Polycyclic cap-dependent endonuclease inhibitors for treating or preventing influenza

Abstract

The present invention provides compounds of formula (I), pharmaceutical compositions containing one or more of the compounds, and methods of using the compounds to treat or prevent influenza. The compounds are inhibitors of cap-dependent endonucleases. [Formula 1] TIFF2023535771000108.tif28146

Description

Background Art

[0001] Influenza virus, a member of the Orthomyxoviridae family, is classified into the following types: influenza A, influenza B, influenza C, or influenza D. Seasonal epidemic diseases caused by influenza A and influenza B are widespread globally, and these diseases are the most significant concerns for human public health. Influenza A virus is characterized by the combination of hemagglutinin (HA, H) and neuraminidase (NA, N), which are surface proteins present on the virion. Both H1N1 virus and H3N2 virus can infect humans and cause diseases. Influenza B virus is classified into either of two lineages, Victoria-like or Yamagata-like, both of which cause human diseases.

[0002] Virus particles of influenza A and influenza B are composed of a cell-derived lipid membrane lined with the viral M1 matrix protein. This envelope contains eight segments of the negative-strand RNA genome, each encoding one or more viral proteins. The hemagglutinin protein, M2 protein, and neuraminidase protein exposed on the surface are involved in the entry into host cells, uncoating, and release of newly formed virus particles from infected cells, respectively. The segmented genome is packaged as a ribonucleoprotein complex composed of RNA coated with a nucleoprotein associated with the heterotrimeric polymerase. The polymerase composed of the PA subunit, PB1 subunit, and PB2 subunit is important for both the replication of the viral genome and the transcription of mRNA. The PB1 subunit has a polymerase active site, and in addition to their roles in genome replication, the PB2 subunit and PA subunit work together to capture (PB2) and remove (PA) the cap of host cell pre-mRNA to facilitate the transcription of viral mRNA.

[0003] Seasonal influenza is a respiratory disease characterized by sudden fever, cough, sore throat, headache, muscle pain, and fatigue. Symptoms range from mild to severe, and infected humans can die. Worldwide, 3 to 5 million people contract severe influenza each year, and about 500,000 people die. Humans with weakened immunity, such as very young humans and those over 65 years old, are at the highest risk of influenza-related morbidity and mortality.

[0004] Vaccines for the prevention of influenza disease are available. However, the effectiveness of such vaccines varies from year to year, and the pooled effectiveness in healthy adults is estimated to be 59% (Osterholm et al, CIDRAP report (2012)). Influenza virus strains that can evade the host's immunity are selectively transmitted. Therefore, in order to provide protection against currently circulating viruses, seasonal influenza vaccines need to be re-prescribed and re-administered annually. Vaccines with permanence and providing protection across multiple seasons or broad spectra are not currently available.

[0005] Several small molecules targeting influenza virus have been approved for therapeutic use and / or limited prophylactic use in one or more countries. Such small molecules include M2 ion channel inhibitors, NA inhibitors, nucleoside analogs, and recently approved inhibitors targeting the endonuclease activity of the PA protein. As a treatment, small molecule inhibitors of influenza must be administered within 48 hours of onset in order to be effective in reducing viral shedding and the duration of respiratory symptoms. Currently circulating influenza virus strains are resistant to approved M2 inhibitors, and thus the use of M2 inhibitors is no longer recommended. The purine analog favipiravir is approved for use only in Japan and its use is restricted due to safety concerns. In the past, the effectiveness of several neuraminidase inhibitors has been limited by an increase in the level of circulating drug-resistant virus mutants. However, since the 2009 H1N1 pandemic, the level of drug resistance has been low. Resistance to baloxavir marboxil, a cap-dependent endonuclease inhibitor, was high in late-stage clinical trials, and viruses with a mutation at amino acid 38 of the PA protein were isolated from 9.7% of adults and 23.4% of pediatric trial participants (Hayden et al, N Engl J Med., 379(10):913-923(2018); Hirotsu et al, Clin Infect Dis., ciz908(2019)). Whether these mutant viruses, which significantly reduce antiviral efficacy in vitro, can be efficiently transmitted from human to human is unknown (Omoto et al, Sci Rep., 8(1):9633(2018); Noshi et al, Antiviral Res., 160:109‐117(2018)).

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0007] Currently, the options for treating and preventing influenza diseases are limited, and there are significant concerns about drug resistance to approved therapies. Furthermore, due to the inability of seasonal vaccines to provide a consistent, robust, and persistent broad-spectrum defense against influenza diseases, combined with the threat of the emergence of new zoonotic influenza viruses that have the potential to cause pandemics, there is a continuous need to develop both preventive and therapeutic agents targeting influenza viruses.

Means for Solving the Problems

[0008] The present invention relates to formula I:

Chemical Formula

BEST MODE FOR CARRYING OUT THE INVENTION

[0009] The present invention relates to a compound of formula (I):

Chemical formula

[0010] In one embodiment of the present invention, X is N. In another embodiment of the present invention, X is CH.

[0011] In one embodiment of the present invention, Y is absent. In another embodiment of the present invention, Y is CHR 5 . In one class of this embodiment, Y is CH2. In another embodiment of the present invention, Y is -CH2-CHR 5 -. In one class of this embodiment, Y is -CH2-CH2-. In another embodiment of the present invention, Y is -CH2-CHR 5 -CH2-. In one class of this embodiment, Y is -CH2-CH2-CH2-. In another embodiment of the present invention, Y is S. In another embodiment of the present invention, Y is SO. In another embodiment of the present invention, Y is SO2.

[0012] In one embodiment of the present invention, R 1 is methyl.

[0013] In one embodiment of the present invention, R 2 is phenyl, where the phenyl may be substituted with one or two substituents independently selected from the group consisting of halo, CH3, CF3, OCHF2, and OCH3.

[0014] In one embodiment of the present invention, R 3 is hydrogen, methyl, ethyl, or hydroxy.

[0015] In one embodiment of the present invention, R 4 is hydrogen, methyl, ethyl, propyl, trifluoroethyl, CH2CH2OH, CH2CH2OCH3, or cyclopropylmethyl.

[0016] In one embodiment of the present invention, R 5 is hydrogen. In another embodiment of the present invention, R 5 is methyl.

[0017] In one embodiment of the present invention, R 6 is hydrogen.

[0018] In one embodiment of the present invention, R 4 and R 6 together with the carbon atom to which they are attached can form a C 4-6 cycloalkyl group.

[0019] In one embodiment of the present invention, R 7 is hydrogen. In another embodiment of the present invention, R 7 is methyl.

[0020] References to the preferred classes and subclasses described above are intended to encompass all combinations of specific groups and preferred groups unless otherwise indicated.

[0021] Certain embodiments of the present invention include, but are not limited to, Compounds 1A - 167 or pharmaceutically acceptable salts thereof identified herein as Examples 1 - 40.

[0022] Within the scope of the present invention, further included are pharmaceutical compositions comprising a compound represented by Formula I described above and a pharmaceutically acceptable carrier. The present invention is also intended to include pharmaceutical compositions comprising a pharmaceutically acceptable carrier and any of the compounds specifically disclosed in the present application. These and other aspects of the present invention will be apparent from the teachings contained herein.

[0023] The present invention further includes compositions comprising a compound of the present invention in a pharmaceutically acceptable carrier for inhibiting cap - dependent endonuclease in a virus, treating a disease caused by a virus having cap - dependent endonuclease, treating influenza in a mammal, and preventing influenza. These compositions may optionally contain another antiviral agent. The compositions can be added to blood, blood products, or mammalian organs to effect the desired inhibition.

[0024] The compounds of the present invention can be administered in the form of pharmaceutically acceptable salts. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids, which include inorganic or organic bases and inorganic or organic acids. The salts of basic compounds included in the term "pharmaceutically acceptable salts" are generally non-toxic salts of the compounds of the present invention prepared by reacting the free base with a suitable organic or inorganic acid.Representative salts of the basic compound of the present invention include, but are not limited to, the following: acetate, ascorbate, adipate, alginate, aspirate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, camphorate, camphorsulfonate, cantharidate, carbonate, chloride, clubranate, citrate, cyclopentanepropionate, diethylacetic, digluconate, dihydrochloride, dodecylsulfanate, edetate, edisylic acid salt, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptanoate, gluconate, glutamate, glycerophosphate, glycollylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, 2-hydroxyethanesulfonate, hydroxynaphthoate, iodide, isonicotinic, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, methanesulfonate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, phosphate / diphosphate, pimelic, phenylpropionic, polygalacturonate, propionate, salicylate, stearate, sulfate, basic acetate, succinate, tannate, tartrate, theophylline salt, thiocyanate, tosylate, triethiodide, trifluoroacetate, undeconate, valerate, etc.Furthermore, when the compound of the present invention has an acidic moiety, suitable pharmaceutically acceptable salts include, but are not limited to, salts derived from inorganic bases such as aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, mangamous, potassium, sodium, zinc, etc. Furthermore, ammonium salts, calcium salts, magnesium salts, potassium salts and sodium salts are also included. Salts derived from pharmaceutically acceptable non-toxic organic bases include salts of primary amines, secondary amines and tertiary amines, salts of cyclic amines, salts of dicyclohexylamine, and basic ion exchange resins such as arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. Furthermore, the basic nitrogen-containing group can be quaternized with agents such as the following: lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate and diamyl sulfate; long-chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides such as benzyl and phenethyl bromides, etc.

[0025] These salts can be obtained by known methods, for example, by mixing the compounds of the present invention with a solution containing an equivalent amount of a desired acid or base, etc., and then filtering the salt or distilling off the solvent to collect the desired salt. The compounds of the present invention and their salts can form solvates with solvents such as water, ethanol or glycerol. Depending on the type of substituents on the side chain, the compounds of the present invention can also form salts of acid addition salts and bases simultaneously.

[0026] When the compound represented by formula I contains an acidic group and a basic group simultaneously in its molecule, the present invention further includes, in addition to the above salt forms, inner salts or betaines (zwitterions).

[0027] The present invention encompasses all stereoisomeric forms of the compounds represented by formula I. Unless a specific stereochemistry is indicated, the present invention is intended to encompass all such isomeric forms of these compounds. All of the asymmetric centers present in the compounds represented by formula I can independently have the (R) configuration or the (S) configuration. When the bond to a chiral carbon in the structural formula of the present invention is depicted as a straight line, it is understood that both the (R) configuration and the (S) configuration of the chiral carbon are included within the scope of the formula, and thus both individual enantiomers and their mixtures are included within the scope of the formula. When a specific configuration is depicted, the enantiomer (either (R) or (S) at its center) is intended. Similarly, when a compound name is described without explicitly indicating chirality for a chiral carbon, it is understood that both the (R) configuration and the (S) configuration of the chiral carbon, and thus individual enantiomers and their mixtures, are included by that name. Regarding the preparation of a specific stereoisomer or its mixture, it can be confirmed in the examples in which such stereoisomer or mixture is obtained, but this in no way limits the fact that it is within the scope of the present invention to encompass all stereoisomers and their mixtures.

[0028] Unless a specific enantiomer or diastereomer is indicated, the present invention encompasses all possible enantiomers and diastereomers, as well as mixtures of two or more stereoisomers in all ratios (e.g., mixtures of enantiomers and / or diastereomers). Thus, enantiomers in enantiomerically pure form (both as the levorotatory and dextrorotatory enantiomers), enantiomers in the form of racemic compounds, and enantiomers in the form of mixtures of two enantiomers in all ratios are the subject of the present invention. In the case of cis / trans isomerism, the present invention encompasses both the cis and trans forms and mixtures of these forms in all ratios. The preparation of individual stereoisomers can be carried out, if necessary, by separating the mixture by conventional methods (e.g., chromatography or crystallization), or by using stereochemically homogeneous starting materials in the synthesis, or by stereoselective synthesis. Optionally, derivatization can be carried out prior to the separation of the stereoisomers. The separation of mixtures of stereoisomers can be carried out at the intermediate stage during the synthesis of the compounds of formula I or on the final racemic product. The absolute stereochemistry can be confirmed by subjecting crystalline products or crystalline intermediates (which are derivatized, if necessary, with reagents containing stereocenters of known configuration) to X-ray crystallography. If the compounds of the present invention are tautomerizable, all individual tautomers and their mixtures are included within the scope of the present invention. The present invention includes all such isomers, as well as salts, solvates (which include hydrates) and solvated salts of such racemic compounds, enantiomers, diastereomers and tautomers and their mixtures.

[0029] In the compounds of the present invention, the atoms may exhibit their natural isotope abundances, or one or more of the atoms may be artificially enriched in a specific isotope having the same number of atoms but a different atomic mass or mass number than the atomic mass or mass number predominantly found in nature. The present invention is intended to encompass all suitable isotopic variations of the specifically and generically described compounds. For example, various isotopic forms of hydrogen (H) include protium ( 1 H) and deuterium ( 2 H), etc. Protium is the main hydrogen isotope found in nature. Enriching deuterium may result in certain therapeutic advantages, such as an increase in the in vivo half-life or a reduction in the required dosage, or may provide compounds useful as standards for the characterization of biological samples. Isotope-enriched compounds can be prepared without undue experimentation by conventional methods well known to those skilled in the art or by methods similar to those described in the general process schemes and examples herein using suitable isotope-enriched reagents and / or intermediates.

[0030] If any variable part is present more than once in any component, its definition in each occurrence is independent of all other occurrences. Also, combinations of substituents and variable parts are permitted only if such combinations result in stable compounds. A line drawn from a substituent to a ring system represents that the indicated bond can be attached to any of the ring atoms that can be substituted. When the ring system is bicyclic, the bond is intended to be attached to any appropriate atom on either ring of the bicyclic moiety.

[0031] It will be understood that one of ordinary skill in the art can incorporate one or more silicon (Si) atoms in place of one or more carbon atoms into the compounds of the invention to provide compounds that are chemically stable and can be readily synthesized by techniques known to one of ordinary skill in the art from readily available starting materials. Carbon and silicon have different covalent radii, which results in differences in bond distances and steric arrangements when comparing similar C and Si element bonds. These differences result in subtle changes in the size and shape of silicon-containing compounds compared to carbon. One of ordinary skill in the art will understand that differences in size and shape can result in subtle or dramatic changes in potency, solubility, lack of off-target activity, packaging properties, etc. (Diass, J. O. et al. Organometallics (2006) 5:1188-1198; Showell, G.A. et al. Bioorganic & Medicinal Chemistry Letters (2006) 16:2555-2558).

[0032] It is understood that one of ordinary skill in the art can select substituents and substitution patterns in the compounds of the invention to provide compounds that are readily synthesizable by techniques known in the art and the methods described below from readily available starting materials and are chemically stable. It is understood that when the substituent itself is substituted with two or more groups, these multiple groups can be on the same carbon or different carbons as long as a stable structure is obtained. The expression "(optionally substituted) with one or more substituents" should be understood to mean that the group can be unsubstituted or substituted with one or more substituents.

[0033] Furthermore, the compounds of the present invention can exist in amorphous form and / or one or more crystalline forms, and all such amorphous and crystalline forms of the compounds represented by Formula I, as well as mixtures thereof, are intended to be included within the scope of the present invention. Additionally, some of the compounds of the present invention may form solvates (i.e., hydrates) with water or solvates with common organic solvents. Such solvates and hydrates of the compounds of the present invention, particularly pharmaceutically acceptable solvates and hydrates, are likewise included within the scope of the present invention, together with the unsolvated anhydrous forms of the compounds.

[0034] Furthermore, when a carboxylic acid (-COOH) group or an alcohol group is present in the compounds of the present invention, pharmaceutically acceptable esters of carboxylic acid derivatives such as methyl, ethyl, or pivaloyloxymethyl, or acyl derivatives of alcohols such as O-acetyl, O-pivaloyl, O-benzoyl, and O-aminoacyl can also be used. Esters and acyl groups known in the art for modifying solubility or hydrolysis characteristics for use in sustained-release formulations or prodrug formulations are included.

[0035] Any pharmaceutically acceptable prodrug modification of the compounds of the present invention that results in in vivo conversion to a compound within the scope of the present invention is within the scope of the present invention. For example, esters can optionally be made by esterification of available carboxylic acid groups or by formation of esters with available hydroxy groups within the compound. Similarly, readily convertible amides can also be made. Pharmaceutically acceptable esters or amides of the compounds of the present invention can be prepared to act as prodrugs that are hydrolyzable, particularly in vivo, to the acid form (or the -COO- form depending on the pH of the body fluid or tissue in which the conversion occurs) or the hydroxy form, and as such are included within the scope of the present invention. Examples of pharmaceutically acceptable prodrug modifications include, but are not limited to, -C 1-6 substituted with alkyl esters and phenyl esters -C 1-6 alkyl, etc.

[0036] Accordingly, within the scope of the general structural formula, compounds, embodiments, and the specific compounds described and claimed herein include salts, all possible stereoisomers and tautomers, physical forms (e.g., amorphous and crystalline forms), their solvated and hydrated forms, and any mixtures of these forms, as well as their salts, their prodrug forms, and their prodrug forms, where such forms are possible unless otherwise specified.

[0037] The terms used herein have their ordinary meanings, and the meaning of such terms is independent for each occurrence. Nevertheless, and unless otherwise indicated, the following definitions apply throughout this specification and the claims. Chemical names, common names, and chemical structures may be used interchangeably to describe the same structure. These definitions apply regardless of whether the term is used alone or in combination with other terms, unless otherwise indicated. Thus, the definition of "alkyl" applies to "alkyl" and to "alkyl" moieties such as "hydroxyalkyl", "haloalkyl", "-O-alkyl", etc.

[0038] As used herein and throughout the disclosure, the following terms should be understood to have the following meanings unless otherwise indicated.

[0039] "Subject" is a human or non-human mammal. In one embodiment, the subject is a human. In another embodiment, the subject is a primate. In another embodiment, the subject is a monkey. In another embodiment, the subject is a chimpanzee. In yet another embodiment, the subject is a rhesus monkey.

[0040] As used herein, the terms "treatment" and "treating" refer to all processes in which there may be a slowing, interruption, arrest, control, or cessation of the progression of a disease or disorder described herein. This term does not necessarily indicate complete elimination of all symptoms of the disease or disorder.

[0041] As used herein, the term "preventing" or "prophylaxis" refers to reducing the likelihood of contracting a disease or disorder described herein, or reducing the severity of a disease or disorder described herein.

[0042] As used herein, the term "alkyl" refers to an aliphatic hydrocarbon group in which one of its hydrogen atoms is replaced by a bond. The alkyl group can be straight-chain or branched-chain and can contain from about 1 to about 20 carbon atoms. In one embodiment, the alkyl group contains from about 1 to about 12 carbon atoms. In different embodiments, the alkyl group contains 1 to 6 carbon atoms (C1-C6 alkyl) or from about 1 to about 4 carbon atoms (C1-C4 alkyl). Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, isopentyl, n-hexyl, isohexyl, and neohexyl. In one embodiment, the alkyl group is straight-chain. In another embodiment, the alkyl group is branched-chain. Unless otherwise indicated, the alkyl group is unsubstituted.

[0043] As used herein, the term "haloalkyl" refers to the alkyl group defined above in which one or more of the hydrogen atoms of the alkyl group are replaced by halogen. In one embodiment, the haloalkyl group has 1 to 6 carbon atoms. In another embodiment, the haloalkyl group is substituted with 1 to 3 F atoms. Non-limiting examples of haloalkyl groups include -CH2F, -CHF2, -CF3, -CH2Cl, and -CCl3. The term "C1-C6 haloalkyl" refers to a haloalkyl group having 1 to 6 carbon atoms.

[0044] As used herein, the term "halo" means -F, -Cl, -Br or -I.

[0045] The term "cycloalkyl" means a monocyclic or bicyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms. For example, "cycloalkyl" includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. Bicyclic cycloalkyl ring systems include fused ring systems in which two rings share two atoms and spiro ring systems in which two rings share one atom.

[0046] As used herein, the term "aryl" represents a stable bicyclic or tricyclic ring system having up to 10 atoms in each ring, where at least one ring is aromatic and all ring atoms are carbon. Bicyclic and tricyclic ring systems include fused ring systems in which two rings share two atoms and spiro ring systems in which two rings share one atom.

[0047] As used herein, the term "heteroaryl" refers to a stable monocyclic or bicyclic ring system having up to 10 atoms in each ring, wherein at least one ring is aromatic and at least one ring contains 1 to 4 heteroatoms selected from the group consisting of O, N, and S. Bicyclic heteroaryl ring systems include fused ring systems in which two rings share two atoms and spiro ring systems in which two rings share one atom. Heteroaryl groups within the scope of this definition include, but are not limited to, the following: azaindolyl, benzimidazolyl, benzisoxazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, dihydroindenyl, furanyl, indolinyl, indolyl, indradinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthalenyl, naphthyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, pyranyl, pyrazinyl, pyrazolyl, pyrazolopyrimidinyl, pyridazinyl, pyridopyridinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydroindolyl, dihydroquinolinyl, dihydrobenzodioxinyl, dihydropyrazolooxazinyl, dihydropyrazolothiadiazinedioxidyl, methylenedioxybenzene, benzothiazolyl, benzothienyl, quinolinyl, isoquinolinyl, oxazolyl, tetrahydroquinoline, and 3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine. It is understood that when heteroaryl contains a nitrogen atom, its corresponding N-oxide is also included in this definition.

[0048] As used herein, the terms "heterocycle", "heterocycloalkyl", or "heterocyclyl" are intended to mean a stable non-aromatic monocyclic or bicyclic ring system having up to 10 atoms in each ring and containing 1 to 4 heteroatoms selected from the group consisting of O, N, S, SO, or SO2, unless otherwise indicated. Bicyclic heterocyclic ring systems include fused ring systems in which two rings share two atoms and spiro ring systems in which two rings share one atom. Thus, examples of "heterocyclyl" include, but are not limited to, azaspirononanyl, azaspirooctanyl, azetidinyl, dioxanyl, oxazazaspirodecenyl, oxaspirooctanyl, oxazolidinonyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydropyranyl, dihydropiperidinyl, and tetrahydrothiophenyl. It is understood that when a heterocycle contains nitrogen, its corresponding N-oxide is also included in this definition.

[0049] "Celite®" (Fluka) diatomite is diatomaceous earth and can be referred to as "celite".

[0050] The term "substituted" means that one or more hydrogens on the designated atom are replaced with a moiety selected from the indicated groups, provided that the normal valence of the designated atom is not exceeded in the existing environment and that a stable compound is obtained by the substitution. Combinations of substituents and / or variable portions are permitted only if such combinations result in a stable compound. A "stable compound" or "stable structure" means a compound that is sufficiently robust to withstand isolation to a useful degree of purity from a reaction mixture and formulation into an effective therapeutic agent.

[0051] As used herein, the term "in substantially purified form" refers to the physical state of a compound after it has been isolated from a synthetic process (e.g., from a reaction mixture), or from a natural source, or a combination thereof. The term "in substantially purified form" further refers to the physical state of a compound that is of a purity sufficient to be characterized by standard analytical techniques described herein or analytical techniques well known to those skilled in the art, after the compound has been obtained from one or more purification processes described herein or one or more purification processes well known to those skilled in the art (e.g., chromatography, recrystallization, etc.).

[0052] It should also be noted that any carbon and heteroatoms in the text, schemes, examples and tables herein that do not have a satisfied valence are presumed to have a sufficient number of hydrogen atoms to satisfy that valence.

[0053] When a functional group in a compound is said to be "protected", this means that the group is in a modified form to prevent an undesired side reaction at that protected site when the compound is subjected to a reaction. Suitable protecting groups will be recognized by those skilled in the art and by reference to standard textbooks such as, for example, "T. W. Greene et al, Protective Groups in Organic Synthesis (1991), Wiley, New York".

[0054] In any of the components or in Formula I, if any substituent or variable part (e.g., R x ) appears more than once, its definition in each occurrence is independent of its definition in all other occurrences unless otherwise indicated.

[0055] As used herein, the term "composition" is intended to encompass a product containing the specified components in the specified amounts, and any product obtained from a combination of the specified amounts of the specified components.

[0056] The present invention further relates to a medicament comprising at least one compound represented by formula I and / or a pharmaceutically acceptable salt of a compound represented by formula I and / or a stereoisomeric form in case of a compound represented by formula I or a pharmaceutically acceptable salt of a stereoisomeric form of a compound represented by formula I, together with a pharmaceutically suitable and pharmaceutically acceptable vehicle, additive and / or other active substance and adjuvant.

[0057] As used herein, the term "patient" is to be construed to mean a mammal such as a primate, human, sheep, horse, cow, pig, dog, cat, rat and mouse.

[0058] The term "influenza" encompasses seasonal influenza, pandemic influenza, avian influenza, swine influenza and influenza diseases in humans or animals. Seasonal influenza is caused by influenza A virus and / or influenza B virus.

[0059] The medicament according to the present invention can be administered by oral administration, inhalation administration, rectal administration or transdermal administration, or by subcutaneous injection, intra-articular injection, intraperitoneal injection or intravenous injection. Oral administration is preferred. It is possible to coat stents and other surfaces that come into contact with blood in the body with the compound represented by formula (I).

[0060] The present invention further relates to a method for manufacturing a medicament, wherein the method comprises using a pharmaceutically suitable and pharmaceutically acceptable carrier and optionally further suitable active substances, additives or adjuvants to convert at least one compound represented by formula (I) into a suitable dosage form.

[0061] Suitable solid pharmaceutical forms or galenic pharmaceutical forms are, for example, granules, powders, coated tablets, tablets, (micro)capsules, suppositories, syrups, solutions, suspensions, emulsions, drops or injection solutions, and formulations with sustained release of the active substance, where, in these formulations, customary excipients such as vehicles, disintegrants, binders, coating agents, swelling agents, flow promoters or lubricants, flavoring agents, sweetening agents and solubilizing agents are used. Frequently used adjuvants that can be mentioned are magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, lactose, gelatin, starch, cellulose and its derivatives, animal and vegetable oils such as cod liver oil, sunflower oil, peanut oil or sesame oil, polyethylene glycol, and solvents such as sterile water, and monohydric or polyhydric alcohols such as glycerol.

[0062] The dosing schedule using the cap-dependent endonuclease inhibitor of the present invention is selected according to various factors including the following: type, species, age, weight, sex and medical condition of the patient; severity of the condition to be treated; route of administration; renal and hepatic function of the patient; and the specific compound or its salt used. A physician or veterinarian having ordinary skill can readily determine and prescribe an effective amount of the drug required to prevent, arrest or stop the progression of the condition.

[0063] The oral dosage of the cap-dependent endonuclease inhibitor, when used for the indicated effect, ranges from about 0.01 mg / kg body weight / day (mg / kg / day) to about 30 mg / kg / day, preferably from 0.025 mg / kg / day to 7.5 mg / kg / day, more preferably from 0.1 mg / kg / day to 2.5 mg / kg / day, and most preferably from 0.1 mg / kg / day to 0.5 mg / kg / day (the amount of the active ingredient is based on the free base unless otherwise specified). For example, an 80 kg patient is administered about 0.8 mg / day to 2.4 g / day, preferably 2 mg / day to 600 mg / day, more preferably 8 mg / day to 200 mg / day, and most preferably 8 mg / kg / day to 40 mg / kg / day. Accordingly, a properly prepared pharmaceutical for once-daily administration will contain 0.8 mg to 2.4 g, preferably 2 mg to 600 mg, more preferably 8 mg to 200 mg, and most preferably 8 mg to 40 mg (e.g., 8 mg, 10 mg, 20 mg, and 40 mg). Advantageously, the cap-dependent endonuclease inhibitor can be administered in divided doses two, three, or four times a day. In the case of twice-daily administration, a properly prepared medicament will contain 0.4 mg to 4 g, preferably 1 mg to 300 mg, more preferably 4 mg to 100 mg, and most preferably 4 mg to 20 mg (e.g., 4 mg, 5 mg, 10 mg, and 20 mg).

[0064] For intravenous injection, a patient will be administered an amount of the active ingredient sufficient to deliver from 0.025 mg / kg / day to 7.5 mg / kg / day, preferably from 0.1 mg / kg / day to 2.5 mg / kg / day, more preferably from 0.1 mg / kg / day to 0.5 mg / kg / day. Such amounts can be administered in many suitable ways (e.g., a large volume of a low concentration of the active ingredient over a single long period, or several low volume high concentration administrations of the active ingredient per day over a short period, e.g., once a day). Typically, conventional intravenous formulations can be prepared containing the active ingredient at a concentration of about 0.01 mg / mL to 1.0 mg / mL (e.g., 0.1 mg / mL, 0.3 mg / mL, and 0.6 mg / mL), and the formulation can be administered in an amount of 0.01 mL / kg patient body weight to 10.0 mL / kg patient body weight (e.g., 0.1 mL / kg, 0.2 mL / kg, 0.5 mL / kg) per day. In one example, an 80 kg patient is administered 8 mL of an intravenous formulation having an active ingredient at a concentration of 0.5 mg / mL twice a day, or 8 mg of the active ingredient per day. Glucuronic acid, L-lactic acid, acetic acid, citric acid, or a pharmaceutically acceptable acid / conjugate base having suitable buffering capacity in the pH range acceptable for intravenous administration can be used as a buffer. The appropriate choice of buffer and pH for the formulation can be readily carried out by one skilled in the art depending on the solubility of the drug being administered.

[0065] The compound represented by Formula I can be administered as monotherapy and in combination with other therapeutic agents, which include other antiviral agents or treatments for influenza.

[0066] The cap-dependent endonuclease inhibitor can also be co-administered with a suitable antiviral agent, where the suitable antiviral agent includes, but is not limited to, M2 ion channel inhibitors, neuraminidase inhibitors, nucleoside analogs, and inhibitors that target the endonuclease activity of the PA protein.

[0067] Alternatively, or in addition, one or more additional pharmacologically active agents can be administered in combination with the compounds of the invention. The additional active agent(s) is / are intended to mean one or more pharmaceutically active agents that are different from the compounds of the invention and that are active in the body (including prodrugs that are converted to a pharmaceutically active form after administration), and the free acids, free bases and pharmaceutically acceptable salts of such additional active agents are also included where such forms are commercially available or chemically possible. In general, any suitable active agent(s) (including, but not limited to, M2 ion channel inhibitors, neuraminidase inhibitors, nucleoside analogs and inhibitors targeting the endonuclease activity of PA protein) can be used in any combination with the compounds of the invention in a single-dose formulation (fixed-dose drug combination), or can be administered to a patient in one or more separate dosage forms that allow for co-administration or sequential administration (co-administration of separate active agents) of the active agent(s).

[0068] Typical dosages of the cap-dependent endonuclease inhibitors of the invention in combination with other suitable M2 ion channel inhibitors, neuraminidase inhibitors, nucleoside analogs and inhibitors targeting the endonuclease activity of PA protein can be the same as the dosage of the cap-dependent endonuclease inhibitor administered without co-administration of the additional M2 ion channel inhibitor, neuraminidase inhibitor, nucleoside analog and inhibitor targeting the endonuclease activity of PA protein, or can be substantially less than the dosage of the thrombin inhibitor administered without co-administration of the M2 ion channel inhibitor, neuraminidase inhibitor, nucleoside analog and inhibitor targeting the endonuclease activity of PA protein, depending on the patient's treatment needs.

[0069] The compound is administered to a mammal in a therapeutically effective amount. "Therapeutically effective amount" means an amount of a compound of the invention that is effective, alone or in combination with additional therapeutic agents, when administered to a mammal, to treat (i.e., prevent, inhibit or ameliorate) a viral disease or to treat the progression of the disease in the host.

[0070] The compounds of the invention are preferably administered to a mammal alone in a therapeutically effective amount. However, the compounds of the invention can also be administered to a mammal in a therapeutically effective amount in combination with additional therapeutic agents, as defined below. When administered in combination, the combination of compounds is preferably, but not necessarily, a synergistic combination. Synergism occurs when the effect of the compounds (in this case, inhibition of a desired target) when administered in combination is greater than the sum of the individual effects of the compounds when administered separately as individual agents, as described, for example, by Chou and Talalay, "Adv. Enzyme Regul. 1984, 22, 27-55". In general, synergistic effects are most clearly demonstrated at concentrations below the optimal concentration of the compounds. Synergism can relate to lower cytotoxicity, increased anticoagulant effect or some other beneficial effect of the combination as compared to the individual components.

[0071] "Administered in combination" or "combination therapy" means that the compound of the invention and one or more additional therapeutic agents are administered simultaneously to the mammal being treated. When administered in combination, the components can be administered simultaneously or sequentially in any order at different times. Thus, each component can be administered separately, but sufficiently close in time, so as to produce the desired therapeutic effect.

[0072] The present invention is not limited in scope by the specific embodiments disclosed in the examples which are intended as illustrations of only a few aspects of the invention, and any embodiments that are functionally equivalent are within the scope of the present invention. Indeed, various modifications of the present invention in addition to those shown and described herein will be apparent to those of ordinary skill in the relevant art and are intended to be within the scope of the appended claims.

[0073] General method Several methods for preparing the compounds of the present invention are illustrated in the following schemes and examples. The starting materials and necessary intermediates can optionally be commercially available or can be prepared according to literature procedures or as shown herein. The compounds of the present invention can be prepared by using the reactions shown in the following schemes in addition to other standard procedures known in the literature or exemplified in experimental procedures. The numbering of the substituents shown in the schemes does not necessarily correlate with the numbering used in the claims, and in many cases, for clarity, a single substituent is shown attached to the compound for which multiple substituents are allowed under the above definitions. The reactions used to produce the compounds of the present invention are carried out using the reactions shown in the schemes and examples herein in addition to other standard procedures such as ester hydrolysis, cleavage of protecting groups, etc. known in the literature or exemplified in experimental procedures. The starting materials are prepared according to procedures known in the art or as shown herein.

[0074] The compounds of the present invention can be prepared by various methods. Optionally, the final product can be further modified, for example, by manipulating substituents. These operations include, but are not limited to, reduction, oxidation, alkylation, acylation, and hydrolysis reactions generally known to those skilled in the art. Optionally, the order in which the foregoing reaction schemes are carried out can be changed to facilitate the reaction or to avoid unwanted reaction products. Since the scheme is illustrative, the present invention should not be construed as being limited by the chemical reactions and conditions presented. The preparation of the various starting materials used herein is within the skill of those in the art. The following examples are provided so that the present invention may be more fully understood. These examples are for illustrative purposes only and should in no way be construed as limiting the present invention. The absolute stereochemistry of the individual stereoisomers in the examples and intermediates is not determined unless otherwise indicated in the examples or clearly indicated by the nomenclature.

[0075] When chiral resolution was achieved by chromatography using a chiral column, the chiral columns used for SFC chiral resolution are listed in the table. Some of the chiral columns used were CHIRALPAK AD, CHIRALCEL OJ, CHIRALPAK AS, CHIRALPAK AY, CHIRALPAK IA, CHIRALPAK AD-H, and CHIRALPAK AS-H. Hereinafter, they are represented by two-letter or three-letter abbreviations. By convention, in this table, the isomer that elutes earlier from the chiral resolution is always listed first, immediately followed by the isomer that elutes later from the same resolution. When more than three isomers are separated, they are always listed in the order in which they eluted, such as peak 1, followed by peak 2, peak 3, etc. in the table.

[0076] The catalyst is used in the following procedure. "Grubbs II" is also known as "Grubbs catalyst 2nd generation" and (1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene) dichloro(phenylmethylene)(tricyclohexylphosphine)ruthenium; carbonyltris(triphenylphosphine)rhodium(I) hydride; Wilkinson's catalyst is also known as chloridotris(triphenylphosphine)rhodium(I); all of these are available from Millipore Sigma.

[0077] General scheme Method for producing the compound represented by formula (I) The compound represented by formula (I) can be prepared from known starting materials or starting materials that are easily prepared according to methods known to those skilled in the art of organic synthesis. Methods useful for producing the compound represented by formula (I) are described in the following examples and are also generalized in Schemes 1, 2, 3, 4, 5 and 6 below. Alternative synthetic routes and similar structures will be apparent to those skilled in the art of organic synthesis.

[0078] Scheme 1

Chemical formula

[0079] Scheme 2

Chem.

[0080] Scheme 3

Chem.

[0081] Scheme 4

Chem.

[0082] Scheme 5

Chem.

[0083] Scheme 6

Chem.

[0084] In the method for preparing the compounds of the present invention described in the foregoing scheme, in addition to those already clearly described in the foregoing scheme, functional groups in various moieties and substituents may be sensitive or reactive under the reaction conditions used and / or in the presence of the reagents used. Such sensitivity / reactivity may interfere with the progress of the desired reaction, reducing the yield of the desired product, or even preventing its formation. Thus, it may be necessary or desirable to protect any sensitive or reactive groups of the molecules involved. Protection can be achieved using conventional protecting groups such as those described in " Protective Groups in Organic Chemistry , ed. J.F.W. McOmie, Plenum Press, 1973" and "T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis , John Wiley & Sons, 3 rd edition, 1999, and 2 nd edition, 1991". The protecting groups can be removed using methods known in the art at a convenient subsequent stage. Alternatively, interfering groups can be introduced into the molecule after the reaction step of interest.

[0085] Those skilled in the art of organic synthesis will recognize that the synthesis of compounds having multiple reactive functional groups (e.g., -OH and NH2) may require the protection of specific functional groups (i.e., derivatization for the purpose of chemical compatibility with the reaction conditions). Suitable protecting groups for the various functional groups of these compounds, as well as methods for their introduction and removal, are well known in the field of organic chemistry. A summary of many of these methods can be found in "Greene & Wuts, Protecting Groups in Organic Synthesis, John Wiley & Sons, 3rd edition (1999)".

[0086] One skilled in the art of organic synthesis will also recognize that, depending on the choice of substituents to be added, one route for the synthesis of the compounds represented by formula (I) may be more desirable. Further, one skilled in the art will also recognize that, in some cases, the order of reactions may differ from that shown herein in order to avoid incompatibility of functional groups, and thus the synthetic route can be adapted accordingly.

[0087] The compounds represented by formula vii, formula x, formula xv and formula xvii can be further synthesized using methods well known to those skilled in the art of organic synthesis for producing the entire range of compounds represented by formula (I), or, for example, using the methods described in the following examples.

[0088] The starting materials used and the intermediates prepared using the methods described in Schemes 1 - 6 can be isolated and purified, if necessary, using conventional techniques (where such conventional techniques include, but are not limited to, filtration, distillation, crystallization, chromatography, etc.). Such substances can be characterized using conventional means (which include physical constants and spectral data).

[0089] The following abbreviations may be used in the following experiments:

[0090]

Table 1

[0091]

Examples

[0092] The following examples are for illustrative purposes only with respect to the present invention and its implementation. These examples should not be construed as limitations on the scope or spirit of the present invention. In these examples, all temperatures are in degrees Celsius unless otherwise indicated, and "room temperature" indicates a temperature within the range of about 20°C to about 25°C. Reactions sensitive to moisture or air were carried out under nitrogen using anhydrous solvents and anhydrous reagents. The progress of the reaction was confirmed by either analytical thin-layer chromatography (TLC) using an E.Merck pre-coated TLC plate (silica gel 60F-254, layer thickness 0.25 mm) or liquid chromatography-mass spectrometry (LC-MS). Regarding the HPLC / MS data, the two HPLC conditions used were as follows: (1) LC2 (Waters C18 XTerra TM 3.5 μm 2.1×20 mm column [gradient from 10:90 - 98:2 (v / v) CH3CN / H2O + v0.05% TFA over 1.25 minutes, then held at 98:2 (v / v) CH3CN / H2O + v0.05% TFA for 0.75 minutes]; flow rate 1.5 mL / min, UV wavelength 254 nm); and, (2) LC4 (Waters C18 XTerra 3.5 μm 2.1×20 mm column [gradient from 10:90 - 98:2 (v / v) CH3CN / H2O + v0.05% TFA over 3.25 minutes, then held at 98:2 (v / v) CH3CN / H2O + v0.05% TFA for 0.75 minutes]; flow rate 1.5 mL / min, UV wavelength 254 nm).

[0093] Mass spectrometry was carried out using electrospray ionization in the positive ion detection mode. 1 1H NMR spectra were recorded at 400 - 500 MHz on a Varian instrument or a Bruker instrument. Chemical shifts are reported in ppm downfield and upfield from tetramethylsilane (TMS), and are referenced to either internal TMS or the solvent resonance ( 1 1H NMR: δ 7.27 for CDCl3 and δ 2.50 for (CD3)(CHD2)SO; and, 1313C NMR: δ 77.02 for CDCl3 and δ 39.51 for (CD3)2SO. Coupling constants (J) are expressed in Hertz (Hz), and spin multiplicities are given as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), m (multiplet), and br (broad line). Chiral resolution was carried out on either a "Waters Thar 80 SFC" or a "Berger MGII preparative SFC system".

[0094] Concentration of the solution was carried out using a rotary evaporator under reduced pressure or by freeze-drying. Flash chromatography was carried out on a prepacked silica gel column using a commercially available MPLC system. The compounds described in this specification were synthesized as racemic mixtures unless otherwise indicated in the experimental procedures.

[0095] Example 1 Preparation of Compound 1A and Compound 1B 7-Hydroxy-5-methyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chemical Structure

[0096] Step B - Synthesis of Compound Int-1b To a solution of Int-1a (8 g, 45.4 mmol) in DCM (50 mL) were added acetyl chloride (5.34 g, 68.1 mmol) and TEA (18.98 mL, 136 mmol) at 0 °C. The mixture was slowly warmed to room temperature over 16 h. Upon completion, 50 mL of aqueous NH4Cl solution was added to the above reaction. The mixture was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by an 80 g flash silica gel column using 0 - 5% EtOAc / PE as the eluting solvent to give Int-1b. 1 1H NMR (500 MHz, chloroform-d) δ 7.27 - 7.38 (m, 5H), 5.72 - 5.81 (m, 2H), 4.93 - 5.04 (m, 2H), 2.06 - 2.10 (m, 5H), 1.88 - 1.97 (m, 1H), 1.75 - 1.84 (m, 1H), 1.45 (ttd, J = 5.23, 7.51, 18.18 Hz, 1H), 1.30 - 1.39 (m, 1H).

[0097] Step C - Synthesis of Compound Int-1c A solution of Int-1b (6g, 27.5 mmol) in t-BuOH (120 mL) and water (30 mL) was added with NaHCO3 (23.09 g, 275 mmol) and sodium periodate (35.3 g, 165 mmol) at 20 °C. Then, osmium tetroxide (6.99 mL, 0.275 mmol) was added at 20 °C. The reaction mixture was stirred at 20 °C for 3 h. Upon completion, 200 mL of EtOAc was added to the reaction. The mixture was filtered. The filtrate was washed with water (3 × 50 mL), brine (100 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by an 80 g flash silica gel column using 0-10% EtOAc / PE as the eluting solvent to obtain Int-1c. 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.63 (t, J = 1.37 Hz, 1H), 7.26-7.39 (m, 5H), 5.65 (dd, J = 5.87, 7.83 Hz, 1H), 2.42-2.48 (m, 2H), 2.05 (s, 3H), 1.67-1.89 (m, 2H), 1.36-1.59 (m, 2H).

[0098] Step D - Synthesis of Compound Int-1e In a 40 mL sealed tube, a solution of Int-1d (1 g, 3.66 mmol) in DMF (10 mL) and AcOH (1 mL) was added with Int-1c (1.3 g, 5.90 mmol) at 25 °C. The reaction mixture was stirred at 120 °C for 5 h. Upon completion, the reaction was concentrated and purified by an 80 g flash silica gel column using 1-10% MeOH in DCM as the eluting solvent to obtain Int-1e. LCMS analysis C 27 H 29 Calculated for C17H15N3O5: 475.21; Found: 476.2 (M+H) + .

[0099] Step E - Synthesis of Compound Int-1f To a solution of Int-1e (1.11 g, 2.334 mmol) in DCM (10 mL) was added SEMCl (0.828 mL, 4.67 mmol) and TEA (0.976 mL, 7.00 mmol) at 25 °C. The reaction mixture was stirred at 40 °C for 16 h. Upon completion, the reaction mixture was directly purified by an 80 g silica gel column using 0 - 5% MeOH in DCM as the eluent solvent to give Int-1f. LCMS analysis C 33 H 43 Calculated for C18H21N3O6Si: 605.3; Found: 606.4 (M+H) + .

[0100] Step F - Synthesis of Compound Int-1g To a solution of Int-1f (110 mg, 0.182 mmol) in MeOH (5 mL) was added K2CO3 (25.10 mg, 0.182 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 3 h. Upon completion, it was concentrated and purified by preparative silica gel TLC plate using 6% MeOH in DCM as the eluent solvent to give Int-1g. LCMS analysis C 31 H 41 Calculated for C16H19N3O5Si: 563.3; Found: 564.2 (M+H) + .

[0101] Step G - Synthesis of Compound Int-1h To a solution of Int-1g (100 mg, 0.177 mmol) in DCM (5 mL) were added methanesulfonyl chloride (0.429 mL, 5.50 mmol) and triethylamine (0.099 mL, 0.710 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was concentrated to give the crude product.

[0102] To a solution of the above crude product (103 mg, 0.177 mmol) in THF (1 mL) was added TBAF (1 mL, 1.000 mmol) at 25 °C. The reaction mixture was stirred at 50 °C for 16 h. Upon completion, the reaction mixture was concentrated. The residue was purified by preparative silica gel TLC plate using 6% MeOH in DCM as the eluent solvent to afford Int-1h. LCMS analysis C 25 H 25 Calculated for C N3O3: 415.2; Found: 416.3 (M+H) + .

[0103] Step H - Synthesis of Compound Int-1i and Compound Int-1j Int-1h (150 mg, 0.361 mmol) was separated by SFC to afford the first elution peak Int-1i and the second elution peak Int-1j. SFC conditions: Daicel@ chiralpak AS column (250×30 mm, 10 μm); 45% EtOH (containing 0.1% NH3H2O); 70 mL / min. LCMS analysis C 25 H 25 Calculated for C N3O3: 415.2; Found: 416.3 (M+H) + .

[0104] Step I - Synthesis of Compound 1A and Compound 1B To a stirred mixture of Int-1i (15 mg, 0.036 mmol) in ACN (1 mL) was added magnesium bromide (6.65 mg, 0.036 mmol) at 20 °C. The mixture was stirred at 20 °C for 2 h under N2 atmosphere. Upon completion, it was purified by HPLC on a C18 column (YMC-Actus Triart 150×30 mm×5um) using 0 - 100% ACN in water (adjusted with 0.1% TFA regulator) as the eluent solvent to afford Compound 1A. LCMS analysis C 18 H 19 Calculated for C N3O3: 325.1; Found: 326.0 (M+H) + . 1 H NMR (500 MHz, methanol-d 4) δ 7.08 - 7.73 (m, 5H), 7.03 (d, J = 7.32 Hz, 1H), 5.84 (d, J = 7.32 Hz, 1H), 5.30 (t, J = 2.90 Hz, 1H), 4.22 (dd, J = 2.75, 11.60 Hz, 1H), 3.24 (s, 3H), 2.53 (td, J = 2.75, 15.26 Hz, 1H), 2.16 - 2.33 (m, 2H), 1.85 - 2.01 (m, 2H), 1.66 - 1.79 (m, 1H).

[0105] Compound 1B was prepared from Int-1j essentially according to the method used to prepare Compound 1A of Example 1. LCMS analysis C 18 H 19 Calculated for N3O3: 325.1; Found: 326.0 (M + H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 7.29 (br s, 5H), 6.96 (d, J = 7.53 Hz, 1H), 5.73 (d, J = 7.53 Hz, 1H), 5.28 (s, 1H), 4.20 (dd, J = 2.76, 11.80 Hz, 1H), 3.23 (s, 3H), 2.49 - 2.54 (m, 1H), 2.17 - 2.27 (m, 2H), 1.86 - 1.97 (m, 2H), 1.74 (br t, J = 13.55 Hz, 1H).

[0106] Example 2 Preparation of Compound 2A and Compound 2B 1-(4-Fluorophenyl)-7-hydroxy-5-methyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chemical Structure

[0107] Step B - Synthesis of Compound Int-2b A solution of Int-2a (2 g, 10.09 mmol) in DCM (50 mL) was added to Dess-Martin periodinane (8.56 g, 20.18 mmol). The resulting mixture was stirred at room temperature for 1 hour. It was concentrated to remove most of the DCM. 20 mL of DCM was added to the above residue and the mixture was filtered. The filtrate was subjected to an 80 g silica gel column using 0-100% EtOAc in hexane as the eluting solvent to obtain Int-2b. LCMS analysis C 11 H 11 Calculated for FO2: 194.1; Found: 195.3 (M+H) + .

[0108] Step C - Synthesis of Compound Int-2c Int-1d (1.4 g, 7.21 mmol) was added to a solution of Int-2b (1.8 g, 6.59 mmol) in DMF (5 mL) and AcOH (0.5 mL) at 25 °C. The reaction was stirred at 120 °C for 2 hours. Upon completion, it was concentrated. The residue was purified by an 80 g silica gel column using 1-5% MeOH in DCM as the eluting solvent to obtain Int-2c. LCMS analysis of C 25 H 24 Calculated value for FN3O4: 449.2; Measured value: 450.4 (M+H) + .

[0109] Step D - Synthesis of Compound Int-2d Sodium borohydride (42.1 mg, 1.112 mmol) was added to a stirred solution of Int-2c (500 mg, 1.112 mmol) in DCM (8 mL) and MeOH (40 mL) at room temperature. It was stirred at this temperature for 30 minutes. Upon completion, the reaction was quenched with 1 mL of 1N aqueous HCl and concentrated. The residue was subjected to an 80 g silica gel column using 0 - 100% 30% EtOH / hexane in EtOAc as the eluting solvent to obtain Int-2d. LCMS analysis of C 25 H 26 Calculated value for FN3O4: 451.5; Measured value: 452.4 (M+H) + .

[0110] Step E - Synthesis of Compound Int-2e A mixture of Int-2d (410 mg, 0.908 mmol) and 2-(tributyl-l5-phosphanylidene)acetonitrile (438 mg, 1.816 mmol) in toluene (10 mL) was heated at 120 °C for 4 hours. Upon completion, it was cooled and the resulting reaction was subjected to an 80 g silica gel column using 0 - 100% 30% EtOH / hexane in EtOAc as the eluent to remove phosphine impurities. Then the mixture was subjected to a C18 column (100 g) using 0 - 100% ACN in water as the eluent to obtain minor isomer Int-2e and major isomer Int-2f. LCMS analysis of C 25 H 24 Calculated value for FN3O3: 433.2; Measured value: 434.4 (M+H) + .

[0111] Step F - Synthesis of Compound Int-2g and Compound Int-2h Int-2h (185 mg, 0.427 mmol) was separated by SFC to obtain the first elution peak Int-2g and the second elution peak Int-2h. SFC conditions: Daicel@ chiralpak AS-H column (250×21 mm, 10 μm); 45% EtOH (containing 0.2% DIPA); 70 mL / min. LCMS analysis C 25 H 24 Calculated value for FN3O3: 433.2; Measured value: 434.4 (M+H) + .

[0112] Step G - Synthesis of Compound 2A and Compound 2B To a solution of Int-2g (52 mg, 0.120 mmol) in MeOH (3 mL) was added palladium on carbon (25.5 mg, 0.024 mmol). The resulting mixture was stirred at room temperature for 30 minutes under a hydrogen balloon. Upon completion, it was filtered through celite, and the filtrate was applied to a 50 g ISCO@ C18 column using 0 - 100% ACN in water (adjusted with 0.01% TFA regulator) as the elution solvent to obtain Compound 2A. LCMS analysis C 18 H 18 Calculated value for FN3O3: 343.1; Measured value: 344.3 (M+H) + . 1 H NMR (500 MHz, Methanol-d 4 ) δ 7.19 (d, J = 7.3 Hz, 1H), 7.05 (broad, 5 H), 6.14 (d, J = 7.3 Hz, 1H), 5.35 (s, 1H), 4.30 (dd, J = 11.7, 2.7 Hz, 1H), 3.27 (s, 3H), 2.59 - 2.52 (m, 1H), 2.32 - 2.19 (m, 2H), 1.96 (dd, J = 22.6, 14.9 Hz, 2H), 1.90 (s, 1H), 1.82 - 1.70 (m, 1H).

[0113] Compound 2B was prepared from Int-2h essentially according to the method used to produce Compound 2A of Example 2. LCMS analysis of C 18 H 18 Calculated value for FN3O3: 343.1; Measured value: 344.3 (M+H) + . 1 H NMR (500 MHz, Methanol-d 4 ) δ 7.20 (d, J = 7.3 Hz, 1H), 7.04 (broad, 5H), 6.15 (d, J = 7.3 Hz, 1H), 5.36 (s, 1H), 4.29 (dd, J = 11.7, 2.5 Hz, 1H), 3.26 (s, 2H), 2.56 (d, J = 15.4 Hz, 1H), 2.31 - 2.19 (m, 1H), 2.01 - 1.94 (m, 1H), 1.91 (d, J = 13.9 Hz, 1H), 1.81 - 1.69 (m, 1H).

[0114] The examples in Table 1 were prepared from appropriate starting materials using procedures similar to those described in Example 2.

Table 2

[0115] Example 3 Preparation of Compound 17A and Compound 17B 1-(2-(Difluoromethoxy)-5-fluorophenyl)-7-hydroxy-5-methyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chemical Structure

[0116] Step B - Synthesis of Compound Int-17b To a stirred solution of Int-17a (500 mg, 1.123 mmol) in dry THF (18 mL) at 0 °C was added 4-penten-1-magnesium bromide (0.5 M in 2-Me-THF) (6736 μL, 3.37 mmol). The mixture was stirred at this temperature for 14 minutes. Upon completion, the reaction was quenched by adding water (ca. 30 mL) and extracted with EtOAc (3 × 60 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by an 80 g silica gel column using an Isco system (using 0 - 100% EtOH-EtOAc (3:1) / hexane as the eluting solvent) to give Int-17b. LCMS analysis C 27 H 28 Calculated for F3N3O4: 515.2; Found 516.4 (M+H) + .

[0117] Step C - Synthesis of Compound Int-17c Int-17b (210 mg, 0.407 mmol) was dissolved in THF (3 mL) and water (1 mL), and osmium tetroxide (2.5% in t-BuOH) (102 μL, 8.15 μmol) was added to a 40 mL vial containing the stirred solution at room temperature. Subsequently, sodium periodate (261 mg, 1.222 mmol) was added. The vial was sealed with a pressure relief cap and stirred at room temperature for 7 hours. Upon completion, the mixture was diluted with EtOAc (ca. 100 mL), the solid was filtered off and washed with EtOAc (ca. 20 mL). The filtrate was washed twice with 10% aqueous Na2S2O3 solution (30 mL × 2). The organic layer was separated, dried over Na2SO4, filtered and concentrated under reduced pressure to give Int-17c. LCMS analysis C 26 H 26 Calculated for F3N3O5: 517.2; Found 518.4 (M+H) + .

[0118] Step D - Synthesis of Compound Int-17d AcOH (539 μL) was added to a 40 mL vial containing a stirred solution of Int-17c (184 mg, 0.356 mmol) dissolved in DMF (5 mL) at room temperature. The vial was sealed with a pressure relief cap and stirred at 120 °C for 8 hours. The mixture was cooled to room temperature and then purified by reverse phase 100 g Gold C18 column on an Isco system using 0 - 100% acetonitrile - water (containing 0.05% TFA buffer) as the elution solvent. The fractions containing the desired material were combined, lyophilized, then redissolved in DCM (ca. 15 mL), TEA (49.6 μL, 0.356 mmol) was added, and the mixture was passed through a 40 g silica gel column using an Isco system (using EtOH / EtOAc (3:1) / hexane as the elution solvent) to give Int-17d. LCMS analysis C 26 H 24 Calculated for F3N3O4: 499.2; Found 500.3 (M+H) + .

[0119] Step E - Synthesis of Compound Int-17e and Compound Int-17f The compound Int-17d (48.5 mg, 0.097 mmol) was resolved by chiral SFC to give the first elution peak Int-17e (21.5 mg, 0.043 mmol) and the second elution peak Int-17f (21.8 mg, 0.044 mmol). SFC conditions: Daicel@ chiralpak AD-H column (250×21 mm, 10 μm); 20% IPA (containing 0.2% DIPA); 70 mL / min. LCMS analysis C 26 H 24 Calculated for F3N3O4: 499.2; Found: 500.3 (M+H) + .

[0120] Step F - Synthesis of Compound 17A and Compound 17B Pd on carbon (22.37 mg, 0.021 mmol) was added to a round-bottom flask (50 mL), followed by the addition of Int-17f (21 mg, 0.042 mmol) in EtOH (12 mL). A hydrogen balloon was attached to the flask. The system was degassed and refilled with H2 twice. The mixture was then stirred at room temperature under H2 for 45 minutes. At the end, the catalyst was filtered off. The filtrate was concentrated under reduced pressure. The residue was purified by a 30 g Isco C18 gold column using 0-100% acetonitrile / water (buffered with 0.05% TFA) as the eluent to give compound 17A. LCMS analysis C 19 H 18 Calculated for F3N3O4: 409.1; Found 410.2 (M+H) + . 11H NMR (500 MHz, Methanol-d4) δ 7.61 (d, J = 9.1 Hz, 1H), 7.18 (d, J = 7.3 Hz, 1H), 7.12 (d, J = 7.7 Hz, 1H), 7.05 (d, J = 4.2 Hz, 1H), 6.58 (t, J = 73.2 Hz, 1H), 6.05 (d, J = 7.6 Hz, 1H), 5.33 (s, 1H), 4.84 (s, 1H), 3.26 (s, 3H), 2.55 (d, J = 15.4 Hz, 1H), 2.24 (t, J = 14.6 Hz, 2H), 1.95 - 1.93 (m, 2H), 1.77 - 1.74 (m, 1H).

[0121] Compound 17A was prepared from Int-17e essentially according to the method used to produce Compound 17B of Example 3. LCMS analysis C 19 H 18 Calculated for F3N3O4: 409.1; Found 410.2 (M+H) + . 1 1H NMR (500 MHz, Methanol-d 4 ) δ 7.61 (d, J = 8.6 Hz, 1H), 7.31 - 6.96 (m, 3H), 6.57 (t, J = 73.2 Hz, 1H), 6.03 (d, J = 7.2 Hz, 1H), 5.33 (s, 1H), 4.84 (s, 1H), 3.26 (s, 3H), 2.55 (d, J = 15.7 Hz, 1H), 2.24 (t, J = 14.1 Hz, 2H), 2.0 - 1.9 (m, 2H), 1.77 - 1.74 (m, 1H).

[0122] Example 4 Preparation of Compound 18A and Compound 18B 7-Hydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chemical Structure

[0123] Step B - Synthesis of Compound Int-18b To a stirred solution of Int-18a (5380 mg, 14.33 mmol) in dry THF (18 mL) was added allylmagnesium bromide (1.0 M in diethyl ether) (4.01E+04 μL, 40.1 mmol) at 0 °C. The mixture was stirred at this temperature for 15 minutes. Upon completion, the reaction was quenched by adding saturated NH4Cl (ca. 50 mL) and partitioned between EtOAc (100 mL) and water (ca. 50 mL). The aqueous layer was further extracted with EtOAc twice (2×80 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by a 220 g silica gel flash column using 0 - 100% EtOH-EtOAc (3:1) / hexane as the eluting solvent to obtain Int-18b. LCMS analysis C 25 H 27 Calculated for C N3O3: 417.2; Found: 418.2 (M+H) + .

[0124] Step C - Synthesis of Compound Int-18c To a 350 mL high-pressure vessel containing dicyclohexyl(2’,6’-dimethoxy-[1,1’-biphenyl]-2-yl)phosphane (S-phos) (703 mg, 1.731 mmol), chloro(1,5-cyclooctadiene)iridium(I) dimer [Ir(cod)Cl]2 (240 mg, 0.357 mmol), and DCC (3682 mg, 17.84 mmol) under N2 at room temperature were added Int-18b (2980 mg, 7.14 mmol) and formic acid (1.1 mL, 28.6 mmol) in dry acetonitrile (70 mL). The vial was sealed with a pressure relief cap. The mixture was heated to 75 °C overnight and then to 90 °C for 3 days. At completion, the reaction mixture was cooled to room temperature, diluted with EtOAc (ca. 60 mL), and then filtered. The solid was washed with EtOAc / DCM (20 mL / 20 mL). The combined filtrates were concentrated. The residue was purified by 120 g silica gel flash column using 0 - 100% EtOH-EtOAc(3:1) / hexane as the eluting solvent to give Int-18c and Int-18d. LCMS analysis C 26 H 27 Calculated for C18H23N3O3: 429.2; Found: 430.2 (M+H) + .

[0125] Step D - Synthesis of Compound Int-18d To a 40 mL vial containing a stirred solution of Int-18c (230 mg, 0.535 mmol) dissolved in DMF (6 mL) was added AcOH (609 μL) at room temperature. The vial was sealed with a pressure relief cap. The mixture was stirred at 120 °C for 3 days. At completion, the mixture was cooled to room temperature and then purified by 100 g reverse-phase C18 column using 0 - 100% acetonitrile-water (containing 0.05% TFA buffer) to give Int-18d. LCMS analysis C 26 H 27 Calculated for C18H23N3O3: 429.2; Found: 430.3 (M+H) + .

[0126] Step E - Synthesis of Compound Int-18e and Compound Int-18f Int-18d (974 mg, 2.268 mmol) was separated by SFC to obtain the first elution peak Int-18e and the second elution peak Int-18f. SFC conditions: Chiral OD-H column (250×21 mm, 10 μm); 45% EtOH; 70 mL / min. LCMS analysis C 26 H 27 Calculated value for N3O3: 429.2; Measured value: 430.3 (M+H) + .

[0127] Step F - Synthesis of Compound 18A and Compound 18B 10% Palladium on carbon (312 mg, 0.293 mmol) was added to a round-bottom flask (150 mL) containing Int-18e (420 mg, 0.978 mmol), and then EtOH (80 mL) was added under N2. A hydrogen balloon was attached to the flask. The system was degassed and refilled with H2 twice. Then the mixture was stirred at room temperature for 50 minutes. At the end, the catalyst was filtered off. The filtrate was concentrated under reduced pressure. The residue was purified by a 150 g C18 reverse-phase column using 0-100% ACN in water as the elution solvent to obtain 18A. LCMS analysis C 19 H 21 Calculated value for N3O3: 339.2; Measured value: 340.6 (M+H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 7.58 (d, J = 5.7 Hz, 2H), 7.38 (d, J = 7.3 Hz, 3H), 6.52 (d, J = 7.1 Hz, 1H), 5.73 (d, J = 7.1 Hz, 1H), 5.30 (s, 1H), 3.23 (s, 3H), 2.60 (d, J = 13.3 Hz, 2H), 2.30 (t, J = 13.0 Hz, 1H), 1.89 (dd, J = 24.2, 13.9 Hz, 3H), 1.65 (s, 3H).

[0128] Compound 18B was prepared from Int-18f essentially according to the method used to produce Compound 18A of Example 4. LCMS analysis C 19 H 21 Calculated for N3O3: 339.2; Found: 340.6 (M+H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 7.59 (d, J = 5.7 Hz, 2H), 7.40 (d, J = 7.8 Hz, 3H), 6.71 (d, J = 6.9 Hz, 1H), 6.13 (d, J = 7.0 Hz, 1H), 5.36 (s, 1H), 3.33 (s, 3H), 3.26 (s, 2H), 2.67 - 2.59 (m, 1H), 2.34 (t, J = 14.0 Hz, 1H), 1.99 - 1.87 (m, 3H), 1.65 (s, 3H).

[0129] The examples in Table 2 were prepared from the appropriate starting materials using procedures similar to those described in Example 4.

Table 3

[0130] Example 5 Preparation of Compound 56A, Compound 56B, Compound 56C and Compound 56D 5-(Cyclopropylmethyl)-7-hydroxy-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chem.

[0131] The above intermediate (5 g, 12.09 mmol) in DCM (50 mL) was added to TFA (12 mL) at 0 °C. The reaction solution was stirred at 30 °C for 1 h. Upon completion, the mixture was concentrated and purified by a 220 g silica gel flash column eluting with 0 - 10% MeOH in DCM to give Int-56a. LCMS analysis C 17 H 19 Calculated for C₁₅H₁₇N₃O₃: 313.1; Found: 314.1 (M+H) + .

[0132] Step B - Synthesis of Compound Int-56b To a solution of Int-56a (200 mg, 0.638 mmol) in DMF (2.0 mL) and AcOH (0.2 mL) in a 40 mL sealed tube, Int-1c (211 mg, 0.957 mmol) was added at 20 °C. The reaction was stirred at 120 °C for 4 h. Upon completion, it was concentrated and purified by a 50 g silica gel flash column using 0 - 10% MeOH in DCM to give Int-56b. LCMS analysis C 30 H 33Calculated value for N3O5: 515.2; Measured value: 515.3 (M+H) + .

[0133] Step C - Synthesis of Compound Int-56c To a solution of Int-56b (600 mg, 1.164 mmol) in THF (10 mL) was added NaHMDS (2.327 mL, 2.327 mmol) at -78 °C under N2. The resulting mixture was stirred at this temperature for 30 minutes, and then SEMCl (0.248 mL, 1.396 mmol) was added. The resulting reaction was slowly warmed to room temperature over 2 hours. Upon completion, it was quenched with NH4Cl (1 N, 20 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by an 80 g silica gel flash column using 100% EtOAc as the eluting solvent to give Int-56c and Int-56d. LCMS analysis C 36 H 47 Calculated value for N3O6Si: 645.3; Measured value: 646.4 (M+H) + .

[0134] Step D - Synthesis of Compound Int-56d To a solution of Int-56c (160 mg, 0.248 mmol) in MeOH (3 mL) was added K2CO3 (41.1 mg, 0.297 mmol) at 20 °C. The resulting mixture was stirred at this temperature for 3 hours. Upon completion, water (10 mL) was added. The mixture was extracted with EtOAc (3 × 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated to give Int-56d. LCMS analysis C 34 H 45 Calculated value for N3O5Si: 603.3; Measured value: 604.3 (M+H) + .

[0135] Step E - Synthesis of Compound Int-56e To a solution of Int-56d (145 mg, 0.240 mmol) in DCM (3 mL) was added triethylamine (0.1 mL, 0.720 mmol) and methanesulfonyl chloride (0.054 g, 0.480 mmol) at 0 °C. The resulting reaction mixture was stirred at room temperature for 30 minutes. Upon completion, 10 mL of DCM was added and the mixture was washed with 15 mL of 0.5 N HCl solution and brine. The organic layer was dried over Na2SO4 and concentrated to give the crude product. The crude product was used in the next reaction without purification.

[0136] The above crude product (210 mg, 0.337 mmol) in THF (2 mL) was added to TBAF (2.70 mL, 2.70 mmol) at 25 °C. The reaction mixture was stirred at 50 °C for 16 hours. Upon completion, it was concentrated and purified by a C18 column (YMC-Actus Triart C18 150×30 mm×5 μm) using 10%-100% ACN in water (containing 0.5% TFA regulator) as the elution solvent to give Int-56e. LCMS analysis C 28 H 29 Calculated for C18H19N3O3: 455.2; Found: 456.2 (M+H) + .

[0137] Step F - Synthesis of Compound Int-56 and Compound Int-56g Int-23e (20 mg, 0.044 mmol) was separated by SFC to give the first elution peak Int-56f and the second elution peak Int-56g. SFC conditions: Regis@ Whelk O1 column (250×21 mm, 5 μm); 50% EtOH (containing 0.1% NH3H2O as the regulator); 70 mL / min. LCMS analysis C 28 H 29 Calculated for C18H19N3O3: 455.2; Found: 456.2 (M+H) + .

[0138] Step G - Synthesis of Compound 56A and Compound 56B To a stirred mixture of Int-56f (7 mg, 0.015 mmol) in acetonitrile (1 mL), magnesium bromide (2.83 mg, 0.015 mmol) was added at 20 °C. The mixture was stirred at 20 °C for 2 h under a N2 atmosphere. Upon completion, it was purified by a C18 column (YMC-Actus Triart C18 150×30 mm×5 μm) using 10%-100% ACN in water (containing 0.5% TFA regulator) as the elution solvent to give 23A. LCMS analysis C 21 H 23 Calculated for C18H17N3O3: 365.2; Found: 366.2 (M+H) + . 1 H NMR (400 MHz, methanol-d 4 ) δ 7.30 (br s, 5H), 7.10 (d, J = 7.34 Hz, 1H), 5.95 (d, J = 7.34 Hz, 1H), 5.53 (br s, 1H), 4.22 (dd, J = 2.69, 11.74 Hz, 1H), 4.15 (dd, J = 8.07, 14.67 Hz, 1H), 3.25 - 3.29 (m, 1H), 2.58 (br d, J = 15.41 Hz, 1H), 2.24 - 2.32 (m, 2H), 1.97 (br d, J = 13.69 Hz, 1H), 1.85 - 1.92 (m, 1H), 1.72 - 1.81 (m, 1H), 1.14 - 1.21 (m, 1H), 0.54 - 0.62 (m, 2H), 0.41 - 0.46 (m, 2H).

[0139] Compound 56B was prepared from Int-56g essentially according to the method used to prepare compound 56A of Example 5. LCMS analysis C 21 H 23 Calculated for C18H17N3O3: 365.2; Found: 366.2 (M+H) + . 1 H NMR (400 MHz, methanol-d 4) δ 7.30 (broad singlet, 5H), 7.07 (doublet, J = 7.58 Hz, 1H), 5.89 (doublet, J = 7.58 Hz, 1H), 5.52 (broad singlet, 1H), 4.12 - 4.23 (multiplet, 2H), 3.24 - 3.30 (multiplet, 1H), 2.57 (broad doublet, J = 15.41 Hz, 1H), 2.24 - 2.32 (multiplet, 2H), 1.96 (broad doublet, J = 13.69 Hz, 1H), 1.88 (broad doublet, J = 13.69 Hz, 1H), 1.74 (quartet, J = 13.78 Hz, 1H), 1.12 - 1.21 (multiplet, 1H), 0.53 - 0.63 (multiplet, 2H), 0.41 - 0.46 (multiplet, 2H).

[0140] Step H - Synthesis of Compound Int-56h To a solution of Int-56b (350 mg, 0.679 mmol) in MeOH (5 mL) was added K2CO3 (113 mg, 0.815 mmol), and the mixture was stirred at 20 °C for 3 h. At the completion of the reaction, water (10 mL) was added. The mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by an 80 g silica gel flash column using 0 - 10% MeOH in DCM as the elution solvent to give Int-56h. LCMS analysis C 28 H 31 Calculated for C N3O4: 473.2; Found: 474.2 (M + H) + .

[0141] Step I - Synthesis of Compound Int-56i To a solution of Int-56h (295 mg, 0.623 mmol) in T3P (1586 mg, 2.492 mmol) was added methanesulfonic acid (59.9 mg, 0.623 mmol) under N2. The mixture was stirred at 50 °C for 16 h. At completion, the reaction mixture was purified by a C18 column (YMC-Actus Triart C18 150 × 30 mm × 5 um) using 10% - 100% ACN in water (containing 0.5% TFA modifier) as the elution solvent to give Int-56i. LCMS analysis of C 28 H 29 Calculated value for N3O3: 455.2; Measured value: 456.2 (M+H) + .

[0142] Step J - Synthesis of Compound Int-56j and Compound Int-56k Int-56i (80 mg, 0.176 mmol) was separated by SFC to obtain the first elution peak Int-56j and the second elution peak Int-56k. SFC conditions: Daicel@ chiralpak AD-H column (250×30 mm, 10 μm); 30% EtOH (containing 0.1% NH3H2O as modifier); 70 mL / min. LCMS analysis of C 28 H 29 Calculated value for N3O3: 455.2; Measured value: 456.2 (M+H) + .

[0143] Step K - Synthesis of Compound 56C and Compound 56D To a stirred mixture of Int-56j (30 mg, 0.066 mmol) in acetonitrile (1 mL), magnesium bromide (12.12 mg, 0.066 mmol) was added at 20 °C and it was stirred at 20 °C for 2 hours under N2 atmosphere. Upon completion, the reaction mixture was purified on a C18 column (YMC-Actus Triart C18 150×30 mm×5um) using 10%-100% ACN in water (containing 0.5% TFA modifier) as the elution solvent to obtain 56C. LCMS analysis of C 21 H 23 Calculated value for N3O3: 365.2; Measured value: 366.2 (M+H) + . 1 H NMR (400 MHz, methanol-d 4) δ 8.29 (d, J = 7.09 Hz, 1H), 7.20-7.27 (m, 1H), 7.15 (br t, J = 7.46 Hz, 2H), 7.05 (br d, J = 7.58 Hz, 2H), 6.89 (d, J = 7.09 Hz, 1H), 5.39-5.44 (m, 1H), 4.71 (br d, J = 6.36 Hz, 1H), 3.73 (dd, J = 7.58, 14.43 Hz, 1H), 3.28 (br d, J = 6.85 Hz, 1H), 2.67 (br d, J = 13.45 Hz, 1H), 2.46-2.59 (m, 1H), 2.19-2.43 (m, 3H), 1.87-1.97 (m, 1H), 1.06-1.17 (m, 1H), 0.49-0.62 (m, 2H), 0.38 (br d, J = 2.93 Hz, 2H).

[0144] Compound 56D was prepared from Int-56k essentially according to the method used to produce Compound 56C of Example 5. LCMS analysis C 21 H 23 Calculated for N3O3: 365.2; Found: 366.2 (M+H) + . 1 H NMR (400 MHz, methanol-d 4) δ 8.31 (d, J = 7.34 Hz, 1H), 7.20 - 7.27 (m, 1H), 7.15 (br t, J = 7.46 Hz, 2H), 7.04 (br d, J = 7.58 Hz, 2H), 6.92 (d, J = 7.34 Hz, 1H), 5.40 - 5.44 (m, 1H), 4.71 (br d, J = 6.36 Hz, 1H), 3.73 (dd, J = 7.58, 14.67 Hz, 1H), 3.26 - 3.30 (m, 1H), 2.67 (br d, J = 13.45 Hz, 1H), 2.47 - 2.59 (m, 1H), 2.19 - 2.43 (m, 3H), 1.87 - 1.97 (m, 1H), 1.05 - 1.17 (m, 1H), 0.48 - 0.63 (m, 2H), 0.33 - 0.43 (m, 2H).

[0145] Example 6 Preparation of Compound 57A and Compound 57B 4-Hydroxy-6-methyl-11-phenyl-6a,7,8,9,10,11-hexahydro-3H-pyrido[1’,2’:1,6][1,2,4]triazino[2,3-a]azepine-3,5(6H)-dione

Chem.

[0146] Step B - Synthesis of Compound Int-57b A solution of Int-1c (1 g, 3.66 mmol) in EtOH (10 mL) was added benzaldehyde (0.777 g, 7.32 mmol) under N2. The reaction was stirred at 80 °C for 10 h. Upon completion, it was concentrated. The residue was purified by an 80 g silica gel flash column using 0 - 100% MeOH in DCM as the eluting solvent to give Int-57b. LCMS analysis C 21 H 19 Calculated for N3O3: 361.1; Found: 362.2 (M + H) + .

[0147] Step C - Synthesis of Compound Int-57c A solution of Int-57b (100 mg, 0.277 mmol) in THF (5 mL) was added Int-57a (1 mL, 2.147 mmol, 2.147 M in THF) at 0 °C and stirred at this temperature for 1 h. Upon completion, it was quenched with aqueous NH4Cl (10 mL). The mixture was extracted with 2 × 50 mL of EtOAc. The combined organic layers were dried over MgSO4 and concentrated. The residue was purified by an 80 g silica gel flash column using 0 - 10% MeOH in DCM as the eluting solvent to give Int-57c. LCMS analysis C 27 H 31 Calculated for N3O3: 445.2; Found: 446.3 (M + H) + .

[0148] Step D - Synthesis of Compound Int-57d To a solution of Int-57c (630 mg, 1.414 mmol) in water (15 mL) and t-BuOH (15 mL) was added sodium periodate (1512 mg, 7.07 mmol), and then potassium osmate(VI) dihydrate (130 mg, 0.353 mmol) was added at 25 °C. The mixture was stirred at room temperature for 30 minutes. Upon completion, it was quenched with aqueous Na2SO3 (50 mL) and extracted with DCM (3 × 50 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by a 120 g silica gel flash column using 0-10% MeOH in DCM as the eluting solvent to give Int-57d. LCMS analysis C 26 H 29 Calculated for C19H17N3O4: 447.2; Found: 448.1 (M+H) + .

[0149] Step E - Synthesis of Compound Int-57e To a solution of Int-57d (400 mg, 0.894 mmol) in ACN (30 mL) was added MsOH (0.174 mL, 2.68 mmol). The resulting reaction mixture was stirred at 70 °C for 2.5 h. Upon completion, the mixture was concentrated and purified by preparative HPLC using a Boston Green ODS column (150×30 mm×5um) with aqueous ACN in water (0.1% TFA modifier) to give Int-57e (150 mg, 0.349 mmol). LCMS analysis C 26 H 27 Calculated for C19H17N3O3: 447.2; Found: 430.2 (M+H) + .

[0150] Step F - Synthesis of Compound Int-57f and Compound Int-57g Int-57e (250 mg, 0.582 mmol) was resolved by SFC to give the first eluting peak Int-57f and the second eluting peak Int-57g. SFC conditions: Daicel@ chiralpak IG column (250×30 mm, 10μm); 55% EtOH (containing 0.1% NH3H2O); 80 mL / min. LCMS analysis C26 H 27 Calculated value for N3O3: 447.2; Measured value: 430.2 (M+H) + .

[0151] Step G - Synthesis of Compound 57A and Compound 57B To a solution of Int-57f (80 mg, 0.186 mmol) in ACN (5 mL) was added MgBr2 (171 mg, 0.931 mmol) at 25 °C, and the mixture was stirred for 4 h. At completion, MeOH (1 mL) was added. The mixture was purified by preparative HPLC using a Phenomenex Synergi C18 column (150×30 mm×4um) with 0 - 100% ACN in water (containing 0.1% TFA regulator) as the elution solvent to give 57A (55.57 mg, 0.164 mmol). LCMS analysis C 19 H 21 Calculated value for N3O3: 339.1; Measured value: 340.2 (M+H) + . 1 H NMR (400 MHz, methanol-d 4 ) δ 7.85 (d, J = 7.3 Hz, 1H), 7.17 (br s, 5H), 6.29 (d, J = 7.3 Hz, 1H), 5.33 (dd, J = 7.6, 9.05 Hz, 1H), 4.79 (br d, J = 10.8 Hz, 1H), 3.28 (s, 3H), 2.53 - 2.67 (m, 2H), 2.10 - 2.27 (m, 2H), 2.05 (br dd, J = 3.67, 13.94 Hz, 1H), 1.90 - 1.99 (m, 2H), 1.59 - 1.72 (m, 1H).

[0152] Compound 57B was prepared from Int-57g essentially according to the method used to produce compound 57A of Example 6. LCMS analysis C 19 H 21 Calculated value for N3O3: 339.1; Measured value: 340.2 (M+H) + . 11H NMR (400 MHz, methanol-d 4 ) δ 7.85 (d, J = 7.34 Hz, 1H), 7.17 (br s, 5H), 6.27 (d, J = 7.3 Hz, 1H), 5.33 (dd, J = 7.5, 9.2 Hz, 1H), 4.79 (br d, J = 10.5 Hz, 1H), 3.28 (s, 3H), 2.51 - 2.67 (m, 2H), 2.09 - 2.25 (m, 2H), 2.05 (br dd, J = 3.8, 13.8 Hz, 1H), 1.89 - 2.00 (m, 2H), 1.60 - 1.73 (m, 1H).

[0153] Example 7 Preparation of Compound 58A, Compound 58B, Compound 58C and Compound 58D 4-(Benzyloxy)-6-methyl-12-phenyl-6,6a,7,8,9,10,11,12-octahydropyrido[1’,2’:1,6][1,2,4]triazino[2,3-a]azocine-3,5-dione

Chem.

[0154] Step B - Synthesis of Compound Int-58b To a solution of 1-phenylbut-3-en-1-ol (500 mg, 3.37 mmol) in DCM (10 mL) were added TEA (0.940 mL, 6.75 mmol) and methanesulfonyl chloride (0.394 mL, 5.06 mmol) at 0 °C, and the mixture was stirred at 0 °C for 1 hour. Upon completion, the mixture was quenched with water (10 mL) and extracted with MTBE (3 × 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated to give 1-phenylbut-3-en-1-yl methanesulfonate. This was used in the next step without further purification.

[0155] Step C - Synthesis of Compound Int-58c To a solution of Int-1d (350 mg, 0.859 mmol) in DMF (1 mL) and AcOH (0.1 mL) was added pent-4-enal (38.7 mg, 1.288 mmol) at 25 °C. The solution was stirred at 120 °C for 4 hours. Upon completion, the mixture was filtered and concentrated. The crude product was purified by a 12 g flash silica gel column using 0 - 10% MeOH in DCM as the eluting solvent to give Int-58c. LCMS analysis C 19 H 21 Calculated for C15H17N3O3: 339.2; Found: 340.2 (M+H) + .

[0156] Step D - Synthesis of Compound Int-58d To a solution of Int-58c (750 mg, 2.210 mmol) in DMF (10 mL) were added NaH (265 mg, 6.63 mmol) and 1-phenylbut-3-en-1-yl methanesulfonate (750 mg, 3.31 mmol) at 0 °C. The solution was stirred at 0 °C for 1 hour. Upon completion, the reaction was quenched with aqueous NH4Cl (5 mL) and diluted with water (20 mL). The mixture was extracted with DCM (3 × 30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by a 40 g flash silica gel column using 0 - 10% MeOH in DCM as the eluting solvent to give Int-58d. LCMS analysis C29 H 31 Calculated value for N3O3: 469.2; Measured value: 470.2 (M+H) + .

[0157] Step E - Synthesis of Compounds Int-58d-P1, Int-58d-P2, Int-58d-P3 and Int-58d-P4 The mixture Int-58d (200 mg, 0.426 mmol) was divided, and the mixture of Int-58d-P1 and Int-58d-P2 (110 mg, 0.223 mmol) was obtained as the isomer eluting first, and the mixture of Int-58d-P3 and Int-58d-P4 (90 mg, 0.168 mmol) was obtained as the isomer eluting second. SFC conditions: REGIS (s,s) WHELK-O1 column (250 mm × 30 mm, 5 μm); 55% EtOH (containing 0.1% NH3H2O); 60 mL / min.

[0158] The mixture of Int-58d-P1 and Int-58d-P2 (110 mg, 0.234 mmol) was further separated by SFC to obtain the first elution peak Int-25d-P1 and the second elution peak Int-58d-P2. SFC conditions: DAICEL CHIRALPAK AD-H (250 mm × 30 mm, 5 μm); 55% EtOH (containing 0.1% NH3H2O); 65 mL / min. LCMS analysis C 29 H 31 Calculated value for N3O3: 469.2; Measured value: 470.2 (M+H) + .

[0159] The mixture of Int-58d-P3 and Int-58d-P4 (90 mg, 0.192 mmol) was further separated by SFC to obtain the first elution peak Int-58d-P3 and the second elution peak Int-58d-P4. SFC conditions: Phenomenex-Amylose-1 (250 mm × 30 mm, 5 μm); 55% EtOH (containing 0.1% NH3H2O); 50 mL / min. LCMS analysis C 29 H 31 Calculated value for N3O3: 469.2; Measured value: 470.2 (M+H)+ .

[0160] Step F - Synthesis of Compounds Int-58e-P1, Int-58e-P2, Int-58e-P3 and Int-58e-P4 To a solution of Int-58d-P2 (50 mg, 0.106 mmol) in DCE (8 mL) was added (1,3-dimesitylimidazolidin-2-ylidene)(2-isopropoxybenzylidene)ruthenium(VI) chloride (13.34 mg, 0.021 mmol). The reaction mixture was stirred at 45 °C for 16 h. Upon completion, the reaction was quenched with water and extracted with DCM (3 × 10 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by pre-TLC eluting with 0→10% MeOH in DCM to afford (Z)-4-(benzyloxy)-6-methyl-12-phenyl-6,6a,7,8,11,12-hexahydropyrido[1’,2’:1,6][1,2,4]triazino[2,3-a]azocin-3,5-dione as a mixture.

[0161] The above mixture was combined with another batch (150 mg) and purified on a YMC-Actus Pro C18 (150 mm × 30 mm 5 μm) using ACN in water (containing 0.1% TFA modifier) as the elution solvent to afford a mixture. This was further purified by pre-TLC eluting with 0→10% MeOH in DCM to afford Int-58e-P1 as the first peak and Int-58e-P2 as the second peak. LCMS analysis C 27 H 27 Calculated for C23H25N3O3: 441.2; Found: 442.4 (M+H) + .

[0162] Compounds Int-58e-P3 and Int-58e-P4 were prepared from Int-58d-P3 essentially according to the method used for the preparation of compounds Int-58e-P1 and Int-58e-P2 of Example 7. LCMS analysis C 27 H 27Calculated value for N3O3: 441.2; Measured value: 442.4 (M+H) + .

[0163] Step G - Synthesis of Compounds 58A, 58B, 58C and 58D To a solution of Int-58e-P4 (25 mg, 0.057 mmol) in MeOH (2 mL) was added carbon-supported Pd (24.10 mg, 0.011 mmol). The reaction mixture was degassed under reduced pressure and purged with H2 three times. The reaction mixture was stirred at 20 °C for 1 hour under an H2 balloon. Upon completion, the reaction was filtered and purified by preparative HPLC using a YMC-Actus Pro C18 (150 mm × 30 mm 5 μm) column with ACN in water (containing 0.1% TFA regulator) as the elution solvent to obtain 58D. LCMS analysis C 20 H 23 Calculated value for N3O3: 353.2; Measured value: 354.2 (M+H) + . 1 H NMR (500 MHZ, methanol-d 4 ) δ 7.96 (dd, J = 7.2, 3.7 Hz, 1H), 6.78 - 7.61 (m, 5H), 6.64 (br t, J = 7.2 Hz, 1H), 5.15 (br d, J = 11.6 Hz, 1H), 4.57 - 4.66 (m, 1H), 3.00 (s, 3H), 2.32 - 2.48 (m, 1H), 1.55 - 2.09 (m, 8H), 1.23 - 1.39 (m, 1H).

[0164] Compounds 58A, 58B, and 58C were prepared from Int-58e-P1, Int-58e-P2, and Int-58e-P3, respectively, essentially following the method used to produce compound 58D of Example 7. 58A: LCMS analysis C 20 H 23 Calculated value for N3O3: 353.2; Measured value: 354.2 (M+H) + . 1 H NMR (500 MHz, methanol-d 4) δ 7.83 - 8.06 (m, 1H), 7.02 - 7.36 (m, 5H), 6.37 - 6.66 (m, 1H), 5.13 (br d, J = 9.6 Hz, 1H), 4.39 - 4.65 (m, 1H), 2.97 - 3.13 (m, 3H), 2.24 - 2.48 (m, 1H), 1.51 - 2.07 (m, 9H), 58B: LCMS analysis C 20 H 23 Calculated for C₁₈H₂₅N₃O₃: 353.2; Found: 354.2 (M+H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 7.84 - 7.99 (m, 1H), 6.98 - 7.54 (m, 5H), 6.49 - 6.75 (m, 1H), 5.15 (br dd, J = 11.4, 3.0 Hz, 1H), 4.43 - 4.64 (m, 1H), 2.98 - 3.10 (m, 3H), 2.30 - 2.46 (m, 1H), 1.54 - 2.10 (m, 8H), 1.31 (br d, J=10.7 Hz, 1H), 58C: LCMS analysis C 20 H 23 Calculated for C₁₈H₂₅N₃O₃: 353.2; Found: 354.2 (M+H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 7.65 - 7.87 (m, 1H), 6.87 - 7.27 (m, 5H), 6.29 - 6.55 (m, 1H), 4.90 - 5.11 (m, 1H), 4.20 - 4.49 (m, 1H), 2.83 - 3.01 (m, 3H), 2.12 - 2.32 (m, 1H), 1.38 - 1.92 (m, 9H),

[0165] Example 8 Preparation of Compounds 59A and 59B 7-Hydroxy-4a,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chemical Structure

[0166] Step B - Synthesis of Compound Int-59c To a mixture of Int-59b (200 mg, 0.611 mmol) in DCE (10 mL) were added ethyl acrylate (0.455 mL, 4.28 mmol) and Grubbs II (207 mg, 0.244 mmol) under N2. The mixture was stirred at 58 °C for 15 h. Upon completion, the mixture was concentrated. The residue was purified by ISCO® column on 12 g silica gel using 0 - 10% MeOH in DCM as the eluting solvent to afford Int-59c. LCMS analysis C 21 H 25 Calculated for C17H21N3O5: 399.2; Found: 400.2 (M+H) + .

[0167] Step C - Synthesis of Compound Int-59d To a mixture of Int-59c (200 mg, 0.501 mmol) in MeOH (4 mL) was added palladium on carbon (53.3 mg, 0.501 mmol). The mixture was degassed, refilled with H2, and stirred at 20 °C for 2 h. Upon completion, the mixture was filtered through celite. The filtrate was concentrated. The residue was purified by ISCO® on a 24 g silica gel column using 0 - 10% MeOH in DCM as the eluting solvent to give Int-59d. LCMS analysis C 21 H 27 Calculated for C N3O5: 401.2; Found: 402.2 (M+H) + .

[0168] Step D - Synthesis of Compound Int-59e To a mixture of Int-59d (200 mg, 0.498 mmol) in THF (4 mL) was added methylmagnesium bromide (1.495 mL, 1.495 mmol, 3 M in Et2O) under N2. The mixture was stirred at 20 °C for 0.5 h. Upon completion, the reaction mixture was quenched with aqueous NH4Cl (10 mL). It was extracted with DCM (2 × 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated to give Int-59e. The crude product was used in the next step without further purification. LCMS analysis C 20 H 24 Calculated for C N3O4: 371.2; Found: 372.2 (M+H) + .

[0169] Step E - Synthesis of Compound Int-59f To a mixture of Int-59e (7 mg, 0.019 mmol) in acetonitrile (1 mL) was added methanesulfonic acid (20 mg, 0.208 mmol) under N2. The mixture was stirred at 70 °C for 1.5 h. Upon completion, the mixture was concentrated. The residue was purified by prep HPLC using a Phenomenex Synergi C18 column (150 mm × 30 mm × 4 μm) with 0 - 100% ACN in water (containing 0.1% TFA modifier) as the eluting solvent to give Int-59f. LCMS analysis of C 20 H 23 Calculated value for N3O3: 353.2; Measured value: 354.2 (M+H) + .

[0170] Step F - Preparation of Compounds Int-59f-P1 and Int-59f-P2 Int-59f (33.5 mg, 0.095 mmol) was separated by SFC to obtain the first elution peak Int-59f-P1 and the second elution peak Int-59f-P2. SFC conditions: Daicel@ chiralpak AD column (250×30 mm, 10 μm); 40% IPA (containing 0.1% NH3H2O as modifier); 60 mL / min. LCMS analysis of C 20 H 23 Calculated value for N3O3: 353.2; Measured value: 354.2 (M+H) + .

[0171] Step G - Synthesis of Compounds 59A and 59B Magnesium bromide (23.44 mg, 0.127 mmol) was added to a stirred mixture of Int-59f-P1 (15 mg, 0.042 mmol) in ACN (3 mL) at 20 °C. The mixture was stirred at 20 °C for 2 h under a N2 atmosphere. At completion, the reaction was quenched with MeOH (0.2 mL). It was purified using prep HPLC on a Phenomenex Synergi C18 column (150 mm×30 mm×4 μm) with 0-100% ACN in water (containing 0.1% TFA modifier) as the elution solvent to obtain 59A. LCMS analysis of C 19 H 21 Calculated value for N3O3: 339.2; Measured value: 340.2 (M+H) + . 1 H NMR (500 MHz, methanol-d 4) δ 7.01 - 7.50 (m, 5H), 6.01 (d, J = 7.32 Hz, 1H), 4.12 (dd, J = 2.90, 11.75 Hz, 1H), 3.26 (s, 3H), 2.59 (br dd, J = 2.52, 15.34 Hz, 1H), 2.17 - 2.30 (m, 1H), 2.03 - 2.15 (m, 1H), 1.98 (br d, J = 13.73 Hz, 1H), 1.86 - 1.94 (m, 1H), 1.72 (tq, J = 3.47, 13.61 Hz, 1H), 1.43 (s, 3H).

[0172] Compound 59B was prepared from Int-59f-P2 essentially according to the method used to produce Compound 59A of Example 8. LCMS analysis C 19 H 21 Calculated for N3O3: 339.2; Found: 340.2 (M + H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 7.07 - 7.46 (m, 5H), 6.01 (d, J = 7.32 Hz, 1H), 4.12 (dd, J = 2.82, 11.83 Hz, 1H), 3.20 - 3.29 (m, 3H), 2.59 (dd, J = 2.67, 15.34 Hz, 1H), 2.25 (dq, J = 4.04, 12.84 Hz, 1H), 2.05 - 2.16 (m, 1H), 1.98 (td, J = 2.86, 13.66 Hz, 1H), 1.85 - 1.93 (m, 1H), 1.66 - 1.78 (m, 1H), 1.40 - 1.46 (m, 3H).

[0173] Example 9 Preparation of Compounds 60A and 60B 6-Hydroxy-4-methyl-1-phenyl-2,3,3a,4-tetrahydro-1H-pyrido[2,1-f]pyrrolo[1,2-b][1,2,4]triazine-5,7-dione

Chemical Structure

[0174] Step B - Synthesis of Compound Int-60b To a solution of 10-(3,5-dimethoxyphenyl)-9-mesityl-1,3,6,8-tetramethoxyacridin-10-ium tetrafluoroborate (8.87 mg, 0.014 mmol), Na2CO3 (73.3 mg, 0.692 mmol) and Int-60a (50 mg, 0.138 mmol) in dioxane (1384 μL), N2 was bubbled for 2 min, followed by the addition of 1,1,1,3,3,3-hexamethyl-2-(trimethylsilyl)trisilan-2-ol (36.6 mg, 0.138 mmol) and 3-bromo-1,1-dimethoxypropane (101 mg, 0.553 mmol). The resulting mixture was irradiated with Kessil Lamps (Kessil KSH150B Grow Light Blue (34w), 7 cm away from the reaction vessel at 20 °C for 15 h) for 16 h. Upon completion, the crude product was purified by a C18 reverse-phase column (YMC-Actus Triart C18 150×30mm×5um) using 30 - 100% ACN in water (containing 0.1% TFA regulator) to obtain Int-60b. LCMS analysis C 26 H 31 Calculated for C22H28N3O5: 465.2; Found: 466.2 (M+H) + .

[0175] Step C - Synthesis of Compound Int-60c To a solution of Int-60b (10 mg, 0.021 mmol) in ACN (1 mL) and H2O (0.2 mL), MsOH (4.18 μL, 0.064 mmol) was added. The mixture was stirred at 60 °C for 2 hours. Upon completion, the reaction mixture was directly purified by preparative HPLC using 30 - 100% ACN in water (containing 0.1% TFA regulator) (column: YMC-Actus Triart C18 150×30 mm×5um) to obtain Int-60c. LCMS analysis C 24 H 23 Calculated for N3O3: 401.2; Found: 402.0 (M+H) + .

[0176] Step D - Synthesis of Compounds Int-60d and Int-60e Int-27c (35 mg, 0.087 mmol) was separated by SFC to obtain the first elution peak Int-60d and the second elution peak Int-60e. SFC conditions: Daicel@ chiralpak AD-H column (250×30 mm, 5 μm); 30% EtOH (containing 0.1% NH3H2O); 60 mL / min. LCMS analysis C 24 H 23 Calculated for N3O3: 401.2; Found: 402.0 (M+H) + .

[0177] Step E - Synthesis of Compound 60A To a solution of Int-60d (17 mg, 0.042 mmol) in ACN (2 mL), magnesium bromide (39.0 mg, 0.212 mmol) was added at 25 °C and it was stirred for 16 hours. Upon completion, the mixture was diluted with MeOH (0.2 mL) and purified by reverse phase HPLC using 0 - 100% ACN in water (containing 0.1% TFA regulator) as the elution solvent (Agela DuraShell C18 150 mm×25 mm×5μm) to obtain 60A. LCMS analysis C 17 H 17 Calculated for N3O3: 311.2; Found: 312.1 (M+H) + . 11H NMR (500 MHz, methanol-d 4 ) δ 7.34 - 7.46 (m, 1H), 7.38 (br d, J = 8.24 Hz, 4H), 7.07 (d, J = 7.32 Hz, 1H), 6.23 (d, J = 7.32 Hz, 1H), 5.41 (d, J = 3.97 Hz, 1H), 4.30 - 4.52 (m, 1H), 2.50 - 2.72 (m, 3H), 2.12 - 2.19 (m, 1H).

[0178] Compound 60B was prepared essentially according to the method used to produce Compound 60A of Example 9. LCMS analysis C 17 H 17 Calculated for C₁₇H₁₅N₃O₃: 311.2; Found: 312.1 (M+H) + . 1 1H NMR (500 MHz, methanol-d 4 ) δ 7.33 - 7.49 (m, 5H), 7.09 (d, J = 7.48 Hz, 1H), 6.27 (d, J = 7.32 Hz, 1H), 5.42 (d, J = 4.12 Hz, 1H), 4.36 - 4.51 (m, 1H), 2.50 - 2.74 (m, 3H), 2.05 - 2.25 (m, 1H).

[0179] Example 10 Preparation of Compounds 61A and 61B 6-Hydroxy-1,4-dimethyl-1-phenyl-2,3,3a,4-tetrahydro-1H-pyrido[2,1-f]pyrrolo[1,2-b][1,2,4]triazine-5,7-dione

Chemical Structure

[0180] Step B - Synthesis of Compound Int-61b To a stirred suspension of Int-61a (240 mg, 0.551 mmol) in DCM (9184 μL) at 0 °C was added Dess-Martin periodinane (280 mg, 0.661 mmol). The mixture was stirred at room temperature for 2 h. Upon completion, the reaction mixture was diluted with DCM (200 mL), washed with saturated NaHCO3 (ca. 50 mL), and then washed with 10% Na2S2O3 in water (ca. 50 mL). The organic layer was separated, dried over Na2SO4, filtered, and concentrated under reduced pressure to afford Int-61b. LCMS m / z C 25 H 27Calculated value for N3O4: 433.2; Measured value 434.4 (M+H) + .

[0181] Step C - Synthesis of Compound Int-61c To a stirred solution of Int-61b (100 mg, 0.231 mmol) in DMF (3 mL), AcOH (262 μL) was added at room temperature. The mixture was then stirred at 120 °C for 2 hours. Upon completion, the mixture was cooled to room temperature and then purified by reverse-phase HPLC using an Isco system (RediSep Rf Gold C18 column 100 g) with 0 - 100% acetonitrile - water (buffered with 0.05% TFA) as the elution solvent to afford the TFA salt form of Int-61c (88 mg, 0.166 mmol). The above product was then redissolved in DCM, neutralized by the addition of TEA (32 uL, 0.23 mmol), and repurified by flash chromatography using an Isco system (40 g silica gel gold column) with 0 - 100% EtOAc - EtOH (3:1) / hexane as the elution solvent to afford the free base form of Int-61c. LCMS m / z C 25 H 25 Calculated value for N3O3: 415.2; Measured value 416.3 (M+H) + .

[0182] Step D - Synthesis of Compounds Int-61d and Int-61e Int-28c (32 mg, 0.077 mmol) was separated by SFC to afford the first elution peak Int-61d and the second elution peak Int-61e. SFC conditions: Daicel@ chiralpak OJ-H column (250×21 mm, 5 μm); 30% IPA (containing 0.2% DIPA); 50 mL / min. LCMS m / z C 25 H 25 Calculated value for N3O3: 415.2; Measured value 416.3 (M+H) + .

[0183] Step E - Synthesis of Compound 61A 10% palladium on carbon (17.93 mg, 0.017 mmol) was added to a round-bottom flask, followed by the addition of Int-61d (14 mg, 0.034 mmol) in EtOH (12 mL). A hydrogen balloon was attached to the flask. The system was degassed and flushed twice with H2. The mixture was stirred at room temperature under H2 for 40 minutes. Upon completion, the catalyst was filtered off and the filtrate was concentrated. The residue was purified by reverse-phase HPLC using a Gilson System (Sunfire Prep 30×150 mm column) with 5-100% acetonitrile-water (buffered with 0.05% TFA) as the eluting solvent to afford 61A. LCMS m / z C 18 H 19 Calculated for C H N3O3: 325.1; Found 326.3 (M+H) + . 1 H NMR (500 MHz, methanol-d4) δ 7.65 (d, J = 8.1 Hz, 2H), 7.45 (t, J = 7.5 Hz, 2H), 7.35 (t, J = 7.0 Hz, 1H), 7.16 (d, J = 7.4 Hz, 1H), 6.35 (d, J = 7.4 Hz, 1H), 5.47 (d, J = 3.7 Hz, 1H), 3.17 (s, 3H), 2.59 (dd, J = 14.0, 7.2 Hz, 1H), 2.51 - 2.41 (m, 1H), 2.40 - 2.30 (m, 1H), 2.23 (dd, J = 12.5, 7.8 Hz, 1H), 1.44 (s, 3H).

[0184] Compound 61B was prepared from Int-61e essentially according to the method used for the preparation of compound 61A of Example 10. LCMS m / z C 18 H 19 Calculated for C H N3O3: 325.1; Found 326.3 (M+H) + . 11H NMR (500 MHz, methanol-d4) δ 7.65 (d, J = 8.1 Hz, 2H), 7.46 (t, J = 7.5 Hz, 2H), 7.36 (t, J = 7.1 Hz, 1H), 7.23 (d, J = 7.4 Hz, 1H), 6.48 (d, J = 7.3 Hz, 1H), 5.49 (d, J = 3.7 Hz, 1H), 3.33 (s, 9H), 3.18 (s, 3H), 2.60 (dd, J = 14.2, 7.3 Hz, 1H), 2.53 - 2.41 (m, 1H), 2.41 - 2.31 (m, 1H), 2.24 (dd, J = 12.6, 7.9 Hz, 1H), 1.44 (s, 3H).

[0185] The examples in Table 3 were prepared from appropriate starting materials using a procedure similar to the procedure described in Example 10.

[0186]

Table 4

[0187] Example 11 Preparation of Compounds 64A, 64B, 64C and 64D 2-Allyl-1-(2-chlorophenyl)-7-hydroxy-5-methyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chemical formula

[0188] Step B - Synthesis of Compound Int-64b To a solution of Int-64a (4.64 g, 19.28 mmol) in THF (20 mL) was added potassium bis(trimethylsilyl)amide (19.33 mL, 19.33 mmol, 1 M) dropwise at room temperature under N2, followed by the addition of 3-bromoprop-1-ene (3.50 g, 28.9 mmol). The reaction mixture was stirred at room temperature for 16 h. At completion, methanol (10 mL) was added. The mixture was concentrated and saturated aqueous ammonium chloride solution (50 mL) was added. The mixture was extracted with ethyl acetate (2 × 100 mL). The combined organic fractions were dried over Na2SO4, filtered, and concentrated. The residue was purified by an 80 g silica gel flash column using 0-100% EtOAc in hexane as the eluent solvent to give methyl 4-(2-chlorobenzoyl)hepta-6-enoate. The substance was then resolved on a chiral AD-H column (50 × 250 mm, 5 um) using 10% MeOH as the eluent solvent to give Int-64b-peak 1 and Int-64b-peak 2. LCMS analysis C 15 H 17 Calculated value for ClO3: 280.1; Measured value: 281.2 (M+H) + .

[0189] Step C - Synthesis of Compound Int-64c - Peak 1 To a solution of Int-64b-peak 1 (993 mg, 3.54 mmol) in THF (35.4 mL) was added LAH (189 mg, 4.98 mmol) portionwise at 0 °C and stirred for 1 h. At completion, water (0.38 mL), 15% NaOH (0.19 mL), and water (0.95 mL) were sequentially added to the above reaction. It was then diluted with EtOAc (10 mL) and anhydrous MgSO4 (4 g) was added. The mixture was stirred at room temperature for 0.5 h, filtered, and concentrated to give Int-64c-peak 1. LCMS analysis C 14 H 19Calculated value for ClO2: 254.1; Measured value: 277.3 (M+Na) + .

[0190] Compound Int-64c-peak 2 was prepared essentially according to the method used to produce Compound Int-64c-peak 1 of Example 11. LCMS analysis C 14 H 19 Calculated value for ClO2: 254.1; Measured value: 277.2 (M+Na) + .

[0191] Step D - Synthesis of Compound Int-64d - Peak 1 Dess-Martin periodinane (3090 mg, 7.29 mmol) was added to a solution of Int-64c-peak 1 (928 mg, 3.64 mmol) in dichloromethane (36.40 mL) at 0 °C. The mixture was stirred at room temperature for 3 hours. Upon completion, the reaction was filtered and concentrated. The residue was purified by a 120 g silica gel flash column using 0 - 100% EtOAc in hexane as the eluting solvent to give Int-64d-peak 1. LCMS analysis C 14 H 15 Calculated value for ClO2: 250.08; Measured value: 251.15 (M+H) + .

[0192] Compound Int-64d-peak 2 was prepared essentially according to the method used to produce Compound Int-64d-peak 1 of Example 11. LCMS analysis C 14 H 15 Calculated value for ClO2: 250.0; Measured value: 250.9 (M+H) + .

[0193] Step E - Synthesis of Compound Int-64e - Peak 1 In a sealed tube, Int-64d-peak 1 (710 mg, 2.83 mmol) was added to 1-amino-3-(benzyloxy)-N-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide (774 mg, 2.83 mmol) in DMF (5.5 mL) and AcOH (0.550 mL). The resulting reaction mixture was stirred at 120 °C for 1.5 h. Upon completion, the reaction was cooled and concentrated. The residue was purified by an 80 g silica gel flash column using 0-10% MeOH in DCM as the elution solvent to afford Int-64e-peak 1 (1902 mg, 2.158 mmol). LCMS analysis C 28 H 28 Calculated for ClN3O4: 505.18; Found: 506.26 (M+H) + .

[0194] Compound Int-64e-peak 2 was prepared essentially according to the method used for the preparation of compound Int-64e-peak 1 of Example 11. LCMS analysis C 28 H 28 Calculated for ClN3O4: 505.2; Found: 506.3 (M+H) + .

[0195] Step F - Synthesis of Compound Int-64f - Peak 1 To a stirred solution of Int-64e-peak 1 (199 mg, 0.393 mmol) in DCM (10 mL) was added DIEA (0.137 mL, 0.787 mmol), followed by the addition of SEMCl (0.070 mL, 0.393 mmol) at 0 °C. The resulting reaction mixture was stirred at room temperature for 16 h. Upon completion, it was concentrated. The residue was purified by a 40 g silica gel flash column using 0-10% MeOH in DCM as the elution solvent to afford Int-64f-peak 1. LCMS analysis C 34 H 42 Calculated for ClN3O5Si: 635.26; Found: 636.52 (M+H) + .

[0196] Compound Int-64f-peak 2 was prepared essentially according to the method used to produce Compound Int-64f-peak 1 of Example 11. LCMS analysis C 34 H 42 Calculated for ClN3O5Si: 635.3; Found: 636.4 (M+H) + .

[0197] Step G - Synthesis of Compound Int-64g - Peak 1 Under N2, Int-64f-peak 1 (193 mg, 0.303 mmol) in a mixture of dichloromethane (4 mL) and methanol (2 mL) was cooled to -5 °C, then NaBH4 (9.18 mg, 0.243 mmol) was added and the mixture was stirred for 1.5 h. At completion, the reaction was quenched with 0.4 mL of 1N HCl and concentrated. The residue was purified by a 40 g silica gel flash column using 0-10% MeOH in DCM as the eluting solvent to afford Int-64g-peak 1. LCMS analysis C 34 H 44 Calculated for ClN3O5Si: 637.27; Found: 638.54 (M+H) + .

[0198] Compound Int-64g-peak 2 was prepared essentially according to the method used to produce Compound Int-64g-peak 1 of Example 11. LCMS analysis C 34 H 44 Calculated for ClN3O5Si: 637.3; Found: 638.4 (M+H) + .

[0199] Step H - Synthesis of Compound Int-64h - Peak 1 A solution of Int-64g-peak 1 (166 mg, 0.260 mmol) in 5.0 mL of DCM was cooled to 0 °C, triethylamine (0.181 mL, 1.30 mmol) was added, followed by methanesulfonyl chloride (0.07 mL, 0.898 mmol). The reaction was stirred at room temperature for 1.5 h. Upon completion, the reaction was diluted with dichloromethane (20 mL) and washed with water. The organic layer was separated, dried over Na2SO4, filtered, and concentrated to give Int-64h-peak 1. LCMS analysis C 35 H 46 Calculated for ClN3O7SSi: 715.25; Found: 716.36 (M+H) + .

[0200] Compound Int-64h-peak 2 was prepared essentially according to the method used to produce Compound Int-64h-peak 1 of Example 11. LCMS analysis C 35 H 46 Calculated for ClN3O7SSi: 715.3; Found: 716.4 (M+H) + .

[0201] Step I - Synthesis of Compound Int-64i - Peak 1a Tetrabutylammonium fluoride (1.577 mL, 1.577 mmol, 1 M) was added to a solution of Int-64h-peak 1 (226 mg, 0.315 mmol) in 5 mL of THF. The resulting reaction was heated at 60 °C for 2 h. Additional tetrabutylammonium fluoride (1.577 mL, 1.577 mmol, 1 M) was added and the mixture was heated for 1.5 h. Upon completion, the resulting reaction was concentrated. The residue was purified by a 24 g silica gel flash column using 0 - 10% MeOH in DCM as the eluting solvent to give the racemic material. The material was resolved using a chiral AS-H column (21×250 mm, 5 μM) with 40% IPA as the eluting solvent to give Int-64i-peak 1a and Int-64i-peak 1b. LCMS analysis C 28 H 28Calculated value for ClN3O3: 489.18; Measured value: 490.35 (M+H) + .

[0202] Compound Int-64i-peak 2a and compound Int-64i-peak 2b were prepared essentially according to the method used to produce compound Int-31i-peak 1 of Example 11. LCMS analysis C 28 H 28 Calculated value for ClN3O3: 489.2; Measured value: 490.3 (M+H) + .

[0203] Step J - Synthesis of Compound 64A To a solution of Int-64i-peak 1a (20 mg, 0.041 mmol) in 1 mL of DMF was added lithium chloride (17.30 mg, 0.408 mmol), and it was stirred at 100 °C for 2.5 hours. Upon completion, the reaction was concentrated. The residue was purified by preparative HPLC using a reverse-phase C18 column with 0 - 100% acetonitrile in water (0.05% TFA regulator) as the elution solvent to obtain compound 64A. LCMS analysis C 21 H 22 Calculated value for ClN3O3: 399.1; Measured value: 400.3 (M+H) + . 1 H NMR (500 MHz, methanol-d 4) δ 7.83 (dd, J = 7.9, 1.6 Hz, 1H), 7.48 (td, J = 7.5, 1.4 Hz, 1H), 7.41 - 7.25 (m, 2H), 7.06 (d, J = 7.4 Hz, 1H), 5.95 (d, J = 7.5 Hz, 1H), 5.65 (dddd, J = 16.8, 10.2, 7.9, 6.5 Hz, 1H), 5.33 (t, J = 3.0 Hz, 1H), 5.02 - 4.95 (m, 1H), 4.95 - 4.87 (m, 1H), 4.79 (d, J = 10.6 Hz, 1H), 3.26 (s, 3H), 2.58 (dq, J = 15.4, 3.2 Hz, 1H), 2.44 - 2.22 (m, 2H), 2.07 - 1.93 (m, 1H), 1.89 - 1.72 (m, 2H), 1.48 (qd, J = 14.1, 3.4 Hz, 1H).

[0204] Compound 64B was prepared essentially according to the method used to produce Compound 64A of Example 11. LCMS analysis C 21 H 22 Calculated for ClN3O3: 399.13; Found: 400.34 (M+H) + . 1 H NMR (500 MHz, methanol-d 4) δ 7.84 (dd, J = 7.8, 1.6 Hz, 1H), 7.49 (td, J = 7.6, 1.4 Hz, 1H), 7.39 - 7.25 (m, 2H), 7.10 (d, J = 7.4 Hz, 1H), 6.02 (dd, J = 7.4, 1.3 Hz, 1H), 5.65 (dddd, J = 16.8, 10.2, 7.9, 6.5 Hz, 1H), 5.35 (t, J = 3.1 Hz, 1H), 4.98 (ddd, J = 10.1, 2.1, 1.0 Hz, 1H), 4.92 (dd, J = 17.0, 1.8 Hz, 1H), 4.79 (d, J = 10.6 Hz, 1H), 3.27 (s, 3H), 2.59 (dq, J = 15.6, 3.2 Hz, 1H), 2.33 (dddt, J = 48.0, 15.4, 14.0, 4.0 Hz, 2H), 2.07 - 1.93 (m, 1H), 1.89 - 1.73 (m, 2H), 1.48 (tdd, J = 14.0, 11.9, 3.5 Hz, 1H).

[0205] Compound 64C was prepared essentially according to the method used to prepare compound 64A of Example 11. LCMS analysis C 21 H 22 Calculated for ClN3O3: 399.1; Found: 400.2 (M+H) + . 1 H NMR (500 MHz, methanol-d 4) δ 7.84 (dd, J = 7.9, 1.6 Hz, 1H), 7.49 (td, J = 7.5, 1.4 Hz, 1H), 7.35 (ddd, J = 8.8, 7.2, 1.6 Hz, 1H), 7.29 (dd, J = 8.1, 1.4 Hz, 1H), 7.14 (dd, J = 7.4, 1.0 Hz, 1H), 6.08 (dd, J = 7.4, 1.6 Hz, 1H), 5.65 (dddd, J = 16.8, 10.2, 7.9, 6.5 Hz, 1H), 5.36 (t, J = 3.1 Hz, 1H), 4.98 (ddt, J = 10.1, 2.0, 1.0 Hz, 1H), 4.92 (dd, J = 16.8, 1.7 Hz, 1H), 4.80 (d, J = 10.6 Hz, 1H), 3.27 (s, 3H), 2.59 (dq, J = 15.6, 3.2 Hz, 1H), 2.38 (dtt, J = 10.5, 8.1, 4.1 Hz, 1H), 2.29 (ddt, J = 17.3, 15.5, 3.7 Hz, 1H), 2.08 - 1.93 (m, 1H), 1.89 - 1.79 (m, 1H), 1.84 - 1.73 (m, 1H), 1.49 (tdd, J = 14.1, 11.9, 3.5 Hz, 1H). ).

[0206] Compound 64D was prepared essentially according to the method used to produce compound 64C of Example 11. LCMS analysis C 21 H 22 Calculated for ClN3O3: 399.1; Found: 400.2 (M+H) + . 11H NMR (500 MHz, methanol-d4) δ 7.84 (dd, J = 7.9, 1.6 Hz, 1H), 7.49 (td, J = 7.6, 1.4 Hz, 1H), 7.35 (ddd, J = 8.8, 7.2, 1.6 Hz, 1H), 7.29 (dd, J = 8.2, 1.3 Hz, 1H), 7.15 (d, J = 7.4 Hz, 1H), 6.09 (d, J = 7.4 Hz, 1H), 5.65 (dddd, J = 16.8, 10.2, 7.9, 6.5 Hz, 1H), 5.36 (t, J = 3.1 Hz, 1H), 5.02 - 4.94 (m, 1H), 4.99 - 4.87 (m, 1H), 4.80 (d, J = 10.6 Hz, 1H), 3.27 (s, 3H), 2.60 (dq, J = 15.5, 3.2 Hz, 1H), 2.39 (dtt, J = 10.5, 8.1, 4.1 Hz, 1H), 2.29 (ddt, J = 15.5, 14.0, 3.6 Hz, 1H), 2.08 - 1.93 (m, 1H), 1.89 - 1.79 (m, 1H), 1.84 - 1.73 (m, 1H), 1.49 (tdd, J = 14.1, 11.9, 3.5 Hz, 1H).

[0207] Example 12 Preparation of Compound 65A and Compound 65B 1-(2-Chlorophenyl)-7-hydroxy-5-methyl-2-propyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chem.

[0208] Compound 65B was prepared from Int-64i-peak 1b essentially according to the method used to produce compound 65A of Example 12. LCMS analysis C 21 H 24 Calculated value for ClN3O3: 401.2; Measured value: 402.3 (M+H) + . 1 H NMR (500 MHz, methanol-d 4) δ 7.82 (dd, J = 7.9, 1.6 Hz, 1H), 7.48 (td, J = 7.5, 1.4 Hz, 1H), 7.34 (ddd, J = 8.7, 7.2, 1.6 Hz, 1H), 7.28 (dd, J = 8.1, 1.4 Hz, 1H), 7.15 (d, J = 7.4 Hz, 1H), 6.09 (d, J = 7.4 Hz, 1H), 5.36 (t, J = 3.1 Hz, 1H), 4.77 (d, J = 10.5 Hz, 1H), 3.28 (s, 3H), 2.60 (dq, J = 15.5, 3.3 Hz, 1H), 2.35 - 2.23 (m, 2H), 2.08 (dq, J = 13.9, 3.7 Hz, 1H), 1.51 - 1.29 (m, 2H), 1.16 (dddd, J = 13.2, 10.0, 7.4, 6.2 Hz, 1H), 1.05 (dtd, J = 14.4, 9.6, 4.7 Hz, 1H), 0.95 (dddd, J = 13.7, 10.0, 6.2, 3.3 Hz, 1H), 0.76 (t, J = 7.3 Hz, 3H).

[0209] Example 13 Preparation of Compound 66A and Compound 66B 1-(2-Chlorophenyl)-7-hydroxy-2-(2-hydroxyethyl)-5-methyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chem.

[0210] Int-66a2 was prepared essentially according to the method used to produce the compound Int-66a1 of Example 13. LCMS analysis C 27 H 26 Calculated value for ClN3O4: 491.2; Measured value: 492.1 (M+H) + .

[0211] Step B - Synthesis of Compound Int-66b1 Sodium borohydride (2.307 mg, 0.061 mmol) was added to a solution of Int-66a1 (30 mg, 0.061 mmol) in a mixture of DCM (4 mL) and MeOH (2 mL) at 0 °C and stirred for 1.5 h. At completion, the reaction was quenched with 1N HCl (0.06 mL) and concentrated. The residue was purified by silica gel flash chromatography using 0 - 10% MeOH in DCM as the eluting solvent to give Int-66b1. LCMS analysis C 27 H 28 Calculated value for ClN3O4: 493.2; Measured value: 494.4 (M+H) + .

[0212] Int-66b2 was prepared essentially according to the method used to produce the compound Int-66b1 of Example 13. LCMS analysis C 27 H 28 Calculated value for ClN3O4: 493.2; Measured value: 494.1 (M+H) + .

[0213] Step C - Synthesis of Compound 66A Lithium chloride (45.3 mg, 1.06 mmol) was added to a solution of Int-66b1 (26.4 mg, 0.053 mmol) in 2 mL of DMF. The reaction mixture was stirred at 100 °C for 4.5 h. Upon completion, the mixture was cooled and purified by preparative reverse-phase HPLC on a C18 column using acetonitrile in water (containing 0.05% TFA regulator) as the eluting solvent to afford Compound 66A. LCMS analysis C 20 H 22 Calculated for C18H19ClN3O4: 403.1; Found 404.2: (M+H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 7.82 (t, J = 7.9 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 7.41 - 7.25 (m, 2H), 7.12 (dd, J = 9.5, 7.5 Hz, 1H), 6.02 (dd, J = 20.0, 7.4 Hz, 1H), 5.36 (d, J = 2.7 Hz, 1H), 4.82 (dd, J = 14.9, 10.7 Hz, 1H), 4.34 - 4.24 (m, 1H), 3.47 (t, J = 6.5 Hz, 1H), 3.28 (s, 3H), 2.61 (ddd, J = 15.4, 7.0, 3.3 Hz, 1H), 2.44 (dt, J = 10.5, 5.3 Hz, 1H), 2.36 - 2.22 (m, 1H), 2.12 (dq, J = 10.1, 3.4 Hz, 1H), 1.68 - 1.38 (m, 2H), 1.25 (q, J = 6.6 Hz, 1H).

[0214] Compound 66B was prepared essentially according to the method used to produce Compound 66A of Example 13. LCMS analysis C 20 H 22 Calculated for C18H19ClN3O4: 403.1; Found: 404.2 (M+H) + . 11H NMR (500 MHz, methanol-d4) δ 7.85 - 7.78 (m, 1H), 7.52 - 7.42 (m, 1H), 7.42 - 7.26 (m, 2H), 7.13 (dd, J = 10.0, 7.4 Hz, 1H), 6.08 - 5.99 (m, 1H), 5.36 (q, J = 2.8 Hz, 1H), 4.82 (dd, J = 14.8, 10.6 Hz, 1H), 4.29 (dd, J = 7.5, 5.5 Hz, 1H), 3.50 - 3.42 (m, 1H), 3.28 (s, 3H), 2.61 (ddq, J = 13.7, 6.4, 3.2 Hz, 1H), 2.44 (qt, J = 9.9, 5.1 Hz, 1H), 2.36 - 2.24 (m, 1H), 2.12 (ddq, J = 13.8, 7.1, 3.7 Hz, 1H), 1.62 - 1.39 (m, 2H), 1.33 - 1.21 (m, 1H).

[0215] Example 14 Preparation of Compound 67 1-(2-Chlorophenyl)-7-hydroxy-2-(2-methoxyethyl)-5-methyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione [Chemical formula] Step A - Synthesis of Compound Int-67a To a solution of compound 66A (3.6 mg, 6.95 μmol) in DMF (1 mL) was added sodium hydride (1.668 mg, 0.042 mmol) under N2, followed by addition of iodomethane (1.912 μL, 0.03 mmol), and the mixture was stirred at room temperature for 16 h. Upon completion, the resulting reaction mixture was filtered and purified by preparative reverse-phase HPLC on a C18 column using 0 - 100% acetonitrile in water (containing 0.05% TFA modifier) as the elution solvent to obtain Int-67a. LCMS analysis C 22 H 26 Calculated for C18H19ClN3O4: 431.2; Found: 432.2 (M + H) + .

[0216] Step B - Synthesis of Compound 67 Lithium chloride (4.658 mg, 0.110 mmol) was added to a solution of Int-67a (2 mg, 0.0037 mmol) in 2 mL of DMF, and the mixture was heated at 100 °C for 2.5 h. Upon completion, the mixture was concentrated. The residue was purified by preparative reverse-phase HPLC on a C18 column using 0 - 100% acetonitrile in water (containing 0.05% TFA regulator) as the elution solvent to obtain Compound 67. LCMS analysis C 21 H 24 Calculated for C18H19ClN3O4: 417.2; Found: 418.2 (M+H) + . 1 1H NMR (500 MHz, methanol-d 4 ) δ 7.80 (dd, J = 7.8, 1.6 Hz, 1H), 7.46 (td, J = 7.4, 1.3 Hz, 1H), 7.32 (td, J = 7.7, 7.2, 1.6 Hz, 1H), 7.28 (dd, J = 8.1, 1.4 Hz, 1H), 7.02 (d, J = 7.5 Hz, 1H), 5.87 (d, J = 7.5 Hz, 1H), 5.32 (t, J = 3.1 Hz, 1H), 4.78 (d, J = 10.7 Hz, 1H), 3.28 - 3.18 (m, 4H), 2.58 (dd, J = 15.4, 3.2 Hz, 1H), 2.40 (d, J = 11.2 Hz, 1H), 2.33 - 2.21 (m, 1H), 2.08 (dd, J = 13.8, 3.8 Hz, 1H), 1.54 - 1.42 (m, 1H), 1.31 (s, 2H), 1.31 - 1.20 (m, 1H).

[0217] Example 15 Preparation of Compound 68A, Compound 68B, Compound 68C, Compound 68D 3,3-Difluoro-7’-hydroxy-5’-methyl-1’-phenyl-3’,4’,4a’,5’-tetrahydro-1’H-spiro[cyclopentane-1,2’-dipyrido[1,2-b:2’,1’-f][1,2,4]triazine]-6’,8’-dione

Chemical Structure

[0218] Step B - Synthesis of Compound Int-68b Under N2, Int-68a (10.5 g, 54.3 mmol) was dissolved in 200 mL of THF, and then phenylmagnesium bromide (70.7 mL, 1 M, 70.7 mmol) was added at 0 °C. The reaction was stirred at room temperature for 24 h. Upon completion, saturated NH4Cl (ca. 100 mL) was added, and the organic layer was extracted with EtOAc (200 mL). The organic layer was washed with brine (50 mL), dried (NaSO4), filtered, and concentrated. The residue was purified by a 220 g silica gel column using 0 - 80% EtOAc / hexane as the elution solvent to afford Int-68b. LCMS analysis C 12 H 12 Calculated for C H F2O: 210.22; Found: 211.1 (M+H) +

[0219] Step C - Synthesis of Compound Int-68c Under N2, to a solution of Int-68b (2.508 g, 11.93 mmol) in THF (100 mL) at 0 °C was added tert-butyl acrylate (1.77 g, 13.50 mmol), followed by sodium tert-butoxide (6.75 mL, 2 M, 13.50 mmol). The resulting reaction mixture was stirred for 2 h. Upon completion, the mixture was poured into an aqueous NH4Cl solution and extracted with EtOAc (30 mL). The organic layer was washed with brine (15 mL), dried (Na2SO4), filtered, and concentrated. The residue was purified by an 80 g silica gel column using 0 - 50% EtOAc in hexane as the eluting solvent to give Int-68c. LCMS analysis C 19 H 24 Calculated for C: 338.39; Found: 361.01 (M+Na) + .

[0220] Step D - Synthesis of Compound Int-68d To a solution of Int-68c (1.705 g, 5.04 mmol) in THF (45 mL) at 0 °C was added LAH (0.269 g, 7.09 mmol). The reaction mixture was stirred at room temperature for 90 min. Upon completion, it was quenched with 0.54 mL of H2O, 0.27 mL of 15% NaOH, and 1.35 mL of H2O, then diluted with EtOAc (25 mL) and stirred with MgSO4 (5 g) at room temperature for 1 h. It was filtered and concentrated to give Int-68d. LCMS analysis C 15 H 20 Calculated for C: 270.32; Found: 253.2 (M-OH) +

[0221] Step E - Synthesis of Compound Int-68e Des-martin periodinane (4971 mg, 11.72 mmol) was added to a solution of Int-68d (1440 mg, 5.33 mmol) in dichloromethane (50 mL) at 0 °C. The mixture was stirred at room temperature for 2 h. Upon completion, the reaction was filtered and concentrated. The residue was purified by an 80 g silica gel flash column using 0 - 100% EtOAc in hexane as the elution solvent to give Int-68e. LCMS analysis C 15 H 16 Calculated for F2O2: 266.11; Found: 267.2 (M+H) + .

[0222] Step F - Synthesis of Compound Int-68f Acetic acid (0.9 mL) was added to a solution of Int-68e (528 mg, 1.98 mmol) in DMF (8.5 mL), followed by 1-amino-3-methoxy-N-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide (391 mg, 1.983 mmol), and the mixture was heated at 120 °C for 2.5 h. Upon completion, the mixture was concentrated. The residue was purified by an 80 g silica gel column using 0 - 10% MeOH in CH2Cl2 to give Int-1f. LCMS analysis C 23 H 25 Calculated for F2N3O4: 445.18; Found: 446.2 (M+H) + .

[0223] Step G - Synthesis of Compound Int-68g To a solution of Int-68f (633 mg, 1.42 mmol) in trifluoroethanol (15 mL) was added triethylsilane (1.362 mL, 8.53 mmol) and triflic acid (0.757 mL, 8.53 mmol), and then the mixture was stirred at 35 °C for 16 h. The reaction was cooled and neutralized with saturated aqueous NaHCO3 (16 mL). The mixture was extracted with DCM. The organic layer was dried over MgSO4, filtered, and concentrated. The residue was first purified by a 120 g silica gel column using 0 - 10% MeOH in CH2Cl2 and then by preparative HPLC using a SunFire C18 OBD Prep column (19×100 mm, 5 μm) with ACN in water (containing 0.05% TFA modifier) to give racemic Int-68g. The substance was resolved using an AS-H (21×250 mm, 5 μm) chiral column with 25% EtOH (containing 0.2% DIPA) at 70 mL / min as the eluent to give Int-68g-peak 1, Int-68g-peak 2, Int-68g-peak 3, and Int-68g-peak 4. LCMS analysis C 23 H 25 Calculated for F2N3O3: 429.19; found: 430.2 (M+H) + .

[0224] Step H - Synthesis of Compounds 68A, 68B, 68C and 68D To a stirred mixture of Int-68g-peak 1 (4 mg, 9.31 μmol) in DMF (1 mL) was added lithium chloride (47 mg, 1.1 mmol) at room temperature. The mixture was stirred at 100 °C for 5 h. Upon completion, the mixture was cooled. The residue was purified by preparative HPLC using a SunFire C18 OBD Prep column (19×100 mm, 5 μm) with ACN in water (containing 0.05% TFA modifier) as the eluent to give compound 68A. LCMS analysis C 22 H 23 Calculated for F2N3O3: 415.17; found: 416.17 (M+H) + . 11H NMR (500 MHz, methanol-d4) δ 7.93 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 7.4 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 7.35 (t, J = 7.4 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 6.02 (d, J = 7.4 Hz, 1H), 5.37 (t, J = 2.8 Hz, 1H), 4.40 (s, 1H), 3.26 (s, 3H), 2.91 (ddd, J = 13.6, 9.0, 4.0 Hz, 1H), 2.50 - 2.34 (m, 2H), 2.09 (t, J = 13.0 Hz, 1H), 2.00 - 1.88 (m, 3H), 1.88 - 1.68 (m, 3H).

[0225] 68B was prepared essentially according to the method used to produce 68A of Example 15. LCMS analysis C 22 H 23 Calculated for F2N3O3: 415.17; Found: 416.17 (M+H) + . 1 1H NMR (500 MHz, methanol-d4) δ 7.95 - 7.79 (m, 2H), 7.41 (t, J = 7.6 Hz, 1H), 7.33 (t, J = 7.4 Hz, 1H), 7.21 (t, J = 7.4 Hz, 1H), 7.00 (d, J = 7.5 Hz, 1H), 6.06 (d, J = 7.4 Hz, 1H), 5.37 (t, J = 2.9 Hz, 1H), 4.40 (s, 1H), 3.26 (s, 3H), 3.22 - 2.97 (m, 1H), 2.51 (dq, J = 15.8, 3.1 Hz, 1H), 2.44 - 2.22 (m, 2H), 1.97 - 1.71 (m, 3H), 1.60 (t, J = 7.5 Hz, 2H), 1.11 (tdd, J = 21.0, 13.0, 8.6 Hz, 1H).

[0226] 68C was prepared essentially according to the method used to produce 68A of Example 15. LCMS analysis C 22 H 23 Calculated for F2N3O3: 415.17; Found: 416.17 (M+H) + . 1 H NMR (500 MHz, methanol-d4) δ 7.93 (d, J = 7.7 Hz, 1H), 7.77 (d, J = 7.4 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.35 (t, J = 7.4 Hz, 1H), 7.21 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 7.5 Hz, 1H), 6.01 (d, J = 7.4 Hz, 1H), 5.37 (t, J = 2.8 Hz, 1H), 4.40 (s, 1H), 3.25 (s, 3H), 2.91 (ddd, J = 13.6, 9.1, 4.0 Hz, 1H), 2.52 - 2.32 (m, 2H), 2.18 - 2.03 (m, 1H), 2.01 - 1.90 (m, 2H), 1.90 - 1.64 (m, 4H).

[0227] 68D was prepared essentially according to the method used to produce 68A of Example 15. LCMS analysis C 22 H 23 Calculated for F2N3O3: 415.17; Found: 416.19 (M+H) + . 11H NMR (500 MHz, methanol-d4) δ 7.86 (d, J = 7.4 Hz, 2H), 7.41 (t, J = 7.5 Hz, 1H), 7.34 (t, J = 7.4 Hz, 1H), 7.21 (t, J = 7.3 Hz, 1H), 7.00 (d, J = 7.5 Hz, 1H), 6.07 (d, J = 7.4 Hz, 1H), 5.37 (t, J = 2.8 Hz, 1H), 4.41 (s, 1H), 3.27 (s, 3H), 3.18 - 3.06 (m, 1H), 2.51 (dq, J = 15.9, 3.1 Hz, 1H), 2.39 - 2.25 (m, 2H), 1.91 - 1.74 (m, 3H), 1.60 (t, J = 7.6 Hz, 2H), 1.11 (tdd, J = 21.1, 13.3, 8.7 Hz, 1H).

[0228] The examples in Table 4 were prepared from appropriate starting materials using a procedure similar to the procedure described in Example 15.

[0229] [Table 5] TIFF0007695339000047.tif246168TIFF0007695339000048.tif219170TIFF0007695339000049.tif236170TIFF0007695339000050.tif243168TIFF0007695339000051.tif245169TIFF0007695339000052.tif243168TIFF0007695339000053.tif241168TIFF0007695339000054.tif227169TIFF0007695339000055.tif239169TIFF0007695339000056.tif247169TIFF0007695339000057.tif248169TIFF0007695339000058.tif245170TIFF0007695339000059.tif217168TIFF0007695339000060.tif236168TIFF0007695339000061.tif250167TIFF0007695339000062.tif232167TIFF0007695339000063.tif231170TIFF0007695339000064.tif237168TIFF0007695339000065.tif232168TIFF0007695339000066.tif158167

[0230] Example 16 Preparation of Compounds 130A and 130B 7-Hydroxy-3-isobutyl-5-methyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chem.

[0231] Step B - Synthesis of Compound Int-130b TMS-diazomethane (3.56 mL, 7.13 mmol) was added to Int-130a (885 mg, 3.56 mmol) in a mixture of DCM (9.0 mL) and MeOH (3.0 mL) at 0 °C. The resulting reaction was stirred for 1 hour. Upon completion, it was concentrated. The residue was purified by silica gel flash chromatography using 0 - 100% EtOAc in hexane as the eluent to give 510 mg of the racemic material. The racemic compound was resolved on a chiral AD-H (21 × 250 mm, 5 μm) column using 5% IPA (0.1% DIPA) as the eluent to give Int-130b-peak 1 and Int-130b-peak 2. LCMS analysis C 16 H 22 Calculated for C: 262.2; Found: 263.3 (M+H) + .

[0232] Step C - Synthesis of Compound Int-130c A solution of Int-130b-peak 1 (181.4 mg, 0.691 mmol) in THF (10 mL) was added with lithium aluminum hydride (39 mg, 1.028 mmol) at 0 °C under N₂. The mixture was stirred at this temperature for 1 hour. Upon completion, water (0.08 mL), 15% NaOH solution (0.04 mL), and water (0.2 mL) were added sequentially. It was diluted with ethyl acetate (50 mL), and MgSO₄ (2 g) was added. The mixture was stirred at room temperature for 0.5 hour. It was filtered and concentrated to obtain Int-130c. LCMS analysis C 15 H 24 Calculated for C H O₂: 236.2; Found: 260.2 (M+Na) +. .

[0233] Step D - Synthesis of Compound Int-130d Dess-Martin periodinane (607 mg, 1.430 mmol) was added to a solution of Int-130c (169 mg, 0.715 mmol) in DCM (10 mL) at 0 °C. The resulting reaction mixture was stirred at room temperature for 1 hour. Upon completion, the reaction mixture was filtered and concentrated. The residue was purified by silica gel flash chromatography using 0 - 100% EtOAc in hexane as the eluent solvent to obtain Int-130d. LCMS analysis C 15 H 20 Calculated for C H O₂: 232.2; Found: 233.3 (M+H) +. .

[0234] Step E - Synthesis of Compound Int-130e A solution of Int-130d (0.0536 g, 0.231 mmol) and 1-amino-3-(benzyloxy)-N-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide (0.063 g, 0.231 mmol) in DMF (2.5 mL) containing acetic acid (0.25 mL) was heated in a sealed tube at 120 °C for 2 hours. Upon completion, the reaction mixture was concentrated. The residue was purified by silica gel flash chromatography using 0 - 10% MeOH in DCM as the eluent solvent to obtain Int-130e. LCMS analysis of C 29 H 33 Calculated value for N3O4: 487.3; Measured value: 488.4 (M+H) + .

[0235] Step F - Synthesis of Compound Int-130f To a solution of Int-130e (41 mg, 0.084 mmol) in DCM (5.0 mL) was added DIEA (0.032 mL, 0.185 mmol), followed by the addition of SEMCl (0.016 mL, 0.092 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 1.5 h. Upon completion, the reaction mixture was concentrated. The residue was purified by silica gel flash chromatography using 0-10% MeOH in DCM as the eluent to afford Int-130f. LCMS analysis of C 35 H 47 Calculated value for N3O5Si: 617.3; Measured value: 618.5 (M+H) + .

[0236] Step G - Synthesis of Compound Int-130g Int-130f (39.5 mg, 0.064 mmol) in a mixture of DCM (2 mL) and MeOH (1 mL) was cooled to 0 °C and NaBH4 (4.84 mg, 0.128 mmol) was added. The resulting mixture was stirred at this temperature for 2 h. Upon completion, the reaction mixture was concentrated. The residue was purified by silica gel flash chromatography using 0-10% MeOH in DCM as the eluent to afford Int-130g. LCMS analysis of C 35 H 49 Calculated value for N3O5Si: 619.3; Measured value: 620.3 (M+H) + .

[0237] Step H - Synthesis of Compound Int-130h Int-130g (37.2 mg, 0.060 mmol) in DCM (3.0 mL) was cooled to 0 °C, and then triethylamine (0.042 mL, 0.300 mmol) and methanesulfonyl chloride (0.014 mL, 0.180 mmol) were added. The reaction mixture was stirred at room temperature for 1.5 h. Upon completion, the reaction mixture was diluted with DCM (15 mL), washed with water and brine, dried over Na2SO4, filtered, and concentrated to give Int-130h. LCMS analysis C 35 H 48 Calculated for ClN3O4Si: 437.3; Found: 638.3 (M+H) + .

[0238] Step I - Synthesis of Compound Int-130i Tetrabutylammonium fluoride (0.501 mL, 0.501 mmol) was added to a solution of Int-130h (64 mg, 0.100 mmol) in THF (5 mL) at room temperature. The resulting mixture was heated at 60 °C for 16 h. Upon completion, the reaction mixture was concentrated. The residue was purified by silica gel flash chromatography using 0 - 10% MeOH in DCM as the eluting solvent to give Int-130i (20 mg, 0.057 mmol). Int-130i was resolved using a chiral AD-H column (21×250 mm, 5 μm) with 30% IPA (0.2% DIPA) as the eluting solvent to give Int-130i-peak 1 and Int-130i-peak 2. LCMS analysis C 29 H 33 Calculated for N3O3: 471.2; Found: 472.3 (M+H) + .

[0239] Step J - Synthesis of Compound 130A To a solution of Int-130i-peak 1 (3 mg, 0.0064 mmol) in DMF (1 mL) was added lithium chloride (8.08 mg, 0.19 mmol), and the mixture was stirred at 100 °C for 3 h. Upon completion, the reaction was concentrated. The residue was purified by preparative reverse-phase HPLC on a C18 column using ACN in water (0.05% TFA modifier) as the elution solvent to give Compound 130A. LCMS analysis C 22 H 27 Calculated for C18H19N3O3: 381.2; Found: 382.3 (M+H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 7.30 (s, b, 5H), 7.13 (d, J = 7.4 Hz, 1H), 5.86 (d, J = 7.4 Hz, 1H), 5.35 - 5.25 (m, 1H), 4.40 (dd, J = 11.6, 2.5 Hz, 1H), 3.20 (s, 3H), 2.59 - 2.41 (m, 3H), 2.21 (s, b, 1H), 1.95 - 1.85 (m, 1H), 1.70 (dt, J = 13.6, 6.6 Hz, 1H), 1.61 (ddd, J = 15.2, 9.2, 6.1 Hz, 1H), 1.49 (dt, J = 13.9, 7.2 Hz, 1H), 0.98 (dd, J = 19.8, 6.5 Hz, 6H).

[0240] Compound 130B was prepared essentially according to the method used to produce Compound 130A of Example 16. LCMS analysis C 22 H 27 Calculated for C18H19N3O3: 381.2 Found: 382.3 (M+H) + . 1 H NMR (500 MHz, methanol-d 4) δ 7.72 (s, 1H), 7.46 (s, 1H), 7.32 (t, J = 7.3 Hz, 1H), 7.14 (s, 1H), 7.07 (d, J = 7.4 Hz, 1H), 6.75 (s, 1H), 5.92 (d, J = 7.3 Hz, 1H), 5.34 (s, 1H), 4.31 (d, J = 10.5 Hz, 1H), 3.26 (s, 3H), 2.54 (d, J = 12.6 Hz, 1H), 2.07-1.86 (m, 4H), 1.76 (tt, J = 13.7, 6.8 Hz, 1H), 1.31 (t, J = 6.4 Hz, 2H), 0.98 (t, J = 6.3 Hz, 6H).

[0241] Example 17 Preparation of Compounds 131A, 131B, 131C, 131D, 131E and 131F 5-Ethyl-7-hydroxy-10-(methoxymethyl)-1-phenyl-2-(2,2,2-trifluoroethyl)-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chem.

[0242] Step B - Synthesis of Compound Int-131b To a solution of Int-131a (5.1 g, 13.62 mmol) in MeOH (15 mL) was added hydrogen chloride (10.22 mL, 40.9 mmol) in dioxane at room temperature under N2. The reaction mixture was stirred at this temperature for 1 h. Upon completion, it was concentrated. The residue was diluted with 100 mL of EtOAc and subsequently washed with 100 mL of saturated aqueous NaHCO3. The organic layer was dried over MgSO4 and concentrated to give Int-131b. LCMS analysis C 15 H 14 Calculated for C: 290.2; Found: 291.0 (M+H) + .

[0243] Step C - Synthesis of Compound Int-131c To a stirred solution of Int-131b (5 g, 17.23 mmol) in DMF (150 mL) was added iodomethane (2.93 g, 20.67 mmol) at 0 °C, followed by sodium hydride (0.827 g, 20.67 mmol). The resulting mixture was stirred at this temperature for 1 h. Upon completion, it was quenched slowly with 100 mL of water. The mixture was extracted with 2 × 100 mL of EtOAc. The combined organic layers were dried over MgSO4 and concentrated. The residue was purified by a 120 g silica gel column using 0 - 10% MeOH in DCM as the eluting solvent to give Int-131c. LCMS analysis C 16 H 16 Calculated for C: 304.3; Found: 305.0 (M+H) + .

[0244] Step D - Synthesis of Compound Int-131d A solution of pyridinium - p - toluenesulfonate (4.68 g, 18.63 mmol) and Int - 131d (2.1 g, 6.90 mmol) in DMA (40 mL) was heated to 60 °C. Then, a solution of tert - butyl carbazate (1.277 g, 9.66 mmol) in DMA (10 mL) was slowly added dropwise to the reaction solution over 10 minutes. The reaction solution was stirred at 60 °C for 48 hours. Upon completion, 50 mL of water was added to the mixture. It was extracted with 2 × 50 mL of EtOAc. The combined organic layers were dried over MgSO4, filtered, and concentrated. The residue was purified by a 120 g silica gel column using 0 - 100% EtOAc / hexane as the eluting solvent to obtain Int - 131d. LCMS analysis C 21 H 26 Calculated for C N2O7: 418.4; Found: 419.2 (M + H) + .

[0245] Step E - Synthesis of Compound Int-131e A mixture of Int - 131d (1.2 g, 2.87 mmol) and ethanamine in THF (14.34 mL, 28.7 mmol) was heated at 80 °C overnight. Upon completion, it was concentrated. To the above residue, 5 mL of DCM and 5 mL of TFA were added. After stirring it at room temperature for 2 hours, it was concentrated. The resulting residue was subjected to a 100 g C18 reverse - phase column using 0 - 40% ACN in water (0.05% TFA modifier) to obtain Int - 131e. LCMS analysis C 17 H 21 Calculated for C N3O4: 331.4 Found: 332.1 (M + H) + .

[0246] Step F - Synthesis of Compound Int-131f Int - 131e (240 mg, 0.724 mmol) and 4 - benzoyl - 6,6,6 - trifluorohexanal were mixed in a sealed 20 mL capacity microwave tube. The reactant was stirred at 120 °C for 2.5 hours. Upon completion, the reactant was cooled and concentrated. The residue was purified by MPLC on an ISCO Combi-flash with an ISCO Redi-Sep 24 column eluting with 0 - 10% CH2Cl2 / MeOH to afford Int-131f. LCMS analysis C 30 H 32 Calculated for F3N3O5: 571.6; Found: 572.4 (M+H) + .

[0247] Step G - Synthesis of Compound Int-131g Under N2, to a solution of Int-131f (590 mg, 1.032 mmol) in DCM (20 mL) was added DIEA (0.397 mL, 2.271 mmol) and SEM-Cl (0.201 mL, 1.135 mmol) at 0 °C. The reaction mixture was stirred at 25 °C overnight and then concentrated. The residue was purified by MPLC on an ISCO Combi-flash with an ISCO Redi-Sep 40 g column eluting with 0 - 10% CH2Cl2 / MeOH to afford Int-131g. LCMS analysis C 36 H 46 Calculated for F3N3O6Si: 304; Found: 305.0 (M+H) + .

[0248] Step H - Synthesis of Compound Int-131h Under N2, to a solution of Int-131g (680 mg, 0.969 mmol) in DCM (10 mL) / MeOH (5 mL) was added NaBH4 (36.7 mg, 0.969 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 1.5 hours. Upon completion, the reaction mixture was cooled to 0 °C, quenched with 1N HCl (0.5 mL), and then concentrated. The residue was purified by MPLC on an ISCO Combi-flash with an ISCO Redi-Sep 40 g column eluting with 0 - 10% CH2Cl2 / MeOH to afford Int-131h. LCMS analysis C 36 H 48Calculated value for F3N3O6Si: 703.8; Measured value: 704.6 (M+H) + .

[0249] Step I - Synthesis of Compound Int-131i Under N2, TEA (599 μL, 4.30 mmol) was added to a solution of Int-131h (605 mg, 0.860 mmol) in DCM (10 mL), and then Ms-Cl (201 μL, 2.58 mmol) was added at 0 °C. The reaction mixture was stirred at room temperature for 1.0 h. Upon completion, the reaction mixture was diluted with DCM (15 mL), then washed with water, brine, dried over sodium sulfate, filtered, and concentrated to obtain Int-131i. This was used in the next step without purification. LCMS analysis C 37 H 50 Calculated value for F3N3O8SSi: 781.9 Measured value: 782.4 (M+H) + .

[0250] Step J - Synthesis of Compound Int-131j Under N2, TBAF (3.821 μL, 3.821 mmol) in THF was added to a solution of Int-131i (712 mg, 0.911 mmol) in THF (10 mL) at 60 °C. The reaction mixture was stirred at this temperature for 4.0 h, cooled, and concentrated. The residue was purified by MPLC on an ISCO Redi-Sep 40 g column eluting with 0 - 20% CH2Cl2 / MeOH using an ISCO Combi-flash to give two fractions. Both fractions were subjected to SFC fractionation. The less polar fraction gave two peaks (Int-131j-peak 1 and Int-131j-peak 2) from the SFC fractionation. The more polar fraction gave four peaks (Int-131j-peak 3, Int-131j-peak 4, Int-131j-peak 5, and Int-131j-peak 6) from the SFC fractionation. (SFC conditions: Daicel@ chiralpak OD-H column (250×21 mm, 5 μm); 35% EtOH (containing 0.1% DIEA); 70 mL / min). LCMS analysis C 30H 32 Calculated value for F3N3O4: 555.6 Measured value: 556.5 (M+H) + .

[0251] Step K - Synthesis of Compound Int-131h Int-131j-peak 1 (7 mg, 0.013 mmol) was dissolved in MeOH (1 mL). The flask was purged twice with nitrogen, and then Pd / C (9.39 mg, 8.82 μmol) was added. It was purged three times with hydrogen and stirred under a H2 balloon for 1 hour. The resulting reaction mixture was filtered through a syringe filter and then concentrated. The residue was purified by preparative HPLC Reverse phase (C-18 column) eluting with 0-90% acetonitrile / water (containing 0.1% TFA regulator) to obtain 131A. LCMS analysis C 23 H 26 Calculated value for F3N3O4: 465.4; Measured value: 466.3 (M+H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 7.34 - 6.85 (m, 6 H), 5.27 (s, 1 H), 4.93 (d, J = 13.2 Hz, 1 H), 4.63 (d, J = 13.2 Hz, 1 H), 4.57 (s, 1 H), 3.59 (s, 3 H), 2.93 - 2.81 (m, 2 H), 2.51 (s, 3 H), 1.97 (d, J = 9.7 Hz, 1H).

[0252] Compound 131B was prepared essentially according to the method used to produce compound 131A of Example 17. LCMS analysis C 23 H 26 Calculated value for F3N3O4: 465.4; Measured value: 466.3 (M+H) + . 1 H NMR (500 MHz, methanol-d 4) δ 7.33 - 6.92 (m, 6 H), 5.29 (s, 1 H), 4.95 (d, J = 13.4 Hz, 1 H), 4.68 (d, J = 13.4 Hz, 1 H), 4.56 (s, 1 H), 3.60 (s, 3 H), 2.93 - 2.68 (m, 3 H), 2.52 (s, 3 H), 1.98 (d, J = 9.2 Hz, 1 H).

[0253] Compound 131C was prepared essentially according to the method used to produce compound 131A of Example 17. LCMS analysis C 23 H 26 Calculated for F3N3O4: 465.4; Found: 466.3 (M+H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 7.84 - 6.57 (m, 6 H), 6.18 (s, 1 H), 5.36 (s, 1 H), 4.52 (d, J = 14.6 Hz, 1 H), 4.14 (d, J = 10.9 Hz, 1 H), 3.90 (d, J = 14.6 Hz, 1 H), 3.26 (s, 3 H), 2.86 (q, J = 10.4 Hz, 1 H), 2.64 - 1.52 (m, 7 H).

[0254] Compound 131D was prepared essentially according to the method used to produce compound 131A of Example 17. LCMS analysis C 23 H 26 Calculated for F3N3O4: 465.4; Found: 466.3 (M+H) + . 1 H NMR (500 MHz, methanol-d 4) δ 7.53 - 7.22 (m, 7 H), 6.29 (s, 1 H), 5.42 (s, 1 H), 4.76 - 4.64 (m, 2 H), 4.22 (d, J = 14.3 Hz, 1 H), 3.38 (s, 4 H), 3.33 (s, 5 H), 2.84 - 2.67 (m, 2 H), 2.68 - 2.44 (m, 4 H), 2.10 - 1.96 (m, 2 H).

[0255] Compound 131E was prepared essentially according to the method used to produce compound 131A of Example 17. LCMS analysis C 23 H 26 Calculated for F3N3O4: 465.4; Found: 466.3 (M+H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 7.30 (tt, J = 15.8, 6.8 Hz, 5 H), 6.24 (s, 1 H), 5.41 (s, 1 H), 4.76 - 4.64 (m, 2 H), 4.22 (d, J = 14.2 Hz, 1 H), 3.50 - 3.34 (m, 5 H), 2.83 - 2.66 (m, 2 H), 2.65 - 2.43 (m, 3 H), 2.10 - 1.92 (m, 2 H).

[0256] Compound 131F was prepared essentially according to the method used to produce compound 131A of Example 17. LCMS analysis C 23 H 26 Calculated for F3N3O4: 465.4; Found: 466.3 (M+H) + . 11H NMR (500 MHz, chloroform-d) δ 7.79 (d, J = 7.7 Hz, 1 H), 7.54 (d, J = 6.9 Hz, 1 H), 7.42 (d, J = 7.5 Hz, 1 H), 7.17 (d, J = 7.1 Hz, 1 H), 6.73 (d, J = 7.2 Hz, 1 H), 6.29 (d, J = 15.0 Hz, 1 H), 5.42 (s, 1 H), 4.63 - 4.38 (m, 2 H), 4.19 (t, J = 12.5 Hz, 1 H), 3.96 (t, J = 14.8 Hz, 1 H), 3.36 - 3.26 (m, 6 H), 2.92 (d, J = 10.1 Hz, 1 H), 2.61 (d, J = 15.0 Hz, 1 H), 2.48 - 1.48 (m, 5 H).

[0257] The examples in Table 5 were prepared from appropriate starting materials using a procedure similar to the procedure described in Example 17.

[0258]

Table 6

[0259] Example 18 Preparation of Compound 133 7-Hydroxy-5-methyl-6,8-dioxo-1-phenyl-2-(2,2,2-trifluoroethyl)-1,2,3,4,4a,5,6,8-octahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-9-carbonitrile

Chemical Structure

[0260] Step B - Synthesis of Compound Int-133b Int-133a (255 mg, 0.605 mmol) was dissolved in anhydrous DCM (10 mL) under N2 atmosphere. N-Bromosuccinimide (162 mg, 0.908 mmol) was added to this solution and the reaction was stirred at room temperature for 90 minutes. Further, N-bromosuccinimide (115 mg) was added and the reaction was stirred at room temperature for an additional 45 minutes. Upon completion, the reaction volume was reduced under reduced pressure and directly applied to a normal-phase silica gel (24 g ISCO gold column) eluting under gradient conditions 0 - 40% (26% EtOH - EtOAc / EtOAc). Appropriate fractions were collected, concentrated under reduced pressure, and then dried under high vacuum to give Int-133b. LCMS analysis of C 21 H 21 Calculated value for BrF3N3O3: 499.1; Measured value: 500.0 (M+H) + .

[0261] Step C - Synthesis of Compound Int-133c In a microwave reaction vial, under N2 atmosphere, Int-133b (175 mg, 0.350 mmol) and zinc cyanide (205 mg, 1.749 mmol) were mixed in anhydrous DMF (4 mL) and purged with a stream of N2 gas. Bis(tri-t-butylphosphine)palladium(0) (89 mg, 0.175 mmol) was added, the reaction was sealed, and heated at 100 °C for 3 hours in a preheated block. The reaction was cooled and partitioned between DCM and water. The mixture was extracted with DCM (4×), the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was dissolved in DCM / EtOAc and purified on a normal phase silica gel (12 g ISCO gold column) eluting with gradient conditions 0 - 20% (26% EtOH - EtOAc / EtOAc). Appropriate fractions were collected, concentrated under reduced pressure, and then dried under high vacuum to obtain Int-133c. LCMS analysis of C 22 H 21 Calculated value for F3N4O3: 446.4; Measured value: 447.2 (M+H) + .

[0262] Step D - Synthesis of Compound 133 Int-133c (9 mg, 0.020 mmol) and magnesium bromide (12 mg, 0.065 mmol) were mixed in anhydrous THF (3 mL) and stirred at room temperature for 2 hours. Then the reaction was concentrated under reduced pressure. The residue was redissolved in 5:1 DMF / water and purified on a Sunfire Prep column (C18, 30×150 mm) by reverse phase gradient elution (25 - 75% CH3CN / water (adjusted with 0.1% TFA)) for 12 minutes. Appropriate fractions were lyophilized to obtain 133. LCMS analysis of C 21 H 19Calculated value for F3N4O3: 432.4; Measured value: 433.2 (M+H) + . 1 1H NMR (500 MHz, acetonitrile-d3) δ 12.36 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 7.13 (s, 1H), 6.81 (d, J = 7.3 Hz, 1H), 5.19 (s, 1H), 4.00 (d, J = 10.6 Hz, 1H), 3.14 (s, 3H), 2.63 - 2.54 (m, 1H), 2.50 - 2.43 (m, 1H), 2.23 - 2.14 (m, 1H), 2.13 - 2.07 (m, 1H), 2.04 - 1.96 (m, 1H), 1.78 - 1.66 (m, 1H), 1.57 - 1.47 (m, 1H).

[0263] Example 19 Preparation of Compound 134 7-Hydroxy-5-methyl-1-phenyl-9-(1H-tetrazol-5-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4,4a,5-hexahydrodipyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chemical Structure

[0264] Example 20 Preparation of Compound 135A and Compound 135B 2,7-Dihydroxy-5-methyl-1-phenyl-1,2,3,4,4a,5-hexahydrodipyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chemical Structure

[0265] Step B - Synthesis of Compound Int-135b and Compound Int-135c A mixture of Int-135a (10.00 g, 22.648 mmol), TFA (2.59 g, 22.715 mmol), and Hoveyda-Grubbs 2nd generation catalyst (5.79 g, 6.820 mmol) was stirred at 80 °C for 4 h. After cooling, the reaction mixture was concentrated. The residue was applied to a silica gel column using 0 - 15% dichloromethane / methanol to give a mixture (110 g). This was further purified on a Sunfire Prep C18 column using 30 - 80% ACN in water (0.05% TFA regulator) as the eluent to give 40 g of a mixture. The mixture was further purified by Prep-SFC using a CHIRAL ART Cellulose-SB column (S-5um 50×250 mm, 50 mm×250 mm, 5um; mobile phase, CO2 (50%) and MeOH) to give Int-1b (22.52 g, 5.562 mmol) and Int-1c (3.17 g, 0.783 mmol). LCMS analysis C 19 H 19 Calculated value for N3O3: 337.2; Measured value: 338.3 (M+H) + .

[0266] Step C - Synthesis of Compound Int-135d To a solution of Int-135c (0.300 g, 0.89 mmol) in dioxane (9 mL) was added selenium dioxide (0.197 g, 1.78 mmol). The resulting reaction mixture was stirred at 95 °C overnight. After cooling, the mixture was concentrated and purified by ISCO reverse phase C18 column chromatography using 0 - 50% H2O / ACN (containing 0.05% TFA regulator) as the eluent to give Int-135d (0.09 g, 0.276 mmol). LCMS analysis C 19 H 19 Calculated value for N3O4: 353.1; Measured value: 354.1 (M+H) + .

[0267] Step D - Synthesis of Compound Int-135e To a solution of Int-135d (0.142 g, 0.40 mmol) in MeOH (5 mL) was added 10% Pd-C (0.070 g) with stirring at 25 °C. The resulting black suspension was placed under a hydrogen (balloon) atmosphere for 3 hours. The reaction mixture was filtered through a pad of Celite, and the solid was washed thoroughly with MeOH. The combined filtrates were concentrated to give Int-135e. LCMS analysis C 19 H 21 Calculated for C N3O4: 355.2; Found: 356.3 (M+H) + .

[0268] Step E - Preparation of Compound Int-135f and Compound Int-135g Int-135e (0.57 g) was separated by SFC using an AD-H column (50 × 250 mm) and 40% MeOH as the co-solvent to give Int-135f (peak 1) and Int-135g (peak 2). LCMS analysis C 19 H 21 Calculated for C N3O4: 355.2; Found: 356.3 (M+H) + .

[0269] Step F - Synthesis of Compound 135A and Compound 135B To a solution of Int-135f (10.0 mg, 0.04 mmol) in ACN (1 mL) was added MgBr2 (36.8 mg, 0.20 mmol) at 25 °C. The reaction was stirred at 25 °C for 3 hours. Volatiles were removed under reduced pressure. The residue was purified by HPLC (Sunfire Prep C18 column) using 0.05% TFA as the modifier to give 135A (10.6 mg). LCMS analysis C 18 H 19 Calculated for C N3O4: 341.2; Found: 342.6 (M+H) + .

[0270] Compound 135B was prepared essentially according to the method used to produce compound 135A in step F. LCMS analysis C 18 H 19 Calculated for N3O4: 341.2; Found: 342.6 (M+H) + .

[0271] Example 21 Preparation of Compound 136A and Compound 136B 2,7-Dihydroxy-5-methyl-1-phenyl-1,2,3,4,4a,5-hexahydrodipyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chem.

[0272] Step B - Synthesis of Compound Int-136b Potassium carbonate (1 mg) was added to a solution of Int-136a (48.8 mg, 0.097 mmol) in MeOH (1 mL) at room temperature. The resulting reaction mixture was stirred overnight at room temperature. The volatile substances were removed under reduced pressure. The residue was purified by silica gel column chromatography (ISCO 12 g column) using 0→10% MeOH in dichloromethane as the eluent to obtain Int-136b. LCMS analysis C 19 H 19Calculated value for N3O4: 353.1; Measured value: 354.3 (M+H) + .

[0273] Steps C, D, E - Preparation of Compound 136A and Compound 136B Compounds 136A and 136B were prepared essentially according to the method used to produce Compounds 135A and 135B. LCMS analysis C 18 H 19 Calculated value for N3O4: 341.1; Measured value: 342.3 (M+1) + .

[0274] Example 22 Preparation of Compound 137A, Compound 137B, Compound 137C and Compound 137D 2,7-Dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydrodipyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chemical Structure

[0275] Step B - Synthesis of Compound Int-137b Into a 2L high-pressure tank reactor, ACN (680.00 mL), Int-137a (34.00 g, 95.129 mmol), BiPhePhos (1.50 g, 1.906 mmol), carbonyltris(triphenylphosphine)rhodium(I) hydride (1.75 g, 1.903 mmol), and CO (0.5 Mpa) were added. H2(g) (0.5 MPa) was introduced into the above reactor at 80 °C. The resulting mixture was stirred at 80 °C for 20 hours. It was cooled upon completion. The reaction mixture was concentrated to 10V and used directly in the next step.

[0276] Step C - Synthesis of Compound Int-137c and Compound Int-137d Into a 1L four-necked round-bottom flask, the reaction mixture from the previous step and AcOH (68 g) were added. The resulting solution was stirred at 80 °C for 3 days. Upon completion, the resulting mixture was concentrated. The residue was diluted with ACN and purified by Flash-Prep-HPLC using reverse-phase C18 (220 g) with 10 - 100% ACN in H2O as the elution solvent to obtain Int-137c and Int-137d. LCMS analysis C 20 H 23 Calculated for C18H15N3O4: 369.2; Found: 370.2 (M+H) + .

[0277] Step D - Preparation of Compound Int-137e and Compound Int-137f Int-137c (1.0 g) was separated by SFC using an OD-H column (21×250 mm) with 35% MeOH as the co-solvent to obtain Int-137e (peak 1: 0.433 g) and Int-137f (peak 2: 0.423 g). LCMS analysis C 20 H 23 Calculated for C18H15N3O4: 369.2; Found: 370.2 (M+H) + .

[0278] Step E - Preparation of Compound Int-137g and Compound Int-137h Int-137d (1.0 g) was separated by SFC using an OJ-H column (21 × 250 mm) and 30% MeOH and 0.2% DIPA as co-solvents, and Int-137g (peak 1) and Int-137f (peak 2) were obtained. LCMS analysis C 20 H 23 Calculated for N3O4: 369.2; Found: 370.3 (M+H) + .

[0279] Step F - Synthesis of Compound 137A, Compound 137B, Compound 137C and Compound 137D Compound 137A, Compound 137B, Compound 137C and Compound 137D were prepared essentially according to the method used to produce Compound 135A and Compound 135B. LCMS analysis C 19 H 21 Calculated for N3O4: 355.2; Found: 356.2 (M+H) + .

[0280] Example 23 Preparation of Compound 138A and Compound 138B 7-Hydroxy-1,5-dimethyl-1-phenyl-3,4,4a,5-tetrahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-2,6,8(1H)-trione

Chemical Structure

[0281] Step B - Preparation of Compound Int-138b and Compound Int-138c Int-138a (0.083 g) was separated by SFC using an AD-H column (21 × 250 mm) and 30% IPA and 0.2% DIPA as co-solvents, and Int-138b (peak 1) and Int-138c (peak 2) were obtained. LCMS analysis C 20 H 21 Calculated for N3O4: 367.2; Found: 368.3 (M+1) + .

[0282] Step C - Synthesis of Compound 138A and Compound 138B Compound 138A and compound 138B were prepared essentially according to the method used to produce compound 135A and compound 135B. LCMS analysis C 19 H 19 Calculated for N3O4: 355.2; Found: 354.2 (M+H) + .

[0283] Example 24 Preparation of Compound 139 (1R,2S,4aS)-2-Azido-7-hydroxy-5-methyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chemical Structure

[0284] Step B - Synthesis of Compound Int-139b Sodium azide (0.113 g, 1.74 mmol) was added to a solution of Int-139a (0.250 g, 0.579 mmol) in anhydrous DMF (1 mL). The resulting reaction mixture was stirred at 40 °C overnight. After cooling, the volatile substances were removed under reduced pressure. The residue was purified by silica gel column chromatography using 0→5% MeOH in dichloromethane as the eluent to obtain Int-139b. LCMS analysis C 19 H 18 Calculated value for N6O3: 378.2; Measured value: 379.3 (M+H) + .

[0285] Step C - Synthesis of Compound Int-139c Wilkinson's catalyst (0.012 g, 1.74 mmol) was added to a solution of Int-139b (0.024 g, 0.063 mmol) in a mixture of THF (1 mL) and methanol (1 mL). The resulting reaction mixture was stirred at room temperature overnight under a hydrogen atmosphere. The volatile substances were removed under reduced pressure. The residue was purified by silica gel column chromatography using 0→5% MeOH in dichloromethane as the eluent to obtain Int-139c. LCMS analysis C 19 H 20 Calculated value for N6O3: 380.3; Measured value: 381.3 (M+H) + .

[0286] Step D - Synthesis of Compound 139 Compound 139 was prepared essentially according to the method used to produce Compound 135A and Compound 135B. LCMS analysis C 18 H 18 Calculated value for N6O3: 366.2; Measured value: 367.3 (M+H) + .

[0287] Example 25 Preparation of Compound 140

Chem.

[0288] Step B - Synthesis of Compound Int-140b Trifluoroacetic anhydride (0.007 mL, 0.053 mmol) was added to a solution of Int - 140a (12.5 mg, 0.035 mmol) and triethylamine (0.009 mL, 0.07 mmol) in anhydrous dichloromethane (1 mL). The resulting reaction mixture was stirred overnight at room temperature. The volatile substances were removed under reduced pressure. The residue was purified by silica gel column chromatography using 0 → 10% MeOH in dichloromethane as the eluent to give Int - 140b. LCMS analysis C 21 H 21 Calculated for C 21 H 21 F3N4O4: 450.2; Found: 451.3 (M + H) + .

[0289] Step C - Synthesis of Compound 140 Compound 140 was prepared essentially according to the method used for preparing Compound 135A and Compound 135B. LCMS analysis C 20 H 19 Calculated for C 20 H 19 F3N4O4: 436.2; Found: 437.3 (M + H) +.

[0290] Example 26 Preparation of Compound 141 (1R,2S,4aS)-7-Hydroxy-2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)-5-methyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione [Chem.] Step A - Synthesis of Compound Int-141a A mixture of copper(II) sulfate (264 mg, 1.05 mmol) and sodium (R)-2-((S)-1,2-dihydroxyethyl)-4-hydroxy-5-oxo-2,5-dihydrofuran-3-olate (2.094 mg, 10.57 μmol) in water (1 mL) was added to a mixture of Int-5b (40.0 mg, 0.106 mmol) and propargyl alcohol (6.79 mL, 0.116 mmol) in tBuOH (1 mL) at room temperature. The resulting reaction mixture was stirred overnight. Additional copper(II) sulfate (264 mg, 1.05 mmol) and sodium (R)-2-((S)-1,2-dihydroxyethyl)-4-hydroxy-5-oxo-2,5-dihydrofuran-3-olate (2.094 mg, 10.57 μmol) were added to water (1 mL), and stirring was continued for an additional 24 hours. Volatiles were removed under reduced pressure. The residue was purified by silica gel column chromatography using 0→10% MeOH in dichloromethane as the eluent to give Int-141a. LCMS analysis C 22 H 22 Calculated for C6H4N6O4: 434.2; Found: 435.3 (M+H) + .

[0291] Step B - Synthesis of Compound Int-141b Pd-C (10%, 10 mg) was added to a solution of Int-141a (20 mg, 0.046 mmol) in methanol (3 mL). The resulting suspension was placed under a hydrogen (balloon) atmosphere overnight. The reaction was filtered through a pad of celite and washed thoroughly with methanol. The combined filtrates were concentrated under reduced pressure to give Int-141b. LCMS analysis C 22 H24 Calculated value for N6O4: 436.2; Measured value: 437.3 (M+H) + .

[0292] Step C - Synthesis of Compound 141 Compound 141 was prepared essentially according to the method used to produce Compound 135A and Compound 135B. LCMS analysis C 21 H 22 Calculated value for N6O4: 422.2; Measured value: 423.3 (M+H) + .

[0293] Example 27 Preparation of Compound 142 (1R,2S,4aS)-2-Azido-7-hydroxy-5-methyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chemical Structure

[0294] Step B - Synthesis of Compound 142 Compound 142 was prepared essentially according to the method used to produce Compound 135A and Compound 135B. LCMS analysis C 18 H 18 Calculated value for N6O3: 366.1; Measured value: 367.3 (M+1) + .

[0295] Example 28 Preparation of Compound 143A and Compound 143B

Chem.

[0296] Step B - Synthesis of Compound Int-143b Wilkinson's catalyst (112 mg, 0.122 mmol) was added to a solution of Int-143a (50 mg, 0.122 mmol) in methanol (4 mL) and THF (4 mL). The resulting mixture was left overnight under a hydrogen (balloon) atmosphere. Volatiles were removed under reduced pressure. The residue was purified by silica gel column chromatography using 0→5% MeOH in dichloromethane as the eluent to give Int-143b. LCMS analysis C 21 H 23 Calculated for C₁₈H₁₇N₃O₄S: 413.1; Found: 414.2 (M+H) + .

[0297] Step C - Preparation of Compound Int-143c and Compound Int-143d Int-143b (0.016 g) was resolved by SFC using an AD-H column (21×250 mm) and 30% EtOH as the co-solvent to give Int-9c (peak 1) and Int-9d (peak 2). LCMS analysis of C 21 H 23 Calculated value for N3O4S: 413.1; Measured value: 414.2 (M+H) + .

[0298] Step D - Synthesis of Compound Int-143e and Compound Int-143f Aqueous 1M sodium hydroxide (0.034 mL, 0.034 mmol) was added to a solution of Int-143c (7 mg, 0.017 mmol) in THF (1 mL). The resulting mixture was heated at 40 °C overnight. Volatiles were removed under reduced pressure to give Int-143e. The residue was used in the next step without purification. LCMS analysis of C 22 H 27 Calculated value for N3O3S: 413.2; Measured value: 414.2 (M+H) + .

[0299] Compound Int-143f was prepared essentially according to the method used to produce compound Int-143e. LCMS analysis of C 22 H 27 Calculated value for N3O3S: 413.2; Measured value: 414.2 (M+1) + .

[0300] Step E - Synthesis of Compound 143A and Compound 143B Compounds 143A and 143B were prepared essentially according to the method used to produce compounds 135A and 135B. LCMS analysis of C 21 H 25 Calculated value for N3O3S: 399.1; Measured value: 400.1 (M+H) + .

[0301] Example 29 Preparation of Compound 144 and Compound 145 N-((1S,4S,4aS)-7-Hydroxy-5-methyl-6,8-dioxo-1-phenyl-1,2,3,4,4a,5,6,8-octahydropyrido[1,2-b:2’,1’-f][1,2,4]triazin-4-yl)acetamide, and N-((1S,4S,4aS)-7-hydroxy-5-methyl-6,8-dioxo-1-phenyl-1,2,3,4,4a,5,6,8-octahydropyrido[1,2-b:2’,1’-f][1,2,4]triazin-4-yl)-N-methylacetamide

Chemical Structure

[0302] Step B - Synthesis of Compound Int-144b and Compound Int-144c Sodium azide (25.8 mg, 0.396 mmol) was added to a solution of Int-144a (57 mg, 0.707 mmol) in anhydrous DMF (2 mL). The resulting reaction mixture was stirred at 40 °C overnight. Volatiles were removed under reduced pressure. The residue was purified by reverse-phase HPLC using 0-100% ACN in water (0.05% TFA as modifier) as the eluent to give Int-144b and Int-144c. LCMS analysis C 19 H 18 Calculated for C N6O3: 378.1; found: 379.2 (M+H) + .

[0303] Step C - Synthesis of Compound Int-144d and Compound Int-145a Pd-C (10%, 5 mg) was added to a solution of Int-144b (14 mg, 0.037 mmol) in methanol (3 mL). The resulting suspension was left under a hydrogen (balloon) atmosphere overnight. The reaction was filtered through a pad of celite and the solid was washed thoroughly with methanol. The combined filtrates were concentrated under reduced pressure to give a mixture of Int-144d and Int-145a. It was used in the next step without purification.

[0304] Step D - Synthesis of Compound Int-144e and Compound Int-145b Acetic anhydride (1 drop from a pipette) was added to a mixture of Int-144d, Int-145a (13 mg), and triethylamine (0.05 mL) in anhydrous dichloromethane (1 mL). The resulting reaction mixture was stirred at room temperature overnight. The reaction was concentrated under reduced pressure. The residue was purified by reverse-phase HPLC using 0 - 100% ACN in water (0.05% TFA as a modifier) as the eluent to obtain Int-144e and Int-145b. Int-144e: LCMS analysis C 21 H 24 Calculated for C H N4O4: 386.2; Found: 397.2 (M+H) + , and, Int-145b: LCMS analysis C 22 H 26 Calculated for C H N4O4: 410.2; Found: 411.3 (M+H) + ,

[0305] Step E - Synthesis of Compound 144 and Compound 145 Compounds 144 and 145 were prepared essentially according to the method used for preparing Compounds 135A and 135B. 144: LCMS analysis C 20 H 22 Calculated for C H N4O4: 382.2; Found: 383.3 (M+1) + 145: LCMS analysis C 21 H 24 Calculated for C H N4O4: 396.2; Found: 397.3 (M+H) + .

[0306] Example 30 Preparation of Compound 146A and Compound 146B 2-(Isopropylthio)-7-methoxy-5-methyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione [Chemical Structure] Step A - Synthesis of Compound Int-146a and Compound Int-146b Potassium thioacetate (144 mg, 1.259 mmol) was added to a solution of Int-144a (181 mg, 0.420 mmol) in anhydrous DMF (8 mL). The resulting reaction mixture was stirred at 40 °C for 4 h. After cooling, the volatile substances were removed under reduced pressure. The residue was purified by silica gel column chromatography using 0→10% MeOH in dichloromethane as the eluent to give Int-146a and Int-146b. LCMS analysis C 21 H 21 Calculated for C N3O4S: 411.2; Found: 412.3 (M+H) + .

[0307] Step B - Synthesis of Compound Int-146c Wilkinson's catalyst (117 mg, 0.126 mmol) was added to a solution of Int-146b (52 mg, 0.126 mmol) in methanol (4 mL) and THF (4 mL). The resulting mixture was left under a hydrogen (balloon) atmosphere overnight. The volatile substances were removed under reduced pressure. The residue was purified by silica gel column chromatography using 0→10% MeOH in dichloromethane as the eluent to give Int-146c. LCMS analysis C 21 H 23 Calculated for C N3O4S: 413.2; Found: 414.3 (M+H) + .

[0308] Step C - Preparation of Compound Int-146d and Compound Int-146e Int-146c (0.048 g) was resolved by SFC using an OD column (21×250 mm) and 35% EtOH as the co-solvent to give Int-146d (peak 1) and Int-146e (peak 2). LCMS analysis C 21 H 23 Calculated for C N3O4S: 413.2; Found: 414.3 (M+H) + .

[0309] Step D - Synthesis of Compound Int-146f and Compound Int-146g Aqueous 1 M sodium hydroxide (0.076 mL, 0.076 mmol) was added to a solution of Int-146d (15.7 mg, 0.038 mmol) and isopropyl iodide (4 mL, 0.038 mmol) in THF (1 mL). The resulting mixture was stirred overnight. Volatiles were removed under reduced pressure to afford Int-146f. The residue was used in the next step without purification. LCMS analysis C 22 H 22 Calculated for C N3O3S: 413.2; Found: 414.3 (M+H) + .

[0310] Compound Int-146g was prepared essentially according to the method used to produce compound Int-146f. LCMS analysis C 22 H 22 Calculated for C N3O3S: 413.2; Found: 414.3 (M+H) + .

[0311] Step E - Synthesis of Compound 146A and Compound 146B Compounds 146A and 146B were prepared essentially according to the method used to produce compounds 135A and 135B. LCMS analysis C 21 H 25 Calculated for C N3O3S: 399.2; Found: 400.2 (M+H) + .

[0312] Example 31 Preparation of Compound 147 (1S,4S,4aS)-7-Hydroxy-5-methyl-4-(methylthio)-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chemical Structure

[0313] Step B - Synthesis of Compound Int-147b Wilkinson's catalyst (74.8 mg, 0.081 mmol) was added to a solution of Int-147a (crude product from step A) in methanol (3 mL) and THF (3 mL). The resulting mixture was left overnight under a hydrogen (balloon) atmosphere. Volatiles were removed under reduced pressure. The residue was purified by reverse phase HPLC using 0 - 100% ACN in water (0.05% TFA as modifier) as eluent to afford Int-147b. LCMS analysis C 20 H 23 Calculated for C N3O3S: 385.2; Found: 386.2 (M+H) + .

[0314] Step C - Synthesis of Compound 147 Compound 147 was prepared essentially according to the method used for the preparation of Compound 135A and Compound 135B. LCMS analysis C 19 H 21 Calculated for C N3O3S: 371.2; Found: 372.3 (M+H) + .

[0315] Example 32 Preparation of Compound 148 7-Hydroxy-5,12a-dimethyl-2a,3,4,4a,5,12a-hexahydro-1H-isochromeno[3’,4’:5,6]pyrido[1,2-b]pyrido[2,1-f][1,2,4]triazine-6,8-dione

Chemical Structure

[0316] Step B - Synthesis of Compound 148 Compound 148 was prepared essentially according to the method used to prepare Compound 135A and Compound 135B. LCMS analysis C 18 H 19 Calculated for C16H17N3O3S: 367.2; Found: 368.3 (M+H) + .

[0317] Example 33 Preparation of Compound 149 (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chemical Structure

[0318] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione Compound 148 was prepared essentially according to the method used to produce Compound 135A and Compound 135B. LCMS analysis C 26 H 25 Calculated value for N3O4: 443.2; Measured value: 444.3 (M+H) + .

[0319] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chemical Structure

[0320] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione Int-150a (1.60 g, 7.83 mmol) in anhydrous DMF (10 mL) was added to sodium hydride (0.94 g, 60% suspension in mineral oil, 23.5 mmol) under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 1 hour, and 4-bromobut-1-ene was added in one portion. The resulting reaction mixture was stirred at 40 °C overnight. After cooling, the mixture was partitioned between dichloromethane and saturated aqueous ammonium chloride. The aqueous layer was separated and further extracted with dichloromethane (2×). The combined organic layers were dried (MgSO4), filtered, and concentrated. The residue was purified by silica gel column chromatography using 0 - 100% EtOAc in hexane as the eluent to give Int-150b. LCMS analysis C 16 H 18 Calculated for C: 258.1; Found: 259.2 (M+H) + .

[0321] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione Aqueous 1.0 M sodium hydroxide (9.68 mL, 9.68 mmol) was added to a solution of Int-150b (0.500 g, 1.94 mmol) in THF (15 mL). The resulting reaction mixture was heated at 100 °C overnight. After cooling, the mixture was partitioned between dichloromethane and aqueous 1N HCl. The aqueous layer was separated and further extracted with dichloromethane (2×). The combined organic layers were dried (MgSO4), and the volatiles were removed under reduced pressure. The residue was purified by silica gel column chromatography using 0→10% EtOAc in hexane as the eluent to give Int-150c. LCMS analysis C 14 H 16 Calculated for C: 200.1; Found: 201.2 (M+H) + .

[0322] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione Trifluoroacetic acid (0.141 mL, 1.59 mmol) was added under nitrogen to a solution of 1-amino-3-methoxy-N-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide (0.157 g, 0.80 mmol), Int-150c (0.159 g, 0.80 mmol), and palladium diacetate (0.65 mg, 2.89 mmol) in anhydrous dioxane (5 mL). The resulting reaction mixture was stirred at room temperature overnight. Volatiles were removed under reduced pressure. The residue was purified by silica gel column chromatography using 0→10% MeOH in dichloromethane as the eluent to give Int-150d. LCMS analysis C 22 H 25 Calculated for C13H13N3O3: 379.2; Found: 380.3 (M+H) + .

[0323] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione Sodium borohydride (61.8 mg, 1.63 mmol) was added under a nitrogen atmosphere to a solution of Int-150d (0.062 g, 0.334 mmol) in methanol (1 mL). The resulting reaction mixture was stirred at room temperature overnight. Volatiles were removed under reduced pressure. The residue was purified by silica gel column chromatography using 0→10% MeOH in dichloromethane as the eluent to give Int-150e. LCMS analysis C 22 H 27 Calculated for C13H15N3O3: 381.2; Found: 382.4 (M+H) + .

[0324] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione To a solution of Int-150e (27 mg, 0.07 mmol) in a mixture of THF (1 mL) and water (0.3 mL) was added osmium tetroxide (9 mL, 4% solution in tBuOH, 1.41 mmol) at room temperature under nitrogen, followed by the addition of sodium periodate (45.4 mg, 0.212 mmol). The resulting reaction mixture was stirred overnight and then quenched by the addition of aqueous 10% sodium thiosulfate and stirred for 1 hour. Volatiles were removed under reduced pressure. The residue was extracted thoroughly with 20% MeOH in dichloromethane (50 mL). The combined extracts were dried (sodium sulfate). Volatiles were removed under reduced pressure. The residue was purified by silica gel column chromatography using 0→10% MeOH in dichloromethane as the eluent to afford Int-150f. LCMS analysis C 21 H 25 Calculated for C H N3O4: 383.2; Found: 384.4 (M+H) + .

[0325] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione Int-150f (13 mg, 0.034 mmol) in a mixture of DMF (1 mL) and AcOH (0.1 mL) was heated at 120 °C for 1 hour. After cooling, volatiles were removed under reduced pressure. The residue was purified by silica gel column chromatography using 0→10% MeOH in dichloromethane as the eluent to afford Int-150g. LCMS analysis C 21 H 23 Calculated for C H N3O3: 365.2; Found: 366.1 (M+H) + .

[0326] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione Compound 150 was prepared essentially according to the method used for the preparation of Compound 135A and Compound 135B. LCMS analysis C 20 H 21 Calculated for C H N3O3: 351.2; Found: 352.1 (M+1) + .

[0327] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chem.

[0328] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione Compound 151 was prepared essentially according to the method used to prepare Compound 135A and Compound 135B. LCMS analysis C 22 H 23 Calculated for F2N3O4: 431.2; Found: 432.3 (M+1) + .

[0329] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chem.

[0330] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione Pd-C (10%, 12.6 mg) was added to a solution of Int-151a (45 mg, 0.118 mmol) in MeOH (2.4 mL). The resulting suspension was placed under a hydrogen (balloon) atmosphere for 5 hours. The mixture was filtered and washed with MeOH (1 mL). The filtrate was concentrated to obtain Int-151b. This was used in the next step without further purification.

[0331] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione Int-151b (crude product from Step B) was separated by SFC using an AD-H column (21×250 mm) and 20% MeOH as the co-solvent to obtain Int-151c and Int-151d. LCMS analysis C 21 H 25 Calculated for C H N3O4: 383.2; Found: 384.3 (M+H) + .

[0332] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione Lithium chloride (1.66 mg, 0.039 mmol) was added to a solution of Int-151c (3 mg, 7.82 mmol) in DMF (0.16 mL). The resulting reaction mixture was heated at 100 °C for 3 hours. After cooling, the solution was purified directly by reverse-phase HPLC using 0 - 100% ACN in water (0.05% TFA as regulator) as the eluent to obtain 152A. LCMS analysis C 20 H 23 Calculated for C N3O4: 369.2; Found: 370.3 (M+H) + .

[0333] Compound 152B was prepared essentially according to the method used to produce compound 152A. LCMS analysis C 20 H 23 Calculated for C N3O4: 369.2; Found: 370.3 (M+H) + .

[0334] The examples in Table 6 were prepared from the appropriate starting materials using a procedure similar to that described in Example 36.

[0335]

Table 7

[0336] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione

Chem.

[0337] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione To a stirred mixture of N-methyl-2-nitrobenzenesulfonamide (447 mg, 2.066 mmol), Int-165a (360 mg, 2.066 mmol), and 2-(diphenylphosphanyl)pyridine (816 mg, 3.10 mmol) in DCM (20 mL) was added DIAD (603 μL, 3.10 mmol). The mixture was stirred at room temperature for 4 hours. Without further workup, the mixture was applied to a 100 g silica gel column using 0 - 90% EtOAc in hexane as the eluting solvent to give Int-165b. LCMS analysis C 19 H 20 Calculated for C H N2O4S: 372.4; Found: 395.2 (M+Na) + .

[0338] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione Benzenethiol (1302 mg, 11.81 mmol) was added to a mixture of Int-165b (2.2 g, 5.89 mmol) and potassium carbonate (3266 mg, 23.63 mmol) in acetonitrile (100 mL) at room temperature. The mixture was stirred at room temperature overnight. Upon completion, it was concentrated. The residue was redissolved in DCM (100 mL), filtered, concentrated, and purified by a 100 g silica gel column using 0 - 5% EtOAc in MeOH as the eluting solvent to obtain Int-165c. LCMS analysis C 13 H 17 Calculated for C H N: 187.3; Found: 188.2 (M+H) + .

[0339] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione To a stirred solution of Int-165c (300 mg, 1.6 mmol) and 4-(benzyloxy)-5-bromo-3-methoxypicolinic acid (650 mg, 1.922 mmol) in DMF (16 mL), N-ethyl-N-isopropylpropan-2-amine (621 mg, 4.81 mmol) was added, followed by the addition of HATU (914 mg, 2.403 mmol) at room temperature. The mixture was stirred at room temperature for 1 hour. Upon completion, the reaction was subjected to a 100 g silica gel column using 0 - 100% 30% EtOH in EtOAc / hexane as the eluting solvent to obtain Int-165d. LCMS analysis C 27 H 27 Calculated for C BrN₂O₃: 507.4; Found: 507.3, 509.3 (M+H) + .

[0340] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione It should be noted that in the translation of the chemical formula calculation part in and , the chemical formula in the original text seems to be incomplete. The correct chemical formula should be provided for a more accurate translation. Here, I translated it according to the existing text.To a stirred solution of Int-165d (670 mg, 1.320 mmol) in THF (11 mL) and water (2.2 mL) were added 4-methylmorpholine 4-oxide (309 mg, 2.64 mmol) and osmium(VIII) oxide (839 μL, 0.132 mmol). The resulting solution was heated at 50 °C for 24 h. Upon completion, it was cooled, 0.5 g of sodium metabisulfite was added, and the mixture was stirred for 1 h and then filtered. The filtrate was concentrated. The residue was subjected to a 120 g silica gel column using 0 - 100% 30% EtOH in EtOAc / hexane as the eluting solvent to afford Int-165e. LCMS analysis C 27 H 29 Calculated for BrN2O5: 541.4; Found: 541.3, 543.3 (M+H) + .

[0341] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione To a stirred solution of Int-165e (650 mg, 1.20 mmol) in MeOH (12 mL) was added palladium on carbon (128 mg, 0.120 mmol). The resulting mixture was flushed with N2 and connected to an H2 balloon for 1 h. Upon completion, the reaction mixture was filtered through a pad of celite. The filtrate was concentrated to afford Int-165f. LCMS analysis C 20 H 24 Calculated for N2O5: 372.4; Found: 373.4 (M+H) + .

[0342] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione To a stirred solution of Int-165f (410 mg, 1.101 mmol) in DCM (11 mL) were added triethylamine (334 mg, 3.30 mmol) and methanesulfonyl chloride (277 mg, 2.422 mmol). The resulting mixture was stirred at room temperature for 3 h. Upon completion, it was concentrated to remove most of the DCM. To the residue were added 20 mL of EtOAc and 5 mL of 0.2 N aqueous HCl. The organic layer was separated, dried over MgSO4, and concentrated to afford the crude product.

[0343] To the crude product, 10 mL of DMF and potassium carbonate (761 mg, 5.50 mmol) were added. The mixture was stirred at 100 °C for 4 h. At completion, it was filtered. The filtrate was subjected to a C18 column (220 g) using 0 - 100% ACN in water (containing 0.05% TFA regulator) as the elution solvent to obtain the racemic substance (82 mg, 0.231 mmol). The racemic substance was further separated by SFC using an AD - H (4.6×250 mm, 5 μm) column with 30% MeOH as the elution solvent, and the faster - eluting peak Int - 165g and the slower - eluting peak Int - 165h were obtained. LCMS analysis C 20 H 22 Calculated for N2O4: 354.4; Found: 355.4 (M + H) + .

[0344] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione To a stirred mixture of Int - 165g (10 mg, 0.028 mmol) in DMF (1 mL) at room temperature, lithium chloride (11.96 mg, 0.282 mmol) was added. The mixture was stirred at 100 °C overnight. At completion, the mixture was cooled. It was purified by reverse - phase preparative HPLC using a 40 g C18 column with ACN in water (containing 0.1% TFA regulator) to obtain Compound 165A. LCMS analysis C 19 H 20 Calculated for N2O4: 340.4; Found: 341.4 (M + H) + . 1 H NMR (500 MHz, methanol - d 4 ) δ 8.3 - 8.1 (br, 1H), 7.3 - 6.95 (m, 6H), 4.85 (s,1H), 4.37 (s, 1H), 2.98 (s, 3H), 2.75 - 2.62(br, 1H), 2.62 - 2.39 (br,1H), 2.27 - 2.02(m, 4H).

[0345] Compound 165B was prepared from Int-165h essentially according to the method used to produce Compound 165A. LCMS analysis C 19 H 20 Calculated for N2O4: 340.4; Found: 341.3 (M+H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 8.27 - 8.07 (br, 1H), 7.3 - 6.9 (m, 6H), 4.83 (s,1H), 4.36 (s, 1H), 2.97 (s, 3H), 2.73 - 2.62 (br, 1H), 2.62 - 2.39 (br, 1H), 2.26 - 2.04 (m, 4H).

[0346] (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione (1R,2R)-4-((E)-benzylidene)-2,7-dihydroxy-1,5-dimethyl-1-phenyl-1,2,3,4,4a,5-hexahydropyrido[1,2-b:2’,1’-f][1,2,4]triazine-6,8-dione 1,7-Dihydroxy-5-methyl-1-phenyl-2-propyl-1,3,4,4a,5,11a-hexahydro-2H-pyrido[1,2-a]quinoxaline-6,8-dione

Chem.

[0347] Step B - Synthesis of Compound Int-166b To a solution of Int-166a (3.3 g, 18.11 mmol) in THF (91 mL) was added phenylmagnesium bromide (23.54 mL, 23.54 mmol) at -78 °C. The resulting mixture was stirred at this temperature for 10 minutes and then warmed to 0 °C over 30 minutes. At completion, it was quenched with 100 mL of aqueous NH4Cl solution. The mixture was extracted with 3 × 100 mL of EtOAc. The combined organic layers were dried over MgSO4 and concentrated to give 5 g of the crude product.

[0348] The above crude product was added to 100 mL of MeOH and 50 mL of water, followed by the addition of 2 mL of TFA. The resulting mixture was stirred at room temperature for 24 hours. At completion, it was concentrated to remove most of the MeOH. The residue was added to 100 mL of EtOAc. The organic layer was separated, dried over MgSO4, and concentrated. The residue was subjected to a 220 g silica gel column using 20% EtOAc in hexane as the eluent to give Int-166b. LCMS analysis C 15 H 18 Calculated for: 214.3; Found: 215.6 (M+H) + .

[0349] Step C - Synthesis of Compound Int-166c A mixture of methanamine (40.8 mL, 82 mmol) and Int-166b (3.5 g, 16.33 mmol) in MeOH was added to 0.2 mL of TFA and 5 g of anhydrous MgSO4. The resulting reaction was stirred at room temperature for 4 hours. It was filtered and the filtrate was concentrated. To the above residue was added 100 mL of DCM and 10 mL of MeOH, followed by the addition of sodium borohydride (0.989 g, 26.1 mmol) in small portions. At completion, it was quenched with 10 mL of 2N aqueous HCl solution. The reaction mixture was dried over MgSO4 and concentrated. The residue was purified by a 120 g silica gel column using 0 - 20% MeOH in DCM as the eluent to give Int-166c. LCMS analysis C16 H 23 Calculated value for N: 229.4 Measured value: 230.4 (M+H) + .

[0350] Step D - Synthesis of Compound Int-166d A mixture of Int-166c (1700 mg, 7.41 mmol), 4-(benzyloxy)-5-bromo-3-methoxypicolinic acid (2506 mg, 7.41 mmol), triethylamine (1500 mg, 14.82 mmol), and HATU (3382 mg, 8.89 mmol) in DCM (100 mL) was stirred at room temperature for 5 h. At completion, 100 mL of 0.2 HCl aqueous solution was added. The organic layer was separated, dried over MgSO4, and concentrated. The residue was subjected to a 120 g silica gel column using 0 - 100% EtOAc in hexane to obtain Int-166d. LCMS analysis C 30 H 33 Calculated value for BrN2O3: 549.5 Measured value: 550.4 (M+H) + .

[0351] Step E - Synthesis of Compound Int-166e To a stirred solution of Int-166d (2.3 g, 4.19 mmol) in THF (35.9 mL) / water (5.98 mL) were added 4-methylmorpholine 4-oxide (0.981 g, 8.37 mmol) and osmium(VIII) oxide (2.66 mL, 0.419 mmol). The resulting mixture was stirred at 50 °C overnight. At completion, 5 g of solid sodium metabisulfite and 5 g of MgSO4 were added. The resulting mixture was stirred at room temperature for 1 h. It was filtered and the filtrate was concentrated. The residue was purified by a 120 g C18 column using 0 - 100% ACN in water (containing 0.05% TFA modifier) to obtain Int-166e. LCMS analysis C 30 H 35 Calculated value for BrN2O5: 583.5; Measured value: 584.4 (M+H) + .

[0352] Step F - Synthesis of Compound Int-166f To a stirred solution of Int-166e (1.1 g, 1.885 mmol) in methanol (20 mL) / DCM (2 mL), palladium on carbon (0.201 g, 0.189 mmol) was added. The resulting mixture was hydrogenated at room temperature for 1 hour under a H2 balloon. Upon completion, it was filtered and the filtrate was concentrated to give Int-166f. LCMS analysis C 23 H 30 Calculated for N2O5: 414.5; Found: 415.4 (M+H) + .

[0353] Step G - Synthesis of Compound Int-166g To a stirred solution of Int-166f (760 mg, 1.834 mmol) in 20 mL of THF, triethylamine (649 mg, 6.42 mmol) and methanesulfonyl chloride (462 mg, 4.03 mmol) were added at 0 °C. The resulting mixture was stirred at this temperature for 1 hour. Upon completion, it was filtered and the filtrate was concentrated and 50 mL of EtOAc was added. It was washed with 0.1 N aqueous HCl and dried over MgSO4. It was concentrated to give the crude product.

[0354] To the above crude product, DMF (20 mL) and potassium carbonate (1267 mg, 9.17 mmol) were added. The resulting mixture was stirred at 100 °C overnight. Upon completion, it was cooled and 100 mL of water and 100 mL of EtOAc were added. The organic layer was separated and dried over MgSO4. It was concentrated and the residue was subjected to a 100 g C18 column using 0 - 100% ACN in water (0.05% TFA modifier) as the eluent to give Int-166g (320 mg, 0.807 mmol). This was subjected to SFC separation (AD-H, 4.6×250 mm, EtOH) to give Int-166g-peak 1 and Int-166g-peak 2. LCMS analysis C 23 H 28 Calculated for N2O4: 396.5; Found: 397.4 (M+H) + .

[0355] Step H - Synthesis of Compound 166A and Compound 166B Int-166g-peak 1 (15 mg, 0.038 mmol) was dissolved in a suspension of LiCl (16.04 mg, 0.378 mmol) in DMF (1000 μL), and the mixture was stirred at 100 °C for 1 hour. The suspension was filtered. The filtrate was purified by reverse-phase preparative HPLC (SunFire C18 OBD Prep column) eluting with 10 - 90% acetonitrile / water (containing 0.1% TFA modifier) to give 166A. LCMS analysis C 22 H 26 Calculated for N2O4: 382.5; Found: 383.2 (M+H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 8.14 (s, 1H), 7.26 - 6.94 (m, 5H), 4.68 (s, 1H), 4.38 (s, 1H), 3.06 (s, 3H), 2.67 (d, J = 12.4 Hz, 1H), 2.30 - 2.00 (m, 5H), 1.68 - 1.26 (m, 5H).

[0356] Compound 166B was prepared from Int-166g-peak 2 essentially according to the method used to produce compound 166A. LCMS analysis C 22 H 26 Calculated for N2O4: 382.5; Found: 383.2 (M+H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 8.14 (s, 1H), 7.26 - 6.94 (m, 5H), 4.68 (s, 1H), 4.38 (s, 1H), 3.06 (s, 3H), 2.67 (d, J = 12.4 Hz, 1H), 2.30 - 2.00 (m, 5H), 1.68 - 1.28 (m, 5H).

[0357] Example 40 Preparation of Compound 167 7-Hydroxy-1-methoxy-5-methyl-1-phenyl-1,3,4,4a,5,11a-hexahydro-2H-pyrido[1,2-a]quinoxaline-6,8-dione [Chemistry] Step A - Synthesis of Compound Int-167a To a stirred solution of dibenzyl (3-hydroxycyclohexane-1,2-diyl) dicarbamate (2 g, 5.02 mmol) in CH2Cl2 (50.2 mL) was added Dess-Martin periodinane (3.19 g, 7.53 mmol). The resulting mixture was stirred at room temperature for 2 h. One drop of water was added and the precipitate was filtered through a pad of celite. The filtrate was concentrated. The residue was subjected to a 120 g silica gel column using 0 - 100% 30% EtOH / hexane in EtOAc as the eluent to afford Int-167a. LCMS analysis C 22 H 24 Calculated for N2O5: 396.4; Found: 397.2 (M + H) + .

[0358] Step B - Synthesis of Compound Int-167b To a stirred solution of Int-167a (1800 mg, 4.54 mmol) in THF (50 mL) was added phenyllithium (9559 μL, 18.16 mmol) at -78 °C. The resulting mixture was slowly warmed to 0 °C over 1 h. Upon completion, it was quenched with 20 mL of aqueous NH4Cl solution. The mixture was extracted with 2 × 30 mL of EtOAc. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The residue was purified by a 100 g silica gel column using 0 - 100% 30% EtOH in EtOAc / hexane to afford Int-167b. This was carried on to the next step without further purification. LCMS analysis C 28 H 30 Calculated for N2O5: 474.5; Found: 475.3 (M + H) + .

[0359] Step C - Synthesis of Compound Int-167c To a stirred solution of Int-167b (700 mg, 1.475 mmol) in MeOH (20 mL) was added palladium on carbon (157 mg, 0.148 mmol). The resulting mixture was hydrogenated under a H2 balloon for 3 h. It was filtered through a pad of celite. The filtrate was concentrated to give Int-167c. LCMS analysis C 12 H 18 Calculated for N2O: 206.3; found 207.3 (M+H) + .

[0360] Step D - Synthesis of Compound Int-167d A stirred solution of Int-167c (399 mg, 1.454 mmol) and 2,3-diamino-1-phenylcyclohexan-1-ol (300 mg, 1.454 mmol) in n-methylimidazole (5 mL) was heated at 80 °C overnight. Upon completion, it was cooled. The reaction was subjected to a 100 g C18 column using 0 - 100% ACN in water as eluent to give the crude product. The crude product was then subjected to Gilson using a SunFire C18 OBD Prep column using 0 - 100% ACN in water (0.1% TFA regulator) as eluent to give Int-167d. LCMS analysis C 25 H 24 Calculated for N2O4: 416.5; found 417.2 (M+H) + .

[0361] Step E - Synthesis of Compound Int-167e To a stirred solution of Int-167d (40 mg, 0.096 mmol) in DMF (960 μL) were added iodomethane (40.9 mg, 0.288 mmol) and sodium hydride (11.52 mg, 0.288 mmol) at 0 °C. The resulting mixture was stirred at this temperature for 1 h. Upon completion, it was quenched with 2 drops of 1N aqueous ammonium chloride solution. The mixture was subjected to a 50 g C18 column using 0 - 100% ACN in water (0.05% TFA as regulator) as eluent to give Int-167e. LCMS analysis C27 H 28 Calculated value for N2O4: 444.5; Measured value 445.2 (M+H) + .

[0362] Step F - Synthesis of Compound 167 To a stirred and cooled to room temperature mixture of Int-167e (24 mg, 0.054 mmol) in methanol, Pd / C (5.75 mg, 5.40 μmol) was added. The mixture was stirred at room temperature for 3 h. The reaction mixture was then filtered. The filtrate was applied to a 50 g C18 column and eluted with 0-100% acetonitrile / water (0.05% TFA) to give 167. LCMS analysis C 20 H 22 Calculated value for N2O4: 354.4; Measured value: 355.2 (M+H) + . 1 H NMR (500 MHz, methanol-d 4 ) δ 7.38 (s, 2H), 4.73 (s, 1H), 4.41 (s, 1H), 3.33 (s, 13H), 3.25 (s, 3H), 3.13 (s, 3H), 2.05 (s, 2H).

[0363] Flu A Neuraminidase Antiviral Assay To evaluate anti-viral compounds against Flu A, a fluorescence assay was developed to monitor the activity of influenza-derived neuraminidase (NA) enzyme in infected cells. NA activity not only enables the release of influenza virions from infected cells, but also functions in cell culture systems that retain infectious virus on the cell surface, such as Madin-Darby canine kidney (MDCK) epithelial cells, which require chemical intervention for virus release. NA activity can be monitored by the increase in fluorescence of MUNAN (4-methylumbelliferone) released as a product from the enzymatic cleavage of the substrate 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA). The amount of fluorescence is proportional to the amount of NA enzyme activity that increases with virus replication.

[0364] The MDCK (Sigma) cells are incubated at 37 °C in an atmosphere of 5% CO2 and humidity > 85% in a growth medium of DMEM (Thermo Fisher) containing Glutamax and pyruvate supplemented with 5% heat-inactivated fetal bovine serum (Thermo Fisher) and 1% Pen-Strep (Thermo-Fisher). On the day of the assay, the cells are washed with 15 - 20 mL of PBS (Thermo Fisher), followed by addition of 1.5 mL of 0.25% trypsin-EDTA (Thermo Fisher) solution and incubation at 37 °C for 2 - 5 minutes. After the cells are detached from the plate, 6 - 8 mL of growth medium is added to resuspend the cells. The cells are counted with a ViCell Counter (Beckman), and growth medium is added to adjust the cell density to 80,000 cells / mL. FLUA (PR / 8 / 34) at 3.4E+08 pfu / mL is diluted 1:25,000 with the MDCK cell suspension.

[0365] The compound diluted with DMSO is titrated (10 points, 3-fold dilution), and added to a 384-well black polystyrene tissue culture-treated microplate (Corning) by acoustic dispensing (200 nL, Labcyte Echo). A suspension of cells and virus (25 μL) is dispensed into each well of the assay plate. The plate is centrifuged briefly (300 rpm × 30 seconds) and incubated at 37 °C for 48 hours in an atmosphere of 5% CO2 and humidity > 85%. To detect NA activity, the MUNANA substrate (MP Biomedical) is diluted with dH2O to a concentration of 2.5 mM. The substrate is further diluted with assay buffer (66.6 mM MES, 8 mM CaCl2, pH 6.5) to a concentration of 200 μM. 6 μL of the substrate dilution of this volume is added to each well of the assay plate, shaken for 1 minute to mix, and returned to the incubator at 37 °C for 1 hour. Then, 25 μL of MUNANA Stop Solution (0.2 M sodium carbonate, Fisher) in dH2O is added to each well, followed by shaking for 1 minute. The fluorescence intensity (Ex = 355 nm, Em = 460 nm) of each well is measured using an Envision plate reader (Perkin Elmer).

[0366] The raw data from each test well is normalized against the average signal of wells inhibited 100% (maximum effect; 100% inhibition) and virus-infected cells only (minimum effect; 0% inhibition), and the % inhibition is calculated using the following formula: % inhibition = 100 × (test Cmpd - maximum effect) / (minimum effect - maximum effect). The data is analyzed using Activity Base (IDBS), and a dose-response curve is created by plotting % inhibition (Y-axis) against Log 10 compound concentration (X-axis). The IC 50 value is calculated using a non-linear regression 4-parameter sigmoid dose-response model.

[0367] The compounds of the present invention were tested in the described assay. The results are shown in the following table.

[0368]

Table 8

Claims

1. formula 【Chemical 1】 [wherein, X is N or CH; Y is absent or CHR ５ , -CH ２ -CHR ５ -, -CH ２ -CHR ５ -CH ２ -, S, SO or SO ２ ; Z is NR １ or CR １ R １ａ ; R １ is selected from the group consisting of hydrogen, C １－６ alkyl, C １－３ alkyl (C ３－７ cycloalkyl), C １－３ alkyl (heterocyclyl), (C １－６ alkyl) OR ｘ and C １－３ haloalkyl, wherein the cycloalkyl group can be monocyclic or bicyclic and may be substituted with one or two substituents independently selected from the group consisting of halo and R ｘ ; R １ａ is selected from the group consisting of hydrogen, C １－６ alkyl, C １－３ alkyl (cyclopropyl), (C １－６ alkyl) OR ｘ and C １－３ haloalkyl; R ２ is aryl, heteroaryl, cycloalkyl or heterocyclyl, wherein the aryl can be monocyclic or bicyclic, and wherein the aryl, heteroaryl, cycloalkyl and heterocyclyl may be substituted with 1 to 3 substituents independently selected from the group consisting of halo, cyano, cyclopropyl, R ｘ , R ｙ , SR ｘ and OR ｘ ; R ３ is hydrogen, hydroxy, C １－６ alkyl, OR ｘ or C １－３ haloalkyl; or, R ２ and R ３ together with the carbon or heteroatom to which they are attached can form dihydroindene, 1,2,3,4 - tetrahydronaphthalene, chroman, 2,3 - dihydrobenz[b]thiophene, dihydrobenzofuran or thiochroman; R ４ is hydrogen, hydroxy, N ３ , NH(C = O)R ｘ , SR ｘ , C １－６ alkyl, C ２－６ alkenyl, O(C １－６ alkyl), O(C ２－６ alkenyl), (C １－３ alkyl)R ｙ , O(C １－３ alkyl)R ｙ , R ｙ and heteroaryl, where the alkyl group may be substituted with 1 - 3 substituents independently selected from the group consisting of halo, R ｘ , R ｙ , OR ｘ , cyano and phenyl, where the heteroaryl group and the alkenyl group may be substituted with 1 - 3 substituents independently selected from the group consisting of halo, R ｘ and (C １－３ alkyl)OR ｘ ; or, R ２ and R ４ together with the carbon or heteroatom to which they are attached can form dihydroindene, 1,2,3,4 - tetrahydronaphthalene, 2,3 - dihydrobenz[b]thiophene, dihydrobenzofuran, chroman or thiochroman; R ４ａ is hydrogen and C １－６Selected from the group consisting of alkyl, wherein the alkyl group may be substituted with 1 to 3 substituents independently selected from the group consisting of halo and OR ｘ ; or R ４ and R ４ａ together with the carbon or heteroatom to which they are attached can form an oxo group or a C ４－６ cycloalkyl group [wherein the cycloalkyl group can be monocyclic or bicyclic and may be substituted with 1 to 3 substituents independently selected from the group consisting of halo, C １－３ alkyl and C １－３ haloalkyl]; R ５ is hydrogen, C １－６ alkyl, C １－３ alkyl(cyclopropyl) and (C １－６ alkyl)OR ｘ ; R ６ is hydrogen, C １－６ alkyl, SR ｘ , NR ｘ (C=O)C １－６ alkyl or (C ２－３ alkenyl)R ｙ ; or R ４ and R ６ together with the carbon atom to which they are attached can form a C ４－６ cycloalkyl group; R ７ is hydrogen or C １－３ alkyl; R ８ is hydrogen, C １－６ alkyl or (C １－６ alkyl)OR ｘ ; R ９ is hydrogen, cyano or C １－６ alkyl, wherein the alkyl may be substituted with 1 to 3 halo; R ｘ is hydrogen and C １－６Selected from the group consisting of alkyl, wherein the alkyl may be substituted with 1 to 3 halos; R ｙ is selected from the group consisting of phenyl and C ３－６ cycloalkyl, wherein the phenyl and cycloalkyl groups may be substituted with 1 to 3 substituents independently selected from the group consisting of halo, cyano, and R ｘ ; a compound represented by the formula or a pharmaceutically acceptable salt thereof. **Claim 2** The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein X is N. **Claim 3** R １ is methyl, the compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2. **Claim 4** Y is CH ２ , the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3. **Claim 5** R ２ is phenyl, wherein the phenyl may be substituted with 1 or 2 substituents independently selected from the group consisting of halo, CH ３ CF ３ OCHF ２ and OCH ３ ; the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4. **Claim 6** R ３ is hydrogen, methyl, ethyl or hydroxy, the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5. **Claim 7** R ４ is hydrogen, methyl, ethyl, propyl, trifluoroethyl, CH ２ CH ２ OH, CH ２ CH ２ OCH ３ or cyclopropylmethyl, the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6.

8. R ４ and R ６ together with the carbon atom to which they are attached form a C ４－６ cycloalkyl group, a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

9. The following, 【Chemical Formula 2】 【Chemical Formula】 【Chemical Formula】 【Chemical Formula】 【Chemical Formula】 【Chemical Formula】 【Chemical Formula】 【Chemical Formula】 A compound selected from or a pharmaceutically acceptable salt thereof.

10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 10 for the treatment of a disease caused by a virus having a cap-dependent endonuclease.

12. The pharmaceutical composition according to claim 10 for inhibiting a cap-dependent endonuclease in a virus.

13. The pharmaceutical composition according to claim 10 for the treatment of influenza in a mammal.

14. The pharmaceutical composition according to claim 10 for the prevention of influenza in a mammal.

15. The pharmaceutical composition according to claim 13, wherein the mammal is a human.

16. The pharmaceutical composition according to claim 14, wherein the mammal is a human.

17. Use of a compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating or preventing influenza.

18. A compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof for use in therapy.

Images