Tricyclic fused heterocyclic PDE3 / 4 dual inhibitor, and preparation and use therefor

Abstract

A tricyclic fused heterocyclic PDE3 / 4 dual inhibitor, and a preparation and use therefor. Specifically provided is a compound having structural formula (I) or a pharmaceutically acceptable form thereof, wherein each substituent is as defined in the description, respectively. The compound or the pharmaceutically acceptable form thereof can be used as a phosphodiesterase inhibitor and has high activity.

Description

Tricyclic fused heterocyclic PDE3 / 4 dual inhibitors and their preparation method and application Technical Field

[0001] The present invention relates to a tricyclic fused heterocyclic compound and a preparation method and application thereof, and in particular to a tricyclic fused heterocyclic compound and a preparation method thereof and application thereof as a phosphodiesterase PDE3 / 4 dual inhibitor. Background Art

[0002] Phosphodiesterases (PDEs) belong to a superfamily of enzymes encompassing at least 11 families and 22 subtypes. They are involved in intracellular and extracellular information transmission and functional regulation. PDEs catalyze the hydrolysis of the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) to generate AMP and GMP, respectively.

[0003] The PDE3 family consists of two genes, PDE3A and PDE3B. PDE3 activity in the respiratory system is primarily concentrated in alveolar macrophages, endothelial cells, and platelets. PDE3 is involved in regulating numerous physiological activities in the body, such as vasodilation of vascular smooth muscle, antiplatelet aggregation, antithrombotic, cardiotonic, and anti-cell proliferation. Excessive use of PDE3 inhibitors can cause adverse reactions such as hypotension and tachycardia, significantly limiting their clinical application.

[0004] PDE4 is an enzyme that specifically hydrolyzes cAMP. Its family consists of four subtypes: PDE4A, PDE4B, PDE4C, and PDE4D. Each subtype is encoded by a corresponding gene and has distinct cellular distribution and roles. PDE4 is primarily distributed in airway smooth muscle cells, inflammatory cells, and immune cells, regulating intracellular cAMP levels. Currently, most PDE4 inhibitors used in clinical practice have some degree of side effects, such as gastrointestinal reactions like nausea and vomiting, and even depression.

[0005] Given the limitations of using either PDE3 or PDE4 inhibitors alone and the side effects of PDE inhibitors, dual inhibition of inhaled PDE3 / 4 appears to be a more attractive approach to target the key pathological features of COPD and asthma. Evidence has shown that inhaled dual-target PDE3 / 4 inhibitors have synergistic inhibitory effects, including synergistic anti-inflammatory and bronchodilatory effects.

[0006] CN100415743C discloses a pyrimido[6,1a]isoquinolin-4-one derivative:

[0007] The compound of the general formula is used as a PDE inhibitor for treating respiratory diseases such as asthma, has a longer duration of action than troquinecin, and does not have the very bitter taste of troquinecin.

[0008] CN112368281A discloses a class of tricyclic compounds as PDE3 / PDE4 dual inhibitors:

[0009] The compound of the general formula can be used to prepare drugs for PDE3 / PDE4 related diseases, especially chronic obstructive pulmonary disease (COPD).

[0010] There is still an urgent need in the art for new PDE3 / PDE4 inhibitors, especially PDE3 / PDE4 inhibitors with high activity and good drugability. Summary of the Invention

[0011] An object of the present invention is to provide a novel compound as a PDE inhibitor.

[0012] Another object of the present invention is to provide a method for preparing the compound.

[0013] Another object of the present invention is to provide the use of the compound.

[0014] Another object of the present invention is to provide a pharmaceutical composition comprising the compound and use thereof.

[0015] Another object of the present invention is to provide an intermediate for preparing the compound.

[0016] Another object of the present invention is to provide a method for preparing the intermediate.

[0017] <First Aspect>

[0018] The present invention provides a compound having structural formula I or a pharmaceutically acceptable form thereof,

[0019] in:

[0020] R1 and R2 are each independently selected from H, C 1-6 Straight chain alkyl, C 3-6 Branched alkyl and C 3-6 Cycloalkyl; the linear alkyl, branched alkyl or cycloalkyl is optionally further substituted by 0 to 4 groups selected from H, F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1- 4 substituted by an alkoxy substituent;

[0021] R3, R4, R5 are each independently selected from H, halogen, CN, C 1-6 Alkoxy, C 1-6 Straight chain alkyl, C 3-6 Branched alkyl, and C 3-6Cycloalkyl; the alkoxy, linear alkyl, branched alkyl or cycloalkyl group is optionally further substituted by 0 to 4 groups selected from H, F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent;

[0022] R6 is selected from The H in R6 is optionally further replaced by 0 to 4 selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent;

[0023] R7 is selected from H, =O, NH2, CN, C 1-6 Straight chain alkyl, C 3-6 Branched alkyl, C 3-6 Cycloalkyl and The H in R7 is optionally further replaced by 0 to 4 selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent;

[0024] L is selected from n is 0, 1 or 2; k is 0, 1 or 2; the H in L is optionally further replaced by 0 to 4 selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent;

[0025] X is N;

[0026] Y is C 1-3 Alkylene or not present; said alkylene is optionally further substituted by 0 to 4 groups selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent;

[0027] Optionally, R1 and R2 are connected to form a 5- or 6-membered ring together with the connected O; the H in the 5- or 6-membered ring is optionally further replaced by 0 to 4 selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent;

[0028] Optionally, X and L are connected by C 1-2Alkylene groups are connected to form a 5- or 6-membered ring; the H in the 5- or 6-membered ring is optionally further replaced by 0 to 4 groups selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent;

[0029] Optionally, R6 and L together form a 5- or 6-membered ring; the H in the 5- or 6-membered ring is optionally further replaced by 0 to 4 selected from F, Cl, Br, I, OH, ═O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent;

[0030] Optionally, H of the compound of formula I is optionally further substituted with 0 to 6 D;

[0031] Furthermore, the compound of formula I is not:

[0032] According to some specific embodiments of the present invention, in the compound of formula I of the present invention or a pharmaceutically acceptable form thereof, R1 and R2 are each independently selected from H, CH3, CHF2, and CD3.

[0033] According to some specific embodiments of the present invention, in the compound of formula I of the present invention or a pharmaceutically acceptable form thereof, R1 and R2 respectively correspond to the groups or values ​​shown in any one of the compounds in Table 1 (Compounds 1 to Compounds 33).

[0034] According to some specific embodiments of the present invention, in the compound of formula I of the present invention or a pharmaceutically acceptable form thereof, R3, R4, and R5 are each independently selected from H, CH3, i-Pr, OMe, CN, CD3 or halogen.

[0035] According to some specific embodiments of the present invention, in the compound of formula I of the present invention or a pharmaceutically acceptable form thereof, R3, R4, and R5 respectively correspond to the groups or values ​​shown in any one of the compounds in Table 1 (Compounds 1 to Compounds 33).

[0036] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, R6 is

[0037] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, R7 is H, =O

[0038] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, L is selected from n is 0, 1 or 2; k is 1 or 2.

[0039] According to some specific embodiments of the present invention, in the compound of formula I or a pharmaceutically acceptable form thereof, L corresponds to the group or value shown in any one of the compounds in Table 1 (Compound 1-Compound 33).

[0040] According to some specific embodiments of the present invention, in the compound of formula I of the present invention or a pharmaceutically acceptable form thereof, Y is absent.

[0041] According to some specific embodiments of the present invention, in the compound of formula I of the present invention or a pharmaceutically acceptable form thereof, R1, R2, R3, R4, R5, R6, R7, L, X, and Y respectively correspond to the groups or values ​​shown in any one of the compounds in Table 1 (Compounds 1 to Compounds 33).

[0042] According to some specific embodiments of the present invention, the compound of formula I or a pharmaceutically acceptable form thereof of the present invention, wherein the compound has the structure shown in formula Ia:

[0043] in:

[0044] R1 and R2 are each independently selected from CH3, CHF2 and CD3;

[0045] R3, R4, R5 are each independently selected from CH3, F, methoxy and isopropyl;

[0046] L is selected from

[0047] According to some specific embodiments of the present invention, in Formula Ia:

[0048] R1, R2, R3, R4, and R5 are CH3 respectively;

[0049] L is

[0050] According to some specific embodiments of the present invention, the compound of formula I or a pharmaceutically acceptable form thereof of the present invention is selected from one or more of the compounds shown in Table 1:

[0051] Table 1

[0052] According to some specific embodiments of the present invention, the compound of formula I of the present invention or a pharmaceutically acceptable form thereof, the pharmaceutically acceptable form is selected from a pharmaceutically acceptable salt or cocrystal, stereoisomer, tautomer, deuterated form, solvate, chelate, non-covalent complex or prodrug.

[0053] <Second Aspect>

[0054] The present invention also provides an intermediate compound having a structure shown in Formula II:

[0055] Wherein, R1, R2, R3, R4, R5, R7, X, and Y are as defined in any one of the embodiments of the compound of formula I or its pharmaceutically acceptable form according to the <First Aspect> of the present invention;

[0056] L1 is selected from n is 0, 1 or 2; k is 0, 1 or 2; the H in L1 is optionally further replaced by 0 to 4 selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent.

[0057] According to some specific embodiments of the present invention, the intermediate compound of the present invention has the following structure:

[0058] <Third Aspect>

[0059] The present invention also provides a method for preparing the compound of the first aspect of the present invention or a pharmaceutically acceptable form thereof. A synthetic route for preparing the compound of Formula I or a pharmaceutically acceptable form thereof can be designed based on the chemical structure of the compound of Formula I or a pharmaceutically acceptable form thereof, with reference to methods known in the art.

[0060] According to some specific embodiments of the present invention, the method of preparing the compound of the first aspect of the present invention or a pharmaceutically acceptable form thereof comprises:

[0061] The carboxyl end of the intermediate compound shown in Formula II described in the <Second Aspect> of the present invention is subjected to a modification reaction to prepare a compound having structural formula I.

[0062] According to some specific embodiments of the present invention, the method of preparing the compound described in the <first aspect> of the present invention or a pharmaceutically acceptable form thereof also includes a process for preparing the intermediate compound described in the <second aspect> of the present invention.

[0063] According to some specific embodiments of the present invention, the method of preparing the compound described in the <first aspect> of the present invention or a pharmaceutically acceptable form thereof comprises the steps shown in any one of the reaction schemes in Examples 1 to 18.

[0064] According to some specific embodiments of the present invention, the method of preparing the compound of the first aspect of the present invention or a pharmaceutically acceptable form thereof comprises:

[0065] <Fourth Aspect>

[0066] The present invention also provides a pharmaceutical composition comprising: the compound described in the <First Aspect> of the present invention or a pharmaceutically acceptable form thereof (preferably a pharmaceutically acceptable salt), and a pharmaceutically acceptable carrier, excipient and / or one or more other therapeutic agents.

[0067] <Fifth Aspect>

[0068] The present invention also provides the use of the compound described in the first aspect of the present invention or a pharmaceutically acceptable form thereof (preferably a pharmaceutically acceptable salt) or the pharmaceutical composition described in the fourth aspect of the present invention in the preparation of a formulation for inhibiting phosphodiesterase. Preferably, the phosphodiesterase includes PDE3 and / or PDE4.

[0069] <Sixth Aspect>

[0070] The present invention also provides the use of the compound described in the <first aspect> of the present invention or a pharmaceutically acceptable form thereof (preferably a pharmaceutically acceptable salt) or the pharmaceutical composition described in the <fourth aspect> of the present invention in the preparation of a drug for treating phosphodiesterase-related diseases.

[0071] The present invention also provides a method for treating phosphodiesterase-related diseases, which comprises administering to a subject an effective amount of the compound described in the <first aspect> of the present invention or a pharmaceutically acceptable form thereof (preferably a pharmaceutically acceptable salt) or the pharmaceutical composition described in the <fourth aspect> of the present invention.

[0072] According to some specific embodiments of the present invention, the phosphodiesterase comprises PDE3 and / or PDE4.

[0073] According to some specific embodiments of the present invention, the phosphodiesterase-related disease includes a respiratory disease such as asthma.

[0074] According to some specific embodiments of the present invention, the subject is a mammal or a human, preferably, the subject is a human.

[0075] The compound of the present invention having structural formula I or a pharmaceutically acceptable form thereof can be used as a phosphodiesterase inhibitor, has a highly effective inhibitory activity against phosphodiesterase, particularly PDE3 and / or PDE4, and has practical value.

[0076] Definition and Description

[0077] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be construed as indefinite or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0078] Unless otherwise specified, use a solid wedge key. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond and straight dashed key Indicate the relative configuration of stereocenters with a wavy line Indicates a wedge-shaped solid key or dotted wedge key Or use a wavy line Indicates a straight solid bond and straight dashed bond

[0079] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0080] The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salts thereof" refers to salts of the compounds of the present invention prepared by reacting the compounds of the present invention with relatively nontoxic acids or bases, as discovered herein. When the compounds of the present 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 neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When the compounds of the present 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 neat solution or in 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, dihydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, and the like; 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 salts of amino acids (such as arginine) and organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and can be converted into either base or acid addition salts.

[0081] 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 a suitable base or acid in water or an organic solvent or a mixture of the two.

[0082] The term "eutectic" refers to a crystalline material comprising two or more distinct solids at room temperature, each solid having different physical properties, such as structure, melting point, and heat of fusion.

[0083] The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a perpendicular asymmetric plane due to at least one chiral factor (including a chiral center, chiral axis, chiral plane, etc.), thereby being able to rotate plane-polarized light. Because the compounds of the present invention contain asymmetric centers and other chemical structures that may lead to stereoisomerism, the present invention also includes these stereoisomers and mixtures thereof. Because the compounds of the present invention and their salts may include asymmetric carbon atoms, they can exist as single stereoisomers, racemates, enantiomers, and mixtures of diastereomers. Typically, these compounds can be prepared as racemic mixtures. However, if desired, such compounds can be prepared or isolated to obtain pure stereoisomers, i.e., single enantiomers or diastereomers, or mixtures enriched in a single stereoisomer (purity ≥98%, purity ≥95%, ≥93%, ≥90%, ≥88%, ≥85%, or ≥80%). A single stereoisomer of a compound is synthesized from an optically active starting material containing the desired chiral center, or by preparing a mixture of enantiomeric products followed by separation or resolution, for example, by conversion to a mixture of diastereomers followed by separation or recrystallization, chromatography, use of a chiral resolving agent, or direct separation of the enantiomers on a chiral chromatographic column. Starting compounds with a specific stereochemistry are either commercially available or prepared as described herein and resolved by methods well known in the art. Unless otherwise indicated, all stereoisomeric forms of the compounds of the present invention are within the scope of the compounds of the present invention.

[0084] The term "tautomer" (or "tautomeric form") refers to structural isomers with different energies that can be interconverted through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (or prototropic tautomers) include (but are not limited to) interconversions via proton migration, such as keto-enol isomerization, imine-enamine isomerization, amide-iminoalcohol isomerization, etc. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are within the scope of the compounds of the present invention.

[0085] Unless otherwise indicated, the compounds represented by the structural formulae of the present invention may be in the form of a purified single stereoisomer or tautomer, or in the form of a mixture containing a plurality of stereoisomers or tautomers.

[0086] The term "solvate" refers to a substance formed by the combination of a compound of the present invention or a pharmaceutically acceptable salt thereof with at least one solvent molecule through non-covalent intermolecular forces. Common solvates include (but are not limited to) hydrates, ethanolates, acetonides, etc.

[0087] The term "chelate" refers to a complex having a cyclic structure, which is obtained by the chelation of two or more ligands with the same metal ion to form a chelate ring.

[0088] The term "non-covalent complex" is formed by the interaction of a compound with another molecule, wherein no covalent bond is formed between the compound and the molecule. For example, complexation can occur through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also known as ionic bonding).

[0089] The term "prodrug" refers to a derivative compound that, upon application to a patient, is capable of providing, directly or indirectly, a compound of the invention. Particularly preferred derivative compounds or prodrugs are compounds that, when administered to a patient, can increase the bioavailability of the compound of the invention (e.g., more readily absorbed into the bloodstream) or compounds that facilitate delivery of the parent compound to the site of action (e.g., the lymphatic system). Unless otherwise indicated, all prodrug forms of the compounds of the invention are within the scope of the invention, and various prodrug forms are well known in the art.

[0090] The term "each independently" means that at least two groups (or ring systems) present in a structure with the same or similar value ranges may have the same or different meanings in specific circumstances. For example, if substituent X and substituent Y are each independently hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl, then when substituent X is hydrogen, substituent Y may be either hydrogen, or halogen, hydroxyl, cyano, alkyl, or aryl. Similarly, when substituent Y is hydrogen, substituent X may be either hydrogen, or halogen, hydroxyl, cyano, alkyl, or aryl.

[0091] The terms "including" and "comprising" are used in their open, non-limiting sense.

[0092] "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.

[0093] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include 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 oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen 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 any on the basis of chemical achievable.

[0094] 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, with each occurrence of R being an independent choice. Furthermore, combinations of substituents and / or their variants are permissible only if such combinations result in stable compounds.

[0095] When a variable in a structural formula is selected as missing, it means that it does not exist. For example, when R in CR is selected as missing, it means that the structure is actually C.

[0096] When a variable connecting two groups in a structural formula is selected from a bond or does not exist, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a bond or does not exist, it means that the structure is actually AZ.

[0097] When the substituents listed do not specify through which atom they are connected to the substituted group, such substituents can be bonded through any atom thereof. For example, a phenyl substituent can be connected to the substituted group through any carbon atom on the benzene ring.

[0098] Unless otherwise specified, the term "alkyl" is used to refer to a straight or branched saturated hydrocarbon group, which may be monosubstituted (e.g., -CH2F) or polysubstituted (e.g., -CF3), and may be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like.

[0099] Unless otherwise specified, the term "alkylene" refers to a divalent straight or branched chain alkane group consisting only of carbon atoms and hydrogen atoms, containing no saturation, and connected to other fragments by two single bonds, including (but not limited to) methylene, 1,1-ethylene and 1,2-ethylene. For example, "C 1-3 "Alkylene" refers to a saturated divalent straight or branched chain alkyl group containing from 1 to 3 carbon atoms.

[0100] Unless otherwise specified, "cycloalkyl" includes any stable cyclic or polycyclic hydrocarbon radical, any carbon atom of which is saturated, which may be monosubstituted or polysubstituted, and which may be monovalent, divalent, or polyvalent. Examples of such cycloalkyl radicals include, but are not limited to, cyclopropyl, norbornyl, [2.2.2]bicyclooctane, [4.4.0]bicyclodecane, and the like.

[0101] Unless otherwise specified, the term "alkoxy" means an alkyl group attached to the rest of the molecule via an oxygen atom, wherein the alkyl group has the meaning as defined herein. 1-5 Alkoxy groups include C1, C2, C3, C4, and C5 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. The alkoxy group may be optionally substituted with one or more substituents described herein.

[0102] Unless otherwise specified, the term "3-6 membered ring" means a saturated or unsaturated monocyclic ring with or without heteroatoms, which contains 3, 4, 5 or 6 C, O, S, N atoms in the ring; the "3-6 membered ring" can be connected to the rest of the structural formula through any carbon atom or, if present, a nitrogen atom.

[0103] Unless otherwise specified, the term "halogen," by itself or as part of another substituent, means a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom. The term "hydroxy" refers to -OH. The term "cyano" refers to -CN. The term "amino" refers to -NH2.

[0104] Unless otherwise specified, the term "pharmaceutical composition" refers to a mixture of one or more compounds of the present invention or a pharmaceutically acceptable form thereof with other chemical components, wherein "other chemical components" refers to a pharmaceutically acceptable carrier, excipient and / or one or more other therapeutic agents. "Carrier" refers to a material that does not significantly irritate the organism and does not eliminate the biological activity and properties of the administered compound. "Excipient" refers to an inert substance added to a pharmaceutical composition to facilitate the administration of a compound. Non-limiting examples include calcium carbonate, calcium phosphate, sugars, starch, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oils, polyethylene glycols, diluents, granulating agents, lubricants, binders and disintegrants.

[0105] The compound of the present invention has good PDE inhibitory activity and has potential application value in treating diseases related to PDE, especially diseases related to PDE3 / PDE4. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] FIG1A to FIG1D show the results of the study on the effect of each compound on the viability of bronchial smooth muscle cells.

[0107] FIG2 shows the results of the TNF-α level detection in the cell supernatant in the pharmacodynamic study of each compound on the LPS-induced inflammation model.

[0108] FIG3 shows the effects of the compounds of the present invention on the relaxation function of isolated trachea. DETAILED DESCRIPTION

[0109] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all commercially available conventional products.

[0110] The structures of the compounds were determined by nuclear magnetic resonance or mass spectrometry. Nuclear magnetic resonance was measured using a BRUKER 400M nuclear magnetic spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6) or deuterated chloroform (CDCl3) as the solvent and tetramethylsilane (TMS) as the internal standard. Chemical shifts (δ) were measured in 10 -6 The unit is ppm. Mass spectra were measured using a Waters ACQUITY Arc / ACQUITY QDa or a Thermo U3000-ISQ EC LC / MS instrument.

[0111] High-performance liquid chromatography (HPLC) analysis was performed using a Thermo U3000 HPLC, and preparative HPLC was performed using a Hanbon DAC-50 or Shimadzu LC-20AP preparative chromatograph.

[0112] Reaction monitoring was performed using thin-layer chromatography (TLC) or liquid chromatography-mass spectrometry (LC-MS). The developing solvents used for TLC included dichloromethane and methanol, or petroleum ether and ethyl acetate. The solvent volume ratio was adjusted based on the polarity of the compound or by adding a small amount of triethylamine. LC-MS was performed using a Waters ACQUITY Arc / ACQUITY QDa or Thermo Fisher Scientific U3000-ISQ EC LC / MS instrument.

[0113] Column chromatography generally uses 200-300 mesh silica gel as a carrier. Eluent systems include: dichloromethane and methanol system, petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound, or a small amount of triethylamine can be added to adjust the ratio.

[0114] Unless otherwise specified in the following examples, the reaction temperature was room temperature (20°C-30°C) and the solvents were dried and purified according to standard methods.

[0115] Example 1, Compound 1

[0116] Under air protection, to a solution of (2E)-3-(3-aminocyclobutyl)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (100 mg, 0.2 mmol, 1 eq) and TEA (0.1 mL, 0.6 mmol, 3 eq) in DCM (2 mL) was added isocyanotrimethylsilane (37 mg, 0.3 mmol, 1.5 eq) at 0°C. Under nitrogen protection, the resulting residue was stirred at room temperature for 1.5 hours. The resulting residue was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (HPLC) under the following conditions (column type: CHIRALPAK IG 2*25 cm, 5 μm; mobile phase A: ETOH:DCM=1:1, mobile phase B: MtBE (0.1% DEA)-HPLC; flow rate: 20 mL / min; gradient: isocratic; wavelength: 220 nm; RT1 (min): 11.5; RT2 (min): 14.5; injection solvent: methanol; injection volume: 0.6 mL; number of runs: 4).

[0117] Two isomeric compounds were obtained: compound 1-1 and compound 1-2.

[0118] Compound 1-1 (isomer 1, RT1 (min): 11.5, 10.36 mg, 9% yield)

[0119] LCMS (ESI): [M+H] + =504.30

[0120] 1 H NMR(400MHz, Methanol-d4)δ7.13(s,2H),6.93(s,1H),6.68(s,1H),5.45(s,1H),5.25–5.16(m,1H),4.14–4.07(m,2 H),3.98–3.93(m,1H),3.89(s,3H),3.68(s,3H),2.96(t,J=6.5Hz,2H),2.38(s,3H),2.36–2.27(m,4H),2.16(s,6H).

[0121] Compound 1-2 (isomer 2, RT2 (min): 14.5, 40.48 mg, 36% yield)

[0122] LCMS (ESI): [M+H] + =504.30

[0123] 1H NMR(400MHz, Methanol-d4)δ7.13(s,2H),6.93(s,1H),6.68(s,1H),5.45(s,1H),5.25–5.16(m,1H),4.14–4.07(m,2 H),3.98–3.93(m,1H),3.89(s,3H),3.68(s,3H),2.96(t,J=6.5Hz,2H),2.38(s,3H),2.36–2.27(m,4H),2.16(s,6H).

[0124] Example 2, Compound 2

[0125] Step 1.

[0126] To a three-necked flask, potassium tert-butoxide (22.8 g, 203.4 mmol, 1.0 equiv) and n-pentane (200 mL) were added sequentially. Under nitrogen, the temperature was lowered to -30°C. At this temperature, 1,3-butadiene (toluene solution, 11.0 g, 203.4 mmol, 20%, 1.0 equiv) and bromoform (51.4 g, 203.4 mmol, 1.0 equiv) were added dropwise. After the additions were complete, the mixture was allowed to react at this temperature for 1.5 hours, then gradually returned to room temperature and allowed to react overnight. Water (30 mL) was added dropwise under an ice bath, and the mixture was extracted with n-pentane (3 x 100 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness at no higher than 30°C to yield crude 1,1-dibromo-2-vinylcyclopropane (70.0 g).

[0127] Step 2.

[0128] In a single-necked flask, 1,1-dibromo-2-vinylcyclopropane (74.0 g, 327.6 mmol, 1.0 equivalent), cetrimonium chloride (3.1 g, 9.8 mmol, 0.03 equivalent), DCM (150 mL) and water (150 mL) were added sequentially. Concentrated sulfuric acid (106.0 g, 1080.9 mmol, 3.3 equivalents) was added dropwise under an ice bath. After completion, potassium permanganate (155.3 g, 982.7 mmol, 3.0 equivalents) was slowly added at below 5°C, the temperature was gradually restored to room temperature and the reaction was continued for 12 h. 50% aqueous sulfuric acid solution and anhydrous sodium sulfate (100.0 g) were added dropwise under an ice bath, gradually returned to room temperature and reacted for 10 minutes. The aqueous phase was extracted with DCM (3 x 500 mL), and the combined organic phases were dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness at no more than 30° C. to obtain a crude product 2,2-dibromocyclopropane-1-carboxylic acid (40.0 g, crude).

[0129] Step 3.

[0130] To a three-necked flask, 2,2-dibromocyclopropane-1-carboxylic acid (20.0 g, 81.3 mmol, 1.0 equivalent) and diethyl ether (100 mL) were added sequentially. Methyllithium (60 mL, 90.3 mmol, 1.1 equivalent, 1 M in THF) was added dropwise under an ice bath. The mixture was gradually warmed to room temperature and allowed to react for 2 h. Water (100 mL) was added dropwise under an ice bath. After separation, the aqueous phase was adjusted to pH 6 with dilute hydrochloric acid (2 M) and extracted with DCM (3 x 100 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated to dryness at no higher than 30°C to yield crude 2-bromocyclopropane-1-carboxylic acid (10.0 g).

[0131] LCMS (ESI, m / z): [M+H] - =162.9.

[0132] Step 4.

[0133] 2-Bromocyclopropane-1-carboxylic acid (10.0 g, crude), potassium carbonate (7.5 g, 5.5 mmol, 3.0 equiv), DMF (20 mL), and iodomethane (6.0 g, 36.3 mmol, 1.0 equiv) were added to a single-necked flask and reacted at room temperature for 5 h. Water (100 mL) was added and the mixture was extracted with DCM (3 x 50 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated to dryness at no higher than 30°C to yield the crude product, 2-bromocyclopropane-1-carboxylate (20.0 g, crude).

[0134] Step 5.

[0135] In a single-necked flask, 2-bromocyclopropane-1-carboxylate (200 mg, 1.1 mmol, 1.0 equivalent), (2E)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-3H,6H,7H-pyrimido[4,3-a]isoquinolin-4-one (437 mg, 1.1 mmol, 1.0 equivalent), cesium carbonate (1.1 g, 3.4 mmol, 3.0 equivalent), Xantphos (106.5 mg, 0.2 mmol, 0.2 equivalent) and 1.4-dioxane (80 mL) were added in sequence. Pd2(dba)3 (64.2 mg, 0.1 mmol, 0.1 equivalent) was added under nitrogen protection, and the reaction was carried out at 100°C overnight. The solvent was evaporated and the crude product was purified by normal phase purification using MeOH / DCM (1 / 10) as the mobile phase, which was further purified by reverse phase chromatography (column type (YMC Triart C18 ExRs 5m, 30mm*150mm; mobile phase A: water (10mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 39%B to 62%B in 10min; wavelength: 254nm / 220nm; RT1(min): 27)) to obtain methyl 2-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimidinyl[4,3-a]isoquinolin-3-yl]cyclopropane-1-carboxylate (100mg, 18.3% yield).

[0136] LCMS (ESI, m / z): [M+H] + =490.2.

[0137] Step 6.

[0138] In a single-necked flask, methyl 2-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimidinyl[4,3-a]isoquinolin-3-yl]cyclopropane-1-carboxylate (500 mg, 1.0 mmol, 1.0 equivalent), methanol (10 mL) and lithium hydroxide (85 mg, 2.0 mmol, 2.0 equivalent) in water (2 mL) were added in sequence and reacted at room temperature for 2 h. The solvent was dried and the pH was adjusted to 6 with 4 M hydrochloric acid solution. The mixture was diluted with DCM (3x The mixture was extracted with 50 mL of 4% paraformaldehyde (50 mL), and the combined organic phases were dried over anhydrous sodium sulfate. The organic phase was concentrated to give 2-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclopropane-1-carboxylic acid (450 mg, 92.7% yield).

[0139] LCMS (ESI, m / z): [M+H] + =476.2.

[0140] Step 7.

[0141] In a three-necked flask, 2-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclopropane-1-carboxylic acid (500 mg, 1.1 mmol, 1.0 equivalent), 1.4-dioxane (156 mL), triethylamine (319 mg, 3.2 mmol, 3.0 equivalent) and DPPA (347.2 mg, 1.3 mmol, 1.2 equivalent) were added in sequence. The mixture was protected by nitrogen and reacted at room temperature for 2 h. The mixture was then heated to 100 ° C and reacted for 1 h. The temperature was cooled to room temperature, and ammonia solution in tetrahydrofuran (1.4 M) (100 mL) was added. The reaction was continued at room temperature for 2 h. The solvent was dried and the product was subjected to HPLC (column type: YMC Triart C18 ExRs 5m,30mm*150mm; mobile phase A: water (10mmol / LNH4HCO3), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 39% B to 62% B in 10min; wavelength: 254nm / 220nm; RT1(min): 27) was purified to obtain 2-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclopropylurea (120mg, 20.0% yield)

[0142] LCMS (ESI, m / z): [M+H] + =490.2.

[0143] Step 8.

[0144] The crude product 2-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclopropylurea (120 mg) was separated by Chiral-HPLC (column type: CHIRAL ART Cellulose-SB, 2*25 cm, 5 μm; mobile phase A: DCM-HPLC, mobile phase B: IPA (0.2% DEA); flow rate: 16 mL / min; gradient: isocratic 50; wavelength: 220 nm; RT1 (min): 4.9; RT2 (min): 7.0; Sample Solvent: MeOH:DCM=1:1-HPLC; injection volume: 0.6 mL; Number Of Runs: 5) to give two isomeric compounds: compound 2-1 and compound 2-2.

[0145] Compound 2-1 (isomer 1, RT1 (min): 4.9, 52.0 mg, 47.6% yield):

[0146] LCMS (ESI, m / z): [M+H] + =490.45

[0147] 1 H NMR(400MHz, Methanol-d4)δ7.12(s,1H),7.07(s,1H),6.94(s,1H),6.68(s,1H),5.47(s,1H),4.19–4.13(m,1H),4.10–4.05(m,1H),3 .89(s,3H),3.67(s,3H),2.99–2.93(m,3H),2.84(s,1H),2.37(s,3H),2.22(s,3H),2.02(s,3H),0.99–0.92(m,1H),0.75–0.72(m,1H).

[0148] Compound 2-2 (isomer 2, RT2 (min): 7.0, 51.7 mg, 47.6% yield):

[0149] LCMS (ESI, m / z): [M+H] + =490.45

[0150] 1H NMR(400MHz, Methanol-d4)δ7.12(s,1H),7.07(s,1H),6.94(s,1H),6.68(s,1H),5.47(s,1H),4.19–4.13(m,1H),4.10–4.05(m,1H),3 .89(s,3H),3.67(s,3H),2.99–2.93(m,3H),2.84(s,1H),2.37(s,3H),2.22(s,3H),2.02(s,3H),0.99–0.92(m,1H),0.75–0.72(m,1H).

[0151] Example 3, Compound 3

[0152] Step 1.

[0153] In a single-necked flask, tert-butyl N-(4-hydroxycyclohexyl)carbamate (3.0 g, 10.9 mmol, 1.0 equiv), DCM (80 mL), p-toluenesulfonyl chloride (5.0 g, 13.2 mmol, 1.2 equiv), triethylamine (3.3 g, 32.9 mmol, 3.0 equiv), and DMAP (134 mg, 1.1 mmol, 0.1 equiv) were added sequentially and reacted at room temperature overnight. The solvent was dried, EA (300 mL) was added, and the mixture was washed with saturated sodium bicarbonate solution (3 x 100 mL). The organic phase was dried over anhydrous sodium sulfate and dried to give tert-butyl N-{4-[(4-methylbenzenesulfonyl)oxy]cyclohexyl}carbamate (5.0 g, 61.7%).

[0154] LCMS (ESI, m / z): [M+H] + =370.2

[0155] Step 2.

[0156] To a single-necked flask, (2E)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-3H,6H,7H-pyrimidin[4,3-a]isoquinolin-4-one (1.0 g, 2.6 mmol, 1.0 equiv), dioxane (100 mL), cesium carbonate (1.7 g, 5.1 mmol, 2.0 equiv), and (tert-butyl N-{4-[(4-methylbenzenesulfonyl)oxy]cyclohexyl}carbamate) (1.4 g, 3.8 mmol, 1.5 equiv) were added sequentially and reacted at 100°C overnight. The mixture was filtered, the filtrate was concentrated to dryness, and the resulting residue was purified by reverse-phase column chromatography using the following conditions: C18 column, mobile phase: water and acetonitrile, gradient from 10% to 1000% over 30 minutes, UV 220 nm detection. Tert-butyl N-{4-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimidin[4,3-a]isoquinolin-3-yl]cyclohexyl}carbamate (110 mg, 7.3%) was obtained.

[0157] LCMS (ESI, m / z): [M+H] + =589.3

[0158] Step 3.

[0159] To a single-necked flask, tert-butyl N-{4-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimidin[4,3-a]isoquinolin-3-yl]cyclohexyl}carbamate (100 mg, 0.2 mmol, 1.0 equiv) and a 1,4-dioxane solution of HCl (4 M, 15 mL) were added sequentially and reacted at room temperature for 2 h. The solvent was then reduced to dryness to obtain (2E)-3-(4-aminocyclohexyl)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimidin[4,3-a]isoquinolin-4-one (80 mg, 96.4%).

[0160] LCMS (ESI, m / z): [M+H] + =489.3

[0161] Step 4.

[0162] (2E)-3-(4-Aminocyclohexyl)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimidin[4,3-a]isoquinolin-4-one (140 mg, 0.3 mmol, 1.0 equiv) was dissolved in dichloromethane (8 mL). Triethylamine (145 mg, 1.5 mmol, 5.0 equiv) and trimethylsilyl isocyanate (40 mg, 0.4 mmol, 1.2 equiv) were added to the above mixture at room temperature, and the mixture was stirred at room temperature for 3 h. The crude product was purified by Prep-HPLC under the following conditions (column: Xbridge BEH Shield RP18, 5 μm, 19*250 mm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: from 50% B to 57% B in 10 minutes; wavelength: 254 nm / 220 nm; RT1 (min): 8.93) to give 4-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclohexylurea (32.52 mg, yield 21.24%).

[0163] LCMS (ESI): [M+H] + =532.30

[0164] 1 H NMR(400MHz, Methanol-d4)δ6.93(s,2H),6.88(s,1H),6.74(s,1H),5.50(s,1H),5.36(s,1H),4.03–3.96(m,2H),3.93(s,1H),3.87(s,3 H),3.69(s,3H),2.95(t,J=6.2Hz,2H),2.79(s,2H),2.29(s,3H),2.05(s,6H),1.98-1.90(m,2H),1.80–1.70(m,2H),1.68–1.62(m,2H).

[0165] Example 4, Compound 4

[0166] Step 1.

[0167] To a single-necked flask, tert-butyl 4-hydroxypiperidine-1-carboxylate (3.0 g, 14.9 mmol, 1.0 equiv), p-toluenesulfonyl chloride (3.4 g, 17.9 mmol, 1.2 equiv), dichloromethane (100 mL), triethylamine (4.5 g, 44.7 mmol, 3.0 equiv), and 4-dimethylaminopyridine (182 mg, 1.5 mmol, 0.1 equiv) were added sequentially and allowed to react overnight at room temperature. The solvent was concentrated, ethyl acetate (300 mL) was added, and the mixture was washed with saturated sodium bicarbonate solution (3 x 100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to afford tert-butyl 4-[(4-methylbenzenesulfonyl)oxy]piperidine-1-carboxylate (4.3 g, 81.2% yield).

[0168] LCMS (ESI, m / z): [M+H] + =356.1

[0169] Step 2.

[0170] To a single-necked bottle, (2E)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-3H,6H,7H-pyrimido[4,3-a]isoquinolin-4-one (1.0 g, 2.6 mmol, 1.0 equiv), 1.4-dioxane (100 mL), cesium carbonate (2.5 g, 7.7 mmol, 3.0 equiv), and tert-butyl 4-[(4-methylbenzenesulfonyl)oxy]piperidine-1-carboxylate (1.0 g, 2.8 mmol, 1.1 equiv) were added sequentially and reacted at 100°C overnight. The mixture was filtered, the filtrate was concentrated, and the resulting residue was purified by reverse-phase column chromatography using a mobile phase of water and acetonitrile, with a gradient from 10% to 100% over 30 minutes, and a UV 220 nm detector. The resulting mixture was tert-butyl 4-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]piperidine-1-carboxylate (120 mg, 8.17% yield).

[0171] LCMS (ESI, m / z): [M+H] + =575.3

[0172] Step 3.

[0173] To a single-necked flask, tert-butyl 4-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]piperidine-1-carboxylate (100 mg, 0.2 mmol, 1.0 equiv) and a 1,4-dioxane solution of hydrogen chloride (2N, 17 mL) were added sequentially and reacted at room temperature for 2 h. The solvent was concentrated to afford (2E)-9,10-dimethoxy-3-(piperidin-4-yl)-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (80 mg, 96.9% yield).

[0174] LCMS (ESI, m / z): [M+H] + =475.3

[0175] Step 4.

[0176] To a solution of (2E)-9,10-dimethoxy-3-(piperidin-4-yl)-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (78 mg, 0.2 mmol, 1.0 equiv) in dichloromethane (2 mL) were added triethylamine (50 mg, 0.5 mmol, 3.0 equiv) and trimethylsilyl isocyanate (28 mg, 0.3 mmol, 1.5 equiv). The mixture was stirred at room temperature for 2 h. The mixture was concentrated by rotary evaporation under reduced pressure, and the resulting residue was purified by reverse phase column chromatography under the following conditions: chromatographic column: XBridge BEH Shield RP18, 5 μm, 19*250 mm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 46% B to 55% B in 10 min; wavelength: 254 nm / 220 nm; RT1 (min): 8.57 to obtain 4-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]piperidine-1-carboxamide (34.40 mg, yield 40.36%).

[0177] LCMS (ESI): [M+H] + =518.30

[0178] 1H NMR(400MHz,Methanol-d4)δ6.89(s,2H),6.85(s,1H),6.72(s,1H),5.50(s,1H),5.46(s,1H),4.19–4.13(m,2H), 3.94(t,J=6.1Hz,2H),3.85(s,3H),3.66(s,3H),2.99–2.69(m,6H),2.26(s,3H),2.02(s,6H),1.75–1.68(m,2H).

[0179] Example 5, Compound 5

[0180] Step 1.

[0181] (2E)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-3H,6H,7H-pyrimido[4,3-a]isoquinolin-4-one (5 g, 12.7 mmol, 1 equiv) and tert-butyl 3-bromoazoidine-1-carboxylate (3.62 g, 15.3 mmol, 1.2 equiv) were stirred in DMF (50 mL) and stirred at 100°C overnight under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. Purification by silica gel column chromatography using CH2Cl2 / MeOH (10 / 1) as the eluent gave tert-butyl 3-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]azidoidine-1-carboxylate (1.3 g, 18.6%).

[0182] Step 2.

[0183] Tert-butyl 3-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]azepine-1-carboxylate (1.0 g, 1.8 mmol, 1.0 equiv) was stirred in 1,4-dioxane (20 mL) at room temperature under a nitrogen atmosphere for 1 hour. The resulting mixture was concentrated under reduced pressure. This yielded (2E)-3-(azidobutan-3-yl)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (0.7 g, 85.6%). The crude product was used directly in the next step without further purification.

[0184] LCMS (ESI): [M+H] + =447.25

[0185] Step 3.

[0186] (2E)-3-(azetidin-3-yl)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-A]isoquinoline-4-1 (50 mg, 0.112 mmol, 1.0 eq) and trimethylsilyl isocyanate (14.19 mg, 0.12 mmol, 1.1 eq) were stirred in DCM (6 mL) at room temperature under a nitrogen atmosphere overnight. The resulting mixture was concentrated under vacuum. Column type: Xbridge BEH Shield RP18, 5μm, 19*250mm; mobile phase A: water (10mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 25mL / min; gradient: 40% B to 48% B, 10 minutes; wavelength: 254nm / 220nm; RT1 (min): 9.15 to obtain 3-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imine]-6H,7H-pyrimidin[4,3-a]isoquinolin-3-yl]azepine-1-carboxamide (14.69mg, 25.59%).

[0187] LCMS (ESI): [M+H] + =490.25

[0188] 1 H-NMR(400MHz, Methanol-d4)δ7.23(s,1H),7.13(d,J=3.9Hz,2H),7.03(s,1H),6.00(s,1H),5.15–5.08(m,1H),4.39–4.32(m,1H),4.28–4.20(m, 2H),4.22–4.19(m,1H),4.19–4.15(m,1H),3.95(s,3H),3.79(s,3H),3.6 2–3.56(m,1H),3.18–3.05(m,2H),2.38(s,3H),2.35(s,3H),2.27(s,3H).

[0189] Example 6, Compound 6

[0190] Step 1.

[0191] To a stirred solution of (2E)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-3H,6H,7H-pyrimido[4,3-a]isoquinolin-4-one (1.4 g, 3.5 mmol, 1 equiv) and Cs2CO3 (2.3 g, 7.1 mmol, 2 equiv) in 1,4-dioxane (15 mL) was added tert-butyl N-(3-bromocyclobutyl)carbamate (1.3 g, 5.3 mmol, 1.5 equiv). The resulting mixture was stirred at 100°C overnight under a nitrogen atmosphere. The filtrate was concentrated under reduced pressure, and the residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, acetonitrile-water (10 mmol / L NH4HCO3), gradient 50%-58% over 10 minutes; detection, UV 254 nm. Tert-butyl N-{3-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclobutyl}carbamate was obtained (700 mg, 32.% yield).

[0192] LCMS (ESI): [M+H] + =561.2

[0193] Step 2.

[0194] Tert-butyl N-{3-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclobutyl}carbamate (700 mg, 1.2 mmol, 1 equivalent) was added to a solution of hydrochloric acid in 1,4-dioxane (10 ml, 4 M), and the resulting mixture was stirred under nitrogen atmosphere at room temperature for 1.5 hours. The resulting mixture was concentrated under reduced pressure to give (2E)-3-(3-aminocyclobutyl)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (800 mg, crude product).

[0195] LCMS (ESI): [M+H] + =461.2

[0196] Step 3.

[0197] To a solution of (2E)-3-(3-aminocyclobutyl)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (800 mg, 1.7 mmol, 1 eq) and TEA (0.7 mL, 5.2 mmol, 3 eq) in DCM (10 mL) was added isocyanotrimethylsilane (300 mg, 2.6 mmol, 1.5 eq) under nitrogen at 0° C. The resulting mixture was stirred at room temperature overnight under nitrogen. The residue was purified by silica gel column chromatography eluting with dichloromethane / methanol (10 / 1) to give 3-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclobutylurea (400 mg, 42% yield).

[0198] LCMS (ESI): [M+H] + =504.2

[0199] Step 4.

[0200] Under air protection, to a solution of 3-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclobutylurea (400 mg, 0.7 mmol, 1 equivalent) and acetic anhydride (3 mL) in acetic acid (3 mL) was added an aqueous solution of HBr (3 mL). The resulting residue was stirred at 120°C under nitrogen protection for 2 hours. The reaction mixture was quenched with water at room temperature. The reaction mixture was extracted with dichloromethane / methanol (10 / 1) (2 x 30 mL). The combined organic phases were backwashed with saturated brine (2 x 100 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. 3-[(2E)-9,10-dihydroxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclobutylurea (120 mg, crude product) was obtained.

[0201] LCMS (ESI): [M+H] + =476.2

[0202] Step 5.

[0203] To a solution of 3-[(2E)-9,10-dihydroxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclobutylurea (100 mg, 0.2 mmol, 1 equiv) and potassium carbonate (87 mg, 0.6 mmol, 3 equiv) in DMF (1 mL) was added CD3I (76 mg, 0.5 mmol, 2.5 equiv) at room temperature under air protection. The resulting residue was stirred and reacted overnight at room temperature under nitrogen protection. The reaction mixture was quenched with water at room temperature. The reaction mixture was extracted with dichloromethane (2 x 30 ml). The combined organic phases were backwashed with saturated brine (2 x 50 ml) and dried over sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by chiral-SFC to obtain the product under the following conditions (column type: CHIRALPAK IH 2*25 cm, 5 μm; mobile phase A: MeOH:DCM=1:1--HPLC, mobile phase B: MtBE (0.1% DEA)-HPLC--; flow rate: 20 mL / min; gradient: isocratic; wavelength: 220 nm; RT1 (min): 13.9; RT2 (min): 17.2; solvent: methanol; injection volume: 0.5 mL; number of runs: 4).

[0204] Two isomeric compounds were obtained: compound 6-1 and compound 6-2.

[0205] Compound 6-1 (isomer 1, RT1 (min): 13.9, 25.91 mg, 24% yield):

[0206] LCMS (ESI): [M+H] + =510.30

[0207] 1 H NMR (400MHz, Methanol-d4) δ7.12(s,2H),6.92(s,1H),6.66(s,1H),5.43(s,1H),4.66–4.60(m,1H),4.09(t,J=6.4H z,2H),3.89(d,J=5.5Hz,1H),2.96(t,J=6.5Hz,2H),2.82–2.72(m,2H),2.37(s,3H),2.16(s,6H),1.96–1.90(m,2H).

[0208] Compound 6-2 (isomer 2, RT2 (min): 17.2, 9.41 mg, 8.5% yield):

[0209] LCMS (ESI): [M+H] +=510.2

[0210] 1 H NMR (400MHz, Methanol-d4) δ7.12(s,2H),6.92(s,1H),6.66(s,1H),5.43(s,1H),4.66–4.60(m,1H),4.09(t,J=6.4H z,2H),3.89(d,J=5.5Hz,1H),2.96(t,J=6.5Hz,2H),2.82–2.72(m,2H),2.37(s,3H),2.16(s,6H),1.96–1.88(m,2H).

[0211] Example 7, Compound 7

[0212] Under air protection, to a solution of (E)-1-(3-(9,10-dihydroxy-2-(methyltrimethylimino)-4-oxo-6,7-dihydro-2H-pyrimido[6,1-a]isoquinolin-3(4H)-yl)cyclobutyl)urea (100 mg, 0.2 mmol, 1 equiv) and K2CO3 (87 mg, 0.6 mmol, 3 equiv) in DMSO (2 mL) was added sodium 2-chloro-2,2-difluoroacetate (80 mg, 0.5 mmol, 2.5 equiv) at room temperature. Under nitrogen protection, the resulting residue was stirred and reacted overnight at room temperature. The reaction mixture was quenched with 50 mL of water at room temperature. The reaction mixture was extracted with dichloromethane (2 x 30 ml). The organic phases were combined, backwashed with saturated brine (2 x 50 ml), and dried over sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography to give 3-[(2E)-9,10-bis(difluoromethoxy)-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclobutylurea (6.77 mg, 5.3% yield) under the following conditions (column: CHIRALPAK IH 2*25 cm, 5 μm; mobile phase A: MeOH:DCM=1:1--HPLC, mobile phase B: MtBE (0.1% DEA)-HPLC--; flow rate: 20 mL / min; gradient: isocratic; wavelength: 220 nm; RT1 (min): 13.9; RT2 (min): 17.2; solvent: methanol; injection volume: 0.5 mL; number of runs: 4).

[0213] LCMS (ESI): [M+H] + =576.25

[0214] 1H NMR (400MHz, Methanol-d4) δ7.31(s,1H),7.13(d,J=2.3Hz,3H),6.95–6.55(m,2H),5.51(d,J=7.1Hz,1H),4.66–4.60(m,1H),4.13(t,J =6.4Hz,2H),4.02–3.88(m,1H),3.04(t,J=6.4Hz,2H),2.85–2.70(m,1H),2.50–2.15(m,5H),2.16(d,J=2.5Hz,6H),1.96–1.90(m,1H).

[0215] Example 8, Compound 8

[0216] Step 1.

[0217] 2-[(2,6-diisopropyl-4-methylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (500 mg, 1.1 mmol, 1 equiv), cesium carbonate (1.3 g, 3.3 mmol, 3 equiv), and tert-butyl(3-bromocyclobutyl)carbamate (560 mg, 2.3 mmol, 1.5 equiv) were dissolved in 1,4-dioxane (10 mL) and stirred at 100°C overnight under nitrogen. The mixture was cooled to room temperature, dissolved in 20 mL of water, and extracted with ethyl acetate (3 x 10 mL). The organic layer was washed with 20 mL of saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by reverse phase column chromatography under the following conditions (chromatographic column: C18; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 50% B to 80% B in 20 min; wavelength: 254 nm;) to give tert-butyl (E)-(3-(2-((2,6-diisopropyl-4-methylphenyl)imino)-9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido[6,1-a]isoquinolin-3(4H)-yl)cyclobutyl)carbamate (300 mg, 43.5% yield).

[0218] LCMS (ESI): [M+H] + =617.55

[0219] Step 2.

[0220] Tert-butyl (E)-(3-(2-((2,6-diisopropyl-4-methylphenyl)imino)-9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido[6,1-a]isoquinolin-3(4H)-yl)cyclobutyl)carbamate (300 mg, 0.49 mmol, 1 equivalent) was dissolved in 10 mL of a 1,4-dioxane solution of hydrochloric acid and stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure, dissolved in 10 mL of water, extracted with ethyl acetate (3×10 mL), and the organic layer was washed with 20 mL of saturated sodium chloride, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give 2-((1-aminopropan-2-yl)(2,6-diisopropyl-4-methylphenyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (300 mg, crude).

[0221] LCMS (ESI): [M+H] + =517.55

[0222] Step 3.

[0223] 2-((1-aminopropan-2-yl)(2,6-diisopropyl-4-methylphenyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (300 mg, 0.59 mmol, 1.0 eq.), triethylamine (180 mg, 1.8 mmol, 3 eq.), and trimethylsilyl isocyanate (103 mg, 1.2 mmol, 2 eq.) were dissolved in 10 mL of DCM and stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure, dissolved in 10 mL of water, and extracted with ethyl acetate (3×10 mL). The organic layer was washed with 20 mL of saturated sodium chloride, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product (150 mg) was purified by Chiral-SFC under the following conditions (chromatographic column: CHIRALPAK IH, 2*25 cm, 5 μm; mobile phase A: methanol: dichloromethane = 1:1--HPLC, mobile phase B: methyl tert-ether (0.1% ethylenediamine)--HPLC; flow rate: 20 mL / min; gradient: isocratic; wavelength: 220 nm; RT1 (min): 8; RT2 (min): 13.0; sample solvent: methanol--HPLC; injection volume: 1 mL) to give (E)-1-(3-(2-((2,6-diisopropyl-4-methylphenyl)imino)-9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido[6,1-a]isoquinolin-3(4H)-yl)cyclobutyl)urea (33.18 mg, yield 14.96%).

[0224] LCMS (ESI): [M+H] +=560.30

[0225] 1 H NMR(400MHz, Methanol-d4)δ7.21(s,2H),6.93(s,1H),6.64(s,1H),5.46(s,1H),4.49–4.44(m,1H),4.14–4.06(m,2H),3.97–3.88(m,1H),3 .89(s,3H),3.64(s,3H),2.94-2.90(m,4H),2.74-2.78(m,2H),2.44(s ,3H),2.20–2.08(m,2H),1.30(d,J=6.8Hz,7H),1.11(d,J=6.8Hz,6H).

[0226] Example 9, Compound 9

[0227] Step 1.

[0228] (2E)-2-[(2,6-Dimethoxy-4-methylphenyl)imino]-9,10-dimethoxy-3H,6H,7H-pyrimido[4,3-a]isoquinolin-4-one (500 mg, 1.2 mmol, 1 equiv) and tert-butyl N-(3-bromocyclobutyl)carbamate (354 mg, 1.4 mmol, 1.2 equiv) were added to DMF (1 mL), and cesium carbonate (769 mg, 2.3 mmol, 2 equiv) was added. The resulting mixture was stirred under nitrogen at 100°C overnight. The filter cake was filtered and washed with ethyl acetate (2 x 100 mL). The filtrate was concentrated under reduced pressure, and the residue was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, acetonitrile in water (10 mmol / L NH4HCO3), gradient 45%-56% over 10 minutes; detection, UV 254 nm. As a result, tert-butyl N-{3-[(2E)-2-[(2,6-dimethoxy-4-methylphenyl)imino]-9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclobutyl}carbamate (400 mg, 45% yield) was obtained.

[0229] LCMS (ESI): [M+H] + =593.2

[0230] Step 2.

[0231] Tert-butyl N-{3-[(2E)-2-[(2,6-dimethoxy-4-methylphenyl)imino]-9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclobutyl}carbamate (400 mg, 0.7 mmol, 1 equivalent) was added to a solution of hydrochloric acid in 1,4-dioxane (4 mL, 4 M), and the resulting mixture was stirred at room temperature under a nitrogen atmosphere for 1.5 hours. The resulting mixture was then concentrated under reduced pressure. The residue was purified by Prep-TLC (CH2Cl2 / MeOH=10 / 1) to give (2E)-3-(3-aminocyclobutyl)-2-[(2,6-dimethoxy-4-methylphenyl)imino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (130 mg, 37% yield).

[0232] LCMS (ESI): [M+H] + =493.2

[0233] Step 3.

[0234] Under nitrogen atmosphere at 0°C, (2E)-3-(3-aminocyclobutyl)-2-[(2,6-dimethoxy-4-methylphenyl)imino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (100 mg, 0.2 mmol, 1 eq) and triethylamine (0.1 mL, 0.6 mmol, 3 eq) were added to a DCM (2 mL) solution, followed by the addition of trimethylsilyl isocyanate (35 mg, 0.3 mmol, 1.5 eq). The resulting mixture was stirred at room temperature under nitrogen atmosphere overnight. The crude product was purified by high performance liquid chromatography under the following conditions (chromatographic column: XBridge BEH C18 5.0mmol: XBridge BEH C18 5μm, 30*150mm; mobile phase A: water (10mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60mL / min; column temperature: 25°C; flow rate: 60mL / min; gradient: from 26% B to 37% B in 7 minutes; wavelength: 254nm / 220nm; RT1 (min): 6.37; RT2 (min): 7.05 to obtain two isomeric compounds: compound 9-1 and compound 9-2.

[0235] Compound 9-1 (isomer 1, RT1 (min): 6.37, 1.18 mg, 0.8% yield).

[0236] LCMS (ESI): [M+H] + =536.25

[0237] 1 H NMR(400MHz, Methanol-d4)δ6.92(s,1H),6.73–6.69(m,3H),5.57(s,1H),5.31–5.25(m,1H),4.10–4.05(m, 2H),3.89(s,3H),3.81(d,J=1.6Hz,6H),3.70(s,3H),2.95(t,J=6.4Hz,2H),2.48(s,3H),2.26–2.20(m,4H).

[0238] Compound 9-2 (isomer 2, RT2 (min): 7.05, 34.31 mg, 22% yield)

[0239] LCMS (ESI): [M+H] + =536.30

[0240] 1 H NMR(400MHz, Methanol-d4)δ6.92(s,1H),6.73–6.69(m,3H),5.57(s,1H),5.31–5.25(m,1H),4.10–4.05(m, 2H),3.89(s,3H),3.81(d,J=1.6Hz,6H),3.70(s,3H),2.95(t,J=6.4Hz,2H),2.48(s,3H),2.26–2.20(m,4H).

[0241] Example 10, Compound 10

[0242] Step 1.

[0243] Under nitrogen protection, 2-chloro-9,10-dimethoxy-2H,3H,6H,7H-pyrimido[4,3-a]isoquinolin-4-one (1 g, 3.4 mmol, 1 equiv) and 2,6-difluoro-4-methylaniline (582 mg, 4.0 mmol, 1.2 equiv) were reacted in isopropanol (15 mL) at 80°C overnight. The mixture was concentrated under reduced pressure to give (E)-2-((2,6-difluoro-4-methylphenyl)imino)-9,10-dimethoxy-2,3,6,7-tetrahydro-4H-pyrimido[6,1-a]isoquinolin-4-one (1 g, 73.80%).

[0244] LCMS(ESI):[M+H] + =400.14

[0245] Step 2.

[0246] Under nitrogen protection, (E)-2-((2,6-difluoro-4-methylphenyl)imino)-9,10-dimethoxy-2,3,6,7-tetrahydro-4H-pyrimido[6,1-a]isoquinolin-4-one (1 g, 2.5 mmol, 1 equivalent), tert-butyl 3-bromocyclobutyl)carbamate (625 mg, 2.5 mmol, 1 equivalent), cesium carbonate (2.4 g, 7.5 mmol, 3 equivalents), and a 1,4-dioxane solution (20 mL) were stirred at 100°C overnight. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (3 × 100 mL). The organic phases were combined, backwashed with saturated sodium chloride (1 × 100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure. The resulting residue was purified by reverse phase column chromatography under the following conditions: C18 chromatography column, mobile phase, water and acetonitrile, gradient from 10% to 50% over 20 minutes, UV254 nm detector to give tert-butyl (E)-(3-(2-((2,6-difluoro-4-methylphenyl)imino)-9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido[6,1-a]isoquinolin-3(4H)-yl)cyclobutyl)carbamate (400 mg, 28.10%).

[0247] LCMS(ESI):[M+H] + =569.25

[0248] Step 3.

[0249] Tert-butyl (E)-(3-(2-((2,6-difluoro-4-methylphenyl)imino)-9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido[6,1-a]isoquinolin-3(4H)-yl)cyclobutyl)carbamate (400 mg, 0.52 mmol, 1 equivalent) was dissolved in 10 ml of a 1,4-dioxane solution (4 M) of hydrochloric acid and stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure, dissolved in 10 ml of water, extracted with ethyl acetate (3×10 mL), and the organic layer was washed with 20 mL of saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give (E)-3-(3-aminocyclobutyl)-2-((2,6-difluoro-4-methylphenyl)imino)-9,10-dimethoxy-2,3,6,7-tetrahydro-4H-pyrimido[6,1-a]isoquinolin-4-one (400 mg, crude).

[0250] LCMS(ESI):[M+H] + =469.20

[0251] Step 4.

[0252] Under nitrogen protection, a solution of (E)-3-(3-aminocyclobutyl)-2-((2,6-difluoro-4-methylphenyl)imino)-9,10-dimethoxy-2,3,6,7-tetrahydro-4H-pyrimido[6,1-a]isoquinolin-4-one (100 mg, 0.2 mmol, 1 equiv), trimethylsilyl isocyanate (36 mg, 0.3 mmol, 1.5 equiv), and triethylamine (64 mg, 0.6 mmol, 3 equiv) in dichloromethane (2 mL) was stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure. The crude product was purified by HPLC under the following conditions (chromatographic column: YMC Triart C18 ExRs5 μm, 20 mm × 250 mm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: 30% B to 38% B in 10 min; wavelength: 254 nm / 220 nm; RT1 (min): 9.28 / 10.10) to obtain two isomeric compounds: compound 10-1 and compound 10-2.

[0253] Compound 10-1 (isomer 1, RT (min): 9.28, 16.86 mg, yield 15.38%)

[0254] LCMS(ESI):[M+H] + =512.25

[0255] 1 H NMR (400MHz, CDCl3) δ7.26-6.72(m,5H),5.49(s,1H),4.65-4.15(m,3H),4.14-4.00( m,2H),3.92(s,1H),3.74(s,3H),3.65(s,3H),2.96(m,4H),2.63(s,2H),2.42(s,5H).

[0256] Compound 10-2 (isomer 2, RT (min): 10.10, 4.74 mg, yield 4.19%)

[0257] LCMS(ESI):[M+H] + =512.25

[0258] 1 H NMR (400MHz, CD3OD) δ7.13-6.82(m,4H),5.65–5.35(m,2H),4.21–4.11(m,2H),4. 10–3.73(m,5H),3.61(s,2H),3.16-2.88(m,2H),2.67(s,3H),2.48–2.19(m,4H).

[0259] Example 11, Compound 11

[0260] Step 1.

[0261] Tert-butyl (E)-(2-(2-(methanesulfonylimido)-9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido[6,1-a]isoquinolin-3(4H)-yl)propyl)carbamate (500 mg, 0.911 mmol) was stirred in a solution of hydrochloric acid in dioxane (10 mL, 4 M) at room temperature for 0.5 hours. The resulting mixture was concentrated under reduced pressure to afford the product (300 mg, 73.39% yield).

[0262] LCMS(ESI):[M+H] + =449.16

[0263] Step 2.

[0264] (2E)-3-(1-aminopropan-2-yl)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (200 mg, 0.45 mmol), triethylamine (135 mg, 1.338 mmol), and trimethylsilyl isocyanate (102.73 mg, 0.9 mol) in DCM (10 ml) were stirred at room temperature for 2 hours. The resulting mixture was concentrated under reduced pressure. The resulting mixture was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (3 x 50 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by reverse phase flash evaporation under the following conditions (chromatographic column: Xselect CSH C185 m, 30 mm × 150 mm; mobile phase A: water (0.1% FA), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: from 28% B to 38% B in 8 minutes; wavelength: 254 nm / 220 nm nm; RT1 (min): 5.85 / 7.65) to obtain the product (100 mg, yield 45.62%).

[0265] LCMS(ESI):[M+H] + =492.35

[0266] Step 3.

[0267] The product (100 mg) was purified by CHIRAI-HPLC under the following conditions (chromatographic column: CHIRALPAK IE-3, 4.6*50 mm, 3 um; mobile phase A: DCM--HPLC, mobile phase B: ETOH:DCM=1:1; flow rate: 18 mL / min; gradient: isocratic 20; wavelength: 220 nm; sample solvent: MeOH-HPLC injection volume: 0.45 mL; number of runs: 5) to obtain two isomeric compounds of the product: compound 11-1 and compound 11-2.

[0268] Compound 11-1 (isomer 1, RT (min) = 17.4 min, 6.9 mg, yield 6.28%)

[0269] LCMS(ESI):[M+H] + =492.35

[0270] 1 H NMR(400MHz, Methanol-d4)δ7.14–7.06(m,2H),6.92(s,1H),6.67(s,1H),5.50(s,1H),4.58–4.50(m,1H),4.20–4.10(m,1H),4.11–4.00(m,1H), 3.95–3.87(m,1H),3.87(s,3H),3.66(s,3H),3.28–3.19(m,1H),2.97(t, J=6.5Hz,2H),2.35(s,3H),2.17(d,J=8.1Hz,6H),1.15(d,J=6.3Hz,3H).

[0271] Compound 11-2 (isomer 2, RT (min) = 21.8 min, 17.1 mg, yield 37.07)

[0272] LCMS(ESI):[M+H] + =492.35

[0273] 1H NMR(400MHz, Methanol-d4)δ7.14–7.06(m,2H),6.92(s,1H),6.67(s,1H),5.50(s,1H),4.58–4.50(m,1H),4.20–4.10(m,1H),4.11–4.00(m,1H), 3.95–3.87(m,1H),3.87(s,3H),3.66(s,3H),3.28–3.19(m,1H),2.97(t, J=6.5Hz,2H),2.35(s,3H),2.17(d,J=8.1Hz,6H),1.15(d,J=6.3Hz,3H).

[0274] Example 12, Compound 12

[0275] Step 1.

[0276] To a stirred solution of 2-chloro-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (2.0 g, 6.8 mmol) in DCM (20 ml) was added boron tribromide (8.5 g, 34.1 mmol) dropwise at 0°C. The resulting mixture was stirred at room temperature for 2 hours. The reaction was quenched with water / ice at room temperature. The residue was purified by reverse phase flash chromatography under the following conditions: chromatographic column, C18 silica gel; mobile phase, acetonitrile water (10 mmol / L NH4HCO3), gradient from 10% to 50% in 10 minutes; detector, UV 254 nm. 2-chloro-9,10-dihydroxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (1.8 g, yield 69.6%) was obtained.

[0277] LCMS(ESI):[M+H] + =265.0

[0278] Step 2.

[0279] A solution of 2-chloro-9,10-dihydroxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (1.8 g, 6.8 mmol) in DMF (20 mL) was treated with CD3I (2.4 g, 17.0 mmol) at room temperature overnight. The resulting mixture was diluted with water (50 mL). The aqueous layer was extracted with EtOAc (3 x 50 mL). The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, MeCN in water (10 mmol / L NH4HCO3), gradient from 10% to 50% over 10 minutes; detector, UV 254 nm. This afforded 2-chloro-9,10-di(2H3)methoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (700 mg, 34.4% yield).

[0280] LCMS(ESI):[M+H] + =299.1

[0281] Step 3.

[0282] 2-Chloro-9,10-bis(2H3)methoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (700 mg, 2.3 mmol) was reacted with 2,4,6-trimethylaniline (1.5 g, 11.7 mmol) in 10 mL of isopropyl alcohol (IPA) at 90°C overnight under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was washed with EtOAc (20 mL). This afforded (2E)-9,10-bis(2H3)methoxy-2-[(2,4,6-trimethylphenyl)imino]-3H,6H,7H-pyrimido[4,3-a]isoquinolin-4-one (900 mg, 77.3% yield).

[0283] LCMS(ESI):[M+H] + =398.2

[0284] Step 4.

[0285] (2E)-9,10-bis(2H3)methoxy-2-[(2,4,6-trimethylphenyl)imino]-3H,6H,7H-pyrimido[4,3-a]isoquinolin-4-one (900 mg, 2.2 mmol) was mixed with tert-butyl N-(2-bromoethyl)carbamate (761 mg, 3.3 mmol), Pd2(dba)3 (207 mg, 0.2 mmol) and cesium carbonate (2.2 g, 6.7 mmol) in 1,4-dioxane (10 mL) at 100°C overnight under nitrogen. The resulting mixture was diluted with water (20 mL). The aqueous layer was extracted with EtOAc (3 x 50 mL). The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, MeCN in water (10 mmol / L NH4HCO3), gradient from 10% to 50% over 10 minutes; detector, UV 254 nm. Tert-butyl N-{2-[(2E)-9,10-bis(2H3)methoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]ethyl}carbamate (350 mg, 28.5% yield) was obtained.

[0286] LCMS(ESI):[M+H] + =541.3

[0287] Step 5.

[0288] A mixture of tert-butyl N-{2-[(2E)-9,10-bis(2H3)methoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]ethyl}carbamate (350 mg, 0.6 mmol) and HCl in 1,4-dioxane (10 mL, 4 M) was stirred at room temperature for 1 hour. The resulting mixture was concentrated under reduced pressure to yield 350 mg of crude product. The crude product was used directly in the next step without further purification.

[0289] LCMS(ESI):[M+H] + =441.3

[0290] Step 6.

[0291] (2E)-3-(2-Aminoethyl)-9,10-bis(2H3)methoxy-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (350 mg, 0.8 mmol) in DCM (5 mL) was treated with isocyanotrimethylsilane (137.2 mg, 1.2 mmol) at room temperature overnight. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with water (10 mL). The aqueous layer was extracted with EtOAc (3 x 50 mL). The residue was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, MeCN in water (10 mmol / L NH4HCO3), gradient from 10% to 50% over 10 minutes; detection, UV 254 nm. As a result, 2-[(2E)-9,10-bis(2H3)methoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]ethyl urea (30.6 mg, yield 7.9%) was obtained.

[0292] LCMS(ESI):[M+H] + =484.40

[0293] 1 H NMR(400MHz, Methanol-d4)δ7.11(s,2H),6.94(d,J=6.6Hz,1H),6.70(s,1H),5.51(s,1H),4.16–4.09 (m,2H),3.95(t,J=6.8Hz,2H),3.44(t,J=6.8Hz,2H),2.98(t,J=6.4Hz,2H),2.36(s,3H),2.18(s,6H).

[0294] Example 13, Compound 13

[0295] Step 1.

[0296] Tert-butyl carbamate (0.8 g, 6.8 mmol) was reacted with NaH (0.4 g, 17.1 mmol) in THF (20 mL) at 0 ° C for 30 minutes under a nitrogen atmosphere, and then stirred at 0 ° C for 30 minutes. 2-Chloro-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (1 g, 3.4 mmol, 1.0 equivalent) was then added in portions at 0 ° C. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. The resulting mixture was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (2 x 50 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with CH2Cl2 / MeOH to give benzyl N-[(2E)-9,10-dimethoxy-4-oxo-3H,6H,7H-pyrimido[4,3-a]isoquinolin-2-ylidene]carbamate (750 mg, 53.9% yield).

[0297] LCMS (ESI, m / z): [M+H] + =374.2

[0298] Step 2.

[0299] To a stirred solution of tert-butyl N-[(2E)-9,10-dimethoxy-4-oxo-3H,6H,7H-pyrimido[4,3-a]isoquinolin-2-ylidene]carbamate (850 mg, 2.3 mmol) and tert-butyl N-(2-bromoethyl)carbamate (612.1 mg, 2.7 mmol) in dioxane (3 mL) was added CsCO (1483.3 mg, 4.6 mmol, 2.0 equiv) and Pd(dba) (208.5 mg, 0.2 mmol) in portions at room temperature under nitrogen. The resulting mixture was stirred at 100°C under nitrogen overnight. The resulting mixture was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (2 x 50 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, aqueous MeCN, gradient from 10% to 50% over 10 minutes; detection, UV 254 nm. The result was tert-butyl N-[(2E)-3-(2-[(tert-butoxycarbonyl)amino]ethyl-9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-ylidene]carbamate (450 mg, 38.3% yield).

[0300] LCMS (ESI, m / z): [M+H] + =517.3

[0301] Step 3.

[0302] A mixture of tert-butyl N-[(2E)-3-(2-[(tert-butoxycarbonyl)amino]ethyl-9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-ylidene]carbamate (450 mg, 0.9 mmol) and HCl in 1,4-dioxane (10 mL) was stirred at room temperature under nitrogen for 4 hours. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, MeCN in water (0.1% FA), 10%-90% gradient, 30 minutes; detector, UV 254 nm. The result was 3-(2-aminoethyl)-2-imino-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (200 mg, 72.6% yield).

[0303] LCMS (ESI, m / z): [M+H] + =317.2

[0304] Step 4.

[0305] To a solution of 3-(2-aminoethyl)-2-imino-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (200 mg, 0.6 mmol) and triethylamine (319.9 mg, 3.2 mmol) in DCM (3 mL) was added trimethylsilyl isocyanate (145.7 mg, 1.3 mmol) portionwise under nitrogen at room temperature. The resulting mixture was stirred overnight under nitrogen at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, acetonitrile in water, gradient from 10% to 50% over 20 minutes; detection, UV 254 nm. As a result, 2-(2-imino-9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinoline-3-ethylurea) was obtained (150 mg, yield 66.1%).

[0306] LCMS (ESI, m / z): [M+H] + =360.2

[0307] Step 5.

[0308] To a stirred solution of 2-(2-imino-9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinoline-3-ethylurea (150 mg, 0.417 mmol) and K2CO3 (115.4 mg, 0.8 mmol) in DMF (5 mL) was added 2-(bromomethyl)-1,3,5-trimethylbenzene (177.9 mg, 0.8 mmol) in portions at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 70°C overnight under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography under the following conditions: (column: YMC Triart C18 ExRs 5m, 30mm*150mm; mobile phase A: water (10mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: from 43% B to 45% B in 10 minutes). B; wavelength: 254nm / 220nm nm; RT1 (min): 9.38), to give 2-[(2E)-9,10-dimethoxy-4-oxo-2-([(2,4,6-trimethylphenyl)methyl]imino-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]ethyl urea (4.8 mg, 2.32%).

[0309] LCMS (ESI, m / z): [M+H] + =492.50

[0310] 1 H NMR (400MHz, Methanol-d4) δ7.41(s,1H),6.90(s,1H),6.79(s,2H),6.42(s,1H),5.24(s,2H),3.96(t,J=6.2Hz,1H),3.95(s,4H), 3.91(s,3H),3.48(d,J=6.2Hz,2H),3.29(t,J=6.2Hz,2H),2.92(t,J=6.2Hz,2H),2.33(s,6H),2.23(s,3H),0.12(d,J=2.2Hz,1H).

[0311] Example 14, Compound 14

[0312] Step 1.

[0313] H₂O₂ (14.8 mL, 190.6 mmol, 5.0 eq, 30%) was added to a solution of 2,6-dimethylbenzonitrile (5.0 g, 38.1 mmol) and KOH (4.28 g, 76.2 mmol, 2.0 eq) in MeOH (50 mL) and DMSO (5 mL). The mixture was stirred at 0°C under an air atmosphere. The resulting mixture was stirred at room temperature overnight under a nitrogen atmosphere. The resulting mixture was extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with brine (3 × 50 mL) and dried over anhydrous Na₂SO₄. The filtered filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography using PE / EA (3 / 2) as the eluent to afford 2,6-dimethylbenzylamine (4.8 g, 80.19% yield).

[0314] LCMS (ESI): [M+H]+=150.1

[0315] Step 2.

[0316] XantPhos (100 mg, 0.2 mmol, 0.1 eq) and Cs2CO3 (1.7 g) were added portionwise to a stirred solution of 2,6-dimethylbenzamide (250 mg, 1.7 mmol) and 2-chloro-9,10-dimethoxy-6h,7h-pyrimido[4,3-a]isoquinolin-4-one (500 mg, 1.7 mmol) in dioxane (5 mL). The resulting mixture was stirred at 105°C under a nitrogen atmosphere for 6 h. The resulting mixture was filtered, and the filter cake was washed with methanol (3 x 15 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with EA / MeOH (8 / 1) to give N-[(2E)-9,10-dimethoxy-4-oxo-3h,6H,7h-pyrimidin[4,3-a]isoquinolin-2-yl]-2,6-dimethylbenzamide (450 mg, yield 64.98%).

[0317] LCMS (ESI): [M+H]+=406.4

[0318] Step 3.

[0319] N-[(2E)-9,10-dimethoxy-4-oxo-3h,6H,7h-pyrimidin[4,3-a]isoquinolin-2-yl]-2,6-dimethylbenzylamine (200 mg, 0.5 mmol) was added separately to a stirred solution of tert-butyl N-(2-bromoethyl)carbamate (120 mg, 0.5 mmol) and Cs2CO3 (200 mg, 0.6 mmol) in DMF (4 ml). The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 14 h. The resulting mixture was filtered, and the filter cake was washed with methanol (3 × 50 mL). The filtrate was concentrated under reduced pressure. Purification was performed by reverse-phase flash chromatography using the following conditions: a C18 silica gel column; a mobile phase of acetonitrile in water (10 mmol / L NH4HCO3) with a gradient of 60% to 70% over 10 min; and a UV detector at 254 nm. As a result, tert-butyl N-{2-[(2E)-2-(2,6-dimethylbenzoyl)-9,10-dimethoxy-4-oxo-6h,7h-pyrimidin[4,3-a]isoquinolin-3-yl]ethylcarbamate (200 mg, yield 73.90%) was obtained.

[0320] LCMS (ESI): [M+H]+=549.3

[0321] Step 4.

[0322] N-{2-[(2E)-2-(2,6-dimethylbenzoyl)-9,10-dimethoxy-4-oxo-6h,7h-pyrimidin[4,3-a]isoquinolin-3-yl]ethylcarbamate (200 mg, 0.4 mmol) was stirred in 1,4-dioxane (4 mL, 4 M) at room temperature under air for 2 hours. The resulting mixture was concentrated under reduced pressure. The resulting mixture was concentrated under vacuum. This resulted in N-[(2E)-3-(2-aminoethyl)-9,10-dimethoxy-4-oxo-6h,7h-pyrimidin[4,3-a]isoquinolin-2-yl]-2,6-dimethylbenzamide (120 mg, 73.39% yield).

[0323] LCMS(ESI):[M+H] + =449.2

[0324] Step 5.

[0325] Trimethylsilyl isocyanate (33 mg, 0.3 mmol, 1.2 equiv.) was added dropwise to a stirred solution of N-[(2E)-3-(2-aminoethyl)-9,10-dimethoxy-4-oxo-6h,7h-pyrimido[4,3-a]isoquinolin-2-ethyl]-2,6-dimethylbenzylamine (110 mg, 0.2 mmol) and TEA (55 mg, 0.5 mmol) in DCM (2 mL) at room temperature. The resulting mixture was stirred at room temperature under air for 2 hours. The resulting mixture was concentrated under reduced pressure. Pre-HPLC (column: XBridge BEH C18 OBD Prep column 130, 5m, 30mm*150mm; mobile phase A: water (10mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60ml / min mL / min; gradient: 30% B to 48% B, 10 minutes; wavelength: 254nm / 220nm nm; RT1 (min): 9.6.) was used to obtain N-[(2E)-3-[2-(carbamoylamino)ethyl]-9,10-dimethoxy-4-oxo-6h,7h-pyrimidin[4,3-a]isoquinoline-2-ethyl]-2,6-dimethylbenzamide (22.7 mg, yield 18.77%).

[0326] LCMS(ESI):[M+H] + =492.35

[0327] 1 H NMR (400MHz, DMSO-d6) δ7.17–7.09(m,3H),7.06(s,1H),7.03(d,J=7.5Hz,2H),6.05(t,J=6.0Hz,1H),5.3 4(s,2H),4.10–4.02(m,4H),3.87–3.83(m,6H),3.24(q,J=6.4Hz,2H),2.99(t,J=6.3Hz,2H),2.30(s,6H).

[0328] Example 15, Compound 15

[0329] Step 1.

[0330] To an 8 mL vial, 2-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6h,7h-pyrimidin[4,3-a]isoquinolin-3-yl]ethylurea (100 mg, 0.2 mmol) and HBr (0.5 mL) were added. The resulting mixture was stirred at 110°C under a nitrogen atmosphere for 3 hours. Purification was performed by reverse-phase flash chromatography using a C18 silica gel column, a mobile phase of acetonitrile (0.1% FA) in water, a 10% to 50% gradient over 10 minutes, and a UV detector at 254 nm. The resulting product was 2-[(2E)-9,10-dihydroxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6h,7h-pyrimidin[4,3-a]isoquinolin-3-yl]ethylurea (50 mg, 53.12% yield).

[0331] LCMS(ESI):[M+H] + =450.2

[0332] Step 2.

[0333] 2-[(2E)-9,10-Dihydroxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6h,7h-pyrimido[4,3-a]isoquinolin-3-yl]ethylurea (100 mg, 0.2 mmol) and ethyl 2-bromo-2,2-difluoroacetate (59 mg, 0.3 mmol) were stirred in DMF (0.5 mL). K2CO3 (92 mg, 0.7 mmol) was added at 25°C under a nitrogen atmosphere. The reaction mixture was stirred at 60°C under a nitrogen atmosphere for 2 hours. Purification was performed by reverse-phase flash chromatography using the following conditions: a C18 silica gel column; a mobile phase of acetonitrile (0.1% FA) in water, with a gradient from 10% to 50% over 10 minutes; and a UV detector at 254 nm. As a result, 2-[(2E)-9-(difluoromethoxy)-10-hydroxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6h,7h-pyrimidin[4,3-a]isoquinolin-3-yl]ethyl urea (12 mg, yield 10.80%) was obtained.

[0334] LCMS(ESI):[M+H] + =500.2

[0335] Step 3.

[0336] 2-[(2E)-9-(Difluoromethoxy)-10-hydroxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6h,7h-pyrimido[4,3-a]isoquinolin-3-yl]ethylurea (50 mg, 0.1 mmol) and (bromoethyl)cyclopropane (14 mg, 0.1 mmol) were stirred in DMF (0.5 mL). K2CO3 (17 mg, 0.1 mmol) was added at 25°C under a nitrogen atmosphere. The reaction mixture was stirred at 50°C under a nitrogen atmosphere for 2 hours. Purification was performed by reversed-phase flash chromatography using a C18 silica gel column, a mobile phase of acetonitrile (0.1% FA) in water, a gradient of 50% to 80% over 10 minutes, and a UV detector at 254 nm. As a result, 2-[(2E)-10-(cyclopropylmethoxy)-9-(difluoromethoxy)-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6h,7h-pyrimidin[4,3-a]isoquinolin-3-yl]ethyl urea (4.7 mg, yield 8.37%) was obtained.

[0337] LCMS(ESI):[M+H] + =555.41

[0338] 1 H NMR(400MHz,Methanol-d4)δ7.14(s,1H),7.11(s,2H),6.96–6.88(m,1H),6.86(s,1H) ),5.56(s,1H),4.59(s,1H),4.13(t,J=6.3Hz,2H),3.99–3.93(m,2H),3.78(d,J=6.8 Hz,2H),3.46(t,J=6.8Hz,2H),2.97(t,J=6.3Hz,2H),2.37(s,3H),2.18(s,6H),2.14 (s,1H),1.34–1.28(m,1H),1.18–1.05(m,1H),0.63–0.52(m,2H),0.28–0.22(m,2H).

[0339] Example 16, Compound 16

[0340] Step 1.

[0341] 4-Bromo-2,6-dimethylaniline (1.0 g, 5.0 mmol) was dissolved in DMF (15 ml) at room temperature. KCO (2.07 g, 15.0 mmol) and KI (2.07 g, 12.5 mmol) were added sequentially. BnBr (2.14 g, 12.5 mmol) was added dropwise under a nitrogen atmosphere. After addition, the mixture was heated to 130°C and stirred for 16 h. After completion of the reaction, water (20 ml) was added to the mixture and extracted with ethyl acetate (30 ml x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, the solvent removed, and concentrated under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (eluent: n-heptane) to yield the product (1.6 g, 84.2%).

[0342] LCMS (ESI, m / z): [M+H] + =380.0

[0343] Step 2.

[0344] At room temperature, N,N-dibenzyl-4-bromo-2,6-dimethylaniline (1.5 g, 3.9 mmol, 1 eq) was dissolved in THF (15 ml). Under a nitrogen atmosphere, the temperature was lowered to -80°C, and 1.6 M n-BuLi solution (3.75 ml, 5.85 mmol) was added. After addition, the mixture was stirred at -80°C for 16 h. After completion of the reaction, saturated aqueous ammonium chloride (30 ml) was added to the mixture, and the mixture was extracted with ethyl acetate (60 ml x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, the solvent removed, and concentrated under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (eluent: n-heptane / ethyl acetate = 50:1-10:1) to afford the product (0.78 g, 62.1%).

[0345] LCMS (ESI, m / z): [M+H] + =319.2

[0346] Step 3.

[0347] N,N-dibenzyl-2,6-dimethyl-4-(methyl-d3)aniline (1.4 g, 4.4 mmol) was dissolved in ethyl acetate (70 ml) at room temperature. Pd / C (0.14 g, 10% w / w) and Pd(OH)2 (0.14 g, 10% w / w) were added sequentially. The mixture was pressurized with a hydrogen balloon and stirred at 40°C for 16 h. After completion of the reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure to yield the product (0.65 g, 62.1%).

[0348] LCMS (ESI, m / z): [M+H] + =139.12

[0349] Step 4.

[0350] At room temperature, 2,6-dimethyl-4-(methyl-d3)aniline (6.4 g, 5.0 mmol, 5 eq) and 2-chloro-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (3.0 g, 12.5 mmol) were added to IPA (30 ml). Under a nitrogen atmosphere, the temperature was raised to 90°C and stirred for 18 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (eluent: n-heptane / ethyl acetate = 30:1 to 1:1) to obtain the product (3.4 g, 84.2%).

[0351] LCMS (ESI, m / z): [M+H] + =395.21

[0352] Step 5.

[0353] At room temperature, (E)-2-((2,6-dimethyl-4-(methyl-d3)phenyl)imino)-9,10-dimethoxy-2,3,6,7-tetrahydro-4H-pyrimido[6,1-a]isoquinolin-4-one (3.4 g, 8.6 mmol) and Nphth-I (13.20 g, 51.6 mmol) were added to MIBK (120 ml). K2CO3 (10.77 g, 77.4 mmol) was also added. Under a nitrogen atmosphere, the temperature was raised to 85°C and stirred for 64 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The resulting residue was added to methanol (50 ml), stirred, and filtered to obtain the product (2.26 g, 84.2%).

[0354] LCMS (ESI, m / z): [M+H] + =568.2579

[0355] Step 6.

[0356] At room temperature, (E)-2-(2-((2,6-dimethyl-4-(methyl-d3)phenyl)imino)-9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido[6,1-a]isoquinolin-3(4H)-yl)ethyl)isoindoline-1,3-dione (2.2 g, 3.9 mmol) was dissolved in chloroform (22 ml) and EtOH (22 ml). Hydrazine hydrate (2.2 ml, 85%) was added, and the mixture was stirred at 25°C under a nitrogen atmosphere for 16 hours. After completion of the reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent to obtain the product (1.56 g, 92%).

[0357] LCMS (ESI, m / z): [M+H] + =438.2580

[0358] Step 7.

[0359] Compound (E)-3-(2-aminoethyl)-2-((2,6-dimethyl-4-(methyl-d3)phenyl)imino)-9,10-dimethoxy-2,3,6,7-tetrahydro-4H-pyrimido[6,1-a]isoquinolin-4-one (1.35 g, 3.1 mmol) was dissolved in 1,2-dichloroethane (27 ml) at room temperature. Trimethylsilyl isocyanate (1.24 g, 3.1 mmol) was added dropwise. Under a nitrogen atmosphere, the mixture was stirred at 25°C for 16 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (eluent: dichloromethane / methanol = 100:1 to 10:1) to obtain the product (0.8 g, 92%).

[0360] LCMS (ESI, m / z): [M+H] + =481.10

[0361] 1 H NMR (400MHz, CDCl3) δ6.88(s,2H),6.69(s,1H),6.67(s,1H),5.44(s,1H),4.41-4.38(t,J=7.2Hz,2H),4.05-4.0 2(t,J=6.1Hz,2H),3.90(s,3H),3.76(s,3H),3.53-3.52(d,J=7.5Hz,2H),2.92–2.89(t,2H),2.05-2.04(s,6H).

[0362] Example 17, Compound 17

[0363] Step 1.

[0364] To a 50 mL flask at room temperature, (E)-3-(2-aminoethyl)-2-(methyltrimethylimino)-9,10-dimethoxy-2,3,6,7-tetrahydro-4H-pyrimido[6,1-a]isoquinolin-4-one (0.5 g, 1.2 mmol) and methanol (5 mL) were added and stirred. 3,4-dimethoxycyclobut-3-ene-1,2-one (0.17 g, 1.2 mmol) was added and stirred at room temperature under nitrogen for 24 hours. After the reaction was complete, the system was cooled to 0°C and stirred for 1 hour. The reaction was then filtered and the filter cake was rinsed with 1 mL of methanol. The product (0.4 g, 63.8%) was purified on a silica gel column.

[0365] LCMS (ESI, m / z): [M+H] + =545.2

[0366] Step 2.

[0367] To a 50 mL flask at room temperature was added (E)-3-((2-(2-(methyltrimethylimino)-9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido[6,1-a]isoquinolin-3(4H)-yl)ethyl)amino)-4-methoxycyclobut-3-ene-1,2-one (1 g, 1.84 mmol) and methanol (15 mL), stirred. A 7 M ammonia-methanol solution (0.39 mL) was added, and the mixture was stirred at room temperature under nitrogen for 24 h. After completion of the reaction, the system was evaporated to dryness under reduced pressure and purified on a silica gel column to obtain the product (711 mg, 73.1%).

[0368] LCMS (ESI, m / z): [M+H] + =530.1

[0369] 1 H NMR(400MHz,DMSO-d6)δ7.90-7.15(broad,3H),6.95(s,1H),6.85(s,2H),6.65(s,1H),5.35 (s,1H),4.35(m,2H),3.95-3.75(m,6H),3.62(s,3H),2.88(s,2),2.23(s,3H),1.96(s,6H).

[0370] Example 18, Compound 18

[0371] Step 1.

[0372] Under a nitrogen atmosphere, (E)-2-(Methylimino)-9,10-dimethoxy-2,3,6,7-tetrahydro-4H-pyrimido[6,1-a]isoquinolin-4-one (20.61 g) and tert-butyl 3-(2-(methylphenyloxy)ethyl)imidazolidine-1-carboxylate (3.5 g) were dissolved in DMF (70 ml). Sodium carbonate (11.13 g) and sodium iodide (8.17 g) were added sequentially. The temperature was raised to 80°C and the reaction was maintained for 72 h. After completion of the reaction, water (140 ml) and ethyl acetate (100 ml x 3) were added. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (eluent: n-heptane:ethyl acetate = 20:1 to 5:1) to obtain the product (1.4 g).

[0373] LCMS (ESI, m / z): [M+H]+ =604.31

[0374] Step 2.

[0375] At room temperature, tert-butyl (E)-3-(2-(2-(methyltrimethylimino)-9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido[6,1-a]isoquinolin-3(4H)-yl)ethyl)-2-oxoimidazolidine-1-carboxylate (1.3 g) was dissolved in ethyl acetate (13 ml), and a solution of hydrogen chloride in ethyl acetate (4N, 5.4 ml) was added dropwise. The reaction was stirred for 4 h, and a saturated aqueous sodium bicarbonate solution was added dropwise to adjust the pH to about 8-9. The mixture was separated and washed with water (15 ml × 3). The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure. The residue was purified by high pressure to give the product (30 mg, 2.8%).

[0376] LCMS (ESI, m / z): [M+H] + =504.25

[0377] 1 H NMR (400MHz, CDCl3) δ6.87(s,2H),6.72(s,1H),6.65(s,1H),5.45(s,1H),4.49-4.40(m,2H),4.21(s,1H),4.08-4.01 (m,2H),3.90(s,3H),3.74(s,3H),3.71-3.52(m,4H),3.43-3.36(m,2H),2.92-2.85(m,2H),2.27(s,3H),2.08(s,6H).

[0378] Biological test evaluation

[0379] 1. PDE enzyme activity inhibition experiment 1

[0380] The FP method was used to test the PDE enzyme activity inhibition experiment of the compounds in each example, and the compound RPL-554 in Example 1 of CN100415743C was used as a positive control.

[0381] Prepare PDE enzyme and substrate (FAM-cyclic AMP / cyclic AMP) solutions in reaction buffer (1× IMAP Reaction Buffer containing 0.1% BSA supplemented with 1 mM DTT). The positive control has a starting concentration of 1 / 10 μM in PDE, with 3-fold dilutions and a 10+0 dose. 0.05 μL of compound in 100% DMSO was delivered to a 384-well plate (Corning 4514) using acoustic liquid delivery technology (Echo 655) and centrifuged at 1000 rpm for 1 minute. 2.5 μL of PDE enzyme solution was transferred to the 384-well plate and centrifuged at 1000 rpm for 1 minute, followed by incubation at 25°C for 10 minutes. 2.5 μL of Sub solution was transferred to the 384-well plate and centrifuged at 1000 rpm for 1 minute, followed by incubation at 25°C for 60 minutes. 15 μL of binder mixture was transferred to the 384-well plate and centrifuged at 1000 rpm for 1 minute, followed by incubation at 25°C for 60 minutes. FP signals were read using a BMG (PHERAstar FSX). IC50 values ​​and nonlinear regression curve fitting were obtained using GraphPad Prism software.

[0382] Table 2 shows the enzyme activity test IC of each compound in the comparative examples and examples 50 IC 50 The smaller the value, the smaller the amount of compound required to achieve half-inhibition of different PDE enzymes, indicating that its inhibitory activity is stronger.

[0383] Table 2

[0384] The results show that the compound of the present invention has a highly effective inhibitory activity on PDE3 / 4 and has practical value.

[0385] 2. PDE enzyme activity inhibition experiment 2

[0386] Compounds were tested for PDE enzyme activity inhibition using the FP method, with control compounds RPL-554 and Compound D1 used as controls. The specific testing method was the same as in PDE enzyme activity inhibition experiment 1. The structural formulas of Compounds RPL-554 and D1 are shown below.

[0387] Table 3 shows the enzyme activity test IC values ​​of the control compound RPL-554, compound D1, compound 2-1, compound 2-2 and compound 1-2 of the present invention. 50 value.

[0388] Table 3

[0389] 3. Study on the Effect of Compounds on Bronchial Smooth Muscle Cell Viability

[0390] Human bronchial smooth muscle cells were taken from the logarithmic growth phase and plated at 1×10 5 Cells were seeded at a density of cells / mL in a 96-well plate and cultured in an incubator. When the cells adhered to the wall and grew to a density of 80%, they were divided into 9 groups, with 6 parallel controls set up in each group. Cells were treated with 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 μmol / L of compounds, respectively. Basic culture medium was added to the blank control group, and culture was continued for 24 hours. The liquid in the wells was aspirated and discarded. MTS reagent: basic culture medium was prepared in a ratio of 1:4, 100 μL of MTS working solution was added to each well, and a Blank control well was set up. The cells were incubated at 37°C in the dark for 2 hours. The wavelengths of 490 nm and 630 nm were selected, and the light absorbance of each well was measured on a microplate reader. The results were recorded and the IC was calculated. 50 value.

[0391] Human bronchial smooth muscle cells were treated with 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 μmol / L of the compound for 24 hours, and cell viability was assessed by the MTS assay. The results are shown in Figures 1A to 1D. Compared with the control group, all concentrations of the compound inhibited the growth of human bronchial smooth muscle cells in a dose-dependent manner.

[0392] 4. Pharmacodynamic Study of Compounds in LPS-Induced Inflammation Model

[0393] Human monocytes were resuspended in RPMI 1640 complete medium containing 1% fetal bovine serum (FBS) at a concentration of 1×10 6 Cells were plated at a density of 100 μL / ml in a 96-well plate with 100 μL of cell suspension per well. The cells were divided into 8 groups, each with 6 parallel controls, including: a normal control group, a model control group, low- and high-dose groups of the positive control compound RPL-554 (0.78 and 1.56 μmol / L), low- and high-dose groups of compound 2-2 (0.78 and 1.56 μmol / L), and low- and high-dose groups of compound 1-2 (0.78 and 1.56 μmol / L). The LPS working concentration in the model control group and the compound group was 1 μg / ml. The cells were incubated in the absence or presence of LPS and the compound (5% CO2, 37°C). After 24 hours, the cell culture supernatant was collected and centrifuged at 2000-3000g for 5 minutes. The precipitate was removed, and the supernatant was collected for the detection of TNF-α levels.

[0394] The results of TNF-α level detection in cell supernatants are shown in Figure 2. TNF-α levels in the model control group were significantly higher than those in the normal control group. However, levels in the RPL-554 positive control group and compound group were significantly lower than those in the model group.

[0395] 5. Effects of Compounds on Airway Constriction Induced by Aerosolized Acetylcholine in Mice

[0396] Test sample preparation: The test sample is of high purity. Ignore impurities when weighing and calculate as pure. The test drug concentration is 10 mg / mL, and the solvent is 25% DMSO in pH 3.2 citric acid-sodium hydrogen phosphate buffer solution.

[0397] Administration of test article: The NAM system of DSI Company was used for oral and nasal aerosol administration of the drug to mice, 400 μL / mouse, aerosolized twice, each aerosolization time was 5 minutes, and the model group was aerosolized with the same amount of solvent.

[0398] Newly received experimental animals were quarantined for 3 days and grouped as described above. Airway nebulization was performed using the NAM system. The nebulization period was 5 minutes (200 μL), followed by a 3-minute interval and a further 5 minutes (200 μL). Approximately 0.5 hours after the completion of nebulization, acetylcholine (15 mg / ml, 20 μL, 30 seconds, in 0.9% saline) was nebulized using the RC system to induce airway constriction. Changes in airway resistance (RL) were simultaneously measured using an RC instrument. The results are shown in Table 4.

[0399] Table 4

[0400] VI. Oral bioavailability study in rats

[0401] Experimental purpose: To investigate the bioavailability and other pharmacokinetic parameters of 1 mg / kg RPL-554 and compound 2-2 of the present invention after oral administration to rats.

[0402] Test drugs: RPL-554, compound 2-2 of the present invention.

[0403] Experimental animals: 12 male SD rats.

[0404] Dosing solution for the RPL-554-iv-1 mg / kg and RPL-554-po-1 mg / kg groups: Weigh approximately 3.11 mg of RPL-554, add 0.3 mL of DMSO, then add 29.7 mL of normal saline, and vortex for 2 minutes. Prepare the solution immediately for use.

[0405] Dosing solution for the 1 mg / kg compound 2-2-iv and 1 mg / kg compound 2-2-po groups: Weigh approximately 3.0 mg of 2-2, add 0.3 mL of DMSO, mix thoroughly, then add 29.7 mL of normal saline, and vortex for 2 minutes until completely dissolved. Prepare the solution for immediate use.

[0406] Dosage regimen: The tail vein group received tail vein administration, while the gavage group received gavage administration. See Table 5.

[0407] Table 5

[0408] Rats: Both groups received a 10 mL / kg dose via tail vein or oral gavage. Blood samples (0.2 mL) were collected 5, 15, 30, 1, 2, 4, 6, 8, 12, 24, and 48 hours after administration. The blood was transferred to a disposable anticoagulant tube and centrifuged at 3500 rpm at 4°C for 10 minutes. The supernatant was stored at -20°C until analysis.

[0409] Plasma processing and LC / MS analysis: 50 μL of plasma sample was placed in a 1.5 mL centrifuge tube, 200 μL of internal standard working solution was added, vortexed for 5 min, and centrifuged at 12,000 rpm in a high-speed centrifuge for 10 min. The supernatant was collected and added to the injector vial for LC / MS analysis, and the chromatogram was recorded.

[0410] Pharmacokinetic parameter results: The main PK characteristics of compound 2-2 in rats were basically consistent with those of the original compound RPL-554, and the oral bioavailability of both compounds was extremely low. See Tables 6 and 7.

[0411] Table 6

[0412] Table 7

[0413] VII. Human in vitro PK experiment - plasma protein binding rate

[0414] Experimental purpose: To investigate the in vitro human plasma protein binding rate of RPL-554 and compound 2-2.

[0415] Test drugs: RPL-554, compound 2-2. Test plasma: human plasma.

[0416] Incubation conditions: test substance incubation time 5 hours; test substance final incubation concentration 0.5 μM; species human; temperature 37.0°C.

[0417] Test steps:

[0418] Prepare test substance and warfarin stock solutions, dilute, and add to the plasma matrix to a final concentration of 10 μM for the test substance and 1 μM for warfarin. After preparation, remove 50 μL and add to the stop buffer. Add 50 μL of blank buffer to serve as the T0 sample for recovery calculation.

[0419] Test group and system control group: 100 μL of plasma solution containing warfarin or test substance or 100 μL of buffer solution containing test substance was added to the administration end of the equilibrium dialysis device;

[0420] The dosing end and the receiving end were placed at 37°C and 100 rpm for 5 hours;

[0421] Stability test group: the test substance was incubated with plasma or blank buffer for 0 and 5 hours;

[0422] After the incubation, take 50 μL of solution sample from the administration end according to the group and add 50 μL of blank buffer; take 50 μL of solution sample from the receiving end and add 50 μL of blank plasma, so that the final volume of all samples is 100 μL;

[0423] All samples were added with methanol containing internal standard to precipitate proteins. After centrifugation, the supernatant was collected and the content of the test substance or warfarin in the samples was determined by LC-MS / MS relative quantitative analysis.

[0424] Pharmacokinetic parameter results: RPL-554 and compound 2-2 both exhibited high plasma protein binding in human plasma (see Table 8).

[0425] Table 8

[0426] 8. In vitro PK study - CYP450 DDI

[0427] Purpose of the experiment: To investigate the inhibitory effects of RPL-554 and compound 2-2 on CYP3A4, CYP2C9, and CYP2D6.

[0428] Test drugs: RPL-554, compound 2-2. CYP enzymes: CYP3A4, CYP2C9, CYP2D6.

[0429] Incubation conditions: Test substance incubation time: CYP3A4, 5 minutes; CYP2C9 and CYP2D6, 10 minutes; test substance final incubation concentration 10 μM; microsomal protein concentration 0.5 mg / mL; NADPH concentration 1.0 mM; temperature 37.0°C.

[0430] Test steps:

[0431] Prepare working solutions of substrates and inhibitors of CYP2C9, CYP2D6, and CYP3A4 respectively;

[0432] Prepare NADPH solution and pre-warm it in a water bath shaker at 37°C before use;

[0433] Liver microsomes were thawed and diluted with buffer;

[0434] Add the corresponding solution to each well of the incubation plate and pre-incubate for 15 minutes. Grouping: Test group = test substance at various concentrations and liver microsome solution. Negative control group (NC) = microsomes + buffer. Positive control group (PC) = microsomes + selective inhibitors of the respective subenzyme.

[0435] After pre-incubation, the corresponding solution was added again for reaction (CYP3A4: 5 minutes; CYP2C9 and CYP2D6: 10 minutes). Test group = substrate + coenzyme of each subenzyme. Negative control group (NC) = substrate + coenzyme of each subenzyme. Positive control group (PC) = substrate + coenzyme of each subenzyme.

[0436] All samples were incubated at 37°C, and pre-cooled methanol was added at the termination time to terminate the reaction and the time was recorded;

[0437] All samples were mixed and centrifuged at 4000 rpm for 10 min, and the supernatant was analyzed by LC-MS / MS.

[0438] Pharmacokinetic Parameters: At a concentration of 10 μM, both RPL-554 and compound 2-2 exhibited some inhibitory activity against CYP2C9, but had less inhibitory activity against CYP2D6. At a concentration of 10 μM, RPL-554 had no inhibitory effect on CYP3A4, but compound 2-2 exhibited varying degrees of inhibition. See Table 9.

[0439] Table 9

[0440] IX. Human in vitro PK experiment - liver microsome incubation test

[0441] Experimental purpose: To investigate the in vitro metabolic stability of RPL-554 and compound 2-2.

[0442] Test drugs: RPL-554, compound 2-2.

[0443] Test system: human liver microsomes.

[0444] Incubation conditions: test substance incubation time 0, 30, 60, 120 min; test substance final incubation concentration 0.5 μM; microsomal protein concentration 0.5 mg / mL; NADPH and UDPGA concentrations 1.0 mM; temperature 37.0°C.

[0445] Test steps:

[0446] Prepare a mixed testosterone and 7-hydroxycoumarin working solution;

[0447] Prepare a mixed NADPH and UDPGA solution and pre-warm it in a water bath shaker at 37°C before use;

[0448] Liver microsomes were thawed and diluted with buffer;

[0449] Add the prepared test substance and liver microsome solution to each well of the incubation plate;

[0450] The test was divided into three groups and sample addition was performed. Test substance test group = test substance + microsomes + coenzyme. Test substance negative control group (NC) = test substance + microsomes + buffer. Positive control group = Phase I and Phase II substrate + microsomes + coenzyme.

[0451] All samples were incubated at 37°C, and pre-cooled methanol containing internal standard was added at each termination time to terminate the reaction and the time was recorded;

[0452] All samples were mixed and centrifuged at 4000 rpm for 10 min, and the supernatant was analyzed by LC-MS / MS.

[0453] Pharmacokinetic parameter results: The metabolic types of RPL-554 and compound 2-2 are shown in Table 10.

[0454] Table 10

[0455] 10. Human in vitro PK experiment-Caco2 cell permeability test

[0456] Experimental purpose: To investigate the Caco2 cell permeability of RPL-554 and compound 2-2.

[0457] Test drugs: RPL-554, compound 2-2. Test cell line: human colorectal adenocarcinoma cell line.

[0458] Incubation conditions: test system Caco-2; incubation time 2 hours; final test substance concentration 1 μM; temperature 37.0°C.

[0459] Test steps:

[0460] Quality control test before permeability test: Before the start of the test, the cell transmembrane resistance value is measured with a resistance meter, and the apparent transmembrane resistance value of the monolayer cell membrane is calculated;

[0461] Permeability test: Before the test, remove the cell culture medium from the culture plate and add 37°C pre-warmed HBSS buffer to wash three times (Apical end and Basal end); remove the buffer in the plate, add 800μL 37°C pre-warmed HBSS buffer to the B end, and add 500μL to the A end*. Pre-warm propranolol solution (PC), nadolol solution (SC), and test substance solution* at 37°C, and remove 100 μL of solution from end A as the 0-hour end A sample, which is then stored at -20°C for testing. Place the culture plate in a 37°C incubator and incubate for 120 minutes. After the incubation period, remove 100 μL of solution from both end A and end B of all samples as 120-minute samples, which are then stored at -20°C for testing. All samples are mixed with methanol containing the internal standard at a ratio of 1:4 and detected by LC-MS / MS. After the incubation period, aspirate 100 μL from end B into a black 96-well plate to prepare an LFY standard curve. Analyze using a fluorescence microplate reader at an excitation wavelength of 485 nm.

[0462] Note: The A-terminal contains 10 μM Lucifer Yellow, and 0.1% BSA is added to the test buffer.

[0463] Pharmacokinetic parameter results: At a test concentration of 1 μM, the Papp(AB) values ​​of the test substance RPL-554 and compound 2-2 were 0.643×10 -6 cm / s, 0.656×10 -6 cm / s. Based on the permeability criteria, the test objects RPL-554 and compound 2-2 are low permeability compounds (see Table 11).

[0464] Table 11

[0465] 11. Effects of Compounds on Isolated Tracheal Relaxation

[0466] Preparation of the nutrient solution: The composition of the modified KH solution (Krebs-Henseleit solution) is (mmol / L): NaCl 133, KCl 4.7, MgSO4 0.61, NaH2PO4 1.35, CaCl2 2.52, NaHCO3 16.3, and glucose 7.8. With the exception of NaHCO3, CaCl2, and glucose, which are added immediately before the experiment, all other components are prepared as highly concentrated stock solutions (stored at room temperature). On the day of the experiment, appropriate amounts of each stock solution are diluted to the desired concentration with ultrapure water. The prepared nutrient solution is adjusted to a pH of 7.2-7.4 with hydrochloric acid and fully presaturated with a mixture of 95% O2 and 5% CO2 before use.

[0467] Experimental animals: Male guinea pigs, weighing 300–350 g, were provided by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with feed also provided by the center. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Guinea pigs were housed at the New Drug Evaluation Center of Hebei Yiling Pharmaceutical Research Institute Co., Ltd. under a 12-hour light cycle, a temperature of 20–26°C, and a relative humidity of 40–70%, with 5 animals per cage. Corncob bedding was provided by Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd. and sterilized by high temperature and pressure. Animals were fed SPF-grade breeding feed and supplied with clean, bottled water, which was freely available and changed daily. After one week of acclimatization, subsequent experimental procedures were performed.

[0468] During the experiment, the weight was recorded and the animals were hit on the head with a wooden stick. After being knocked unconscious, the chest was quickly opened and the complete trachea from the thyroid cartilage to the tracheal branch of the lung parenchyma was taken and immersed in KH solution with a mixture of 95% O2 and 5% CO2.

[0469] The connective tissue around the trachea was stripped off, and the trachea was cut transversely every two cartilage rings. Every four rings were ligated to form a tracheal ring. Two tungsten wire rings were inserted parallel to the lumen of the tracheal ring, and the specimen was placed in a bath containing 10 mL of modified KH solution. One side of the tracheal ring was fixed to the lower end of the stainless steel bracket with the help of a tungsten wire ring, and the other side was connected to the tension transducer with the help of a tungsten wire ring through a silk thread. The tension signal of the trachea was recorded in the data acquisition system through the tension transducer. The bath temperature was maintained at 37°C, oxygen was continuously supplied, the initial load was adjusted to 2g, the nutrient solution was replaced every 15 minutes, the specimen was balanced for 1 hour, and the solution was replaced 4 times during this period. The experiment was started after the experimental conditions were stable. A normal curve was recorded, and acetylcholine chloride (Ach) was added to a final concentration of 1×10 -5 mol / L, observe when the contraction height reaches the maximum, and then add the compound with a final concentration of 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, and 300 μmol / L in sequence, with an interval of 5 minutes between each concentration, and record the value when the relaxation height is the minimum. The tracheal relaxation function is expressed as the tracheal relaxation rate (the percentage of the compound relaxation amplitude to the ACh pre-contraction amplitude), and the EC of each compound is calculated. 50 value.

[0470] The results are shown in Figure 3 and Table 12. Compared with the positive control group, compound 2-2 can effectively alleviate the contraction of guinea pig isolated organ smooth muscle induced by acetylcholine.

[0471] Table 12

[0472] 12. hERG Experimental Evaluation

[0473] The experimental materials are shown in Table 13.

[0474] Table 13

[0475] Human embryonic kidney (HEK293) cells stably expressing the hERG channel were cultured in DMEM medium in a 37°C cell culture incubator containing 5% CO2. The cell culture medium was supplemented with 10% fetal bovine serum (FBS) and G418 at a final concentration of 400 μg / ml. Prior to the experiment, cells were observed under an inverted microscope until they reached approximately 75% confluency. After digestion with 0.25% trypsin, a single cell suspension was obtained. The cell suspension was pipetted onto a cell slide and cultured in a cell culture incubator. Once adhered, the cells were used for patch clamp analysis. The positive drug, terfenadine, was used.

[0476] Voltage clamp parameters were set using Clampex 10.6 software. In whole-cell mode, cells were clamped at -80 mV for 125 ms, then depolarized to +50 mV for 5000 ms to activate hERG channels. The cells were then repolarized to -50 mV for 5000 ms to elicit the characteristic hERG tail current, which was then recorded. This tail current was used for data acquisition, measurement, and analysis. Results are shown in Table 14.

[0477] Table 14

[0478] Conclusion: RPL-554 has an inhibitory effect on hERG current at a concentration of 10 μM, while compound 2-2 has no effect on hERG current at both 1 μM and 10 μM concentrations.

[0479] 13. Mutagenicity of Compounds to Salmonella Typhimurium

[0480] The bacterial toxicity and potential mutagenicity of compound 2-2 against Salmonella typhimurium were evaluated to determine the optimal dosage range of the test article in subsequent Salmonella typhimurium mutagenicity tests.

[0481] The experiment was conducted using five histidine-deficient Salmonella typhimurium strains, TA97a, TA98, TA100, TA102, and TA1535 (provided by Shao Yan (Suzhou) New Drug Research Center Co., Ltd.). Under non-metabolic activation and metabolic activation conditions, each strain system was treated with the test substance (Compound 2-2) at doses of 5000, 1500, 500, 150, and 50 μg / dish, respectively. A vehicle control (DMSO) was also established. At each test point, two replicate plates were incubated at 37°C for approximately 48 hours for TA97a, TA98, TA100, and TA1535, and for approximately 72 hours for TA102, under metabolic activation (+S9) and non-metabolic activation (-S9) conditions. The number of revertant colonies on each plate was counted, and the background bacterial lawn was observed under a microscope. Furthermore, the presence of turbidity or precipitation was observed upon addition of the control or test substance or at the end of the incubation period.

[0482] Results: Under both metabolic activation and non-metabolic activation conditions, turbidity or precipitation was observed for each strain at doses of 1500 to 5000 μg / dish upon addition of the test sample and at the end of incubation. At all other doses, no turbidity or precipitation was observed for each strain at the time of addition or at the end of incubation. The test sample was bacteriologically toxic to histidine-deficient Salmonella Typhimurium strains TA97a, TA100, and TA1535 at a dose of 5000 μg / dish, and to histidine-deficient Salmonella Typhimurium strains TA97a and TA100 at a dose of 1500 μg / dish. Due to precipitation, the background bacterial lawn of strains TA98 and TA102 could not be observed at a dose of 5000 μg / dish, and strain TA98 at a dose of 1500 μg / dish, making toxicity indeterminate. No reduction in the number of revertant colonies was observed under the other conditions. Given the normal background bacterial lawn for each strain, no significant bacteriological toxicity was observed. The number of revertant colonies in the vehicle control group for each strain was within the historical background data of this laboratory. Compared with the vehicle control group, no biologically significant increase in the number of revertant colonies was observed in the test article groups.

[0483] Conclusion: In this study, under both metabolic and non-metabolic activation conditions, the test article was bacteriotoxic to strains TA97a, TA100, and TA1535 at a dose of 5000 μg / dish, and to strains TA97a and TA100 at a dose of 1500 μg / dish. Due to precipitation, background bacterial lawns could not be observed in each culture dish for strains TA98 and TA102 at a dose of 5000 μg / dish, and for strain TA98 at a dose of 1500 μg / dish, making toxicity impossible to assess. No significant bacteriotoxicity was observed under the other conditions. Compound 2-2 was non-mutagenic to histidine-deficient Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA1535 under both metabolic and non-metabolic activation conditions.

[0484] The above description is only a specific embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A compound of formula I or a pharmaceutically acceptable form thereof, wherein the pharmaceutically acceptable form is selected from a pharmaceutically acceptable salt or cocrystal, stereoisomer, tautomer, deuterated form, solvate, chelate, non-covalent complex or prodrug: in: R1 and R2 are each independently selected from H, C 1-6 Straight chain alkyl, C 3-6 Branched alkyl and C 3-6 Cycloalkyl; the linear alkyl, branched alkyl or cycloalkyl is optionally further substituted by 0 to 4 groups selected from H, F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1- 4 is substituted by an alkoxy substituent; R3, R4, R5 are each independently selected from H, halogen, CN, C 1-6 Alkoxy, C 1-6 Straight chain alkyl, C 3-6 Branched alkyl, and C 3-6 Cycloalkyl; the alkoxy, linear alkyl, branched alkyl or cycloalkyl may be further substituted by 0 to 4 groups selected from H, F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent; R6 is selected from The H in R6 is optionally further replaced by 0 to 4 selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent; R7 is selected from H, =O, NH2, CN, C 1-6 Straight chain alkyl, C 3-6 Branched alkyl, C 3-6 Cycloalkyl and The H in R7 is optionally further replaced by 0 to 4 selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent; L is selected from n is 0, 1 or 2; k is 0, 1 or 2; the H in L is optionally further replaced by 0 to 4 selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent; X is N; Y is C 1-3 Alkylene or absent; the alkylene is optionally further substituted by 0 to 4 groups selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent; Optionally, R1 and R2 are connected to form a 5- or 6-membered ring together with the connected O; the H in the 5- or 6-membered ring is optionally further replaced by 0 to 4 selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent; Optionally, X and L are connected through C 1-2 The alkylene groups are connected to form a 5- or 6-membered ring; the H in the 5- or 6-membered ring is optionally further replaced by 0 to 4 groups selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent; Optionally, R6 and L together form a 5- or 6-membered ring; the H in the 5- or 6-membered ring is optionally further replaced by 0 to 4 selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent; Optionally, H of the compound of formula I is optionally further substituted with 0 to 6 D; Furthermore, the compound of formula I is not:

2. The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein: R1 and R2 are each independently selected from H, CH3, CHF2, and CD3.

3. A compound according to claim 1 or 2, or a pharmaceutically acceptable form thereof, wherein: R3, R4, and R5 are each independently selected from H, CH3, i-Pr, OMe, CN, CD3 or halogen.

4. A compound according to any one of claims 1 to 3 or a pharmaceutically acceptable form thereof, wherein: R6 is and / or R7 is H, =O or 5. A compound according to any one of claims 1 to 4, or a pharmaceutically acceptable form thereof, wherein: L is selected from n is 0, 1 or 2; k is 1 or 2.

6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable form thereof, wherein: Y does not exist.

7. The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein the compound has a structure shown in Formula Ia: in: R1, R2 are each independently selected from CH3, CHF2 and CD3; R3, R4, R5 are each independently selected from CH3, F, methoxy and isopropyl; L is selected from 8. The compound according to claim 7 or a pharmaceutically acceptable form thereof, wherein: R1, R2, R3, R4, and R5 are CH3 respectively; L is 9. The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein: The compound is selected from one or more of the following compounds:

10. An intermediate compound having a structure shown in Formula II: in, R1, R2, R3, R4, R5, R7, X, and Y are as defined in any one of claims 1 to 9; L1 is selected from n is 0, 1 or 2; k is 0, 1 or 2; the H in L1 is optionally further replaced by 0 to 4 selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent; Preferably, the intermediate compound has the following structure:

11. A method for preparing a compound according to any one of claims 1 to 9 or a pharmaceutically acceptable form thereof, the method comprising: The carboxyl end of the intermediate compound of formula II described in claim 10 is subjected to a modification reaction to prepare a compound having structural formula I.

12. The method according to claim 11, further comprising a process for preparing the intermediate compound according to claim 10.

13. A pharmaceutical composition comprising: A compound according to any one of claims 1 to 9, or a pharmaceutically acceptable form thereof, and a pharmaceutically acceptable carrier, excipient and / or one or more other therapeutic agents.

14. Use of the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable form thereof or the pharmaceutical composition according to claim 13 in the preparation of a preparation for inhibiting phosphodiesterase.

15. Use of the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable form thereof or the pharmaceutical composition according to claim 13 in the preparation of a medicament for treating a phosphodiesterase-related disease; Preferably, the phosphodiesterase-related disease comprises a respiratory disease such as asthma.

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