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The enones which displayed the highest potencies towards the D6
and C235 malarial parasites (5b) and the lowest IC50 value in the
ILSDA screen (5d) were examined using MDCK cells. The apparent
permeability (Papp) values of the compounds from the apical to the
basal compartment (A–B) and from the basal to the apical com-
partment (B–A) were determined and the results are presented
in Table 2. The permeability ratio (PR) viz PappA–B/PappB–A for 5d
is 57.4 and compounds with a PR value of 10 or more are predicted
to be well absorbed. The efflux ratio (ER) figure should be low
namely 0.2 or less and the data in Table 2 reveals that both 5b
and 5d achieve this criterion. The PR ratios and efflux ratios of
5b and 5d compare favourably with chloroquine and mefloquine.
investigation is a modification of a literature procedure.16 In prep-
aration for the liver cell culture, sterile, white 384 well culture
plates (Nunc Cat #164610) were first coated with 19 lL of a 1:50
dilution of ECL Cell Attachment Matrix in HBSS (ECL—Millipore
#08-110, HBSS-Gibco-Invitrogen #14175-095) using a Tecan EVO
Freedom robotics system, and the plates were then incubated for
1 h at 37 °C or overnight at 4 °C. The residual ECL matrix solution
was subsequently removed from each plate using the Tecan robot-
ics system. HepG2 cells were cultured in complete Minimal Essen-
tial Medium (MEM) (Gibco-Invitrogen, #11090-099) prepared by
supplementing MEM with 0.22% sodium bicarbonate (Gibco-BRL
Cat #25080-094), 10% heat inactivated FBS (Gibco-Invitrogen
#16000-036), 2 mM L-glutamine (Gibco-Invitrogen #25030-081),
0.1 mM MEM nonessential amino acids (Gibco-Invitrogen
#11140-050), 0.01 mg/mL insulin (Sigma #I1882), 0.2% bovine ser-
um albumin (Sigma #A1470), 20 units/mL penicillin–streptomycin
4. Conclusions
In summary, the 3,5-bis(benzylidene)-4-piperidones 5a–d and
related N-acyl analogs in series 1–4 are clusters of novel antimalar-
ial agents. With few exceptions the compounds in series 1–5 sig-
nificantly inhibit the growth of a drug-sensitive and a drug-
resistant strain of P. falciparum. The IC50 values towards both
strains are virtually identical. Of particular note is 5b which dis-
played slightly better potency than chloroquine against the drug-
resistant strain C235 of P. falciparum. Several compounds inhibited
the liver stage development of P. berghei and the lead compounds
are 2b, 3b, 5c and 5d. The most promising compound in this bioas-
say is 5d which also has very good permeability characteristics.
Most of the active compounds are nontoxic to human HepG2 cells
that is, the IC50 values are in excess of 2000 ng/mL. The enone 5b
displayed excellent metabolic stability in human plasma. These re-
sults warrants further development of 3,5-bis(benzylidene)-4-pip-
eridones as novel antimalarials. Various suggestions for analog
development were made based on these initial results.
(Gibco-Invitrogen #15140-148), and 5 lg/mL gentamycin (Gibco-
Invitrogen #15710-064). HepG2 cells cultured in complete MEM
were trypsonized using a 0.25% trypsin/EDTA solution (Invitrogen
#25200-106), washed in complete MEM, and resuspended at a
concentration of 125,300 cells/mL, dispensed in a volume of
38.3 lL to yield a final concentration of 4800 HepG2 cells per well
in ECL coated 384 well plates (384 well optical Bottom Plates Poly-
base Black w/lid Cell Culture Sterile PS, Nunc Cat #142761) and
incubated overnight in 5% CO2 at 37 °C. After overnight incubation,
the media was removed from each well using the Tecan EVO Free-
dom robot, and sporozoites were added to each well and allowed
to invade the HepG2 cells. Luciferase-expressing P. berghei spor-
ozoites were obtained using
a modification of a literature
method.17 The sporozoites were isolated through microscopic dis-
section of the salivary glands from mosquitoes that had been fed
on mice infected with the luciferase-expressing strain of P. berghei,
and centrifuged through Ozaki tubes to yield a smooth suspension
of sporozoites. The sporozoites were suspended at 204,500 spor-
ozoites per mL and 3900 sporozoites were dispensed per well in
5. Experimental
a volume of 19.1 lL. The plates were incubated at 37 °C for 3 h to
allow for sporozoite invasion of the HepG2 cell layer. After 3 h of
5.1. Synthesis of series 1–5
incubation, the media was removed with the Tecan EVO Freedom
The preparation of the compounds in series 1–5 has been de-
robot, and two wash cycles were conducted with 38.3 lL complete
scribed previously.3
MEM media to remove sporozoites that did not invade the HepG2
cells. The Tecan EVO Freedom robot was programmed to add the
wash media in one corner of the well with slow speed aspiration
to avoid disturbing the HepG2 cell layer. A final volume of
5.2. Evaluation against Plasmodium falciparum
The compounds in series 1–5 were evaluated against the D6 and
C235 strains of P. falciparum using the malaria SYBR green I-based
fluorescence (MSF) assay which has been described previously.13 In
brief, different concentrations of the compounds were added to the
parasites cultured in a supplemental RPMI 1640 medium with an
initial parasitemia of 0.3% and a hematocrit of 2%. The cultures
were incubated at 37 °C for 72 h in an atmosphere of oxygen
(5%), carbon dioxide (5%) and nitrogen (90%). The lysis buffer con-
taining SYBR green I was added to each well and incubated for 24 h
at room temperature in the dark. The plates were examined for
their relative fluorescence. From these data, the IC50 values were
33.3 lL of media was added to the cells after the second wash step.
Drug plates were prepared with the Tecan EVO Freedom robot
using sterile 96 well plates containing twelve duplicate twofold se-
rial dilutions of each test compound suspended in DMSO. Five
microlitres of diluted test compound was added to the 33.3 lL of
media present in each well providing a 7.6-fold final dilution of
compound. The final concentration range tested was 0.5 to
10,000 ng/mL for all assays. Atovaquone (Sigma Aldrich #A7986)
was used as a plate control with a concentration ranging from
0.03 to 62.5 ng/mL. After 48 h of incubation, 3.8
lL of a luciferin
solution (Caliper Life Science) diluted to 150 g/mL was added to
l
calculated using
a nonlinear regression (sigmoidal dose–re-
each well, and the plates were incubated for 30 min at 37 °C, in
the dark. Each plate was read using a Genios plate reader. The
50% inhibitory concentrations (IC50) were then generated for each
dose response test using GraphPad Prism (GraphPad Software Inc.,
San Diego, CA) using the nonlinear regression (sigmoidal dose–re-
sponse/variable slope) equation.
sponse/variable slope) equation with the aid of a GraphPad
Prism.14
5.3. Inhibition of liver stage development assay (ILSDA)
The compounds 1b, 2b, 3b–d, 4b, c, 5a–d and the reference
drugs, chloroquine and mefloquine were screened for the inhibi-
tion of the liver stage development of the malarial parasite. The
luciferase-expressing P. berghei parasite used in this assay is a
genetically modified parasite of clone cl15cy1 of the ANKA strain
first described by Janse et al.15 The assay used in the present
5.4. Cytotoxicity assay against human HepG2 cells
The cytotoxicity of the compounds 1b, 2b, 3b–d, 4b, 4c, 5a–d
and the reference drugs, chloroquine and mefloquine was evalu-
ated using the MTT assay. The 384 well MTT cytotoxicity assay is