CN109651362B

CN109651362B - Swainsonine derivative and preparation method and application thereof

Info

Publication number: CN109651362B
Application number: CN201811559615.2A
Authority: CN
Inventors: 梁剑平; 刘宇; 梁妍; 郝宝成; 王保海; 王学红; 郭文柱; 赵凤舞; 杨珍; 贾忠; 张新刚; 陶蕾
Original assignee: Lanzhou Institute of Animal Husbandry and Veterinary Medicine CAAS
Current assignee: BEIJING ZHONGLIAN HUAKANG TECHNOLOGY Co.,Ltd.
Priority date: 2018-12-20
Filing date: 2018-12-20
Publication date: 2021-06-01
Anticipated expiration: 2038-12-20
Also published as: CN109651362A

Abstract

The invention discloses a swainsonine derivative which has the following chemical structural formula:

Description

Swainsonine derivative and preparation method and application thereof

Technical Field

The invention relates to a swainsonine derivative and a preparation method and application thereof.

Background

Locoweed is distributed in the western part of semiarid regions and western countries for the first time, and can generate a large amount of toxic herbs which can cause animals to generate lochia, and then the toxic herbs of leguminous acanthaceae (oxotropis) and Astragalus (Astragalus) are collectively called Locoweed, and swainsonine SW (swainsonine) is the main toxic component. In 2006, Tanaka and other researches show that SW competitively inhibits alpha-mannosidase activity in endoplasmic reticulum, destroys endoplasmic reticulum function, blocks expression of HPIV3 surface protein fusion protein (F) and hemagglutinin-neuraminidase (HN), and weakens the capability of infection of cells by human parainfluenza virus type 3 (HPIV3) virus, thereby inhibiting the activity of SW. The domestic scholars use the established SW to treat the newcastle disease virus model to research the immunotherapy effect of the SW on the newcastle disease virus vaccine, and the research result shows that the antibodies of the SW treatment group are obviously increased and a certain inhibition effect on the Newcastle Disease Virus (NDV) is generated. The therapeutic effect of swainsonine on chicken Marek's Disease (MD) is also studied by scholars, and the results show that SW has a regulating effect on WBC, AKP and LDH levels in chicken and has a certain inhibiting effect on the formation and metastasis of MD tumors. Long-term research shows that the swainsonine has unstable property, and the popularization and application of the swainsonine in clinic are limited.

Aspirin (Aspirin), also known as Aspirin or acetylsalicylic acid, has been used clinically for over 80 years and is an ancient antipyretic and analgesic drug. With the recent intensive research on prostaglandins, new insights have been made into aspirin. Clinically, the traditional Chinese medicine composition is used for relieving fever, easing pain and resisting inflammation, and is also used for resisting platelet aggregation and the like. Also has important value in preventing and treating myocardial infarction, cerebrovascular disease and other vascular embolic diseases, regulating renal blood flow and sodium balance and preventing cancer.

Disclosure of Invention

The invention aims to provide a novel swainsonine derivative, a preparation method and application thereof according to the current situation of the background technology.

In order to solve the technical problems, the invention provides the following technical scheme:

a swainsonine derivative has the following chemical structural formula:

the preparation method of the swainsonine derivative comprises the following steps: the aspirin and the swainsonine react by an acyl chloride esterification method to obtain the swainsonine derivative.

Preferably, the specific process of the acyl chloride esterification method comprises the following steps: aspirin and dichlorosulfoxide are firstly subjected to acyl chlorination reaction, and then swainsonine is added for alcoholysis.

Preferably, the acid chloride esterification method employs N, N-Dimethylformamide (DMF) as a solvent. The N, N-dimethylformamide can also play a role of an acid binding agent while being used as a solvent.

Preferably, the amount ratio of aspirin to N, N-dimethylformamide is 0.05-0.3 g/ml.

Preferably, the mole ratio of the aspirin to the melanterite is 1:0.5-1.5, and the mole ratio of the aspirin to the swainsonine is 1: 0.5-1.5.

Preferably, the mole ratio of the aspirin to the melanterite is 1:0.85-1, and the mole ratio of the aspirin to the swainsonine is 1: 1-1.15.

Preferably, the acyl chlorination reaction time is 0.5-2.5h, and the alcoholysis reaction time is 4-12 h.

The swainsonine derivative of the invention has simple preparation process and easy operation. Compared with the swainsonine, the swainsonine derivative has stronger antiviral activity and can be used for preparing antiviral drugs, such as canine distemper virus resistant drugs.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.

EXAMPLE 1 preparation of swainsonine derivatives

1.1 Experimental part

1.1.1 Main instruments and reagents

An Avance model 400 nuclear magnetic resonance spectrometer, a Tensor model 27 infrared spectrometer (Bruker, Germany); DSQ type II mass spectrometer (Thermo corporation, usa); x-6 type precise micro melting point tester (Beijing ya Leien electromechanical technical research institute).

Aspirin, DMF, SOCl₂(analytical purity, chemical reagents of national drug group, Ltd.); swainsonine (not less than 99%, industrial product, Shanghai-Yiyan Biotech Co., Ltd.); the reagents such as ethyl acetate, petroleum ether (60-90 ℃) and the like are all commercially available analytical purifiers; and (4) carrying out common column chromatography on silica gel (200-300 meshes).

1.1.2 Experimental methods

1.1.2.1 chemical reactions of swainsonine derivatives

1.1.2.2 Synthesis procedure of swainsonine derivatives

1.260g (7mmol) aspirin and 5mL DMF were added to a single-neck flask, stirred at room temperature and dissolved, and 0.507mL (7mmol) SOCl was slowly added dropwise with cooling in an ice-water bath₂Acyl chlorination reaction is carried out for 1h at 25 ℃, and then 1.38g (8mmol) of swainsonine is added for alcoholysis for 10h at 25 ℃. After completion of the reaction, 40mL of ethyl acetate and 40mL of distilled water were added to the reaction mixture, followed by liquid separation, extraction of the aqueous layer with 40mL of ethyl acetate, combination of the organic layers, washing with saturated brine (40 mL. times.4), and anhydrous MgSO₄Drying, filtering, removing ethyl acetate and then carrying out column chromatography (V (CH)₂Cl₂)∶V(CH₃OH) ═ 20: 1) to give a white powdery solid in 88.20% yield, m.p.164-166 ℃.

¹HNMR(DMSO-d6，400MHz)，δ：11.08(s，1H，2′-OH)；8.37(s，1H，3′-OH)；7.20(d，2H，J＝8.0Hz，-C₆H₄)；7.10(d，2H，J＝8.0Hz，-C₆H₄)；5.32(s，1H，5′-OH)；4.47(d，1H，J＝1.6Hz，4′-H)；4.14～4.05(m，2H，6′-H)；4.01～3.29(m，1H，5′-H)；3.79(q，1H，J＝7.12，2-H)；2.41(d，2H，J＝7.12Hz，4-H)；1.85～1.74(m，1H，5-H)；1.40(d，3H，J＝7.12Hz，3-H)；0.84(d，6H，J＝6.4Hz，6-H，7-H)。

¹³CNMR(DMSO-d6，100MHz)，δ：174.18，170.76，152.58，152.51，140.28，138.24，129.55，127.57，118.63，75.29，65.89，65.73，65.21，65.08，44.68，44.57，30.06，22.61，22.58，18.90。

IR(KBr)，v，cm^-1：3213，2955，1760，1683，1338，848，692。ESI-MS，m/z：365.5[M+H]；387.1[M+Na]。

1.1.2.3 optimization of Synthesis conditions

1.1.2.3.1 Effect of DMF dosage on reaction yield

1.260g (7mmol) aspirin and different amount of DMF were added into a single-neck flask, stirred and dissolved at room temperature, and 0.507mL (7mmol) SOCl was slowly added dropwise while cooling in an ice-water bath₂Acyl chlorination reaction is carried out for 1h at 25 ℃, then 0.865g (5mmol) of swainsonine is added, and alcoholysis is carried out for 10h at 25 ℃. The effect of DMF content on the reaction yield was examined and the results are shown in Table 1.

TABLE 1 influence of the amount of DMF on the reaction yield

As can be seen from Table 1, DMF has a significant promoting effect on the reaction, and the yield is the greatest when the amount of DMF reaches 10 mL; the yield is rather decreased by increasing the amount of DMF, and the possible reason for this is that the collision efficiency is decreased by increasing the amount of DMF, and the system concentration becomes thinner. In general, the ratio of aspirin to DMF may be controlled to be 0.05-0.3g/ml, more preferably 0.12-0.25 g/ml.

1.1.2.3.2 Effect of acyl chlorination reaction time on yield

1.260g (7mmol) aspirin and 5mL DMF were added to a single-neck flask, stirred at room temperature and dissolved, and 0.507mL (7mmol) SOCl was slowly added dropwise with cooling in an ice-water bath₂Acyl chlorination reaction is carried out at 25 ℃ for different times, then 0.865g (5mmol) of swainsonine is added, and alcoholysis is carried out for 10h at 25 ℃. The effect of the acid chlorination reaction time on the reaction yield was examined and the results are shown in Table 2.

TABLE 2 Effect of acylchlorination reaction time on reaction yield

As can be seen from Table 2, the reaction yield does not vary significantly with the duration of the acid chlorination under the given conditions, and two possible causes are: firstly, DMF is taken as an acid-binding agent to absorb byproduct HCl generated in the acyl chlorination reaction in time, so that the acyl chlorination reaction is facilitated to be carried out, and meanwhile, the absorbed HCl has a catalytic effect on subsequent alcoholysis; secondly, since the acid chloride is not isolated, it is still possible to continue the acid chlorination reaction while the alcoholysis is taking place. In general, the acyl chlorination reaction is preferably carried out for 0.5 to 2.5 hours, more preferably for 1 hour.

1.1.2.3.3 Effect of alcoholysis time on reaction yield

1.260g (7mmol) of aspirin and 5mL of DMF were added to a single-neck flask, stirred at room temperature to dissolve, and 0.435mL (6mmol) of SOCl was slowly added dropwise with cooling in an ice-water bath₂Performing acyl chlorination at 25 ℃ for 1h, adding 0.865g (5mmol) of swainsonine, performing alcoholysis reaction at 25 ℃ for different times, and inspecting the influence of the alcoholysis reaction time on the reaction yield, wherein the results are shown in Table 3.

TABLE 3 influence of alcoholysis time on reaction yield

As can be seen from Table 3, the reaction yield increased and then decreased with the increase of the reaction time. This may be caused by residual SOCl after completion of the acylchlorination reaction₂Not separated from the reaction system, and not separated SOCl along with the prolonging of alcoholysis time₂It is likely to react with the upper hydroxyl group in the swainsonine molecule to generate corresponding chlorohydrocarbon, so that the reaction system becomes more complicated, and the ester forming time is too long, so that the reaction yield is reduced. In general, alcoholysis is preferably carried out for 4 to 12 hours, in particular for 10 hours.

1.1.2.3.4SOCl₂Effect of the amount on the reaction yield

1.260g (7mmol) of aspirin and 5mL of DMF are added into a single-neck flask, stirred and dissolved at room temperature, and cooled in an ice-water bath to be slowly releasedSlowly dropwise adding different amounts of SOCl₂Acyl chlorination reaction at 25 deg.C for 1 hr, adding 0.865g (5mmol) swainsonine, alcoholysis at 25 deg.C for 10 hr, and inspecting SOCl₂The effect of the amount on the reaction yield is shown in Table 4.

TABLE 4 SOCl₂Effect of the amount on the reaction yield

As can be seen from Table 4, with SOCl₂The reaction yield increases first and then decreases with increasing amount. The possible cause of this phenomenon is SOCl which does not participate in the reaction in the acylation reaction₂Probably, the reaction with-OH of swainsonine generates corresponding chlorinated hydrocarbon, and the side products are increased. And from the appearance color of the product, with SOCl₂The product color deepens when the dosage is increased. Taking into account n (aspirin):n (SOCl)₂) Can be controlled at 1:0.5-1.5, preferably at 1:0.85-1, and optimally at a molar ratio of 1: 1.

1.1.2.3.4 Effect of swainsonine dosage on reaction yield

1.260g (7mmol) of aspirin and 5mL of DMF are added into a single-neck flask, stirred and dissolved at room temperature, and 0.435mL (6mmol) of SOCl is slowly added dropwise under cooling of an ice-water bath₂Acyl chlorination reaction is carried out for 0.5h at 25 ℃, swainsonine with different gram numbers is added for reaction for 10h at 25 ℃, the influence of the swainsonine dosage on the reaction yield is examined, and the result is shown in table 5.

TABLE 5 Effect of swainsonine usage on reaction yield

As can be seen from Table 5, the reaction yield gradually increased with the increase in the amount of swainsonine used. In the immobilization of SOCl₂Under the condition of using amount, increasing the using amount of the swainsonine means increasing the concentration of alcoholysate, which is beneficial to the generation of products; as the consumption of swainsonine continues to increase, the system becomes more viscous, thereby affecting mass transferThe yield decreased slightly. Therefore, n (aspirin) and n (swainsonine) can be controlled at 1:0.5-1.5, preferably at 1:1-1.15, and the optimal molar ratio is 7: 8.

1.2 conclusion

Using DMF and SOCl₂Formation of salts

Therefore, aspirin is promoted to generate acyl chloride, DMF has good solubility, other solvents are not needed to be added, the acyl chloride generated in the reaction is not separated, and swainsonine is directly added to react with the acyl chloride to generate swainsonine and aspirin ester.

Example 2 in vitro Activity assay of swainsonine derivatives against Canine distemper Virus

Canine distemper is a highly-contagious disease caused by canine distemper virus, has the characteristics of high morbidity, high mortality and the like, is mainly clinically used for high fever, respiratory symptoms and neurological symptoms, and is one of epidemic diseases which have the greatest harm to the pet industry at present. At present, the immunoprophylaxis of canine distemper virus mainly depends on attenuated vaccine, but the attenuated vaccine can damage the nervous system to a certain extent and even cause immunosuppression. Therefore, the search for safer and more effective vaccines or drugs is urgent. The test compares the in vitro activity of swainsonine and swainsonine derivatives against canine distemper virus.

2.1 materials and methods

2.1.1 test drugs

Swainsonine and swainsonine derivatives.

Vero, a gift from national sea biology, languis, gansu.

Canine Distemper Virus (CDV), purchased from the institute of veterinary medicine in china.

MEM medium, fetal bovine serum, purchased from HyClone, USA; 100mL/L fetal bovine serum cell growth medium and trypsin, which are purchased from Difco corporation, Shanghai Biochemical reagents, USA, and dispensed separately; sodium bicarbonate and the like are all domestic analytical pure reagents.

2.1.2 viral infection of half cells (TCID)₅₀)

According to the conventional method, the Vero cells are subjected to digestion and passage and added into a 96-well cell culture plate with the concentration of 0.1 mL/well and the concentration of 50mL/LCO₂And cultured at 37 ℃. After the cells grew into a monolayer, the dilution of the inoculation was 10^-1-10^-10The virus solution of (4) was inoculated into 6 wells per dilution, and cell control was additionally provided, 50mL/L CO₂Incubated at 37 ℃ for 72 hours, and observed daily for cytopathic effect (CPE). Calculating TCID₅₀。

2.1.3 the cell-innocuous concentration (TC) of the drug₀) Measurement of (2)

Diluting the stock solution in multiple times, adding into Vero cell 96-well culture plate with 0.1 mL/well, repeating 6 wells for each dilution, setting cell control, and placing in 50mL/L CO₂Incubate at 37 ℃ for 72h in an incubator, and observe CPE every day.

2.1.4 Swainsonine and Swainsonine + Aspirin and Swainsonine derivatives in vitro CDV test

Using 100TCID₅₀The CDV of (1) infects Vero cells, and a cell control group, a virus control group, a swainsonine + aspirin group (equal-equivalent physical mixing), a swainsonine group and a swainsonine derivative group are arranged. Dosing is carried out by adopting 3 modes: 1. blocking action of the drug (first drug and then toxin): firstly, adding the medicament into Vero cells, culturing for 2h at 37 ℃, and then inoculating viruses; 2. inhibition of virus by drugs (first toxic then drug): infecting the virus with Vero cells, culturing at 37 ℃ for 2h, and then adding medicines with various concentrations; 3. direct action of the drugs (drug toxicity is added): the virus was added to Vero cells at 37 ℃ simultaneously with the drug. Thereafter, each group of cells was placed in 50mL/L CO₂Incubations were carried out at 37 ℃ and the CPE was observed daily and recorded when the CPE reached ++++ in the virus control group.

2.2 results

2.2.1 Virus TCID₅₀Measurement of (2)

The TCID of CDV was measured 72h after inoculating Vero cells with the virus₅₀Is 10^-5.68/0.1mL。

2.2.2 drug-on-cell TC₀Measurement of (2)

The TC of the drug to the cells is measured 72h after the drug is added into the Vero cells₀It was 0.312 g/L. The results are shown in Table 6.

TABLE 6 non-toxic concentration of drug To Cells (TC)₀) Measurement of (2)

2.2.3 cell culture and Observation of cytopathic effects

After Vero cells are added into a 96-well plate, compact single layers can be grown in 24h, normal cells are in various shapes such as polygonal shapes and circular shapes, and the cell outline is clear. At 72h and 96h after CDV inoculation, the cells gradually lose the polygonal contour and begin to fall off, become round and gather into a pile.

2.2.4 Effect of drugs on viruses under different modes of administration

2.2.4.1 the result of the blocking effect of the drug on the virus shows that round cells or dead cells are seen everywhere in the field of view of the virus control group at 72h, and the pathological features are very obvious. The swainsonine group showed a large number of round cells or dead cells in the visual field as the swainsonine + aspirin group, and the cell monolayer was severely damaged. At 96h, the cell monolayers in the two groups were almost completely destroyed. The swainsonine derivatives have a certain antiviral effect, which is shown in Table 7.

TABLE 7 blocking Effect of drugs on viruses

2.2.4.2 results of the drug-induced viral inhibition showed that at 72h the test cell monolayer was essentially intact, most of the cells grew well, and occasionally round or dead cells appeared. At 96h, the cells of the virus control group are all diseased, while the cells of the test group are damaged by the virus, but the cell monolayer is basically good, and the refractive index of the cells is basically consistent with that of normal cells. The swainsonine derivatives have more obvious antiviral effects than swainsonine, swainsonine and aspirin, and are shown in Table 8.

TABLE 8 inhibitory Effect of drugs on viruses

2.2.4.3 direct killing effect of the drug on the virus shows that most of the cells in the test group at 72h are identical to those in the virus control group and are seriously damaged, the cytopathic effect is typical, and the cell monolayer is seriously damaged and basically does not form a monolayer at 96 h. The swainsonine derivatives have antiviral effect, as shown in Table 9.

TABLE 9 direct killing of viruses by drugs

2.3 discussion

In recent years, with the great increase of the feeding amount of military dogs, police dogs, test dogs and pet dogs and the change of ecological environment in China, the morbidity and the mortality of canine distemper in canine animals in China are obviously increased. No specific medicine exists in the current treatment of canine distemper, the symptomatic treatment is mainly carried out by injecting canine distemper hyperimmune serum in a large dose and combining antibacterial drugs, corticoid drugs, vitamins and the like, and meanwhile, a good nursing effect can be obtained for early sick dogs, so that the research and development of novel antiviral drugs for treating canine distemper have very important significance.

In the test, the effects of swainsonine, swainsonine + aspirin and swainsonine derivatives on canine distemper virus resistance are observed through three different administration modes: the inhibition effect of the medicine on the virus (first poison and then medicine) aims to observe whether the medicine can act on the virus in cells and inhibit the biosynthesis and mature release of the virus; the drug exhibits an effect against virus-infected cells (drug first and then toxin) by enhancing the resistance of the cells, aiming to observe whether the drug can enter the cells or adsorb to the cell surface to prevent adsorption and proliferation of the virus; the medicine and virus are added into cells simultaneously (the medicine and the virus are added simultaneously), aiming at observing the direct killing effect of the medicine on the virus. Test results show that under the condition that the swainsonine, the swainsonine plus the aspirin and the swainsonine derivative are firstly infected with the virus and then administered, cells of a virus control group are obviously diseased, but cells of a test group grow well, the refractive index of the cells is basically consistent with that of normal cells, and particularly the swainsonine derivative group shows good inhibition effect on the virus; under the condition of infecting viruses after first administration, the protective effect of the swainsonine, the swainsonine plus aspirin and the swainsonine derivative on Vero cells is weaker, and the swainsonine derivative has a certain antiviral effect compared with other groups, possibly related to the reason that the action time of the medicament is too short, the medicament cannot be completely absorbed by the cells, and the action concentration of the added medicament is indirectly reduced.

In conclusion, the swainsonine and the aspirin are physically mixed, the swainsonine derivative has stronger inhibitory action on CDV in-vitro cell tests, and the swainsonine derivative is obviously stronger than equivalent physical mixtures of swainsonine groups, swainsonine and aspirin and the like.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A swainsonine derivative has the following chemical structural formula:

。

2. a process for the preparation of swainsonine derivatives as claimed in claim 1 which comprises: the aspirin and the swainsonine react by an acyl chloride esterification method to obtain the swainsonine derivative.

3. The method of claim 2, wherein: the specific process of the acyl chloride esterification method comprises the following steps: aspirin and dichlorosulfoxide are firstly subjected to acyl chlorination reaction, and then swainsonine is added for alcoholysis.

4. The production method according to claim 2 or 3, characterized in that: the acyl chloride esterification method adopts N, N-dimethylformamide as a solvent.

5. The method of claim 4, wherein: the dosage ratio of aspirin and N, N-dimethylformamide is 0.05-0.3 g/ml.

6. The production method according to claim 3 or 5, characterized in that: the molar ratio of the aspirin to the melanterite is 1:0.5-1.5, and the molar ratio of the aspirin to the swainsonine is 1: 0.5-1.5.

7. The method of claim 4, wherein: the molar ratio of the aspirin to the melanterite is 1:0.5-1.5, and the molar ratio of the aspirin to the swainsonine is 1: 0.5-1.5.

8. The method of claim 6, wherein: the molar ratio of the aspirin to the melanterite is 1:0.85-1, and the molar ratio of the aspirin to the swainsonine is 1: 1-1.15.

9. The method of claim 7, wherein: the molar ratio of the aspirin to the melanterite is 1:0.85-1, and the molar ratio of the aspirin to the swainsonine is 1: 1-1.15.

10. The production method according to any one of claims 3, 5, and 7 to 9, wherein: the time of the acyl chlorination reaction is 0.5-2.5h, and the time of the alcoholysis reaction is 4-12 h.

11. The method of claim 4, wherein: the time of the acyl chlorination reaction is 0.5-2.5h, and the time of the alcoholysis reaction is 4-12 h.

12. The method of claim 6, wherein: the time of the acyl chlorination reaction is 0.5-2.5h, and the time of the alcoholysis reaction is 4-12 h.

13. Use of swainsonine derivatives as claimed in claim 1 wherein: the swainsonine derivative is applied to the preparation of antiviral drugs.

14. Use according to claim 13, characterized in that: the swainsonine derivative is applied to the preparation of the medicine for resisting canine distemper virus.