CN105218447B - Sclerotiorin derivatives and preparation method thereof and the application as anti-influenza A H 1 N 1 virus agent - Google Patents

Abstract

A kind of Sclerotiorin derivative and its preparation method and the application as the anti-influenza A (H1N1) virus agent, during the preparation, the fungus Penicillium sp. (TA33-1) is first carried out strain culture, and then the fungus is fermented and cultivated, and the obtained bacteria The silk was extracted 3 times with chloroform-methanol mixture (1:1) and concentrated under reduced pressure, then extracted 3 times with ethyl acetate to obtain crude extract; followed by normal phase silica gel column chromatography, Sephadex LH-20 gel column Chromatography, HPLC high-performance liquid chromatography, obtain yellow powder, be (+)-Sclerotiorin; In the dichloromethane solution that dissolves (+)-Sclerotiorin, add organic primary amine and potassium carbonate, obtain formula I compound after reaction . The invention provides an anti-type A (H1N1) influenza virus agent, which is characterized in that the compound of formula I of the invention or a pharmaceutically acceptable salt thereof is used for treating influenza caused by type A (H1N1) virus.

Description

Sclerotiorin derivatives and preparation method thereof and as anti-H1N1 influenza virus application of poison

technical field

The invention relates to a sclerotiorin derivative and its preparation method and application, in particular to a sclerotiorin derivative with strong inhibitory activity against influenza A (H1N1) virus, its preparation method and application.

Background technique

Influenza A (H1N1) virus is a type A influenza virus, carrying H1N1 subtype swine influenza virus strains, including ribonucleic acid gene fragments of three influenza viruses: avian influenza, swine influenza and human influenza, as well as Asian swine influenza and African swine Influenza virus characteristics. Since the outbreak of H1N1 influenza virus in Mexico on March 18, 2009, cases of infection and death have been found successively. Therefore, finding a safe and efficient anti-H1N1 influenza virus agent has become an urgent problem in the world. Marine natural products are considered an important source of novel antiviral agents. Marine microorganisms have become the most important source of active marine compounds because they can be fermented on a large scale in the laboratory and are not easy to damage the natural environment. However, the application of natural compounds or their derivatives directly from marine microorganisms as anti-influenza A (H1N1) virus agents has not been seen in recent years.

(Centers for Disease Control and Prevention. Update: novel influenza A(H1N1) virus infections-worldwide, May 6, 2009. MMWR Morb Mortal Wkly Rep. 2009, 58, 453-458; Scalera, N.M.; Mossad, S.B. Postgrad Med. 2009, 121, 43-47; Medina, R.A.; Garcia-Sastre, A. Nat. Rev. Microbiol. 2011, 9, 590-603.).

Contents of the invention

The purpose of the present invention is to provide a preparation method of Sclerotiorin derivative derived from marine fungus and its application as an anti-H1N1 influenza virus agent, which can meet the above-mentioned requirements of the prior art. Strain preservation information: name of preservation unit: General Microbiology Center of China Committee for the Preservation of Microbial Strains; address of preservation unit: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; preservation date: April 3, 2014 date; deposit number: CGMCCNo.8994; classification name: Penicillium sp.

The present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof:

or a pharmaceutically acceptable salt thereof. In formula I, R is The present invention provides a preparation method for the compound of formula I, which is characterized in that the endophytic fungus Penicillium sp. (TA33-1) isolated from Gorgonian coral is first cultured in the culture medium, and then in the fermentation medium. The fungus was fermented and cultured, and the obtained mycelium was extracted 3 times with chloroform-methanol mixed solution (1:1) and then concentrated under reduced pressure, then extracted 3 times with ethyl acetate to obtain a crude extract; the ethyl acetate phase crude extract was separated After normal-phase silica gel column chromatography and Sephadex LH-20 gel column chromatography, HPLC high-performance liquid phase preparative chromatography is performed, and the obtained eluent is concentrated to obtain a yellow powder, which is (+)-Sclerotiorin; In the dichloromethane solution of (+)-Sclerotiorin, add organic primary amine and potassium carbonate, and react to obtain the compound of formula I.

In the above-mentioned preparation method, the culture medium preferably contains 0.1%-5.0% of glucose (percentage by weight, the same below), 0.01%-1% of yeast extract, 0.01%-1% of peptone, 0.1%-3.0% of agar, and 0.05% of sodium chloride. %-5%, the rest is water, the culture temperature is preferably 0-30°C, and the culture time is preferably 3-15 days; the fermentation medium preferably contains rice 1.0%-80.0% (percentage by weight, the same below), sodium chloride 0.05% %-5%, the rest is water, the culture temperature is preferably 0-30°C, and the culture time is preferably 10-60 days; the stationary phase used in the normal phase silica gel column chromatography is preferably 200-300 mesh silica gel, and the mobile phase is preferably Volume ratio is the ethyl acetate-petroleum ether mixed solvent of 15%-60%; The mobile phase preferred volume ratio that described Sephadex LH-20 gel column chromatography adopts is sherwood oil: chloroform: methyl alcohol=2: 1: 1 Mixed solvent; the chromatographic column adopted in the HPLC preparative liquid phase chromatography is a conventional ODS C18 column in the field, preferably Kromasil 10×250mm, 5 μm, the flow rate is preferably 1.0-5.0mL/min, and the mobile phase preferably has a volume ratio of 50 %-80% methanol-water mixed solvent; the organic primary amine is

The sclerotiorin derivative obtained from the marine fungus of the invention has extremely strong inhibitory activity on influenza A (H1N1) virus, can be used to develop anti-influenza A (H1N1) virus agents, and has broad application prospects.

Another embodiment of the present invention provides the use of the compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of an anti-Influenza A (H1N1) virus agent.

The term "pharmaceutically acceptable salt" in the present invention refers to non-toxic inorganic or organic acid and/or base addition salts. See "Salt selection for basic drugs", Int. J. Pharm. (1986), 33, 201-217.

detailed description

In order to facilitate a further understanding of the present invention, the examples provided below illustrate it in more detail. However, these examples are only for a better understanding of the invention and are not used to limit the scope or implementation principle of the invention, and the embodiments of the invention are not limited to the following content.

Example 1

(1) The culture of the endophytic fungus Penicillium sp. (TA33-1) of Gorgonian coral

The culture medium used for strain culture contains glucose 1.0% (percentage by weight, the same below), yeast extract 0.2%, peptone 0.2%, agar 1.0%, sodium chloride 3.0%, and the rest is water. Strains were cultured at 30°C for 3 days.

(2) Fermentation of the endophytic fungus Penicillium sp. (TA33-1) from Gorgonian coral

The culture medium used in the fermentation culture contains 40.0% rice (percentage by weight, the same below), 3.0% sodium chloride, and the rest is water; the fungal strain is cultured at 28° C. for 30 days.

(3) Extraction and separation of (+)-Sclerotiorin

Get 60 bottles of mycelia obtained in step (2), extract 3 times with chloroform-methanol mixed solution (1:1) and concentrate under reduced pressure, then extract 3 times with ethyl acetate to obtain crude extract; Extraction extract is carried out normal phase silica gel column chromatography (stationary phase is 200-300 order silica gel; Mobile phase is 30% ethyl acetate/petroleum ether mixed solvent, volume ratio), Sephadex LH-20 gel column chromatography (petroleum ether : chloroform: methanol = 2: 1: 1 mixed solvent, volume ratio), then HPLC high performance liquid phase preparative chromatographic separation (chromatographic column is Kromasil 10 × 250mm, 5 μ m, flow velocity is 2.0mL/min, mobile phase is 65 % methanol-water mixed solvent, volume ratio), the obtained eluate was concentrated to obtain a yellow powder, namely (+)-Sclerotiorin.

Example 2

(1) The culture of the endophytic fungus Penicillium sp. (TA33-1) of Gorgonian coral

The culture medium used for strain cultivation contains 0.1%-5.0% of glucose (percentage by weight, the same below), 0.01%-1% of yeast extract, 0.01%-1% of peptone, 0.1%-3.0% of agar, and 0.05% of sodium chloride- 5%, and the rest is water. When used, it is made into a test tube slant, and the fungal strains are cultivated at 0-30°C for 3-15 days.

(2) Fermentation of the endophytic fungus Penicillium sp. (TA33-1) from Gorgonian coral

The culture medium used in the fermentation culture contains 1.0%-80.0% (weight percentage, the same below) of rice, 0.05%-5% of sodium chloride and the rest is water, and the fungal strain is cultivated at 0-30°C for 10-60 days.

(3) Extraction and separation of (+)-Sclerotiorin compound

Take 10-300 bottles of the obtained mycelium obtained in step (2), extract the obtained mycelium with chloroform-methanol mixed solution (1:1) 3 times, concentrate under reduced pressure, extract 3 times with ethyl acetate to obtain crude extract; ethyl acetate After the crude extract of the ester phase is concentrated, carry out normal phase silica gel column chromatography (the stationary phase is conventional normal phase silica gel in this field, and the mobile phase is 15%-40% ethyl acetate-petroleum ether mixed solvent, volume ratio), Sephadex LH After -20 gel column chromatography (mobile phase is sherwood oil: chloroform: the mixed solvent of methyl alcohol=2: 1: 1, volume ratio), then through HPLC high performance liquid phase preparative chromatography (chromatographic column is this area conventional ODS C18 post , the flow rate is 1.0-5.0mL/min, the mobile phase is 50%-80% methanol-water mixed solvent, volume ratio), and the obtained eluate is concentrated to obtain a yellow powder solid, which is (+)-Sclerotiorin compound.

Other bacterial strain cultivation, fermentation conditions not specified in embodiment 1-2, and normal phase silica gel column chromatographic separation, Sephadex LH-20 gel column chromatographic separation, high performance liquid chromatographic separation and other experimental operating conditions are all in this Those skilled in the art can make reasonable selections of conventional experimental operating conditions in the field according to actual needs.

Example 3

Weigh the dried (+)-Sclerotiorin (0.1mol) and dissolve it in dichloromethane. Under normal temperature, add the organic primary amine (0.12mol) dropwise to the reaction solution under full stirring, and react for 1 hour Finally, add distilled water (200mL) to the reactant to terminate the reaction, extract with ethyl acetate (500mL), concentrate the extract, perform column chromatography, elute with ethyl acetate, concentrate the eluate, and recrystallize After obtaining the compound of formula I.

Other organic chemical reaction conditions not specifically specified in Example 3, and other experimental operating conditions such as normal phase silica gel column chromatography separation are all conventional experimental operating conditions in this field, and those skilled in the art can make reasonable choices according to actual needs .

Specific compound structure examples of compounds of formula I:

The structural confirmation data of the compound of formula I:

1: ¹ H NMR (600MHz, CDCl ₃ ) δ _H 7.84 (1H, s, H-1), 7.16 (1H, s, H-4), 6.98 (1H, d, J=15.6Hz, H-10) , 6.58(1H, t, J=2.4Hz), 6.41(2H, d, J=1.8Hz), 5.70(1H, d, J=15.6Hz, H-9), 5.68(1H, d, J=9.6 Hz, H-12), 3.83(6H, s), 2.42(1H, m, H-13), 2.18(3H, s, H-20), 1.59(3H, s, H-17), 1.57(3H , s, H-18), 1.42 (1H, m, H-14), 1.32 (1H, m, H-14), 1.00 (3H, d, J=6.6Hz, H-16), 0.85 (3H, t, J=7.2Hz, H-15). ¹³ C NMR (150MHz, CDCl ₃ ) δ _C 193.9 (C-6), 184.7 (C-8), 170.3 (COCH ₃ ), 161.8, 161.8, 147.9 (C -1), 147.4, 144.2, 143.2, 141.8, 141.1, 132.0 (C-11), 116.2 (C-9), 114.3 (C-5), 109.6, 109.6, 109.6, 103.3 (C-8a), 101.8 ( C-4), 84.9 (C-7), 56.0 (-OCH ₃ ), 55.9 (-OCH ₃ ), 35.1 (C-13), 30.1 (C-14), 23.3 (C-18), 20.4 (C -16), 20.3(COCH ₃ ), 12.5(C-17), 12.1(C-15).ESIMSm/z 526.05/528.05[M+H] ⁺ /[M+H+2] ⁺ (3:1) , 547.99/550.01[M+Na] ⁺ /[M+Na+2] ⁺ (3:1), 1072.84/1072.77[2M+Na] ⁺ /[2M+Na+2] ⁺ (3:1).HRESIMSm /z526.1981[M+H] ⁺ (calcd for C ₂₉ H ₃₃ O ₆ NCl, 526.1991).

2: ¹ H NMR (600MHz, CDCl ₃ ) δ _H 7.85 (1H, s, H-1), 7.16 (1H, s, H-4), 6.99 (1H, d, J=15.6Hz, H-10) , 6.50(2H, s), 5.68(2H, oVerlapped, H-9and H-12), 3.91(3H, s), 3.87(6H, s), 2.42(1H, m, H-13), 2.19(3H , s, H-20), 1.59 (3H, s, H-17), 1.57 (3H, s, H-18), 1.42 (1H, m, H-14), 1.32 (1H, m, H-14 ), 1.00 (3H, d, J=6.6Hz, H-16), 0.85 (3H, t, J=7.2Hz, H-15). ¹³ CNMR (150MHz, CDCl ₃ ) δ _C 193.9 (C-6) , 184.7(C-8), 170.4(COCH ₃ ), 154.1, 148.0, 148.0, 147.6, 144.1, 143.3, 141.2, 141.2, 139.2, 135.8, 131.9(C-11), 116.2, 114.3(C-5), 109.6, 104.2, 103.4(C-8a), 84.8(C-7), 61.2( _-OCH3 ), 56.7(-OCH3), 56.6( _-OCH3 ), 35.1(C-13), 30.1(C-14 ), 23.3(C-18), 20.4(C-16), 20.3(COCH ₃ ), 12.5(C-17), 12.0(C-15).ESIMSm/z556.07/558.06[M+H] ⁺ / [M+H+2] ⁺ (3:1), 578.05/580.04[M+Na] ⁺ /[M+Na+2] ⁺ (3:1), 1132.83[2M+Na] ⁺ .HRESIMSm/z556. 2087[M+H] ⁺ (calcd for C ₃₀ H ₃₅ O ₇ NCl, 556.2097).

3: ¹ H NMR (600MHz, CDCl ₃ ) δ _H 8.84 (1H, d, J = 2.4Hz), 8.24 (1H, d, J = 8.4Hz), 8.20 (1H, d, J = 2.4Hz), 7.94 (1H, d, J = 7.8Hz), 7.91 (1H, ddd, J = 8.4, 6.6, 1.2Hz), 7.88 (1H, s, H-1), 7.74 (1H, ddd, J = 7.8, 7.2, 1.2Hz), 7.19(1H, s, H-4), 7.04(1H, d, J=15.6Hz, H-10), 5.70(1H, d, J=9.6Hz, H-12), 5.58(1H , d, J=15.6Hz, H-9), 2.38 (1H, m, H-13), 2.19 (3H, s, H-20), 1.61 (3H, s, H-17), 1.46 (3H, s, H-18), 1.42 (1H, m, H-14), 1.32 (1H, m, H-14), 0.98 (3H, d, J=6.6Hz, H-16), 0.84 (3H, t , J=7.2Hz, H-15); ¹³ C NMR (150MHz, CDCl ₃ ) δ _C 193.6 (C-6), 185.1 (C-8), 170.4 (COCH3), 148.7, 147.9, 147.6, 147.4, 144.3 , 143.5, 141.1, 134.1, 133.1, 131.9, 131.7, 129.9, 128.9, 128.2, 127.3, 115.7, 115.1(C-5), 110.4, 104.6, 84.8(C-7), 35.1(C-13), 30.1( C-14), 23.2(C-18), 20.4(C-16), 20.3(COCH ₃ ), 12.5(C-17), 12.1(C-15).ESIMSm/z 517.05/519.10[M+H] ⁺ /[M+H+2] ⁺ (3∶1), 539.01/541.01[M+Na] ⁺ /[M+Na+2] ⁺ (3∶1), 1054.85[2M+Na] ⁺ .HRESIMSm/ z517.1885[M+H] ⁺ (calcd for C ₃₀ H ₃₀ O ₄ N ₂ Cl, 517.1889).

4: ¹ H NMR (600MHz, CDCl ₃ ) δ _H 7.88 (2H, d, J = 8.4Hz), 7.76 (1H, s, H-1), 7.49 (2H, d, J = 8.4Hz), 7.13 ( 1H, s, H-4), 6.98 (1H, d, J=15.6Hz, H-10), 5.71 (1H, d, J=9.6Hz, H-12), 5.49 (1H, d, J=15.6 Hz, H-9), 2.42 (1H, m, H-13), 2.18 (3H, s, H-20), 1.58 (3H, s, H-17), 1.54 (3H, s, H-18) , 1.42(1H, m, H-14), 1.32(1H, m, H-14), 1.00(3H, d, J=6.6Hz, H-16), 0.86(3H, t, J=7.2Hz, H-15). ¹³ C NMR (150MHz, CDCl ₃ ) δ _C 193.4 (C-6), 185.1 (C-8), 170.3 (COCH ₃ ), 148.7, 146.6, 144.0, 143.9, 143.1, 140.2, 134.2, 134.2, 131.7, 127.7, 127.7, 117.2, 115.8, 114.9, 114.1, 110.5, 104.9, 84.8(C-7), 35.1(C-13), 30.1(C-14), 23.1(C-18), 20.3( C-16), 20.3 (COCH ₃ ), 12.4 (C-17), 12.1 (C-15).ESIMSm/z 491.04/493.07[M+H] ⁺ /[M+H+2] ⁺ (3:1 ), 513.04/514.99[M+Na] ⁺ /[M+Na+2] ⁺ (3:1), 1118.99[2M+Na] ⁺ ; HRESIMSm/z 491.1728[M+H] ⁺ (calcd for C ₂₈ H ₂₈ O ₄ N ₂ Cl, 491.1732).

5: ¹ H NMR (600MHz, CDCl ₃ ) δ _H 7.89 (1H, s, H-1), 7.00 (1H, s, H-4), 6.96 (1H, d, J=15.6Hz, H-10) , 6.31(1H, d, J=15.6Hz, H-9), 5.72(1H, d, J=9.6Hz, H-12), 4.60(2H, s, H-21), 2.63(1H, t, 2.4Hz, H-23), 2.50(1H, m, H-13), 2.17(3H, s, H-20), 1.88(3H, s, H-17), 1.55(3H, s, H-18 ), 1.45(1H, m, H-14), 1.35(1H, m, H-14), 1.03(3H, d, J=6.6Hz, H-16), 0.89(3H, t, J=7.2Hz , H-15). ¹³ C NMR (150MHz, CDCl ₃ ) δ _C 193.5 (C-6), 184.7 (C-8), 170.2 (COCH ₃ ), 148.4, 147.7, 145.4, 144.2, 140.8, 132.0, 114.9 , 114.6, 111.4, 103.0, 85.0 (C-7), 77.5, 75.3, 43.8, 35.2 (C-13), 30.1 (C-14), 23.3 (C-18), 20.4 (C-16), 20.3 ( COCH ₃ ), 12.7 (C-17), 12.1 (C-15). ESIMS m/z 428.03/430.09[M+H] ⁺ /[M+H+2] ⁺ (3:1), 450.01/452.02[ M+Na] ⁺ /[M+Na+2] ⁺ (3∶1), 876.80/878.80[2M+Na] ⁺ /[2M+Na+2] ⁺ .HRESIMS m/z428.1621[M+H] ⁺ (calcd for C ₂₄ H ₂₇ O ₄ NCl, 428.1623).

Example 4

The method for testing the inhibitory activity of the compound of formula I of the present invention on influenza A (H1N1) virus is as follows: the antiviral activity is studied by using cytopathic inhibition (CPE). The test virus is: H1N1 (influenza virus), and the canine kidney cells (MDCK) are H1N1 sensitive cells. MDCK cells cultured into a single layer were digested with trypsin, seeded in a 96-well plate, and grown into a single layer for use. Inoculate H1N1 disease on MDCK cells, add 2% serum 1640 culture solution, and then culture at 37°C and 5% _CO2 . After more than 90% of the lesions appear, freeze and thaw 3 times, blow and centrifuge, and quantitatively aliquot. Store in -80°C refrigerator for later use. After each tube of the test sample was dissolved in 10 μL DMSO, 200 μL of 2% 1640 culture solution was added, and 10 consecutive 2-fold dilutions were made, with a total of 10 dilutions, and then horizontally inoculated on the monolayer cells in 96 plate wells. Column 11 is the virus control, column 12 is the cell control, cultured at 37°C, 5% CO ₂ , observed lesions every hour, and observed continuously for 24 hours. After more than 90% of the lesions appeared in the virus control, the liquid in the wells was aspirated and discarded, stained with 1% neutral red, the OD value was measured at a wavelength of 540nm, and the half effective concentration (IC ₅₀ ) of the drug was calculated by the Reed-Muench method.

The compound of formula I of the present invention shows important inhibitory activity to influenza A H1N1 virus, which is far stronger than the positive drug ribavirin. This shows that the compound of formula I or its pharmaceutically acceptable salt can be used to prepare the antiviral agent of high efficiency and low toxicity, and marine source fungus Penicillium sp. (TA33-1) can carry out large-scale fermentation production, has guaranteed the source of compound of formula I ,have a broad vision of application.

Claims (7)

1. a kind of Selerotiorin derivatives or its pharmaceutically acceptable salt, it is characterised in that there is the structure shown in Formulas I：

Wherein R is

2. the preparation method of the compound of formula I described in claim 1, it is characterised in that first to fungi in bacterium culture medium Penicillium sp. carry out Spawn incubation, then carry out fermented and cultured to the fungi in the fermentation medium, by gained mycelium Extracted 3 times with chloroform-methanol mixed liquor after being concentrated under reduced pressure, coarse extract is extracted with ethyl acetate 3 times to obtain；Ethyl acetate coarse extract , will after carrying out normal-phase silica gel column chromatography, Sephadex LH-20 gel filtration chromatographies respectively, then through HPLC high performance liquid preparative chromatographies Gained eluent concentrates, and obtains yellow powder, is (+)-Sclerotiorin；In the dichloromethane dissolved with (+)-Sclerotiorin In alkane solution, organic primary amine and potassium carbonate are added, Formulas I chloro polyketide is obtained after reaction；Wherein described bacterium culture medium In containing glucose, yeast extract, peptone, agar, coarse sea salt, water；In described fermentation medium containing rice, coarse sea salt, Water；Described chromatographic isolation is normal phase silica gel column chromatography separation, gel filtration chromatography separation and high performance liquid chromatography separation；Described Organic primary amine is

3. preparation method as claimed in claim 2, it is characterised in that the bacterium culture medium contains glucose 0.1%- 5.0%th, yeast extract 0.01%-1%, peptone 0.01%-1%, agar 0.1%-3.0%, sodium chloride 0.05%-5%, remaining For water, above-mentioned percentage composition is weight percentage, and cultivation temperature is 0-30 DEG C, and incubation time is 3-15 days.

4. preparation method as claimed in claim 2, it is characterised in that the fermentation medium contain rice 1.0%-80.0%, Sodium chloride 0.05%-5%, remaining is water, and above-mentioned percentage composition is weight percentage, and cultivation temperature is 0-30 DEG C, during culture Between be 10-60 days.

5. the preparation method as described in claim any one of 2-4, it is characterised in that what described normal-phase silica gel column chromatography used Stationary phase is 200-300 mesh silica gel, and mobile phase is the ethyl acetate-light petrol mixed solvent that volume ratio is 15%-60%；It is described The mobile phase that Sephadex LH-20 gel filtration chromatographies use is petroleum ether for volume ratio: chloroform: methanol=2: 1: 1 mixing is molten Agent；The chromatographic column used in the HPLC high performance liquid preparative chromatographies is 10 × 250mm of Kromasil, 5 μm, flow velocity 1.0- 5.0mL/min, mobile phase are volume ratio 50%-80% Methanol+Water.

6. a kind of anti-influenza A H 1 N 1 virus agent, it is characterised in that it contains the compound of formula I or its medicine described in claim 1 Acceptable salt is as active ingredient on.

7. the compound of formula I or its pharmaceutically acceptable salt described in claim 1 are preparing anti-influenza A H 1 N 1 virus agent In application.