Medicine for treating severe malaria and synthesis method thereof

Abstract

The invention relates to a medicine for treating severe malaria and a synthesis method thereof, the medicine has a remarkable treatment effect on severe malaria (cerebral malaria), and compared with an existing first-line treatment medicine artesunate for severe malaria (cerebral malaria), the medicine can effectively remove plasmodium, reduce the death rate, relieve nerve injury and effectively reduce the malaria recontamination rate. Chemical structure of medicine as follows: # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and relates to a class of artemisinin / ligustrazine complexes, and pharmaceutically acceptable salts, hydrates, optical isomers thereof, and pharmaceutical compositions with the complexes as active ingredients, as well as uses thereof for treating severe malaria. The present invention is aimed at severe malaria and its pathological characteristics such as nervous system damage, and uses the principle of drug chemistry complexation to design and synthesize a class of novel artemisinin / ligustrazine complexes, whose chemical structures are determined by 1NMR, 13NMR, and HRMS-ESI characterization. Background Art

[0002] Cerebral malaria (CM) is the most serious complication caused by Plasmodium falciparum infection and the leading cause of death in children under 5 years old in Africa. It can lead to long-term cognitive and motor dysfunction in surviving children and is a clinical problem that needs to be urgently addressed in the field of malaria treatment.

[0003] 74% of cerebral malaria cases occur in African children under five years old, with a mortality rate of 15-20% (World Health Organization, 2016. de Miranda AS, 2010). Cerebral malaria can cause acute diffuse encephalitis, accompanied by various neurological symptoms, clinically coma, impaired consciousness, meningeal irritation signs, hemiplegia, convulsions, ataxia and even death, and 10-17% of surviving children have persistent neurological deficits, cognitive and motor dysfunction (Birbeck GL, 2010; de Miranda AS, 2010). Among them, the motor ability, language ability and social interaction ability of children under five years old can be delayed for more than 6 months, and the delay is more significant in those with obvious abnormalities in MRI.

[0004] The "mechanical obstruction" theory of cerebral malaria holds that the adhesion and blockage of parasite-infected red blood cells (pRBC) in the brain microvessels is the initiating factor leading to cerebral malaria. A large number of pRBCs are "retained" in the microvessels, resulting in reduced blood perfusion and hypoxia and ischemia of the brain tissue, which is the basis for the onset of cerebral malaria and subsequent neurological damage.

[0005] The local embolic changes in brain microvessels, microcirculation disorders, olfactory bulb bleeding, blood rheology changes, and platelet dysfunction that occur during cerebral malaria meet the diagnostic criteria of "blood stasis syndrome" in traditional Chinese medicine. Its neurological symptoms such as high fever, irritability, coma, delirium, and cerebral hemorrhage are all secondary lesions based on "blood stasis".

[0006] Artesunate (AS) is also called dihydroartemisinin-12-α-succinate. Artesunate has good solubility and bioavailability, is water-soluble, can pass through the blood-brain barrier, and can be administered through multiple routes such as intravenous injection, intramuscular injection, oral administration, and rectal administration. Artesunate can maintain high concentrations in the nervous system and has the characteristics of high efficiency and low toxicity. Intravenous artesunate is the first choice for the treatment of cerebral malaria recommended by the WHO "Practical Manual for the Management of Severe Malaria". As the current first-line treatment drug, it has a market share of more than 90% in the global severe malaria market. More and more evidence shows that artesunate has multiple pharmacological activities such as anti-inflammatory, antioxidant, blood-brain barrier protection, immunomodulatory, antibacterial, and anti-tumor.

[0007] The following problems exist in the clinical treatment of cerebral malaria with artesunate: ① According to the WHO Practical Manual for the Management of Severe Malaria (third edition), due to the reduced sensitivity of Plasmodium falciparum to artemisinin drugs, the recommended dosage of artesunate for the treatment of cerebral malaria has been greatly increased. The first-day treatment dose for adults has increased from 120 mg to 432 mg, and the 7-day full-course treatment dose has increased from 480 mg to 1296 mg. The treatment cost, the risk of drug resistance when using a single drug, and the risk of side effects such as hemolysis are increasing. ② Artesunate has poor efficacy in late cerebral malaria where the host has already developed inflammatory reactions and blood-brain barrier damage. ③ After artesunate kills the malarial parasite, the parasite still blocks the local brain microvessels and cannot effectively alleviate the ischemic and hypoxic state. ④ Children who survive cerebral malaria have persistent neurological deficits and long-term cognitive and motor dysfunction, which artesunate cannot effectively intervene in. ⑤ In clinical practice, intravenous artesunate is used to treat severe malaria such as cerebral malaria. In areas with a high incidence of cerebral malaria in Africa, extremely backward medical conditions have largely limited the timely and effective clinical application of artesunate. ⑥ Infants and young children under 5 years old, a high-risk population, have poor compliance with intravenous artesunate.

[0008] Tetramethylpyrazine (TMP) is considered to be the characteristic alkaloid of Ligusticum chuanxiong. Its role in the acute injury of ischemic stroke has been confirmed in many studies. For example, it has a protective effect on brain damage caused by reperfusion after ischemic stroke, can effectively improve behavioral and learning and memory disorders after brain injury, and can also alleviate the deterioration of stroke and reduce mortality or disability. Studies have reported that Ligustrazine nasal spray can effectively increase the concentration of Ligusticum chuanxiong in brain tissue and give full play to its therapeutic effect.

[0009] Previous studies have confirmed that the combined nasal administration of artesunate and ligustrazine is effective in treating cerebral malaria, significantly reducing the mortality and parasitemia levels in mice with cerebral malaria, and effectively improving nerve damage in mice. Its effects are related to increasing cerebral blood perfusion and improving nerve blood oxygen supply. The mutation of the nitrosylation site (cysteine ​​C263) was used to determine that regulating the nitrosylation of PDHB to affect the balance of the NO-related redox system is one of its key links.

[0010] However, the poor chemical compatibility of artesunate and ligustrazine brings difficulties to the development of compound antimalarial preparations and affects their drugability. In terms of chemical structure, artesunate is a monoester formed by dihydroartemisinin and succinic acid, and there is also a free carboxyl group in the molecule. In order to improve the water solubility of the compound, its sodium salt, sodium artesunate, is used clinically as an injection, which is alkaline; ligustrazine is an oily compound that is insoluble in water. Similarly, in order to improve its water solubility and stability, its hydrochloride, ligustrazine hydrochloride, is used clinically, which is strongly acidic. When ligustrazine and artesunate are co-dissolved in an aqueous or organic solvent system, the ester bond of artesunate is rapidly hydrolyzed, resulting in a rapid decrease in content. When co-dissolved in an alkaline aqueous solvent, a violent acid-base neutralization reaction will occur, artesunate will precipitate immediately, and an insoluble ligustrazine oily substance will be produced, and a uniform and stable preparation cannot be formed. To solve this problem, different buffer systems and nanoformulation technology have been tried, but none of them can effectively solve this problem.

[0011] The present invention obtains a new compound by chemically splicing artemisinin and ligustrazine, solves the problems existing in the compound preparation of artesunate and ligustrazine, and retains the efficacy advantage of the combination of the two. Summary of the invention

[0012] The object of the present invention is to provide a class of artemisinin / ligustrazine complexes and pharmaceutically acceptable salts, hydrates and prodrugs thereof. These compounds can be used to prepare new drugs for treating severe malaria.

[0013] To this end, the present invention provides a drug represented by general formula I or a pharmaceutically acceptable salt thereof:

[0014]

[0015] Wherein, X and Y are each selected from CH2, O, S, NH,

[0016] L is a linker arm selected from:

[0017]

[0018] Here, n is an integer from 0 to 6.

[0019] Preferably, the drug of the present invention or a pharmaceutically acceptable salt thereof has the following structure:

[0020]

[0021] Here, n is an integer from 0 to 6.

[0022] Most preferably, the drug of the present invention or a pharmaceutically acceptable salt thereof has the following structure:

[0023]

[0024] The most preferred drug of the present invention is a new compound, which is referred to as Artesunazine in this specification, also known as Compound Ia-1, also known as Artemisinin / ligustrazine complex, and its chemical name is (3R, 5aS, 6R, 9R, 10R, 12R, 12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isobenzopyran-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)succinate. The NMR and high-resolution mass spectrometry data of the compound are as follows: 1 H NMR(600MHz,cdcl3)δ5.79(d,J=9.9Hz,1H),5.43(s,1H),5.21(s,2H),2.82–2.73(m,3 H),2.73–2.66(m,1H),2.54–2.49(m,9H),2.39(d,J=13.5Hz,1H),2.03(d,J=15.0Hz,2H ),1.89(s,1H),1.80–1.70(m,2H),1.63-1.60(m,1H),1.50-1.47(m,1H),1.43(s,3H),1 .41–1.23(m,4H),1.03(t,J=10.6Hz,1H),0.97(d,J=6.2Hz,3H),0.83(d,J=7.1Hz,3H). 13 C NMR (151MHz, cdcl3) δ171.80,171.00,151.34,149.08,148.98,144.52,104.45,92.16,91.47,80.08,65.34,51.52,45. 19,37.24,36.18,34.06,31.76,29.13,28.69,25.94,24.55,21.96,21.69,21.45,20.49,20.21,12.01.HRMS(ESI)calcd for[C 27 H 38N2O8+H]+519.2628,found519.2487.

[0025]

[0026] Compound Ia-2, chemical name: (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isobenzopyran-10-yl((3,5,6-trimethylpyrazine-2-yl)methyl)malonate. The NMR and high-resolution mass spectrometry data of the compound are as follows: 1 H NMR(600MHz,cdcl3)δ5.60(d,J=9.7Hz,1H),5.35(s,1H),5.21(s,2H),3.40(s,2H),2.82– 2.73(m,3H),2.73–2.66(m,1H),2.54–2.49(m,5H),2.39(d,J=13.5Hz,1H),2.03(d,J=14.0 Hz,2H),1.89(s,1H),1.80–1.70(m,2H),1.63-1.60(m,1H),1.50-1.47(m,1H),1.43(s,3H ),1.41–1.23(m,3H),1.03(t,J=10.6Hz,1H),0.97(d,J=6.2Hz,3H),0.83(d,J=7.1Hz,3H). 13 C NMR (151MHz, cdcl3) δ171.60,171.20,151.24,149.32,148.87,144.50,104.32,92.18,91.47,80.08,65.34,51.52, 47.32,45.19,37.24,36.18,34.06,28.52,25.84,24.35,21.91,21.70,21.42,20.42,20.12,12.07.HRMS(ESI)calcd for[C 26 H 36 N2O8+H]+505.2550,found505.2547.

[0027]

[0028] Compound Ia-3, chemical name: (3R, 5aS, 6R, 9R, 10R, 12R, 12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isobenzopyran-10-yl((3,5,6-trimethylpyrazine-2-yl)methyl)maleate. The NMR and high-resolution mass spectrometry data of the compound are as follows: 1H NMR (600MHz, cdcl3) δ6.98 (d, J = 10.2Hz, 1H), 5.79 (d, J = 9.9Hz, 1H), 5.43 (s, 1H), 5.33 (d , J=10.7Hz,1H), 5.21(s,2H),2.54–2.49(m,9H),2.39(d,J=13.5Hz,1H),2.03(d,J=13.0H z,2H),1.89(s,1H),1.80–1.70(m,2H),1.63-1.60(m,1H),1.50-1.47(m,1H),1.43(s,3H) ,1.41–1.23(m,3H),1.03(t,J=10.6Hz,1H),0.97(d,J=6.2Hz,3H),0.83(d,J=7.1Hz,3H). 13 C NMR(151MHz,cdcl3)δ164.37,163.50,151.64,149.52,147.61,144.01,134.06,133.71,104.50,92.86,91.53,80.01,65.85 ,51.49,45.16,37.24,36.16,34.03,31.72,29.68,25.89,24.54,21.95,21.69,21.44,20.48,20.19,12.06.HRMS(ESI)calcd for[C 27 H 36 N2O8+H]+517.2550,found517.2550.

[0029]

[0030] Compound Ia-4, Compound Ia-3, chemical name: (3R, 5aS, 6R, 9R, 10R, 12R, 12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isobenzopyran-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)pentanedioate. The NMR and high-resolution mass spectrometry data of the compound are as follows: NMR(600MHz,cdcl3)δ5.78(d,J=9.9Hz,1H),5.45(s,1H),5.21(s,2H),2.82–2.73(m,3 H),2.73–2.66(m,1H),2.53–2.47(m,9H),2.39(d,J=13.7Hz,1H),2.02(d,J=14.5Hz,2H ),1.89(s,1H),1.80–1.70(m,2H),1.63-1.60(m,1H),1.50-1.47(m,3H),1.41(s,3H),1 .41–1.23(m,3H),1.03(t,J=10.6Hz,1H),0.97(d,J=6.0Hz,3H),0.84(d,J=7.0Hz,3H). 13 C NMR (150MHz, cdcl3) δ172.35,171.53,151.05,148.90,148.65,104.23,91.75,91.27,79.92,64.67,51.38,45.06, 37.04,36.06,33.94,32.97,32.81,31.58,29.54,25.74,24.44,21.79,21.43,21.24,20.24,20.10,19.69,11.98.

[0031] HRMS(ESI)calcd for[C 28 H 40 N2O8+H]+533.2863, found 533.2851.

[0032]

[0033] Compound Ia-5, chemical name: (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isobenzopyran-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)heptanedioate. The NMR and high-resolution mass spectrometry data of the compound are as follows: 1H NMR(600MHz,cdcl3)δ5.79(d,J=9.8Hz,1H),5.44(s,1H),5.19(s,2H),2.82–2.73(m,3H), 2.73–2.66(m,1H),2.54–2.49(m,9H),2.40(d,J=14.5Hz,1H),2.04(d,J=15.3Hz,2H),1.89 (s,1H),1.80–1.70(m,2H),1.63-1.60(m,1H),1.51-1.44(m,1H),1.41(s,3H),1.41–1.36( m,3H),1.31–1.24(m,4H),10.5-1.02(m,3H),0.97(d,J=6.2Hz,3H),0.83(d,J=7.1Hz,3H). 13 C NMR(151MHz,cdcl3)δ172.92,172.01,151.03,148.79,148.77,144.72,104.22,91.58,91.30,79.94,64.75,51.43,45.11,37.08, 36.09,33.98,33.87,33.68,31.66,28.36,25.81,24.47,24.14,21.84,21.54,21.32,20.91,20.36,20.13,12.02.HRMS(ESI)calcd for[C 30 H 44 N2O8+H]+561.3175, found 561.3181.

[0034]

[0035] Compound Ia-6, chemical name: (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isobenzopyran-10-yl((3,5,6-trimethylpyrazine-2-yl)methyl)adipate. The NMR and high-resolution mass spectrometry data of the compound are as follows: 1H NMR(600MHz,cdcl3)δ5.79(d,J=9.5Hz,1H),5.44(s,1H),5.19(s,2H),2.82–2.73(m,3H),2 .73–2.66(m,1H),2.52–2.50(m,9H),2.42(d,J=14.0Hz,1H),2.04(d,J=14.0Hz,2H),1.89(s ,1H),1.80–1.70(m,2H),1.63-1.60(m,1H),1.50-1.47(m,1H),1.43(s,3H),1.41–1.27(m, 3H),1.27–1.23(m,4H)1.03(t,J=10.6Hz,1H),0.97(d,J=6.2Hz,3H),0.83(d,J=7.5Hz,3H). 13 C NMR (151MHz, cdcl3) δ172.66,171.76,151.02,149.36,148.80,144.66,104.21,91.64,91.29,79.92,64.75,51.42,45.10,37. 07,36.08,33.97,33.50,31.63,29.56,25.79,24.46,24.16,23.90,21.83,21.52,21.31,20.34,20.11,12.01.HRMS(ESI)calcd for[C 29 H 42 N2O8+H]+547.3019,found 547.3007.

[0036]

[0037] Compound Ia-7, chemical name: (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isobenzopyran-10-yl((3,5,6-trimethylpyrazine-2-yl)methyl) suberate. The NMR and high-resolution mass spectrometry data of the compound are as follows: 1H NMR(600MHz,cdcl3)δ5.79(d,J=9.0Hz,1H),5.44(s,1H),5.19(s,2H),2.82–2.73(m,3 H),2.73–2.66(m,1H),2.53–2.42(m,9H),2.39(d,J=13.5Hz,1H),2.03(d,J=15.0Hz,2H ),1.89(s,1H),1.80–1.70(m,2H),1.63-1.60(m,5H),1.50-1.47(m,1H),1.43(s,3H),1 .41–1.23(m,7H),1.03(t,J=10.6Hz,1H),0.97(d,J=6.2Hz,3H),0.83(d,J=7.1Hz,3H). 13 C NMR(151MHz,cdcl3)δ173.16,172.24,151.10,149.42,148.84,144.78,91.60,91.36,80.01,64.80,51.47,45.16,37.14,36.13,34.0 7,31.71,28.64,28.59,25.87,24.64,24.50,24.35,21.90,21.59,21.37,21.28,21.17,20.40,20.17,19.29,12.07.HRMS(ESI)calcd for[C 31 H 46 N2O8+H]+575.3332, found 575.3342.

[0038]

[0039] Compound Ia-8, chemical name: 2,3,5-trimethyl-6-[(2-(((3R,5aS,6R,9R,12R,12aR)-3,6,9-trimethylbicyclo[3.2.1]heptane-12H-3,12-epoxide[1,2]dioxepane[4,3-i]isoorientin-10-yl)oxy)ethoxy)methyl]pyrazine. The NMR and high-resolution mass spectrometry data of the compound are as follows: 1H NMR(600MHz,cdcl3)δ5.79(d,J=9.1Hz,1H),5.43(s,1H),5.21(s,2H),3.72–3.54 (m,4H),2.54–2.49(m,9H),2.39(d,J=14.5Hz,1H),2.03(d,J=15.0Hz,2H),1.89(s ,1H),1.80–1.70(m,2H),1.63-1.60(m,1H),1.50-1.47(m,1H),1.43(s,3H),1.41– 1.23(m,3H),1.03(t,J=10.6Hz,1H),0.97(d,J=6.2Hz,3H),0.83(d,J=7.1Hz,3H). 13 C NMR (150MHz, cdcl3) δ171.80,171.00,151.34,149.08,148.98,144.52,104.45,92.16,91.47,80.08,70.1,70.3 4,65.34,51.52,45.19,37.24,36.18,25.94,24.55,21.96,21.69,21.45,20.49,20.21,12.01.HRMS(ESI)calcd for[C 25 H 38 N2O8+H]+463.2808,found 463.2810.

[0040]

[0041] Compound Ia-9, chemical name: (3,5,6-trimethylpyrazine-2-yl)methyl 1-(2-oxy-2-(((3R,5aS,6R,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepane-10-yl)oxy)ethyl)piperidine-4-carboxylate. The NMR and high-resolution mass spectrometry data of the compound are as follows: 1H NMR(600MHz,cdcl3)δ5.79(d,J=9.7Hz,1H),5.43(s,1H),5.21(s,2H),3.32(s,2H ),2.54–2.49(m,13H),2.45-2.37(m,2H),2.03(d,J=15.0Hz,2H),1.97-1.89(m,5 H),1.80–1.70(m,2H),1.63-1.60(m,1H),1.50-1.47(m,1H),1.43(s,3H),1.41–1 .23(m,3H),1.03(t,J=10.6Hz,1H),0.97(d,J=6.2Hz,3H),0.83(d,J=7.1Hz,3H). 13 C NMR (150MHz, cdcl3) δ171.80,171.00,151.34,149.08,148.98,144.52,104.45,92.16,91.47,80.08,65.34,57.44,51.52,45.19,37. 24,36.18,34.06,31.76,47.44,47.02,41.2,29.21,28.37,25.94,24.55,21.96,21.69,21.45,20.49,20.21,12.01.HRMS(ESI)calcd for[C 31 H 45 N3O8+H]+588.3285,found588.3287.

[0042]

[0043] The chemical name of compound Ib-1 is: (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isobenzopyran-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)ether. The NMR and high-resolution mass spectrometry data of the compound are as follows: 1H NMR (600MHz, CDCl3) δ5.30(s,1H),4.63(s,2H),4.21-4.15(m,1H),3.64-3.60(m,2H),2.71-2.62(m,4H),2.57-2.54(m,3H),2.34-2. 28(m,1H),2.06-2.00(m,1H),1.96-1.81(m,2H),1.78-1.77(m,1H),1.68-1.33(m,7H),1.32-1.25(m,7H),0.97(d,3H),0.86(d,3H). 13 C NMR (151MHz, cdcl3) δ149.71, 148.21, 147.62, 147.45, 121.28, 96.44, 89.23, 81.87, 72.72, 71.37, 51.77, 4 3.58,37.01,36.48,32.93,29.72,29.51,26.03,25.12,20.27,19.26,19.12,19.01,18.86.HRMS(ESI)calcd for[C 26 H 40 N2O8+H]+461.2915,found461.2927.

[0044] The present invention further provides the drug salt of the present invention, which is an addition salt formed by the drug of the present invention and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalene disulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, etc. Hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, maleic acid, benzenesulfonic acid, succinic acid.

[0045] The present invention further provides a pharmaceutical composition comprising the drug of the present invention or a pharmaceutically acceptable salt thereof. The pharmaceutical composition is in the form of a pharmaceutical preparation and may further comprise auxiliary materials required for the preparation as required. The pharmaceutical composition of the present invention is selected from any edible pharmaceutical dosage form, such as a nasal preparation, an injection, or an oral preparation.

[0046] The present invention further provides a method for preparing the drug of the present invention or a pharmaceutically acceptable salt thereof.

[0047] Among them, the preferred drug of the present invention, the preparation method comprises the following steps:

[0048]

[0049]

[0050] in,

[0051] Step a: Compound Ia-1-1 is reacted with hydrogen peroxide under glacial acetic acid to obtain compound Ia-1-2;

[0052] Step b: Compound Ia-1-2 reacts with acetic anhydride to obtain compound Ia-1-3;

[0053] Step c: Ia-1-3 is hydrolyzed under sodium hydroxide to obtain compound Ia-1-4;

[0054] Step d: Compound Ia-1-5 reacts with succinic anhydride to obtain compound Ia-1-6;

[0055] Step e: Compound Ia-1-6 and Ia-1-4 undergo condensation reaction in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole to obtain a preferred drug of the present invention.

[0056] The synthesis can also be carried out by following the steps outlined in the general schemes, which include different orders of synthesis of the intermediates. Starting materials can be purchased commercially or prepared by known methods reported in the literature.

[0057] The present invention further provides use of the drug of the present invention or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing severe malaria.

[0058] The drugs or pharmaceutically acceptable salts thereof of the present invention further include their solvates, such as hydrates, optical isomers, such as racemates or isomers thereof, and also include polymorphs, such as different crystalline forms produced by crystallization from different solvents.

[0059] The compound or its pharmaceutically acceptable salt, hydrate, prodrug involved in the present invention can be used alone as the only antimalarial drug, or can be used in combination with the antimalarial drugs currently on the market for the treatment and prevention of severe malaria.

[0060] The present invention solves the problems in the prior art and produces beneficial effects, which are mainly manifested in:

[0061] 1. By combining the two, the core chemical structures are retained to obtain a new composite (i.e., the compound of the present invention), which improves the stability of the original artesunate-ligustrazine compound in the same solvent system and provides a good foundation for the development of new antimalarial drugs.

[0062] 2. The complex of the present invention has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range, which is comparable to the effect of artesunate, the first-line drug for cerebral malaria recommended by the WHO.

[0063] 3. The combination of the present invention has an outstanding protective effect on the mouse cerebral malaria model, and is superior to artesunate in improving mouse survival rate, neurobehavioral scores, and inhibiting relapse. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 is the effect of drugs on the survival curve of mice with cerebral malaria,

[0065] ***p<0.001, indicating statistical difference compared with the Model group; ###p<0.001, indicating statistical difference compared with the AS group.

[0066] Figure 2 The effect of drugs on the RMCBS score of mice with cerebral malaria model.

[0067] * indicates comparison with the M group, # indicates comparison with the AS group, *** and ### indicate significant P < 0.001, ** and ## indicate significant P < 0.01.

[0068] Figure 3 The effect of drugs on the body temperature of mice with cerebral malaria model.

[0069] * indicates comparison with the M group, # indicates comparison with the AS group, *** and ### indicate significant P<0.001, and ** indicates significant P<0.01.

[0070] Figure 4 The effect of drugs on the body weight of mice with cerebral malaria model.

[0071] * indicates comparison with the M group, # indicates comparison with the AS group, *** and ### indicate significant P<0.001, and ** indicates significant P<0.01.

[0072] Figure 5 Artesunazine and AS inhibit the growth of Plasmodium falciparum standard strain 3D7.

[0073] Figure 6 It is the change of the content of the complex and artesunate in the stability experiment (the drug content on day 0 is 100%).

[0074] Figure 7 It is the change of the content of ligustrazine hydrochloride in the stability experiment (the drug content on day 0 is 100%). DETAILED DESCRIPTION

[0075] The examples and preparation examples provided below further illustrate and illustrate the compounds of the present invention and their preparation methods. It should be understood that the following examples and preparation examples do not limit the scope of the present invention in any way. Without further elaboration, it is believed that those skilled in the art can make the best use of the present invention with the help of the foregoing description. Therefore, the examples provided below are only intended to further illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0076] Starting materials may be obtained from commercial sources or prepared by known methods or as described herein.

[0077] The structures of the compounds were determined by nuclear magnetic resonance ( 1 H-NMR, 13 C-NMR) and high resolution mass spectrometry (HRMS-MS). The H NMR spectrum was measured using an AVANCE-600MHz NMR spectrometer, the measuring solvent was deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (DMSO-d6), and TMS was used as the internal standard. The mass spectrum was measured using an Agilent 1100LC / MSD. Column chromatography used 200-300 mesh silica gel (produced by Qingdao Ocean Chemical Plant).

[0078] Example 1, Preparation of Compound Ia-1:

[0079] (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)succinate

[0080] Step 1. Preparation of intermediate Ia-1-2: 2,3,5,6-tetramethylpyrazine-1-oxide

[0081]

[0082] At 0°C, glacial acetic acid AcOH (20 mL) was slowly added to 2,3,5,6-tetramethylpyrazine (6.86 g, 50 mmol). The reaction solution was warmed to room temperature and H2O2 (30%, 20 mL) was added. The mixture was heated to 50-60°C, H2O2 (30%, 20 mL) was added continuously, and stirred for 16 h. After the reaction was completed, it was cooled to room temperature and H2O (50 mL) was added. The mixture was concentrated to about 10% of its original volume, and 50 mL of H2O was added, and repeated three times. Saturated aqueous potassium carbonate solution was added until the pH value of the mixture reached 9, and then extracted with dichloromethane 4 times. The organic phases were combined, washed with saturated brine, dried over anhydrous MgSO4, and then concentrated to obtain 6.20 g of the target compound as a white crystalline solid with a yield of 82%.

[0083] Step 2. Preparation of intermediate Ia-1-3: 3,5,6-trimethylpyrazine-2-methylol acetate

[0084] Add excess Ac2O (20mL) to 2,3,5,6-tetramethylpyrazine-1-oxide (3.01g, 19.7mmol) and react at 100°C for 16 hours. After the reaction is completed, cool to room temperature, pour the reaction solution into ice water, and adjust its pH value to 9 with K2CO3. Extract with Et2O three times, combine the organic phases, wash with saturated brine, dry with anhydrous MgSO4, and concentrate under reduced pressure to obtain a crude product, which is separated by silica gel chromatography and eluted with 60% ethyl acetate / petroleum ether to obtain 2.36g of colorless oily product with a yield of 61%.

[0085] Step 3. Preparation of intermediate Ia-1-4: (3,5,6-trimethylpyrazine-2-yl)methanol

[0086]

[0087] 5M NaOH (10 mL, 50 mmol) was added to 2.4 g of the prepared intermediate Ia-1-3, and the mixture was stirred at room temperature for 12 hours. The reaction solution was extracted three times with dichloromethane. The organic phases were combined, washed with saturated brine, dried over MgSO4, and concentrated to give the colorless oily product Ia-1-4 with a yield of 100%.

[0088] Step 4. Preparation of target product Ia-1: (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isobenzopyran-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)succinate

[0089]

[0090] The intermediate Ia-1-4 (0.242 g), Ia-1-5 (1.153 g), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.592 g), 1-hydroxybenzotriazole (0.183 g) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was washed with 20 mL of water and saturated brine in turn, dried with MgSO4, and concentrated to obtain a crude light yellow crystalline solid, which was separated by silica gel column chromatography and eluted with 50% ethyl acetate / petroleum ether to obtain 0.76 g of a colorless crystalline solid with a yield of 97%.

[0091] Example 2, Preparation of Compound Ia-2:

[0092] (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)malonate

[0093] Step 1: Preparation of intermediate Ia-2-6: dihydroartemisinin-10-α-malonic acid monoester. Dissolve intermediate Ia-1-5 (0.284 g) and N,N-dimethylaminopyridine (0.122 g) in 10 mL of dichloromethane, add malonic anhydride (0.090 g), and stir at room temperature for 6 hours. After the reaction is complete, the reaction solution is washed with 20 mL of water and saturated sodium chloride, dried with MgSO4, and concentrated to obtain 0.326 g of a light yellow crystalline solid and 0.314 g of the pure product after recrystallization, with a yield of 85%.

[0094] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0095] Step 2. Preparation of target product Ia-2: (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isobenzopyran-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)malonate

[0096]

[0097] The intermediate Ia-1-4 (0.152 g), Ia-2-6 (0.370 g), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.229 g), 1-hydroxybenzotriazole (0.162 g) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was washed with 20 mL of water and saturated brine in turn, dried with MgSO4, and concentrated to obtain a crude light yellow crystalline solid, which was separated by silica gel column chromatography and eluted with 50% ethyl acetate / petroleum ether to obtain 0.48 g of a colorless crystalline solid with a yield of 95%.

[0098] Example 3, Preparation of Compound Ia-3:

[0099] (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)maleate

[0100] Step 1: Preparation of intermediate Ia-3-6: dihydroartemisinin-10-α-maleic acid monoester. Dissolve intermediate Ia-1-5 (0.284 g) and N,N-dimethylaminopyridine (0.122 g) in 10 mL of dichloromethane, add maleic anhydride (0.098 g), and stir at room temperature for 6 hours. After the reaction is complete, the reaction solution is washed with 20 mL of water and saturated sodium chloride, dried over MgSO4, and concentrated to obtain 0.351 g of a light yellow crystalline solid and 0.347 g of a pure product after recrystallization, with a yield of 91%.

[0101] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0102] Step 2. Preparation of target product Ia-3: (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)maleate.

[0103] The intermediate Ia-1-4 (0.152 g), Ia-2-6 (0.382 g), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.229 g), 1-hydroxybenzotriazole (0.162 g) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was washed with 20 mL of water and saturated brine in turn, dried with MgSO4, and concentrated to obtain a crude light yellow crystalline solid, which was separated by silica gel column chromatography and eluted with 50% ethyl acetate / petroleum ether to obtain 0.49 g of a colorless crystalline solid with a yield of 98%.

[0104] Example 4, Preparation of Compound Ia-4:

[0105] (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl) glutarate

[0106] Step 1: Preparation of intermediate Ia-4-6: dihydroartemisinin-10-α-glutaric acid monoester. Dissolve intermediate Ia-1-5 (0.284 g) and N,N-dimethylaminopyridine (0.122 g) in 10 mL of dichloromethane, add glutaric anhydride (0.115 g), and stir at room temperature for 6 hours. After the reaction is complete, the reaction solution is washed with 20 mL of water and saturated sodium chloride, dried over MgSO4, and concentrated to obtain 0.361 g of a light yellow crystalline solid and 0.346 g of the pure product after recrystallization, with a yield of 87%.

[0107] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0108] Step 2. Preparation of target product Ia-4: (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)pentanedioate

[0109] The intermediate Ia-1-4 (0.152 g), Ia-4-6 (0.398 g), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.229 g), and 1-hydroxybenzotriazole (0.162 g) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was washed with 20 mL of water and saturated brine in turn, dried with MgSO4, and concentrated to obtain a crude light yellow crystalline solid, which was separated by silica gel column chromatography and eluted with 50% ethyl acetate / petroleum ether to obtain 0.49 g of a colorless crystalline solid with a yield of 92%.

[0110] Example 5, Preparation of Compound Ia-5:

[0111] (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)heptanedioate

[0112] Step 1: Preparation of intermediate Ia-5-6: Dihydroartemisinin-10-α-heptanedioic acid monoester. Dissolve intermediate Ia-1-5 (0.284 g) and N,N-dimethylaminopyridine (0.122 g) in 10 mL of dichloromethane, add heptane dicarboxylic anhydride (0.142 g), and stir at room temperature for 6 hours. After the reaction is complete, the reaction solution is washed with 20 mL of water and saturated sodium chloride, dried over MgSO4, and concentrated to obtain 0.384 g of a light yellow crystalline solid and 0.362 g of the pure product after recrystallization, with a yield of 85%.

[0113] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0114] Step 2. Preparation of target product Ia-5: (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)heptanedioate

[0115] The intermediate Ia-1-4 (0.152 g), Ia-5-6 (0.462 g), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.229 g), 1-hydroxybenzotriazole (0.162 g) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was washed with 20 mL of water and saturated brine in turn, dried with MgSO4, and concentrated to obtain a crude light yellow crystalline solid, which was separated by silica gel column chromatography and eluted with 50% ethyl acetate / petroleum ether to obtain 0.526 g of a colorless crystalline solid with a yield of 94%.

[0116] Example 6, Preparation of Compound Ia-6:

[0117] (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl) adipate

[0118] Step 1: Preparation of intermediate Ia-6-6: dihydroartemisinin-10-α-adipic acid monoester. Dissolve intermediate Ia-1-5 (0.284 g) and N,N-dimethylaminopyridine (0.122 g) in 10 mL of dichloromethane, add adipic anhydride (0.129 g), and stir at room temperature for 6 hours. After the reaction is complete, the reaction solution is washed with 20 mL of water and saturated sodium chloride, dried over MgSO4, and concentrated to obtain 0.391 g of a light yellow crystalline solid and 0.367 g of the pure product after recrystallization, with a yield of 89%.

[0119] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0120] Step 2. Preparation of target product Ia-6: (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isobenzopyran-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl) adipate

[0121] The intermediate Ia-1-4 (0.152 g), Ia-6-6 (0.412 g), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.229 g), and 1-hydroxybenzotriazole (0.162 g) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was washed with 20 mL of water and saturated brine in turn, dried with MgSO4, and concentrated to obtain a crude light yellow crystalline solid, which was separated by silica gel column chromatography and eluted with 50% ethyl acetate / petroleum ether to obtain 0.496 g of a colorless crystalline solid with a yield of 91%.

[0122] Example 7, Preparation of Compound Ia-7:

[0123] (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl) suberate

[0124] Step 1: Preparation of intermediate Ia-7-6: Dihydroartemisinin-10-α-suberate. Dissolve intermediate Ia-1-5 (0.284 g) and N,N-dimethylaminopyridine (0.122 g) in 10 mL of dichloromethane, add suberic anhydride (0.156 g), and stir at room temperature for 6 hours. After the reaction is complete, the reaction solution is washed with 20 mL of water and saturated sodium chloride, dried over MgSO4, and concentrated to obtain 0.412 g of a light yellow crystalline solid and 0.387 g of the pure product after recrystallization, with a yield of 88%.

[0125] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0126] Step 2. Preparation of target product Ia-7: (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl) suberate

[0127] The intermediate Ia-1-4 (0.152 g), Ia-7-6 (0.440 g), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.229 g), 1-hydroxybenzotriazole (0.162 g) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was washed with 20 mL of water and saturated brine in turn, dried with MgSO4, and concentrated to obtain a crude light yellow crystalline solid, which was separated by silica gel column chromatography and eluted with 50% ethyl acetate / petroleum ether to obtain 0.545 g of a colorless crystalline solid with a yield of 95%.

[0128] Example 8, Preparation of Compound Ia-8:

[0129] 2,3,5-Trimethyl-6-[(2-(((3R,5aS,6R,9R,12R,12aR)-3,6,9-trimethylbicyclo[3.2.1]heptane-12H-3,12-epoxy[1,2]dioxepane[4,3-i]isoorientin-10-yl)oxy)ethoxy)methyl]pyrazine

[0130]

[0131] Step 1: Preparation of intermediate Ia-8-6: dihydroartemisinin-10-α-pimelic acid monoester:

[0132] The intermediate Ia-1-5 (0.284 g) and ethylene glycol (0.062 g) were dissolved in 10 mL of dichloromethane, K2CO3 was added, and the mixture was stirred at room temperature for 12 hours. After the reaction was complete, the reaction solution was washed with 20 mL of water and saturated sodium chloride, dried with MgSO4, and concentrated to obtain 0.210 g of a white solid. After recrystallization, 0.187 g of the pure product was obtained, with a yield of 57%.

[0133] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0134] Step 2. Preparation of target product Ia-8: (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)heptanedioate;

[0135] The intermediate Ia-1-4 (0.152 g), Ia-8-6 (0.328 g), K2CO3 (0.229 g) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was washed with 20 mL of water and saturated brine, dried with MgSO4, and concentrated to obtain a crude light yellow crystalline solid, which was separated by silica gel column chromatography and eluted with 50% ethyl acetate / petroleum ether to obtain 0.282 g of a colorless crystalline solid with a yield of 61%.

[0136] Example 9, Preparation of Compound Ia-9:

[0137] (3,5,6-Trimethylpyrazin-2-yl)methyl 1-(2-oxy-2-(((3R,5aS,6R,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepan-10-yl)oxy)ethyl)piperidine-4-carboxylate

[0138]

[0139] Step 1: Preparation of intermediate Ia-9-6: dihydroartemisinin-10-α-1-(carboxymethyl)-4-piperidinic acid monoester. Dissolve intermediate Ia-1-5 (0.284 g), 1-(carboxymethyl)-4-piperidinic acid (0.187 g), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.229 g), and 1-hydroxybenzotriazole (0.162 g) in 20 mL of dichloromethane, and stir at room temperature for 6 hours. After the reaction is complete, the reaction solution is washed with 20 mL of water and saturated sodium chloride in turn, dried over MgSO4, and concentrated to obtain 0.399 g of a light yellow crystalline solid and 0.385 g of a pure product after recrystallization, with a yield of 85%.

[0140] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0141] Step 2. Preparation of target product Ia-9: (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)suberate. Dissolve the intermediate Ia-1-4 (0.152 g), Ia-9-6 (0.453 g), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.229 g) and 1-hydroxybenzotriazole (0.162 g) in 20 mL of dichloromethane and stir at room temperature for 2 hours. After the reaction was completed, the reaction solution was washed with 20 mL of water and saturated brine, dried over MgSO4, and concentrated to obtain a crude light yellow crystalline solid. The crude product was separated by silica gel column chromatography and eluted with 50% ethyl acetate / petroleum ether to obtain 0.511 g of a colorless crystalline solid with a yield of 87%.

[0142] Example 10. Preparation of Compound Ib-1 (3R,5aS,6R,9R,10R,12R,12aR)-3,6,9-trimethyldecahydro-12h-3,12-epoxy[1,2]dioxypyrano[4,3-i]isochromene-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)ether

[0143] Step 1:

[0144] Preparation of intermediate Ib-1-1:

[0145] Dihydroartemisinin (2g, 7.034mmol, 1equiv) and pyridine (2.78g, 35.170mmol, 5equiv) were dissolved in DCM (20mL), and benzoyl chloride (1.19g, 8.441mmol, 1.2equiv) was slowly added dropwise at 0°C. After the addition was complete, the reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, 20mL of water was added to quench the reaction, the reaction solution was extracted with DCM, the organic phases were combined, and washed with saturated NaHCO3 solution, and the organic phase was dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to obtain 2g of white crystals of intermediate Ib-1-1 with a yield of 73%.

[0146] Preparation of intermediate Ib-1-2:

[0147] The intermediate (2 g, 5.149 mmol, 1 equiv) was dissolved in DCM (20 mL), and zinc chloride (0.70 g, 5.149 mmol, 1 equiv) and 4A-MS (1.14 g, 0.676 mmol, 0.13 equiv) were added in sequence at 0 ° C. After the addition was completed, the reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, the resulting mixture was diluted with DCM, washed with 5% citric acid aqueous solution, saturated NaHCO3 aqueous solution and brine. The organic phase was separated, dried over anhydrous Na2SO4, and concentrated under reduced pressure to evaporate the solvent to obtain a crude product, which was purified by silica gel column chromatography to obtain 1.06 g of intermediate Ib-1-2 as a colorless oil with a yield of 68%.

[0148] Preparation of intermediate Ib-1-3:

[0149] The intermediate Ib-1-2 (900 mg, 2.918 mmol, 1 equiv) was dissolved in 15 mL THF, cooled to -20 ° C, and 5 mL of THF solution of borane-dimethyl sulfide complex (265.99 mg, 3.502 mmol, 1.2 equiv, 1 M) was slowly added dropwise. After the addition was complete, the temperature was raised to room temperature and stirred for 2 hours. After the reaction was completed, the reaction was quenched with saturated Na2CO3 solution (10 ml), and then 30% H2O2 solution (5 ml) was added and stirred at room temperature for 30 min. After the reaction was completed, the solvent was removed in vacuo. The residue was extracted with DCM and washed with water and saturated brine. The organic phase was dried over Na2SO4, and the solvent was evaporated to obtain a crude product, and the intermediate Ib-1-3 was a brown solid 960 mg, which was used directly without separation.

[0150] Preparation of Compound Ib-1

[0151] Ib-1-3 (960 mg, crude product, 1 equiv) was dissolved in 10 mL THF, NaH (85 mg, 3.529 mmol, 1.2 equiv) was added at 0 ° C, and the mixture was stirred for 30 minutes. The intermediate Ib-1-4 (760 mg, 3.529 mmol, 1.2 equiv) was added, and the mixture was warmed to room temperature and stirred for 2 hours. After the reaction was completed, water was added to quench the reaction, and the reaction solution was extracted with DCM. The organic phases were combined, washed with water and saturated brine, and concentrated under reduced pressure to evaporate the solvent to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain 720 mg of compound Ib-1 as a colorless oil, and the total yield of the previous two steps was 53%.

[0152] Example 11: The following test data further illustrate the beneficial effects of the present invention.

[0153] Artesunazine (Example 1a-1) in vitro and in vivo antimalarial activity test study

[0154] 1. Artesunazine protects mice from infection with Plasmodium berghei ANKA strain

[0155] 1. Experimental materials:

[0156] 1. Animals: C57BL / 6J, body weight 16-18g, 10 animals per group, 50 animals in total.

[0157] 2. Plasmodium strains: Plasmodium berghei ANKA strain, kindly donated by Professor Zhao Ya of the Air Force Medical University (Fourth Military Medical University) of the Chinese People's Liberation Army, was blood-transmitted or cryopreserved in our laboratory.

[0158] 3. Drugs and preparation methods: Artesunazine, a compound, and artesunazine, a positive control drug, were dissolved in a mixed solvent of polyethylene glycol 400 and water (PEG400: water = 4:1) and prepared before use.

[0159] (II) Experimental methods

[0160] 1. Recovery and passage of malarial parasites: Take out the malarial parasite Plasmodium berghei ANKA strain frozen in liquid nitrogen, place it in a 37℃ water bath, wait for it to thaw, and immediately inoculate it intraperitoneally into C57BL / 6J mice (0.2mL / mouse). When the infection rate of the breeder mice reaches 15-30%, remove the eyeballs and collect blood in an anticoagulant tube. After shaking well, use a 1mL syringe to pass the blood to the offspring C57BL / 6J mice at a rate of 0.2mL / mouse.

[0161] 2. Plasmodium inoculation: C57BL / 6J mice were weighed and fed adaptively. When the infection rate of offspring C57BL / 6J mice reached 15-30%, the total number of red blood cells and the number of red blood cells infected with Plasmodium (pRBC) were counted after removing the eyeballs and taking blood. 0.2 mL (containing 1×10 7 pRBC) were intraperitoneally injected into C57BL / 6J mice in the model group, which was the inoculation day 0 (D0).

[0162] 3. Animal grouping: The experimental mice were randomly divided into 5 groups after inoculation, including the model group (M), high-, medium-, and low-dose groups of the combination (Artesunazine 40 mg / kg, 20 mg / kg, and 10 mg / kg), and artesunate group (AS, 15.6 mg / kg, clinical equivalent dose). The doses of the medium-dose combination group (Artesunazine 20 mg / kg) and the artesunate group (15.6 mg / kg) were equivalent. There were 10 mice in each group, for a total of 50 mice.

[0163] 4. Survival rate determination

[0164] Starting from the 0th day of inoculation, the mortality of mice was observed every day for 28 consecutive days.

[0165]

[0166] 5. Determination of re-ignition rate

[0167] After inoculation, the mice were randomly divided into groups and nasal drops were administered 3 hours later. After four days of continuous administration, blood smears were measured on D4. A small drop of blood was taken from the tip of the mouse tail and dropped on a glass slide. The blood drop was pushed to one end at a certain angle with the edge of another glass slide to form a thin blood film of appropriate thickness. After the thin blood film was air-dried, it was fixed with methanol and left to air-dry completely. After 20 minutes of staining with Giemsa stain at a dilution of 1:9 (completely covering the fixed thin blood film), it was placed under the tap for rinsing and the surface moisture was quickly absorbed with filter paper. A drop of cedar oil was dropped on the glass slide, and the number of infected red blood cells contained in 1000 red blood cells was counted under a 100x oil microscope. The mouse relapse rate was calculated by the ratio of the number of relapsed mice to the total number of mice.

[0168]

[0169] 6. RMCBS score determination

[0170] The rapid coma and behavior scale (RMCBS) of mice was used to evaluate the neurological function of experimental mice. Starting from D0, the mice were scored from 10 aspects: gait, balance, movement-related behavior, body position, limb strength, touch reaction, auricle reflex, toe touch reaction, offensive reaction, offensive behavior, and hygiene behavior. Normal mice have smooth fur, stable gait, stretch their bodies when walking, can explore the four corners of the grid within 15 seconds, and have obvious exploration behavior, touch behavior, auricle reaction, toe reaction, touch reflex, etc., while the model mice have less exploration behavior within 120 seconds, messy fur, ataxia, and arched back. When the model mice are stimulated, their reflexes are reduced or disappeared. The mice are scored according to their performance, with each score ranging from 0 to 2 points, for a total of 20 points. The tests were performed at a fixed time and in a fixed order every day. During the test, the mouse was placed from the upper left corner into a box with a grid paper bottom (length, width and height of 31.8cm×19.8cm×10.5cm). A long rod with a diameter of 3mm was used to touch the mouse. The test time for one mouse was about 3 minutes. After each test, the mark left by the previous mouse needed to be wiped off.

[0171] 7. Temperature measurement

[0172] The rectal temperature of mice was measured starting from D0 of inoculation and observed until the 14th day.

[0173] 8. Body weight measurement

[0174] The body weight of mice was measured starting from the inoculation D0 and observed until the 14th day.

[0175] (III) Experimental results

[0176] 1. Effect of the complex on the survival rate of mice with cerebral malaria model

[0177] The survival rates of the groups were compared after the administration for 28 days. 15 All died. Each drug-treated group could significantly prolong the survival time of mice with cerebral malaria. The survival rate of the high-dose (Artesunazine 40 mg / kg) group of the compound was 100%, the survival rate of the medium-dose (Artesunazine 20 mg / kg) group was 90%, the survival rate of the low-dose (Artesunazine 10 mg / kg) group was 50%, and the survival rate of the artesunate (AS) group was 40%. The therapeutic effect of the high- and medium-dose groups of the compound on mice with cerebral malaria was better than that of the artesunate group (P<0.001).

[0178] 2. Effect of the complex on relapse of cerebral malaria model in mice

[0179] After the end of the administration, the relapse rates of each group were compared after observation for 28 days. The results showed that the infection rate of the model group was 100% on D4. After 4 days of administration, Artesunazine 40 mg / kg and Artesunazine 20 mg / kg groups could completely kill malarial parasites, and Artesunazine 10 mg / kg and AS groups had 10% relapses. On D7, there was no relapse in Artesunazine 40 mg / kg and Artesunazine 20 mg / kg groups, and the relapse rates of Artesunazine 10 mg / kg and AS groups were 80% and 90%, respectively. After D28, the relapse rates of Artesunazine 40 mg / kg and Artesunazine 20 mg / kg were 10%, and the relapse rates of Artesunazine 10 mg / kg and AS groups were 100%. The high and medium dose Artesunazine groups were better than the AS group in inhibiting the relapse of malarial parasites.

[0180] Table 1 Effects of drugs on relapse of cerebral malaria model mice

[0181]

[0182] 3. Effects of the complex on neurological function in mice with cerebral malaria model (RMCBS score)

[0183] The RMCBS scoring method, a classic neurological function evaluation method for cerebral malaria, was used for experimental observation. The results showed that the RMCBS score of the model group began to gradually decrease on D6 of inoculation, and by the end of observation, the score dropped to 14.00±0.26; during the observation period, the RMCBS scores of Artesunazine 40 mg / kg and Artesunazine 20 mg / kg did not decrease, and both remained stable at around 20 points; the RMCBS scores of the AS group and Artesunazine 10 mg / kg group gradually decreased on D9 and D10 of inoculation, and by the end of observation, the RMCBS scores dropped to 16.22±0.55 and 18.40±0.48, respectively. At the end of observation, the RMCBS scores of the combination groups were significantly higher than those of the model group (P<0.001), and the RMCBS scores of the AS group were significantly higher than those of the model group (P<0.01); the RMCBS scores of the high, medium, and low dose Artesunazine groups were significantly higher than those of the AS group (P<0.001, P<0.001, P<0.01). The combination groups and the AS group can effectively improve the behavioral and neurological pathological changes of malaria mice in the late stage, such as decreased exploration desire, messy fur, ataxia, arched back, weak limbs, reduced or absent reflexes, and the overall improvement of the behavior of mice in the combination groups is better than that in the AS group.

[0184] 4. Effects of the complex on body temperature of mice with cerebral malaria model

[0185] During the observation period, the body temperature of the mice in the model group decreased, and at the end of observation, the body temperature dropped to 36.82±0.42. The body temperatures of the mice in the Artesunazine10 mg / kg, Artesunazine20 mg / kg, Artesunazine40 mg / kg groups, and AS group were significantly decreased (P<0.001, P<0.001, P<0.001, P<0.01). Each drug-treated group could significantly inhibit the abnormal decrease in body temperature of the cerebral malaria model mice, and Artesunazine20 mg / kg and Artesunazine40 mg / kg had better improvements on the body temperature of cerebral malaria mice than the AS group (P<0.001, P<0.001).

[0186] 5. Effect of the complex on the body weight of mice with cerebral malaria model

[0187] In the first 6 days of the observation period, the weight of mice in each group showed a trend of gradual increase, and the weight gain of mice in each group was basically the same. The weight of mice in the model group began to decrease on D6, and at the end of observation, their weight dropped to 13.15±0.29. The weight of the Artesunazine 10 mg / kg group and the AS group began to gradually decrease on D7 and D9 days of inoculation, respectively, and at the end of observation, their weight dropped to 16.66±0.45 and 15.41±0.50, respectively. The weight of mice in the Artesunazine 40 mg / kg and Artesunazine 20 mg / kg groups did not decrease significantly during the entire observation period. All drug-treated groups could significantly improve the weight loss of mice with cerebral malaria (P<0.001, P<0.001, P<0.001, P<0.01), and the Artesunazine 40 mg / kg and Artesunazine 20 mg / kg groups had better improvement on the weight loss of mice with cerebral malaria than the AS group (P<0.001, P<0.001).

[0188] 2. Pharmacodynamic study of the combination in treating Plasmodium falciparum cultured in vitro

[0189] 1. Experimental Materials

[0190] Experimental drugs and reagents: bovine serum albumin Plasmodium falciparum complete medium (see medium preparation for details). 0.16% saponin (Sigma, LOT: BCBR4223V): saponin 0.016g, add 10ml deionized water, mix well, prepare before use. 0.1M EDTA (Sigma, LOT: BCBR3854V): EDTA (MW: 292.24g / mol) 0.5844g, add 20ml deionized water, mix well. 0.4M Tris-Base (Sigma, LOT: SLBR9609V): Tris-Base (MW = 121.14g / mol) 0.969g, add 20ml deionized water, mix well. 0.8% Triton X-100 (Sigma, LOT: SLBR3411V): Triton X-100 liquid 80μl, add 9.92ml deionized water, mix well. Lysis solution: 2 ml each of 0.16% saponin, 0.1 M EDTA, 0.4 M Tris-Base, 0.8% TritonX-100 and deionized water, 10 μl of SYBR Green I (Invitrogen, LOT: 1797921), mix well. Experimental equipment: incubator, fluorescence microplate reader, biosafety cabinet, centrifuge, drying oven, microscope, pressure cooker. Experimental consumables: pipettes (1000, 200, 100, 10, 2.5 μl) and tips, 15 ml centrifuge tubes, slides, culture dishes

[0191] (II) Experimental methods

[0192] Synchronize the ring stage Plasmodium falciparum. Smear and count the infection rate. Rationally match the synchronized infected red blood cells, healthy red blood cells and culture medium to obtain a Plasmodium in vitro culture system with a hematocrit of 2% and an infection rate of 1% (ring body, worm age 2-4 hours). RPMI 1640 is plated (50μl / well). A1-A10 is plated with 25μl of drug (8 duplicate wells for each drug), mixed and then diluted in multiples (Ax-Hx). Except A12-H12, 50μl of Plasmodium culture medium is plated in other wells, and A12-H12 is plated with 50μl of healthy red blood cells (hematocrit is 2% culture medium). Under conventional in vitro culture conditions for Plasmodium falciparum (5% O2, 3% CO2, 92% N2, 37°C), culture for 72 hours. Add 100μl of lysis solution to each well and place in a drawer away from light for 1 hour. Lysis solution (final concentration): 40 mM Tris, pH 7.5; 10 mM EDTA; 0.016% saponin (fresh); 0.08% Triton X-100; Sybr Green I (Invitrogen 10000×) 1000-fold dilution. Fluorescence microplate reader determination: Ex = 485 nm; Em = 525 nm.

[0193] In this experiment, the SYBR Green assay was used to determine the half-maximal inhibitory concentration (IC50) value of the drug for the treatment of Plasmodium falciparum 3D7, and the values ​​were calculated by nonlinear regression using Graphpad Prism 8.0 software (values ​​are mean ± standard error). At the same time, a four-parameter logistic fitting curve was used, and each value in the curve was the mean ± standard deviation of eight biological replicates.

[0194] (III) Experimental results

[0195] (1) Analysis of the efficacy of compound Ia-1 in treating human Plasmodium falciparum standard strain 3D7

[0196] Compound Ia-1 showed a strong inhibitory effect on the proliferation of parasites in the nanomolar concentration range (IC50 = 7.358nM), indicating that the compound has a strong inhibitory effect on Plasmodium although it is comparable to standard antimalarial drugs (such as chloroquine (about 2.360nM), mefloquine (about 3.962nM) and artemisinin (about 11.768nm)). At the same time, the control drug artesunate has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7, with an IC50 value of 8.825nM (such as Figure 5 ).

[0197] (2) Analysis of the efficacy of compound Ia-2 in treating human Plasmodium falciparum standard strain 3D7

[0198] Compound Ia-2 has a significant inhibitory effect on the proliferation of parasites in the nanomolar concentration range (IC50=12.52nM).

[0199] (3) Analysis of the efficacy of compound Ia-3 in treating human Plasmodium falciparum standard strain 3D7

[0200] Compound Ia-3 has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range (IC50=6.863nM).

[0201] (4) Analysis of the efficacy of compound Ia-4 in treating human Plasmodium falciparum standard strain 3D7

[0202] Compound Ia-4 has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range (IC50=33.92nM).

[0203] (5) Analysis of the efficacy of compound Ia-5 in treating human Plasmodium falciparum standard strain 3D7

[0204] Compound Ia-5 has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range (IC50=2.953nM).

[0205] (6) Analysis of the efficacy of compound Ia-6 in treating human Plasmodium falciparum standard strain 3D7

[0206] Compound Ia-6 has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range (IC50=14.29nM).

[0207] (7) Analysis of the efficacy of compound Ia-7 in treating human Plasmodium falciparum standard strain 3D7

[0208] Compound Ia-7 has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range (IC50=50.68nM).

[0209] (8) Analysis of the efficacy of compound Ia-8 in treating human Plasmodium falciparum standard strain 3D7

[0210] Compound Ia-8 has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range (IC50=107.2nM).

[0211] (9) Analysis of the efficacy of compound Ia-9 in treating human Plasmodium falciparum standard strain 3D7

[0212] Compound Ia-9 has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range (IC50=32.73nM).

[0213] In summary, compounds Ia-2 to Ia-9 all have significant inhibitory effects on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range, and their efficacy is comparable to that of Ia-1.

[0214] 3. Study on the stability of the composite

[0215] 1. Experimental methods

[0216] 1. Study on the stability of the complex

[0217] 160.2 mg of the complex was weighed into a vial, 8 mL of 80% polyethylene glycol 400 solution was added, and the complex was completely dissolved by ultrasonic treatment to obtain a complex solution of about 20.25 mg / mL; the vial containing the solution was then sealed, covered with aluminum foil to avoid light, and placed in a 40°C stability test chamber; 0.5 mL of the sample was taken into a 25 mL volumetric flask on the 0th day, 3rd day, 7th day and 10th day, respectively, and the content of the complex was determined by UPLC after dilution with methanol and shaking.

[0218] The UPLC assay conditions for the complex are as follows: Instrument: Waters ACQUITY UPLC ultra-high performance liquid chromatograph, chromatographic column: ACQUITY UPLC HSS T3 Column ( 1.8μm, 2.1mm×100mm), detection wavelength: 295nm, mobile phase: acetonitrile-0.1% phosphoric acid water (35:65), flow rate: 0.3ml / min, column temperature: 30℃, sample chamber temperature: 25℃, injection volume: 3μL.

[0219] 2. Study on the stability of artesunate and ligustrazine hydrochloride in the same solution

[0220] According to the molar concentration of the complex of 20 mg / mL, a mixed solution of artesunate and ligustrazine hydrochloride was prepared, specifically as follows: 148.5 mg of artesunate and 66.2 mg of ligustrazine hydrochloride were weighed into the same vial, 10 mL of 80% polyethylene glycol 400 aqueous solution was added and ultrasonicated until completely dissolved to obtain a mixed solution with a concentration of 14.85 mg / mL artesunate and 6.62 mg / mL ligustrazine hydrochloride; the vial containing the solution was sealed, covered with aluminum foil to avoid light, and placed in a 40°C stability test chamber; 0.5 ml of the sample was taken into a 25 mL volumetric flask on the 0th day, 3rd day, 7th day and 10th day, respectively, and the content of the complex was determined by UPLC method after dilution with methanol and shaking.

[0221] (1) The conditions for UPLC determination of artesunate are as follows: Instrument: Waters ACQUITY UPLC ultra-high performance liquid chromatograph, chromatographic column: ACQUITY UPLC HSS T3 Column ( 1.8μm, 2.1mm×100mm), detection wavelength: 216nm, mobile phase: acetonitrile-0.1% phosphoric acid water (44:56), flow rate: 0.3ml / min, column temperature: 30℃, sample chamber temperature: 25℃, injection volume: 4μL.

[0222] (2) The UPLC determination conditions of ligustrazine hydrochloride are as follows: Instrument: Waters ACQUITY UPLC ultra-high performance liquid chromatograph, chromatographic column: ACQUITY UPLC HSS T3 Column ( 1.8μm, 2.1mm×100mm), detection wavelength: 295nm, mobile phase: acetonitrile-0.1% phosphoric acid water (10:90), flow rate: 0.3ml / min, column temperature: 30℃, sample chamber temperature: 25℃, injection volume: 1μL.

[0223] (II) Experimental results

[0224] The stability of the artesunate-ligustrazine hydrochloride compound in 80% polyethylene glycol 400 solution is poor. The content of artesunate decreases rapidly in a light-proof environment at 40°C, and the color of the mixture solution gradually turns yellow. In contrast, the stability of the composite in 80% polyethylene glycol 400 solution is significantly improved, and the color of the composite solution remains basically unchanged. This result provides a good basis for the development of new antimalarial drugs ( Figure 6 and Figure 7 ).

Claims

1. The drug represented by general formula I or a pharmaceutically acceptable salt thereof: in, X, Y are each selected from CH2, O, S, NH, L is a linker arm selected from: Here, n is an integer from 0 to 6.

2. The drug according to claim 1 or a pharmaceutically acceptable salt thereof, having the following structure: in, n is an integer from 0 to 6.

3. The drug according to claim 1 or a pharmaceutically acceptable salt thereof, having the following structure:

4. The drug according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein: The pharmaceutically acceptable salts are addition salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalene disulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, etc. Hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, maleic acid, benzenesulfonic acid, succinic acid.

5. A pharmaceutical composition comprising the drug according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition according to claim 5 is in the form of a pharmaceutical preparation and may further contain auxiliary materials required for the preparation as required.

7. The pharmaceutical composition of claim 5, selected from any edible pharmaceutical dosage form.

8. The pharmaceutical composition according to claim 7, selected from nasal preparations, injections, and oral preparations.

9. A method for preparing the drug or a pharmaceutically acceptable salt thereof according to claim 2, comprising the following steps: or 。 10. Use of the drug according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing severe malaria.

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