Novel thiazole compound and its preparation method and use

Abstract

A novel thiazole compound and its prodrug, pharmaceutically acceptable salt, crystal and pharmaceutical composition thereof, as well as preparation method and use thereof. The compound can not only significantly improve biological activity, but also effectively reduce the loss of biological activity due to glycosylation, significantly increase the exposure of effective drugs, and expand its clinical application.

Description

Technical Field

[0001] The present invention relates to a novel thiazole compound and its prodrug, pharmaceutically acceptable salt, crystal and pharmaceutical composition, as well as preparation method and use thereof. Background Art

[0002] Viral infection has always been a major challenge to human health. Chronic hepatitis B virus (HBV) infection is a common cause of severe liver disease worldwide. In China, the number of new HBV infections is as high as 1 million per year, and the total number of infections is as high as 90 million. According to the World Health Organization (WHO), there are approximately 257 million HBV-infected patients worldwide. Each year, more than 650,000 people die from HBV infection-related end-stage liver disease, including liver failure, cirrhosis, and hepatocellular carcinoma (HCC). Currently, vaccines, nucleoside (NA) or nucleotide (NUC) drugs can effectively reduce the rate of new infections, and for HBV patients who adhere to long-term viral suppression therapy, they can delay the progression of liver disease. Hepatitis B surface antigen (HBsAg) negative conversion is associated with improved liver function, improved tissue pathology, and improved long-term prognosis. It is the ideal treatment goal recommended by the latest domestic and international chronic hepatitis B prevention and treatment guidelines, that is, the functional cure of hepatitis B or the goal of clinical cure. However, direct antiviral drugs (DAA) [such as nucleoside (acid) analogs (NA)] or immunomodulators [such as pegylated interferon α (PEG-IFN)] have limited effects in achieving clinical cure when used alone. In theory, NA and PEG-IFN play different antiviral roles against HBV, and their reasonable combination can produce synergistic and complementary effects, but the cure rate is still very low. At present, some drugs with new mechanisms of action have begun to enter clinical trials, such as immune checkpoint inhibitors (anti-PD-1 or anti-CTLA-4 therapy), capsid assembly inhibitors (AT-130, NVR-3778, JNJ6379, etc.), RNAi therapy (JNJ3989 or ARO-HBV) and inhibitors of apoptosis proteins (IAP, Inhibitors of Apoptosi s Proteins) inhibitors (such as APG-1387), etc. (Fanning et al., volume 18, pages 827–844 (2019)). None of these can achieve the goal of functional cure of hepatitis B. Therefore, it is necessary to discover more effective drugs for the treatment of hepatitis B and improve its functional cure rate.

[0003] Since the COVID-19 outbreak in the world at the end of 2019, as of March 17, 2021, the number of infections has reached 117 million and the death toll has exceeded 2.6 million, with no clear end in sight. Although several vaccines have been granted emergency use rights, no effective drugs have been officially approved for marketing. There is an urgent need to discover effective therapeutic drugs to address the current health crisis caused by COVID-19.

[0004] The incidence of fatty liver is increasing, along with the incidence of liver fibrosis caused by it, but there is no effective drug treatment. Therefore, there is an urgent need to find effective therapeutic drugs.

[0005] Recent studies have found that nitazoxanide has a broad-spectrum antiviral effect (J. Genetic Engineering and Biotechology 2020, 18, 1), and can effectively inhibit the replication of viruses including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), coronavirus (COVID-19), influenza virus (Influenza A / B), etc. Compound 1 has a significant inhibitory effect on multiple proteins of hepatitis B virus, including HBsAg, HBeAg, HBcAg, HBV RI, HBV RNA, cccDNA and HBV variants (Antiviral Res. 2008, 77, 56). Nitazoxanide can effectively kill multidrug-resistant Gram-negative bacteria (CID 2013, 56, 1685), significantly inhibit the new coronavirus (J. Infect. Dev. Ctries. 2020, 14, 982), is also effective in treating respiratory infections (DOI: 10.1093 / cid / ciz100), can effectively inhibit influenza virus (Antimicrob. Agents Chemother. 2015, 59, 1061), has broad-spectrum antiviral activity including anti-HIV activity (J. Genetic Engineer. Biotech. 2020, 18, 35), is effective against tuberculosis (J. Med. Chem. 2009, 52, 5789), is effective in treating fibrosis caused by fatty hepatitis (US Pat. 2018 / 0092885A1), and is also effective against various cancers (Mutation Research / Fundamental and Molecular Mechanisms of Mutagenesis, Volume 768, October 2014, Pages 16-21). Chinese patent CN201610329100.8 also discloses the use of compound 1 for Alzheimer's disease and Parkinson's disease.

[0006] Nitazoxanide and its metabolically active drug tizoxanide (2) have poor water solubility, with only 31.5% absorbed orally and 66.2% excreted from the stool (Int. J. Clin. Pharmacol. Ther. 2000, 38, 387). This is feasible for the treatment of gastrointestinal diseases. However, for systemic treatment of viral infections, the drug must be effectively absorbed to exert its drug effect. The preparation of prodrugs with basic groups can form salts, increase their water solubility, and may improve their bioavailability. Stachulski et al. reported that the bioavailability of amino acid ester hydrochloride (RM5061) increased from 2.8% of compound 1 to 20% (Eur. J. Med. Chem. 2017, 126, 154).

[0007] It is reported that the active ingredient of compound 1 is easily glycosylated to form compound 5 (TZG, Tizoxanide Glucuronide) with no biological activity, and the drug concentration in plasma is sometimes difficult to reach an effective therapeutic concentration. Any form of prodrug of compound 2 cannot change the property that its hydroxyl group is glycosylated and loses its biological activity.

[0008] According to literature reports, the activity of 2-methyl substituted compound (2-Me-2) in inhibiting HCV is significantly lower than that of compound 1, EC 90 The anti-HBV activity of 2-methyl substituted compounds (2-Me-2) was also significantly reduced, EC 50 Reduced by 4 times (J. Med. Chem. 2011, 54, 4119).

[0009] Summary of the invention

[0010] One of the objectives of the present invention is to provide a novel thiazole compound with stronger biological activity. Another objective of the present invention is to provide a novel thiazole compound that significantly reduces the glycosylation of the active ingredient or significantly increases the drug exposure.

[0011] The inventors found that RM5061 could not effectively increase the exposure of the drug. The inventors conceived that the H at the ortho position of compound 1 or 2 could be replaced by different substituents to prevent the glycosidation of the hydroxyl group and effectively increase the exposure of the drug. The inventors surprisingly found that the anti-HBsAg activity was improved after the ortho position was replaced by ethyl (I-1a-8) and isopropyl (I-1a-12). The EC values ​​of I-1a-8 and I-1a-12 were 50 They are 9.5 and 2.7 times that of compound 2. The drug exposure of compound I-1a-12 is 12.5 times that of compound 2 after oral administration in rats.

[0012]

[0013] On this basis, a compound of formula I is provided; a prodrug thereof; a pharmaceutically acceptable salt of the compound or its prodrug; or a crystal of the compound, the prodrug or the salt.

[0014]

[0015] in:

[0016] X is selected from NO2, Cl, Br;

[0017] R 1 Selected from, but not limited to: alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, heterocyclic or substituted heterocyclic, aryl or substituted aryl, alkoxy or substituted alkoxy, halogen, cyano, nitro, hydroxy, -OCOR', -OCOOR', -OCONHR', amino, -NHCOR', -NHCOOR', -NHCONHR', modified alkyl, amino acid ester

[0018] R 2 , R 3 , R 4 Each independently, but not limited to, is selected from: hydrogen, alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, heterocyclic or substituted heterocyclic, aryl or substituted aryl, alkoxy or substituted alkoxy, halogen, cyano, nitro, hydroxy, -OCOR', -OCOOR', -OCONHR', amino, -NHCOR', -NHCOOR', -NHCONHR', modified alkyl, amino acid ester

[0019] Optionally, R 1 , R 2 When it is an alkyl group, any one or more hydrogens on the alkyl group are replaced by deuterium;

[0020] Optionally, R 1 and R 2 , or R 2 and R 3 , or R 3 and R 4 Forming a 3-7 membered saturated or unsaturated ring;

[0021] R is selected from hydrogen, -COR', -COOR', -CONHR', amino, -NHCOR', -NHCOOR', -NHCONHR', alkyl, modified alkyl, or

[0022] Each R' is independently selected from, but not limited to, hydrogen, alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, heterocyclyl or substituted heterocyclyl, aryl or substituted aryl;

[0023] in The two R" are each independently selected from: hydrogen, alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, heterocyclyl or substituted heterocyclyl, aryl or substituted aryl;

[0024] The substituents in the substituted alkyl, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted aryl, and substituted heterocyclic group are independently selected from, but not limited to: 1-3 halogens, cyano, amino, nitro, hydroxyl, alkyl, alkoxy, modified alkyl, amino acid ester;

[0025] The amino acid ester groups include racemic and optically pure amino acid groups and pharmaceutically acceptable salts thereof.

[0026] The "halogen" is selected from: F, Cl, Br or I;

[0027] The "alkyl" in the "alkyl" and "alkoxy" is C 1-12 Straight or branched chain alkyl, optionally C 1-8 Straight or branched chain alkyl, optionally C 1-5 Straight or branched alkyl; can be selected from: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, or n-pentyl;

[0028] The "cycloalkyl" is C 3-8 monocyclic or bicyclic cycloalkyl;

[0029] The "alkenyl" is a C2-C6 alkenyl;

[0030] The "alkynyl" is a C2-C6 alkynyl;

[0031] The "heterocyclyl" is a 3-10 membered non-aromatic heterocyclic ring containing 1, 2 or 3 heteroatoms selected from N, O and S;

[0032] The "aryl" is a 6-10 membered aromatic group; it may be phenyl or naphthyl;

[0033] The "modified alkyl" is a group obtained by replacing any carbon atom of an alkyl group and, if present, the hydrogen atom thereon with one or more groups selected from -O-, -CO-, -NH2, -OH, halogen, -CN, -NO2;

[0034] The compound of formula I is not the following compounds 3 and 4:

[0035]

[0036] Optionally, any one or more hydrogens are replaced by deuterium.

[0037] Optionally,

[0038] R 1 Selected from, but not limited to: alkyl, halogen, cyano, nitro, or haloalkoxy;

[0039] R 2 Selected from, but not limited to: hydrogen, alkyl, or alkoxy;

[0040] R 3 Selected from, but not limited to: hydrogen, hydroxy, alkoxy, halogen, amino, or -NHCOR';

[0041] R 4 Selected from, but not limited to: hydrogen, hydroxy, alkoxy, -NHCOR', or amino;

[0042] R is selected from, but not limited to: hydrogen, -COR', -COOR', -CONHR', amino, -NHCOR', -NHCOOR', -NHCONHR',

[0043] Optionally,

[0044] Each R' and R" is independently selected from, but not limited to, hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, benzyl, sec-butyl, n-pentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, phenyl, and substituted phenyl.

[0045] Optionally, the compound of formula I; its prodrug; a pharmaceutically acceptable salt of the compound or its prodrug; or a crystal of the compound, the prodrug or the salt, the compound of formula I is selected from the following compounds I-1:

[0046]

[0047] Where: R 1 , R 2 , R 3 , R 4 and R are defined as above.

[0048] Optionally, the compound of formula I; its prodrug; the pharmaceutically acceptable salt of the compound or its prodrug; or the crystal of the compound, the prodrug or the salt, the compound of formula I is selected from compound I-1a or I-1b:

[0049]

[0050] In compounds I-1a and I-1b: R 1 , R 2 , R 3 and R 4 The definition of is as above.

[0051] Optionally, the compound of formula I; its prodrug; the pharmaceutically acceptable salt of the compound or its prodrug; or the crystal of the compound, the prodrug or the salt, the compound of formula I is selected from compound I-1a or I-1b:

[0052]

[0053] In compounds I-1a and I-1b:

[0054] R 1 Selected from, but not limited to: alkyl, halogen, cyano, nitro, or haloalkoxy;

[0055] R 2 Selected from, but not limited to: hydrogen, alkyl, or alkoxy;

[0056] R 3 Selected from, but not limited to: hydrogen, hydroxy, alkoxy, halogen, amino, or -NHCOR';

[0057] R 4 Selected from, but not limited to: hydrogen, hydroxy, alkoxy, -NHCOR', or amino;

[0058] Optionally,

[0059] R 1 Selected from: methyl, ethyl, isopropyl, F, Br, cyano, nitro, or trifluoromethyl;

[0060] R 2 Selected from: hydrogen, methyl, or methoxy;

[0061] R 3 is selected from: hydrogen, hydroxy, methoxy, Br, amino, or -NHAc;

[0062] R 4 Selected from: hydrogen, hydroxy, methoxy, -NHAc, or amino.

[0063] Optionally, the compound of formula I; its prodrug; the pharmaceutically acceptable salt of the compound or its prodrug; or the crystal of the compound, the prodrug or the salt, the compound of formula I is selected from compound I-1a or I-1b:

[0064]

[0065] Wherein, compounds I-1a and I-1b are selected from the following compounds I-1a-3 to I-1a-39, and I-1b-3 to I-1b-39, respectively:

[0066]

[0067]

[0068]

[0069] Optionally, the pharmaceutically acceptable salt comprises an anionic salt or a cationic salt of the compound of formula I;

[0070] Optionally, the pharmaceutically acceptable salts include metal salts and ammonium salts of the compound of formula I; the metal includes Na, K, Mg, Ca, Li, or Zn;

[0071] Optionally, the pharmaceutically acceptable salt includes a salt formed by the compound of formula I and an inorganic acid or an organic acid;

[0072] Optionally, the inorganic acid includes hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, or carbonic acid;

[0073] Optionally, the organic acid comprises formic acid, ascorbic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, citric acid, tartaric acid, gluconic acid, tartaric acid, glucuronic acid, carbonic acid, picric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzoic acid, benzenesulfonic acid, p-bromobenzenesulfonic acid, glutamic acid, salicylic acid, or pamoic acid;

[0074] Optionally, the pharmaceutically acceptable salt is the hydrochloride salt of the compound of formula I;

[0075] Optionally, the pharmaceutically acceptable salt is an NH4 salt or a metal salt formed with a phenolic hydroxyl group at the R group of the compound of formula I, and the metal is selected from, but not limited to, Na, K, Mg, Ca, Li, or Zn.

[0076] The present invention also provides crystalline forms A, B, C, D, E, F, and H of compound I-1a-12, wherein, using Cu-Kα radiation, the crystalline form A has characteristic peaks at diffraction angles of 2θ values ​​= 11.32°, 12.01°, 12.76°, 16.39°, 18.81°, 19.61°, 20.46°, 22.74°, 23.58°, 24.55°, 25.84°, 27.15°, 29.15°, and 35.24° in the X-ray powder diffraction pattern;

[0077] Using Cu-Kα radiation, the X-ray powder diffraction pattern of the crystalline form B has characteristic peaks at diffraction angles of 2θ values ​​= 7.70°, 9.32°, 13.04°, 15.41°, 16.31°, 23.25°, 25.15°, 25.57°, 31.62°, 39.20°, and 39.60°;

[0078] Using Cu-Kα radiation, the XRPD spectrum of the crystalline form C has characteristic peaks at diffraction angles of 2θ values ​​= 7.69°, 13.05°, 15.43°, 16.31°, 23.23°, 25.57°, 31.62°, and 39.61°;

[0079] Using Cu-Kα radiation, the X-ray powder diffraction pattern of the crystalline form D has characteristic peaks at diffraction angles of 2θ values ​​= 10.45°, 10.68°, 12.96°, 16.73°, 17.97°, 18.26°, 23.97°, 24.45°, and 25.38°;

[0080] Using Cu-Kα radiation, the XRPD spectrum of the crystalline form E has characteristic peaks at diffraction angles of 2θ values ​​= 9.63°, 10.08°, 12.40°, 13.33°, 23.38°, 23.89°, and 24.30°;

[0081] Using Cu-Kα radiation, the crystalline form F has characteristic peaks at diffraction angles of 2θ values ​​= 7.64°, 9.27°, 13.00°, 15.38°, 16.27°, 22.14°, 23.19°, 24.24°, 25.53°, 31.56°, and 39.55° in the X-ray powder diffraction pattern;

[0082] Using Cu-Kα radiation, the crystalline form H has characteristic peaks at diffraction angles of 2θ values ​​= 10.60°, 14.04°, 15.7°, 18.42°, 19.70°, 25.73°, and 26.44° in the X-ray powder diffraction pattern.

[0083] In another aspect, the present invention provides a compound of formula I; a prodrug thereof; a pharmaceutically acceptable salt of the compound or its prodrug; or a method for preparing a crystal of the compound, the prodrug or the salt, comprising the step of reacting compound II-1 with compound II-2 to obtain a compound of formula I;

[0084]

[0085] Among them, R, R 1 , R 2 , R 3 , R 4 and X are defined as above.

[0086] In another aspect, the present invention provides a pharmaceutical composition comprising the compound of formula I; a prodrug thereof; a pharmaceutically acceptable salt of the compound or its prodrug; or one or more crystals of the compound, the prodrug or the salt, and a pharmaceutically acceptable carrier;

[0087] Optionally, the dosage form of the pharmaceutical composition includes an oral solid preparation or a liquid preparation;

[0088] Optionally, the solid preparation includes tablets, powders, granules, or capsules;

[0089] Optionally, the liquid preparation includes water or saline or oil suspension, or syrup.

[0090] Optionally, the pharmaceutical composition further comprises an active compound other than the compound of formula I; a prodrug thereof; a pharmaceutically acceptable salt of the compound or its prodrug; or a crystal of the compound, the prodrug or the salt.

[0091] Another aspect of the present invention provides a compound of formula I as described above; a prodrug thereof; a pharmaceutically acceptable salt of the compound or its prodrug; a crystal of the compound, the prodrug or the salt; or a use of the pharmaceutical composition as described above in the preparation of a drug for preventing or treating viral infection.

[0092] Another aspect of the present invention provides a compound of formula I as described above; a prodrug thereof; a pharmaceutically acceptable salt of the compound or its prodrug; or a crystal of the compound, the prodrug or the salt; or use of the pharmaceutical composition as described above in the preparation of a drug for preventing or treating hepatitis B.

[0093] In another aspect, the present invention provides a compound of formula I as described above; a prodrug thereof; a pharmaceutically acceptable salt of the compound or its prodrug; or a crystal of the compound, the prodrug or the salt; or a use of the pharmaceutical composition as described above in the preparation of a drug for preventing or treating coronavirus infection;

[0094] Optionally, the coronavirus includes COVID-19, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.

[0095] Another aspect of the present invention provides a compound of formula I as described above; a prodrug thereof; a pharmaceutically acceptable salt of the compound or its prodrug; a crystal of the compound, the prodrug or the salt; or a use of the pharmaceutical composition as described above in the preparation of a drug for preventing or treating liver fibrosis.

[0096] Another aspect of the present invention provides a compound of formula I as described above; a prodrug thereof; a pharmaceutically acceptable salt of the compound or its prodrug; a crystal of the compound, the prodrug or the salt; or the pharmaceutical composition as described above in combination with other preparations, for use as a drug for preventing or treating diseases.

[0097] In another aspect, the present invention provides a method for preventing or treating viral infection, comprising administering to a patient in need thereof a preventive or therapeutically effective amount of the compound of formula I; a prodrug thereof; a pharmaceutically acceptable salt of the compound or its prodrug; a crystal of the compound, the prodrug or the salt; or the pharmaceutical combination described above.

[0098] In another aspect, the present invention provides a method for preventing or treating hepatitis B, comprising administering to a patient in need thereof an effective amount of the compound of formula I; a prodrug thereof; a pharmaceutically acceptable salt of the compound or its prodrug; or a crystal of the compound, the prodrug or the salt; or the pharmaceutical composition described above.

[0099] In another aspect, the present invention provides a method for preventing or treating coronavirus infection, comprising administering to a patient in need thereof an effective amount of the compound of formula I; a prodrug thereof; a pharmaceutically acceptable salt of the compound or its prodrug; or a crystal of the compound, the prodrug or the salt; or the pharmaceutical composition described above. Optionally, the coronavirus includes COVID-19, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.

[0100] In another aspect, the present invention provides a method for preventing or treating liver fibrosis, comprising administering to a patient in need thereof a preventive or therapeutically effective amount of the compound of formula I; a prodrug thereof; a pharmaceutically acceptable salt of the compound or its prodrug; a crystal of the compound, the prodrug or the salt; or the pharmaceutical combination described above.

[0101] Deuterated forms of any of the compounds of the present invention are within the scope of the present invention.

[0102] Beneficial technical effects:

[0103] In one aspect, Compound I or its prodrug or pharmaceutically acceptable salt or crystal thereof can significantly improve biological activity.

[0104] On the other hand, Compound I or its prodrug or their pharmaceutically acceptable salts or crystals can effectively reduce the loss of biological activity due to glycosylation, or significantly increase the exposure of effective drugs, thereby expanding clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1This is the XRPD spectrum of Form A

[0106] Figure 2 This is the XRPD spectrum of Form B

[0107] Figure 3 This is the XRPD spectrum of Form C

[0108] Figure 4 This is the XRPD spectrum of Form D

[0109] Figure 5 This is the XRPD spectrum of Form E

[0110] Figure 6 This is the XRPD spectrum of Form F

[0111] Figure 7 This is the XRPD spectrum of Form H

[0112] Figure 8 This is the DSC spectrum of Form H

[0113] Fig. 9 This is the TGA spectrum of Form H

[0114] Fig.10 It is the crystal form H 1 H NMR spectrum

[0115] Fig.11 This is the XRPD spectrum of Form B in stability study

[0116] Fig.12 This is the HPLC chart of the stability study of Form B

[0117] Fig.13 and Fig.14 This is the XRPD spectrum of Form H in the stability study

[0118] Fig.15 and Fig.16 This is the HPLC chart of the stability study of Form H.

[0119] Fig.17 This is the XRPD spectrum of Form B in the grinding and tableting test

[0120] Fig.18 This is the XRPD spectrum of Form H in the grinding and tableting test

[0121] Fig.19 This is the DVS test result of crystal form B

[0122] Fig. 20 This is the XRPD spectrum of Form B before and after DVS testing

[0123] Fig.21 This is the DVS test result of crystal form H

[0124] Fig. 22 This is the XRPD spectrum of Form H before and after DVS testing

[0125] Fig.23 This is the DVS retest result of another batch of Form H samples

[0126] Fig.24 This is the XRPD spectrum of another batch of Form H samples before and after DVS retesting

[0127] Fig.25 This is the standard curve of Form B

[0128] Fig.26 This is the XRPD spectrum of the filter cake sample after the solubility of Form B in water was tested.

[0129] Fig. 27 This is the HPLC chart of the solubility test of Form B in water

[0130] Fig.28 is the standard curve of Form H

[0131] Fig.29 This is the XRPD spectrum of the filter cake sample after the solubility of Form H in water was tested.

[0132] Fig.30 This is the HPLC chart of the solubility test of Form H in water

[0133] Fig.31 This is the solubility curve of crystal form B and crystal form H in water DETAILED DESCRIPTION

[0134] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate the present invention by way of example and are not used to limit the present invention.

[0135] Example 1 Preparation of Compounds

[0136] 1. Preparation of I-1a-2

[0137]

[0138] Add compound 1a (3.0 g, 17.8 mmol) to a dry round-bottom flask, then add glacial acetic acid (40 mL), slowly add acetic anhydride (6.8 mL, 71.4 mmol, 4.0 eq) and concentrated sulfuric acid (1.46 mL, 26.8 mmol, 1.5 eq) in turn at 0°C, move to room temperature and react overnight. Add water, extract with ethyl acetate, extract the aqueous phase with EA once, combine the organic phases, dry the organic phases with anhydrous sodium sulfate, concentrate, and vacuum dry to obtain a white solid, which is directly used in the next step with a yield of 80%. Compound 1b 1 H NMR (400MHz, CDCl3) δ7.67(d,J=7.9Hz,1H),7.30(t,J=8.0Hz,1H),7.22(d,J=8.2Hz,1H),3.89(s,3H),2.39(s,3H); m / z(ES+)(M+Na) + =233.1.

[0139] Add compound 1b (3.01 g, 14.3 mmol) to a dry round-bottom flask, then add ether (30 mL), pyridine (1.16 mL, 14.3 mmol, 1.0 eq) and thionyl chloride (1.25 mL, 17.2 mmol, 1.2 eq), and react at room temperature for one hour. Filter and remove the solvent, and the residue is directly used in the next step. The obtained acid chloride was dissolved in DMF (40 mL), 2-amino-5-nitrothiazole (2.08 g, 14.3 mmol, 1.0 eq) and triethylamine (12 mL, 85.9 mmol, 6.0 eq) were added, and the mixture was reacted at 60°C for three hours, quenched with 1M HCl at low temperature, extracted with ethyl acetate, and the organic solution was washed with 1M HCl to remove the unreacted 2-amino-5-nitrothiazole, and then washed with saturated sodium bicarbonate, water and saturated brine, respectively, dried over anhydrous sodium sulfate, concentrated, and recrystallized with petroleum ether: ethyl acetate = 4:1 to obtain a yellow solid I-1a-2 (123 mg). Compound I-1a-2 1 H NMR (400MHz, DMSO) δ: 13.60 (br s, 1H), 8.71 (s, 1H), 7.43-7.37 (m, 3H), 3.84 (s, 3H), 2.25 (s, 3H); m / z (ES+) (M+Na) + =360.1.2, the following compounds were prepared by the method for preparing compound I-1a-2:

[0140] Compound 1: 1H NMR(400MHz,DMSO)δ13.60(br s,1H),8.70(s,1H),7.67(d,J=7.7Hz,1H),7.59(d,J=7.6Hz,1H),7.36(t,J=7.7Hz,1H),2.28(s,3H),2.21(s,3H); m / z(ES+)(M+Na) + =344.0.

[0141] Compound I-1a-3: 1 H NMR(400MHz,DMSO)δ13.90(br s,1H),8.74(s,1H),8.37(dd,J=6.7,8.3Hz,1H),8.21(dd,J=6.3,7.8Hz,1H),7.73(t,J=8.0Hz,1H),2.32(s,3H); m / z(ES+)(MH) - =351.0.

[0142] Compound I-1a-4: 1 H NMR (400MHz, DMSO) δ13.80(br s,1H),8.71(s,1H),8.00(d,J=8.0Hz,1H),7.88(d,J=7.7Hz,1H),7.42(t,J=8.0Hz,1H),2.32(s,3H); m / z(ES+)(MH) - =385.9.

[0143] Compound I-1a-5: 1 H NMR (400MHz, DMSO) δ13.78(br s,1H),8.73(s,1H),7.72-7.67(m,2H),7.54-7.48(m,1H),2.33(s,3H); 19 F NMR (400MHz, CDCl3) δ-128.53; m / z (ES+) (MH)-=324.0.

[0144] Compound I-1a-6: 1 H NMR (400MHz, DMSO) δ13.83(br s,1H),8.73(s,1H),7.91(dd,J=6.4,7.8Hz,1H),7.85-7.82(m,1H),7.60(t,J=8.0Hz,1H),2.32(s,3H); 19 F NMR (400MHz, CDCl3) δ-57.07; m / z (ES+) (MH) - =390.0.

[0145] Compound I-1a-7: 1 H NMR (400MHz, DMSO) δ13.90 (br s, 1H), 8.74 (s, 1H), 8.24-8.21 (m, 2H), 7.69 (t, J = 7.8Hz, 1H), 2.38 (s, 3H); m / z (ES+) (MH) - =331.0.

[0146] Compound I-1a-8: 1 H NMR (400MHz, DMSO) δ13.60(br s,1H),8.71(s,1H),7.69-7.66(m,2H),7.43(t,J=7.8Hz,1H),2.28(s,3H),1.19(s,3H),1.17(s,3H); m / z(ES+)(MH) + =348.1.

[0147] Compound I-1a-9: 1 H NMR(400MHz,DMSO)δ13.53(br s,1H),8.70(s,1H),7.61(d,J=7.9Hz,1H),7.26(d,J=8.0Hz,1H),2.35(s,3H),2.29(s,3H),2.09(s,3H); m / z(ES+)(M+Na) + =358.0.

[0148] Compound I-1a-10: 1 H NMR (400MHz, DMSO) δ13.68(br s,1H),8.71(s,1H),7.90(d,J=2.2Hz,1H),7.84(d,J=1.8Hz,1H),2.28(s,3H),2.20(s,3H); m / z(ES+)(MH) - =399.9.

[0149] Compound I-1a-11: 1 H NMR(400MHz,DMSO)δ13.48(br s,1H),8.70(s,1H),7.69(d,J=8.8Hz,1H),7.14(d,J=8.9Hz,1H),3.93(s,3H),3.73(s,3H),2.30(s,3H); m / z(ES+)(M+Na) + =390.1.

[0150] Compound I-1a-12: 1H NMR(400MHz,DMSO)δ13.62(br s,1H),8.71(s,1H),7.68(d,J=7.6Hz,1H),7.61(d,J=7.6Hz,1H),7.40(t,J=7.7Hz, 1H),2.58(dd,J=7.6,15.2Hz,2H),2.28(s,3H),1.15(t,J=7.5Hz,3H); m / z(ES+)(MH) - =334.1.

[0151] Compound I-1b-12: Dissolve compound I-1a-12 (1.0 g, 2.98 mmol) in THF (5 mL), add 2 mL of concentrated hydrochloric acid, reflux for 16 h, cool to room temperature, add EA (100 mL) for extraction, wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, and purify the residue by silica gel column chromatography to obtain I-1b-12 (500 mg, 57%). 1 H NMR(400MHz,DMSO)δ8.75(s,1H),7.94(d,J=7.6Hz,1H),7.41(d,J=7.0Hz,1H),6 .91(t,J=7.6Hz,1H),2.64(q,J=7.4Hz,2H),1.17(t,J=7.5Hz,3H); m / z(ESI)[MH] - =292.1.

[0152] Example 2 Anti-HBV Activity Test

[0153] 1. Purpose of the experiment: The purpose of this experiment is to evaluate the in vitro anti-HBV activity of the compound. HepG2.2.15 cells were used to evaluate the in vitro anti-HBV activity of the test compound. The HBV DNA content in the supernatant of HepG2.2.15 cells was detected by real-time fluorescence quantitative PCR (qPCR), and the HBsAg content was detected by ELISA to determine the anti-HBV activity of the compound. At the same time, CellTiter-Glo detected the effect of the test compound on the viability of HepG2.2.15 cells. LAM was used as a reference compound in the experiment to monitor the quality of the experiment. The active compound was then used to verify the in vitro anti-HBV efficacy of the test compound using human primary hepatocytes (PHH).

[0154] 2. Materials

[0155] Test compounds and control compounds:

[0156] The test compound was provided by Henan Meitaibao Biopharmaceutical Co., Ltd. and prepared into a 20 mM stock solution using 100% DMSO.

[0157] Control compound: Lamivudine (LAM), provided by WuXi AppTec, prepared into a 20 mM stock solution using 100% DMSO.

[0158] 3. Cell lines

[0159] HepG2.2.15 cells were provided by WuXi AppTec.

[0160] 4. Reagents and consumables:

[0161] The main reagents used in this experiment include DNA extraction kit (Qiagen, catalog number 51162), HBsAg ELISA kit (catalog number: Antu-CL 0310), FastStart Universal Probe Master (Roche, catalog number 04914058001), CellTiter-Glo detection reagent (Promega, catalog number G7573), 96-well plate (Costar 3599) and 96-well V-plate (Axygen WIPP02280).

[0162] 5. Instruments:

[0163] The main instruments used in this experiment include a fluorescent qPCR instrument (Applied Biosystems, model QuantStudio(TM)6 Flex System) and an ELISA reader (BioTek, model Synergy 2).

[0164] 6. Test methods

[0165] 6.1 Cell plating and compound treatment

[0166] On day 0, HepG2.2.15 cells were seeded into 96-well plates at a density of 60,000 cells per well and cultured overnight at 37°C, 5% CO2. Fresh culture medium containing different concentrations of compounds was added on day 1, and fresh culture medium containing different concentrations of compounds was replaced every day from day 4, with a final DMSO concentration of 0.5%. The final test concentrations (μM) of the anti-HBV activity test compounds were: 100, 33.3, 11.1, 3.70, 1.23, 0.41, 0.14.

[0167] 6.2. Sample collection and testing

[0168] Antiviral activity test: On the 7th day, the cell supernatant was collected and DNA was extracted from the supernatant using a DNA extraction kit (Qiagen-51162). HBV DNA in the sample was quantified by qPCR. HBV plasmid DNA was used as a standard and serially diluted 10-fold. The standard range was from 1.0×10 7The PCR reaction program was: 95°C for 10 minutes, then enter the cycle mode, 95°C for 15 seconds, then 60°C for 1 minute, for a total of 40 cycles. The HBV DNA content in the sample was calculated based on the standard curve and the Ct value of each sample.

[0169] Refer to the instructions of Antu Biotechnology HBsAg ELISA kit and use an ELISA instrument to detect the HBsAg content.

[0170] 6.3 Data processing and statistical analysis

[0171] HBV DNA inhibition rate (%) = (HBV DNA content in DMSO control group - HBV DNA content in sample) / HBV DNA content in DMSO control group × 100%

[0172] HBsAg inhibition rate (%) = (1-HBsAg value of sample / HBsAg value of DMSO control group) × 100%

[0173] The data were processed using the GraphPad Prism software log(agonist) vs.response-Variable slope(fourparameters) analysis method to obtain EC 50 .

[0174] 6.4 Results and Conclusions

[0175] The results of the inhibition of the test compound and the control compound LAM on HBsAg and HBV DNA in the cell culture supernatant are shown in Table 1 below:

[0176] Table 1. Anti-HBV activity of compounds (EC 50 )

[0177]

[0178] The control compound LAM inhibited the EC of HBV DNA in HepG2.2.15 cells 50 The value is 0.11uM, which is in line with the expected results. Among the tested compounds, compounds I-1a-8, I-1a-9 and I-1a-12 have the same HBV DNA inhibition activity as compound 1, but their HBsAg inhibition activity is 2.5 to 9.5 times that of compound 1. Because existing clinical nucleoside hepatitis B drugs, such as entecavir and tenofovir alafenamide fumarate (TAF), can very effectively inhibit HBV DNA, but there are no drugs that effectively inhibit HBsAg on the market. Therefore, the discovery of I-1a-8, I-1a-9, I-1a-12 and their analogs is of great significance.

[0179] Example 3. Anti-new coronavirus activity test

[0180] (I) Experimental materials and reagents

[0181] 1. Cell line: Vero-6 cells, preserved in our laboratory;

[0182] 2. Virus strain: 2019-nCoV (COVID-19);

[0183] 3. Test drugs, representative compounds

[0184] 4. Positive control drugs: Remdesivir, Chloroquine and Lopinavir;

[0185] 5. Reagents: DMEM medium (Gibco), fetal bovine serum (Gibco), double antibody, trypsin, MTT (Amresco), etc.;

[0186] 6. Kits: QIAamp viral RNA mini kit (52906, Qiagen), One Step TB Green PrimeScript PLUS RT-PCR Kit (Perfect Real Time) (RR096A TaKaRa)

[0187] 7. Consumables: cell culture plates, 96-well ELISA plates, etc.

[0188] 8. Instruments: multifunctional microplate reader, StepOnePlus fluorescence quantitative PCR instrument, carbon dioxide incubator, etc.

[0189] (II) Experimental steps

[0190] 1. Cytotoxicity assay of representative compounds

[0191] The MTT method was used to detect the toxicity of the test compound to Vero-6 cells. The full name of MTT is 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide, which is a yellow dye. The MTT colorimetric method is a method for detecting cell survival and growth. The detection principle is that the succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in the cells, while dead cells do not have this function. 10% SDS (dissolved in 0.01 mol / L HCl solution) can dissolve the formazan in the cells, and its light absorption value is measured at a wavelength of 570nm using a multifunctional microplate reader, which can indirectly reflect the number of living cells. Within a certain range of cell numbers, the amount of MTT crystals formed is proportional to the number of cells. By detecting the light absorption value at a wavelength of 570nm under different interferon concentrations, the cell survival rate at the drug concentration can be calculated, thereby calculating the half toxic concentration (CC) of the drug. 50 ).

[0192] Vero-6 cells were seeded in a 96-well plate one day in advance, with 1×10 cells per well. 4 (Note that the wells at the edge of the 96-well plate should not be used as experimental wells, and PBS should be added to prevent the volatilization of the culture medium in other wells); observe the cell state, and when it reaches about 50%, dilute the test compound with DMEM culture medium containing 2% FBS by 2 times, and add it to the cell plate at 100 μL / well, with 6 replicates for each concentration; set up a control group (group without drug) and a blank group (group without cells) at the same time, and culture them in an incubator at 37°C and 5% CO2; 48 hours after drug addition, add 25 μL of MTT solution (5 mg / mL) to each well, continue to culture for 4 hours, add 125 μL of 10% SDS (dissolved in 0.01 mol / L HCl solution) to each well, gently blow, and place for 2 hours to fully dissolve the crystals, adjust to zero with the blank group, measure OD570, and calculate according to the following formula: Survival rate (%) = OD570 of drug group / OD570 of control group × 100%. At the same time, calculate the half toxic concentration (CC50) of the drug.

[0193] 2. Evaluation of the inhibitory effect of compounds on 2019-nCoV (COVID-19) virus

[0194] The antiviral activity was determined on the Vero-6 cell model, with 3 replicates per test, repeated 3 times in total.

[0195] 1) 5×10 cells were seeded in each well of a 24-well cell culture plate. 4Vero-6 cells were cultured at 37°C and 5% CO2. When the confluence reached 60%, 200 μL of 2019-nCoV (COVID-19) virus solution diluted with DMEM medium containing 2% FBS was added to each well according to the multiplicity of infection MOI of 0.005. After adsorption in a 37°C, 5% CO2 incubator for 1 hour, the virus solution was discarded, and the positive control drug and compound were diluted 2-fold from the maximum non-toxic concentration with DMEM medium containing 2% FBS, added to the cell plate at 500 μL / well, and a control group (drug-free group) was set up at the same time. The supernatant virus solution was collected 48 hours after infection.

[0196] 2) Quantify the RNA of the collected viruses using real-time RT-PCR (qRT-PCR):

[0197] 140 μL of the collected supernatant virus solution was taken and RNA was extracted according to the instructions of the QIAamp viral RNA mini kit. qRT-PCR detection was performed using the One Step TB Green PrimeScript PLUS RT-PCR Kit (Perfect RealTime).

[0198] The total volume of the reaction system was 20 μL: 10 μL 2X One Step TB Green RT-PCR Buffer 4, 1.2 μL TaKaRa Ex Taq HS Mix, 0.4 μL PrimeScript PLUS RTase Mix, 0.8 μL each of RBD-qF1 and RBD-qR1, 0.4 μL ROX Reference Dye (50X), 2 μL viral RNA, 4.4 μL RNase Free dH2O. The reaction parameters were: reverse transcription at 42°C for 5 min, pre-denaturation at 95°C for 10 s, and PCR for 40 cycles including denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s.

[0199] 3) Calculate the drug inhibition rate at each concentration. Inhibition rate (%) = 1-the number of viral RNA copies in the experimental group / the number of viral RNA copies in the drug-free group × 100%. At the same time, calculate the half effective concentration (EC 50 ) and therapeutic index (TI) = median toxic concentration (CC 50 ) / half effective concentration (EC 50 ).

[0200] (III) Calculation of experimental results (Table 2)

[0201] Table 2. Representative compounds inhibiting the activity of COVID-19

[0202]

[0203]

[0204] The results in Table 2 show that the anti-COVID-19 activity of the series of new compounds provided by the present invention is significantly improved compared with nitazoxanide (1). The anti-COVID-19 activity of compounds I-1a-12 and I-1b-12 (EC 50 The EC50 is 1.48 and 1.30uM respectively), which is 3.80 and 4.33 times that of compound 1 (EC50 is 5.63), and 5.33 and 6.1 times that of remdesivir.

[0205] Example 4: Animal Pharmacokinetics Test

[0206] The compounds are administered in the form of acetate prodrugs and are hydrolyzed by esterase in vivo to release active compounds, such as conversion of compound 1 to compound 2, and conversion of compounds I-1a-12 to compounds I-1b-12.

[0207]

[0208] The pharmacokinetic properties of the metabolites of the test compounds were determined in male SD rats after a single oral administration. Three male SD rats were given a single oral administration. Plasma samples were collected from all animals at 0.083h (5 minutes), 0.167h (10 minutes), 0.25h (15 minutes), 0.5h (30 minutes), 1h, 2h, 4h, 8h and 12h after administration. The concentration of metabolites (active ingredients) in plasma samples was determined by LC-MS / MS method.

[0209] 1. Drug administration

[0210] On the day of administration, the actual body weight of SD rats was weighed and the administration volume was calculated. In this experiment, the body weight of male SD rats at the time of administration ranged from 230.65 to 259.01 g. All SD rats were administered a single intragastric administration.

[0211] 2. Sample collection and preparation

[0212] About 0.2 mL of blood sample was collected from the jugular vein of the experimental animals / animal / time point, and the actual blood collection time was recorded. The points within 1 hour after administration were within the range of ±1 minute, and the other times were within 5% of the theoretical time as the acceptable deviation range.

[0213] After blood sample collection, it was immediately transferred to a labeled commercial sample tube containing K2-EDTA (0.85-1.15 mg) (Jiangsu Kangjian Medical Supplies Co., Ltd.), and then centrifuged (3200g, 4°C, 10 minutes) to obtain plasma. The plasma was transferred to a pre-cooled centrifuge tube, and 5 times the volume of ACN precipitate containing 6 internal standards was added, such as 100 μL plasma sample + 500 μL of ACN containing 6 internal standards. After precipitation, centrifuge (4°C, 12000rpm, 10 minutes) to obtain the supernatant (completed within 20 minutes after centrifugation to obtain plasma), quick-frozen in dry ice, and then stored in an ultra-low temperature refrigerator at -60°C or lower until LC-MS / MS analysis.

[0214] 3. Analysis methods

[0215] Plasma concentrations of Compound 2 and I-1b-12 were determined using LC-MS / MS methods.

[0216] 4. Pharmacokinetic data analysis

[0217] The plasma drug concentration data of compound 2 and I-1b-12 were processed using WinNonlin Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software with a non-compartmental model. The relevant pharmacokinetic parameters were calculated using the linear-log trapezoidal method (Table 3).

[0218] Table 3. Pharmacokinetic parameters of compound 1

[0219]

[0220]

[0221] The results in Table 3 show that after a single oral administration of 30 mg / kg of Compound 1 to male SD rats, the peak concentration (C max ) was 19498±3831nmol / L, and the peak time (T max ) appeared at 0.111±0.0485h after administration. AUC 0-last It is 8778±4722nmol·h / L.

[0222] Table 4. Pharmacokinetic parameters of compound I-1a-12

[0223]

[0224] The results in Table 4 show that after a single oral administration of 32.74 mg / kg of I-1a-12 to male SD rats, the peak concentration (C max ) was 46329±5890nmol / L, and the peak time (Tmax ) appears 0.500h after administration. AUC 0-last It is 109758±6007nmol·h / L.

[0225] Comparison of the pharmacokinetic parameters in Table 3 and Table 4 shows that the half-life of compound 2 (1.3 h) is comparable to that of I-1b-12 (1.22 h); the C max (46329) is 2.4 times that of compound 2 (19498); the AUC of compound I-1b-12 0-last (109758) is 12.5 times that of compound 2 (8778).

[0226] According to the results of the study on the activity and pharmacokinetics of inhibiting hepatitis B surface antigen (HBsAg), the above-mentioned example compounds not only have significantly improved antiviral activity (increased by 2.5-10 times), but also have greatly improved the exposure of effective drugs (increased by >10 times).

[0227] Study on the crystal form of compound I-1a-12

[0228] Crystalline Form A

[0229] The solid compound I-1a-12 (300 mg) was recrystallized from isopropanol (3 mL) and 2-MeTHF (1 mL) and filtered to obtain Form A. TGA, DSC and 1 The results of H-NMR studies show that Form A is anhydrous crystalline with a melting point of 176.75°C. Using Cu-Kα radiation, Form A has an X-ray powder diffraction pattern ( Figure 1 ) 2θ value = 11.32°, 12.01°, 12.76°, 16.39°, 18.81°, 19.61°, 20.46°, 22.74°, 23.58°, 24.07°, 24.55°, 24.97°, 25.84°, 27.15°, 29.15°, 29.86°, 30.53°, 35.24° diffraction angle has characteristic peaks, wherein the 2θ value error range is ± 0.2. More detailed peak data are shown in Tables 5 and Figure 1 .

[0230] Table 5. XRPD diffraction peak data of Form A

[0231] Pos.[°2θ] Rel.Int.[%] 11.32 26.9％ 12.01 32.7％ 12.76 25.4％ 16.39 27.5％ 18.81 65.3％ 19.61 41.0％ 20.46 37.4％ 22.74 47.3％ 23.58 100.0％ 24.07 16.9％ 24.55 39.0％ 24.97 13.1％ 25.84 20.1％ 27.15 53.0％ 29.15 41.7％ 29.86 15.6％ 30.53 11.0％ 35.24 25.0％

[0232] Form A is converted into Form B at 25°C or 30°C in different solvents.

[0233] Form B

[0234] Weigh about 300 mg of compound I-1a-12 solid into a 40 mL glass bottle, add hexane (6.0 mL) and EtOAc (4.0 mL), stir at 25°C for 3 days and then filter. After the filter cake is vacuum dried at 50°C for 12 hours, XRPD is measured and the resulting crystals are Form B.

[0235] After being placed at room temperature / sealed for 20 days, Form A transformed into Form B. TGA, DSC and 1 The results of H-NMR studies show that Form B is anhydrous crystalline. The melting point is 181.61°C. Using Cu-Kα radiation, the X-ray powder diffraction pattern of Form B has characteristic peaks at 2θ values ​​of 7.70°, 9.32°, 13.04°, 15.41°, 16.31°, 17.48°, 22.20°, 23.25°, 23.84°, 24.29°, 25.15°, 25.57°, 31.62°, 39.20°, and 39.60°, where the error range of 2θ value is ±0.2. More detailed peak data are shown in Tables 6 and Figure 2 .

[0236] Table 6. XRPD diffraction peak data of Form B

[0237] Pos.[°2θ] Rel.Int.[%] 7.70 54.0％ 9.32 26.3％ 13.04 51.8％ 15.41 99.4％ 16.31 100.0％ 17.48 11.1％ 22.20 16.8％ 23.25 74.4％ 23.84 16.5％ 24.29 15.9％ 25.57 26.8％ 31.62 25.0％ 39.20 27.0％ 39.60 36.1％

[0238] Crystal forms C, D and E

[0239] About 40 mg of Form A was weighed into a sample bottle, and the solvent shown in Table 7 was added to dissolve it at 50°C. It was then filtered, and the filtrate was cooled to about 25°C and stirred at 25°C. For samples that did not precipitate, stirring was continued for 1 day. Anti-solvent was added to samples that still did not precipitate. The results are summarized in Table 7. Form B, Form C, and Form D were obtained by cooling crystallization. Form D was vacuum dried at 50°C for 18 hours to convert to Form E.

[0240] Table 7. Cooling crystallization experimental parameters

[0241]

[0242] Crystalline Form C

[0243] Crystal form C was subjected to TGA, DSC, and NMR analysis, and its DSC results showed complex thermal phenomena. The TGA temperature corresponding to the melting point peak had a small step weight loss (0.5%), and the NMR results showed that the sample had 1.00% tetrahydrofuran remaining. Crystal form C was heated to 150°C by DSC (10°C / min) and kept at 150°C for 10 minutes. The heated sample was tested by XRPD and 1H NMR; The results show that the crystal form of Form C does not change after heating. The XRPD spectrum of Form C is similar to that of Form B. Form C is a solvent compound of THF or Form B containing residual THF solvent. Using Cu-Kα radiation, the XRPD spectrum of Form C has characteristic peaks at 2θ values ​​= 7.69°, 9.32°, 13.05°, 15.43°, 16.31°, 17.46°, 22.19°, 22.52°, 23.23°, 23.84°, 24.28°, 24.86°, 25.57°, 31.62°, 39.21°, and 39.61°, where the error range of 2θ value is ±0.2. More detailed peak data are shown in Tables 8 and Figure 3 .

[0244] Table 8. XRPD diffraction peak data of Form C

[0245] Pos.[°2θ] Rel.Int.[%] 7.69 35.0％ 9.32 19.7％ 13.05 34.3％ 15.43 72.3％ 16.31 100.0％ 17.46 12.1％ 22.19 18.7％ 22.52 3.2％ 23.23 33.7％ 23.84 14.8％ 24.28 17.0％ 24.86 12.6％ 25.57 20.6％ 31.62 24.2％ 39.21 15.2％ 39.61 30.0％

[0246] Crystal form D

[0247] TGA, DSC, and NMR analyses were performed on Form D. The DSC results showed complex thermal phenomena. Within the temperature range corresponding to the first endothermic peak, there was an 11.191% weight loss on the TGA curve. The NMR results showed that 11.52% of dioxane remained in the sample, indicating that Form D was a solvate, which would convert into Form E or Form G after vacuum drying at 50°C for 18 hours. Using Cu-Kα radiation, the X-ray powder diffraction pattern of Form D has characteristic peaks at diffraction angles of 2θ values ​​= 9.59°, 10.45°, 10.68°, 12.96°, 15.30°, 16.73°, 17.25°, 17.97°, 18.26°, 21.45°, 23.42°, 23.97°, 23.42°, 23.97°, 24.45°, 25.38°, 25.69°, 27.17°, 27.84°, 28.50°, 29.26°, 29.86°, 30.20°, and 30.84°, wherein the error range of 2θ value is ±0.2. More detailed peak data are shown in Tables 9 and Figure 4 .

[0248] Table 9. XRPD diffraction peak data of Form D

[0249] Pos.[°2θ] Rel.Int.[%] 9.59 18.2％ 10.45 55.4％ 10.68 100.0％ 12.96 35.9％ 15.30 17.2％ 16.73 30.7％ 17.25 12.3％ 17.97 70.5％ 18.26 35.2％ 21.45 14.4％ 23.42 16.7％ 23.97 73.9％ 24.45 33.9％ 25.38 81.1％ 25.69 10.2％ 27.17 18.9％ 30.84 16.4％

[0250] Crystalline Form E

[0251] The crystal form E was analyzed by TGA, DSC, NMR, etc., and it was confirmed that it was anhydrous crystal form. Its Onset was 177.42℃, and the peak temperature T peakThe sample was heated to 150°C by DSC (10°C / min) and kept at 150°C for 10 min. The heated sample was tested by XRPD and 1 H NMR. The results show that Form E turns into Form B after heating; very little dioxane solvent remains. Using Cu-Kα radiation, the XRPD spectrum of Form E has characteristic peaks at diffraction angles of 2θ values ​​= 9.63°, 10.08°, 12.40°, 13.33°, 14.10°, 16.14°, 23.38°, 23.89°, and 24.30°, where the error range of 2θ values ​​is ±0.2. More detailed peak data are shown in Tables 10 and Figure 5 .

[0252] Table 10. XRPD diffraction peak data of Form E

[0253]

[0254]

[0255] Form F

[0256] The crystal form F was analyzed by TGA, DSC, and NMR. Its Onset was 178.59°C, T peak The TGA spectrum corresponding to the DSC endothermic peak temperature showed a 1.1% weight loss, and the NMR results showed 0.76% dioxane residue. Form F was heated to 150°C by DSC (10°C / min) and kept at 150°C for 10 min. The heated sample was tested by XRPD and 1 H NMR. The results show that after heating, the crystal form F does not change, and there is residual dioxane solvent. The XRPD spectrum of crystal form F is similar to that of crystal form B. Crystal form F is a solvent compound or crystal form B containing residual solvent. Using Cu-Kα radiation, crystal form F has characteristic peaks at diffraction angles of 2θ values ​​= 7.64°, 9.27°, 12.42°, 13.00°, 15.38°, 16.27°, 17.42°, 19.48°, 22.14°, 23.19°, 23.79°, 24.24°, 24.81°, 25.53°, 31.56°39.17°, and 39.55° in the X-ray powder diffraction spectrum, and the error range of 2θ value is ±0.2. More detailed peak data are shown in Tables 11 and Figure 6 .

[0257] Table 11. XRPD diffraction peak data of Form F

[0258] Pos.[°2θ] Rel.Int.[%] 7.64 44.4％ 9.27 32.2％ 12.42 11.4％ 13.00 62.0％ 15.38 77.0％ 16.27 100.0％ 17.42 18.5％ 19.48 13.2％ 22.14 24.5％ 23.19 32.5％ 23.79 14.3％ 24.24 22.1％ 24.81 16.7％ 25.53 24.9％ 31.56 20.2％ 39.17 12.5％ 39.55 24.1％

[0259] Crystal form H

[0260] Preparation of Form H from Form B: Weigh about 20 mg of Form B into an 8 mL glass bottle, add ethanol (3.0 mL) to completely dissolve it, filter it after the solution is clear, stir it for 15 minutes to precipitate solids. Stir it for 6 hours and then filter it, and dry the filter cake in vacuum at 50°C for 14 hours; XRPD was measured, and the obtained crystals were Form H.

[0261] About 800 mg of Form B was weighed into a 250 mL round-bottom flask, 120.0 mL of ethanol was added to completely dissolve it at 50 °C, and then the temperature was naturally cooled to 25 °C in the air. A small amount of solid precipitated, and solvent n-heptane (60.0 mL) was added at a rate of 3.33 mL / min. After stirring for 1 h, the mixture was filtered and the filter cake was vacuum dried at 50 °C for 15 hrs. XRPD detection confirmed that the obtained crystals were Form H.

[0262] Form D / E was competitively slurried in n-heptane (25°C), or Form G was ground in ethanol for 3 minutes to obtain Form H.

[0263] The crystal form H was analyzed by TGA, DSC, and NMR, confirming that it is an anhydrous crystal form. Its Onset is 179.01℃, T peak The temperature of the sample was 181.10℃. TGA and NMR results showed that there was no residual solvent in the sample. XRPD used Cu-Kα radiation, and the crystal form H had characteristic peaks at the diffraction angles of 2θ values ​​= 9.74°, 10.60°, 14.04°, 14.49°, 15.7°, 18.42°, 19.70°, 20.02°, 25.73°, 26.44°, and 29.33° in the X-ray powder diffraction pattern, and the error range of the 2θ value was ±0.2. For more detailed data, see Tables 12 and Figures 7 to 10 .

[0264] Table 12. XRPD diffraction peak data of Form H

[0265] Pos.[°2θ] Rel.Int.[%] 9.74 12.2％ 10.60 45.3％ 14.04 57.7％ 14.49 10.0％ 15.75 29.6％ 18.42 49.3％ 19.70 23.4％ 20.02 16.6％ 25.73 100.0％ 26.44 33.3％ 29.33 14.3％

[0266] Stability study of crystal form B and crystal form H

[0267] Weigh appropriate amounts of Form B and Form H into 2 mL vials, place at 80°C for 1 day, and take samples at 0 and 1 day to analyze their physical and chemical stability. Place at 25°C / 60%RH and 40°C / 75%RH for 7 days, and take samples at 0, 1 and 7 days to analyze their physical and chemical stability.

[0268] The results show that the physical and chemical properties of Form B and Form H are stable after being placed at 80°C for one day and at 25°C / 60%RH and 40°C / 75%RH for 7 days. The results are shown in Tables 13, 14 and Figures 11 to 16 .

[0269] Table 13. Stability study of Form B and Form H

[0270]

[0271] Table 14. 80°C stability test of Form B and Form H

[0272]

[0273] Grinding and tableting experiments of Form B and Form H

[0274] In order to verify whether Form B and Form H are stable under grinding and tableting conditions, Form B and Form H were put into an agate mortar and ground for 3 minutes. After 3 minutes, their XRPD results were detected respectively. As follows: (1) Dry grinding, weigh about 20 mg, grind for 3 minutes, the crystal form did not change, and the crystallinity decreased slightly; (2) Wet grinding, weigh about 20 mg respectively, add 20 microliters of water and ethanol respectively, grind for 3 minutes, the crystal form did not change, and the crystallinity decreased slightly. After Form B and Form H were tableted at 20 MPa, the crystal form did not change, and the crystallinity decreased slightly. The specific results are as follows: Fig.17 and 18 shown.

[0275] DVS test of Form B and Form H

[0276] A DVS test was performed on Form B, and the results showed that the weight gain of the sample at 80% RH was 0.525%, so Form B was slightly hygroscopic; at the same time, XRPD was measured before and after the DVS test of Form B, and the results showed that the crystal form of the sample was consistent before and after the DVS test. A DVS test was performed on Form H, and the results showed that the weight gain of the sample at 80% RH was 0.007%, so Form H was not hygroscopic; XRPD was measured before and after the DVS test of Form H, and the results showed that the crystal form of the sample was consistent before and after the DVS test. Another batch of Form H samples was used to retest Form H with DVS, which further verified that Form H was not hygroscopic. The results are as follows: Figures 19 to 24 shown.

[0277] Solubility test of Form B in water

[0278] Preparation of standard solution: Weigh 10.12 mg of Form B into a 20 mL volumetric flask, add diluent (water-acetonitrile (V / V) = 1:1 solution), sonicate to dissolve the solid, and then add diluent to the mark. (The solution concentration is 506 μg / mL). The standard curve concentration is shown in Table 15 and Fig.25 .

[0279] Solubility test: Weigh about 20 mg of Form B and add 3 mL of water to prepare 2 parallel samples. Take samples at 1 hour, 4 hours, and 24 hours, and filter. The filtrate is tested by HPLC to calculate the solubility. The results are shown in Table 16. Fig.26 , Fig. 27 and Fig.31 shown.

[0280] Table 15. Standard curve data of Form B

[0281]

[0282] Table 16. Solubility test results of Form B in water

[0283]

[0284] Solubility test of Form H in water

[0285] Weigh about 20 mg of Form H and add 3 mL of water to prepare two parallel samples. Take samples at 1 hour, 4 hours, and 24 hours, and filter. The solubility of the obtained filtrate is tested by HPLC, and the XRPD of the filter cake sample after 24 hours is tested.

[0286] The concentrations of the standard curve are shown in Table 17 and Fig.28 .

[0287] The solubility test results are shown in Table 18. Figure 29 to Figure 31 shown.

[0288] Table 17 Standard curve data of Form H

[0289]

[0290] Table 18 Solubility test results of Form H in water

[0291]

[0292] Among the eight crystal forms, the stability and hygroscopic properties of crystal form B and crystal form H are relatively good. The solubility of crystal form B in water is 1.31 μg / mL, and the solubility of crystal form H reaches 4.98-8.19 μg / mL.

Claims

1. The following compound I-1a or compound I-1b, or a pharmaceutically acceptable salt thereof: In compounds I-1a and I-1b: R 1 is: alkyl; R 2 is: hydrogen, or alkyl; R 3 For: Hydrogen; R 4 For: Hydrogen; The "alkyl" is C 1-12 Straight or branched chain alkyl; And not the following compound 3, compound 5 2. Compound I-1a or Compound I-1b according to claim 1, or a pharmaceutically acceptable salt thereof: The "alkyl" is C 1-8 Straight chain or branched chain alkyl.

3. Compound I-1a or Compound I-1b according to claim 1, or a pharmaceutically acceptable salt thereof: The "alkyl" is C 1-5 Straight chain or branched chain alkyl.

4. Compound I-1a or Compound I-1b according to claim 1, or a pharmaceutically acceptable salt thereof: The "alkyl" is ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, or n-pentyl.

5. Compound I-1a or Compound I-1b according to claim 1, or a pharmaceutically acceptable salt thereof: in, The compounds I-1a and I-1b are selected from the following compounds:

6. The compound I-1a or compound I-1b according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, characterized in that: The pharmaceutically acceptable salts include anionic salts or cationic salts of Compound I-1a or Compound I-1b.

7. The compound I-1a or compound I-1b according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, characterized in that: The pharmaceutically acceptable salts include metal salts and ammonium salts of Compound I-1a or Compound I-1b; the metal includes Na, K, Mg, Ca, Li, or Zn.

8. The compound I-1a or compound I-1b according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, characterized in that: The pharmaceutically acceptable salts include salts formed by compound I-1a or compound I-1b and an inorganic acid or an organic acid; The inorganic acid includes hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, or carbonic acid; The organic acid includes formic acid, ascorbic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, citric acid, tartaric acid, gluconic acid, carbonic acid, picric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzoic acid, benzenesulfonic acid, p-bromobenzenesulfonic acid, glutamic acid, salicylic acid, or pamoic acid.

9. The compound I-1a or compound I-1b according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, characterized in that: The pharmaceutically acceptable salt includes the hydrochloride of Compound I-1a or Compound I-1b.

10. The compound I-1a or compound I-1b according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, characterized in that: The pharmaceutically acceptable salt is an NH4 salt or a metal salt formed at the -OH or -OAc group of the compound of formula I with a phenolic hydroxyl group, wherein the metal is selected from Na, K, Mg, Ca, Li, or Zn.

11. Form A of compound I-1a-12, characterized in that: Using Cu-Kα radiation, the crystalline form A has characteristic peaks at diffraction angles of 2θ values ​​= 11.32°, 12.01°, 12.76°, 16.39°, 18.81°, 19.61°, 20.46°, 22.74°, 23.58°, 24.55°, 25.84°, 27.15°, 29.15°, and 35.24° in the X-ray powder diffraction pattern.

12. Form B of compound I-1a-12, characterized in that: Using Cu-Kα radiation, the X-ray powder diffraction pattern of the crystalline form B has characteristic peaks at diffraction angles of 2θ values ​​= 7.70°, 9.32°, 13.04°, 15.41°, 16.31°, 23.25°, 25.15°, 25.57°, 31.62°, 39.20°, and 39.60°.

13. Form C of compound I-1a-12, characterized in that: Using Cu-Kα radiation, the XRPD spectrum of the crystalline form C has characteristic peaks at diffraction angles of 2θ values ​​= 7.69°, 13.05°, 15.43°, 16.31°, 23.23°, 25.57°, 31.62°, and 39.61°.

14. Form D of compound I-1a-12, characterized in that: Using Cu-Kα radiation, the X-ray powder diffraction pattern of the crystalline form D has characteristic peaks at diffraction angles of 2θ values ​​= 10.45°, 10.68°, 12.96°, 16.73°, 17.97°, 18.26°, 23.97°, 24.45°, and 25.38°.

15. Form E of compound I-1a-12, characterized in that: Using Cu-Kα radiation, the XRPD spectrum of the crystalline form E has characteristic peaks at diffraction angles of 2θ values ​​= 9.63°, 10.08°, 12.40°, 13.33°, 23.38°, 23.89°, and 24.30°.

16. Form F of compound I-1a-12, characterized in that: Using Cu-Kα radiation, the crystalline form F has characteristic peaks at diffraction angles of 2θ values ​​= 7.64°, 9.27°, 13.00°, 15.38°, 16.27°, 22.14°, 23.19°, 24.24°, 25.53°, 31.56°, and 39.55° in the X-ray powder diffraction pattern.

17. Form H of compound I-1a-12, characterized in that: Using Cu-Kα radiation, the crystalline form H has characteristic peaks at diffraction angles of 2θ values ​​= 10.60°, 14.04°, 15.7°, 18.42°, 19.70°, 25.73°, and 26.44° in the X-ray powder diffraction pattern.

18. Preparation of Compound I-1a or Compound I-1b according to any one of Claims 1 to 10, or a pharmaceutically acceptable salt thereof: It is characterized in that The method comprises reacting compound II-1 with compound II-2 to obtain compound I-1a or compound I-1b; Among them, R 1 , R 2 , R 3 , R 4 The definition is as described in any one of claims 1 to 10.

19. A pharmaceutical composition, characterized in that Comprising the compound according to any one of claims 1 to 17.

20. The pharmaceutical composition according to claim 19, characterized in that The dosage form of the pharmaceutical composition includes an oral solid preparation or a liquid preparation; The solid preparation includes tablets, powders, granules, or capsules; The liquid preparations include water or saline or oil suspensions, or syrups.

21. The pharmaceutical composition according to claim 19 or 20, characterized in that The pharmaceutical composition further comprises an active compound other than Compound I-1a or Compound I-1b or a pharmaceutically acceptable salt thereof.

22. Use of the compound according to any one of claims 1 to 17, or the pharmaceutical composition according to claim 19 or 20, in the preparation of a medicament for preventing or treating viral infection.

23. Use of the compound according to any one of claims 1 to 17; or the pharmaceutical composition according to claim 19 or 20 in the preparation of a medicament for preventing or treating hepatitis B.

24. Use of a compound according to any one of claims 1 to 17; or a pharmaceutical composition according to claim 19 or 20 in the preparation of a medicament for preventing or treating coronavirus infection.

25. The use according to claim 24, wherein the coronavirus comprises COVID-19, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV or MERS-CoV.

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