CN112920194B - Chromene coumarin derivative containing fluorine functional group and preparation method and application thereof - Google Patents

Abstract

The invention discloses a coumarin derivative containing fluorine functional groups of chromenes and a preparation method and application thereof, which are characterized in that the structure of the coumarin derivative is shown as a formula I, the preparation method comprises the specific steps of putting 2 mmol of 4-hydroxycoumarin, 3 mmol of p-fluorobenzaldehyde, 2 mmol of malononitrile and 0.1 mmol of sodium dodecyl sarcosine into a round-bottom flask, adding 10 mL of water as a solvent, stirring for 5 hours at the temperature of 60-65 ℃, filtering and removing water after the reaction is finished to obtain a crude product, and recrystallizing the crude product by adopting dichloromethane to obtain 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H, 5H-pyrone [3,2-c ] chromene Easy to transform.

Description

A kind of coumarin derivative of chromene fluorine-containing functional group and its preparation method and application

technical field

The present invention relates to coumarin derivatives, in particular to a chromene fluorine-containing functional group coumarin derivative and its preparation method and application.

Background technique

Coumarin is a class of heterocyclic compounds belonging to the benzopyrrolone class, with antibacterial, anti-inflammatory, antioxidant, anti-tumor, anti-viral and other biological activities, and is easy to be synthesized and converted into a variety of functional coumarin, Widely used in the fields of medicine and pesticides. Vitiligo is listed as one of the animal diseases that must be reported by the World Organization for Animal Health (Office international des épizooties, OIE). Since the isolation and identification of the white spot syndrome virus, researchers have been working hard to find treatments that can effectively prevent and control the virus. In general, the research, application and basic innovation research and industrialization of high-efficiency, low-toxicity, low-residue, and pollution-free aquatic and fishery drugs for fishery vaccines and industrialization are the current methods to control the occurrence of diseases and ensure the safety of aquatic products and ecological safety. means. However, since the immune system of animal larvae is not yet fully developed, the variation of pathogens and the diversity of antigens are also one of the most important factors affecting the use of fishing vaccines. Therefore, the research on drug control technology is of great significance for the prevention and control of white spot syndrome and other aquatic virus diseases and healthy breeding.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a chromene-based fluorine-containing functional group coumarin derivative with excellent anti-white spot syndrome virus effect, relatively simple synthesis process, high yield and easy conversion, and the same. Preparation method and application.

The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a chromene-based coumarin derivative containing a fluorine functional group, and the structure of the coumarin derivative is shown in formula I:

Formula I.

The preparation method of the coumarin derivatives of the chromene-based fluorine-containing functional group comprises the following steps: the Biginelli reaction of 4-hydroxycoumarin, p-fluorobenzaldehyde and malononitrile to obtain 4-fluorophenyl -3-cyano-2-amino-5-deoxy-4H,5H-pyrone[3,2-c]chromene.

The specific steps are as follows: put 2 mmol of 4-hydroxycoumarin, 3 mmol of p-fluorobenzaldehyde, 2 mmol of malononitrile and 0.1 mmol of sodium lauryl sarcosinate in a round-bottomed flask, add 10 mL of water as a solvent, Stir at 60-65 °C for 5 h. After the reaction, remove water by filtration to obtain the crude product, and recrystallize the crude product with dichloromethane to obtain 4-fluorophenyl-3-cyano-2-amino-5-deoxy- 4H,5H-pyrone[3,2-c]chromene, the structure of which is shown in formula I:

Formula I.

The application of the coumarin derivatives of the chromene type fluorine functional group in the preparation of a drug for inhibiting the vitiligo syndrome virus.

Compared with the prior art, the advantages of the present invention are: a chromene fluorine-containing functional group-containing coumarin derivative, a preparation method and an application thereof, the coumarin derivative is effective against vitiligo syndrome. The virus has good killing activity, excellent antiviral effect, and the synthesis process is relatively simple, the yield is high, and the transformation is easy, and it has the application value of preventing and controlling aquatic virus diseases.

Description of drawings

Fig. 1 is the general synthetic route schematic diagram of 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H,5H-pyrone[3,2-c]chromene;

Figure 2 is the copy number map of WSSV and the mortality map of larvae, where A is 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H,5H-pyrone at different concentrations [ Inhibition of WSSV copy number by 3,2-c]chromene, B is 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H,5H-pyrone[3] ,2-c]chromene survival rate of larvae at 72 h, C is 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H,5H-pyrone[3,2 -c] The copy number map of WSSV after soaking larvae with chromene and WSSV for 24, 48 and 72 h at the same time;

Figure 3 is the copy number map of WSSV and the mortality map of larvae, where A is WSSV pre-incubated 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H,5H-pyrone[ 3,2-c]chromene infection of larvae after 1, 2 and 4 h, copy number map of WSSV, B is WSSV pre-incubated 4-fluorophenyl-3-cyano-2-amino-5-deoxy -4H, 5H-pyrone[3,2-c]chromene 1, 2 and 4 hours after infection of larvae, the mortality of larvae;

Figure 4 is the copy number map of WSSV and the mortality map of larvae, where A is the pre-incubated 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H,5H-pyrone of larvae [3,2-c]chromene was reinfected with WSSV after 1, 4 and 8 h, copy number map of WSSV, B is larvae pre-incubated with 4-fluorophenyl-3-cyano-2-amino-5-de Mortality of larvae after 1, 4 and 8 hours of oxy-4H,5H-pyrone[3,2-c]chromene infection with WSSV;

Figure 5 is the copy number map of WSSV and the death curve of larvae, where A is no replacement of 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H,5H-pyrone[3, 2-c]chromene, the mortality of WSSV-infected larvae, B is a replacement of 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H, 5H-pyrone[3, 2-c]chromene, the mortality chart of WSSV-infected larvae, C is two replacements of 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H,5H-pyrone[3 ,2-c]chromene, the mortality of WSSV-infected larvae, D is three replacements of 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H,5H-pyrone[3 ,2-c]chromene, mortality of WSSV-infected larvae, E is four replacements of 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H,5H-pyrone[ 3,2-c]chromene, mortality of WSSV-infected larvae, F is five replacements of 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H,5H-pyrone [3,2-c]chromene, mortality of WSSV-infected larvae, G is five replacements of 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H,5H-pyran Keto[3,2-c]chromene, copy number map of WSSV;

Figure 6 is the copy number map of WSSV and the mortality map of larvae, where A is 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H,5H-pyrone[3,2 -c]chromene soaked larvae simultaneously with WSSV after standing in aquaculture water for 0 h, copy number map of WSSV, B is 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H , 5H-pyrone[3,2-c]chromene was left standing in aquaculture water for 0 h and soaked larvae at the same time with WSSV, the mortality chart of larvae, C is 4-fluorophenyl-3-cyano- 2-Amino-5-deoxy-4H, 5H-pyrone[3,2-c]chromene was left standing in aquaculture water for 24 h and soaked larvae with WSSV at the same time, copy number map of WSSV, D is 4- Fluorophenyl-3-cyano-2-amino-5-deoxy-4H, 5H-pyrone[3,2-c]chromene was left standing in aquaculture water for 24 h and soaked larvae simultaneously with WSSV. Mortality diagram of shrimp, E is 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H,5H-pyrone[3,2-c]chromene standing in aquaculture water 48 h soaked larvae at the same time with WSSV, copy number map of WSSV, F is 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H, 5H-pyrone[3,2-c ]chromene was left standing in aquaculture water for 48 h and soaked larvae at the same time with WSSV. Mortality chart of larvae, G is 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H, 5H -Pyranone[3,2-c]chromene was left standing in aquaculture water for 72 h and soaked larvae with WSSV at the same time, the copy number map of WSSV, H is 4-fluorophenyl-3-cyano-2-amino -5-Deoxy-4H, 5H-pyrone[3,2-c]chromene was left standing in aquaculture water for 72 h and soaked larvae with WSSV at the same time, the mortality of larvae, I was 4-fluorobenzene Alkyl-3-cyano-2-amino-5-deoxy-4H,5H-pyrone[3,2-c]chromene was left standing in aquaculture water for 96 h while soaking larvae with WSSV, a copy of WSSV Digital map, J is 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H, 5H-pyrone[3,2-c]chromene in aquaculture water for 96 h and WSSV soaked larvae at the same time, the mortality chart of larvae.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments of the accompanying drawings.

Specific embodiment one

A chromene fluorine-containing functional group-containing coumarin derivative, the structure of the coumarin derivative is shown in formula I:

Formula I.

Specific embodiment two

1. The preparation method of coumarin derivatives in the above-mentioned specific embodiment 1, as shown in Figure 1, the steps are as follows

Place 2 mmol of 4-hydroxycoumarin, 3 mmol of p-fluorobenzaldehyde, 2 mmol of malononitrile and 0.1 mmol of sodium lauryl sarcosinate in a round-bottomed flask, add 10 mL of water as a solvent, and mix at 60-65 Stir at ℃ for 5 h. After the reaction, remove water by filtration to obtain the crude product, and recrystallize the crude product with dichloromethane to obtain 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H, 5H- Pyranone[3,2-c]chromene, whose structure is shown in formula I:

Formula I.

2. The structural data of the target compounds are listed in Tables 1 and 2:

Table 1. Properties of the compounds

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Table 2. ¹ H NMR, ¹³ C NMR and ESI-MS data of compounds

As can be seen from Table 1, the preparation method of the target compound provided by the present invention has a high yield, and the structure of the target compound can be determined from Table 2. The target compounds such as 4.50 in the hydrogen spectrum and 36.26 in the carbon spectrum in Table 2 are special chemical shift, which proves the correctness of the reaction product structure.

Specific embodiment three

1. Determination of anti-white spot syndrome virus activity

(1) Test material

Virus material: White spot syndrome virus (WSSV), sourced from Zhejiang Institute of Marine Aquaculture; Experimental animal: Pacific white shrimp post-larvae, sourced from Zhejiang Institute of Marine Aquaculture Qingjiang Base.

Preparation of the drug solution to be tested: Accurately weigh 500 mg of the coumarin derivatives with the structure shown in formula I (referred to as the compound of formula I or C5), put them in a 10 mL volumetric flask, add dimethyl sulfoxide (DMSO). ) and dissolved to constant volume to obtain the test solution with a concentration of 50 mg/mL, which was stored in a refrigerator at 4°C for later use.

(2) WSSV challenge concentration detection

P. vannamei larvae were randomly added to six-well plates, each well containing 6 mL of culture water and 10 larvae. The WSSV group was soaked with different concentrations of virus diluent, the concentrations were 1.6×10 ⁴ , 1.6×10 ⁵ , 1.6×10 ⁶ , 1.6×10 ⁷ and 1.6×10 ⁸ copies/μL, respectively. The blank control group had only aquaculture water. The temperature was maintained at 28 ± 0.5°C during the test. The experiment lasted for 3 days, and the mortality of larvae was recorded every 24 hours. According to the test results, the WSSV concentration at which the mortality of larvae reached 100% within 3 days was selected as the challenge concentration for the subsequent experiments.

(3) Toxicity detection of compounds of formula I to larvae of Penaeus vannamei

P. vannamei larvae were randomly added to six-well plates, each well containing 6 mL of culture water and 10 larvae. The larvae were soaked in different concentrations of the test solution (0.1-40 mg/L), and a solvent control group (soaked in 0.08% DMSO) and a blank control group were set. The temperature was kept at 28 ± 0.5 °C, and the survival status of larvae was observed and recorded for 72 hours.

(4) Detection of antiviral activity of compounds of formula I

A. P. vannamei larvae were randomly added to six-well plates, each well containing 6 mL of culture water and 10 larvae. The larvae were soaked in the WSSV dilution solution (final concentration of 1.6×10 ⁶ copies/μL) and the test solution (0.32, 0.63, 1.25, 25, 5 and 10 mg/L) at the same time. The control group was added with WSSV virus dilution and 0.02% DMSO. Observe the survival of larvae every 12 hours;

b. C5 (10 mg/L) and WSSV dilution solution (8×10 ⁷ copies/μL) were pre-incubated for 1, 2 or 4 h, and the control group was incubated with 0.04% DMSO, and then the mixture was diluted 50 times and soaked in larvae. The concentration of C5 was 0.4 mg/L (C5 did not work at this concentration), the copy concentration of WSSV dilution was 1.6×10 ⁶ copies/μL, and the cells were cultured at 28°C for 144 h, and the survival of larvae was recorded every 12 h. In addition, the larvae were treated as above, and the larvae were collected at 72 h;

c. The larvae were first soaked in 10 mg/L C5 and incubated at 28 °C for 1, 4 or 8 h. The control group was added with 0.04% DMSO, then the solution was aspirated and discarded. ⁶ copies/μL), cultured at 28 °C for 144 h, and the survival of larvae was recorded every 12 h. In addition, the larvae were treated as above, and the larvae were collected at 72 h;

D. The larvae of Penaeus vannamei were randomly added to the six-well plate, each well containing 6 mL of culture water and 10 larvae, and six experimental groups a, b, c, d, e and f were set up, and WSSV dilution solution (final) was added. The concentration was 1.6×10 ⁶ copies/μL), the solution was sucked and discarded after 24 h, the culture water was rinsed 3 times, 10 mg/L C5 or 0.02% DMSO was added, and fresh C5 or DMSO was replaced every 24 h, and the control group was replaced with culture Water body. Group a was replaced 0 times, group b was replaced once, group c was replaced twice, group d was replaced 3 times, group e was replaced 4 times, group f was replaced 5 times, cultured at 28 °C for 144 h, and recorded larvae every 12 h death rate. The experimental method was the same as above, with five consecutive drug changes, cultured at 28 °C for 144 h, and three larvae were harvested every 8 h. DNA was extracted with a marine animal tissue genomic DNA rapid extraction kit (Tiangen), and stored at -80°C for later use.

(5) Water stability of the compound of formula I

The compound of formula I was dissolved in aquaculture water, and placed at 28°C for 0, 1, 2, 3 and 4 days, respectively, and then each water sample containing the compound of formula I and virus liquid were mixed and soaked in larvae. At this time, the compound of formula I and WSSV The concentrations of the diluents were 10 mg/L and 1.6×10 ⁶ copies/μL, respectively, cultured at 28°C for 72 h, collected larvae, extracted DNA, and stored at -80°C until use.

(6) Detection of WSSV genomic DNA copy number

After DNA extraction is complete, measure its concentration and purity using an ultra-micro spectrophotometer. Dilute the DNA with sterile water to uniformly adjust the concentration to 30 ng/μL, and use it as a template for RT-qPCR. The detection primer was VP28 ₁₄₁ (VP28-F: 5'-AAACCTCCGCATTCCTGTGA-3', VP28-R: 5'-TCCGCATCTTCTTCCTTCAT-3'). The RT-qPCR reaction system and reaction procedure are shown in Tables 3 and 4. The quantitative PCR results were converted according to the standard curve made by the pMD19T-VP28 ₁₄₁ standard to obtain the virus copy number.

Table 3 PCR reaction system

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Table 4 PCR reaction program

The compound of formula I was crystallized at 20-40 mg/L, and had no effect on the survival rate of larvae at 10 mg/L. When the larvae were soaked in 1.6×10 ⁶ copies/μL of WSSV dilution solution, the mortality rate of the larvae within 3 days was 100%, so this virus concentration was selected as the challenge concentration in the subsequent experiments.

The inhibitory effect of the compound of formula I on the replication of WSSV in larvae is shown in Figure 2A. C5 can significantly inhibit the replication of WSSV at 1.25 mg/L, and the inhibition rate of virus replication at 10 mg/L is more than 90%. The protection rate of C5 against WSSV-infected larvae increased in a concentration-dependent manner, and at 10 mg/L, the protection rate of larvae exceeded 50% within 72 h (Fig. 2B). In addition, C5 significantly inhibited the proliferation of WSSV in larvae at 24, 48 and 72 h. Compared with the WSSV control group, C5 pre-incubated WSSV virions for 1, 2, and 4 _h , and the WSSV copy number decreased by 2.3, 2.4, and 2.7 times, respectively (Fig. 3A). The cumulative mortality was 100%, while in the WSSV _{C5-4 h} group, the cumulative mortality of larvae within 84 h was 40%, and the larvae were still alive at 144 h (Fig. 3B). It can be seen that C5 can effectively reduce the viral particles. Infectivity, and this weakening effect is more pronounced with the prolongation of drug preincubation virus time. In addition, C5 pre-incubated larvae did not effectively prevent WSSV infection of larvae (Fig. 4A-B).

The effect of continuous dressing change on the antiviral effect of C5 is shown in Figure 5. 24 h after larvae were infected with WSSV, without changing the culture water and drugs, the larvae in the WSSV _DMSO treatment group began to die from 12 h, and the cumulative mortality reached 100% at 60 h, while the C5-WSSV did not. In the dressing-change treatment group, larvae began to die from 36 h, and the cumulative mortality reached 100% at 108 h. Subsequently, the drugs were changed once, twice, 3 times, 4 times and 5 times, respectively, every 24 h. The cumulative mortality of WSSV-infected larvae in the C5-WSSV treatment group decreased from 100% to 25% at 108 h. And after 5 consecutive drug changes, the cumulative mortality of larvae at 144h was 50%. Live shrimp were sampled every 8 hours after continuous dressing change. In the WSSV _DMSO treatment group, the virus content in the larvae gradually increased with time. In the C5-WSSV treatment group, the virus in the larvae increased. The content was always lower than that in the WSSV _DMSO treatment group, and showed an overall downward trend.

The results of the water stability experiment of C5 are shown in Figure 6. After C5 was dissolved in aquaculture water and placed at 28°C for 1 d or 2 d, there was no significant difference in the inhibition rate of virus copy number between C5 and freshly prepared C5. In the WSSV treatment group, the cumulative mortality of larvae at 72 h was 100%, and the cumulative mortality of larvae in WSSV _{C5-0 d} , WSSV _{C5-1 d} , WSSV _{C5-2 d} , WSSV _{C5-3 d} , and WSSV _{C5-4 d} In the treatment group, the time when the cumulative mortality of larvae was 100% was 120 h, 120 h, 108 h, 96 h, and 72 h, respectively. It can be seen that C5 may be degraded in the aquaculture water, and with the increase in the aquaculture water. Over time, its antiviral effect gradually decreased.

The above description does not limit the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the essential scope of the present invention should also belong to the protection scope of the present invention.

Claims (4)

1. the application of the coumarin derivatives of a chromene fluorine-containing functional group in the preparation of a drug that suppresses the vitiligo syndrome, is characterized in that the structure of the coumarin derivatives is as shown in formula I:

. 2. the application of the coumarin derivatives of a kind of chromene fluorine-containing functional group according to claim 1 in the preparation of drugs for inhibiting Vitiligo Syndrome virus, it is characterized in that described coumarin derivatives The preparation method comprises the following steps: the Biginelli reaction of 4-hydroxycoumarin, p-fluorobenzaldehyde and malononitrile to obtain 4-fluorophenyl-3-cyano-2-amino-5-deoxy-4H, 5H-pyrone[3,2-c]chromene. 3. the application of the coumarin derivatives of a kind of chromene fluorine-containing functional group according to claim 2 in the preparation of drugs for inhibiting vitiligo syndrome, it is characterized in that described coumarin derivatives The specific steps of the preparation method are as follows: 2 mmol 4-hydroxycoumarin, 3 mmol p-fluorobenzaldehyde, 2 mmol malononitrile and 0.1 mmol sodium lauryl sarcosinate were placed in a round-bottom flask, and 10 mL of water was added. As a solvent, stir at 60-65 °C for 5 h. After the reaction, remove water by filtration to obtain a crude product, and recrystallize the crude product with dichloromethane to obtain 4-fluorophenyl-3-cyano-2-amino-5- Deoxy-4H,5H-pyrone[3,2-c]chromene, the structure of which is shown in formula I:

. 4. The application of the chromene fluorine-containing functional group-containing coumarin derivative of claim 1 in the preparation of a drug for inhibiting Vitiligo Syndrome Virus.

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