Patent Application
20230286930
The present invention relates to N-substituted 4-(1,3-aryloxazolo-2-yl)phenyl compounds of formula (I) and their corresponding derivatives useful for therapy and/or prophylaxis of HBV infection in a mammal, and in particular to HBsAg (HBV Surface antigen) and HBeAg (HBV e antigen) inhibitors as well as their manufacture and pharmaceutical compositions containing them.
The present invention relates to compounds of formula (I) wherein A1 to A4 and R1 are as described below, or a pharmaceutically acceptable salt thereof.
Hepatitis B virus (HBV) is one of the most dangerous human pathogens. A safe and effective vaccine has been available for longer than two decades; however, WHO estimated that approximately 257 million people are chronically infected with HBV. Chronic Hepatitis B (CHB) infection predisposes its host to severe liver disease, including liver cirrhosis and hepatocellular carcinoma, if left untreated. HBV infection is ranked among the top unmet medical need worldwide. The currently approved drugs have contributed to substantial progress in CHB treatment; however, the cure rate remains less than 10%.
The control of viral infection needs an effective immune surveillance. Upon recognition of viral infection, the host innate immune system could respond within minutes to impede viral replication and limits the development of a chronic and persistent infection. The secretion of antiviral cytokines from infected hepatocytes and intra-hepatic immune cells is critically important for the clearance of viral infection. However, chronically infected patients only display a weak immune response due to various escape strategies adopted by the virus to counteract the host cell recognition systems and the subsequent antiviral responses.
Many observations showed that several HBV viral proteins could counteract the initial host cellular response by interfering with the viral recognition signaling system and subsequently the interferon (IFN) antiviral activity. Among these, the excessive secretion of HBV empty subviral particles (SVPs, HBsAg) may contribute to immune tolerant state observed in CHB patients. The persistent exposure to HBsAg and other viral antigens can lead to HBV-specific T-cell functional impairment and depletion (Kondo et al. Journal of Immunology (1993), 150, 4659-4671; Kondo et al. Journal of Medical Virology (2004), 74, 425-433; Fisicaro et al. Gastroenterology, (2010), 138, 682-693;). Moreover, HBsAg has been reported to suppress immune cell functions, including monocytes, dendritic cells (DCs) and natural killer (NK) cells (Op den Brouw et al. Immunology, (2009b), 126, 280-289; Woltman et al. PLoS One, (2011), 6, e15324; Shi et al. J Viral Hepat. (2012), 19, e26-33; Kondo et al. ISRN Gasteroenterology, (2013), Article ID 935295).
HBsAg is an important biomarker for prognosis and treatment response in CHB. However, the achievement of HBsAg loss and seroconversion is rarely achieved in CHB patients. HBsAg loss with or without anti-HBsAg seroconversion remains the ideal clinical treatment endpoints. Current therapies, such as nucleos(t)ide analogues, are effective in supressing HBV DNA, but are not effective in reducing HBsAg level. Nucleos(t)ide analogs, even with prolonged therapy, have demonstrated HBsAg clearance rates comparable to those observed naturally (Janssen et al. Lancet, (2005), 365, 123-129; Marcellin et al. N. Engl. J. Med., (2004), 351, 1206-1217; Buster et al. Hepatology, (2007), 46, 388-394). Therefore, there is an urgent need for the development of novel therapeutic agents that could efficiently reduce HBsAg. (Wieland, S. F. & F. V. Chisari. J Virol, (2005), 79, 9369-9380; Kumar et al. J Virol, (2011), 85, 987-995; Woltman et al. PLoS One, (2011), 6, e15324; Op den Brouw et al. Immunology, (2009b), 126, 280-289).
Objects of the present invention are novel compounds of formula (I), their manufacture, medicaments based on a compound in accordance with the invention and their production as well as the use of compounds of formula (I) as HBV inhibitors and for the treatment or prophylaxis of HBV infection. The compounds of formula (I) show superior anti-HBV activity. In addition, the compounds of formula (I) also show good safety and good PK profiles.
The present invention relates to a compound of formula (I)
wherein
As used herein, the term “C1-6alkyl” alone or in combination signifies a saturated, linear- or branched chain alkyl group containing 1 to 6, particularly 2 to 6 or 1 to 4 carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl and the like. Particular “C1-6alkyl” groups are methyl, ethyl, propyl, isopropyl, isobutyl and tert-butyl.
The term “C1-6alkoxy” alone or in combination signifies a group C1-6alkyl-O—, wherein the “C1-6alkyl” is as defined above; for example methoxy, ethoxy, propoxy, iso-propoxy, n-butoxy, iso-butoxy, 2-butoxy, tert-butoxy, pentoxy, hexyloxy and the like. Particular “C1-6alkoxy” groups are methoxy, ethoxy, and propoxy.
The term “halogen” denotes fluoro, chloro, bromo, or iodo.
The compounds according to the present invention may exist in the form of their pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of formula (I) and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Acid-addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide. The chemical modification of a pharmaceutical compound into a salt is a technique well known to pharmaceutical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. It is for example described in Bastin R. J., et al., Organic Process Research & Development 2000, 4, 427-435. Particular are the sodium salts of the compounds of formula (I).
The present invention provides (i) a compound having the general formula (I):
wherein
A further embodiment of the present invention is (ii) a compound of formula (I) according to (i), wherein
A further embodiment of the present invention is (iii) a compound of formula (I) according to (i), or a pharmaceutically acceptable salt thereof, wherein A2 is CH.
A further embodiment of the present invention is (iv) a compound of formula (I) according to (i), or a pharmaceutically acceptable salt thereof, wherein A3 is CR4; wherein R4 is H or halogen.
A further embodiment of the present invention is (v) a compound of formula (I) according to (i), or a pharmaceutically acceptable salt thereof, wherein A3 is CR4; wherein R4 is H, F or Cl.
A further embodiment of the present invention is (vi) a compound of formula (I) according to (i), or a pharmaceutically acceptable salt thereof, wherein A4 is CR5; wherein R5 is H or halogen.
A further embodiment of the present invention is (vii) a compound of formula (I) according to (i), or a pharmaceutically acceptable salt thereof, wherein A4 is CR5; wherein R5 is H or F.
A further embodiment of the present invention is (viii) a compound of formula (I) according to (i), or a pharmaceutically acceptable salt thereof, wherein R1 is tetrahydrofuran-3-yl, 3-methyloxetan-3-yl, 1-hydroxy-1-methyl-ethyl, 1-bicyclo[1.1.1]pentanyl, 3-fluoro-1-bicyclo[1.1.1]pentanyl, 1,1-dioxothiolan-3-yl, 1,1-dioxothian-4-yl, tert-butyl, cyclopropyl, 3-methyltetrahydrofuran-3-yl, 8-oxabicyclo[3.2.1]octan-3-yl, 1,4-dioxan-2-yl, 1-hydroxyethyl, cyclobutyl, tetrahydropyran-4-yl, oxazol-2-yl, 5-methyloxazol-2-yl, 2-pyridyl or 4-methyloxazol-2-yl.
A further embodiment of the present invention is (ix) a compound of formula (I) according to (i), or a pharmaceutically acceptable salt thereof, wherein
A further embodiment of the present invention is (x) a compound of formula (I) according to (i), or a pharmaceutically acceptable salt thereof, wherein
In another embodiment (xi) of the present invention, particular compounds of the present invention are selected from:
In another embodiment (xii) of the present invention, particular compounds of the present invention are selected from:
The compounds of the present invention can be prepared by any conventional means. Suitable processes for synthesizing these compounds as well as their starting materials are provided in the schemes below and in the examples. All substituents, in particular, A1 to A4 and R1 are as defined above unless otherwise indicated. Furthermore, and unless explicitly otherwise stated, all reactions, reaction conditions, abbreviations and symbols have the meanings well known to a person of ordinary skill in organic chemistry.
wherein R2 is OH or halogen; LG is OH or halogen.
A compound of formula IV reacts with an acyl chloride V in the presence of a base, such as DIPEA, in a solvent, such as THF, to give a compound of formula VI. The compound of formula VI can also be formed with heating in the presence of a solvent such as pyridine. Cyclization of a compound of formula VI with POCl3, affords a compound of formula VII. The compound of formula VII can also be formed in the presence of CuI, N,N′-dimethylethylenediamine and a base, such as Cs2CO3, in a suitable solvent such as 1,4-dioxane. Hydrogenation of a compound of formula VII in the presence of a catalyst, such as palladium on carbon, under a hydrogen atmosphere, in a suitable solvent, such as MeOH or a mixed solution of THF and EtOH, affords a compound of formula IX. The compound of formula IX can also be prepared by reaction of a compound of formula IV and a carboxylic acid VIII in the presence of an acid, such as polyphosphoric acid. Then a compound of formula IX reacts with a compound of formula X in the presence of a coupling reagent, such as HATU, EDCI or T3P, and a base, such as TEA or DIPEA, in a solvent such as DMF or DCM, to afford a compound of formula I. The compound of formula I can also be prepared by reaction of a compound of formula IX and a compound of formula Xin the presence of a base, such as TEA or DIPEA, in a suitable solvent, such as DCM or pyridine. The compound of formula I can also be prepared by reaction of a compound of formula IX and a compound of formula X by heating under a neat condition.
This invention also relates to a process for the preparation of a compound of formula (I) comprising one of the following steps:
with a compound of formula (X),
in the presence of a base and optionally in the presence of a coupling reagent;
A compound of formula (I) when manufactured according to the above process is also an object of the invention.
The compound of this invention also shows good safety and PK profile.
The invention also relates to a compound of formula (I) for use as therapeutically active substance. Another embodiment provides pharmaceutical compositions or medicaments containing the compounds of the invention and a therapeutically inert carrier, diluent or excipient, as well as methods of using the compounds of the invention to prepare such compositions and medicaments. In one example, compounds of formula (I) may be formulated by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form. The pH of the formulation depends mainly on the particular use and the concentration of compound, but preferably ranges anywhere from about 3 to about 8. In one example, a compound of formula (I) is formulated in an acetate buffer, at pH 5. In another embodiment, the compounds of formula (I) are sterile. The compound may be stored, for example, as a solid or amorphous composition, as a lyophilized formulation or as an aqueous solution.
Compositions are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The “effective amount” of the compound to be administered will be governed by such considerations, and is the minimum amount necessary to reduction of HBsAg and HBeAg in HBV patients. For example, such amount may be below the amount that is toxic to normal cells, or the mammal as a whole.
In one example, the pharmaceutically effective amount of the compound of the invention administered parenterally per dose will be in the range of about 0.1 to 100 mg/kg, alternatively about 0.1 to 50 mg/kg of patient body weight per day, with the typical initial range of compound used being 0.3 to 15 mg/kg/day. In another embodiment, oral unit dosage forms, such as tablets and capsules, preferably contain from about 25 to about 1000 mg of the compound of the invention.
The compounds of the invention may be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
The compounds of the present invention may be administered in any convenient administrative form, e.g., tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain components conventional in pharmaceutical preparations, e.g., diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents.
A typical formulation is prepared by mixing a compound of the present invention and a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel, Howard C., et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. The formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament).
An example of a suitable oral dosage form is a tablet containing about 25 to 500 mg of the compound of the invention compounded with about 90 to 30 mg anhydrous lactose, about 5 to 40 mg sodium croscarmellose, about 5 to 30 mg polyvinylpyrrolidone (PVP) K30, and about 1 to 10 mg magnesium stearate. The powdered ingredients are first mixed together and then mixed with a solution of the PVP. The resulting composition can be dried, granulated, mixed with the magnesium stearate and compressed to tablet form using conventional equipment. An example of an aerosol formulation can be prepared by dissolving the compound, for example 5 to 400 mg, of the invention in a suitable buffer solution, e.g. a phosphate buffer, adding a tonicifier, e.g. a salt such sodium chloride, if desired. The solution may be filtered, e.g., using a 0.2 micron filter, to remove impurities and contaminants.
An embodiment, therefore, includes a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof.
In a further embodiment includes a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or excipient.
Another embodiment includes a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof for use in the treatment of HBV infection.
The compounds of the invention have anti-HBV activity. Accordingly, the compounds of the invention are useful for the treatment or prophylaxis of HBV infection.
The invention also relates to the use of a compound of formula (I) for the inhibition of HBeAg.
The invention further relates to the use of a compound of formula (I) for the inhibition of HBsAg.
The invention relates to the use of a compound of formula (I) for the inhibition of HBV DNA.
The invention relates to the use of a compound of formula (I) for use in the treatment or prophylaxis of HBV infection.
The use of a compound of formula (I) for the preparation of medicaments useful in the treatment or prophylaxis diseases that are related to HBV infection is an object of the invention.
The invention relates in particular to the use of a compound of formula (I) for the preparation of a medicament for the treatment or prophylaxis of HBV infection.
Another embodiment includes a method for the treatment or prophylaxis of HBV infection, which method comprises administering an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
The invention relates in particular to a compound of formula (I) for use in the treatment or prophylaxis of HBV infection.
The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.
Intermediates and final compounds were purified by flash chromatography using one of the following instruments: i) Biotage SP1 system and the Quad 12/25 Cartridge module. ii) ISCO combi-flash chromatography instrument. Silica gel Brand and pore size: i) KP-SIL 60 Å, particle size: 40-60 μm; ii) CAS registry NO: Silica Gel: 63231-67-4, particle size: 47-60 micron silica gel; iii) ZCX from Qingdao Haiyang Chemical Co., Ltd, pore: 200-300 or 300-400.
Intermediates and final compounds were purified by preparative HPLC on reversed phase column using X Bridge™ Perp C18 (5 μm, OBD™ 30×100 mm) column or SunFire™ Perp C18 (5 μm, OBD™ 30×100 mm) column.
Chiral Separation was conducted on Thar 350 preparative SFC using ChiralPak AD-10μ (200×50 mm I.D.) with mobile phase A for CO2 and B for ethanol. LC/MS spectra were obtained using a Waters UPLC-SQD Mass. Standard LC/MS conditions were as follows (running time: 3 minutes):
Acidic condition: A: 0.1% formic acid and 1% acetonitrile in H2O; B: 0.1% formic acid in acetonitrile;
Basic condition: A: 0.05% NH3·H2O in H2O; B: acetonitrile.
Mass spectra (MS): generally only ions which indicate the parent mass are reported, and unless otherwise stated the mass ion quoted is the positive mass ion (M+H)+.
NMR Spectra were obtained using Bruker Avance 400 MHz.
All reactions involving air-sensitive reagents were performed under an argon atmosphere. Reagents were used as received from commercial suppliers without further purification unless otherwise noted.
To PPA (10 g, 3.15 mmol) at 110° C. were added simultaneously 2-amino-5-fluorophenol (0.40 g, 3.15 mmol) and 4-aminobenzoic acid (0.43 g, 3.15 mmol). The resulting mixture was then heated to 180° C. for 3h. The solution was then poured into water. The resulting precipitate was collected by filtration and washed with water (20 mL), a mix solution of methanol and water (40 mL, methanol/water=1/3) and saturated aqueous NaHCO3 (40 mL). The filter cake was collected and purified by silica gel column eluted with DCM to DCM/MeOH=40/1 to give 4-(6-fluoro-1,3-benzoxazol-2-yl)aniline (496 mg) as a light yellow solid.
MS obsd. (ESI+) [(M+H)+]: 229.3. 1H NMR (400 MHz, DMSO-d6) δ: 7.80-7.86 (m, 2H), 7.64-7.69 (m, 2H), 7.19 (ddd, J=10.0, 8.8, 2.5 Hz, 1H), 6.66-6.72 (m, 2H), 6.01 (s, 2H).
The title compound was prepared in analogy to the procedure described for the preparation of Int-1, by using 2-amino-4-fluorophenol instead of 2-amino-5-fluorophenol. The product was purified by preparative HPLC to afford Int-2 as a light yellow solid.
MS obsd. (ESI+) [(M+H)+]: 229.6. 1H NMR (400 MHz, DMSO-d6) δ: 7.85 (d, J=7.6 Hz, 2H), 7.62-7.77 (m, 1H), 7.52 (d, J=7.6 Hz, 1H), 7.15 (s, 1H), 6.69 (d, J=7.3 Hz, 2H), 6.06 (br, 2H).
The title compound was prepared in analogy to the procedure described for the preparation of Int-1, by using 2-amino-5-chlorophenol instead of 2-amino-5-fluorophenol. The product was purified by preparative HPLC to afford Int-3 as a light yellow solid.
MS obsd. (ESI+) [(M+H)+]: 245.9. 1H NMR (400 MHz, DMSO-d6) δ: 7.82-7.87 (m, 3H), 7.66 (d, J=8.4 Hz, 1H), 7.37 (dd, J=8.4, 2.1 Hz, 1H), 6.69 (d, J=8.1 Hz, 2H), 6.06 (s, 2H).
The title compound was prepared in analogy to the procedure described for the preparation of Int-1, by using 2-amino-6-fluorophenol instead of 2-amino-5-fluorophenol. The product was purified by silica gel column to afford Int-4 as a light yellow solid.
MS obsd. (ESI+) [(M+H)+]: 229.3. 1H NMR (400 MHz, DMSO-d6) δ: 7.86-7.91 (m, 2H), 7.51 (dd, J=7.9, 0.9 Hz, 1H), 7.21-7.36 (m, 2H), 6.68-6.73 (m, 2H), 6.10 (s, 2H).
The title compound was prepared in analogy to the procedure described for the preparation of Int-1, by using 2-amino-3-fluorophenol instead of 2-amino-5-fluorophenol. The product was purified by silica gel column to afford Int-5 as a pink solid.
MS obsd. (ESI+) [(M+H)+]: 229.3. 1H NMR (400 MHz, DMSO-d6) δ: 7.85-7.91 (m, 2H), 7.55 (dd, J=8.1, 0.7 Hz, 1H), 7.32 (td, J=8.2, 5.1 Hz, 1H), 7.21 (t, J=9.3 Hz, 1H), 6.68-6.73 (m, 2H), 6.07 (s, 2H).
To a solution of 4-nitrobenzoic acid chloride (3.71 g, 20.0 mmol) and DIPEA (7.04 g, 54.5 mmol) in THF (30 mL) was added 3-amino-2-hydroxypyridine (2.0 g, 18.2 mmol). The mixture was stirred at 25° C. for 12 h. Then the resulting mixture was filtered and washed with MTBE (50 mL) to give N-(2-hydroxy-3-pyridyl)-4-nitro-benzamide (1.50 g) as a light grey solid.
MS obsd. (ESI+) [(M+H)+]:260.1
A mixture of N-(2-hydroxy-3-pyridyl)-4-nitro-benzamide (600.0 mg, 2.31 mmol) in POCl3 (10.0 mL) was heated to 120° C. for 12 h. The mixture was concentrated and then poured into ice-cold saturated aqueous NaHCO3. The resulting precipitate was collected by filtration and washed with ACN (40 mL). The solid was collected to afford 2-(4-nitrophenyl)oxazolo[5,4-b]pyridine (320 mg) as a light grey solid.
MS obsd (ESI+) [(M+H)+]: 242.2
To a solution of 2-(4-nitrophenyl)oxazolo[5,4-b]pyridine (150.0 mg, 0.620 mmol) in THF (10 mL) and ethanol (10 mL) was added Pd/C (6.6 mg, 0.060 mmol) under nitrogen atmosphere. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 balloon at 25° C. for 12 h. The mixture was then filtered to remove Pd/C and concentrated to afford the crude product. The crude product was purified by preparative HPLC to give 4-oxazolo[5,4-b]pyridin-2-ylaniline (Int-6) (29.6 mg) as a white solid.
MS obsd (ESI+) [(M+H)+]: 211.9. 1H NMR (400 MHz, DMSO-d6) δ: 8.24 (dd, J=4.9, 1.4 Hz, 1H), 8.08 (dd, J=7.8, 1.4 Hz, 1H), 7.89 (d, J=8.7 Hz, 2H), 7.41 (dd, J=7.7, 4.9 Hz, 1H), 6.71 (d, J=8.7 Hz, 2H), 6.14 (s, 1H), 6.21-6.06 (m, 1H).
To a solution of 4-nitrobenzoic acid chloride (1.78 g, 9.62 mmol) in pyridine (20 mL) was added 6-bromo-2,3-difluoro-aniline (1.0 g, 4.81 mmol) in one portion. The reaction mixture was stirred at 120° C. for 16 h and concentrated. The residue was diluted with DCM (50 mL) and was washed with saturated aqueous NaHCO3 (10 mL×2). The organic layer was concentrated to give the crude product. The crude product was purified by silica gel column (PE/EtOAc=5/1) to give N-(6-bromo-2,3-difluoro-phenyl)-4-nitro-benzamide (540 mg) as a white solid.
MS obsd. (ESI+)[(M+H)+]: 367.0.
To a solution of N-(6-bromo-2,3-difluoro-phenyl)-4-nitro-benzamide (300.0 mg, 0.840 mmol), N,N′-dimethylethylenediamine (14.8 mg, 0.170 mmol) and Cs2CO3 (547.4 mg, 1.68 mmol) in 1,4-dioxane (60 mL) was added copper(I) iodide (32.0 mg, 0.170 mmol) in one portion under nitrogen atmosphere. The reaction mixture was stirred at 80° C. for 16 h and filtered. The filtrate was concentrated to give the crude product. The crude product was purified by silica gel column (PE/EtOAc=15/1) to give 4,5-difluoro-2-(4-nitrophenyl)-1,3-benzoxazole (240 mg) as a white solid.
MS obsd. (ESI+)[(M+H)+]: 277.1.
To a solution of 4,5-difluoro-2-(4-nitrophenyl)-1,3-benzoxazole (480.0 mg, 1.74 mmol) in methanol (50 mL) was added palladium on carbon (184.9 mg, 0.170 mmol) in one portion under nitrogen atmosphere. The reaction system was degassed and purged with H2 (15 psi) and stirred at 20° C. for 16 h. The reaction mixture was filtered, and the filtrate was concentrated to give the crude product. The crude product was purified by preparative HPLC to give 4-(4,5-difluoro-1,3-benzoxazol-2-yl)aniline (Int-7) (185 mg) as a white solid.
MS obsd. (ESI+)[(M+H)+]: 247.1. 1H NMR (400 MHz, DMSO-d6) δ: 7.87 (d, J=8.7 Hz, 2H), 7.53 (ddd, J=1.1, 3.4, 8.9 Hz, 1H), 7.36 (ddd, J=7.8, 8.8, 11.5 Hz, 1H), 6.70 (d, J=8.8 Hz, 2H), 6.14 (s, 2H).
To a solution of 2, 4-difluorophenol (5.0 g, 38.4 mmol) in DCM (20 mL) was added nitric acid (5.0 mL) dropwise under nitrogen atmosphere at 0° C. After addition, the resulting reaction was stirred at 0° C. for 2 h. The mixture was poured into ice-water (20 mL), extracted with DCM (30 mL×3). The combined organic layer was washed with brine (30 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column to give 2, 4-difluoro-6-nitro-phenol (3.4 g) as a yellow solid.
A mixture of 2, 4-difluoro-6-nitro-phenol (3.4 g, 19.4 mmol) and Pd/C (0.34 g) in ethanol (50 mL) was stirred at 20° C. for 16 h under H2 atmosphere (1520 mmHg). The mixture was filtered and the filtrate was concentrated to give 2-amino-4, 6-difluoro-phenol (2.7 g) as a light yellow solid.
The title compound was prepared in analogy to the procedure described for the preparation of Int-1, by using 2-amino-4, 6-difluoro-phenol instead of 2-amino-5-fluorophenol. The product was purified by preparative HPLC to afford Int-8 as an off-white solid.
MS obsd. (ESI+) [(M+H)+]: 247.0. 1H NMR (400 MHz, DMSO-d6) δ: 7.87 (d, J=8.6 Hz, 2H), 7.44 (dd, J=8.6, 2.07 Hz, 1H), 7.31 (td, J=10.3, 2.26 Hz, 1H), 6.70 (d, J=8.8 Hz, 2H), 6.16 (s, 2H).
The title compound was prepared in analogy to the procedure described for the preparation of Int-7, by using 3-amino-2-chloro-6-picoline instead of 6-bromo-2,3-difluoro-aniline. The product was purified by silica gel column to afford Int-9 as a yellow solid.
MS obsd. (ESI+) [(M+H)+]: 226.0. 1H NMR (400 MHz, DMSO-d6) δ: 7.95 (d, J=7.9 Hz, 1H), 7.85 (d, J=8.7 Hz, 2H), 7.26 (d, J=8.0 Hz, 1H), 6.70 (d, J=8.7 Hz, 2H), 6.07 (s, 2H), 2.55 (s, 3H).
To a solution of 2-chloro-6-methoxy-3-nitropyridine (4.0 g, 21.2 mmol) and ammonium chloride (5.67 g, 106.1 mmol) in ethanol (100 mL) and water (20 mL) was added iron (5.92 g, 106.1 mmol) in one portion under nitrogen atmosphere. The reaction mixture was stirred at 70° C. for 3 h. The reaction mixture was filtered, and the filtrate was extracted with DCM (100 mL×3). The combined organic layers were dried over Na2SO4 and concentrated to give the crude product. The crude product was purified by silica gel column to give 2-chloro-6-methoxy-pyridin-3-amine (2.95 g) as yellow oil.
MS obsd. (ESI+)[(M+H)+]: 159.1.
The title compound was prepared in analogy to the procedure described for the preparation of Int-7, by using 2-chloro-6-methoxy-pyridin-3-amine instead of 6-bromo-2,3-difluoro-aniline. The product was purified by preparative HPLC to afford Int-10 as a yellow solid.
MS obsd. (ESI+) [(M+H)+]: 242.1. 1H NMR (400 MHz, DMSO-d6) δ: 8.02 (d, J=8.5 Hz, 1H), 7.81 (d, J=8.7 Hz, 2H), 6.83 (d, J=8.5 Hz, 1H), 6.69 (d, J=8.7 Hz, 2H), 5.99 (s, 2H), 3.91 (s, 3H).
The title compound was prepared in analogy to the procedure described for the preparation of Int-1, by using 2-amino-4-chlorophenol instead of 2-amino-5-fluorophenol. The product was purified by trituration to afford Int-11 as a light brown solid.
MS obsd. (ESI+) [(M+H)+]: 245.9. 1H NMR (400 MHz, DMSO-d6) δ: 7.82-7.90 (m, J=8.7 Hz, 2H), 7.74 (d, J=2.1 Hz, 1H), 7.70 (d, J=8.6 Hz, 1H), 7.34 (dd, J=8.6, 2.1 Hz, 1H), 6.66-6.74 (m, J=8.7 Hz, 2H), 6.07 (s, 2H).
The title compound was prepared in analogy to the procedure described for the preparation of Int-1, by using 6-amino-2,3-difluoro-phenol instead of 2-amino-5-fluorophenol The product was collected by filtration to afford Int-12 as a black solid.
MS obsd. (ESI+) [(M+H)+]: 247.0. 1H NMR (400 MHz, DMSO-d6) δ: 7.84 (d, J=8.7 Hz, 2H), 7.35-7.50 (m, 2H), 6.69 (d, J=8.7 Hz, 2H), 6.10 (s, 2H).
The title compound was prepared in analogy to the procedure described for the preparation of Int-7, by using 2-bromo-4,6-difluoro-aniline instead of 6-bromo-2,3-difluoro-aniline. The product was purified by silica gel column to afford Int-13 as a yellow solid.
MS obsd. (ESI+) [(M+H)+]: 247.0. 1H NMR (400 MHz, DMSO-d6) δ: 7.81-7.87 (m, 2H), 7.59 (dd, J=8.2, 2.0 Hz, 1H), 7.29 (td, J=10.5, 2.3 Hz, 1H), 6.67-6.73 (m, 2H), 6.10 (s, 2H).
The title compound was prepared in analogy to the procedure described for the preparation of Int-1, by using 2-amino-6-chlorophenol instead of 2-amino-5-fluorophenol. The product was purified by silica gel column to afford Int-14 as a yellow solid.
MS obsd. (ESI+) [(M+H)+]: 246.0. 1H NMR (400 MHz, DMSO-d6) δ: 7.84-7.90 (m, 2H), 7.64 (dd, J=7.7, 1.1 Hz, 1H), 7.31-7.42 (m, 2H), 6.68-6.73 (m, 2H), 6.10 (s, 2H).
A solution of 4-hydroxytetrahydro-2H-pyran-4-carboxylic acid (300.0 mg, 2.05 mmol) was mixed with pyridine (195.0 mg, 199 μL, 2.46 mmol) and DCM (10 mL). After cooling to 0° C., acetyl chloride (177.0 mg, 161 μL, 2.26 mmol) was added dropwise to the mixture. The resulting mixture was stirred at room temperature for 3 h. The reaction was diluted with DCM (15 mL) and washed with saturated aqueous NH4Cl (5 mL) and brine (5 mL). The organic phase was dried over Na2SO4 and concentrated in vacuum. The crude product (Int-15) was used for next step without further purification.
MS obsd. (ESI+) [(M−H)+]: 187.0.
The title compound was prepared in analogy to the procedure described for the preparation of Int-15, by using 1-hydroxycyclopropanecarboxylic acid instead of 4-hydroxytetrahydro-2H-pyran-4-carboxylic acid. The product Int-16 was used for next step without further purification.
To a solution of triphosgene (842.0 mg, 2.84 mmol) in DCM (2.5 mL) was added a solution of tetrahydrofuran-3-ol (500.0 mg, 5.68 mmol) and pyridine (978.0 mg, 1 mL, 12.4 mmol) in DCM (2.5 mL) dropwise at 0° C. The mixture became orange and a yellow precipitate appeared. The mixture was allowed to warm to room temperature and stirred for 1 h. Aqueous HCl (0.1N, 10 mL) was added and the organic layer was separated. The organic layer was washed with 0.1 N HCl (3×5 mL), brine (5 mL), dried over Na2SO4 and concentrated in vacuum to give light brown oil which became solid upon standing in the freezer for 24 h. The crude product Int-17 was used for next step without further purification.
To a solution of 4-(6-fluorobenzo[d]oxazol-2-yl)aniline (Int-1, as the “AMINE” in Table 1) (50.0 mg, 0.219 mmol) and tetrahydrofuran-3-carboxylic acid (30.5 mg, 0.263 mmol, as the “ACID” or “ACYL CHLORIDE” in Table 1) in DCM (2 mL) was added HATU (100.0 mg, 0.263 mmol) and DIPEA (56.6 mg, 76.5 μL, 0.438 mmol). Then the mixture was stirred at 50° C. for 18 h. The reaction mixture was concentrated in vacuum and the residue was triturated in water (5 mL). The resulting mixture was filtered and the filter cake was washed with a mixed solution of water (10 mL) and methanol (10 mL). The cake was collected and dried in vacuum to give N-[4-(6-fluoro-1,3-benzoxazol-2-yl)phenyl]tetrahydrofuran-3-carboxamide (Example 1)(69 mg) as a pink solid.
MS obsd. (ESI+) [(M+H)+]: 327.3. 1H NMR (400 MHz, DMSO-d6) δ: 10.40 (s, 1H), 8.12 (d, J=8.2 Hz, 2H), 7.75-7.87 (m, 4H), 7.28 (ddd, J=10.0, 8.7, 2.6 Hz, 1H), 3.96 (t, J=8.2 Hz, 1H), 3.70-3.83 (m, 3H), 3.16-3.30 (m, 1H), 2.05-2.17 (m, 2H).
The following Example 2 to Example 85 were prepared in analogy to the procedure described for the preparation of Example 1, replacing 4-(6-fluorobenzo[d]oxazol-2-yl)aniline (Int-1) with “AMINE”, tetrahydrofuran-3-carboxylic acid with “ACID” or “ACYL CHLORIDE”. The “AMINE”, “ACID” and “ACYL CHLORIDE” are the reagents indicated in Table 1.
1H NMR and (ESI+)
1H NMR (400 MHz, DMSO-d6)
1H NMR (400 MHz, DMSO-d6)
1H NMR (DMSO-d6,
1H NMR (DMSO-d6,
1H NMR (DMSO-d6, 400 MHz)
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1H NMR (DMSO-d6, 400 MHz)
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1H NMR (DMSO-d6, 400 MHz)
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1H NMR (DMSO-d6, 400 MHz)
1H NMR (DMSO-d6, 400 MHz)
1H NMR (DMSO-d6, 400 MHz)
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1H NMR (DMSO-d6, 400 MHz)
1H NMR (DMSO-d6, 400 MHz)
1H NMR (DMSO-d6, 400 MHz)
1H NMR (DMSO-d6, 400 MHz)
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1H NMR (DMSO-d6, 400 MHz)
1H NMR (DMSO-d6, 400 MHz)
1H NMR (DMSO-d6, 400 MHz)
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1H NMR (DMSO-d6, 400 MHz)
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1H NMR (DMSO-d6, 400 MHz)
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1H NMR (DMSO-d6, 400 MHz)
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The two enantiomers (Example 86-a, Example 86-b) were obtained through SFC [Instrument: Thar 200 preparative SFC (SFC-7), Phenomenex Lux Cellulose-2, 300×50 mm I.D., 10 μm; Mobile phase: A for CO2 and B for MEOH; Gradient: B 50%; Flow rate: 200 mL/min; Back pressure: 100 bar; Column temperature: 38° C.; elution order was Example 86-a, Example 86-b] chiral separation of N-(4-oxazolo[5,4-b]pyridin-2-ylphenyl)-1,1-dioxo-thiolane-3-carboxamide (Example 63).
Example 86-a: MS obsd. (ESI+) [(M+H)+]: 358.1. 1H NMR (DMSO-d6, 400 MHz) δ: 10.60 (s, 1H), 8.36 (d, J=5.0 Hz, 1H), 8.18-8.26 (m, 3H), 7.87 (d, J=8.7 Hz, 2H), 7.47-7.52 (m, 1H), 3.39-3.52 (m, 2H), 3.12-3.33 (m, 3H), 2.44-2.48 (m, 1H), 2.19-2.31 (m, 1H).
Example 86-b: MS obsd. (ESI+) [(M+H)+]: 358.1. 1H NMR (DMSO-d6, 400 MHz) δ: 10.60 (s, 1H), 8.36 (d, J=5.0 Hz, 1H), 8.18-8.26 (m, 3H), 7.87 (d, J=8.1 Hz, 2H), 7.50 (dd, J=7.8, 5.0 Hz, 1H), 3.39-3.52 (m, 2H), 3.12-3.32 (m, 3H), 2.44-2.48 (m, 1H), 2.19-2.31 (m, 1H).
The two enantiomers (Example 87-a, Example 87-b) were obtained through SFC [Instrument: SFC 80, Column: ChiralCel OJ, 250×20 mm I.D., 5 μm; Mobile phase: A for CO2 and B for Methanol (0.1% NH4OH); Gradient: B 40%; Flow rate: 50 mL/min; Back pressure: 100 bar; Column temperature: 35° C.; elution order was Example 87-a, Example 87-b] chiral separation of N-[4-(5-chloro-1,3-benzoxazol-2-yl)phenyl]tetrahydrofuran-3-carboxamide (Example 75).
Example 87-a: MS obsd. (ESI+) [(M+H)+]: 343.2. 1H NMR (DMSO-d6, 400 MHz) δ: 10.43 (s, 1H), 8.15 (d, J=8.1 Hz, 2H), 7.79-7.90 (m, 4H), 7.45 (d, J=8.7 Hz, 1H), 3.96 (t, J=8.3 Hz, 1H), 3.70-3.83 (m, 3H), 3.21 (quin, J=7.5 Hz, 1H), 2.07-2.17 (m, 2H).
Example 87-b: MS obsd. (ESI+) [(M+H)+]: 343.2. 1H NMR (DMSO-d6, 400 MHz) δ: 10.43 (s, 1H), 8.15 (d, J=8.1 Hz, 2H), 7.79-7.90 (m, 4H), 7.45 (d, J=8.7 Hz, 1H), 3.96 (t, J=8.2 Hz, 1H), 3.69-3.83 (m, 3H), 3.16-3.25 (m, 1H), 2.05-2.17 (m, 2H).
Detailed procedures regarding primary human hepatocyte (PHH) HBV natural infection assay are described as below. One tube of frozen PHH (10 million cells) is thawed in 37° C. water bath and then transferred to 20 mL of PHH thawing medium (Sigma, InVitroGRO HT Medium, Cat. S03319) with gently mixing. The cells were then centrifuged at 80 g/min for 5 min, the supernatant was discarded and the tube was refilled with 25 mL of PHH plating medium (Sigma, InVitroGRO CP Medium, Cat. S03317). The tube was shaken very gently to re-suspend all cells, and then 50 μL of cells were transferred to each well 384-well collagen I coated plate with appropriate liquid handling equipment, e.g. Integra VIAFLO384 or Agilent Bravo. The cells were then cultured for 24 hours in a cell incubator. For HBV infection, after PHH attachment on the culture plate, the plating medium was removed and replenished with PHH culture medium containing HBV virus. The PHH culture medium was prepared with Dulbecco's Modified Eagle Medium (DMEM)/F12 (1:1 in volume ratio) containing 10% fetal bovine serum (Gibco, Cat. 10099141), 5 ng/mL human epidermal growth factor (Gibco, Cat.PHG0311L), 20 ng/mL dexamethasone (Sigma, Cat.D4902-100 mg), 250 ng/mL human recombinant insulin (Gibco, Cat.41400045) and 100 U/mL penicillin. HBV virus at 200 genome equivalent (GE) per cell with 4% PEG8000 (Sigma, Cat.P1458) containing culture medium were added to the PHH culture medium for infection. The cells were then cultured for 24 hours in cell incubator. Then the cell culture supernatant was removed. The HBV-infected PHH were cultured with sandwich culture method with PHH culture medium containing 1% DMSO and 0.25 mg/mL matrix gel for 72 hours. The supernatant was then refreshed with PHH culture medium containing different concentrations of testing compounds for two times with 72-hour interval. At the end of treatment, the supernatant was collected for viral markers measurements, including HBsAg, HBeAg, HBV DNA and cytotoxicity. HBsAg and HBeAg were detected using alphalisa method using their specific antibodies. For HBV DNA detection, HBV DNA Quantitative Fluorescence Diagnostic Kit (Sansure Biotech Inc.) was used following the manufacture's protocol. Cytotoxicity was determined using Cell Counting Kit-8 (CCK8, Dojindo Molecular Technologies, Inc.).
The compounds of the present invention were tested for their capacity to inhibit HBsAg and HBeAg as described herein. The Examples were tested in the above assay and found to have IC50 below 10 μM. Results of PHH assay are given in Table 1.
This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.
It is to be understood that the invention is not limited to the particular embodiments and aspects of the disclosure described above, as variations of the particular embodiments and aspects may be made and still fall within the scope of the appended claims. All documents cited to or relied upon herein are expressly incorporated by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/130578 | Nov 2020 | WO | international |
This application is a continuation of International Application No. PCT/EP2021/082237 having an International Filing Date of Nov. 19, 2021 and which claims benefit under 35 U.S.C. § 119 to International Application No. PCT/CN2020/1350578 having an International Filing Date of Nov. 20, 2020. The entire contents of both are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2021/082237 | Nov 2021 | US |
| Child | 18318914 | US |
