WO2025228404A1 - Oxamide compound and use thereof in pharmaceuticals - Google Patents

Abstract

Provided in the present invention are an oxamide compound having a structure as shown in formula (I), or a pharmaceutically acceptable salt, isotope derivative or solvate thereof, or a stereoisomer, geometric isomer or tautomer thereof, or a prodrug molecule or metabolite thereof, and a pharmaceutical composition thereof and the use thereof. The compound involved in the present invention can efficiently inhibit STAT6 phosphorylation, can be used for preparing drugs for preventing and treating inflammatory diseases, and can be used for preparing anti-tumor drugs.

Description

Oxalide compounds and their applications in medicine Technical Field

This invention belongs to the field of pharmaceutical technology and relates to oxalamide compounds, their preparation methods and their pharmaceutical applications. Specifically, it relates to the compound shown in formula (I) or its pharmaceutically acceptable salts, isotope derivatives, solvates, or its stereoisomers, geometric isomers, tautomers, or its prodrug molecules, metabolites, and their pharmaceutical applications.

Background Technology

Transcription factors play a crucial role in eukaryotic gene expression by binding to specific DNA sites and regulating the transcription of virtually every gene in the cellular genome. It is estimated that there are over 1600 transcription factors in the human genome, and nearly 20% have been associated with different disease phenotypes. Many transcription factors have been identified as being associated with inflammatory and oncological diseases.

STAT6 is a key component of the Jak-STAT signaling pathway, which connects extracellular signals from various cytokines, hormones, and growth factors with nuclear transcription mechanisms. Four JAK (Janus tyrosine kinase) proteins (JAK1, JAK2, JAK3, TYK2) and seven STAT members (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6) have been identified in mammals. STAT proteins regulate the expression of numerous genes, including those involved in cell survival, development, differentiation, migration, apoptosis, and immune responses.

STAT6, primarily stimulated by IL-4 and IL-13, plays a crucial role in type II inflammation dominated by helper T cells (Th2). Therefore, it is closely related to the pathophysiology of various allergic diseases, such as atopic dermatitis, bullous pemphigoid, nodular prurigo, chronic spontaneous urticaria, eosinophilic esophagitis and food allergies, chronic rhinosinusitis with nasal polyps (CRSwNP), chronic rhinosinusitis without nasal polyps (CRSsNP), and nonsteroidal anti-inflammatory drug-induced respiratory diseases. This includes conditions such as drug-exacerbated respiratory disease/aspirin-exacerbated respiratory disease (NSAID-ERD/AERD), allergic rhinitis, asthma, chronic obstructive pulmonary disease (COPD), eosinophilic granulomatous polyangiitis (EGPA), and allergic bronchopulmonary aspergillosis. The JAK-STAT6 transduction pathway can induce tumor-associated macrophages (TAMs) to polarize towards M2-type TAMs, which plays a role in forming an immunosuppressive tumor microenvironment and promoting intratumoral angiogenesis. Furthermore, STAT6 is also involved in the regulation of the tumor microenvironment. In addition, certain forms of lymphoma, particularly Hodgkin's lymphoma, primary mediastinal and primary central nervous system lymphomas, as well as some follicular and T-cell lymphomas, are associated with STAT6 pathway dysregulation. Therefore, STAT6 has broad application prospects in inflammatory and tumor diseases. Because STAT6 is downstream of the JAK-STAT pathway, regulating STAT6 may be safer than using JAK inhibitors.

Currently, there are no STAT6 inhibitors on the market, but targeting the STAT6 signaling pathway may provide a better treatment strategy for these diseases.

Summary of the Invention

The technical problem to be solved by the present invention is to provide a novel oxalamide compound that can be used as a STAT6 inhibitor to prepare drugs for treating STAT6-mediated diseases or conditions and related diseases or conditions.

On the one hand, the present invention provides the compound of formula (I) or its pharmaceutically acceptable salt, isotope derivative, solvate, or its stereoisomer, geometric isomer, tautomer, or its prodrug molecule or metabolite:

in,

It can be a single bond or a double bond;

q and t are each independently selected from 0, 1 or 2, and q and t are not both selected from 0 at the same time;

p is selected from 1 or 2;

Z is selected from S, O, -S(=O)-, -S(=O) _2- , -S(=O)=NH, CHR ¹⁰ , CR ^{9R 10} or NR ¹⁰ ;

Y is selected from CHR ⁹ , CR ⁹ R ¹⁰ or NR ⁹ ;

^R1 is selected from _C6-12 aryl, 5-12 heteroaryl, 4-12 heterocyclic, 4-12 cycloalkenyl, _C3 - _C12 cycloalkyl, _C1-4 alkylene _C6-12 aryl, or _C1-4 alkenyl C6-12 aryl substituted with one or _more substituents, ^wherein the substituent is selected from ^Rg , ^CR1aR2aP ( ^O ) ^OR1bOR2b , ^CR1aR2aP (O) ^OR1bNHR2b , CR1aR2aP ⁽ O)( ^OR1b )(NH(AA)C(O) ^{OR1c), CR1aR2aP(O)(NHR2c)(NH(AA)C(O)OR1c ) , CR1aR2aP ( O ) ( NH (} AA)C(O) ^OR1c ... ^1c ), P(O)OR ^1b OR ^2b , P(O)(OR ^1b )(NH(AA)C(O)OR ^1c ), P(O)(NHR ^2c )(NH(AA)C(O)OR ^1c ) or P(O)(NH(AA)C(O)OR ^1c )(NH(AA)C(O)OR ^1c );

^R1a and ^R2a are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, _C1-4 alkyl, _C1-4 haloalkyl or _C1-4 hydroxyalkyl; or, ^R1a and ^R2a form a C3 _{- C6} cycloalkyl or a 3-6 membered heterocyclic group with the atom to which they are attached.

R ^1b and R ^2b are each independently selected from -R ^1aa , -R ^1aa -OC(O)-R ^1ab , -R ^1aa -C(O)OR ^1ab , -R ^1aa -OC(O)OR ^1ab , -R ^1aa -OR ^1ab , -R ^1aa -SC(O)OR ^1ab , -R ^1aa -SC(O)-R ^1ab , -R ^1aa -OC(O)NH-R ^1ab , -R ^1aa -OC(O)NR ^1ab R ^1ac , -R ^1aa -OC(O)-R ^1ab -OR ^1ac , -R ^1aa -OC(O)OR ^1ab -OR ^1ac , -R ^1aa -SC(O)OR ^1ab -OR ^1ac , -R ^1aa -SC(O)-R ^1ab -OR ^1ac or -R ^1aa -OC(O)-(NH(AA)C(O)OR ^1c );

^R1aa , ^R1ab , and ^R1ac are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, _C1-4 alkyl, _C1-4 haloalkyl, _C1-4 hydroxyalkyl, 5-7 membered heterocyclic, _C6-12 aryl, 5-12 membered heteroaryl, _C1-4 alkylene C6-12 aryl, or _C1-4 alkylene _C2-10 heteroaryl. The 5-7 membered heterocyclic, _C6-12 aryl, 5-12 membered _heteroaryl , C1-4 alkylene _{C6-12 aryl} , or _C1-4 alkylene _C2-10 heteroaryl may optionally be further substituted by one or more ^Rh or C(O) ^ORh .

^R1c and ^R2c are each independently selected from hydrogen, deuterium, or _C1-6 alkyl, 5-7 heterocyclic, _C6-12 aryl, 5-12 heteroaryl, C1-4 alkylene _C6-12 aryl or _C1-4 alkylene C2-10 _heteroaryl groups, which may be optionally substituted with one or more substituents, wherein the substituents are selected from deuterium, halogen, cyano, hydroxyl, amino, _C1-4 alkyl, _C1-4 alkoxy, _C1-4 haloalkyl, and _C1-4 haloalkoxy groups _;

AA is selected from residues of natural or non-natural amino acids, wherein the residues of the natural or non-natural amino acids are in the α or β configuration;

^R2 is selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, or C1-6 alkyl, _C1-6 alkoxy, _C2-6 alkenyl, _C2-6 alkynyl, _C3-6 cycloalkyl, _C1-6 alkylsulfonyl, _C1-6 alkylthio, _C1-6 alkylamine, or 4-10 membered heterocyclic groups, wherein the substituent is selected from deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, _C1-4 alkyl, _C1-4 alkoxy, _{C1-4 haloalkyl} , or _C1-4 haloalkoxy.

^R3 and ^R4 are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, or optionally substituted with one or more ^RY _C1-6 alkyl, _C1-6 alkoxy, _C1-6 alkylacyl, _C2-6 alkenyl, _C2-6 alkynyl, _C3-6 cycloalkyl, _C1-6 alkylsulfonyl, _C1-6 alkylthio, C1-6 alkylamine, 4-10 membered heterocyclic, _C6-12 aryl, _5-12 membered heteroaryl, _C1-6 alkylene _C6-12 aryl, or _C1-6 alkylene _C2-10 heteroaryl;

Alternatively, ^R3 and ^R4 and the atoms they are attached to together form a _{C3 - C6} cycloalkyl or a _3-6 membered heterocyclic group, which may optionally be substituted by one or more R ^Y1 or R ^Y2 ;

^RY1 and ^RY2 are each independently selected from deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, _C1-4 alkyl, _C1-4 alkoxy, _C1-4 haloalkyl or _C1-4 haloalkoxy;

^R5 and ^R6 are each independently selected from hydrogen, deuterium, _C1-6 alkyl, or _C1-6 haloalkyl;

^R7 and ^R8 are each independently selected from _C1-6 alkyl, 5-12 heterocyclic, 5-12 cycloalkyl, C6-12 aryl, _5-12 heteroaryl, _C1-4 alkylene _C6-12 aryl or _C1-4 alkylene _C2-10 heteroaryl, which may be optionally substituted by one or more ^R2 .

Alternatively, ^R7 and ^R8 and the atoms they are attached to together form 4-14 membered heterocyclic groups and 5-12 membered heteroaryl groups, which may optionally be further substituted by one or more ^R2 groups.

^R9 and ^R10 are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, or optionally substituted with one or more ^RY _C1-6 alkyl, _C1-6 alkoxy, _C1-6 alkylacyl, _C2-6 alkenyl, _C2-6 alkynyl, _C3-6 cycloalkyl, _C1-6 alkylsulfonyl, _C1-6 alkylthio, C1-6 alkylamine, 4-10 membered heterocyclic, _C6-12 aryl, _5-12 membered heteroaryl, _C1-6 alkylene _C6-12 aryl, or _C1-6 alkylene _C2-10 heteroaryl;

Alternatively, ^R9 and ^R10 and the atoms they are attached to together form a _C3 - _C6 cycloalkyl or a _{3-6 membered} heterocyclic group, which may optionally be substituted by one or more R ^Y1 or R ^Y2 ;

R Z  and RY are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, imino, or optionally substituted with one or more RQ , and may be C 1-6 alkyl, C  1-6  alkoxy, C 2-6  alkenyl, C2-6 alkynyl, C 3-6  cycloalkyl, C 1-6  alkylsulfonyl, C1-6 alkylthio, C 1-6  alkylamine, 4-10 membered heterocyclic, C 6-12  aryl, 5-12 membered heteroaryl, -NR a R b , -OR a , -C(O)R a , -C(O)ORa, -NHC(O)OR a  , -NR b C(O)OR a , -NR  aC(O)NR b Rc, -C(O)NRaRb , -S ( O)R c  , -S(O)  2  R c  -S(O) = NHR c , -S(O)NR c R d or -S(O) 2 NR c R d ;

R and ^Q are selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, imino, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, C1-6 haloalkoxy, C2-6 alkenyl, _C2-6 alkynyl _{, C3-6} cycloalkyl, _C1-6 alkylsulfonyl, _C1-6 alkylthio, _C1-6 alkylamine, 4-10 membered heterocyclic, _C6-12 aryl, 5-12 membered heteroaryl, -NR ^e R ^f , -OR ^e , -C(O)R ^e , _-C (O)OR ^e , -NHC(O)OR ^e , -NHC(O)R ^e , -NR ^e C(O)OR ^f , -NR ^e C(O)R ^f , -NR ^g C(O)NR ^e R ^f , -C(O)NR ^e R ^f , -S(O)R ^e -S(O) ₂ ^Re , -S(O) = NHR ^Re , -S(O)NR ^Re R ^f or -S(O) ₂ NR ^Re R ^f ;

^Ra , ^Rb , ^Rc , ^Rd , ^Re , ^Rf , ^Rg , and ^Rh are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, imino, or optionally substituted with one or more substituents, namely _C1-6 alkyl, _C1-6 alkoxy, _C2-6 alkenyl, _C2-6 ynyl, _C3-6 cycloalkyl, C1-6 alkylsulfonyl, _C1-6 alkylthio, _C1-6 alkylamine, 4-10 membered heterocyclic, _C6-12 aryl, 5-12 membered heteroaryl, C1-4 _{alkyleneC6-12} aryl, or _C1-4 alkyleneC2-10 _heteroaryl , wherein the substituents _are selected from deuterium, halogen, cyano, hydroxyl, amino, _C1-4 alkyl, C1-4 alkoxy, _C1-4 haloalkyl, C2-6 alkylyl, C3-6 cycloalkyl, C4-6 alkylsulfonyl, C1-6 alkylthio, _C1-6 alkylamine, _C3-6 cycloalkyl, C4-6 alkylyl ... _1-4 haloalkoxy, phenyl, or benzyl.

In some embodiments, the compound has the structure shown in formula (II):

The definitions of p, t, q, Z, Y, ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , ^R6 , ^R7 , and ^R8 are as described in general formula (I).

In some embodiments, the compound has a structure as shown in formula (III), (IV), (V), (VI), (VII), or (VIII):

The definitions of p, Z, Y, ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , ^R6 , ^R7 , and ^R8 are as described in general formula (I).

In some embodiments, the compound has a structure as shown in formula (IIIa), (IVa), (Va), (VIa), (VIIa), (VIIIa), (IIIb), (IVb), (Vb), (VIb), (VIIb), or (VIIIb):

The definitions of Z, Y, ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , ^R6 , ^R7 , and ^R8 are as described in general formula (I).

In some embodiments, the compound has a structure as shown in formulas (IIIa-1), (IIIa-2), (IIIa-3), (IIIa-4), (IIIa-5), (IVa-1), (IVa-2), (IVa-3), (IVa-4), (IVa-5), (IVa-6), (IVa-7), (IVa-8), (IVa-9), (IVa-10), (Va-1), (Via-1), or (IVb-1):

The definitions of ^R1 , ^R2 , ^R5 , ^R6 , ^R7 , ^R8 , ^R9 , ^R10 , ^RY1 , and ^RY2 are as described in general formula (I).

In some embodiments, ^R2 is selected from hydrogen or hydroxyl.

In some embodiments, ^R3 and ^R4 are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, or optionally substituted with one or more ^RYs , namely _C1-6 alkyl, _C1-6 alkoxy, _C1-6 alkylacyl, C3-6 cycloalkyl, _4-10 heterocyclic, _C6-12 aryl, 5-12 heteroaryl, _C1-6 alkylene _C6-12 aryl, or _C1-6 alkylene _C2-10 heteroaryl, wherein each ^RY is independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, or imino.

In some embodiments, ^R3 and ^R4 and the atoms they are attached to together form a _C3 - _{C6 cycloalkyl} group, which may optionally be substituted by one or more ^RY1 or ^RY2 groups; each of ^{the RY1} _and ^RY2 groups is independently selected from deuterium, halogen, cyano, hydroxyl, amino, _mercapto , oxo, C1-4 alkyl, _C1-4 alkoxy, _C1-4 haloalkyl, or _C1-4 haloalkoxy.

In some embodiments, ^R3 and ^R4 are each independently selected from hydrogen, deuterium, hydroxyl, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkylene, _C2-10 heteroaryl, or _C1-6 alkylacyl.

In some embodiments, ^R9 and ^R10 are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, or optionally substituted with one or more ^RYs , namely _C1-6 alkyl, _C1-6 alkoxy, C1-6 alkylacyl, _C2-6 alkenyl, _C2-6 alkynyl, _C3-6 cycloalkyl, _C1-6 alkylsulfonyl, _C1-6 alkylthio, _C1-6 alkylamine, 4-10 membered heterocyclic, _C6-12 aryl, 5-12 membered heteroaryl, _C1-6 alkylene _{C6-12 aryl} , or _C1-6 alkylene _C2-10 heteroaryl; and each ^RY is independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, _C1-6 alkyl, or imino.

In some embodiments, ^R9 and ^R10 are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, or optionally substituted with one or more ^RYs , namely _C1-6 alkyl, _C1-6 alkoxy, C1-6 alkylacyl, _C2-6 alkenyl, _C2-6 alkynyl, _C3-6 cycloalkyl, _C1-6 alkylsulfonyl, _C1-6 alkylthio, _C1-6 alkylamine, 4-10 membered heterocyclic, _C6-12 aryl, 5-12 membered heteroaryl, _C1-6 alkylene _{C6-12 aryl} , or _C1-6 alkylene _C2-10 heteroaryl; and each ^RY is independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, or imino.

In some embodiments, ^R9 and ^R10 and the atoms to which they are attached together form a _{C3 - C6} cycloalkyl or a _3-6 membered heterocyclic group, which may optionally be substituted by one or _more ^RY1 or ^RY2 groups, each of which is independently selected from ^deuterium , halogen, cyano, hydroxyl, amino, mercapto, oxo, _C1-4 alkyl, ^C1-4 alkoxy, _C1-4 haloalkyl, or _C1-4 haloalkoxy.

In some embodiments, ^R9 and ^R10 are each independently selected from hydrogen, deuterium, or _C1-6 alkyl, C1-6 alkylacyl, _5-12 heteroaryl, or _C1-6 alkylene _C2-10 heteroaryl, which may optionally be substituted with one or more ^RY .

In some embodiments, the ^R9 , ^R10 and the atoms to which they are attached together form a cyclopropyl group, which may optionally be substituted by one or more ^RY1 or ^{RY2 groups} , each of which is independently selected from deuterium, ^halogen , cyano, ^hydroxyl , amino, mercapto, oxo, _C1-4 alkyl, _C1-4 alkoxy, _C1-4 haloalkyl, or _C1-4 haloalkoxy.

In some embodiments, ^R5 and ^R6 are each independently selected from hydrogen or _C1-6 alkyl.

In some embodiments, ^R1 is selected from _C6-12 aryl or 5-12 heteroaryl groups substituted with one or more substituents, wherein the substituents are selected ^{from Rg , CR1aR2aP} (O) ^OR1bOR2b , ^CR1aR2aP (O ^{) OR1bNHR2b} , ^{or CR1aR2aP} (O) ^{( OR1b} )(NH(AA)C(O) ^OR1c ).

In some embodiments, ^R1 is selected from compounds substituted with one or more substituents. The substituent group is selected from R ^g , CR ^1a R ^2a P(O)OR ^1b OR ^2b , CR ^1a R ^2a P(O)OR ^1b NHR ^2b or CR ^1a R ^2a P(O)(OR ^1b )(NH(AA)C(O)OR ^1c ).

In some embodiments, ^R1a or ^R2a is each independently selected from hydrogen, halogen or oxo, wherein the halogen is preferably F or Cl.

In some embodiments, the CR ^1a R ^2a P(O)OR ^1b OR ^2b , CR ^1a R ^2a P(O)OR ^1b NHR ^2b , or CR ^1a R ^2a P(O)(OR ^1b )(NH(AA)C(O)OR ^1c ) is selected from...

In some embodiments, the CR ^1a R ^2a P(O)OR ^1b OR ^2b , CR ^1a R ^2a P(O)OR ^1b NHR ^2b , or CR ^1a R ^2a P(O)(OR ^1b )(NH(AA)C(O)OR ^1c ) is selected from...

In some embodiments, ^R7 and ^R8 are each independently selected from _C1-6 alkyl, 5-12 heterocyclic, 5-12 cycloalkyl, C6-12 aryl, _5-12 heteroaryl, _C1-4 alkylene _C6-12 aryl, or _C1-4 alkylene _C2-10 heteroaryl groups that may be optionally substituted with one or more R2 ^groups .

The R and ^Z are independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, oxo, or C1-6 _alkyl , _C3-6 cycloalkyl, 4-10 heterocyclic, _C6-12 aryl, or 5-12 heteroaryl groups that may be optionally substituted with one or more R and ^Q.

In some embodiments, ^R7 and ^R8 and the atoms to which they are attached together form a 4-14 membered heterocyclic group, which may optionally be substituted by one or more ^R2 groups;

The R and ^Z are independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, oxo, or C1-6 _alkyl , _C3-6 cycloalkyl, 4-10 heterocyclic, _C6-12 aryl, or 5-12 heteroaryl groups that may be optionally substituted with one or more R and ^Q.

In a further embodiment, ^RQ is selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo _, imino, _C1-6 alkyl, _C1-6 haloalkyl, C1-6 alkoxy, _C1-6 haloalkoxy, _C1-6 alkylamine, 4-10 membered heterocyclic, _C6-12 aryl, 5-12 membered heteroaryl, ^-NReRf , -C(O) ^Re , -NHC(O) ^Re , or ^-NReC (O) ^{Rf ,} wherein ^Re and ^Rf are each independently selected from hydrogen, deuterium, or _C1-6 alkyl.

In some implementations, the Selected from:

In some embodiments, the compound of formula (I) is selected from compounds with the following structures, or pharmaceutically acceptable salts, isotope derivatives, solvates, or stereoisomers, geometric isomers, tautomers, or prodrug molecules or metabolites thereof:

On the other hand, the present invention provides a pharmaceutical composition comprising a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IIIa), (IVa), (Va), (VIa), (VIIa), (VIIIa), (IIIb), (IVb), (Vb), (VIb), (VIIb), (VIIIb), (IIIa-1), (IIIa-2), (IIIa-3), (IIIa-4), (IIIa-5), (IVa-1), (IVa-2), (IVa-3), (IVa-4), (IVa-5), (IVa-6), (IVa-7), (IVa-8), (IVa-9), (IVa-10), (Va-1), (Via-1), or (IVb-1) or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof.

In another aspect, the present invention provides compounds of formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IIIa), (IVa), (Va), (VIa), (VIIa), (VIIIa), (IIIb), (IVb), (Vb), (VIb), (VIIb), (VIIIb), (IIIa-1), (IIIa-2), (IIIa-3), (IIIa-4), (IIIa-5), (IVa-1), (IVa-2), (IVa-3), (IVa-4), (IVa-5), (IVa-6), (IVa-7), (IVa-8), (IVa-9), (IVa-10), (Va-1), (Via-1), or (IVb-1) or their pharmaceutically acceptable salts, isotopic derivatives, solvates, or their stereoisomers, geometric isomers, tautomers, or their prodrug molecules, metabolites, or... The use of the pharmaceutical composition described above in the preparation of a medicament for treating STAT6-mediated diseases or conditions and related diseases or conditions, wherein the STAT6-mediated diseases or conditions are preferably atopic dermatitis, bullous pemphigoid, nodular prurigo, chronic spontaneous urticaria, eosinophilic esophagitis, food allergy, chronic rhinosinusitis with nasal polyps (CRSwNP), chronic rhinosinusitis without nasal polyps (CRSsNP), and nonsteroidal anti-inflammatory drug-induced respiratory disease (NSAID-ERD/ AERD), allergic rhinitis, asthma, chronic obstructive pulmonary disease (COPD), eosinophilic granulomatous polyangiitis (EGPA), or allergic bronchopulmonary aspergillosis; the tumor is selected from lymphoma, solitary fibrous tumor, colon cancer, esophageal cancer, breast cancer, bile duct cancer, liver cancer, kidney cancer, gastric cancer, head and neck squamous cell carcinoma, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), acute B-lymphoblastic leukemia, bladder cancer, pancreatic cancer, osteosarcoma, myeloma, glioma, ovarian cancer, or skin cancer.

The present invention also provides a method for treating a disease or symptom, the method comprising administering to a patient in need a therapeutically effective amount of the formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IIIa), (IVa), (Va), (VIa), (VIIa), (VIIIa), (IIIb), (IVb), (Vb), (VIb), (VIIb), (VIIIb), (IIIa-1), (IIIa-2), (IIIa-3) as described above. The compounds (IIIa-4), (IIIa-5), (IVa-1), (IVa-2), (IVa-3), (IVa-4), (IVa-5), (IVa-6), (IVa-7), (IVa-8), (IVa-9), (IVa-10), (Va-1), (Via-1), or (IVb-1), or their pharmaceutically acceptable salts, isotopic derivatives, solvates, or their stereoisomers, geometric isomers, tautomers, or their prodrug molecules, metabolites, or pharmaceutical compositions described above.

In some implementations, the disease being treated and/or prevented is a STAT6-mediated disease, which is a tumor or a type II inflammation-related disease selected from atopic dermatitis, bullous pemphigoid, nodular prurigo, chronic spontaneous urticaria, eosinophilic esophagitis, food allergy, chronic rhinosinusitis with nasal polyps (CRSwNP), chronic rhinosinusitis without nasal polyps (CRSsNP), nonsteroidal anti-inflammatory drug-induced respiratory disease (NSAID-ERD/AERD), allergic rhinitis, asthma, chronic obstructive pulmonary disease (COPD), eosinophilic granulomatous polyangiitis (EGPA), or allergic bronchopulmonary aspergillosis.

Furthermore, the tumor is selected from lymphoma, solitary fibrous tumor, colon cancer, esophageal cancer, breast cancer, bile duct cancer, liver cancer, kidney cancer, gastric cancer, head and neck squamous cell carcinoma, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), acute B-lymphoblastic leukemia, bladder cancer, pancreatic cancer, osteosarcoma, myeloma, glioma, ovarian cancer, or skin cancer.

In some embodiments, the present invention provides an intermediate compound M2, chemically named (3R,4S or 3S,4R)-1-((3S,6S,10aS)-6-amino-5-oxodecahydropyrrolo[1,2-a]azopyren-3-carbonyl)-4-phenylpyrrolidine-3-carbamate, with the following structural formula:

In some embodiments, the intermediate compound M2 provided by the present invention is a mixture of 3R,4S and 3S,4R; other similar structures described by thick solid lines in the present invention are mixtures of 3R,4S and 3S,4R.

In some embodiments, the present invention provides an intermediate compound M3 or a pharmaceutically acceptable salt, isotopic derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule thereof, wherein the chemical name of M3 is (3S,6S,10aS)-6-amino-3-((R)-2-phenylmorpholine-4-carbonyl)octahydropyrrolo[1,2-a]zosin-5(1H)-one trifluoroacetate, and its structural formula is as follows:

In some embodiments, the present invention provides an intermediate compound M1-1 or a pharmaceutically acceptable salt, isotopic derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule thereof:

The definitions of ^R1a , ^R2a , ^R6 , and ^Rg are as described in general formula (I).

In some embodiments, the present invention provides intermediate compound M1-3 or its pharmaceutically acceptable salt, isotope derivative, solvate, or its stereoisomer, geometric isomer, tautomer, or prodrug molecule:

The definitions of ^R1a , ^R2a , ^R5 , ^R6 , ^R7 , ^R8 , ^Rg , Z and Y are as described in general formula (I).

In some embodiments, the present invention provides an intermediate compound M1 or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule thereof:

The definitions of ^R1a , ^R2a , ^R5 , ^R6 , ^R7 , ^R8 , ^Rg , Z and Y are as described in general formula (I).

In some embodiments, the present invention provides an intermediate compound M4 or a pharmaceutically acceptable salt, isotopic derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule thereof:

Unless otherwise stated, the general chemical terms used in the structural formulas have their usual meanings.

For example, unless otherwise stated, the term "halogen" as used in this invention refers to fluorine, chlorine, bromine, or iodine.

In this invention, unless otherwise stated, "alkyl" includes a straight-chain or branched monovalent saturated hydrocarbon group. For example, alkyl includes methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 3-(2-methyl)butyl, 2-pentyl, 2-methylbutyl, neopentyl, n-hexyl, 2-hexyl, 2-methylpentyl, etc. Similarly, " _1-6 " in " _C1-6 alkyl" refers to a group containing 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight-chain or branched form.

The term " _alkylene " refers to the group formed by removing one hydrogen atom _from the aforementioned "alkyl". Examples include methylene, _{-CH2CH2- , -CH2CH2CH2- , -CH2CH2CH2CH2- , -CH2CH} ( _{CH3 )} -, and _-CH2CH ( _CH3 ) _CH2- .

The term "alkoxy" refers to the oxygen ether form of the aforementioned straight-chain or branched alkyl group, i.e., -O-alkyl.

The term "halogenated alkyl" refers to an alkyl group in which one or more H atoms have been replaced by halogen atoms.

The term "haloalkoxy" refers to a group consisting of -O-haloalkyl groups.

The term "oxo" or "oxo group" refers to an oxygen atom in the form of a divalent substituent, which forms a carbonyl group when attached to a carbon atom, and a sulfoxide group, sulfone group, or N-oxide group when attached to a heteroatom.

The term "cycloalkyl" refers to a ring system having at least one cycloalkyl group. Preferably, it is a _C3-12 cycloalkyl group, more preferably a _C3-6 group, where " _C3-12 " means that the cycloalkyl group can have 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cyclic atoms. The cycloalkyl group can include monocyclic and polycyclic groups (e.g., having 2, 3, or 4 fused rings, spirocyclic, bridged rings, etc.). In some embodiments, the cycloalkyl group includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, etc.; the cycloalkyl group can also be fused to an aryl, heterocyclic, or heteroaryl ring, wherein the ring connected to the parent structure is a cycloalkyl group.

The term "alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds, such as vinyl, propenyl, 1,3-butadiene, cis-butenyl, trans-butenyl, etc.

The term "alkenyl" refers to the group formed by removing one hydrogen atom from the aforementioned "alkenyl" group, such as -CH=CH-, -CH ₂ CH=CH-, -CH ₂ CH=CHCH _2- , etc.

The term "imino" refers to the divalent group remaining after removing two hydrogen atoms from an ammonia molecule, with the structural formula: HN= (or -NH-). When it is attached to a hydrocarbon group, it forms a secondary amine.

The term "alkynyl" refers to an alkyl group having one or more carbon-carbon triple bonds, such as ethynyl, propynyl, etc.

The term "aryl" in this invention, unless otherwise stated, refers to an unsubstituted or substituted monocyclic or fused-ring aromatic group comprising a carbon ring atom. Preferably, it is a _C6-12 aryl group, more preferably a _C6-10 monocyclic or bicyclic aromatic ring group. Phenyl or naphthyl are preferred. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl group, wherein the ring attached to the parent structure is an aryl ring; non-limiting examples include, but are not limited to, benzocyclopentyl.

The term "heteroaryl" in this invention, unless otherwise stated, refers to a monocyclic or polycyclic (e.g., fused bicyclic) aromatic heterocycle having at least one heteroatom selected from N, O, and/or S, wherein the nitrogen or sulfur heteroatom is selectively oxidized, and the nitrogen heteroatom is selectively quaternized. Preferably, it is a 5-14 membered heteroaryl, wherein "5-14" in 5-14 membered heteroaryl refers to a heteroaryl containing 5-14 cyclic atoms of C, N, O, or S. More preferably, it is a 5-10 membered heteroaryl, and even more preferably, it is a 5-6 membered heteroaryl. Examples of heteroaryl groups include, but are not limited to, thienyl, furanyl, imidazolyl, isoxazolyl, oxazolyl, pyrazolyl, pyrroloyl, thiazolyl, thiadiazolyl, triazolyl, pyridinyl, pyridazinyl, indolyl, azaindolyl, indolyl, benzimidazolyl, benzofuranyl, benzothiophene, benzoisoxazolyl, benzothiazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyladenine, quinolinyl, or isoquinolinyl. The heteroaryl group may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring.

The term "heterocyclic group" refers to a ring system having at least one cyclic alkyl or cyclic alkenyl group containing a heterocycle, wherein the heteroatom is selected from N, O, and/or S. The heterocyclic group can include monocyclic or polycyclic groups (e.g., having 2, 3, or 4 fused rings, spirocyclic, bridged rings, etc.). The heterocyclic group can be connected to other parts of the compound via cyclic carbon atoms or cyclic heteroatoms. Preferably, it is a 3-14 membered heterocyclic group, where "3-14" refers to a heterocyclic group consisting of 3-14 cyclic atoms of C, N, O, or S; more preferably, it is a 3-6 membered heterocyclic group, and even more preferably, a 5-6 membered heterocyclic group; wherein the nitrogen or sulfur heteroatom can be selectively oxidized, and the nitrogen heteroatom can be selectively quaternized. Examples of these heterocyclic groups include, but are not limited to, aza-butyl, pyrrolidinyl, piperidinyl, piperazinyl, oxoperazinyl, oxoperridinyl, tetrahydrofuranyl, dioxopentyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydrooxazolyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, and tetrahydrooxadiazolyl. Spiroheterocycles can be 6- to 12-membered spiroheterocycles, including, but not limited to, 4-azaspiro[2,4]heptane and 4-azaspiro[2,4]heptane. Heterocyclic groups also include cyclic systems in which the above-mentioned heterocyclic rings are fused with one or more cycloalkyl, aryl, or heteroaryl groups, including, but not limited to, dihydroindolyl, isodihydroindolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinone, tetrahydroquinolinyl, tetrahydroisoquinolinyl, tetrahydrobenzopyranyl, and tetrahydropyranopyridyl.

The term " _benzyl " refers to the group formed by removing a hydrogen atom _{from the methyl carbon in a toluene molecule (C6H5CH2- )} .

The term "amino acid residue" refers to the unit of amino acids that make up a polypeptide. When these amino acids are linked together, some of their groups participate in the formation of peptide bonds, resulting in the loss of a water molecule. Therefore, the amino acid unit in a polypeptide is called an amino acid residue; that is, the remaining part after the amino acids linked by peptide bonds lose water. For example, the glycine residue is: -NH- _CH2 -CO-.

The term "α-amino acid" refers to any natural (encoded) and non-natural α-aminocarboxylic acid, including their D-isomers, which are called α-amino acids when the amino group is attached to a carbon atom (also called the α-carbon atom) directly bonded to the carboxyl carbon.

The term "β-amino acid" refers to any β-aminocarboxylic acid, that is, when the amino group is attached to a carbon atom (also called the β-carbon atom) that is one carbon atom away from the carboxyl carbon. Examples include β-alanine and isoserine.

The term "alkylamino" refers to an open-chain alkyl group containing a nitrogen atom, such as _C1 - _C6 alkylamino, including but not limited to methylamino, ethylamino, isopropylamino, dimethylamino, methylethylamino, diethylamino, etc.

The term "alkylthio" refers to a straight-chain or branched alkyl group linked by a sulfur atom, i.e., -S-alkyl, such as _C1-6 alkylthio groups, including but not limited to methylthio, ethylthio, propylthio (including n-propylthio and isopropylthio), butylthio (including n-butylthio, isobutylthio, sec-butylthio, and tert-butylthio), penthio (including n-pentylthio, isopentylthio, and neopentylthio), and hexylthio (n-hexylthio, 2-methylpentylthio, 3-methylpentylthio, 2,3-dimethylbutylthio, and 2,2-dimethylbutylthio).

The term "alkyl sulfone" refers to a straight-chain or branched alkyl group linked by a sulfone group, i.e., _-SO2 -alkyl, such as _C1-6 alkyl sulfones, including but not limited to methyl sulfone, ethyl sulfone, propane sulfone (including n-propane sulfone and isopropane sulfone), butyl sulfone (including n-butyl sulfone, isobutyl sulfone, sec-butyl sulfone, and tert-butyl sulfone), pentyl sulfone (including n-pentyl sulfone, isopentyl sulfone, and neopentyl sulfone), and hexyl sulfone (n-hexyl sulfone, 2-methylpentyl sulfone, 3-methylpentyl sulfone, 2,3-dimethylbutyl sulfone, and 2,2-dimethylbutyl sulfone), etc.

The term "cyano" refers to the -CN group.

The term "medicinal salt" refers to salt prepared from a pharmaceutically acceptable, non-toxic alkali or acid.

The "compound" described in this invention includes, but is not limited to, compounds in the following forms: free base, stereoisomer, geometric isomer, tautomer, isotope, pharmaceutically acceptable salt, solvate, hydrate, prodrug (ester), etc.

The "compound" described in this invention can be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, for example, enantiomers and diastereomers. Compounds containing asymmetric carbon atoms in this invention can be isolated in optically active pure form or in racemic form. Optically active pure form can be obtained by resolution of racemic mixtures, synthesis using chiral starting materials or chiral reagents.

The term "pharmaceutically acceptable salt" refers to a salt of the compounds of this invention, prepared by reacting a compound having specific substituents discovered in this invention with a relatively non-toxic acid or base. When the compounds of this invention contain relatively acidic functional groups, a base addition salt can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. When the compounds of this invention contain relatively basic functional groups, an acid addition salt can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Certain specific compounds of this invention contain both basic and acidic functional groups, and thus can be converted into either a base or an acid addition salt.

The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof.

When the compounds provided by this invention are acids, their corresponding salts can be conveniently prepared from pharmaceutically acceptable, non-toxic bases, including inorganic and organic bases. Salts derived from inorganic bases include salts of aluminum, ammonium, calcium, copper (high and low valence), ferric iron, ferrous iron, lithium, magnesium, manganese (high and low valence), potassium, sodium, zinc, etc. Salts of ammonium, calcium, magnesium, potassium, and sodium are particularly preferred. Non-toxic organic bases capable of being derived into pharmaceutically acceptable salts include primary, secondary, and tertiary amines, as well as cyclic amines and amines containing substituents, such as naturally occurring and synthetic amines containing substituents. Other pharmaceutically acceptable non-toxic organic bases that can form salts include ion exchange resins, as well as arginine, betaine, caffeine, choline, N',N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, reduced glucosamine, glucosamine, histidine, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, polyamine resins, procaine, chloroprocaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.

When the compound provided by this invention is a base, pharmaceutically acceptable non-toxic acids, including inorganic and organic acids, can be used to conveniently prepare their corresponding salts. Such acids include, for example, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, formic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucilage, nitric acid, pyric acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, oxalic acid, propionic acid, glycolic acid, hydroiodic acid, perchloric acid, cyclohexanesulfonic acid, salicylic acid, 2-naphthalenesulfonic acid, saccharinic acid, trifluoroacetic acid, tartaric acid, and p-toluenesulfonic acid. More preferably, citric acid, hydrobromic acid, formic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid, and tartaric acid. More preferably, formic acid and hydrochloric acid.

Unless otherwise stated, the term "isomer" is intended to include geometric isomers, cis-trans isomers, stereoisomers, enantiomers, optical isomers, diastereomers and tautomers.

In addition to the salt form, the compounds provided by this invention also exist in prodrug form. The prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to be converted into the compounds of this invention. Furthermore, the prodrugs can be converted into the compounds of this invention in the in vivo environment via chemical or biochemical methods.

The compounds of this invention may contain non-natural proportions of atomic isotopes on one or more of the atoms constituting the compound. For example, the compounds may be labeled with radioactive isotopes such as tritium ( ^3H ), iodine-125 ( ^125I ), or C-14 ( ^14C ). As another example, deuterium may be used to replace hydrogen to form deuterated drugs. The bond between deuterium and carbon is stronger than that between ordinary hydrogen and carbon, and compared to undeuterated drugs, deuterated drugs have advantages such as reduced toxicity, increased drug stability, enhanced efficacy, and prolonged biological half-life. All variations in the isotopic composition of the compounds of this invention, regardless of radioactivity, are included within the scope of this invention.

The drug prodrugs of the compounds of this invention are included within the scope of protection of this invention. Generally, a drug prodrug refers to a functional derivative that is readily converted into the desired compound in vivo. For example, any pharmaceutically acceptable salt, ester, salt of ester, or other derivative of the compounds of this application, which, upon administration to a receptor, can directly or indirectly provide the compound of this application or its pharmaceutically active metabolites or residues.

The compounds described in this invention may contain one or more asymmetric centers, and may thereby produce diastereomers and optical isomers. This invention includes all possible diastereomers and their racemic mixtures, their substantially pure enantiomers, all possible geometric isomers, and their pharmaceutical salts.

When the compound represented by formula (I) has tautomers, unless otherwise stated, the present invention includes any possible tautomers and their pharmaceutical salts, and mixtures thereof.

This invention also includes atoms of all isotopes, whether in intermediates or final compounds. Isotopic atoms include those having the same number of atoms but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

The term "pharmaceutical composition" refers to a mixture of one or more compounds of this application or their pharmaceutical salts with pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the compounds of this application to an organism.

In this invention, the terms "a," "an," "the," "at least one," and "one or more" are used interchangeably. Thus, for example, a mixture comprising "a" pharmaceutically acceptable excipient can be interpreted as indicating that the pharmaceutical composition includes "one or more" pharmaceutically acceptable excipients.

The term "pharmaceuticalally acceptable excipient" refers to excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

The pharmaceutical compositions of the present invention can be prepared by combining the compounds of this application with suitable pharmaceutically acceptable excipients, for example, in solid, semi-solid, liquid or gaseous formulations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalers, gels, microspheres and aerosols.

Typical routes of administration for the compounds of the present invention or their pharmaceutical salts or pharmaceutical compositions include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, vaginal, nasal, ocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

The term "treatment" generally refers to achieving the desired pharmacological and/or physiological effect. This effect can be therapeutic, depending on whether it partially or completely stabilizes or cures the disease and/or causes side effects due to the disease. As used herein, "treatment" encompasses any treatment of a patient's disease, including: (a) suppressing the symptoms of the disease, i.e., preventing its progression; or (b) alleviating the symptoms of the disease, i.e., causing the disease or symptoms to regress.

The term "effective amount" means (i) the amount of the compound of this application used to treat or prevent a particular disease, condition, or disorder; (ii) to reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) to prevent or delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the compound of this application constituting a "therapeutic effective amount" varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and the present disclosure.

The term "STAT6" refers to member 6 of the family of signal transduction and transcription activator factors.

This invention, based on the STAT6 target, has developed a series of novel compounds. This invention is the first to apply oxalamide-based core compounds in the development of STAT6 inhibitors. Biological experimental results show that the compounds of this invention can significantly inhibit STAT6 phosphorylation, while possessing good physicochemical properties, excellent pharmacokinetic characteristics, and good metabolic stability across different species. They also exhibit low hERG inhibition rates, demonstrating great promise for clinical application.

Detailed Implementation

To make the above content clearer and more explicit, the technical solution of the present invention will be further illustrated by the following embodiments. The following embodiments are only used to illustrate specific implementation methods of the present invention so that those skilled in the art can understand the present invention, but are not intended to limit the scope of protection of the present invention. In the specific implementation methods of the present invention, the technical means or methods, etc., not specifically described, are conventional technical means or methods in the art.

Unless otherwise stated, all temperatures in this invention refer to degrees Celsius.

This invention uses the following abbreviations:

DCM: Dichloromethane; DMF: N,N-Dimethylformamide; DIPEA: N,N-Diisopropylethylamine; EA: Ethyl acetate; LC-MS: Liquid Chromatography-Mass Spectrometry; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-Tetramethylurea hexafluorophosphate; TFA: Trifluoroacetic acid; Cd: Cadmium powder; CuCl: Cuprous chloride; LiOH: Lithium hydroxide; THF: Tetrahydrofuran; TMSBr: Trimethylbromosilane; _AgNO3 : Silver nitrate; NaOH: Sodium hydroxide; _CH3I : Iodomethane; NaH: Sodium hydride; DMSO: Dimethyl sulfoxide; LC-MS: Liquid Chromatography-Mass Spectrometry; HEPES: Hydroxyethylpiperazine ethanesulfonic acid; Tween 20: Tween 20; EDTA: Ethylenediaminetetraacetic acid; DTT: Dithiothreitol; _ddH2 O: Double-distilled water; IPTG: Isopropyl-β-D-thiogalactoside; 5-FAM: 5-FAM-ApYKPFQDLI- _NH2 ; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide; PBST: Phosphate-buffered saline solution; PVDF membrane: Polyvinylidene fluoride membrane; TBST: Tris-buffered saline containing Tween 20; FBS: Fetal bovine serum; DMEM: Durbico-modified avian culture medium; IL-4: Interleukin-4; BSA: Bovine serum albumin; PBS: Phosphate-buffered saline solution; PMSF: Benzyl sulfonyl fluoride.

In the embodiments of this invention, x mL × y means that the extraction is repeated y times, with x mL each time. For example, if ethyl acetate (20 mL × 3) is used for extraction, it means that 20 mL of ethyl acetate is used for extraction each time, and the extraction is repeated 3 times. In the embodiments of this invention, the amount of eluent is a volume ratio. For example, petroleum ether: ethyl acetate = 40:60 means that the volume ratio of petroleum ether to ethyl acetate is 40:60.

Furthermore, all operations involving easily oxidized or hydrolyzed raw materials are performed under nitrogen protection. Unless otherwise stated, the raw materials used in this invention are commercially available and can be used directly without further purification.

All reaction raw materials and common intermediates involved in the embodiments of the present invention can be purchased commercially or obtained in-house. The preparation process of raw materials and common intermediates that need to be obtained in-house is detailed below.

Preparation route: Preparation of key intermediate M1

Compound M1-1 and compound M1-2 undergo an amide condensation reaction under alkaline conditions to generate compound M1-3. Compound M1-3 is then deprotected in the presence of trimethylbromosilane to give compound M1.

The definitions of ^R1a , ^R2a , ^R5 , ^R6 , ^R7 , ^R8 , ^Rg , Z and Y are as described in general formula (I).

Synthesis of intermediate M2: (3R,4S or 3S,4R)-1-((3S,6S,10aS)-6-amino-5-oxodecahydropyrrolo[1,2-a]azobenzene-3-carbonyl)-4-phenylpyrrolidin-3-carbamate

Step 1: Synthesis of ((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]azo-6-yl) tert-butyl carbamate (M2-2)

(3S,6S,10aS)-6-((tert-Butoxycarbonyl)amino)-5-oxodecahydropyrrolo[1,2-a]azo-3-carboxylic acid (M2-1, 326 mg, 1 mmol) and (3R,4S or 3S,4R)-4-phenylpyrrolidine-3-carbamate (206 mg, 1.2 mmol, according to WO2023133336) were added to a flask, along with 5 mL of DMF as solvent. The flask was cooled to 0°C in an ice bath, and then HATU (570 mg, 1.5 mmol) and DIPEA (193 mg, 1.5 mmol) were added while maintaining this temperature. The mixture was brought to room temperature and reacted. After 10 min, LC-MS showed that the reaction was complete. 40 mL of EA was added to dissolve the product, which was then washed three times with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and passed through a reverse-phase column (methanol/water = 5-95% gradient elution) to obtain the target product ((3S,6S,10aS)-3-((3R,4 or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]azo-6-yl)carbamate tert-butyl ester. After lyophilization, M2-2 (290 mg) was obtained, with a yield of 60%.

LC-MS(m/z):481.1[M+H] ⁺ .

¹ H NMR(400MHz,Chloroform-d)δ7.28-7.44(m,5H),4.94-5.37(m,1H),4.18-4.72(m,4H),3.97-4.17 (m,2H),3.51-3.82(m,2H),3.09-3.36(m,1H),1.89-2.20(m,6H),1.54-1.76(m,5H),1.43(s,9H).

Step 2: Synthesis of (3R,4S or 3S,4R)-1-((3S,6S,10aS)-6-amino-5-oxodecahydropyrrolo[1,2-a]azopyren-3-carbonyl)-4-phenylpyrrolidine-3-carbamate (M2)

((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]azo-6-yl) tert-butyl carbamate (M2-2, 290 mg, 0.6 mmol) was dissolved in 12 mL of DCM, cooled to 0 °C in an ice bath, and then 3 mL of trifluoroacetic acid was added. After restoring to room temperature for 1 h, LC-MS monitoring showed that the reaction was complete. The organic phase was concentrated under reduced pressure and used directly for the next step of amide condensation reaction without purification.

LC-MS(m/z): 381.2[M+H] ⁺ .

Synthesis of intermediate M3: (3S,6S,10aS)-6-amino-3-((R)-2-phenylmorpholine-4-carbonyl)octahydropyrrolo[1,2-a]zosin-5(1H)-one trifluoroacetate

Step 1: Synthesis of tert-butyl ((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]zozin-6-yl)carbamate (M3-2)

Compound M2-1 (326.00 mg, 1.00 mmol) and (R)-2-phenylmorpholine (163.00 mg, 1.00 mmol) were dissolved in N,N-dimethylformamide (5 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (322.00 mg, 2.50 mmol) and N,N,N’,N’-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (570.00 mg, 1.50 mmol) were added sequentially. The mixture was then reacted at 25 °C for 30 minutes. After the reaction was monitored by LC-MS to be complete, water (20 mL) was added, and the mixture was extracted with ethyl acetate (3 times, 20 mL each time). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 40:60 as eluent) to give compound M3-2 (470.00 mg), yield 99.8%.

LC-MS(m/z):472.0[M+H] ⁺ .

Step 2: Synthesis of compound (3S,6S,10aS)-6-amino-3-((R)-2-phenylmorpholine-4-carbonyl)octahydropyrrolo[1,2-a]zosin-5(1H)-one trifluoroacetate (M3)

Compound M3-2 (470.00 mg, 1.00 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (5 mL) was added. The reaction mixture was placed at 25 °C and reacted for 1 hour. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure to give compound M3 (370.00 mg), with a yield of 99.7%.

LC-MS(m/z):372.0[M+H] ⁺ .

Synthesis of intermediate M4: (3S,6S,10aS)-6-amino-3-((S)-2-phenylmorpholine-4-carbonyl)octahydropyrrolo[1,2-a]zosin-5(1H)-one trifluoroacetate (M4)

Step 1: Synthesis of tert-butyl ((3S,6S,10aS)-5-oxo-3-((S)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]zozin-6-yl)carbamate (M4-2)

Compound M2-1 (0.326 g, 1.00 mmol) and (S)-2-phenylmorpholine (0.163 g, 1.00 mmol) were dissolved in N,N-dimethylformamide (5 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (0.322 g, 2.50 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.570 g, 1.50 mmol) were added sequentially. The mixture was reacted at 25 °C for 30 min. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 40:60 as eluent) to give compound M4-2 (0.470 g), with a yield of 99.8%.

LC-MS(m/z):472.0[M+H] ⁺ .

Step 2: Synthesis of compound (3S,6S,10aS)-6-amino-3-((S)-2-phenylmorpholine-4-carbonyl)octahydropyrrolo[1,2-a]zosin-5(1H)-one trifluoroacetate (M4)

Compound M4-2 (0.470 g, 1.00 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (5 mL) was added. The reaction mixture was placed at 25 °C and reacted for 1 hour. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure to give compound M4 (0.370 g), with a yield of 99.7%.

LC-MS(m/z):372.0[M+H] ⁺ .

Example 1: Synthesis of ((((4-(2-((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zosin-6-yl)amino)-2-oxoacetamyl)phenyl)difluoromethyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (Compound 1)

Step 1: Synthesis of compound 2-((4-iodophenyl)amino)-2-oxoacetic acid methyl ester (1-2)

Compound 1-1 (3.00 g, 13.70 mmol) was dissolved in anhydrous dichloromethane (60 mL), and diisopropylethylamine (2.65 g, 20.54 mmol) was added. The reaction solution was cooled to 0 °C, and oxaloyl chloride monomethyl ester (2.00 g, 16.39 mmol) was added dropwise. The reaction was carried out at 25 °C for 1 hour. After the reaction was monitored by LC-MS to be complete, water (50 mL) was added, and the mixture was extracted with dichloromethane (3 times, 50 mL each time). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 50:50 as eluent) to give compound 1-2 (4.02 g), yield 96.2%.

LC-MS(m/z):306.0[M+H] ⁺ .

Step 2: Synthesis of compound 2-((4-(diethoxyphosphoryl)difluoromethyl)phenyl)amino)-2-oxoacetic acid methyl ester (1-3)

Cadmium powder (1.61 g, 14.38 mmol) was added to a 50 mL three-necked flask, followed by 5 mL of anhydrous N,N-dimethylformamide solution containing diethyl bromofluoromethyl phosphate (3.50 g, 13.11 mmol). The mixture was then reacted at 40 °C for 2 hours. In a separate three-necked flask, cuprous chloride (0.97 g, 9.80 mmol) and compound 1-2 (2.00 g, 6.56 mmol) were dissolved in 15 mL of anhydrous N,N-dimethylformamide. The yellow solution from the cadmium powder flask was filtered through a filter and slowly injected into the reaction mixture in the second flask. The reaction mixture was then reacted at 40 °C for 16 hours. Add ethyl acetate (50 mL) and water (50 mL), filter with diatomaceous earth, extract the filtrate with ethyl acetate (3 times, 50 mL each time), wash the combined organic phases with saturated sodium chloride solution (50 mL), dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure, and purify by silica gel column chromatography (petroleum ether:ethyl acetate = 40:60 as eluent) to give compounds 1-3 (1.50 g), yield 62.7%.

LC-MS(m/z):366.0[M+H] ⁺ .

Step 3: Synthesis of compound 2-((4-(diethoxyphosphoryl)difluoromethyl)phenyl)amino)-2-oxoacetic acid (1-4)

Compounds 1-3 (200.00 mg, 0.55 mmol) were dissolved in tetrahydrofuran (3 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of lithium hydroxide (46.00 mg, 1.10 mmol) (3 mL) was added dropwise. The mixture was then reacted at 0 °C for 15 minutes. After the reaction was confirmed to be complete by LC-MS, 1.0 M dilute hydrochloric acid was added to the reaction solution to adjust the pH of the aqueous phase to acidic. The aqueous phase was extracted with ethyl acetate (3 times, 20 mL each time). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compounds 1-4 (189.00 mg), with a yield of 97.9%.

LC-MS(m/z):350.0[MH] ^- .

Step 4: Synthesis of compound diethyl(4-(2-(((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]azoazine-6-yl)amino)-2-oxoacetamyl)phenyl)difluoromethyl)phosphonate diethyl ester (1-5)

Compounds 1-4 (189.00 mg, 0.54 mmol) and M2 (257.00 mg, 0.54 mmol) were dissolved in a mixed solution of tetrahydrofuran (8 mL) and N,N-dimethylformamide (2 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (209.00 mg, 1.62 mmol) and N,N,N’,N’-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (308.00 mg, 0.81 mmol) were added sequentially. The mixture was then reacted at 25 °C for 1 hour. After the reaction was monitored by LC-MS to be complete, water (20 mL) was added, and the mixture was extracted with ethyl acetate (3 times, 20 mL each time). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 96:4 as eluent) to give compounds 1-5 (350.00 mg), yield 90.9%.

LC-MS(m/z):714.0[M+H] ⁺ .

Step 5: Synthesis of compound (4-(2-(((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphonic acid (93)

Compounds 1-5 (220.00 mg, 0.31 mmol) were dissolved in anhydrous dichloromethane (10 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (1.42 g, 9.30 mmol) was added dropwise. The reaction solution was then incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure. A mixture of acetonitrile (8 mL) and water (2 mL) was added, and the reaction solution was incubated at 25 °C for 40 minutes. The solution was then concentrated under reduced pressure and purified by reversed-phase column chromatography (methanol:water = 50:50 as eluent) to give compound 93 (129.00 mg), with a yield of 63.3%.

LC-MS(m/z):658.0[M+H] ⁺ .

Step 6: Synthesis of compound ((((4-(2-((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zosin-6-yl)amino)-2-oxoacetamyl)phenyl)difluoromethyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (1)

Compound 93 (129.00 mg, 0.20 mmol) was dissolved in water (2 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (15.00 mg, 0.38 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (79.00 mg, 0.46 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (152.00 mg, 0.63 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by HPLC to obtain compound 1 (10.40 mg), with a yield of 5.9%.

LC-MS(m/z):886.0[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ11.1-10.9(m,1H),8.81-8.67(m,1H),8.10-7.93(m,2H),7.58-7.27 (m,7H),5.75-5.62(m,4H),4.86-4.68(m,1H),4.59-4.45(m,1H),4.41- 4.18(m,2H),4.15-3.94(m,1H),3.94-3.74(m,2H),3.74-3.60(m,1H),3 .60-3.44(m,1H),2.37-2.17(m,1H),2.10-1.48(m,11H),1.15(s,18H).

Example 2 Synthesis of ((((4-(2-((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zosin-6-yl)amino)-N-methyl-2-oxoacetamyl)phenyl)difluoromethyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (Compound 2)

Step 1: Synthesis of compound 2-((4-(diethoxyphosphoryl)difluoromethyl)phenyl)(methyl)amino)-2-oxoacetic acid methyl ester (2-1)

Sodium hydride (41.00 mg, 1.02 mmol) was dissolved in N,N-dimethylformamide (5 mL). 1-3 (250.00 mg, 0.68 mmol) of N,N-dimethylformamide solution (3 mL) was slowly added dropwise under ice bath conditions. The reaction was carried out at 25 °C for 30 minutes. The reaction solution was cooled to 0 °C, and iodomethane (127.00 mg, 0.88 mmol) was slowly added dropwise. The reaction was carried out at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (3 times, 20 mL each time). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 50:50 as eluent) to give compound 2-1 (200.00 mg), yield 77.6%.

LC-MS(m/z):380.0[M+H] ⁺ .

Step 2: Synthesis of compound 2-((4-(diethoxyphosphoryl)difluoromethyl)phenyl)(methyl)amino)-2-oxoacetic acid (2-2)

Compound 2-1 (200.00 mg, 0.53 mmol) was dissolved in tetrahydrofuran (5 mL), and an aqueous solution of lithium hydroxide monohydrate (45.00 mg, 1.06 mmol) (5 mL) was added. The mixture was reacted at 25 °C for 10 minutes. After the reaction was confirmed to be complete by LC-MS, 1.0 M dilute hydrochloric acid was added to the reaction solution to adjust the pH of the aqueous phase to acidic. The aqueous phase was extracted with ethyl acetate (3 times, 20 mL each time). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 2-2 (130.00 mg), with a yield of 67.2%.

LC-MS(m/z):366.0[M+H] ⁺ .

Step 3: Synthesis of compound diethyl(4-(2-(((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]azoazine-6-yl)amino)-N-methyl-2-oxoacetamido)phenyl)difluoromethylphosphonic acid diethyl ester (2-3)

Compound 2-2 (130.00 mg, 0.36 mmol) and M2 (137.00 mg, 0.36 mmol) were dissolved in N,N-dimethylformamide (10 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (140.00 mg, 1.08 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (205.00 mg, 0.54 mmol) were added sequentially. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (3 times, 20 mL each time). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 90:10 as eluent) to give compound 2-3 (230.00 mg), with a yield of 87.9%.

LC-MS(m/z):728.0[M+H] ⁺ .

Step 4: Synthesis of compound (4-(2-(((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-N-methyl-2-oxoacetamyl)phenyl)difluoromethyl)phosphonic acid (94)

Compound 2-3 (230.00 mg, 0.32 mmol) was dissolved in anhydrous dichloromethane (10 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (1.47 g, 9.6 mmol) was added dropwise. The reaction solution was incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure, and a mixture of acetonitrile (8 mL) and water (2 mL) was added. The reaction solution was incubated at 25 °C for 40 minutes, concentrated under reduced pressure, and purified by reversed-phase column chromatography (methanol:water = 80:20 as eluent) to give compound 94 (60.00 mg), with a yield of 27.9%.

LC-MS(m/z): 672.0[M+H] ⁺ .

Step 5: Synthesis of compound ((((4-(2-((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zosin-6-yl)amino)-N-methyl-2-oxoacetamido)phenyl)difluoromethyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate))(2)

Compound 94 (60.00 mg, 0.09 mmol) was dissolved in water (2 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (6.48 mg, 0.16 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (34.00 mg, 0.20 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (65.34 mg, 0.27 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by thin-layer chromatography to obtain compound 2 (7.80 mg), with a yield of 9.8%.

LC-MS(m/z):900.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ8.85-8.70(m,1H),7.65-7.28(m,9H),5.68(d,J＝22.1Hz,4H),4.64-4 .46(m,2H),4.41-3.93(m,2H),3.89-3.47(m,3H),3.24(d,J＝22.7Hz,4H) ,2.42-2.07(m,2H),1.99(dt,J＝18.8,7.1Hz,2H),1.89-1.56(m,5H),1. 46(d,J＝9.6Hz,2H),1.41-1.24(m,1H),1.16(s,18H),1.08-0.81(m,1H).

Example 3 Synthesis of ((((4-(N-butyl-2-(((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zosin-6-yl)amino)-2-oxoacetamyl)phenyl)difluoromethyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (3)

Step 1: Synthesis of compound 2-(butyl(4-(diethoxyphosphoryl)difluoromethyl)phenyl)amino)-2-oxoacetic acid methyl ester (3-1)

Compounds 1-3 (340.00 mg, 0.93 mmol) were dissolved in N,N-dimethylformamide (10 mL), followed by the sequential addition of potassium carbonate (386.00 mg, 2.80 mmol), tetrabutylammonium iodide (69.00 mg, 0.19 mmol), and n-iodobutane (515.00 mg, 2.80 mmol). The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (3 times, 20 mL each time). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (using dichloromethane:methanol = 98:2 as eluent) to give compound 3-1 (300.00 mg), in a yield of 76.6%.

LC-MS(m/z):422.0[M+H] ⁺ .

Step 2: Synthesis of compound 2-(butyl(4-(ethoxy(hydroxy)phosphoryl)difluoromethyl)phenyl)amino)-2-oxoacetic acid (3-2)

Compound 3-1 (300.00 mg, 0.71 mmol) was dissolved in tetrahydrofuran (5 mL), and an aqueous solution of lithium hydroxide monohydrate (60.00 mg, 1.43 mmol) (5 mL) was added. The mixture was reacted at 25 °C for 10 minutes. After the reaction was confirmed to be complete by LC-MS, 1.0 M dilute hydrochloric acid was added to the reaction solution to adjust the pH of the aqueous phase to acidic. The aqueous phase was extracted with ethyl acetate (3 times, 20 mL each time). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 3-2 (178.00 mg), with a yield of 66.1%.

LC-MS(m/z):380.0[M+H] ⁺ .

Step 3: Synthesis of compound ethylhydro(4-(N-butyl-2-(((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]azoazine-6-yl)amino)-2-oxoacetamyl)phenyl)difluoromethyl)phosphonate ethyl ester (3-3)

Compounds 3-2 (178.00 mg, 0.47 mmol) and M2 (224.00 mg, 0.47 mmol) were dissolved in a mixed solution of tetrahydrofuran (8 mL) and N,N-dimethylformamide (2 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (363.00 mg, 2.81 mmol) and N,N,N’,N’-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (267.00 mg, 0.70 mmol) were added sequentially. The mixture was then reacted at 25 °C for 1 hour. After the reaction was monitored by LC-MS to be complete, water (20 mL) was added, and the mixture was extracted with ethyl acetate (3 times, 20 mL each time). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 90:10 as eluent) to give compound 3-3 (340.00 mg), yield 97.6%.

LC-MS(m/z):742.0[M+H] ⁺ .

Step 4: Synthesis of compound (4-(N-butyl-2-(((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-2-oxoacetamyl)phenyl)difluoromethyl)phosphonic acid (95)

Compound 3-3 (340.00 mg, 0.46 mmol) was dissolved in anhydrous dichloromethane (10 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (2.16 g, 14.1 mmol) was added dropwise. The reaction solution was then incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure. A mixture of acetonitrile (8 mL) and water (2 mL) was added, and the reaction solution was incubated at 25 °C for 40 minutes. The solution was then concentrated under reduced pressure and purified by reversed-phase column chromatography (methanol:water = 80:20 as eluent) to give compound 95 (162.00 mg), with a yield of 49.4%.

LC-MS(m/z):714.0[M+H] ⁺ .

Step 5: Synthesis of compound ((((4-(N-butyl-2-(((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zosin-6-yl)amino)-2-oxoacetamyl)phenyl)difluoromethyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate))(3)

Compound 95 (162.00 mg, 0.23 mmol) was dissolved in water (2 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (17.00 mg, 0.41 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (86.00 mg, 0.51 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (167.00 mg, 0.69 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by HPLC to obtain compound 3 (29.20 mg), with a yield of 13.5%.

LC-MS(m/z): 942.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ8.88-8.68(m,1H),7.62-7.31(m,9H),5.76-5.57(m,4H),4.63-4.41(m,2H),4 .41-4.17(m,1H),4.16-3.92(m,2H),3.91-3.44(m,6H),3.31-3.21(m,1H),2.29- 2.13(m,1H),2.04-1.91(m,1H),1.90-1.69(m,4H),1.68-1.59(m,1H),1.59-1.4 9(m,1H),1.49-1.34(m,4H),1.33-1.22(m,4H),1.16(s,18H),0.88-0.80(m,2H).

Example 4 Synthesis of (((difluoro(4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]zozin-6-yl)amino)acetamido)phenyl)methyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropionate) (compound 4)

Step 1: Synthesis of compound difluoro(4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azoazine-6-yl)amino)acetamyl)phenyl)methyl)phosphonate diethyl ester (4-1)

Compounds 1-4 (200.00 mg, 0.57 mmol) and M3 (257.00 mg, 0.55 mmol) were dissolved in a mixed solution of tetrahydrofuran (8 mL) and N,N-dimethylformamide (2 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (213.00 mg, 1.65 mmol) and N,N,N’,N’-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (314.00 mg, 0.83 mmol) were added sequentially. The mixture was then placed at 25 °C and reacted for 1 hour. After the reaction was monitored by LC-MS to be complete, water (20 mL) was added, and the mixture was extracted with ethyl acetate (3 times, 20 mL each time). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 98:2 as eluent) to give compound 4-1 (350.00 mg), yield 87.2%.

LC-MS(m/z):705.0[M+H] ⁺ .

Step 2: Synthesis of compound (difluoro(4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]zozin-6-yl)amino)acetamido)phenyl)methyl)phosphonic acid (52)

Compound 4-1 (350.00 mg, 0.50 mmol) was dissolved in anhydrous dichloromethane (10 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (2.30 g, 15.03 mmol) was added dropwise. The reaction solution was then incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure, and a mixture of acetonitrile (8 mL) and water (2 mL) was added. The reaction solution was then incubated at 25 °C for 40 minutes, concentrated under reduced pressure, and purified by reversed-phase column chromatography (methanol:water = 80:20 as eluent) to give compound 52 (100.00 mg) as a yellow liquid, with a yield of 30.9%.

LC-MS(m/z): 649.0[M+H] ⁺ .

Step 3: Synthesis of compound (((difluoro(4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]zosin-6-yl)amino)acetamido)phenyl)methyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropionate))(4)

Compound 52 (100 mg, 0.15 mmol) was dissolved in water (2 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (11.00 mg, 0.28 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (58.00 mg, 0.34 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (112.00 mg, 0.46 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by HPLC to obtain compound 4 (14.5 mg), with a yield of 11.0%.

LC-MS(m/z):877.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.99(d,J＝9.2Hz,1H),8.71(dd,J＝34.1,7.0Hz,1H),7.99(t,J＝7.4Hz,2H),7.5 6-7.48(m,2H),7.46-7.27(m,5H),5.75-5.58(m,4H),4.95-4.71(m,2H),4.60-4.25 (m,3H),4.21-3.96(m,2H),3.56(td,J＝11.9,2.7Hz,1H),3.16-2.80(m,1H),2.66-2 .59(m,1H),2.33-2.12(m,1H),2.11-1.97(m,2H),1.94-1.46(m,9H),1.15(s,18H).

Example 7 Synthesis of ((difluoro(4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((S)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]zozin-6-yl)amino)acetamido)phenyl)methyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropionate) (Compound 7)

Step 1: Synthesis of compound difluoro(4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((S)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azoazine-6-yl)amino)acetamyl)phenyl)methyl)phosphonate diethyl ester (7-1)

Compounds 1-4 (0.242 g, 0.689 mmol) and M4 (0.257 g, 0.693 mmol) were dissolved in N,N-dimethylformamide (6 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (0.224 g, 1.73 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.394 g, 1.04 mmol) were added sequentially. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 96:4 as eluent) to give compound 7-1 (0.400 g), with a yield of 82.3%.

LC-MS(m/z):705.0[M+H] ⁺ .

Step 2: Synthesis of compound (difluoro(4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((S)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]zozin-6-yl)amino)acetamido)phenyl)methyl)phosphoric acid (96)

Compound 7-1 (0.400 g, 0.568 mmol) was dissolved in anhydrous dichloromethane (10 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (2.62 g, 17.1 mmol) was added dropwise. The reaction solution was then incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure. A mixture of acetonitrile (8 mL) and water (2 mL) was added, and the reaction solution was incubated at 25 °C for 40 minutes. The solution was then concentrated under reduced pressure and purified by reversed-phase column chromatography (methanol:water = 50:50 as eluent) to give compound 96 (0.300 g), with a yield of 81.1%.

LC-MS(m/z): 649.0[M+H] ⁺ .

Step 3: Synthesis of compound ((difluoro(4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((S)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]zosin-6-yl)amino)acetamido)phenyl)methyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropionate))(7)

Compound 96 (0.200 g, 0.309 mmol) was dissolved in water (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (23.0 mg, 0.575 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (0.116 g, 0.682 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (0.225 g, 0.930 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by thin-layer chromatography to obtain compound 7 (70.0 mg), with a yield of 25.7%.

LC-MS(m/z):877.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d6) δ10.97(d,J＝20.5Hz,1H),8.69(dd,J＝68.7,7.0Hz,1H),7.98(dd,J＝17.3,8.4Hz,2H),7.51(t,J＝9.8Hz,2H),7.44(d,J ＝7.6Hz,1H),7.38(qd,J＝8.5,7.5,2.9Hz,3H),7.32(td,J＝7.0,4.3Hz,1H),5.68(ddt,J＝18.2,11.1,5.4Hz,4H),4.98–4.66(m,2H),4.39(dd, J＝10.5,2.6Hz,1H),4.37–4.22(m,2H),4.16–3.90(m,2H),3.62(dtd,J＝109.6,11.9,2.4Hz,1H),3.16(dd,J＝13.5,10.7Hz,1H),2.88–2.66(m ,1H),2.37–2.14(m,1H),2.13–1.99(m,2H),1.90(qd,J＝12.0,7.3,6.3 Hz,3H),1.85–1.44(m,5H),1.28–1.22(m,1H),1.15(d,J＝4.4Hz,18H).

Example 21 Synthesis of ((4-(2-(((5S,8S,10aR)-3-acetyl-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazooctyl-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropionate) (Compound 21)

Step 1: Synthesis of ((4-(2-(((5S,8S,10aR)-3-acetyl-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazooctyl-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropionate))(21)

Compound 53 (0.150 g, 0.217 mmol) was dissolved in water (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (16.0 mg, 0.400 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (82.0 mg, 0.482 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (0.160 g, 0.661 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by preparative thin-layer chromatography to obtain compound 21 (32.0 mg), with a yield of 16.0%.

LC-MS(m/z): 920.0[M+H] ⁺ .

Example 22 Synthesis of ((difluoro(4-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionylhydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)acetamido)phenyl)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (Compound 22)

Step 1: Synthesis of compound ((difluoro(4-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionylhydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)acetamido)phenyl)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate)(22)

Compound 56 (0.198 g, 0.281 mmol) was dissolved in water (2 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide was added dropwise. After the addition was complete, the pH of the solution was approximately 9-10. Silver nitrate (0.105 g, 0.618 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (0.205 g, 0.847 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by thin-layer chromatography (using ethyl acetate as eluent) to give compound 22 (68.7 mg), with a yield of 26.0%.

LC-MS(m/z): 934.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d6) δ11.17(d,J＝10.9Hz,1H),8.68(dd,J＝39.6,6.0Hz,1H),8.02(t,J＝8.2Hz,2H),7.53–7.45(m,2H),7.43–7 .31(m,5H),5.76–5.60(m,4H),5.05–4.76(m,2H),4.68–4.50(m,1H),4.38(dd,J＝11.2,2.8Hz,1H),4.31–4.13(m,2H),4.03(dt,J ＝18.8,9.5Hz,3H),3.86(d,J＝13.5Hz,1H),3.57(td,J＝11.7,2.8Hz,1H),3.17–3.05(m,2H),2.92–2.73(m,2H),2.67(dd,J＝13.7, 10.9Hz,1H),2.40(ddd,J＝9.8,7.1,3.1Hz,1H),2.11–1.86(m,3H),1.78–1.53(m,2H),1.15(s,18H),1.04(dd,J＝7.3,4.3Hz,3H).

Example 23 Synthesis of ((((4-(2-((5S,8S,10aR)-3-(2,2-difluoroethyl)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (Compound 23)

Step 1: Synthesis of compound methyl(5S,8S,10aR)-5-((tert-butoxycarbonyl)amino)-3-(2,2-difluoroethyl)-6-oxodecahydropyrrolo[1,2-a][1,5]diazo-8-carboxylic acid (23-2)

Compound 22-SM (0.400 g, 1.17 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL), and triethylamine (0.238 g, 2.34 mmol) was added. The reaction solution was cooled to 0 °C, and 2,2-difluoroethyltrifluoromethanesulfonate (0.501 g, 2.34 mmol) was added dropwise. The reaction was carried out at 25 °C for 2 hours. After the reaction was confirmed to be complete by LC-MS, water (50 mL) was added, and the mixture was extracted with EA (10 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 23-2 (0.370 g), with a yield of 78.0%.

LC-MS(m/z):406.0[M+H] ⁺ .

Step 2: Synthesis of compound (5S,8S,10aR)-5-((tert-butoxycarbonyl)amino)-3-(2,2-difluoroethyl)-6-oxodecahydropyrrolo[1,2-a][1,5]diazo-8-carboxylic acid (23-3)

Compound 23-2 (0.370 g, 0.914 mmol) was dissolved in tetrahydrofuran (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of lithium hydroxide (76.0 mg, 1.82 mmol) (4 mL) was added dropwise. The mixture was allowed to react at room temperature for 2 hours. After the reaction was confirmed to be complete by LC-MS, 1.0 M dilute hydrochloric acid was added to the reaction solution to adjust the pH of the aqueous phase to acidic. The aqueous phase was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 23-3 (0.350 g), with a yield of 98.3%.

LC-MS(m/z):392.0[M+H] ⁺ .

Step 3: Synthesis of compound (5S,8S,10aR)-3-(2,2-difluoroethyl)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)tert-butyl carbamate (23-4)

Compound 23-3 (0.350 g, 0.895 mmol) and R-2-phenylmorpholine (0.166 g, 1.02 mmol) were dissolved in N,N-dimethylformamide (5 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (0.287 g, 2.23 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.507 g, 1.36 mmol) were added sequentially. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 96:4 as eluent) to give compound 23-4 (0.400 g), yield 84.0%.

LC-MS(m/z):537.0[M+H] ⁺ .

Step 4: Synthesis of compound (5S,8S,10aR)-5-amino-3-(2,2-difluoroethyl)-8-((R)-2-phenylmorpholine-4-carbonyl)octahydropyrrolo[1,2-a][1,5]diazoazine-6(1H)-one trifluoroacetate (23-5)

Compound 23-4 (0.400 g, 0.743 mmol) was dissolved in anhydrous dichloromethane (7 mL). The reaction solution was cooled to 0 °C, and trifluoroacetic acid (1.28 g, 11.3 mmol) was added. The mixture was then reacted at 25 °C for 1 hour. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure to give compound 23-5 (0.300 g), with a yield of 91.5%.

LC-MS(m/z):437.0[M+H] ⁺ .

Step 5: Synthesis of compound (4-(2-(((5S,8S,10aR)-3-(2,2-difluoroethyl)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphonate diethyl ester (23-6)

Compounds 1-4 (0.241 g, 0.688 mmol) and 23-5 (0.300 g, 0.688 mmol) were dissolved in N,N-dimethylformamide (6 mL). The reaction mixture was cooled to 0 °C, and diisopropylethylamine (0.224 g, 1.73 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.394 g, 1.04 mmol) were added sequentially. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 96:4 as eluent) to give compound 23-6 (0.350 g), with a yield of 66.0%.

LC-MS(m/z):770.0[M+H] ⁺ .

Step 6: Synthesis of compound (4-(2-(((5S,8S,10aR)-3-(2,2-difluoroethyl)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphonic acid (103)

Compound 23-6 (0.350 g, 0.455 mmol) was dissolved in anhydrous dichloromethane (10 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (2.10 g, 13.5 mmol) was added dropwise. The reaction solution was then incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure. A mixture of acetonitrile (8 mL) and water (2 mL) was added, and the reaction solution was incubated at 25 °C for 40 minutes. The solution was then concentrated under reduced pressure and purified by reversed-phase column chromatography (methanol:water = 50:50 as eluent) to give compound 103 (0.220 g), with a yield of 68.5%.

LC-MS(m/z):714.0[M+H] ⁺ .

Step 7: Synthesis of compound ((((4-(2-((5S,8S,10aR)-3-(2,2-difluoroethyl)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (23)

Compound 103 (0.220 g, 0.309 mmol) was dissolved in water (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (22.0 mg, 0.550 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (0.116 g, 0.682 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (0.225 g, 0.930 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by thin-layer chromatography to obtain compound 23 (85.0 mg), with a yield of 29.1%.

LC-MS(m/z): 942.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ11.07(d,J＝10.3Hz,1H),8.52(dd,J＝39.7,6.5Hz,1H),8.00(t,J＝7.9Hz,2H),7.51(dd,J＝8.8,3.8Hz,2H),7.45(d,J＝7.7Hz, 1H),7.42–7.35(m,3H),7.32(t,J＝7.3Hz,1H),6.29–5.95(m,1H),5.75–5.61(m,4H),4.88(dt,J＝115.3,8.6Hz,1H),4.65(dd,J ＝85.1,9.9Hz,2H),4.37(t,J＝9.6Hz,2H),4.22(dd,J＝64.1,13.2Hz,1H),4.03(t,J＝14.5Hz,2H),3.57(q,J＝11.4Hz,1H),3.26– 3.09(m,4H),3.09–3.00(m,1H),2.94–2.59(m,2H),2.43–2.17(m,1H),2.13–1.66(m,4H),1.54(q,J＝14.7Hz,1H),1.15(s,18H).

Example 26 Synthesis of ((difluoro(3-fluoro-4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholin-4-carbonyl)decahydropyrrolo[1,2-a]zosin-6-yl)amino)acetamido)phenyl)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (compound 26)

Step 1: Synthesis of compound 2-((2-fluoro-4-iodophenyl)amino)-2-oxoacetic acid methyl ester (26-2)

Compound 26-1 (3.00 g, 12.7 mmol) was dissolved in anhydrous dichloromethane (60 mL), and diisopropylethylamine (2.45 g, 19.0 mmol) was added. The reaction solution was cooled to 0 °C, and oxaloyl chloride monomethyl ester (1.86 g, 15.2 mmol) was added dropwise. The reaction was carried out at 25 °C for 1 hour. After the reaction was monitored by LC-MS to be complete, water (50 mL) was added, and the mixture was extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 50:50 as eluent) to give compound 26-2 (3.90 g), yield 95.5%.

LCMS (ESI): m/z=324.0[M+H] ⁺ .

Step 2: Synthesis of compound 2-((4-(diethoxyphosphoryl)difluoromethyl)-2-fluorophenyl)amino)-2-oxoacetic acid methyl ester (26-3)

Cadmium powder (1.53 g, 13.6 mmol) was added to a 50 mL three-necked flask (first reaction flask), and an anhydrous N,N-dimethylformamide solution (5 mL) of diethyl bromofluoromethyl phosphate (3.31 g, 12.4 mmol) was added. The mixture was placed at 40 °C for 2 hours. In a separate three-necked flask (second reaction flask), cuprous chloride (0.920 g, 9.28 mmol) and compound 26-2 (2.00 g, 6.19 mmol) were dissolved in anhydrous N,N-dimethylformamide (15 mL). The reaction solution from the first reaction flask was filtered through a filter and slowly injected into the reaction system in the second reaction flask. The reaction solution was placed at 40 °C for 16 hours. Add ethyl acetate (50 mL) and water (50 mL), filter with diatomaceous earth, extract the filtrate with ethyl acetate (50 mL × 3), wash the combined organic phases with saturated sodium chloride solution (50 mL), dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure, and purify by silica gel column chromatography (petroleum ether:ethyl acetate = 40:60 as eluent) to give compound 26-3 (1.80 g), yield 75.9%.

LC-MS(m/z):384.0[M+H] ⁺ .

Step 3: Synthesis of compound 2-((4-((diethoxyphosphoryl)difluoromethyl)-2-fluorophenyl)amino)-2-oxoacetic acid (26-4)

Compound 26-3 (1.80 g, 4.69 mmol) was dissolved in tetrahydrofuran (8 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of lithium hydroxide (0.394 g, 9.38 mmol) (8 mL) was added dropwise. The mixture was then reacted at 0 °C for 15 minutes. After the reaction was confirmed to be complete by LC-MS, 1.0 M dilute hydrochloric acid was added to the reaction solution to adjust the pH of the aqueous phase to acidic. The aqueous phase was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 26-4 (1.23 g), with a yield of 71.0%.

LC-MS(m/z): 370.0[MH] ⁺ .

Step 4: Synthesis of compound difluoro(3-fluoro-4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azoazine-6-yl)amino)acetamyl)phenyl)methyl)phosphonate diethyl ester (26-5)

Compound 26-4 (0.200 g, 0.542 mmol) and M3 (0.201 g, 0.542 mmol) were dissolved in N,N-dimethylformamide (5 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (0.174 g, 1.35 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.308 g, 0.811 mmol) were added sequentially. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 96:4 as eluent) to give compound 26-5 (0.280 g), with a yield of 71.7%.

LC-MS(m/z):723.0[M+H] ⁺ .

Step 5: Synthesis of compound (difluoro(3-fluoro-4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]zozin-6-yl)amino)acetamido)phenyl)methyl)phosphoric acid (102)

Compound 26-5 (0.280 g, 0.388 mmol) was dissolved in anhydrous dichloromethane (10 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (1.74 g, 11.4 mmol) was added dropwise. The reaction solution was then incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure. A mixture of acetonitrile (8 mL) and water (2 mL) was added, and the reaction solution was incubated at 25 °C for 40 minutes. The solution was then concentrated under reduced pressure and purified by reversed-phase column chromatography (methanol:water = 50:50 as eluent) to give compound 102 (0.120 g), with a yield of 47.4%.

LC-MS(m/z): 667.0[M+H] ⁺ .

Step 6: Synthesis of compound ((difluoro(3-fluoro-4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]zosin-6-yl)amino)acetamido)phenyl)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (26)

Compound 102 (0.120 g, 0.180 mmol) was dissolved in water (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (13.0 mg, 0.325 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (67.0 mg, 0.394 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (0.131 g, 0.541 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by HPLC to obtain compound 26 (31.8 mg), with a yield of 19.7%.

LC-MS(m/z):895.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.47(d,J＝8.3Hz,1H),8.72(dd,J＝35.4,7.1Hz,1H),7.91(q,J＝8.0Hz,1H),7. 56–7.24(m,7H),5.72(ddd,J＝18.2,12.6,5.5Hz,4H),5.05–4.67(m,2H),4.46–4. 24(m,3H),4.21–3.95(m,2H),3.56(t,J＝11.7Hz,1H),3.17–2.78(m,1H),2.71–2. 55(m,1H),2.35–2.11(m,1H),2.10–1.97(m,2H),1.96–1.41(m,8H),1.16(s,18H).

Example 29 Synthesis of ((difluoro(3-methoxy-4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azoazine-6-yl)amino)acetamido)phenyl)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (29)

Step 1: Synthesis of compound 2-((4-iodo-2-methoxyphenyl)amino)-2-oxoacetic acid methyl ester (29-2)

Compound 29-1 (3.00 g, 12.0 mmol) was dissolved in anhydrous dichloromethane (60 mL), and diisopropylethylamine (2.33 g, 18.1 mmol) was added. The reaction solution was cooled to 0 °C, and oxaloyl chloride monomethyl ester (1.77 g, 14.4 mmol) was added dropwise. The reaction was carried out at 25 °C for 1 hour. After the reaction was monitored by LC-MS to be complete, water (50 mL) was added, and the mixture was extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 50:50 as eluent) to give compound 29-2 (3.90 g), yield 96.7%.

LC-MS(m/z):336.0[M+H] ⁺ .

Step 2: Synthesis of compound 2-((4-(diethoxyphosphoryl)difluoromethyl)-2-methoxyphenyl)amino)-2-oxoacetic acid methyl ester (29-3)

Cadmium powder (1.47 g, 13.1 mmol) was added to a 50 mL three-necked flask (first reaction flask), and an anhydrous N,N-dimethylformamide solution (5 mL) of diethyl bromofluoromethyl phosphate (3.18 g, 11.9 mmol) was added. The mixture was placed at 40 °C for 2 hours. In a separate three-necked flask (second reaction flask), cuprous chloride (0.890 g, 8.95 mmol) and compound 29-2 (2.00 g, 5.97 mmol) were dissolved in anhydrous N,N-dimethylformamide (15 mL). The reaction solution from the first reaction flask was filtered through a filter and slowly injected into the reaction system in the second reaction flask. The reaction solution was placed at 40 °C for 16 hours. Add ethyl acetate (50 mL) and water (50 mL), filter with diatomaceous earth, extract the filtrate with ethyl acetate (50 mL × 3), wash the combined organic phases with saturated sodium chloride solution (50 mL), dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure, and purify by silica gel column chromatography (petroleum ether:ethyl acetate = 40:60 as eluent) to give compound 29-3 (1.80 g), yield 76.2%.

LC-MS(m/z):396.0[M+H] ⁺ .

Step 3: Synthesis of compound 2-((4-((diethoxyphosphoryl)difluoromethyl)-2-methoxyphenyl)amino)-2-oxoacetic acid (29-4)

Compound 29-3 (1.80 g, 4.55 mmol) was dissolved in tetrahydrofuran (8 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of lithium hydroxide (0.382 g, 9.10 mmol) (8 mL) was added dropwise. The mixture was then reacted at 0 °C for 15 minutes. After the reaction was confirmed to be complete by LC-MS, 1.0 M dilute hydrochloric acid was added to the reaction solution to adjust the pH of the aqueous phase to acidic. The aqueous phase was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 29-4 (1.40 g), with a yield of 81.0%.

LC-MS(m/z):382.0[M+H] ⁺ .

Step 4: Synthesis of compound difluoro(3-methoxy-4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]zosin-6-yl)amino)acetamyl)phenyl)methyl)phosphonate diethyl ester (29-5)

Compound 29-4 (0.200 g, 0.522 mmol) and M3 (0.193 g, 0.522 mmol) were dissolved in N,N-dimethylformamide (5 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (0.168 g, 1.30 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.297 g, 0.782 mmol) were added sequentially. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 96:4 as eluent) to give compound 29-5 (0.300 g), with a yield of 78.5%.

LC-MS(m/z):735.0[M+H] ⁺ .

Step 5: Synthesis of compound (difluoro(3-methoxy-4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]zozin-6-yl)amino)acetamido)phenyl)methyl)phosphoric acid (29-6)

Compound 29-5 (0.300 g, 0.409 mmol) was dissolved in anhydrous dichloromethane (10 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (1.84 g, 12.0 mmol) was added dropwise. The reaction solution was incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure, and a mixture of acetonitrile (8 mL) and water (2 mL) was added. The reaction solution was incubated at 25 °C for 40 minutes, concentrated under reduced pressure, and purified by reversed-phase column chromatography (methanol:water = 50:50 as eluent) to give compound 29-6 (0.170 g), with a yield of 62.7%.

LC-MS(m/z): 679.0[M+H] ⁺ .

Step 6: Synthesis of compound ((difluoro(3-methoxy-4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azoazine-6-yl)amino)acetamido)phenyl)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (29)

Compound 29-6 (0.170 g, 0.251 mmol) was dissolved in water (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (18.0 mg, 0.450 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (94.0 mg, 0.553 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (0.182 g, 0.744 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by HPLC to obtain compound 29 (43.3 mg), with a yield of 19.1%.

LC-MS(m/z): 907.0[M+H] ⁺ .

^1H NMR (600MHz, DMSO-d6 ₎ )δ9.85(d,J＝9.2Hz,1H),8.81(d,J＝34.2Hz,1H),8.29(d,J＝9.5Hz,1H),7.38( td,J＝30.6,27.7,14.3Hz,5H),7.18(d,J＝33.3Hz,2H),5.70(t,J＝13.6Hz,4H), 5.04–4.48(m,2H),4.43–4.13(m,3H),4.03(s,2H),3.96(s,3H),3.55(d,J=12. 1Hz,1H),3.12(s,1H),1.91(ddd,J＝201.3,131.2,83.4Hz,13H),1.14(s,18H).

Example 52 Synthesis of difluoro(3-methoxy-4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]zozin-6-yl)amino)acetamido)phenyl)methyl)phosphoric acid (52)

The synthesis method is described in Example 4.

LC-MS(m/z): 649.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.91–10.80(m,1H),8.75–8.60(m,1H),7.95–7.81(m,2H),7.52–7.27(m,7H),4.98–4.71(m,2H),4.61–4.13(m,4H),4.07–3 .91(m,1H),3.62–3.52(m,1H),3.39–3.05(m,1H),2.89–2.58(m,1H),2.33–2.11(m,1H),2.10–1.96(m,2H),1.94–1.48(m,9H).

Example 53 Synthesis of (4-(2-(((5S,8S,10aR)-3-acetyl-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphonic acid (compound 53)

Step 1: Synthesis of compound 3-benzyl-8-methyl(5S,8S,10aR)-5-((tert-butoxycarbonyl)amino)-6-oxooctahydropyrrolo[1,2-a][1,5]diazo-3,8(4H)-dicarboxylate (53-1)

Compound 22-SM (1.40 g, 4.11 mmol) was dissolved in dioxane (10 mL), and sodium bicarbonate (1.03 g, 12.3 mmol) was added. The reaction system was placed at 10 °C, and benzyl chloroformate (0.907 g, 5.33 mmol) was added, resulting in the precipitation of a white solid. The reaction system was then placed at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was concentrated under reduced pressure and purified by silica gel column chromatography (ethyl acetate: petroleum ether = 2:1 as eluent) to give compound 53-1 (1.65 g), with a yield of 84.6%.

LC-MS(m/z):476.0[M+H] ⁺ .

Step 2: Synthesis of compound (5S,8S,10aR)-3-((benzyloxy)carbonyl)-5-(tert-butoxycarbonyl)amino)-6-oxodecahydropyrrolo[1,2-a][1,5]diazo-8-carboxylic acid (53-2)

Compound 53-1 (1.65 g, 3.47 mmol) was dissolved in dioxane (10 mL), and an aqueous solution of lithium hydroxide (0.167 g, 6.94 mmol) (5 mL) was added. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, the solution was concentrated under reduced pressure to remove most of the dioxane. 1.0 M dilute hydrochloric acid was added to the reaction solution to adjust the pH of the aqueous phase to 5. The aqueous phase was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 53-2 (1.60 g), with a yield of 99.8%.

LC-MS(m/z):462.0[M+H] ⁺ .

Step 3: Synthesis of the compound benzyl(5S,8S,10aR)-5-((tert-butoxycarbonyl)amino)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)octahydropyrrolo[1,2-a][1,5]diazoazine-3(4H)-carboxylic acid ester (53-3)

Compound 53-2 (1.60 g, 3.47 mmol) and R-2-phenylmorpholine (0.566 g, 3.47 mmol) were dissolved in DMF (8 mL). Diisopropylethylamine (1.34 g, 10.4 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (1.58 g, 4.16 mmol) were added sequentially, and the mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate as eluent) to give compound 53-3 (1.90 g) in 90.4% yield.

LC-MS(m/z): 607.0[M+H] ⁺ .

Step 4: Synthesis of the compound benzyl(5S,8S,10aR)-5-amino-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)octahydropyrrolo[1,2-a][1,5]diazo-3(4H)-carboxylic acid ester (53-4).

Compound 53-3 (1.70 g, 2.80 mmol) was dissolved in anhydrous dichloromethane (4 mL). The reaction solution was cooled to 0 °C, and trifluoroacetic acid (4 mL) was added dropwise. The reaction solution was then placed at 25 °C for 1 hour. After the reaction was monitored by TLC (alkalinization) to confirm its completion, the solution was concentrated under reduced pressure to obtain crude compound 53-4 (1.70 g).

LC-MS(m/z):507.0[M+H] ⁺ .

Step 5: Synthesis of the compound benzyl(5S,8S,10aR)-5-(2-((4-(diethoxyphosphoryl)difluoromethyl)phenyl)amino)-2-oxoacetamido)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)octahydropyrrolo[1,2-a][1,5]diazoazine-3(4H)-carboxylic acid ester (53-5)

Compounds 53-4 (1.70 g, 2.80 mmol) and 1-4 (0.983 g, 2.80 mmol) were dissolved in N,N-dimethylformamide (10 mL), followed by the sequential addition of diisopropylethylamine (1.30 g, 10.8 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (1.27 g, 3.36 mmol). The mixture was reacted at 25 °C for 1 hour. The reaction was monitored by LC-MS and TLC until complete. Water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate as eluent) to give compound 53-5 (1.40 g), yield 59.5%.

LC-MS(m/z):840.0[M+H] ⁺ .

Step 6: Synthesis of compound difluoro(4-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)acetamyl)phenyl)methyl)phosphonate diethyl ester (53-6)

Compound 53-5 (0.400 g, 0.477 mmol) was dissolved in THF (10 mL), and palladium on carbon (10%, 0.200 g, 0.189 mmol) was added. The mixture was purged three times with hydrogen balloons, and the reaction solution was placed under a hydrogen atmosphere at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, and the filtrate was concentrated under reduced pressure to give compound 53-6 (0.290 g), with a yield of 86.3%.

Step 7: Synthesis of compound (4-(2-(((5S,8S,10aR)-3-acetyl-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphonate diethyl ester (53-7)

Compound 53-6 (0.150 g, 0.213 mmol) was dissolved in anhydrous dichloromethane (5 mL). The reaction solution was cooled to 0 °C, and triethylamine (43.0 mg, 0.426 mmol) and acetyl chloride (25.0 mg, 0.318 mmol) were added sequentially. The mixture was reacted at 25 °C for 0.5 h. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol = 96:4 as eluent) to give compound 53-7 (0.120 g), with a yield of 76.4%.

LC-MS(m/z):748.0[M+H] ⁺ .

Step 8: Synthesis of compound (4-(2-(((5S,8S,10aR)-3-acetyl-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphonic acid (53)

Compound 53-7 (0.120 g, 0.161 mmol) was dissolved in anhydrous dichloromethane (5 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (0.735 g, 4.80 mmol) was added dropwise. The reaction solution was incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure, and a mixture of acetonitrile (8 mL) and water (2 mL) was added. The reaction solution was incubated at 25 °C for 40 minutes, concentrated under reduced pressure, and purified by reversed-phase column chromatography (methanol:water = 50:50 as eluent) to give compound 53 (80.0 mg), with a yield of 72.1%.

LC-MS(m/z): 692.0[M+H] ⁺ .

Example 54 Synthesis of ((difluoro(4-(2-(((3S,6S,10aS)-3-(indole-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]azoazine-6-yl)amino)-2-oxoacetamido)phenyl)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate (54))

Step 1: Synthesis of tert-butyl ((3S,6S,10aS)-3-(indole-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)carbamate (54-1)

Compound M2-1 (0.300 g, 0.920 mmol) and dihydroindole (0.163 g, 1.01 mmol) were dissolved in N,N-dimethylformamide (5 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (0.297 g, 2.30 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.525 g, 1.38 mmol) were added sequentially. The mixture was reacted at 25 °C for 30 min. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 40:60 as eluent) to give compound 54-1 (0.300 g), with a yield of 76.3%.

LC-MS(m/z):428.0[M+H] ⁺ .

Step 2: Synthesis of compound (3S,6S,10aS)-6-amino-3-(indole-1-carbonyl)octahydropyrrolo[1,2-a]zosin-5(1H)-one trifluoroacetate (54-2)

Compound 54-1 (0.300 g, 0.704 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (1.20 g, 10.5 mmol) was added. The reaction mixture was placed at 25 °C for 1 hour. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure to give compound 54-2 (0.200 g), with a yield of 87.3%.

LC-MS(m/z):328.0[M+H] ⁺ .

Step 3: Synthesis of diethyl difluoro(4-(2-(((3S,6S,10aS)-3-(indole-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-2-oxoacetamido)phenyl)methyl)phosphonate (54-3)

Compounds 1-4 (0.215 g, 0.613 mmol) and 54-2 (0.200 g, 0.613 mmol) were dissolved in N,N-dimethylformamide (6 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (0.197 g, 1.53 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.348 g, 0.916 mmol) were added sequentially. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 96:4 as eluent) to give compound 54-3 (0.300 g), with a yield of 74.4%.

LC-MS(m/z): 661.0[M+H] ⁺ .

Step 4: Synthesis of compound ((difluoro(4-(2-(((3S,6S,10aS)-3-(indole-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-2-oxoacetamyl)phenyl)methyl)phosphonic acid (97)

Compound 54-3 (0.300 g, 0.455 mmol) was dissolved in anhydrous dichloromethane (10 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (2.06 g, 13.5 mmol) was added dropwise. The reaction solution was then incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure. A mixture of acetonitrile (8 mL) and water (2 mL) was added, and the reaction solution was incubated at 25 °C for 40 minutes. The solution was then concentrated under reduced pressure and purified by reversed-phase column chromatography (methanol:water = 50:50 as eluent) to give compound 97 (0.171 g), with a yield of 62.9%.

LC-MS(m/z): 605.0[M+H] ⁺ .

Step 5: Synthesis of compound ((difluoro(4-(2-(((3S,6S,10aS)-3-(indole-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]azoazine-6-yl)amino)-2-oxoacetamido)phenyl)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (54)

Compound 97 (0.171 g, 0.283 mmol) was dissolved in water (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (20.0 mg, 0.500 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (0.105 g, 0.618 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (0.204 g, 0.843 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by thin-layer chromatography to obtain compound 54 (55.0 mg), with a yield of 23.6%.

LC-MS(m/z):833.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.98(s,1H),8.71(d,J＝7.0Hz,1H),8.08(d,J＝8.1Hz,1H),7.98(d,J＝8.5Hz,2H),7.51(d,J＝8.5Hz,2H),7.25(d ,J＝7.4Hz,1H),7.14(t,J＝7.7Hz,1H),7.01(t,J＝7.4Hz,1H),5.68(ddd,J＝18.2,12.6,5.5Hz,4H),4.82(dt,J＝12.2, 6.1Hz,1H),4.64(t,J＝8.7Hz,1H),4.35(p,J＝9.2Hz,2H),4.18(td,J＝10.1,6.5Hz,1H),3.19(q,J＝8.9Hz,2H),2.37 (dt,J＝14.2,7.4Hz,1H),2.14–2.04(m,1H),2.03–1.85(m,5H),1.83–1.47(m,3H),1.35–1.18(m,2H),1.15(s,18H).

Example 55 Synthesis of (((difluoro(4-(2-((3S,6S,10aS)-3-(methyl(phenyl)carbamoyl)-5-oxodecahydropyrrolo[1,2-a]azoazine-6-yl)amino)-2-oxoacetamido)phenyl)methyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropionate) (compound 55)

Step 1: Synthesis of tert-butyl ((3S,6S,10aS)-3-(methyl(phenyl)carbamoyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)carbamate (55-1)

Compound M2-1 (0.200 g, 0.613 mmol) and N-methylaniline (65.0 mg, 0.607 mmol) were dissolved in N,N-dimethylformamide (5 mL). The reaction solution was cooled to 10 °C, and diisopropylethylamine (0.237 g, 1.84 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.279 g, 0.734 mmol) were added sequentially. The mixture was reacted at 25 °C for 30 min. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 40:60 as eluent) to give compound 55-1 (0.130 g), yield 51.3%.

LC-MS(m/z):416.0[M+H] ⁺ .

Step 2: Synthesis of compound (3S,6S,10aS)-6-amino-N-methyl-5-oxo-N-phenyldecahydropyrrolo[1,2-a]azo-3-carboxamide (55-2)

Compound 55-1 (0.130 g, 0.313 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (1.50 g, 13.2 mmol) was added. The reaction mixture was placed at 25 °C for 1 hour. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure to give compound 55-2 (0.125 g), with a yield of 97.6%.

LC-MS(m/z):316.0[M+H] ⁺ .

Step 3: Synthesis of diethyl difluoro(4-(2-(((3S,6S,10aS)-3-(methyl(phenyl)carbamoyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-2-oxoacetamido)methyl)phosphonate (55-3)

Compounds 55-2 (0.125 g, 0.303 mmol) and 1-4 (0.109 g, 0.311 mmol) were dissolved in N,N-dimethylformamide (6 mL). The reaction mixture was cooled to 10 °C, and diisopropylethylamine (0.120 g, 0.930 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.140 g, 0.368 mmol) were added sequentially. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (EA as eluent) to give compound 55-3 (0.150 g), with a yield of 77.3%.

LC-MS(m/z): 649.0[M+H] ⁺ .

Step 4: Synthesis of compound ((difluoro(4-(2-(((3S,6S,10aS)-3-(methyl(phenyl)carbamoyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-2-oxoacetamido)phenyl)methyl)phosphoric acid (98)

Compound 55-3 (0.150 g, 0.231 mmol) was dissolved in anhydrous dichloromethane (5 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (1.05 g, 6.86 mmol) was added dropwise. The reaction solution was incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure, and a mixture of acetonitrile (8 mL) and water (2 mL) was added. The reaction solution was incubated at 25 °C for 40 minutes, concentrated under reduced pressure, and purified by reversed-phase column chromatography (methanol:water = 50:50 as eluent) to give compound 98 (55.0 mg), with a yield of 40.1%.

LC-MS(m/z):593.0[M+H] ⁺ .

Step 5: Synthesis of compound (((difluoro(4-(2-((3S,6S,10aS)-3-(methyl(phenyl)carbamoyl)-5-oxodecahydropyrrolo[1,2-a]azoazine-6-yl)amino)-2-oxoacetamido)phenyl)methyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropionate) (55)

Compound 98 (55.0 mg, 0.0929 mmol) was dissolved in water (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide was added dropwise. After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (34.6 mg, 0.204 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervaline (67.0 mg, 0.277 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by thin-layer chromatography to obtain compound 55 (17.9 mg), with a yield of 23.7%.

LC-MS(m/z):821.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ ¹ H NMR(600MHz,DMSO-d6)δ10.96(s,1H),8.67(d,J＝6.9Hz,1H),7.98(d,J＝8.5Hz,2H), 7.45(ddd,J＝55.0,20.9,8.1Hz,7H),5.68(ddd,J＝18.3,12.6,5.5Hz,4H),4.81–4.67 (m,1H),4.16(d,J＝6.2Hz,2H),3.16(s,3H),2.03(s,1H),1.96–1.82(m,5H),1.70(t, J＝4.2Hz,1H),1.62(s,2H),1.52(t,J＝12.2Hz,2H),1.27–1.20(m,1H),1.15(s,18H).

Example 56 Synthesis of difluoro(4-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionylpyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)acetamido)phenyl)methyl)phosphoric acid (compound 56)

Step 1: Synthesis of compound methyl(5S,8S,10aR)-5-((tert-butoxycarbonyl)amino)-6-oxodecahydropyrrolo[1,2-a][1,5]diazo-8-carboxylic acid (56-1)

Compound 22-SM (0.200 g, 0.580 mmol) was dissolved in THF (5 mL), followed by the sequential addition of triethylamine (0.118 g, 1.17 mmol) and propionyl chloride (65.0 mg, 0.700 mmol) at 0 °C. A white solid precipitated, and the mixture was reacted at 0 °C for 1 hour. After the reaction was confirmed to be complete by LC-MS, water (20 mL) was added, and the aqueous phase was extracted with dichloromethane (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 90:10 as eluent) to give compound 56-1 (0.227 g), with a yield of 98.7%.

LC-MS(m/z):398.0[M+H] ⁺ .

Step 2: Synthesis of compound (5S,8S,10aR)-5-((tert-butoxycarbonyl)amino)-6-oxo-3-propionyldecahydropyrrolo[1,2-a][1,5]diazo-8-carboxylic acid (56-2)

Compound 56-1 (0.227 g, 0.572 mmol) was dissolved in dioxane (4 mL), and an aqueous solution of lithium hydroxide (48.0 mg, 1.15 mmol) (2 mL) was added. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, most of the dioxane was removed by concentration under reduced pressure. 1.0 M dilute hydrochloric acid was added to the reaction solution to adjust the pH of the aqueous phase to 5. The aqueous phase was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 56-2 (0.222 g), with a yield of 99.0%.

LC-MS(m/z):384.0[M+H] ⁺ .

Step 3: Synthesis of compound (5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionylhydropyrrolo[1,2-a][1,5]diazoazine-5-yl)carbamate tert-butyl ester (56-3)

Compound 56-2 (0.222 g, 0.579 mmol) and (R)-2-phenylmorpholine (0.112 g, 0.687 mmol) were dissolved in N,N-dimethylformamide (3 mL). Diisopropylethylamine (0.221 g, 1.71 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.260 g, 0.684 mmol) were added sequentially, and the mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate as eluent) to give compound 56-3 (0.293 g), with a yield of 97.6%.

LC-MS(m/z):529.0[M+H] ⁺ .

Step 4: Synthesis of compound (5S,8S,10aR)-5-amino-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionylheptahydropyrrolo[1,2-a][1,5]diazoazine-6(1H)-one (56-4)

Compound 56-3 (0.293 g, 0.555 mmol) was dissolved in anhydrous dichloromethane (10 mL). The reaction solution was cooled to 0 °C, and trifluoroacetic acid (2 mL) was added dropwise. The reaction solution was then placed at 25 °C for 1 hour. After the reaction was monitored by TLC (alkalinization) to confirm its completeness, the solution was concentrated under reduced pressure to obtain compound 56-4 (0.310 g, crude product).

LC-MS(m/z):429.0[M+H] ⁺ .

Step 5: Synthesis of compound difluoro(4-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionylhydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)acetamyl)phenyl)methyl)phosphonate diethyl ester (56-5)

The crude compounds 56-4 (0.310 g) and 1-4 (0.203 g, 0.578 mmol) were dissolved in N,N-dimethylformamide (3 mL) solution. Diisopropylethylamine (0.250 g, 1.93 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.244 g, 0.642 mmol) were added sequentially, and the mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate as eluent) to give compound 56-5 (0.200 g), with a yield of 44.7%.

LC-MS(m/z):762.0[M+H] ⁺ .

Step 6: Synthesis of compound (difluoro(4-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionylpyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)acetamido)phenyl)methyl)phosphoric acid (56)

Compound 56-5 (0.200 g, 0.263 mmol) was dissolved in anhydrous dichloromethane (10 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (1.3 mL) was added dropwise. The reaction solution was then incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure. A mixture of acetonitrile (8 mL) and water (2 mL) was added, and the reaction solution was incubated at 25 °C for 40 minutes. The solution was then concentrated under reduced pressure and purified by reversed-phase column chromatography (methanol:water = 80:20 as eluent) to obtain compound 56 (0.198 g), crude product.

LC-MS(m/z):704.0[MH] ^– .

¹ H NMR (600MHz, DMSO-d ₆ )δ11.05(d,J＝11.8Hz,1H),8.68(dd,J＝39.2,6.2Hz,1H),8.00–7.74(m,2H),7.60 –7.27(m,7H),5.09–4.71(m,2H),4.63–4.33(m,2H),4.32–3.97(m,5H),3.93–3.7 6(m,1H),3.66–3.54(m,2H),3.17–3.03(m,2H),2.91–2.73(m,2H),2.71–2.61(m, 1H),2.43–2.21(m,1H),2.11–1.85(m,2H),1.77–1.51(m,2H),1.08–1.01(m,3H).

Example 57 Synthesis of ((difluoro(4-(2-(((3S,6S,10aS)-3-(morpholin-4-carbonyl)-5-oxodecahydropyrrolo[1,2-a]azoazine-6-yl)amino)-2-oxoacetamido)phenyl)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate (compound 57))

Step 1: Synthesis of tert-butyl ((3S,6S,10aS)-3-(morpholino-4-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)carbamate (57-1)

Compound M2-1 (0.300 g, 0.920 mmol) and morpholine (88.0 mg, 1.01 mmol) were dissolved in N,N-dimethylformamide (5 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (0.297 g, 2.30 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.525 g, 1.38 mmol) were added sequentially. The mixture was reacted at 25 °C for 30 min. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 40:60 as eluent) to give compound 57-1 (0.350 g), with a yield of 96.2%.

LC-MS(m/z):396.0[M+H] ⁺ .

Step 2: Synthesis of compound (3S,6S,10aS)-6-amino-3-(morpholin-4-carbonyl)octahydropyrrolo[1,2-a]zosin-5(1H)-one trifluoroacetate (57-2)

Compound 57-1 (0.350 g, 0.886 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (1.50 g, 13.2 mmol) was added. The reaction mixture was placed at 25 °C for 1 hour. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure to give compound 57-2 (0.205 g), with a yield of 78.8%.

LC-MS(m/z):296.0[M+H] ⁺ .

Step 3: Synthesis of diethyl difluoro(4-(2-(((3S,6S,10aS)-3-(morpholin-4-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-2-oxoacetamido)phenyl)methyl)phosphonate (57-3)

Compounds 1-4 (0.242 g, 0.689 mmol) and 57-2 (205.00 mg, 0.695 mmol) were dissolved in N,N-dimethylformamide (6 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (0.224 g, 1.73 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.394 g, 1.04 mmol) were added sequentially. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 96:4 as eluent) to give compound 57-3 (0.400 g), with a yield of 92.2%.

LC-MS(m/z): 629.0[M+H] ⁺ .

Step 4: Synthesis of compound (difluoro(4-(2-(((3S,6S,10aS)-3-(morpholin-4-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-2-oxoacetamyl)phenyl)methyl)phosphonic acid (99)

Compound 57-3 (0.400 g, 0.637 mmol) was dissolved in anhydrous dichloromethane (10 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (2.10 g, 19.2 mmol) was added dropwise. The reaction solution was then incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure. A mixture of acetonitrile (8 mL) and water (2 mL) was added, and the reaction solution was incubated at 25 °C for 40 minutes. The solution was then concentrated under reduced pressure and purified by reversed-phase column chromatography (methanol:water = 50:50 as eluent) to give compound 99 (0.130 g), with a yield of 35.5%.

LC-MS(m/z):573.0[M+H] ⁺ .

Step 5: Synthesis of compound ((difluoro(4-(2-(((3S,6S,10aS)-3-(morpholin-4-carbonyl)-5-oxodecahydropyrrolo[1,2-a]azoazine-6-yl)amino)-2-oxoacetamido)phenyl)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (57)

Compound 99 (0.130 g, 0.227 mmol) was dissolved in water (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (16.6 mg, 0.415 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (86.0 mg, 0.506 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (0.167 g, 0.690 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by thin-layer chromatography to obtain compound 57 (16.4 mg), with a yield of 9.0%.

LC-MS(m/z):801.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.98(s,1H),8.69(d,J＝7.0Hz,1H),7.99(d,J＝8.5Hz,2H),7.50(t,J＝7.2Hz,2H),5.68(ddd ,J＝18.4,12.6,5.5Hz,4H),5.41(d,J＝10.4Hz,1H),4.86–4.67(m,2H),4.29(dd,J＝11.8,8.1Hz, 1H),3.67(d,J＝11.4Hz,1H),3.62–3.56(m,3H),3.55–3.47(m,3H),2.22(dt,J＝12.0,7.6Hz,1H ), 2.02(tt,J＝11.6,8.0Hz,2H),1.89(dt,J＝12.3,7.4Hz,2H),1.84–1.47(m,7H),1.15(s,18H).

Example 58 Synthesis of ((((4-(2-((3S,6S,10aS)-3-((2S,6R)-2,6-dimethylmorpholino-4-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zosin-6-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (Compound 58)

Step 1: Synthesis of tert-butyl ((3S,6S,10aS)-3-((2S,6R)-2,6-dimethylmorpholino-4-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)carbamate (58-1)

Compound M2-1 (0.300 g, 0.920 mmol) and cis-2,6-dimethylmorpholine (0.116 g, 1.01 mmol) were dissolved in N,N-dimethylformamide (5 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (0.297 g, 2.30 mmol) and N,N,N’,N’-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.525 g, 1.38 mmol) were added sequentially. The mixture was then reacted at 25 °C for 30 minutes. After the reaction was monitored by LC-MS to be complete, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 40:60 as eluent) to give compound 58-1 (0.330 g), yield 84.6%.

LC-MS(m/z):424.0[M+H] ⁺ .

Step 2: Synthesis of compound (3S,6S,10aS)-6-(l2-azacycloyl)-3-((2S,6R)-2,6-dimethylmorpholin-4-carbonyl)octahydropyrrolo[1,2-a]azazole-5(1H)-one (58-2)

Compound 58-1 (0.330 g, 0.780 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (3 mL) was added. The reaction mixture was placed at 25 °C and reacted for 1 hour. After the reaction was completed as monitored by LC-MS, the solution was concentrated under reduced pressure to obtain crude product 58-2 (0.330 g), which was directly proceeded to the next step without purification.

LC-MS(m/z):324.0[M+H] ⁺ .

Step 3: Synthesis of diethyl difluoro(4-(2-(((3S,6S,10aS)-3-(morpholin-4-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-2-oxoacetamido)phenyl)methyl)phosphonate (58-3)

Compounds 1-4 (0.270 g, 0.780 mmol) and 58-2 (0.330 g, 0.780 mmol) were dissolved in N,N-dimethylformamide (3 mL). The reaction mixture was cooled to 10 °C, and diisopropylethylamine (0.302 g, 2.34 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.356 g, 0.937 mmol) were added sequentially. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (EA as eluent) to give compound 58-3 (0.190 g), with a yield of 37.1%.

LC-MS(m/z):657.0[M+H] ⁺ .

Step 4: Synthesis of compound (4-(2-(((3S,6S,10aS)-3-((2S,6R)-2,6-dimethylmorpholino-4-carbonyl)-5-oxodecahydropyrrolo[1,2-a]azazoline-6-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphonic acid (100)

Compound 58-3 (0.190 g, 0.290 mmol) was dissolved in anhydrous dichloromethane (1.5 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (1.5 mL) was added dropwise. The reaction solution was then incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure. A mixture of acetonitrile (8 mL) and water (2 mL) was added, and the reaction solution was incubated at 25 °C for 40 minutes. The solution was then concentrated under reduced pressure and purified by reversed-phase column chromatography (methanol:water = 50:50 as eluent) to give compound 100 (0.110 g), with a yield of 63.2%.

LC-MS(m/z):601.0[M+H] ⁺ .

Step 5: Synthesis of compound ((((4-(2-((3S,6S,10aS)-3-((2S,6R)-2,6-dimethylmorpholino-4-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zosin-6-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (58)

Compound 100 (0.110 g, 0.183 mmol) was dissolved in water (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide was added dropwise. After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (68.2 mg, 0.401 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (0.133 g, 0.550 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by thin-layer chromatography to obtain compound 58 (66.7 mg), with a yield of 44.5%.

LC-MS(m/z):829.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ ¹ H NMR (600MHz, DMSO-d6) δ11.03–10.93(m,1H),8.68(dd,J＝19.5,7.0Hz,1H),7.99(d,J＝8.6Hz,2H),7.51(d, J＝8.5Hz,2H),5.75–5.61(m,4H),4.88–4.66(m,2H),4.35–4.19(m,2H),3.97(dd,J＝41.9,13.2Hz,1H),3.6 5–3.43(m,1H),2.83–2.67(m,1H),2.30–2.18(m,2H),2.02(td,J＝17.0,15.4,8.5Hz,2H),1.89(d,J＝4.7Hz ,2H),1.82–1.66(m,4H),1.61–1.49(m,3H),1.30–1.19(m,1H),1.15(s,18H),1.10(dd,J＝11.9,6.9Hz,6H).

Example 59 Synthesis of ((((4-(2-((5S,8S,10aR)-8-((2S,6R)-2,6-dimethylmorpholine-4-carbonyl)-3-(5-methylbenzo[d]isoxazole-3-carbonyl)-6-oxodecahydropyrrole[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (Compound 59)

Step 1: Synthesis of compound methyl(5S,8S,10aR)-5-((tert-butoxycarbonyl)amino)-3-(5-methylbenzo[d]isoxazole-3-carbonyl)-6-oxodecahydropyrrolo[1,2-a][1,5]diazo-8-carboxylic acid (59-2)

Compound 22-SM (0.250 g, 0.733 mmol) and 5-methylbenzo[d]isoxazole-3-carboxylic acid (168.00 mg, 0.949 mmol) were dissolved in anhydrous dichloromethane (10 mL). The reaction mixture was cooled to 0 °C, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.211 g, 1.10 mmol) and 1-hydroxybenzotriazole (0.109 g, 0.807 mmol) were added. The mixture was reacted at 25 °C for 2 hours. After the reaction was complete as monitored by LC-MS, water (50 mL) was added, and the mixture was extracted with DCM (10 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 50:50 as eluent) to give compound 59-2 (0.350 g), yield 95.6%.

LC-MS(m/z):501.0[M+H] ⁺ .

Step 2: Synthesis of compound ((5S,8S,10aR)-5-((tert-butoxycarbonyl)amino)-3-(5-methylbenzo[d]isoxazole-3-carbonyl)-6-oxodecahydropyrrolo[1,2-a][1,5]diazo-8-carboxylic acid (59-3)

Compound 59-2 (0.350 g, 0.700 mmol) was dissolved in tetrahydrofuran (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of lithium hydroxide (59.0 mg, 1.40 mmol) (4 mL) was added dropwise. The reaction was allowed to proceed at room temperature for 2 hours. After the reaction was confirmed to be complete by LC-MS, 1.0 M dilute hydrochloric acid was added to the reaction solution to adjust the pH of the aqueous phase to acidic. The aqueous phase was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 59-3 (0.330 g), with a yield of 96.7%.

LC-MS(m/z):487.0[M+H] ⁺ .

Step 3: Synthesis of tert-butyl((5S,8S,10aR)-8-((2S,6R)-2,6-dimethylmorpholino-4-carbonyl)-3-(5-methylbenzo[d]isoxazole-3-carbonyl)-6-oxodecahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)carbamate (59-4)

Compound 59-3 (0.330 g, 0.679 mmol) and cis-2,6-dimethylmorpholine (94.0 mg, 0.817 mmol) were dissolved in N,N-dimethylformamide (5 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (0.220 g, 1.70 mmol) and N,N,N’,N’-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.388 g, 1.02 mmol) were added sequentially. The mixture was then reacted at 25 °C for 1 hour. After the reaction was monitored by LC-MS to be complete, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 96:4 as eluent) to give compound 59-4 (0.380 g), yield 95.7%.

LC-MS(m/z): 584.0[MH] ⁺ .

Step 4: Synthesis of compound (5S,8S,10aR)-5-amino-8-((2S,6R)-2,6-dimethylmorpholin-4-carbonyl)-3-(5-methylbenzo[d]isoxazole-3-carbonyl)octahydropyrrolo[1,2-a][1,5]diazoazine-6(1H)-one trifluoroacetate (59-5)

Compound 59-4 (0.380 g, 0.652 mmol) was dissolved in anhydrous dichloromethane (7 mL). The reaction solution was cooled to 0 °C, and trifluoroacetic acid (1.11 g, 9.75 mmol) was added. The mixture was then reacted at 25 °C for 1 hour. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure to give compound 59-5 (0.300 g), with a yield of 95.2%.

LC-MS(m/z):484.0[M+H] ⁺ .

Step 5: Synthesis of compound (4-(2-(((5S,8S,10aR)-8-((2S,6R)-2,6-dimethylmorpholine-4-carbonyl)-3-(5-methylbenzo[d]isoxazole-3-carbonyl)-6-oxodecahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamyl)phenyl)difluoromethyl)phosphonate diethyl ester (59-6)

Compounds 1-4 (0.218 g, 0.621 mmol) and 59-5 (0.300 g, 0.621 mmol) were dissolved in N,N-dimethylformamide (6 mL). The reaction solution was cooled to 0 °C, and diisopropylethylamine (0.200 g, 1.55 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.354 g, 0.932 mmol) were added sequentially. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 96:4 as eluent) to give compound 59-6 (0.400 g), with a yield of 79.1%.

LC-MS(m/z):817.0[M+H] ⁺ .

Step 6: Synthesis of compound (4-(2-(((5S,8S,10aR)-8-((2S,6R)-2,6-dimethylmorpholine-4-carbonyl)-3-(5-methylbenzo[d]isoxazole-3-carbonyl)-6-oxodecahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamyl)phenyl)difluoromethyl)phosphonic acid (101)

Compound 59-6 (0.400 g, 0.490 mmol) was dissolved in anhydrous dichloromethane (10 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (2.25 g, 14.7 mmol) was added dropwise. The reaction solution was then incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure, and a mixture of acetonitrile (8 mL) and water (2 mL) was added. The reaction solution was then incubated at 25 °C for 40 minutes, concentrated under reduced pressure, and purified by reversed-phase column chromatography (methanol:water = 50:50 as eluent) to give compound 101 (0.250 g), with a yield of 67.0%.

LC-MS(m/z):761.0[M+H] ⁺ .

Step 7: Synthesis of compound ((((4-(2-((5S,8S,10aR)-8-((2S,6R)-2,6-dimethylmorpholino-4-carbonyl)-3-(5-methylbenzo[d]isoxazole-3-carbonyl)-6-oxodecahydropyrrole[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamyl)phenyl)difluoromethyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (59)

Compound 101 (0.150 g, 0.197 mmol) was dissolved in water (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (14.0 mg, 0.350 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (75.0 mg, 0.441 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (0.145 g, 0.599 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by preparative thin-layer chromatography to obtain compound 59 (18.2 mg), with a yield of 9.2%.

LC-MS(m/z): 989.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.92(s,1H),8.94(dd,J＝21.0,6.8Hz,1H),8.05–7.94(m,2H),7.83(d,J＝5.8Hz,1H),7.74–7.69(m,1H),7.52(dd,J＝24.7 ,8.7Hz,3H),5.68(ddt,J＝18.3,12.3,5.4Hz,4H),4.97(d,J＝23.2Hz,1H),4.88(dt,J＝17.4,8.2Hz,1H),4.74(q,J＝8.4,7.6Hz ,1H),4.43(d,J＝9.2Hz,2H),4.22–4.06(m,2H),3.94(td,J＝33.5,31.8,20.4Hz,3H),3.65–3.37(m,1H),2.73(dq,J＝25.7,13. 1,12.6Hz,1H),2.45(s,3H),2.39–2.15(m,3H),2.14–1.92(m,3H),1.79(dd,J＝24.6,13.0Hz,1H),1.16(s,6H),1.14(s,18H).

Example 61 Synthesis of ((((4-(2-((5S,8S,10aR)-3-acetyl-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphoryl)bis(oxy))bis(methylene)diisopropylbis(carbonate) (compound 61)

Step 1: Synthesis of compound 3-benzyl-8-methyl(5S,8S,10aR)-5-((tert-butoxycarbonyl)amino)-6-oxooctahydropyrrolo[1,2-a][1,5]diazo-3,8(4H)-dicarboxylate (61-1)

Compound 22-SM (1.40 g, 4.11 mmol) was dissolved in dioxane (10 mL), and sodium bicarbonate (1.03 g, 12.3 mmol) was added. The reaction system was placed at 10 °C, and benzyl chloroformate (0.907 g, 5.33 mmol) was added, resulting in the precipitation of a white solid. The reaction system was then placed at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was concentrated under reduced pressure and purified by silica gel column chromatography (ethyl acetate: petroleum ether = 2:1 as eluent) to give compound 61-1 (1.65 g), with a yield of 84.6%.

LC-MS(m/z):476.0[M+H] ⁺ .

Step 2: Synthesis of compound (5S,8S,10aR)-3-((benzyloxy)carbonyl)-5-(tert-butoxycarbonyl)amino)-6-oxodecahydropyrrolo[1,2-a][1,5]diazo-8-carboxylic acid (61-2)

Compound 61-1 (1.65 g, 3.47 mmol) was dissolved in dioxane (10 mL), and an aqueous solution of lithium hydroxide (0.167 g, 6.94 mmol) (5 mL) was added. The mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, most of the dioxane was removed by concentration under reduced pressure. 1.0 M dilute hydrochloric acid was added to the reaction solution to adjust the pH of the aqueous phase to 5. The aqueous phase was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 61-2 (1.60 g), with a yield of 99.8%.

LC-MS(m/z):462.0[M+H] ⁺ .

Step 3: Synthesis of the compound benzyl(5S,8S,10aR)-5-((tert-butoxycarbonyl)amino)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)octahydropyrrolo[1,2-a][1,5]diazoazine-3(4H)-carboxylic acid ester (61-3)

Compound 61-2 (1.60 g, 3.47 mmol) and R-2-phenylmorpholine (0.566 g, 3.47 mmol) were dissolved in DMF (8 mL). Diisopropylethylamine (1.34 g, 10.4 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (1.58 g, 4.16 mmol) were added sequentially, and the mixture was reacted at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate as eluent) to give compound 61-3 (1.90 g) in 90.4% yield.

LC-MS(m/z): 607.0[M+H] ⁺ .

Step 4: Synthesis of the compound benzyl(5S,8S,10aR)-5-amino-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)octahydropyrrolo[1,2-a][1,5]diazo-3(4H)-carboxylic acid ester (61-4)

Compound 61-3 (1.70 g, 2.80 mmol) was dissolved in anhydrous dichloromethane (4 mL). The reaction solution was cooled to 0 °C, and trifluoroacetic acid (4 mL) was added dropwise. The reaction solution was then placed at 25 °C for 1 hour. After the reaction was monitored by TLC (alkalinization) to confirm its completion, the solution was concentrated under reduced pressure to obtain crude compound 61-4 (1.70 g).

LC-MS(m/z):507.0[M+H] ⁺ .

Step 5: Synthesis of the compound benzyl(5S,8S,10aR)-5-(2-((4-(diethoxyphosphoryl)difluoromethyl)phenyl)amino)-2-oxoacetamido)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)octahydropyrrolo[1,2-a][1,5]diazoazine-3(4H)-carboxylic acid ester (61-5).

Compounds 61-4 (1.70 g, 2.80 mmol) and 1-4 (0.983 g, 2.80 mmol) were dissolved in N,N-dimethylformamide (10 mL), followed by the sequential addition of diisopropylethylamine (1.30 g, 10.8 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (1.27 g, 3.36 mmol). The mixture was reacted at 25 °C for 1 hour. The reaction was monitored by LC-MS and TLC until complete. Water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate as eluent) to give compound 61-5 (1.40 g), yield 59.5%.

LC-MS(m/z):840.0[M+H] ⁺ .

Step 6: Synthesis of compound difluoro(4-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)acetamyl)phenyl)methyl)phosphonate diethyl ester (61-6)

Compound 61-5 (0.400 g, 0.477 mmol) was dissolved in THF (10 mL), and palladium on carbon (10%, 0.200 g, 0.189 mmol) was added. The mixture was purged three times with hydrogen balloons, and the reaction solution was placed under a hydrogen atmosphere at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, and the filtrate was concentrated under reduced pressure to give compound 61-6 (0.290 g), with a yield of 86.3%.

Step 7: Synthesis of compound (4-(2-(((5S,8S,10aR)-3-acetyl-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphonate diethyl ester (61-7)

Compound 61-6 (0.150 g, 0.213 mmol) was dissolved in anhydrous dichloromethane (5 mL). The reaction solution was cooled to 0 °C, and triethylamine (43.0 mg, 0.426 mmol) and acetyl chloride (25.0 mg, 0.318 mmol) were added sequentially. The mixture was reacted at 25 °C for 0.5 h. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol = 96:4 as eluent) to give compound 61-7 (0.120 g), with a yield of 76.4%.

LC-MS(m/z):748.0[M+H] ⁺ .

Step 8: Synthesis of compound (4-(2-(((5S,8S,10aR)-3-acetyl-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphonic acid (53)

Compound 61-7 (0.120 g, 0.161 mmol) was dissolved in anhydrous dichloromethane (5 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (0.735 g, 4.80 mmol) was added dropwise. The reaction solution was incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure, and a mixture of acetonitrile (8 mL) and water (2 mL) was added. The reaction solution was incubated at 25 °C for 40 minutes, concentrated under reduced pressure, and purified by reversed-phase column chromatography (methanol:water = 50:50 as eluent) to give compound 53 (80.0 mg), with a yield of 72.1%.

LC-MS(m/z): 692.0[M+H] ⁺ .

Step 9: Synthesis of compound ((((4-(2-((5S,8S,10aR)-3-acetyl-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphoryl)bis(oxy))bis(methylene)diisopropylbis(carbonate) (61)

Compound 53 (80.0 mg, 0.116 mmol) was dissolved in water (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (9.00 mg, 0.225 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (45.0 mg, 0.265 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (87.0 mg, 0.360 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by preparative thin-layer chromatography to obtain compound 61 (6.80 mg), with a yield of 6.1%.

LC-MS(m/z): 924.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ11.17(d,J＝10.8Hz,1H),8.66(dd,J＝38.3,6.3Hz,1H),8.03(t,J＝8.2Hz,2H),7.53–7.44(m,3H),7.39(dt,J＝10.3,7.6Hz,3H),7.33 (dt,J＝9.5,4.4Hz,1H),5.73–5.62(m,4H),5.05–4.52(m,5H),4.44–4.23(m,3H),4.19–3.96(m,3H),3.84(d,J＝13.6Hz,1H),3.66–3.4 9(m,1H),3.43–3.33(m,1H),3.12(dt,J＝25.3,12.8Hz,1H),2.92–2.59(m,1H),2.41(ddd,J＝16.6,8.3,5.3Hz,1H),2.28(d,J＝5.7Hz,3 H),2.12–1.97(m,1H),1.97–1.85(m,1H),1.69(ddd,J＝39.7,11.6,6.6Hz,2H),1.56(dd,J＝27.9,13.4Hz,1H),1.26(d,J＝6.2Hz,12H).

Example 62 Synthesis of ((difluoro(4-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionylhydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)acetamido)phenyl)methyl)phosphoryl)bis(oxy))bis(methylene)diisopropylbis(carbonate) (Compound 62)

Step 1: Synthesis of compound difluoro(4-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionylhydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)acetamyl)phenyl)methyl)phosphonate diethyl ester (62-1)

Compound 61-6 (0.150 g, 0.213 mmol) was dissolved in anhydrous dichloromethane (5 mL). The reaction solution was cooled to 0 °C, and triethylamine (43.0 mg, 0.426 mmol) and propionyl chloride (30.0 mg, 0.323 mmol) were added sequentially. The mixture was reacted at 25 °C for 0.5 h. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol = 96:4 as eluent) to give compound 62-1 (0.120 g), with a yield of 75.0%.

LC-MS(m/z):762.0[M+H] ⁺ .

Step 2: Synthesis of compound (difluoro(4-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionylpyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)acetamido)phenyl)methyl)phosphonic acid (56)

Compound 62-1 (0.120 g, 0.158 mmol) was dissolved in anhydrous dichloromethane (5 mL). The reaction solution was cooled to 0 °C, and trimethylbromosilane (0.735 mg, 4.80 mmol) was added dropwise. The reaction solution was then incubated at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure. A mixture of acetonitrile (8 mL) and water (2 mL) was added, and the reaction solution was incubated at 25 °C for 40 minutes. The solution was then concentrated under reduced pressure and purified by reversed-phase column chromatography (methanol:water = 50:50 as eluent) to give compound 56 (80.0 mg), with a yield of 71.0%.

LC-MS(m/z):706.0[M+H] ⁺ .

Step 3: Synthesis of compound ((difluoro(4-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionylhydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)acetamido)phenyl)methyl)phosphoryl)bis(oxy))bis(methylene)diisopropylbis(carbonate) (62)

Compound 56 (80.0 mg, 0.113 mmol) was dissolved in water (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (8.00 mg, 0.200 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (41.0 mg, 0.241 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (3 mL), and methyl iodide tervastatin (80.0 mg, 0.331 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by thin-layer chromatography to obtain compound 62 (5.10 mg), with a yield of 5.0%.

LC-MS(m/z): 938.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ11.17(d,J＝10.6Hz,1H),8.68(dd,J＝39.7,6.2Hz,1H),8.03(t,J＝8.0Hz,2H),7.53 –7.48(m,2H),7.46(d,J＝7.7Hz,1H),7.39(dt,J＝14.6,7.5Hz,3H),7.33(dt,J＝8.6,4 .1Hz,1H),5.68(dq,J＝13.0,5.7Hz,4H),5.36–4.96(m,1H),4.82(tt,J＝14.6,7.3Hz, 3H),4.58(d,J＝10.6Hz,1H),4.38(d,J＝11.3Hz,1H),4.21(dd,J＝72.6,12.9Hz,2H),4 .10–3.95(m,3H),3.86(d,J＝13.6Hz,1H),3.58(q,J＝11.8Hz,1H),3.11(dt,J＝35.6,1 3.2Hz,1H),2.93–2.73(m,1H),2.72–2.59(m,1H),2.45–2.16(m,1H),2.11–1.84(m,2 H),1.73(dd,J＝11.6,7.2Hz,1H),1.70–1.41(m,1H),1.33(d,J＝32.3Hz,1H),1.26(d, J＝6.3Hz,12H),1.23(s,1H),1.04(qd,J＝8.1,7.6,5.1Hz,3H),0.85(t,J＝6.9Hz,1H).

Example 65 Synthesis of ((difluoro(4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azoazine-6-yl)amino)acetamido)phenyl)methyl)phosphoryl)bis(oxy))bis(methylene)diisopropylbis(carbonate) (compound 65)

Step 1: Synthesis of compound ((difluoro(4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azoazine-6-yl)amino)acetamido)phenyl)methyl)phosphoryl)bis(oxy))bis(methylene)diisopropylbis(carbonate) (65)

Compound 52 (0.162 g, 0.250 mmol) was dissolved in water (4 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (18.0 mg, 0.450 mmol) was added dropwise (2 mL). After the addition was complete, the pH of the solution was approximately 9. Silver nitrate (93.5 mg, 0.550 mmol) was added, and the mixture was reacted at 0 °C for 2 hours. The mixture was filtered, and the solid was collected and dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in toluene (4 mL), and iodomethyl isopropyl carbonate (0.183 g, 0.756 mmol) was added. The mixture was reacted at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and purified by thin-layer chromatography to obtain compound 65 (55.0 mg), with a yield of 25.0%.

LC-MS(m/z):881.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ11.01(d,J＝9.3Hz,1H),8.71(dd,J＝34.1,7.0Hz,1H),8.00(t,J＝7.4Hz,2H),7.50(dd,J＝8. 7,3.1Hz,2H),7.39(ddt,J＝20.4,13.2,7.3Hz,4H),7.32(td,J＝7.0,3.8Hz,1H),5.68(dq,J＝13 .0,5.8Hz,4H),4.92(t,J＝8.5Hz,1H),4.82(dhept,J＝13.2,6.2Hz,3H),4.75(t,J＝8.8Hz,1H) ,4.60–4.52(m,1H),4.40–4.34(m,1H),4.29(t,J＝11.3Hz,1H),4.16(d,J＝13.3Hz,1H),4.03(t ,J＝13.8Hz,2H),3.56(td,J＝11.8,2.7Hz,1H),3.35(d,J＝12.4Hz,1H),3.13(dd,J＝13.5,10.6 Hz,1H),2.85(td,J＝12.9,3.6Hz,1H),2.63(dd,J＝12.7,10.2Hz,1H),2.31(dd,J＝11.9,7.2Hz, 1H),2.22–2.12(m,1H),2.02(dd,J＝35.2,8.3Hz,1H),1.89(dt,J＝12.2,8.2Hz,2H),1.81(t,J ＝12.6Hz,1H),1.71(ddd,J＝37.4,14.7,7.2Hz,1H),1.64–1.45(m,1H),1.26(d,J＝6.2Hz,12H).

Example 67 Synthesis of difluoro(4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azo-6-yl)amino)acetamido)phenyl)methyl)(hydroxy)phosphoryl)oxy)methylneopentate (67) and its ammonium salt

Step 1: Synthesis of compound ((difluoro(4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azo-6-yl)amino)acetamido)phenyl)methyl)(hydroxy)phosphoryl)oxy)methylneopentate (67)

Compound 4 (25.0 mg, 28.5 μmol) was dissolved in methanol (3 mL) and reacted at 25 °C for 36 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was concentrated under reduced pressure to give compound 67.

LC-MS(m/z):763.0[M+H] ⁺ .

Step 2: Synthesis of the compound ((difluoro(4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azo-6-yl)amino)acetamido)phenyl)methyl)(hydroxy)phosphoryl)oxy)methylneopentate ammonium salt)

Compound 67 was purified by preparative HPLC to obtain its ammonium salt (9.0 mg), with a yield of 41.5%. Preparation method: A C18 column was used, with a mobile phase of 10 mmol/L ammonium bicarbonate aqueous solution (A) and acetonitrile (B). Gradient elution was performed at a flow rate of 15 mL/min, based on the volume content of phase B: 0–10 min (15% → 100%), 10.01–15 min (100%), and 15.01–20 min (100% → 15%).

LC-MS(m/z):763.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.78(d,J＝9.4Hz,1H),8.67(dd,J＝33.6,7.1Hz,1H),7.81(t,J＝7.6Hz,2H),7.48(dd,J＝8.9,3.3Hz,2H),7.45–7.28(m,5H ),7.10(t,J＝51.0Hz,4H),5.38(d,J＝10.6Hz,2H),5.02–4.68(m,2H),4.57(d,J＝10.4Hz,1H),4.36(t,J＝12.8Hz,1H),4.29(t, J＝10.7Hz,1H),4.17(d,J＝13.4Hz,1H),4.03(t,J＝12.3Hz,2H),3.56(t,J＝12.2Hz,1H),3.12(dd,J＝13.5,10.5Hz,1H),2.99–2 .79(m,1H),2.72–2.55(m,1H),2.30(d,J＝10.1Hz,1H),2.22–1.96(m,3H),1.96–1.78(m,3H),1.77–1.45(m,3H),1.14(s,9H).

Example 68 Synthesis of (((4-(2-(((5S,8S,10aR)-3-acetyl-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazooctyl-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)(hydroxy)phosphoryl)oxy)methylneopentate (compound 68) and its ammonium salt

Step 1: Synthesis of (((4-(2-(((5S,8S,10aR)-3-acetyl-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazooctyl-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)(hydroxy)phosphoryl)oxy)methylneopentate (68)

Compound 21 (32.0 mg, 34.8 μmol) was dissolved in methanol (3 mL) and reacted at 25 °C for 36 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was concentrated under reduced pressure to obtain compound 68.

LC-MS(m/z):806.0[M+H] ⁺ .

Step 2: Synthesis of (((4-(2-(((5S,8S,10aR)-3-acetyl-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazooctyl-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)(hydroxy)phosphoryl)oxy)methylneopentate ammonium salt)

Compound 68 was purified by HPLC to obtain its ammonium salt (12.0 mg), with a yield of 42.9%. Preparation method: A C18 column was used, with a mobile phase of 10 mmol/L ammonium bicarbonate aqueous solution (A) and acetonitrile (B). Gradient elution was performed at a flow rate of 15 mL/min, based on the volume content of phase B: 0 to 10 min (15% → 100%), 10.01 to 15 min (100%), and 15.01 to 20 min (100% → 15%).

LC-MS(m/z):806.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.96(d,J＝11.9Hz,1H),8.65(dd,J＝39.2,6.3Hz,1H),7.84(t,J＝7.9Hz,2H),7.52–7.45(m,3H),7.43–7.35(m,3 H),7.35–7.30(m,1H),7.10(t,J＝51.0Hz,4H),5.38(d,J＝10.5Hz,2H),5.09–4.73(m,2H),4.68–4.23(m,4H),4.21–3 .95(m,3H),3.90–3.77(m,1H),3.58(dt,J＝21.1,12.0Hz,1H),3.11(ddd,J＝25.6,14.4,10.7Hz,2H),2.97–2.62(m,1 H),2.46–2.31(m,1H),2.28(d,J=5.9Hz,3H),2.14–1.98(m,2H),1.97–1.85(m,1H),1.78–1.45(m,2H),1.14(s,9H).

Example 69 Synthesis of difluoro(4-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionyldecahydropyrrolo[1,2-a][1,5]diazo-5-yl)amino)acetamido)phenyl)methyl)(hydroxy)phosphoryl)oxy)methylneopentate (compound 69) and its ammonium salt

Step 1: Synthesis of (((difluoro(4-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionyldecahydropyrrolo[1,2-a][1,5]diazo-5-yl)amino)acetamido)phenyl)methyl)(hydroxy)phosphoryl)oxy)methylneopentate (69)

Compound 22 (82.0 mg, 87.9 μmol) was dissolved in methanol (2 mL) and reacted at 25 °C for 48 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was concentrated under reduced pressure to give compound 69.

LC-MS(m/z):820.0[M+H] ⁺ .

Step 2: Synthesis of (((difluoro(4-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionyldecahydropyrrolo[1,2-a][1,5]diazo-5-yl)amino)acetamido)phenyl)methyl)(hydroxy)phosphoryl)oxy)methylneopentate ammonium salt)

Compound 69 was purified by preparative HPLC to obtain its ammonium salt (21.1 mg), with a yield of 29.3%.

LC-MS(m/z):820.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.97(d,J＝11.8Hz,1H),8.67(dd,J＝40.3,6.2Hz,1H),7.84(t,J＝7.8Hz,2H),7.56–7.43(m,3H),7.38(dq,J＝15.7,8.0Hz,3H),7.32(t,J =7.3Hz,1H),7.10(t,J＝51.0Hz,4H),5.38(d,J＝10.4Hz,2H),5.05–4.75(m,2H),4.48(dd,J＝128.3,11.1Hz,2H),4.21(dd,J＝72.4,13.2Hz, 2H),4.03(dd,J＝19.3,11.9Hz,3H),3.85(d,J＝13.5Hz,1H),3.61–3.53(m,1H),3.10(dq,J＝26.2,12.3Hz,2H),2.90–2.63(m,2H),2.58–2.5 2(m,1H),2.43–2.24(m,1H),2.11–1.79(m,3H),1.72(dd,J＝11.9,7.2Hz,1H),1.66–1.54(m,1H),1.14(s,9H),1.05(dt,J＝11.0,5.6Hz,3H).

Example 93 Synthesis of compound (4-(2-(((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphonic acid (compound 93)

The synthesis method is described in Example 1.

LC-MS(m/z):658.0[M+H] ⁺ .

Example 94 Synthesis of (4-(2-(((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-N-methyl-2-oxoacetamyl)phenyl)difluoromethyl)phosphonic acid (94)

The synthesis method is described in Example 2.

LC-MS(m/z): 672.0[M+H] ⁺ .

Example 95 Synthesis of (4-(N-butyl-2-(((3S,6S,10aS)-3-((3R,4S or 3S,4R)-3-cyano-4-phenylpyrrolidine-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-2-oxoacetamyl)phenyl)difluoromethyl)phosphonic acid (95)

The synthesis method is described in Example 3.

LC-MS(m/z):714.0[M+H] ⁺ .

Example 96 Synthesis of difluoro(4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((S)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azozin-6-yl)amino)acetamido)phenyl)methyl)phosphoric acid (96)

The synthesis method is described in Example 7.

LC-MS(m/z): 649.0[M+H] ⁺ .

Example 97 Synthesis of ((difluoro(4-(2-(((3S,6S,10aS)-3-(indole-1-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-2-oxoacetamyl)phenyl)methyl)phosphonic acid (97)

The synthesis method is described in Example 54.

LC-MS(m/z): 605.0[M+H] ⁺ .

Example 98 Synthesis of ((difluoro(4-(2-(((3S,6S,10aS)-3-(methyl(phenyl)carbamoyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-2-oxoacetamido)phenyl)methyl)phosphoric acid (98)

The synthesis method is described in Example 55.

LC-MS(m/z):593.0[M+H] ⁺ .

Example 99 Synthesis of difluoro(4-(2-(((3S,6S,10aS)-3-(morpholin-4-carbonyl)-5-oxodecahydropyrrolo[1,2-a]zozin-6-yl)amino)-2-oxoacetamyl)phenyl)methyl)phosphonic acid (99)

The synthesis method is described in Example 57.

LC-MS(m/z):573.0[M+H] ⁺ .

Example 100 Synthesis of (4-(2-(((3S,6S,10aS)-3-((2S,6R)-2,6-dimethylmorpholino-4-carbonyl)-5-oxodecahydropyrrolo[1,2-a]azazoline-6-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphonic acid (100)

The synthesis method is described in Example 58.

LC-MS(m/z):601.0[M+H] ⁺ .

Example 101 Synthesis of (4-(2-(((5S,8S,10aR)-8-((2S,6R)-2,6-dimethylmorpholine-4-carbonyl)-3-(5-methylbenzo[d]isoxazole-3-carbonyl)-6-oxodecahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamyl)phenyl)difluoromethyl)phosphonic acid (101)

The synthesis method is described in Example 59.

LC-MS(m/z):761.0[M+H] ⁺ .

Example 102 Synthesis of difluoro(3-fluoro-4-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azozin-6-yl)amino)acetamido)phenyl)methyl)phosphoric acid (102)

The synthesis method is described in Example 26.

LC-MS(m/z): 667.0[M+H] ⁺ .

Example 103 Synthesis of (4-(2-(((5S,8S,10aR)-3-(2,2-difluoroethyl)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a][1,5]diazoazine-5-yl)amino)-2-oxoacetamido)phenyl)difluoromethyl)phosphonic acid (103)

The synthesis method is described in Example 23.

LC-MS(m/z):714.0[M+H] ⁺ .

Example 121 Synthesis of difluoro(5-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azo-6-yl)amino)acetamido)thiophene-3-yl)methyl)phosphonic acid (121)

Step 1: Synthesis of compound 2-((4-((diethoxyphosphoryl)difluoromethyl)thiophene-2-yl)amino)-2-oxoacetate methyl ester (121-1)

Compound 121-SM (1.50 g, 5.20 mmol) was dissolved in anhydrous dichloromethane (30 mL), and diisopropylethylamine (0.80 g, 6.20 mmol) was added. The reaction solution was cooled to 0 °C, and oxaloyl chloride monomethyl ester (0.96 g, 7.89 mmol) was added dropwise. The reaction was carried out at 25 °C for 1 hour. After the reaction was monitored by LC-MS to be complete, water (20 mL) was added to quench the reaction. The aqueous phase was extracted with dichloromethane (30 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 50:50 as eluent) to give compound 121-1 (1.40 g), yield 73.9%.

LC-MS(m/z):372.0[M+H] ⁺ .

Step 2: Synthesis of compound 2-((4-((diethoxyphosphoryl)difluoromethyl)thiophen-2-yl)amino)-2-oxoacetic acid (121-2)

Compound 121-1 (1.20 g, 3.23 mmol) was dissolved in tetrahydrofuran (10 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of lithium hydroxide (0.34 g, 8.08 mmol) was added dropwise (10 mL). The reaction solution was kept at 0 °C for 30 minutes. After the reaction was confirmed to be complete by LC-MS, a 1.0 M hydrochloric acid solution was added to the reaction solution to adjust the pH of the aqueous phase to acidic. The aqueous phase was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 121-2 (830.0 mg), with a yield of 55.3%.

LC-MS(m/z):356.0[MH] ^- .

Step 3: Synthesis of compound difluoro(5-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azo-6-yl)amino)acetamyl)thiophene-3-yl)methyl)phosphonate diethyl ester (121-3)

Compound 121-2 (0.83 g, 2.32 mmol) and (3S,6S,10aS)-6-amino-3-((R)-2-phenylmorpholine-4-carbonyl)octahydropyrrolo[1,2-a]azolin-5(1H)-one (0.86 g, 2.32 mmol) were dissolved in N,N-dimethylformamide (40 mL). Diisopropylethylamine (1.00 g, 4.48 mmol) and N,N,N’,N’-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (1.02 g, 2.68 mmol) were added sequentially at 0 °C. The reaction solution was placed at 25 °C and reacted for 1 hour. After the reaction was monitored by LC-MS to be complete, water (50 mL) was added to quench the reaction. The aqueous phase was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (EA as eluent) to give compound 121-3 (882.0 mg), with a yield of 55.0%.

LC-MS(m/z):709.0[MH] ^- .

Step 4: Synthesis of compound (difluoro(5-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azo-6-yl)amino)acetamido)thiophene-3-yl)methyl)phosphonic acid (121)

Compound 121-3 (0.31 g, 4.36 mmol) was dissolved in anhydrous dichloromethane (3 mL), and trimethylbromosilane (3 mL) was added. The reaction mixture was incubated at 25 °C for 8 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure. A mixture of acetonitrile (4 mL) and water (1 mL) was added, and the reaction mixture was incubated at 25 °C for 20 minutes. The solution was then concentrated under reduced pressure, and the residue was purified by reversed-phase column chromatography (methanol:water = 80:20 as eluent) to give compound 121 (195.0 mg), with a yield of 68.4%.

LC-MS(m/z): 655.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ12.23(d,J＝10.3Hz,1H),8.69(dd,J＝32.2,7.1Hz,1H),7.48–7.29(m,6H),7.25–7.18(m,1H),4.96–4.72(m,2H),4.59–4.24(m,3H), 4.21–3.95(m,2H),3.62–3.52(m,1H),3.16–3.09(m,1H),2.90–2.58(m,1H),2.33–2.13(m,1H),2.10–1.96(m,2H),1.94–1.47(m,9H).

Example 122 Synthesis of difluoro(5-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azo-6-yl)amino)acetamyl)thiophen-3-yl)methyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropionate) (122)

Step 1: Synthesis of compound (((difluoro(5-(2-oxo-2-(((3S,6S,10aS)-5-oxo-3-((R)-2-phenylmorpholine-4-carbonyl)decahydropyrrolo[1,2-a]azo-6-yl)amino)acetamyl)thiophen-3-yl)methyl)phosphoryl)bis(oxy)bis(methylene)bis(2,2-dimethylpropionate) (122)

Compound 121 (0.16 g, 0.25 mmol) was suspended in water (2 mL). An aqueous solution of sodium hydroxide (19.6 mg, 0.49 mmol) was added dropwise (2 mL) at 0 °C. After the addition was complete, the pH of the aqueous phase was approximately 10. Silver nitrate (124.9 mg, 0.73 mmol) was then added, and the reaction mixture was kept at 0 °C for 2 hours. The mixture was filtered, and the filter cake was dried under reduced pressure using an oil pump (rotary vane vacuum pump). The dried solid was dissolved in acetonitrile (10 mL), and methyl iodide tervastatin (592.9 mg, 2.45 mmol) was added. The reaction mixture was kept at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the mixture was filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 2:1 as eluent) to give compound 122 (38.5 mg), with a yield of 17.4%.

LC-MS(m/z):883.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ12.35(d,J＝10.4Hz,1H),8.74(dd,J＝34.1,7.0Hz,1H),7.51(s,1H),7.40(ddd,J＝17.3,15.2,7.3Hz,4H),7.32(dp,J＝7 .3,5.1,3.4Hz,1H),7.19(dd,J＝6.4,1.8Hz,1H),5.69(td,J＝14.9,5.6Hz,4H),4.95–4.83(m,1H),4.82–4.52(m,2H),4.4 0–4.24(m,3H),4.19–3.98(m,2H),3.56(t,J＝11.4Hz,1H),3.16–2.80(m,1H),2.23(dd,J＝86.7,10.1Hz,1H),2.10–2.00( m,2H),1.91–1.85(m,2H),1.80(t,J＝13.2Hz,1H),1.70(ddt,J＝32.6,21.8,11.3Hz,3H),1.62–1.51(m,3H),1.15(s,18H).

Example 123 Synthesis of difluoro(5-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionyldecahydropyrrolo[1,2-a][1,5]diazacyclooctyl-5-yl)amino)acetamido)thiophene-3-yl)methyl)phosphonic acid (123)

Step 1: Synthesis of diethyl(difluoro(5-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionyldecahydropyrrolo[1,2-a][1,5]diazacyclooctyl-5-yl)amino)acetamido)thiophene-3-yl)methyl)phosphonate (123-1)

Compounds 121-2 (0.22 g, 0.62 mmol) and 56-3 (0.26 g, 0.62 mmol) were dissolved in N,N-dimethylformamide (5 mL). Diisopropylethylamine (0.16 g, 1.24 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (0.28 g, 0.74 mmol) were added sequentially at 0 °C. The reaction mixture was incubated at 25 °C for 1 hour. After the reaction was complete as monitored by LC-MS, water (10 mL) was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (EA as eluent) to give compound 123-1 (60.0 mg), with a yield of 12.6%.

LC-MS(m/z):768.0.0[M+H] ⁺ .

Step 2: Synthesis of (difluoro(5-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionyldecahydropyrrolo[1,2-a][1,5]diazacyclooctyl-5-yl)amino)acetamido)thiophene-3-yl)methyl)phosphonic acid (123)

Compound 123-1 (60.0 mg, 0.078 mmol) was dissolved in anhydrous dichloromethane (2 mL), and trimethylbromosilane (1 mL) was added. The reaction mixture was incubated at 25 °C for 8 hours. After the reaction was confirmed to be complete by LC-MS, the solution was concentrated under reduced pressure. A mixture of acetonitrile (3 mL) and water (1 mL) was added, and the reaction mixture was incubated at 25 °C for 20 minutes. The solution was then concentrated under reduced pressure, and the residue was purified by reversed-phase column chromatography (methanol:water = 80:20 as eluent) to give compound 123 (22.0 mg), with a yield of 39.6%.

LC-MS(m/z):712.0[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ12.39(d,J＝12.8Hz,1H),8.61(dd,J＝41.1,6.3Hz,1H),7.49–7.44(m,1H),7.42–7.3 5(m,3H),7.35–7.30(m,2H),7.28–7.21(m,1H),5.06–4.77(m,2H),4.64–4.32(m,2H), 4.32–4.11(m,2H),4.08–3.79(m,4H),3.65–3.53(m,2H),3.18–2.99(m,2H),2.91–2.6 3(m,2H),2.44–2.20(m,1H),2.10–1.84(m,3H),1.77–1.48(m,2H),1.10–0.99(m,3H).

Example 124 Synthesis of difluoro(5-(2-oxo-2-(((5S,8S,10aR)-6-oxo-8-((R)-2-phenylmorpholine-4-carbonyl)-3-propionyldecahydropyrrolo[1,2-a][1,5]diazacyclooctyl-5-yl)amino)acetamido)thiophen-3-yl)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionate) (124)

Compound 123 (20.0 mg, 0.028 mmol) was suspended in water (1 mL). The reaction solution was cooled to 0 °C, and an aqueous solution of sodium hydroxide (2.8 mg, 0.07 mmol) was added dropwise (1 mL). After the addition was complete, the pH of the solution was approximately 10. Then, silver nitrate (17.9 mg, 0.11 mmol) was added, and the reaction was carried out at 0 °C for 2 hours. After the reaction was completed, the solid was filtered and collected. The solid was then placed under a rotary vane vacuum pump to remove water. The resulting dry solid was suspended in toluene (1 mL), and methyl iodide tervastatin (85.1 mg, 0.35 mmol) was added. The reaction solution was carried out at 25 °C for 16 hours. After the reaction was confirmed to be complete by LC-MS, the solid was filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 2:1 as eluent) to give compound 124 (3.1 mg), with a yield of 11.8%.

LC-MS(m/z): 940.0[M+H] ⁺ .

^1H NMR (600MHz, DMSO-d6 ₎ )δ12.62–12.25(m,1H),8.62(dd,J＝42.8,6.3Hz,1H),7.59–7.17(m,7H),5 .76–5.62(m,4H),5.04–4.74(m,2H),4.62–4.33(m,2H),4.30–4.07(m,2H) ,4.08–3.78(m,4H),3.64–3.49(m,1H),3.20–2.84(m,2H),2.80–2.60(m,2 H),2.12–1.86(m,4H),1.77–1.52(m,3H),1.15(s,18H),1.08–1.00(m,3H).

Bioactivity test

Test Example 1: Fluorescence polarization screening experiment of the compounds of this invention

1. Main reagents and consumables

2. Reagent preparation

1) Prepare the test buffer (10mL system) according to the table below:

2) Prepare the working concentrations of each component using the test buffer according to the table below (final volume: 50 μL):

"—" indicates that there is no relevant data.

3) In this experiment, the STAT6 protein expression plasmid pT7-TrxA-6×His-STAT6 (human-derived) was used. The Rosetta (DE3) strain was used to induce protein expression at a concentration of 0.2 mM IPTG at 16 ℃ for 20 h. The protein was purified by the Ni NTA Beads kit to obtain the purified STAT6 protein.

3. Fluorescence polarization activity detection

1) Compound dilution: Add 2 μL of 10 mM compound stock solution to 198 μL of DMSO to obtain a 100 μM compound dilution. Then, perform a 3-fold serial dilution by adding 30 μL of the 100 μM compound dilution to 60 μL of DMSO to obtain 8 concentrations. Then, add 5 μL of each concentration of the test compound to a 96-well black plate, and set up two negative controls (DMSO final concentration 10%).

2) Compound and protein incubation: Then add 30 μL of test buffer and 10 μL of 2.5 μM STAT6 protein to the 96-well black plate, centrifuge at 1000 rpm for 1 min at room temperature, and incubate at 25 °C with shaking for 45 min.

3) Substrate reaction: Add 5 μL of 125 nM substrate 5-FAM-ApYKPFQDLI-NH ₂ to the 96-well black plate, centrifuge at 1000 rpm for 1 min at room temperature, and then incubate at 25 °C in the dark with shaking for 30 min.

4) Transfer the reaction system from the 96-well black plate to the 384-well black plate at a rate of 20 μL per well. Detect the absorbance at 485/535 nm using a microplate reader and calculate the fluorescence polarization mP value using software.

5) Data processing: The group containing only 5-FAM substrate (without STAT6 protein) was used as the negative control, and the value of the DMSO control group was used as the positive control. The inhibition rate (%) of the compound on the binding of STAT6 to 5-FAM-ApYKPFQDLI- _NH2 was calculated using the formula "Inhibition rate (%) = (1 - (Experimental group value - Negative control group value) / (Positive control group value - Negative control group value)) * 100%". Then, the _IC50 value of the compound was obtained by fitting the data using Graphpad Prism 8 software.

Table 1 shows the fluorescence polarization experiment results of the compounds of this invention on STAT6 protein.

Table 1. Fluorescence polarization experiment results of the compounds of the present invention.

Test Example 2: p-STAT6 experiment with STAT6 inhibitors

(1) Cells

(2) Reagents and Consumables

(3) Compound preparation

Take an appropriate amount of the test compound and dissolve it in DMSO to 10 mM. Take an appropriate amount of the 10 mM test compound stock solution and perform a 4-fold serial dilution with DMSO to obtain compound stock solutions (2, 0.5, 0.125, 0.03125, 0.008, 0.002, 0.0005, 0.00012, 0.00003 μM).

(4) Experimental methods

BEAS-2B cells were seeded at 70%-80% confluence in 24-well plates at a density of 2* ^10⁵ cells per well and cultured overnight in DMEM medium (containing 2% FBS). Cells in the drug-treated groups were pretreated for 3 h with different concentrations of the drug (final concentrations of 2, 0.5, 0.125, 0.03125, 0.008, 0.002, 0.0005, 0.00012, and 0.00003 μM). The blank control and IL-4 single-drug groups received an equal volume of DMSO. After 3 h of pretreatment, except for the blank control group, each well was treated with 2 ng/ml IL-4 for 30 min, followed by cell harvesting. The supernatant was discarded, and the cells were washed once with PBS. 50 μL of RIPA lysis buffer (containing a protease inhibitor mixture, PMSF, and 1.5x protein loading buffer) was added to each well. The cell lysis buffer was boiled at 100°C for 5 min and stored at -20°C.

Equal volumes of protein samples and protein molecular weight standards were loaded into the wells of an SDS-PAGE gel and electrophoresed at 100V. After electrophoresis, the membrane was transferred at a constant current of 300mA. After transfer, the PVDF membrane was removed and blocked with 5% skim milk PBST solution at room temperature for 1 hour. After blocking, the corresponding primary antibody incubation solution (primary antibody dilution ratio 1:1000, 5% BSA + PBST) was added, and the membrane was incubated overnight at 4°C with gentle shaking. After incubation, the membrane was washed four times with PBST for 5 minutes each time. Secondary antibody incubation solution (secondary antibody dilution ratio 1:2000, 5% skim milk + PBST) was added, and the membrane was incubated at room temperature for 1 hour. The membrane was washed four times with PBST for 5 minutes each time. Development and exposure were performed according to the method provided by the ECL chemiluminescence detection kit. The development results were analyzed using ImagJ software to calculate the grayscale molecular weight, inhibition rate, and _IC50 .

The inhibition rate is calculated as follows:

Inhibition rate (%) = 100 - (gray value of the treated group - gray value of the blank group) / (gray value of the IL-4 monotherapy group - gray value of the blank group) × 100%

Table 2 shows the results of the degree of inhibition of STAT6 phosphorylation in cells by the tested compounds.

Table 2. Inhibition of cellular pSTAT6 activity by the compounds of this invention

As can be seen from the data in Table 2, compound 4 can strongly inhibit the phosphorylation of STAT6.

Test Example 3: Detection of p-STAT6 in BEAS-2B cells

(1) Cells: Refer to test case 2;

(2) Reagents and consumables: Refer to test example 2;

(3) Compound preparation

Take an appropriate amount of the compound to be tested, dissolve it in DMSO to 10 mM, and obtain the compound stock solution.

(4) Experimental methods

BEAS-2B cells were seeded at 70%-80% confluence in 24-well plates at a density of 2* ^10⁵ cells per well and cultured overnight in DMEM medium (containing 2% FBS). Cells in the drug-treated groups were pretreated with different concentrations of the drug for 3 hours, while the control group and the IL-4 single-drug group received an equal volume of DMSO. After 3 hours of pretreatment, except for the control group, each well was treated with 2 ng/ml IL-4 for 30 minutes, followed by cell harvesting. The supernatant was discarded, and the cells were washed once with PBS. 50 μL of RIPA lysis buffer (containing a protease inhibitor mixture, PMSF, and 1.5x protein loading buffer) was added to each well. The cell lysis buffer was boiled at 100°C for 5 minutes and stored at -20°C.

Equal volumes of protein samples and protein molecular weight standards were loaded into the wells of an SDS-PAGE gel and electrophoresed at 100V. After electrophoresis, the membrane was transferred at a constant current of 300mA. After transfer, the PVDF membrane was removed and blocked with 5% skim milk PBST solution at room temperature for 1 hour. After blocking, the corresponding primary antibody incubation solution (primary antibody dilution ratio 1:1000, 5% BSA + PBST) was added, and the membrane was incubated overnight at 4°C with gentle shaking. After incubation, the membrane was washed four times with PBST for 5 minutes each time. Secondary antibody incubation solution (secondary antibody dilution ratio 1:2000, 5% skim milk + PBST) was added, and the membrane was incubated at room temperature for 1 hour. The membrane was washed four times with PBST for 5 minutes each time. Development and exposure were performed according to the method provided by the ECL chemiluminescence detection kit. The development results were analyzed using ImagJ software to calculate the grayscale molecular weight, inhibition rate, and _IC50 .

The inhibition rate is calculated as follows:

Inhibition rate (%) = 100 - (gray value of the treatment group - gray value of the blank group) / (gray value of the IL-4 monotherapy group - gray value of the blank group) × 100%, the results are shown in Table 3:

Table 3. Inhibition rate of compounds at different concentrations on pSTAT6 in BEAS-2B cells


A: Indicates a test compound concentration of 2 μM; B: Indicates a test compound concentration of 1 μM; C: Indicates a test compound concentration of 0.25 μM.
μM; D: indicates the concentration of the test compound is 0.125 μM; E: indicates the concentration of the test compound is 0.1 μM.

Test Example 4: Detection of p-STAT6 in PBMCs

The compounds of this invention were prepared according to the methods described in the examples.

(1) Cells

(2) Reagents and consumables: Refer to test example 2;

(3) Compound preparation

Take an appropriate amount of the test compound and dissolve it in DMSO to 10 mM. Take an appropriate amount of the 10 mM test compound stock solution and perform a 3-fold serial dilution with DMSO to obtain the compound stock solutions (1, 0.333, 0.111, 0.037, 0.0123, 0.004 mM).

(4) Experimental methods

After PBMC cell resuscitation, cells were plated at 1* ^10⁶ cells per well in 24-well plates and cultured in PRIM-1640 (containing 2% FBS) for 2 h. Cells in the drug-treated groups were pretreated for 3 h with different concentrations of the drug (final concentrations of 1, 0.333, 0.111, 0.037, 0.0123, and 0.004 μM). The control group and the IL-4 single-drug group received an equal volume of DMSO. After 3 h of pretreatment, except for the control group, each well was treated with 2 ng/ml of IL-4 for 10 min, followed by cell harvesting. 50 μL of RIPA lysis buffer (containing a protease inhibitor mixture, PMSF, and 1.5x protein loading buffer) was added to each well, and the cell lysis buffer was boiled at 100°C for 5 min and stored at -20°C.

Equal volumes of protein samples and protein molecular weight standards were loaded into the wells of an SDS-PAGE gel and electrophoresed at 100V. After electrophoresis, the membrane was transferred at a constant current of 300mA. After transfer, the PVDF membrane was removed and blocked with 5% skim milk PBST solution at room temperature for 1 hour. After blocking, the corresponding primary antibody incubation solution (primary antibody dilution ratio 1:1000, 5% BSA + PBST) was added, and the membrane was incubated overnight at 4°C with gentle shaking. After incubation, the membrane was washed four times with PBST for 5 minutes each time. Secondary antibody incubation solution (secondary antibody dilution ratio 1:2000, 5% skim milk + PBST) was added, and the membrane was incubated at room temperature for 1 hour. The membrane was washed four times with PBST for 5 minutes each time. Development and exposure were performed according to the method provided by the ECL chemiluminescence detection kit. The development results were analyzed using ImagJ software to calculate the grayscale molecular weight, inhibition rate, and _IC50 .

The inhibition rate is calculated as follows:

Inhibition rate (%) = 100 - (gray value of the treatment group - gray value of the blank group) / (gray value of the IL-4 monotherapy group - gray value of the blank group) × 100%, the results are shown in Table 4:

Table 4. Compounds inhibit pSTAT6 activity in PBMC cells.

Test Example 5: Western Blot Test for p-STAT3 in BEAS-2B Cells

The compound of the present invention was prepared according to the method described in the examples.

(1) Cells

(2) Reagents and Consumables

For other reagents and consumables, refer to Test Example 2.

(3) Compound preparation

Take an appropriate amount of the test compound and dissolve it in DMSO to 10 mM. Take an appropriate amount of the 10 mM test compound stock solution and perform a 4-fold serial dilution with DMSO to obtain compound stock solutions (10, 2.5, 0.625, 0.156 mM).

(4) Experimental methods

BEAS-2B cells were seeded at 70%-80% confluence in 24-well plates at a density of 2* ^10⁵ cells per well and cultured overnight in DMEM medium (containing 2% FBS). Cells in the drug-treated groups were pretreated for 3 h with different concentrations of the drug (final concentrations of 10, 2.5, 0.625, and 0.156 μM), while the control group and the IL-6 monotherapy group received an equal volume of DMSO. After 3 h of pretreatment, except for the control group, each well was treated with 10 ng/ml IL-6 for 60 min, followed by cell harvesting. The supernatant was discarded, and the cells were washed once with PBS. 50 μL of RIPA lysis buffer (containing a protease inhibitor mixture, PMSF, and 1.5x protein loading buffer) was added to each well. The cell lysis buffer was boiled at 100°C for 5 min and stored at -20°C.

Equal volumes of protein samples and protein molecular weight standards were loaded into the wells of an SDS-PAGE gel and electrophoresed at 100V. After electrophoresis, the membrane was transferred at a constant current of 300mA. After transfer, the PVDF membrane was removed and blocked with 5% skim milk PBST solution at room temperature for 1 hour. After blocking, the corresponding primary antibody incubation solution (primary antibody dilution ratio 1:1000, 5% BSA + PBST) was added, and the membrane was incubated overnight at 4°C with gentle shaking. After incubation, the membrane was washed four times with PBST for 5 minutes each time. Secondary antibody incubation solution (secondary antibody dilution ratio 1:2000, 5% skim milk + PBST) was added, and the membrane was incubated at room temperature for 1 hour. The membrane was washed four times with PBST for 5 minutes each time. Development and exposure were performed according to the method provided by the ECL chemiluminescence detection kit. The development results were analyzed using ImagJ software to calculate the grayscale molecular weight, inhibition rate, and _IC50 .

The inhibition rate is calculated as follows:

Inhibition rate (%) = 100 - (gray value of the treatment group - gray value of the blank group) / (gray value of IL-6 monotherapy group - gray value of the blank group) × 100%, the results are shown in Table 5:

Table 5. Compounds inhibiting pSTAT3 activity in BEAS-2B cells

The results showed that compound 4 did not inhibit pSTAT3 levels in BEAS-2B cells, demonstrating high selectivity.

Test Example 6: Inhibitory effect of the compound of the present invention on human hERG ion channels

The effects of compounds on hERG ion channels were tested on HEK293 cells stably expressing hERG ion channels. Cells were transferred to a perfusion tank and perfused with extracellular fluid. The intracellular fluid (mM) consisted of: K aspartate, 130; MgCl2, 5; EGTA, 5; HEPES, 10; Tris-ATP, 4; pH 7.2 (titrated with KOH). Intracellular fluid was stored in batches at -80°C and thawed on the day of the experiment. Electrodes were fabricated using PC-10 (Narishige). Whole-cell patch-clamp recording was performed, with noise filtered at one-fifth of the sampling frequency. Cells were clamped at -80 mV, then depolarized to 40 mV with a 4-second square wave, followed by hyperpolarization to -40 mV with a 2-second square wave to obtain the hERG tail current. This procedure was repeated every 20 seconds. The maximum current induced by the second square wave was measured, and after stabilization, the test compounds were perfused using a gravity-fed perfusion system, with at least one cell tested for each concentration. After the reaction stabilized, the change in current before and after the compound was used was compared to calculate the blocking effect. Data were collected and analyzed using a pCLAMP 10 (Molecular Devices), and the results were reviewed by the experimenter. The inhibitory effect of the compound of this invention on human hERG ion channels is shown in the table below:

Table 6. Inhibition rate of the compounds of this invention on human hERG ion channels

As shown in Table 6, the representative compounds of this invention do not have a significant inhibitory effect on human hERG ion channels at high concentrations, which can reflect to some extent that the compounds of this invention have low cardiotoxicity and have positive significance for drug safety assessment.

Test Example 7: Pharmacokinetic Characteristics of Rats

Main reagents and materials:

SPF-grade male SD rats (n=3 per group) were administered compound 4 via a single gavage dose of 3.7 mg/kg. Blood samples were collected at specified time points, plasma was separated, and stored at -80°C for later use. The plasma samples were thawed at room temperature, vortexed (2500 rpm, 1 min), and 30.0 μL of plasma sample was transferred to a 1.5 mL centrifuge tube. 6.00 μL of internal standard (ramelteinamide, 500.0 ng/mL) was added, followed by 1000 μL of methanol. The tube was vortexed (2500 rpm, 1 min) and centrifuged (17000 g, 4°C) for 10 min. 180 μL of the supernatant was transferred to a 96-well plate, sealed, and analyzed by LC-MS/MS with an injection volume of 1.00 μL. Compounds 4 and 52 were also detected simultaneously. Experimental data are shown in the table below.

Table 7. In vivo pharmacokinetic parameters (po) of the compounds

"-" indicates that the corresponding value cannot be calculated.

As shown in Table 7, compound 4 of the present invention rapidly decomposes into active metabolite 52 after oral administration, exhibiting good plasma exposure and demonstrating excellent oral administration potential.

Test Example 8: In vitro metabolic stability

Main reagents and materials:

Incubation system:

Experimental methods:

1 μM of the test sample and the positive control testosterone were incubated with microsomes in the presence of NADPH for 60 min, and a negative control group was set up (the test sample was incubated with microsomes in the absence of any coenzyme for 60 min). Samples were taken at 0 min and 60 min, and 300 μL of pre-cooled methanol solution containing the internal standard (rameltein: 1.000 ng/mL) was added to the sample. The sample was vortexed (2500 rpm, 1 min), centrifuged (4700 rpm, 4℃) for 10 min, and 50.0 μL of the supernatant was added to 200 μL of pure water in a 96-well plate. The plate was vortexed (1000 rpm, 10 min), sealed, and analyzed by LC-MS/MS. The parent remaining amount of the test substance or probe substrate was detected by LC-MS/MS. The metabolic stability of the test substance was expressed as the percentage of the parent remaining amount of the test substance at each time point relative to the parent amount before incubation (0 min). The data were calculated according to the following formula: Parent remaining percentage (% of 0 min) = T _x parent amount / T ₀ parent amount × 100; T _x : any incubation time point; T ₀ : 0-minute incubation time point. The test results are shown in Table 8 below.

Table 8 shows the in vitro metabolic stability results of the compounds.

As can be seen from the data in Table 8, the compounds of the present invention have good in vitro metabolic stability and little species difference.

Claims (30)

Oxalide compounds having the structure shown in formula (I), or their pharmaceutically acceptable salts, isotope derivatives, solvates, or their stereoisomers, geometric isomers, tautomers, or their prodrug molecules or metabolites:
in, It can be a single bond or a double bond; q and t are each independently selected from 0, 1 or 2, and q and t are not both selected from 0 at the same time; p is selected from 1 or 2; Z is selected from S, O, -S(=O)-, -S(=O) 2- , -S(=O)=NH, CHR 10 , CR 9R 10 or NR 10 ; Y is selected from CHR 9 , CR 9 R 10 or NR 9 ; R1 is selected from C6-12 aryl, 5-12 heteroaryl, 4-12 heterocyclic, 4-12 cycloalkenyl, C3 - C12 cycloalkyl, C1-4 alkylene C6-12 aryl, or C1-4 alkenyl C6-12 aryl substituted with one or more substituents, wherein the substituent is selected from Rg , CR1aR2aP ( O ) OR1bOR2b , CR1aR2aP (O) OR1bNHR2b , CR1aR2aP ( O)( OR1b )(NH(AA)C(O) OR1c), CR1aR2aP(O)(NHR2c)(NH(AA)C(O)OR1c ) , CR1aR2aP ( O ) ( NH ( AA)C(O) OR1c ... 1c ), P(O)OR 1b OR 2b , P(O)(OR 1b )(NH(AA)C(O)OR 1c ), P(O)(NHR 2c )(NH(AA)C(O)OR 1c ) or P(O)(NH(AA)C(O)OR 1c )(NH(AA)C(O)OR 1c ); R1a and R2a are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, C1-4 alkyl, C1-4 haloalkyl or C1-4 hydroxyalkyl; or, R1a and R2a form a C3 - C6 cycloalkyl or a 3-6 membered heterocyclic group with the atom to which they are attached. R 1b and R 2b are each independently selected from -R 1aa , -R 1aa -OC(O)-R 1ab , -R 1aa -C(O)OR 1ab , -R 1aa -OC(O)OR 1ab , -R 1aa -OR 1ab , -R 1aa -SC(O)OR 1ab , -R 1aa -SC(O)-R 1ab , -R 1aa -OC(O)NH-R 1ab , -R 1aa -OC(O)NR 1ab R 1ac , -R 1aa -OC(O)-R 1ab -OR 1ac , -R 1aa -OC(O)OR 1ab -OR 1ac , -R 1aa -SC(O)OR 1ab -OR 1ac , -R 1aa -SC(O)-R 1ab -OR 1ac or -R 1aa -OC(O)-(NH(AA)C(O)OR 1c ); R1aa , R1ab , and R1ac are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, C1-4 alkyl, C1-4 haloalkyl, C1-4 hydroxyalkyl, 5-7 membered heterocyclic, C6-12 aryl, 5-12 membered heteroaryl, C1-4 alkylene C6-12 aryl, or C1-4 alkylene C2-10 heteroaryl. The 5-7 membered heterocyclic, C6-12 aryl, 5-12 membered heteroaryl , C1-4 alkylene C6-12 aryl , or C1-4 alkylene C2-10 heteroaryl may optionally be further substituted by one or more Rh or C(O) ORh . R1c and R2c are each independently selected from hydrogen, deuterium, or C1-6 alkyl, 5-7 heterocyclic, C6-12 aryl, 5-12 heteroaryl, C1-4 alkylene C6-12 aryl or C1-4 alkylene C2-10 heteroaryl groups, which may be optionally substituted with one or more substituents, wherein the substituents are selected from deuterium, halogen, cyano, hydroxyl, amino, C1-4 alkyl, C1-4 alkoxy, C1-4 haloalkyl, and C1-4 haloalkoxy groups ; AA is selected from residues of natural or non-natural amino acids, wherein the residues of the natural or non-natural amino acids are in the α or β configuration; R2 is selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, or C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, C1-6 alkylsulfonyl, C1-6 alkylthio, C1-6 alkylamine, or 4-10 membered heterocyclic groups, wherein the substituent is selected from deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, C1-4 alkyl, C1-4 alkoxy, C1-4 haloalkyl , or C1-4 haloalkoxy. R3 and R4 are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, or optionally substituted with one or more RY C1-6 alkyl, C1-6 alkoxy, C1-6 alkylacyl, C2-6 alkenyl, C2-6 alkynyl , C3-6 cycloalkyl, C1-6 alkylsulfonyl, C1-6 alkylthio, C1-6 alkylamine, 4-10 membered heterocyclic, C6-12 aryl, 5-12 membered heteroaryl, C1-6 alkylene C6-12 aryl, or C1-6 alkylene C2-10 heteroaryl; Alternatively, R3 and R4 and the atoms they are attached to together form a C3 - C6 cycloalkyl or a 3-6 membered heterocyclic group, which may optionally be substituted by one or more R Y1 or R Y2 ; RY1 and RY2 are each independently selected from deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, C1-4 alkyl, C1-4 alkoxy, C1-4 haloalkyl or C1-4 haloalkoxy; R5 and R6 are each independently selected from hydrogen, deuterium, C1-6 alkyl, or C1-6 haloalkyl; R7 and R8 are each independently selected from C1-6 alkyl, 5-12 heterocyclic, 5-12 cycloalkyl, C6-12 aryl, 5-12 heteroaryl, C1-4 alkylene C6-12 aryl or C1-4 alkylene C2-10 heteroaryl, which may be optionally substituted by one or more R2 . Alternatively, R7 and R8 and the atoms they are attached to together form 4-14 membered heterocyclic groups and 5-12 membered heteroaryl groups, which may optionally be further substituted by one or more R2 groups. R9 and R10 are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, or optionally substituted with one or more RY C1-6 alkyl, C1-6 alkoxy, C1-6 alkylacyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, C1-6 alkylsulfonyl, C1-6 alkylthio, C1-6 alkylamine, 4-10 membered heterocyclic, C6-12 aryl, 5-12 membered heteroaryl, C1-6 alkylene C6-12 aryl, or C1-6 alkylene C2-10 heteroaryl; Alternatively, R9 and R10 and the atoms they are attached to together form a C3 - C6 cycloalkyl or a 3-6 membered heterocyclic group, which may optionally be substituted by one or more R Y1 or R Y2 ; R Z  and RY are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, imino, or optionally substituted with one or more RQ , and may be C 1-6 alkyl, C  1-6  alkoxy, C 2-6  alkenyl, C2-6 alkynyl, C 3-6  cycloalkyl, C 1-6  alkylsulfonyl, C1-6 alkylthio, C 1-6  alkylamine, 4-10 membered heterocyclic, C 6-12  aryl, 5-12 membered heteroaryl, -NR a R b , -OR a , -C(O)R a , -C(O)ORa, -NHC(O)OR a  , -NR b C(O)OR a , -NR  aC(O)NR b Rc, -C(O)NRaRb , -S ( O)R c  , -S(O)  2  R c  -S(O) = NHR c , -S(O)NR c R d or -S(O) 2 NR c R d ; R and Q are selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, imino, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, C2-6 alkenyl, C2-6 alkynyl , C3-6 cycloalkyl, C1-6 alkylsulfonyl, C1-6 alkylthio, C1-6 alkylamine, 4-10 membered heterocyclic, C6-12 aryl, 5-12 membered heteroaryl, -NR e R f , -OR e , -C(O)R e , -C (O)OR e , -NHC(O)OR e , -NHC(O)R e , -NR e C(O)OR f , -NR e C(O)R f , -NR g C(O)NR e R f , -C(O)NR e R f , -S(O)R e -S(O) 2 Re , -S(O) = NHR Re , -S(O)NR Re R f or -S(O) 2 NR Re R f ; Ra , Rb , Rc , Rd , Re , Rf , Rg , and Rh are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, imino, or optionally substituted with one or more substituents, namely C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 ynyl, C3-6 cycloalkyl, C1-6 alkylsulfonyl, C1-6 alkylthio, C1-6 alkylamine, 4-10 membered heterocyclic, C6-12 aryl, 5-12 membered heteroaryl, C1-4 alkyleneC6-12 aryl, or C1-4 alkyleneC2-10 heteroaryl , wherein the substituents are selected from deuterium, halogen, cyano, hydroxyl, amino, C1-4 alkyl, C1-4 alkoxy, C1-4 haloalkyl, C2-6 alkylyl, C3-6 cycloalkyl, C4-6 alkylsulfonyl, C1-6 alkylthio, C1-6 alkylamine, C3-6 cycloalkyl, C4-6 alkylyl ... 1-4 haloalkoxy, phenyl, or benzyl. The compound of claim 1, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, is characterized in that the compound has the structure shown in formula (II):
The definitions of p, t, q, Z, Y, R1 , R2 , R3 , R4 , R5 , R6 , R7 , and R8 are as described in claim 1. The compound as claimed in claim 1 or 2, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, is characterized in that the compound has a structure as shown in formula (III), (IV), (V), (VI), (VII), or (VIII):
The definitions of p, Z, Y, R1 , R2 , R3 , R4 , R5 , R6 , R7 , and R8 are as described in claim 1. The compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, is characterized in that the compound has a structure as shown in formula (IIIa), (IVa), (Va), (VIa), (VIIa), (VIIIa), (IIIb), (IVb), (Vb), (VIb), (VIIb), or (VIIIb):

The definitions of Z, Y, ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , ^R6 , ^R7 , and ^R8 are as described in claim 1. The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, is characterized in that the compound has a structure as shown in formula (IIIa-1), (IIIa-2), (IIIa-3), (IIIa-4), (IIIa-5), (IVa-1), (IVa-2), (IVa-3), (IVa-4), (IVa-5), (IVa-6), (IVa-7), (IVa-8), (IVa-9), (IVa-10), (Va-1), (Via-1), or (IVb-1):

The definitions of ^R1 , ^R2 , ^R5 , ^R6 , ^R7 , ^R8 , ^R9 , ^R10 , ^RY1 , and ^RY2 are as described in claim 1. The compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, characterized in that ^R2 is selected from hydrogen or hydroxyl. The compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, is characterized in that ^R3 and ^R4 are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, or optionally substituted with one or more ^RYs of the following _C1-6 alkyl, _C1-6 alkoxy, _C1-6 alkylacyl, C3-6 cycloalkyl, _4-10 heterocyclic, _C6-12 aryl, _5-12 heteroaryl, C1-6 _{alkyleneC6-12} aryl, or _{C1-6 alkyleneC2-10} heteroaryl, wherein each ^RY is independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, or imino. The compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, is characterized in that ^R3 and ^R4 and the atoms to which they are attached together form a _C3 - _C6 cycloalkyl group, wherein the C3 _{- C6} cycloalkyl group may optionally be substituted by one or more ^RY1 or ^RY2 groups; wherein each ^RY1 and ^RY2 is independently selected from deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, _C1-4 alkyl, _C1-4 alkoxy, _C1-4 haloalkyl or _C1-4 haloalkoxy. The compound of claim 7, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or its stereoisomer, geometric isomer, tautomer, or its prodrug molecule or metabolite, is characterized in that ^R3 and ^R4 are each independently selected from hydrogen, deuterium, hydroxyl, _C1-6 alkyl, C1-6 haloalkyl _{, C1-6 alkylene} , _C2-10 heteroaryl, or _C1-6 alkylacyl. The compound as claimed in any one of claims 1 to 9, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, characterized in that ^R9 and ^R10 are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo _{, or} optionally substituted with one or more ^{Ry groups} _, namely _C1-6 alkyl, _C1-6 alkoxy, _C1-6 alkylthio, _C1-6 alkylamine, _4-10 membered heterocyclic, _C6-12 aryl, _5-12 membered heteroaryl, _{C1-6 alkyleneC6-12} aryl, or _{C1-6 alkyleneC2-10} heteroaryl; ^Y is independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, _C1-6 alkyl or imino. The compound as claimed in any one of claims 1 to 9, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, characterized in that ^R9 and ^R10 are each independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo _{, or} optionally substituted with one or more ^{Ry groups} _, namely _C1-6 alkyl, _C1-6 alkoxy, _C1-6 alkylthio, _C1-6 alkylamine, _4-10 membered heterocyclic, _C6-12 aryl, _5-12 membered heteroaryl, _{C1-6 alkyleneC6-12} aryl, or _{C1-6 alkyleneC2-10} heteroaryl; ^Y is independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, or imino. The compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, is characterized in that ^R9 and ^R10 and the atoms to which they are attached together form a C3 _{- C6} cycloalkyl or a 3-6 membered heterocyclic group, wherein the C3 _{- C6} cycloalkyl or 3-6 membered heterocyclic group may optionally be substituted by one or more ^RY1 or ^RY2 , wherein each ^RY1 or ^RY2 is independently selected from deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, _C1-4 alkyl, _C1-4 alkoxy, _C1-4 haloalkyl or _C1-4 haloalkoxy. The compound of claim 12, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, is characterized in that ^R9 , ^R10 and the atoms to which they are attached together form a cyclopropyl group, wherein the cyclopropyl group may optionally be substituted by one or more ^RY1 or RY2 ^groups , wherein each ^RY1 or ^{RY2 group} is independently selected from deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, _C1-4 alkyl, _C1-4 alkoxy, _C1-4 haloalkyl or _C1-4 haloalkoxy. The compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, characterized in that ^R5 and ^R6 are each independently selected from hydrogen or _C1-6 alkyl. The compound as claimed in any one of claims 1 to 14, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, characterized in that ^R1 is selected from _C6-12 aryl or 5-12 heteroaryl groups substituted with one or more substituents, wherein the substituent is selected from ^Rg , ^CR1aR2aP (O) ^{OR1bOR2b , CR1aR2aP(O)OR1bNHR2b , or CR1aR2aP (} O) ^{( OR1b} )(NH(AA) ^C (O) ^OR1c ). The compound of claim 15, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, characterized in that ^R1 is selected from compounds substituted with one or more substituents. The substituent group is selected from R ^g , CR ^1a R ^2a P(O)OR ^1b OR ^2b , CR ^1a R ^2a P(O)OR ^1b NHR ^2b or CR ^1a R ^2a P(O)(OR ^1b )(NH(AA)C(O)OR ^1c ). The compound of claim 16, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, characterized in that R ^1a or R ^2a is each independently selected from hydrogen, halogen or oxo, wherein the halogen is preferably F or Cl. The compound according to any one of claims 1-17, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, characterized in that CR ^1a R ^2a P(O)OR ^1b OR ^2b , CR ^1a R ^2a P(O)OR ^1b NHR ^2b , or CR ^1a R ^2a P(O)(OR ^1b )(NH(AA)C(O)OR ^1c ) is selected from...
The compound according to any one of claims 1-17, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, characterized in that CR ^1a R ^2a P(O)OR ^1b OR ^2b , CR ^1a R ^2a P(O)OR ^1b NHR ^2b , or CR ^1a R ^2a P(O)(OR ^1b )(NH(AA)C(O)OR ^1c ) is selected from...
The compound according to any one of claims 1 to 19, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or its stereoisomer, geometric isomer, tautomer, or its prodrug molecule or metabolite, characterized in that ^R7 and ^R8 are each independently selected from _C1-6 alkyl, 5-12 heterocyclic, 5-12 cycloalkyl, _C6-12 aryl, 5-12 heteroaryl, _C1-4 alkylene _C6-12 aryl or _C1-4 alkylene _C2-10 heteroaryl, which may be optionally substituted by one or more ^R2 . The R and ^Z are independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, oxo, or C1-6 _alkyl , _C3-6 cycloalkyl, 4-10 heterocyclic, _C6-12 aryl, or 5-12 heteroaryl groups that may be optionally substituted with one or more R and ^Q. The compound as claimed in any one of claims 1 to 19, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, characterized in that ^R7 and ^R8 and the atoms to which they are attached together form a 4-14 membered heterocyclic group, wherein the 4-14 membered heterocyclic group may optionally be substituted by one or more ^{R2 groups} ; The R and ^Z are independently selected from hydrogen, deuterium, halogen, cyano, hydroxyl, oxo, or C1-6 _alkyl , _C3-6 cycloalkyl, 4-10 heterocyclic, _C6-12 aryl, or 5-12 heteroaryl groups that may be optionally substituted with one or more R and ^Q. The compound of claim 20 or 21, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or its stereoisomer, geometric isomer, tautomer, or its prodrug molecule or metabolite, is characterized in that ^RQ is selected from hydrogen, deuterium, halogen, cyano, hydroxyl, amino, mercapto, oxo, imino, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, C1-6 haloalkoxy, _C1-6 alkylamino, 4-10 membered heterocyclic, _C6-12 aryl, _5-12 membered heteroaryl, ^-NReRf , -C(O) ^Re , -NHC(O) ^Re , or ^-NReC (O) ^Rf , wherein ^Re and ^Rf are each independently ^selected from hydrogen, deuterium, or _C1-6 alkyl. The compound as claimed in any one of claims 1 to 22, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, characterized in that, the Selected from:
The compound according to any one of claims 1 to 23, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule or metabolite thereof, is characterized in that the compound is selected from the following structural compounds:






A pharmaceutical composition, characterized in that the pharmaceutical composition contains a therapeutically effective amount of the compound of any one of claims 1-24 or its pharmaceutically acceptable salt, isotope derivative, solvate, or its stereoisomer, geometric isomer, tautomer, or its prodrug molecule or metabolite. The use of any one of the compounds of claims 1-24, or pharmaceutically acceptable salts, isotope derivatives, solvates, or stereoisomers, geometric isomers, tautomers, or prodrug molecules, metabolites, or pharmaceutical compositions of claim 25 in the preparation of a medicament for treating STAT6-mediated diseases or conditions and related diseases or conditions, wherein the STAT6-mediated diseases or conditions are tumors or type II inflammation-related diseases, and the type II inflammation-related diseases are selected from atopic dermatitis, bullous pemphigoid, nodular prurigo, chronic spontaneous urticaria, eosinophilic esophagitis, food allergy, and chronic rhinosinusitis with nasal polyps (CRS). The tumor is selected from lymphoma, solitary fibroma, colon cancer, esophageal cancer, breast cancer, bile duct cancer, liver cancer, kidney cancer, gastric cancer, head and neck squamous cell carcinoma, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), acute B-lymphocytic leukemia, bladder cancer, pancreatic cancer, osteosarcoma, myeloma, glioma, ovarian cancer, or skin cancer. A method of treating and/or preventing a disease, comprising administering to a subject a therapeutically effective amount of any one of claims 1-24, or a pharmaceutically acceptable salt, isotope derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule, metabolite thereof, or pharmaceutical composition of claim 25 thereof. The method according to claim 27, characterized in that the disease being treated and/or prevented is a STAT6-mediated disease, the disease being a tumor or a type II inflammation-related disease, wherein the type II inflammation-related disease is selected from atopic dermatitis, bullous pemphigoid, nodular prurigo, chronic spontaneous urticaria, eosinophilic esophagitis, food allergy, chronic rhinosinusitis with nasal polyps (CRSwNP), chronic rhinosinusitis without nasal polyps (CRSsNP), and nonsteroidal anti-inflammatory drug-induced respiratory disease (NSAID-Aggravated Respiratory Disease). D-ERD/AERD), allergic rhinitis, asthma, chronic obstructive pulmonary disease (COPD), eosinophilic granulomatous polyangiitis (EGPA), or allergic bronchopulmonary aspergillosis; the tumor is selected from lymphoma, solitary fibrous tumor, colon cancer, esophageal cancer, breast cancer, bile duct cancer, liver cancer, kidney cancer, gastric cancer, head and neck squamous cell carcinoma, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), acute B-lymphoblastic leukemia, bladder cancer, pancreatic cancer, osteosarcoma, myeloma, glioma, ovarian cancer, or skin cancer. Intermediate compounds M1-1, M1-3, or M1, or their pharmaceutically acceptable salts, isotopic derivatives, solvates, or their stereoisomers, geometric isomers, tautomers, or prodrug molecules:
The definitions of ^R1a , ^R2a , ^R5 , R6, ^R7 , ^R8 , ^Rg , Z, and Y are as described in claim ¹ . Intermediate compound M3 or M4, or its pharmaceutically acceptable salt, isotopic derivative, solvate, or its stereoisomer, geometric isomer, tautomer, or prodrug molecule: