CN112745334B

CN112745334B - Prodrugs of Barosavir and derivatives thereof, preparation method and application thereof

Info

Publication number: CN112745334B
Application number: CN202110179302.XA
Authority: CN
Inventors: 顾世海; 丁延辉
Original assignee: Xiamen Weiyang Pharmaceutical Co ltd; Xiamen Yixian Pharmaceutical Co ltd
Current assignee: Xiamen Weiyang Pharmaceutical Co ltd
Priority date: 2021-02-07
Filing date: 2021-02-07
Publication date: 2022-03-25
Anticipated expiration: 2041-02-07
Also published as: CN112745334A

Abstract

The invention provides a compound with a structure shown in a formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a preparation method and application thereof, wherein X, Y, Z, R in the formula (I)₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、(R₉)_mM is as defined in the description and claims of the invention. The compound is a prodrug of the baroxavir and derivatives thereof, and the bioavailability of the compound is greatly improved compared with that of the existing baroxavir marboxil. The compound of the invention has better effect than Baloxavir marboxil in reducing dosage, reducing side effect of medicine and improving medicine effect, and can be used as a medicine for treating and/or preventing influenza virus infectious diseases.

Description

Prodrugs of Barosavir and derivatives thereof, preparation method and application thereof

Technical Field

The invention relates to the field of pharmaceutical chemistry, in particular to a prodrug of baroxavir and derivatives thereof, and a preparation method and application of the prodrug.

Background

Barosavirenz (Baloxavir marboxil) belongs to the third-generation anti-influenza chemical medicine, and the original company adopts Japanese salt wild pharmacy and is developed by Roche globally; the drug has been marketed in japan in 2 months of 2018, and is also the first anti-influenza drug approved by the FDA in the united states in the last 20 years, which has an innovative mechanism of action, is an initial, chemical book single-dose oral drug, has a completely new anti-influenza mechanism of action, and is intended to combat influenza a and B viruses, including tamiflu (oseltamivir) drug-resistant influenza strains and avian influenza strains (H7N 9, H5N 1). Research shows that the new medicine can take effect only by oral administration once when used for treating acute influenza patients over 12 years old and with the duration of influenza symptoms not more than 48 hours.

。

Baloxavir marboxil is a prodrug that is metabolized in vivo to the active compound Baloxavir (Baloxavir). Barosavir acts by inhibiting the cap-dependent endonuclease present in influenza virus by the mechanism that the genetic material of influenza virus is carried in RNA, the primary role of which is to aid genome replication, i.e., to aid influenza virus replication of progeny chemical book. Barosavir acts as an inhibitor of cap-dependent endonucleases, inhibiting enzymatic reactions, rendering the viral genome incapable of replication, thereby blocking viral proliferation upon entry into the cell. By means of the biological mechanism, the balosavir moves forward to prevent and control the entrance, and virus is killed in the cradle, so that the influenza can be cured quickly.

However, the bioavailability of the baroxavir in the body is low, which affects the absorption and utilization of the medicine, and the bioavailability of the Baloxavir marboxil is improved compared with that of the baroxavir, but the Baloxavir marboxil is still at a low level, which cannot meet the clinical requirement.

Therefore, there is a need to develop a novel prodrug of baroxavir and its derivatives to improve the bioavailability of the drug.

Disclosure of Invention

Accordingly, it is an object of the present invention to provide prodrugs of baroxavir and derivatives thereof having a greatly improved bioavailability of the macbecylate relative to baroxavir and derivatives thereof.

Another object of the present invention is to provide a process for preparing the prodrug of baroxavir and its derivatives.

It is a further object of the present invention to provide the use of prodrugs of said baroxavir and derivatives thereof.

It is a further object of the present invention to provide a pharmaceutical composition comprising a prodrug of baroxavir and its derivatives of the present invention.

The purpose of the invention is realized by the following technical scheme: .

In one aspect, the present invention provides a compound having the structure of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

。

Wherein, the flow rate of the water is controlled by the control unit.

X is O or S; .

Y is a C1-C6 straight chain or branched chain alkylene; .

Z is N or C, wherein when Z is C, a substituent R is attached thereto₈；。

R₁、R₂、R₃、R₄Each independently selected from H or C1-C6 straight chain or branched chain alkyl; .

R₅、R₆、R₇、R₈Each independently selected from H, halogen, trifluoromethyl, trifluoroethyl, trifluoromethoxy, hydroxyl, amino, carboxyl, cyano, nitro, C1-C6 linear or branched alkyl, C1-C6 linear or branched alkoxy, or C1-C6 linear or branched alkylthio; .

(R₉)_mRepresents m identical or different radicals selected from the following groups: H. halogen, cyano, hydroxyl, carboxyl, amino, alkyl, alkoxy, alkylthio, nitro, haloalkyl, hydroxyalkyl, haloalkoxy or alkylamino, preferably the alkyl is a C1-C6 straight chain or branched alkyl; .

Wherein m is an integer of 0 to 10.

In certain embodiments of the invention, the compound has the structure of formula (I'): .

。

Wherein, the flow rate of the water is controlled by the control unit.

X is O or S; .

Y is a C1-C6 straight chain or branched chain alkylene; .

Z is N or C, wherein when Z is C, a substituent R is attached thereto₈；。

R₅、R₆、R₇、R₈Each independently selected from H, halogen, trifluoromethyl, trifluoroethyl, trifluoromethoxy, hydroxyl, amino, carboxyl, cyano, nitro, C1-C6 linear or branched alkyl, C1-C6 linear or branched alkoxy, or C1-C6 linear or branched alkylthio.

In certain embodiments of the invention, in a compound of the invention, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, Y is a C1-C3 straight or branched chain alkylene; r₁、R₂、R₃、R₄Each independently selected from H or C1-C3 straight chain or branched chain alkyl; and/or R₅、R₆、R₇、R₈Each independently selected from H, F, Cl, Br, I, trifluoromethyl, trifluoroethyl, trifluoromethoxy, hydroxyl, amino, carboxyl, cyano, nitro, C1-C3 linear or branched alkyl, C1-C3 linear or branched alkoxy, or C1-C3 linear or branched alkylthio.

In certain embodiments of the invention, the pharmaceutically acceptable salt is a salt of a compound of formula (I) with an acid; preferably, the acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, or benzoic acid.

In certain embodiments of the invention, the compounds of the present invention are compounds having the following structure, stereoisomers thereof, or pharmaceutically acceptable salts thereof:

、

、

、

、

、

、

、

、

、

、

or

。

In another aspect, the present invention provides a method of preparing a compound having the structure of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as described herein, comprising the steps of:

。

(1) carrying out amidation reaction on the compound of the formula (1) and the compound of the formula (2) to generate a compound of a formula (3); .

(2) Carrying out an esterification reaction on the compound of the formula (3) and the compound of the formula (4) to generate a compound of a formula (5); .

(3) Subjecting a compound of formula (5) to a nucleophilic substitution reaction with a compound of formula (6) to produce a compound of formula (7); .

(4) Removing R from the compound of formula (7)₁₀Protecting groups to produce compounds of formula (I); optionally, reacting a compound of formula (I) with an acid to form a pharmaceutically acceptable salt; .

Wherein, X, Y, Z, R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、(R₉)_mM is as defined in claims 1 to 5, R₁₀Preferably Boc as amino protecting group.

In certain embodiments of the invention, wherein step (1) is carried out in the presence of an organic base; preferably, the organic base is selected from one or more of the following: DIPEA, triethylamine, DMAP, and DBU; the esterification reaction in the step (2) is carried out in the presence of a condensing agent and a catalyst; preferably, the condensing agent is selected from one or more of the following: EDCI, DCC, BOP and PyBOP, the catalyst being selected from the group consisting of: DMAP or DBU; step (3) is carried out in the presence of a base and a catalyst; preferably, the base is potassium carbonate, sodium carbonate, cesium carbonate, sodium bicarbonate and/or sodium phosphate, and the catalyst is sodium iodide, potassium iodide, and/or cesium iodide; and/or the deprotection reaction in step (4) is carried out under acidic conditions, and the salt-forming reaction is carried out in one step.

In certain embodiments of the present invention, wherein the compound of formula (6) is

Or

(ii) a Preferably, the

Is obtained by

Prepared by reaction with lawson's reagent.

In yet another aspect, the present invention provides the use of a compound described herein, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment and/or prevention of an influenza viral infection.

In yet another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient; preferably, the pharmaceutical composition is an oral formulation.

The inventor unexpectedly finds that the pro-drug of the baroxavir and the derivatives thereof can greatly improve the bioavailability of the parent compound, and the bioavailability is improved by more than 2.5 times compared with that of the Baloxavir marboxil. Higher bioavailability means that the dosage of the compounds of the invention can be correspondingly reduced by several times, and the corresponding side effects of the drug can be significantly reduced, for the same efficacy. Therefore, the compound of the invention is more excellent than Baloxavir marboxil in reducing dosage, reducing side effects of drugs and improving drug effect, and can provide a better choice for patients. Therefore, the compound of the present invention can be a drug for treating and/or preventing influenza virus infectious diseases, which has excellent oral absorbability.

Detailed Description

The present invention will be described in further detail with reference to specific examples so that those skilled in the art can more fully understand the present invention. The specific examples are only for illustrating the technical solutions of the present invention, and do not limit the present invention in any way.

The reagents and starting materials used in the following examples are all commercially available unless otherwise specified.

Example 1.

Step 1.

。

Y-1 (1 g) was added to methylene chloride at-10 ℃ followed by DIPEA (1.21 g) and Y-2 (1.14 g) for 1 hour, TLC showed no starting material, Y-4 (1.5 g), EDCI (1.52 g) and DMAP (0.265 g) were added to the reaction mixture, and the mixture was reacted at-10 ℃ for 2 hours, slowly warmed to room temperature, and quenched with water after 0.5 hour. The organic phase was washed successively with 0.1N HCl, water, aqueous sodium bicarbonate, brine, dried and spin dried to give 1.86g of a pale yellow oil Y-5. The HPLC purity is about 90%, and the product is directly used in the next reaction. MS (M/z):416.1(M + H)⁺。

And 2, step 2.

。

Lawson's reagent (2.63 g) was added to a toluene mixture of Y-7 (4.83 g) under nitrogen and stirred at 80 ℃ for 3 h. TLC showed no starting material, spin-dried, and column chromatographed to give Y-82.65 g as a yellow solid in 53.0% yield.

¹H-NMR(400MHz，CDCl₃)δ11.51(s，1H)，7.00～7.19 (m，4H)，6.83～6.88(m，2H)，6.68(d，1H)，5.35(d，2H)，5.26(d，1H)，4.73(d，1H)，4.64(d，1H)，4.11(d，1H)，3.98(m，1H)，3.83(m，1H)，3.58(t，1H)，3.48(m，1H)，3.02(m，1H)。MS(m/z):500.1(M+H)⁺。

And 3, step 3.

。

Y-8 (500 mg) was added to DMA under nitrogen followed by Y-5, potassium carbonate and sodium iodide and reacted at 60 ℃ for 4 hours with TLC showing no starting material. Ethyl acetate and water were added, the mixture was extracted and separated, and the organic phase was washed with water, dried over anhydrous sodium sulfate, and spin-dried to give 850mg of a yellow solid. Separating by column chromatography to obtain Y-9460 mg with yield 53.0%. MS (M/z) 879.2(M + H)⁺。

And 4, step 4.

。

Adding Y-9 (200 mg) into ethyl acetate hydrochloride, reacting for two hours at room temperature, carrying out suction filtration, washing a filter cake with ethyl acetate, and drying to obtain a product A171 mg with a yield of 92.1%.¹H-NMR(400MHz，DMSO) δ8.58 (m，1H)，7.96 (m，1H)，7 .42～7.53 (m，1H)，7 .07～7.26 (m，4H)，6.74～6.98(m，3H)，6.21(m，1H)，5.85(m，1H)，5.43(m，1H)，5.17(m，3H)，4.54(m，1H)，4.41(m，1H)，4.02～4.19 (m，4H)，3.94(m，1H)，3.66(m，1H)，3.55(m，1H) ，3.33(m，1H)，2.83～2.95 (m，4H)， 2.58(m，3H)，1.61～1.75 (m，2H)。MS(m/z):779.2(M+H)⁺。

Preparation of chiral Compounds B and C.

。

HPLC preparative separation of pure A (100 mg) (preparative column: Waters Symmetry C18, mobile phase A: water; B: methanol) gave compound B32 mg and C36 mg MS (M/z):779.2(M + H)⁺。

Example 2.

Step 1.

。

Y-1 (1 g) was added to methylene chloride at-10 ℃ followed by DIPEA (1.21 g) and Y-6 (1.02 g) and reacted for 1 hour, and after TLC showed no starting material, Y-4 (1.5 g), EDCI (1.52 g) and DMAP (0.265 g) were added to the reaction mixture, reacted at-10 ℃ for 2 hours, slowly warmed to room temperature, and quenched with water after 0.5 hour. The organic phase was washed successively with 0.1N HCl, water, aqueous sodium bicarbonate, brine, dried and spin-dried to give 1.78g of Y-11 as a pale yellow oil. The HPLC purity is about 92%, and the product is directly used in the next reaction. MS (M/z) 402.1(M + H)⁺。

And 2, step 2.

。

Y-8 (450 mg) was added to DMA under nitrogen followed by Y-11, potassium carbonate and sodium iodide and reacted at 60 ℃ for 4 hours with TLC showing no starting material. Adding ethyl acetate and water, extracting, separating, washing the organic phase with water, drying with anhydrous sodium sulfate, spin-drying, and separating by column chromatography to obtain solid Y-12459 mg with yield of 59.1%. MS (M/z) 865.2(M + H)⁺。

And 3, step 3.

。

Adding Y-12 (200 mg) into ethyl acetate hydrochloride, reacting for two hours at room temperature, carrying out suction filtration, washing a filter cake with ethyl acetate, and drying to obtain a product D170 mg with the yield of 92.0%.¹H-NMR(400MHz，DMSO) δ8.52 (m，1H)，7.89 (m，1H)，7.31～7.53 (m，1H)，7.10～7.29 (m，4H)，6.76～6.99(m，3H)，5.58～6.03 (m，3H)，5.41(m，1H)，5.19(m，3H)，4.56(m，1H)，4.39(m，1H)，4.02～4.21 (m，4H)，3.96(m，1H)，3.68(m，1H)，3.53(m，1H)，2.83～2.97(m，4H)，2.65(m，3H)。MS(m/z):765.2(M+H)⁺。

Example 3.

Step 1.

。

Y-7 (500 mg) was added to DMA under nitrogen followed by Y-5, potassium carbonate and sodium iodide and reacted at 60 ℃ for 4 hours with TLC showing no starting material. Adding ethyl acetate and water, extracting, separating liquid, washing an organic phase with water, drying with anhydrous sodium sulfate, spin-drying, and separating by column chromatography to obtain Y-13582 mg with a yield of 65.2%. MS (M/z):863.3(M + H)⁺。

And 2, step 2.

。

Adding Y-13 (200 mg) into ethyl acetate hydrochloride, reacting for two hours at room temperature, carrying out suction filtration, washing a filter cake with ethyl acetate, and drying to obtain a product E176 mg with the yield of 95.1%.¹H-NMR(400MHz，DMSO) δ8.51 (m，1H)，7.88(m，1H)，7 .33～7.54(m，3H)，7 .08～7.29 (m，3H)，6.75～6.99(m，2H)，6.21(m，1H)，5.58～6.03 (m，3H)，5.42(m，1H)，5.17(m，1H)，4.53(m，1H)，4.43(m，1H)，4.05～4.25 (m，4H)，3.96(m，1H)，3.68(m，1H)，3.36～3.49 (m，2H)，2.85～2.99(m，4H) ，2.69(m，3H) ,1.63～1.76 (m，2H)。MS(m/z):763.2(M+H)⁺。

Preparation of chiral Compounds F and G.

。

HPLC preparative separation of pure E (100 mg) (preparative column: Waters Symmetry C18, mobile phase A: water; B: methanol) gave 35 mg and G38 mg of MS (M/z):763.2(M + H)⁺。

Example 4.

Step 1.

。

Y-7 (500 mg) was added to DMA under nitrogen followed by Y-11, potassium carbonate and sodium iodide and reacted at 60 ℃ for 4 hours with TLC showing no starting material. Adding ethyl acetate and water, extracting, separating liquid, washing an organic phase with water, drying with anhydrous sodium sulfate, spin-drying, and separating by column chromatography to obtain Y-14554 mg with the yield of 63.1%. MS (M/z) 849.3(M + H)⁺。

And 2, step 2.

。

Adding Y-14 (200 mg) into ethyl acetate hydrochloride, reacting for two hours at room temperature, carrying out suction filtration, washing a filter cake with ethyl acetate, and drying to obtain a product H168 mg with the yield of 91.0%.¹H-NMR(400MHz，DMSO) δ8.53 (m，1H)，7.91 (m，1H)，7 .31～7.53 (m，3H)，7 .13～7.29 (m，3H)，6.73～6.99(m，2H)，5.72～5.75 (m，3H)，5.63(m，1H)，5.19～5.35 (m，3H)，4.53(m，1H)，4.35(m，1H)，4.02～4.26 (m，4H)，3.91(m，1H)，3.73(m，1H)，3.57(m，1H)，2.83～2.97(m，4H)，2.65(m，3H)。MS(m/z):749.2(M+H)⁺。

Example 5.

。

Y-8 (500 mg) was added to DMA under nitrogen followed by Y-15, potassium carbonate and sodium iodide and reacted at 60 ℃ for 4 hours with TLC showing no starting material. Adding ethyl acetate and water, extracting, separating, washing the organic phase with water, drying with anhydrous sodium sulfate, spin-drying, and separating by column chromatography to obtain compound I514 mg with yield 83.5%.¹H-NMR(400MHz，DMSO) 7.38～7.47 (m，2H)，7.08～7.21 (m，3H)，7.03(d，1H)，6.87(d，1H)，6.74(m，1H)，5.83(s，1H)，5.55～5.68 (m，1H)，5.35～5.43 (m，1H)，4.53(m，1H)，4.37(m，1H)，4.09 (m，1H)，3.90(m，1H)，3.72～3.83 (m，2H)，3.57(m，1H)，3.35～3.47 (m，2H),2.83 (m，2H), 1.61-1.69 (m，2H), 1.32 (t，3H)。MS(m/z):616.2(M+H)⁺。

Preparation of chiral Compounds J and K.

。

HPLC preparative separation of pure product I (100 mg) (preparative column: Waters Symmetry C18, mobile phase A: water; B: methanol) gave compound J38 mg and K40 mg MS (M/z):616.2(M + H)⁺。

Example 6.

。

Y-8 (500 mg) was added to DMA under nitrogen followed by Y-16, potassium carbonate and sodium iodide and reacted at 60 ℃ for 4 hours with TLC showing no starting material. Adding ethyl acetate and water, extracting, separating, washing the organic phase with water, drying with anhydrous sodium sulfate, spin-drying, and separating by column chromatography to obtain compound L500 mg with yield of 85.1%.¹H-NMR(400MHz，DMSO) 7.38～7.45 (m，2H)，7.09～7.23 (m，3H)，7.00(d，1H)，6.85(d，1H)，6.78(m，1H)，5.81(s，1H)，5.43～5.5 (m，2H)，5.34～5.41 (m，1H)，4.51(m，1H)，4.39(m，1H)，4.09 (m，1H)，3.91(m，1H)，3.73(s，3H)，3.59(m，1H)，3.36～3.48 (m，2H)，2.82 (m，1H)。MS(m/z):588.2(M+H)⁺。

Example 7.

Step 1.

。

Y-8 (450 mg) was added to DMA under nitrogen followed by Y-17, potassium carbonate and sodium iodide at 6Reaction was carried out at 0 ℃ for 4 hours and TLC showed no starting material. Adding ethyl acetate and water, extracting, separating, washing the organic phase with water, drying with anhydrous sodium sulfate, spin-drying, and separating by column chromatography to obtain solid Y-18618 mg with yield of 78.2%. MS (M/z) 878.3(M + H)⁺。

And 2, step 2.

。

Adding Y-18 (200 mg) into ethyl acetate hydrochloride, reacting for two hours at room temperature, carrying out suction filtration, washing a filter cake with ethyl acetate, and drying to obtain a product M151 mg with the yield of 81.6%.¹H-NMR(400MHz，DMSO) δ7.65 (m，1H)，7.07～7.29 (m，7H)，6.71～6.95(m，3H)，6.15(m，1H)，5.83(m，1H)，5.41(m，1H)，5.14(m，3H)，4.57(m，1H)，4.39(m，1H)，4.02～4.25 (m，4H)，3.95(m，1H)，3.67(m，1H)，3.57(m，1H)，3.41(m，1H)，2.85～2.99 (m，4H)，2.64(m，3H)，1.62～1.76 (m，2H)。MS(m/z):778.3(M+H)⁺。

Using the methods of examples 1 to 7, the following compounds N, MS (M/z):796.3(M + H)⁺(ii) a Compound O, MS (M/z):812.2(M + H)⁺(ii) a Compound P, MS (M/z):846.3(M + H)⁺(ii) a Compound Q, MS (M/z):792.3(M + H)⁺；。

The compound R is obtained by measuring,¹H-NMR(400MHz，DMSO) δ8.05 (m，2H)，7.29～7.53 (m，4H)，7.09～7.23 (m，3H)，6.71～6.98(m，2H)，5.71～5.76 (m，1H)，5.61(m，1H)，5.42(m，1H)，4.48(m，1H)，4.35(m，1H)，4.00～4.06 (m，2H)，3.59～3.69 (m，3H)，3.45(m，1H)，3.31～3.35(m，1H)，2.91～2.97(m，1H)，2.41 (d，6H)。MS(m/z):645.2(M+H)⁺(ii) a And (c).

The compound S is obtained by measuring the content of the compound S,¹H-NMR(400MHz，DMSO) 7.36～7.43 (m，2H)，7.07～7.24 (m，3H)，7.01(d，1H)，6.86(d，1H)，6.79(m，1H)，5.83(m，1H)，5.35～5.43 (m，1H)，4.53(m，1H)，4.41(m，1H)，4.11 (m，1H)，3.92(m，1H)，3.59(m，1H)，3.06～3.26 (m，4H)，2.62～2.69 (m，4H)，2.82 (m，1H)，2.35 (s，3H)。MS(m/z):626.2(M+H)⁺。

example 8: anti-influenza virus activity (CPE inhibition effect) assay.

< materials >.

1. 2% FCS E-MEM (prepared by adding kanamycin and FCS to MEM (minimum essential medium)).

2. 0.5% BSA E-MEM (in MEM (minimum essential medium) adding kanamycin and BSA to prepare).

3. HBSS (Hanks Balanced salt solution).

4. MDBK cells: the number of cells was adjusted to an appropriate number (3X 10) with 2% FCS E-MEM⁵/mL）。

5. MDCK cells: the cells were washed 2 times with HBSS and then adjusted to the appropriate number of cells (5X 10) with 0.5% BSA E-MEM⁵/mL)。

6. Trypsin solution: trypsin (SIGMA) from porcine pancreas was dissolved in PBS (-) and filtered through a 0.45 μm filter.

7. EnVision (microplate reader) (PerkinElmer).

8. WST-8 kit (Kishida chemical).

9. 10% SDS solution.

< operational procedure >.

1. Diluting and dispensing the test sample.

2% FCS E-MEM was used for MDBK cells, and 0.5% BSA E-MEM was used for MDCK cells. The same culture medium was used for dilution of viruses, cells, and test samples.

The test sample was diluted in a culture medium to an appropriate concentration in advance, and a 2-to 5-fold serial dilution series (50. mu.L/well) was prepared in a 96-well plate. Two blocks for measuring influenza resistance and cytotoxicity were prepared. Triplicate assays were performed for each drug.

In the case of MDCK cells, trypsin was added to the cells to a final concentration of 3. mu.g/mL only for measurement of anti-influenza activity.

2. Diluting and dispensing influenza virus.

Influenza virus culture medium was diluted to an appropriate concentration in advance, and each was dispensed into a 96-well plate to which test samples were added at 50. mu.L/well. The culture medium was dispensed at 50. mu.L/well into the plate for measuring cytotoxicity.

3. Diluting and dispensing the cells.

Cells adjusted to an appropriate number of cells were dispensed into a 96-well plate containing a test sample at 100. mu.L/well.

Mixed with a plate mixer (plate mixer) and cultured in a CO2 incubator. The cells were cultured for 3 days for both influenza resistance and cytotoxicity.

4. And (5) performing separated injection of WST-8.

The 96-well plate cultured for 3 days was observed with the naked eye under a microscope to confirm the morphology of the cells, the presence or absence of crystals, and the like. The supernatant was removed from the plate without aspiration of the cells.

The WST-8 kit was diluted 10-fold with a culture medium, and the WST-8 solution was dispensed into each well at 100. mu.L each. Mixing with a hole plate mixer, and then culturing in a CO2 incubator for 1-3 hours.

After the plate for measuring anti-influenza activity was cultured, 10. mu.L of 10% SDS solution was dispensed into each well, and the virus was inactivated.

5. And (4) measuring the absorbance.

For the mixed 96-well plate, absorbance was measured at 450nm/620nm of two wavelengths using EnVision.

< calculation of each measurement item value >.

CPE inhibition and cell viability were calculated using GraphPad Prism or a program with equivalent computational processing power, and EC was obtained from the fitted curve₅₀And CC₅₀The value is obtained.

Wherein, the flow rate of the water is controlled by the control unit.

CPE inhibition ═ 100% (addition well absorbance-virus control well absorbance)/(cell control well absorbance-virus control well absorbance).

The cell survival rate is (light absorption value of dosing well-light absorption value of medium control well)/(light absorption value of cell control well-light absorption value of medium control well) × 100%.

The measurement results of example 8 are shown in table 1.

。

As can be seen from Table 1, Y-7 and Y-8 both have very good in vitro activity, and Y-8 has twice the activity of Y-7, which indicates that sulfur substituted oxygen atom can greatly improve the in vitro antiviral activity of the compound. The compound D, H, I, L has low in vitro activity because they are prodrugs, and they are stable in vitro and can not release active molecules basically, and can only enter human body and release active molecules through the degradation of enzymes in human body, so as to generate drug effect, which completely accords with the prodrug design theory. CC of Compound₅₀The values show that the molecules are low in toxicity to cells and reliable in safety.

Example 9: bioavailability (BA) assay.

Oral absorbability study test materials and methods.

1. Animals were used: mice or SD rats were used.

2. Feeding conditions are as follows: the solid feed and the sterilized tap water were freely ingested by the mice or SD rats.

3. Dose amount and group setting: the drug is administered orally or intravenously in a specific dose. In the following manner.

A group is set. (the amount of each compound administered was varied).

1-30 mg/kg (n is 2-3) is orally administered.

The intravenous administration is 0.5-10 mg/kg (n is 2-3).

4. Preparation of a dosing solution: oral administration is in the form of a solution or suspension. Intravenous administration is administration after solubilization.

5. The administration method comprises the following steps: oral administration is forcibly administered into the stomach through a feeding tube. Intravenous administration is via a syringe with a needle attached from the tail vein.

6. Evaluation items: blood was collected over time and the concentration of the compound of the present invention in plasma was measured using LC/MS/MS.

7. Statistical analysis: for the concentration change in plasma, the area under the concentration-time curve (AUC) in plasma was calculated using a non-linear least squares procedure, and the Bioavailability (BA) was calculated from the AUC of the oral administration group and the intravenous administration group.

The measurement results of example 9 are shown in table 2.

。

From the above results, it can be seen that the bioavailability of the prodrug of the present invention is significantly improved over the parent compound. Wherein, the bioavailability of the compound A, D, E, H is improved by more than ten times compared with the parent compound, and is also improved by 4 to 5 times compared with the Baloxavir marboxil, the bioavailability of the compound M, N, O, P is improved by more than 7 times compared with the parent compound, and is also improved by more than 2.5 times compared with the Baloxavir marboxil, and the effect is very obvious. However, compounds R and S, although they also contain an amino group capable of forming a salt with an acid, do not significantly improve the bioavailability of the parent compound, even if the bioavailability is greatly reduced compared to Baloxavir marboxil.

The higher bioavailability means that the dosage of the compound of the invention can be correspondingly reduced by several times under the same drug effect condition, and the corresponding side effect of the drug can be also obviously reduced. Therefore, the compound of the invention is more excellent than Baloxavir marboxil in reducing dosage, reducing side effects of drugs and improving drug effect, and can provide a better choice for patients. Therefore, the compound of the present invention can be a drug for treating and/or preventing influenza virus infectious diseases, which has excellent oral absorbability.

Claims

1. A compound having the structure of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof,

wherein,

x is O or S;

y is a C1-C6 linear or branched alkylene;

z is N or C, wherein when Z is C, a substituent R is attached thereto₈；

R₁、R₂、R₃、R₄Each independently selected from H or C1-C6 straight chain or branched chain alkyl;

R₅、R₆、R₇、R₈each independently selected from H, halogen, trifluoromethyl, trifluoroethyl, trifluoromethoxy, hydroxy, amino, carboxy, cyano, nitro, C1-C6 linear or branched alkyl, C1-C6 linear or branched alkoxy, or C1-C6 linear or branched alkylthio;

(R₉)_mrepresents m identical or different radicals selected from the following groups: H. halogen, cyano, hydroxyl, carboxyl, amino, alkyl, alkoxy, alkylthio, nitro, haloalkyl, hydroxyalkyl, haloalkoxy or alkylamino, wherein the alkyl in the alkyl, alkoxy, alkylthio, haloalkyl, hydroxyalkyl, haloalkoxy or alkylamino is a C1-C6 straight chain or branched chain alkyl;

wherein m is an integer of 0 to 10.

2. A compound according to claim 1, a stereoisomer or pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (Γ):

wherein,

x is O or S;

y is a C1-C6 linear or branched alkylene;

z is N or C, wherein when Z is C, a substituent R is attached thereto₈；

R₅、R₆、R₇、R₈each independently selected from H, halogen, trifluoromethyl, trifluoroethyl, trifluoromethoxy, hydroxy, amino, carboxy, cyano, nitro, C1-C6 linear or branched alkyl, C1-C6 linear or branched alkoxy, or C1-C6 linear or branched alkylthio.

3. The compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein,

y is a C1-C3 linear or branched alkylene;

R₁、R₂、R₃、R₄each independently selected from H or C1-C3 straight chain or branched chain alkyl; and/or

R₅、R₆、R₇、R₈Each independently selected from H, F, Cl, Br, I, trifluoromethyl, trifluoroethyl, trifluoromethoxy, hydroxyl, amino, carboxyl, cyano, nitro, C1-C3 linear or branched alkyl, C1-C3 linear or branched alkoxy, or C1-C3 linear or branched alkylthio.

4. The compound according to claim 1 or 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is a salt of the compound represented by formula (I) with an acid.

5. A compound, a stereoisomer, or a pharmaceutically acceptable salt thereof, according to claim 4, wherein the acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, or benzoic acid.

6. The compound according to claim 1 or 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which is a compound having the structure:

7. a process for preparing a compound having the structure of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 6, comprising the steps of:

(1) carrying out amidation reaction on the compound of the formula (1) and the compound of the formula (2) to generate a compound of a formula (3);

(2) carrying out an esterification reaction on the compound of the formula (3) and the compound of the formula (4) to generate a compound of a formula (5);

(3) subjecting a compound of formula (5) to a nucleophilic substitution reaction with a compound of formula (6) to produce a compound of formula (7);

(4) removing R from the compound of formula (7)₁₀Protecting groups to produce compounds of formula (I);

wherein, X, Y, Z, R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、(R₉)_mM is as defined in any one of claims 1 to 6, R₁₀Is an amino protecting group.

8. The method of claim 7, wherein:

the step (1) is carried out in the presence of organic base;

the esterification reaction in the step (2) is carried out in the presence of a condensing agent and a catalyst;

step (3) is carried out in the presence of a base and a catalyst; and/or

The reaction of removing the protecting group in the step (4) is carried out under an acidic condition, and is carried out in one-step reaction with a salt-forming reaction.

9. The method of claim 8, wherein:

the organic base in step (1) is selected from one or more of the following: DIPEA, triethylamine, DMAP, and DBU;

the condensing agent in step (2) is selected from one or more of the following: EDCI, DCC, BOP and PyBOP, the catalyst being selected from the group consisting of: DMAP or DBU; and/or

In the step (3), the alkali is potassium carbonate, sodium carbonate, cesium carbonate, sodium bicarbonate and/or sodium phosphate, and the catalyst is sodium iodide, potassium iodide and/or cesium iodide.

10. The process of claim 7, wherein the amino protecting group is Boc.

11. The method of claim 7, further comprising the step of reacting the compound of formula (I) with an acid to form a pharmaceutically acceptable salt.

12. The method according to any one of claims 7 to 11, wherein the compound of formula (6) is

13. The method of claim 12, wherein the

Is obtained by

Prepared by reaction with lawson's reagent.

14. Use of a compound of any one of claims 1 to 6, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment and/or prevention of influenza viral infection.

15. A pharmaceutical composition comprising a compound of any one of claims 1 to 6, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

16. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is an oral formulation.