Tricyclic fused heterocyclic pde3 / 4 dual inhibitor, and preparation and use thereof

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

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 phosphodiesterase inhibitor (especially a PDE3 / 4 dual inhibitor) and its preparation method and application. 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, 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;

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

[0022] L is selected from and, n is 0, 1 or 2; k is 0, 1, 2 or 3; 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 Alkoxy is substituted by a substituent; wherein R9, R 10 Each independently selected from H, C 1-6 Straight chain alkyl, C 3- 6 branched alkyl, C 3-6 Cycloalkyl, C 3-6 Heterocycloalkyl, C 6-10 Aryl, C 5-10 Heteroaryl and COOCH3, and R9, R 10 are not H at the same time; the heteroaryl group contains 1-3 heteroatoms selected from N, O and S;

[0023] R6 is selected from Among them, R 11 Selected from amino, C 1-6 Alkoxy, C 3-12 Cycloalkyl, C 6-10 Aryl, C 5-10 Heterocyclic group, the heterocyclic group contains 1-3 heteroatoms selected from N, O and S, the amino, alkoxy, cycloalkyl, aryl, heteroaryl are optionally further substituted by 0 to 4 atoms selected from H, F, Cl, Br, I, OH, =O, NH2, CN, COOH, CF3, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 substituted by a cycloalkyl substituent;

[0024] Optionally, R6 forms with L

[0025] R7 and R8 are each independently selected from H, =O, halogen, NH2, CN, C 1-6 Straight chain alkyl, C 3-6 Branched alkyl, C 3-6 Cycloalkyl and

[0026] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, R1 and R2 are each independently selected from CH3, CHF2, CD3 or C 3-6 Cycloalkyl.

[0027] According to some specific embodiments of the present invention, in the compound of formula I or a pharmaceutically acceptable form thereof, R1 and R2 are respectively CD3.

[0028] 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, one of R1 and R2 is CH3, and the other is CHF2.

[0029] 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 CH3, i-Pr, OMe, CD3 or halogen.

[0030] According to some specific embodiments of the present invention, in the compound of formula I or a pharmaceutically acceptable form thereof, R1 and R2 correspond to the groups or values ​​shown in any compound in Table 1.

[0031] 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, R5 is CH3, and the CH3 is optionally further substituted by 0 to 3 Ds; R3 and R4 are the same and are selected from CH3, i-Pr, OMe or halogen.

[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, R3, R4, and R5 correspond to the groups or values ​​shown in any compound in Table 1.

[0033] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, L is n is 0 or 1, preferably 0. Preferably, k is 0, 1 or 2. Preferably, R9, R 10 One of them is H, and the other is selected from H, C 1-6 Straight chain alkyl, C 3-6 Branched alkyl, C 3-6 Cycloalkyl, C 3-6 Heterocycloalkyl, C 6-10 Aryl, C 5-10 Heteroaryl and COOCH3; the heteroaryl contains 1 or 2 heteroatoms N; the H in L is optionally further replaced by 0 to 4 atoms selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, C 1-4 Alkyl, C 1-4 substituted by an alkoxy substituent.

[0034] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, L is

[0035] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, L is n is 0 or 1, and k is 1.

[0036] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, L is n is 0 or 1, and k is 1.

[0037] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, L is n is 2 and k is 2.

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

[0039] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, L is n is 2 and k is 2.

[0040] According to some specific embodiments of the present invention, in the compound of formula I or a pharmaceutically acceptable form thereof, L corresponds to a group or value shown in any compound in Table 1.

[0041] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, R6 is Among them, R 11 is an amino group, which is optionally further substituted by 0, 1 or 2 groups selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, CF3, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 The cycloalkyl group is substituted with a substituent.

[0042] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form, R6 is Among them, R 11 C 6-10 Aryl or C 5-10Heterocyclic group, the heterocyclic group contains 1, 2 or 3 heteroatoms N; the aryl and heteroaryl groups are optionally further substituted by 0, 1, 2 or 3 groups selected from H, F, Cl, Br, I, OH, =O, NH2, CN, COOH, CF3, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 The cycloalkyl group is substituted with a substituent.

[0043] According to some specific embodiments of the present invention, in the compound of formula I or a pharmaceutically acceptable form thereof, R6 corresponds to a group or value shown in any compound in Table 1.

[0044] 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, R7 is H or CH3, and R8 is H or F.

[0045] According to some specific embodiments of the present invention, in the compound of formula I or a pharmaceutically acceptable form thereof, R7 and R8 correspond to the groups or values ​​shown in any compound in Table 1.

[0046] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form:

[0047] R1 and R2 are each independently selected from CH3 or CD3;

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

[0049] L is

[0050] R6 is Among them, R 11 For amino, methoxy, Wherein, the amino group is optionally further substituted by 0 or 1 methyl or cyclopropyl groups; Optionally further substituted with 0 or 1 methyl or NH2;

[0051] R7 and R8 are H respectively.

[0052] According to some specific embodiments of the present invention, in the compound of formula I or its pharmaceutically acceptable form:

[0053] R1 and R2 are CH3 respectively;

[0054] R3, R4, and R5 are CH3 respectively;

[0055] L is

[0056] R6 is Among them, R 11 For amino,

[0057] R7 and R8 are H respectively.

[0058] 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, R8, and L respectively correspond to the groups or values ​​shown in any compound in Table 1.

[0059] According to some specific embodiments of the present invention, the compound of formula I or its pharmaceutically acceptable form of the present invention is selected from one or more of the compounds shown in Table 1:

[0060] Table 1

[0061] 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 of any one or more compounds shown in Table 1.

[0062] <Second Aspect>

[0063] The present invention also provides an intermediate compound having a structure shown in Formula II:

[0064] Wherein, R1, R2, R3, R4, R5, R7, and R8 are as defined in any one of the embodiments of the compound of formula I or a pharmaceutically acceptable form thereof according to the first aspect of the present invention;

[0065] L1 is selected from R9, R 10 , n, k 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; R 12 、R 13 Each independently represents H, Boc, Cbz, SEM, Fmoc, Alloc, Pht, OTs, PMB, Bn, and Trt.

[0066] According to some specific embodiments of the present invention, the intermediate compound of the present invention has the following structure:

[0067] R 12 、R 13 One of them is H, and the other is Boc, Cbz, SEM, Fmoc, Alloc, Pht, OTs, PMB, Bn, Trt.

[0068] <Third Aspect>

[0069] 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.

[0070] According to some specific embodiments of the present invention, the method of the present invention for preparing the compound of the first aspect of the present invention or a pharmaceutically acceptable form thereof comprises:

[0071] 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.

[0072] 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.

[0073] 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 includes the steps shown in any reaction scheme in Examples 1 to 38.

[0074] According to some specific embodiments of the present invention, the method of the present invention for preparing the compound of the first aspect of the present invention or a pharmaceutically acceptable form thereof comprises:

[0075] <Fourth Aspect>

[0076] 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.

[0077] <Fifth Aspect>

[0078] 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.

[0079] <Sixth Aspect>

[0080] 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.

[0081] 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.

[0082] According to some specific embodiments of the present invention, the phosphodiesterase comprises PDE3 and / or PDE4.

[0083] According to some specific embodiments of the present invention, the phosphodiesterase-related disease includes a respiratory disease such as asthma.

[0084] According to some specific embodiments of the present invention, the subject is a mammal or a human, preferably, the subject is a human.

[0085] 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.

[0086] Definition and Description

[0087] 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.

[0088] 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 bond 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

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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 comprising a plurality of stereoisomers or tautomers.

[0096] 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.

[0097] 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.

[0098] 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).

[0099] 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.

[0100] 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.

[0101] The terms "including" and "comprising" are used in their open, non-limiting sense.

[0102] "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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] Unless otherwise specified, the term "amino" refers to -NH2, -NH(alkyl), or -N(alkyl)(alkyl).

[0114] Unless otherwise specified, the term "aromatic ring" means a polyunsaturated aromatic alkane monocyclic ring which may be mono- or polysubstituted.

[0115] Unless otherwise specified, the term "4-6 membered heterocycloalkyl" refers to a saturated monovalent monocyclic hydrocarbon ring containing 3, 4 or 5 carbon atoms and one or more radicals selected from O, NR a heteroatom group, wherein R a Represents hydrogen atom or C 1-6 Alkyl; the "4-6 membered heterocycloalkyl" may be attached to the rest of the molecule via any carbon atom or, if present, a nitrogen atom.

[0116] Unless otherwise specified, the term "heteroaromatic ring" refers to an aromatic ring containing one to four heteroatoms selected from one or more of N, O and S.

[0117] Unless otherwise specified, the term "heterocyclyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, for example, fused, bridged or spiro) non-aromatic group, the ring atoms of which are composed of carbon atoms and at least one heteroatom selected from N, O and S, wherein the S atom is optionally substituted to form S(=O), S(=O)2 or S(=O)(=NR x ), R x Independently selected from H or C 1-4Alkyl. If the valence bond requirements are met, the heterocyclic group can be attached to the rest of the molecule through any one of the ring atoms. For example, the term "3-8 membered heterocyclic group" as used in the present invention refers to a heterocyclic group having 3 to 8 ring atoms. For example, the heterocyclic group can be an oxiranyl, aziridine, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, pyrrolidonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithianyl or trithianyl.

[0118] Unless otherwise specified, the term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated π electron system. For example, the term "C 6-10 The term "aryl" refers to an aromatic group having 6 to 10 carbon atoms. For example, the aromatic group may be phenyl, naphthyl, anthracenyl, phenanthrenyl, acenaphthenyl, azulenyl, fluorenyl, indenyl, pyrenyl, and the like.

[0119] Unless otherwise specified, the term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic group having a conjugated π electron system, the ring atoms of which are composed of carbon atoms and at least one heteroatom selected from N, O and S. If the valence bond requirements are met, the heteroaryl group can be connected to the rest of the molecule through any one of the ring atoms. For example, the term "5-10 membered heteroaryl" as used in the present invention refers to a heteroaryl group having 5 to 10 ring atoms. For example, the heteroaryl group can be thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and benzo derivatives thereof, pyrrolopyridinyl, pyrrolopyrazinyl, pyrazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purinyl, etc.

[0120] Unless otherwise specified, the term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I). The term "hydroxy" refers to -OH. The term "cyano" refers to -CN. The term "amino" refers to -NH2.

[0121] 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.

[0122] 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

[0123] FIG1 is a single crystal diffraction pattern of compound 1-2 of the present invention.

[0124] FIG2A to FIG2G show the results of the study on the effect of each compound on the viability of bronchial smooth muscle cells.

[0125] FIG3 shows the results of the detection of TNF-α levels in the cell supernatant in the pharmacodynamic study of each compound on the LPS-induced inflammation model.

[0126] FIG4A and FIG4B respectively show the effects of compound 10 of the present invention on airway resistance (RL, cmH2O) and the amount of change in guinea pig airway resistance.

[0127] FIG5A and FIG5B respectively show the effects of compounds 1-2 of the present invention on guinea pig airway resistance (RL, cmH2O) and the amount of change.

[0128] FIG6 shows the effects of the compounds of the present invention on the relaxation function of isolated trachea. DETAILED DESCRIPTION

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] Unless otherwise specified in the following examples and comparative examples, the reaction temperature was room temperature (20° C. to 30° C.), and the solvents were dried and purified according to standard methods.

[0135] The comparative example compounds are shown in Table 2.

[0136] Table 2, Comparative Example Compounds

[0137] Preparation of Comparative Example Compound D1:

[0138] Preparation steps:

[0139] 1.

[0140] To a solution of 2,4,6-trimethylaniline (2.0 g, 14.7 mmol, 1 eq) and tert-butyl N-(3-bromopropyl)carbamate (14.1 g, 59.1 mmol, 4 eq) in DMF (20 mL, 258.4 mmol) were added K2CO3 (6.1 g, 44.3 mmol, 3 eq) and KI (2.5 g, 14.7 mmol, 1 eq), respectively, under nitrogen at room temperature. The reaction mixture was allowed to react at 80°C overnight. The reaction mixture was extracted with EA (3 × 50 ml). The combined organic phases were washed with saturated sodium chloride solution (2 × 30 ml) and dried over anhydrous sodium sulfate. The mixture was filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with EA / PE (1 / 4) to give tert-butyl N-(3-[(2,4,6-trimethylphenyl)amino]propylcarbamate (2.2 g, yield 50.86%).

[0141] LCMS (ESI, m / z): [M+H] + =293.2

[0142] 2.

[0143] Under nitrogen protection at room temperature, to a solution of tert-butyl N-(3-[(2,4,6-trimethylphenyl)amino]propylcarbamate (1.2 g, 4.1 mmol, 1 equivalent) and 2-chloro-9,10-dimethoxy-6,7-dihydropyrimido[4,3-a]isoquinolin-4-one (1.8 g, 6.1 mmol, 1.5 equivalents) in dioxane (20 mL) were added Cs2CO3 (2.7 g, 8.2 mmol, 2 equivalents), APhos Pd G3 (0.3 g, 0.4 mmol, 0.1 equiv) and 4-(di-tert-butylphosphonyl)-N,N-dimethylaniline (0.1 g, 0.4 mmol, 0.1 equiv). The reaction was stirred at 100°C overnight under nitrogen. The reaction mixture was extracted with EA (3 x 50 mL). The combined organic phases were washed with saturated sodium chloride solution (2 x 30 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. 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 90% over 30 minutes, UV: 254 nm. This afforded tert-butyl N-[3-((9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl(2,4,6-trimethylphenyl)amino)propyl]carbamate (200 mg, 8.88% yield).

[0144] LCMS (ESI, m / z): [M+H] + =549.4

[0145] 3.

[0146] Under nitrogen protection, TFA (5 mL) was added dropwise to a solution of tert-butyl N-[3-((9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl(2,4,6-trimethylphenyl)amino)propyl]carbamate (290 mg, 1 equivalent) in DCM (5 mL) at 0°C. The resulting residue was stirred and reacted for 2 h at room temperature under nitrogen protection. The reaction mixture was extracted with DCM (3×20 ml). The combined organic phases were washed with saturated sodium chloride solution (2×10 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered and the filtrate was concentrated under reduced pressure to give 2-[(3-aminopropyl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (200 mg, crude product).

[0147] LCMS (ESI, m / z): [M+H] + =449.3

[0148] 4.

[0149] Under nitrogen protection, trimethylsilyl isocyanate (128.4 mg, 1.1 mmol, 2.5 equiv) was added dropwise to a solution of 2-[(3-aminopropyl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (200 mg, 0.4 mmol, 1.0 equiv) and TEA (135 mg, 1.3 mmol, 3 equiv) in DCM (5 mL) at 0°C. The resulting mixture was stirred at 50°C under nitrogen protection for 2 days. The resulting residue was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography to give 3-((9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl(2,4,6-trimethylphenyl)amino)propanurea (7.5 mg, yield 3.4%) under the following conditions (column type: Xbridge BEH Phenyl 5 μm, 19*250 mm; mobile phase A: water (0.1% FA), mobile phase B: MeOH; flow rate: 25 mL / min, gradient: 43% B to 53% B in 10 minutes; wavelength: 254 nm / 220 nm; RT1 (minute): 9.6

[0150] LCMS (ESI, m / z): [M+H] + =492.25

[0151] 1 H NMR (400MHz, Methanol-d4) δ8.22(broad,1H),7.26(s,1H),7.01(s,2H),6.94(s,1H),6.24(s,1H),4.05(t,J=6.4Hz,2H ),3.91(s,6H),3.61(t,J=7.8Hz,2H),3.45(t,J=6.6Hz,2H),3.01–2.89(m,2H),2.29(s,3H),2.24(s,6H),1.88(s,2H).

[0152] Preparation of Comparative Example Compound D2:

[0153] To a 50 mL flask at room temperature, (2E)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-3H,6H,7H-pyrimido[4,3-a]isoquinolin-4-one (1.0 g, 2.55 mmol, 1.0 equiv) and dichloromethane (20 mL) were added and stirred. Acetic anhydride (0.29 g, 2.81 mmol, 1.1 equiv) was added and stirred at room temperature under nitrogen for 24 h. After completion of the reaction, the system was cooled to 0°C and 10 mL of water was added dropwise. The mixture was stirred for 1 h and extracted three times with dichloromethane. The organic phases were combined, dried, rotary evaporated, and purified on a silica gel column to afford N-(9,10-dimethoxy-4-oxo-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-2-yl)-N-m-methylacetamide (0.11 g).

[0154] LCMS (ESI, m / z): [M+H] + =434.21

[0155] 1 H NMR (400MHz, CDCl3) δ7.72(s,1H),7.18(s,1H),6.94(s,2H),6.74(s,1H),4.19(t,J=6 .4Hz,2H),3.92(s,6H),2.95(t,J=6.4Hz,2H),2.31(s,3H),2.12(s,6H),2.05(s,3H).

[0156] Preparation of Comparative Example Compound D3:

[0157] (2E)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-3H,6H,7H-pyrimido[4,3-a]isoquinolin-4-one (1.00 g, 2.554 mmol, 1.0 eq), 2-bromopropane (0.47 g, 3.831 mmol, 1.5 eq), Pd2(dba)3 (0.23 g, 0.255 mmol, 0.1 eq), Cs2CO3 (2.50 g, 7.662 mmol, The product was dissolved in 1,4-dioxane (3.0 eq) and refluxed under nitrogen with stirring for 12 h. After completion of the reaction, water (50 ml) and ethyl acetate (50 ml × 3) were added, and the mixture was extracted with saturated sodium chloride (50 ml × 1). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2-(isopropyl(methyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (7.8 g, 71.43%).

[0158] LCMS (ESI, m / z): [M+H]+ =434.25

[0159] 1 H NMR (400MHz, Methanol-d4) δ7.08(s,2H),6.89(s,1H),6.58(s,1H),5.35(s,1H),4.82–4.74(m,1H),4.06(t,J=6 .4Hz,2H),3.86(s,3H),3.63(s,3H),2.93(t,J=6.4Hz,2H),2.34(s,3H),2.14(s,3H),1.25(s,3H),1.23(s,3H).

[0160] Preparation of Comparative Example Compound D4:

[0161] 9,10-Dimethoxy-2-[(2,4,6-trimethylphenyl)amino]-6h,7h-pyrimido[4,3-a]isoquinoline-4-1 (200 mg, 0.5 mmol, 1.0 equiv) was dissolved in DMF (2 mL). NaH (40 mg, 1.0 mmol, 2.0 equiv, 60% purity) was added at 0°C, and the resulting mixture was stirred under nitrogen for 0.5 h. (2-Bromoethyl)diethylamine hydrobromide (400 mg, 1.5 mmol, 3.0 equiv) was added to the above mixture, and stirred at room temperature for 1 h. The reaction was quenched by the addition of water (10 mL) at room temperature. The mixture was extracted with ethyl acetate (2 × 50 mL), and the organic layer was washed with saturated brine (3 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by column chromatography (EA / PE=1 / 2) gave 2-{[2-(diethylamino)ethyl](2,4,6-trimethylphenyl)amino}-9,10-dimethoxy-6h,7h-pyrimidin[4,3-a]isoquinoline-4-1 (37.2 mg, 14% yield).

[0162] LCMS(ESI):[M+H] + =491.25

[0163] 1 H NMR(400MHz, Methanol-d4)δ6.95–6.85(m,3H),6.77(s,1H),5.50(s,1H),4.44–4.35(m,2H),4.06–3.99(m,2H),3. 87(s,3H),3.69(s,3H),2.99–2.90(m,4H),2.74(q,J=7.2Hz,4H),2.28(s,3H),2.05(s,6H),1.16(t,J=7.2Hz,6H).

[0164] Preparation of Comparative Example Compound D5:

[0165] Preparation steps:

[0166] 1.

[0167] Under nitrogen, 9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)amino]-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (500 mg, 1.2 mmol, 1.0 equiv) and Cs2CO3 (1248 mg, 3.8 mmol, 3.0 equiv) were added to a DMF (10 mL) solution at room temperature. Tert-butyl N-(2-bromoethyl)carbamate (715 mg, 3.1 mmol, 2.5 equiv) and Pd2(dba)3 (116 mg, 0.1 mmol, 0.1 equiv) were added portionwise. The resulting residue was stirred at 100°C overnight under nitrogen. The reaction mixture was extracted with ethyl acetate (3 x 50 mL). The combined organic phases were washed with saturated sodium chloride solution (2 x 30 mL) and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with dichloromethane / methanol (20 / 1) to give tert-butyl N-[2-((9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl(2,4,6-trimethylphenyl)amino)ethyl]carbamate (200 mg, 29.29%).

[0168] LCMS(ESI):[M+H] + =535.2

[0169] 2.

[0170] Under nitrogen protection, tert-butyl N-[2-((9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl(2,4,6-trimethylphenyl)amino)ethyl]carbamate (180 mg, 0.3 mmol, 1.0 equivalent) was added to a solution of HCl in dioxane (4 mL, 4 M) at room temperature and stirred for 1 hour. The reaction mixture was basified to pH = 8 with saturated aqueous sodium bicarbonate. The reaction mixture was extracted with ethyl acetate (3×20 mL). The organic phases were combined, backwashed with saturated sodium chloride solution (2×10 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain 2-[(2-aminoethyl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (160 mg, crude product).

[0171] LCMS(ESI):[M+H] + =435.2

[0172] 3.

[0173] Under nitrogen, 2-[(2-aminoethyl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (100 mg, 0.2 mmol, 1.0 equiv) and TEA (69 mg, 0.6 mmol, 3.0 equiv) were added to a DCM (5 mL) solution at room temperature, followed by the dropwise addition of trimethylsilyl isocyanide (53 mg, 0.4 mmol, 2.0 equiv). The resulting residue was stirred under nitrogen at room temperature for 1.5 h. The reaction mixture was extracted with dichloromethane (3 x 20 mL). The combined organic phases were backwashed with saturated brine (2 x 50 mL) and dried over anhydrous 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 2-((9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl(2,4,6-trimethylphenyl)amino)ethyl urea (26.4 mg, 24.02%) under the following conditions (column type: YMC Triart C18 ExRs 5m, 19mm*250mm; mobile phase A: water (10mmol / LNH4HCO3), mobile phase B: acetonitrile; flow rate: 25mL / min; gradient: 32% B to 55% B in 10min; wavelength: 254nm / 220nm; RT1(min):9.17).

[0174] LCMS(ESI):[M+H] + =478.20

[0175] 1 H NMR(400MHz, Methanol-d4)δ7.11(s,2H),6.94(s,1H),6.70(s,1H),5.51(s,1H),4.15–4.08(m,2H),3.99–3. 92(m,2H),3.89(s,3H),3.67(s,3H),3.44(t,J=6.4Hz,2H),2.97(t,J=6.4Hz,2H),2.36(s,3H),2.18(s,6H).

[0176] Preparation of Comparative Example Compound D6:

[0177] (2E)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-3H,6H,7H-pyrimido[4,3-a]isoquinolin-4-one (500 mg, 1.28 mmol, 1.0 eq), 1-bromobutane (263 mg, 1.92 mmol, 1.5 eq), Pd2(dba)3 (119 mg, 0.13 mmol, 0.1 eq), Cs2CO3 (1.25 g, 3.84 mmol, 3.0 eq), Xantphos (150 The product was dissolved in 1,4-dioxane (10 ml), and the temperature was raised to reflux with stirring under nitrogen for 12 h. After the reaction was completed, water (50 ml) and ethyl acetate (50 ml × 3) were added, and the product was extracted with saturated sodium chloride (50 ml × 1), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified on a silica gel column to obtain 2-(butyl(m-methyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (121 mg, 21.1%).

[0178] LCMS(ESI):[M+H] + =448.26

[0179] 1 H NMR (400MHz, CDCl3) δ7.01(s,2H),6.71(s,1H),6.64(s,1H),5.34(s,1H),4.17(t,J=6.4Hz,2H),3.93–3.85(m,5H),3 .73(s,3H),2.90(t,J=6.4Hz,2H),2.35(s,3H),2.16(s,6H),1.62–1.51(m,2H),1.40–1.27(m,2H),0.89(t,J=8.0Hz).

[0180] Example 1, Compound 1-1 and Compound 1-2

[0181] Preparation steps:

[0182] 1.

[0183] Tert-butyl N-(2-bromopropyl)carbamate (0.91 g, 3.8 mmol, 1.5 equiv), cesium carbonate (2.50 g, 7.662 mmol, 3 equiv), Pd2(dba)3 (0.23 g, 0.26 mmol, 0.1 equiv), and (E)-2-(methylsulfonylimino)-9,10-dimethoxy-2,3,6,7-tetrahydro-4H-pyrimido[6,1-a]isoquinolin-4-one (1 g, 2.54 mmol, 1.0 equiv) were dissolved in 20 mL of 1,4-dioxane, stirred at 110°C overnight under nitrogen atmosphere, concentrated under reduced pressure, dissolved in 10 mL of water, extracted with ethyl acetate (3×10 mL), and the organic layer 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: 30% B to 50% B in 20 min; wavelength: 254 nm;) to give tert-butyl (E)-(2-(2-(methylsulfonylimino)-9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido[6,1-a]isoquinolin-3(4H)-yl)propyl)carbamate (500 mg, 35.6% yield).

[0184] LCMS(ESI):[M+H] + =549.06

[0185] 2.

[0186] Tert-butyl (E)-(2-(2-(methylsulfonylimino)-9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido[6,1-a]isoquinolin-3(4H)-yl)propyl)carbamate (500 mg, 0.91 mmol, 1 equivalent) was dissolved in 10 mL of a 4M hydrochloric acid solution in 1,4-dioxane and stirred at room temperature for 1 hour. 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 sodium sulfate, filtered, and concentrated under reduced pressure to afford 2-((1-aminopropan-2-yl)(methylsulfonyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (300 mg, 73.3% yield).

[0187] LCMS(ESI):[M+H] + =449.16

[0188] 3.

[0189] Dissolve 2-((1-aminopropan-2-yl)(methylsulfonyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (200 mg, 0.45 mmol, 1.0 eq.), triethylamine (135 mg, 1.34 mmol, 3 eq.), and trimethylsilyl isocyanate (102.73 mg, 0.9 mol, 2 eq.) in 10 mL of dichloromethane and stir at room temperature for 2 hours. Concentrate under reduced pressure, dissolve in 10 mL of water, and extract with ethyl acetate (3 x 10 mL). The organic layer is washed with 20 mL of saturated sodium chloride, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by reverse phase column chromatography under the following conditions (column: Xselect CSH C185 m, 30 mm x 150 mm; mobile phase A: water (0.1% FA), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 28% B to 38% B in 8 minutes; wavelength: 254 nm / 220 nm; RT1 (min): 5.85 / 7.65) to give 1-(2-((9,10-dimethoxy-4-oxo-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-2-yl)(methanesulfonamido)propyl)urea (100 mg, 45.6% yield).

[0190] LCMS(ESI):[M+H] + =492.35

[0191] 4.

[0192] The racemate (100 mg) was purified by chiral-HPLC using the following conditions: CHIRALPAK IE-3 column, 4.6*50 mm, 3 μm; mobile phase A: DCM-HPLC, mobile phase B: ETOH:DCM = 1:1; flow rate: 18 mL / min; gradient: isocratic 20; wavelength: 220 nm; RT1 (min): 17.4; RT2 (min): 21.8; sample solvent: MeOH-HPLC; injection volume: 0.45 mL; number of runs: 5. The product was obtained.

[0193] Compound 1-1 (isomer 1, RT1 (min): 17.4, 13.8 mg, yield 22.14%)

[0194] LCMS(ESI):[M+H] + =492.35

[0195] 1H NMR(400MHz, Methanol-d4)δ7.14–7.11(d,J=10.1Hz,2H),6.93(s,1H),6.64(s,1H),5.40(s,1H),4.49(broad,1H),4. 20–4.04(m,2H),3.89(s,3H),3.66(s,3H),2.97(t,J=6.5Hz,2H),2.37(s,3H),2.22(s,3H),2.10(s,3H),1.05(s,3H).

[0196] Compound 1-2 (isomer 2, RT2 (min): 21.8, 13.0 mg, yield 22.0%)

[0197] LCMS(ESI):[M+H] + =492.35

[0198] 1 H NMR(400MHz,Methanol-d4)δ7.12(d,J=10.1Hz,2H),6.93(s,1H),6.64(s,1H),5.40(s,1H),4.49(broad,1H),4.20 –4.04(m,2H),3.89(s,3H),3.66(s,3H),2.97(t,J=6.5Hz,2H),2.37(s,3H),2.22(s,3H),2.08(s,6H),1.05(s,3H).

[0199] The single crystal diffraction pattern of compound 1-2 is shown in Figure 1.

[0200] Example 2, Compound 2

[0201] Preparation steps:

[0202] 1.

[0203] 9,10-Dimethoxy-2-[(2,4,6-trimethylphenyl)amino]-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (3.0 g, 7.7 mmol, 1.0 equiv) and tert-butyl 3-bromopyrrolidine-1-carboxylate (2.3 g, 9.2 mmol, 1.2 equiv) were dissolved in 1,4-dioxane (100 mL), and Xantphos (0.9 g, 1.5 mmol, 0.2 equiv), cesium carbonate (5.0 g, 15.3 mmol, 2.0 equiv) and Pd2(dba)3 (0.7 g, 0.7 mmol, 0.1 equiv) were added. After stirring at 100°C under nitrogen atmosphere for 18 h, the resulting mixture was concentrated under reduced pressure. The residue was purified by reverse phase chromatography under the following conditions: chromatographic column, C18 silica gel column; mobile phase, acetonitrile, water, 0%-100% gradient, 30 minutes; detector, UV 254 nm to obtain tert-butyl 3-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)pyrrolidine-1-carboxylate (1.4 g, yield 32.6%).

[0204] LCMS(ESI):[M+H] + =561.3

[0205] 2.

[0206] Tert-butyl 3-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)pyrrolidine-1-carboxylate (300 mg, 0.5 mmol, 1.0 equivalent) was mixed with a solution of hydrochloric acid in 1,4-dioxane (8 mL), and the resulting mixture was stirred at room temperature for 1 hour. The resulting mixture was concentrated under reduced pressure to provide 9,10-dimethoxy-2-[pyrrolidin-3-yl(2,4,6-trimethylphenyl)amino]-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (300 mg, crude). The crude product was used in the next step without further purification.

[0207] LCMS(ESI):[M+H] + =461.2

[0208] 3.

[0209] 9,10-Dimethoxy-2-[pyrrolidin-3-yl(2,4,6-trimethylphenyl)amino]-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (100 mg, 0.2 mmol, 1.0 equiv) was dissolved in dichloromethane (4 mL) at room temperature, and triethylamine (109 mg, 1.1 mmol, 5.0 equiv) and isocyanatotrimethylsilane (38 mg, 0.3 mmol, 1.5 equiv) were added. The resulting mixture was stirred at room temperature for 2 h. The resulting mixture was concentrated under reduced pressure. The crude product was purified by reverse phase purification under the following conditions (chromatographic column: YMC-Triart C8 5.5 mmol / L, mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: from 32% B to 50% B in 7 minutes; wavelength: 254 nm / 220 nm; RT1 (min): 6.23) to give 3-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)pyrrolidine-1-carboxamide (30.5 mg, yield 27.4%).

[0210] LCMS (ESI, m / z): [M+H] + =504.25

[0211] 1 H NMR(400MHz, Methanol-d4)δ7.13(s,2H),6.92(s,1H),6.64(s,1H),5.41(s,1H),5.08–4.99(m,1H),4.19–4.03(m,3H),3.88(s,3H),3.66(s,3H),3 .58–3.52(m,1H),3.29(t,J=7.3Hz,1H),3.25–3.18(m,1H),2.96(t,J=6. 5Hz,2H),2.38(s,3H),2.29–2.22(m,1H),2.17–2.15(m,6H),1.77(s,1H).

[0212] Example 3, Compound 3-1 and Compound 3-2

[0213] Preparation steps:

[0214] 1.

[0215] Dimethoxyphenethylamine (50.0 g, 275.8 mmol, 1.0 equiv) and ethyl cyanoacetate were added sequentially to a single-necked flask under nitrogen atmosphere. The reaction was carried out at 100°C for 16 h, then the temperature was lowered to 70°C and ethanol (80 mL) was added. The mixture was filtered, and the filter cake was rinsed with ethanol (3 x 30 mL). The residual ethanol in the filter cake was evaporated to obtain 2-cyano-N-[2-(3,4-dimethoxyphenyl)ethyl]acetamide (50.0 g, 73.0% yield).

[0216] LCMS (ESI, m / z): [M+H] + =249.2

[0217] 2.

[0218] 2-Cyano-N-[2-(3,4-dimethoxyphenyl)ethyl]acetamide (50.0 g, 201.3 mmol, 1.0 equivalent) and phosphorus oxychloride (500 mL) were added sequentially to a single-necked bottle under nitrogen and reacted overnight at 85°C. The solvent was dried, water (300 mL) was added, and the mixture was extracted with DCM (3 x 400 mL). The combined organic phases were dried over anhydrous sodium sulfate and dried to yield 2-[(1E)-6,7-dimethoxy-3,4-dihydro-2H-isoquinolin-1-ylidene]acetonitrile (45.0 g, 97.0%).

[0219] LCMS (ESI, m / z): [M+H] + =231.2

[0220] 3.

[0221] 2-[(1E)-6,7-dimethoxy-3,4-dihydro-2H-isoquinolin-1-ylidene]acetonitrile (45.0 g, 195.4 mmol, 1.0 equivalent) and concentrated sulfuric acid (320 mL) were added sequentially to a single-necked flask and allowed to react at room temperature for 3 h. The reaction solution was slowly poured into ice water (1 L), adjusted to pH 7 with sodium hydroxide (4 M) at 0°C, and extracted with EtOAc (3 x 600 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated to dryness to yield 2-[(1E)-6,7-dimethoxy-3,4-dihydro-2H-isoquinolin-1-ylidene]acetamide (40.0 g, 82.4%).

[0222] LCMS (ESI, m / z): [M+H] + =249.2

[0223] 4.

[0224] To a single-necked flask, 2-[(1E)-6,7-dimethoxy-3,4-dihydro-2H-isoquinolin-1-ylidene]acetamide (40.0 g, 161.1 mmol, 1.0 equiv), ethanol (600 mL), and an ethanolic solution of sodium ethoxide (127 mL, 21%) were added sequentially. The mixture was reacted at 80°C for 0.5 h. Diethyl carbonate (57.1 g, 483.3 mmol, 3.0 equiv) was then added at the same temperature and the mixture was reacted at 80°C overnight. The mixture was cooled to room temperature, water (500 mL) was added, and the pH was adjusted to 7 with dilute hydrochloric acid. The mixture was filtered, and the filter cake was rinsed with ethanol (3 x 100 mL). The residual ethanol in the filter cake was evaporated to obtain 9,10-dimethoxy-3H,6H,7H-pyrimido[4,3-a]isoquinoline-2,4-dione (40.0 g, 90.5%).

[0225] LCMS (ESI, m / z): [M+H] + =275.2

[0226] 5.

[0227] 9,10-Dimethoxy-3H,6H,7H-pyrimido[4,3-a]isoquinoline-2,4-dione (40.0 g, 145.8 mmol, 1.0 equivalent) and phosphorus oxychloride (240 mL) were added sequentially to a single-necked flask under nitrogen atmosphere and reacted overnight at 100°C. The solvent was dried, dissolved in water (500 mL), and extracted with DCM (3 x 500 mL). The combined organic phases were dried over anhydrous sodium sulfate and dried to yield 2-chloro-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (30.0 g, 70.2%).

[0228] LCMS (ESI, m / z): [M+H] + =293.2

[0229] 6.

[0230] To a single-necked flask, 2-chloro-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (2.0 g, 6.8 mmol, 1.0 equiv), isopropanol (50 mL), and trimethylaniline (4.6 g, 34.1 mmol, 5.0 equiv) were added sequentially. The mixture was reacted at 90°C overnight under nitrogen. The solvent was evaporated, and EA (20 mL) and saturated aqueous sodium bicarbonate (200 mL) were added. The mixture was filtered, and the filter cake was rinsed with EA (3 x 50 mL). The residual EA in the filter cake was evaporated to obtain (2E)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-3H,6H,7H-pyrimido[4,3-a]isoquinolin-4-one (2.0 g, 74.8%).

[0231] LCMS (ESI, m / z): [M+H] + =392.2

[0232] 7.

[0233] In a single-necked flask, (2E)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-3H,6H,7H-pyrimido[4,3-a]isoquinolin-4-one (2.0 g, 5.1 mmol, 1.0 equivalent), ethyl 4-bromovalerate (2.1 g, 10.2 mmol, 2.0 equivalent), 1,4-dioxane (100 mL), cesium carbonate (5.0 g, 15.3 mmol, 3.0 equivalent), Xantphos (0.6 g, 1.0 mmol, 0.2 equivalent) and Pd2(dba)3 (0.5 g, 0.5 mmol, 0.1 equivalent) were added in sequence and reacted at 100°C for 12 h under nitrogen protection. Normal phase purification was performed using DCM / MeOH (10 / 1) as the mobile phase to give ethyl 4-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)pentanoate (500 mg, 18.8% yield).

[0234] LCMS (ESI, m / z): [M+H] + =520.3

[0235] 8.

[0236] In a single-necked flask, ethyl 4-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)pentanoate (500 mg, 1.0 mmol, 1.0 equivalent), methanol (50 mL) and a solution of sodium hydroxide (77 mg, 2.0 mmol, 2.0 equivalent) in water (10 mL) were added in sequence and reacted at room temperature for 3 h. The solvent was dried, water (20 mL) was added, and the pH was adjusted to 5 with dilute hydrochloric acid. The mixture was extracted with EA (3 x 50 mL), and the combined organic phases were dried over anhydrous sodium sulfate. The organic phase was dried to give 4-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)pentanoic acid (450 mg, 95.1% yield).

[0237] LCMS (ESI, m / z): [M+H] + =492.2

[0238] 9.

[0239] In a three-necked flask, 4-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)pentanoic acid (300 mg, 0.6 mmol, 1.0 equiv), 1.4-dioxane (100 mL), triethylamine (182 mg, 1.8 mmol, 3.0 equiv) and DPPA (336 mg, 1.2 mmol, 2.0 equiv) were added in sequence. The mixture was protected by nitrogen and reacted at room temperature for 1 h. The mixture was then heated to 100 °C and reacted for 1 h. The temperature was then lowered to 80 °C and a 1.4 M ammonia solution in tetrahydrofuran (100 mL) was added. The mixture was reacted at room temperature for 2 h. The solvent was dried and reverse chromatographic chromatography (column type: X-Bridge BEH C18 5 μm, 30*150 mm; mobile phase A: water (10 mmol / L The product was purified by HPLC-MS / MS (5% NH4HCO3), mobile phase B: acetonitrile; flow rate: 60 mL / min mL / min; gradient: 39% B to 48% B in 9 min; wavelength: 254 nm / 220 nm nm; RT1 (min): 8) to obtain 3-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)butaneurea (94 mg, 30.46% yield).

[0240] LCMS (ESI, m / z): [M+H] + =506.3

[0241] 10.

[0242] The crude product 3-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)butaneurea (50 mg) was separated by Chiral-HPLC (column type: CHIRAL ART Cellulose-SB, 2*25 cm, 5 μm; mobile phase A: MtBE (0.1% DEA)-HPLC, mobile phase B: MeOH:DCM=1:1--HPLC; Flow rate: 20 mL / min; gradient: isocratic; wavelength: 254 nm; RT1 (min): 5.3; RT2 (min): 7.2; Sample Solvent: MeOH:DCM=1:1--HPLC; injection volume: 1 mL; Number Of Runs: 5) to obtain the product.

[0243] Compound 3-1 (isomer 1, RT1 (min): 5.3, 17.6 mg, 34.78% yield)

[0244] LCMS (ESI, m / z): [M+H] + =506.50

[0245] 1 H NMR(400MHz,Methanol-d4)δ7.16–7.08(m,2H),6.93(s,1H),6.64(s,1H),5.4 1(s,1H),4.59–4.55(m,1H),4.19–4.02(m,2H),3.89(s,3H),3.66(s,3H),3.38 –3.34(m,1H),3.25–3.21(m,1H),2.97(t,J=6.4Hz,2H),2.38(s,3H),2.34–2. 30(m,1H),2.23(s,3H),2.08(s,3H),1.80–1.76(m,1H),0.99(d,J=6.9Hz,3H).

[0246] Compound 3-2 (isomer 2, RT2 (min): 7.2, 17.8 mg, 34.78% yield)

[0247] LCMS (ESI, m / z): [M+H] + =506.50

[0248] 1H NMR(400MHz,Methanol-d4)δ7.16–7.08(m,2H),6.93(s,1H),6.64(s,1H),5.4 1(s,1H),4.59–4.45(m,1H),4.19-4.02(m,2H),3.89(s,3H),3.66(s,3H),3.38 -3.34(m,1H),3.25-3.21(m,1H),2.97(t,J=6.4Hz,2H),2.38(s,3H),2.34-2. 30(m,1H),2.23(s,3H),2.08(s,3H),1.80-1.76(m,1H),0.99(d,J=6.9Hz,3H).

[0249] Example 4, Compound 4-1 and Compound 4-2

[0250] Preparation steps:

[0251] 1.

[0252] 2,4,6-Trimethylaniline (3.0 g, 22.2 mmol, 1.0 equiv) was dissolved in MeOH (30 mL) at room temperature under air, and NaBH3CN (1.0 g, 44.3 mmol, 2.0 equiv) and acetic acid (1 mL, 17.4 mmol, 0.7 equiv) were added. The resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The reaction was quenched with water at room temperature. The resulting mixture was extracted with ethyl acetate (2 x 100 mL). The combined organic phases were washed with saturated brine (3 x 200 mL) and dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure to provide tert-butyl N-{3-[(2,4,6-trimethylphenyl)amino]cyclobutyl}carbamate (1.2 g, 16% yield).

[0253] LCMS (ESI): [M+H]+=305.1

[0254] 2.

[0255] Tert-butyl N-{3-[(2,4,6-trimethylphenyl)amino]cyclobutyl}carbamate (1.2 g, 3.9 mmol, 1.0 equiv) and 2-chloro-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (1.3 g, 4.7 mmol, 1.2 equiv) were added to isopropanol (15 ml) at room temperature. The reaction mixture was heated to 100°C and stirred at 100°C under a nitrogen atmosphere overnight. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using petroleum ether / ethyl acetate (2 / 1) as the eluent to afford 2-[(3-aminocyclobutyl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (150 mg, 6% yield).

[0256] LC-MS (ESI): [M+H] + =461.2

[0257] 3.

[0258] N-(2,4,6-Trimethylphenyl)cyclobutane-1,3-diamine (150 mg, 0.7 mmol, 1.0 equiv) was dissolved in DCM (1 ml) at room temperature in air, and solid phosgene (193 mg, 0.6 mmol, 2.0 equiv) was added to the solution. The resulting mixture was stirred at room temperature under nitrogen for 1 hour. A 7 M solution of ammonia in methanol (15 mL) was added to the mixture at room temperature. The resulting mixture was stirred at room temperature overnight. 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 (10 mmol / L NH4HCO3), gradient 30%-47% over 7 minutes; detection, UV 254 nm. The crude product was purified by pre-HPLC under the following conditions (chromatographic column: CHIRAL ART Cellulose-SB, 2*25 cm, 5 μm; mobile phase A: MtBE (0.1% DEA)-HPLC, mobile phase B: MeOH:DCM=1:1-HPLC; flow rate: 20 mL / min; gradient: isocratic; wavelength: 220 nm; RT1 (min): 6.5; RT2 (min): 9.4; Sample Solvent: injection volume: 0.7 mL; number of runs: 3) to give the product.

[0259] Compound 4-1 (isomer 1, RT1 (min): 6.5, 16.3 mg, 9% yield)

[0260] LCMS(ESI):[M+H] + =504.30

[0261] 1 H NMR(400MHz, Methanol-d4)δ7.20(s,1H),7.05(s,2H),6.93(s,1H),6.11(s,1H),4.56–4.47(m,1H),4.28–4.15(m,1H),4.03 (t,J=6.2Hz,2H),3.90–3.88(m,6H),2.93(t,J=6.2Hz,2H),2.78–2.66(m,2H),2.32(s,3H),2.23(s,6H),1.88–1.73(m,2H).

[0262] Compound 4-2 (isomer 2, RT2 (min): 9.4, 15.5 mg, 10% yield)

[0263] LCMS(ESI):[M+H] + =504.40

[0264] 1 H NMR(400MHz,Methanol-d4)δ7.26(s,1H),7.05(s,2H),6.95(s,1H),6.25(s,1H),5.05–4.90(m,1H),4.28 –4.15(m,1H),4.04(t,J=6.4Hz,2H),3.92(s,6H),2.95(t,J=6.4Hz,2H),2.34–2.32(m,7H),2.22(s,6H).

[0265] Example 5, Compound 5-1 and Compound 5-2

[0266] Preparation steps:

[0267] 1.

[0268] 2-Chloro-9,10-dimethoxy-6h,7h-pyrimido[4,3-a]isoquinoline-4-1 (200 mg, 0.7 mmol, 1.0 equiv) and 2,4,6-trimethylaniline (369 mg, 2.7 mmol, 4.0 equiv) were dissolved in isopropanol (4 mL) and stirred at 90°C under nitrogen for 3 hours. The resulting mixture was concentrated under reduced pressure. Purification by column chromatography using DCM / MeOH (10 / 1) afforded (2E)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-3h,6H,7h-pyrimido[4,3-a]isoquinoline-4-1 (100 mg, 37.39% yield).

[0269] LCMS (ESI, m / z): [M+H] + =392.1

[0270] 2.

[0271] Tert-butyl 2-bromopropylcarbamate (304 mg, 1.3 mmol, 1.0 equiv) and Cs2CO3 (1.0 g, 3.9 mmol, 3.0 equiv) were added to a mixed solution of (2E)-9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)imino]-3h,6H,7H-pyrimido[4,3-a]isoquinoline-1 (500 mg, 1.3 mmol, 1.0 equiv) and dioxane (10 mL). Pd2(dba)3 (117 mg, 0.1 mmol, 0.1 equiv) and Xantphos (74 mg, 0.1 mmol, 0.1 equiv) were then added under nitrogen. The resulting mixture was stirred at 80°C under nitrogen for 6 hours. The resulting mixture was concentrated under reduced pressure. Purification by reverse-phase column chromatography using C18 silica gel as the mobile phase, water, MeCN (0.1% FA), gradient 30% to 70% over 15 min, and UV at 254 nm afforded tert-butyl N-[2-({9,10-dimethoxy-4-oxo-6h,7h-pyrimidin[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]carbamate (400 mg, 57.08% yield).

[0272] LCMS (ESI, m / z): [M+H] + =549.6

[0273] 3.

[0274] A 25 mL round-bottom flask was charged with N-[2-({9,10-dimethoxy-4-oxo-6h,7h-pyrimidin[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]carbamate (100 mg, 0.2 mmol, 1.0 equiv) and a solution of HCl in 1,4-dioxane (2 mL, 4 M). The reaction mixture was stirred at 25°C under nitrogen for 1 hour. The resulting mixture was concentrated under reduced pressure. This afforded 2-[(1-aminopropen-2-yl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6h,7h-pyrimidin[4,3-a]isoquinolin-4-1 (80 mg, crude product).

[0275] LCMS (ESI, m / z): [M+H] + =449.2

[0276] 4.

[0277] 2-[(1-Aminopropan-2-yl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6h,7h-pyrimido[4,3-a]isoquinoline-4-1 (100 mg, 0.2 mmol, 1.0 equiv) and isocyanotrimethylsilane (38 mg, 0.3 mmol, 1.5 equiv) were stirred in DCM (5 mL). TEA (60 mg, 0.6 mmol, 3.0 equiv) was added dropwise under nitrogen at 25°C. The reaction mixture was stirred at 25°C under nitrogen for 2 hours. The resulting mixture was concentrated under reduced pressure. The product was purified by column chromatography using C18 silica gel; the mobile phase was aqueous ACN (0.1% NH3.H2O) in a gradient of 25% to 75% over 20 minutes; UV was detected at 254 nm. 2-({9,10-dimethoxy-4-oxo-6h,7h-pyrimidin[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propylurea (80 mg, yield 73.0%) was obtained.

[0278] LCMS (ESI, m / z): [M+H] + =492.2

[0279] 5.

[0280] A 50 mL round-bottom flask was charged with 2-({9,10-dimethoxy-4-oxo-6h,7h-pyrimidin[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propylurea (100 mg, 0.2 mmol, 1.0 equiv) and an aqueous solution of hydrobromic acid (5 mL). The resulting mixture was stirred at 120°C under nitrogen for 3 hours. The mixture was extracted with ethyl acetate (2 x 50 mL), and the organic layer was washed with saturated brine (3 x 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. This afforded 2-({9,10-dihydroxy-4-oxo-6h,7h-pyrimidin[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propylurea (80 mg, crude product).

[0281] LCMS (ESI, m / z): [M+H] + =464.2

[0282] 6.

[0283] 2-({9,10-dihydroxy-4-oxo-6h,7h-pyrimidin[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propylurea (100 mg, 0.2 mmol, 1.0 equiv) and K2CO3 (149 mg, 1.0 mmol, 5.0 equiv) were added to DMF (1.5 mL), and ethyl 2-bromo-2,2-difluoroacetate (44 mg, 0.2 mmol, 1.0 equiv) was added dropwise at room temperature. The reaction mixture was stirred at 55°C under nitrogen for 1.5 hours. The resulting mixture was concentrated under reduced pressure. Purification was performed by reverse-phase column chromatography using the following conditions: C18 silica gel column; mobile phase: water, ACN (0.1% FA), gradient 20% to 70% over 20 min; UV illumination: 254 nm. 2-{[9-(difluoromethoxy)-10-hydroxy-4-oxo-6h,7h-pyrimidin[4,3-a]isoquinolin-2-yl](2,4,6-trimethylphenyl)amino}propylurea (10 mg, yield 9.03%) was obtained.

[0284] LCMS (ESI, m / z): [M+H] + =514.2

[0285] 7.

[0286] 2-{[9-(Difluoromethoxy)-10-hydroxy-4-oxo-6h,7h-pyrimidin[4,3-a]isoquinolin-2-yl](2,4,6-trimethylphenyl)amino}propylurea (10 mg, 0.02 mmol, 1.0 equiv) and K2CO3 (4 mg, 0.03 mmol, 1.5 equiv) were added dropwise in DMF (0.5 mL) at room temperature to iodomethane (4 mg, 0.03 mmol, 1.5 equiv). The resulting mixture was stirred at 60°C under nitrogen for 1.5 hours. The resulting mixture was concentrated under reduced pressure. The racemic product was purified by high-performance liquid chromatography (HPLC) using an XBridge BEH C18 5μm column (30x150mm); mobile phase A: water (10mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60mL / min; gradient: 40% B to 50% B over 8 minutes; wavelength: 254nm / 220nm; RT1 (min): 7.3. The racemic product was then purified by chiral high-performance liquid chromatography (HPLC) using a chiral ART Cellulose-SB column (2x25cm, 5μm); mobile phase A: MeOH:DCM = 1:1 (HPLC); mobile phase B: MtBE (0.1% DEA) (HPLC); flow rate: 20ml / min; gradient: equidistant 50; wavelength: 220nm; RT1 (min): 4.4; RT2 (min): 10.7; sample solvent: MeOH (HPLC); injection volume: 1ml).

[0287] Compound 5-1 (isomer 1, RT1 (min): 4.4, 1.54 mg, yield 14.87%).

[0288] LCMS (ESI, m / z): [M+H] + =529.40.

[0289] 1 H NMR(400MHz, Methanol-d4)δ7.20–7.05(m,3H),6.95–6.82(m,1H),6.80(s,1H),5.49(s,1H),4.49(s,1H),4.16–4.1 0(m,2H),3.72(s,3H),2.97(t,J=6.4Hz,2H),2.37(s,3H),2.23(s,3H),2.10(s,3H),1.35–1.28(m,2H),1.06(s,3H).

[0290] Compound 5-2 (isomer 2, RT2 (min): 10.7, 2.19 mg, yield 20.9%)

[0291] LCMS (ESI, m / z): [M+H] + =529.45

[0292] 1 H NMR(400MHz, Methanol-d4)δ7.20–7.05(m,3H),6.95–6.82(m,1H),6.80(s,1H),5.49(s,1H),4.49(s,1H),4.16–4.1 0(m,2H),3.72(s,3H),2.96(t,J=6.4Hz,2H),2.37(s,3H),2.22(s,3H),2.11(s,3H),1.35–1.28(m,2H),1.06(s,3H).

[0293] Example 6, Compound 6

[0294] Preparation steps:

[0295] 1.

[0296] Dissolve trimethylaniline (1.0 g, 7.4 mmol, 1.0 equiv), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (3.4 g, 8.8 mmol, 1.2 equiv), and N,N-diisopropylethylamine (3.9 mL, 22.2 mmol, 3.0 equiv) in 20 mL of dichloromethane and stir at room temperature for 2 hours. Concentrate under reduced pressure, dissolve in 10 mL of water, and extract with ethyl acetate (3×10 mL). The organic layer is 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 normal phase column chromatography under the following conditions (mobile phase A: petroleum ether, mobile phase B: ethyl acetate; flow rate: 60 mL / min; gradient: 10% B to 50% B in 20 min; wavelength: 254 nm;) to give 2-(1,3-dioxoisoindolin-2-yl)-N-methylsulfonylacetamide (600 mg, yield 25.17%).

[0297] LCMS(ESI):[M+H] + =323.14

[0298] 2.

[0299] 2-(1,3-Dioxoisoindol-2-yl)-N-(2,4,6-trimethylphenyl)acetamide (600 mg, 1.8 mmol, 1.0 equivalent), 2-chloro-9,10-dimethoxy-6,7-dihydro-4H-pyrimidin[6,1-A]isoquinolin-4-one (653 mg, 2.2 mmol, 1.2 equivalent), O,O-(1-acetoxy-2,2,2-trimethylphenyl)acetamide (600 mg, 1.8 mmol, 1.0 equivalent), 10 mL of 1,2-diphenyl chloroethylphosphonate (118.40 mg, 0.186 mmol, 0.1 equiv), {[4-(N,N-(dimethylamino)phenyl]di-tert-butylphosphino}(2'-amino-1,1'-biphenyl-2-yl)palladium(II)methanesulfonate (49.39 mg, 0.186 mmol, 0.1 equiv) and cesium carbonate (1.5 g, 4.6 mmol, 2.5 equiv) were added. The product was dissolved in 1,4-dioxane, stirred at 110°C overnight under nitrogen atmosphere. The product was concentrated under reduced pressure, dissolved in 10 mL of water, and extracted with ethyl acetate (3 × 20 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 (column: C18; mobile phase A: water (10 mmol / L ammonium bicarbonate); mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 30% B to 50% B in 20 min; wavelength: 254 nm) to afford 2-amino-N-(9,10-dimethoxy-4-oxo-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-2-yl)-N-methylsulfonylacetamide (50 mg, 5.04% yield).

[0300] LCMS(ESI):[M+H] + =449.16

[0301] 3.

[0302] 2-Amino-N-(9,10-dimethoxy-4-oxo-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-2-yl)-N-methylsulfonylacetamide (20 mg, 0.05 mmol, 1.0 equiv), triethylamine (14 mg, 0.15 mmol, 3.0 equiv) and N,N'-carbonyldiimidazole (10.9 mg, 0.07 mmol, 1.5 equiv) were dissolved in 2 mL of dichloromethane under nitrogen protection. 2 mL of amine solution in 1.4-dioxane was added dropwise at 0°C and stirred at room temperature for 2 hours. The crude product was purified by reverse phase column chromatography under the following conditions (column: SunFire C18 5m, 30mmX150mm; mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile; flow rate: 60mL / minmL / min; gradient: 45% B to 59% B in 7 minutes; wavelength: 254nm / 220nm; time: 5.92) to give N-(9,10-dimethoxy-4-oxo-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-2-yl)-N-methylsulfonyl-2-ureidoacetamide (2.9 mg, 13.10% yield).

[0303] LCMS (ESI, m / z): [M+H] + =475.20

[0304] 1 H NMR(400MHz,DMSO-d6)δ8.45(s,1H),7.71(s,1H),7.18(s,1H),7.08-7.01(m,3H),4.77(s,2H),4.0 8(t,J=6.6Hz,2H),3.86(s,3H),3.79(s,3H),2.97(t,J=6.6Hz,2H),2.30(s,3H),2.12–2.10(s,6H).

[0305] Example 7, Compound 7-1, Compound 7-2 and Compound 7-3

[0306] Preparation steps:

[0307] 1.

[0308] To methyl 2-bromocyclopropane-1-carboxylate (200 mg, 1.1 mmol, 1.0 equiv) and (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 equiv) in 1,4-dioxane (80 mL) were added cesium carbonate (1.1 g, 3.4 mmol, 3.0 equiv), Xantphos (106 mg, 0.2 mmol, 0.2 equiv), and Pd2(dba)3 (64 mg, 0.1 mmol, 0.1 equiv). After stirring at 100°C under nitrogen for 12 h, the resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using methanol / dichloromethane (1 / 10) as the eluent to give the crude product. The crude product was purified by chromatography on a YMC Triart C18ExRs 5m, 30mm*150mm; mobile phase A: water (10mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 39%B to 62%B, 10 minutes; wavelength: 254nm / 220nm; RT1(min): 8.33) to give methyl 2-[(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-6H,7H-pyrimido[4,3-a]isoquinolin-3-yl]cyclopropane-1-carboxylate (100 mg, yield 18.3%).

[0309] LCMS(ESI):[M+H] + =490.2

[0310] 2.

[0311] To methyl 2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)cyclopropane-1-carboxylate (160 mg, 0.3 mmol, 1.0 equiv) in methanol (20 mL) was added an aqueous solution of sodium hydroxide (65 mg, 1.6 mmol, 5.0 equiv) in water (5 mL) at room temperature. The mixture was stirred at 50°C for 18 h. After completion of the reaction, the reaction solution was concentrated under reduced pressure. The resulting mixture was extracted with ethyl acetate (3 x 50 mL). The aqueous layer was acidified to pH = 4 with 6M aqueous hydrochloric acid. The resulting mixture was extracted with dichloromethane (3 x 100 mL). The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give 2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)cyclopropane-1-carboxylic acid (170 mg, crude product).

[0312] LCMS(ESI):[M+H] + =476.2

[0313] 3.

[0314] To 2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)cyclopropane-1-carboxylic acid (170 mg, 0.4 mmol, 1.0 equiv) in dichloromethane (10 mL) was added oxalyl chloride (0.1 mL, 1.1 mmol, 3.0 equiv) dropwise. The resulting mixture was stirred at 0°C for 1 h under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The crude product was used directly without further purification. The crude product was dissolved in dichloromethane (1 mL) and added to a solution of ammonia in tetrahydrofuran (60 mL, 1.3 M) at 0°C. The resulting mixture was stirred at 0°C for 1 h. The resulting mixture was extracted with ethyl acetate (3 x 60 mL). The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give 2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)cyclopropane-1-carboxamide (180 mg, crude product).

[0315] LCMS(ESI):[M+H] + =475.2

[0316] 4.

[0317] A mixture of 2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)cyclopropane-1-carboxamide (180 mg, 0.4 mmol, 1.0 equiv) in tetrahydrofuran (20 mL) was cooled to 0°C, and sodium borohydride (143 mg, 3.8 mmol, 10.0 equiv) was added portionwise. Iodine pellets (963 mg, 3.8 mmol, 10.0 equiv) dissolved in tetrahydrofuran (3 mL) were added to the resulting mixture. The resulting mixture was stirred at 70°C for 18 h. The reaction was quenched by the addition of water (30 mL) at room temperature. The resulting mixture was extracted with ethyl acetate (2 x 50 mL). The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by Prep-TLC (dichloromethane / methanol = 10 / 1) to give 2-{[2-(aminomethyl)cyclopropyl](2,4,6-trimethylphenyl)amino}-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (45 mg, 25.8% yield).

[0318] LCMS(ESI):[M+H] + =461.2

[0319] 5.

[0320] A mixture of 2-{[2-(aminomethyl)cyclopropyl](2,4,6-trimethylphenyl)amino}-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (45 mg, 0.1 mmol, 1.0 equiv), triethylamine (30 mg, 0.3 mmol, 3.0 equiv) and trimethylsilyl isocyanate (23 mg, 0.2 mmol, 2.0 equiv) in DCM (5 mL) was stirred at room temperature for 3 h. The resulting mixture was concentrated under reduced pressure. The crude product (45 mg) was purified by Prep-HPLC under the following conditions (chromatographic column: XBridge BEH C18 OBD Prep Column 130, 5 m, 19 mm * 250 mm; mobile phase A: water (10 mmol / L ammonium bicarbonate); flow rate: 25 mL / min; gradient: 45% B to 67% B 10 minutes; wavelength: 254 nm / 220 nm; RT1 (min): 7.93) to give [2- ({9,10-dimethoxy-4-oxo-6H,7H-pyrimido [4,3-a] isoquinolin-2-yl} (2,4,6-trimethylphenyl) amino) cyclopropyl] methyl urea (20 mg, yield 40.65%).

[0321] LCMS(ESI):[M+H] +=504.3

[0322] 6.

[0323] Compound 7-1, isomer 1 (chromatographic column: CHIRAL ART Amylose-SA, 2*25 cm, 5 μm; mobile phase A: methanol: dichloromethane = 1:1, mobile phase B: methyl tert-butyl ether (0.1% diethylamine) flow rate: 20 mL / min; gradient: isocratic; wavelength: 254 nm; RT1 (min): 6.7; sample solvent: methanol (0.1% diethylamine); injection volume: 1 mL; number of runs: 3) obtained the product (3.8 mg, yield 12.43%).

[0324] LCMS(ESI):[M+H] + =504.30

[0325] 1 H NMR(400MHz, Methanol-d4)δ7.10(s,1H),7.07(s,1H),6.95(s,1H),6.72(s,1H),5.54(s,1H),4.25–4.06(m,2H),3.90( s,4H),3.68(s,3H),2.99(t,J=6.6Hz,3H),2.48–2.40(m,1H),2.36(s,3H),2.23(s,3H),2.03(s,3H),1.43–1.31(m,2H).

[0326] Compound 7-2, isomer 2 (chromatographic column: CHIRAL ART Amylose-SA, 2*25 cm, 5 μm; mobile phase A: methanol: dichloromethane = 1:1, mobile phase B: methyl tert-butyl ether (0.1% diethylamine) flow rate: 20 mL / min; gradient: isocratic; wavelength: 254 nm; RT2 (min): 10.9; sample solvent: methanol (0.1% diethylamine); injection volume: 1 mL; number of runs: 3) obtained the product (3.5 mg, yield 11.29%).

[0327] LCMS(ESI):[M+H] + =504.35

[0328] 1H NMR(400MHz, Methanol-d4)δ7.10(s,1H),7.07(s,1H),6.95(s,1H),6.72(s,1H),5.54(s,1H),4.25–4.06(m,2H),3.90( s,4H),3.68(s,3H),2.99(t,J=6.6Hz,3H),2.48–2.40(m,1H),2.36(s,3H),2.23(s,3H),2.03(s,3H),1.43–1.31(m,2H).

[0329] Compound 7-3, isomer 3 (chromatographic column: XBridge BEH C18 OBD Prep Column 130, 5m, 19mm*250mm; mobile phase A: water (10mmol / L ammonium bicarbonate); mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 39% B to 61% B 9 minutes; wavelength: 254nm / 220nm; RT1(min): 5.45), to obtain the product (20 mg, yield 40.6%).

[0330] LCMS(ESI):[M+H] + =504.35

[0331] 1 H NMR(400MHz, Methanol-d4)δ7.10(s,1H),7.07(s,1H),7.02–6.89(m,2H),6.70(s,1H),5.57(s,1H),4.61(s,6H),4.27–4.04(m,2H),4 .05–3.91(m,2H),3.90(s,3H),3.85–3.72(m,1H),3.68(s,3H),3.05–2.95(m,2H),2.92–2.87(m,1H),2.36(s,3H),2.31–2.14(m,8H).

[0332] Example 8, Compound 8

[0333] Preparation steps

[0334] 1.

[0335] After stirring tert-butyl N-(2-hydroxypropyl)carbamate (15.0 g, 85.6 mmol, 1.0 equiv) and TEA (26.0 g, 256.8 mmol, 1.5 equiv) in DCM (80 mL), DMAP (1.1 g, 8.6 mmol, 0.1 equiv) and TsCl (21.2 g, 111.3 mmol, 1.3 equiv) were added portionwise at 0°C. The resulting mixture was stirred at room temperature for 2 h. The resulting mixture was extracted with dichloromethane (3 x 200 mL). The organic layer was washed with brine and dried over anhydrous sodium sulfate. The filtered filtrate was concentrated under reduced pressure and purified by silica gel column chromatography using PE / EA (5 / 1) as the eluent to afford N-[2-[(4-methylbenzenesulfonyl)oxy]propyl]carbamate (20.3 g, 71.9% yield).

[0336] LC-MS(ESI):[M+H] + =330.1

[0337] 2.

[0338] To a solution of 9,10-dimethoxy-2-[(2,4,6-trimethylphenyl)amino]-6h,7h-pyrimido[4,3-a]isoquinolin-4-one (1.6 g, 4.1 mmol, 1.0 equiv) in 1,4-dioxane (20 mL) were added tert-butyl N-[2-[(4-methylphenylsulfonyl)oxy]propyl]carbamate (2.0 g, 6.1 mmol, 1.5 equiv), cesium carbonate (2.7 g, 8.2 mmol, 2.0 equiv), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (0.5 g, 0.8 mmol, 0.2 equiv), and tris(dibenzylideneacetone)dipalladium (0.4 g, 0.4 mmol, 0.1 equiv), and the mixture was purged with nitrogen and heated to 100°C. The mixture was stirred at 100°C under nitrogen for 5 h. The reaction system was extracted with ethyl acetate (3 x 200 mL) and water (150 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporated. The resulting residue was purified by reverse-phase column chromatography using the following conditions: XBridge BEH C18, 5 μm, 30 x 150 mm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 55% B to 65% B in 7 min; wavelength: 254 nm / 220 nm; RT (min): 5.67 / 6.05. This afforded tert-butyl N-[2-({9,10-dimethoxy-4-oxo-6h,7h-pyrimidin[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]carbamate (35.33 mg, 1.55% yield).

[0339] LC-MS(ESI):[M+H] + =549.35

[0340] 1 H NMR (400MHz, Methanol-d4) δ7.11 (d, J = 10.2Hz, 2H), 6.93 (s, 1H), 6.64 (s, 1H) ),5.41(s,1H),4.57–4.39(m,1H),4.18–4.02(m,2H),3.89(s,3H),3.66(s,3H ),3.55–3.48(m,1H),3.40(t,J=8.2Hz,1H),2.97(t,J=6.5Hz,2H),2.37(s,3H ), 2.22 (s, 3H), 2.11 (s, 3H), 1.46 (s, 9H), 1.31 (s, 1H), 1.08 (d, J = 6.9Hz, 3H).

[0341] Example 9, Compound 9-1 and Compound 9-2

[0342] Preparation steps:

[0343] 1.

[0344] 2-((1-aminopropan-2-yl)(methylsulfonyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (150 mg, 0.33 mmol, 1.0 equiv), 3H-1,2,3-triazole-4-carboxylic acid (56 mg, 0.5 mmol, 1.5 equiv), 1-(3-dimethylaminopropyl)-3-ethylcarbodiamine (96 mg, 0.5 mmol, 1.5 equiv), and 1-hydroxybenzotriazole (67 mg, 0.5 mmol, 1.5 equiv) were dissolved in 2 ml of DMF, protected by nitrogen, stirred at room temperature for 1 hour, and concentrated under reduced pressure. The crude product was purified by reverse phase column chromatography under the following conditions (column: Xselect CSH C185 m, 30 mm x 150 mm; mobile phase A: water (0.1% FA), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 27% B to 55% Bin over 9 min; wavelength: 254 nm / 220 nm; RT1 (min): 6.77 / 8.43) to give N-(2-((9,10-dimethoxy-4-oxo-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-2-yl)(methanesulfonamido)propyl)-1H-1,2,3-triazole-5-carboxamide (30 mg, 16.5% yield).

[0345] LCMS(ESI):[M+H] + =544.25

[0346] 2.

[0347] The racemate (30 mg) was purified by Chiral-SFC under the following conditions (chromatographic column: CHIRALPAK IH 3*25 cm, 5 μm; mobile phase A: CO2, mobile phase B: methanol (0.1% 2M NH3-MEOH); flow rate: 85 mL / min; gradient: isocratic 35% B; column temperature (°C): 35; pressure (bar): 100; wavelength: 220 nm; RT1 (min): 2.92; RT2 (min): 4.42; sample solvent: DCM (0.1% 2M NH3-MeOH)--HPLC; injection volume: 1 mL) to obtain the product.

[0348] Compound 9-1 (isomer 1, RT1 (min): 2.92, 9.2 mg, yield 28.58%)

[0349] LCMS(ESI):[M+H] + =544.25

[0350] 1 H NMR(400MHz, Methanol-d4)δ8.25(s,1H),7.15–7.11(m,2H),6.99–6.92(m,1H),6.65(s,1H),5.44(s,1H),4.74(s,1H),4.23–4.04(m,2H),3.95-3.8 8(m,4H),3.85–3.75(m,1H),3.67(s,3H),2.99(t,J=6.4Hz,2H),2.38(s,3 H),2.27(s,3H),2.13(s,3H),1.31(d,J=4.0Hz,1H),1.11(d,J=6.8Hz,3H).

[0351] Compound 9-2 (isomer 2, RT2(min):4.42, 8.8 mg, yield 24.5%)

[0352] LCMS(ESI):[M+H] + =544.25

[0353] 1H NMR(400MHz, Methanol-d4)δ8.25(s,1H),7.15–7.11(m,2H),6.99–6.92(m,1H),6.65(s,1H),5.44(s,1H),4.74(s,1H),4.23–4.04(m,2H),3.95-3.8 8(m,4H),3.85–3.75(m,1H),3.67(s,3H),2.99(t,J=6.4Hz,2H),2.38(s,3 H), 2.27 (s, 3H), 2.13 (s, 3H), 1.31 (d, J = 4.0Hz, 1H), 1.11 (d, J = 6.8Hz, 3H).

[0354] Example 10, Compound 10-1 and Compound 10-2

[0355] Preparation steps:

[0356] 1.

[0357] 2-((1-aminopropyl-2-yl)(methylsulfonyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (150 mg, 0.33 mmol, 1.0 equivalent), imidazole-2-carboxylic acid (56 mg, 0.5 mmol, 1.5 equivalent), 1-(3-dimethylaminopropyl)-3-ethylcarbodiamine (96 mg, 0.5 mmol, 1.5 equivalent), and 1-hydroxybenzotriazole (67 mg, 0.5 mmol, 1.5 equivalent) were dissolved in 2 ml of DMF, protected by nitrogen, stirred at room temperature for 1 hour, and concentrated under reduced pressure. The crude product was purified by reverse phase column chromatography under the following conditions (column: Xselect CSH C185 m, 30 mm x 150 mm; mobile phase A: water (0.1% FA), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 27% B to 55% B in 9 minutes; wavelength: 254 nm / 220 nm; RT1 (min): 6.77 / 8.43) to give N-(2-((9,10-dimethoxy-4-oxo-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-2-yl)(methanesulfonamido)propyl)-1H-imidazole-2-carboxamide (70 mg, 44.0% yield).

[0358] LCMS(ESI):[M+H] + =544.25

[0359] 2.

[0360] The racemate (70 mg) was purified by Chiral-SFC under the following conditions (chromatographic column: CHIRAL ART Cellulose-SB, 3*25 cm, 5 μm; mobile phase A: CO2, mobile phase B: MeOH (0.1% 2M NH3-MeOH); flow rate: 100 mL / min; gradient: isocratic 25% B; column temperature (°C): 35; pressure (bar): 100; wavelength: 220 nm; RT1 (min): 9.4; RT2 (min): 10.72; sample solvent: MeOH (0.1% DEA)--HPLC; injection volume: 1 mL) to obtain the product.

[0361] Compound 10-1 (isomer 1, RT1 (min): 9.4, 24.9 mg, yield 34.5%).

[0362] LCMS(ESI):[M+H] + =543.25

[0363] 1 H NMR(400MHz, Methanol-d4)δ7.21(s,3H),7.16–7.08(m,2H),6.94(s,1H),6.65(s,1H),5.43(s,1H),4.70(s,1H),4.21–4.02(m,2H),3.97–3.91(m ,1H),3.89(s,4H),3.82–3.84(m,1H),3.67(s,3H),3.05–2.94(m,2H),2. 38(s,3H),2.27(s,3H),2.14(s,3H),1.31(s,1H),1.15(d,J=6.8Hz,3H).

[0364] Compound 10-2 (isomer 2, RT2 (min): 10.72, 24.4 mg, yield 34.5%)

[0365] LCMS(ESI):[M+H] + =543.25

[0366] 1H NMR(400MHz, Methanol-d4)δ7.21(s,3H),7.16–7.08(m,2H),6.94(s,1H),6.65(s,1H),5.43(s,1H),4.70(s,1H),4.21–4.02(m,2H),3.97–3.91(m ,1H),3.89(s,4H),3.84–3.76(m,1H),3.67(s,3H),3.05–2.94(m,2H),2. 38(s,3H),2.27(s,3H),2.14(s,3H),1.31(s,1H),1.15(d,J=6.8Hz,3H).

[0367] Compounds 98-125 (derivative structures and deuterated structures of compound 10) were synthesized respectively. The structures of the compounds are shown in Table 1, and the MS data are shown in Table 3.

[0368] Table 3

[0369] Example 11, Compound 11-1 and Compound 11-2

[0370] Preparation steps:

[0371] 1.

[0372] 2H-Pyrazole-3-carboxylic acid (25 mg, 0.2 mmol, 1.0 equiv) was dissolved in SOCl2 (1 mL) and stirred at room temperature under nitrogen for 2 h. The resulting mixture was concentrated in vacuo to dryness. This mixture was dissolved in DCM (1 mL) and added dropwise to a solution of 2-[(1-aminopropan-2-yl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (100 mg, 0.2 mmol, 1.0 equiv) and N,N-diisopropylethylamine (86 mg, 0.7 mmol, 3.0 equiv) in DCM (5 mL) at 0°C under nitrogen. The reaction mixture was stirred at room temperature under nitrogen for 2 h. The reaction mixture was quenched with water (5 mL) at room temperature, extracted with ethyl acetate (3 x 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Reverse phase purification was performed using the following conditions: C18 silica gel column; mobile phase: ACN (0.1% FA) in water, gradient 10% to 50% over 10 min; UV detector at 254 nm. The product was purified to yield N-[2-({9,10-dimethoxy-4-oxo-6h,7h-pyrimidin[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]-2H-pyrazole-3-carboxamide (40 mg, 32.0% yield).

[0373] 2.

[0374] The crude product (40 mg) was purified by column chromatography: column: CHIRALPAK IH 3*25 cm, 5 μm; mobile phase A: CO2, mobile phase B: MEOH (0.1% DEA); flow rate: 80 mL / min; gradient: isocratic 40% B; column temperature (℃): 10℃; column head temperature (℃): 10℃: 80 mL / min; gradient: 40% B isocratic; column temperature (℃): 35; pressure (bar): 100; wavelength: 220 nm; RT1 (min): 2.73; RT2 (min): 4.17; sample solvent: injection volume: 4.8 mL to obtain the product.

[0375] Compound 11-1 (isomer 1, RT1 (min): 2.73, 12.1 mg, yield 9.6%)

[0376] LCMS(ESI):[M+H] + =543.55

[0377] 1 H NMR (400MHz, Methanol-d4) δ7.71(d,J=2.4Hz,1H),7.13(d,J=13.6Hz,2H),6.93(s,1H),6.78(d,J=2.4Hz,1H),6.65(s,1H),5.43(s,1H),4.70( s,1H),4.16–4.10(m,2H),3.91–3.78(m,5H),3.67(s,3H),2.98(t,J=6. 4Hz,2H),2.38(s,3H),2.27(s,3H),2.13(s,3H),1.11(d,J=6.8Hz,3H).

[0378] Compound 11-2 (isomer 2, RT2(min):4.17, 10.9 mg, yield 26.6%)

[0379] LCMS(ESI):[M+H] + =543.35

[0380] 1H NMR (400MHz, Methanol-d4) δ7.71(d,J=2.4Hz,1H),7.18–7.12(m,2H),6.93(s,1H),6.78(d,J=2.4Hz,1H),6.65(s,1H),5.43(s,1H),4.75–4.55 (m,1H),4.16–4.10(m,2H),3.91–3.78(m,5H),3.67(s,3H),2.98(t,J=6 .4Hz,2H),2.38(s,3H),2.27(s,3H),2.13(s,3H),1.11(d,J=6.8Hz,3H).

[0381] Example 12, Compound 12-1 and Compound 12-2

[0382] Preparation steps:

[0383] 1.

[0384] In an autoclave, palladium on carbon (10%, 2.90 g) was added to ethyl 1-benzyl-5-methyl-1,2,3-triazole-4-carboxylate (850 mg, 3.5 mmol, 1.0 equiv) in 150 mL of methanol. The mixture was stirred at room temperature under 20 bar of hydrogen pressure for 18 hours, filtered through celite, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using PE / EA (1 / 1) as the eluent to give ethyl 5-methyl-1-hydrogen-1,2,3-triazole-4-carboxylate (260 mg, 48.4% yield).

[0385] LCMS (ESI, m / z): [M+H] + =156.1

[0386] 2.

[0387] A mixture of ethyl 5-methyl-1H-1,2,3-triazole-4-carboxylate (260 mg, 1.7 mmol, 1.0 equiv) and sodium hydroxide (201 mg, 5.1 mmol, 3.0 equiv) in methanol (10 mL) and water (10 mL) was stirred at 60°C for 18 hours. The resulting mixture was concentrated under reduced pressure. The mixture was acidified to pH 5 with 2 M aqueous hydrochloric acid. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse phase chromatography using the following conditions: column, C18 silica gel; mobile phase, acetonitrile, water, 0%-10% gradient over 10 minutes; detector, UV 254 nm, to afford 5-methyl-3H-1,2,3-triazole-4-carboxylic acid (200 mg, 93.9% yield).

[0388] LCMS(ESI):[M+H] + =128.0

[0389] 3.

[0390] 2-[(1-aminopropan-2-yl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (100 mg, 0.2 mmol, 1.0 equiv) and 5-methyl-3H-1,2,3-triazole-4-carboxylic acid (28.3 mg, 0.2 mmol, 1.0 equiv) were dissolved in DMF (3 mL) at room temperature. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (64 mg, 0.3 mmol, 1.5 equiv) and 1-hydroxybenzotriazole (45 mg, 0.3 mmol, 1.5 equiv) were added. The resulting mixture was stirred at room temperature for 2 h. The resulting mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by reverse phase purification under the following conditions (chromatographic column: XBridge BEH Shield RP18 5m, 30mm*150mm; mobile phase A: water (10mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60ml / min; gradient: from 36% B to 54% B in 10 minutes; wavelength: 254nm / 220nm nm; RT1(min): 6.18) to give N-[2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]-5-methyl-3H-1,2,3-triazole-4-carboxamide (30mg, yield 24.1%).

[0391] LCMS(ESI):[M+H] + =558.3

[0392] 4.

[0393] The crude product (30 mg) was purified by Chiral-HPLC under the following conditions: CHIRALPAK IH 3*25 cm, 5 μm; mobile phase A: CO2, mobile phase B: methanol (0.1% diethylamine); flow rate: 85 mL / min; gradient: isocratic 35% B; column temperature (℃): 25℃; back pressure (bar): 100; wavelength: 254 nm; RT1 (min): 2.85; RT2 (min): 4.5 sample solvent: methanol: dichloromethane = 1:1--HPLC; injection volume: 1 mL) to obtain the product.

[0394] Compound 12-1 (isomer 1, RT1 (min): 2.85, 1.6 mg, yield 5.14%)

[0395] LCMS (ESI, m / z): [M+H] + =558.35

[0396] 1 H NMR(400MHz, Methanol-d4)δ7.17–7.10(m,2H),6.93(s,1H),6.65(s,1H),5.43(s,1H),4.78–4.69(m,1H),4.20–4.04(m,2H),3.97 –3.77(m,5H),3.67(s,3H),2.98(t,J=6.5Hz,2H),2.59–2.52(m,3H),2.38(s,3H),2.27(s,3H),2.13(s,3H),1.11(d,J=6.9Hz,3H).

[0397] Compound 12-2 (isomer 2, RT2(min):4.5, 3.2 mg, yield 9.86%)

[0398] LCMS (ESI, m / z): [M+H] + =558.46

[0399] 1 H NMR(400MHz, Methanol-d4)δ7.17–7.10(m,2H),6.93(s,1H),6.65(s,1H),5.43(s,1H),4.78–4.69(m,1H),4.20–4.04(m,2H),3.97 –3.77(m,5H),3.67(s,3H),2.98(t,J=6.5Hz,2H),2.59–2.52(m,3H),2.38(s,3H),2.27(s,3H),2.13(s,3H),1.11(d,J=6.9Hz,3H).

[0400] Example 13, Compound 13-1 and Compound 13-2

[0401] Preparation steps:

[0402] 1.

[0403] 2-[(1-Aminopropan-2-yl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (100 mg, 0.2 mmol, 1.0 equiv), 3H-imidazole-4-carboxylic acid (25 mg, 0.2 mmol, 1.0 equiv), HOBT (45 mg, 0.3 mmol, 1.5 equiv), and EDCI (64 mg, 0.3 mmol, 1.5 equiv) were dissolved in DMF (5 mL) and stirred under nitrogen at room temperature for 2 h. The reaction was quenched with water (10 mL) at room temperature. The aqueous layer was extracted with ethyl acetate (3 x 20 mL), and the organic layer was washed with saturated brine (3 x 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Reverse phase purification was performed under the following conditions: column, C18 silica gel; mobile phase, acetonitrile-water (0.1% FA), gradient from 10% to 50% over 10 minutes; detection, UV 254 nm. The product was purified to yield N-[2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]-3H-imidazole-4-carboxamide (20 mg, 15.7% yield).

[0404] 2.

[0405] The crude product (20 mg) was purified by Chiral-HPLC: CHIRALPAK IH 3*25 cm, 5 μm; mobile phase A: CO2, mobile phase B: MeOH:DCM=2:1 (0.1% 2M NH3-MeOH); flow rate: 100 mL / min; gradient: isocratic 18% B; column temperature (℃): 20℃; column head temperature (℃): 20℃: 100 mL / min; gradient: isocratic 18% B; column temperature (℃): 35; back pressure (bar): 100; wavelength: 220 nm; RT1 (min): 14.2; RT2 (min): 21.37; Sample Solvent: DCM--HPLC; injection volume: 2 mL to obtain the product.

[0406] Compound 13-1 (isomer 1, RT1 (min): 14.2, 2.04 mg, yield 1.6%)

[0407] LCMS(ESI):[M+H] + =543.20

[0408] 1H NMR(400MHz, Methanol-d4)δ7.75–7.70(m,2H),7.15–7.11(m,2H),6.98–6.92(m,1H),6.65(s,1H),5.43(s,1H),4.75–4.55(m,1H),4.15–4.09( m,2H),3.90(s,3H),3.85–3.80(m,2H),3.67(s,3H),2.98(t,J=6.4Hz,2 H),2.38(s,3H),2.33–2.27(m,3H),2.15(s,3H),1.16(d,J=6.9Hz,3H).

[0409] Compound 13-2 (isomer 2, RT2 (min): 21.37, 2.72 mg, yield 13.0%)

[0410] LCMS(ESI):[M+H] + =543.20

[0411] 1 H NMR(400MHz, Methanol-d4)δ7.75–7.70(m,2H),7.15–7.11(m,2H),6.98–6.92(m,1H),6.65(s,1H),5.43(s,1H),4.75–4.55(m,1H),4.15–4.09( m,2H),3.90(s,3H),3.85–3.80(m,2H),3.67(s,3H),2.98(t,J=6.4Hz,2 H), 2.38 (s, 3H), 2.33–2.27 (m, 3H), 2.15 (s, 3H), 1.16 (d, J = 6.9Hz, 3H).

[0412] Example 14, Compound 14

[0413] Preparation steps:

[0414] 1.

[0415] In a single-necked flask, 2-[(1-aminopropan-2-yl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (250 mg, 0.6 mmol, 1.0 equiv), DCM (5 mL), DIEA (216 mg, 1.7 mmol, 3.0 equiv), cyanoacetic acid (57 mg, 0.7 mmol, 1.2 equiv) and HATU (318 mg, 0.8 mmol, 1.5 equiv) were added in sequence and reacted at room temperature overnight. The solvent was dried and the reaction was carried out by reverse phase chromatography (column type, C18 silica gel; mobile phase, acetonitrile water (10 mmol / L NH4HCO3), gradient 10% to 50% for 30 minutes; detection wavelength 254 nm) to give 2-cyano-N-[2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]acetamide (200 mg, 69.6% yield).

[0416] LCMS (ESI, m / z): [M+H] + =516.2

[0417] 2.

[0418] In a single-necked flask, 2-cyano-N-[2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]acetamide (200 mg, 0.4 mmol, 1.0 equiv), 1.4-dioxane (30 mL), sodium acetate (95 mg, 1.2 mmol, 3.0 equiv) and acetic anhydride (48 mg, 0.5 mmol, 1.2 equiv) were added in sequence and reacted at 100°C overnight. The solvent was dried and reverse phase chromatography (column type: XBridge BEH Shield RP18 5m, 30mm*150mm; mobile phase A: water (10mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 21% B to 44% B 9 minutes; wavelength: 254nm / 220nm; RT1(min): 6.82) was performed to obtain (2Z)-2-cyano-N-[2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]-3-hydroxy-2-enamide (13.27 mg, 6.1% yield).

[0419] LCMS (ESI, m / z): [M+H] + =558.25

[0420] 1 H NMR (400MHz, Methanol-d4) δ7.15–7.05(m,2H),7.07–6.88(m,2H),6.63(s,1H),5.40(s,1H),4.68–4.59(m,1H),4.21–4.12(m,2H),4.11–4. 05(m,1H),3.89(s,3H),3.66(s,3H),2.98(t,J=6.5Hz,2H),2.37(s,3H ), 2.33 (s, 1H), 2.25–2.23 (m, 6H), 2.14 (s, 3H), 1.09 (d, J = 6.7Hz, 3H).

[0421] Example 15, Compound 15-1 and Compound 15-2

[0422] Preparation steps:

[0423] 1.

[0424] Under air at 0°C, tert-butyl N-(2-hydroxybutyl)carbamate (500 mg, 2.6 mmol, 1.0 equiv) and TEA (1.1 mL, 7.9 mmol, 3.0 equiv) were dissolved in DCM (5 mL). TsCl (1.2 g, 3.9 mmol, 1.5 equiv) and DMAP (32 mg, 0.3 mmol, 0.1 equiv) were added, respectively. The resulting mixture was stirred under nitrogen at room temperature for 1.5 hours. The reaction was quenched with water at room temperature. The resulting mixture was extracted with dichloromethane (2 x 50 mL). The combined organic layers were washed with saturated brine (2 x 100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse phase purification under the following conditions: chromatographic column, C18 silica gel; mobile phase, ACN water (10 mmol / L NH4HCO3), gradient from 10% to 50% in 10 minutes; detector, UV 254 nm to give tert-butyl N-{2-[(4-methylbenzenesulfonyl)oxy]butyl}carbamate (700 mg, 69% yield).

[0425] LCMS(ESI):[M+H] + =361.2

[0426] 2.

[0427] 9,10-Dimethoxy-2-[(2,4,6-trimethylphenyl)amino]-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (1.0 g, 2.5 mmol, 1.0 equiv), tert-butyl N-{2-[(4-methylbenzenesulfonyl)oxy]butyl}carbamate (1.3 g, 3.8 mmol, 1.5 equiv), and cesium carbonate (1.7 g, 5.108 mmol, 2.0 equiv) were dissolved in N,N-dimethylformamide (10 mL). XantPhos (295 mg, 0.5 mmol, 0.2 equiv) and Pd2(dba)3 (233 mg, 0.3 mmol, 0.1 equiv) were added at room temperature under air. The resulting mixture was stirred at 100°C overnight under nitrogen. The reaction was quenched with water at room temperature. The resulting mixture was extracted with ethyl acetate (2 x 50 mL). The combined organic layers were washed with saturated brine (2 x 100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse phase chromatography using the following conditions: column, C18 silica gel; mobile phase, acetonitrile-water (10 mmol / L NH4HCO3), 50%-60% gradient over 10 minutes; detector, UV 254 nm. This yielded tert-butyl N-[2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)butyl]carbamate (330 mg, crude product).

[0428] LCMS(ESI):[M+H] + =563.2

[0429] 3.

[0430] Under nitrogen at room temperature, tert-butyl N-[2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)butyl]carbamate (330 mg, 0.6 mmol, 1.0 equiv) was dissolved in 1,4-dioxane (2 mL), and a solution of hydrochloric acid in 1,4-dioxane (2 mL, 4 M) was added. The resulting mixture was stirred under nitrogen at room temperature for 1.5 hours. The reaction was quenched with water at room temperature. The mixture was neutralized to pH 9 with saturated sodium carbonate solution. The resulting mixture was extracted with ethyl acetate (2 x 50 mL). The combined organic layers were washed with saturated brine (2 x 100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by Prep-TLC (DCM / MeOH=20 / 1) to give 2-[(1-aminobutan-2-yl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (150 mg, 49% yield).

[0431] LCMS(ESI):[M+H] + =463.2

[0432] 4.

[0433] 2-[(1-Aminobutan-2-yl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (100 mg, 0.2 mmol, 1 eq) and triethylamine (65 mg, 0.6 mmol, 3.0 eq) were dissolved in DCM (1 mL). Isocyanotrimethylsilane (37 mg, 0.3 mmol, 1.5 eq) was added at 0°C under air. The resulting mixture was stirred at room temperature under nitrogen for 2 hours. The resulting mixture was concentrated under reduced pressure. The crude product (50 mg) was purified by pre-liquid chromatography under the following conditions (chromatographic column: XBridge BEH C185, 19*250 mm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 25 mL / min; gradient: from 41% B to 48% B in 10 minutes; wavelength: 254 nm / 220 nm; RT (min): 7.25 / 8.32) to obtain the product.

[0434] Compound 15-1 (isomer 1, RT (min): 7.25, 4.02 mg, yield 4%)

[0435] LCMS (ESI): [M+H]+=506.25

[0436] 1 H NMR(400MHz, Methanol-d4)δ7.15–7.06(m,2H),6.93(s,1H),6.63(s,1H),5.41(s,1H),4.21–4.02(m,3H),3.89(s,3H) ,3.66(s,4H),2.97(t,J=8.0.Hz,2H),2.37(s,3H),2.21(s,3H),2.11(s,3H),1.66–1.52(m,2H),0.98(t,J=7.4Hz,3H).

[0437] Compound 15-2 (isomer 2, RT (min): 8.32, 4.02 mg, yield 4%)

[0438] LCMS (ESI): [M+H]+=506.25

[0439] 1 H NMR(400MHz, Methanol-d4)δ7.15–7.06(m,2H),6.93(s,1H),6.63(s,1H),5.41(s,1H),4.21–4.02(m,3H),3.89(s,3H) ,3.66(s,4H),2.97(t,J=8.0.Hz,2H),2.37(s,3H),2.21(s,3H),2.11(s,3H),1.66–1.52(m,2H),0.98(t,J=7.4Hz,3H).

[0440] Example 16, Compound 16

[0441] Preparation steps

[0442] 1.

[0443] To a solution of 2-[(1-aminopropan-2-yl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (200 mg, 0.4 mmol, 1.0 equiv) and triethylamine (135 mg, 1.3 mmol, 3.0 equiv) in dichloromethane (5 mL) was added trimethylsilyl isocyanate (77 mg, 0.7 mmol, 1.5 equiv) at room temperature. The resulting reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was extracted with dichloromethane (3 x 20 mL). The combined organic phases were backwashed with saturated brine and dried over anhydrous sodium sulfate. The obtained mixture was filtered, and the filtrate was concentrated under reduced pressure to give 2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propaneurea (200 mg, crude product).

[0444] LCMS(ESI):[M+H] + =492.3

[0445] 2.

[0446] To an 8 mL vial at room temperature was added 2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propaneurea (200 mg, 0.4 mmol, 1.0 equiv), acetic acid (1 mL), acetic anhydride (1 mL), and hydrobromic acid (1 mL). The resulting reaction mixture was stirred at 120°C overnight. The reaction mixture was basified to pH 6 with saturated aqueous sodium bicarbonate. The reaction mixture was extracted with dichloromethane and methanol (3 x 50 mL). The combined organic phases were backwashed with saturated brine (3 x 50 mL), and dried over anhydrous sodium sulfate. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure to give 2-({9,10-dihydroxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propaneurea (180 mg, yield 95.4%).

[0447] LCMS(ESI):[M+H] + =464.2

[0448] 3.

[0449] To a solution of 2-({9,10-dihydroxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propaneurea (150 mg, 0.3 mmol, 1.0 equiv) in N,N-dimethylformamide (9 mL) at room temperature were added potassium carbonate (268 mg, 1.8 mmol, 6.0 equiv) and deuterated iodomethane (234 mg, 1.5 mmol, 5.0 equiv). The reaction mixture was stirred at room temperature overnight. The crude product was purified by reverse phase column chromatography to give 2-{[9,10-bis(2H3)methoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl](2,4,6-trimethylphenyl)amino}propanurea (25.92 mg, 15.52% yield) under the following conditions: (Column: Xbridge BEH Shield RP185μm, 30*150mm; mobile phase A: water (10mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 35% B to 45% B in 7min; wavelength: 254nm / 220nm; RT1(min): 6.23.

[0450] LCMS(ESI):[M+H] + =498.30

[0451] 1 H NMR(400MHz, Methanol-d4)δ7.11(d,J=10.6Hz,2H),6.93(s,1H),6.63(s,1H),5.40(s,1H),4.60(s,2H),4.4 9(s,1H),4.20–4.01(m,2H),2.97(t,J=6.5Hz,2H),2.37(s,3H),2.22(s,3H),2.09(s,3H),1.04–0.96(m,3H).

[0452] Example 17, Compound 17

[0453] To a solution of 2-[(1-aminopropan-2-yl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (60 mg, 0.18 mmol, 1.0 equiv) in DCM (2 mL) was added triethylamine (41 mg, 0.40 mmol, 3.0 equiv) and dimethyl dicarbonate (36 mg, 0.27 mmol, 2.0 equiv). After stirring for two hours, the mixture was concentrated by rotary evaporation under reduced pressure. The crude product (80 mg) was purified by Prep-TLC normal phase, EA / PE = 1 / 3 to give the product methyl N-[2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]carbamate (23.01 mg, 32.94% yield).

[0454] LCMS(ESI):[M+H] + =507.25

[0455] 1 H NMR(400MHz, Methanol-d4)δ7.09(m,2H),6.90(s,1H),6.61(s,1H),5.38(s,1H),4.48–4.42(m,1H),4.20–4.02(m,2H),3.86(s,3H),3 .64(s,6H),3.60–3.53(m,1H),3.48–3.38(m,1H),2.94(t,J=6.5Hz,2H),2.35(s,3H),2.19(s,3H),2.09(s,3H),1.06(d,J=6.9Hz,3H).

[0456] Example 18, Compound 18

[0457] To a solution of benzoic acid (76 mg, 0.62 mmol, 3.5 equiv) in DMF (1 mL) were added 2-[(1-aminopropan-2-yl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (80 mg, 0.18 mmol, 1.0 equiv), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (103 mg, 0.53 mmol, 3.0 equiv), and 1-hydroxybenzotriazole (72 mg, 0.53 mmol, 3.0 equiv). The mixture was stirred for 2 h. The mixture was concentrated under reduced pressure and the crude product (100 mg) was purified by Prep-TLC with EA / PE = 1 / 5 to give N-[2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]benzamide) (18.91 mg, 19.11% yield).

[0458] LC-MS(ESI):[M+H] + =553.3

[0459] 1 H NMR(400MHz,Methanol-d4)δ7.92–7.87(m,2H),7.59–7.45(m,3H),7.17–7 .08(m,2H),6.91(s,1H),6.63(s,1H),5.42(s,1H),4.69–4.60(m,1H),4.19 –4.02(m,2H),3.93–3.82(m,4H),3.79–3.72(m,1H),3.64(s,3H),2.96(t,J =6.5Hz,2H),2.36(s,3H),2.25(s,3H),2.11(s,3H),1.12(d,J=7.0Hz,3H).

[0460] Example 19, Compound 19

[0461] To a solution of 2-picolinic acid (41 mg, 0.34 mmol, 1.0 equiv) in DMF (1 mL) were added 2-[(1-aminopropan-2-yl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (100 mg, 0.22 mmol, 1.0 equiv), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (64 mg, 0.34 mmol, 1.5 equiv), and 1-hydroxybenzotriazole (45 mg, 0.34 mmol, 1.5 equiv). The mixture was stirred for 2 h. The mixture was concentrated under reduced pressure and the crude product (100 mg) was purified by Prep-HPLC to give N-[2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]pyridine-2-carboxamide (17.35 mg, 13.93% yield).

[0462] LCMS(ESI):[M+H] + =554.25

[0463] 1 H NMR(400MHz, Methanol-d4)δ8.69–8.60(m,1H),8.06(d,J=7.7Hz,1H),8.02–7.90(m, 1H),7.58–7.50(m,1H),7.15–7.09(m,2H),6.91(s,1H),6.62(s,1H),5.40(s,1H),4. 83–4.76(m,1H),4.18–4.04(m,2H),3.86(s,3H),3.85(d,J=6.4Hz,2H),3.64(s,3H), 2.95(t,J=6.5Hz,2H),2.36(s,3H),2.25(s,3H),2.12(s,3H),1.09(d,J=6.9Hz,3H).

[0464] Example 20, Compound 20

[0465] 9,10-Dimethoxy-2-[(1-amino-2-propyl)(2,4,6-trimethylphenyl)amino]-6h,7h-pyrimido[4,3-a]isoquinoline-4-one (70 mg, 0.2 mmol, 1.0 equiv) and pyrimidine-4-carboxylic acid (58 mg, 0.5 mmol, 3.0 equiv) were dissolved in DMF (2 mL). HOBT (63 mg, 0.5 mmol, 3.0 equiv) and EDCI (90 mg, 0.5 mmol, 3.0 equiv) were added to the mixture at room temperature and stirred at room temperature for 2 h. The crude product was purified by Prep-HPLC under the following conditions (column: XBridge BEH C18 5 μm, 30*150 mm; mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: from 40% B to 47% B in 7 minutes; wavelength: 254 nm / 220 nm; RT1 (min): 6.45) to give N-[2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimidin[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]pyrimidine-4-carboxamide (19.00 mg, yield 21.34%).

[0466] LCMS(ESI):[M+H] + =555.35

[0467] 1 H NMR(400MHz, Methanol-d4)δ9.32(s,1H),9.03(d,J=4.8Hz,1H),8.16–8.02(m,1H),7.18–7.10(m,2H),6.94(s,1H),6.64(s,1H),5.43(s,1H),4.6 0(s,1H),4.23–4.08(m,2H),3.89(d,J=6.5Hz,5H),3.67(s,3H),2.98(t, J=6.5Hz,2H),2.38(s,3H),2.27(s,3H),2.14(s,3H),1.23–0.98(m,3H).

[0468] Example 21, Compound 21

[0469] 9,10-Dimethoxy-2-[(1-amino-2-propyl)(2,4,6-trimethylphenyl)amino]-6h,7h-pyrimido[4,3-a]isoquinoline-4-1 (50 mg, 0.1 mmol, 1.0 equiv) was dissolved in DCM (3 mL). Isocyanatocyclopropane (14 mg, 0.2 mmol, 1.5 equiv) and triethylamine (34 mg, 0.3 mmol, 3.0 equiv) were added to the above mixture at room temperature and stirred at room temperature for 2 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: 60 mL / min; gradient: from 36% B to 50% B in 7 minutes; wavelength: 254 nm / 220 nm; RT1 (min): 6.05 / 6.5) to give 3-cyclopropyl-1-[2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimidin[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]urea (17.19 mg, yield 28.92%).

[0470] LCMS(ESI):[M+H] + =532.30

[0471] 1 H NMR(400MHz,Methanol-d4)δ7.15–7.05(m,2H),6.93(s,1H),6.63(s,1H),5.40 (s,1H),4.57-4.49(m,1H),4.17-4.04(m,2H),3.88(s,3H),3.75-3.60(m,4H),3 .55-3.45(m,1H),2.96(t,J=6.5Hz,2H),2.57-2.48(m,1H),2.37(s,3H),2.23(s ,3H),2.11(s,3H),1.09(d,J=6.9Hz,3H),0.79-0.67(m,2H),0.57-0.46(m,2H).

[0472] Example 22, Compound 22

[0473] 9,10-Dimethoxy-2-[(1-amino-2-propyl)(2,4,6-trimethylphenyl)amino]-6h,7h-pyrimido[4,3-a]isoquinoline-4-1 (70 mg, 0.2 mmol, 1.0 equiv) and triethylamine (47 mg, 0.5 mmol, 3.0 equiv) were dissolved in DCM (3 mL) at room temperature. N-Methylcarbamoyl chloride (22 mg, 0.2 mmol, 1.5 equiv) was added to the mixture at 0°C and stirred at room temperature for 2 h. The crude product was purified by Prep-HPLC under the following conditions (column: XBridge BEH C18 5μm, 30*150mm; mobile phase A: water (10mmol / L ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: from 36% B to 48% B in 7 minutes; wavelength: 254nm / 220nm; RT1(min): 6.37) to give 1-[2-({9,10-dimethoxy-4-oxo-6H,7H-pyrimidin[4,3-a]isoquinolin-2-yl}(2,4,6-trimethylphenyl)amino)propyl]-3-methylurea (16.82mg, yield 21.25%).

[0474] LCMS(ESI):[M+H] + =506.30

[0475] 1 H NMR (400MHz, Methanol-d4) δ7.16–7.07(m,2H),6.93(s,1H),6.63(s,1H),5.39(s,1H),4.60(s,1H),4.46(t,J=7.6Hz,1H),4.21 –4.00(m,2H),3.95–3.85(m,4H),3.66(s,3H),3.05–2.80(m,5H),2.37(s,3H),2.21(s,3H),2.08(s,3H),1.02(d,J=6.9Hz,3H).

[0476] Example 23, Compound 23-1 and Compound 23-2

[0477] Preparation steps:

[0478] 1.

[0479] 4-Bromo-2,6-diisopropylaniline (5 g, 20 mmol, 1.0 equiv), cesium carbonate (20.0 g, 60 mmol, 3 equiv), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (1.6 g, 2 mmol, 0.1 equiv), methylboronic acid (5.8 g, 98 mmol, 5 equiv), and 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (1.6 g, 4 mmol, 0.2 equiv) were dissolved in 1,4-dioxane (90 mL) and water (15 mL), stirred at 100°C overnight under nitrogen. The mixture was cooled to room temperature, dissolved in 100 mL of water, and extracted with ethyl acetate (3 x 100 mL). The organic layer was washed with 100 mL of saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by normal-phase column chromatography under the following conditions (mobile phase A: dichloromethane, mobile phase B: methanol; flow rate: 100 mL / min; gradient: 0% B to 10% B in 30 min; wavelength: 254 nm) to afford 2,6-diisopropyl-4-methylaniline (2.1 g, 56.2% yield).

[0480] LCMS(ESI):[M+H] + =192.17

[0481] 2.

[0482] Under nitrogen, a solution of 2,6-diisopropyl-4-methylaniline (1 g, 5.2 mmol, 1 equivalent) and 2-chloro-9,10-dimethoxy-6,7-dihydro-4H-pyrimido-6,1-a]isoquinolin-4-one (1.68 g, 5.8 mmol, 1.1 equivalent) in isopropanol (2 mL) was stirred and reacted overnight at 90°C. The resulting reaction solution was concentrated under reduced pressure, filtered, and the filter cake was collected and washed with ethyl acetate (2 x 10 mL) to afford 2-[(2,6-diisopropyl-4-methylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (1.0 g, 42.7% yield).

[0483] LCMS(ESI):[M+H] + =448.25

[0484] 3.

[0485] 2-[(2,6-diisopropyl-4-methylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (700 mg, 1.6 mmol, 1 equivalent), cesium carbonate (1.6 g, 4.8 mmol, 3 equivalents), and tert-butyl N-(2-bromopropyl)carbamate (560 mg, 2.3 mmol, 1.5 equivalents) were dissolved in 1,4-dioxane (10 mL), protected by nitrogen, and stirred at 100° C. overnight. The mixture was cooled to room temperature, dissolved in 20 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 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 N-{2-[(2,6-diisopropyl-4-methylphenyl)({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl})amino]propyl}carbamate (600 mg, 63.4% yield).

[0486] LCMS(ESI):[M+H] + =605.35

[0487] 4.

[0488] Tert-butyl N-{2-[(2,6-diisopropyl-4-methylphenyl)({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl})amino]propyl}carbamate (500 mg, 0.91 mmol, 1 equivalent) was dissolved in 10 mL of a 4M hydrochloric acid solution in 1,4-dioxane and stirred at room temperature for 1 hour. 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 to obtain 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, 73.3% yield).

[0489] LCMS(ESI):[M+H] + =505.30

[0490] 5.

[0491] 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 NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by reverse phase column chromatography under the following conditions (column: XBridge BEH C18 5 μm, 30*150 mm; mobile phase A: water (10 mmol / L ammonium bicarbonate); mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 43% B to 55% B, 8 minutes; wavelength: 254 / 220 nm; RT1 (min): 5.77) to give 1-(2-((2,6-diisopropyl-4-methylphenyl)(9,10-dimethoxy-4-oxo-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-2-yl)amino)propyl)urea (150 mg, 45.0% yield).

[0492] 6.

[0493] The racemate (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: tert-methyl ether (0.1% ethylenediamine)--HPLC; flow rate: 20 mL / min; gradient: isocratic; column temperature (°C): 35; pressure (bar): 100; wavelength: 220 nm; RT1 (min): 10; RT2 (min): 5; sample solvent: methanol--HPLC; injection volume: 1 mL) to obtain the product.

[0494] Compound 23-1 (isomer 1, RT1 (min): 10, 16.5 mg, yield 6.4%).

[0495] LCMS(ESI):[M+H] + =548.30

[0496] 1H NMR (400MHz, Methanol-d4) δ7.25–7.18(m,2H),6.94(s,1H),6.61(s,1H),5.45(s,1H),4.48(s,1H),4.15–4.08(m,2H),3.89(s, 3H),3.63(s,3H),3.11–2.93(m,4H),2.75(s,1H),2.44(s,3H),1.36–1.25(m,8H),1.17(d,J=6.8Hz,3H),1.02(d,J=6.6Hz,4H).

[0497] Compound 23-2 (isomer 2, RT2 (min): 5, 16.42 mg, yield 6.4%).

[0498] LCMS(ESI):[M+H] + =548.30

[0499] 1 H NMR(400MHz, Methanol-d4)δ7.26–7.18(m,2H),6.94(s,1H),6.61(s,1H),5.45(s,1H),4.48(s,1H),4.16–4.08(m,2H),3.89(s, 3H),3.63(s,3H),3.11–2.93(m,3H),2.75(s,1H),2.44(s,3H),1.35–1.28(m,7H),1.17(d,J=6.8Hz,3H),1.02(d,J=6.6Hz,3H).

[0500] Example 24, Compound 24-1 and Compound 24-2

[0501] Preparation steps:

[0502] 1.

[0503] 1,3-Difluoro-5-methyl-2-nitrobenzene (200 mg, 1.1 mmol, 1 equivalent) was dissolved in MeOH (5 mL) at room temperature in the presence of air, and a methanolic solution of sodium methoxide (3 mL, 60%) was added. The mixture was allowed to react overnight at room temperature. The mixture was evaporated to dryness under reduced pressure, 50 mL of water was added, and extraction was performed with ethyl acetate (2 x 50 mL). The combined organic layers were washed with saturated brine (2 x 100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield 1,3-dimethoxy-5-methyl-2-nitrobenzene (200 mg, crude product).

[0504] 2.

[0505] 1,3-Dimethoxy-5-methyl-2-nitrobenzene (190 mg, 0.9 mmol, 1 equivalent) and H₂O (1 mL) were added to an EtOH (5 mL) solution at room temperature in the presence of air. NHCl (154 mg, 3 mmol, 3 equivalents) and Fe (8.5 mg, 0.2 mmol, 3 equivalents) were then added. The reaction was allowed to proceed at 80°C for two hours. The resulting mixture was filtered, and the filter cake was washed with ethyl acetate (2 x 30 mL). The reaction was quenched with water at room temperature. The resulting mixture was extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with saturated sodium chloride (2 x 150 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to yield 2,6-dimethoxy-4-methylaniline (150 mg, crude product).

[0506] LCMS(ESI):[M+H] + =168.2

[0507] 3.

[0508] 2-Chloro-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (273 mg, 0.9 mmol, 1.2 equivalents) was dissolved in isopropanol (3 ml) at room temperature in air, and 2,6-dimethoxy-4-methylaniline (152 mg, 0.9 mmol, 1.2 equivalents) was added. The resulting mixture was concentrated under reduced pressure to give 2-[(2,6-dimethoxy-4-methylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (300 mg, crude product).

[0509] LCMS(ESI):[M+H] + =424.2

[0510] 4.

[0511] 2-[(2,6-Dimethoxy-4-methylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (300 mg, 0.7 mmol, 1 eq.), tert-butyl N-(2-bromopropyl)carbamate (168 mg, 0.7 mmol, 1 eq.) and Cs2CO3 (461 mg, 1.4 mmol, 2 eq.) were dissolved in 1,4-dioxane (5 ml) solution, and XantPhos (81 mg, 0.1 mmol, 0.1 eq.) and Pd2(dba)3 (64 mg, 0.1 mmol, 0.1 eq.) were added. The resulting mixture was stirred at 100°C under nitrogen atmosphere overnight. 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 from 10% to 50% over 10 minutes; detector, UV 254 nm. This afforded tert-butyl N-{2-[(2,6-dimethoxy-4-methylphenyl)({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl})amino]propyl}carbamate (270 mg, 59% yield).

[0512] LCMS(ESI):[M+H] + =581.2

[0513] 5.

[0514] Under nitrogen at room temperature, tert-butyl N-{2-[(2,6-dimethoxy-4-methylphenyl)({9,10-dimethoxy-4-oxo-6H,7H-pyrimido[4,3-a]isoquinolin-2-yl})amino]propyl}carbamate (300 mg, 0.6 mmol, 1.0 equiv) was dissolved in 1,4-dioxane (2 mL), and a solution of hydrochloric acid in 1,4-dioxane (2 mL) was added. The resulting mixture was stirred under nitrogen at room temperature for 1.5 hours. The reaction was quenched with water at room temperature. The mixture was neutralized to pH 9 with saturated sodium carbonate solution. The resulting mixture was extracted with ethyl acetate (2 x 50 mL). The combined organic layers were washed with saturated brine (2 x 100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by Prep-TLC (CH2Cl2 / MeOH=20 / 1) to give 2-[(1-aminopropan-2-yl)(2,6-dimethoxy-4-methylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (150 mg, 49% yield).

[0515] LCMS(ESI):[M+H] +=481.2

[0516] 6.

[0517] 2-[(1-Aminopropyl-2-yl)(2,6-dimethoxy-4-methylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinolin-4-one (400 mg, 0.8 mmol, 1 eq) and TEA (0.4 mL, 2.5 mmol, 3 eq) were added to the solution in DCM (4 mL). Isocyanotrimethylsilane (191 mg, 1.6 mmol, 2.0 eq) was added to the solution. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 2 hours, and the reaction mixture was concentrated under reduced pressure. The crude product (420 mg) was purified by HPLC under the following conditions (column: Sunfire C18 5 m, 30 mm × 4.5 mm): mobile phase A: water (0.1% FA), mobile phase B: acetonitrile; flow rate: 60 ml / min; gradient: from 51% B to 74% B in 10 minutes; wavelength: 254 nm / 220 nm; RT1 (min): 6.03; 6.62) to give 1-(2-((2,6-dimethoxy-4-methylphenyl)(9,10-dimethoxy-4-oxo-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-2-yl)amino)propyl)urea (130 mg), which was then purified by Chiral-HPLC under the following conditions (column: JW-CHIRALPAK IG-3, 3.0*50mm; 3um; 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): 12; RT2 (min): 16.5; sample solvent: sample solvent: MeOH:DCM=1:1--HPLC; injection volume: 0.7 mL; number of runs: 5 times) to obtain the product.

[0518] Compound 24-1 (isomer 1, RT1 (min): 12, 30.12 mg, 6.8% yield)

[0519] LCMS(ESI):[M+H] + =524.25

[0520] 1H NMR(400MHz,Methanol-d4)δ6.92(s,1H),6.72–6.66(m,3H),5.53(s,1H),4.11–4.05(m,2 H),3.89(s,3H),3.81(d,J=10.1Hz,6H),3.69(s,3H),2.95(t,J=6.5Hz,2H),2.47(s,3H).

[0521] Compound 24-2 (isomer 2, RT2 (min) 16.5, 44.03 mg, 10% yield)

[0522] LCMS(ESI):[M+H] + =524.25

[0523] 1 H NMR(400MHz,Methanol-d4)δ6.92(s,1H),6.72–6.66(m,3H),5.53(s,1H),4.11–4.05(m,2 H), 3.89 (s, 3H), 3.81 (d, J = 10.1Hz, 6H), 3.69 (s, 3H), 2.95 (t, J = 6.5Hz, 2H), 2.47 (s, 3H).

[0524] Example 25, Compound 25-1 and Compound 25-2

[0525] Preparation steps:

[0526] 1.

[0527] A mixture of 2,6-difluoro-4-methylaniline (1.0 g, 6.9 mmol, 1 equiv) and 2-chloro-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinoline-4-one (2.6 g, 9.0 mmol, 1.3 equiv) in IPA (40 mL) was stirred at 90°C overnight under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The mixture was basified to pH 8 with saturated NaHCO₃. The resulting mixture was extracted with EtOEt (3 x 50 mL). The combined organic layers were washed with brine (2 x 100 mL) and dried over anhydrous Na₂SO₄. The filtered filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using CH2Cl2 / MeOH (80 / 1 to 60 / 1) as eluent to give 2-[(2,6-difluoro-4-methylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimidin[4,3-a]isoquinoline-4-1 (2.0 g, 71.6%).

[0528] LCMS(ESI):[M+H]+ =400.15

[0529] 2.

[0530] 2-[(2,6-difluoro-4-methylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimidin[4,3-a]isoquinolin-4-one (440 mg, 1.1 mmol, 1 equiv) and tert-butyl N-(2-bromopropyl)carbamate (288.5 mg, 1.2 mmol, 1.1 equiv) were stirred in DMF (15 mL) at 100°C under a nitrogen atmosphere overnight. The resulting mixture was concentrated under vacuum. Purification by silica gel column chromatography using CH2Cl2 / MeOH (10 / 1) as the eluent gave tert-butyl N-{2-[(2,6-difluoro-4-methylphenyl)({9,10-dimethoxy-4-oxo-6H,7H-pyrimidin[4,3-a]isoquinolin-2-yl})amino]propylcarbamate (200 mg, 32.6%).

[0531] LCMS(ESI):[M+H] + =557.25

[0532] 3.

[0533] N-{2-[(2,6-difluoro-4-methylphenyl)({9,10-dimethoxy-4-oxy-6H,7H-pyrimidin[4,3-a]isoquinolin-2-yl})amino]propyl}carbamate (200 mg, 0.359 mmol, 1 equivalent) was stirred in 1,4-dioxane (15 mL) at room temperature under a nitrogen atmosphere for 3 hours. The resulting mixture was concentrated under reduced pressure. This yielded 2-[(1-aminopropen-2-yl)(2,6-difluoro-4-methylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimidin[4,3-a]isoquinolin-4-yl (150 mg, 91.45%). The crude product was used directly in the next step without further purification.

[0534] LCMS(ESI):[M+H] + =457.2

[0535] 4.

[0536] 2-[(1-Aminopropen-2-yl)(2,6-difluoro-4-methylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimido[4,3-a]isoquinoline-4-one (50 mg, 0.11 mmol, 1.0 equiv) and trimethylsilyl isocyanate (12.6 mg, 0.11 mmol, 1.0 equiv) were stirred in DCM (5 mL) at room temperature under a nitrogen atmosphere overnight. The resulting mixture was concentrated under reduced pressure. The crude product (60 mg) was purified by Prep-HPLC under the following conditions (column: XSelect CSH Fluoro Phenyl 5m, 19 mm*250 mm; mobile phase A: water (0.1% FA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 42% B to 65% B, 10 minutes; wavelength: 254 nm / 220 nm; RT1 (min): 5.67) to give (2R)-2-[(2,6-difluoro-4-methylphenyl)({9,10-dimethoxy-4-oxo-6H,7H-pyrimidin[4,3-a]isoquinolin-2-yl})amino]propyl urea (50 mg). The product was then purified by Chiral-HPLC under the following conditions (chromatographic 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: equidistant; wavelength: 220 nm; RT1 (min): 11.2; RT2 (min): 15.0; sample solvent: MeOH:DCM = 1:1—HPLC; injection volume: 1.5 mL; number of runs: 5) to obtain the product.

[0537] Compound 25-1 (isomer 1, RT1 (min): 11.2, 13.41 mg, 24.49%).

[0538] LCMS(ESI):[M+H] + =500.25

[0539] 1 H NMR (400MHz, Methanol-d4) δ6.90(s,1H),6.87(s,1H),6.85(s,1H),6.83(s,1H),5.70(t,J=1.7Hz,1H),5.63(s,1H),4.00(t,J=6.3 Hz,2H),3.96–3.90(m,1H),3.88(s,3H),3.73(s,3H),3.65–3.56(m,1H),2.95(t,J=6.2Hz,2H),2.35(s,3H),1.54(d,J=6.9Hz,3H).

[0540] Compound 25-2 (isomer 2, RT2 (min): 15.0, 15.09 mg, 21.59%)

[0541] LCMS(ESI):[M+H] + =500.25

[0542] 1 H-NMR (400MHz, Methanol-d4) δ6.89(s,1H),6.87(s,1H),6.86–6.84(m,1H),6.84–6.81(m,1H),5.70(t,J=1.6Hz,1H),5.63(s, 1H),4.00(t,J=6.3Hz,2H),3.96–3.90(m,1H),3.88(s,3H),3.73(s,3H),2.97–2.93(m,2H),2.35(s,3H),1.54(d,J=6.9Hz,3H).

[0543] Example 26, Compound 26

[0544] Preparation steps

[0545] 1.

[0546] 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 100% over 30 minutes, UV 220 nm detection. Tert-butyl (4-((9,10-dimethoxy-4-oxo-6,7-dihydro-4H-pyrimidin[6,1-a]isoquinolin-2-yl)(methylsulfonyl)amino)cyclohexyl)carbamate (110 mg, 7.3%) was obtained.

[0547] LCMS (ESI, m / z): [M+H] + =589.3

[0548] 2.

[0549] To a single-necked flask, tert-butyl (4-((9,10-dimethoxy-4-oxo-6,7-dihydro-4H-pyrimidin[6,1-a]isoquinolin-2-yl)(methylsulfonyl)amino)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 evaporated to obtain 2-[(4-aminocyclohexyl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimidin[4,3-a]isoquinolin-4-one (80 mg, 96.4%).

[0550] LCMS (ESI, m / z): [M+H] + =489.3

[0551] 3.

[0552] 2-[(4-Aminocyclohexyl)(2,4,6-trimethylphenyl)amino]-9,10-dimethoxy-6H,7H-pyrimidin[4,3-a]isoquinolin-4-one (140 mg, 0.3 mmol, 1.0 equiv) was dissolved in DCM (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 39% B to 4% B in 10 minutes; wavelength: 254 nm / 220 nm; RT1 (min): 9.4) to give 4-({9,10-dimethoxy-4-oxo-6H,7H-pyrimidin[4,3-a]isoquinolin-2-yl}(2,6-dimethylphenyl)amino)cyclohexylurea (35.71 mg, yield 22.81%).

[0553] LCMS (ESI): [M+H]+=532.25

[0554] 1H NMR (400MHz, Methanol-d4) δ7.10(s,2H),6.92(s,1H),6.62(s,1H),5.39(s,1H),4.18–4.03(m,3H),3.88(s,4H),3. 66(s,3H),2.96(t,J=6.4Hz,2H),2.36(s,3H),2.21(s,6H),2.05–1.92(m,4H),1.88–1.80(m,2H),1.75–1.62(m,2H).

[0555] Example 27, Compound 27

[0556] Preparation steps

[0557] 1.

[0558] In a single-necked flask, (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 equivalent), 1.4-dioxane (100 mL), cesium carbonate (2.5 g, 7.7 mmol, 3.0 equivalent) and tert-butyl 4-[(4-methylbenzenesulfonyl)oxy]piperidine-1-carboxylate (1.0 g, 2.8 mmol, 1.1 equivalent) were added in sequence and reacted at 100°C overnight. Filter and concentrate the filtrate. The resulting residue was purified by reverse phase column chromatography under the following conditions: mobile phase, water and acetonitrile, 10% to 100% gradient over 30 minutes, UV220 nanometer detector to give tert-butyl 4-((9,10-dimethoxy-4-oxo-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-2-yl)(amidino)amino)piperidine-1-carboxylate (120 mg, 8.17% yield).

[0559] LCMS (ESI, m / z): [M+H] + =575.3

[0560] 2.

[0561] To a single-necked flask, tert-butyl 4-((9,10-dimethoxy-4-oxo-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-2-yl)(amidino)amino)piperidine-1-carboxylate (100 mg, 0.2 mmol, 1.0 equiv) and hydrogen chloride in 1,4-dioxane (17 mL) were added sequentially and reacted at room temperature for 2 h. The solvent was concentrated to afford 2-(methylsulfonyl(piperidin-4-yl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (80 mg, 96.9% yield).

[0562] LCMS (ESI, m / z): [M+H] + =475.3

[0563] 3.

[0564] To a solution of 2-(methylsulfonyl(piperidin-4-yl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (1050 mg, 2.2 mmol, 1.0 equiv) in dichloromethane (20 mL) were added triethylamine (640 mg, 6.3 mmol, 3.0 equiv) and trimethylsilyl isocyanate (364 mg, 3.2 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: 35% B to 40% 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) (39.74 mg, yield 3.63%).

[0565] LCMS(ESI):[M+H] + =518.40

[0566] 1H NMR(400MHz, Methanol-d4)δ7.10(s,2H),6.92(s,1H),6.62(s,1H),5.39(s,1H),4.73–4.62(m,1H),4.18 –3.97(m,4H),3.88(s,3H),3.66(s,3H),3.02–2.90(m,4H),2.36(s,3H),2.17(s,8H),1.43–1.29(m,2H).

[0567] Example 28, Compound 28

[0568] 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: tert-methyl 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 1-(3-((2,6-diisopropyl-4-methylphenyl)(9,10-dimethoxy-4-oxo-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-2-yl)amino)cyclobutyl)urea (13.00 mg, yield 14.9%).

[0569] LCMS(ESI):[M+H] + =560.30

[0570] 1 H NMR(400MHz, Methanol-d4)δ7.21(s,2H),6.93(s,1H),6.64(s,1H),5.46(s,1H),4.51–4.44(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).

[0571] Example 29, Compound 29

[0572] The crude product (30 mg) was purified by Chiral-HPLC under the following conditions: CHIRALPAK IH 3*25 cm, 5 μm; mobile phase A: CO2, mobile phase B: methanol (0.1% diethylamine); flow rate: 85 mL / min; gradient: isocratic 35% B; column temperature (℃): 25℃; back pressure (bar): 100; wavelength: 254 nm; RT1 (min): 2.85; RT2 (min): 4.5 sample solvent: methanol: dichloromethane = 1:1--HPLC; injection volume: 1 mL) to obtain the product.

[0573] Compound 29 (isomer 1, RT1 (min): 2.85, 3.2 mg, yield 10.3%)

[0574] LCMS (ESI, m / z): [M+H] + =558.35

[0575] 1 H NMR(400MHz, Methanol-d4)δ7.17–7.10(m,2H),6.93(s,1H),6.65(s,1H),5.43(s,1H),4.78–4.69(m,1H),4.20–4.04(m,2H),3.97 –3.77(m,5H),3.67(s,3H),2.98(t,J=6.5Hz,2H),2.59–2.52(m,3H),2.38(s,3H),2.27(s,3H),2.13(s,3H),1.11(d,J=6.9Hz,3H).

[0576] Example 30, Compound 30

[0577] To a 25 mL single-necked vial was added 2-((1-aminopropan-2-yl)(methyltrimethyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (100 mg, 0.22 mmol), 6-methylpyridine-2-carboxylic acid (45.3 mg, 0.33 mmol), EDCI (63.3 mg, 0.33 mmol), HOBT (44.6 mg, 0.33 mmol), and DMF (2 mL) at room temperature for 24 h. After completion of the reaction, the reaction system was filtered and purified by reverse-phase flash chromatography (mobile phase A: 0.5% FA in water, mobile phase B: acetonitrile; flow rate: 40 mL / min; gradient: 0% B to 55% B over 20 min; product at 50% B; wavelength: 254 nm / 220 nm) to afford the product (19.2 mg, 10.3%).

[0578] LC-MS(ESI):[M+H] + =568.2

[0579] 1 H NMR(400MHz, CDCl3)8.76-8.74(m,1H),7.86(d,J=8.0Hz,1H),7.63(t,J=12.0Hz,1H),7 .19-7.16(m,1H),6.91(d,J=8.0Hz,1H),6.62(s,1H),6.52(s,1H),5.22(s,1H),4.96-4 .95(m,1H),4.16–4.10(m,2H),3.83(s,3H),3.82-3.75(m,2H),3.63(s,3H),2.84(t,J= 8.0Hz,2H),2.54(s,3H),2.26(s,3H),2.17(s,3H),2.05(s,3H),1.05(d,J=8.0Hz,3H).

[0580] Example 31, Compound 31

[0581] To a 25 mL single-necked vial was added 2-((1-aminopropan-2-yl)(methyltrimethyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (150 mg, 0.33 mmol), 5-methyl-2-pyridine carboxylate (68.6 mg, 0.50 mmol), EDCI (95.9 mg, 0.50 mmol), HOBT (67.6 mg, 0.50 mmol), and DMF (3 mL) at room temperature for 24 h. After completion of the reaction, the reaction system was filtered and purified by reverse-phase flash chromatography (mobile phase A: 0.5% FA aqueous solution, mobile phase B: acetonitrile; flow rate: 40 mL / min; gradient: 0% B to 55% B over 20 min; product at 50% B; wavelength: 254 nm / 220 nm) to afford the product (37.5 mg, 19.7%).

[0582] LC-MS(ESI):[M+H] + =568.2

[0583] 1H NMR(400MHz, CDCl3)8.81(s,1H),8.35(s,1H),7.95(d,J=8.0Hz,1H),7.54-7.51(m ,1H),6.93(d,J=8.0Hz,1H),6.62(s,1H),6.52(s,1H),5.21(s,1H),4.92-4.87(m,1 H),4.17–4.07(m,1H),3.83(s,3H),3.82-3.75(m,2H),3.63(s,3H),2.84(t,J=8.0 Hz, 2H), 2.31 (s, 3H), 2.25 (s, 3H), 2.17 (s, 3H), 2.05 (s, 3H), 1.09 (d, J = 8.0Hz, 3H).

[0584] Example 32, Compound 32

[0585] To a 25 mL single-necked vial was added 2-((1-aminopropan-2-yl)(methyltrimethyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (150 mg, 0.33 mmol), 4-methyl-2-pyridinecarboxylic acid (68.6 mg, 0.50 mmol), EDCI (95.9 mg, 0.50 mmol), HOBT (67.6 mg, 0.50 mmol), and DMF (3 mL) at room temperature for 24 h. After completion of the reaction, the reaction system was filtered and purified by reverse-phase flash chromatography (mobile phase A: 0.5% FA aqueous solution, mobile phase B: acetonitrile; flow rate: 40 mL / min; gradient: 0% B to 55% B over 20 min; product at 50% B; wavelength: 254 nm / 220 nm) to afford the product (32.3 mg, 17.3%).

[0586] LC-MS(ESI):[M+H] + =568.2

[0587] 1H NMR(400MHz, CDCl3)8.87(s,1H),8.37(d,J=8.0Hz,1H),8.21(s,1H),7.91(s,1H),7.1 5(d,J=8.0Hz,1H),6.91(d,J=12Hz,1H),6.62(s,1H),6.51(s,1H),5.23(s,1H),4.87-4 .82(m,1H),4.19–4.07(m,2H),3.83(s,3H),3.82-3.75(m,2H),3.64(s,3H),2.85(t,J= 8.0Hz,2H),2.34(s,3H),2.26(s,3H),2.17(s,3H),1.98(s,3H),1.07(d,J=4.0Hz,3H).

[0588] Example 33, Compound 33

[0589] To a 25 mL single-necked vial was added 2-((1-aminopropan-2-yl)(methyltrimethyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (150 mg, 0.33 mmol), 3-methyl-2-pyridinecarboxylic acid (68.6 mg, 0.50 mmol), EDCI (95.9 mg, 0.50 mmol), HOBT (67.6 mg, 0.50 mmol), and DMF (3 mL) at room temperature for 24 h. After completion of the reaction, the reaction system was filtered and purified by reverse-phase flash chromatography (mobile phase A: 0.5% FA aqueous solution, mobile phase B: acetonitrile; flow rate: 40 mL / min; gradient: 0% B to 55% B over 20 min; product at 50% B; wavelength: 254 nm / 220 nm) to afford the product (12.8 mg, 6.8%).

[0590] LC-MS(ESI):[M+H] + =568.2

[0591] 1H NMR(400MHz, CDCl3)8.86(s,1H),8.35(d,J=4.0Hz,1H),7.47(d,J=8.0Hz,1H),7.22- 7.20(m,1H),6.91(d,J=8.0Hz,1H),6.62(s,1H),6.52(s,1H),5.22(s,1H),4.96-4.91 (m,1H),4.19–4.03(m,2H),3.83(s,3H),3.82-3.75(m,2H),3.63(s,3H),2.84(t,J=8 .0Hz,2H),2.64(s,3H),2.26(s,3H),2.18(s,3H),2.02(s,3H),1.08(d,J=4.0Hz,3H).

[0592] Example 34, Compound 34

[0593] To a 25 mL single-necked vial was added 2-((1-aminopropan-2-yl)(methyltrimethyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (100 mg, 0.22 mmol), pyrimidine-2-carboxylic acid (40.9 mg, 0.33 mmol), EDCI (63.3 mg, 0.33 mmol), HOBT (44.6 mg, 0.33 mmol), and DMF (2 mL) at room temperature for 24 h. After completion of the reaction, the reaction mixture was filtered and purified by reverse-phase flash chromatography (mobile phase A: 0.5% FA aqueous solution, mobile phase B: acetonitrile; flow rate: 40 mL / min; gradient: 0% B to 55% B over 20 min; product at 50% B; wavelength: 254 nm / 220 nm) to afford the product (20 mg, 10.9%).

[0594] LC-MS(ESI):[M+H] + =555.2

[0595] 1H NMR(400MHz, DMSO-d6)9.56(t,J=8.0Hz,1H),9.03-9.00(m,2H),8.16(s,1H) ,7.87(s,1H),7.74-7.71(m,1H),7.12-7.10(m,2H),7.02(s,1H),4.74–4.70 (m,1H),4.16(s,3H),4.15-4.10(m,2H),3.98-3.95(m,2H),3.89(s,3H),3.0 5-3.00(m,2H),2.31(s,3H),2.19(s,3H),2.03(s,3H),0.83(d,J=8.0Hz,3H).

[0596] Example 35, Compound 35

[0597] To a 25 mL single-necked vial was added 2-((1-aminopropyl-2-yl)(methyltrimethyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (100 mg, 0.22 mmol), 5-methylpyrimidine-2-carboxylic acid (45.6 mg, 0.33 mmol), EDCI (63.3 mg, 0.33 mmol), HOBT (44.6 mg, 0.33 mmol), and DMF (2 mL) at room temperature for 24 h. After completion of the reaction, the reaction mixture was filtered and purified by reverse-phase flash chromatography (mobile phase A: 0.5% FA aqueous solution, mobile phase B: acetonitrile; flow rate: 40 mL / min; gradient: 0% B to 55% B over 20 min; product at 50% B; wavelength: 254 nm / 220 nm) to afford the product (27.6 mg, 14.7%).

[0598] LC-MS(ESI):[M+H] + =569.2

[0599] 1 H NMR(400MHz, CDCl3)9.28(s,1H),8.64(s,1H),6.91(d,J=8.0Hz,1H),6.62(s,1H),6.51(s,1H),5.22(s,1H),4.93-4.90(m,1H),4.13–4.10(m,2 H),3.83(s,3H),3.82-3.75(m,2H),3.63(s,3H),2.85-2.83(m,2H),2.3 1(s,3H),2.26(s,3H),2.16(s,3H),2.04(s,3H),1.05(d,J=8.0Hz,3H).

[0600] Example 36, Compound 36

[0601] To a 25 mL single-necked vial was added 2-((1-aminopropan-2-yl)(methyltrimethyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (100 mg, 0.22 mmol), pyrazine-2-carboxylic acid (40.9 mg, 0.33 mmol), EDCI (63.3 mg, 0.33 mmol), HOBT (44.6 mg, 0.33 mmol), and DMF (2 mL) at room temperature for 24 h. After completion of the reaction, the reaction mixture was filtered and purified by reverse-phase flash chromatography (mobile phase A: 0.5% FA in water, mobile phase B: acetonitrile; flow rate: 40 mL / min; gradient: 0% B to 55% B over 20 min; product at 50% B; wavelength: 254 nm / 220 nm) to afford the product (9.5 mg, 5.2%).

[0602] LC-MS(ESI):[M+H] + =555.2

[0603] 1 H NMR(400MHz, DMSO-d6)9.51(t,J=8.0Hz,1H),9.13-9.12(m,1H),8.92(d,J=4.0Hz,1H ),8.78-8.77(m,1H),7.98(s,1H),7.60(s,1H),7.10-7.08(m,1H),7.05(s,1H),7.02( s,1H),4.74–4.70(m,1H),4.08(s,3H),4.07-4.05(m,2H),3.98-3.95(m,2H),3.89(s, 3H), 3.05-3.00 (m, 2H), 2.31 (s, 3H), 2.19 (s, 3H), 2.03 (s, 3H), 0.83 (d, J = 8.0Hz, 3H).

[0604] Example 37, Compound 37

[0605] To a 25 mL single-necked vial was added 2-((1-aminopropyl-2-yl)(methyltrimethyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (100 mg, 0.22 mmol), 6-methylpyrazine-2-carboxylic acid (45.6 mg, 0.33 mmol), EDCI (63.3 mg, 0.33 mmol), HOBT (44.6 mg, 0.33 mmol), and DMF (2 mL) at room temperature for 24 h. After completion of the reaction, the reaction system was filtered and purified by reverse-phase flash chromatography (mobile phase A: 0.5% FA aqueous solution, mobile phase B: acetonitrile; flow rate: 40 mL / min; gradient: 0% B to 55% B over 20 min; product at 50% B; wavelength: 254 nm / 220 nm) to afford the product (29.3 mg, 15.6%).

[0606] LC-MS(ESI):[M+H] + =569.2

[0607] 1 H NMR(400MHz, CDCl3)9.17(s,1H),8.97(s,1H),8.59(s,1H),7.00(d,J=8.0 Hz,2H),6.70(s,1H),6.60(s,1H),5.31(s,1H),5.08-5.07(m,1H),4.23–4. 17(m,2H),3.92(s,3H),3.92-3.85(m,2H),3.72(s,3H),2.94-2.91(m,2H), 2.68(s,3H),2.34(s,3H),2.25(s,3H),2.12(s,3H),1.11(d,J=8.0Hz,3H).

[0608] Example 38, Compound 38

[0609] To a 25 mL single-necked vial was added 2-((1-aminopropyl-2-yl)(methyltrimethyl)amino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (150 mg, 0.33 mmol), 5-methylpyrazine-2-carboxylic acid (69.1 mg, 0.50 mmol), EDCI (95.8 mg, 0.50 mmol), HOBT (67.6 mg, 0.50 mmol), and DMF (3 mL) at room temperature for 24 h. After completion of the reaction, the reaction mixture was filtered and purified by reverse-phase flash chromatography (mobile phase A: 0.5% FA aqueous solution, mobile phase B: acetonitrile; flow rate: 40 mL / min; gradient: 0% B to 55% B over 20 min; product at 50% B; wavelength: 254 nm / 220 nm) to afford the product (50.2 mg, 26.8%).

[0610] LC-MS(ESI):[M+H] + =569.2

[0611] 1 H NMR(400MHz, CDCl3)9.23(s,1H),8.48(s,1H),8.59(s,1H),7.00(d,J=12.0 Hz,2H),6.70(s,1H),6.60(s,1H),5.31(s,1H),4.98-4.93(m,1H),4.23–4. 17(m,2H),3.92(s,3H),3.92-3.85(m,2H),3.72(s,3H),2.94-2.91(m,2H), 2.65(s,3H),2.35(s,3H),2.25(s,3H),2.12(s,3H),1.14(d,J=8.0Hz,3H).

[0612] Biological test evaluation

[0613] 1. PDE enzyme activity inhibition experiment 1

[0614] 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.

[0615] 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.

[0616] Table 4 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.

[0617] Table 4

[0618] Conclusion: The compound of the present invention has a highly effective inhibitory activity against PDE3 / 4, is superior to the existing technology, and has good development prospects.

[0619] 2. PDE enzyme activity inhibition experiment 2

[0620] Compounds were tested for PDE enzyme activity inhibition using the FP method, with control compounds RPL-554 and Compound D7 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 D7 are shown below.

[0621] Table 5 shows the enzyme activity test IC of the reference compound RPL-554, compound D7 and the present compounds 1-1 and 1-2. 50 value.

[0622] Table 5

[0623] 3. Study on the Effect of Compounds on Bronchial Smooth Muscle Cell Viability

[0624] 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.

[0625] 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 MTS assay. The results are shown in Figures 2A to 2G. Compared with the control group, all concentrations of the compound inhibited the growth of human bronchial smooth muscle cells in a dose-dependent manner.

[0626] 4. Pharmacodynamic Study of Compounds in LPS-Induced Inflammation Model

[0627] 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 96-well plates. The cells were divided into 14 groups, each with six parallel controls: 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 1-2 (0.78 and 1.56 μmol / L), low- and high-dose groups of compound 10-2 (0.78 and 1.56 μmol / L), low- and high-dose groups of compound 12-1 (0.78 and 1.56 μmol / L), and low- and high-dose groups of compound 19 (0.78 and 1.56 μmol / L). The LPS working concentration in the model control group and the compound groups was 1 μg / ml. The cells were incubated in the absence or presence of LPS and the compound (5% CO2, 37°C). After 24 h, the cell culture supernatant was collected and centrifuged at 2000-3000 g for 5 min. The precipitate was removed and the supernatant was collected to detect the level of TNF-α.

[0628] The results of TNF-α level detection in cell supernatants are shown in Figure 3. 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.

[0629] 5. Efficacy of the Compounds on the Guinea Pig Airway Constriction Model Induced by Acetylcholine Chloride

[0630] (1) Observe the inhibitory effect of inhalation administration of the test sample (compound 10-2 of the present invention) on guinea pig airway contraction caused by acetylcholine chloride. 40 male Hartley guinea pigs were randomly divided into a negative control group (clean air, inhalation time 60 min), a low-dose test sample group (inhalation time 10 min), a medium-dose test sample group (inhalation time 20 min), and a high-dose test sample group (inhalation time 60 min), 10 animals per group, single inhalation. About 2 hours after the inhalation administration, the animals were anesthetized, the trachea and jugular vein were surgically separated, the trachea was intubated, and the jugular vein was catheterized. The animals were placed on the support board of the scanning box and connected to the airway. The distal end of the venous catheter was pulled outside the scanning box, and then the scanning box was closed. 25 μg / kg, 50 μg / kg, and 100 μg / kg of acetylcholine chloride were administered through the jugular vein catheter, and airway resistance was detected using the AniRes2005 animal pulmonary function analysis system.

[0631] The test results are shown in Figures 4A and 4B. A single nebulized inhalation of the test substance (Compound 10-2 of the present invention) at actual delivered doses of 0.171 mg / kg, 0.341 mg / kg, and 1.024 mg / kg significantly inhibited acetylcholine-induced airway constriction at doses of 25 μg / kg to 100 μg / kg 2 hours after administration, demonstrating a dose-response relationship.

[0632] (2) Observe the inhibitory effect of inhalation administration of the test sample (compound 1-2 of the present invention) on guinea pig airway contraction caused by acetylcholine chloride. 40 male Hartley guinea pigs were randomly divided into a negative control group (clean air, inhalation time 30 minutes), a low-dose test sample group (inhalation time 6 minutes), a medium-dose test sample group (inhalation time 12 minutes), and a high-dose test sample group (inhalation time 30 minutes), 10 animals per group, single inhalation. About 2 hours after the inhalation administration, the animals were anesthetized, the trachea and jugular vein were surgically separated, a tracheal cannula was inserted, and a jugular vein catheter was inserted. The animals were placed on the support board of the scanning box and connected to the airway. The distal end of the venous catheter was pulled outside the scanning box, and then the scanning box was closed. Different doses of acetylcholine chloride were administered through the jugular vein catheter, and airway resistance was detected using the AniRes2005 animal pulmonary function analysis system.

[0633] The test results are shown in Figures 5A and 5B. A single nebulized inhalation of the test product (Compound 1-2 of the present invention) at actual delivered doses of 0.235 mg / kg, 0.470 mg / kg, and 1.174 mg / kg significantly inhibited acetylcholine-induced airway constriction at doses of 25 μg / kg to 100 μg / kg two hours after administration, demonstrating a dose-response relationship.

[0634] VI. Effects of Compounds on Airway Constriction Induced by Aerosolized Acetylcholine in Mice

[0635] 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-disodium hydrogen phosphate buffer solution.

[0636] 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.

[0637] Newly received experimental animals were quarantined for 3 days and grouped as described above. Airway nebulization was performed using the NAM system. The nebulization process lasted 5 minutes (200 μL), followed by a 3-minute interval and a further 5-minute interval (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 6.

[0638] Table 6

[0639] VII. Oral bioavailability study in rats

[0640] Experimental purpose: To investigate the bioavailability and other pharmacokinetic parameters of 1 mg / kg RPL-554, compound 1-2 of the present invention, compound 10-2, compound 67, and compound 69 administered orally to rats.

[0641] Test drugs: RPL-554, compound 1-2 of the present invention, compound 10-2, compound 67, and compound 69.

[0642] Experimental animals: 30 male SD rats.

[0643] 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.

[0644] Dosing solution for the 1 mg / kg compound 10-2-iv group and the 1 mg / kg compound 10-2-po group: Weigh approximately 3.0 mg of 10-2, add 0.3 mL of DMSO, mix thoroughly, then add 29.7 mL of 10% sulfobutyl-β-cyclodextrin in saline. Vortex for 2 minutes until completely dissolved. Prepare the solution for immediate use.

[0645] Dosing solution for the 1 mg / kg compound 1-2-iv group and the 1 mg / kg compound 1-2-po group: Weigh approximately 3.0 mg of 1-2, 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.

[0646] Dosing solution for the 1 mg / kg compound 67-iv group and the 1 mg / kg compound 67-po group: Weigh approximately 3.0 mg of 67, add 1.5 mL of purified polyoxyethylene 35 castor oil, then add 28.5 mL of normal saline. Vortex for 2 minutes and sonicate until completely dissolved. Prepare the solution for immediate use.

[0647] Dosing solution for the 1 mg / kg compound 69-iv group and the 1 mg / kg compound 69-po group: Weigh approximately 3.0 mg of 69, add 1.5 mL of PEG-400, mix thoroughly, then add 28.5 mL of normal saline. Vortex for 2 minutes and sonicate until completely dissolved. Prepare the solution for immediate use.

[0648] Dosage regimen: The tail vein group received tail vein administration, while the gavage group received gavage administration. See Table 7.

[0649] Table 7

[0650] Rats: All 10 groups of animals were administered a 10 mL / kg dose via tail vein or oral gavage. Blood (0.2 mL) was 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 for analysis.

[0651] 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.

[0652] Pharmacokinetic parameter results: The main PK characteristics of compounds 1-2, 10-2, 67, and 69 in rats were basically consistent with those of the original compound RPL-554, and their oral bioavailability was extremely low. See Tables 8 and 9.

[0653] Table 8

[0654] Table 9

[0655] 8. Human in vitro PK experiment - plasma protein binding rate

[0656] Experimental purpose: To investigate the in vitro human plasma protein binding rate of RPL-554, compound 1-2, compound 10-2, compound 67, and compound 69.

[0657] Test drugs: RPL-554, compound 1-2, compound 10-2, compound 67, compound 69.

[0658] Test plasma: human plasma.

[0659] Incubation conditions: test substance incubation time 5 hours; test substance final incubation concentration 0.5 μM; species human; temperature 37.0°C.

[0660] Test steps:

[0661] 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.

[0662] 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;

[0663] The dosing end and the receiving end were placed at 37°C and 100 rpm for 5 hours;

[0664] Stability test group: the test substance was incubated with plasma or blank buffer for 0 and 5 hours;

[0665] 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;

[0666] 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.

[0667] Pharmacokinetic parameter results: RPL-554, compound 1-2, compound 10-2, compound 67, and compound 69 all exhibited high plasma protein binding in human plasma (see Table 10).

[0668] Table 10

[0669] IX. Human in vitro PK study - CYP450 DDI

[0670] Experimental purpose: To investigate the inhibitory effects of RPL-554, compound 1-2, compound 10-2, compound 67, and compound 69 on CYP3A4, CYP2C9, and CYP2D6.

[0671] Test drugs: RPL-554, compound 1-2, compound 10-2, compound 67, compound 69.

[0672] CYP enzymes: CYP3A4, CYP2C9, CYP2D6.

[0673] 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.

[0674] Test steps:

[0675] Prepare working solutions of substrates and inhibitors of CYP2C9, CYP2D6, and CYP3A4 respectively;

[0676] Prepare NADPH solution and pre-warm it in a water bath shaker at 37°C before use;

[0677] Liver microsomes were thawed and diluted with buffer;

[0678] 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.

[0679] 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.

[0680] 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;

[0681] All samples were mixed and centrifuged at 4000 rpm for 10 min, and the supernatant was analyzed by LC-MS / MS.

[0682] Pharmacokinetic Parameters: At a concentration of 10 μM, RPL-554, compound 1-2, compound 10-2, compound 67, and compound 69 all exhibited some inhibitory activity against CYP2C9, but exhibited less inhibitory activity against CYP2D6. At a concentration of 10 μM, RPL-554 and compound 10-2 had no inhibitory effect on CYP3A4, but compounds 1-2, compound 67, and compound 69 exhibited varying degrees of inhibition. See Table 11.

[0683] Table 11

[0684] 10. Human in vitro PK experiment - liver microsome incubation test

[0685] Experimental purpose: To investigate the in vitro metabolic stability of RPL-554, compound 1-2, compound 10-2, compound 67, and compound 69.

[0686] Test drugs: RPL-554, compound 1-2, compound 10-2, compound 67, compound 69.

[0687] Test system: human liver microsomes.

[0688] 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.

[0689] Test steps:

[0690] Prepare a mixed testosterone and 7-hydroxycoumarin working solution;

[0691] Prepare a mixed NADPH and UDPGA solution and pre-warm it in a water bath shaker at 37°C before use;

[0692] Liver microsomes were thawed and diluted with buffer;

[0693] Add the prepared test substance and liver microsome solution to each well of the incubation plate;

[0694] 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.

[0695] 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;

[0696] All samples were mixed and centrifuged at 4000 rpm for 10 min, and the supernatant was analyzed by LC-MS / MS.

[0697] Pharmacokinetic parameter results: The metabolic types of RPL-554, compound 1-2, compound 10-2, compound 67, and compound 69 are shown in Table 12.

[0698] Table 12

[0699] 11. Human in vitro PK experiment-Caco2 cell permeability test

[0700] Experimental purpose: To investigate the Caco2 cell permeability of RPL-554, compound 1-2, compound 10-2, compound 67, and compound 69.

[0701] Test drugs: RPL-554, compound 1-2, compound 10-2, compound 67, compound 69.

[0702] Test cell line: human colorectal adenocarcinoma cell line.

[0703] Incubation conditions: test system Caco-2; incubation time 2 hours; final test substance concentration 1 μM; temperature 37.0°C.

[0704] Test steps:

[0705] 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;

[0706] 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.

[0707] Note: The A-terminal contains 10 μM Lucifer Yellow, and 0.1% BSA is added to the test buffer.

[0708] Pharmacokinetic parameter results: At a test concentration of 1 μM, the Papp(AB) values ​​of the test substances RPL-554, compound 1-2, compound 10-2, compound 67, and compound 69 were 0.643×10 -6 cm / s, 0.412×10 -6 cm / s, 0.0477×10 -6 cm / s, 0.422×10 -6 cm / s and 0.594×10 -6 cm / s. Based on the permeability criteria, the test objects RPL-554, compound 1-2, compound 10-2, compound 67, and compound 69 are low permeability compounds. See Table 13.

[0709] Table 13

[0710] 12. Effects of Compounds on Isolated Tracheal Relaxation

[0711] 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.

[0712] 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.

[0713] 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.

[0714] 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 -5mol / 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.

[0715] The results are shown in Figure 6 and Table 14. Compared with the positive control group, compound 1-2, compound 10-2, compound 12-1 and compound 19 can effectively alleviate the contraction of guinea pig isolated organ smooth muscle induced by acetylcholine.

[0716] Table 14

[0717] 13. hERG Experimental Evaluation

[0718] The experimental materials are shown in Table 15.

[0719] Table 15

[0720] 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.

[0721] 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 induce the characteristic hERG tail current, which was then recorded. This tail current was used for data acquisition, measurement, and analysis. The experimental results are shown in Table 16.

[0722] Table 16

[0723] Conclusion: RPL-554, compound 1-2 and compound 19 have inhibitory effects on hERG current at a concentration of 10 μM, while the other drugs have no effect on hERG current at concentrations of 1 μM and 10 μM.

[0724] 14. Mutagenicity of Compounds to Salmonella Typhimurium

[0725] The bacterial toxicity and potential mutagenicity of compounds 1-2, 10-2, 12-1, and 19 to Salmonella typhimurium were evaluated respectively to determine the optimal dosage range of the test articles in subsequent Salmonella typhimurium mutagenicity tests.

[0726] Five histidine-deficient Salmonella typhimurium strains, TA97a, TA98, TA100, TA102, and TA1535 (provided by Shao Yan (Suzhou) New Drug Research Center Co., Ltd.), were tested. Under non-metabolic activation and metabolic activation conditions, the test sample was added to each strain at doses of 5000, 1500, 500, 150, and 50 μg / dish, respectively. A vehicle control (DMSO) was also established. For 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 sample or at the end of the incubation period.

[0727] ① Compound 1-2: In this test, bacterial toxicity was observed for strains TA97a and TA100 at a dose of 5000 μg / dish under both metabolic and non-metabolic activation conditions. Toxicity could not be determined for strains TA98, TA102, and TA1535 at a dose of 5000 μg / dish due to precipitation. No significant bacterial toxicity was observed under other conditions. Compound 1-2 showed no mutagenicity against histidine-deficient Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA1535 under both non-metabolic and metabolic activation conditions.

[0728] ② Compound 10-2: In this test, bacterial toxicity was observed against strain TA100 at a dose of 1500 μg / dish of the test article under both metabolic and non-metabolic activation conditions. Due to the effects of precipitation, the number of culture plates containing TA97a, TA98, TA100, TA102, and TA1535 at a dose of 5000 μg / dish of the test article, TA97a, TA98, TA102, and TA1535 at a dose of 1500 μg / dish of the test article, and TA102 at a dose of 500 μg / dish of the test article, the number of culture plates could not be counted, making toxicity indeterminate. No significant bacterial toxicity was observed under the other conditions. Compound 10-2 showed no mutagenicity against histidine-deficient Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA1535 under both non-metabolic and metabolic activation conditions.

[0729] ③ Compound 12-1: In this test, at doses of 1500-5000 μg / dish, the background bacterial lawn of strains TA97a, TA98, TA100, TA102, and TA1535 was unobservable due to precipitation. Counts of strains TA97a, TA98, and TA102 under metabolic activation and non-metabolic activation conditions, and TA100 under metabolic activation conditions, could not be counted due to precipitation, making toxicity impossible to assess. No significant bacterial toxicity was observed under the remaining conditions. Compound 12-1 showed no mutagenicity against histidine-deficient Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA1535 under both non-metabolic and metabolic activation conditions.

[0730] ④ Compound 19: In this test, under both non-metabolic and metabolic activation conditions, background bacterial lawns were obscured in the culture dishes of strains TA97a, TA98, TA100, TA102, and TA1535 at doses of 1500-5000 μg / dish due to precipitation, making toxicity impossible to assess. No significant bacterial toxicity was observed under other conditions. Compound 19 showed no mutagenicity against histidine-deficient Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA1535 under both non-metabolic and metabolic activation conditions.

[0731] 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 D, 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 D, 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 and, n is 0, 1 or 2; k is 0, 1, 2 or 3; 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; Among them, R9, R 10 Each independently selected from H, C 1-6 Straight chain alkyl, C 3-6 Branched alkyl, C 3-6 Cycloalkyl, C 3-6 Heterocycloalkyl, C 6-10 Aryl, C 5-10 heteroaryl and COOCH3, and R9, R 10 are not H at the same time; the heteroaryl group contains 1-3 heteroatoms selected from N, O and S; R6 is selected from Among them, R 11 Selected from amino, C 1-6 Alkoxy, C 3-12 Cycloalkyl, C 6-10 Aryl, C 5-10 A heterocyclic group, wherein the heterocyclic group contains 1-3 heteroatoms selected from N, O and S, and the amino, alkoxy, cycloalkyl, aryl, heteroaryl are optionally further substituted by 0 to 4 atoms selected from H, F, Cl, Br, I, OH, =O, NH2, CN, COOH, CF3, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Substituted by a cycloalkyl substituent; Optionally, R6 forms with L R7 and R8 are each independently selected from H, =O, halogen, NH2, CN, C 1-6 Straight chain alkyl, C 3-6 Branched alkyl, C 3-6 Cycloalkyl and 2. The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein: R1 and R2 are each independently selected from CH3, CHF2, CD3 or C 3-6 Cycloalkyl; Preferably, R1 and R2 are CD3 respectively; Preferably, one of R1 and R2 is CH3, and the other is CHF2.

3. A compound according to claim 1 or 2, or a pharmaceutically acceptable form thereof, wherein: R3, R4, R5 are each independently selected from CH3, i-Pr, OMe, CD3 or halogen; Preferably, R5 is CH3, and the CH3 is optionally further substituted by 0 to 3 D; R3 and R4 are the same and are selected from CH3, i-Pr, OMe or halogen.

4. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable form thereof, wherein: L is selected from one of the following options: L is n is 0 or 1, preferably 0; Preferably, k is 0, 1 or 2; Preferably, R9, R 10 One of them is H, and the other is selected from H, C 1-6 Straight chain alkyl, C 3-6 Branched alkyl, C 3-6 Cycloalkyl, C 3-6 Heterocycloalkyl, C 6-10 Aryl, C 5-10 Heteroaryl and COOCH3; the heteroaryl contains 1 or 2 heteroatoms N; the H in L is optionally further replaced by 0 to 4 atoms 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 L is n is 0 or 1, k is 1; L is n is 0 or 1, k is 1; L is n is 2, k is 2; L is n is 1, k is 1 or 2; or L is n is 2, k is 2.

5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable form thereof, wherein: R6 is selected from one of the following options: R6 is Among them, R 11 is an amino group, which is optionally further substituted by 0, 1 or 2 groups selected from F, Cl, Br, I, OH, =O, NH2, CN, COOH, CF3, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Substituted by a cycloalkyl substituent; R6 is Among them, R 11 C 6-10 Aryl or C 5-10 The heterocyclic group contains 1, 2 or 3 heteroatoms N; the aryl and heteroaryl groups are optionally further substituted by 0, 1, 2 or 3 groups selected from H, F, Cl, Br, I, OH, =O, NH2, CN, COOH, CF3, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 The cycloalkyl group is substituted with a substituent.

6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable form thereof, wherein: R7 is H or CH3, and R8 is H or F.

7. The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein: R1 and R2 are each independently selected from CH3 or CD3; R3, R4, and R5 are CH3 respectively; L is R6 is Among them, R 11 Amino, methoxy, Wherein, the amino group is optionally further substituted by 0 or 1 methyl or cyclopropyl group; Optionally further substituted with 0 or 1 methyl or NH2; R7 and R8 are H respectively.

8. The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein: R1 and R2 are CH3 respectively; R3, R4, and R5 are CH3 respectively; L is R6 is Among them, R 11 For amino, R7 and R8 are H respectively.

9. The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein: The compound or its pharmaceutically acceptable form is selected from one or more of the compounds shown in Table 1.

10. An intermediate compound having a structure shown in Formula II: in, R1, R2, R3, R4, R5, R7, R8 are as defined in any one of claims 1 to 9; L1 is selected from R9, R 10 , n, k as defined in any one of claims 1 to 9; R 12 , R 13 each independently is H, Boc, Cbz, SEM, Fmoc, Alloc, Pht, OTs, PMB, Bn, Trt; Preferably, the intermediate compound is R 12 , R 13 One of them is H, and the other is Boc, Cbz, SEM, Fmoc, Alloc, Pht, OTs, PMB, Bn, Trt.

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: For the intermediate compound of claim 10, R 12 , R 13 The end is subjected to 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.