CN111393391A

CN111393391A - Antiviral agent for hepatitis B virus infection

Info

Publication number: CN111393391A
Application number: CN202010301579.0A
Authority: CN
Inventors: 杨正; 王婕; 李进; 刘浏; 杨民民
Original assignee: Nanjing Anakang Biotechnology Co ltd
Current assignee: Nanjing Anakang Biotechnology Co ltd
Priority date: 2020-04-16
Filing date: 2020-04-16
Publication date: 2020-07-10
Anticipated expiration: 2040-04-16
Also published as: CN111393391B

Abstract

The invention discloses an antiviral agent for hepatitis B virus infection, comprising pharmaceutically acceptable salt, ester, prodrug, complex, solvate, hydrate or isomer form thereof, and application thereof in preparing a medicament for treating or inhibiting hepatitis B virus infection.

Description

Antiviral agent for hepatitis B virus infection

Technical Field

The invention relates to the field of pharmaceutical chemistry, in particular to an antiviral agent for hepatitis B virus infection.

Background

Hepatitis B Virus (HBV) infection remains a major public health problem. Currently, there are estimated 3.5 million people worldwide, of which 140 million in the united states have chronic HBV. Approximately one third of these people will die of serious liver diseases, such as cirrhosis and liver cancer, if left untreated.

Currently, there are seven drugs available for the treatment of chronic hepatitis b, including two α -interferon formulations (standard and peg-modified) and five nucleoside analogs that inhibit HBV DNA polymerase (lamivudine, adefovir, entecavir, telbivudine, and tenofovir.) currently, the first line of treatment option of choice is entecavir, tenofovir, or pegylated interferon α -2 a. however, even with the first line of treatment option, pegylated interferon α -2a may only be effective in one third of the patients receiving treatment for serological milestones, often accompanied by severe side effects.

Therefore, there is still a need for new antiviral drugs that are clinically effective and effective for the treatment of disease relief in patients infected with hepatitis b virus. The invention provides a new choice for treating the disease remission of patients infected with hepatitis B virus and effective new antiviral drugs.

Disclosure of Invention

In one aspect, the present invention discloses compounds having the structure:

in some embodiments, it is a pharmaceutically acceptable salt, ester, prodrug, complex, solvate, hydrate, or isomer form.

In another aspect, the invention discloses the use of the aforementioned compounds for the manufacture of a medicament for the treatment or inhibition of hepatitis b virus infection.

In another aspect, the invention discloses the use of the aforementioned compounds for the preparation of a medicament for the prophylactic treatment of hepatitis b virus infection.

In another aspect, the invention discloses the use of the aforementioned compounds for the manufacture of a medicament for reducing the recurrence of hepatitis b virus infection.

In another aspect, the invention discloses the application of the compound in preparing a medicament for inducing and relieving liver injury caused by hepatitis B virus infection

In another aspect, the present invention discloses the use of the aforementioned compounds for the manufacture of a medicament for reducing adverse physiological effects caused by hepatitis b virus infection.

The compounds in the foregoing uses may be in the form of pharmaceutically acceptable salts, esters, prodrugs, complexes, solvates, hydrates, or isomers.

The term "isomer" as used herein includes enantiomeric, diastereomeric and geometric (or conformational) isomeric forms of a given structure. For example, the present application includes R and S configurations for each asymmetric center, Z and E double bond isomers, Z and E conformers, single stereochemical isomers and mixtures of enantiomers, diastereomers and geometric (or conformational) isomers. Unless otherwise indicated, this application includes all tautomeric forms of the structures described herein. "salts" include acid and base addition salts. It is understood that when the compounds or examples of the present application are shown as particular salts, the present application includes the corresponding free base as well as other salts of the corresponding free base (including pharmaceutically acceptable salts of the corresponding free base). "solvate" refers to an association or complex of one or more solvent molecules and a compound of the present application. Examples of the solvent include water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" refers to a complex in which the solvent molecule is water.

In another aspect, the invention discloses a pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 and a pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable carrier or excipient" as used herein refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated.

In some embodiments, the aforementioned pharmaceutical composition further comprises an additional therapeutic agent that is an HBV polymerase inhibitor, an interferon, or a reverse transcriptase inhibitor.

Detailed Description

Example 1

Step one preparation of 3- (chlorosulfonyl) ethyl benzoate

Adding 3- (chlorosulfonyl) benzoic acid (15.18g, 0.069mol, 1.0eq.) into DCM (150m L) at room temperature, cooling to 0-5 ℃ with an ice-water bath, adding thionyl chloride (32.74g, 0.27mol, 4.0eq.) and a catalytic amount of DMF (1.5 m L), heating to room temperature to react overnight for 16 hours, evaporating the reaction solution to dryness, removing the solvent with toluene for 2 times, pouring into THF (150m L), cooling to 0-5 ℃ with an ice-water bath, dropwise adding ethanol (30m L), reacting for 4-6 hours at room temperature, directly evaporating the reaction solution to dryness, and purifying by column chromatography (200-300 mesh silica gel, Heptane/EtOAc (10: 1-5: 1) to obtain 12.23g of yellow oily liquid with a yield of 71.5%.

Preparation of step-bis-3-sulfamoylethyl benzoate

Ethyl 3- (chlorosulfonyl) benzoate (12.20g, 4.9mmol, 1.0eq.) was added to THF (150m L) at room temperature, the mixture was cooled to-10 ℃ or lower, aqueous ammonia (13.67g, 24.5mmol, 5.0eq.) was added dropwise and reacted for 1 hour, the reaction mixture was adjusted to pH 4 to 5 with 4N hydrochloric acid, part of THF was distilled off, the precipitated solid was filtered, the filter cake was washed with water to neutrality, and dried to obtain 10.53g of a white solid, the yield was 93.8%.

Step three preparation of ethyl 3- (N- (tert-butyldimethylsilyl) sulfamoyl) benzoate

Ethyl 3-sulfamoylbenzoate (3.00g, 13.1mmol, 1.0eq.) was added to DCM (30m L) and THF (15m L) at room temperature, and triethylamine (3.97g, 39.3mmol, 3.0eq.) and TBS-Cl (2.37g, 15.7mmol, 1.20eq.) were added and reacted for 12 hours.

Step four preparation of 3- (N- (tert-butyldimethylsilyl) -S-chlorosulfonylimido) Ethyl benzoate

Triphenylphosphine dichloride (5.24g, 15.7mmol, 1.2eq.) was added to the reaction solution of the previous step at room temperature (4.50g, 13.1mmol, 1.0eq.) and the reaction was completed at 30-35 ℃ for 10 hours. The reaction solution was directly fed to the next reaction.

Step five preparation of Ethyl 3- (N' - (tert-butyldimethylsilyl) -N- ((S) -tetrahydrofuran-3-yl) sulfonamide diimide) benzoate

(3S) -Oxolan-3-amine (967.0mg, 11.1mmol, 3.0eq.) was added to the reaction solution of the previous step (1.34g, 3.7mmol, 1.0eq.) at room temperature, and the reaction was carried out overnight for 12 hours at room temperature after the addition. The reaction was quenched by addition of saturated brine, extracted with DCM, separated and dried, filtered and evaporated to dryness to give a sand, which was purified by silica gel column chromatography (200-300 mesh silica gel, Heptane/EtOAc ═ 10: 1-5: 1) to give 430.0mg of a pale yellow oily liquid.

Preparation of step hexa 3- (N' - (tert-butyldimethylsilyl) -N- ((S) -tetrahydrofuran-3-yl) sulfonamide diimide-based) benzoic acid

Ethyl 3- (N' - (tert-butyldimethylsilyl) -N- ((S) -tetrahydrofuran-3-yl) sulfonamide imidoyl) benzoate (430.0mg, 1.04mmol, 1.0eq.) was added to THF (10m L) at room temperature, solid L iOH (87.45mg, 3.2mmol, 2.0eq.) and 5m L water were added and reacted for 12 hours, the reaction solution was washed with 2N diluted brine to pH 3-4, extracted with EA, the organic phase was washed with saturated brine once, separated and dried, filtered and evaporated to dryness to give 400.0mg of a yellow liquid with a yield of 100%.

Preparation of step hepta 3- (N' - (tert-butyldimethylsilyl) -N- ((S) -tetrahydrofuran-3-yl) sulfonamidoiminoimidoacyl) -N- (3, 4-difluorophenyl) benzamide

Adding 3- (N' - (tert-butyldimethylsilyl) -N- ((S) -tetrahydrofuran-3-yl) sulfonamide diimide) benzoic acid (400.0mg, 1.04mmol, 1.0eq.) to DM (8m L) F at room temperature, cooling the reaction solution to 0-5 ℃ with an ice water bath, adding 3, 4-difluoroaniline (140.0mg, 1.08m mol, 1.1eq.) and DIPEA (410.0mg, 3.12mmol, 3.0eq.) to the reaction solution, reacting the mixture for 30 minutes, adding HATU (429.5mg, 1.2mmol, 1.2eq.) to the reaction solution, reacting the mixture at room temperature overnight for 12 hours, adding 5m L saturated NaHCO to the reaction solution, and reacting the mixture for 12 hours at room temperature₃Quenching the aqueous solution, pouring water (30m L), extracting with EA, washing the organic phase with saturated salt water once, separating, drying, filtering, evaporating to dryness to prepare sand, and purifying by silica gel column chromatography (200-300 mesh silica gel, Heptane/EtOAc 10: 1-5: 1) to obtain 350.0mg of yellow liquid with the yield of 63.6%.

Step preparation of octa-N- (3, 4-difluorophenyl) -3- (N- (((S) -tetrahydrofuran-3-yl) sulfonamide diimidazinyl) benzamide

3- (N' - (tert-butyldimethylsilyl) -N- ((S) -tetrahydrofuran-3-yl) sulfonamidoiminoacyl) -N- (3, 4-difluorophenyl) benzamide (350.0mg, 0.7mmol, 1.0eq.) was added to dioxane (5m L) at room temperature, a 4N dioxane HCl solution (0.7m L, 2.8mmol, 4.0eq.) was added and the reaction was allowed to react for 6 hours, the reaction mixture was evaporated to dryness, EA was dissolved, the mixture was washed with water and saturated brine in this order, the mixture was separated and dried, and 150.6mg of a yellow solid was obtained by filtration and evaporation to dryness, with the yield being 56.3%.¹HNMR(400MHz,DMSO)(ppm)1.59-1.84(m,2H),3.58-3.68(m,4H),4.47(m,1H),7.31(brs,1H),7.42-7.49(m,1H),7.55(m,1H),7.71-7.74(t,1H),7.91-7.96(m,1H),8.09-8.15(m,2H),8.44(s,1H),10.66(s,1H)。MS(ESI):m/z([M+H]⁺)382.50。

Experimental example 1HBV viral replication inhibition experiment

DMEM/F12(1:1) medium, PBS (1X), penicillin-streptomycin double antibody, 0.5% pancreatin (10X), qPCRSYBR Green Mix from ThermoFisher (Waltham, MA, USA), certified Fetal Bovine Serum (FBS) from Biological Industries (Israel), hydrocortisone from alfa. insulin from Sigma. doxycycline from Clontech.96 well, and 384 well cell culture plates from CORNI (USA) primers from Nanjing Sisley Biotech Inc. qPCR 384 from Roche. QuickExtract DNA extraction reagents from L ucigen.

"growth medium" was DMEM/F12(1:1), 10% FBS, 1 XP, 350nM hydrocortisone, 5ug/m L insulin, 1ug/m L doxycycline "treatment medium" was DMEM/F12(1:1), 2% FBS, 1 XP, 350nM hydrocortisone, 5ug/m L insulin.

To evaluate the inhibition of HBV viral replication by the synthetic compounds, HepAD38 cells grown in growth medium were trypsinized, resuspended in treatment medium after centrifugation, and plated at 6000/well in 384-well plates, 40. mu. L per well, placed at 37 ℃ and 5% CO₂Was diluted to 12 points in DMSO, 3-fold gradient dilutions, starting at 2 mm.1 μ L DMSO solution from compound stock plates was added to 199 μ L treatment medium (final maximum concentration of compound in assay is 10 μ M, and final concentration of DMSO is 0.5%). medium in cell culture plates was discarded, 40 μ L compound solution was added per well according to gradient, and 384 well plates were placed at 37 ℃, 5% CO₂After 3 days of incubation in the incubator, 3 days of incubation, the culture medium in the wells is discarded, the freshly prepared culture medium of the compound is added, the 384-well plate is placed in the incubator again for 3 days of incubation, after 6 days of total administration, 4ul of culture medium is taken out from each well of the 384-well cell culture plate and transferred to the 384-well qPCR plate, 4ul of Quickextract DNA extraction reagent is added into each well, after centrifugal mixing, DNA is extracted at 65 ℃ for 30min and 95 ℃ for 17min in Roche fluorescence quantitative PCR L ightcycler 480 II.

The status of virus replication was detected by quantitative qPCR. The detection primers are forward 5'-GAGTGTGGATTCGCACTCC-3' and backward 5'-GAGGCGAGGGAGTTCTTCT-3'. Upwards toIn the 384 well plate containing the sample, 10ul of qPCR SYBR Green Mix, 1ul of forward primer, 1ul of backward primer, incubation at 95 ℃ for 10min, Fluorogenic quantitative PCR was performed in Roche Fluorogenic quantitative PCR L light cycler 480 II, program set to 95 ℃ for 15S,60 ℃ for 1min,40 cycles, viral load was calculated from the standard curve, concentration of 50% inhibition of viral load by compound was determined using sigmoidal dose reaction model (variable slope, four parameters) in Prism 7 (L a Jolla, CA) (IC, four parameters)₅₀Value).

IC of the representative Compounds of the invention₅₀The values are shown in Table 1.

TABLE 1

Claims

1. A compound having the structure:

2. the compound of claim 1, which is a pharmaceutically acceptable salt, ester, prodrug, complex, solvate, hydrate, or isomer form.

3. Use of a compound of claim 1 for the manufacture of a medicament for treating or inhibiting hepatitis b virus infection.

4. Use of a compound of claim 1 for the manufacture of a medicament for the prophylactic treatment of hepatitis b virus infection.

5. Use of a compound of claim 1 for the manufacture of a medicament for reducing the recurrence of hepatitis b virus infection.

6. Use of a compound of claim 1 for the manufacture of a medicament for inducing remission of liver damage caused by hepatitis b virus infection.

7. Use of a compound of claim 1 for the manufacture of a medicament for reducing adverse physiological effects caused by hepatitis b virus infection.

8. The use according to any one of claims 3-7, wherein the compound is a pharmaceutically acceptable salt, ester, prodrug, complex, solvate, hydrate or isomer form.

9. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1 and a pharmaceutically acceptable carrier or excipient.

10. The pharmaceutical composition of claim 9, further comprising an additional therapeutic agent that is an HBV polymerase inhibitor, an interferon, or a reverse transcriptase inhibitor.