Structure and Properties of Artemisinin

CHAPTE R 10

Structure and Properties of Artemisinin Chapter Outline 1 Overview of Structure of Artemisinin  213 2 Chemical Properties and Reactions of Artemisinin  215 2.1 2.2 2.3 2.4 2.5 2.6

Reaction of Peroxy Group  215 Borohydride Reduction  215 Other Reduction Reactions  215 Reactions With Bases  216 Reactions With Acids  217 Electrochemical Properties  217

3 Color Reactions of Artemisinin  218 3.1 Condensation Reaction of PDAB  218 3.2 Ferric Hydroxamate Test  218 3.3 2,4-Dinitrophenylhydrazine Test  219 3.4 Alkaline m-Dinitrobenzene Test  219 3.5 Vanillin-Sulfuric Acid Color Reaction  219 3.6 Acidic Potassium Iodide Solution Test  219

References  219

1  Overview of Structure of Artemisinin [1–5] Artemisinin is an innovative compound derived from Artemisia annua L., which is first found and named by the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (ICMM-CACMS). The CAS Registry Number of artemisinin (Qinghaosu, QHS) is 63968-64-9, and its molecular formula is C15H22O5, which is a novel sesquiterpene lactone with a structural unit of 1,2,4-trioxane including a peroxy group rare in nature. Its molecule consists of seven chiral centers, and biogenetically falls into amorphane type; it is characterized by cis-connection between A and B rings, trans relationship between isopropyl and bridgehead hydrogen, and interruption of A-ring carbon skeleton by an oxygen atom in artemisinin. The name of artemisinin was originally Qinghaosu in Pinyin. Later, it was named as artemisinine due to its relation to the generic name, Artemisia; however, “ine” is generally the postfix of such nitrogen compounds as alkaloids and amino acids, whereas artemisinin is a nonnitrogen compound, so Chemical Abstracts recommend “artemisinin” as its English name. Today, artemisinin and Qinghaosu are frequently used. From Artemisia annua L. to Artemisinins http://dx.doi.org/10.1016/B978-0-12-811655-5.00010-6

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214  Chapter 10

Figure 10.1: The Structure of Artemisinin.

The structure of artemisinin is illustrated in Fig. 10.1, and there are two structure numberings as follows. In recent years, the latter numbering is accepted in the Chinese Pharmacopoeia, and thus the name of artemisinin is (3R,5αS,6R,8αS,9R,12S,12αR)-octahydro-3,6,9-trimethyl-3,12epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10(3H)-one. Artemisinin is white needle crystal: melting point 156–157°C, [α ]17 D = +66.3° (c = 1.64, CCl4), + HRMS m/e 282.1472M , microanalysis (C 63.72%, H 7.86%), without double bond and UV absorption. It is freely soluble in chloroform (CCl4), acetone, ethyl acetate, and benzene; soluble in ethanol and ether; slightly soluble in cold petroleum ether; and insoluble in water. Owing to its special peroxy group, it is susceptible to reducing substances [5]. Artemisinin is decomposed at more than 190°C. Its IR spectrum (KBr) has revealed that it contains a δ-lactone (1745 cm−1) and peroxy groups (831, 881 cm and 1115 cm−1). H-NMR (CCl4, 100 MHz (Me3Si)2O, δ): 0.93 (d, 3H, J = 6 Hz, 15-CH3); 1.06 (d, 3H, J = 6 Hz, 16-CH3); 1.36 (s, 3H, 14-CH3); 3.26 (m, 1H, 9-H, irradiation on this proton causes signal at 1.06 ppm to change from doublet to singlet); 5.68 (s, 1H, 12-H). 1

C-NMR (CHCl3, 22.63 MHz): a broadband decoupled spectrum reveals signals of 15 carbon atoms equivalent to sesquiterpenoid skeleton; an off-resonance decoupled spectrum (δ) reveals: 79.5, 105 (s, 2-C); 32.5, 33, 45, 50, 93.5 (d, 5-CH); 25, 25.1, 35.5, 37 (t, 4-CH2); 12, 19, 23 (q, 3-CH3); 172 (s, ─C═O). 13

Different research groups reported the NMR data and signal assignments for the artemisinin in literature [6–12]. After measuring crystallographic parameters with X-ray diffractometry, artemisinin is orthorhombic: space group D24 -P212121, unit cell parameters a = 24.077 Å, b = 9.443 Å, c = 6.356 Å; experimental d0 = 1.30 g/cm3, calculated dc = 1.296 g/cm3, number of molecules in unit cell Z = 4.

Structure and Properties of Artemisinin  215

2  Chemical Properties and Reactions of Artemisinin [1,4,6,13] Structurally, artemisinin molecule contains such active groups as peroxy, acetal, ketal, and lactone groups, which can react under different conditions, including reduction reactions of different types, and reactions with acids and bases.

2.1  Reaction of Peroxy Group Artemisinin is refluxed in triphenylphosphine and xylene solution under nitrogen atmosphere, agitated with formaldehyde and water. The organic layer is rinsed with water. Water layer and acidic solution are then combined. After alkalization, the combined solution is extracted with peroxide-free ether and dried with anhydrous sodium sulfate to remove ether. Triphenylphosphine is weighed. The result shows that the mole number of triphenylphosphine consumed is close to that of artemisinin, demonstrating that artemisinin molecule contains a peroxy fragment.

2.2  Borohydride Reduction [1,2,4,6,13] Artemisinin is dissolved in methanol. Solid sodium borohydride is slowly added successively under stirring at 0–5°C, and then continues to stir for 0.5 h. The reaction solution is neutralized with glacial acetic acid, and the solvent is evaporated under vacuum to obtain macrocrystalline dihydroartemisinin, which is a hemiacetal compound derived by sodium borohydride reducing artemisinin. Such mild reductants as lithium borohydride and potassium borohydride may also reduce artemisinin to dihydroartemisinin. General lactone structures cannot be reduced by borohydrides under reaction conditions of artemisinin (0–5°C, methanol solution). It is neither clear how the peroxy group in the artemisinin molecule promotes the borohydride reduction of δ-lactone, nor clear why it is only reduced to hemiacetal, but not further reacted to alcohols.

2.3  Other Reduction Reactions In catalytic hydrogenation of artemisinin over Pd/CaCO3 in methanol at room temperature and atmospheric pressure, the peroxide is reduced to the epoxide, that is, deoxoartemisinin. During this reaction process, what is originally derived from the reaction is oily matter. If it is dissolved in a small quantity of acetone in n-hexane for 4–5 days, it will spontaneously change into deoxoartemisinin crystal, with a yield of 75%; if it is dissolved in benzene containing a small quantity of p-toluenesulfonic acid, it will entirely change into

216  Chapter 10

Figure 10.2: Structures of Several Reduction Products of Artemisinin.

deoxoartemisinin crystal rapidly, with a yield up to 96%; when in diazomethane, it will change into methyl ester compounds by methyl esterification. Catalytic hydrogenation of dihydroartemisinin over Pd/CaCO3 at room temperature and atmospheric pressure will lead to a loss of the peroxy group and obtain an epoxide, that is, deoxydihydroartemisinin. Zinc/acetic acid reduction may also convert artemisinin into deoxoartemisinin with a high yield. For deoxoartemisinin, the lactone structure can be reduced to deoxy dihydroartemisinin over diisobutylaluminium hydride at low temperatures. Deoxoartemisinin can be recovered from oxidation of deoxy dihydroartemisinin over chromic anhydride/pyridine (Fig. 10.2). A restudy of chemistry of artemisinin by Sy et al. [14] in 1997 found four products illustrated in Fig. 10.3 during reduction of artemisinin by lithium aluminum hydride (LiAlH4). Of them, those illustrated in Fig. 10.3A–B are newly found products.

2.4  Reactions With Bases [1,4,15,16] Artemisinin is dissolved in methanol. In addition, potassium carbonate is dissolved in water. The resulting solution is slowly added to the artemisinin/methanol solution while stirring, mixing well to a clear liquid. At a constant temperature of 20–22°C for 1 h, add water to the solution, extract with ether twice; the ether layer is rinsed with a little water twice, while the water layer is acidized with 10% hydrochloric acid to a PH of 2, then extract with ether thrice; the ether layer is rinsed with water until neutral, dried with anhydrous sodium sulfate

Structure and Properties of Artemisinin  217

Figure 10.3: Structures of Reduction Products of Artemisinin by LiAlH4.

for 2–3 h, and evaporated under vacuum; the resulting residues are stored in a refrigerator overnight; when semisolids are precipitated, add a little methanol, cool down, precipitate needle crystal, that is, compound 1 in Fig. 10.4, filter, recrystallize once, and obtain more purified crystal. Dissolve artemisinin in ethanol, add 0.2% sodium hydroxide solution, react in a water bath at a constant temperature of 50°C for 0.5 h, and thus quantitatively produce Q292, with a maximum UV absorbance at 292 nm and an absorptivity of 1.65 × 104 L/(mol·cm). At pH 5.38–6.04, Q292 can be quantitatively converted into the compound Q260, with a maximum UV absorbance at 260 nm and an absorptivity of 1.12 × 104 L/(mol·cm).

2.5  Reactions With Acids [1,4,17] Shake artemisinin with a mixture of concentrated H2SO4 to dissolve, and stand at 25°C for 16–17 h. The resulting solution is sundown, with mild fluorescence. Pour the reaction solution into isometric ice water, mix well, and extract with CCl4 thrice; the CCl4 layer is rinsed with water until neutral, dried with anhydrous sodium sulfate, and evaporated under vacuum; recrystallize the resulting coarse crystal twice, and obtain the flaky crystal of the compound 2 in Fig. 10.4, with a melting point of 144–146°C, [α ]10 D = −16.2° (c = 2.1, CCl4). Add hydrochloric acid to artemisinin in ethanol, and react in a water bath at a constant temperature of 55°C for 5 h. The resulting reaction products have maximum absorbance at 254 nm. NMR indicates destruction of the oxygen bridge and into a product with α,βunsaturated carbonyl structure in acidic solution, but its chemical structure remains unclear.

2.6  Electrochemical Properties Chen et al. [18,19] used multiple electrochemical methods to investigate the reduction of the peroxy group of artemisinin molecule on the mercury electrode, and found that when the reduction potential was around 0.0 V (vs Ag/AgCl), the electrode process was an irreversible reduction; number of electrons for reduction reaction n = 2, half-wave potential E1/2 = 0.012 V, electron transfer coefficient α = 0.66, apparent standard rate constant for the

218  Chapter 10

Figure 10.4: Structures of Some Acid-Base Reaction Products of Artemisinin.

electrode reaction ks′ = 6.34 × 10 −6 cm / s, and diffusion coefficient D = 4.3 × 10−6 cm2/s; reaction products had adsorbability on the electrode surface. Also, they used electrochemical methods to investigate the interaction between artemisinin and hemin. For artemisinin, an irreversible reduction, in which two electrons transferred, occurred at −1.08 V on a glassy carbon electrode. Chan et al. [20] also used HPLC-ECD to investigate electrochemical properties of artemisinin.

3  Color Reactions of Artemisinin [1,6,21,22] Color reactions are simple and feasible methods for qualitative analysis and identification of artemisinin. Test tube reactions or TLCs are commonly used, and there are several methods as follows.

3.1  Condensation Reaction of PDAB Dissolve approximately 10 mg of artemisinin in 2 mL of ethanol, add 1 mL of PDAB reagent, and heat in water bath. The solution should be bluish violet.

3.2  Ferric Hydroxamate Test Dissolve approximately 10 mg of artemisinin in 1 mL of methanol, add 4–5 drops of solution of 7% hydroxylamine hydrochloride in methanol, heat in water bath to boil, cool down, adjust to be acidic with hydrochloric acid, and then add 1–2 drops of 1% FeCl3 in ethanol. The solution should be purplish red.

Structure and Properties of Artemisinin  219

3.3  2,4-Dinitrophenylhydrazine Test Dissolve approximately 10 mg of artemisinin in 1 mL of CCl4, apply it to a thin layer plate, spray with 2,4-dinitrophenylhydrazine reagent, and bake in an oven at 80°C for 10 min. A yellow spot should be produced.

3.4 Alkaline m-Dinitrobenzene Test Dissolve approximately 10 mg of artemisinin in 2 mL of ethanol, separately add several drops of 2% m-dinitrobenzene in ethanol and KOH in ethanol, and slightly heat in water bath. The solution should be red.

3.5  Vanillin-Sulfuric Acid Color Reaction Apply a certain amount of artemisinin TS to the silica gel G plate, using petroleum ether–ethyl acetate (8:2) or benzene–ether (4:1) as the mobile phase, 1% vanillin in concentrated sulfuric acid as the chromogenic reagent. The spot of artemisinin should be yellow, and then turn to blue.

3.6  Acidic Potassium Iodide Solution Test It is a color reaction of the peroxy group. After reaction of artemisinin with potassium iodide solution, add the starch solution, and bluish violet is produced immediately.

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