TW201717956A - Medical use of hydroxypurine compound - Google Patents

Abstract

The invention discloses applications of a series of hydroxypurine compounds in preparation of drugs for treating or preventing liver diseases, and specifically discloses uses of a compound having a structure as shown in Formula (I), tautomers and pharmaceutically acceptable salts thereof in drugs of liver diseases.

Description

Medical use of hydroxy steroids

The present invention relates to the use of a series of hydroxy steroids in the preparation of a medicament for treating liver diseases, and in particular to the use of a compound of the formula (I), a tautomer thereof or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating liver diseases.

Phosphodiesterase (PDE) catalyzes the hydrolysis of cyclized nucleotides cGMP and cAMP, and regulates various physiological responses by controlling the intramolecular concentrations of these two important secondary signaling factors. The abnormal regulation of cyclized nucleotides cGMP and cAMP molecules is the cause of many diseases. Several drugs have been used to improve and treat diseases by inhibiting PDE activity, such as PDE5 inhibitors for pulmonary hypertension, and PDE4 inhibitors for PDE4 inhibitors. Arthritis caused by psoriasis. There are currently eleven major classes of phosphodiesterase genes, each of which can express several subtypes, for a total of more than 100 PDE subtypes. Different subtypes have different structures, different tissue distributions, and the activities of cyclized nucleotides cGMP and cAMP are also very different, and the physiological functions of regulation are also very different.

PDE2 phosphodiesterase can catalyze the hydrolysis of cyclized nucleotides cGMP and cAMP, while cAMP activity is regulated by cGMP, which plays a key role in the balance of cGMP and cAMP functions in cells. PDE2 is widely expressed in human tissues and is mainly distributed in the heart, central nervous system, liver, adrenal gland, endothelial cells, and platelets. PDE2 is involved in the regulation of various physiological activities, such as central learning, memory and cognition, maintaining the basic rhythm of the heart, smooth muscle and endothelial cells, permeability of endothelial cells, and regulating inflammation. PDE2 knockout mice directly cause embryonic death. It can be used in a variety of central, cardiovascular, and inflammatory responses by inhibiting PDE2 activity.

Non-selective PDE inhibitory activities of various natural and synthetic terpenoids have long been discovered, such as caffeine, theophylline, pentoxifylline and the like. Pentoxifylline (PDE2 activity) is clinically approved for lower limb paralysis caused by peripheral vascular occlusion, the main role is to reduce blood viscosity, increase red blood cell deformation, inhibit platelet aggregation and so on. Novel highly selective PDE2 inhibitors have also been reported to control endothelial cell division and angiogenesis, and to improve central cognitive impairment. However, the development and application of the new novel selective PDE2 inhibitors are still very limited, and the discovery and application of new PDE2 inhibitors have broad prospects.

Tumor necrosis factor alpha (TNF-α) is a cytokine with many biological activities, which has an important impact on the occurrence, development and treatment of various diseases, especially immune and inflammation related diseases. TNF-α is mainly produced by monocytes and macrophage cell lines, and participates in the body's immune regulation and cytokine network coordination. Under normal circumstances, TNF-α plays an important role in immune defense and immune surveillance, but in some cases it has an adverse effect. Studies have shown that overexpression of TNF-α can induce the expression of proinflammatory cytokines such as interleukon-1, IL-1, IL-6, increase endothelial cell permeability, up-regulate adhesion molecule expression, and activate neutrophils. And eosinophils, and induce the secretion of acute phase substances and tissue degrading enzymes by bone synovial cells and chondrocytes to promote the occurrence of inflammation. These pathological reactions play a very important role in the development of many immune-mediated inflammatory diseases (IMID), such as rheumatoid arthritis (RA) and psoriatic arthritis. , PsA), ankylosing spondylitis (AS), inflammatory bowel disease (IBD), juvenile chronic arthritis (JCA), and vasculitis. Studies have shown that TNF-α is an ideal target for many of the above IMIDs, and for some diseases caused by chronic chronic inflammatory damage, such as steatohepatitis, chronic obstructive pneumonia, etc., TNF-α inhibitors are used. And excessive TNF-α is also an effective prevention and treatment route. TNF-α monoclonal antibody has been clinically proven to inhibit TNF-α as a very effective means of treating the above-mentioned inflammation-related diseases. PDE2 can regulate the expression of TNF-α by mechanism, so it can control the level of TNF-α by regulating PDE2 activity, thus controlling inflammatory response.

The present invention provides a compound of the formula (I), a tautomer thereof or a pharmaceutically acceptable salt thereof for use in the preparation of a medicament for treating or preventing liver disease, (I) where the structural unit can be Replace with , specifically replaced by ; L _{11 is} selected from the group consisting of N, C(R)(R'); R and R' are each independently selected from H, halogen, OH, NH ₂ , CN, optionally substituted 1 to 6 alkyl or heteroalkyl And optionally, R, R' may be cyclized to a 3-6 membered cycloalkyl, heterocycloalkyl; A is vacant or selected from optionally substituted cycloalkyl, heterocycloalkyl, aryl, a heteroaryl group; L _{12 is} selected from an optionally substituted 1 to 6 membered alkyl or heteroalkyl group; and R _{1 is} selected from an optionally substituted 1 to 6 membered alkyl group, a 3 to 6 membered cycloalkyl group or a heteroalkyl group. group; "heteroaryl" represents N, O, S, C ( = O), S (= O), S (= O) 2, each of the number of heteroatoms selected from the group 2, 3 or 4.

In some aspects of the invention, the liver disease is selected from the group consisting of nonalcoholic fatty liver and liver fibrosis.

In some embodiments of the invention, the substituents in the above R, R', A, L ₁₂ and R ₁ are each independently selected from the group consisting of halogen, OH, NH ₂ , CN, optionally substituted 1 to 6 alkyl, 3 A 6-membered cycloalkyl or heteroalkyl group, the number of each group substituent being independently selected from 1, 2 or 3.

In some embodiments of the invention, the substituents in the above R, R', A, L ₁₂ and R ₁ are each independently selected from the group consisting of halogen, CF ₃ , CN, OH, Me, Et, n-propyl, isopropyl, and ring. Propyl, , .

In some aspects of the invention, R and R' are each independently selected from the group consisting of H, Me, CF ₃ , and Et.

In some embodiments of the invention, the above L _{11 is} selected from , , , , , , .

In some embodiments of the invention, the above A is selected from the group consisting of: a 3 to 12 membered cycloalkyl or heterocycloalkyl group, a 5 to 12 membered aryl group or a heteroaryl group.

In some embodiments of the invention, the above A is selected from the group consisting of: optionally substituted: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pentyl, phenyl, pyridyl, pyrazinyl, oxazolyl , isoxazolyl, thiazolyl, bicyclo[1.1.1]pentane, or a bicyclic group, a spiro group or a bicyclic group selected from any two of the above groups.

In some aspects of the invention, the above A is selected from the group consisting of: , , , , , , , , , , , , , , .

In some aspects of the invention, the above A is selected from the group consisting of , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

In some embodiments of the invention, the above L _{12 is} selected from the group consisting of methylene, , , , , .

In some embodiments of the invention, the above R _{1 is} selected from the group consisting of Me, CHF ₂ , CF ₃ , Et, CH ₂ CF ₃ , isopropyl, , cyclopropyl, , , , .

The invention is selected from the group consisting of the following compounds in the preparation of a medicament for treating or preventing liver diseases: ; ; .

Further, the present invention is selected from the group consisting of the following compounds in the preparation of a medicament for treating or preventing liver diseases: .

Related definitions.

Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered undefined or unclear without a particular definition, but should be understood in the ordinary sense. When a trade name appears in this document, it is intended to refer to its corresponding commodity or its active ingredient.

The term "pharmaceutically acceptable" as used herein is intended to mean that those compounds, materials, compositions and/or dosage forms are within the scope of sound medical judgment and are suitable for use in contact with human and animal tissues. Without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" refers to a salt of a compound of the invention prepared from a compound having a particular substituent found in the present invention and a relatively non-toxic acid or base. When a relatively acidic functional group is contained in the compound of the present invention, a base addition salt can be obtained by contacting a neutral amount of such a compound with a sufficient amount of a base in a neat solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When a relatively basic functional group is contained in the compound of the present invention, an acid addition salt can be obtained by contacting a neutral form of such a compound with a sufficient amount of an acid in a neat solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogencarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and an organic acid salt, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; and salts of amino acids (such as arginine, etc.) And salts of organic acids such as glucuronic acid (see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds of the invention contain both basic and acidic functional groups which can be converted to any base or acid addition salt.

Preferably, the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated, thereby regenerating the neutral form of the compound. The parent form of the compound differs from the form of its various salts by certain physical properties, such as differences in solubility in polar solvents.

As used herein, a "pharmaceutically acceptable salt" is a derivative of a compound of the invention wherein the parent compound is modified by salt formation with an acid or with a base. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of bases such as amines, alkali metal or organic salts of acid groups such as carboxylic acids, and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts or quaternary ammonium salts of the parent compound, for example salts formed from non-toxic inorganic or organic acids. Conventional non-toxic salts include, but are not limited to, those derived from inorganic acids and organic acids selected from the group consisting of 2-ethyloxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid. , benzenesulfonic acid, benzoic acid, hydrogencarbonate, carbonic acid, citric acid, edetic acid, ethane disulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptose, gluconic acid, glutamic acid, glycolic acid , hydrobromic acid, hydrochloric acid, hydroiodide, hydroxyl, hydroxynaphthalene, isethionethane, lactic acid, lactose, dodecyl sulfonic acid, maleic acid, malic acid, mandelic acid, methane sulfonic acid, nitric acid, oxalic acid , pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturaldehyde, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, sulfamic acid, p-aminobenzenesulfonic acid, sulfuric acid, tannin , tartaric acid and p-toluenesulfonic acid.

The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing an acid group or a base by conventional chemical methods. In general, such salts are prepared by reacting these compounds in water or an organic solvent or a mixture of the two via a free acid or base form with a stoichiometric amount of a suitable base or acid. Generally, a nonaqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is preferred.

In addition to the form of the salt, the compounds provided herein also exist in the form of prodrugs. Prodrugs of the compounds described herein are readily chemically altered under physiological conditions to convert to the compounds of the invention. Furthermore, prodrugs can be converted to the compounds of the invention by chemical or biochemical methods in an in vivo setting.

Certain compounds of the invention may exist in unsolvated or solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are included within the scope of the invention.

Certain compounds of the invention may have asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereomers, geometric isomers and individual isomers are included within the scope of the invention.

Wedge and dashed keys are used unless otherwise stated ) indicates the absolute configuration of a stereocenter, used Represents the relative configuration of a stereocenter. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, they include the E , Z geometric isomers unless otherwise specified. Likewise, all tautomeric forms are included within the scope of the invention.

The compounds of the invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including the cis and trans isomers, the (-)- and (+)-enantiomers, the ( R )- and ( S )-enantiomers, diastereoisomers , ( D )-isomer, ( L )-isomer, and racemic mixtures thereof and other mixtures, such as enantiomerically or diastereomeric enriched mixtures, all of which belong to the present Within the scope of the invention. Additional asymmetric carbon atoms may be present in the substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the invention.

The optically active ( R )- and ( S )-isomers as well as the D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If an enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary wherein the resulting mixture of diastereomers is separated and the auxiliary groups cleaved to provide a pure Enantiomers are required. Alternatively, when a molecule contains a basic functional group (e.g., an amino group) or an acidic functional group (e.g., a carboxyl group), a salt of a diastereomer is formed with a suitable optically active acid or base, and then by a conventional method known in the art. The diastereomers are resolved and the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomers is generally accomplished by the use of chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (eg, formation of an amino group from an amine). Formate).

The compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound. For example, radiolabeled compounds can be used, such as tritium ⁽³ H), iodine -125 ⁽¹²⁵ I) or ^C-14 (14 C). Alterations of all isotopic compositions of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.

The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium that is capable of delivering an effective amount of an active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects to the host or patient, including water, oil, vegetables. And minerals, cream bases, lotion bases, ointment bases, and the like. These bases include suspending agents, tackifiers, transdermal enhancers, and the like. Their formulations are well known to those skilled in the cosmetic or topical pharmaceutical arts. For additional information on the vector, reference is made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the disclosure of which is incorporated herein by reference.

The term "excipient" generally refers to the carrier, diluent and/or vehicle required to formulate an effective pharmaceutical composition.

The term "effective amount" or "therapeutically effective amount" with respect to a pharmaceutical or pharmacologically active agent refers to a sufficient amount of a drug or agent that is non-toxic but that achieves the desired effect. For oral dosage forms in the present invention, an "effective amount" of an active substance in a composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount will vary from person to person, depending on the age and general condition of the recipient, and also on the particular active substance, and a suitable effective amount in a case can be determined by one skilled in the art based on routine experimentation.

The term "active ingredient", "therapeutic agent", "active substance" or "active agent" refers to a chemical entity that is effective in treating a target disorder, disease or condition.

"Optional" or "optionally" means that the subsequently described event or condition may, but is not necessarily, to occur, and that the description includes instances in which the event or condition occurs and the event or condition does not occur. Case.

The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, including variants of heavy hydrogen and hydrogen, as long as the valence of the particular atom is normal and the substituted compound is stable. . When the substituent is a keto group (ie, =0), it means that two hydrogen atoms are substituted. Ketone substitution does not occur on the aryl group. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the kind and number of substituents may be arbitrary on the basis of chemically achievable.

When any variable (eg, R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0-2 R, the group may optionally be substituted with at most two R, and each case has an independent option. Furthermore, combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

When the number of one linking group is 0, such as -(CRR) ₀ -, it indicates that the linking group is a single bond.

When one of the variables is selected from a single bond, it means that the two groups to which it is attached are directly linked. For example, when L represents a single bond in A-L-Z, the structure is actually A-Z.

When a substituent is vacant, it means that the substituent is absent. For example, when X is vacant in A-X, the structure is actually A.

When a bond of a substituent can be cross-linked to two atoms on a ring, the substituent can be bonded to any atom on the ring. When the recited substituents do not indicate which atom is attached to a compound included in the chemical structural formula including but not specifically mentioned, such a substituent may be bonded through any atomic phase thereof. Combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds. For example, a structural unit or It is indicated that it can be substituted at any position on the cyclohexyl or cyclohexadiene.

Unless otherwise specified, the term "halo" or "halogen", by itself or as part of another substituent, denotes a fluorine, chlorine, bromine or iodine atom. Further, the term "haloalkyl" is intended to include both monohaloalkyl and polyhaloalkyl. For example, the term "halo(C ₁ -C ₄ )alkyl" is intended to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. Wait.

Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl. "Alkoxy" represents the above alkyl group having a specified number of carbon atoms attached through an oxygen bridge. The C _1-6 alkoxy group includes a C ₁ , C ₂ , C ₃ , C ₄ , C ₅ and C ₆ alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, bis-butoxy, tert-butoxy, n-pentyloxy, and S -pentyloxy. "Cycloalkyl" includes saturated cyclic groups such as cyclopropyl, cyclobutyl or cyclopentyl. The 3-7 cycloalkyl group includes C ₃ , C ₄ , C ₅ , C ₆ and C ₇ cycloalkyl groups. "Alkenyl" includes hydrocarbon chains in a straight or branched configuration wherein one or more carbon-carbon double bonds, such as vinyl and propylene groups, are present at any stable site on the chain.

The term "halo" or "halogen" refers to fluoro, chloro, bromo and iodo.

Unless otherwise specified, the term "hetero" denotes a hetero atom or a hetero atomic group (ie, a radical containing a hetero atom), including atoms other than carbon (C) and hydrogen (H), and radicals containing such heteroatoms, including, for example, oxygen (O). ), nitrogen (N), sulfur (S), antimony (Si), germanium (Ge), aluminum (Al), boron (B), -O-, -S-, =O, =S, -C (= O) O-, -C(=O) -, -C(=S)-, -S(=O), -S(=O) ₂ -, and optionally substituted -C(=O)N (H)-, -N(H)-, -C(=NH)-, -S(=O) ₂ N(H)- or -S(=O) N(H)-.

Unless otherwise specified, "ring" means substituted or unsubstituted cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, aryl or heteroaryl. So-called rings include single rings, interlocking rings, spiral rings, parallel rings or bridge rings. The number of atoms on the ring is usually defined as the number of elements of the ring. For example, "5 to 7-membered ring" means 5 to 7 atoms arranged in a circle. Unless otherwise specified, the ring optionally contains from 1 to 3 heteroatoms. Thus, "5- to 7-membered ring" includes, for example, phenylpyridine and acridinyl; on the other hand, the term "5- to 7-membered heterocycloalkyl ring" includes pyridyl and acridinyl, but does not include phenyl. The term "ring" also includes ring systems containing at least one ring, each of which "ring" independently conforms to the above definition.

Unless otherwise specified, the term "heterocycle" or "heterocyclyl" means a stable monocyclic, bicyclic or tricyclic ring containing a hetero atom or a heteroatom group which may be saturated, partially unsaturated or unsaturated ( Aromatic) which comprise a carbon atom and 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S, wherein any of the above heterocycles may be fused to a phenyl ring to form a bicyclic ring. The nitrogen and sulfur heteroatoms can be optionally oxidized (i.e., NO and S(O)p, p is 1 or 2). The nitrogen atom can be substituted or unsubstituted (i.e., N or NR, wherein R is H or other substituents as already defined herein). The heterocyclic ring can be attached to the side groups of any hetero atom or carbon atom to form a stable structure. If the resulting compound is stable, the heterocycles described herein can undergo substitutions at the carbon or nitrogen sites. The nitrogen atom in the heterocycle is optionally quaternized. A preferred embodiment is that when the total number of S and O atoms in the heterocycle exceeds 1, these heteroatoms are not adjacent to each other. Another preferred embodiment is that the total number of S and O atoms in the heterocycle does not exceed one. The term "aromatic heterocyclic group" or "heteroaryl" as used herein means a stable 5, 6, or 7 membered monocyclic or bicyclic or aromatic ring of a 7, 8, 9 or 10 membered bicyclic heterocyclic group, It contains carbon atoms and 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S. The nitrogen atom can be substituted or unsubstituted (i.e., N or NR, wherein R is H or other substituents as already defined herein). The nitrogen and sulfur heteroatoms can be optionally oxidized (i.e., NO and S(O)p, p is 1 or 2). It is worth noting that the total number of S and O atoms on the aromatic heterocycle does not exceed one. Bridged rings are also included in the definition of heterocycles. A bridged ring is formed when one or more atoms (ie, C, O, N, or S) join two non-adjacent carbon or nitrogen atoms. Preferred bridged rings include, but are not limited to, one carbon atom, two carbon atoms, one nitrogen atom, two nitrogen atoms, and one carbon-nitrogen group. It is worth noting that a bridge always converts a single ring into a three ring. In the bridged ring, a substituent on the ring can also be present on the bridge.

Examples of heterocyclic compounds include, but are not limited to, acridinyl, octanoyl, benzimidazolyl, benzofuranyl, benzofuranylfuranyl, benzindenylphenyl, benzoxazolyl, benzimidin Oxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolyl, oxazolyl, 4a H -carbazolyl , porphyrin, chroman, chromene, porphyrin-decahydroquinolinyl, 2 H , 6 H -1, 5,2-dithiazinyl, dihydrofuran [2,3 - b ] tetrahydrofuranyl, furyl, furfuryl, imidazolidinyl, imidazolinyl, imidazolyl, 1 H -carbazolyl, nonenyl, indanyl, mesoindolyl, fluorenyl , 3 H -indenyl, isatino, isobenzofuranyl, isodecyl, isoindoline, isoquinolyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl , morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5- Diazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, hydroxydecyl, pyrimidinyl, phenanthryl, phenanthrene Orolinyl, phenazine, phenothiazine, benzoxanthyl, phenolzinyl, pyridazinyl, pyridazinyl, acridinyl, acridone, 4-acridone, piperonyl, pteridine Base, fluorenyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridylthiazole, pyridyl, pyrrolidinyl , pyrrolinyl, 2 H -pyrrolyl, pyrrolyl, quinazolinyl, quinolyl, 4 H -quinazinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, Tetrahydroquinolyl, tetrazolyl, 6 H -1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2, 5-thiadiazolyl, 1,3,4-thiadiazolyl, thioxyl, thiazolyl, isothiazolylthiophenyl, thienyl, thienooxazolyl, thienothiazolyl, thienoimidazolyl, Triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthene. Also included are fused ring and spiro compounds.

Unless otherwise specified, the term "hydrocarbyl" or its subordinate concept (such as alkyl, alkenyl, alkynyl, aryl, etc.), by itself or as part of another substituent, is meant to be straight-chain, branched or cyclic. The hydrocarbon atom group or a combination thereof may be fully saturated (such as an alkyl group), a unit or a polyunsaturated (such as an alkenyl group, an alkynyl group, an aryl group), may be monosubstituted or polysubstituted, and may be monovalent (such as Methyl), divalent (such as methylene) or polyvalent (such as methine), may include divalent or polyvalent radicals with a specified number of carbon atoms (eg, C ₁ -C ₁₂ represents 1 to 12 carbons) , C _{1-12 is} selected from C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ and C ₁₂ ; C _{3-12 is} selected from C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ and C ₁₂ .). "Hydrocarbyl" includes, but is not limited to, aliphatic hydrocarbyl groups including chain and cyclic, including but not limited to alkyl, alkenyl, alkynyl groups including, but not limited to, 6-12 members. An aromatic hydrocarbon group such as benzene, naphthalene or the like. In some embodiments, the term "hydrocarbyl" means a straight or branched chain radical or a combination thereof, which may be fully saturated, unitary or polyunsaturated, and may include divalent and multivalent radicals. Examples of saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, secondary butyl, isobutyl, cyclohexyl, (ring Hexyl) methyl, cyclopropylmethyl, and homologs or isomers of radicals such as n-pentyl, n-hexyl, n-heptyl, n-octyl. The unsaturated hydrocarbon group has one or more double or triple bonds, and examples thereof include, but are not limited to, a vinyl group, a 2-propenyl group, a butenyl group, a crotyl group, a 2-isopentenyl group, and a 2-(butadienyl group). , 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers body.

Unless otherwise specified, the term "heterohydrocarbyl" or its subordinate concept (such as heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, etc.), by itself or in combination with another term, means a stable straight chain, branched chain. Or a cyclic hydrocarbon radical or a combination thereof having a number of carbon atoms and at least one heteroatom. In some embodiments, the term "heterohydrocarbyl" by itself or in conjunction with another term denotes a stable straight chain, branched hydrocarbon radical or combination thereof, having a number of carbon atoms and at least one heteroatom. In a typical embodiment, the heteroatoms are selected from the group consisting of B, O, N, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen heteroatoms are optionally quaternized. The heteroatoms B, O, N and S can be located at any internal position of the heterohydrocarbyl group (including where the hydrocarbyl group is attached to the rest of the molecule). Examples include, but are not limited to, -CH ₂ -CH ₂ -O-CH ₃ , -CH ₂ -CH ₂ -NH-CH ₃ , -CH ₂ -CH ₂ -N(CH ₃ )-CH ₃ , -CH ₂ -S -CH ₂ -CH ₃ , -CH ₂ -CH ₂ , -S(O)-CH ₃ , -CH ₂ -CH ₂ -S(O) ₂ -CH ₃ , -CH=CH-O-CH ₃ , - CH ₂ -CH=N-OCH ₃ and -CH=CH-N(CH ₃ )-CH ₃ . Up to two heteroatoms may be consecutive, for example, -CH ₂ -NH-OCH _3.

The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are conventionally used to refer to those alkane which are attached to the remainder of the molecule through an oxygen atom, an amino group or a sulfur atom, respectively. Base group.

Unless otherwise specified, the term "alkyl" is used to denote a straight or branched saturated hydrocarbon group, which may be monosubstituted (eg, -CH ₂ F) or polysubstituted (eg, -CF ₃ ), and may be monovalent (eg, Methyl), divalent (such as methylene) or polyvalent (such as methine). Examples of the alkyl group include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl). , t-butyl), pentyl (eg, n-pentyl, isopentyl, neopentyl) and the like.

Unless otherwise specified, the term "halo" or "halogen", by itself or as part of another substituent, denotes a fluorine, chlorine, bromine or iodine atom. Further, the term "haloalkyl" is intended to include both monohaloalkyl and polyhaloalkyl. For example, the term "halo(C ₁ -C ₄ )alkyl" is intended to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. Wait. Unless otherwise specified, examples of haloalkyl include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl.

The above-described alkyl groups having the specified number of carbon atoms, "alkoxy" represents attached through an oxygen bridge, unless otherwise specified, C _1-6 alkoxy groups include _{C 1, C 2, C 3} , C 4, C 5 , and C ₆ alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, bis-butoxy, tert-butoxy, n-pentyloxy, and S -pentyloxy.

Unless otherwise specified, the term "cycloalkyl", "heterocycloalkyl" or its subordinate concept (such as aryl, heteroaryl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl) A heterocyclic alkynyl group, etc., by itself or in combination with other terms, denotes a cyclized "hydrocarbyl group" or "heterohydrocarbyl group", respectively. Further, in the case of a heterohydrocarbyl group or a heterocycloalkyl group (such as a heteroalkyl group or a heterocycloalkyl group), a hetero atom may occupy a position at which the hetero ring is attached to the rest of the molecule. Examples of cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Non-limiting examples of heterocyclic groups include 1-(1,2,5,6-tetrahydropyridyl), 1-acridinyl, 2-acridinyl, 3-acridinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-pyridazinyl and 2-pyridazinyl.

Unless otherwise specified, the term "aryl" denotes a polyunsaturated, aromatic hydrocarbon substituent which may be monosubstituted, disubstituted or polysubstituted, may be monovalent, divalent or polyvalent, it may be monocyclic or Polycyclic (preferably 1 to 3 rings) which are fused together or covalently linked. The term "heteroaryl" refers to an aryl (or ring) containing one to four heteroatoms. In an illustrative example, the heteroatoms are selected from the group consisting of B, N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl or heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyridyl Azyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxan Azyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thiophene , 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, indolyl, 2-benzimidazolyl, 5-indenyl, 1-isoquinolyl, 5-isoquinolinyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl and 6-quinolinyl. The substituents of any of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

For simplicity, aryl groups, when used in conjunction with other terms (eg, aryloxy, arylthio, aralkyl), include aryl and heteroaryl rings as defined above. Thus, the term "aralkyl" is intended to include those radicals to which an aryl group is attached to an alkyl group (eg, benzyl, phenethyl, pyridylmethyl, and the like), including wherein the carbon atom (eg, methylene) has been, for example, oxygen. Those alkyl groups substituted by an atom, such as phenoxymethyl, 2-pyridyloxymethyl 3-(1-naphthyloxy)propyl and the like.

The term "leaving group" refers to a functional group or atom which may be substituted by another functional group or atom by a substitution reaction (for example, an affinity substitution reaction). For example, representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, p-toluenesulfonic acid Ester and the like; an oxime group such as an ethoxy group, a trifluoroacetoxy group or the like.

The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxy protecting group" or "thiol protecting group". The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to, indolyl; fluorenyl, such as alkane fluorenyl (e.g., ethoxymethyl, trichloroethyl or trifluoroethyl); alkoxycarbonyl, such as tertiary Butoxycarbonyl (Boc); arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr , 1,1-di-(4'-methoxyphenyl)methyl; methoxyalkyl, such as trimethylmethanyl (TMS) and tertiary butyl dimethyl methacrylate (TBS), etc. Wait. The term "hydroxy protecting group" refers to a protecting group suitable for use in preventing hydroxy side reactions. Representative hydroxy protecting groups include, but are not limited to, alkyl groups such as methyl, ethyl and tertiary butyl groups; fluorenyl groups such as alkane fluorenyl groups (e.g., ethenyl); arylmethyl groups such as benzyl (Bn) , p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); formyl, such as trimethylmethanyl (TMS) And tertiary butyl dimethylformamidine (TBS) and the like.

The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, combinations thereof with other chemical synthesis methods, and equivalents well known to those skilled in the art. Alternatively, preferred embodiments include, but are not limited to, embodiments of the invention.

All solvents used in the present invention are commercially available and can be used without further purification. The present invention employs the following abbreviations: Compound 1 is Example 51 isomer 2; Pen. is pentoxifylline; INT-747 is 6-ethyl chenodeoxycholic acid; aq stands for water; HATU stands for O- (7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; EDC stands for N-(3-dimethylaminopropyl)-N '-Ethylcarbodiimide hydrochloride; m-CPBA stands for 3-chloroperoxybenzoic acid; eq stands for equivalent, equivalent; CDI stands for carbonyldiimidazole; DCM stands for dichloromethane; PE stands for petroleum ether; DIAD stands for Diisopropyl azodicarboxylate; DMF stands for N,N-dimethylformamide; DMSO stands for dimethyl hydrazine; EtOAc stands for ethyl acetate; EtOH stands for ethanol; MeOH stands for methanol; CBz stands for benzyloxycarbonyl, Is an amine protecting group; BOC represents a tertiary carbonyl group which is an amine protecting group; HOAc represents acetic acid; NaCNBH ₃ represents sodium cyanoborohydride; rt represents room temperature; O/N represents overnight; THF represents tetrahydrofuran; Representing ₂ O - tert.butyl dicarbonate; Representative TFA trifluoroacetic acid; Representing DIPEA diisopropylethylamine; SOCl ₂ represents an alkylene chloride sulfone; CS ₂ on behalf of the carbon disulfide; TsOH A representative Acid; NFSI Representative fluoro-N- -N- (benzenesulfonamide acyl) benzenesulfonamide Amides; the NCS represents a 1-chloro-2,5-dione; -Bu ₄ NF Representative n-tetrabutylammonium fluoride; iPrOH represents 2-propanol; mp represents melting point; LDA represents lithium diisopropylamide; TMSCF ₃ represents trifluoromethyltrimethylnonane; Ti(Oi-Pr) ₄ represents tetraisopropyl titanate; MSCl represents Methane sulfonium chloride; DMAP stands for N,N-dimethyl-4-aminopyridine; TEA stands for triethylamine; BnBr stands for benzyl bromide; DIEA stands for diisopropylethylamine; BH ₃ DMS stands for borane dimethyl sulfide DMP stands for Des Martin Sodium Periodane; TBAF stands for tetrabutylammonium fluoride; HOBT stands for 1-hydroxybenzotriazole; AIBN stands for azobisisobutyronitrile; NBS stands for N-bromosuccinimide; RT buffer represents reverse transcription buffer; dNTP represents deoxyribonucleoside triphosphate; PBS represents phosphate buffer.

Compounds are named by hand or by ChemDraw® software, and commercial compounds are listed under the supplier's catalogue.

The invention is described in detail below by the examples, but is not intended to limit the invention.

Example 1.

3,7-Dimethyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -indole-2,6(3H,7H)-dione.

first step.

3,7-Dimethyl-1-(6,6,6-trifluoro-5-methyl-5-(trimethyldecyloxy)hexyl)-1 H -indole-2,6(3 H , 7 H )-dione.

3,7-Dimethyl-1-(5-oxohexyl)-1 H -indole-2,6(3 H ,7 H )-dione (200 mg, 0.719 mmol), cesium fluoride (10.9 M, 0.0719 mmol) was dissolved in tetrahydrofuran (2 mL) and trifluoromethyltrimethyldecane (153 mg, 1.08 mmol) was then evaporated. The reaction mixture was stirred at 20 ° C for 2 hr. The organic layer was washed with brine (100 mL EtOAc) Drying in vacuo gave 3,7-dimethyl-1-(6,6,6-trifluoro-5-methyl-5-(trimethyldecyloxy)hexyl)-1 H -indole-2,6 ( 3 H , 7 H )-dione (200 mg, white solid), yield: 66%. MS-ESI calcd [M + H] ⁺ 422.

The second step.

3,7-Dimethyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -indole-2,6(3 H ,7 H )-dione.

3,7-Dimethyl-1-(6,6,6-trifluoro-5-methyl-5-(trimethyldecyloxy)hexyl)-1 H -indole-2,6 (3H, 7H)-Dione (200 mg, 0.476 mmol) was dissolved in tetrahydrofuran (2 mL), and 1 M hydrochloric acid (0.5 mL) was added dropwise at 0 ° C and then stirred at 20 ° C for 1 hour. The mixture was cooled to 0 ° C and quenched with sodium bicarbonate (30 mL). The mixture was extracted with ethyl acetate (100 mL x 3). The organic layer was washed with brine (100 mL EtOAc) Separation and purification by preparative high performance liquid chromatography gave 3,7-dimethyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -嘌呤-2,6 (3) H , 7H )-dione (50.0 mg, white solid), yield: 30%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.85 (s, 1H), 4.02-3.98 (m, 2H), 3.96 (s, 3H), 3.52 (s, 3H), 1.69-1.64 (m, 4H), 1.52-1.48 (m, 2H), 1.28 (s, 3H). MS-ESI calcd [M + H] ⁺ 495.

Example 2.

1-(5-Hydroxy-5-methylheptyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione.

first step.

Ethyl 5-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)pentanoic acid ethyl ester.

3,7-Dimethyl-1 H -indole-2,6(3 H ,7 H )-dione (5.00 g, 28.0 mmol), ethyl bromopentanoate (7.51 g, 33.4 mmol), potassium carbonate (7.73 g, 56.0 mmol) and potassium iodide (500 mg, 2.80 mmol) were dissolved in N , N -dimethylformamide (62 mL). The reaction solution was heated to 110 ° C and stirred for two hours. The reaction was poured into water and extracted with ethyl acetate (20 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give ethyl 5- (3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro -1 H -嘌呤-1-yl)ethyl valerate (5.00 g, yellow solid), yield: 50%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.51 (s, 1H), 4.14-4.09 (m, 2H), 4.04-4.01 (m, 2H), 3.97 (s, 3H), 3.57 (s, 3H) ), 2.37-2.33 (m, 2H), 1.72-1.69 (m, 4H), 1.25 (t, J = 7.2 Hz, 3H).

The second step.

1-(5-Ethyl-5-hydroxyheptyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione.

Ethyl 5-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)pentanoic acid ethyl ester (0.500 g, 1.62 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL). N.sub.2. The reaction solution was stirred at -78 ° C for 0.5 hour, then slowly raised to 0 ° C and reacted for 0.5 hour. The reaction solution was poured into water and extracted with ethyl acetate (30 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 1- (5-ethyl-5-hydroxy-3,7-dimethyl-heptyl -1 H - purine -2,6 (3 H , 7 H )-dione (0.300 g, colorless oil), yield: 57%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.50 (s, 1H), 4.05-4.01 (m, 2H ), 3.99 (s, 3H), 3.57 (s, 3H), 1.70-1.37 (m, 10H), 0.86 (t, J = 7.6 Hz, 6H). MS-ESI calculated [M + H] ⁺ 323, Found 323.

Example 3.

1-(4-(1-Hydroxycyclopropyl)butyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione.

Ethylmagnesium bromide (3 M ether solvent, 1.1 mL, 3.24 mmol) was added to ethyl 5-(3,7-dimethyl-2,6-dioxo-2 at -35 °C under nitrogen. Ethyl 3,6,7-tetrahydro-1 H -indol-1-yl)pentanoate (500 mg, 1.62 mmol) and tetraisopropyl titanate (461 mg, 1.62 mmol) in tetrahydrofuran (10 mL) in. The reaction solution was slowly heated to 25 ° C and stirred for 2 hours. The reaction was quenched by water (10 mL). The organic phase was combined, dried over anhydrous sodium sulfate, filtered, and then filtered and evaporated. 1 H - purine -2,6 (3 H, 7 H) - dione (90.0 mg, white solid), yield: 19%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.86 (s, 1H), 4.03 - 3.90 (m, 5H), 3.51 (s, 3H), 1.72-1.53 (m, 6H), 0.68-0.59 (m, 2H), 0.46-0.38 (m, 2H). MS-ESI calcd for [M ⁺ H] + 293, found 293.

Example 4.

3,7-Dimethyl-1-((1-(1,1,1-trifluoro-2-hydroxypropan-2-yl)cyclopropyl)methyl)-1 H -嘌呤-2,6- (3 H , 7 H )-dione.

first step.

Ethyl 1-ethenylcyclopropane.

Ethyl-3-oxobutanoic acid (10.0 g, 76.8 mmol) and 1,2-dibromoethane (21.7 g, 115 mmol) were dissolved in dimethyl hydrazine (300 mL) under nitrogen Potassium carbonate (42.5 g, 307 mmol) was added in portions. The reaction solution was stirred at 25 ° C for 24 hours. After adding water (500 mL), the reaction mixture was extracted with ethyl acetate (300 mL×3), and the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (10:1 petroleum ether) Ethyl acetate (Rf = 0.4) gave ethyl 1-ethylhydrazinocyclopropane (6.00 g, white oil), yield: 50%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 4.25 - 4.20 (m, 2H), 2.44 (s, 3H), 1.47-1.42 (m, 4H), 1.32-1.28 (m, 3H).

The second step.

1-(1,1,1-Trifluoro-2-hydroxypropan-2-yl)cyclopropanecarboxylic acid.

Ethyl 1-ethenylcyclopropane (2.00 g, 12.8 mmol), cesium fluoride (195 mg, 1.28 mmol) was dissolved in tetrahydrofuran (30 mL), then trifluoromethyltrimethyl decane was added at 0 °C. 3.64 g, 25.6 mmol). The reaction solution was reacted under nitrogen for 20 hours at 20 °C. Then 4 N diluted hydrochloric acid (7 mL) was added. The mixture was reacted under nitrogen for 6 hours at room temperature. The reaction was quenched with EtOAc EtOAc (EtOAc (EtOAc) 1 petroleum ether / ethyl acetate, Rf = 0.4) to give 1-(1,1,1-trifluoro-2-hydroxypropan-2-yl)cyclopropanecarboxylic acid (1.70 g, white oil) : 59%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 4.14-4.10 (m, 2H), 1.64 (s, 3H), 1.29-1.24 (m, 3H), 1.23-1.22 (m, 2H), 0.92 -0.90 (m, 2H).

third step.

1,1,1-Trifluoro-2-(1-(hydroxymethyl)cyclopropyl)propan-2-ol.

1-(1,1,1-Trifluoro-2-hydroxypropan-2-yl)cyclopropanecarboxylic acid (400 mg, 1.77 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL). Lithium (81.0 mg, 2.12 mmol). The reaction solution was warmed to 25 ° C and stirred for 1 hour. It was quenched with water (10 mL), EtOAc (EtOAc (EtOAc)EtOAc. = 0.2) gave 1,1,1-trifluoro-2-(1-(hydroxymethyl)cyclopropyl)propan-2-ol (200 mg, yellow oil). ¹ H NMR: (400 MHz, DMSO- d ₆ ) δ 5.64 (s, 1H), 4.63-4.60 (m, 1H), 3.64 - 3.60 (m, 1H), 3.23 - 3.17 (m, 1H), 1.36 (s, 3H), 0.83-0.91 (m, 1H), 0.56-0.55 (m, 1H), 0.39-0.35 (m, 2H).

the fourth step.

(1-(1,1,1-trifluoro-2-hydroxypropan-2-yl)cyclopropyl)methyl methanesulfonate

Dissolve 1,1,1-trifluoro-2-(1-(hydroxymethyl)cyclopropyl)propan-2-ol (100 mg, 0.543 mmol) in dichloromethane (5 mL) at 0 ° C Triethylamine (110 mg, 1.08 mmol) and methanesulfonium chloride (62.2 mg, 0.543 mmol) were added. The reaction solution was reacted at 0 ° C for 2 hours. The organic layer was extracted with aq. EtOAc (EtOAc) The filtrate was concentrated under reduced pressure to give (1-(1,1,1-trifluoro-2-hydroxypropan-2-yl)cyclopropyl)methyl methanesulfonate (80.0 mg, yellow oil). : 56%.

the fifth step.

3,7-Dimethyl-1-((1-(1,1,1-trifluoro-2-hydroxypropan-2-yl)cyclopropyl)methyl)-1 H -嘌呤-2,6- (3 H , 7 H )-dione.

(1-(1,1,1-Trifluoro-2-hydroxypropan-2-yl)cyclopropyl)methyl methanesulfonate (80.0 mg, 0.305 mmol), 3,7-dimethyl- 1H -嘌呤-2,6-(3 H ,7 H )-dione (54.9 mg, 0.305 mmol), potassium iodide (5.10 mg, 0.0305 mmol) and potassium carbonate (126 mg, 0.915 mmol) dissolved in anhydrous N , N - In dimethylformamide (5 mL). The reaction solution was heated to 120 ° C and allowed to react for 2 hours. The reaction solution was cooled to 20 ° C, filtered, and purified by preparative HPLC to give 3,7-dimethyl-1-((1-(1,1,1-trifluoro-2-hydroxypropan-2-yl) ) cyclopropyl) methyl) -1 H - purine -2,6- (3 H, 7 H) - dione (40.0 mg, white solid), yield: 38%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.88 (s, 1H), 4.45 (d, J = 6.8 Hz, 1H), 4.24 (d, J = 6.8 Hz, 1H), 3.97 (s, 3H), 3.53 (s, 3H), 1.53 (s, 3H), 0.92-0.88 (m, 1H), 0.64-0.63 (m, 1H), 0.41-0.38 (m, 1H), 0.15-0.12 (m, 1H). MS-ESI calculated [M + H] ⁺ 347 found 347.

Example 5.

1-((3-hydroxy-3-(trifluoromethyl)cyclobutyl)methyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione .

first step.

Methyl 3-oxocyclobutanecarboxylate.

The 3-oxo-cyclobutane carboxylic acid (25.0 g, 0.220 mmol), methanol (14 mL) and N, N - dimethyl-4-aminopyridine (3.00 g, 353 mmol) was dissolved in dichloromethane (500 In mL), stirring at 25 ° C, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (64.0 g, 340 mmol) was slowly added dropwise and stirred overnight. The reaction solution was washed successively with aqueous hydrochloric acid (1.5 N, 72 mL), water (150 mL x 2) and brine (75 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated, evaporated.

The second step.

Methyl 5,8-dioxaspiro[3,4]octane-2-carboxylate.

Methyl-3-oxocyclobutanecarboxylic acid (25.0 g, 195 mmol), ethylene glycol (35.0 g, 564 mmol) and p-toluenesulfonic acid (3.50 g, 20.0 mmol) in toluene (250 mL) In the middle, the water separator was added and heated to reflux overnight. The reaction solution was cooled to 25 ° C and washed successively with water (300 mL×2) and saturated sodium hydrogen carbonate (500 mL×2). The organic phase was dried over anhydrous magnesium sulfate, filtered and evaporated, : 90%.

third step.

5,8-Dioxaspiro[3,4]octane-2-methanol.

Lithium tetrahydrogenate (5.20 g, 136 mmol) was slowly dissolved in tetrahydrofuran (240 mL) under nitrogen at 0 ° C, then 5,8-dioxospirate dissolved in tetrahydrofuran (60 mL) was added dropwise. Methyl [3,4]octane-2-carboxylate (19.5 g, 113 mmol). The reaction was slowly raised to 25 ° C and stirred for 3.5 hours. The reaction solution was cooled to 0 ° C, and water (5.20 g, 289 mmol), 15% sodium hydroxide (5.20 g, 19.5 mmol) and water (15.6 g, 867 mmol) were added slowly. Filtration, the filter cake was washed with tetrahydrofuran (10 mL×3), and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (1:1 petroleum ether/ethyl acetate, Rf = 0.4) to give product 5,8-dioxaspiro[ 3,4]octane-2-methanol (10.0 g, yellow liquid), yield: 62%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 3.90-3.87 (m, 4H), 3.67 (d, J = 6.4 Hz, 2H), 2.45-2.40 (m, 2H), 2.38-2.26 (m, 1H) ), 2.13 - 2.08 (m, 2H).

the fourth step.

5,8-Dioxaspiro[3,4]octane-2-ylmethyl methanesulfonate.

5,8-Dioxaspiro[3,4]octane-2-methanol (500 mg, 53.1 mmol) and triethylamine (896 mg, 6.90 mmol) were dissolved in dichloromethane (23 mL). Methane sulfonium chloride (1.40 g, 12.6 mmol) was slowly added at °C. The reaction solution was raised to 25 ° C and stirred overnight. The reaction was quenched with water (50 mL)EtOAcEtOAc The organic phase was combined, dried over anhydrous sodium sulfate, filtered, and then evaporated. .

MS-ESI calcd for [M ⁺ H] + 223, found 223.

the fifth step.

(1-(5,8-Dioxaspiro[3,4]octane-2-ylmethyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H ) -dione

5,8-Dioxaspiro[3,4]octane-2-ylmethyl methanesulfonate (1.00 g, 4.50 mmol), 3,7-dimethyl-1 H -indole-2,6 (3 H ,7 H )-dione (810 mg, 4.50 mmol), potassium carbonate (1.20 g, 13.5 mmol) and potassium iodide (75.0 mg, 0.45 mmol) dissolved in N , N -dimethylformamide (20 In mL). The reaction was heated to 130 ° C and stirred for 3.5 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give 1-(5,8-dioxaspiro[3,4]octane-2-ylmethyl)-3,7-dimethyl- 1H -indole-2 , 6(3 H , 7 H )-dione (1.50 g, brown liquid), yield: 93%. MS-ESI calcd [M + H] ⁺ 303.

The sixth step.

3,7-Dimethyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione.

1-(5,8-Dioxaspiro[3,4]octane-2-ylmethyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H ) The diketone (1.50 g, 5.00 mmol) was dissolved in acetone (18 mL) and aqueous hydrochloric acid (4 N, 3 mL). The reaction was heated to 30 ° C and stirred overnight. It was diluted with water, and the mixture was taken to EtOAc EtOAc (EtOAc) The organic phase was combined, dried over anhydrous sodium sulfate, filtered, and then evaporated, evaporated, evaporated. -1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (180 mg, white solid), yield: 14%.

¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.49 (s, 1 H), 4.25 (d, J = 7.6 Hz, 2 H), 3.95 (s, 3 H), 3.55 (s, 3 H), 3.13-2.96 (m, 4 H), 2.95-2.84 (m, 1 H). MS-ESI calcd for [M + H] ⁺ 263.

The seventh step.

1-((3-hydroxy-3-(trifluoromethyl)cyclopentyl)methyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione .

3,7-Dimethyl-1-((3-oxocyclopentyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione (100 mg, 0.382 mmol) The ruthenium fluoride (11.5 mg, 0.0763 mmol) was dissolved in anhydrous tetrahydrofuran (3 mL) and trifluoromethyltrimethyl decane (95.0 mg, 0.640 mmol). The reaction solution was slowly heated to 30 ° C and stirred for 12 hours. Aqueous hydrochloric acid (1 N, 5 mL) was then added to the reaction and stirring was continued for 0.5 h. Diluted with water (50 mL), and the mixture was adjusted to pH 7 with saturated aqueous sodium hydrogen carbonate (10 mL), and concentrated under reduced pressure and purified by preparative HPLC to give 1-((3-hydroxy-3) -(Trifluoromethyl)cyclopentyl)methyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione (80.0 mg, white solid) Rate: 64%. 1H NMR: (400 MHz, Mehonal- d ₄ ) δ 8.54 (s, 1H), 4.13-4.07 (m, 5H), 3.56 (s, 3H), 2.58-2.48 (m, 3H), 2.14-2.10 ( m, 2H). MS-ESI calcd [M + H] ⁺ 333.

Example 6.

1-((3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl)-3-hydroxycyclobutyronitrile .

first step.

(((1,3-Dibromopropan-2-yl)oxy)methyl)benzene.

2-(Bromomethyl)oxirane (8.40 g, 61.3 mmol) was added to benzyl bromide (8.74 g, 51.1 mmol) eluting with cuprous chloride (6.87 g, 51.1 mmol). The reaction was stirred at 150 ° C for 11 hours. The reaction solution was cooled to room temperature, water (100 mL) was slowly added, and ethyl acetate (100 mL? The organic phase was combined, dried over anhydrous sodium sulfate, filtered and evaporated, evaporated, evaporated, )oxy)methyl)benzene (8.60 g, yellow oil), yield: 44%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.39 - 7.31 (m, 5H), 4.67 (s, 2H), 3.82-3.78 (m, 1H), 3.58 (d, J = 5.2 Hz, 4H).

The second step.

Ethyl ethyl 3-(benzyloxy)-1-cyanocyclobutanecarboxylate.

Ethyl cyanoacetate (2.76 g, 24.3 mmol) was slowly added to the solution (((1,3-dibromopropyl-2-yl)oxy)methyl)benzene (7.00 g, 18.2 mmol) and Potassium carbonate (10.0 g, 72.7 mmol) in N , N -dimethylformamide (35 mL). The reaction was stirred at 90 ° C for 4 hours. It was cooled to room temperature, filtered, and theEtOAc was evaporated. The organic phase obtained was washed with a saturated aqueous solution of ammonium chloride (20 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and then filtered,jjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj Ethyl-1-cyanocyclobutanecarboxylate (3.80 g, colorless oil), yield: 81%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.40-7.28 (m, 5H), 4.48-4.44 (m, 2H), 4.37-4.31 (m, 1H), 4.30-4.24 (m, 2H) , 2.97-2.80 (m, 2H), 2.73 - 2.65 (m, 2H), 1.37-1.30 (m, 3H).

third step.

3-(Benzyloxy)-1-(hydroxymethyl)cyclobutanenitrile.

Sodium borohydride (1.39 g, 36.6 mmol) was dissolved in tetrahydrofuran and water (20 mL: 2 mL), and ethyl 3-(benzyloxy)-1-cyanide was slowly added dropwise at 0 ° C over 20 min. A solution of ethylcyclobutanecarboxylate (3.80 g, 14.6 mmol) in tetrahydrofuran (22 mL). The reaction was stirred at room temperature for 2 hours. The organic layer was washed with water (30 mL) and brine (30 mL). -(Hydroxymethyl)cyclobutyronitrile (3.70 g, colorless oil). ¹ H NMR: (400 MHz, DMSO- d ₆ ) δ 7.38-7.25 (m, 5H), 5.57-5.52 (m, 1H), 4.39-4.36 (m, 2H), 4.13-4.04 (m, 1H) , 3.57-3.51 (m, 2H), 2.58-2.51 (m, 1H), 2.49-2.45 (m, 1H), 2.31-2.09 (m, 2H).

the fourth step.

(3-(Benzyloxy)-1-cyanocyclobutyl)methyl methanesulfonate.

3-(Benzyloxy)-1-(hydroxymethyl)cyclobutanenitrile (3.70 g, 15.3 mmol), triethylamine (3.10 g, 30.6 mmol) dissolved in dichloromethane (35 mL) Methanesulfonyl chloride (3.29 g, 28.7 mmol) was slowly added at °C. The reaction mixture was stirred at room temperature for 4 hr. EtOAc (EtOAc m. (Benzyloxy)-1-cyanocyclobutyl)methyl methanesulfonate (4.56 g, dark brown oil). ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.36-7.26 (m, 5H), 4.47-4.45 (m, 2H), 4.44-4.38 (m, 2H), 3.21-3.18 (m, 1H) , 3.17-3.14 (m, 3H), 2.81-2.60 (m, 2H), 2.53-2.26 (m, 2H).

the fifth step.

3-(Benzyloxy)-1-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl ) Cyclobutyronitrile.

(3-(Benzyloxy)-1-cyanocyclobutyl)methyl methanesulfonate (4.50 g, 15.2 mmol), 3,7-dimethyl-1 H -indole-2,6-( 3 H ,7 H )-dione (2.75 g, 15.2 mmol) and potassium iodide (1.26 g, 7.62 mmol) were dissolved in N , N -dimethylformamide (100 mL), and potassium carbonate (6.32 g, 45.7 mmol), the reaction was heated to reflux at 120 ° C for 4 hours. The reaction solution was cooled to room temperature, filtered and evaporated, evaporated, evaporated, The organic phase was combined, dried over anhydrous sodium 3,6,7-Tetrahydro-1 H -indol-1-yl)methyl)cyclobutyronitrile (4.60 g, yellow solid). MS-ESI calcd [M + H] ⁺ 380.

The sixth step.

1-((3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl)-3-hydroxycyclobutyronitrile .

3-(Benzyloxy)-1-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)- The cyclobutyronitrile (100 mg, 0.263 mmol) was dissolved in dichloromethane (10 mL) and ferric chloride (128 mg, 0.790 mmol). The reaction was stirred at room temperature for 12 hours. Water (10 mL) was added and extracted with dichloromethane (40 mL x 3). The organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the obtained product was purified by preparative high performance liquid chromatography to give the product 1-((3,7-dimethyl-2,6-dioxo-2) , 3,6,7-tetrahydro-1 H -indol-1-yl)methyl)-3-hydroxycyclobutyronitrile (12.0 mg, yellow solid), yield: 16%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.56 (s, 1H), 4.66-4.49 (m, 1H), 4.45-4.37 (m, 2H), 4.01 (s, 3H), 3.62 (s, 3H) ), 2.96-2.85 (m, 2H), 2.60-2.49 (m, 2H). MS-ESI calcd for [M + H] ⁺ 290.

Example 7.

first step.

Methyl 3-(hydroxymethyl)bicyclo[1.1.1]pentane-1-carboxylate.

3-(Methoxycarbonyl)bicyclo[1.1.1]pentan-1-carboxylic acid (100 mg, 0.587 mmol) and triethylamine (71.0 mg, 0.705 mmol) in tetrahydrofuran (20 mL), -10 Methyl chloroformate (56.0 mg, 0.587 mmol) was slowly added dropwise at °C. The reaction solution was stirred at 0 ° C for half an hour, then sodium borohydride (33.0 mg, 0.881 mmol) was added and the reaction was continued for 2 hr. Water (10 mL), and ethyl acetate (10 mL×3) was evaporated, evaporated, evaporated, evaporated, evaporated. Methyl 3-(hydroxymethyl)bicyclo[1.1.1]pentane-1-carboxylate (80.0 mg, colorless oil), yield: 87%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 3.65 (s, 3H), 3.60 (s, 2H), 2.00 (s, 6H).

The second step.

Methyl 3-(((methylsulfonyl)oxy)methyl)bicyclo[1.1.1]pentan-1-carboxylate.

Methyl 3-(hydroxymethyl)bicyclo[1.1.1]pentan-1-carboxylate (40.0 mg, 0.256 mmol) and triethylamine (39.0 mg, 0.384 mmol) were dissolved in dichloromethane (15) In mL), methanesulfonium chloride (35.0 mg, 0.307 mmol) was slowly added dropwise at 0 °C. The reaction mixture was stirred at 0 ° C for 2 hours. The mixture was diluted with methylene chloride (10 mL). Methyl (-((methylsulfonyl)oxy)methyl)bicyclo[1.1.1]pentan-1-carboxylate (50.0 mg, yellow oil).

third step.

Methyl 3-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl)bicyclo[1.1.1] Methyl pentane-1-carboxylate.

Methyl 3-(((methylsulfonyl)oxy)methyl)bicyclo[1.1.1]pentan-1-carboxylate (100 mg, 0.426 mmol) and 3,7-dimethyl -1 H -嘌呤-2,6-(3 H ,7 H )-dione (77.0 mg, 0.427 mmol) was dissolved in N , N -dimethylformamide (20 mL). Potassium carbonate (88.0 mg, 0.640 mmol) and potassium iodide (8.00 mg, 0.0430 mmol). The reaction mixture was stirred at 100 ° C for 2 hours. The reaction mixture was evaporated to dryness EtOAc. Concentration, separation and purification by gel column chromatography (1:1 petroleum ether / ethyl acetate, Rf = 0.2) to give methyl 3-((3,7-dimethyl-2,6-dioxo-2,3) , 6,7-Tetrahydro-1 H -indol-1-yl)methyl)bicyclo[1.1.1]pentan-1-carboxylic acid methyl ester (100 mg, yellow solid), yield: 73%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.50 (s, 1H), 4.12 (s, 2H), 3.95 (s, 3H), 3.60 (s, 3H), 3.53 (s, 3H), 1.95 ( s, 6H). MS-ESI calcd for [M ⁺ H] + 319, found 319.

the fourth step.

3-((3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl)bicyclo[1.1.1]pentane 1-carboxylic acid.

Methyl 3-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl)bicyclo[1.1.1 Methyl pentane-1-carboxylate (100 mg, 0.314 mmol) was dissolved in tetrahydrofuran (15 mL) and water (5 mL). After stirring at room temperature for 2 hours, the reaction mixture was added with 2N diluted hydrochloric acid (10 mL) to adjust pH to 4, ethyl acetate (15 mL x 3), and the organic phase was washed with saturated sodium chloride solution (20 mL x 2) Dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to 3-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl) Methyl)bicyclo[1.1.1]pentane-1-carboxylic acid (90.0 mg, white solid), yield: 94%.

MS-ESI calcd [M + H] ⁺ 303.

the fifth step.

3-((3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl) -N -methoxy- N -Methylbicyclo[1.1.1]pentan-1-indoleamine.

3-((3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl)bicyclo[1.1.1]penta Alkyl-1-carboxylic acid (30.0 mg, 0.0986 mmol) and N , O -dimethylhydroxylamine (10.0 mg, 0.0986 mmol) were dissolved in dichloromethane (20 mL) Nitrobenzotriazole) -N , N , N ', N' -tetramethylurea hexafluorophosphate (75.0 mg, 0.197 mmol) and diisopropylethylamine (19.0 mg, 0.148 mmol). After stirring at room temperature for 12 hours, the reaction mixture was evaporated w~~~~~~~~~~~~~~~~~~~~~~~ Drying over anhydrous sodium sulfate, filtration, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (1:2 petroleum ether / ethyl acetate, Rf = 0.2) to give 3-((3,7-dimethyl-2, 6-Dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl) -N -methoxy- N -methylbicyclo[1.1.1]pentan-1- Guanidine (30.0 mg, white solid), yield: 88%.

MS-ESI calcd for [M ⁺ H] + 348, found 348.

The sixth step.

1-((3-Ethylbicyclo[1.1.1]pentan-1-yl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H ) - Diketone.

3-((3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl) -N -methoxy- N -Methylbicyclo[1.1.1]pentan-1-indoleamine (20.0 mg, 0.0575 mmol) was dissolved in tetrahydrofuran (20 mL), and the reaction mixture was added methyl magnesium bromide (3 M) at -78 °C. Ether solution, 0.040 mL, 0.120 mmol), stirring was continued for 30 min and then warmed to room temperature for 4 hours. The reaction mixture was extracted with saturated aqueous ammonium chloride (10 mL), ethyl acetate (15 mL×3), and the organic phase was washed with saturated sodium chloride solution (20 mL×2) and dried over anhydrous sodium sulfate Filtered and the filtrate was concentrated under reduced pressure. Separation and purification by gel column chromatography (1:1 petroleum ether / ethyl acetate, Rf = 0.5) affords 1-((3-ethylhydrazinobicyclo[1.1.1]pentan-1-yl)methyl)- 3,7-Dimethyl-1 H -indole-2,6-( 3H , 7H )-dione (15.0 mg, mp. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.55 (s, 1H), 4.17 (s, 2H), 3.99 (s, 3H), 3.59 (s, 3H), 2.07 (s, 3H), 1.97 ( s, 6H). MS-ESI calcd [M + H] ⁺ 303, Found 303.

The seventh step.

3,7-Dimethyl-1-((3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)bicyclo[1.1.1]pentan-1-yl)methyl)- 1 H -嘌呤-2,6-(3 H ,7 H )-dione.

1-((3-Ethylbicyclo[1.1.1]pentan-1-yl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )-Dione (20.0 mg, 0.0660 mmol) and trifluoromethyltrimethylchloromethane (19.0 mg, 0.132 mmol) were dissolved in tetrahydrofuran (15 mL). Mg, 00660 mmol), and the reaction was continued for 12 hours at room temperature. 2N dilute hydrochloric acid (10 mL) was added to the reaction mixture, and the mixture was stirred for 30 minutes, ethyl acetate (20 mL×2) was evaporated. The filtrate was concentrated under reduced pressure and purified by high-performance liquid chromatography to give 3,7-dimethyl-1-((3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)bicyclo[1.1. 1]pentan-1-yl)methyl)-1 H -indole-2,6-( 3H , 7H )-dione (5.00 mg, colorless oil), yield: 20%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.59 (s, 1H), 4.20 (s, 2H), 4.02 (s, 3H), 3.59 (s, 3H), 1.97 (s, 6H), 1.79 ( s, 3H). MS-ESI calcd for [M ⁺ H] + 373, found 373.

Example 8. first step

3,7-Dimethyl-1-[[3-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]cyclobutyl]methyl]indole-2, 6-diketone.

3,7-Dimethyl-1-[[3-(2,2,2-trifluoro-1,1-dihydroxy-ethyl)cyclobutyl]methyl]indole-2,6-dione (60.0 mg, 0.165 mmol), cesium fluoride (25.2 mg, 0.165 mmol) dissolved in tetrahydrofuran (10 mL), trifluoromethyltrimethylchloromethane (70.6 mg, 0.496 mmol) hour. The reaction was quenched by the addition of water (20 mL). Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Purification by preparative high performance liquid chromatography gave product 1 (8.00 mg, yellow solid) (isomer, first peak), yield: 12%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.01 (s, 1H), 4.22-4.17 (m, 2H), 4.01 (s, 3H), 3.54 (s, 3H), 3.55-3.19 (m , 1H), 2.63-2.56 (m, 1H), 2.50-2.42 (m, 2H), 1.82-1.78 (m, 2H). MS-ESI calcd for [M + H] ⁺ 415.

And product 2 (isomer 2, second peak), yield: 6%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.20 (s, 1H), 4.04-4.00 (m, 5H), 3.55 (s, 3H) 2.70-2.65 (m, 1H), 2.55-2.53 ( m, 1H), 2.17-2.12 (m, 2H), 2.02-1.98 (m, 2H). MS-ESI calcd for [M + H] ⁺ 415.

Example 9.

first step.

3-methylenecyclobutanecarboxylic acid.

3-Methylenecyclobutyronitrile (10.0 g, 107 mmol) and potassium hydroxide (18.1 g, 322 mmol) were dissolved in ethanol (100 mL) and water (50 mL) and reacted at 100 ° C for 2 hours. 1 N hydrochloric acid (120 mL) was added. Dichloromethane (30 mL x 3), dried over anhydrous sodium sulfate, filtered and evaporated. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 4.83-4.76 (m, 2H), 3.15-2.96 (m, 1H), 2.95 - 2.92 (m, 4H).

The second step.

Methyl-3-methylenecyclobutanecarboxylic acid.

3-Methylenecyclobutanecarboxylic acid (11.0 g, 98.1 mmol) and potassium carbonate (27.1 g, 196 mmol) were dissolved in acetone (100 mL) and dimethyl sulfate (14.8 g, 117 was added at 25 ° C) Methyl), after reacting at 70 ° C for 12 hours. The reaction was quenched with EtOAc (EtOAc) (EtOAc (EtOAc) Yellow oil), Yield: 97%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 4.83-4.79 (m, 2H), 3.96 (s, 2H), 3.68 (s, 3H), 3.17-3.15 (m, 1H), 2.95-2.92 (m, 2H).

third step.

Ethyl methyl 3-(hydroxymethyl)cyclobutanecarboxylate.

Methyl-3-methylenecyclobutanecarboxylic acid (2.00 g, 15.8 mmol) was dissolved in tetrahydrofuran (30 mL), and borane dimethyl sulfide (3.61 g, 47.5 mmol) was added dropwise at -10 °C. Then react at -10 ° C for 3 hours, add 3 N aqueous sodium hydroxide solution (10 mL) and hydrogen peroxide (5 mL), continue the reaction for 1 hour, and then add the saturated sodium thiosulfate aqueous solution (30 mL) to quench the reaction, dichloro Methane extraction (10 mL x 3), dried over anhydrous sodium sulfate, filtered, evaporated, evaporated, evaporated,jjjjjjjjjjj %. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 3.70 (s, 3H), 3.58 (d, J = 6.8 Hz, 1H), 3.49 (d, J = 6.8 Hz, 1H), 3.10-3.05 ( m, 1H), 2.32-2.26 (m, 3H), 2.03-1.98 (m, 2H).

the fourth step.

Ethyl 3-(methylsulfonyloxymethyl)cyclobutanecarboxylate.

Ethyl 3-(hydroxymethyl)cyclobutanecarboxylate (1.00 g, 6.94 mmol) and triethylamine (2.11 g, 20.8 mmol) were dissolved in dichloromethane (20 mL). Chlorofluorene (1.59 g, 13.9 mmol). The reaction solution was slowly warmed to room temperature and stirred for 2 hours. The reaction was quenched by the addition of aqueous sodium bicarbonate (50 mL). Extract with dichloromethane (10 mL x 3). The organic phase was combined, washed with brine, dried over anhydrous sodium sulfatessssssssssssssssssssssssssss , Yield: 91%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 4.28 (d, J = 6.8 Hz, 1H), 4.19 (d, J = 6.8 Hz, 1H), 3.70 (s, 3H), 3.20-3.08 ( m, 4H), 2.40-2.34 (m, 3H), 2.13 - 2.09 (m, 2H). MS-ESI calcd for [M ⁺ H] + 223, found 223.

the fifth step.

Ethyl 3-[(3,7-dimethyl-2,6-dioxo-indol-1-yl)methyl]cyclobutanecarboxylate

Ethyl 3-(methylsulfonyloxymethyl)cyclobutanecarboxylate (1.40 g, 6.30 mmol), 3,7-dimethyl- 1H -indole-2,6-( 3H ,7 H )-Dione (1.13 g, 6.30 mmol), potassium iodide (209 mg, 1.26 mmol) and potassium carbonate (2.61 g, 18.90 mmol) were dissolved in N , N -dimethylformamide (100 mL). The reaction solution was warmed to 120 ° C and stirred for 3 hours. It was cooled to room temperature, filtered, and water (100 mL) was evaporated. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Ethyl cyclobutanecarboxylate (1.50 g, yellow solid), yield: 78%.

¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.51 (s, 1H), 4.18-4.10 (m, 2H), 3.99 (s, 3H), 3.67 (s, 3H), 3.55 (s, 3H) ), 3.26-2.65 (m, 2H), 2.29-2.13 (m, 4H). MS-ESI calcd [M + H] ⁺ 303.

The sixth step.

3-[(3,7-Dimethyl-2,6-dioxo-indol-1-yl)methyl]cyclobutanecarboxylic acid

Ethyl 3-[(3,7-dimethyl-2,6-dioxo-indol-1-yl)methyl]cyclobutanecarboxylate (1.00 g, 3.26 mmol), potassium hydroxide (548) Mg, 9.78 mmol) was dissolved in methanol (10 mL) and water (5 mL). The reaction solution was warmed to 90 ° C and stirred for 3 hours. After cooling to room temperature, it was neutralized by adding 1 N hydrochloric acid (20 mL), and extracted with dichloromethane (10 mL x 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Cyclobutanecarboxylic acid (800.00 mg, yellow solid), yield: 84%. MS-ESI calcd for [M ⁺ H] + 293, found 293.

The seventh step.

3-[(3,7-Dimethyl-2,6-dioxo-indol-1-yl)methyl] -N -methoxy- N -methylcyclobutanecarbamidine.

3-[(3,7-Dimethyl-2,6-dioxo-indol-1-yl)methyl]cyclobutanecarboxylic acid (300 mg, 1.03 mmol), N , O -dimethyl Hydroxylamine hydrochloride (200 mg, 2.05 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (394 mg, 2.06 mmol), 1-hydroxybenzotriazole ( 27.8 mg, 0.206 mmol) and triethylamine (312 mg, 3.09 mmol) were dissolved in dichloromethane (10 mL). Stir at 25 ° C for 12 hours. The reaction mixture was concentrated under reduced pressure and purified (yield ethyl acetate, Rf value = 0.3) to afford 3-[(3,7-dimethyl-2,6-dioxo-indol-1-yl). Base] -N -methoxy- N -methylcyclobutanecarbamide (200 mg, yellow solid), yield: 58%. MS-ESI calculated [M + H] ⁺ 356 found 336.

The eighth step.

1-[(3-Ethylcyclobutyl)methyl]-3,7-dimethylindole-2,6-dione

3-[(3,7-Dimethyl-2,6-dioxo-indol-1-yl)methyl] -N -methoxy- N -methylcyclobutanecarbamamine (300 mg , 0.894 mmol) was dissolved in tetrahydrofuran (10 mL). Methylmagnesium bromide (3 M in diethyl ether, 1.49 mL, 4.47 mmol) was added dropwise at 0 ° C for 3 hours. The reaction mixture was quenched with EtOAc EtOAc (EtOAc) The organic phase was combined, washed with brine, dried over anhydrous sodium sulfate, filtered, evaporated, evaporated, evaporated, evaporated Methyl]-3,7-dimethylindole-2,6-dione (200 mg, yellow solid), yield: 77%.

MS-ESI calcd for [M + H] ⁺ 291.

The ninth step.

1-[(3-Ethylcyclobutyl)methyl]-3,7-dimethylindole-2,6-dione (250 mg, 0.861 mmol), cesium fluoride (130 mg, 0.861 mmol) Dissolved in tetrahydrofuran (10 mL), trimethyl-trifluoromethyl-decane (244 mg, 1.72 mmol) was added at room temperature and stirred for 12 hours. The reaction was quenched by the addition of water (20 mL). Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Purification by preparative high performance liquid chromatography gave product 1 (65.0 mg, yellow solid) (isomer 1, first peak), yield: 20%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.21 (s, 1H), 4.23 (d, J = 7.6 Hz, 2H), 4.03 (s, 3H), 3.55 (s, 3H), 3.26- 3.19 (m, 2H), 2.63-2.56 (m, 2H), 2.55-2.42 (m, 2H), 1.82-1.78 (m, 3H). MS-ESI calcd for [M ⁺ H] + 361, found 361.

Product 2 (isomer 2, second peak), yield: 22%. 1H NMR: (400 MHz, Methonal-d4) δ 8.05 (s, 1H), 4.01-3.99 (m, 5H), 4.03 (s, 3H), 3.54 (s, 3H), 2.71-2.66 (m, 1H) ), 2.55-2.54 (m, 1H), 2.17-2.12 (m, 2H), 2.02-1.98 (m, 2H). MS-ESI calcd for [M ⁺ H] + 361, found 361.

Example 10.

first step.

Methyl 1,4-dioxaspiro[4.4]decane-7-carboxylate.

Methyl 3-oxo-cyclopentanoate (16.0 g, 110 mmol), p-toluenesulfonic acid (14.0 g, 220 mmol) and ethylene glycol (969 mg, 5.60 mmol) dissolved in anhydrous toluene (160 mL) In the middle, the water separator was heated and refluxed for 4 hours. The reaction was quenched with EtOAc (EtOAc)EtOAc. The combined organic phases were washed with water (200 mL EtOAc) EtOAc. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (5:1 petroleum ether/ethyl acetate, Rf = 0.3) 1,4-dioxaspiro[4.4]decane-7-carboxylic acid methyl ester (6.20) g, yellow oil), yield: 29%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 3.93 - 3.89 (m, 4H), 3.69 (s, 3H), 2.91-2.89 (m, 1H), 2.11-1.82 (m, 6H). MS-ESI calcd for [M + H] ⁺ 187.

The second step.

(1,4-Dioxaspiro[4.4]decane-7-yl)-methanol.

Methyl 1,4-dioxaspiro[4.4]decane-7-carboxylate (1.00 g, 10.7 mmol) was dissolved in anhydrous tetrahydrofuran (30 mL), nitrogen was added, and tetrahydro aluminum hydride was slowly added at -10 °C. (531 mg, 13.9 mmol). The reaction solution was slowly warmed to 25 ° C and stirred for 3 hours. Water (0.5 mL), 15% sodium hydroxide solution (0.5 mL), and water (1.5 mL) were sequentially added to the mixture. The insoluble material was removed by filtration, and the filtrate was concentrated under reduced pressure to give (4-dioxaspiro[4.4]decane-7-yl)-methanol (m. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 3.94 - 3.89 (m, 4H), 3.58 - 3.57 (m, 2H), 2.31-1.48 (m, 7H). MS-ESI calcd [M + H] ⁺ 159, Found 159.

third step.

1,4-Dioxaspiro[4.4]decane-7-ylmethyl methanesulfonate.

(1,4-Dioxaspiro[4.4]decane-7-yl)-methanol (500 mg, 53.1 mmol) and triethylamine (800 mg, 7.92 mmol) were dissolved in anhydrous dichloromethane (5 mL) In a nitrogen atmosphere, methanesulfonyl chloride (433 mg, 3.80 mmol) was slowly added at 0 °C. The reaction solution was raised to 25 ° C and stirred for 2 hours. The reaction was quenched with water (40 mL)EtOAcEtOAc. The combined organic phases were washed with water (20 mL×2), EtOAc (EtOAc) [4.4] decane-7-ylmethyl ester (800 mg, yellow oil). MS-ESI calcd for [M + H] ^{+ s} .

the fourth step.

(1,4-Dioxaspiro[4.4]decane-7-ylmethyl)-3,7-dimethyl 1 H -indole-2,6(3 H ,7 H )-dione.

Dissolving 1,4-dioxaspiro[4.4]decane-7-ylmethyl methanesulfonate (300 mg, 1.27 mmol) in anhydrous N , N -dimethylformamide (10 mL), nitrogen Protection, potassium carbonate (350 mg, 2.54 mmol), potassium iodide (21.0 mg, 0.130 mmol), 2,6-hydroxy-3,7-dimethylindole (275 mg, 1.52 mmol) was added at 25 °C. The reaction solution was heated to 130 ° C and stirred for 3 hours. The reaction mixture was cooled to 25 ° C, then quenched with water (40 mL). The organic phase was combined, washed with a saturated sodium chloride solution (100 mL×2), dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to give (1,4-dioxaspiro[4.4]decane-7-yl. yl) -3,7-dimethyl-1 H - purine -2,6 (3 H, 7 H) - dione (200 mg, white solid), yield: 45%. MS-ESI calculated [M + H] ⁺ 353 found 321.

the fifth step.

3,7-Dimethyl-1-(3-oxo-cyclopentylmethyl)-1 H -indole-2,6(3 H ,7 H )-dione.

(1,4-Dioxaspiro[4.4]decane-7-ylmethyl)-3,7-dimethyl 1 H -indole-2,6(3 H ,7 H )-dione (200 The mg, 0.620 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL). The reaction solution was stirred at 25 ° C for 1 hour. It was diluted with water (60 mL), and the mixture was extracted with ethyl acetate (20 mL). The organic phase was combined, washed with aq. EtOAc EtOAc (EtOAc (EtOAc) 0.3), 3,7-dimethyl-1-(3-oxo-cyclopentylmethyl)-1 H -indole-2,6(3 H ,7 H )-dione (100 mg, yellow) Oily), yield: 57%. MS-ESI calcd for [M + H] ⁺ 277.

The sixth step.

1,3 trans-1-((3-hydroxy-3-(trifluoromethyl)cyclopentyl)methyl)-3,7-dimethyl-1 H -indole-2,6( 3H , 7 H )-dione.

1,3 cis-1-((3-hydroxy-3-(trifluoromethyl)cyclopentyl)methyl)-3,7-dimethyl-1 H -indole-2,6(3 H , 7 H )-dione.

3,7-Dimethyl-1-((3-oxocyclopentyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione (100 mg, 0.362 mmol) The ruthenium fluoride (11.0 mg, 0.0725 mmol) was dissolved in anhydrous tetrahydrofuran (3 mL) and trifluoromethyltrimethyldecane (95.0 mg, 0.640 mmol). The reaction solution was slowly heated to 30 ° C and stirred for 12 hours. Aqueous hydrochloric acid (1 N, 5 mL) was added to the mixture and the mixture was stirred at 30 ° C for 0.5 hour. Water (50 mL) was added to the mixture and the mixture was evaporated. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and evaporated.

Product 1 (isomer 1, first peak) (40.0 mg, white solid), yield: 32%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.68 (s, 1H), 4.13-4.08 (m, 2H), 4.05 (s, 3H), 3.61 (s, 3H), 2.80-2.78 (m) , 1H), 2.40-2.24 (m, 1H), 2.04-2.03 (m, 1H), 2.01-1.87 (m, 2H), 1.84-1.76 (m, 1H), 1.62-1.60 (m, 1H). MS-ESI calculated [M + H] ⁺ 347 found 347.

Product 2 (isomer 2, second peak) (20.0 mg, white solid), yield: 16%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.62 (s, 1H), 4.22-4.18 (m, 1H), 4.05-4.04 (m, 1H), 4.00 (s, 3H), 3.63 (s) , 3H), 2.65-2.63 (m, 1H), 2.09-2.01 (m, 4H), 1.70-1.68 (m, 1H), 1.67-1.65 (m, 1H). MS-ESI calculated [M + H] ⁺ 347 found 347.

Example 11.

L - ((4-hydroxy-4- (trifluoromethyl) cyclohexyl) methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

first step.

Ethyl ethyl 1,4-dioxaspiro[4,5]decane-8-carboxylate.

Ethyl 4-oxocyclohexanecarboxylic acid (30.0 g, 176 mmol), ethylene glycol (22.0 g, 353 mmol) and p-toluenesulfonic acid (304 mg, 1.70 mmol) were dissolved in toluene (315 mL) After adding a water separator, the mixture was heated to reflux overnight. The reaction solution was cooled to 25 ° C, and washed with water (300 mL×2) and saturated sodium hydrogen carbonate (500 mL×2). The organic phase was dried over anhydrous magnesium sulfate and filtered. (1:1 petroleum ether / ethyl acetate, Rf = 0.3) gave the product ethyl 1,4-dioxaspiro[4,5]nonane-8-carboxylic acid ethyl ester (37.2 g, yellow liquid). Rate: 99%. MS-ESI calcd for [M + H] ⁺ 215.

The second step.

1,4-Dioxaspiro[4,5]decane-8-ylmethanol.

Lithium tetrahydroaluminum (2.30 g, 61.0 mmol) was slowly added to tetrahydrofuran (60 mL) under nitrogen at 0 ° C, and ethyl 1,4-dioxaspiro[4,5]decane-8 was added dropwise. A solution of ethyl carboxylate (10.0 g, 42.0 mmol) in tetrahydrofuran (40 mL). The reaction was slowly raised to 25 ° C and stirred for 3.5 hours. The reaction was cooled to 0 ° C, then water (2.3 g, 127 mmol), 15% sodium hydroxide (2.3 g, 8.60 mmol) and water (6.9 g, 383 mmol). Filtration, the filter cake was washed with tetrahydrofuran (50 mL×3), and the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 1,4-dioxaspiro[4,5]nonane-8- Methanol (6.22 g, yellow liquid), yield: 89%. MS-ESI calcd for [M + H] ⁺ 173.

third step.

1,4-Dioxaspiro[4,5]decane-8-ylmethyl methanesulfonate.

1,4-Dioxaspiro[4,5]decane-8-ylmethanol (2.00 g, 12.0 mmol) and diisopropylethylamine (3.10 g, 24.0 mmol) were dissolved in dichloromethane (40) In mL), methanesulfonium chloride (3.90 g, 30.0 mmol) was slowly added at 0 °C. The reaction solution was raised to 25 ° C and stirred overnight. The reaction was quenched with EtOAc (EtOAc)EtOAcEtOAc The organic phase was combined, dried over anhydrous magnesium sulfate, filtered, and then evaporated, evaporated, evaporated, evaporated. 4,5]decane-8-ylmethyl methanesulfonate (1.80 g, yellow liquid), yield: 60%. Calcd MS-ESI [M ⁺ H] + 251, found 251.

the fourth step.

1- (1,4-dioxaspiro [4,5] decan-8-yl-methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - Dione.

1,4-Dioxaspiro[4,5]nonane-8-ylmethyl methanesulfonate (1.50 g, 6.00 mmol), 3,7-dimethyl-1 H -indole-2,6 (3 H ,7 H )-dione (1.00 g, 6.00 mmol) and potassium carbonate (2.50 g, 18.0 mmol), potassium iodide (100 mg, 0.600 mmol) dissolved in N , N -dimethylformamide (20 In mL), the reaction solution was heated to 130 ° C and stirred for 3 hours. The reaction mixture was cooled to 25 ° C, then EtOAc (EtOAc) The organic phase was combined, dried over anhydrous magnesium sulfate, filtered, evaporated, evaporated, evaporated, evaporated, hetero-spiro [4,5] decan-8-yl-methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (1.75 g, white solid) , Yield: 63%. MS-ESI calcd for [M + H] ⁺ 335.

the fifth step.

3,7-Dimethyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione.

1-(1,4-Dioxaspiro[4,5]decane-8-ylmethyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H ) The diketone (1.50 g, 4.50 mmol) was dissolved in acetone (15 mL) and aqueous hydrochloric acid (2N, 2.5 mL). The reaction was stirred at rt EtOAc (EtOAc)EtOAc. The organic phase was combined, dried over anhydrous sodium sulfate, filtered and evaporated, evaporated, evaporated. (4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (1.02 g, white solid), yield: 78%. MS-ESI calcd for [M + H] ⁺ 291.

The sixth step.

L - ((4-hydroxy-4- (trifluoromethyl) cyclohexyl) methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

3,7-Dimethyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione (100 mg, 0.330 mmol) and fluorine Plutonium (60.0 mg, 0.350 mmol) was dissolved in tetrahydrofuran (5 mL) and trifluoromethyltrimethyldecane (75.0 mg, 0.500 mmol) was slowly added under nitrogen. The reaction solution was stirred at 30 ° C for 3 hours. After cooling to 25 ° C, aqueous hydrochloric acid (4 N, 3 mL) was added and stirred at 25 ° C for half an hour, pH was adjusted to 7, diluted with water and extracted with ethyl acetate (20 mL x 3). The organic phase was combined, dried over anhydrous sodium sulfate, filtered, evaporated, evaporated. 7-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (24.0 mg, white solid), yield: 39%.

¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.86 (s, 1H), 4.04 (d, J = 7.2 Hz, 1H), 3.97 (s, 3H), 3.89 (d, J = 7.6 Hz, 1H), 3.53 (s, 3H), 2.06-1.97 (m, 2H), 1.88-1.77 (m, 3H), 1.62-1.43 (m, 4H). MS-ESI calcd for [M ⁺ H] + 361, found 361.

Example 12.

first step.

Ethyl 4-hydroxy-4-(trifluoromethyl)cyclohexanecarboxylate.

Ethyl 4-oxocyclohexanecarboxylate (10.0 g, 58.7 mmol) was dissolved in tetrahydrofuran (100 mL), trifluoromethyltrimethyldecane (12.5 g, 88.1 mmol) and fluorinated at room temperature铯 (8.92 g, 58.7 mmol). The reaction mixture was stirred at room temperature for 12 hrs, then EtOAc (EtOAc (EtOAc,EtOAc) x 2) Washing, drying over anhydrous sodium sulfate, concentrating by filtration and purification by EtOAc (EtOAc:EtOAc:EtOAc: Ethyl hexanecarboxylate (12.0 g, colorless oil), yield: 85%. ¹ H NMR: (400 MHz, Methanol- d ₄ ) δ 4.20 - 4.12 (m, 2H), 2.03-1.86 (m, 9H), 1.29 - 1.25 (m, 3H).

MS-ESI calcd [M + H] ⁺ 241, Found 241.

The second step.

4-(Hydroxymethyl)-1-(trifluoromethyl)cyclohexanol.

Ethyl 4-hydroxy-4-(trifluoromethyl)cyclohexanecarboxylate (12.00 g, 49.9 mmol) was dissolved in tetrahydrofuran (20 mL). EtOAc (3. ), reacted for 2 hours. The reaction was quenched by the addition of water (30 mL). It is extracted with ethyl acetate (50 mL×3), dried over anhydrous sodium sulfate, filtered, filtered, evaporated, evaporated, evaporated. -(Hydroxymethyl)-1-(trifluoromethyl)cyclohexanol (9.00 g, colorless oil), yield: 91%. ¹ H NMR: (400 MHz, Methanol- d ₄ ) δ 3.58 -3.40 (m, 2H), 1.90-1.40 (m, 9H).

third step.

(4-Hydroxy-4-(trifluoromethyl)cyclohexyl)methyl methanesulfonate.

(4-(Hydroxymethyl)-1-(trifluoromethyl)cyclohexanol (11.0 g, 55.5 mmol) and triethylamine (1.18 g, 11.6 mmol) were dissolved in dichloromethane (80 mL) Methanesulfonium chloride (14.4 g, 125 mmol) was added at 0 ° C. The reaction mixture was stirred at room temperature for 2 hr then diluted with methylene chloride (60 mL). Dry over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure and purified by silica gel column chromatography (4:1 petroleum ether / ethyl acetate, Rf = 0.5) to give (4-hydroxy-4-(trifluoromethyl) ring Hexyl)methanesulfonate (13.00 g, colorless oil), yield: 85%. ¹ H NMR: (400 MHz, Methanol- d ₄ ) δ 4.25-4.01 (m, 2H), 3.10- 3.07 (m, 3H), 2.03-1.24 (m, 9H).

the fourth step.

1-((( S ,4 S )-4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1 H -indole-2,6(3 H , 7 H )-dione.

(4-Hydroxy-4-(trifluoromethyl)cyclohexyl)methyl methanesulfonate (10.0 g, 36.2 mmol) was dissolved in N , N -dimethylformamide (100 mL). 3,7-Dimethyl-1 H -indole-2,6(3 H ,7 H )-dione (6.52 g, 36.2 mmol), potassium carbonate (7.50 g, 54.3 mmol) and Potassium iodide (184 mg, 1.11 mmol). The reaction mixture was heated to 100 ° C, and the reaction was concentrated for 5 hours. The reaction mixture was concentrated, evaporated with ethyl acetate (100 mL). Concentration, separation by preparative SFC, separation conditions: column: Chiralpak AD-3 150 x 4.6 mm ID, 3um mobile phase: ethanol (0.05% diethylamine) in CO ₂ from 5% to 40% at 2.5mL/ Min Wavelength: 220 nm gave product 1 (2.5 g, white solid) (isomer 1, first peak), yield: 19%. ¹ H NMR: (400 MHz, Methanol- d ₄ ) δ 7.88 (s, 1H), 4.02 (d, J = 7.6 Hz, 2H), 3.98 (s, 3H), 3.53 (s, 3H), 2.16- 2.02 (m, 1H), 1.99-1.98 (m, 2H), 1.87-1.80 (m, 2H), 1.60-1.49 (m, 2H), 1.48-1.46 (m, 2H). MS-ESI calcd for [M ⁺ H] + 361, found 361.

Product 2 (2.40 g, white solid) (isomer 2, second peak), yield: 19%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.88 (s, 1H), 3.99 (s, 3H), 3.90 (d, J = 7.6 Hz, 2H), 3.54 (s, 3H), 1.84-1.81 ( m, 3H), 1.58-1.46 (m, 6H). MS-ESI calcd for [M ⁺ H] + 361, found 361.

Example 13.

1-((4-Hydroxy-4-methylcyclohexyl)methyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione.

Dissolve 3,7-dimethyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione (50.0 mg, 0.170 mmol) In tetrahydrofuran (2 mL). Under nitrogen, slowly add methyl Grignard reagent (3 M ether solvent, 0.4 mL, 1.20 mmol) at -78 °C. The reaction solution was stirred at -78 ° C for 0.5 hours, slowly warmed to 0 ° C and stirred for 0.5 hours. It was quenched by the addition of a saturated solution of ammonium chloride, and the pH was adjusted to 7. It was extracted with ethyl acetate, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and purified by preparative high-performance liquid chromatography to give 1-((4-hydroxy-4-methylcyclohexyl)methyl)-3,7-dimethyl- 1H -indole-2,6 (3) H , 7H )-dione (8.0 mg, white solid), yield: 16%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.86 (s, 1H), 3.97 (s, 3H), 3.88 (d, J = 7.6 Hz, 2H), 3.52 (s, 3H), 1.85- 1.78 (m, 1H), 1.73-1.57 (m, 3H), 1.46-1.33 (m, 2H), 1.32-1.15 (m, 6H). MS-ESI calcd for ^{_{[M + HH 2 O] +}} 289, found 289.

Example 14.

L - ((4-ethyl-4-hydroxy-cyclohexyl) methyl) -1H- purine-3,7-dimethyl -2,6 (3 H, 7 H) - dione.

Dissolve 3,7-dimethyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione (50.0 mg, 0.170 mmol) In tetrahydrofuran (2 mL). Under a nitrogen atmosphere, ethyl Grignard reagent (3M in ether solvent, 0.4 mL, 1.20 mmol) was slowly added at -78 °C. The reaction solution was stirred at -78 ° C for 0.5 hours, slowly warmed to 0 ° C and stirred for 0.5 hours. It was quenched by the addition of a saturated solution of ammonium chloride, and the pH was adjusted to 7. It was extracted with ethyl acetate, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and purified by preparative high performance liquid chromatography to give 1-((4-ethyl-4-hydroxycyclohexyl)methyl)-3,7-dimethyl-1H-indole-2,6 ( 3H) , 7 H )-dione (42.0 mg, white solid), yield: 77%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.86 (s, 1H), 3.97 (s, 3H), 3.88 (d, J = 7.6 Hz, 2H), 3.54-3.50 (m, 3H), 1.93-1.80(m,1H),1.76-1.72(m,2H),1.66-1.51(m,3H),1.38-1.28(m,3H),1.27-1.13(m,2H),0.89(t, J = 7.2 Hz, 3H). MS-ESI calcd for [M + H H ₂ O] ⁺ 303.

Example 15.

first step.

Methyl 2-(1,4-dioxaspiro[4.5]decane-8-ylidene)acetate.

Triethyl phosphinoacetate (12.2 g, 54.4 mmol) was dissolved in tetrahydrofuran (100 mL), and sodium hydride (1.92 g, 48.0 mmol) was added portionwise at 0 ° C. A solution of 1,4-cyclohexanedione monoethylene ketal (5.00 g, 32.0 mmol) dissolved in tetrahydrofuran (15 mL) was added dropwise to the reaction solution at 0 ° C, and the reaction solution was stirred at 25 ° C. hour. The reaction was quenched with water (25 mL)EtOAcEtOAc The organic phase was combined, washed with brine (20 mL), dried over anhydrous sodium sulfatesssssssssssssssssssssssssssssssss Methyl 2-(1,4-dioxaspiro[4.5]decane-8-ylidene)acetate (6.30 g, colourless oil), yield: 93%. ^{1 H NMR: (400MHz, CDCl} 3) δ5.67 (s, 1H), 4.15 (q, J = 7.2 Hz, 2H), 3.98 (s, 4H), 3.00 (t, J = 6.4 Hz, 2H), 2.38 (t, J = 6.4 Hz, 2H), 1.84-1.68 (m, 4H), 1.28 (t, J = 7.2 Hz, 3H). MS-ESI calcd for [M ⁺ H] + 227, found 227.

The second step.

Ethyl 2-(1,4-dioxaspiro[4.5]decane-8-yl)acetate.

Methyl 2-(1,4-dioxaspiro[4.5]decane-8-ylidene)acetate (3.80 g, 17.9 mmol) was dissolved in methanol (50 mL) and dry palladium carbon (palladium 10) %, water 1%, 400 mg), the reaction was allowed to react under hydrogen (50 psi) for 18 hours at room temperature. The reaction mixture was filtered, and the filtrate was evaporated tolulujjjjjjjjjjj .

¹ H NMR: (400MHz, CDCl ₃ ) δ 4.12 (q, J = 7.2 Hz, 2H), 3.93 (s, 4H), 2.22 (d, J = 7.2 Hz, 2H), 1.90-1.64 (m, 5H) ), 1.63-1.48 (m, 2H), 1.40-1.16 (m, 5H). MS-ESI calcd for [M + H] ⁺ 229.

third step.

2-(1,4-Dioxaspiro[4.5]decane-8-yl)ethanol.

Ethyl ethyl 2-(1,4-dioxaspiro[4.5]decane-8-yl)acetate (1.00 g, 4.38 mmol) was dissolved in tetrahydrofuran (20 mL), and tetrahydrogen was added portionwise at 0 °C. Lithium aluminum (216 mg, 5.69 mmol) was stirred under nitrogen for 18 hours. The reaction solution was cooled to 0 ° C, and water (0.2 mL), 15% aqueous sodium hydroxide (0.2 mL) and water (0.6 mL). Filtration and concentration of the filtrate under reduced pressure afforded 2-(1,4-dioxaspiro[4.5]decane-8-yl)ethanol (780 mg,yield of yellow oil).

^{1 H NMR: (400MHz, CDCl} 3) δ3.94 (s, 4H), 3.69 (t, J = 6.4 Hz, 2H), 1.79-1.65 (m, 4H), 1.59-1.38 (m, 5H), 1.34 -1.17 (m, 2H). MS-ESI calcd for [M + H] ⁺ 187.

the fourth step.

2-(1,4-Dioxaspiro[4.5]decane-8-yl)ethyl methanesulfonate

2-(1,4-Dioxaspiro[4.5]decane-8-yl)ethanol (400 mg, 2.15 mmol) and triethylamine (435 mg, 4.30 mmol) in dichloromethane (10 mL) Methanesulfonium chloride (369 mg, 3.23 mmol) was slowly added at 0 °C. The reaction solution was stirred at 0 ° C for 4 hours. The reaction was quenched with water (10 mL)EtOAcEtOAc The organic layer was combined, washed with aq. NaHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH Ethyl mesylate (500 mg crude, yellow oil).

¹ H NMR: (400 MHz, CDCl ₃ ) δ 4.28 (t, J = 6.4 Hz, 2H), 3.94 (s, 4H), 3.01 (s, 3H), 1.76-1.63 (m, 6H), 1.60-1.43 (m, 3H), 1.37-1.21 (m, 2H). MS-ESI calcd for [M + H] ⁺ 265.

the fifth step.

1-(2-(1,4-Dioxaspiro[4.5]decane-8-yl)ethyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )-dione.

Dissolve 3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )-dione (204 mg, 1.13 mmol) in N , N -dimethylformamide (15 mL) Add 2-(1,4-dioxaspiro[4.5]decane-8-yl)ethyl methanesulfonate (300 mg, 1.13 mmol), potassium carbonate (312 mg, 2.26 mmol) and potassium iodide ( 225 mg, 1.36 mmol). The reaction solution was heated to 120 ° C and stirred for 3 hours. Concentration under reduced pressure, the residue was purified by silica gel chromatography (1:1 petroleum ether/ethyl acetate, Rf = 0.2) to give 1-[2-(1,4-dioxaspiro[4.5]decane-8 -yl)ethyl]-3,7-dimethylindole-2,6-dione (190 mg, white solid), yield: 48%.

^{1 H NMR: (400MHz, CDCl} 3) δ7.50 (s, 1H), 4.09-4.03 (m, 2H), 4.02 (s, 3H), 3.99 (s, 4H), 3.57 (s, 1H), 1.90 -1.70 (m, 5H), 1.68-1.47 (m, 6H), 1.45-1.31 (m, 2H). MS-ESI calcd [M + H] ⁺ 495.

The sixth step.

3,7-Dimethyl-1-(2-(4-oxocyclohexyl)ethyl)-1 H -indole-2,6-(3 H ,7 H )-dione.

1-[2-(1,4-Dioxaspiro[4.5]decane-8-yl)ethyl]-3,7-dimethylindole-2,6-dione (190 mg, 545 umol) Dissolved in tetrahydrofuran (3 mL) and concentrated hydrochloric acid (1 mL). The reaction solution was stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced EtOAc. EtOAc (EtOAc m. Filtration, concentration under reduced pressure, and the residue was purified by silica gel chromatography (ethyl acetate, Rf = 0.3) to give 3,7-dimethyl-1-(2-(4-oxocyclohexyl)ethyl) -1 H - purine -2,6- (3 H, 7 H) - dione (150 mg, colorless oil). yield: 90%.

MS-ESI calcd [M + H] ⁺ 303.

The seventh step.

3,7-Dimethyl-1-(2-(4-(trifluoromethyl)-4-((trimethylcarbinyl)oxy)cyclohexyl)ethyl)-1-indole-2, 6-(3H,7H)-dione.

3,7-Dimethyl-1-[2-(4-oxocyclohexyl)ethyl]indole-2,6-dione (145 mg, 0.476 mmol) and cesium fluoride (7.2 mg, 0.0476 mmol) Dissolved in tetrahydrofuran (10 mL) and slowly added trifluoromethyltrimethylnonane ₍ 203 mg, 1.43 mmol) under nitrogen. The reaction solution was stirred at 25 ° C for 18 hours. The mixture was diluted with water (20 mL), EtOAc (EtOAc)EtOAc. ,7-Dimethyl-1-(2-(4-(trifluoromethyl)-4-((trimethylcarbinyl)oxy)cyclohexyl)ethyl)-1-in-2,6 -( 3H , 7H )-dione (170 mg, colorless liquid), yield: 80%.

MS-ESI calcd [M+H] ⁺ 447.

The eighth step.

3,7-Dimethyl-1-[2-[4-(trifluoromethyl)-4-trimethyldecyloxy-cyclohexyl]ethyl]indole-2,6-dione (160 mg , 0.358 mmol) was dissolved in tetrahydrofuran (3 mL) and concentrated hydrochloric acid (12 M, 0.107 mL). The reaction solution was stirred at 25 ° C for 18 hours. Diluted with water, aq. sodium hydrogen carbonate (10 mL) was adjusted to pH 7 and ethyl acetate (10 mL x 2). The organic phase was combined, dried over anhydrous sodium sulfate, filtered, evaporated, evaporated, evaporated,,,,,,,,,,,,,,,,,,,, . ^{1 H NMR: (400MHz, CDCl} 3) δ8.01 (s, 1H), 4.09-3.94 (m, 5H), 3.53 (s, 3H), 1.97-1.79 (m, 4H), 1.76-1.62 (m, 3H), 1.61-1.45 (m, 4H). MS-ESI calcd [M + H] ⁺ 372. And product 2 (15.0 mg, white solid) (isomer 2, second peak), yield: 10%. ^{1 H NMR: (400MHz, CDCl} 3) δ8.01 (s, 1H), 4.09-3.95 (m, 5H), 3.53 (s, 3H), 1.87-1.68 (m, 4H), 1.64-1.48 (m, 4H), 1.46-1.25 (m, 3H). MS-ESI calcd [M+H]+ 375.

Example 16.

1-((4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1H-indole-2,6-(3 H ,7 H )-dione.

first step.

Ethyl-8-methyl-1,4-dioxaspiro[4.5]decane-8-carboxylic acid tert-butyl butyl ester

Ethyl ethyl 1,4-dioxaspiro[4.5]decane-8-carboxylate (5.00 g, 23.3 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL). A solution of lithium diisopropylamide (2 M in tetrahydrofuran, 14.0 mL, 28.0 mmol) was added and the mixture was stirred at -78 ° C for one hour. Methyl iodide (6.62 g, 46.7 mmol) was added slowly and stirring was continued for 1 hour. The reaction was quenched by the addition of water (100 mL). The reaction mixture was extracted with EtOAc (EtOAc (EtOAc)EtOAc. 0.4) Ethyl 8-methyl-1,4-dioxaspiro[4.5]decane-8-carboxylic acid tert-butyl ester (5.00 g, yellow oil). Yield: 94%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 4.16-4.10 (m, 2H), 3.93-3.86 (m, 4H), 2.13-2.06 (m, 2H), 1.61-1.48 (m, 6H) , 1.25-1.22 (m, 3H), 1.15 (s, 3H).

The second step.

Ethyl-1-methyl-4-oxocyclohexanecarboxylic acid.

Ethyl 8-methyl-1,4-dioxaspiro[4.5]decane-8-carboxylic acid tert-butyl butyl ester (5.00 g, 21.9 mmol) was dissolved in tetrahydrofuran (50 mL) and added dropwise at 0 °C. After 1 N aqueous hydrochloric acid (20 mL) was stirred at 20 ° C for 1 hour. The mixture was cooled to 0 ° C and quenched with sodium bicarbonate (50 mL). The mixture was extracted with ethyl acetate (100 mL x 3). The organic layer was washed with brine (100 mL EtOAc) Purification by gel column chromatography (10:1 petroleum ether / ethyl acetate, Rf = 0.4) afforded ethyl-1-methyl-4-oxocyclohexanecarboxylic acid (3.00 g, colorless oil) Rate: 74%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 4.26-4.11 (m, 2H), 2.46-2.29 (m, 5H), 1.74-1.55 (m, 3H), 1.33-1.26 (m, 6H) .

third step.

Ethyl 4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexanecarboxylic acid.

Ethyl-1-methyl-4-oxocyclohexanecarboxylic acid (3.00 g, 16.3 mmol), cesium fluoride (247 mg, 1.63 mmol) was dissolved in tetrahydrofuran (50 mL), then added at 0 °C Methyl fluorenium trifluoromethyl (4.63 g, 35.3 mmol). The reaction solution was reacted under nitrogen for 20 hours at 20 °C. Then 4 N aqueous hydrochloric acid (4 mL) was added. The mixture was reacted under nitrogen for 6 hours at room temperature. The reaction was quenched with EtOAc EtOAc (EtOAc (EtOAc) 1 petroleum ether / ethyl acetate, Rf = 0.3) gave ethyl 4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexanecarboxylic acid (3.00 g, colourless oil). 73%. ¹ H NMR: (400 MHz, Methanol- d ₄ ) δ 4.20 - 4.12 (m, 2H), 2.03-1.31 (m, 8H), 1.29-1.23 (m, 6H).

the fourth step.

4-(Hydroxymethyl)-4-methyl-1-(trifluoromethyl)cyclohexanol.

Ethyl 4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexanecarboxylic acid (3.00 g, 11.8 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL), and lithium tetrahydroaluminum was added at 0 °C. (896 mg, 23.6 mmol). The reaction solution was warmed to 25 ° C and stirred for 1 hour. Quenched with water (20 mL), EtOAc (EtOAc (EtOAc)EtOAc. = 0.2) to give 4-(hydroxymethyl)-4-methyl-1-(trifluoromethyl)cyclohexanol (2.00 g, m. ¹ H NMR: (400 MHz, Methanol- d ₄ ) δ 3.25 (s, 2H), 1.76-1.64 (m, 6H), 1.29-1.26 (m, 2H), 0.93-0.91 (m, 3H).

the fifth step.

(4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl methanesulfonate. 4-(Hydroxymethyl)-4-methyl-1-(trifluoromethyl)cyclohexanol (2.00 g, 9.42 mmol) was dissolved in dichloromethane (30 mL). Amine (953 mg, 9.42 mmol) and methanesulfonyl chloride (1.08 g, 9.42 mmol). The reaction solution was reacted at 0 ° C for 2 hours. The organic layer was extracted with aq. EtOAc (EtOAc (EtOAc) The filtrate was concentrated under reduced pressure to give (4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl methanesulfonate (2.00 g, yellow oil).

The sixth step.

1-((4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )-dione.

(4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl methanesulfonate (100 mg, 0.344 mmol), 3,7-dimethyl- 1H -indole-2, 6-(3 H ,7 H )-dione (62.1 mg, 0.344 mmol), potassium iodide (5.70 mg, 0.0344 mmol) and potassium carbonate (47.6 mg, 0.344 mmol) dissolved in anhydrous N , N -dimethylformamidine In the amine (5 mL). The reaction solution was heated to 150 ° C in the microwave for 4 hours. The reaction solution was cooled to 20 ° C, filtered, and purified by preparative HPLC to give 1-((4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7- Dimethyl- 1H -indole-2,6-( 3H , 7H )-dione (3.0 mg, white solid), yield: 2%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.88 (s, 1H), 3.98 (s, 3H), 3.96 (s, 2H), 3.54 (s, 3H), 1.81-1.64 (m, 6H) ), 1.63-1.34 (m, 2H), 1.00 (s, 3H). MS-ESI calcd [M + H] ⁺ 372.

Example 17.

1-((4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )-dione.

first step.

Ethyl 8-(methoxymethyl)-1,4-dioxaspiro[4.5]decane-8-carboxylic acid ethyl ester.

Ethyl ethyl 1,4-dioxaspiro[4.5]decane-8-carboxylate (5.00 g, 23.3 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL). A solution of lithium diisopropylamide (2 M n-hexane solution, 14.0 mL, 28.0 mmol) was added and the mixture was stirred at -78 ° C for one hour. Methoxybromomethane (5.83 g, 46.7 mmol) was added slowly and stirring was continued for 1 hour. The reaction was quenched by the addition of water (100 mL). The reaction mixture was extracted with EtOAc (EtOAc (EtOAc)EtOAc. 0.3) Ethyl ethyl 8-(methoxymethyl)-1,4-dioxaspiro[4.5]decane-8-carboxylate (5.00 g, yellow oil), yield: 83% . ¹ H NMR: (400 MHz, Methanol- d ₄ ) δ 4.18 (q, J = 6.8 Hz, 2H), 3.94 (s, 4H), 3.55 (s, 2H), 3.33 (s, 3H), 2.14 2.12 (m, 2H), 1.65-1.57 (m, 6H), 1.26 (t, J = 6.8 Hz, 3H).

The second step.

Ethyl ethyl-1-(methoxymethyl)-4-oxocyclohexanecarboxylate.

Ethyl ethyl 8-(methoxymethyl)-1,4-dioxaspiro[4.5]decane-8-carboxylate (5.00 g, 19.4 mmol) was dissolved in tetrahydrofuran (50 mL). 1 N diluted hydrochloric acid (10 mL) was added dropwise, and the mixture was stirred at 20 ° C for 1 hour. The mixture was cooled to 0 ° C and quenched with sodium bicarbonate (50 mL). The mixture was extracted with ethyl acetate (100 mL x 3). The organic layer was washed with brine (100 mL EtOAc) Purification by column chromatography (10:1 petroleum ether / ethyl acetate, Rf = 0.4) afforded ethyl ethyl 1-(methoxymethyl)-4-oxocyclohexanecarboxylate (3.00 g, White oil), Yield: 73%. ¹ H NMR: (400 MHz, Methanol- d ₄ ) δ 4.25 (q, J = 6.8 Hz, 2H), 3.52 (s, 2H), 3.34 (s, 3H), 2.52-2.30 (m, 6H), 1.82-1.78 (m, 2H), 1.30 (t, J = 6.8 Hz, 3H).

third step.

Ethyl 4-hydroxy-1-(methoxymethyl)-4-(trifluoromethyl)cyclohexanecarboxylate.

Ethyl ethyl-1-(methoxymethyl)-4-oxocyclohexanecarboxylate (3.00 g, 14.0 mmol), cesium fluoride (243 mg, 1.40 mmol) dissolved in tetrahydrofuran (50 mL) Then, trimethylsulfonium trifluoromethyl (3.98 g, 28.0 mmol) was added at 0 °C. The reaction solution was reacted under nitrogen for 20 hours at 20 °C. Then 4 N dilute hydrochloric acid (7 mL) was added. The mixture was reacted under nitrogen for 6 hours at room temperature. The reaction was quenched with EtOAc EtOAc (EtOAc (EtOAc) 1 petroleum ether / ethyl acetate, Rf = 0.4) to give ethyl 4-hydroxy-1-(methoxymethyl)-4-(trifluoromethyl)cyclohexanecarboxylate (1.7 g, colorless oil ), yield: 43%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) 4.18-4.09 (m, 2H), 3.61 (s, 2H), 3.33 (s, 3H), 1.84-1.71 (m, 8H), 1.28-1.25 (m) , 3H).

the fourth step.

4-(Hydroxymethyl)-4-(methoxymethyl)-1-(trifluoromethyl)cyclohexanol. Ethyl 4-hydroxy-1-(methoxymethyl)-4-(trifluoromethyl)cyclohexanecarboxylate (1.50 g, 5.28 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL). Lithium tetrahydroaluminum (220 mg, 5.81 mmol) was added. The reaction solution was warmed to 25 ° C and stirred for 1 hour. Quenched with water (20 mL), EtOAc (EtOAc)EtOAc = 0.2) to give 4-(hydroxymethyl)-4-(methoxymethyl)-1-(trifluoromethyl)cyclohexanol (1.20 g, m. ¹ H NMR: (400 MHz, Methanol- d ₄ ) δ 3.33 - 3.32 (m, 7H), 1.67-1.63 (m, 4H), 1.52-1.48 (m, 4H).

the fifth step.

(4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl methanesulfonate.

4-(Hydroxymethyl)-4-(methoxymethyl)-1-(trifluoromethyl)cyclohexanol (1.20 g, 4.95 mmol) was dissolved in dichloromethane (20 mL) Triethylamine (851 mg, 9.91 mmol) and methanesulfonium chloride (851 mg, 7.43 mmol) were added at °C. The reaction solution was reacted at 0 ° C for 2 hours. The organic layer was extracted with aq. EtOAc (EtOAc (EtOAc) The filtrate was concentrated under reduced pressure to give (4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl methanesulfonate (1.30 g, yellow oil).

The sixth step.

1-((4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )-dione.

(4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl methanesulfonate (300 mg, 1.05 mmol), 3,7-dimethyl- 1H -indole-2, 6-(3 H ,7 H )-dione (189 mg, 1.05 mmol), potassium iodide (17.4 mg, 0.105 mmol) and potassium carbonate (435 mg, 3.15 mmol) dissolved in anhydrous N , N -dimethylformamide In the amine (5 mL). The reaction solution was heated to 150 ° C in the microwave for 2 hours. The reaction solution was cooled to 20 ° C, filtered, and purified by preparative HPLC to give 1-((4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7- Dimethyl- 1H -indole-2,6-( 3H , 7H )-dione (12.0 mg, white solid), yield: 3%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.87 (s, 1H), 4.06 (s, 2H), 3.83 (s, 3H), 3.98 (s, 3H), 3.53 (s, 2H), 3.42 (s, 3H), 1.69-1.58 (m, 8H). MS-ESI calcd for [M + H] ⁺ 405.

Example 18.

1-((4-(3-Hydroxypenta-3-yl)-cyclohexyl)methyl)-3,7-dimethyl 1 H -indole-2,6(3 H ,7 H )-dione.

first step.

Methyl 4-hydroxymethylcyclohexanecarboxylate.

Methyl 1,4-cyclohexanecarboxylate (1.20 g, 6.45 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL). N.sub.2. 10.3 mmol), the reaction solution was stirred at 0 ° C for 0.5 hour, slowly raised to 25 ° C, and stirring was continued for 1 hour. The reaction was quenched by water (40 mL). The combined organic phases were washed with EtOAcqqqHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH 91%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 3.67 (s, 3H), 3.48-3.46 (m, 2H), 2.26-2.25 (m, 1H), 2.05-2.01 (m, 2H), 1.89-1.85 (m, 2H), 1.47-1.43 (m, 2H), 1.31 (s, 1H), 1.01 - 0.98 (m, 2H). MS-ESI calcd for [M + H] ⁺ 173.

The second step.

Methyl 4-methanesulfonyloxymethyl-cyclohexanecarboxylate.

Methyl 4-hydroxymethylcyclohexanecarboxylate (900 mg, 5.20 mmol) and triethylamine (1.58 g, 15.6 mmol) were dissolved in anhydrous dichloromethane (5 mL). Methane sulfonium chloride (720 mg, 6.30 mmol). The reaction solution was raised to 25 ° C and stirred for 2 hours. The reaction was quenched by the addition of water (60 mL). The organic phase was combined, washed sequentially with EtOAc EtOAc EtOAc (HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH Methyl 4-methanesulfonyloxymethyl-cyclohexanecarboxylate (1.00 g, white solid), yield: 91%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 3.67 (s, 3H), 3.48-3.46 (m, 2H), 3.01 (s, 3H), 2.26-2.25 (m, 1H), 2.05-2.01 (m) , 2H), 1.89-1.85 (m, 2H), 1.47-1.43 (m, 2H), 1.31 (s, 1H), 1.01 - 0.98 (m, 2H). Calcd MS-ESI [M ⁺ H] + 251, found 251.

third step.

4-((3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl)-cyclohexanecarboxylic acid ester.

Methyl 4-methanesulfonyloxymethyl-cyclohexanecarboxylate (580 mg, 2.32 mmol) was dissolved in 5 mL of anhydrous N , N -dimethylformamide and potassium carbonate was added at 25 ° C under nitrogen. (640 mg, 4.64 mmol), potassium iodide (38.0 mg, 0.230 mmol), 2,6-hydroxy-3,7-dimethylindole (501 mg, 2.80 mmol). The reaction solution was stirred at 130 ° C for 3 hours. 40 mL of water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was combined, washed sequentially with water and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. ((3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl)-cyclohexanecarboxylic acid methyl ester ( 400 mg, white solid), yield: 52%. MS-ESI calcd for [M + H] ⁺ 335.

the fourth step.

1-((4-(3-Hydroxypenta-3-yl)-cyclohexyl)methyl)-3,7-dimethyl 1 H -indole-2,6(3 H ,7 H )-dione.

Methyl 4-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)-cyclohexanecarboxylic acid ( 100 mg, 0.30 mmol) was dissolved in 5 mL of anhydrous tetrahydrofuran. Ethyl magnesium bromide solution (3 M diethyl ether solution, 1 mL, 3.00 mmol) was slowly added dropwise at -65 ° C under nitrogen atmosphere. The reaction mixture was at -65 ° C. After the mixture was stirred for 2 hours, the reaction mixture was poured with EtOAc EtOAc EtOAc. Purification by high performance liquid chromatography gave the product 1-((4-(3-hydroxypenta-3-yl)-cyclohexyl)methyl)-3,7-dimethyl- 1H -indole-2,6( 3H ,7 H )-dione (20.0 mg, white solid), yield: 19%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.52 (s, 1H), 4.00 (s, 3H), 3.90 - 3.88 ( m, 2H), 3.59 (s, 3H), 1.80-1.74 (m, 6H), 1.50-1.45 (m, 4H), 1.11-1.10 (m, 4H), 0.86-0.82 (m, 6H). MS ESI Calculated [M + H] ⁺ 363, found 363.

Example 19.

3,7-Dimethyl-1-[[trans-4-(2,2,2-trifluoro-1-hydroxy-1-methyl-ethyl)cyclohexyl]methyl]indole-2,6 - Diketone.

first step.

Trans-4-methylmethylcyclohexanecarboxylic acid methyl ester.

The trans-cyclohexane-1,4-dicarboxylic acid monomethyl ester (5.00 g, 26.8 mmol) was dissolved in tetrahydrofuran (100 mL), and borane dimethyl sulfide (3.06 g, 40.3 mmol) was added at 0 °C. The reaction was carried out at room temperature for 2 hours. The reaction was quenched by the addition of saturated methanol (50 mL). After concentrating, water (50 mL), EtOAc (EtOAc m. Yellow oil), Yield: 87%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 3.67 (s, 3H), 3.43-3.38 (m, 2H), 2.31-2.54 (m, 1H), 2.03-1.98 (m, 2H), 1.90 -1.82 (m, 2H), 1.45-1.38 (m, 3H), 1.03-0.99 (m, 2H). MS-ESI calcd for [M + H] ⁺ 173.

The second step.

Trans-4-methylsulfonyloxymethyl-cyclohexanecarboxylic acid methyl ester.

Methyl trans-4-hydroxymethylcyclohexanecarboxylate (4.00 g, 23.2 mmol) and triethylamine (7.05 g, 69.6 mmol) were dissolved in dichloromethane (50 mL). Sulfonium chloride (7.98 g, 69.6 mmol). The reaction solution was slowly warmed to room temperature and stirred for 2 hours. The reaction was quenched by the addition of aqueous sodium bicarbonate (50 mL). Extract with dichloromethane (20 mL x 3). The organic layer was combined, washed with brine (30 mL) Yellow oil), yield: 99%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 4.10-4.03 (m, 2H), 3.65 (s, 3H), 3.07 (s, 3H), 2.42-2.31 (m, 1H), 2.10-2.03 (m, 2H), 1.90-1.82 (m, 2H), 1.75-1.66 (m, 1H), 1.48-1.42 (m, 2H), 1.21-1.10 (m, 2H). Calcd MS-ESI [M ⁺ H] + 251, found 251.

third step.

Trans-methyl 4-[(3,7-dimethyl-2,6-dioxo-indol-1-yl)methyl]cyclohexanecarboxylic acid.

Methyl trans-4-methylsulfonyloxymethyl-cyclohexanecarboxylate (1.00 g, 4.00 mmol), 3,7-dimethyl- 1H -indole-2,6( 3H ,7 H )-Dione (719 mg, 4.00 mmol), potassium iodide (66.0 mg, 0.397 mmol) and potassium carbonate (1.10 g, 7.96 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was warmed to 120 ° C and stirred for 3 hours. It was cooled to room temperature, filtered, and the filtrate was concentrated. Separation and purification by column chromatography (ethyl acetate, Rf value = 0.1) gave trans-methyl 4-[(3,7-dimethyl-2,6-dioxo-indol-1-yl) Methyl]cyclohexanecarboxylic acid (800 mg, yellow solid), yield: 60%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.88 (s, 1H), 3.98 (s, 3H), 3.90-3.82 (m, 2H), 3.72 (s, 3H), 3.51 (s, 3H) ), 2.33 - 2.25 (m, 1H), 2.03-1.98 (m, 2H), 1.80-1.74 (m, 3H), 1.42-1.36 (m, 2H), 1.21-1.10 (m, 2H). MS-ESI calcd for [M + H] ⁺ 335.

the fourth step.

1-(trans-4-Ethylcyclohexylmethyl)-3,7-dimethyl-3,7-dihydro-indole-2,6-dione

Trans-methyl 4-[(3,7-dimethyl-2,6-dioxo-indol-1-yl)methyl]cyclohexanecarboxylic acid (300 mg, 0.897 mmol) and O , N -Dimethylhydroxylamine hydrochloride (114 mg, 1.17 mmol) was dissolved in tetrahydrofuran (25 mL). The reaction solution was slowly warmed to room temperature and stirred for 12 hours. The reaction was quenched by the addition of saturated aqueous ammonium chloride (10 mL). Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with EtOAc EtOAc. Purification by preparative TLC (ethyl acetate, Rf = 0.4) afforded 1-(trans-4- ethylcyclohexylmethyl)-3,7-dimethyl-3,7-dihydro-indole- 2,6-dione (80.0 mg, yellow oil), yield: 29%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.88 (s, 1H), 3.98 (s, 3H), 3.92-3.84 (m, 2H), 3.55 (s, 3H), 2.42-2.33 (m) , 1H), 2.15 (s, 3H), 1.98-1.88 (m, 2H), 1.85-1.75 (m, 3H), 1.32-1.10 (m, 4H). MS-ESI calcd for [M ⁺ H] + 319, found 319.

the fifth step.

3,7-Dimethyl-1-[[trans-4-(2,2,2-trifluoro-1-hydroxy-1-methyl-ethyl)cyclohexyl]methyl]indole-2,6 - Diketone.

1-(trans-4-Ethylcyclohexylmethyl)-3,7-dimethyl-3,7-dihydro-indole-2,6-dione (80.0 mg, 0.251 mmol), fluorine The hydrazine (11.5 mg, 0.753 mmol) was dissolved in tetrahydrofuran (10 mL), and trimethyl-trifluoromethyl-decane (71.6 mg, 0.502 mmol) was added at room temperature and stirred for 12 hours. After adding 1 N hydrochloric acid (10 mL), the mixture was stirred at room temperature for 1 hour, and then the mixture was then evaporated and evaporated. Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with EtOAc EtOAc. Purification by preparative high performance liquid chromatography to give 3,7-dimethyl-1-[[trans-4-(2,2,2-trifluoro-1-hydroxy-1-methyl-ethyl) Cyclohexyl]methyl]anthracene-2,6-dione (35.0 mg, yellow solid), yield: 70%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.88 (s, 1H), 3.98 (s, 3H), 3.88 (d, J = 6.8 Hz, 2H), 3.53 (s, 3H), 1.96– 1.67 (m, 6H), 1.22 (s, 3H), 1.15 - 1.06 (m, 4H). MS-ESI calcd [M + H] ⁺ </RTI>

Example 20.

3,7-Dimethyl-1-[trans-4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-cyclohexylmethyl]-3,7 - Dihydro-indole-2,6-dione.

first step.

3,7-Dimethyl-1-[trans-4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-cyclohexylmethyl]-3,7 - Dihydro-indole-2,6-dione.

Fluorine-trans-methyl 4-[(3,7-dimethyl-2,6-dioxo-indol-1-yl)methyl]cyclohexanecarboxylic acid (200 mg, 0.598 mmol), fluorinated The hydrazine (45.4 mg, 0.299 mmol) was dissolved in tetrahydrofuran (10 mL), and trimethyl-trifluoromethyl-decane (340 mg, 2.39 mmol) was added at room temperature and stirred for 12 hours. After adding 1 N hydrochloric acid (10 mL), the mixture was stirred at room temperature for 1 hour, and then the mixture was then evaporated and evaporated. Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with EtOAc EtOAc. Purification by preparative high performance liquid chromatography to give 3,7-dimethyl-1-[trans-4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl -cyclohexylmethyl]-3,7-dihydro-indole-2,6-dione (35.0 mg, yellow solid), yield: 41%.

¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.88 (s, 1H), 3.98 (s, 3H), 3.88 (d, J = 6.8 Hz, 2H), 3.53 (s, 3H), 2.08- 1.79 (m, 6H), 1.30-1.24 (m, 2H), 1.11-1.08 (m, 2H). MS-ESI calcd for [M + H] ⁺ 443.

Example 21.

1-[[trans-4-(1-hydroxycyclopropyl)cyclohexyl]methyl]-3,7-dimethylindole-2,6-dione.

first step.

1-[[trans-4-(1-hydroxycyclopropyl)cyclohexyl]methyl]-3,7-dimethylindole-2,6-dione.

Trans-methyl 4-[(3,7-dimethyl-2,6-dioxo-indol-1-yl)methyl]cyclohexanecarboxylic acid (200 mg, 0.598 mmol), four different The propyltitanium oxide (340 mg, 1.20 mmol) was dissolved in tetrahydrofuran (10 mL). EtOAc (EtOAc m. The reaction was quenched by the addition of saturated aqueous ammonium chloride (50 mL). Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with EtOAc EtOAc. The residue was purified by preparative high performance liquid chromatography to give 1-[[trans-4-(1-hydroxycyclopropyl)cyclohexyl]methyl]-3,7-dimethylindole-2,6-di Ketone (70.0 mg, yellow solid), yield: 35%.

¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.87 (s, 1H), 3.98 (s, 3H), 3.88 (d, J = 6.8 Hz, 2H), 3.53 (s, 3H), 1.79- 1.71 (m, 5H), 1.29-1.07 (m, 5H), 0.60-0.57 (m, 2H), 0.42-0.39 (m, 2H). MS-ESI calcd [M + H] ⁺ 333.

Example 22.

1-(2-(3-Ethyl-3-hydroxycyclohexyl)ethyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione.

first step.

2-(3-Ethyl-3-hydroxycyclohexyl)ethyl methanesulfonate.

1-Ethyl-3-(2-hydroxyethyl)cyclohexanol (450 mg, 2.61 mmol) and diisopropylethylamine (500 mg, 3.92 mmol) were dissolved in dichloromethane (10 mL) Methanesulfonium chloride (600 mg, 5.40 mmol) was slowly added at 0 °C. The reaction solution was stirred at 0 ° C for 0.5 hours. The reaction was quenched with water and EtOAc (EtOAc) The organic phase was combined, dried over anhydrous sodium sulfate, filtered and evaporated, evaporated, evaporated. Cyclohexyl)ethyl methanesulfonate (450 mg, yellow oil), yield: 69%. Calcd MS-ESI [M ⁺ H] + 251, found 251.

The second step.

1-(2-(3-Ethyl-3-hydroxycyclohexyl)ethyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione.

2-(3-Ethyl-3-hydroxycyclohexyl)ethyl methanesulfonate (200 mg, 0.790 mmol), 3,7-dimethyl- 1H -indole-2,6( 3H ,7 H )-Dione (144 mg, 0.790 mmol) and potassium carbonate (220 mg, 1.60 mmol), potassium iodide (13.1 mg, 0.0790 mmol) were dissolved in N , N -dimethylformamide (3 mL). The reaction solution was heated to 130 ° C and stirred for 3 hours. The reaction solution was cooled to 25 ° C, and brine (EtOAc) The organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by preparative high-performance liquid chromatography to give the product 1-(2-(3-ethyl-3-hydroxycyclohexyl)ethyl)-3. 7-Dimethyl-1 H -indole-2,6(3 H ,7 H )-dione (70.0 mg, white solid), yield: 26%. ¹ H NMR _: (400 MHz, Methonal- d ₄ ) δ 7.86 (s, 1H), 4.09-3.94 (m, 5H), 3.52 (s, 3H), 1.88-1.84 (m, 1H), 1.80-1.40 (m, 10H), 1.26-1.16 (m, 1H), 1.02-0.95 (m, 1H), 0.91 (t, J = 7.2 Hz, 3H). MS-ESI calcd for ^{[M + H-18] +} 317, found 317.

Example 23.

first step.

Ethyl 3-trifluoromethyl-3-trimethyldecyloxy-cyclohexanecarboxylate.

Ethyl-3-oxocyclohexanecarboxylic acid (1.00 g, 5.88 mmol), cesium fluoride (446 mg, 2.94 mmol) was dissolved in tetrahydrofuran (30 mL). Methyl-decane (1.67 g, 11.7 mmol) was stirred for 12 hours. The reaction was quenched by the addition of water (20 mL). Extract with ethyl acetate (20 mL x 3). The organic phase was combined, washed with brine (EtOAc) g, yellow oil), yield: 76%. MS-ESI calcd for [M ⁺ H] + 313, found 313.

The second step.

(3-Trifluoromethyl-3-trimethyldecaneoxycyclohexyl)methanol.

Ethyl 3-trifluoromethyl-3-trimethyldecyloxy-cyclohexanecarboxylate (1.00 g, 3.20 mmol) was dissolved in tetrahydrofuran (10 mL). Mg, 6.40 mmol), reaction for 1 hour. The reaction was quenched by the addition of water (10 mL). Extracted with ethyl acetate (20 mL x 3), dried over anhydrous sodium sulfate, filtered and evaporated. Shape), yield: 92%. MS-ESI calcd [M + H] ⁺ 271.

third step.

[3-(Trifluoromethyl)-3-trimethyldecyloxycyclohexyl]methyl methanesulfonate.

3-Trifluoromethyl-3-trimethyldecaneoxycyclohexyl)methanol (850 mg, 3.14 mmol) and triethylamine (953 mg, 9.42 mmol) were dissolved in dichloromethane (15 mL) Methanesulfonyl chloride (719 mg, 6.28 mmol) was added at °C. The reaction solution was slowly warmed to room temperature and stirred for 2 hours. The reaction was quenched by the addition of aqueous sodium bicarbonate (10 mL). Extract with dichloromethane (20 mL x 3). The organic phase was combined, washed with brine (20 mL) Sulfonate (900 mg, yellow oil), yield: 82%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 4.36-4.32 (m, 1H), 4.17 - 4.13 (m, 1H), 3.08 (s, 3H), 2.12-1.60 (m, 9H), 0.16 (s, 9H). MS-ESI calcd [M + H] ⁺ 495.

the fourth step.

3,7-Dimethyl-1-[[3-(trifluoromethyl)-3-trimethyldecyloxy-cyclohexyl]methyl]indole-2,6-dione.

[3-(Trifluoromethyl)-3-trimethyldecyloxycyclohexyl]methyl methanesulfonate (200 mg, 0.573 mmol), 3,7-dimethyl- 1H -indole-2 ,6(3 H ,7 H )-dione (103 mg, 0.574 mmol), potassium iodide (28.6 mg, 0.172 mmol) and potassium carbonate (374 mg, 1.15 mmol) dissolved in N , N -dimethylformamide (30 mL). The reaction solution was warmed to 120 ° C and stirred for 3 hours. It was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure and purified by preparative high-purity liquid chromatography to give 3,7-dimethyl-1-[[3-(trifluoromethyl)-3-trimethyloxyl. -cyclohexyl]methyl]anthracene-2,6-dione (150 mg, yellow solid), yield: 60%.

MS-ESI calcd for [M + H] ⁺ 437.

the fifth step.

3,7-Dimethyl-1-[[3-(trifluoromethyl)-3-trimethyldecyloxy-cyclohexyl]methyl]indole-2,6-dione (200 mg, 0.462) Methyl acetate was dissolved in THF (10 mL). Extract with ethyl acetate (20 mL x 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Purification by preparative high performance liquid chromatography gave product 1 (10.0 mg, yellow solid) (isomer 1, first peak), yield: 6%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.87 (s, 1H), 3.97 (s, 3H), 3.89-3.83 (m, 2H), 3.52 (s, 3H), 2.23-2.22 (m) , 1H), 1.76-1.07 (m, 8H). MS-ESI calcd for [M ⁺ H] + 361, found 361.

Product 2 (85.0 mg of yellow solid) (isomer 2, second peak), yield: 51%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.87 (s, 1H), 4.31-4.26 (m, 1H), 3.99-3.95 (m, 4H), 3.55 (s, 3H), 2.26-1.88 (m, 3H), 1.79-1.47 (m, 6H). MS-ESI calcd for [M ⁺ H] + 361, found 361.

Example 24.

1-((5-Hydroxy-5-(trifluoromethyl)tetrahydro- 2H -pyran-2-yl)methyl)-3,7-dimethyl-1 H -indole-2,6- (3 H , 7 H )-dione.

first step.

(3,4-Dihydro-2 H -pyran-2-yl)methanol.

3,4-Dihydro-2 H -pyran-2-carbaldehyde (3.00 g, 26.7 mmol) was dissolved in methanol (20 mL), sodium borohydride (2.02 g, 53.5 mmol) was added at 0 ° C, reaction 2 hour. The reaction was quenched by the addition of saturated aqueous ammonium chloride (30 mL). Extracted with dichloromethane (20 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give (3,4-dihydro -2 H - pyran-2-yl) methanol (1.50 g, yellow oil ), yield: 49%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 6.40 (d, J = 6.0 Hz, 1H), 4.71-4.68 (m, 1H), 3.86-3.83 (m, 1H), 3.82-3.61 (m) , 2H), 2.13-2.12 (m, 1H), 2.10-2.08 (m, 1H), 2.02-2.01 (m, 1H), 1.68-1.63 (m, 1H).

The second step.

(3,4-Dihydro-2 H -pyran-2-yl)methyl methanesulfonate.

(3,4-Dihydro- 2H -pyran-2-yl)methanol (1.50 g, 13.1 mmol) and triethylamine (2.66 g, 26.3 mmol) were dissolved in dichloromethane (20 mL) Methanesulfonyl chloride (3.01 g, 26.3 mmol) was added at °C. The reaction solution was slowly warmed to room temperature and stirred for 2 hours. The reaction was quenched by the addition of aqueous sodium bicarbonate (10 mL). Extract with dichloromethane (20 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give (3,4-dihydro -2 H - pyran-2-yl) methyl methanesulfonate (1.70 g, Yellow oil), Yield: 67%. MS-ESI calcd for [M + H] ⁺ 193.

third step.

1-((3,4-Dihydro-2 H -pyran-2-yl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )- Dione.

(3,4-Dihydro-2 H -pyran-2-yl)methyl methanesulfonate (1.70 g, 8.84 mmol), 3,7-dimethyl-1 H -indole-2,6- (3 H ,7 H )-dione (1.59 g, 8.84 mmol), potassium iodide (146 mg, 0.884 mmol) and potassium carbonate (2.44 g, 17.7 mmol) dissolved in N , N -dimethylformamide (50 In mL). The reaction solution was warmed to 120 ° C and stirred for 3 hours. Cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (ethyl acetate, Rf = 0.4) to give l - ((3,4-dihydro -2 H - pyran-2-yl) methyl -3,7-Dimethyl- 1H -indole-2,6-( 3H , 7H )-dione (1.30 g, yellow solid), yield: 53%. MS-ESI calcd for [M + H] ⁺ 277.

the fourth step.

1-((5-Hydroxytetrahydro-2 H -pyran-2-yl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )- ketone.

1-(3,4-Dihydro- 2H -pyran-2-ylmethyl)-3,7-dimethylindole-2,6-dione (600 mg, 2.17 mmol) was dissolved in tetrahydrofuran ( In 30 mL), borane dimethyl sulfide (825 mg, 10.7 mmol) was added at 0 °C. The reaction solution was slowly warmed to room temperature and stirred for 12 hours. A 3 N aqueous sodium hydroxide solution (30 mL) and hydrogen peroxide (10 mL) were added and the reaction was continued for one hour. The reaction was quenched with EtOAc (EtOAc) (EtOAc)EtOAc. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Separation and purification by preparative TLC plate (20:1 dichloromethane/methanol, Rf = 0.3) afforded 1-((5-hydroxytetrahydro- 2H -pyran-2-yl)methyl)-3,7- Methyl- 1H -indole-2,6-( 3H , 7H )-dione (130 mg, yellow oil), yield: 20%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.88 (s, 1H), 4.25-4.23 (m, 1H), 4.20 (s, 3H), 3.98-3.67 (m, 5H), 3.54 (s) , 3H), 2.10 - 1.77 (m, 2H), 1.49-1.31 (m, 2H). MS-ESI calcd for [M + H] ⁺ 295.

the fifth step.

3,7-Dimethyl-1-((5-oxotetrahydro-2 H -pyran-2-yl)methyl)-1 H -嘌呤-2,6-(3 H ,7 H )- Dione.

1-((5-Hydroxytetrahydro-2 H -pyran-2-yl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )- The diketone (130 mg, 0.441 mmol) was dissolved in dichloromethane (10 mL). EtOAc (EtOAc) The reaction was quenched with EtOAc EtOAc (EtOAc)EtOAc. Separation and purification by preparative TLC (20:1 dichloromethane/methanol, Rf = 0.4) afforded 3,7-dimethyl-1-((5-oxotetrahydro-2H-pyran-2-yl) methyl) -1 H - purine -2,6- (3 H, 7 H) - dione (60.0 mg, yellow solid), yield: 47%. MS-ESI calcd for [M ⁺ H] + 293, found 293.

The sixth step.

1-((5-Hydroxy-5-(trifluoromethyl)tetrahydro- 2H -pyran-2-yl)methyl)-3,7-dimethyl-1 H -indole-2,6- (3 H , 7 H )-dione.

3,7-Dimethyl-1-((5-oxotetrahydro-2 H -pyran-2-yl)methyl)-1H-indole-2,6-(3 H ,7 H )- Diketone (60.0 mg, 0.205 mmol), cesium fluoride (6.24 mg, 0.0411 mmol) in tetrahydrofuran (10 mL), trimethyltrifluoromethyl decane (87.5 mg, 0.615 mmol) 5 hours. After adding 1 N hydrochloric acid (10 mL), the mixture was stirred at room temperature for 1 hour, and then the mixture was stirred and evaporated. Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Purification by preparative high performance liquid chromatography to give 1-((5-hydroxy-5-(trifluoromethyl)tetrahydro- 2H -pyran-2-yl)methyl)-3,7-dimethyl -1 H - purine -2,6- (3 H, 7 H) - dione (15.0 mg, yellow solid), yield: 30%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.28 (s, 1H), 4.39-4.10 (m, 2H), 4.05 (s, 3H), 3.93-3.89 (m, 2H), 3.55 (s) , 3H), 3.32-3.27 (m, 1H), 1.89-1.65 (m, 4H). MS-ESI calcd for [M + H] ⁺ 363.

Example 25.

1-(4-(3-Hydroxypentan-3-yl)benzyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione.

first step.

Methyl 4-((3,7-dimethyl-2,6-oxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl)benzoate.

Nitrogen protection, 3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione (180 mg, 1.00 mmol), 4-(bromomethyl) at 25 ° C Methyl benzoate (251 mg, 1.10 mmol), potassium iodide (55.0 mg, 0.33 mmol) and potassium carbonate (179 mg, 1.30 mmol) were dissolved in anhydrous N , N -dimethylformamide (4 mL). Heat to 110 ° C and stir for 3 hours. After cooling to 25 ° C, it was diluted with water and extracted with ethyl acetate (30 mL x 2). The organic phase was combined, dried over anhydrous sodium sulfate, filtered and evaporated. Methyl 6-oxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl)benzoate (300 mg, white solid), yield: 91%. MS-ESI calcd for [M + H] ⁺ 329.

The second step.

1-(4-(3-Hydroxypentan-3-yl)benzyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione.

Methyl 4-((3,7-dimethyl-2,6-oxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl)benzoate (200 mg , 0.610 mmol) dissolved in dry tetrahydrofuran (3 mL). Under nitrogen, ethylmagnesium bromide (3 M in diethyl ether, 1.2 mL, 3.60 mmol) was added dropwise at -78 °C. The reaction solution was stirred at this temperature for 0.5 hour, and naturally raised to 25 ° C to continue the reaction for 1 hour. Quenched (5 mL) with EtOAc (EtOAc) The organic phase was combined, dried over anhydrous sodium sulfate, filtered, evaporated, evaporated, evaporated. - yl) benzyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (190 mg, white solid), yield: 87%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.85 (s, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 5.25 (s, 2H), 3.96 (s, 3H), 3.52 (s, 3H), 1.82-1.72 (m, 4H), 0.69 (t, J = 7.2 Hz, 6H). MS-ESI calcd [M + H] ⁺ 357.

Example 26.

3,7-Dimethyl-1-(3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzyl)-1 H -indole-2,6-(3 H ,7 H )-dione.

first step.

Ethyl 3-ethylmercaptobenzoate.

3-Ethyl benzoic acid (500 mg, 3.05 mmol) was dissolved in N , N -dimethylformamide (20 mL) and ethyl iodide (475 mg, 3.05 mmol) Potassium (632 mg, 4.57 mmol), EtOAc (3 mL), EtOAc (EtOAc)EtOAc. Concentration by pressure and purification by EtOAc (5:1 petroleum ether / ethyl acetate, Rf = 0.5) afforded ethyl 3-ethyl propyl benzoate (530 mg, white solid). ¹ H NMR: (400 MHz, CDCl ₃ ) δ 8.60 (s, 1H), 8.24 (d, J = 7.6 Hz, 1H), 8.15 (d, J = 7.6 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 4.41 (q, J = 7.2 Hz, 2H), 2.66 (s, 3H), 1.42 (t, J = 7.2 Hz, 3H). MS-ESI calcd for [M + H] ⁺ 193.

The second step.

Ethyl ethyl 3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzoate.

Ethyl 3-ethylmercaptobenzoate (500 mg, 2.60 mmol) was dissolved in tetrahydrofuran (20 mL). Trifluoromethyltrimethyldecane (370 mg, 2.60 mmol) and cesium fluoride were added at room temperature. (79.0 mg, 0.520 mmol). After stirring at room temperature for 12 hours, the reaction mixture was diluted with ethyl acetate (30 mL). Ethyl ethyl 3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzoate (600 mg, yellow solid), m.p. Yield: 88%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 8.26 (s, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 1.82 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H). MS-ESI calcd for [M + H] ⁺ 263.

third step.

1,1,1-Trifluoro-2-(3-hydroxymethyl)phenyl)propan-2-ol.

Ethyl ethyl 3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzoate (500 mg, 1.91 mmol) was dissolved in tetrahydrofuran (20 mL). Lithium aluminum hydride (108 mg, 2.87 mmol) was added thereto, and the mixture was stirred at room temperature for 2 hours, and water (0.1 mL), 15% sodium hydroxide (0.1 mL) and water (0.3 mL) were added to the mixture and stirred for 20 minutes. The reaction mixture was diluted with ethyl acetate (30 mL). EtOAc. Hydroxymethyl)phenyl)propan-2-ol (400 mg, yellow solid), yield: 95%.

¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.62 (s, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.41-7.37 (m, 2H), 4.73 (s, 2H), 1.80 ( s, 3H). MS-ESI calcd for [M ⁺ H] + 221, found 221.

the fourth step.

3-(1,1,1-Trifluoro-2-hydroxypropan-2-yl)benzyl methanesulfonate.

1,1,1-Trifluoro-2-(3-hydroxymethyl)phenyl)propan-2-ol (400 mg, 1.82 mmol) and triethylamine (275 mg, 2.72 mmol) were dissolved in dichloromethane (20 mL), the reaction solution was added with methanesulfonium chloride (250 mg, 2.18 mmol) at 0 ° C, stirred for 2 hours, diluted with dichloromethane (30 mL), and washed with water (20 mL x 2) Drying over anhydrous sodium sulfate, filtration, EtOAcqqqHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH Yield: 92%.

MS-ESI calcd [M + H] ⁺ 299.

the fifth step.

3,7-Dimethyl-1-(3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzyl)-1 H -indole-2,6-(3 H ,7 H )-dione.

3-(1,1,1-Trifluoro-2-hydroxypropan-2-yl)benzyl mesylate (100 mg, 0.335 mmol) and 3,7-dimethyl-1 H -indole-2 ,6-(3 H ,7 H )-dione (60.0 mg, 0.335 mmol) was dissolved in N , N -dimethylformamide (20 mL), and potassium carbonate (70.0 mg, 0.502) Methyl) and potassium iodide (6.00 mg, 0.0335 mmol), heated at 100 ° C for 2 hours, the reaction mixture was cooled and concentrated, diluted with ethyl acetate (30 mL), and the organic phase was washed with water (20 mL? Filtered and the filtrate was concentrated under reduced pressure. Purification by high performance liquid chromatography gave 3,7-dimethyl-1-(3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzyl)-1 H -indole-2. 6-( 3H , 7H )-Dione (30.0 mg, white solid), yield: 23%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.90 (s, 1H), 7.70 (s, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.38-7.32 (m, 2H), 5.21. s, 2H), 4.00 (s, 3H), 3.55 (s, 3H), 1.71 (s, 3H). MS-ESI calcd [M + H] ⁺ 383.

Example 27.

3,7-Dimethyl-1-(4-(1,1,1-trifluoro-2-hydroxypropyl-2-yl)phenyl)-1 H -嘌呤-2,6(3 H ,7 H )-dione.

first step.

Methyl 4-(1,1,1-trifluoro-2-hydroxypropyl-2-yl)benzoate.

Methyl 4-ethylmercaptobenzoate (10.0 g, 56.1 mmol) and trimethyl(trifluoromethyl)decane (16.0 g, 112 mmol) were dissolved in anhydrous tetrahydrofuran (150 mL). Tetrabutylammonium fluoride (22.0 g, 84.2 mmol) was slowly added dropwise. The reaction was slowly warmed to room temperature and stirred overnight. The reaction was quenched by the addition of water (50 mL). Extract with ethyl acetate (50 mL x 3). The organic phase was combined, washed with aq. Methyl benzoate (7.00 g, yellow liquid), yield: 50%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 8.04 (d, J = 8.0 Hz, 2H), 7.68 (d, J = 8.0 Hz, 2H), 3.92 (s, 3H), 3.27 (s, 1H) , 1.80 (s, 3H). MS-ESI calcd [M + H] ⁺ 495.

The second step.

1,1,1-Trifluoro-2-(4-(hydroxymethyl)phenyl)propyl-2-ol.

Lithium aluminum hydride (1.61 g, 42.3 mmol) was slowly added to methyl 4-(1,1,1-trifluoro-2-hydroxypropyl-2-yl)benzoate (7.00 g) at 0 ° C under nitrogen. , 28.2 mmol) in tetrahydrofuran (150 mL). The reaction solution was stirred at 0 ° C for 3 hours. At 0 ° C, water (1.60 mL), 15% sodium hydroxide solution (1.60 mL) and water (4.80 mL) were added slowly. Filtration and concentration of the filtrate under reduced pressure gave the product 1,1,1-trifluoro-2-(4-(hydroxymethyl)phenyl)propyl-2-ol (2.40 g,yel.). ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.55 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 4.66 (s, 2H), 3.37 (s, 1H) , 2.39 (s, 1H), 1.75 (s, 3H). MS-ESI calcd for [M ⁺ H] + 221, found 221.

third step.

4-(1,1,1-Trifluoro-2-hydroxypropyl-2-yl)phenyl methanesulfonate.

1,1,1-Trifluoro-2-(4-(hydroxymethyl)phenyl)propyl-2-ol (5.80 g, 26.3 mmol) and diisopropylethylamine (10.2 g, 79.0 mmol) Dissolved in dichloromethane (80 mL) and slowly added methanesulfonium chloride (4.53 g, 39.5 mmol) at 0 °C. The reaction solution was stirred at 0 ° C for 0.5 hour. The reaction was quenched with EtOAc (EtOAc)EtOAcEtOAc The combined organic layers were washed with EtOAc EtOAc EtOAc EtOAc (HHHHHHHHHHH The product 4-(1,1,1-trifluoro-2-hydroxypropyl-2-yl)phenyl methanesulfonate (3.45 g, m. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.66 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 5.26 (s, 2H), 2.96 (s, 3H) , 2.84 (s, 1H), 1.80 (s, 3H). MS-ESI calcd [M + H] ⁺ 299.

the fourth step.

3,7-Dimethyl-1-(4-(1,1,1-trifluoro-2-hydroxypropyl-2-yl)phenyl)-1 H -嘌呤-2,6(3 H ,7 H )-dione.

4-(1,1,1-Trifluoro-2-hydroxypropyl-2-yl)phenyl methanesulfonate (1.95 g, 10.8 mmol), 3,7-dimethyl- 1H -indole- 2,6(3 H ,7 H )-dione (652 mg, 3.62 mmol) and potassium carbonate (2.99 g, 21.6 mmol) and potassium iodide (180 mg, 1.08 mmol) dissolved in N , N -dimethylformamidine Amine (30 mL). The reaction solution was heated to 130 ° C and stirred for 3 hours. The reaction solution was cooled to room temperature and brine (20 mL) The organic phase was combined, dried over anhydrous sodium sulfate, filtered, filtered, evaporated, evaporated, evaporated,,,,,,,,,,,,,,,, -(4-(1,1,1-Trifluoro-2-hydroxypropyl-2-yl)phenyl)-1 H -indole-2,6(3 H ,7 H )-dione (1.27 g, White solid), Yield: 31%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.57-7.55 (m, 5H), 5.20 (s, 2H), 3.99 (s, 3H), 3.58 (s, 3H), 2.60 (s, 1H), 1.74 (s, 3H). MS-ESI calcd [M + H] ⁺ 383.

Example 28.

first step.

6-bromonicotinic acid methyl ester.

6-Bromonicotinic acid (1.00 g, 4.95 mmol) was dissolved in N , N -dimethylformamide (30 mL), iodomethane (0.703 g, 4.95 mmol) and potassium carbonate (1.03 g, 7.43 mmol) . The reaction solution was stirred at 20 ° C for 12 hours. The reaction mixture was diluted with water (100 mL), EtOAc (EtOAc (EtOAc)EtOAc. Ether/ethyl acetate, Rf = 0.5) gave 6-bromonicotinic acid methyl ester (1.00 g, white solid), yield: 94%. MS-ESI calculated [M + H] ⁺ 216 and 218, found 216 and 218.

The second step.

(6-Bromopyridin-3-yl)methanol.

Methyl 6-bromoinotinic acid (1.00 g, 4.63 mmol) was dissolved in tetrahydrofuran (20 mL). EtOAc (EtOAc, m. The reaction was quenched by the addition of water (10 mL). It was extracted with ethyl acetate (20 mL×3), dried over anhydrous sodium sulfate, filtered, evaporated, evaporated, evaporated. -Bromopyridin-3-yl)methanol (600 mg, yellow oil), yield: 69%. MS-ESI calculated [M + H] ⁺ 188 and 190, found 188 and 190.

third step.

(6-Bromopyridin-3-yl)methyl methanesulfonate.

(6-Bromopyridin-3-yl)methanol (1.00 g, 5.32 mmol) and triethylamine (1.18 g, 11.6 mmol) were dissolved in dichloromethane (20 mL). (1.38 g, 12.0 mmol). After the reaction mixture was stirred at room temperature for 2 hr, EtOAc EtOAc (EtOAc m. Separation and purification (4:1 petroleum ether / ethyl acetate, Rf = 0.5) gave (6-bromopyridin-3-yl)methyl methanesulfonate (1.20 g, colorless oil), yield: 85% . MS-ESI calculated [M + H] ⁺ 266 and 268, found 266 and 268.

the fourth step.

1-((6-Bromopyridin-3-yl)methyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione.

(6-Bromopyridin-3-yl)methyl methanesulfonate (500 mg, 1.88 mmol) was dissolved in N , N -dimethylformamide (20 mL). 3,7-Dimethyl-1 H -indole-2,6(3 H ,7 H )-dione (338 mg, 1.88 mmol), potassium carbonate (389 mg, 2.82 mmol) and potassium iodide (184 mg, 1.11) Mm). The reaction mixture was heated to 100 ° C, and the reaction was stirred for 2 hr. EtOAc (EtOAc) (EtOAc) Separation and purification by column chromatography (1:1 petroleum ether / ethyl acetate, Rf = 0.3) gave 1-((6-bromopyridin-3-yl)methyl)-3,7-dimethyl-1 H - purine -2,6 (3 H, 7 H) - dione (300 mg, yellow solid), yield: 46%. MS-ESI calculated [M + H] ⁺ 350 and 352, found 350 and 352.

the fifth step.

1-((6-Ethylpyridin-3-yl)methyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione.

1 - ((6-bromo-3-yl) methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (2.00 g, 5.71 mmol Soluble in 1,4-dioxane (50 mL), the reaction solution was added tributyl(1-ethoxyvinyl)stannane (8.25 g, 22.8 mmol) and tetratriphenyl at room temperature. Palladium phosphine (329 mg, 0.285 mmol). The reaction mixture was heated to 120 ° C and stirred for 2 hours. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (70 mL), washed with saturated sodium hydrogen sulfate (20 mL) (30 mL? , concentrated, and purified by silica gel column chromatography (3:1 petroleum ether / ethyl acetate, Rf = 0.3) to give 1-((6-ethylpyridin-3-yl)methyl)-3,7- dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (1.00 g, yellow solid), yield: 56%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 8.83 (s, 1H), 8.00-7.98 (m, 1H), 7.95-7.93 (m, 1H), 7.54 (s, 1H), 5.27 (s, 2H) ), 4.01 (s, 3H), 3.59 (s, 3H), 2.71 (s, 3H). MS-ESI calcd for [M + H] ⁺ 314.

The sixth step.

3,7-Dimethyl-1-((6-(1,1,1-trifluoro-2-hydroxypropan-2-yl)pyridin-3-yl)methyl)-1 H -indole-2, 6(3 H ,7 H )-dione.

1-((6-Ethylpyridin-3-yl)methyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione (150 mg, 0.478 mmol) was dissolved in tetrahydrofuran (30 mL). Trifluoromethyltrimethyldecane (102 mg, 0.718 mmol) and yttrium fluoride (73.0 mg, 0.478 mmol) were added at room temperature. The reaction was stirred at room temperature for 12 hrs, then EtOAc (50.0 mg, EtOAc, EtOAc) x 2) Washing, drying over anhydrous sodium sulfate, filtering, and concentrating the filtrate under reduced pressure and purifying by HPLC to give 3,7-dimethyl-1-((6-(1,1,1-trifluoro-2-hydroxy) propan-2-yl) pyridin-3-yl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (50 mg, white solid), yield: 27%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 8.76 (s, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.53 (s, 1H), 7.44 (d, J = 8.0 Hz, 1H) , 5.23 (s, 2H), 3.99 (s, 3H), 3.58 (s, 3H), 1.68 (s, 3H). MS-ESI calcd for [M + H] ⁺ 384.

Example 29.

3,7-Dimethyl-1-[[5-(2,2,2-trifluoro-1-hydroxy-1-methyl-ethyl)-2-pyridyl]methyl]indole-2,6 - Diketone.

first step.

1-[6-(Bromomethyl)-3-pyridyl]ethanone.

1-(6-Methyl-3-pyridyl)ethanone (500 mg, 3.70 mmol), N -bromosuccinimide (658 mg, 3.70 mmol), azobisisobutyronitrile (182 mg) , 1.11 mmol) was dissolved in carbon tetrachloride (20 mL) and reacted at 90 ° C for 12 hours. The reaction was quenched by the addition of saturated sodium thiosulfate solution (30 mL). Extracted with dichloromethane (10 mL x 3), dried over anhydrous sodium sulfate, filtered and evaporated. ), yield: 16%. MS-ESI calcd for [M ⁺ H] + 214,216, 214, 216 found.

The second step.

1-[(5-Ethyl-2-pyridyl)methyl]-3,7-dimethylindole-2,6-dione.

1-[6-(Bromomethyl)-3-pyridyl]ethanone (100 mg, 0.467 mmol), 3,7-dimethylindole-2,6-dione (84.2 mg, 0.467 mmol), Potassium iodide (7.70 mg, 0.0467 mmol) and potassium carbonate (194 mg, 1.40 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was warmed to 120 ° C and stirred for 3 hours. It was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure and purified by preparative TLC (ethyl acetate, Rf value = 0.3) to give 1-[(5-ethyl-purinyl-2-pyridyl)methyl]-3,7 - dimethylindole-2,6-dione (50.0 mg, yellow solid), yield: 34%. MS-ESI calcd for [M + H] ⁺ 314.

third step.

3,7-Dimethyl-1-[[5-(2,2,2-trifluoro-1-hydroxy-1-methyl-ethyl)-2-pyridyl]methyl]indole-2,6 - Diketone.

1-[(5-Ethyl-2-pyridyl)methyl]-3,7-dimethylindole-2,6-dione (50.0 mg, 0.159 mmol), cesium fluoride (24.2 mg, 0.159 mmol) was dissolved in tetrahydrofuran (10 mL). Trimethyl-trifluoromethyl-decane (113 mg, 0.798 mmol) was added at room temperature and stirred for 12 hours. The reaction was quenched by the addition of water (20 mL). Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Purification by preparative high performance liquid chromatography to give 3,7-dimethyl-1-[[5-(2,2,2-trifluoro-1-hydroxy-1-methyl-ethyl)-2-pyridine Methyl] fluorene-2,6-dione (10.0 mg, yellow solid), yield: 16%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.96 (s, 1H), 8.75 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.97 (s, 1H), 5.55 (s, 2H), 4.00 (s, 3H), 3.57 (s, 3H), 1.86 (s, 3H). MS-ESI calcd for [M + H] ⁺ 384.

Example 30.

3,7-Dimethyl-1-((5(1,1,1-trifluoro-2-hydroxypropan-2-yl)pyrazin-2-yl)methyl)-indole-2,6(3) H , 7 H )-dione.

first step.

N -methoxy- N ,5-dimethylpyrazine-2-carboxamide

5-Methylpyrazine-2-carboxylic acid (2.00 g, 14.5 mmol), 1-hydroxybenzotriazole (391 mg, 2.90 mmol), 1-(3-dimethylaminopropyl)-3-B The carbodiimide hydrochloride (1.69 g, 17.4 mmol) was dissolved in anhydrous dichloromethane (10 mL) and trichloromethane (30 mL). , 17.4 mmol), and the reaction solution was stirred at 25 ° C for 12 hours. The reaction was quenched by the addition of water (50 mL). The reaction mixture was extracted with EtOAc (EtOAc (EtOAc)EtOAc. Rf = 0.1) gave N -methoxy- N ,5-dimethylpyrazine-2-carboxamide (2.00 g, yellow oil). ¹ H NMR: (400 MHz, CDCl ₃ ) δ 8.80 (s, 1H), 8.45 (s, 1H), 3.73 (s, 3H), 3.40 (s, 3H), 2.61 (s, 3H).

The second step.

1-(5-methylpyrazin-2-yl)ethanone.

N -Methoxy- N ,5-dimethylpyrazine-2-carboxamide (1.50 g, 8.28 mmol) was dissolved in tetrahydrofuran (30 mL), and methyl magnesium bromide (3 M) was added dropwise at 0 °C. A solution of diethyl ether, 13.3 mL, 39.9 mmol) was then stirred at 25 ° C for one hour. The mixture was cooled to 0 ° C and quenched by water (10 mL). The mixture was extracted with EtOAc (30 mLEtOAc) The residue was purified by silica gel column chromatography (20:1 petroleum ether/ethyl acetate, Rf = 0.2) to give 1-(5-methylpyrazin-2-yl)ethyl (700 mg, yellow oil) Rate: 62%. MS-ESI calcd for [M + H] ⁺ 137.

third step.

1-(5(bromomethyl)pyrazin-2-yl)ethanone.

1-(5-Methylpyrazin-2-yl)ethyl (700 mg, 5.14 mmol) was dissolved in carbon tetrachloride (20 mL) then azobisisobutyronitrile (169 mg, 1.03 mmol) And N-bromosuccinimide (1.14 g, 6.43 mmol). The reaction solution was reacted under nitrogen at 100 ° C for 5 hours. The reaction mixture was directly filtrated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (20:1 petroleum ether/ethyl acetate, Rf = 0.5) to give 1-(5(bromomethyl)pyrazin-2-yl) (300 mg, yellow oil), yield: 27%. MS-ESI calculated [M + H] ⁺ 215 and 217, found 215 and 217.

the fourth step.

1-((5-Ethylpyrazin-2-yl)methyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione

1- (5 (bromomethyl) pyrazin-2-yl) acetate (300 mg, 1.40 mmol), 3,7- dimethyl -1 H - purine -2,6 (3 H, 7 H) - Diketone (251 mg, 1.40 mmol), potassium iodide (23.2 mg, 0.140 mmol) and potassium carbonate (578 mg, 4.19 mmol) were dissolved in anhydrous N , N -dimethylformamide (20 mL). The reaction solution was heated to 120 ° C and allowed to react for 3 hours. The reaction solution was cooled to 20 ° C, filtered, and the filtrate was evaporated, evaporated,jjjjjjjjjjjjjjjjjjjjj ) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (300 mg, yellow solid), yield: 68%. MS ESI calculated [M + H] ⁺ 315, found 315.

the fifth step.

3,7-Dimethyl-1-((5(1,1,1-trifluoro-2-hydroxypropan-2-yl)pyrazin-2-yl)methyl)-indole-2,6(3) H , 7 H )-dione.

1-((5-Ethylpyrazin-2-yl)methyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione (300 mg , 0.954 mmol), cesium fluoride (14.5 mg, 0.0954 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL) then trimethylsulfonium trifluoromethyl (407 mg, 2.86 mmol). The reaction solution was reacted under nitrogen at 25 ° C for 2 hours. Then hydrochloric acid (4 N, 4 mL) was added. The mixture was reacted for 1 hour under a nitrogen atmosphere at room temperature. The reaction was quenched with EtOAc (EtOAc) (EtOAc (EtOAc) 1: 1 petroleum ether / ethyl acetate, Rf = 0.3) to give 3,7-dimethyl-1-((5(1,1,1-trifluoro-2-hydroxypropan-2-yl)pyrazine- 2- yl) methyl) - purin -2,6 (3 H, 7 H) - dione (100 mg, white solid), yield: 40%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.85 (s, 1H), 8.65 (s, 1H), 7.92 (s, 1H), 5.40 (s, 2H), 3.99 (s, 3H), 3.56 (s, 3H), 1.78 (s, 3H). MS ESI calcd [M + H] ⁺ 385.

Example 31.

1-((3-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)isoxazole-5-yl)methyl)-3,7-dimethyl -1 H -嘌呤-2,6-(3 H ,7 H )-dione.

first step.

Methyl 5-(bromomethyl)isoxazol-3-carboxylate.

Ethyl methyl 5-methylisoxazole-3-carboxylate (5.00 g, 35.4 mmol), N -bromosuccinimide (6.31 g, 35.4 mmol), benzamidine peroxide (858 mg) , 3.54 mmol) was dissolved in carbon tetrachloride (20 mL) and reacted at 80 ° C for 12 hours. The reaction was quenched by the addition of saturated sodium thiosulfate solution (30 mL). It is extracted with dichloromethane (20 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure and purified by silica gel column chromatography (3:1 petroleum ether / ethyl acetate, Rf = 0.5) Ethyl 5-(bromomethyl)isoxazole-3-carboxylate (2.00 g, yellow oil), yield: 26%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 6.88 (s, 1H), 4.73 (s, 2H), 3.97 (s, 3H). MS-ESI calcd for [M + H] ⁺ 220, 222.

The second step.

Methyl 5-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl)isoxazole-3 - Ethyl carboxylate.

Ethyl 5-(bromomethyl)isoxazol-3-carboxylate (2.00 g, 9.09 mmol), 3,7-dimethyl- 1H -indole-2,6-( 3H , 7H ) Di-one (1.64 g, 9.09 mmol), potassium iodide (151 mg, 0.909 mmol) and potassium carbonate (2.51 g, 18.2 mmol) were dissolved in N , N -dimethylformamide (50 mL). The reaction solution was warmed to 120 ° C and stirred for 3 hours. After cooling to room temperature, filtration, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (ethyl acetate, Rf value = 0.4) to give methyl 5-((3,7-dimethyl-2,6-dioxo) Ethyl 2-(3,6,7-tetrahydro-1 H -indol-1-yl)methyl)isoxazole-3-carboxylate (1.70 g, yellow solid), yield: 59%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.06 (s, 1H), 6.82 (s, 1H), 5.22 (s, 2H), 3.87 (s, 3H), 3.83 (s, 3H), 3.45 (s, 3H). MS-ESI calcd for [M+H] ⁺ 320, found 320.

third step.

1-((3-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)isoxazole-5-yl)methyl)-3,7-dimethyl -1 H -嘌呤-2,6-(3 H ,7 H )-dione

Methyl 5-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)methyl)isoxazole- 3-carboxylic acid ethyl ester (200 mg, 0.626 mmol), cesium fluoride (95.0 mg, 0.626 mmol) dissolved in tetrahydrofuran (10 mL), trimethyltrifluoromethyl decane (445 mg, 3.13) Methyl), stirred for 12 hours. After adding 1 N hydrochloric acid (10 mL), the mixture was stirred at room temperature for 1 hour, and then the mixture was stirred and evaporated. Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with EtOAc EtOAc. Purification by preparative high performance liquid chromatography to give 1-((3-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)isoxazole-5-yl) -3,7-Dimethyl- 1H -indole-2,6-( 3H , 7H )-dione (10.0 mg, yellow solid), yield: 4%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.95 (s, 1H), 6.52 (s, 1H), 5.37 (s, 2H), 4.40 (s, 3H), 3.57 (s, 3H). MS-ESI calcd for [M ⁺ H] + 428, found 428.

Example 32.

3,7-Dimethyl-1-((3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)isoxazol-5-yl)methyl)-1 H -嘌呤- 2,6-(3 H ,7 H )-dione.

first step.

1-(5-Methylisoxazol-3-yl)ethanone.

Ethyl methyl 5-methylisoxazole-3-carboxylate (5.00 g, 35.4 mmol) and triethylamine (21.5 g, 213 mmol) were dissolved in tetrahydrofuran (80 mL). Magnesium bromide (3 M in diethyl ether, 35 mL, 105 mmol) was reacted for three hours. The reaction was quenched by the addition of saturated aqueous ammonium chloride (30 mL). It was extracted with ethyl acetate (30 mL×3), dried over anhydrous sodium sulfate, filtered, filtered, evaporated, evaporated, evaporated. (5-Methylisoxazol-3-yl)ethanone (1.00 g, yellow oil), yield: 23%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 6.39 (s, 1H), 2.58 (s, 3H), 2.49 (s, 3H). MS-ESI calcd [M+H] ⁺ 126, Found 126.

The second step.

1-(5-(Bromomethyl)isoxazol-3-yl)ethanone.

1-(5-Methylisoxazol-3-yl)ethanone (100 mg, 0.799 mmol), N -bromosuccinimide (142 mg, 0.799 mmol), benzamidine peroxide (19.3) Mg, 0.0800 mmol) was dissolved in carbon tetrachloride (10 mL) and reacted at 90 ° C for 12 hours. The reaction was quenched by the addition of saturated sodium thiosulfate solution (30 mL). Extracted with dichloromethane (10 mL x 3), dried over anhydrous sodium sulfate, filtered and evaporated. Shape), yield: 93%. MS-ESI calculated [M + H] ⁺ 204 and 206, found 204 and 206.

third step.

1-((3-Ethylisoxazole-5-yl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )-dione.

1-(5-(Bromomethyl)isoxazol-3-yl)ethanone (150 mg, 0.735 mmol), 3,7-dimethyl- 1H -indole-2,6-( 3H , 7 H )-Diketone (132 mg, 0.735 mmol), potassium iodide (61.0 mg, 0.367 mmol) and potassium carbonate (305 mg, 2.21 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was warmed to 120 ° C and stirred for 3 hours. It was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure and purified by preparative TLC (ethyl acetate, Rf value = 0.3) to give 1-((3-ethylhydrazylisoxazole-5-yl)methyl) -3,7-Dimethyl- 1H -indole-2,6-( 3H , 7H )-dione (50.0 mg, yellow solid), yield: 22%. MS-ESI calcd for [M + H] ⁺ 303.

the fourth step.

3,7-Dimethyl-1-((3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)isoxazol-5-yl)methyl)-1 H -嘌呤- 2,6-(3 H ,7 H )-dione

1-[(3-Ethylisoxazole-5-yl)methyl]-3,7-dimethylindole-2,6-dione (50.0 mg, 0.164 mmol), cesium fluoride (25.0 The solution was dissolved in tetrahydrofuran (10 mL). Trimethyltrifluoromethyl decane (70.3 mg, 0.494 mmol). After adding 1 N hydrochloric acid (10 mL), the mixture was stirred at room temperature for 1 hour, and then the mixture was stirred and evaporated. Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Purification by preparative high performance liquid chromatography to give 3,7-dimethyl-1-((3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)isoxazole-5-yl Methyl)-1 H -indole-2,6-( 3H , 7H )-dione (22.0 mg, yellow solid), yield: 36%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.98 (s, 1H), 6.48 (s, 1H), 5.33 (s, 2H), 4.01 (s, 3H), 3.57 (s, 3H), 1.71 (s, 3H). MS-ESI calcd [M + H] ⁺ 374.

Example 33.

3,7-Dimethyl-1-((2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl)methyl)-1 H -indole-2, 6-(3 H ,7 H )-dione.

first step.

1-(4-(Bromomethyl)thiazol-2-yl)ethanone.

1-(4-Methylthiazol-2-yl)ethanone (200 mg, 1.42 mmol), N -bromosuccinimide (252 mg, 1.42 mmol), azobisisobutyronitrile (46.6 mg , 0.284 mmol) was dissolved in carbon tetrachloride (20 mL) and reacted at 80 ° C for 12 hours. The reaction was quenched by the addition of saturated sodium thiosulfate solution (30 mL). Extracted with dichloromethane (10 mL x 3), dried over anhydrous sodium sulfate, filtered and evaporated. ), yield: 64%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.97 (s, 1H), 4.73 (s, 2H), 2.66 (s, 3H). MS-ESI calcd for [M + H] ⁺ 220, 222.

The second step.

1-((2-Ethylthiazol-4-yl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )-dione.

1-(4-(Bromomethyl)thiazol-2-yl)ethanone (100 mg, 0.454 mmol), 3,7-dimethyl- 1H -indole-2,6-( 3H , 7H )-Dione (81.9 mg, 0.454 mmol), potassium iodide (7.50 mg, 0.0454 mmol) and potassium carbonate (125 mg, 0.908 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was warmed to 120 ° C and stirred for 3 hours. It was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure and purified by preparative TLC (ethyl acetate, Rf = 0.3) to give 1-((2-ethylthiothiazol-4-yl)methyl)-3,7 -Dimethyl- 1H -indole-2,6-( 3H , 7H )-dione (80.0 mg, yellow solid), yield: 55%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.92 (s, 1H), 7.73 (s, 1H), 5.38 (s, 2H), 4.00 (s, 3H), 3.57 (s, 3H), 2.64(s, 3H). MS-ESI calcd for [M+H] ⁺ 320, found 320.

third step.

3,7-Dimethyl-1-((2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl)methyl)-1 H -indole-2, 6-(3 H ,7 H )-dione.

1-((2-Ethylthiazol-4-yl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )-dione (200 mg , 0.626 mmol), cesium fluoride (95.0 mg, 0.626 mmol) was dissolved in tetrahydrofuran (10 mL), and trimethyl-trifluoromethyl-decane (267 mg, 1.88 mmol) was added at room temperature and stirred for 12 hours. The reaction was quenched by the addition of water (20 mL). Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Purification by preparative high performance liquid chromatography to give 3,7-dimethyl-1-((2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl)- yl) -1 H - purine -2,6- (3 H, 7 H) - dione (100 mg, yellow solid), yield: 41%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.10 (s, 1H), 7.33 (s, 1H), 5.32 (s, 2H), 4.03 (s, 3H), 3.57 (s, 3H), 1.80 (s, 3H). MS-ESI calcd for [M + H] ⁺ 390.

Example 34.

3,7-Dimethyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl)methyl)-1 H -嘌呤-2,6-(3 H ,7 H )-dione.

first step.

1-(4-(Bromomethyl)-5-methylthiazol-2-yl)ethanone.

1-(4,5-Dimethylpyridin-2-yl)ethanone (200 mg, 1.29 mmol), N -bromosuccinimide (229 mg, 1.29 mmol), azobisisobutyronitrile (21.1 mg, 0.129 mmol) was dissolved in carbon tetrachloride (10 mL) and reacted at 80 ° C for 12 hours. The reaction was quenched by the addition of saturated sodium thiosulfate solution (30 mL). It was extracted with dichloromethane (10 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to give 1-(4-(bromomethyl)-5-methylthiazol-2-yl)ethanone (200) Mg, yellow oil), yield: 66%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 4.88 (s, 2H), 2.65 (s, 3H), 2.47 (s, 3H). MS-ESI calcd for [M+H] ⁺ 234, 236.

The second step.

1-((2-Ethyl-5-methylthiazol-4-yl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )- ketone.

1-(4-(Bromomethyl)-5-methylthiazol-2-yl)ethanone (200 mg, 0.854 mmol), 3,7-dimethyl- 1H -indole-2,6-( 3 H ,7 H )-dione (154 mg, 0.854 mmol), potassium iodide (14.0 mg, 0.0854 mmol) and potassium carbonate (354 mg, 2.56 mmol) dissolved in N , N -dimethylformamide (10 mL) )in. The reaction solution was warmed to 120 ° C and stirred for 3 hours. It was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure and purified by preparative TLC (ethyl acetate, Rf value = 0.3) to give 1-((2- ethinyl-5-methylthiazol-4-yl)methyl -3,7-Dimethyl- 1H -indole-2,6-( 3H , 7H )-dione (200 mg, yellow solid), yield: 70%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.90 (s, 1H), 5.35 (s, 2H), 4.00 (s, 3H), 3.55 (s, 3H), 2.66 (s, 3H), 2.61 (s, 3H). MS-ESI calcd for [M ⁺ H] + 334, found 334.

third step.

3,7-Dimethyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl)methyl)-1 H -嘌呤-2,6-(3 H ,7 H )-dione.

1-((2-Ethyl-5-methylthiazol-4-yl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )- Diketone (80.0 mg, 0.240 mmol), cesium fluoride (18.2 mg, 0.120 mmol) in tetrahydrofuran (10 mL), trimethyl-trifluoromethyl-decane (102 mg, 0.720 mmol) Stir for 12 hours. The reaction was quenched by the addition of water (20 mL). Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Purification by preparative high performance liquid chromatography to give 3,7-dimethyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazole- 4-yl)methyl)-1 H -indole-2,6-( 3H , 7H )-dione (35.0 mg, yellow solid), yield: 36%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.27 (s, 1H), 5.36 (s, 2H), 4.06 (s, 3H), 3.57 (s, 3H), 2.73 (s, 3H), 1.90 (s, 3H). MS-ESI calculated [M + H] ⁺ 404, found 404.

Example 35.

3,7-Dimethyl-1-((2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-5-yl)methyl)-1 H -indole-2, 6-(3 H ,7 H )-dione.

first step.

1-(5-methylthiazol-2-yl)ethylcyclohexanone.

5-Methylthiazole (2.00 g, 20.2 mmol) was dissolved in tetrahydrofuran (50 mL), and n-butyllithium (2.5 M tetrahydrofuran solution, 9.68 mL, 24.2 mmol) was slowly added dropwise at -78 °C under nitrogen. . The reaction mixture was stirred at -78 ° C for 0.5 hr, and N -methoxy- N -methylacetamide (2.50 g, 24.2 mmol) dissolved in THF (1 mL). The reaction solution was heated to 0 ° C and stirred for 1.5 hours. Water (10 mL) was slowly added to the reaction mixture at 0 ° C, and extracted with ethyl acetate (30 mL x 3). The organic phase was combined, dried over anhydrous sodium sulfate, filtered and evaporated evaporated evaporated. 2-yl)ethylcyclohexanone (1.45 g, yellow solid), yield: 51%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.73 (s, 1H), 2.61 (s, 3H), 2.57 (s, 3H). MS-ESI calcd for [M + H] ⁺ 422.

The second step.

1-(5-(Bromomethyl)thiazol-2-yl)ethylcyclohexanone.

1-(5-Methylthiazol-2-yl)ethylcyclohexanone (200 mg, 1.42 mmol) and azoisobutylidene (2.33 mg, 0.0142 mmol) were dissolved in chloroform (5 mL). Bromobutadiene imine (252 mg, 1.42 mmol) was added. The reaction solution was heated to 78 ° C and stirred for 16 hours. The reaction solution was cooled to room temperature, water (30 mL) was slowly added and extracted with chloroform (30 mL x 3). The combined organic layers were dried with EtOAc EtOAc EtOAcjHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH MS-ESI calculated [M + H] ⁺ 220 and 222, found 220 and 222.

third step.

1-((2-Ethylthiazol-5-yl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )-dione.

1-(5-(Bromomethyl)thiazol-2-yl)ethylcyclohexanone (290 mg, 1.05 mmol), 3,7-dimethyl-1H-indole-2,6-(3H,7 H) - dione (284 mg, 1.58 mmol) and potassium iodide (17.5 mg, 0.105 mmol) was dissolved in N, N - dimethylformamide (5 mL) was added potassium carbonate (437 mg, 3.16 mmol), The reaction was carried out at 130 ° C for 2.5 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was evaporated, evaporated, mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj -3,7-Dimethyl- 1H -indole-2,6-( 3H , 7H )-dione (292 mg, yellow solid), yield: 87%. MS-ESI calcd for [M+H] ⁺ 320, found 320.

the fourth step.

3,7-Dimethyl-1-((2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-5-yl)methyl)-1 H -indole-2, 6-(3 H ,7 H )-dione.

1-((2-Ethylthiazol-5-yl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )-dione (280 mg , 0.438 mmol) and cesium fluoride (6.66 mg, 0.0438 mmol) were dissolved in tetrahydrofuran (6 mL) and trifluoromethyltrimethyl decane (75.0 mg, 0.500 mmol) was slowly added under nitrogen. The reaction was stirred at 25 ° C for 1.5 hours. After adding 4 N aqueous hydrochloric acid (0.2 mL) and stirring at room temperature for half an hour, the pH was adjusted to 7 with saturated sodium bicarbonate (10 mL), water (20 mL) and ethyl acetate (50 mL x 3), organic The phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product which was purified by preparative HPLC to give the product 3,7-dimethyl-1-((2-(1,1,1-trifluoro-2-hydroxy) propan-2-yl) thiazol-5- yl) methyl) -1 H - purine -2,6- (3 H, 7 H) - dione (32.0 mg, white solid), yield: 19%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.89 (s, 1H), 7.82 (s, 1H), 5.35 (s, 2H), 4.00 (s, 3H), 3.56 (s, 3H), 1.76 (s, 3H). MS-ESI calcd for [M + H] ⁺ 390.

Example 36.

3,7-Dimethyl-1-(2-(4-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-5-yl)ethyl)- 1 H -嘌呤-2,6-(3 H ,7 H )-dione.

first step.

1-(5-(2-Hydroxyethyl)-4-methylthiazol-2-yl)ethanone.

2-(4-Methylthiazol-5-yl)ethanol (500 mg, 3.49 mmol) was dissolved in tetrahydrofuran (100 mL), and n-butyllithium (3 M n-hexane solution, 2.33 mL, 6.98 mmol), after half an hour of reaction, N -methoxy- N -methyl-acetamide (432 mg, 4.19 mmol) was added and stirring was continued for 3 hours. The reaction was quenched by the addition of saturated aqueous ammonium chloride (50 mL). Ethyl acetate extraction (10 mL x 3), dried over anhydrous sodium sulfate, filtered, evaporated, evaporated, evaporated, evaporated, (5-(2-Hydroxyethyl)-4-methylthiazol-2-yl)ethanone (200 mg, yellow oil), yield: 31%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 3.78 (t, J = 6.4 Hz, 2H), 3.18 (t, J = 6.4 Hz, 2H), 2.67 (s, 3H), 2.47 (s, 3H) . MS-ESI calcd [M + H] ⁺ 186.

The second step.

2-(2-Ethyl-4-methyl-thiazol-5-yl)ethyl methanesulfonate.

1-(5-(2-Hydroxyethyl)-4-methylthiazol-2-yl)ethanone (120 mg, 0.647 mmol) and triethylamine (196 mg, 1.94 mmol) were dissolved in dichloromethane. Methanesulfonyl chloride (148 mg, 1.30 mmol) was added at 0 °C in 10 mL). The reaction solution was slowly warmed to room temperature and stirred for 2 hours. The reaction was quenched by the addition of aqueous sodium bicarbonate (50 mL). Extract with dichloromethane (10 mL x 3). The organic phase was combined, washed with brine, dried over anhydrous sodium sulfate, filtered, evaporated, evaporated, evaporated. Ethyl 4-methyl-thiazol-5-yl)ethyl methanesulfonate (150 mg, yellow oil), yield: 88%. ¹ H NMR: (400 MHz, CDCl3) δ 4.41 (t, J = 6.4 Hz, 2H), 3.27 (t, J = 6.4 Hz, 2H), 3.01 (s, 3H), 2.67 (s, 3H), 2.46 (s, 3H). MS-ESI calcd [M + H] ⁺ 264.

third step.

1-(2-(2-Ethyl-4-methylthiazol-5-yl)ethyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H ) - Diketone.

2-(2-Ethyl-4-methyl-thiazol-5-yl)ethyl methanesulfonate (150 mg, 0.569 mmol), 3,7-dimethyl- 1H -indole-2, 6-(3 H ,7 H )-dione (102 mg, 0.569 mmol), potassium iodide (18.9 mg, 0.114 mmol) and potassium carbonate (236 mg, 1.71 mmol) dissolved in N , N -dimethylformamide (10 mL). The reaction solution was warmed to 120 ° C and stirred for 3 hours. It was cooled to room temperature, filtered, and the filtrate was concentrated. Separation and purification by preparative TLC (ethyl acetate, Rf value = 0.5) afforded 1-(2-(2- ethanemethyl-4-methylthiazol-5-yl)ethyl)-3,7-dimethyl Base-1 H -indole-2,6-( 3H , 7H )-dione (30.0 mg, yellow solid), yield: 15%. MS-ESI calcd for [M ⁺ H] + 348, found 348.

the fourth step .

3,7-Dimethyl-1-(2-(4-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-5-yl)ethyl)- 1 H -嘌呤-2,6-(3 H ,7 H )-dione.

1-(2-(2-Ethyl-4-methylthiazol-5-yl)ethyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H - diketone (40.0 mg, 0.115 mmol), cesium fluoride (17.5 mg, 0.115 mmol) dissolved in tetrahydrofuran (10 mL), trimethyltrifluoromethyl decane (49.0 mg, 0.345 mmol) Stir for 12 hours. The reaction was quenched by the addition of water (20 mL). Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Purification by preparative high performance liquid chromatography to give 3,7-dimethyl-1-(2-(4-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)) thiazol-5-yl) ethyl) -1 H - purine -2,6- (3 H, 7 H) - dione (15.0 mg, yellow solid), yield: 31%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.37 (s, 1H), 4.25 (t, J = 6.4 Hz, 2H), 4.01 (s, 3H), 3.54 (s, 3H), 3.26 ( t, J = 6.4 Hz, 2H), 2.50 (s, 3H), 1.90 (s, 3H). MS-ESI calcd for [M ⁺ H] + 418, found 418.

Example 37.

1-(3-hydroxy-2-(hydroxymethyl)-2-methylpropyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )-dione .

3,7-Dimethyl-1-[(3-methyloxetan-3-yl)methyl]indole-2,6-dione (20.0 mg, 0.0757 mmol) was dissolved in 0.16% hydrochloric acid (0.5 mL), the reaction mixture was stirred at room temperature for 6 hours, and the pH was adjusted to 7 with a saturated aqueous sodium hydrogen carbonate aqueous solution and purified by high-performance liquid chromatography to give 1-(3-hydroxy-2-(hydroxymethyl) 2-Methylpropyl)-3,7-dimethyl- 1H -indole-2,6-( 3H , 7H )-dione (12.0 mg, white solid), yield: 56%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.58 (s, 1H), 4.25-3.94 (m, 7H), 3.62 (s, 3H), 3.35-3.26 (m, 2H), 3.25-3.14 (m, 2H), 1.01 (s, 3H). MS-ESI calcd for [M + H] ⁺ 283.

Example 38.

1-(2-(2-Hydroxy-2-methylcyclopropyl)ethyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione.

first step.

2-(2-(3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indolyl)ethoxy) acetate.

To a solution of sodium hydride (21.0 mg, 0.890 mmol) in N , N -dimethylformamide (10 mL), 1-(2-hydroxyethyl)-3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (100 mg, 0.446 mmol), the reaction was stirred at 25 ℃ 1 hour. Additional ethyl 2-bromoacetate (149 mg, 0.890 mmol) was added. The reaction solution was stirred for further 16 hours. The insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure, and purified by preparative high-performance liquid chromatography to give 2-(2-(3,7-dimethyl-2,6-dioxo-2,3,6,7- Hydrogen-1 H -indenyl)ethoxy)acetate (60.0 mg, white solid), yield: 43%.

MS-ESI calculated [M + H] ⁺ 311, found 311.

The second step.

1-(2-(2-Hydroxy-2-methylcyclopropyl)ethyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione.

2-(2-(3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indolyl)ethoxy)acetic acid at -78 °C A solution of the ester (100 mg, 0.322 mmol) in tetrahydrofuran (5 mL) was slowly added dropwise to a solution of methylmagnesium bromide (3M in tetrahydrofuran, 0.43 mL, 1.29 mmol). The reaction solution was stirred at -78 ° C for 2 hours. The reaction was quenched by the addition of saturated aqueous ammonium chloride (20 mL). The mixture was extracted with ethyl acetate (20 mL x 3). The organic phase was combined, concentrated under reduced pressure and purified by preparative HPLC to give 1-(2-(2-hydroxy-2-methylcyclopropyl)ethyl)-3,7-dimethyl-1 H - purine -2,6 (3 H, 7 H) - dione (40.0 mg, colorless oil). Yield: 42%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.88 (s, 1H), 4.23 (t, J = 5.8 Hz, 2H), 3.98 (s, 3H), 3.72 (t, J = 5.8 Hz, 2H), 3.53 (s, 3H), 3.32 (s, 2H), 1.13 (s, 6H). MS-ESI calcd for [M + H] ⁺ 296.

Example 39.

1- (2 - ((1-hydroxy-cyclobutyl) methoxy) ethyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

first step.

1-(Hydroxymethyl)cyclobutanol.

A solution of 1-hydroxycyclobutyric acid (1.16 g, 10.0 mmol) in tetrahydrofuran (10 mL) was added dropwise to a solution of lithium tetrahydroaluminum (1.52 g, 40.0 mmol) in tetrahydrofuran (30 mL). The reaction solution was heated to reflux and allowed to react for 1 hour. The reaction mixture was cooled to 25 ° C, EtOAc (EtOAc) (EtOAc) Cyclobutanol (0.800 g, colorless oil), yield: 80%.

The second step.

2-(3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)ethylmethanesulfonate.

To 2-(2-hydroxyethyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione (448 mg, 2.00 mmol) in dichlorobenzene at 0 °C Triethylamine (600 mg, 6.00 mmol) and methanesulfonium chloride (342 mg, 3.00 mmol) were added to methane (25 mL). The reaction solution was stirred at 0 ° C for 0.5 hours. The reaction was quenched with saturated aqueous sodium hydrogen sulfate (30 mL). The organic phase was washed with brine (20 mL EtOAc) -tetrahydro-1 H -indol-1-yl)ethyl methanesulfonate (650 mg, yellow oil), yield: 100%.

third step.

1- (2 - ((1-hydroxy-cyclobutyl) methoxy) ethyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

To the mixture 1-(hydroxymethyl)cyclobutanol (102 mg, 1.00 mmol) and 2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 Potassium carbonate (414 mg, 3.00 mmol) and potassium iodide were added to a solution of H -indol-1-yl)ethylmethanesulfonate (450 mg, 1.50 mmol) in N , N -dimethylformamide (5 mL). (16.0 mg, 0.100 mmol). The reaction solution was heated to 60 ° C and stirred overnight. It was then slowly cooled to room temperature and quenched with water (20 mL). The mixture was extracted with EtOAc (20 mL EtOAc)EtOAc. Filtration, concentration of the filtrate under reduced pressure, and purification by preparative high performance liquid chromatography to give 1-(2-((1-hydroxycyclobutyl)methoxy)ethyl)-3,7-dimethyl-1 H - purine -2,6 (3 H, 7 H) - dione (50.0 mg, white solid), yield: 16%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.88 (s, 1H), 4.57-4.59 (m, 2H), 4.21-4.24 (m, 2H), 3.98 (s, 3H), 3.80 (s, 2H), 3.54 (s, 3H), 2.07-1.95 (m, 4H), 1.52-1.54 (m, 2H). MS-ESI calcd [M+H] ⁺ 303.

Example 40.

(S) -1- (2 - ( (2- hydroxypropyl) amino) ethyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

first step.

1-(3-chloropropyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione

The 3,7-dimethyl -1 H - purine -2,6 (3H, 7H) - dione (1.00 g, 5.56 mmol) was dissolved in methanol (20 mL), was added 30% sodium methoxide (9.64 g, 49.9 mmol), the reaction solution was refluxed for 1 hour. Further, 1-bromo-2-chloroethane (47.2 g, 299 mmol) was added, and the mixture was stirred for 16 hr. The reaction was quenched with EtOAc (EtOAc (EtOAc)EtOAc. purification chromatography (1: 2 petroleum ether / ethyl acetate) to give 1- (3-chloropropyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) -Dione (230 mg, white solid), Yield: 17%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.50 (s, 1H), 4.36 (t, J = 6.4 Hz, 2H), 3.97 (s, 3H), 3.75 (t, J = 6.4 Hz, 2H) , 3.56 (s, 3H).

The second step.

(S) -1- (2 - ( (2- hydroxypropyl) amino) ethyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

To the mixture 1-(3-chloropropyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )-dione (62.4 mg, 0.826 mmol) at 25 °C Potassium carbonate (138 mg, 1.03 mmol) and potassium iodide (86.3 mg, 0.517 mmol) were added to a solution of ( S )-1-aminopropan-2-ol (50.0 mg, 0.207 mmol) in EtOAc (2 mL). The reaction solution was stirred at 90 ° C for 4 hours. The reaction was quenched with water (10 mL)EtOAcEtOAcEtOAc The organic layer was washed with brine (20 mL EtOAc) Separation and purification by preparative high performance liquid chromatography gave ( S )-1-(2-((2-hydroxypropyl)amino)ethyl)-3,7-dimethyl-1 H -indole-2,6 (3) H , 7H )-dione (10.0 mg, white solid), yield: 17%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.86 (s, 1H), 4.16 (t, J = 6.4 Hz, 2H), 3.97 (s, 3H), 3.84 (m, 1H), 3.56 (s, 3H), 2.93 (m, 2H), 2.64 (m, 2H), 1.14 (d, J = 6.4 Hz, 3H). MS-ESI calcd for [M + H] ⁺ 282.

Example 41.

first step.

1-(1,4-Dioxaspiro[4.5]decane-8-ylmethyl)-7-(difluoromethyl)-3-methyl-1 H -indole-2,6( 3H , 7 H )-dione.

1-(1,4-Dioxaspiro[4.5]decane-8-ylmethyl)-9-(difluoromethyl)-3-methyl-1 H -indole-2,6(3 H , 7 H )-dione.

7-(Difluoromethyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione and 9-(difluoromethyl)-3-methyl-1 H - purine -2,6 (3 H, 7 H) - dione mixture (200 mg, 0.930 mmol) was dissolved in N, N - dimethylformamide (20 mL), and the reaction solution under a room temperature Add 1,4-dioxaspiro[4.5]decane-8-ylmethyl methanesulfonate (245 mg, 1.10 mmol), potassium iodide (183 mg, 1.10 mmol) and potassium carbonate (303 mg, 2.20 mmol) . The reaction solution was heated to 100 ° C and stirred for 2 hours. The reaction mixture was diluted with ethyl acetate (30 mL). EtOAc (EtOAc m. Methyl)-7-(difluoromethyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione and 1-(1,4-dioxaspiro[ 4.5] a mixture of decane-8-ylmethyl)-9-(difluoromethyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione (234 mg, Yellow oil), Yield: 68%.

MS-ESI calcd for [M + H] ⁺ 372.

The second step.

7-(Difluoromethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione.

9-(Difluoromethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione.

1-(1,4-Dioxaspiro[4.5]decane-8-ylmethyl)-7-(difluoromethyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione, 1-(1,4-dioxaspiro[4.5]decane-8-ylmethyl)-9-(difluoromethyl)-3-methyl-1 H -indole a mixture of -2,6(3 H ,7 H )-dione (230 mg, 0.750 mmol) was dissolved in tetrahydrofuran (15 mL), 10% hydrochloric acid (5 mL) was added at room temperature, and the mixture was heated to 50 Stir at °C for 1 hour. After cooling to room temperature, it was diluted with ethyl acetate (20 mL). EtOAc (EtOAc m. (2:1 petroleum ether / ethyl acetate, Rf = 0.3) afforded 7-(difluoromethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H - oxime-2 ,6(3 H ,7 H )-dione,9-(difluoromethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -嘌呤-2,6( Mixture of 3 H , 7 H )-dione (200 mg, white solid), yield: 81%.

MS-ESI calcd for [M + H] ⁺ 327.

third step.

7-(Difluoromethyl)-1-(4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione.

9-(Difluoromethyl)-1-(4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione.

7-(Difluoromethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione, 9- a mixture of (difluoromethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione (168 mg, 0.515 mmol) was dissolved in tetrahydrofuran (30 mL) and trifluoromethyltrimethyldecane (109 mg, 0.773 mmol) and cesium fluoride (15.7 mg, 0.103 mmol) were added at room temperature. The reaction was stirred at room temperature for 12 hrs, then EtOAc (50.0 mg, EtOAc, EtOAc) x 2) Washing, drying over anhydrous sodium sulfate, filtration, and concentration of the filtrate under reduced pressure and purified by high-performance liquid chromatography to give 7-(difluoromethyl)-1-(4-hydroxy-4-(trifluoromethyl)cyclohexyl) Methyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione (54 mg, white solid), yield: 23%, ¹ H NMR: (400 MHz, Methanol _{- d 4) δ8.46 (s,} 1H), 7.89-7.74 (m, 1H), 4.06 (d, J = 7.2 Hz, 2H), 3.59 (s, 3H), 2.19-2.17 (m, 1H), 2.05-1.99 (m, 2H), 1.88-1.81 (m, 2H), 1.61-1.58 (m, 2H), 1.51-1.47 (m, 2H). MS-ESI calcd [M + H] ⁺ 397.

9- (difluoromethyl) -1- (4-hydroxy-4- (trifluoromethyl) cyclohexyl) methyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H )-Dione (12 mg, white solid), Yield: 10%. ¹ H NMR: (400 MHz, Methanol- d ₄ ) δ 8.49 (s, 1H), 7.93-7.89 (m, 1H), 4.07 (d, J = 7.2 Hz, 2H), 3.59 (s, 3H), 2.20-2.19 (m, 1H), 2.05-1.99 (m, 2H), 1.88-1.85 (m, 2H), 1.61-1.58 (m, 2H), 1.51-1.48 (m, 2H). MS-ESI calcd [M + H] ⁺ 397.

Example 42.

7-Ethyl-1-(5-ethyl-5-hydroxyheptyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione.

first step.

7-Ethyl-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione.

3-Methyl-1 H -嘌呤-2,6(3 H ,7 H )-dione (500 mg, 3.00 mmol), potassium carbonate (414 mg, 3.00 mmol) and potassium iodide (4.0 mg, 0.300 mmol) Dissolved in N , N -dimethylformamide (15 mL). The reaction solution was heated to 80 ° C for half an hour. Iodoethane (470 mg, 4.50 mmol) was added. Continue the reaction for 5 hours. The reaction mixture was poured into aq. EtOAc (50 mL). The aqueous phase was adjusted to pH to pH 7 with 1 N dilute hydrochloric acid (10 mL), filtered, and the filter cake was dried to give 7-ethyl-3-methyl -1 H - purine -2,6 (3 H, 7 H )-dione (500 mg, pale yellow solid), yield: 86%. ¹ H NMR: (400 MHz, DMSO- d ₆ ): δ 8.05 (s, 1H), 4.25 - 4.19 (m, 2H), 3.34 (s, 3H), 1.37 (t, J = 7.2 Hz, 3H) . MS-ESI calculated [M + H] ⁺ 195, found 195.

The second step.

Ethyl 5-(7-ethyl-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)pentanoic acid ethyl ester.

7-Ethyl-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione (0.300 g, 1.55 mmol), ethyl bromopentanoate (480 mg, 2.32 mmol), Potassium carbonate (430 mg, 3.10 mmol) and potassium iodide (26.0 mg, 0.155 mmol) were dissolved in N , N -dimethylformamide (4 mL). The reaction solution was heated to 110 ° C for 2 hours. The reaction was poured into water (20 mL). The combined organic layers were dried with anhydrous sodium sulfate, filtered and evaporated Ethyl 1 H -indol-1-yl)pentanoate (320 mg, yellow solid), yield: 62%. MS-ESI calcd [M + H] ⁺ 323.

third step.

7-Ethyl-1-(5-ethyl-5-hydroxyheptyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione.

Ethyl 5-(7-ethyl-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)pentanoic acid ethyl ester (0.100 g, 0.310 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL). EtOAc (3 M THF, EtOAc (EtOAc) The reaction solution was reacted at -78 ° C for 0.5 hour, and slowly raised to 0 ° C for 0.5 hour. After the reaction was completed, the mixture was poured into water (20 mL) The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, to give 7-ethyl-1- (5-ethyl-5-hydroxy-hept-yl) -1 H -3-methyl-purified by column chromatography with silica gel - purin - 2,6( 3H , 7H )-dione (30.0 mg, colorless oil), yield: 30%.

¹ H NMR: (400 MHz, CDCl ₃ ): δ 7.56 (s, 1H), 4.37 - 4.32 (m, 2H), 4.05 (t, J = 7.2 Hz, 2H), 3.60 (s, 3H), 1.68 –1.37 (m, 13H), 0.86 (t, J = 7.2 Hz, 6H). MS-ESI calcd for [M ⁺ H] + 337, found 337.

Example 43.

7-Ethyl-3-methyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -indole-2,6(3 H ,7 H )- ketone.

first step.

7-Ethyl-3-methyl-1-(5-oxohexyl)-1 H -indole-2,6(3 H ,7 H )-dione

7-Ethyl-3-methyl-1H-indole-2,6(3 H ,7 H )-dione (0.100 g, 0.515 mmol), 6-chloro-2-pentanone (90.0 mg, 0.670 mmol) ), potassium carbonate (140 mg, 1.03 mmol) and potassium iodide (8.5 mg, 0.0155 mmol) was dissolved in DMF (2 mL), the reaction was heated to 110 ^o C for two hours. The reaction was poured into water and extracted with ethyl acetate (20 mL x 3). The organic phases were combined, dried, filtered, concentrated and washed with tert.butyl methyl ether, the solid was dried to give the title compound 7-ethyl-3-methyl-1- (5-oxo-hexyl) -1 H - purin-2,6 ( 3 H , 7 H )-dione (100 mg, white solid), yield: 70%. MS-ESI calcd for [M ⁺ H] + 293, found 293.

The second step.

7-Ethyl-3-methyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -indole-2,6(3 H ,7 H )- ketone.

Dissolve 7-ethyl-3-methyl-1-(5-oxohexyl)-1 H -indole-2,6(3 H ,7 H )-dione (100 mg, 0.340 mmol) in 1 mL tetrahydrofuran, were added trifluoromethyltrimethylsilane Silane (53.0 mg, 0.370 mmol) and cesium fluoride (10.0 mg, 0.0340 mmol), the reaction at 30 ^o C 3 hours. The reaction solution was poured into dilute hydrochloric acid (10%, 10 mL) and stirred for half an hour. Extract with ethyl acetate (20 mL x 3). The organic phase was combined, dried and concentrated, and the title compound was purified by preparative chromatography column 7-ethyl-3-methyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)- 1 H - purine -2,6 (3 H, 7 H) - dione (20.0 mg, white solid), yield: 79%. ¹ H NMR: (400MHz, CDCl ₃ ): δ 7.95 (s, 1H), 4.39 - 4.33 (m, 2H), 4.04 - 4.00 (m, 2H), 3.53 (s, 3H), 1.71 - 1.64 (m) , 4H), 1.50–1.46 (m, 5H), 1.28 (s, 3H). MS-ESI calcd for [M + H] ⁺ 363.

Example 44.

1-((4-Hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-7-(2,2,2-trifluoroethyl)-1 H -indole-2, 6(3 H ,7 H )-dione.

first step.

1-(1,4-Dioxaspiro[4,5]decane-8-ylmethyl)-3-methyl-7-(2,2,2-trifluoroethyl)-1 H -嘌呤-2,6(3 H ,7 H )-dione

1,4-Dioxaspiro[4,5]nonane-8-ylmethyl methanesulfonate (200 mg, 0.800 mmol), 3-methyl-7-(2,2,2-trifluoro Ethyl)-1 H -嘌呤-2,6(3 H ,7 H )-dione (200 g, 0.800 mmol) and potassium carbonate (334 mg, 2.42 mmol), potassium iodide (14.0 mg, 0.0800 mmol) In N , N -dimethylformamide (3 mL), the reaction solution was heated to 130 ° C and stirred for 3.5 hours. The reaction solution was filtrated, and the filtrate was concentrated under reduced pressure to obtain a crude product of 1- (1,4-dioxaspiro [4,5] decan-8-yl-methyl) -3,7-dimethyl -1 H -嘌呤-2,6(3 H ,7 H )-dione. MS-ESI calcd [M + H] ⁺ 403.

The second step.

3-methyl-1-((4-oxocyclohexyl)methyl)-7-(2,2,2-trifluoroethyl)-1 H -indole-2,6(3 H ,7 H ) - Diketone.

1-(1,4-Dioxaspiro[4,5]decane-8-ylmethyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H ) The diketone (2.50 g, 6.00 mmol) was dissolved in acetone (18 mL) and aqueous hydrochloric acid (4 N, 2.5 mL). The reaction was stirred at 30 ° C overnight, EtOAc (EtOAc)EtOAc. The organic phase was combined, dried over anhydrous sodium sulfate and filtered and evaporated. Oxocyclohexyl)methyl)-7-(2,2,2-trifluoroethyl)-1 H -indole-2,6(3 H ,7 H )-dione (220 mg, white solid) Yield: 11%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.68 (s, 1H), 5.08 - 4.99 (m, 2H), 4.00 (d, J = 7.0 Hz, 2H), 3.61 (s, 3H), 2.46- 2.24 (m, 5H), 2.04-1.96 (m, 2H), 1.63-1.56 (m, 2H). MS-ESI calcd [M + H] ⁺ 359.

third step.

1-((4-Hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-7-(2,2,2-trifluoroethyl)-1 H -indole-2, 6(3 H ,7 H )-dione.

3-Methyl-1-((4-oxocyclohexyl)methyl)-7-(2,2,2-trifluoroethyl)-1 H -嘌呤-2,6(3 H ,7 H )-dione (128 mg, 0.360 mmol) and cesium fluoride (6.0 mg, 0.0360 mmol) dissolved in tetrahydrofuran (3 mL), slowly added trifluoromethyltrimethyl decane (77.0 mg, 0.540) under nitrogen. Mm). The reaction solution was stirred at 30 ° C for 3 hours. After cooling to room temperature, 4 N aqueous hydrochloric acid (2.5 mL) was added and stirred at 25 ° C for half an hour, pH was adjusted to 7, diluted with water and extracted with ethyl acetate (20 mL x 3). The organic phase was combined, dried over anhydrous sodium sulfate, filtered and evaporated methyl-7- (2,2,2-trifluoroethyl) -1 H - purine -2,6 (3 H, 7 H) - dione (14.0 mg, white solid), yield: 10%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 8.09 (s, 1H), 5.27-5.20 (m, 2H), 4.08-3.91 (m, 2H), 3.58 (s, 3H), 2.07-1.98 (m) , 2H), 1.89-1.80 (m, 2H), 1.62-1.46 (m, 5H). MS-ESI calcd [M + H] ⁺ 422.

Example 45.

1-((4-Hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-7-(2,2,2-trifluoroethyl)-1 H -indole-2, 6( 3H , 7H )-dione (500 mg, 1.17 mmol) was isolated by preparative SFC to afford two isomers. Separation conditions: column: AD 250 mm x 30 mm, 10 um mobile phase: A: supercritical carbon dioxide, B: ethanol (0.05% aqueous ammonia), A: B = 550: 45 flow rate: 80 mL/min wavelength: 220 nm.

Product 1 (isomer 1, first peak) (300 mg, white solid), yield: 90%. ¹ H NMR: (400 MHz, DMSO- d ₆ ) δ 8.23 (s, 1H), 5.64 (s, 1H), 5.31-5.24 (m, 2H), 3.89 (d, J = 3.6 Hz, 2H), 3.43 (s, 3H), 2.06-2.05 (m, 1H), 1.87-1.81 (m, 2H), 1.73-1.61 (m, 2H), 1.49-1.45 (m, 2H), 1.33-1.31 (m, 2H) ).MS ESI calc'd.[M + H] ⁺ 429,found 429.

Product 2 (isomer 2, second peak) (150 mg, white solid), yield 90%. ¹ H NMR: (400 MHz, DMSO- d ₆ ) δ 8.22 (s, 1H), 5.63 (s, 1H), 5.29-5.23 (m, 2H), 3.74 (d, J = 3.6 Hz, 2H), 3.42 (s, 3H), 1.68-1.66 (m, 3H), 1.45-1.31 (m, 6H). MS ESI calc'd. [M + H] ⁺ 429, found 429.

Example 46.

first step.

1-(1,4-Dioxaspiro[4,5]decane-8-ylmethyl)-3-methyl-7-(2,2,2-trifluoroethyl)-1 H -嘌呤-2,6-(3 H ,7 H )-dione.

1,4-Dioxaspiro[4,5]decane-8-ylmethyl methanesulfonate (603 mg, 2.41 mmol), 3-methyl-7-(2,2,2-trifluoro Ethyl)-1 H -嘌呤-2,6-(3 H ,7 H )-dione (500 mg, 2.01 mmol) and potassium iodide (33.3 mg, 0.201 mmol) dissolved in N , N -dimethylformamidine Potassium carbonate (555 mg, 4.02 mmol) was added to the amine (8 mL), and the mixture was heated and refluxed at 130 ° C for 4 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was evaporated evaporated. -(2,2,2-Trifluoroethyl)-1 H -indole-2,6-(3 H ,7 H )-dione (980 mg, yellow oil). MS-ESI calcd [M + H] ⁺ 403.

The second step.

3-methyl-1-((4-oxocyclohexyl)methyl)-7-(2,2,2-trifluoroethyl)-1 H -indole-2,6-(3 H ,7 H )-dione.

1-(1,4-Dioxaspiro[4,5]decane-8-ylmethyl)-3-methyl-7-(2,2,2-trifluoroethyl)-1 H - Indole-2,6-( 3H , 7H )-dione (980 mg, 1.51 mmol) was dissolved in EtOAc (EtOAc) (EtOAc) The reaction was stirred at room temperature overnight, EtOAc (EtOAc)EtOAc. 1:1 petroleum ether / ethyl acetate, Rf = 0.3) gave the product 3-methyl-1-((4-oxocyclohexyl)methyl)-7-(2,2,2-trifluoroethyl) -1 H - purine -2,6- (3 H, 7 H) - dione (78.0 mg, yellow solid), yield: 15%. MS-ESI calcd [M + H] ⁺ 359.

3-methyl-1-((4-oxocyclohexyl)methyl)-7-(2,2,2-trifluoroethyl)-1 H -嘌呤-2,6-(3 H ,7 H )-Diketone (66.0 mg, 0.184 mmol) was dissolved in tetrahydrofuran (2 mL). Methyld. reagent (3 M diethyl ether solution, 0.184 mL, 0.552 mmol) was slowly added at -78 ° C under nitrogen. The mixture was stirred at ° C for half an hour, followed by a reaction at 0 ° C for 2 hours. Add water (10 mL), extract with ethyl acetate (30 mL×3), dry over anhydrous sodium sulfate. , white solid), Yield: 12%, ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.08 (s, 1H), 5.27-5.19 (m, 2H), 3.92 (d, J = 7.2 Hz , 2H), 3.58 (s, 3H), 1.71-1.62 (m, 4H), 1.46-1.38 (m, 2 H), 1.32-1.18 (m, 6H). MS-ESI calcd for _{[M + H - H 2 O} ] + 357, found 357.

Product 2 (12.0 mg, white solid) (isomer 2, second peak), yield: 17%. ¹ H NMR: (400 MHz, Methonal-d4) δ 8.08 (s, 1H), 5.27-5.21 (m, 2H), 3.91 (d, J = 7.2 Hz, 2H), 3.57 (s, 3H), 1.69 -1.66 (m, 2H), 1.49-1.44 (m, 3H), 1.37-1.28 (m, 4H), 1.17 (s, 3H). MS-ESI calcd for _{[M + H - H 2 O} ] + 357, found 357.

Example 47.

7-Cyclopropyl-3-methyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -indole-2,6(3 H ,7 H )- Dione.

first step.

6-Amino-5-bromo-1-methylpyrimidine-2,4(1 H ,3 H )-dione.

Mixture 6-Amino-1-methylpyrimidine-2,4(1 H ,3 H )-dione (5.46 g, 40.0 mmol) and bromobutaneimide (7.56 g, 42.0 mmol) in acetonitrile (100) The mL) solution was heated to reflux under nitrogen for 1.5 hours. The reaction solution was cooled to room temperature, filtered, and the solvent was evaporated, and the obtained solid was washed with water (20 mL) and dried to give 6-amino-5-bromo-1-methylpyrimidine-2,4( 1H , 3H )-dione (8.6 g, white solid), Yield: 98%. ^{1 H NMR: (400 MHz,} DMSO- d6) δ10.90 (s, 1H), 7.04 (s, 2H), 3.28 (s, 3H).

The second step.

6-amino-5- (cyclopropylamine) -1-methyl-pyrimidine -2,4 (1 H, 3 H) - dione.

6-Amino-5-bromo-1-methylpyrimidine-2,4(1 H ,3 H )-dione (2.19 g, 10.0 mmol) dissolved in a mixture of cyclopropylamine (20 mL) and water (5 mL) In the solvent. The reaction solution was heated to reflux for 5 hours. The reaction solution was filtered to remove the solvent to give the crude product 6-amino-5-(cyclopropylamine)-1-methylpyrimidine-2,4( 1H , 3H )-dione.

third step.

7-Cyclopropyl-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione.

6-Amino-5-(cyclopropylamine)-1-methylpyrimidine-2,4(1 H ,3 H )-dione (1.96 g, 10.0 mmol), trimethyl orthoformate under N2 (2.12 g, 20.0 mmol) and p-toluenesulfonic acid (86.0 mg, 0.500 mmol) dissolved in anhydrous N , N -dimethylformamide (20 mL). The reaction solution was heated to 100 ° C overnight. The reaction was filtered, the solvent was removed to give crude product 7-cyclopropyl-3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione was used directly in the next reaction.

the fourth step.

7-Cyclopropyl-3-methyl-1-(5-oxohexane)-1 H -indole-2,6(3 H ,7 H )-dione.

7-Cyclopropyl-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione (100 mg, 0.480 mmol), 6-chlorohexane-2- under a nitrogen atmosphere Ketone (97.0 mg, 0.730 mmol) and potassium carbonate (132 mg, 0.960 mmol) in N , N -dimethylformamide (5 mL). The reaction solution was heated to 120 ° C for 3 hours. After the reaction was cooled to room temperature, it was diluted with water (20 mL) and ethyl acetate (10 mL), ethyl acetate (30 mL) Propyl-3-methyl-1-(5-oxohexane)-1 H -indole-2,6(3 H ,7 H )-dione was used directly in the next step. MS-ESI calcd [M + H] ⁺ 303.

the fifth step.

7-Cyclopropyl-3-methyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -indole-2,6(3 H ,7 H )- Dione.

7-Cyclopropyl-3-methyl-1-(5-oxohexane)-1 H -indole-2,6(3 H ,7 H )-dione (200 mg, 0.660 under nitrogen) Methyl acetate was dissolved in anhydrous tetrahydrofuran (3 mL), then trifluoromethyltrimethyldecane (0.2 mL, 0.990 mmol) and yttrium fluoride (20.0 mg, 0.130 mmol). The resulting reaction solution was reacted at 30 ° C for 2 hours. The reaction solution was then diluted with water (30 mL), EtOAc (EtOAc)EtOAc. Ester, Rf = 0.3) gives 7-cyclopropyl-3-methyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -indole-2,6 ( 3 H , 7 H )-dione (150 mg, white solid), yield: 61%. ¹ H NMR: (400 MHz, CDCl ₃ ) δ 7.55 (s, 1H), 4.13-4.02 (m, 2H), 3.63-3.61 (m, 1H), 3.55 (s, 3H), 2.96 (s, 1H) ), 1.90- 1.68 (m, 2H), 1.67-1.64 (m, 2H), 1.47-1.45 (m, 2H), 1.28 (s, 3H), 1.18-1.16 (m, 2H), 1.06-1.04 (m , 2H). MS-ESI calcd [M + H] ⁺ 372.

Example 48.

7-(Cyclopropylmethyl)-1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -indole-2,6( 3H , 7 H )-dione.

first step.

1-(1,4-Dioxaspiro[4.5]decane-8-ylmethyl)-7-isopropyl-3-methyl-1 H -indole-2,6(3 H ,7 H ) - Diketone.

1,4-Dioxaspiro[4.5]decane-8-ylmethyl methanesulfonate (250 mg, 1.00 mmol), 7-isopropyl-3-methyl- 1H -indole-2, 6(3 H ,7 H )-dione (208 mg, 1.00 mmol), potassium iodide (15.8 mg, 0.100 mmol) and potassium carbonate (276 mg, 2.00 mmol) dissolved in anhydrous N , N -dimethylformamide (8 mL). The reaction solution was heated to 120 ° C and stirred for 3 hours. The reaction was cooled to 20 ° C, and the mixture was filtered. -1 H - purine -2,6 (3 H, 7 H) - dione (300 mg, white oil), yield: 83%. MS-ESI calcd for [M + H] ⁺ 372.

The second step.

7-Isopropyl-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione

1-(1,4-Dioxaspiro[4.5]decane-8-ylmethyl)-7-isopropyl-3-methyl-1 H -indole-2,6(3 H ,7 H )-Dione (300 mg, 0.828 mmol) was dissolved in acetone (10 mL) and hydrochloric acid (0.5 mL) was added. The reaction was stirred at room temperature for 30 minutes. Water was added to the reaction mixture, and the mixture was stirred with EtOAc EtOAc EtOAc (EtOAc) The mixture was washed with anhydrous sodium sulfate and filtered, and the filtrate was evaporated, evaporated, evaporated,,,,,,,,,,,,,,,, oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (180 mg, white oil), yield: 68%. MS-ESI calcd for [M ⁺ H] + 319, found 319.

third step.

7-Isopropyl-3-methyl-1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )- Dione.

7-Isopropyl-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione (122 mg, 0.382 mmol And cesium fluoride (11.5 mg, 0.0763 mmol) were dissolved in anhydrous tetrahydrofuran (3 mL) and trifluoromethyltrimethyl decane (95.0 mg, 0.640 mmol). The reaction solution was heated to 30 ° C and stirred for 12 hours. Then aqueous hydrochloric acid (1 N, 5 mL) was added and stirred for additional 30 min. Water was added to the reaction mixture, and the mixture was stirred with EtOAc EtOAc EtOAc (EtOAc) Washing, drying over anhydrous sodium sulfate, filtration, concentration of the filtrate under reduced pressure, and purification by preparative high-performance liquid chromatography to give 7-isopropyl-3-methyl-1-((4-hydroxy-4-(trifluoro)) yl) cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (80.0 mg, white solid), yield: 53%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.10 (s, 1H), 5.06-5.00 (m, 1H), 4.08-3.91 (m, 2H), 3.55 (s, 3H), 2.17-2. (m, 2H), 1.88-1.84 (m, 2H), 1.61-1.40 (m, 6H), 1.59-1.57 (m, 5H). MS-ESI calcd [M + H] ⁺ </RTI>

Example 49.

7-(Cyclopropylmethyl)-1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -indole-2,6( 3H , 7 H )-dione.

first step.

1- (1,4-dioxaspiro [4.5] decan-8-ylmethyl) -7- (cyclopropylmethyl) -3-methyl -1 H - purin-2,6 (3 H, 7 H )-dione.

(cyclopropylmethyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione (220 mg, 1.00 mmol), 1,4-dioxospiro[4.5 ] decane-8-ylmethyl methanesulfonate (250 mg, 1.00 mmol), potassium iodide (15.8 mg, 0.100 mmol) and potassium carbonate (276 mg, 2.00 mmol) dissolved in anhydrous N , N -dimethyl In the guanamine (8 mL), the reaction solution was heated to 120 ° C and stirred for 3 hours. The reaction was cooled to 20 ° C, and the mixture was filtered and evaporated tolulululululululululululululululululu - methyl - -1 H - purine -2,6 (3 H, 7 H) - dione (300 mg, white oil), yield: 80%. MS-ESI calcd [M + H] ⁺ 372.

The second step.

7-(Cyclopropylmethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione.

1-(1,4-Dioxaspiro[4.5]decane-8-ylmethyl)-3,7-dimethyl-1 H -indole-2,6(3 H ,7 H )- The ketone (300 mg, 0.802 mmol) was dissolved in acetone (10 mL). Water (30 mL) was added to the reaction mixture, EtOAc (EtOAc m. Filtration and concentration of the filtrate under reduced pressure and purification by chromatography (ethyl acetate, Rf = 0.3) affords 7-(cyclopropylmethyl)-3-methyl-1-((4-oxocyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (180 mg, white oil), yield: 75%. MS-ESI calcd for [M ⁺ H] + 331, found 331.

third step.

7-(Cyclopropylmethyl)-1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -indole-2,6( 3H , 7 H )-dione.

7-(Cyclopropylmethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione (126 Mg, 0.382 mmol) and cesium fluoride (11.5 mg, 0.0763 mmol) were dissolved in anhydrous tetrahydrofuran (3 mL) and trifluoromethyltrimethyl decane (95.0 mg, 0.640 mmol). The mixture was stirred at 30 ° C for 12 hours. Then 1 N aqueous hydrochloric acid (5 mL) was added and stirring was continued for further 30 min. Water (30 mL) was added to the reaction mixture, EtOAc (EtOAc) (EtOAc) 30 mL x 2) Washed, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by preparative HPLC to give 7-(cyclopropylmethyl)-1-((4-hydroxy-4-( trifluoromethyl) cyclohexyl) methyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (80.0 mg, white solid), yield: 53%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.16-8.15 (m, 1H), 4.24-4.22 (m, 2H), 4.08-3.91 (m, 2H), 3.57 (s, 3H), 2.18- 2.07 (m, 2H), 1.85-1.82 (m, 2H), 1.61-1.47 (m, 6H), 0.64-0.60 (m, 2H), 0.50-0.48 (m, 2H). MS-ESI calculated [M + H] ⁺ 401, found 401.

Example 50.

7-(Cyclopropylmethyl)-1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -indole-2,6( 3H , 7 H) - dione (900 mg, 2.25 mmol), separated by preparative SFC to give two isomers. Separation conditions: Column: AD 250mm x 30mm, 5um mobile phase: A: supercritical carbon dioxide, B: methanol (0.05% ammonia), A: B = 55:45 Flow rate: 40 mL/min Wavelength: 220 nm. Product 1 (isomer 1, first peak) (600 mg, white solid), yield: 100%. ¹ H NMR: (400 MHz, DMSO- d ₆ ) δ 8.11 (s, 1H), 5.62 (s, 1H), 4.08 (d, J = 7.6 Hz, 2H), 3.88 (d, J = 7.6 Hz, 2H), 3.43 (s, 3H), 2.05-2.04 (m, 1H), 1.85-1.82 (m, 2H), 1.48-1.45 (m, 2H), 1.33-1.32 (m, 2H), 1.30-1.28 ( m, 3H), 0.48-0.46 (m, 2H), 0.41 - 0.39 (m, 2H). MS-ESI calculated [M + H] ⁺ 401, found 401.

Product 2 (isomer 2, second peak) (300 mg, white solid), yield 100%. ^{1 H NMR: (400 MHz,} DMSO- d 6) δ8.11 (s, 1H), 5.62 (s, 1H), 4.09 (d, J = 7.6 Hz, 2H), 3.74 (d, J = 7.6 Hz, 2H), 3.42 (s, 3H), 1.69 - 1.45 (m, 3H), 1.45 - 1.29 (m, 7H), 0.48 - 0.46 (m, 2H), 0.41 - 0.39 (m, 2H). MS-ESI calculated [M + H] ⁺ 401, found 401.

Example 51.

first step.

1-(1,4-Dioxaspiro[4,5]decane-8-ylmethyl-7-(cyclopropylmethyl)-3-methyl-1 H -indole-2,6-( 3 H , 7 H )-dione.

1,4-Dioxaspiro[4,5]nonane-8-ylmethyl methanesulfonate (682 mg, 2.72 mmol), 7-(cyclopropylmethyl)-3-methyl-1 H -嘌呤-2,6-(3 H ,7 H )-dione (500 mg, 2.27 mmol) and potassium iodide (37.7 mg, 0.227 mmol) dissolved in N , N -dimethylformamide (10 mL) Potassium carbonate (627 mg, 4.54 mmol) was added, and the reaction was heated to reflux at 130 ° C for 4 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to give the crude product 1-(1,4-dioxaspiro[4,5]decane-8-ylmethyl-7-(cyclopropylmethyl) 3-methyl- 1H -indole-2,6-( 3H , 7H )-dione (1.10 g, yellow oil). MS-ESI calc. [M + H] ⁺ 375 The value is 375.

The second step.

7-(Cyclopropylmethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6-(3 H ,7 H )-dione.

1-(1,4-Dioxaspiro[4,5]decane-8-ylmethyl-7-(cyclopropylmethyl)-3-methyl-1 H -indole-2,6- ( 3H , 7H )-Diketone (1.20 g, 2.09 mmol) was dissolved in EtOAc (EtOAc) (EtOAc)EtOAc. The ethyl ester (30 mL x 3) was extracted, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained product was purified by silica gel column chromatography (1:1 petroleum ether / ethyl acetate, Rf = 0.3) The product 7-(cyclopropylmethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6-(3 H ,7 H )-dione ( 52.0 mg, yellow solid), yield: 8% .MS-ESI calcd for [M ⁺ H] + 331, found 331.

third step.

7-(Cyclopropylmethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6-(3 H ,7 H )-dione ( 100 mg, 0.303 mmol) dissolved in tetrahydrofuran (5 mL), slowly added methyl Grignard reagent (3 M diethyl ether solution, 0.600 mL, 1.81 mmol) under nitrogen at -78 ° C, and stirred at -78 ° C for half an hour, then The reaction was carried out at 0 ° C for 2 hours. Add water (10 mL), extract with ethyl acetate (30 mL×3), EtOAc (EtOAc)EtOAc. , white solid) (isomer 1, first peak), yield: 25%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.99 (s, 1H), 4.19 (d, J = 7.6 Hz, 2H), 3.90 (d, J = 7.6 Hz, 2H), 3.54 (s, 3H), 1.90-1.79 (m, 1H), 1.70-1.61 (m, 4H), 1.45-1.36 (m, 3H), 1.27-1.16 (m, 5H), 0.65-0.55 (m, 2H), 0.49- 0.42 (m, 2H). MS-ESI calcd for _{[M + H - H 2 O} ] + 329, found 329.

Product 2 (42.0 mg, white solid) (isomer 2, second peak), yield: 40%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.99 (s, 1H), 4.19 (d, J = 7.6 Hz, 2H), 3.89 (d, J = 7.6 Hz, 2H), 3.54 (s, 3H), 1.81-1.70 (m, 1H), 1.69-1.62 (m, 2H), 1.51-1.41 (m, 4H), 1.39-1.25 (m, 3H), 1.15 (s, 3H), 0.63-0.56 ( m, 2H), 0.48-0.42 (m, 2H). MS-ESI calcd for _{[M + H - H 2 O} ] + 329, found 329.

Example 52.

1-((4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )-dione.

first step.

1-((4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H )-dione.

(4-Hydroxy-1-(methoxymethyl)-4-(trifluoromethyl)cyclohexyl)methyl methanesulfonate (100 mg, 0.349 mmol, 7-(cyclopropylmethyl)-3 -Methyl-1 H -indole-2,6-(3 H ,7 H )-dione (76.9 mg, 0.349 mmol), potassium iodide (5.8 mg, 0.0349 mmol) and potassium carbonate (149 mg, 1.05 mmol) In anhydrous N , N -dimethylformamide (5 mL), the reaction solution was heated to 150 ° C in the microwave for 2 hours. The reaction solution was cooled to 20 ° C, filtered, and purified by preparative high performance liquid chromatography to obtain 1- ((4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1 H -indole-2,6-(3 H ,7 H ) -dione (10.0 mg, white solid), yield: 6%. ¹ H NMR: (400 MHz, DMSO- d ₆ ) δ 8.13 (s, 1H), 4.12 (s, 2H), 3.94 (s, 1H), 3.43-3.38 (m, 4H), 3.31 (s, 3H), 3.19 (s, 3H), 1.56-1.45 (m, 8H), 1.43-1.31 (m, 1H), 0.51-0.49 (m, 2H), 0.44-0.42 (m, 2H).

MS-ESI calcd for [M + H] ⁺ 445. .

Example 53.

7-(Cyclopropylmethyl)-1-((4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -indole-2, 6-(3 H ,7 H )-dione.

first step.

7-(Cyclopropylmethyl)-1-((4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -indole-2, 6-(3 H ,7 H )-dione.

(4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl methanesulfonate (100 mg, 0.344 mmol), 7-(cyclopropylmethyl)-3-methyl- 1 H -嘌呤-2,6-(3 H ,7 H )-dione (75.9 mg, 0.344 mmol), potassium iodide (5.70 mg, 0.0344 mmol) and potassium carbonate (47.6 mg, 0.344 mmol) dissolved in anhydrous N. N -dimethylformamide (5 mL). The reaction solution was heated to 150 ° C and microwaved for 4 hours. The reaction solution was cooled to 20 ° C, filtered, concentrated, and then purified by preparative HPLC to give 7-(cyclopropylmethyl)-1-((4-hydroxy-1-methyl-4-(trifluoro) Cyclo)yl)methyl)-3-methyl- 1H -indole-2,6-( 3H , 7H )-dione (10.0 mg, white solid). ¹ H NMR: (400 MHz, DMSO- d ₆ ) δ 8.13 (s, 1H), 4.13-4.09 (m, 2H), 3.83 (s, 1H), 3.43 (s, 3H), 3.34 (s, 2H) ), 1.7-1.53 (m, 6H), 1.23-1.20 (m, 3H), 0.88 (s, 3H), 0.50-0.42 (m, 4H). MS-ESI calcd for [M + H] ⁺ 415.

Example 54.

7-(cyclopropylmethyl)-3-methyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl )methyl)-1H-indole 2,6-(3H,7H)-dione.

first step.

1-(4-(Bromomethyl)-5-methylthiazol-2-yl)ethanone.

1-(4,5-Dimethylpyridin-2-yl)ethanone (200 mg, 1.29 mmol), N -bromosuccinimide (229 mg, 1.29 mmol), azobisisobutyronitrile (21.2 mg, 0.129 mmol) was dissolved in carbon tetrachloride (20 mL) and reacted at 80 ° C for 12 hours. The reaction was quenched by the addition of saturated sodium thiosulfate solution (30 mL). It was extracted with dichloromethane (10 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to give 1-(4-(bromomethyl)-5-methylthiazol-2-yl)ethanone (200) Mg, yellow oil), yield: 46%. MS-ESI calcd for [M+H] ⁺ 234, 236.

The second step.

1-((2-Ethyl-5-methylthiazol-4-yl)methyl)-7-(cyclopropylmethyl)-3-methyl-1 H -indole-2,6-(3H , 7H)-dione.

1-(4-(Bromomethyl)-5-methylthiazol-2-yl)ethanone (200 mg, 0.598 mmol), 7-(cyclopropylmethyl)-3-methyl-1 H - Indole-2,6-dione (132 mg, 0.598 mmol), potassium iodide (19.8 mg, 0.119 mmol) and potassium carbonate (248 mg, 1.79 mmol) dissolved in N , N -dimethylformamide (10 mL) in. The reaction solution was warmed to 120 ° C and stirred for 3 hours. After cooling to room temperature, filtration, the filtrate was concentrated under reduced pressure and purified by preparative TLC (1:1 petroleum ether / ethyl acetate, Rf value = 0.4) to give 1-((2- ethane--5-methylthiazole - 4-yl)methyl)-7-(cyclopropylmethyl)-3-methyl-1 H -indole-2,6-(3 H ,7 H )-dione (100 mg, yellow solid) Yield: 45%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.02 (s, 1H), 5.35 (s, 2H), 4.21. (d, J = 7.6 Hz, 2H), 3.56 (s, 3H), 2.66 ( s, 3H), 2.60 (s, 3H), 1.46-1.41 (m, 1H), 0.65-0.61 (m, 2H), 0.60-0.48 (m, 2H). MS-ESI calcd [M + H] ⁺ 374.

third step.

7-(cyclopropylmethyl)-3-methyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl )methyl)-1 H -嘌呤2,6-(3 H ,7 H )-dione.

1-((2-Ethyl-5-methylthiazol-4-yl)methyl)-7-(cyclopropylmethyl)-3-methyl-1 H -indole-2,6-( 3 H ,7 H )-dione (100 mg, 0.267 mmol), cesium fluoride (40.6 mg, 0.267 mmol) dissolved in tetrahydrofuran (10 mL), trimethyl-trifluoromethyl-decane at room temperature (114 mg, 0.803 mmol), stirred for 12 hours. The reaction was quenched by the addition of water (20 mL). Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Purification by preparative high performance liquid chromatography to give 7-(cyclopropylmethyl)-3-methyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropane) 2-yl) thiazol-4-yl) methyl) -1 H - purin-2,6- (3 H, 7 H) - dione (50.0 mg, yellow solid), yield: 42%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.11 (s, 1H), 5.33 (s, 2H), 4.23 (d, J = 7.6 Hz, 2H), 3.57 (s, 3H), 2.64 ( s, 3H), 1.81 (s, 3H), 1.45-1.41 (m, 1H), 0.65-0.61 (m, 2H), 0.60-0.49 (m, 2H). MS-ESI calcd for [M + H] ⁺ 444.

Example 55.

7-(Cyclopropylmethyl)-3-methyl-1-((6-(1,1,1-trifluoro-2-hydroxypropan-2-yl)pyridin-3-yl)methyl)- 1 H -嘌呤2,6-(3 H ,7 H )-dione.

first step.

1-[5-(Bromomethyl)-2-pyridyl]ethanone.

1-(5-Methyl-2-pyridyl)ethanone (500 mg, 3.70 mmol), N -bromosuccinimide (658 mg, 3.70 mmol), azobisisobutyronitrile (182 mg , 1.11 mmol) was dissolved in carbon tetrachloride (20 mL) and reacted at 90 ° C for 12 hours. The reaction was quenched by the addition of saturated sodium thiosulfate solution (30 mL). Extracted with dichloromethane (10 mL x 3), dried over anhydrous sodium sulfate, filtered and evaporated. ), yield: 16%. MS-ESI calculated [M + H] ⁺ 214 and 216, found 214 and 216.

The second step.

1-[(6-Ethyl-3-pyridyl)methyl]-7-(cyclopropylmethyl)-3-methyl-1 H -indole-2,6-dione.

1-[5-(Bromomethyl)-2-pyridyl]ethanone (100 mg, 0.467 mmol), 7-(cyclopropylmethyl)-3-methyl- 1H -indole-2,6 Diketone (103 mg, 0.467 mmol), potassium iodide (15.5 mg, 0.0934 mmol) and potassium carbonate (193 mg, 1.40 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was warmed to 120 ° C and stirred for 3 hours. It was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure and purified by preparative TLC (ethyl acetate, Rf value = 0.4) to give 1-[(6-ethylamino-3-pyridyl)methyl]-7- ( Cyclopropylmethyl)-3-methyl- 1H -indole-2,6-dione (50.0 mg, yellow solid), yield: 30%. MS-ESI calcd for [M + H] ⁺ 352.

third step.

7-(Cyclopropylmethyl)-3-methyl-1-((6-(1,1,1-trifluoro-2-hydroxypropan-2-yl)pyridin-3-yl)methyl)- 1 H -嘌呤2,6-(3 H ,7 H )-dione.

1-[(6-Ethyl-3-pyridyl)methyl]-7-(cyclopropylmethyl)-3-methyl-1 H -indole-2,6-dione (100 mg, 0.283 mmol), ruthenium fluoride (43.0 mg, 0.283 mmol) was dissolved in tetrahydrofuran (10 mL). Trimethyl-trifluoromethyl-decane (60.4 mg, 0.424 mmol) was added at room temperature and stirred for 12 hours. The reaction was quenched by the addition of water (20 mL). Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Purification by preparative high performance liquid chromatography to give 7-(cyclopropylmethyl)-3-methyl-1-((6-(1,1,1-trifluoro-2-hydroxypropan-2-yl)) pyridin-3-yl) methyl) -1 H - purin-2,6- (3 H, 7 H) - dione (40.0 mg, yellow solid), yield: 32%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 9.00 (s, 1H), 8.80-8.72 (m, 1H), 8.46 (s, 1H), 8.35-8.29 (m, 1H), 5.43 (s) , 2H), 4.28 (d, J = 7.6 Hz, 2H), 3.58 (s, 3H), 1.95 (s, 3H), 1.50-1.46 (m, 1H), 0.68-0.64 (m, 2H), 0.53- 0.51 (m, 2H). MS-ESI calcd for [M ⁺ H] + 424, found 424.

Example 56.

7-(cyclopropylmethyl)-3-methyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl )methyl)-1 H -嘌呤2,6-(3 H ,7 H )-dione.

first step.

1-[(5-Ethyl-2-pyridyl)methyl]-7-(cyclopropylmethyl)-3-methyl-1 H -indole-2,6-dione.

1-[6-(Bromomethyl)-3-pyridyl]ethanone (100 mg, 0.467 mmol), 7-(cyclopropylmethyl)-3-methyl- 1H -indole-2,6 Diketone (103 mg, 0.467 mmol), potassium iodide (15.5 mg, 0.0934 mmol) and potassium carbonate (193 mg, 1.40 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was warmed to 120 ° C and stirred for 3 hours. It was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure and purified by preparative TLC (ethyl acetate, Rf value = 0.5) to give 1-[(5-ethyl-purinyl-2-pyridyl)methyl]-7- Cyclopropylmethyl)-3-methyl- 1H -indole-2,6-dione (50.0 mg, yellow solid), yield: 30%. MS-ESI calcd for [M + H] ⁺ 352.

The second step.

7-(cyclopropylmethyl)-3-methyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl )methyl)-1 H -嘌呤2,6-(3 H ,7 H )-dione.

1-[(5-Ethyl-2-pyridyl)methyl]-7-(cyclopropylmethyl)-3-methyl-1 H -indole-2,6-dione (100 mg, 0.283 mmol), ruthenium fluoride (43.0 mg, 0.283 mmol) was dissolved in tetrahydrofuran (10 mL). Trimethyl-trifluoromethyl-decane (60.4 mg, 0.424 mmol) was added at room temperature and stirred for 12 hours. The reaction was quenched by the addition of water (20 mL). Extract with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate Purification by preparative high performance liquid chromatography to give 7-(cyclopropylmethyl)-3-methyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropane) 2-yl) thiazol-4-yl) methyl) -1 H - purin-2,6- (3 H, 7 H) - dione (10.0 mg, yellow solid), yield: 8%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 8.92 (s, 1H), 8.75 (d, J = 7.6 Hz, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.97 (s, 1H), 5.54 (s, 2H), 4.21 (d, J = 7.6 Hz, 2H), 3.58 (s, 3H), 1.85 (s, 3H), 1.45-1.42 (m, 1H), 0.64-0.59 (m , 2H), 0.50-0.46 (m, 2H). MS-ESI calcd for [M ⁺ H] + 424, found 424.

Example 57.

1-((4-Hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-7-isobutyl-3-methyl-1 H -indole-2,6(3 H ,7 H )- Dione.

first step.

1-(1,4-Dioxaspiro[4,5]decane-8-ylmethyl)-7-isobutyl-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione.

1,4-Dioxaspiro[4,5]decane-8-ylmethyl methanesulfonate (1.07 g, 4.81 mmol), 7-isobutyl-3-methyl- 1H -indole- 2,6(3 H ,7 H )-dione (1.00 g, 4.01 mmol) and potassium carbonate (647 mg, 4.81 mmol) were dissolved in N , N -dimethylformamide (14 mL), and potassium iodide was added. (66.5 mg, 0.401 mmol), and the reaction was heated to reflux at 130 ° C for 3 hours. The reaction solution was directly filtered, and the filtrate was concentrated under reduced pressure to give the crude product 1-(1,4-dioxaspiro[4,5]decane-8-ylmethyl)-7-isobutyl-3-methyl- 1 H - purine -2,6 (3 H, 7 H) - dione (crude 2.56 g, brown oil). MS-ESI calcd for [M + H] ⁺ 377.

The second step.

7-Isobutyl-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione.

1-(1,4-Dioxaspiro[4,5]decane-8-ylmethyl)-7-isobutyl-3-methyl-1 H -indole-2,6( 3H , 7H )-Dione (2.50 g, 6.00 mmol) was dissolved in EtOAc (EtOAc) (EtOAc) The reaction was stirred at 30 ° C overnight, and aq. Water (100 mL) was added to the reaction mixture, and the mixture was evaporated, evaporated, evaporated, evaporated 2 petroleum ether / ethyl acetate, Rf = 0.3) gives the product 7-isobutyl-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -嘌呤-2,6 (3 H , 7 H )-dione (150 mg, white solid), yield: 13%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.94 (s, 1H), 4.14 (d, J = 7.6 Hz, 2H), 3.99 (d, J = 7.6 Hz, 2H), 3.55 (s, 3H), 2.29-2.38 (m, 5H), 2.20-2.13 (m, 1H), 2.03-1.98 (m, 2H), 1.53-1.47 (m, 2H), 0.92 (d, J = 6.4 Hz, 6H) . MS-ESI calcd [M + H] ⁺ 333.

third step.

1-((4-Hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-7-isobutyl-3-methyl-1 H -indole-2,6(3 H ,7 H )- Dione.

7-Isobutyl-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -indole-2,6(3 H ,7 H )-dione (150 mg, 0.450 mmol And cesium fluoride (8.0 mg, 0.0450 mmol) was dissolved in tetrahydrofuran (3 mL) and trifluoromethyltrimethyl decane (950 mg, 0.640 mmol) was slowly added under nitrogen. The reaction was stirred at 30 ° C for 16 hours, added 4N aqueous HCl (3 mL) and stirred at 25 ° C for half an hour, then aq. (50 mL x 3), the organic phase was dried over anhydrous sodium sulfate and evaporated. methyl) -7-isobutyl-3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (86.0 mg, white solid), yield: 48%. ¹ H NMR: (400 MHz, Methanol- d ₄ ) δ 7.93 (s, 1H), 4.15-4.04 (m, 2H), 3.89 (d, J = 7.6 Hz, 1H), 3.54 (s, 3H), 2.20-1.98 (m, 3H), 1.86-1.79 (m, 2H), 1.61-1.42 (m, 5H), 0.92 (d, J = 6.4 Hz, 6H). MS-ESI calcd [M + H] ⁺ 403.

Example 58.

1-((4-Hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-7-isobutyl-3-methyl-1 H -indole-2,6(3 H ,7 H )- The diketone (900 mg, 2.24 mmol) was isolated by preparative SFC to afford two isomers. Isomer 1 Separation conditions: Column: AD 250mm x 30mm, 5 um Mobile phase: A: supercritical carbon dioxide, B: ethanol (0.05% ammonia), A: B = 80:20 Flow rate: 50 mL/min Wavelength: 220 nm. Isomer 2 separation conditions: column: WEEK-1 300mm x 25 mm, 5 um mobile phase: A: supercritical carbon dioxide, B: ethanol (0.05% ammonia), A: B = 60:40, flow rate: 60 mL /min, wavelength: 220 nm.

Product 1 (isomer 1, first peak) (216 mg, white solid), yield: 36%. ¹ H NMR: (400 MHz, DMSO- d ₆ ) δ 8.09 (s, 1H), 5.65 (s, 1H), 4.07 (d, J = 7.2 Hz, 2H), 3.90 (d, J = 7.2 Hz, 2H), 3.43 (s, 3H), 2.14 - 2.00 (m, 2H), 1.92-1.80 (m, 2H), 1.77-1.66 (m, 2H), 1.52-1.44 (m, 2H), 1.37-1.30 ( m, 2H), 0.84 (d, J = 6.4 Hz, 6H). MS-ESI calcd [M + H] ⁺ 403.

Product 2 (isomer 2, second peak) (101 mg, white solid), yield 37%. ¹ H NMR: (400 MHz, DMSO- d ₆ ) δ 8.07 (s, 1H), 5.64 (s, 1H), 4.05 (d, J = 7.6 Hz, 2H), 3.75 (d, J = 7.6 Hz, 2H), 3.42 (s, 3H), 2.16-2.03 (m, 1H), 1.71-1.66 (m, 3H), 1.48-1.30 (m, 6H), 0.83 (d, J = 6.4 Hz, 6H). MS-ESI calcd [M + H] ⁺ 403.

Example 59.

7-(2,3-Dihydroxypropyl)-1-(5-ethyl-5-hydroxyheptyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )- ketone.

first step.

7-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )- Dione.

3-Methyl-1 H -嘌呤-2,6(3 H ,7 H )-dione (200 mg, 1.20 mmol), sodium carbonate (128 mg, 1.20 mmol), 4-(chloromethyl)- 2,2-Dimethyl-1,3-dioxolane (217 mg, 1.44 mmol) and potassium iodide (20.0 mg, 0.120 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was heated to 110 ° C and reacted for 36 hours. The reaction was quenched with EtOAc (EtOAc) (EtOAc (EtOAc) Ethyl ester, Rf = 0.5) gives 7-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-3-methyl-1 H -indole-2,6 (3 H , 7 H )-dione (169 mg, yellow solid), yield: 50%. MS-ESI calcd [M + H] ⁺ 281.

The second step.

Ethyl 5-(7-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-3-methyl-2,6-dioxo-2,3 , 6,7-tetrahydro-1 H -decane-1-yl)pentanoic acid ethyl ester.

7-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-3-methyl-1 H -indole-2,6(3 H ,7 H ) -dione (169 mg, 0.604 mmol), ethyl bromopentanoate (178 mg, 0.906 mmol), potassium carbonate (167 mg, 1.21 mmol) and potassium iodide (20.0 mg, 0.0600 mmol) dissolved in N , N -dimethyl Carbenamide (10 mL). The reaction mixture was heated to 130 ° C, and the mixture was reacted for 3 hours, filtered, concentrated, and purified by preparative TLC (ethyl acetate, Rf = 0.4) to give ethyl 5-(7-((2,2-dimethyl-1). 3-dioxolan-4-yl)methyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -decane-1-yl)pentyl Ethyl acetate (124 mg, yellow solid), yield: 50%. MS-ESI calcd for [M + H] ⁺ 409.

third step.

7-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-1-(5-ethyl-5-hydroxyheptyl)-3-methyl-1 H -嘌呤-2,6(3 H ,7 H )dione.

Ethyl 5-(7-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-3-methyl-2,6-dioxo-2, Ethyl 3,6,7-tetrahydro-1 H -decane-1-yl)pentanoate (30.0 mg, 0.0750 mmol) was dissolved in anhydrous tetrahydrofuran (1 mL) and then slowly dropped at -65 °C Ethyl magnesium bromide (3 M in tetrahydrofuran, 0.15 mL, 0.450 mmol). The reaction was allowed to react at -65 ° C for 0.5 hours and then at 0 ° C for 0.5 hour. The reaction mixture was poured into EtOAc EtOAc (EtOAc m. 0.5) to give 7-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-1-(5-ethyl-5-hydroxyheptyl)-3-methyl yl -1 H - purin--2,6 (3 H, 7 H) -dione (20.0 mg, yellow oil). yield: 63%. MS-ESI calcd for [M ⁺ H] + 423, found 423.

the fourth step.

7-(2,3-Dihydroxypropyl)-1-(5-ethyl-5-hydroxyheptyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )- ketone.

7-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-1-(5-ethyl-5-hydroxyheptyl)-3-methyl- 1 H - purine -2,6 (3 H, 7 H) -dione (20.0 mg, 0.0470 mmol) was dissolved in anhydrous tetrahydrofuran (1 mL) and dilute hydrochloric acid (0.3 mL), the reaction at 25 ℃ 36 hours. After the reaction was completed, it was concentrated under reduced pressure and purified by preparative TLC (ethyl acetate / methanol, Rf = 0.3) to give 7-(2,3-dihydroxypropyl)-1-(5-ethyl) -5-hydroxy-hept-yl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (5.0 mg, white solid), yield: 28%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.90 (s, 1H), 4.57-4.53 (m, 1H), 4.26-4.22 (m, 1H), 4.03-3.96 (m, 3H), 3.58 -3.55 (m, 2H), 3.54 (s, 3H), 1.66-1.61 (m, 2H), 1.48-1.44 (m, 6H), 1.34-1.29 (m, 2H), 0.85 (t, J = 8.0 Hz , 6H). MS-ESI calcd [M + H] ⁺ 383.

Example 60.

1- (5-ethyl-5-hydroxy-heptyl) -7- (2-hydroxyethyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione.

first step.

7-(2-Hydroxyethyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione.

3-Methyl-1 H -嘌呤-2,6(3 H ,7 H )-dione (1.00 g, 6.00 mmol), potassium carbonate (830 mg, 6.00 mmol) dissolved in N , N -dimethyl In tolamine (10 mL). The reaction solution was heated to 80 ° C for 0.5 hour, and 2-bromoethanol (900 mg, 7.20 mmol) was added. The reaction solution was heated to 130 ° C and allowed to react overnight. The reaction mixture was concentrated to give crude 7- (2-hydroxyethyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione was used directly in the next step.

The second step.

Ethyl 5-(7-(2-hydroxyethyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)pentanoic acid Ethyl ester.

7-(2-Hydroxyethyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione (1.05 g, 5.00 mmol), ethyl 5-bromopentanoate (1.25 g, 6.00 mmol) and potassium carbonate (1.66 g, 12.0 mmol) were dissolved in N , N -dimethylformamide (3 mL). The reaction solution was heated to 130 ° C for 3 hours. The reaction mixture was poured into water (20 mL). The organic phase was combined, dried over anhydrous sodium sulfate, filtered, evaporated, evaporated, evaporated,,,,,,,,,,,,,,,,,,,,,,,,,, Ethyl 2-(3,6,7-tetrahydro-1 H -indol-1-yl)pentanoate (700 mg, yellow oil), yield: 35%. MS-ESI calcd [M + H] ⁺ 339.

third step.

1- (5-ethyl-5-hydroxy-heptyl) -7- (2-hydroxyethyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione.

Ethyl 5-(7-(2-hydroxyethyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -indol-1-yl)pentyl Ethyl acetate (700 mg, 2.07 mmol) was dissolved in anhydrous tetrahydrofuran (7 mL). EtOAc (3M, THF, The reaction solution was reacted at -78 ° C for 1 hour. The reaction mixture was poured into water (20 mL The organic phase was dried over anhydrous sodium sulfate, filtered, evaporated, evaporated, evaporated,,,,,,,,,,,,,,,,,,,,,,, methyl -1 H - purine -2,6 (3 H, 7 H) - dione (110 mg, white solid), yield: 15%. ¹ H NMR: (400 MHz, Methonal- d ₄ ): δ 7.90 (s, 1H), 4.41 (t, J = 5.0 Hz, 2H), 4.00 (t, J = 7.6 Hz, 2H), 3.87 (t , J = 5.0 Hz, 2H), 3.54 (s, 3H), 1.68-1.59 (m, 2H), 1.50-1.42 (m, 5H), 1.39-1.29 (m, 3H), 0.95-0.77 (m, 6H) ). MS-ESI calcd for _{[M + H - H 2 O} ] + 335, found 335.

Example 61.

1-(5-ethyl-5-hydroxyheptanol)-7-(2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)-3-methyl-1 H -嘌呤-2,6(3 H ,7 H )-dione.

first step.

1-Bromo-3-((2-hydroxyethyl)(methyl)amino)propan-2-ol.

2-(Methylamino)ethanol (135 mg, 1.80 mmol) was dissolved in N , N -dimethylformamide (5 mL) and 2-(bromomethyl)oxirane (206 mg, 1.51 mmol) was reacted at room temperature for 1.5 hours. After the reaction was completed, the reaction solution was used directly for the next reaction.

The second step.

7-(2-Hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )-dione .

Add 3-methyl-1 H -嘌呤-2,6(3 H ,7 H ) to a solution of 1-bromo-3-((2-hydroxyethyl)(methyl)amino)propan-2-ol - Dione (100 mg, 0.602 mmol), sodium carbonate (64.0 mg, 0.602 mmol) and potassium iodide (10.0 mg, 0.0600 mmol). The reaction solution was heated to 80 ° C and allowed to react for 10 hours. After completion of the reaction, filtered and concentrated under reduced pressure to give crude 7- (2-hydroxy-3 - ((2-hydroxyethyl) (methyl) amino) propyl) -3-methyl -1 H - purin -2 , 6(3 H , 7 H )-dione was used directly in the next step. MS-ESI calcd [M + H] ⁺ 298.

third step.

Ethyl 5-(7-(2-hydroxy-3-((2-hydroxyethyl))(methyl)amino)propyl)-3-methyl-2,6-dioxo-2,3,6 , 7-Tetrahydro-1 H -decane-1-yl)pentanoic acid ethyl ester.

7-(2-Hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)-3-methyl-1 H -indole-2,6(3 H ,7 H )- Ketone (170 mg, 0.572 mmol), ethyl bromopentanoate (169 mg, 0.858 mmol), potassium carbonate (158 mg, 1.14 mmol) and potassium iodide (10.0 mg, 0.0570 mmol) dissolved in N , N -dimethyl In the guanamine (5 mL). The reaction solution was heated to 130 ° C and reacted for 3 hours. The reaction mixture was poured into water (5 mL) and evaporated and evaporated. The organic phase was combined, dried over anhydrous sodium sulfate, filtered, evaporated, evaporated, evaporated, (2-hydroxyethyl)(methyl)amino)propyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -decane-1-yl Ethyl valerate (118 mg, yellow oil), yield: 49%. MS-ESI calcd for [M ⁺ H] + 426, found 426.

the fourth step.

1-(5-ethyl-5-hydroxyheptanol)-7-(2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)-3-methyl-1 H -嘌呤-2,6(3 H ,7 H )-dione.

Ethyl 5-(7-(2-hydroxy-3-((2-hydroxyethyl))(methyl)amino)propyl)-3-methyl-2,6-dioxo-2,3, Ethyl 6,7-tetrahydro-1 H -decane-1-yl)pentanoate (100 mg, 0.235 mmol) was dissolved in anhydrous tetrahydrofuran (4 mL). Magnesium bromide (3 M in tetrahydrofuran, 0.47 mL, 1.41 mmol). The reaction was reacted at -65 ° C for 0.5 hour and then reacted at 0 ° C for 0.5 hours. After the reaction was completed, the reaction mixture was evaporated to mjjjjjjjjjjjjjjjj 1 ethyl acetate / methanol, Rf = 0.3) purified 1-(5-ethyl-5-hydroxyheptanol)-7-(2-hydroxy-3-((2-hydroxyethyl))(methyl)amino ) propyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (3.0 mg, white solid), yield: 3%. ¹ H NMR: (400 MHz, Methonal- d ₄ ) δ 7.92 (s, 1H), 4.91-4.59 (m, 2H), 4.52-4.39 (m, 3H), 4.30-4.25 (m, 2H), 4.03 -3.99 (m, 2H), 3.88-3.84 (m, 2H), 3.55 (s, 3H), 2.90 (s, 3H), 1.68-1.59 (m, 4H), 1.48-1.45 (m, 6H), 0.88 -0.84 (m, 6H). MS-ESI calcd for [M + H] ⁺ 440.

Experimental Example 1: PDE2 phosphodiesterase inhibitory activity was evaluated in vitro.

EXPERIMENTAL OBJECTIVE: To detect the AMP/GMP concentration produced in the reaction system by detecting the AlexaFluor 633 fluorescent dye substituted on the AMP/GMP antibody by fluorescence polarization analysis, and calculate the IC ₅₀ value of the PDE2 phosphodiesterase inhibition of the test compound.

Experimental Materials.

Assay buffer solution: 10 mM Tris buffer, pH 7.5, 5 mM magnesium chloride, 0.01% polyoxyethylene lauryl ether, 1 mM dithiothreitol and 1% DMSO. Enzyme: Recombinant full length human PDE2A protein was expressed in insect Sf9 cells using a baculovirus using an N-terminal GST tag. Substrate: 1 μM cGMP.

Detection method.

Transcreener® AMP ² / GMP ² Antibody, AMP2/GMP2 AlexaFluor 633 Fluorescent Dye.

Experimental operation.

The freshly prepared buffer solution was placed in an enzyme solution, and then added to the reaction well, and the DMSO solution of the test compound was added through an Echo 550 non-contact nano-sound sonic pipetting system, and then pre-incubated for 10 minutes at room temperature, and the substrate was added ( The reaction was initiated by 1 mM cGMP) and allowed to react at room temperature for one hour. Then, a detection system (Transcreener® AMP ² / GMP ² antibody, AMP2/GMP2 AlexaFluor 633 fluorescent dye) was added, and reacted at room temperature for 90 minutes, and then fluorescence polarization was detected using Ex/Em 620/688.

Fluorescence polarization intensity was converted to nM concentration by AMP/GMP standard curve, then relative enzyme activity inhibition relative to DMSO blank was calculated, and IC50 values and curves were calculated using the Prism software package (GraphPad Software, San Diego California, USA).

Experimental results: See Table 1. Table 1 PDE2 phosphodiesterase inhibitory activity test results Note: 10 mM ≦ "+"< 50 mM; 1 mM ≦ "++"< 10 mM; "+++"< 1 mM; -- N/A

Conclusion: The compounds of the invention have significant and unexpected PDE2A protease inhibitory activity.

Experimental Example 2: The effect of compounds on the TNF-a in the blood of rats induced by LPS was evaluated in vitro.

EXPERIMENTAL OBJECTIVE: To examine the effects of compounds on the TNF-a in rat blood induced by LPS in vitro, and to evaluate the inhibitory effect of the compounds on LPS-induced TNF-a in rat blood.

Experimental materials: Sprague Dawley rats (male, 210-260 g, 8-10 weeks old, Shanghai Slack); Rat TNF-alpha Quantikine ELISA Kit (R&D, #SRTA00).

Experimental procedure: A test compound solution having a concentration of 1 mM was prepared, and 40 μL (the final concentration of the compound was 100 μM) was added to a 48-well cell culture plate. After the rats were anesthetized with isoflurane, blood was collected from the heart (heparin anticoagulation). Blood was added to a 48-well plate to which the test compound had been added, 320 μL per well. The 48-well plate was placed in a cell culture incubator, and after 30 minutes of incubation, it was taken out, 40 μL of LPS solution (100 ug/mL) was added, mixed, and placed in an incubator to continue incubation. After 5 hours, remove the 48-well plate, transfer the blood sample to a 1.5 mL centrifuge tube, centrifuge in a centrifuge (4,500 rpm, 4 ° C, 5 minutes), separate the upper layer of plasma, and then freeze it after packaging. Store at -80 °C. refrigerator. The next day, according to the kit instructions, the R&D ELISA kit was used to detect TNF-a levels in plasma samples.

Experimental results: See Table 2. Table 2 TNF-a inhibitory activity test results Note: 60% ≦ “+” <80%; 80% ≦ “++” <100%; -- N/A

Conclusion: The compounds of the invention have significant and unexpected TNF-a inhibitory activity.

Experimental Example 3: Evaluation of compound pharmacokinetics.

Experimental Objective: To test the pharmacokinetics of compounds in SD rats.

Experimental material: Sprague Dawley rats (male, 200-300 g, 7-9 weeks old, Shanghai Slack).

Experimental procedure: The rodent pharmacological characteristics of the compound after intravenous injection and oral administration were tested by a standard protocol. In the experiment, the candidate compound was formulated into a clear solution, and the rats were administered a single intravenous injection and oral administration. The intravenous and oral vehicles are a certain proportion of aqueous hydroxypropyl β-cyclodextrin or physiological saline solution. Collect whole blood samples within 24 hours, centrifuge at 3000g for 15 minutes, separate the supernatant to obtain plasma samples, add 4 times volume of acetonitrile solution containing internal standard to precipitate protein, centrifuge to remove the supernatant, add equal volume of water and centrifuge to remove the supernatant. For example, the plasma concentration was quantitatively analyzed by LC-MS/MS analysis, and the pharmacokinetic parameters such as peak concentration, peak time, clearance rate, half-life, area under the curve of the drug, and bioavailability were calculated.

Experimental results: See Table 3. Table 3 Pharmacokinetic test results

Conclusion: The compounds of the present invention can significantly increase the single or partial index of rat pharmacokinetics.

Experimental Example 4: In vivo pharmacological study of the MCD mouse model.

Main task: To evaluate the liver histology, TG level, TNF-a, mRNA level and inflammatory factor (IL-1b level; plasma ALT/AST level, inflammatory factor (TNF-) of compound 1 in non-alcoholic steatohepatitis model MCD mice a, IL-1b) level of influence.

Proposed problem: Test compound 1 for liver histology, TG level, TNF-a mRNA level and inflammatory factor (IL-1b) level in MCD mice; plasma ALT/AST level, inflammatory factor (TNF-a, IL-1b) The effect of the level of the compound on the liver function protection of the non-alcoholic steatohepatitis model mice and the reduction of liver inflammation.

Experimental model: Methionine-choline-deficient diet (MCD mice) induced by methionine and choline-deficient diet in mice

Experimental animals: C57BL/6J mice (male, purchased at 4 weeks old, Shanghai Slack)

Experimental reagents: 1. MCD feed (Research Diets, # A02082002B) 2. MCD control feed (Research Diets, # A02082003B) 3. RNeasy® Mini RNA Extraction Kit (Qiagen, #74104) 4. High Capacity cDNA Reverse Transcription Kit (ABI, # 4368814) 5. TaqMan® Gene Expression Assay TNF-a (ABI, Assay ID: Mm00443258_m1) 6. TaqMan® Endogenous Control 18S (ABI, Assay ID: Mm03928990_g1) 7. TaqMan® Fast Universal PCR Mater Mix (ABI) , # 4366072) 8. One cOmplete, EDTA-free protease inhibitor tablet (Roche, # 11873580001) 9. BCA Protein Quantitation Kit (Pierce, # 23227) 10. MSD Inflammatory Factor Detection Kit (Meso Scale Discovery, #K15A0H- 2) 11. Triglyceride Quantification Kit (BioVision, #K622-100) 12. Hematoxylin (Baso, # BA4097) 13. Eosin (Baso, # BA4099)

experimental method.

1. Experimental process.

2. In vivo experimental methods.

Four-week-old C57BL/6J mice were purchased and special feeds were started after 4 weeks of adaptation to the environment. Pre-adaptation of oral administration was started 1 week after the special feed was raised. Preconditioning method: 0.5% (w/v) hydroxypropyl methylcellulose according to 5 mL/kg oral vehicle control. After a week of preconditioning, randomization was performed orally, and the grouping and administration information was as follows: MCD control diet + vehicle control (po tid, 2wk) n=8 MCD diet + vehicle control (po tid, 2wk) n=8 MCD diet + pentoxifylline (po 200mg/kg, tid, 2wk) n=8 MCD diet + INT-747 (po 30mg/kg, qd 2wk) n=8 MCD diet + compound 1 (po 30mg/kg, bid, 2wk ) n=8

Two weeks after the administration, the mice were sacrificed by CO ₂ , and the hearts were bled 500 μL to 600 μL (K ₂ -EDTA anticoagulation). The blood samples were centrifuged in a centrifuge (4,500 rpm, 4 ° C, 5 minutes), and the upper plasma was separated. After quick-packing, it is stored in a refrigerator at -80 °C. Liver tissue was dissected for histological analysis and biomarker detection.

Frozen plasma samples were tested for ALT and AST the following day.

3. Histological analysis methods.

3.1 HE staining method.

3.1.1 Organizational processing.

Using the Leica ASP200S fully automatic dewatering machine, the relevant dehydration steps are as follows: a) 10% neutral buffered formalin for 5 minutes to 6 hours b) 70% ethanol for 45 minutes c) 80% ethanol for 45 minutes d) 95% ethanol 45 Minute e) 95% ethanol 45 minutes f) 100% ethanol 60 minutes g) 100% ethanol 60 minutes h) 100% ethanol 60 minutes i) xylene 60 minutes j) xylene 60 minutes k) paraffin 60 minutes l) paraffin 60 Minute m) paraffin 60 minutes

3.1.2 Organizational embedding. a) Preparation before embedding. Composition and function of the Leica tissue embedding system: The Leica tissue embedding system consists of two parts: the embedding table (Leica EG1150 H) and the freezing station (Leica EG1150 C). The embedding station includes a left metal mold storage cylinder, a right side tissue cassette storage cylinder and a paraffin bath on the upper part of the instrument. There is a small cold stage at the front of the embedding station that can quickly cool a single embedding mold. The freezing station is mainly composed of a cold stage, and rapidly cools the paraffin embedded in the mold. After the cassette is cooled, the wax block is stored at room temperature. b) Turn on the cold stage. c) Take out the cassette in the dehydrator and place it in the embedding wax bath. d) Embedding. The hot tweezers are taken out of the tissue cassette in the embedding machine wax bath and placed on the hot stage of the embedding machine. Place the embedding box corresponding to the tissue on the mold. The tissue is placed in the embedding mold with hot tweezers, and a little dissolved paraffin is added to the embedding mold, and the embedding mold is placed on the cold table until the paraffin becomes completely solid. Finally, the solid wax block is taken out of the embedding mold.

3.1.3 Preparation of tissue sections. a) Confirm the status before using the Leica RM2235 paraffin slicer. Before loading the wax block, make sure that the slice wheel handle is loosely locked, open the knife holder cover, and clean the wax on the knife holder. Carefully insert an unused disposable knife to confirm that the blade is in place at home and lock the knife holder according to the manufacturer's instructions. Confirm the inclination of the blade, which should be set to 3-6 degrees. b) Fix the tissue wax block to the desired position in the clipper slot. Follow the manufacturer's instructions for roughing with the roughing wheel handles so that the wax block slowly advances until the desired tissue plane is exposed. c) Start the slicing, first set the slice thickness, start slicing, and rotate the slicing wheel forward until the flat wax sheet is formed. Lock the slice rotation handle and use a forceps or brush to move the tissue piece into the spreader (Leica HI1210) and flatten it onto the slide. d) After roughing or slicing, lock the wheel handle and carefully remove the wax block.

3.1.4 HE staining.

HE staining was performed using a fully automated staining workstation consisting of Leica ST5010, Leica TS5010 and Leica CV5030.

Press the <Stain> key in the main menu of the dyeing machine. Conventional HE staining is performed by the up and down arrow selection procedure 15 of the button area. Open the load compartment, place the slicer, and close the load compartment. Start the dye program after pressing the <Load> button. The procedure is as follows: a) Oven 55 ° C for 2 minutes b) Xylene I 10 minutes c) Xylene II 10 minutes d) Xylene III 10 minutes e) Anhydrous ethanol I 2 minutes f) Anhydrous ethanol II 1.5 minutes g) 95% ethanol I 1 min h) 80% Ethanol I 0.5 min i) Washed for 3 minutes j) Hematoxylin dye solution for 3 minutes k) Washed for 2 minutes l) 1% Hydrochloric acid-ethanol 5 seconds m) Washed for 2 minutes n) Washed for 20 minutes o) 80% ethanol II 1 minute p) Eosin 5 seconds q) 95% ethanol II 30 seconds r) absolute ethanol III 2 minutes s) absolute ethanol IV 2 minutes t) absolute ethanol V 2 minutes u) absolute ethanol VI 2 minutes v) xylene IV 3 minutes w) xylene V 3 minutes

3.2 Histological non-alcoholic fatty liver disease activity score (NAS) related scoring criteria: histological diagnosis and clinical efficacy evaluation of nonalcoholic fatty liver disease (NAFLD) refer to the National Institutes of Health NASH Clinical Research Network Pathology Working Group Guide, General Perform NAFLD activity score (NAS).

NAS score (0-8 points): a) Hepatocyte steatosis: 0 points (<5%); 1 point (5% to 33%); 2 points (33% to 66%); 3 points (>66%) ). b) Inflammation in the lobules (20-fold microscopic count necrosis): 0 points, none; 1 point (<2); 2 points (2 to 4); 3 points (>4). c) Hepatocyte balloon-like changes: 0 points, no; 1 point, rare; 2 points, more common.

4. Liver TG detection method.

4.1 Sample preparation: Add 5% NP40 lysate to each liver tissue to a concentration of 100 mg/mL. The tissue was lysed on a tissue homogenizer. The sample was heated in an 80-100 degree water bath for 5 min and then cooled to room temperature. Repeat the heating once. Centrifuge at maximum speed for 2 min and take the supernatant. The supernatant was diluted 60-fold and 30 μL was added to each well of the assay plate.

4.2 Standard preparation: 1 mM triglyceride was diluted to 0.2 mM, and 0, 10, 20, 30, 40, 50 μL of 0.2 mM triglyceride standard was added to the standard well column, and the test solution was added to 50 μL.

4.3 Add 2 μL of lipase to each well and mix for 20 min at room temperature.

4.4 Proportionally configure the enzyme reaction solution: 46 μL of triglyceride detection solution + 2 μL of triglyceride probe + 2 μL of triglyceride reaction solution. Add 50 μL of enzyme reaction solution to all wells, mix and incubate for 30-60 min at room temperature.

4.5 Detection: The absorbance at 570 nm was measured with a microplate reader.

5. Detection method of TNF-a mRNA in liver tissue.

RNA was extracted using the RNeasy® Mini RNA Extraction Kit (Qiagen #74104) according to the operating manual. Reverse transcription was performed on the same day. The reverse transcription system ratio (ABI # 4368814) was as follows: 10× reverse transcription buffer 2 μL 25× deoxyribonucleoside Triphosphate solution (100 mM) 0.8 μL 10× reverse transcription random primer 2 μL MultiScribe reverse transcriptase 1 μL Template RNA 1 μg RNase-free H ₂ O to 20 μL

Reverse transcription program:

The cDNA was stored in a refrigerator at -20 °C.

Q-PCR was performed according to the instructions of TaqMan® Fast Universal Kit (ABI, # 4366072). The system is equipped with the following formula: TaqMan® Gene Expression Master Mix (2✕) 5μL TaqMan® Gene Expression Assay (20✕) 0.5μL cDNA template 0.8μL H ₂ O 3.7μL

Q-PCR program:

6. Methods for detecting inflammatory factors.

6.1 Liver tissue homogenate and sample preparation.

6.1.1 Start the pre-cooling centrifuge.

6.1.2 A piece of One cOmplete, EDTA-free protease inhibitor tablet was dissolved in 50 mL of phosphate buffer, and PBS was placed in wet ice for 1:10 (w/v).

6.1.3 Centrifuge 1000g, 10min, 4°C, transfer 220μL to the ultracentrifuge tube, and prepare two tubes for ultracentrifugation in each supernatant.

6.1.4 The sample was placed in an ultracentrifuge OptimaTM L-80 XP (Beckman) which had been pre-cooled and ultracentrifuged at 100,000 g, 4 ° C, 50 min.

6.1.5 Pre-cool the V-bottom 96-well plate on dry ice, and combine the two tube supernatants corresponding to the same sample into a 1.5 mL EP tube. After mixing with light bomb, take 120 μL and dispense into a 96-well plate at the bottom of the V. Dry ice is frozen. A total of three sample plates were prepared (one plate for BCA protein quantification, one plate for MSD detection, and one remaining spare).

6.2 Determination of protein concentration in tissue homogenate by BCA method.

6.2.1 Preparation of BCA working solution: Mix reagents A and B according to reagent A: reagent B=50:1.

6.2.2 Preparation of BSA (bovine serum white egg) standard solution A~I.

6.2.3 Dilute the homogenate with PBS, 20 μL homogenate + 80 μL PBS.

6.2.4 Add 10 μL of BSA standard solution A~I and diluted sample to be tested in sequence in a 96-well plate.

6.2.5 Add 200 μL of LBCA working solution to each well to the standard solution and the sample to be tested, and mix on the platen.

6.2.6 Incubate for 30 minutes in a 37 ° C incubator.

6.2.7 Remove the plate and let it cool to room temperature.

6.2.8 Measure the absorbance at 562 nm on a microplate reader.

6.3 MSD test samples for inflammatory factors.

6.3.1 Remove the MSD plate from the refrigerator and store at room temperature.

6.3.2 Dilute the standard solution in a 1:3 gradient.

6.3.3 Add 50 μL of standard and sample solution to each well, fix on a vibrating plate, and incubate at 300-1000 rpm for 2 hr at room temperature.

6.3.4 Add 300 μL of Wash buffer to each well and wash the plate three times. Add 25 μL of 1× detection antibody solution to each well, fix it on the vibrating plate, and incubate at 300-1000 rpm for 2 hr at room temperature.

6.3.5 Add 300 μL Wash buffer to each well and wash the plate three times. Add 150 μL of 1× reading solution to each well and insert the signal value into the Sector Imager 6000 Model 1200.

Experimental results:

1. Histological results.

1.1 HE stained picture.

The results of HE staining showed that the liver fat of mice increased significantly and the inflammation of hepatic lobule increased significantly after MCD feeding; pentoxifylline (200mpk, po., tid) and INT-747 (30mpk, po., qd) were administered. Week, MCD mice showed no significant improvement in liver lesions, while compound 1 (30mpk, po., bid) administration significantly improved liver lobular inflammation in MCD mice for two weeks. The experimental results are shown in Figure 1.

1.2 NAS score. Liver histology of NAS and lobular inflammation in MCD mice was significantly increased. The three compounds tested in this experiment had no significant effect on NAS, but compound 1 could significantly reduce liver lobular inflammation in MCD mice. The experimental results are shown in Fig. 2.

2. Liver TG test results. The liver TG level of MCD mice was significantly increased. The three compounds tested in this experiment had no significant effect on liver TG levels in MCD mice. The experimental results are shown in Figure 3.

3. Results of liver TNF-a mRNA levels. Compound 1 (30mpk, po., b.i.d.) There was a downward trend in liver TNF-a mRNA levels in MCD mice administered for two weeks, but there was no statistically significant difference. The experimental results are shown in Figure 4.

4. Hepatic inflammatory factor test results.

After 4 weeks of MCD feeding, the level of IL-1b protein in the liver of mice was significantly increased. Pentoxifylline (200mpk, po., tid) and INT-747 (30mpk, po., qd) were administered for two weeks to the liver of MCD mice. There was no significant effect on IL-1b protein level; the level of IL-1b protein in liver of MCD mice was significantly decreased in compound 1 (30mpk, po., bid) for two weeks. The experimental results are shown in Figure 5.

5. Plasma inflammatory factor test results.

Plasma levels of TNF-a and IL-1b were significantly elevated in MCD mice, and pentoxifylline (200mpk, po., tid) and INT-747 (30mpk, po., qd) were administered to MCD mouse plasma for two weeks. There was no significant effect on the levels of TNF-a and IL-1b protein. The levels of TNF-a and IL-1b in plasma of MCD mice were significantly decreased in compound 1 (30mpk, po., bid) for two weeks. The experimental results are shown in Figure 6.

6. Plasma ALT and AST test results.

Plasma ALT and AST levels were significantly increased in MCD mice. The three compounds tested in this experiment could not reduce plasma ALT and AST levels in MCD mice. The experimental results are shown in Figure 7.

to sum up.

1. MCD mice are widely used animal models of non-alcoholic steatohepatitis. The liver fatty degeneration of the model mice is obvious, and the TG level is significantly increased. The histological HE staining picture shows obvious lobular inflammation and the liver TNF-a mRNA is significantly elevated. High, liver inflammatory factors IL-1b, plasma TNF-a and IL-1b protein levels were significantly higher than normal; plasma ALT and AST levels were significantly higher than normal mice.

2. Compound 1 (30mpk, po., b.i.d.) After two weeks of administration, liver lobular inflammation was significantly improved in MCD mice, and liver inflammatory factors IL-1b, plasma TNF-a and IL-1b protein levels were significantly decreased.

3. Compound 1 (30mpk, po., b.i.d.) administered twice a week had no significant effect on liver TG levels, liver TNF-a mRNA, plasma ALT and AST levels in MCD mice.

Experimental Example 5 In vivo pharmacological study of the nSTZ+HFD mouse model.

main mission.

The effects of Compound 1 on liver histology, TNF-a mRNA, TGF-b1 mRNA and collagen 1a1 mRNA levels, serum ALT, AST, TG and TC levels in non-alcoholic steatohepatitis model nSTZ+HFD mice were evaluated.

The problem to be solved.

The effects of test compound 1 on liver histology, TNF-a mRNA, TGF-b1 mRNA and Collagen 1a1 mRNA levels, serum ALT, AST, TG and TC levels in nSTZ+HFD mice were evaluated for small non-alcoholic steatohepatitis models. The liver function of the mouse protects the liver from inflammation and fibrosis.

Experimental model: STZ injection of neonatal mice with a non-alcoholic steatohepatitis model induced by a high-fat diet (nSTZ+HFD mice).

Experimental animals: C57BL/6J mice (female pregnant mice, purchased 14-15 days pregnant, Shanghai Lingchang Biotechnology Co., Ltd.).

Experimental reagents: 1. Streptozotocin (STZ) (Sigma, #S0130-1G) 2. High-fat diet (Research Diets, # D12492i) 3. RNeasy® Mini RNA Extraction Kit (Qiagen, #74104) 4 High Capacity cDNA Reverse Transcription Kit (ABI, # 4368814) 5. TaqMan® Gene Expression Assay TNF-a (ABI, Assay ID: Mm00443258_m1) 6. TaqMan® Gene Expression Assay TGF-b1 (ABI, Assay ID: Mm00441724_m1) 7. TaqMan® Gene Expression Assay Collagen 1a1 (ABI, Assay ID: Mm00801666_g1) 8. TaqMan® Endogenous Control 18S (ABI, Assay ID: Mm03928990_g1) 9. TaqMan® Fast Universal PCR Mater Mix (ABI, # 4366072) 10. Hematoxylin (Baso, # BA4097) 11. Eosin (Baso, # BA4099) 12. Sirius red (Bogoo, # PT036)

experimental method.

1. Experimental process.

2. In vivo experimental methods.

14 to 15 days of gestational age C57BL/6J mice were purchased, and newborn rats were born for 2 to 3 days for STZ injection (200 ug, s.c.). Oral diets are raised until 4 weeks of age to start a high-fat diet. Animals were randomized after 1 week of high fat feeding and started oral administration.

Grouping and dosing information were as follows: Control group (STZ vehicle control + normal diet) n=12 Model group (STZ + high fat diet) + vehicle control (0.5% HPMC, potid) n=20 Model group (STZ + high fat diet) + pentoxifylline (100 mg/kg, po, tid) n=20 model group (STZ + high fat diet) + compound 1 (30 mg/kg, po, bid) n=19 banned for 5 weeks after continuous administration food. The mice were sacrificed by CO ₂ , and the heart was taken for 2 hours at room temperature. The blood samples were centrifuged (2000 g, 4 ° C, 15 minutes), and the supernatant was separated, frozen, and stored at -80 °. C refrigerator. Liver tissue was dissected for histological analysis and biomarker detection.

Frozen serum samples were tested for ALT, AST, TG and TC the following day.

3. Histological analysis method.

3.1 HE staining method.

3.1.1 Organizational processing.

Using the Leica ASP200S automatic dehydrator, the relevant dehydration steps are as follows: a) 10% neutral buffered formalin for 5 minutes to 6 hours b) 70% ethanol for 45 minutes c) 80% ethanol for 45 minutes d) 95% ethanol 45 Minute e) 95% ethanol 45 minutes f) 100% ethanol 60 minutes g) 100% ethanol 60 minutes h) 100% ethanol 60 minutes i) xylene 60 minutes j) xylene 60 minutes k) paraffin 60 minutes l) paraffin 60 Minute m) paraffin 60 minutes

3.1.2 Organizational embedding. a) Preparation before embedding. Composition and function of the Leica tissue embedding system: The Leica tissue embedding system consists of two parts: the embedding table (Leica EG1150 H) and the freezing station (Leica EG1150 C). The embedding station includes a left metal mold storage cylinder, a right side tissue cassette storage cylinder and a paraffin bath on the upper part of the instrument. There is a small cold stage at the front of the embedding station that can quickly cool a single embedding mold. The freezing station is mainly composed of a cold stage, and rapidly cools the paraffin embedded in the mold. After the cassette is cooled, the wax block is stored at room temperature. b) Turn on the cold stage. c) Remove the cassette from the dehydrator and place it in the wax bath of the embedding machine d) Embed. The hot tweezers are taken out of the tissue cassette in the embedding machine wax bath and placed on the hot stage of the embedding machine. Place the embedding box corresponding to the tissue on the mold. The tissue is placed in the embedding mold with hot tweezers, and a little dissolved paraffin is added to the embedding mold, and the embedding mold is placed on the cold table until the paraffin becomes completely solid. Finally, the solid wax block is taken out of the embedding mold.

3.1.3 Preparation of tissue sections. a) Confirm the status before using the Leica RM2235 paraffin slicer. Before loading the wax block, make sure that the slice wheel handle is loosely locked, open the knife holder cover, and clean the wax on the knife holder. Carefully insert an unused disposable knife to confirm that the blade is in place at home and lock the knife holder according to the manufacturer's instructions. Confirm the inclination of the blade, which should be set to 3-6 degrees. b) Fix the tissue wax block to the desired position in the clipper slot. Follow the manufacturer's instructions for roughing with the roughing wheel handles so that the wax block slowly advances until the desired tissue plane is exposed. c) Start the slicing, first set the slice thickness, start slicing, and rotate the slicing wheel forward until the flat wax sheet is formed. Lock the slice rotation handle and use a forceps or brush to move the tissue piece into the spreader (Leica HI1210) and flatten it onto the slide. d) After roughing or slicing, lock the wheel handle and carefully remove the wax block.

3.1.4 HE staining.

HE staining was performed using a fully automated staining workstation consisting of Leica ST5010, Leica TS5010 and Leica CV5030.

Press the <Stain> key in the main menu of the dyeing machine. Conventional HE staining is performed by the up and down arrow selection procedure 15 of the button area. Open the load compartment, place the slicer, and close the load compartment. Start the dye program after pressing the <Load> button. The procedure is as follows: a) Oven 55 ° C for 2 minutes b) Xylene I 10 minutes c) Xylene II 10 minutes d) Xylene III 10 minutes e) Anhydrous ethanol I 2 minutes f) Anhydrous ethanol II 1.5 minutes g) 95% ethanol I 1 min h) 80% Ethanol I 0.5 min i) Washed for 3 minutes j) Hematoxylin dye solution for 3 minutes k) Washed for 2 minutes l) 1% Hydrochloric acid-ethanol 5 seconds m) Washed for 2 minutes n) Washed for 20 minutes o) 80% ethanol II 1 minute p) Eosin 5 seconds q) 95% ethanol II 30 seconds r) absolute ethanol III 2 minutes s) absolute ethanol IV 2 minutes t) absolute ethanol V 2 minutes u) absolute ethanol VI 2 minutes v) xylene IV 3 minutes w) xylene V 3 minutes x) xylene V 3 minutes y) EXIT xylene 3 minutes z) Leica CV5030 Sections were mounted automatic capping machine

3.2 Sirius red staining method.

The tissue white sheets prepared in 3.1.3 were hand-made into Sirius red stained sections by the following steps: a) xylene I 15 min b) xylene II 15 min c) absolute ethanol I 3 min d) absolute ethanol II 3 min e 95% ethanol I 3 minutes f) 95% ethanol II 3 minutes g) 80% ethanol 3 minutes h) 70% ethanol 3 minutes i) distilled water I 3 minutes j) distilled water II 3 minutes k) distilled water III 3 minutes l) Sirius Red dye solution 1 hour m) distilled water for 5 minutes n) 95% ethanol III 30 seconds o) absolute ethanol III 2 minutes p) absolute ethanol IV 2 minutes q) absolute ethanol V 2 minutes r) absolute ethanol VI 2 minutes s) Toluene IV 15 minutes t) xylene V 15 minutes u) neutral gum seal

3.3 Histological non-alcoholic fatty liver disease activity score (NAS) related scoring criteria.

The histological diagnosis and clinical efficacy evaluation of nonalcoholic fatty liver disease (NAFLD) was performed according to the National Institutes of Health's NASH Clinical Research Network Pathology Working Group Guidelines, and the NAFLD activity score (NAS) was routinely performed.

NAS score (0-8 points): a) Hepatocyte steatosis: 0 points (<5%); 1 point (5% to 33%); 2 points (33% to 66%); 3 points (>66%) ). b) Inflammation in the lobules (20-fold microscopic count necrosis): 0 points, none; 1 point (<2); 2 points (2 to 4); 3 points (>4). c) Hepatocyte balloon-like changes: 0 points, no; 1 point, rare; 2 points, more common.

3.4 Fibrosis scoring criteria.

Stage of liver fibrosis (0 ~ 4): a) 0 points: no fibrosis; b) 1 point: 1A, mild sinus fibrosis in the liver acinar 3 area; 1B, moderate sinus fibers in the liver acinar 3 area 1C, only fibrosis around the portal vein; c) 2 points: fibrosis around the anterior segment of the hepatic acinar 3 area and fibrosis around the portal vein; d) 3 points: bridging fibrosis; and e) 4 points: highly suspicious Or diagnose liver cirrhosis.

4. Method for detecting liver tissue biomarker mRNA.

RNA was extracted using the RNeasy® Mini RNA Extraction Kit (Qiagen #74104) according to the operating manual. Reverse transcription was performed on the same day. The reverse transcription system ratio (ABI # 4368814) was as follows: 10×RT buffer 2 μL 25×dNTP mixture (100 mM) ) 0.8μL 10×RT random primer 2μL MultiScribe reverse transcriptase 1μL template RNA 1μg RNase-free H ₂ O added to 20μL

Reverse transcription program:

The cDNA was stored in a refrigerator at -20 °C.

Q-PCR was performed according to the instructions of TaqMan® Fast Universal Kit (ABI, # 4366072). The system is equipped with the following formula: TaqMan® Gene Expression Master Mix (2✕) 5μL TaqMan® Gene Expression Assay (20✕) 0.5μL cDNA template 0.8μL H ₂ O 3.7μL

Q-PCR program:

Experimental results.

1. Histological results.

1.1 HE stained picture.

HE staining results showed that liver steatosis was significantly increased in nSTZ+HFD mice, balloon-like changes in hepatocytes, and increased hepatic lobular inflammation, pentoxifylline (100mpk, po., tid) and compound 1 (30mpk, po., Bid) 5 weeks of treatment did not significantly improve the pathological changes in liver tissue, such as fatty degeneration, balloon degeneration and inflammatory aggregation in nSTZ+HFD mice. The experimental results are shown in Figure 8.

1.2 NAS score.

The liver histology of NAS in nSTZ+HFD mice was significantly increased, liver fatification was significantly increased, a small number of hepatocytes were balloon-like, and hepatic lobular inflammation was significantly increased. Treatment with pentoxifylline (100 mpk, po., t.i.d.) and Compound 1 (30 mpk, po., b.i.d.) for 5 weeks did not significantly affect liver NAS in nSTZ+HFD mice. The experimental results are shown in Figure 9.

1.3 Sirius red stained picture.

The results of Sirius red staining showed that the liver tissue of nSTZ+HFD mice showed more fibrotic changes around the sinus and 3 areas combined with portal vein. See also suspected bridging fibrosis in individual samples. The 5-week treatment group of pentoxifylline (100mpk, po., tid) and compound 1 (30mpk, po., bid) alleviated the degree of fibrosis in the above-mentioned liver tissue of STZ+HFD mice. 1 (30mpk, po., bid) The 5-week treatment group significantly improved the morphological changes of fibrosis around the sinus and portal vein in the liver tissue. The experimental results are shown in Figure 10.

1.4 Fibrosis score.

The liver fibrosis score of nSTZ+HFD mice increased significantly. The 5-week treatment of pentoxifylline (100mpk, po., tid) reduced the liver fibrosis trend of nSTZ+HFD mice, but statistically, there was no significantity. Compound 1 (30mpk, po., bid) 5 weeks of treatment significantly reduced liver fibrosis scores in nSTZ+HFD mice. The experimental results are shown in Figure 11.

2. Liver biomarker mRNA detection results. The TNF-a and TGF-b1 mRNA in the liver of nSTZ+HFD mice increased significantly, and the levels of TNF-a and TGF-b1 mRNA in the liver of nSTZ+HFD mice were not significantly treated with pentoxifylline (100mpk, po., tid) for 5 weeks. Sexual effects, Compound 1 (30mpk, po., bid) The levels of TNF-a and TGF-b1 mRNA in the liver of nSTZ+HFD mice were significantly decreased after 5 weeks of administration. The mRNA of Collagen 1a1 in nSTZ+HFD mice increased, and the growth of Collagen mRNA in nSTZ+HFD mice decreased after compound 5 (30mpk, po., bid) for 5 weeks, but the statistical analysis was not significant. Further research is needed. analysis. The experimental results are shown in Figure 12.

3. Results of serum biochemical indicators.

The serum ALT and TC levels of nSTZ+HFD mice were significantly increased. The treatment with pentoxifylline (100mpk, po., tid) and compound 1 (30mpk, po., bid) for 5 weeks did not significantly affect the serum of nSTZ+HFD mice. ALT and TC levels. The experimental results are shown in Figure 13.

to sum up. 1. Newborn rats were injected with low-dose streptozotocin (STZ) and fed a high-fat diet for 1 week to form diabetes with fatty liver. A persistent high-fat diet results in liver fat deposition, increased hepatic lobular inflammation, and is accompanied by foam-like macrophage infiltration. The nSTZ+HFD mouse model mimics the pathological progression of human diabetes-nonalcoholic steatohepatitis-hepatocellular carcinoma, and is a widely used animal model of nonalcoholic steatohepatitis. Liver histology HE staining images of nSTZ+HFD model mice showed obvious steatosis, balloon-like changes and lobular inflammation, Sirius red staining showed obvious fibrosis, liver inflammation biomarker TNF-a mRNA increased significantly, fibrosis The biomarker TGF-b1 mRNA was significantly increased, and serum ALT and TG levels were significantly higher than normal mice. 2. Compound 1 (30mpk, po., b.i.d.) After 5 weeks of administration, liver fibrosis was significantly improved in nSTZ+HFD mice, and liver TNF-a and TGF-b1 mRNA levels were significantly decreased. 3. Compound 1 (30mpk, po., b.i.d.) 5 weeks of administration had no significant effect on liver NAS, serum ALT, AST, TG and TC levels in nSTZ+HFD mice.

Experimental Example 6: Efficacy study of anti-rat cholestasis liver fibrosis.

Main task: To evaluate the effect of Compound 1 on liver histology in rat cholestasis-type liver fibrosis model, as well as serum ALT, AST, TG and TC levels.

The proposed problem: the effect of test compound 1 on liver histology, serum ALT, AST, TG and TC levels in rat cholestasis liver fibrosis model, and evaluation of compound liver function protection in rat cholestasis liver fibrosis model, Reduces the effects of liver inflammation and fibrosis.

Experimental model: (ANIT) induced rat model of cholestasis-induced liver fibrosis.

Experimental animals: SD rats (male, Shanghai Slack Laboratory Animals Co., Ltd.)

Experimental reagents: ANIT (1-NAPHTHYL ISOTHIOCYANATE, Aldrich-N4525); HPMC (Hydroxypropyl methyl cellulose, Sigma-H7509.

experimental method.

1. Experimental process.

2. In vivo experimental methods.

90 rats were ordered, and the animals were grouped on the same day according to the principle of the smallest difference between the groups, with 12 rats in each group and 4 cages per cage. After the animals arrive at the facility, the adaptation period is 5 days. During the adaptation period, animals are provided with normal diet and drinking water, and the health of the animals is monitored daily. If any abnormalities or infections are found, the rats need to be removed from the test group.

On day 0, the drug was administered first. After 2 hours, the normal feed in all the cages except the first group was changed to the ANIT supplement, which was the normal three-day intake of the rats (this amount is based on the previous experimental results). The ANIT additive is updated every three days. The first group of rats consumed normal feed throughout the experiment.

All rats were withdrawn from food on the morning of the 8th day until the end of the experiment, with a fasting time of more than 5 hours. Euthanasia method: Cardiac blood was sacrificed after anesthesia with pentobarbital sodium. (Collect as much blood as possible). 1) Collect 2 mL of blood; collect 750 μL of serum and dispense in 3 centrifuge tubes. (Serum collection method: 10000 rpm, centrifugation at 4 ° C for 10 minutes, collect serum, transfer to dry ice and transfer to -80 ° C for storage); 2) Collect animal liver, rinse with PBS; 3) Wipe dry with a sterile paper towel, weigh and Record; 4) Take a picture of the entire isolated liver; 5) From the largest diameter of the left lobe of the liver, cut a piece, about 3 mm wide, and immerse 10 times the tissue volume of 10% formalin solution (pure water configuration, need to adjust PH in advance) The value is neutral; 6) The remaining left hepatic lobe, take 100mg of two pieces, cut into small pieces and divide into two cryotubes for liver TG/TC detection; 7) Liver lobe, one point For the second part, each part is placed in two cryotubes.

3. Histological analysis method.

3.1 Organizational processing.

Dehydration of liver tissue, paraffin block production, paraffin section, HE stained paraffin piece thickness 3 μm, Sirius red stained paraffin piece thickness 4 μm. HE staining and Sirius red staining were performed according to the KCI pathological standard staining SOP. The HE stained films were scored by two pathologists (the criteria were found in the pathological report), and the mean values of the two groups were used to analyze the liver damage of each group. Degree; fibrosis stained sections were scanned on a Digital Pathscope 4S scanner and subjected to whole analysis.

3.2 rat cholestasis model inflammatory pathology scoring standard

Experimental results.

1. Histological results.

1.1 HE stained pictures and results.

According to the HE staining results (Fig. 14), the liver inflammation levels were scored according to the inflammatory pathology scoring criteria (Fig. 15), and the results were subjected to t-test. The healthy control group was significantly lower than the model group, but the score of the positive control INT-747 administration group was significantly increased. No improvement in hepatic inflammation was observed in pentoxifylline-100 mg/kg and Compound 1.

1.2 Sirius red staining picture and fibrosis score.

Liver tissue sections were stained by Sirius red (Fig. 16), and subjected to a full-scale scan of Digital pathscope 4S. The panoramic sections were analyzed to calculate the percentage of collagen fibers in the sections to determine the degree of fibrosis of each group of animals after compound treatment. Change (Figure 17). The results showed that the scores of the model group were significantly higher than those of the healthy control group, indicating that the liver fibrosis was successfully modeled. Compared with the model group, Compound 1 had an inhibitory effect on liver fibrosis (p=0.03) and inhibition rate of 17% in the highest dose group of the study at 10 mg/kg, and INT-747 for liver fibrosis. The degree of improvement was similar (p=0.008). However, pentoxifylline did not improve liver fibrosis at a dose of 100 mg/kg.

2. Results of serum biochemical indicators (Table 4).

Compared with the normal group, the ALT, AST, ALP, TBA, TB, DB and IB of the model group were significantly increased, indicating that the modeling was successful. Compared with the model group, the INT-747 group can reduce serum ALT and AST levels to 18% and 25%, respectively (mean comparison), but there is no statistical difference in this trial; Both ketomatole and Compound 1 did not significantly reduce the effects of AST and ALT. None of the drug-administered groups reduced the level of ALP in the serum.

The level of total bile acid (TBA) was significantly higher in the model group than in the normal group, which was about 9 times higher. Compared with the model group, the TBA level of the INT-747, 20 mg/kg treatment group was significantly reduced by about 75%. The Compound 1 treatment group had a certain effect on reducing total bile acid, which was 37%, 26%, and 33%, respectively, but was not statistically significant.

The results of TB, DB, and IB showed that the values of the model group were significantly higher than those of the normal control group, which were 53-fold, 62-fold, and 36-fold higher, respectively, while the drug-treated group was positive compared with the model group. The control group INT-747 significantly reduced the effects of TB, DB, IB, respectively, by 53%, 54%, 49%; Compound 1 decreased the levels of TB, DB, IB under high dose, and can The corresponding dose dependence was seen, but there was no statistically significant experimental result compared to the model group.

Summary: 1) In the ANIT-induced cholestasis-induced liver fibrosis rat model, the serum ALT/AST/ALP levels in the model group were significantly higher in the serum than in the healthy control group; TBA/TB/DB/IB The results in the serum were also significantly increased; in the inflammation scores, the degree of fibrosis was significantly increased in the model group compared with the healthy control group; in the pathological fibrosis results, compared with the healthy control group, the model group The degree of fibrosis is significantly increased. 2) Compound 1 (10mpk, po., b.i.d.) treatment group showed significant anti-fibrotic effect on the degree of pathological fibrosis. 3) Compound 1 treatment groups had no significant effect on serum ALT, AST, TG and TC levels.

Experimental Example 7: Study on the efficacy of a carbon tetrachloride-induced mouse liver fibrosis model.

Main Task: To evaluate the effect of Compound 1 on liver histology of serum carbon tetrachloride-induced liver fibrosis in mice, as well as serum ALT, AST, TG and TC levels.

Proposed problem: Test compound 1 on carbon tetrachloride-induced liver fibrosis model in mice liver histology, serum ALT, AST, TG and TC levels, evaluation of compounds on carbon tetrachloride-induced mouse liver fibers The liver function protection of the model reduces the effects of liver damage and fibrosis.

Experimental model: a model of liver fibrosis induced by carbon tetrachloride in mice.

Experimental animals: C57BL/6 mice (male, Shanghai Lingchang Biotechnology Co., Ltd.)

Experimental reagents: 1. CCl ₄ (Jiangsu Qiangsheng Functional Chemical Co., Ltd.) 2. Olive oil (Aladdin Industries, 0108686-500ml) 3. HPMC (Hydroxypropyl methyl cellulose, Sigma-H7509)

experimental method.

1. Experimental process.

2. In vivo experimental methods.

108 mice were ordered, of which 3 were reserved. On the day the animals arrived, they were grouped according to the principle of the smallest difference between the groups, with 15 in each group. After the animals arrive at the facility, the adaptation period is 5 days. During the adaptation period, the health of the animals was monitored daily. If any abnormalities or infections are found, the mice will need to be removed from the test group. The 25% CCl ₄ solution is prepared in pure olive oil and is ready for use. Except for the animals of the first group, the injection time was on day 0, day 4, day 8, and day 12, and all other groups of mice were intraperitoneally injected, and administered according to the body weight, 2 mL/kg. The first group of mice was injected intraperitoneally with pure olive oil.

All mice were withdrawn from food on the morning of the 14th day until the end of the experiment, with a fasting time of more than 5 hours. Euthanasia method: After anesthesia, blood was collected from the posterior venous plexus, and the neck was removed to confirm death. (Collect as much blood as possible). 1) Collect as much blood as possible; operate to collect 250 ul of serum and dispense into the appropriate centrifuge tube. (Serum collection: 10000 rpm, centrifugation at 4 degrees for 10 minutes, collect serum, transfer to dry ice and transfer to -80 °C.) 2) Collect animal liver, rinse with PBS 3) Wipe dry with sterile paper towel, weigh and record 4) Take a picture; 5) Cut a piece from the largest diameter of the left lobe of the liver, about 3mm wide, and immerse it in 10 times volume of 10% formalin solution (pure water configuration, need to adjust PH value in advance) Placed vertically and fixed; 6) The remaining left hepatic lobe, cut into small pieces and divided into two cryotubes for detecting liver TG/TC; 7) The middle lobe of the liver, divided into two, placed in two cryotubes In, as a sample for protein analysis and mRNA detection.

It should be noted that the samples of samples 6) and 7) need to be quickly immersed in liquid nitrogen after collection, and it is necessary to ensure that the cryotubes are completely immersed in liquid nitrogen after being thrown into liquid nitrogen.

3. Histological analysis method.

3.1 Organizational processing.

Dehydration of liver tissue, paraffin block production, paraffin section, HE stained paraffin piece thickness 3 μm, Sirius red stained paraffin piece thickness 4 μm. HE staining and Sirius red staining were performed according to the KCI pathological standard staining SOP. HE-stained films were scored by two pathologists, and the mean values of the two groups were used to analyze the degree of liver damage in each group; fibrosis stained sections. After a full scan of the Digital Pathscope 4S scanner, a full analysis was performed.

3.2 Carbon tetrachloride-induced pathological scoring criteria for animal models of liver injury.

Experimental results.

1. Histological results.

1.1 HE stained pictures and results.

Liver pathological sections were scored by HE staining (Fig. 18) according to pathological scoring criteria for inflammation, ballooning, watery degeneration and necrosis. The results showed that compared with the healthy control group, the scores of the four indicators in the model group were extremely significantly increased, which proved that the modeling was successful (Fig. 19, Fig. 20, Fig. 21, Fig. 22).

Compared with the model group, the scores of watery degeneration and necrosis in the three dose groups of Compound 1 were lower than those in the model group (both statistically significant), and the effect showed a dose-dependent manner. The improvement of watery degeneration and necrosis by the compound 1-10 mg/kg group was comparable to that of the pentoxifylline-100 mg/kg group. Compared with the model group, the water-like degeneration and necrosis scores of the compound 1-10 mg/kg were decreased by 0.93 and 0.86, respectively, and the water-like degeneration and necrosis scores of the pentoxifylline-100 mg/kg group were decreased by 0.90 and 0.67, respectively.

The sum of the scores of the four indicators showed that each test compound had a significant improvement in the sum score. There was a significant improvement in liver damage in the three test doses of Compound 1, with a dose-related effect comparable to pentoxifylline - 100 mg/kg (Figure 23). 1.2 Sirius red staining picture and fibrosis score.

The Sirius red staining results (Fig. 24) read the fibrosis area (Fig. 25). The scores of the model group were significantly higher than those of the healthy control group, indicating that the liver fibrosis was successfully modeled. Compared with the model group, the high, medium and low dose groups of Compound 1, pentoxifylline and INT-747 all significantly reduced fibrosis of liver tissue.

2. Results of serum biochemical indicators (Table 5).

Compared with the normal group, the ALT, AST, TG, and TC of the model group were significantly increased, indicating that the modeling was successful.

Compared with the model group, Compound 1 had a certain effect of lowering ALT and AST, and it was dose-dependent. Compound 1 reduced ALT by 24%, 52% and 56% at doses of 1, 3 and 10 mg/kg, respectively. AST decreased by 23%, 42%, and 52%, respectively. However, only the 1-10 mg/kg dose of the compound had a statistically significant change in AST (p=0.02). INT-747 significantly reduced serum ALT and AST levels by 75% and 64%, respectively, and INT-747 also decreased TG and TC by 11% and 12%, respectively. Compared with the model group, pentoxifylline did not improve ALT, AST, TG, and TC.

Summary : 1) Three doses of Compound 1 can reduce ALT/AST in serum to a certain extent, and show a dose-dependent manner, especially the highest dose of 10 mg/kg can significantly reduce AST; 2) staining with HE As a result, Compound 1 can significantly improve liver damage, including inflammation level, degree of watery degeneration, and degree of necrosis. The improvement effect of these indicators on the 10 mg/kg treatment group is the strongest among the three doses; 3) Compound 1 At the three test doses, the degree of liver fibrosis can be significantly reduced.

Figure 1: Liver HE staining of MCD mice; Figure 2: Liver histology scores of MCD mice, data expressed as mean ± standard error, n = 8, ####p<0.0001, ###p<0.001 vs. MCD control diet + vehicle control group, ***p<0.001 vs. MCD diet + vehicle control group, one-way repeated variance analysis and Tukey's group comparison; Figure 3: Effect of compound 1 on liver TG level in MCD mice , the data is expressed as mean ± standard error, n = 7 ~ 8. ####p<0.0001 vs. MCD control diet + vehicle control group. Single factor repeat analysis of variance and Tukey's group comparison, the overflow value (> mean + 2 ✕ standard deviation, or < mean - 2 ✕ standard deviation) has been removed; Figure 4: Compound 1 on MCD mouse liver TNF-α mRNA levels Impact. Data are expressed as mean ± standard error, n = 7 ~ 8. #p<0.05 vs. MCD control diet + vehicle control group. Single factor repeat analysis of variance and Tukey's group comparison, the overflow value (> mean + 2 ✕ standard deviation, or < mean - 2 ✕ standard deviation) has been removed; Figure 5: Compound 1 inhibits liver IL-1b levels in MCD mice , the data is expressed as mean ± standard error, n = 7 ~ 8. ###p<0.001 vs. MCD control diet + vehicle control group, *p<0.001 vs. MCD diet + vehicle control group. Single factor repeat analysis of variance and Tukey's group comparison, the overflow value (> mean + 2 ✕ standard deviation, or < mean - 2 ✕ standard deviation) has been removed; Figure 6: Compound 1 inhibits MCD mouse plasma TNF-a and IL-1b level, the data is expressed as mean ± standard error, n = 7 ~ 8. ####p<0.0001 vs. MCD control diet + vehicle control group. *p<0.05, **p<0.01 vs. MCD diet + vehicle control group, one-way repeat analysis of variance and pairwise comparison between Uncorrected Fisher's LSD group, overflow value (>mean + 2 ✕ standard deviation, or < mean - 2 ✕ standard deviation) has been removed; Figure 7: Effect of Compound 1 on plasma ALT and AST levels in MCD mice, data are expressed as mean ± standard error, n = 7-8. ####p<0.0001 vs. MCD control diet + vehicle control group. Single factor repeat analysis of variance and Tukey's group comparison, the overflow value (> mean + 2 ✕ standard deviation, or < mean - 2 ✕ standard deviation) has been removed; Figure 8: nSTZ + HFD mouse liver HE staining picture: A Control group (STZ solvent control + normal diet) B. Model group (STZ + high fat diet) + vehicle control (tid); C. Model group (STZ + high fat diet) + pentoxifylline (100 mg/kg, Po, tid); and D. Model group (STZ + high fat diet) + Compound 1 (30 mg/kg, po, bid); Figure 9: Liver histology NAS score of nSTZ+HFD mice. Data are expressed as mean ± standard error, n = 12 ~ 17. ####p<0.0001, ##p<0.01 vs. control group. Univariate repeated analysis of variance and pairwise comparison between Uncorrected Fish's LSD groups; Figure 10: Liver Sirius red staining of STZ+HFD mice; Figure 11: Liver fibrosis score of nSTZ+HFD mice. Data are expressed as mean ± standard error, n = 3, ##p < 0.01 vs. control group, *p < 0.05 vs. model group + vehicle control. Univariate repeated analysis of variance and a pairwise comparison between Uncorrected Fish's LSD groups; Figure 12: Effect of Compound 1 on liver biomarker mRNA levels in nSTZ+HFD mice. Data are expressed as mean ± standard error, n = 4 to 20. #p <0.05, ##p <0.01 vs. control group, *p <0.05 vs. model group + vehicle control. One-way repeated analysis of variance and pairwise comparisons between Uncorrected Fish's LSD groups, spillover values (>mean + 2✕ standard deviation, or <mean-2✕ standard deviation) were removed; Figure 13: Compound 1 vs. nSTZ+HFD mouse serum The impact of biochemical indicators. Data are expressed as mean ± standard error, n = 15~17. ###p<0.001,####p<0.0001 vs. control group. One-way repeated analysis of variance and pairwise comparisons between Uncorrected Fish's LSD groups, spillover values (>mean + 2✕ standard deviation, or <mean-2✕ standard deviation) were removed; Figure 14: Cholestatic liver fibrosis model rats Liver HE staining image, 100x magnification. A: group-1, healthy control; B: group-2, model control; C: group-3 INT-747-20 mg/kg, QD; D: group-4, pentoxifylline-100 mg/kg, TID; E: Group-5, compound 1-1 mg/kg, BID; F: group -6, compound 1-3 mg/kg, BID; G: group -7, compound 1-10 mg/kg, BID. Figure 15: Liver pathological inflammation score in cholestasis-type liver fibrosis model rats. Data are indicated as mean ± SEM, n = 12. ***p<0.001 vs model group; Figure 16: Liver Sirius red staining picture of cholestasis-type liver fibrosis model, 50-fold magnification. A: group-1, healthy control; B: group-2, model control; C: group-3 INT-747-20 mg/kg, QD; D: group-4, pentoxifylline-100 mg/kg, TID; E: Group-5, compound 1-1 mg/kg, BID; F: group -6, compound 1-3 mg/kg, BID; G: group -7, compound 1-10 mg/kg, BID. Figure 17: Liver fibrosis area in a rat model of cholestasis liver fibrosis. Data were expressed as mean ± SEM, n = 12, compared with the Vechile control group, *p < 0.05, *** p <0.001; Figure 18: Liver HE staining of mice with liver fibrosis induced by CCl ₄ , 100 times magnification. A: group-1, healthy control; B: group-2, model control; C: group-3 INT-747-20 mg/kg, QD; D: group-4, pentoxifylline-100 mg/kg, TID; E: Group-5, compound 1-1 mg/kg, BID; F: group -6, compound 1-3 mg/kg, BID; G: group -7, compound 1-10 mg/kg, BID; : CCl ₄ induced liver pathological inflammation score in mice with liver fibrosis model. Data are indicated as mean ± SEM, n = 15. Compared with model group, *** p <0.001, ** p <0.01, * p <0.05; FIG. 20: CCl ₄ induced liver fibrosis model mouse liver ballooning degeneration scores. Data were expressed as mean ± SEM, n = 15, compared to the model group, ***p < 0.001, **p < 0.01, *p <0.05; Figure 21: CCl ₄ induced mouse liver fibrosis model mice Liver watery degeneration score. Data were expressed as mean ± SEM, n = 15, compared to the model group, ***p < 0.001, **p < 0.01, *p <0.05; Figure 22: CCl ₄ induced mouse liver fibrosis model mice Liver necrosis score. Data were expressed as mean ± SEM, n = 15, compared to the model group, ***p < 0.001, **p < 0.01, *p <0.05; Figure 23: CCl ₄ induced mouse liver fibrosis model mice Comprehensive score for liver damage. Data were expressed as mean ± SEM, n = 15, compared to the model group, ***p < 0.001, **p < 0.01, *p <0.05; Figure 24: CCl ₄ induced mouse liver fibrosis model mice Liver Sirius red staining picture, 50x magnification: A: group-1, healthy control; B: group-2, model control; C: group-3 INT-747-20mg/kg, QD; D: group-4, Ketocomroline-100mg/kg, TID; E: Group-5, compound 1-1 mg/kg, BID; F: Group-6, compound 1-3 mg/kg, BID; G: Group-7, Compound 1 -10 mg / kg, BID; and FIG. 25: CCl ₄ induced liver and mouse liver fibrosis model the area of fibrosis. Data are presented as mean ± SEM, n = 15, compared to the model group, *p < 0.05, *** p < 0.001.

Claims (8)

The use of a compound of the formula (I), a tautomer thereof or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment or prevention of liver disease, (I) where the structural unit can be Replace with , specifically replaced by L _{11 is} selected from the group consisting of N, C(R)(R'); R and R' are each independently selected from H, halogen, OH, NH ₂ , CN, optionally substituted 1 to 6 alkyl or heteroalkyl Optionally, R and R' may be cyclized to a 3-6 membered cycloalkyl or heterocycloalkyl group; A is vacant or is selected from optionally substituted cycloalkyl, heterocycloalkyl, aryl, a heteroaryl group; L _{12 is} selected from an optionally substituted 1 to 6 membered alkyl or heteroalkyl group; and R _{1 is} selected from an optionally substituted 1 to 6 membered alkyl group, a 3 to 6 membered cycloalkyl group or a heteroalkyl group. And "hetero" represents N, O, S, C(=O), S(=O), S(=O) ₂ , and the number of heteroatoms on each group is selected from 1, 2, 3 or 4 Specifically, the disease is selected from the group consisting of nonalcoholic steatohepatitis and liver fibrosis. The use of claim 1, wherein the substituents in R, R', A, L ₁₂ and R ₁ are each independently selected from the group consisting of halogen, OH, NH ₂ , CN, optionally substituted 1 to 6 alkyl groups. a 3-6 membered cycloalkyl or heteroalkyl group, the number of each group substituent being independently selected from 1, 2 or 3; specifically, a substituent in R, R', A, L ₁₂ , R ₁ Individually selected from the group consisting of halogen, CF ₃ , CN, OH, Me, Et, n-propyl, isopropyl, cyclopropyl, , . The application of claim 1 or 2, wherein R, R' are each independently selected from the group consisting of H, Me, CF ₃ , Et; and specifically, L _{11 is} selected from , , , , , , . The use according to claim 1 or 2, wherein A is selected from the group consisting of optionally substituted: a 3- to 12-membered cycloalkyl or heterocycloalkyl group, a 5- to 12-membered aryl group or a heteroaryl group; Optional substituted: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, epoxypentyl, phenyl, pyridyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, bicyclic [1.1.1] Pentane, or a bicyclo, spiro or anthracene ring group selected from any two of the above groups; specifically, A is selected from the group consisting of: , , , , , , , , , , , , , , And specifically, A is selected from , , , , , , , , , , , , , , , , , , , , , , , , , , , , . The application of claim 1 or 2, wherein L _{12 is} selected from the group consisting of methylene, , , , , , . The use of claim 1 or 2, wherein R _{1 is} selected from the group consisting of Me, CHF ₂ , CF ₃ , Et, CH ₂ CF ₃ , isopropyl, , cyclopropyl, , , , . The use of the compound of the formula in the preparation of a medicament for treating or preventing liver disease: ; ;as well as . The use of the compound of the formula in the preparation of a medicament for treating or preventing liver disease: .