jnp 2015

1 Syntheses and in Vitro Antiplasmodial Activity of Aminoalkylated2 Chalcones and Analogues3 Anke Wilhelm,*,† Pravin Kendrekar,† Anwar E. M. Noreljaleel,† Efrem T. Abay,‡ Susan L. Bonnet,†

4 Lubbe Wiesner,‡ Carmen de Kock,‡ Kenneth J. Swart,§ and Jan Hendrik van der Westhuizen*,⊥

5†Department of Chemistry and ⊥Directorate: Research Development, University of the Free State, Nelson Mandela Drive 205,

6 Bloemfontein 9301, South Africa

7‡Department of Pharmacology, University of Cape Town, Medical School, Observatory 7925, South Africa

8§PAREXEL International Clinical Research Organization, Private Bag X09, Brandhof 9324, Bloemfontein 339, South Africa

9 *S Supporting Information

10 ABSTRACT: A series of readily synthesized and inexpensive aminoalkylated chalcones and diarylpropane analogues (1−55)11 were synthesized and tested against chloroquinone-sensitive (D10 and NF54) and -resistant (Dd2 and K1) strains of Plasmodium12 falciparum. Hydrogenation of the enone to a diarylpropane moiety increased antiplasmodial bioactivity significantly. The13 influence of the structure of the amine moiety, A-ring substituents, propyl vs ethyl linker, and chloride salt formation on further14 enhancing antiplasmodial activity was investigated. Several compounds have IC50 values similar to or better than chloroquine15 (CQ). The most active compound (26) had an IC50 value of 0.01 μM. No signs of resistance were detected, as can be expected16 from compounds with structures unrelated to CQ and other currently used antimalarial drugs. Toxicity tests (in vitro CHO cell17 assay) gave high SI indices.

18Malaria is one of the leading causes of death, and 300−50019 million new clinical cases and 660 000 deaths are20 reported annually. Almost 90% of cases and deaths occur in21 sub-Saharan Africa, where malaria is the leading cause of22 morbidity of children younger than 5 years and pregnant23 women.1 Despite extensive research, malaria remains a serious24 threat, places a substantial strain on health services, and costs25 Africa at least $12 billion in lost production annually. This is26 attributed to the emergence of drug resistance by Plasmodium27 falciparum, the main cause of human malaria infections.1−3

28 Artemisone, synthesized from dihydroartemisinin, is cur-29 rently the only drug devoid of resistance problems. However,30 disconcerting indications of resistance to artemisinin have been31 reported from Southeast Asia.4,5 Owing to its short half-life (t1/232 ≈ 2−5 h), it cannot be used as a prophylaxis and is used in33 combination therapies with existing drugs to increase the half-34 life. These existing drugs cannot be used alone, as P. falciparum35 have developed various degrees of resistance, depending on the36 malaria strain and the region. Efforts to develop antimalarial37 vaccines have, despite so-called promising results, failed to38 produce vaccines.6−8 The quest thus remains to develop new39 antimalarial drugs to replace those that have already partially or40 fully succumbed to resistance and are expected to become41 ineffective.42 A plethora of in vitro biological activities have been reported43 for flavonoids including antimicrobial,9 anti-inflammatory,10

44 anticancer,11 and antioxidant12 properties. Many flavonoid-45 containing extracts, particularly bark extracts, are used tradi-

46tionally to treat malaria. Chalcones (1,3-diaryl-2-propen-1-47ones) are precursors in the biosynthesis of flavonoids and occur48widely in medicinal plants. Bioactivity of chalcones includes in49vivo activity against skin carcinogenesis13 and limiting cell50proliferation. The in vivo efficacy and mode of action of51flavonoids, including chalcones, is however controversial since52polar polyphenols are poorly absorbed, do not conform to the53Lipinski rules,14 and are rapidly metabolized by liver enzymes in54the plasma,15 leading to insignificant bioavailability. It thus55remains a challenge to reconcile their poor bioavailability with56putative health effects.57Most drugs contain nitrogen, and the introduction of58nitrogen into molecules has often led to enhanced bioactivity.59Dimmock and co-workers reviewed the biological activity of60Mannich bases,16 obtained via the Mannich reaction, and found61properties such as antimalarial,17,18 antiviral,19 and antibacte-62rial20 activity. Flavonoids have also served as scaffolds and63inspiration to design new molecules with potential biological64activity. Little research has been reported on the synthesis and65biological activity of chalcones with nitrogen moieties. Reddy66and co-workers21 reported the syntheses and in vitro biological67evaluation of heterocyclic nitrogen-containing chalcones with68different substitution patterns in the B-ring, together with a69discussion of structure−acitivity relationships. We postulated an

Received: February 4, 2015

Article

pubs.acs.org/jnp

© XXXX American Chemical Society andAmerican Society of Pharmacognosy A DOI: 10.1021/acs.jnatprod.5b00114

J. Nat. Prod. XXXX, XXX, XXX−XXX

deb00 | ACSJCA | JCA10.0.1465/W Unicode | research.3f (R3.6.i9:4386 | 2.0 alpha 39) 2015/05/14 15:05:00 | PROD-JCAVA | rq_4776202 | 6/12/2015 12:59:11 | 11 | JCA-DEFAULT

70 increase in the bioactivity and efficacy of chalcones by reducing71 the number of OH groups, removing the enone moiety, and72 introducing a nitrogen functionality. Herein we report the73 syntheses of a series of novel aminoalkylated chalcones and74 analogues via the Mannich reaction and the evaluation of their75 in vitro antiplasmodial bioactivity. Most of the analogues are76 molecules where the enone moiety has been reduced to yield a77 diarylpropane. Since the Mannich reaction with aromatic78 compounds requires a hydroxy group on the aromatic ring,79 the synthetic compounds are classified as α-aminoalkyl80 phenols.22,23

81 ■ RESULTS AND DISCUSSION82 Chemistry. The amino functionality was introduced with83 the Mannich reaction into a series of chalcones, available via the

s1 84 aldol reaction (Scheme 1). The Mannich reaction can be85 applied to aromatic rings provided one hydroxy group is

s2 86 available in the ortho-position (Scheme 2).

87 It is well known that aromatic OH groups are the targets for88 enzymatic removal of phenols from plasma, via either89 degradation or conjugation.24−26 The enoyl moiety is90 associated with toxicity via conjugated nucleophilic attack91 with DNA and consequent alkylation.27 Enone moieties are92 rigid compared to their propane counterparts and would not fit93 as readily into enzyme active sites.28−30 Medicinal chemistry94 considerations thus suggest that changing the enoyl moiety into95 a propane moiety and removal of aromatic OH groups would96 enhance the efficacy and bioavailability and reduce toxicity of97 chalcones and aminoalkylated chalcones.98 Hydrogenation of the aminoalkylated chalcones gave the99 corresponding propanes in poor yields (5−10%), and products100 where the benzylic aminoalkyl groups had been lost were

s3 101 isolated (Scheme 3). The chalcones were subsequently102 hydrogenated prior to performing the Mannich aminoalkylation103 reaction to secure high yields for both the hydrogenation and104 aminoalkylation steps. Yields ranged between 80% and 90% for

s4 105 the dihydrochalcones and in excess of 90% for the diary-

106 s4lpropanes (Scheme 4). Salient is the fact that yields of the107aminoalkylation steps of both the dihydrochalcone and108diarylpropanes were superior to those obtained upon amino-109alkylation of the chalcones. The unconjugated B-ring is thus110more nucleophilic in the Mannich reaction, as removal of111conjugation probably increased the energy of the highest112occupied aromatic π-orbital and, thus, nucleophilicity.113The ratio between partially and fully reduced chalcones could114be controlled by the reaction time of hydrogenation (24−48 h115vs 48−72 h) and the reaction conditions. Hydrogenation was116initially performed at 20 bar, but it was subsequently found that117the presence of catalytic amounts of 10% HCl(aq) gave high118yields of the fully reduced chalcone at atmospheric pressure.119The carbonyl group on chalcone-type compounds with120heterocyclic A-rings, particularly those containing nitrogen121and sulfur, e.g., compound 16−18, was rather resistant to122catalytic hydrogenation. This was attributed to conjugation of123the carbonyl with the lone-pair electrons on the sulfur and124nitrogen atoms of the heterocyclic A-ring. By using Wolff−125Kisher reduction (NH2−NH2 and KOH/NaOH)31 the target126arylpropanes with nitrogen- and sulfur-containing A-rings were127subsequently obtained.128Some aminoalkylated phenols, e.g., compounds 46 and 47129 t1� t9(Tables 8 and 9), were obtained from reacting phenols with130 s5CH2O/piperidine (Scheme 5). Compounds 46 and 47 are131similar to 2-aminomethyl-3,5-di-tert-butylphenol (MK-4815)132 f1(Figure 1), which has been investigated by Merck Research133Laboratories as a potential treatment for malaria.32 The134diarylethane analogue 49 was synthesized via the sequence135 s6s7depicted in Scheme 7, using the Wittig reaction for the136formation of the 1,2-diarylethene functionality.137Structure Elucidation. E-Chalcones are characterized by138the large Jα,β (trans)

1H NMR coupling constants of 15−16 Hz139in the aromatic region. These change to two two-proton triplets140(J = 7 Hz) at about 3.24 and 2.96 ppm in the aliphatic region141upon hydrogenation to form the dihydrochalcones. The142diarylpropanes exhibit two two-proton triplets (J = 7 Hz) at143about 2.49 and 2.55 ppm and a two-proton multiplet at about1441.85 ppm. Salient in the aminoalkylated chalcones and145diarylpropanes is the benzylic aminomethylene moiety that146resonates as a two-proton singlet at about 3.60 ppm. The147heterocyclic amine substituents often require elevated temper-148atures for well-resolved resonances. This is attributed to149hydrogen bonding between nitrogen and the o-hydroxy150function that restricts rotation via the presence of a six-151membered ring. Ortho-aminoalkylated produts were observed152in this study. The o-aminoalkylated product is probably153stabilized by the aforementioned hydrogen bond. This explains154the exclusive formation of o-substituted products under mild155reaction conditions (Scheme 2). Para-substitution was observed156in only one case subsequent to ortho-substitution to yield an157o,p-diaminoalkylated product. This, however, required extended158reaction times.159From the HMBC data (compound 24, used as an example),160H-3 correlates with both C-2″ and C-6″. The aminomethylene161protons (−N-CH2) correlate with C-3″, C-4″, and C-5″. The

Scheme 1. Synthesis of Aminoalkylated Chalcones

Scheme 2. Aminomethylation of Phenols via the MannichReaction

Scheme 3. Hydrogenation of Aminoalkylated Chalcones

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.5b00114J. Nat. Prod. XXXX, XXX, XXX−XXX

B

162correlation between the aminomethylene protons and C-3″163 f2confirms o-substitution relative to the phenolic hydroxy group.164Biological Evaluations. Initial results for compounds 1−16511 indicated that the introduction of an aminoalkyl group into166chalcones enhanced bioactivity against the chloroquine-167sensitive Plasmodium falciparum strain, D1033 (ca. 10-fold),168supporting our hypothesis that introduction of a nitrogen169moiety would enhance bioactivity. Replacing the piperidine170moiety with morpholine (6), 1-methylpiperazine (7), pyrroli-171dine (8), and 1-ethylpiperazine (9) did not increase bioactivity172significantly. Thus, only analogues with piperidine as the amino173moiety were synthesized.174Structural modifications to the A-ring indicate that A-ring175substituents have the potential to further enhance bioactivity176(Table 4). Replacing the 4′-methoxy group with CF3 (12), Br177(13), CH2CH3 (14), and F (15) led to a ca. 100-fold increase178in activity. The chloroquine-resistant P. falciparum Dd234 gave179similar results to the chloroquine sensitive D10 strains with18012−14. This suggests that aminoalkylated chalcones use a181different parasite inhibitory mechanism than chloroquine,182which usually has an RI value of between 5 and 10. Since183dihydrochalcones 20−22 (Table 4) did not show increased184bioactivity compared to the chalcone precursors, no further185dihydrochalcone analogues were synthesized.186The best bioactivities were obtained with the fully reduced187diarylpropanes (Table 5, 23−32), where the rigidity associated188with the planar conjugated enone was removed. This correlated189with the finding of Lovering and co-workers29 that molecules190with a higher degree of saturation and more stereogenic centers191have lower melting points, higher solubility, and a better chance

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 δH (J in Hz) δH (J in Hz) δH (J in Hz) δH (J in Hz) δH (J in Hz)

H-1 2.57−2.51, m 2.56, t (7.6)H-2 7.69, d (15.6) 7.68, d (15.5) 3.24, t (7.6) 1.89−1.83, m 1.91−1.82, mH-3 7.80, d (15.6) 7.81, d (15.5) 2.96, t (7.6) 2.57−2.51, m 2.53, t (7.6)H-2′/6′ 8.16, d (8.9) 8.18, d (8.9) 7.97, dd (8.9,b 5.4,c) 7.06, d (8.5) 7.10, d (8.7)H-3′/5′ 7.07, d (8.9) 7.07, d (8.9) 7.09, t (8.9,b 8.9c) 6.82, d (8.6) 6.82, d (8.7)H-2″ 7.30−7.25,a m 7.17, d (1.5) 6.69, d (1.5) 6.64, d (1.5) 6.56, d (1.5)H-3″H-4″ 7.30−7.25,a m 6.72, d (7.6)H-5″ 7.30−7.25,a m 7.09, d (7.5) 6.86, d (7.5) 7.10, t (8.0) 6.86, d (7.2)H-6″ 6.95−6.93,a m 7.16, dd (7.5, 1.5) 6.64, dd (7.6, 1.5) 6.64, d (1.5) 6.86, dd (7.3, 1.5)H-2‴/6‴ 2.53, br s 2.47, br s 2.45, br sH-3‴/5‴ 1.66−1.60, m 1.61, s 1.57, pH-4‴ 1.52, br s 1.61, s 1.46, br sOCH3 3.91, s 3.91, s 3.76, s 3.73, sCH2 3.74, s 3.62, s 3.59, s

aInterchangeable. b3JH−H.c4JH−F.

Table 2. 13C NMR (150 MHz) Data for Compounds 1(Acetone-d6), 3 (Acetone-d6), 20 (CDCl3), 23 (CDCl3), and24 (CDCl3) [δC, ppm]

1 3 20 23 24

carbon δC δC δC δC δC

C-1 187.4 188.2 197.8 34.4 35.2C-2 121.9 122.3 40.3 32.9 34.2C-3 143.3 144.1 30.0 35.2 35.8C-1′ 131.1 132.1 133.3a (3.1) 134.5 135.0C-2′ 130.7 131.6 130.7b (9.1) 129.3 130.1C-3′ 113.9 114.7 115.7c (21.8) 113.7 114.5C-4′ 163.6 164.5 165.7d (254.4) 157.5 159.0C-5′ 113.9 114.7 115.7c (21.8) 113.7 114.5C-6′ 130.7 131.6 130.7b (9.1) 129.3 130.1C-1″ 136.7 136.5 141.7 144.3 143.7C-2″ 117.4 120.7 115.8 115.4 116.4C-3″ 157.8 159.6 158.1 155.6 26.7C-4″ 114.9 125.6 119.5 112.7 24.7C-5″ 129.9 130.0 128.6 129.4 26.7OCH3 55.1 56.0 55.3 54.4CH2 62.4 61.9 62.3C-2‴ 54.5 53.9 55.4C-3‴ 26.7 25.9 26.7C-4‴ 24.6 24.0 24.7C-5‴ 26.7 25.9 26.7C-6‴ 54.4 53.9 55.1

a4JC−F.b3JC−F.

c1JC−F.d(2JC−F)



C

192 of clinical success. The chloroquine-resistant strain Dd2 was193 replaced with the K135 strain in Table 3. Similar to the sensitive194 strains D10 and NF54,36 there are only minor genetic195 differences between Dd2 and K1, but the results are considered196 similar.37

197 The influence of the structure of the amine moiety on198 antiplasmodial activity (Table 6) correlates with the results199 obtained with aminoalkylated chalcones (Tables 3 and 4). In200 the case of diarylpropanes, the pyrrolidine moiety as in 37201 enhances activity slightly more than the piperidine unit as in 33.202 To improve solubility in the solvents required by the in vitro203 bioactivity assays and future bioanalytical quantifications, N-204 hydrochlorides were synthesized. Bubbling dry HCl gas205 through a solution of the free amine containing aminoalkylated206 compounds (Table 7, 40−44) in dry DCM gave the salts as207 precipitates. Salt formation does not interfere with the in vitro208 bioactivity and in fact enhances it for most of the compounds209 tested (Table 7).210 The bioactivity of a small number of diverse related211 analogues (Table 8) may be useful to direct future research.212 Aminoalkylphenols have been reported to have antiplasmodial213 properties.32 However, removal of the arylpropane moiety,214 derived from the chalcone A-ring, led to much reduced215 antiplasmodial activity. A larger moiety on the A-ring (50, 53)216 and shortening of the propyl to an ethyl linker (49) seem to217 enhance bioactivity, while esterification (54) or removal of the218 aromatic o-hydroxy group (52 compared to 39) reduces219 bioactivity considerably. Compound 53, which has two220 aminoalkylphenol moieties, shows good bioactivity.221 The toxicity of a representative sample of the synthetic222 compounds was determined with the CHO bioassay, and the223 selectivity indices (SI) were calculated (Table 9). The most224 active compounds showed relatively low CHO cytotoxicity and225 relatively high SI values, indicating that these compounds226 selectively inhibit malaria parasites compared to healthy cells.

227The unreduced aminoalkylated chalcones have much lower SI228values.229In conclusion, it was established that chalcones with an230aminoalkyl moiety on the aromatic B-ring exhibit promising in231vitro antiplasmodial activity. This supports the hypothesis that232nitrogen-containing flavonoids would enhance biological233activity compared to naturally occurring non-nitrogen ana-234logues. Reduction of the enone moiety increased antimalarial235bioactivity significantly. The structure of the amino moiety, A-236ring substituents, shortening of the propyl to an ethyl linkage,237and chloride salt formation further enhanced antiplasmodial238activity. The most active compound (26) has an IC50 value of2390.01 μM (10 nM). Many of the compounds have similar or240better IC50 values than CQ against CQ-sensitive malaria strains241(D10 and NF54) and showed little difference in activity against242CQ-resistant strains (Dd2 and K1). This is to be expected since243the new synthetic compounds possess structures unrelated to244CQ and other currently used antimalarial drugs. In vitro245cytotoxicity tests suggest that the compounds are relatively246nontoxic with high SI indices. Thus, we succeeded in247synthesizing antiplasmodial compounds that are relatively248uncomplicated and inexpensive to manufacture and are249structurally unrelated to existing antimalarial drugs.

250■ EXPERIMENTAL SECTION251General Experimental Procedures. Melting points were252determined with a Reichert Thermopan microscope with a Koffler253hot-stage and are uncorrected. Solid-state FT-IR spectra were recorded254as neat compound on a Bruker Tensor 27 spectrometer. A 600 MHz255Bruker Avance spectrometer was used to record the 1H NMR, COSY,256HMBC, HMQC (600 MHz) and 13C, APT (150 MHz) experiments257in either CDCl3 (δH = 7.24; δC = 77.2), acetone-d6, (δH = 2.04; δC =25829.8), or methanol-d4, (δH = 4.87 and 3.31; δC = 49.2) with TMS as259internal standard. Chemical shifts were expressed as parts per million260(ppm) on the delta (δ) scale, and coupling constants (J) are accurate261to 0.01 Hz. High-resolution mass spectral data (HRMS) were262collected using a Waters Micromass LCT Premier TOF-MS

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

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



D

263 spectrometer. All samples were dissolved and diluted to ∼2 ng/μL and264 infused without additives. Thin-layer chromatography (TLC) was265 performed on Merck aluminum sheets (silica gel 60 F254, 0.25 mm).266 Reactions were monitored by TLC on silica gel, with detection by UV267 light (254 nm). Thin-layer chromatograms were sprayed with a 2% (v/268 v) solution of formaldehyde (40% solution in H2O) in concentrated269 H2SO4 and subsequently heated to 110 °C to effect maximum270 development of color. Purity was measured with Shimadzu HPLC271 systems using a Phenomenex C18 (100 mm × 4.6 mm) 2.6 μm272 column; 2.0 μL injection volume; flow, 0.2 mL/min; isocratic system,273 mobile phase A, 0.1% HCO2H in H2O, and mobile phase B, MeCN,274 with a Shimadzu LC-20AD pump SPD-M20A UV detector set at 254275 nm. Chemicals purchased from commercial vendors were used without276 purification.277 General Procedure for the Synthesis of Chalcones via Aldol278 Condensation. A mixture of acetophenone (1 equiv) and aryl279 aldehyde (1 equiv) was stirred in EtOH (50 mL) at room temperature.280 A KOH solution (50%, 25 mL) was added after 10 min, which turned281 the reaction mixture bright yellow. The reaction mixture was left to stir282 overnight, after which it was quenched with ice-cold 1 N HCl (100283 mL) solution and extracted with EtOAc (2 × 50 mL). The organic284 layer was washed with water (1 × 50 mL) and dried over Na2SO4, and285 the solvent evaporated under reduced pressure. This is demonstrated286 for the synthesis of (E)-3-(3-hydroxyphenyl)-1-(4-methoxyphenyl)-

287prop-2-en-1-one (1) using 4-methoxyacetophenone (3.1356 g; 20.9288mmol) and 3-hydroxybenzaldehyde (3.0547 g; 25.0 mmol).289(E)-3-(3-Hydroxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one290(1): yellow crystals38 (EtOH); mp 163−164 °C; IR (neat) νmax2913323.24, 1583.50, 1168.39, 830.84, 666.33 cm−1; 1H NMR (acetone-292d6, TMS, 600 MHz) δ 8.16 (2H, d, J = 8.9 Hz, H-2′, H-6′), 7.80 (1H,293d, J = 15.6 Hz, H-3), 7.69 (1H, d, J = 15.6 Hz, H-2), 7.30−7.25 and2946.95−6.93 (4H, m, H-5″, H-2″, H-6″, H-4″), 7.07 (2H, d, J = 8.9 Hz,295H-3′, H-5′), 3.91 (3H, s, OCH3);

13C NMR (acetone-d6, TMS, 150296MHz) δ 187.4 (C-1), 163.6 (C-4′), 157.8 (C-3″), 143.3 (C-3), 136.7297(C-1″), 131.1 (C-1′), 130.7 (C-2′, C-6′), 129.9 (C-5″), 121.9 (C-2),298120.0 (C-6″), 117.4 (C-2″), 114.9 (C-4″), 113.9 (C-3′, C-5′), 55.1299(OCH3); HRESMS [M + H]+ m/z 255.1965 (calcd for C16H14O3 +300H+, 255.1960); Rf = 0.37, toluene/acetone (5:5), 3.506 g, 66%.301General Procedure for the Synthesis of Aminoalkylated302Chalcones via the Mannich Reaction. A mixture of the appropriate303chalcone (1 equiv), paraformaldehyde (1.5 equiv), and the appropriate304amine (2 equiv) was dissolved in EtOH (2 mL) and concentrated HCl305(5 drops). The reaction mixture was refluxed for about 9 h until TLC306showed the disappearance of the starting material. The reaction307mixture was quenched with solid NaHCO3 and extracted with EtOAc308(2 × 50 mL), and the extract washed with water (2 × 50 mL). The309organic layer was dried over Na2SO4, and the solvent evaporated under310reduced pressure. This is demonstrated for the synthesis of (E)-3-[3-

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

aNF54: alternative chloroquine-sensitive Plasmodium falciparum strain. bDd2: chloroquine-resistant Plasmodium falciparum strain. cRI: resistanceindex: IC50(Dd2)/IC50(D10).

dND: not determined.



E

311 hydroxy-4-(piperidin-1-ylmethyl)phenyl]-1-(4-methoxyphenyl)prop-312 2-en-1-one (3) using (E)-3-(3-hydroxyphenyl)-1-(4-methoxyphenyl)-313 prop-2-en-1-one (1) (0.609 g; 2.4 mmol), paraformaldehyde (0.145 g;314 4.8 mmol), and piperidine (0.50 mL; 5.1 mmol).315 (E)-3-(3-Hydroxy-4-[piperidin-1-ylmethyl)phenyl]-1-(4-316 methoxyphenyl)prop-2-en-1-one (3): beige crystals39 (EtOH); mp317 120−121 °C; IR (KBr) νmax 2945.44, 2159.13, 2031.94, 1598.90,318 1256.25 cm−1; 1H NMR (acetone-d6, TMS, 600 MHz) δ 8.18 (2H, d, J319 = 8.9 Hz, H-2′, H-6′), 7.81 (1H, d, J = 15.5 Hz, H-3), 7.68 (1H, d, J =320 15.5 Hz, H-2), 7.17 (1H, d, J = 1.5 Hz, H-2″), 7.16 (1H, dd, J = 7.5,321 1.5 Hz, H-6″), 7.09 (1H, d, J = 7.5 Hz, H-5″), 7.07 (2H, d, J = 8.9 Hz,322 H-3′, H-5′), 3.91 (3H, s, OCH3), 3.74 (2H, s, CH2), 2.53 (4H, br s, H-323 2‴, H-6‴), 1.66−1.60 (4H, m, H-3‴, H-5‴), 1.52 (2H, br s, H-4‴);324

13C NMR (acetone-d6, TMS, 150 MHz) δ 188.2 (C-1), 164.5 (C-4′),325 159.6 (C-3″), 144.1 (C-3), 136.5 (C-1″), 132.1 (C-1′), 131.6 (C-2′,326 C-6′), 130.0 (C-5″), 125.6 (C-4″), 122.3 (C-2), 120.7 (C-2″), 115.6327 (C-6″), 114.7 (C-3′, C-5′), 62.4 (CH2), 56.0 (OCH3), 54.5 (C-2‴, C-328 6‴), 26.7 (C-3‴, C-5‴), 24.6 (C-4‴); HREIMS m/z 351.1826 (calcd329 for C22H25NO3, 351.1824); HPLC purity 99.1%, tR = 1.54 min;330 column chromatography [toluene/acetone (5:5), 1.5 cm × 20 cm]; Rf331 = 0.42; 0.520 g, 62%.332 General Procedure for the Synthesis of the Dihydrochal-333 cones. The appropriate chalcone (1 equiv) was dissolved in a 1:3 (v/334 v) solution of EtOAc/H2O. Pd(OH)2/C (0.060 g) was added, and the335 system flushed with hydrogen. The reaction mixture was left to stir at336 room temperature for 24−48 h under H2 at atmospheric pressure.337 After completion of the reaction (TLC) the reaction mixture was

338filtered through silica gel, and the filtrate was extracted with EtOAc (2339× 50 mL) and washed with water (1 × 30 mL) and brine (1 × 20 mL).340The organic layer was dried over anhydrous MgSO4, and the solvent341evaporated under reduced pressure. Column chromatography342[hexanes/EtOAc (7:3), 1.5 cm × 15 cm] yielded the pure343dihydrochalcones in good yield. This is demonstrated for the synthesis344of 1-(4-fluorophenyl)-3-[3-hydroxy-4-(piperidin-1-ylmethyl)phenyl]-345propan-1-one (20) using 1-(4-fluorophenyl)-3-(3-hydroxyphenyl)-346propan-1-one (0.100 g; 0.41 mmol), paraformaldehyde (0.024 g;3470.80 mmol), and piperidine (0.09 mL; 0.88 mmol).3481-(4-Fluorophenyl)-3-(3-hydroxy-4-(piperidin-1-ylmethyl)phenyl)-349propan-1-one (20): light yellow oil; 1H NMR (CDCl3, TMS, 600350MHz) δ 7.97 (2H, dd, 3JH−H = 8.9 Hz; 4JH−F = 5.4 Hz, H-2′, H-6′),3517.09 (2H, t, 3JH−H = 8.9 Hz; 4JH−F = 8.9 Hz, H-3′, H-5′), 6.86 (1H, d, J352= 7.6 Hz, H-5″), 6.69 (1H, d, J = 1.5 Hz, H-2″), 6.64 (1H, dd, J = 7.6,3531.5 Hz, H-6″), 3.62 (2H, s, CH2), 3.24 (2H, t, J = 7.6 Hz, H-2), 2.96354(2H, t, J = 7.6 Hz, H-3), 2.47 (4H, H-2‴, H-6‴), 1.61 (6H, s, H-3‴,355H-4‴, H-5‴); 13C NMR (CDCl3, TMS, 150 MHz) δ 197.8 (C-1),356165.7 (1C, d, 1JC−F = 254.4 Hz, C-4′), 158.1 (C-3″), 141.7 (C-1″),357133.3 (1C, d, 4JC−F = 3.1 Hz, C-1′), 130.7 (2C, d, 3JC−F = 9.1 Hz, C-2′,358C-6′), 128.6 (C-5″), 119.5 (C-4″), 119.0 (C-6″), 115.8 (C-2″), 115.7359(2C, d, 2JC−F = 21.8 Hz, C-3′, C-5′), 61.9 (CH2), 53.9 (C-2‴, C-6‴),36040.3 (C-2), 30.0 (C-3), 25.9 (C-3‴, C-5‴), 24.0 (C-4‴); column361chromatography [hexanes/EtOAc (6:4), 1.5 cm × 15 cm]; Rf = 0.52;3620.118 g; 84%.363General Procedure for the Synthesis of the Diarylpropanes.364The appropriate chalcone (1 equiv) was dissolved in a 1:3 (v/v)

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



F

365 solution of EtOAc/H2O. Ten percent HCl(aq) (10 mL) with366 Pd(OH)2/C (0.060 g) was added, and the system flushed with367 hydrogen. The reaction mixture was left to stir at room temperature368 for 48−72 h under H2 at atmospheric pressure. After completion of369 the reaction (TLC) the reaction mixture was filtered through silica gel,370 and the filtrate was extracted with EtOAc (2 × 50 mL) and washed371 with water (1 × 30 mL) and brine (1 × 20 mL). The organic layer was372 dried over anhydrous MgSO4, and the solvent evaporated under373 reduced pressure. Column chromatography [hexanes/EtOAc (7:3),374 1.5 cm × 20 cm] yielded the pure diarylpropanes in good yield. This is375 demonstrated for the synthesis of 3-[3-(4-methoxyphenyl)propyl]-

376phenol (23) using (E)-3-(3-hydroxyphenyl)-1-(4-methoxyphenyl)-377prop-2-en-1-one (1) (0.200 g; 0.80 mmol).3783-[3-(4-Methoxyphenyl)propyl]phenol (23): yellow oil; IR (KBr)379νmax 2933.38, 1586.44, 1510.27, 1241.05 cm−1; 1H NMR (CDCl3,380TMS, 600 MHz) δ 7.10 (1H, t, J = 8.0 Hz, H-5″), 7.06 (2H, d, J = 8.5,381H-2′, H-6′), 6.82 (2H, d, J = 8.6 Hz, H-3′, H-5′), 6.72 (1H, d, J = 7.6382Hz, H-4″), 6.64 (2H, d, J = 1.5 Hz, H-2″, H-6″), 3.76 (3H, s, OCH3),3832.57−2.51 (4H, m, H-1, H-3), 1.89−1.83 (2H, m, H-2); 13C NMR384(CDCl3, TMS, 150 MHz) δ 157.5 (C-4′), 155.6 (C-3″), 144.3 (C-1″),385134.5 (C-1′), 129.4 (C-5″), 129.3 (C-2′, C-6′), 120.8 (C-6″), 115.4386(C-2″), 113.7 (C-3′, C-5′), 112.7 (C-4″), 55.3 (OCH3), 35.2 (C-3),

Table 6. Antiplasmodial Activity of Diarylpropanes with Different Amine Moieties (33−39)

Table 7. Antiplasmodial Activity of Diarylpropane HCl Salts (40−44)



G

387 34.4 (C-1), 32.9 (C-2); HREIMS m/z 242.1306 (calcd for C16H18O2,388 242.1307); Rf = 0.55, 0.185 g; 97%.389 General Procedure for the Synthesis of Aminoalkylated390 Diarylpropanes. A mixture of the appropriate diarylpropane (1391 equiv), paraformaldehyde (1.5 equiv), and the appropriate amine (2392 equiv) was dissolved in EtOH (2 mL) and concentrated HCl (5393 drops). The reaction mixture was refluxed for 9 h until TLC showed394 the disappearance of the starting material. The reaction mixture was395 quenched with solid NaHCO3 and extracted with EtOAc (2 × 50 mL),396 and the extract was washed with water (2 × 50 mL). The organic layer397 was dried over Na2SO4, and the solvent evaporated under reduced398 pressure. This is demonstrated for the synthesis of 5-[3-(4-399 methoxyphenyl)propyl]-2-(piperidin-1-ylmethyl)phenol (24) using400 3-[3-(4-methoxyphenyl)propyl]phenol (23) (0.165 g; 0.68 mmol),401 paraformaldehyde (0.037 g; 1.23 mmol), and piperidine (0.1 mL; 1.0402 mmol).403 5-[3-(4-Methoxyphenyl)propyl]-2-(piperidin-1-ylmethyl)phenol404 (24): light yellow oil; IR (KBr) νmax 2932.41, 2360.34, 1510.49,405 1242.60 cm−1; 1H NMR (acetone-d6, TMS, 600 MHz) δ 7.10 (2H, d, J406 = 8.7 Hz, H-2′, H-6′), 6.86 (1H, d, J = 7.2 Hz, H-5″), 6.82 (2H, d, J =407 8.7 Hz, H-3′, H-5′), 6.58−6.54 (1H, d, J = 1.5 Hz, H-2″, 1H, dd, J =408 7.3, 1.5 Hz, H-6″), 3.73 (3H, s, OCH3), 3.59 (2H, s, CH2), 2.56 (2H,409 t, J = 7.6 Hz, H-1), 2.53 (2H, t, J = 7.6 Hz, H-3), 2.45 (4H, br s, H-2‴,410 H-6‴), 1.91−1.82 (2H, m, H-2), 1.57 (4H, p, H-3‴, H-5‴), 1.46 (2H,411 br s, H-4‴); 13C NMR (acetone-d6, TMS, 150 MHz) δ 159.0 (C-4′),

412158.8 (C-3″), 143.7 (C-1″), 135.0 (C-1′), 130.1 (C-2′, C-6′), 129.3413(C-5″), 120.0 (C-4″), 119.6 (C-6″), 116.4 (C-2″), 114.5 (C-3′, C-5′),41462.3 (CH2), 55.4 (C-2‴, C-6‴), 54.4 (OCH3), 35.8 (C-3), 35.2 (C-1),41534.2 (C-2), 26.7 (C-3‴, C-5‴), 24.7 (C-4‴); HREIMS m/z 339.2198416(calcd for C22H29NO2, 339.2200); flash column chromatography417[hexanes/EtOAc (6:4), 1.5 cm × 15 cm]; Rf = 0.52; 0.095 g; 41%.418General Synthesis of HCl Salts of the Aminoalkylated419Diarylpropanes. The appropriate aminoalkyldiarylpropane was420dissolved in dry DCM (10 mL) at 0 °C. HCl gas was bubbled421through the reaction mixture for 60 min. Precipitation indicated the422formation of the salt. The excess solvent was removed under a stream423of N2 gas, and the product was lyophilized overnight. This is424demonstrated for the synthesis of 1-[2-hydroxy-4-{3-(4-425methoxyphenyl)propyl}benzyl]piperidinium chloride (40) using 5-426[3-(4-methoxyphenyl)propyl]-2-(piperidin-1-ylmethyl)phenol (26)427(0.200 g, 0.59 mmol).4281-[2-Hydroxy-4-{3-(4-methoxyphenyl)propyl}benzyl]piperidinium429chloride (40): white, amorphous solid; IR (neat) νmax 2935.94,4301511.06, 1242.36, 1033.16, 827.06 cm−1; 1H NMR (acetone-d6, TMS,431600 MHz) δ 7.47 (1H, d, J = 7.8 Hz, H-5″), 7.13 (2H, d, J = 8.6 Hz,432H-2′, H-6′), 6.98 (1H, d, J = 1.2 Hz, H-2″), 6.84 (2H, d, J = 8.6 Hz, H-4333′, H-5′), 6.76 (1H, dd, J = 7.6, 1.5 Hz, H-6″), 4.18 (2H, d, J = 4.7 Hz,434CH2), 3.76 (3H, s, OCH3), 3.42 (2H, d, J = 11.6 Hz, H-2‴), 2.96−4352.87 (6H, m, H-3‴, H-4‴, H-5‴), 2.63−2.53 (4H, m, H-1, H-3),4361.94−1.85 (2H, m, H-2‴, H-6‴), 1.82 (2H, d, J = 14.5 Hz, H-6‴); 13C

Table 8. Antiplasmodial Activity of Diverse Analogues (45−55)



H

Table 9. Toxicity Values (IC50) of the Most Promising Analogues against CHO Cell Lines and SI Values

aSI: selectivity index = IC50(CHO)/IC50(D10).

Scheme 5. Synthesis of Compound 46 via the MannichReaction

Figure 1. Stucture of MK-4815.



I

437 NMR (acetone-d6, TMS, 150 MHz) δ 158.9 (C-4′), 157.8 (C-3″),438 147.0 (C-1″), 134.9 (C-1′), 134.7 (C-2′, C-6′), 130.1 (C-5″), 121.3439 (C-6″), 119.3 (C-4″), 115.7 (C-2″), 114.5 (C-3′, C-5′), 55.4 (C-2‴,440 C-6‴), 52.7 (OCH3, CH2), 35.8 (C-3), 35.1 (C-1), 34.0 (C-2), 23.5441 (C-3‴, C-5‴), 22.7 (C-4‴); HRTOFESMS [M + H]+ m/z 340.2270442 (calcd C22H29NO2 + H+, 340.2277); HPLC purity 86.7%, tR = 1.61443 min; 0.185 g; 84%.444 Bioassays. Antiplasmodial Assay. Continuous in vitro cultures of445 asexual erythrocyte stages of P. falciparum were maintained using the446 modified method of Trager and Jensen.40 Quantitative assessment of447 antiplasmodial activity in vitro was determined via the parasite lactate448 dehydrogenase assay using a modified method described by Makler.41

449 The test samples were tested in triplicate on one or two separate450 occasions. Compounds were initially screened for antiplasmodial451 activity against the chloroquine-sensitive (CQS) D10 and NF54452 strains. The most promising compounds were subsequently tested453 against the chloroquine-resistant (CQR) strains, Dd2 and K1.454 CQ was used as an internal standard to monitor the experimental455 conditions and showed IC50 values within an acceptable range of456 0.018−0.060 μM for the CQS strains and 0.470−0.780 μM for the457 CQR strains. The 50% inhibitory concentration (IC50) values were458 obtained from full dose−response curves (Supporting Information),459 using a nonlinear dose−response curve-fitting analysis via GraphPad460 Prism v.4 software.461 Cytotoxicity Assay. The MTT assay as described by Mosmann462 (with minor modifications) was used to determine cell viability using463 the Chinese hamster ovarian (CHO) cell line.42 The sample464 preparation was done in the same manner as for the antiplasmodial465 testing. Emetine was used as the reference drug in all experiments and466 showed IC50 values within an acceptable range (0.080−0.120 μM).467 The initial concentration of emetine was 100 μg/mL, which was468 serially diluted in complete medium with 10-fold dilutions to give six469 concentrations, the lowest being 0.001 μg/mL. The same dilution470 technique was applied to all the test samples. The highest

471concentration of solvent (0.5%) to which the cells were exposed had472no measurable effect on the cell viability (data not shown).

473■ ASSOCIATED CONTENT474*S Supporting Information475Experimental detail, 1D and 2D NMR data, MS, IR, melting476points, and HPLC data. The Supporting Information is477available free of charge on the ACS Publications website at478DOI: 10.1021/acs.jnatprod.5b00114.

479■ AUTHOR INFORMATION480Corresponding Authors481*Tel: +27 (0)51 401 9305. Fax: +27 (0)51 401 7295. E-mail:[email protected] (A. Wilhelm).483*Tel: +27 (0)51 401 2782. Fax: +27 (0)51 401 7295. E-mail:[email protected] (J. H. van der Westhuizen).485Notes486The authors declare no competing financial interest.

487■ ACKNOWLEDGMENTS488We thank Dr. C. Edlin, previously from iThemba Pharmaceut-489icals, for his valuable input toward the structural modifications490of compounds in this study. Financial support by the University491of the Free State is acknowledged.

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Scheme 6. Synthesis of Compound 47 via the Mannich Reaction

Scheme 7. Synthesis of Compound 49 via the WittigReaction

Figure 2. Selected HMBC correlations of compound 24.



J

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