Medication for treating severe malaria and synthesis method therefor

Abstract

The present invention relates to a medication for treating severe malaria and a synthesis method therefor. The medication has a remarkable treatment effect on severe malaria (cerebral malaria). Compared with existing first-line treatment medication artesunate against severe malaria (cerebral malaria), the medication can effectively eliminate plasmodium, reduce mortality, relieve nerve damage, and effectively reduce the malaria reinfection rate. The chemical structure of the medication is shown as a formula (I).

Description

A medicine for treating severe malaria and its synthesis method Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and relates to a class of artemisinin / ligustrazine complexes, as well as pharmaceutically acceptable salts, hydrates, and optical isomers thereof, pharmaceutical compositions containing the complexes as active ingredients, and their use in treating severe malaria. This invention, targeting severe malaria and its often associated pathological features such as neurological damage, utilizes the principles of medicinal chemistry to design and synthesize a class of novel artemisinin / ligustrazine complexes. Their chemical structures have been confirmed 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 among 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 of age, with a mortality rate of 15-20% (World Health Organization, 2016; de Miranda AS, 2010). Cerebral malaria presents with acute diffuse encephalitis, accompanied by various neurological symptoms. Clinically, coma, impaired consciousness, meningeal irritation, hemiplegia, convulsions, ataxia, and even death may occur. Among surviving children, 10-17% experience persistent neurological deficits, cognitive impairment, and motor dysfunction (Birbeck GL, 2010; de Miranda AS, 2010). In children under five years of age, motor, language, and social development can be delayed for more than six months, with even more significant delays in those with significant MRI abnormalities.

[0004] The "mechanical obstruction" theory of cerebral malaria holds that the adhesion and blockage of parasitoid-infected red blood cells (pRBCs) in 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 hemorrhage, 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), also known as dihydroartemisinin-12-α-succinate, has good solubility and bioavailability. It is water-soluble and can penetrate the blood-brain barrier. It can be administered via multiple routes, including intravenous, intramuscular, oral, and rectal. Artesunate can maintain high concentrations in the nervous system and is highly effective and low in toxicity. Intravenous artesunate is the preferred treatment for cerebral malaria recommended in the WHO's "Practical Manual for the Management of Severe Malaria." As the current first-line treatment, it holds over 90% of the global market share for severe malaria. Growing evidence indicates that artesunate has multiple pharmacological activities, including anti-inflammatory, antioxidant, blood-brain barrier protection, immunomodulatory, antibacterial, and anti-tumor activities.

[0007] The clinical treatment of cerebral malaria with artesunate presents the following challenges: ① According to the WHO's "Practical Manual for the Management of Severe Malaria" (3rd edition), the recommended dosage of artesunate for cerebral malaria has been significantly increased due to factors such as decreased susceptibility of Plasmodium falciparum to artemisinin-based drugs. The first-day treatment dose for adults has increased from 120 mg to 432 mg, and the full 7-day treatment dose has increased from 480 mg to 1296 mg. This has led to increased costs, the risk of drug resistance when used alone, and the risk of side effects such as hemolysis. ② Artesunate is less effective in late-stage cerebral malaria, when the host has already developed inflammatory responses and blood-brain barrier disruption. ③ After artesunate kills the parasites, they remain locally occluded in the brain's microvasculature, failing to effectively alleviate the ischemic and hypoxic state. ④ Children who survive cerebral malaria experience persistent neurological deficits and long-term cognitive and motor impairments, which artesunate is not effective in addressing. ⑤ In clinical practice, intravenous artesunate is used to treat severe malaria, including cerebral malaria. In areas of Africa with a high incidence of cerebral malaria, extremely poor medical conditions significantly limit the timely and effective use of artesunate. ⑥ Infants and young children under five years of age, a high-risk population, have poor compliance with intravenous artesunate.

[0008] Tetramethylpyrazine (TMP), considered the characteristic alkaloid of Ligusticum chuanxiong, has been confirmed in multiple studies to play a role in the acute injury phase of ischemic stroke. For example, it has a protective effect against brain damage caused by reperfusion after ischemic stroke, effectively improving behavioral and learning and memory impairments after brain injury, and can also alleviate stroke progression and reduce mortality and disability. Studies have reported that Ligustrazine nasal spray can effectively increase the concentration of Ligusticum chuanxiong in brain tissue, fully exerting its therapeutic effects.

[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 oxygen supply. Through the mutation of the nitrosylation site (cysteine ​​C263), it was determined that one of its key links is to affect the balance of the NO-related redox system by regulating PDHB nitrosylation.

[0010] However, the poor chemical compatibility between artesunate and ligustrazine presents difficulties in the development of compound antimalarial formulations, hindering their drugability. Chemically, artesunate is a monoester of dihydroartemisinin and succinic acid, with a free carboxyl group in the molecule. To improve its water solubility, its sodium salt, artesunate sodium, is clinically used as an injectable formulation. Ligustrazine is a poorly water-soluble oily compound. Similarly, to improve its water solubility and stability, its hydrochloride, ligustrazine hydrochloride, is used clinically. When ligustrazine and artesunate are co-dissolved in aqueous or organic solvent systems, the ester bond of artesunate is rapidly hydrolyzed, resulting in a rapid decrease in its content. However, when co-dissolved in alkaline aqueous solvents, a vigorous acid-base neutralization reaction occurs, immediately precipitating artesunate and producing an insoluble ligustrazine oil, preventing the formation of a uniform and stable formulation. To solve this problem, different buffer systems and nanoformulation technology have been tried, but none of them have been able to effectively solve this problem.

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

[0012] Summary of the Invention

[0013] The present invention aims 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.

[0014] To this end, the present invention provides a drug represented by general formula I or a pharmaceutically acceptable salt thereof:

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

[0016] L is a linker arm selected from:

[0017] Here, n is an integer from 0 to 6.

[0018] Preferably, the drug of the present invention or a pharmaceutically acceptable salt thereof has the following structure:

[0019] Here, n is an integer from 0 to 6.

[0020] Most preferably, the drug of the present invention or a pharmaceutically acceptable salt thereof has the following structure:

[0021] The most preferred drug of the present invention is a novel compound, referred to herein as Artesunazine, also known as Compound Ia-1, and also known as an artemisinin / ligustrazine complex. 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]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)succinate. The NMR and high-resolution mass spectrometry data for this 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 38 N2O8+H]+519.2628,found 519.2487.

[0022] Compound Ia-2, chemically named (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. The NMR and high-resolution mass spectrometry data for this 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, found 505.2547.

[0023] 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]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)maleate. The NMR and high-resolution mass spectrometry data of this 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, found 517.2550.

[0024] Compound Ia-4 and 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]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl) glutarate. The NMR and high-resolution mass spectrometry data of the compound are as follows: H 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.

[0025] HRMS(ESI)calcd for[C 28 H 40 N2O8+H]+533.2863, found 533.2851.

[0026] Compound Ia-5, chemically named (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)pimelate. The NMR and high-resolution mass spectrometry data for this 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.

[0027] 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]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)adipate. The NMR and high-resolution mass spectrometry data for this 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.

[0028] 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]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)suberate. The NMR and high-resolution mass spectrometry data for this 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, 148.84,144.78,91.60,91.36,80.01,64.80,51.47,45.16,37.14,36.13,34.07,31.71,28.64,28.59,25. 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.

[0029] 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-epoxy[1,2]dioxepane[4,3-i]isoorientin-10-yl)oxy)ethoxy)methyl]pyrazine. The NMR and high-resolution mass spectrometry data of this 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.

[0030] Compound Ia-9, chemical name: (3,5,6-trimethylpyrazin-2-yl)methyl 1-(2-oxyl-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. The NMR and high-resolution mass spectrometry data of this 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,found 588.3287.

[0031] 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]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)ether. The NMR and high-resolution mass spectrometry data for this compound are as follows: 1 H 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). 13C 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,found 461.2927.

[0032] The present invention further provides salts of the drugs of the present invention, which are addition salts formed between the drugs 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.

[0033] The present invention further provides a pharmaceutical composition comprising the drug described herein or a pharmaceutically acceptable salt thereof. The pharmaceutical composition is in the form of a pharmaceutical preparation and, if necessary, may also contain excipients required for the preparation. The pharmaceutical composition described herein can be selected from any edible pharmaceutical dosage form, such as a nasal preparation, an injection, or an oral preparation.

[0034] The present invention further provides a method for preparing the drug of the present invention or a pharmaceutically acceptable salt thereof.

[0035] Among them, the preferred drug of the present invention has a preparation method comprising the following steps:

[0036] in,

[0037] Step a: Compound Ia-1-1 reacts with hydrogen peroxide in the presence of glacial acetic acid to obtain compound Ia-1-2;

[0038] Step b: Compound Ia-1-2 reacts with acetic anhydride to obtain compound Ia-1-3;

[0039] Step c: Ia-1-3 is hydrolyzed in the presence of sodium hydroxide to obtain compound Ia-1-4;

[0040] Step d: Compound Ia-1-5 reacts with succinic anhydride to obtain compound Ia-1-6;

[0041] Step e: Compound Ia-1-6 and Ia-1-4 are subjected to a condensation reaction in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole to obtain a preferred drug of the present invention.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] The compound of the present invention or its pharmaceutically acceptable salt, hydrate, or prodrug can be used alone as a sole antimalarial drug, or can be used in combination with an antimalarial drug currently on the market for the treatment and prevention of severe malaria.

[0046] The present invention solves the problems in the prior art and produces beneficial effects, which are mainly manifested in:

[0047] 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.

[0048] 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 potency of artesunate, the first-line drug recommended by the WHO for cerebral malaria.

[0049] 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

[0050] Figure 1 Effects of drugs on the survival curve of mice with cerebral malaria

[0051] ***p<0.001, indicating statistical difference compared with the Model group; ###p<0.001, indicating statistical difference compared with the AS group

[0052] Figure 2 Effects of drugs on RMCBS scores in cerebral malaria model mice

[0053] * indicates comparison with the M group, # indicates comparison with the AS group, *** and ### indicate significant P < 0.001, ** and ## indicate significant P < 0.01

[0054] Figure 3 Effects of drugs on body temperature of mice with cerebral malaria model

[0055] * indicates comparison with the M group, # indicates comparison with the AS group, *** and ### indicate significant P < 0.001, and ** indicates significant P < 0.01

[0056] Figure 4 Effects of drugs on body weight of mice with cerebral malaria model

[0057] * indicates comparison with the M group, # indicates comparison with the AS group, *** and ### indicate significant P < 0.001, and ** indicates significant P < 0.01

[0058] Fig. 5 Inhibitory effects of artesunazine and AS on the growth of Plasmodium falciparum standard strain 3D7.

[0059] Figure 6: Changes in the content of the complex and artesunate during the stability experiment (drug content on day 0 is 100%)

[0060] Figure 7: Changes in the content of ligustrazine hydrochloride in the stability experiment (drug content on day 0 is 100%) DETAILED DESCRIPTION

[0061] The following examples and preparations further illustrate and illustrate the compounds of the present invention and their preparation methods. It should be understood that the following examples and preparations are not intended to limit the scope of the present invention in any way. Without further elaboration, it is believed that one skilled in the art can utilize the present invention to its fullest extent with the aid of the foregoing description. Therefore, the following examples are provided merely to further illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0062] Starting materials can be obtained from commercial sources or prepared by known methods or as described herein.

[0063] The structure of the compound was determined by nuclear magnetic resonance ( 1 H-NMR, 13 C-NMR) and high-resolution mass spectrometry (HRMS-MS). Proton nuclear magnetic resonance spectra were measured using an AVANCE-600 MHz nuclear magnetic resonance spectrometer, using deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (DMSO-d6) as the solvent and TMS as the internal standard. Mass spectra were measured using an Agilent 1100 LC / MSD. Column chromatography utilized 200-300 mesh silica gel (produced by Qingdao Ocean Chemical Plant).

[0064] Example 1. Preparation of Compound Ia-1:

[0065] (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

[0066] Step 1. Preparation of intermediate Ia-1-2: 2,3,5,6-tetramethylpyrazine-1-oxide

[0067] 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 mixture was warmed to room temperature, and H₂O₂ (30%, 20 mL) was added. The mixture was heated to 50-60°C, and H₂O₂ (30%, 20 mL) was further added. Stir for 16 h. After the reaction was complete, the mixture was cooled to room temperature, and H₂O (50 mL) was added. The mixture was concentrated to approximately 10% of its original volume, and 50 mL of H₂O was added three times. Saturated aqueous potassium carbonate was added until the mixture reached a pH of 9, and then extracted four times with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous MgSO₄, and concentrated to yield 6.20 g of the target compound as a white crystalline solid in an 82% yield.

[0068] Step 2. Preparation of Intermediate Ia-1-3: 3,5,6-Trimethylpyrazine-2-methylol acetate

[0069] Excess Ac2O (20 mL) was added to 2,3,5,6-tetramethylpyrazine-1-oxide (3.01 g, 19.7 mmol) and the reaction was allowed to proceed at 100°C for 16 hours. After completion of the reaction, the mixture was cooled to room temperature and poured into ice water. The pH was adjusted to 9 with K2CO3. The mixture was extracted three times with Et2O. The combined organic phases were washed with saturated brine, dried over anhydrous MgSO4, and concentrated under reduced pressure to obtain the crude product. The crude product was separated by silica gel chromatography using 60% ethyl acetate / petroleum ether as the eluent to afford 2.36 g of the product as a colorless oil in a 61% yield.

[0070] Step 3. Preparation of intermediate Ia-1-4: (3,5,6-trimethylpyrazin-2-yl)methanol

[0071] 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 afford the product Ia-1-4 as a colorless oil in 100% yield.

[0072] 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]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)succinate

[0073] Intermediate Ia-1-4 (0.242 g), Ia-1-5 (1.153 g), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.592 g), and 1-hydroxybenzotriazole (0.183 g) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 2 hours. After completion of the reaction, the reaction solution was washed sequentially with 20 mL of water and saturated brine, dried over MgSO₄, and concentrated to afford a crude pale yellow crystalline solid. This was separated by silica gel column chromatography using 50% ethyl acetate / petroleum ether as the eluent to afford 0.76 g of a colorless crystalline solid in a 97% yield.

[0074] Example 2, Preparation of Compound Ia-2:

[0075] (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

[0076] Step 1: Preparation of Intermediate Ia-2-6: Dihydroartemisinin-10-α-malonate

[0077] Intermediate Ia-1-5 (0.284 g) and N,N-dimethylaminopyridine (0.122 g) were dissolved in 10 mL of dichloromethane, and malonic anhydride (0.090 g) was added. The mixture was stirred at room temperature for 6 hours. After the reaction was complete, the reaction solution was washed with 20 mL of water and saturated sodium chloride, dried over MgSO4, and concentrated to obtain 0.326 g of a light yellow crystalline solid. 0.314 g of the pure product was recrystallized, with a yield of 85%.

[0078] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0079] 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]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl)malonate

[0080] Intermediate Ia-1-4 (0.152 g), Ia-2-6 (0.370 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 completion of the reaction, the reaction solution was washed sequentially with 20 mL of water and saturated brine, dried over MgSO₄, and concentrated to afford a crude pale yellow crystalline solid. This was separated by silica gel column chromatography using 50% ethyl acetate / petroleum ether as the eluent to afford 0.48 g of a colorless crystalline solid in a 95% yield.

[0081] Example 3, Preparation of Compound Ia-3:

[0082] (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

[0083] Step 1: Preparation of Intermediate Ia-3-6: Dihydroartemisinin-10-α-maleic acid monoester

[0084] Intermediate Ia-1-5 (0.284 g) and N,N-dimethylaminopyridine (0.122 g) were dissolved in 10 mL of dichloromethane, and maleic anhydride (0.098 g) was added. The mixture was stirred at room temperature for 6 hours. After the reaction was complete, the reaction solution was 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. 0.347 g of the pure product was recrystallized, with a yield of 91%.

[0085] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0086] 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.

[0087] Intermediate Ia-1-4 (0.152 g), Ia-2-6 (0.382 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 completion of the reaction, the reaction solution was washed sequentially with 20 mL of water and saturated brine, dried over MgSO₄, and concentrated to afford a crude pale yellow crystalline solid. This was separated by silica gel column chromatography using 50% ethyl acetate / petroleum ether as the eluent to afford 0.49 g of a colorless crystalline solid in a 98% yield.

[0088] Example 4, Preparation of Compound Ia-4:

[0089] (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

[0090] Step 1: Preparation of Intermediate Ia-4-6: Dihydroartemisinin-10-α-glutaric acid monoester

[0091] Intermediate Ia-1-5 (0.284 g) and N,N-dimethylaminopyridine (0.122 g) were dissolved in 10 mL of dichloromethane, and glutaric anhydride (0.115 g) was added. The mixture was stirred at room temperature for 6 hours. After the reaction was complete, the reaction solution was 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. 0.346 g of the pure product was recrystallized, with a yield of 87%.

[0092] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0093] 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) glutarate

[0094] 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 completion of the reaction, the reaction solution was washed sequentially with 20 mL of water and saturated brine, dried over MgSO₄, and concentrated to afford a crude pale yellow crystalline solid. This was separated by silica gel column chromatography using 50% ethyl acetate / petroleum ether as the eluent to afford 0.49 g of a colorless crystalline solid in a 92% yield.

[0095] Example 5, Preparation of Compound Ia-5:

[0096] (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)pimelate

[0097] Step 1: Preparation of Intermediate Ia-5-6: Dihydroartemisinin-10-α-pimelic acid monoester

[0098] Intermediate Ia-1-5 (0.284 g) and N,N-dimethylaminopyridine (0.122 g) were dissolved in 10 mL of dichloromethane, and pimelic anhydride (0.142 g) was added. The mixture was stirred at room temperature for 6 hours. After the reaction was complete, the reaction solution was 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. 0.362 g of the pure product was recrystallized, with a yield of 85%.

[0099] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0100] 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)pimelate

[0101] Intermediate Ia-1-4 (0.152 g), Ia-5-6 (0.462 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 completion of the reaction, the reaction solution was washed sequentially with 20 mL of water and saturated brine, dried over MgSO₄, and concentrated to afford a crude pale yellow crystalline solid. This was separated by silica gel column chromatography using 50% ethyl acetate / petroleum ether as the eluent to afford 0.526 g of a colorless crystalline solid in a 94% yield.

[0102] Example 6, Preparation of Compound Ia-6:

[0103] (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

[0104] Step 1: Preparation of intermediate Ia-6-6: dihydroartemisinin-10-α-adipate monoester

[0105] Intermediate Ia-1-5 (0.284 g) and N,N-dimethylaminopyridine (0.122 g) were dissolved in 10 mL of dichloromethane, and adipic anhydride (0.129 g) was added. The mixture was stirred at room temperature for 6 hours. After the reaction was complete, the reaction solution was 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. The pure product was recrystallized to obtain 0.367 g, with a yield of 89%.

[0106] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0107] 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]isochromen-10-yl((3,5,6-trimethylpyrazin-2-yl)methyl) adipate

[0108] 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 completion of the reaction, the reaction solution was washed sequentially with 20 mL of water and saturated brine, dried over MgSO₄, and concentrated to afford a crude pale yellow crystalline solid. This was separated by silica gel column chromatography using 50% ethyl acetate / petroleum ether as the eluent to afford 0.496 g of a colorless crystalline solid in a 91% yield.

[0109] Example 7, Preparation of Compound Ia-7:

[0110] (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

[0111] Step 1: Preparation of intermediate Ia-7-6: dihydroartemisinin-10-α-suberate

[0112] Intermediate Ia-1-5 (0.284 g) and N,N-dimethylaminopyridine (0.122 g) were dissolved in 10 mL of dichloromethane, and suberic anhydride (0.156 g) was added. The mixture was stirred at room temperature for 6 hours. After the reaction was complete, the reaction solution was 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. The pure product was recrystallized to obtain 0.387 g, with a yield of 88%.

[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-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

[0115] Intermediate Ia-1-4 (0.152 g), Ia-7-6 (0.440 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 completion of the reaction, the reaction solution was washed sequentially with 20 mL of water and saturated brine, dried over MgSO₄, and concentrated to afford a crude pale yellow crystalline solid. This was separated by silica gel column chromatography using 50% ethyl acetate / petroleum ether as the eluent to afford 0.545 g of a colorless crystalline solid in a 95% yield.

[0116] Example 8, Preparation of Compound Ia-8:

[0117] 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]dioxepan[4,3-i]isoorientin-10-yl)oxy)ethoxy)methyl]pyrazine

[0118] Step 1: Preparation of intermediate Ia-8-6: dihydroartemisinin-10-α-pimelic acid monoester:

[0119] Intermediate Ia-1-5 (0.284 g) and ethylene glycol (0.062 g) were dissolved in 10 mL of dichloromethane, and K2CO3 was added. 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 over MgSO4, and concentrated to obtain 0.210 g of a white solid. 0.187 g of the pure product was obtained by recrystallization, with a yield of 57%.

[0120] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0121] 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)pimelate;

[0122] Intermediate Ia-1-4 (0.152 g), Ia-8-6 (0.328 g), and KCO (0.229 g) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 2 hours. After completion, the reaction solution was washed sequentially with 20 mL of water and saturated brine, dried over MgSO, and concentrated to afford a crude pale yellow crystalline solid. This was separated by silica gel column chromatography using 50% ethyl acetate / petroleum ether as the eluent to afford 0.282 g of a colorless crystalline solid in a 61% yield.

[0123] Example 9, Preparation of Compound Ia-9:

[0124] (3,5,6-Trimethylpyrazin-2-yl)methyl 1-(2-oxyl-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

[0125] 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 then in 10 mL of dichloromethane. 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 0.399 g of a light yellow crystalline solid and 0.385 g of the pure product after recrystallization, with a yield of 85%.

[0126] The preparation of intermediate Ia-1-4 was carried out according to the method of Example 1.

[0127] 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

[0128] 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) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 2 hours. After completion of the reaction, the reaction solution was washed sequentially with 20 mL of water and saturated brine, dried over MgSO₄, and concentrated to afford a crude pale yellow crystalline solid. This was separated by silica gel column chromatography using 50% ethyl acetate / petroleum ether as the eluent to afford 0.511 g of a colorless crystalline solid in an 87% yield.

[0129] Example 10. Preparation of Compound Ib-1

[0130] (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)ether

[0131] Step 1:

[0132] Preparation of intermediate Ib-1-1:

[0133] Dihydroartemisinin (2 g, 7.034 mmol, 1 equiv) and pyridine (2.78 g, 35.170 mmol, 5 equiv) were dissolved in DCM (20 mL). Benzoyl chloride (1.19 g, 8.441 mmol, 1.2 equiv) was slowly added dropwise at 0°C. After completion, the reaction mixture was stirred at room temperature for 16 hours. After completion, the reaction was quenched by adding 20 mL of water. The reaction mixture was extracted with DCM, and the organic phases were combined, washed with saturated NaHCO₃ solution, and 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 2 g of intermediate Ib-1-1 as white crystals in a 73% yield.

[0134] Preparation of intermediate Ib-1-2:

[0135] The intermediate (2 g, 5.149 mmol, 1 equiv) was dissolved in DCM (20 mL). Zinc chloride (0.70 g, 5.149 mmol, 1 equiv) and 4A-MS (1.14 g, 0.676 mmol, 0.13 equiv) were added sequentially at 0°C. After addition, the reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction, the resulting mixture was diluted with DCM and washed with 5% aqueous citric acid, saturated aqueous NaHCO₃, and brine. The organic phase was separated, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to dryness to afford the crude product. The crude product was purified by silica gel column chromatography to afford 1.06 g of intermediate Ib-1-2 as a colorless oil in a 68% yield.

[0136] Preparation of intermediate Ib-1-3:

[0137] Intermediate Ib-1-2 (900 mg, 2.918 mmol, 1 equiv) was dissolved in 15 mL of THF and cooled to -20°C. A 5 mL solution of borane-dimethyl sulfide complex (265.99 mg, 3.502 mmol, 1.2 equiv, 1 M) in THF was slowly added dropwise. After the addition was complete, the mixture was warmed to room temperature and stirred for 2 hours. After completion, the reaction was quenched with saturated Na2CO3 solution (10 mL), followed by the addition of 30% H2O2 solution (5 mL) and stirring at room temperature for 30 minutes. After completion, 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 dryness to yield the crude product, Intermediate Ib-1-3, as a brown solid (960 mg), which was used directly without further isolation.

[0138] Preparation of compound Ib-1

[0139] Ib-1-3 (960 mg, crude product, 1 equiv) was dissolved in 10 mL of THF, and NaH (85 mg, 3.529 mmol, 1.2 equiv) was added at 0°C and stirred for 30 minutes. 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 dryness 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%.

[0140] Example 11: The following test data further illustrates the beneficial effects of the present invention.

[0141] Study on the Antimalarial Activity of Artesunazine (Example 1a-1) in Vivo and In Vitro

[0142] 1. Artesunazine protects mice from infection with Plasmodium berghei ANKA strain

[0143] (1) Experimental materials:

[0144] 1. Animals: C57BL / 6J mice, weighing 16-18 g, 10 animals per group, for a total of 50 animals.

[0145] 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 maintained in our laboratory by blood transfusion or cryopreservation.

[0146] 3. Drugs and preparation methods: Artesunazine, a compound, and artesunate, a positive control drug, were dissolved in a mixed solvent of polyethylene glycol 400 and water (PEG400:water=4:1) and prepared immediately before use.

[0147] (2) Experimental methods

[0148] 1. Plasmodium berghei ANKA strain frozen in liquid nitrogen was removed and placed in a 37°C water bath. After thawing, the parasite was immediately inoculated intraperitoneally into C57BL / 6J mice (0.2 mL / mouse). When the infection rate of the breeder mice reached 15-30%, blood was collected from the eyeballs into an anticoagulant tube. After shaking, the blood was passed to the offspring C57BL / 6J mice at a rate of 0.2 mL / mouse using a 1 mL syringe.

[0149] 2. Plasmodium inoculation: Weigh C57BL / 6J mice and feed them adaptively. When the infection rate of offspring C57BL / 6J mice reaches 15-30%, remove the eyeballs and collect blood. Count the total number of red blood cells and the number of red blood cells infected with Plasmodium (pRBC). 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).

[0150] 3. Animal Grouping: After inoculation, experimental mice were randomly divided into five groups: a model group (M), high-, medium-, and low-dose combination groups (Artesunazine 40 mg / kg, 20 mg / kg, and 10 mg / kg), and an artesunate group (AS, 15.6 mg / kg, clinically equivalent). The medium-dose combination group (Artesunazine 20 mg / kg) was dosed at the same level as the artesunate group (15.6 mg / kg). Each group contained 10 mice, for a total of 50 mice.

[0151] 4. Survival Rate Determination

[0152] Starting from the 0th day of inoculation, the death of mice was observed every day for 28 consecutive days.

[0153] 5. Determination of re-ignition rate

[0154] After inoculation, the mice were randomly divided into groups. Three hours after inoculation, intranasal administration was performed. Blood smears were performed starting on day 4 after four consecutive days of administration. A small drop of blood was collected from the tip of the mouse's tail and placed on a glass slide. The drop was then pushed toward one side at an angle using the edge of another glass slide to create a thin blood film of appropriate thickness. After the film air-dried, it was fixed with methanol and allowed to air-dry completely. A 1:9 dilution of Giemsa stain was used to completely cover the fixed blood film for 20 minutes. The film was then rinsed under the tap and the surface moisture was quickly blotted with filter paper. A drop of cedar oil was placed on the glass slide, and the infected erythrocytes were counted per 1000 erythrocytes under a 100x oil immersion microscope. The relapse rate was calculated by the ratio of the number of relapsed mice to the total number of mice.

[0155] 6. RMCBS score determination

[0156] The rapid mouse coma and behavior scale (RMCBS) was used to evaluate the neurological function of experimental mice. Starting from D0, the mice were scored in 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, and a stretched body when walking. They can explore the four corners of the grid within 15 seconds and have obvious exploratory behavior, touch behavior, auricle reaction, toe reaction, touch reflex, etc., while the model mice have less exploratory behavior within 120 seconds, and have messy fur, ataxia, and arched backs. 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. The mice were placed from the upper left corner into a box with a grid paper bottom (31.8 cm × 19.8 cm × 10.5 cm in length, width and height). A 3 mm diameter long rod was used to touch the box. The testing time for one mouse was about 3 minutes. After each test, the mark left by the previous mouse needed to be wiped off.

[0157] 7. Body temperature measurement

[0158] The rectal temperature of mice was measured starting from D0 of inoculation and was observed until the 14th day.

[0159] 8. Weight measurement

[0160] The body weight of mice was measured starting from the inoculation D0 and observed until the 14th day.

[0161] (3) Experimental results

[0162] 1. Effect of the complex on the survival rate of mice with cerebral malaria model

[0163] The survival rates of the groups were compared after the administration for 28 days. 15All mice died. All drug-treated groups significantly prolonged the survival of mice with cerebral malaria. The survival rates were 100% in the high-dose (40 mg / kg) combination group, 90% in the medium-dose (20 mg / kg) combination group, 50% in the low-dose (10 mg / kg) combination group, and 40% in the artesunate (AS) group. The high- and medium-dose combination groups showed superior therapeutic effects on mice with cerebral malaria compared to the artesunate group (P < 0.001).

[0164] 2. Effects of the complex on relapse of cerebral malaria in mice

[0165] The relapse rates among the groups were compared 28 days after the end of drug administration. Results showed that on day 4, the infection rate in the model group was 100%. Four days after drug administration, Artesunazine 40 mg / kg and Artesunazine 20 mg / kg groups completely eliminated Plasmodium parasites, while Artesunazine 10 mg / kg and AS groups each experienced 10% relapse. On day 7, no relapse occurred in the Artesunazine 40 mg / kg and Artesunazine 20 mg / kg groups, while relapse rates in the Artesunazine 10 mg / kg and AS groups were 80% and 90%, respectively. After day 28, relapse rates were 10% in the Artesunazine 40 mg / kg and Artesunazine 20 mg / kg groups, while relapse rates in the Artesunazine 10 mg / kg and AS groups were 100%. High- and medium-dose Artesunazine groups were more effective in inhibiting Plasmodium relapse than AS.

[0166] Table 1 Effects of drugs on relapse of cerebral malaria model mice

[0167] 3. Effects of the complex on neurological function in mice with cerebral malaria (RMCBS score)

[0168] Experimental observations were conducted using the RMCBS score, a classic neurological function assessment method for cerebral malaria. Results showed that the RMCBS score in the model group began to gradually decline on day 6 of inoculation, reaching 14.00±0.26 by the end of observation. During the observation period, the RMCBS scores of the artesunazine 40 mg / kg and artesunazine 20 mg / kg groups remained stable at around 20 points. The RMCBS scores in the AS and artesunazine 10 mg / kg groups gradually decreased on day 9 and day 10 of inoculation, reaching 16.22±0.55 and 18.40±0.48, respectively, by the end of observation. At the end of observation, RMCBS scores were significantly higher in all combination-dose groups compared with the model group (P<0.001), and significantly higher in the AS group compared with the model group (P<0.01). RMCBS scores in the high-, medium-, and low-dose artesunazine groups were significantly higher than in the AS group (P<0.001, P<0.001, and P<0.01, respectively). All combination-dose groups and the AS group effectively improved behavioral and neurological pathological changes in malaria-infected mice, including decreased exploratory drive, matted fur, ataxia, arched backs, limb weakness, and decreased or absent reflexes in the late stage. The overall improvement in behavioral characteristics in mice was superior to that in the AS group in all combination-dose groups.

[0169] 4. Effect of the complex on body temperature in mice with cerebral malaria model

[0170] During the observation period, the body temperature of the mice in the model group decreased to 36.82±0.42 at the end of observation. The body temperature of the mice in the Artesunazine 10 mg / kg, Artesunazine 20 mg / kg, Artesunazine 40 mg / kg groups, and AS group decreased significantly (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 Artesunazine 20 mg / kg and Artesunazine 40 mg / kg showed better improvement in the body temperature of the cerebral malaria mice than the AS group (P<0.001, P<0.001).

[0171] 5. Effect of the complex on the body weight of mice with cerebral malaria model

[0172] During the first six days of the observation period, the weight of mice in all groups showed a gradual increase, with similar weight gains across all groups. The weight of mice in the model group began to decrease on day 6, reaching 13.15±0.29 by the end of observation. The weight of mice in the artesunazine 10 mg / kg group and the AS group began to decrease gradually on day 7 and day 9 of inoculation, respectively, reaching 16.66±0.45 and 15.41±0.50 by the end of observation. The weight of mice in the artesunazine 40 mg / kg and artesunazine 20 mg / kg groups did not decrease significantly throughout the 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 a better effect on improving the weight loss of mice with cerebral malaria than the AS group (P<0.001, P<0.001).

[0173] 2. Pharmacodynamic Study of the Compound in Treating Plasmodium falciparum Cultures in Vitro

[0174] (1) Experimental materials

[0175] Experimental drugs and reagents: Bovine serum albumin complete Plasmodium falciparum medium (see medium preparation for details). 0.16% saponin (Sigma, LOT: BCBR4223V): Add 0.016 g of saponin to 10 ml of deionized water, mix thoroughly, and prepare immediately before use. 0.1 M EDTA (Sigma, LOT: BCBR3854V): Add 0.5844 g of EDTA (MW: 292.24 g / mol) to 20 ml of deionized water, mix thoroughly. 0.4 M Tris-Base (Sigma, LOT: SLBR9609V): Add 0.969 g of Tris-Base (MW = 121.14 g / mol) to 20 ml of deionized water, mix thoroughly. 0.8% Triton X-100 (Sigma, LOT: SLBR3411V): Add 80 μl of Triton X-100 to 9.92 ml of deionized water, mix thoroughly. Lysis buffer: 2 ml each of 0.16% saponin, 0.1 M EDTA, 0.4 M Tris-Base, 0.8% Triton X-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, autoclave. Experimental supplies: Pipettes (1000, 200, 100, 10, 2.5 μl) and tips, 15 ml centrifuge tubes, slides, culture dishes.

[0176] (2) Experimental methods

[0177] Synchronize ring-stage Plasmodium falciparum. Smear and count parasite infestation. Adequately proportion synchronized infected red blood cells (RBCs), healthy RBCs, and culture medium to obtain an in vitro Plasmodium culture system with a hematocrit of 2% and a parasite infestation rate of 1% (ring stage, 2-4 hours old). Plate with RPMI 1640 (50 μl / well). Plate 25 μl of drug in wells A1-A10 (eight replicates per well), mix thoroughly, and perform serial dilutions (Ax-Hx). Plate 50 μl of parasite culture medium in each well except wells A12-H12, which are then plated with 50 μl of healthy RBCs (with a hematocrit of 2%). Incubate under standard in vitro Plasmodium falciparum culture conditions (5% O2, 3% CO2, 92% N2, 37°C) for 72 hours. Add 100 μl of lysis buffer to each well and store in a dark drawer for 1 hour. Lysis buffer (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×) diluted 1000-fold. Fluorescence microplate reader: Ex = 485 nm; Em = 525 nm.

[0178] 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 GraphPad Prism 8.0 software was used for nonlinear regression calculation (values ​​are mean ± standard error). At the same time, a four-parameter logistic fit curve was used, and each value in the curve was the mean ± standard deviation of eight biological replicates.

[0179] (3) Experimental results

[0180] (1) Analysis of the efficacy of compound Ia-1 in treating human Plasmodium falciparum standard strain 3D7

[0181] Compound Ia-1 exhibited a strong inhibitory effect on parasite proliferation at nanomolar concentrations (IC50 = 7.358 nM), demonstrating its potent inhibitory effect against Plasmodium falciparum, despite being comparable in potency to standard antimalarial drugs such as chloroquine (approximately 2.360 nM), mefloquine (approximately 3.962 nM), and artemisinin (approximately 11.768 nM). Furthermore, the control drug artesunate significantly inhibited the proliferation of Plasmodium falciparum 3D7, with an IC50 value of 8.825 nM (Figure 5).

[0182] (2) Analysis of the efficacy of compound Ia-2 in treating human Plasmodium falciparum standard strain 3D7

[0183] Compound Ia-2 has a significant inhibitory effect on parasite proliferation in the nanomolar concentration range (IC50=12.52nM).

[0184] (3) Analysis of the efficacy of compound Ia-3 in treating human Plasmodium falciparum standard strain 3D7

[0185] Compound Ia-3 has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range (IC50=6.863nM).

[0186] (4) Analysis of the efficacy of compound Ia-4 in treating human Plasmodium falciparum standard strain 3D7

[0187] Compound Ia-4 has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range (IC50=33.92nM).

[0188] (5) Analysis of the efficacy of compound Ia-5 in treating human Plasmodium falciparum standard strain 3D7

[0189] Compound Ia-5 has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range (IC50=2.953nM).

[0190] (6) Analysis of the efficacy of compound Ia-6 in treating human Plasmodium falciparum standard strain 3D7

[0191] Compound Ia-6 has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range (IC50=14.29nM).

[0192] (7) Analysis of the efficacy of compound Ia-7 in treating human Plasmodium falciparum standard strain 3D7

[0193] Compound Ia-7 has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range (IC50=50.68nM).

[0194] (8) Analysis of the efficacy of compound Ia-8 in treating human Plasmodium falciparum standard strain 3D7

[0195] Compound Ia-8 has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range (IC50=107.2nM).

[0196] (9) Analysis of the efficacy of compound Ia-9 in treating human Plasmodium falciparum standard strain 3D7

[0197] Compound Ia-9 has a significant inhibitory effect on the proliferation of Plasmodium falciparum 3D7 in the nanomolar concentration range (IC50=32.73nM).

[0198] 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.

[0199] 3. Stability Study of the Composite

[0200] (1) Experimental methods

[0201] 1. Study on the stability of the complex

[0202] 160.2 mg of the complex was weighed into a vial, 8 mL of 80% polyethylene glycol 400 solution was added, and sonication was performed to completely dissolve the complex, yielding a complex solution at approximately 20.25 mg / mL. The vial containing the solution was then sealed, covered with aluminum foil to protect from light, and placed in a 40°C stability chamber. On days 0, 3, 7, and 10, 0.5 mL of the sample was transferred to a 25 mL volumetric flask, the volume was adjusted to the desired volume with methanol, and the solution was shaken thoroughly. The content of the complex was then determined by UPLC.

[0203] The UPLC assay conditions for the complex were 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.

[0204] 2. Study on the Stability of Artesunate and Ligustrazine Hydrochloride in the Same Solution

[0205] A mixed solution of artesunate and ligustrazine hydrochloride was prepared according to a molar concentration of 20 mg / mL of the complex, 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 the mixture was sonicated until completely dissolved, thereby obtaining 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 protect from light, and placed in a 40°C stability test chamber; 0.5 mL of the sample was transferred to a 25 mL volumetric flask on days 0, 3, 7, and 10, respectively. The volume was made up with methanol, and the mixture was shaken well. The content of the complex was then determined by UPLC.

[0206] (1) Artesunate UPLC assay conditions 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.

[0207] (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.

[0208] (2) Experimental results

[0209] The stability of the artesunate-ligustrazine hydrochloride compound in 80% polyethylene glycol 400 solution was poor. At 40°C in a dark environment, the artesunate content decreased rapidly and the color of the mixture solution gradually turned yellow. In contrast, the stability of the composite in 80% polyethylene glycol 400 solution was significantly improved, and the color of the composite solution remained basically unchanged. This result provides a good foundation for the development of new antimalarial drugs (Figures 6 and 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:

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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