Ai-designed benzodiazepine derivative and use thereof

Abstract

Disclosed in the present invention are an AI-designed benzodiazepine derivative and a use thereof. The benzodiazepine derivative has a structure of Formula I, wherein the definition of each group is as described in the description. The benzodiazepine derivative provided by the present invention, as an active component of a pharmaceutical composition, can significantly improve selectivity for an M2 receptor and significantly reduce the inhibitory effect on an M3 receptor, resulting in a significant increase in the IC50 ratio of M3 / M2. In addition, the benzodiazepine derivative has specific pharmacological properties, can effectively treat myopia without affecting pupil size, and facilitates improvement of treatment specificity and patient comfort.

Description

A benzodiazepine derivative designed based on AI and its uses Technical Field

[0001] This invention belongs to the field of chemical and pharmaceutical technology, and relates to a drug for the prevention and / or treatment of myopia and its application, specifically to an AI-designed benzodiazepine derivative and its uses. Background Technology

[0002] Myopia, a common refractive error characterized by decreased distance vision, has become a major public health problem in modern society (PNBaird et al., Nature reviews, 2020, 6(99): 1-20). The high incidence and younger age of onset of myopia is closely related to the widespread use of video tools such as computers, mobile phones, and tablets, which leads to a significant increase in screen time and poses a serious threat to the visual health of children and adolescents.

[0003] According to a 2020 study by PNBaird et al. published in *Nature Reviews*, and the WHO's 2020 *World Report on Vision*, the number of people with myopia worldwide has reached 2.6 billion. The *China Eye Health White Paper* shows that the overall incidence of myopia among children and adolescents in my country is 53.6%, and the overall incidence among university students exceeds 90%.

[0004] Myopia disrupts the balance between accommodation and convergence, leading to symptoms such as double vision, blurred vision, eye pain, headache, nausea, eye fatigue, and dryness. Based on disease progression and pathological changes, myopia can be classified into simple myopia and pathological myopia. Simple myopia usually does not cause changes in the fundus, while pathological myopia may be accompanied by symptoms such as protruding eyeballs, a deeper anterior chamber, a large pupil with a slow response, and a narrow palpebral fissure. It also increases the risk of serious eye diseases such as macular degeneration and retinal detachment, further severely damaging vision (Haarman A. et al., Invest Ophthalmol Vis Sci, 2020, 61(4):49).

[0005] Currently, drug-based prevention and control is an effective means of controlling myopia (JHHuang et al., American Academy of Ophthalmology, 2016, 123(4):697-708). A 2020 study by Walline JJ et al. indicated that atropine is an effective drug for preventing and / or inhibiting the progression of myopia (Walline JJ et al., Cochrane Database Syst Rev, 2020, 1:D4916). However, a 2021 study by Joachimsen L. et al. found that when using atropine, adverse reactions such as pupillary dilation and changes in accommodation occurred after using 0.01%, 0.025%, and 0.05% atropine (Joachimsen L. et al., Int Ophthalmol, 2021, 41(6):2001-2008).

[0006] In conclusion, there is an urgent need for myopia prevention and control, but existing drugs present a trade-off between effectiveness and safety. Summary of the Invention

[0007] The purpose of this invention is to develop a new treatment strategy to provide a safer and more effective solution for the prevention and / or treatment of myopia.

[0008] To achieve the above objectives, the present invention provides an AI-designed benzodiazepine derivative having the structure of Formula I:

[0009] Wherein, R1 is any one of H, deuterium, halogen, or C1-C3 alkoxy group;

[0010] R2 and R3 are each independently one of H, deuterium, and C1-C5 alkyl groups;

[0011] R4 is a polycyclic cycloalkyl group or a polycyclic heterocyclic group;

[0012] L1 is -(CH2) m -, where m is an integer from 1 to 3;

[0013] L2 is a single bond or -(CH2). n - where n is 1 or 2.

[0014] Optionally, L2 is a single bond, and R3 and R4 can form a C4-C bond together with the N bonded to them. 20 The polycyclic heterocyclic structure.

[0015] Optionally, one of the methylene groups of R2 and L1 can form a C4-C7 heterocyclic structure together with the N group to which it is attached.

[0016] Optionally, the benzodiazepine derivative has the structure of formula II:

[0017] Wherein, R3 is any one of H, deuterium, and C1-C5 alkyl groups;

[0018] R4 is a polycyclic cycloalkyl group;

[0019] L2 is a single bond or -(CH2) n - where n is 1 or 2.

[0020] Optionally, L2 is a single bond, and R3 and R4 can form a C7-C bond together with the N bonded to them. 20 The heterocyclic structure.

[0021] Optionally, the benzodiazepine derivative comprises:

[0022] Any one of them.

[0023] The present invention also provides a pharmaceutical composition comprising: the above-mentioned AI-designed benzodiazepine derivative as an active ingredient.

[0024] Optionally, the pharmaceutical composition further comprises: pharmaceutical excipients.

[0025] The present invention also provides the use of AI-designed benzodiazepine derivatives for the preparation of drugs for preventing myopia and / or inhibiting the progression of myopia.

[0026] Optionally, the myopia includes at least one of the following: axial myopia, refractive myopia, pseudomyopia, pathological myopia, simple myopia, extremely high myopia, severe myopia, high myopia, moderate myopia, low myopia, myopia combined with glaucoma, myopia with risk of developing glaucoma, or myopia accompanied by high intraocular pressure.

[0027] Compared with the prior art, the technical solution of the present invention has at least the following beneficial effects:

[0028] Experiments have demonstrated that the AI-designed benzodiazepine derivative provided in this invention exhibits high selectivity for M2 cells, significantly inhibiting Human M2 cells while showing little inhibition of Human M3 cells. The IC50 ratio of M3 / M2 is [missing information]. 50The ratio is significantly higher than that of AFDX-116, an existing clinical drug for myopia. Therefore, the benzodiazepine derivative of the present invention holds promise for preventing the onset of myopia and / or inhibiting its further deterioration without causing mydriatic side effects. Attached Figure Description

[0029] Figure 1 is a schematic diagram showing the effect of compound 2 of the present invention on the refractive power of a myopic guinea pig model. Detailed Implementation

[0030] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] M2 receptors are mainly distributed in the ciliary muscle and retina of the eye and are related to regulating the eye's focusing and refractive state. Barathi et al. (Barathi V.A. et al. Dis. Model. Mech. 2013, 6(5): 1146-1158) knocked out the muscarinic M2 receptor gene in mice. After 4-8 weeks of negative lens induction, the control group mice showed significant increases in axial length, lens depth, and vitreous cavity depth, exhibiting myopia symptoms; while the M2 gene knockout mice did not show myopia symptoms. In M2 receptor gene knockout mice, scleral collagen type I increased and type V decreased, i.e., scleral fibrosis increased, thereby inhibiting axial elongation of the eye. It can be seen that myopia can be treated or prevented by inhibiting the activity of M2 receptors. The literature also disclosed two examples of selective M2 receptor inhibitors used to inhibit the development of myopia, including AFDX-116.

[0032] M3 receptors, or muscarinic type 3 receptors, are mainly distributed on the pupillary sphincter muscle of the eye. M3 receptors are associated with the contraction of the pupillary sphincter muscle, affecting pupil size. When these receptors are activated, they cause the pupillary sphincter muscle to contract, thus constricting the pupil—a process called mydriasis. Conversely, when the activity of M3 receptors is reduced or inhibited, the contraction of the pupillary sphincter muscle weakens, causing the pupil to dilate—a process called mydriasis.

[0033] Therefore, in treating or preventing myopia, this invention aims to minimize mydriasis by maximizing the selectivity of the drug for the M2 receptor. Ideally, the drug should primarily act on the M2 receptor while having minimal impact on other muscarinic receptors (such as the M3 receptor), i.e., the IC50 ratio of M3 / M2 should be minimized. 50 The higher the ratio, the better.

[0034] In the latest developments in AI technology, artificial intelligence can be used for the discovery, screening, and optimization of small molecule drugs. By integrating AI computing tools, databases (such as the CAS compound library), and receptor-ligand complex interaction information into efficient molecular / atomic training sets and AI digital workflows, and combining them with BT technologies (bioinformatics, in vitro target cell bioactivity detection, etc.), new therapeutic drugs can be invented.

[0035] As is well known, in the process of drug development, traditional small molecule compounds (drugs) require a lot of time to go from seed compounds to lead compounds and then to candidate compounds (usually taking 5-6 years or even longer). However, drug development driven by artificial intelligence (AIDrug Discovery & Design, AIDD) can perform data cross-comparison, molecular docking, molecular dynamics simulation, etc. in a shorter time (usually only 3-5 months), thereby accelerating the screening or de novo design of new compounds. Its core value lies in pioneering innovation and efficiency improvement.

[0036] Based on relatively well-defined mechanisms (such as the Schrödinger equation and Gibbs free energy change), AI can search multiple, broader, and more comprehensive databases of different compounds and proteomics, quickly completing searches and cross-matches, saving significant resources previously used for wet experiments. Wet experimental data can then be fed back into the AI ​​for iterative optimization of compound structures.

[0037] This invention is based on a computational structural biology technology platform built independently using an AI (dry experiment) + BT (wet experiment) interactive fusion R&D model. It combines various cutting-edge algorithms in medicinal chemistry and quantum chemistry with deep learning, natural language processing, and pre-trained models of various neural network frameworks to generate novel molecular structures with expected functional properties. Then, based on the target-drug related binding interface, molecular modeling, molecular docking, and molecular dynamics simulation are performed to obtain the optimal receptor-ligand binding free energy function, binding constant, and dissociation constant. This optimizes the screening of candidate compounds with the best in vitro cell activity and the most suitable overall evaluation. Meanwhile, through in-depth analysis of disease / drug correlation networks, AI servers, and workstations, drug deep learning and multi-threaded collaborative simulation can be performed simultaneously and independently. Alternatively, an AI computing power cluster can be formed to process large data of libraries and candidate compounds, deeply mining libraries, structure-pharmacodynamic group relationships (QSAR), and novel drug targets, saving innovative drug development costs and shortening R&D time. This forms a new R&D path of "dry and wet combination, seamless connection" for new drug development, which is expected to become a scientific paradigm for future innovative drug development, bringing a new breakthrough in thinking and experimental innovation to the past drug development process that was extremely dependent on wet experiments, which was time-consuming, laborious, and cumbersome.

[0038] Through multiple rounds of iterative dry and wet closed-loop experiments, this invention ultimately yielded a series of benzodiazepine derivatives with the structure of Formula I:

[0039] Wherein, R1 is any one of H, deuterium, halogen, or C1-C3 alkoxy group; R2 and R3 are each independently any one of H, deuterium, or C1-C5 alkyl group; R4 is a polycyclic cycloalkyl group or a polycyclic heterocyclic group; L1 is -(CH2). m - where m is an integer from 1 to 3; L2 is a single bond or -(CH2). n - where n is 1 or 2.

[0040] Optionally, L2 is a single bond, and R3 and R4 can form a C4-C bond together with the N bonded to them. 20 The polycyclic heterocyclic structure.

[0041] Optionally, one of the methylene groups of R2 and L1 can form a C4-C7 heterocyclic structure together with the N group to which it is attached.

[0042] Optionally, the benzodiazepine derivative has the structure of formula II:

[0043] Wherein, R3 is any one of H, deuterium, or C1-C5 alkyl groups; R4 is a polycyclic alkyl group; and L2 is a single bond or -(CH2). n - where n is 1 or 2.

[0044] The present invention also provides a pharmaceutical composition comprising: the above-mentioned AI-designed benzodiazepine derivative as an active ingredient, and pharmaceutical excipients.

[0045] The present invention also provides the use of AI-designed benzodiazepine derivatives, which can be used to prepare drugs for preventing myopia and / or inhibiting the progression of myopia.

[0046] Terminology Definition

[0047] The "single bond" mentioned in this invention refers to R4 being directly connected to N connected by R3, that is, L2 is a compound bond connecting N and R4.

[0048] The "halogen" mentioned in this invention refers to fluorine, chlorine, bromine, and iodine.

[0049] The “C1-C3 alkoxy” mentioned in this invention refers to a group formed by an alkyl group having 1 to 3 carbon atoms bonded to an oxygen atom, wherein the oxygen atom has the ability to form bonds freely, such as methoxy, ethoxy, and propoxy.

[0050] The "C1-C5 alkyl" as used in this invention refers to straight-chain, branched, or cyclic alkyl groups having 1 to 5 carbon atoms. Examples of straight-chain alkyl groups include methyl, ethyl, n-propyl, n-butyl, and n-pentyl; examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl, and isopentyl; examples of cyclic alkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, and cyclopentyl.

[0051] The term "polycyclic cycloalkyl" as used in this invention refers to saturated or partially unsaturated polycyclic cyclic substituents, wherein the polycyclic cycloalkyl ring contains 4-20 carbon atoms, preferably 7-12 carbon atoms. Polycyclic cycloalkyl groups include spirocyclic, fused, and bridged rings.

[0052] The term "spirocyclic" as used in this invention refers to a polycyclic group consisting of 5 to 20 quintiles, where the monocyclic rings share a single carbon atom (called a spiro atom), and may contain one or more double bonds. Preferably, it is 6 to 14 quintiles, more preferably 7 to 10 quintiles (e.g., 7, 8, 9, or 10 quintiles). Spirocyclic alkyl groups are classified into monospirocyclic alkyl groups, bispirocyclic alkyl groups, or polyspirocyclic alkyl groups based on the number of shared spiro atoms between the rings, with monospirocyclic alkyl groups and bispirocyclic alkyl groups being preferred. More preferably, they are 3 / 5 quintile, 3 / 6 quintile, 4 / 4 quintile, 4 / 5 quintile, 4 / 6 quintile, 5 / 5 quintile, or 5 / 6 quintile monospirocyclic alkyl groups. Non-limiting examples of spirocyclic alkyl groups include:

[0053] At least one of them.

[0054] The "fused ring" described in this invention refers to a 5- to 20-membered ring, wherein each ring shares an adjacent pair of carbon atoms with other rings in the structure as a fully carbon polycyclic group, and one or more rings may contain one or more double bonds. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused alkyl, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic alkyl. Non-limiting examples of fused alkyl groups include:

[0055] At least one of them.

[0056] The "bridged ring" described in this invention refers to a 5- to 20-membered polycyclic carbon group in which any two rings share two non-directly connected carbon atoms, and may contain one or more double bonds. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged alkyl groups, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged alkyl groups include:

[0057] At least one of them.

[0058] The polycyclic cycloalkyl group can be substituted or unsubstituted. When substituted, it can be substituted at any usable connection point. The substituent is preferably independently selected independently from one or more substituents selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclicoxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0059] The "polycyclic heterocyclic group" described in this invention refers to a saturated or partially unsaturated polycyclic cyclic hydrocarbon substituent containing 4 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, wherein the sulfur may optionally be oxidized (i.e., forming sulfoxide or sulfone), but does not include the -OO-, -OS-, or -SS- ring moiety, and the remaining ring atoms are carbon. Preferably, it contains 7 to 12 ring atoms, wherein 1 to 4 (e.g., 1, 2, 3, and 4) are heteroatoms; the polycyclic heterocyclic group includes spirocyclic, fused-ring, and bridged-ring heterocyclic groups.

[0060] The "spirocyclic heterocyclic group" described in this invention refers to a 5- to 20-membered polycyclic heterocyclic group in which the monocyclic rings share a single atom (called a spiro atom), wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, and the sulfur may optionally be oxidized (i.e., forming sulfoxide or sulfone), and the remaining ring atoms are carbon. It may contain one or more double bonds. Preferably, it is 6- to 14-membered. Spirocyclic heterocyclic groups are classified into monospirocyclic, bispirocyclic, or polyspirocyclic groups according to the number of shared spiro atoms between rings, with monospirocyclic and bispirocyclic groups being preferred. More preferably, it is a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospirocyclic group. Non-limiting examples of spirocyclic heterocyclic groups include:

[0061] At least one of them.

[0062] The "fused-ring heterocyclic group" described in this invention refers to a 4- to 20-membered polycyclic heterocyclic group in which each ring shares an adjacent pair of atoms with other rings in the structure. One or more rings may contain one or more double bonds, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, wherein the sulfur may optionally be oxidized (i.e., forming sulfoxide or sulfone), and the remaining ring atoms are carbon. Preferably, it is 6 to 14-membered, more preferably 7 to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of constituent rings, it can be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic fused-ring heterocyclic group, preferably bicyclic or tricyclic, more preferably 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, and 6-membered / 6-membered bicyclic fused-ring heterocyclic groups. Non-limiting examples of fused-ring heterocyclic groups include:

[0063] At least one of them.

[0064] The "bridged heterocyclic group" described in this invention refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two non-directly connected atoms. It may contain one or more double bonds, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, and the sulfur may optionally be oxidized (i.e., forming sulfoxide or sulfone), with the remaining ring atoms being carbon. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of rings, it can be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic group, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

[0065] At least one of them.

[0066] The polycyclic heterocyclic group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably independently and arbitrarily selected from one or more substituents selected from hydrogen, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclicoxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0067] The "C4-C7 heterocycle" mentioned in this invention refers to a saturated or partially unsaturated monocyclic hydrocarbon substituent with a total of 4 to 7 ring atoms, one or two of which are heteroatoms selected from nitrogen, oxygen and sulfur. The sulfur may be optionally substituted with oxygen (i.e., to form sulfoxide or sulfone), and the remaining ring atoms are carbon.

[0068] In this invention, "substituted" means that an organic group (containing one or more bonds bonded to a hydrogen atom) as defined herein is replaced by one or more bonds bonded to a non-hydrogen atom or group of atoms, wherein the non-hydrogen atom or group of atoms is a substituent.

[0069] This invention further provides isotopically labeled compounds of the compounds of this invention. The term "isotopically labeled compound" or "isotopically labeled" in this invention refers to the compound described herein, wherein one or more atoms are replaced by isotopic atoms whose atomic mass or mass number differs from that of naturally occurring atoms. Suitable radionuclides may include, but are not limited to, 2H (deuterium, also written as D), 3H (tritium, also written as T), 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 18F, 35S, 36Cl, 82Br, 75Br, 76Br, 77Br, 123I, 124I, 125I, and 131I. The type of radioisotope contained in the isotopically labeled compound will depend on the specific application of the isotopically labeled compound. For example, for labeling and competition assays of in vitro IDO enzymes, compounds containing 3H, 14C, 82Br, 125I, 131I, or 35S are generally most useful. For isotope imaging applications, 11C, 18F, 125I, 123I, 124I, 131I, 75Br, 76Br, or 77Br are generally most useful.

[0070] The methods known in the prior art for labeling organic compounds with radioactive isotopes are also applicable to the compounds of the present invention.

[0071] When administered as a drug, the compound can be delivered in the form of a pharmaceutical composition. Therefore, in another aspect, this application provides a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0072] The term "composition" as used herein means a product containing a compound disclosed in this application or a pharmaceutically acceptable salt thereof as a specific active ingredient, as well as any other product directly or indirectly combined with said active ingredient.

[0073] Typically, the pharmaceutical composition contains at least one pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable" means that the carrier or excipient is compatible with other components in the formulation and is harmless to the subject. The carrier referred to herein is a substance used to improve the selectivity, efficacy, and / or safety of the drug during delivery. The carrier is primarily used to control drug release and may also be used to improve the pharmacokinetic properties of the drug, particularly bioavailability. The excipient refers to any substance in the pharmaceutical formulation other than the active ingredient, primarily used for long-term stability, filling solid dosage forms (therefore, it is often used specifically to refer to "filler"), or enhancing product efficacy (e.g., promoting absorption, reducing viscosity, or increasing solubility).

[0074] The term "myopia" as used in this article includes at least one of the following: axial myopia, refractive myopia, pseudomyopia, pathological myopia, simple myopia, high myopia, severe myopia, high myopia, moderate myopia, low myopia, myopia combined with glaucoma, myopia with a risk of developing glaucoma, or myopia accompanied by high intraocular pressure. Among these, high myopia, severe myopia, high myopia, moderate myopia, and low myopia are categories of myopia based on differences in refractive error. In this article, high myopia is defined as having a refractive error of -10.00D or lower; severe myopia as having a refractive error between -6.00D and -9.99D; high myopia as having a refractive error between -4.00D and -5.99D; moderate myopia as having a refractive error between -2.00D and -3.99D; and low myopia as having a refractive error between -0.50D and -1.99D.

[0075] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0076] Unless otherwise specified, the experimental methods used in the following examples were performed under conventional or manufacturer-recommended conditions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0077] The full names of the reagent abbreviations used in the examples are as follows:

[0078] TEA: Triethylamine

[0079] DMSO: Dimethyl sulfoxide

[0080] TFA: Trifluoroacetic acid

[0081] HATU: 2-(7-Azobenzotriazole)-N,N,N',N'-Tetramethylurea hexafluorophosphate

[0082] DIEA: N,N-Diisopropylethylamine.

[0083] Example 1: Preparation of Compound 1

[0084] Synthetic route of compound 1:

[0085] Step 1: Preparation of intermediate 1a

[0086] (2-Aminoethyl)(ethyl)carbamate tert-butyl ester (226 mg, 1.20 mmol) was dissolved in dichloroethane (10 mL), and then spiro[5.5]undecane-3-one (200 mg, 1.20 mmol) and acetic acid (72 mg, 1.20 mmol) were added. The resulting system was stirred at room temperature for 1 hour under nitrogen protection, followed by the addition of sodium triacetoxyborohydride (1.27 g, 6.01 mmol), and the reaction was continued at room temperature for 3 hours.

[0087] The reaction was quenched by adding 20 mL of saturated sodium bicarbonate aqueous solution. The aqueous phase was extracted three times with 30 mL of dichloromethane. The organic phases were combined and washed twice with 50 mL of brine. The mixture was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness.

[0088] The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1) to give 350 mg of intermediate 1a (yield: 85.9%).

[0089] LCMS:[Ms+H] + =339.4.

[0090] Step 2: Preparation of intermediate 1b

[0091] Intermediate 1a (350 mg, 1.03 mmol) was dissolved in methanol (10 mL), and then paraformaldehyde (310 mg, 10.34 mmol), acetic acid (310 mg, 5.17 mmol), and sodium triacetoxyborohydride (260 mg, 414 mmol) were added. The mixture was reacted at room temperature for 16 hours.

[0092] The reaction was quenched by adding 20 mL of saturated sodium bicarbonate aqueous solution. The aqueous phase was extracted three times with 30 mL of dichloromethane. The organic phases were combined and washed twice with 50 mL of brine. The mixture was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness.

[0093] The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1) to give 320 mg of intermediate 1b (yield: 87.8%).

[0094] LCMS:[Ms+H]+ =353.3.

[0095] Step 3: Preparation of intermediate 1c

[0096] Intermediate 1b (160 mg, 0.45 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1 mL) was added at 0 °C. The mixture was reacted at room temperature for 3 hours.

[0097] The reaction solution was evaporated to dryness, and the residue was azeotropically reacted with dichloromethane (30 mL) three times.

[0098] Without further purification, the crude product was used directly in the next step.

[0099] LCMS:[Ms+H] + =253.2.

[0100] Step 4: Preparation of intermediate 1d

[0101] 5,11-dihydro-6H-benzopyrido[3,2-b][1,4]diazaphen-6-one (2 g, 12 mmol) and triethylamine (1.6 mL, 6 mmol) were dissolved in 1,4-dioxane (40 mL), then heated to 80 °C and ethyl 4-chloro-4-oxobutyrate (2.4 g, 11.3 mmol) was added. The mixture was reacted at 100 °C for 4 hours under nitrogen atmosphere.

[0102] The reaction was quenched with water (30 mL), followed by extraction three times with ethyl acetate (20 mL), the organic phases were combined and washed three times with brine (20 mL), dried over anhydrous sodium sulfate, and then evaporated to dryness.

[0103] The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1) to give 1.1 g of intermediate 1d (yield: 27%).

[0104] LCMS:[Ms+H] + =340.2.

[0105] Step 5: Preparation of intermediate 1e

[0106] Intermediate 1d (155 mg, 0.46 mmol) was dissolved in ethanol (3 mL), and 1N sodium hydroxide aqueous solution (1.5 mmol, 1.5 mL) was added. The mixture was reacted at room temperature for 1 hour.

[0107] Add 1.5 mL of 1N hydrochloric acid aqueous solution to neutralize the reaction, then evaporate the reaction solution to dryness, add N,N-dimethylformamide, filter, and obtain an N,N-dimethylformamide solution containing 140 mg of intermediate 1e, which can be used directly in the next reaction.

[0108] LCMS:[Ms+H]+ =312.0.

[0109] Step 6: Preparation of Compound 1

[0110] Intermediate 1e (140 mg, 0.45 mmol) and intermediate 1c (114 mg, 0.45 mmol) were dissolved in N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (292 mg, 2.26 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (206 mg, 0.54 mmol) were added sequentially. The reaction was carried out at room temperature for 16 hours under nitrogen protection.

[0111] Dilute the reaction system with water (20 mL) and extract three times with dichloromethane (20 mL). Combine the organic phases, wash with brine (30 mL), dry with anhydrous sodium sulfate, and concentrate.

[0112] The residue was purified by Pre-HPLC to give 56.56 mg of compound 1 (yield: 23.0%).

[0113] LCMS:[Ms+H] + =546.4.

[0114] 1 H NMR(400MHz, CDCl3)δ8.34(s,1H),7.91(d,J=7.1Hz,1H),7.78–7.57(m,2H),7.52(d, J=7.2Hz,1H),7.42(t,J=7.0Hz,1H),7.35(s,1H),4.60(s,3H),4.12–3.62(m,1H),3.5 9–3.28(m,3H),3.24–3.00(m,3H),2.96–2.64(m,5H),2.61–2.25(m,2H),2.00–1.67(m ,3H),1.65–1.47(m,2H),1.46–1.25(m,8H),1.16(t,J=7.0Hz,3H),1.09–0.85(m,2H).

[0115] Example 2: Preparation of Compound 2

[0116] Synthetic route of compound 2:

[0117] Step 1: Preparation of intermediate 2a

[0118] Oxaloyl chloride (563 mg, 4.44 mmol) was dissolved in dichloromethane (15 mL), and then a solution of dimethyl sulfoxide (421 mg, 5.39 mmol) in dichloromethane (3 mL) was added dropwise at -78 °C. The resulting system was stirred at -78 °C for 25 min under nitrogen protection, and then a solution of ethyl (2-hydroxyethyl) tert-butyl carbamate (600 mg, 3.17 mmol) in dichloromethane (3 mL) was added dropwise. The resulting mixture was stirred at -78 °C for 25 min under nitrogen protection, and then a solution of triethylamine (1.12 g, 11.10 mmol) in dichloromethane (3 mL) was added dropwise. The mixture was then allowed to react at room temperature for 2 hours. The reaction solution was then cooled to 0°C, and 3-azaspiro[5.5]undecane (202 mg, 1.32 mmol), acetic acid (1.11 g, 18.45 mmol), and sodium triacetoxyborohydride (978 mg, 4.61 mmol) were added. The reaction was then carried out at room temperature for 1 hour.

[0119] The reaction was quenched by adding saturated sodium bicarbonate aqueous solution (10 mL), the aqueous phase was extracted three times with dichloromethane (30 mL), the organic phases were combined and washed twice with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness.

[0120] The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1) to give 330 mg of intermediate 2a (yield: 80.6%).

[0121] LCMS:[Ms+H] + =325.5.

[0122] Step 2: Preparation of intermediate 2b

[0123] Intermediate 2b was prepared from intermediate 2a (160 mg, 0.49 mmol) according to step 3 of Example 1, yielding 110 mg of crude product, which was directly used in the next step.

[0124] LCMS:[Ms+H] + =225.5.

[0125] Step 3: Preparation of Compound 2

[0126] Compound 2 was prepared from intermediate 1e (110 mg, 0.49 mmol) and intermediate 2b (153 mg, 0.49 mmol) according to step 6 of Example 1, with a yield of 15.9%.

[0127] LCMS:[Ms+H] + =518.6.

[0128] 1H NMR (400MHz, CDCl3) δ9.41 (s, 1H), 8.34 (s, 1H), 7.89 (d, J = 7.7Hz, 1H), 7.75 –7.56(m,2H),7.51(d,J=7.1Hz,1H),7.46–7.30(m,2H),4.05–3.67(m,2H), 3.56–3.13(m,6H),3.03–2.74(m,4H),2.74–2.62(m,1H),2.62–2.48(m,1H) ,2.32(s,6H),1.89–1.62(m,4H),1.37–1.21(m,4H),1.13(t,J=7.0Hz,3H).

[0129] Example 3: Preparation of Compound 3

[0130] Synthetic route of compound 3:

[0131] Step 1: Preparation of intermediate 3a

[0132] Spiro[5.5]undecane-3-one (450 mg, 2.71 mmol) was dissolved in ethylene glycol dimethyl ether (10 mL), and then p-toluenesulfonylmethylisocyanate (687 mg, 3.52 mmol), ethanol (249 mg, 5.41 mmol), and potassium tert-butoxide (729 mg, 6.50 mmol) were added at 0 °C. The resulting system was stirred at 0 °C for 1 hour under nitrogen protection, and then reacted at room temperature for 3 hours.

[0133] The reaction solution was evaporated to dryness, then 20 mL of ether was added and stirred for 0.5 hours before filtration. This operation was repeated twice, and the filtrates were combined and evaporated to dryness.

[0134] The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 5 / 1) to give 240 mg of intermediate 3a (yield: 50.0%).

[0135] 1 H NMR (400MHz, CDCl3) δ2.56 (dq, J = 12.6, 4.2Hz, 1H), 1.91–1.66 (m, 4H), 1.66–1.54 (m, 2H), 1.50–1.10 (m, 12H).

[0136] Step 2: Preparation of intermediate 3b

[0137] Intermediate 3a (240 mg, 1.35 mmol) was dissolved in tetrahydrofuran (10 mL), and then a 2.5 N solution of lithium aluminum hydride in tetrahydrofuran (1.08 mL, 2.71 mmol) was added dropwise at 0 °C. The resulting system was stirred at 70 °C for 4 hours under nitrogen protection.

[0138] The reaction was quenched by adding 10 mL of saturated 1 N sodium hydroxide aqueous solution. The aqueous phase was extracted three times with 30 mL of ethyl acetate. The organic phases were combined and washed twice with 50 mL of brine. The mixture was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness. The crude product did not require purification and was used directly in the next step.

[0139] LCMS:[Ms+H] + =182.2.

[0140] Step 3: Preparation of intermediate 3c

[0141] Intermediate 3c was prepared from tert-butyl ethyl (2-hydroxyethyl)carbamate (313 mg, 1.65 mmol) and intermediate 3b (240 mg, 1.32 mmol) according to the method in step 1 of Example 2, with a yield of 21.4%.

[0142] LCMS:[Ms+H] + =353.3.

[0143] Step 4: Preparation of intermediate 3D

[0144] Intermediate 3c (100 mg, 0.28 mmol) was dissolved in methanol (5 mL), and then paraformaldehyde (85 mg, 2.84 mmol), acetic acid (85 mg, 1.42 mmol), and sodium triacetoxyborohydride (71 mg, 1.13 mmol) were added. The mixture was reacted at room temperature for 16 hours.

[0145] The reaction was quenched by adding 20 mL of saturated sodium bicarbonate aqueous solution. The aqueous phase was extracted three times with 30 mL of dichloromethane. The organic phases were combined and washed twice with 50 mL of brine. The mixture was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness. The resulting residue, intermediate 3d, did not require purification and was used directly in the next step.

[0146] LCMS:[Ms+H] + =367.3.

[0147] Step 5: Preparation of intermediate 3e

[0148] Intermediate 3e was prepared from intermediate 3d (103 mg, 0.28 mmol) according to step 3 of Example 1, yielding 75 mg of crude product, which was directly used in the next step.

[0149] LCMS:[Ms+H]+ =267.5.

[0150] Step 6: Preparation of Compound 3

[0151] Compound 3 was prepared from intermediate 1e (88 mg, 0.28 mmol) and intermediate 3e (75 mg, 0.28 mmol) according to step 6 of Example 1, with a yield of 48.6%.

[0152] LCMS:[Ms+H] + =560.4.

[0153] 1 H NMR(400MHz, CDCl3)δ8.35(s,1H),8.09–7.82(m,1H),7.77–7.57(m,2H),7.52(d,J=7.0Hz,1H),7.46–7.30(m,2H),4.07–3.70(m,2H),3 .49–3.27(m,3H),3.24–2.81(m,7H),2.81–2.25(m,3H),1.78–1.46(m,4H),1.45–1.27(m,8H),1.16(t,J=6.9Hz,7H),1.09–0.78(m,3H).

[0154] Example 4: Preparation of Compound 4

[0155] Synthetic route of compound 4:

[0156] Step 1: Preparation of intermediate 4a

[0157] tert-butyl-2-aminoethyl(ethyl)carbamate (403 mg, 2.14 mmol) was dissolved in N,N-dimethylformamide (5 mL), and then bicyclo[2.2.1]heptane-1-carboxylic acid (250 mg, 1.78 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (848 mg, 2.23 mmol) and N,N-diisopropylethylamine (1.15 mg, 8.92 mmol) were added. The resulting system was reacted at room temperature for 16 hours under nitrogen protection.

[0158] The reaction was quenched with water (10 mL), the aqueous phase was extracted three times with ethyl acetate (20 mL), the organic phases were combined and washed twice with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness.

[0159] The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 30 / 1) to give 550 mg of intermediate 4a (yield: 99.3%).

[0160] LCMS:[Ms+H] + =311.2.

[0161] Step 2: Preparation of intermediate 4b

[0162] Intermediate 4a (400 mg, 1.29 mmol) was dissolved in tetrahydrofuran (15 mL), and 1 N of borane tetrahydrofuran complex (6.44 mL, 6.44 mmol) was added at 0 °C. The reaction was carried out at 70 °C for 2 hours under nitrogen protection.

[0163] The reaction was quenched by slowly adding methanol (5 mL), and then stirred at 70 °C for 16 hours. The reaction solution was evaporated to dryness, and the residue was azeotropically reacted twice with methanol (30 mL). No further purification was required; the resulting crude product, intermediate 4b, was used directly in the next step.

[0164] LCMS:[Ms+H] + =297.2.

[0165] Step 3: Preparation of intermediate 4c

[0166] Intermediate 4c was prepared from intermediate 4b (380 mg, 1.28 mmol) and paraformaldehyde (385 mg, 12.82 mmol) according to step 2 of Example 1, yielding 560 mg of a colorless oily substance.

[0167] LCMS:[Ms+H] + =311.2.

[0168] Step 4: Preparation of intermediate 4d

[0169] Intermediate 4d was prepared from intermediate 4c (280 mg, 0.90 mmol) according to step 3 of Example 1. The crude product was not further purified and was used directly in the next step.

[0170] LCMS:[Ms+H] + =211.2.

[0171] Step 5: Preparation of Compound 4

[0172] Compound 4 was prepared from intermediate 1e (252 mg, 0.81 mmol) and intermediate 4d (189 mg, 0.90 mmol) according to step 6 of Example 1, with a yield of 16.6%.

[0173] LCMS:[Ms+H] + =504.3.

[0174] 1 H NMR(400MHz, CDCl3)δ8.35(s,1H),7.96(s,1H),7.74–7.49(m,3H),7.42(s,1H),7.31(dd,J=7.7,4.7Hz,1H),3.56–3.22(m,4H),2.98(brs,1H),2. 86–2.71(m,1H),2.69–2.39(m,6H),2.30(s,2H),2.15(s,1H),1.57(t,J= 12.0Hz, 2H), 1.51–1.33 (m, 2H), 1.36–1.11 (m, 9H), 1.05 (t, J = 7.1Hz, 1H).

[0175] Example 5: Preparation of Compound 5

[0176] Synthetic route of compound 5:

[0177] Step 1: Preparation of intermediate 5a

[0178] Benzyl(1R,3r,5S)-3-hydroxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid ester (2 g, 7.66 mmol) was dissolved in pyridine (15 mL), and then methanesulfonyl chloride (2.644 mg, 23.0 mmol) was added at 0 °C. The resulting system was reacted at room temperature for 3 h under nitrogen protection.

[0179] The reaction solution was evaporated to dryness to obtain 2.6 g of crude intermediate 5a, which could be used directly in the next step without further purification.

[0180] LCMS:[Ms+H] + =340.0.

[0181] Step 2: Preparation of intermediate 5b

[0182] Intermediate 5a (2.6 g, 7.66 mmol) was dissolved in ultradry toluene (15 mL), followed by the addition of 1,8-diazacyclo[5,4,0]undecene-7 (2.389 mg, 15.32 mmol). The resulting system was reacted overnight at 100 °C under nitrogen protection.

[0183] The reaction solution was evaporated to dryness. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1) to give 260 mg of intermediate 5b (yield: 13.95%).

[0184] LCMS:[Ms+H] + =244.1.

[0185] Step 3: Preparation of intermediate 5c

[0186] Intermediate 5b (260 mg, 1.07 mmol) was dissolved in methanol (15 mL), and then palladium on carbon (130 mg, 1.22 mmol) was added. The resulting system was reacted overnight at room temperature under hydrogen atmosphere.

[0187] The reaction solution was filtered through diatomaceous earth, and the filtrate was evaporated to dryness to obtain 139 mg of crude intermediate 5c. Without further purification, it was used directly in the next step.

[0188] LCMS:[Ms+H] + =112.3.

[0189] Step 4: Preparation of intermediate 5d

[0190] Intermediate 5d was prepared from tert-butyl ethyl (2-hydroxyethyl)carbamate (579 mg, 3.06 mmol) and intermediate 5c (170 mg, 1.53 mmol) according to the method in step 1 of Example 2, with a yield of 50.72%.

[0191] LCMS:[Ms+H] + =283.2.

[0192] Step 5: Preparation of intermediate 5e

[0193] Intermediate 5e was prepared from intermediate 5d (100 mg, 0.355 mmol) according to step 3 of Example 1, yielding 65 mg of crude product, which was directly used in the next step.

[0194] LCMS:[Ms+H] + =183.2.

[0195] Step 6: Preparation of Compound 5

[0196] Compound 5 was prepared from intermediate 1e (64.6 mg, 0.355 mmol) and intermediate 5e (110 mg, 0.355 mmol) according to step 6 of Example 1, with a yield of 17.92%.

[0197] LCMS:[Ms+H] + =476.2.

[0198] 1H NMR (400MHz, CDCl3) δ8.96 (s, 1H), 8.36 (dd, J = 4.7, 1.5Hz, 1H), 7.94 (d, J = 7.2Hz, 1H), 7. 63(d,J=7.0Hz,2H),7.54(d,J=7.8Hz,1H),7.48–7.29(m,2H),4.04–3.87(m,2H),3.75(s, 1H),3.55–3.35(m,3H),3.17–2.84(m,4H),2.46(t,J=12.8Hz,2H),2.25–2.11(m,2H),1. 82(s,3H),1.65(d,J=10.4Hz,2H),1.49(s,2H),1.42–1.32(m,1H),1.20(t,J=6.9Hz,3H).

[0199] Example 6: Preparation of Compound 6

[0200] Synthetic route of compound 6:

[0201] Step 1: Preparation of intermediate 6a

[0202] Intermediate 6a was prepared from tert-butyl ethyl (2-hydroxyethyl)carbamate (450 mg, 2.38 mmol) and adamantane (300 mg, 1.98 mmol) according to the method in step 1 of Example 2, with a yield of 51.5%.

[0203] LCMS:[Ms+H] + =323.2.

[0204] Step 2: Preparation of intermediate 6b

[0205] Intermediate 6b was prepared from intermediate 6a (330 mg, 1.02 mmol) and paraformaldehyde (307 mg, 10.23 mmol) according to step 2 of Example 1, yielding 300 mg of a colorless oily substance.

[0206] LCMS:[Ms+H] + =337.3.

[0207] Step 3: Preparation of intermediate 6c

[0208] Intermediate 6c was prepared from intermediate 6b (150 mg, 0.44 mmol) according to step 3 of Example 1, yielding 105 mg of crude product, which was directly used in the next step.

[0209] LCMS:[Ms+H] + =237.1.

[0210] Step 4: Preparation of Compound 6

[0211] Compound 6 was prepared from intermediate 1e (138 mg, 0.44 mmol) and intermediate 6c (105 mg, 0.44 mmol) according to step 6 of Example 1, with a yield of 11.4%.

[0212] LCMS:[Ms+H]+=530.4.

[0213] 1 H NMR (400MHz, CDCl3) δ8.33(d,J=3.8Hz,1H),7.91(d,J=8.5Hz,1H),7.75(d,J=7.3Hz,1 H),7.68–7.57(m,1H),7.54(d,J=7.8Hz,1H),7.49–7.36(m,1H),7.36–7.28(m,1H),3. 99(s,1H),3.74(s,1H),3.45(d,J=9.1Hz,2H),3.11(s,1H),2.98–2.55(m,6H),2.53–2 .34(m,2H),2.19(s,3H),2.07–1.79(m,10H),1.20(t,J=7.1Hz,3H),0.97–0.73(m,2H).

[0214] Example 7: Preparation of Compound 7

[0215] Synthetic route of compound 7:

[0216] Step 1: Preparation of intermediate 7a

[0217] Intermediate 7a was prepared from tert-butyl ethyl (2-hydroxyethyl)carbamate (500 mg, 2.65 mmol) and 3-azabicyclo[3.1.0]hexane (132.5 mg, 1.1 mmol) according to step 1 of Example 2, with a yield of 98%.

[0218] LCMS:[Ms+H] + =255.1.

[0219] Step 2: Preparation of intermediate 7b

[0220] Intermediate 7b was prepared from intermediate 7a (227 mg, 0.895 mmol) according to step 3 of Example 1, yielding 140 mg of crude product, which was directly used in the next step.

[0221] LCMS:[Ms+H] + =155.3.

[0222] Step 3: Preparation of Compound 7

[0223] Compound 7 was prepared from intermediate 1e (139 mg, 0.448 mmol) and intermediate 7b (107 mg, 0.895 mmol) according to step 6 of Example 1, with a yield of 40.55%.

[0224] LCMS:[Ms+H] + =448.1.

[0225] 1 H NMR (400MHz, CDCl3) δ9.42(s,1H),8.35(s,1H),7.91(d,J=7.0Hz,1H),7.62(dd,J=15.6,8.1Hz,2H),7.53( d,J=7.3Hz,1H),7.42(t,J=6.8Hz,1H),7.38–7.29(m,1H),4.01(d,J=10.2Hz,1H),3.79(d,J=10.4Hz,2H),3 .57(s,1H),3.43–3.18(m,5H),3.07(s,1H),2.97–2.83(m,1H),2.82–2.68(m,1H),2.59–2.45(m,1H),2.37 (d,J=15.9Hz,1H),1.69(d,J=3.9Hz,2H),1.13(t,J=7.1Hz,3H),0.88(s,1H),0.74(dd,J=14.6,7.5Hz,1H).

[0226] Example 8: Preparation of Compound 8

[0227] Synthetic route of compound 8:

[0228] Step 1: Preparation of intermediate 8a

[0229] Intermediate 8a was prepared from tert-butyl ethyl (2-hydroxyethyl)carbamate (351 mg, 1.86 mmol) and 7-azaspiro[3.5]nonane hydrochloride (200 mg, 1.24 mmol) according to step 1 of Example 2, with a yield of 81.80%.

[0230] LCMS:[Ms+H] + =297.2.

[0231] Step 2: Preparation of intermediate 8b

[0232] Intermediate 8b was prepared from intermediate 8a (150 mg, 0.51 mmol) according to step 3 of Example 1, yielding 99 mg of crude product, which was directly used in the next step.

[0233] LCMS:[Ms+H] + =197.2.

[0234] Step 3: Preparation of Compound 8

[0235] Compound 8 was prepared from intermediate 1e (105 mg, 0.34 mmol) and intermediate 8b (99 mg, 0.51 mmol) according to step 6 of Example 1, with a yield of 12.20%.

[0236] LCMS:[Ms+H] + =490.2.

[0237] 1 H NMR (400MHz, CDCl3) δ8.34(s,1H),7.97(d,J=7.2Hz,1H),7.70–7.52(m,3H),7.42(s,1H),7.30(dd,J=7.9,4.7Hz,1H),3.49–3.26(m,4H),2.97 (s,1H),2.81–2.72(m,1H),2.68–2.58(m,1H),2.52–2.29(m,7H),1.88 –1.81(m,2H),1.73–1.67(m,4H),1.60–1.53(m,4H),1.18–1.00(m,3H).

[0238] Example 9: Preparation of Compound 9

[0239] Synthetic route of compound 9:

[0240] Step 1: Preparation of intermediate 9a

[0241] Intermediate 9a was prepared from tert-butyl-2-aminoethyl(ethyl)carbamate (427 mg, 2.27 mmol) and spiro[3.3]heptane-2-one (250 mg, 2.27 mmol) according to step 1 of Example 1, with a yield of 78.01%.

[0242] LCMS:[Ms+H] + =283.2.

[0243] Step 2: Preparation of intermediate 9b

[0244] Intermediate 9b was prepared from intermediate 9a (500 mg, 1.77 mmol) and paraformaldehyde (532 mg, 17.70 mmol) according to step 2 of Example 1, with a yield of 74.31%.

[0245] LCMS:[Ms+H] + =297.3.

[0246] Step 3: Preparation of intermediate 9c

[0247] Intermediate 9c was prepared from intermediate 9b (100 mg, 0.34 mmol) according to step 3 of Example 1, yielding 67 mg of crude product, which was directly used in the next step.

[0248] LCMS:[Ms+H] + =197.2.

[0249] Step 4: Preparation of Compound 9

[0250] Compound 9 was prepared from intermediate 1e (106 mg, 0.34 mmol) and intermediate 9c (67 mg, 0.34 mmol) according to step 6 of Example 1, with a yield of 33.32%.

[0251] LCMS:[Ms+H] + =490.2.

[0252] 1 H NMR (400MHz, CDCl3) δ8.36(d,J=3.7Hz,1H),7.92(s,1H),7.65(dt,J=14.1,7.0Hz,2H),7.52(d,J=7.7Hz,1H),7.44(s,1H),7.37(dd,J=7.9,4.8Hz,1H ),4.67(s,3H),3.88(s,1H),3.48–3.31(m,3H),2.94–2.69(m,5H),2.46–2. 16(m,5H),2.05–1.88(m,4H),1.84(d,J=7.4Hz,3H),1.16(t,J=6.8Hz,3H).

[0253] Example 10: Preparation of Compound 10

[0254] Synthetic route of compound 10:

[0255] Step 1: Preparation of intermediate 10a

[0256] Intermediate 10a was prepared from tert-butyl-2-aminoethyl(ethyl)carbamate (341 mg, 1.81 mmol) and spiro[3.5]nonane-2-one (250 mg, 1.81 mmol) according to step 1 of Example 1, with a yield of 71.22%.

[0257] LCMS:[Ms+H] + =311.3.

[0258] Step 2: Preparation of intermediate 10b

[0259] Intermediate 10b was prepared from intermediate 10a (400 mg, 1.29 mmol) and paraformaldehyde (387 mg, 12.88 mmol) according to step 2 of Example 1, with a yield of 71.76%.

[0260] LCMS:[Ms+H] + =325.3.

[0261] Step 3: Preparation of intermediate 10c

[0262] Intermediate 10c was prepared from intermediate 10b (150 mg, 0.46 mmol) according to step 3 of Example 1, yielding 104 mg of crude product, which was directly used in the next step.

[0263] LCMS:[Ms+H] + =225.2.

[0264] Step 4: Preparation of Compound 10

[0265] Compound 10 was prepared from intermediate 1e (144 mg, 0.46 mmol) and intermediate 10c (104 mg, 0.46 mmol) according to step 6 of Example 1, with a yield of 26.61%.

[0266] LCMS:[Ms+H] + =518.3.

[0267] 1H NMR (400MHz, CDCl3) δ8.97–8.48(m,1H),8.35(s,1H),7.96(s,1H),7.73–7.51(m,3H),7.43(s,1H),7.30(dd,J=7.8,4.7Hz,1H),3.58–3.24(m,4H), 2.97(s,1H),2.87–2.67(m,2H),2.66–2.57(m,1H),2.54–2.28(m,3H),2.2 6–2.09(m,2H),1.98–1.87(m,2H),1.49–1.30(m,11H),1.21–1.01(m,3H).

[0268] Example 11: Preparation of Compound 11

[0269] Synthetic route of compound 11:

[0270] Step 1: Preparation of intermediate 11a

[0271] Methylenecyclopentane (1.50 g, 18.26 mmol) was dissolved in diethyl ether (15 mL), and zinc powder (2.39 g, 36.52 mmol) was added at 0 °C, followed by the slow dropwise addition of trichloroacetyl chloride (4.98 g, 27.39 mmol). The resulting system was stirred at 40 °C for 1.5 h under nitrogen protection.

[0272] The reaction solution was filtered through diatomaceous earth and washed twice with diethyl ether (30 mL). The organic phases were combined and then washed successively with water (100 mL), saturated sodium bicarbonate aqueous solution (100 mL), and brine (100 mL), dried over anhydrous sodium sulfate, and concentrated. The concentrated product yielded 3.00 g of a colorless, oily crude intermediate 11a, which was directly used in the next reaction step.

[0273] TLC:Rf=0.7(PE / EA=10 / 1).

[0274] Step 2: Preparation of intermediate 11b

[0275] Zinc powder (25.40 g, 388.47 mmol) was dissolved in acetic acid (50 mL), and a solution of intermediate 11a (3.00 g, 15.54 mmol) in acetic acid (10 mL) was slowly added dropwise at room temperature. The resulting mixture was reacted at 60 °C for 2 hours under nitrogen atmosphere.

[0276] The reaction solution was filtered through diatomaceous earth, washed twice with n-pentane (100 mL), and the organic phases were combined. Water (100 mL) was added, followed by extraction with n-pentane (100 mL). The organic phase was washed twice with 1 N sodium hydroxide aqueous solution (50 mL), washed with brine (100 mL), dried over anhydrous sodium sulfate, and concentrated.

[0277] The residue was purified by normal-phase column chromatography (eluent: petroleum ether / ethyl acetate = 20 / 1) to give 300 mg of intermediate 11b (yield: 15.55%).

[0278] 1 H NMR (400MHz, CDCl3) δ2.91 (s, 4H), 1.83–1.65 (m, 8H).

[0279] Step 3: Preparation of intermediate 11c

[0280] Potassium hydroxide (1.08 g, 19.33 mmol) was dissolved in methanol (5 mL) and water (1 mL). A methanol solution (5 mL) of intermediate 11b (200 mg, 1.61 mmol) was slowly added dropwise at 0 °C. After the addition was complete, N-methyl-N-nitroso-p-toluenesulfonamide (449 mg, 2.09 mmol) was added. The resulting mixture was reacted at room temperature under nitrogen atmosphere for 0.5 h.

[0281] Add acetic acid (1 mL) and water (10 mL) to the reaction solution, then extract three times with n-pentane (30 mL), combine the organic phases, wash once with brine (100 mL), dry with anhydrous sodium sulfate, and evaporate to dryness.

[0282] The residue was purified by normal-phase column chromatography (eluent: petroleum ether / ethyl acetate = 20 / 1) to give 130 mg of intermediate 11c (yield: 58.40%).

[0283] 1 H NMR (400MHz, CDCl3) δ1.78–1.47(m,4H),1.38–1.00(m,5H),0.92–0.81(m,5H).

[0284] Step 4: Preparation of intermediate 11d

[0285] Intermediate 11d was prepared from tert-butyl-2-aminoethyl (ethyl)carbamate (180 mg, 0.96 mmol) and intermediate 11c (132 mg, 0.96 mmol) according to step 1 of Example 1, with a yield of 67.38%.

[0286] LCMS:[Ms+H] + =311.2.

[0287] Step 5: Preparation of intermediate 11e

[0288] Intermediate 11e was prepared from intermediate 11d (170 mg, 0.55 mmol) and paraformaldehyde (164 mg, 5.48 mmol) according to step 2 of Example 1, with a yield of 88.50%.

[0289] LCMS:[Ms+H] + =325.2.

[0290] Step 3: Preparation of intermediate 11f

[0291] Intermediate 11f was prepared from intermediate 11e (100 mg, 0.31 mmol) according to step 3 of Example 1, yielding 69 mg of crude product, which was directly used in the next step.

[0292] LCMS:[Ms+H] + =225.2.

[0293] Step 4: Preparation of Compound 11

[0294] Compound 11 was prepared from intermediate 1e (105 mg, 0.34 mmol) and intermediate 11f (69 mg, 0.34 mmol) according to step 6 of Example 1, with a yield of 28.88%.

[0295] LCMS:[Ms+H] + =518.2.

[0296] 1 H NMR (400MHz, CDCl3) δ9.28 (s, 1H), 8.34 (s, 1H), 7.97 (d, J = 7.7Hz, 1H), 7.73 –7.52(m,3H),7.42(s,1H),7.30(dd,J=7.8,4.7Hz,1H),3.55–3.25(m,4H),3 .00(s,1H),2.90–2.71(m,2H),2.69–2.39(m,4H),2.34–2.19(m,3H),1.84( d,J=10.3Hz,1H),1.75–1.65(m,1H),1.60–1.34(m,12H),1.20–1.03(m,3H).

[0297] Example 12: Preparation of Compound 12

[0298] Synthetic route of compound 12:

[0299] Step 1: Preparation of intermediate 12a

[0300] Bicyclo[3.3.0]octane-3,7-dione (2.00 g, 14.48 mmol) was dissolved in toluene (40 mL), followed by the addition of 9H-fluorene-9,9-diethanol (3.28 g, 14.48 mmol) and p-toluenesulfonic acid (249 mg, 1.45 mmol). The resulting system was stirred at 110 °C for 4 hours under nitrogen protection.

[0301] The reaction solution was directly evaporated to dryness. The resulting residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 8 / 1) to give 2.50 g of intermediate 12a (yield: 49.85%).

[0302] TLC:Rf=0.5(PE / EA=4 / 1).

[0303] Step 2: Preparation of intermediate 12b

[0304] Intermediate 12a (2.50 g, 7.22 mmol) was dissolved in diethylene glycol (30 mL), and hydrazine hydrate (4.34 g, 84.60 mmol) and potassium hydroxide (2.02 g, 36.08 mmol) were added. The resulting mixture was reacted at 160 °C for 1 h under nitrogen atmosphere, followed by reaction at 205 °C for 2 h.

[0305] The reaction was quenched with water (50 mL), followed by extraction three times with methyl tert-butyl ether (50 mL), the organic layers were combined, washed once with brine (100 mL), dried over anhydrous sodium sulfate, and then evaporated to dryness.

[0306] The residue was purified by normal-phase column chromatography (eluent: petroleum ether / ethyl acetate = 20 / 1) to give 2.25 g of intermediate 12b (yield: 93.79%).

[0307] TLC:Rf = 0.8 (PE:EA = 10 / 1).

[0308] Step 3: Preparation of intermediate 12c

[0309] Intermediate 12b (450 mg, 1.352 mmol) was dissolved in tetrahydrofuran (10 mL), and 3N hydrochloric acid aqueous solution (4.51 mL, 13.54 mmol) was added. The resulting mixture was reacted at 70 °C for 2 hours under nitrogen atmosphere.

[0310] The reaction was cooled to room temperature, then the pH was adjusted to 8 with a saturated sodium bicarbonate aqueous solution. The mixture was then extracted three times with ethyl acetate (30 mL), the organic layers were combined, washed once with brine (100 mL), dried over anhydrous sodium sulfate, and evaporated to dryness. 158 mg of crude intermediate 12c was obtained and used directly in the next step.

[0311] TLC:Rf = 0.7 (PE:EA = 10 / 1).

[0312] Step 4: Preparation of intermediate 12d

[0313] Intermediate 12d was prepared from tert-butyl-2-aminoethyl (ethyl)carbamate (300 mg, 1.59 mmol) and intermediate 12c (237 mg, 1.91 mmol) according to step 1 of Example 1, with a yield of 52.29%.

[0314] LCMS:[Ms+H] + =297.2.

[0315] Step 5: Preparation of intermediate 12e

[0316] Intermediate 12e was prepared from intermediate 12d (250 mg, 0.84 mmol) and paraformaldehyde (253 mg, 8.43 mmol) according to step 2 of Example 1, with a yield of 99.30%.

[0317] LCMS:[Ms+H] + =311.2.

[0318] Step 3: Preparation of intermediate 12f

[0319] Intermediate 12f was prepared from intermediate 12e (100 mg, 0.32 mmol) according to step 3 of Example 1, yielding 67 mg of crude product, which was directly used in the next step.

[0320] LCMS:[Ms+H] + =211.2.

[0321] Step 4: Preparation of Compound 12

[0322] Compound 12 was prepared from intermediate 1e (99 mg, 0.32 mmol) and intermediate 12f (67 mg, 0.32 mmol) according to step 6 of Example 1, with a yield of 38.41%.

[0323] LCMS:[Ms+H] + =504.3.

[0324] 1H NMR(400MHz, CDCl3)δ8.35(s,1H),7.97(d,J=7.9Hz,1H),7.70–7.51(m,3H),7.42(s,1H),7.30(dd,J=7.9,4.7Hz,1H),3.53–3.26(m ,4H),2.97(s,1H),2.81–2.45(m,6H),2.36(s,3H),2.15–2.00(m,2H),1.81–1.51(m,6H),1.39(d,J=5.0Hz,2H),1.20–0.93(m,5H).

[0325] Example 13: Preparation of Compound 13

[0326] Synthetic route of compound 13:

[0327] Step 1: Preparation of intermediate 13a

[0328] Cyclopentene (3.07 g, 45 mmol) and rhodium dimer acetate (59.7 mg, 0.003 mmol) were dissolved in ether (60 mL), and then a solution of ethyl diazonate (8.85 g, 77.6 mmol) in ether (15 mL) was slowly added dropwise. The addition was completed after 5 hours.

[0329] The reaction solution was evaporated to dryness. The residue was purified by silica gel column chromatography to give 1.2 g of intermediate 13a (yield: 17.27%).

[0330] 1 H NMR (400MHz, CDCl3) δ4.13–4.06(m,2H),1.88–1.51(m,6H),1.41–1.20(m,5H),1.16–0.85(m,1H).

[0331] Step 2: Preparation of intermediate 13b

[0332] Intermediate 13a (600 mg, 3.9 mmol) was dissolved in methanol (20 mL), and sodium hydroxide solution (4 mol / L, 10 mL) was added. The resulting system was reacted overnight at room temperature under nitrogen atmosphere.

[0333] The reaction solution was evaporated to dryness, and the residue was dissolved in water (50 mL). The solution was extracted with ether (50 mL), and the pH of the aqueous phase was adjusted to 2 with 2 mol / L hydrochloric acid. The solution was extracted with ether three times, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to obtain 333 mg of crude intermediate 13b. No further purification was required, and it was used directly in the next step.

[0334] LCMS:[Ms-H]=125.1.

[0335] Step 3: Preparation of intermediate 13c

[0336] Intermediate 13b (360 mg, 2.86 mmol) was dissolved in tert-butanol (15 mL), and triethylamine (317 mg, 3.14 mmol) and diphenyl azidophosphate (864 mg, 3.14 mmol) were added. The resulting mixture was reacted at 80 °C for 48 hours under nitrogen atmosphere.

[0337] The reaction solution was evaporated to dryness, and the residue was added to water (50 mL). The mixture was extracted three times with diethyl ether, and the organic phases were combined. The mixture was then washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness.

[0338] The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10%) to give 140 mg of intermediate 13c (yield: 24.87%).

[0339] LCMS:[Ms+H-56]=142.2.

[0340] Step 4: Preparation of intermediate 13d

[0341] Intermediate 13c (100 mg, 0.5 mmol) was dissolved in N,N-dimethylformamide (5 mL), and sodium hydride (100 mg, 2.5 mmol) was added at 0 °C. The resulting mixture was stirred at room temperature for 1 hour, and then iodomethane (355 mg, 2.5 mmol) was added at 0 °C. The resulting system was then reacted at room temperature under nitrogen atmosphere for another 1 hour.

[0342] The reaction was quenched by adding 10 mL of saturated ammonium chloride solution to the reaction solution, extracted with 200 mL of ethyl acetate, washed twice with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness.

[0343] The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10:1) to give 68 mg of intermediate 13d (yield: 63.49%).

[0344] LCMS:[Ms+H-56]=156.2.

[0345] Step 5: Preparation of intermediate 13e

[0346] Intermediate 13d (88 mg, 0.417 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (1 mL) was added dropwise at 0 °C. The reaction was carried out at room temperature for 1 hour.

[0347] The reaction mixture was evaporated to dryness, and the residue was azeotropically reacted three times with dichloromethane (20 mL). Without further purification, the residue was evaporated to dryness to give 50 mg of intermediate 13e, which was used directly in the next step.

[0348] LCMS:[Ms+H]=112.3.

[0349] Step 6: Preparation of intermediate 13f

[0350] Intermediate 13f was prepared from tert-butyl ethyl (2-hydroxyethyl)carbamate (158 mg, 0.834 mmol) and intermediate 13e (46 mg, 0.417 mmol) according to the method in step 1 of Example 2, with a yield of 98%.

[0351] LCMS:[Ms+H] + =283.3.

[0352] Step 7: Preparation of 13g of intermediate

[0353] Intermediate 13g was prepared from intermediate 13f (130 mg, 0.46 mmol) according to step 3 of Example 1, yielding 85 mg of crude product, which was directly used in the next step.

[0354] LCMS:[Ms+H] + =183.3.

[0355] Step 8: Preparation of Compound 13

[0356] Compound 13 was prepared from intermediate 1e (286 mg, 0.92 mmol) and intermediate 13g (84 mg, 0.46 mmol) according to step 6 of Example 1, with a yield of 44.3%.

[0357] LCMS:[Ms+H] + =476.1.

[0358] 1 H NMR(400MHz, CDCl3)δ8.36(s,1H),8.07–7.84(m,1H),7.75–7.59(m,2H),7. 53(d,J=6.3Hz,1H),7.43(s,1H),7.36(dd,J=7.3,4.8Hz,1H),3.90(s,1H), 3.52–3.08(m,5H),3.05–2.63(m,5H),2.60–2.32(m,2H),2.30–2.12(m,1H) ,1.96(s,2H),1.86–1.58(m,5H),1.17(t,J=7.0Hz,3H),1.09–0.95(m,1H).

[0359] Example 14: Preparation of Compound 14

[0360] Synthetic route of compound 14:

[0361] Step 1: Preparation of intermediate 14a

[0362] 2-Formylpiperidine-1-carboxylic acid tert-butyl ester (213 mg, 1 mmol) was dissolved in methanol (10 mL), and 3-azaspiro[5.5]undecane (153 mg, 1 mmol), acetic acid (120 mg, 2 mmol), and sodium cyanoborohydride (189 mg, 3 mmol) were added sequentially. The mixture was reacted at room temperature under nitrogen atmosphere for 4 hours.

[0363] The reaction was quenched by adding 20 mL of saturated sodium bicarbonate aqueous solution. The aqueous phase was extracted three times with 100 mL of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness.

[0364] The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1) to give 290 mg of intermediate 14a (yield: 82.84%).

[0365] LCMS:[Ms+H] + =351.3.

[0366] Step 2: Preparation of intermediate 14b

[0367] Intermediate 14b was prepared from intermediate 14a (150 mg, 0.428 mmol) according to step 3 of Example 1, yielding 110 mg of crude product, which was directly used in the next step.

[0368] LCMS:[Ms+H] + =251.2.

[0369] Step 3: Preparation of Compound 14

[0370] Compound 14 was prepared from intermediate 1e (133 mg, 0.428 mmol) and intermediate 14b (107 mg, 0.428 mmol) according to step 6 of Example 1, with a yield of 26.24%.

[0371] LCMS:[Ms+H] + =544.2.

[0372] 1H NMR (400MHz, CDCl3) δ9.38(d,J=27.3Hz,1H),8.34(s,1H),7.97(d,J=7.4Hz,1H),7.60( d,J=6.9Hz,3H),7.42(s,1H),7.29(dd,J=7.8,4.7Hz,1H),4.86(s,0.5H),4.44(d,J=13. 1Hz,0.5H),4.05(s,0.5H),3.71(d,J=13.1Hz,0.5H),3.14–2.96(m,1H),2.94–2.55(m,3 H),2.54–2.29(m,7H),1.83–1.68(m,1H),1.65–1.46(m,4H),1.37(s,10H),1.26(s,5H).

[0373] Example 15: Preparation of Compound 15

[0374] Synthetic route of compound 15:

[0375] Step 1: Preparation of intermediate 15a

[0376] 1-(tert-Butoxycarbonyl)pyrrolidine-2-carboxylic acid (300 mg, 1.39 mmol) and 3-azaspiro[5.5]undecane (214 mg, 1.39 mmol) were dissolved in N,N-dimethylformamide (5 mL), followed by the sequential addition of N,N-diisopropylethylamine (721 mg, 5.57 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (636 mg, 3.19 mmol). The reaction was carried out at room temperature for 16 hours under nitrogen protection.

[0377] Dilute the reaction system with water (50 mL) and extract three times with dichloromethane (30 mL). Combine the organic phases, wash with brine (50 mL), dry with anhydrous sodium sulfate, and concentrate.

[0378] The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1) to give 450 mg of intermediate 15a (yield: 92.12%).

[0379] LCMS:[Ms+H] + =351.2.

[0380] Step 2: Preparation of intermediate 15b

[0381] Intermediate 15a (450 mg, 1.28 mmol) was dissolved in tetrahydrofuran (10 mL), and then a 1 N solution of boranetetrahydrofuran (6.42 mL, 6.42 mmol) was added dropwise at room temperature. The resulting system was stirred at 70 °C for 2 hours under nitrogen protection.

[0382] The reaction was quenched by adding methanol (10 mL), and the mixture was stirred overnight at 70 °C. The reaction solution was then directly evaporated to dryness and azeotropically reacted twice with methanol (20 mL). 300 mg of crude intermediate 15b was obtained, which, without further purification, was used directly in the next step.

[0383] LCMS:[Ms+H] + =337.2.

[0384] Step 3: Preparation of intermediate 15c

[0385] Intermediate 15c was prepared from intermediate 15b (100 mg, 0.30 mmol) according to step 3 of Example 1, yielding 70 mg of crude product, which was directly used in the next step.

[0386] LCMS:[Ms+H] + =237.2.

[0387] Step 3: Preparation of Compound 15

[0388] Compound 15 was prepared from intermediate 1e (92 mg, 0.30 mmol) and intermediate 15c (70 mg, 0.30 mmol) according to step 6 of Example 1, with a yield of 65.03%.

[0389] LCMS:[Ms+H] + =530.2.

[0390] 1 H NMR (400MHz, CDCl3) δ8.35(d,J=3.3Hz,1H),8.03–7.79(m,3H),7.72–7.60(m,2H),7.52(d,J=7.2Hz,1H),7.41–7.35(m,1H),4.52–4.26(m,1H) ),3.85–3.38(m,4H),3.20–2.57(m,6H),2.41(t,J=11.7Hz,2H),2.20– 2.05(m,1H),2.01–1.85(m,2H),1.84–1.61(m,4H),1.57–1.21(m,11H).

[0391] Example 16: Detection of the inhibitory activity of the compound against Human M2 and M3 receptors

[0392] Experimental Principle

[0393] Using CHO cell lines stably expressing Human M2 and Human M3 respectively, the effects of compounds on the activity of Human M2 and Human M3 receptors were determined using the FLIPR Calcium 6 Assay Kit. The effects of the test substances on Human M2 and Human M3 were studied based on changes in cell signal intensity, and the corresponding concentration-effect curves were calculated.

[0394] Experimental reagents, consumables and instruments are shown in Tables 1 and 2.

[0395] Table 1: Information on the Sources of Reagents and Consumables

[0396] Table 2: Instrument Information

[0397] Cell Information

[0398] 1. Human M2-CHO and Human M3-CHO cell lines were cultured in F-12 medium containing 10% fetal bovine serum (Fetal Bovine Serum, AusGeneX, FBS500-S) and 0.2 mg / mL Hygromycin B (Solarbio, H8080-1g) at 37°C and 5% carbon dioxide.

[0399] 2. Cell passage: Remove the old culture medium in a biosafety cabinet and wash once with PBS. Then add 1 mL of 0.25% Trypsin-EDTA (Gibco, 25200-072) solution and incubate at 37°C for approximately 2 minutes. When the cells detach from the bottom of the dish, add approximately 5 mL of preheated (37°C) complete culture medium. Gently pipette the cell suspension to separate any aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

[0400] 3. To maintain the physiological activity of the cells, the degree of cell confluence was controlled at around 80% under an inverted microscope (Olympus, CKX53).

[0401] Experimental steps

[0402] 1. Cell Plating: Human M2-CHO and Human M3-CHO cells were digested and collected, resuspended, counted, and seeded into 384-well cell culture plates (Corning, 3764) at a density of 1.2 × 10⁻⁶ cells / well. 4 Cells / 25μL / well. Then incubate the cell plate at 37°C in a 5% CO2 incubator for approximately 16-20 hours;

[0403] 2. Day 2: Prepare the Assay Buffer (20mM HEPES + 1×HBSS) according to the FLIPR Calcium 6 Assay Kit (Molecular Devices, R8191) instructions. After freezing and thawing 20×Component A to room temperature, dilute it to 1×loading buffer with the Assay Buffer and store at room temperature.

[0404] 3. Remove the culture medium from the cell plate, quickly add 35 μL of the above 1× loading buffer to each well, centrifuge using a microplate centrifuge at low speed, and then incubate the cell plate at 37°C in the dark for 120 minutes.

[0405] 4. Prepare working solutions of positive and test compounds in 96-well dilution plates (Biosen, P-0.6-BSA-96-S), and transfer 5 μL into the corresponding cell wells. Incubate at 37°C in the dark for 30 minutes.

[0406] 5. Prepare the agonist working solution and transfer 20 μL / well to a 384-well compound plate (Corning, 264573);

[0407] 6. Place the cell culture plate (Corning, 3764), the 384-well compound plate (Corning, 264573), and the pipette tip into the corresponding positions on the FLIPR instrument, and use... Penta added the 10 μL of agonist diluted in step 5 to each experimental well and collected data at wavelengths of 515 nm to 575 nm.

[0408] 7. By plotting the signal value against the compound concentration, curve fitting and IC50 analysis were performed using the nonlinear regression method in GraphPad Prism software. 50 calculate.

[0409] Data Analysis

[0410] 1) Z'factor = 1 - 3 * (SD) Max +SD Min ) / (AVG Max -AVG Min )

[0411] 2) CV Max = (SD Max / AVG Max )*100%

[0412] 3) CV Min = (SD Min / AVG Min )*100%

[0413] 4) Signal-to-noise ratio S / B = AVG Max / AVG Min

[0414] 5) Calculate the compound IC using the GraphPad nonlinear fitting formula. 50 :

[0415] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope))

[0416] 6) Inhibition rate formula:

[0417] The average value of the positive control group.

[0418] The mean value of the negative control (0.1% DMSO).

[0419] Experimental results

[0420] Based on the above experimental method, using AFDX-116 as a positive control compound, the inhibitory activity of the compound described in this invention on Human M2 and M3 receptors was detected. The data summary is shown in Table 3 below.

[0421] Table 3: Inhibitory effects and selectivity of compounds 1-15 prepared in Examples 1-15 on Human M2 and M3

[0422] As can be seen from Table 3, compounds 1-15 prepared in the embodiments of the present invention all have excellent M2 inhibition and M3 / M2 selectivity.

[0423] Example 17: In vivo efficacy evaluation

[0424] Objective: To evaluate the inhibitory effect of compound 2 on the development of myopia (refractive error) using a guinea pig myopia model. Young guinea pigs are born with hyperopia, which gradually progresses to emmetropia during eye development. Form deprivation accelerates this process. This experiment investigates the effect of compound 2 on delaying the emmetropization process in model young guinea pigs.

[0425] Sample preparation: 0.5 wt% of Compound 2 eye drops were prepared by compound 2 and 10 wt% of sulfobutyl ether-belta-cyclodextrin aqueous solution.

[0426] Grouping: Guinea pigs were divided into three groups based on the refractive value of their right eyes: a negative control group, a model control group, and two compound groups. In the negative control group, no intervention was performed on the eyes of the guinea pigs, and their right eyes developed normally.

[0427] Modeling: Except for the negative control group, all other animals underwent modeling. A myopia model was created using the right eyelid suture technique. The procedure was as follows: After removing hair from the right eye of all animals, a U-shaped suture was used to deprive them of visual perception and accelerate the process of emmetropization. Animals were fed for 28 days after the initial suture.

[0428] Administration: Starting from the day of suturing and modeling, the right eye of each group of animals was treated with eye drops according to the set method (the administration samples are shown in Table 4), 20 μL / eye, once a day, for 28 consecutive days.

[0429] Table 4: Summary of Dosage and Administration Design for Each Group

[0430] Testing: Refractive error was measured using an infrared refractometer (ST-PR.01, Striatech) on days 15 and 29 after modeling.

[0431] The experimental results are shown in Figure 1. Young guinea pigs are born with hyperopia (positive refractive value), which gradually progresses towards emmetropia during eye development, i.e., the refractive value decreases from positive to zero (as shown in the negative control group). The modeling (form deprivation) of this invention accelerates the emmetropia process, eventually resulting in a negative refractive value (myopia). As shown in Figure 1, the slope of emmetropia development in the model control group is significantly greater than that in the negative control group, proving the effectiveness of the modeling, i.e., the modeling accelerates emmetropia. The compound 2 group showed an increase in positive refractive value on day 15, indicating a significant therapeutic effect. On day 15 of administration, the compound 2 group showed a better degree of slowing down the progression of refractive decline than the negative control group. On day 29 of administration, the compound 2 group showed a similar degree of slowing down the progression of refractive decline as the negative control group. It is evident that the compound 2 group accelerated the emmetropia process through modeling, but slowed it down after medication. After 29 days of administration, the degree of emmetropia remained comparable to that of natural eye development (negative group), proving that compound 2 can effectively inhibit the development of myopia.

[0432] In summary, the benzodiazepine derivatives provided by this invention, when used as active components in pharmaceutical compositions, can significantly improve selectivity for M2 receptors, significantly reduce inhibitory effects on M3 receptors, and increase the IC50 of M3 / M2. 50 The ratio increases significantly, exhibiting specific pharmacological properties that can effectively prevent or inhibit the development of myopia without affecting pupil size, and also help improve the targeted nature of treatment and patient comfort.

[0433] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

A benzodiazepine derivative designed based on AI, characterized in that, It has the structure of Formula I: in, R1 is any one of H, deuterium, halogen, or C1-C3 alkoxy group; R2 and R3 are each independently H, deuterium, or a C1-C5 alkyl group; R4 is a polycyclic cycloalkyl group or a polycyclic heterocyclic group; L1 is -(CH2) m -, where m is an integer from 1 to 3; L2 is a single bond or -(CH2) n - where n is 1 or 2. The benzodiazepine derivative designed based on AI as described in claim 1 is characterized in that, L2 is a single bond, and R3 and R4 can form a C4-C bond together with the N bonded to them. 20 The polycyclic heterocyclic structure. The benzodiazepine derivative designed based on AI as described in claim 1 is characterized in that, One of the methylene groups in R2 and L1 can form a C4-C7 heterocyclic structure together with the N group to which it is attached. The AI-designed benzodiazepine derivative as described in any one of claims 1-3 is characterized in that, It has the structure of Formula II: in, R3 is any one of H, deuterium, and C1-C5 alkyl groups; R4 is a polycyclic cycloalkyl group; L2 is a single bond or -(CH2) n - where n is 1 or 2. The benzodiazepine derivative designed based on AI as described in claim 4 is characterized in that, L2 is a single bond, and R3 and R4 can form a C7-C bond together with the N bonded to them. 20 The heterocyclic structure. The benzodiazepine derivative designed based on AI as described in claim 1 is characterized in that, Include: Any one of them. A pharmaceutical composition, characterized in that, It contains: an AI-designed benzodiazepine derivative as described in any one of claims 1-6 as an active ingredient. The pharmaceutical composition according to claim 7, characterized in that, It also includes: pharmaceutical excipients. The use of an AI-designed benzodiazepine derivative, characterized in that, It is used to prepare drugs for preventing and / or inhibiting the development of myopia. The use as described in claim 9, characterized in that, The myopia includes at least one of the following: axial myopia, refractive myopia, pseudomyopia, pathological myopia, simple myopia, extremely high myopia, severe myopia, high myopia, moderate myopia, low myopia, myopia combined with glaucoma, myopia with risk of developing glaucoma, or myopia accompanied by high intraocular pressure.

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