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Liezl Gibhard , Kendrekar Pravin , Efrem Abay , Anke Wilhelm , Kenneth Swart , Nina Lawrence , Rosal Khoury , Jan van der Westhuizen , Peter Smith , L...
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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.

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tionally to treat malaria. Chalcones (1,3-diaryl-2-propen-1ones) 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 antibacterial20 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

alaria is one of the leading causes of death, and 300−500 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 currently 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 halflife. 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 flavonoidcontaining extracts, particularly bark extracts, are used tradi© XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 4, 2015

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aminoalkylation steps of both the dihydrochalcone and diarylpropanes were superior to those obtained upon aminoalkylation 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. 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 temperatures 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 sixmembered 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. 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

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

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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

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 diarylpropanes (Scheme 4). Salient is the fact that yields of the Scheme 3. Hydrogenation of Aminoalkylated Chalcones

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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)] proton H-1 H-2 H-3 H-2′/6′ H-3′/5′ H-2″ H-3″ H-4″ H-5″ H-6″ H-2‴/6‴ H-3‴/5‴ H-4‴ OCH3 CH2 a

1

3

20

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

7.69, d (15.6) 7.80, d (15.6) 8.16, d (8.9) 7.07, d (8.9) 7.30−7.25,a m

7.68, 7.81, 8.18, 7.07, 7.17,

7.30−7.25,a m 7.30−7.25,a m 6.95−6.93,a m

b3

(15.5) (15.5) (8.9) (8.9) (1.5)

7.09, d (7.5) 7.16, dd (7.5, 1.5) 2.53, br s 1.66−1.60, m 1.52, br s 3.91, s 3.74, s

3.91, s

Interchangeable.

d d d d d

d (7.5) dd (7.6, 1.5) br s s s

6.72, d (7.6) 7.10, t (8.0) 6.64, d (1.5)

24 δH (J in Hz) 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)

6.86, 6.86, 2.45, 1.57, 1.46, 3.73, 3.59,

d (7.2) dd (7.3, 1.5) br s p br s s s

JH−H. c4JH−F.

1

3

20

23

24

δC

δC

δC

δC

δC

C-1 C-2 C-3 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‴

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 62.4 54.5 26.7 24.6 26.7 54.4

197.8 40.3 30.0 133.3a (3.1) 130.7b (9.1) 115.7c (21.8) 165.7d (254.4) 115.7c (21.8) 130.7b (9.1) 141.7 115.8 158.1 119.5 128.6

34.4 32.9 35.2 134.5 129.3 113.7 157.5 113.7 129.3 144.3 115.4 155.6 112.7 129.4 55.3

35.2 34.2 35.8 135.0 130.1 114.5 159.0 114.5 130.1 143.7 116.4 26.7 24.7 26.7 54.4 62.3 55.4 26.7 24.7 26.7 55.1

b3

6.86, 6.64, 2.47, 1.61, 1.61,

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)

3.76, s

carbon

JC−F.

t (7.6) t (7.6) dd (8.9,b 5.4,c) t (8.9,b 8.9c) d (1.5)

3.62, s

Table 2. 13C NMR (150 MHz) Data for Compounds 1 (Acetone-d6), 3 (Acetone-d6), 20 (CDCl3), 23 (CDCl3), and 24 (CDCl3) [δC, ppm]

a4

3.24, 2.96, 7.97, 7.09, 6.69,

23

61.9 53.9 25.9 24.0 25.9 53.9

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 chloroquinesensitive 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), pyrrolidine (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. 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

JC−F. c1JC−F. d(2JC−F)

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Table 3. Antiplasmodial Activity (IC50) of Aminoalkylated Chalcones (3−11) Compared with Chalcones Devoid of a Nitrogen Functionality (1, 2)

a

Data shown as means ± SD where applicable. bD10: chloroquine-sensitive Plasmodium falciparum strain.

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 analogues. Reduction of the enone moiety increased antimalarial bioactivity significantly. The structure of the amino moiety, Aring 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.

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 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, Nhydrochlorides 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. 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.

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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 D

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Table 4. Antiplasmodial Activity of A-Ring-Substituted Chalcones (12−19) and Dihydrochalcones (20−22)

a

NF54: alternative chloroquine-sensitive Plasmodium falciparum strain. bDd2: chloroquine-resistant Plasmodium falciparum strain. cRI: resistance index: IC50(Dd2)/IC50(D10). dND: 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 H2 O) 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 3323.24, 1583.50, 1168.39, 830.84, 666.33 cm−1; 1H NMR (acetoned6, 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-[3E

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Table 5. Antiplasmodial Activity of Aminolalkylated Diarylpropanes (23−32)

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). 1-(4-Fluorophenyl)-3-(3-hydroxy-4-(piperidin-1-ylmethyl)phenyl)propan-1-one (20): light yellow oil; 1H NMR (CDCl3, TMS, 600 MHz) δ 7.97 (2H, dd, 3JH−H = 8.9 Hz; 4JH−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), 165.7 (1C, d, 1JC−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 (2C, d, 2JC−F = 21.8 Hz, C-3′, C-5′), 61.9 (CH2), 53.9 (C-2‴, C-6‴), 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)

hydroxy-4-(piperidin-1-ylmethyl)phenyl]-1-(4-methoxyphenyl)prop2-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). (E)-3-(3-Hydroxy-4-[piperidin-1-ylmethyl)phenyl]-1-(4methoxyphenyl)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, H2‴, H-6‴), 1.66−1.60 (4H, m, H-3‴, H-5‴), 1.52 (2H, br s, H-4‴); 13 C 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‴, C6‴), 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%. General Procedure for the Synthesis of the Dihydrochalcones. 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 F

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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

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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%. 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-(4methoxyphenyl)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′),

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-(4methoxyphenyl)propyl}benzyl]piperidinium chloride (40) using 5[3-(4-methoxyphenyl)propyl]-2-(piperidin-1-ylmethyl)phenol (26) (0.200 g, 0.59 mmol). 1-[2-Hydroxy-4-{3-(4-methoxyphenyl)propyl}benzyl]piperidinium chloride (40): white, amorphous solid; IR (neat) νmax 2935.94, 1511.06, 1242.36, 1033.16, 827.06 cm−1; 1H 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

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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

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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

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ASSOCIATED CONTENT

S Supporting Information *

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.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +27 (0)51 401 9305. Fax: +27 (0)51 401 7295. E-mail: [email protected] (A. Wilhelm). *Tel: +27 (0)51 401 2782. Fax: +27 (0)51 401 7295. E-mail: [email protected] (J. H. van der Westhuizen). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. C. Edlin, previously from iThemba Pharmaceuticals, 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.

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C 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 13

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