Identification of Compounds with Efficacy against Malaria Parasites

Dec 21, 2015 - Four known kaempferol 3-O-rhamnosides were isolated from the methanolic supercritical fluid extract of Platanus occidentalis L. (Platan...
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Identification of Compounds with Efficacy against Malaria Parasites from Common North American Plants Shengxin Cai,†,‡,# April L. Risinger,§,⊥,# Shalini Nair,∥ Jiangnan Peng,§ Timothy J. C. Anderson,∥ Lin Du,†,‡ Douglas R. Powell,† Susan L. Mooberry,*,§,⊥ and Robert H. Cichewicz*,†,‡ †

Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, and ‡Natural Products Discovery Group, and Institute for Natural Products Applications and Research Technologies, University of Oklahoma, Norman, Oklahoma 73019, United States § Department of Pharmacology and ⊥Cancer Therapy and Research Center, University of Texas Health Science Center, San Antonio, Texas 78229, United States ∥ Department of Genetics, Texas Biomedical Research Institute, San Antonio, Texas 78227, United States S Supporting Information *

ABSTRACT: Some of the most valuable antimalarial compounds, including quinine and artemisinin, originated from plants. While these drugs have served important roles over many years for the treatment of malaria, drug resistance has become a widespread problem. Therefore, a critical need exists to identify new compounds that have efficacy against drug-resistant malaria strains. In the current study, extracts prepared from plants readily obtained from local sources were screened for activity against Plasmodium falciparum. Bioassay-guided fractionation was used to identify 18 compounds from five plant species. These compounds included eight lupane triterpenes (1−8), four kaempferol 3-O-rhamnosides (10−13), four kaempferol 3-O-glucosides (14−17), and the known compounds amentoflavone and knipholone. These compounds were tested for their efficacy against multi-drug-resistant malaria parasites and counterscreened against HeLa cells to measure their antimalarial selectivity. Most notably, one of the new lupane triterpenes (3) isolated from the supercritical extract of Buxus sempervirens, the common boxwood, showed activity against both drug-sensitive and -resistant malaria strains at a concentration that was 75-fold more selective for the drug-resistant malaria parasites as compared to HeLa cells. This study demonstrates that new antimalarial compounds with efficacy against drug-resistant strains can be identified from native and introduced plant species in the United States, which traditionally have received scant investigation compared to more heavily explored tropical and semitropical botanical resources from around the world.

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infectious organism. While several promising new antimalarial compounds are being explored,5,6 the high rate of attrition for candidate molecules under clinical investigation suggests that additional efforts are needed to develop alternative therapeutic strategies. The majority of approved treatment options for malaria are either plant-derived natural products or synthetic analogues of plant-derived compounds. Quinine, originally obtained from the bark of the South American cinchona tree, was introduced to Europe in the 17th century as an antimalarial treatment by Jesuit priests returning from Peru.7 Chloroquine has been widely used for over 50 years before resistance became widespread. Artemisinin, derived from sweet wormwood (Artemisia annua), clears parasites from the blood of patients

n 2013, malaria infections impacted an estimated 198 million people across 97 different countries, resulting in an estimated 584 000 deaths.1 While several related Plasmodium spp. are implicated in the disease, Plasmodium falciparum is responsible for the majority of deaths. No effective vaccine has been developed for malaria, which has meant that smallmolecule therapeutics must continue to fulfill the treatment needs of infected patients and at-risk individuals. There are four major classes of compounds that are used clinically to treat malaria: (1) quinolines (e.g., chloroquine, quinine, mefloquine), (2) antifolates (e.g., sulfadoxine, pyrimethamine), (3) artemisinin derivatives (e.g., artesunate, artemether), and (4) antimicrobials (e.g., doxycycline); however, resistance to the most widely used agents from the first three major drug classes is now widespread.2 Specifically, resistance to artemisinin derivatives, which form the basis of widely used combination therapies for malaria, is now common across Southeast Asia.3 The further spread of artemisinin resistance is anticipated to be disastrous for malaria control efforts.4 It is therefore imperative that new classes of drugs be developed for the treatment of this © XXXX American Chemical Society and American Society of Pharmacognosy

Special Issue: Special Issue in Honor of John Blunt and Murray Munro Received: October 1, 2015

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DOI: 10.1021/acs.jnatprod.5b00874 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

more rapidly than any other known antimalarial.8 Artemisinin derivatives are now widely used as part of combination therapies with other drugs such as lumefantrine or mefloquine. Even atovaquone, which is combined with proguanil in the drug Malarone, is an analogue of the plant-derived compound lapachol from Tabebuia sp. (Bignoniaceae). The recent identification of the alkaloid tazopsine from a Madagascan plant (Strychnopsis thousarsii), which kills the liver stage of malaria, further supports the continued exploration of plants for antimalarial leads.9 Despite these successes, only a small fraction of the world’s plant species have been screened for antimalarial activity. Furthermore, the plants that have been tested tend to have been obtained solely from the flora of malaria-endemic countries or have been selected based on a history of medicinal use against the disease.10,11 The intent of this study was to expand the diversity of plants tested for bioactive compounds that are active against malaria by focusing on readily available botanical resources from our local environment in the south-central United States. Specifically, we have focused exclusively on plants that grow well in the sometimes harsh environment of southern Texas.12 Our studies have yielded several potent and selective inhibitors of drug-resistant P. falciparum, which highlight the value of local North American plants as a resource for bioactive natural product leads for malaria.

guided fractionation based on their capacity to specifically inhibit the growth of the malaria parasite with minimal or no toxicity toward human cells. It is worth noting the important role that the extraction methods played in liberating the active compounds from the freeze-dried plant material; among the hits, only a single extract from a given plant yielded a bioactive hit. In other words, the different extraction methods performed on a given plant captured unique sets of metabolites that were distinct enough from one another so as to preclude the possibility that a single extraction method would have sufficed to produce the hits detected in this project. Our investigation of the compounds responsible for the antimalarial activities of the active extracts was led by an HPLCbased microtiter plate fractionation process13 in which approximately 5 mg of extract was separated into 92 wells of a 96-well plate. The fractions were split with half the material used for bioassay testing, and the other half was retained for LCESIMS analysis of the active wells. This approach enabled us to use the LCESIMS data to rapidly guide the follow-up metabolite purification process. We used this tactic to purify a total of 18 compounds that exhibited varying degrees of antimalarial activity, including knipholone,14 amentoflavone,15 eight lupane triterpenes [1−8; compounds 3−5 and 7−8 are new, whereas 1 (23-O-(Z)-p-coumaroyl-23-hydroxybetulin), 2 (23-O-(E)-p-coumaroyl-23-hydroxybetulin), and 6 (3-O-(E)-pcoumaroyl-23-hydroxybetulin) were reported earlier this year by the Kinghorn group],16 four known kaempferol 3-Orhamnosides (10−13),17 and four known kaempferol 3-Oglucosides (14−17).18−20 Eight lupane triterpenes (1−8, of which 3−5 and 7−8 are new analogues) were purified from the methanolic supercritical extract of the common boxwood, Buxus sempervirens L. (Buxaceae), after their detection using our microtiter plate fractionation process. Compound 1 was obtained as colorless, block-shaped crystals, and its molecular formula was established as C39H56O5 based on HRESIMS data. The planar structure of

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RESULTS AND DISCUSSION A collection of 1281 plant extracts was generated from 463 species of plants that grow in Texas, USA. The extracts were generated using supercritical fluid (CO2) extraction with or without methanol, as well as traditional aqueous extractions to capture as broad a range of metabolites as possible from each sample. For the initial screening, extracts were tested for their abilities to inhibit the growth of the drug-sensitive HB3 strain of P. falciparum at a concentration of 20 μg/mL. Using this delimiting parameter, five extracts were prioritized for bioassayB

DOI: 10.1021/acs.jnatprod.5b00874 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR Spectroscopic Data for Compounds 1, 3−5, 7, and 8 (δ in ppm, J in Hz) 1a,b

no. 1

3c,d

4c,d

5a,d

2

1.50, m 0.75, m 1.45, m

1.74, m 1.00, m 1.67, m

1.63, m 0.83, m 1.58, m

3 5 6

3.29, dd (9.6, 6.0) 0.86, m 1.25, m

3.61, dd (4.9, 11.2) 1.20, m 1.46, m

3.46, dd (5.2, 11.8) 0.95, m 1.35, m

7

1.12, 1.06, 1.12, 1.29, 1.12, 1.55, 0.95, 1.54, 1.55, 0.78, 1.85, 0.98, 1.42, 2.33, 1.83, 1.23, 1.83, 0.85, 3.91, 3.87, 0.60, 0.75, 0.91, 0.74, 3.47, 3.04, 4.63, 4.51, 1.60, 7.57, 6.72, 6.72, 7.57, 6.82, 5.75, 9.82,

1.41, m 1.40, m 1.46, m

1.45, 1.20, 1.23, 1.40,

1.70, m

1.65, m

1.70, 1.76, 1.01, 1.92, 1.14, 1.58, 2.41, 1.95, 1.37, 1.89, 0.99, 4.09, 4.04, 0.77, 0.92, 1.08, 0.97, 3.73, 3.27, 4.68, 4.57, 1.68, 7.21,

m m m m m t (11.7) m m m m m d (11.4) d (11.4) s s s s d (10.4) d (11.1) d (2.2) brs s d (1.9)

1.66, 1.58, 0.91, 1.92, 1.12, 1.57, 2.41, 1.92, 1.33, 1.90, 0.98, 4.08, 3.99, 0.73, 0.87, 1.03, 0.83, 3.72, 3.27, 4.68, 4.57, 1.69, 7.66,

m m m m m t (11.5) m m m m m d (11.4) d (11.4) s s s s d (10.8) d (10.8) d (2.2) brs s d (2.0)

6.83, 7.08, 7.63, 6.38,

d (8.2) dd (8.3, 1.9) d (15.9) d (15.9)

6.77, 7.08, 6.89, 5.81,

d (8.2) dd (2.0, 8.3) d (12.9) d (12.8)

9 11 12 13 15 16 18 19 21 22 23 24 25 26 27 28 29 30 2′ 3′ 5′ 6′ 7′ 8′ OH-4′ OMe-3′

m m m m m m m m m m m m m m m m m m d (11.5) d (11.5) s s s s d (10.7) d (10.7) d (2.3) brs s d (8.7) d (8.7) d (8.7) d (8.7) d (12.9) d (12.9) s

3.91, s

m m m m

3.88, s

1.60, 0.88, 1.67, 1.59, 4.78, 1.21, 1.40, 1.28, 1.42, 1.27, 1.31, 1.35, 1.16, 1.60,

m m m m dd (5.0, 11.8) m m m m m m m m m

1.60, m 1.68, m 0.91, m 1.90, m 1.05, m 1.48, t (11.8) 2.39 m 1.85, m 1.25, m 1.84, m 0.88, m 3.11, d (10.9) 2.93, d (10.9) 0.57, s 0.82, s 0.98, s 0.94, s 3.51, d (10.8) 3.07, d (10.8) 4.66, d (2.0) 4.53, brs 1.64, s 7.62, d (8.7) 6.73, d (8.7) 6.73, d (8.7) 7.62, d (8.7) 6.80, d (13.0) 5.71, d (12.8) 9.34, brs

7c,d

8c,d

1.73, m 1.02, m 1.76, m

1.73, m 1.02, m 1.76, m

4.95, m 1.32, m 1.48, m

4.90, m 1.32, m 1.48, m

1.60, 1.38, 1.44, 1.45,

1.60, 1.38, 1.44, 1.45,

m m m m

m m m m

1.69, m

1.69, m

1.68, m 1.78, m 1.05, m 1.96, m 1.19, m 1.61, m 2.42, m 1.95, m 1.35, m 1.91, m 0.99, m 3.32e 3.14, d (12.0) 0.80, s 0.95, s 1.09, s 1.04, s 3.74, d (11.5) 3.29e 4.69, brs 4.57, brs 1.70, s 7.18, d (1.8)

1.68, m 1.78, m 1.05, m 1.96, m 1.19, m 1.61, m 2.42, m 1.95, m 1.35 m, 1.91, m 0.99, m 3.28e 3.10, d (11.5) 0.66, s 0.92, s 1.08, s 1.04, s 3.74, d (11.5) 3.29e 4.69, brs 4.57, brs 1.70, s 7.73, d (1.9)

6.80, 7.05, 7.57, 6.33,

6.75, 7.08, 6.84, 5.75,

d (8.2) dd (8.3, 1.8) d (15.9) d (15.9)

3.89, s

d (8.2) dd (8.2, 1.9) d (12.8) d (12.9)

3.87, s

a

Solvent was DMSO-d6. bPerformed on a 400 MHz Varian instrument. cSolvent was MeOH-d4. dPerformed on a 500 MHz Varian instrument. e Signal was overlapped with solvent peak.

compound 1 was hydrolyzed overnight in 1 M NaOH at room temperature, and the triterpene portion of the molecule (23-hydroxybetulin [9]) was purified from the hydrolysate. NMR (Figures S45 and S46, Supporting Information) and optical rotation ([α]20D +16) data obtained for the hydrolysis product were consistent with that reported for 9 ([α]20D +22).22 The proposed absolute configuration is consistent with an in silico derived specific rotation value for 9 (calcd +68).23,24 Compound 2 was obtained as a white, amorphous powder, and the planar structure was determined by comparing its HRESIMS and NMR data with published data.16 The relative

compound 1 was determined by de novo analysis of its NMR data (Tables 1 and 2 and Figure 1) and, later, by comparing its NMR data to structurally related metabolites.16 The relative configuration of the compound was determined based on ROESY experimental data (Figure 1), which were consistent with reported lupane triterpene analogues.16 During the course of the NMR studies, we obtained crystals of compound 1 that were suitable for X-ray crystallography, which served to confirm the compound’s planar structure, as well as its relative configuration (Figure 2). The absolute configuration of 1 was determined by refinement of the Hooft parameter.21 To further substantiate the absolute configuration of the metabolite, C

DOI: 10.1021/acs.jnatprod.5b00874 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 13C NMR Spectroscopic Data for Compounds 1, 3−5, 7, and 8 no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ OMe-3′ a

1a,b 38.4 27.1 70.4 41.9 47.7 18.2 33.9 40.9 50.5 36.9 20.8 25.3 37.2 42.5 26.9 29.5 47.9 48.6 47.8 150.9 29.8 34.3 65.6 12.6 16.7 16.2 14.7 58.4 110.1 19.2 126.0 132.7 115.4 159.3 115.4 132.7 142.9 116.2 166.7

CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH2 CH C CH2 CH2 C CH CH C CH2 CH2 CH2 CH3 CH3 CH3 CH3 CH2 CH2 CH3 C CH CH C CH CH CH CH C

3b,c 38.4 26.1 71.3 41.9 47.8 17.9 33.7 40.7 50.7 36.7 20.6 25.2 37.3 42.4 26.7 28.9 47.5 48.6 47.7 150.4 29.4 33.6 65.3 11.1 15.6 15.1 13.8 58.9 108.8 17.8 126.2 110.2 148.0 149.3 115.1 122.8 145.4 114.1 167.7 55.0

CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH2 CH C CH2 CH2 C CH CH C CH2 CH2 CH2 CH3 CH3 CH3 CH3 CH2 CH2 CH3 C CH C C CH CH CH CH C CH3

4b,c 38.2 26.0 71.1 41.7 47.2 17.9 33.7 40.6 50.4 36.6 20.5 25.1 37.2 42.3 26.5 28.9 47.5 48.5 47.7 150.4 29.4 33.6 65 CH2 11.1 15.5 15.0 13.6 58.9 108.8 17.7 126.8 113.1 147.0 148.1 114.4 124.7 142.9 115.6 167.1 54.9

5a,b CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH2 CH C CH2 CH2 C CH CH C CH2 CH2 CH3 CH3 CH3 CH3 CH2 CH2 CH3 C CH C C CH CH CH CH C CH3

38.1 23.1 74.2 41.5 46.6 17.6 33.7 40.8 50.1 36.7 20.8 25.2 37.1 42.7 27.1 29.5 47.8 48.6 47.7 150.8 29.7 34.3 63.1 13.6 16.7 16.1 14.9 58.3 110.1 19.2 125.9 132.9 115.3 159.2 115.3 132.9 143.1 116.6 166.1

CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH2 CH C CH2 CH2 C CH CH C CH2 CH2 CH2 CH3 CH3 CH3 CH3 CH2 CH2 CH3 C CH CH C CH CH CH CH C

7c,d 37.9 22.9 74.4 41.6 46.5 17.3 33.3 40.6 50.3 36.5 20.6 25.1 37.2 42.4 26.7 28.9 47.8 48.5 47.9 150.3 29.4 33.7 63.1 12.3 15.6 15.1 13.8 58.9 108.8 17.9 126.2 110.0 148.0 149.0 115.0 122.7 145.0 114.3 167.7 55.0

CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH2 CH C CH2 CH2 C CH CH C CH2 CH2 CH2 CH3 CH3 CH3 CH3 CH2 CH2 CH3 C CH C C CH CH CH CH C CH3

8c,d 37.9 22.9 74.2 41.6 46.5 17.3 33.3 40.6 50.3 36.5 20.6 25.1 37.2 42.4 26.7 28.9 47.8 48.5 47.9 150.3 29.4 33.7 63.1 12.1 15.6 15.1 13.8 58.9 108.8 17.9 126.3 113.3 147.0 148.0 114.0 124.9 144.1 115.8 166.6 55.0

CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH2 CH C CH2 CH2 C CH CH C CH2 CH2 CH2 CH3 CH3 CH3 CH3 CH2 CH2 CH3 C CH C C CH CH CH CH C CH3

Solvent was DMSO-d6. bPerformed on a 100 MHz Varian instrument. cSolvent was MeOH-d4. dCarbon data were determined by a combination of C (100 MHz), HSQC (500 MHz), and HMBC (500 MHz) experiments.

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Figure 1. Key 1H−1H COSY, HMBC, and ROESY correlations for compound 1.

Figure 2. ORTEP structure generated from the X-ray diffraction data for a single crystal of 1.

D

DOI: 10.1021/acs.jnatprod.5b00874 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 3. Bioassay Data of Amentoflavone, Knipholone, and Compounds 1−17 compound amentoflavone knipholone 1 2 3 4 and 3 (3:1) 5 and 6 (4:1) 6 7 and 8 (2:1) 9 10 11 12 13 14 15 16 17

HB3 IC50 ± SE (μM)a 25 4.9 0.8 0.9 0.5 1.0 2.3 1.9 3.0 3.4 0.6 2.0 0.50 1.8 0.6 0.9 0.8 2.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2 0.6 0.4 0.1 0.2 0.1 0.1 0.2 0.3 0.5 0.2 0.6 0.03 0.4 0.1 0.2 0.1 0.9

NHP1337 IC50 ± SE (μM)a

relative resistance (NHP1337/HB3)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.8 1.0 1.9 0.9 0.5 0.3 0.7 0.7 0.8 1.5 12 2.0 8.2 3.9 3.5 5.6 5.0 1.8

19 5 1.53 0.85 0.27 0.31 1.6 1.4 2.3 5.2 7 4 4.1 7 2.1 5 4 3.8

7 2 0.04 0.05 0.03 0.05 0.1 0.2 0.4 0.3 1 1 0.5 1 0.6 1 1 0.6

HeLa IC50 ± SE (μM)b

fold selectivity (HeLa/HB3)

fold selectivity (HeLa/NHP1337)

± ± ± ±

1.9 6.6 8.8 31 >40 20 9.6 >11 >6.7 >5.9 34 6.0 19 8.9 3.8 2.9 3.0 2.3 2.3 20 20.6 11.9 9.3 16