Organocatalytic asymmetric vinylogous 1,4-addition of α,α-Dicyanoolefins to chalcones under a bio-based reaction media: Discovery of new Michael adducts with antiplasmodial activity

Article abstract of DOI:10.1016/j.tet.2019.05.022

The organocatalysed asymmetric vinylogous Michael addition of α,α-dicyanoolefins to α,β-unsaturated aldehydes and ketones have been reported in the last decade, however, chalcones have been poorly explored. Moreover, a considerable part of the publications in this theme still employs undesirable solvents, such as toluene and THF, with concerns related to health and environmental safety. We report herein the use of a bifunctional catalyst derived from a Cinchona alkaloid to perform the enantio- and diastereoselective Michael addition of α,α-dicyanoolefins to chalcones using 2-MeTHF as solvent. The Michael adducts were obtained in moderate to good yields and were evaluated for their antiplasmodial and cytotoxic activity.

Full text of DOI:10.1016/j.tet.2019.05.022

Accepted Manuscript
Organocatalytic asymmetric vinylogous 1,4-addition of α,α-Dicyanoolefins to
chalcones under a bio-based reaction media: Discovery of new Michael adducts with
antiplasmodial activity
Lorena S.R. Martelli, Lucas C.C. Vieira, Márcio W. Paixão, Julio Zukerman-
Schpector, Juliana O. de Souza, Anna Caroline C. Aguiar, Glaucius Oliva, Rafael
V.C. Guido, Arlene G. Corrêa
PII:
S0040-4020(19)30561-7
https://doi.org/10.1016/j.tet.2019.05.022
TET 30343
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 9 April 2019
Revised Date: 7 May 2019
Accepted Date: 10 May 2019
Please cite this article as: Martelli LSR, Vieira LCC, Paixão MáW, Zukerman-Schpector J, de Souza JO,
Aguiar ACC, Oliva G, Guido RVC, Corrêa AG, Organocatalytic asymmetric vinylogous 1,4-addition of
α,α-Dicyanoolefins to chalcones under a bio-based reaction media: Discovery of new Michael adducts
with antiplasmodial activity, Tetrahedron (2019), doi: https://doi.org/10.1016/j.tet.2019.05.022.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Organocatalytic Asymmetric Vinylogous 1,4-addition of α,α-
Dicyanoolefins to Chalcones Under a Bio-based Reaction
Media: Discovery of New Michael Adducts with Antiplasmodial
Activity
Lorena S. R. Martelli,1 Lucas C. C. Vieira,1,2 Márcio W. Paixão,1 Julio
Zukerman-Schpector,1 Juliana O. de Souza,3 Anna Caroline C. Aguiar,3
Glaucius Oliva,3 Rafael V. C. Guido3 and Arlene G. Corrêa1*
1Centre of Excellence for Research in Sustainable Chemistry,
Department of Chemistry, Federal University of São Carlos, 13565-905 São
Carlos, SP. 2 Institute of Engineering, Federal University of Mato Grosso,
78060-900 Cuiabá, MT. 3Center for Innovation in Biodiversity and Drug
Discovery, São Carlos Institute of Physics, University of São Paulo, 13563-120
São Carlos, SP, Brazil. *agcorrea@ufscar.br
Abstract
The organocatalysed asymmetric vinylogous Michael addition of α,α-
dicyanoolefins to α,β-unsaturated aldehydes and ketones have been reported in
the last decade, however, chalcones have been poorly explored. Moreover, a
considerable part of the publications in this theme still employs undesirable
solvents, such as toluene and THF, with concerns related to health and
environmental safety. We report herein the use of a bifunctional catalyst derived
from a Cinchona alkaloid to perform the enantio- and diastereoselective Michael
addition of α,α-dicyanoolefins to chalcones using 2-MeTHF as solvent. The
Michael adducts were obtained in moderate to good yields and were evaluated
for their antiplasmodial and cytotoxic activity.
1. Introduction
Development of green products and processes is one of the most
important challenges of the present society [1]. In this sense, new metal-free
catalytic protocols using bio-based solvents to the asymmetric synthesis of
structurally diverse compounds is of great interest [2].
1

ACCEPTED MANUSCRIPT
Organocatalytic asymmetric vinylogous Michael addition to α,β-
unsaturated carbonyl derivatives has attracted major attention in the organic
chemistry community [3]. This method provides highly functionalized
compounds, which allow many further transformations [4,5].
One of the pioneering works in this field, employing α,α-dicyanoolefins
[6] as nucleophilic species, was described by Chen & Deng [7]. In addition,
several vinylogous systems [8], such as γ-butenolides [9], γ-butyrolactams [10],
angelica lactones [11], 3,5-dialkyl-substituted 4-nitroisoxazoles [12] and 3-
alkylidene oxindoles [13] have also been explored. Furthermore, a number of
Michael acceptors, as 3‐aroyl acrylates and 1,2‐diaroylethylenes [14], 2-
enoylpyridines [15] and nitrostyrenes [16] for instance, have been described.
Nevertheless, α,β-unsaturated aldehydes and ketones [17] have been the most
extensively explored ones.
Nayak et al. [18] have reviewed the Michael addition of various
nucleophiles such as nitroalkane, malonate, cyanoacetate, hydroxyamine,
malononitrile, thiol, cyanomethylphosphate, butenolide, coumarin, 1-fluoro-1-
nitro(phenylsulfonyl)methane to chalcones using a variety of organocatalysts.
Recently, Sharma et al. have employed a Cinchona derived squaramide for
catalysing the enantioselective 1,4-Michael addition reaction of 2-enoylpyridines
with pyrazolin-5-ones [19]. However, to the best of our knowledge, chalcones
have been poorly explored towards asymmetric Michael reaction with α,α-
dicyanoolefins. The only report by Singh’s group described the asymmetric
direct vinylogous Michael addition of α,α-dicyanoolefins to 2‐enoylpyridine N‐
oxides catalysed by bifunctional thiourea (Cat. A) having toluene as solvent
media (Scheme 1a) [20]. Thus, this transformation still represents a challenging
task regarding the reactivity and stereoselectivity of the two reactants.
Moreover, a considerable amount of publications in this theme still employs
undesirable solvents, such as toluene and THF, with concerns related to health
and environmental safety. In this sense, we report herein the use of a
bifunctional catalyst derived from the Cinchona alkaloid to perform the
enantioselective Michael addition of α,α-dicyanoolefins to chalcones using 2-
MeTHF as solvent (Scheme 1b).
2

ACCEPTED MANUSCRIPT
Although several publications emphasized that the vinylogous Michael
addition to α,β-unsaturated carbonyl derivatives provides a facile access to
important biological scaffolds, no report on the biological or pharmacological
evaluation of the α,α-dicyanoolefin adducts has been published so far.
Interestingly, Kiruthika et al. [21] described the antimicrobial activity of
spirocarbocycles obtained through vinylogous Michael addition of alkylidene
malononitriles to isoxazolone- or isatin-chalcones followed by in situ
intramolecular cyclization. Therefore, in this work we also report the
antiplasmodial and cytotoxic activity of the Michael adducts.
Scheme 1. Synthetic approaches for the asymmetric Michael addition of α,α-
dicyanoolefins to chalcones.
2. Results and discussion
Based on a previous report [22], we initiated our study of the Michael
addition reaction of chalcone 1a with α,α-dicyanoolefin 2a using 9-amino-9-
deoxyepiquinine 3a as organocatalyst in THF, with TFA (20 mol%) as co-
catalyst (Scheme 2). The reaction was monitored by TLC and after 96 h at room
temperature the desired product 4a was obtained with 56% isolated yield and
97% enantiomeric excess (e.e.). Looking for greener solvents, we have tried to
perform this reaction in ethanol, however the desired product was obtained in
3

ACCEPTED MANUSCRIPT
only 6% yield and 71% e.e. Hydrogen bond donor organocatalysts 3b and 3c
were also evaluated furnishing 4a in low yields and e.e.
O
O
Cat. 3 (20 mol%)
CN
CN
*
*
+
CN
Solvent t, TFA,
r.t., 96 h
1a
CN
4a
2a
N
O
O
H
CF3
NH2
O
N
CF3
H
O
N
H
O
N
S
N
H
NH
CF3
N
H
N
CF3
N
H
3a
3b
N
Scheme 2. Organocatalysts evaluated in the Michael addition reaction
Having defined 3a as the catalyst of choice, we then investigated the
influence of reaction time, temperature and co-catalyst (Table 1). The reaction
was performed up to five days at room temperature and the best results were
still achieved in 4 days (Table 1, entries 1-5). Unfortunately, no changes in
chemical yield was observed using three equivalents of α,α-dicyanoolefin 2a
(Table 1,entry 6). We further evaluated the influence of the reaction
temperature, however, carrying out the Michael addition at 4 or -20 oC (Table 1,
entries 7 and 8) no improvement on chemical yield and/or e.e. could be
observed. Microwave irradiation was then tested, but at 60 or 80ºC most of the
starting materials were recovered, and at 100°C pro duct degradation and
formation of a complex mixture was observed (Table 1, entries 9-11). Finally,
we have examined the influence of different co-catalysts, and no improvements
in terms of yield and selectivity were observed when benzoic acid or 3,5-
dinitrobenzoic acid have been used (Table 1, entries 12 and 13).
In the next step, we turned our attention to the catalyst loading. A
decrease in the reaction rate was observed by decreasing the catalyst loading
to 10 mol% (both organocatalyst 3a and TFA) – however, when 15 mol% of
4

ACCEPTED MANUSCRIPT
catalyst were evaluated, the Michael adduct could be obtained in similar results
as for 20 mol% (Table 2, entries 1 and 2).
Table 1. Influence of co-catalyst, reaction time and temperature.a
Co-
catalyst
TFA
Temp.
(°C)
e.e.c
(%)
94
Entry
Time (h)
Yieldb (%)
1
2
r.t.
24
48
18
47
49
57
31
14
40
13
11
16
12
12
TFA
TFA
TFA
TFA
TFA
TFA
TFA
TFA
TFA
TFA
BzOH
DNBA
r.t.
r.t.
r.t.
60
r.t.
4
97
94
94
0
3
72
4
120
48
5
6d
96
91
91
92
90
80
0
7
96
8
-20
60
80
100
r.t.
r.t.
96
9e
10e
11e
12f
13g
0.5
0.5
0.5
96
70
95
96
32
a
b
Conditions: 1a (0.5 mmol), 2a (0.75 mmol; 1.5 eq), THF (2.5 mL), isolated yield after
c
d
e
purification by chromatography column determined by chiral HPLC. 3 eq of 2a. MW
irradiation (30W).
f BzOH = benzoic acid. g DNBA=3,5-dinitrobenzoic acid.
Towards the development of a more sustainable protocol, we then
evaluated greener solvents. In this sense, we first tested polar solvents such as
water [23], ethanol and acetonitrile (Table 2, entries 3-6), which afforded 4a in
low yield and e.e. On the other hand, ethyl acetate and ethyl carbonate
provided the desired product with moderate yields and excellent e.e. (Table 2,
entries 7 and 8). The best results were obtained with the bio-based solvent 2-
5

ACCEPTED MANUSCRIPT
MeTHF [24], using either 20 or 15 mol% of both 3a and TFA (Table 2, entries 9
and 10). When the reaction was carried out in the absence of TFA, low yield
and e.e. were observed (Table 2, entry 11).
Table 2. Screening of solvents and influence of catalyst loading.a
3a and TFA
(x mol%)
Entry
Solvent
Yield (%)b
e.e. (%)c
1
2
THF
THF
15
59
33
13
13
6
94
95
0
10
20
20
20
20
20
20
20
15
20
3
H2O
4d
5e
6
H2O
82
71
0
EtOH
MeCN
AcOEt
CO3(Et)2
2-MeTHF
2-MeTHF
2-MeTHF
22
56
47
61
61
31
7
93
97
95
96
40
8
9
10
11f
a Conditions: 1a (0.5 mmol), 2a (0.75 mmol; 1.5 eq), solvent (2.5 mL). b isolated yield
c
after purification by chromatography column. enantiomeric excess determined by chiral-phase
d
e
f
HPLC analysis. benzoic acid (30 mol%) instead of TFA for 3 days. TFA (40 mol%). without
co-catalyst.
On the basis of these results, we initiated the study of the scope and
limitations of the vinylogous Michael addition. Firstly, we evaluated the influence
of electron donor and withdrawing groups on the para position on both aromatic
rings of chalcones with the α,α-dicyanoolefin 2a (Table 3). The desired products
were successfully isolated in moderate yields and good to excellent
enantiomeric excess. We have observed that the electronic nature of the
6

ACCEPTED MANUSCRIPT
substituents has no or very little effect in the reaction yield, as for example when
nitro (compounds 4b and 4f), trifluoromethyl (4k), halogens (compounds 4c, 4h
and 4l) as well as methoxy groups (compounds 4d and 4g) were employed.
Table 3. Michael addition of different chalcones with α,α-dicyanoolefin 2a.
Yield
(%)a
61
e.e.
(%)b
96
Compound
R1
R2
R3
R4 R5
R6
d.r.c
4a
4b
4c
4d
4e
4f
H
H
H
H
H
H
H
H
H
H
H
H
NO2
Br
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
20:1
20:1
20:1
20:1
56:44
20:1
20:1
20:1
20:1
20:1
20:1
20:1
20:1
66
97
H
H
59
98
H
OMe
H
H
60
95
84d
OMe
H
H
32
H
NO2
OMe
H
62
98
4g
4h
4i
H
H
40
71
H
H
58
96
H
Cl
I
45
96
4j
H
-O-CH2-O-
Br
42
95
4k
4l
H
H
H
H
CF3
Br
H
70
97e
94e
97e
H
OMe
55
4m
H
Br
-O-CH2-O-
47
b
a isolated yield after purification by chromatography column. enantiomeric excess calculated
c
d
using chiral-phase HPLC analysis, diastereoisomeric ratio measured by 1 H NMR, for the
major diastereoisomer. e using 3a (20 mol%), TFA (20 mol%) in THF.
Interestingly, when a chalcone with a methoxy substituent in the ortho
position was employed, the reaction furnished a mixture of two
1
diastereoisomers in 56:44 ratio measured by H NMR. The minor isomer 4eb,
isolated in 19% yield, had its structure unambiguously determined by X-ray
structure analysis (Figure 1a) as being the syn-adduct while the major one,
7

ACCEPTED MANUSCRIPT
which was isolated in 32% yield, was characterized by NMR experiments as the
anti-adduct 4ea (see support information).
Furthermore, chalcones containing two or three substituents in the meta
and para positions furnished the corresponding products 4i, 4j, 4l and 4m in 42-
55% yield and high e.e. The single crystal X-ray analysis of vinylogous product
4i confirmed the absolute stereochemistry to be (6S, 11R) (Figure 1b).
a)
b)
Figure 1. X-ray structures of Michael adducts a) 4eb and b) 4i.
We then evaluated the influence of heteroaromatic rings in the chalcone
(Scheme 3). To our delight, products containing pyridine and thiophene as
moieties (4n-4p) were obtained with moderate yield and good e.e, however the
furan derivative did not afford the desired product 4q.
Continuing with the scope and limitation study, we turned our attention to
the α,α-dicyanoolefins. In this sense, the cyclohexane derivative 4r was
efficiently obtained in 55% and 98% e.e. The cyclopentanone 4s and
dehydroepiandrosterone 4t derivatives were also isolated however in lower
yields (10 and 23%, respectively). Interestingly, when the acetone derivative
was employed, the Michael adduct underwent a 6-exo-trig cyclization furnishing
the 4u in 36% yield and 96% e.e.
Additionally, trans,trans-dibenzylideneacetone was successfully used
with the α,α-dicyanoolefin 2a, furnishing the corresponding adduct 4v in 45%
yield and excellent d.r. and e.e. Finally, aiming to expand our protocol to α,β-
unsaturated ketones, we employed the (E)-1-phenylbut-2-en-1-one, instead of a
chalcone, and the desired Michael adduct 4w was obtained in 34% yield and
8

ACCEPTED MANUSCRIPT
91% e.e. It is worth noting that this transformation has been previously reported
by Xie et al. [22] with 60% yield and same e.e. but using DIPEA (15 mol%) as
base and 20 mol% of catalyst 3a in THF as solvent.
Scheme 3. Scope & limitations of the Michael addition.
According to previous reports, this organocatalysed Michael addition
reaction could proceed via activation by hydrogen bonding [25,26] or formation
of an iminium ion [22,27]. To contribute with the mechanism proposal, an
analysis using an infrared probe of the mixture of chalcone 2a, catalyst 1a and
TFA in 2-MeTHF was performed every 30 minutes over a period of 16 hours.
The spectra were measured using the blank of the sample with 2-MeTHF and
TFA, in order to follow the decrease of the band at 1670 cm-1 corresponding to
the chalcone. After the addition of chalcone in 40 minutes of reaction, the
9

ACCEPTED MANUSCRIPT
immobilized ketone-ion balance could be observed due to the slight decrease of
the carbonyl band, demonstrating evidence that the reaction occurs via iminium
ion (see supporting information for further details).
Therefore, we proposed that this Michael addition occurs via nucleophilic
addition of the primary amine of the catalyst 3a to the carbonyl of chalcone 1
furnishing the corresponding iminium ion. In parallel, the tertiary amine of the
catalyst deprotonates the α,α-dicyanoolefin 2 and then a 1,4-addition occurs.
The steric hindrance caused by the aromatic ring of the iminium ion with the
quinoline moiety makes the E configuration more favourable. Thus, the
nucleophilic approach would occur by the less hindered Si-face (Figure 2) [28].
Figure 2. Proposed transition state for Michael addition.
The Michael adducts 4 were tested against the chloroquine sensitive 3D7
strain of Plasmodium falciparum for determination of the antiplasmodial
potency. Malaria is one of the most prevalent parasitic infections in the world
and its control still relies on chemotherapy; however, drug resistance is a
limiting factor. Therefore, the discovery and development of new and efficacious
drugs for the treatment and prevention of malaria are urgently needed to both
overcome resistance and meet the challenge of eradicating malaria [29].
Briefly, a culture of P. falciparum was incubated for 72 hours with serial
dilutions of each compound (concentration range 10 – 0.156 µM), after which
parasite viability was indirectly determined by the addition of the SYBR green
stain and fluorescence determination. Viability controls (non-treated parasitized
RBCs and non-parasitized cells) were used for signal normalization, and
concentration-response curves were used to determine IC50 values.
Compounds with IC50 below 10 µM were considered active in this screening
[30]. Table 4 shows the IC50 values determined for each compound. Ten out of
10

ACCEPTED MANUSCRIPT
the fifteen compounds tested were active against the parasite, with IC50 values
between 1.8 and 9.3 µM.
The antiplasmodial potencies for each compound were then analysed
regarding the structure-activity relationships. The IC50 value of the unsubstituted
derivative (4a) was 8 µM. Substitutions on the A- or B-ring were favourable for
the antiplasmodial activity of the series. For instance, methoxy (4d, IC50 = 4.8
µM) or nitro (4b, IC50 = 5.7 µM) substituents in the para position of A-ring
improved the antiplasmodial potency, while a bromide on the same position
caused loss of activity (4c, IC50 > 10 µM). In the ortho position, a methoxy
substituent increased 3-fold the inhibitory activity (4e, IC50 = 2.9 µM). As for B-
ring, both methoxy (4g, IC50 = 3.4 µM) and nitro (4f, IC50 = 3.3 µM) substituents
in the para position resulted in compounds with improved inhibitory potency.
The replacement of A- or B-ring with thiophene (4o and 4p) or pyridine (4n)
decreased log P of the compounds. However, the new derivatives were inactive
(IC50 > 10 µM). Finally, the replacement of the tetrahydronaphthalene ring with a
smaller cyclohexane moiety caused loss of antiplasmodial activity (4r, IC50 > 10
µM), while the dehydroepiandrosterone derivative (4t, IC50 = 1.8 µM) resulted in
the most potent compound of the series. The preliminary SAR is summarized in
Figure 3.
The cytotoxicity of the active compounds was assayed against HepG2
cells (human hepatoma cell line) using the MTT assay [31]. In short,
compounds are incubated with HepG2 cells for 24 hours, after which the MTT
salt is added. After another 24 hours of incubation, cell viability for each
compound concentration is obtained through a colorimetric assay. The active
compounds 4i, 4g, 4u, and 4t had selectivity index (SI) values higher than 10,
which indicate good safety profile. For some compounds (4a, 4l, 4b, 4d, and
4e), the low solubility in assay medium prevented the accurate determination of
the SI, and for 4f no EC50 value was determined due to compound precipitation
in the assay condition. However, we did not observe a reduction in cells viability
at the highest tested concentration, thereby suggesting that this series has
reasonable cytotoxicity.
11

ACCEPTED MANUSCRIPT
Table 4. Antiplasmodial and cytotoxic activity of the Michael adducts.
Compound
LSPN645 (4a)
LSPN651 (4b)
R1
R2
IC50 (µM) (CI95%)a
HepG2 EC50 (µM) Selectivity index
H
H
8 (5 - 11)
> 6.3
> 25
> 0.8
> 4
H
H
H
5.7 (5.6 – 5.8)
> 10
LSPN650 (4c)
LSPN652 (4d)
n.d.
n.d.
> 5
4.8 (4.6 – 5.0)
> 25
LSPN653 (4ea)
LSPN654 (4f)
H
2.9 (2.1 – 3.7)
3.3 (2.6 – 4.0)
> 3.1
n.d.b
> 1
H
H
n.d.b
LSPN656 (4g)
LSPN655 (4i)
LSPN649 (4l)
LSPN657 (4n)
3.4 (3.3 – 3.5)
3.9 (3.5 – 4.3)
4 (3 – 5)
> 50
> 100
> 25
n.d.
> 15
> 26
> 6
n.d.
H
> 10
12

ACCEPTED MANUSCRIPT
n.d.
n.d.
LSPN658 (4o)
H
> 10
n.d.
n.d.
LSPN659 (4p)
H
> 10
LSPN661 (4r)
LSPN665 (4t)
> 10
> 10
n.d.
1.8 (1.3 – 2.4)
1.8 (1.3 – 2.4)
> 400
LSPN664 (4u)
9.3 (9.0 – 9.6)
9.3 (9.0 – 9.6)
> 100
39655
Artesunate
0.0056 (0.004 – 0.008)
230 (150 – 320)
a P. falciparum 3D7 strain. b compound precipitated in all bioassay tested concentrations. n.d.: not determined.
13

ACCEPTED MANUSCRIPT
Figure 3. Preliminary SAR analysis of Michael adducts with antiplasmodial
activity.
3. Conclusions
We report herein the use of a bifunctional catalyst derived from the
Cinchona alkaloid to perform the enantioselective addition of α,α-dicyanoolefins
to chalcones. The Michael addition reaction was optimized by investigating
time, acid additive, temperature, catalyst loading and solvent. Looking for more
environmentally sustainable conditions, we have tested a series of greener
solvents and 2-MeTHF at 15 mol% of catalyst loading proved to be the best
condition for this transformation. We also investigated the scope and limitations
of the developed protocol, where 22 new compounds were synthesized with
yields in the range of 10-70% and 71-98% enantiomeric excesses.
The compounds had their antiplasmodial and cytotoxic effect evaluated
against P. falciparum and human cells, respectively. The biological evaluation
indicated that the new compounds are inhibitors of the parasite growth in the
low micromolar range and have an attractive safety profile. A preliminary SAR
investigation suggested that the addition of methoxy substituent on A- or B-ring
is favourable for antiplasmodial activity.
14

ACCEPTED MANUSCRIPT
4. Experimental section
4.1. General information
All reagents used were commercially available from Sigma-Aldrich. The
solvents were from commercial sources and when required were treated and
dried as according to the literature. Purification of the products was performed
by flash chromatographic column, using silica gel 60, 230-400 mesh ASTM
Merck and silica gel 60 A, 70-230 mesh Aldrich Co. TLC analyses were
performed using Macherey-Nagel ALUGRAM® Xtra aluminium sheets with 254
nm fluorescent indicator and staining using a solution of vanillin in ethanol.
Chemical yields refer to pure and dry products after chromatographic
purification or solvent washing. The NMR analyses were performed in Bruker
ARX 400 MHz spectrometers and chemical shifts (δ) are expressed in ppm
referenced by the residual solvent signal and the coupling constants (J) in Hertz
(Hz). To indicate the multiplicity of the signals the following abbreviations were
used: s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), dd (doublet
of doublets), ddd (doublet of doublet of doublets), ddt (doublet of doublet of
triplets), dt (doublet of triplets), td (triplet of doublets) and tt (triplet of triplets).
The enantiomeric ratios of the optimization study were determined using high
performance liquid chromatography (HPLC), Shimadzu, LC-10ATVp pump, UV-
vis detector model: 10AVP with SCL-10 AVP model controller. The
enantiomeric ratios of the reaction scope were determined employing Waters
ACQUITY's Ultrafast chromatography (UPC²). The values of [α]D were
determined using the Schmidt Haensch polarimeter, model Polatronic H100, at
589 nm and 22ºC. The IR spectra were generated on a Shimadzu
spectrophotometer, model IR Prestigie-21. The samples were macerated in KBr
to generate the pellets. The infrared probe experiment was performed in a
Mettler Toledo spectrometer, model ReactIR15 and analysed in situ. The
absorptions were expressed in wave number (cm-1). High resolution mass
spectra were obtained in a Bruker Daltonics mass spectrometer by ESI-TOF
spray electron ionization at the Faculty of Pharmaceutical Sciences of Ribeirão
Preto, University of São Paulo. The melting points were obtained using a Büchi
apparatus, model M-560 and reported in degrees Celsius (°C). Reactions
performed under microwave irradiation were done using a CEM Discovery®
15

ACCEPTED MANUSCRIPT
oven. The x-ray analysis was performed on a Bruker APEX-II CCD
diffractometer at the São Carlos Institute of Chemistry, University of São Paulo.
4.2. Materials
Chalcone derivatives 1a-q [32,33], α,α-dicyanoolefins 2a-f [34,35], and catalyst
3a [36] were synthesized by following the literature procedures (see support
information for details).
4.3. Synthesis of Michael adducts 4a-x
In a 10 mL vial was added catalyst 3a (24 mg, 0.075 mmol), chalcone 1a (104
mg, 0.5 mmol) and 2-MeTHF (2.5 mL). This mixture was stirred for 30 min and
then added α,α-dicyanoolefin 2a (146 mg, 0.75 mmol). After 15 min, TFA (5.74
ꢀL, 0.075 mmol) was added and the reaction mixture was stirred for 4 days at
room temperature.
2-((S)-2-((R)-3-oxo-1,3-diphenylpropyl)-3,4-dihydronaphthalen-1(2H)-
ylidene)malononitrile (4a). The product was obtained as a white solid in 61%
yield (122 mg, 0.3 mmol) after purification by chromatography column using 95:
1
5 Hexane-EtOAc as eluent. H NMR (400 MHz, CDCl3) δ: 8.03 (d, J = 7.4 Hz,
1H), 7.74 (dd, J = 8.3, 1.1 Hz, 2H), 7.62 - 7.48 (m, 2H), 7.43-7.34 (m, 3H), 7.32
(dd, J = 4.2, 3.4 Hz, 5H), 7.25-7.19 m, 1H), 3.66 (dt, J = 11.2, 3.5 Hz, 1H), 3.53
(dd, J = 16.4, 9.6 Hz, 1H), 3.40 (ddd, J = 4.14, 9.70, 11.17 Hz, 1H), 3.08 (ddd, J
= 16.5, 11.9, 4.8 Hz, 2H), 2.83 (dd, J = 19.0, 6.5 Hz, 1H), 2.03-1.86 (m, 1H),
1.77 (ddd, J = 9.0, 6.5, 3.2 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ: 197.1; 177.3;
140.9; 140.1; 136.4; 134.1; 133.3; 130.0; 128.9; 128.8; 128.7; 128.6; 128.0;
127.9; 127.4; 127.0; 113.8; 113.6; 80.5; 47.5; 43.2; 41.7; 25.7; 24.4. ee: 96%
measured by UPC2 on a Trefoil AMY1 column, CO2/iPrOH gradient [CO2 (1
min), CO2 at 60:40 (4 min), 60:40 (3 min)], 2 mL/min, 35 °C, 137.89 bar, λ =
250.0 nm, tmajor= 3.448 min, tminor= 3.136 min. [α]D22 = -140.3 (c = 0.068, CHCl3).
HRMS-ES+ m/z: [M + H]+ calcd for C24H22N2O 403.1805; found 403.1804. IR
(λmax): 2233, 1687, 1546 cm-1. mp: 161.4-162.9 °C.
16

ACCEPTED MANUSCRIPT
2-((S)-2-((R)-3-(4-nitrophenyl)-3-oxo-1-phenylpropyl)-3,4-dihydronaphthalen-
1(2H)-ylidene)malononitrile (4b). The product was obtained as a white solid in
66% yield (149 mg, 0.33 mmol) after purification by chromatography column
using Hexane-EtOAc 95 : 5 as eluent. 1H NMR (400 MHz, CDCl3) δ: 8.24 – 8.16
(m, 2H), 8.06 (dd, J = 8.0, 0.9 Hz, 1H), 7.91 – 7.82 (m, 2H), 7.59 (td, J = 7.6, 1.3
Hz, 1H), 7.40 (td, J = 8.1, 1.1 Hz, 1H), 7.36 – 7.26 (m, 5H), 7.23 (dt, J = 3.3, 2.0
Hz, 1H), 3.68 (dt, J = 11.3, 3.6 Hz, 1H), 3.56 (dd, J = 16.5, 9.7 Hz, 1H), 3.36
(ddd, J= 4.32, 9,.88, 11.34 Hz, 1H), 3.17 – 2.98 (m, 2H), 2,85 (dd, J = 18.5, 6.1
13
Hz, 1H), 2.06 – 1.87 (m, 1H), 1.86 – 1.70 (m, 1H). C NMR (100 MHz, CDCl3)
δ: 196.0; 176.9; 150.4; 140.8; 140.2; 140.1; 134.3; 130.1; 129.1; 129.0; 128.7;
128.4; 127.8; 127.7; 127.1; 123.9; 113.8; 113.5; 80.6; 47.2; 43.8; 42.0; 25.6;
24.3. ee: 97%, measured by UPC2 on a Trefoil AMY1 column, CO2/iPrOH
gradient [CO2 (1 min), CO2 at 60:40 (4 min), 60:40 (3 min)], 1 mL/min, 35ºC,
137.89 bar, λ= 258.8 nm, tmaJor = 5.454 min, tminor = 5.314 min. [α]D22 = - 75.7 (c
= 0.103; CHCl3). HRMS–ES+ m/z: [M + Na]+ calcd para C28H21N3NaO3
470.1475; found 470.1464. IR (νmax): 2229, 1697, 1525, 1342 cm-1. mp: 186.7-
188.2ºC.
2-((S)-2-((R)-3-(4-bromophenyl)-3-oxo-1-phenylpropyl)-3,4-dihydronaphthalen-
1(2H)-ylidene)malononitrile (4c). The product was obtained as a light brown
solid in 59% yield (141 mg, 0.29 mmol) after purification by chromatography
1
column using 95: 5 Hexane-EtOAc as eluent. H NMR (400 MHz, CDCl3) δ:
8.04 (d, J = 7.8 Hz, 1H), 7.62-7.45 (m, 5H), 7.42-7.28 (m, 6H), 7.25-7.17 (m,
1H), 3.65 (dt, J = 11.2, 3.3 Hz, 1H), 3.47 (dd, J = 16.2, 9.6 Hz, 1H), 3.41-3 , 30
(m, 1H), 3.15-2.96 (m, 2H), 2.84 (dd, J = 18.2, 6.3 Hz, 1H), 2.02-1.88 (m ,1H),
1.85-1.71 (m, 1H).13C NMR (100 MHz, CDCl3) δ: 197.1; 177.3; 140.9; 140.1;
136.4; 134.1; 133.3; 130.0; 128.9; 128.8; 128.6; 128.6; 128.0; 127.9; 127.4;
127.0; 113.8; 113.6; 80.5; 47.5; 43.2; 41.7; 25.7; 24.4. ee: 98%, measured by
UPC² on a Trefoil AMY1 column, CO2/iPrOH gradient [CO2 (1 min), CO2 at
60:40 (4 min), 60:40 (3 min)], 1 mL/min, 137.89 bar, λ = 326.3 nm, tmaJor = 5.511
min, tminor= 5.374 min. [α]D 20 = -84.7 (c = 0.127; CHCl3). HRMS–ES+ m/z: [M +
H]+ calcd for C28H21BrN2O 481.0910 found 481.0906. IR (νmax): 2232, 1691,
1571, 698 cm-1. mp: 202.3 - 203.8 ºC.
17

ACCEPTED MANUSCRIPT
2-((S)-2-((R)-3-(4-methoxyphenyl)-3-oxo-1-phenylpropyl)-3,4-
dihydronaphthalen-1(2H)-ylidene)malononitrile (4d). The product was obtained
as a white solid in 60% yield (130 mg, 0.3 mmol) after purification by
1
chromatography column using 9:1 Hexane-EtOAc as eluent. H NMR (400
MHz, CDCl3) δ: 8.02 (d, J = 7.6 Hz, 1H), 7.76 – 7.71 (m, 2H), 7.55 (td, J = 7.6,
1.0 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.34 – 7.28 (m, 5H), 7.22 (dt, J = 5.8, 4.1
Hz, 1H), 6.91 – 6.80 (m, 2H), 3.84 (s, 3H), 3.65 (dt, J = 11.0, 3.5 Hz, 1H), 3.53 –
3.34 (m, 2H), 3.17 – 2.95 (m, 2H), 2.82 (dd, J = 18.2, 6.0 Hz, 1H), 2.04 – 1.86
(m, 1H), 1.82 – 1.71 (m, 1H). 13C NMR (100 MHz, CDCl3) δ: 195.5; 177.4;
163.6; 141.0; 140.1; 134.1; 130.3; 129.9; 129.6; 128.9; 128.8; 128.6; 127.9;
127.4; 126.9; 113.8; 113.6; 80.4; 55.5; 47.5; 42.8; 41.8; 25.7; 24.4. ee: 95%,
measured by UPC² on a Trefoil AMY1 column, CO2/iPrOH gradient [CO2 (1
min), CO2 at 60:40 (4 min), 60:40 (3 min)], 1 mL/min, 35ºC, 137.89 bar, λ=
327.9 nm, tmajor = 5.568 min, tminor = 5.387 min. [α]D 22 = -100 (c = 0.119; CHCl3).
HRMS–ES+ m/z: [M + H]+ calcd for C29H24N2O2 433.1911; found 433.1907. IR
(νmax): 2231, 1664, 1602 cm-1. mp: 119.7 - 121.1 ºC.
2-((S)-2-((R)-3-(2-methoxyphenyl)-3-oxo-1-phenylpropyl)-3,4-
dihydronaphthalen-1(2H)-ylidene)malononitrile (4ea). The product was obtained
as a white in 32% yield (71 mg, 0.16 mmol) after purification by chromatography
column using Hexane-EtOAc 95: 5 as eluent. 1H NMR (400 MHz, CDCl3) δ: 8.00
(dd, J = 8.0, 0.8 Hz, 1H), 7.47 (td, J = 7.6, 1.3 Hz, 1H), 7.37-7.28 (m, 3H), 7.27-
7.19 (m, 5H), 7.18-7.10 (m, 1H), 6.85 (d, J = 8.2Hz, 1H), 6.86 (td, J = 7.8, 0.9
Hz, 1H), 3.85 (s, 3H), 3.62 (dd, J = 17.7,10.1Hz, 1H), 3.51 (dt, J = 11.4, 3.5 Hz,
1H), 3.27 (ddd, J = 11.2, 10.3, 4.0 Hz, 1H), ), 3.11 (dd, J = 17.7, 1 H), 3.14-2.87
(m, 1H), 2.74 (dd, J = 18.5, 6.4 Hz, 1H), 2.02-1.76 (m, 1H) , 1.76-1.58 (m, 1H).
13C NMR (100 MHz, CDCl3) δ: 198.9; 177.6; 158.8; 141.5; 140.3; 134.2; 134.0;
130.4; 130.0; 128.9; 128.8; 128.7; 128.2; 127.5; 127.2; 127.0; 120.7; 114.0;
113, 9; 111.6; 80.4; 55.6; 48.8; 47.8; 41.6; 25.7; 24.6. ee: 84%, measured by
UPC² on a Trefoil AMY1 column, CO2/iPrOH gradient [CO2 (1 min), CO2 at
60:40 (4 min), 60:40 (3 min)], 1 mL/min, 35ºC, 137.89 bar, λ= 240 nm, tmajor
=
22
5.10 min, tminor= 5.59 min. [α]D = -117.4 (c =0.084; CHCl3). HRMS–ES+ m/z:
18

ACCEPTED MANUSCRIPT
[M + H]+ calcd for C29H24N2O2 433.1811; found 433.1913. IR (νmax): 2216, 1670,
1597cm-1. mp: 161.3 - 162.1 ºC.
2-((R)-2-((R)-3-(2-methoxyphenyl)-3-oxo-1-phenylpropyl)-3,4-
dihydronaphthalen-1(2H)-ylidene)malononitrile (4eb). The product was obtained
as a white solid in 19% yield (42 mg, 0.1 mmol) after purification by
1
chromatography column using Hexane-EtOAc 95: 5 as eluent. H NMR (600
MHz, CDCl3) δ 7.54 – 7.48 (m, 1H), 7.44 – 7.40 (m, 1H), 7.36 (dd, J = 7.7, 1.7
Hz, 1H), 7.29 (dt, J = 9.9, 6.7 Hz, 1H), 7.22 – 7.15 (m, 1H), 6.93 (d, J = 8.4 Hz,
1H), 6.91 – 6.89 (m, 1H), 6.89 – 6.85 (m, 1H), 3.90 (s, 1H), 3.58 (dt, J = 10.7,
3.6 Hz, 1H), 3.54 (dd, J = 17.4, 8.7 Hz, 1H), 3.46 (dd, J = 17.3, 4.7 Hz, 1H),
3.41 (ddd, J = 10.8, 8.8, 4.7 Hz, 1H), 3.24 (ddd, J = 17.8, 11.2, 6.3 Hz, 1H),
13
2.95 (dd, J = 18.1, 6.4 Hz, 1H), 2.40 – 2.29 (m, 1H), 2.18 – 2.10 (m, 1H). C
NMR (100 MHz, CDCl3) δ: 200.3; 177.8; 158.2; 141.1; 139.7; 133.7; 133.4;
130.2; 129.6; 129.4; 128.7; 128.4; 128.2; 127.8; 127.3; 126.5; 120.7; 113.5;
113.4; 111.4; 80.5; 55.6; 48.1; 47.4; 41.3; 25.4; 24.4. ee: 72%, measured by
UPC² on a Trefoil AMY1 column, CO2/iPrOH gradient [CO2 (1 min), CO2 at
60:40 (4 min), 60:40 (3 min)], 1 mL/min, 35ºC, 137.89 bar, λ= 240 nm, tmajor
=
4.930 min, tminor= 5.19 min. HRMS–ES+ m/z: [M + H]+ calcd for C29H24N2O2
433,1911; found 433,1913. IR (νmax): 2214, 1676, 1593 cm-1. mp: 138.3 - 139.4
ºC.
2-((S)-2-((R)-1-(4-nitrophenyl)-3-oxo-3-phenylpropyl)-3,4-dihydronaphthalen-
1(2H)-ylidene)malononitrile (4f). The product was obtained as a light yellow
solid in 62% yield (138 mg, 0.31 mmol) after purification by chromatography
column using 9: 1 Hexane-EtOAc as eluent. 1H NMR (400 MHz, CDCl3) δ: 8.26-
8.17 (m, 2H), 8.05 (d, J = 8.1 Hz, 1H), 7.76 (dd, J = 8.2, 1.1 Hz, 2H), 7.64-7.49
(m, 4H), 7.46-7.32 (m, 4H), 3.69 (dt, J = 11.0, 3.5 Hz, 1H), 3.62-3.52(m, 2H),
3.10 (dd, J = 16.3, 2.9 Hz, 1H), 3.06-2.99 (m, 1H), 2.90 (dd, J = 18.2, 7.4 Hz,
1H), 2.10 – 1. 90 (m, 1H), 1.80 – 1.64 (m, 1H).2. 7.4 Hz, 1H), 2.10-1.90 (m, 1H),
13
1.80-1.64 (m, 1H). C NMR (100 MHz, CDCl3) δ: 196.2; 175.8; 148.7; 147.2;
139.6; 135.9; 134.5; 133.8; 130.1; 129.0; 128.8; 128.8; 128.2; 127.9; 127.3;
124.2; 113.7; 113.3; 81.0; 46.8; 42.8; 41.4; 25.5; 24.2. ee: 98%, measured by
UPC² on a Trefoil CEL 2 column, CO2/iPrOH gradient [CO2 at 60:40 (5 min),
60:40 (2 min), CO2 (1 min)], 2 mL/min, 35ºC, 137.89 bar, λ= 230 nm, tmajor
=
19

ACCEPTED MANUSCRIPT
3.69 min, tminor = 3.42 min. [α]D 22 = -143.4 (c = 0.075; CHCl3). HRMS–ES+ m/z:
[M + H]+ calcd for C28H21N3O3 448.1656; found 448.1654. IR (νmax): 2218, 1683,
1523, 1348 cm-1. mp: 180.0 - 181.6 ºC.
2-((S)-2-((R)-1-(4-methoxyphenyl)-3-oxo-3-phenylpropyl)-3,4-
dihydronaphthalen-1(2H)-ylidene)malononitrile (4g). The product was obtained
as a white solid in 40% yield (88 mg, 0.2 mmol) after purification by
1
chromatography column using Hexane-EtOAc 95: 5 as eluent. H NMR (400
MHz, CDCl3) δ: 7.96 (d, J = 7.1 Hz, 1H), 7.68-7.64 (m, 2H), 7.49 (td, J = 7.6, 1.2
Hz, 1H), 7.44 (dt, J = 2.5, 1.6 Hz, 1H), 7.34-7.26 (m, 3H), 7.24 (d, J = 7.8 Hz,
1H) 7.17 – 7.08 (m, 2H), 6.81 – 6.73 (m, 2H), 3.70 (s, 3H), 3.54 (dt, J = 11.0, 3.5
Hz, 1H), 3.42 (dd, J = 10.9, 10.1, 4.0 Hz, 1H), 3.06 (dd, J = 16.4, 9.8 Hz, - 2.89
(m, 2H), 2.76 (dd, J = 18.3, 6.7 Hz, 1H), 1.94-1.81 (m, 1H), 1.77-1.67 (m,
1H).13C NMR (100 MHz, CDCl3) δ: 197.3; 177.5; 158.7; 140.1; 136.5; 134.1;
133.3; 132.7; 130.0; 128.9; 128.7; 128.6; 128.0; 126.9; 114.3; 113.8; 113.6;
80.4; 55.2; 47.6; 43.3; 41.0; 25.7; 24.3. ee: 71%, measured by UPC² on a
Trefoil CEL1 column, CO2/iPrOH gradient [CO2 (1 min), CO2 at 60:40 (4 min),
60:40 (3 min)], 1 mL/min, 35ºC, 137.89 bar, λ= 328.5 nm, tmajor = 5.115 min,
tminor = 5.024 min. [α]D = -71.7 (c = 0.099; CHCl3). HRMS–ES+ m/z: [M + H]+
22
calcd for C29H24N2O2 433.1911; found 433.1909. IR (νmax): 2227, 1680, 1510
cm-1. mp: 156.0 - 156.6 ºC.
2-((S)-2-((R)-1-(2-chlorophenyl)-3-oxo-3-phenylpropyl)-3,4-dihydronaphthalen-
1(2H)-ylidene)malononitrile (4h). The product was obtained as a white solid in
128 mg in 58% yield (0.29 mmol) after purification by chromatography column
using Hexane-EtOAc 90:10 as eluent. 1H NMR (400 MHz, CDCl3) δ 8.04 (d, J =
8.0 Hz, 1H), 7.70 (d, J = 7.5 Hz, 2H), 7.60-7.47(m, 2H), 7.49-7.28 (m, 7H), 7.19-
7.15 (m, 1H), 4.03 (td, J = 10.4, 4.7 Hz, 1H), 3.68 (dt, J = 10.9, 3.3 Hz, 1H),
3.45-3.27 (m, 2H), 3.15 (dd, J = 16.3, 4.8 Hz, 1H), 2.87 (dd, J = 18.6, 6.7 Hz,
1H), 2.08-1.90 (m, 1H), 1.87-1.75 (m, 1H). 13C NMR (100 MHz, CDCl3) δ: 196.6;
176.9; 140.4; 138.7; 136.2; 135.0; 134.2; 133.8; 133.4; 130.1; 130.0; 128.7;
128.6; 128.4; 128.0; 127.8; 127.7; 126.9; 113.8; 113.5; 80.7; 47.9; 43.6; 37.4;
25.4; 24.8. ee: 96%, measured by UPC² on a Trefoil AMY1 column, CO2/iPrOH
gradient [CO2 (1 min), CO2 at 60:40 (4 min), 60:40 (3 min)], 1 mL/min, 35ºC,
20

ACCEPTED MANUSCRIPT
22
137.89 bar, λ= 250 nm, tmajor = 4.16 min, tminor = 4.04 min. [α]D = -105.5 (c =
0.11; CHCl3). HRMS–ES+ m/z: [M + H]+ calcd for C28H21ClN2O 437.1415; found
437. 1416. IR (νmax): 2229, 1695, 1570, 744 cm-1. mp: 147.0-148.3ºC.
2-((S)-2-((R)-1-(4-chlorophenyl)-3-(4-iodophenyl)-3-oxopropyl)-3,4-
dihydronaphthalen-1(2H)-ylidene)malononitrile (4i). The product was obtained
as a light-yellow solid in 45% yield (126 mg, 0.22 mmol) after purification by
1
chromatography column using Hexane-EtOAc 95:5 as eluent. H NMR (400
MHz, CDCl3) δ: 8.03 (dd, J = 8.1, 1.0 Hz, 1H), 7.78 – 7.72 (m, 2H), 7.57 (td, J =
7.6, 1.2 Hz, 1H), 7.44 – 7.40 (m, 2H), 7.40 – 7.35 (m, 1H), 7.34 – 7.30 (m, 1H),
7.30 – 7.27 (m, 2H), 7.25 – 7.20 (m, 2H), 3.60 (dt, J = 11.1, 3.5 Hz, 1H), 3.43
(dd, J = 16.1, 9.9 Hz, 1H), 3.38 – 3.31 (m, 1H), 3.08 – 2.93 (m, 2H), 2.85 (dd, J
13
= 18.5, 6.2 Hz, 1H), 2.04 – 1.91 (m, 1H), 1.79 – 1.71 (m, 1H). C NMR (100
MHz, CDCl3) δ: 196.2; 176.6; 139.9; 139.1; 138.1; 135.5; 134.3; 133.3; 130.0;
129.3; 129.2; 128.7; 128.4; 127.1; 113.7; 113.4; 101.6; 80.7; 47.1; 43.0; 41.2;
25.5; 24.3. ee: 96%, measured by UPC² on a Trefoil CEL1 column, CO2/iPrOH
gradient [CO2 (1 min), CO2 at 60:40 (4 min), 60:40 (3 min)], 1 mL/min, 35ºC,
137.89 bar, λ= 321.0 nm, tmajor= 5.656 min, tminor = 5.474 min. [α]D22 = -69.4 (c =
0.108; CHCl3). HRMS–ES+ m/z: [M + H]+ calcd for C28H20ClIN2O 563.0382;
found 563.0400. IR (νmax): 2227, 1683,1579,1487, 769, 578 cm-1. mp: 159.8 -
161.2 ºC.
2-((S)-2-((R)-3-(benzo[d][1,3]dioxol-5-yl)-1-(4-bromophenyl)-3-oxopropyl)-3,4-
dihydronaphthalen-1(2H)-ylidene)malononitrile (4j). The product was obtained
as a white solid in 42% yield (44.1 mg, 0.084 mmol) after purification by
1
chromatography column using Hexane-EtOAc 95:5 as eluent. H NMR (400
MHz, CDCl3) δ: 1.72-1.78 (m, 1H), 1.90-2.00 (m, 1H), 2.84 (dd, J=18.7, 6.6 Hz,
1H), 2.90-2.97 (m, 1H), 2.99-3.07 (m, 1H), 3.32-3.43 (m, 2H), 3.58 (dt, J=11.0,
3.4 Hz, 1H), 6.01 (d, J=2.1 Hz, 2H), 6.78 (d, J=8.2 Hz, 1H), 7.17-7.19 (m, 3H),
7.30-7.38 (m, 3H), 7.43 (d, J=8.5 Hz, 2H), 7.55 (dt, J= 1.6, 7.0 Hz, 1H), 8.02 (d,
J=8.1 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ: 194.6; 176.7; 152.1; 148.3; 139.9;
139.8; 134.2; 132.0; 131.1; 129.9; 129.6; 128.7; 128.4; 127.0; 124.4; 121.2;
113.7; 113.4; 107.9; 107.6; 101.9; 80.6; 47.1; 42.7; 41.3; 25.6; 24.3. ee: 95%,
21

ACCEPTED MANUSCRIPT
measured by HPLC on a Chiralpak AS-H column (20% 2-propanol/hexane, 1
mL/min) tmajor = 32.61 min, tminor = 26.11 min. [α]D 20= -81.2 (c = 2.7; CHCl3).
2-((S)-2-((R)-3-oxo-1-phenyl-3-(4-(trifluoromethyl)phenyl)propyl)-3,4-
dihydronaphthalen-1(2H)-ylidene)malononitrile (4k). The product was obtained
as a white solid in 70% yield (65.8 mg, 0.14 mmol) after purification by
1
chromatography column using Hexane-EtOAc 95: 5 as eluent. H NMR (400
MHz, CDCl3) δ: 1.70-1.77 (m, 1H); 1.90-2.00 (m, 1H); 2.84 (dd, J=17.9; 6.3 Hz,
1H); 3.04 (dd, J=16.6; 4.3 Hz, 1H); 3.07 (dd, J=18.5; 6.1 Hz, 1H); 3.34-3.40 (m,
1H); 3.54 (dd, J=16.6; 9.6 Hz, 1H); 3.67 (dt, J=11.3; 3.5 Hz, 1H); 7.21-7.26 (m,
2H); 7.29-7.33 (m, 4H); 7.36-7.40 (m, 1H); 7.57 (td, J=7.5; 1.1 Hz, 1H); 7.63 (d,
13
J=8.3 Hz, 2H); 7.80 (d, J=8.1 Hz, 2H); 8.04 (d, J=7.8 Hz, 1H). C NMR (100
MHz, CDCl3) δ: 196.3; 176.9; 140.3; 140.0; 134.7; 134.3; 134.1; 132.4; 129.9;
128.9; 128.6; 128.4; 128.2; 127.8; 127.6; 126.9; 125.6 (q, J = 4.5 Hz); 122.0;
113.7; 113.4; 80.5; 47.2; 43.5; 41.8; 25.6; 24.3. ee: 97%, measured by HPLC
on a column Chiralpak AD-H (10% 2-propanol/hexane, 1 mL/min) tmajor = 11.50
min, tminor = 12.85 min. [α]D20 = -56.4 (c = 5.0, CHCl3).
2-((S)-2-((R)-3-(4-bromophenyl)-1-(4-methoxyphenyl)-3-oxopropyl)-3,4-
dihydronaphthalen-1(2H)-ylidene)malononitrile (4l). The product was obtained
as a white solid in 60% yield (56.2 mg, 0.11 mmol) after chromatography
1
column purification using Hexane-EtOAc 95: 5 as eluent. H NMR (400 MHz,
CDCl3) δ: 8.04 (dd, J = 8.0, 0.9 Hz, 1H), 7.60-7.53 (m, 3H), 7.53-7.47 (m, 2H),
7.38 (t, J = 7.2Hz, 1H), 7.31 (d, J = 7.7Hz, 1H), 7.20-7.16 (m, 2H), 6.91-6.77
2H), 3.77 (s, 3H), 3.60 (dt, J = 11.2, 3.5 Hz, 1H), 3.43 (dd, J = 16.2, 9.9 Hz, 1H),
3.35-3.26 (m, 1H), 3.04 (ddd, J = 19.0, 12.1, 6.5 Hz, 1H), 2.96 (dd, J = 2, 4.0
Hz, 1H), 2.83 (dd, J = 18.1, 6.8 Hz, 1H), 2.00-1.88 (m, 1H), 1.83-1.74 (m, 1 H).
13C NMR (100 MHz, CDCl3) δ: 196.4; 177.3; 158.8; 140.1; 135.2; 134.2; 132.3;
131.9; 130.0; 129.5; 128.8; 128.7; 128.5; 127.0; 114.3; 113.8; 113.5; 80.4; 55.2;
47.6; 43.3; 41.2; 25.6; 24.3. ee: 98%, measured by UPC² on a Trefoil AMY1
column, CO2/iPrOH gradient [CO2 (1 min), CO2 at 60:40 (4 min), 60:40 (3 min)],
0.8 mL/min, 35ºC, 137.89 bar, λ= 234.8 nm, tmajor = 6.392 min, tminor = 6.525
22
min. [α]D = - 65.4 (c = 0.088; CHCl3). HRMS–ES+ m/z: [M + Na]+ calcd para
22

ACCEPTED MANUSCRIPT
C29H23BrN2O2 533.0835; found 533.0842. IR (νmax): 2229, 1678, 1585, 1516,
572 cm-1. mp: 160.0-161.1ºC.
2-((S)-2-((R)-1-(benzo[d][1,3]dioxol-4-yl)-3-(4-bromophenyl)-3-oxopropyl)-3,4-
dihydronaphthalen-1(2H)-ylidene)malononitrile (4m). The product was obtained
as a white solid in 47% yield (49.3 mg, 0.094 mmol) after purification by
1
chromatography column using Hexane-EtOAc 95:5 as eluent. H NMR (400
MHz, CDCl3) δ: 1.79-1.87 (m, 1H); 1.91-2.00 (m, 1H); 2.84 (dd, J = 18.7, 6.4 Hz,
1H); 2.93 (dd, J = 16.3, 4.0 Hz, 1H); 3.00-3.09 (m, 1H); 3.28 (td, J = 10.6, 3.6
Hz, 1H); 3.38 - 3.44 (m, 1H); 3.56 (ddd, J = 11.5, 3.8, 3.6 Hz, 1H); 5.93 (s, 2H);
6.71-6.76 (m, 3H); 7.30-7.39 (m, 2H); 7.50-7.59 (m, 5H); 8.03 (d, J = 7.9 Hz,
1H).13C NMR (100 MHz, CDCl3) δ: 196.2; 177.0; 148.1; 146.8; 140.0; 135.1;
134.2; 134.1; 132.0; 131.9; 130.0; 129.5; 129.3; 128.7; 128.6; 128.4; 127.0;
113.7; 113.5; 108.5; 101.2; 80.5; 47.5; 43.2; 41.6; 25.6; 24.3. ee: 97%,
measured by HPLC on a Chiralpak ADH column (2% 2-propanol/hexane, 0.5
mL/min) tmajor = 180.53 min, tminor = 197.46 min. [α]D20= -48.1 (c = 3.4, CHCl3).
2-((S)-2-((R)-3-oxo-3-phenyl-1-(pyridin-2-yl)propyl)-3,4-dihydronaphthalen-
1(2H)-ylidene)malononitrile (4n).The product was obtained as a light brown
solid in 33% yield (67 mg, 0.17 mmol) after purification by chromatography
1
column using Hexane-AcOEt 85:15 as eluent. H NMR (400 MHz, CDCl3) δ
8.58-8.54 (m, 1H), 8.00 (dd, J = 7.9, 0.7 Hz, 1H), 7.83-7.79 (m, 2H), 7.67 - 7.58
(m, 1H), 7.56-7.49 (m, 2H), 7.44-7.36 (m, 2H), 7.35-7.28 (m, 3H) 7.19 - 7.12 (m,
1H), 4.00 - 3.87 (m, 2H), 3.55 (td, J = 10.7, 3.5 Hz, 1H), 3.24 – 3.07 (m, 1H),
2,99 (dd, J = 17.2, 3.6 Hz, 1H), 2.84 (dd, J = 18.2, 5.4 Hz, 1H), 2.05-1.91 (m,
1H), 1.72-1.61 (m, 1H). 13C NMR (100 MHz, CDCl3) δ: 197.6; 177.0; 160.0;
140.1; 136.3; 134.0; 133.4; 129.9; 128.8; 128.63; 128.58; 128.2; 128.1; 126.9;
124.5; 122.3; 113.7; 113.5; 80.9; 46.2; 42.9; 42.0; 25.5; 24.7. ee: 76%,
measured by UPC² on a Trefoil CEL 2 column, CO2/iPrOH gradient [CO2 at
60:40 (5 min), 60:40 (2 min), CO2 (1 min)], 1 mL/min, 40ºC, 137.89 bar, λ= 235
22
nm, tmajor = 4.440 min, tminor = 4.570 min. [α]D = -142.4 (c = 0.071; CHCl3).
HRMS–ES+ m/z: [M + H]+ calcd para C27H21N3O 404.1757; found 404.1758. IR
(νmax): 2222, 1687,1543 cm-1. mp: 126.2 - 127.5 ºC.
23

ACCEPTED MANUSCRIPT
2-((S)-2-((R)-3-oxo-3-phenyl-1-(thiophen-2-yl)propyl)-3,4-dihydronaphthalen-
1(2H)-ylidene)malononitrile (4o).The product was obtained as a white solid in
58% yield (118 mg, 0.29 mmol) after purification by chromatography column
using Hexane-EtOAc 9: 1 as eluent. 1H NMR (400 MHz, CDCl3) δ: 7.98 (dd, J =
8.0, 0.9 Hz, 1H), 7.81-7.78 (m, 2H), 7.58-7.52 (m, 2H), 7.45 - 7.38 (m, 2H),
7.38-7.29 (m, 2H), 7.17 (ddd, J = 5.1, 1.2, 0.5 Hz, 1H), 6.95 (dd, J = 3.5, 1.0 Hz,
1H), 6.91 (dd, J = 5.1, 3.5 Hz, 1H), 3.79 (ddd, J = 5, 4.4 Hz, 1H), 3.61 (dt, J =
11.1, 3.7 Hz, 1H), 3.54 (dd, J = 16.7, 9.4 Hz, 1H), 3.20-3.10 (m, 1H), 3.06 (dd, J
= 16.7, 4.4 Hz, 1H), 2.87 (ddd, J = 18.2, 6.2 ,2.4 Hz, 1H), 2.09-1.93 (m, 2H). 13C
NMR (100 MHz, CDCl3) δ: 196.7; 176.7; 143.8; 140.0; 136.3; 134.1; 133.5;
129.9; 128.7; 128.5; 128.0; 127.0; 126.9; 125.8; 124.3; 113.6; 113.5; 80.8; 48.5;
44.1; 37.2; 25.8; 24.5. ee: 93%, measured by UPC² on a Trefoil CEL 2 column,
CO2/iPrOH gradient [CO2 at 60:40 (10 min), 60:40 (2 min), CO2 (1 min)], 1
mL/min, 40ºC, 137.89 bar, λ= 300 nm, tmajor = 5.960 min, tminor = 6.130 min.
22
[α]D = -126.9 (c = 0.081; CHCl3). HRMS–ES+ m/z: [M + H]+ calcd para
C26H20N2OS 409.1369; found 409.1369. IR (νmax): 2239, 1687, 1579 cm-1. mp:
119.8 - 120.9 ºC.
2-((S)-2-((R)-3-oxo-1-phenyl-3-(thiophen-2-yl)propyl)-3,4-dihydronaphthalen-
1(2H)-ylidene)malononitrile (4p). The product was obtained as a light orange
solid in 27% yield (56 mg, 0.14 mmol) after chromatography column purification
1
using Hexane-EtOAc 9:1 as eluent. H NMR (400 MHz, CDCl3) δ: 7.97 (d, J =
7.9 Hz, 1H), 7.57-7.47 (m, 3H), 7.35-7.26 (m, 4H), 7.25 (s, 1H) , 7.23-7.17 (m,
2H), 7.02 (dd, J = 4.7, 4.1 Hz, 1H), 3.61 (dt, J = 10.9, 3.5 Hz, 1H), 3.41-3.29 (m,
2H), 3.09-2.94 (m, 2H), 2.78 (dd, J = 18.2, 6.1 Hz, 1H), 1.94 - 1.83 (m, 1H), 1.77
- 1.67 (m, 1H).13C NMR (100 MHz, CDCl3) δ: 189.8; 177.0; 143.9; 140.6; 140.0;
134.3; 134.1; 132.2; 129.9; 129.0; 128.8; 128.2; 127.9; 127.5; 127.0; 113.8;
113.5; 80.6; 47.4; 44.0; 41.9; 25.8; 24.4. ee: 89%, measured by UPC² on a
Trefoil AMY1 column, CO2/iPrOH gradient [CO2 (1 min), CO2 at 60:40 (4 min),
60:40 (3 min)], 1 mL/min, 35ºC, 137.89 bar, λ= 279,2 nm, tmajor = 5.096 min,
22
tminor = 4.938 min. [α]D = -51.6 (c =0.126; CHCl3). HRMS–ES+ m/z: [M + H]+
calcd for C26H20N2OS 409.1369; found 409.1367. IR (νmax): 2227, 1664, 1554
cm-1. mp: 133.7 - 135.0 ºC.
24

ACCEPTED MANUSCRIPT
2-((S)-2-((R)-3-oxo-1,3-diphenylpropyl)cyclohexylidene)malononitrile (4r). The
product was obtained as a light yellow solid in 55% yield (49 mg, 0.14 mmol)
after purification by chromatography column using Hexane-EtOAc 97:3 as
1
eluent. H NMR (400 MHz, CDCl3) δ: 7.87-7.82 (m, 2H), 7.59-7.52 (m, 1H),
7.47-7.40 (m, 2H), 7.38-7.28 (m, 4H), 7.23 (ddd, J = 6.9, 5.9, 2.5 Hz, 1H), 3.90
(ddd, J = 11.6, 7.7, 5.5 Hz, 1H), 3.40 (dd, J = 7.71, 17.24 Hz, 1 H) 3.38-3.33 (m,
1H), 3.07 (dd, J = 17.2, 5.4 Hz, 1H), 2.70 (d, J = 13.7, 5.8 Hz, 1H), 2.24-2.13
(m, 1H) 91 - 1.73 (m, 1H), 1.56 - 1.44 (m, 3H), 1.38 (tt, J = 4.09, 13.70 Hz, 1H).
13C NMR (100 MHz, CDCl3) δ: 197.5; 188.1; 141.3; 136.4; 133.6; 129.1; 128.8;
128.0; 127.4; 112.06; 111.3; 84.3; 48.9; 43.3; 41.6; 31.7; 30.5; 28.7; 19.6. ee:
98%, measured by UPC² on a Trefoil AMY column, CO2/iPrOH gradient [CO2 (1
min), CO2 at 60:40 (4 min), 60:40 (3 min)], 1 mL/min, 35ºC, 137.89 bar, λ=
22
287.7 nm, tmajor = 4.330 min, tminor = 4.525 min. [α]D = 55.6 (c = 0.1; CHCl3).
HRMS–ES+ m/z: [M + H]+ calcd for C24H22N2O 355.1805; found 355.1804. IR:
2232, 1674, 1579 cm-1. mp: 145.9 - 146.7 ºC.
2-((S)-2-((R)-3-oxo-1,3-diphenylpropyl)cyclopentylidene)malononitrile (4s). The
product was obtained as a pale orange solid in 10% yield (11 mg, 0.03 mmol)
after purification by chromatography column using Hexane-AcOEt 9: 1 as
1
eluent. H NMR (400 MHz, CDCl3) δ: 7.97 - 7.88 (m, 2H), 7.63 - 7.53 (m, 1H),
7.46 (t, J = 7.6 Hz, 2H), 7.39 - 7.26 (m, 5H), 3.82-3.69 (m, 1H), 3.63 (dd, J =
17.7, 7.9 Hz, 1H), 3.46-3.28 (m, 1H), 2.92-2.68 (m, 2H), 2.17-2.00 (m, 1H),
1.99-1.90 (m, 1H), 1.90-1.69 (m, 3H).13C NMR (100 MHz, CDCl3) δ: 197.8;
194.0; 141.5; 136.4; 133.6; 129.0; 128.8; 128.1; 128.1; 127.5; 112.6; 111.4;
82.7; 53.1; 43.2; 42.9; 34.9; 29.8; 21.9. ee: 83%, measured by UPC² on a
Trefoil CEL 2 column, CO2/iPrOH gradient [CO2 at 80:20 (10 min), 80:20 (2
min), CO2 (1 min)], 1 mL/min, 35ºC, 137.89 bar, λ= 225 nm, tmajor = 6.90 min,
22
tminor = 6.69 min. [α]D = 33.4 (c = 0.125; CHCl3). HRMS–ES+ m/z: [M + H]+
calcd for C23H20N2O 341.1648; found 341.1648. IR (νmax): 2231, 1681, 1600,
1446 cm-1. mp: 64.9 - 66.0 ºC.
(3S,8R,9S,10R,13S,14S,16S)-17-(dicyanomethylene)-10,13-dimethyl-16-((R)-3-
oxo-1,3-diphenylpropyl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-
25

ACCEPTED MANUSCRIPT
1H-cyclopenta[a]phenanthren-3-yl acetate (4t). The product was obtained as a
light yellow solid in 23% yield (37 mg, 0.06 mmol) after purification by
1
chromatography column using Hexane-EtOAc 9: 1 as eluent. H NMR (400
MHz, CDCl3) δ: 7.99-7.94 (m, 2H), 7.61-7.53 (m, 1H), 7.51-7.42 (m, 2H), 7.37-
7.26 (m, 3H ), 7.23-7.17 (m, 2H), 5.32 (t, J = 6.1 Hz, 1H), 4.63-4.51 (m, 1H),
4.23 (td, J = 7.3, 5.0 Hz, 1H), 3.57-3.48 (m, 2H), 2.42 (dt, J = 12.4, 3.6 Hz, 1H),
2.36– 2.20 (m, 2H), 2.03 (s, 3H), 1.94-1.71 (m, 4H), 1.61-1.27 (m, 7H), 1.13 (dd,
J = 4.47, 12.49 Hz, 1H), 1.09 - 1.03 (m, 1), 0.99 (s, 3H), 0.95 (s, 3H), 0.59 - 0.48
(m, 1H), 0.48-0.34 (m, 1H).13C NMR (100 MHz, CDCl3) δ: 199.5; 197.8; 170.6;
140.5; 139.9; 136.5; 133.5; 128.9; 128.8; 128.2; 128.1; 127.9; 121.6; 113.0;
111.5; 82.1; 73.6; 51.9; 50.8; 49.7; 49.3; 43.5; 42.1; 38.0; 36.7; 36.5; 32.7; 31.2;
30.7; 27.6; 27.2; 21.4; 20.5; 19.2; 17.8. rd: 92:8 %, measured by UPC² on a
Trefoil CEL 2 column, CO2/iPrOH gradient [CO2 at 60:40 (5 min), 60:40 (2 min),
CO2 (1 min)], 1 mL/min, 45ºC, 137.89 bar, λ= 250 nm, tmajor = 5.89 min, tminor
=
6.41 min. [α]D22 = -36.3 (c = 0.151; CHCl3). HRMS–ES+ m/z: [M + H]+ calcd for
C39H42N2O3 587.3268; found 587.3269. IR (νmax): 2230, 1728,1681, 1600, 1598,
1448, 1247 cm-1. mp: 90.1 -92.0 ºC.
(R)-2-(1',6'-dihydro-[1,1':3',1''-terphenyl]-5'(2'H)-ylidene)malononitrile (4u). The
product was obtained as a yellow solid in 36% yield (27 mg, 0.17 mmol) after
1
purification by chromatography column using Hexane-AcOEt 9: 1 as eluent. H
NMR (400 MHz, CDCl3) δ: 7.67 - 7.60 (m, 1H), 7.51 - 7.36 (m, 3H), 7.36 - 7.25
(m, 2H), 3.36 - 3.08 (m, 2H), 3.00-2.79 (m, 1H). 13C NMR (100 MHz, CDCl3) δ:
169.5; 157.6; 142.2; 137.8; 131.1; 129.1; 127.6; 126.8; 126.5; 120.6; 113.1;
112.5; 79.4; 40.0; 36.4; 36.1. ee: 96%, measured by UPC² on a Trefoil CEL1
column, CO2/iPrOH gradient [CO2 (1 min), CO2 at 60:40 (4 min), 60:40 (3 min)],
1 mL/min, 35ºC, 137.89 bar, λ= 286,1 nm, tmajor = 5.825 min, tminor = 5.685 min.
22
[α]D = -78.8 (c = 0.064; CHCl3). HRMS–ES+ m/z: [M + H]+ calcd for C21H16N2
297,1386; found 297,1387. IR (νmax): 2213, 1572 cm-1. mp:113.1 - 114.6 ºC.
2-((S)-2-((R,E)-3-oxo-1,5-diphenylpent-4-en-1-yl)-3,4-dihydronaphthalen-1(2H)-
ylidene)malononitrile (4v). The product was obtained as a yellow solid in 45%
yield (96 mg, 0.22 mmol) after purification by chromatography column using
26

ACCEPTED MANUSCRIPT
1
Hexane-EtOAc 9:1 as eluent. H NMR (400 MHz, CDCl3) δ: 7.99 (dd, J = 8.0,
0.9 Hz, 1H), 7.48 (td, J = 7.6, 1.2 Hz, 1H), 7.39 – 7.34 (m, 2H), 7.33 – 7.26 (m,
5H), 7.25 – 7.21 (m, 4H), 7.20 – 7.14 (m, 2H), 6.44 (d, J = 16.2 Hz, 1H), 3.56
(dt, J = 11.2, 3.5 Hz, 1H), 3.30 – 3.19 (m, 1H), 3.12 (dd, J = 16.0, 9.7 Hz, 1H),
2.99 (ddd, J = 18.0, 11.7, 6.2 Hz, 1H), 2.83 – 2.65 (m, 2H), 1.93 – 1.80 (m, 1H),
1.74 – 1.66 (m, 1H). 13C NMR (100 MHz, CDCl3) δ: 197.0, 177.3, 143.2, 140.8,
140.1, 134.1, 134.1, 130.7, 129.9, 128.9, 128.8, 128.6, 128.4, 127.9, 127.5,
127.0, 125.8, 113.8, 113.6, 80.5, 47.4, 45.6, 41.9, 25.7, 24.4. ee: 96%,
measured by HPLC on a Chiralpak AD-H column (5% 2-propanol/hexane, 0.8
mL/min) tmajor = 27.67 min, tminor = 23.82 min. [α]D21 = -96.04 (c = 0.104; CHCl3).
HRMS–ES+ m/z: [M + H]+ calcd for C30H25N2O 429,1967; found 429.1963. IR:
2227, 1697, 1620, 1573 cm-1. mp: 196.8 – 197.8 ºC.
2-((S)-2-((R)-3-oxo-1-phenylbutyl)-3,4-dihydronaphthalen-1(2H)-
ylidene)malononitrile (4w) [22]. The product was obtained as a brown solid in
34% yield (58mg, 0.17 mmol) after purification by chromatography column using
1
Hexane-EtOAc 9:1 as eluent. H NMR (400 MHz, CDCl3) δ: 8.03 (dd, J = 8.0,
0.9 Hz, 1H), 7.54 (td, J = 7.6, 1.2 Hz, 1H), 7.40 – 7.26 (m, 6H), 7.26 – 7.23 (m,
1H), 3.54 (dt, J = 11.3, 3.5 Hz, 1H), 3.15 (td, J = 10.7, 4.2 Hz, 1H), 3.09 – 2.92
(m, 2H), 2.81 (dd, J = 18.3, 6.0 Hz, 1H), 2.49 (dd, J = 16.4, 4.2 Hz, 1H), 1.96 –
13
1.85 (m, 1H), 1.89 (s, 3h), 1.77 – 1.69 (m, 1H). C NMR (100 MHz, CDCl3) δ:
13C NMR (101 MHz, CDCl3) δ 206.0, 177.2, 140.6, 140.0, 134.1, 129.9, 129.0,
128.7, 128.5, 127.8, 127.5, 127.0, 113.7, 113.6, 80.3, 48.0, 47.2, 41.5, 30.9,
25.6, 24.3. ee: 91%,measured by HPLC on a Chiralpak AS-H column (3%
ethanoll/hexane, 0.5 mL/min) tmajor = 24.61 min, tminor = 20.58 min. [α]D 21= -179.5
(c = 0.103; CHCl3). HRMS–ES+ m/z: [M + Na]+ calcd for C23H20N2ONa
363.1473; found 363.1468. IR: 2920, 2223, 1713, 1600, 1575 cm-1. mp: 151.0 –
151.9 ºC.
4.4. In vitro evaluation of antiplasmodial activity
The antiplasmodial activity of the compounds was tested against the P.
falciparum 3D7 clone, obtained from the MR4 source. The 3D7 clone was
derived from NF54 strain (originally isolated from a patient living in Amsterdam)
27

ACCEPTED MANUSCRIPT
by limiting dilution. This clone is sensitive to all antimalarials and routinely used
for screening new lead candidates for malaria. A continuous parasite culture
was maintained as described by Jansen and Trager [37]. Compounds were
solubilized in dimethyl sulfoxide (DMSO). A parasite ring-stage culture
synchronized using the sorbitol method [38] was added to serial dilutions of
each compound (to a maximal final DMSO concentration of 0.05% v/v) in 96-
well plates. Inhibition controls included artesunate, pyrimethamine and
chloroquine.
The plates were then incubated for 72 hours and growth inhibition was
evaluated using the SYBR green assay. Briefly, the assay plate was
centrifuged, and the medium was removed. The pelleted red blood cells were
resuspended in PBS and incubated for 30 minutes with SYBR green I DNA
stain (1:20000) diluted in lysis buffer (2.4228 g TRIS, ultra-pure (for 20 mM
solution), pH 7.5; 1.8612 g of EDTA (for 5 mM solution); 800 µL of Triton X-100
(0.08% v/v); 80 mg Saponin (0.008% w/v)). Plate fluorescence was read in a
fluorimeter (λabs = 485 nm and λemi = 535 nm). Fluorescence values of controls
wells for 0% inhibition (infected RBCs without compound addition) and 100%
inhibition (uninfected RBCs) were used to normalize viability in each test well.
Concentration-response curves were plotted for each compound and the IC50
values were obtained from sigmoidal curve fitting in the Origin software.
4.5. Cytotoxicity evaluation against human hepatoma (HepG2) cells
Compound toxicity was tested in human hepatoma cells (HepG2 cell
line). Human HepG2 cells are used for anticipating toxic effects on human liver,
an important cause of drug failure in the clinic. In addition, the cell line is easy to
handle, retain many of the morphological characteristics of liver parenchymal
cells, and contain several enzymes responsible for the activation of various
xenobiotics. To evaluate the viability of the cell the MTT assay was performed
[31]. Cell culture was maintained in 75 cm2 flasks in complete RPMI medium
supplemented with 10% Fetal Bovine Serum and 40 mg/mL gentamicin, at 37
ºC in a humidified atmosphere of 5% CO2. When cell monolayer reached
confluency, the flask was trypsinized and cells were resuspended in culture
medium and distributed in 96-well plates, to a final count of 5x104 cells per well.
The plate was then incubated overnight at culture conditions for cell adhesion.
28