K. Kumar et al. / European Journal of Medicinal Chemistry 87 (2014) 801e804
803
Table 1
W2 strains of P. falciparum. The improvement in antimalarial effi-
cacy of N-alkylbromo-isatins with the introduction of ferrocene
nucleus clearly validated the potential of introducing organome-
tallics for improving biological efficacies in present day drug dis-
covery paradigm. The non-cytotoxic conjugates 5f and 5h proved to
be the most potent among the series with IC50 values of 3.76 and
Antiplasmodial activity against the 3D7 and W2 strains of P. falciparum.
Compound
R
N
Strains
3D7
W2
IC50
IC50
(
m
M) 95% CI
(mM) 95% CI
5a
5b
5c
5d
5e
5f
5g
5h
CQ
H
F
CH3
Cl
H
F
CH3
Cl
2
2
2
2
3
3
3
3
24.43
>100
>100
>100
20.70e29.90
_
_
_
4.78e8.44
2.75e7.13
13.70e19.20
7.83e9.21
26.50
>100
>100
>100
21.10e33.40
_
_
4.58 mM against 3D7 and W2 strain of P. falciparum respectively.
4. Experimental section
_
6.35
7.41
6.13e8.96
5.53e6.45
9.05e10.36
3.86e5.44
0.37e0.63
3.76
16.20
8.49
5.97
9.68
4.58
0.49
Ethynylferrocene was procured from SigmaeAldrich. Melting
points were determined by open capillary using Veego Precision
Digital Melting Point apparatus (MP-D) and are uncorrected. 1H
NMR spectra were recorded in deuterochloroform and dime-
thylsulfoxide-d6 with Jeol 300 (300 MHz) spectrometers using TMS
as an internal standard. Chemical shift values are expressed as parts
per million downfield from TMS and J values are in hertz. Splitting
patterns are indicated as s: singlet, d: doublet, t: triplet, m: multi-
plet, dd: double doublet, ddd: doublet of a doublet of a doublet, and
br: broad peak. 13C NMR spectra were recorded on Jeol 300
(75 MHz) spectrometers in deuterochloroform and dimethylsulf-
oxide-d6 using TMS as internal standard. Elemental analyses were
performed on Heraus CHNeO-Rapid Elemental Analyzer. Mass
spectra were recorded on a BRUCKER high resolution mass spec-
trometer (micrOTOF-QII). Column chromatography was performed
using silica gel (60e120 mesh).
0.021 0.018e0.025
IC50 values are means of 5 experiments. 95%CI: 95% confident interval.
an IC50 value of 24.43 mM. Increasing the chain length from ethyl to
propyl seemed to have considerable influence on the activity pro-
files as the conjugates 5eeh displayed IC50s in the range
3.76e16.20
stituent at C-5 position and a propyl linker displayed an IC50 value
of 3.76 M against 3D7 strain. Similar comparison of structure ac-
mM. The most potent conjugate 5f with a fluoro sub-
m
tivity relationship of the synthesized conjugates against W2 strain
revealed the ethyl linked conjugates (5aed) to be inactive even at
highest tested concentration. The replacement of the ethyl chain
with propyl chain again improved the activity profiles with little
influence of the nature of substituent introduced at C-5 position of
the isatin ring. The conjugate 5n with electron withdrawing Cl-
substituent at C-5 position and n ¼ 3 proved to be the most
4.1. Procedure for the preparation of isatin ferrocene hybrids
(5aeh)
potent with an IC50 of 4.58 mM.
Copper sulfate (0.05 mmol) and sodium ascorbate (0.13 mmol)
was added to a stirred solution of ethynyl-ferrocene 4 (1 mmol) and
The improvement in activity profiles of conjugates 5eeh, over
precursors' viz. N-alkylbromo-isatins against both CQ-S and CQ-R
strains of P. falciparum clearly established the importance of
amalgamating a ferrocene nucleus among the well established
isatin family via triazole-linkers with reputed physicochemical
profiles. Although the in vitro antimalarial activities of the syn-
thesized isatin-ferrocene conjugates is lower than ferroquine in
laboratory strains (2.1e13.4 nM) [30], the conjugates are equipotent
as that of ferrocene-ciprofloxacin conjugates [31,32] and
ferrocenyl-chalcones such as 1-ferrocenyl-3-(4-nitrophenyl)prop-
N-alkylated azido-isatins
3 (1 mmol) in ethanol-water (9:1)
mixture. The reaction mixture was allowed to stir at room tem-
perature for 7e10 h. After completion of reaction as evidenced by
TLC, water (20 mL) was added and the reaction mixture was
extracted twice with dichloromethane (2 ꢁ 30 mL). The combined
organic layers were dried over anhydrous sodium sulfate, concen-
trated under reduced pressure and purified via column chroma-
tography using 40:60 (EtOAc: hexane) mixture.
2-en-1-one [33,34] or mono- and bis-ferrocenylchalcones-
conjugates [26].
b-lactam
4.2. Methods for assessment of antimalarial activity of test
compounds
Cytotoxicity of two most potent conjugates viz. 5f and 5h was
also determined against mammalian HeLa cells and is listed in
Table 2. As evident, the conjugates were non-cytotoxic against
mammalian cells and therefore had the selectivity for inhibition of
P. falciparum.
The two strains viz. 3D7, the CQ-susceptible strain (isolated in
West Africa; obtained from MR4, VA, USA), and W2 (isolated in
Indochina; obtained from MR4, VA, USA), the CQ-resistant strain,
were maintained in culture in RPMI 1640 (Invitrogen, Paisley,
United Kingdom), supplemented with 10% human serum (Abcys
S.A. Paris, France) and buffered with 25 mM HEPES and 25 mM
NaHCO3. Parasites were grown in A-positive human blood (Eta-
blissement Français du Sang, Marseille, France) under controlled
atmospheric conditions that consisted of 10% O2, 5% CO2 and 85% N2
at 37 ꢀC with a humidity of 95%.
The two strains were synchronized twice with sorbitol before
use [35], and clonality was verified every 15 days through PCR
genotyping of the polymorphic genetic markers msp1 and msp2
and microsatellite loci [36,37]; additionally, clonality was verified
each year by an independent laboratory from the Worldwide Anti-
malarial Resistance Network (WWARN).
3. Conclusion
In conclusion, a series of isatin-ferrocene conjugates have been
synthesized via Cu-promoted azide-alkyne cyloaddition reactions
and evaluated for their antimalarial activities against the 3D7 and
Table 2
Cytotoxicity and selective index of conjugates 5f and 5h.
Strains
Compound
Cytotoxicity
(IC50 M)
3D7
W2
Chloroquine diphosphate (CQ) was purchased from Sigma (Saint
Louis, MO). CQ was resuspended in water in concentrations ranging
between 5 and 3200 nM. The synthetic compounds were resus-
pended in DMSO and then diluted in RPMI-DMSO (99v/1v) to
m
(IC50
m
M)
SIa
(IC50
m
M)
SIa
5f
5h
82.14
86.68
3.76
8.49
21.85
10.21
5.97
4.58
13.76
18.93
a
Selectivity index.
obtain final concentrations ranging from 1 nM to 100 mM.