log P) in the case of the compounds with a 7-Cl group (38e44) as
shown in Fig. 1a. Thus, in the case of the 7-Cl series, in vitro anti-
malarial activity simply increases with lipophilicity, probably
reflecting the ease with which the compound reaches the digestive
vacuole of the parasite. Of interest in this respect is compound 40.
This compound has two cyclohexyl groups attached to the amide N,
resembling the reversed chloroquine motifs reported by Andrews
et al. [6]. Indeed, identical terminal amide side chains with ethyl or
The successful synthesis of a series of quinolineetriazole amides
using click chemistry has produced several compounds with good
in vitro antimalarial activity. More importantly, these compounds
have proved to be valuable probes of activity and resistance in the 4-
aminoquinolines. Several observations are particularly noteworthy.
Firstly, replacement of the 7-Cl group with a 7-CN group, which
results in significantly more polar compounds, does not render the
compound inactive. Furthermore, the CN group decouples the tight
correlation between activity and lipophilicity seen in the 7-Cl series.
This is important, because reduced lipophilicity is often accompa-
nied by decreased adverse drug effects and is also beneficial for oral
bioavailability [9]. Attempting to reduce lipophilicity in 7-Cl ana-
logues simply decreases activity, whereas some of the hydrophilic
CN compounds retain strong activity. Secondly, the observation of
much lower levels of cross-resistance in the 7-CN series suggests
that the 7-chloroquinoline ring itself is involved in resistance. Many
studies have pointed to the importance of the side-chain in deter-
mining resistance, while the role of the 4-amino-7-chloroquinoline
core has been less clear. It seems from this work that this group is
itself part of the pharmacophore involved in the resistance mech-
anism. This highlights the need to move away from this motif.
Thirdly, the 4-amino-7-cyanoquinoline series exhibits much lower
levels of resistance in hydrophilic analogues than the 7-Cl series.
This suggests that strongly active hydrophilic analogues with rela-
tively little cross resistance with CQ can be prepared.
It may be possible to improve the activities of these compounds by
reducing the amide groups to amines, increasing pH trapping to
produce compounds with activities similar to chloroquine, but not

M.C. Joshi et al. / European Journal of Medicinal Chemistry 69 (2013) 338e347
341
Table 2
IC50 data for in vitro antimalarial activity against the CQS D10 and CQR K1 strains of P. falciparum and
index, IC50(K1)/IC50(D10), calculated log P values. Values for CQ are given for comparison.
b-haematin (b-H) inhibition of compounds 38e51, together with resistance
Compound
Structure
Parasite IC50 (nM)
D10
RI
b
-H IC50
(
mM)
log P
K1
38
1440
1592
10,498
9518
7.3
20.3
32.7
2.45
39
40
6.0
1.5
3.27
4.93
348.8
518.6
14.7
41
42
43
2473
1910
8387
26,681
9077
10.8
4.8
30.5
21.7
28.7
2.70
3.54
1.42
69,624
8.3
44
45
2063
11,287
2399
5.5
4.1
8.9
2.48
1.74
584.5
46.3
46
47
3969
2599
14,440
6563
3.6
2.5
23.7
26.5
2.57
4.23
48
49
12,131
1247
37,297
15,057
3.1
16.1
15.9
2.00
2.84
12.1
(continued on next page)

342
M.C. Joshi et al. / European Journal of Medicinal Chemistry 69 (2013) 338e347
Table 2 (continued )
Compound
Structure
Parasite IC50 (nM)
D10
RI
b-H IC50
(
mM)
log P
K1
50
27,518
130,625
4.7
44.6
0.72
51
13,619
17.4
43,439
354.7
3.2
32.7
18.2
1.78
3.81
CQ
20.3
exhibiting cross-resistance with chloroquine. However there is a
caveat. The finding that secondary amides with either 7-Cl or 7-CN
groups show high-level resistance may indicate that N-dealkylation
of the tertiary amines would result in a secondary amine metabolite
withsimilarhigh-levelresistance. Thiswillneedcarefulinvestigation.
6.2.1.1. 2-Chloro-N,N-diethylacetamide (8) [24]. Light-yellow liquid;
yield (78%); 1H NMR (400 MHz, CDCl3):
1.13 (t, J ¼ 7.2 Hz, 3H), 1.22
d
(t, J ¼ 7.2 Hz, 3H), 3.36 (q, J ¼ 7.2 Hz, 2H), 3.38 (q, J ¼ 7.2 Hz, 2H), 4.04
(s, 2H); 13C NMR (400 MHz, CDCl3):
165.8.
d 12.7, 14.5, 40.7, 41.3, 42.6,
6.2.1.2. 2-Chloro-N,N-diisopropylacetamide (9). Dark-brown liquid;
6. Experimental
yield (96%); 1H NMR (300 MHz, CDCl3):
d
1.24 (d, J ¼ 7.6 Hz, 6H),
1.39 (d, J ¼ 7.6 Hz, 6H), 3.44 (m, 1H), 3.96 (m, 1H), 4.01 (s, 2H); 13
C
6.1. Chemistry
NMR (400 MHz, CDCl3):
d 20.4, 21.0, 43.3, 46.5, 50.0, 165.2.
Solvents, acids, and common salts were obtained from
Saarchem, South Africa, while 4,7-dichloroquinoline 36 and other
chemicals were obtained from SigmaeAldrich. 4-Chloro-7-
cyanoquinoline 37 was synthesized according to a literature pro-
cedure [30]. Pre-coated silica-gel plates as well as silica-gel for
column chromatography were obtained from Merck. 1H and 13C
NMR spectra were recorded on a Varian Mercury spectrometer at
300 MHz and a Bruker spectrometer at 400 MHz. All spectra were
recorded in either d-chloroform (references ¼ 7.26 ppm for 1H and
77.16 ppm for 13C NMR), d4-methanol (references ¼ 3.31 ppm for 1H
and 49.00 ppm for 13C NMR), or d6-dimethylsulphoxide
(references ¼ 2.50 for 1H and 39.52 ppm for 13C NMR), which were
obtained from SigmaeAldrich. Mass spectra were recorded on a VG
Micromass 16F spectrometer operating at 70 eV with an acceler-
ating voltage of 4 kV and a variable temperature source. Accurate
mass determinations were performed on a Kratos Limited MS9/50
spectrometer. All mass spectra were obtained using electron-
impact techniques. The elemental analysis (HRMS-ESþ) of final
compounds were recorded using a Waters Synapt G2 instrument.
All the final compounds were subjected to HPLC to evaluate purity
using an Agilent Technologies 1220 Infinity LC Gradient system
6.2.1.3. 2-Chloro-N,N-dicyclohexylacetamide
powder; yield (82%); 1H NMR (300 MHz, CDCl3):
20H), 3.02 (m, 1H), 3.43 (m, 1H), 4.01 (s, 2H); 13C NMR (400 MHz,
CDCl3): 25.4 (2), 26.0, 26.6, 29.7, 31.4, 43.6, 56.6, 59.1, 165.6.
(10). Brown-solid
d
1.21e1.81 (m,
d
6.2.1.4. 2-Chloro-N-cyclopentylacetamide (11) [25]. Brown, crystal-
line solid; yield (92%); 1H NMR (300 MHz, CDCl3):
1.38e1.47 (m,
d
2H), 1.59e1.74 (m, 4H), 1.97e2.05 (m, 2H), 4.01 (s, 2H), 4.25 (m, 1H),
6.47 (br. s, 1H); 13C NMR (400 MHz, CDCl3):
165.5.
d 23.8, 33.1, 42.8, 51.7,
6.2.1.5. N-Benzyl-2-chloro-N-methylacetamide
Dark-yellow liquid; yield (w100%); 1H NMR (400 MHz, CDCl3):
(major isomer) 2.98 (s, 3H), 4.12 (s, 2H), 4.58 (s, 2H), 7.17e7.35 (m,
5H); (minor isomer) 2.95 (s, 3H), 4.08 (s, 2H), 4.58 (s, 2H), 7.17e7.35
(m, 5H); 13C NMR (400 MHz, CDCl3):
(major isomer) 35.2, 41.5,
(12)
[26].
d
d
51.5, 126.6, 128.1, 128.9, 136.6, 166.8; (minor isomer) 34.5, 41.2, 53.8,
126.6, 127.8, 129.2, 135.9, 167.0.
6.2.1.6. 2-Chloro-1-(morpholin-4-yl)ethanone
(13)
[27].
3.52
Light-reddish solid; yield (92%); 1H NMR (300 MHz, CDCl3):
d
with an Eclipse Plus C18 column (5
exceeded 95% except where otherwise stated. Melting points for all
m
m, 4.6 ꢂ 150 mm). Purity
(m, 2H), 3.61 (m, 2H), 3.66e3.74 (m, 4H), 4.05 (s, 2H); 13C NMR
(400 MHz, CDCl3):
d 40.7, 42.6, 46.9, 66.6, 66.7, 165.4.
final products were measured using a BioCote SMP-10 instrument.
6.2.1.7. 2-Chloro-1-(piperidin-1-yl)ethanone
(14)
[28].
1.55e
Dark-yellow liquid; yield (92%); 1H NMR (300 MHz, CDCl3):
d
6.2. Synthesis
1.60 (m, 2H), 1.61e1.68 (m, 4H), 3.45 (t, J ¼ 5.7 Hz, 2H), 3.57 (t,
6.2.1. General method for the synthesis of 8e14
To a series of substituted amines (1e7) in dichloromethane
(0.05 g/mL) was added chloroacetyl chloride (1.1 equiv.) followed
by the addition of triethylamine (2.5 equiv.) at 0e10 ꢀC. The reac-
tion was continued at the same temperature for 30 min to 1 h. The
progress of reaction was monitored by TLC. After completion of the
reaction, the reaction mixture was washed with saturated NaHCO3
solution and washed with brine solution The excess organic solvent
was removed under reduced pressure and the crude compound
used for further reaction without purification.
Table 3
Cytotoxicity and selectivity index values of compounds 40 and 45. The cytotoxicity
of emetine is given for comparison.
Compound
D10 IC50
M)
K1 IC50
M)
CHO IC50
M)
SI
(D10)
SI
(K1)
(
m
(m
(
m
40
45
Emetine
0.349
0.585
e
0.519
2.40
e
10.7
73.2
0.041
30.7
125
e
20.6
30.5
e

M.C. Joshi et al. / European Journal of Medicinal Chemistry 69 (2013) 338e347
343
Fig. 1. Quantitative structureeactivity relationships were observed in the QTA series. (a) The 7-Cl series (D10 strain) with ꢁlog IC50 ¼ 0.34(7)log P þ 4.7(2), r2 ¼ 0.84, P ¼ 0.0039. (b)
In the 7-CN series there is no relationship between biological activity and lipophilicity (r2 ¼ 0.23, P ¼ 0.28). (c) In the 7-Cl series RI ¼ ꢁ1.9(3)log P þ 11(1), r2 ¼ 0.90, P ¼ 0.0039. (d)
In the 7-CN series RI ¼ ꢁ0.6(2)log P þ 4.8(4), r2 ¼ 0.70, P ¼ 0.038. The open squares in (c) and (d) represent secondary amide compounds that show much higher levels of cross-
resistance with chloroquine and were not included in correlation analyses.
J ¼ 5.7 Hz, 2H), 4.06 (s, 2H); 13C NMR (400 MHz, CDCl3):
d
24.4, 25.5,
1.74 (m, 4H),1.95e2.04 (m, 2H), 3.92 (s, 2H), 4.21 (m,1H), 6.20 (br. s,
26.4, 41.3, 43.4, 47.5, 165.0.
1H); 13C NMR (400 MHz, CDCl3):
d 23.8, 33.1, 51.3, 52.9,166.1; EI-MS
(m/z) %: 168 (Mþ, 18), 69 (100).
6.2.2. General method for the synthesis of 15e21
To a mixture of 8e14 in dimethylformamide (0.067 g/mL) was
added sodium azide (3.0 equiv.) at room temperature and the re-
action allowed to proceed for 8e12 h. The progress of the reaction
was monitored by TLC. Upon completion, the reaction mixture was
subjected to an extraction with ethyl acetate with saturated
NaHCO3 solution, and the organic extracts washed with brine so-
lution and finally water. The organic solvent was removed under
reduced pressure and the crude compounds were used for further
reaction without any purification, based on a high-level purity
(>95%) by 1H NMR spectroscopy.
6.2.2.5. 2-Azido-N-benzyl-N-methylacetamide (19). Brown liquid;
yield (97%); 1H NMR (300 MHz, CDCl3):
(major isomer) 2.87 (s,
3H), 3.97 (s, 2H), 4.61 (2H, s), 7.15 (m, 1H), 7.23e7.42 (m, 4H);
d
(minor isomer) 3.01 (s, 3H), 3.95 (s, 2H), 4.46 (s, 2H), 7.15 (m, 1H),
7.23e7.42 (m, 4H); 13C NMR (400 MHz, CDCl3):
d (major isomer)
34.2, 50.7, 51.4, 126.3, 128.7, 129.2,136.5,167.2; (minor isomer) 34.5,
50.8, 53.0, 126.3, 128.2, 129.7, 135.6, 167.6; EI-MS (m/z) %: 204 (17),
91 (100).
6.2.2.6. 2-Azido-1-(morpholin-4-yl)ethanone (20) [27]. Yellow
powder; yield (50%); 1H NMR (400 MHz, CDCl3):
d
3.38 (m, 2H),
3.64 (m, 2H), 3.68 (m, 4H), 3.92 (s, 2H); 13C NMR (400 MHz, CDCl3):
42.4, 45.7, 50.7, 66.5, 66.8, 165.9; EI-MS (m/z) %: 170 (Mþ, 17), 113
6.2.2.1. 2-Azido-N,N-diethylacetamide (15). Dark-yellow liquid;
yield (99%); 1H NMR (400 MHz, CDCl3):
d
1.14 (t, J ¼ 7.2 Hz, 3H), 1.18
d
(t, J ¼ 7.2 Hz, 3H), 3.22 (q, J ¼ 7.2 Hz, 2H), 3.40 (q, J ¼ 7.2 Hz, 2H), 3.88
(100).
(s, 2H); 13C NMR (400 MHz, CDCl3):
d 13.0, 14.4, 40.8, 41.7, 50.5,
166.5; EI-MS (m/z) %: 156 (Mþ, 12), 100 (100), 72 (80).
6.2.2.7. 2-Azido-1-(piperidin-1-yl)ethanone (21) [29]. Brown liquid;
yield (97%); 1H NMR (300 MHz, CDCl3):
d
1.53e1.70 (m, 6H), 3.30
(m, 2H), 3.57 (m, 2H), 3.91 (s, 2H); 13C NMR (400 MHz, CDCl3):
24.5, 25.6, 26.5, 43.3, 46.3, 50.9, 165.2; EI-MS (m/z) %: 168 (Mþ,
6.2.2.2. 2-Azido-N,N-diisopropylacetamide (16). Dark-brown liquid;
yield(92%); 1H NMR (400 MHz, CDCl3):
d
1.22 (d, 6H),1.39 (d, 6H), 3.53
20.6,
d
(m, 1H), 3.73 (m, 1H), 3.85 (s, 2H); 13C NMR (400 MHz, CDCl3):
d
33), 112 (100).
21.1, 46.5, 48.4, 52.3,165.8; (EI-MS (m/z) %:185 (M þ H)þ, 9), 86 (100).
6.2.3. General method for the synthesis of 22e28
6.2.2.3. 2-Azido-N,N-dicyclohexylacetamide (17). Brown solid; yield
To each of the azides 15e21 in t-BuOH (0.067 g substrate/mL)
was added N-Boc-propargylamine (1.6 equiv.) and the mixture
vigorously stirred at room temperature in a round-bottomed flask.
In a second round-bottomed flask a yellow suspension was pre-
pared by vigorously stirring a solution of sodium ascorbate
(0.40 equiv.) and CuSO4$5H2O (0.22 equiv.) in distilled water. This
yellow suspension was then poured into the first round-bottomed
flask with vigorous stirring such that the ratio of t-BuOH to water
(82%); 1H NMR (300 MHz, CDCl3):
d 1.10e1.90 (m, 20H), 3.05 (m,
1H), 3.18 (m,1H), 3.86 (s, 2H); 13C NMR (400 MHz, CDCl3):
d 25.3 (2),
26.1, 26.6, 30.1, 31.5, 52.3, 56.5, 57.7, 166.1; EI-MS (m/z) %: 264 (Mþ,
5), 83 (100).
6.2.2.4. 2-Azido-N-cyclopentylacetamide (18) [25]. Brown solid;
yield (88%); 1H NMR (300 MHz, CDCl3):
d 1.36e1.46 (m, 2H), 1.58e

344
M.C. Joshi et al. / European Journal of Medicinal Chemistry 69 (2013) 338e347
was 1:1. The reaction temperature was then increased from room
temperature to 60e80 ꢀC and the reaction vigorously stirred for 4e
8 h. The progress of the reaction was monitored by TLC. Upon re-
action completion, excess solvent was removed from the reaction
mixture and the crude mixture dissolved in ethyl acetate, which
was washed with 80% brine solution. The organic solvent was dried
over anhydrous MgSO4 and excess organic solvent was removed
under reduced pressure. The compounds were purified by flash
column chromatography using ethyl acetate/methanol/triethyl-
amine as eluent.
(s, 2H), 7.67 (s, 1H); 13C NMR (400 MHz, CDCl3):
d 24.3, 25.4, 26.4,
28.5, 36.4, 43.6, 46.6, 51.2, 79.7, 123.7, 145.8, 155.9, 163.1; EI-MS (m/
z) %: 323 (Mþ, 14), 125 (100).
6.2.4. General method for the synthesis of 29e35
To each of 22e28 in 2:1 dichloromethane/methanol (0.1 g
compound/mL) was added trifluoroacetic acid (5.0 equiv.) at 0 ꢀC
and the reaction left at room temperature for 2e4 h. The progress of
the reaction was monitored by TLC. After completion of the reac-
tion, excess solvent was removed, the crude mixture dissolved in
anhydrous methanol (0.1 g substrate/mL), and dioxaneeHCl solu-
tion added at 0 ꢀC to give a final concentration of 0.067 g substrate/
mL. After 0.5 h, the excess solvent was removed under high vacuum
and the crude solid washed several times with 80% ethyl acetate/
petroleum ether to remove organic by-products. The residues 29e
35 were each obtained as a hydrochloride salt, which were pure
enough (>90%) by 1H NMR spectroscopy for the final SNAr reaction.
6.2.3.1. tert-Butyl ({1-[2-(diethylamino)-2-oxoethyl]-1H-1,2,3-
triazol-4-yl}methyl)carbamate (22). Dark-brown oil; yield (93%);
1H NMR (400 MHz, CDCl3):
d
1.13 (t, J ¼ 7.2 Hz, 3H),1.25 (t, J ¼ 7.2 Hz,
3H), 1.42 (s, 9H), 3.41 (m, 4H), 4.42 (s, 2H), 5.28 (s, 2H), 5.37 (br. s,
1H), 7.77 (s, 1H); 13C NMR (400 MHz, CDCl3):
12.9, 14.5, 28.5, 36.2,
d
41.1, 42.1, 51.2, 79.7, 124.1, 146.3, 155.9, 164.0; EI-MS (m/z) %: 311
(Mþ, 10), 255 (98), 100.02 (100).
6.2.5. General method for the synthesis of 38e51 with 4,7-
dichloroquinoline (36) or 4-chloro-7-cyanoquinoline (37)
To a stirred solution of each of 29e35 in anhydrous EtOH
6.2.3.2. tert-Butyl ({1-[2-(diisopropylamino)-2-oxoethyl]-1H-1,2,3-
triazol-4-yl}methyl)carbamate (23). Light-yellow powder; yield
(84%); 1H NMR (400 MHz CDCl3):
d
1.18e1.40 (m, 12H), 1.44 (s, 9H),
3.57 (m, 1H), 3.97 (m, 1H), 4.41 (d, J ¼ 5.6 Hz, 2H), 5.07 (br. s, 1H),
5.14 (s, 2H), 7.67 (s, 1H); 13C NMR (400 MHz CDCl3):
20.6, 20.8,
(5.0 mL) under
(5.0 equiv.) and the reaction mixture was stirred for 15e20 min at
ꢀC. 4,7-Dichloroquinoline (2.5 equiv) (36) or 4-chloro-7-
a N2 atmosphere was added triethylamine
d
0
28.6, 36.7, 46.8, 48.8, 52.7, 79.8, 123.6, 145.8, 155.9, 163.5; EI-MS (m/
cyanoquinoline (37) was added and the mixture allowed to reach
room temperature before being heated under pressure in a sealed
cycloaddition tube at 130e135 ꢀC for 8 h. The progress of the re-
action was monitored by TLC. On completion of the reaction the
mixture was decanted into a round-bottomed flask and excess
solvent removed under reduced pressure. The crude reaction
mixture was then washed with 25% ethyl acetate/petroleum ether
to remove excess 36. The remaining crude mixture was then pu-
rified twice by column chromatography using mixtures of ethyl
acetate/methanol/triethylamine (100% ethyl acetate to 80:15:5) as
an eluent. Owing to the highly polar nature of all of these com-
pounds, impurities were always included in the product. Thus, for
analytical and biological purposes they were further purified by
prep-TLC using ethyl acetate/methanol/triethylamine (92:5:3) as
mobile phase (double development of plate) to give 38e51.
z) %: 339 (Mþ, 10), 86 (100).
6.2.3.3. tert-Butyl ({1-[2-(dicyclohexylamino)-2-oxoethyl]-1H-1,2,3-
triazol-4-yl}methyl)carbamate
(w100%); 1H NMR (300 MHz, DMSOd6):
20H), 3.06 (m, 1H), 3.53 (m, 1H), 4.18 (d, J ¼ 6.0 Hz, 2H), 5.34 (s, 2H),
7.19 (br. s, 1H), 7.71 (s, 1H); 13C NMR (400 MHz, DMSOd6):
24.6,
(24). Brown
powder;
yield
d
1.39 (s, 9H),1.25e1.80 (m,
d
25.1 (2), 25.7, 28.2, 29.4, 30.3, 35.6, 51.8, 54.9, 56.1, 77.8, 124.1, 145.1,
155.5, 164.1; EI-MS (m/z) %: 419 (Mþ, 2), 69 (100).
6.2.3.4. tert-Butyl ({1-[2-(cyclopentylamino)-2-oxoethyl]-1H-1,2,3-
triazol-4-yl}methyl)carbamate (25). Light-yellow powder; yield
(93%); 1H NMR (400 MHz, CDCl3):
d
1.34 (s, 2H),1.44 (s, 9H),1.61 (m,
4H), 1.94 (m, 2H), 4.17 (m, 1H), 4.42 (s, 2H), 4.98 (s, 2H), 5.18 (br. s,
1H), 6.05 (br. s, 1H), 7.67 (s, 1H); 13C NMR (400 MHz, CDCl3):
23.8,
d
28.5, 32.9, 36.3, 51.8, 53.3, 79.9, 124.0, 146.7, 156.0, 164.5; EI-MS (m/
6.2.5.1. 2-(4-{[(7-Chloroquinolin-4-yl)amino]methyl}-1H-1,2,3-
z) %: 323 (Mþ, 8), 113 (100).
triazol-1-yl)-N,N-diethylacetamide (38). White powder; yield
(22%); Mp: 213e216 ꢀC; 1H NMR (300 MHz, CD3OD):
d 1.12 (t,
6.2.3.5. tert-Butyl ({1-[2-(N-benzyl-N-methylamino)-2-oxomethyl]-
J ¼ 7.2 Hz, 3H), 1.27 (t, J ¼ 7.2 Hz, 3H), 3.40 (q, J ¼ 7.2 Hz, 2H), 3.47 (q,
J ¼ 7.2 Hz, 2H), 4.71 (s, 2H), 5.40 (s, 2H), 6.65 (d, J ¼ 5.4 Hz, 1H), 7.43
(dd, J ¼ 2.1 Hz, J ¼ 9.0 Hz, 1H), 7.80 (d, J ¼ 2.1 Hz, 1H), 7.93 (s, 1H),
8.12 (d, J ¼ 9.0 Hz, 1H), 8.37 (br. s, 1H); 13C NMR (400 MHz, DMSO-
1H-1,2,3-triazol-4-yl}methyl)carbamate (26). Yellow powder; yield
(76%); 1H NMR (300 MHz, DMSOd6):
d (major isomer) 1.40 (s, 9H),
3.02 (s, 3H), 4.20 (d, J ¼ 5.7 Hz, 2H), 4.54 (s, 2H), 5.49 (s, 2H), 7.20e
7.43 (m, 5H), 7.79 (s, 1H); (minor isomer) 1.40 (s, 9H), 2.83 (s, 3H),
4.20 (d, J ¼ 5.7 Hz, 2H), 4.67 (s, 2H), 5.44 (s, 2H), 7.20e7.43 (m, 5H),
7.81 (s, 1H); 13C NMR (400 MHz, DMSOd6): (major isomer only)
d6): d 12.8, 13.9, 37.8, 39.8, 40.7, 50.4, 99.3, 117.4, 124.1, 124.4, 124.6,
126.9, 133.7, 143.8, 148.3, 150.1, 151.2, 164.5; HRMS-ESþ: Observed:
373.1551; Calculated: 373.1544 [M þ H]þ for C18H22ClN6O; HPLC:
97.6%.
d
28.0, 33.7, 35.6, 50.3, 51.6, 77.6, 123.9, 126.8, 127.3, 128.5, 136.9,
145.2, 155.5, 165.7; EI-MS (m/z) %: 359 (Mþ, 10), 91(100).
6.2.5.2. 2-(4-{[(7-Chloroquinolin-4-yl)amino]methyl}-1H-1,2,3-
6.2.3.6. tert-Butyl ({1-[2-(morpholin-1-yl)-2-oxoethyl]-1H-1,2,3-
triazol-1-yl)-N,N-diisopropylacetamide (39). Off-white powder;
triazol-4-yl}methyl)carbamate (27). Yellow powder; yield (92%);
yield (13%); Mp: 135e141 ꢀC; 1H NMR (300 MHz, CD3OD):
d 1.30e
1H NMR (300 MHz CDCl3):
d
1.44 (s, 9H), 3.56 (m, 2H), 3.64 (m, 2H),
1.36 (m, 6H), 3.61 (m, 1H), 4.08 (m, 1H), 4.88 (s, 2H), 5.42 (s, 2H),
7.00 (d, J ¼ 5.6 Hz, 1H), 7.68 (dd, J ¼ 1.5 Hz, J ¼ 6.6 Hz, 1H), 7.88 (d,
J ¼ 1.5 Hz,1H), 8.01 (s,1H), 8.34 (d, J ¼ 6.6 Hz,1H), 8.42 (d, J ¼ 5.6 Hz,
3.70 (m, 4H), 4.42 (d, J ¼ 5.9 Hz, 2H), 5.13 (br. s,1H), 5.21 (s, 2H), 7.68
(s, 1H); 13C NMR (400 MHz CDCl3):
d 28.5, 36.4, 42.7, 45.9, 51.0, 66.5,
66.7, 79.8, 123.6, 146.0, 155.9, 163.7; EI-MS (m/z) %: 325 (Mþ, 10),
1H); 13C NMR (400 MHz, CD3OD):
d
20.8 (4ꢂ Me), 39.8, 47.7, 50.0,
269 (100).
53.3, 100.5, 121.8, 125.6, 126.7, 128.4, 138.0, 140.3, 141.9, 145.7, 149.3,
166.0; HRMS-ESþ: Observed: 401.1862; Calculated: 401.1857
[M þ H]þ for C20H26ClN6O; HPLC: 90.1%.
6.2.3.7. tert-Butyl ({1-[2-oxo-2-(piperidin-1-yl)ethyl]-1H-1,2,3-
triazol-4-yl}methyl)carbamate (28). Yellow powder; yield (44%);
1H NMR (400 MHz, CDCl3):
d
1.43 (s, 9H), 1.54e1.75 (m, 6H), 3.46
6.2.5.3. 2-(4-{[(7-Chloroquinolin-4-yl)amino]methyl}-1H-1,2,3-
(m, 2H), 3.56 (m, 2H), 2H, 4.41 (d, J ¼ 7.8 Hz, 2H), 5.13 (br. s,1H), 5.20
triazol-1-yl)-N,N-dicyclohexylacetamide (40). Light-yellow powder;

M.C. Joshi et al. / European Journal of Medicinal Chemistry 69 (2013) 338e347
345
Mp: 165e171 ꢀC; yield (20%); 1H NMR (300 MHz, CD3OD):
d
1.12e
6.2.5.9. 2-(4-{[(7-Cyanoquinolin-4-yl)amino]methyl}-1H-1,2,3-
1.84 (m, 20H), 3.12 (m, 1H), 3.57 (m, 1H), 4.75 (s, 2H), 5.40 (s, 2H),
6.74 (d, J ¼ 4.5 Hz, 1H), 7.50 (dd, J ¼ 1.5 Hz, 6.8 Hz, 1H), 7.82 (d,
J ¼ 1.5 Hz,1H), 7.92 (s,1H), 8.17 (d, J ¼ 6.8 Hz,1H), 8.38 (d, J ¼ 4.5 Hz,
triazol-1-yl)-N,N-diisopropylacetamide (46). Off-white powder;
Mp: 198e201 ꢀC; yield (25%); 1H NMR (300 MHz, CD3OD):
d 1.28 (d,
J ¼ 6.9 Hz, 6H), 1.36 (d, J ¼ 6.9 Hz, 6H), 3.61 (m, 1H), 4.08 (m, 1H),
4.73 (s, 2H), 5.39 (s, 2H), 6.79 (d, J ¼ 5.7 Hz, 1H), 7.68 (dd, J ¼ 1.5 Hz,
8.7 Hz, 1H), 7.93 (s, 1H), 8.20 (d, J ¼ 1.5 Hz, 1H), 8.31 (d, J ¼ 8.7 Hz,
1H); 13C NMR (400 MHz, CD3OD):
d 26.2, 26.5, 26.8, 27.3, 30.9, 32.1,
39.5, 53.6, 57.8, 59.1, 100.5, 124.6, 126.1 (2), 126.9, 137.5, 145.4, 147.6,
150.6, 153.5, 166.3; HRMS-ESþ: Observed 481.2470; Calculated
481.2483 [M þ H]þ for C26H34ClN6O; HPLC: 95.9%.
1H), 8.50 (br. s, 1H); 13C NMR (400 MHz, CD3OD):
d 20.8 (4), 39.5,
47.7, 50.0, 53.3, 102.0, 114.3, 119.2, 122.7, 124.5, 126.3, 126.6, 134.1,
145.4, 147.6, 152.5, 152.7, 166.0; HRMS-ESþ: Observed 392.2192;
Calculated 392.2199 [M þ H]þ for C21H26N7O; HPLC: 98.8%.
6.2.5.4. 2-(4-{[(7-Chloroquinolin-4-yl)amino]methyl}-1H-1,2,3-
triazol-1-yl)-N-cyclopentylacetamide (41). Off-white powder; Mp:
215e218 ꢀC (decomp.); yield (10%); 1H NMR (300 MHz, CD3OD):
6.2.5.10. 2-(4-{[(7-Cyanoquinolin-4-yl)amino]methyl}-1H-1,2,3-
d
1.46e1.75 (m, 6H), 1.90e1.97 (m, 2H), 4.11 (m, 1H), 4.74 (s, 2H),
triazol-1-yl)-N,N-dicyclohexylacetamide (47). Light-yellow powder;
5.09 (s, 2H), 6.71 (d, J ¼ 3.9 Hz, 1H), 7.49 (dd, J ¼ 1.5 Hz, 6.5 Hz, 1H),
Mp: 147e151 ꢀC; yield (10%); 1H NMR (300 MHz, CD3OD):
d 1.36e
7.82 (d, J ¼ 1.5 Hz,1H), 7.97 (s, 1H), 8.17 (d, J ¼ 6.5 Hz, 1H), 8.39 (br. s,
1.85 (m, 18H), 2.33 (m, 2H), 3.12 (m, 1H), 3.56 (m, 1H), 4.75 (s, 2H),
5.40 (s, 2H), 6.82 (d, J ¼ 5.9 Hz,1H), 7.72 (dd, J ¼ 1.5, 8.8 Hz,1H), 7.93
(s, 1H), 8.20 (d, J ¼ 1.5 Hz, 1H), 8.33 (d, J ¼ 8.8 Hz, 1H), 8.50 (d,
1H); 13C NMR (400 MHz, CD3OD):
d 24.7, 33.4, 39.5, 52.8, 53.2,
100.4, 124.8 (2), 125.9. 127.3, 133.9, 139.5, 145.0, 148.9, 149.0, 166.9;
HRMS-ESþ: Observed 385.1544; Calculated: 385.1544 [M þ H]þ for
J ¼ 5.9 Hz, 1H); 13C NMR (400 MHz, CD3OD):
d 26.2, 26.4, 26.8, 27.3,
C
19H22ClN6O; HPLC: 95.4%.
30.9, 32.1, 39.5, 53.6, 57.8, 59.1, 101.9, 114.8, 119.1, 122.6, 124.7, 126.2,
126.9, 133.3, 145.3, 146.8, 152.0, 153.0, 166.3; HRMS-ESþ: Observed
472.2833; Calculated 472.2825 [M þ H]þ for C27H34N7O; HPLC:
96.2%.
6.2.5.5. N-Benzyl-2-(4-{[(7-chloroquinolin-4-yl)amino]methyl}-1H-
1,2,3-triazol-1-yl)-N-methylacetamide (42). Off-white powder; Mp:
152e156 ꢀC; yield (24%); 1H NMR (300 MHz, CD3OD):
d (major
isomer) 3.08 (s, 3H), 4.60 (s, 2H), 4.82 (s, 2H), 5.53 (s, 2H), 6.84 (d,
J ¼ 5.7 Hz, 1H), 7.24e7.33 (m, 5H), 7.56 (dd, J ¼ 1.8, 9.0 Hz, 1H), 7.83
(d, J ¼ 1.8 Hz, 1H), 8.00 (s, 1H), 8.25 (d, J ¼ 9.0 Hz, 1H), 8.38 (br. s,
6.2.5.11. 2-(4-{[(7-Cyanoquinolin-4-yl)amino]methyl}-1H-1,2,3-
triazol-1-yl)-N-cyclopentylacetamide (48). Light-yellow powder;
Mp: 250e252 ꢀC; yield (25%); 1H NMR (300 MHz, CD3OD):
d 1.45e
1H); 13C NMR (300 MHz,DMSO-d6):
d
(major isomer) 33.9, 38.0,
1.97 (m, 8H), 4.10 (qu, J ¼ 6.6 Hz, 1H), 4.73 (s, 2H), 5.08 (s, 2H), 6.77
50.4, 50.7, 99.2, 116.6, 124.7, 124.9, 125.5, 127.0, 127.1, 127.5, 128.4,
135.6, 137.0, 143.0, 144.1, 147.6, 152.4, 165.9; HRMS-ESþ: Observed
421.1538; Calculated: 421.1544 [M þ H]þ for C22H22ClN6O; HPLC:
97.65%.
(d, J ¼ 5.7 Hz, 1H), 7.68 (dd, J ¼ 1.5, 8.7 Hz, 1H), 7.97 (s, 1H), 8.19 (d,
J ¼ 1.5 Hz, 1H), 8.31 (d, J ¼ 8.7 Hz, 1H), 8.48 (d, J ¼ 5.7 Hz, 1H); 13
C
NMR (400 MHz, CD3OD-CDCl3):
d 24.6, 33.4, 39.4, 52.8, 53.2, 101.9,
114.4, 119.1, 122.6, 124.4, 125.7, 126.6, 133.9, 145.4, 147.3, 152.4 (2),
166.9; HRMS-ESþ: Observed 376.1891; Calculated 376.1886
[M þ H]þ for C20H22N7O; HPLC: 98.0%.
6.2.5.6. 2-(4-{[(7-Chloroquinolin-4-yl)amino]methyl}-1H-1,2,3-
triazol-1-yl)-1-(morpholine-1-yl)ethanone (43). Light-yellow pow-
der; Mp: 170e175 ꢀC; yield (12%); 1H NMR (300 MHz, CD3OD):
6.2.5.12. N-Benzyl-2-(4-{[(7-cyanoquinolin-4-yl)amino]methyl}-1H-
d
3.55e3.62 (m, 4H), 3.63e3.74 (m, 4H), 4.77 (d, J ¼ 2.5 Hz, 2H), 5.46
1,2,3-triazol-1-yl)-N-methylacetamide (49). Light-yellow powder;
(s, 2H), 6.74 (d, J ¼ 6.0 Hz, 1H), 7.50 (dd, J ¼ 2.1, 9.0 Hz, 1H), 7.82 (d,
J ¼ 2.1 Hz, 1H), 7.94 (s, 1H), 8.17 (d, J ¼ 9.0 Hz, 1H), 8.38 (br. s, 1H);
Mp: 175e178 ꢀC; yield (25%); 1H NMR (300 MHz, CD3OD):
d (major
isomer) 3.06 (s, 3H), 4.60 (s, 2H), 4.74 (s, 2H), 5.51 (s, 2H), 6.77 (t,
13C NMR (400 MHz, CD3OD):
d
39.5, 43.6, 46.5, 51.6, 67.3, 100.3,
J ¼ 5.7 Hz 1H), 7.28 (m, 5H), 7.67 (dd, J ¼ 1.5 Hz,1H), 7.97 (s,1H), 8.18
112.2, 118.5, 124.6, 126.0, 127.2, 132.4, 139.5, 141.2, 148.9, 154.2,
166.0; HRMS-ESþ: Observed 387.1339; Calculated 387.1336
[M þ H]þ for C18H20ClN6O; HPLC: 94.1%.
(d, J ¼ 1.5 Hz,1H), 8.30 (d, J ¼ 8.7 Hz,1H), 8.48 (d, J ¼ 5.7 Hz,1H); 13
C
NMR (400 MHz, CD3OD):
d (major isomer) 34.6, 39.4, 52.0, 52.4,
101.7, 114.4, 119.0, 122.6, 124.3, 125.9, 126.5, 128.5, 128.8, 129.6,
133.9, 134.0, 145.3, 147.3, 152.4, 152.5, 167.6; HRMS-ESþ: Observed
412.1891; Calculated 412.1886 [M þ H]þ for C23H22N7O; HPLC:
100.00%.
6.2.5.7. 2-(4-{[(7-Chloroquinolin-4-yl)amino]methyl}-1H-1,2,3-
triazol-1-yl)-1-(piperidin-1-yl)ethanone (44). Off-white powder;
Mp: 238e242 ꢀC; yield (20%); 1H NMR (300 MHz, CD3OD):
d 1.51e
1.58 (m, 2H), 1.62e1.73 (m, 4H), 3.52 (m, 4H), 4.72 (s, 2H), 5.42 (s,
2H), 6.66 (d, J ¼ 5.7 Hz, 1H), 7.45 (dd, J ¼ 2.1, 9.0 Hz, 1H), 7.80 (d,
J ¼ 2.1 Hz, 1H), 7.91 (s,1H), 8.13 (d, J ¼ 9.0 Hz,1H), 8.37 (d, J ¼ 5.7 Hz,
6.2.5.13. 4-[({4-[2-(Morpholin-4-yl)-2-oxoethyl]-1H-1,2,3-triazol-1-
yl}methyl)amino]quinoline-7-carbonitrile (50). Off-white powder;
Mp: 193e196 ꢀC; yield (23%); 1H NMR (300 MHz, CD3OD):
d 3.58
1H); 13C NMR (400 MHz, CD3OD):
d
25.0, 26.3, 27.1, 39.4, 44.5, 47.2,
(m, 4H), 3.65e3.73 (m, 4H), 4.75 (s, 2H), 5.45 (s, 2H), 6.79 (d,
J ¼ 6.0 Hz, 1H), 7.69 (dd, J ¼ 1.5, 9.0 Hz, 1H), 7.94 (s, 1H), 8.18 (d,
52.0, 100.3, 118.6, 124.2, 125.9, 126.5, 126.9, 136.9, 143.0, 145.5, 151.3
(2), 165.5; HRMS-ESþ: Observed 385.1546; Calculated 385.1544
[M þ H]þ for C19H22ClN6O; HPLC: 95.8%.
J ¼ 1.5 Hz, 1H), 8.31 (d, J ¼ 9.0 Hz, 1H), 8.48 (d, J ¼ 6.0 Hz, 1H); 13
C
NMR (300 MHz, CD3OD-CDCl3):
d 39.4, 43.6, 46.4, 51.7, 67.2, 101.6,
114.5, 118.7, 122.1, 124.4, 125.9, 126.6, 132.7, 146.7, 145.8, 151.1, 152.7,
165.7; HRMS-ESþ: Observed 378.1678; Calculated 378.1678
[M þ H]þ for C19H20N7O2; HPLC: 100.00%.
6.2.5.8. 2-(4-{[(7-Cyanoquinolin-4-yl)amino]methyl}-1H-1,2,3-
triazol-1-yl)-N,N-diethylacetamide (45). Off-white powder; Mp:
207e210 ꢀC; yield (31%); 1H NMR (300 MHz, CD3OD):
d 1.12 (t,
J ¼ 7.2 Hz, 3H), 1.27 (t, J ¼ 7.2 Hz, 3H), 3.41 (q, J ¼ 7.2 Hz, 2H), 3.47 (q,
J ¼ 7.2 Hz, 2H), 4.74 (s, 2H), 5.41 (s, 2H), 6.79 (d, J ¼ 5.7 Hz, 1H), 7.69
(dd, J ¼ 1.4, 8.8 Hz, 1H), 7.95 (s, 1H), 8.19 (d, J ¼ 1.4 Hz, 1H), 8.32 (d,
6.2.5.14. 4-[({4-[2-Oxo-2-(piperidin-1-yl)ethyl]-1H-1,2,3-triazol-1-
yl}methyl)amino]quinoline-7-carbonitrile (51). Light-yellow pow-
der; Mp: 220e223 ꢀC; yield (21%); 1H NMR (300 MHz, CD3OD):
J ¼ 8.8 Hz, 1H), 8.49 (br. s, 1H); 13C NMR (400 MHz, CD3OD):
d
13.0,
d 1.51e1.71 (m, 6H), 3.52 (m, 4H), 4.72 (s, 2H), 5.42 (s, 2H), 6.76 (d,
14.3, 39.4, 42.0, 42.8, 51.8, 101.8, 114.3, 118.9, 122.7, 124.3, 126.0,
126.6, 133.5, 145.0, 146.8, 152.0, 152.5, 166.3; HRMS-ESþ: Observed
364.1888; Calculated 364.1886 [M þ H]þ for C19H22N7O; HPLC:
99.2%.
J ¼ 5.7 Hz, 1H), 7.45 (dd, J ¼ 2.4, 9.0 Hz), 7.80 (d, J ¼ 2.4 Hz, 1H), 7.91
(s, 1H), 8.13 (d, J ¼ 9.0 Hz, 1H), 8.37 (d, J ¼ 5.7 Hz, 1H); 13C NMR
(400 MHz, CD3OD):
d 25.0, 26.3, 27.1, 39.4, 44.5, 47.2, 52.0, 101.8,
114.1, 119.1, 122.7, 124.3, 125.9, 126.4, 134.2, 145.4, 147.7, 152.1, 152.7,

346
M.C. Joshi et al. / European Journal of Medicinal Chemistry 69 (2013) 338e347
165.5; HRMS-ESþ: Observed 376.1883; Calculated 376.1886
had no measurable effect on the cell viability (data not shown). The
50% inhibitory concentration (IC50) values were obtained from full
doseeresponse curves, using a non-linear doseeresponse curve
fitting analysis via GraphPad Prism v.4 software.
[M þ H]þ for C20H22N7O; HPLC: 100.00%.
6.3. Biology
Haemin (ꢃ98%; Fluka), chloroquine diphosphate salt, amodia-
quine, dimethyl sulfoxide (DMSO), sodium acetate trihydrate, acetic
acid, acetone, HEPES and pyridine were purchased from Sigmae
Aldrich. NP-40 was obtained from Pierce Biotechnology, Rockford,
IL. Flat bottom, 96 well plates (catalogue number 655 180; Cellstar)
were purchased from Greiner Bio-One.
6.3.2. Biophysical testing
6.3.2.1. Detergent mediated assay for
b-haematin inhibition.
The -haematin formation inhibition assay method described by
b
Carter et al. [31] was modified for manual liquid delivery [41]. Stock
solutions of the test compounds were prepared at 10 mM, 2 mM and
0.4 mM by dissolving each sample in DMSO with sonication. Test
compounds were delivered to a 96 well plate in triplicate from 0 to
6.3.1. Cell culture
500
mM (final concentration) with a total DMSO volume of 10
mL in
6.3.1.1. Parasite survival assay. The samples were tested in triplicate
each well. Deionized H2O (70
m
L) and NP-40 (20 L; 30.6 M) were
m
m
on one occasion against
a
chloroquine-sensitive strain of
then added. A 25 mM haematin stock solution was prepared by
sonicating haemin in DMSO, for complete dissolution, and then sus-
P. falciparum (D10) and a chloroquine-resistant strain K1. Contin-
uous in vitro cultures of asexual erythrocyte stages of P. falciparum
were maintained using a modified method of Trager and Jensen
[37]. Quantitative assessment of antimalarial activity in vitro was
determined via the parasite lactate dehydrogenase assay using a
modified method described by Makler [38].
The test samples were prepared as 20 mg/ml stock solutions in
100% DMSO and sonicated to enhance solubility. Samples were tested
as a suspension if not completely dissolved. Stock solutions were
stored at ꢁ20 ꢀC. Further dilutions were prepared on the day of the
experiment. Briefly, samples were serially diluted with complete
medium to produce solutions of varying concentration across 96-well
pending 178
enous suspension (100
buffer and haematin concentrations of 1 M and 100
m
L of this in a 2 M acetate buffer (pH 4.8). The homog-
L) was then added to the wells to give final
M respectively.
m
m
The plate was covered and incubated at 37 ꢀC for 5e6 h in a water
bath. Analysis was carried out using the pyridineeferrichrome
method developed by Ncokazi and Egan [32]. A solution of 50% (v/v)
pyridine, 30% (v/v) H2O, 20% (v/v) acetoneand 0.2 M HEPES buffer (pH
7.4) was prepared and 32
mL added to each well to give a final pyridine
concentration of w5% (v/v). Acetone (60
mL) was then added to assist
with haematin dispersion. The UVevis absorbance of the plate wells
was read on a SpectaMax plate reader. Sigmoidal doseeresponse
curves were fitted tothe absorbance data using GraphPadPrismv3.02
to obtain a 50% inhibitory concentration (IC50) for each compound.
plates (100
2% haematocrit) were added to each well (test and control). Unpar-
asitized red blood cells (100 L) were added to blank wells. The plates
were incubated for 48 h at 37 ꢀC under 3% O2, 4% CO2 and 93% N2.
Thereafter, the contents of each well were resuspended and 15 L was
transferred to other plates containing Malstat reagent in each well
(100 L). Finally, 25 L of nitroblue tetrazolium (NBT)/phenazine
mL). Parasitized red blood cells (100 mL, 2% parasitaemia,
m
Acknowledgements
m
This work was supported by the South African Malaria Initiative
(SAMI), the National Research Foundation, the Medical Research
Council of South Africa and the University of Cape Town. Any
opinion, findings and conclusions or recommendations expressed
in this material are those of the authors and do not necessarily
reflect the views of the funding agencies involved.
m
m
ethosulfate (PES) was added to each well and the plates were allowed
to develop in the dark for 5e10 min and the absorbance of the blue
formazan product in each well was measured at 620 nm on a
microplate reader. Chloroquine (CQ) was used as a reference drug in
all experiments. A full doseeresponse was performed for all com-
pounds to determine the concentration inhibiting 50% of parasite
growth (IC50). Test samples were used at a starting concentration of
1000 ng/ml, which was then serially diluted 2-fold in complete me-
dium to give 10 concentrations, with the lowest concentration being
2 ng/ml. The same dilutiontechniquewas used forall samples. CQwas
tested at a starting concentration of 100 ng/ml. The highest concen-
tration of solvent to which the parasites were exposed had no
measurable effect on the parasite viability (data not shown). The IC50
values were obtained using a non-linear least-squares dosee
response curvefitting analysis via GraphPadPrismv.4.0software[39].
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.08.046.
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