WO2022116998A1

WO2022116998A1 - Fused heterocyclic derivatives as hbv inhibitors

Info

Publication number: WO2022116998A1
Application number: PCT/CN2021/134799
Authority: WO
Inventors: Lianzhu LIU; Gang Deng; Chunliang Lu; Koen Vandyck; Zhiguo Liu; Tim Hugo Maria Jonckers; Sandrine Céline Grosse; Scott D Kuduk; Edgar Jacoby; Lindsey Graham DERATT; Pierre Jean-Marie Bernard Raboisson
Original assignee: Janssen Sciences Ireland Unlimited Company; Johnson & Johnson (China) Investment Ltd.
Priority date: 2020-12-02
Filing date: 2021-12-01
Publication date: 2022-06-09

Abstract

The application describes fused heterocycle derivative compounds, pharmaceutical compositions comprising these compounds, chemical processes for preparing these compounds and their use in the treatment of diseases associated with HBV infection.

Description

[Title established by the ISA under Rule 37.2] FUSED HETEROCYCLIC DERIVATIVES AS HBV INHIBITORS

FIELD

The application relates to fused heterocyclic derivative compounds, pharmaceutical compositions comprising these compounds, chemical processes for preparing these compounds and their use in the treatment of diseases associated with HBV infection.

BACKGROUND

Chronic hepatitis B virus (HBV) infection is a significant global health problem, affecting over 5%of the world population (over 350 million people worldwide and 1.25 million individuals in the U.S. ) .

Despite the availability of a prophylactic HBV vaccine, the burden of chronic HBV infection continues to be a significant unmet worldwide medical problem, due to suboptimal treatment options and sustained rates of new infections in most parts of the developing world. Current treatments do not provide a cure and are limited to only two classes of agents (interferon alpha and nucleoside analogues/inhibitors of the viral polymerase) ; drug resistance, low efficacy, and tolerability issues limit their impact. The low cure rates of HBV are attributed at least in part to the fact that complete suppression of virus production is difficult to achieve with a single antiviral agent. However, persistent suppression of HBV DNA slows liver disease progression and helps to prevent hepatocellular carcinoma. Current therapy goals for HBV-infected patients are directed to reducing serum HBV DNA to low or undetectable levels, and to ultimately reducing or preventing the development of cirrhosis and hepatocellular carcinoma.

The HBV capsid protein plays essential functions during the viral life cycle. HBV capsid/core proteins form metastable viral particles or protein shells that protect the viral genome during intercellular passage, and also play a central role in viral replication processes, including genome encapsidation, genome replication, and virion morphogenesis and egress. Capsid structures also respond to environmental cues to allow un-coating after viral entry. Consistently, the appropriate timing of capsid assembly and dis-assembly, the appropriate capsid stability and the function of core protein have been found to be critical for viral infectivity.

The crucial function of HBV capsid proteins imposes stringent evolutionary constraints on the viral capsid protein sequence, leading to the observed low sequence variability and high conservation. Consistently, mutations in HBV capsid that disrupt its assembly are lethal, and mutations that perturb capsid stability severely attenuate viral replication. The high functional constraints on the multi-functional HBV core/capsid protein is consistent with a high sequence conservation, as many mutations are deleterious to function. Indeed, the core/capsid protein sequences are >90%identical across HBV genotypes and show only a small number of polymorphic residues. Resistance selection to HBV core/capsid protein binding compounds may therefore be difficult to select without large impacts on virus replication fitness.

Reports describing compounds that bind viral capsids and inhibit replication of HIV, rhinovirus and HBV provide strong pharmacological proof of concept for viral capsid proteins as antiviral drug targets.

There is a need in the art for therapeutic agents that can increase the suppression of virus production and that can treat, ameliorate, and/or prevent HBV infection. Administration of such therapeutic agents to an HBV infected patient, either as monotherapy or in combination with other HBV treatments or ancillary treatments, will lead to significantly reduced virus burden, improved prognosis, diminished progression of the disease and enhanced seroconversion rates.

In view of the clinical importance of HBV, the identification of compounds that can increase the suppression of virus production and that can treat, ameliorate, and/or prevent HBV infection represents an attractive avenue into the development of new therapeutic agents. Such compounds are provided herein.

SUMMARY

The present disclosure is directed to the general and preferred embodiments defined, respectively, by the independent and dependent claims appended hereto, which are incorporated by reference herein. The present invention is directed to compounds capable of capsid assembly modulation. The compounds of the present invention may provide a beneficial balance of properties with respect to prior art compounds, e.g. they may display a different profile, display improved solubility, etc. Thus, in particular, the present disclosure is directed to a compound of Formula (I) :

or a stereoisomeric or a tautomeric form thereof, wherein

R ^1a represents halo;

R ^1b is selected from the group consisting of CN, CF ₃, CHF ₂, OCHF ₂ and OCF ₃;

R ^1c represents hydrogen or a substituent selected from the group consisting of halo, CF ₃, C _1-4alkyl and C _3-6cycloalkyl;

R ² is hydrogen or a substituent selected from the group consisting of CHF ₂, CF ₃, C _1- ₄alkyl, C _1-4alkylOC1-4alkyl and C _3-6cycloalkyl;

Q represents a ring selected from the group consisting of phenyl, a five-membered aromatic heterocyclic ring, and a six-membered aromatic heterocyclic ring;

n represents 1, 2 or 3;

each R ³ independently represents a substituent selected from the group consisting of CF ₃, CHF ₂, CH ₂F, C _1-6alkyl, OC _1-6alkyl, OCF ₃, OCHF ₂, and C _3-6cycloalkyl;

in the event that n represents 2 or 3, two R ³ on adjacent ring atoms, together with said ring atoms, optionally form a 5-membered ring or 6-membered ring, said ring optionally comprising 1, 2, or 3 heteroatoms each independently selected from N, O and S, said ring optionally carrying one or more fluoro or oxo substituents;

or a pharmaceutically acceptable salt or a solvate thereof,

with the proviso that the compound is not:

Further embodiments include pharmaceutically acceptable salts and solvates of compounds of Formula (I) , and stereoisomeric and tautomeric forms of the compounds of Formula (I) , as well as pharmaceutically acceptable salts thereof.

In embodiments, the compounds of Formula (I) are compounds selected from those species described or exemplified in the detailed description below.

The present disclosure is also directed to pharmaceutical compositions comprising one or more compounds of Formula (I) , and pharmaceutically acceptable salts and solvates of compounds of Formula (I) . Pharmaceutical compositions may further comprise one or more pharmaceutically acceptable excipients or one or more other agents or therapeutics.

The present disclosure is also directed to methods of using or uses of compounds of Formula (I) . In embodiments, compounds of Formula (I) are used to treat or ameliorate hepatitis B viral (HBV) infection, increase the suppression of HBV production, interfere with HBV capsid assembly or other HBV viral replication steps or products thereof. The methods comprise administering to a subject in need of such method an effective amount of at least one compound of Formula (I) , and pharmaceutically acceptable salts and solvates of compounds of Formula (I) . Additional embodiments of methods of treatment are set forth in the detailed description.

DETAILED DESCRIPTION

Additional embodiments, features, and advantages of the subject matter of the present disclosure will be apparent from the following detailed description of such disclosure and through its practice. For the sake of brevity, the publications, including patents, cited in this specification are herein incorporated by reference.

In one embodiment, provided herein are compounds of Formula (I) ,

or a stereoisomeric or a tautomeric form thereof, wherein

R ^1a represents halo;

R ² is hydrogen or a substituent selected from the group consisting of CHF ₂, CF ₃, C _1- ₄alkyl, C _1-4alkylOC _1-4alkyl and C _3-6cycloalkyl;

n represents 1, 2 or 3;

in the event that n represents 2 or 3, two R ³ on adjacent ring atoms, together with said ring atoms, optionally form a 5-membered ring or 6-membered ring, said ring optionally comprising 1, 2, or 3 heteroatoms each independently selected from N, O and S, said ring optionally carrying one or more fluoro or oxo subsituents;

or a pharmaceutically acceptable salt or a solvate thereof,

with the proviso that the compound is not:

In an additional embodiment, the present disclosure provides compounds of Formula (I) , and the stereoisomers or tautomers thereof, and the pharmaceutically acceptable salts thereof, as described herein, wherein Q is a phenyl or pyridyl ring.

In another embodiment, the present disclosure provides compounds of Formula (I) , and the stereoisomers or tautomers thereof, and the pharmaceutically acceptable salts thereof, as described herein, wherein n is 2, and wherein Q- (R ³) _n is an imidazopyridinyl group, such as a group satisfying formula (QR1) , with *indicating the attachment point of the group:

In a further embodiment, the present disclosure provides compounds of Formula (IA) , and the stereoisomers or tautomers thereof, and the pharmaceutically acceptable salts thereof, as described herein:

wherein W and X each independently are CR ³ or N, and wherein R ^1a, R ^1b, R ^1c, and R ², are as defined for Formula (I) , and wherein R ³ represents a substituent selected from the group consisting of CF ₃, CHF ₂, CH ₂F, C _1-6alkyl, OC _1-6alkyl, OCF ₃, OCHF ₂, and C _3-6cycloalkyl; or, in the event that W and X are both CR ³, the R ³ groups, together with the carbon atoms to which they are attached, form a 5-membered ring comprising 1, 2 or 3 heteroatoms each independently selected from N and O, said ring optionally carrying one or more fluor substituents.

In another embodiment, the present disclosure provides compounds of Formula (IB) , and the stereoisomers or tautomers thereof, and the pharmaceutically acceptable salts thereof, as described herein:

wherein R ^1a, R ^1b, R ^1c, and R ², are as defined for Formula (I) .

In another embodiment, the present disclosure provides compounds of Formula (IC) , and the stereoisomers or tautomers thereof, and the pharmaceutically acceptable salts thereof, as described herein:

wherein R ^1a, R ^1b, R ^1c, and R ², are as defined for Formula (I) , and wherein R ³ is selected from the group consisting of CF ₃, OCF ₂H, and OCF ₃. In an embodiment hereof, R ³ is in the meta or para position.

In a still further embodiment, the present disclosure provides compounds of any one of Formulae (I) , (IA) , (IB) , or (IC) , wherein R ^1a is chloro, R ^1b is cyano, and R ^1c is hydrogen.

In some embodiments, in the event that n represents 2 or 3, two R ³ on adjacent ring atoms, together with said ring atoms, optionally form a 5-membered ring or 6-membered ring, said ring optionally comprising 1, 2, or 3 heteroatoms each independently selected from N, O and S, said ring optionally carrying one or more fluoro or oxo substituents. It will be understood that in these embodiments the resulting ring is fused to ring Q. Said fused ring can be an unsaturated ring or an at least partially saturated ring, which unsaturated ring can be an aromatic or a non-aromatic ring. As indicated, said ring optionally comprises 1, 2, or 3 heteroatoms each independently selected from N, O and S. Accordingly, said fused ring optionally is selected from the group consisting of heteroaryl and heterocyclyl.

In an embodiment, the present disclosure provides compounds of any one of the formulae (I) , (IA) , (IB) , or (IC) , wherein R ² is H or CH ₃.

A further embodiment of the present disclosure is a compound selected from the group consisting of the compounds described in Table 1 below, a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt thereof.

TABLE 1

Pharmaceutical Compositions

Also disclosed herein are pharmaceutical compositions comprising (A) at least one compound of any one of Formula (I) , (IA) , (IB) , or (IC) , in any one of the

embodiments defined above, or a pharmaceutically acceptable salt thereof, and (B) at least one pharmaceutically acceptable excipient.

In embodiments, the pharmaceutical composition comprises at least one additional active or therapeutic agent. Additional active therapeutic agents may include, for example, an anti-HBV agent such as an HBV polymerase inhibitor, interferon, viral entry inhibitor, viral maturation inhibitor, capsid assembly modulator, reverse transcriptase inhibitor, immunomodulatory agent such as a TLR-agonist, or any other agents that affect the HBV life cycle and/or the consequences of HBV infection. The active agents of the present disclosure are used, alone or in combination with one or more additional active agents, to formulate pharmaceutical compositions of the present disclosure.

As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one compound useful within the present disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the present disclosure within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the present disclosure, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the present disclosure and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the present disclosure are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA) , which is incorporated herein by reference.

A “pharmaceutically acceptable excipient” refers to a substance that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to a subject, such as an inert substance, added to a pharmacological composition or otherwise used as a vehicle, carrier, or diluent to facilitate administration of an agent and that is compatible therewith. Examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

Delivery forms of the pharmaceutical compositions containing one or more dosage units of the active agents may be prepared using suitable pharmaceutical excipients and compounding techniques known or that become available to those skilled in the art. The compositions may be administered in the inventive methods by a suitable route of delivery, e.g., oral, parenteral, rectal, topical, or ocular routes, or by inhalation.

The preparation may be in the form of tablets, capsules, sachets, dragees, powders, granules, lozenges, powders for reconstitution, liquid preparations, or suppositories. Preferably, the compositions are formulated for intravenous infusion, topical administration, or oral administration.

For oral administration, the compounds of the present disclosure can be provided in the form of tablets or capsules, or as a solution, emulsion, or suspension. To prepare the oral compositions, the compounds may be formulated to yield a dosage of, e.g., from about 0.05 to about 100 mg/kg daily, or from about 0.05 to about 35 mg/kg daily, or from about 0.1 to about 10 mg/kg daily. For example, a total daily dosage of about 5 mg to 5 g daily may be accomplished by dosing once, twice, three, or four times per day.

Oral tablets may include a compound according to the present disclosure mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservative agents. Suitable inert fillers include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, and the like. Exemplary liquid oral excipients include ethanol, glycerol, water, and the like. Starch, polyvinyl-pyrrolidone (PVP) , sodium starch glycolate, microcrystalline cellulose, and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin. The lubricating agent, if present, may be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract or may be coated with an enteric coating.

Capsules for oral administration include hard and soft gelatin capsules. To prepare hard gelatin capsules, compounds of the present disclosure may be mixed with a solid, semi-solid, or liquid diluent. Soft gelatin capsules may be prepared by mixing the compound of the present disclosure with water, an oil such as peanut oil or olive oil, liquid paraffin, a mixture of mono and di-glycerides of short chain fatty acids, polyethylene glycol 400, or propylene glycol.

Liquids for oral administration may be in the form of suspensions, solutions, emulsions or syrups or may be lyophilized or presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid compositions may optionally contain: pharmaceutically-acceptable excipients such as suspending agents (for example, sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel and the like) ; non-aqueous vehicles, e.g., oil (for example, almond oil or fractionated coconut oil) , propylene glycol, ethyl alcohol, or water; preservatives (for example, methyl or propyl p-hydroxybenzoate or sorbic acid) ; wetting agents such as lecithin; and, if desired, flavoring or coloring agents.

The active agents of this present disclosure may also be administered by non-oral routes. For example, the compositions may be formulated for rectal administration as a suppository. For parenteral use, including intravenous, intramuscular, intraperitoneal, or subcutaneous routes, the compounds of the present disclosure may be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity or in parenterally acceptable oil. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Such forms will be presented in unit-dose form such as ampules or disposable injection devices, in multi-dose forms such as vials from which the appropriate dose may be withdrawn, or in a solid form or pre-concentrate that can be used to prepare an injectable formulation. Illustrative infusion doses may range from about 1 to 1000 μg/kg/minute of compound, admixed with a pharmaceutical carrier over a period ranging from several minutes to several days.

For topical administration, the compounds may be mixed with a pharmaceutical carrier at a concentration of about 0.1%to about 10%of drug to vehicle. Another mode of administering the compounds of the present disclosure may utilize a patch formulation to affect transdermal delivery.

Compounds of the present disclosure may alternatively be administered in methods of this present disclosure by inhalation, via the nasal or oral routes, e.g., in a spray formulation also containing a suitable carrier.

Methods of Use

The disclosed compounds are useful in the prevention or treatment of an HBV infection or of an HBV-induced disease in mammal in need thereof, more particularly in a human in need thereof.

In a non-limiting aspect, these compounds may (i) modulate or disrupt HBV assembly and other HBV core protein functions necessary for HBV replication or the generation of infectious particles, (ii) inhibit the production of infectious virus particles or infection, or (iii) interact with HBV capsid to effect defective viral particles with reduced infectivity or replication capacity acting as capsid assembly modulators. In particular, and without being bound to any particular mechanism of action, it is believed that the disclosed compounds are useful in HBV treatment by disrupting, accelerating, reducing, delaying and/or inhibiting normal viral capsid assembly and/or disassembly of immature or mature particles, thereby inducing aberrant capsid morphology leading to antiviral effects such as disruption of virion assembly and/or disassembly, virion maturation, virus egress and/or infection of target cells. The disclosed compounds may act as a disruptor of capsid assembly interacting with mature or immature viral capsid to perturb the stability of the capsid, thus affecting its assembly and/or disassembly. The disclosed compounds may perturb protein folding and/or salt bridges required for stability, function and/or normal morphology of the viral capsid, thereby disrupting and/or accelerating capsid assembly and/or disassembly. The disclosed compounds may bind capsid and alter metabolism of cellular polyproteins and precursors, leading to abnormal accumulation of protein monomers and/or oligomers and/or abnormal particles, which causes cellular toxicity and death of infected cells. The disclosed compounds may cause failure of the formation of capsids of optimal stability, affecting efficient uncoating and/or disassembly of viruses (e.g., during infectivity) . The disclosed compounds may disrupt and/or accelerate capsid assembly and/or disassembly when the capsid protein is immature. The disclosed compounds may disrupt and/or accelerate capsid assembly and/or disassembly when the capsid protein is mature. The disclosed compounds may disrupt and/or accelerate capsid assembly and/or disassembly during viral infectivity which may further attenuate HBV viral infectivity and/or reduce viral load. The disruption, acceleration, inhibition, delay and/or reduction of capsid assembly and/or disassembly by the disclosed compounds may eradicate the virus from the host organism. Eradication of HBV from a subject by the disclosed compounds advantageously obviates the need for chronic long-term therapy and/or reduces the duration of long-term therapy.

An additional embodiment of the present disclosure is a method of treating a subject suffering from an HBV infection, comprising administering to a subject in need of such treatment an effective amount of at least one compound of Formula (I) .

In another aspect, provided herein is a method of reducing the viral load associated with an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound of Formula (I) , or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method of reducing reoccurrence of an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound of Formula (I) , or a pharmaceutically acceptable salt thereof.

Additionally, HBV acts as a helper virus to hepatitis delta virus (HDV) , and it is estimated that more than 15 million people may be HBV/HDV co-infected worldwide, with an increased risk of rapid progression to cirrhosis and increased hepatic decompensation, than patients suffering from HBV alone (Hughes, S.A. et al. Lancet 2011, 378, 73-85) . HDV, infects therefore subjects suffering from HBV infection. In a particular embodiment, the compounds of the invention may be used in the treatment and/or prophylaxis of HBV/HDV co-infection, or diseases associated with HBV/HDV co infection. Therefore, in a particular embodiment, the HBV infection is in particular HBV/HDV co-infection, and the mammal, in particular the human, may be HBV/HDV co-infected, or be at risk of HBV/HDV co infection.

In another aspect, provided herein is a method of inhibiting or reducing the formation or presence of HBV DNA-containing particles or HBV RNA-containing particles in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound of Formula (I) , or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method of reducing an adverse physiological impact of an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound of Formula (I) , or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method of inducing remission of hepatic injury from an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound of Formula (I) , or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method of reducing the physiological impact of long-term antiviral therapy for HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound of Formula (I) , or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method of prophylactically treating an HBV infection in an individual in need thereof, wherein the individual is afflicted with a latent HBV infection, comprising administering to the individual a therapeutically effective amount of a compound of Formula (I) , or a pharmaceutically acceptable salt thereof.

In embodiments, the disclosed compounds are suitable for monotherapy. In embodiments, the disclosed compounds are effective against natural or native HBV strains. In embodiments, the disclosed compounds are effective against HBV strains resistant to currently known drugs.

In another embodiment, the compounds provided herein can be used in methods of modulating (e.g., inhibiting or disrupting) the activity, stability, function, and viral replication properties of HBV cccDNA.

In yet another embodiment, the compounds of the present disclosure can be used in methods of diminishing or preventing the formation of HBV cccDNA.

In another embodiment, the compounds provided herein can be used in methods of modulating (e.g., inhibiting or disrupting) the activity of HBV cccDNA.

In yet another embodiment, the compounds of the present disclosure can be used in methods of diminishing the formation of HBV cccDNA.

In another embodiment, the disclosed compounds can be used in methods of modulating, inhibiting, or disrupting the generation or release of HBV RNA particles from within the infected cell.

In a further embodiment, the total burden (or concentration) of HBV RNA particles is modulated. In a preferred embodiment, the total burden of HBV RNA is diminished.

In another embodiment, the methods provided herein reduce the viral load in the individual to a greater extent or at a faster rate compared to the administering of a compound selected from the group consisting of an HBV polymerase inhibitor, interferon, viral entry inhibitor, viral maturation inhibitor, distinct capsid assembly modulator, antiviral compounds of distinct or unknown mechanism, and any combination thereof.

In another embodiment, the methods provided herein cause a lower incidence of viral mutation and/or viral resistance than the administering of a compound selected from the group consisting of an HBV polymerase inhibitor, interferon, viral entry inhibitor, viral maturation inhibitor, distinct capsid assembly modulator, antiviral compounds of distinct or unknown mechanism, and combination thereof.

In another embodiment, the methods provided herein further comprise administering to the individual at least one HBV vaccine, a nucleoside HBV inhibitor, an interferon or any combination thereof.

In an aspect, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising reducing the HBV viral load by administering to the individual a therapeutically effective amount of a compound of Formula (I) , or a pharmaceutically acceptable salt thereof, alone or in combination with a reverse transcriptase inhibitor; and further administering to the individual a therapeutically effective amount of HBV vaccine.

In an embodiment, the methods provided herein further comprise monitoring the HBV viral load of the subject, wherein the method is carried out for a period of time such that the HBV virus is undetectable.

Combinations

Provided herein are combinations of one or more of the disclosed compounds with at least one additional therapeutic agent. In embodiments, the methods provided herein can further comprise administering to the individual at least one additional therapeutic agent. In embodiments, the disclosed compounds are suitable for use in combination therapy. The compounds of the present disclosure may be useful in combination with one or more additional compounds useful for treating HBV infection. These additional compounds may comprise compounds of the present disclosure or compounds known to treat, prevent, or reduce the symptoms or effects of HBV infection.

In an exemplary embodiment, additional active ingredients are those that are known or discovered to be effective in the treatment of conditions or disorders involved in HBV infection, such as another HBV capsid assembly modulator or a compound active against another target associated with the particular condition or disorder involved in HBV infection, or the HBV infection itself. The combination may serve to increase efficacy (e.g., by including in the combination a compound potentiating the potency or effectiveness of an active agent according to the present disclosure) , decrease one or more side effects, or decrease the required dose of the active agent according to the present disclosure. In a further embodiment, the methods provided herein allow for administering of the at least one additional therapeutic agent at a lower dose or frequency as compared to the administering of the at least one additional therapeutic agent alone that is required to achieve similar results in prophylactically treating an HBV infection in an individual in need thereof.

Such compounds include but are not limited to HBV combination drugs, HBV vaccines, HBV DNA polymerase inhibitors, immunomodulatory agents, toll-like receptor (TLR) modulators, interferon alpha receptor ligands, hyaluronidase inhibitors, hepatitis b surface antigen (HBsAg) inhibitors, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors, cyclophilin inhibitors, HBV viral entry inhibitors, antisense oligonucleotide targeting viral mRNA, short interfering RNAs (siRNA) and ddRNAi endonuclease modulators, ribonucleotide reductase inhibitors, HBV E antigen inhibitors, covalently closed circular DNA (cccDNA) inhibitors, famesoid X receptor agonists, HBV antibodies, CCR2 chemokine antagonists, thymosin agonists, cytokines, nucleoprotein modulators, retinoic acid-inducible gene 1 simulators, NOD2 stimulators, phosphatidylinositol 3-kinase (PI3K) inhibitors, indoleamine-2, 3-dioxygenase (IDO) pathway inhibitors, PD-1 inhibitors, PD-L1 inhibitors, recombinant thymosin alpha-1, bruton’s tyrosine kinase (BTK) inhibitors, KDM inhibitors, HBV replication inhibitors, arginase inhibitors, and any other agent that affects the HBV life cycle and/or affect the consequences of HBV infection or combinations thereof.

In embodiments, the compounds of the present disclosure may be used in combination with an HBV polymerase inhibitor, immunomodulatory agents, interferon such as pegylated interferon, viral entry inhibitor, viral maturation inhibitor, capsid assembly modulator, reverse transcriptase inhibitor, a cyclophilin/TNF inhibitor, immunomodulatory agent such as a TLR-agonist, an HBV vaccine, and any other agent that affects the HBV life cycle and/or affect the consequences of HBV infection or combinations thereof. In particular, the compounds of the present disclosure may be used in combination with one or more agents (or a salt thereof) selected from the group consisting of

HBV reverse transcriptase inhibitors, and DNA and RNA polymerase inhibitors, including but not limited to: lamivudine (3TC, Zeffix, Heptovir, Epivir, and Epivir-HBV) , entecavir (Baraclude, Entavir) , adefovir dipivoxil (Hepsara, Preveon, bis-POM PMEA) , tenofovir disoproxil fumarate (Viread, TDF or PMPA) ;

interferons, including but not limited to interferon alpha (IFN-α) , interferon beta (IFN- β) , interferon lambda (IFN-λ) , and interferon gamma (IFN-γ) ;

viral entry inhibitors;

viral maturation inhibitors;

literature-described capsid assembly modulators, such as, but not limited to BAY 41-4109;

reverse transcriptase inhibitor;

an immunomodulatory agent such as a TLR-agonist; and

agents of distinct or unknown mechanism, such as but not limited to AT-61 ( (E) -N- (1-chloro-3-oxo-1-phenyl-3- (piperidin-1-yl) prop-1-en-2-yl) benzamide) , AT-130 ( (E) -N- (1-bromo-1- (2-methoxyphenyl) -3-oxo-3- (piperidin-1-yl) prop-1-en-2-yl) -4-nitrobenzamide) , and similar analogs.

In embodiments, the additional therapeutic agent is an interferon. The term “interferon” or “IFN” refers to any member the family of highly homologous species-specific proteins that inhibit viral replication and cellular proliferation and modulate immune response. Human interferons are grouped into three classes; Type I, which include interferon-alpha (IFN-α) , interferon-beta (IFN-β) , and interferon-omega (IFN-ω) , Type II, which includes interferon-gamma (IFN-γ) , and Type III, which includes interferon-lambda (IFN-λ) . Recombinant forms of interferons that have been developed and are commercially available are encompassed by the term “interferon” as used herein. Subtypes of interferons, such as chemically modified or mutated interferons, are also encompassed by the term “interferon” as used herein. Chemically modified interferons include pegylated interferons and glycosylated interferons. Examples of interferons also include, but are not limited to, interferon-alpha-2a, interferon-alpha-2b, interferon-alpha-n1, interferon-beta-1a, interferon-beta-1b, interferon-lamda-1, interferon-lamda-2, and interferon-lamda-3. Examples of pegylated interferons include pegylated interferon-alpha-2a and pegylated interferon alpha-2b.

Accordingly, in one embodiment, the compounds of Formula I, can be administered in combination with an interferon selected from the group consisting of interferon alpha (IFN-α) , interferon beta (IFN-β) , interferon lambda (IFN-λ) , and interferon gamma (IFN-γ) . In one specific embodiment, the interferon is interferon-alpha-2a, interferon-alpha-2b, or interferon-alpha-n1. In another specific embodiment, the interferon-alpha-2a or interferon-alpha-2b is pegylated. In a preferred embodiment, the interferon-alpha-2a is pegylated interferon-alpha-2a (PEGASYS) .

In another embodiment, the additional therapeutic agent is selected from immune modulator or immune stimulator therapies, which includes biological agents belonging to the interferon class.

Further, the additional therapeutic agent may be an agent that disrupts the function of other essential viral protein (s) or host proteins required for HBV replication or persistence.

In another embodiment, the additional therapeutic agent is an antiviral agent that blocks viral entry or maturation or targets the HBV polymerase such as nucleoside or nucleotide or non-nucleos (t) ide polymerase inhibitors. In a further embodiment of the combination therapy, the reverse transcriptase inhibitor and/or DNA and/or RNA polymerase inhibitor is Zidovudine, Didanosine, Zalcitabine, ddA, Stavudine, Lamivudine, Abacavir, Emtricitabine, Entecavir, Apricitabine, Atevirapine, ribavirin, acyclovir, famciclovir, valacyclovir, ganciclovir, valganciclovir, Tenofovir, Adefovir, PMPA, cidofovir, Efavirenz, Nevirapine, Delavirdine, or Etravirine.

In an embodiment, the additional therapeutic agent is an immunomodulatory agent that induces a natural, limited immune response leading to induction of immune responses against unrelated viruses. In other words, the immunomodulatory agent can affect maturation of antigen presenting cells, proliferation of T-cells and cytokine release (e.g., IL-12, IL-18, IFN-alpha, -beta, and -gamma and TNF-alpha among others) .

In a further embodiment, the additional therapeutic agent is a TLR modulator or a TLR agonist, such as a TLR-7 agonist or TLR-9 agonist. In further embodiment of the combination therapy, the TLR-7 agonist is selected from the group consisting of SM360320 (9-benzyl-8-hydroxy-2- (2-methoxy-ethoxy) adenine) and AZD 8848 (methyl [3- ( { [3- (6-amino-2-butoxy-8-oxo-7, 8-dihydro-9H-purin-9-yl) propyl] [3- (4-morpholinyl) propyl] amino} methyl) phenyl] acetate) .

In any of the methods provided herein, the method may further comprise administering to the individual at least one HBV vaccine, a nucleoside HBV inhibitor, an interferon or any combination thereof. In an embodiment, the HBV vaccine is at least one of RECOMBIVAX HB, ENGERIX-B, ELOVAC B, GENEVAC-B, or SHANVAC B.

In another aspect, provided herein is method of treating an HBV infection in an individual in need thereof, comprising reducing the HBV viral load by administering to the individual a therapeutically effective amount of a compound of the present disclosure alone or in combination with a reverse transcriptase inhibitor; and further administering to the individual a therapeutically effective amount of HBV vaccine. The reverse transcriptase inhibitor may be one of Zidovudine, Didanosine, Zalcitabine, ddA, Stavudine, Lamivudine, Abacavir, Emtricitabine, Entecavir, Apricitabine, Atevirapine, ribavirin, acyclovir, famciclovir, valacyclovir, ganciclovir, valganciclovir, Tenofovir, Adefovir, PMPA, cidofovir, Efavirenz, Nevirapine, Delavirdine, or Etravirine.

For any combination therapy described herein, synergistic effect may be calculated, for example, using suitable methods such as the Sigmoid-E _max equation (Holford & Scheiner, 19981, Clin. Pharmacokinet. 6: 429-453) , the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22: 27-55) . Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

Methods

The application relates to a method for the preparation of a compound of Formula (I) as described herein.

The method comprises at least one step among steps a) , b) , c) , d) , e) and f) :

a) reacting a compound of Formula (II) ,

with a compound of Formula (III) ,

in the presence of NaBH ₃CN, to form a compound of Formula (IV) ,

wherein

G ¹ is substituted phenyl;

G ³ is hydrogen or a group selected from CHF ₂, CF ₃, C _1-4alkyl, C _1-4alkylOC _1- ₄alkyl and C _3-6cycloalkyl;

G ⁴ is a substituted phenyl, or a 6-or a 5-membered aromatic heterocyclic ring;

b) reacting a compound of Formula (V) ,

with a compound of Formula (VI) ,

in the presence of potassium iodide (KI) , to form a compound of Formula (VII) ,

wherein G ¹ is substituted phenyl;

G ⁵ is hydrogen or a group selected from CHF ₂, CF ₃, C _1-4alkyl, C _1-4alkylOC _1- ₄alkyl and C _3-6cycloalkyl;

G ⁶ is a substituted phenyl or a 5-or a 6-membered aromatic heterocyclic ring;

c) reacting a compound of Formula (VIII) ,

with 1, 5, 7-triazabicyclo [4.4.0] dec-5-en (TBD) or trimethylaluminium (Al (CH ₃) ₃) , optionally in the presence of 1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU) , to form a compound of Formula (IX) ,

wherein

R is hydrogen or

G ¹ is substituted phenyl;

G ⁷ is a substituted phenyl or a 5-or a 6-membered aromatic heterocyclic ring;

G ⁸ is hydrogen, CHF ₂, CF ₃, C _1-4alkyl, C _1-4alkylOC _1-4alkyl or C _3-6cycloalkyl;

d) reacting a compound of Formula (X) ,

with a compound of Formula (XI) ,

in the presence of a non-nucleophilic base, more particularly sodium hydroxide, cesium carbonate (Cs ₂CO ₃) or sodium hydride (NaH) to form a compound or Formula (XII) ,

wherein

G ¹ is substituted phenyl;

G ⁹ is hydrogen, CHF ₂, CF ₃, C _1-4alkyl, C _1-4alkylOC _1-4alkyl or C _3-6cycloalkyl,

G ¹⁰ is a substituted phenyl or a 5-or a 6-membered aromatic heterocyclic ring;

L is bromide, chloride or iodide;

e) reacting a compound of Formula (XIII) ,

with a strong acid, more particularly hydrochloric acid (HCl) or trifluoroacetic acid (TFA) , to form a compound of Formula (XIV) ,

wherein G ¹¹ is H, a substituted phenyl or a 5-or a 6-membered aromatic heterocyclic ring;

f) reacting a compound of Formula (XIV) ,

with a compound of Formula (XV) ,

in the presence of a non-nucleophilic base, more particularly sodium carbonate (Na ₂CO ₃) , triethylamine (Et ₃N) or diisopropylethylamine (DIPEA) , to form a compound of Formula (XVI) ,

wherein R ₁ is as defined for a compound of Formula (I) - (IC) and wherein G ¹¹ has been defined in step e) ,

provided that

when the process comprises step a, the process further comprises step c,

when the process comprises step b, the process further comprises step c, and

when the process comprises step e, the process further comprises step f.

Definitions

Listed below are definitions of various terms used to describe this present disclosure. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the applicable art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

As used herein, the articles “a” and “an” refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include, ” “includes, ” and “included, ” is not limiting.

As used in the specification and in the claims, the term “comprising” can include the embodiments “consisting of” and “consisting essentially of. ” The terms “comprise (s) , ” “include (s) , ” “having, ” “has, ” “can, ” “contain (s) , ” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients/steps and permit the presence of other ingredients/steps. However, such description should be construed as also describing compositions or processes as “consisting of” and “consisting essentially of” the enumerated compounds, which allows the presence of only the named compounds, along with any pharmaceutically acceptable carriers, and excludes other compounds. All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 300 mg” is inclusive of the endpoints, 50 mg and 300 mg, and all the intermediate values) . The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and/or values.

As used herein, approximating language can be applied to modify any quantitative representation that can vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “substantially, ” cannot be limited to the precise value specified, in some cases. In at least some instances, the approximating language can correspond to the precision of an instrument for measuring the value.

The term “alkyl” as a group or as part of another group, refers to a straight-or branched-chain alkyl group having carbon and hydrogen atoms in the chain. Examples of alkyl groups include methyl (Me, which also may be structurally depicted by the symbol, “/” ) , ethyl (Et) , n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl (tBu) , pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and groups that in light of the ordinary skill in the art and the teachings provided herein would be considered equivalent to any one of the foregoing examples. The term C _1- ₄alkyl as used here refers to a straight-or branched-chain alkyl group having from 1 to 4 carbon atoms in the chain. The term C _1-6alkyl as used here refers to a straight-or branched-chain alkyl group having from 1 to 6 carbon atoms in the chain.

The term “C _3-6cycloalkyl” refers to a saturated, monocyclic carbocycle having from 3 to 6 ring atoms. Illustrative examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term “phenyl” represents the following moiety:

The term “heteroaryl” refers to an aromatic monocyclic or bicyclic aromatic ring system having 5 to 10 ring members and which contains carbon atoms and from 1 to 4 heteroatoms independently selected from the group consisting of N, O, and S. Included within the term heteroaryl are five-membered aromatic heterocyclic rings, and six-membered aromatic heterocyclic rings, which are defined as aromatic rings of 5, respectively 6 members wherein the ring consists of carbon atoms and has at least one heteroatom member. Suitable heteroatoms include nitrogen, oxygen, and sulfur. In the case of 5 membered rings, the heteroaryl ring preferably contains one member of nitrogen, oxygen or sulfur and, in addition, up to 3 additional nitrogens. In the case of 6 membered rings, the heteroaryl ring preferably contains from 1 to 4, e.g. tetrazolyl, more in particular from 1 to 3 nitrogen atoms. For the case wherein the 6 membered ring has 3 nitrogens, at most 2 nitrogen atoms are adjacent. Examples of heteroaryl groups include furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, isoindolyl, benzofuryl, benzothienyl, indazolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzothiadiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl and quinazolinyl. Unless otherwise noted, the heteroaryl is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure.

The term “heterocyclyl” represents a non-aromatic monocyclic or bicyclic system, unless otherwise specified, having for example, 3 to 8 ring members, more usually 5 to 6 ring members. Examples of monocyclic groups are groups containing 3 to 8 ring members, more usually, 5 or 6 ring members. Non-limiting examples of monocyclic heterocyclyl systems containing at least one heteroatom selected from nitrogen, oxygen or sulfur (N, O, S) include, but are not limited to 3-to 8-membered heterocyclyl systems such as azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, pyranyl, dihydropyranyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl. Unless otherwise specified, each can be bound to the remainder of the molecule through any available ring carbon atom or nitrogen atom, and may optionally be substituted, where possible, on carbon and/or nitrogen atoms according to the embodiments.

Those skilled in the art will recognize that the species of heteroaryl groups listed or illustrated above are not exhaustive, and that additional species within the scope of these defined terms may also be selected.

The term “cyano” refers to the group -CN.

The terms “halo” or “halogen” represent chloro, fluoro, bromo or iodo.

The term “oxo” represents =O.

The term “5-membered ring or 6-membered ring, said ring optionally comprising 1, 2, or 3 heteroatoms each independently selected from N, O and S, said ring optionally carrying one or more fluoro or oxo substituents” refers herein to a C ₅cycloalkyl or C ₆cycloalkyl group when the rings do not contain any heteroatoms, or alternatively a 5-or 6-membered heterocyclyl which may optionally be partly unsaturated, or a 5-or 6-membered aromatic heterocyclyl (or also referred to as heteroaryl) as described herein. In particular, the 5-or 6-membered ring is a 5-or 6-membered heterocyclyl. Alternatively, the 5-or 6-membered ring is a 5-or 6-membered aromatic heterocyclyl (or heteroaryl) .

The term “substituted” means that the specified group or moiety bears one or more substituents. The term “unsubstituted” means that the specified group bears no substituents. The term “optionally substituted” means that the specified group is unsubstituted or substituted by one or more substituents. Where the term “substituted” is used to describe a structural system, the substitution is meant to occur at any valency-allowed position on the system. In cases where a specified moiety or group is not expressly noted as being optionally substituted or substituted with any specified substituent, it is understood that such a moiety or group is intended to be unsubstituted.

The terms “para” , “meta” , and “ortho” have the meanings as understood in the art. Thus, for example, a fully substituted phenyl group has substituents at both “ortho” (o) positions adjacent to the point of attachment of the phenyl ring, both “meta” (m) positions, and the one “para” (p) position across from the point of attachment. To further clarify the position of substituents on the phenyl ring, the 2 different ortho positions will be designated as ortho and ortho’ and the 2 different meta positions as meta and meta’ as illustrated below.

When referring to substituents on a pyridyl group, the terms “para” , “meta” , and “ortho” refer to the placement of a substituent relative to the point of attachment of the pyridyl ring. For example, the structure below is described as 3-pyridyl with the X ¹ substituent in the ortho position, the X ² substituent in the meta position, and X ³ substituent in the para position:

To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term “about” . It is understood that, whether the term “about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value. Whenever a yield is given as a percentage, such yield refers to a mass of the entity for which the yield is given with respect to the maximum amount of the same entity that could be obtained under the particular stoichiometric conditions. Concentrations that are given as percentages refer to mass ratios, unless indicated differently.

The terms “buffered” solution or “buffer” solution are used herein interchangeably according to their standard meaning. Buffered solutions are used to control the pH of a medium, and their choice, use, and function is known to those of ordinary skill in the art. See, for example, G.D. Considine, ed., Van Nostrand’s Encyclopedia of Chemistry, p. 261, 5 ^th ed. (2005) , describing, inter alia, buffer solutions and how the concentrations of the buffer constituents relate to the pH of the buffer. For example, a buffered solution is obtained by adding MgSO ₄ and NaHCO ₃ to a solution in a 10: 1 w/w ratio to maintain the pH of the solution at about 7.5.

Any formula given herein is intended to represent compounds having structures depicted by the structural formula as well as certain variations or forms. In particular, compounds of any formula given herein may have asymmetric centers and therefore exist in different enantiomeric forms. All optical isomers of the compounds of the general formula, and mixtures thereof, are considered within the scope of the formula. Thus, any formula given herein is intended to represent a racemate, one or more enantiomeric forms, one or more diastereomeric forms, one or more atropisomeric forms, and mixtures thereof. Furthermore, certain structures may exist as geometric isomers (i.e., cis and trans isomers) , as tautomers, or as atropisomers.

It is also to be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers. ”

Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers. ” When a compound has an asymmetric center, for example, it is bonded to four different groups, and a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R-and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) -or (-) -isomers respectively) . A chiral compound can exist as either an individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture. ”

“Tautomers” refer to compounds that are interchangeable forms of a particular compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of π electrons and an atom (usually H) . For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Another example of tautomerism is the aci-and nitro-forms of phenyl nitromethane, that are likewise formed by treatment with acid or base.

Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.

The compounds of this present disclosure may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R) -or (S) -stereoisomers or as mixtures thereof.

Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art.

Certain examples contain chemical structures that are depicted as an absolute enantiomer but are intended to indicate enantiopure material that is of unknown configuration. In these cases (R*) or (S*) or (*R) or (*S) is used in the name to indicate that the absolute stereochemistry of the corresponding stereocenter is unknown. Thus, a compound designated as (R*) or (*R) refers to an enantiopure compound with an absolute configuration of either (R) or (S) . In cases where the absolute stereochemistry has been confirmed, the structures are named using (R) and (S) .

The symbols

and

are used as meaning the same spatial arrangement in chemical structures shown herein. Analogously, the symbols

and

are used as meaning the same spatial arrangement in chemical structures shown herein.

Certain compounds of Formula (I) , or pharmaceutically acceptable salts of compounds of Formula (I) , may be obtained as solvates. Solvates include those formed from the interaction or complexation of compounds of the present disclosure with one or more solvents, either in solution or as a solid or crystalline form. In some embodiments, the solvent is water and the solvates are hydrates.

Reference to a compound herein stands for a reference to any one of: (a) the actually recited form of such compound, and (b) any of the forms of such compound in the medium in which the compound is being considered when named. For example, reference herein to a compound such as R-COOH, encompasses reference to any one of, for example, R-COOH _(s) , R-COOH _(sol) , and R-COO ^- _(sol) . In this example, R-COOH _(s) refers to the solid compound, as it could be for example in a tablet or some other solid pharmaceutical composition or preparation; R-COOH _(sol) refers to the undissociated form of the compound in a solvent; and R-COO ^- _(sol) refers to the dissociated form of the compound in a solvent, such as the dissociated form of the compound in an aqueous environment, whether such dissociated form derives from R-COOH, from a salt thereof, or from any other entity that yields R-COO ^-upon dissociation in the medium being considered. In another example, an expression such as “exposing an entity to compound of formula R-COOH” refers to the exposure of such entity to the form, or forms, of the compound R-COOH that exists, or exist, in the medium in which such exposure takes place. In still another example, an expression such as “reacting an entity with a compound of formula R-COOH” refers to the reacting of (a) such entity in the chemically relevant form, or forms, of such entity that exists, or exist, in the medium in which such reacting takes place, with (b) the chemically relevant form, or forms, of the compound R-COOH that exists, or exist, in the medium in which such reacting takes place. In this regard, if such entity is for example in an aqueous environment, it is understood that the compound R-COOH is in such same medium, and therefore the entity is being exposed to species such as R-COOH _(aq) and/or R-COO ^- _(aq) , where the subscript “ (aq) ” stands for “aqueous” according to its conventional meaning in chemistry and biochemistry. A carboxylic acid functional group has been chosen in these nomenclature examples; this choice is not intended, however, as a limitation but it is merely an illustration. It is understood that analogous examples can be provided in terms of other functional groups, including but not limited to hydroxyl, basic nitrogen members, such as those in amines, and any other group that interacts or transforms according to known manners in the medium that contains the compound. Such interactions and transformations include, but are not limited to, dissociation, association, tautomerism, solvolysis, including hydrolysis, solvation, including hydration, protonation, and deprotonation. No further examples in this regard are provided herein because these interactions and transformations in a given medium are known by any one of ordinary skill in the art.

In another example, a zwitterionic compound is encompassed herein by referring to a compound that is known to form a zwitterion, even if it is not explicitly named in its zwitterionic form. Terms such as zwitterion, zwitterions, and their synonyms zwitterionic compound (s) are standard IUPAC-endorsed names that are well known and part of standard sets of defined scientific names. In this regard, the name zwitterion is assigned the name identification CHEBI: 27369 by the Chemical Entities of Biological Interest (ChEBI) dictionary of molecular entities. As generally well known, a zwitterion or zwitterionic compound is a neutral compound that has formal unit charges of opposite sign. Sometimes these compounds are referred to by the term “inner salts” . Other sources refer to these compounds as “dipolar ions” , although the latter term is regarded by still other sources as a misnomer. As a specific example, aminoethanoic acid (the amino acid glycine) has the formula H ₂NCH ₂COOH, and it exists in some media (in this case in neutral media) in the form of the zwitterion ⁺H ₃NCH ₂COO ^-. Zwitterions, zwitterionic compounds, inner salts and dipolar ions in the known and well established meanings of these terms are within the scope of this present disclosure, as would in any case be so appreciated by those of ordinary skill in the art. Because there is no need to name each and every embodiment that would be recognized by those of ordinary skill in the art, no structures of the zwitterionic compounds that are associated with the compounds of this present disclosure are given explicitly herein. They are, however, part of the embodiments of this present disclosure. No further examples in this regard are provided herein because the interactions and transformations in a given medium that lead to the various forms of a given compound are known by any one of ordinary skill in the art.

Any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²⁵I, respectively. Such isotopically labeled compounds are useful in metabolic studies (preferably with ¹⁴C) , reaction kinetic studies (with, for example deuterium (i.e., D or ²H) ; or tritium (i.e., T or ³H) ) , detection or imaging techniques such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. In particular, an ¹⁸F or ¹¹C labeled compound may be particularly preferred for PET or SPECT studies. Further, substitution with heavier isotopes such as deuterium (i.e., ²H) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements. Isotopically labeled compounds of this present disclosure can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

When referring to any formula given herein, the selection of a particular moiety from a list of possible species for a specified variable is not intended to define the same choice of the species for the variable appearing elsewhere. In other words, where a variable appears more than once, the choice of the species from a specified list is independent of the choice of the species for the same variable elsewhere in the formula, unless stated otherwise.

According to the foregoing interpretive considerations on assignments and nomenclature, it is understood that explicit reference herein to a set implies, where chemically meaningful and unless indicated otherwise, independent reference to embodiments of such set, and reference to each and every one of the possible embodiments of subsets of the set referred to explicitly.

By way of a first example on substituent terminology, if substituent S ¹ _example is one of S ₁ and S ₂, and substituent S ² _example is one of S ₃ and S ₄, then these assignments refer to embodiments of this present disclosure given according to the choices S ¹ _example is S ₁ and S ² _example is S ₃; S ¹ _example is S ₁ and S ² _example is S ₄; S ¹ _example is S ₂ and S ² _example is S ₃; S ¹ _example is S ₂ and S ² _example is S ₄; and equivalents of each one of such choices. The shorter terminology “S ¹ _example is one of S ₁ and S ₂, and S ² _example is one of S ₃ and S ₄” is accordingly used herein for the sake of brevity, but not by way of limitation. The foregoing first example on substituent terminology, which is stated in generic terms, is meant to illustrate the various substituent assignments described herein. The foregoing convention given herein for substituents extends, when applicable, to members such as R ¹, R ², R ³, R ⁴, R ⁵, G ¹, G ², G ³, G ⁴, G ⁵, G ⁶, G ⁷, G ⁸, G ⁹, G ¹⁰, G ¹¹, n, L, R, T, Q, W, X, Y, and Z and any other generic substituent symbol used herein.

Furthermore, when more than one assignment is given for any member or substituent, embodiments of this present disclosure comprise the various groupings that can be made from the listed assignments, taken independently, and equivalents thereof. By way of a second example on substituent terminology, if it is herein described that substituent S _example is one of S ₁, S ₂, and S ₃, this listing refers to embodiments of this present disclosure for which S _example is S ₁; S _example is S ₂; S _example is S ₃; S _example is one of S ₁ and S ₂; S _example is one of S ₁ and S ₃; S _example is one of S ₂ and S ₃; S _example is one of S ₁, S ₂ and S ₃; and S _example is any equivalent of each one of these choices. The shorter terminology “S _example is one of S ₁, S ₂, and S ₃” is accordingly used herein for the sake of brevity, but not by way of limitation. The foregoing second example on substituent terminology, which is stated in generic terms, is meant to illustrate the various substituent assignments described herein. The foregoing convention given herein for substituents extends, when applicable, to members such as R ¹, R ², R ³, R ⁴, R ⁵, G ¹, G ², G ³, G ⁴, G ⁵, G ⁶, G ⁷, G ⁸, G ⁹, G ¹⁰, G ¹¹, n, L, R, T, Q, W, X, Y, and Z and any other generic substituent symbol used herein.

The nomenclature “C _i-j” with j > i, when applied herein to a class of substituents, is meant to refer to embodiments of this present disclosure for which each and every one of the number of carbon members, from i to j including i and j, is independently realized. By way of example, the term C _1-4 refers independently to embodiments that have one carbon member (C ₁) , embodiments that have two carbon members (C ₂) , embodiments that have three carbon members (C ₃) , and embodiments that have four carbon members (C ₄) .

The term C _n-malkyl refers to an aliphatic chain, whether straight or branched, with a total number N of carbon members in the chain that satisfies n ≤ N ≤ m, with m > n. Any disubstituent referred to herein is meant to encompass the various attachment possibilities when more than one of such possibilities are allowed. For example, reference to disubstituent –A-B-, where A ≠ B, refers herein to such disubstituent with A attached to a first substituted member and B attached to a second substituted member, and it also refers to such disubstituent with A attached to the second substituted member and B attached to the first substituted member.

The present disclosure includes also pharmaceutically acceptable salts of the compounds of Formula (I) , preferably of those described above and of the specific compounds exemplified herein, and methods of treatment using such salts.

The term “pharmaceutically acceptable” means approved or approvable by a regulatory agency of Federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans.

A “pharmaceutically acceptable salt” is intended to mean a salt of a free acid or base of compounds represented by Formula (I) that are non-toxic, biologically tolerable, or otherwise biologically suitable for administration to the subject. It should possess the desired pharmacological activity of the parent compound. See, generally, G.S. Paulekuhn, et al., “Trends in Active Pharmaceutical Ingredient Salt Selection based on Analysis of the Orange Book Database” , J. Med. Chem., 2007, 50: 6665–72, S. M. Berge, et al., “Pharmaceutical Salts” , J Pharm Sci., 1977, 66: 1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth, Eds., Wiley-VCH and VHCA, Zurich, 2002. Examples of pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of patients without undue toxicity, irritation, or allergic response. A compound of Formula (I) may possess a sufficiently acidic group, a sufficiently basic group, or both types of functional groups, and accordingly react with a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt.

As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one compound provided herein with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound provided herein within or to the patient such that it can perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound provided herein, and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound provided herein, and are physiologically acceptable to the patient. Supplementary active compounds can also be incorporated into the compositions. The “pharmaceutically acceptable carrier” can further include a pharmaceutically acceptable salt of the compound provided herein. Other additional ingredients that can be included in the pharmaceutical compositions provided herein are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA) , which is incorporated herein by reference.

The term “stabilizer, ” as used herein, refers to polymers capable of chemically inhibiting or preventing degradation of a compound disclosed herein. Stabilizers are added to formulations of compounds to improve chemical and physical stability of the compound.

The term “tablet, ” as used herein, denotes an orally administrable, single-dose, solid dosage form that can be produced by compressing a drug substance or a pharmaceutically acceptable salt thereof, with suitable excipients (e.g., fillers, disintegrants, lubricants, glidants, and/or surfactants) by conventional tableting processes.

As used herein, the term “capsule” refers to a solid dosage form in which the drug is enclosed within either a hard or soft soluble container or “shell. ” The container or shell can be formed from gelatin, starch and/or other suitable substances.

As used herein, the terms “effective amount, ” “pharmaceutically effective amount, ” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

The term “combination, ” “therapeutic combination, ” “pharmaceutical combination, ” or “combination product” as used herein refer to a non-fixed combination or a kit of parts for the combined administration where two or more therapeutic agents can be administered independently, at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g., synergistic, effect.

The term “modulators” include both inhibitors and activators, where “inhibitors” refer to compounds that decrease, prevent, inactivate, desensitize, or down-regulate HBV assembly and other HBV core protein functions necessary for HBV replication or the generation of infectious particles.

As used herein, the term “capsid assembly modulator” refers to a compound that disrupts or accelerates or inhibits or hinders or delays or reduces or modifies normal capsid assembly (e.g., during maturation) or normal capsid disassembly (e.g., during infectivity) or perturbs capsid stability, thereby inducing aberrant capsid morphology and function. In one embodiment, a capsid assembly modulator accelerates capsid assembly or disassembly, thereby inducing aberrant capsid morphology. In another embodiment, a capsid assembly modulator interacts (e.g. binds at an active site, binds at an allosteric site, modifies and/or hinders folding and the like) with the major capsid assembly protein (CA) , thereby disrupting capsid assembly or disassembly. In yet another embodiment, a capsid assembly modulator causes a perturbation in structure or function of CA (e.g., ability of CA to assemble, disassemble, bind to a substrate, fold into a suitable conformation, or the like) , which attenuates viral infectivity and/or is lethal to the virus.

As used herein, the term “treatment” or “treating, ” is defined as the application or administration of a therapeutic agent, i.e., a compound of the present disclosure (alone or in combination with another pharmaceutical agent) , to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications) , who has an HBV infection, a symptom of HBV infection or the potential to develop an HBV infection, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the HBV infection, the symptoms of HBV infection or the potential to develop an HBV infection. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

As used herein, the term “prevent” or “prevention” means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.

As used herein, the term “patient, ” “individual” or “subject” refers to a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the patient, subject or individual is human.

In treatment methods according to the present disclosure, an effective amount of a pharmaceutical agent according to the present disclosure is administered to a subject suffering from or diagnosed as having such a disease, disorder, or condition. An “effective amount” means an amount or dose sufficient to generally bring about the desired therapeutic or prophylactic benefit in patients in need of such treatment for the designated disease, disorder, or condition. Effective amounts or doses of the compounds of the present disclosure may be ascertained by routine methods such as modeling, dose escalation studies or clinical trials, and by taking into consideration routine factors, e.g., the mode or route of administration or drug delivery, the pharmacokinetics of the compound, the severity and course of the disease, disorder, or condition, the subject's previous or ongoing therapy, the subject's health status and response to drugs, and the judgment of the treating physician. An example of a dose is in the range of from about 0.001 to about 200 mg of compound per kg of subject's body weight per day. An example of a dose of a compound is from about 1 mg to about 2,500 mg.

Once improvement of the patient's disease, disorder, or condition has occurred, the dose may be adjusted for preventative or maintenance treatment. For example, the dosage or the frequency of administration, or both, may be reduced as a function of the symptoms, to a level at which the desired therapeutic or prophylactic effect is maintained. Of course, if symptoms have been alleviated to an appropriate level, treatment may cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of symptoms.

HBV infections that may be treated according to the disclosed methods include HBV genotype A, B, C, and/or D infections. However, in an embodiment, the methods disclosed may treat any HBV genotype ( “pan-genotypic treatment” ) . HBV genotyping may be performed using methods known in the art, for example,

HBV Genotyping, Innogenetics N.V., Ghent, Belgium) .

In an attempt to help the reader of the present application, the description has been separated in various paragraphs or sections. These separations should not be considered as disconnecting the substance of a paragraph or section from the substance of another paragraph or section. To the contrary, the present description encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated.

Each of the relevant disclosures of all references cited herein is specifically incorporated by reference. The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES

Exemplary compounds useful in methods of the present disclosure will now be described by reference to the illustrative synthetic schemes for their general preparation below and the specific examples that follow. Artisans will recognize that, to obtain the various compounds herein, starting materials may be suitably selected so that the ultimately desired substituents will be carried through the reaction scheme with or without protection as appropriate to yield the desired product. Alternatively, it may be necessary or desirable to employ, in the place of the ultimately desired substituent, a suitable group that may be carried through the reaction scheme and replaced as appropriate with the desired substituent. Unless otherwise specified, the variables are as defined above in reference to Formula (I) , (IA) , (IB) , and (IC) . Reactions may be performed between the melting point and the reflux temperature of the solvent, and preferably between 0 ℃ and the reflux temperature of the solvent. Reactions may be heated employing conventional heating or microwave heating. Reactions may also be conducted in sealed pressure vessels above the normal reflux temperature of the solvent.

Compounds of any one of Formula (I) , (IA) , (IB) , and (IC) , may be converted to their corresponding salts using methods known to one of ordinary skill in the art. For example, an amine of Formula (I) is treated with trifluoroacetic acid, HCl, or citric acid in a solvent such as Et ₂O, CH ₂Cl ₂, THF, MeOH, chloroform, or isopropanol to provide the corresponding salt form. Alternately, trifluoroacetic acid or formic acid salts are obtained as a result of reverse phase HPLC purification conditions. Crystalline forms of pharmaceutically acceptable salts of compounds of Formula (I) , (IA) , (IB) , and (IC) , may be obtained in crystalline form by recrystallization from polar solvents (including mixtures of polar solvents and aqueous mixtures of polar solvents) or from non-polar solvents (including mixtures of non-polar solvents) .

Where the compounds according to this present disclosure have at least one chiral center, they may accordingly exist as enantiomers. Where the compounds possess two or more chiral centers, they may additionally exist as diastereomers. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present disclosure.

Compounds represented as “stereomeric mixture” (means a mixture of two or more stereoisomers and includes enantiomers, diastereomers and combinations thereof) are separated by SFC resolution.

Compounds may be obtained as single forms, such as single enantiomers, by form-specific synthesis, or by resolution. Compounds may alternately be obtained as mixtures of various forms, such as racemic (1: 1) or non-racemic (not 1: 1) mixtures. Where racemic and non-racemic mixtures of enantiomers are obtained, single enantiomers may be isolated using conventional separation methods known to one of ordinary skill in the art, such as chiral chromatography, recrystallization, diastereomeric salt formation, derivatization into diastereomeric adducts, biotransformation, or enzymatic transformation. Where regioisomeric or diastereomeric mixtures are obtained, as applicable, single isomers may be separated using conventional methods such as chromatography or crystallization.

1. GENERAL INFORMATION

CHEMICAL NAMES

Chemical names were generated using the chemistry software: ACD/ChemSketch, and may follow preferably the IUPAC rules.

GENERAL PROCEDURE FOR LCMS METHODS

The High Performance Liquid Chromatography (HPLC) measurement was performed using a LC pump, a diode-array (DAD) or a UV detector and a column as specified in the respective methods.

Flow from the column was brought to the Mass Spectrometer (MS) which was configured with an atmospheric pressure ion source. It is within the knowledge of the skilled person to set the tune parameters (e.g. scanning range, dwell time…) in order to obtain ions allowing the identification of the compound’s nominal monoisotopic molecular weight (MW) . Data acquisition was performed with appropriate software.

Compounds are described by their experimental retention times (Rt) and ions. If not specified differently, the reported molecular ion corresponds to the [M+H] ⁺ (protonated molecule) and/or [M-H] ^- (deprotonated molecule) . In case the compound was not directly ionizable the type of adduct is specified (i.e. [M+NH ₄] ⁺, [M+HCOO] ^-, etc. ) . All results were obtained with experimental uncertainties that are commonly associated with the method used. Hereinafter, “SQD” means Single Quadrupole Detector, “MSD” Mass Selective Detector, “RT” room temperature, “BEH” bridged ethylsiloxane/silica hybrid, “DAD” Diode Array Detector, “HSS” High Strength silica., “Q-Tof” Quadrupole Time-of-flight mass spectrometers, “CLND” , ChemiLuminescent Nitrogen Detector, “ELSD” Evaporative Light Scanning Detector.

NMR ANALYSIS

¹H NMR spectra were recorded on 1) a Bruker DPX 400 MHz spectrometer or 2) a Bruker Avance 400 MHz spectrometer or c) Bruker Avance III 400 MHz spectrometer or d) Bruker Avance 600 MHz spectrometer or e) a Bruker Avance NEO 400 MHz spectrometer or f) Bruker model AVIII 400 spectrometer g) ZKNJ BIXI-1 300 MHz, Bruker Avance III 400 MHz or h) Bruker AVANCE Neo 400 MHz.

NMR spectra were recorded at ambient temperature unless otherwise stated. Data are reported as follow: chemical shift in parts per million (ppm) relative to TMS (δ = 0 ppm) on the scale, integration, multiplicity (s= singulet, d = doublet, t = triplet, q = quartet, quin = quintet, sext = sextet, sept = septet, m = multiplet, b = broad, or a combination of these) , coupling constant (s) J in Hertz (Hz) .

MS ANALYSIS

Mass spectra were obtained on a Shimadzu LCMS-2020 MSD or Agilent 1200/G6110A MSD using electrospray ionization (ESI) in positive mode unless otherwise indicated.

2. Abbreviations

Table 7

μw	Microwave
ADDP	1, 1’- (Azodicarbonyl) dipiperidine
AlMe ₃	Trimethylaluminium
aq.	Aqueous
atm	Atmosphere
Boc ₂O	Di-tert-butyl dicarbonate
BOC	tert-Butyloxycarbonyl
BODIPY	Boron-dipyrromethene
br	broad
CA	Capsid assembly
DAST	(Diethylamino) sulfur trifluoride
DBU	1, 8-Diazabicyclo [5.4.0] undec-7-ene
DCE	1, 2-Dichloroethane
DCM	Dichloromethane

dd	Doublet of doublets
DEA	Diethylamine
DIPE	Diisopropyl ether
DIPEA/DIEA	N, N-diisopropylethylamine
DMF	Dimethylformamide
DMF-DMA	Dimethylformamide-dimethylacetal
DMSO	Dimethylsulfoxide
DNA	Deoxyribonucleic Acid
EDCI	N- (3-Dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride
ESI	Electrospray ionization
Et ₃N	Triethylamine
Et ₂O	Ether, diethyl ether
EtOAc/EA	Ethyl acetate
EtOH	Ethanol
FCC	Normal-phase silica gel chromatography
g	grams
h/hr	hour
HBV	Hepatitis B virus
HOAc	Acetic acid
HPLC	High Performance Liquid Chromatography
Hz	Hertz
i-PrNH ₂	Isopropylamine
i-PrOH/IPA	Isopropyl alcohol
KOtBu	Potassium tert-butoxide
LAH	Lithium aluminum hydride
LCMS	Liquid Chromatography Mass Spectrometry
LHMDS, LiHMDS	Lithium bis (trimethylsilyl) amide
M	molar
m	multiplet
MeCN/ACN	Acetonitrile
MeOH	Methanol
mg	milligrams
MHz	megahertz
min	Minutes
mL	Milliliters
μL	microliters
mmol	Millimole

μmol	micromole
MS	Mass spectra
MsCl	Mesityl chloride
m/z	Mass to charge ratio
N	normal
NaOAc/AcONa	Sodium acetate
NMR	Nuclear Magnetic Resonance
o/n	Overnight
PCR	Polymerase chain reaction
PE	Petroleum ether
PMPA	9- (2-Phosphonyl-methoxypropyl) adenine
ppm	Parts per million
ppt	precipitate
Py	pyridine
RNA	Ribonucleic Acid
R _t	Retention time
rt	Room temperature
s	singlet
sat.	Saturated
SFC	Supercritical Fluid Chromatography
t	triplet
T ₃P	Propanephosphonic acid anhydride
TBAI	Tetrabutylammonium iodide
TBD	1, 5, 7-Triazabicyclo [4.4.0] dec-5-ene
TEA	triethylamine
TFA	Trifluoroacetic acid
THF	Tetrahydrofuran
TLC	Thin Layer Chromatography
TLR	Toll-like receptor
TNF	Tumor necrosis factor
V or volumes	Volume in milliliters of solvent per gram of substrate
Δ	Heating under reflux

EXAMPLES

Compound 1:

2-chloro-5- ( (R) -3-methyl-10-oxo-9- ( (S) -1- (4- (trifluoromethoxy) phenyl) ethyl) - 1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

Intermediate 1-2:

ethyl (R) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylate hydrochloride

A mixture of 5- (tert-butyl) 3-ethyl (R) -6-methyl-2, 4, 6, 7-tetrahydro-5H-pyrazolo [4, 3-c] pyridine-3, 5-dicarboxylate 1-1 (CAS number: 2171134-54-4) (10.0 g, 100 %purity, 32.3 mmol) in 4 M hydrochloride in dioxane (100 mL) was stirred at room temperature for 2 hours under nitrogen atmosphere. LCMS showed the material was consumed. The mixture was concentrated under reduced pressure to give the title compound (8.0 g, 95%purity from HNMR, 96 %yield) as yellow solids without further purification. LC-MS (ESI) : R _T = 0.5 min, mass calcd. for C ₁₀H ₁₅N ₃O ₂ 209.1, m/z found 210.2 [M+H] ⁺. ¹HNMR (400 MHz, DMSO-d ₆) δ 9.68 (br s, 1H) , 9.50 (br s, 1H) , 4.36 -4.17 (m, 5H) , 3.75 -3.50 (m, 1H) , 3.05 (d, J = 16.0 Hz, 1H) , 2.75 -2.69 (m, 1H) , 1.45 -1.37 (m, 3H) , 1.34 -1.26 (m, 3H) .

Intermediate 1-4:

(R) -Ethyl 5- (4-chloro-3-cyanobenzoyl) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylate

To a mixture of 4-chloro-3-cyanobenzoic acid 1-3 (5.9 g, 100 %purity, 32.5 mmol) in dichloromethane (60 mL) was added dropwise N, N-dimethylformamide (235 mg, 3.22 mmol) and oxalyl dichloride (4 mL, 47.3 mmol) at 0 ℃. After stirred at room temperature for 2 hours under nitrogen atmosphere, the mixture became clear until no gas escaped, then concentrated under reduced pressure. The resulting residue and (R) -ethyl 6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylate hydrochloride 1-2 (8.0 g, 95 %purity, 30.9 mmol) was dissolved in dichloromethane (60 mL) , N-ethyl-N-isopropylpropan-2-amine (16 mL, 96.8 mmol) was added dropwise at 0 ℃. After stirred for 1 hour, the mixture was poured into water (60 mL) and adjusted to pH 5 ～ 6 with 1M hydrochloride aqueous solution, extracted with dichloromethane (60 mL) twice. The combined organic layers were concentrated under reduced pressure. The resulting residue was purified by C18 column (acetonitrile : water (+ 0.02 %ammonium acetate) = 10 %to 75 %) to give the title compound (9.3 g, 90 %purity, 69.1 %yield) as yellow solids. LC-MS (ESI) : R _T = 1.38 min, mass calcd. for C ₁₈H ₁₇ClN ₄O ₃ 372.1, m/z found 373.1 [M+H] ⁺. ¹HNMR (400 MHz, CDCl ₃) δ 10.81 (br s, 1H) , 7.75 (S, 1H) , 7.61 (S, 2H) , 5.76 -5.36 (m, 1H) , 4.82 -4.17 (m, 4H) , 3.19 -2.95 (m, 1H) , 2.71 (d, J = 16.0 Hz, 1H) , 1.38 (S, 3H) , 1.26 (S, 3H) .

Intermediate 1-6:

(S) -1- (4- (Trifluoromethoxy) phenyl) ethanamine hydrochloride

A solution of (S) -tert-butyl (1- (4- (trifluoromethoxy) phenyl) ethyl) carbamate 1-5 (2 g, 90 % purity, 5.90 mmol) in 4 M hydrochloride in dioxane (20 mL, 4 mmol) was stirred at 25 ℃ for 3 hours. Then the mixture was concentrated to give the title compound (1.4 g, 100 %purity, 98 %yield) as light yellow solids.

LC-MS (ESI) : R _T = 1.49 min, mass calcd. for C ₉H ₁₁ClF ₃NO 241.1, m/z found 189.1 [M-HCl-16] ⁺.

Intermediate 1-7:

(S) -tert-Butyl (2- ( (tert-butyldimethylsilyl) oxy) ethyl) (1- (4- (trifluoromethoxy) phenyl) ethyl) carbamate

To a mixture of (2-bromoethoxy) (tert-butyl) dimethylsilane (5.1 g, 21.3 mmol) in N, N-dimethylformamide (40 mL) was added (S) -1- (4- (trifluoromethoxy) phenyl) ethylamine hydrochloride 1-6 (4.5 g, 18.6 mmol) , potassium carbonate (7.74 g, 56.0 mmol) , and sodium iodide (0.42 g, 2.80 mmol) at room temperature. After stirred at 95 ℃ for 16 hours under nitrogen atmosphere, the mixture was cooled down to room temperature, di-tert-butyl dicarbonate (12.2 g, 55.9 mmol) , water (20 mL) and sodium carbonate (5.9 g, 55.7 mmol) was added at 0 ℃, then the mixture was stirred at at room temperature for 3 hours, and poured into water (250 mL) , extracted with ethyl acetate (200 mL) twice. The combined organic layers were concentrated. The resulting residue was purified by C18 (acetonitrile: water (+ 0.02 %ammonium acetate) = 15 %to 100 %) to give the title compound (5.5 g, 80 %purity, 51 %yield) as colorless oil. LC-MS (ESI) : R _T = 1.68 min, mass calcd. for C ₂₂H ₃₆F ₃NO ₄Si 463.2, m/z found 464.2 [M+H] ⁺.

Intermediate 1-8:

(S) -tert-Butyl (2-hydroxyethyl) (1- (4- (trifluoromethoxy) phenyl) ethyl) carbamate

To a mixture of (S) -tert-butyl (2- ( (tert-butyldimethylsilyl) oxy) ethyl) (1- (4- (trifluoromethoxy) phenyl) ethyl) carbamate 1-7 (5.5 g, 80 %purity, 9.49 mmol) in tetrahydrofuran (25 mL) was added 1 M tetrabutylammonium fluoride in tetrahydrofuran (28 mL) at room temperature. After stirred at room temperature for 2 hours, the mixture was concentrated, water (150 mL) was added, then the mixture was extracted with ethyl acetate (150 mL) twice. The combined organic layers were concentrated. The resulting residue was purified by C18 (acetonitrile : water (+ 0.02 %ammonium acetate) = 10 %to 75 %) to give the desired product (3.25 g, 100 %purity, 98 %yield) colourless oil. LC-MS (ESI) : R _T = 1.74 min, mass calcd. for C ₁₆H ₂₂F ₃NO ₄ 349.2, m/z found 294.2 [M+H-56] ⁺.

Intermediate 1-9:

(R) -Ethyl 2- (2- ( (tert-butoxycarbonyl) ( (S) -1- (4- (trifluoromethoxy) phenyl) ethyl) amino) ethyl) -5- (4-chloro-3-cyanobenzoyl) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3- c] pyridine-3-carboxylate

To a mixture of (S) -tert-butyl (2-hydroxyethyl) (1- (4- (trifluoromethoxy) phenyl) ethyl) carbamate 1-8 (3.0 g, 100 %purity, 8.59 mmol) , (R) -ethyl 5- (4-chloro-3-cyanobenzoyl) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylate 1-4 (3.8 g, 100 %purity, 10.2 mmol) and triphenylphosphine (3.4 g, 13.0 mmol) in dry tetrahydrofuran (30 mL) was added di-tert-butyl azodicarboxylate (3.0 g, 13.0 mmol) was added at 0 ℃. After stirred for 2 hours at 50 ℃ under N ₂ atmosphere, the mixture was cooled down to room temperature and concentrated under reduced pressure. The resulting residue was diluted with water (150 mL) , extracted with ethyl acetate (150 mL) twice. The combined organic layers were concentrated under reduced pressure, The resulting residue was purified by C18 (acetonitrile : water (+ 0.02 %ammonium acetate) = 15 %to 85 %) to give the title compound (4.0 g, 95 %purity, 63 %yield) as white solids. LC-MS (ESI) : R _T = 2.01 min, mass calcd. for C ₃₄H ₃₇ClF ₃N ₅O ₆ 703.2, m/z found 704.0 [M+H] ⁺.

Intermediate 1-10:

(R) -2- (2- ( (tert-Butoxycarbonyl) - ( (S) -1- (4- (trifluoromethoxy) phenyl) ethyl) amino) ethyl) -5- (4-chloro-3-cyanobenzoyl) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylic acid

To a mixture of (R) -ethyl 2- (2- ( (tert-butoxycarbonyl) ( (S) -1- (4- (trifluoromethoxy) phenyl) ethyl) amino) ethyl) -5- (4-chloro-3-cyanobenzoyl) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylate 1-9 (4.0 g, 95 %purity, 5.40 mmol) in tetrahydrofuran (48 mL) and methanol (16 mL) was added dropwise lithium hydroxide hydrate (684 mg, 16.3 mmol) in water (16 mL) at 0 ℃. After stirred for 2 hours at room temperature, the mixture was concentrated under reduced pressure. The resulting residue was diluted with water (60 mL) and adjusted to pH 4 ～ 5 with 1 M hydrochloride aqueous solution, extracted with ethyl acetate (60 mL) twice. The combined organic layers were concentrated under reduced pressure to give the title compound (3.6 g, 100 %purity, 99 %yield) as white solids. LC-MS (ESI) : R _T = 1.50 min, mass calcd. for C ₃₂H ₃₃ClF ₃N ₅O ₆ 675.2, m/z found 676.0 [M+H] ⁺.

Intermediate 1-11:

(R) -5- (4-Chloro-3-cyanobenzoyl) -6-methyl-2- (2- ( ( (S) -1- (4- (trifluoromethoxy) phenyl) ethyl) amino) ethyl) -4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylic acid

A mixture of (R) -2- (2- ( (tert-butoxycarbonyl) ( (S) -1- (4- (trifluoromethoxy) phenyl) ethyl) amino) ethyl) -5- (4-chloro-3-cyanobenzoyl) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylic acid 1-10 (3.6 g, 100 %purity, 5.33 mmol) in 4 M hydrochloride in dioxane (70 mL) was stirred at room temperature for 2 hours under nitrogen atmosphere. LCMS showed the material was consumed. The mixture was concentrated under reduced pressure to give the title compound (3.1 g, 97 %purity, 92 %yield) as white solids without further purification. LC-MS (ESI) : R _T = 1.46 min, mass calcd. for C ₂₇H ₂₅ClF ₃N ₅O ₄ 575.2, m/z found 576.0 [M+H] ⁺.

Compound 1:

2-Chloro-5- ( (R) -3-methyl-10-oxo-9- ( (S) -1- (4- (trifluoromethoxy) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

To a mixture of (R) -5- (4-chloro-3-cyanobenzoyl) -6-methyl-2- (2- ( ( (S) -1- (4- (tri fluoromethoxy) phenyl) ethyl) amino) ethyl) -4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylic acid hydrochloride 1-11 (3.0 g, 97 %purity, 4.75 mmol) and N-ethyl-N-isopropylpropan-2-amine (2.4 mL, 14.5 mmol) in N, N-dimethylformamide (35 mL) was added 2- (7-azabenzotriazol-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate (2.7 g, 7.10 mmol) at 0 ℃. After stirred for 1 hour at room temperature, the mixture was poured into water (200 mL) and adjusted to pH 4 ～ 5 with 1 M hydrochloride aqueous solution, extracted with ethyl acetate (200 mL) twice. The combined organic layers were concentrated under reduced pressure. The resulting residue was purified by C18 (acetonitrile : water (+ 0.02 %ammonium acetate) = 10 %to 70 %) to give the title compound (2.5 g, 98.7 %purity, 93 %yield) as white solids, which was combined with another batch (850 mg, 95.9 %purity, 1.46 mmol) , dissolved in ethyl acetate (5 mL) at 0 ℃. After stirred at 0 ℃ for 10 minutes, the mixture was added petroleum ether (25 mL) . The resulting mixture was filtered, and the filter cake was concentrated to remove ethyl acetate and petroleum ether. The resulting white solids was dissolved in acetonitrile (7 mL) and stirred for 10 minutes at 0 ℃, the white solids were collected by filtration to give title compound (3.151 g, 99.1 %purity, 94.4 %yield) . LC-MS (ESI) : R _T = 8.644 min, mass calcd. for C ₂₇H ₂₃ClF ₃N ₅O ₃ 557.1, m/z found 558.1 [M+H] ⁺. ¹H NMR (400 MHz, CDCl ₃) δ 7.75 (S, 1H) , 7.65 -7.57 (m, 2H) , 7.44 -7.33 (m, 2H) , 7.25 -7.18 (m, 2H) , 6.23 -5.93 (m, 1H) , 5.83 -5.29 (m, 1H) , 4.94 -4.42 (m, 2H) , 4.33 -4.22 (m, 1H) , 4.21 -4.08 (m, 1H) , 3.64 -3.56 (m, 1H) , 3.36 -3.23 (m, 1H) , 3.19 -2.93 (m, 1H) , 2.67 (d, J = 16.4 Hz, 1H) , 1.60 (d, J = 6.8 Hz, 3H) , 1.40 -1.19 (m, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -57.89 (S, 3F) .

Compound 2A and 2B:

2, 6-Dichloro-3- ( (R) -9- ( (R*) -1- (4- (difluoromethoxy) phenyl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile and

2, 6-dichloro-3- ( (R) -9- ( (S*) -1- (4- (difluoromethoxy) phenyl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

Intermediate 2-2:

2- ( (tert-Butyldimethylsilyl) oxy) -N- (1- (4- (difluoromethoxy) phenyl) ethyl) ethanamine

To a solution of 1- (4- (difluoromethoxy) phenyl) ethanone 2-1 (2.0 g, 10.74 mmol) and 2- ( (tert-butyldimethylsilyl) oxy) ethanamine (3.8 g, 21.7 mmol) in tetrahydrofuran (10 mL) was added titanium (IV) propan-2-olate (9.2 g, 33.4 mmol) . After stirred at 70 ℃ for 16 hours, the reaction mixture was cooled to 0 ℃, added sodium borohydride (815 mg, 21.5 mmol) and stirred at room temperature for 1 hour. The reaction was quenched with methanol (10 mL) , poured into water (10 mL) , filtered with kieselguhr. The filtrate was concentrated to give the crude, which was purified by C18 column (acetonitrile: water = 50%to 95 %) to give the compound (2.51 g, 90 %purity from NMR, 60.9 %yield) as yellow oil. LC-MS (ESI) : R _T = 2.047 min, mass calcd. for C ₁₇H ₂₉F ₂NO ₂Si 345.5, m/z found 346.2 [M+H] ⁺. ¹H NMR (400 MHz, CDCl ₃) δ 7.31 (d, J = 8.4 Hz, 2H) , 7.06 (d, J = 8.4 Hz, 2H) , 6.49 (t, J = 74.4 Hz, 1H) , 3.78 (q, J = 6.8 Hz, 1H) , 3.71 -3.63 (m, 2H) , 2.61 -2.47 (m, 2H) , 1.33 (d, J = 6.8 Hz, 3H) , 0.89 (S, 9H) , 0.05 -0.04 (m, 6H) .

Intermediate 2-3:

2- ( (1- (4- (Difluoromethoxy) phenyl) ethyl) amino) ethanol

The a solution of 2- ( (tert-butyldimethylsilyl) oxy) -N- (1- (4- (difluoromethoxy) phenyl) ethyl) ethanamine 2-2 (2.51 g, 90 %purity, 6.54 mmol) in tetrahydrofuran (5 mL) was added 1 M tetrabutylammonium fluoride solution (5 mL) . After stirred at room temperature for 1 hour, the mixture was diluted with water (30 mL) , extracted with ethyl acetate (30 mL) twice. The combined organic layers were washed with brine (30 mL) , dried over Na ₂SO _4 (S) , filtered. The filtrate was concentrated, purified by C18 column (acetonitrile: water = 45 %to 55 %) to give the title compound (2 g, 65 %purity from NMR, 86.0 %yield) as colorless oil. LC-MS (ESI) : R _T = 1.36 min, mass calcd. for C ₁₁H ₁₅F ₂NO ₂ 231.2, m/z found 232.2 [M+H] ⁺. ¹H NMR (400 MHz, CDCl ₃) δ 7.30 (d, J = 8.0 Hz, 2H) , 7.07 (d, J = 8.4 Hz, 2H) , 6.49 (t, J = 74.0 Hz, 1H) , 3.77 (q, J = 6.4 Hz, 1H) , 3.64 -3.53 (m, 2H) , 2.69 -2.55 (m, 2H) , 1.35 (d, J = 6.4 Hz, 3H) .

Intermediate 2-4:

(6R) -5-tert-Butyl 3-ethyl 2- (2- ( (1- (4- (difluoromethoxy) phenyl) ethyl) amino) ethyl) -6-methyl-6, 7-dihydro-2H-pyrazolo [4, 3-c] pyridine-3, 5 (4H) -dicarboxylate

To a solution of 2- ( (1- (4- (difluoromethoxy) phenyl) ethyl) amino) ethanol 2-3 (1.0 g, 65 %purity, 2.81 mmol) , 5- (tert-butyl) 3-ethyl (R) -6-methyl-2, 4, 6, 7-tetrahydro-5H-pyrazolo [4, 3-c] pyridine-3, 5-dicarboxylate 1-1 (CAS number: 2171134-54-4) (1.9 g, 92 %purity, 5.66 mmol) and triphenylphosphine (1.5 g, 5.72 mmol) in tetrahydrofuran (10 mL) was added di-tert-butyl azodicarboxylate (1.3 g, 5.65 mmol) at 0 ℃. After stirred at room temperature for 3 hours, the reaction mixture was quenched with water (50 mL) , extracted with ethyl acetate (50 mL) twice. The combined organic layers were washed with brine (30 mL) , dried over Na ₂SO _4 (S) , filtered. The filtrate was concentrated, purified by C18 column (acetonitrile: water = 70 %to 90 %) to give the title compound (1.2 g, 71.0 %purity from LCMS, 58.0 %yield) as colorless oil. LC-MS (ESI) : R _T = 1.943 min, mass calcd. for C ₂₆H ₃₆F ₂N ₄O ₅ 522.6, m/z found 523.3 [M+H] ⁺.

Intermediate 2-5:

(6R) -5- (tert-Butoxycarbonyl) -2- (2- ( (1- (4- (difluoromethoxy) phenyl) ethyl) amino) ethyl) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylic acid

To a solution of (6R) -5-tert-butyl 3-ethyl 2- (2- ( (1- (4- (difluoromethoxy) phenyl) ethyl) amino) ethyl) -6-methyl-6, 7-dihydro-2H-pyrazolo [4, 3-c] pyridine-3, 5 (4H) -dicarboxylate 2-4 (1.2 g, 71 %purity, 1.63 mmol) in tetrahydrofuran (2 mL) and methanol (2 mL) was added a solution of lithium hydroxide monohydrate (500 mg, 11.9 mmol) in water (2 mL) at 0 ℃. After stirred 0 ℃ for 1 hours, the mixture was acidified to pH = 6 with 0.1M hydrochloride aqueous solution and extracted with ethyl acetate (50 mL) twice. The combined organic layers were washed with water (50 mL) for three times and brine (30 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated under reduced pressure to give the title compound (1.18 g, 66.8 %purity from LCMS, 97.8 %yield) as yellow solids. LC-MS (ESI) : R _T = 1.456 min, mass calcd. for C ₂₄H ₃₂F ₂N ₄O ₅ 494.5, m/z found 495.3 [M+H] ⁺.

Intermediate 2-6:

(3R) -tert-Butyl 9- (1- (4- (difluoromethoxy) phenyl) ethyl) -3-methyl-10-oxo-3, 4, 7, 8, 9, 10-hexahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2 (1H) -carboxylate

A mixture of (6R) -5- (tert-butoxycarbonyl) -2- (2- ( (1- (4- (difluoromethoxy) phenyl) ethyl) amino) ethyl) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylic acid 2-5 (1.18 g, 66.8 %purity, 1.59 mmol) , N-ethyl-N-isopropylpropan-2-amine (1.12 g, 8.67 mmol) and 2- (3H- [1, 2, 3] triazolo [4, 5-b] pyridin-3-yl) -1, 1, 3, 3-tetramethylisouronium hexafluorophosphate (1.0 g, 2.63 mmol) in N, N-dimethylformamide (2 mL) was stirred at 30 ℃ overnight. The mixture was purified by C18 column (acetonitrile: water = 55 %to 85 %) to give the title compound (540 mg, 90 %purity from NMR, 64.0 %yield) as yellow solids. LC-MS (ESI) : R _T = 1.74 min, mass calcd. for C ₂₄H ₃₀F ₂N ₄O ₄ 476.5, m/z found 477.2 [M+H] ⁺. ¹H NMR (400 MHz, CDCl ₃) δ 7.39 -7.36 (m, 2H) , 7.12 (d, J = 8.0 Hz, 2H) , 6.52 (t, J = 73.6 Hz, 1H) , 6.09 (q, J = 7.2 Hz, 1H) , 5.23 -5.08 (m, 1H) , 4.90 (br s, 1H) , 4.30 -4.21 (m, 2H) , 4.15 -4.06 (m, 1H) , 3.65 -3.56 (m, 1H) , 3.29 -3.22 (m, 1H) , 2.98 -2.93 (m, 1H) , 2.58 (d, 16 Hz, 1H) , 1.60 (S, 3H) , 1.49 (d, J = 1.6 Hz, 9H) , 1.14 (t, J = 5.6 Hz, 3H) .

Intermediate 2-7:

(3R) -9- (1- (4- (Difluoromethoxy) phenyl) ethyl) -3-methyl-1, 2, 3, 4, 8, 9-hexahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazin-10 (7H) -one hydrochloride

To a solution of (3R) -tert-butyl 9- (1- (4- (difluoromethoxy) phenyl) ethyl) -3-methyl-10-oxo-3, 4, 7, 8, 9, 10-hexahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2 (1H) -carboxylate 2-6 (140 mg, 90 %purity, 0.26 mmol) in 1, 4-dioxane (1 mL) was added 3 M hydrochloride in 1, 4-dioxane (2 mL, 6 mmol) . After stirred at room temperature for 2 hours under nitrogen atmosphere, the mixture was filtrated to give the title compound (100 mg, 91.3 %purity from LCMS, 83.6 %yield) as white solids. LC-MS (ESI) : R _T = 1.49 min, mass calcd. for C ₁₉H ₂₂F ₂N ₄O ₂ 376.4, m/z found 377.2 [M+H] ⁺.

Intermediate 2-9:

1-Bromo-2, 4-dichloro-3-nitrobenzene

To a solution of 1, 3-dichloro-2-nitrobenzene 2-8 (5 g, 26.0 mmol) in sulfuric acid (120 mL) was added N-bromosuccinimide (5.13 g, 28.8 mmol) at 0 ℃. After stirred at 40 ℃ for 14 hours, the mixture was added into water (500 mL) and extracted with ethyl acetate (100 mL) twice. The combined organic layers were washed with water (50 mL) for three times and brine (50 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated under reduced pressure to get a residue, which was purified by flash silica gel chromatography ( (120 g) ; petroleum ether in ethyl acetate, 10 : 90, 45 mL /min) to give the title compound (5.1 g, 90 %purity from H NMR, 65 %yield) as white solids. ¹HNMR (400 MHz, DMSO-d ₆) δ 8.10 (d, J = 8.8 Hz, 1H) , 7.78 (d, J = 9.2 Hz, 1H) .

Intermediate 2-10:

Methyl 3-amino-2, 4-dichlorobenzoate

To a solution of 1-bromo-2, 4-dichloro-3-nitrobenzene 2-9 (3 g, 90 %purity, 9.97 mmol) and triethylamine (3 g, 29.6 mmol) in methanol (20 mL) and acetonitrile (5 mL) was added 1, 1'-bis (diphenylphosphino) ferrocene (551 mg, 0.994 mmol) and palladium (II) acetate (180 mg, 0.802 mmol) . After stirred at 100 ℃ under carbon monoxide atmosphere (5 Mpsi) for 14 hours, the mixture was concentrated under reduced pressure to give a residue, which was purified by flash silica gel chromatography ( (40 g) ; petroleum ether in ethyl acetate, 20 : 80 to 30 : 70, 40mL /min) to give the title compound (800 mg, 90 %purity from HNMR, 33 %yield) as yellow solids. ¹HNMR (400 MHz, DMSO-d ₆) δ 7.33 (d, J = 8.8 Hz, 1H) , 6.94 (d, J = 8.0 Hz, 1H) , 5.80 (br s, 2H) , 3.83 (S, 3H) .

Intermediate 2-11:

Methyl 2, 4-dichloro-3-cyanobenzoate

A solution of copper (I) cyanide (880 mg, 9.83 mmol) in dimethyl sulfoxide (10 mL) was added tert-butyl nitrite (2.02 g, 19.6 mmol) at 60 ℃. After stirred at 60 ℃ for 1 hour, methyl 3-amino-2, 4-dichlorobenzoate 2-10 (800 mg, 90 %purity, 3.27 mmol) in dimethyl sulfoxide (10 mL) was added into the mixture. After stirred at 60 ℃ for 14 hours, the mixture was added water (50 mL) and 30 %ammonium hydroxide in water (6 mL) and extracted with ethyl acetate (50 mL) twice. The combined organic layers were washed with water (50 mL) for three times and brine (50 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated under reduced pressure to get a residue, which was purified by flash silica gel chromatography ( (40 g) ; petroleum ether in ethyl acetate, 10 : 90, 35 mL /min) to give the title compound (260 mg, 90 %purity from H NMR, 31 %yield) as yellow solids. ¹HNMR (400 MHz, CDCl ₃) δ 7.00 (d, J = 8.8 Hz, 1H) , 7.51 (d, J = 8.8 Hz, 1H) , 3.97 (S, 3H) .

Intermediate 2-12:

2, 4-Dichloro-3-cyanobenzoic acid

To a solution of methyl 2, 4-dichloro-3-cyanobenzoate 2-11 (260 mg, 90 %purity, 1.02 mmol) in tetrahydrofuran (10 mL) and methanol (3 mL) was added a solution of lithium hydroxide monohydrate (256 mg, 6.10 mmol) in water (3 mL) at 0 ℃. After stirred 0 ℃ for 1 hours, the mixture was acidified to pH = 6 with 0.1M hydrochloride aqueous solution and extracted with ethyl acetate (30 mL) twice. The combined organic layers were washed with water (30 mL) for three times and brine (30 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated under reduced pressure to give the title compound (220 mg, 90 %purity from HNMR, 90 %yield) as yellow solids. ¹H NMR (400 MHz, DMSO-d ₆) δ 14.04 (br s, 1H) , 8.11 (d, J = 8.4 Hz, 1H) , 7.84 (d, J = 8.8 Hz, 1H) .

Compound 2:

2, 6-Dichloro-3- ( (3R) -9- (1- (4- (difluoromethoxy) phenyl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

A mixture of (3R) -9- (1- (4- (difluoromethoxy) phenyl) ethyl) -3-methyl-1, 2, 3, 4, 8, 9-hexahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazin-10 (7H) -one hydrochloride 2-7 (310 mg, 99 %purity, 0.743 mmol) , N-ethyl-N-isopropylpropan-2-amine (781 mg, 6.04 mmol) , 2, 4-dichloro-3-cyanobenzoic acid 2-12 (178 mg, 0.742 mmol) and 2- (3H- [1, 2, 3] triazolo [4, 5-b] pyridin-3-yl) -1, 1, 3, 3-tetramethylisouronium hexafluorophosphate (V) (383 mg, 1.01 mmol) in N, N-dimethylformamide (6 mL) was stirred at 30 ℃ under nitrogen for 14 hours. The mixture was acidified to pH = 6 with 0.05M hydrochloride aqueous solution and extracted with ethyl acetate (20 mL) twice. The combined organic layers were washed with water (30 mL) for three times and brine (30 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated under reduced pressure to give a residue. Which was purified by C18 column (acetonitrile: water = 65 %to 70 %) to give the title compound (310 mg, 98 %purity from LCMS, 71 %yield) as yellow solids. R _T = 1.67 min, mass calcd. for C ₂₇H ₂₃Cl ₂F ₂N ₅O ₃ 573.1 m/z found 574.0 [M+H] ⁺.

Compounds 2A and 2B:

A racemate of 2, 6-dichloro-3- ( (3R) -9- (1- (4- (difluoromethoxy) phenyl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 2 (310 mg, 98 %purity, 0.529 mmol) was separated by chiral Prep. HPLC (Separation condition: Column: Chiralpak OD 10 μm 25 *250 mm; Mobile Phase: Hexane : EtOH = 70 : 30 at 30 mL/min; Temp: 38 ℃; Wavelength: 254 nm) to afford the title compounds 2A (51.1 mg, 99.4 %purity, 17 %yield) as white solids and 2B (53 mg, 99.7 %purity, 17 %yield) as white solids.

2A:

R _T = 2.871 min, mass calcd. for C ₂₇H ₂₃Cl ₂F ₂N ₅O ₃ 573.1 m/z found 574.1 [M+H] ⁺.

Chiral HPLC (Column: Chiralpak OD-H 5 μm 4.6 *150 mm; Mobile Phase: Hexane : EtOH = 70 : 30 at 1.0 mL/min; Temp: 30 ℃; Wavelength: 254 nm, R _T = 6.436 min) .

¹H NMR (400 MHz, CDCl ₃) δ 7.54 -7.29 (m, 4H) , 7.15 -7.09 (m, 2H) , 6.52 (t, J = 74.0, 0.6H) , 6.51 (d, J = 74.0, 0.4H) , 6.15 -6.08 (m, 0.4H) , 5.96 -5.81 (m, 1H) , 5.57 -5.49 (m, 0.6H) , 4.57 -4.27 (m, 2H) , 4.25 -3.95 (m, 2H) , 3.65 -3.56 (m, 1H) , 3.34 -3.22 (m, 1H) , 3.15 -3.06 (m, 1H) , 2.75 -2.54 (m, 1H) , 1.61 -1.57 (m, 3H) , 1.32 -1.17 (m, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -81.07 --81.20 (m, 2F) .

2B:

R _T = 2.945 min, mass calcd. for C ₂₇H ₂₃Cl ₂F ₂N ₅O ₃ 573.1 m/z found 574.1 [M+H] ⁺.

Chiral HPLC (Column: Chiralpak OD-H 5 μm 4.6 *150 mm; Mobile Phase: Hexane : EtOH = 70 : 30 at 1.0 mL/min; Temp: 30 ℃; Wavelength: 254 nm, R _T = 8.202 min) .

¹H NMR (400 MHz, CDCl ₃) δ 7.54 -7.33 (m, 4H) , 7.15 -7.11 (m, 2H) , 6.52 (t, J = 73.6, 0.4H) , 6.51 (t, J = 73.6, 0.6H) 6.11 -5.94 (m, 1H) , 5.87 -5.78 (m, 0.4H) , 5.57 -5.50 (m, 0.6H) , 4.64 -3.95 (m, 4H) , 3.70 -3.56 (m, 1H) , 3.34 -3.28 (m, 1H) , 3, 15 -3.04 (m, 1H) , 2.77 -2.56 (m, 1H) , 1.62 -1.54 (m, 3H) , 1.34 -1.19 (m, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -81.07 --81.17 (m, 2F) .

Compounds 3A and 3B:

6-Chloro-3- ( (R) -9- ( (R*) -1- (4- (difluoromethoxy) phenyl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) -2-methylbenzonitrile and

6-chloro-3- ( (R) -9- ( (S*) -1- (4- (difluoromethoxy) phenyl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) -2-methylbenzonitrile

Intermediate 3-2:

4-Chloro-3-hydroxy-2-methylbenzoic acid

To a suspension of 3-hydroxy-2-methylbenzoic acid 3-1 (5.0 g, 32.9 mmol) in water (50 mL) was added 3 M sodium hydroxide aqueous solution (15 mL, 45 mmol) at 0 ℃. Then sodium hypochlorite (50 mL, 5.5%purity, 46.2 mmol) was added dropwise over about 30 minutes. After the addition was completed, the mixture was added 3 M hydrochloride aqueous solution (70 mL) , then filtrated and washed with water to give the title compound (2.0 g, 90 %purity from ¹H NMR, 29.4 %yield) as brown solids. LC-MS (ESI) : R _T = 0.411 min, mass calcd. for C ₈H ₇ClO ₃ 186.0, m/z found 185.1 [M-H] ^-. ¹H NMR (400 MHz, CDCl ₃) δ 7.32 -7.29 (m, 1H) , 7.21 -7.19 (m, 1H) , 2.46 (S, 3H) .

Intermediate 3-3:

Methyl 4-chloro-3-hydroxy-2-methylbenzoate

To a solution of 4-chloro-3-hydroxy-2-methylbenzoic acid 3-2 (2.0 g, 90 %purity, 9.65 mmol) in methanol (30 mL) was added sulfuric acid (3 mL) at 0 ℃. After stirred at 60 ℃ for 3 hours, the mixture was cooled, concentrated under reduced pressure to remove methanol, dissolved with ethyl acetate (30 mL) and washed with saturated sodium bicarbonate solution (30 mL) twice, brine (30 mL) . The organic layers were dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated to give the compound (1.8 g, 90 %purity from NMR, 83.7 %yield) as brown solids. LC-MS (ESI) : R _T = 1.55 min, mass calcd. for C ₉H ₉ClO ₃ 200.2, m/z found 199.1 [M-H] ^-. ¹H NMR (400 MHz, CDCl ₃) δ 7.38 (d, J = 8.4 Hz, 1H) , 7.21 (d, J = 8.4 Hz, 1H) , 3.89 (S, 3H) , 2.51 (S, 3H) .

Intermediate 3-4:

Methyl 4-chloro-2-methyl-3- ( ( (trifluoromethyl) sulfonyl) oxy) benzoate

To a solution of methyl 4-chloro-3-hydroxy-2-methylbenzoate 3-3 (1.8 g, 90 %purity, 8.06 mmol) and triethylamine (3 mL, 21.6 mmol) in dichloromethane (10 mL) was added slowly trifluoromethanesulfonic anhydride (2 mL, 11.9 mmol) at 0 ℃. The mixture was warmed to room temperature and stirred for 3 hours. The mixture was concentrated and purified The mixture was purified by C18 column (acetonitrile: water = 90 %to 95 %) to give the compound (2 g, 90 %purity from NMR, 55.8 %yield) as brown oil. ¹H NMR (400 MHz, CDCl ₃) δ 7.85 (d, J = 8.4 Hz, 1H) , 7.42 (d, J = 8.4 Hz, 1H) , 3.92 (S, 3H) , 2.62 (S, 3H) .

Intermediate 3-5:

Methyl 4-chloro-3-cyano-2-methylbenzoate

A mixture of methyl 4-chloro-2-methyl-3- ( ( (trifluoromethyl) sulfonyl) oxy) benzoate 3-4 (1.0 g, 75 %purity, 2.25 mmol) , tetrakis (triphenylphosphine) palladium (280 mg, 0.24 mmol) and zinc cyanide (200 mg, 1.70 mmol) in N, N-dimethylformamide (10 mL) . After stirred at 120 ℃ for 3 hours, the mixture was cooled down, diluted with water (100 mL) and extracted with ethyl acetate (30 mL) twice. The combined organic layers were washed with brine (30 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated under reduced pressure to give the crude compound, which was purified by silica gel column chromatography (petroleum ether : ethyl acetate = 4 : 1) to give the title compound (313 mg, 90 %purity from HNMR, 59.6 %yield) as yellow oil. ¹H NMR (400 MHz, CDCl ₃) δ 8.02 (d, J = 8.4 Hz, 1H) , 7.16 (d, J = 8.4 Hz, 1H) , 3.93 (S, 3H) , 2.84 (S, 3H) .

Intermediate 3-6:

4-Chloro-3-cyano-2-methylbenzoic acid

To a solution of methyl 4-chloro-3-cyano-2-methylbenzoate 3-5 (313 mg, 90 %purity, 1.34 mmol) in tetrahydrofuran (2 mL) and methanol (2 mL) was added a solution of lithium hydroxide monohydrate (320 mg, 7.63 mmol) in water (2 mL) at 0 ℃. After stirred 0 ℃ for 1 hour, the mixture was acidified to pH = 6 with 0.1M hydrochloride aqueous solution and extracted with ethyl acetate (20 mL) twice. The combined organic layers were washed with water (20 mL) for three times and brine (20 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated under reduced pressure to give the title compound (260 mg, 100 %purity from LCMS, 98.9 %yield) as yellow solids. LC-MS (ESI) : R _T = 0.67 min, mass calcd. for C ₉H ₆ClNO ₂ 195.0, m/z found 194.0 [M-H] ^-.

Compound 3:

6-Chloro-3- ( (3R) -9- (1- (4- (difluoromethoxy) phenyl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) -2-methylbenzonitrile

A mixture of (3R) -9- (1- (4- (difluoromethoxy) phenyl) ethyl) -3-methyl-1, 2, 3, 4, 8, 9-hexahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazin-10 (7H) -one hydrochloride 2-7 (150 mg, 97.3 %purity, 0.35 mmol) , N-ethyl-N-isopropylpropan-2-amine (330 mg, 2.55 mmol) , 4-chloro-3-cyano-2-methylbenzoic acid 3-6 (90 mg, 100 %purity, 0.46 mmol) and 2- (3H- [1, 2, 3] triazolo [4, 5-b] pyridin-3-yl) -1, 1, 3, 3-tetramethylisouronium hexafluorophosphate (330 mg, 0.87 mmol) in N, N-dimethylformamide (1 mL) was stirred at 30 ℃ overnight. The mixture was purified by C18 column (acetonitrile: water = 55 %to 95 %) to give the compound (160 mg, 91.1 %purity from LCMS, 74.4 %yield) as yellow solids. LC-MS (ESI) : R _T = 1.66 min, mass calcd. for C ₂₈H ₂₆ClF ₂N5O ₃ 553.2, m/z found 554.0 [M+H] ⁺.

Compounds 3A and 3B:

A racemic mixture of 6-chloro-3- ( (R) -9- (1- (4- (difluoromethoxy) phenyl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) -2-methylbenzonitrile 3 (160 mg, 91.1 %purity, 0.26 mmol) was separated by chiral Prep. HPLC (Separation conditon: Column: Chiralpak IB N-5 5 μm 20 *250 mm; Mobile Phase: CO ₂ : EtOH = 75 : 25 at 25 g/min; Temp: 40 ℃; Wavelength: 230 nm, Back pressure: 100 bar) ) to give the title compounds 3A (50.6 mg, 98.7 %purity from LCMS, 34.3 %yield, 100 %ee) and 3B (53.7 mg, 99.1 %puirty from LCMS, 36.5 %yield, 100 %ee) as white solids.

3A:

LC-MS (ESI) : R _T = 3.698 min, mass calcd. for C ₂₈H ₂₆ClF ₂N ₅O ₃ 553.9, m/z found 554.2 [M+H] ⁺. Chiral analysis (Column: Chiralpak IB N-5 5 μm 4.6 *250 mm; Mobile Phase: CO ₂: MeOH (0.2 %DEA) = 75 : 25 at 3 mL/min; Col. Temp: 40 ℃; Wavelength: 230 nm, Back pressure: 100 bar; R _T = 8.51 min) . ¹H NMR (400 MHz, CDCl ₃) δ 7.45 -7.28 (m, 4H) , 7.14 -7.10 (m, 2H) , 6.52 (t, J = 74.0 Hz, 1H) , 6.16 -6.10 (m, 0.4H) , 5.99 -5.85 (m, 1H) , 5.60 -5.53 (m, 0.6H) , 4.61 -3.97 (m, 4H) , 3.63 -3.57 (m, 1H) , 3.32 -3.24 (m, 1H) , 3.11 -3.06 (m, 0.6H) , 2.88 -2.81 (m, 0.4H) , 2.75 -2.40 (m, 4H) , 1.70 -1.61 (m, 2H) , 1.56 -1.54 (m, 1H) , 1.29 -1.19 (m, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -81.07 --81.22 (m, 2F) .

3B:

LC-MS (ESI) : R _T = 3.729 min, mass calcd. for C ₂₈H ₂₆ClF ₂N ₅O ₃ 553.9, m/z found 554.2 [M+H] ⁺. Chiral analysis (Column: Chiralpak IB N-5 5 μm 4.6 *250 mm; Mobile Phase: CO ₂: MeOH (0.2 %DEA) = 75 : 25 at 3 mL/min; Col. Temp: 40 ℃; Wavelength: 230 nm, Back pressure: 100 bar; R _T = 10.5 min) . ¹H NMR (400 MHz, CDCl ₃) δ 7.45 -7.29 (m, 4H) , 7.15 -7.10 (m, 2H) , 6.52 (t, J = 73.6 Hz, 0.4H) , 6.51 (t, J = 73.6 Hz, 0.6H) , 6.12 -6.07 (m, 0.4H) , 6.00 -5.95 (m, 0.6H) , 5.88 -5.82 (m, 0.4H) , 5.60 -5.53 (m, 0.6H) , 4.56 -3.96 (m, 4H) , 3.72 -3.56 (m, 1H) , 3.31 -3.26 (m, 1H) , 3.11 -3.06 (m, 0.6H) , 2.89 -2.50 (m, 3.4H) , 2.38 (S, 1H) , 1.65 -1.60 (m, 2H) , 1.57 -1.50 (m, 1H) , 1.30 -1.20 (m, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -81.08 --81.17 (m, 2F) .

Compounds 4A and 4B:

2-Chloro-5- ( (R) -3-methyl-10-oxo-9- ( (R*) -1- (4- (trifluoromethyl) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile and

2-Chloro-5- ( (R) -3-methyl-10-oxo-9- ( (S*) -1- (4- (trifluoromethyl) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

Intermediate 4-2:

(R) -5-tert-Butyl 3-ethyl 2- (2- ( ( (benzyloxy) carbonyl) amino) ethyl) -6-methyl-6, 7-di hydro-2H-pyrazolo [4, 3-c] pyridine-3, 5 (4H) -dicarboxylate

To a solution of (R) -5-tert-butyl 3-ethyl 6-methyl-6, 7-dihydro-2H-pyrazolo [4, 3-c] pyri dine-3, 5 (4H) -dicarboxylate 1-1 (30 g, 90 %purity, 87.3 mol) , triphenylphosphine (34.3 g, 131 mmol) and benzyl (2-hydroxyethyl) carbamate 4-1 (20.5 g, 105 mmol) in tetrahydrofuran (500 mL) was added (E) -diisopropyl diazene-1, 2-dicarboxylate (26.5 g, 131 mmol) dropwise at 0 ℃. After stirred at 50 ℃ under nitrogen atmosphere, the reaction mixture was poured into water (300 mL) and extracted with ethyl acetate (300 mL) for three times. The combined organic phases were washed with brine (200 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4 : 1) to give the title compound (37 g, 90 %purity from HNMR, 78 %yield) as yellow oil. LC-MS (ESI) : R _T = 1.78 min, mass calcd. for C ₂₅H ₃₄N ₄O ₆ 486.2, m/z found 431.0 [M-56] ⁺. ¹H NMR (400 MHz, CDCl ₃) δ 7.37 -7.29 (m, 5H) , 5.28 -5.24 (m, 1H) , 5.08 -4.96 (m, 3H) , 4.75 -4.58 (m, 2H) , 4.39 -4.28 (m, 2H) , 4.15 -4.06 (m, 2H) , 3.66 -3.65 (m, 2H) , 2.90 (dd, J = 15.6 Hz, 6.0 Hz, 1H) , 2.56 (d, J = 16.0 Hz, 1H) , 1.49 (S, 9H) , 1.38 (t, J = 6.8 Hz, 3H) , 1.10 (d, J = 7.2 Hz, 3H) .

Intermediate 4-3:

(R) -tert-Butyl 3-methyl-10-oxo-3, 4, 7, 8, 9, 10-hexahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2 (1H) -carboxylate

To the solution of (R) -5-tert-butyl 3-ethyl 2- (2- ( ( (benzyloxy) carbonyl) amino) ethyl) -6-methyl-6, 7-dihydro-2H-pyrazolo [4, 3-c] pyridine-3, 5 (4H) -dicarboxylate 4-2 (37.0 g, 90 %purity, 68.4 mmol) in methanol (400 mL) was added 10 %wt. palladium on activated carbon (10.0 g, 9.40 mmol) . After stirred at 45 ℃ under hydrogen atmosphere 30 psi for 16 hours, the reaction mixture was filtered. The filtrate was concentrated under reduced pressure to give the title compound (23 g, 90 %purity from HNMR, 98 %yield) as whitle solids.

LC-MS (ESI) : R _T = 1.39 min, mass calcd. for C ₁₅H ₂₂N ₄O ₃ 306.1, m/z found 307.1 [M+H] ⁺. ¹H NMR (400 MHz, CDCl ₃) δ 5.95 (br s, 1H) , 5.13 -5.09 (m, 1H) , 4.94 -4.82 (m, 1H) , 4.34 (t, J = 6.0 Hz, 2H) , 4.24 -4.19 (m, 1H) , 3.79 -3.75 (m, 2H) , 2.97 (dd, J = 16.0, 6.0 Hz, 1H) , 2.59 (d, J = 16.0 Hz, 1H) , 1.48 (S, 9H) , 1.12 (d, J = 6.8 Hz, 3H) .

Intermediate 4-4:

(R) -3-Methyl-1, 2, 3, 4, 8, 9-hexahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazin-10 (7H) -one hydrochloride

To a solution of (R) -tert-butyl 3-methyl-10-oxo-3, 4, 7, 8, 9, 10-hexahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2 (1H) -carboxylate 4-3 (20 g, 90 %purity, 58.8 mmol) was added 4 M hydrochloride in dioxane (150 mL, 600 mmol) . The reaction mixture was stirred at room temperature under nitrogen atmosphere for 2 hours. The reaction mixture was concentrated under reduced pressure to give the title compound (16.0 g, 84 %purity from LCMS, 94 %yield) as white solids. LC-MS (ESI) : R _T = 0.420 min, mass calcd. for C ₁₀H ₁₄N ₄O 206.1, m/z found 207.0 [M+H] ⁺.

Intermediate 4-5:

(R) -2-Chloro-5- (3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

To a mixture of 4-chloro-3-cyanobenzoic acid 1-3 (10.0 g, 55.1 mmol) and oxalyl chloride (7.0 mL, 82.7 mmol) in dichloromethane (150 mL) was added N, N-dimethylformamide (100 mg, 1.37 mmol) at 0 ℃ under nitrogen atmosphere. After stirred at 25 ℃ for 2 hours, the reaction mixture was concentrated to give a crude A. To a mixture of A and (R) -3-methyl-1, 2, 3, 4, 8, 9-hexahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazin-10 (7H) -one hydrochloride 4-4 (16 g, 84 %purity, 55.4 mmol) in dichloromethane (150 mL) was added N, N-diisopropylethylamine (27 mL, 163 mmol) dropwise at 0 ℃. After stirred at 25 ℃ for 1 hour, the reaction mixture was acidized with 0.5 M hydrochloride aqueous solution to pH ～ 6 and extracted with ethyl acetate (200 mL) twice. The combined organic layers were washed with brine (100 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated to get a residue, which was purified by C18 column (acetonitrile: water = 20 %to 70 %) to give the title compound (16 g, 90 %purity from HNMR, 70 %yield) as white solids. LC-MS (ESI) : R _T = 1.37 min, mass calcd. for C ₁₈H ₁₆ClN ₅O ₂ 369.1, m/z found 370.0 [M+H] ⁺. ¹H NMR (400 MHz, CDCl ₃) δ 7.74 -7.72 (m, 1H) , 7.62 -7.55 (m, 2H) , 5.94 -5.30 (m, 2H) , 4.87 -4.16 (m, 4H) , 3.83 -3.73 (m, 2H) , 3.17 -2.93 (m, 1H) , 2.68 (d, J = 19.2 Hz, 1H) , 1.32 -1.18 (m, 3H) .

Intermediate 4-7:

1- (4- (Trifluoromethyl) phenyl) ethanol

To a solution of 1- (4- (trifluoromethyl) phenyl) ethanone 4-6 (2.5 g, 13.3 mmol) in methanol (25 mL) was slowly added sodium borohydride (1.0 g, 26.4 mmol) at 0 ℃. After stirred at room temperature for 3 hours, the reaction was quenched with acetone (20 mL) dropwise and concentrated to give a residue, which was dissolved into dichloromethane (50 mL) and washed with brine (50 mL) twice, dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated to give the title compound (2.6 g, 90 %purity from ¹HNMR, 93 %yield) as yellow oil. ¹H NMR (400 MHz, CDCl ₃) δ 7.60 (d, J = 8.0 Hz, 2H) , 7.49 (d, J = 8.0 Hz, 2H) , 5.01 -4.92 (m, 1H) , 1.92 (d, J = 2.8 Hz, 1H) , 1.50 (d, J = 6.8 Hz, 3H) .

Intermediate 4-8:

1- (1-Bromoethyl) -4- (trifluoromethyl) benzene

To a solution of 1- (4- (trifluoromethyl) phenyl) ethanol 4-7 (5.0 g, 90 %purity, 23.6 mmol) in dichloromethane (100 mL) was slowly added phosphorus (III) bromide (10 g, 36.9 mmol) at 0 ℃. After stirred at room temperature for 2 hours, the reaction mixture was poured into ice water (200 mL) and extracted with dichloromethane (200 mL) twice. The combined organic layers were washed with saturated sodium bicarbonate solution (200 mL) twice and brine (500 mL) twice, dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated at below 7 ℃ under reduced pressure to give title crude compound (5.0 g, 90 %purity from ¹HNMR, 75 %yield) as colorless oil. ¹H NMR (400 MHz, CDCl ₃) δ 7.60 (d, J = 8.4 Hz, 2H) , 7.54 (d, J = 8.4 Hz, 2H) , 5.19 (q, J = 7.2 Hz, 1H) , 2.04 (d, J = 6.8 Hz, 3H) .

Intermediate 4:

2-Chloro-5- ( (3R) -3-methyl-10-oxo-9- (1- (4- (trifluoromethyl) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

To a mixed solution of 60 %sodium hydride in minearal oil (500 mg, 12.5 mmol) in N, N-dimethylformamide (15 mL) was added (R) -2-chloro-5- (3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 4-5 (1.5 g, 95 %purity, 3.85 mmol) and 1- (1-bromoethyl) -4- (trifluoromethyl) benzene 4-8 (3.0 g, 90 %prity, 10.6 mmol) at 0 ℃. After stirred at room temperature for 3 hours, the reaction mixture was quenched with 2 M hydrochloride aqueous solution (50 mL) , then extracted with dichloromethane (100 mL) for three times. The combined organic layers were washed with brine (300 mL) , dried over anhydrous Na ₂SO _4 (S) and filtrated, and the solvent was evaporated under reduced pressure to give the crude, which was purified by C18 column (acetonitrile : water = 50 %to 60 %) to give the title compound (1.9 g, 90 %purity from ¹HNMR, 81.8 %yield) as offwhite solids. LC-MS (ESI) : R _T = 1.71 min, mass calcd. for C ₂₇H ₂₃ClF ₃N ₅O ₂, 541.9, m/z [M+H] ⁺ found 542.1. ¹H NMR (400 MHz, CDCl ₃) δ 7.75 (S, 1H) , 7.65 -7.58 (m, 4H) , 7.50 -7.46 (m, 2H) , 6.07 (br s, 1H) , 5.72 -5.40 (m, 1H) , 4.87 -4.09 (m, 4H) , 3.72 -3.58 (m, 1H) , 3.32 -3.28 (m, 1H) , 3.19 -2.06 (m, 1H) , 2.70 -2.66 (m, 1H) , 1.63 (d, J = 6.8 Hz, 3H) , 1.33 -1.25 (m, 3H) .

Compounds 4A and 4B:

A racemic mixture of 2-chloro-5- ( (3R) -3-methyl-10-oxo-9- (1- (4- (trifluoromethyl) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 4 (4.8 g, 90 %purity, 7.97 mmol) was separated by chiral. HPLC (Separation condition: Column: Chiralpak IB 5 μm 20 *250 mm; Mobile Phase: Hex: EtOH = 60 : 40 at 30 mL/min; Temp: 30 ℃; Wavelength: 214 nm) , then further purified by C18 column (acetonitrile : water = 50 %to 70 %) to give the title compounds 4A (1.556 g, 98.8 %purity from LCMS, 94.8 %yield, 100 %stereopure) as white solids and 4B (1.356 g, 99.42 %purity from LCMS, 99.8 %yield, 99.8 %stereopure) as white solids.

4A:

LC-MS (ESI) : R _T = 9.234 min, mass calcd. for C ₂₇H ₂₃ClF ₃N ₅O ₂, 541.9, m/z [M+H] ⁺ found 542.1. Chiral analysis (Column: Chiralpak IB 5 μm 4.6 *250 mm; Mobile Phase: Hex : EtOH = 60 : 40 at 1 mL/min; Temp: 30 ℃; Wavelength: 254 nm; R _T = 6.121 min) . ¹H NMR (400 MHz, CDCl ₃) δ 7.75 (S, 1H) , 7.64 -7.60 (m, 4H) , 7.47 -7.45 (m, 2H) , 6.19 -6.07 (m, 1H) , 5.98 -5.25 (m, 1H) , 4.98 -4.10 (m, 4H) , 3.68 -3.58 (m, 1H) , 3.31 -3.26 (m, 1H) , 3.23 -2.95 (m, 1H) , 2.70 -2.66 (m, 1H) , 1.63 (d, J = 7.2 Hz, 3H) , 1.29 -1.25 (m, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -62.67 (S, 3F) .

4B:

LC-MS (ESI) : R _T = 9.319 min, mass calcd. for C ₂₇H ₂₃ClF ₃N ₅O ₂, 541.9, m/z [M+H] ⁺ found 542.1. Chiral analysis (Column: Chiralpak IB 5 μm 4.6 *250 mm; Mobile Phase: Hex : EtOH = 60 : 40 at 1 mL/min; Temp: 30 ℃; Wavelength: 254 nm; R _T = 7.508 min) . ¹H NMR (400 MHz, CDCl ₃) δ 7.74 (S, 1H) , 7.65 -7.58 (m, 4H) , 7.50 -7.48 (m, 2H) , 6.18 -6.08 (m, 1H) , 5.99 -5.31 (m, 1H) , 4.87 -4.26 (m, 3H) , 4.17 -4.10 (m, 1H) , 3.74 -3.61 (m, 1H) , 3.32 -3.26 (m, 1H) , 3.22 -3.05 (m, 1H) , 2.91 -2.66 (m, 1H) , 1.63 (d, J = 6.8 Hz, 3H) , 1.30 -1.25 (m, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -62.67 (S, 3F) .

Compound 5:

2-Bromo-5- ( (R) -3-methyl-10-oxo-9- ( (S) -1- (4- (trifluoromethoxy) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

Intermediate 5-2:

(R) -Ethyl 5- (4-bromo-3-cyanobenzoyl) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylate

To a solution of 4-bromo-3-cyanobenzoic acid 5-1 (700 mg, 3.10 mmol) and N, N-dimethylformamide (20 mg, 0.274 mmol) in dichloromethane (20 mL) was added oxalyl chloride (0.4 mL, 4.73 mmol) at 0 ℃ under nitrogen atmosphere. After stirred at room temperature for 2 hours, the solution was concentrated to give yellow solids A. To the solution of (R) -ethyl 6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylate hydrochloride 1-2 (650 mg, 100 %purity, 2.65 mmol) and triethylamine (1.5 mL, 10.8 mmol) in dichloromethane (15 mL) was added the solution of A in dichloromethane (5 mL) at 0 ℃dropwise. After stirred at room for 1 hour, the reaction mixture was quenched with saturated sodium bicarbonate solution (10 mL) , extracted with ethyl acetate (20 mL) twice. The combined organic layers were washed with brine (10 mL) , dried over Na ₂SO _4 (S) , filtered. The filtrate was concentrated, purified by C18 column (acetonitrile : water = 50 %to 60 %) to give the title compound (1.0 g, 91 %purity from LCMS, 82 %yield) as white solids. LC-MS (ESI) : R _T =1.48 min, mass calcd. for C ₁₈H ₁₇BrN ₄O ₃ 416.1, m/z found 417.0 [M+H] ⁺. ¹H NMR (400 MHz, CDCl ₃) δ 7.78 (d, J = 8.0 Hz, 1H) , 7.73 (d, J = 1.2 Hz, 1H) , 7.52 (dd, J = 8.4, 1.6 Hz, 1H) , 5.74 -5.33 (m, 1H) , 4.79 -4.19 (m, 4H) , 3.21 -2.92 (m, 1H) , 2.74 (d, J = 16.0 Hz, 1H) , 1.52 -1.31 (m, 3H) , 1.26 (t, J = 6.8 Hz, 3H) .

Intermediate 5-3:

(R) -Ethyl 5- (4-bromo-3-cyanobenzoyl) -2- (2- ( (tert-butoxycarbonyl) ( (S) -1- (4- (trifluoromethoxy) phenyl) ethyl) amino) ethyl) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylate

To a solution of (R) -ethyl 5- (4-bromo-3-cyanobenzoyl) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylate 5-2 (300 mg, 91 %purity, 0.654 mmol) , (S) -tert-butyl (2-hydroxyethyl) (1- (4- (trifluoromethoxy) phenyl) ethyl) carbamate 1-8 (200 mg, 95 %purity, 0.544 mmol) and triphenylphosphine (200 mg, 0.763 mmol) in tetrahydrofuran (3 mL) was added di-tert-butyl azodicarboxylate (190 mg, 0.825 mmol) at 0 ℃. After stirred at room temperature for 3 hours, the reaction mixture was diluted with water (10 mL) , extracted with ethyl acetate (10 mL) twice. The combined organic layers were washed with brine (10 mL) , dried over Na ₂SO _4 (S) , filtered. The filtrate was concentrated, purified by C18 column (acetonitrile: water = 70 %to 90 %) to give the title compound (100 mg, 100 %purity from LCMS, 25 %yield) as white solids. LC-MS (ESI) : R _T = 2.02 min, mass calcd. for C ₃₄H ₃₇BrF ₃N ₅O ₆ 747.2, m/z found 747.9 [M+H] ⁺.

Intermediate 5-4:

(R) -5- (4-Bromo-3-cyanobenzoyl) -2- (2- ( (tert-butoxycarbonyl) ( (S) -1- (4- (trifluoromethoxy) phenyl) ethyl) amino) ethyl) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylic acid

To the solution of (R) -ethyl 5- (4-bromo-3-cyanobenzoyl) -2- (2- ( (tert-butoxycarbonyl) ( (S) -1- (4- (trifluoromethoxy) phenyl) ethyl) amino) ethyl) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylate 5-3 (100 mg, 100 %purity, 0.134 mmol) in tetrahydrofuran (1 mL) , methanol (0.3 mL) and water (0.3 mL) , was added sodium hydroxide monohydrate (12 mg, 0.286 mmol) . After stirred at room temperature for 2 hours, the reaction mixture was acidized with 1 M hydrochloride solution to pH ～ 5, extracted with chloromethane (5 mL) three times. The combined organic layers were dried over Na ₂SO _4 (S) , concentrated to give the title compound (85 mg, 96 %purity from LCMS, 85 %yield) as white solids. LC-MS (ESI) : R _T = 1.51 min, mass calcd. for C ₃₂H ₃₃BrF ₃N ₅O ₆ 719.2, m/z found 719.8 [M+H] ⁺.

Intermediate 5-5:

(R) -5- (4-Bromo-3-cyanobenzoyl) -6-methyl-2- (2- ( ( (S) -1- (4- (trifluoromethoxy) phenyl) ethyl) amino) ethyl) -4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylic acid

To a solution of (R) -5- (4-bromo-3-cyanobenzoyl) -2- (2- ( (tert-butoxycarbonyl) ( (S) -1- (4- (trifluoromethoxy) phenyl) ethyl) amino) ethyl) -6-methyl-4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylic acid 5-4 (85 mg, 96 %purity, 0.113 mmol) in dichloromethane (1 mL) was added 2 M hydrochloride solution in 1, 4-dioxane (2 mL) . After stirred at room temperature for 2 hours, the mixture was concentrated under reduced pressure to give the title compound (70 mg, 100 %purity from LCMS, 94 %yield) as white solids. LC-MS (ESI) : R _T = 1.49 min, mass calcd. for C ₂₇H ₂₅BrF ₃N ₅O ₄ 655.1, m/z found 618.6 [M-HCl-H] ^-.

Compound 5:

To a solution of (R) -5- (4-bromo-3-cyanobenzoyl) -6-methyl-2- (2- ( ( (S) -1- (4- (trifluoromethoxy) phenyl) ethyl) amino) ethyl) -4, 5, 6, 7-tetrahydro-2H-pyrazolo [4, 3-c] pyridine-3-carboxylic acid hydrochloride 5-5 (70 mg, 100 %purity, 0.107 mmol) and N, N-diisopropylethylamine (55 mg, 0.426 mmol) in dichloromethane (5 mL) was added O- (7-azabenzotriazol-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate (60 mg, 0.158 mmol) . After stirred at 0 ℃ for 10 minutes, the mixture was poured into water (3 mL) , extracted with dichloromethane (5 mL) twice. The combined organic layers were washed with brine (5 mL) , dried over Na ₂SO _4 (S) , filtrated. The filtrate was concentrated, purified by C18 column (acetonitrile: water = 60 %to 75 %) to give the title compound (40 mg, 98.8 %purity from LCMS, 62 %yield) as white solids. LC-MS (ESI) : R _T = 7.523 min, mass calcd. for C ₂₇H ₂₃BrF ₃N ₅O ₃ 601.1, m/z found 602.1 [M+H] ⁺. ¹H NMR (400 MHz, CDCl ₃) δ 7.77 (d, J = 8.4 Hz, 1H) , 7.72 (d, J = 2.0 Hz, 1H) , 7.52 (dd, J = 8.4, 1.6 Hz, 1H) , 7.37 (d, J = 7.2 Hz, 2H) , 7.21 (d, J = 8.4 Hz, 2H) , 6.23 -5.92 (m, 1H) , 5.86 -5.25 (m, 1H) , 4.91 -4.31 (m, 2H) , 4.30 -4.24 (m, 1H) , 4.20 -4.07 (m, 1H) , 3.64 -3.58 (m, 1H) , 3.30 -3.24 (m, 1H) , 3.07 (br s, 1H) , 2.68 (d, J = 16.0 Hz, 1H) , 1.61 -1.59 (m, 3H) , 1.35 -1.21 (m, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -57.88 (S, 3F) .

Compound 6:

(R) -2-Chloro-5- (9- ( (2, 2-difluorobenzo [d] [1, 3] dioxol-5-yl) methyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2- carbonyl) benzonitrile

Intermediate 6-2:

(2, 2-Difluorobenzo [d] [1, 3] dioxol-5-yl) methanol

To a solution of 2, 2-difluorobenzodioxole-5-carboxaldehyde 6-1 (300 mg, 1.61 mmol) in methanol (10 mL) was added sodium borohydride (90 mg, 2.38 mmol) at 0 ℃. After stirred at the temperature for 5 minutes, the mixture was alkalified with 1 N hydrochloride aqueous to pH ～ 6, extracted with ethyl acetate (8 mL) three times. The combined organic layers were dried over Na ₂SO _4 (S) , filtered. The filtrate was concentrated, purified by silca gel column chromatography (petroleum ether: ethyl acetate = 4: 1) to give the title compound (300 mg, 90 %purity from ¹H NMR, 89 %yield) as colorless oil. ¹H NMR (400 MHz, CDCl ₃) δ 7.11 (S, 1H) , 7.06 -7.01 (m, 2H) , 4.67 (S, 2H) .

Intermediate 6-3:

5- (Bromomethyl) -2, 2-difluorobenzo [d] [1, 3] dioxole

To a solution of (2, 2-difluorobenzo [d] [1, 3] dioxol-5-yl) methanol 6-2 (300 mg, 90 %purity, 1.44 mmol) in dichloromethane (3 mL) was added phosphorus tribromide (0.1 mL, 1.06 mmol) at 0 ℃. After stirred at room temperature for 2 hours. The mixture was poured into water (10 mL) , extracted with dichloromethane (10 mL) twice. The combined organic layers were washed with brine (10 mL) , dried over Na ₂SO _4 (S) , filtered. The filtrate was concentrated to give the title compound (360 mg, 90 %purity from ¹H NMR, 90 %yield) as yellow oil. ¹H NMR (400 MHz, CDCl ₃) δ 7.14 -7.09 (m, 2H) , 7.01 (d, J = 8.0 Hz, 1H) , 4.47 (S, 2H) .

Compound 6:

To a solution of (R) -2-chloro-5- (3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 4-5 (2.8 g, 90 %purity, 6.81 mmol) in N, N-dimethylformamide (25 mL) was added sodium hydride (840 mg, 60 %purity, 21.0 mmol) at 0 ℃. After stirred at 0 ℃ for 30 minutes, followed to add 5- (bromomethyl) -2, 2-difluorobenzo [d] [1, 3] dioxole 6-3 (3.8 g, 90 %purity, 13.6 mmol) in N, N-dimethylformamide and stirring continued at room temperature for 2 hours, the mixture was quenched with water (100 mL) , extracted with ethyl acetate (50 mL) twice. The combined organic layers were washed with brine (50 mL) , dried over Na ₂SO _4 (S) , filtered. The filtrate was concentrated, purified by C18 column (acetonitrile : water = 70 %to 90 %) to give the crude, which was purified by silica gel column chromatography (dichloromethane : methanol =15 : 1) and C18 column (acetonitrile : water = 70 %to 90 %) to give the title compound (2.2 g, 98.5 %purity from LCMS, 59 %yield) as white solids. LC-MS (ESI) : R _T = 10.143 min, mass calcd. for C ₂₆H ₂₀ClF ₂N ₅O ₄ 539.1, m/z found 540.1 [M+H] ⁺. ¹H NMR (400 MHz, CDCl ₃) δ 7.74 (S, 1H) , 7.60 (S, 2H) , 7.06 -7.03 (m, 3H) , 5.88 -5.27 (m, 1H) , 4.78 -4.29 (m, 6H) , 3.75 -3.61 (m, 2H) , 3.06 (br s, 1H) , 2.68 (d, J = 16.0 Hz, 1H) , 1.28 (d, J = 4.4 Hz, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -49.94 (S, 2F) .

Compound 7A and Compound 7B:

2-Chloro-5- ( (R) -9- ( (R*) -1- (2, 2-difluorobenzo [d] [1, 3] dioxol-5-yl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile and

2-chloro-5- ( (R) -9- ( (S*) -1- (2, 2-difluorobenzo [d] [1, 3] dioxol-5-yl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

Intermediate 7-1:

1- (2, 2-Difluorobenzo [d] [1, 3] dioxol-5-yl) ethanol

To a soluion of 2, 2-difluorobenzo [d] [1, 3] dioxole-5-carbaldehydee 6-1 (6 g, 32.2 mmol) in tetrahydrofuran (40 mL) was added 3 M methylmagnesium bromide in tetrahydrofuran (16 mL, 48.0 mmol) . After stirred at 0 ℃ for 2 hours, the reaction mixture was quenched with saturated ammonium chloride aqueous solution (100 mL) and extracted with ethyl acetate (100 mL) twice. The combined organic layers were washed with water (100 mL) for three times, brine (100 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated under reduced pressure to get a residue, which was purified by C18 column (acetonitrile: water = 50 %to 65 %) to give the title compound (5.0 g, 90 %purity from HNMR, 69 %yield) as yellow oil. ¹H NMR (400 MHz, CDCl ₃) δ 7.13 (S, 1H) , 7.06 -6.99 (m, 2H) , 5.89 (q, J = 6.8 Hz, 1H) , 1.47 (d, J = 6.4 Hz, 3H) .

Intermediate 7-2:

5- (1-Bromoethyl) -2, 2-difluorobenzo [d] [1, 3] dioxole

To a solution of 1- (2, 2-difluorobenzo [d] [1, 3] dioxol-5-yl) ethanol 7-1 (5.0 g, 90 %purity, 22.3 mmol) in dichloromethane (50 mL) was added tribromophosphine (2.5 g, 9.24 mmol) at 0 ℃. After stirred at 0 ℃ for 0.5 hour, the mixture was added into saturated sodium bicarbonate aqueous solution (100 mL) and extracted with dichloromethane (100 mL) twice. The combined organic layers were washed with water (100 mL) , brine (100 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated under reduced pressure to give the title compound (6.0 g, 90 %purity from HNMR, 92 %yield) as yellow oil. ¹H NMR (400 MHz, CDCl ₃) δ 7.20 (S, 1H) , 7.13 (dd, J = 8.0, 1.6 Hz, 1H) , 6.99 (d, J = 8.0 Hz, 1H) , 5.18 (q, J = 6.8 Hz, 1H) , 2.02 (d, J = 6.8 Hz, 3H) ,

Intermediate 7:

2-Chloro-5- ( (3R) -9- (1- (2, 2-difluorobenzo [d] [1, 3] dioxol-5-yl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

To a solution of (R) -2-chloro-5- (3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 4-5 (5 g, 95 %purity, 12.8 mmol) in N, N-dimethylformamide (50 mL) was added 60 %wt. sodium hydride in mineral oil (1.5 g, 37.5 mmol) , After stirred at 0 ℃ for 0.5 hour, 5- (1-bromoethyl) -2, 2-difluorobenzo [d] [1, 3] dioxole 7-2 (6.0 g, 90 %purity, 20.4 mmo) in N, N-dimethylformamide (10 mL) was added into the mixture. After stirred at 0 ℃ for 1 hour, the mixture was diluted with water (300 mL) and extracted with ethyl acetate (10 mL) trice. The combined organic layers were washed with brine (30 mL) , dried over Na ₂SO _4 (S) and filtered, which was purified by C18 column (acetonitrile : water = 50 %to 55 %) to give the title compound (6.0 g, 100 %purity from LCMS, 84 %yield) as yellow solids. LCMS: R _T = 1.56 min, mass calcd. for C ₂₇H ₂₂ClF ₂N ₅O ₄ 553.1 m/z found 554.0 [M+H] ⁺.

Compound 7A and Compound 7B:

2-chloro-5- ( (R) -9- ( (S*) -1- (2, 2-difluorobenzo [d] [1, 3] dioxol-5-yl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2- carbonyl) benzonitrile

A racemic 2-Chloro-5- ( (3R) -9- (1- (2, 2-difluorobenzo [d] [1, 3] dioxol-5-yl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 7 (5 g, 100 %purity, 9.03 mmol) was separated by chiral Prep. HPLC (Separation condition: Column: Chiralpak IB 5 μm 20 *250 mm; Mobile Phase: Hexane -EtOH = 70 : 30 at 12 mL/min; Temp: 30 ℃; Wavelength: 254 nm) to afford the title compound 7A (1765.2 mg, 99.3 %purity, 35 %yield, 100 %stereopure) as white solids and 7B (1675.2 mg, 99.2 %purity, 33 %yield, 99.8 %stereopure) as white solids.

7A:

LCMS: R _T = 8.321 min, mass calcd. for C ₂₇H ₂₂ClF ₂N ₅O ₄ 553.1 m/z found 554.1 [M+H] ⁺. Chiral HPLC (Column: Chiralpak IB 5 μm 4.6 *250 mm; Mobile Phase: Hexane : EtOH = 70 : 30 at 1.0 mL/min; Temp: 30 ℃; Wavelength: 254 nm, R _T = 10.443 min) . ¹H NMR (400 MHz, CDCl ₃) δ 7.74 (S, 1H) , 7.62 -7.57 (m, 2H) , 7.06 -7.02 (m, 3H) , 6.01 (br s, 1H) , 5.67 -5.38 (m, 1H) , 4.79 -4.14 (m, 4H) , 3.64 -3.57 (m, 1H) , 3.30 -3.26 (m, 1H) , 3.07 (br s, 1H) , 2.69 -2.65 (m, 1H) , 1.58 (d, J = 6.8 Hz, 3H) , 1.27 (d, J = 4.8 Hz, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -49.89 (S, 2F) .

7B:

LCMS: R _T = 8.458 min, mass calcd. for C ₂₇H ₂₂ClF ₂N ₅O ₄ 553.1 m/z found 554.1 [M+H] ⁺. Chiral HPLC (Column: Chiralpak IB 5 μm 4.6 *250 mm; Mobile Phase: Hexane : EtOH = 70 : 30 at 1.0 mL/min; Temp: 30 ℃; Wavelength: 254 nm, R _T = 13.336 min) . ¹H NMR (400 MHz, CDCl ₃) δ 7.74 (S, 1H) , 7.62 -7.57 (m, 2H) , 7.08 -7.03 (m, 3H) , 6.02 (br s, 1H) , 5.70 -5.41 (m, 1H) , 4.73 -4.24 (m, 3H) , 4.16 -4.09 (m, 1H) , 3.66 -3.61 (m, 1H) , 3.32 -3.26 (m, 1H) , 3.06 (br s, 1H) , 2.69 -2.65 (m, 1H) , 1.57 (d, J = 6.4 Hz, 3H) , 1.29 (d, J = 4.8 Hz, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -49.90 (S, 2F) .

Compound 8:

(R) -5- (9- ( (1H-Benzo [d] [1, 2, 3] triazol-5-yl) methyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) -2-chlorobenzonitrile

Intermediate 8-2:

Ethyl 1- ( (2- (trimethylsilyl) ethoxy) methyl) -1H-benzo [d] [1, 2, 3] triazole-5-carboxylate

To a mixture of ethyl 1H-benzo [d] [1, 2, 3] triazole-5-carboxylate 8-1 (1 g, 5.23 mmol) in N, N-dimethylformamide (10 mL) was added 60 %wt. sodium hydride (314 mg, 7.85 mmol) at 0 ℃under nitrogen atmosphere. The resluting mixture was stirred at 25 ℃ for 30 minutes, then (2- (chloromethoxy) ethyl) trimethylsilane (1.05 g, 6.30 mmol) was added and the mixture was stirred at 25 ℃ for another 16 hours. The reaction was quenched with water (100 mL) , and extracted with ethyl acetate (30 mL) for three times. The combined organic phases were washed with brine (30 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated to give a residue, which was purified by gel column chromatography (petroleum ether: ethyl acetate = 5 %to 15 %) to give the title compound (900 mg, 90 %purity from ¹HNMR, 48 %yield) as colorless oil. ¹H NMR (400 MHz, CDCl ₃) δ 8.82 (S, 0.5H) , 8.44 (S, 0.5H) , 8.24 -8.22 (m, 0.5H) , 8.13 -8.08 (m, 1H) , 7.72 -7.70 (m, 0.5H) , 6.04 -6.01 (m, 2H) , 4.48 -4.42 (m, 2H) , 3.61 -3.56 (m, 2H) , 1.46-1.42 (m, 3H) , 0.93 -0.87 (m, 2H) , -0.065 --0.075 (m, 9H) .

Intermediate 8-3:

(1- ( (2- (Trimethylsilyl) ethoxy) methyl) -1H-benzo [d] [1, 2, 3] triazol-5-yl) methanol

To a solution of ethyl 1- ( (2- (trimethylsilyl) ethoxy) methyl) -1H-benzo [d] [1, 2, 3] triazole-5-carboxylate 8-2 (900 mg, 90 %purity, 2.52 mmol) in tetrahydrofuran (10 mL) was added lithium aluminium hydride (200 mg, 5.27 mmol) at 0 ℃. After stirred at 0 ℃ for 1 hour under nitrogen atmosphere, the reaction mixture was quenched with water (0.2 mL) , 10%～ 15%wt aqueous sodium hydroxide solution (0.6 mL) and water (0.2 mL) in turn, then filtered. The filtrate was concentrated to give the title compound (760 mg, 90 %purity from ¹HNMR, 97 %yield) as colorless oil. ¹H NMR (400 MHz, CDCl ₃) δ 8.11 -8.07 (m, 0.6H) , 7.95 -7.91 (m, 0.8H) , 7.76 -7.70 (m, 0.6H) , 7.61 -7.56 (m, 0.3H) , 7.47 -7.41 (m, 0.7H) , 6.02 -5.99 (m, 2H) , 4.94 -4.88 (m, 2H) , 3.77 -3.73 (m, 1H) , 3.63-3.58 (m, 1H) , 2.18 -1.96 (m, 1H) , 1.01 -0.90 (m, 2H) , -0.00 --0.034 (m, 9H) .

Intermediate 8-4:

5- (Bromomethyl) -1- ( (2- (trimethylsilyl) ethoxy) methyl) -1H-benzo [d] [1, 2, 3] triazole

To a solution of (1- ( (2- (trimethylsilyl) ethoxy) methyl) -1H-benzo [d] [1, 2, 3] triazol-5-yl) methanol 8-3 (360 mg, 90 %purity, 1.16 mmol) and carbon tetrabromide (615 mg, 1.85 mmol) in dichloromethane (7 mL) was added triphenylphosphine (457 mg, 1.74 mmol) at 0 ℃. After stirred at 25 ℃ for 0.5 hour, the reaction was concentrated to give a residue, which was purified by gel column chromatography (petroleum ether : ethyl acetate = 2 %to 8 %) to give the title compound (200 mg, 90 %purity from ¹HNMR, 45 %yield) as white solids. ¹H NMR (400 MHz, CDCl ₃) δ 8.08 -8.04 (m, 1H) , 7.71 -7.67 (m, 1H) , 7.60 -7.58 (m, 0.5H) , 7.46 -7.44 (m, 0.5H) , 5.98 -5.97 (m, 2H) , 4.68 -4.65 (m, 2H) , 3.60 -3.55 (m, 2H) , 0.93 -0.88 (m, 2H) , 0.059 (S, 9H) .

Intermediate 8-5:

(R) -2-Chloro-5- (3-methyl-10-oxo-9- ( (1- ( (2- (trimethylsilyl) ethoxy) methyl) -1H-benzo [d] [1, 2, 3] triazol-5-yl) methyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

To a solution of 5- (bromomethyl) -1- ( (2- (trimethylsilyl) ethoxy) methyl) -1H-benzo [d] [1, 2, 3] triazole 8-4 (256 mg, 90 %purity, 0.673 mmol) , (R) -2-chloro-5- (3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 4-5 (230 mg, 90 %purity, 0.56 mmol) and benzyltriethylammonium (16 mg, 0.07 mmol) in 2-methyltetrahydrofuran (5 mL) was added 50%sodium hydroxide aqueous solution (2.5 mL) . After stirred at 50 ℃ for 2 hours, the reaction was diluted with water (10 mL) and extracted with ethyl acetate (5 mL) for three times. The combined organic phases were wahsed with brine (5 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated to give a residue, which was purified by gel column chromatography (dichloromethane : methanol = 1 %to 5 %) to give the title compound (250 mg, 87 %purity from LCMS, 62 %yield) as white solids. LC-MS (ESI) : R _T = 1.73 min, mass calcd. for C ₃₁H ₃₅ClN ₈O ₃Si 630.2, m/z found 631.0 [M+H] ⁺.

Compound 8:

To a solution of (R) -2-chloro-5- (3-methyl-10-oxo-9- ( (1- ( (2- (trimethylsilyl) ethoxy) methyl) -1H-benzo [d] [1, 2, 3] triazol-5-yl) methyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 8-5 (200 mg, 87 %purity, 0.276 mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (3 mL) . The reaction was stirred at 25 ℃ for 3 hours. The reaction mixture was concentrated to give a residue, which was purified by C18 (acetonitrile : water (0.1%ammonium bircarbonate) = 20 %to 50 %) to give the title compound (65.3 mg, 98.6 %purity, 46.6 %yield) as white solids. LC-MS (ESI) : R _T = 3.652 min, mass calcd. for C ₂₅H ₂₁ClN ₈O ₂ 500.2, m/z found 501.1 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.22 -8.09 (m, 1H) , 7.96 -7.78 (m, 4H) , 7.47 -7.31 (m, 1H) , 5.48 -5.44 (m, 0.6H) , 5.31 -5.16 (m, 0.4H) , 4.94 -4.49 (m, 3H) , 4.31 -4.15 (m, 3H) , 3.83 -3.65 (m, 2H) , 3.06 -2.87 (m, 1H) , 2.70 -2.58 (m, 1H) , 1.29 -1.04 (m, 3H) .

Compound 9A and Compound 9B:

2-Chloro-5- ( (R) -3-methyl-10-oxo-9- ( (R*) -1- (3- (trifluoromethoxy) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile and

2-chloro-5- ( (R) -3-methyl-10-oxo-9- ( (S*) -1- (3- (trifluoromethoxy) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

Intermediate 9-2:

1- (3- (Trifluoromethoxy) phenyl) ethanol

To the solution of 1- (3- (trifluoromethoxy) phenyl) ethanone 9-1 (5.00 g, 24.5 mmol) in tetrahydrofuran (45 mL) and methanol (15 mL) was added sodium borohydride (500 mg, 13.2 mmol) at 0 ℃. After stirred at 0 ℃ for 1 hour and at room temperature for another 30 minutes, the mixture was diluted 0.5 M hydrochloric acid aqueous solution (50 mL) and extracted with ethyl acetate (50 mL) for three times. The combined organic layers were washed with brine (50 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated to give the title compound (5.3 g, 95 %purity from H NMR, 99.7 %yield) as yellow oil. ¹HNMR (400 MHz, CDCl ₃) δ 7.39 -7.35 (m, 1H) , 7.30 (d, J = 7.6 Hz, 1H) , 7.25 -7.23 (m, 1H) , 7.12 (d, J = 8.0 Hz, 1H) , 4.93 (q, J = 6.4 Hz, 1H) , 1.98 -1.71 (m, 1H) , 1.50 (d, J = 6.4 Hz, 3H) .

Intermediate 9-3:

1- (1-Bromoethyl) -3- (trifluoromethoxy) benzene

To the solution of 1- (3- (trifluoromethoxy) phenyl) ethanol 9-2 (9.53 g, 95 %purity, 43.9 mmol) in dichloromethane (100 mL) was added phosphorus tribromide (6.41 g, 23.7 mmol) at 0 ℃. After stirred at 0 ℃ for 20 minutes and at room temperature for another 1 hour, the mixture was quenched with saturated aqueous sodium bicarbonate solution (200 mL) and extrated with dichloromethane (300 mL) twice. The combined organic layers were washed with brine (200 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated to give the title compound (8.62 g, 95 %purity from HNMR, 69.3 %yield) as yellow oil. ¹HNMR (400 MHz, CDCl ₃) δ 7.36 (d, J = 5.2 Hz, 2H) , 7.28 (S, 1H) , 7.15 (S, 1H) , 5.16 (q, J = 6.8 Hz, 1H) , 2.03 (d, J = 6.8 Hz, 3H) .

Compound 9:

2-Chloro-5- ( (3R) -3-methyl-10-oxo-9- (1- (3- (trifluoromethoxy) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

To the solution of (R) -2-chloro-5- (3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 4-5 (2.00 g, 95 %purity, 5.14 mmol) in N, N-dimethylformamide (15 mL) was added 60 %sodium hydride in mineral oil (650 mg, 16.3 mmol) at 0 ℃. The mixture was stirred at 0 ℃ for 30 minutes and added 1- (1-bromoethyl) -3- (trifluoromethoxy) benzene 9-3 (3.5 g, 95 %purity, 12.4 mmol) . After stirred at 0 ℃ for 2 hours, the mixture was quenched with 0.5 M hydrochloride aqueous solution (50 mL) and extracted with ethyl acetate (80 mL) twice. The combined organic layers were washed with brine (50 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated and purified by silica gel column chromatography (dichloromethane : methanol = 20: 1) to give the title compound (2.75 g, 100 %purity, 95.9 %yield) as yellow solids. LC-MS (ESI) : R _T = 1.71 min, mass calcd. for C ₂₇H ₂₃ClF ₃N ₅O ₃ 557.1, m/z found 558.0 [M+H] ⁺.

Compound 9A and Compound 9B:

A racemate of 2-chloro-5- ( (3R) -3-methyl-10-oxo-9- (1- (3- (trifluoromethoxy) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 9 (6.5 g, 100 %purity, 11.7 mmol) was separated by chiral Prep. SFC (Separation condition : Column : Chiralpak IB N-5 5 μm 20 *250 mm, Mobile Phase: CO ₂ : MeOH (0.2 %DEA) = 80 : 20 at 60 g/min; Col. Temp: 40 ℃; Wavelength: 214 nm, Back pressure: 100 bar) to afford the crude Peak 1 and Peak 2.

Peak 1 was purified by C18 column (acetonitrile : water (+ 0.2 %ammonium bicarbonate) = 60 %to 70 %) to give the title compound 9A (2.5 g, 99.6 %purity, 38.3 %yield, 100 %stereopure) as white solids.

Peak 2 was added acetonitrile (3 mL) , stirred at room temperature for 20 minutes and filtered. The white solids were collected by filtration and vacuum drying oven at 55 ℃ for 2 hours, then cooled to room temperature to give the title compound 9B (2.1 g, 99.4 %purity, 32.1 %yield, 100 %stereopure) as white solids.

9A:

LC-MS (ESI) : R _T = 5.576 min, mass calcd. for C ₂₇H ₂₃ClF ₃N ₅O ₃ 557.1, m/z found 558.1 [M+H] ⁺. Chiral analysis (Column: Chiralpak IB N-5 5 μm 4.6 *250 mm; Mobile Phase: CO ₂ : MeOH (0.2 %DEA) = 80 : 20 at 3 mL/min; Col. Temp: 40 ℃; Wavelength: 254 nm, RT = 7.37 min) . ¹H NMR (400 MHz, CDCl ₃) δ 7.76 (S, 1H) , 7.62 -7.58 (m, 2H) , 7.42 -7.38 (m, 1H) , 7.32 -7.29 (m, 1H) , 7.18 (d, J = 6.8 Hz, 2H) , 6.02 (br s, 1H) , 5.81 -5.31 (m, 1H) , 4.80 -4.10 (m, 4H) , 3.66 -3.59 (m, 1H) , 3.29 -3.23 (m, 1H) , 3.16 -2.99 (m, 1H) , 2.68 (d, J = 14.8 Hz, 1H) , 1.61 (d, J = 7.2 Hz, 3H) , 1.28 (d, J = 4.8 Hz, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -57.79 (S, 3F) .

9B:

LC-MS (ESI) : R _T = 5.765 min, mass calcd. for C ₂₇H ₂₃ClF ₃N ₅O ₃ 557.1, m/z found 558.1 [M+H] ⁺. Chiral SFC (Column: Chiralpak IB N-5 5 μm 4.6 *250 mm; Mobile Phase: CO ₂ : MeOH (0.2 %DEA) = 80 : 20 at 3 mL/min; Col. Temp: 40 ℃; Wavelength: 254 nm, RT = 8.51 min) . ¹H NMR (400 MHz, CDCl ₃) δ 7.74 (S, 1H) , 7.62 -7.58 (m, 2H) , 7.43 -7.39 (m, 1H) , 7.31 -7.30 (m, 1H) , 7.19 (d, J = 7.2 Hz, 2H) , 6.04 (br s, 1H) , 5.82 -5.34 (m, 1H) , 4.74 -4.09 (m, 4H) , 3.70 -3.60 (m, 1H) , 3.30 -3.24 (m, 1H) , 3.16 -2.99 (m, 1H) , 2.69 (d, J = 16.0 Hz, 1H) , 1.60 -1.58 (m, 3H) , 1.31 -1.26 (m, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -57.79 (S, 3F) .

Compounds 10A and 10B:

2-Chloro-5- ( (R) -9- ( (R*) -1- (3- (difluoromethoxy) phenyl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile and

2-chloro-5- ( (R) -9- ( (S*) -1- (3- (difluoromethoxy) phenyl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

Intermediate 10-2:

1- (3- (Difluoromethoxy) phenyl) ethanol

To a solution of 1- (3- (difluoromethoxy) phenyl) ethanone 10-1 (1.0 g, 5.37 mmol) in methanol (10 mL) was slowly added sodium borohydride (600 mg, 15.9 mmol) at 0 ℃. After stirred at room temperature for 3 hours, the reaction was quenched with acetone (10 mL) dropwise and concentrated to give a residue, which was dissolved into ethyl acetate (20 mL) and washed with brine (50 mL) twice, dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated to give the title compound P1 (1.1 g, 90 %purity from HNMR, 98 %yield) as colourless oil. ¹H NMR (400 MHz, CDCl ₃) δ 7.34 (t, J = 7.6 Hz, 1H) , 7.21 (d, J = 7.2 Hz, 1H) , 7.15 (S, 1H) , 7.02 (d, J = 8.4 Hz, 1H) , 6.52 (t, J = 74.4 Hz, 1H) , 4.91 (q, J = 6.0 Hz, 1H) , 1.93 (br s, 1H) , 1.50 (d, J = 6.4 Hz, 3H) .

Intermediate 10-3:

1- (1-Bromoethyl) -3- (difluoromethoxy) benzene

To a solution of 1- (3- (difluoromethoxy) phenyl) ethanol 10-2 (1.1 g, 90 %purity, 5.26 mmol) in dichloromethane (30 mL) was slowly added phosphorus (III) bromide (2.0 g, 7.39 mmol) at 0 ℃. After stirred at room temperature for 2 hours, the reaction mixture was poured into ice water (20 mL) and extracted with dichloromethane (20 mL) twice. The combined organic layers were washed with saturated sodium bicarbonate solution (50 mL) twice and brine (50 mL) twice, dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated under reduced pressure to give the crude title compound (1.0 g, 95 %purity from HNMR, 72 %yield) as yellow oil. ¹H NMR (400 MHz, CDCl ₃) δ 7.36 -7.27 (m, 2H) , 7.19 (S, 1H) , 7.04 (d, J = 7.6 Hz, 1H) , 6.52 (t, J = 73.6 Hz, 1H) , 5.16 (q, J = 7.2 Hz, 1H) , 2.03 (d, J = 7.2 Hz, 3H) .

Compound 10:

2-Chloro-5- ( (3R) -9- (1- (3- (difluoromethoxy) phenyl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

To a solution of (R) -2-chloro-5- (3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 4-5 (200 mg, 95 %purity, 0.514 mmol) , 1- (1-bromoethyl) -3- (difluoromethoxy) benzene 10-3 (200 mg, 95 %purity, 0.757 mmol) and benzyltriethylammonium chloride (15 mg, 0.066 mmol) in 2-methyltetrahydrofuran (4 mL) and 50 %sodium hydroxide aqueous solution (2 mL) . After stirred at 50 ℃ for 2 hours, the reaction mixture was quenched with ice water (30 mL) and extracted with ethyl acetate (30 mL) twice. The combined organic layers were washed with brine (50 mL) twice, dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated under reduced pressure to give crude, which was purified by C18 column (acetonitrile : water = 60 %to 80 %) to give the title compound (160 mg, 91 %purity from LCMS, 52 %yield) as white solids. LC-MS (ESI) : R _T = 1.52 min, mass calcd. for C ₂₇H ₂₄ClF ₂N ₅O ₃ 539.2, m/z found 540.3 [M+H] ⁺.

Compounds 10A and 10B:

A racemate of 2-chloro-5- ( (3R) -9- (1- (3- (difluoromethoxy) phenyl) ethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 10 (160 mg, 91 %purity, 0.27 mmol) was separated by chiral prep. HPLC (Separation condition: Column: Chiralpak IG 5 μm 20 *250 mm; Mobile Phase: MeOH : DCM = 60 : 40 at 15 mL/min; Temp: 30 ℃; Wavelength: 254 nm) , then further purified by C18 column (acetonitrile: water (0.2 %ammonium bicarbonate) = 40 %to 60 %) to give the title compounds 10A (47.0 mg, 99.5 %purity, 91 %yield, 100 %stereopure) as white solids as white solids and 10B (45.7 mg, 99.3 %purity, 88 %yield, 99.8 %stereopure) as white solids.

10A:

LC-MS (ESI) : R _T = 9.289 min, mass calcd. for C ₂₇H ₂₄ClF ₂N ₅O ₃ 539.2, m/z found 540.1 [M+H] ⁺. Chiral analysis (Column: Chiralpak IG 5 μm 4.6 *250 mm; Mobile Phase: MeOH : DCM = 60 : 40 at 1.0 mL/min; Temp: 30 ℃; Wavelength: 254 nm; R _T = 4.216 min) . ¹H NMR (400 MHz, CDCl ₃) δ 7.74 (S, 1H) , 7.62 -7.58 (m, 2H) , 7.37 (t, J = 7.6 Hz, 1H) , 7.23 -7.19 (m, 1H) , 7.11 -7.07 (m, 2H) , 6.52 (t, J = 73.6 Hz, 1H) , 6.08 -5.96 (m, 1H) , 5.70 -5.34 (m, 1H) , 4.73 -4.13 (m, 4H) , 3.67 -3.61 (m, 1H) , 3.32 -3.26 (m, 1H) , 3.12 -2.97 (m, 1H) , 2.70 -2.62 (m, 1H) , 1.65 -1.63 (m, 3H) , 1.31 -1.25 (m, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -81.02 (S, 2F) .

10B:

LC-MS (ESI) : R _T = 9.244 min, mass calcd. for C ₂₇H ₂₄ClF ₂N ₅O ₃ 539.2, m/z found 540.1 [M+H] ⁺. Chiral analysis (Column: Chiralpak IG 5 μm 4.6 *250 mm; Mobile Phase: MeOH : DCM = 60 : 40 at 1.0 mL/min; Temp: 30 ℃; Wavelength: 254 nm; R _T = 5.331 min) . ¹H NMR (400 MHz, CDCl ₃) δ 7.76 (S, 1H) , 7.63 -7.58 (m, 2H) , 7.36 (t, J = 7.6 Hz, 1H) , 7.20 -7.18 (m, 1H) , 7.08 -7.03 (m, 2H) , 6.51 (t, J = 73.6 Hz, 1H) , 6.12 -5.93 (m, 1H) , 5.67 -5.28 (m, 1H) , 4.77 -4.15 (m, 4H) , 3.65 -3.58 (m, 1H) , 3.31 -3.25 (m, 1H) , 3.13 -2.98 (m, 1H) , 2.70 -2.62 (m, 1H) , 1.67 -1.61 (m, 3H) , 1.29 -1.25 (m, 3H) . ¹⁹F NMR (376 MHz, CDCl ₃) δ -81.05 (S, 2F) .

Compound 11A and Compound 11B:

2-Chloro-5- ( (R) -3-methyl-10-oxo-9- ( (R*) -1- (3- (trifluoromethyl) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile and

2-chloro-5- ( (R) -3-methyl-10-oxo-9- ( (S*) -1- (3- (trifluoromethyl) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

Compound 11:

2-Chloro-5- ( (3R) -3-methyl-10-oxo-9- (1- (3- (trifluoromethyl) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

To a solution of (R) -2-chloro-5- (3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 4-5 (300 mg, 95 %purity, 0.77 mmol) in 2-methyltetrahydrofuran (4 mL) was added 1- (1-bromoethyl) -3- (trifluoromethyl) benzene 11-1 (300 mg, 97 %purity, 1.15 mmol) and N-benzyl-N, N-diethylethanaminium chloride (24 mg, 0.11 mmol) and 50 %wt. sodium hydroxide solution (2mL) . After stirred at 50 ℃ under nitrogen atmosphere for 3 hours, the reaction mixture was quenched with water (4 mL) and extracted with ethyl acetate (10 mL) twice. The combined organic layer was dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated under reduced pressure to give a crude, which was purified by C18 column (acetonitrile: water = 5 %to 90 %) to give the title compound (320 mg, 90 %purity from HNMR, 69 %yield) as white solids. LC-MS (ESI) : R _T = 1.763 min, mass calcd. for C ₂₇H ₂₃ClF ₃N5O2 541.2, m/z found 542.1 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.12 (S, 1H) , 7.87 -7.80 (m, 2H) , 7.69 -7.63 (m, 4H) , 5.90 -5.74 (m, 1H) , 5.47 -5.23 (m, 1H) , 4.56 -4.13 (m, 4H) , 3.76 (S, 1H) , 3.36 (S, 1H) , 2.99 -2.62 (m, 2H) , 1.61 (S, 3H) , 1.16 -1.13 (m, 3H) .

Compounds 11A and Compound 11B:

2-Chloro-5- ( (R) -3-methyl-10-oxo-9- ( (R*) -1- (3- (trifluoromethyl) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2- carbonyl) benzonitrile and

A racemate of 2-chloro-5- ( (3R) -3-methyl-10-oxo-9- (1- (3- (trifluoromethyl) phenyl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 11 (320 mg, 90 %purity, 0.531 mmol) was separated by chiral Prep. HPLC (Separation condition: Column: Chiralpak IG 5 μm 20 *250 mm; Mobile Phase: MeOH : DCM = 80 : 20 at 25 mL/min; Temp: 30 ℃; Wavelength: 230 nm) to afford the title compounds 11A (75.5 mg, 26 %yield, 99.7 %purity, 100 %stereopure) as white solids and 11B (69.6 mg, 24 %yield, 99.5 %purity, 100 %stereopure) as white solids.

11A:

LC-MS (ESI) : R _T = 4.013 min, mass calcd. for C ₂₇H ₂₃ClF ₃N ₅O ₂ 541.15, m/z found 542.2 [M+H] ⁺. Chiral analysis (Column: Chiralpak IG 5 μm 4.6 *250 mm; Mobile Phase: MeOH: DCM = 80: 20 at 1.0 mL/min; Temp: 30 ℃; Wavelength: 254 nm, R _T = 6.641 min) . ¹H NMR (400 MHz, DMSO-d ₆) δ 8.12 (S, 1H) , 7.87 -7.80 (m, 2H) , 7.69 -7.62 (m, 4H) , 5.89 -5.78 (m, 1H) , 5.47 -5.23 (m, 1H) , 4.56 -4.13 (m, 4H) , 3.74 (S, 1H) , 3.37 -3.32 (m, 1H) , 2.95 -2.62 (m, 2H) , 1.61-1.56 (m, 3H) , 1.20 -1.13 (m, 3H) . ¹⁹F NMR (376 MHz, DMSO-d ₆) δ -60.86 (S, 3F) .

11B:

LC-MS (ESI) : R _T = 3.953 min, mass calcd. for C ₂₇H ₂₃ClF ₃N ₅O ₂ 541.15, m/z found 542.2 [M+H] ⁺. Chiral analysis (Column: Chiralpak IG 5 μm 4.6 *250 mm; Mobile Phase: MeOH: DCM = 80: 20 at 1.0 mL/min; Temp: 30 ℃; Wavelength: 254 nm, R _T = 11.346 min) . ¹H NMR (400 MHz, DMSO-d ₆) δ 8.11 (S, 1H) , 7.87 -7.80 (m, 2H) , 7.69 -7.62 (m, 4H) , 5.91 -5.74 (m, 1H) , 5.47 -5.23 (m, 1H) , 4.56 -4.13 (m, 4H) , 3.75 (S, 1H) , 3.38 -3.31 (m, 1H) , 2.94 -2.60 (m, 2H) , 1.61 (S, 3H) , 1.20 -1.12 (m, 3H) . ¹⁹F NMR (376 MHz, DMSO-d ₆) δ -60.91 (S, 3F) .

Compound 12:

(R) -5- (9- (Benzo [d] oxazol-6-ylmethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) -2-chlorobenzonitrile

Intermediate 12-2:

6- (Bromomethyl) benzo [d] oxazole

To a solution of 6-methylbenzo [d] oxazole (1.1 g, 8.26 mmol) in chloroform (20 ml) was added 1-bromopyrrolidine-2, 5-dione 12-1 (1.54 g, 8.65 mmol) , (E) -2, 2'- (diazene-1, 2-diyl) bis (2-methylpropanenitrile) (121 mg, 0.74 mmol) at room temperature. After stirred at 65 ℃ for 2 hours, the reaction mixture was poured into water (50 ml) and extracted with dichloromethane (50 ml) for three times. The combined organic phases were washed with brine (50 ml) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated and the residue was purified by silica gel column chromatography (petroleum ether : ethyl acetate = 15 : 1 to 10 : 1) to give the title compound (900 mg, 90 %purity from NMR, 46 %yield) as white solids. LC-MS (ESI) : R _T = 1.52 min, mass calcd. for C ₈H ₆BrNO 211.0, m/z found 212.0 [M+H] ⁺. ¹H NMR (400 MHz, CDCl ₃) δ 8.13 (S, 1H) , 7.75 (d, J = 8.4 Hz, 1H) , 7.64 (S, 1H) , 7.42 (d, J = 9.2 Hz, 1H) , 4.64 (S, 2H) .

Compound 12:

To a solution of (R) -2-chloro-5- (3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 4-5 (150 mg, 90 %purity, 0.37 mmol) in N, N-dimethylformamide (10 ml) was added 6- (bromomethyl) benzo [d] oxazole 12-2 (258 mg, purity 90 %, 1.10 mmol) , sodium hydride (44 mg, purity 60 %, 1.10 mmol) at room temperature. After stirred at room temperature for 2 hours, the mixture was quenched with water (30 ml) and extracted with ethyl acetate (30 ml) for three times. The combined organic layers were washed with brine (20 ml) , dried over anhydrous Na ₂SO _4 (S) and filtered. The filtrate was concentrated and purified by C18 column (acetonitrile : water (0.1 %ammonium bicarbonate) = 5 %to 80 %) to give the title compound (55 mg, 97.3 %purity from LCMS, 29 %yield) as white soilds. LC-MS (ESI) : R _T = 3.629 min, mass calcd. for C ₂₆H ₂₁ClN ₆O ₃ 500.1, m/z found 501.2 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.73 (S, 1H) , 8.13 (S, 1H) , 7.87 -7.70 (m, 4H) , 7.40 -7.34 (m, 1H) , 5.48 -5.24 (m, 1H) , 4.83 -4.44 (m, 3H) , 4.31-4.14 (m, 3H) , 3.74 (br s, 2H) , 2.96 (br s, 1H) , 2.43 -2.25 (m, 1H) , 1.23 -1.13 (m, 3H) .

Compound 13:

(R) -2-Chloro-5- (9- (imidazo [1, 2-a] pyridin-6-ylmethyl) -3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

Intermediate 13-2:

6- (Bromomethyl) imidazo [1, 2-a] pyridine

To a solution of imidazo [1, 2-a] pyridin-6-ylmethanol 13-1 (300 mg, 97 %purity, 1.96 mmol) in 1, 4-dioxane (6 mL) was added tribromophosphine (1.0 g, 3.69 mmol) at 0 ℃. After stirred at room temperature under nitrogen for 2 hours, the reaction mixture was concentrated to give the title compound (150 mg, 92 %purity from LCMS, 33 %yield) as yellow oil. LC-MS (ESI) : R _T = 1.36 min, mass calcd. for C ₈H ₇BrN ₂, 210.0, m/z [M+H] found 211.1.

Compound 13:

To a solution of (R) -2-chloro-5- (3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 4-5 (100 mg, 95 %purity, 0.26 mmol) , 6- (bromomethyl) imidazo [1, 2-a] pyridine 13-2 (100 mg, 92 %purity, 0.436 mmol) in 2-methyltetrahydrofuran (4 mL) at room temperature was added 50 %sodium hydroxide (2 mL) and benzyltriethylammonium chloride (10 mg, 0.044 mmol) . After stirred at 60 ℃ under nitrogen atmosphere for 2 hours, the reaction was quenched with water (30 mL) and extracted with ethyl acetate (50 mL) for three times. The combined organic layers were washed with brine (50 mL) , dried over anhydrous Na ₂SO _4 (S) and filtered. The filtrate was evaporated under reduced pressure to afford yellow crude solids, which was purified by Prep. HPLC (Column: waters Xbridge C18 (5 μm 19 *150 mm) , Mobile Phase A: Water (+ 0.02 %ammonium acetate) , Mobile Phase B: acetonitrile, UV: 214 nm, Flow rate: 15 mL/min, Gradient: 05 -95 % (%B) ) to give the title compound (27 mg, 99 %purity from ¹HNMR, 20.8 %yield) as white solids, which combined with another batch (total 47 mg, 99.0 %purity) were dissolved in acetonitrile (10 mL) and water (20 mL) . The mixture was dried under lioflization to give the title compound (41 mg, 99.4 %purity from LCMS, 87.5 %yield) as white solids. LC-MS (ESI) : R _T = 3.119 min, mass calcd. for C ₂₆H ₂₂ClN ₇O ₂, 499.2, m/z found 500.2 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.58 -8.51 (m, 1H) , 8.21 -8.11 (m, 1H) , 7.94 (S, 1H) , 7.87 -7.80 (m, 2H) , 7.57 -7.52 (m, 2H) , 7.26 -7.13 (m, 1H) , 5.47 -5.43 (m, 0.6H) , 5.29 -5.16 (m, 0.4H) , 4.69 -4.33 (m, 5H) , 4.17 -4.13 (m, 1H) , 3.78 (br s, 2H) , 3.03 -2.92 (m, 1H) , 2.67 -2.59 (m, 0.5H) , 2.41 -2.33 (m, 0.5H) , 1.26 -1.22 (m, 3H) .

Compound 14A and Compound 14B:

2-Chloro-5- ( (R) -3-methyl-10-oxo-9- ( (R*) -1- (2- (trifluoromethyl) pyridin-4-yl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile and

2-chloro-5- ( (R) -3-methyl-10-oxo-9- ( (S*) -1- (2- (trifluoromethyl) pyridin-4-yl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

Intermediate 14-2:

1- (2- (Trifluoromethyl) pyridin-4-yl) ethanol

To the solution of 4-bromo-2- (trifluoromethyl) pyridine 14-1 (500 mg, 2.21 mmol) in tetrahydrofuran (15 mL) at 0 ℃ was slowly added isopropylmagnesium chloride (2 mL, 2.60 mmol) . The reaction mixture was stirred at room temperature for 30 minutes then acetaldehyde (0.2 ml, 3.56 mmol) was added and the resulting mixture was stirred at 0 ℃ for 1 hour. It was poured into ammonium chloride aqueous (20 mL) and extracted with ethyl acetate (50 mL) for three times. The combined organic phases were washed with brine (20 mL) , dried over Na ₂SO _4 (S) and filtered. The filtrate was concentrated and the residue was purified by C18 (acetonitrile : water (0.1%ammonium bicarbonate) = 30 %to 80 %) to give the title compound (300 mg, 73 %purity from LCMS, 52 %yield) as white solids. LC-MS (ESI) : R _T = 1.33 min, mass calcd. for C ₈H ₈F ₃NO 191.1 m/z found 192.1 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.72 (d, J = 5.2 Hz, 1H) , 7.86 (S, 1H) , 7.69 (d, J = 4.8 Hz, 1H) , 5.64 (br, s, 1H) , 4.90 (q, J = 6.4 Hz, 1H) , 1.39 (d, J = 6.8 Hz, 3H) .

Intermediate 14-3:

4- (1-Bromoethyl) -2- (trifluoromethyl) pyridine

To the solution of 1- (2- (trifluoromethyl) pyridin-4-yl) ethanol 14-2 (1.7 g, 73 %purity, 6.49 mmol) in tetrahydrofuran (50 mL) at 0 ℃ was slowly added perbromomethane (4.3 g, 12.9 mmol) . The reaction mixture was stirred at room temperature then triphenylphosphine (3.4 g, 12.9 mmol) was added and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was filtered. The filtrate was concentrated and the residue was purified by silica gel column chromatography (petroleum ether : ethyl acetate = 20 : 1 to 10 : 1) to give the title compound (1.3 g, 90 %purity from NMR, 71 %yield) as white solids. ¹H NMR (400 MHz, CDCl ₃) δ 8.71 (d, J = 5.2 Hz, 1H) , 7.73 (S, 1H) , 7.54 (d, J = 5.2 Hz, 1H) , 5.12 (q, J = 6.8 Hz, 1H) , 2.05 (d, J = 6.8 Hz, 3H) .

Compound 14:

2-Chloro-5- ( (3R) -3-methyl-10-oxo-9- (1- (2- (trifluoromethyl) pyridin-4-yl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile

To a solution of (R) -2-chloro-5- (3-methyl-10-oxo-1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile (350 mg, 95 %purity, 0.90 mmol) in 2-methyltetrahydrofuran 4-5 (10 mL) was added 4- (1-bromoethyl) -2- (trifluoromethyl) pyridine 14-3 (329 mg, 90 %purity, 1.17 mmol) , N-benzyl-N, N-diethylethanaminium chloride (74 mg, 0.33 mmol) and sodium hydroxide (0.6 ml) at room temperature. After addition, the mixture stirred at 55 ℃ for 4 hours. The mixture was quenched with water (30 mL) and extracted with ethyl acetate (30 mL) three times. The combined organic layers were washed with brine (30 mL) , dried over anhydrous Na ₂SO _4 (S) and filtered. The filtrate was concentrated and purified by C18 column (acetonitrile : water (0.1 %ammonium bicarbonate) = 5 %to 80 %) to give the title compound (280 mg, 97.4 %purity from LCMS, 56 %yield) as white soilds. LC-MS (ESI) : R _T = 1.639 min, mass calcd. for C ₂₆H ₂₂ClF ₃N ₆O ₂ 542.1 m/z found 543.2 [M+H] ⁺.

30 mg (97.4 %purity) was further purified by prep-TLC (dichloromethane : ethyl acetate = 2 : 1) and C18 column (acetonitrile : water (0.1 %ammonium bicarbonate) = 5 %to 80 %) to give the title compound (15 mg, 98.8 %purity from LCMS) as white soilds. LC-MS (ESI) : R _T = 3.870 min, mass calcd. for C ₂₆H ₂₂ClF ₃N ₆O ₂ 542.1 m/z found 543.2 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.78 -8.69 (m, 1H) , 8.11 (S, 1H) , 7.87 -7.64 (m, 4H) , 5.83 -5.73 (m, 1H) , 5.47 -5.19 (m, 1H) , 4.53 -4.11 (m, 4H) , 3.79 (S, 1H) , 3.51 (S, 1H) , 2.97 (br s, 1H) , 2.78 -2.61 (m, 1H) , 1.68 -1.52 (m, 3H) , 1.23 -1.07 (m, 3H) . ¹⁹F NMR (376 MHz, DMSO-d ₆) δ -66.26 --66.29 (m, 3F) .

Compounds 14A and Compound 14B:

2-Chloro-5- ( (R) -3-methyl-10-oxo-9- ( (R*) -1- (2- (trifluoromethyl) pyridin-4-yl) ethyl) - 1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile and

A racemate of 2-chloro-5- ( (3R) -3-methyl-10-oxo-9- (1- (2- (trifluoromethyl) pyridin-4-yl) ethyl) -1, 2, 3, 4, 7, 8, 9, 10-octahydropyrido [4', 3': 3, 4] pyrazolo [1, 5-a] pyrazine-2-carbonyl) benzonitrile 14 (250 mg, 97.4 %purity, 0.45 mmol) was separated by chiral. SFC (Column: Chiralpak IA 5 um 20 *250 mm; Mobile Phase: CO ₂ : MeOH = 60 : 40 at 30 g/min; Co-solvent: MeOH (0.2 ℃) ; Col. Temp: 40 ℃; Wavelength: 220 nm, Back pressure: 100 bar) to afford the title compound 14A (60 mg, 99.3 %purity from LCMS, 24 %yield, 100 %stereopure) as white solids and 14B (50 mg, 98.9 %purity from LCMS, 20 %yield, 100 %stereopure) as white solids.

14A:

LC-MS (ESI) : R _T = 3.876 min, mass calcd. for C ₂₆H ₂₂ClF ₃N ₆O ₂ 542.1 m/z found 543.1 [M+H] +. Chiral analysis (Column (Chiralpak IA 5 um 4.6 *250 mm; Mobile Phase: CO2 : MeOH = 60 : 40 at 3 g/min; Col. Temp: 40 ℃; Wavelength: 230 nm, Back pressure: 100 bar; R _T = 7.25 min) . ¹H NMR (400 MHz, DMSO-d ₆) δ 8.74 (S, 1H) , 8.17 -8.12 (m, 1H) , 7.87 -7.68 (m, 4H) , 5.87 -5.70 (m, 1H) , 5.46 -5.22 (m, 1H) , 4.58 -4.32 (m, 3H) , 4.17 -4.07 (m, 1H) , 3.78 (br s, 1H) , 3.51 (br s, 1H) , 2.95 (br s, 1H) , 2.67 -2.56 (m, 1H) , 1.65 -1.53 (m, 3H) , 1.23 -1.11 (m, 3H) . ¹⁹F NMR (376 MHz, DMSO-d ₆) δ -66.25 (S, 3F) .

14B:

LC-MS (ESI) : R _T = 3.855 min, mass calcd. for C ₂₆H ₂₂ClF ₃N ₆O ₂ 542.1 m/z found 543.1 [M+H] ⁺. Chiral analysis (Column (Chiralpak IA 5 um 4.6 *250 mm; Mobile Phase: CO2 : MeOH = 60 : 40 at 3 g/min; Col. Temp: 40 ℃; Wavelength: 230 nm, Back pressure: 100 bar; R _T = 8.77 min) . ¹H NMR (400 MHz, DMSO-d ₆) δ 8.76 -8.65 (m, 1H) , 8.18 -8.02 (m, 1H) , 7.87 -7.57 (m, 4H) , 5.88 -5.63 (m, 1H) , 5.50 -5.16 (m, 1H) , 4.64 -4.01 (m, 4H) , 3.78 -3.58 (m, 1H) , 3.38 (br s, 1H) , 3.01 -2.75 (m, 1H) , 2.49 -2.38 (m, 1H) , 1.71 -1.45 (m, 3H) , 1.25 -1.00 (m, 3H) . ¹⁹F NMR (376 MHz, DMSO-d ₆) δ -66.29 --66.42 (m, 3F) .

EXAMPLE 1: Anti-HBV viral activity in HepG2.117 cells

Procedure

The anti HBV activity was measured using the HepG2.117 cell line, a stable, inducibly HBV producing cell line, which replicates HBV in the absence of doxicycline (Tet-off system) . The HepG2 cell line is available from ATCCR under number HB-8065. Transfection of the HepG2 cell line can be as described in Sun and Nassal 2006 Journal of Hepatology 45 (2006) 636-645 “Stable HepG2-and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus” .

For the antiviral assay, HBV replication was induced, followed by a treatment with serially diluted compound in 96-well plates. After 3 days of treatment, the antiviral activity was determined by quantification of intracellular HBV DNA using real-time PCR and an HBV specific primer set and probe.

Cytotoxicity of the compounds was tested using HepG2 or HepG2.117 cells, incubated for 3 or 4 days in the presence of compounds. The viability of the cells was assessed using the PERKIN ELMER ATPlite Luminescence Assay System. ”

Results

N.D = not determined

CC ₅₀ values: 3-days incubation unless marked with * (*= 4-days incubation)

Induction or Non-induction of HBc speckling

HepG2.117 cells were cultured in the presence of DMSO or test compound in absence of doxycycline.

After formaldehyde fixation and Triton-X-100 permeabilization, Hepatitis B virus core protein (HBc) was immunolabeled with a primary anti-HBc antibody. ALEXA 488-conjugated secondary antibody was used for fluorescent detection of the primary HBV Core signal. CELLMASK Deep Red and HOECHST 33258 were used for the detection of cytoplasm and nucleus respectively, which allowed the segmentation of cellular compartments.

An image analysis software that allows to detect different morphological phenotypes was used to determine the level of HBV core in the cytoplasm or nucleus (high content imaging assay) .

Claims

A compound of Formula (I) ,

or a stereoisomeric or a tautomeric form thereof, wherein

R ^1a represents halo;

R ^1b is selected from the group consisting of CN, CF ₃, CHF ₂, OCHF ₂ and OCF ₃;

R ^1c represents hydrogen or a substituent selected from the group consisting of halo, CF ₃, C _1- ₄alkyl and C _3-6cycloalkyl;

R ² is hydrogen or a substituent selected from the group consisting of CHF ₂, CF ₃, C _1-4alkyl, C _1-4alkylOC _1-4alkyl and C _3-6cycloalkyl;

Q represents a ring selected from the group consisting of phenyl, a five-membered aromatic heterocyclic ring, and a six-membered aromatic heterocyclic ring;

n represents 1, 2 or 3;

each R ³ independently represents a substituent selected from the group consisting of CF ₃, CHF ₂, CH ₂F, C _1-6alkyl, OC _1-6alkyl, OCF ₃, OCHF ₂, and C _3-6cycloalkyl;

in the event that n represents 2 or 3, two R ³ on adjacent ring atoms, together with said ring atoms, optionally form a 5-membered ring or 6-membered ring, said ring optionally comprising 1, 2, or 3 heteroatoms each independently selected from N, O and S, said ring optionally carrying one or more fluoro or oxo substituents;

or a pharmaceutically acceptable salt or a solvate thereof,

with the proviso that the compound is not:
The compound of claim 1, wherein Q is a phenyl or pyridyl ring.
The compound of claim 1 or 2, wherein n is 2, and wherein Q- (R ³) _n is an imidazopyridinyl group, such as a group satisfying formula (QR1) , with *indicating the attachment point of the group:
The compound of claim 1, wherein the structure of Formula (I) satisfies Formula (IA) :

wherein W and X each independently are CR ³ or N, and wherein R ^1a, R ^1b, R ^1c, and R ², are as defined for Formula (I) , and wherein R ³ represents a substituent selected from the group consisting of CF ₃, CHF ₂, CH ₂F, C _1-6alkyl, OC _1-6alkyl, OCF ₃, OCHF ₂, and C _3-6cycloalkyl; or wherein,

in the event that W and X are both CR ³, the R ³ groups, together with the carbon atoms to which they are attached, form a 5-membered ring comprising 1, 2 or 3 heteroatoms each independently selected from N and O, said ring optionally carrying one or more fluoro or oxo substituents.
The compound of claim 1, wherein the structure of Formula (I) satisfies Formula (IB) :
The compound of claim 1, wherein the structure of Formula (I) satisfies Formula (IC) :

wherein R ³ is selected from the group consisting of CF ₃, OCF ₂H, and OCF ₃.
The compound of claim 6, wherein R ³ is in the meta or para position.
The compound of any one of the preceding claims, wherein R ^1a is chloro, R ^1b is cyano, and R ^1c is hydrogen.
A compound selected from the group consisting of the following compounds 1-14 or a stereoisomeric or a tautomeric form thereof:

or a pharmaceutically acceptable salt, an N-oxide, or a solvate thereof.
A pharmaceutical composition, which comprises the compound of any one of claims 1 to 9, and which further comprises at least one pharmaceutically acceptable excipient.
The compound of any one of claims 1 to 9, or the pharmaceutical composition of claim 10, for use as a medicament.
The compound of any one of claims 1 to 9, or the pharmaceutical composition of claim 10, for use in the prevention or treatment of an HBV infection or of an HBV-induced disease in a subject in need thereof.
The compound of any one of claims 1 to 9, or the pharmaceutical composition of claim 10, for use in the prevention or treatment of chronic hepatitis B.
A method of treating an HBV infection or an HBV-induced disease in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of the compound of any one of claims 1 to 9 or the pharmaceutical composition of claim 10.
A product comprising a first compound and a second compound as a combined preparation for simultaneous, separate or sequential use in the prevention or treatment of an HBV infection or of an HBV-induced disease in a subject in need thereof, wherein said first compound is different from said second compound, wherein said first compound is the compound of any one of claims 1 to 9 or the pharmaceutical composition of claim 10, and wherein said second compound is another HBV inhibitor.
The product of claim 15, wherein said second compound is another HBV inhibitor which is selected from the group consisting of: therapeutic agents selected from HBV combination drugs, HBV vaccines, HBV DNA polymerase inhibitors, immunomodulatory agents, toll-like receptor (TLR) modulators, interferon alpha receptor ligands, hyaluronidase inhibitors, hepatitis b surface antigen (HBsAg) inhibitors, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors, cyclophilin inhibitors, HBV viral entry inhibitors, antisense oligonucleotide targeting viral mRNA, short interfering RNAs (siRNA) and ddRNAi endonuclease modulators, ribonucleotide reductase inhibitors, HBV E antigen inhibitors, covalently closed circular DNA (cccDNA) inhibitors, famesoid X receptor agonists, HBV antibodies, CCR2 chemokine antagonists, thymosin agonists, cytokines, nucleoprotein modulators, retinoic acid-inducible gene 1 simulators, NOD2 stimulators, phosphatidylinositol 3-kinase (PI3K) inhibitors, indoleamine-2, 3-dioxygenase (IDO) pathway inhibitors, PD-1 inhibitors, PD-L1 inhibitors, recombinant thymosin alpha-1, bruton’s tyrosine kinase (BTK) inhibitors, KDM inhibitors, HBV replication inhibitors, arginase inhibitors, and other HBV drugs.
A compound as defined in any one of claims 1 to 9 or the pharmaceutical composition of claim 10 for use in the prevention or treatment of an HBV infection or an HBV-induced disease in a subject, wherein the compound or pharmaceutical composition is administered to the subject in combination with another HBV inhibitor.