WO2003022855A1 - Tert-butoxy dihydro artimisinin, its production and pharmaceutical composition - Google Patents

Abstract

The present invention provides a new artemisinin derivative, tert-butoxy dihydro artimisinin. It is produced by using dihydro-artimisinin as a starting material, acylating with bi(tert-butyl) dicarbonate in organic solvents. The present invention also relates to an antiparasitic pharmaceutical composition comprising therapeutically effective amount of tert-butoxy dihydro-artimisinin and pharmaceutically acceptable carriers. Comparing with the artemisinin derivatives that had already been synthesized, the tert-butoxy dihydro-artimisinin has a highest Therapy Index (> 1700). It is a kind of antiparasitic medicament with higher effective and lower toxicity. It can reduce the toxicity and side-effect when it is used in the process of preventing and treating the parasitic diseases such as schistosomiasis and malaria. It has significant meaning in lowering the puerile mortality rate especially in treating the malignant malaria.

Description

 Tert-butoxycarbonyl dihydroartemisinin, preparation method and pharmaceutical composition thereof

 The present invention relates to terpenoids, in particular to artemisinin derivatives, a preparation method thereof, and an antiparasitic drug composition containing the derivatives. Background technique

annua L. ) 中提取出青蒿素， 证明它具有强大的抗疟作用， 能治愈抗药性 疟疾。 为了改善它的溶解度和生物利用度， 国内外研究人员曾制备了各 种青蒿素的衍生物提供筛选， 其中蒿甲醚、 青蒿琥酯、 二氢青蒿素和蒿 乙醚己经先后上市。 According to World Health Organization statistics, malaria is one of the most serious infectious diseases today, with more than 2 billion people living in malaria areas and 500 million people suffering from malaria each year. As Plasmodium develops resistance to chloroquine, mechloroquine, halofantrine, quinine, and sulfa antimalarials, more than 2 million deaths occur each year, half of which are children. In 1972 Chinese researchers took the Chinese herbal medicine Artemisia annua (plant Artemisia annua

annua L.) has been extracted from artemisinin, which proves that it has a strong antimalarial effect and can cure drug-resistant malaria. In order to improve its solubility and bioavailability, researchers at home and abroad have prepared various artemisinin derivatives for screening, among which artemether, artesunate, dihydroartemisinin and artemether have been listed on the market. .

Artemisinin dihydroartemisinin R = H (α + β) R '= Me, Et, Pr (n), Pr (i), artemether R = Me (β) Bu (n), Bu (i ), Phenyl, artemether R = Et (β) 1-adamantyl artesunate R = COCH ₂ CH ₂ COOH (α) The present inventors have also developed a class of carbonates of dihydroartemisinin Derivatives (R 'is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, phenyl, and adamantyl. See Li Ying et al., Pharmaceutical Journal, 1981, 16: 429; Chemistry Journal of 1982, 40: 557; Lee ZL 93112454.9). Their antimalarial activity is also high, but because their solubility in oil is not as good as artemether, they have not been studied further.

 The above artemisinin-based antimalarial drugs can quickly and safely cure drug-resistant malaria and have been widely used in clinical applications. However, in some patients, reticulocytes were transiently reduced, and aspartate aminotransferase and alanine aminotransferase were slightly increased. Very few patients may have arrhythmias (eg, ventricular premature beats, etc.). Summary of invention

 An object of the present invention is to provide an artemisinin derivative which is more active and less toxic. Another object of the present invention is to provide a method for preparing such an artemisinin derivative.

 Yet another aspect of the present invention is to provide a pharmaceutical composition containing such an artemisinin derivative. The artemisinin derivative provided by the present invention is tert-butoxycarbonyl dihydroartemisinin, and its chemical formula is as follows:

 The method for preparing tert-butoxycarbonyl dihydroartemisinin provided by the present invention is an acylation reaction of dihydroartemisinin and di-tert-butyl dicarbonate in an organic solvent.

 The antiparasitic pharmaceutical composition provided by the present invention comprises a therapeutically effective amount of tert-butoxycarbonyl dihydroartemisinin and a pharmaceutically acceptable carrier. Detailed description of the invention

The tert-butoxycarbonyl dihydroartemisinin of the present invention is prepared by using dihydroartemisinin as a starting material and performing an acylation reaction with di-tert-butyl dicarbonate. Dissolve 10 mg of dihydroartemisinin in 30-100 ml of organic solvent, add bis-tert-butyl dicarbonate (molecular ratio is 1.0-3.0), and perform the reaction with stirring. The organic solvent may be selected from dichloromethane, dichloroacetamidine, tetrahydrofuran, acetonitrile, dimethylformamide, and the like.

 The reaction temperature can be controlled between -10 ° C and 85 ° C.

 'An acylation catalyst such as dimethylaminopyridine (1 to 10% molar ratio) can be added to accelerate the reaction.

 After the reaction is basically completed, a general post-treatment is performed to obtain a crude product of t-butoxycarbonyldihydroartemisinin, which is composed of two epimers of the 12-α form and the 12-β form, and is recrystallized or column chromatography. They can get their pure products.

 Their physical data are as follows:

 Tert-Butoxycarbonyl dihydroartemisinin (12-alpha form):

Melting point: 157—158. C, optical rotation: [a] _D ²⁵ = +13.2. (C = 1.14, CHC1 ₃ ) Elemental analysis (C _2. H ₃₂ 0 ₇ ) _:

 Theoretical values: C 62.48, H 8.39

 Found: C 62.30, H 8.21

 NMR spectrum: 5.54 (IH, d, J = 9.7 Hz), 5.41 (IH, s), 1.47 (9H, s), 1.41 (3H, s), 0.94 (3H, d, J = 6.0 Hz), 0.88 (3H, d, J = 7.0 Hz)

 Infrared spectrum: 2951 -2870, 1751, 1456, 1373, 1279, 1257, 1 165, 1146, 1040, 928, 864.

 Tert-Butoxycarbonyl dihydroartemisinin 02-β body):

Melting point: 154-6. C, optical rotation: [a] _D ²⁵ = + 137.2 ° (C = 0.95, CHC1 ₃ ) Elemental analysis: (C ₂₀ H ₃₂ O ₇ ) _:

 Theoretical values: C 62.48, H 8.39

 Found: C 62.54, H 8.40

 NMR spectrum: 6.01 (IH, d, J = 3.5 Hz), 5.49 (IH, s), 1.47. (9H, s), 1.38 (3H, s), 0.93 (3H, d, J = 6.3 Hz), 0.88 (3H, d, J = 7.2 Hz)

 Infrared spectrum: (cm-1): 2937-2870, 1736, 1458, 1373, 1283, 1161, 1113,

1043, 920, 837. The pharmaceutical composition of the present invention contains 0.1-99.9% by weight of tert-butoxycarbonyldihydroartemisinin and an appropriate amount of a pharmaceutically acceptable carrier, and can be prepared into a preparation form suitable for oral, injection or enteral administration. .

 The solid pharmaceutical composition for oral administration may be powder, tablet, hard capsule, soft capsule, drip pill and the like. It may contain the following excipients: sugar (lactose, glucose, sucrose, etc.), starch (corn starch, potato starch, modified starch, etc.), cellulose and its derivatives (microcrystalline cellulose, sodium carboxymethylcellulose, carboxylate Propyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), polyethylene glycol, polyvinyl pyrrolidone, mannitol, sorbitol, calcium hydrogen phosphate, micronized silica gel, stearic acid, stearic acid Magnesium acid, glyceryl monostearate, gelatin, insect wax, vegetable oil, hydrogenated vegetable oil, stearyl alcohol (stearyl alcohol), cetyl alcohol (cetyl alcohol), semi-synthetic fatty acid ester, talc, stabilizer, flavoring Agents, preservatives, etc.

 Liquid pharmaceutical compositions (including suspending agents) for oral administration may include suspending agents Tween, Span, sorbitol, sugar solution, sodium carboxymethyl cellulose, carboxypropyl cellulose, etc .; emulsifier sorbitan monooleate Acid esters, etc .; solvent vegetable oils, glycerides, etc .; preservatives, parabens, pharmaceutical compositions for injection, soluble in vegetable oils (such as olive oil, sesame oil, soybean oil, peanut oil, tea oil, etc.) .

 '' For intestinal use, it can be made into a suppository. Its excipients can be polyethylene glycol, Tween, Span, glyceryl laurate, sodium carboxymethyl cellulose, methyl cellulose, stearyl alcohol, stearic acid, aluminum monostearate, gelatin Wait.

 When the pharmaceutical composition of the present invention is used to treat malaria, the dosage of an adult patient is generally 25-150 mg of active substance per day. When used for schistosomiasis prevention, the dosage for adults is usually 50-200 mg of active substance every 2-3 weeks. The actual dosage depends on a number of factors, such as patient age, general health, disease severity and duration, route of administration, individual sensitivity to the drug, and epidemic situation in the affected area.

The pharmaceutical composition of the present invention can choose different modes of administration for different patients, and it is preferred to use injections and suppositories for patients with severe coma. It is suitable to use sugar-containing powder and suspension for sick children And suppositories. detailed description

 The specific implementations of the present invention are further described below with reference to the examples, but these examples are by no means any limitation to the present invention.

 Synthesis Example 1

 Dissolve dihydroartemisinin (2.84 g, 10 mg molecules) in 40 ml of dichloroformamidine, add bis-tert-butyl dicarbonate (2.84 g, 12.8 mg molecules), and then add the acylation catalyst, dimethylamine Pyridine (20 mg), and the reaction was stirred at room temperature. After the reaction was basically completed, the reaction solution was washed with water and then dried over magnesium sulfate. The solvent was distilled off, and the obtained crude product was recrystallized from ethyl acetate-petroleum ether to obtain 3.00 g (yield 78%) of t-butoxycarbonyldihydroartemisinin (yield 78%) and t-butoxycarbonyldihydrocyanine. Artemisinin (12-beta body) 0.19 g (5% yield). Synthesis Example 2

 First, dihydroartemisinin (2.84 g, 10 mg molecules) was dissolved in 50 ml of anhydrous acetonitrile, and di-tert-butyl dicarbonate (2.5 g, 1 1.3 mg molecules) was added, and the reaction was heated at reflux. After the reaction was basically completed, the reaction solution was evaporated under reduced pressure to remove the solvent. The obtained crude product was separated by column chromatography (silica gel, 6% ethyl acetate / petroleum ether as eluent) to obtain tert-butoxycarbonyldihydroartemisinin 12-alpha form) 1.46 g (yield 38%) and tert-butoxycarbonyl dihydroartemisinin (12-beta form) 1.54 g (yield 40%). Preparation Example 1

 100 grams of tert-butoxycarbonyl dihydroartemisinin (12-alpha form) and 100 grams of lactose are ground, sieved with 100 mesh, and mixed evenly into 1000 bags (200 mg each, containing 100 mg of active substance) . Formulation example 2 tablets

500 g of tert-butoxycarbonyl dihydroartemisinin (body 12-alpha) was sieved through a 100-mesh sieve, and 40 g of starch Pass through a 100-mesh sieve, mix well, add 170 g of starch paddles, stir well, make granules through a 16-mesh sieve, and after drying, sieve, add 3 g of magnesium stearate, mix well and press into 10,000 tablets. Each tablet contains 50 mg of effective substance. Test example 1 in vitro antimalarial test

 The experimental data was provided by the World Health Organization.

 Antimalarial activity was measured using the methods described in the following literature:

 Trager W, Jensen JB. Human malaria parasites in continuous culture. Science, 1976, 193: 673-677.

 Makler MT, Ries JM, Williams JA, Bancroft JE, Piper RC, Gibbins BL,

Hinrichs DJ. Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. American Journal of Tropical Medicine and Hygiene, 1993, 48: 739-41.

The test sample was dissolved with DMSO and formulated to a certain concentration. Use deionized water for a 1: 2 serial dilution from serial 1/2 to 1/16 concentration. Two microliters of different concentrations of samples were added to the wells of a 96-well microassay plate, which already contained 190 microliters of the Plasmodium inoculum. A protozoa growth control group without test samples was also set up. After 72 hours of incubation at 37 ° C, the assay plate was deep frozen at -20 ° C. After thawing, remove 20 microliters from each well and transfer to another plate, and mix with 100 microliters of Malstat [TM] and 10 microliters of PES and NBT (1: 1) in the dark for 2 hours, then use 655 A nano spectrophotometer measures the change in color. Compared with the control group, the deworming rate of different dose groups was obtained, and the IC ₅ of the test sample was finally calculated. (See Table 1). Table 1 Anti-malarial effects of tert-butoxycarbonyldihydroartemisinin in vitro Malaria parasite strains t-butoxycarbonyldihydroartemisinin t-butoxycarbonyldihydroartemisinin

 (12-α body) (12- β body)

P. falciparum K1 (resistant strain)

 0. 15 <0. 5

P. falciparum NF54 (Sensitive strain)

0. 44 <0.5 Test Example 2 Murine malaria (^ oe /// ssp NS, chloroquine-resistant strain;) test

 The experimental data was provided by the World Health Organization.

Peters' four-day inhibition test was used to determine the ED _5Q and ED _9Q of t-butoxycarbonyldihydroartemisinin (12-alpha form). D _Q days after mice were injected intravenously 0.2 ml inoculum ^(2χ 10 δ protozoal erythrocytes / ml of blood), divided into treatment and control groups. The test samples were formulated into several concentrations, and 0.2 ml was administered orally or subcutaneously 2 hours later, and Di-D was administered continuously for ₃ days, once a day. _On day ₄ D, blood was taken from mice in each group, thin blood film was applied, and Giemron staining was performed. At least 1,000 red blood cells were examined under a microscope, and the number of protozoa-infected cells was counted. Compared with the control group, the protozoan inhibition rates of different dose groups were obtained, and the ED _5Q and ED _{9 ()} of the drug were finally calculated (see Table 2 for specific data). Table 2 Anti-mouse malaria effect of tert-butoxycarbonyl dihydroartemisinin (12-alpha body)

Note: SSV suspension is prepared from carboxymethyl cellulose sodium salt (a-CMC), benzyl alcohol, Tween 80 and 0.9% aqueous sodium chloride solution. It can be seen from Table 2 that the antimalarial activity of t-butoxycarbonyldihydroartemisinin (body 12-alpha) is equivalent to or slightly higher than that of artesunate, a known antimalarial drug, when tested by oral or subcutaneous injection in different routes . Test example 3 schistosomiasis prevention Take Kunming mice (18-22 grams), infect 40 to 60 Schistosoma japonicum cercariae from the abdomen, randomly divide them into groups 7 days later, and administer the test samples once through the gastric tube. Check the number of schistosomiasis that survive in the abdomen of the mouse, calculate the reduction rate, and evaluate the preventive effect (see Table 3 for the results). Table 3 Preventive effects of tert-butoxycarbonyl dihydroartemisinin (body 12-a) on schistosomiasis

表 3的结果表明， 在同样剂量 （300 mg/kg) 下叔丁氧羰基二氢青蒿 素 （12— α 体） 的减虫率高于已知血吸虫病预防药蒿甲醚， 在同样减虫 效果的情况下叔丁氧羰基二氢青蒿素 （12— α 体） 的剂量 （200mg/kg ) 低于蒿甲醚的剂量（300mg/kg)。可见叔丁氧羰基二氢青蒿素（12— α 体) 的血吸虫病预防作用高于蒿甲醚。 试验例 4 急性毒性 （小鼠， 口服， CMC混悬液）

The results in Table 3 indicate that at the same dose (300 mg / kg), the deworming rate of t-butoxycarbonyldihydroartemisinin (body 12-α) is higher than that of the known schistosomiasis preventive drug artemether. In the case of insecticidal effects, the dose of t-butoxycarbonyldihydroartemisinin (body 12-alpha) (200 mg / kg) is lower than the dose of artemether (300 mg / kg). It can be seen that the prevention of schistosomiasis of t-butoxycarbonyl dihydroartemisinin (body 12-α) is higher than that of artemether. Test Example 4 Acute toxicity (mouse, oral, CMC suspension)

 Forty Kunming mice, half male and half female, were randomly divided into two groups (control group and medication group), given 0.5% CMC (sodium carboxymethylcellulose) and 12.79% (the highest concentration) in a single gavage. Butoxycarbonyl dihydroartemisinin (12- ot body), the intragastric volume was 70 tnl / kg (maximum volume), and its acute toxicity to mice was observed. As a result, after 7 days of observation, the activities, diet and weight gain of the two groups of animals were normal. No animal deaths were seen. After 7 days in the treatment group, the mice were sacrificed by cervical dislocation, and no obvious lesions were found in the naked eyes of all major organs at necropsy. Therefore, the acute toxicity test results of t-butoxycarbonyldihydroartemisinin (body 12-α) are:

Male rats: LD _{5 ()} 〉 8950 mg / kg; Female rats: LD ₅ o> 8950 mg / kg The above examples and test examples merely illustrate the preparation of the compounds and pharmaceutical compositions of the present invention and the results of pharmacological experiments, but those skilled in the art can make various modifications and changes without departing from the spirit and The scope, the appended claims, cover all such modifications within the scope of the invention. Possibility of industrial use

 Further experimental results show that the tert-butoxycarbonyl dihydroartemisinin (12-α body and 12-β body) of the present invention has antimalarial activity equivalent to or slightly higher than that of the known antimalarial drug artesunate and higher Methyl ether schistosomiasis prevention.

In the acute toxicity test performed under the same conditions as the present invention, the LD _5Q of artemether was 895 mg / kg, and the LD _{5 of} artesunate. At 1409 mg / kg, the LD _{5 of} artemisinin. It is 4228 mg / kg, and the LD _{5Q of} tert-butoxycarbonyl dihydroartemisinin is> 8950 mg / kg, which is much less toxic than artemisinin, artesunate and artemether.

Therapeutic index (Tl = LD ED _5Q ) is often used to judge the safety of drugs. Compared with the known artemisinin antimalarial therapeutic index (artemether-447, artesunate-793, artemisinin-384), t-butoxycarbonyldihydroartemisinin (12-alpha form And 12-beta) have the largest therapeutic index (> 1700).

 Therefore, the tert-butoxycarbonyl dihydroartemisinin of the present invention is a highly effective and low toxic antiparasitic drug. It can reduce the occurrence of toxic and side effects in the prevention and treatment of parasitic diseases such as schistosomiasis and malaria, especially in the treatment of malignant malaria, which has great significance in reducing child mortality.

Claims

Rights request 1. tert-butoxycarbonyl dihydroartemisinin having the structure:

 2. The method for preparing tert-butoxycarbonyldihydroartemisinin according to claim 1, characterized in that: the dihydroartemisinin is acylated with di-tert-butyl dicarbonate in an organic solvent.  3. The method according to claim 2, wherein the organic solvent is selected from the group consisting of dichloromethane, dichloroacetamidine, tetrahydrofuran, acetonitrile, and dimethylformamide.  4. The method according to claim 2, wherein the acylation reaction is performed at a temperature of -10 ° C ~ 85 ° C.  5. The method according to claim 2, wherein an acylation catalyst, dimethylaminopyridine, is also used. 6. The method according to claim 2, further comprising separating t-butoxycarbonyldihydroartemisinin into 12-α and 12-β isomers by recrystallization or column chromatography. Pure body P 7. An antiparasitic pharmaceutical composition comprising a therapeutically effective amount of tert-butoxycarbonyldihydroartemisinin and a pharmaceutically acceptable carrier.