Indazole pyridone compounds and uses thereof

Abstract

The present application provides novel tetracyclic pyridone compounds according to Formula I: # imgabs0 # wherein R and X are defined herein. Also provided are pharmaceutical compositions containing such compounds, methods of using these compounds and compositions in the treatment and prophylaxis of HBV infection.

Description

Technical Field

[0001] The present invention relates to novel fused tetrahydropyridone compounds which are hepatitis virus expression inhibitors selectively acting on the liver and can thus be used for treating viral infections, particularly hepatitis B virus (HBV) and hepatitis D virus (HDV) infections. The present invention provides novel tetrahydropyridone compounds as disclosed herein, pharmaceutical compositions containing such compounds, and methods of using these compounds and compositions for treating and preventing HBV infections. Background Art

[0002] Currently, there are many methods and manufacturing processes for processing light guide layers for backlight applications. Generally, a transparent polymer is injection molded to form a thin layer with surface features. These surface features interfere with total internal reflection of light, and thus can controllably emit the light guided within the plate.

[0003] Hepatitis B virus (HBV) infection is one of the most common infectious diseases in the world. Chronic hepatitis B (CHB) represents a serious unmet medical need, with over 240 million people globally suffering from chronic infection. Chronic HBV carriers may develop into severe liver diseases such as chronic hepatitis, cirrhosis, and primary hepatocellular carcinoma (HCC). Due to the impact of CHB, the annual death toll is approximately 650,000 [(Chisari, Isogawa et al., 2010, 2016), (GBD Mortality Causes of Death Collaborators, 2016; World Health Organization (WHO), 2018)].

[0004] The goal of CHB therapy is to improve the quality of life and extend survival by preventing the disease from progressing to cirrhosis and HCC. Functional cure of HBV, also known as loss of hepatitis B surface antigen (HBsAg), with or without seroconversion to anti-HBsAg, is recognized as the endpoint of anti-HBV therapy (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). Multiple previous studies have shown that the loss of HBsAg is associated with improvement in liver histology (including reversal of cirrhosis, reduced risk of HTC, and extended survival), and is regarded as evidence of functional cure (Fattovich, Giustina et al., 1998; Benias and Min, 2011; Kim, Lim et al., 2013).

[0005] Nucleoside (nucleotide) analogues are the standard of care for CHB treatment, which inhibit viral replication and confer long-term clinical benefits, reducing the risk of liver complications (Dienstag, Goldin et al., 2003; Liaw, 2011; Lok, 2013). However, functional cure is rarely achieved with nucleoside (nucleotide) inhibitors (Kwon and Lok, 2011). Therefore, new treatment options that improve HBsAg clearance are needed to provide treatment options with a limited duration for functional cure.

[0006] Eliminating HBV-infected hepatocytes requires a broad immune response against HBV (Rehermann and Nascimbeni 2005; Das and Maini 2010; Burton, Pallett et al., 2018). Previous studies have shown that in chronic HBV infection, high levels of dominant viral antigens such as HBsAg may lead to antiviral CD8 + T cell exhaustion (Frebel, Richter et al., 2010; Isogawa, Chung et al., 2013; Ochel, Cebula et al., 2016; Zhu, Liu et al., 2016). Additionally, several other reports have described the negative regulation of HBV-specific immune responses by HBsAg through direct regulation of the functions 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, a preclinical study has shown that in a chronic HBV mouse model, reducing extracellular HBsAg with monoclonal HBsAg antibodies and then vaccinating can clear HBV from both the serum and the liver (Zhu, Liu et al., 2016).

[0007] Collectively, these studies suggest that in CHB, antiviral agents such as PAPD5 / 7 inhibitors (HBV RNA destabilizers / degraders / HBsAg secretion inhibitors) have potential therapeutic roles in reducing HBsAg levels and restoring virus-specific immune responses.

[0008] Hepatitis D virus (HDV) is the pathogen of chronic hepatitis D (CHD), which is the most severe form of viral hepatitis. At least 12 million people worldwide are co-infected with HBV / HDV, but the global number may be underestimated due to suboptimal detection. HDV infection can occur simultaneously with HBV or as a superinfection in patients already chronically infected with HBV. The dependence of HDV on HBV is mainly due to the use of HBV-encoded envelope protein (HBsAg) for its release and de novo infection. Effective antiviral therapies are urgently needed to prevent its progressive course leading to cirrhosis, end-stage liver disease, and hepatocellular carcinoma infection (Dandri, Volmari et al., 2022).

[0009] The minimum acceptable endpoint for new anti-HDV therapies is a ≥2-log normalized decrease in HDV RNA and alanine transaminase (ALT) during treatment 10 (FDA 2019; Yurdaydin, Abbas et al., 2019). A previous study confirmed that a 2-log decrease in HDV RNA in patients treated with conventional interferon 10 was associated with a survival benefit in CHD (Farci, Roskams et al., 2004).

[0010] Pegylated interferon alpha (pegIFNα) has been used as an off-label treatment for HDV, despite associated side effects, low response rates, and high relapse rates (Bahcecioglu, Ispiroglu et al., 2015; Rizzetto and Smedile, 2015). In the new anti-HDV strategies, bulevirtide can effectively block the entry of HBV and HDV by targeting the host receptor NTCP and is the first HDV-specific drug to obtain conditional marketing authorization in Europe (Masetti and Aghemo 2021; Lampertico, Roulot et al., 2022). Lonafarnib is currently being evaluated in clinical trials and is a farnesyltransferase inhibitor and thus inhibits the release of HDV (Yurdaydin, Keskin et al., 2022). A recent preclinical study confirmed that anti-HBsAg monoclonal antibodies neutralize HDV in vitro and significantly reduce HDV RNA levels in a CHD mouse model (Lempp 2021).

[0011] Several previous clinical studies have evaluated the intravenous or subcutaneous administration of anti-HBsAg monoclonal antibodies in CHB patients and have shown that these antibodies are safe and well tolerated, significantly reducing peripheral HBsAg levels in almost all subjects (Galun, Eren et al., 2002; Lee, Park et al., 2020; Agarwal, Yuen et al., 2022).

[0012] In summary, these data confirm the potential role of HBsAg reduction in the development of anti-HBV / anti-HDV therapies. We describe herein our novel liver-targeted PAPD5 / 7 inhibitors (HBV RNA destabilizer / degrader / HBsAg secretion inhibitor) designed to reduce HBsAg in patients with HBV or HBV / HDV, which can achieve functional cure either alone or in combination with other anti-HBV / anti-HBD agents.

[0013] A recent study confirmed that the hepatitis B virus (HBV) PAPD5 / 7 inhibitor (HBV RNA destabilizer / degrader / HBsAg secretion inhibitor), specifically GS-8873, which is structurally closely related to the first inhibitor of the compound RO7834 class, has neurotoxicity, where GS-8873 differs by replacing carbon with nitrogen to form a hydrazine core [Lake, April D. et al. Toxicological Sciences (2022) 186(2), pp. 298-308), structure shown below].[[]END]]

[0014]

[0015] It was found that starting at 4 weeks, GS-8873 prolonged the nerve conduction velocity (NCV) of several peripheral nerves in both rats and monkeys, with a more pronounced effect at 13 weeks. Rats were found to be the more sensitive species, in which, at daily doses of 20 MPK / day and 60 MPK / day of GS-8873, the changes in rat tail nerve NCV, digital nerve NCV, cauda equina nerve latency, digital nerve latency, and tibial nerve latency compared to baseline at week 13 all exceeded 20%. These changes were considered to corroborate the peripheral neuropathy that occurred in monkeys during this 13-week study. Additionally, starting at 4 weeks, GS-8873 had a significant effect on rat functional tests. Whether this peripheral CNS toxicology is driven by a chemotype-specific toxin or a target-based toxin is unclear. Several compounds in this class have entered clinical trials but have been discontinued or put on hold (Roche RO7834, Enanta EDP-721), and the reasons for the discontinuation have not been fully disclosed. Therefore, the prioritization of the systemic safety signal of HBV RNA destabilizers in this class has led us to focus on liver-targeted mechanisms that can provide elevated drug levels in the liver and lower systemic drug exposure to avoid systemic-based safety signals.

[0016] Although ACC inhibitors have the potential to address the pathogenic factors leading to NASH, the re-generation of fat in human bone marrow is important for platelet production, which limits the degree of safe systemic ACC inhibition tolerance during long-term treatment. The ACC inhibitors of Pfizer and Gilead-Nimbus are designed to incorporate structural features for recognition by organic anion transporting polypeptides (OATPs), which are members of the solute carrier organic anion (SLCO) superfamily of exogenous transporters. Among 11 human OATP transporters, OATP1B1 and OATP1B3 are expressed on the sinusoidal membrane of hepatocytes and can facilitate 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). Pfizer and Gilead-Nimbus sought ACC inhibitors as OATP1B1 / 1B3 transport substrates to drive liver selectivity. Thus, having both ACC inhibitors and statins preferentially utilize hepatic uptake via OATP1B1 and OATP1B3 to drive liver selectivity means that these same transporters would be well utilized for HBV RNA destabilizers. Summary of the Invention

[0017] A first aspect of the invention is a compound of formula (I):

[0018]

[0019] or a pharmaceutically acceptable salt thereof, its racemic mixture, and a pharmaceutical composition thereof, wherein:

[0020] R is selected from hydrogen, ethyl or a straight-chain, cyclic or branched C 3 -C 8 alkyl group, and X is a linker selected from a straight-chain, cyclic or branched C 5 -C 10 alkylene group.

[0021] A second aspect of the present invention is a method for treating a patient infected with a virus (such as chronic hepatitis B infection or chronic hepatitis D infection) by administering an effective amount of a pharmaceutical composition containing a compound of formula (I):

[0022]

[0023] or a pharmaceutically acceptable salt thereof, or its racemic mixture, wherein:

[0024] R is selected from hydrogen, ethyl or a straight-chain, cyclic or branched C 3 -C 8 alkyl group, and X is a linker selected from a straight-chain, cyclic or branched C 5 -C 10 alkylene group.

[0025] A third aspect of the present invention is a method for preventing a patient from developing downstream liver diseases due to chronic hepatitis B infection or chronic hepatitis D infection by administering an effective amount of a pharmaceutical composition containing a compound of formula (I):

[0026]

[0027] or a pharmaceutically acceptable salt thereof, or its racemic mixture, wherein:

[0028] R is selected from hydrogen, ethyl or a straight-chain, cyclic or branched C 3 -C 8 alkyl group, and X is a linker selected from a straight-chain, cyclic or branched C 5 -C 10 alkylene group.

[0029] A fourth aspect of the present invention is a compound of formula (I):

[0030]

[0031] or a pharmaceutically acceptable salt thereof, or its racemic mixture in the manufacture of a medicament for treating a patient infected with a virus, wherein:

[0032] R is selected from hydrogen, ethyl or a straight-chain, cyclic or branched C 3 -C 8 alkyl group, and X is a linker selected from a straight-chain, cyclic or branched C 5 -C 10 alkylene group.

[0033] A fifth aspect of the present invention is a compound of formula (I):

[0034]

[0035] or a racemic mixture thereof for use in the manufacture of a medicament for preventing a patient from suffering from downstream liver diseases due to chronic hepatitis B infection or chronic hepatitis D infection, wherein:

[0036] R is selected from hydrogen, ethyl or a straight-chain, cyclic or branched C 3 -C 8 alkyl group, and X is a linker selected from a straight-chain, cyclic or branched C 5 -C 10 alkylene group.

[0037] A sixth aspect of the present invention is a method for treating a patient infected with a virus by administering an effective amount of a pharmaceutical composition comprising a compound of formula (I):

[0038]

[0039] or a pharmaceutically acceptable salt thereof, or a racemic mixture thereof, wherein:

[0040] R is selected from hydrogen, ethyl or a straight-chain, cyclic or branched C 3 -C 8 alkyl group, and X is a linker selected from a straight-chain, cyclic or branched C 5 -C 10 alkylene group; the method further comprises: administering to the subject an additional therapeutic agent selected from: HBV replication inhibitors, including but not limited to nucleoside (nucleotide) analogue polymerase inhibitors, non-nucleoside (nucleotide) analogue polymerase inhibitors; HBsAg targeting agents, including but not limited to siRNAs and antisense molecules targeting HBV transcripts, HBV capsid inhibitors, cccDNA inhibitors, HBx inhibitors and 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 and TLR-8) and immunomodulator vaccines.

[0041] The seventh aspect of the present invention is a method for preventing a patient from developing upstream and downstream liver diseases due to chronic hepatitis B infection or chronic hepatitis D infection by administering an effective amount of a pharmaceutical composition comprising a compound of formula (I):

[0042]

[0043] or a pharmaceutically acceptable salt thereof, or a racemic mixture thereof, wherein:

[0044] R is selected from hydrogen, ethyl or a straight-chain, cyclic or branched C 3 -C 8 alkyl, and X is a linker selected from a straight-chain, cyclic or branched C 5 -C 10 alkylene; the method further comprises: administering to the subject an additional therapeutic agent selected from: HBV replication inhibitors, including but not limited to nucleoside (nucleotide) analogue polymerase inhibitors, non-nucleoside (nucleotide) analogue polymerase inhibitors; HBsAg targeting agents, including but not limited to siRNAs and antisense molecules targeting HBV transcripts, HBV capsid inhibitors, cccDNA inhibitors, HBx inhibitors and 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 and TLR-8), and immunomodulatory vaccines.

[0045] In some embodiments of the compound of formula (I), X is a straight-chain C 5 -C 8 alkylene.

[0046] In some embodiments of the compound of formula (I), X is selected from straight-chain -C 5 H 10 -, straight-chain -C 6 H 12 -, straight-chain -C 7 H 14 -, and straight-chain -C 8 H 16 .

[0047] In some embodiments of the compound of formula (I), R is selected from n-C 2 H 5 , n-C 3 H 7 , i-C 3 H 7 , n-C 4 H 9 , n-C 5 H 11 , n-C 6 H13 , 2-ethylbutyl, n-C 7 H 15、 and n-C 8 H 17 。

[0048] In some embodiments of the compounds of formula (I), R is H.

[0049] In some embodiments of the compounds of formula (I), R is i-C 3 H 7 。

[0050] In some embodiments of the compounds of formula (I), R is n-C 4 H 9 。

[0051] In some embodiments, the compound of formula (I) is one of the following:

[0052]

[0053]

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 is the efficacy of certain compounds of formula I in reducing the level of HBV S antigen when administered in an in vivo AAV mouse model. DETAILED DESCRIPTION

[0056] The present invention provides novel compounds that inhibit the secretion of HBsAg from hepatitis B virus-infected cells, thereby reducing the viral load and viral replication in patients with chronic HBV infection. Thus, the compounds of the present invention are suitable for treating patients with HBV (including chronic HBV). The compounds of the present invention are also suitable for treating patients with HBD, including those co-infected with HBV.

[0057] Each compound in the compounds of the examples (including each compound listed in Table 1) is a specific embodiment of the compounds of the present invention. The following description of the preferred embodiments of the present invention does not limit the scope of the present invention.

[0058] In some embodiments, there is provided a compound of the following formula (I):

[0059]

[0060] Wherein, R is hydrogen, ethyl or a straight-chain, cyclic or branched C 3 -C 8 alkyl, and X is a straight-chain, cyclic or branched C5 -C 8 Alkylene. Also included are its pharmaceutically acceptable salts, its racemic mixtures, and its pharmaceutical compositions.

[0061] Preferably, a compound of formula (I), wherein X is selected from straight-chain -C 5 H 10 -, straight-chain -C 6 H 12 -, and straight-chain -C 7 H 14 -; its pharmaceutically acceptable salts; and its pharmaceutical compositions.

[0062] Preferably, a compound of formula (I), wherein n-C 2 H 5 、n-C 3 H 7 、i-C 3 H 7 、n-C 4 H 9 、n-C 5 H 11 、n-C 6 H 13、 2-ethylbutyl, n-C 7 H 15、 and n-C 8 H 17 ; its pharmaceutically acceptable salts; its racemic mixtures; and its pharmaceutical compositions.

[0063] Preferably, a compound of formula I, wherein R is H; its pharmaceutically acceptable salts; its racemic mixtures; and its pharmaceutical compositions.

[0064] Preferably, a compound of formula I, wherein R is i-C 3 H 7 ; its pharmaceutically acceptable salts; its racemic mixtures; and its pharmaceutical compositions.

[0065] Preferably, a compound of formula I, wherein R is n-C 4 H 9 ; its pharmaceutically acceptable salts; its racemic mixtures; and its pharmaceutical compositions.

[0066] Preferably, the compounds of formula (I) illustrated in Table 1 below; its pharmaceutically acceptable salts; its racemic mixtures; and its pharmaceutical compositions.

[0067] Preferably, the compounds I.6, I.9 or I.10 illustrated in Table 1 below; its pharmaceutically acceptable salts; its racemic mixtures; and its pharmaceutical compositions.

[0068] Preferably, a method for treating a patient with chronic HBV infection by administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of formula (I) or a racemic mixture thereof; preferably, a compound of formula (I), wherein R is selected from n-C 2 H 5 、n-C 3 H 7 、i-C 3 H 7 、n-C 4 H 9 、n-C 5 H 11 、n-C 6 H 13 、2-ethylbutyl, n-C 7 H 15、 and n-C 8 H 17 , and preferably, wherein R is H, i-C 3 H 7 or n-C 4 H 9 ; and X is a straight-chain C 5 -C 8 alkylene, preferably straight-chain -C 5 H 10 -, straight-chain -C 6 H 12 - and straight-chain -C 7 H 14 ; and preferably, wherein the compound of formula (I) is a compound exemplified in Table 1 below; preferably Compound I.6, I.9 or I.10 exemplified in Table 1 below.

[0069] Preferably, the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a patient with HBV infection; preferably, a compound of formula (I), wherein R is selected from n-C 2 H 5 、n-C 3 H 7 、i-C 3 H 7 、n-C 4 H 9 、n-C 5 H 11 、n-C 6 H 13 、2-ethylbutyl, n-C 7 H 15、 and n-C 8 H 17 , and preferably, wherein R is H, i-C3 H 7 or n-C 4 H 9 ; and X is a straight-chain C 5 -C 8 alkylene group, preferably straight-chain -C 5 H 10 -, straight-chain -C 6 H 12 -, and straight-chain -C 7 H 14 ; and preferably, wherein the compound of formula (I) is a compound exemplified in Table 1 below; preferably a compound exemplified in Table 1 below, Compound I.6, I.9 or I.10.

[0070] Preferably, a method for treating a patient with chronic HBV infection by administering an effective amount of a compound of formula (I) or a pharmaceutical composition comprising an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof; preferably, a compound of formula (I), wherein R is selected from n-C 2 H 5 , n-C 3 H 7 , i-C 3 H 7 , n-C 4 H 9 , n-C 5 H 11 , n-C 6 H 13 , 2-ethylbutyl, n-C 7 H 15、 , and n-C 8 H 17 , and preferably, wherein R is H, i-C 3 H 7 or n-C 4 H 9 ; and X is a straight-chain C 5 -C 8 alkylene group, preferably straight-chain -C 5 H 10 -, straight-chain -C 6 H 12 -, and straight-chain -C 7 H 14; and preferably, wherein the compound of formula (I) is a compound illustrated in Table 1 below; preferably compound I.6, I.9 or I.10 illustrated in Table 1 below; the method further comprises: administering to the subject an additional therapeutic agent selected from: HBV replication inhibitors, including but not limited to nucleoside (nucleotide) analog polymerase inhibitors, non-nucleoside (nucleotide) analog polymerase inhibitors; HBsAg targeting agents, including but not limited to siRNA and antisense molecules targeting HBV transcripts, HBV capsid inhibitors, cccDNA inhibitors, HBx inhibitors, and 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, and TLR-8), and immunomodulator vaccines.

[0071] Although one enantiomer of the formula compound is generally more active than the other corresponding isomer, both isomers exhibit activity against HBsAg, as confirmed by the published PCT applications WO 2018 / 198079 and US10,301,312B2 (patent date: May 28, 2019), the contents of the above applications are incorporated herein by reference in their entirety.

[0072] The term "optical isomer" or "stereoisomer" refers to any one of the various stereoisomeric configurations that may exist for a given compound of the present invention and includes geometric isomers. It should be understood that substituents may be attached to the chiral centers of carbon atoms. The term "chiral" refers to a molecule that has the property of non-superimposability on its mirror image partner, while the term "achiral" refers to a molecule that is superimposable on its mirror image partner. Thus, the present invention includes enantiomers, diastereomers, and racemates of the compound. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture. This term is used, where appropriate, to designate a racemic mixture. "Diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. Absolute stereochemistry is designated according to the Cahn-Ingold-Prelog R-S system. When a compound is a pure enantiomer, the stereochemistry at each chiral carbon can be designated as R or S.

[0073] A resolved compound of unknown absolute configuration can be designated as (+) or (-), depending on its direction of rotation of plane-polarized light (right-handed or left-handed) at the wavelength of the sodium D line. Certain compounds described herein contain one or more asymmetric centers or axes and can therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined in the sense of absolute stereochemistry as (R)- or (S)-.

[0074] As used herein, a "prodrug" is a molecule that is converted in vivo to an active parent drug, typically involving physical properties of the active parent that are disadvantageous for improving oral bioavailability. A "prodrug" has an inherent structural lability, whether accidental or designed, that permits biotransformation in vivo to the active parent drug. This transformation can occur by chemical or enzymatic processes or a combination of both. The transformation releases the active drug from a masking "promoiety" or drug carrier, such that the resulting molecule (active metabolite) exhibits full complement of the desired therapeutic effect. For a more detailed review of prodrug strategies and examples, see Rautio, J., Meanwell, N., Di, L. et al. Nat Rev Drug Discov 2018, 17, 559 - 587.

[0075] Table 1. Table of Compounds.

[0076]

[0077]

[0078]

[0079]

[0080] Some of the compounds of formula (I) in Table 1 are "prodrugs", i.e., they are converted in vivo to some other compounds of formula (I) (the "parent drugs") also listed in Table 1.

[0081]

[0082] Each of the compounds of the examples (including each of the "prodrugs" and "parent drugs" listed in Table 2) is a specific embodiment of the compounds of the present invention.

[0083] Table 2. Examples of "prodrugs" and "parent drugs" selected from the compounds of formula (I).

[0084]

[0085]

[0086]

[0087] For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form.

[0088] Unless the context clearly indicates otherwise, the terms used in this specification have the following meanings.

[0089] As used herein, the term "subject" refers to an animal. In certain aspects, the animal is a mammal. The subject also refers to, for example, a primate (e.g., a human), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In certain embodiments, the subject is a human. As used herein, "patient" refers to a human subject.

[0090] As used herein, the term "inhibition" ("inhibition" or "inhibiting") refers to a significant reduction or suppression of the baseline activity of a given condition, symptom, or disorder, or disease, or biological activity or biological process.

[0091] As used herein, the term "treating" ("treating" or "treatment") of any disease or disorder in one embodiment refers to alleviating the disease or disorder (i.e., slowing or preventing or reducing the development of the disease or at least one of its clinical symptoms). In another embodiment, "treating" refers to alleviating or reducing at least one physical parameter, including those that may not be distinguishable to the patient. In yet another embodiment, "treating" refers to modulating the disease or disorder physically (e.g., stabilizing a distinguishable symptom), psychologically (e.g., stabilizing a physical parameter), or both ways. In yet another embodiment, "treating" refers to preventing or delaying the onset or development or progression of a disease or disorder.

[0092] As used herein, unless otherwise specified herein or clearly inconsistent with the context, the terms "a", "an", and "the" and similar terms used in the context of the present invention (especially in the context of the claims) shall be construed to cover both the singular and the plural.

[0093] Unless otherwise stated herein or unless clearly inconsistent with the context, all methods described herein can be carried out in any suitable order. Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is only intended to better illustrate the present invention and does not limit the scope of the present invention. "Optionally substituted" means that the indicated group can be substituted at one or more positions by any one or any combination of the radicals listed thereafter. The number, placement, and selection of substituents are understood to include only those that are expected by a professional chemist to be reasonably stable; thus, for example, "oxo" would not be a substituent on an aryl ring or a heteroaryl ring, and a single carbon atom would not have three hydroxyl and amino substituents. Unless otherwise stated, optional substituents are generally up to 4 groups selected from: halo, oxo, CN, amino, hydroxy, -C 1-3Alkyl, -OR*, -NR* 2 , -SR*, -SO 2 R*, -COOR* and -CONR* 2 , wherein each R* is independently H or C 1-3 alkyl.

[0094] Unless otherwise specified, "aryl" as used herein refers to a phenyl or naphthyl group.

[0095] Unless otherwise specified, the aryl group may optionally be substituted with up to 4 groups selected from: halo, CN, amino, hydroxy, -C 1-3 alkyl, -OR*, -NR* 2 , -SR*, -SO 2 R*, -COOR* and -CONR* 2 , wherein each R* is independently H or C 1-3 alkyl.

[0096] As used herein, "halo" or "halogen" may be fluorine, chlorine, bromine or iodine.

[0097] As used herein, "C 3-8 alkyl" or "C 3 -C 8 alkyl" represents a straight-chain or branched-chain alkyl having 3 - 8 carbon atoms. If a different number of carbon atoms is specified, such as C 5 or C 5 , then the definition should be modified accordingly. For example, "C 1-4 alkyl" will represent methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl.

[0098] As used herein, "C 5-10 alkylene" or "C 5 -C 10 alkylene" represents a straight-chain or branched-chain alkyl having 5 - 10 carbon atoms and two open valences for connecting to other two groups. If a different number of carbon atoms is specified, such as C 4 or C 3 , then the definition should be modified accordingly. For example, "C 1-4 alkylene" will represent methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), straight-chain or branched-chain propylene (-CH 2 CH 2 CH 2 - or -CH 2 -CHMe-CH 2 -), etc.

[0099] As used herein, "C 5-8 alkoxy" means a straight or branched chain alkoxy (-O-alkyl) having 5 to 8 carbon atoms. If a different number of carbon atoms is specified, such as C4 or C3, the definition shall be modified accordingly, such that "C 1-4 alkoxy" would represent methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, and tert-butoxy.

[0100] As used herein, "C 1-4 haloalkyl" or "C 1 -C 4 haloalkyl" means a straight or branched chain alkyl having 1 to 4 carbon atoms, wherein at least one hydrogen has been replaced by a halogen. The number of halogen substitutions can range from 1 to the number of hydrogen atoms on the unsubstituted alkyl group. If a different number of carbon atoms is specified, such as C6 or C3, the definition shall be modified accordingly. Thus, "C 1-4 haloalkyl" would represent methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, and tert-butyl in which at least one hydrogen has been replaced by a halogen, such as where the halogen is fluorine: CF 3 CF 2 -, (CF 3 ) 2 CH-, CH 3 -CF 2 -, CF 3 CF 2 -, CF 3 -, CF 2 H-, CF 3 CF 2 CH(CF 3 )-, or CF 3 CF 2 CF 2 CF 2 -.

[0101] As used herein, "C 3-8 cycloalkyl" refers to a saturated monocyclic hydrocarbon ring having 3 to 8 carbon atoms. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. If a different number of carbon atoms is specified, such as C 3 -C 6 , the definition shall be modified accordingly.

[0102] "4- to 8-membered heterocyclic group", "5- to 6-membered heterocyclic group", "3- to 10-membered heterocyclic group", "3- to 14-membered heterocyclic group", "4- to 14-membered heterocyclic group" and "5- to 14-membered heterocyclic group" respectively refer to 4- to 8-membered, 5- to 6-membered, 3- to 10-membered, 3- to 14-membered, 4- to 14-membered and 5- to 14-membered heterocycles; unless otherwise specified, such rings further contain 1 to 7, 1 to 5 or 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur as ring members, and these rings can be saturated, or partially saturated but not aromatic. The heterocyclic group can be attached to another group at a nitrogen atom or a carbon atom. The term "heterocyclic group" includes monocyclic groups, fused ring groups and bridged groups. Examples of such heterocyclic groups include, but are not limited to: pyrrolidine, piperidine, piperazine, pyrrolidone, morpholine, tetrahydrofuran, tetrahydrothiophene, tetrahydrothiophene, tetrahydropyran, tetrahydropyran, 1,4-dioxane, 1,4-thioxane, 8-aza-bicyclo[3.2.1]octane, 3,8-diazabicyclo[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-diazabicyclo[2.2.1]heptane, azetidine, ethylenedioxy, oxetane or thiazole. In certain embodiments, unless otherwise specified, the heterocyclic group has 1 to 2 heteroatoms selected from N, O and S as ring members and 4 to 7 ring atoms, and is optionally substituted by up to four groups selected from: halo, oxo, CN, amino, hydroxy, C 1-3 alkyl, -OR*, -NR* 2 、-SR*, -SO 2 R*, -COOR* and -CONR* 2 ,wherein each R* is independently H or C 1-3 alkyl. In particular, the heterocyclic group containing a sulfur atom is optionally substituted by one or two oxo groups on the sulfur.

[0103] As used herein, "4- to 6-membered cyclic ether" refers to a 4- to 6-membered ring containing one oxygen atom as a ring member. Examples include oxetane, tetrahydrofuran and tetrahydropyran.

[0104] "Heteroaryl" refers to a completely unsaturated (aromatic) ring. The term "heteroaryl" refers to a 5- to 14-membered monocyclic, bicyclic, or tricyclic aromatic ring system having 1 to 8 heteroatoms selected from N, O, or S. Generally, heteroaryl is a 5- to 10-membered ring or ring system (e.g., a 5- to 7-membered monocyclic group or an 8- to 10-membered bicyclic group), and a 5- to 6-membered ring often contains up to 4 heteroatoms selected from N, O, and S, but the heteroaryl ring often contains no more than one divalent O or S in the 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, 2-, 4-, or 5-pyrimidinyl. The heteroaryl group is optionally substituted with up to 4 groups selected from: halogenated, oxo, CN, amino, hydroxy, C 1-3 alkyl, -OR*, -NR* 2 、-SR*, -SO 2 R*, -COOR* and -CONR* 2 , where each R* is independently H or C 1-3 alkyl. The term "hydroxy" or "hydroxyl" refers to the -OH group.

[0105] In addition, the compounds of the present invention (including their salts) can also be obtained in the form of their hydrates, or include other solvents used for their crystallization. The compounds of the present invention can inherently or by design form solvates with pharmaceutically acceptable solvents (including water); thus, the present invention is intended to cover solvated and non-solvated forms. The term "solvate" 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 those known in the pharmaceutical field that are harmless to recipients, such as water, ethanol, etc. The term "hydrate" refers to a complex in which the solvent molecule is water.

[0106] As used herein, the terms "salt" or "salts" refer to acid addition salts or base addition salts of the compounds of the present invention. "Salts" particularly include "pharmaceutically acceptable salts". The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the compound of the present invention and is generally biologically or otherwise desirable. In many cases, the compounds of the present invention are capable of forming acid salts and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto.

[0107] Pharmaceutically acceptable acid addition salts can be formed using inorganic acids and organic acids, for example, acetates, aspartates, benzoates, benzenesulfonates, bromides / hydrobromides, bicarbonates / carbonates, bisulfates / sulfates, camphorsulfonates, chlorides / hydrochlorides, chlortheophyllonates, citrates, edisylates, fumarates, glucoheptonates, gluconates, glucuronates, hippurates, hydroiodides / iodides, isethionates, lactates, lactobionates, lauryl sulfates, malates, maleates, malonates, mandelates, mesylates, methyl sulfates, naphthoates, naphthalenesulfonates, nicotinates, nitrates, octadecanoates, oleates, oxalates, palmitates, pamoates, phosphates / monohydrogen phosphates / dihydrogen phosphates, polygalacturonates, propionates, stearates, succinates, basic salicylates, tartrates, toluenesulfonates, and trifluoroacetates.

[0108] Inorganic acids from which the salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

[0109] Organic acids from which the salts can be derived include, for example, 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, sulfosalicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed using inorganic bases and organic bases.

[0110] Inorganic bases from which the salts can be derived include, for example, ammonium salts and metals from Groups I - XII of the Periodic Table. In certain embodiments, the 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.

[0111] Organic bases from which the salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, and the like. Some organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, glucosamine, piperazine, and tromethamine.

[0112] The 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 forms of these compounds with a stoichiometric amount of a suitable base (e.g., hydroxides, carbonates, bicarbonates, etc. of Na, Ca, Mg or K), or by reacting the free base forms of these compounds with a stoichiometric amount of a suitable acid. Such reactions are typically carried out in water or an organic solvent or a mixture of both. Generally, when feasible, a non-aqueous medium such as (diethyl) ether, ethyl acetate, ethanol, isopropanol or acetonitrile is used. A listing of additional suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 20th Edition, Mack Publishing Co., Easton, Pa., (1985) and Stahl and Wermuth's Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, Weinheim, Germany, 2002).

[0113] Any chemical formula given herein is intended to represent both the unlabeled form and the isotopically labeled form of the compounds of the present invention, which have up to three atoms in a non-natural isotopic distribution, e.g., deuterium or 13 C or 15 N-enriched sites. The isotopically labeled compounds have the structures depicted by the chemical formulas given herein, except that one or more atoms are replaced by atoms having a selected atomic mass and mass number, rather than the natural abundance mass distribution. Examples of isotopes that can be effectively incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 18 F 31 P, 32 P, 35 S, 36 Cl, 125 I. The present invention includes various isotopically labeled compounds of the present invention, e.g., those in which radioactive isotopes (such as 3 H and 14 C) or non-radioactive isotopes (such as 2 H and 13 C) are present at levels substantially higher than the normal isotopic distribution. Such isotopically labeled compounds can be used in metabolic studies (e.g., using 14C), reaction kinetics studies (using, for example 2 H or 3 H), detection or imaging techniques (such as positron emission tomography (PET) or single photon emission computed tomography (SPECT) including determination of drug or matrix tissue distribution), or radiotherapy of a patient. In particular, the 18 F-labeled compounds of the present invention may be particularly suitable for PET or SPECT studies. The isotopically labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art, or by methods similar to those described in the attached "Examples" and "Preparations", using appropriate isotopically labeled reagents in place of the unlabeled reagents normally employed. In cases where the isotope incorporation is quite low, the labeled sample can be useful, such as in the case of using radioactive labels to detect trace compounds.

[0114] Furthermore, site-specific substitution with heavier isotopes, particularly deuterium (i.e., 2 H or D), can provide certain therapeutic advantages due to higher metabolic stability, such as an extended in vivo half-life or reduced dose requirements or an improved therapeutic index. It should be understood that in this context, deuterium is considered a substituent of the compounds of the present invention, and a sample of a compound with deuterium as a substituent typically has at least 50% deuterium incorporation at the labeled position. The concentration of such heavier isotopes (particularly deuterium) can be defined by the isotope enrichment factor. As used herein, the term "isotope enrichment factor" means the ratio of the isotopic abundance of a particular isotope to its natural abundance. If a substituent in a compound of the present invention represents deuterium, the isotope enrichment factor for each designated deuterium atom of the compound is at least 3500 (52.5% deuterium incorporation for 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).

[0115] Pharmaceutically acceptable solvates according to the present invention include those solvates in which the crystallization solvent can be isotopically substituted, such as isotopically substituted solvates, such as D 2 O, d 6 -acetone, d 6 -dimethyl sulfoxide (DMSO).

[0116] The compounds of the present invention contain groups that can act as donors and / or acceptors of hydrogen bonds and can form 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 processes. Such processes include grinding, heating, co-sublimation, co-melting, or contacting the compounds of the present invention with co-crystal formers in solution under crystallization conditions and separating the co-crystals thus formed. Suitable co-crystal formers include those described in WO 2004 / 078163. Accordingly, the present invention also provides co-crystals comprising the compounds of the present invention.

[0117] Method of Use

[0118] Unless otherwise specified herein or unless clearly contradicted by the context, all methods described herein can be carried out in any suitable order. Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the invention and does not limit the scope of the invention.

[0119] The compounds of the present invention can be administered by known methods, including orally, parenterally, by inhalation, etc. In certain embodiments, the compounds of the present invention are administered orally in the form of pills, lozenges, troches, capsules, solutions, or suspensions. In other embodiments, the compounds of the present invention are administered by injection or infusion. Infusion is typically via the vein and is often carried out over a period of about 15 minutes to 4 hours. In other embodiments, the compounds of the present invention are administered intranasally or by inhalation, and the inhalation method is particularly useful for treating respiratory infections. The compounds of the present invention exhibit oral bioavailability, and thus oral administration is sometimes preferred.

[0120] 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 indicated herein).

[0121] For the term "combination", it means a fixed combination in a unit dosage form, as separate dosage forms suitable for simultaneous or sequential use together, or as a kit for combined administration (wherein the compounds of the present invention and the combination partner can be administered simultaneously independently, or separately at time intervals that particularly allow the combination partner to exhibit coordinated (e.g., synergistic) effects), or any combination thereof.

[0122] The second antiviral agent can be administered in combination with the compounds of the present invention, wherein the second antiviral agent is administered before, simultaneously with, or after one or more compounds of the present invention. When it is desired to administer the compounds of the present invention and the second agent simultaneously and the routes of administration are the same, the compounds of the present invention and the second agent can be formulated into the same dosage form. Examples of dosage forms containing the compounds of the present invention and the second agent are tablets and capsules.

[0123] In some embodiments, the combination of the compounds of the present invention and a second antiviral agent can provide synergistic activity. The compounds of the present invention and the second antiviral agent can be administered together, separately but simultaneously, or sequentially.

[0124] An "effective amount" of a compound is an amount necessary or sufficient to treat or prevent a viral infection and / or the diseases or conditions described herein. In one example, an effective amount of a compound of Formula I is an amount sufficient to treat a viral infection in a subject. In another example, the effective amount is an amount sufficient to treat HBV in a subject in need of such treatment. The effective amount can 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 the compound of the present invention will affect what constitutes an "effective amount". A person of ordinary skill in the art will be able to study the factors included herein and, without undue experimentation, make a determination regarding the effective amount of the compounds of the present invention.

[0125] The dosing regimen affects what constitutes an "effective amount". The compounds of the present invention can be administered to a subject before or after the onset of a viral infection. Further, multiple divided doses and staggered dosing can be administered daily or sequentially, or the dose can be by continuous infusion or can be a bolus. Further, the dosing of the compounds of the present invention can be increased or decreased proportionally depending on the urgency of the treatment or prevention situation.

[0126] The compounds of the present invention can be used to treat the states, conditions or diseases described herein, or for the manufacture of a pharmaceutical composition for treating these diseases. The present invention provides a method of using the compounds of the present invention to treat these diseases, or a method of preparing a pharmaceutical composition for treating these diseases having the compounds of the present invention.

[0127] The term "pharmaceutical composition" includes formulations suitable for administration to a mammal (e.g., a human). When the compounds of the present invention are administered as a medicament to a mammal (e.g., a human), they can be administered by themselves or as a pharmaceutical composition containing, as an active ingredient, for example, from 0.1% - 99.5% (more preferably 0.5% - 90%) of at least one compound of Formula (I) or any subgenus thereof in combination with a pharmaceutically acceptable carrier (or optionally two or more pharmaceutically acceptable carriers).

[0128] The phrase "pharmaceutically acceptable carrier" is well recognized in the art 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 involved in carrying or transporting the principal agent 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 injurious to the patient. Some examples of materials which can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients, such as cocoa butter or suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Generally, pharmaceutically acceptable carriers are sterile and / or substantially pyrogen-free.

[0129] Wetting agents, emulsifying agents, and lubricants (such as sodium lauryl sulfate and magnesium stearate) may also be present in the composition, as well as coloring agents, demolding agents, coating agents, sweetening agents, flavoring agents, and perfuming agents, preservatives, and antioxidants.

[0130] Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0131] The formulations of the present invention include formulations suitable for oral, nasal, inhalation, topical, transdermal, buccal, sublingual, rectal, vaginal, or parenteral administration. These formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the pharmaceutical art. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of the compound that produces a therapeutic effect. Generally speaking, on a 100% basis, this amount ranges from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0132] The methods for preparing these formulations or compositions include the following steps: associating the compounds of the present invention with a carrier and optionally one or more auxiliary ingredients. Generally, the formulations are prepared by uniformly and intimately associating the compounds of the present invention with a liquid carrier or a subdivided solid carrier or both carriers, and then (if necessary) shaping the product.

[0133] The formulations suitable for oral administration of the present invention can be capsules, oblong capsules, pills, tablets, lozenges (using a flavoring base, such as usually sucrose and gum arabic or tragacanth), powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as water-in-oil or oil-in-water liquid emulsions, or as elixirs or syrups, or as confectionery lozenges (using an inert base, such as gelatin and glycerin, or sucrose and gum arabic) and / or as mouthwashes, etc., each containing a predetermined amount of the compound of the present invention as an active ingredient. The compound of the present invention can also be used as boluses, troches or pastes.

[0134] In the solid dosage forms (capsules, tablets, pills, dragees, powders, granules, etc.) for oral administration of the present invention, the active ingredient is mixed with one or more pharmaceutically acceptable carriers (such as sodium citrate and dibasic calcium phosphate) and / or any of the following: fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; binders, such as, for example, carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / or gum arabic; humectants, such as glycerol; disintegrants, such as agar, calcium carbonate, potato and tapioca starch, alginic acid, certain silicates and sodium carbonate; solution blockers, such as paraffin wax; absorption promoters, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; absorbents, such as kaolin and bentonite clays; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical composition can also contain buffering agents. Solid compositions of a similar type can also be used as fillers for soft and hard gelatin capsules, which use excipients such as lactose or milk sugar and high molecular weight polyethylene glycols, etc.

[0135] Tablets can be made by compression and molding, optionally using one or more auxiliary ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or croscarmellose sodium), surfactants or dispersants. Molded tablets can be made by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0136] Tablets and other solid dosage forms (such as dragees, capsules, pills, and granules) of the pharmaceutical composition of the present invention can optionally be provided with or prepared with coatings or shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation field. They can also be formulated, for example, using different proportions of hydroxypropyl methylcellulose (to provide the desired release profile), other polymer matrices, liposomes, and / or microspheres to provide sustained or controlled release of the active ingredient therein. They can be sterilized by, for example, filtration through a bacteria-retaining filter or by incorporating a sterilizing agent immediately before use in the form of a sterile solid composition soluble in sterile water or some other sterile injectable medium. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in the form of microcapsules, optionally with one or more of the above excipients.

[0137] Liquid dosage forms for oral administration of the compounds of the present invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof.

[0138] In addition to the inert diluent, the oral compositions may also contain adjuvants, such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, coloring agents, perfuming agents, and preservatives.

[0139] In addition to the active compound, the suspensions may contain suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar, and tragacanth, and mixtures thereof. The pharmaceutical compositions of the present invention for rectal or vaginal administration can be presented as suppositories, which can be prepared by mixing one or more compounds of the present invention with one or more suitable non-irritating excipients or carriers (including, for example, cocoa butter, polyethylene glycol, suppository wax, or salicylates), and the suppositories are solid at room temperature but liquid at body temperature, and thus will melt in the rectal or vaginal cavity and release the active compound.

[0140] The formulations of the present invention suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing carriers known in the art.

[0141] The dosage forms for external or transdermal administration of the compounds of the present invention include powders, sprays, ointments, pastes, creams, liniments, gels, solutions, patches and inhalants. The active compounds can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers or propellants as may be required.

[0142] In addition to the active compounds of the present invention, ointments, pastes, creams and gels may also contain excipients such as animal and vegetable fats, oils, waxes, paraffin wax, starch, gum tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0143] In addition to the compounds of the present invention, powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0144] Transdermal patches have the additional advantage of providing the compounds of the present invention to the body in a controlled delivery manner. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. Penetration enhancers can also be used to increase the flux of the compound through the skin. The rate of such flux can be controlled by providing a rate controlling membrane or by dispersing the active compound in a polymer matrix or gel.

[0145] The pharmaceutical compositions of the present invention suitable for parenteral administration may comprise one or more compounds of the present invention in combination with one or more pharmaceutically acceptable carriers such as sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which can be reconstituted into sterile injectable solutions or dispersions before use, said sterile powders may contain antioxidants, buffers, bacteriostatic agents, solutes rendering the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0146] Examples of suitable aqueous or non-aqueous carriers that can be employed in the pharmaceutical compositions of the present invention include water, ethanol, ethylene glycol ethers, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). For example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants, appropriate fluidity can be maintained.

[0147] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents, etc. By including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc., the action of microorganisms can be ensured to be prevented. It may also be necessary to include in the composition isotonic agents such as sugars, sodium chloride, etc. Additionally, by including agents that delay absorption such as aluminum monostearate and gelatin, the absorption of injectable pharmaceutical forms can be prolonged.

[0148] In some cases, in order to prolong the drug action, it is desirable to slow down the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a poorly water-soluble crystalline or amorphous material. Then, the absorption rate of the drug depends on its dissolution rate, which in turn may depend on the crystal size and crystalline form. Alternatively, the delayed absorption of parenteral pharmaceutical forms is achieved by dissolving or suspending the drug in an oily solvent.

[0149] Injectable depot forms are made by forming a microcapsule matrix of the subject compound in a biodegradable polymer such as polylactide - polyglycolide. Depending on the ratio of the drug to the polymer and the nature of the specific polymer employed, the drug release rate can be controlled. Examples of other biodegradable polymers include polyorthoesters and polyanhydrides. Depot injection formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0150] The formulations of the present invention can be administered orally, parenterally, topically, or rectally. Of course, they are administered in forms suitable for each administration route. For example, they are administered in the form of tablets or capsules by injection, inhalation, eye drops, ointments, suppositories, etc., administered by injection, infusion, or inhalation; topically administered by lotions or ointments; and rectally administered by suppositories.

[0151] As used herein, the phrases "parenteral administration" and "administered parenterally" mean a mode of administration that is usually by injection other than enteral and topical administration, and includes but is not limited to intravenous, intramuscular, intra - arterial, intracavitary, intracapsular, intra - orbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra - articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. Intravenous infusion is sometimes the preferred delivery method for the compounds of the present invention. Infusion can be used to deliver single daily doses and multiple doses. In some embodiments, the compounds of the present invention are administered by infusion at intervals of 15 minutes to 4 hours, usually 0.5 hour to 3 hours. Such infusions can be used once a day, twice a day, or up to three times a day.

[0152] As used herein, the phrases "systemic administration", "administered systemically", "peripheral administration" and "administered peripherally" mean the administration of a compound, drug or other material indirectly into the central nervous system such that it enters the patient's system and is thereby subject to metabolism and other similar processes, such as subcutaneous administration.

[0153] These compounds can be administered to humans and other animals for treatment by any suitable route of administration, including oral, nasal (e.g., by spray), rectal, intravaginal, parenteral, intracisternal and topical (e.g., by powder, ointment or drops) administration, including buccal and sublingual administration.

[0154] Regardless of the route of administration selected, the compounds of the present invention (which can be used in a suitable hydrated form) and / or the pharmaceutical compositions of the present invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0155] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0156] The selected dosage level will depend upon various factors including the activity of the particular compound or its ester, salt or amide employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0157] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the required pharmaceutical composition. For example, the physician or veterinarian may start the dosage level of the compounds of the present invention employed in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0158] In general, a suitable daily dosage of the compounds of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such effective doses will generally depend upon the factors described above. In general, when used for the indicated effects, the intravenous and subcutaneous dosage ranges for the compounds of the present invention for a patient will be from about 0.0001 mg to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 mg to about 50 mg per kilogram per day, and still more preferably from about 0.1 mg to about 20 mg per kilogram per day. An effective amount is an amount that prevents or treats infection by a virus such as HBV.

[0159] Treatment with the compounds or compositions described herein can be repeated daily, either alone or in combination with other therapeutic agents, for a period sufficient to reduce the period of detectable HBsAg, whether used alone or in combination with other therapeutic agents.

[0160] The daily dose of the compound can be administered once, twice, or three times a day, to be determined.

[0161] The compounds of the present invention can be administered alone or in combination with other therapeutic agents (sequentially or simultaneously). Accordingly, methods of using the compounds of the present invention include administering the compound as a pharmaceutical composition, wherein, prior to administration, at least one compound of the present invention is mixed with a pharmaceutically acceptable carrier.

[0162] Combined use of the compounds of the present invention

[0163] The compounds and compositions described herein can be used or administered in combination with one or more therapeutic agents, including but not limited to: immunomodulators [(i.e., checkpoint inhibitors (small molecule inhibitors or blocking antibodies), natural or modified cytokines, TLR agonists, vaccines, etc.); and / or HBsAg targeting agents (i.e., siRNAs and antisense molecules targeting HBV transcripts, HBV capsid inhibitors, cccDNA inhibitors, HBx inhibitors, and antibodies targeting HBV and HDV proteins, etc.); and / or HBV replication inhibitors (nucleoside (nucleotide) analog polymerase inhibitors, non-nucleoside (nucleotide) analog polymerase inhibitors, etc.).

[0164] Generally, it is expected that the level of use of each therapeutic agent used in combination will not exceed its level of use alone. In some embodiments, the dose used in combination can be lower than the dose used alone.

[0165] Treatment with the compounds or compositions described herein can be repeated daily, either alone or in combination with other therapeutic agents, for a period sufficient to reduce the period of detectable HBsAg, whether used alone or in combination with other therapeutic agents.

[0166] The daily dose of the compound can be administered once, twice, or three times a day, to be determined.

[0167] The compounds of the present invention can be administered alone or in combination with other therapeutic agents (sequentially or simultaneously). Accordingly, methods of using the compounds of the present invention include administering the compound as a pharmaceutical composition, wherein, prior to administration, at least one compound of the present invention is mixed with a pharmaceutically acceptable carrier.

[0168] The compounds described herein can be synthesized by the following general synthetic routes, and specific examples of these general synthetic routes are described in more detail in the "Examples".

[0169] General synthetic procedure

[0170] All starting materials, structural units, reagents, acids, bases, dehydrating agents, solvents, and catalysts used in the synthesis of 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). The general synthesis methods of the compounds of the present invention are illustrated by the general methods in the following "Examples" and "Scheme 1", as well as the methods disclosed in the published PCT applications WO2018 / 198079 and US10,301,312B2 (Patent Date: May 28, 2019), the contents of which are incorporated herein by reference in their entirety.

[0171] Preparation of common intermediate 3:

[0172] Synthetic route:

[0173]

[0174] Preparation 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).

[0175]

[0176] The reaction was carried out in two identical batches. In each batch, at 0 °C, BBr 2 was added dropwise to a mixture 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) (45 g, 99.22 mmol) in DCM (1 L). 3 (99.43 g, 397 mmol, 38.24 mL, 4.0 eq). The mixture was warmed to 40 °C and stirred for 12 h. A solid precipitate appeared. TLC (petroleum ether:ethyl acetate = 0:1, R f = 0.1) showed that the reaction was complete. The two batches of reactants were combined for inspection. The mixture was diluted with dichloromethane (DCM, 1 L) and stirred for 5 min. The solid was collected by filtration and washed with DCM (3 x 500 mL). The filter cake was dried in vacuo. THF (450 mL), H 2 O (450 mL), and LiOH.H 2O (16.65 g, 416.2 mmol, 4.2 eq). The solution was stirred at 25 °C for 2 h. The residue was adjusted to pH = 4 with HCl (2 M). The residue was filtered to give the yellow solid (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%).

[0177] 1 H NMR (400 MHz, DMSO-d 6 ) 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).

[0178] 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).

[0179]

[0180] At 0 °C, SOCl 2 (101.00 g, 848.97 mmol, 61.6 mL, 5.0 eq) was added 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) (60 g, 169.79 mmol) in EtOH (600 mL). The mixture was stirred at 60 °C for 12 h. LCMS showed that the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:EtOH = 1:0 to 5:1) to give the yellow solid 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%).

[0181] LCMS: RT = 0.194 min, MS calculated: 381.2, MS result: [M+H] + = 382.3

[0182] 11H NMR (400 MHz, DMSO-d 6 ) 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).

[0183] Compound 3M was prepared by using methanol instead of ethanol with this method.

[0184] Compound I.1

[0185] Synthetic route:

[0186]

[0187] Preparation process 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)

[0188]

[0189] At 20 °C, Cs 2 CO 3 (4.48 g, 13.76 mmol, 3.5 eq.) was added in one portion 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 eq.) and tert-butyl 6-bromohexanoate (2.47 g, 9.83 mmol, 2.5 eq.) in DMF (15 mL). The mixture was stirred at 50 °C for 4 h. LCMS showed that the reaction was complete. The mixture was poured into water (20 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with brine (10 mL x 3), dried over Na 2 SO 4 filtered and concentrated in vacuo. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 100*30 mm*10 um; mobile phase: [water (NH 4 HCO 3)-ACN]; B%: 40%-70%, 8 min) purification to obtain a yellow solid 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, yield: 64.5%).

[0190] 1 H NMR (400 MHz, DMSO-d 6 ) 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).

[0191] Preparation process 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)

[0192]

[0193] At 20 °C, trifluoroacetic acid (TFA, 5 mL) was added in one portion to a mixture 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) (1.2 g, 2.18 mmol, 1 eq.) in DCM (10 mL). The mixture was stirred at 20 °C for 2 h. LCMS showed that the reaction was complete. The mixture was concentrated and adjusted to pH = 7 by saturated NaHCO 3 The aqueous phase was extracted with DCM (10 mL x 3). The combined organic phases were washed with brine (5 mL x 3), dried over Na 2 SO 4 dried, filtered and concentrated in vacuo. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 250*50 mm*10 um; mobile phase: [water (NH4 HCO 3 )-ACN]; B%: 10% - 40%, 10 min) for purification to obtain the yellow solid (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, yield: 56.02%). 1 H NMR (400 MHz, DMSO-d 6 ) 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).

[0194] Preparation process 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)

[0195]

[0196] To a mixture 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 (0.15 g, 303 umol, 1 eq.) in CH 3 CN (1.5 mL) and H 2 O (0.3 mL), add LiOH.H 2 O (57 mg, 1.36 mmol, 4.5 eq.). The mixture is stirred at 35 °C for 1 hour. LCMS shows that the reaction is complete. The mixture is adjusted to pH = 6 - 7 with 1N HCl and concentrated in vacuo. The residue is purified by preparative HPLC (column: Waters Xbridge C18 150*50 mm*10 um; mobile phase: [water (NH 4 HCO 3)-ACN]; B%: 1%-30%, 8 min) for purification to obtain a yellow solid, (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, yield: 54%).

[0197] 1 H NMR (400 MHz, DMSO-d 6 ) 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).

[0198] Compound I.2

[0199] Synthetic route:

[0200]

[0201] 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)

[0202]

[0203] At 25 °C, to 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 (3) (1.5 g, 3.93 mmol, 1 eq.) and tert-butyl 7-bromoheptanoate (1.56 g, 5.9 mmol, 1.5 eq.) in DMF (20 mL) was added Cs 2 CO 3 (4.48 g, 13.76 mmol, 3.5 eq.). The mixture was stirred at 50 °C for 12 h. LCMS showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was extracted with EtOAc (3 x 60 mL). The combined organic layers were washed with brine (3 x 30 mL) and dried over Na 2 SO 4Dry, filter and concentrate under reduced pressure. The residue was purified by column chromatography (SiO 2 , petroleum ether: ethyl acetate = 100:1 to 0:1) to give the yellow solid 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, yield: 71.92%).

[0204] Preparation 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)

[0205]

[0206] At 25 °C, to a solution 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) (1.5 g, 2.65 mmol, 1 eq.) in DCM (10 mL) and TFA (5 mL). The mixture was stirred at 25 °C for 1 h. LCMS showed that the reaction was complete. Adjust the pH to 7 with saturated NaHCO 3 The residue was extracted with DCM (3 x 15 mL). The combined organic layers were washed with brine (15 mL) and dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Welch Xtimate C18 250*70mm*10um; mobile phase: [water (NH 4 HCO 3 )-ACN]; B%: 10% - 40%, 20 min) to give the yellow solid (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, yield: 84%, purity: 98.76%).

[0207] 1 1H NMR (400 MHz, DMSO-d 6) 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).

[0208] Preparation process of (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)

[0209]

[0210] 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 eq) in CH 3 CN (4 mL) and H 2 O (2 mL) was added LiOH·H 2 O (123.52 mg, 2.94 mmol, 3 eq). The mixture was stirred at 20 °C for 12 h. LCMS showed that the reaction was complete. The mixture was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex C18 80*40 mm*3 μm; mobile phase: [water (NH 4 HCO 3 )-ACN]; B%: 1% - 30%, 8 min) to give a light yellow solid (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, yield: 29%, purity: 99.23%).

[0211] 1 H NMR (400 MHz, DMSO-d 6) 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).

[0212] Compound I.3

[0213] Synthetic route:

[0214]

[0215] Preparation process 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)

[0216]

[0217] 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 Cs 2 CO 3 (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 mixture was monitored by TLC (SiO 2 , ethyl acetate:methanol = 10:1). The reaction mixture was quenched by adding H 2 O (180 mL), and extracted with EtOAc (60 mL x 3). The combined organic layers were washed with brine (50 mL x 3), dried over Na 2 SO 4 , and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 1:0 to 0:1) to give the yellow solid 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, yield: 69%).

[0218] Preparation Process 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)

[0219]

[0220] To 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) in H 2 O (60 mL) and CH 3 CN (60 mL) was added LiOH.H 2 O (472.83 mg, 11.27 mmol), and the mixture was stirred at 20 °C for 2 h. The mixture was monitored by LCMS. The mixture was adjusted to pH = 4 with dilute HCl (1 N), and extracted with a solution of 5% EtOH in DCM (50 mL x 3). The combined organic layers were washed with brine (50 mL), dried over Na 2 SO 4 dried, filtered and concentrated under reduced pressure. The mixture was purified by preparative HPLC (column: Welch Xtimate C18 250*70 mm #10um; mobile phase: [water (NH 4 HCO 3 )-ACN]; B%: 33% - 63%, 20 min) to give the yellow solid (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, purity: 98%).

[0221] 1 H NMR (400 MHz, DMSO-d 6) 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).

[0222] MS calculated value: 510.3, result: [M + H] + = 510.3.

[0223] Compound I.4

[0224] Synthetic route:

[0225]

[0226] Preparation process 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)

[0227]

[0228] The reaction was carried out in two identical batches. In each batch, Cs was added 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) 2 CO 3 (665 mg, 2.04 mmol), and the mixture was stirred at 60 °C for 10 min. Then, heptyl 7-bromoheptanoate (313.6 mg, 1.02 mmol, 1.5 eq) was added to the mixture, and the mixture was stirred at 60 °C for 3 h. The mixture was monitored by LCMS. The two batches were combined for inspection. The reaction mixture was quenched by adding H 2 O (3 mL), and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2, purified with ethyl acetate:methanol = 10:1 to obtain yellow solid 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).

[0229] Preparation process 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)

[0230]

[0231] To 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 MeCN (3.5 mL) and H 2 O (3.5 mL) was added LiOH.H 2 O (25 mg, 0.589 mmol), and the reaction mixture was stirred at 25 °C for 2 h. The mixture was monitored by LCMS. The mixture was added 1N HCl to pH = 4 and extracted with a solution of 5% EtOH in DCM (5 mL x 3). The combined organic layers were washed with brine (10 mL), dried over Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 75*30 mm*3 um; mobile phase: [water (FA)-ACN]; B%: 50%-90%, 8 min). After lyophilization, the residue was repurified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40 mm*10 um; mobile phase: [water (NH 4 HCO 3 )-ACN]; B%: 40%-95%, 8 min) to obtain yellow solid (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%).

[0232] 11H NMR (400 MHz, DMSO-d 6 ) 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).

[0233] Mass spectrometry calculated value: 579.3, result: [M + H] + = 580.3.

[0234] Compound I.5

[0235] (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) Preparation process

[0236]

[0237] 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 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, R f = 0.2) showed that the reaction was complete. The mixture was poured into dilute HCl (0.1 M, 70 mL) and filtered. The filtrate was extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried over Na 2 SO 4 dried, filtered and concentrated under reduced pressure. The mixture was purified by preparative HPLC (Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water (NH 4 HCO3 )-ACN]; B%: 35% - 65%, 8 min) was purified to obtain a yellow solid (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, yield: 8%, purity: 99%).

[0238] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 8.96 (s, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.28 - 7.21 (m, 2H), 6.82 (d, J = 7.6 Hz, 1H), 5.23 - 5.07 (m, 2H), 4.97 (d, J = 4.8 Hz, 1H), 4.88 (td, J = 6.4, 12.4 Hz, 1H), 4.19 - 4.13 (m, 2H), 2.26 (t, J = 7.2 Hz, 2H), 1.86 - 1.77 (m, 2H), 1.60 - 1.43 (m, 4H), 1.41 - 1.32 (m, 2H), 1.17 (d, J = 6.4 Hz, 6H), 0.71 (s, 9H).

[0239] Mass spectrometry calculated value: 523.2, result: [M + H] + = 524.3.

[0240] Compound I.6

[0241] Synthetic route:

[0242]

[0243] Preparation process 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 (10)

[0244]

[0245] At 25 °C, Cs was added 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) (1 g, 2.62 mmol) in DMF (10 mL). 2 CO 3(2.56 g, 7.87 mmol) and ethyl 8-bromooctanoate (856.02 mg, 3.41 mmol). The mixture was stirred at 60 °C for 3 h. The mixture was monitored by LCMS. The mixture was quenched by adding H 2 O (30 mL), and extracted with EtOAc (15 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 1:0 to 0:1) to give the yellow solid 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, yield: 72%).

[0246] 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)

[0247]

[0248] To 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 (10) (1.20 g, 2.18 mmol) in CH 3 CN (10 mL) and H 2 O (10 mL) was added LiOH.H 2 O (274 mg, 6.53 mmol), and the mixture was stirred at 20 °C for 2 h. The mixture was monitored by LCMS. The mixture was adjusted to pH = 4 by adding HCl (1 N), and extracted with a 5% EtOH in DCM (10 mL x 3) solution. The combined organic layers were washed with brine (10 mL), dried over Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 250*50 mm*10 um; mobile phase: [water (NH 4 HCO 3)-ACN]; B%: 5% - 45%, 10 min) for purification to obtain a white solid (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, purity: 100%).

[0249] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 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).

[0250] Mass spectrometry calculated value: 496.2, result: [M + H] + = 496.3.

[0251] Compound I.7

[0252] Synthetic route:

[0253]

[0254] Preparation process 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)

[0255]

[0256] 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 Cs 2 CO 3 (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 mixture was monitored by TLC (SiO 2 , ethyl acetate:methanol = 10:1, R f= 0.5). The reaction mixture was quenched by adding H 2 O (150 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (50 mL x 3) and dried over Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 1:0 to 0:1) to give the yellow solid 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, yield: 65.6%).

[0257] 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)

[0258]

[0259] To 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) in H 2 O (50 mL) and MeCN (50 mL) was added LiOH.H 2 O (374.61 mg, 8.93 mmol). The reaction mixture was stirred at 20 °C for 2 h. The mixture was monitored by LCMS. The mixture was quenched by adding 1N HCl to pH = 4 and extracted with 5% EtOH in DCM (50 mL x 3). The combined organic layers were washed with brine (50 mL) and dried over Na 2 SO 4 filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 250*70mm*10um; mobile phase: [water (NH 4 HCO 3)-ACN]; B%: 30% - 75%, 18 min) for purification to obtain a yellow solid, (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, purity: 98%).

[0260] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 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).

[0261] MS calculated value: 523.3, result: [M + H] + = 524.3.

[0262] Compound I.8

[0263] Synthetic route:

[0264]

[0265] Preparation process of 8-bromooctyl propionate (13)

[0266]

[0267] To a solution of 8-bromooctanoic acid (12) (3.00 g, 13.45 mmol, 1 eq) in 1-pentanol (30 mL), add SOCl 2 (3.20 g, 26.89 mmol, 1.95 mL, 2 eq). The mixture was stirred at 80 °C for 2 h. TLC (petroleum ether:ethyl acetate = 5:1, R f = 0.5) indicated that the reaction was complete. After removing the solvent under reduced pressure, the residue was dissolved in EtOAc (50 mL) and water (50 mL). The organic layer was separated, dried over Na 2 SO 4 filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2, purified with petroleum ether:ethyl acetate = 50:1 to 20:1), to obtain colorless oily 8-bromooctyl propionate (3.37 g, 12.71 mmol, yield: 94%).

[0268] 1 H NMR (400 MHz, CDCl 3 ) 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).

[0269] Preparation of methyl (R)-6-(tert-butyl)-2-oxo-10-((8-oxooctyloxy)octyl)oxy)-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (14)

[0270]

[0271] 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 eq) in DMF (20 mL) was added Cs 2 CO 3 (7.76 g, 23.82 mmol, 3.5 eq) and 8-bromooctyl propionate (13) (1.98 g, 7.49 mmol, 1.1 eq). The mixture was stirred at 50 °C for 2 h. LCMS indicated that the reaction was complete. The reaction mixture was poured into water (30 mL), and extracted with EtOAc (50 mL x 2). The organic layer was washed with brine (30 mL x 2), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 2:1 to 0:1), to obtain white solid methyl (R)-6-(tert-butyl)-2-oxo-10-((8-oxooctyloxy)octyl)oxy)-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate (14) (2.5 g, 4.53 mmol, yield: 66%).

[0272] 11H NMR (400 MHz, CDCl 3 ) 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).

[0273] Preparation process 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)

[0274]

[0275] 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 eq) in CH 3 CN (12 mL) and H 2 O (12 mL) was added LiOH·H 2 O (247.22 mg, 5.89 mmol, 1.3 eq). The mixture was stirred at 20 °C for 1 h. LCMS indicated that the reaction was complete. The reaction mixture was poured into water (30 mL), and the pH was adjusted to 6 - 7 using 1 M HCl. The mixture was extracted with EtOAc (20 mL x 2). The organic layer was washed with brine (10 mL x 2), dried over Na 2 SO 4 and filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (column: Welch Xtimate C18 250*70 mm #10 um; mobile phase: [water (NH 4 HCO 3)-ACN]; B%: 40%-65%, 20 min) purification to obtain a white solid (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, yield: 51%).

[0276] 1 H NMR (400 MHz, DMSO-d 6 ) 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).

[0277] Compound I.9

[0278] Synthetic route:

[0279]

[0280] Preparation process of isopropyl 8-bromooctanoate (15)

[0281]

[0282] To a solution of 8-bromooctanoic acid (12) (3.5 g, 15.69 mmol) in i-PrOH (35 mL) was added SOCl 2 (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). At 25 °C, the reaction mixture was quenched by adding H 2 O (25 mL), and extracted with EtOAc (30 mL x 3). The combined organic extracts were washed with brine and dried over Na 2 SO 4 filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2, purified with petroleum ether:ethyl acetate = 1:0 to 5:1), to obtain colorless oily 8-bromoisopropyl octanoate (15) (5.7 g, 21.49 mmol, yield: 95.91%).

[0283] 1 H NMR (400 MHz, CDCl 3 ) 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),

[0284] Preparation process 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)

[0285]

[0286] 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) (3.52 g, 9.57 mmol) in DMF (35 mL) was added Cs 2 CO 3 (9.36 g, 28.72 mmol) and 8-bromoisopropyl octanoate (15) (3.3 g, 12.44 mmol), and the mixture was stirred at 60 °C for 12 h. The reaction was monitored by TLC (petroleum ether:THF = 10:1, Rf = 0.33). At 25 °C, the reaction mixture was quenched with H 2 O (30 mL), and extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (25 mL), dried over Na 2 SO 4 dried, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (THF:petroleum ether = 1:1 to 1:0) to obtain yellow solid 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) (3.56 g, 6.45 mmol, yield: 67%).

[0287] MS calculated value: 551.3, result: [M+H] + = 552.3

[0288] (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) preparation process

[0289]

[0290] 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 H 2 O (20 mL) was added LiOH.H 2 O (200.81 mg, 4.79 mmol), and the mixture was stirred at 25 °C for 1 h. The reaction was monitored by LCMS. The mixture was added 1N HCl to pH = 4 and extracted with 5% EtOH in DCM (3 x 50 mL). The combined organic layers were washed with brine (50 mL), dried over Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Agela DuraShell C18 250*70 mm*10 um; mobile phase: [water (NH 4 HCO 3 )-ACN]; B%: 40%-70%, 20 min) to give the yellow solid (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, yield: 94%).

[0291] 1 H NMR (400 MHz, DMSO-d 6)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).

[0292] Mass spectrometry calculated value: 537.3, result: [M + H] + = 538.3.

[0293] Compound I.10

[0294] Synthesis route:

[0295]

[0296] Preparation process of butyl 8 - bromooctanoate (17)

[0297]

[0298] At 60 °C, SOCl 2 (16.00 g, 134.46 mmol) was added to a mixture of 8 - bromooctanoic acid (12) (10 g, 44.82 mmol) in n - BuOH (100 mL) and stirred for 3 h. The mixture was monitored by TLC (SiO 2 , petroleum ether: ethyl acetate = 5:1, R f = 0.5). The mixture was concentrated under reduced pressure. The mixture was purified by column chromatography (SiO 2 , petroleum ether: ethyl acetate = 1:0 to 5:1) to obtain butyl 8 - bromooctanoate (17) (12 g, crude product) as a colorless oil.

[0299] Preparation process 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)

[0300]

[0301] At 20 °C, a mixture of 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) in DMF (35 mL) was added Cs 2 CO 3 (9.31 g, 28.58 mmol, 3 eq) and butyl 8-bromooctanoate (17) (3.46 g, 12.38 mmol, 1.3 eq). The mixture was stirred at 60 °C for 3 h. The mixture was monitored by LCMS. The mixture was quenched by adding H 2 O (100 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (50 mL x 3), dried over Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 1:0 to 0:1) to give the yellow solid 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, yield: 45.25%).

[0302] (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) Preparation Process

[0303]

[0304] A mixture 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) (2.5 g, 4.42 mmol) in CH 3 CN (20 mL) and H 2 O (20 mL) was added LiOH.H 2 O (185.45 mg, 4.42 mmol), and the reaction mixture was stirred at 20 °C for 1 h. The mixture was monitored by LCMS. The mixture was adjusted to pH = 4 with HCl (1 N) and extracted with 5% EtOH in DCM (30 mL x 3). The combined organic layers were washed with brine (30 mL), dried over Na 2 SO 4Dry under reduced pressure and concentrate to obtain a residue. The residue was purified by preparative HPLC (column: Welch Xtimate C18 250*70mm #10um; mobile phase: [water (NH 4 HCO 3 )-ACN]; B%: 42%-72%, 20 min) to obtain the yellow solid (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%).

[0305] 1 H NMR (400 MHz, DMSO-d 6 ) 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).

[0306] MS calculated value: 551.3, result: [M+H] + = 552.3.

[0307] Compound I.11

[0308] Synthetic route:

[0309]

[0310] Preparation process of pentyl 8-bromooctanoate (19)

[0311]

[0312] At 0 °C, SOCl was added dropwise to a mixture of 8-bromooctanoic acid (12) (3.00 g, 13.45 mmol, 1 eq.) in 1-pentanol (30 mL) 2 (3.20 g, 26.89 mmol, 1.95 mL, 2 eq.). The mixture was stirred at 100 °C for 1 hour. TLC (petroleum ether:ethyl acetate = 5:1, R f= 0.5) indicates that the reaction is complete. The mixture is concentrated in vacuo and poured into saturated NaHCO 3 (30 mL). The aqueous phase is extracted with ethyl acetate (30 mL x 3). The organic phase is washed with brine (20 mL x 2), dried over Na 2 SO 4 dried, filtered and concentrated in vacuo. The residue is purified by silica gel chromatography (SiO 2 , petroleum ether:ethyl acetate = 1:0 to 5:1) to give pentyl 8-bromooctanoate as a colorless oil (3.7 g, 12.62 mmol, yield: 94%).

[0313] 1 1H NMR (400 MHz, CDCl 3 ) 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).

[0314] 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)

[0315]

[0316] At 25 °C, cesium carbonate (7.76 g, 23.82 mmol, 3.5 eq.) is added in one portion to a mixture of pentyl 8-bromooctanoate (19) (2.39 g, 8.17 mmol, 1.2 eq.) 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 eq.) in DMF (30 mL). The mixture is stirred at 50 °C for 12 h. LCMS indicates that the reaction is complete. The combined mixture is poured into water (50 mL). The aqueous phase is extracted with ethyl acetate (30 mL x 3). The combined organic phases are washed with brine (40 mL x 2), dried over Na 2 CO 3 liver, filtered and concentrated under reduced pressure. The residue is purified by silica gel chromatography (SiO 2 SO 4 liver, filtered and concentrated under reduced pressure. The residue is purified by silica gel chromatography (SiO 2, purified with ethyl acetate:MeCN = 1:0 to 0:1), to obtain a yellow solid, 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, yield: 58%).

[0317] 1 H NMR (400 MHz, DMSO-d 6 ) 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).

[0318] 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)

[0319]

[0320] At 25 °C, to a mixture 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) (2.4 g, 4.14 mmol, 1 eq.) in CH 3 CN (14 mL) and H 2 O (14 mL) was added LiOH·H 2 O (261 mg, 6.21 mmol, 1.5 eq.) in one portion. The mixture was stirred at 25 °C for 1 hour. LCMS showed that the reaction was complete. The mixture was adjusted to pH = 6 - 7 with 1 M HCl. The mixture was extracted with ethyl acetate (30 mL x 3). The organic phase was washed with brine (10 mL x 2), and dried over Na 2 SO 4Dry, filter and concentrate in vacuo. The residue was purified by preparative HPLC (column: Agela DuraShell C18 250*70mm*10um; mobile phase: [water (NH 4 HCO 3 )-ACN]; B%: 45%-80%, 20 min) to give the yellow solid (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, yield: 57%).

[0321] 1 1H NMR (400 MHz, DMSO-d 6 ) 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).

[0322] Compound I.12

[0323] Synthetic route:

[0324]

[0325] Preparation process of butyl 2-ethyl-8-bromooctanoate (21)

[0326]

[0327] To a solution of 8-bromooctanoic acid (12) (3 g, 13.45 mmol, 1 eq) in 2-ethyl-1-butanol (20 mL) was added SOCl 2 (3.20 g, 26.89 mmol, 1.95 mL, 2 eq). The mixture was stirred at 100 °C for 2 h. TLC (petroleum ether:ethyl acetate = 5:1, R f = 0.80) indicated that the reaction was complete. The reaction mixture was diluted with H 2 O (30 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (30 mL) and dried over Na 2 SO4 Dry, filter and concentrate under reduced pressure. The residue was purified by column chromatography (SiO 2 , petroleum ether: ethyl acetate = 100:1 to 20:1) to give 2-ethyl-8-bromooctyl butyrate (21) as a colorless oil (4.1 g, 13.34 mmol, yield: 99%).

[0328] 1 H NMR (400 MHz, CDCl 3 ) 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).

[0329] 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)

[0330]

[0331] At 25 °C, to a solution of 2-ethyl-8-bromooctyl butyrate (21) (2.51 g, 8.17 mmol, 1.2 eq.) 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 eq.) in DMF (30 mL) was added Cs 2 CO 3 (7.76 g, 23.82 mmol, 3.5 eq.). The mixture was stirred at 60 °C for 12 h. LCMS showed that the reaction was complete. The reaction mixture was diluted with H 2 O (100 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (50 mL x 3), dried over Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2, purified with petroleum ether: ethyl acetate = 20:1 to 0:1) to obtain a yellow solid, 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, yield: 71.7%).

[0332] 1 H NMR (400 MHz, CDCl 3 ) δ 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).

[0333] Preparation process 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)

[0334]

[0335] At 25 °C, 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 eq.) in acetonitrile (15 mL) and H 2 O (15 mL) was added LiOH.H 2 O (266.45 mg, 6.35 mmol, 1.3 eq.). The mixture was stirred at 25 °C for 1 h. LCMS showed that the reaction was complete. The reaction mixture was treated with H 2Diluted with O (20 mL), adjusted to pH = 7 with dilute HCl (1 M), and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (30 mL) and dried over Na 2 SO 4 filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Agela DuraShell C18 250*70 mm*10 um; mobile phase: [water (NH 4 HCO 3 ) - ACN]; B%: 45% - 80%, 20 min) to obtain the yellow solid (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 mol, yield: 41%).

[0336] 1 1H NMR (400 MHz, DMSO-d 6 ) δ 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).

[0337] Compound I.13

[0338] Route:

[0339]

[0340] 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)

[0341]

[0342] To 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 eq) in DMF (10 mL) was added Cs 2 CO 3 (2.56 g, 7.86 mmol, 3 eq) and ethyl 9-bromononanoate (903.22 mg, 3.41 mmol, 1.3 eq), and the mixture was stirred at 60 °C for 4 h. The mixture was diluted with H 2 O (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with brine (100 mL × 2), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 1:0 to 0:1) to give the yellow solid 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%).

[0343] Mass calculated: 565.71, found: [M+H] + = 566.4.

[0344] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 8.53 (s, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 6.87 - 6.76 (m, 2H), 5.15 - 4.97 (m, 2H), 4.70 (d, J = 4.4 Hz, 1H), 4.24 (q, J = 7.2 Hz, 2H), 4.14 (dt, J = 2.8, 6.4 Hz, 2H), 4.04 (q, J = 7.2 Hz, 2H), 2.26 (t, J = 7.2 Hz, 2H), 1.80 (quin, J = 6.8 Hz, 2H), 1.58 - 1.41 (m, 4H), 1.39 - 1.24 (m, 9H), 1.16 (t, J = 7.2 Hz, 3H), 0.70 (s, 9H).

[0345] Preparation of (R)-6-(tert-butyl)-10-((8-carboxyloctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (Compound I.13)

[0346]

[0347] 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 eq) in a mixture of CH 3 CN (6 mL) and H 2 O (6 mL) was added LiOH.H 2 O (133.51 mg, 3.18 mmol, 3 eq), and the mixture was stirred at 25 °C for 2 h. At 0 °C, the pH of the reaction solution was adjusted to 7 with 1 M HCl solution. The reaction solution was purified by preparative HPLC (column: Waters XbridgeBEH C18 250*70 mm*10 um; mobile phase: [water (NH 4 HCO 3 )-ACN]; B%: 10%-40%, 20 min) to give a green solid, (R)-6-(tert-butyl)-10-((8-carboxyloctyl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid (Compound I.13) (400 mg, yield: 71%, purity: 96.35%).

[0348] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 8.94 (s, 1H), 7.62 (br d, J = 8.4 Hz, 1H), 7.33 - 7.15 (m, 2H), 6.82 (d, J = 7.6 Hz, 1H), 5.24 - 5.08 (m, 2H), 4.96 (br d, J = 4.4 Hz, 1H), 4.23 - 4.11 (m, 2H), 2.18 (t, J = 7.2 Hz, 2H), 1.88 - 1.76 (m, 2H), 1.57 - 1.42 (m, 4H), 1.40 - 1.23 (m, 6H), 0.71 (s, 9H).

[0349] MS calculated value: 509.6, result: [M+H] + = 510.0.

[0350] Compound I.14

[0351] (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) Preparation Process

[0352]

[0353] At 25 °C, 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 eq) and LiOH·H 2 O (36 mg, 870.08 μmol, 1 eq) in a mixture of MeCN (4.8 mL) and H 2 O (4.8 mL) was stirred for 1 h. At 0 °C, the pH of the reaction solution was adjusted to 7 with 1 M HCl solution. 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 (NH 4 HCO 3 )-ACN]; B%: 10% - 40%, 20 min) to obtain the green solid (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%).

[0354] 1 H NMR (400 MHz, DMSO-d 6 ) 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).

[0355] Mass spectrometry calculated value: 537.66, result: [M + H] + = 538.1.

[0356] Although compound (1) was prepared according to the methods disclosed in the published PCT application WO 2018 / 198079 and US10,301,312B2 (patent date: May 28, 2019), the contents of which are incorporated herein by reference in their entirety, for clarity, we include the following general procedure. Compound (25) was obtained by alkylation of commercially available (24) with potassium carbonate in acetonitrile. The nitro group of compound (25) was reduced using hydrogen over a palladium on carbon catalyst to give compound (26). Compound (26) was treated with isoamyl nitrite and acetic anhydride to give vermizole (27). Alkylation of (27) was achieved by treatment with (28) in the presence of lithium carbonate and heating in dioxane to give (29) as the major regioisomer (the regioisomer separated by chromatography). Compound (29) was formylated using N-formylmorpholine to give (30). Deprotection and concomitant cyclization were accomplished by treatment with trifluoroacetic acid to give compound (31). Compound (31) was concentrated with compound (32) to give the tetracycle (33) in quantitative yield. Desaturation of compound (33) was achieved by treatment with DDQ to give compound (1).

[0357] Synthesis of Intermediate 1.

[0358] General Scheme:

[0359]

[0360] Preparation of 1-(3-methoxypropoxy)-3-methyl-2-nitrobenzene (25)

[0361]

[0362] Charge acetonitrile (5v) into a 100 L reactor, then add 24 (10.0 kg, 65.3 mol, 1 eq) at 25 °C. At 22 °C, add K 2 CO 3 (10.8 kg, 78.4 mol, 1.2 eq) in one portion, and the color changes from yellow to red. At 25 °C, add 1-bromo-3-methoxypropane (11.0 kg, 71.8 mol, 1.1 eq) dropwise over 5 min while maintaining the reaction temperature. Heat the resulting mixture to 70 °C and stir under nitrogen for 16 h. Then cool the mixture to 25 °C, filter and wash the solid with methyl tert-butyl ether (MTBE, 2v). Concentrate the filtrate in vacuo to give a crude oil. Dissolve the oil in MTBE (2v), then wash with 2N NaOH solution (0.3 eq), and extract the aqueous phase with MTBE (1v x 2). Combine the organic phases, wash with brine (1V), and dry over anhydrous Na 2 SO 4Dry, filter and concentrate in vacuo to obtain a green oil 25 (13.9 kg, yield: 94.5%).

[0363] Preparation of 2-(3-methoxypropoxy)-6-methylaniline (26)

[0364]

[0365] Charge ethanol (10 v) into a 200 L reactor, then add 25 (13.9 kg, 61.71 mol, 1 eq) at 25 °C. At 25 °C, add palladium on carbon (278 g, 2% wt) in one portion. Degas the suspension in vacuo and purge with H 2 three times. Heat the resulting mixture to 50 °C and stir in hydrogen for 48 h. Cool the mixture to 25 °C, filter and wash the solid with ethanol (2 v). Concentrate the filtrate in vacuo to obtain a brown oil 26 (11.5 kg, yield: 93.8%).

[0366] Preparation of 7-(3-methoxypropoxy)-1H-indazole (27)

[0367]

[0368] Charge toluene (10 v) into a 250 L reactor, then add 26 (9.0 kg, 46.09 mol, 1 eq) at 25 °C. At 25 °C, add potassium acetate (5.4 kg, 55.31 mol, 1.2 eq) in one portion. At 25 °C, add acetic anhydride (14.1 kg, 138.28 mol, 12.95 L, 3 eq) dropwise. Heat the mixture to 50 °C and stir in N 2 for 1 h. At 50 °C, add tert-butyl nitrite (11.9 kg, 115.23 mol, 13.7 L, 2.5 eq) dropwise. Heat the resulting mixture to between 60 °C and 65 °C and stir under nitrogen for 16 h. Cool the mixture to 25 °C and quench with water (3 V). Extract the product with ethyl acetate (2 V x 2). Combine the organic phases, wash with brine (2 V), and dry with anhydrous Na 2 SO 4 dry, filter and concentrate in vacuo at 40 °C to obtain the crude product. Dissolve the crude product in MeOH (4 V), then add 3N HCl solution (3 V) dropwise while maintaining the reaction temperature at no higher than 35 °C, then stir at 45 °C. At 40 °C, concentrate the mixture in vacuo to remove MeOH, and extract the aqueous phase with ethyl acetate (2 v x 3). Combine the organic phases, wash with brine (2 V), and dry with anhydrous Na 2 SO 4Dry, filter and concentrate in vacuo at 40 °C to obtain 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 obtain crude product 27 as a yellow oil. The oil was dissolved in MTBE (1.5 v) and stirred for 1 h, and a yellow solid precipitated. The mixture was filtered, and the filter cake was washed twice with n-heptane / MTBE (1.6 V:0.4 V) and dried in vacuo to obtain 27 (7.0 kg, yield: 73%).

[0369] Preparation of tert-butyl (R)-(1-(7-(3-methoxypropoxy)-2H-indazol-2-yl)-3,3-dimethylbutan-2-yl)carbamate (29)

[0370]

[0371] Charge dioxane (10 v) into a 50 L reactor, then add 27 (2.8 kg, 13.58 mol, 1 eq) and 28 (5.7 kg, 20.36 mol, 1.5 eq) at 25 °C. At 25 °C, lithium carbonate (2.0 kg, 27.15 mol, 2 eq) was added in one portion. The resulting mixture was heated to 100 °C and stirred in N 2 for 90 h. The reaction mixture was filtered and the filter cake was washed with ethyl acetate (0.5 v x 2). The filtrate was concentrated under reduced pressure to remove most of the dioxane to obtain the crude product. Then, at 25 °C, the crude product was triturated with NaOH (1 M, 4 v) for 16 h. The reaction mixture was filtered to obtain the crude product (∼5.5 kg, crude). At 25 °C, the crude product was triturated with NaOH (1 M, 4 v) for 16 h. The reaction mixture was filtered to obtain the crude product (∼5.1 kg, crude). At 25 °C, the crude product was triturated with MTBE / n-heptane (2 v:1 v) for 1 h. The reaction mixture was filtered to obtain 29 (3.8 kg, 69.0%).

[0372] Preparation of tert-butyl (R)-(1-(3-formyl-7-(3-methoxypropoxy)-2H-indazol-2-yl)-3,3-dimethylbutan-2-yl)carbamate (30)

[0373]

[0374] The following process is carried out in nine parallel batches. In each batch, tetrahydrofuran (10 v) is charged into a 3 L glass flask, and then 29 (540 g, 1.33 mol, 1 eq) is added at 25 °C. The mixture is degassed and purged with nitrogen three times. The mixture is cooled to -60 °C using an ethanol and dry ice bath. n-BuLi (2.5 M, 1.86 L, 3.5 eq) is added dropwise at -60 °C in nitrogen over 1.5 h. The mixture is stirred at -60 °C in nitrogen for 0.5 h. N-Formylmorpholine (460 g, 3.99 mol, 400 mL, 3 eq) is added dropwise at -60 °C in nitrogen over 1 h. The mixture is stirred at -60 °C in nitrogen for 2 h. Saturated NH 4 Cl (2 v) is slowly added dropwise to the reaction mixture at -60 °C in nitrogen. The reaction mixture is warmed to 25 °C. At this stage, all nine batches are combined. The product is extracted with ethyl acetate (2 v x 3). The combined organic phases are washed with brine (2 V x 2), dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo at 50 °C to obtain the crude product. At 25 °C, the crude product is triturated with heptane (4 v) for 1 h. The mixture is filtered and the filter cake is washed twice with heptane (2 v) and dried in vacuo to obtain 30 (4.3 kg, 82.8%).

[0375] Preparation of (R)-3-(tert-butyl)-7-(3-methoxypropoxy)-3,4-dihydropyrazino[1,2-b]indazole (31).

[0376]

[0377] Dichloromethane (10 v) is charged into a 50 L glass flask, and then 30 (3.5 kg, 8.07 mol, 1 eq) is added at 20 °C. Trifluoroacetic acid (TFA, 2 v, 7 L) is added dropwise at 20 °C. The mixture is stirred at 20 °C for 2 h. The reaction mixture is concentrated in vacuo at 50 °C to remove dichloromethane and most of the TFA. The reaction mixture is diluted with dichloromethane (10 v). Saturated NaHCO 3 (~10 v) is slowly added to the reaction mixture, and the pH is adjusted to 7 - 8. The product is extracted with dichloromethane (2 v x 2). The combined organic phases are concentrated in vacuo at 50 °C to obtain 31 (2.5 kg, 98.2%).

[0378] 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)

[0379]

[0380] Ethanol (10 v) and water (1 v) were charged into a 50 L reactor. At 25 °C, 31 (2.7 kg, 8.56 mol, 1 eq) was added within 30 min. In N 2 , compound 32 (3.2 kg, 17.12 mol, 2 eq) was added at 25 °C. After the addition, the reaction mixture was heated to 50 °C. In N 2 , the resulting mixture was stirred at 50 °C for 16 h. The reaction mixture was concentrated under reduced pressure to remove EtOH and H 2 O, and crude 33 (4.3 kg) was obtained.

[0381] 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)

[0382]

[0383] Dimethoxyethane (DME, 10 V) was charged into a 50 L reactor. At 25 °C, 33 (4.3 kg, 9.44 mol, 1 eq) was added within 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. The mixture was stirred at 25 °C for 2 h. The mixture was concentrated to obtain a residue. Then the residue was dissolved in ethyl acetate (10 V). The solution was neutralized with saturated Na 2 CO 3 (0.5 V), the alkalized reaction mixture was diluted with water (0.6 V), and the organic layer was separated. The aqueous phase was extracted with ethyl acetate (3 V x 2). The combined organic phases were washed with water (0.6 V) and brine (0.6 V) and concentrated to obtain the crude product. The crude product was dissolved in ethyl acetate (0.3 V), and then added dropwise at 25 °C to a 2N HCl solution in ethyl acetate (9.4 L, 18.88 mol, 2 eq), and stirred for 12 h. The mixture was filtered to obtain a yellow solid. Residual water was removed to obtain yellow solid 1 (2.4 kg, yield: 56.0%, purity: 95.69%, obtained by HPLC).

[0384] 1 H NMR (400 MHz, DMSO-d 6 )

[0385] d 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).

[0386] Preparation of tert-butyl (4R)-4-(tert-butyl)-1,2,3-oxathiazolidine-3-carboxylate 2-oxide (35)

[0387]

[0388] Charge tetrahydrofuran (1.5 v) into a 50 L glass flask, and then add pyridine (7.6 kg, 96.64 mol, 7.8 L, 7 eq) at 25 °C. Cool the reaction mixture to 0 °C. At 0 to 10 °C, add thionyl chloride (4.9 kg, 41.42 mol, 3.0 L, 3 eq) dropwise over 1 hour. At 0 to 10 °C, add a solution of 34 (3.0 kg, 13.81 mol, 1 eq) in THF (3 v) dropwise over 2 hours. The resulting mixture is stirred at 25 °C for 16 h in nitrogen. In an ice bath, cool the reaction mixture to 0 °C, and slowly add the reaction mixture to ice water. The reaction mixture is extracted with EtOAc (2 v x 2). Separate the organic phase and wash it twice with brine (1 v x 2). The organic phase is dried over Na 2 SO 4 dry, filter and concentrate under reduced pressure to obtain a residue (3.6 kg, crude).

[0389] Preparation of tert-butyl (R)-4-(tert-butyl)-1,2,3-oxathiazolidine-3-carboxylate 2,2-dioxide (28)

[0390]

[0391] The reaction is carried out in four batches. In each batch, charge acetonitrile (5 v) and water (5 v) into a 50 L reactor, and then add 35 (2.5 kg, 9.49 mol, 1 eq) at 25 °C over 20 min. Cool the reaction mixture to 0 °C over 30 min. Under nitrogen, add RuCl 3 (19.7 g, 94.93 mmol, 6.33 mL, 0.01 eq) at 0 °C. At 0 - 10 °C, add NaIO 4(3.05 kg, 14.24 mol, 789 mL, 1.5 eq) (At 20 °C, the solubility of NaIO 4 is 80 g / L (for 4 hours). Under nitrogen, the resulting mixture was stirred at 25 °C for 1.5 h. At this stage, four batches were combined. The mixture was diluted with methyl tert-butyl ether (MTBE, 1.7 v). The reaction mixture was filtered through a diatomaceous earth pad. The diatomaceous earth pad was washed with MTBE (1 v x 3). The combined filtrates were extracted with MTBE (1.3 v x 3). The combined organic phases were washed with saturated Na 2 SO 3 solution (1.7 v x 2). The organic phase was separated and washed with brine (1.3 v x 2). The organic phase was dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give the crude product. At 20 °C, the crude product was triturated with n-heptane / ethyl acetate (2 v / 0.4 v) for 30 min. The reaction mixture was filtered and the filter cake was washed with n-heptane (0.33 v). Dried in vacuo to give 28 (7.9 kg, 75%).

[0392] HBsAg determination

[0393] HepG2.2.15 cells were grown in flasks under a microscope and seeded into 96-well plates at a concentration of 6.0 x 10 4 cells / well and incubated overnight at 37 °C, 5% CO 2 .

[0394] Stock concentrations of reference compounds and test compounds for source plate preparation were 20 mM. First, the compounds were manually diluted to obtain the first concentration points of 10 μM and 200 μM respectively. Then, three-fold 8-point serial dilutions were performed to obtain a 200× source plate.

[0395] The following pipetting was performed on the source plate compounds: 3 μL of 200× source plate compounds were pipetted into a 2 mL sterile 96-well plate containing 297 μL of 2% FBS medium, mixed well and transferred to the corresponding cell culture plate at 100 μL / well. Therefore, the final test conditions for the compounds were an initial concentration of 1 μM, three-fold serial dilution and 8 concentration points, in duplicate.

[0396] The cells were incubated at 37 °C, 5% CO2 for three days.

[0397] Twenty-four hours after seeding, the cells were treated with medium at 200 μl / well containing compounds serially diluted in DMSO. DMSO alone was used as the drug-free control. The final DMSO concentration in all wells was 0.5%.

[0398] The secreted HBsAg levels were determined using an HBsAg kit (Auto Biology - CL 0310). The HBSAg assay was performed as follows:

[0399] a) Equilibrate the ELISA kit at room temperature for 30 minutes.

[0400] b) Add 50 μL of the standard solution, samples, positive control, and negative control to each well of the microplate in the kit.

[0401] c) Add 50 μL of the HBsAg enzyme conjugate to each well.

[0402] d) Install the plate membrane, shake for 60 seconds, and then incubate at 37 °C for 60 minutes.

[0403] e) Add 350 μL of the wash buffer to each well, shake gently, discard the liquid, and repeat 6 times. Then, tap 5 times on absorbent paper to dry the wells.

[0404] f) Add 50 μL of the mixture of Reagents A and B to each well. Shake the microplate for 10 seconds.

[0405] g) Cover the plate membrane and incubate at room temperature for 10 minutes. Read the luminescence signal using a Biotek - Synergy 2.

[0406] Data Analysis

[0407] The inhibition of the compound on HBsAg at different concentrations was calculated by the following formula:

[0408] Inhibition % = (1 - sample value / control average) × 100.

[0409] The 50% effective concentration (EC 50 ) was calculated using GraphPad Prism software.

[0410] Table 3. Activities of selected compounds of formula (I) in the HepG2 HBV S antigen secretion bioassay (geometric mean ± standard deviation, if applicable):

[0411] Compound <![CDATA[EC 50 , nM]]> Number of repetitions, n I.1 25.5 1 I.2 6.5±2.5 3 I.3 11.8 1 I.4 14.0 1 I.6 2.6±0.6 3 I.7 7.1 1 I.9 5.3±0.9 3 I.10 4.0±0.8 3 I.13 1.2±0.5 3

[0412] Uptake Transporters

[0413] The OATP1B1 and OATP1B3 substrate assays were performed in Wuxi using the human embryonic kidney cell line HEK293 stably transfected with the human transporter gene. Potential transporter substrates were identified by measuring the uptake fold values in the transfected HEK293 - OATP1B1 and OATP1B3 cell lines and the HEK293 - MOCK cell line in the presence and absence of a positive inhibitor.

[0414] The purpose of this study was to determine whether Compound I.2 and Compound I.6 are potential uptake substrates of OATP1B1 and OATP1B3.

[0415] Details of the substrates, positive inhibitors, and internal standards are shown in the following table.

[0416]

[0417] HEK-293 cells stably expressing human OATP1B1 and OATP1B3 transporters and the HEK293-MOCK cell line were obtained under license from GenoMembrane (Kanagawa, Japan). The 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. The cells were incubated at 5.0% CO 2 ₂, 37.0 °C, and saturated humidity. HEK293-OATP1B1 (passage 19), OATP1B3 (passage 19), and HEK293-MOCK (passages 14 and 16) were grown to 80%-90% confluence in culture flasks. Trypsin / EDTA (0.05% / 0.02%, w / v) was added to detach the cells from the culture flasks. The cells were seeded onto 96-well plates at a density of 5.00×10 4 ⁵ cells / well and then incubated at 5.0% CO 2 ₂, 37.0 °C, and saturated humidity for 24 hours before being used for uptake studies.

[0418] Pre-incubation: The cell medium was removed from the 96-well plates seeded with HEK293-OATP1B1 and OATP1B3 or HEK293-MOCK cells, and then the cells were rinsed twice with warm (37.0 °C) transport buffer. Then, the cells were pre-incubated with transport buffer for 30 minutes at 5.0% CO 2 ₂, 37.0 °C, and saturated humidity in the presence and absence of a positive inhibitor.

[0419] Uptake incubation: At the end of the pre-incubation, the buffer was removed, and the cells were treated with 0.100 μM or 1.00 μM of Compound I.6 or Compound I.2 in the presence and absence of a positive inhibitor. In separate wells, the radiolabeled substrates of the transporter cells were incubated with the dosing solution (Table A), and all treatments were performed in triplicate in the wells at 5.0% CO 2 ₂, 37.0 °C, and saturated humidity.

[0420] Table A Inhibitor and Substrate Information for Individual Transporters.

[0421]

[0422] Cell Lysis: At the end of incubation, the dosing solution was removed. After removing the remaining dosing solution, the cells were rinsed three times with ice-cold transport buffer (2.0 - 8.0 °C), and then 100 μL of cold acetonitrile:methanol (95:5, v:v) containing an internal standard was added to the cells treated with the test compound for bioanalysis. For cells with a positive control added, 100 μL of cold acetonitrile:methanol (95:5, v:v) containing an internal standard was added to the cells for bioanalysis. All samples were gently shaken for 30 min. Then, for the test compound and the positive control, 75 μL of cell lysate was mixed with 75 μL of transport buffer and 150 μL of cold acetonitrile:methanol (95:5, v:v) containing an internal standard. The lysed samples were centrifuged at 3220×g for 10 minutes, and then the supernatant, defined as the final sample, was taken to determine the intracellular uptake of the test compound or substrate by LC-MS / MS.

[0423] The uptake fold was calculated using Equation (1).

[0424]

[0425] Table 4. Uptake Rates of Selected Formula (I) Compounds in the Uptake Transporter Assay:

[0426]

[0427] In Vivo Pharmacokinetic Studies

[0428] According to the IACUC guidelines, the compounds of the present invention were tested by orally administering a formulation of 25% PEG 400:10% solutol:65% water (5 mL per kg of solution) to CD-1 mice, C57BL / J6 mice, or Sprague Dawley rats across species. The rat PK of Compound I.9 and Compound I.10 (PO dose of 32 mpk) illustrate the method of use across species.

[0429] The purpose of this rat PK study was to determine the pharmacokinetic properties of Compound I.9, Compound I.6, and Compound I.10 in plasma after oral gavage administration of Compound I.9 and Compound I.10 to female Sprague-Dawley rats. For the PO group, liver and plasma concentrations were measured at 1, 2, 6, and 10 hours after dosing. The concentrations of Compound I.9, Compound I.6, and Compound I.10 in plasma and tissue samples were determined by liquid chromatography tandem mass spectrometry (LC-MS / MS). Oral (PO) dose: Compound I.9 in Groups 1 and 2 was prepared as a clear solution in 25% PEG400 + 10% Solutol + 65% water (final pH adjusted to 7 - 8). Compound I.10 in Groups 3 and 4 was prepared as a homogeneous suspension in 25% PEG400 + 10% Solutol + 65% water (final pH adjusted to 7 - 8). At each time point, blood (approx. 0.2 mL) was collected from each study animal via jugular vein puncture into Tube A. The following 4-component mixed stabilizer for esterase inhibition was added in advance to a pre-cooled commercial Tube B containing potassium ethylenediaminetetraacetate (K2 EDTA) (0.85 - 1.15 mg). Then, the blood in Tube A was precisely transferred (4-component mixed stabilizer: blood, 1:10 (v:v)) to Tube B and placed on wet ice until centrifugation.

[0430]

[0431] Tissue Processing

[0432] The following 4-component mixed stabilizer was added in advance to cold homogenization buffer (MeOH / 15 mM PBS: 1:2). Then, each tissue was weighed and cold homogenization buffer containing the stabilizer was added at a ratio of 1:9 (1 g of tissue to 9 mL of buffer), and then homogenized on wet ice. The tissue homogenate was stored at -60 °C or lower until LC-MS / MS analysis.

[0433]

[0434] Bioanalysis: The concentrations of Compound I.9, Compound I.6, and Compound I.10 in plasma and tissue were determined using the LCMSMS / MS method. Instruments and conditions for Compound I.9, Compound I.10, and Compound I.6 in plasma and tissue homogenates.

[0435] LC Parameters

[0436] Equipment System: ACQUITY UPLC System

[0437] Analytical Column: ACQUITY UPLC HSS T3 1.8μm 2.1×50mm

[0438] Sample volume: 5 μL for plasma; 3.5 μL for tissue homogenate

[0439] Mobile phase A: 0.1% FA & 2 mmol / L HCOONH 4 in water / CAN (v / v, 95:5)

[0440] Mobile phase B: 0.1% FA & 2 mmol / L HCOONH 4 in ACN / water (v / v, 95:5)

[0441] Elution mode: gradient

[0442] Compound I.9, Compound I.10 and Compound I.6 in plasma and tissue homogenate

[0443]

[0444] Mass spectrometer: Triple Quad 6500plus

[0445] Ionization mode: ESI(+)

[0446] Detection mode: MRM

[0447] After single PO 1 and PO 2 administrations of Compound I.9 at 32 mg / kg in male SD rats, the PK parameters of Compound I.9 in plasma and tissues were not determined because most concentrations were below the lower limit of quantification. After single PO 3 and PO 4 administrations of Compound I.10 at 32 mg / kg in male SD rats, the PK parameters of Compound I.10 in plasma and tissues were not determined because most concentrations were below the lower limit of quantification.

[0448] Table 5. Compounds of Formula I can preferentially deliver HBV S antigen inhibitors to the liver rather than plasma (SD rats, n = 3 animals):

[0449]

[0450] Table 6. Compounds of Formula I can preferentially deliver HBV S antigen inhibitors to the liver rather than plasma (mice, n = 3, ND = not determined, if the observed metabolite plasma levels for one or more animals are BQL, then below the quantification level):

[0451]

[0452] *Based on the detection limit of 1 ng / mL in plasma, Compounds I.3, I.5, I.7 and I.14 have good liver / plasma ratios of >186, >343, >193 and >533, respectively.

[0453] In vivo efficacy

[0454] The purpose of this study was to investigate the in vivo pharmacological efficacy of Compound I.9 and Compound I.10 in a mouse model transfected with adeno-associated virus hepatitis B virus (AAV-HBV). On day 0 before dosing, each mouse was injected via the tail vein with 1×10 11 recombinant AAV-HBV vector genomes in 200 μL of phosphate-buffered saline. On days 21 and 28 before dosing (21 and 28 days after injection of AAV-HBV), the mice were bled and 10 μL of serum was prepared from each mouse. The serum samples were stored at -70 °C and transferred to the clinical pathology department for quantitative detection of HBsAg (as the baseline). Based on the body weight and serum levels on day 35 before dosing, 48 mice were selected and randomly divided into seven groups of 6 mice each.

[0455] From day 0 to 13, the vehicle was administered twice daily at 12-hour intervals. I.10 or I.9 was administered twice daily at doses of 16 mg / kg / dose, 8 mg / kg / dose, and 4 mg / kg / dose, or once daily at a dose of 16 mg / kg / dose. In the case of the twice-daily regimen, dosing was performed at 12-hour intervals from day 0 to 13, and once on day 14. All test articles were administered by oral gavage at 5 mL / kg / dose. The clinical signs of the animals were monitored once daily, and body weights were measured twice weekly from day 0 to 14. On days 0, 3, 7, 10, and 14, the mice were bled, and 10 μL of serum was prepared from each mouse. The serum samples were stored at -70 °C and transferred to the Clinical Pathology Department for viral marker detection. HBsAg was detected on days 0, 3, 7, 10, and 14. For the groups administered compound I.10 or compound I.9, after the first dose on day 0, the first 3 mice in each group were bled at 0.5 h and 4 h, and the last 3 mice in each group were bled at 1 h and 8 h. 7 μL of plasma samples were prepared from each mouse at each time point, stored at -70 °C, and transferred to the Metabolism Department for bioanalysis (data reports were sent separately by the Labcorp Metabolism Department to the sponsor). On day 14, the vehicle control group mice were sacrificed without collecting blood and tissues. After dosing on day 14, the first 3 mice and the last 3 mice in each group treated with I.10 and I.9 were sacrificed at 2 h and 6 h, respectively. For these mice, after local perfusion with saline, the liver was obtained. The liver was removed from the abdominal cavity, rinsed with saline, and placed on a soft absorbent paper to drain all remaining fluid. The liver was weighed, cut into small pieces, placed in a test tube, and quickly frozen in liquid nitrogen. Under ice-cold conditions, the liver pieces were homogenized at a ratio of 1:9 with a methanol solution (MeOH:15 mM PBS = 1:2). The homogenate was stored at -70 °C and transferred to the Metabolism Department for bioanalysis (data reports were sent separately by the Labcorp Metabolism Department to the sponsor). Serum hepatitis B surface antigen (HBsAg) was measured using ARCHITECT i2000 (Abbott Laboratories, Lake Bluff, Illinois, USA) and ancillary reagents.

[0456] Compared with the vehicle control group, the serum HBsAg levels in the groups treated with compound I.9 and compound I.10 decreased by 0.42 - 0.69 Log 10 units after two weeks of treatment, and the maximum efficacy was observed after administration of 64 MPK BID, 32 MPK BID, and 16 MPK BID (plotted). Based on the initial maximum efficacy at the high dose, the results after administration of the compound at 16 MPK BID, 16 MPK QD, 8 MPK BID, and 4 MPK BID 16 were as Figure 1 shown.

[0457] Glossary of Abbreviations and Terms

[0458]

Claims

1. A compound of formula 1: wherein, R is hydrogen, ethyl or a straight-chain, cyclic or branched C 3 -C 8 alkyl group, and X is a straight-chain, cyclic or branched C 5 -C 10 alkylene group; or a pharmaceutically acceptable salt thereof.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein, X is a straight-chain C 5 -C 8 alkylene group.

3. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein, X is selected from linear - C 5 H 10 -, linear - C 6 H 12 - and linear - C 7 H 14 -.

4. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein, R is selected from n-C 2 H 5 、n-C 3 H 7 、i-C 3 H 7 、n-C 4 H 9 、n-C 5 H 11 、n-C 6 H 13 、2-ethylbutyl, n-C 7 H 15 、and n-C 8 H 17 。 5. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein, R is H.

6. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein, R is i-C 3 H 7 。 7. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein, R is n-C 4 H 9 。 8. The compound or a pharmaceutically acceptable salt thereof according to claim 1, selected from: and pharmaceutically acceptable salts of each of the foregoing compounds.

9. A compound having the following structure:

10. A compound having the following structure:

11. A compound having the following structure:

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

13. A method for treating human hepatitis B in a human subject, which comprises: administering to the subject the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, or the pharmaceutical composition according to claim 12.

14. The method according to claim 13, which further comprises: administering to the subject an additional therapeutic agent selected from HBV replication inhibitors, HBsAg targeting agents, and immunomodulators.

15. A method for treating human hepatitis D in a human subject, which comprises: administering to the subject the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, or the pharmaceutical composition according to claim 12.

16. The method according to claim 15, which further comprises: administering to the subject an additional therapeutic agent selected from DNA polymerase inhibitors, HBV capsid inhibitors, HBV targeting antibodies, and HBV targeting therapeutic vaccines.

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

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

19. The compound according to any one of claims 1 to 11 for use in therapy.

20. The compound according to claim 19, wherein, the therapy is for treating a bacterial infection.

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

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