Flavonolignan glycoside for preparing a PTR1 inhibitor, preparation method and application

Abstract

The present invention discloses a flavonolignan glycoside and a pharmaceutically acceptable salt thereof, and the flavonolignan glycoside is shown as structural formula I; the present invention also discloses a preparation method of the flavonolignan glycoside and its application in the preparation of pteridine reductase-1 inhibitors. The flavonolignan glycoside provided by the present invention can effectively inhibit the activity of pteridine reductase-1 and can be used to prepare drugs for alleviating leishmaniasis.

Description

Technical Field

[0001] The present invention relates to the field of pteridine reductase-1 inhibitors. More specifically, the present invention relates to a flavonolignan glycoside for preparing a PTR1 inhibitor, a preparation method and applications thereof. Background Art

[0002] Leishmania is the pathogen of various forms of leishmaniasis. The life cycle of Leishmania has two stages. The first stage consists of the motile, flagellated promastigote stage, which exists in the digestive tract of sand fly species. The second stage is the non-motile amastigote stage, which occurs in its mammalian host. Leishmania is pterin auxotrophic; the parasite takes up pterin from the host and reduces it to dihydro and tetrahydro forms. For this purpose, the parasite utilizes pteridine reductase-1 (PTR1, an enzyme of the short-chain dehydrogenase family), which can reduce biopterin to its dihydro (H2B) and tetrahydro forms (H4B) respectively, to achieve the inhibition of dihydrofolate reductase-thymidylate synthase (DHFR-TS). PTR1 also provides a bypass mechanism for reducing folate (conjugated pterin). Therefore, the parasite is resistant to antifolates while ensuring the continuous synthesis of thymidine (a component of DNA) required for its survival. This makes PTR1 an important target for anti-leishmaniasis drug discovery, and drug compositions capable of inhibiting pteridine reductase-1 activity have a certain alleviating effect on leishmaniasis.

[0003] Plants of the genus Lumnitzera are deciduous trees. Their barks can be used to extract tannins, the stem bark fibers can be woven into coarse cloth, and the wood can be used to make furniture. The medicinal value of plants of the genus Lumnitzera lies in their barks, which have the effects of scavenging free radicals, anti-tumor and inhibiting cancer cells. So far, there has been no report that plants of the genus Lumnitzera and their extracts can be used to prepare pteridine reductase-1 inhibitors and alleviate leishmaniasis. Summary of the Invention

[0004] One object of the present invention is to solve at least the above problems and provide at least the advantages described hereinafter.

[0005] Another object of the present invention is to provide a flavonolignan glycoside for preparing a pteridine reductase-1 inhibitor, which can effectively inhibit pteridine reductase-1 and is expected to be used in the preparation of drugs for alleviating leishmaniasis.

[0006] Another object of the present invention is to provide a preparation method of the flavonolignan glycoside.

[0007] Another object of the present invention is to provide the application of the flavonolignan glycoside in the preparation of a pteridine reductase-1 inhibitor.

[0008] Another object of the present invention is to provide a pharmaceutical composition comprising the flavonolignan glycoside.

[0009] To achieve these objects and other advantages according to the present invention, there is provided a flavonolignan glycoside and a pharmaceutically acceptable salt thereof, and the flavonolignan glycoside is shown as structural formula Ⅰ;

[0010]

[0011] The present invention further claims the preparation method of the flavonolignan glycoside for relieving leishmaniasis, including:

[0012] Step 1: Extract the dried stem bark of Lannea acida A.Rich with an ethanol solution having a volume fraction of 70-90%, filter to obtain the supernatant, and concentrate it under reduced pressure to obtain a paste extract. Suspend the obtained paste extract in water, and extract it once with n-hexane and ethyl acetate. Take the ethyl acetate extract and separate it by silica gel column chromatography, and elute it with a gradient of n-hexane-ethyl acetate-methanol to obtain fractions E1-E11;

[0013] Step 2: Separate the obtained fraction E4 by silica gel column chromatography, and elute it with a gradient of n-hexane-acetone-methanol to obtain fractions E4-1-E4-8;

[0014] Step 3: Purify E4-4 by RP-HPLC using a mixed solution of acetonitrile and water with a mass ratio of 7:3 to obtain the flavonolignan glycoside.

[0015] Preferably, in Step 1, the extraction time is 5-9 days, the extraction times are 2-4 times, and the supernatants obtained by multiple extractions and filtrations are combined and concentrated under reduced pressure.

[0016] The present invention further claims the application of the flavonolignan glycoside and a pharmaceutically acceptable salt thereof in the preparation of a pteridine reductase-1 inhibitor.

[0017] The present invention further claims a pharmaceutical composition, which includes the flavonolignan glycoside or a pharmaceutically acceptable salt thereof as claimed in claim 1 and excipients.

[0018] The present invention has at least the following beneficial effects: The present invention provides a flavonolignan glycoside and a pharmaceutically acceptable salt thereof, which can effectively inhibit pteridine reductase-1 and can be used to prepare a drug for relieving leishmaniasis.

[0019] Other advantages, objectives and features of the present invention will be partially reflected by the following description, and will also be understood by those skilled in the art through the research and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For the flavonolignan glycoside of the present invention 1 1H NMR spectrum;

[0021] Figure 2 13C NMR spectrum of the flavonolignan glycoside of the present invention 13 13C NMR spectrum

[0022] Figure 3 DEPT spectrum of the flavonolignan glycoside of the present invention

[0023] Figure 4 1H– 1 H– 1 1H–1H COSY spectrum

[0024] Figure 5 HSQC spectrum of the flavonolignan glycoside of the present invention

[0025] Figure 6 HMBC spectrum of the flavonolignan glycoside of the present invention

[0026] Figure 7 NOESY spectrum of the flavonolignan glycoside of the present invention

[0027] Figure 8 Key correlation signal diagrams of HSQC, HMBC and NOESY spectra of the flavonolignan glycoside of the present invention 1 HSQC, HMBC and NOESY spectra Detailed implementation manners

[0028] The following further describes the present invention in detail with specific embodiments, so that those skilled in the art can implement it according to the text of the specification.

[0029] It should be understood that the terms such as "having", "comprising" and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0030] A new flavonolignan glycoside was isolated from the stem bark of the plant Lannea acida A.Rich of the genus Lannea, and it has the structure shown in Formula I:

[0031]

[0032] The following is a specific embodiment of the present application:

[0033] Plant raw materials:

[0034] The stem bark of Lannea acida A.Rich was collected in January 2020 from the bank of Agbaragun River in Egbe forest, Yagba West, Kogi State, Nigeria by the University of Ilorin. The material was identified and specimens were collected in the Herbarium Unit of the Department of Plant Biology, Forestry Research Institute of Nigeria (FRIN), with the specimen number (UILH / 001 / 1414 / 2019).

[0035] Extraction and Isolation

[0036] Step 1: Soak the stem bark of Lannea acida A.Rich (10.0 kg) in ethanol:water (90:10) for 7 days, then filter and concentrate under reduced pressure. This process was repeated three times. The extract (1.2 kg) was suspended in water and successively extracted with n-hexane and ethyl acetate. The ethyl acetate fraction (300 g) was subjected to silica gel column chromatography and eluted with a gradient solvent system of n-hexane:ethyl acetate:methanol (100:0:0 - 0:100:0 - 0:0:100) to obtain 11 fractions E1 - E11. The solvent concentrations corresponding to each fraction of E1 - E11 are shown in Table 1.

[0037] Table 1 Solvent Concentrations and Fractions

[0038]

[0039] Step 2: Subject the E4 fraction to silica gel column chromatography again and elute with a gradient solvent system of n-hexane:acetone:methanol (100:0:0 - 0:100:0 - 0:0:100) to obtain fractions E4-1–E4-8. The solvent concentrations corresponding to each fraction of E4-1–E4-8 are shown in Table 2.

[0040] Table 2 Solvent Concentrations and Fractions

[0041]

[0042] Step 3: Purify E4-4 by RP-HPLC using ACN:H 2 O(7:3), and the resulting compound is the flavonolignan glycoside (50 mg).

[0043] Characterization of Flavonolignan Glycoside

[0044] 1H NMR (500 MHz, DMSO-d 6 ), 13C NMR (125 MHz, DMSO-d 6 ), DEPT spectra, two-dimensional NMR 1 1H– 1 1H COSY spectra, two-dimensional NMR HSQC spectra, two-dimensional NMR HMBC spectra, and two-dimensional NMR NOESY spectra are shown as Figures 1 to 7 follows. The key relevant signals of the 1 1H– 1 1H COSY, HMBC, and NOESY spectra of the flavonolignan glycoside are shown as Figure 8 follows. The 1 1H (500 MHz) and 13 13C (125 MHz) NMR data of the flavonolignan glycoside are shown in Table 3.

[0045] Table 3 1 1H (500 MHz) and 13 13C (125 MHz) NMR data (deuterated methanol)

[0046]

[0047]

[0048] The flavonolignan glycoside prepared in the present invention is a light yellow powder; melting point: 270–272 °C; as Figures 1 to 7 shown, its spectral information is as follows: Vλ max (logε) 229 (3.92);

[0049] IR (KBr disk) υ max (cm -1 ): 3392 (OH), 1689 (C═O); FAB-MS m / z: 593.1 [M-H] - ; HRFAB-MS m / z: 593.1266 [M-H] - (calcd. for 593.1295, C 30 H 25 O 13 ).

[0050] As Figure 1 and Table 1 shown, 1 1H-NMR (CD 3 3OD, 500 MHz) spectral information is as follows: δ H5.10 (d, J = 6.0 Hz, H-2), 4.17 (q, J = 5.5 Hz, H-3), 2.91 (dd, J = 16.5, 6.0 Hz, H-4a); 2.76 (dd, J = 16.5, 5.0 Hz, H-4b), 6.70 (s, H-8), 6.72 (d, J = 2.0 Hz, H-2′), 6.70 (d, J = 8.5 Hz, H-5′), 6.61 (dd, J = 8.5, 2.0 Hz, H-6′), 6.75 (s, H-3″), 7.33 (s, H-6″), 6.10 (s, H-8″), 4.51 (br s, H-1″′), 3.38 (overlapped, H-2″′), 3.30 (overlapped, H-3″′), 3.11 (t, J = 9.5 Hz, H-4″′), 3.33 (overlapped, H-5″′), 1.07 (d, J = 6.5 Hz, H-6″′).

[0051] As Figure 2 shown in and Table 1, 13 C-NMR (CD 3 OD, 125 MHz) spectrum information is as follows: δ C 79.2 (C-2), 71.0 (C-3), 23.8 (C-4), 151.6 (C-5), 99.7 (C-6), 148.7 (C-7), 97.2 (C-8), 157.5 (C-9), 102.9 (C-10), 128.8 (C-1′), 113.6 (C-2′), 145.2 (C-3′), 145.2 (C-4′), 115.5 (C-5′), 117.5 (C-6′), 106.0 (C-1″), 152.5 (C-2″), 103.2 (C-3″), 146.6 (C-4″), 143.7 (C-5″), 109.1 (C-6″), 141.5 (C-7″), 92.0 (C-8″), 161.0 (C-9″), 100.1 (C-1″′), 70.4 (C-2″′), 70.5 (C-3″′), 71.8 (C-4″′), 69.1 (C-5″′), 17.7 (C-6″′).

[0052] As Figure 2 shown in and Table 1, the molecular formula was deduced by HRFAB-MS analysis to be C 30 H 26 O 13 ([M-H] + m / z 593.1266, calculated value is 593.1295 C 30 H 25 O 13), showing an unsaturation degree of 18, 13 The 13C NMR spectrum (Table 1) shows 30 carbon atoms, which can be differentiated into 14 quaternary carbons, 14 methines, 1 methylene, and 1 methyl by the DEPT spectrum. In the 1 1H NMR spectrum of I, 7 aromatic hydrogen signals can be seen, including one ABX coupling system at δ H 6.72 (d, J = 2.0 Hz), 6.70 (d, J = 8.5 Hz), 6.61 (dd, J = 8.5, 2.0 Hz), and 4 singlet signals at δ H 7.33, 6.75, 6.70, 6.10. From the 1 1H NMR spectrum at δ H 5.10 (d, J = 6.0 Hz), 4.17 (q, J = 5.5 Hz), 2.91 (dd, J = 16.5, 6.0 Hz), and 2.76 (dd, J = 16.5, 5.0 Hz), it can be inferred that there is a flavan-3-ol ring in compound I. The 4 hydrogen signals correspond to H-2, H-3, H-4a, and H-4b on the C ring of flavan-3-ol respectively, and the COSY correlation signals between them can also confirm this inference. The ABX coupling system should come from the B ring. Combining with the carbon spectrum and two-dimensional spectra, the presence of the catechin fragment can be confirmed (Dubeler et al, Phytochemistry, 1997; Chao et al, J. Agric. Food Chem., 2010). According to the correlation signals with C-9 (δ C 157.5), C-10 (δ C 102.9) in the HMBC spectrum, the singlet at δ H 6.70 can be located at H-8. Through the correlation signals of H-8 in the HMBC spectrum, δ C 99.7 and δ C 148.7 are determined as C-6 and C-7 respectively. In addition to the data of the C6-C3-C6 skeleton, more aromatic carbons can be observed in the 13 13C NMR spectrum, including a tetrasubstituted benzene ring (δ C 106.0, 152.5, 103.2, 146.6, 143.7, and 109.1) and an α,β-unsaturated ketone (δC 161.0, 93.5, 142.4), indicating the presence of a C6-C3 unit similar to the caffeic acid skeleton (Rong et al, Magn. Reson. Chem., 2005). According to the HSQC and HMBC correlation information, 1 the three remaining aromatic hydrogen singlet signals at δ H 7.33, 6.75, and 6.10 in the 1H NMR spectrum are located within the caffeic acid skeleton. δ H6.10 and δ C The HMBC correlation signals between 99.7 (C-6) confirm its position at H-8″, H-3″ (δ H 6.75) and the positioning of H-6″ (δ H 7.33) are also determined by the HMBC long-range correlation signals of the nearby quaternary carbons. The trans relationship between C-2 / C-3 in ring C is determined by the large coupling constant (6.0 Hz) between H-2 (δ H 5.10) and H-3 (δ H 4.17). In the case of the cis relationship between C-2 / C-3, the coupling constant is very small. All the above data are close to those of mururin A previously isolated from Brosimum acutifolium (Takashima et al., Planta Med., 2002).

[0053] In addition, 13 A set of carbon signals of rhamnose (δ C 100.1, 70.4, 70.5, 71.8, 69.1 and 17.7) appears in the 13C NMR spectrum, 1 The anomeric proton signal of the sugar is observed at δ H 4.51 (br s) in the 1H NMR spectrum, and the four oxygenated methylene proton signals at δ H 3.38, 3.33, 3.30, 3.11 and the methyl signal at δ H 1.07 (d, J = 6.5 Hz). After acid hydrolysis, it is determined to be L-rhamnose (Dai et al, Molecules, 2023). According to the NOESY correlation of Rha-H-1″′ (δ H 4.51) and Rha-H-2″′) (δ H 3.38), the anomeric configuration is inferred to be α-configuration (Wang et al, Planta Med., 2014). The HMBC spectrum shows that H-3 (δ H 4.17) is correlated with the anomeric carbon signal Rha-C-1″′ (δ C 100.1), indicating that rhamnose is linked to C-3 (δ C 71.0). Flavonolignan glycoside is a novel flavonolignan rhamnopyranoside with highly fused rings. Therefore, the structure of flavonolignan glycoside is inferred to be mururin A-3-O-α-L-rhamnopyranoside.

[0054] Based on the mass spectrometry and the above spectral information, its molecular formula is deduced as C 30 H 26 O 13 ([M-H]- The theoretical value of the mass number is 593.1295, C 30 H 25 O 13 ), and the spectrum shows m / z 593.1266 [M-H] - , and the flavonolignan glycoside prepared in the present invention has the molecular formula C 30 H 26 O 13 , which is consistent with the theoretical value.

[0055] Determination method for pteridine reductase-1 inhibitory activity:

[0056] The in vitro PTR1 inhibitory activity of the obtained flavonolignan glycoside was analyzed using a cytochrome-c coupled assay, which was carried out in a buffer containing 20 mM sodium acetate and 20 mM NaCl at pH 4.5 (obtained by adding glacial acetic acid). Each test compound was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 500 μM and serially diluted to obtain a series of concentrations (i.e., 3.906, 7.812, 15.625, 31.25, 62.5, and 125 μM). The diluted test compounds were dispensed into a 96-well plate, and the final DMSO level in all samples (including the control) was 5%. Then, the final reaction mixture containing LmPTR1 (5 μM), biopterin (0.35 μM), and cytochrome C (81 μM) was added to each well using a multi-channel pipette (Eppendorf plus). Each reaction was performed in triplicate. After addition, the 96-well plate was incubated at 30 °C for 10 minutes using an ELISA spectrophotometer to obtain a prereading at 550 nm. In the second stage, NADPH (final concentration: 500 μM) was added to initiate the reaction, and then it was incubated at 30 °C for 40 minutes with moderate shaking for 1 min between each reading. The results were converted to the percentage of inhibition (%), and the determination results of pteridine reductase-1 inhibitory activity are shown in Table 4.

[0057] Table 4 Inhibitory rates of flavonolignan glycosides with different concentrations on PTR1

[0058]

[0059] The blank control group refers to the treatment of the sample without using any inhibitory drugs. The trimethoprim group serves as the positive control. The trimethoprim molecule has a 2,4-diaminopyrimidine skeleton, and molecules with such pharmacophores are potent inhibitors of enzymes such as dihydrofolate reductase-thymidylate synthase (DHFR-TS) and pteridine reductase-1 (PTR1). As can be seen from Table 4, flavonolignan glycoside showed significant inhibitory effects on PTR1. As the concentration of flavonolignan glycoside increased, the inhibition rate of flavonolignan glycoside on pteridine reductase-1 also increased. When the concentration reached 62.5 μM, the inhibition rate of flavonolignan glycoside on pteridine reductase-1 reached 81.45%, even exceeding the inhibition rate of the positive control trimethoprim on pteridine reductase-1 (76.8%). Further, the Prism-Graphpad software was used for interpretation to calculate the IC 50 value. The IC 50 value of flavonolignan glycoside was 56.43 ± 2.14 μM. Therefore, flavonolignan glycoside with a concentration greater than 56.43 μM had a significant inhibition rate on pteridine reductase-1.

[0060] Cytotoxicity experiment (MTT method):

[0061] Further, we conducted toxicity experiments on flavonolignan glycoside with a concentration greater than 56.43 μM. Considering the rationality of medical concentrations, 100 μM flavonolignan glycoside was selected for the toxicity experiment. Human normal cells BJ were obtained from the American Type Culture Collection (ATCC). They were cultured in DMEM medium supplemented with 5% FBS, 100 IU / mL penicillin, and 100 μg / mL streptomycin and stored in a 5% CO 2 incubator at 37°C. 100 μL / well cell solution (5×10 4 cells / mL BJ cells) was added to a 96-well plate and incubated at 37°C for 24 hours, and then treated with 100 μM flavonolignan glycoside or the negative control DMSO for 48 hours. Then, 20 μL of MTT (5 mg / mL) dye was added to each well, and the plate was incubated at 37°C for 4 hours. After incubating for 100 μL, DMSO was added to each well to dissolve the purple formazan crystals, and then the absorbance was measured at 570 nm using an ELISA plate reader, and the growth inhibition rate of BJ cells was calculated. Five parallel experiments were designed, and the results obtained from the five parallel experiments are shown in Table 5.

[0062] Table 5 Growth inhibition rate of BJ cells

[0063]

[0064] Generally speaking, a drug with a growth inhibition rate of less than 50% on normal human cells BJ is considered non-toxic. The results of the cytotoxicity experiment showed that flavonolignan glycoside did not show cytotoxicity on normal human cells BJ at the 100 μM level (the growth inhibition rate of BJ cells < 5%). At a safe concentration above the IC 50 concentration, flavonolignan glycoside showed a significant inhibitory effect on dihydropteridine reductase-1, even superior to the inhibitory effect of trimethoprim on dihydropteridine reductase-1. However, many complications are associated with the clinical use of trimethoprim, such as susceptibility to certain infections, slow healing, and gum bleeding. The flavonolignan glycoside provided in this application is derived from natural herbs and has no side effects, and can be used as a potential candidate for preparing dihydropteridine reductase-1 inhibitors.

[0065] In summary, flavonolignan glycoside can be used as a potential candidate for inhibiting dihydropteridine reductase-1, and can be used to prepare dihydropteridine reductase-1 inhibitors and drugs for relieving leishmaniasis.

[0066] The present invention further claims a pharmaceutical composition, which comprises the flavonolignan glycoside or a pharmaceutically acceptable salt thereof. The pharmaceutical forms of the pharmaceutical composition include but are not limited to pills, granules, tablets, capsules, or injections.

[0067] As described above, according to the present invention, there is provided a flavonolignan glycoside for relieving leishmaniasis, which can effectively inhibit dihydropteridine reductase-1 and can be used to prepare drugs for relieving leishmaniasis.

[0068] The number of devices and the processing scale described here are used to simplify the description of the present invention. Modifications and variations to the flavonolignan glycoside, preparation method, and application for preparing dihydropteridine reductase-1 inhibitors of the present invention will be obvious to those skilled in the art.

[0069] Although the embodiments of the present invention have been disclosed as above, it is not limited to only the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details and the specific embodiments shown and described here.

Claims

1. Flavonolignan glycosides and pharmaceutically acceptable salts thereof, characterized in that: The flavonolignan glycosides are shown in structural formula Ⅰ; Formula Ⅰ.

2. The method for preparing the flavonolignan glycosides and pharmaceutically acceptable salts thereof according to claim 1, characterized in that: include: Step 1: Lannea acida The dried stem bark of A. Rich was extracted with 70-90% ethanol solution by volume, the supernatant was filtered and concentrated under reduced pressure to obtain a paste extract, the obtained paste extract was suspended in water, and extracted with n-hexane and ethyl acetate in sequence, the ethyl acetate extract was separated by silica gel column chromatography, and fractions E1-E11 were obtained by gradient elution with n-hexane-ethyl acetate-methanol; Step 2, fraction E4 was separated by silica gel column chromatography, and fractions E4-1 to E4-8 were obtained by gradient elution with n-hexane-acetone-methanol; Step 3: Use a mixed solution of acetonitrile and water in a mass ratio of 7:3 to purify E4-4 by RP-HPLC to obtain the flavonolignan glycoside.

3. The method for preparing the flavonolignan glycosides and pharmaceutically acceptable salts thereof according to claim 2, characterized in that: In step 1, the extraction time is 5 to 9 days, the number of extractions is 2 to 4 times, and the supernatants filtered from multiple extractions are combined and concentrated under reduced pressure.

4. Use of the flavonolignan glycosides and pharmaceutically acceptable salts thereof as claimed in claim 1 in the preparation of pteridine reductase-1 inhibitors.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the flavonolignan glycoside or a pharmaceutically acceptable salt thereof according to claim 1 and excipients.

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