Use of triazoloquinoline derivatives for the preparation of a medicament for the prevention or treatment of malaria

Abstract

The application discloses a new application of a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof. The triazoloquinoline derivative can inhibit the expression of an invasion gene PfAP2-O5, block the invasion of Plasmodium falciparum into red blood cells, affect the growth and development of the Plasmodium falciparum, and achieve a killing effect of the Plasmodium falciparum, so that the triazoloquinoline derivative can be used as an inhibitor of PfAP2-O5, a drug for inhibiting Plasmodium falciparum, or a drug for preventing or treating malaria.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, more particularly, to the application of triazoloquinoline derivatives in the preparation of drugs for preventing or treating malaria. BACKGROUND

[0002] Malaria is a parasitic disease caused by infection of protozoan Plasmodium, which can seriously threaten human health and life safety, and its morbidity and mortality are high among infectious diseases. According to the World Malaria Report released by WHO in 2021, there are 87 countries and regions in the world where malaria is prevalent, and there were 241 million new cases and about 627,000 deaths in 2020.

[0003] The life cycle of Plasmodium is complex, mainly including sexual reproduction in the final host and asexual reproduction in the intermediate host. In the human body, Plasmodium (sporozoite) first invades the liver, undergoes asexual multiplication by one-time schizogony, and the released merozoites invade red blood cells, starting the schizogony phase in red blood cells, which is the period of clinical onset of malaria, and the invasion of merozoites into red blood cells is the key link of malaria pathogenesis.

[0004] At present, drug treatment is the main means of malaria prevention and control, among which the artemisinin-based combination therapy effectively curbs the spread of malaria worldwide. However, with the long-term and extensive use of artemisinin drugs, there have been gradually emerging strains with reduced sensitivity in Southeast Asia and even Africa. The emergence and global spread of such strains pose a serious challenge to the world's malaria control and elimination strategy. Therefore, it is of great significance to find new targets for antimalarial drugs, especially to develop safe and effective transmission blocking drugs targeting the key link of Plasmodium pathogenesis to curb the spread of malaria. SUMMARY

[0005] The present application provides a new drug for preventing or treating malaria to overcome the above-mentioned deficiencies.

[0006] Another object of the present application is to provide a new application of triazoloquinoline derivatives.

[0007] The above-mentioned object of the present application is achieved by the following technical solutions:

[0008] The application of triazoloquinoline derivatives or pharmaceutically acceptable salts or hydrates thereof in the preparation of an inhibitor of transcription factor PfAP2-O5 of Plasmodium falciparum, wherein the triazoloquinoline derivative has a structure as shown in formula (I):

[0009]

[0010] The inventors first discovered that PfAP2-O5 positively regulates the invasion of red blood cells in the course of in-depth research on the mechanism of invasion of Plasmodium falciparum into red blood cells. The triazoloquinoline derivative shown in formula (I) can block the invasion of Plasmodium falciparum into red blood cells by inhibiting the expression of the invasion gene PfAP2-O5, affect the growth and development of Plasmodium falciparum, and achieve the killing effect of Plasmodium falciparum.

[0011] The PfAP2-O5 inhibitor of the present application refers to a compound that achieves the effect of killing Plasmodium falciparum by inhibiting the function of AP2-O5.

[0012] Meanwhile, the triazoloquinoline derivative shown in formula (I) has shown good growth inhibition effect on Plasmodium falciparum in vitro experiments, and has almost no toxicity to mammalian cells, making it possible to be developed into a medicine. It also has good killing effect on resistant insect strains, helps to delay the occurrence and spread of drug resistance, and will be more efficient in anti-malaria treatment.

[0013] The application of a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof in the preparation of a medicine for inhibiting Plasmodium falciparum; the triazoloquinoline derivative has a structure shown in formula (I):

[0014]

[0015] The application of a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof in the preparation of a medicine for preventing or treating malaria caused by Plasmodium falciparum; the triazoloquinoline derivative has a structure shown in formula (I):

[0016]

[0017] In the present application, the "pharmaceutically acceptable salt" includes a salt formed by the triazoloquinoline derivative and an inorganic acid or an organic acid.

[0018] The inorganic acid is, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc. The organic acid is, for example, acetic acid, hydroxyacetic acid, propionic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclic acid, cyclic salicylic acid, p-aminosalicylic acid, etc.

[0019] In the present application, the "hydrate" refers to a salt containing water molecules combined in a certain proportion as a component of a crystal.

[0020] A pharmaceutical composition containing a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof having a structure shown in formula (I) as an active ingredient, and a pharmaceutically acceptable carrier, diluent or excipient as an aid;

[0021]

[0022] In preparing these compositions, the active ingredient will usually be mixed with an excipient, or diluted by an excipient, or enclosed within such a carrier, which can be in the form of a capsule or sachet. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. The compositions can be in unit dosage form.

[0023] Examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, arabic gum, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. In addition, lubricating agents such as talc, magnesium stearate, and mineral oil, wetting agents, emulsifying and suspending agents, preservatives such as methyl and propylhydroxybenzoates, sweetening agents, or flavoring agents can be included.

[0024] Therefore, the pharmaceutical composition can be prepared into various known dosage forms, and preferably, the pharmaceutical composition is prepared into an oral dosage form or an injection dosage form.

[0025] The oral dosage form includes a solid dosage form such as a tablet, a drop pill, a fast-release drop pill, a capsule (e.g., a liquid gel capsule and a solid gel capsule), a granule, or a powder. The tablet can be a compressed tablet, a ground tablet, an enteric-coated tablet, a sugar-coated tablet, a film-coated tablet, or a multi-layered compressed tablet. As a suitable type of an aid for preparing the tablet, there are included, but not limited to, a binder, a lubricant, a diluent, a disintegrant, a coloring agent, a flavoring agent, a fluidizing agent, or a melting agent. The oral dosage form can also be a liquid oral dosage form such as an aqueous solution, an emulsion, a suspension, a solution and / or a suspension prepared from a non-effervescent granule, or an effervescent prepared from an effervescent granule. As a suitable type of an aid for preparing the liquid oral dosage, there are included a solvent, a preservative, an emulsifying agent, a suspending agent, a diluent, a sweetening agent, a melting agent, a coloring agent, or a flavoring agent.

[0026] The injection dosage form includes an injection solution (ordinary), a lyophilized powder for injection, a powder for injection (ordinary), a tablet for injection, etc.

[0027] Specifically, the pharmaceutical composition can be a tablet, a pill, a powder, an elixir, a suspension, an emulsion, a solution, a syrup, a soft and hard gelatin capsule, a suppository, a sterilized injectable solution, and a sterilized packaged powder.

[0028] By the growth inhibition experiment of the malignant malaria parasite, the EC50 value of the growth inhibition of the malignant malaria parasite of the present application reached 2.632 μM, showing a good growth inhibition function, as compared with artemisinin.

[0029] The pharmaceutical composition can be a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof shown in the structure of formula (I) as the only active ingredient, and can also have a known artemisinin compound as a co-active ingredient.

[0030] Preferably, the active ingredient further comprises an artemisinin compound. The artemisinin compound has been proved to have a therapeutic effect on malaria. More preferably, the artemisinin compound is a dihydroartemisinin compound. The dihydroartemisinin compound has a better effect on various drug-resistant Plasmodium than the general artemisinin compound.

[0031] Preferably, in the composition, the molar ratio of the triazoloquinoline derivative or the pharmaceutically acceptable salt or hydrate thereof shown in the structure of formula (I) to the artemisinin compound is 1:500-500:1.

[0032] Preferably, the pharmaceutical composition is prepared into an oral dosage form or an injection dosage form.

[0033] The pharmaceutical composition is used for preparing a medicine for preventing and / or treating malaria.

[0034] Preferably, the pharmaceutical composition is used for preparing a medicine for treating malaria resistant to artemisinin and its analogues.

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] The present application provides a new application of a triazoloquinoline derivative or a pharmaceutically acceptable salt or hydrate thereof, which can inhibit the expression of an invasion gene PfAP2-O5, block the invasion of Plasmodium falciparum into red blood cells, affect the growth and development of Plasmodium falciparum, and achieve a killing effect on Plasmodium falciparum, so that the triazoloquinoline derivative can be used as an inhibitor of PfAP2-O5, a medicine for inhibiting Plasmodium, or a medicine for preventing or treating malaria. Based on the above finding, the present application provides a new pharmaceutical composition, which can have a triazoloquinoline derivative as an active ingredient alone, or can have a triazoloquinoline derivative and an artemisinin compound as co-active ingredients; the pharmaceutical composition can be used for preparing a medicine for treating malaria, especially a medicine for treating malaria resistant to artemisinin and its analogues. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 In, Figure 1 A shows the WB identification results of the PfAP2-O5 gene knockdown strain, and the expression level of the PfAP2-O5 protein is reduced after the PfAP2-O5 gene is knocked down; Figure 1 B shows the growth curve test of the PfAP2-O5 gene knockdown strain.

[0038] Figure 2 ChIP-seq analysis of PfAP2-O5 distribution on the whole genome; and the target gene types of PfAP2-O5 binding, wherein most of them are invasion gene families.

[0039] Figure 3 A schematic diagram of the invasion gene expression inhibition effect of triazoloquinoline derivatives. Figure 3 A RNA-Seq technique was used to determine the invasion gene expression of the PfAP2-O5 gene knockdown worm strain; Figure 3 B shows the invasion gene expression after the triazoloquinoline derivative inhibits the function of the PfAP2-O5 gene; Figure 3 C shows the invasion gene expression inhibition effect of PfAP2-O5 gene knockdown and triazoloquinoline derivative treatment.

[0040] Figure 4 A schematic diagram of the quantitative growth inhibition effect of triazoloquinoline derivatives on in vitro cultured Plasmodium. It shows that its quantitative growth inhibition effect on in vitro cultured Plasmodium is 2.632 μM, and the 3-day SYBR Green I cell growth determination method is used to determine the Plasmodium growth.

[0041] Figure 5 A schematic diagram of the cytotoxic effect of the triazoloquinoline derivative on the human renal epithelial cell line 293T and the human hepatoma cell line HepG2. It also shows the selectivity index between the inhibition effect of the small molecule inhibitor on Plasmodium and the toxicity on different cell lines, and the 3-day CCK8 method is used to determine the cell growth.

[0042] Figure 6 A schematic diagram of the triazoloquinoline derivative as a compatible drug with dihydroartemisinin for killing resistant worm strains, and a ring body survival experiment is used to determine the growth inhibition effect of combined drug on resistant worm strains. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present application will be described clearly and completely below, but the embodiments of the present application are not limited thereto.

[0044] The reagents, methods and equipment used in the present application are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0045] 1. Triazoloquinoline derivative represented by formula (I): purchased from ChemDiv ID. V003-8956

[0046] 2. Malignant Plasmodium (3D7_G7): purchased from ATCC

[0047] 3. 293T cell strain: purchased from ATCC

[0048] 4. HepG2 cell line: purchased from ATCC

[0049] 5. SYBR Green I: purchased from Invitrogen Cat. No. S-7585

[0050] 6. Trypsin-EDTA: purchased from Gbico Cat. No. 25200-072

[0051] 7. DMEM medium: purchased from Gbico Cat. No. 11965-092

[0052] 8. FBS: purchased from Gbico Cat. No. 10082-147

[0053] 9. Double-stranded DNA quantification kit: purchased from ThermoFisher Cat. No. P7581

[0054] 10. Protein A / G magnetic beads: purchased from ThermoFisher Cat. No. 88803

[0055] 11. Mouse monoclonal antibody: purchased from Sigma Cat. No. SAB4800032

[0056] 12. 37% formaldehyde solution: purchased from Sigma Cat. No. 47608-250ML-F

[0057] 13. 96-well plate: purchased from Corning

[0058] 14. Red blood cells: purchased from Shanghai Blood Center

[0059] 15. Dihydroartemisinin (CAS: 71939-50-9)

[0060] 16. Various buffer components are as follows:

[0061] (1) Lysis buffer

[0062] Reagents (stock solutions) Final concentration 1 M Hepes pH 7.9 10 mM 0.5 M EDTA pH 8.0 0.1 mM 0.5 M EGTA pH 8.0 0.1 mM 2 M KC1 10 mM 1 M DTT (add fresh) 1 mM 100 x protease inhibitors (add fresh) 1×

[0063] (2) Sonication buffer

[0064] Reagents (stock solutions) Final concentration 10% SDS 0.1％ 0.5 M EDTA pH 8.0 1 mM 1 M Tris-HCl pH 8.0 10 mM

[0065] (3) ChIP dilution buffer

[0066] Reagents (stock solutions) Final concentration 10% SDS 0.01％ 100% Triton X-100 1.1％ 5 M NaCl 150 mM 0.5 M EDTA pH 8.0 1.2 mM 1 M Tris-HCl pH 8.0 16.7 mM

[0067] (4) Low-salt immune complex wash buffer

[0068] Reagents (stock solutions) Final concentration 10% SDS 0.1％ 100% Triton X-100 1％ 5 M NaCl 150 mM 0.5 M EDTA pH 8.0 2 mM 1 M Tris-HCl pH 8.0 20 mM

[0069] (5) High salt immune complex wash buffer

[0070]

[0071]

[0072] (6) Lithium chloride immune complex wash buffer

[0073] Reagents (stock solutions) Final concentration 10% NP-40 1％ 10% deoxycholic acid 1％ 5 M LiCl 250 mM 0.5 M EDTA pH 8.0 1 mM 1 M Tris-HCl pH 8.0 10 mM

[0074] (7) TE buffer

[0075] Reagents (stock solutions) Final concentration 1 M Tris-HCl pH 8.0 10 mM 0.5 M EDTA pH 8.0 1 mM

[0076] (8) Elution buffer

[0077] Reagents (stock solutions) Final concentration 10% SDS 1％ 1 M NaHCO3 100 mM

[0078] Example 1. Cultivation of Plasmodium

[0079] The P. falciparum was cultivated in a 37 °C incubator (5% CO2, 5% O2 and 90% N2) using RPMI 1640 complete medium.

[0080] 0.5 L RPMI 1640 complete medium contains: 5.22 g RPMI-1640 powder; 1.0 g sodium bicarbonate; 5 g Albmax I; 2.98 g 25 mM HEPES; 13.6 mg hypoxanthine; 10 mg gentamicin sulfate.

[0081] Example 2. Construction of transgenic parasite strain: PfAP2-O5 conditional knockdown parasite strain

[0082] In this experiment, the CRISPR / Cas9 gene editing system was used to clone the guide RNA into the vector to construct the PfAP2-O5 conditional knockdown parasite strain.

[0083] The specific construction steps are as follows:

[0084] 1. Construction of transgenic parasite knockdown vector (named pL6cs-ap2-o5-glmS)

[0085] Using the CRISPR / Cas9 gene editing system, the guide RNA was cloned into the vector, and the specific method is as follows:

[0086] (1) Design of gRNA

[0087] The sequence of the PfAP2-O5 gene of the Plasmodium falciparum 3D7-G7 strain was downloaded from the PlasmoDB website (from the database at https: / / plasmodb.org / plasmo / app, Plasmodium falciparum: PF3D7_1449500 (715 aa PfAP2-O5). The sequence is also disclosed at ncbi: Sequence ID: XM_001348609.1). A specific sequence of 20 bp in length was selected as a guide RNA (gRNA) through BLAST alignment, and the sequence was as follows: 5'-ACCAAGTCATATGTCATTAA-3'.

[0088] (2) Design of primers

[0089] The SnapGene software was used to design the primers.

[0090] P1: 5'-GCCCTAGTCTAG GGcGCGCC CAATTTTAATAACAATTTGGCG-3';

[0091] P2: 5'-CAAGTAAAATAATTGGTCTTTAAGTGACATATGACTTGGTAGTTTACTG-3';

[0092] P3: 5'-AGCGGCCGCAAAAAAAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATATATATTATAC-3';

[0093] P4: 5'-TTTTTTACAAAATG CTTAAG AACTAGCGTATGTGATAACTTAG-3' was used to amplify the homologous arm of the PfAP2-O5 gene and introduce a synonymous mutation site. The synonymous mutation site was introduced to prevent self-cleavage of the gRNA to the plasmid;

[0094] P7: 5'-TATTTTACTTGGACAAATTAGGTACCGGAAGTGGTTCTGG-3';

[0095] P8: 5'-TTTATTTTTTTTGCGGCCGCGCGGCCGCGTCCCCTC-3' was used to amplify the exogenous green fluorescent protein (GFP) gene.

[0096] (3) Gene amplification

[0097] The DNA of the 3D7-G7 strain of Plasmodium falciparum was extracted, and P1+P2 and P3+P4 primers were used to amplify the left and right homologous sequences of the PfAP2-O5 gene respectively and introduce mutation sites, and P7+P8 primers were used to amplify the glmS ribozyme sequence (the glmS sequence is from the article: doi:10.1371 / journal.pone.0073783).

[0098] (4) Construction of the ligation product

[0099] The left and right homologous sequences and the glmS ribozyme sequence were connected by primers to obtain the ligation product. The 3' end of the PfAP2-O5 gene was labeled with the glmS ribozyme sequence.

[0100] The reaction system was as follows: 2×Phanta Max Master Mix (Dye Plus): 25 μL, P1 primer (10 μM): 2 μL, P4 primer (10 μM): 2 μL, left and right homologous sequences and glmS ribozyme sequence: 1 μL each, and ddH2O was added to 50 μL system.

[0101] The PCR conditions were as follows: 95℃ for 3 min, 95℃ for 15 s, 65℃ for 15 s, 65℃ for 30 s / 6b, 65℃ for 5 min, a total of 30 cycles; 72℃ for 10 min.

[0102] (5) Construction of the knockdown vector

[0103] The plasmid pL6cs (the plasmid was self-built in the laboratory, and the specific sequence is reported in the article: doi:10.3389 / fmicb.2020.625862) carrying the screening drug dihydrofolate reductase inhibitor (WR) resistance gene was digested with Xho I and Avr II, the product was recovered and purified by gel recovery kit, and the gRNA fragment was connected by DNA ligase and then transformed into Escherichia coli XL-10 competent cells, and inoculated on LB agar plate containing 100 μg / ml ampicillin and cultured overnight. A single colony was picked and cultured at 34℃, 200 rpm / s, and the plasmid was extracted and sequenced.

[0104] The plasmid with gRNA was determined by sequencing, and was digested with Asc I and Afl II, and the product was recovered and connected with the homologous arm containing the glmS ribozyme sequence, and a single colony was picked and cultured, and the correct plasmid was identified by PCR and sequenced, and the strain was stored at-80℃.

[0105] The transgenic strain knockdown vector was obtained and named pL6cs-ap2-o5-glmS.

[0106] 2. Construction of PfAP2-O5 conditional knockdown strain

[0107] Construct transgenic strain of PfAP2-O5 conditional knockdown strain by using the method of electroporation combined with drug screening.

[0108] (1) Preparation of plasmid for electroporation

[0109] Inoculate the bacteria with correct sequence into LB agar plate containing 100 μg / ml ampicillin and incubate overnight. The next day, pick a single colony and inoculate into LB medium, and incubate at 34°C, 200 rpm / s for 8h. Dilute 1:1000 into 400ml LB medium, and incubate for 14h. Collect the bacteria and purify the plasmid using plasmid DNA purification kit.

[0110] Take the transgenic strain knockdown vector pL6cs-ap2-o5-glmS plasmid constructed above, and 100 μg of plasmid pUF1-Cas9 (plasmid is self-built in the laboratory, see the literature for specific sequence: doi:10.3389 / fmicb.2020.625862) carrying the drug resistance gene of blasticidin (BSD) and expressing Cas9 protein. Add 1 / 10 volume of 3M CH3COONa (pH 5.2) solution to the plasmid solution, mix well, then add 2.5 times volume of absolute ethanol to the plasmid solution, mix well, centrifuge at 13000 x g for 30 min, discard the supernatant, wash the precipitate with 75% ethanol twice, then air dry in a clean bench and dissolve the precipitate with 150 μl of sterile water.

[0111] (2) Electroporation and screening of transgenic strains

[0112] Set the electroporation instrument to the following conditions: shock parameters 310V, 950μF, resistance ∞, slit 2mm. Add an equal volume (150 μl) of 2x cell mixing solution to the prepared plasmid and mix well, then add 150 μl of red blood cells, mix well, and then transfer into the electroporation cup, place in the electroporation instrument, and shock. After shocking, transfer the mixture into a culture bottle, add an appropriate amount of culture medium, then add purified schizonts of Plasmodium.

[0113] Incubate in a 37°C three-gas incubator. From the second day, replace the fresh culture medium every day, and on the fourth day, when the electroporation density is about 10% (parasite rate, the proportion of Plasmodium-infected red blood cells to all red blood cells counted in a certain field of view (~5000)), add 200 μl of fresh red blood cells, replace the culture medium and add the screening drugs WR and BSD (blasticidin). The screening drugs can kill wild-type Plasmodium falciparum, while the transgenic Plasmodium falciparum carrying the plasmid expressing the drug resistance gene is not affected by the drugs, so the transgenic Plasmodium falciparum can be screened out.

[0114] After the addition of the drug until most of the wild malaria parasites are killed, the culture medium is replaced and the drug is added every 3-4 days to maintain the culture. Smear microscopy is performed on day 21 after transfection, and transgenic strains are obtained 3-6 weeks later.

[0115] In summary, the transgenic strain PfAP2-O5 conditional knockdown strain is constructed.

[0116] 3. Gradient dilution cloning

[0117] The transgenic strain (PfAP2-O5 conditional knockdown strain) obtained by transgenesis is an integrated strain, which needs to be gradient diluted and cloned, i.e. through limited dilution method, a single clone strain completely integrated is screened, and the experiment is carried out in a 96-well cell culture plate. Through gradient dilution, the infected red blood cells are diluted to contain 10, 1, 0.5 and 0.1 worms per 1 ml in theory, and the specific experimental steps are as follows:

[0118] (1) The transgenic strain is sorbitol synchronized to obtain ring stage parasites with relatively uniform properties;

[0119] (2) Smear microscopy is performed on the trophozoite stage of the parasites, and the rate of primitive protozoa P x is strictly counted;

[0120] (3) The trophozoite stage culture is collected and centrifuged at 2000 rpm at room temperature for 5 min, and the supernatant is discarded to obtain about 200 μl of infected red blood cell packed volume (iRBC);

[0121] (4) Add 800 μl of fresh culture medium to the iRBC, mix gently, and transfer to a 1.5 ml centrifuge tube with a final volume of 1 ml;

[0122] (5) 1500 rpm, room temperature centrifugation for 2-3 min, discard the supernatant, take 50 μl iRBC to a new centrifuge tube for standby;

[0123] (6) According to the formula 50 μl x P x = 1% x (50 μl + V x), the protozoa rate of the above 50 μl infected red blood cells is diluted to 1%, and the culture A (V x is the volume of fresh red blood cells added) is obtained, and smear microscopy is performed to confirm the protozoa rate after dilution;

[0124] (7) Preheat fresh culture medium, and according to the principle of gradient dilution, dilute the above prepared infected red blood cells with protozoa rate of 1% in proportion to obtain a gradient corresponding to each centrifuge tube:

[0125] Prepare 9 centrifuge tubes of 15ml and number them as 1, 2, 3…9, add 10ml fresh medium to tube 1, add 9ml fresh medium to tube 2-7 and 9, add 5ml fresh medium to tube 8, all centrifuge tubes are preheated in 37℃ incubator for standby. Take 100μl of culture A with 1% parasites in (6) and add to tube 1, obtain culture B with 1×10 6 parasites / ml iRBC;

[0126] 1) Take 1ml of culture B to tube 2, obtain culture C with 1×10 5 parasites / ml iRBC;

[0127] 2) Take 1ml of culture C to tube 3, obtain culture D with 1×10 4 parasites / ml iRBC;

[0128] 3) Take 1ml of culture D to tube 4, obtain culture E with 1×10 3 parasites / ml iRBC;

[0129] 4) Take 1ml of culture E to tube 5, obtain culture F with 1×10 2 parasites / ml iRBC;

[0130] 5) Take 1ml of culture F to tube 6, obtain culture G with 10 parasites / ml iRBC;

[0131] 6) Take 1ml of culture G to tube 7, obtain culture H with 1 parasite / ml iRBC;

[0132] 7) Take 5ml of culture H to tube 8, obtain culture I with 0.5 parasite / ml iRBC;

[0133] 8) Take 1ml of culture H to tube 9, obtain culture J with 0.1 parasite / ml iRBC.

[0134] (8) Transfer the different gradient cultures G, H, I, J obtained above to 96-well cell culture plates, 200μl / well, finally 96-well plates with 12 wells in each row correspond to one gradient.

[0135] (9) The late culture is replaced with fresh medium every 2 days, and fresh red blood cells (2μl / well) are added every 6 days to maintain the culture;

[0136] After about 2 weeks, smear and microscopic examination, until live parasites are observed under the microscope, then slowly expand the culture in 24-well, 12-well, 6-well plates, after genome detection is correct, obtain completely integrated single clone PfAP2-O5 transgenic parasite strain (PfAP2-O5 conditional knockdown parasite strain), preserve and carry out subsequent experiments.

[0137] Example 3 PfAP2-O5 transgenic strain (PfAP2-O5 conditional knockdown strain) identification and growth and development test

[0138] The ring stage (10-15h), trophozoite stage (28-32h), and schizont stage (40-44h) cultures of the PfAP2-O5 transgenic strain (PfAP2-O5 conditional knockdown strain) were collected. The infected erythrocyte protein samples were lysed with 0.15% saponin, and the proteins were separated by SDS-PAGE electrophoresis and then transferred to a nitrocellulose membrane by semi-dry transfer, with the transfer parameters being 0.1 A and 2h. After the transfer, an appropriate volume of blocking solution (5% skim milk powder: dissolved in PBST solution) was added, and the membrane was blocked at room temperature for 2h. The primary antibody (mouse-derived anti-Ty1 antibody) was diluted with 5% skim milk powder at a ratio of 1:1000, and the membrane was incubated overnight at 4°C. The secondary antibody (goat anti-mouse) was diluted with 5% skim milk powder at a ratio of 1:5000, and the membrane was incubated at room temperature for 2h. Color development: equal volumes of color development solution A and B were mixed (note: avoid light), and a certain amount of color development solution was added to the membrane, and the exposure picture was saved by taking a photo. The results are shown in Figure 1 A, Figure 1 A shows the western blot identification of the PfAP2-O5 conditional knockdown strain before and after the addition of 5mM glucosamine (GlcN), indicating that PfAP2-O5 is effectively knocked down.

[0139] PfAP2-O5 conditional knockdown strain growth curve test: the strain was synchronized to the ring stage for several times, and the initial parasite rate was strictly counted and determined. The strain was divided into two bottles (one bottle was added with the corresponding concentration of GlcN, and the other bottle was used as a control without addition of GlcN). The initial parasite rate of each bottle was about 0.1%. The culture was continuously maintained for 4 life cycles, and the parasite rate was counted by smear examination in the middle and late stages of each life cycle (the culture medium was replaced when the parasite rate was high, and the drug was added to the drug group). The parasite rates of the drug group and the control group were recorded, and the growth curve was drawn. The results are shown in Figure 1 B, Figure 1 B shows the growth curve test of the wild-type strain and the PfAP2-O5 conditional knockdown strain, indicating that the growth of Plasmodium is significantly inhibited after the PfAP2-O5 conditional knockdown.

[0140] Example 4 Chromatin immunoprecipitation (ChIP) sequencing

[0141] Chromatin immunoprecipitation (ChIP) sequencing is a method that uses the specificity of antigen-antibody reaction to truly reflect the distribution of AP2-O5 binding target genes in the whole genome at the chromatin level, and to reflect the binding of proteins and target genes. The specific method is as follows:

[0142] The synchronized schizont stage culture was collected, 1% formaldehyde solution was added, and incubated at 37°C for 10 min; 0.125M pre-cooled glycine solution was added and incubated on ice for 5 min; centrifuged at 2500 rpm, 4°C for 5 min, the supernatant was discarded, 30 mL of 1xPBS was added to resuspend the red blood cells, 0.15% saponin was added to lyse the red blood cells and collect the parasites; 2 mL of pre-cooled Lysis Buffer was added to resuspend the parasites and transferred to a pre-cooled grinding tube for ice bath for 30 min; 200 μL of ultrasonic buffer was added, and the liquid phase ultrasonic was resuspended after precipitation, the parameters were set as: duty cycle 5%, peak incident power 75W, cycle number 200, temperature 7°C, time 30 min; 10 μL of the mixed solution after ultrasonic was added to 90 μL of ChIP dilution buffer, 4 μL of 5M NaCl, 2.5 μL of protease K, mixed and incubated at 50°C for 2 h, 2 μL of RNase A was added and incubated at 37°C for 45 min, QIAGEN PCR kit was used for extraction, and 2% agarose gel electrophoresis was used for detection of ultrasonic effect; the ultrasonic product of 14000 pm with ideal ultrasonic effect was collected after centrifugation at 4°C for 10 min, and 10 times the volume of ChIP dilution buffer was added; protein A / G magnetic beads were added to the above solution, and incubated at 4°C for 2 h, the supernatant was transferred to a 1.5 mL centrifuge tube, and the corresponding antibody and protein A / G magnetic beads were added, and incubated at 4°C overnight; the antibody / magnetic bead / chromatin complex was washed with different buffers in the following order:

[0143] A: low-salt immune complex washing buffer: 1 mL per tube, 4°C rotation for 5 min;

[0144] B: high-salt immune complex washing buffer: 1 mL per tube, 4°C rotation for 5 min;

[0145] C: lithium chloride immune complex washing buffer: 1 mL per tube, 4°C rotation for 5 min;

[0146] D: TE buffer was washed twice, 1 mL per tube, 37°C rotation for 5 min;

[0147] Elution was performed by adding 200 μL elution buffer, rotating incubation at 37 °C for 30 min, and collecting the supernatant, while 180 μL elution buffer was added to the DNA tube before immunoprecipitation; 16 μL of 5M NaCl was added to each tube, mixed, and incubated at 45 °C overnight; 0.8 μL of RNase A (20 mg / mL) was added to each tube, and incubated at 37 °C for 30 min; 3 μL of proteinase K was added to each tube, and incubated at 45 °C for 2 h; the sample DNA was extracted using a QIAGEN PCR purification kit, eluted twice with 10 μL of deionized water, and incubated at room temperature for 10 min before each elution; the concentration was determined, and the library was constructed. To prepare the sequencing library, 1.5 ng of ChIP-DNA was subjected to end repair, 3' adenylation, and adapter ligation; after purification with Agencourt AMPure XP magnetic beads, the library was amplified using a KAPA HiFi PCR kit under the following conditions: 95 °C for 1 min, 98 °C for 10 s, 65 °C for 1 min, 12 cycles, 65 °C for 5 min, and 4 °C for ∞. The library was sequenced on an Illumina HiSeq X Ten platform; each ChIP-seq data set was performed in biological duplicate.

[0148] The results, as shown in Figure 2 , show the distribution of PfAP2-O5 protein target genes across the whole genome, such as the invasion gene families msps, raps, rhophs, ralp1, ama1, etc., proving that most of the target genes of PfAP2-O5 are related to invasion

[0149] Example 5 Transcriptome sequencing (RNA-seq) analysis of the effect of triazoloquinoline derivatives on PfAP2-O5

[0150] The wild-type strain and the PfAP2-O5 conditional knockdown strain were treated with triazoloquinoline derivatives represented by formula (I), respectively, and the strains were strictly synchronized, 1 μM triazoloquinoline derivatives were added one life cycle in advance, and the trophozoites (28-32 h), schizonts (40-44 h), and ring forms (10-15 h) of the wild-type strain and the PfAP2-O5 conditional knockdown strain treated with triazoloquinoline derivatives were collected after one life cycle, total RNA was purified with a Direct-zol RNA kit, and a strand-specific RNA sequencing library was prepared with KAPA mRNA beads, and the RNA was fragmented; the library was sequenced on an Illumina HiSeq X Ten system, and the differential gene expression of the two strains after treatment with the inhibitor was analyzed.

[0151] The results, as shown in Figure 3 , Figure 3A RNA-Seq was used to determine the expression of invasion genes after conditional knockdown of PfAP2-O5; Figure 3 B RNA-Seq was used to determine the expression of invasion genes after inhibition of PfAP2-O5 function by triazoloquinoline derivatives. The results showed that after conditional knockdown of PfAP2-O5, the invasion-related target genes were down-regulated, and after the wild-type strain was treated with triazoloquinoline derivatives, the PfAP2-O5 target genes were down-regulated. For example, the expression levels of Rhoptry Nec6, Peripheral surface protein, Rhoptry protein, Rhoptry bulb, Micronemes protein, and GPI-Anchored MSP, a family of malarial merozoite surface structure protein genes, were significantly reduced by 1.5 times or more (mRNA level). (The family of malarial merozoite surface structure protein genes mainly regulates the invasion of malarial merozoites into red blood cells. The main references are as follows: DOI: 10.1016 / j.chom.2017.07.003; DOI: 10.1016 / j.chom.2017.05.006); Figure 3 C shows the intersection of invasion-related target genes after conditional knockdown of PfAP2-O5 and inhibition of PfAP2-O5 function by triazoloquinoline derivatives.

[0152] Example 6 In vitro growth inhibition EC50 determination of triazoloquinoline derivatives on Plasmodium

[0153] 100 μL of complete medium was added to a 96-well plate, 200 μL of compound stock solution with a concentration of 200 μM was added to the first well, and gradient dilution was performed according to a 1 / 2 ratio (11 concentration gradients). Dihydroartemisinin (DHA) was used as a positive control, complete medium was used as a negative control, and red blood cells without Plasmodium and compounds were used as a background control.

[0154] Accurately add 100 μL of wild-type Plasmodium culture (1% parasitemia, 4% hematocrit) to each well, so that the final parasitemia is 0.5% and the hematocrit is 2% per well, and the final concentration gradient of the compound is (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM, 0.390625 μM, 0.195313 μM, 0.097656 μM), and all samples are set in triplicate; after the addition is completed, the 96-well plate is placed in a 37°C incubator (5% CO2, 5% O2) for accurate incubation for 72 h. After the incubation is completed, 100 μL of Lysis buffer (1x SYBR Green I, 0.12 mg / mL saponin, 0.12% v / v Triton X-100, 30 mM Tris-HCl, and 7.5 mM EDTA) is added to each well, mixed well, and then incubated at room temperature in the dark for 2 h. After the incubation is completed, the fluorescence intensity is recorded on a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm, and the inhibition rate of the small molecule inhibitor on the growth of Plasmodium is calculated according to the fluorescence intensity.

[0155] Survival rate = (Fl drug treatment - Fl background) / (Fl control - Fl background)

[0156] wherein Fl drug treatment, Fl background, and Fl control represent the fluorescence intensity values of the candidate small molecule drug, the red blood cell control, and the no-drug treatment control, respectively.

[0157] The inhibition rate of the drug is calculated according to the survival rate at different concentrations, and the growth inhibition curve is summarized in terms of inhibition rate-drug concentration, and the IC50 is calculated by SPSS 18.0 software.

[0158] The results are shown in Table 1. Figure 4 The quantitative growth inhibition effect IC50 of the triazoloquinoline derivative on the in vitro cultured Plasmodium is 2.63 μM.

[0159] Example 7 Determination of IC50 of cytotoxicity of small molecule inhibitors

[0160] After the mammalian cells 293T and HepG2 are cultured and passaged, they are trypsinized, resuspended in DMEM medium, counted by a hemocytometer, and diluted to 10 5Cells were cultured at 100 μL per well in 96-well plates at 37°C (5% CO2) for 24 hours. Then, the appropriate concentration of inhibitor was added, and the plates were serially diluted (11 concentration gradients) at a 1 / 2 ratio. Complete culture medium was used as a negative control, and DMEM medium as a background control. After 72 hours of incubation at 37°C (5% CO2), 10 μL of CCK8 reagent was added to each well, and the plates were incubated at 37°C (5% CO2) for 1 hour. The absorbance was read at 450 nm using a microplate reader, and the viability was calculated based on the absorbance.

[0161] Survival rate = (Ab drug treatment - Ab background) / (Ab control - Ab background)

[0162] Wherein, Ab drug treatment, Ab background, and Ab control represent the absorbance values ​​of the small molecule drug, DMEM control, and no-drug control group, respectively. The drug inhibition rate was calculated based on the survival rate at different concentrations, and the growth inhibition curve was summarized as inhibition rate-drug concentration. The IC50 was calculated using SPSS 18.0 software.

[0163] The results are as follows Figure 5 As shown. Figure 5 It can be seen that the triazoloquinoline derivative of formula (I) described in this invention has an IC50 of 2.63 μM against the novel merozoon protein of Plasmodium falciparum strain 3D7. Meanwhile, Figure 5 Triazoloquinoline derivatives reflecting the structure of formula (I) of this invention exhibit low cytotoxicity to mammalian cells and good biosafety.

[0164] Example 8: Pharmacokinetic Analysis of Small Molecule Inhibitors

[0165] This experiment aims to investigate the species differences in the metabolic rate of triazoloquinoline derivatives with the structure of formula (Ⅰ) in the liver microsomes of mice, rats, and humans.

[0166] The microsomal metabolism assay involved incubating the inhibitor with liver microsomes of different species, terminating the reaction at different time points, and determining the remaining amount of inhibitor in the samples at each time point using LC-MS / MS. The percentage of the original drug remaining was calculated, and the intrinsic hepatic clearance rate (CL) was ultimately obtained. int(liver) Testosterone was used as a control in this study to test the reliability of the experimental system. The specific procedure is as follows:

[0167] The incubation system consists of 0.1 M Tris-HCl buffer (pH 7.4), 1 mM MgCl2, 1 mM NADPH and 0.5 mg mL-1microsomes, and the final volume of the reaction is 200 μL. After pre-incubation of 1 M inhibitor and testosterone with liver microsomes of different species of animals in a 37 °C water bath for 5 min, 100 μL of NADPH is added to start the reaction, and 400 μL of termination solution is added to terminate the reaction at 0 min, 5 min, 15 min, 30 min, 45 min and 60 min, respectively. After vortexing for 5 min, 40 μL of internal standard is added, and the mixture is centrifuged at 20000 g and 4 °C for 5 min. 100 μL of supernatant is taken and diluted with 100 μL of water, and then the sample is analyzed. The retention time of the compound and internal standard, chromatogram acquisition and chromatogram integration are processed by software Analyst 1.6.3, and the compound is linearly regressed with 1 / X 2 The concentration at each time point is calculated. The natural logarithm of the percentage remaining at each time point is linearly regressed against the incubation time to obtain the in vitro elimination rate constant 6. The half-life (T 1 / 2 ) and the liver intrinsic clearance CL int(liver) of the test sample are calculated according to the following formula.

[0168] The liver microsomal clearance C Lint(mic) = 0.693 / half-life / mg microsomal protein per mL; the liver intrinsic clearance CL int(liver) = liver microsomal clearance x mg microsomal protein / g liver weight x liver weight / body weight ratio, and the liver weight / body weight ratios of mice, rats and humans are 88, 40 and 20 g / 6 g, respectively; the average value of mg microsomal protein / g liver weight in different species is 45. The results of the metabolism of the triazoloquinoline derivatives in mouse / rat / human microsomes are shown in Table 1

[0169] Table 1

[0170]

[0171] The results of the liver microsomal metabolism show that the inhibitor is a medium clearance compound in rat and human liver microsomes and a high clearance compound in mice.

[0172] Example 9. Cyclic body survival experiment of triazoloquinoline derivatives on resistant insect strains

[0173] This experiment aims to evaluate the killing effect of triazoloquinoline derivatives as a compatible drug on drug-resistant insect strains by the drug resistance gold standard method. The specific implementation steps are as follows:

[0174] 1. When the parasitic rate of Plasmodium is greater than 4% and most of them are in the R phase, Sorbitol is synchronized for 10 min, and vortexed for 5 s;

[0175] 2. 30-48h (depending on the stage of the parasite) after the Sorbitol synchronization is repeated;

[0176] 3. After about 30h of culture, smear microscopy: if the proportion of mature S phase (10-12 merozoites) is greater than 0.5%, proceed to step 4; if the proportion of mature S phase (10-12 merozoites) is less than 0.5%, repeat step 2;

[0177] 4. Collect the culture for late stage enrichment of the parasites (Plasmion or Percoll); smear microscopy of the enriched parasites, S phase must be greater than 10% and R phase must be less than 10%;

[0178] 5. Add 10ml of culture medium, 200ul (appropriate amount) of fresh red blood cells, maintain the culture for 3h (must be precise);

[0179] 6. After mixing the culture, take about 200ul, centrifuge and smear microscopy (can be done about 10min in advance), quickly calculate the rate of protozoa (must be greater than 0.5%);

[0180] 7. Collect the culture for Sorbitol synchronization for 10min, vortex for 5s, centrifuge and discard the supernatant;

[0181] 8. In the 24-well plate: control group: add 100ul of DMSO control solution to each well

[0182] Experimental group: add 100ul of 7mM DHA solution and 20μM triazoloquinoline derivative solution (dissolved in culture medium) to each well;

[0183] 9. Add 900ul of resuspended iRBC culture to each well, mix well; maintain the culture for 6h precisely;

[0184] 10. After 6h, transfer the culture in each well to a 1.5ml EP tube, centrifuge to remove the supernatant;

[0185] 11. Add 1ml of preheated culture medium for washing, centrifuge to remove the supernatant; repeat the washing once, a total of two times;

[0186] 12. Add 1ml of preheated culture medium, mix well, transfer to a new well plate, continue to maintain the culture for 66h;

[0187] 13. After 66h of culture, smear microscopy to calculate the rate of protozoa, and calculate the survival rate.

[0188] The results are as follows Figure 6As shown, triazoloquinoline derivative (inhibitor) represents, "-" represents no corresponding material, "+" represents containing the corresponding material, the results show that the combination of DHA can kill the artemisinin-resistant strain more effectively, suggesting that small molecules can be used as a compatible drug to improve the spread of artemisinin resistance.

[0189] Obviously, the above-mentioned embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can also be made by those skilled in the art. Here, it is not necessary and impossible to enumerate all the embodiments. Any modification, equivalent replacement and improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. Use of a triazoloquinoline derivative or a pharmaceutically acceptable salt thereof having a structure as shown in formula (I) for the manufacture of an inhibitor of transcription factor PfAP2-O5 of Plasmodium falciparum: ###0001### Formula (I).

2. Use of a triazoloquinoline derivative or a pharmaceutically acceptable salt thereof having a structure as shown in formula (I) for the manufacture of a medicament for inhibiting Plasmodium falciparum: ###0002### Formula (I).

3. Use of a triazoloquinoline derivative or a pharmaceutically acceptable salt thereof having a structure as shown in formula (I) for the manufacture of a medicament for preventing or treating malaria caused by Plasmodium falciparum: ###0003### Formula (I).

4. A pharmaceutical composition comprising a triazoloquinoline derivative or a pharmaceutically acceptable salt thereof having a structure as shown in formula (I) as an active ingredient, and a pharmaceutically acceptable carrier, diluent or excipient as an adjuvant: ###0004### Formula (I).

5. The pharmaceutical composition according to claim 4, wherein the active ingredient further comprises an artemisinin compound.

6. The pharmaceutical composition according to claim 5, wherein the artemisinin compound is a dihydroartemisinin compound.

4. A pharmaceutical composition, characterized by, 7. The pharmaceutical composition according to claim 4, wherein the molar ratio of the triazoloquinoline derivative or a pharmaceutically acceptable salt thereof having a structure as shown in formula (I) to the artemisinin compound in the composition is 1:500 to 500:

1.

8. The pharmaceutical composition according to claim 4, wherein the dosage form is an oral dosage form or an injectable dosage form.

5. The pharmaceutical composition according to claim 4, wherein 9. Use of the pharmaceutical composition according to claim 4 for the manufacture of a medicament for preventing and / or treating malaria.

6. The pharmaceutical composition according to claim 5, wherein 10. Use of the pharmaceutical composition according to claim 4 for the manufacture of a medicament for treating malaria resistant to artemisinin and artemisinin compounds.

7. The pharmaceutical composition according to claim 5, wherein ​ 8. The pharmaceutical composition according to claim 4, wherein ​ ​ ​

Images