C−H Activation Enables a Concise Total Synthesis of Quinine and Analogues with Enhanced Antimalarial Activity

Abstract We report a novel approach to the classical natural product quinine that is based on two stereoselective key steps, namely a C−H activation and an aldol reaction, to unite the two heterocyclic moieties of the target molecule. This straightforward and flexible strategy enables a concise synthesis of natural (−)‐quinine, the first synthesis of unnatural (+)‐quinine, and also provides access to unprecedented C3‐aryl analogues, which were prepared in only six steps. We additionally demonstrate that these structural analogues exhibit improved antimalarial activity compared with (−)‐quinine both in vitro and in mice infected with Plasmodium berghei.


Synthesis of 4a-j General Procedure for the C−H Arylation Step
To a solution of 3 (1 equiv) and iodoaryl (3 equiv) in DMF (0.3 M) were added successively pivalic acid (1 equiv), Pd(OAc)2 (15 mol%) and Ag2CO3 (1 equiv). The resulting mixture was slowly heated to 100 °C and stirred at this temperature for 16 h. After 16 h, the reaction was cooled to room temperature, diluted with 5 M NaOH solution and extracted three times with dichloromethane. The combined organic phase was dried over Na2SO4, concentrated and purified by flash chromatography using a gradient of 10 to 30% DMA (CH2Cl2/MeOH/NH4OH 80:20:3) in dichloromethane to afford the pure product.

Synthesis of 5
A 250 ml round bottomed flask was charged with (−)-4 (1.35 g, 4.00 mmol), ruthenium(III) chloride (331 mg, 1.60 mmol) and sodium periodate (20.55 g, 96.03 mmol) to which H2O was added (53 ml) followed by EtOAc (13.5 ml) and acetonitrile (13.5 ml). The flask was sealed with a septum pierced with a cannula to allow gases to escape and the reaction was stirred at room temperature overnight. After 18 h, the reaction was filtered through celite, and the pad was washed with 200 ml H2O. The filtrate was transferred to a separating funnel and washed three times with DCM (3 × 50 ml). The halogenated phases were discarded, while the aqueous phase was collected and evaporated to a volume of ca. 20 ml in vacuo. To this residue was added 10 ml of 1 M HCl and the mixture was loaded onto a wet column of DOWEX-50WX8 (20 g) which had been pre-washed with 150 ml water. The column was washed with 4 × 500 ml portions of H2O, during which time the NaIO4 eluted. The column was then washed with 5 × 250 ml portions of 1 M NH4OH; the product eluted in the first four fractions. Water was evaporated from the collected fractions in vacuo  3240,3059,3015,1655,1586,1568,1509,1464,1434,1376,1292,1088,1043,996,911

Synthesis of 9
8 (85 mg, 0.33 mmol) was suspended in 6 ml H2O to which conc. HCl was added (0.23 ml), followed by Zn(OTf)2 (120 mg, 0.33 mmol). After stirring at room temperature for 5 min, Zn dust (324 mg, 4.95 mmol) was added. After 1.5 h, the reaction mixture was filtered through celite and the pad was washed with 50 ml H2O. The filtrate was cooled in an ice-water bath and 40 ml DCM was added with stirring, followed by slow addition of 10 M aq. NaOH (20 ml). After 15 min, the mixture was poured into a separating funnel and extracted three times DCM (3 × 60 ml). The combined organic phase was dried over Na2SO4, filtered, and evaporated at 150 mbar/35 °C. The crude material was purified by chromatography over silica gel using a gradient of 30 to 50% DMA (CH2Cl2/MeOH/NH4OH 80:20:3) in dichloromethane. After chromatography, solvents were removed under the same conditions as before, at 150 mbar/35 °C. The purified amine (clear oil, purity >90%, 43 mg, 77%) was stored under argon at −78 °C (in a sealed vessel immersed in dry ice) and used in the subsequent reaction within 36 h to minimize decomposition. On a 1.28 mmol scale (330 mg), the product was obtained in 35% yield (purity >90%, 75 mg, 0.44 mmol). Concerning the chemical instability of 9, upscaling is not recommended as bigger scale setups require more time for work-up and purification.  3274,3072,2931,2867,1633,1592,1454,1320,1265,1069,1048,998,974,907,814,732

Synthesis of Quinine (1)
Lithium aluminium hydride solution (1.0 M in THF,116 µl,0.116 mmol) was diluted with THF (0.3 ml) in a Schlenk flask under argon. Methanol (14 µl, 0.348 mmol) was added dropwise at 0°C and stirred at this temperature for 10 min. In another Schlenk flask, a solution of hydrazone 14 (10 mg, 0.023 mmol) was diluted in THF (0.3 ml). The aluminium hydride solution was added dropwise to the solution of hydrazone at 0°C. The reaction mixture was then warmed to room temperature and stirred for an additionnal 10 min before being quenched by sat. aq. NaHCO3 solution at 0°C. The mixture was extracted from sat. aq. NaHCO3 with three volumes of DCM. The combined organic phase was dried over Na2SO4 and filtered. The crude was purified by chromatography using a gradient of 10 to 30% DMA (CH2Cl2/MeOH/NH4OH 80:20:3) in dichloromethane to afford the product as a white powder (4 mg, 53%). Data in accordance with the literature.

Synthesis of C3-Aryl Analogues (±)-15b and (±)-15c
Synthesis of (±)-16b (±)-4b (650 mg, 1.73 mmol) was suspended in 50 ml H2O to which 5 ml conc. HCl was added slowly with stirring. The reaction was stirred at room temperature for 5 min, before Zn dust (1.70 g, 26.00 mmol) was added. After 16 h, 65 ml DCM was added, and the reaction mixture was cooled using an ice-bath followed by slow addition of 5 M NaOH (100 ml). The mixture was filtered through celite and the pad was washed with 30 ml H2O and 30 ml DCM. The combined filtrate was extracted three times with DCM (3 × 100 ml). The combined organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. The crude material was purified by chromatography over silica gel. The combined organic phase was dried over Na2SO4 and filtered. The crude was purified by chromatography using a gradient of 20 to 40% DMA (CH2Cl2/MeOH/NH4OH 80:20:3) in dichloromethane to afford the free amine as a colorless oil (361 mg, 77%).  2929,2872,1616,1326,1163,1117,1069,1016,842,807

In vitro assays
The in vitro antiprotozoal activities against P. falciparum and cytotoxicity assessment against L6 cells were determined as reported elsewhere. 1 The following strains, parasite forms and positive controls were used: P. falciparum, NF54 erythrocytic stages, chloroquine, IC50 of 6 nM and L6 cells, rat skeletal myoblasts, podophyllotoxin, IC50 of 0.010 .

In vivo assays
The in vivo antimalarial activity was assessed basically as previously described. 2 Groups of three female NMRI mice (20-22 g) intravenously infected with 2 × 10 7 parasitized erythrocytes on day 0 with GFP-transfected P. berghei strain ANKA. 3 Compounds were formulated in Tween 80/Ethanol (70%/30%), diluted 10-fold in distilled water and administered orally in a volume of 10 ml kg -1 as a single dose (24 h post infection). Parasitaemia was determined on day 3 post infection by FACS analysis. Activity was calculated as the difference between the mean per cent parasitaemia for the control (n = 5 mice) and treated groups expressed as a per cent relative to the control group. The survival time in days was also recorded up to 30 days after infection. A compound was considered curative if the animal survived to day 30 after infection with no detectable parasites. In vivo efficacy studies in mice were conducted at the Swiss Tropical and Public Health Institute (Basel) according to the rules and regulations for the protection of animal rights ("Tierschutzverordnung") of the Swiss "Bundesamt für

S54
Veterinärwesen". They were approved by the veterinary office of Canton Basel-Stadt, Switzerland.

X-Ray Analysis
The X-ray intensity data were measured on Bruker X8 APEXII diffractometers equipped with multilayer monochromators, Mo K/a INCOATEC micro focus sealed tube and Kryoflex cooling devices. The structures were solved by direct methods and refined by full-matrix leastsquares techniques. Non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were inserted at calculated positions and refined with a riding model or as rotating groups. The following software was used: Frame integration, Bruker SAINT software package i using a narrow-frame algorithm, Absorption correction, SADABS ii , structure solution, SHELXL-2013 iii , refinement, SHELXL-2013 iii , OLEX2 iv , ShelXle v , molecular diagrams, OLEX2 iv . Experimental data and CCDC-Code can be found in Supplementary Table 2.

Hydrazone TBS-13
Metrical parameters for structure TBS-13 are available free of charge from the Cambridge Crystallographic Data Centre (CCDC) under reference number 1500944. Crystal data, data collection parameters, and structure refinement details are given in Supplementary Table 3 and  Supplementary Table 4. Molecular Structure in "Ortep View" is displayed in Supplementary  Figure 2.