Application of an alkaloid compound in the preparation of a drug with anti-herpes simplex virus type II effect

Abstract

The present invention relates to the field of pharmaceutical chemistry technology and specifically discloses the use of an alkaloid compound in the preparation of a drug having an anti-herpes simplex virus type II effect. The alkaloid compound has a structure represented by Formula I. Studies have shown that the alkaloid compound having the structure represented by Formula I has highly significant anti-herpes simplex virus type II activity, and its anti-herpes simplex virus type II activity is superior to that of the positive drug acyclovir; therefore, using it as an active ingredient in the preparation of a drug having an anti-herpes simplex virus type II effect has important application value.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular to the use of an alkaloid compound in the preparation of a drug having an anti-herpes simplex virus type II effect. Background Art

[0002] Herpes simplex virus type 2 (HSV-2) is a linear, double-stranded DNA virus of the Alphavirinae subfamily of the Herpesviridae family. It easily infects the mucous membranes of the urinary tract, genitals, and perianal areas, causing genital herpes, a chronic, inflammatory, recurrent, and difficult-to-cure sexually transmitted disease. According to the World Health Organization, 536 million people aged 15 to 49 are infected with HSV-2 globally, with up to 23 million new cases each year. Both men and women are susceptible to infection, with HSV-2 infection rates as high as 30% to 80% in women and 10% to 50% in men. Furthermore, genital mucosal damage caused by HSV-2 infection increases the risk of infection with other pathogens, such as HIV and Treponema pallidum. HSV-2 infection is one of the leading causes of sexually transmitted infections worldwide, and its incidence in my country is also increasing, posing a serious threat to human health. Acyclovir is the primary antiviral drug used clinically to treat genital herpes. However, long-term clinical use of this drug has led to mutations in the UL23 gene encoding thymidine kinase, resulting in the emergence of drug-resistant viral variants. As a result, an increasing number of patients have developed resistance to acyclovir, resulting in suboptimal treatment outcomes. Therefore, the development of new alternative anti-HSV-2 drugs is needed.

[0003] Natural medicines, with their wide range of effects and safety, are becoming a research hotspot for antiviral drugs. Peganum harmala L., the dried, mature seeds of the plant Peganum harmala L., belongs to the Tribulus terrestris family. It is clinically used to treat cough, asthma, rheumatic pain, swelling, and itchy skin. Modern pharmaceutical research indicates that Peganum harmala extracts possess multiple pharmacological activities, including antiviral, antitumor, antibacterial, anti-inflammatory, and antioxidant properties. The alkaloids they contain are the primary pharmacological basis for their effects. Summary of the Invention

[0004] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides a new alkaloid compound; the alkaloid compound has a very excellent anti-herpes simplex virus type II effect, and its anti-herpes simplex virus type II effect is stronger than acyclovir.

[0005] The technical solutions of the present invention are as follows:

[0006] An alkaloid compound having a structure shown in Formula I:

[0007]

[0008] Formula I.

[0009] The alkaloid compound of formula I (abbreviated as PF) is a new compound. Studies have shown that the alkaloid compound of formula I has an IC of 1.0 against respiratory syncytial virus. 50 0.90 ± 0.10 μM, which is significantly lower than the IC of the positive drug acyclovir against respiratory syncytial virus. 50 (1.12 ± 0.15 μM); this indicates that the alkaloid compound of the structure shown in Formula I has very significant anti-herpes simplex virus type II activity, and its anti-herpes simplex virus type II activity is significantly better than the positive drug acyclovir; this technical effect is unpredictable by those skilled in the art.

[0010] The present invention also provides a method for preparing the alkaloid compounds, which uses Peganum harmala as a raw material and extracts and separates the alkaloid compounds from it.

[0011] The present invention separates the alkaloid compound with the structure shown in formula I from the fruit of Peganum harmala for the first time, and provides a new method for preparing the alkaloid compound with the structure shown in formula I.

[0012] Preferably, the preparation method comprises the following steps:

[0013] (1) Peganum harmala seeds were taken and crushed, and cold-infused with ethanol to obtain the total extract;

[0014] (2) subjecting the extract to an acid extraction and alkali precipitation step to obtain the total alkaloid components;

[0015] (3) Separate the total alkaloids on a D101 macroporous resin column to obtain a macroporous resin elution fraction;

[0016] (4) The macroporous resin elution fraction is subjected to an ODS column for separation to obtain an ODS elution fraction;

[0017] (5) The ODS eluted fraction was subjected to HPLC again to prepare the alkaloid compound with the structure shown in Formula I.

[0018] The present invention also provides an extract containing an alkaloid compound with a structure shown in Formula I.

[0019] Since the alkaloid compound with the structure shown in Formula I has excellent anti-herpes simplex virus type II effect, those skilled in the art can expect that the extract containing the structure shown in Formula I also has anti-herpes simplex virus type II effect.

[0020] Preferably, the mass fraction of the alkaloid compound with the structure shown in Formula I in the extract is 1% to 80%.

[0021] Preferably, the mass fraction of the alkaloid compound of the structure represented by formula I in the extract is 5% to 50%;

[0022] Further preferably, the mass fraction of the alkaloid compound with the structure shown in Formula I in the extract is 10% to 30%.

[0023] Preferably, the extract is an extract prepared using Peganum harmala as raw material.

[0024] The present invention also provides a use of the above alkaloid compound or extract in preparing a medicine having an anti-herpes simplex virus type II effect.

[0025] Preferably, the drug contains a therapeutically effective amount of a compound having the structure shown in Formula I and a pharmaceutically acceptable carrier.

[0026] Preferably, the dosage form of the drug is powder, pill, tablet, capsule, oral solution, aerosol or injection.

[0027] Beneficial Effects: The present invention provides an alkaloid compound with a novel structure. Studies have shown that the alkaloid compound represented by Formula I has highly significant anti-HSV-II activity, and its anti-HSV-II activity is significantly superior to that of the positive drug acyclovir. Furthermore, the alkaloid compound represented by Formula I has an SI value of 86.2, indicating a wider safety margin. Therefore, it has significant application value as an active ingredient in the preparation of drugs with anti-HSV-II activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the chemical structural formula of compound PF.

[0029] Figure 2 is the molecular number of compound PF.

[0030] Figure 3 The figure shows the experimental results of the inhibitory effect of compound PF on herpes simplex virus type II (HSV-2); Figure 3 A in the table represents the cytotoxic effect of compound PF on Vero cells; Figure 3 B and Figure 3 C in the figure is the inhibitory effect of compound PF and positive control drug acyclovir on herpes simplex virus type 2 (HSV-2) at different concentrations; Figure 3 D in the figure is the effect of compound PF on the proliferation of herpes simplex virus type Ⅱ (HSV-2) at different times;

[0031] Figure 4 This is the high-resolution mass spectrum of compound PF.

[0032] Figure 5 This is the UV spectrum of compound PF (dissolved in methanol).

[0033] Figure 6 This is the infrared spectrum of compound PF (KBr pellet).

[0034] Figure 7 The NMR of compound PF 1 H spectrum (600 MHz, dissolved in DMSO).

[0035] Figure 8 The NMR of compound PF 13 C spectrum (150 MHz, dissolved in DMSO).

[0036] Figure 9 The DEPT-135 nuclear magnetic resonance spectrum of compound PF (150 MHz, dissolved in DMSO) is shown in FIG.

[0037] Figure 10 The NMR of compound PF 1 H- 1 H COSY spectrum (150 MHz, dissolved in DMSO).

[0038] Figure 11 The NMR HSQC spectrum of compound PF (150 MHz, dissolved in DMSO) is shown.

[0039] Figure 12 The HMBC NMR spectrum of compound PF (150 MHz, dissolved in DMSO) is shown in FIG.

[0040] Figure 13 The NMR NOESY spectrum of compound PF (150 MHz, dissolved in DMSO) is shown. DETAILED DESCRIPTION

[0041] To more clearly understand the present invention, the present invention is further described with reference to the following examples and accompanying drawings. The examples are intended to illustrate the present invention only and are not intended to limit the present invention in any way. In the examples, all raw materials and reagents are commercially available. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0042] Example 1

[0043] (1) 15.0 kg of dried Peganum harmala seeds were crushed and cold-extracted with 95% ethanol to prepare 1.6 kg of total extract;

[0044] (2) To further concentrate the alkaloid components, the total extract was subjected to an "acid extraction and alkali precipitation" treatment step, ultimately obtaining 378 grams of total alkaloid components; the "acid extraction and alkali precipitation" step was specifically as follows: the total extract was dispersed in 2L of water, the pH of the solution was adjusted to 2 with dilute hydrochloric acid, and then two volumes of dichloromethane were added, and the extraction was performed three times, and the extract was discarded. The pH of the acid solution was then adjusted to 9 with 25% ammonia water, and then two volumes of dichloromethane were added and the extraction was performed three times to obtain the extract, i.e., the total alkaloids;

[0045] (3) The total alkaloids were loaded onto a D101 macroporous resin column and eluted with a mixed solvent of ethanol / water at a volume ratio of 10:90, and the eluate was discarded; then the total alkaloids were eluted with a mixed solvent of ethanol / water at a volume ratio of 30:70, and the eluate was collected and concentrated and dried to obtain a macroporous resin elution fraction;

[0046] (4) The macroporous resin elution fraction was loaded onto an ODS column and eluted with a methanol / water solvent at a volume ratio of 80:20, and the eluate was discarded; then the macroporous resin was eluted with a methanol / water solvent at a volume ratio of 90:10, and the eluate was collected and concentrated to obtain an ODS elution fraction;

[0047] (5) The ODS eluted fraction was again subjected to HPLC (using a C18 reverse phase preparative column, methanol-water-ammonia as the mobile phase, and isocratic elution at a volume ratio of methanol:water:ammonia = 38:62:0.01), and the fractions with retention time t R = 21.2min) to prepare compound PF, an alkaloid compound with a structure shown in Formula I.

[0048] Structural identification basis:

[0049] The structure of compound PF was obtained by analyzing high-resolution mass spectrometry (HRESIMS), infrared spectroscopy (IR), and one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy (NMR).

[0050] Molecular weight determined by high resolution mass spectrometry (HRESIMS): m / z 355.1276 [M + Na] + (Theoretical value: 355.1264), the molecular formula is C 17 H 20 N2O5.

[0051] The infrared spectroscopy showed that there were amino groups (3392 cm -1 ) and benzene ring (1612, 1464cm -1 ).

[0052] Table 1. One-dimensional and two-dimensional NMR spectra of compound PF (DMSO, δ in ppm, J in Hz)

[0053]

[0054] H NMR spectroscopy ( 1 H NMR) data and assignments are shown in Table 1. The data show that compound PF has an AMX spin system [δ H 7.20 (1H, d, J = 8.2), 6.52 (1H, dd, J = 8.2, 2.0), 6.39 (1H, d, J =2.0)], a hydrogen proton signal of a methoxy group [δ H 3.86 (3H, s)] and two methyl proton signals [δ H 1.14 (1H,s), 1.12 (1H, s)].

[0055] According to the carbon nuclear magnetic resonance spectroscopy ( 13 C NMR data and their assignments are shown in Table 1. The data showed that compound PF has 8 quaternary carbons (including five connected oxygens), 3 tertiary carbons, 3 secondary carbons, and 3 primary carbons (including one connected oxygen), for a total of 17 carbon signals.

[0056] The data of the two-dimensional NMR spectra and the binding relationships are shown in Table 1 .

[0057] The molecular number of compound PF is as follows Figure 2 shown.

[0058] Experimental Example 2: Anti-Herpes Simplex Virus Type II Activity Test of Compound PF

[0059] 1. The cytotoxicity of the compound was determined by MTT assay. 1.5 × 10 4 Cells were placed in 96-well plates and cultured at 37 °C in a 5% CO2 incubator for 18 h;

[0060] 2. Add agar overlays containing different concentrations of compound (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM) for culture;

[0061] 3. After 48 hours of cell culture, the cells were treated with MTT assay for 4 hours;

[0062] 4. Then, remove the supernatant and add dimethyl sulfoxide (DMSO) to dissolve the blue-purple crystalline formazan;

[0063] 5. Measure the absorbance at 570 nm and calculate the 50% cytotoxic concentration (CC) based on the nonlinear regression analysis of cell viability. 50 );

[0064] 6. Using the maximum non-cytotoxic concentration (MNCC) as the first concentration, test the antiviral activity of the compound;

[0065] 7. Cell suspension (1.5 × 10 per well) 4 cells) were seeded into each well of a 96-well plate;

[0066] 8. After 18 hours, the Vero cell monolayers were treated with HSV-2 virus and different concentrations of compound PF and cultured for 72 hours;

[0067] 9. Observe the degree of syncytial pathology in each well under a microscope and evaluate the concentration that inhibits half of the CPE, which is the half-maximal inhibitory concentration (IC 50 );

[0068] 10. Finally, the selectivity index of the compound for inhibiting HSV-2 (SI = CC 50 / IC 50 ).

[0069] Table 2. Anti-herpes simplex virus activity of compound PF

[0070]

[0071] From the experimental data in Table 2, it can be seen that the IC of the compound PF shown in Formula I against respiratory syncytial virus 50 0.90 ± 0.10 μM, which is less than the IC of the positive drug acyclovir against respiratory syncytial virus. 50 (1.12 ± 0.15 μM); this shows that the compound PF with the structure shown in Formula I has very significant anti-herpes simplex virus type II activity, and its anti-herpes simplex virus type II activity is significantly better than the positive drug acyclovir; this technical effect is unpredictable for those skilled in the art.

[0072] In addition, it can be seen from the experimental data in Table 2 that the SI value of compound PF against respiratory syncytial virus reached 86.2, which is much larger than the minimum standard of 10.0. This shows that compound PF has a wider safety margin when used to prepare drugs against herpes simplex virus type 2.

[0073] Furthermore, Figure 3 A in the figure shows the cytotoxicity of compound PF to Vero cells (CC 50) was 77.59 ± 1.33 μM. Vero cells were infected with HSV-2 virus at different titers (MOI = 0.1, 1 or 5) to evaluate the tolerance of compound PF to high titer virus infection ( Figure 3 Then, the inhibition of compound PF on respiratory syncytial virus was tested in a plaque inhibition assay at a non-toxic concentration ( Figure 3 The results showed that compound PF and HSV-2 virus acted on Vero cells simultaneously, which could significantly reduce the virus yield in a concentration-dependent manner. Virus yield determination ( Figure 3 D) shows that the number of progeny viruses reached a plateau on day 3 after HSV-2 infection. Compound PF (40 μM) exhibited significant inhibitory effects on HSV-2 infection over a period of 1-4 days. These data demonstrate that compound PF significantly inhibits HSV-2 infection at varying titers.

[0074] It will be understood by those skilled in the art that the use of the present invention is not limited to the specific applications described above. With respect to the specific elements and / or features described or depicted herein, the present invention is not limited to its preferred embodiments. It will be understood that the present invention is not limited to the disclosed embodiment examples or embodiments, and that many rearrangements, modifications, and substitutions are possible without departing from the scope of the present invention as set forth and defined by the following claims.

Claims

1. An alkaloid compound, characterized in that It has the structure shown in formula I: Formula I.

2. The method for preparing the alkaloid compound according to claim 1, wherein The preparation method comprises the following steps: (1) Peganum harmala seeds were taken and crushed, and cold-infused with ethanol to obtain the total extract; (2) subjecting the extract to an acid extraction and alkali precipitation step to obtain the total alkaloid components; (3) The total alkaloids were loaded onto a D101 macroporous resin column and eluted with a mixed solvent of ethanol / water at a volume ratio of 10:90, and the eluate was discarded; then the total alkaloids were eluted with a mixed solvent of ethanol / water at a volume ratio of 30:70, and the eluate was collected and concentrated and dried to obtain a macroporous resin elution fraction; (4) The macroporous resin elution fraction was loaded onto an ODS column and eluted with a methanol / water solvent at a volume ratio of 80:20, and the eluate was discarded; then the macroporous resin was eluted with a methanol / water solvent at a volume ratio of 90:10, and the eluate was collected and concentrated to obtain an ODS elution fraction. (5) The ODS eluted fraction was subjected to HPLC again using a C18 reverse phase preparative column with methanol-water-ammonia as the mobile phase and isocratic elution at a volume ratio of methanol:water:ammonia = 38:62:0.

01. The retention time was collected. t R = 21.2 min, the fraction corresponding to the chromatographic peak, was used to prepare compound PF, which is the alkaloid compound with the structure shown in formula I according to claim 1.

3. An extract, characterized in that An alkaloid compound containing the structure represented by formula I according to claim 1.

4. The extract according to claim 3, characterized in that The mass fraction of the alkaloid compound with the structure shown in formula I according to claim 1 in the extract is 1% to 80%.

5. The extract according to claim 4, characterized in that The mass fraction of the alkaloid compound with the structure shown in formula I according to claim 1 in the extract is 5% to 50%.

6. Use of the alkaloid compound according to claim 1 or the extract according to any one of claims 3 to 5 in the preparation of a medicament having an anti-herpes simplex virus type II effect.

7. The use according to claim 6, characterized in that The medicine contains a therapeutically effective amount of a compound having a structure shown in formula I and a pharmaceutically acceptable carrier.

8. The use according to claim 6, characterized in that The dosage form of the medicine is powder, pill, tablet, capsule, oral liquid, aerosol or injection.

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