Antiplasmodial activity of novel keto-enamine chalcone-chloroquine based hybrid pharmacophores

Article abstract of DOI:10.1016/j.bmc.2012.03.011

A series of novel keto-enamine chalcone-chloroquine based hybrids were synthesized following new methodology developed in our laboratory. The synthesized compounds were screened against chloroquine sensitive strain (3D7) of Plasmodium falciparum in an in

Full text of DOI:10.1016/j.bmc.2012.03.011

Bioorganic & Medicinal Chemistry 20 (2012) 2971–2981
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antiplasmodial activity of novel keto-enamine chalcone-chloroquine
based hybrid pharmacophores q
Koneni V. Sashidhara a, , Manoj Kumar a, Ram K. Modukuri a, Rajeev Kumar Srivastava b, Awakash Soni b,
⇑
Kumkum Srivastava b, Shiv Vardan Singh c, J.K. Saxena c, Harsh M. Gauniyal d, Sunil K. Puri b
a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Lucknow 226 001, India
b Parasitology Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Lucknow 226 001, India
c Biochemistry Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Lucknow 226 001, India
d SAIF Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel keto-enamine chalcone-chloroquine based hybrids were synthesized following new
methodology developed in our laboratory. The synthesized compounds were screened against chloro-
quine sensitive strain (3D7) of Plasmodium falciparum in an in vitro model. Some of the compounds were
showing comparable antimalarial activity at par with chloroquine. Compounds with significant in vitro
antimalarial activity were then evaluated for their in vivo efficacy in Swiss mice against Plasmodium yoelii
(chloroquine resistant N-67 strain), wherein compounds 25 and 27 each showed an in vivo suppression of
99.9% parasitaemia on day 4. Biochemical studies reveal that inhibition of hemozoin formation is the pri-
mary mechanism of action of these analogues.
Received 13 December 2011
Revised 1 March 2012
Accepted 2 March 2012
Available online 10 March 2012
Keywords:
Synthesis
Quinoline
Keto-enamine
Antimalarial agents
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
and consequences of malaria internationally.6 The biochemical
studies on CQ suggested that accumulation of the chemotherapeu-
The problem of widespread malaria continues unabated world-
wide, as the disease is present in over 100 countries and threatens
half of the world’s population. Even with decades of research and
the victorious progress of combination therapy, malaria remains
one of the most serious health problem worldwide, especially in
developing countries where it has huge economic and social costs.1
The resistance of Plasmodium falciparum to chloroquine and other
antimalarial drugs and the adverse effects of some of the available
antimalarial drugs have created an urgent need for new drugs that
are safe and effective for the prophylaxis and treatment of
malaria.2,3
Chloroquine (CQ) and other quinoline antimalarials have been
mainstays of antimalarial chemotherapy for more than past
40 years. The achievement of these drugs was based on excellent
clinical efficacy, limited host toxicity, ease of use, and simple, cost
effective synthesis.4 Figure 1 shows chemical structure of some
clinically useful quinoline based antimalarial agents. Although
chloroquine is most widely used clinical agent, but overcoming
drug resistance5 has helped fuel a strong increase in occurrence
tic agents in the parasite vacuole is critical for their antimalarial
activity.7 CQ resistance had largely been recognized to mutations
in the CQ resistance transporter (pfcrt) gene that encodes for the
protein believed to mediate efflux of the drug from the digestive
vacuole of the parasite, leading to sub-optimal drug
concentrations.8
To overcome the challenges of multi-drug resistance in P. falci-
parum, many approaches currently being adopted contain optimi-
sation of treatment with available drugs including combination
therapy, developing analogues of the existing drugs and evaluation
of drug resistance reversers (chemo sensitizers) as well as explor-
ing new chemotherapeutic targets.9 Among these approaches, the
medicinal chemistry hybridization strategy, involves the rational
design of new chemical entities by the fusion (usually via a cova-
lent linker) of two drugs, both active compounds and/or pharmaco-
phoric units recognized and derived from known bioactive
molecules. In the context of attempting to circumvent antimalarial
drug resistance, hybridization is quite an attractive strategy,
particularly when the pharmacophores being merged possess
independent modes of antimalarial action. Thus the hybrid mole-
cules are chemical entities with two or more structural domains
having different biological functions and dual activity.10 Despite
the worldwide distribution of resistance of Plasmodium falciparum
to chloroquine (CQ), the 4-aminoquinoline analogues continue to
q
Part XVII in the series, ‘Advances in drug design and discovery’. CSIR-CDRI
#8213.
⇑
Corresponding author. Tel.: +91 9919317940; fax: +91 522 2628493.
E-mail
addresses:
kv_sashidhara@cdri.res.in,
sashidhar123@gmail.com
(K.V. Sashidhara).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.011

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K. V. Sashidhara et al. / Bioorg. Med. Chem. 20 (2012) 2971–2981
Figure 1. Chemical structures of some clinically useful antimalarials.
attract interest because the resistance seems to be compound spe-
cific and not related to changes in the structure of the drug target.
Structure–activity relationship studies on CQ-Hematin binding re-
veals the importance of 7-chloro-4-aminoquinoline scaffold for
activity.11 Based on these facts several groups have reported hybrid
molecules by coupling 7-chloro-4-aminoquinolines (lateral side
chain modification) with different bioactive molecules like: perox-
ide based trioxaquine derivatives,12 aminoquinoline–imipramine
hybrid,13 ferrocene–chloroquine analogs,14 chloroquine-isatin
derivatives,15 chloroquine–astemizole hybrids,16 etc.
wish to report the synthesis and biological evaluation of novel
keto-enamine chalcone-chloroquine hybrid analogues.
2. Synthesis
The synthesis of target and intermediate compounds were per-
formed as outlined in Scheme 1. Synthesis of compounds 2–4 was
achieved by the nucleophilic substitution at the 4th position of 4,7-
dichloroquinoline (1) with aliphatic and aromatic amines. On the
other hand regio selective chalcones were synthesized by utilizing
our previously reported protocol.21 The Duff reaction on ortho alkyl
substituted phenols (5–7) in the presence of hexamethylenetetra-
amine (HMTA) and TFA at 120 °C gave aromatic dicarbaldehydes
(8–10), which on coupling with appropriate acetophenones in
refluxing dioxane, in the presence of a catalytic amount of concd
HCl afforded regio selective para-condensed chalcones in excellent
yields. Furthermore, these synthesized chalcones were condensed
with 4-aminoquinolines (2–4) in presence of ethanol as a solvent
to furnish final compounds (11–38) in which 4-aminoquinoline
and chalcones were attached by a keto-enamine linker. The syn-
thetic methodology has been perfected by us in which the reac-
tions were regio selective and the products exists in the keto-
enamine form.22
On the other hand antimalarial activity of chalcones was first
noted when licochalcone
A was reported to exhibit potent
antimalarial activity.17 Subsequently, several synthetic chalcone
derivatives were reported as alternatives in antimalarial chaemo-
therapy.18 Recently, Chibale et al. have reported chalcone-chloro-
quinoline hybrid analogues wherein, these hybrids have shown
significant antimalarial activity and biochemical studies reveal that
inhibition of hemozoin formation is the primary mechanism of ac-
tion of these analogues.19 Based on these observations, and also
considering that free heme within the food vacuole of the parasite
is still an attractive target, we wanted to design and develop a un-
ique class of antimalarials that interact with free heme on one
hand and other part of the molecule inhibit cysteine protease, also
this pharmacophore because of extended conjugation is also envis-
aged to reverse chloroquine resistance (act as chemosensitizer) in
Plasmodium falciparum. We envisaged that this strategy would re-
sult in compounds that could overcome Plasmodium falciparum
resistance to chloroquine (CQ) and offer a viable strategy for the
discovery of new antimalarial therapies.20
3. Results and discussion
All the synthesized hybrid compounds having chalcone and
quinoline scaffolds in one frame (11–38) were evaluated for their
antimalarial activity against CQ sensitive 3D7 strain of P. falcipa-
rum in vitro according to the procedure reported in the literature.
The IC50 values are calculated from experiments carried out in trip-
licate. Some of the selected compounds, which have shown activity
comparable to CQ, were also evaluated against the N-67 strain of P.
yoelli in Swiss mice.
Thus, we were interested in pursuing a bio inspired hybridiza-
tion strategy that combines the core portions of the two structur-
ally distinct moieties of CQ and ketoenamine chalcone, each of
which possesses significant antiplasmodial activities, via an appro-
priate linker. It is also possible that hybrid molecules will possess
superior bioavailability and/or different mode of action from that
of individual drugs in combination. The structure of the designed
novel prototype is shown in Figure 2. In this communication, we
Among the 28 compounds tested, compounds 12 and 27 exhib-
ited antiplasmodial activity comparable to chloroquine with IC50
3.63 and 4.64 ng/mL, respectively. Additionally, 5 compounds (11,
18, 23, 24 and 25) showed IC50 lower than 10 ng/mL. The difference
in the IC50 values can be attributed to factors such as number of car-
bon atoms in the side chain, ring size, and the substitutions on the
chalcone ring. The significant activity of the above mentioned com-
pounds underscores the importance of the two carbon linker be-
tween the two pharmacophores, as the compounds having
aromatic linker between quinoline and chalcones (34–38) showed
drastic decrease in activity. While on the other hand the com-
pounds with propyl linker (28–33) exhibited only mild inhibition
in comparison to standard drug. The activity data (Table 1) suggest
that two-carbon atoms in the side chain linker are appropriate for
the antimalarial activity of compounds with five or six-membered
chalcone rings (like 24 and 12), as an increase in carbon chain
length results in reduced activity. To elucidate the role of keto-en-
amine based chalcones in antimalarial activity we have synthesized
compounds (1b–1d) without quinoline pharmacophore, which is
outlined in Scheme 2. However, the synthesized compounds
showed moderate in vitro activity (IC50 >100 ng/ml) which
Figure 2. Designing of keto-enamine chalcone-chloroquine hybrids.

K. V. Sashidhara et al. / Bioorg. Med. Chem. 20 (2012) 2971–2981
2973
Scheme 1. Synthesis of keto-enamine chalcone-chloroquine hybrids. Reagents and conditions: (i) diamines, 110 °C, neat or ethanol; (ii) hexamethylenetramine/TFA, 120 °C,
3 h; (iii) 10% H2SO4, 90–100 °C, 2 h; (iv) concd HCl, p-R1COCH3, dioxane, 80–90 °C, 1.0–1.5 h; (v) 2/3, ethanol, rt, 10 min; (vi) 4, ethanol, rt, 10 min.
underscores the role of the quinoline pharmacophore for antimalar-
ial activity. The idea of introducing an aryl linker was not without
reason, it was envisaged that since these molecules are fluorescent
(due to extended conjugation) they would enhance the antimalarial
activity due to their conjugation because many dyes with extended
conjugation like xanthenes, azines, oxazines, thiazines, etc. are re-
ported in literature for their antimalarial potential.23 However, on
evaluation these compounds (34–38) they were found to be less ac-
tive than compounds with aliphatic linker. The reason for the high-
er IC50 values for these compounds is probably due to less
accumulation in the parasite food vacuole by pH trappings for high-
er pKa values. Furthermore, in vivo results strongly suggests that
halogen atom at para position of the chalcone aryl ring improves
the in vivo antimalarial activity as compound 25 with chloro and
compound 27 with bromo group showed 99.9% parasite inhibition
on day four while compound 14 with fluoro group and 16 with bro-
mo group illustrated 99.5% and 99.78% inhibition. Introduction of
heteroaryl group like furan and thiophene (compound 18, 23 and
24) also increased the in vivo activity.
Furthermore, all compounds were tested for their cytotoxicity
against VERO cell line and they were devoid of cytotoxicity as these
compounds have high SI values, particularly compounds 12 with a
SI value of 27548.21 which was around threefold more as com-
pared to chloroquine while rest of compounds showed SI values
ranging between 300 and 3755.
Ten compounds with noteworthy activity in vitro were evalu-
ated for in vivo efficacy in Swiss mice against Plasmodium yoelii
(chloroquine resistant N-67 strain). Under these conditions, the
survival time of untreated animals (control) was 12 days. Treated
animals were orally dosed daily for 4 days (do to d3 post infection)
at a dose of 100 mg/kg/d. On day 4, parasitaemia was below detect-
able levels. This result indicates a rapid parasite clearance. How-
ever, the number of cured mice at 30 days was 0/5. The mean
survival time of these animals was around 18 days, with recrudes-
cence between 17 and 21 days. The synthesized compounds
showed significant activity against P. yoelii infections in mice, par-
ticularly the compounds 25 and 27 exhibited 99.9% suppression on
day four compared to 99% suppression displayed by CQ (at 20 mg/
kg/d dose), while compounds 11, 17, and 18 suppressed 99.78%
parasitaemia (Table 3). Additionally compound 25 and 27 showed
97.0% and 96.7% suppression on day 6. These data show that the
synthesized derivatives are highly active and able to clear parasite-
mia below detectable levels in mice orally at 100 mg/kg. However,
a curative effect (no recrudescence, cured mice) was not obtained
at the tested dose.
As the quinoline based antimalarials like chloroquine act
through the inhibition of hemozoin formation, therefore inhibition
of b-hematin formation was demonstrated in target hybrids. Most
of the synthesized compounds showed b-hematin inhibitory activ-
ities (IC50 <3.5
lg/ml) better than the reference drug chloroquine
(IC50 3.65 g/ml) (Table 2). Although most of the compounds illus-
l
trated good hemozoin inhibitory activities yet there was no steady
correlation in comparison to their in vitro antimalarial potency.
This difference may occur due to factors like degree of accumula-
tion in parasite food vacuole which is structure and pH dependent.
In spite of a major modification in the side chain, this series of

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K. V. Sashidhara et al. / Bioorg. Med. Chem. 20 (2012) 2971–2981
Table 1
In vitro antimalarial activity against chloroquine sensitive strain 3D7 of P. falciparum, in vitro cytotoxicity of compounds on VERO cell line and LogP values
Compound
(Linker)
R
R1
IC50 (ng/ml)
SI
LogP
CH3
OH
OMe
F
11
1,2-Ethylenediamine
Methyl
9.75
1034.23
4.15
12
13
14
15
16
1,2-Ethylenediamine
1,2-Ethylenediamine
1,2-Ethylenediamine
1,2-Ethylenediamine
1,2-Ethylenediamine
Methyl
Methyl
Methyl
Methyl
Methyl
3.63
27548.21
1137.97
1034.23
3755.43
1114.59
3.28
3.54
3.82
4.22
4.49
16.67
10.85
22.21
18.85
Cl
Br
OMe
OMe
OMe
17
1,2-Ethylenediamine
Methyl
10.68
1392.22
3.29
S
18
19
20
1,2-Ethylenediamine
1,2-Ethylenediamine
1,2-Ethylenediamine
Methyl
9.75
1414.59
498.54
3.65
4.83
4.44
sec Butyl
sec Butyl
13.63
22.03
OH
OMe
Cl
1071.27
21
1,2-Ethylenediamine
sec Butyl
18.26
1058.05
4.70
22
23
24
25
1,2-Ethylenediamine
1,2-Ethylenediamine
1,2-Ethylenediamine
1,2-Ethylenediamine
sec Butyl
sec butyl
sec Butyl
tert Butyl
17.91
7.09
9.1
1014.51
1138.22
1373.63
1499.3
5.39
3.45
4.81
5.04
O
S
F
7.19
Cl
26
27
28
29
30
31
32
33
34
1,2-Ethylenediamine
1,2-Ethylenediamine
1,3-Propanediamine
1,3-Propanediamine
1,3-Propanediamine
1,3-Propanediamine
1,3-Propanediamine
1,3-Propanediamine
1,4-Diaminobenzene
tert Butyl
tert Butyl
Methyl
28.17
4.64
868.3
5.44
5.71
3.38
3.68
4.33
4.55
4.81
5.49
5.38
Br
1883.62
505.98
295.66
513.99
486.57
549.12
491.77
315.33
OH
OMe
Cl
35.12
38.76
42.86
42.44
41.84
41.93
200.55
Methyl
Methyl
OH
OMe
Cl
sec Butyl
sec Butyl
sec Butyl
Methyl
OMe

K. V. Sashidhara et al. / Bioorg. Med. Chem. 20 (2012) 2971–2981
2975
Table 1 (continued)
Compound
(Linker)
R
R1
IC50 (ng/ml)
160.3
SI
LogP
F
35
36
37
1,4-Diaminobenzene
Methyl
407.52
5.67
4.12
7.28
7.55
O
1,4-Diaminobenzene
1,4-Diaminobenzene
1,4-Diaminobenzene
sec Butyl
tert Butyl
tert Butyl
80.01
678.11
196.70
ND
Cl
Br
379.82
38
>100
CQ
2.45 1.08
IC50: Concentration corresponding to 50% growth inhibition of the parasite.
SI: IC50 values of cytotoxic activity/IC50 values of antimalarial activity.
LogP: Calculated by ChemDraw Ultra software.
ND: Not done.
Scheme 2. Synthesis of keto-enamine based chalcones.
compounds exhibits antimalarial activity by the same mode of
action as that of CQ, namely, inhibition of hemozoin formation.
5.2. General procedure for the synthesis of compounds 8–10
and chalcones
Synthesis of dicarbaldehyde substrates and regioselective para
condensed chalcones were achieved by our previously reported
protocol.21
4. Conclusion
In conclusion, we have synthesized keto-enamine based quino-
line-chalcone hybrids using an uncommon method established in
our laboratory. Interestingly, this class of compounds exhibits
acceptable selectivity against the malaria parasite and shows anti-
malarial activity in vivo against the MDR rodent malaria parasite P.
yoelii. This study has identified new class of compounds showing
good antimalarial activity in both in vitro and in vivo models.
Furthermore, mechanistic studies reveal that said compound act
through heme polymerization target. Further structural optimiza-
tion of these analogues may lead to the development of the more
potent molecules.
5.3. General procedure for the synthesis of compounds 11–38
A mixture of 2–4 (1.0 equiv) and appropriate para condensed
chalcones (1 equiv) in ethanol were stirred for 10 min at room
temperature. The solvent was evaporated under vacuum and the
solid was purified directly with column chromatography to obtain
the respective compounds 11–38 in excellent yields.
5.3.1. (E)-6-((2-(7-Chloroquinolin-4-ylamino) ethylamino) meth
ylene)-2-methyl-4-((E)-3-oxo-3-p-tolylprop-1-enyl)cyclohexa-
2,4-dienone (11)
5. Experimental section
Yellow solid, yield: 87%; mp 235–236 °C; IR (KBr): 3213, 2841,
1711, 1625, 1599, 1015 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
5.1. General procedure for the synthesis of compounds 2, 3 and
4
14.32 (s, 1H), 8.51 (s, 1H), 8.42 (d, J = 5.4 Hz, 1H), 8.25 (d,
J = 9.1 Hz, 1H), 8.02 (d, J = 8.1 Hz, 2H), 7.85 (s, 1H), 7.79 (d,
J = 2.0 Hz, 1H), 7.73–7.59 (m, 3H), 7.53–7.44 (m, 2H), 7.35 (d,
J = 8.1 Hz, 2H), 6.63 (d, J = 5.4 Hz, 1H), 3.92 (t, J = 5.3 Hz, 2H), 3.66
(d, J = 5.5 Hz, 2H), 2.39 (s, 3H), 2.19 (s, 3H); 13C NMR (DMSO-d6,
75 MHz) d: 188.7, 167.2, 166.3, 152.4, 150.4, 149.5, 144.2, 143.7,
135.9, 134.0, 133.2, 129.7, 128.9, 128.0, 124.7, 124.5, 123.8,
118.7, 117.9, 117.1, 99.5, 54.9, 43.3, 21.6, 15.9; ESI-MS: (m/z):
484 (M+H)+; HRMS m/z calcd for C29H26ClN3O2 (M+H)+ 484.1793,
found 484.1796.
A mixture of 4,7-dichloroquinoline (1 equiv) and 1,2-diaminoe-
thane/1,3-diaminopropane/p-phenylenediamine (1.5–2 equiv) in
ethanol/neat were heated for 8 h with continued stirring to drive
the reaction to completion. After completion of the reaction the ex-
cess solvent was removed under vacuum. The reaction mixture
was then poured into ice cold water and precipitate was filtered
and washed with ethyl acetate to furnish compound 2, 3 and 4 in
quantitative yields.

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K. V. Sashidhara et al. / Bioorg. Med. Chem. 20 (2012) 2971–2981
Table 2
5.3.3. (E)-6-((2-(7-Chloroquinolin-4-ylamino)ethylamino)
methylene)-4-((E)-3-(4-methoxyphenyl)-3-oxoprop-1-enyl)-2-
methylcyclohexa-2,4-dienone (13)
Inhibition of hemozoin formation
Compounds
Inhibition of b-hematin formation IC50 (lg/mL)
Yellow solid, yield: 91%; mp 238–239 °C; IR (KBr): 3199, 2851,
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
CQ
2.91
2.89
3.01
2.91
2.99
7.6
9.61
3.10
3.15
2.71
12.6
11.2
2.89
2.62
13.8
8.06
21.9
3.48
5.34
2.9
1723, 1621, 1588, 1010 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.32 (s, 1H), 8.51 (s, 1H), 8.41 (d, J = 5.4 Hz, 1H), 8.25 (d,
J = 9.0 Hz, 1H), 8.13 (d, J = 8.8 Hz, 2H), 7.87 (s, 1H), 7.79 (d,
J = 2.1 Hz, 1H), 7.75–7.69 (m, 1H), 7.66 (d, J = 1.8 Hz, 1H), 7.63 (s,
1H), 7.57–7.54 (m, 1H), 7.45 (dd, J = 2.1 Hz and J = 8.9 Hz, 1H),
7.06 (d, J = 8.8 Hz, 2H), 6.63 (d, J = 5.5 Hz, 1H), 3.92 (t, J = 5.5 Hz,
3H), 3.66 (d, J = 5.6 Hz, 2H), 2.19 (s, 3H); 13C NMR (DMSO-d6,
75 MHz) 187.0, 166.5, 164.7, 162.8, 151.6, 149.9, 148.9, 142.9,
133.3, 132.6, 132.0, 130.8, 130.4, 127.3, 127.0, 124.0, 123.8,
118.4, 117.4, 116.7, 113.8, 98.9, 55.4, 54.7, 15.1; ESI-MS: (m/z):
500 (M+H)+; HRMS m/z calcd for C29H26ClN3O3 (M+H)+ 500.1742,
found 500.1776.
5.3.4. (E)-6-((2-(7-Chloroquinolin-4-ylamino)ethylamino)
methylene)-4-((E)-3-(4-fluorophenyl)-3-oxoprop-1-enyl)-2-
methylcyclohexa-2,4-dienone (14)
2.93
6.59
6.08
4.35
3.78
3.21
7.88
Yellow solid, yield: 89%; mp 235–236 °C; IR (KBr): 3212, 2855,
1724, 1633, 1598, 1014 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.32 (s, 1H), 8.5 (s, 1H), 8.43 (d, J = 5.4 Hz, 1H), 8.28–8.18 (m,
3H), 7.87 (s, 1H), 7.79 (d, J = 2.2 Hz, 1H), 7.73–7.61 (m, 4H), 7.47
(dd, J = 2.1 Hz and J = 6.9 Hz, 1H), 7.37 (t, J = 8.8 Hz, 2H), 6.65 (d,
J = 5.5 Hz, 1H), 3.93 (t, J = 5.3 Hz, 2H), 3.68–3.66 (m, 2H), 2.19 (s,
3H); 13C NMR (DMSO-d6, 75 MHz) d: 187.7, 167.2, 166.8, 152.0,
150.7, 149.0, 144.8, 135.1, 134.2, 133.6, 133.2, 131.8, 131.6,
128.1, 127.6, 124.8, 124.5, 123.6, 118.3, 117.9, 117.0, 116.3,
116.0, 99.5, 54.8, 43.3, 15.9; ESI-MS: (m/z): 488 (M+H)+; HRMS
m/z calcd for C28H23ClFN3O2 (M+H)+ 488.1542, found 488.1534.
13.9
3.65
Data are the mean of three different experiments in triplicate.
The IC50 represents the concentration of compound that inhibit b-
hematin formation by 50%.
Table 3
5.3.5. (E)-4-((E)-3-(4-Chlorophenyl)-3-oxoprop-1-enyl)-6-
((2-(7-chloroquinolin-4-ylamino)ethylamino)methylene)-2-
methylcyclohexa-2,4-dienone (15)
In vivo antimalarial activity against chloroquine resistant of P. yoelii strain N-67 in
Swiss mice at 100 mg/kg/day dose by oral route
Compounds
% Suppression
Mean survival time (days)
Yellow solid, yield: 88%; mp 230–231 °C; IR (KBr): 3211, 2839,
Day 4
Day 6
1722, 1634, 1583, 1018 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
11
14
16
17
18
19
23
24
99.78
99.5
99.70
99.78
99.78
99.6
99.5
99.4
99.9
99.9
88.74
83.5
80.60
82.06
82.31
83.2
85.8
86.7
97.0
96.7
17.6 0.98
15.6 2.30
18.0 1.97
17.0 1.91
16.2 2.03
14.6 2.19
17.6 1.78
18.2 1.32
18.4 1.94
17.6 0.98
12.4 0.68
>28
14.33 (s, 1H), 8.49 (s, 1H), 8.42 (d, J = 3 Hz, 1H), 8.32 (s, 1H), 8.24
(d, J = 4.5 Hz, 1H), 8.13 (d, J = 4.5 Hz, 2H), 7.87 (s, 1H), 7.79 (d,
J = 1.05 Hz, 1H), 7.68–7.60 (m, 5H), 7.46 (dd, J = 1.5 Hz and
J = 4.5 Hz, 1H), 6.63 (d, J = 1.5 Hz, 1H), 3.92 (t, J = 3 Hz, 2H), 3.66
(d, J = 3 Hz, 2H), 2.18 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) d:
188.0, 167.1, 152.2, 150.4, 149.4, 145.1, 138.2, 137.1, 134.0,
133.8, 133.2, 130.7, 129.7, 128.3, 124.8, 124.5, 123.5, 118.1,
117.9, 116.8, 99.5, 19.6, 54.6, 43.2, 15.9; ESI-MS: (m/z): 504
(M+H)+; HRMS m/z calcd for C28H23Cl2N3O2 (M+H)+ 504.1246,
found 504.1234.
25
27
Control
CQ (20 mg/kg)
(10 mg/kg)
99.0
99.0
93.9
93.6
13.6 1.72
5.3.6. (E)-4-((E)-3-(4-Bromophenyl)-3-oxoprop-1-enyl)-6-
((2-(7-chloroquinolin-4-ylamino)ethylamino)methylene)-2-
methylcyclohexa-2,4-dienone (16)
5.3.2. (E)-6-((2-(7-Chloroquinolin-4-ylamino)ethylamino)
methylene)-4-((E)-3-(4-hydroxyphenyl)-3-oxoprop-1-enyl)-2-
methylcyclohexa-2,4-dienone (12)
Yellow solid, yield: 92%; mp 245–246 °C; IR (KBr): 3201, 2840,
1715, 1621, 1597, 1013 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
Yellow solid, yield: 90%; mp 265–266 °C; IR (KBr): 3207, 2854,
1711, 1634, 1589, 1028 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.32 (s, 1H), 8.49 (s, 1H), 8.43 (d, J = 4.5 Hz 1H), 8.26 (d,
J = 9.1 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.86 (s, 1H), 7.80–7.74 (m,
3H), 7.66–7.58 (m, 4H), 7.47 (dd, J = 2.1 Hz and 8.9 Hz, 1H), 6.65
(d, J = 5.5 Hz, 1H), 3.93 (t, J = 5.3 Hz 2H), 3.67 (d, J = 5.4 Hz, 2H),
2.18 (s, 3H); 13C (DMSO-d6, 100 MHz) d: 188.3, 167.2, 167.0,
152.0, 150.6, 149.1, 145.2, 137.5, 134.1, 133.7, 133.3, 132.2,
130.8, 128.3, 127.7, 127.4, 124.8, 124.5, 123.5, 118.2, 117.9,
116.9, 99.5, 54.7, 15.9; ESI-MS: (m/z): 548 (M+H)+; HRMS m/z calcd
for C28H23BrClN3O2 (M+H)+ 548.0741, found 548.0745.
14.27 (s, 1H), 10.37–10.29 (m, 1H), 8.51 (s, 1H), 8.42 (d, J = 6 Hz,
1H), 8.30–8.25 (m, 1H), 8.03 (d, J = 9.0 Hz, 2H), 7.81 (d, J = 6.0 Hz,
2H), 7.64 (m, 4H), 7.46 (d, J = 9.0 Hz, 1H), 6.88 (d, J = 4.5 Hz, 2H),
6.64 (d, J = 3 Hz, 1H), 3.92 (s, 2H), 3.39 (s, 2H), 2.19 (s, 3H); 13C
(DMSO-d6, 75 MHz) d: 187.7, 167.6, 165.8, 162.8, 152.3, 151.1,
149.4, 143.6, 134.6, 133.5, 133.1, 131.7, 130.3, 128.1, 128, 125.3,
124.9, 124.6, 119.3, 118.3, 117.6, 116.1, 99.9, 80.0, 55.6, 43.8,
16.3; ESI-MS: (m/z): 486 (M+H)+; HRMS m/z calcd for C28H24ClN3O3
(M+H)+ 486.1585, found 486.1591.

K. V. Sashidhara et al. / Bioorg. Med. Chem. 20 (2012) 2971–2981
2977
5.3.7. (E)-6-((2-(7-Chloroquinolin-4-ylamino)ethylamino)
methylene)-2-methyl-4-((E)-3-oxo-3-(3,4,5-trimethoxyphenyl)
prop-1-enyl)cyclohexa-2,4-dienone (17)
5.3.11. (E)-2-sec-Butyl-6-((2-(7-chloroquinolin-4-ylamino)
ethylamino)methylene)-4-((E)-3-(4-methoxyphenyl)-3-
oxoprop-1-enyl)cyclohexa-2,4-dienone (21)
Yellow solid, yield: 87%; mp 241–242 °C; IR (KBr): 3211, 2856,
Yellow solid, yield: 85%; mp 210–212 °C; IR (KBr): 3201, 2839,
1724, 1625, 1596, 1013 cmÀ1
;
1H NMR (DMSO-d6, 400 MHz) d:
1722, 1623, 1579, 1010 cmÀ1 1H NMR (DMSO-d6, 300 MHz)
;
14.32 (s, 1H), 8.49 (s, 1H), 8.40 (d, J = 5.2 Hz 1H), 8.23 (d,
J = 8.8 Hz, 1H), 7.83 (s, 1H), 7.78 (d, J = 2 Hz, 1H), 7.69–7.61 (m,
3H), 7.49 (t, J = 5.2 Hz, 1H), 7.43 (dd, J = 2 Hz and J = 8.8 Hz, 1H),
7.37 (s, 2H), 6.61 (d, J = 5.1 Hz, 1H), 3.93–3.88 (m, 9H), 3.75 (s,
2H), 3.65 (t, J = 5.2, 2H), 2.18 (s, 3H); 13C (DMSO-d6, 100 MHz) d:
188.0, 167.1, 166.3, 153.3, 152.3, 150.4, 149.5, 144.4, 142.2,
133.95, 133.9, 133.3, 128.0, 124.7, 124.7, 123.8, 118.5, 117.9
117.0, 113.9, 112.4, 106.5, 99.5, 60.7, 56.7, 54.9, 43.3, 15.9; ESI-
MS: (m/z): 560 (M+H)+; HRMS m/z calcd for C31H30ClN3O5 (M+H)+
560.1953, found 560.1943.
d:14.31 (s, 1H), 8.51 (s, 1H), 8.42 (d, J = 5.4 Hz, 1H), 8.24 (d,
J = 9.1 Hz, 1H), 8.13 (d, J = 8.9 Hz, 2H), 7.80–7.77 (m, 2H), 7.71–
7.67 (m, 3H), 7.55 (t, J = 5.3 Hz, 1H), 7.46 (dd, J = 2.2 Hz and
J = 6.75 Hz, 1H), 7.06 (d, J = 9.1 Hz, 2H), 6.64 (d, J = 5.4 Hz, 1H),
3.94–3.90 (m, 2H), 3.85 (s, 3H), 3.69–3.65 (m, 2H), 3.09 (q,
J = 7.1 Hz, 1H), 1.16–1.59 (m, 2H), 1.21 (d, J = 6.9 Hz, 3H), 0.81 (t,
J = 7.3 Hz, 3H); 13C (DMSO-d6, 300 MHz) d: 187.6, 167.4, 165.0,
163.4, 152.3, 150.4, 149.5, 144.0, 136.9, 133.9, 132.7, 131.2,
130.3, 128.0, 124.7, 124.5, 124.2, 118.7, 117.9, 117.5, 114.4, 99.5,
56.0, 55.3, 43.3, 33.5, 29.3, 20.6, 12.7; ESI-MS: (m/z): 542 (M+H)+;
HRMS m/z calcd for
C
32H32ClN3O3 (M+H)+ 542.2211, found
542.2215
5.3.8. (E)-6-((2-(7-Chloroquinolin-4-ylamino)ethylamino)
methylene)-2-methyl-4-((E)-3-oxo-3-(thiophen-2-yl)prop-1-
enyl)cyclohexa-2,4-dienone (18)
5.3.12. (E)-2-sec-Butyl-4-((E)-3-(4-chlorophenyl)-3-oxoprop-1-
enyl)-6-((2-(7-chloroquinolin-4-ylamino)ethylamino)
methylene)cyclohexa-2,4-dienone (22)
Yellow solid, yield: 87%; mp 222–223 °C; IR (KBr): 3201, 2839,
1727, 1635, 1596, 1002 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
Yellow solid, yield: 87%; mp 220–221 °C; IR (KBr): 3198, 2841,
1707, 1622, 1596, 1008 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.32 (s, 1H), 8.50 (s, 1H), 8.43 (d, J = 5.5 Hz, 1H), 8.29–8.23 (m,
2H), 8.01 (d, J = 4.6 Hz, 1H), 7.85 (s, 1H), 7.80 (d, J = 2.1 Hz, 1H),
7.69–7.62 (m, 4H), 7.48 (dd, J = 2 Hz and 6.9 Hz 1H), 7.30 (t,
J = 4.5 Hz, 1H), 6.66 (d, J = 4.5 Hz, 1H), 3.93 (t, J = 5.2 Hz, 2H), 3.68
(d, J = 5.4 Hz, 2H), 2.19 (s, 3H); 13C NMR (DMSO-d6, 300 MHz) d:
181.8, 167.2, 166.6, 151.8, 150.8, 148.8, 146.4, 143.7, 135.4,
134.3, 133.5, 133.3, 133.2, 129.2, 128.1, 127.4, 124.9, 124.6,
123.5, 118.5, 117.8, 117.0, 99.5, 54.8, 43.3, 15.9; ESI-MS: (m/z):
476 (M+H)+.
14.43 (s, 1H), (s, 1H), 8.49 (s, 1H), 8.40 (d, J = 6.0 Hz, 1H), 8.28 (d,
J = 9.1 Hz, 1H), 8.16–8.13 (m, 2H), 7.8–7.79 (m, 2H), 7.69–7.68
(m, 3H), 7.63–7.60 (m, 2H), 7.54 (t, J = 5.3 Hz, 1H), 7.45 (dd,
J = 2.2 Hz and J = 9.1 Hz, 1H), 6.63 (d, J = 5.4 Hz, 1H), 3.92 (t,
J = 5.2 Hz, 2H), 3.67 (t, J = 5.19 Hz, 2H), 3.07 (q, J = 6.9 Hz, 1H),
1.69–1.57 (m, 2H), 1.2 (d, J = 6.7 Hz, 3H), 0.81 (d, J = 7.3 Hz, 3H);
13C (DMSO-d6, 300 MHz) d: 188.6, 167.8, 166.5, 152.7, 150.9,
149.9, 145.9, 138.6, 137.7, 137.5, 134.4, 133.8, 131.1, 130.8,
129.6, 128.3, 125.1, 124.9, 124.1, 118.6, 118.3, 117.7, 99.9, 55.3,
43.6, 33.9, 29.7, 20.9, 132.0; ESI-MS: (m/z): 546 (M+H)+; HRMS
m/z calcd for C31H29Cl2N3O2 (M+H)+ 546.1716, found 546.1742.
5.3.9. (E)-2-sec-Butyl-6-((2-(7-chloroquinolin-4-ylamino)
ethylamino)methylene)-4-((E)-3-oxo-3-phenylprop-1-
enyl)cyclohexa-2,4-dienone (19)
5.3.13. (E)-2-sec-Butyl-6-((2-(7-chloroquinolin-4-ylamino)
ethylamino)methylene)-4-((E)-3-(furan-2-yl)-3-oxoprop-1-
enyl)cyclohexa-2,4-dienone (23)
Yellow solid, yield: 90%; mp 215–216 °C; IR (KBr): 3210, 2853,
1714, 1622, 1588, 1004 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.42 (s, 1H), 8.51 (s, 1H), 8.41 (d, J = 4.9 Hz, 1H), 8.26 (d,
J = 7.1 Hz, 1H), 8.12 (d, J = 8.6 Hz, 2H), 7.80–7.77 (m, 2H), 7.71–
7.70 (m, 3H), 7.64–7.62 (m, 1H), 7.58–7.53 (m, 3H), 7.45 (dd,
J = 2.1 Hz and 6.8 Hz, 1H), 6.63 (d, J = 5.5 Hz, 1H), 3.92 (t,
J = 5.3 Hz, 2H), 3.68 (t, J = 5.3 Hz 2H), 3.1–3.07 (m, 1H), 1.72–1.56
(m, 2H), 1.21 (d, J = 6.9 Hz, 3H), 0.811 (t, J = 7.3 Hz, 3H); 13C NMR
(DMSO-d6, 300 MHz) d: 189.3, 167.4, 165.5, 152.3, 150.5, 149.5,
144.9, 138.5, 137.0, 134.0, 133.2, 133.0, 130.4, 129.1, 128.8,
127.9, 124.7, 124.5, 124.0, 118.7, 118.0, 117.5, 99.5, 55.2, 43.3,
33.5, 29.3, 20.5, 12.7; ESI-MS: (m/z): 512 (M+H)+; HRMS m/z calcd
for C31H30ClN3O2 (M+H)+ 512.2106, found 512.2133.
Yellow solid, yield: 87%; mp 220–221 °C; IR (KBr): 3201, 2836,
1710, 1618, 1595, 1004 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.42 (s, 1H), 8.52 (s, 1H), 8.41 (d, J = 5.4 Hz, 1H), 8.26 (d,
J = 8.9 Hz, 1H), 8.03–8.02 (m, 1H), 7.7 (d, J = 2.1 Hz, 1H), 7.72–
7.64 (m, 4H), 7.53–7.43 (m, 3H), 6.78–6.76 (m, 1H), 6.63 (d,
J = 5.4 Hz, 1H), 3.92 (t, J = 5.4 Hz, 2H), 3.67 (t, J = 5.4 Hz, 2H),
3.09–3.06 (m, 1H), 1.67–1.57 (m, 2H), 1.20 (d, J = 6.1 Hz, 3H),
0.80 (t, J = 7.2 Hz, 3H); 13C (DMSO-d6, 300 MHz) d: 177.1, 167.4,
165.8, 153.6, 152.3, 150.4, 149.5, 148.4, 143.6, 137.1, 133.9,
133.0, 130.3, 128.0, 124.7, 124.5, 123.6, 119.1, 118.4, 117.9,
117.4, 113.0, 99.5, 54.9, 43.2, 33.5, 29.2, 20.5, 12.7; ESI-MS: (m/
z): 502 (M+H)+; HRMS m/z calcd for C29H28ClN3O3 (M+H)+
502.1898, found 502.1904.
5.3.10. (E)-2-sec-Butyl-6-((2-(7-chloroquinolin-4-ylamino)
ethylamino)methylene)-4-((E)-3-(4-hydroxyphenyl)-3-
oxoprop-1-enyl)cyclohexa-2,4-dienone (20)
5.3.14. (E)-2-sec-Butyl-6-((2-(7-chloroquinolin-4-ylamino)
ethylamino)methylene)-4-((E)-3-oxo-3-(thiophen-2-yl)prop-
1-enyl)cyclohexa-2,4-dienone (24)
Yellow solid, yield: 91%; mp 255–256 °C; IR (KBr): 3212, 2844,
1711, 1619, 1582, 1021 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.38 (s, 1H), 10.37 (s, 1H), 8.51 (s, 1H), 8.41 (d, J = 5.4 Hz, 1H),
8.31–8.25 (m, 1H), 8.04 (d, J = 8.7 Hz, 2H), 7.80–7.45 (m, 6H),
7.46 (dd, J = 2.1 Hz and J = 11.2 Hz, 1H), 6.89 (d, J = 8.7 Hz, 2H),
6.65 (d, J = 6.0 Hz, 1H), 3.92–3.91 (m, 2H), 3.67 (d, J = 6.1 Hz, 2H),
3.09–3.06 (m, 1H), 1.69–1.56 (m, 2H), 1.20 (d, J = 6.9 Hz, 3H),
0.80 (t, J = 7.3 Hz, 3H); 13C (DMSO-d6, 300 MHz) d: 187.8, 167.9,
164.9, 162.8, 152.0, 151.3, 149.0, 143.9, 137.1, 134.7, 132.8,
131.8, 130.7, 130.3, 127.7, 125.3, 125.0, 124.9, 119.3, 118.2,
118.0, 116.1, 99.9, 55.8, 43.8, 33.9, 29.7, 20.9, 13.0; ESI-MS: (m/
z): 528 (M+H)+; HRMS m/z calcd for C31H30ClN3O3 (M+H)+
528.2055, found 528.2068.
Yellow solid, yield: 91%; mp 226–227 °C; IR (KBr): 3202, 2842,
1714, 1618, 1585, 1014 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.43 (s, 1H), 8.50 (s, 1H), 8.41 (d, J = 5.4 Hz, 1H), 8.28–8.24 (m,
2H), 8.0 (d, J = 4.9 Hz, 1H), 7.80–7.77 (m, 2H), 7.68–7.65 (m, 3H),
7.52 (t, J = 5.4 Hz, 1H), 7.45 (dd, J = 2.1 Hz and J = 8.9 Hz, 1H),
7.29 (t, J = 4.7 Hz, 1H), 6.63 (d, J = 5.4 Hz, 1H), 3.92 (t, J = 5.6 Hz,
2H), 3.67 (t, J = 5.3 Hz, 2H), 3.08 (q, J = 7.1 Hz, 1H), 1.70–1.58 (m,
2H), 1.21 (d, J = 6.9 Hz, 3H), 0.81 (t, J = 7.3 Hz, 3H); 13C NMR
(DMSO-d6, 300 MHz) d: 181.9, 167.4, 165.7, 152.4, 150.4, 149.6,
146.4, 144.0, 137.1, 135.3, 133.9, 133.4, 133.1, 130.4, 129.2,
128.0, 124.7, 124.5, 123.8, 118.5, 118.0, 117.4, 99.5, 55.1, 43.3,

2978
K. V. Sashidhara et al. / Bioorg. Med. Chem. 20 (2012) 2971–2981
33.5, 29.3, 20.5, 12.7; ESI-MS: (m/z): 518 (M+H)+; HRMS m/z calcd
5.3.19. (E)-6-((3-(7-Chloroquinolin-4-ylamino)propylamino)
methylene)-4-((E)-3-(4-methoxyphenyl)-3-oxoprop-1-enyl)-2-
methylcyclohexa-2,4-dienone (29)
for C29H28ClN3O2S (M+H)+ 518.1670, found 518.1658.
5.3.15. (E)-2-tert-Butyl-6-((2-(7-chloroquinolin-4-ylamino)
ethylamino)methylene)-4-((E)-3-(4-fluorophenyl)-3-oxoprop-
1-enyl)cyclohexa-2,4-dienone (25)
Yellow solid, yield: 92%; mp 198–199 °C; IR (KBr): 3212, 2841,
1712, 1618, 1598, 1013 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.45 (s, 1H), 8.55 (s, 1H), 8.39 (d, J = 5.4 Hz, 1H), 8.26 (d,
J = 9.1 Hz, 1H), 8.13 (d, J = 8.9 Hz, 2H), 7.86 (s, 1H), 7.78–7.75 (m,
1H), 7.70 (s, 1H), 7.66–7.60 (m, 2H), 7.45–7.37 (m, 2H), 7.07 (d,
J = 8.9 Hz, 1H), 3.86 (s, 3H), 3.75 (t, J = 6.4 Hz, 2H), 2.18 (s, 3H),
2.07 (t, J = 6.5 Hz, 2H); 13C (DMSO-d6, 300 MHz) d: 187.5, 166.5,
166.4, 163.4, 152.3, 150.5, 149.4, 143.8, 133.9, 133.1, 133.0,
131.3, 131.1, 128.0, 127.9, 124.6, 124.5, 123.8, 118.5, 117.9,
116.9, 114.4, 99.2, 56.0, 54.3, 29.2, 15.9; ESI-MS: (m/z): 514
(M+H)+; HRMS m/z calcd for C30H28ClN3O3 (M+H)+ 514.1898, found
514.1889.
Yellow solid, yield: 89%; mp 210–211 °C; IR (KBr): 3211, 2838,
1714, 1622, 1596, 1012 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.69 (s, 1H), 8.49 (s, 1H), 8.41 (d, J = 5.4 Hz, 1H), 8.27–8.16 (m,
3H), 7.79 (d, J = 2.1 Hz, 1H), 7.73–7.63 (m, 4H), 7.53 (t, J = 5.6 Hz
1H), 7.45 (dd, J = 2.1 Hz and J = 8.9 Hz, 1H), 7.37 (t, J = 8.8 Hz, 2H),
6.65 (d, J = 5.5 Hz 1H), 3.92 (t, J = 5.5 Hz, 2H), 3.68 (d, J = 5.2 Hz
2H), 1.41 (s, 9H); 13C NMR (DMSO-d6, 300 MHz) d: 187.8, 168.3,
167.6, 152.3, 150.4, 149.5, 145.4, 139.6, 135.2, 133.9, 131.8,
131.6, 130.4, 128.0, 124.7, 124.5, 122.8, 118.0, 117.7, 116.3, 99.6,
54.5, 43.2, 35.1, 29.6; ESI-MS: (m/z): 530 (M+H)+; HRMS m/z calcd
for C31H29ClFN3O2 (M+H)+ 530.2011, found 530.2002.
5.3.20. (E)-4-((E)-3-(4-Chlorophenyl)-3-oxoprop-1-enyl)-6-
((3-(7-chloroquinolin-4-ylamino)propylamino)methylene)-2-
methylcyclohexa-2,4-dienone (30)
5.3.16. (E)-2-tert-Butyl-4-((E)-3-(4-chlorophenyl)-3-oxoprop-1-
enyl)-6-((2-(7-chloroquinolin-4-ylamino)ethylamino)
methylene)cyclohexa-2,4-dienone (26)
Yellow solid, yield: 93%; mp 216–217 °C; IR (KBr): 3201, 2845,
1714, 1621, 1599, 1013 cmÀ1 1H NMR (TFA-d, 300 MHz) d: 8.94
;
Yellow solid, yield: 93%; mp 240–241 °C; IR (KBr): 3202, 2839,
(s, 1H), 8.4 (d, J = 4.2 Hz, 1H), 8.24 (d, J = 8.3 Hz, 1H), 8.26–7.96
(m, 6H), 7.79–7.63 (m, 4H), 6.96 (d, J = 4.9 Hz, 1H), 4.35 (s, 2H),
3.99 (s, 2H), 2.66 (s, 2H), 2.54 (s, 3H); 13C (DMSO-d6, 300 MHz) d:
187.7, 166.6, 166.5, 163.6, 152.5, 150.6, 149.6, 143.9, 134.0,
133.3, 133.2, 131.4, 131.3, 128.1, 128.0, 124.7, 124.6, 124.0,
118.7, 118.1, 117.1, 114.5, 99.3, 54.5, 29.3, 16.1; ESI-MS: (m/z):
518 (M+H)+; Anal. Calcd for C29H25Cl2N3O2: C, 67.19; H, 4.86; N,
8.11. Found: C, 67.15; H, 4.81; N, 8.07.
1714, 1622, 1596, 1009 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.68 (s, 1H), 8.48 (s, 1H), 8.41 (d, J = 6 Hz, 1H), 8.25 (d,
J = 9.0 Hz, 1H), 8.11 (d, J = 9.0 Hz, 2H), 7.78 (d, J = 3.0 Hz, 1H),
7.73 (s, 1H), 7.69–7.67 (m, 2H), 7.62–7.51 (m, 4H), 7.45 (dd,
J = 3 Hz and J = 9 Hz, 1H), 6.65 (d, J = 3 Hz, 1H), 3.91 (d, J = 6 Hz,
2H), 3.68 (d, J = 6 Hz, 2H), 1.41 (s, 9H); 13C NMR (DMSO-d6,
300 MHz) d: 188.2, 168.7, 167.6, 152.3, 150.4, 149.5, 145.8, 139.7,
138.1, 137.2, 134.1, 133.9, 30.7, 130.4, 129.2, 128.0, 124.7, 124.5,
122.7, 117.9, 117.8, 117.7, 99.6, 54.4, 43.1, 35.1, 29.6; ESI-MS:
(m/z): 546 (M+H)+; Anal. Calcd for C31H29Cl2N3O2: C, 68.13; H,
5.35; N, 7.69. Found: C, 68.19; H, 5.31; N, 7.60.
5.3.21. (E)-2-sec-Butyl-6-((3-(7-chloroquinolin-4-ylamino)
propylamino)methylene)-4-((E)-3-(4-hydroxyphenyl)-3-
oxoprop-1-enyl)cyclohexa-2,4-dienone (31)
5.3.17. (E)-4-((E)-3-(4-Bromophenyl)-3-oxoprop-1-enyl)-2-tert-
butyl-6-((2-(7-chloroquinolin-4-ylamino)ethylamino)
methylene)cyclohexa-2,4-dienone (27)
Yellow solid, yield: 88%; mp 158–159 °C; IR (KBr): 3216, 2854,
1722, 1634, 1595, 1013 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.52 (s, 1H), 10.34 (s, 1H), 8.57 (s, 1H), 8.39 (d, J = 5.4 Hz, 1H),
8.27 (d, J = 9.1 Hz, 1H), 8.05 (d, J = 8.7 Hz, 2H), 7.78–7.75 (m, 2H),
7.69–7.60 (m, 3H), 7.45–7.38 (m, 2H), 6.89 (d, J = 8.7 Hz, 2H),
6.50 (d, J = 5.5 Hz, 1H), 3.76 (t, J = 6.4 Hz, 2H), 3.42–3.38 (m, 2H),
3.07 (q, J = 7.0 Hz, 1H), 2.10–2.04 (m, 2H), 1.72–1.56 (m, 2H),
1.21 (d, J = 6.9 Hz, 3H), 0.82 (t, J = 7.3 Hz, 3H); 13C (DMSO-d6,
300 MHz) d: 187.7, 166.8, 165.4, 163.5, 152.3, 150.7, 149.4, 144.2,
137.1, 134.0, 132.8, 131.4, 131.3, 130.3, 127.9, 124.7, 124.2,
118.8, 118.0, 117.5, 114.5, 99.3, 54.7, 33.6, 29.4, 20.6, 12.8; ESI-
MS: (m/z): 542 (M+H)+; HRMS m/z calcd for C32H32ClN3O3 (M+H)+
542.2211, found 542.2199.
Yellow solid, yield: 88%; mp 260–261 °C; IR (KBr): 3201, 2844,
1715, 1632, 1585, 1018 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.5 (s, 1H), 8.51 (s, 1H), 8.40 (d, J = 4 Hz, 1H), 8.22 (d, J = 8.8 Hz,
1H), 7.97 (d, J = 8.4 Hz, 2H), 7.78–7.64 (m, 6H), 7.51–7.47 (m 1H),
7.4 (dd, J = 2.4 Hz and J = 6.8 Hz, 1H), 7.30 (s, 1H), 6.62 (d,
J = 5.4 Hz, 1H), 3.94 (t, J = 6 Hz, 2H), 3.7 (t, J = 5.3 Hz, 2H), 1.41 (s,
9H); 13C NMR (DMSO-d6, 300 MHz) d: 187.9, 167.7, 167.0, 151.8,
149.9, 149, 145.1, 139.1, 137.0, 133.4, 133.2, 131.6, 130.2, 129.9,
127.4, 126.6, 124.1, 123.9, 122.3, 117.4, 117.2, 116.2, 114.7,
113.3, 99.05, 54.0, 42.6, 34.5, 29; ESI-MS: (m/z): 590 (M+H)+; Anal.
Calcd for C31H29BrClN3O2: C, 63.01; H, 4.95; N, 7.11. Found: C,
62.97; H, 4.90; N, 7.21.
5.3.22. (E)-2-sec-Butyl-6-((3-(7-chloroquinolin-4-ylamino)
propylamino)methylene)-4-((E)-3-(4-methoxyphenyl)-3-
oxoprop-1-enyl)cyclohexa-2,4-dienone (32)
5.3.18. (E)-6-((3-(7-Chloroquinolin-4-ylamino)propylamino)
methylene)-4-((E)-3-(4-hydroxyphenyl)-3-oxoprop-1-enyl)-2-
methylcyclohexa-2,4-dienone (28)
Yellow solid, yield: 88%; mp 128–129 °C; IR (KBr): 3199, 2843,
1712, 1626, 1596, 1013 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
Yellow solid, yield: 85%; mp 159–160 °C; IR (KBr): 3217, 2841,
14.54 (s, 1H), 8.57 (s, 1H), 8.39 (d, J = 4.9 Hz, 1H), 8.26 (d,
J = 7.1 Hz, 1H), 8.14 (d, J = 8.6 Hz, 2H), 7.77–7.68 (m, 5H), 7.45–
7.37 (m, 2H), 7.07 (d, J = 8.6 Hz, 2H), 6.5 (d, J = 5.3 Hz, 1H), 3.86–
3.76 (m, 2H), 3.40–3.32 (m, 6H), 3.12–3.05 (m, 1H), 2.09 (t,
J = 6.1 Hz, 2H), 1.72–1.56 (m, 2H), 1.21 (d, J = 6.8 Hz, 3H), 0.81 (t,
J = 7.1 Hz, 3H); 13C NMR (DMSO-d6, 300 MHz) d: 187.1, 166.2,
164.8, 162.9, 151.7, 150.1, 143.6, 136.5, 133.4, 132.2, 130.8,
130.7, 129.7, 127.3, 124.1, 124.0, 123.6, 118.2, 116.9, 113.9, 98.7,
55.5, 54.1, 33.0, 28.8, 28.7, 20.0, 12.2; ESI-MS: (m/z): 556 (M+H)+;
Anal. Calcd for C33H34ClN3O3: C, 71.27; H, 6.16; N, 7.56; Found:
C, 71.21; H, 6.20; N, 7.61.
1720, 1622, 1585, 1006 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.43 (s, 1H), 10.45–10.44 (m, 1H), 8.55 (s, 1H), 8.39 (d,
J = 5.5 Hz, 1H), 8.31–8.26 (m, 2H), 8.04 (d, J = 8.8 Hz, 2H), 7.84 (s,
1H), 7.77 (d, J = 2.2 Hz, 1H), 7.68–7.62 (m, 3H), 7.47–7.42 (m,
2H), 6.89 (d, J = 8.7 Hz, 2H), 6.51 (d, J = 5.6 Hz, 1H), 3.75 (t,
J = 6.4 Hz, 2H), 2.18 (s, 3H), 2.08 (t, J = 6.5 Hz, 3H); 13C (DMSO-d6,
300 MHz) d: 187.8, 166.9, 166.4, 162.8, 152.4, 151.1, 149.4, 143.7,
134.5, 133.4, 133.2, 131.8, 130.4, 128.3, 127.9, 125.0, 124.4,
119.2, 118.3, 117.4, 116.2, 99.6, 80.0, 54.9, 29.6, 16.3; ESI-MS:
(m/z): 500 (M+H)+; HRMS m/z calcd for C29H26ClN3O3 (M+H)+
500.1742, found 500.1732.

K. V. Sashidhara et al. / Bioorg. Med. Chem. 20 (2012) 2971–2981
2979
5.3.23. (E)-2-sec-Butyl-4-((E)-3-(4-chlorophenyl)-3-oxoprop-1-
enyl)-6-((3-(7-chloroquinolin-4-ylamino)propylamino)
methylene)cyclohexa-2,4-dienone (33)
5.3.27. (E)-2-tert-Butyl-4-((E)-3-(4-chlorophenyl)-3-oxoprop-1-
enyl)-6-((4-(7-chloroquinolin-4-ylamino)phenylamino)
methylene)cyclohexa-2,4-dienone (37)
Yellow solid, yield: 91%; mp 131–132 °C; IR (KBr): 3212, 2838,
Red solid, yield: 87%; mp 145–146 °C; IR (KBr): 3202, 2841,
1712, 1621, 1582, 1001 cmÀ1
;
1H NMR (DMSO-d6, 300 MHz) d:
1712, 1619, 1592, 1008 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.57 (s, 1H), 8.55 (s, 1H), 8.39 (s, 1H), 8.27 (t, J = 8.0 Hz, 1H),
8.15 (d, J = 6.3 Hz, 2H), 7.78–7.61 (m, 7H), 7.40 (t, J = 9.0 Hz, 2H),
6.49 (s, 1H), 3.76 (s, 2H), 3.35 (s, 6H), 3.08 (d, J = 5.1 Hz, 1H), 2.08
(s, 2H), 1.68–1.55 (m, 2H), 1.21 (d, J = 3.7 Hz, 3H), 0.81 (s, 3H);
13C NMR (DMSO-d6, 300 MHz) d: 187.7, 166.2, 166, 151.8, 150,
149, 145, 137.7, 136.9, 136.7, 133.4, 132.9, 130.2, 129.8, 128.7,
127.4, 124.1, 124, 123.1, 117.6, 117.5, 116.7, 98.7, 53.7, 33, 28.8,
28.7, 20, 12.2; ESI-MS: (m/z): 560 (M+H)+; Anal. Calcd for
C31H31Cl2N3O2: C, 68.57; H, 5.57; N, 7.50. Found: C, 68.52; H,
5.62; N, 7.56.
14.89 (s, 1H), 9.23 (s, 1H), 9.02 (s, 1H), 8.52 (d, J = 6 Hz, 1H), 8.42
(d, J = 9.1 Hz, 1H), 8.14 (d, J = 9.0 Hz, 2H), 8.05 (s, 1H), 7.91 (d,
J = 3.1 Hz, 1H), 7.80–7.76 (m, 3H), 7.64–7.54 (m, 5H), 7.47 (d,
J = 9.0 Hz, 2H), 7.04 (d, J = 6.0 Hz, 1H), 1.47 (s, 9H); 13C NMR
(DMSO-d6, 300 MHz) d: 193.1, 168.0, 167.6, 157.2, 154.8, 152.6,
149.9, 147.2, 145.0, 143.1, 142.9, 141.7, 139.2, 137.8, 135.9,
135.5, 134.0, 132.9, 130.3, 130.1, 129.7, 128.0, 127.7, 124.3,
124.2, 123.8, 107.7, 39.9, 34.4; ESI-MS: (m/z): 594 (M+H)+; Anal.
Calcd for C35H29Cl2N3O2: C, 70.71; H, 4.92; N, 7.07. Found: C,
70.66; H, 4.95; N, 7.12.
5.3.24. (E)-6-((4-(7-Chloroquinolin-4-ylamino)phenylamino)
methylene)-4-((E)-3-(4-methoxyphenyl)-3-oxoprop-1-enyl)-2-
methylcyclohexa-2,4-dienone (34)
5.3.28. (E)-4-((E)-3-(4-Bromophenyl)-3-oxoprop-1-enyl)-2-tert-
butyl-6-((4-(7-chloroquinolin-4-ylamino)phenylamino)
methylene)cyclohexa-2,4-dienone (38)
Red solid, yield: 93%; mp 165–166 °C; IR (KBr): 3200, 2854,
Red solid, yield: 92%; mp 166–167 °C; IR (KBr): 3214, 2839,
1725, 1621, 1582, 1003 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
1714, 1623, 1598, 1014 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.26 (s, 1H), 9.23 (s, 1H), 9.0 (s, 1H), 8.51 (d, J = 6 Hz, 1H), 8.42
(d, J = 9.0 Hz, 1H), 8.14 (d, J = 9.0 Hz, 2H), 7.93–7.91 (m, 3H),
7.84–7.78 (m, 1H), 7.69–7.64 (m, 1H), 7.60–7.53 (m, 3H), 7.46 (d,
J = 9.0 Hz, 2H), 7.09–7.03 (m, 3H), 3.86 (s, 3H), 2.29 (s, 3H); 13C
NMR (DMSO-d6, 300 MHz) d:187.0, 163.0, 161.7, 161.3, 152,
151.8, 149.5, 147.4, 142.3, 139.7, 133.9, 133.3, 132.2, 130.6,
127.6, 126.2, 125.4, 125.0, 124.5, 124.3, 122.7, 122.4, 120.1,
119.3, 118.5, 118.3, 114.5, 113.7, 113.3, 112.5, 109.2, 102.6,
102.2, 101.8, 55.8, 55.3, 55.1, 15.3, 15.1; ESI-MS: (m/z): 548
(M+H)+; HRMS m/z calcd for C33H26ClN3O3 (M+H)+ 548.1742, found
548.1725.
14.89 (s, 1H), 9.2 (s, 1H), 9.02 (s, 1H), 8.52 (d, J = 5.3 Hz, 1H), 8.43
(d, J = 9.1 Hz, 1H), 8.08–8.04 (m, 3H), 7.91 (d, J = 2.1 Hz, 1H),
7.80–7.74 (m, 5H), 7.60–7.55 (m, 3H), 7.47 (d, J = 8.6 Hz, 2H),
7.04 (d, J = 5.4 Hz 1H), 1.47 (s, 9H); 13C NMR (DMSO-d6,
300 MHz) d: 188.6, 163.3, 162.8, 152.4, 150.0, 147.9, 145.2, 142.5,
140.2, 138.2, 137.3, 134.5, 133.0, 132.2, 131.2, 130.9, 128.1,
127.5, 125.6, 125.3, 124.9, 123.2, 122.9, 119.6, 119.4, 119.0,
103.0, 35.2, 29.6; ESI-MS: (m/z): 638 (M+H)+; HRMS m/z calcd for
C
35H29BrClN3O2 (M+H)+ 638.1211, found 638.1224.
5.4. General procedure for the synthesis of compounds 1a–1d
5.3.25. (E)-6-((4-(7-Chloroquinolin-4-
Synthesis of 1a was achieved by our previously reported
ylamino)phenylamino)methylene)-4-((E)-3-(4-fluorophenyl)-3-
oxoprop-1-enyl)-2-methylcyclohexa-2,4-dienone (35)
Red solid, yield: 91%; mp 150–151 °C; IR (KBr): 3213, 2839,
protocol.21
1718, 1620, 1590, 1017 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
5.4.1. 3-sec-Butyl-2-hydroxy-5-(3-oxo-3-p-tolyl-propenyl)-
benzaldehyde (1a)
14.34–14.29 (m, 1H), 9.24 (s, 1H), 9.02 (s, 1H), 8.52 (d, J = 5.4 Hz,
1H), 8.43 (d, J = 9.1 Hz, 1H), 8.26–8.21 (m, 2H), 7.97–7.92 (m,
3H), 7.85–7.68 (m, 2H), 7.61–7.54 (m, 3H), 7.49–7.37 (m, 4H),
7.04 (d, J = 5.3 Hz, 1H), 2.29 (s, 3H); 13C NMR (DMSO-d6,
300 MHz) d: 187.9, 162.2, 152.4, 150.0, 148.0, 144.3, 142.8, 140.2,
134.9, 134.5, 133.9, 133.1, 131.8, 131.7, 128.1, 126.9, 125.7,
125.6, 124.9, 123.3, 123.0, 119.6, 119.0, 118.8, 116.4, 116.1,
102.9, 15.7; ESI-MS: (m/z): 536 (M+H)+; HRMS m/z calcd for
White solid: mp 85–86 °C; IR (KBr, cmÀ1): 3433, 3022, 2846,
1654, 1591; 1H NMR (CDCl3, 300 MHz): d 11.60 (s, 1H), 9.94 (s,
1H), 7.94 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 15.7 Hz, 1H), 7.69 (d,
J = 2.4 Hz, 1H), 7.45 (d, J = 15.7 Hz, 1H), 7.31 (d, J = 8.1 Hz, 2H),
3.23–3.11 (m, 1H), 2.43 (s, 3H), 1.74–1.61 (m, 2H), 1.28 (d,
J = 6.6 Hz, 3H), 0.88 (t, J = 8.0 Hz, 3H); 13C NMR (75 MHz): d
196.8, 189.9, 161.5, 143.8, 143.3, 137.3, 135.8, 133.8, 132.1,
129.5, 128.8, 127.0, 120.9, 120.4, 33.5, 29.5, 21.8, 20.2, 12.2; ESI-
MS: m/z: 323 (M+H)+; Anal. Calcd for C21H22O3: C, 78.23; H, 6.88;
O, 14.89. Found: C, 78.21; H, 6.90; O, 14.88.
C
32H23ClFN3O2 (M+H)+ 536.1542, found 536.1567.
5.3.26. (E)-2-sec-Butyl-6-((4-(7-chloroquinolin-4-ylamino)
phenylamino)methylene)-4-((E)-3-(furan-2-yl)-3-oxoprop-1-
enyl)cyclohexa-2,4-dienone (36)
A mixture of 1a (1.0 equiv) and appropriate amines (1.0 equiv)
in ethanol were stirred for 10 min at room temperature. The sol-
vent was evaporated under vacuum and the solid was purified di-
rectly with column chromatography to obtain the respective
compounds 1b–1d in excellent yields.
Red solid, yield: 92%; mp 170–171 °C; IR (KBr): 3212, 2838,
1711, 1624, 1582, 1015 cmÀ1 1H NMR (DMSO-d6, 300 MHz) d:
;
14.43 (s, 1H), 9.23 (s, 1H), 9.04 (s, 1H), 8.52 (d, J = 5.2 Hz, 1H),
8.43 (d, J = 9.1 Hz, 1H), 8.06 (s, 1H), 7.95–7.92 (m, 2H), 7.85 (s,
1H), 7.77–7.73 (m, 2H), 7.62–7.55 (m, 4H), 7.47 (d, J = 8.7 Hz,
2H), 7.04 (d, J = 5.3 Hz 1H), 6.80–6.79 (m, 1H), 3.16 (q, J = 6.7 Hz,
1H), 1.79–1.59 (m, 2H), 1.21 (d, J = 6.9 Hz, 3H), 0.81 (t, J = 7.3 Hz,
3H); 13C NMR (DMSO-d6, 300 MHz) d: 177.1, 162.6, 161.5, 153.6,
152.4, 150.0, 148.5, 147.9, 143.2, 142.8, 140.2, 136.0, 134.5,
132.5, 131.0, 128.1, 125.7, 125.6, 124.9, 123.3, 123.0, 119.7,
119.4, 119.1, 113.1, 102.9, 33.5, 29.3, 20.6, 12.6; ESI-MS: (m/z):
550 (M+H)+; Anal. Calcd for C33H28ClN3O3: C, 72.06; H, 5.13; N,
7.64, Found: C, 72.10; H, 5.17; N, 7.68.
5.4.2. (E)-2-sec-Butyl-6-((methylamino)methylene)-4-((E)-3-
oxo-3-p-tolylprop-1-enyl)cyclohexa-2,4-dienone (1b)
Red solid, yield: 92%; mp 91–92 °C; IR (KBr): 3220, 2834, 1711,
1620, 1598, 1014 cmÀ1 1H NMR (CDCl3, 300 MHz) d: 14.57–14.54
;
(m, 1H), 8.36 (s, 1H), 7.94 (d, J = 9.0 Hz, 2H), 7.81–7.76 (m, 1H),
7.53 (d, J = 2.1 Hz, 1H), 7.42–7.30 (m, 4H), 3.67 (q, J = 6.5 Hz, 2H),
3.20 (q, J = 6.9 Hz, 1H), 2.45 (s, 3H), 1.77–1.62 (m, 2H), 1.37 (t,
J = 6 Hz, 3H), 1.28 (d, J = 6.9 Hz, 3H), 0.94–0.89 (m, 3H); ESI-MS:
(m/z): 350 (M+H)+.

2980
K. V. Sashidhara et al. / Bioorg. Med. Chem. 20 (2012) 2971–2981
5.4.3. (E)-2-sec-Butyl-6-((ethylamino)methylene)-4-((E)-3-oxo-
3-p-tolylprop-1-enyl)cyclohexa-2,4-dienone (1c)
6.4. In vivo antimalarial assay
Red solid, yield: 91%; mp 86–87 °C; IR (KBr): 3211, 2830, 1705,
1624, 1591, 1011 cmÀ1 1H NMR (CDCl3, 300 MHz) d: 14.62–14.57
(m, 1H), 8.35 (s, 1H), 7.95 (d, J = 9.1 Hz, 2H), 7.81–7.76 (m, 1H),
7.53 (d, J = 2.1 Hz, 1H), 7.42–7.70 (m, 4H), 3.59 (t, J = 6.8 Hz, 2H),
3.20 (q, J = 6.9 Hz, 1H), 2.45 (s, 3H), 1.80–1.59 (m, 4H), 1.28 (d,
J = 6 Hz, 3H), 1.02 (t, J = 7.4 Hz, 3H), 0.93 (d, J = 7.3 Hz, 3H); ESI-
MS: (m/z): 364 (M+H)+.
The in vivo drug response was evaluated in Swiss mice infected
with P. yoelii (N-67 strain) which is innately resistant to CQ. The
mice (24 2 g) were inoculated with 1 Â 105 parasitized RBC on
day 0 and treatment was administered to a group of five mice from
day 0 to 3, once daily. The aqueous suspensions of compounds
were prepared with a few drops of Tween 80. The efficacy of test
compounds was evaluated at 100 mg/kg/day by oral route. Para-
sitaemia levels were recorded from thin blood smears on days 4
and day 6. The mean value determined for a group of five mice
was used to calculate the percent suppression of parasitaemia with
respect to the untreated control group. The animals were followed
further till day 28 to record the day of death. Mice treated with CQ
served as reference controls.27
5.4.4. (E)-2-sec-Butyl-4-((E)-3-oxo-3-p-tolylprop-1-enyl)-6-
((phenylamino)methylene)cyclohexa-2,4-dienone (1d)
Red solid, yield: 88%; mp 95–96 °C; IR (KBr): 3202, 2844, 1712,
1621, 1598, 1002 cmÀ1 1H NMR (CDCl3, 300 MHz) d: 14.20–14.16
;
(m, 1H), 8.70 (s, 1H), 7.96 (d, J = 9.0 Hz, 2H), 7.84–7.79 (m, 1H), 7.60
(d, J = 3 Hz, 1H), 7.55 (d, J = 2 Hz, 1H), 7.49–7.43 (m, 3H), 7.34–7.31
(m, 5H), 3.30–3.21 (m, 1H), 2.45 (s, 3H), 1.82–1.67 (m, 3H), 1.31 (t,
J = 6.9 Hz, 3H), 0.96 (d, J = 7.3 Hz, 3H); ESI-MS: (m/z): 398 (M+H)+.
Acknowledgments
Instrumentation facilities from SAIF, CDRI are gratefully
acknowledged. M.K., R.K.M., R.K.S. and A.S. are thankful to CSIR
and ICMR respectively, for financial support. This work was sup-
ported by EMPOWER grant (OLP0007) of CSIR-CDRI to KVS. This
is CSIR-CDRI publication number 8213.
6. Biological materials and methods
6.1. In vitro antimalarial assay
The compounds were dissolved in DMSO at 5 mg/mL. Twofold
serial dilutions of test samples were made in 96 well plates and
incubated with 1.0% parasitized cell suspension containing 0.8%
parasitaemia (asynchronous culture with more than 80% ring
stages). The plates were incubated at 37 °C in CO2 incubator in
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.03.011.
an atmosphere of 5% CO2 and air mixture. Later (72 h) 100 ll of ly-
References and notes
sis buffer containing 1Â concentration of SYBR Green-I (Invitrogen)
was added to each well and incubated for 1 h at 37 °C. The plates
were examined at 485 20 nm of excitation and 530 20 nm of
emission for relative fluorescence units (RFUs) per well using the
fluorescence plate reader (FLUO star, BMG lab technologies). Data
was transferred into a graphic programme (EXCEL) and IC50 values
were obtained by Logit regression analysis.24 Chloroquine was
used as the standard reference drug.
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8. Cravo, P.; Culleton, R.; Afonso, A.; Ferreira, I. D.; Do Rosario, V. E. Anti-Infect.
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6.2. Cytotoxicity assay
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