Application of hexahydroquinoline derivatives in preparing drugs for preventing or treating malaria

Abstract

This invention discloses novel applications of hexahydroquinoline derivatives or their pharmaceutically acceptable salts or hydrates. These hexahydroquinoline derivatives can inhibit the expression of the invading gene PfAP2-O5, blocking the invasion of erythrocytes by Plasmodium falciparum, affecting its growth and development, and achieving a killing effect on Plasmodium falciparum. Therefore, these hexahydroquinoline derivatives can serve as inhibitors of PfAP2-O5, drugs for inhibiting Plasmodium falciparum, or drugs for the prevention or treatment of malaria.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the use of hexahydroquinoline derivatives in the preparation of drugs for the prevention or treatment of malaria. Background Technology

[0002] Malaria is a parasitic disease caused by infection with the protozoan Plasmodium parasite. It poses a serious threat to human health and life, with a high morbidity and mortality rate among infectious diseases. According to the 2021 WHO World Malaria Report, malaria is endemic in 87 countries and regions worldwide, with 241 million new cases and approximately 627,000 deaths in 2020.

[0003] The life cycle of Plasmodium is complex, mainly consisting of sexual reproduction in the definitive host and asexual reproduction in the intermediate host. In humans, Plasmodium (spores) first invades the liver, undergoes one cycle of schizogony for asexual reproduction, and releases merozoites into the bloodstream to invade red blood cells, beginning the schizogony phase within the red blood cells. This phase is the clinical onset of malaria, and the invasion of red blood cells by merozoites is a key step in the pathogenesis of malaria.

[0004] Currently, drug therapy is the primary means of malaria prevention and control, with artemisinin-based combination therapies effectively curbing the spread of malaria worldwide. However, with the long-term and widespread use of artemisinin-based drugs, strains with decreased susceptibility to these drugs have gradually emerged in Southeast Asia and even Africa. The emergence and global spread of these strains pose a serious challenge to global malaria control and elimination efforts. Therefore, finding new targets for antimalarial drugs, especially developing safe and effective transmission-blocking drugs targeting key pathogenic mechanisms of Plasmodium, is of paramount importance in curbing the spread of malaria. Summary of the Invention

[0005] This invention provides a new drug for the prevention or treatment of malaria to overcome the above-mentioned shortcomings.

[0006] Another object of the present invention is to provide new applications for hexahydroquinoline derivatives.

[0007] The above-mentioned objectives of the present invention are achieved through the following technical solutions:

[0008] The use of hexahydroquinoline derivatives or pharmaceutically acceptable salts or hydrates thereof in the preparation of inhibitors of the transcription factor PfAP2-O5 of Plasmodium falciparum, wherein the hexahydroquinoline derivatives have the structure shown in formula (I):

[0009]

[0010] Equation (Ⅰ).

[0011] The inventors conducted in-depth research on the mechanism of Plasmodium falciparum invading erythrocytes and discovered for the first time the positive regulatory function of PfAP2-O5 on erythrocyte invasion. The hexahydroquinoline derivative shown in formula (I) can block the invasion of Plasmodium falciparum into erythrocytes by inhibiting the expression of the invading gene PfAP2-O5, thereby affecting its growth and development and achieving the killing effect of Plasmodium falciparum.

[0012] The PfAP2-O5 inhibitor described in this invention refers to a compound that achieves the effect of killing Plasmodium falciparum by inhibiting the function of AP2-O5.

[0013] Meanwhile, in in vitro experiments, the hexahydroquinoline derivative shown in formula (I) exhibited good growth-inhibiting effects on Plasmodium falciparum, while being virtually non-toxic to mammalian cells, making drug development possible. It also showed good killing effects against resistant strains, which is helpful in delaying the development and spread of drug resistance, and will allow for more efficient antimalarial treatment.

[0014] The use of hexahydroquinoline derivatives or pharmaceutically acceptable salts or hydrates thereof in the preparation of drugs for inhibiting Plasmodium falciparum; said hexahydroquinoline derivative having a structure as shown in formula (I):

[0015]

[0016] Equation (Ⅰ).

[0017] The use of hexahydroquinoline derivatives or pharmaceutically acceptable salts or hydrates thereof in the preparation of medicaments for the prevention or treatment of malaria caused by Plasmodium falciparum, wherein the hexahydroquinoline derivative has a structure as shown in formula (I):

[0018]

[0019] Equation (Ⅰ).

[0020] In this invention, the "pharmaceutically acceptable salt" includes salts formed by the hexahydroquinoline derivative and inorganic or organic acids.

[0021] The inorganic acids are, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid. The organic acids are, for example, acetic acid, glycolic 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 acids, cyclic salicylic acid, and para-aminosalicylic acid.

[0022] In this invention, "hydrate" refers to a salt containing water molecules combined in a certain proportion as a component of a crystal.

[0023] A pharmaceutical composition comprising a hexahydroquinoline derivative comprising a pharmaceutically acceptable salt or hydrate thereof having the structure shown in formula (I) as an active ingredient, and a pharmaceutically acceptable carrier, diluent or excipient as an adjuvant;

[0024]

[0025] Equation (Ⅰ).

[0026] In preparing these compositions, the active ingredient is typically mixed with an excipient, diluted with an excipient, or encapsulated in a carrier that may be in the form of a capsule or pouch. When the excipient acts as a diluent, it can be a solid, semi-solid, or liquid material serving as the medium for the excipient, carrier, or active ingredient.

[0027] Examples of suitable excipients include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. In addition, the composition may include lubricants (such as talc, magnesium stearate, and mineral oil), humectants, emulsifiers and suspending agents, preservatives (such as methylparaben and propylparaben), sweeteners, or flavoring agents.

[0028] Therefore, the pharmaceutical composition can be prepared into various known dosage forms, preferably, the dosage form of the pharmaceutical composition is an oral dosage form or an injectable dosage form.

[0029] Oral dosage forms include solid dosage forms such as tablets, pellets, immediate-release pellets, capsules (such as liquid gel capsules and solid gel capsules), granules, or powders. Tablets can be compressed tablets, ground tablets, enteric-coated tablets, sugar-coated tablets, film-coated tablets, or multilayer compressed tablets. Suitable adjuvants for tablet preparation include, but are not limited to, binders, lubricants, diluents, disintegrants, colorants, flavoring agents, flow diverters, or melting agents. Oral dosage forms can also be liquid oral dosage forms, such as aqueous solutions, emulsions, suspensions, solutions and / or suspensions made from non-effervescent particles, or effervescent tablets made from effervescent particles. Suitable adjuvants for liquid oral dosage forms include solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, melting agents, colorants, or flavoring agents.

[0030] The injectable dosage forms include: injection solutions (ordinary), lyophilized powder for injection, powder for injection (ordinary), and tablets for injection.

[0031] Specifically, the pharmaceutical composition may be tablets, pills, powders, elixirs, suspensions, emulsions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0032] Through growth inhibition experiments on Plasmodium falciparum, with artemisinin as a control, the EC50 value of this invention for inhibiting the growth of Plasmodium falciparum reached 2.158 μM, demonstrating good growth inhibition function.

[0033] The pharmaceutical composition may be a hexahydroquinoline derivative of the structure shown in formula (I) or a pharmaceutically acceptable salt or hydrate thereof as the sole active ingredient, or may include known artemisinin-like compounds as common active ingredients.

[0034] Preferably, the active ingredient further includes artemisinin compounds. Artemisinin compounds have been shown to have therapeutic effects against malaria. More preferably, the artemisinin compound is a dihydroartemisinin compound. Dihydroartemisinin compounds are more effective against various drug-resistant Plasmodium parasites than conventional artemisinin compounds.

[0035] Preferably, in the composition, the molar ratio of the hexahydroquinoline derivative of formula (I) or its pharmaceutically acceptable salt or hydrate to the artemisinin-like compound is 1:500 to 500:1.

[0036] Preferably, the dosage form of the pharmaceutical composition is an oral dosage form or an injectable dosage form.

[0037] The use of the pharmaceutical composition in the preparation of drugs for the prevention and / or treatment of malaria.

[0038] Preferably, the pharmaceutical composition is used in the preparation of a medicament for treating malaria resistant to artemisinin and its analogues.

[0039] Compared with the prior art, the beneficial effects of the present invention are:

[0040] This invention provides a novel application of hexahydroquinoline derivatives or their pharmaceutically acceptable salts or hydrates, which can inhibit the expression of the invading gene PfAP2-O5, block the invasion of erythrocytes by Plasmodium falciparum, affect its growth and development, and achieve the killing effect of Plasmodium falciparum. Therefore, the hexahydroquinoline derivative can be used as an inhibitor of PfAP2-O5, a drug for inhibiting Plasmodium falciparum, or a drug for the prevention or treatment of malaria. Based on the above findings, this invention provides a novel pharmaceutical composition, which can use the hexahydroquinoline derivative alone as an active ingredient, or combine it with artemisinin-like compounds as a common active ingredient; this pharmaceutical composition can be used to prepare drugs for treating malaria, especially drugs for treating malaria resistant to artemisinin and its analogues. Attached Figure Description

[0041] Figure 1 middle, Figure 1A shows the WB identification results of the PfAP2-O5 gene knockout strain. After the PfAP2-O5 gene was knocked down, the expression level of PfAP2-O5 protein was reduced. Figure 1 B shows the growth curve test of the PfAP2-O5 gene knockout strain.

[0042] Figure 2 The results show the distribution of PfAP2-O5 across the entire genome using ChIP-seq analysis, as well as the types of target genes that PfAP2-O5 binds to, most of which are invasive gene families.

[0043] Figure 3 This is a schematic diagram illustrating the inhibitory effect of hexahydroquinoline derivatives on the expression of invading genes. Figure 3 A used RNA-Seq technology to determine the expression of the invasive gene in the PfAP2-O5 gene knockout strain; Figure 3 B shows the expression of the invading gene after the hexahydroquinoline derivative inhibited the function of the PfAP2-O5 gene; Figure 3 C shows the inhibitory effect of PfAP2-O5 gene knockdown and hexahydroquinoline derivative treatment on the expression of invading genes.

[0044] Figure 4 This is a schematic diagram illustrating the quantitative growth inhibitory effect of hexahydroquinoline derivatives on cultured Plasmodium malariae. The EC50 value for its quantitative growth inhibitory effect on cultured Plasmodium malariae is shown to be 2.158 μM. The growth of Plasmodium malariae was determined using a 3-day SYBR Green I cell growth assay.

[0045] Figure 5 This is a schematic diagram illustrating the cytotoxic effects of the hexahydroquinoline derivative on the human renal epithelial cell line 293T and the human hepatocellular carcinoma cell line HepG2.

[0046] Figure 6 To investigate the antagonistic insecticidal effect of the hexahydroquinoline derivative in combination with dihydroartemisinin, a cyclic survival experiment was used to clarify the inhibitory effect of the combined drug on the growth of antagonistic insecticidal strains. Detailed Implementation

[0047] The technical solutions in the embodiments of the present invention will be clearly and completely described below, but the implementation of the present invention is not limited thereto.

[0048] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0049] 1. The hexahydroquinoline derivative with the structure shown in formula (Ⅰ): purchased from ChemDiv ID. 4023-0687

[0050] 2. Plasmodium falciparum (3D7_G7): Purchased from ATCC

[0051] 3. 293T cell line: purchased from ATCC

[0052] 4. HepG2 cell line: purchased from ATCC

[0053] 5. SYBR Green I: Purchased from Invitrogen Cat. No. S-7585

[0054] 6. Trypsin-EDTA: Purchased from Gbico Cat. No. 25200-072

[0055] 7. DMEM culture medium: purchased from Gbico Cat. No. 11965-092

[0056] 8. FBS: Purchased from Gbico Cat. No. 10082-147

[0057] 9. Double-stranded DNA Quantitative Detection Kit: Purchased from ThermoFisher, Cat. No. P7581

[0058] 10. Protein A / G magnetic beads: purchased from ThermoFisher, Cat. No. 88803

[0059] 11. Mouse monoclonal antibody: purchased from Sigma Cat. No. SAB4800032

[0060] 12. 37% formaldehyde solution: purchased from Sigma, Cat. No. 47608-250ML-F

[0061] 13. 96-well plate: purchased from Corning

[0062] 14. Red blood cells: purchased from Shanghai Blood Center

[0063] 15. Dihydroartemisinin (CAS: 71939-50-9)

[0064] 16. The components of various buffer solutions are as follows:

[0065] (1) Lysis buffer

[0066]

[0067] (2) Ultrasonic buffer solution

[0068]

[0069] (3) ChIP dilution buffer

[0070]

[0071] (4) Low-salt immune complex washing buffer

[0072]

[0073] (5) High-salt immune complex washing buffer

[0074]

[0075] (6) Lithium chloride immune complex washing buffer

[0076]

[0077] (7) TE buffer

[0078]

[0079] (8) Elution buffer

[0080]

[0081] Example 1: Culture of Plasmodium

[0082] Plasmodium falciparum was cultured in RPMI 1640 complete medium in a 37°C incubator (5% CO2, 5% O2 and 90% N2).

[0083] 0.5L RPMI 1640 complete culture medium contains: 5.22g RPMI-1640 powder; 1.0g sodium bicarbonate; 5g Albmax I; 2.98g 25 mM HEPES; 13.6 mg hypoxanthine; 10 mg gentamicin sulfate.

[0084] Example 2: Construction of transgenic insect strain: PfAP2-O5 conditional knockout strain

[0085] This experiment used the CRISPR / Cas9 gene editing system to clone guide RNA into a vector and construct a PfAP2-O5 conditional knockout strain.

[0086] The specific construction steps are as follows:

[0087] 1. Construction of the transgenic insect knockdown vector (named pL6cs-ap2-o5- glmS )

[0088] The guide RNA was cloned into a vector using the CRISPR / Cas9 gene editing system, as follows:

[0089] (1) gRNA design

[0090] The sequence of the PfAP2-O5 gene of Plasmodium falciparum strain 3D7-G7 was downloaded from the PlasmoDB website (from the database https: / / plasmodb.org / plasmo / app, Plasmodium falciparum: PF3D7_1449500 (plasmodium falciparum) 715aaPfAP2-O5. This sequence is also disclosed in NCBI: Sequence ID: XM_001348609.1). A 20bp specific sequence was selected as the guide RNA (gRNA) through BLAST alignment, as follows: 5′-ACCAAGTCATATGTCATTAA -3′.

[0091] (2) Primer design

[0092] Primer design using SnapGene software:

[0093] P1: 5′-GCCCTAGTCTAG GGcGCGCC CAATTTTAATAACAATTTGGCG-3′;

[0094] P2: 5′-CAAGTAAAATAATTGGTCTTTAAGTGACATATGACTTGGTAGTTTACTG-3′;

[0095] P3: 5′-AGCGGCCGCAAAAAAAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATATATATTATAC-3′;

[0096] 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 introduction of the synonymous mutation site was to prevent the gRNA from self-cutting the plasmid.

[0097] P7: 5′-TATTTTACTTGGACAAATTAGGTACCGGAAGTGGTTCTGG-3′;

[0098] P8: 5′- TTTATTTTTTTTGCGGCCGCGCGGCCGCGTCCCCTC-3′ is used to amplify the exogenous green fluorescent protein (GFP) gene.

[0099] (3) Gene amplification

[0100] DNA was extracted from the Plasmodium falciparum 3D7-G7 strain. The left and right homologous sequences of the PfAP2-O5 gene were amplified using primers P1+P2 and P3+P4, respectively, with mutation sites introduced. Primer P7+P8 was then used for amplification. glmS Ribozyme sequence (glmS sequence is from the literature report: doi:10.1371 / journal.pone.0073783).

[0101] (4) Construction of the linker product

[0102] Use primers to connect the left and right homologous sequences to... glmS Ribozyme sequences are ligated to obtain ligation products.

[0103] The 3' end marker of the PfAP2-O5 gene is... glmS Ribozyme sequence.

[0104] The reaction system is as follows: 2 × Phanta Max Master Mix (Dye Plus): 25 μL, P1 primer (10 μM): 2 μL, P4 primer (10 μM): 2 μL, 1 μL each of the left and right homologous sequences and the glmS ribozyme sequence, and ddH2O to make up to 50 μL.

[0105] The PCR conditions were as follows: 95℃ for 3 min, 95℃ for 15 s, 65℃ for 15 s, 65℃ for 30 s / kb, 65℃ for 5 min, for a total of 30 cycles; 72℃ for 10 min.

[0106] (5) Construction of knockdown vector

[0107] The plasmid pL6cs carrying the resistance gene for the screening drug dihydrofolate reductase inhibitor (WR) was digested with XhoⅠ and AvrⅡ (the plasmid was constructed in our laboratory; the specific sequence can be found in the literature: doi:10.3389 / fmicb.2020.625862). The product was recovered and purified using a gel extraction kit. The gRNA fragment was ligated with DNA ligase and transformed into Escherichia coli XL-10 competent cells. The cells were inoculated onto LB agar plates containing 100 μg / ml ampicillin and cultured overnight. Single colonies were picked and cultured overnight at 34 ℃ with shaking at 200 rpm / s. The plasmid was then extracted and sequenced.

[0108] The plasmid containing gRNA was identified by sequencing, and then double-digested with AscⅠ and AflⅡ. The product was recovered and ligated to a homologous arm containing the glmS ribozyme sequence for transformation. Single clones were selected for culture, and the correct plasmid was identified by PCR and sequenced. The bacterial strain was stored at -80℃.

[0109] The transgenic insect knockdown vector was obtained and named pL6cs-ap2-o5- glmS .

[0110] 2. Constructing a PfAP2-O5 conditional knockout strain

[0111] A transgenic insect strain, PfAP2-O5 conditional knockout strain, was constructed using electrotransfection combined with drug screening.

[0112] (1) Preparation of electroporation transfection plasmids

[0113] The correctly sequenced bacterial strain was inoculated onto LB agar plates containing 100 μg / ml ampicillin and cultured overnight. The next day, single clones were picked and cultured in LB medium at 34°C and 200 rpm / s for 8 hours with shaking. The culture was then diluted 1:1000 to 400 ml of LB medium and cultured with shaking for 14 hours. The bacterial cells were then collected, and the plasmids were collected using a plasmid DNA purification kit.

[0114] The transgenic insect knockdown vector pL6cs-ap2-o5- constructed above was used. glmS 100 μg each of plasmid and plasmid pUF1-Cas9 (a self-constructed plasmid, specific sequence can be found in the literature: doi:10.3389 / fmicb.2020.625862) carrying the resistance gene of the screening drug blast fungicide (BSD) and expressing the Cas9 protein were added to the plasmid solution. After mixing, 2.5 times the volume of anhydrous ethanol was added to the plasmid solution and mixed thoroughly. The mixture was centrifuged at 13000×g for 30 min, the supernatant was discarded, the precipitate was washed twice with 75% ethanol, dried in a clean bench, and dissolved in 150 μl of sterile water.

[0115] (2) Electroporation and screening of transgenic insect strains

[0116] The electroporator was set to the following conditions: electroporation parameters of 310V, 950μF, ∞ resistance, and 2mm slit. An equal volume (150μl) of 2× cell mixture was added to the prepared plasmid and mixed well. Then, 150μl of red blood cells were added, mixed well, and transferred to an electroporation cuvette. The cuvette was then placed in the electroporator for electroporation. After electroporation, the mixture was transferred to a culture flask, and an appropriate amount of culture medium was added. Subsequently, purified schizont-stage Plasmodium was added.

[0117] The culture was carried out in a 37°C tri-gas incubator. Starting from the second day, the culture medium was changed daily with fresh medium. On the fourth day, when the transfection density reached about 10% (the protozoan rate, the proportion of red blood cells infected with Plasmodium to all red blood cells in a certain field of view (~5000 cells), 200 μl of fresh red blood cells were added, the culture medium was changed, and the selection drugs WR and BSD (methamidophos) were added. The selection drugs can kill wild-type Plasmodium falciparum, while the successfully transgenic Plasmodium is unaffected by the drugs because it carries a plasmid expressing the drug resistance gene. Therefore, transgenic Plasmodium can be screened out.

[0118] After adding screening drugs until most wild-type Plasmodium parasites were killed, the culture medium was changed and drugs were added every 3-4 days to maintain the culture. Smear microscopy was started on day 21 post-transfection, and transgenic parasite strains were obtained in 3-6 weeks.

[0119] In summary, the transgenic insect strain PfAP2-O5 conditional knockout strain was constructed.

[0120] 3. Gradient dilution cloning

[0121] The transgenic parasite strain obtained from the transgene (PfAP2-O5 conditional knockdown strain) is an integrated strain and requires serial dilution cloning, i.e., screening for completely integrated monoclonal strains using a limiting dilution method. This experiment was conducted in 96-well cell culture plates. Through serial dilution, the parasite-infected red blood cells were diluted to theoretically contain 10, 1, 0.5, and 0.1 parasites per ml, respectively. The specific experimental steps are as follows:

[0122] (1) Synchronize the transgenic insect strains with sorbitol to obtain annular stage insects with relatively uniform properties;

[0123] (2) Examine smears under a microscope during the trophozoite stage of the parasite and strictly count the original protozoan rate Px;

[0124] (3) Collect trophoblastic culture, centrifuge at 2000 rpm at room temperature for 5 min, discard the supernatant, and obtain about 200 µl of infected erythrocyte hematocrit (iRBC).

[0125] (4) Add 800 µl of fresh culture medium to iRBC, mix gently, and then transfer to a 1.5 ml centrifuge tube to a final volume of 1 ml.

[0126] (5) Centrifuge at 1500 rpm at room temperature for 2-3 min, discard the supernatant, and transfer 50 µl of iRBC to a new centrifuge tube for later use;

[0127] (6) Dilute the above 50 µl of protozoa infecting red blood cells to 1% according to the formula 50 µl × P x = 1% × (50 µl + Vx) to obtain culture A (Vx is the volume of fresh red blood cells added), and confirm the protozoa rate after dilution by microscopic examination of the smear.

[0128] (7) Preheat the fresh culture medium, and according to the principle of gradient dilution, dilute the infected red blood cells with a protozoan rate of 1% prepared above in sequence so that each centrifuge tube corresponds to a gradient:

[0129] Prepare nine 15 ml centrifuge tubes and number them 1, 2, 3...9. Add 10 ml of fresh culture medium to tube 1, 9 ml of fresh culture medium to tubes 2-7 and 9, and 5 ml of fresh culture medium to tube 8. Incubate all centrifuge tubes at 37°C. o Preheat in incubator C for later use. Add 100 µl of culture A (6) with a protozoan content of 1% to tube 1 above to obtain 1×10⁻⁶ cells / mL. 6 Culture B of 1 / mlliRBC;

[0130] 1) Take 1 ml of culture B into tube 2 to obtain 1×10 5 Culture C of iRBCs / ml;

[0131] 2) Take 1 ml of culture C into tube 3 to obtain 1×10 4 Culture D of iRBCs / ml;

[0132] 3) Take 1 ml of culture D into tube 4 to obtain 1×10⁻⁶ 3 Culture E of iRBCs / ml;

[0133] 4) Transfer 1 ml of culture E to tube 5 to obtain 1×10⁻⁶ 2 Culture F of iRBCs / ml;

[0134] 5) Take 1 ml of culture F into tube 6 to obtain culture G with 10 iRBCs / ml;

[0135] 6) Take 1 ml of culture G into tube 7 to obtain culture H with 1 iRBC / ml;

[0136] 7) Take 5 ml of culture H into tube 8 to obtain culture I with 0.5 iRBCs / ml;

[0137] 8) Take 1 ml of culture H into tube 9 to obtain culture J with 0.1 cells / ml iRBC.

[0138] (8) Transfer the different gradient cultures G, H, I and J obtained above to 96-well cell culture plates, 200 µl / well, so that each row of 12 wells in the 96-well plate corresponds to one gradient.

[0139] (9) During the later stage of culture, fresh culture medium was changed every 2 days and fresh red blood cells (2 µl / well) were added every 6 days to maintain the culture.

[0140] About two weeks later, smears were examined under a microscope until live insects were detected. The culture was then gradually expanded using 24-well, 12-well, and 6-well plates. After the genome was confirmed to be correct, a fully integrated monoclonal PfAP2-O5 transgenic insect strain (PfAP2-O5 conditional knockout strain) was obtained. The strain was preserved and subsequent experiments were conducted.

[0141] Example 3: Identification and growth and development test of PfAP2-O5 transgenic insect strain (PfAP2-O5 conditional knockout strain)

[0142] Cultures of PfAP2-O5 transgenic parasites (PfAP2-O5 conditional knockout strain) at the circumvallate (10-15h), trophozoite (28-32h), and schizont (40-44h) stages were collected. Protein samples were collected by lysing infected erythrocytes with 0.15% saponin, and proteins were separated by SDS-PAGE electrophoresis. The proteins were then transferred to nitrocellulose membranes using a semi-dry transfer method with the following parameters: 0.1A, 2h. After transfer, add an appropriate volume of blocking buffer (5% skim milk powder: skim milk powder dissolved in PBST solution) and block at room temperature for 2 hours; incubate with primary antibody (mouse-derived anti-Ty1 antibody): the primary antibody (anti-Ty1) is diluted with 5% skim milk powder at a ratio of 1:1000, and the membrane is sealed and incubated overnight at 4°C; incubate with secondary antibody: the secondary antibody (goat anti-mouse) is diluted with 5% skim milk powder at a ratio of 1:5000 and incubated at room temperature for 2 hours; develop color: mix equal volumes of color development solutions A and B (protect from light), add a certain amount of color development solution to the membrane, and take a photograph to save the exposed image. The results are as follows. Figure 1 As shown in A, Figure 1 A shows the Western blot analysis of the PfAP2-O5 conditionally knocked-down strain before and after the addition of 5 mM glucosamine (GlcN), indicating that PfAP2-O5 was effectively knocked down.

[0143] The growth curve test of PfAP2-O5 conditional knockout strains involved several synchronizations to the annular stage, with strict counting to determine the initial protozoan percentage. The strains were divided into two bottles (one with the appropriate concentration of GlcN, and the other as a control without GlcN), each with an initial protozoan percentage of approximately 0.1%. Four life cycles were continuously cultured, and late-stage smears from each life cycle were examined microscopically to count the protozoan percentage (if the percentage was high, the culture medium was changed, and the drug-treated group was supplemented with the drug). The protozoan percentages in the drug-treated and untreated groups were recorded, and growth curves were plotted. Results are as follows: Figure 1 As shown in B, Figure 1 B shows the growth curve tests of wild-type and PfAP2-O5 conditional knockout strains, indicating that the growth of Plasmodium is significantly inhibited after PfAP2-O5 conditional knockout.

[0144] Example 4: Chromatin Immunoprecipitation (ChIP) Sequencing

[0145] Chromatin immunoprecipitation (ChIP) sequencing utilizes the specificity of antigen-antibody reactions to accurately reflect the distribution of AP2-O5-bound target genes across the entire genome at the chromatin level, while also reflecting the binding of proteins to target genes. The specific procedure is as follows:

[0146] Collect the schizont stage culture after synchronization treatment, add 1% formaldehyde solution, and incubate at 37℃ for 10 min; add pre-chilled 0.125 M glycine solution and incubate on ice for 5 min; centrifuge at 2500 rpm, 4℃ for 5 min, discard the supernatant, add 30 mL of 1×PBS to resuspend the red blood cells, add 0.15% saponin to lyse the red blood cells and collect the parasites; add 2 mL of pre-chilled Lysis Buffer to resuspend the parasites, transfer to pre-chilled grinding tubes, and incubate on ice for 30 min; add 200 μL of sonication buffer, resuspend the precipitate, and sonicate in liquid chromatography with the following parameters: duty cycle 5%, peak incident power 75 W, cycle count 200, temperature 7℃, and time 30 min; take 10 μL of the sonicated mixture, add 90 M ChIP dilution buffer, 4 μL 5 M NaCl, and 2.5 μL proteinase K, mix well, and incubate at 50℃ for 2 h; add 2 μL of LRNase A, and incubate at 37℃ for 45 min; QIAGEN PCR was used to extract the chromatin, and the sonication effect was detected by 2% agarose gel electrophoresis. For products with ideal sonication effect, centrifugation at 14000 pm at 4°C for 10 min was performed, and the supernatant was collected. Ten volumes of ChIP dilution buffer were added. Protein A / G magnetic beads were added to the above solution, and the mixture was incubated at 4°C with shaking 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. The mixture was incubated overnight at 4°C. The antibody / magnetic bead / chromatin complex was washed with different buffers in the following order:

[0147] A: Low-salt immune complex washing buffer: 1 mL per tube, wash by rotating at 4°C for 5 min;

[0148] B: High-salt immune complex washing buffer: 1 mL per tube, wash by rotation at 4°C for 5 min;

[0149] C: Lithium chloride immune complex washing buffer: 1 mL per tube, wash by rotation at 4°C for 5 min;

[0150] D: Wash twice with TE buffer, 1 mL per tube, and rotate at 37°C for 5 min.

[0151] Add 200 μL of elution buffer for elution, incubate at 37°C for 30 min by rotation, collect the supernatant, and simultaneously add 180 μL of elution buffer to the DNA tube before immunoprecipitation; add 16 μL of 5M NaCl to each of the above tubes, mix well, and incubate overnight at 45°C; after incubation, add 0.8 μL of RNase A (20 mg / mL) to each tube, incubate at 37°C for 30 min; add 3 μL of proteinase K to each tube, and incubate at 45°C for 2 h; extract sample DNA using a QIAGEN PCR purification kit, elute twice with 10 μL of deionized water, and incubate at room temperature for 10 min before each elution; determine the concentration and construct the library. To prepare sequencing libraries, 1.5 ng of ChIP-DNA underwent end repair, 3' adenosineting, and adapter ligation. After purification with Agencourt AMPure XP magnetic beads, the libraries were amplified using the KAPA HiFi PCR kit under the following conditions: 95℃ for 1 min, 98℃ for 10 s, 65℃ for 1 min, 12 cycles, 65℃ for 5 min, and 4℃ at infinity. The libraries were sequenced on the Illumina HiSeq X Ten platform; each ChIP-seq dataset was subjected to two biological replicates.

[0152] The results are as follows Figure 2 The diagram shows the distribution of PfAP2-O5 protein target genes across the entire genome, including invasion gene families such as msps, raps, rhophs, ralp1, and ama1, demonstrating that most of PfAP2-O5 target genes are related to invasion.

[0153] Example 5: Transcriptome sequencing (RNA-seq) analysis of the effect of hexahydroquinoline derivatives on PfAP2-O5

[0154] Wild-type insect strains and PfAP2-O5 conditional knockout strains were treated with the hexahydroquinoline derivative shown in formula (I). After strict synchronization of the insect strains, 1 μM of hexahydroquinoline derivative was added one life cycle in advance. After one life cycle, the circumvallate (10-15h), trophozoite (28-32h), and schizont (40-44h) cultures of the wild-type and PfAP2-O5 conditional knockout strains were collected. Total RNA was purified using the Direct-zol RNA kit, and strand-specific RNA sequencing libraries were prepared using KAPA mRNA beads. The RNA was then fragmented. The libraries were sequenced on the Illumina HiSeq X Ten system, and the differential gene expression of the two strains after inhibitor treatment was analyzed.

[0155] The results are as follows Figure 3 Show, Figure 3 A used RNA-Seq technology to determine the expression of invading genes after PfAP2-O5 conditional knockout; Figure 3 B used RNA-Seq technology to determine the inhibitory effect of hexahydroquinoline derivatives on PfAP2-O5 function and the expression of invasion genes. The results showed that conditional knockdown of PfAP2-O5 downregulated invasion-related target genes. In wild-type strains treated with hexahydroquinoline derivatives, the expression levels of PfAP2-O5 target genes, such as Rhoptry Neck, Peripheral surface protein, Rhoptry protein, Rhoptry bulb, Micronemes protein, and GPI-Anchored MSP, showed a significant decrease of more than 1.5-fold (mRNA level). (The P. malaria merozoite surface structural protein family genes mainly regulate the invasion of P. malaria merozoites into erythrocytes. 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 hexahydroquinoline derivatives.

[0156] Example 6: Determination of EC50 for the in vitro growth inhibition of Plasmodium by hexahydroquinoline derivatives.

[0157] 100 μL of complete culture medium was added to a 96-well plate. 200 μL of 200 μM stock solution of the compound was added to the first well. The solution was serially diluted in a 1 / 2 ratio (11 concentration gradients). Dihydroartemisinin (DHA) was used as a positive control, complete culture medium as a negative control, and red blood cells without malaria parasites and the compound as a background control.

[0158] Accurately add 100 μL of wild-type Plasmodium culture (1% parasite rate, 4% hematocrit) to each well, resulting in a final parasite rate of 0.5% and a hematocrit of 2% per well. The final compound concentration gradient 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). All samples are tested in triplicate. After adding the samples, the 96-well plate is incubated at 37°C (5% CO2, 5% O2) for 72 h. After incubation, 100 μL of Lysis buffer (1xSYBRGreen I, 0.12 mg / mL saponin, 0.12% v / v Triton X-100, 30 mM Tris-HCl, and 7.5 mM EDTA) was added to each well, mixed thoroughly, and incubated at room temperature in the dark for 2 h. After incubation, fluorescence intensity was recorded using a microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The inhibition rate of the small molecule inhibitor on the growth of Plasmodium was calculated based on the fluorescence intensity.

[0159] Survival rate = (Fl drug treatment - Fl background) / (Fl control - Fl background)

[0160] Wherein, Fl drug treatment, Fl background, and Fl control represent the fluorescence intensity values ​​of the candidate small molecule drug, the erythrocyte control, and the control group without drug treatment, respectively.

[0161] 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.

[0162] The results are as follows Figure 4 As shown, the IC50 of the quantitative growth inhibition effect of the hexahydroquinoline derivative on cultured Plasmodium in vitro was 2.158 μM.

[0163] Example 7: Determination of IC50 for the cytotoxicity of small molecule inhibitors

[0164] After passage of mammalian 293T and HepG2 cells, they were trypsinized, resuspended in DMEM medium, counted using a hemocytometer, and diluted to 10⁻⁶. 5 Cells 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.

[0165] Survival rate = (Ab drug treatment - Ab background) / (Ab control - Ab background)

[0166] 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.

[0167] The results are as follows Figure 5 As shown. Figure 5 The hexahydroquinoline derivatives reflecting the structure of formula (I) of this invention exhibit low cytotoxicity to mammalian cells and good biosafety.

[0168] Example 8: Pharmacokinetic Analysis of Small Molecule Inhibitors

[0169] This experiment aims to investigate the species differences in the metabolic rate of hexahydroquinoline derivatives with the structure of formula (Ⅰ) in the liver microsomes of mice, rats, and humans.

[0170] 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:

[0171] The incubation system consisted of 0.1 M Tris-HCl buffer (pH 7.4), 1 mM MgCl2, 1 mM NADPH, and 0.5 mg mL⁻¹ microsomes, with a final reaction volume of 200 μL. 1 M inhibitors and testosterone were reacted with liver microsomes from different animal species at 37°C. oAfter preheating and incubating in a C water bath for 5 min, 100 μL of NADPH was added to start the reaction. The reaction was terminated by adding 400 μL of stop solution at time points of 0 min, 5 min, 15 min, 30 min, 45 min, and 60 min, respectively. 40 μL of internal standard was added and the mixture was vortexed for 5 min. The reaction was then carried out at 20000 g and 4... o Centrifuge at C for 5 min, take 100 μL of the supernatant and dilute with 100 μL of water, then inject for analysis. Retention times of compounds and internal standards, chromatogram acquisition, and chromatogram integration were processed using Analyst 1.6.3 software. Compounds were analyzed as 1 / X. 2 Linear regression was performed using weighted coefficients to calculate the concentration at each time point. The natural logarithm of the percentage residue at each time point was then linearly regressed against the incubation time to obtain the in vitro elimination rate constant k. The half-life (T0) of the test sample was calculated using the following formula. 1 / 2 ) and hepatic intrinsic clearance CL int（liver） .

[0172] Half-life T 1 / 2 (min) = Liver microsomal clearance rate C Lint（mic） = 0.693 / half-life / mg microsomal protein per mL; hepatic intrinsic clearance CL int（liver） =Hepatic microsomal clearance rate × mg microsomal protein / g liver weight × liver weight to body weight ratio. The liver weight to body weight ratios for mice, rats, and humans were 88, 40, and 20 g / kg, respectively; the mean value of mg microsomal protein / g liver weight across different species was 45. Table 1 shows the microsomal metabolism results of hexahydroquinoline derivatives in mice, rats, and humans.

[0173] Table 1

[0174]

[0175] The results of liver microsomal metabolism showed that the inhibitor was a medium-clearance compound in mouse and human liver microsomes, and a high-clearance compound in rats.

[0176] Example 9: Survival Experiment of Hexahydroquinoline Derivative Resistant Insect Strains in Ring Formation

[0177] This experiment aims to evaluate the killing effect of hexahydroquinoline derivatives as a combination drug against drug-resistant insect strains using the gold standard method for drug resistance. The specific implementation steps are as follows:

[0178] 1. For Plasmodium culture, when the parasite rate is greater than 4% and most of them are in the R stage, use Sorbitol for synchronization for 10 min and vortex for 5 s;

[0179] 2. Repeat Sorbitol synchronization after 30-48 hours (depending on the stage of the parasite);

[0180] 3. After culturing for about 30 hours, examine the smears under a microscope: if the proportion of mature S-stage (10-12 merozoites) is greater than 0.5%, proceed to step 4; if the proportion of mature S-stage (10-12 merozoites) is less than 0.5%, repeat step 2.

[0181] 4. Collect cultures for late-stage parasite enrichment (Plasmion or Percoll); for microscopic examination of smears, the enriched parasites must have a S-stage content greater than 10% and a R-stage content less than 10%;

[0182] 5. Add 10ml of culture medium and 200ul (appropriate amount) of fresh red blood cells, and maintain culture for 3h (must be precise);

[0183] 6. After mixing the culture, take about 200 μL, centrifuge, and then smear the sample for microscopic examination (this can be done about 10 minutes in advance). Quickly calculate the protozoan rate (it must be greater than 0.5%).

[0184] 7. Collect the culture and synchronize with Sorbitol for 10 min, vortex for 5 s, centrifuge and discard the supernatant;

[0185] 8. In a 24-well plate: Control group: Add 100 μL of DMSO control solution to each well.

[0186] Experimental group: Add 100 μL of 7 mM DHA solution and 20 μM hexahydroquinoline derivative solution (dissolved in culture medium) to each well.

[0187] 9. Add 900 μL of resuspended iRBC culture to each well and mix well; maintain culture precisely for 6 hours;

[0188] 10. After 6 hours, transfer the culture from each well to a 1.5 ml EP tube, centrifuge to remove the supernatant;

[0189] 11. Add 1 ml of preheated culture medium and wash, centrifuge to remove supernatant; repeat washing once, for a total of two washes;

[0190] 12. Add 1 ml of preheated culture medium, mix well, transfer to a new well plate, and continue incubation for 66 h;

[0191] 13. After culturing for 66 hours, examine the smears under a microscope to calculate the protozoan rate and survival rate.

[0192] The results are as follows Figure 6 As shown, hexahydroquinoline derivatives (inhibitors) are indicated by "-" indicating the absence of the corresponding substance and "+" indicating the presence of the corresponding substance. The results show that combined use with DHA can more effectively kill artemisinin-resistant strains, suggesting that small molecules can be used as synergistic drugs to improve the spread of artemisinin resistance.

[0193] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The use of a hexahydroquinoline derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting Plasmodium falciparum, said hexahydroquinoline derivative having a structure as shown in formula (I): Equation (Ⅰ).

2. The use of a hexahydroquinoline derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of malaria caused by Plasmodium falciparum, said hexahydroquinoline derivative having a structure as shown in formula (I): Equation (Ⅰ).

3. A pharmaceutical composition, characterized in that, The active ingredient is a hexahydroquinoline derivative or a pharmaceutically acceptable salt thereof with the structure shown in formula (I), and a pharmaceutically acceptable carrier is used as an adjuvant; the artemisinin compound is a dihydroartemisinin compound; Equation (Ⅰ).

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutically acceptable carrier is a diluent or excipient.

5. The pharmaceutical composition according to claim 3, characterized in that, In the composition, the molar ratio of the hexahydroquinoline derivative of formula (I) or its pharmaceutically acceptable salt to the artemisinin-like compound is 1:500 to 500:

1.

6. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition is formulated into an oral dosage form or an injectable dosage form.

7. Use of the pharmaceutical composition of claim 3 in the preparation of a malaria prevention and / or treatment drug.

8. Use of the pharmaceutical composition of claim 3 in the preparation of a medicament for treating artemisinin-resistant malaria.

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