CN114591352B

CN114591352B - Triazolopyridazine compound and application thereof

Info

Publication number: CN114591352B
Application number: CN202210505202.6A
Authority: CN
Inventors: 王非; 孙勇; 吴金华; 陈南阳
Original assignee: Shanghai Semerode Biotechnology Co ltd; Shanghai Simr Biotechnology Co ltd
Current assignee: Shanghai Semerode Biotechnology Co ltd; Shanghai Simr Biotechnology Co ltd
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2022-09-09
Anticipated expiration: 2042-05-11
Also published as: CN114591352A

Abstract

The invention discloses a triazolopyridazine compound and application thereof. The present invention provides a substance X or a pharmaceutically acceptable salt thereof; the substance X is a triazolopyridazine compound I or a stereoisomer thereof. The compound pair alpha ₅ ‑GABA _A The receptor has the characteristics of good selective reverse regulation activity, bioavailability and the like.

Description

Triazolopyridazine compound and application thereof

Technical Field

The invention relates to a triazolopyridazine compound and application thereof.

Background

Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system of mammals, and two types of GABA receptors exist in nature, one is GABA _A Receptors which are members of the ligand-gated ion channel superfamily, the other class being GABA _B Receptors, which are members of the superfamily of G protein-coupled receptors. GABA in mammals _A The receptor subunits were found to be alpha 1-6, beta 1-4, gamma 1-3, delta, epsilon, theta, and rho 1-3 subunits, among which the alpha, beta, and gamma subunit pairs form an integral functional GABA _A The receptor is essential, and the alpha subunit is terephthal azepine and GABA _A Receptor binding is crucial.

GABA containing alpha 5 _A Receptor (alpha 5-GABA _A Receptor) in mammalian brain _A The receptor accounts for less than 5%, and the expression level in cerebral cortex is very low, but GABA in cerebral hippocampal tissue _A The proportion of receptors is greater than 20%, and other brain regions are hardly expressed. Considering alpha 5-GABA _A Study of receptor specific distribution and function in hippocampal tissues of brain, many pharmaceutical companies including Roche, Merck, etc. have been engaged in alpha 5-GABA _A Research on receptor ligands, and a large number of compounds are synthesized successively, especially for alpha 5-GABA in hippocampal tissues of the brain _A Inverse modulators of the receptor, wherein α 5IA and MRK-016 show good cognitive improvement effects in animal disease models. It is generally accepted that alpha 5-GABA _A Receptor inverse modulators may be used for the treatment of cognitive diseases, in particular Alzheimer's disease (Wallace, T.L. et al, Pharmacology Biochemistry and Behavior, 2011.99 (2): 130-. Patent application US20110224278 discloses alpha 5-GABA _A Inverse modulators of the receptor are useful for the treatment of multi-infarct dementia and stroke related diseases.

Depression is a serious mental illness that can endanger life (e.g., suicide). The current standard therapy for antidepressant treatment is a selective 5-hydroxytryptamine reuptake inhibitor (SSRI), but the drugs can achieve the maximum drug effect after being taken for 6-8 weeks, and not only the maximum drug effect is achieved, but also the effective rate is limited, and the therapeutic effect is achieved on nearly half of patients. Other marketed and clinically under-developed antidepressant drugs also have varying degrees of side effects, limiting the use of antidepressant drugs. Therefore, an antidepressant with a rapid onset and lower side effects is urgently needed in clinic to relieve the burden of depressed patients and society. Fishell et al reported alpha 5-GABA _A Antidepressant effects of receptor inverse modulators (J. Fischell et al, Neuropsychopharmacology, 2015, 40(11), 2499-2509), considered selective for alpha 5-GABA _A Receptor-inverse modulators may have a rapid onset of action, unlike the slow onset of action of traditional antidepressants, and are safer than existing ketamine therapies with the same rapid onset of action. Thus selective alpha 5-GABA _A The receptor reversal regulator is possible to become a brand new mechanism of anti-depression therapy with quick effect, and meets the serious unmet requirements of the existing clinical depression treatment.

Detecting whether a compound is directed against GABA comprising an alpha 5 subunit _A Inverse modulators or antagonists of the receptor, for which much research has been done, for example in the international applications WO 1992022652 and WO1994013799, using GABA _A The combination of α 5, β 3 and γ 2 of the receptor to detect whether a compound binds to the receptor; in the course of performing drug screening, Goeders et al (Goeders, N.E. and Kuhar, M.J.) are commonly used.Life Sci.1985, 37(4), 345- & 355). Detection of an energy and GABA _A The study of whether a ligand bound by the α 5 subunit of the receptor is an antagonist, agonist or inverse modulator is also numerous and reference can be made to waford et al (Wafford, k. a., Whiting, p.j. and Kemp, j. a).Mol. Pharmacol. 1993, 43, 240-.

Recent research results show that GABA _A Receptors mediate at least 2 modes of inhibition, phasic inhibition and tonic inhibition. Intrasynaptic GABA _A The receptor causes the GABA after the synapse to be increased sharply due to the synchronous release of vesicles containing GABA in the synapse caused by action potential, so that the GABA concentration in millimole level appears in the synaptic cleft _A Simultaneous activation and rapid desensitization of the receptor results in a phase-type inhibition. And located extrasynaptic GABA _A Receptors, usually in low concentrations of tens of nanomolar to several micromolar, that persist _A High affinity GABA in the environment _A The receptor is continuously activated asynchronously, forming a tonic type of suppression. Both phase-type and tonic-type inhibition regulate neuronal excitability and signaling. (Farrant, M. et al, Nat Rev Neurosci, 2005, 6, 215-. Yeung JY et al discloses that low concentrations of GABA more readily activate alpha 5-GABA _A Receptor (Yeung JY et al.Mol Pharmacol, 2003, 63, 2-8). K.y. Lee reports that sustained high affinity GABA currents of low concentration GABA activation were detected on isolated DRG cells cultured for 24 hours. (Lee, K.Y. et al,Neuroscience2012, 208, 133-142). 2013I, Lecker et al disclose alpha 5-GABA _A The receptor inverse modulator L-655,708 dose-dependently inhibited the current induced by low concentrations of GABA (5, 50 and 500 nM), with the highest concentration of L-655,708 inhibiting only 15% of the current when the GABA concentration was increased to 1 μ M and with L-655,708 having no inhibitory effect on the current induced by GABA when the GABA concentration continued to increase. (the power of the light source in the Lecker, i. et al,British Journal of Anaesthesia, 2013, 110 (S1), i73-i81)。

disclosure of Invention

The technical problem to be solved by the invention is to solve the existing alpha pair ₅ -GABA _A The receptor has a single structure of a compound with selective reverse regulation activity, so the invention provides a triazolopyridazine compound and application thereof. The compound pair alpha ₅ -GABA _A The receptor has the characteristics of good selective reverse regulation activity, bioavailability and the like.

The present invention provides a substance X or a pharmaceutically acceptable salt thereof; the substance X is a triazolopyridazine compound I or a stereoisomer thereof;

wherein R is ¹ Is as a quilt R ^1-2 A substituted 5-to 6-membered heteroaryl; in the 5-6 membered heteroaryl, the number of heteroatoms is 1,2, 3 or 4, and the heteroatoms are selected from one or more of N, O and S;

R ^1-2 independently is C ₁ ~C ₆ An alkyl group;

R ² is hydrogen, R ³ Is C ₁ ~C ₆ An alkyl group; r ^X Is composed of

M and n are independently 1 or 2, and m + n =2 or 3, Z ¹ is-N (R) ^4-1 )-，R ^4-1 Independently a 4-membered heterocycloalkyl group; in the 4-membered heterocycloalkyl group, the number of heteroatoms is 1, and the heteroatoms are selected from the group consisting of N, O, S and-S (= O) ₂ -one or more of;

or, R ² 、R ³ Together with the carbon atom to which they are attached form a benzene ring, R ^X Is composed of

Ring a is 6-membered heteroaryl; in the 6-membered heteroaryl, the number of heteroatoms is 1 or 2, and the heteroatoms are N.

In one embodiment, in the substance X or a pharmaceutically acceptable salt thereof, some groups of the triazolopyridazine compound I are defined as follows, and the remaining groups are defined as described in any one of the other embodiments (hereinafter referred to as "in one embodiment"):

R ¹ in the 5-to 6-membered heteroaryl group, the number of heteroatoms may be 2 or 3.

In a certain embodiment, R ¹ In (A), theThe hetero atom in the 5-to 6-membered heteroaryl group of (a) may be one or more selected from N and O.

In a certain embodiment, R ^1-2 In (b), the C ₁ ~C ₆ The alkyl group may be C ₁ ~C ₃ Alkyl, in turn, can be methyl, ethyl, n-propyl or isopropyl.

In a certain embodiment, R ¹ (may be)

。

In a certain embodiment, R ³ In (b), the C ₁ ~C ₆ The alkyl group may be C ₁ ~C ₄ Alkyl, in turn, can be methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl.

In a certain embodiment, R ² Can be hydrogen, R ³ (may be)

(ii) a Or, R ² 、R ³ Together with the carbon atoms to which they are attached form a benzene ring.

In one embodiment, the number of heteroatoms in the 6-membered heteroaryl group in ring a may be 1.

In one embodiment, the heteroatom in the 6 membered heteroaryl in ring a may be N.

In one embodiment, the 6-membered heteroaryl in ring a can be pyridyl.

In one embodiment, all of the atoms in substance X or a pharmaceutically acceptable salt thereof are atoms that comprise isotopes at natural ratios.

The invention also provides a substance X or a pharmaceutically acceptable salt thereof; the substance X is a triazolopyridazine compound I or a stereoisomer thereof; the triazolopyridazine compound I is of any one of the following structures:

。

the invention also provides a pharmaceutical composition comprising substance Y and a pharmaceutical excipient;

the substance Y is the substance X or pharmaceutically acceptable salt thereof.

The invention also provides a method for preparing alpha 5-GABA by using the substance Y _A Use in receptor inverse modulators;

the substance Y is the substance X or pharmaceutically acceptable salt thereof.

In said use, said alpha 5-GABA _A The receptor inverse modulator may be alpha 5-GABA for use in vivo or in vitro _A A receptor inverse modulator.

In said use, the α 5-GABA is present relative to α 1 and/or α 2 _A The receptor inverse modulator may be selective.

In said use, said α 5-GABA is relative to α 2 _A The receptor inverse modulator may be selective.

The invention also provides the application of the substance Y in preparing medicaments;

the substance Y is the substance X or pharmaceutically acceptable salt thereof;

the medicine is used for preventing or treating alpha 5-GABA _A A medicament for a receptor mediated disease.

In said use, the medicament can act on alpha 5-GABA relative to alpha 1 and/or alpha 2 _A The receptor is selective.

In such applications, the drug may be selective with respect to α 2.

In said application, said compound is prepared from alpha 5-GABA _A The receptor-mediated disorder can be depression, pain, Alzheimer's disease, multi-infarct dementia, stroke, anxiety, panic disorder, agoraphobia, post-traumatic stress disorder, premenstrual dysphoric disorder, attention deficit disorder, obsessive compulsive disorder, autism, schizophrenia, obesity, bulimia or deficiency, Tourette's syndrome, vasomotor flushing, sexual dysfunction, borderline personality disorder, chronic fatigue syndrome, Raynaud's syndrome, stroke, anxiety, panic disorder, stress disorder, stress disorder, stress disorder, stress disorder, stress disorder, stress disorder, stress disorderNori syndrome, parkinson's disease, or, epilepsy and mood disorders following head injury.

In said application, said compound is prepared from alpha 5-GABA _A The receptor-mediated disease may be depression.

In the use, the depression may be bipolar depression, postpartum depression, major depression, dysthymia, atypical depression, melancholia, treatment resistant depression, huntington's disease related depression, multiple sclerosis related depression or anxiety related depression.

In the use, the depression can be mild depression, moderate depression or major depression.

In said use, said anxiety disorder may be generalized anxiety disorder or social anxiety disorder.

In said use, said pain may be fibromyalgia.

the substance Y is the substance X or pharmaceutically acceptable salt thereof;

the medicine is a medicine for preventing or treating any one of the following diseases:

depression, pain, alzheimer's disease, multi-infarct dementia, stroke, anxiety disorders, panic disorder, agoraphobia, post-traumatic stress disorder, premenstrual dysphoric disorder, attention deficit disorder, obsessive compulsive disorder, autism, schizophrenia, obesity, bulimia nervosa or deficiency, tourette's syndrome, vasomotor flushing, sexual dysfunction, borderline personality disorder, chronic fatigue syndrome, raynaud's syndrome, parkinson's disease, and mood disorders following epilepsy and head injury.

In the application, the medicament is a medicament for preventing or treating depression.

In said use, said pain may be fibromyalgia.

Definition of

The following terms used in the present invention are intended to have the following definitions, unless otherwise indicated. A particular term should not be considered as unclear unless it is specifically defined, but rather construed according to ordinary meaning.

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

The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from the compounds of the present invention found to have particular substituents, with relatively nontoxic acids or bases. When compounds of the invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the free forms of such compounds with a sufficient amount of a base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium calcium, ammonium, organic amines or magnesium salts or similar salts. When compounds of the present invention contain relatively basic functional groups, base addition salts can be obtained by contacting the free forms of such compounds with a sufficient amount of an acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include salts with inorganic acids including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like, as well as salts with organic acids including similar acids 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, methanesulfonic acid, and the like; also included are salts of amino acids such as arginine and the like, and salts of organic acids such as glucuronic acid and the like. Certain specific compounds of the invention contain both basic and acidic functionalities and can thus be converted to either base or acid addition salts.

The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains an acid or base, by conventional chemical methods. In general, such salts are prepared by the following method: prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid, in water or an organic solvent or a mixture of the two.

“

By "is meant that the group is attached to the rest of the compound through this site.

The term "stereoisomers" includes the cis and trans isomers, (-) -and (+) -enantiomers, (R) and (C)R) -and (a)S) Enantiomers, diastereomers, (D) The isomers (A), (B), (C) and C)L) -isomers. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups.

The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds, said isotopes having the same number of atoms, but differing in atomic mass or mass number from the atomic mass or mass number predominantly found in nature. For example, the compounds may be labelled with radioisotopes, such as deuterium (g) ((R)) ² H) Tritium (a) ³ H) Iodine-125 ( ¹²⁵ I) Or C-14 ( ¹⁴ C) In that respect All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention. By conventional techniques well known to those skilled in the art, or by routes consistent with the present inventionIsotopically enriched compounds within formula (I) can be prepared by processes analogous to those described in the examples using appropriate isotopically enriched reagents and/or intermediates without undue experimentation.

The naming convention used in the present invention is based on IUPAC systematic naming, generated by ChemDraw software. Any open valency appearing on a carbon, oxygen, sulfur or nitrogen atom in the structures given in this invention indicates the presence of a hydrogen atom.

The term "substituted with … …" means that any one or more hydrogen atoms on a particular atom are replaced with a substituent, provided that the valency of the particular atom is normal and the substituted compound is stable. The number of substituents is 1,2 or 3.

When any variable (e.g., R) occurs more than one time in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0-2R, the group may optionally be substituted with up to two R, and there are separate options for R in each case. Furthermore, combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

When the number of one linking group is 0, e.g. - (CRR) ₀ -, represents that the linking group is a single bond. When one of the variables is selected from a single bond, it means that the two groups to which it is attached are directly connected, for example when L represents a single bond in A-L-Z, it means that the structure is actually A-Z.

When the direction of attachment of the linking group is not indicated in the list, the direction of attachment is arbitrary, for example,

wherein the linking group L is

At this time

Can be in the same order as the reading from left to rightIn the same direction connecting benzene ring and cyclopentyl

Alternatively, the phenyl group and the cyclopentyl group may be linked in the reverse order of reading from left to right

. Combinations of the linking groups, substituents, and/or variants thereof are permissible only if such combinations result in stable compounds.

The number of atoms in a ring is generally defined as the number of ring members, e.g., "3-7 membered ring" means a "ring" around which 3-7 atoms are arranged.

The term "halogen" refers to fluorine, chlorine, bromine and iodine.

The term "C _1- C ₆ Alkyl "is used to denote a straight or branched saturated hydrocarbon group consisting of 1 to 6 carbon atoms. Said C is _1- C ₆ The alkyl group comprising C _1-5 、C _1-4 、C _1-3 、C _1-2 、C _2-6 、C _2-4 、C ₆ And C ₅ Alkyl groups and the like; it may be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). C _1- C ₆ Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (includingn-a butyl group, an isobutyl group,s-butyl andt-butyl), pentyl (includingnPentyl, isopentyl and neopentyl), hexyl and the like.

The term "C _1- C ₃ Alkyl "is used to denote a straight or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. Said C is _1- C ₃ The alkyl group comprising C _1- C ₂ And C _2- C ₃ Alkyl groups and the like; it may be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). C _1- C ₃ Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

The term "C _1- C ₆ Alkoxy "refers to those alkyl groups containing 1 to 6 carbon atoms that are attached to the rest of the molecule through an oxygen atom. Said C is _1- C ₆ The alkyl group comprising C _1- C ₄ 、C _1-3 、C _1-2 、C _2-6 、C _2-4 、C ₆ 、C ₅ 、C ₄ And C ₃ Alkoxy, etc.; c _1- C ₆ Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (includingn-a butoxy group, an isobutoxy group,s-butoxy andt-butoxy), pentyloxy (includingnPentoxy, isopentoxy and neopentoxy), hexoxy, and the like.

The term "C _1- C ₃ Alkoxy "denotes those alkyl groups containing 1 to 3 carbon atoms which are attached to the rest of the molecule through an oxygen atom. Said C is _1- C ₆ The alkyl group comprising C _1- C ₂ And C _2- C ₃ Alkoxy, etc.; c _1- C ₃ Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.

The term "C _3- C ₆ Cycloalkyl "or" 3-6 membered cycloalkyl "represents a saturated cyclic hydrocarbon group consisting of 3 to 6 carbon atoms, which is a monocyclic ring system, C _3-6 Cycloalkyl radicals including C _3-5 、C _4-5 And C _5-6 Cycloalkyl groups and the like; it may be monovalent, divalent or polyvalent. C _3-6 Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

The term "3-6 membered heterocycloalkyl" by itself or in combination with other terms denotes a saturated monocyclic group consisting of 3 to 6 ring atoms, 1,2, 3 or 4 of which are heteroatoms independently selected from O, S and N, the remainder being carbon atoms. Furthermore, with respect to the "3-6 membered heterocycloalkyl", the heteroatom may occupy the position of the heterocycloalkyl linkage to the rest of the molecule. The 3-6 membered heterocycloalkyl group includes 4-6 membered, 5-6 membered, 4-membered, 5-membered and 6-membered heterocycloalkyl groups and the like, and examples of the 3-6 membered heterocycloalkyl group include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl, tetrahydrofuranyl (including tetrahydrofuran-2-yl), piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl and the like.

The term "6-10 aryl" denotes a monovalent aromatic carbocyclic ring system containing 6-10 carbon atoms and each ring being aromatic. Examples of aryl groups are, but not limited to, phenyl, naphthyl, and the like.

The term "5-6 membered heteroaryl" denotes a compound consisting of 5 to 6 ring atoms having conjugationπCyclic group of an electron system, 1,2, 3 or 4 ring atoms of which are heteroatoms independently selected from O, S and N, the remainder being carbon atoms. The 5-6 membered heteroaryl group can be attached to the rest of the molecule through a heteroatom or carbon atom, and includes 5-and 6-membered heteroaryl groups, and the like. Examples of the 5-6 membered heteroaryl group include, but are not limited to, pyrrolyl (includingNPyrrolyl, 2-pyrrolyl, 3-pyrrolyl and the like), pyrazolyl (including 2-pyrazolyl, 3-pyrazolyl and the like), imidazolyl (includingNAn imidazolyl group, a 2-imidazolyl group, a 4-imidazolyl group, a 5-imidazolyl group and the like), an oxazolyl group (including a 2-oxazolyl group, a 4-oxazolyl group, a 5-oxazolyl group and the like), a triazolyl group (1)H-1, 2, 3-triazolyl, 2H-1, 2, 3-triazolyl, 1H-1, 2, 4-triazolyl and 4H-1, 2, 4-triazolyl), tetrazolyl, isoxazolyl (including 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, and the like), thiazolyl (including 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, and the like), furyl (including 2-furyl, 3-furyl, and the like), thienyl (including 2-thienyl, 3-thienyl, and the like), pyridyl (including 2-pyridyl, 3-pyridyl, 4-pyridyl, and the like), pyrazinyl, pyrimidinyl (including 2-pyrimidinyl, 4-pyrimidinyl, and the like), and the like.

Unless otherwise specified, C _n-n+m Or C _n -C _n+m Including any one particular case of n to n + m carbons, e.g. C _1- C ₇ Comprising C ₁ 、C ₂ 、C ₃ 、C ₄ 、C ₅ 、C ₆ And C ₇ Also included are any ranges of n to n + m, e.g. C _1-7 Comprising C _1-3 、C _1-6 、C _3-6 、C _4-7 And C _5-7 Etc.; similarly, n-to n + m-members represent n to n + m ring atoms, and for example, 3-to 7-membered rings include 3-to 4-to 5-to 6-and 7-membered rings, and any range of n to n + m, for example, 3-to 7-membered rings include 3-to 6-membered rings, 4-to 7-membered rings, 5-to 7-membered rings, 6-to 7-membered rings, and the like.

The term "treatment" refers to the administration of one or more pharmaceutical substances, in particular the compounds of formula (I) according to the invention and or pharmaceutically acceptable salts thereof, to an individual suffering from a disease or having symptoms of said disease, for the cure, alleviation, modification, cure, amelioration, improvement or influence of said disease or symptoms of said disease.

The term "prevention" refers to the administration of one or more pharmaceutical substances, in particular the compounds of formula (I) according to the invention and or their pharmaceutically acceptable salts, to an individual having a predisposition to the disease, in order to prevent the individual from suffering from the disease.

The terms "treating", "contacting" and "reacting" when referring to a chemical reaction, refer to the addition or mixing of two or more reagents under appropriate conditions to produce the indicated and or desired product it is to be understood that the reaction that produces the indicated and/or desired product may not necessarily result directly from the combination of the two reagents that were initially added, i.e., there may be one or more intermediates in the mixture that are produced that ultimately result in the formation of the indicated and or desired product.

As used herein, "patient" is defined as any warm-blooded animal, such as, but not limited to, a mouse, guinea pig, dog, horse or human, with the patient preferably being a human.

The term "effective amount" as used herein refers to an amount generally sufficient to produce a beneficial effect in an individual. An effective amount of a compound of the invention can be determined by conventional methods (e.g., modeling, dose escalation studies, or clinical trials) in combination with conventional influencing factors (e.g., mode of administration, pharmacokinetics of the compound, severity and course of the disease, medical history of the individual, health of the individual, degree of responsiveness of the individual to the drug, etc.).

Technical and scientific terms used herein that are not specifically defined have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The solvent used in the present invention can be commercially available. The invention employs the following abbreviations: ac for acetyl, ACN for acetonitrile, B2pin2 for bis-pinacolato borate, CbzCl for benzyl chloroformate, DAST for diethylaminosulfur trifluoride, DCDMH for dichlorohydantoin, DCM for dichloromethane, DEAD for diethyl azodicarboxylate, DIBAL-H for diisobutylaluminum hydride, DIEA for diisopropylethylamine, DMAP for 4-dimethylaminopyridine, DMF for N, N-dimethylformamide, DMSO for dimethyl sulfoxide, EDCI for 1-ethyl- (3-dimethylaminopropyl) carbodiimidate hydrochloride, HATU for 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, HOBt for 1-hydroxybenzotriazole, HPLC for high performance liquid chromatography, MDS for bis (trimethylsilyl) amino potassium, MDS for bis (trimethylsilyl) amide, LAH for lithium aluminium hydride, MsCl for methanesulfonyl chloride, NCS for chlorosuccinimide, Pd (dppf) Cl ₂ Represents 1, 1' -bis-diphenylphosphino ferrocene palladium dichloride and Pd ₂ (dba) ₃ Represents tris-dibenzylidene acetone dipalladium and PhNTf ₂ Represents N-phenylbis (trifluoromethanesulfonimide), PMBCl represents p-methoxybenzyl chloride, pyr represents pyridine, Ruphos represents 2-dicyclohexylphos-2 ', 6' -diisopropoxy-1, 1' -biphenyl, Ruphos-Pd-G3 represents methanesulfonic acid (2-dicyclohexylphosphino-2 ', 6' -diisopropoxy-1, 1' -biphenyl) (2-amino-1, 1' -biphenyl-2-yl) palladium (II), TBAF represents tetrabutylammonium fluoride, TBSCl represents tert-butyldimethylchlorosilane, THF represents tetrahydrofuran, TEA represents triethylamine, TfF represents triethylamine trihydrofluoride, Tf represents trifluoromethanesulfonyl, TFA represents trifluoroacetic anhydride, TLC represents thin-layer chromatography, TMS represents trimethylsilyl, TMA represents trimethylsilyl, Tol. stands for toluene, Ts for p-toluenesulfonyl, Xantphos for 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene, LCMS for liquid chromatography-mass spectrometry, h for hour, min for minute.

The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the compound pair alpha ₅ -GABA _A The receptor has the characteristics of good selective reverse regulation activity, bioavailability and the like.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.

Intermediate 2

3- (7- (tert-butyl) -6-chloro- [1,2, 4)]Triazolo [4,3-b ] s]Pyridazin-3-yl) -5-methylisoxazoles

4- (tert-butyl) -3, 6-dichloropyridazine (3g, 14.6mmol, WO9967245A1) and 5-methylisoxazole-3-carbohydrazide (2.1g, 14.6mmol) were added to n-butanol (30mL) and the reaction mixture 120 ^o C stirring for 16 hours. The reaction was cooled to room temperature, and the precipitate was collected by filtration and dried in vacuo to give the title compound as a white solid (1.4g, yield 33%). ¹ H NMR (400MHz, CDCl ₃ ) δ 8.20 (s, 1 H), 6.86 (s, 1 H), 2.58 (s, 3 H), 1.57 (s, 9 H)；LC-MS: m/z [M-55] ⁺ =292.

Intermediate 4

3- (6-chloro- [1,2, 4)]Triazolo [3,4-a]Phthalazin-3-yl) -5-methylisoxazole

Intermediate 4 was synthesized according to the method described in patent WO2005041971a 1.

Intermediate 38

2- (((((7- (tert-butyl) -3- (5-methylisoxazol-3-yl)) - [1,2, 4)]Triazolo [4,3-b]Pyridazine-6- Yl) oxy) methyl) -5H-pyrrolo [3,4-b]Pyridine-6 (7H) -carboxylic acid tert-butyl ester

2- (hydroxymethyl) -5, 7-dihydro-6H-pyrrolo [3, 4-b)]Tert-butyl pyridine-6-carboxylate (386mg, 1.55mmol, WO2021121294A1) was dissolved in anhydrous tetrahydrofuran (5mL), 60% sodium hydride (74mg, 3.1 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. Then adding 3- (7- (tert-butyl) -6-chloro- [1,2, 4%]Triazolo [4,3-b]Pyridazin-3-yl) -5-methylisoxazole (300mg, 1.03mmol) and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched by dropwise addition of an appropriate amount of 1M aqueous sodium hydroxide solution, and the mixture was concentrated and purified by preparative thin layer chromatography (dichloromethane/methanol =30/1) to give the title compound (500mg, yield 96%). LC-MS M/z [ M + H ]] ⁺ =506.

3- (7- (tert-butyl) -6- (((6, 7-dihydro-5H-pyrrolo [3, 4-b))]Pyridin-2-yl) methoxy) - [1,2,4] Triazolo [4,3-b]Pyridazin-3-yl) -5-methylisoxazoles

Mixing 2- ((((7- (tert-butyl) -3- (5-methyl isoxazol-3-yl)) - [1,2, 4%]Triazolo [4,3-b]Pyridazin-6-yl) oxy) methyl) -5H-pyrrolo [3,4-b]Pyridine-6 (7H) -carboxylic acid tert-butyl ester (500mg, 0.99mmol) was added to a mixed solvent of dichloromethane/methanol (5mL/2mL), followed by trifluoroacetic acid (4mL), and the mixture was stirred at room temperature overnight. The reaction was concentrated directly, and the residue was dissolved in methanol (5mL), followed by addition of macroporous resin (2g) and stirring for 20 minutes. The mixture was filtered and the filtrate was concentrated to give the title compound (300mg, yield 75%). LC-MS M/z [ M + H ]] ⁺ =406.

Referring to the following table, the following intermediates 34a and 37a were prepared with reference to the preparation of intermediate 38 except that the starting materials described in the column "starting materials" were used instead of the corresponding starting materials.

Intermediate 46

2- (((((7- (tert-butyl) -3- (5-methylisoxazol-3-yl)) - [1,2, 4)]Triazolo [4,3-b]Pyridazine-6- Yl) oxy) methyl) -7, 8-dihydro-1, 6-naphthyridine-6 (5H) -carboxylic acid tert-butyl ester

2- (hydroxymethyl) -7, 8-dihydro-1, 6-naphthyridine-6 (5H) -carboxylic acid tert-butyl ester (408mg, 1.55mmol, WO2016014463A1) was dissolved in tetrahydrofuran (5mL), 60% sodium hydride (74mg, 3.1 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. Then 3- (7- (tert-butyl) -6-chloro- [1,2,4 ] is added]Triazolo [4,3-b]Pyridazin-3-yl) -5-methylisoxazole (intermediate 2, 300mg, 1.03mmol) and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched by dropwise addition of an appropriate amount of 1M aqueous sodium hydroxide solution, and the reaction mixture was directly concentrated and purified by preparative thin layer chromatography (dichloromethane/methanol =30/1) to give the title compound (320mg, yield 60%). LC-MS M/z [ M + H ]] ⁺ =520.

Intermediate 48

3- (7- (tert-butyl) -6- (((5,6,7, 8-tetrahydro-1, 6-naphthyridin-2-yl) methoxy)) - [1,2,4]A triazole [4 ] or a triazole derivative, 3 -b]pyridazin-3-yl) -5-methylisoxazoles

2- (((((7- (tert-butyl) -3- (5-methylisoxazol-3-yl)) - [1,2, 4)]Triazolo [4,3-b]Pyridazin-6-yl) oxy) methyl) -7, 8-dihydro-1, 6-naphthyridine-6 (5H) -carboxylic acid tert-butyl ester (intermediate 46, 320mg, 0.616mmol) was dissolved in methanol (5mL), followed byTrifluoroacetic acid (2mL) was added and the mixture was stirred at room temperature overnight. The reaction was concentrated directly, and the residue was dissolved in methanol (5mL), followed by addition of a macroporous resin (2g) and stirring for twenty minutes, filtration, and concentration of the filtrate to give the title compound (300 mg). LC-MS M/z [ M + H ]] ⁺ =420.

Intermediate 55

6-Ethyl-2- (hydroxymethyl) -7, 8-dihydro-1, 6-naphthyridin-5 (6H) -one

(6-Ethyl-5, 6,7, 8-tetrahydro-1, 6-naphthyridin-2-yl) methanol (200mg, 1.02mmol) was dissolved in a mixed solvent of tetrahydrofuran/water (2.5/1, 40mL), and sodium hydrogencarbonate (900mg, 10.2mmol) and iodine (2g, 7.5mmol) were added in this order to stir the mixture at room temperature overnight. Neutralization with sodium thiosulfate to remove the color, extraction with dichloromethane multiple times, combination of the organic phases, concentration, and column chromatography gave the title compound as a colorless oil (60mg, 29% yield). LC-MS M/z [ M + H ]] ⁺ =207.

Intermediate 63

(6-Ethyl-6, 7-dihydro-5H-pyrrolo [3, 4-b)]Pyridin-2-yl) methanol

Intermediate 63 was synthesized according to the method described in patent WO2021121294a 1.

Intermediate 64

4- (6- ((((3- (5-methylisoxazol-3-yl) - [1,2,4 ]))]Triazole [3,4-a ]]Phthalazin-6-yl) oxy]First of all 1, 3-dihydro-2H-pyrrolo [3, 4-c) -yl]Pyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester

3- (6- ((2, 3-dihydro-1H-pyrrolo [3, 4-c)]Pyridin-6-yl) methoxy) - [1,2,4]Triazolo [3,4-a]Phthalazin-3-yl) -5-methylisoxazole (intermediate 37a, 50mg, 0.125mmol) was added to a mixed solvent of dichloromethane (2mL) and methanol (1mL), sodium cyanoborohydride (24mg, 0.375mmol) and tert-butyl 4-oxopiperidine-1-carboxylate (74mg, 0.375mmol) were added in portions, and the mixture was stirred at room temperature for half an hour. The reaction was quenched by the addition of 1M aqueous sodium hydroxide solution, extracted three times with dichloromethane (20mL), the organic phases were combined, washed three times with water (10mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the title compound (73mg, yield 100%). LC-MS M/z [ M + H ]] ⁺ =583.

5-methyl-3- (6- ((2- (piperidin-4-yl) -2, 3-dihydro-1H-pyrrolo [3, 4-c)]Pyridin-6-yl) methoxy Base) - [1,2,4]Triazolo [3,4-a]Phthalazin-3-yl) isoxazoles

4- (6- ((((3- (5-methylisoxazol-3-yl) - [1,2,4 ]))]Triazole [3,4-a ]]Phthalazin-6-yl) oxy]Methyl) -1, 3-dihydro-2H-pyrrolo [3,4-c]Pyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester (73mg, 0.125mmol) was dissolved in a mixed solvent of dichloromethane (5mL) and methanol (1mL), an ethyl hydrogen chloride solution (1mL) was added, and the mixture was stirred at room temperature for 1 hour. The reaction was concentrated directly, the residue was dissolved in methanol (5mL), the mixture was stirred for 16 hours after addition to a macroporous resin, the mixture was filtered, and the filtrate was concentrated to give the title compound (60mg, yield 99%). LC-MS M/z [ M + H ]] ⁺ =483.

Example 1

Method A

3- (7- (tert-butyl) -6- ((6-methyl-6, 7-dihydro-5H-pyrrolo [3, 4-b)]Pyridin-2-yl) methoxy) - [1,2,4]Triazolo [4,3-b]Pyridazin-3-yl) -5-methylisoxazoles

3- (7- (tert-butyl) -6- (((6, 7-dihydro-5H-pyrrolo [3, 4-b))]Pyridin-2-yl) methoxy) - [1,2,4]Triazolo [4,3-b]Pyridazin-3-yl) -5-methylisoxazole (100mg, 0.25mmol, intermediate 38) was dissolved in methanol (1.5mL), sodium cyanoborohydride (46mg, 0.75mmol) was added and the mixture stirred at room temperature for 10 minutes, then 37% aqueous formaldehyde (1mL) was added and the reaction mixture stirred at room temperature for 3 hours. The reaction was quenched by the addition of an appropriate amount of 1M aqueous sodium hydroxide solution, extracted three times with dichloromethane (20mL), the organic phases were combined, washed three times with water (10mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the title compound (50mg, yield 48%). ¹ HNMR (400MHz, CDCl ₃ ) δ 7.96 (s, 1 H), 7.57 - 7.52 (m, 1 H), 7.52 - 7.47 (m, 1 H), 6.84 (s, 1 H), 5.65 (s, 2 H), 3.98 (d, J = 6.8 Hz, 4 H), 2.63 (s, 3 H), 2.58 (s, 3 H), 1.45 (s, 9 H)；LC-MS: m/z [M+H] ⁺ =420.

Example 6

Method B

3- (7- (tert-butyl) -6- ((6- (1-ethylpyrrolidin-3-yl) pyridin-2-yl) methoxy) - [1,2,4]Triazole compounds And [4,3-b ]]Pyridazin-3-yl) -5-methylisoxazoles

Reacting 3- (7- (tert-butyl) -6-chloro- [1,2, 4)]Triazolo [4,3-b]Pyridazin-3-yl) -5-methylisoxazole (100mg, 0.34mmol), (6- (1-ethylpyrrolidin-3-yl) pyridin-2-yl) methanol (60mg, 0.34mmol) and cesium carbonate (225mg, 0.7mmol) were added successively to acetonitrile (5mL) and the mixture was reacted overnight at 50 ℃. The reaction was added to water (20mL), extracted three times with dichloromethane (20mL), dried, concentrated, and purified by preparative thin layer chromatography (dichloromethane/methanol =10/1) to give the title compound as a white solid (5mg, yield 4%). ¹ HNMR (400MHz, DMSO-d6) δ 8.15 (s, 1 H), 7.86 (t, J=7.58 Hz, 1 H), 7.55 (d, J=7.83 Hz, 1 H), 7.40 (d, J=7.34 Hz, 1 H), 6.97 (s, 1 H), 5.61 (s, 2 H), 3.68 - 3.78 (m, 1 H), 3.59 (br. s., 2 H), 3.15 - 3.24 (m, 2 H), 3.06 (d, J=5.87 Hz, 2 H), 2.56 (s, 3 H), 2.37 (dd, J=12.72, 5.87 Hz, 1 H), 2.03 - 2.13 (m, 1 H), 1.35 -1.53 (m, 9 H), 1.20 (t, J=7.09 Hz, 3 H)；LC-MS: m/z [M+H] ⁺ =462.

Example 23

Method E

3- (7- (tert-butyl) -6- (((2-methyl-2, 3-dihydro-1H-pyrrolo [3, 4-c))]Pyridin-6-yl) methoxy) - [1,2,4 ]Triazolo [4,3-b]Pyridazin-3-yl) -5-methylisoxazole hydrochloride

3- (7- (tert-butyl) -6- ((2, 3-dihydro-1H-pyrrolo [3, 4-c)]Pyridin-6-yl) methoxy) - [1,2,4]Triazolo [4,3-b]Pyridazin-3-yl) -5-methylisoxazole (60mg, 0.15mmol) was dissolved in a mixed solvent of dichloromethane and methanol (1mL/1mL), sodium cyanoborohydride (30mg, 0.45mmol) and 37% formaldehyde solution (40mg, 0.45mmol) were added in portions, and the mixture was stirred at room temperature for half an hour. The reaction mixture was quenched by addition of 1M aqueous sodium hydroxide solution, extracted three times with an appropriate amount of dichloromethane, the organic phases were combined, 4M ethyl hydrogen chloride solution (0.2mL) was added, and the precipitated solid was collected by filtration and dried to give the title compound (24mg, yield 32%). ¹ HNMR (400MHz, DMSO-d6) δ 8.48 (br. s., 1 H), 8.12 (br. s., 1 H), 7.53 (br. s., 1 H), 6.97 (br. s., 1 H), 5.57 (br. s., 2 H), 3.85 (d, J=9.29 Hz, 5 H), 2.55 (br. s., 4 H), 1.39 (br. s., 10 H)；LC-MS: m/z [M+H] ⁺ =420.

Example 46

Method H

3- (6- ((6-ethyl-6, 7-dihydro-5H-pyrrolo [3, 4-b)]Pyridin-2-yl) methoxy) - [1,2,4]Triazole compounds And [3,4-a ]]Phthalazin-3-yl) -5-methylisoxazole

Reacting (6-ethyl-6, 7-dihydro-5H-pyrrole [3, 4-b)]Pyridin-2-yl) methanol (intermediate 63, 38mg, 0.21mmol) was dissolved in anhydrous tetrahydrofuran (1mL), 0 ^o Lithium bis (trimethylsilyl) amide (1M, 0.21mL, 0.21mmol) was added dropwise under C, maintaining 0 ^o Stirring for 30 minutes under the condition of C. Then 3- (6-chloro- [1,2,4 ] is added]Triazole [3,4-a ]]Phthalazin-3-yl) -5-methylisoxazole (intermediate 4, 61mg, 0.21mmol), the mixture was stirred at room temperature overnight. Diluting with water, extracting with dichloromethane multiple times, combining organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, concentrating, and separating by column chromatography (dichloromethane: methanol =100:1 to 20:1) to obtain the title compound (14mg, yield 15%) as a pale yellow solid. ¹ HNMR (400MHz, DMSO-d6) δ 8.59-8.57 (m, 1H), 8.32-8.30 (m, 1H), 8.14-8.10 (m, 1H), 7.99-7.97 (m, 1H), 7.74-7.73 (m, 1H), 7.59-7.57 (m, 1H), 7.06 (s, 1H), 5.67 (s, 2H), 3.90 (s, 2H), 3.88 (s, 2H), 2.76-2.70 (m, 2H), 2.59 (s, 3H), 1.13-1.10 (m, 3H)；LC-MS: m/z [M+H] ⁺ =428.

The examples listed in the table below were prepared by a method similar to that described in the above examples, starting from the corresponding intermediates:

the biological experimental method comprises the following steps:

cell line construction and subculture

The alpha subunit, beta subunit and gamma subunit form a complete functional GABA _A Receptors are essential. In this example, the invention was performed by lipofection (Felgner, p.l.,et alproceedings of the National Academy of Sciences, 1987, 84: 7413- _A Receptor (comprising. alpha.5. beta.3. gamma.2 subunit), alpha.1-GABA _A Receptor (comprising. alpha.1. beta.3. gamma.2 subunit) and. alpha.2-GABA _A HEK293 cell line for the receptor (containing the α 2 β 3 γ 2 subunit). The specific subunit protein sequences are as follows: the alpha 5 subunit (protein sequence is shown in GenBank accession No.: NM-000810.3); the alpha 1 subunit (protein sequence is shown in GenBank accession No.: NM-000806.5); the alpha 2 subunit (protein sequence is shown in GenBank accession No.: NM-000807.4); beta 3 subunit (protein sequence see GenBank accession No.: NM-000814.5); gamma 2 subunit (protein sequence see GenBank accession No.: NM-000816.3).

The above cell lines were subcultured. Amplification of alpha 5 beta 3 gamma 2-HEK293 cells for compound on GABA _A Benzodiazepine site (BZD) affinity assay of the receptor. α 5 β 3 γ 2-HEK293, α 1 β 3 γ 2-HEK293 and α 2 β 3 γ 2-HEK293 cells were partially suspended during passaging and plated on slides pretreated with Poly-D-Lysine for electrophysiological testing (methods see patent CN 107344936A, 0586).

Effect example 1 Compound of the present invention on alpha 5-GABA _A Affinity activity of receptor

Flunitrazepam (Flunitrazepam) is an agent that binds the BZD site non-specifically and efficiently. By reacting a compound with ³ H-flunitrazepam competition binding human alpha 5 beta 3 gamma 2 receptor measurable compound to alpha 5-GABA _A The affinity of the receptor.

HEK293 cells stably expressing human alpha 5 beta 3 gamma 2 receptors are collected, and cell membranes are extracted for competition binding tests. The cells were suspended in 50mM Tris-HCl buffer (pH =7.4, Sigma;) and homogenized in a homogenizer for 20 seconds 10 times on ice, at 4 ℃,1000g for 10 min. And taking the supernatant, and repeating the steps. The supernatant was centrifuged at 4 ℃ for 60 min (33800g; Thermo, rotor: A27-8X50), and the pellet was resuspended in Tris buffer (50 mM Tris-HCl, 10 mM MgCl) ₂ 0.5 mM EDTA, 10% glycerol), the pellet is the cell membrane. Use of BCA (Bic)inchoninic Acid) method (Pierce BCA Protein Assay Kit, Thermo;) to determine Protein content. Cell membranes were prepared in 1ml aliquots and stored at-80 ℃.

The radioligand competition binding assay was performed in a 200. mu.L system (96-well plate, Agilent ™) with 100. mu.L of cell membrane. ³ The concentration of H-flunitazepam (PerkinElmer;, cat # NET567250 UC) was 1 nM and the concentration of test compound was 1X 10 ^-5 -10 ^-6 And M is in the range. Flumazenil (Tocris. TM. protein, cat # 1318) was used as a control. Low signal control wells (Low control, LC) were loaded with 1. mu.L of 2mM flumazenil (final concentration 10. mu.M), and High signal control wells (High control, HC) were loaded with 1. mu.L of DMSO (Sigma, cat # 472301). The final concentration of target membrane protein was 5. mu.g/well. All test compound samples were dissolved in DMSO at a stock concentration of 10 mM. The working concentration of the samples was that all samples were diluted to 0.2mM with DMSO and then diluted in 4-fold serial gradients for a total of 8 concentration gradients. The 96-well plate was sealed with a sealing plate membrane (PerkinElmer, cat #6050185), and then incubated at room temperature for 1 hour in a shaker. GF/C filter plates were also soaked in dip-plate buffer (0.3% PEI, Sigma, cat # P3143, 4 ℃ C.) for at least 0.5 hours. After completion of the binding incubation, the cells were harvested from the GF/C filter plates and washed 4 times with wash buffer (50 mM Tris-HCl, pH 7.4, 4 ℃ C.). After drying in an oven at 50 ℃ for 1 hour, the bottom of the dried GF/C filter plate is sealed, the residual radioactivity in the filter plate is detected by liquid scintillation counting, 50. mu.L of scintillation fluid is added to each well, the wells are sealed and read using Microbeta2 (Perkin Elemer). Calculating the pairs of samples to be measured ³ H-flunitrazepam and GABA _A Inhibitory Activity against receptor Membrane protein binding, IC of each test sample was calculated by dose-response Curve fitting (GraphPad Prism 5 software) ₅₀ And through the IC ₅₀ Calculating Ki of the sample to evaluate the sample's interaction with alpha 5-GABA _A The binding capacity of the receptor.

By the above determination of compounds and expression of human GABA _A Representative results of the measurement of the binding affinity of the receptor to HEK293 cells are shown in Table 1, and the products of examples 1,6, 13, 18, 23, 46, 47, 48, 58, 62, 75 and 81, in which the compounds are not shown in Table 1, are shown in Table 1Object pair alpha 5-GABA _A The receptors also all have affinity activity.

TABLE 1 EXAMPLE product vs. alpha 5-GABA _A Affinity activity of receptor

Effect example 2 Compound of the present invention on alpha 5/1/2-GABA _A Inverse modulatory activity of receptors

The inventor detects the test compound pair alpha 5/1/2-GABA through an electrophysiological method _A Functional activity of the receptor. This method preferentially tests compounds for alpha 5-GABA _A Efficiency of inverse modulation of the receptor, selection of compounds with potent activity therein and retesting on α 1/2-GABA _A Functional activity of the receptor. The specific method is as follows:

compound concentration setting: drug screening was performed using 100 nM of drug final concentration. GABA concentrations were varied according to different cell line characteristics: for expression of alpha 5-GABA _A The GABA concentration of the receptor cell line is 0.05-0.06 μ M (about EC) _7~8 ) (ii) a For expression of alpha 1-GABA _A The GABA concentration of the receptor cell line is 0.05-0.07 μ M (about EC) _2~4 ) (ii) a For expression of alpha 2-GABA _A Cell line of receptor, GABA concentration is 0.10-0.11 μ M (about EC) _7~8 ）。

Electrophysiological experiments using the whole-cell patch-clamp technique are described in the literature (i. Lecker, y. Yin, d.s. Wang and b.a. orer,British Journal of Anaesthesia2013, 110 (S1), i73-i 81). The extracellular fluid for Electrophysiology (ECS) had the following composition: 150 mM NaCl, 5mM KCl, 2.5 mM CaCl ₂ ，1 mM MgCl ₂ 10 mM HEPES and 10 mM glucose (pH 7.4); the formulation of the electrophysiological electrode internal solution (ICS) is as follows: 140 mM CsCl, 11 mM EGTA, 10 mM HEPES, 2mM CaCl ₂ ，1 mM MgCl ₂ 4mM MgATP, 2mM TEA (pH 7.3). Preparing GABA (Sigma, cat # A5835) powder into mother liquor with pure water, and diluting in ECS; compounds were first prepared in DMSO (Sigma, cat # 34869) as 4mM stock solution and gradually dilutedCorresponding concentrations of GABA-ECS. The solutions were all freshly prepared prior to electrophysiological testing.

The signals were collected using EPC 10 amplifier and PatchMaster software (HEKA) or Axon700B amplifier and Clampex software (AXON). The recording electrode is formed by drawing borosilicate glass, and has an electrode resistance of 4-6M omega. Extracellular administration was carried out using the ALA-VC-8PG system. When recorded, individual, independently growing cells were selected. The glass electrode and the cell form good sealing and then rupture of the membrane to form a whole cell mode. Cell membrane potential was clamped and recorded at-60 mV, Gap-free mode. In the test, extracellular fluid is applied extracellularly for about 10 to 20 seconds. After the baseline stabilized, the extracellular fluid was switched to GABA solution. At this time, the current caused by GABA can be detected. And (4) switching the extracellular fluid to a corresponding medicine solution after the current is stabilized for about 20-40 seconds, and detecting the effect of the compound. Finally, the solution was switched to extracellular fluid and the test was terminated when the baseline returned to the pre-dose level. Only cells with a current greater than 50 pA after addition of GABA solution and a relatively stable current over time can be used for the determination of the compound.

Experimental results analysis used either the PatchMaster v2x90.1 or PatchMaster v2x90.3 software (EPC 10 amplifier) and the claudex 10.6 software (Axon 700B amplifier). The current value of each stage is calculated according to the average value of the stabilized current under the corresponding condition. During the analysis, the leakage currents (I) are measured separately _leak ) GABA current before dosing (I) _pre ) And GABA current after dosing (I) _post ) The functional activity of the compound is calculated by the following formula: reverse regulation efficiency (%) =100- _post - I _leak )/(I _pre - I _leak ). The details are shown in Table 2.

TABLE 2 product of the examples vs. alpha 5/alpha 1/2-GABA _A Inverse modulatory activity of receptors

TABLE 3 products of part of the examples and comparative compounds are directed against alpha 5/1/2-GABA _A Inverse modulatory activity of receptors

From the point of view of electrophysiological activity, the products of examples 1,6, 13, 18, 23, 46, 47, 48, 58, 62, 75, 81 of the present application are directed against α 5-GABA _A The receptor has better selectivity, and can achieve better drug effect and generate less side effects.

Effect example 3 comparison of pharmacological Properties of the Compound of the present invention and existing Compound

(1) Solubility of Compounds in simulated intestinal fluid (FaSSIF)

Experimental materials and equipment:

50mM phosphate buffer pH = 7.4: 0.39g NaH was weighed out ₂ PO ₄ •2H ₂ O，1.4025 g Na ₂ HPO ₄ Placed in an Erlenmeyer flask, added with 240 ml of water, dissolved and mixed, adjusted to pH 7.4 with 10M NaOH solution, transferred to a 250ml volumetric flask and diluted to the mark with water.

Simulated intestinal fluid FaSSIF 54.76 mg of FaSSIF-V2 (Biorelevant, batch. V2FAS-0619-A, lot. V2FAS 01) was weighed out and added to 15 ml of buffer solution, and after dissolution, the solution was made up to 30ml and left to stand at room temperature for 1 hour.

Waters e2695 HPLC high Performance liquid chromatography, Mettler XSE105 analytical balance.

The experimental method comprises the following steps:

10 mM stock solution was prepared with DMSO and diluted with diluent (ACN: PB buffer50: 50) to a standard solution of 1. mu.M to 200. mu.M.

Simulating thermodynamic solubility in intestinal fluid (TS in FaSSIF). Weighing about 0.3 mg of each sample, adding the sample into 1.5mL of FaSSIF solution, paralleling the sample and the FaSSIF solution, shaking at 1000 rpm at 37 ℃ for 4 hours, filtering, discarding 1mL of primary filtrate, taking 400 mu L of subsequent filtrate, and detecting the filtrate by using a high performance liquid chromatograph (UV). The test results of the compounds of the present invention are shown in table 4 below.

(2) Blood concentration of compound after oral absorption in SD rat

Maximum blood concentration (C) in rat pharmacokinetic experiment _max ) To evaluate the absorption of the compound in rats. Male SD rats were orally (po) gavaged with test compounds dissolved and 3 animals per group were fasted overnight prior to dosing. Approximately 0.25ml of each sample was collected by jugular venipuncture at 0.25, 0.5, 1,2,4 and 8 hours after administration of the test compound. Plasma drug concentrations were determined by LC-MS/MS method and pharmacokinetic parameters were calculated using Phoenix winnonlin 7.0. The dosage is 3mg/kg, and the solvent is 5% CMC-Na water solution. The test results of the compounds of the present invention are shown in table 4 below.

Table 4 solubility and pharmacokinetic parameters in SD rats for the products of the examples and comparative compounds of part 4

Simulated intestinal poor solubility of the comparative Compounds, exposure to oral absorption in SD rats (plasma maximum drug concentration C) _max ) Low, increases the difficulty of the development of the preparation, and limits the potential clinical application. The simulated intestinal juice solubility of the compounds 13 and 46 in the embodiment of the patent is better, the compounds are well absorbed by oral administration, and the patent medicine property is better.

Claims

1. A substance X or a pharmaceutically acceptable salt thereof; the substance X is a triazolopyridazine compound I;

R ¹ is as a quilt R ^1-2 A substituted 5-to 6-membered heteroaryl; in the 5-6-membered heteroaryl, the number of heteroatoms is 1,2, 3 or 4, and the heteroatoms are selected from one or more of N, O and S;

R ^1-2 independently is C ₁ ~C ₆ An alkyl group;

R ² is hydrogen, R ³ Is C ₁ ~C ₆ An alkyl group; r ^X Is composed of

M and n are independently 1 or 2, and m + n =2 or 3, Z ¹ is-N (R) ^4-1 )-，R ^4-1 Independently is a 4-membered heterocycloalkyl group; in the 4-membered heterocyclic alkyl, the number of the heteroatoms is 1, and the heteroatoms are O or S.

2. Substance X according to claim 1, or a pharmaceutically acceptable salt thereof, which fulfils one or more of the following conditions:

a)R ¹ wherein the number of heteroatoms in the 5-to 6-membered heteroaryl is 2 or 3;

b)R ¹ in the 5-6 membered heteroaryl, the heteroatom is selected from one or more of N and O;

c)R ^1-2 in (b), the C ₁ ~C ₆ Alkyl is C ₁ ~C ₃ An alkyl group;

d)R ³ in (b), the C ₁ ~C ₆ Alkyl is C ₁ ~C ₄ An alkyl group;

e)R ^4-1 wherein the heteroatom in said 4-membered heterocycloalkyl is O;

f) all atoms in substance X or a pharmaceutically acceptable salt thereof are atoms comprising isotopes of natural proportions.

3. Substance X according to claim 2, or a pharmaceutically acceptable salt thereof, which fulfils one or more of the following conditions:

g)R ^1-2 in (b), the C ₁ ~C ₆ Alkyl is methyl, ethyl, n-propyl or isopropyl;

h)R ³ in (b), the C ₁ ~C ₆ Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl.

4. Substance X according to claim 3, or a pharmaceutically acceptable salt thereof, which fulfils one or more of the following conditions:

i） R ¹ is composed of

；

j）R ³ Is composed of

。

5. A substance X or a pharmaceutically acceptable salt thereof, wherein the substance X is a triazolopyridazine compound I; the triazolopyridazine compound I is of any one of the following structures:

。

6. a pharmaceutical composition comprising substance Y and a pharmaceutical excipient;

the substance Y is the substance X or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5.

7. Preparation of alpha 5-GABA from substance Y _A Use in receptor inverse modulators;

8. Use according to claim 7, which satisfies one or more of the following conditions:

k) said alpha 5-GABA _A The receptor inverse modulator is alpha 5-GABA used in vivo or in vitro _A A receptor inverse modulator;

l) said α 5-GABA relative to α 1 and/or α 2 _A Receptor inverse modulators are selective.

9. Use of a substance Y for the preparation of a medicament;

the substance Y is the substance X or the pharmaceutically acceptable salt thereof according to any one of claims 1 to 5;

10. Use according to claim 9, which satisfies one or more of the following conditions:

m) the drug para to alpha 5-GABA relative to alpha 1 and/or alpha 2 _A The receptor is selective;

n) said compound consisting of alpha 5-GABA _A The receptor mediated diseases are depression, pain, alzheimer's disease, multi-infarct dementia, stroke, anxiety disorders, panic disorder, agoraphobia, post-traumatic stress disorder, premenstrual dysphoric disorder, attention deficit disorder, obsessive compulsive disorder, autism, schizophrenia, obesity, bulimia nervosa or deficiency, tourette's syndrome, vasomotor flushing, sexual dysfunction, borderline personality disorder, chronic fatigue syndrome, raynaud's syndrome, parkinson's disease or epilepsy and mood disorders following head injury.

11. The use of claim 10, which satisfies one or more of the following conditions:

o) the drug pair alpha 5-GABA relative to alpha 2 _A The receptor is selective;

p) said depression is bipolar depression, postpartum depression, dysthymia, atypical depression, melancholia, treatment-resistant depression, depression associated with Huntington's disease, depression associated with multiple sclerosis or depression associated with anxiety;

q) said depression is mild depression, moderate depression or major depression;

r) said anxiety disorder is generalized anxiety disorder or social anxiety disorder;

s) the pain is fibromyalgia.

12. Use of a substance Y for the preparation of a medicament;

depression, pain, alzheimer's disease, multi-infarct dementia, stroke, anxiety disorders, panic disorder, agoraphobia, post-traumatic stress disorder, premenstrual dysphoric disorder, attention deficit disorder, obsessive compulsive disorder, autism, schizophrenia, obesity, bulimia nervosa or deficiency, tourette's syndrome, vasomotor flushing, sexual dysfunction, borderline personality disorder, chronic fatigue syndrome, raynaud's syndrome, parkinson's disease, and mood disorders following head injury.

13. Use according to claim 12, which satisfies one or more of the following conditions:

t) said depression is bipolar depression, postpartum depression, dysthymia, atypical depression, melancholia, treatment-resistant depression, depression associated with Huntington's disease, depression associated with multiple sclerosis or depression associated with anxiety;

u) said depression is mild depression, moderate depression or major depression;

v) said anxiety disorder is generalized anxiety disorder or social anxiety disorder;

w) the pain is fibromyalgia.