WO2025007853A1 - 2-indolinone compound serving as vegfr and pka dual-target inhibitor, and preparation method therefor and use thereof - Google Patents

Abstract

The present invention relates to a 2-indolinone compound serving as a VEGFR and PKa dual-target inhibitor, and a preparation method therefor and a use thereof. Specifically, the present invention relates to a 2-indolone compound represented by general formula (I), and a preparation method therefor and a use thereof, particularly used for treating or preventing microangiopathy-related diseases mediated by high levels of VEGF protein and PKa protein, such as diabetic macular edema (DME) and age-related macular degeneration (AMD).

Description

2-Indole ketone compounds as dual-target inhibitors of VEGFR and PKa, preparation methods thereof, and uses thereof Technical Field

The present invention belongs to the field of biomedicine, and relates to 2-indolone compounds as dual-target inhibitors of vascular endothelial growth factor receptor (VEGFR) and human plasma kallikrein (PKa), a preparation method thereof, and uses thereof. Specifically, the present invention relates to a 2-indolone compound represented by general formula (I), a preparation method thereof, and uses thereof, especially for treating or preventing microangiopathy-related diseases mediated by high levels of VEGF protein and PKa protein, such as diabetic macular edema (DME) and age-related macular degeneration (AMD).

Background Art

Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes. It is caused by chronic progressive diabetes mellitus, which leads to leakage and blockage of retinal microvessels, resulting in a series of fundus lesions, such as microaneurysms, hard exudates, cotton wool spots, neovascularization, vitreous proliferation, macular edema and even retinal detachment. Long-term hyperglycemia can cause glycosylation of proteins and lipids, and its development changes the molecular structure, resulting in reduced or lost function of enzymes and important cellular components. Hyperglycemia also upregulates the level of intracellular diacylglycerol (DAG), which is a second messenger of lipid signals. DAG acts as a physiological activator of protein kinase C (PKC). Activation of PKC leads to upregulation of VEGF. Once VEGF expression increases, it becomes a driver of the disease, leading to accelerated disease because it stimulates leukocyte arrest, leading to the progression of retinal nonperfusion (RNP), further increasing VEGF expression, leading to a positive feedback loop of disease progression that changes vascular permeability, leading to hypoxia and blood-retinal barrier (BRB) damage and macular edema. According to statistics from the World Health Organization, approximately 285 million people worldwide are visually impaired, of which 4.8% of patients have visual impairment caused by diabetic retinopathy (DR). Diabetic macular edema (DME), as a late symptom of DR, is the main cause of irreversible vision loss in diabetic patients. The number of patients with diabetic macular edema (DME) in China increased from 6.1535 million in 2016 to 6.9201 million in 2020. The compound annual growth rate from 2016 to 2020 is 3.0%; it is expected to grow to 7.890 million in 2025 at an average compound annual growth rate of 2.7% starting from 2020.

The VEGF-VEGFR family is currently the most studied angiogenic regulatory factor, inducing endothelial cell differentiation and angiogenesis. There are five main receptors that bind to VEGF ligands: VEGFR-1, VEGFR-2, VEGFR-3, neuropilin-1 (NRP-1) and NRP-2, of which three vascular endothelial growth factor receptors (VEGFR-1, VEGFR-2, VEGFR-3) are receptor tyrosine kinases.

VEGFR-1 binds to VEGF-A, VEGF-B and PlGF, and is only involved in the differentiation of endothelial cells, not in early embryonic angiogenesis; VEGFR-2 has a higher affinity with low molecular weight VEGF-A isoforms (110-165 amino acid residues) and VEGF-E, and a lower affinity with VEGF-C and VEGF-D; VEGFR-3 preferentially binds to VEGF-C and VEGF-D, and activates downstream signaling pathways, thereby regulating the development and function of lymphatic vessels. VEGFR-2 is a VEGF signal transduction that causes endothelial cell proliferation and increases vascular VEGFR-2 is an important regulatory factor for permeability effect and promotion of angiogenesis, and the affinity between VEGFR-2 and VEGF is greater than that of VEGFR-1. Studies have shown that only VEGFR-2 is expressed in endothelial cells, and activation of VEGFR-2 can effectively stimulate angiogenesis. Therefore, VEGFR-2 is the main target for the development of anti-angiogenic drugs. VEGFR-2 can bind to a variety of VEGF ligands (VEGF-A, C, D, E) to form a homodimer complex, which changes its protein conformation, autophosphorylates tyrosine residues and transmits signals to the downstream. Among them, the main signaling pathways include Ras/Raf/MAPK and PI3K/AKT. When these signaling pathways are activated, they will lead to the activation of transcription factors, interference with the cell cycle, promotion of cell migration, maintenance of cell proliferation, and inhibition of cell apoptosis. On the other hand, cells can activate HIF-1α protein in a hypoxic state, induce VEGF-A transcriptional expression and secretion, bind to VEGFR-2 on the vascular endothelial cells around the cells, and stimulate angiogenic response. Therefore, the effect of reducing angiogenesis can be achieved by inhibiting VEGFR-2 signaling through antibody binding to VEGF-A or VEGFR-2 protein, or by inhibiting VEGFR-2 receptor kinase activity through small molecule inhibitors.

Under normal physiological conditions, angiogenesis is in a relatively dynamic equilibrium state due to the strict regulation of pro-angiogenic factors (VEGF) and anti-angiogenic factors (anti-VEGF). Under pathological conditions, the so-called angiogenesis switch is turned on, the balance of pro-angiogenic factors/anti-angiogenic factors is broken, and pro-angiogenic factors play a leading role. They can cause the proliferation of vascular endothelial cells, promote the formation of new blood vessels, and have the effect of increasing vascular permeability. The VEGF level in the eyes of patients with diabetic macular edema (DME) and age-related macular degeneration AMD will increase abnormally, which will lead to the formation of dysplastic and fragile new blood vessels in the choroid of the eye. This abnormal new blood vessel will quickly enter between the retina and the choroid, causing fundus hemorrhage and fluid leakage, further damaging photoreceptor cells and retinal pigment epithelial cells, causing severe vision loss in patients.

The onset of diabetic macular edema (DME) and age-related macular degeneration (AMD) is also related to the kallikrein-kinin system (KKS) signaling pathway. Plasma prekallikrein (PPK) in the KKS signaling pathway is an abundant serine protease zymogen, which can be converted into catalytically active plasma kallikrein (PKa) by factor XIIa (FXIIa) and participate in inflammatory responses. PKa cleaves kininogen to produce bradykinin. Bradykinin (BK) is a non-peptide that stimulates BK receptors, which are expressed in large quantities in vascular cells, glial cells, and neuronal cells. BK is a strong inflammatory factor, and BK receptor activation can cause (it binds to _B1R and _B2R) to activate BK receptors. R binding can promote the increase of bradykinin levels by stimulating the release of mediators such as eicosanoids, endothelial-derived hyperpolarizing factor, NO, propylene glycol, tissue plasminogen activator (tPA) and glucose transporter-1/2 (glu1/2), further mediating multiple signal cascades. These mediators are related to inflammation, angiogenesis, vasodilation, and increased vascular permeability, which can induce DME formation. Proteomic analysis of vitreous samples from patients with DME showed that PPK and PKa were increased 11.0 times compared with the control vitreous of patients with macular holes (p<0.0001). Research It has been shown that systemic continuous administration of PKa inhibitors can reduce retinal vascular permeability and retinal blood flow abnormalities in diabetic mice. Additional preclinical evidence suggests that plasma kallikrein (PKa) may cause VEGF-mediated retinal edema. Currently, the treatment of DME mainly relies on anti-VEGF therapy, but some patients (about 50%) do not respond to VEGF therapy. This means that 50% of patients cannot receive effective treatment. According to literature reports, systemic administration of PKa inhibition has a significant effect on the treatment of VEGF-induced retinal thickening in mice. The present invention provides an effective method for VEGFR and 2-Indole one compounds, which are dual-targeted small molecule inhibitors of PKa, provide an effective therapeutic solution for diseases such as diabetic macular edema (DME) and age-related macular degeneration (AMD) caused by retinal microvascular disorders.

Summary of the invention

The present invention provides a 2-indolone compound as a dual-target inhibitor of VEGFR and PKa, a preparation method thereof, and uses thereof. Specifically, the present invention provides a preparation method of a 2-indolone compound as a dual-target small molecule inhibitor of VEGFR2 and PKa, and a VEGFR2 ligand is combined with a PKa ligand through a chemical bond to prepare a dual-target small molecule inhibitor of VEGFR2 and PKa. This dual-target inhibitor has the ability to target the "active core" of VEGFR2 and the "active core" of PKa, so that such compounds present a "dual efficacy" against VEGFR2 and PK. The compound is a dual inhibitor of VEGFR2 protein and PKa protein, and can be used as a therapeutic agent for treating conditions characterized by abnormal VEGFR2 and PK expression (e.g., cardiovascular and cerebrovascular diseases, diabetic retinopathy (e.g., diabetic macular edema and age-related macular degeneration, etc.)). In fact, by targeting VEGFR2 and PKa, the compound of the present invention is suitable for treating subjects with high expression of VEGF protein and PKa protein.

Specifically, the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof:

in,

L ₁ is selected from

L ₂ is absent or selected from a linear -(CH ₂ ) _1-22 - group, wherein one or more -(CH ₂ )- in the linear -(CH ₂ ) _1-22 - group is optionally and independently substituted by one or more groups selected from -C(O)-, -O-, -(NR ² )-, -S-, -(C＝C)-, -(C≡C)-, a divalent cycloalkyl group, a divalent aryl group, a divalent heterocyclic group and a divalent heteroaryl group, and L ₂ is optionally substituted by 1, 2 or 3 R ¹ ;

Each R ¹ is independently selected from C ₁ -C ₆ alkyl, halogenated C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogenated C ₁ -C ₆ alkyl, halogen, cyano and hydroxyl,

Each R ² is independently selected from hydrogen, C ₁ -C ₆ alkyl and acyl, wherein said C ₁ -C ₆ alkyl is optionally substituted with 1, 2 or 3 R ¹ ;

The group Ar ₁ is selected from -(CH ₂ )-aryl, -(CH ₂ )-heteroaryl and Wherein ring a is a 5-6 membered heterocyclic ring, the aryl, heteroaryl and Optionally, one or more selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, halogen, cyano, C ₁ -C ₆ alkoxy, halogenated C ₁ -C ₆ alkoxy, amino, hydroxyl, The group substitution;

The group Ar ₂ is selected from -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, cycloalkyl, aryl and heteroaryl, wherein the group Ar ₂ is optionally substituted by one or more selected from halogen, cyano, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ cycloalkyl, C ₁ -C ₆ cycloalkylalkyl, 5-6 membered heterocyclyl and substituted by a group, a 5-6 membered heterocyclic group or Optionally substituted with one or more halogens.

In a particular embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof:

wherein X is selected from an oxygen atom or NR ² ;

L ₂ , R ² , the group Ar ₁ and the group Ar ₂ are as defined above for formula (I).

In a particular embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is a compound represented by formula (III) or a pharmaceutically acceptable salt thereof:

L ₂ , the group Ar ₁ and the group Ar ₂ are as defined above for formula (I).

In a preferred embodiment, the compound represented by formula (I), formula (II) or formula (III) or its pharmaceutical preparation In the above acceptable salts, Ar ₁ is selected from -(CH ₂ )-phenyl, -(CH ₂ )-pyridyl and Wherein ring a is a 5-6 membered heteroaryl group, preferably imidazolyl or pyrazolyl, more preferably for

The phenyl, pyridyl and Optionally, one or more selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, halogen, cyano, C ₁ -C ₆ alkoxy, halogenated C ₁ -C ₆ alkoxy, amino, hydroxyl, The group substitution;

R is selected from hydrogen and C ₁ -C ₆ alkyl.

In another preferred embodiment, in the compound represented by formula (I), formula (II) or formula (III) or a pharmaceutically acceptable salt thereof:

Ar ₂ is selected from C _6-10 aryl, preferably phenyl, 5 to 10 membered heteroaryl, -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, C ₃ -C ₆ cycloalkyl, wherein the C _6-10 aryl, 5 to 10 membered heteroaryl is optionally substituted by one or more selected from halogen, cyano, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ cycloalkyl, C ₁ -C ₆ cycloalkylalkyl, 5-6 membered heterocyclyl and The 5-6 membered heterocyclic group or Optionally substituted with one or more halogens.

In another preferred embodiment, in the compound represented by formula (I), formula (II) or formula (III) or a pharmaceutically acceptable salt thereof, L ₂ is selected from C _1-2 alkylene;

Alternatively, L ₂ is selected from -(CH ₂ ) _t -(C=C)-* and -(CH ₂ ) _t -(C≡C)-*; wherein t is an integer from 0 to 6, preferably an integer from 1 to 4, more preferably 1 or 2; * is the position of connection to the pyrazole ring;

Alternatively, _L2 is selected from wherein X ₁ , X ₂ , X ₃ , and X ₄ are each independently selected from a bond, —O—, —(NR ² )—, —C(O)NH—, —NHC(O)—, —S—, —(C═C)—, —(C≡C)—, a 4-6 membered divalent heterocyclyl, a 3-6 membered divalent cycloalkyl, a 5-10 membered divalent heteroaryl, and a 6-10 membered divalent aryl, wherein the 4-6 membered divalent heterocyclyl, the 3-6 membered divalent cycloalkyl, the 5-10 membered divalent heteroaryl, or the 6-10 membered divalent aryl is optionally substituted by 1, 2, or 3 R ^1s ; each R ² is independently selected from hydrogen, a C ₁ -C ₆ alkyl, and an acyl group, wherein the C 1 -C 6 alkyl is optionally substituted by 1, 2, or 3 R ^1s , each R ¹ is independently selected from a C ₁ -C ₆ alkyl, a halogenated C ₁ -C ₆ alkyl, a C 1 -C 6 alkyl, a C ₁ -C ₆ alkyl, _a -C ₆ alkoxy, halogenated C ₁ -C ₆ alkyl, halogen, cyano and hydroxyl; t ₁ , t ₂ , t ₃ , t ₄ , t ₅ are each independently an integer of 0 to 6; * is the position of connection to the pyrazole ring.

In another preferred embodiment, in the compound represented by formula (I), formula (II) or formula (III) or a pharmaceutically acceptable salt thereof, the divalent heterocyclic group is selected from:

The divalent heteroaryl group is selected from:

In another preferred embodiment, in the compound represented by formula (I), formula (II) or formula (III) or a pharmaceutically acceptable salt thereof, -L ₂ - is selected from:

Wherein: G is a 5-6 membered heteroaryl group, preferably More preferred

_G1 and _G2 are each independently selected from a 4-6 membered heterocyclic group, preferably

p is 1 or 2;

s is an integer from 0 to 6, preferably 0 or 1;

_s1 is an integer from 1 to 10;

s ₂ is 1 or 2, preferably 1;

_s3 and _s4 are each independently selected from an integer of 1 to 6, preferably an integer of 1 to 4, more preferably 1 or 2;

v is an integer from 0 to 4, preferably 0 or 1;

* is the position of connection to the pyrazole ring.

In another preferred embodiment, in the compound represented by formula (I), formula (II) or formula (III) or a pharmaceutically acceptable salt thereof, Ar ₁ is selected from

Each R ³ is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, halogen, C ₁ -C ₆ alkoxy, halogenated C ₁ -C ₆ alkoxy, Preferred are halogen, C ₁ -C ₆ alkoxy, Each R ⁴ is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, halogen, C ₁ -C ₆ alkoxy, halogenated C ₁ -C ₆ alkoxy, amino, hydroxyl, preferably C ₁ -C ₆ alkyl, halogen, C ₁ -C ₆ alkoxy, amino;

R ⁵ is selected from C ₁ -C ₆ alkyl;

m is 0, 1, 2 or 3, preferably 2 or 3, more preferably 3;

n is 0, 1, 2 or 3, preferably 2 or 3, more preferably 3.

In another preferred embodiment, in the compound represented by formula (I), formula (II) or formula (III) or a pharmaceutically acceptable salt thereof, Ar ₂ is selected from C ₁ -C ₆ alkyl,

^R6 is hydrogen or halogen;

R ⁷ is hydrogen or C ₃ -C ₆ cycloalkyl;

R ⁸ is C ₃ -C ₆ cycloalkyl;

Each R ⁹ is independently selected from hydrogen, halogen, cyano, 5-6 membered heterocyclyl, the 5-6 membered heterocyclyl being optionally substituted with one or more halogens;

q is 0, 1, or 2.

Examples of compounds represented by formula (I) include, but are not limited to:

or a pharmaceutically acceptable salt thereof.

The present invention also provides a pharmaceutical composition, which comprises a compound represented by formula (I), formula (II) or formula (III) as described above or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

The present invention also relates to the use of the compound represented by formula (I), formula (II) or formula (III) as described above or its pharmaceutically acceptable salt or a pharmaceutical composition comprising the same in the preparation of a drug for treating or preventing vascular disease-related diseases.

The present invention also relates to a compound represented by formula (I), formula (II) or formula (III) as described above or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same, which is used as a drug.

The present invention also relates to a compound represented by formula (I), formula (II) or formula (III) as described above or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same, which is used for treating or preventing vascular disease-related diseases.

The present invention also relates to a method for treating or preventing vascular disease-related diseases, which comprises administering to a subject in need thereof a therapeutically effective amount of a compound represented by formula (I), formula (II) or formula (III) as described above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same.

In a particular embodiment, the vasculopathy-related condition is selected from diabetic retinopathy (DR), diabetic macular edema (DME), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), macular degeneration, wet age-related macular degeneration (wet AMD), retinopathy of prematurity (ROP), neovascular glaucoma, retinitis pigmentosa (RP), proliferative and non-proliferative retinopathy, retinal angiomatous proliferation, macular telangiectasia, ischemic retinopathy, iris neovascularization, intraocular neovascularization, corneal neovascularization, retinal neovascularization, polypoidal choroidal vasculopathy (PCV), choroidal neovascularization and retinal degeneration. In particular, the vasculopathy-related condition is selected from diabetic retinopathy (DR), diabetic macular edema (DME), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), wet age-related macular degeneration (wet AMD). In particular, the vasculopathy-related condition is selected from hereditary angioedema (HAE) and brain edema after stroke. In particular, the vasculopathy-related disorder is selected from diabetic complications of retinal vascular permeability associated with diabetic retinopathy and diabetic macular edema.

Preferably, the vascular disease-related condition is selected from diabetic macular edema (DME), wet age-related Macular degeneration (AMD), non-exudative choroidal neovascularization (CNV), hereditary angioedema (HAE), and brain edema after stroke.

Terminology

Unless stated otherwise, the terms used in the specification and claims have the following meanings.

The term "alkyl" refers to a saturated straight or branched aliphatic hydrocarbon group having 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) carbon atoms (i.e., _C1-20 alkyl). The alkyl group is preferably an alkyl group having 1 to 12 carbon atoms (i.e., _C1-12 alkyl), and more preferably an alkyl group having 1 to 6 carbon atoms (i.e., _C1-6 alkyl). Non-limiting examples include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2 ,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched chain isomers thereof.

The term "alkylene" refers to a divalent alkyl group, wherein the alkyl group is as defined above, and has 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) carbon atoms (i.e., _C1-20 alkylene). The alkylene group is preferably an alkylene group having 1 to 12 carbon atoms (i.e., _C1-12 alkylene), and more preferably an alkylene group having 1 to 6 carbon atoms (i.e., _C1-6 alkylene). Non-limiting examples include: _-CH2- , -CH( _CH3 )-, _-C ( _CH3 ) _2- , _-CH2CH2- , -CH _{( CH2CH3 ) - , -CH2CH} ( _CH3 )-, _-CH2C (CH3) _{2- , -CH2CH2CH2- , -CH2CH2CH2CH2- ,} and the like.

The term "alkenyl" refers to an alkyl group containing at least one carbon-carbon double bond in the molecule, wherein the definition of alkyl is as described above, and it has 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) carbon atoms (i.e., _C2-12 alkenyl). The alkenyl group is preferably an alkenyl group having 2 to 6 carbon atoms (i.e., _C2-6 alkenyl). Non-limiting examples include: vinyl, propenyl, isopropenyl, butenyl, etc.

The term "alkynyl" refers to an alkyl group containing at least one carbon-carbon triple bond in the molecule, wherein the alkyl group is as defined above and has 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) carbon atoms (i.e., _C2-12 alkynyl). The alkynyl group is preferably an alkynyl group having 2 to 6 carbon atoms (i.e., _C2-6 alkynyl). Non-limiting examples include: ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc.

The term "alkoxy" refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples include methoxy, ethoxy, propoxy, butoxy, and the like.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic all-carbon ring (i.e., monocyclic cycloalkyl) or polycyclic ring system (i.e., polycyclic cycloalkyl) having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., _C3-20 cycloalkyl). The cycloalkyl is preferably a cycloalkyl having 3 to 12 ring atoms (i.e., _C3-12 cycloalkyl), more preferably a cycloalkyl having 3 to 8 ring atoms (i.e., _C3-8 cycloalkyl), a cycloalkyl having 4 to 7 ring atoms (i.e., _C4-7 cycloalkyl), or a cycloalkyl having 3 to 6 ring atoms (i.e., _C3-6 cycloalkyl).

Non-limiting examples of the monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl and cyclooctyl.

The polycyclic cycloalkyl group includes: spirocycloalkyl group, fused cycloalkyl group and bridged cycloalkyl group.

The term "divalent cycloalkyl" refers to a cycloalkyl group with one hydrogen atom removed, which has two sites of attachment.

The term "spirocycloalkyl" refers to a polycyclic system in which one carbon atom (called spiro atom) is shared between the rings, and the rings may contain one or more double bonds, or the rings may contain one or more heteroatoms selected from nitrogen, oxygen and sulfur (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxidized, i.e., to form sulfoxides or sulfones, but not including -OO-, -OS- or -SS-), provided that at least one all-carbon ring is contained and the connection point is on the all-carbon ring, and it has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., 5 to 20-membered spirocycloalkyl). The spirocycloalkyl preferably has 6 to 14 ring atoms (i.e., 6 to 14-membered spirocycloalkyl), and more preferably has 7 to 10 ring atoms (i.e., 7 to 10-membered spirocycloalkyl). The spirocycloalkyl includes monospirocycloalkyl and polyspirocycloalkyl (such as bispirocycloalkyl, etc.), preferably monospirocycloalkyl or bispirocycloalkyl, more preferably 3-yuan/4-yuan, 3-yuan/5-yuan, 3-yuan/6-yuan, 4-yuan/4-yuan, 4-yuan/5-yuan, 4-yuan/6-yuan, 5-yuan/3-yuan, 5-yuan/4-yuan, 5-yuan/5-yuan, 5-yuan/6-yuan, 5-yuan/7-yuan, 6-yuan/3-yuan, 6-yuan/4-yuan, 6-yuan/5-yuan, 6-yuan/6-yuan, 6-yuan/7-yuan, 7-yuan/5-yuan or 7-yuan/6-yuan monospirocycloalkyl. Non-limiting examples include:

The term "fused cycloalkyl" refers to a polycyclic system in which two adjacent carbon atoms are shared between the rings, which is a monocyclic cycloalkyl fused to one or more monocyclic cycloalkyls, or a monocyclic cycloalkyl fused to one or more heterocyclyls, aryls or heteroaryls, wherein the point of attachment is on the monocyclic cycloalkyl, which may contain one or more double bonds within the ring, and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., 5 to 20-membered fused cycloalkyl). The fused cycloalkyl preferably has 6 to 14 ring atoms (i.e., 6 to 14-membered fused cycloalkyl), and more preferably has 7 to 10 ring atoms (i.e., 7 to 10-membered fused cycloalkyl). The fused cycloalkyl includes bicyclic fused cycloalkyl and polycyclic fused cycloalkyl (such as tricyclic fused cycloalkyl, tetracyclic fused cycloalkyl, etc.), preferably bicyclic fused cycloalkyl or tricyclic fused cycloalkyl, more preferably 3 yuan/4 yuan, 3 yuan/5 yuan, 3 yuan/6 yuan, 4 yuan/4 yuan, 4 yuan/5 yuan, 4 yuan/6 yuan, 5 yuan/3 yuan, 5 yuan/4 yuan, 5 yuan/5 yuan, 5 yuan/6 yuan, 5 yuan/7 yuan, 6 yuan/3 yuan, 6 yuan/4 yuan, 6 yuan/5 yuan, 6 yuan/6 yuan, 6 yuan/7 yuan, 7 yuan/5 yuan or 7 yuan/6 yuan bicyclic fused cycloalkyl. Non-limiting examples include:

The term "bridged cycloalkyl" refers to a full carbon polycyclic system that shares two carbon atoms that are not directly connected between the rings, which may contain one or more double bonds in the ring and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) carbon atoms (i.e., 5 to 20-membered bridged cycloalkyl). The bridged cycloalkyl preferably has a bridged cycloalkyl of 6 to 14 carbon atoms (i.e., 6 to 14-membered bridged cycloalkyl), and more preferably has a bridged cycloalkyl of 7 to 10 carbon atoms (i.e., 7 to 10-membered bridged cycloalkyl). The bridged cycloalkyl includes bicyclic bridged cycloalkyl and polycyclic bridged cycloalkyl (e.g., tricyclic bridged cycloalkyl, tetracyclic bridged cycloalkyl, etc.), preferably bicyclic bridged cycloalkyl or tricyclic bridged cycloalkyl. Non-limiting examples include:

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic heterocycle (i.e., a monocyclic heterocyclyl) or a polycyclic heterocyclic ring system (i.e., a polycyclic heterocyclyl), which contains at least one (e.g., 1, 2, 3 or 4) heteroatoms selected from nitrogen, oxygen and sulfur (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxidized, i.e., to form sulfoxides or sulfones, but does not include -O-O-, -O-S- or -S-S-), and has 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., a 3- to 20-membered heterocyclyl). The heterocyclic group is preferably a heterocyclic group having 3 to 12 ring atoms (i.e., a 3- to 12-membered heterocyclic group); further preferably a heterocyclic group having 3 to 8 ring atoms (i.e., a 3- to 8-membered heterocyclic group) or a heterocyclic group having 4 to 7 ring atoms (i.e., a 4- to 7-membered heterocyclic group); more preferably a heterocyclic group having 3 to 6 ring atoms (i.e., a 3- to 6-membered heterocyclic group) or a heterocyclic group having 5 or 6 ring atoms (i.e., a 5- or 6-membered heterocyclic group).

Non-limiting examples of the monocyclic heterocyclic group include pyrrolidinyl, tetrahydropyranyl, dihydropyridyl, tetrahydropyridyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl and homopiperazinyl.

The polycyclic heterocyclic group includes a spiro heterocyclic group, a fused heterocyclic group and a bridged heterocyclic group.

The term "divalent heterocyclic group" refers to a heterocyclic group with one hydrogen atom removed, which has two attachment sites.

The term "spiroheterocyclyl" refers to a polycyclic heterocyclic ring system in which the rings share one atom (called a spiro atom), which may contain one or more double bonds in the ring and at least one (e.g., 1, 2, 3 or 4) heteroatom selected from nitrogen, oxygen and sulfur in the ring (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxidized, i.e., to form sulfoxides or sulfones, but excluding -OO-, -OS- or -SS-), provided that it contains at least one monocyclic heterocyclic group and the point of attachment is on the monocyclic heterocyclic group, which has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., a 5- to 20-membered spiroheterocyclyl). The spiro heterocyclic group preferably has 6 to 14 ring atoms (i.e., 6 to 14-membered spiro heterocyclic group), and more preferably has 7 to 10 ring atoms (i.e., 7 to 10-membered spiro heterocyclic group). The spiro heterocyclic group includes a monospiro heterocyclic group and a polyspiro heterocyclic group (such as a bispiro heterocyclic group, etc.), preferably a monospiro heterocyclic group or a bispiro heterocyclic group, and more preferably a 3-membered/4-membered, 3-membered/5-membered, 3-membered/6-membered, 4-membered/4-membered, 4-membered/5 ... 6-membered, 5-membered, 3-membered, 5-membered, 4-membered, 5-membered, 5-membered, 5-membered, 5-membered, 7-membered, 6-membered, 3-membered, 6-membered, 4-membered, 6-membered, 5-membered, 6-membered, 6-membered, 7-membered, 7-membered, 5-membered, or 7-membered monospiro heterocyclic group. Non-limiting examples include:

wait.

The term "fused heterocyclyl" refers to a polycyclic heterocyclic ring system that shares two adjacent atoms between the rings, which may contain one or more double bonds in the ring, and which contains at least one (e.g., 1, 2, 3 or 4) heteroatoms selected from nitrogen, oxygen and sulfur in the ring (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxidized, i.e., to form sulfoxides or sulfones, but does not include -O-O-, -O-S- or -S-S-), which is a monocyclic heterocyclyl fused to one or more monocyclic heterocyclyls, or a monocyclic heterocyclyl fused to one or more of cycloalkyl, aryl or heteroaryl, wherein the point of attachment is on the monocyclic heterocyclyl, and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., a 5- to 20-membered fused heterocyclyl). The fused heterocyclic group preferably has a fused heterocyclic group of 6 to 14 ring atoms (i.e., a 6 to 14-membered fused heterocyclic group), and more preferably has a fused heterocyclic group of 7 to 10 ring atoms (i.e., a 7 to 10-membered fused heterocyclic group). The fused heterocyclic group includes bicyclic and polycyclic fused heterocyclic groups (such as tricyclic fused heterocyclic groups, tetracyclic fused heterocyclic groups, etc.), preferably bicyclic fused heterocyclic groups or tricyclic fused heterocyclic groups, more preferably 3 yuan/4 yuan, 3 yuan/5 yuan, 3 yuan/6 yuan, 4 yuan/4 yuan, 4 yuan/5 yuan, 4 yuan/6 yuan, 5 yuan/3 yuan, 5 yuan/4 yuan, 5 yuan/5 yuan, 5 yuan/6 yuan, 5 yuan/7 yuan, 6 yuan/3 yuan, 6 yuan/4 yuan, 6 yuan/5 yuan, 6 yuan/6 yuan, 6 yuan/7 yuan, 7 yuan/5 yuan or 7 yuan/6 yuan bicyclic fused heterocyclic groups. Non-limiting examples include:

wait.

The term "bridged heterocyclic group" refers to a polycyclic heterocyclic ring system that shares two atoms that are not directly connected between the rings, which may contain one or more double bonds in the ring, and at least one (e.g., 1, 2, 3 or 4) heteroatoms selected from nitrogen, oxygen and sulfur in the ring (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxidized, i.e., to form sulfoxides or sulfones, but does not include -OO-, -OS- or -SS-), and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., a 5- to 20-membered bridged heterocyclic group). The bridged heterocyclic group preferably has a bridged heterocyclic group with 6 to 14 ring atoms. Cyclic group (i.e. 6 to 14-membered bridged heterocyclic group), more preferably a bridged heterocyclic group having 7 to 10 ring atoms (i.e. 7 to 10-membered bridged heterocyclic group). According to the number of constituent rings, it can be divided into bicyclic bridged heterocyclic group and polycyclic bridged heterocyclic group (such as tricyclic bridged heterocyclic group, tetracyclic bridged heterocyclic group, etc.), preferably bicyclic bridged heterocyclic group or tricyclic bridged heterocyclic group. Non-limiting examples include:

wait.

The term "aryl" refers to a monocyclic all-carbon aromatic ring (i.e., monocyclic aromatic group) or a polycyclic aromatic ring system (i.e., polycyclic aromatic group) having a conjugated π electron system, which has 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13 or 14) ring atoms (i.e., 6 to 14-membered aromatic group). The aryl group is preferably an aromatic group having 6 to 10 ring atoms (i.e., 6 to 10-membered aromatic group), and more preferably an aromatic group having 8 to 10 ring atoms (i.e., 8 to 10-membered polycyclic aromatic group). The monocyclic aromatic group is, for example, phenyl. Non-limiting examples of the polycyclic aromatic group include: naphthyl, anthracenyl, phenanthrenyl, etc.

The term "divalent aryl" refers to an aryl group with one hydrogen atom removed, which has two sites of attachment.

The term "heteroaryl" refers to a monocyclic heteroaromatic ring (i.e., a monocyclic heteroaryl) or a polycyclic heteroaromatic ring system (i.e., a polycyclic heteroaryl) having a conjugated π electron system, which contains at least one (e.g., 1, 2, 3 or 4) heteroatoms selected from nitrogen, oxygen and sulfur (the nitrogen may be optionally oxidized, i.e., to form a nitrogen oxide; the sulfur may be optionally oxidized, i.e., to form a sulfoxide or sulfone, but does not include -O-O-, -O-S- or -S-S-), and has 5 to 14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14) ring atoms (i.e., a 5- to 14-membered heteroaryl). The heteroaryl group is preferably a heteroaryl group having 5 to 10 ring atoms (i.e. a 5- to 10-membered heteroaryl group), more preferably a heteroaryl group having 5 or 6 ring atoms (i.e. a 5- or 6-membered monocyclic heteroaryl group), or preferably a heteroaryl group having 8 to 10 ring atoms (i.e. an 8- to 10-membered polycyclic heteroaryl group).

The monocyclic heteroaryl group includes, but is not limited to, furanyl, thienyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furazanyl, pyrrolyl, N-alkylpyrrolyl, pyridyl, pyrimidinyl, pyridonyl, N-alkylpyridone (e.g. etc.), pyrazinyl, pyridazinyl, etc.

Non-limiting examples of the polycyclic heteroaryl group include indolyl, indazolyl, quinolyl, isoquinolyl, quinoxalinyl, phthalazinyl, benzimidazolyl, benzothiophenyl, benzofuranyl, quinazolinyl, carbazolyl, pyrrolotriazinyl and the like.

The term "divalent heteroaryl" refers to a heteroaryl group with one hydrogen atom removed, which has two sites of attachment.

The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

The term "haloalkoxy" refers to an alkoxy group substituted with one or more halogens, wherein alkoxy is as defined above.

The term "hydroxyalkyl" refers to an alkyl group substituted with one or more hydroxy groups, wherein alkyl is as defined above.

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

The term "hydroxy" refers to -OH.

The term "thiol" refers to -SH.

The term "amino" refers to _-NH2 .

The term "cyano" refers to -CN.

The term "nitro" refers to _-NO2 .

The term "oxo" or "oxo" refers to "=0".

The term "carbonyl" refers to C=O.

The term "carboxy" refers to -C(O)OH.

The compounds of the present invention may exist in specific stereoisomeric forms. The term "stereoisomer" refers to isomers with the same structure but different arrangements of atoms in space. It includes cis and trans (or Z and E) isomers, (-)- and (+)-isomers, (R)- and (S)-enantiomers, diastereomers, (D)- and (L)-isomers, tautomers, atropisomers, conformers and mixtures thereof (such as racemates, mixtures of diastereomers). The substituents in the compounds of the present invention may have additional asymmetric atoms. All of these stereoisomers and their mixtures are included within the scope of the present invention. Optically active (-)- and (+)-isomers, (R)- and (S)-enantiomers and (D)- and (L)-isomers may be prepared by chiral synthesis, chiral reagents or other conventional techniques. An isomer of a compound of the present invention can be prepared by asymmetric synthesis or chiral auxiliary, or, when the molecule contains a basic functional group (such as amino) or an acidic functional group (such as carboxyl), it can be formed into a diastereoisomer salt with an appropriate optically active acid or base, and then the diastereoisomers can be separated by conventional methods known in the art to obtain pure isomers. In addition, the separation of enantiomers and diastereoisomers is usually achieved by chromatography.

In the chemical structure of the compound described in the present invention, the bond Indicates that the configuration is not specified, that is, if there are chiral isomers in the chemical structure, the bond Can be or or include both and Two configurations.

"Optional" or "optional" means that the event or environment described later can but does not necessarily occur, and includes both situations where the event or environment occurs or does not occur. For example, "alkyl optionally substituted by halogen or cyano" includes the situation where alkyl is substituted by halogen or cyano and the situation where alkyl is not substituted by halogen and cyano.

"Substitution" or "substituted" refers to one or more hydrogen atoms in a group, preferably 1-6, more preferably 1-3, and even more preferably 1-2 hydrogen atoms, which are independently replaced by a corresponding number of substituents. Those skilled in the art can determine possible or impossible substitutions (by experiment or theory) without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated bond (such as an alkene).

"Pharmaceutical composition" means a mixture containing one or more compounds described herein or their pharmaceutically acceptable salts and other chemical components, as well as other components such as pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

"Pharmaceutically acceptable salts" refer to salts of the compounds of the present invention, which can be selected from inorganic salts or organic salts. Such salts are safe and effective when used in mammals and have the desired biological activity. The pharmaceutically acceptable salts may be prepared during the final separation and purification of the compound, or separately by reacting a suitable group with a suitable base or acid. Bases commonly used to form pharmaceutically acceptable salts include inorganic bases, such as sodium hydroxide and potassium hydroxide, and organic bases, such as ammonia. Acids commonly used to form pharmaceutically acceptable salts include inorganic acids and organic acids.

As used herein, the term "treat" includes treating a disease or condition in a mammal, particularly a human, and includes: (a) inhibiting an infection, disease or condition, i.e., arresting or delaying the development of an infection, disease or condition; (b) alleviating an infection, disease or condition, i.e., causing regression of the disease or condition, and/or (c) curing an infection, disease or condition.

As used herein, the term "prevention" includes prophylactic therapy in mammals, especially humans, to reduce the likelihood of infection, disease or condition. Patients who receive prophylactic therapy can be selected based on their increased risk of infection or disease or condition compared to the general population. "Prevention" can include treatment of subjects who have not yet presented with an infection or clinical condition, and prevention of a second occurrence of the same or similar infection or clinical condition.

With respect to a drug or pharmacologically active agent, the term "therapeutically effective amount" refers to an amount of the drug or agent sufficient to achieve or at least partially achieve the desired effect. The determination of a therapeutically effective amount varies from person to person, depending on the age and general condition of the recipient and on the specific active substance, and the appropriate therapeutically effective amount in an individual case can be determined by a person skilled in the art based on routine experiments.

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

As used herein, the singular form of "a," "an," and "the" include plural references and vice versa unless the context clearly dictates otherwise.

When the term "about" is applied to a parameter such as pH, concentration, temperature, etc., it indicates that the parameter can vary by ±10%, and sometimes more preferably within ±5%. As will be understood by those skilled in the art, when a parameter is not critical, numbers are generally given only for illustrative purposes and not for limitation.

It should be noted that, unless otherwise explicitly stated, the description "independently selected" used in the present invention should be understood in a broad sense, meaning that each individual described is selected independently of each other. Therefore, each substituent may be the same as or different from other substituents. In more detail, the description "independently selected" can mean that in different groups, the specific options expressed by the same symbols do not affect each other; it can also mean that in the same group, the specific options expressed by the same symbols do not affect each other.

DETAILED DESCRIPTION

The following examples are only used to illustrate the present invention, but not to limit the present invention. Modifications, changes, variations, etc. made within the protection scope of the present invention are all within the protection scope of the present invention.

The compounds provided by the present invention can be synthesized from readily available The starting materials can be prepared by using or synthesizing methods known in the art, or can be purchased commercially. The experimental methods in the following examples without specifying specific conditions can be determined by those skilled in the art through conventional optimization procedures according to conventional methods and conditions.

The structure of the compound is determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). NMR shifts are given in units of 10 ^-6 (ppm). NMR measurements are performed using a Brukerdps400 NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ), deuterated methanol (CD ₃ OD) as the measuring solvent, and tetramethylsilane (TMS) as the internal standard.

LC-MS measurements were performed using an 1100 Series LC/MSD Trap (ESI) mass spectrometer (manufacturer: Agilent).

GC-MS determination was performed using GCMS-QP2010 SE.

The preparative liquid chromatography method used LC3000 high performance liquid chromatograph and LC6000 high performance liquid chromatograph (manufacturer: Chuangxin Tongheng). The column chromatography method used Daisogel C18 10μm 60A (20mm×250mm).

High performance liquid chromatography (HPLC) was performed using Shimadzu LC-20AD high pressure liquid chromatograph (Agilent TC-C18 250×4.6mm 5μm column chromatography) and Shimadzu LC-2010AHT high pressure liquid chromatograph (Phenomenex C18 250×4.6mm 5μm column chromatography).

The thin layer chromatography silica gel plate used was Qingdao Ocean Chemical GF254 silica gel plate. The silica gel plate used in thin layer chromatography (TLC) had a specification of 0.15 mm to 0.2 mm, and the specification used for thin layer chromatography separation and purification products was 0.4 mm to 0.5 mm.

Column chromatography generally uses Qingdao marine silica gel 100-200 mesh and 200-300 mesh silica gel as the carrier.

The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from online shopping malls, Beijing Coupling, Sigma, Bailingwei, Yishiming, Shanghai Shuya, Inokai, Nanjing Yaoshi, Anaiji Chemical and other companies.

Unless otherwise specified in the examples, the reactions can be carried out under an argon atmosphere or a nitrogen atmosphere.

Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1L.

Microwave reactions were performed using a CEM Discover SP microwave reactor.

Unless otherwise specified in the examples, the solution refers to an aqueous solution.

Unless otherwise specified in the examples, the reaction temperature is room temperature, particularly 20°C to 30°C.

The reaction progress in the embodiment is monitored by thin layer chromatography (TLC), and the developing solvent systems used in the reaction are: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether and ethyl acetate system, D: acetone, and the volume ratio of the solvent is adjusted according to the polarity of the compound.

The eluent system of column chromatography and the developing solvent system of thin layer chromatography used for purifying compounds include: A: dichloromethane and methanol system, B: petroleum ether, ethyl acetate and dichloromethane system, C: petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.

Example

Preparation Example 1: Preparation of tert-butyl 2-(2-hydroxyethoxy)ethoxyacetate (Intermediate 1)

Step 1: Under nitrogen atmosphere, compound Int1-1 (10.0 g, 94.34 mmol) was added to THF (50 mL), cooled to 0°C, sodium hydride (8.3 g, 0.21 mol, 60% purity) was added in batches, and stirred at 0°C for 1 h. Benzyl bromide (16.1 g, 94.34 mmol) dissolved in THF (50 mL) was slowly added dropwise, and the temperature was raised to 70°C and stirred for 16 h. The mixture was cooled to room temperature, saturated aqueous ammonium chloride solution (200 mL) was added, and the THF was removed by concentration under reduced pressure. The mixture was extracted with ethyl acetate (100 mL×3), the organic phases were combined, washed with water (200 mL) and saturated aqueous sodium chloride solution (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (ethyl acetate: petroleum ether = 0-100%) to obtain compound Int1-2 (10.0 g, yield: 56%), a yellow oily liquid.

MS(ESI)M/Z: 219.10[M+Na] ⁺ .

¹ H NMR (400MHz, CHCl ₃ -d) δ7.38–7.26(m,5H), 4.58(s,2H), 3.76–3.68(m,4H), 3.66–3.60(m,4H).

Step 2: Under nitrogen atmosphere, compound Int1-2 (5.0 g, 25.51 mmol) was added to THF (25 mL), cooled to 0°C, sodium hydride (8.3 g, 30.61 mmol) was added in batches, and stirred at 0°C for 1 h. Tert-butyl 2-bromoacetate (1.2 g, 94.34 mmol) dissolved in THF (50 mL) was slowly added dropwise, and stirred at room temperature for 16 h. Saturated aqueous ammonium chloride solution (100 mL) was added, and THF was removed by concentration under reduced pressure. The organic phases were combined, washed with water (200 mL) and saturated aqueous sodium chloride solution (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (ethyl acetate: petroleum ether = 0-100%) to obtain compound Int1-3 (3.0 g, yield: 38%), a yellow oily liquid.

MS (ESI) M/Z: 311.2 [M+H] ⁺ .

¹ H NMR (400MHz, CHCl ₃ -d) δ7.37–7.25(m,5H),4.57(s,2H),4.03(s,2H),3.81–3.58(m,8H),1.47(s,9H) ).

Step 3: Under nitrogen atmosphere, compound Int1-3 (3.0 g, 9.68 mmol), acetic acid (580 mg, 9.68 mmol) and Pd/C (10%, 300 mg) were added to ethanol in sequence, replaced with hydrogen three times, and reacted at room temperature for 16 h. Filter, collect the filtrate, and concentrate under reduced pressure to obtain a residue, which was separated and purified by column chromatography (ethyl acetate: petroleum ether = 0-100%) to obtain intermediate 1 (2.0 g, yield: 94%), a yellow oily liquid.

MS(ESI)M/Z: 243.10[M+Na] ⁺ .

¹ H NMR (400MHz, CDCl3-d) δ4.01 (s, 2H), 3.75–3.68 (m, 6H), 3.64–3.60 (m, 2H), 1.47 (s, 9H).

Preparation Example 2: 3-(Bromomethyl)-1-(4-((2-oxopyridin-1(2H)yl)methyl)benzyl)-1H-pyrazole-4-carboxylic acid Preparation of ester (Intermediate 2)

Step 1: At room temperature, compound Int2-1 (10.0 g, 42.02 mmol) and DMF-DMA (5.0 g, 42.02 mmol) were added to DMF (100 mL) in sequence and stirred at 110°C for 16 h. The mixture was cooled to room temperature and concentrated under reduced pressure to obtain a crude compound Int2-2 (13.0 g) as a yellow oily liquid. The mixture was used directly in the next step without further purification.

MS (ESI) M/Z: 292.1 [M+H] ⁺ .

Step 2: At room temperature, compound Int2-2 (13.0 g, 42.02 mmol), HOAc (2.5 g, 42.02 mmol) and N-tert-butyl carbonyl hydrazine (80%, 2.6 g, 42.02 mmol) were added to EtOH (100 mL) in sequence, and the temperature was raised to 80° C. and stirred for 16 h. The mixture was cooled to room temperature and concentrated under reduced pressure to obtain a residue, which was separated and purified by column chromatography (ethyl acetate: petroleum ether = 0-100%) to obtain compound Int2-3 (8.9 g, yield: 82%), a yellow oily liquid.

MS (ESI) M/Z: 261.1 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ13.42(d,J＝70.4Hz,1H),8.06(d,J＝153.2Hz,1H),7.44–7.20(m,5H),4.74(s,2H),4.56(s,2H), 4.19(q,J=7.2Hz,2H), 1.24(t,J=7.2Hz,3H).

Step 3: Under nitrogen atmosphere, compound Int2-3 (8.0 g, 30.77 mmol), acetic acid (1.8 g, 30.77 mmol) and Pd/C (10%, 1.0 g) were added to ethanol in sequence, replaced with hydrogen three times, and reacted at room temperature for 16 h. Filter, collect the filtrate, and concentrate under reduced pressure to obtain a residue, which was separated and purified by column chromatography (EA: PE = 3: 1) to obtain compound Int2-4 (4.8 g, yield: 92%) as a yellow solid.

MS (ESI) M/Z: 171.1 [M+H] ⁺ .

¹ H NMR (400MHz, CHCl ₃ -d) δ7.94 (s, 1H), 4.89 (s, 2H), 4.29 (q, J = 7.2Hz, 2H), 2.04 (s, 3H), 1.34 (t, J=7.2Hz,3H).

Step 4: At room temperature, compound Int2-4 (4.8 g, 28.24 mmol) and potassium carbonate (19.5 g, 141.20 mmol) were added to DMF (100 mL) in sequence, stirred at room temperature for 5 minutes, compound Int2-5 (9.3 g, 33.89 mmol) was added, and stirred at room temperature for 16 hours. Water (500 mL) and dichloromethane (200 mL×3) were added. After extraction, the organic phases were combined, washed with water (200 mL) and saturated aqueous sodium chloride solution (200 mL), respectively, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (EA: PE = 0-100%) to obtain compound Int2-6 (7.6 g, yield: 74%) as a white solid.

MS (ESI) M/Z: 368.1 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.37(s,1H),7.79–7.73(m,1H),7.47–7.33(m,1H),7.25(s,4H),6.43–6.36(m,1H),6.22(td,J＝ 6.4,1.2Hz,1H),5 .28(s,2H),5.06(s,2H),4.84(t,J＝6.0Hz,1H),4.56(d,J＝6.0Hz,2H),4.18(q,J＝7.2Hz,2H) ,1.25(t,J=7.2Hz,3H).

Step 5: At room temperature, compound Int2-6 (1.0 g, 2.72 mmol) and phosphorus tribromide (1.1 g, 4.09 mmol) were added to acetonitrile (10 mL) in sequence, and the temperature was raised to 70°C and stirred for 1 h. The mixture was cooled to room temperature and poured into an ice-cold saturated aqueous sodium bicarbonate solution (100 mL), extracted with dichloromethane (50 mL×3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain intermediate 2 (1.1 g, yield: 92%) as a white solid.

MS (ESI) M/Z: 429.1 [M+H] ⁺ .

Preparation Example 3: Preparation of (Z)-N-(2-aminoethyl)-5-((5-fluoro-2-oxoindole-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide (Intermediate 3)

Step 1: Under nitrogen atmosphere, compound Int3-1 (780 mg, 8.0 mmol) was added to H ₂ O (18 mL), cooled to 0°C, potassium hydroxide (500 mg, 16.0 mmol) was added in batches, and the temperature was raised to 100°C and stirred for 4 h. HCl (10 mL) was added at 0°C, and DCM (30 mL×3) was used for washing. The mixture was filtered under reduced pressure to obtain compound Int3-2 (640 mg, crude product) as a yellow oily solid.

MS (ESI) M/Z: 167.1 [M+H] ⁺ .

Step 2: At room temperature, compound Int3-2 (1.8 g, 11.9 mmol) was added to ethanol (18 mL), and compound Int3-3 (1.92 g, 11.9 mmol) and piperidine (2.4 mL, 24 mmol) were added to the mixture in sequence, stirred at room temperature for 0.5 h, and heated to 90 ° C and stirred for 16 h. HCl (10 mL) was added at 0 ° C, and the mixture was filtered under reduced pressure to obtain a solid, which was slurried with a mixture of DCM (30 mL) and MeOH (3 ml). Filtered, the filter residue was washed with EtOH (30 ml) to obtain a yellow solid Int3-4 (2.4 g, 63%).

MS (ESI) M/Z: 301.1 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-D6) δ13.84(s,1H),12.14(s,1H),10.92(s,1H),7.85 –7.63(m,2H),6.98–6.76(m,2H),2.47(d,J=2.2Hz,6H).

Step 3: At room temperature, compound Int3-4 (100 mg, 0.33 mmol), compound Int3-5 (64 mg, 0.40 mmol), HOBT (67.5 mg, 0.50 mmol), EDCI (95.05 mg, 0.50 mmol) and triethylamine (67.30 mg, 0.66 mmol) were added to DMF (2 mL) in sequence and stirred at room temperature for 12 h. Water (10 mL) was added, and DCM (10 mL×3) was used for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (dichloromethane: methanol = 0-10%) to obtain compound Int3-6 (39 mg, yield: 26.7%) as a yellow solid.

MS (ESI) M/Z: 443.6 [M+H] ⁺ .

Step 4: At room temperature, compound Int3-6 (29 mg, 0.07 mmol) was added to a hydrochloric acid/dioxane solution (3 mL), cooled to 0°C, and stirred at room temperature for 1 hour. The pH of the reaction solution was adjusted to alkaline with saturated sodium bicarbonate aqueous solution, extracted with DCM (10 mL×3), and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain intermediate 3 (30 mg, hydrochloride) as a white solid.

MS(ESI)M/Z: 343.14[M+1] ⁺ .

Preparation Example 4: Preparation of (S,Z)-5-((5-fluoro-2-oxoindole-3-ylidene)methyl)-2,4-dimethyl-N-(pyrrolidin-3-yl)-1H-pyrrole-3-carboxamide (Intermediate 4)

Step 1: At room temperature, compound Int3-4 (500 mg, 1.67 mmol), compound Int4-1 (372 mg, 2.00 mmol), HOBT (338 mg, 2.50 mmol), EDCI (477.5 mg, 2.50 mmol) and triethylamine (336.00 mg, 3.34 mmol) were added to DMF (5 mL) in sequence and stirred at room temperature for 12 h. Water (60 mL) was added, and DCM (60 mL×3) was used for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (PE: EA = 0-100%) to obtain compound Int4-2 (491 mg, yield: 62.9%) as a yellow solid.

MS (ESI) M/Z: 469.6 [M+H] ⁺ .

Step 2: At room temperature, compound Int4-2 (80 mg, 0.17 mmol) was added to a hydrochloric acid/dioxane solution (10 mL), cooled to 0°C, and stirred at room temperature for 1 hour. The pH of the reaction solution was adjusted to alkaline with saturated sodium bicarbonate aqueous solution, extracted with DCM (10 mL×3), and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain intermediate 4 (80 mg, hydrochloride) as a white solid.

MS(ESI)M/Z: 369.14[M+1] ⁺ .

Preparation Example 5: Preparation of (S,Z)-3-((2-(4-(3-(5-((5-fluoro-2-oxoindole-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide)pyrrolidine-1-carbonyl)piperazin-1-yl)ethoxy)methyl)-1-(4-((2-oxopyridin-1(2H)-yl)methyl)benzyl)-1H-pyrazole-4-carboxylic acid (Intermediate 5)

Step 1: Under nitrogen atmosphere, compound Int5-1 (386 mg, 1.68 mmol) was added to THF (5 mL), cooled to 0°C, sodium hydride (67 mg, 1.68 mmol) was added, stirred at 0°C for 1 h, intermediate 2 (600 mg, 1.40 mmol) dissolved in DMF (5 mL) was slowly added dropwise, and stirred at room temperature for 16 h. Saturated aqueous ammonium chloride solution (50 mL) was added, and THF was removed by concentration under reduced pressure, and ethyl acetate (50 mL×3) was used for extraction. The organic phases were combined, washed with water (100 mL) and saturated aqueous sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (methanol: dichloromethane = 0-100%) to obtain compound Int5-2 (115 mg, yield: 15%), a yellow oily liquid.

MS (ESI) M/Z: 580.3 [M+H] ⁺ .

Step 2: Add compound Int5-2 (115 mg, 0.20 mmol) to a solution of hydrogen chloride/dioxane (4.0 M, 5 mL), react at room temperature for 1 hour, and concentrate under reduced pressure to obtain compound Int5-3 (95 mg, yield: 100%) as a white solid.

MS (ESI) M/Z: 480.3 [M+H] ⁺ .

Step 3: Under nitrogen atmosphere, compound Int5-3 (95 mg, 0.2 mmol) was dissolved in DCM (2 mL), cooled to 0°C, a solution of triphosgene (0.1 mmol) dissolved in DCM (2 mL) was added dropwise, stirred at 0°C for 30 minutes, intermediate 4 (73 mg, 0.2 mmol) dissolved in DMF (2 mL) was added dropwise, and the mixture was naturally restored to room temperature for 1 hour. Water (20 mL) was added and extracted with DCM (20×3), the organic phases were combined, washed with water (30 mL) and saturated sodium chloride aqueous solution (30 mL), dried over anhydrous sulfuric acid, filtered, and concentrated under reduced pressure to obtain a residue, which was separated and purified by column chromatography (methanol: dichloromethane = 0-100%) to obtain compound Int5-4 (100 mg, yield: 57%) as a yellow solid.

MS (ESI) M/Z: 874.4 [M+H] ⁺ .

Step 4: Compound Int5-4 (100 mg, 0.11 mmol) was added to ethanol (3 mL) at room temperature. A solution of sodium hydroxide (23 mg, 0.57 mmol) dissolved in water (0.6 mL) was added and stirred at room temperature for 24 hours. The pH of the reaction solution was adjusted to acidic with 1N aqueous hydrogen chloride solution, extracted with DCM (10 mL×3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain intermediate 5 (60 mg, yield: 62%) as a yellow solid.

MS (ESI) M/Z: 846.4 [M+H] ⁺ .

Preparation Example 6: Preparation of tert-butyl 2-(prop-2-en-1-oxy)acetate (Intermediate 6)

Under nitrogen atmosphere, tert-butyl 2-hydroxyacetate (6.7 g, 50.85 mmol) was added to THF (25 mL), cooled to 0°C, sodium hydride (2.0 g, 50.85 mmol) was added in batches, stirred at 0°C for 1 h, 3-bromo-1-propyne (5.0 g, 42.37 mmol) dissolved in THF (25 mL) was slowly added dropwise, and stirred at room temperature for 16 h. The temperature was cooled to 0°C, saturated aqueous ammonium chloride solution (100 mL) was added, and the THF was removed by concentration under reduced pressure. The organic phases were combined, washed with water (200 mL) and saturated aqueous sodium chloride solution (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (ethyl acetate: petroleum ether = 0-100%) to obtain intermediate 6 (2.3 g, yield: 32%), a yellow oily liquid.

MS (ESI) M/Z: 171.1 [M+H] ⁺ .

Preparation Example 7: Preparation of tert-butyl 4-(prop-2-en-1-oxy)piperidine-1-carboxylate (Intermediate 7)

Under nitrogen atmosphere, compound intermediate 7-1 (6 g, 30 mmol) was added to THF (40 mL), cooled to 0°C, sodium hydride (1.21 g, 30 mmol) was added in batches, and stirred at 0°C for 1 h. 3-Bromo-1-propyne (3.77 g, 32 mmol) dissolved in THF (25 mL) was slowly added dropwise, and stirred at room temperature for 16 h. The temperature was lowered to 0°C, saturated aqueous ammonium chloride solution (100 mL) was added, and the THF was removed by concentration under reduced pressure. The organic phases were combined, washed with water (200 mL) and saturated aqueous sodium chloride solution (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (ethyl acetate: petroleum ether = 0-100%) to obtain compound intermediate 7 (4.3 g, yield: 60%), a yellow oily liquid.

MS (ESI) M/Z: 240.2 [M+H] ⁺ .

Preparation Example 8: Preparation of tert-butyl 3-(prop-2-en-1-oxy)azetidine-1-carboxylate (Intermediate 8)

Under nitrogen atmosphere, compound intermediate 8-1 (6.92 g, 40 mmol) was added to THF (60 mL), cooled to 0°C, sodium hydride (1.62 g, 40 mmol) was added in batches, and stirred at 0°C for 1 h. 3-Bromo-1-propyne (4.77 g, 40 mmol) dissolved in THF (25 mL) was slowly added dropwise, and stirred at room temperature for 16 h. The temperature was lowered to 0°C, saturated aqueous ammonium chloride solution (100 mL) was added, and the THF was removed by concentration under reduced pressure. The organic phases were combined, washed with water (200 mL) and saturated aqueous sodium chloride solution (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (ethyl acetate: petroleum ether = 0-100%) to obtain compound intermediate 8 (5.5 g, yield: 65%), a yellow oily liquid.

MS (ESI) M/Z: 212.1 [M+H] ⁺ .

Preparation Example 9: Preparation of (R)-tert-butyl 3-(prop-2-yn-1-yloxy)pyrrolidine-1-carboxylate (Intermediate 9)

Under nitrogen atmosphere, compound intermediate 9-1 (5.61 g, 30 mmol) was added to THF (50 mL), cooled to 0°C, sodium hydride (1.22 g, 30 mmol) was added in batches, and stirred at 0°C for 1 h. 3-Bromo-1-propyne (4.17 g, 35 mmol) dissolved in THF (25 mL) was slowly added dropwise, and stirred at room temperature for 16 h. The temperature was lowered to 0°C, saturated aqueous ammonium chloride solution (100 mL) was added, and the THF was removed by concentration under reduced pressure. The organic phases were combined, washed with water (200 mL) and saturated aqueous sodium chloride solution (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (ethyl acetate: petroleum ether = 0-100%) to obtain compound intermediate 9 (3.92 g, yield: 58%), a yellow oily liquid.

MS (ESI) M/Z: 226.1 [M+H] ⁺ .

Preparation Example 10: Preparation of tert-butyl (R)-3-(prop-2-yn-1-yloxy)pyrrolidine-1-carboxylate (Intermediate 10)

Intermediate 10 was prepared by the same synthetic method as Intermediate 9.

MS (ESI) M/Z: 226.1 [M+H] ⁺ .

Preparation Example 11: Preparation of ethyl 3-bromo-1-(4-(2-oxopyridin-1(2H)-yl)methyl)benzyl)-1H-pyrazole-4-carboxylate (Intermediate 11)

Step 1: To a solution of pyridin-2(H)-one (25.0 g, 262 mmol, 1.00 eq) in MeCN (500 mL) was added 1,4-bis(bromomethyl)benzene (104.08 g, 394.33 mmol, 1.50 eq) and K ₂ CO ₃ (72.7 g, 525 mmol, 2.00 eq). The reactants were stirred at 80 °C for 12 hours. The reaction mixture was filtered and the filter cake was washed with DCM (200 mL). It was then diluted with H ₂ O (500 mL) and extracted with DCM 1500 mL (500 mL×3). The combined organic layers were washed with brine (500 mL×3), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ , PE/EtOAc=1/0 to 1/1, plate 2, PE:EtOAc=1:1, Rf=0.45) to obtain intermediate 11-1 (8.00 g, 28.8 mmol, 10.9% yield) as a white solid.

LCMS(ESI): m/z 278[M+1] ⁺ .

¹ H NMR (400MHz, CDCl ₃ ) δ7.70-7.69 (d, J = 1.6Hz, 1H), 7.68-7.53 (m, 1H), 7.40 -7.38(d,J＝8Hz,2H),7.28-7.26(d,J＝8Hz,2H),6.59-6.56(d,J＝8.8Hz,1H),6.41-6.38(m,1H),5.19( s,2H),4.53(s,2H).

Step 2: Add intermediate 11-1 (200 mg, 0.72 mmol), ethyl 3-bromo-1H-pyrazole-4-carboxylate (157 mg, 0.72 mmol) and potassium carbonate (197 mg, 1.44 mmol) to DMF (5 mL) in sequence, and react at room temperature overnight. Filter, wash the filter cake with ethyl acetate, and concentrate the filtrate under reduced pressure to obtain a residue, which is separated and purified by column chromatography (petroleum ether/ethyl acetate = 0-100%) to obtain intermediate 11 (250 mg, yield: 83.7%) as a white solid.

MS (ESI) M/Z: 416.0 [M+H] ⁺ .

Example 1: Preparation of (S,Z)-N-((6-amino-2,4-dimethylpyridin-3-yl)methyl)-3-((2-(2-(2-(3-(5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamido)pyrrolidin-1-yl)-2-oxoethoxy)ethoxyethoxymethyl)-1-(4-((2-oxopyridin-1(2H)-yl)methyl)benzyl)-1H-pyrazole-4-carboxamido (Compound 46)

Step 1: Under nitrogen atmosphere, intermediate 2 (466 mg, 2.33 mmol) was added to THF (5 mL), cooled to 0°C, sodium hydride (93 mg, 2.33 mmol) was added, stirred at 0°C for 1 h, intermediate 1 (500 mg, 1.16 mmol) dissolved in DMF (5 mL) was slowly added dropwise, and stirred at room temperature for 16 h. Saturated aqueous ammonium chloride solution (50 mL) was added, and THF was removed by concentration under reduced pressure, and ethyl acetate (50 mL×3) was used for extraction. The organic phases were combined, washed with water (100 mL) and saturated aqueous sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue, which was separated and purified by column chromatography (methanol: dichloromethane = 0-100%) to obtain compound 46-1 (150 mg, yield: 23%), a yellow oily liquid.

MS (ESI) M/Z: 569.3 [M+H] ⁺ .

Step 2: Add compound 46-1 (150 mg, 0.26 mmol) to a solution of hydrogen chloride/dioxane (4.0 M, 5 mL), react at room temperature for 1 hour, and concentrate under reduced pressure to obtain compound 46-2 (150 mg, yield: 100%) as a white solid.

MS (ESI) M/Z: 514.2 [M+H] ⁺ .

Step 3: At room temperature, compound 46-2 (150 mg, 0.29 mmol), HATU (133 mg, 0.35 mmol) and triethylamine (59 mg, 0.58 mmol) were added to DMF (2 mL) in sequence, stirred at room temperature for 0.5 h, and intermediate 4 (128 mg, 0.35 mmol) was added. Stirred at room temperature for 0.5 h, water (10 mL) was added, and extracted with DCM (10 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a residue, which was separated and purified by column chromatography (methanol: dichloromethane = 0-100%) to obtain compound 46-3 (85 mg, yield: 33%) as a yellow solid.

MS (ESI) M/Z: 864.4 [M+H] ⁺ .

Step 4: At room temperature, compound 46-3 (85 mg, 0.10 mmol) was added to ethanol (3 mL), and a solution of sodium hydroxide (20 mg, 0.40 mmol) dissolved in water (0.6 mL) was added, and stirred at room temperature for 24 hours. The pH of the reaction solution was adjusted to acidic with 1N aqueous hydrogen chloride solution, extracted with DCM (10 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue, which was separated and purified by column chromatography (methanol: dichloromethane = 0-100%) to obtain compound 46-4 (58 mg, yield: 69%) as a yellow solid.

MS (ESI) M/Z: 836.3 [M+H] ⁺ .

Step 5: Compound 46-4 (58 mg, 0.07 mmol), HATU (31 mg, 0.08 mmol) and triethylamine (21 mg, 0.21 mmol) were added to DMF (2 mL) in sequence at room temperature, stirred at room temperature for 0.5 h, and compound 46-5 (16 mg, 0.08 mmol) was added. The mixture was stirred at room temperature for 0.5 h, water (10 mL) was added, and extracted with DCM (10 mL×3). The organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to obtain a residue, which was separated and purified by column chromatography (methanol: dichloromethane=0-100%) to obtain compound 46 (47 mg, yield: 70%) as a yellow solid.

MS (ESI) M/Z: 969.4 [M+H] ⁺ .

Example 2: Preparation of (S,Z)-N-(4-aminoiminoylbenzyl)-3-((2-(2-(3-(5-((5-fluoro-2-oxoindole-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamido)pyrrolidin-1-yl)-2-oxoethoxy)ethoxy)ethoxy)methyl)-1-(4-((2-oxopyridin-1(2H)-yl)methyl)benzyl)-1H-pyrazole-4-carboxamide (Compound 47)

At room temperature, compound 46-4 (24 mg, 0.03 mmol), HATU (15 mg, 0.04 mmol) and triethylamine (15 mg, 0.15 mmol) were added to DMF (2 mL) in sequence, stirred at room temperature for 0.5 h, and compound 47-1 (6 mg, 0.04 mmol) was added. Stirred at room temperature for 0.5 h, water (10 mL) was added, extracted with DCM (10 mL×3), the organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (methanol: dichloromethane=0-100%) to obtain compound 47 (21 mg, yield: 92%) as a yellow solid.

MS(ESI)M/Z: 877.41[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ13.68(s,1H),10.91(s,1H),9.27(s,2H),8.93(s,1H),8.56–8.47m,1H),8.33(s,1H),7.87–7.60( m,4H),7.50(d,J＝8.4Hz,2H), 7.43–7.31(m,1H),7.26(s,5H),7.23–7.14(m,1H),6.67(s,1H),6.40–6.37(m,1H),6.24–6.19(m,1H), 5.33–5.3 0(m,2H),5.06(s,2H),4.65(s,2H),4.50–4.42(m,4H),3.98(s,2H),3.28–3.24(m,4H),2.42(s, 3H),2.41(s,3H).

Example 3: Preparation of (Z)-N-(4-aminoiminoylbenzyl)-3-(1-(5-((5-fluoro-2-oxoindole-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrol-3-yl)-5-methyl-1-oxo-8,11-dioxa-2,5-diazadodecane-12-yl)-1-(4-((2-oxopyridin-1(2H)-yl)methyl)benzyl)-1H-pyrazole-4-carboxamide (Compound 49)

Step 1: Under nitrogen atmosphere, intermediate 2 (148 mg, 1.40 mmol) was added to THF (5 mL), cooled to 0°C, sodium hydride (56 mg, 1.40 mmol) was added, and stirred at 0°C for 1 h. Diethylene glycol (300 mg, 0.70 mmol) dissolved in DMF (5 mL) was slowly added dropwise, and stirred at room temperature for 16 h. Saturated aqueous ammonium chloride solution (50 mL) was added, and the THF was removed by concentration under reduced pressure. DCM (50 mL×3) was used for extraction, and the organic phases were combined, washed with water (100 mL) and saturated aqueous sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (methanol: dichloromethane = 0-100%) to obtain compound 49-1 (70 mg, yield: 22%), a colorless oily liquid.

MS (ESI) M/Z: 456.2 [M+H] ⁺ .

Step 2: Compound 49-1 (70 mg, 0.15 mmol) and Dess-Martin reagent (98 mg, 0.23 mmol) were added to DCM (3 mL) in sequence and stirred at room temperature for 16 hours. The mixture was filtered, the filtrate was collected, and the residue was concentrated under reduced pressure. The residue was separated and purified by column chromatography (methanol: dichloromethane = 0-100%) to obtain compound 49-2 (35 mg, yield: 50%), a colorless oily liquid.

MS (ESI) M/Z: 454.2 [M+H] ⁺ .

Step 3: Under nitrogen atmosphere, compound 49-2 (35 mg, 0.08 mmol), compound 49-3 (27 mg, 0.08 mmol, prepared by the same method as in Preparation Example 3) and acetic acid (5 mg, 0.08 mmol) were added sequentially to In a mixed solution of DCM (2 mL) and methanol (0.2 mL), the temperature was lowered to 0°C, STAB (34 mg, 0.16 mmol) was added, and the mixture was naturally returned to room temperature and stirred for 3 hours. Water (10 mL) was added, and DCM (10 mL×3) was used for extraction. The organic phases were combined, washed with water (20 mL) and saturated sodium chloride aqueous solution (20 mL), respectively, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (methanol: dichloromethane = 0-100%) to obtain compound 49-4 (35 mg, yield: 57%) as a yellow solid.

MS (ESI) M/Z: 794.4 [M+H] ⁺ .

Step 4: At room temperature, compound 49-4 (35 mg, 0.04 mmol) was added to ethanol (3 mL), and a solution of sodium hydroxide (9 mg, 0.22 mmol) dissolved in water (0.6 mL) was added, and stirred at room temperature for 24 hours. The pH of the reaction solution was adjusted to acidic with 1N aqueous hydrogen chloride solution, extracted with DCM (10 mL×3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 49-5 (30 mg, yield: 94%) as a yellow solid.

MS (ESI) M/Z: 766.3 [M+H] ⁺ .

Step 5: Compound 49-5 (30 mg, 0.04 mmol), HATU (18 mg, 0.05 mmol) and triethylamine (12 mg, 0.12 mmol) were added to DMF (2 mL) at room temperature, stirred at room temperature for 0.5 h, 4-aminomethylbenzylamine dihydrochloride (compound 47-1) (9 mg, 0.05 mmol) was added, and stirred at room temperature for 0.5 h. Water (10 mL) was added, and DCM (10 mL×3) was used for extraction. The organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (methanol: dichloromethane=0-100%) to obtain compound 49 (20 mg, yield: 55%) as a yellow solid.

MS (ESI) M/Z: 897.4 [M+H] ⁺ .

Example 4: Preparation of (S,Z)-N-(6-amino-2,4-dimethylpyridin-3-yl)-3-((2-(4-(3-(5-((5-fluoro-2-oxoindole-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamido)pyrrolidine-1-carboxyl)piperazin-1-yl)ethoxy)methyl)-1-(4-((2-oxopyridin-1(2H)-yl)methyl)benzyl)-1H-pyrazole-4-carboxamide (Compound 50)

At room temperature, intermediate 5 (30 mg, 0.03 mmol), HATU (16 mg, 0.0 4 mmol) and triethylamine (10 mg, 0.10 mmol) were added to DMF (2 mL) in sequence, stirred at room temperature for 0.5 h, compound 1-5 (8 mg, 0.04 mmol) was added, and stirred at room temperature for 0.5 h. Water (10 mL) was added, and DCM (10 mL×3) was used for extraction. The organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (methanol: dichloromethane=0-100%) to obtain compound 50 (21 mg, yield: 62%) as a yellow solid.

MS (ESI) M/Z: 979.5 [M+H] ⁺ .

¹ H NMR (400MHz, CH ₃ OH-d ₄ )δ8.11(s,1H),7.66(dd,J＝6.8,2.0Hz,1H),7.55(d,J＝2.4Hz,1H),7.52–7.46(m,1H),7.43–7.39(m ,1H),7. 30–7.22(m,4H),6.88–6.84(m,2H),6.54–6.50(m,1H),6.47(s,1H),6.38–6.32m,1H),5.36–5.32(m,1H) ,5.2 8(s,2H),5.14(s,2H),4.65(s,2H),4.44(s,3H),3.71–3.57(m,4H),3.51–3.41(m,2H),3.29–3.22( m,4H),2 .59–2.48(m,6H),2.47(s,3H),2.43(s,3H),2.40(s,3H),2.34(s,3H),2.25–2.17(m,2H),2.08–1.98 (m,3H).

Example 5: Preparation of (Z)-N-((6-amino-2,4-dimethylpyridin-3-yl)methyl)-3-(3-(2-((2-(5-((5-fluoro-2-indolyl-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamido)ethyl)amino)-2-oxoethoxy)prop-1-yn-1-yl)-1-(4-((2-pyridone-1(2H)-yl)methyl)benzyl)-1H-pyrazole-4-carboxamido (Compound 48)

Step 1: Under nitrogen atmosphere, add intermediate 11 (500 mg, 1.20 mmol), intermediate 6 (408 mg, 2.40 mmol), cuprous iodide (23 mg, 0.12 mmol) and tetrakistriphenylphosphine palladium (138 mg, 0.12 mmol) in sequence to a mixed solution of triethylamine (2.5 mL) and DMF (2.5 mL), heat to 100 ° C and stir for 3 hours. Cool to room temperature, add dichloromethane (25 mL), filter, collect the filtrate, and concentrate under reduced pressure to obtain a residue, which is separated and purified by column chromatography (dichloromethane: methanol = 0-100%) to obtain compound 48-2 (500 mg, purity 50%), a yellow oily liquid, which is directly used in the next step.

MS (ESI) M/Z: 506.2 [M+H] ⁺ .

Step 2: Add compound 48-2 (600 mg, 0.59 mmol) to a solution of hydrogen chloride/dioxane (4.0 M, 5 mL). React at room temperature for 1 hour, concentrate under reduced pressure to obtain a residue, and suspend the residue in dichloromethane (50 mL). Adjust the pH to about 8 with a saturated sodium bicarbonate aqueous solution, let stand, separate the layers, and use DCM to separate the aqueous phase. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (dichloromethane: methanol = 0-100%) to obtain compound 48-3 (97 mg, yield: 37%) as a white solid.

MS (ESI) M/Z: 450.1 [M+H] ⁺ .

Step 3: At room temperature, compound 48-3 (43 mg, 0.10 mmol), HATU (44 mg, 0.11 mmol) and triethylamine (20 mg, 0.20 mmol) were added to DMF (2 mL) in sequence, stirred at room temperature for 0.5 h, compound Int3-5 (18 mg, 0.11 mmol) was added, and stirred at room temperature for 0.5 h. Water (10 mL) was added, and DCM (10 mL×3) was used for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (methanol: dichloromethane = 0-100%) to obtain compound 48-4 (53 mg, yield: 88%) as a yellow solid.

MS (ESI) M/Z: 592.3 [M+H] ⁺ .

Step 4: At room temperature, compound 48-4 (53 mg, 0.09 mmol) was added to ethanol (3 mL), and a solution of sodium hydroxide (18 mg, 0.45 mmol) dissolved in water (0.6 mL) was added, and stirred at room temperature for 48 hours. The pH of the reaction solution was adjusted to acidic with 1N aqueous hydrogen chloride solution, extracted with DCM (10 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a residue, and the residue was separated and purified by column chromatography (methanol: dichloromethane = 0-100%) to obtain compound 48-5 (47 mg, yield: 94%) as a yellow solid.

MS (ESI) M/Z: 563.2 [M+H] ⁺ .

Step 5: Compound 48-5 (47 mg, 0.08 mmol), HATU (38 mg, 0.10 mmol) and triethylamine (24 mg, 0.24 mmol) were added to DMF (2 mL) at room temperature, stirred at room temperature for 0.5 h, compound 1-5 (19 mg, 0.10 mmol) was added, and stirred at room temperature for 0.5 h. Water (10 mL) was added, and DCM (10 mL×3) was used for extraction. The organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to obtain a residue, which was separated and purified by column chromatography (methanol: dichloromethane=0-100%) to obtain compound 48-6 (50 mg, yield: 89%) as a yellow solid.

MS (ESI) M/Z: 697.3 [M+H] ⁺ .

Step 6: Add compound 48-6 (50 mg, 0.07 mmol) to a solution of hydrogen chloride/dioxane (4.0 M, 5 mL), react at room temperature for 1 hour, and concentrate under reduced pressure to obtain compound 48-7 (42 mg, yield: 100%) as a white solid.

MS (ESI) M/Z: 597.3 [M+H] ⁺ .

Step 7: Compound 48-7 (42 mg, 0.07 mmol), HATU (32 mg, 0.08 mmol) and triethylamine (21 mg, 0.24 mmol) were added to DMF (2 mL) at room temperature, stirred at room temperature for 0.5 h, compound Int3-4 (24 mg, 0.08 mmol) was added, and stirred at room temperature for 0.5 h. Water (10 mL) was added, and DCM (10 mL×3) was used for extraction. The organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to obtain a residue, which was separated and purified by column chromatography (methanol: dichloromethane=0-100%) to obtain compound 48 (20 mg, yield: 33%) as a yellow solid.

MS (ESI) M/Z: 879.4 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.67(s,1H),10.89(s,1H),8.27(s,1H),7.96(t, J＝5.2Hz,1H),7.87(s,1H),7.78–7.73(m,2H),7.71(s,1H),7.68–7.61(m,1H),7.42-7 .37(m,1H),7.27–7.20(m,5H),6.96–6.88(m,1H),6.86–6.80(m,1H),6.38(d,J＝10.4H z,2H),6.24–6.16(m,1H),5.28(s,2H),5.05(s,2H),4.42(s,2H),4.24(d,J＝4.8Hz,2H ),3.99(s,2H),3.32–3.27(m,4H),2.43(s,3H),2.41(s,3H),2.39(s,3H),2.26(s,3H).

The compounds listed in the table below were prepared using procedures similar to those described in Examples 1, 2, 3, 4 and 5 using appropriate starting materials.

Table 1.

Example 6: Preparation of (Z)-N-(4-aminoiminoylbenzyl)-3-(3-(2-(2-(5-((5-fluoro-2-oxoindole-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamido)ethyl)amino)-2-oxoethoxy)prop-1-yn-1-yl)-1-(4-((2-oxopyridin-1(2H)-yl)methyl)benzyl)-1H-pyrazole-4-carboxamide (Compound 60)

Step 1: At room temperature, intermediate 3 (97 mg, 0.22 mmol), HATU (99 mg, 0.26 mmol) and triethylamine (67 mg, 0.67 mmol) were added to DMF (2 mL) in sequence, stirred at room temperature for 0.5 h, compound 48-2 (85 mg, 0.22 mmol) was added, and stirred at room temperature for 0.5 h. Water (10 mL) and DCM (10 The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by column chromatography (methanol: dichloromethane = 0-100%) to obtain compound 60-1 (110 mg, yield: 65%) as a yellow solid.

MS (ESI) M/Z: 774.3 [M+H] ⁺ .

Step 2: At room temperature, compound 60-1 (105 mg, 0.14 mmol) was added to ethanol (3 mL), and a solution of sodium hydroxide (27 mg, 0.68 mmol) dissolved in water (0.6 mL) was added, and stirred at room temperature for 48 hours. The pH of the reaction solution was adjusted to acidic with 1N aqueous hydrogen chloride solution, extracted with DCM (10 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue, which was separated and purified by column chromatography (methanol: dichloromethane = 0-100%) to obtain compound 60-2 (24 mg, yield: 23%) as a yellow solid.

MS (ESI) M/Z: 746.3 [M+H] ⁺ .

Step 3: At room temperature, compound 60-2 (24 mg, 0.03 mmol), HATU (15 mg, 0.04 mmol) and triethylamine (9 mg, 0.09 mmol) were added to DMF (1 mL) in sequence, stirred at room temperature for 0.5 h, compound 2-1 (9 mg, 0.04 mmol) was added, and stirred at room temperature for 0.5 h. Water (10 mL) was added, and DCM (10 mL×3) was used for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue, which was separated and purified by column chromatography (methanol: dichloromethane=0-100%) to obtain compound 60 (21 mg, yield: 75%) as a yellow solid.

MS (ESI) M/Z: 877.3 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ13.68(s,1H),10.91(s,1H),9.27(s,2H),8.93(s,1H),8.55–8.50(m,1H),8.33(s,1H),7.8 7–7.60(m,4H),7.50(d,J＝8.4Hz,2H),7.43–7.31(m,1H),7.26(s,5H),7.23–7.14(m,1H),6.86 –6.81(m,1H),6.67(s,1H),6.40–6.37(m,1H),6.24-6.19(m,1H),5.32–5.31(m,2H),5.06(s,2 H),4.65(s,2H),4.50–4.42(m,4H),3.98(s,2H),3.27–3.24(m,4H),2.42(s,3H),2.41(s,3H).

Example 7: Preparation of (S,Z)-N-(4-aminoiminoylbenzyl)-3-((2-(4-(3-(5-((5-fluoro-2-oxoindole-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamido)pyrrolidine-1-carboxyl)piperazin-1-yl)ethoxy)methyl)-1-(4-((2-oxopyridin-1(2H)-yl)methyl)benzyl)-1H-pyrazole-4-carboxamide (Compound 50)

At room temperature, intermediate 5 (30 mg, 0.03 mmol), HATU (16 mg, 0.04 mmol) and triethylamine (10 mg, 0.10 mmol) were added to DMF (2 mL) in sequence and stirred at room temperature for 0.5 h. 4-Aminomethylbenzylamine dihydrochloride (Compound 47-1) (9 mg, 0.04 mmol) was added and stirred at room temperature for 0.5 h. Water (10 mL) was added and extracted with DCM (10 mL×3). The organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, and decompressed. The residue was concentrated and separated and purified by column chromatography (methanol: dichloromethane = 0-100%) to give compound 50 (19 mg, yield: 56%) as a yellow solid.

MS (ESI) M/Z: 977.4 [M+H] ⁺ .

¹ H NMR (400MHz, CH ₃ OH-d ₄ )δ8.15(s,1H),7.79–7.75(m,2H),7.69–7.65(m,1H),7.59–7.54(m,3H),7.52–7.47(m,1H),7.43–7.39( m,1H) ,7.31–7.25(m,4H),6.89–6.84(m,2H),6.55–6.50(m,1H),6.38–6.33(m,1H),5.36–5.33(m,1H),5.31(s, 2H),5 .15(m,2H),4.70(s,2H),4.63(s,2H),4.47–4.40(m,1H),3.68–3.56(m,4H),3.50–3.37(m,2H),3.18 –3.11(m, 4H),2.55(t,J＝4.8Hz,2H),2.43(s,3H),2.40(s,3H),2.38–2.36(m,2H),2.22–2.16(m,2H),2.05–2.00 (m,2H).

Example 8: Preparation of (S,Z)-N-(4-aminoiminoylbenzyl)-3-(3-((1-(3-(5-((5-fluoro-2-oxoindole-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamido)pyrrolidin-1-carboxyl)piperidin-4-yl)oxy)prop-1-yn-1-yl)-1-(4-((2-oxopyridin-1(2H)-yl)methyl)benzyl)-1H-pyrazole-4-carboxamide (Compound 61)

Step 1: Under nitrogen atmosphere, add intermediate 11 (500 mg, 1.20 mmol), intermediate 7 (574 mg, 2.40 mmol), cuprous iodide (23 mg, 0.12 mmol) and tetrakistriphenylphosphine palladium (138 mg, 0.12 mmol) in sequence to a mixed solution of triethylamine (2.5 mL) and DMF (2.5 mL), heat to 100° C. and stir for 3 hours. Cool to room temperature, add dichloromethane (25 mL), filter, collect the filtrate, and concentrate under reduced pressure to obtain a residue, which is separated by column chromatography (dichloromethane: methanol = 0-100%) to obtain compound 61-1 (470 mg, 68%), a yellow oily liquid, which is directly used in the next step.

MS (ESI) M/Z: 575.3 [M+H] ⁺ .

Step 2: Add compound 61-1 (470 mg, 0.82 mmol) to a solution of hydrogen chloride/dioxane (4.0 M, 5 mL) and react at room temperature for 1 hour. Concentrate under reduced pressure to obtain a residue, suspend the residue in dichloromethane (50 mL), adjust the pH to about 8 with a saturated sodium bicarbonate aqueous solution, let stand, separate the layers, extract the aqueous phase with DCM (50 mL × 2), combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain a residue. The residue is separated and purified by column chromatography (dichloromethane: methanol = 0-100%) to obtain compound 61-2 (237 mg, yield: 65%) as a white solid.

MS (ESI) M/Z: 447.2 [M+H] ⁺ .

Step 3: Under nitrogen atmosphere, compound 61-2 (178 mg, 0.4 mmol) was dissolved in DCM (2 mL), cooled to 0°C, triphosgene (0.1 mmol) solution dissolved in DCM (4 mL) was added dropwise, stirred at 0°C for 30 minutes, intermediate 4 (147 mg, 0.2 mmol) dissolved in DMF (2 mL) was added dropwise, and the mixture was naturally restored to room temperature for 1 hour. Water (20 mL) was added, extracted with DCM (20×3), the organic phases were combined, washed with water (30 mL) and saturated sodium chloride aqueous solution (30 mL), dried over anhydrous sulfuric acid, filtered, and concentrated under reduced pressure to obtain a residue, which was separated and purified by column chromatography (methanol: dichloromethane = 0-100%) to obtain compound 61-3 (191 mg, yield: 55%) as a yellow solid.

MS (ESI) M/Z: 869.4 [M+H] ⁺ .

Step 4: At room temperature, compound 61-3 (190 mg, 0.22 mmol) was added to ethanol (3 mL), and a solution of sodium hydroxide (46 mg, 1.14 mmol) dissolved in water (1 mL) was added, and stirred at room temperature for 24 hours. The pH of the reaction solution was adjusted to acidic with 1N aqueous hydrogen chloride solution, extracted with DCM (10 mL×3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 61-4 (111 mg, yield: 60%) as a yellow solid.

MS (ESI) M/Z: 839.3 [M+H] ^- .

Step 5: Compound 61-4 (50 mg, 0.06 mmol), HATU (32 mg, 0.08 mmol) and triethylamine (20 mg, 0.20 mmol) were added to DMF (4 mL) at room temperature in sequence, stirred at room temperature for 0.5 h, 4-aminomethylbenzylformamidine dihydrochloride (compound 47-1) (18 mg, 0.08 mmol) was added, and stirred at room temperature for 0.5 h. Water (10 mL) was added, and the mixture was extracted with DCM (10 mL×3). The organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to obtain a residue, which was separated and purified by column chromatography (methanol: dichloromethane=0-100%) to obtain compound 61 (31 mg, yield: 53%) as a yellow solid.

MS (ESI) M/Z: 972.4 [M+H] ⁺ .

The compounds listed in the table below were prepared using procedures similar to those described in Examples 6, 7 and 8 using appropriate starting materials.

Table 2.

Test Example 1: In vitro inhibitory activity test of the compounds of the present invention on plasma kallikrein (PKa)

The ability of the compounds of the present invention to inhibit plasma kallikrein (PKK) was determined in the presence of 1% (v/v) DMSO using the following biochemical assay in assay buffer (100 mM Tris, 150 mM NaCl, adjusted to pH 7.8 with HCl, and containing 0.1% (w/v) BSA and 0.05% (v/v) Tween 20): The FI method was used to test the inhibition of PKa enzyme activity by the compounds. PKK (R&D, Cat#2497-SE) and Pro-Phe-Arg-AMC (Nanjing Peptide Industry, Cat#NJP23946) solutions were prepared in reaction buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl). The final concentrations of enzyme protein (R&D, Cat#2497-SE) and Pro-Phe-Arg-AMC in the reaction mixture were 10 nM and 60 μM, respectively. The positive reference Avoralstat (MCE, Cat#HY-16735) started at 100 nM, 3-fold dilution, 10+0 dose. 0.05 μL of compound in 100% DMSO was delivered to a 384-well plate by acoustic liquid delivery technology (Echo665; nanoliter range). Compounds were tested in duplicate wells. 5 μL of 2× enzyme solution was transferred to a 384 reaction plate and centrifuged at 1000 rpm for 1 minute, incubated at 25°C for 15 minutes; 5 μL of 2× Pro-Phe-Arg-AMC solution was transferred to a 384 reaction plate, centrifuged at 1000 rpm for 1 minute, and incubated at 25°C for 3 hours. Finally, BMG was used and the wavelength excitation was set to 355 nm and the wavelength was set to 100 nm. Fluorescence was measured with an Envision reader with long emission set to 460 nm, reading the FI signal (ex380/em460). _IC50 values and nonlinear regression curve fitting were obtained using GraphPad Prism software.

These data were fitted to generate best fit _IC50 values and the results of these assays are shown below in Table 2, with the numbers corresponding to the compound numbers.

Test Example 2: Evaluation of the inhibition of PKa of the compounds of the present invention in kaolin-activated human PPP

Platelet-poor plasma (PPP) obtained from human whole blood and anticoagulated with sodium citrate was incubated with various concentrations of test compounds and 25, 75, 250 or 750 μg/mL kaolin in assay buffer for 20 minutes at 37° C., so that for each kaolin dose used, a concentration response of the test compound was obtained. Positive drug Avoralstat (MCE, Cat#HY-16735) was diluted according to the reference concentration, and the dilution was used as the first point and diluted 3 times.

50 nL of compound was then transferred to a 384 assay plate, with each well containing 2 replicates. The 384 assay plate was centrifuged at 1000 RPM. 2.5 μL of human plasma working solution was added to each assay well. The 384 assay plate was centrifuged at 1000 RPM. 2.5 μL of kaolin working solution was added to each assay well. The 384 assay plate was centrifuged at 1000 RPM and incubated at 25°C for 20 minutes. 5 μL of Pro-Phe-Arg-AMC working solution was added to each assay well. The 384 assay plate was centrifuged at 1000 RPM and incubated at 25°C for 60 minutes. The fluorescence signal was read by BMG.

The inhibition percentage of the compound was calculated according to the following formula:

Compound (inhibition %) = 100 × (average of negative control well values - compound well values) / (average of negative control well values - average of positive control well values)

These data were fitted to generate best fit _IC50 values and the results of these assays are shown below in Table 2, with the numbers corresponding to the compound numbers.

Test Example 3: In vitro VEGFR2 and PDGFRβ inhibitory activity test of compounds

Vortex the test compound to mix thoroughly and dilute it with DMSO (Cat. No.: D4540, Manufacturer: Sigma) to 100× the test concentration. 655SYSTEM) to transfer 50 nL of compound to 384 detection plate (Cat. No.: 784075, Manufacturer: Greiner). Prepare 2× kinase solution with 1× kinase reaction buffer (1×Buffer, 5mM MgCl ₂ , 1mM DTT, 1mM MnCl ₂ ), transfer 2.5 μL of VEGFR2 (0.07nM, Manufacturer: Carna, Cat. No.: 08-191) solution to 384 reaction plate. Centrifuge at 1000 rpm for 60 seconds using a microplate centrifuge (Model: Plate pro3200, Manufacturer: Monad), and incubate in a biochemical incubator (Model: LRH-250F, Manufacturer: bluepard) at 25°C for 10 minutes. Prepare a 2× mixture of substrate (TK: 1 μM) and ATP (4 μM, manufacturer: Promega, catalog number: V915B) using kinase reaction buffer. Add 2.5 μL of the mixture of substrate and ATP to the reaction plate to start the reaction. Centrifuge at 1000 rpm for 1 minute. Incubate at 25°C for 50 minutes. Add 5 μL of kinase detection reagent to each well of the reaction plate, centrifuge at 1000 rpm for 1 minute, and incubate at 25°C for 60 minutes. Read the plate using an ELISA reader (factory The fluorescence signals were read at 620 nm (Cryptate) and 665 nm (XL665) by BMG (BMG, Model: PHERAstar FSX). Each reaction was tested in duplicate, and IC ₅₀ was analyzed using GraphPad Prism 8.0 software.

Vortex the test compound to mix thoroughly and dilute it with DMSO (Cat. No.: D4540, Manufacturer: Sigma) to 100× the test concentration. 655 SYSTEM) to transfer 50 nL of compound to 384 detection plate (Cat. No.: 784075, Manufacturer: Greiner). Prepare 2× kinase solution with 1× kinase reaction buffer (1×Buffer, 5mM MgCl ₂ , 1mM DTT, 1mM MnCl ₂ , 12.5nM SEB), and transfer 2.5μL of PDGFRβ (0.23nM, Manufacturer: Carna, Cat. No.: 08-158) solution to 384 reaction plate. Centrifuge at 1000rpm for 60 seconds using a microplate centrifuge (Model: Plate pro3200, Manufacturer: Monad), and incubate in a biochemical incubator (Model: LRH-250F, Manufacturer: bluepard) at 25°C for 10 minutes. Prepare a 2× mixture of substrate (TK: 1 μM) and ATP (1 μM, manufacturer: Promega, catalog number: V915B) in kinase reaction buffer, add 2.5 μL of the mixture of substrate and ATP to the reaction plate to start the reaction, centrifuge at 1000 rpm for 1 minute. Incubate at 25°C for 50 minutes. Add 5 μL of kinase detection reagent to each well of the reaction plate, centrifuge at 1000 rpm for 1 minute, and incubate at 25°C for 60 minutes. Read the fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) using an ELISA reader (manufacturer: BMG, model: PHERAstar FSX). Each reaction was tested in duplicate, and IC ₅₀ was analyzed using GraphPad Prism 8.0 software.

These data were fitted to generate best fit _IC50 values and the results of these assays are shown below in Table 3, with the numbers corresponding to the compound numbers.

Table 3.

The above results indicate that the compounds of the present invention have a strong inhibitory effect on plasma kallikrein (PKa), VEGFR2 and PDGFRβ.

For those skilled in the art, the present disclosure is not limited to the foregoing illustrative embodiments, but can be embodied in other specific forms without departing from its essential attributes. It is therefore desired to regard all aspects as illustrative and not restrictive, and the embodiments referred to in the appended claims rather than the foregoing embodiments, and all changes that fall within the meaning and scope of equivalents of the claims, are therefore intended to be included herein.

Claims (12)

A compound represented by formula (I) or a pharmaceutically acceptable salt thereof:
in, L ₁ is selected from L ₂ is absent or selected from a linear -(CH ₂ ) _1-22 - group, wherein one or more -(CH ₂ )- in the linear -(CH ₂ ) _1-22 - group is optionally and independently substituted by one or more groups selected from -C(O)-, -O-, -(NR ² )-, -S-, -(C＝C)-, -(C≡C)-, a divalent cycloalkyl group, a divalent aryl group, a divalent heterocyclic group and a divalent heteroaryl group, and L ₂ is optionally substituted by 1, 2 or 3 R ¹ ; Each R ¹ is independently selected from C ₁ -C ₆ alkyl, halogenated C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogenated C ₁ -C ₆ alkyl, halogen, cyano and hydroxyl, Each R ² is independently selected from hydrogen, C ₁ -C ₆ alkyl and acyl, wherein said C ₁ -C ₆ alkyl is optionally substituted with 1, 2 or 3 R ¹ ; The group Ar ₁ is selected from -(CH ₂ )-aryl, -(CH ₂ )-heteroaryl and Wherein ring a is a 5-6 membered heterocyclic ring, the aryl, heteroaryl and Optionally, one or more selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, halogen, cyano, C ₁ -C ₆ alkoxy, halogenated C ₁ -C ₆ alkoxy, amino, hydroxyl, The group substitution; The group Ar ₂ is selected from -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, cycloalkyl, aryl and heteroaryl, wherein the group Ar ₂ is optionally substituted by one or more selected from halogen, cyano, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ cycloalkyl, C ₁ -C ₆ cycloalkylalkyl, 5-6 membered heterocyclyl and substituted by a group, a 5-6 membered heterocyclic group or Optionally substituted with one or more halogens. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, which is a compound of formula (II) or a pharmaceutically acceptable salt thereof:
wherein X is selected from an oxygen atom, NR ² ; L ₂ , R ² , the group Ar ₁ and the group Ar ₂ are as defined in claim 1 . The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, which is a compound of formula (III) or a pharmaceutically acceptable salt thereof:
wherein L ₂ , group Ar ₁ and group Ar ₂ are as defined in claim 1 . The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein: Ar ₁ is selected from -(CH ₂ )-phenyl, -(CH ₂ )-pyridyl and Wherein ring a is a 5-6 membered heteroaryl, preferably imidazolyl or pyrazolyl, more preferably for The phenyl, pyridyl and Optionally, one or more selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, halogen, cyano, C ₁ -C ₆ alkoxy, halogenated C ₁ -C ₆ alkoxy, amino, hydroxyl, The group substitution; R is selected from hydrogen and C ₁ -C ₆ alkyl. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein: Ar ₂ is selected from C _6-10 aryl, preferably phenyl, 5 to 10 membered heteroaryl, -C ₁ -C ₆ alkyl, -C ₁ -C ₆ haloalkyl, C ₃ -C ₆ cycloalkyl, wherein the C _6-10 aryl, 5 to 10 membered heteroaryl is optionally substituted by one or more selected from halogen, cyano, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ cycloalkyl, C ₁ -C ₆ cycloalkylalkyl, 5-6 membered heterocyclyl and The 5-6 membered heterocyclic group or Optionally substituted with one or more halogens. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein L ₂ is selected from C _1-2 alkylene; Alternatively, L ₂ is selected from -(CH ₂ ) _t -(C=C)-* and -(CH ₂ ) _t -(C≡C)-*; wherein t is an integer from 0 to 6, preferably an integer from 1 to 4, more preferably 1 or 2; * is the position of connection to the pyrazole ring; Alternatively, _L2 is selected from wherein X ₁ , X ₂ , X ₃ , and X ₄ are each independently selected from a bond, —O—, —(NR ² )—, —C(O)NH—, —NHC(O)—, —S—, —(C═C)—, —(C≡C)—, a 4-6 membered divalent heterocyclyl, a 3-6 membered divalent cycloalkyl, a 5-10 membered divalent heteroaryl, and a 6-10 membered divalent aryl, wherein the 4-6 membered divalent heterocyclyl, the 3-6 membered divalent cycloalkyl, the 5-10 membered divalent heteroaryl, or the 6-10 membered divalent aryl is optionally substituted by 1, 2, or 3 R ^1s ; each R ² is independently selected from hydrogen, a C ₁ -C ₆ alkyl, and an acyl group, wherein the C 1 -C 6 alkyl is optionally substituted by 1, 2, or 3 R ^1s , each R ¹ is independently selected from a C ₁ -C ₆ alkyl, a halogenated C ₁ -C ₆ alkyl, a C 1 -C 6 alkyl, a C ₁ -C ₆ alkyl, _a -C ₆ alkoxy, halogenated C ₁ -C ₆ alkyl, halogen, cyano and hydroxyl; t ₁ , t ₂ , t ₃ , t ₄ and t ₅ are each independently an integer from 0 to 6; * is the position of connection to the pyrazole ring. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein the divalent heterocyclic group is selected from: The divalent heteroaryl group is selected from: The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein -L ₂ - is selected from:
Wherein: G is a 5-6 membered heteroaryl group, preferably More preferred _G1 and _G2 are each independently selected from a 4-6 membered heterocyclic group, preferably p is 1 or 2; s is an integer from 0 to 6, preferably 0 or 1; _s1 is an integer from 1 to 10; s ₂ is 1 or 2, preferably 1; _s3 and _s4 are each independently selected from an integer of 1 to 6, preferably an integer of 1 to 4, more preferably 1 or 2; v is an integer from 0 to 4, preferably 0 or 1; * is the position of connection to the pyrazole ring. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, which is selected from:





or a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising a compound represented by formula (I) according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients. Use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating or preventing vascular disease-related diseases. The use according to claim 11, wherein the vascular disease-related condition is selected from diabetic retinopathy (DR), diabetic macular edema (DME), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), macular degeneration, wet age-related macular degeneration (wet AMD), retinopathy of prematurity (ROP), neovascular glaucoma, retinitis pigmentosa (RP), proliferative and non-proliferative retinopathy, retinal angiomatous proliferation, macular telangiectasia, ischemic retinopathy, iris neovascularization, intraocular neovascularization, corneal neovascularization, retinal neovascularization, polypoidal choroidal vasculopathy (PCV), choroidal neovascularization. and retinal degeneration, in particular selected from diabetic retinopathy (DR), diabetic macular edema (DME), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), wet age-related macular degeneration (wet AMD), in particular selected from hereditary angioedema (HAE) and brain edema after stroke, in particular selected from diabetic complications of retinal vascular permeability associated with diabetic retinopathy and diabetic macular edema, preferably selected from diabetic macular edema (DME), wet age-related macular degeneration (AMD), non-exudative choroidal neovascularization (CNV), hereditary angioedema (HAE) and brain edema after stroke.