Use of hydroxypurine compounds for treating skin diseases

Abstract

The present invention discloses the use of a series of hydroxypurine compounds in preparing drugs for treating skin diseases, and specifically discloses the use of a compound represented by formula (I), its tautomers or pharmaceutically acceptable salts thereof in preparing drugs for treating skin diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicines for treating skin diseases, and in particular to the application of a series of hydroxypurine compounds in preparing medicines for treating skin diseases, in particular atopic dermatitis. Technical Background

[0002] Phosphodiesterase (PDE) catalyzes the hydrolysis of cyclic nucleotides cGMP and cAMP, and regulates various physiological reactions by controlling the intramolecular concentrations of these two important secondary signaling factors. Abnormal regulation of cyclic nucleotides cGMP and cAMP molecules is the cause of many diseases. Now there are many drugs that improve and treat diseases by inhibiting PDE activity, such as PDE5 inhibitors for pulmonary hypertension and PDE4 inhibitors for arthritis caused by psoriasis. There are eleven major categories of phosphodiesterase genes known so far, and each category can express several subtypes, with a total of more than 100 PDE subtypes. Different subtypes have different structures, different tissue distributions, and very different catalytic activities for cyclic nucleotides cGMP and cAMP, and the physiological functions they regulate are also very different.

[0003] Tumor necrosis factor alpha (TNF-α) is a cytokine with multiple biological activities, which has an important influence on the occurrence, development and treatment of many diseases, especially those related to immunity and inflammation. TNF-α is mainly produced by monocytes and macrophages, 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 has adverse effects in some cases. Studies have shown that excessive expression of TNF-α can induce the expression of proinflammatory cytokines such as interleukin-1 (IL-1) and IL-6, increase endothelial cell permeability, upregulate adhesion molecule expression, activate neutrophils and eosinophils, and induce synovial cells and chondrocytes to secrete acute phase substances and tissue degrading enzymes, which promote the occurrence of inflammation. These pathological reactions play a very important role in the occurrence and development of many immune-mediated inflammatory diseases (IMIDs), such as rheumatoid arthritis (RA), 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. At the same time, for some diseases caused by long-term chronic inflammatory damage, such as fatty hepatitis and chronic obstructive pulmonary disease, the use of TNF-α antagonists (TNF-αinhibitors) to neutralize excessive TNF-α is also an effective prevention and treatment approach. TNF-α monoclonal antibody drugs have been clinically proven to be very effective in treating the above-mentioned inflammatory-related diseases by inhibiting TNF-α.

[0004] PDE can regulate the expression of TNF-α from a mechanistic perspective, so the level of TNF-α can be controlled by regulating PDE activity, thereby controlling the inflammatory response.

[0005] Cyclic nucleotides cGMP and cAMP transduce external signals by activating key cell effectors such as protein kinase A (PKA). PKA inhibits the activity of many transcription factors through phosphorylation, including nuclear factor Kappa-light-chain-enhancer of activated T-cells (NFAT). These factors control the expression of inflammatory mediators such as interleukins IL-2, IL-4, IL-6, IL-31 and tumor necrosis factor TNF-α, and then regulate the inflammatory response of cells such as T lymphocytes and helper T lymphocytes (T-helper, Th) 2 cells, such as neutrophil degranulation, chemotaxis and endothelial cell adhesion. Therefore, the reduction of intracellular cAMP levels is related to the secretion of pro-inflammatory mediators.

[0006] Atopic dermatitis (AD) is a common pruritic skin disease with a relapsing-remitting process, which seriously affects the patient's physical and mental health and quality of life. Epidemiological studies show that the global incidence of AD in children is 20% and that in adults is 3%. The level of PDE4 in inflammatory cells in AD patients is significantly increased. PDE4 is one of the effective targets for the treatment of AD. PDE4 inhibitors reduce the release of downstream cytokines and chemokines by inducing an increase in intracellular cAMP and inhibiting NFAT and NFκB signaling pathways through PKA activation. Multiple in vitro experiments have shown that the production of prostaglandins E2, IL-4, and IL-10 in atopic leukocytes exposed to highly effective PDE4 inhibitors is significantly reduced. Subsequent in vivo experiments also confirmed this prediction. After PDE4 inhibition was applied to AD patients, the relevant inflammatory parameters were significantly reduced. This effective anti-inflammatory effect in vivo is the basic principle for the application of PDE4 inhibitors in the treatment of AD.

[0007] Crisaborole (AN2728) is a small molecule borylbenzoxolane PDE4 inhibitor that has been shown to improve the severity of the disease in AD patients. Currently, Crisaborole was approved by the FDA for the first time in 2016 and is the first new molecular entity approved by the US FDA in the past 15 years for the treatment of atopic dermatitis (eczema). In March 2020, the US FDA approved the expansion of the indications of Crisaborole ointment to children with mild to moderate atopic dermatitis aged 3 months. This supplemental approval makes it the first and only topical prescription drug suitable for children with mild to moderate AD aged 3 months.

[0008]

[0009] OPA-15406, another ointment formulation of PDE4 inhibitor, has achieved good efficacy in Phase I and II clinical trials for AD in patients aged 10 to 70 years. The pharmacokinetics of OPA-15406 were also evaluated in Phase I and Phase II clinical trials. The compound is highly selective for a PDE4B subtype, but also has an inhibitory effect on PDE2. Overall, OPA-15406 has been proven to be safe and well tolerated in all studies.

[0010]

[0011] European Patent EP544391, applied by Teva Pharmaceuticals on August 19, 1992, discloses the use of pentoxifylline for topical treatment of psoriasis and atopic dermatitis.

[0012]

[0013] Chinese patent ZL201580054840.9, applied by Guangdong Zhongsheng Ruichuang Biotechnology Co., Ltd., disclosed a series of hydroxypurine compounds on August 1, 2017, which have the function of inhibiting PDE2. The inventors were pleasantly surprised to find in subsequent studies that this series of compounds also have the function of inhibiting PDE4 and highly inhibit the PDE4B isoenzyme, thereby achieving the effect of treating skin diseases such as atopic dermatitis. Summary of the invention

[0014] The present invention provides the use of a compound represented by formula (I), its tautomer or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating skin diseases.

[0015]

[0016] in,

[0017] The structural unit can be Replaced with its tautomer derivative

[0018] L 11 Selected from absent, C(R)(R');

[0019] R, R' are independently selected from H, halogen, OH, NH2, CN, optionally substituted C 1～6 Alkyl or heteroalkyl;

[0020] Optionally, R and R' can be cyclized together with the carbon atom to which they are attached to form a 3-6 membered cycloalkyl or heterocycloalkyl group;

[0021] A is absent or optionally substituted cycloalkyl, heterocycloalkyl, aryl, heteroaryl;

[0022] L 12 Selected from optionally substituted C 1～6 Alkyl or heteroalkyl;

[0023] R1 is selected from optionally substituted C 1～6 Alkyl, 3-6 membered cycloalkyl or heteroalkyl;

[0024] "Hetero" represents N, O, S, C(=O), S(=O), S(=O)2, and the number of heteroatoms in each group is selected from 1, 2, 3 or 4.

[0025] In some embodiments of the present invention, the above R, R', A, L 12 , R1 substituents are independently selected from halogen, OH, NH2, CN, C 1～6 Alkyl, 3-6 membered cycloalkyl or heteroalkyl, the number of substituents in each group is independently selected from 1, 2 or 3.

[0026] In some embodiments of the present invention, Preferably

[0027] In some embodiments of the present invention, the above R, R', A, L 12 The substituents in R1 are independently selected from halogen, CF3, CN, OH, Me, Et, n-propyl, isopropyl, cyclopropyl,

[0028] In some embodiments of the present invention, the above R and R' are independently selected from H, Me, CF3, and Et.

[0029] In some embodiments of the present invention, the above L 11 Selected from

[0030] In some embodiments of the present invention, the above A is selected from optionally substituted: 3-12 membered cycloalkyl or heterocycloalkyl, 5-12 membered aryl or heteroaryl.

[0031] In some embodiments of the present invention, the above A is selected from the group consisting of optionally substituted: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, epoxypentyl, phenyl, pyridyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, bicyclo[1.1.1]pentane, or a bicyclic group, spirocyclic group or cyclocyclic group consisting of any two of the above groups.

[0032] In some embodiments of the present invention, the above A is selected from optionally substituted:

[0033] In some embodiments of the present invention, the above A is selected from

[0034] In some embodiments of the present invention, the above L 12 Selected from methylene,

[0035] In some embodiments of the present invention, the above R1 is selected from Me, CHF2, CF3, Et, CH2CF3, isopropyl, Cyclopropyl,

[0036] The present invention specifically relates to the use of the following compound in the preparation of drugs for treating skin diseases:

[0037]

[0038]

[0039] Furthermore, the present invention relates to the use of the compound of the following formula in the preparation of drugs for treating skin diseases:

[0040]

[0041]

[0042] The present invention relates to the use of the compound in preparing a drug for treating skin diseases, wherein the skin diseases are preferably atopic dermatitis, psoriasis, contact dermatitis, other eczematous dermatitis, and delayed hypersensitivity reaction; phytotoxic and solar dermatitis; seborrheic dermatitis, dermatitis herpetiformis; lichen planus, lichen sclerosus, lichen sclerosus atrophicus, pyoderma gangrenosum, cutaneous sarcoidosis, angioedema, vasculitis, toxic erythema, cutaneous eosinophilia, localized alopecia, male pattern baldness, Sweet's syndrome, Weyne-Kraft syndrome, multiple erythema; infectious or non-infectious cellulitis; panniculitis; cutaneous lymphoma, non-melanoma skin cancer or other dysplastic lesions; and drug-induced diseases, including fixed drug eruptions.

[0043] More preferably, the above-mentioned skin disease is selected from atopic dermatitis.

[0044] Furthermore, the present invention relates to the use of the compound in the preparation of a drug for treating skin diseases, wherein the compound is compound 1 as shown in the following formula, and the skin disease is atopic dermatitis.

[0045]

[0046] Related definitions

[0047] Unless otherwise specified, the terms and phrases used herein have the following meanings. A particular term or phrase should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0048] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0049] The term "pharmaceutically acceptable salt" refers to salts of compounds of the invention, prepared from compounds of the invention having specific substituents with relatively nontoxic acids or bases. When the compounds of the invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid and the like; also include 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 present invention contain basic and acidic functional groups, and thus can be converted into either base or acid addition salts.

[0050] Preferably, the neutral form of the compound is regenerated by contacting the salt with a base or acid in a conventional manner and isolating the parent compound. The parent form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents.

[0051] As used herein, "pharmaceutically acceptable salts" are derivatives of the compounds of the invention, wherein the parent compound is modified by salification with an acid or alkali. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of bases such as amines, alkali or organic salts of acid radicals such as carboxylic acids, and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound, such as salts formed with non-toxic inorganic or organic acids. Conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from the group consisting of 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptose, gluconic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxy, hydroxynaphthalene, isethionic acid, lactic acid, lactose, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, sulfamic acid, p-aminobenzenesulfonic acid, sulfuric acid, tannin, tartaric acid, and p-toluenesulfonic acid.

[0052] The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferably used.

[0053] In addition to the form of salts, compounds provided by the present invention also exist in prodrug forms. Prodrugs of compounds described herein easily undergo chemical changes under physiological conditions to be converted into compounds of the present invention. In addition, prodrugs can also be converted to compounds of the present invention by chemical or biochemical methods in vivo.

[0054] Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the present invention.

[0055] Certain compounds of the present invention may possess asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereomers, geometric isomers and individual isomers are all within the scope of the present invention.

[0056] Unless otherwise specified, wedge-shaped keys and dashed keys are used. To express the absolute configuration of a stereocenter, use Represents the relative configuration of a stereocenter. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, they include E and Z geometric isomers. Similarly, all tautomeric forms are also included within the scope of the present invention.

[0057] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention relates to all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present invention.

[0058] Optically active (R)- and (S)-isomers as well as D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), diastereomeric salts are formed with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is usually accomplished by using chromatography, which employs a chiral stationary phase and is optionally combined with chemical derivatization (e.g., carbamate formation from an amine).

[0059] The compounds of the invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

[0060] The term "pharmaceutically acceptable carrier" refers to any preparation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient. Representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, tackifiers, transdermal enhancers, etc. Their preparations are well known to those skilled in the art of cosmetics or topical drugs. For additional information about the carrier, reference may be made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0061] The term "excipient" generally refers to carriers, diluents and / or other media required to formulate an effective pharmaceutical composition.

[0062] With respect to a drug or pharmacologically active agent, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a drug or agent that is non-toxic but can achieve the desired effect. For oral dosage forms of the present invention, an "effective amount" of an active substance in the composition refers to the amount required to achieve the desired effect. The determination of the effective amount varies from person to person, depending on the age and general condition of the recipient, and also on the specific active substance. The appropriate effective amount in each case can be determined by a person skilled in the art based on routine experiments.

[0063] The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating a target disorder, disease, or condition.

[0064] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0065] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, including deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is a keto group (i.e., =O), it means that two hydrogen atoms are replaced. Keto substitution does not occur on aromatic groups. The term "optionally substituted" means that it may be substituted or not substituted, and unless otherwise specified, the type and number of substituents can be arbitrary on the basis of chemical achievable.

[0066] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, and each occurrence of R is an independent choice. In addition, combinations of substituents and / or variants thereof are permitted only if such combinations result in stable compounds.

[0067] When the number of a linking group is 0, such as -(CRR)0-, it means that the linking group is a single bond.

[0068] When one of the variables is selected from a single bond, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a single bond, it means that the structure is actually AZ.

[0069] When a substituent is absent, such as AX, where X is absent, it means that the structure is actually A.

[0070] When a substituent's bond can cross-link to two atoms on a ring, the substituent can be bonded to any atom on the ring. When a substituent is listed without indicating the atom through which it is bonded to the compound included in the chemical formula but not specifically mentioned, the substituent can be bonded through any atom. Combinations of substituents and / or their variants are permitted only if such combinations result in stable compounds. For example, the structural unit It means that it can be substituted at any position on the cyclohexyl group or cyclohexadiene.

[0071] Unless otherwise specified, the term "halo" or "halogen" by itself or as part of another substituent represents a fluorine, chlorine, bromine or iodine atom. In addition, the term "haloalkyl" is intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo (C1-C4) alkyl" is intended to include but is not limited to trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl and 3-bromopropyl, etc.

[0072] Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl.

[0073] "Alkoxy" represents an alkyl radical as defined above having the specified number of carbon atoms attached through an oxygen bridge. 1-6 Alkoxy groups include C1, C2, C3, C4, C5 and C6 alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy and S-pentoxy.

[0074] "Cycloalkyl" includes saturated cyclic groups, such as cyclopropyl, cyclobutyl or cyclopentyl. 3-7 cycloalkyl includes C3, C4, C5, C6 and C7 cycloalkyl. "Alkenyl" includes hydrocarbon chains of straight or branched configuration, wherein one or more carbon-carbon double bonds are present at any stable position on the chain, such as ethenyl and propenyl.

[0075] Unless otherwise specified, the term "hetero" means a heteroatom or heteroatom group (i.e., an atom group containing heteroatoms), including atoms other than carbon (C) and hydrogen (H) and atom groups containing these heteroatoms, for example, including oxygen (O), nitrogen (N), sulfur (S), silicon (Si), germanium (Ge), aluminum (Al), boron (B), =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)2N(H)- or -S(=O)N(H)-.

[0076] Unless otherwise specified, "ring" means substituted or unsubstituted cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, aryl or heteroaryl. The so-called ring includes a monocyclic, linked ring, spirocyclic, cyclic or bridged ring. The number of atoms on the ring is usually defined as the number of members of the ring, for example, "5-7 membered ring" means 5 to 7 atoms arranged around. Unless otherwise specified, the ring optionally contains 1 to 3 heteroatoms. Therefore, "5-7 membered ring" includes, for example, phenyl, pyridyl and piperidyl; on the other hand, the term "5-7 membered heterocycloalkyl ring" includes pyridyl and piperidyl, but does not include phenyl. The term "ring" also includes a ring system containing at least one ring, each of which "rings" independently meets the above definition.

[0077] Unless otherwise specified, the term "heterocycle" or "heterocyclyl" means a stable monocyclic, bicyclic or tricyclic ring containing a heteroatom or heteroatom group, which may be saturated, partially unsaturated or unsaturated (aromatic), and which contains carbon atoms 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 carbon ring to form a bicyclic ring. The nitrogen and sulfur heteroatoms may be optionally oxidized (i.e., N=O and S(=O)p, p is 1 or 2). The nitrogen atom may be substituted or unsubstituted (i.e., N or NR, where R is H or other substituents as defined herein). The heterocycle may be attached to any heteroatom or carbon atom side group to form a stable structure. The heterocycles described herein may be substituted at the carbon or nitrogen position if the resulting compound is stable. 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 heterocyclic ring does not exceed 1. As used herein, the term "aromatic heterocyclic group" or "heteroaryl" means a stable aromatic 5-, 6-, 7-membered monocyclic or bicyclic or 7-, 8-, 9- or 10-membered bicyclic heterocyclic group, which 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, where R is H or other substituents defined herein). The nitrogen and sulfur heteroatoms can be optionally oxidized (i.e., N=O and S(=O)p, where p is 1 or 2). It is worth noting that the total number of S and O atoms in the aromatic heterocyclic ring does not exceed 1. Bridged rings are also included in the definition of heterocyclic rings. A bridged ring is formed when one or more atoms (i.e., C, O, N or S) connect two non-adjacent carbon atoms or nitrogen atoms. The "bridge" in the preferred bridged ring includes, but is not limited to: one carbon atom, two carbon atoms, one nitrogen atom, two nitrogen atoms and a carbon-nitrogen group. It is worth noting that a bridge always converts a monocyclic ring into a tricyclic ring. In bridged rings, substituents on the ring may also appear on the bridge.

[0078] Examples of heterocyclic compounds include, but are not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromene, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuranyl , furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolene, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isoindolyl, isoindolyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxadiazolyl oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazine, phenothiazine, benzoxanthinyl, phenoloxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinine Cycyl, tetrahydrofuranyl, tetrahydroisoquinolyl, tetrahydroquinolyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, isothiazolylthienyl, thienyl, thienooxazolyl, thienothiazolyl, thienoimidazolyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl. Also included are fused ring and spirocyclic compounds.

[0079] Unless otherwise specified, the term "hydrocarbyl" or its subordinate concepts (such as alkyl, alkenyl, alkynyl, aryl, etc.) by itself or as part of another substituent means a straight-chain, branched or cyclic hydrocarbon radical or combination thereof, which may be fully saturated (such as alkyl), mono- or poly-unsaturated (such as alkenyl, alkynyl, aryl), mono- or poly-substituted, monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine), and may include divalent or polyvalent radicals, having a specified number of carbon atoms (such as C1-C 12 Indicates 1 to 12 carbons, C 1-12 Selected from C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C11 and C 12 ; C 3-12 Selected from C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 and C 12 . ). "Hydrocarbon" includes but is not limited to aliphatic hydrocarbon and aromatic hydrocarbon, the aliphatic hydrocarbon includes chain and ring, specifically but is not limited to chain and ring alkyl, alkenyl, alkynyl, the aromatic hydrocarbon includes but is not limited to 6-12 yuan aromatic hydrocarbon, such as benzene, naphthalene, etc. In some embodiments, the term "hydrocarbon" means a linear or branched atomic group or a combination thereof, which may be completely saturated, mono- or poly-unsaturated, and may include divalent and polyvalent atomic groups. Examples of saturated hydrocarbon atomic groups include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, isobutyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, and homologues or isomers of atomic groups such as n-pentyl, n-hexyl, n-heptyl, and n-octyl. Unsaturated hydrocarbon groups have one or more double or triple bonds, examples of which include, but are not limited to, ethenyl, 2-propenyl, butenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers.

[0080] Unless otherwise specified, the term "heteroalkyl" or its subordinate concepts (such as heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, etc.) by itself or in combination with another term refers to a stable straight-chain, branched or cyclic hydrocarbon radical or combination thereof, consisting of a certain number of carbon atoms and at least one heteroatom. In some embodiments, the term "heteroalkyl" by itself or in combination with another term refers to a stable straight-chain, branched hydrocarbon radical or combination thereof, consisting of a certain number of carbon atoms and at least one heteroatom. In a typical embodiment, the heteroatom is selected from B, O, N and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. The heteroatoms B, O, N and S can be located at any interior position of the heteroalkyl (including the position where the alkyl is attached to the rest of the molecule). Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(=O)-CH3, -CH2-CH2-S(=O)2-CH3, -CH=CH-O-CH3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, for example, -CH2-NH-OCH3.

[0081] The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are customary expressions referring to those alkyl groups which are attached to the remainder of the molecule via an oxygen atom, an amino group or a sulfur atom, respectively.

[0082] Unless otherwise specified, the term "alkyl" is used to refer to a straight or branched saturated hydrocarbon group, which may be monosubstituted (such as -CH2F) or polysubstituted (such as -CF3), and may be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (such as n-propyl and isopropyl), butyl (such as n-butyl, isobutyl, s-butyl, t-butyl), pentyl (such as n-pentyl, isopentyl, neopentyl), etc.

[0083] Unless otherwise specified, the terms "cycloalkyl", "heterocycloalkyl" or their subordinate concepts (such as aryl, heteroaryl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, etc.) by themselves or in combination with other terms refer to a cyclized "alkyl", "heteroalkyl", respectively. In addition, in the case of heteroalkyl or heterocycloalkyl (such as heteroalkyl, heterocycloalkyl), a heteroatom can occupy the position at which the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl 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-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuranindol-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl and 2-piperazinyl.

[0084] Unless otherwise specified, the term "aryl" means a polyunsaturated aromatic hydrocarbon substituent, which may be monosubstituted, disubstituted or polysubstituted, monovalent, divalent or polyvalent, and which may be monocyclic or polycyclic (preferably 1 to 3 rings), fused together or covalently linked. The term "heteroaryl" refers to an aryl (or ring) containing one to four heteroatoms. In an exemplary embodiment, the heteroatoms are selected from B, N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. The heteroaryl may be connected to the rest of the molecule via 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-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl and 6-quinolyl. Substituents for any of the above aryl and heteroaryl ring systems are selected from the acceptable substituents described below.

[0085] For simplicity, aryl when used in conjunction with other terms (e.g., aryloxy, arylthio, aralkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "aralkyl" is intended to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, etc.), including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced, for example, by an oxygen atom, e.g., phenoxymethyl, 2-pyridyloxymethyl 3-(1-naphthyloxy)propyl, etc.

[0086] The term "leaving group" refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (e.g., an affinity substitution reaction). For example, representative leaving groups include trifluoromethanesulfonate; chlorine, bromine, iodine; sulfonate groups, such as mesylate, tosylate, p-brosylate, p-toluenesulfonate, etc.; acyloxy groups, such as acetoxy, trifluoroacetoxy, etc.

[0087] 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: formyl; acyl, such as alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl, such as tert-butyloxycarbonyl (Boc); arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-bis-(4'-methoxyphenyl)methyl; silyl, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), etc. The term "hydroxy protecting group" refers to a protecting group suitable for preventing side reactions of the hydroxyl group. Representative hydroxy protecting groups include, but are not limited to, alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl (e.g., acetyl); arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like.

[0088] 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, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention.

[0089] All solvents used in the present invention were commercially available and used without further purification.

[0090] The present invention adopts the following abbreviations: Compound 1 is isomer 2 in Example 51 of WO2016054971; Pen. is pentoxifylline; INT-747 is 6-ethylchenodeoxycholic acid; aq represents water; HATU represents O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; EDC represents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; m-CPBA represents 3-chloroperbenzoic acid; eq represents equivalent; CDI represents carbonyldiimidazole; DCM represents dichloromethane; PE represents petroleum ether ; DIAD represents diisopropyl azodicarboxylate; DMF represents N,N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOAc represents ethyl acetate; EtOH represents ethanol; MeOH represents methanol; CBz represents benzyloxycarbonyl, which is an amine protecting group; BOC represents tert-butylcarbonyl, which is an amine protecting group; HOAc represents acetic acid; NaCNBH3 represents sodium cyanoborohydride; rt represents room temperature; O / N represents overnight; THF represents tetrahydrofuran; Boc2O represents di-tert-butyl dicarbonate; TFA represents trifluoroacetic acid; DIPEA represents diisopropylethylamine; SOCl2 stands for thionyl chloride; CS2 stands for carbon disulfide; TsOH stands for p-toluenesulfonic acid; NFSI stands for N-fluoro-N-(phenylsulfonyl)benzenesulfonamide; NCS stands for 1-chloropyrrolidine-2,5-dione; n-Bu4NF stands for tetrabutylammonium fluoride; i-PrOH stands for 2-propanol; mp stands for melting point; LDA stands for lithium diisopropylamide; TMSCF3 stands for trifluoromethyltrimethylsilane; Ti(Oi-Pr)4 stands for tetraisopropyl titanate; MSCl stands for methanesulfonyl chloride; DMAP stands for N,N-dimethyl-4-aminopyridine; TEA stands for triethylamine; B nBr stands for benzyl bromide; DIEA stands for diisopropylethylamine; BH3DMS stands for borane dimethyl sulfide; DMP stands for Dess-Martin periodinane; TBAF stands for tetrabutylammonium fluoride; HOBT stands for 1-hydroxybenzotriazole; AIBN stands for azobisisobutyronitrile; NBS stands for N-bromosuccinimide; RT buffer stands for reverse transcription buffer; dNTP stands for deoxyribonucleoside triphosphates; PBS stands for phosphate-buffered saline; LPS stands for bacterial lipopolysaccharide; TNF-α stands for tumor necrosis factor alpha; PBMC stands for peripheral blood mononuclear cells; and ELISA stands for enzyme-linked immunosorbent assay.

[0091]

[0092]

[0093] Compounds are manually or The software names were used, and commercially available compounds were named using the supplier's catalog names. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 : TNF-α levels in PBMC and whole blood culture supernatants, data are expressed as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA, new multiple range test, compared with blank group (DMSO); #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001, t test, compared with LPS group;

[0095] Figure 2 :Analysis of the inhibition rate of compounds on TNF-α detected by PBMC and whole blood culture;

[0096] Figure 3 : Ear thickness inhibition rate of mouse ear edema model, compared with the model group, ***p<0.001, **p<0.01;

[0097] Figure 4 : Ear weight inhibition rate of mouse ear edema model, compared with the model group, ***p<0.001, **p<0.01;

[0098] Figure 5 : Body weight change rate of mice in each group, ***p<0.001, **p<0.01, *p<0.05;

[0099] Figure 6 : Skin lesion scores of mice in each group, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05;

[0100] Figure 7 : Changes in subiliac lymph node weights of mice in each group, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. DETAILED DESCRIPTION

[0101] All compounds in the invention of this scheme were prepared by referring to the preparation method disclosed in WO2016054971.

[0102] Experimental Example 1: In vitro evaluation of PDE4B phosphodiesterase inhibitory activity

[0103] Experimental purpose: The reaction product AMP or GMP was detected by fluorescence polarization method (BellBrook, Madison WI). The fluorescent tracer competes with AMP or GMP for binding with the antibody, resulting in a change in the fluorescence polarization value. The inhibitory activity (IC 50 value).

[0104] Experimental materials: 10 mM Tris-HCl buffer, pH 7.5, 5 mM magnesium chloride, 0.01% polyoxyethylene lauryl ether, 1 mM dithiothreitol and 1% DMSO.

[0105] Reagents:

[0106] AMP2 / GMP2 assay kit:BellBrook Cat#3015-1K PDE1A:

[0107] SignalChem(Richmond,Canada)Cat#P89-30H PDE2A:

[0108] SignalChem Cat# P90-30G

[0109] PDE4B:SignalChem Cat#P92-31BG

[0110] IBMX:EMD Millipore(Billerica,MA)Cat#410957

[0111] Methoxyquinazoline:EMD Millipore Cat#475250

[0112] BRL-50481:R&D Systems(Minneapolis,MN)Cat#2237

[0113] Zaprinast:R&D Systems Cat#0947

[0114] Pentoxifylline

[0115] All reagents were purchased from Sigma-Aldrich (St. Louis, MO).

[0116] Reaction concentration:

[0117] Enzymes Amount of enzyme in the assay (pM) Substrate Amount of substrate in the assay (uM) PDE4B 40 cAMP 1

[0118] Background: No enzymes, only all other components

[0119] Time: 1 hour at room temperature

[0120] Conversion rate: 5% to 20%

[0121] Sample preparation and dilution:

[0122] Compound 1 or pentoxifylline was added to DMSO to prepare a 25 mM sample for later use. 6 μL of the 25 mM sample to be tested was transferred and added to the DMSO solution (6 μL) of the Echo shoot plate (LabCyte cat#LP-0200) to prepare a 2-fold diluted sample. The 2-fold serially diluted compound samples were directly added to the reaction as follows without any other intermediate dilution steps.

[0123] Experimental process:

[0124] 1. Prepare the required enzyme solution with freshly prepared reaction buffer;

[0125] 2. Transfer 1.5X enzyme solution into the reaction well; 10μL / well;

[0126] 3. Using Acoustic technology (Echo550; LabCyte Inc. Sunnyvale, CA), transfer 60 nL of the test compound, add it to the enzyme solution, mix slowly, and incubate at room temperature for 20 min;

[0127] 4. Prepare and transfer 3X substrate solution and add it to the reaction well to initiate the reaction; 5μL / well;

[0128] 5. React at room temperature for 1 hour, add detection agent, 5μL / well, mix slowly at room temperature, and incubate for 90 minutes;

[0129] 6. EnVision 2102 reads the fluorescence polarization signal (Perkin Elmer, Waltham MA; Ex / Em=FP620 / S 688 / P 688nm);

[0130] 7. Prepare the standard curve of AMP and GMP by 3-fold dilution of 10 μM concentration, add the detection agent, and the signal detection method is as described above;

[0131] 8. The fluorescence polarization intensity was converted into nM concentration using the AMP / GMP standard curve, and then the enzyme inhibition activity % relative to the DMSO blank control (no inhibitor) was calculated. The IC was calculated using the Prism software package (GraphPad Software, San Diego California, USA). 50 Values ​​and curves.

[0132] Experimental results:

[0133] Table 1 PDE4B phosphodiesterase inhibitory activity test results

[0134] Test sample (compound prepared in each example) <![CDATA[PDE4B phosphodiesterase inhibitory activity (IC 50 )]]> Compound 1 16.6μM Pentoxifylline 78.8μM

[0135] Conclusion: The compounds of the present invention have significant and even unexpected PDE4B protease inhibitory activity, and the inhibitory activity is significantly higher than that of pentoxifylline.

[0136] Experimental Example 2: In vitro evaluation of the effects of compounds on LPS-induced TNF-α secretion in human PBMC and whole blood

[0137] Experimental purpose: To detect the effects of compounds and pentoxifylline on the secretion of TNF-α by human blood and human peripheral blood mononuclear cells induced by bacterial lipopolysaccharide (LPS) in order to evaluate their in vitro anti-inflammatory activity.

[0138] Experimental Materials:

[0139] Normal whole blood, donated by staff of Shanghai WuXi AppTec Pharmaceuticals Co., Ltd. on April 1, 2016, 30 mL, anticoagulated with sodium heparin, with signed informed consent;

[0140] Heparin sodium anticoagulation tube, purchased from BD, catalog number: 367878, batch number: 4314542;

[0141] RPMI 1640, purchased from Gibco, catalog number: 224400-089, batch number: 1699742;

[0142] Fetal bovine serum (FBS) was purchased from Gibco, catalog number: 10099-141, batch number: 1618863;

[0143] The culture medium was RPMI 1640 containing 10% FBS;

[0144] LPS, purchased from Sigma, catalog number: L2630, batch number 114M4009U, stock solution (1 mg / mL), stored in a −80 °C refrigerator;

[0145] Dexamethasone (Dex), purchased from J&K Chemical Company, product number: 308890, batch number: LMA0N14, stock solution (500 μM), stored in a -20°C refrigerator;

[0146] PBS, purchased from Corning, catalog number: 21-031-CVR, batch number: 21031469R;

[0147] Lymphoprep, a lymphocyte separation medium, was purchased from STEMCELL, with the product number: 07851, batch number: 12HES21;

[0148] DMSO, purchased from Sigma, catalog number: D2650, batch number: RNBD9495;

[0149] U-bottom 96-well plate, purchased from Costar, catalog number: 3799.

[0150] Experimental operation:

[0151] (1) PBMC isolation

[0152] a) Strictly follow the method provided in the Lymphprep product manual: dilute an equal volume of whole blood with PBS containing 2% FBS and layer it on the lymphocyte separation solution to maintain a good interface and avoid mixing of blood and lymphocyte separation solution:

[0153] b) Centrifuge PBMC at 800g for 30 minutes at room temperature with both the centrifuge speeds set to the lowest setting;

[0154] c) collecting the cloud layer between the plasma layer and the lymphocyte separation liquid layer, and washing it twice with culture medium;

[0155] d) Resuspend the culture medium to 10 mL, count with Vi-CELL, and adjust the cell concentration to 2.5x10 6 / mL.

[0156] (2) Preparation of test and control compounds

[0157] a) Preparation of compound stock solutions: Compound 1 and pentoxifylline were added to corresponding volumes of DMSO to prepare 100 mM stock solutions;

[0158] b) Preparation of compound intermediate solutions: The stock solutions of compound 1 and pentoxifylline were 100 mM in concentration, and DMSO was used to dilute the compound stock solutions to 30 mM, 10 mM, 3 mM, 1 mM, 300 μM, and 100 μM, respectively;

[0159] c) Preparation of compound working solutions: Stock solutions and intermediate solutions of compound 1 and pentoxifylline, including 100mM, 30mM, 10mM, 3mM, 1mM, 300μM, and 100μM, were diluted 250 times in cell culture medium to prepare concentrations of 400μM, 120μM, 40μM, 12μM, 4μM, 1200nM, and 400nM, which were 4 times the final culture concentration.

[0160] (3) Preparation of LPS working solution

[0161] First, the LPS stock solution (1 mg / mL) was diluted 1000-fold with PBS to a 1 μg / mL solution;

[0162] a) For PBMC stimulation (4 ng / mL): dilute the 1 ug / mL solution 250 times with culture medium to 4 ng / mL, which is 4 times the final concentration in culture;

[0163] b) For whole blood stimulation (4 ng / mL): Dilute the 1 μg / mL solution 50-fold using culture medium to 20 ng / mL, which is 4 times the final culture concentration.

[0164] (4) Preparation of dexamethasone (Dex) and vehicle working solution

[0165] Dexamethasone (Dex) 500 μM stock solution was diluted 1250 times to 400 nM using cell culture medium to serve as Dex working solution, which was 4 times the final concentration in culture.

[0166] (5) PBMC and whole blood culture

[0167] a) PBMC culture:

[0168] Add 50 μL / well of the test compound 1, the control compound pentoxifylline, the dexamethasone (Dex) working solution, and the vehicle (DMSO) working solution;

[0169] LPS (4 nM) working solution 50 μL / well was added;

[0170] PBMC suspension (2.5x10 6 / mL), 100 μL / well added;

[0171] Each sample was plated in duplicate, with a final volume of 200 μL in each well. If the volume was less than 200 μL, culture medium was added to make it up to 200 μL / well. 200 μL of PBS was added to each well marked with PBS.

[0172] b) Whole blood culture:

[0173] Add 50 μL / well of the test compound 1, the control compound pentoxifylline, the dexamethasone (Dex) working solution, and the vehicle (DMSO) working solution;

[0174] LPS (20 nM) working solution 50 μL / well was added;

[0175] Blood, 50 μL / well added; serum-free RPMI 1640 50 μL / well added;

[0176] Each sample was plated in duplicate, and the final volume of each well was 200 μL. If the volume was less than 200 μL, culture medium was added to make it up to 200 μL / well. 200 μL of PBS was added to each well marked with PBS;

[0177] c) Cultivation:

[0178] The culture plate was placed in a CO2 incubator for 24 hours.

[0179] (6) Supernatant collection and TNF-α level detection

[0180] a) After culturing PBMC and whole blood for 24 hours, the culture supernatant was collected, and about 150 μL of supernatant was collected from each well, collected into a 96-well plate, and stored at -80°C for ELISA detection;

[0181] b) Before ELISA testing, the supernatant samples were taken out of the -80°C freezer, thawed at room temperature, diluted 3 times after thawing, and the TNF-α level was tested according to the instructions of the ELISA kit.

[0182] Experimental results:

[0183] Table 2. TNF-a detection value (pg / ml)

[0184]

[0185] Table 3. Inhibition rate results (%)

[0186]

[0187] Conclusion: As shown in Table 2, dexamethasone (Dex) at a concentration of 100 nM has a significant inhibitory effect on the secretion of TNF-α in LPS-stimulated whole blood (inhibition rate 98.3%). At the same concentration, it also has a certain inhibitory effect on the secretion of TNF-α in PBMC, but the inhibitory effect is lower than that of whole blood (inhibition rate 37.2%). As shown in Table 3, Figure 1 and Figure 2 As shown, in whole blood culture, pentoxifylline at a concentration of 100 μM significantly inhibited the secretion of whole blood TNF-α (inhibition rate 39.8%), with significant differences, while no significant inhibitory effect was observed at concentrations of 30 μM and below. The inhibition rates of compound 1 at 100 μM and 30 μM were 80.1% and 48.4%, respectively, with significant differences, and no significant inhibition was observed at concentrations of 10 μM and below. In PBMC culture, pentoxifylline at 100 μM significantly inhibited the secretion of TNF-α, with an inhibition rate of 27.1%, with significant differences, and no significant inhibitory effect was observed at doses of 30 μM and below. In this system, compound 1 had a significant inhibitory effect at 100 μM and 30 μM, with inhibition rates of 83.1% and 68.1%, respectively, with significant differences, and an inhibition rate of 27.7% was detected at 10 μM. It can be seen that the minimum effective dose of compound 1 is lower than that of pentoxifylline, and at the same dose, the effect of compound 1 in inhibiting TNF-α is stronger than that of pentoxifylline.

[0188] Experimental Example 3 Pharmacodynamic study of PMA-induced mouse ear edema model

[0189] Experimental purpose: PMA stimulation can induce the release of inflammatory factors TNF-α, IL-6 and IL-1β and increase vascular permeability, causing acute inflammatory edema in the mouse ears. The mouse model was used to evaluate the anti-inflammatory effect of the test drug.

[0190] Experimental model: PMA-induced mouse ear edema model

[0191] Experimental animals: CD-1 female mice (Chengdu Dashuo Animal Technology Co., Ltd.)

[0192] Experimental reagents:

[0193] 1. Acetone (Chengdu Changlian Chemical Reagent Co., Ltd.);

[0194] 2. Anhydrous ethanol (Chengdu Kelong Chemical Reagent Factory);

[0195] 3. Phorbol 12-myristate 13-acetate (phorbol ester, PMA, TPA)

[0196] 4. Crisaborole (AN2728) (Chengdu Zhipulai Biopharmaceutical Technology Co., Ltd.)

[0197] Experimental methods:

[0198] Twenty CD-1 female mice were ordered, with three spares. On the day of arrival, the animals were randomly divided into groups of five according to their weight. After the animals arrived at the facility, they were acclimated for five days. During the acclimation period, the health of the animals was monitored daily. If any abnormality or infection was found, the mice were removed from the experimental group.

[0199] Specific grouping information is shown in the table below. The grouping time is before the animal modeling begins. Normal mice are selected to start grouping modeling and drug administration. Table 4 is a table of experimental grouping after the formal drug administration begins.

[0200] Table 4 Experimental grouping

[0201]

[0202] PMA (20 μL / mouse, 0.25 mg / mL dissolved in acetone) was applied to stimulate the skin on the front and back of the right ear of the mice. Each group was treated with the same position on the front and back of the right ear of the mice according to Table 4 30 minutes before and 30 minutes after PMA stimulation (60 μL / mouse, dissolved in acetone: ethanol (=2:3)). After 6 hours of PMA stimulation, the mice were killed by cervical dislocation, and the same part of the left and right ears of the mice was obtained using a puncher (d=8 mm), and the thickness of the left and right ears of the mice was measured using a micrometer.

[0203] Experimental results:

[0204] Table 5 Mouse ear edema inhibition rate

[0205]

[0206]

[0207] The results showed that after local application of PMA stimulation to induce the release of inflammatory factors TNF-α, IL-6 and IL-1β and increase vascular permeability, causing acute inflammatory edema in the ears of mice, both compound 1 and AN2728 could inhibit inflammatory edema. Figure 3 and Figure 4 As shown, at a dose of 1 mg / ear, the ear thickness inhibition rate and weight inhibition rate of compound 1 in the mouse ear edema model were greater than those of AN2728, indicating that the inhibitory effect of compound 1 was significantly better than that of AN2728, showing that compound 1 has good anti-inflammatory effect and potential treatment effect for atopic dermatitis.

[0208] Example 4 Pharmacodynamic study in DNCB-induced atopic dermatitis model in mice

[0209] Experimental purpose: Pharmacodynamic study of the test drug in the DNCB-induced mouse atopic dermatitis model

[0210] Experimental model: DNCB-induced atopic dermatitis model in female BALB / c mice

[0211] Experimental animals: Female BALB / c mice (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.)

[0212] Experimental reagents:

[0213] 1. Compound 1 (Guangdong Zhongsheng Ruichuang Technology Biological Co., Ltd.)

[0214] 2. Acetone (Chengdu Changlian Chemical Reagent Co., Ltd.)

[0215] 3. Olive oil (Chengdu Kelong Chemical Co., Ltd.)

[0216] 4.1-Chloro-2,4-dinitrobenzene (DNCB) (Sigma-Aldrich)

[0217] 5. Tacrolimus (MedChemExpress)

[0218] 6. Crisaborole (AN2728) (Chengdu Zhipulai Biopharmaceutical Technology Co., Ltd.)

[0219] Experimental methods:

[0220] Animal grouping: Animals were randomly grouped by body weight. The specific grouping information is shown in the table below. The grouping time was before the animal modeling began. Normal mice were selected to start grouping and modeling and drug administration. The table below shows the experimental grouping situation after the formal drug administration began.

[0221] Table 6 Experimental grouping

[0222]

[0223]

[0224] AD mouse modeling: 50 BALB / c female mice were randomly divided into 6 groups according to their body weight, with 6 mice in the normal group and 8 mice in each of the other groups. The hair on the back of the mice was shaved 2 days before the start of the experiment, with an area of ​​about 8cm 2 On D1, D2, and D3, 200 μL (0.5%) DNCB solution was applied to the back skin of mice for sensitization, once a day; from D14, 200 μL (1%) DNCB solution was applied to the back skin of mice for stimulation, twice a week, for 4 weeks. The normal group was treated with the same volume of solvent (acetone: olive oil = 3:1).

[0225] AD mice were administered: 200 μL 1.5% AN2728 or 0.5% or 1.5% compound 1 solution was applied to the back skin of mice starting from D14, twice a day, for 4 weeks, and the model group was treated with the same volume of solvent (acetone: olive oil = 3:1). The tacrolimus group was given 200 μL, 0.1% bid in the first and second weeks, and the dose was adjusted to 200 μL, 0.5% bid in the third and fourth weeks.

[0226] Anatomical sampling: 48 hours after the last stimulation with 200 μL 1% DNCB solution, blood was collected from the orbits of the AD mice, and then the mice were killed by cervical dislocation. The back skin tissue was obtained and fixed with 10% neutral formalin solution.

[0227] Mouse skin tissue inflammation score (EASI): The skin condition was observed and scored before each sensitization and photographed. The skin was judged based on 4 symptoms: dryness / scaling, bleeding / rash, ulceration / exfoliation, and edema. Each symptom was scored as 0 (none), 1 (mild), 2 (moderate), and 3 (severe). The final score was the sum of the 4 symptoms. The skin tissue inflammation score was 0-12 points.

[0228] Subiliac lymph node size and weight: Subiliac lymph node size and weight of mice were obtained during dissection.

[0229] Data processing: The experimental data of each group of animals were described by mean ± standard error (X ± SD). The data were analyzed by Kruskal–Wallis One-way ANOVA. P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS11 software.

[0230] Experimental results:

[0231] 1. Clinical observation and body weight

[0232] like Figure 5 As shown in the figure, compared with the normal group, the body weight of mice in the model group showed a slight downward trend, and there were significant differences at D25, D32, and D35. The weight loss was related to the DNCB modeling stimulation. Compared with the model group, the body weight of mice in the Tacrolimus group showed a downward trend in the first 2 weeks of administration (significantly decreased on D28), and began to recover in the 3rd and 4th weeks of administration. There was no difference in body weight changes between the other drug-administered groups and the model group.

[0233] 2. Skin lesion score (EASI)

[0234] like Figure 6 As shown, the mice were scored for skin lesions before each DNCB sensitization. 30% of the animals in the D14 model group (sensitization stage) had skin lesion scores of 1-3 points, and the degree of skin lesions showed a downward trend compared with D7, and the score in the sensitization stage was reduced, which was consistent with the previous modeling data. D21 (1 week of administration), compared with the model group, the compound 1 1.5% group had a significant downward trend; D28 (2 weeks of administration), compared with the model group, there was no significant difference in each administration group; D35 (3 weeks of administration), compared with the model group, the Tacrolimus group, AN2728 1.5% and compound 1 0.5% / 1.5% all showed a significant downward trend. D41 (4 weeks of administration), compared with the model group, the Tacrolimus group, AN2728 1.5% and compound 1 high-dose group 1.5% all showed a significant downward trend.

[0235] 3. Organ coefficient - subiliac lymph nodes

[0236] like Figure 7 As shown in the figure, after 4 weeks of administration, the subiliac lymph nodes in the model group were significantly enlarged compared with the normal group. Compared with the model group, the Tacrolimus group showed a downward trend, but it was not statistically significant. Compared with the model group, the AN2728 1.5% and compound 10.5% and 1.5% groups all showed a significant downward trend. See the table below for details.

[0237] Table 7 Subiliac lymph node weight and skin thickness of animals in each group (Mean±SD)

[0238]

[0239] Note: One-way ANONA, compared to AD model*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0240] In summary, in the pharmacodynamic study in the DNCB-induced mouse atopic dermatitis model, compound 1, Tacrolimus and AN2728 all had an improvement effect on atopic dermatitis, and under the 1.5% condition, the skin lesion score of compound 1 was lower than that of AN2728, indicating that the inhibitory effect of compound 1 on atopic dermatitis was significantly better than that of AN2728, showing that compound 1 has a significant improvement effect on the symptoms of DNCB-induced atopic dermatitis in mice, and has potential therapeutic effects on atopic dermatitis.

Claims

1. Use of compound 1 in the preparation of a drug for treating skin diseases, characterized in that: The skin disease is atopic dermatitis ear inflammatory edema,

Images