CN107513085B

CN107513085B - Compound and preparation method and application thereof

Info

Publication number: CN107513085B
Application number: CN201610431943.9A
Authority: CN
Inventors: 萧伟; 李海波; 姚新生; 于洋; 房卉; 孟兆青; 黄文哲; 王振中
Original assignee: Jiangsu Kanion Pharmaceutical Co Ltd
Current assignee: Jiangsu Kanion Pharmaceutical Co Ltd
Priority date: 2016-06-16
Filing date: 2016-06-16
Publication date: 2020-06-30
Anticipated expiration: 2036-06-16
Also published as: CN107513085A

Abstract

The invention provides a compound with a structure shown in a formula (I), which can be used for preparing a medicament for treating hand-foot-and-mouth disease and/or respiratory syncytial virus infection.

Description

Compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a compound and a preparation method and application thereof.

Background

The hand-foot-and-mouth disease is an infectious disease caused by enteroviruses, and the enteroviruses causing the hand-foot-and-mouth disease are more than 20, wherein the most common enteroviruses are coxsackievirus A16 type (Cox A16) and enterovirus 71 type (EV 71). The disease mostly occurs in children under 5 years old, and shows that the parts of stomachache, anorexia, low fever, hands, feet, oral cavity and the like have small herpes or small ulcer, most children can self-heal for about one week, and few children can cause complications such as myocarditis, pulmonary edema, aseptic meningoencephalitis and the like. The disease of some serious children will develop quickly and lead to death. At present, effective treatment medicines are lacked for symptomatic treatment.

Respiratory Syncytial Virus (RSV) belongs to the genus pneumonias of the family paramyxoviridae, and Morris isolated a first RSV strain from nasopharyngeal secretions of one experimental animal chimpanzee with cold symptoms in 1956. Chanock was isolated from 2 pharyngeal swabs of Baltimore, 2 infants with pneumonia and wheezing symptoms, respectively, in 1957. It is named because it can form special cell fusion lesion in tissue culture. Respiratory syncytial virus is the most common pathogen of infant respiratory infection, and particularly serious bronchiolitis and pneumonia frequently occur after RSV infection of infants of 2-6 months. The row is usually throttled in winter and spring. The infant patients who need hospitalization due to RSV infection in different areas of the world each year are 1-5 per thousand, and the fatality rate of the inpatients is 1-3 per thousand.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a compound, a preparation method and an application thereof, wherein the prepared compound can be used for preparing a medicine for treating hand-foot-and-mouth disease and respiratory syncytial virus infection.

The invention provides a compound represented by the formula (I):

the invention also provides a preparation method of the compound shown in the formula (I), which comprises the following steps:

A) performing column chromatography separation on the injection of the toxic heat removing agent, sequentially performing gradient elution by using water, 25-35% of ethanol and 90-100% of ethanol, and collecting 90-100% of ethanol eluent;

B) performing column chromatography separation on the 90-100% ethanol eluent, and collecting components with a chloroform-methanol ratio of 9:1 by using a chloroform-methanol mixed solvent as an eluent;

C) performing column chromatography separation on the components collected in the step B), performing gradient elution by using a methanol-water mixed solvent as an eluent, and collecting the components with the methanol-water ratio of 1: 1;

D) separating the components collected in the step C) by semi-preparative liquid chromatography to obtain a compound shown in a formula (I);

preferably, the semi-preparative liquid chromatography specifically comprises: acetonitrile-water in a ratio of 25:75 is used as a mobile phase, detection wavelengths are 254nm and 220nm, the flow rate is 4mL/min, and the retention time on a semi-preparation liquid phase is 21.4 min.

Preferably, the column chromatography in step a) is a macroporous adsorbent resin.

Preferably, the column chromatography in step B) is silica gel column chromatography.

Preferably, the column chromatography in step C) is ODS column chromatography.

The invention also provides application of the compound shown in the formula (I) in preparing a medicament for resisting EV71 virus and/or RSV virus,

the invention also provides application of the compound shown in the formula (I) in preparing a medicament for preventing and treating hand-foot-and-mouth disease and/or respiratory tract infection,

the invention also provides a medicament which comprises a compound shown as a formula (I),

the invention also provides application of the medicine in preparing products for preventing and treating hand-foot-and-mouth diseases and/or respiratory tract infection.

Compared with the prior art, the invention provides a compound with a structure shown in formula (I), which can be used for preparing a medicine for treating hand-foot-and-mouth disease and/or respiratory syncytial virus infection.

Drawings

FIG. 1 is HR-ESI-Q-TOF-MS of a compound;

FIG. 2 is a drawing of a compound¹H-NMR spectrum;

FIG. 3 is a drawing of a compound¹³C-NMR spectrum and DEPT-135 spectrum;

FIG. 4 is a drawing of a compound¹H-NMR spectrum and seco-Strycyrronidate and (1S,7R,8 R.10S); process for producing (E) -7,8, 11-trihydroxy-1-hydroxy-4-guaien-3-one¹Comparison of H-NMR spectra;

FIG. 5 is an HMBC spectrum of a compound;

FIG. 6 is H of compound¹-H¹A COSY spectrum;

FIG. 7 is a ROSEY spectrum of a compound;

FIG. 8 is the HSQC spectra of the compounds.

Detailed Description

The invention provides a compound represented by the formula (I):

the compound is a new compound formed by polymerizing sesquiterpene and iridoid glycoside, and in the structural formula, the omitted radicals are supplemented completely as follows:

the invention takes a Reduning injection as a raw material, the Reduning injection (Chinese medicine standard Z20050217) is a new primary traditional Chinese medicine II which is independently researched and developed by Jiangsu Kangyuan pharmaceutical industry GmbH, the prescription of the injection is sweet wormwood, honeysuckle and gardenia, and the auxiliary material is polysorbate 80. The injection can be widely applied to the clinical treatment of diseases such as cold, cough, fever, upper respiratory tract infection and the like caused by external wind heat, and has rapid action and obvious effect.

According to the invention, column chromatography separation is firstly carried out on the toxic heat removing injection, and preferably, macroporous adsorption resin column chromatography separation is adopted in the invention, and more preferably, HP-20 macroporous adsorption resin column chromatography separation is adopted.

Specifically, water, 25-35% ethanol and 90-100% ethanol are adopted for gradient elution in sequence, and the eluates are respectively collected. The 25-35% ethanol and the 90-100% ethanol are ethanol water solutions.

In certain embodiments of the invention, the 25% to 35% ethanol may be a 25% to 30% aqueous ethanol solution, a 25% aqueous ethanol solution, a 30% aqueous ethanol solution, or a 35% aqueous ethanol solution; the 90-100% ethanol can be 90-95% ethanol water solution, 90% ethanol water solution, 95% ethanol water solution or 100% ethanol.

Then, the obtained 90 to 100 percent ethanol eluent is concentrated until no alcohol smell exists, and is subjected to column chromatography, and the invention preferably adopts silica gel column chromatography for separation.

Specifically, a chloroform-methanol mixed solvent is used as an eluent, and components with the chloroform-methanol ratio of 9:1 are collected; the ratio of the chloroform-methanol mixed solvent and the methanol mixed solvent can be adjusted according to the need, and the invention is not limited to this.

Then, the above-collected fractions are subjected to column chromatography, and ODS column chromatography is preferably used in the present invention.

Specifically, a methanol-water mixed solvent is used as an eluent for gradient elution, and components with the methanol-water ratio of 1:1 are collected; the ratio of the methanol-water mixed solvent and the solvent can be adjusted according to the need, and the invention is not limited to this.

And then separating the collected components by semi-preparative liquid chromatography to obtain the compound shown in the formula (I).

In the present invention, the semi-preparative liquid chromatography is preferably performed under the following specific conditions: acetonitrile-water in a ratio of 25:75 is used as a mobile phase, detection wavelengths are 254nm and 220nm, the flow rate is 4mL/min, and the retention time on a semi-preparation liquid phase is 21.4 min.

The invention adopts physical and chemical properties and modern wave spectrum means, such as UV, IR, MS,¹H-NMR、¹³And C-NMR and 2D-NMR, and carrying out structural identification on the separated compound, and confirming that the compound is the novel compound with the structure shown as the formula I as shown in the results of the structural identification shown in figures 1 to 8.

The invention also provides application of the compound shown in the formula (I) in preparing a medicament for resisting EV71 virus and/or RSV virus.

The embodiment of the invention shows that the compound provided by the inventor has a certain inhibition effect on hand-foot-and-mouth disease EV71 virus and respiratory syncytial virus RSV.

The invention also provides application of the compound shown in the formula (I) in preparation of medicines for preventing and treating hand-foot-and-mouth diseases and/or respiratory tract infection.

The invention also provides a medicament which comprises the compound shown in the formula (I).

In the medicine, only the compound shown in the formula I is used as an active ingredient, or other active ingredients are further included to be compounded with the compound shown in the formula I. The compound compounded with the compound of the formula I is not particularly limited, and the compound can be compounded with the compound of the formula I to play a role in drug synergy or synergism.

Preferably, the medicament comprises the compound shown in the formula I and one or more pharmaceutically acceptable auxiliary materials.

The content of the compound represented by formula I is not particularly limited in the present invention, and a therapeutically effective amount may be sufficient.

The dosage form of the above-mentioned medicament is not particularly limited in the present invention, and may be a dosage form well known in the art, such as an oral preparation or an injection preparation, the oral preparation is preferably a tablet, a capsule, a pill, a granule, a decoction, a paste, a lotion, an oral liquid, a drop pill or a syrup, and the injection preparation is preferably an injection or a powder injection.

The hand-foot-and-mouth disease is a disease caused by EV71, and also comprises complications such as myocarditis, pulmonary edema, aseptic meningoencephalitis and the like.

The respiratory tract infection is respiratory tract disease caused by RSV virus, including pneumonia, bronchiolitis, upper respiratory tract infection, etc.

The invention obtains the compound shown in the formula (I) by extracting and researching the Reduining injection, and discovers that the compound has obvious inhibition effect on hand-foot-mouth disease EV71 virus and respiratory syncytial virus RSV by performing cell experiments on the compound, and further has potential application in the field of preparing medicines for preventing and treating hand-foot-mouth disease and/or respiratory tract infection. Experiments show that the compound provided by the invention is used for inhibiting EV71 virus, EC₅₀The value can reach 15.6 mu mol; for inhibition of RSV virus, EC₅₀The value can reach 12.6 mu mol.

In order to further illustrate the present invention, the following examples are provided to describe the compounds provided by the present invention in detail, as well as the preparation and use thereof.

Example 1

(1) Separating the finished product of the injection of the toxic heat removing agent by using an HP-20 macroporous adsorption resin chromatographic column, sequentially carrying out gradient elution by using water, 25-35% of ethanol and 95% of ethanol, respectively collecting eluates, and concentrating under reduced pressure until no ethanol smell exists to obtain a water elution component, a 25-35% of ethanol elution component and a 95% of ethanol elution component;

(2) and (2) separating the 95% ethanol elution component obtained in the step (1) by silica gel column chromatography, carrying out gradient elution by using a chloroform-methanol mixed solvent, and collecting the component with the chloroform-methanol ratio of 9:1 to obtain 2.2g of a product.

Then separating the obtained components by ODS column chromatography, gradient eluting with methanol-water mixed solvent, and collecting the components with methanol-water ratio of 5:5 to obtain 85mg product.

The above collected fractions were separated by semi-preparative liquid phase HPLC using acetonitrile-water as mobile phase in a ratio of 25:75, detection wavelengths of 254nm and 220nm, a flow rate of 4mL/min, a retention time on the semi-preparative liquid phase of 21.4min, the resulting solution was separated and dried to give 11.6mg of the compound of formula (i) of the present invention, 98% purity, as a yellow colloidal solid.

Example 2 structural testing

The structure identification of the compound obtained above is shown in figures 1-8, wherein figure 1 is HR-ESI-Q-TOF-MS (ultra high performance liquid phase high resolution mass spectrometry) diagram of the compound shown in formula (I) provided by the invention, FIG. 2 is a nuclear magnetic hydrogen spectrum of the compound of formula (I), FIG. 3 is a nuclear magnetic carbon spectrum and DEPT-135 spectrum of the compound of formula (I), FIG. 4 is a comparison of nuclear magnetic hydrogen spectrum of the compound represented by formula (I) provided by the present invention with nuclear magnetic hydrogen spectrum of secoisolaricide and (1S,7R,8R.10S) -7,8, 11-trihydroxy-1-hydroxy-4-guaien-3-one, FIG. 5 is a HMBC spectrum of the compound of formula (I) provided by the present invention, and FIG. 6 is a H spectrum of the compound of formula (I) provided by the present invention.¹-H¹COSY spectrum; FIG. 7 is a ROSEY spectrum of the compound of formula (I) provided by the invention, and FIG. 8 is an HSQC spectrum of the compound of formula (I) provided by the invention.

The compound was shown to have an absorption peak M/z 625.2866[ M + H ] by HR-ESI-Q-TOF-MS of FIG. 1]⁺(calculated value 625.2860), the molecular formula of the compound was determined to be C₃₁H₄₄O₁₃The unsaturation was calculated to be 10.

Analysis of the Compound of formula (I) in FIG. 2¹H-NMR(600MHz,in CD₃OD), 4 distinct methyl signals were observed in the high field region: δ 1.05(3H, d, J ═ 7.1Hz), 1.31(3H, s), 1.42(3H, s) and 1.68(3H, d, J ═ 2.2Hz) and the proton signals at the 3-position of 1 typical iridoid: δ 7.41(1H, s) and hemiacetal proton signal at position 1: 5.54(1H, d, J ═ 6.9Hz), 1 glucose end group proton signal: δ 4.70(3H, d, J ═ 7.9 Hz).

FIG. 3 Process for preparing compounds of formula (I)¹³C-NMR(150MHz,in CD₃OD) combined with DEPT-135 spectrum showed 31 carbon signals including 4 methyl groups (. delta.23.9, 21.0,20.6,8.8), 6 methylene groups (. delta.119.2, 62.8,38.5,36.2,33.4,31.4), 7 quaternary carbon signals [2 carbonyl groups (. delta. 211.5,171.8), 2 vicinal oxygen SP' s³Hybrid quaternary carbon (. delta.80.7, 74.0), 1-to-tetrasubstituted double bond (. delta. 141.9,174.8)]And 14 methine carbon signals.

The hydrogen and carbon nuclear magnetic data of the compound show two groups of signals, wherein one structural fragment A is very similar to Secolinic acid, the other structural fragment B is similar to (1S,7R,8R.10S) -7,8, 11-dihydroxy-1-hydroxide-4-guaien-3-one (see figure 4), and the nuclear magnetic data of the structural fragment A is compared with Secolinic acid, and only the carboxyl (-COOH) at the position 7 is replaced by oxygen-containing methine [ delta ]_H5.07(IH,d,J＝5.4,3.6Hz),δ_C96.2]. And the other structure judges that B has 15 residual carbon signals, and the structural fragment B is supposed to be 1 sesquiterpene compound by combining NMR data, wherein the structure contains 4 methoxyl groups, 2 rings, 1 ketocarbonyl group and 1 double bond.

In the HMBC spectrum (see FIG. 5), the relevant peaks H-15 '/C-3', 4 ', 5' and H-2 '/C-1', 3 ', 5', derive α the structural fragment a of the unsaturated cyclopentanone.¹H-¹In the H COSY spectrum (see FIG. 6), H-8 "/H-9"/H-10 "/H-1", H-10 "/CH can be seen₃-14 "correlation peak, HMBC remote correlation with H-6"/C-1 ", 4", 5 ", 7"; CH (CH)₃HMBC remote from-14 '/C-1', 10 ', 9' and H-8 '/C-7', 9 ', 10' are related to construct structural fragment b of cycloheptane. Two unimodal methyl radicals CH₃-12 "and CH₃The-13 "has HMBC remote association with both C-7" and C-11 ", presumably containing fragment C in the structure. At the already presumed 3 knotsIn the structural fragment, we can find that the 1,5,7 carbon atoms are common carbons, thus connecting them into 1 guaiane type sesquiterpene skeleton B. H-8'/C-7 is visible in the HMBC spectrum; CH (CH)₃Remote correlation of-12'/C-7, in combination with formula C₃₁H₄₄O₁₃And unsaturation, it is concluded that the structural fragments A and B are linked by C (C-7) -O-C (C-8 "), C (C-7) -O-C (C-11").

The atom numbers are as follows:

the relative configuration of the above-mentioned compounds provided by the present invention is determined by the ROESY spectrum, in which H-7/H can be seen in the ROSEY spectrum (see FIG. 7)₃-12″，H-8″，H-5；H-5/H-9,H-8″/H₃-14 ", H-1"; H-1/H-10 indicates H-7, H-5, H-9, H₃-12″，H-8″,H₃14 'and H-1' are coplanar and are in β configuration, and H-1 is in α configuration.

The overall carbon and hydrogen signals of the compound were assigned using a combination of 1D and HSQC spectra (see FIG. 8) (see Table 1). No relevant report is found through SciFinder Scholar network search, and the compound is a new compound named as japonica side C.

Nuclear magnetic data for the compounds of table 1 (deuterated methanol,¹H-NMR 600MHz,¹³C-NMR 150MHz)

the detection results show that the compound provided by the invention has the structure shown in the formula (I).

Example 3 Activity test

1. Material

1.1 strains of hand-foot-and-mouth disease EV71 virus and respiratory syncytial virus RSV, and carrying out passage preservation in a laboratory.

1.2 cell model monkey kidney cell lines Vero and Hep-2 cells, both of which were maintained by passage in this laboratory. The culture conditions are DMEM + 10% fetal calf serum, RPMI-1640 medium + 10% fetal calf serum, 37 deg.C, 5% CO₂。

2. Principles and methods

2.1 detection of drug cytotoxicity

Adopt

(Invitrogen) the kit detects the toxic effect of the drug on the cells.

The experimental principle is as follows:

is a redox indicator which can generate absorbance change and fluorescence signals according to metabolic activity.

Is easy to dissolve in water, and the oxidized form of the compound enters cells and is reduced by mitochondrial enzyme to generate measurable fluorescence and color change, so that the compound can be used for quantitative analysis of cell activity and cell proliferation and in vitro cytotoxicity research. This assay is based on the ability of metabolically active cells to convert reagents into fluorescent and colorimetric indicators, with damaged and inactive cells having lower native metabolic activity and corresponding lower signals. Therefore, the fluorescence signal is strong and weak, and can reflect the activity of the cells.

The method comprises the following steps: vero cells and Hep-2 cells are respectively inoculated in a 96-hole cell culture plate, and the cells are kept for later use after being attached to the wall. The drug was diluted in 6 gradients from 2-fold initial concentration (100 μmol) in 3-fold serial gradients with cell maintenance media (DMEM + 5% serum and RPMI-1640 medium + 2% fetal bovine serum), respectively, for single well detection per concentration gradient. Adding medicine and culturing for 48h, adding

Incubating at 37 ℃ for 2h, and detecting by fluorescence

In the reduction case of (1), excitation light is 570nm and emission light is 595 nm.

Cell activity (%) ═ (sample well-blank)/(cell control-blank) × 100%

2.2 drug inhibition assay for EV71 and RSV viruses

2.2.1 inhibition assay of EV-71 Virus by drugs

After the EV71 containing the reporter gene GFP infects Vero cells, the infected cells express green fluorescent protein, and the proliferation condition of the EV71 virus can be reflected by observing the number of the cells expressing GFP green fluorescence under a fluorescence microscope.

The method comprises the following steps:

the Vero cells are inoculated in a 96-hole cell culture plate and are reserved after the cells are attached to the wall. The drug was serially diluted 3-fold in gradient 6 from 4-fold the highest concentration tested (100 μmol); adding the diluted medicine into the hole, adding virus supernatant after 4h for infection, placing the cell culture box at 37 ℃ for culture for 24h, taking a picture under a fluorescent microscope, and counting fluorescent cells.

The experiment was set with no drug control wells (no drug added wells after viral infection) and positive drug control wells (guanidine hydrochloride GuHCl).

Inhibition (%) - (no drug control well-sample well)/no drug control well × 100%

2.2.2 inhibition assay of RSV viruses by drugs

Hep-2 cells are inoculated in a 96-well cell culture plate and are ready for use after the cells are attached to the wall. After the cells grew into a monolayer, 100. mu.l of maintenance medium containing 2X drug and 100. mu.l of virus solution diluted in RPMI-1640 medium were added and cultured at 37 ℃. Cells were observed and recorded daily for specific CPE and cell viability was measured after 5 days of culture (75% -100% lesions in virus control wells).

The experiment was set up with a cell control (no viral infection), a virus control (no drug added wells after viral infection) and a positive drug (ribavirin) control well.

The inhibition rate (%) was × 100% (drug group-virus control group)/(cell control group-virus control group) 100%

3. Results

3.1 detection of toxicity of drug samples to Vero cells and detection of EV71 inhibitory activity

Solvent used for drug sample dilution, highest concentration dissolved, highest concentration tested, CC₅₀，EC₅₀And SI (selection index) are shown in table 2.

TABLE 2EV71 test results for inhibitory Activity

3.2 detection of toxicity of drug samples to Hep-2 cells and detection of RSV inhibitory Activity

Solvent used for drug sample dilution, highest concentration dissolved, highest concentration tested, CC₅₀，EC₅₀And SI (selection index) are shown in table 3.

TABLE 3 detection of RSV inhibitory Activity

4. Conclusion

The experiments show that the compound shown in the formula (I) has no obvious toxicity, the SI values of the compound for resisting EV-71 virus and RSV are respectively 10.1 and 19.5, and the compound has a certain inhibiting effect on hand-foot-mouth disease EV71 virus and respiratory syncytial virus RSV.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims

1. A compound of formula (I):

2. a process for the preparation of a compound of formula (i) which comprises:

the 90-100% ethanol is 90-95% ethanol water solution, 90% ethanol water solution, 95% ethanol water solution or 100% ethanol;

the semi-preparative liquid chromatography specifically comprises the following steps: acetonitrile-water with the ratio of 25:75 is taken as a mobile phase, the detection wavelength is 254nm and 220nm, the flow rate is 4mL/min, and the retention time on a semi-prepared liquid phase is 21.4 min;

3. the method according to claim 2, wherein the column chromatography in step a) is a macroporous adsorbent resin separation.

4. The process according to claim 2, wherein the column chromatography in step B) is silica gel column chromatography.

5. The production method according to claim 2, wherein the column chromatography in step C) is ODS column chromatography.

6. The application of the compound shown in the formula (I) in preparing medicines for resisting EV71 virus and/or RSV virus,

7. the application of the compound shown in the formula (I) in preparing the medicine for preventing and treating hand-foot-and-mouth disease and/or respiratory tract infection,

8. a medicine comprises a compound shown as a formula (I),

9. use of the medicament of claim 8 for the manufacture of a product for the prevention and treatment of hand-foot-and-mouth disease and/or respiratory tract infections.