Indazole pyridone compounds and uses thereof

Abstract

Novel tetracyclic pyridone compounds are provided according to formula 1: (I), where R and X are defined herein. Pharmaceutical compositions containing these compounds and methods of using these compounds and compositions for the treatment and prevention of HBV infection are also provided. [Formula 1] JPEG2025533792000113.jpg3451

Description

[Technical Field]

[0001] The present invention relates to novel fused tetracyclic pyridone compounds, which are inhibitors of hepatitis virus expression that act selectively in the liver and are useful in the treatment of viral infections, particularly hepatitis B virus (HBV) and hepatitis D virus (HDV). The present invention provides the novel tetracyclic pyridone compounds disclosed herein, pharmaceutical compositions containing them, and methods of using these compounds and compositions for the treatment and prevention of HBV infection. [Background technology]

[0002] Hepatitis B virus (HBV) infection is one of the most common infectious diseases worldwide. Chronic hepatitis B (CHB) represents a significant unmet medical need, with over 240 million people chronically infected worldwide. Chronic HBV carriers can develop serious liver diseases, including chronic hepatitis, cirrhosis, and even primary hepatocellular carcinoma (HCC). It is estimated that approximately 650,000 people die each year from CHB [(Chisari, Isagawa et al. 2010, 2016), (GBD Mortality Causes of Death Collaborators 2016, World Health Organization (WHO) 2018)].

[0003] The goal of chronic hepatitis B (CHB) treatment is to improve quality of life and survival by preventing disease progression to cirrhosis and hepatocellular carcinoma (HCC). Functional cure of HBV, i.e., loss of HBV surface antigen (HBsAg) with or without seroconversion to anti-HBsAg, is recognized as the goal of anti-HBV treatment (Lok and McMahon 2009, EASL 2012, Sarin, Kumar et al. 2015, Terrault, Bzowej et al. 2016, European Association for the Study of the Liver 2017). Previous studies have shown that HBsAg loss is associated with improved liver tissue, including resolution of cirrhosis, reduced HCC risk, and prolonged survival, which is considered evidence of functional cure (Fattovich, Giustina et al. 1998, Benias and Min 2011, Kim, Lim et al. 2013).

[0004] Nucleoside(t)ide analogs are the standard of care for chronic hepatitis B (CHB) treatment, providing long-term clinical benefits by suppressing viral replication and reducing the risk of liver complications (Dienstag, Goldin et al. 2003, Liaw 2011, Lok 2013). However, treatment with nucleoside(t)ide inhibitors rarely leads to functional cure (Kwon and Lok 2011). Therefore, new treatment options that improve HBsAg clearance rates and provide time-limited treatment options aimed at functional cure are needed.

[0005] Clearance of HBV-infected hepatocytes requires a broad immune response to HBV (Rehermann and Nascimbeni 2005, Das and Maini 2010, Burton, Pallett et al. 2018). Previous studies have suggested that the presence of high concentrations of major viral antigens, such as HBsAg, during chronic HBV infection can exhaust antiviral T cells (Frebel, Richter et al. 2010, Isagawa, Chung et al. 2013, Ochel, Cebula et al. 2016, Zhu, Liu et al. 2016). Furthermore, several reports have described that HBsAg suppresses HBV-specific immune responses by directly modulating the function of dendritic cells (DCs), monocytes, and natural killer cells (NKs) (Chen, Wei et al. 2005, Op den Brouw, Binda et al. 2009, Woltman, Op den Brouw et al. 2011, Kondo, Ninomiya et al. 2013, Mueller, Wildum et al. 2017). Furthermore, preclinical studies have shown that reduction of extracellular HBsAg with monoclonal HBsAg antibodies followed by vaccination can eliminate HBV in serum and liver in a mouse model of chronic HBV infection (Zhu, Liu et al. 2016).

[0006] Taken together, these studies suggest that antiviral agents such as PAPD5 / 7 inhibitors (HBV RNA destabilizers / degraders / HBsAg secretion inhibitors) may have a therapeutic role in reducing HBsAg (hepatitis B virus surface antigen) levels and restoring virus-specific immune responses in CHB (chronic hepatitis B).

[0007] Hepatitis delta virus (HDV) is the causative agent of chronic hepatitis delta (CHD), the most severe form of viral hepatitis. At least 12 million people worldwide are HBV / HDV coinfected, although this number may be underestimated due to suboptimal testing. HDV infection can occur simultaneously with HBV or as a superinfection in patients already chronically infected with HBV. HDV is dependent on HBV because it uses the HBV-derived envelope protein (HBsAg) for release and de novo infection. Effective antiviral treatment is urgently needed to prevent progression to cirrhosis, end-stage liver disease, and hepatocellular carcinoma (HCC) (Oandri, Volmari et al., 2022).

[0008] The minimum acceptable endpoints for new anti-HOV treatments are a 2-log reduction in HOV RNA and normalization of alanine translocaserin (ALT) levels during treatment (FDA 2019; Yurdaydin, Abbas et al. 2019). Previous studies have shown a 2-log reduction in HOV RNA in patients treated with conventional interferon. 10 It has been shown that a reduction in β-glucan is associated with a survival benefit in CHD (Farci, Roskams et al., 2004).

[0009] Pegylated interferon alpha (pegIFNα) has been used as an off-label treatment for HOV, but it is known to have associated side effects, low response rates, and high relapse rates (Bahcecioglu, Ispiroglu et al. 2015; Rizzetto and Smedile 2015). Among novel anti-HOV strategies, brevirtide targets the host receptor NTCP to efficiently inhibit HBV and HOV entry and is the first HOV-specific drug to receive conditional market approval in Europe (Masetti and Aghemo 2021; Lampertico, Roulot et al. 2022). Currently undergoing clinical trials, lonafarnib is a farnesyltransferase inhibitor that suppresses HOV release (Yurdaydin, Keskin et al. 2022). More recently, preclinical studies have shown that anti-HBsAg monoclonal antibodies neutralize HOV in vitro and significantly reduce HOV RNA levels in a mouse model of CHO (Lempp 2021).

[0010] Several previous clinical studies have evaluated the administration of anti-HBsAg monoclonal antibodies by intravenous or subcutaneous injection in CHB patients and shown that it was safe and well tolerated, with significant reductions in peripheral HBsAg levels in almost all subjects ( Galun, Eren et al., 2002 ; Lee, Park et al., 2020 ; Agarwal, Yuen et al., 2022 ).

[0011] These data suggest a potential role for HBsAg lowering in the development of anti-HBV and anti-HOV therapies. Here, we describe novel liver-targeted PAP05 / 7 inhibitors (HBV RNA destabilizers / degraders / HBsAg secretion inhibitors) aimed at lowering HBsAg in patients with HBV or HBV / HOV. These, alone or in combination with other anti-HBV / anti-HOV drugs, may lead to functional cures.

[0012] Recent studies have demonstrated the neurotoxicity of hepatitis B virus (HBV) PAPDS / 7 inhibitors (HBV RNA destabilizers / degraders / HBsAg secretion inhibitors). Specifically, GS-8873, which is structurally closely related to the first class of compound, RO7834, differs by replacing carbon with nitrogen to form a hydrazine-based core (Lake, 5 April D. et al., Toxicological Sciences (2022) 186(2), pp. 298-308; see structure diagram below).

[0013] [ka] GS-8873 was confirmed to prolong nerve conduction velocity (NCV) in several peripheral nerves in both rats and monkeys. These effects began to appear at week 4 and became more pronounced by week 13. Rats are a more sensitive species, and rat caudal nerve NCV, digital nerve NCV, cauda equina nerve conduction delay, digital nerve delay, and tibial nerve delay all changed by more than 20% from baseline after 13 weeks of daily administration of GS-8873 at doses of 20 MPK and 60 MPK. These changes are believed to support the peripheral neuropathy observed in monkeys during the 13-week study. Furthermore, GS-8873 significantly affected functional tests in rats starting at week 4. It is unclear whether this peripheral and central neurotoxicity is due to chemotype-specific or target-specific effects. Several compounds in this class have advanced to clinical trials but have been withdrawn or discontinued (Roche's RO7834, Enanta's EDP-721). The reasons for its discontinuation have not been fully disclosed. Therefore, the precedent of systemic safety signals in this class of HBV RNA destabilizing agents has led us to focus our efforts on the mechanism of liver targeting, allowing us to avoid systemic safety signals by increasing drug concentrations in the liver and keeping systemic drug exposure low.

[0014] While ACC inhibitors have the potential to address factors contributing to the pathogenesis of NASH, the importance of de novo lipogenesis in human bone marrow for platelet production limits the degree of systemic ACC inhibition that can be safely tolerated during chronic treatment. Pfizer and Giliado Nimbus ACC inhibitors are designed to incorporate structural features intended for recognition by organic anion transporting polypeptides (OATPs), members of the solute carrier organic anion (SLCO) superfamily of xenobiotic transporters. Of the 11 human OATP transporters, OATP1B1 and OATP1B3 are expressed in the venous membranes of hepatocytes and can promote the hepatic uptake of their respective substrates (e.g., statins such as atorvastatin and rosuvastatin) (Kalliokoski, A. et al., Br. J. Pharmacol. 2009, 158, 693-705).

[0015] Pfizer and Gili A. Nimbus sought ACC inhibitors that are substrates of the OATP1B1 / 1B3 transporters to enhance liver selectivity. Thus, the precedent for ACC inhibitors and statins utilizing OATP1B1- and OATP1B3-mediated hepatic uptake to enhance liver selectivity bodes well for the possibility of utilizing these same transporters for HBV RNA destabilizing factors. Summary of the Invention

[0016] [ka] A first aspect of the present invention is a compound of Formula 1, or a pharmaceutically acceptable salt thereof, wherein R is selected from a hydrogen group, an ethyl group, or a linear, cyclic, or branched (3-6 carbon alkyl) group, and X is a linker selected from a linear, cyclic, or branched 5-10 carbon alkene group, including lactimic mixtures thereof, and pharmaceutical compositions thereof.

[0017] [ka] A second aspect of the present invention is a method of treating a patient suffering from a viral infection, such as chronic hepatitis B or hepatitis D, by administering an effective amount of a pharmaceutical composition comprising a compound of Formula 1, or a pharmaceutically acceptable salt thereof. where R is selected from hydrogen, ethyl, or linear, cyclic, or branched (3-6 carbon alkyl) groups, and X is a linker selected from linear, cyclic, or branched 5-10 carbon alkene groups, or lactimic mixtures thereof.

[0018] A third aspect of the present invention is a method for preventing the development of downstream liver disease due to chronic hepatitis B or hepatitis D infection by administering an effective amount of a pharmaceutical composition comprising a compound of Formula 1, or a pharmaceutically acceptable salt thereof.

[0019] [ka] where R is selected from hydrogen, ethyl, or linear, cyclic, or branched (3-6 carbon alkyl) groups, and X is a linker selected from linear, cyclic, or branched 5-10 carbon alkene groups, or lactimic mixtures thereof.

[0020] [ka] A fourth aspect of the present invention is the use of a compound of Formula 1, or a pharmaceutically acceptable salt thereof, where R is selected from hydrogen, ethyl, or a linear, cyclic, or branched (3-6 carbon alkyl) group, and X is a linker selected from a linear, cyclic, or branched 5-10 carbon alkene group, or a lactimic mixture thereof, in the manufacture of a medicament for the treatment of a patient with a viral infection, such as hepatitis B or hepatitis D.

[0021] [ka] A fifth aspect of the present invention is the use of a compound of formula 1: wherein R is selected from hydrogen, ethyl, or linear, cyclic, or branched (3-6 carbon alkyl) groups, and X is a linker selected from linear, cyclic, or branched 5-10 carbon alkene groups, or lactimic mixtures thereof. The compound is used in the manufacture of a medicament for the prophylaxis of patients with chronic hepatitis B or hepatitis D infection from developing downstream liver disease. A sixth aspect of the current invention is a method of treating a patient with a viral infection by administering an effective amount of a pharmaceutical composition comprising the following compound:

[0022] A sixth aspect of the present invention is a method of treating a patient suffering from a viral infection by administering an effective amount of a pharmaceutical composition comprising a compound of Formula 1. [ka] where R is selected from hydrogen, ethyl, or linear, cyclic, or branched (C3-C8 alkyl), and X is linear, cyclic, or branched C5-C 10 or a racemic mixture thereof. The method further comprises administering to the patient an additional HBV replication inhibitor (e.g., including but not limited to, nucleoside (t) analog polymerase inhibitors, non-nucleoside (t) analog polymerase inhibitors), an HBsAg-targeting agent (e.g., including but not limited to, siRNA and antisense targeted to HBV transcripts, HBV capsid inhibitors, cccDNA inhibitors, HBx inhibitors, and antibodies targeted to HBV proteins), an immunomodulator (e.g., including but not limited to, immune checkpoint inhibitors (small molecule inhibitors or blockade antibodies), natural or modified cytokines - interferon-α, TLR agonists - TLR-7, TLR-9, TLR-8, and immunomodulatory vaccines).

[0023] [ka] A seventh aspect of the current invention is a method for preventing the progression of liver disease resulting from chronic hepatitis B or hepatitis D infection by administering an effective amount of a pharmaceutical composition comprising: A compound of formula 1 or a pharmaceutically acceptable salt thereof, wherein R is selected from hydrogen, ethyl, or a linear, cyclic, or branched C3-C8 alkyl group; and X is a linear, cyclic, or branched C5-C8 alkyl group. 10 The linker is selected from an alkylene group, or a racemic mixture thereof. The method further includes administering to the patient an HBV replication inhibitor (e.g., including but not limited to, nucleoside (t) analog polymerase inhibitors, non-nucleoside (t) analog polymerase inhibitors), an HBsAg-targeting agent (e.g., including but not limited to, siRNA and antisense targeted to HBV transcripts, HBV capsid inhibitors, cccDNA inhibitors, HBx inhibitors, and antibodies targeted to HBV proteins), an immunomodulator (e.g., including but not limited to, immune checkpoint inhibitors (small molecule inhibitors or blockade antibodies), natural or modified cytokines - interferon-α, TLR agonists - TLR-7, TLR-9, TLR-8, and immunomodulatory vaccines).

[0024] In some embodiments of the compound of Formula 1, X is a linear C5-C8 alkylene group. In some embodiments of the compound of Formula 1, X is selected from the group consisting of: In some embodiments of the compound of Formula 1, R is selected from the group consisting of: In some embodiments of the compound of Formula 1, R is H. In some embodiments of the compound of Formula 1, R is i-C3H7. In some embodiments of the compound of Formula 1, R is n-C4H9. In some embodiments, the compound of Formula 1 is any of the following: Linear -C5H 10 -, linear -CH 12 -, linear -C7H 14- and linear -CH 16 - n-C2H5, n-C3H7, i-C3H7, n-C4H9, n-C5H 11 , n-CH 13 2-Ethylbutyl, n-CH 15 , n-C8H 17 etc.

[0025] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [Brief explanation of the drawings]

[0026] Figure 1 shows the effect of some compounds of Formula 1 on reducing HBV S antigen levels when administered in an in vivo AAV mouse model.

[0027] The present invention provides novel compounds that inhibit the secretion of HBsAg from cells infected with hepatitis B virus (HBV), thereby reducing viral load and viral replication in patients with chronic HBV infection. Therefore, the compounds of the present invention are suitable for treating HBV patients, particularly chronic HBV patients. The compounds of the present invention are also suitable for treating HBV-associated patients who are co-infected with HBV.

[0028] Each compound shown in the examples, including each compound listed in Table 1, is a specific embodiment of the compounds of the invention. The following description of preferred embodiments of the invention is not intended to limit the scope of the invention.

[0029] In some embodiments, a compound of Formula 1 is provided: [ka] where R is a hydrogen group, an ethyl group, or a linear, cyclic, or branched C3-C8 alkyl group, and X is a linear, cyclic, or branched C5-C8 alkylene group. Also included are pharmacologically acceptable salts, racemic mixtures, and formulations thereof.

[0030] Preferably, X is linear CH 10 , linear CH 12 , and linear CH 14 and pharmacologically acceptable salts thereof, and formulations thereof.

[0031] Preferably, R is n-C2H5, n-C3H7, i-C3H7, n-C4H9, n-C5H 11 , n-CH 13 , 2-ethylbutyl, n-CH 15, and n-CH 17 and pharmacologically acceptable salts thereof, racemic mixtures thereof, and formulations thereof. Further preferred are compounds of formula 1 in which R is a hydrogen group, their pharmacologically acceptable salts, racemic mixtures, and formulations thereof.

[0032] Preferred are compounds of formula 1 wherein R is i-C3H7, their pharmacologically acceptable salts, racemic mixtures, and formulations thereof.

[0033] Preferred are compounds of Formula 1 where R is n-C4H9, their pharmacologically acceptable salts, racemic mixtures, and formulations thereof.

[0034] Preferred are the compounds of Formula 1 shown in Table 1 below, their pharmacologically acceptable salts, racemic mixtures, and formulations thereof.

[0035] Compounds I.6, I.9, or I.10 shown in Table 1 below are preferred, including pharmaceutically acceptable salts thereof, or racemic mixtures thereof, and pharmaceutical compositions thereof.

[0036] A preferred method is to treat a patient with chronic HBV infection by administering a therapeutic amount of a pharmaceutical composition comprising a compound of formula 1 or a racemic mixture thereof, preferably a compound of formula 1 wherein R is n-C2H5, n-C3H7, i-C3H7, n-C4H9, n-C5H 11 , n-CH 13 , 2-ethylhyptane, n-CH 15 , n-C8H 17 More preferably, R is H, i-C3H7 or n-C4H9, and X is a linear C5-C8 alkylene group, more preferably a linear -C5H 10 -, -C6H 12 -, and -CH 14and more preferably the compound of formula 1 is a compound shown in Table 1 below, in particular compound I.6, I.9 or I.10 shown in Table 1.

[0037] Preferred is the use of a compound of formula 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of patients with HBV infection, preferably a compound of formula 1, wherein R is n-C2H5, n-C3H7, i-C3H7, n-C4H9, n-C5H 11 , n-CH 13 , 2-ethylhyptane, n-CH 15 , n-C8H 17 More preferably, R is H, i-C3H7 or n-C4H9, and X is a linear C5-C8 alkylene group, more preferably a linear -C5H 10 -, -C6H 12 -, and -CH 14 and more preferably the compound of formula 1 is a compound shown in Table 1 below, in particular compound I.6, I.9 or I.10 shown in Table 1.

[0038] A preferred method is a method of treating a patient with chronic HBV infection by administering an effective amount of a compound of Formula 1 or a pharmaceutical composition comprising a compound of Formula 1, or a pharmaceutically acceptable salt thereof, preferably a compound of Formula 1, wherein R is n-C2H5, n-C3H7, i-C3H7, n-C4H9, n-C5H 11 , n-CH 13 , 2-ethylhyptane, n-CH 15 , n-C8H 17 More preferably, R is H, i-C3H7 or n-C4H9, and X is a linear C5-C8 alkylene group, more preferably a linear -C5H 10 -, -C6H 12- , and -CH 14and more preferably, the compound of formula 1 is a compound shown in Table 1 below, and in particular compound I.6, I.9, or I.10 shown in Table 1. The method further comprises administering to the subject an additional therapeutic agent selected from the group consisting of: HBV replication inhibitors (including but not limited to nucleoside (analog) polymerase inhibitors and non-nucleoside (analog) polymerase inhibitors), HBsAg targeting agents (including but not limited to siRNA and antisense targeting against HBV transcripts), HBV capsid inhibitors, cccDNA inhibitors, HBx inhibitors, antibodies targeting HBV proteins, immunomodulators (including but not limited to immune checkpoint inhibitors (small molecule inhibitors or blocking antibodies), natural or modified cytokines (interferon-α), TLR agonists (TLR-7, TLR-9, TLR-8), and immunomodulatory vaccines).

[0039] Although one enantiomer of compounds of this formula is typically more active than the other, both isomers exhibit activity against HBsAg, as shown in published PCT applications WO 2018 / 198079 and US 10,301,312 B2 (issued May 28, 2019), the contents of which are hereby incorporated by reference in their entireties.

[0040] The terms "optical isomer" or "stereoisomer" refer to the various stereoisomeric structures that may exist for a particular compound of the present invention, including geometric isomers. It is understood that substituents may be attached to chiral centers of carbon atoms. The term "chiral" refers to a molecule that has the property that it is not superimposable on its mirror image, while the term "achiral" refers to a molecule that is superimposable on its mirror image. Thus, the present invention includes enantiomers, diastereomers, or racemates of a compound. "Enantiomers" refers to a pair of non-superimposable mirror images of one another. A 1:1 mixture of a pair of enantiomers is called a "racemic" mixture.

[0041] The term is used to refer to racemic mixtures when appropriate. "Diastereomers" refer to stereoisomers with at least two asymmetric atoms that are not mirror images of each other. Absolute stereochemistry is specified according to the Cahn-Engle-Prillog RS system. When compounds are pure enantiomers, the stereochemistry at each chiral carbon can be specified as either R or S.

[0042] Compounds of unknown absolute configuration may be designated as (+) or (-) depending on the direction (right- or left-handed) that they rotate plane-polarized light at the wavelength of the sodium D line. Certain compounds described herein may contain one or more asymmetric centers or axes and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, which may be defined in terms of absolute stereochemistry as (R) or (S).

[0043] As used herein, a "prodrug" is a molecule that is converted into an active parent drug in the body and corresponds to physical properties of the active parent drug that typically detract from high oral bioavailability. A "prodrug" possesses, by chance or design, built-in structural features that allow it to bioconvert to the active parent drug in the body. This conversion may occur through chemical or enzymatic processes, or a combination of both. Upon conversion, the active drug is released from the masked "promoiety" or drug carrier, and the resulting molecule (the active metabolite) exerts the full desired therapeutic effect. For a detailed review of prodrug strategies and examples, see Rautio, J., Meanwell, N., Di, L. et al., Nat Rev Drug Discov 2018, 17, 559-587.

[0044] [Table 1-1] [Table 1-2] [Table 1-3] Some of the compounds of Formula 1 in Table 1 are "prodrugs," i.e., they are converted in the body to other compounds of Formula 1 ("parent drugs"), and these parent drugs are also listed in Table 1. [ka] Each compound shown in the examples, including the "prodrug" and "parent drug" listed in Table 2, is a specific example of a compound of the present invention.

[0045] Table 2. Examples of "prodrugs" and "parent drugs" selected from compounds of formula 1. [Table 2-1] [Table 2-2]

[0046] For the purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall also include the plural. Terms used in this specification have the following meanings unless the context clearly indicates otherwise.

[0047] As used herein, the term "subject" refers to an animal. In certain embodiments, the animal is a mammal. The subject may be, for example, a primate (e.g., a human), cow, sheep, goat, horse, dog, cat, rabbit, rat, mouse, fish, bird, etc. In certain embodiments, the subject is a human. The term "patient" as used herein refers to a human subject. The term "inhibition" or "inhibiting" as used herein refers to the reduction or suppression of a particular condition, symptom, or disorder, disease, or a significant decrease in the baseline activity of a biological activity or process.

[0048] As used herein, the term "treatment" or "therapy" refers, in one embodiment, to ameliorating a disease or disorder (i.e., slowing or halting the progression of the disease or reducing at least some of its clinical symptoms). In another embodiment, "treatment" or "therapy" refers to alleviating or improving at least one physical parameter, which may not be discernible by the patient. In yet another embodiment, "treatment" or "therapy" refers to modulating a disease or disorder physically (e.g., stabilizing discernible symptoms), physiologically (e.g., stabilizing physical parameters), or both. In yet another embodiment, "treatment" or "therapy" refers to preventing or delaying the onset, progression, or development of a disease or disorder.

[0049] As used herein, the terms "a," "an," "the," and similar terms used in the context of the present invention (particularly in the context of the claims) are to be construed as including both the singular and the plural, unless otherwise expressly indicated herein or clearly contradicted by context.

[0050] All methods described herein can be performed in any suitable order unless otherwise expressly indicated herein or clearly indicated to the contrary by context. All examples provided herein and the use of exemplary language (e.g., "for example") are intended to more clearly illustrate the invention and do not limit the scope of the invention. "Optionally substituted" means that the referenced group can be substituted at one or more positions with any of the subsequently listed radicals, or a combination thereof. It is understood that the number, arrangement, and choice of substituents include only those substitutions that a skilled chemist would predict to be reasonably stable. For example, "oxo" cannot be a substituent on an aryl or heteroaryl ring, and a single carbon atom cannot have three hydroxy or amino groups as substituents. Unless otherwise specified, the optional substituents are typically halo, oxo, CN, amino, hydroxy, -C 1-3up to four groups selected from alkyl, -OR*, -NR*2, -SR*, -SOR*, -COOR*, and -CONR*2, where each R is independently H or C 1-3 It is alkyl.

[0051] As used herein, "aryl" refers to a phenyl or naphthyl group unless otherwise specified. Aryl groups include halo, CN, amino, hydroxy, C, unless otherwise specified. 1-3 Optionally substituted with up to four groups selected from alkyl, -OR*, -NR*2, -SR*, -SOR*, -COOR*, and -CONR*2, where each R is independently H or C 1-3 It is alkyl.

[0052] As used herein, "halo" or "halogen" refers to fluorine, chlorine, bromine, or iodine. "C3-C8 alkyl" or "C3-C8 alkyl" refers to a straight or branched chain alkyl having from 3 to 8 carbon atoms. If a different number of carbon atoms is specified, e.g., C5 or C5, the definition should be modified accordingly. For example, "C 1-4 "Alkyl" refers to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl.

[0053] "C5- 10 alkylene" or "C5-C 10 The term "alkylene," as used herein, refers to a straight or branched alkyl group having 5 to 10 carbon atoms and having two open valencies that connect it to two other groups. If a different number of carbon atoms (e.g., C4 or C3) is specified, the definition should be modified accordingly. For example, "C 1-4 "Alkylene" is meant to represent methylene (-CH2), ethylene (-CH2CH2), straight or branched propylene (-CH2CH2CH2 or -CH2CHMe-CH2-), and the like.

[0054] "C5-8 The term "alkoxy" as used herein means 5-8 It refers to a straight or branched chain alkoxy (-O-alkyl) having 4 or 3 carbon atoms. If a different number of carbon atoms (e.g., C4 or C3) is specified, the definition should be modified accordingly. For example, "C 1-4 "Alkoxy" refers to methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy and tert-butoxy.

[0055] "C 1-4 The term "haloalkyl" or "C1-C4 haloalkyl," as used herein, refers to a straight or branched chain alkyl having from 1 to 4 carbon atoms in which at least one hydrogen atom is replaced by a halogen. The number of halogen substitutions may be from one to the number of hydrogen atoms on the unsubstituted alkyl group. If a different number of carbon atoms is specified (e.g., C6 or C3), the definition should be modified accordingly. Thus, "C 1-4 "Haloalkyl" refers to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl in which at least one hydrogen has been replaced with a halogen. For example, if the halogen is fluorine, the following structures are possible: CF3CH2-, (CF3)2CH-, CH3-CF2-, CF3CF2-, CF3-, CF2H-, CF3CF2CH(CF3)-, or CF3CF2CH2CF2-.

[0056] "C 38 The term "cycloalkyl" refers to a saturated monocyclic hydrocarbon ring having from 3 to 8 carbon atoms. Examples of such groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. If a different number of carbon atoms is specified (e.g., C3-C6), the definition should be modified accordingly.

[0057] The terms "4-8-membered heterocyclo," "5-6-membered heterocyclo," "3-10-membered heterocyclo," "3-14-membered heterocyclo," "4-14-membered heterocyclo," and "5-14-membered heterocyclo" refer to 4-8-, 5-6-, 3-10-, 3-14-, 4-14-, and 5-14-membered heterocyclo rings, respectively. Unless otherwise specified, these rings contain 1-7, 1-5, or 1-3 heteroatoms selected from nitrogen, oxygen, and sulfur as ring members, and the rings are saturated or partially saturated but not aromatic. Heterocyclo groups can be attached to other groups at either a nitrogen or a carbon atom. The term "heterocyclo" includes monocyclic, polycyclic and bridged groups. Examples of such heterocyclos include pyrrolidine, piperidine, piperazine, pyrrolidinone, morpholine, tetrahydrofuran, tetrahydrothiophene, tetrahydrothiopyran, tetrahydropyran, 1,4-dioxane, 1,4-oxathiane, 8-aza-bicyclo[3.2.1]octane, 3,8-diaza-bicyclo[3.2.1]octane, 3-oxa-8-aza-bicyclo[3.2.1]octane, 8-oxa-3-aza-bicyclo[3.2.1]octane, 2-oxa-5-aza-bicyclo[2.2.1]heptane, 2,5-diaza-bicyclo[2.2.1]heptane, azetidine, ethylenedioxo, oxetane, or thiazole. In certain embodiments, unless otherwise specified, heterocyclo groups have 1-2 heteroatoms selected from N, O, and S as ring members, and 4-7 ring atoms, and can be optionally substituted with up to 4 groups, including halo, oxo, CN, amino, hydroxy, C 1-3 alkyl, -OR*, -NR*2, -SR*, -SOR*, -COOR*, and -CONR*2, where R* is H or C 1-3 In particular, heterocyclo groups containing a sulfur atom may be substituted on the sulfur with one or two oxo groups.

[0058] As used herein, the term "4- to 6-membered cyclic ether" refers to a 4- to 6-membered ring containing one oxygen atom as a ring component. Examples include oxetane, tetrahydrofuran, and tetrahydropyran. "Heteroaryl" refers to a fully unsaturated (aromatic) ring. The term "heteroaryl" refers to a 5- to 14-membered monocyclic, bicyclic, or tricyclic aromatic ring system containing 1 to 8 heteroatoms selected from N, O, or S. Heteroaryls are typically 5- to 10-membered rings or ring systems (e.g., 5- to 7-membered monocyclic or 8- to 10-membered bicyclic rings), often 5- or 6-membered, and typically contain up to four heteroatoms (selected from N, O, and S), although heteroaryl rings often contain no more than one divalent O or S ring. Typical heteroaryl groups include furan, isothiazole, thiadiazole, oxadiazole, indazole, indole, quinoline, 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-imidazolyl, 3-, 4-, or 5-pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-, 4-, or 5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or 5-isoxazolyl, 3- or 5-(1,2,4-triazolyl), 4- or 5-(1,2,3-triazolyl), tetrazolyl, triazine, pyrimidine, 2-, 3-, or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-, or 5-pyrazinyl, 2-pyrazinyl, and 2-, 4-, or 5-pyrimidinyl. Heteroaryl groups include halogen, CN, amino, hydroxy, C 1-3 It may be substituted with up to four groups from the group consisting of alkyl, -OR*, -NR*2, -SR*, -SOR*, -COOR*, and -CONR*2, where each R is independently hydrogen or C 1-3 The term "hydroxy" or "hydroxyl" refers to an -OH group.

[0059] Furthermore, the compounds of the present invention (including their salts) may be obtained in the form of their hydrates or may contain other solvents used in crystallization. The compounds of the present invention may, inherently or by design, form solutions with pharmaceutically acceptable solvents (e.g., water). Therefore, the present invention is intended to encompass both dissolved and undissolved forms. The term "dissolved" refers to a molecular complex of a compound of the present invention (including its pharmaceutically acceptable salts) with one or more solvent molecules. Such solvent molecules are commonly used in the pharmaceutical field and are known to be harmless to the recipient, such as water, ethanol, etc. The term "hydrate" refers to a complex in which the solvent molecule is water.

[0060] As used herein, the term "salt" or "salts" refers to acid addition salts or base addition salts of the compounds of the present invention. "Salts" specifically includes "pharmaceutically acceptable salts." A "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the compounds of the present invention and generally does not have biologically or otherwise undesirable properties. In many cases, the compounds of the present invention are capable of forming acid or base salts by virtue of the presence of amino or carboxyl groups or groups similar thereto.

[0061] Pharmaceutically acceptable acid addition salts can be formed by reaction with inorganic and organic acids, including, for example, acetate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlortheophyllonate, citrate, ethanesulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, and hydroiodide / iodide. Hydroiodide / iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methylsulfate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphatephosphate, polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate, and trifluoroacetate.

[0062] Inorganic acids from which salts may be formed include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

[0063] Examples of organic acids from which salts can be formed include acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, and sulfosalicylic acid.

[0064] Pharmaceutically acceptable base addition salts can be formed by reaction with inorganic and organic bases. Inorganic bases from which salts can be formed include, for example, ammonium salts and metals from Groups I to XII of the periodic table. In certain embodiments, salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper. Particularly suitable salts include ammonium, potassium, sodium, calcium, and magnesium salts.

[0065] Organic bases from which salts may be formed include, for example, primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, etc. Particular organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0066] Pharmaceutically acceptable salts of the present invention can be synthesized from basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid form of the compound with a stoichiometric amount of a suitable base (e.g., hydroxide, carbonate, bicarbonate, etc., of sodium (Na), calcium (Ca), magnesium (Mg), or potassium (K)). Alternatively, the free base form of the compound can be prepared by reacting the free base form of the compound with a stoichiometric amount of a suitable acid. Such reactions are typically carried out in water, an organic solvent, or a mixture thereof. When possible, the use of non-aqueous solvents such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is preferred. Further lists of suitable salts can be found, for example, in "Remington's Pharmaceutical Sciences" (20th ed., Mack Publishing Company, Easton, Pa., 1985) and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Stahl and Wermuth, Wiley-VCH, Weinheim, Germany, 2002).

[0067] Any chemical formula given herein is intended to encompass both unlabeled forms (compounds with normal isotopic distributions) as well as isotopically labeled compounds where up to three atoms have an unnatural isotopic distribution. For example, deuterium ( 2 H) and carbon-13 ( 13 C), nitrogen-15( 15 Isotopically labeled compounds have the same structure as the depicted chemical formula, except that one or more atoms are replaced by an atom having a selected atomic mass or mass number other than their natural abundance. Examples of isotopes that can be usefully incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine. Specific examples include: 2 H (deuterium), 3 H (tritium), 11 C (carbon-11), 13 C (carbon-13), 14C (carbon-14), 15 N (nitrogen-15), 18 F (fluorine-18), 31 P(phosphorus-31), 32 P(phosphorus-32), 35 S (sulfur-35), 36 Cl (chlorine-36), 125 I (iodine-125), etc. The present invention includes various isotopically labeled compounds of the present invention. For example, 3 H (tritium) 14 C (carbon-14) is introduced, or it is a non-radioactive isotope. 2 H (deuterium) 13 These are compounds containing C (carbon-13) in excess of the normal isotopic distribution. Such isotope-labeled compounds are useful for metabolic studies (e.g. 14 C), reaction rate studies (e.g. 2 H or 3 H), are useful in detection or imaging techniques such as positron emission tomography (PET) and single photon emission computed tomography (SPECT), in tissue distribution assays of drugs or substrates, or in radiation treatment of patients. 18 Compounds labeled with F (fluorine-18) may be particularly desirable for PET and SPECT studies. The isotopically labeled compounds of the present invention can generally be prepared using conventional techniques known to those skilled in the art or processes similar to those described in the accompanying Examples and Preparations, where appropriate isotopically labeled reagents are substituted for commonly used non-labeled reagents. Isotopically labeled samples may be useful even with relatively low isotope incorporation rates, such as when radiolabeling is used for trace detection of compounds.

[0068] Furthermore, certain sites contain heavy isotopes, especially deuterium (i.e. 2Substitution of deuterium (H or D) may confer certain therapeutic advantages due to increased metabolic stability, such as increased in vivo half-life, reduced dosage requirements, or improved therapeutic index. In this context, deuterium is considered a substituent of the compounds of the present invention. Typically, a sample of a compound bearing deuterium as a substituent is understood to contain at least 50% or more deuterium incorporation at the labeled position. The concentration of such a heavy isotope, specifically deuterium, is defined by the isotopic enrichment factor. As used in this context, the term "isotopic enrichment factor" refers to the ratio between the isotopic abundance and the natural abundance of a specified isotope. If a substituent in a compound of the invention is designated as deuterium, the compound has a RI of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation) for each designated deuterium atom.

[0069] Pharmaceutically acceptable solubilization solutions according to the present invention include those in which the crystallization solvent is isotopically substituted, for example, D2O, d6-acetone, d6-DMSO, and the like.

[0070] The compounds of the present invention may contain functional groups that can act as hydrogen bond donors and / or acceptors and may therefore be capable of forming co-crystals with suitable co-crystal formers. These co-crystals can be prepared from the compounds of the present invention by known co-crystal formation techniques, including grinding, heating, co-sublimation, co-melting, or contacting a compound of the present invention with a co-crystal former in solution under crystallization conditions and isolating the co-crystal formed therefrom. Suitable co-crystal formers include those described in WO 2004 / 078163. Accordingly, the present invention further provides co-crystals, which include a compound of the present invention. How to use

[0071] All methods described herein can be performed in any suitable order unless otherwise indicated or clearly contradicted by context. The examples provided herein and the use of exemplary language (e.g., "etc.") are intended to more clearly illustrate the invention and are not intended to impose limitations on the scope of the invention.

[0072] The compounds of the present invention can be administered by known methods, such as orally, by injection, or by inhalation. In one embodiment, the compounds of the present invention are administered orally and in the form of tablets, lozenges, troches, capsules, solutions, or suspensions. In another embodiment, the compounds of the present invention are administered by injection or infusion. Infusion is usually administered intravenously and usually over a period of 15 minutes to 4 hours. In another embodiment, the compounds of the present invention are administered intranasally or by inhalation. Inhalation methods are particularly useful for treating respiratory infections. Because the compounds of the present invention exhibit oral bioavailability, oral administration may be preferred.

[0073] In certain embodiments of the present invention, the compounds of the present invention are used in combination with a second antiviral agent, such as those named herein.

[0074] The term "combination" refers to a fixed combination in one dosage unit form, to separate dosage forms for simultaneous or sequential use together, or to a combined administration kit in which a compound of the invention and a combination partner are administered simultaneously or separately with an interval of time, including when the combination partners are administered within a time interval which particularly allows them to exhibit a coordinated, e.g. synergistic, effect, or any combination thereof.

[0075] A second antiviral agent may be administered in combination with a compound of the present invention, where the second antiviral agent is administered before, simultaneously with, or after the compound or compounds of the present invention. When simultaneous administration of the compound of the present invention and the second agent is desired and the administration route is the same, the compound of the present invention may be formulated with the second agent in the same dosage form. Examples of dosage forms containing the compound of the present invention and the second agent include tablets and capsules.

[0076] In some embodiments, the combination of a compound of the present invention and a second antiviral agent may exhibit a synergistic effect. The compound of the present invention and the second antiviral agent may be administered simultaneously, separately but simultaneously, or sequentially.

[0077] An "effective amount" refers to an amount necessary or sufficient to treat or prevent a viral infection or a disease or condition described herein. For example, an effective amount of a compound of Formula 1 is an amount sufficient to treat a viral infection in a subject. In another example, an effective amount is an amount sufficient to treat HBV in a subject in need of treatment. An effective amount may vary depending on factors such as the size and weight of the subject, the type of disease, or the particular compound of the present invention. For example, the choice of compound of the present invention may affect what constitutes an "effective amount." One of ordinary skill in the art can consider the factors included herein and determine an effective amount of a compound of the present invention without undue trial and error.

[0078] The method of administration may affect what constitutes an effective amount. The compounds of the present invention may be administered to a subject before or after the onset of viral infection. Furthermore, the compounds may be administered in multiple daily doses, spaced apart, or by continuous infusion or bolus injection. The dosage of the compounds of the present invention may be proportionally increased or decreased depending on the therapeutic or prophylactic situation.

[0079] The compounds of the invention can be used to treat the conditions, disorders, or diseases described herein, and can also be used to prepare pharmaceutical compositions for use in treating these diseases. The invention provides methods of using the compounds of the invention in treating these diseases, or methods for preparing pharmaceutical compositions containing the compounds of the invention for treating these diseases.

[0080] The term "pharmaceutical composition" includes preparations suitable for administration to mammals (e.g., humans). When the compounds of the present invention are administered to mammals (e.g., humans) as pharmaceuticals, they can be administered on their own or as pharmaceutical compositions. For example, the active ingredient contains 0.1 to 99.5% (more preferably 0.5 to 90%) of at least one compound of Formula 1 or any of its subgeneric groups, and is used in combination with a pharmaceutically acceptable carrier, or in some cases, two or more pharmaceutically acceptable carriers.

[0081] The expression "pharmaceutically acceptable carrier" is recognized in the pharmaceutical arts and includes pharmaceutically acceptable materials, compositions, or vehicles suitable for administering the compounds of the present invention to mammals. Carriers include liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials that are involved in transporting the drug of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Examples of materials used as pharmaceutically acceptable carriers include the following: Examples of suitable carriers include sugars (lactose, glucose, sucrose, etc.), starches (corn starch, potato starch, etc.), cellulose and its derivatives (sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, etc.), powdered tragacanth, malt, gelatin, talc, excipients (cocoa butter, suppository wax, etc.), oils (peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, etc.), glycols (propylene glycol, etc.), polyols (glycerin, sorbitol, mannitol, polyethylene glycol, etc.), esters (ethyl oleate, ethyl laurate, etc.), agar, buffers (magnesium hydroxide, aluminum hydroxide, etc.), alginic acid, apyrogenic water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffer, and other non-toxic and compatible substances used in pharmaceutical preparations. Pharmaceutically acceptable carriers are usually required to be sterile or substantially apyrogenic.

[0082] Surfactants, emulsifiers, lubricants (e.g., sodium lauryl sulfate and magnesium stearate), colorants, release agents, coating agents, sweeteners, flavors, fragrances, preservatives, antioxidants, and the like may also be included in these compositions.

[0083] Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants (e.g., ascorbic acid, cysteine ​​hydrochloride, sodium sulfite, sodium metabisulfite, sulfite salts, etc.), oil-soluble antioxidants (e.g., ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, tocopherol, etc.), and metal chelating agents (e.g., citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.).

[0084] The formulations of the present invention include those suitable for oral, nasal, inhalation, topical, transdermal, submucosal, sublingual, rectal, vaginal, and / or parenteral administration. These formulations may be conveniently provided in unit dosage form and may be prepared by any method well known in the art of pharmacy. When an active ingredient is combined with a carrier material to produce a single dosage form, the amount of active ingredient will generally be that amount of compound that produces a therapeutic effect. Typically, when expressed as 100%, this amount will range from about 1% to about 99%, preferably from about 5% to about 70%, and more preferably from about 10% to about 30%.

[0085] Methods of preparing these formulations and compositions include the step of bringing into association a compound of the present invention with the carriers, and, optionally, adding one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0086] Formulations of the present invention suitable for oral administration may be provided in the form of capsules, cachets, tablets, lozenges (e.g., using a flavored base, typically sucrose and acacia or tragacanth), powders, granules, or solutions or suspensions in aqueous or non-aqueous liquids, water-in-oil or oil-in-water liquid emulsions, elixirs or syrups, pastilles using inert bases such as gelatin and glycerin, or sucrose and acacia, and even mouthwashes, all of which contain a predetermined amount of a compound of the present invention as an active ingredient. The compounds of the present invention can also be administered as a bolus, electuary, or paste.

[0087] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.) of the present invention, the active ingredient is mixed with a pharmaceutically acceptable carrier (e.g., sodium citrate or calcium hydrogen phosphate) and / or any of the following: fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, or silicic acid; binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, or acacia; humectants such as glycerin; disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, or sodium carbonate; solution retarders such as paraffin; absorption accelerators such as quaternary ammonium compounds; wetting agents such as cetyl alcohol or glycerin monostearate; absorbents such as kaolin or bentonite clay; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof; and colorants. In the case of capsules, tablets, or pills, the pharmaceutical compositions may also contain buffers. Similar solid compositions may also be employed as gelatin fillers in soft and hard-filled capsules using such excipients as lactose or lactose-based sugars, as well as high molecular weight polyethylene glycols.

[0088] A tablet may be made by compression or molding, and may optionally contain one or more additional ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrating agents (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agents, or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0089] Tablets and other solid preparations (dragées, capsules, pills, granules, etc.) of the pharmaceutical composition of the present invention can be scored, enteric coated, or coated or shelled as is well known in the pharmaceutical technology. Hydroxypropylmethylcellulose can also be used in various proportions to adjust the desired release profile, or other polymer matrices, liposomes, and / or microspheres can be used to produce sustained- or controlled-release formulations of the active ingredient. Furthermore, sterilization can be achieved by passing through a bacterial filtration filter, or by formulating a sterilizing agent as a sterile solid composition and dissolving it in sterile water or other sterile injectable medium immediately before use. Carrier compositions such as polymeric substances and waxes can also be used, and, if necessary, the active ingredient can be microencapsulated with one or more of the above-mentioned excipients.

[0090] Liquid dosage forms for oral administration of the compounds of the present invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. These liquid dosage forms may contain, in addition to the active ingredient, commonly used inert diluents (e.g., water or other solvents), solubilizers, and emulsifiers. Examples include ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, various oils (especially cottonseed oil, peanut oil, corn oil, wheat germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, fatty acid esters of sorbitan, and mixtures thereof.

[0091] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0092] Suspensions may contain, in addition to the active ingredient, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, and mixtures thereof.

[0093] Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be provided as suppositories, which are prepared by mixing one or more ingredients of the invention with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol, a suppository wax, or a salicylate, which are solid at room temperature and turn liquid at body temperature, thus melting in the rectum or vagina and releasing the active ingredient.

[0094] Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or spray formulations using carriers known in the art to be appropriate.

[0095] Dosage forms for topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and any preservatives, buffers, or propellants which may be required.

[0096] The ointments, pastes, creams, and gels may contain, in addition to the active ingredient of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, zinc oxide, or mixtures thereof.

[0097] Powders and sprays can contain, in addition to the compounds of this invention, lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, polyamide powder, or mixtures of these substances. Sprays can additionally contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane or propane.

[0098] Transdermal patches have the added advantage of providing a controlled delivery of the compounds of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of this flux can be controlled by either providing a rate-controlling membrane or dispersing the active ingredient in a polymer matrix or gel.

[0099] Formulations of the present invention suitable for hydrophilic administration may comprise one or more components of the invention combined with a pharmaceutically acceptable carrier, such as a sterile isotonic aqueous or non-aqueous solution, dispersion, suspension, or emulsion. They may also be provided as sterile powders that can be reconstituted into a sterile injectable solution or dispersion immediately before use. These formulations may contain antioxidants, buffers, bacteriostatic agents, solutes that render the drug isotonic with the blood of the recipient, or suspending or thickening agents.

[0100] Examples of suitable aqueous and non-aqueous carriers that can be used in the formulations of the present invention include water, ethanol, glycol ether, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of particle size in the case of dispersions, or by the use of surfactants.

[0101] These compositions may contain auxiliary agents such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action can be ensured by including antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to add isotonic agents such as sugars and sodium chloride to the compositions. Furthermore, the absorption of injectable preparations can be prolonged by adding agents that delay absorption, such as aluminum monostearate and gelatin.

[0102] In some cases, it may be desirable to slow the absorption of a drug from subcutaneous or intramuscular injection in order to prolong its effect. This can be accomplished by using a liquid suspension containing crystalline or amorphous material that is poorly soluble in water. The rate of absorption of the drug depends on its rate of dissolution, which may depend on crystal size and crystalline form. Alternatively, the absorption of hydrophilic drugs can be delayed by dissolving or suspending the drug in an oily carrier.

[0103] Injectable depot formulations are made by forming a microencapsule matrix of the target compound in biodegradable polymers such as polylactic acid-polyglycolic acid. The drug release rate can be controlled depending on the drug-to-polymer ratio and the properties of the polymer used. Other examples of biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping drugs in liposomes or microemulsions that are compatible with body tissues.

[0104] The preparations of the present invention can be administered orally, by injection, topically, or rectally. They are administered in a form suitable for each administration route. For example, they are administered in the form of tablets, capsules, injections, inhalants, eye drops, ointments, suppositories, etc., and can be administered by injection, infusion, or inhalation. It is used topically as a lotion or ointment and rectally as a suppository.

[0105] As used herein, the phrases "direct administration" and "directly administered" refer to methods of administration other than oral and topical administration, typically by injection. This includes intravenous, intramuscular, intra-arterial, epidural, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, intra-articular, subcutaneous, intrathecal, intraspinal, and intrasternal injections and infusions. Intravenous infusion may be the preferred method of administration of the compounds of the invention. Infusions may be used for a single daily dose or for multiple doses. In some embodiments, the compounds of the invention are administered in infusions at intervals of between 15 minutes and 4 hours, typically between 0.5 and 3 hours. Such infusions may be used once daily, twice daily, or up to three times daily.

[0106] As used herein, the phrases "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered" mean that a compound, drug, or other substance is administered not directly to the central nervous system, but rather is administered so that it enters the patient's body and is therefore subject to metabolic and other similar processes, such as by subcutaneous injection. These compounds can be administered to humans or other animals for therapeutic purposes by any suitable route of administration, including oral, nasal (e.g., spray), rectal, vaginal, intraoral, intracerebroventricular, and topical administration (including buccal or sublingual administration, as powders, ointments, eye drops, etc.).

[0107] Whatever the route of administration selected, the compounds of the present invention may be used in a suitable hydrated form, or the pharmaceutical compositions of the present invention may be prepared into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0108] The actual dosage of the active ingredient in the pharmaceutical composition of the present invention may be adjusted to obtain an amount effective to obtain the desired therapeutic response for the patient, composition, and administration method, but is adjusted within a range that is not toxic to the patient.

[0109] The selected dosage will depend on a variety of factors well known in the medical arts, including the activity of the particular compound of the present invention, or its ester, salt, or amide, being used, the route of administration, the timing of administration, the rate of excretion of the particular compound being used, the duration of treatment, the use of other drugs, compounds, and / or substances in combination, and the age, sex, weight, condition, general health, and past medical history of the patient.

[0110] A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start dosages of the compounds of the present invention in the pharmaceutical composition at levels lower than required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0111] Generally, the appropriate daily dose of the compound of the present invention is the lowest effective dose to achieve a therapeutic effect. This effective dose generally depends on the factors mentioned above. Typically, the intravenous or subcutaneous dose of the compound of the present invention administered to a patient is in the range of about 0.0001 mg to about 100 mg per kilogram of body weight per day to achieve the desired effect, more preferably about 0.01 mg to about 50 mg, and even more preferably about 0.1 mg to about 20 mg. The effective amount refers to the amount for preventing or treating viral infections such as HBV.

[0112] Treatment with the compounds or compositions described in this invention can be repeated daily, alone or in combination with other therapeutic agents, for a period sufficient to produce a measurable reduction in HBsAg.

[0113] The daily dose of the compound may be administered in divided doses, one, two, or three times daily, the number of times being to be determined.

[0114] The compounds of the present invention may be administered alone or in combination (sequentially or simultaneously) with other therapeutic agents. Thus, methods of using the compounds of the present invention include administering the compounds as pharmaceutical compositions in which at least one compound is mixed with a pharmaceutically acceptable carrier prior to administration.

[0115] Combination Use of Compounds of the Invention The compounds and compositions described herein may be used or administered in combination with one or more therapeutic agents, including, but not limited to, immunomodulators (e.g., checkpoint inhibitors (small molecule inhibitors or antibodies)), natural or modified cytokines, TLR agonists, vaccines, etc., and may also be used in combination with HBsAg targeting agents (e.g., siRNA and antisense molecules targeted to HBV transcripts, HBV capsid inhibitors, cccDNA inhibitors, HBx inhibitors, antibodies targeted to HBV or HDV proteins, etc.) and / or HBV replication inhibitors (e.g., nucleoside(t) analog polymerase inhibitors, non-nucleoside(t) analog polymerase inhibitors, etc.).

[0116] In general, it is expected that therapeutic agents used in combination will be used in ranges that do not exceed the levels at which they are used individually, and in some embodiments, the dosages used in combination may be lower than when they are used individually. Treatment with the compounds or compositions described herein can be administered once or twice daily, or three times daily, in combination with other therapeutic agents or alone for as long as necessary to achieve sufficient reduction in HBsAg.

[0117] The compounds of the invention can be administered alone or in combination (sequentially or simultaneously) with other therapeutic agents. Thus, methods of using the compounds of the invention include administering the compounds as a pharmaceutical composition, wherein at least one compound of the invention is mixed with a pharmaceutically acceptable carrier prior to administration.

[0118] The compounds described herein can be synthesized by the following general synthetic routes, specific examples of which are described in detail in the Examples.

[0119] General synthetic procedure All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts used to synthesize the compounds of the present invention are commercially available or can be prepared by organic synthesis methods known to those of ordinary skill in the art (Houben-Weyl, 4th Edition, 1952, Methods of Organic Synthesis, Thieme, Volume 21). General methods for the synthesis of the compounds of the present invention are illustrated by the following examples, the general method of Scheme 1, and methods described in published PCT applications WO 2018 / 198079 and US 10,301,312 B2 (patent date: May 28, 2019), the contents of which are hereby incorporated in their entirety.

[0120] Preparation of common intermediate 3 Synthetic Route: [ka]

[0121] The reaction was divided into two equal batches. In each batch, ethyl (R)-6-(tert-butyl)-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (1) (45 g, 99.22 mmol) was dissolved in dichloromethane (DCM, 1 L). To this mixture, BBr3 (99.43 g, 397 mmol, 38.24 mL, 4.0 eq) was added dropwise at 0 °C under a nitrogen atmosphere. The mixture was then heated to 40 °C and stirred for 12 h. A solid precipitate appeared. TLC (petroleum ether:ethyl acetate = 0:1, R f= 0.1), indicating the reaction was complete. The two reactions were combined and worked up. The mixture was diluted with dichloromethane (DCM, 1 L) and stirred for 5 min. The solid was collected by filtration and washed with DCM (500 mL x 3). The filter cake was dried under vacuum. The residue was added with THF (450 mL), water (450 mL), and hydrated lithium hydroxide (LiOH HO) (16.65 g, 416.2 mmol, 4.2 eq) and stirred at 25 °C for 2 h. The residue was adjusted to pH = 4 with HCl (2 M). The residue was then filtered to give a yellow solid as (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (2) (60 g, yield: 86%). 1 H NMR (400 MHz, DMSO-d6)δ ppm 10.36(s, 1H), 8.94(s, 1H), 7.49(d, J = 8.4 Hz, 1H), 7.23(s, 1H), 7.25 - 7.12(m, 1H), 7.27 - 7.11(m, 1H), 7.16(t, J = 8.0 Hz, 1H), 6.70(d, J = 7.2 Hz, 1H), 5.14 - 5.06(m, 2H), 4.96(br d, J = 3.6 Hz, 1H), 0.71(s, 9H).

[0122] Preparation of ethyl (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2:1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3). [ka]

[0123] (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (2) (60 g, 169.79 mmol) was dissolved in ethanol (600 mL) and SOCl2 (101.00 g, 848.97 mmol, 61.6 mL, 5.0 eq) was added at 0 °C. The mixture was stirred at 60 °C for 12 h. LCMS showed the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (dichloromethane:ethanol = 1:0 to 5:1) to give ethyl (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3) (55 g, yield: 84.92%) as a yellow solid.

[0124] LCMS: RT = 0.194 min, MS calculated: 381.2, MS observed: [M+H] + = 382.3 1 H NMR(400 MHz, DMSO-d6)δ ppm 8.77(s, 1H), 7.39(d, J = 8.4 Hz, 1H), 7.28(s, 1H), 7.16(t, J = 8.0 Hz, 1H), 6.70(d, J = 7.2 Hz, 1H), 5.18 - 4.99(m, 2H), 4.87(d, J = 2.8 Hz, 1H), 4.29(q, J = 7.2 Hz, 2H), 1.30(t, J = 7.2 Hz, 3H), 0.72(s, 9H). Compound 3M was prepared using this method, using methanol instead of ethanol.

[0125] Compound I.1 Synthetic Route: [ka]

[0126] Preparation of ethyl (R)-10-((6-(tert-butoxy)-6-oxohexyl)oxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2,1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (4) [ka]

[0127] To a mixture of ethyl (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3) (1.5 g, 3.93 mmol, 1 equiv.) and tert-butyl 6-bromohexanoate (2.47 g, 9.83 mmol, 2.5 equiv.) in DMF (15 ml) was added Cs2CO3 (4.48 g, 13.76 mmol, 3.5 equiv.) in one portion at 20 °C. The mixture was then stirred at 50 °C for 4 h. LCMS confirmed the reaction was complete. The mixture was poured into water (20 ml) and extracted with ethyl acetate (10 ml x 3). The resulting organic layer was washed with brine (10 ml × 3), dried over NaSO, filtered, and concentrated in vacuo. The residue was purified by prep-HPLC (Waters Xbridge BEH C18 100 30 mm 10 μm column; mobile phase: [water (NH4HCO3)]-ACN; B%: 40%-70%, 8 min) to give ethyl (R)-10-((6-(tert-butoxy)-6-oxohexyl)oxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (4) (1.4 g, 2.54 mmol, 64.5% yield) as a yellow solid.

[0128] 1H NMR(400 MHz, DMSO-d6)δ ppm 8.53(s, 1H), 7.50(d, J = 8.4 Hz, 1H), 7.18(t, J = 8.0 Hz, 1H), 6.83 - 6.75(m, 2H), 5.13 - 4.98(m, 2H), 4.70(d, J = 4.0 Hz, 1H), 4.24(q, J = 6.8 Hz, 2H), 4.18 - 4.08(m, 2H), 2.23(t, J = 7.2 Hz, 2H), 1.81 - 1.79(m, 2H), 1.63 - 1.53(m, 2H), 1.52 - 1.41(m, 2H), 1.38(s, 9H), 1.28(t, J = 6.8 Hz, 3H), 0.69(s, 9H).

[0129] Preparation of (R)-6-((6-(tert-butyl)-3-(ethoxycarbonyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazol-10-yl)oxy)hexanoic acid (5) [ka]

[0130] To ethyl (R)-10-((6-(tert-butoxy)-6-oxohexyl)oxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (4) (1.2 g, 2.18 mmol, 1 equiv.) in dichloromethane (DCM, 10 mL) was added trifluoroacetic acid (TFA, 5 mL) in one portion at 20 °C. The mixture was stirred at 20 °C for 2 h. LCMS confirmed the reaction was complete. The mixture was concentrated, and the pH was adjusted to 7 with saturated sodium bicarbonate (NaHCO3). The aqueous layer was extracted with DCM (10 mL × 3). The resulting organic layer was washed with brine (5 mL × 3), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 250 × 50 mm, 10 μm; mobile phase: [water(NH4HCO3)-ACN]; 8%-10%-40%, 10 min), to give (R)-6-((6-(tert-butyl)-3-(ethoxycarbonyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazol-10-yl)oxy)hexanoic acid (5) (0.61 g, 1.22 mmol, 56.02% yield) as a yellow solid.

[0131] 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.53 (s, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 6.85 - 6.73 (m, 2H), 5.16 - 4.98 (m, 2H), 4.70 (d, J = 4.4 Hz, 1H), 4.24 (q, J = 6.8 Hz, 2H), 4.19 - 4.08 (m, 2H), 2.24 (t, J = 7.2 Hz, 2H), 1.82 - 1.79 (m, 2H), 1.65 - 1.54 (m, 2H), 1.53 - 1.41 (m, 2H), 1.28 (t, J = 6.8 Hz, 3H), 0.70 (s, 9H).

[0132] Procedure for the preparation of (R)-6-(tert-butyl)-10-((5-carboxypentyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.1) [ka]

[0133] (R)-6-((6-(tert-butyl)-3-(ethoxycarbonyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazol-10-yl)oxy)hexanoic acid (0.15 g, 303 μmol, 1 equiv.) was placed in a mixture of CHCN (1.5 mL) and HO (0.3 mL), and LiOH HO (57 mg, 1.36 mmol, 4.5 equiv.) was added. The mixture was stirred at 35 °C for 1 h. LCMS showed the reaction was complete. The pH of the mixture was adjusted to 6–7 with 1 N HCl and concentrated in vacuo. The residue was purified by preparative HPLC (column: Waters Xbridge C18 150 × 50 mm, 10 μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 1%-30%, 8 min), to give (R)-6-(tert-butyl)-10-((5-carboxypentyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.1) (81 mg, 163 μmol, 54% yield) as a yellow solid.

[0134] 1H NMR (400 MHz, DMSO-d5) δ ppm 8.95 (s, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.33 - 7.18 (m, 2H), 6.83 (d, J = 7.6 Hz, 1H), 5.27 - 5.06 (m, 2H), 4.96 (d, J = 4.4 Hz, 1H), 4.22 - 4.09 (m, 2H), 2.24 (t, J = 7.2 Hz, 2H), 1.82 - 1.79 (m, 2H), 1.64 - 1.55 (m, 2H), 1.54 - 1.42 (m, 2H), 0.71 (s, 9H).

[0135] Compound I.2 Synthetic Route: [ka]

[0136] Procedure for the preparation of ethyl (R)-10-((7-(tert-butoxy)-7-oxoheptyl)oxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (6) [ka]

[0137] Ethyl (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3) (1.5 g, 3.93 mmol, 1 eq.) and tert-butyl 7-bromoheptanoate (1.56 g, 5.9 mmol, 1.5 eq.) were added to DMF (20 mL). CsCO (4.48 g, 13.76 mmol, 3.5 eq.) was added at 25 °C. The mixture was stirred at 50 °C for 12 h. Real-time mass spectrometry (LCMS) confirmed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was extracted with ethyl acetate (60 mL x 3). The resulting organic layer was washed with saturated brine (30 mL x 3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 100:1 to 0:1) to give ethyl (R)-10-((7-(tert-butoxy)-7-oxoheptyl)oxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (6) (1.6 g, 71.92% yield) as a yellow solid.

[0138] Preparation procedure for (R)-7-((6-(tert-butyl)-3-(ethoxycarbonyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazol-10-yl)oxy)heptanoic acid (7) [ka]

[0139] Ethyl (R)-10-((7-(tert-butoxy)-7-oxoheptyl)oxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (6) (1.5 g, 2.65 mmol, 1 eq.) was dissolved in DCM (10 mL) and TFA (5 mL) at 25 °C. The mixture was stirred at 25 °C for 1 h. Real-time mass spectrometry (LCMS) confirmed the reaction was complete. The pH was adjusted to 7 with saturated NaHCO3. The residue was extracted with DCM (3 x 15 mL). The resulting organic layer was washed with brine (15 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Welch Xtimate C18 25070 mm 10 μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 10%-40%, 20 min) to give (R)-7-((6-(tert-butyl)-3-(ethoxycarbonyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazol-10-yl)oxy)heptanoic acid (7) (1.15 g, 84% yield, 98.76% purity) as a yellow solid.

[0140] 1 H NMR(400 MHz, DMSO-d6)δ ppm:8.52(s, 1H), 7.50(d, J = 8.4 Hz, 1H), 7.18(t, J = 8.0 Hz, 1H), 6.86 - 6.72(m, 2H), 5.14 - 5.00(m, 2H), 4.70(d, J = 4.4 Hz, 1H), 4.24(q, J = 7.2 Hz, 2H), 4.15 - 4.12(m, 2H), 2.19(t, J = 7.2 Hz, 2H), 1.80(q, J = 6.8 Hz, 2H), 1.57 - 1.42(m, 4H), 1.40 - 1.33(m, 2H), 1.28(t, J = 7.2 Hz, 3H), 0.70(s, 9H).

[0141] [ka]

[0142] To a solution of (R)-7-((6-(tert-butyl)-3-(ethoxycarbonyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazol-10-yl)oxy)heptanoic acid 7 (0.5 g, 981.18 μmol, 1 equiv.) in CHCN (4 mL) and HO (2 mL) was added LiOH HO (123.52 mg, 2.94 mmol, 3 equiv.). The mixture was stirred at 20 °C for 12 h. LCMS showed the reaction was complete. The mixture was filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Phenomenex C18 80 × 40 mm × 3 μm; mobile phase: water (NH4H2O3)-ACN; B%: 1%-30%, 8 min) to give (R)-6-(tert-butyl)-10-((6-carboxyhexyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.2) (137 mg, 281.9 μmol, 29% yield, 99.23% purity) as a pale yellow solid.

[0143] 1 H NMR(400 MHz, DMSO-d6)δ ppm:8.95(s, 1H), 7.63(d, J = 8.4 Hz, 1H), 7.37 - 7.11(m, 2H), 6.82(d, J = 7.6 Hz, 1H), 5.31 - 5.03(m, 2H), 4.96(d, J = 4.8 Hz, 1H), 4.23 - 4.03(m, 2H), 2.21(t, J = 7.2 Hz, 2H), 1.81(quin, J = 6.8 Hz, 2H), 1.62 - 1.41(m, 4H), 1.41 - 1.30(m, 2H), 0.71(s, 9H).

[0144] Compound I.3 Synthetic Route: [ka]

[0145] Procedure for the preparation of methyl (R)-6-(tert-butyl)-10-((7-ethoxy-7-oxoheptyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2,1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (8) [ka]

[0146] To a mixture of methyl (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3M) (6 g, 16.33 mmol) in DMF (60 ml) was added Cs2CO3 (15.96 g, 48.99 mmol) and ethyl 7-bromoheptanoate (5.03 g, 21.23 mmol). The mixture was stirred at 60 °C for 3 h. The reaction was monitored by TLC (SiO2, ethyl acetate:methanol = 10:1). The reaction mixture was quenched by adding H2O (180 ml) and extracted with EtOAc (60 ml x 3). The combined organic layers were washed with brine (50 ml x 3), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 1:0 to 0:1) to give methyl (R)-6-(tert-butyl)-10-((7-ethoxy-7-oxoheptyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (8) (5.9 g, 69% yield) as a yellow solid.

[0147] [ka] Procedure for the preparation of (R)-6-(tert-butyl)-10-((7-ethoxy-7-oxoheptyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.3)

[0148] A mixture of methyl (R)-6-(tert-butyl)-10-((7-ethoxy-7-oxoheptyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (8) (5.9 g, 11.27 mmol) was dissolved in HO (60 ml) and CHCN (60 ml), LiOH HO (472.83 mg, 11.27 mmol) was added, and the mixture was stirred at 20 °C for 2 h. The mixture was monitored by LCMS. The pH was then adjusted to 4 with dilute HCl (1 N), filtered through dry NaSO, and concentrated under reduced pressure. This mixture was purified by preparative HPLC using a Welch Xtimate C18 (250 × 70 mm, #10 μm) column with a mobile phase (water (NH4HCO3))-acetonitrile:B%: 33%-63%, 20 min) to give (R)-6-(tert-butyl)-10-((7-ethoxy-7-oxoheptyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.3) (2.5 g, 98% purity) as a yellow solid.

[0149] 1 H NMR(400 MHz, DMSO-d6)δ ppm 8.95(s, 1H), 7.63(d, J = 8.4 Hz, 1H), 7.35 - 7.17(m, 2H), 6.82(d, J = 7.6 Hz, 1H), 5.28 - 5.05(m, 2H), 4.96(d, J = 4.4 Hz, 1H), 4.16(t, J = 5.6 Hz, 2H), 4.04(q, J = 7.2 Hz, 2H), 2.30(t, J = 7.2 Hz, 2H), 1.89 - 1.78(m, 2H), 1.62 - 1.42(m, 4H), 1.41 - 1.31(m, 2H), 1.17(t, J = 7.2 Hz, 3H), 0.71(s, 9H). Mass spectrum calculated: 510.3, found: [M+H] + = 510.3.

[0150] Compound I.4 Synthetic Route: [ka]

[0151] Procedure for the preparation of methyl (R)-6-(tert-butyl)-10-((7-(heptyloxy)-7-oxoheptyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (9) [ka]

[0152] The reaction was divided into two equal batches. To each was added Cs2CO3 (665 mg, 2.04 mmol) to a mixture of methyl (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3M) (250 mg, 0.680 mmol) in DMF (1 ml) and stirred at 60 °C for 10 min. Heptyl 7-bromoheptanoate (313.6 mg, 1.02 mmol, 1.5x volume) was then added and stirred at 60 °C for 3 h. The reaction was monitored by LCMS. The two batches were combined and worked up. The reaction mixture was quenched with H2O (3 ml) and extracted with ethyl acetate (10 ml x 3). The combined organic layers were washed with brine (10 ml × 3), dried over NaSO, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, ethyl acetate:methanol = 10:1) to give methyl (R)-6-(tert-butyl)-10-((7-(heptyloxy)-7-oxoheptyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (9) (400 mg, crude) as a yellow solid.

[0153] Procedure for the preparation of (R)-6-(tert-butyl)-10-((7-(heptyloxy)-7-oxoheptyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.4) [ka]

[0154] A mixture of methyl (R)-6-(tert-butyl)-10-((7-(heptyloxy)-7-oxoheptyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (9) (350 mg, 0.589 mmol) in acetonitrile (MeCN, 3.5 ml) and water (HO, 3.5 ml) was added. LiOH HO (25 mg, 0.589 mmol) was added, and the reaction mixture was stirred at 25 °C for 2 h. The reaction was monitored by LCMS. The mixture was then adjusted to pH = 4 with 1N hydrochloric acid and extracted with 5% ethanol (EtOH) in dichloromethane (DCM) (5 ml x 3). The combined organic layers were washed with brine (10 ml), dried over NaSO, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 75 × 30 mm, 3 μm, mobile phase: [water (FA)]-acetonitrile (ACN); B%: 50%-90%, 8 min). After lyophilization, the residue was purified again by preparative HPLC (column: Waters Xbridge Prep OBD C18 150 × 40 mm, 10 μm, mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-95%, 8 min) to give (R)-6-(tert-butyl)-10-((7-(heptyloxy)-7-oxoheptyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.4) (60 mg, purity: 98.7%) as a yellow solid.

[0155] 1HH NMR(400 MHz, DMSO-d6)δ ppm 8.95(s, 1H), 7.63(d, J = 8.4 Hz, 1H), 7.32 - 7.18(m, 2H), 6.82(d, J = 7.6 Hz, 1H), 5.27 - 5.04(m, 2H), 4.97(d, J = 4.4 Hz, 1H), 4.16(t, J = 5.6 Hz, 2H), 3.99(t, J = 6.8 Hz, 2H), 2.30(t, J = 7.2 Hz, 2H), 1.89 - 1.78(m, 2H), 1.65 - 1.42(m, 6H), 1.41 - 1.32(m, 2H), 1.31 - 1.18(m, 8H), 0.90 - 0.79(m, 3H), 0.71(s, 9H). Mass spectrum calculated: 579.3, Found: [M+H] + = 580.3

[0156] Compound I.5 Procedure for the preparation of (R)-6-(tert-butyl)-10-((7-isopropoxy-7-oxoheptyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2,3-1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.5) [ka]

[0157] To a solution of (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (2) (500 mg, 1.41 mmol, 1 eq) and t-BuOK (476.31 mg, 4.24 mmol, 3 eq) in DMF (7.5 mL) was added a solution of isopropyl 7-bromoheptanoate (284 mg, 1.13 mmol, 0.8 eq) in DMF (2.5 mL). The mixture was stirred at 60 °C for 3 h. TLC (ethyl acetate:methanol = 10:1, Rf = 0.2) confirmed the reaction was complete. The mixture was poured into dilute hydrochloric acid (0.1 M, 70 mL) and filtered. The filtrate was extracted with ethanol (20 mL x 3). The combined organic layer was washed with brine (20 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure. The mixture was purified by prep-HPLC (Waters Xbridge Prep OBD C18 150 40 mm 10 μm; mobile phase: [water (NH4HCO3)-20% ACN], B%: 35%-65%, 8 min) to afford (R)-6-(tert-butyl)-10-((7-isopropoxy-7-oxoheptyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.5) (58.9 mg, 111 μmol, 8% yield, 99% purity) as a yellow solid.

[0158] 1H NMR (400 MHz, DMSO-d6) δ ppm: 8.96 (s, 1 H), 7.64 (d, J = 8.4 Hz, 1 H), 7.28 - 7.21 (m, 2 H), 6.82 (d, J = 7.6 Hz, 1 H), 5.23 - 5.07 (m, 2 H), 4.97 (d, J = 4.8 Hz, 1 H), 4.88 (td, J = 6.4, 12.4 Hz, 1 H), 4.19 - 4.13 (m, 2 H), 2.26 (t, J = 7.2 Hz, 2 H), 1.86 - 1.77 (m, 2 H), 1.60 - 1.43 (m, 4H), 1.41 - 1.32 (m, 2 H), 1.17 (d, J = 6.4 Hz, 6 H), 0.71 (s, 9 H). Mass spectrum calculated: 523.2, found: [M+H] + = 524.3

[0159] Compound I.6 Synthetic Route: [ka]

[0160] Preparation procedure for methyl (R)-6-(tert-butyl)-10-((8-ethoxy-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (10) [ka]

[0161] Methyl (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3M) (1 g, 2.62 mmol) was dissolved in DMF (10 mL), and CsCO (2.56 g, 7.87 mmol) and ethyl 8-bromooctanoate (856.02 mg, 3.41 mmol) were added at 25 °C. The mixture was stirred at 60 °C for 3 h. The reaction was monitored by LCMS. The mixture was quenched with H2O (30 mL) and extracted with EtOAc (15 mL x 3). The combined organic layer was washed with brine (10 mL x 3), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 1:0 to 0:1) to give methyl (R)-6-(tert-butyl)-10-((8-ethoxy-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (10) (1.20 g, 72% yield) as a yellow solid.

[0162] Procedure for the preparation of (R)-6-(tert-butyl)-10-((7-carboxyheptyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.6) [ka] Methyl (R)-6-(tert-butyl)-10-((8-ethoxy-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (10) (1.20 g, 2.18 mmol) was added to a mixture of CHCN (10 mL) and HO (10 mL), and LiOH HO (274 mg, 6.53 mmol) was added and stirred at 20 °C for 2 h. The reaction was monitored by LCMS. The mixture was adjusted to pH = 4 with 1N hydrochloric acid and extracted with 5% ethanol in DCM (10 mL x 3). The combined organic layer was washed with brine (10 mL), dried over NaSO, and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 250 50 mm 10 μm, mobile phase: [water (NH4HCO3)-ACN], B%: 5%-45%, 10 min) to give (R)-6-(tert-butyl)-10-((7-carboxyheptyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.6) (500 mg, 100% purity) as a white solid.

[0163] 1 H NMR(400 MHz, DMSO-d6)δ 8.95(s, 1H), 7.63(d, J = 8.4 Hz, 1H), 7.34 - 7.19(m, 2H), 6.83(d, J = 8.0 Hz, 1H), 5.27 - 5.06(m, 2H), 4.96(d, J = 4.0 Hz, 1H), 4.23 - 4.08(m, 2H), 2.19(t, J = 7.2 Hz, 2H), 1.89 - 1.78(m, 2H), 1.58 - 1.40(m, 4H), 1.39 - 1.21(m, 4H), 0.71(s, 9H). Mass spectrum calculated: 496.2, found: [M+H] + = 496.3.

[0164] Compound I. 7 Synthetic Route: [ka]

[0165] Procedure for the preparation of methyl (R)-6-(tert-butyl)-10-((8-ethoxy-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2,1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (11) [ka]

[0166] To a mixture of methyl (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3M) (5.00 g, 13.61 mmol) in DMF (50 ml) was added CsCO (13.30 g, 40.83 mmol) and ethyl 8-bromooctanoate (4.44 g, 17.69 mmol). The mixture was stirred at 60 °C for 3 h. The reaction was monitored by TLC (SiO, ethyl acetate:methanol = 10:1, R f The reaction mixture was monitored at RT (pH 7.5). The reaction mixture was quenched by the addition of HO (150 ml) and extracted with EtOAc (50 ml × 3). The resulting organic layer was washed with brine (50 ml × 3), dried over NaSO, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 1:0 to 0:1) to give methyl (R)-6-(tert-butyl)-10-((8-ethoxy-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (11) (4.8 g, 65.6% yield) as a yellow solid.

[0167] Procedure for the preparation of (R)-6-(tert-butyl)-10-((8-ethoxy-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2,1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.7) [ka]

[0168] A mixture of methyl (R)-6-(tert-butyl)-10-((8-ethoxy-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (11) (4.8 g, 8.93 mmol) was dissolved in HO (50 mL) and MeCN (50 mL), and LiOH HO (374.61 mg, 8.93 mmol) was added. The reaction mixture was stirred at 20 °C for 2 h. The reaction progress was monitored by LCMS. After the reaction, the pH was adjusted to 4 with 1N HCl and extracted with 5% ethanol in dichloromethane (DCM) (50 mL × 3). The combined organic layers were washed with brine (50 mL × 10 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 25070mm 10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 30%-75%, 18 min) to give (R)-6-(tert-butyl)-10-((8-ethoxy-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.7) (2.80 g, 98% purity) as a yellow solid.

[0169] 1H NMR(400 MHz, DMSO-d6)δ 8.96(s, 1H), 7.63(d, J = 8.4 Hz, 1H), 7.34 - 7.15(m, 2H), 6.83(d, J = 7.2 Hz, 1H), 5.28 - 5.06(m, 2H), 4.97(d, J = 4.4 Hz, 1H), 4.16(t, J = 6.0 Hz, 2H), 4.04(q, J = 7.2 Hz, 2H), 2.28(t, J = 7.2 Hz, 2H), 1.81(quin, J = 6.8 Hz, 2H), 1.59 - 1.41(m, 4H), 1.41 - 1.26(m, 4H), 1.17(t, J = 7.2 Hz, 3H), 0.71(s, 9H). Mass spectrum calculated: 523.3, observed: [M+H] + = 524.3.

[0170] Compound I.8 Synthetic Route: [ka] Method for preparing propyl 8-bromooctanoate (13) 8-Bromooctanoic acid (12) (3.00 g, 13.45 mmol, 1 equiv.) was dissolved in propan-1-ol (30 ml) and SOCl2 (3.20 g, 26.89 mmol, 1.95 ml, 2 equiv.) was added. The mixture was stirred at 80 °C for 2 h. TLC (petroleum ether:ethyl acetate = 5:1, Rr = 0.5) confirmed the reaction was complete. After removing the solvent under reduced pressure, the residue was dissolved in ethyl acetate (50 ml) and water (50 ml). The organic layer was separated, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 50:1 to 20:1) to give propyl 8-bromooctanoate (3.37 g, 12.71 mmol, 94% yield) as a colorless oil.

[0171] 1H NMR(400 MHz, CDCl3)δ ppm 4.03(td, J=6.4, 0.8 Hz, 2H), 3.40(td, J=6.8, 0.8 Hz, 2H), 2.35 - 2.26(m, 2H), 1.93 - 1.79(m, 2H), 1.68 - 1.59(m, 4H), 1.49 - 1.39(m, 2H), 1.34(dt, J=6.8, 3.2 Hz, 4H), 0.94(td, J=7.6, 1.2 Hz, 3H).

[0172] Procedure for the preparation of methyl (R)-6-(tert-butyl)-2-oxo-10-((8-oxo-8-propoxyoctyl)oxy)-6,7-dihydro-2H-pyrido[2,3-β]pyrazino[1,2-b]indazole-3-carboxylate (14) [ka]

[0173] To a solution of methyl (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3M) (2.5 g, 6.80 mmol, 1 equiv.) in DMF (20 mL) was added CsCO (7.76 g, 23.82 mmol, 3.5 equiv.) and propyl 8-bromooctanoate (13) (1.98 g, 7.49 mmol, 1.1 equiv.). The mixture was stirred at 50 °C for 2 h. LCMS confirmed the reaction was complete. The reaction mixture was poured into water (30 mL) and extracted with EtOAc (50 mL × 2). The organic layer was washed with brine (30 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 2:1 to 0:1) to give methyl (R)-6-(tert-butyl)-2-oxo-10-((8-oxo-8-propoxyoctyl)oxy)-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (14) (2.5 g, 4.53 mmol, 66% yield) as a white solid.

[0174] 1 H NMR(400 MHz, CDCl3)δ ppm:8.30(s, 1H), 7.43(d, J=8.4 Hz, 1H), 7.20(t, J=8.0 Hz, 1H), 7.08(s, 1H), 6.67(d, J=7.6 Hz, 1H), 5.17(br d, J=14.8 Hz, 1H), 4.90(br d, J=11.6 Hz, 1H), 4.20(t, J=6.4 Hz, 2H), 4.10(br s, 1H), 4.03(t, J=6.4 Hz, 2H), 3.95(s, 3H), 2.31(t, J=7.6 Hz, 2H), 1.97 (quintet, J=7.2 Hz, 2H), 1.68-1.60(m, 4H), 1.57-1.49(m, 2H), 1.45-1.35(m, 4H), 0.94(t, J=7.6 Hz, 3H), 0.84(s, 9H).

[0175] Procedure for the preparation of (R)-6-(tert-butyl)-2-oxo-10-((8-oxo-8-propoxyoctyl)oxy)-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.8) [ka]

[0176] To a solution of methyl (R)-6-(tert-butyl)-2-oxo-10-((8-oxo-8-propoxyoctyl)oxy)-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (14) (2.5 g, 4.53 mmol, 1 equiv.) in CHCN (12 mL) and HO (12 mL) was added LiOH HO (247.22 mg, 5.89 mmol, 1.3 equiv.). The mixture was stirred at 20 °C for 1 h. H NMR confirmed the reaction was complete. The reaction mixture was poured into water (30 mL) and the pH was adjusted to 6-7 with 1 M HCl. The mixture was extracted with EtOAc (20 mL × 2). The organic layer was washed with brine (10 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (column: Welch Xtimate C18 250*70 mm#10 μm; mobile phase: [water(NH4HCO3)-ACN]; B%: 40%-65%, 20 min) to give (R)-6-(tert-butyl)-2-oxo-10-((8-oxo-8-propoxyoctyl)oxy)-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.8) (1.24 g, 2.31 mmol, 51% yield) as a white solid.

[0177] 1H NMR (400 MHz, DMSO-d6) δ ppm:8.95(s, 1H), 7.62(br d, J=8.4 Hz, 1H), 7.25(br d, J=7.6 Hz, 2H), 6.82(d, J=7.6 Hz, 1H), 5.04-5.27(m, 2H), 4.97(br s, 1H), 4.15(br t, J=5.2 Hz, 2H), 3.95(t, J=6.8 Hz, 2H), 2.29(t, J=7.6 Hz, 2H), 1.81(quin, J=6.8 Hz, 2H), 1.62-1.51(m, 4H), 1.50-1.41(m, 2H), 1.40-1.26(m, 4H), 0.86(t, J=7.6 Hz, 3H), 0.70(s, 9H).

[0178] Compound I.9 Synthetic Route: Preparation of isopropyl 8-bromooctanoate (15) [ka]

[0179] A solution of 8-bromooctanoic acid (12) (3.5 g, 15.69 mmol) in i-PrOH (35 mL) was added with SOCl (3.73 g, 31.38 mmol, 2.28 mL). The mixture was stirred at 60 °C for 12 h. The reaction was monitored by TLC (petroleum ether:ethyl acetate = 10:1, Rf = 0.55). The reaction was quenched by adding H O (25 mL) at 25 °C and extracted with EtOAc (30 mL × 3). The combined organic extracts were washed with brine, dried over Na SO , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO , petroleum ether:ethyl acetate = 1:0 to 5:1) to give isopropyl 8-bromooctanoate (15) (5.7 g, 21.49 mmol, 95.91% yield) as a colorless oil.

[0180] 1H NMR(400 MHz, CDCl3)δ = 5.01 - 4.95(m, 1H), 3.52 - 3.49(m, 1H), 3.39 - 3.36(m, 1H), 2.26 - 2.22(m, 2H), 1.83 - 1.81(m, 1H), 1.78 - 1.71 (m, 1H), 1.59 - 1.57 (m, 2H), 1.45 - 1.38 (m, 2H), 1.33 - 1.30 (m, 4H), 1.29 - 1.20 (m, 6H).

[0181] Preparation of methyl (R)-6-(tert-butyl)-10-(8-isopropoxy-8-oxooctyl)oxy-2-oxo-6,7-dihydro-2H-pyrido[2,3-1':3,4]pyrazino[1,2-b]indazole-3-carboxylate {16} [ka]

[0182] To a 3 M solution of methyl (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3.52 g, 9.57 mmol) in DMF (35 mL) was added CsCO (9.36 g, 28.72 mmol) and isopropyl 8-bromooctanoate (3.3 g, 12.44 mmol). The mixture was stirred at 60 °C for 12 h. The reaction was monitored by TLC (petroleum ether:THF = 10:1, Rf = 0.33). After the reaction, the mixture was quenched with water (30 mL) at 25 °C and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (25 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (THF:petroleum ether = 1:1 to 1:0) to give methyl (R)-6-(tert-butyl)-10-((8-isopropoxy-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3.56 g, 6.45 mmol, 67% yield) as a yellow solid. Mass spectrum calculated: 551.3, Found: [M+H] + = 552.3

[0183] Procedure for the preparation of (R)-6-(tert-butyl)-10-((8-isopropoxy-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2,1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.9) [ka]

[0184] To a solution of methyl (R)-6-(tert-butyl)-10-((8-isopropoxy-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (16) (2.2 g, 3.99 mmol) in MeCN (20 mL) and water (20 mL) was added LiOH HO (200.81 mg, 4.79 mmol) and stirred at 25 °C for 1 h. The reaction was monitored by LCMS. The mixture was adjusted to pH 4 with 1 N HCl and extracted with 5% ethanol / DCM (50 mL × 3). The combined organic layers were washed with brine (50 mL), dried over NaSO, and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Agela DuraShell C18 250 70 mm 10 μm, mobile phase: [water (NH4HCO3)]-ACN; B%: 40%-70%, 20 min) to give (R)-6-(tert-butyl)-10-((8-isopropoxy-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.9) (3.20 g, 5.95 mmol, 94% yield) as a yellow solid.

[0185] 1 H NMR (400 MHz, DMSO-d6)δ ppm: 8.95(s, 1H), 7.63(d, J = 8.4 Hz, 1H), 7.26 - 7.22(m, 2H), 6.83 - 6.80(m, 1H), 5.17 - 5.12(m, 2H), 4.97 - 4.96(m, 1H), 4.89 - 4.86(m, 1H), 4.17 - 4.15(m, 2H), 2.27 - 2.23(m, 2H), 1.89 - 1.75(s, 2H), 1.55 - 1.45(m, 4H), 1.34 - 1.32(m, 4H), 1.17 - 1.16(m, 6H), 0.71(s, 9H). Mass spectrum calculated: 537.3, Found: [M+H] + = 538.3.

[0186] Compound I.10 Synthetic Route: [ka] Method for preparing butyl 8-bromooctanoate (17)

[0187] 8-Bromooctanoic acid (12) (10 g, 44.82 mmol) was dissolved in n-BuOH (100 mL), SOCl2 (16.00 g, 134.46 mmol) was added, and the mixture was stirred at 60 °C for 3 h. The reaction was monitored by TLC (SiO2, petroleum ether:ethyl acetate = 5:1, Rf = 0.5). After the reaction, the mixture was concentrated under reduced pressure. The mixture was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 5:1) to give butyl 8-bromooctanoate (17) (12 g, crude) as a colorless oil.

[0188] Procedure for the preparation of methyl (R)-10-((8-butoxy-8-oxooctyl)oxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (18) [ka]

[0189] Methyl (R)-6-(tert-butyl)-10-hydroxy-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3M) (3.5 g, 9.53 mmol) was dissolved in DMF (35 mL), and CsCO (9.31 g, 28.58 mmol, 3 volumes) and butyl 8-bromooctanoate (17) (3.46 g, 12.38 mmol, 1.3 volumes) were added at 20 °C. The mixture was stirred at 60 °C for 3 h. The reaction was monitored by LCMS. The reaction was then quenched with H2O (100 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic layers were washed with brine (50 mL × 3), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 1:0 to 0:1) to give methyl (R)-10-((8-butoxy-8-oxooctyl)oxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (18) (2.5 g, 45.25% yield) as a yellow solid.

[0190] Procedure for the preparation of (R)-10-((8-butoxy-8-oxooctyl)oxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.10) [ka]

[0191] Methyl (R)-10-((8-butoxy-8-oxooctyl)oxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (18) (2.5 g, 4.42 mmol) was dissolved in CHCN (20 mL) and HO (20 mL). LiOH HO (185.45 mg, 4.42 mmol) was added, and the reaction mixture was stirred at 20 °C for 1 h. The reaction was monitored by LCMS. The pH of the reaction mixture was adjusted to 4 with 1 N HCl and extracted with 5% ethanol / DCM (30 mL × 3). The combined organic layers were washed with brine (30 mL), dried over NaSO, and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Welch Xtimate C18 250*70mm#10μm, mobile phase: [water (NH4HCO3)]-ACN; B%: 42%-72%, 20 min) to give (R)-10-((8-butoxy-8-oxooctyl)oxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.10) (1.80 g, purity 96.1%) as a yellow solid.

[0192] 1 H NMR(400 MHz, DMSO-d6)δ ppm: 8.95(s, 1H), 7.63(d, J = 8.4 Hz, 1H), 7.33 - 7.20(m, 2H), 6.82(d, J = 7.6 Hz, 1H), 5.26 - 5.05(m, 2H), 4.96(d, J = 4.4 Hz, 1H), 4.22 - 4.10(m, 2H), 4.00(t, J = 6.4 Hz, 2H), 2.29(t, J = 7.2 Hz, 2H), 1.81(quin, J = 6.8 Hz, 2H), 1.59 - 1.42(m, 6H), 1.40 - 1.25(m, 6H), 0.87(t, J = 7.6 Hz, 3H), 0.71(s, 9H). Mass spectrum calculated: 551.3, Found: [M+H] + = 552.3.

[0193] Compound I.11 Synthetic Route: [ka] Method for preparing pentyl 8-bromooctanoate (19)

[0194] To a mixture of 8-bromooctauic acid (12) (3.00 g, 13.45 mmol, 1 eq.) was added pentan-1-ol (30 ml), followed by dropwise addition of SOCl2 (3.20 g, 26.89 mmol, 1.95 ml, 2 eq.) at 0 °C. The mixture was stirred at 100 °C for 1 h. TLC (petroleum ether:ethyl acetate = 5:1, R1 = 0.5) confirmed the reaction was complete. The mixture was concentrated in vacuo and poured into saturated NaHCO3 (30 ml). The aqueous phase was extracted with ethyl acetate (30 ml x 3). The organic phase was washed with brine (20 ml x 2), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 5:1) to give pentyl 8-bromooctanoate (3.7 g, 12.62 mmol, 94% yield) as a colorless oil.

[0195] 1 H NMR(400 MHz, CDCl3)δ ppm 4.07(t, J = 6.8 Hz, 2H), 3.41(t, J = 6.8 Hz, 2H), 2.30(t, J = 7.2 Hz, 2H), 1.89 - 1.84(m, 2H), 1.66 - 1.60(m, 4H), 1.48 - 1.42(m, 2H), 1.36 - 1.33(m, 8H), 0.93 - 0.90(m, 3H).

[0196] Procedure for the preparation of methyl (R)-6-(tert-butyl)-2-oxo-10-(8-oxo-8-(pentyloxy)octyl)oxy-6,7-dihydro-2H-pyrido[2,1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (20) [ka]

[0197] A mixture of pentyl 8-bromooctanoate (19) (2.39 g, 8.17 mmol, 1.2 equiv.) and methyl (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3M) (2.5 g, 6.80 mmol, 1 equiv.) was dissolved in DMF (30 ml), and CsCO (7.76 g, 23.82 mmol, 3.5 equiv.) was added in one portion at 25 °C. The mixture was stirred at 50 °C for 12 h, at which point LCMS confirmed the reaction was complete. The reaction mixture was poured into water (50 ml), and the aqueous phase was extracted with ethyl acetate (30 ml × 3). The combined organic phase was washed with brine (40 ml × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (SiO2, ethyl acetate:MeCN = 1:0 to 0:1) to give methyl (R)-6-(tert-butyl)-2-oxo-10-((8-oxo-8-(pentyloxy)octyl)oxy)-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (20) (2.40 g, 3.93 mmol, 58% yield) as a yellow solid.

[0198] 1H NMR(400 MHz, DMSO-d6)δ ppm: 8.57(s, 1H), 7.51(d, J = 8.4 Hz, 1H), 7.18(t, J = 8.0 Hz, 1H), 6.82(s, 1H), 6.78(d, J = 7.6 Hz, 1H), 5.14 - 4.98(m, 2H), 4.71(d, J = 4.4 Hz, 1H), 4.17 - 4.09(m, 2H), 3.99(t, J = 6.4 Hz, 2H), 3.77(s, 3H), 2.29(t, J = 7.2 Hz, 2H), 1.84 - 1.78(m, 2H), 1.59 - 1.43(m, 6H), 1.38 - 1.25(m, 8H), 0.89 - 0.80(m, 3H), 0.70(s, 9H).

[0199] Procedure for the preparation of (R)-6-(tert-butyl)-2-oxo-10-((8-oxo-8-(pentyloxy)octyl)oxy)-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.11) [ka]

[0200] Methyl (R)-6-(tert-butyl)-2-oxo-10-((8-oxo-8-(pentyloxy)octyl)oxy)-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (20) (2.4 g, 4.14 mmol, 1 equiv.) was dissolved in CHCN (14 ml) and HO (14 ml), and LiOH HO (261 mg, 6.21 mmol, 1.5 equiv.) was added in one portion at 25 °C. The mixture was stirred at 25 °C for 1 h, and the reaction was confirmed to be complete by LCMS. The pH of the mixture was adjusted to 6–7 with 1 M HCl and then extracted with ethyl acetate (30 ml × 3). The organic phase was washed with brine (10 ml × 2), dried over NaSO, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC (column: Agela DuraShell C18 250 × 70 mm × 10 μm; mobile phase: [water(NHHCO)-ACN]; 8% to 45%-80%, 20 min). (R)-6-(tert-butyl)-2-oxo-10-((8-oxo-8-(pentyloxy)octyl)oxy)-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.11) (1.38 g, 2.37 mmol, 57% yield) was obtained as a yellow solid.

[0201] 1 H NMR(400 MHz, DMSO-d6)δ ppm: 8.95(s, 1H), 7.62(d, J = 8.0 Hz, 1H), 7.33 - 7.14(m, 2H), 6.81(d, J = 7.2 Hz, 1H), 5.25 - 5.03(m, 2H), 4.96(s, 1H), 4.15(s, 2H), 3.99(t, J = 6.4 Hz, 2H), 2.29(t, J = 7.2 Hz, 2H), 1.86 - 1.74(m, 2H), 1.60 - 1.42(m, 6H), 1.39 - 1.23(m, 8H), 0.87 - 0.80(m, 3H), 0.70(s, 9H).

[0202] Compound I.12 Synthetic Route: [ka]

[0203] Preparation of 2-ethylbutyl 8-bromooctanoate (21) [ka]

[0204] 8-Bromooctanoic acid (12) (3 g, 13.45 mmol, 1 equiv.) was dissolved in 2-ethylbutan-1-ol (20 ml), and SOCl2 (3.20 g, 26.89 mmol, 1.95 ml, 2 equiv.) was added. The mixture was stirred at 100 °C for 2 h. Completion of the reaction was confirmed by TLC (petroleum ether:ethyl acetate = 5:1, Rf = 0.80). The reaction mixture was diluted with water (30 ml) and extracted with ethyl acetate (20 ml × 3). The combined organic layers were washed with saturated brine (30 ml), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 100:1 to 20:1) to give 2-ethylbutyl 8-bromooctanoate (21) (4.1 g, 13.34 mmol, 99% yield) as a colorless oil.

[0205] 1 H NMR(400 MHz, CDCl3)δ ppm: 4.04 - 3.98(m, 2H), 3.48 - 3.33(m, 2H), 2.40 - 2.24(m, 2H), 1.93 - 1.80(m, 2H), 1.63(d, J = 1.2 Hz, 2H), 1.55 - 1.46(m, 2H), 1.40 - 1.33(m, 9H), 0.91 - 0.88(m, 6H).

[0206] Procedure for the preparation of methyl (R)-6-(tert-butyl)-10-((8-(2-ethylbutoxy)-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (22) [ka]

[0207] To a solution of 2-ethylbutyl 8-bromooctanoate (21) (2.51 g, 8.17 mmol, 1.2 volumes) and methyl (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3M) (2.5 g, 6.8 mmol, 1 volume) in DMF (30 mL) was added CsCO (7.76 g, 23.82 mmol, 3.5 volumes) at 25 °C. The mixture was stirred at 60 °C for 12 h. The progress of the reaction was monitored by LCMS. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with brine (50 mL × 3), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 20:1 to 0:1) to give methyl (R)-6-(tert-butyl)-10-((8-(2-ethylbutoxy)-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (22) (2.9 g, 4.88 mmol, 71.7% yield) as a yellow solid.

[0208] 1H NMR(400 MHz, CDCl3)δ 5 ppm 8.28(s, 1H), 7.39(d, J = 8.4 Hz, 1H), 7.17(t, J = 8.0 Hz, 1H), 7.02(s, 1H), 6.65(d, J = 7.2 Hz, 1H), 5.15(d, J = 14.8 Hz, 1H), 4.92(dd, J = 5.4, 14.8 Hz, 1H), 4.18(t, J = 6.4 Hz, 2H), 4.10(d, J = 5.2 Hz, 1H), 3.99(d, J = 6.0 Hz, 2H), 3.93(s, 3H), 2.31(t, J = 7.6 Hz, 2H), 2.02 - 1.92(m, 2H), 1.64(quin, J = 7.2 Hz, 2H), 1.56 - 1.47(m, 3H), 1.43 - 1.31(m, 8H), 0.89(t, J = 7.6 Hz, 6H), 0.83(s, 9H).

[0209] Procedure for the preparation of (R)-6-(tert-butyl)-10-((8-(2-ethylbutoxy)-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.12) [ka]

[0210] To a solution of methyl (R)-6-(tert-butyl)-10-((8-(2-ethylbutoxy)-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (22) (2.9 g, 4.88 mmol, 1 vol) in acetonitrile (15 mL) and water (15 mL) was added LiOH HO (266.45 mg, 6.35 mmol, 1.3 vol) at 25 °C. The mixture was stirred at 25 °C for 1 h. LCMS confirmed the reaction was complete. The reaction mixture was diluted with water (20 mL), adjusted to pH 7 with dilute hydrochloric acid (1 M), and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (Agela DuraShell C18 25070mm 10 μm column; mobile phase: [water (NH4HCO3)-ACN]; B%: 45%-80%, 20 min) to afford (R)-6-(tert-butyl)-10-((8-(2-ethylbutoxy)-8-oxooctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.12) (1.15 g, 1.98 mmol, 41% yield) as a yellow solid.

[0211] 1 H NMR(400 MHz, DMSO-d6)δ 5 ppm 8.95(s, 1H), 7.63(d, J = 8.0 Hz, 1H), 7.29 - 7.18(m, 2H), 6.82(d, J = 7.6 Hz, 1H), 5.24 - 5.06(m, 2H), 4.96(s, 1H), 4.16(s, 2H), 3.93(d, J = 5.6 Hz, 2H), 2.30(d, J = 7.2 Hz, 2H), 1.89 - 1.74(m, 2H), 1.58 - 1.51(m, 2H), 1.50 - 1.41(m, 3H), 1.38 - 1.24(m, 8H), 0.83(t, J = 7.2 Hz, 6H), 0.71(s, 9H).

[0212] Compound I.13 route: [ka]

[0213] Procedure for the preparation of ethyl (R)-6-(tert-butyl)-10-((9-ethoxy-9-oxononyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (23) [ka]

[0214] A solution of ethyl (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (3M) (1 g, 2.62 mmol, 1 vol.) in DMF (10 mL) was added with CsCO (2.56 g, 7.86 mmol, 3 vol.) and ethyl 9-bromononanate (903.22 mg, 3.41 mmol, 1.3 vol.) and stirred at 60 °C for 4 h. The mixture was diluted with water (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with brine (100 mL x 2), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO, petroleum ether:ethyl acetate = 1:0 to 0:1) to give ethyl (R)-6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (23) (600 mg, yield: 40%) as a yellow solid.

[0215] Mass spectrum calculated: 565.7, obtained: [M+H] + = 566.4. 1H NMR (400 MHz, DM SO-d6) δ 5 ppm 8.53 (s, 1 H), 7.50 (d, J = 8.4 Hz, 1 H), 7.18 (t, J = 8.0 Hz, 1 H), 6.87 - 6.76 (m, 2 H), 5.15 - 4.97 (m, 2 H), 4.70 (d, J = 4.4 Hz, 1 H), 4.24 (q, J = 7.2 Hz, 2 H), 4.14 (dt, J = 2.8, 6.4 Hz, 2 H), 4.04 (q, J = 7.2 Hz, 2 H), 2.26 (t, J = 7.2 Hz, 2 H), 1.80 (quin, J = 6.8 Hz, 2 H), 1.58 - 1.41 (m, 4 H), 1.39 - 1.24 (m, 9 H), 1.16 (t, J = 7.2 Hz, 3 H), 0.70 (s, 9 H).

[0216] Procedure for the preparation of (R)-6-(tert-butyl)-10-((8-carboxyoctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2,3-1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.13) [ka]

[0217] To a mixture of methyl (R)-6-(tert-butyl)-10-((9-ethoxy-9-oxononyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (23) (600 mg, 1.06 mmol, 1 equiv.), CHCN (6 mL) and HO (6 mL) were added, followed by LiOH HO (133.51 mg, 3.18 mmol, 3 equiv.) and stirring at 25 °C for 2 h. The pH of the reaction solution was adjusted to 7 with 1 M HCl solution at 0 °C. The reaction solution was purified by prep-HPLC (column: Waters Xbridge BEH C18 250 × 70 mm × 10 μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 10%-40%, 20 min) to give (R)-6-(tert-butyl)-10-((8-carboxyoctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound 1.13) (400 mg, yield: 71%, purity: 96.35%) as a green solid.

[0218] 1 H NMR(400 MHz, DMSO-d6)δ 5 ppm 8.94(s, 1 H), 7.62(br d, J = 8.4 Hz, 1 H), 7.33 - 7.15(m, 2 H), 6.82(d, J = 7.6 Hz, 1 H), 5.24 - 5.08(m, 2 H), 4.96(br d, J = 4.4 Hz, 1 H), 4.23 - 4.11(m, 2 H), 2.18(t, J = 7.2 Hz, 2 H), 1.88 - 1.76(m, 2 H), 1.57 - 1.42(m, 4 H), 1.40 - 1.23(m, 6) H), 0.71(s, 9 H). Mass spectrum calculated: 509.6, observed: [M+H] + = 510.0.

[0219] Compound I.14 Procedure for the preparation of (R)-6-(tert-butyl)-10-((9-ethoxy-9-oxononyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2,1-b:3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.14) [ka]

[0220] Methyl (R)-6-(tert-butyl)-10-((9-ethoxy-9-oxononyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (23) (480 mg, 870 μmol, 1 equiv.) and LiOH HO (36 mg, 870.08 μmol, 1 equiv.) were stirred in MeCN (4.8 mL) and HO (4.8 mL) at 25 °C for 1 h. The pH of the reaction solution was adjusted to 7 with 1 M HCl solution at 0 °C. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 250 × 70 mm × 10 μm; mobile phase: [water(NH4HCO3)]-ACN; B%: 10%-40%, 20 min) to give (R)-6-(tert-butyl)-10-((9-ethoxy-9-oxononyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (compound I.14) (146.5 mg, yield: 31%, purity: 98.77%) as a green solid.

[0221] 1H NMR (400 MHz, DMSO-d6): δ ppm 8.95(s, 1H), 7.63(d, J = 8.0 Hz, 1H), 7.32 - 7.17(m, 2H), 6.82(d, J = 8.0 Hz, 1H), 5.22 - 5.06(m, 2H), 4.96(s, 1H), 4.15(s, 2H), 4.07 - 3.97(m, 2H), 2.27(t, J = 6.8 Hz, 2H), 1.90 - 1.72(m, 2H), 1.57 - 1.41(m, 4H), 1.40 - 1.24(m, 6H), 1.20 - 1.11(m, 3H), 0.70(s, 9H). Mass spectrum calculated: 537.66, found: [M+H] + = 538.1.

[0222] Compound (1) was prepared using the methods described in published PCT application WO 2018 / 198079 and U.S. Patent No. 10,301,312 B2 (issued May 28, 2019), the contents of which are incorporated herein by reference in their entirety. However, for clarity, the following general procedure is described. From commercially available compound (24), compound (25) was obtained by alkylation with potassium carbonate in acetonitrile. The nitro group of compound (25) was reduced with hydrogen in the presence of a palladium / carbon catalyst to give compound (26). Compound (26) was reacted with isoamyl nitrite and acetic anhydride to give indazole (27). Compound (27) was alkylated by reaction with compound (28) in the presence of lithium carbonate in dioxane with warming to give compound (29) as the major regular isomer (the minor regular isomer was separated by chromatography). Compound (29) was formylated with N-formylmorpholine to give compound (30). Deprotection and simultaneous cyclization were achieved by treatment with trifluoroacetic acid to give compound (31). Compound (31) was condensed with compound (32) to give tetracyclic compound (33) in quantitative yield. Dehydrogenation of compound (33) was achieved by treatment with DDQ to give compound (1).

[0223] Synthesis of intermediate 1 General scheme: [ka] Preparation of 1-(3-methoxypropoxy)-3-methyl-2-nitrobenzene (25)

[0224] Acetonitrile (5 v) was charged to a 100 L reactor, followed by the addition of 24 (10.0 kg, 65.3 mol, 1 vol) at 25 °C. K2CO3 (10.8 kg, 78.4 mol, 1.2 vol) was added in one portion at 22 °C, resulting in a color change from yellow to red. 1-Bromo-3-methoxypropane (11.0 kg, 71.8 mol, 1.1 vol) was added dropwise at 25 °C over 5 min, without any change in reaction temperature. The resulting mixture was heated to 70 °C and stirred under nitrogen for 16 h. The mixture was then cooled to 25 °C, filtered, and the solid was washed with methyl tert-butyl ether (MTBE, 2 v). The filtrate was concentrated under reduced pressure to give the crude product as an oil. The oil was dissolved in MTBE (2 v) and then washed with 2 N NaOH solution (0.3 v). The aqueous layer was extracted with MTBE (1 v × 2). The combined organic layers were washed with brine (1 L), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give 25 (13.9 kg, 94.5% yield) as a yellow oil.

[0225] Preparation of 2-(3-methoxypropoxy)-6-methylaniline (26) [ka]

[0226] Ethanol (10 v) was charged to a 200 L reactor, followed by the addition of 25 (13.9 kg, 61.71 mol, 1 vol) at 25 °C. Palladium on carbon (278 g, 2% wt) was added in one portion at 25 °C. The suspension was degassed under vacuum and purged with H2 three times. The reaction mixture was heated to 50 °C and stirred under hydrogen gas for 48 h. The mixture was cooled to 25 °C, filtered, and the solid was washed with ethanol (2 v). The filtrate was concentrated under reduced pressure to give 26 (11.5 kg, 93.8% yield) as a brown oil.

[0227] Preparation of 7-(3-methoxypropoxy)-1H-indazole (27) [ka]

[0228] Toluene (10 vol) was charged to a 250 L reactor, followed by the addition of 26 (9.0 kg, 46.09 mol, 1 vol) at 25 °C. Potassium acetylacetate (5.4 kg, 55.31 mol, 1.2 vol) was added in one portion at 25 °C. Acetic anhydride (14.1 kg, 138.28 mol, 12.95 L, 3 vol) was added dropwise at 25 °C. The mixture was heated to 50 °C and stirred under nitrogen for 1 h. Tert-butyl nitrite (11.9 kg, 115.23 mol, 13.7 L, 2.5 vol) was added dropwise at 50 °C. The reaction mixture was heated to 60–65 °C and stirred under nitrogen for 16 h. The mixture was cooled to 25 °C and refluxed with water (3 vol). The product was extracted with ethyl acetate (2 V × 2). The combined organic phases were washed with brine (2 volumes), dried over anhydrous sodium sulfate (NaSO), filtered, and concentrated in vacuo at 40 °C to give the crude product. The crude product was dissolved in methanol (4 volumes), and then 3N hydrochloric acid solution (3 volumes) was added dropwise so that the reaction temperature did not exceed 35 °C. The mixture was stirred at 45 °C for 1 h. The mixture was concentrated in vacuo at 40 °C to remove the methanol, and the aqueous phase was then extracted with ethyl acetate (2 volumes × 3). The combined organic phases were washed with brine (2 volumes), dried over anhydrous sodium sulfate (NaSO), filtered, and concentrated in vacuo at 40 °C to give the crude product 27 as a yellow oil. The crude product was purified by silica gel column chromatography (n-heptane / ethyl acetate, 10:1 to 5:1) to give the crude product 27 as a yellow oil. The oil was dissolved in MTBE (1.5 vol) and stirred for 1 h, after which a yellow solid precipitated. The mixture was filtered, and the filter cake was washed twice with n-heptane / MTBE (1.6 vol: 0.4 vol) and dried in vacuo to give 27 (7.0 kg, yield: 73%).

[0229] Preparation of tert-butyl (R)-(1-(7-(3-methoxypropoxy)-2H-indazol-2-yl)-3,3-dimethylbutan-2-yl)carbamate (29) [ka]

[0230] Dioxane (10 vol) was charged to a SOL reactor, followed by the addition of 27 (2.8 kg, 13.58 mol, 1 equiv.) and 28 (5.7 kg, 20.36 mol, 1.5 equiv.) at 25 °C. Lithium carbonate (2.0 kg, 27.15 mol, 2 equiv.) was added in one portion at 25 °C. The resulting mixture was heated to 100 °C and stirred under nitrogen (N) for 90 h. The reaction mixture was filtered, and the filter cake was washed with ethyl acetate (0.5 vol. × 2). The filtrate was concentrated under reduced pressure to remove most of the dioxane and give the crude product. The crude product was then stirred with NaOH (1 M, 4 vol.) at 25 °C for 16 h. The reaction mixture was filtered to give the crude product (approximately 5.5 kg). The crude product was stirred with NaOH (1M, 4 vol) at 25 °C for 16 h. The reaction mixture was filtered to give the crude product (approximately 5.1 kg). The crude product was stirred with MTBE / n-heptane (2 vol: 1 vol) at 25 °C for 1 h. The reaction mixture was filtered to give 29 (3.8 kg, 69.0%).

[0231] Preparation of tert-butyl (R)-(1-(3-formyl-7-(3-methoxypropoxy)-2H-indazol-2-yl)-3,3-dimethylbutan-2-yl)carbamate (30) [ka]

[0232] The following procedure was carried out in nine parallel batches. In each batch, tetrahydrofuran (10 volumes) was charged to a 3 L glass flask, followed by the addition of 29 (540 g, 1.33 mol, 1 equiv.) at 25 °C. The mixture was degassed and purged with nitrogen three times. The mixture was cooled to −60 °C in an ethanol and dry ice bath. n-BuLi (2.5 M, 1.86 L, 3.5 equiv.) was added dropwise at −60 °C under nitrogen over 1.5 h. The mixture was stirred at −60 °C under nitrogen for 0.5 h. N-formylmorpholinium (460 g, 3.99 mol, 400 mL, 3 equiv.) was added dropwise at −60 °C under nitrogen over 1 h. The mixture was stirred at −60 °C under nitrogen for 2 h. Saturated ammonium chloride (2 volumes) was slowly added dropwise to the reaction mixture at -60 °C under nitrogen. The reaction mixture was heated to 25 °C. At this stage, all nine batches were combined. The product was extracted with ethyl acetate (2 volumes x 3). The combined organic phase was washed with brine (2 volumes x 2), dried over anhydrous sodium sulfate (Na2SO4), filtered, and concentrated in vacuo at 50 °C to give the crude product. The crude product was stirred with heptane (4 volumes) at 25 °C for 1 h. The mixture was filtered, and the filter cake was washed twice with heptane (2 volumes) and dried in vacuo to give 30 (4.3 kg, 82.8%).

[0233] Preparation of (R)-3-(tert-butyl)-7-(3-methoxypropoxy)-3,4-dihydropyrazino[1,2-b]indazole (31) [ka]

[0234] Dichloromethane (10 volumes) was charged to a SOL glass flask, followed by the addition of 30 (3.5 kg, 8.07 mol, 1 equiv.) at 20 °C. Trifluoroacetic acid (TFA, 2 volumes, 7 L) was added dropwise at 20 °C. The mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated in vacuo at 50 °C to remove dichloromethane and most of the TFA. The reaction mixture was diluted with dichloromethane (10 volumes). Saturated sodium bicarbonate (approximately 10 volumes) was slowly added to the reaction mixture to adjust the pH to 7–8. The product was extracted with dichloromethane (2 volumes × 2). The combined organic phases were concentrated in vacuo at 50 °C to give 31 (2.5 kg, 98.2%).

[0235] Preparation of ethyl (6R)-6-(tert-butyl)-10-(3-methoxypropoxy)-2-oxo-1,6,7,13c-tetrahydro-2H-pyrido[2,1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (33) [ka]

[0236] Ethanol (10 volumes) and water (1 volume) were charged to a 50 L reactor, and 31 (2.7 kg, 8.56 mol, 1 equiv.) was added over 30 min at 25 °C. Compound 32 (3.2 kg, 17.12 mol, 2 equiv.) was added at 25 °C under nitrogen (N). After the addition, the reaction mixture was heated to 50 °C. The resulting mixture was stirred at 50 °C under nitrogen for 16 h. The reaction mixture was concentrated under reduced pressure to remove the ethanol and water, yielding crude product 33 (4.3 kg).

[0237] Preparation of ethyl (R)-6-(tert-butyl)-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2,1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (1) [ka]

[0238] Dimethoxyethane (DME, 10V) was charged to a 50 L reactor. 33 (4.3 kg, 9.44 mol, 1 eq) was then added over 5 min at 25 °C. 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 2.57 kg, 11.33 mol, 1.2 eq) was added at 25 °C. The mixture was stirred at 25 °C for 2 h. The mixture was then concentrated to give a residue. The residue was dissolved in ethyl acetate (10V). The solution was neutralized with saturated sodium carbonate (0.5V), and the alkalized reaction mixture was diluted with water (0.6V), and the organic layer was separated. The aqueous phase was extracted with ethyl acetate (3V × 2). The combined organic layers were washed with water (0.6V) and brine (0.6V) and concentrated to give the crude product. The crude product was dissolved in ethyl acetate (0.3 V), then 2N hydrochloric acid solution in ethyl acetate (9.4 L, 18.88 mol, 2 eq) was added dropwise at 25 °C and stirred for 12 h. The mixture was filtered to give a yellow solid. Residual water was removed to give 1 (2.4 kg, 56.0% yield, 95.69% purity by HPLC) as a yellow solid.

[0239] 1 H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H); 7.53 (d, J = 8.4 Hz, 1H); 7.24 (t, J = 8.0 Hz, 1H); 7.10 (s, 1H); 6.83 (d, J = 7.6 Hz, 1H); 5.18 - 5.09 (m, 2H); 4.81 (d, J = 4.8 Hz, 1H); 4.28 - 4.20 (m, 4H); 3.54 (t, J = 6.2 Hz, 2H); 3.27 (s, 3H); 2.07 (quintet, J = 6.3 Hz, 2H); 1.30 (t, J = 7.0 Hz, 3H) and 0.72 (s, 9H).

[0240] Preparation of tert-butyl (4R)-4-(tert-butyl)-1,2,3-oxathiazolidine-3-carboxylate 2-oxide (35) [ka]

[0241] Tetrahydrofuran (1.5V) was charged into a 50 L glass flask, and then pyridine (7.6 kg, 96.64 mol, 7.8 L, 7 eq) was added at 25 °C. The reaction mixture was cooled to 0 °C. Thionyl chloride (4.9 kg, 41.42 mol, 3.0 L, 3 eq) was added dropwise over 1 h at 0–10 °C. A solution of 34 (3.0 kg, 13.81 mol, 1 eq) was dissolved in tetrahydrofuran (3V) and this solution was added dropwise over 2 h at 0–10 °C. The resulting mixture was then stirred at 25 °C for 16 h under nitrogen. The reaction mixture was cooled to 0 °C in an ice-water bath and slowly added to ice water. The reaction mixture was extracted with ethyl acetate (2V × 2). The organic phase was separated and washed twice with brine (1V × 2). The organic phase was dried over sodium sulfate (Na2SO4), filtered and concentrated under reduced pressure to give a residue (3.6 kg, crude).

[0242] Preparation of tert-butyl (R)-4-(tert-butyl)-1,2,3-oxathiazolidine-3-carboxylate 2,2-dioxide (28) [ka]

[0243] The reaction was carried out in four batches. In each batch, acetonitrile (5V) and water (5V) were charged to a 50 L reactor, followed by the addition of 35 (2.5 kg, 9.49 mol, 1 eq) over 20 min at 25 °C. The reaction mixture was cooled to 0 °C in 30 min. RuCl3 (19.7 g, 94.93 mmol, 6.33 mL, 0.01 eq) was added at 0 °C under nitrogen. NaIO4 (3.05 kg, 14.24 mol, 789 mL, 1.5 eq) was added in approximately 10 portions between 0 and 10 °C (NaIO4 solubility is 80 g / L at 20 °C) over 4 h. The resulting mixture was then stirred under nitrogen at 25 °C for 1.5 h. At this stage, the four batches were combined. The mixture was diluted with methyl tert-butyl ether (MTBE, 1.7 vol). The reaction mixture was filtered through a pad of Celite. The pad was washed with MTBE (1 vol × 3). The combined filtrate was extracted with MTBE (1.3 vol × 3). The combined organic layers were washed with saturated NaSO solution (1.7 vol × 2). The organic layer was separated and washed with brine (1.3 vol × 2). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure to give the crude product. The crude product was stirred with n-heptane / ethyl acetate (2 vol / 0.4 vol) at 25 °C for 30 min with shaking. The reaction mixture was filtered, and the filter cake was washed with n-heptane (0.33 vol). It was then dried under vacuum to give 28 (7.9 kg, 75%).

[0244] HBsAg assay HepG2.2.15 cells were cultured microscopically in flasks and plated in 96-well plates at 6.0 x 10 4 cells / well and incubated overnight at 37°C and 5% CO2.

[0245] Prepare the source plate. The stock concentration of the reference and test compounds is 20 mM. First, manually dilute the compounds to obtain initial concentration points of 10 μM and 200 μM, respectively. Then, perform eight 3-fold dilutions to create a 200x source plate.

[0246] The compounds in the source plate were pipetted as follows: 3 μL of the 200x source plate compound was placed into a sterile 2 mL 96-well plate containing 297 μL of 2% FBS medium, mixed well, and then 100 μL / well was transferred to the corresponding cell culture plate. Therefore, the final compound test conditions were an initial concentration of 1 μM, 3-fold serial dilutions, and eight concentration points in duplicate.

[0247] Cells were incubated at 37°C, 5% CO2 for 3 days. 24 hours after seeding, cells were treated with 200 μL / well of medium containing compounds diluted in DMSO. DMSO alone was used as a no-drug control. The final DMSO concentration in all wells was 0.5%.

[0248] The level of secreted HBVsAg was measured using an HBsAg kit (Auto Biology-CL 0310). The HBsAg assay was performed as follows. a) Equilibrate the ELISA kit at room temperature for 30 minutes. b) Add 50 μL of standard solution, sample, positive and negative controls to each well. c) Add 50 μL of HBsAg enzyme conjugate to each well. d) Attach the membrane to the plate, shake for 60 seconds, and incubate at 37°C for 60 minutes. e) Add 350 μL of wash buffer to each well, shake gently, discard the liquid, and repeat this process 6 times. Then, gently tap the wells 5 times with absorbent paper towels to dry them. f) Add 50 μL of the mixture of reagents A and B to each well. Shake the microplate for 10 seconds. g) Cover the membrane of the plate and incubate at room temperature for 10 minutes, after which the luminescence signal is read using Biotek-Synergy 2.

[0249] Data analysis The inhibitory rate of the compound against HBsAg at different concentrations was calculated using the following formula: Inhibition rate (%) = (1 - sample value / control mean value) × 100 The 50% effective concentration (EC50) was calculated using GraphPad Prism software.

[0250] Table 3. Activity of selected compounds (Formula 1) in the HBV S antigen secretion biological assay in HepG2 cells (geometric mean ± standard deviation, where applicable). [Table 3]

[0251] Uptake transporters OATP1B1 and OATP1B3 substrate assays were performed in Wuxi using the human embryonic kidney cell line HEK293 stably transfected with human transporter genes. Identification of five potential transporter substrates was achieved by measuring fold-uptake values ​​in transfected HEK293-OATP1B1 and OATP1B3 cell lines and HEK293-MOCK cell lines in the presence and absence of positive inhibitors.

[0252] The objective of this study was to determine whether compounds I.2 and I.6 are potential OATP1B1 and OATP1B3 uptake substrates. Detailed information on the substrate, positive inhibitor, and internal standard is provided in the table below. [Table 1]

[0253] HEK-293 and HEK293-MOCK cell lines stably expressing human OATP1B1 and OATP1B3 transporters were supplied by GenoMembrane (Kanagawa, Japan). The culture medium was DMEM (HEK293-OATP1B3 cells were maintained in DMEM / F12) supplemented with 10.0% FBS, 500 μg / mL G418 sulfate solution, 100 U / mL penicillin G, and 100 μg / mL streptomycin. Cells were incubated at 37.0°C in 5.0% CO2 with saturated humidity.

[0254] HEK293-OATP1B1 (passage: 19), OATP1B3 (passage: 19), and HEK293-MOCK (passages: 14 and 16) were grown in culture flasks until they reached 80.0% to 90.0% confluency. Trypsin / EDTA (0.05% / 0.02%, w / v) was added to detach the cells from the flasks. The cells were plated at 5.00 x 10 cells per well in a 96-well plate. 11 They were seeded at a density of 100 cells / well and then incubated in 5.0% CO2 at 37.0°C in saturated humidity for 24 h before being used for uptake experiments.

[0255] Pretreatment: After removing the cell culture medium from 96-well plates seeded with HEK293-OATP1B1 and OATP1B3 or HEK293-MOCK cells, the cells were washed twice with warmed (37.0°C) transport buffer. Next, the cells were pretreated with transport buffer with or without the positive inhibitor for 30 minutes at 37.0°C in 5.0% CO2 and saturated humidity.

[0256] Uptake incubation: At the end of pretreatment, the buffer was removed and cells were treated with compound I.6 or compound I.2 at 0.100 or 1.00 μM, with or without a positive inhibitor. In separate wells, a 15x concentrated solution of the transporter marker substrate (Table A) was incubated. All treatments were performed in triplicate wells at 37°C in 5% CO2 and saturated humidity.

[0257] Table A. Information on inhibitors and substrates for each transporter [Table A]

[0258] Cell lysis: At the end of the incubation period, the dosing solution was removed. After removing the remaining dosing solution, the cells were washed three times with ice-cold transport buffer (2.0-8.0°C). Cells treated with the test compound were then processed for bioanalysis by adding 100 μL of chilled acetonitrile:methanol (95:5, v:v) containing the internal standard. Cells treated with the positive control were added with 100 μL of chilled acetonitrile:methanol (95:5, v:v) containing the internal standard for bioanalysis. All samples were gently shaken for 30 minutes. Then, for the test compound and positive control, 75 μL of cell lysate was mixed with 75 μL of transport buffer and 150 μL of chilled acetonitrile:methanol (95:5, v:v) containing the internal standard. The lysed samples were centrifuged at 3220 x g for 10 minutes, and the supernatant was removed. This was used as the final sample to measure the cellular uptake of the test compound or substrate using LC-MS / MS.

[0259] Fold uptake was calculated using equation (1).

number

[0260] Table 4. Uptake ratios of selected compounds of Formula 1 in the uptake transporter assay. [Table 4-1]

[0261] In vivo pharmacokinetic studies Compounds of the present invention were administered orally in a 25% PEG 400:10% Solutol:65% water formulation and were tested in CD-1 mice, C57BL / J6 mice, or Sprague-Dawley rats according to IACUC guidelines. The pharmacokinetics of compounds I.9 and I.10 following oral administration of 32 mg / kg in rats demonstrates an example of cross-species methodology.

[0262] The purpose of this rat PK study was to determine the pharmacokinetics of compounds I.9, I.6, and I.10 in plasma after oral gavage administration to male SD rats. In the oral administration groups, liver and plasma concentrations were measured at 1, 2, 6, and 10 hours post-dose. Plasma and tissue concentrations of compounds I.9, I.6, and I.10 were measured by liquid chromatography-tandem mass spectrometry (LC-MS / MS). In oral administration (PO) groups 1 and 2, compound I.9 was prepared as a clear solution in 25% PEG400 + 10% solutol + 65% water, with the final pH adjusted to 7-8. In groups 3 and 4, compound I.10 was prepared as a homogenous suspension in 25% PEG400 + 10% solutol + 65% water, with the final pH adjusted to 7-8. Blood (approximately 0.2 ml) was collected from each test animal by jugular vein puncture into tube A. Then, 4-mix stabilizer containing an esterase inhibitor was added to tube B, which was a commercial product containing potassium (K2) EDTA (0.85–1.15 mg) in advance. Next, the blood in tube A was transferred exactly (4-mix stabilizer:blood = 1:10 (v:v)) to tube B and placed on wet ice until centrifugation.

[0263] [Table 4-1]

[0264] Tissue processing The following 4-mix stabilizers were added to pre-chilled homogenization buffer (methanol / 15 mM PBS 1:2). Each tissue was then weighed, and chilled homogenization buffer containing stabilizers was added at a 1:9 ratio (9 ml of buffer for 1 g of tissue) and homogenized on wet ice. Tissue homogenates were stored at -60°C or below until LC-MS / MS analysis.

[0265] [Table 4-2]

[0266] Bioanalytical Analysis Plasma and tissue concentrations of compounds I.9, I.6, and I.10 were measured using an LC-MS / MS method. Instrumentation and conditions used for analysis of compounds I.9, I.10, and I.6 in plasma and tissue homogenates. LC parameters Instrument: ACQUITY UPLC System Analytical column: ACQUITY UPLC HSS T3 1.8 μm 2.1 x 50 mm Injection volume: 5 μL for plasma, 3.5 μL for tissue homogenate Mobile phase A: Water / acetonitrile (ACN) (v / v, 95:5) containing 0.1% formic acid (FA) and 2 mmol / L formamine ammonium (HCOONH4). Mobile phase B: Acetonitrile (ACN) / water (v / v, 95:5) containing 0.1% formic acid (FA) and 2 mmol / L formamine ammonium (HCOONH4).

[0267] Elution mode: Gradient Analysis of Compound I.9, Compound I.10, and Compound I.6 in Plasma and Tissue Homogenates [Table 4-3]

[0268] After single oral administration of compound I.9 at 32 mg / kg (PO 1 and PO 2) to male SD rats, pharmacokinetic (PK) parameters of compound I.9 in plasma and tissues were not measured because most concentrations were below the lower limit of quantification. Similarly, after single oral administration of compound I.10 at 32 mg / kg (PO 3 and PO 4) to male SD rats, pharmacokinetic parameters of compound I.10 in plasma and tissues were not measured because most concentrations were below the lower limit of quantification.

[0269] [Table 5]

[0270] [Table 6] With a detection limit of 1 ng / mL in plasma, compounds 1.3, 1.5, 1.7, and 1.14 showed promising liver-to-plasma ratios of ≥186, ≥343, ≥193, and ≥533, respectively.

[0271] In vivo efficacy The purpose of this study was to investigate the in vivo pharmacological effects of compounds I.9 and I.10 using an adeno-associated virus-hepatitis virus (AAV-HBV) transfected mouse model. On the day of pre-administration (Day 0), mice were administered 1 × 10 11 Recombinant AAV-HBV vector genome was injected via the tail vein. Blood was collected from the mice, and 10 μL of serum was prepared from each mouse on days 21 and 28 after AAV-HBV injection. Serum samples were stored at -70°C and sent to the clinical pathology department for quantitative detection of HBsAg (as a baseline). Based on body weight and serum HBsAg concentration on day 35 of the pre-treatment period, 48 mice were selected and randomly divided into seven groups of six mice each.

[0272] Vehicle was administered twice daily at 12-hour intervals from Days 0 to 13. I.10 or I.9 was administered at 16 mg / kg / dose, 8 mg / kg / dose, or 4 mg / kg / dose twice daily, or 16 mg / kg / dose once daily. For the twice-daily dose, I.10 or I.9 was administered at 12-hour intervals from Days 0 to 13, with a single dose on Day 14. All test drugs were administered orally at 5 mL / kg / dose. Animals were monitored for clinical symptoms once daily, and body weights were measured twice weekly from Days 0 to 14. Mice were bled to obtain 10 μL of serum on Days 0, 3, 7, 10, and 14. Serum samples were stored at -70°C and sent to the clinical pathology department for viral marker detection. HBsAg was detected on Days 0, 3, 7, 10, and 14. In the groups receiving compound I.10 or compound I.9, the first three mice in each group were bled at 0.5 and 4 hours after dosing on Day 0, and the second three mice were bled at 1 and 8 hours. At each time point, 7 μL plasma samples were prepared per mouse, stored at -70°C, and sent to the Metabolic Medicine Department for bioanalysis (data reports were sent separately by Labcorp Metabolic Medicine to the sponsor). Mice in the vehicle control group were sacrificed on Day 14 without blood or tissue collection. The first three and second three mice in the groups receiving compound I.10 or I.9 were sacrificed 2 and 6 hours after dosing on Day 14, respectively. For these mice, livers were harvested after local saline perfusion. The livers were removed from the abdominal cavity, washed with saline, and placed on soft absorbent paper to drain residual fluid. The livers were weighed, cut into small pieces, placed in tubes, and flash-frozen in liquid nitrogen. Liver pieces were homogenized on ice in a 1:9 ratio (9 mL of buffer for 1 g of tissue) of methanol (methanol:15 mM PBS = 1:2). The homogenate was stored at -70°C and sent to the Department of Metabolism for bioanalysis (data reports were sent separately from Labcorp Metabolism to the sponsor). Serum hepatitis B surface antigen (HBsAg) was measured with the corresponding reagent using an ARCHITECT i2000 (Abbott Laboratories, Lake Bluff, IL, USA).

[0273] Compared to the vehicle control group, serum HBsAg levels in the groups administered Compound I.9 and Compound I.10 were reduced by 0.42 to 0.69 Log10 units 2 weeks after administration of 64 MPK bid, 32 MPK bid, and 16 MPK bid. The greatest effect was observed (not shown in the graph). Based on the initial maximal effect at higher doses, Compounds I.9 and I.10 were administered at 16 MPK bid, 16 MPK QD, 8 MPK bid, and 4 MPK bid, the results of which are shown in Figure 1. Abbreviations

[0274] TIFF2025533792000094.tif123111

Claims

1. A compound of the following formula 1, or a pharmaceutically acceptable salt thereof. 【Chemical 1】 where R is a hydrogen group, an ethyl group, or a linear, cyclic, or branched C 3 -C ８ is an alkyl group, and X is a linear, cyclic, or branched C 5 -C 10 It is an alkenyl group.

2. X in the above formula 1 is a linear C 5 -C ８ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is an alkenyl group.

3. X in the above formula 1 is a linear -C 5 H 10 -, linear -C 6 H 12 - and linear -C 7 H 14 2. The compound of claim 1, wherein the compound is selected from the group consisting of:

4. R in the above formula 1 is nC 2 H 5 , nC 3 H 7 ,I C 3 H 7 , nC 4 H 9 , nC 5 H 11 , nC 6 H 13 , 2-ethylbutyl group, nC 7 H 15 , and nC 8 H 17 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

5. 2. The compound according to claim 1, wherein R in formula 1 is hydrogen, or a pharmaceutically acceptable salt thereof.

6. R in the above formula 1 is iC 3 H 7 2. The compound of claim 1, wherein:

7. R in the above formula 1 is nC 4 H 9 2. The compound of claim 1, wherein:

8. The compound according to claim 1, which is selected from the group consisting of the following formulas 2-1 to 2-14, or a pharmaceutically acceptable salt thereof. 【Chemistry 2-1】 【Chemistry 2-2】 【Chemistry 2-3】 【Chemistry 2-4】 【Chemistry 2-5】 【Chemistry 2-6】 【Chemistry 2-7】 【Chemistry 2-8】 【Chemistry 2-9】 【Chemistry 2-10】 【Chemistry 2-11】 【Chemistry 2-12】 【Chemistry 2-13】 【Chemistry 2-14】

9. A compound with the following chemical structure: 【Chemistry 2-6】

10. A compound with the following chemical structure: 【Chemistry 2-9】

11. A compound with the following chemical structure: 【Chemistry 2-10】

12. 12. A formulation comprising a compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

13. 13. A method for treating hepatitis B in a human subject, comprising administering to the subject a compound of any of claims 1 to 11, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable formulation of claim 12.

14. 14. The method of claim 13, further comprising administering to the subject an additional therapeutic agent selected from an HBV replication inhibitor, an HBsAg-targeted therapeutic agent, and an immunomodulator.

15. 13. A method for treating hepatitis D in a human subject, comprising administering to the subject a compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable formulation of claim 12.

16. The method of claim 15, further comprising administering to the subject an additional therapeutic agent selected from a DNA polymerase inhibitor, an HBV capsid inhibitor, an HBV-targeting antibody, and an HBV-targeting therapeutic vaccine.

17. A method for inhibiting replication of hepatitis B virus, comprising contacting the hepatitis B virus in vitro or in vivo with a compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof.

18. A method for inhibiting replication of hepatitis D virus by contacting hepatitis B virus in vitro or in vivo with a compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof.

19. 12. Use of a compound according to any one of claims 1 to 11 for treatment.

20. 20. The compound of claim 19 for use in treating a bacterial infection.

21. Use of a compound according to any one of claims 1 to 11 in the manufacture of a pharmaceutical.

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