Syntheses and in Vitro Antiplasmodial Activity of Aminoalkylated Chalcones and Analogues

Article abstract of DOI:10.1021/acs.jnatprod.5b00114

(Chemical Equqation Presented). A series of readily synthesized and inexpensive aminoalkylated chalcones and diarylpropane analogues (1-55) were synthesized and tested against chloroquinone-sensitive (D10 and NF54) and -resistant (Dd2 and K1) strains of Plasmodium falciparum. Hydrogenation of the enone to a diarylpropane moiety increased antiplasmodial bioactivity significantly. The influence of the structure of the amine moiety, A-ring substituents, propyl vs ethyl linker, and chloride salt formation on further enhancing antiplasmodial activity was investigated. Several compounds have IC₅₀ values similar to or better than chloroquine (CQ). The most active compound (26) had an IC₅₀ value of 0.01 μM. No signs of resistance were detected, as can be expected from compounds with structures unrelated to CQ and other currently used antimalarial drugs. Toxicity tests (in vitro CHO cell assay) gave high SI indices.

Full text of DOI:10.1021/acs.jnatprod.5b00114

Article
pubs.acs.org/jnp
Syntheses and in Vitro Antiplasmodial Activity of Aminoalkylated
Chalcones and Analogues
Anke Wilhelm,*,† Pravin Kendrekar,† Anwar E. M. Noreljaleel,† Efrem T. Abay,‡ Susan L. Bonnet,†
Lubbe Wiesner,‡ Carmen de Kock,‡ Kenneth J. Swart,§ and Jan Hendrik van der Westhuizen*,⊥
⊥
†Department of Chemistry and Directorate: Research Development, University of the Free State, Nelson Mandela Drive 205,
Bloemfontein 9301, South Africa
‡Division of Clinical Pharmacology, Department of Medicine, University of Cape Town, Cape Town, South Africa
§PAREXEL International Clinical Research Organization, Private Bag X09, Brandhof 9324, Bloemfontein 339, South Africa
S
* Supporting Information
ABSTRACT: A series of readily synthesized and inexpensive aminoalkylated chalcones and diarylpropane analogues (1−55)
were synthesized and tested against chloroquinone-sensitive (D10 and NF54) and -resistant (Dd2 and K1) strains of Plasmodium
falciparum. Hydrogenation of the enone to a diarylpropane moiety increased antiplasmodial bioactivity significantly. The
influence of the structure of the amine moiety, A-ring substituents, propyl vs ethyl linker, and chloride salt formation on further
enhancing antiplasmodial activity was investigated. Several compounds have IC50 values similar to or better than chloroquine
(CQ). The most active compound (26) had an IC50 value of 0.01 μM. No signs of resistance were detected, as can be expected
from compounds with structures unrelated to CQ and other currently used antimalarial drugs. Toxicity tests (in vitro CHO cell
assay) gave high SI indices.
alaria is one of the leading causes of death, and 300−500
tionally to treat malaria. Chalcones (1,3-diaryl-2-propen-1-
ones) are precursors in the biosynthesis of flavonoids and occur
widely in medicinal plants. Bioactivity of chalcones includes in
vivo activity against skin carcinogenesis13 and limiting cell
proliferation. The in vivo efficacy and mode of action of
flavonoids, including chalcones, is however controversial since
polar polyphenols are poorly absorbed, do not conform to the
Lipinski rules,14 and are rapidly metabolized by liver enzymes in
the plasma,15 leading to insignificant bioavailability. It thus
remains a challenge to reconcile their poor bioavailability with
putative health effects.
Most drugs contain nitrogen, and the introduction of
nitrogen into molecules has often led to enhanced bioactivity.
Dimmock and co-workers reviewed the biological activity of
Mannich bases,16 obtained via the Mannich reaction, and found
properties such as antimalarial,17,18 antiviral,19 and antibacte-
rial20 activity. Flavonoids have also served as scaffolds and
inspiration to design new molecules with potential biological
activity. Little research has been reported on the synthesis and
biological activity of chalcones with nitrogen moieties. Reddy
and co-workers21 reported the syntheses and in vitro biological
evaluation of heterocyclic nitrogen-containing chalcones with
different substitution patterns in the B-ring, together with a
discussion of structure−acitivity relationships. We postulated an
M
million new clinical cases and 660 000 deaths are
reported annually. Almost 90% of cases and deaths occur in
sub-Saharan Africa, where malaria is the leading cause of
morbidity of children younger than 5 years and pregnant
women.1 Despite extensive research, malaria remains a serious
threat, places a substantial strain on health services, and costs
Africa at least $12 billion in lost production annually. This is
attributed to the emergence of drug resistance by Plasmodium
falciparum, the main cause of human malaria infections.1−3
Artemisone, synthesized from dihydroartemisinin, is cur-
rently the only drug devoid of resistance problems. However,
disconcerting indications of resistance to artemisinin have been
reported from Southeast Asia.4,5 Owing to its short half-life (t1/2
≈ 2−5 h), it cannot be used as a prophylaxis and is used in
combination therapies with existing drugs to increase the half-
life. These existing drugs cannot be used alone, as P. falciparum
have developed various degrees of resistance, depending on the
malaria strain and the region. Efforts to develop antimalarial
vaccines have, despite so-called promising results, failed to
produce vaccines.6−8 The quest thus remains to develop new
antimalarial drugs to replace those that have already partially or
fully succumbed to resistance and are expected to become
ineffective.
A plethora of in vitro biological activities have been reported
for flavonoids including antimicrobial,9 anti-inflammatory,10
anticancer,11 and antioxidant12 properties. Many flavonoid-
containing extracts, particularly bark extracts, are used tradi-
Received: February 4, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00114
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
increase in the bioactivity and efficacy of chalcones by reducing
the number of OH groups, removing the enone moiety, and
introducing a nitrogen functionality. Herein we report the
syntheses of a series of novel aminoalkylated chalcones and
analogues via the Mannich reaction and the evaluation of their
in vitro antiplasmodial bioactivity. Most of the analogues are
molecules where the enone moiety has been reduced to yield a
diarylpropane. Since the Mannich reaction with aromatic
compounds requires a hydroxy group on the aromatic ring,
the synthetic compounds are classified as α-aminoalkyl
phenols.22,23
aminoalkylation steps of both the dihydrochalcone and
diarylpropanes were superior to those obtained upon amino-
alkylation of the chalcones. The unconjugated B-ring is thus
more nucleophilic in the Mannich reaction, as removal of
conjugation probably increased the energy of the highest
occupied aromatic π-orbital and, thus, nucleophilicity.
The ratio between partially and fully reduced chalcones could
be controlled by the reaction time of hydrogenation (24−48 h
vs 48−72 h) and the reaction conditions. Hydrogenation was
initially performed at 20 bar, but it was subsequently found that
the presence of catalytic amounts of 10% HCl(aq) gave high
yields of the fully reduced chalcone at atmospheric pressure.
The carbonyl group on chalcone-type compounds with
heterocyclic A-rings, particularly those containing nitrogen
and sulfur, e.g., compound 16−18, was rather resistant to
catalytic hydrogenation. This was attributed to conjugation of
the carbonyl with the lone-pair electrons on the sulfur and
nitrogen atoms of the heterocyclic A-ring. By using Wolff−
Kisher reduction (NH2−NH2 and KOH/NaOH)31 the target
arylpropanes with nitrogen- and sulfur-containing A-rings were
subsequently obtained.
RESULTS AND DISCUSSION
■
Chemistry. The amino functionality was introduced with
the Mannich reaction into a series of chalcones, available via the
aldol reaction (Scheme 1). The Mannich reaction can be
applied to aromatic rings provided one hydroxy group is
available in the ortho-position (Scheme 2).
Scheme 1. Synthesis of Aminoalkylated Chalcones
Some aminoalkylated phenols, e.g., compounds 46 and 47
(Tables 8 and 9), were obtained from reacting phenols with
CH2O/piperidine (Scheme 5). Compounds 46 and 47 are
similar to 2-aminomethyl-3,5-di-tert-butylphenol (MK-4815)
(Figure 1), which has been investigated by Merck Research
Laboratories as a potential treatment for malaria.32 The
diarylethane analogue 49 was synthesized via the sequence
depicted in Scheme 7, using the Wittig reaction for the
formation of the 1,2-diarylethene functionality.
Structure Elucidation. E-Chalcones are characterized by
the large Jα,β (trans) 1H NMR coupling constants of 15−16 Hz
in the aromatic region. These change to two two-proton triplets
(J = 7 Hz) at about 3.24 and 2.96 ppm in the aliphatic region
upon hydrogenation to form the dihydrochalcones. The
diarylpropanes exhibit two two-proton triplets (J = 7 Hz) at
about 2.49 and 2.55 ppm and a two-proton multiplet at about
1.85 ppm. Salient in the aminoalkylated chalcones and
diarylpropanes is the benzylic aminomethylene moiety that
resonates as a two-proton singlet at about 3.60 ppm. The
heterocyclic amine substituents often require elevated temper-
atures for well-resolved resonances. This is attributed to
hydrogen bonding between nitrogen and the o-hydroxy
function that restricts rotation via the presence of a six-
membered ring. Ortho-aminoalkylated produts were observed
in this study. The o-aminoalkylated product is probably
stabilized by the aforementioned hydrogen bond. This explains
the exclusive formation of o-substituted products under mild
reaction conditions (Scheme 2). Para-substitution was observed
in only one case subsequent to ortho-substitution to yield an
o,p-diaminoalkylated product. This, however, required extended
reaction times.
Scheme 2. Aminomethylation of Phenols via the Mannich
Reaction
It is well known that aromatic OH groups are the targets for
enzymatic removal of phenols from plasma, via either
degradation or conjugation.24−26 The enoyl moiety is
associated with toxicity via conjugated nucleophilic attack
with DNA and consequent alkylation.27 Enone moieties are
rigid compared to their propane counterparts and would not fit
as readily into enzyme active sites.28−30 Medicinal chemistry
considerations thus suggest that changing the enoyl moiety into
a propane moiety and removal of aromatic OH groups would
enhance the efficacy and bioavailability and reduce toxicity of
chalcones and aminoalkylated chalcones.
Hydrogenation of the aminoalkylated chalcones gave the
corresponding propanes in poor yields (5−10%), and products
where the benzylic aminoalkyl groups had been lost were
isolated (Scheme 3). The chalcones were subsequently
hydrogenated prior to performing the Mannich aminoalkylation
reaction to secure high yields for both the hydrogenation and
aminoalkylation steps. Yields ranged between 80% and 90% for
the dihydrochalcones and in excess of 90% for the diaryl-
propanes (Scheme 4). Salient is the fact that yields of the
From the HMBC data (compound 24, used as an example),
H-3 correlates with both C-2″ and C-6″. The aminomethylene
protons (−N-CH2) correlate with C-3″, C-4″, and C-5″. The
Scheme 3. Hydrogenation of Aminoalkylated Chalcones
B
DOI: 10.1021/acs.jnatprod.5b00114
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Scheme 4. Synthesis of Dihydrochalcones and Diarylpropanes Followed by Mannich Aminoalkylation
Table 1. 1H NMR (600 MHz) Data for Compounds 1 (Acetone-d6), 3 (Acetone-d6), 20 (CDCl3), 23 (CDCl3), and 24 (CDCl3)
[δH, ppm, Mult. (J in Hz)]
1
3
20
23
24
proton
H-1
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
2.57−2.51, m
1.89−1.83, m
2.57−2.51, m
7.06, d (8.5)
6.82, d (8.6)
6.64, d (1.5)
2.56, t (7.6)
1.91−1.82, m
2.53, t (7.6)
7.10, d (8.7)
6.82, d (8.7)
6.56, d (1.5)
H-2
7.69, d (15.6)
7.80, d (15.6)
8.16, d (8.9)
7.68, d (15.5)
7.81, d (15.5)
8.18, d (8.9)
7.07, d (8.9)
7.17, d (1.5)
3.24, t (7.6)
2.96, t (7.6)
H-3
b
c
H-2′/6′
H-3′/5′
H-2″
7.97, dd (8.9, 5.4, )
b
c
7.07, d (8.9)
7.09, t (8.9, 8.9 )
6.69, d (1.5)
a
7.30−7.25,
m
H-3″
H-4″
H-5″
H-6″
a
a
a
7.30−7.25,
7.30−7.25,
6.95−6.93,
m
m
m
6.72, d (7.6)
7.10, t (8.0)
6.64, d (1.5)
7.09, d (7.5)
7.16, dd (7.5, 1.5)
2.53, br s
6.86, d (7.5)
6.64, dd (7.6, 1.5)
2.47, br s
6.86, d (7.2)
6.86, dd (7.3, 1.5)
2.45, br s
1.57, p
H-2‴/6‴
H-3‴/5‴
H-4‴
1.66−1.60, m
1.52, br s
1.61, s
1.61, s
1.46, br s
3.73, s
OCH3
3.91, s
3.91, s
3.76, s
CH2
3.74, s
3.62, s
3.59, s
a
b
c
3
Interchangeable. JH−H
.
4JH−F
.
Table 2. 13C NMR (150 MHz) Data for Compounds 1
(Acetone-d6), 3 (Acetone-d6), 20 (CDCl3), 23 (CDCl3), and
24 (CDCl3) [δC, ppm]
correlation between the aminomethylene protons and C-3″
confirms o-substitution relative to the phenolic hydroxy group.
Biological Evaluations. Initial results for compounds 1−
11 indicated that the introduction of an aminoalkyl group into
chalcones enhanced bioactivity against the chloroquine-
sensitive Plasmodium falciparum strain, D1033 (ca. 10-fold),
supporting our hypothesis that introduction of a nitrogen
moiety would enhance bioactivity. Replacing the piperidine
moiety with morpholine (6), 1-methylpiperazine (7), pyrroli-
dine (8), and 1-ethylpiperazine (9) did not increase bioactivity
significantly. Thus, only analogues with piperidine as the amino
moiety were synthesized.
Structural modifications to the A-ring indicate that A-ring
substituents have the potential to further enhance bioactivity
(Table 4). Replacing the 4′-methoxy group with CF3 (12), Br
(13), CH2CH3 (14), and F (15) led to a ca. 100-fold increase
in activity. The chloroquine-resistant P. falciparum Dd234 gave
similar results to the chloroquine sensitive D10 strains with
12−14. This suggests that aminoalkylated chalcones use a
different parasite inhibitory mechanism than chloroquine,
which usually has an RI value of between 5 and 10. Since
dihydrochalcones 20−22 (Table 4) did not show increased
bioactivity compared to the chalcone precursors, no further
dihydrochalcone analogues were synthesized.
1
3
20
23
24
carbon
δC
δC
δC
δC
δC
C-1
187.4
121.9
143.3
131.1
130.7
113.9
163.6
113.9
130.7
136.7
117.4
157.8
114.9
129.9
55.1
188.2
122.3
144.1
132.1
131.6
114.7
164.5
114.7
131.6
136.5
120.7
159.6
125.6
130.0
56.0
197.8
40.3
30.0
34.4
32.9
35.2
34.2
C-2
C-3
35.2
35.8
a
C-1′
C-2′
C-3′
C-4′
C-5′
C-6′
C-1″
C-2″
C-3″
C-4″
C-5″
OCH3
CH2
C-2‴
C-3‴
C-4‴
C-5‴
C-6‴
133.3 (3.1)
134.5
129.3
113.7
157.5
113.7
129.3
144.3
115.4
155.6
112.7
129.4
55.3
135.0
130.1
114.5
159.0
114.5
130.1
143.7
116.4
26.7
b
130.7 (9.1)
c
115.7 (21.8)
d
165.7 (254.4)
c
115.7 (21.8)
b
130.7 (9.1)
141.7
115.8
158.1
119.5
128.6
24.7
26.7
54.4
62.4
61.9
53.9
25.9
24.0
25.9
53.9
62.3
54.5
55.4
26.7
26.7
24.6
24.7
The best bioactivities were obtained with the fully reduced
diarylpropanes (Table 5, 23−32), where the rigidity associated
with the planar conjugated enone was removed. This correlated
with the finding of Lovering and co-workers29 that molecules
with a higher degree of saturation and more stereogenic centers
have lower melting points, higher solubility, and a better chance
26.7
26.7
54.4
(2JC−F
)
55.1
a
b
c
d
4JC−F
.
3JC−F
.
1JC−F
.
C
DOI: 10.1021/acs.jnatprod.5b00114
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 3. Antiplasmodial Activity (IC50) of Aminoalkylated Chalcones (3−11) Compared with Chalcones Devoid of a Nitrogen
Functionality (1, 2)
a
b
Data shown as means SD where applicable. D10: chloroquine-sensitive Plasmodium falciparum strain.
of clinical success. The chloroquine-resistant strain Dd2 was
replaced with the K135 strain in Table 3. Similar to the sensitive
strains D10 and NF54,36 there are only minor genetic
differences between Dd2 and K1, but the results are considered
similar.37
The unreduced aminoalkylated chalcones have much lower SI
values.
In conclusion, it was established that chalcones with an
aminoalkyl moiety on the aromatic B-ring exhibit promising in
vitro antiplasmodial activity. This supports the hypothesis that
nitrogen-containing flavonoids would enhance biological
activity compared to naturally occurring non-nitrogen ana-
logues. Reduction of the enone moiety increased antimalarial
bioactivity significantly. The structure of the amino moiety, A-
ring substituents, shortening of the propyl to an ethyl linkage,
and chloride salt formation further enhanced antiplasmodial
activity. The most active compound (26) has an IC50 value of
0.01 μM (10 nM). Many of the compounds have similar or
better IC50 values than CQ against CQ-sensitive malaria strains
(D10 and NF54) and showed little difference in activity against
CQ-resistant strains (Dd2 and K1). This is to be expected since
the new synthetic compounds possess structures unrelated to
CQ and other currently used antimalarial drugs. In vitro
cytotoxicity tests suggest that the compounds are relatively
nontoxic with high SI indices. Thus, we succeeded in
synthesizing antiplasmodial compounds that are relatively
uncomplicated and inexpensive to manufacture and are
structurally unrelated to existing antimalarial drugs.
The influence of the structure of the amine moiety on
antiplasmodial activity (Table 6) correlates with the results
obtained with aminoalkylated chalcones (Tables 3 and 4). In
the case of diarylpropanes, the pyrrolidine moiety as in 37
enhances activity slightly more than the piperidine unit as in 33.
To improve solubility in the solvents required by the in vitro
bioactivity assays and future bioanalytical quantifications, N-
hydrochlorides were synthesized. Bubbling dry HCl gas
through a solution of the free amine containing aminoalkylated
compounds (Table 7, 40−44) in dry DCM gave the salts as
precipitates. Salt formation does not interfere with the in vitro
bioactivity and in fact enhances it for most of the compounds
tested (Table 7).
The bioactivity of a small number of diverse related
analogues (Table 8) may be useful to direct future research.
Aminoalkylphenols have been reported to have antiplasmodial
properties.32 However, removal of the arylpropane moiety,
derived from the chalcone A-ring, led to much reduced
antiplasmodial activity. A larger moiety on the A-ring (50, 53)
and shortening of the propyl to an ethyl linker (49) seem to
enhance bioactivity, while esterification (54) or removal of the
aromatic o-hydroxy group (52 compared to 39) reduces
bioactivity considerably. Compound 53, which has two
aminoalkylphenol moieties, shows good bioactivity.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
determined with a Reichert Thermopan microscope with a Koffler
hot-stage and are uncorrected. Solid-state FT-IR spectra were recorded
as neat compound on a Bruker Tensor 27 spectrometer. A 600 MHz
Bruker Avance spectrometer was used to record the 1H NMR, COSY,
HMBC, HMQC (600 MHz) and 13C, APT (150 MHz) experiments
in either CDCl3 (δH = 7.24; δC = 77.2), acetone-d6, (δH = 2.04; δC =
29.8), or methanol-d4, (δH = 4.87 and 3.31; δC = 49.2) with TMS as
internal standard. Chemical shifts were expressed as parts per million
(ppm) on the delta (δ) scale, and coupling constants (J) are accurate
to 0.01 Hz. High-resolution mass spectral data (HRMS) were
collected using a Waters Micromass LCT Premier TOF-MS
The toxicity of a representative sample of the synthetic
compounds was determined with the CHO bioassay, and the
selectivity indices (SI) were calculated (Table 9). The most
active compounds showed relatively low CHO cytotoxicity and
relatively high SI values, indicating that these compounds
selectively inhibit malaria parasites compared to healthy cells.
D
DOI: 10.1021/acs.jnatprod.5b00114
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 4. Antiplasmodial Activity of A-Ring-Substituted Chalcones (12−19) and Dihydrochalcones (20−22)
a
b
c
NF54: alternative chloroquine-sensitive Plasmodium falciparum strain. Dd2: chloroquine-resistant Plasmodium falciparum strain. RI: resistance
d
index: IC50(Dd2)/IC50(D10). ND: not determined.
spectrometer. All samples were dissolved and diluted to ∼2 ng/μL and
infused without additives. Thin-layer chromatography (TLC) was
performed on Merck aluminum sheets (silica gel 60 F254, 0.25 mm).
Reactions were monitored by TLC on silica gel, with detection by UV
light (254 nm). Thin-layer chromatograms were sprayed with a 2%
(v/v) solution of formaldehyde (40% solution in H2O) in
concentrated H2SO4 and subsequently heated to 110 °C to effect
maximum development of color. Purity was measured with Shimadzu
HPLC systems using a Phenomenex C18 (100 mm × 4.6 mm) 2.6 μm
column; 2.0 μL injection volume; flow, 0.2 mL/min; isocratic system,
mobile phase A, 0.1% HCO2H in H2O, and mobile phase B, MeCN,
with a Shimadzu LC-20AD pump SPD-M20A UV detector set at 254
nm. Chemicals purchased from commercial vendors were used without
purification.
General Procedure for the Synthesis of Chalcones via Aldol
Condensation. A mixture of acetophenone (1 equiv) and aryl
aldehyde (1 equiv) was stirred in EtOH (50 mL) at room temperature.
A KOH solution (50%, 25 mL) was added after 10 min, which turned
the reaction mixture bright yellow. The reaction mixture was left to stir
overnight, after which it was quenched with ice-cold 1 N HCl (100
mL) solution and extracted with EtOAc (2 × 50 mL). The organic
layer was washed with water (1 × 50 mL) and dried over Na2SO4, and
the solvent evaporated under reduced pressure. This is demonstrated
for the synthesis of (E)-3-(3-hydroxyphenyl)-1-(4-methoxyphenyl)-
prop-2-en-1-one (1) using 4-methoxyacetophenone (3.1356 g; 20.9
mmol) and 3-hydroxybenzaldehyde (3.0547 g; 25.0 mmol).
(E)-3-(3-Hydroxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one
(1): yellow crystals38 (EtOH); mp 163−164 °C; IR (neat) νmax
1
3323.24, 1583.50, 1168.39, 830.84, 666.33 cm−1; H NMR (acetone-
d6, TMS, 600 MHz) δ 8.16 (2H, d, J = 8.9 Hz, H-2′, H-6′), 7.80 (1H,
d, J = 15.6 Hz, H-3), 7.69 (1H, d, J = 15.6 Hz, H-2), 7.30−7.25 and
6.95−6.93 (4H, m, H-5″, H-2″, H-6″, H-4″), 7.07 (2H, d, J = 8.9 Hz,
H-3′, H-5′), 3.91 (3H, s, OCH3); 13C NMR (acetone-d6, TMS, 150
MHz) δ 187.4 (C-1), 163.6 (C-4′), 157.8 (C-3″), 143.3 (C-3), 136.7
(C-1″), 131.1 (C-1′), 130.7 (C-2′, C-6′), 129.9 (C-5″), 121.9 (C-2),
120.0 (C-6″), 117.4 (C-2″), 114.9 (C-4″), 113.9 (C-3′, C-5′), 55.1
(OCH3); HRESMS [M + H]+ m/z 255.1965 (calcd for C16H14O3 +
H+, 255.1960); Rf = 0.37, toluene/acetone (5:5), 3.506 g, 66%.
General Procedure for the Synthesis of Aminoalkylated
Chalcones via the Mannich Reaction. A mixture of the appropriate
chalcone (1 equiv), paraformaldehyde (1.5 equiv), and the appropriate
amine (2 equiv) was dissolved in EtOH (2 mL) and concentrated HCl
(5 drops). The reaction mixture was refluxed for about 9 h until TLC
showed the disappearance of the starting material. The reaction
mixture was quenched with solid NaHCO3 and extracted with EtOAc
(2 × 50 mL), and the extract washed with water (2 × 50 mL). The
organic layer was dried over Na2SO4, and the solvent evaporated under
reduced pressure. This is demonstrated for the synthesis of (E)-3-[3-
E
DOI: 10.1021/acs.jnatprod.5b00114
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 5. Antiplasmodial Activity of Aminolalkylated Diarylpropanes (23−32)
hydroxy-4-(piperidin-1-ylmethyl)phenyl]-1-(4-methoxyphenyl)prop-
2-en-1-one (3) using (E)-3-(3-hydroxyphenyl)-1-(4-methoxyphenyl)-
prop-2-en-1-one (1) (0.609 g; 2.4 mmol), paraformaldehyde (0.145 g;
4.8 mmol), and piperidine (0.50 mL; 5.1 mmol).
filtered through silica gel, and the filtrate was extracted with EtOAc
(2 × 50 mL) and washed with water (1 × 30 mL) and brine (1 × 20
mL). The organic layer was dried over anhydrous MgSO4, and the
solvent evaporated under reduced pressure. Column chromatography
[hexanes/EtOAc (7:3), 1.5 cm × 15 cm] yielded the pure
dihydrochalcones in good yield. This is demonstrated for the synthesis
of 1-(4-fluorophenyl)-3-[3-hydroxy-4-(piperidin-1-ylmethyl)phenyl]-
propan-1-one (20) using 1-(4-fluorophenyl)-3-(3-hydroxyphenyl)-
propan-1-one (0.100 g; 0.41 mmol), paraformaldehyde (0.024 g;
0.80 mmol), and piperidine (0.09 mL; 0.88 mmol).
(E)-3-(3-Hydroxy-4-[piperidin-1-ylmethyl)phenyl]-1-(4-
methoxyphenyl)prop-2-en-1-one (3): beige crystals39 (EtOH); mp
120−121 °C; IR (KBr) νmax 2945.44, 2159.13, 2031.94, 1598.90,
1256.25 cm−1; 1H NMR (acetone-d6, TMS, 600 MHz) δ 8.18 (2H, d, J
= 8.9 Hz, H-2′, H-6′), 7.81 (1H, d, J = 15.5 Hz, H-3), 7.68 (1H, d, J =
15.5 Hz, H-2), 7.17 (1H, d, J = 1.5 Hz, H-2″), 7.16 (1H, dd, J = 7.5,
1.5 Hz, H-6″), 7.09 (1H, d, J = 7.5 Hz, H-5″), 7.07 (2H, d, J = 8.9 Hz,
H-3′, H-5′), 3.91 (3H, s, OCH3), 3.74 (2H, s, CH2), 2.53 (4H, br s, H-
2‴, H-6‴), 1.66−1.60 (4H, m, H-3‴, H-5‴), 1.52 (2H, br s, H-4‴);
13C NMR (acetone-d6, TMS, 150 MHz) δ 188.2 (C-1), 164.5 (C-4′),
159.6 (C-3″), 144.1 (C-3), 136.5 (C-1″), 132.1 (C-1′), 131.6 (C-2′,
C-6′), 130.0 (C-5″), 125.6 (C-4″), 122.3 (C-2), 120.7 (C-2″), 115.6
(C-6″), 114.7 (C-3′, C-5′), 62.4 (CH2), 56.0 (OCH3), 54.5 (C-2‴, C-
6‴), 26.7 (C-3‴, C-5‴), 24.6 (C-4‴); HREIMS m/z 351.1826 (calcd
for C22H25NO3, 351.1824); HPLC purity 99.1%, tR = 1.54 min;
column chromatography [toluene/acetone (5:5), 1.5 cm × 20 cm];
Rf = 0.42; 0.520 g, 62%.
1-(4-Fluorophenyl)-3-(3-hydroxy-4-(piperidin-1-ylmethyl)phenyl)-
1
propan-1-one (20): light yellow oil; H NMR (CDCl3, TMS, 600
3
4
MHz) δ 7.97 (2H, dd, JH−H = 8.9 Hz; JH−F = 5.4 Hz, H-2′, H-6′),
7.09 (2H, t, 3JH−H = 8.9 Hz; 4JH−F = 8.9 Hz, H-3′, H-5′), 6.86 (1H, d,
J = 7.6 Hz, H-5″), 6.69 (1H, d, J = 1.5 Hz, H-2″), 6.64 (1H, dd, J = 7.6,
1.5 Hz, H-6″), 3.62 (2H, s, CH2), 3.24 (2H, t, J = 7.6 Hz, H-2), 2.96
(2H, t, J = 7.6 Hz, H-3), 2.47 (4H, H-2‴, H-6‴), 1.61 (6H, s, H-3‴,
H-4‴, H-5‴); 13C NMR (CDCl3, TMS, 150 MHz) δ 197.8 (C-1),
1
165.7 (1C, d, JC−F = 254.4 Hz, C-4′), 158.1 (C-3″), 141.7 (C-1″),
133.3 (1C, d, 4JC−F = 3.1 Hz, C-1′), 130.7 (2C, d, 3JC−F = 9.1 Hz, C-2′,
C-6′), 128.6 (C-5″), 119.5 (C-4″), 119.0 (C-6″), 115.8 (C-2″), 115.7
2
(2C, d, JC−F = 21.8 Hz, C-3′, C-5′), 61.9 (CH2), 53.9 (C-2‴, C-6‴),
General Procedure for the Synthesis of the Dihydrochal-
cones. The appropriate chalcone (1 equiv) was dissolved in a 1:3
(v/v) solution of EtOAc/H2O. Pd(OH)2/C (0.060 g) was added, and
the system flushed with hydrogen. The reaction mixture was left to stir
at room temperature for 24−48 h under H2 at atmospheric pressure.
After completion of the reaction (TLC) the reaction mixture was
40.3 (C-2), 30.0 (C-3), 25.9 (C-3‴, C-5‴), 24.0 (C-4‴); column
chromatography [hexanes/EtOAc (6:4), 1.5 cm × 15 cm]; Rf = 0.52;
0.118 g; 84%.
General Procedure for the Synthesis of the Diarylpropanes.
The appropriate chalcone (1 equiv) was dissolved in a 1:3 (v/v)
F
DOI: 10.1021/acs.jnatprod.5b00114
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 6. Antiplasmodial Activity of Diarylpropanes with Different Amine Moieties (33−39)
Table 7. Antiplasmodial Activity of Diarylpropane HCl Salts (40−44)
solution of EtOAc/H2O. Ten percent HCl(aq) (10 mL) with
Pd(OH)2/C (0.060 g) was added, and the system flushed with
hydrogen. The reaction mixture was left to stir at room temperature
for 48−72 h under H2 at atmospheric pressure. After completion of
the reaction (TLC) the reaction mixture was filtered through silica gel,
and the filtrate was extracted with EtOAc (2 × 50 mL) and washed
with water (1 × 30 mL) and brine (1 × 20 mL). The organic layer was
dried over anhydrous MgSO4, and the solvent evaporated under
reduced pressure. Column chromatography [hexanes/EtOAc (7:3),
1.5 cm × 20 cm] yielded the pure diarylpropanes in good yield. This is
demonstrated for the synthesis of 3-[3-(4-methoxyphenyl)propyl]-
phenol (23) using (E)-3-(3-hydroxyphenyl)-1-(4-methoxyphenyl)-
prop-2-en-1-one (1) (0.200 g; 0.80 mmol).
3-[3-(4-Methoxyphenyl)propyl]phenol (23): yellow oil; IR (KBr)
νmax 2933.38, 1586.44, 1510.27, 1241.05 cm−1; 1H NMR (CDCl3,
TMS, 600 MHz) δ 7.10 (1H, t, J = 8.0 Hz, H-5″), 7.06 (2H, d, J = 8.5,
H-2′, H-6′), 6.82 (2H, d, J = 8.6 Hz, H-3′, H-5′), 6.72 (1H, d, J = 7.6
Hz, H-4″), 6.64 (2H, d, J = 1.5 Hz, H-2″, H-6″), 3.76 (3H, s, OCH3),
2.57−2.51 (4H, m, H-1, H-3), 1.89−1.83 (2H, m, H-2); 13C NMR
(CDCl3, TMS, 150 MHz) δ 157.5 (C-4′), 155.6 (C-3″), 144.3 (C-1″),
134.5 (C-1′), 129.4 (C-5″), 129.3 (C-2′, C-6′), 120.8 (C-6″), 115.4
(C-2″), 113.7 (C-3′, C-5′), 112.7 (C-4″), 55.3 (OCH3), 35.2 (C-3),
G
DOI: 10.1021/acs.jnatprod.5b00114
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 8. Antiplasmodial Activity of Diverse Analogues (45−55)
34.4 (C-1), 32.9 (C-2); HREIMS m/z 242.1306 (calcd for C16H18O2,
242.1307); Rf = 0.55, 0.185 g; 97%.
158.8 (C-3″), 143.7 (C-1″), 135.0 (C-1′), 130.1 (C-2′, C-6′), 129.3
(C-5″), 120.0 (C-4″), 119.6 (C-6″), 116.4 (C-2″), 114.5 (C-3′, C-5′),
62.3 (CH2), 55.4 (C-2‴, C-6‴), 54.4 (OCH3), 35.8 (C-3), 35.2 (C-1),
34.2 (C-2), 26.7 (C-3‴, C-5‴), 24.7 (C-4‴); HREIMS m/z 339.2198
(calcd for C22H29NO2, 339.2200); flash column chromatography
[hexanes/EtOAc (6:4), 1.5 cm × 15 cm]; Rf = 0.52; 0.095 g; 41%.
General Synthesis of HCl Salts of the Aminoalkylated
Diarylpropanes. The appropriate aminoalkyldiarylpropane was
dissolved in dry DCM (10 mL) at 0 °C. HCl gas was bubbled
through the reaction mixture for 60 min. Precipitation indicated the
formation of the salt. The excess solvent was removed under a stream
of N2 gas, and the product was lyophilized overnight. This is
demonstrated for the synthesis of 1-[2-hydroxy-4-{3-(4-
methoxyphenyl)propyl}benzyl]piperidinium chloride (40) using 5-
[3-(4-methoxyphenyl)propyl]-2-(piperidin-1-ylmethyl)phenol (26)
(0.200 g, 0.59 mmol).
General Procedure for the Synthesis of Aminoalkylated
Diarylpropanes. A mixture of the appropriate diarylpropane
(1 equiv), paraformaldehyde (1.5 equiv), and the appropriate amine
(2 equiv) was dissolved in EtOH (2 mL) and concentrated HCl
(5 drops). The reaction mixture was refluxed for 9 h until TLC
showed the disappearance of the starting material. The reaction
mixture was quenched with solid NaHCO3 and extracted with EtOAc
(2 × 50 mL), and the extract was washed with water (2 × 50 mL). The
organic layer was dried over Na2SO4, and the solvent evaporated under
reduced pressure. This is demonstrated for the synthesis of 5-[3-(4-
methoxyphenyl)propyl]-2-(piperidin-1-ylmethyl)phenol (24) using 3-
[3-(4-methoxyphenyl)propyl]phenol (23) (0.165 g; 0.68 mmol),
paraformaldehyde (0.037 g; 1.23 mmol), and piperidine (0.1 mL;
1.0 mmol).
5-[3-(4-Methoxyphenyl)propyl]-2-(piperidin-1-ylmethyl)phenol
(24): light yellow oil; IR (KBr) νmax 2932.41, 2360.34, 1510.49,
1242.60 cm−1; 1H NMR (acetone-d6, TMS, 600 MHz) δ 7.10 (2H, d,
J = 8.7 Hz, H-2′, H-6′), 6.86 (1H, d, J = 7.2 Hz, H-5″), 6.82 (2H, d, J
= 8.7 Hz, H-3′, H-5′), 6.58−6.54 (1H, d, J = 1.5 Hz, H-2″, 1H, dd, J =
7.3, 1.5 Hz, H-6″), 3.73 (3H, s, OCH3), 3.59 (2H, s, CH2), 2.56 (2H,
t, J = 7.6 Hz, H-1), 2.53 (2H, t, J = 7.6 Hz, H-3), 2.45 (4H, br s, H-2‴,
H-6‴), 1.91−1.82 (2H, m, H-2), 1.57 (4H, p, H-3‴, H-5‴), 1.46 (2H,
br s, H-4‴); 13C NMR (acetone-d6, TMS, 150 MHz) δ 159.0 (C-4′),
1-[2-Hydroxy-4-{3-(4-methoxyphenyl)propyl}benzyl]piperidinium
chloride (40): white, amorphous solid; IR (neat) νmax 2935.94,
1
1511.06, 1242.36, 1033.16, 827.06 cm−1; H NMR (acetone-d6, TMS,
600 MHz) δ 7.47 (1H, d, J = 7.8 Hz, H-5″), 7.13 (2H, d, J = 8.6 Hz,
H-2′, H-6′), 6.98 (1H, d, J = 1.2 Hz, H-2″), 6.84 (2H, d, J = 8.6 Hz,
H-3′, H-5′), 6.76 (1H, dd, J = 7.6, 1.5 Hz, H-6″), 4.18 (2H, d, J = 4.7
Hz, CH2), 3.76 (3H, s, OCH3), 3.42 (2H, d, J = 11.6 Hz, H-2‴),
2.96−2.87 (6H, m, H-3‴, H-4‴, H-5‴), 2.63−2.53 (4H, m, H-1, H-3),
1.94−1.85 (2H, m, H-2‴, H-6‴), 1.82 (2H, d, J = 14.5 Hz, H-6‴);
H
DOI: 10.1021/acs.jnatprod.5b00114
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 9. Toxicity Values (IC50) of the Most Promising Analogues against CHO Cell Lines and SI Values
a
SI: selectivity index = IC50(CHO)/IC50(D10).
Scheme 5. Synthesis of Compound 46 via the Mannich
Reaction
Figure 1. Stucture of MK-4815.
I
DOI: 10.1021/acs.jnatprod.5b00114
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Scheme 6. Synthesis of Compound 47 via the Mannich Reaction
concentration of solvent (0.5%) to which the cells were exposed had
no measurable effect on the cell viability (data not shown).
Scheme 7. Synthesis of Compound 49 via the Wittig
Reaction
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental detail, 1D and 2D NMR data, MS, IR, melting
points, and HPLC data. The Supporting Information is
available free of charge on the ACS Publications website at
DOI: 10.1021/acs.jnatprod.5b00114.
AUTHOR INFORMATION
Corresponding Authors
*Tel: +27 (0)51 401 9305. Fax: +27 (0)51 401 7295. E-mail:
wilhelma@ufs.ac.za (A. Wilhelm).
■
*Tel: +27 (0)51 401 2782. Fax: +27 (0)51 401 7295. E-mail:
vdwestjh@ufs.ac.za (J. H. van der Westhuizen).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. C. Edlin, previously from iThemba Pharmaceut-
icals, for his valuable input toward the structural modifications
of compounds in this study. Financial support by the University
of the Free State is acknowledged.
Figure 2. Selected HMBC correlations of compound 24.
13C NMR (acetone-d6, TMS, 150 MHz) δ 158.9 (C-4′), 157.8 (C-3″),
147.0 (C-1″), 134.9 (C-1′), 134.7 (C-2′, C-6′), 130.1 (C-5″), 121.3
(C-6″), 119.3 (C-4″), 115.7 (C-2″), 114.5 (C-3′, C-5′), 55.4 (C-2‴,
C-6‴), 52.7 (OCH3, CH2), 35.8 (C-3), 35.1 (C-1), 34.0 (C-2), 23.5
(C-3‴, C-5‴), 22.7 (C-4‴); HRTOFESMS [M + H]+ m/z 340.2270
(calcd C22H29NO2 + H+, 340.2277); HPLC purity 86.7%, tR = 1.61
min; 0.185 g; 84%.
Bioassays. Antiplasmodial Assay. Continuous in vitro cultures of
asexual erythrocyte stages of P. falciparum were maintained using the
modified method of Trager and Jensen.40 Quantitative assessment of
antiplasmodial activity in vitro was determined via the parasite lactate
dehydrogenase assay using a modified method described by Makler.41
The test samples were tested in triplicate on one or two separate
occasions. Compounds were initially screened for antiplasmodial
activity against the chloroquine-sensitive (CQS) D10 and NF54
strains. The most promising compounds were subsequently tested
against the chloroquine-resistant (CQR) strains, Dd2 and K1.
CQ was used as an internal standard to monitor the experimental
conditions and showed IC50 values within an acceptable range of
0.018−0.060 μM for the CQS strains and 0.470−0.780 μM for the
CQR strains. The 50% inhibitory concentration (IC50) values were
obtained from full dose−response curves (Supporting Information),
using a nonlinear dose−response curve-fitting analysis via GraphPad
Prism v.4 software.
Cytotoxicity Assay. The MTT assay as described by Mosmann
(with minor modifications) was used to determine cell viability using
the Chinese hamster ovarian (CHO) cell line.42 The sample
preparation was done in the same manner as for the antiplasmodial
testing. Emetine was used as the reference drug in all experiments and
showed IC50 values within an acceptable range (0.080−0.120 μM).
The initial concentration of emetine was 100 μg/mL, which was
serially diluted in complete medium with 10-fold dilutions to give six
concentrations, the lowest being 0.001 μg/mL. The same dilution
technique was applied to all the test samples. The highest
REFERENCES
■
(1) World Health Organizations Media Centre. Malaria Fact Sheet
No. 94; April 2012, Retrieved Dec 18, 2012, from http://www.who.
int/.
(2) Vitoria, M.; Granich, R.; Gilks, C. F.; Gunneberg, C.; Hosseini,
M.; Were, W.; Raviglione, M.; De Cock, K. M. Am. J. Clin. Pathol.
2009, 131, 844−848.
(3) Millennium Project. Global Burden of Malaria; Retrieved Dec 18,
2012, from http://www.unmillenniumproject.org/documents/
GlobalBurdenofMalaria.pdf.
(4) World Health Organization. Global Plan for Artemisinin Resistance
Containment; 2011; p 5.
(5) Wiwanitkit, V. Malaria Research in Southeast Asia; Nova Science
Publishers, Inc.: New York, 2007; pp 3−11.
(6) Al-Hussaieny, N. H. Parasitol. United J. 2010, 3, 1−8.
(7) Gregory, G. A.; Mayfield, S. P. Appl. Microbiol. Biotechnol. 2014,
98, 1983−1990.
(8) Sheehy, S. H.; Douglas, A. D.; Draper, S. J. Hum. Vaccines
Immunother. 2013, 9, 1831−1840.
(9) Cushnie, T. P. T.; Lamb, A. J. Int. J. Microb. Agents 2011, 38, 99−
107.
(10) Yamamoto, Y.; Gaynor, R. B. J. Clin. Invest. 2001, 107, 135−142.
(11) De Sousa, R. R.; Queiroz, K. C.; Souza, A. C.; Gurgueira, S. A.;
Augusto, A. C.; Miranda, M. A.; Peppelenbosch, M. P.; Ferreira, C. V.;
Aoyama, H. J. Enzyme Inhib. Med. Chem. 2007, 22, 439−444.
(12) Pietta, P.-G. J. Nat. Prod. 2000, 63, 1035−1042.
(13) Statomi, Y. Int. J. Cancer 1993, 55, 506−514.
(14) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv.
Drug Delivery Rev. 2001, 46, 3−26.
(15) Walle, T. Free Radical Biol. Med. 2004, 36, 829−837.
(16) Dimmock, J. R.; Kumar, P. Curr. Med. Chem. 1997, 4, 1.
(17) Barlin, G. B.; Jiravinya, C.; Yan, J. H. Aust. J. Chem. 1991, 44,
677−685.
J
DOI: 10.1021/acs.jnatprod.5b00114
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(18) Barlin, G. B.; Jiravinya, C. Aust. J. Chem. 1990, 43, 1175−1181.
(19) Edwards, M. L.; Ritter, H. W.; Stemerick, D. M.; Stewart, K. T. J.
Med. Chem. 1983, 26, 431.
(20) Pandeya, S. N.; Sriram, D.; Nath, G.; De Clercq, E. Eur. J.
Pharm. Sci. 1999, 9, 25.
(21) Reddy, M. V. B.; Su, C.-R.; Chiou, W.-F.; Liu, Y.-N.; Chen, R.
Y.-H.; Bastow, K. F.; Lee, K.-H.; Wu, T.-S. Bioorg. Med. Chem. 2008,
16, 7358−7370.
(22) Karbe, H. Arch. Pharm. 1950, 283, 38.
(23) Thompson, B. B. J. Pharm. Sci. 1968, 57, 715.
(24) Gao, S.; Hu, M. Mini Rev. Med. Chem. 2010, 10, 550−567.
(25) Crozier, A.; Jaganath, I. B.; Clifford, M. N. Nat. Prod. Rep. 2009,
26, 1001−1043.
(26) Galati, G.; O’Brian, P. J. Free Radical Biol. Med. 2004, 37, 287−
303.
(27) Attia, S. M. Oxid. Med. Cell Longev. 2010, 3, 238−253.
(28) Leeson, P.; St-Gallay, S. Med. Chem. Commun. 2011, 2, 91−105.
(29) Lovering, F.; Bikker, J.; Humblet, C. J. Med. Chem. 2009, 52,
6752−6756.
(30) Ritchie, T. J.; Macdonald, S. J. F. Drug Discovery Today 2009, 14,
1011−1020.
(31) Zhang, Z.; Zhang, L.; Guan, X.; Shen, Z.; Xing, G.; Fan, X.;
Zhou, Q. Liq. Cryst. 2010, 37, 69−76.
(32) Powles, M. A.; Allocco, J.; Yeung, L.; Nare, B.; Liberator, P.;
Schmatz, D. Antimicrob. Agents Chemother. 2012, 56, 2414−2419.
(33) Walliker, D.; Quakyi, I. A.; Wellems, T. E.; McCutchan, T. F.;
Szarfman, A.; London, W. T.; Corcoran, L. M.; Burkot, T. R.; Carter,
R. Science 1987, 236, 1661−1666.
(34) Noedl, H.; Wongsrichanalai, C.; Wernsdorfer, W. H. Trends
Parasitol. 2003, 19, 175−181.
(35) Burkot, T. R.; Williams, J. L.; Schneider, I. Trans R. Soc. Trop.
Med. Hyg. 1984, 78, 339−341.
(36) Ponnudurai, T.; Leeuwenberg, A. D.; Meuwissen, J. H. Trop.
Geogr. Med. 1981, 33, 50−54.
(37) Personal communication, Dr. L. Wiesner, University of Cape
Town, Cape Town, South Africa.
(38) Park, E.; Yang, Y. J.; Kwak, J. H.; Jung, Y. H.; Kang, S. C.; Kim, I.
S. Bioorg. Med. Chem. Lett. 2012, 22, 3653−3655.
(39) Van der Westhuizen, J. H.; Noreljaleel, A. E. M.; Bonnet, S. L.;
Wilhelm-Mouton, A. PCT Int. Appl. 2011, WO 211151789A2
20111208.
(40) Trager, W.; Jensen, J. B. Science 1976, 193, 673−675.
(41) Makler, M. T.; Ries, J. M.; Williams, J. A.; Bancroft, J. E.; Piper,
R. C.; Gibbins, B. L.; Hinrichs, D. J. Am. J. Trop. Med. Hyg. 1993, 48,
739−741.
(42) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63.
K
DOI: 10.1021/acs.jnatprod.5b00114
J. Nat. Prod. XXXX, XXX, XXX−XXX