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. Author manuscript; available in PMC: 2010 Feb 26.
Published in final edited form as: J Med Chem. 2009 Feb 26;52(4):1198–1203. doi: 10.1021/jm801484v

Malaria-Infected Mice Are Cured by a Single Oral Dose of New Dimeric Trioxane Sulfones Which Are Also Selectively and Powerfully Cytotoxic to Cancer Cells

Andrew S Rosenthal a, Xiaochun Chen b, Jun O Liu b, Diana C West c, Paul J Hergenrother c, Theresa A Shapiro d,e, Gary H Posner a,e,*
PMCID: PMC2698029  NIHMSID: NIHMS92809  PMID: 19186946

Abstract

A new series of 6 dimeric trioxane sulfones has been prepared from the natural trioxane artemisinin in 5 or 6 chemical steps. One of these thermally and hydrolytically stable new chemical entities (4c) completely cured malaria-infected mice via a single oral dose of 144 mg/kg. At a much lower single oral dose of only 54 mg/kg combined with 13 mg/kg of mefloquine hydrochloride, this trioxane dimer 4c as well as its parent trioxane dimer 4b also completely cured malaria-infected mice. Both dimers 4c and 4b were potently and selectively cytotoxic toward five cancer cell lines.

Introduction

Even in 2009, one child dies of malaria approximately every 30 seconds.1–3 No vaccine is yet available to prevent malaria infection in humans.4 Chemotherapy of malaria-infected patients has been used broadly with great success. Now, unfortunately, there is widespread malaria parasite resistance to many of the standard amine antimalarial drugs like chloroquine.5 During the past few decades,6–13 a new, non-amine class of antimalarial 1,2,4-trioxanes has emerged from traditional Chinese herbal remedies. Natural 1,2,4-trioxane artemisinin (1) and its semi-synthetic daughter trioxanes dihydroartemisinin (1a), artemether (1b), and water-soluble sodium artesunate (1c) are the fastest-acting antimalarial drugs known.6–13 They are now often combined, as recommended by the World Health Organization (WHO), with standard antimalarial amines like lumefantrine or amodiaquine; such artemisinin combination therapy (ACT), the current chemotherapeutic method of choice where malaria is endemic, requires a repeated dosing regimen.14–17 For example, Coartem® combines the trioxane artemether and lumefantrine in a curative 3-day 6-dose regimen.18 A single dose cure would involve better patient compliance and lower cost. Toward this challenging and urgent goal, we have reported a series of trioxane dimers able to cure malaria-infected mice after only a single subcutaneous dose19 as well as a related series of trioxane dimers curative after three oral doses.20–22 Such trioxane dimers are significantly more efficacious in curing malaria-infected mice than twice the dose of the corresponding trioxane monomers, as well as being significantly more cytotoxic than the corresponding monomeric trioxanes toward cancer cells.20–22 Here, as proof of principle, we describe a new series of dimeric trioxane sulfones 4 (Scheme 1), two of which are curative after only a single oral dose to malaria-infected mice.

Scheme I.

Scheme I

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Some of these new dimeric trioxane sulfones 4 have also selective and potent anticancer activity.23 Increasingly widespread evidence indicates that human cancer cells, richer than normal cells in iron-transport transferrin receptors,24,25 selectively activate trioxanes to produce various cytotoxic intermediates; this process is similar to that in the triggering of trioxanes by heme iron in malaria-infected human erythrocytes.8 The anticancer properties of trioxanes have been reviewed.26–29 Here we disclose that some of the new dimeric trioxane sulfones 4 powerfully inhibit the growth (sub-micromolar IC50 values) of various cancer cells in vitro without strongly affecting noncancerous fibroblasts.

Results and Discussion

Chemistry

As outlined in Scheme I, trioxane dimer primary alcohol 2 was prepared in 3 steps from natural artemisinin, as described previously.30 Primary bromide 3 was easily prepared in good yield by treating alcohol 2 with CBr4 and Ph3P.31 Displacement of the bromide anion by a mercaptide anion, followed by sulfide→sulfone oxidation with meta-chloroperbenzoic acid (mCPBA) proceeded also in good yields without disruption of the crucial peroxide pharmacophore. This is an especially noteworthy result because most peroxides are cleaved by mercaptide anions. Jung and coworkers reported the first example of mercaptide displacement of a trioxane primary bromide leading to a dimeric trioxane sulfone.22 We prepared dimeric trioxane sulfones 4a–f from natural artemisinin in 5–6 chemical steps and good overall yields. The calculated octanol/water partition coefficient (log P) of these sulfone dimers ranged from 7.0 – 9.5. Scale up to multigram synthesis and even to kilogram manufacture of sulfones 4 is not expected to be a problem. Some endoperoxide sulfones like synthetic bicyclic sulfone 532 and semi-synthetic sulfone 633 among others34–37 have excellent antimalarial activities. The extraordinary antimalarial efficacy of dimeric trioxane sulfone benzylic alcohol 4b as well as the chemical versatility of its primary hydroxyl group prompted us to prepare the corresponding carbamates 4c and 4d and phosphate 4e as potential prodrugs. It was expected that, in vivo, esterase enzymes would convert carbamates 4c and 4d and phosphate 4e back into their parent alcohol 4b. Benzylic alcohol 4b and dimethyl carbamate 4c are stable in the solid state at 60 °C for ≥24 hours; carbamate 4c is stable in the solid state at 60 °C even for one week. Both dimers 4b and 4c are stable for at least 12 hours at room temperature in 80/20 DMSO/water at pH 7.4.

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Biology

Each trioxane dimer 4a-4f (7.2 mg) was dissolved in 0.11 mL of 7:3 Tween 80:ethanol and then diluted with 1.10 mL of water for oral administration to 5-week old C57BL/6J male mice (from the Jackson Laboratory) weighing about 22 grams that were infected intraperitoneally on day 0 with the Plasmodium berghei, ANKA strain (2 × 107 parasitized erythrocytes).38 Each of 5 mice in a group was treated orally with a single dose of 0.20 mL (0.20 mL/1.21 mL × 7.2 mg = 1.2 mg) of diluted compound solution, corresponding to a dose of 54 mg/kg, 24 hours post-infection. In separate experiments, a single oral dose of 72 mg/kg and a single oral dose of 144 mg/kg were also used. Blood parasitemia levels as well as monitoring the duration of animal survival compared to survival time of animals receiving no drug are both widely accepted as a measure of a drug’s efficacy in antimalarial drug development. Three days after infection, an average of 16.2% blood parasitemia was observed in the control (no drug) group. Animals receiving no drug die typically 6–8 days post-infection. A widely accepted yardstick of cure (i.e. 100% efficacy) is survival of animals to day 30 post-infection, with no detectable malaria parasites in the animal’s blood at that time. Average survival results are summarized in Table 1. The clinically used monomeric trioxane drugs 1b and 1c and the synthetic trioxolane peroxide drug development candidate OZ277 maleate (7) are included as standards.

Table 1.

Antimalarial Efficacy Using a Single Oral Dose of Dimeric Trioxane Sufones 4 in P. berghei-Infected Mice

Dimeric Trioxane Sulfone Average Survival (days) After Infection; Trioxane Dose (mg/kg) % Suppression of Parasitemia (on day 3 post infection)
1 × 54 1 × 72 1 × 144 1 × 54 1 × 72 1 × 144
4a 14.6 – – 97.1 - -
4b 20.4 (11,12,19,30,30)a 25.0 (12,25,27,30,30)a 30.0 (2 sick, 3 cured) 99.8 99.6 >99.9
4c 22.8 (11,24,26,26,27)a 23.0 (11,14,30,30,30)a 30.0 (all cured) 99.5 98.6 99.3
4d 16.4 - - 99.2 - -
4e 14.8 - - 92.4 - -
4f 11.0 - - 2.0 - -
Vehicle (no drug) - 6.4 (6,6,6,7,7) a - - - -
1b 11 (8,10,11,13,13) a 9.8 (9,9,10,10,11) a 12.4 (12,12,12,13,13) a 99.4 98.7 99.8
1c - 7.6 b - - - -
7 - 10.7 b - - 99.95b -
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a

Actual mouse survival until day.

b

Data from Supporting Information in ref. 39 using a single oral dose of 30 mg/kg. A dash in the table means not tested.

It is clear from the data in Table 1 that all of the dimeric trioxane sulfones 4a–4f prolonged average survival time at least as effectively as trioxolane 7, which is in phase II clinical trials.39 It is also apparent from the data in Table 1 that the dimeric trioxane sulfones 4b and 4c, at a single oral dose of 54 mg/kg, were the most efficacious among sulfones 4a–4f at prolonging survival. Therefore, sulfones 4b and 4c were tested further using a higher single oral dose of 72 mg/kg and separately using an even higher single oral dose of 144 mg/kg. As shown in Table 1, dimeric sulfones 4b and 4c at a single oral dose of 72 mg/kg prolonged average survival to days 23–25. At a higher oral dose if 144 mg/kg, the benzyl alcohol sulfone 4b prolonged average survival to day 30, but 2 of the 5 mice had 7–9% parasitemia at day 30 and appeared sick. In contrast, carbamate sulfone 4c at a single oral dose of 144 mg/kg completely cured all of the malaria-infected mice with no adverse effects. Using the same experimental protocol but combining benzylic alcohol dimer 4b (40 mg/kg) with mefloquine hydrochloride (13 mg/kg) in a single oral dose caused a 99.9% suppression of parasitemia on day 3 post infection and raised average survival to 28 days; a slightly higher single oral dose of dimer 4b (54 mg/kg) along with mefloquine hydrochloride (13 mg/kg) caused >99.9% parasitemia suppression on day 3 post infection and completely cured the mice. Similar curative results were obtained by combining sulfone carbamate dimer 4c (54 mg/kg) with mefloquine (13 mg/kg). In a control experiment, a single oral dose of mefloquine hydrochloride (13 mg/kg) alone prolonged average survival to only day 11. Neither overt toxicity nor behavioral change attributable to trioxane drug administration was observed in any of the malaria-infected animals cured by carbamate 4c or cured by the dimer plus mefloquine hydrochloride combination. The standard monomeric trioxane antimalarial drugs 1b and 1c and the trioxolane drug candidate 7, although able to lower parasitemia levels considerably by day 3 post infection, were not efficacious in prolonging the average survival time beyond day 11.

Table 2 summarizes the in vitro cytotoxic activity of dimeric trioxane sufones 4b and 4c in five different cancer cell lines. The IC50 values are shown as an average of at least three experiments with standard error of the mean using a standard MTS assay or sulforhodamine B (SRB) assay. Clearly, both dimers 4b and 4c are strongly cytotoxic, often comparing well with the anticancer potency of doxorubicin, a clinically used anticancer drug.40 Doxorubicin, however, has serious side effects in humans.41 In this study, we have found that doxorubicin is toxic toward the WT-MEF and Hs888Lu noncancerous immortalized fibroblast cell lines (with IC50 values of 3.4 ± 1.3 μM and 1.4 ± 0.7 μM, respectively). Neither dimer 4b or 4c significantly affected these noncancerous fibroblast cell lines (see Table 2). This selective cytotoxicity of both dimers 4b and 4c is of special importance for potential anticancer drug development of this class of dimeric trioxane sulfones.

Table 2.

Anticancer Activity of Dimeric Trioxane Sulfones 4b and 4c in Human Cancer Cell Lines

Cancer cell line IC50 Values (μM)a
Dimer 4b Dimer 4c
U-937-Lymphoma 0.28 ± 0.15 0.28 ± 0.13
HL-60-Leukemia 0.13 ± 0.09 0.06 ± 0.02
SK-MEL-5-Melanoma 0.14 ± 0.09 0.06 ± 0.03
UACC-62-Melanoma 0.03 ± 0.01 0.04 ± 0.02
HeLa-Cervical 1.1 ± 0.95 0.11 ± 0.06
Control: WT-MEF Noncancerous mouse embryonic fibroblasts >50 >50
Hs888Lu Noncancerous human lung fibroblasts >50 >50
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a

Average of at least three independent experiments with standard error of the mean. Compound toxicity was determined by the MTS assay (for U-937 and HL-60) or the SRB assay (for all other cell lines) after 72 h of cell treatment with the trioxane dimer as described in the experimental section. The IC50 value is the concentration at which 50% of the cells are no longer viable (MTS assay), or where 50% of the cell biomass has been reduced (for the SRB assay)

In conclusion, at only a single oral dose of 144 mg/kg, dimeric trioxane sulfone carbamate 4c completely cured malaria-infected mice. Combining a much lower amount (54 mg/kg) of dimer alchol 4b or of dimer carbamate 4c with a small amount of mefloquine hydrochloride (13 mg/kg) in a single oral dose also completely cured the malaria-infected mice. Both dimers 4b and 4c have powerful and selective anticancer activities. This desirable combination42 of high antimalarial activity and high anticancer activity encourages further lead optimization and preclinical drug development of trioxane dimer sulfones like 4b and 4c.

Experimental

Synthesis of Dimer primary bromide 3

Dimeric trioxane primary alcohol30 2 (76 mg, 0.13 mmol) was dissolved in CH2Cl2 (4 mL) under argon. Triphenylphosphine (49 mg, 0.19 mmol) and carbon tetrabromide (62 mg, 0.19 mmol) were added to the solution at room temperature and allowed to stir overnight. After 18 h, the reaction was quenched with distilled water (2 mL). The aqueous layer was extracted with CH2Cl2 (2 × 3 mL) and washed with brine (3 mL). The organic layer was dried with MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash silica gel column chromatography (0→ 20% EtOAc/hexanes) to yield dimeric trioxane bromide 3 as an amorphous white solid (72 mg, 0.11 mmol, 86%). [α]D21.9 +70.8° (c = 0.56, CHCl3); IR (thin film) 2951, 2875, 1451, 1377, 1252, 1206, 1119, 1055, 1007, 942, 879, 735 cm−1; 1H NMR (400 MHz, CDCl3)δ 5.37 (s, 1H), 5.30 (s, 1H), 4.41-4.37 (m, 1H), 4.24-4.20 (m, 1H), 3.91-3.88 (dd, J1 = 10 Hz, J2 = 5.0 Hz, 1H), 3.82-3.78 (dd, J1 = 10 Hz, J2 = 5.0 Hz, 1H), 2.73-2.68 (q, 1H), 2.62-2.57 (q, 1H), 2.36-2.27 (m, 2H), 2.19-2.17 (m, 1H), 2.04 (m, 1H), 2.00 (m, 1H), 1.91-1.23 (m, 27H including singlets for 3H each at 1.41 and 1.39), 0.99-0.95 (m, 7H), 0.88-0.86 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 103.3, 102.9, 89.6, 89.0, 81.2, 81.2, 74.7, 70.6, 52.5, 52.1, 44.6, 44.1, 40.7, 37.5, 37.4, 36.7, 36.6, 36.2, 34.5, 34.4, 32.1, 31.9, 30.6, 30.5, 26.1, 26.1, 24.9, 24.9, 24.8, 24.7, 20.3, 20.1, 13.3, 12.7; HRMS (ESI) m/z calcd for C34H53BrO8Na (M+Na)+ 691.2816, found 691.2797.

Synthesis of dimeric fluorobenzylic sulfone 4a

Bis-trioxane bromide 3 (10 mg, 0.015 mmol) was dissolved in DMF (0.5 mL) under argon. To the solution was added 4-fluorobenzyl mercaptan (2 μL, 0.018 mmol) and NaH (0.5 mg, 0.018 mmol) consecutively and the reaction was heated at 90 °C for 1 h. (Note: The reaction went from colorless to pink almost instantly upon heating. The pink color faded to a very light yellow over the course of the hour) The reaction was allowed to cool and was diluted with EtOAc (1 mL). The organic layer was then washed with ice cold distilled water (1 mL) and brine (1 mL). The organic layer was dried with MgSO4, filtered, and concentrated in vacuo. The crude product was dissolved in CH2Cl2 (0.5 mL) and mCPBA (4 mg, 0.020 mmol) was added. The reaction was stirred at room temperature for 5 h and then washed with saturated sodium bisulfite solution (1 mL) and saturated sodium bicarbonate solution (1 mL) sequentially. The organic layer was dried with MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash silica gel column chromatography (0→ 20% EtOAc/hexanes) to yield ASR-isobu-SO2-CH2Ph-4-F 9 (6.5 mg, 0.010 mmol, 66%) as an amorphous white solid. [α]D21.3 +67.7° (c = 0.35, CHCl3); IR (thin film) 2938, 2876, 1509, 1456, 1377, 1309, 1226, 1118, 1099, 1056, 1007, 941, 878, 843 cm−1; 1H NMR (400 MHz, CDCl3)δ 7.48-7.44 (m, 2H), 7.08-7.04 (m, 2H), 5.48 (s, 1H), 5.31 (s, 1H), 4.40-4.38 (m, 1H), 4.35 (s, 2H), 4.15-4.13 (m, 1H), 3.54-3.50 (dd, J1 = 14.3 Hz, J2 = 10.1 Hz, 1H), 3.04-2.98 (dd, J1 = 14.4 Hz, J2 = 6.4 Hz, 1H), 2.78-2.72 (m, 1H), 2.65-2.56 (m, 2H), 2.39-2.28 (m, 3H), 2.06-1.95 (m, 2H), 1.92-1.88 (m, 3H), 1.81-1.73 (m, 2H), 1.69-1.19 (m, 21H including singlets for 3H each at 1.42 and 1.37), 0.96-0.93 (dd, J1 = 6.1 Hz, J2 = 3.0 Hz, 7H), 0.86 (s, 3H), 0.84 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 133.0, 132.9, 123.7, 123.7, 115.9, 115.7, 103.4, 103.0, 89.5, 88.7, 81.3, 81.2, 77.2, 74.1, 70.5, 58.6, 54.6, 52.6, 52.1, 44.6, 44.2, 37.5, 37.2, 36.6, 34.6, 34.4, 31.5, 31.4, 30.4, 30.3, 30.1, 26.3, 26.1, 24.8, 24.8, 24.7, 24.7, 20.3, 20.1, 13.4, 12.8. HRMS (FAB) m/z calcd for C41H60FO10S (M+H)+ 763.3891, found 763.3850.

Synthesis of dimeric sulfone benzylic alcohol 4b

Bis-trioxane bromide 3 (39 mg, 0.058 mmol) was dissolved in acetonitrile (3 mL) under argon at room temperature. To the solution was added 4-hydroxymethyl thiophenol (16 mg, 0.116 mmol), easily prepared by LAH reduction of the commercially available parent carboxylic acid, and NaH (3.0 mg, 0.116 mmol) consecutively and the reaction was stirred at room temperature overnight. The reaction was quenched with sat. aq. sodium bicarbonate (2 mL) and extracted with EtOAc (2 mL). The organic layer was then washed with ice cold distilled water (2 × 2 mL) and brine (2 mL). The organic layer was dried with MgSO4, filtered, and concentrated in vacuo. The crude product was dissolved in dichloromethane (3 mL). mCPBA (10 mg, 0.057 mmol) was added at room temperature and stirred for 2 h. The reaction was quenched with saturated aqueous sodium bisulfite (2 mL) and the organic layer was extracted with sat. aq. sodium bicarbonate (2 mL). The crude product was purified by flash silica gel column chromatography (20→ 30% EtOAc/hexanes) to yield dimeric sulfone benzylic alcohol 4b (25 mg, 0.033 mmol, 57%) as an amorphous white solid. mp = 109–112 °C; [α]D21.3 +55° (c = 0.55, CHCl3); IR (thin film) 3511, 2948, 2848, 1454, 1408, 1306, 1197, 1142, 1094, 1051, 1008, 936, 880, 837, 763 cm−1; 1H NMR (400 MHz, CDCl3)δ 7.97-7.95 (d, 2H), 7.49-7.47 (d, 2H), 5.44 (s, 1H), 5.32 (s, 1H), 4.76 (s, 2H), 4.11-4.06 (m, 1H), 3.63-3.58 (dd, J1 = 14 Hz, J2 = 8.0 Hz, 1H), 3.34-3.29 (dd, J1 = 14 Hz, J2 = 8.0 Hz, 1H), 2.71-2.64 (m, 1H), 2.57-2.49 (m, 1H), 2.51-2.44 (m, 1H), 2.37- 2.25 (m, 2H), 2.20-1.16 (m, 29H, including singlets at 1.42 and 1.34 for 3H each), 0.95-0.91 (m, 8H), 0.83-0.80 (m, 7H); 13C NMR (100 MHz, CDCl3) δ 146.8, 139.2, 128.3, 127.0, 103.4, 102.9, 89.5, 88.8, 81.3, 81.2, 73.9, 71.0, 62.2, 60.4, 58.9, 52.5, 52.1, 44.6, 44.1, 37.4, 36.6, 36.6, 34.5, 34.4, 31.2, 31.1, 30.7, 30.4, 26.2, 26.1, 24.8, 24.8, 21.1, 20.3, 20.1, 14.2, 13.3, 12.7; HRMS (FAB) m/z calcd for C41H60O11SNa (M+Na)+ 783.3749, found 783.3707.

Synthesis of dimeric sulfone benzylic dimethyl carbamate 4c

Dimeric sulfone benzylic alcohol 4b (20 mg, 0.026 mmol) was dissolved in DMF (1 mL) under argon at room temperature. To the solution was added dimethylcarbamyl chloride (5 μL, 0.052 mmol) and NaH (1.2 mg, 0.052 mmol) consecutively and the reaction was stirred at room temperature for 2 h. The reaction was quenched with sat. aq. sodium bicarbonate (1 mL) and extracted with dichloromethane (2 × 2 mL). The organic layer was dried with MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash silica gel column chromatography (40% EtOAc/hexanes) to yield dimeric sulfone benzylic dimethyl carbamate 4c (16 mg, 0.019 mmol, 73%) as an amorphous white solid. [α]D23.4 +73.7° (c = 0.12, CHCl3); IR (thin film) 2939, 2876, 1711, 1495, 1454, 1394, 1377, 1306, 1181, 1145, 1089, 1054, 1007, 878, 830, 767, 669 cm−1; 1H NMR (400 MHz, CDCl3)δ 8.02-8.00 (d, J = 8.0 Hz, 2H), 7.50-7.48 (d, J = 8.0 Hz, 2H), 5.48 (s, 1H), 5.34 (s, 1H), 5.18 (s, 2H), 4.45-4.43 (m, 1H), 4.14-4.10 (m, 1H), 3.63-3.58 (dd, J1 = 14.4 Hz, J2 = 7.2 Hz, 1H), 3.38-3.32 (dd, J1 = 14.4 Hz, J2 = 7.2 Hz, 1H), 2.95 (s, 6H), 2.71-2.65 (m, 1H), 2.58-2.50 (m, 2H), 2.39-2.26 (m, 2H), 2.24-2.15 (m, 1H), 2.09-1.97 (m, 2H), 1.95-1.84 (m, 3H), 1.82-1.16 (m, 22H including singlets for 3H each at 1.43 and 1.35), 0.95-0.03 (m, 8H), 0.84-0.82 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 174.7, 142.7, 139.9, 128.4, 127.9, 103.3, 102.9, 89.5, 88.8, 81.3, 81.2, 77.2, 74.0, 71.0, 66.0, 58.9, 56.6, 52.5, 52.1, 44.6, 44.1, 37.4, 37.3, 36.7, 36.6, 34.6, 34.4, 31.2, 31.1, 30.7, 30.4, 26.2, 26.1, 24.8, 24.8, 24.7, 24.7, 20.3, 20.1, 13.3, 12.7; HRMS (ESI) m/z calcd for C44H65NO12SNa (M+Na)+ 854.4120, found 854.4100.

Synthesis of dimeric sulfone benzylic diethyl carbamate 4d

Dimeric sulfone benzylic alcohol 4b (18 mg, 0.024 mmol) was dissolved in DMF (1 mL) under argon at room temperature. To the solution was added diethylcarbamyl chloride (4 μL, 0.026 mmol) and NaH (0.62 mg, 0.026 mmol) consecutively and the reaction was stirred at room temperature for 2 h. The reaction was quenched with sat. aq. sodium bicarbonate (2 mL) and extracted with dichloromethane (2 × 2 mL). The organic layer was dried with MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash silica gel column chromatography (30% EtOAc/hexanes) to yield dimeric sulfone benzylic diethyl carbamate 4d (15.5 mg, 0.018 mmol, 76%) as an amorphous white solid. [α]D23.6 +41.6° (c = 0.12, CHCl3); IR (thin film) 2924, 2853, 1703, 1457, 1377, 1313, 1274, 1168, 1146, 1089, 1053, 1007, 878, 825, 766 cm−1; 1H NMR (400 MHz, CDCl3)δ 8.03-8.01 (d, J = 8 Hz, 2H), 7.49-7.47 (d, J = 8 Hz, 2H), 5.48 (s, 1H), 5.34 (s, 1H), 5.19 (s, 2H), 4.47-4.44 (m, 1H), 4.16-4.12 (dd, J = 4.4 Hz, 1H), 3.65-3.60 (dd, J1 = 9.6 Hz, J2 = 7.4 Hz, 1H), 3.38-3.33 (dd, J1 = 9.6 Hz, J2 = 7.4 Hz, 1H), 3.35-3.31 (m, 4H), 2.71-2.67 (m, 1H), 2.58-2.53 (m, 2H), 2.37-2.27 (m, 2H), 2.22-2.17 (m, 1H), 2.05-1.99 (m, 2H), 1.92-1.82 (m, 2H), 1.79-1.73 (m, 4H), 1.65-1.18 (m, 19H including singlets for 3H each at 1.43 and 1.34), 1.15-1.12 (t, J = 6.8 Hz, 6H), 0.96-0.94 (d, J = 6.4 Hz, 8H), 0.84-0.82 (d, J = 7.2 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 156.9, 142.9, 140.0, 128.4, 127.8, 103.3, 102.9, 89.5, 88.8, 81.3, 81.2, 73.9, 71.0, 65.7, 58.8, 52.6, 52.2, 44.6, 44.1, 37.4, 37.3, 36.7, 36.6, 34.6, 34.4, 31.3, 31.2, 31.2, 30.7, 30.4, 29.7, 26.2, 26.1, 24.8, 24.8, 24.7, 24.7, 20.2, 20.1, 13.2, 12.7; HRMS (ESI) m/z calcd for C46H69NO12SNa (M+Na)+ 882.4433, found 882.4419.

Synthesis of dimeric sulfone benzylic diethyl phosphate 4e

Dimeric sulfone benzylic alcohol 4b (20 mg, 0.026 mmol) was dissolved in anhydrous dichloromethane (2 mL) under argon at room temperature. To the solution was added diethyl chlorophosphate (19 μL, 0.13 mmol) and pyridine (11 μL, 0.13 mmol) consecutively and the reaction was stirred at room temperature for 18 h. The reaction was quenched with sat. aq. sodium bicarbonate (2 mL) and extracted with dichloromethane (2 × 2 mL). The organic layer was dried with MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash silica gel column chromatography (50% EtOAc/hexanes) to yield dimeric sulfone benzylic diethyl phosphate 4e (8.2 mg, 0.009 mmol, 37%) as an amorphous white solid. [α]D24.1 +27.1° (c = 0.06, CHCl3); IR (thin film) 2925, 2854, 1699, 1653, 1558, 1541, 1521, 1507, 1457, 1375, 1270, 1036 cm−1; 1H NMR (400 MHz, CDCl3)δ 8.06-8.03 (d, J = 8.0 Hz, 2H), 7.54-7.52 (d, J = 8.0 Hz, 2H), 5.49 (s, 1H), 5.35 (s, 1H), 5.14-5.12 (d, J = 8.0 Hz, 2H), 4.48-4.43 (m, 1H), 4.16-4.08 (m, 5H), 3.66-3.61 (dd, J1 = 14 Hz, J2 = 6.8 Hz, 1H), 3.39-3.34 (dd, J1 = 14 Hz, J2 = 6.8 Hz, 1H), 2.70-2.66 (m, 1H), 2,58-2.53 (m, 2H), 2.38-2.28 (m, 2H), 2.23-2.16 (m, 1H), 2.06-2.00 (m, 2H), 1.93-1.90 (m, 2H), 1.79-1.21 (m, 29H including singlets for 3H each at 1.44 and 1.35), 0.96-0.95 (m, 8H), 0.85-0.82 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 140.5, 138.0, 128.6, 127.7, 103.3, 102.9, 89.6, 88.9, 81.3, 81.2, 74.0, 70.9, 67.8, 67.7, 64.1, 64.1, 58.8, 57.2, 57.1, 52.6, 52.2, 44.6, 44.1, 37.5, 37.4, 36.7, 36.7, 34.4, 31.3, 30.7, 30.4, 29.7, 26.2, 26.1, 24.8, 24.8, 20.2, 20.1, 16.2, 16.1, 14.2, 13.2, 12.6; HRMS (ESI) m/z calcd for C45H69O14PSNa (M+Na)+ 919.4038, found 919.3999.

Synthesis of dimeric sulfone hexanol 4f

Dimeric trioxane bromide 3 (20 mg, 0.030 mmol) was dissolved in anhydrous acetonitrile (2 mL) under argon at room temperature. To the solution was added 6-mercapto-1-hexanol (9 μL, 0.066 mmol) and sodium hydride (NaH, 2.0 mg, 0.066 mmol) consecutively and the reaction was stirred at room temperature for 2 h. The reaction was quenched with sat. aq. sodium bicarbonate (2 mL) and extracted with dichloromethane (2 × 2 mL). The organic layer was dried with MgSO4, filtered, and concentrated in vacuo. The crude product was dissolved in freshly made dimethyldioxirane solution in acetone (DMDO, 2 mL) at 0 °C and stirred for 1 h. The reaction was concentrated the crude product was purified by flash silica gel column chromatography (50% EtOAc/hexanes) to yield dimeric sulfone hexanol 4f (12 mg, 0.015 mmol, 51%) as an amorphous white solid. [α]D22.5 +58.1° (c = 0.15, CHCl3); IR (thin film) 3520 (br), 2925, 2854, 1456, 1377, 1296, 1121, 1055, 1007, 878, 845 cm−1; 1H NMR (400 MHz, CDCl3)δ 5.41 (s, 1H), 5.34 (s, 1H), 4.46-4.42 (m, 1H), 4.20-4.16 (m, 1H), 3.65-3.62 (t, J = 6.4 Hz, 2H), 3.49-3.42 (dd, J1 = 14.2 Hz, J2 = 7.2 Hz, 1H), 3.10-3.05 (m, 3H), 2.74-2.69 (m, 1H), 2.60-2.54 (m, 2H), 2.35-2.27 (m, 3H), 2.03-1.21 (m, 34H including singlets for 3H each at 1.40 and 1.25), 0.96-0.94 (m, 8H), 0.87-0.85 (m, 8H); ); 13C NMR (100 MHz, CDCl3) δ 103.3, 102.9, 89.7, 88.8, 81.3, 81.2, 73.7, 70.3, 62.7, 55.7, 53.0, 52.6, 52.1, 44.5, 44.1, 37.5, 37.3, 36.7, 36.6, 34.6, 34.4, 32.3, 31.9, 31.6, 30.6, 30.3, 29.8, 29.7, 28.2, 26.2, 26.1, 25.1, 24.8, 24.8, 24.7, 24.7, 21.6, 20.3, 20.1, 13.2, 12.7; HRMS (ESI) m/z calcd for C40H66O11SNa (M+Na)+ 777.4218, found 777.4211.

Cell culture conditions and determination of IC-50 values

HL-60, U-937, SK-MEL-5, UACC-62, and HeLa cell lines were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. WT-MEF and Hs888Lu cell lines were cultured in DMEM media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cell lines were obtained from the American Type Culture Collection (Manassas, VA), with the exception of the HL-60 line, which was a generous gift from Professor Huimin Zhao (University of Illinois), and were grown at 37°C with CO2/air (5:95). 99 μL of cells in media were plated in a 96-well plate. Adherent cell lines were incubated at least five hours to allow cells to attach to the plate. A volume of 1 μL of a range of compound concentrations was added in to the cells, and the plates were incubated for 72 hr. Cell viability in HL-60 and U-937 suspension cell lines were evaluated using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Promega, Inc.). The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)/phenazine methosulfate (PMS) solution was added to the cells, and absorbance at 490nm was measured in a Spectra Max Plus 384 plate reader (Molecular Devices, Sunnyvale, CA) following dye development. Biomass was quantitated in SK-MEL-5, UACC-62, HeLa, WT-MEF, and Hs888Lu cells using the sulforhodamine B assay (SRB).43 Briefly, the cells were fixed with 10% trichloroacetic acid overnight at 4°C, washed with water, and 100 μL of 0.057% w/v sulforhodamine in 1% acetic acid was added to each well for 30 minutes. After rinsing the plates in 1% acetic acid, 200 μL of 10 mM tris base (pH>10) was added to each well for 30 minutes and absorbance was read at 510nm. Logistical dose response curves and IC50 values were generated from the MTS and SRB data using TableCurve 2D 5.01 (SYSTAT Software, Inc., Richmond, CA).

Supplementary Material

1_si_001. Supporting Information Available.

1H, 13C NMR spectra for all of the new trioxane dimers. This material is available free of charge via the internet at http://pubs.acs.org.

Acknowledgments

We thank Nirbhay Kumar (JHU) for a gift of the P. berghei malaria parasites, the NIH (AI 34885 to GHP), the Johns Hopkins Malaria Research Institute and the Bloomberg Family Foundation for financial support (to GHP and to JOL).

Footnotes

1

Abbreviations: ACT, artemisinin combination therapy; mCPBA, meta-chloroperbenzoic acid; DMSO, dimethyl sulfoxide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; SRB, sulforhodamine B; DMF, dimethyl formamide; NaH, sodium hydride; EtOAc, ethyl acetate; LAH, lithium aluminum hydride; DMDO, dimethyldioxirane.

References

  • 1.Ridley RG. Medical need, scientific opportunity, and the drive for antimalarial drugs. Nature. 2002;415:686–693. doi: 10.1038/415686a. [DOI] [PubMed] [Google Scholar]
  • 2.Breman JG, Alilio MS, Mills A. Conquering the intolerable burden of malaria: what’s new, what’s needed: a summary. Am J Trop Med Hyg. 2004;71:1–15. [PubMed] [Google Scholar]
  • 3.Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI. The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature. 2005;434:214–217. doi: 10.1038/nature03342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Troye-Blomberg M, Berzins K. Rational vaccine development against malaria. Microbes and Infection. 2007;9:749–750. doi: 10.1016/j.micinf.2008.07.014. [DOI] [PubMed] [Google Scholar]
  • 5.Olliaro PL, Boland PB. Clinical public health implications of antimalarial drug resistance. In: Rosenthal PJ, editor. Antimalarial Chemotherapy: Mechanisms of Action, Resistance, and New Directions in Drug Discovery. Humana Press; Totowa, NJ: 2001. pp. 65–83. [Google Scholar]
  • 6.Shizhen L. Compendium of Materia Medica (Bencao Gangmu) Foreign Languages Press; Beijing, China: first published in Chinese in 1593, translation published 2003. [Google Scholar]
  • 7.Klayman DL. Qinghaosu (artemisinin): an antimalarial drug from China. Science. 1985;228:1049–1055. doi: 10.1126/science.3887571. [DOI] [PubMed] [Google Scholar]
  • 8.O’Neill PM, Posner GH. A medicinal chemistry perspective on artemisinin and related endoperoxides. J Med Chem. 2004;47:2945–2964. doi: 10.1021/jm030571c. [DOI] [PubMed] [Google Scholar]
  • 9.Tang Y, Dong Y, Vennerstrom JL. Synthetic peroxides as antimalarials. Med Res Rev. 2004;24:425–448. doi: 10.1002/med.10066. [DOI] [PubMed] [Google Scholar]
  • 10.Jefford CW. Synthetic peroxides as antimalarials. Curr Opin In Vest Drugs (Thomson Sci) 2004;5:866–872. [PubMed] [Google Scholar]
  • 11.Haynes RK. From artemisinin to new artemisinin antimalarials: Biosynthesis, extraction, old and new derivatives, stereochemistry and medicinal chemistry requirements. Curr Top Med Chem. 2006;6:509–537. doi: 10.2174/156802606776743129. [DOI] [PubMed] [Google Scholar]
  • 12.Bégué J-P, Bonnet-Delpon D. Fluoroartemisinins: metabolically more stable antimalarial artemisinin derivatives. ChemMedChem. 2007;2:608–624. doi: 10.1002/cmdc.200600156. [DOI] [PubMed] [Google Scholar]
  • 13.Gelb MH. Drug discovery for malaria: A very challenging endeavor. Curr Opin Chem Biol. 2007;11:440–445. doi: 10.1016/j.cbpa.2007.05.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ashley EA, White NJ. Artemisinin-based combinations. Curr Opin Infect Dis. 2005;18:531–536. doi: 10.1097/01.qco.0000186848.46417.6c. [DOI] [PubMed] [Google Scholar]
  • 15.(a) Adjuik M, Babiker A, Garner P, Olliaro P, Taylor W, White N. Artesunate combinations for treatment of malaria: meta-analysis. Lancet. 2004;363:9–17. doi: 10.1016/s0140-6736(03)15162-8. [DOI] [PubMed] [Google Scholar]; (b) Guthmann J-P, Cohuet S, Rigutto C, Fortes F, Saraiva N, Kiguli J, Kyomuhendo J, Francis M, Noel F, Mulemba M, Balkan S. High efficacy of two artemisinin-based combinations (artesunate + amodiaquine and artemether + lumefantrine) in Caala, Central Angola. Am J Trop Med Hyg. 2006;75:143–145. [PubMed] [Google Scholar]
  • 16.Guidelines for the Treatment of Malaria. World Health Organization; Switzerland: 2006. [Google Scholar]
  • 17.Myint HY, Ashley EA, Day NPJ, Nosten F, White NJ. Efficacy and safety of dihydroartemisinin-piperaquine. Trans R Soc Trop Med Hyg. 2007;101:858–866. doi: 10.1016/j.trstmh.2007.05.018. [DOI] [PubMed] [Google Scholar]
  • 18.Sagara I, Diallo AD, Kone M, Coulibaly M, Diawara SI, Guindo O, Maiga H, Niambele MB, Sissoko M, Dicko A, Djimde A, Doumbo OK. A randomized trial of artesunate-mefloquine versus artemether-lumefantrine for treatment of uncomplicated Plasmodium falciparum malaria in Mali. Am J Trop Med Hyg. 2008;79:655–661. [PubMed] [Google Scholar]
  • 19.Posner GH, Paik I-H, Chang W, Borstnik K, Sinishtaj S, Rosenthal AS, Shapiro TA. Malaria-infected mice are cured by a single dose of novel artemisinin derivatives. J Med Chem. 2007;50:2516–2519. doi: 10.1021/jm070149m. [DOI] [PubMed] [Google Scholar]
  • 20.Posner GH, Chang W, Hess L, Woodard L, Sinishtaj S, Usera AR, Maio W, Rosenthal AS, Kalinda AS, D’Angelo JG, Petersen KS, Stohler R, Chollet J, Santo-Tomas J, Snyder C, Rottmann M, Wittlin S, Brun R, Shapiro TA. Malaria-infected mice are cured by oral administration of new artemisinin derivatives. J Med Chem. 2008;51:1035–1042. doi: 10.1021/jm701168h. [DOI] [PubMed] [Google Scholar]
  • 21.For other trioxane dimers see Ekthawatchai S, Kamchonwongpaisan S, Kongsaeree P, Tarnchompoo B, Thebtaranonth Y, Yuthavong Y. C-16 artemisinin derivatives and their antimalarial and cytotoxic activities: Syntheses of artemisinin monomers, dimers, trimers, and tetramers by nucleophilic additions to artemisitene. J Med Chem. 2001;44:4688–4695. doi: 10.1021/jm0103007.
  • 22.For additional trioxane dimers see: Jung M, Lee S, Ham J, Lee K, Kim H, Kim SK. Antitumor activity of novel deoxoartemisinin monomers, dimers, and trimer. J Med Chem. 2003;46:987–994. doi: 10.1021/jm020119d.
  • 23.Alagbala AA, McRiner AJ, Borstnik K, Labonte T, Chang W, D-Angelo JG, Posner GH, Foster BA. Biological mechanism of action of Novel C-10 Non-acetal trioxane dimers in prostate cancer cell lines. J Med Chem. 2006;49:7836–7842. doi: 10.1021/jm060803i. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Gatter KC, Brown G, Townbridge IS, Woolston RE, Mason DY. Transferrin receptors in human tissues; their distribution and possible clinical relevance. J Clin Pathol. 1983;36:539–545. doi: 10.1136/jcp.36.5.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Zhou H-J, Wang Z, Li A. Dihydroartemisinin induces apoptosis in human leukemia cells HL60 via downregulation of transferrin receptor expression. Anti-cancer Drugs. 2008;19:247–255. doi: 10.1097/cad.0b013e3282f3f152. [DOI] [PubMed] [Google Scholar]
  • 26.Lai H, Sasaki T, Singh NP. Targeted treatment of cancer with artemisinin and artemisinin-tagged iron-carrying compounds. Expert Opin Ther Targets. 2005;9:995–1007. doi: 10.1517/14728222.9.5.995. [DOI] [PubMed] [Google Scholar]
  • 27.Posner GH, D’Angelo J, O’Neill PM, Mercer A. Anticancer activity of artemisinin-derived trioxanes. Expert Opin Ther Patents. 2006;16:1665–1672. [Google Scholar]
  • 28.Jung M, Lee K, Kim H, Park M. Recent advances in artemisinin and its derivatives as antimalarial and antitumor agents. Curr Med Chem. 2004;11:1265–1284. doi: 10.2174/0929867043365233. [DOI] [PubMed] [Google Scholar]
  • 29.Efferth T. Mechanistic perspectives for 1,2,4-trioxanes in anti-cancer therapy. Drug Res Update. 2005;8:85–97. doi: 10.1016/j.drup.2005.04.003. [DOI] [PubMed] [Google Scholar]
  • 30.Posner GH, Paik I-H, Sur S, McRiner AJ, Borstnik K, Xie S, Shapiro TA. Orally active, antimalarial, anticancer, artemisinin-derived trioxane dimers with high stability and efficacy. J Med Chem. 2003;46:1060–1065. doi: 10.1021/jm020461q. [DOI] [PubMed] [Google Scholar]
  • 31.Kocienski PJ, Cernigliaro G, Feldstein G. A synthesis of methyl n-tetradeca-trans-2,4,5-trienoate, an allenic ester produced by the male dried bean beetleacanthoscelides obtectus (say) J Org Chem. 1977;42:353–355. doi: 10.1039/j39700000859. [DOI] [PubMed] [Google Scholar]
  • 32.Bachi MD, Korshin EE, Hoos R, Szpilman AM, Ploypradith P, Xie S, Shapiro TA, Posner GH. A short synthesis and biological evaluation of potent and nontoxic antimalarial bridged bicyclic sulfonyl-endoperoxides. doi: 10.1021/jm020584a. [DOI] [PubMed] [Google Scholar]
  • 33.Haynes RK, Fugmann B, Stetter J, Rieckmann K, Heilmann H-D, Chan H-W, Cheung M-K, Lam W-L, Wong H-N, Croft SL, Vivas L, Rattray L, Stewart L, Peters W, Robinson BL, Edstein MD, Kotecka B, Kyle DE, Beckermann B, Gerisch M, Radtke M, Schmuck G, Steinke W, Wollborn U, Schmeer K, Römer A. Artemisone–A highly active antimalarial drug of the artemisinin class. Angew Chem Int Ed. 2006;45:2082–2088. doi: 10.1002/anie.200503071. [DOI] [PubMed] [Google Scholar]
  • 34.Posner GH, O’Dowd H, Ploypradith P, Cumming JN, Xie S, Shapiro TA. Antimalarial cyclic peroxy ketals. J Med Chem. 1998;41:2164–2167. doi: 10.1021/jm980088f. [DOI] [PubMed] [Google Scholar]
  • 35.Posner GH, O’Dowd H, Caferro T, Cumming JN, Ploypradith P, Xie S, Shapiro TA. Antimalarial sulfone trioxanes. Tetrahedron Lett. 1998;39:2273–2276. [Google Scholar]
  • 36.Posner GH, Maxwell JP, O’Dowd H, Krasavin M, Xie S, Shapiro TA. Antimalarial sulfide, sulfone, and sulfonamide trioxanes. Bioorg Med Chem. 2000;8:1361–1370. doi: 10.1016/s0968-0896(00)00079-1. [DOI] [PubMed] [Google Scholar]
  • 37.Posner GH, Paik I-H, Sur S, McRiner AJ, Borstnik K, Xie S, Shapiro TA. Orally activeOrally active, antimalarial, anticancer, artemisinin-derived trioxane dimers with high stability and efficacy. J Med Chem. 2003;46:1060–1065. doi: 10.1021/jm020461q. [DOI] [PubMed] [Google Scholar]
  • 38.Chen X, Chong CR, Shi l, Yoshimoto T, Sullivan DJ, Jr, Lin JO. Inhibitors of Plasmodium falciparum methionine aminopeptidase 1b possess antimalarial activity. Proc Natl Acad Sci U S A. 2006;103:14548–14553. doi: 10.1073/pnas.0604101103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Vennerstrom JL, Arbe-Barnes S, Brun R, Charman SA, Chiu FCK, Chollet J, Dong Y, Dorn A, Hunziker D, Matile H, McIntosh K, Padmanilayam M, Santo TJ, Scheurer C, Scorneaux B, Tang Y, Urwyler H, Wittlin S, Charman WN. Identification of an antimalarial synthetic trioxolane drug development candidate. Nature. 2004;430:900–904. doi: 10.1038/nature02779. [DOI] [PubMed] [Google Scholar]
  • 40.Harris KA, Harney e, Small EJ. Liposomal doxorubicin for the treatment of hormone-refractory prostate cancer. Clin Prostate Cancer. 2002;1:37–41. doi: 10.3816/cgc.2002.n.005. [DOI] [PubMed] [Google Scholar]
  • 41.Takara H, Sakaeda T, Yagami T, Kobayashi H, Ohmoto N, Horinouchi M, Nishiguchi K, Okumura K. Cytotoxic Effects of 27 Anticancer Drugs in HeLa and MDR1-Overexpressing Derivative Cell Lines. Biol Pharm Bull. 2002;25:771–778. doi: 10.1248/bpb.25.771. [DOI] [PubMed] [Google Scholar]
  • 42.Paik I-H, Xie S, Shapiro TA, Labonte T, Narducci Sargeant AA, Baege AC, Posner GH. Second generation, orally active, antimalarial, artemisinin-derived trioxane dimers with high stability, efficacy and anticancer activity. J Med Chem. 2006;49:2731–2734. doi: 10.1021/jm058288w. [DOI] [PubMed] [Google Scholar]
  • 43.Vichai V, Kirtikara K. Sulforhodamine B colorimetric assay for cytotoxic screening. Nature Protocols. 2006;3:1112–1116. doi: 10.1038/nprot.2006.179. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1_si_001. Supporting Information Available.

1H, 13C NMR spectra for all of the new trioxane dimers. This material is available free of charge via the internet at http://pubs.acs.org.

NIHMS92809-supplement-1_si_001.pdf (1.5MB, pdf)

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