A brominated alkaloid compound, and a preparation method and application thereof

Abstract

This invention discloses a brominated alkaloid compound, its preparation method, and its application. The structural formula of this brominated alkaloid compound is shown in Formula I. It is obtained by chemical reaction and subsequent separation and purification of Boc-4-bromo-L-tryptophan and L-proline methyl ester hydrochloride. It exhibits significant antithrombotic activity, providing a new lead compound for the research and development of antithrombotic drugs.

Description

Technical Field

[0001] This invention relates to a brominated alkaloid compound, its preparation method, and its application, particularly to the separation and purification of 4-Br-Brevianamide compound obtained by substitution and cyclization chemical reactions of Boc-4-bromo-L-tryptophan (4-Br-N-Boc-L-Try) and L-proline methyl ester hydrochloride (L-Pro methyl ester·HCl), the preparation method thereof, and its application in the preparation of antithrombotic drugs. Background Technology

[0002] In the field of antithrombotic drug development, identifying lead compounds with unique structures and excellent activity from natural products is an important strategy. Brevianamides, a class of indole alkaloids derived from marine fungi, exhibit diverse biological activities, but only Brevianamide F has been definitively proven to possess antithrombotic activity (Zhang, H.; Sun, C.; Xia, Q.; Li, P.; Liu, K.; Zhang, Y. Brevianamide F Exerts Antithrombotic Effects by Modulating the MAPK Signaling Pathway and Coagulation Cascade. Mar. Drugs 2024, 22, 439.). To enhance its pharmacological properties, structural modifications such as halogenation have become a key research direction; however, natural halogenated derivatives are extremely scarce, making chemical synthesis highly dependent on them. Unfortunately, existing synthetic techniques for Brevianamides are generally inefficient. For example, the reported total yields of biomimetic total synthesis or gold-catalyzed routes are not ideal (Expedited Total Synthesis of (±)-Brevianamide A via the Strategic Use of Gold(I) Catalysis. Organic Letters, 24, 7200–7204.). This high synthetic difficulty and low yield directly limit the possibility of obtaining excellent antithrombotic drug candidates through systematic derivatization. To date, no reports have been found on 4-Br-Brevianamide F alkaloids, their efficient synthetic methods, or their antithrombotic activity. Summary of the Invention

[0003] Objectives of the Invention: One objective of this invention is to provide a 4-Br-Brevianamide F alkaloid compound or a pharmaceutically acceptable salt thereof. Another objective of this invention is to provide a method for the chemical synthesis of the 4-Br-Brevianamide F alkaloid compound or a pharmaceutically acceptable salt thereof. A final objective of this invention is to provide the use of the 4-Br-Brevianamide F alkaloid compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the 4-Br-Brevianamide F alkaloid compound or a pharmaceutically acceptable salt thereof, in the preparation of a thrombotic drug.

[0004] Technical solution: The present invention relates to a 4-Br-Brevianamide F alkaloid compound or its pharmaceutical salt, the chemical structure of which is shown in Formula I:

[0005]

[0006] I.

[0007] Pharmaceutically acceptable salts are selected from hydrochloride, hydrobromide, sulfate, nitrate, acetate, oxalate, tartrate, citrate, malate, succinate, aconitate, chlorogenic acid, quinic acid, benzoate, methanesulfonate, p-toluenesulfonate, or trifluoroacetate.

[0008] The method for preparing the brominated alkaloid compound of the present invention includes the separation and purification of Boc-4-bromo-L-tryptophan (4-Br-Brevianamide F) and L-proline methyl ester hydrochloride (L-Pro methyl ester·HCl) through substitution and cyclization chemical reactions.

[0009] The preparation method of the brominated alkaloid compound of the present invention includes the following steps:

[0010] (1) Dissolve Boc-4-bromo-L-tryptophan in DMF, and add L-proline methyl ester hydrochloride, N,N-diisopropylethylamine, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate in sequence. Stir the reaction, then add organic solvent to dilute, extract with saturated brine multiple times, dry with anhydrous sodium sulfate, filter, and obtain solid powder;

[0011] (2) The solid powder was mixed with silica gel and eluted by normal phase silica gel column chromatography to obtain the intermediate;

[0012] (3) Dissolve the intermediate in acetic acid, heat and stir to react, evaporate by rotary evaporation, dissolve in organic solvent, extract with saturated brine multiple times, dry with anhydrous sodium sulfate, filter, mix with silica gel, and elute by normal phase silica gel column chromatography to obtain the product.

[0013] In step (1), the organic solvent is ethyl acetate or dichloromethane, and the molar ratio of Boc-4-bromo-L-tryptophan, L-proline methyl ester hydrochloride, N,N-diisopropylethylamine, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate is 1.0 : (1.8-2.2) : (3.6-4.4) : (1.35-1.65). The stirring reaction is carried out at room temperature for more than 6 hours. In step (2), the mass ratio of solid powder to silica gel is 1:1.5, and the mobile phase used for elution is dichloromethane:methanol = 50:1 by volume. In step (3), the organic solvent is ethyl acetate or dichloromethane, the heating and stirring reaction temperature is 80-100℃, the heating and stirring reaction time is more than 8 hours, and the mobile phase used for elution is dichloromethane:methanol = 25:1 by volume.

[0014] The pharmaceutical composition of the present invention comprises the brominated alkaloid compound of the present invention or a pharmaceutically acceptable salt thereof.

[0015] The present invention also includes the use of the brominated alkaloid compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present invention in the preparation of a drug for the prevention or treatment of thrombosis.

[0016] Among them, the drugs for preventing or treating thrombosis are antiplatelet drugs, anticoagulants, and / or thrombolytic drugs. The drug uses the brominated alkaloid compound described in this invention or a pharmaceutically acceptable salt thereof as a single active ingredient, or in combination with other drug carriers and other antithrombotic drugs.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The present invention is the first to obtain the alkaloid compound 4-Br-Brevianamide F. The in vivo activity test of model animals proved that the compound (I) at a concentration of 20 μM can improve the abnormal distribution of erythrocytes in the heart of zebrafish induced by arachidonic acid, and can significantly improve thrombosis in zebrafish. The effect is better than that of the positive control aspirin and better than that of the compound Brevianamide F without bromide substitution. It can be used to develop and prepare new antithrombotic drugs. It provides a new lead compound for the research and development of new antithrombotic drugs. (2) The 4-Br-Brevianamide F provided by the present invention has fewer synthetic steps, and the synthetic route only requires 2 steps; the yield is high, with a total yield of 75.6%, which is 10.5 times that of the method for synthesizing the analog (+)-Brevianamide A reported in the existing literature. Attached Figure Description

[0018] Figure 1 These are representative images of zebrafish body regions from each treatment group in Example 2;

[0019] Figure 2 This is a diagram showing the erythrocyte staining area in the cardiac region of zebrafish in each treatment group in Example 2;

[0020] Figure 3 The image shows the staining intensity of the zebrafish heart region in each treatment group in Example 2. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] Example 1: Preparation of 4-Br-Brevianamide F

[0023] ① Dissolve 1 g of 4-Br-N-Boc-L-Try raw material in 10 mL of N,N-dimethylformamide (MW 73.09), then add L-Pro methyl ester·HCl (MW 165.6, 2 equiv), N,N-diisopropylethylamine (MW 129.24, 4 equiv), and finally add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexabromophosphate (MW 380.2, 1.5 equiv) and stir at room temperature for 6 h.

[0024] ② After the reaction was completed, 50 mL of ethyl acetate was added for dilution, and the mixture was extracted three times with 150 mL of saturated brine. After drying with anhydrous sodium sulfate, the solid powder was obtained by filtration.

[0025] ③ The solid powder obtained above was mixed with silica gel at a mass ratio of 1:1.5 and subjected to normal phase silica gel column chromatography with dichloromethane:methanol = 50:1 (v:v) as the mobile phase to obtain the intermediate of the compound shown in formula (I).

[0026] ④ Dissolve the intermediate powder obtained above in 50 mL of acetic acid (MW 60.05) and stir in an oil bath at 110 °C for 8 h. After the reaction is complete, remove the acetic acid by rotary evaporation. Then, dissolve in 50 mL of ethyl acetate, extract twice with 150 mL of saturated brine, dry with anhydrous sodium sulfate, and filter to obtain the crude product of the target product.

[0027] ⑤ The crude product was mixed with silica gel at a mass ratio of 1:1.5 and subjected to normal phase silica gel column chromatography with dichloromethane:methanol = 25:1 (v:v) as the mobile phase to obtain 4-Br-Brevianamide F compound, with a yield of 715 mg and a yield of 75.6%.

[0028] The 4-Br-Brevianamide F compound prepared in this embodiment was analyzed by various modern spectroscopic techniques, including NMR and HRESIMS. The results are as follows: HRESIMS m / z [M + Na] + 384.0305 (Calcd. C 16 H 16 BrN3O2), 1 H and 3 The CNMR (nuclear magnetic resonance) spectral data are shown in Table 1.

[0029] Table 1. 4-Br-Brevianamide F compounds 1 H and 3 C10 NMR spectroscopy data

[0030]

[0031] a Measured at 150 MHz in CDCl3; b Measured at 600 MHz in CDCl3.

[0032] As can be seen from the above analysis, the structure of the 4-Br-Brevianamide F compound prepared in this embodiment is shown in Formula I below:

[0033]

[0034] I.

[0035] The existing literature, Godfrey, RC, Green, NJ, Nichol, GS, & Lawrence, AL (2020). Total synthesis of brevianamide A. Nature Chemistry, 12(7), 615–619. reported a biomimetic total synthesis route for (+)-Brevianamide A, which used L-tryptophan methyl ester as the starting material and obtained the target product through seven steps, with a total yield of only 7.2%. In contrast, the synthesis route for compound 4-Br-Brevianamide F in Example 4 of this invention requires only two steps, with a total yield of 75.6% and a final yield of 715 mg, which is 10.5 times that of (+)-Brevianamide A. This indicates that the synthesis method of this invention is significantly superior to the methods reported in the existing literature.

[0036] Example 2 Evaluation of antithrombotic activity based on zebrafish model

[0037] Wild-type AB strain zebrafish were used as experimental animals in this experiment. The experimental groups included a blank control group, a thrombosis model group, a positive control drug group, a Brevianamide F compound treatment group (for the synthesis of Brevianamide F, see Zhang H, Sun C, Xia Q, Li P, Liu K, Zhang Y. Brevianamide F Exerts Antithrombotic Effects by Modulating the MAPK Signaling Pathway and Coagulation Cascade. Marine Drugs. 2024; 22(10):439.), and a 4-Br-Brevianamide F compound treatment group. Ten zebrafish were placed in each well of a 24-well plate, and each group had three replicates. In this study, the blank control group and the thrombosis model group were given 2 mL of embryo culture water, the positive control group was given 2 mL of embryo culture water containing 125 μmol / L aspirin, and the Brevianamide F compound treatment group and the 4-Br-Brevianamide F compound treatment group were given 2 mL of embryo culture water containing 20 μmol / L of the compound. All zebrafish were placed in a constant temperature incubator at 28 ± 0.5℃. After 6 h of culture, except for the blank control group, the other groups were replaced with embryo culture water containing 80 μmol / L arachidonic acid and cultured for another 1.5 h at 28 ± 0.5℃ to induce thrombosis. After treatment, the solution was discarded, and the zebrafish were stained with o-anisidine staining solution in a dark environment for 10 min. They were washed three times with embryo culture water, fixed with 4% paraformaldehyde, and then placed on their sides on slides coated with methylcellulose. Ten zebrafish were randomly selected from each group. Heart images were taken using a Zeiss microscope. Image-Pro Plus software was used to measure the stained area and intensity of erythrocytes in the heart. Statistical analysis was performed using GraphPad Prism v.8.3.0 software. The results are as follows: Figure 1-3 As shown. By Figure 1-3As can be seen, compared with the blank control group, the staining area and staining intensity of zebrafish cardiac erythrocytes in the thrombosis model group were significantly reduced, indicating that the thrombosis model was successfully established. The aspirin treatment group (positive control) effectively improved the above indicators, verifying the reliability of the experimental system. At a concentration of 20 μmol / L, the compound 4-Br-Brevianamide F of this invention exhibited significant antithrombotic activity, as both the staining area and staining intensity of cardiac erythrocytes recovered compared to the model group. This suggests that it may exert its antithrombotic effect by inhibiting arachidonic acid-induced platelet aggregation, with a better effect than the positive control drug. Furthermore, the antithrombotic effect of 4-Br-Brevianamide F is superior to that of Brevianamide F. Therefore, the compound of this invention can serve as a lead compound for antithrombotic drugs. The existing literature, Zhang H, Sun C, Xia Q, Li P, Liu K, Zhang Y. Brevianamide F Exerts Antithrombotic Effects by Modulating the MAPK Signaling Pathway and Coagulation Cascade. Marine Drugs. 2024; 22(10):439, reports the antithrombotic activity evaluation results of the compound Brevianamide F based on zebrafish, showing that Brevianamide F staining intensity on cardiac erythrocytes was 294097 ± 29205 pixels, and the thrombosis prevention rate was 43.13%. However, through... Figure 1 A comparison of data between 4-Br-Brevianamide F and Brevianamide F shows that the 4-Br-Brevianamide F compound prepared in this embodiment has a staining intensity of 445066 ± 22957 pixels on cardiac erythrocytes and a thrombosis prevention rate of 65.27% (P<0.05), which is superior to the compound Brevianamide F reported in existing literature. It can be used as a lead compound for antithrombotic drugs.

Claims

1. A brominated alkaloid compound or a pharmaceutically acceptable salt thereof, characterized in that, The structural formula of the brominated alkaloid compound is shown in Figure I: I 。 2. The brominated alkaloid compound according to claim 1, characterized in that, Pharmaceutically acceptable salts are selected from hydrochloride, hydrobromide, sulfate, nitrate, acetate, oxalate, tartrate, citrate, malate, succinate, aconitate, chlorogenic acid, quinic acid, benzoate, methanesulfonate, p-toluenesulfonate, or trifluoroacetate.

3. The method for preparing the brominated alkaloid compound according to claim 1 or 2, characterized in that, It was obtained by separation and purification of Boc-4-bromo-L-tryptophan and L-proline methyl ester hydrochloride through substitution and cyclization chemical reactions.

4. The method for preparing the brominated alkaloid compound according to claim 3, characterized in that, Includes the following steps: (1) Dissolve Boc-4-bromo-L-tryptophan in DMF, and add L-proline methyl ester hydrochloride, N,N-diisopropylethylamine, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate in sequence. Stir the reaction, dilute with organic solvent, extract with saturated brine several times, dry with anhydrous sodium sulfate, and filter to obtain solid powder; (2) The solid powder was mixed with silica gel and eluted by normal phase silica gel column chromatography to obtain the intermediate; (3) Dissolve the intermediate in acetic acid, heat and stir to react, evaporate by rotary evaporation, dissolve in organic solvent, extract with saturated brine multiple times, dry with anhydrous sodium sulfate, filter, mix with silica gel, and elute by normal phase silica gel column chromatography to obtain the product.

5. The preparation method according to claim 4, characterized in that, in step (1), the organic solvent is ethyl acetate or dichloromethane, the molar ratio of Boc-4-bromo-L-tryptophan, L-proline methyl ester hydrochloride, N,N-diisopropylethylamine, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate is 1.0 : (1.8-2.2) : (3.6-4.4) : (1.35-1.65), and the stirring reaction is carried out at room temperature for more than 6 hours.

6. The preparation method according to claim 4, characterized in that, in step (2), the mass ratio of solid powder to silica gel is 1:1.5, and the mobile phase used during elution is dichloromethane:methanol = 50:1 by volume.

7. The preparation method according to claim 4, characterized in that, in step (3), the organic solvent is ethyl acetate or dichloromethane, the temperature of the heating and stirring reaction is 80-100℃, the heating and stirring reaction time is more than 8h, and the volume ratio of dichloromethane:methanol = 25:1 is used for elution.

8. A pharmaceutical composition, characterized in that, This includes the brominated alkaloid compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof.

9. The use of the brominated alkaloid compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 8, in the preparation of a thrombotic medicament.

10. The application according to claim 9, characterized in that, Drugs used to prevent or treat thrombosis include antiplatelet drugs, anticoagulants, and / or thrombolytic drugs.

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