WO2024250985A1 - Toll-like receptor agonist heterocyclic derivative, preparation thereof, and use thereof - Google Patents

Abstract

The present invention provides a lipidated toll-like receptor agonist derivative, preparation thereof, and a use thereof. Specifically, the present invention provides a compound as shown in formula I, a preparation method therefor, and a use thereof as a TLR7 and/or TLR8 agonist. The compound can be used as an immunostimulant and an adjuvant.

Description

Toll-like receptor agonist heterocyclic derivatives and their preparation and application Technical Field

The present invention belongs to the field of chemical medicine, and in particular relates to pyridine [4,3-d] pyrimidine compounds and pyrimidopyridazinone compounds as TLR7/8 agonists, and preparation methods and applications thereof.

Background Art

Toll-like receptors (TLRs) are a class of structurally conserved proteins that form the first barrier in the innate immune response. By recognizing various conserved pathogen-associated molecular patterns (PAMPs), TLRs can recognize invasive microorganisms and endogenous molecules released after tissue damage or non-physiological cell death, and activate signaling cascades, leading to the production of pro-inflammatory cytokines. The inflammatory process is crucial to the occurrence and development of many diseases, such as type I diabetes, sepsis, cancer, viral infectious diseases, etc. Therefore, the strategy of manipulating the inflammatory response through small molecule TLRs modulators to treat related diseases is promising.

There are 10 known members of the human TLRs family, which are type I transmembrane proteins characterized by a leucine-rich extracellular domain and a cytoplasmic tail containing a conserved Toll/interleukin (IL)-1 receptor (TIR) domain. In this family, TLR3, TLR7, TLR8, and TLR9 are located in the endosomal compartment.

Both TLR7 and TLR8 can recognize RNA molecules (ssRNA) from single-stranded RNA viruses. TLR7 is mainly expressed in plasmacytoid dendritic cells and B cells. TLR8 is mainly distributed in mDC, macrophages and monocytes, and is also expressed in T cells. TLR7 stimulation mainly induces the production of type I interferons, including interferon-α (IFN-α), and causes the transcription of interferon-stimulated genes (ISGs). Interferon α is one of the main drugs for the treatment of chronic hepatitis B or C. TLR8 is mainly distributed in mDC, macrophages and monocytes, and is also expressed in T cells. TLR8 activation mainly produces a pro-inflammatory response, stimulating immune cells to secrete pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α) and IL-6. There is overlap in the distribution and downstream signaling pathways of TLR7/8, so there are functional similarities. After TLR7/8 is activated, it can exert direct antiviral activity through type I interferon response, and can also regulate innate immunity through pro-inflammatory response to promote the activation of NK and NKT cells, as well as induce adaptive immunity, improve antigen presentation and activate dendritic cells, thereby enhancing T cell response and promoting the differentiation of antibody-producing B cells. The development of TLR7/8 agonists has great clinical value in both antiviral and anti-tumor therapy, and can also be used in antibody-drug conjugates and vaccine adjuvants.

Vaccination has been the most effective medical intervention to reduce the incidence, severity and mortality of infectious diseases in the past two centuries. Vaccination causes the immune system to recognize the antigenic components of live pathogens and produce a strong humoral immune response, including the secretion of high levels of protective antibodies through antigen-specific memory B cells and the establishment of long-term immune memory. The immune effect of vaccines varies greatly depending on the vaccine components, including live attenuated vaccines, inactivated vaccines, subunit vaccines, monovalent/multivalent vaccines, protein vaccines or non-protein vaccines (such as polysaccharides). The immune effect of vaccines depends not only on the antigenic components, but also on the adjuvant components. Adjuvants can regulate the type of immune response and increase the immunogenicity of certain antigens that are not strongly immunogenic. Adjuvants can also enhance the immunogenicity of antigens in specific populations (such as infants, the elderly), who often need to enhance their immune response. In some cases, adjuvants can play a role in increasing the scope of protection. Thanks to all the above effects, vaccine adjuvants can often play a role in saving antigens, which is very useful when large quantities of vaccines are needed, such as during influenza pandemics. Adjuvant enhancement The mechanism of immune response has not been fully elucidated, and different adjuvants have different effects. Most adjuvants work by activating the innate and adaptive immune systems to induce humoral and cell-mediated effector mechanisms, involving antigen uptake by antigen-presenting cells, antigen processing, and antigen presentation, thereby inducing long-term memory of B cells and T cells.

In order for the immune system to fight against the invasion of foreign pathogens, it must first identify and distinguish between foreign antigens and self-antigens. This recognition process is completed by innate immune cells, namely dendritic cells or macrophages, through pattern recognition receptors (PRRs). PRRs have multiple receptor families, including Toll-like receptors (TLRs), NOD-like receptors (NLRs), C-type lectin receptors (CLRs), RIG-I-like receptors (RLRs) and cytoplasmic DNA/RNA receptors. These receptors can recognize lipopolysaccharides (LPS), lipoproteins, flagellin and some specific nucleotide sequences. Once the pattern recognition receptors are stimulated by the above substances, they will start the expression of inflammation-related genes and activate the immune system to respond to infection. Among them, TLRs can sense a variety of microbial components, are widely expressed in a variety of immune cells, and play an important role in the host's innate and adaptive immune responses to pathogen infections. Activating TLRs has the potential to enhance the immune effect of vaccines. Many TLRs agonists have been clinically studied or approved for use as vaccine adjuvants. The most successful of these are adjuvants that can activate TLR4, such as AS01, AS02, and AS04, all of which contain MPLA, a ligand for TLR4. The hepatitis B vaccine Heplisav-B, which was launched in 2017, uses the TLR9 ligand CpG 1018, an oligonucleotide with high chemical stability and adjuvant capacity that can induce a th1-type immune response. CpG1018 in Heplisav-B can improve vaccine efficacy, requiring only two doses of the vaccine, while traditional hepatitis B vaccines require three doses for optimal protection.

TLR7 and TLR8 agonists have also received special attention as vaccine adjuvants because they are expressed in plasmacytoid DC and myeloid DC, the major subsets of human dendritic cells (DC), respectively (5), and by human B cells, and are extremely important for antigen processing and presentation and antibody secretion. In vitro studies have shown that TLR7/8 agonists can enhance the maturation, expression of co-stimulatory markers and cytokine secretion of human DCs, and in vivo studies have also shown that TLR7/8 agonists can enhance the cellular and humoral adaptive responses to antigens in experimental animals. The most important representatives of TLR7/8 agonists are some synthetic small molecules, named imiquimod (R837) and resiquimod (R848). Imiquimod is currently approved and licensed for the treatment of genital warts, superficial basal cell carcinoma and actinic keratosis, while resiquimod has been studied for antiviral and anticancer treatment. In addition, they have also been widely used in vaccine adjuvant research. However, these small molecules have been shown to have some inherent limitations. In particular, they can diffuse away from the site of administration and thus away from the antigen, thereby reducing efficacy and inducing systemic side effects. Therefore, it has been shown that directly conjugating these molecules to aluminum adjuvants can improve vaccine efficacy. Some previous studies have directly conjugated imidazoquinolines to HIV-1 Gag protein or whole inactivated influenza virus to increase Th1 responses and the number of antigen-specific T cells. In addition, conjugation to synthetic polymer scaffolds, lipid-polymer amphiphilic molecules, polyethylene glycol (PEG), nanogels, gelatin, and various other synthetic polymers significantly increased the delivery of imidazoquinolines and promoted the maturation of DCs and antigen-specific T cells. In addition, previous studies using imidazoquinolines in combination with one or more other TLR agonists (such as MPLA [TLR4] and MPLA+CpG ODN [TLR4 and TLR9]) have shown that this combination can enhance the innate immune response, significantly produce antigen-specific neutralizing antibodies, and improve Th1 responses. All of these innovative aspects highlight the excellent potential of TLR7/8 agonists as candidate adjuvants.

There are currently several related TLR7/8 agonist patent applications, but there is still a need to continue to develop highly active, safer and highly therapeutically effective TLR7/8 agonists or vaccine adjuvants.

Summary of the invention

The object of the present invention is to provide a highly active, safer and highly therapeutically effective TLR7/8 agonist or vaccine adjuvant.

The first aspect of the present invention provides a compound having a structure as shown in the following formula (I), or a stereoisomer, enantiomer, or a pharmaceutically acceptable salt thereof:

in:

_Y1 is hydrogen or a lipid structure fragment, and the lipid structure fragment has a structure as shown in the following formula:

p, q are each independently selected from the following group: 0, 1, 2, 3, 4, 5, 6, 7 or 8;

m1, m2 and r are each independently selected from the following group: 0, 1, 2, 3, 4, 5 or 6;

p ₁₀₁ is selected from the following group: 0 or 1;

Q ₁ and Q ₂ are each independently selected from the following group: O, -NR ₃₀₃ - or -CR ₃₀₄ R ₃₀₅ -;

R ₃₀₃ is selected from the group consisting of H, substituted or unsubstituted C _1-6 alkyl, and halogenated C _1-6 alkyl;

R ₃₀₄ and R ₃₀₅ are the same or different, and are each independently selected from the group consisting of hydrogen, halogen, cyano, substituted or unsubstituted C _1-6 alkyl, substituted or unsubstituted C _1-6 alkoxy, substituted or unsubstituted C _3-6 cycloalkyl, substituted or unsubstituted 4-6 membered heterocycloalkyl;

Linker is a divalent linking group;

R ₁₀₁ and R ₁₀₂ are each independently selected from the group consisting of a substituted or unsubstituted C _4-20 straight chain or branched chain alkyl group, a substituted or unsubstituted C _4-20 straight chain or branched chain alkenyl group, and a substituted or unsubstituted C _4-20 straight chain or branched chain alkynyl group;

R ₂₀₁ is a GalNAc target head portion and has a structure as shown in the following formula:

wherein L ₂₀₁ , L ₂₀₂ and L ₂₀₃ are each independently a divalent linking group, and R ₂₀₃ , R ₂₀₄ and R ₂₀₅ are each independently a substituted or unsubstituted saccharide group, preferably a group formed by a substituted or unsubstituted N-acetylgalactosamine molecule;

The _L2 is a group selected from the group consisting of -(CHR) _m1- , optionally substituted -(CHR) _m1 -C(O)NH-( _CH2CH2O ) _n3- ( _CHR ) _m2- , optionally substituted - _{( CH2CH2O} ) _n3- (CHR) _m2- , optionally substituted -(CHR) _m1 -C(O)NH-( _CH2CH2O ) _n3- (CHR) _m2 -C(O)-, optionally substituted _- (CHR) _m1- ( _CH2CH2O ) _n3- (CHR) _m2- , -(CHR) _m1 -C( _O )-, optionally substituted -(CHR) _m1 -C(O) _-L4- NH( _CH2CH2O ) _n3- (CHR) _m2- , optionally substituted -(CHR) _m1 -C(O)NH-(CH2CH2O)n3-(CHR) _{m2-, 5-10} membered aromatic heterocyclic ring -( _{CH2CH 2} O) _n3 -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C(O)-L ₄ -NH-(CH ₂ CH ₂ O) _n3 -L ₄ -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C ₃ -C ₉ cycloalkyl-(CHR) _m2 -, optionally substituted -(CHR) _m1 -C ₃ -C ₉ heterocycloalkyl-(CHR) _m2 -;

Wherein, each of the R groups is independently selected from the following group: H, D, (CH ₂ ) _n4 NHC(O)NH ₂ , C ₁ -C ₆ alkyl;

m1, m2 and n3 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

The substitution refers to that one or more hydrogen atoms on the group are replaced by a substituent selected from the group consisting of C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _3-8 cycloalkyl, 3- to 12-membered heterocyclyl, C _3-8 aryl, 5- to 7-membered heteroaryl, halogen, Hydroxyl, carboxyl (-COOH), C _1-8 aldehyde, C _2-10 acyl, C _2-10 ester, amino, C _1-8 alkoxy, C _1-10 sulfonyl; or two substituents located on adjacent ring atoms can form a group selected from the following group together with the connected ring atoms: a 5-7 membered carbon ring or heterocyclic ring, a benzene ring, or a 5-7 membered heteroaromatic ring;

D is a toxin having a structure selected from the group consisting of Formula Ia, Formula Ib, Formula Ic or Formula Id, and the toxin is covalently linked to a divalent linker -LY- via an N, O or S atom in the molecule:

in:

L ₁₁ is selected from the group consisting of -O-, -NH-, -S-, -S(=O)- or -S(=O) ₂ -;

R ₁ is selected from the following group: H, C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _3-12 cycloalkyl or 4-12 membered heterocycloalkyl; wherein said R ₁ may be further substituted by one or more R ^1a substituents, and said R ^1a is selected from the following group: hydrogen, halogen, hydroxyl, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _3-6 cycloalkyl, -NR ^a1 R ^a2 , -NHC(＝O)-R ^a3 , 5-6 membered heteroaryl substituted by one or more R ^a4 , -OC(＝O)R ^a5 , -C(＝O)R ^a5 , -OC(＝O)OR ^a5 , -C(＝O)OR ^a5 ;

Said ^Ra1 , ^Ra2 , ^Ra3 or ^Ra4 is selected from the following group: _C1-6 alkyl, _C1-6 haloalkyl, _C3-6 cycloalkyl;

The ^Ra5 is selected from the following group: _C1-24 alkyl, halogenated _C1-24 alkyl, _C1-24 heteroalkyl having 1 to 10 heteroatoms, wherein the heteroatoms are selected from one or more of NH, N, O and S;

The _R2 and _R3 are each independently selected from the group consisting of hydrogen, halogen, cyano, _C1-6 alkyl, _C1-6 alkoxy, _C3-6 cycloalkyl, _4-6 membered heterocycloalkyl, 5-8 membered heterocyclic aryl, or 5-8 membered aryl; wherein the _R2 may be further substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, or amino;

m is 0, 1, 2, 3, 4, 5, 6, 7 or 8;

B is absent, or B is selected from the group consisting of C _3-12 cycloalkyl, 4-12 membered heterocycloalkyl, C _6-12 aryl, 5-12 membered heteroaryl, or wherein each A is independently selected from C, CH or N, and R ₆ and R ₇ together with their adjacent carbon atoms form a C _4-7 cycloalkylene group or a C _4-7 heterocycloalkylene group, and one or more methylene groups in the C _4-7 cycloalkylene group or the 4-7 membered heterocycloalkylene group may be independently replaced by a carbonyl group or S(═O) ₂ ; in the 4-7 membered heterocycloalkylene group, the heteroatom is selected from N, O, S, and the number of the heteroatoms is 1 to 3;

L ₁₂ is selected from the group consisting of none, -(CR ^1b R ^1c ) _p -(NR ^1d ) _q -, -O-, -S-, -(CR ^1b R ^1c ) _p -C(═O)-, -(CR ^1b R ^1c ) _p -C(═O)NH-, -(CR ^1b R ^1c ) _p -NHC(═O)-, -S(═O)- or -S(═O) ₂ -; wherein R ^1b , R ^1c are selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, C _3-6 cycloalkyl, _4-6 membered heterocycloalkyl or C _1-6 haloalkyl, and R ^1d is selected from the group consisting of hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, 4-6 membered heterocycloalkyl, C _{1-6 haloalkyl or hydroxy-substituted C 1-6} alkyl; _1-6 alkyl, p is 0, 1, 2, 3, 4, 5 or 6, q is 0 or 1;

R ₄ is selected from the group consisting of hydrogen, halogen, cyano, amino, hydroxyl, C _1-12 alkyl, C _1-12 alkoxy, C _2-12 alkenyl, C _2-12 alkynyl, C _3-12 cycloalkyl, 4-12 membered heterocycloalkyl, C _6-12 aryl or 5-12 membered heteroaryl; and said R ₄ may be optionally substituted by one or more R ^1e substituents, wherein R ^1e is selected from the group consisting of hydrogen, halogen, hydroxyl, carboxylic acid, amino, C _1-6 alkyl, C _1-6 alkyl substituted with one or more R ^e1 , C _1-6 alkoxy, C _1-6 haloalkoxy, C _3-12 cycloalkyl, 4-12 heterocycloalkyl, C _6-12 aryl, 5-12 membered heteroaryl, -N(R ^e2 R ^e3 ), -C(═O)OR ^e2 , -C(═O)NH-R ^e2 , -S(═O) ₂ -R ^e2 ;

When L ₁₂ is absent and R ⁴ is H, B is selected from the group consisting of C _3-12 cycloalkyl, 4-12 membered heterocycloalkyl, C _6-12 aryl, 5-12 membered heteroaryl or wherein each A is independently selected from C, CH or N, and _R6 and _R7 together with their adjacent carbon atoms form a _C4-7 cycloalkylene or _C4-7 heterocycloalkylene;

R ^e1 is selected from the group consisting of halogen, hydroxyl;

R ^e2 and R ^e3 are selected from the group consisting of hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, C _1-6 haloalkyl or hydroxy-substituted C _1-6 alkyl;

R ₅ is selected from the group consisting of halogen, hydroxy, cyano, amino, C _1-6 alkyl, C _3-6 cycloalkyl, 4-6 membered heterocycloalkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 haloalkoxy, -C(=O)OR ^f , -C(=O)NH-R ^f , -S(=O) ₂ -R ^f ; wherein R ^f is selected from the group consisting of hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl or C _1-6 haloalkyl;

n is 1, 2, 3, 4, 5 or 6;

Wherein, each of the heterocyclic groups may be saturated or partially unsaturated (but not having an aromatic structure), and in the heterocyclic group, the heteroatom is selected from N, O, S, and the number of heteroatoms is 1, 2, 3 or 4 (preferably 1 or 2); in the heteroaryl group, the heteroatom is selected from N, O, S, and the number of heteroatoms is 1, 2 or 3.

In another preferred embodiment, the Linker has a structure selected from the following group:

C4-C20 alkyl,

wherein n and m are each independently selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

_n1 and _n2 are each independently selected from the following group: 0 or 1;

W is selected from the following group: a single bond, NH, substituted or unsubstituted C _1-16 alkylene, a substituted or unsubstituted 7-12 membered fused bicyclic ring, a substituted or unsubstituted 7-15 membered spirocyclic ring, a substituted or unsubstituted 5-15 membered bridged ring, -NH-substituted or unsubstituted 7-12 membered fused bicyclic ring, -NH-substituted or unsubstituted 7-15 membered spirocyclic ring, -NH-substituted or unsubstituted 5-15 membered bridged ring, -NH-substituted or unsubstituted 7-12 membered fused bicyclic ring-NH-, -NH-substituted or unsubstituted 7-15 membered spirocyclic ring-NH-, -NH-substituted or unsubstituted 5-15 membered bridged ring-NH-, -NH-substituted or unsubstituted C _1-16 alkylene-NH-; wherein the ring atoms of the fused bicyclic ring, spirocyclic ring and bridged ring may be optionally carbon atoms or heteroatoms;

The substitution refers to that one or more hydrogen atoms on the group are replaced by a substituent selected from the group consisting of C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _3-8 cycloalkyl, 3- to 12-membered heterocyclyl, C _6-10 aryl, 5- to 10 _- membered heteroaryl, halogen, hydroxyl, carboxyl (-COOH), C _1-8 aldehyde, C _2-10 acyl, C _2-10 ester, amino, C _1-8 alkoxy, C _1-10 sulfonyl; or two substituents located on adjacent ring atoms can form a group selected from the group consisting of a 5-7-membered carbocyclic or heterocyclic ring, a benzene ring, or a 5-7-membered heteroaromatic ring together with the connected ring atoms;

Or W has a structure selected from the group consisting of:

wherein s1, s2, s, s4, s5 and s6 are each independently selected from the following group: 0, 1 or 2;

s7 is selected from the group consisting of 1, 2, 3, 4, 5 or 6;

Each of M ₁ , M ₂ , M ₃ and M ₄ is independently selected from the group consisting of CHR, C(R)R, C(O), O, S, NR;

_M5 and _M6 are each independently selected from the group consisting of (CHR) _s6 , C(R)R, C(O), (CHR) _s6 -C(O), O, S, NR;

R is selected from the following group: H, halogen, methyl; or two R located on adjacent reducing atoms and the carbon atom to which they are connected together form a substituted or unsubstituted 4-8 membered carbocyclic or heterocyclic ring (including saturated, unsaturated or aromatic ring).

In another preferred embodiment, the W has a structure selected from the following group:

In another preferred embodiment, the Linker has a structure selected from the following group:

-L ₂ -L ₃ -L ₄ -Y-;

wherein _L2 is a group selected from the group consisting of -(CHR) _m1 -, optionally substituted -(CHR) _m1 -C(O)NH-(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -, optionally substituted -(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C(O)NH-(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -C(O)-, optionally substituted -(CHR) _m1 -(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -, -(CHR) _m1 -C(O)-, optionally substituted -(CHR) _m1 -C(O)-L4 _- NH(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C(O)NH-(CHR) _m1 -5-10 membered aromatic heterocyclic ring -(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C(O)-L ₄ -NH-(CH ₂ CH ₂ O) _n3 -L ₄ -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C ₃ -C ₉ cycloalkyl-(CHR) _m2 -, optionally substituted -(CHR) _m1 -C ₃ -C ₉ heterocycloalkyl-(CHR) _m2 -;

The _L4 is an optionally substituted group selected from the group consisting of: a chemical bond, wherein said ^Ra and ^Rb are each independently selected from the following group: hydrogen, optionally substituted _C1 - _C4 alkyl, optionally substituted _C1 - _C4 deuterated alkyl, _CH2COOH ;

The R ₁₀₁ is selected from the following group: hydrogen, halogen, cyano, amino, hydroxyl, carboxyl, ester, C _1-12 alkyl, C _1-12 alkoxy, C _2-12 alkenyl, C _2-12 alkynyl, C _3-12 cycloalkyl, _4-12 membered heterocycloalkyl, C _6-12 aryl or 5-12 membered heteroaryl, -L ₄ -C(O)-(CH ₂ CH ₂ O) _n3 -R, -C(O)-NR-(CHR) _m2 -NR-C(O)-(CH ₂ CH ₂ O) _n3 -R;

L ₃ is a peptide residue [Am] _p1 -: wherein p1 is 0, 1, 2, 3, 4, 5, 6, 7 or 8; Am is an amino acid residue;

Y is selected from the group consisting of -C(O)-, -OC(O)-, -NHC(O)-, -NR-(CHR) _m2 -NR-C(O)-, -NR-(CHR) _m2 -NR-C(O)O-(CHR) _m1 -, a chemical bond;

Wherein, each of the R groups is independently selected from the following group: H, D, (CH ₂ ) _n4 NHC(O)NH ₂ , C ₁ -C ₆ alkyl;

m1, m2 and n3 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

m101 is selected from 0, 1, 2, 3, 4;

Unless otherwise specified, the term “substituted” refers to the substitution of one or more hydrogen atoms on a group with a substituent selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a nitrile group, a nitro group, a hydroxyl group, an amino group, a C ₁ -C ₆ alkyl-NH-, a (C ₁ -C ₆ alkyl) ₂ N-, a C ₁ -C ₆ alkyl, a C ₂ -C ₆ alkenyl, a C ₂ -C ₆ alkynyl, a C ₁ -C ₆ alkoxy group, a halogenated C ₁ -C ₆ alkyl group, a halogenated C ₂ -C ₆ alkenyl group, a halogenated C ₂ -C ₆ alkynyl group, a halogenated C ₁ -C ₆ alkoxy group, an allyl group, a benzyl group, a C ₆ -C ₁₂ aryl group, a C ₁ -C ₆ alkoxy-C ₁ -C ₆ alkyl group, a C ₁ -C ₆ alkoxy-carbonyl group, a phenoxycarbonyl group, a C ₂ -C ₆ alkynyl-carbonyl group, a C ₂ -C ₆ alkenyl-carbonyl group, a C ₃ -C 6 C ₆ cycloalkyl-carbonyl, C ₁ -C ₆ alkyl-sulfonyl, phenyl, 5-7 membered heteroaryl, C ₃ -C ₈ cycloalkyl, 3-12 membered heterocyclyl, or C(O)NH—L ₂ H.

In another preferred embodiment, _L2 is a group selected from the group consisting of optionally substituted -(CHR) _m1- , optionally substituted -(CHR) _m1 -C(O)NH-( _CH2CH2O ) _n3- ( _CHR ) _m2- , optionally substituted -(CHR) _m1- ( _CH2CH2O ) _n3- (CHR) _{m2- ;}

p1 is 0;

The L ₄ is a chemical bond.

In another preferred embodiment, _L2 is a group selected from the group consisting of: optionally substituted -(CHR) _m1 -C(O)NH- (CH ₂ CH ₂ O) _n3 -(CHR) _m2 -;

p1 is 0;

The L ₄ is a chemical bond.

In another preferred embodiment, L ₂ is a group selected from the group consisting of optionally substituted -(CHR) _m1 -, optionally substituted -(CHR) _m1 -(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -;

p1 is 0;

The L ₄ is a chemical bond.

In another preferred embodiment, the L ₂ is a group selected from the group consisting of: optionally substituted -(CHR) _m1 -C(O)-;

p1 is 1, 2, or 3;

The _L4 is an optionally substituted group selected from the following group:

In another preferred embodiment, the L ₂ is a group selected from the following group: optionally substituted (CHR) _m1 -C(O)-;

p1 is 1, 2, or 3;

The _L4 is an optionally substituted group selected from the following group:

In another preferred embodiment, _L2 is a group selected from the following group: optionally substituted -( _CH2 ) _m1 -C(O)-, optionally substituted -( _CH2CH2O ) _n3- (CHR) _m2 - _C (O)-, optionally substituted -(CHR) _m1 - _C3 - _{C9cycloalkyl-} (CHR) _m2- ; preferably, m1 and m2 are each independently selected from 0, 1, 2, 3, 4 or 5; n3 is selected from 0, 1 or 2;

p1 is 0;

The L ₄ is a chemical bond, or an optionally substituted group selected from the following group:

In another preferred embodiment, the L ₂ is a group selected from the group consisting of: optionally substituted -(CHR) _m1 -C(O)-L ₄ - NH(RCH) _m1 -(OCH ₂ CH ₂ ) _n3 -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C(O)NH-(CHR) _m1 -5-10 membered aromatic heterocycle -(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C(O)-L ₄ -NH-(CH ₂ CH ₂ O) _n3 -L ₄ -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C(O)-L ₄ -NH(RCH) _m1 -(OCH ₂ CH ₂ ) _n3 -L ₄ -(CHR) _m2 -; preferably, the 5- to 10-membered aromatic heterocycle is a 5- or 6-membered aromatic heterocycle

p1 is 0;

The L ₄ is a chemical bond, or an optionally substituted group selected from the following group:

In another preferred embodiment, _L3 is a peptide residue consisting of amino acids selected from the following group: phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine.

In another preferred embodiment, the _L3 is a peptide residue consisting of amino acids selected from the following group: glycine, alanine, lysine, phenylalanine, valine and citrulline.

In another preferred embodiment, _L3 is a peptide residue selected from the following group: -Glycine-Phenylalanine-Glycine-(-Gly-Phe-Gly-), -Glycine-Glycine-Phenylalanine-Glycine-(-Gly-Gly-Phe-Gly-), -Valine-Citrulline-(-Val-Cit-), -Citrulline-Valine-(-Cit-Val-), -Citrulline-Alanine-(-Cit-Ala-), -Valine-Alanine-(-Val-Ala-), -Valine-Arginine-(-Val-Arg-), -Valine-Lysine-(-Val-Lys-), -Valine-Lysine (Ac)-(-Val-Lys (Ala-)). c)-), -Lysine-Valine-(-Lys-Val-), -Leucine-Citrulline-(-Leu-Cit-), -Isoleucine-Citrulline-(-Ile-Cit-), -Tryptophan-Citrulline-(-Trp-Cit-), -Phenylalanine-Lysine-(-Phe-Lys-), -Phenylalanine-Lys(Ac)-(-Phe-Lys(Ac)-), -Phenylalanine-Citrulline-(-Phe-Cit-), -Phenylalanine-Ala-(-Phe-Ala-), -Phenylalanine-Arg-(-Phe-Arg-), -Ala-Lysine-(-Ala-Lys-), -Ala- -Ala-Ala-), -Ala-Ala-Ala-Ala-), -Ala-Ala-Asparagine-(-Ala-Ala-Asn-), -Ala-Ala-Aspartic Acid-(Ala-Ala-Asp-), -Lysine-Ala-Ala-Ala-Asparagine-(-Lys-Ala-Ala-Asn-), -Lysine-Ala-Ala-Ala-Aspartic Acid-(-Lys-Ala-Ala-Asp-), -(D)-Valine-Leu-Lysine-(-D-Val-Leu-Lys-), -Glycine-Glycine-Arginine- (-Gly-Gly-Arg-), -Glycine-Glycine-Asparagine-(-Gly-Gly-Asn-), -Glycine-Glycine-Phenylalanine-(-Gly-Gly-Phe-), -Valine-Lysine-Glycine-(-Val-Lys-Gly-), -Glutamic acid-Alanine-Alanine-(-Glu-Ala-Ala-), -Aspartic acid-Alanine-Alanine-(-Asp-Ala-Ala-), -Valine-Lysine-Glycine-Glycine-(-Val-Lys-Gly-Gly-), and -Lysine-Alanine-Asparagine-(-Lys-Ala-Asn-).

In another preferred embodiment, the _L3 is a structure selected from the following group:

In another preferred embodiment, L ₄ is a chemical bond, or an optionally substituted group selected from the following group: Wherein, the ^Ra and ^Rb are each independently selected from the following group: hydrogen, optionally substituted _C1 - _C4 alkyl, and optionally substituted _C1 - _C4 deuterated alkyl.

In another preferred embodiment, _L4 is a chemical bond, or an optionally substituted structure selected from the following group:

In another preferred embodiment, Y is -C(O)-, -OC(O)-, -NHC(O)-, or a chemical bond.

In another preferred embodiment, L ₂₀₁ , L ₂₀₂ and L ₂₀₃ are each independently selected from the following structures:

wherein each u, v and w are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; _u1 and _u2 are independently selected from 0 or 1; Glu is an unmodified or modified 5-6-membered glycosyl;

X and _Y2 are each independently selected from the group consisting of -NH-(C ₁ -C ₆ alkyl)-NH-, -NH-(C ₃ -C ₈ cycloalkyl)-NH-, -NH-(4-10 membered heterocyclyl)-NH-, Wherein, the ring D is a 4-15 membered heterocyclic group;

The cycloalkyl and heterocyclic groups may be monocyclic, condensed, bridged or spirocyclic; the heterocyclic group may be saturated or partially unsaturated (but not aromatic).

In another preferred embodiment, R ₂ and the linker portion include at least one monocyclic, fused, bridged or spirocyclic structural unit.

In another preferred embodiment, R ₂ and the linker portion include at least one fused ring, bridged ring or spiro ring structural unit.

In another preferred embodiment, X and _Y2 each independently have a structure selected from the following group:

-NH-(C ₁ -C ₆ alkyl)-NH-, -NH-(C ₃ -C ₈ cycloalkyl)-NH-, -NH-(4-10 membered heterocyclyl)-NH-, or a structure selected from the following group:

In another preferred embodiment, the sugar group has a structure shown in the following formula III:

Wherein, the R ₂₁₁ is selected from the following group: -NH(C ₂ -C ₆ acyl), -NH(halogenated C ₂ -C ₆ acyl), -NH(C ₂ -C ₆ sulfonyl), -NH(halogenated C ₂ -C ₆ sulfonyl);

R ₂₁₂ , R ₂₁₃ and R ₂₁₄ are each independently selected from the group consisting of H or C ₁ -C ₆ acyl.

In another preferred embodiment, R ₂₁₁ is selected from the following group: -NH (C ₂ -C ₄ acyl); R ₂₁₂ , R ₂₁₃ and R ₂₁₄ are each independently selected from the following group: H or C ₁ -C ₄ acyl.

In another preferred embodiment, R ₂₁₁ is -NHAc; R ₂₁₂ , R ₂₁₃ and R ₂₁₄ are each independently selected from the following group: H or Ac.

In another preferred embodiment, L ₁₁ is selected from the following group: -O-, -NH- or -S-.

In another preferred embodiment, L ₁₁ is -NH-.

In another preferred embodiment, m is 1 or 2.

In another preferred embodiment, R ₁ is selected from the following group: C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _3-12 cycloalkyl or 4-12 membered heterocycloalkyl; and the R ₁ may be further substituted by one or more R ^1a substituents, and the R ^1a is selected from the following group: halogen, hydroxyl, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _3-6 cycloalkyl, -NR ^a1 R ^a2 , -NHC(＝O)-R ^a3 , a 5-6 membered heteroaryl substituted by one or more R ^a4 , -OC(＝O)R ^a5 , -C(＝O)R ^a5 , -OC(＝O)OR ^a5 , -C(＝O)OR ^a5 .

In another preferred embodiment, the R ₁ is selected from the following group: C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _3-12 cycloalkyl or 4-12 membered heterocycloalkyl; and the R ₁ may be further substituted by one or more R ^1a substituents, and the R ^1a is selected from the following group: halogen, hydroxyl, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _3-6 cycloalkyl.

In another preferred embodiment, R ₁ is selected from the following group: H, C _1-8 alkyl, C _1-8 alkoxy, C _2-8 alkenyl, C _2-8 alkynyl, C _3-8 cycloalkyl or 4-8 membered heterocycloalkyl; wherein, said R ₁ may be further substituted by one or more R ^1a substituents, and said R ^1a is selected from the following group: hydrogen, halogen, hydroxyl, cyano, C _1-4 alkyl, C _1-4 alkoxy.

In another preferred embodiment, the R ₁ is selected from the following group: C _1-8 alkyl; and the R ₁ may be further substituted by one or more R ^1a substituents, and the R ^1a is selected from the following group: C _1-6 alkyl, C _3-6 cycloalkyl.

In another preferred embodiment, B is absent, or B is selected from the following group: C _3-8 cycloalkyl, 4-7 membered heterocyclic group, C _6-10 aryl, or

In another preferred embodiment, the B does not exist, or B is selected from the following group: phenyl, pyridyl.

In another preferred embodiment, L ₁₂ is selected from the following group: -(CR ^1b R ^1c ) _p -(NR ^1d ) _q -, -O-, -S-, -(CR ^1b R ^1c ) _p -C(＝O)-, -(CR ^1b R ^1c ) _p -C(＝O)NH-, -(CR ^1b R ^1c ) _p -NHC(＝O)-, -S(＝O)- or -S(＝O) ₂ -; wherein R ^1b , R ^1c are selected from the following group: hydrogen, halogen, C _1-6 alkyl; R ^1d is hydrogen or C _1-6 alkyl; p is 0, 1, 2 or 3, and q is 0 or 1.

In another preferred embodiment, L ₁₂ is selected from the following group: -(CH ₂ ) _p -, -(CH ₂ ) _p -NR ^1d -, -O-, -S-, -(CH ₂ ) _p -C(＝O)-, -(CH ₂ ) _p -C(＝O)NH-, -(CH ₂ ) _p -NHC(＝O)-; wherein R ^1d is hydrogen or C _1-6 alkyl; p is 0, 1, 2 or 3.

In another preferred embodiment, R ₄ is selected from the following groups: hydrogen, halogen, amino, hydroxyl, C _1-12 alkyl, C _1-12 alkoxy, C _2-12 alkenyl, C _2-12 alkynyl, C _3-10 cycloalkyl, 4-10 membered heterocyclyl, C _6-12 aryl, or 5-12 membered heteroaryl; and the R ₄ may be optionally substituted by one or more ^Re substituents; wherein ^Re is selected from the following groups: hydrogen, halogen, hydroxyl, carboxylic acid, amino, C _1-6 alkyl, one or more ^Re1- substituted C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkoxy, C _3-8 cycloalkyl, 4-7 membered heterocyclyl, phenyl, 5-7 membered heteroaryl, -N( ^Re2Re3 ); wherein ^Re1 is selected from the following groups: halogen, ^hydroxyl , -P(O)( ^ORe2 ) ₂ ; ^Re2 and ^Re3 are selected from the following groups: hydrogen, C The present invention can be any of _{C 1-6} alkyl, C _3-6 cycloalkyl, C _1-6 haloalkyl or C _1-6 alkyl substituted with hydroxy.

In another preferred embodiment, R ₅ is selected from the following group: halogen, hydroxyl, cyano, amino, C _1-6 alkyl, C _1-6 alkoxy, C _3-6 cycloalkyl.

In another preferred embodiment, R ₄ is selected from the following group: hydrogen, C _1-12 alkyl, C _1-12 alkoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a group formed by losing a hydrogen atom from a ring selected from the following group: And the R ₄ may be optionally substituted by one or more ^Re substituents.

In another preferred embodiment, R ₅ is selected from the following group: halogen, hydroxyl, cyano, amino, C _1-6 alkyl, C _1-6 alkoxy, C _3-6 cycloalkyl.

In another preferred embodiment, R ₅ is selected from the following group: halogen, C _1-4 alkyl, C _1-4 alkoxy.

In another preferred embodiment, the toxin is selected from the following group:

In another preferred embodiment, the R ₂₀₁ -NHC(O) has a structure selected from the following group:

In another preferred embodiment, the compound is selected from the following group:

In a second aspect of the present invention, there is provided a compound represented by the following formula Ia, Ib, Ic or Id, or a stereoisomer, enantiomer, or a pharmaceutically acceptable salt thereof:

in:

R ₄ is selected from the group consisting of hydrogen, halogen, cyano, amino, hydroxy, C _1-12 alkyl, C _1-12 alkoxy, C _2-12 alkenyl, C _2-12 alkynyl, C _3-12 cycloalkyl, 4-12 heterocycloalkyl, C _6-12 aryl, or 5-12 heteroaryl; and said R ₄ may be optionally substituted by one or more R ^1e substituents, wherein R ^1e is selected from the group consisting of hydrogen, halogen, hydroxy, carboxylic acid, amino, C _1-6 alkyl, one or more R ^e1 substituted C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkoxy, C _3-12 cycloalkyl, 4-12 heterocycloalkyl, C _6-12 aryl, 5-12 heteroaryl, -N(R ^e2 R ^e3 ), -C(＝O)OR ^e2 , -C(＝O)NH-R ^e2 , -S(＝O) ₂ -R ^e2 ;

And the R ₄ is substituted by at least one R ^1e substituent, one; wherein the R ^1e substituent has the following structure:

Wherein, Q ₂ is -CR ₃₀₄ R ₃₀₅ -; R ₃₀₄ and R ₃₀₅ are the same or different, and are independently selected from the following group: hydrogen, halogen, cyano, substituted or unsubstituted C _1-6 alkyl, substituted or unsubstituted C _1-6 alkoxy, substituted or unsubstituted C _3-6 cycloalkyl, substituted or unsubstituted 4-6 membered heterocycloalkyl; preferably, R ₃₀₄ and R ₃₀₅ are: H or F;

In another preferred embodiment, the compound is selected from the following group:

The third aspect of the present invention provides a compound selected from the following group, or a stereoisomer, enantiomer, or a pharmaceutically acceptable salt thereof:

The fourth aspect of the present invention provides a pharmaceutical composition comprising the compound described in the first to third aspects of the present invention, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt, and optionally a pharmaceutically acceptable carrier, diluent or excipient.

The fifth aspect of the present invention provides the use of the compounds described in the first to third aspects of the present invention, or their stereoisomers, enantiomers, or pharmaceutically acceptable salts, and/or the pharmaceutical composition described in the fourth aspect of the present invention in the preparation of drugs for treating or preventing viral infections or tumors.

The sixth aspect of the present invention provides a combined adjuvant, which comprises: an aluminum adjuvant, and a TLR7/8 agonist adsorbed on the aluminum adjuvant; wherein the TLR7/8 agonist comprises the compound as described in the first to third aspects of the present invention, or its stereoisomers, enantiomers, or pharmaceutically acceptable salts thereof.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

DETAILED DESCRIPTION

After long and in-depth research, the inventors have designed a class of drug compounds that can be used as vaccine adjuvants, which can be used to improve the efficacy of vaccines after being mixed with aluminum adjuvants. Based on the above findings, the inventors have completed the present invention.

Preparation method of compound of formula I

The present invention provides a method for preparing the compound as shown in Formula I. Specifically, the compound of the present invention adopts the following process, such as the preparation method of compound III-2 or compound III-11 or compound IV-8, and can be prepared by conventional adjustments in the art:

When the compound of the present invention has the structural formula III-2, or an analog thereof, the preparation method thereof is as follows:

Using 1,20-diiodo-3,6,9,12,15,18-hexaoxoeicosane (III-2-2) and diethyl (difluoromethyl)phosphonate as starting materials, under the action of a base (such as diisopropylamino), compound III-2-3 is prepared; compound III-2-3 and compound II-1-4 can be prepared under the action of a base (such as potassium carbonate) to obtain compound III-2-1; compound III-2-1 is dissolved in an appropriate solvent (such as dichloromethane, dichloroethane), and hydrolyzed under the action of TMSBr (trimethylsilyl bromide) to obtain compound III-2.

The present invention provides a method for preparing a derivative having the structural formula III-11, and the reaction formula is as follows:

Using 2-(2-chloroethoxy)ethoxyethane-1-ol and benzyl bromide as starting materials, under the action of a base (such as sodium hydrogen), compound III-11-1 is prepared; compound III-11-1 can be prepared into compound III-11-5 through substitution reaction, deprotection, condensation reaction, and hydrogenation deprotection; compound III-11-5 and bis(diisopropylamino)(2-cyanoethoxy)phosphine are used as starting materials, under the action of tetrazole, compound III-11-6 is prepared; similarly, compound III-11-6 and compound II-4 are used as starting materials, and are respectively subjected to substitution reaction under the action of tetrazole, iodine oxidation reaction, and DBU deprotection reaction to prepare compound III-11.

The present invention also provides a method for preparing a derivative having the structural formula IV-8, and the reaction formula is as follows:

According to the method for preparing L96 described in patent WO2014025805A1, compound A2 or its similar derivatives can be successfully synthesized by methods well known to those skilled in the art or according to methods described in detail in the prior art.

Using compound A2 and compound III-6-3 as starting materials, compound IV-8-1 is prepared under the action of a base (such as TEA); compound IV-8-1 is deprotected in an ammonia methanol solution to prepare compound IV-8.

Combined adjuvant containing TLR7/8 agonist and preparation thereof

The present invention provides a method for preparing an aluminum adjuvant adsorbing a TLR7/8 agonist as a combined adjuvant. Specifically, the preparation method comprises the following steps:

(1) Dissolve the TLR7/8 agonist in sterile water and then dilute and disperse it in an appropriate pH buffer;

(2) adding aluminum adjuvant to the solution of step (1) and mixing them for sufficient adsorption.

Specifically, when the solubility of the TLR7/8 agonist in step (1) does not reach the target concentration, a small amount of sodium hydroxide solution can be added to assist dissolution.

Specifically, the pH of the buffer solution in step (1) can be between 5.5 and 8.0 (specifically, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0), which can be an integer or a decimal, and the pH is preferably between 6.5 and 7.5.

Specifically, the buffer solution in step (1) can be Tris buffer, histidine buffer, citrate buffer, One of succinate buffer and borate buffer, preferably Tris buffer and histidine buffer.

Specifically, the concentration of the buffer solution in step (1) is 1 to 50 mM (specifically 1, 5, 10, 15, 20, 30, 50 mM), which can be an integer or a decimal, preferably between 5 and 30 mM, and more preferably between 10 and 20 mM.

Specifically, the aluminum adjuvant in step (2) can be aluminum hydroxide adjuvant or aluminum phosphate adjuvant, preferably aluminum hydroxide adjuvant.

Specifically, the mass ratio of the aluminum adjuvant to the TLR7/8 agonist in step (2) can be 0.5-10:1 (0.5:1, 1:1, 2:1, 3:1, 5:1, 10:1), and can be any ratio within the range, and can be an integer or a decimal. Preferably, it is 1 to 3:1, and more preferably, it is 1 to 2:1.

Specifically, in step (2), the temperature of the mixed reaction after the aluminum adjuvant adsorbs the TLR7/8 agonist can be 2-30°C (2, 8, 10, 20, 25, 30°C), which can be an integer or a decimal, preferably between 10-30°C, and more preferably between 15-25°C.

Specifically, in step (2), the mixing time after the aluminum adjuvant adsorbs the TLR7/8 agonist can be 0.5h to 10h (0.5, 1, 2, 3, 4, 5, 10h), which can be an integer or a decimal, preferably between 0.5 and 3h, and more preferably 1 to 2h.

The pharmaceutical composition of the present invention is an injectable composition. In addition to the active compound, the pharmaceutical composition of the present invention may contain one or more excipients. The excipients are conventional pharmaceutical excipients in the pharmaceutical field, and the following ingredients (including but not limited to) can be selected: pH regulators, bulking agents, lyophilization protectants, buffers, tension regulators, isotonic agents, antioxidants, antimicrobial agents, antibacterial agents, antifungal agents, solubilizers, surfactants and wetting agents, etc.

In general, a bulking agent is included in a freeze-dried product to provide volume and structure to the freeze-dried powder, which helps dissolve the active agent. Lyoprotectants help stabilize the active agent during freeze drying and storage and prevent its degradation. Bulking agents and lyoprotectants that may be included in the pharmaceutical composition of the present invention include, but are not limited to, sucrose, lactose, trehalose, mannitol, sorbitol, raffinose, glycine, histidine, polyethylene glycol, and low molecular weight polyvinyl pyrrolidone (i.e., povidone K12 and povidone K17).

The pharmaceutical composition includes a buffer to adjust and stabilize the pH and optimize the solubility and stability of the active agent. The buffer in the pharmaceutical composition of the present invention includes (but is not limited to) Tris buffer, histidine buffer, citrate buffer, succinate buffer, borate buffer, and the pH is between 5.5 and 8.0.

Tonicity adjusters and isotonic agents are included in the pharmaceutical composition to maintain and ensure that the formulation is isotonic with human plasma. Tonicity adjusters and isotonic agents that may be included in the pharmaceutical composition of the present invention include, but are not limited to, dextrose, glycerol, sodium chloride, glycerol, and mannitol.

Antioxidants are used to prevent/minimize any oxidation of the active agent or excipient during storage, while bacteriostatic agents are used to prevent microbial growth. Antioxidants that may be included in the pharmaceutical compositions of the present invention include, but are not limited to, ascorbic acid, acetylcysteine, sulfites (bisulfites, metabisulfites), monothioglycerol (monothioglyercol), butylhydroxytoluene (BHT), butylhydroxyanisole (BHA), and thiourea. Bacteriostatic agents that may be included in the pharmaceutical compositions of the present invention include, but are not limited to, phenol, metacresol, benzyl alcohol, parabens (e.g., methyl, propyl, butyl parabens), benzalkonium chloride, chlorobutanol, thimerosal, and phenylmercuric salts (acetates, borates, nitrates).

Solubilizers, which can be broadly divided into surfactants and cosolvents, help dissolve the active agent into the formulation or increase its solubility. Surfactants that can be included in the pharmaceutical compositions of the present invention include, but are not limited to, polyoxyethylene sorbitan monooleate (Yewon 80), sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20), lecithin, polyoxyethylene-polyoxypropylene copolymer (Pluronic). Surfactants can also act as wetting agents, and surfactants/wetting agents that can be included in the pharmaceutical compositions of the present invention include, but are not limited to, lecithin, polysorbate 20, polysorbate 80, Pluronic F-68, and sorbitan trioleate (Span 85).

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

In the following examples, the structures of the compounds were determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). NMR shift (δ) is given in units of 10 ^-6 (ppm). NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ), deuterated methanol (CD ₃ OD) as the measuring solvent, and tetramethylsilane (TMS) as the internal standard.

SHIMADZU LC system (chromatographic column: CSH ^™ Prep-C18, 19*150 mm, liquid handler LH-40, pump LC-20AP, detector SPD-20A, system controller CBM-20A, solvent system: acetonitrile and 0.05% trifluoroacetic acid aqueous solution).

LC/MS spectra of the compounds were obtained using LC/MS (Agilent Technologies 1200 Series). The LC/MS conditions were as follows (run time was 10 min):

Acidic conditions: A: 0.05% trifluoroacetic acid in water; B: 0.05% trifluoroacetic acid in acetonitrile;

Basic conditions: A: 0.05% NH ₃ ·H ₂ O aqueous solution; B: acetonitrile

Neutral conditions: A: 10 mM NH ₄ OAC in water; B: acetonitrile

Unless otherwise specified, in the following examples, intermediates and final compounds were purified by silica gel column chromatography, or by preparative HPLC on a reverse phase column using XselectOR CSH ^™ Prep-C18 (5 μm, OBD ^™ 19*150 mm) or XBridge™ Prep Phenyl (5 μm, OBD ^™ 30*100 mm).

Silica gel column chromatography generally uses Yantai Huanghai Silica Gel 200-300 mesh silica gel as the carrier.

The CombiFlash rapid preparation instrument uses Combiflash Rf200 (TELEDYNE ISCO).

Thin layer chromatography (TLC) silica gel plates use Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The specifications of silica gel plates used in thin layer chromatography detection products are 0.15mm~0.2mm, and the specifications used in thin layer chromatography separation and purification products are 0.4mm~0.5mm.

The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, and Darui Chemicals.

Abbreviations: MeOH: methanol; DIEA: N,N-diisopropylethylamine; DMAP: 4-dimethylaminopyridine; DCM: dichloromethane; Dioxane: 1,4-dioxane; EA: ethyl acetate; Conc.HCl: concentrated hydrochloric acid; DMF: dimethylformamide; AcOH: acetic acid; DMA: dimethylacetamide; Xantphos: 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene; DCE: 1,2-dichloroethane; BH ₃ : borane; Boc ₂ O: tert-butyl carbonate; t-BuLi: tert-butyl lithium; DMF-DMA: 1,1-dimethoxy-N,N-dimethyl-methylamine; PPA: polyphosphoric acid; NaHMDS: sodium bis(trimethylsilyl)amide; POCl ₃ : phosphorus oxychloride; Pd/C: palladium on carbon; CAN: acetonitrile; EtOH: ethanol; t-butyl nitrite: tert-butyl nitrite; pyrrolidine: pyrrolidine; toluene: toluene; TsOH: p-toluenesulfonic acid; acrylamide: prop-2-enamide; THF: tetrahydrofuran; LDA: lithium diisopropylamide; LAH: lithium aluminum hydride; Pd ₂ (dba) ₃ : tris(dibenzylideneacetone)dipalladium; PMB: p-methoxybenzyl; Xphos: 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl; H ₂ : hydrogen; Pd(dppf)Cl ₂ : [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium; t-BuOK: potassium tert-butoxide; K ₂ CO ₃ : potassium carbonate; HCOONH ₄ : ammonium formate; LiHMDS: lithium bis(trimethylsilyl)amide; Urea: urea; n-BuLi: n-butyllithium; MsCl: methanesulfonyl chloride; Dppf: bis(diphenylphosphinoferrocene); Et ₃ N: triethylamine; AcCl: acetyl chloride; NaH: sodium hydride; TFA: tris(III) Fluoroacetic acid; SOCl ₂ : thionyl chloride; NBS: N-bromosuccinimide; NCS: N-chlorosuccinimide; 1,4-Dioxane: 1,4-dioxane; K ₃ PO ₄ : potassium phosphate; RuPhos Pd G2: chloro(2-dicyclohexylphosphino-2,6-di-isopropoxy-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II); MTBE: methyl tert-butyl ether.

Preparation Example Preparation of Compound A1

Step 1: Preparation of compound A1-1

4A molecular sieve (30 g) and TMSOTf (18.8 g, 84.8 mmol) were slowly added to a dichloromethane (300 ml) solution of compound Int.1 (30 g, 77.1 mmol); after the addition, the reaction mixture was stirred at 55°C for 3 hours. Then the reaction mixture was neutralized with triethylamine, poured into 500 ml of water, and diluted and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a yellow oil compound A1-1 (22.8 g). MS: 330.2 (M+H) ⁺ .

Step 2: Preparation of compound A1-2

Under nitrogen protection, 4A molecular sieve (10 g) and compound Int.02 (3.7 g, 15.2 mmol) were slowly added to a dichloroethane (100 ml) solution of compound A1-1 (11 g, 30.4 mmol). After the addition, the reaction mixture was stirred at 25°C for 30 minutes; then TMSOTf (3.7 g, 15.2 mmol) was slowly added to the above reaction mixture under stirring at 0 degrees, and the reaction mixture was stirred at 25°C for 16 hours. The reaction mixture was filtered, the filter cake was washed with dichloromethane, the filtrate was neutralized with sodium bicarbonate solution and extracted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain a yellow oil compound A1-2 (7 g, 48.8%). MS: 430.2 (M+H) ⁺ .

Step 3: Preparation of Compound A1-3

Under nitrogen protection, a mixed solution of NaIO ₄ (13.0 g, 60.6 mmol) and compound A1-3 (6.5 g, 15.2 mmol) in dichloromethane (30 ml), acetonitrile (30 ml) and water (45 ml) was stirred at 0°C for 30 minutes; then, RuCl ₃ 3H ₂ O (95 mg, 0.45 mmol) was slowly added to the above reaction mixture under stirring at 0 degrees, and the reaction mixture was stirred at 25°C for 16 hours. The reaction mixture was diluted with 100 ml of water and sodium bicarbonate was added to adjust the pH value to 7.5, and extracted with dichloromethane. The aqueous phase was adjusted to pH 3 with citric acid and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a white foam solid compound A1-3 (3.2 g, 45%). MS: 448.2 (M+H) ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ11.98(s,1H),7.81(d,J＝9.2Hz,1H),5.21(d,J＝3.2Hz,1H),4.96(dd,J＝11.2,3.4Hz,1H),4.49(d,J＝8.4Hz,1H),4.02(s,3H),3.87( d,J＝10.0Hz,1H),3.78–3.37(m,2H),2.20(t,J＝6.8Hz,2H),2.10(s,3H),2.00(s,3H),1.89(s,3H),1.77(s,3H),1.49(d,J＝2.8Hz,4H).

Step 4: Preparation of Compound A1

Under nitrogen protection at 0°C, compound Int.3 (10.3 g, 36.8 mmol) was slowly added to a solution of compound A1-3 (11.0 g, 24.6 mmol) and DIEA (6.4 g, 49.2 mmol) in dichloromethane (100 ml), and then The reaction mixture was stirred at 25°C for 3 hours. The reaction mixture was concentrated to obtain a crude product, which was purified to obtain a yellow oily compound A1 (11.2 g, 74.2%). MS: 614.2 (M+H) ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ7.83(d,J＝9.2Hz,1H),5.22(d,J＝3.2Hz,1H),4.96(dd,J＝11.2,3.4Hz,1H),4.51(d,J＝8.4Hz,1H),4.03(s,3H),3.90(dd,J＝20.0,9.0Hz,1H),3.6 2(dtd,J＝15.8,10.2,6.0Hz,2H),2.79(t,J＝7.4Hz,2H),2.11(s,3H),2.0 0(s,3H),1.89(s,3H),1.76(s,3H),1.73–1.64(m,2H),1.63–1.54(m,2H).

Preparation Example Preparation of Compound A2

Step 1: Preparation of compound A2-1

Synthesis of Compound A2-1 Referring to Compound A1, Compound A2-1 was prepared by using Compound Int.2 instead of Compound A1-3. MS: 509.1 (M+Na) ⁺ .

Step 2: Preparation of compound A2-2

Under nitrogen protection, compound A3 (400 mg, 0.223 mmol), compound A2-1 (163 mg, The reaction mixture was stirred at 20 degrees for 40 hours; the reaction mixture was then concentrated to obtain a crude product, which was purified to obtain a white solid compound A2-2 (270 mg, 57.8%). MS: 1049.3 (1/2M+H) ⁺ .

Step 3: Preparation of compound A2-3

Compound A2-2 (270 mg, 0.129 mmol) was dissolved in tetrahydrofuran (10 ml), palladium carbon (14 mg) was added to the reaction mixture under stirring, and then the reaction mixture was stirred under hydrogen pressure at room temperature for 16 hours; the reaction mixture was filtered and concentrated to obtain compound A2-3 (210 mg, 81%). MS: 1003.6 (1/2M+H) ⁺ .

Step 4: Preparation of Compound A2

Under nitrogen protection and stirring at 0°C, compound Int.3 (56.8 mg, 0.209 mmol) was slowly added to a solution of compound A2-3 (210 mg, 0.105 mmol) and DIEA (67.6 mg, 0.523 mmol) in acetonitrile (10 ml), and the reaction mixture was stirred at 20°C for 15 minutes. The reaction mixture was then diluted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give compound A2 (220 mg, 97%). MS: 1087.2 (1/2M+H) ⁺ .

Preparation Example Preparation of Compound A3

Step 1: Preparation of compound A3-1

Tert-butyl acrylate (1060 g, 8260 mmol) and NaOH (5M, 19.8 ml) were added slowly to the DMSO (100 ml) reaction solution of compound Int.6 (100 g, 825.5 mmol) in sequence. After the addition, the reaction mixture was stirred at 20°C for 28 hours. The reaction mixture was then extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified to obtain a light yellow oil compound A3-1 (74 g, 17.7%). MS: 506.4 (M+H) ⁺ .

Step 2: Preparation of compound A3-2

Under stirring conditions at 0 degrees, CbzCl (22.7 g, 133.6 mmol) was slowly added to a dichloromethane (100 ml) reaction solution of compound A3-1 (15 g, 29.7 mmol) and TEA (9.0 g, 89.1 mmol). After the addition, the reaction mixture was stirred at 20°C for 12 hours; then the reaction mixture was diluted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain a yellow oil compound A3-2 (18.8 g). ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.36–7.30 (m, 6H), 4.97 (s, 2H), 3.54 (t, J = 6.4Hz, 6H), 3.51 (s, 6H), 2.38 (t, J = 6.0Hz, 6H), 1.39 (s, 27H).

Step 3: Preparation of compound A3-3

The reaction mixture of compound A3-2 (18.8 g) and formic acid (200 ml) was stirred at 20°C for 12 hours; then the reaction mixture was concentrated to obtain a yellow oil compound A3-3 (16.5 g). 1H NMR (400 MHz, DMSO) δ12.12 (s, 3H), 7.40–7.32 (m, 5H), 5.17 (s, 1H), 4.99 (s, 2H), 3.57 (t, J＝6.4 Hz, 6H), 3.50 (s, 6H), 2.42 (t, J＝6.4 Hz, 6H).

Step 4: Preparation of Compound A3-4

Under nitrogen protection and stirring at room temperature, HOBt (13.2 g, 97.6 mmol) and EDCI (18.7 g, 97.6 mmol) were slowly added to a DMF (120 ml) solution of compound A3-3 (11.5 g, 24.4 mmol), N-tert-butyloxycarbonyl-1,3-propylenediamine (17.0 g, 97.6 mmol) and DIEA (25 g, 193.6 mmol). After the addition, the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with ethyl acetate and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain a yellow oil compound A3-4 (16 g). ¹ H NMR (400MHz, CDCl ₃ )δ7.37–7.28(m,5H),6.83(s,3H),5.55(s,1H),5.16(t,J＝6.4Hz,3H),5.03(s,2H),3.68(t,J＝6.0Hz,6H), 3.65(s,6H),3.27–3.24(m,6H),3.14–3.10(m,6H),2.40(t,J＝5.6Hz,6H),1.70–1.47(m,6H),1.43(s,27H).

Step 5: Preparation of Compound A3-5

A reaction mixture of compound A3-4 (16 g) in dichloromethane (100 ml) and trifluoroacetic acid (20 ml) was stirred at 20°C for 12 hours; the reaction mixture was then concentrated to obtain a yellow oil compound A3-5 (20 g), which was used directly in the next step without purification.

Step 6: Preparation of Compound A3-6

Under stirring and nitrogen protection at 0 degrees, compound A1 (5 g) in dichloromethane (20 ml) was slowly added to the reaction mixture of compound A3-5 (5 g) and DIEA (2.8 g) in acetonitrile (20 ml). After the addition, the reaction mixture was stirred at room temperature for 12 hours; then the reaction mixture was diluted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain white solid compound A3-6 (3.85 g). ¹ H NMR (400 MHz, DMSO-d ₆ )δ7.82-7.79(m,6H),7.71(t,J＝5.6Hz,3H),7.37-7.27(m,5H),6.51(s,1H),5.21(d,J＝7.6Hz ,3H),4.98-4.95(m,5H),4.48(d,J＝7.6Hz,3H),4.04-4.00(m,9H),3.90-3.83(m,3H),3.72-3. 67(m,3H),3.56-3.48(m,12H),3.43-3.37(m,3H),3.05-2.99(m,12H),2.27(t,J＝6.4Hz,6H),2 .10(s,9H),2.04(t,J＝7.6Hz,6H),1.99(s,9H),1.89(s,9H),1.77(s,9H),1.51–1.45(m,18H).

Step 7: Preparation of Compound A3

Compound A3-6 (4.5 g) was dissolved in methanol (25 ml), palladium carbon (1 g) was added to the reaction mixture under stirring, and then the reaction mixture was stirred under hydrogen pressure at room temperature for 1.5 hours; the reaction mixture was filtered and concentrated to obtain white solid compound A3 (4.2 g).

Intermediate Int.A

2-(Bis(4-methoxybenzyl)amino)-4-(butylamino)pyridin[4,3-d]pyrimidin-5(6H)-one

Step 1: Preparation of methyl 4-(butylamino)-2-chloro-6-methylpyrimidine-5-carboxylate (Int. A-1)

2,4-Dichloro-6-methylpyrimidine-5-carboxylic acid methyl ester (10 g, 45.2 mmol) and n-butylamine (3.6 g, 49.8 mmol) were dissolved in DCM (100 ml), and DIEA (8.8 g, 67.9 mmol) was added under stirring at 0 degrees. The reaction mixture was stirred for 3 hours at 0 degrees. The reaction mixture was poured into 10% potassium bicarbonate ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product, which was purified by silica gel column chromatography to give a colorless oily substance 4-(butylamino)-2-chloro-6-methylpyrimidine-5-carboxylic acid methyl ester (11.0 g, 95%). MS: 258.1 [M + H] ⁺

Step 2: Preparation of methyl 2-(bis(4-methoxybenzyl)amino)-4-(butylamino)-6-methylpyrimidine-5-carboxylate (Int. A-2)

Dissolve 4-(Butylamino)-2-chloro-6-methylpyrimidine-5-carboxylic acid methyl ester (11.0 g, 42.8 mmol) and bis(4-methoxybenzyl)amine (13.2 g, 51.4 mmol) in acetonitrile (100 ml), add K ₂ CO ₃ (8.9 g, 64.2 mmol) under stirring at 0°C. Continue stirring the reaction mixture at 70 degrees for 13 hours. Pour the reaction mixture into ice water and extract with ethyl acetate. The organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which is purified by silica gel column chromatography to obtain yellow solid 2-(bis(4-methoxybenzyl)amino)-4-(butylamino)-6-methylpyrimidine-5-carboxylic acid methyl ester (18.4 g, 90%). MS: 479.3[M+H] ⁺

Step 3: Preparation of 2-(bis(4-methoxybenzyl)amino)-4-(butylamino)pyridin[4,3-d]pyrimidin-5(6H)-one (Int.A)

2-(Bis(4-methoxybenzyl)amino)-4-(butylamino)-6-methylpyrimidine-5-carboxylic acid methyl ester (23 g, 48.1 mmol) and 1,3,5-triazine (4.68 g, 57.7 mmol) were dissolved in DMSO (150 ml), and potassium tert-butoxide (8.09 g, 72.1 mmol) was added under stirring at 80 degrees. The reaction mixture was stirred for 2 hours at 80 degrees. The reaction mixture was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain a yellow solid 2-(bis(4-methoxybenzyl)amino)-4-(butylamino)pyridin[4,3-d]pyrimidin-5(6H)-one (17.0 g, 74.7%). MS: 474.2[M+H] ⁺

Intermediate Int.B

(R)-Pentan-2-amine hydrochloride

Step 1: Preparation of (R,E)-2-methyl-N-(2-pentylidene)propane-2-sulfoxide amide (Intermediate Int.B-1)

Pentan-2-one (10 g, 116 mmol) was dissolved in EtOAc (200 ml), and (R)-2-methylpropane-2-sulfoxide amide (14 g, 116 mmol) and tetraisopropyl titanate (66 g, 233 mmol) were added to the reaction mixture under stirring at 10 degrees, and the reaction mixture was stirred at 10 degrees for 15 hours. The reaction mixture was poured into ice water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain a colorless oil intermediate Int.B-1 (7.0 g, 32%). MS: 190.2 [M + H] ⁺

Step 2: Preparation of (R)-2-methyl-N-((R)-pentan-2-yl)propane-2-sulfoxide amide (Intermediate Int. B-2)

The intermediate Int.B-1 (7.0 g, 37 mmol) was dissolved in THF (100 ml), sodium borohydride (2.1 g, 55.6 mmol) was added to the reaction mixture under stirring at -70 degrees, and the reaction mixture was stirred at 20 degrees for 2 hours. Ice water was poured into the reaction mixture to quench the reaction, and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain a colorless oil intermediate Int.B-2 (3.5 g, 49%). MS: 192.2 [M + H] ⁺

Step 3: Preparation of (R)-pentane-2-amine hydrochloride (Intermediate Int. B)

The intermediate Int.B-2 (3.5 g, 18.3 mmol) was stirred in a 4N 1,4-dioxane hydrochloric acid solution (20 ml) at 20 degrees for 12 hours. The reaction mixture was concentrated and evaporated to dryness to obtain a crude product, which was washed with ether to obtain a white solid intermediate Int.B (2.0 g, 90%).

Intermediate Int.C

(R)-2-(Bis(4-methoxybenzyl)amino)-4-(pentan-2-ylamino)pyridinyl[4,3-d]pyrimidin-5(6H)-one

Synthesis of Intermediate Int.C Referring to Intermediate Int.A, Intermediate Int.C was prepared by using Int.B instead of n-butylamine. MS: 502.2[M+H] ⁺

Intermediate Int.D

(S)-2-(Bis(4-methoxybenzyl)amino)-4-(1-hydroxypentan-2-yl)amino)pyridin[4,3-d]pyrimidin-5(6H)-one

Synthesis of Intermediate Int.D Referring to Intermediate Int.A, Intermediate Int.D was prepared by using (S)-2-aminopentane-1-ol instead of n-butylamine. MS: 504.2 [M+H] ⁺

Intermediate Int.G1

Synthesis of Intermediate Int.G1-2 Referring to Intermediate Int.D, Intermediate Int.G1-2 was prepared by using 4-methoxybenzylamine instead of bis(4-methoxybenzyl)amine. MS: 384.3 [M+H] ⁺ .

Synthesis of Intermediate Int.G1 Referring to Intermediate I-1-1, Intermediate Int.G1 was prepared by using 4-(chloromethyl)-3-methoxybenzaldehyde instead of methyl 4-(bromomethyl)benzoate and Intermediate Int.G1-2 instead of Intermediate Int.A. MS: 532.3 [M+H] ⁺ .

Intermediate Int.G2

Synthesis of Intermediate Int.G2 Referring to Intermediate Int.G1, Intermediate Int.C-1 was used instead of Intermediate Int.D-1 to prepare Intermediate Int.G2. MS: 516.3 [M+H] ⁺ .

Intermediate Int.A4

2-(Bis(4-methoxybenzyl)amino)-4-(pentan-2-ylamino)pyrimidin[4,5-d]pyridazin-5(6H)-one

Step 1: Preparation of methyl 2-chloro-4-methyl-6-(pentan-2-ylamino)pyrimidine-5-carboxylate (Int. A4-1)

Dissolve 2,4-dichloro-6-methylpyrimidine-5-carboxylic acid methyl ester (500 mg, 2.262 mmol) and pentane-2-amine (237 mg, 2.71 mmol) in DCM (10 ml), and add DIEA (351 mg, 2.71 mmol) under stirring at 0°C. Continue stirring the reaction mixture at 0°C for 2 hours. Pour the reaction mixture into 10% potassium bicarbonate ice water and extract with ethyl acetate. The organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which is purified by silica gel column chromatography to obtain a yellow solid 2-chloro-4-methyl-6-(pentane-2-ylamino)pyrimidine-5-carboxylic acid methyl ester (560 mg, 91%). MS: 272.1 [M + H] ⁺

Step 2: Preparation of methyl 2-(bis(4-methoxybenzyl)amino)-4-methyl-6-(pentan-2-ylamino)pyrimidine-5-carboxylate (Int. A4-2)

2-Chloro-4-methyl-6-(pentane-2-ylamino)pyrimidine-5-carboxylic acid methyl ester (560 mg, 2.061 mmol) and bis(4-methoxybenzyl)amine (636 mg, 2.473 mmol) were dissolved in DMSO (10 ml), and DIEA (400 mg, 3.09 mmol) was added under stirring at 0°C. The reaction mixture was stirred at 100°C for 2 hours. The reaction mixture was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain a yellow solid 2-(bis(4-methoxybenzyl)amino)-4-methyl-6-(pentane-2-ylamino)pyrimidine-5-carboxylic acid methyl ester (800 mg, 78.8%). MS: 493.3[M+H] ⁺

Step 3: Preparation of methyl 2-(bis(4-methoxybenzyl)amino)-4-formyl-6-(pentan-2-ylamino)pyrimidine-5-carboxylate (Int. A4-3)

Dissolve 2-(bis(4-methoxybenzyl)amino)-4-methyl-6-(pentan-2-ylamino)pyrimidine-5-carboxylic acid methyl ester (800 mg, 1.624 mmol) in 1,4-Dioxane (5 ml) and add selenium dioxide (216 mg, 1.949 mmol) under stirring. Continue stirring the reaction mixture at 100 degrees for 12 hours. Pour the reaction mixture into ice water and extract with ethyl acetate. The organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product is purified by silica gel column chromatography to obtain a yellow solid 2-(bis(4-methoxybenzyl)amino)-4-formyl-6-(pentan-2-ylamino)pyrimidine-5-carboxylic acid methyl ester (360 mg, 43.8%). MS: 507.2[M+H] ⁺

Step 4: Preparation of 2-(bis(4-methoxybenzyl)amino)-4-(pentan-2-ylamino)pyrimidin[4,5-d]pyridazin-5(6H)-one (Int. A4)

Dissolve 2-(bis(4-methoxybenzyl)amino)-4-formyl-6-(pentan-2-ylamino)pyrimidine-5-carboxylic acid methyl ester (360 mg, 0.711 mmol) in ethanol (5 ml) and add hydrazine hydrate (45.5 mg, 1.421 mmol) under stirring at 0 degrees. Continue stirring the reaction mixture at 85 degrees for 1 hour. Concentrate the reaction mixture, filter it, and wash it with sodium carbonate. Washing with oily ether/ethyl acetate gave a white solid 2-(bis(4-methoxybenzyl)amino)-4-(pentan-2-ylamino)pyrimidin[4,5-d]pyridazin-5(6H)-one (230 mg, 66.2%). MS: 489.2 [M+H] ⁺

Intermediate Int.K

(S)-2-(Bis(4-methoxybenzyl)amino)-4-(1-hydroxypentan-2-yl)amino)pyrimidin[4,5-d]pyridazin-5(6H)-one

Synthesis of Intermediate Int.K Referring to Intermediate Int.A4, Intermediate Int.K was prepared by using (S)-2-aminopentane-1-ol instead of pentane-2-amine. MS: 505.2 [M+H] ⁺

Intermediate I-1-4

Step 1: Preparation of intermediate I-1-1

2-(Bis(4-methoxybenzyl)amino)-4-(butylamino)pyridin[4,3-d]pyrimidin-5(6H)-one (4.0 g, 8.45 mmol), methyl 4-(bromomethyl)benzoate (3.87 g, 16.89 mmol), K ₂ CO ₃ (2.335 g, 16.89 mmol) were dissolved in DMF (40 ml), and the reaction mixture was stirred at 60 degrees for 12 hours. The reaction mixture was poured into ice water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain methyl 4-((2-(bis(4-methoxybenzyl)amino)-4-(butylamino)-5-oxopyridin[4,3-d]pyrimidin-6(5H)-yl)methyl)benzoate (4.6 g, 88%) as a yellow solid. MS: 622.2 [M+H] ⁺ .

Step 2: Preparation of intermediate I-1-2

Intermediate I-1-1 (1.4 g, 3.54 mmol) was dissolved in THF (20 ml), and 1.0 M LAH (3.5 ml, 3.54 mmol) tetrahydrofuran solution. The reaction mixture was heated to 20 degrees and stirred for 1 hour. The reaction mixture was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain a white solid intermediate I-1-2 (1 g, 77%). MS: 594.2 [M + H] ⁺

Step 3: Preparation of intermediate I-1-3

Intermediate I-1-2 (1000 mg, 2.72 mmol) was dissolved in DCM (10 ml), and thionyl chloride (486 mg, 4.08 mmol) was added under stirring at 0°C. The reaction mixture was then stirred at 20°C for 1 hour. The reaction mixture was concentrated to give white solid intermediate I-1-3 (1 g, 95%). MS: 612.2 [M+H] ⁺

Step 4: Preparation of intermediate I-1-4

Intermediate I-1-3 (280 mg, 0.457 mmol) and piperazine (79 mg, 0.915 mmol) were dissolved in EtOH (10 ml), and K ₂ CO ₃ (316 mg, 2.28 mmol) was added under stirring at 20°C; then the reaction mixture was stirred at 100 degrees for 1 hour. The reaction mixture was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain white solid intermediate I-1-4 (150 mg, 61.2%). MS: 662.1 [M+H] ⁺ .

Example Compound II-1

Step 1: Preparation of intermediate II-1-3

Synthesis of Intermediate II-1-3 Referring to Intermediate I-1-4, Intermediate II-1-3 was prepared by using Intermediate Int.C instead of Intermediate Int.A. MS: 807.3 [M+H] ⁺ .

Step 2: Preparation of intermediate II-1-4

The intermediate II-1-3 (3.2 g, 4.7 mmol) was dissolved in trifluoroacetic acid (20 ml), and the reaction mixture was stirred at 80 degrees for 1 hour. The reaction mixture was concentrated and evaporated to dryness, and extracted with ethyl acetate. The organic phase was washed with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow solid intermediate II-1-4 (2.1 g, 85%). MS: 466.5 [M + H] ⁺ .

Step 3: Preparation of Compound II-1

Intermediate II-1-4 (1.7 g, 2.6 mmol) and 2-(2-(2-bromoethoxy)ethoxy]ethane-1-ol (575 mg, 2.7 mmol) were dissolved in acetonitrile (30 ml), and K ₂ CO ₃ (706 mg, 5.1 mmol) was added under stirring at 20°C; then the reaction mixture was stirred at 80 degrees for 4 hours. The reaction mixture was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain a white solid compound II-1 (1.2 g, 80%). MS: 599.2 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.39(d,J＝8.1Hz,1H),7.62(d,J＝7.5Hz,1H),7.08–6.63(m,4H),6.12(d,J＝7.5Hz,1H),5.05– 4.91(m,2H),4.64–4.55(m,1H),4.24(dt,J＝13.9,6.8Hz,1H),3.86(s,3H),3.66(s,2H),3.54(d, J＝6.0Hz,6H),3.50(t,J＝5.2Hz,4H),3.43(t,J＝5.0Hz,4H),3.19(d,J＝4.9Hz,1H),3.00(s,4H),1 .50(qd,J＝8.1,7.3,2.4Hz,2H),1.39–1.29(m,2H),1.16(d,J＝6.5Hz,3H),0.90(t,J＝7.3Hz,3H).

Example Compound II-2

Synthesis of Compound II-2 Referring to Compound II-1, Compound II-2 was prepared by using 17-bromo-3,6,9,12,15-pentaoxaheptadecan-1-ol instead of 2-(2-(2-bromoethoxy)ethoxy]ethane-1-ol. MS: 730.1 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.35 (d, J = 8.2 Hz, 1H), 8.16 (s, 1H), 7.59 (d, J = 7.6 Hz, 1H), 6.98 (s, 1H), 6.92–6.84 (m, 2H), 6.62 (s, 2H), 6.10 (d, J = 7.5 Hz, 1H), 5.03–4.91 (m, 2H), 4.23 (dt, J = 14.0, 6.8 Hz ,1H),3.85(s,4H),3.52(d,J＝1.9Hz,18H),3.45–3.41(m,4H),2.66(s,8H),2.47(s,2H ),1.55–1.45(m,2H),1.41–1.24(m,2H),1.16(d,J＝6.5Hz,3H),0.90(t,J＝7.3Hz,3H).

Example Compound II-3

Synthesis of Compound II-3 Referring to Compound II-1, Compound II-3 was prepared by using 2-bromoethanol instead of 2-(2-(2-bromoethoxy)ethoxy]ethane-1-ol. MS: 510.5 [M+H] ⁺ .

Example Compound II-4

Synthesis of Compound II-4 Referring to Compound II-1, Compound II-4 was prepared by using 3-bromopropanol instead of 2-(2-(2-bromoethoxy)ethoxy]ethane-1-ol. MS: 524.7 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ9.35 (d, J=8.2 Hz, 1H), 8.18 (s, 1H), 7.60 (d, J=7.5 Hz, 1H), 6.99 (s, 1H), 6.88 (q, J=7.7 Hz, 2H), 6.67 (s, 2H), 6.11 (d, J=7.5 Hz, 1H), 5.06–4.90 (m, 2H), 4.24 (dt, J=14.0, 6.8 Hz,1H),3.52(s,2H),3.46(t,J＝6.1Hz,3H),2.82–2.62(m,6H),1.66(q,J＝7.4,6.9Hz,2 H), 1.56–1.46 (m, 2H), 1.40–1.27 (m, 2H), 1.16 (d, J = 6.5Hz, 3H), 0.90 (t, J = 7.3Hz, 3H).

Example Compound II-5

Step 1: Preparation of intermediate II-5-1

Dissolve the intermediate II-1-1 (2.1 g, 3.61 mmol) in MeOH (20 ml), add 0.8 M sodium hydroxide aqueous solution (9 ml, 7.2 mmol) under stirring at 0°C. Continue stirring the reaction mixture at 50 degrees for 14 hours. Cool the reaction solution, pour the reaction mixture into ice water, adjust the Ph value to 4 with 1N hydrochloric acid, and extract with ethyl acetate. Wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter, and concentrate to obtain a crude product, which is purified by silica gel column chromatography to obtain the intermediate II-5-1 (1.6 g, 85%). MS: 652.2 [M + H] ⁺ .

Step 2: Preparation of intermediate II-5-2

A mixed solution of intermediate II-5-1 (1.6 g, 3.1 mmol), HATU (2.3 g, 6.2 mmol), DIEA (3.2 ml,) and 2-(piperazine-1-yl)ethane-1-ol (1.2 g, 9.3 mmol) in DMF (20 ml) was stirred at 0 degrees for 2 hours. The reaction mixture was then poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product, which was purified by silica gel column chromatography to give intermediate II-5-2 (2.0 g, 85%). MS: 765.2 [M+H] ⁺ .

Step 3: Preparation of compound II-5

The intermediate II-5-2 (2 g, 2.6 mmol) was dissolved in trifluoroacetic acid (20 ml), and the reaction mixture was stirred at 80 degrees for 1 hour. The reaction mixture was concentrated and evaporated to dryness, and extracted with ethyl acetate. The organic phase was washed with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain compound II-5. MS: 524.7 [M + H] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ9.37(d,J＝8.2Hz,1H),7.66(d,J＝7.5Hz,1H),7.05(s,1H),6.95(d,J＝1.7H z,2H),6.76(s,2H),6.14(d,J＝7.5Hz,1H),5.10–4.94(m,2H),4.23(dt,J＝13 .9,6.8Hz,1H),3.89(s,3H),3.57(s,6H),2.62(s,7H),1.49(ddd,J＝8.6,6.3 ,3.1Hz,2H),1.37–1.25(m,2H),1.16(d,J＝6.5Hz,3H),0.90(t,J＝7.3Hz,3H).

Example Compound II-6

Synthesis of Compound II-6 Referring to Compound II-5, Compound II-6 was prepared by using 3-(piperazin-1-yl)propan-1-ol instead of 2-(piperazin-1-yl)ethan-1-ol. MS: 538.7 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.32(d,J＝8.2Hz,1H),7.62(d,J＝7.5Hz,1H),7.03(s,1H),6.93(s,2H),6.64( s,2H),6.12(d,J＝7.5Hz,1H),5.09–4.93(m,2H),4.23(p,J＝6.7Hz,1H),3.88(s, 3H),3.61(s,2H),3.44(t,J＝6.3Hz,4H),2.39(s,7H),1.60(q,J＝6.8Hz,2H),1.5 7–1.44(m,2H),1.41–1.23(m,2H),1.16(d,J＝6.5Hz,3H),0.90(t,J＝7.3Hz,3H).

Example Compound II-7

Step 1: Preparation of intermediate II-7-1

Synthesis of Intermediate II-7-1 Referring to compound II-5, Intermediate II-7-1 was prepared by using Intermediate Int.G2 instead of Intermediate II-5-2. MS: 396.1 [M+H] ⁺ .

Step 2: Preparation of compound II-7

A solution of 2-amino-2-(hydroxymethyl)propane-1,3-diol (103 mg, 0.9 mmol), acetic acid (34 mg, 0.6 mmol), and intermediate Int.G2 (225 mg, 0.6 mmol) in acetonitrile (8 ml) was stirred at 20 degrees for 10 minutes; then sodium acetate borohydride (127 mg, 0.6 mmol) was added to the above reaction mixture, and the reaction mixture was stirred at 20 degrees for 16 hours. The reaction mixture was poured into ice water to quench and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain compound II-7 (89 mg, 31%). MS: 501.6 [M + H] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ9.35(d,J＝8.2Hz,1H),8.25(s,1H),7.57(d,J＝7.5Hz,1H),7.10(s,1H),6.92(s,2H),6.60(s,2H),6.09(d,J＝7.5Hz,1H),5.05–4.90(m,2H), 4.27–4.20(m,1H),3.85(d,J＝9.8Hz,5H),3.47(s,6H),1.50(s,2H),1.3 4(dt,J=13.5,7.1Hz,2H), 1.17(d,J=6.5Hz,3H), 0.91(t,J=7.3Hz,3H).

Example Compound II-8

The synthesis of compound II-8 was based on compound II-7 by using 2-aminopropane-1,3-diol instead of 2-amino- 2-(Hydroxymethyl)propane-1,3-diol was used to prepare compound II-8. MS: 471.6 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.33 (d, J = 8.3 Hz, 1H), 8.19 (s, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.28 (s, 1H), 7.02 (d, J = 7.8 Hz, 1H), 6.95 (d, J = 7.7 Hz, 1H), 6.62 (s, 2H), 6.11 (d, J = 7.5 Hz, 1H), 5.07–4.92 (m, 2H), 4.24 (dt, J ＝13.9,6.8Hz,1H),4.08(s,2H),3.88(s,3H),3.67–3.54(m,6H),2.95–2.83(m,1H),1.50(q,J =6.8, 5.1Hz, 2H), 1.34 (dt, J = 13.3, 7.1Hz, 2H), 1.16 (d, J = 6.5Hz, 3H), 0.91 (t, J = 7.3Hz, 3H).

Example Compound II-9

Synthesis of Compound II-9 Referring to Compound II-7, Compound II-9 was prepared by using 2-amino-2-methylpropane-1,3-diol instead of 2-amino-2-(hydroxymethyl)propane-1,3-diol. MS: 485.6 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.36(d,J＝8.3Hz,1H),8.19(s,1H),7.56(d,J＝7.6Hz,1H),7.10(s,1H),6.9 4–6.84(m,2H),6.60(s,2H),6.09(d,J＝7.5Hz,1H),5.04–4.89(m,2H),4.24(p ,J＝6.9Hz,1H),3.85(s,3H),3.74–3.66(m,7H),1.55–1.45(m,2H),1.35(dd,J =13.9,7.0Hz,2H),1.17(d,J=6.6Hz,3H),1.00(s,3H),0.91(t,J=7.3Hz,3H).

Example Compound II-10

Synthesis of Compound II-10 Referring to Compound II-1, Compound II-10 was prepared by using (14-bromo-3,6,9,12-tetrahydrotetradecyl)carbamic acid tert-butyl ester instead of 2-(2-(2-bromoethoxy)ethoxy]ethane-1-ol and Intermediate III-6-3 instead of Intermediate II-1-4. MS: 701.4 [M+H] ⁺ .

Example Compound II-11

The synthesis of compound II-11 refers to compound II-1, by using tert-butyl 2-(2-(2-bromoethoxy)ethoxyethyl)carbamate instead of 2-(2-(2-bromoethoxy)ethoxy]ethane-1-ol and intermediate III-6-3 instead of Intermediate II-1-4 was used to prepare compound II-11. MS: 613.6 [M+H] ⁺ .

Example Compound II-12

Synthesis of Compound II-12 Referring to Compound II-7, Compound II-12 was prepared by using 2-methylaminoethanol instead of 2-amino-2-(hydroxymethyl)propane-1,3-diol. MS: 455.1 [M+H] ⁺ .

Example Compound III-1

Synthesis of Compound III-1 Referring to Compound III-2, Compound III-1 was prepared by using 1,14-diiodo-3,6,9,12-tetraoxotetradecane instead of 1,20-diiodo-3,6,9,12,15,18-hexaoxoeicosane. MS: 800.8 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.44(d,J＝8.3Hz,1H),7.65(d,J＝7.5Hz,1H),7.09(s,1H),6.93(q,J＝7.8Hz ,3H),6.14(d,J＝7.5Hz,1H),5.06–4.92(m,2H),3.87(s,4H),3.74–3.58(m,8H ),3.57–3.47(m,10H),2.88(s,6H),2.68(s,4H),2.33–2.11(m,2H),1.50(s,2 H), 1.34 (dt, J = 13.3, 6.8 Hz, 2H), 1.17 (d, J = 6.5 Hz, 3H), 0.90 (t, J = 7.3 Hz, 3H).

Example Compound III-2

Step 1: Preparation of intermediate III-2-3

Under nitrogen protection and stirring at -80 degrees, 2M LDA tetrahydrofuran solution (17.7 ml, 35.5 mmol) was slowly added to a tetrahydrofuran (150 ml) solution of 1,20-diiodo-3,6,9,12,15,18-hexaoxoeicosane (14.9 g, 27.3 mmol) and diethyl (difluoromethyl)phosphonate (6.7 g, 35.5 mmol), and then the reaction mixture was stirred at -80 degrees for 2 hours. The reaction mixture was then poured into ice water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain a yellow oil intermediate III-2-3 (1.9 g, 11%). MS: 607.4 [M + H] ⁺ .

Step 2: Preparation of intermediate III-2-1

Synthesis of Intermediate III-2-1 Referring to compound II-1, Intermediate III-2-1 was prepared by using Intermediate III-2-3 instead of 2-(2-(2-bromoethoxy)ethoxy]ethan-1-ol. MS: 945.1 [M+H] ⁺ .

Step 3: Preparation of compound III-2

The intermediate III-2-1 (109 mg, 0.1 mmol) was dissolved in dichloromethane (3 ml), and TMSBr (3540 mg, 23.12 mmol) was slowly added under stirring at 20°C; the reaction mixture was then stirred at 20°C for 16 hours. The reaction mixture was concentrated to obtain a crude product, which was purified to obtain a white solid compound III-2 (29 mg, 28%). MS: 889.4 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.44(d,J＝8.0Hz,1H),7.65(d,J＝7.5Hz,1H),7.07(s,1H),6.92(q,J＝7.8Hz,3H),6.15(d,J ＝7.5Hz,1H),5.07–4.93(m,2H),4.24(p,J＝6.9Hz,1H),3.87(s,3H),3.65(dd,J＝16.6,9.0Hz, 10H),3.51–3.47(m,20H),2.87(s,6H),2.66(s,4H),2.21(ddt,J＝19.4,13.1,6.8Hz,2H),1.5 1(td,J＝7.7,7.0,4.6Hz,2H),1.41–1.25(m,2H),1.17(d,J＝6.5Hz,3H),0.91(t,J＝7.3Hz,3H).

Example Compound III-3

Step 1: Preparation of intermediate III-3-1

Synthesis of Intermediate III-3-1 Referring to compound II-7, intermediate III-3-1 was prepared by using methylamine instead of 2-amino-2-(hydroxymethyl)propane-1,3-diol. MS: 531.6 [M+H] ⁺ .

Step 2: Preparation of intermediate III-3-2

Synthesis of Intermediate III-3-2 Referring to Intermediate II-7-1, Intermediate III-3-2 was prepared by using Intermediate III-3-1 instead of Intermediate Int.G2. MS: 411.6 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.30 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H), 7.00 (d, J = 5.4 Hz, 2H), 6.85 (s, 2H), 6.72 (d, J = 7.9 Hz, 1H), 6.57 (s, 2H), 6.05 (d, J = 7.5 Hz, 1H), 4.99–4.88 (m, 2H), 4.46 (s, 1H), 3 .81(d,J＝7.8Hz,3H),3.53–3.40(m,16H),2.90(s,2H),2.64–2.57(m,2H),2.02–1.93(m ,H),1.49–1.43(m,2H),1.32–1.27(m,2H),1.12(d,J＝6.4Hz,3H),0.86(t,J＝7.4Hz,3H).

Step 3: Preparation of compound III-3

Synthesis of Compound III-3 Referring to Compound III-1, Compound III-3 was prepared by using Intermediate III-3-2 instead of Intermediate II-1-4. MS: 745.9 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.63 (s, 0H), 7.81 (d, J = 7.5 Hz, 1H), 7.28 (s, 1H), 7.01 (q, J = 7.8 Hz, 2H), 6.27 (d, J = 7.5 Hz, 1H), 5.12–4.97 (m, 2H), 4.31–4.21 (m, 1H), 4.02 (s, 2H), 3.89 (s, 3H), 3.73 (s, 2H),3.66(t,J＝7.5Hz,4H),3.47(d,J＝5.0Hz,6H),3.44(s,3H),2.32–2.11(m,2H),1. 59–1.50(m,2H),1.35(q,J＝7.1Hz,2H),1.19(d,J＝6.5Hz,3H),0.90(d,J＝7.3Hz,3H).

Example Compound III-4

Step 1: Preparation of intermediate III-4-1

Synthesis of Intermediate III-4-1 Referring to compound II-5, intermediate III-4-1 was prepared by using tert-butyl piperazine-1-carboxylate instead of 2-(piperazin-1-yl)ethan-1-ol. MS: 481.2 [M+H] ⁺ .

Step 2: Preparation of compound III-4

Synthesis of Compound III-4 Referring to Compound III-2, Compound III-4 was prepared by using Intermediate III-4-1 instead of Intermediate II-1-4 and Intermediate III-4-3 instead of Intermediate III-2-3. MS: 726.8 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.45 (d, J = 8.4 Hz, 1H), 7.65 (d, J = 7.5 Hz, 1H), 7.07–7.00 (m, 2H), 7.00–6.90 (m, 2H), 6.17 (d, J = 7.4 Hz, 1H), 5.11–4.96 (m, 2H), 4.30–4.18 (m, 2H), 3.88 (s, 3H), 3.6 8(t,J＝7.4Hz,4H),3.62(s,4H),3.51(s,4H),2.74(s,6H),2.26–2.14(m,2H),1.57 –1.47(m,2H),1.34(p,J＝6.8Hz,2H),1.17(d,J＝6.5Hz,3H),0.90(t,J＝7.3Hz,3H).

Example Compound III-5

Step 1: Preparation of intermediate III-5-1

Synthesis of Intermediate III-5-1 Referring to Intermediate III-6-3, Intermediate III-5-1 was prepared by using 4-methylpiperazine instead of tert-butyl piperazine-1-carboxylate. MS: 496.3 [M+H] ⁺ .

Step 2: Preparation of intermediate III-5

Synthesis of Compound III-5 Referring to Compound III-4, Compound III-5 was prepared by using Intermediate III-5-1 instead of Intermediate III-4-1. MS: 742.8 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.42 (d, J = 8.6 Hz, 1H), 7.59 (d, J = 7.5 Hz, 1H), 6.99 (s, 1H), 6.97–6.79 (m, 2H), 6.63 (s, 1H), 6.09 (d, J = 7.5 Hz, 1H), 5.09–4.82 (m, 2H), 4.20 (d, J = 7.3 Hz, 1H), 3.85 (s, 3H), 3.65 (q, J = 7.4, 6.7 Hz, 5H), 3.58 ( d,J＝7.2Hz,4H),3.54–3.47(m,7H),3.15(s,3H),2.84–2.64(m,4H),2.16(ddt,J＝20.6,13.7,6.5Hz,2H ), 1.60 (q, J = 7.2Hz, 1H), 1.46 (q, J = 6.1, 5.4Hz, 1H), 1.32 (p, J = 7.7, 7.2Hz, 2H), 0.89 (t, J = 7.3Hz, 3H).

Example Compound III-6

Step 1: Preparation of intermediate III-6-3

Synthesis of Intermediate III-6-3 Referring to Intermediate II-1-4, Intermediate III-6-3 was prepared by using Intermediate Int.D instead of Intermediate Int.C. MS: 482.3 [M+H] ⁺ .

Step 2: Preparation of intermediate III-6

Synthesis of Compound III-6 Referring to Compound III-2, Compound III-6 was prepared by using Intermediate III-6-3 instead of Intermediate II-1-4 and Intermediate III-4-3 instead of Intermediate III-2-3. MS: 728.8 [M+H] ⁺ . ¹ HNMR (400 MHz, DMSO-d ₆ ) δ 10.07 (d, J = 8.7 Hz, 1H), 7.91 (d, J = 7.5 Hz, 1H), 7.08 (d, J = 7.2 Hz, 2H), 6.92 (d, J = 7.7 Hz, 1H), 6.38 (d, J = 7.4 Hz, 1H), 5.13–4.99 (m, 2H), 4.25 (t, J = 7.4 Hz, 1H), 3.84 (s, 3H), 3.7 5–3.64(m,6H),3.53(d,J＝3.8Hz,6H),3.11(s,6H),2.78(s,4H),2.22(ddt,J＝19.3,12.5 ,6.7Hz,2H),1.57(dt,J＝34.1,7.5Hz,2H),1.33(q,J＝7.6Hz,2H),0.90(t,J＝7.3Hz,3H).

Example Compound III-7

Synthesis of Compound III-7 Referring to Compound III-6, Compound III-7 was prepared by using Intermediate III-9-3 instead of Intermediate III-4-3. MS: 696.8 [M+H] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ9.54(d,J＝8.6Hz,1H),7.65(d,J＝7.5Hz,1H),7.04–6.76(m,4H),6.14(d,J＝7.4Hz,1H),4.98(q,J＝15.3Hz,2H),4.49–4.18(m,3H) ,3.84(s,3H),3.59–3.40(m,4H),2.84(t,J＝74.9Hz,8H),1.92(s,2H),1.74–1.41(m,6H),1.40–1.18(m,6H),0.89(t,J＝7.3Hz,3H).

Example Compound III-8

Synthesis of Compound III-8 Referring to Compound III-2, Compound III-8 was prepared by using Intermediate III-4-3 instead of Intermediate III-2-3. MS: 722.7 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.04 (d, J = 8.3 Hz, 1H), 8.37 (s, 1H), 7.96 (d, J = 7.5 Hz, 1H), 7.16–7.05 (m, 2H), 6.94 (d, J = 7.8 Hz, 1H), 6.44 (d, J = 7.5 Hz, 1H), 5.08 (d, J = 6.1 Hz, 2H), 4.29 (p, J = 6.8 Hz, 1H), 3.85 (s, 3H), 3.78 (s, 2H), 3.73 (q, J = 4.6Hz,2H),3.67(t,J＝7.3Hz,2H),3.53(p,J＝4.7,3.5Hz,4H),3.17(s,6H),2.84(s,4H),2.23(t,J＝6.7H z,2H),1.64–1.50(m,2H),1.35(td,J＝8.7,7.8,5.0Hz,2H),1.22(d,J＝6.5Hz,3H),0.91(t,J＝7.3Hz,3H).

Example Compound III-9

Synthesis of Compound III-9 Referring to Compound III-2, Compound III-9 was prepared by using Intermediate III-9-2 instead of Intermediate III-2-2. MS: 680.75 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.73 (d, J = 8.2 Hz, 1H), 7.85 (d, J = 7.5 Hz, 1H), 7.04–6.93 (m, 2H), 6.86 (d, J = 7.8 Hz, 1H), 6.29 (d, J = 7.4 Hz, 1H), 5.08–4.94 (m, 2H), 4.32–4.19 (m, 1H), 3.83 (s, 3H), 3. 55(s,2H),3.35(t,J＝6.5Hz,1H),2.88(s,2H),1.96(d,J＝32.4Hz,3H),1.67(s,2H), 1.57–1.43(m,5H),1.41–1.22(m,9H),1.18(d,J＝6.5Hz,3H),0.89(t,J＝7.3Hz,3H).

Example Compound III-10

Step 1: Preparation of intermediate III-10-2

Under stirring conditions at 20 degrees, tetrazole (107 mg, 1.53 mmol) was slowly added dropwise to the dichloromethane (30 ml) reaction solution of compound II-4 (400 mg, 0.765 mmol), and the reaction solution was stirred at 20 degrees for 0.5 hours; then the obtained reaction mixture was slowly added dropwise to the dichloromethane (10 ml) solution of intermediate III-10-1 (1175 mg, 1.53 mmol) at 0 degrees, and the obtained reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was poured into ice water and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude yellow solid intermediate III-10-2 (350 mg).

Step 2: Preparation of intermediate III-10-3

Under stirring at 20 degrees, a mixed solution of iodine in tetrahydrofuran/water/pyridine (0.05M, 15 ml, THF/H ₂ O/Py＝70:20:10) was slowly added dropwise to a reaction solution of intermediate III-10-2 (350 mg, 0.276 mmol) in dichloromethane (10 ml), and the reaction solution was stirred at 20 degrees for 3 hours; the reaction mixture was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain a yellow solid intermediate III-10-3 (240 mg). MS: 604.8[1/2M+H] ⁺ .

Step 3: Preparation of compound III-10

Under stirring conditions at 20 degrees, DBU (152 mg, 0.995 mmol) was slowly added dropwise to the dichloromethane (10 ml) reaction solution of the intermediate III-10-3 (240 mg, 0.199 mmol), and the reaction solution was stirred at 20 degrees for 2 hours; the reaction mixture was poured into ice water and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain yellow solid compound III-10 (30 mg). ¹ H NMR(400MHz,Chloroform-d)δ9.79(d,J＝8.2Hz,1H),7.45(d,J＝7.6Hz,1H),7.21(d,J＝7.7Hz,1H),6.93–6.84(m,2H),6.38(d,J＝7.5Hz,1 H),5.23(dt,J＝8.8,4.4Hz,1H),5.04(q,J＝14.5Hz,2H),4.40(dd,J＝12.0,3.1Hz,1H),4.29(dt,J＝14.1,6.9Hz,1H),4.18(dd,J＝12.0,6. 6Hz,1H),3.99(dt,J＝18.5,6.1Hz,4H),3.85(s,3H),3.54–3.43(m,3H),2.78(d,J＝20.9Hz,4H),2.64(s,4H),2.28(q,J＝7.5Hz,4H),1.99 (d,J＝13.0Hz,2H),1.81–1.75(m,1H),1.61–1.52(m,5H),1.43–1.35(m,2H),1.25(s,52H),0.93(t,J＝7.3Hz,3H),0.88(t,J＝6.8Hz,6H).

Example Compound III-11

Step 1: Preparation of intermediate III-11-1

Under stirring conditions at 0 degrees, benzyl bromide (45.7 g, 0.267 mol) and NaH (3.9 g, 0.098 mol) were slowly added to a DMF (200 ml) solution of 2-(2-chloroethoxy)ethoxyethane-1-ol (15 g, 0.089 mol), and the reaction solution was stirred at 20 degrees for 16 hours; the reaction mixture was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain a yellow oil intermediate III-11-1 (13.8 g, 60%). MS: 259.2 [M+H] ⁺ .

Step 2: Preparation of intermediate III-11-2

Under stirring at 0°C, NaH (880 mg, 22 mmol) and TBAI (406 mg, 1.1 mmol) were added in sequence. Slowly add (2,2-dimethyl-1,3-dioxolane-4-yl)methanol (1.2 g, 11 mmol) in tetrahydrofuran (50 ml), and continue to stir the reaction solution at 0 degrees for 1 hour; then slowly add intermediate III-11-1 (1175 mg, 1.53 mmol) to the above reaction solution under stirring at 20 degrees, and continue to stir the reaction solution at 68 degrees for 16 hours. Pour the reaction mixture into ice water and extract with ethyl acetate. The organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which is purified by silica gel column chromatography to obtain yellow oil intermediate III-11-2 (1.4 g, 37%). MS: 377.2 [M + Na] ⁺ .

Step 3: Preparation of intermediate III-11-3

1M hydrochloric acid solution (5 ml) was added to a solution of intermediate III-11-2 (1.4 g, 4 mmol) in methanol (20 ml), and the reaction solution was stirred at 20 degrees for 4 hours; the reaction mixture was concentrated to obtain a white solid intermediate III-11-3 (1.2 g). MS: 315.6 [M+H] ⁺ .

Step 4: Preparation of intermediate III-11-4

Palmitic acid (2.9 g, 11.4 mmol), DMAP (140 mg, 1.14 mmol) and EDCI (2.2 g, 11.4 mmol) were added to a DCM (40 ml) solution of intermediate III-11-3 (1.2 g, 3.8 mmol) under stirring at 20 degrees, and the reaction solution was stirred at 20 degrees for 16 hours. The reaction mixture was poured into ice water and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product, which was purified by silica gel column chromatography to give a white solid intermediate III-11-4 (1.8 g, 60%). ¹ H NMR (400MHz, CDCl ₃ )δ7.36–7.27(m,5H),5.20(m,1H),4.57(s,2H),4.33(m,1H),4.15(m,1H),3.72–3.5 6(m,14H),2.30(m,4H),1.60(d,J＝4.0Hz,4H),1.25(s,48H),0.88(t,J＝6.8Hz,6H).

Step 5: Preparation of intermediate III-11-5

The intermediate III-11-4 (350 mg, 0.44 mmol) was dissolved in a solution of ethyl acetate (20 ml) and methanol (2 ml), palladium carbon (300 mg) was added to the reaction mixture under stirring conditions, and then the reaction mixture was stirred under hydrogen pressure conditions at room temperature for 16 hours; the reaction mixture was filtered and concentrated to give a white solid intermediate III-11-5 (300 mg, 88%). ¹ H NMR (400MHz, CDCl ₃ )δ5.15(s,1H),4.28(dd,J＝13.7,5.4Hz,1H),4.12–4.01(m,1H),3.73–3.47(m,14H),2.31– 2.15(m,5H),1.84–1.71(m,3H),1.54(d,J＝6.7Hz,4H),1.18(s,49H),0.81(t,J＝6.8Hz,6H).

Step 6: Preparation of intermediate III-11-6

Under stirring conditions at 20 degrees, tetrazole (60 mg, 0.86 mmol) was slowly added dropwise to the dichloromethane (20 ml) reaction solution of intermediate III-11-5 (300 mg, 0.43 mmol), and the reaction solution was stirred at 20 degrees for 0.5 hours; then bis(diisopropylamino)(2-cyanoethoxy)phosphine (260 mg, 0.86 mmol) was slowly added dropwise to the above reaction mixture, and the obtained reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was poured into ice water and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain a white solid intermediate III-11-6 (150 mg, 33%). ¹ H NMR (400MHz, CD ₃ CN)δ5.14(dtd,J＝6.7,5.3,3.5Hz,1H),4.28–4.22(m,1H),4.14–4.04(m,1H),3.84–3.66(m,4H),3.63–3.50(m,14H),2.65(m,2 H), 2.27 (td, J = 7.4, 4.5Hz, 4H), 1.56 (m, 4H), 1.27 (s, 48H), 1.18 (d, J = 1.9Hz, 6H), 1.16 (d, J = 1.9Hz, 5H), 0.88 (t, J = 6.8Hz, 6H).

Step 7: Preparation of Compound III-11

Synthesis of Compound III-11 Referring to Compound III-10, Compound III-11 was prepared by using Intermediate III-11-6 instead of Intermediate III-10-1. ¹ H NMR (400 MHz, Chloroform-d) δ9.74 (d, J = 8.2 Hz, 1H), 7.40 (d, J = 7.5 Hz, 1H), 7.15 (d, J = 7.7 Hz, 1H), 6.86 (s, 1H), 6.81 (d, J = 7.7 Hz, 1H), 6.32 (d, J = 7.5 Hz, 1H), 5.19–5.11 (m, 1H), 5.05–4.94 (m, 2H), 4.31–4.23 (m, 1H),4.09(dd,J＝11.9,6.5Hz,1H),4.01–3.89(m,4H),3.80(s,3H),3.66–3. 52(m,12H),3.46(d,J＝13.3Hz,3H),2.89(s,1H),2.78(s,4H),2.61(s,3H),2 .25(q,J＝7.4Hz,4H),1.94(d,J＝12.5Hz,2H),1.64(s,1H),1.60–1.49(m,5H ), 1.35 (dt, J = 14.0, 6.8Hz, 2H), 1.20 (s, 52H), 0.85 (dt, J = 18.2, 7.1Hz, 9H).

Example Compound III-12

Synthesis of Compound III-12 Referring to Compound III-10, Compound III-12 was prepared by using Intermediate III-12-1 instead of Intermediate III-10-1 and Compound II-1 instead of Compound II-4. ¹ H NMR (400 MHz, Chloroform-d) δ11.43 (s, 1H), 9.75 (d, J = 8.1 Hz, 1H), 7.40 (d, J = 7.5 Hz, 1H), 7.15 (d, J = 7.6 Hz, 1H), 6.82 (d, J = 7.5 Hz, 1H), 6.31 (d, J = 7.4 Hz, 1H), 5.17 (s, 1H), 4.97 (q, J = 14.5 Hz, 2H), 4.34 (d, J = 9.7 Hz, 1H), 4.23 (dd, J = 13.6, 6.7 Hz, 1H), 4.12 (dd, J = 11.7 ,6.7Hz,1H),3.96(s,4H),3.79(s,3H),3.72(s,2H),3.62–3.51(m,7H),3.45(dd,J＝15.5,7.5Hz,3H),3.38(s,2H),2.93–2.59(m,9H) ,2.21(q,J＝7.0Hz,4H),2.03–1.93(m,2H),1.64(s,1H),1.50(s,5H),1.36–1.31(m,2H),1.18(s,52H),0.83(dt,J＝18.7,7.1Hz,9H).

Example Compound III-16

Synthesis of Compound III-16 Referring to Compound III-10, Compound III-16 was prepared by using Compound II-1 instead of Compound II-4. ^1H NMR(400MHz,Chloroform-d)δ9.83(d,J＝8.2Hz,1H),7.44(d,J＝7.5Hz,1H),7.20(d,J＝7.6Hz,1H),6.90(s,1H),6.85(d,J＝7.7Hz,1H),6.40(d,J＝7.5Hz ,1H),5.21(dt,J＝8.8,4.4Hz,1H),5.01(q,J＝14.4Hz,2H),4.38(dd,J＝12.0 ,3.2Hz,1H),4.30–4.22(m,1H),4.17(dd,J＝12.0,6.5Hz,1H),4.01(t,J＝5.7 Hz,4H),3.82(s,3H),3.69(d,J＝4.7Hz,2H),3.67–3.45(m,19H),2.78(s,5H),2.60(s,4H),2.25(q,J＝7.2Hz,4H ),1.53(dd,J＝17.8,7.0Hz,5H),1.39–1.33(m,2H),1.22(s,54H),0.90(t,J＝7.3Hz,3H),0.84(t,J＝6.7Hz,6H).

Example Compound III-19

Synthesis of Compound III-19 Referring to Compound III-2, Compound III-19 was prepared by using Compound III-6-3 instead of Compound II-1-4. MS: 904.4 (M+H) ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.66 (d, J = 8.6 Hz, 1H), 7.74 (d, J = 7.5 Hz, 1H), 7.07 (s, 1H), 7.01–6.87 (m, 2H), 6.21 (d, J = 7.5 Hz, 1H), 5.08–4.93 (m, 2H), 4.23 (d, J = 8.2 Hz, 1H), 3.86 (s, 3H), 3.67 (dd, J = 18.2, 10.5 Hz, 9H) ,3.48(dd,J＝12.5,6.2Hz,22H),2.98(s,6H),2.70(s,4H),2.22(ddt,J＝19.5,12.7,6.5Hz,2H) ,1.55(dtd,J＝51.7,13.8,7.4Hz,2H), 1.33(td,J＝9.0,8.5,4.2Hz,2H), 0.90(t,J＝7.3Hz,3H).

Example Compound IV-8

Step 1: Preparation of compound IV-8-1

Under nitrogen protection, a reaction mixture of compound A2 (200 mg, 0.092 mmol), compound III-6-3 (58 mg, 0.12 mmol) and TEA (47 mg, 0.46 mmol) in acetonitrile (10 ml) was stirred at 20 degrees for 4 hours; then the reaction mixture was poured into ice water and extracted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain a white solid compound IV-8-1 (170 mg, 74%). MS: 1236.0 (1/2M+H) ⁺ .

Step 2: Preparation of compound IV-8

Compound IV-8-1 (170 mg, 0.092 mmol) and 7M methanolic ammonia solution (10 ml) were stirred at 20 degrees for 4 hours; the reaction mixture was then concentrated to obtain a crude product, which was purified to obtain a white solid compound IV-8 (58.72 mg, 40%). MS: 1046.8 (1/2M+H) ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.40(m,1H),7.84(m,3H),7.74(m,3H),7.62(m,3H),7.56(d,J＝7.6Hz,1H),6.98(m,2H),6.86(m,2H),6.54(s,2H),6.07(d,J ＝7.5Hz,1H),5.02–4.85(m,2H),4.78(t,J＝5.1Hz,1H),4.61–4.54(m,6H),4.47(d,J＝4.3Hz,3H),4.22(d,J＝8.4Hz,4H),3.83(s ,3H),3.68(m,10H),3.59–3.39(m,32H),3.38–3.34(m,3H),3.29(t,J＝6.2Hz,3H),3.02(m,13H),2.37–2.21(m,13H),2.05(dd, J＝13.6,6.6Hz,9H),1.80(s,9H),1.57(d,J＝7.5Hz,1H),1.47(m,25H),1.35–1.26(m,3H),1.22(s,13H),0.88(t,J＝7.3Hz,3H).

Example Compound IV-6

Synthesis of Compound IV-6 Compound IV-6 was prepared by referring to Compound IV-8. ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.40 (d, J＝8.6 Hz, 1H), 7.85 (t, J＝5.4 Hz, 4H), 7.74 (t, J＝5.6 Hz, 3H), 7.62 (d, J＝9.1 Hz, 3H), 7.56 (d, J＝7.6 Hz, 1H), 7.04 (s, 1H), 6.94 (s, 1H), 6.87 (d, J＝7.7 Hz, 1H), 6.81 (d, J＝8.5 Hz, 1H), 6.54 (s, 2H ),6.06(d,J＝7.5Hz,1H),4.94(dd,J＝30.1,15.3Hz,2H),4.77(t,J＝5.2Hz,1H),4.61–4.51(m,6H),4 .47(d,J＝4.3Hz,3H),4.22(d,J＝8.4Hz,4H),3.82(s,3H),3.67(dt,J＝7.4,6.1Hz,9H),3.57–3.51(m, 15H),3.50–3.45(m,18H),3.45–3.35(m,10H),3.31–3.26(m,6H),3.18(dd,J＝11.5,5.7Hz,3H),3.08–2.99(m,12H),2.41(dd,J＝27.0,2 1.3Hz,10H),2.28(t,J＝6.3Hz,7H),2.03(d,J＝7.3Hz,10H),1.80(s,9H),1.70–1.55(m,4H),1.55–1.25(m,22H),0.88(t,J＝7.3Hz,3H).

Example Compound IV-7

Synthesis of Compound IV-7 Compound IV-7 was prepared by referring to Compound IV-8. ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.40(d,J＝8.5Hz,1H),7.84(t,J＝5.5Hz,3H),7.74(t,J＝5.5Hz,3H),7.62(d, J＝9.0Hz,3H),7.56(d,J＝7.6Hz,1H),6.98(d,J＝10.8Hz,2H),6.86(dd,J＝17.2,7 .8Hz,2H),6.54(s,2H),6.07(d,J＝7.5Hz,1H),5.02–4.85(m,2H),4.78(t,J＝5. 1Hz,1H),4.61–4.54(m,6H),4.47(d,J＝4.3Hz,3H),4.22(d,J＝8.4Hz,4H),3.83( s,3H),3.68(ddd,J＝13.1,7.9,2.9Hz,10H),3.59–3.39(m,32H),3.38–3.34(m, 3H),3.29(t,J＝6.2Hz,3H),3.02(dd,J＝10.9,5.7Hz,13H),2.37–2.21(m,13H),2 .05(dd,J＝13.6,6.6Hz,9H),1.80(s,9H),1.57(d,J＝7.5Hz,1H),1.47(ddd,J＝1 4.9, 12.0, 5.9Hz, 25H), 1.35–1.26 (m, 3H), 1.22 (s, 13H), 0.88 (t, J = 7.3Hz, 3H).

Example Compound III-15

Step 1: Preparation of compound III-15-1

Under nitrogen protection and stirring at 0 degrees, DMAP (0.14 g, 11.5 mmol), TEA (3.8 ml) and TBDPSCl (12.6 g, 46 mmol) were added to a solution of compound III-15-A (3 g, 23 mmol) in dichloromethane (100 ml) in sequence, and then the reaction mixture was stirred at 20 degrees for 16 hours. The reaction mixture was poured into ice water and extracted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain yellow oily compound III-15-1 (4.3 g, 51%). MS: 393.2 (M+Na) ⁺ .

Step 2: Preparation of compound III-15-2

Under stirring conditions at 0 degrees, 1M hydrochloric acid solution (15 ml) was added to a solution of compound III-15-1 (4.3 g, 12 mmol) ethanol (45 ml) and water (15 ml), and then the reaction mixture was stirred at 20 degrees for 1 hour. The reaction mixture was poured into ice water and extracted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain yellow oily compound III-15-2 (1.2 g, 31%). MS: 353.2 (M+Na) ⁺ .

Step 3: Preparation of compound III-15-3

Under stirring conditions at 0 degrees, EDCI (1.73 g, 9 mmol), DMAP (22 mg, 0.2 mmol) and oleic acid (2.54 g, 9 mmol) were added to a solution of compound III-15-2 (1.2 g, 3.6 mmol) in dichloromethane (60 ml), and then the reaction mixture was stirred at 20 degrees for 16 hours. The reaction mixture was poured into ice water and extracted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain yellow oily compound III-15-3 (1.6 g, 51%). ¹ H NMR (400MHz, CDCl ₃ )δ5.40–5.28(m,4H),5.11–5.02(m,1H),4.36–4.06(m,3H),3.77–3.70(m,2H),2.33(dd,J＝16.4,7.6 Hz,4H),2.07–1.95(m,7H),1.62(dd,J＝13.1,6.6Hz,4H),1.39–1.21(m,41H),0.88(t,J＝6.8Hz,6H).

Step 4: Preparation of compound III-15-4

A reaction mixture of compound III-15-3 (1.6 g, 1.8 mmol) and triethylamine hydrogen fluoride salt (5 ml) in acetonitrile (10 ml), dichloromethane (10 ml) and tetrahydrofuran (10 ml) was stirred at 20 degrees for 3 hours. The combined solution was poured into ice water and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified to obtain yellow oily compound III-15-4 (0.9 g, 78%). ¹ HNMR (400MHz, CDCl ₃ )δ5.40–5.28(m,4H),5.12–5.04(m,1H),4.32(dd,J＝11.9,4.5Hz,1H),4.27–4.07(m,2H),3.73(d,J＝5.3Hz,1H) ,2.33(dd,J＝16.4,7.7Hz,4H),2.05–1.96(m,7H),1.68–1.56(m,4H),1.36–1.22(m,42H),0.88(t,J＝6.7Hz,6H).

Step 5: Preparation of Compound III-15

Synthesis of Compound III-15 Compound III-15 was prepared by referring to Compound III-11. ¹ H NMR (400 MHz, CDCl ₃ ) δ9.66 (d, J=8.1 Hz, 1H), 7.34 (d, J=7.5 Hz, 1H), 7.15 (d, J=7.6 Hz, 1H), 6.89 (s, 1H), 6.83 (d, J=7.6 Hz, 1H), 6.26 (d, J=7.4 Hz, 1H), 6.04 (d, J=14.9 Hz, 1H), 5.38–5.24 (m, 4H), 5.20 (s, 1H), 5.00 (dd, J=27.7, 14.6 Hz, 3H), 4.61 (s, 7H), 4.37 (d, J=9.6 Hz, 2H), 4.25 (dd, J= 13.0,6.1Hz,2H),4.15(dd,J＝11.9,6.8Hz,1H),3.99(d,J＝4.8Hz,4H),3.82(s,3H),3.71–3.53(m,9H),3.49(s,2H),2.69(s,6H),2.5 5(s,4H),2.25(dd,J＝15.3,7.7Hz,5H),1.97(d,J＝5.7Hz,8H),1.62–1.47(m,7H),1.42–1.12(m,53H),0.87(dt,J＝13.1,7.1Hz,10H).

Example Compound III-17

Step 1: Preparation of compound III-17-1

Under stirring conditions at -80 to -85 degrees, 2M LDA tetrahydrofuran solution (35.5 ml, 71 mmol) was slowly added to a tetrahydrofuran (300 ml) solution of compound SM1 (15 g, 40.5 mmol) and compound SM2 (13.4 g, 71 mmol), and then the reaction mixture was stirred at -80 to -85 degrees for 2 hours. The reaction mixture was poured into ice water and extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain yellow oily compound III-17-1 (3.7 g, 21%). MS: 431.0 (M+1) ⁺ .

Step 2: Preparation of compound III-17-2

Under stirring conditions at 0 degrees, sodium hydrogen (2.3 g, 58.6 mmol) was slowly added to a tetrahydrofuran (100 ml) solution of compound SM3 (5 g, 39.1 mmol), and then the reaction mixture was stirred at 0 degrees for 1 hour; a tetrahydrofuran (20 ml) solution of compound III-17-1 (6 g, 13.9 mmol) was slowly added to the above reaction solution, and the reaction mixture was stirred at room temperature for 15 hours. The reaction mixture was poured into ice water and extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain a yellow oil compound III-17-2 (1.3 g, 22%). MS: 431.2 (M+1) ⁺ .

Step 3: Preparation of compound III-17-3

Under stirring conditions at 0 degrees, 1M TBAF tetrahydrofuran solution (5 ml, 5 mmol) was slowly added to a tetrahydrofuran (10 ml) solution of compound III-17-2 (1.1 g, 2.6 mmol), and then the reaction mixture was stirred at room temperature for 15 hours. The reaction mixture was poured into ice water and extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified to obtain yellow oily compound III-17-3 (850 mg, 92%). MS: 359.1 (M+1) ⁺ .

Step 4: Preparation of compound III-17-4

Under nitrogen protection, a reaction mixture of compound III-17-3 (800 mg, 2.2 mmol), Pd(PPh ₃ ) ₂ Cl ₂ (78 mg, 0.11 mmol), CuI (85 mg, 0.45 mmol), TEA (451 mg, 4.5 mmol) and methyl 4-iodo-2-methoxybenzoate (783 mg, 2.7 mmol) in DMF (10 ml) was stirred at 100 degrees for 15 hours. The reaction mixture was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified to obtain a yellow oil compound III-17-4 (700 mg, 61%). MS: 523.2 (M+1) ⁺ .

Step 5: Preparation of compound III-17-5

Compound III-17-4 (700 mg, 1.34 mmol) was dissolved in methanol (10 ml), palladium carbon (100 mg) was added to the reaction mixture under stirring, and then the reaction mixture was stirred under 40 degrees hydrogen pressure for 15 hours; the reaction mixture was filtered and concentrated to obtain a crude product, which was purified to obtain a yellow oil compound III-17-5 (460 mg, 66%). MS: 527.2 (M+1) ⁺ .

Step 6: Preparation of compound III-17-8

Synthesis of Compound III-17-8 Compound III-17-8 was prepared by referring to Compound II-1-3. MS: 728.2 (M+1) ⁺ .

Step 7: Preparation of Compound III-17

Synthesis of Compound III-17 Compound III-17 was prepared by referring to Compound III-2. MS: 672.3 (M+1) ⁺ .

Example Compound III-18

Synthesis of Compound III-18 Compound III-18 was prepared by referring to Compound III-2. MS: 816.3 (M+1) ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ9.49 (d, J=8.6 Hz, 1H), 7.62 (d, J=7.5 Hz, 1H), 7.06 (s, 1H), 6.96–6.87 (m, 2H), 6.78 (s, 1H), 6.12 (d, J=7.5 Hz, 1H), 5.04–4.91 (m, 2H), 4.22 (d, J=5.0 Hz, 2H), 3.86 (s, 4H), 3.64 (t, J=7 .4Hz,8H),3.51(d,J＝6.0Hz,13H),2.77(s,6H),2.60(s,4H),2.29–2.09(m,2H),1.93(s,1H) ,1.60(dt,J＝13.3,6.6Hz,1H),1.52–1.42(m,1H),1.39–1.27(m,2H),0.90(t,J＝7.3Hz,3H).

Example Compound III-20

Synthesis of Compound III-20 Compound III-20 was prepared by referring to Compound III-6. MS: 630.6 [M+H] ⁺ .

Example Compound III-21

Synthesis of Compound III-21 Compound III-21 was prepared by referring to Compound III-6. MS: 646.6 [M+H] ⁺ .

Example Compound III-22

Synthesis of Compound III-22 Compound III-22 was prepared by referring to Compound III-24. MS: 829.2 [M+H] ⁺ . 1H NMR (400MHz, DMSO-d ₆ ) δ9.55 (d, J=159.7Hz, -1H), 7.87 (s, 1H), 7.49 (s, 2H), 7.00 (d, J=7.3Hz, 2H), 6.88 (d, J=8.0Hz, 1H), 6.30 (d, J=6.2Hz, 1H), 5.06 (t, J=12.4Hz, 2H), 4.35–4.21 (m, 1H), 3.91–3.83 (m ,3H),3.54(d,J＝18.1Hz,6H),3.00(d,J＝46.1Hz,5H),2.34(d,J＝15.7Hz,1H),1.58(d,J＝3 4.0Hz, 8H), 1.29 (d, J = 25.8Hz, 26H), 1.20 (d, J = 6.5Hz, 3H), 0.89 (dt, J = 13.3, 7.1Hz, 6H).

Example Compound III-23

Synthesis of Compound III-23 Compound III-23 was prepared by referring to Compound III-24. MS: 898.6 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.82(s,1H),8.00–7.85(m,1H),7.56(s,2H),7.02(d,J＝7.3Hz,2H),6.88( d,J＝7.5Hz,1H),6.33(d,J＝7.4Hz,1H),5.13–4.97(m,1H),4.28(p,J＝7.0Hz, 1H),3.85(s,3H),3.51(s,6H),2.99(d,J＝42.7Hz,5H),2.35(s,1H),1.71–1. 43(m,8H),1.25(s,45H),1.20(d,J＝6.5Hz,3H),0.89(dt,J＝14.4,7.1Hz,6H).

Example Compound III-24

Step 1: Preparation of compound III-24-1

Under nitrogen protection and stirring at 0°C, heptadecan-1-amine (360 mg, 1.4 mmol) was slowly added to the reaction mixture of III-24-A (200 mg, 1.4 mmol) in methanol (2 ml), and then the reaction mixture was stirred at 0°C. The reaction was stirred at 20 degrees for 5 hours. The reaction mixture was concentrated to obtain a crude product, compound III-24-1 (511 mg), which was used directly in the next step without purification. MS: 366.6 [M+H] ⁺ .

Step 2: Preparation of compound III-24-2

Compound III-24-1 (200 mg, 0.5 mmol) was dissolved in acetonitrile (4 ml), and K ₂ CO ₃ (113 mg, 0.8 mmol) and 3-chloro-N, N-dimethylpropan-1-amine (100 mg, 0.8 mmol) were added under stirring at 20°C; the reaction mixture was then stirred at 80°C for 2 hours. The reaction mixture was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain compound III-24-2 (150 mg, 60%). MS: 451.7 [M+H] ⁺ .

Step 3: Preparation of compound III-24-6

Synthesis of Compound III-24-3 Compound III-24-3 was prepared by referring to Compound III-3-1. MS: 586.5 [M+H] ⁺ .

Synthesis of Compound III-24-5 Compound III-24-5 was prepared by referring to Compound II-10. MS: 686.4 [M+H] ⁺ .

The reaction mixture of compound III-24-5 (150 mg, 0.22 mmol), compound III-24-2 (148 mg, 0.3 mmol) and DIEA (740 mg, 5.7 mmol) in methanol (4 ml) was stirred at 20 degrees for 3 days. The reaction mixture was concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain compound III-24-6 (228 mg, 94%). MS: 1104.6 [M+H] ⁺ .

Step 4: Preparation of Compound III-24

Synthesis of Compound III-24 Compound III-24 was prepared by referring to Compound II-5. MS: 984.4 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.16–10.01 (m, 1H), 8.09 (d, J = 7.5 Hz, 1H), 7.78–7.66 (m, 1H), 7.15–7.02 (m, 2H), 6.93 (d, J = 7.7 Hz, 1H), 6.45 (d, J = 7.5 Hz, 1H), 5.16–5.01 (m, 2H), 4.42–4.26 (m, 2H), 3.8 5(s,4H),3.80–3.66(m,4H),3.58(d,J＝6.6Hz,3H),3.53–3.32(m,4H),3.17–2.87(m,8 H), 2.80 (d, J = 4.0Hz, 6H), 1.92 (s, 2H), 1.56 (s, 8H), 1.25 (s, 38H), 0.95–0.83 (m, 6H).

Example Compound III-25

Step 1: Preparation of intermediate III-25-3

The synthesis of intermediate III-25-3 refers to intermediate III-6-3 by using intermediate Int.K instead of intermediate Int.D prepared intermediate III-25-3. MS: 453.3 [M+H] ⁺ .

Step 2: Preparation of intermediate III-25

Synthesis of Compound III-25 Referring to Compound III-1, Compound III-6 was prepared by using Intermediate III-25-3 instead of Intermediate II-1-4. MS: 787.8 [M+H] ⁺ .

Preparation of adjuvant pharmaceutical composition

Adjuvant pharmaceutical composition Example 1: Preparation method of aluminum adjuvant adsorbed TLR7/8 agonist

Weigh 1 mg of TLR7/8 agonist (small molecule compound) and dissolve it in 0.25 ml of sterile water to prepare a 4 mg/ml aqueous solution. Add 0.50 ml of Tris-HCl solution (40 mM, pH 7.5) to the solution and mix well. Then add 0.20 ml of aluminum hydroxide gel (InvivoGen) adjuvant 2%, Al content 10 mg/ml), then add 0.05 ml of sterile water, and gently stir at room temperature for 2 hours to prepare the aluminum adjuvant adsorbed TLR7/8 agonist suspension (1 mg/ml TLR7/8 agonist, 2 mg/ml aluminum hydroxide gel, in 20 mM Tris-HCl).

Determination of adsorption rate: TLR7/8 agonists were prepared into concentrations of 0.01 mg/ml, 0.025 mg/ml, 0.05 mg/ml, 0.1 mg/ml, and 0.2 mg/ml, and analyzed by HPLC to calculate the standard curve of TLR7/8 agonists. The suspension was centrifuged in a centrifuge tube (10000 rpm, 5 min), the supernatant was taken, analyzed on HPLC, and compared with the standard curve of the single TLR7/8 agonist to calculate the adsorption rate.

Analytical methods:

a. Instrument: Agilent 1260 series high performance liquid chromatograph (HPLC)

b. Chromatographic column: Agilent reverse phase liquid chromatography column ZORBAX SB-Aq (3.5μm, 4.6*150mm)

c. Column temperature: 30°C

d. The injection volume is 5uL

e. Mobile phase A: 0.05% trifluoroacetic acid (TFA) aqueous solution

f. Mobile phase B: 0.05% trifluoroacetic acid (TFA) in acetonitrile

g. Flow rate: 1ml/min

h. Gradient elution, the elution program is as follows: 90%-60% (0min-10min), 60%-5% (10min-15min), 5%-5% (15min-18min), 5%-90% (18min-18.1min).

i. Data Analysis

The concentration in the supernatant was calculated based on the sample standard curve; the adsorption rate of the TLR7/8 agonist on the aluminum hydroxide gel was calculated as: concentration in the supernatant/theoretical concentration of the entire sample*100%.

Adjuvant pharmaceutical composition Example 2: Investigation of the adsorption efficiency of aluminum adjuvant adsorbing TLR7/8 agonists under different conditions

TLR agonist and aluminum hydroxide gel were subjected to adsorption test in two different buffers (pH 7.5 Tris-HCl buffer, pH 6.5 histidine buffer) at different mass ratios (2.:1, 1:0.85, 1:1, 1:2, 1:3 w/w) to investigate the adsorption efficiency under different conditions. The test was performed according to the method of Example 1. The test results are as follows:

Biological activity evaluation

1. Determination of the agonistic activity of compounds on human TLR7 and TLR8

1) Weigh the compound to be tested and prepare it to a concentration of 10 mM with DMSO (Sigma). Use DMSO to dilute the compound to be tested by 3.16 times in a gradient, with a total of 10 concentration gradients, and the highest concentration is 1 mM.

2) The test compound and DMSO control were diluted 10-fold using DPBS buffer (Gibco) and added to a 96-well cell culture plate (Corning) in a volume of 20 μL.

3) HEK-Blue ^TM hTLR7 cells (Invivogen) were digested with cell separation solution (Gibco) and the cells were counted (TC-20 cell counter, Bio-rad). The cells were diluted to 4.4E5/mL with DMEM cell culture medium (Gibco). 180 μL was added to a 96-well cell culture plate. The plates were incubated in a 37°C, 5% CO2 incubator for 16-22 hours.

4) Prepare QUANTI-Blue detection reagent (Invivogen), take 180 μL and add it to a new 96-well plate. Take 20 μL of cell culture supernatant from the cell culture plate and add it to the 96-well plate, and incubate at 37°C for 1-3 hours.

5) The light absorption at 630 nm was measured by Neo2 multifunctional microplate reader (Bio-tek), and the half effective concentration (EC _{50 )} of the drug was calculated by GraphPad Prism.

2. Determination of the agonist activity of compounds on human peripheral blood mononuclear cells

1) Human peripheral blood mononuclear cells (Miaotong Biotechnology Co., Ltd.) were thawed in a 37°C water bath. After counting, the cells were diluted to 0.2 M/mL and added to a 96-well cell culture plate in a volume of 150 μL and incubated for 40 hours.

2) Weigh the compound to be tested and prepare it to a concentration of 10 mM with DMSO. Use DMSO to dilute the compound to be tested by 10 times in a gradient, with a total of 4 concentration gradients, and the highest concentration is 400 μM.

3) Dilute the test compound, DMSO control and positive control 100-fold using cell culture medium (RPMI 1640 cell culture medium, Gibco) and add 50 μL to a 96-well cell culture plate.

4) Incubate at 37° C. for about 8 hours, collect the cell culture supernatant by centrifugation, and measure the level of cytokines in the supernatant by ELISA, including but not limited to IFN-α, TNF-α and IL-6.

The light absorption at 450 nm was measured by an enzyme-labeled instrument, and the cytokine levels in the culture supernatant were quantitatively determined according to the standard curve.

3. Evaluation of Mouse Immune Competence

1) Prepare a vaccine formulation containing an antigen and with or without a TLR7/8 agonist. Vaccinate 6-8 week old C57BL/6 female mice (10 mice/group), and inject 50-100 μL of the vaccine formulation intramuscularly into each mouse for primary immunization.

2) Two weeks later, the second injection was given for booster immunization, and two weeks later (D28), blood was collected for ELISA specific antibody detection and neutralization test.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the 24 claims attached to this application.

Claims (39)

A compound having a structure shown in the following formula (I), or a stereoisomer, enantiomer, or a pharmaceutically acceptable salt thereof:
in: _Y1 is hydrogen or a lipid structure fragment, and the lipid structure fragment has a structure as shown in the following formula:
p, q are each independently selected from the following group: 0, 1, 2, 3, 4, 5, 6, 7 or 8; m1, m2 and r are each independently selected from the following group: 0, 1, 2, 3, 4, 5 or 6; p ₁₀₁ is selected from the following group: 0 or 1; Q ₁ and Q ₂ are each independently selected from the following group: O, -NR ₃₀₃ - or -CR ₃₀₄ R ₃₀₅ -; R ₃₀₃ is selected from the group consisting of H, substituted or unsubstituted C _1-6 alkyl, and halogenated C _1-6 alkyl; R ₃₀₄ and R ₃₀₅ are the same or different, and are each independently selected from the group consisting of hydrogen, halogen, cyano, substituted or unsubstituted C _1-6 alkyl, substituted or unsubstituted C _1-6 alkoxy, substituted or unsubstituted C _3-6 cycloalkyl, substituted or unsubstituted 4-6 membered heterocycloalkyl; Linker is a divalent linking group; R ₁₀₁ and R ₁₀₂ are each independently selected from the group consisting of a substituted or unsubstituted C _4-20 straight chain or branched chain alkyl group, a substituted or unsubstituted C _4-20 straight chain or branched chain alkenyl group, and a substituted or unsubstituted C _4-20 straight chain or branched chain alkynyl group; R ₂₀₁ is a GalNAc target head portion and has a structure as shown in the following formula: wherein L ₂₀₁ , L ₂₀₂ and L ₂₀₃ are each independently a divalent linking group, and R ₂₀₃ , R ₂₀₄ and R ₂₀₅ are each independently a substituted or unsubstituted saccharide group, preferably a group formed by a substituted or unsubstituted N-acetylgalactosamine molecule; _{The L2} is a group selected from the group consisting of -(CHR) _m1- , optionally substituted -(CHR) _m1 -C(O)NH-( _CH2CH2O ) _n3- ( _CHR ) _m2- , optionally substituted - _{( CH2CH2O} ) _n3- (CHR) _m2- , optionally substituted -(CHR) _m1 -C(O)NH-( _CH2CH2O ) _n3- (CHR) _m2 -C(O)-, optionally substituted _- (CHR) _m1- ( _CH2CH2O ) _n3- (CHR) _m2- , -(CHR) _m1 -C( _O )-, optionally substituted -(CHR) _m1 -C(O) _-L4- NH( _CH2CH2O ) _n3- (CHR) _m2- , optionally substituted -(CHR) _m1 -C(O)NH-(CH2CH2O)n3-(CHR) _{m2-, 5-10} membered aromatic heterocyclic ring -( _{CH2CH 2} O) _n3 -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C(O)-L ₄ -NH-(CH ₂ CH ₂ O) _n3 -L ₄ -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C ₃ -C ₉ cycloalkyl-(CHR) _m2 -, optionally substituted -(CHR) _m1 -C ₃ -C ₉ heterocycloalkyl-(CHR) _m2 -; Wherein, each of the R groups is independently selected from the following group: H, D, (CH ₂ ) _n4 NHC(O)NH ₂ , C ₁ -C ₆ alkyl; m1, m2 and n3 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; The substitution refers to the substitution of one or more hydrogen atoms on the group by a substituent selected from the group consisting of C _1-8 alkyl, C _2-8 alkenyl, _C2-8 alkynyl, _C3-8 cycloalkyl, 3- to 12-membered heterocyclyl, _C3-8 aryl, 5- to 7-membered heteroaryl, halogen, hydroxyl, carboxyl (-COOH), _C1-8 aldehyde, _C2-10 acyl, _C2-10 ester, amino, _C1-8 alkoxy, _C1-10 sulfonyl; or two substituents located on adjacent ring atoms can form a group selected from the following group together with the connected ring atoms: 5-7 membered carbocyclic or heterocyclic ring, benzene ring, or 5-7 membered heteroaromatic ring; D is a toxin having a structure selected from the group consisting of Formula Ia, Formula Ib, Formula Ic or Formula Id, and the toxin is covalently linked to a divalent linker -LY- via an N, O or S atom in the molecule:
in: L ₁₁ is selected from the group consisting of -O-, -NH-, -S-, -S(=O)- or -S(=O) ₂ -; R ₁ is selected from the following group: H, C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _3-12 cycloalkyl or 4-12 membered heterocycloalkyl; wherein said R ₁ may be further substituted by one or more R ^1a substituents, and said R ^1a is selected from the following group: hydrogen, halogen, hydroxyl, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _3-6 cycloalkyl, -NR ^a1 R ^a2 , -NHC(＝O)-R ^a3 , 5-6 membered heteroaryl substituted by one or more R ^a4 , -OC(＝O)R ^a5 , -C(＝O)R ^a5 , -OC(＝O)OR ^a5 , -C(＝O)OR ^a5 ; Said ^Ra1 , ^Ra2 , ^Ra3 or ^Ra4 is selected from the following group: _C1-6 alkyl, _C1-6 haloalkyl, _C3-6 cycloalkyl; The ^Ra5 is selected from the following group: _C1-24 alkyl, halogenated _C1-24 alkyl, _C1-24 heteroalkyl having 1 to 10 heteroatoms, wherein the heteroatoms are selected from one or more of NH, N, O and S; The _R2 and _R3 are each independently selected from the group consisting of hydrogen, halogen, cyano, _C1-6 alkyl, _C1-6 alkoxy, _C3-6 cycloalkyl, _4-6 membered heterocycloalkyl, 5-8 membered heterocyclic aryl, or 5-8 membered aryl; wherein the _R2 may be further substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, or amino; m is 0, 1, 2, 3, 4, 5, 6, 7 or 8; B is absent, or B is selected from the group consisting of C _3-12 cycloalkyl, 4-12 membered heterocycloalkyl, C _6-12 aryl, 5-12 membered heteroaryl, or wherein each A is independently selected from C, CH or N, and R ₆ and R ₇ together with their adjacent carbon atoms form a C _4-7 cycloalkylene group or a C _4-7 heterocycloalkylene group, and one or more methylene groups in the C _4-7 cycloalkylene group or the 4-7 membered heterocycloalkylene group may be independently replaced by a carbonyl group or S(═O) ₂ ; in the 4-7 membered heterocycloalkylene group, the heteroatom is selected from N, O, S, and the number of the heteroatoms is 1 to 3; L ₁₂ is selected from the group consisting of none, -(CR ^1b R ^1c ) _p -(NR ^1d ) _q -, -O-, -S-, -(CR ^1b R ^1c ) _p -C(═O)-, -(CR ^1b R ^1c ) _p -C(═O)NH-, -(CR ^1b R ^1c ) _p -NHC(═O)-, -S(═O)- or -S(═O) ₂ -; wherein R ^1b , R ^1c are selected from the group consisting of hydrogen, halogen, C _1-6 alkyl, C _3-6 cycloalkyl, _4-6 membered heterocycloalkyl or C _1-6 haloalkyl, and R ^1d is selected from the group consisting of hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, 4-6 membered heterocycloalkyl, C _{1-6 haloalkyl or hydroxy-substituted C 1-6} alkyl; _1-6 alkyl, p is 0, 1, 2, 3, 4, 5 or 6, q is 0 or 1; _R4 is selected from the group consisting of hydrogen, halogen, cyano, amino, hydroxyl, _C1-12 alkyl, _C1-12 alkoxy, _C2-12 alkenyl, _C2-12 alkynyl, _C3-12 cycloalkyl, 4-12 membered heterocycloalkyl, _C6-12 aryl or 5-12 membered heteroaryl; and _R4 can be any is optionally substituted with one or more R ^1e substituents, wherein R ^1e is selected from the group consisting of hydrogen, halogen, hydroxy, carboxylic acid, amino, C _1-6 alkyl, C 1-6 alkyl substituted with one or more R ^e1 , C _1-6 alkoxy, C _1-6 haloalkoxy, C _3-12 cycloalkyl, _4-12 heterocycloalkyl, C _6-12 aryl, 5-12 membered heteroaryl, -N(R ^e2 R ^e3 ), -C(＝O)OR ^e2 , -C(＝O)NH-R ^e2 , -S(＝O) ₂ -R ^e2 ; When L ₁₂ is absent and R ⁴ is H, B is selected from the group consisting of C _3-12 cycloalkyl, 4-12 membered heterocycloalkyl, C _6-12 aryl, 5-12 membered heteroaryl or wherein each A is independently selected from C, CH or N, and _R6 and _R7 together with their adjacent carbon atoms form a _C4-7 cycloalkylene or _C4-7 heterocycloalkylene; R ^e1 is selected from the group consisting of halogen, hydroxyl; R ^e2 and R ^e3 are selected from the group consisting of hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, C _1-6 haloalkyl or C _1-6 alkyl substituted with hydroxyl; R ₅ is selected from the group consisting of halogen, hydroxy, cyano, amino, C _1-6 alkyl, C _3-6 cycloalkyl, 4-6 membered heterocycloalkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 haloalkoxy, -C(=O)OR ^f , -C(=O)NH-R ^f , -S(=O) ₂ -R ^f ; wherein R ^f is selected from the group consisting of hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl or C _1-6 haloalkyl; n is 1, 2, 3, 4, 5 or 6; Wherein, each of the heterocyclic groups may be saturated or partially unsaturated (but not having an aromatic structure), and in the heterocyclic group, the heteroatom is selected from N, O, S, and the number of heteroatoms is 1, 2, 3 or 4 (preferably 1 or 2); in the heteroaryl group, the heteroatom is selected from N, O, S, and the number of heteroatoms is 1, 2 or 3. The compound of formula (I) according to claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that the linker has a structure selected from the group consisting of: C4-C20 alkyl, wherein n and m are each independently selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; _n1 and _n2 are each independently selected from the following group: 0 or 1; W is selected from the following group: a single bond, NH, substituted or unsubstituted C _1-16 alkylene, a substituted or unsubstituted 7-12 membered fused bicyclic ring, a substituted or unsubstituted 7-15 membered spirocyclic ring, a substituted or unsubstituted 5-15 membered bridged ring, -NH-substituted or unsubstituted 7-12 membered fused bicyclic ring, -NH-substituted or unsubstituted 7-15 membered spirocyclic ring, -NH-substituted or unsubstituted 5-15 membered bridged ring, -NH-substituted or unsubstituted 7-12 membered fused bicyclic ring-NH-, -NH-substituted or unsubstituted 7-15 membered spirocyclic ring-NH-, -NH-substituted or unsubstituted 5-15 membered bridged ring-NH-, -NH-substituted or unsubstituted C _1-16 alkylene-NH-; wherein the ring atoms of the fused bicyclic ring, spirocyclic ring and bridged ring may be optionally carbon atoms or heteroatoms; The substitution refers to that one or more hydrogen atoms on the group are replaced by a substituent selected from the group consisting of C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _3-8 cycloalkyl, 3- to 12-membered heterocyclyl, C _6-10 aryl, 5- to 10 _- membered heteroaryl, halogen, hydroxyl, carboxyl (-COOH), C _1-8 aldehyde, C _2-10 acyl, C _2-10 ester, amino, C _1-8 alkoxy, C _1-10 sulfonyl; or two substituents located on adjacent ring atoms can form a group selected from the group consisting of a 5-7-membered carbocyclic or heterocyclic ring, a benzene ring, or a 5-7-membered heteroaromatic ring together with the connected ring atoms; Or W has a structure selected from the group consisting of:

wherein s1, s2, s, s4, s5 and s6 are each independently selected from the following group: 0, 1 or 2; s7 is selected from the group consisting of 1, 2, 3, 4, 5 or 6; Each of M ₁ , M ₂ , M ₃ and M ₄ is independently selected from the group consisting of CHR, C(R)R, C(O), O, S, NR; _M5 and _M6 are each independently selected from the group consisting of (CHR) _s6 , C(R)R, C(O), (CHR) _s6 -C(O), O, S, NR; R is selected from the following group: H, halogen, methyl; or two R located on adjacent reducing atoms and the carbon atom to which they are connected together form a substituted or unsubstituted 4-8 membered carbocyclic or heterocyclic ring (including saturated, unsaturated or aromatic ring). The compound of formula (I) according to claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that W has a structure selected from the group consisting of:
The compound of formula (I) according to claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that the linker has a structure selected from the group consisting of:
-L ₂ -L ₃ -L ₄ -Y-; wherein _L2 is a group selected from the group consisting of -(CHR) _m1 -, optionally substituted -(CHR) _m1 -C(O)NH-(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -, optionally substituted -(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C(O)NH-(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -C(O)-, optionally substituted -(CHR) _m1 -(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -, -(CHR) _m1 -C(O)-, optionally substituted -(CHR) _m1 -C(O)-L4 _- NH(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C(O)NH-(CHR) _m1 -5-10 membered aromatic heterocyclic ring -(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C(O)-L ₄ -NH-(CH ₂ CH ₂ O) _n3 -L ₄ -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C ₃ -C ₉ cycloalkyl-(CHR) _m2 -, optionally substituted -(CHR) _m1 -C ₃ -C ₉ heterocycloalkyl-(CHR) _m2 -; The _L4 is an optionally substituted group selected from the group consisting of: a chemical bond, wherein said ^Ra and ^Rb are each independently selected from the following group: hydrogen, optionally substituted _C1 - _C4 alkyl, optionally substituted _C1 - _C4 deuterated alkyl, _CH2COOH ; The R ₁₀₁ is selected from the following group: hydrogen, halogen, cyano, amino, hydroxyl, carboxyl, ester, C _1-12 alkyl, C _1-12 alkoxy, C _2-12 alkenyl, C _2-12 alkynyl, C _3-12 cycloalkyl, _4-12 membered heterocycloalkyl, C _6-12 aryl or 5-12 membered heteroaryl, -L ₄ -C(O)-(CH ₂ CH ₂ O) _n3 -R, -C(O)-NR-(CHR) _m2 -NR-C(O)-(CH ₂ CH ₂ O) _n3 -R; L ₃ is a peptide residue [Am] _p1 -: wherein p1 is 0, 1, 2, 3, 4, 5, 6, 7 or 8; Am is an amino acid residue; Y is selected from the group consisting of -C(O)-, -OC(O)-, -NHC(O)-, -NR-(CHR) _m2 -NR-C(O)-, -NR-(CHR) _m2 -NR-C(O)O-(CHR) _m1 -, a chemical bond; Wherein, each of the R groups is independently selected from the following group: H, D, (CH ₂ ) _n4 NHC(O)NH ₂ , C ₁ -C ₆ alkyl; m1, m2 and n3 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; m101 is selected from 0, 1, 2, 3, 4; Unless otherwise specified, the term “substituted” refers to the substitution of one or more hydrogen atoms on a group with a substituent selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a nitrile group, a nitro group, a hydroxyl group, an amino group, a C ₁ -C ₆ alkyl-NH-, a (C ₁ -C ₆ alkyl) ₂ N-, a C ₁ -C ₆ alkyl, a C ₂ -C ₆ alkenyl, a C ₂ -C ₆ alkynyl, a C ₁ -C ₆ alkoxy group, a halogenated C ₁ -C ₆ alkyl group, a halogenated C ₂ -C ₆ alkenyl group, a halogenated C ₂ -C ₆ alkynyl group, a halogenated C ₁ -C ₆ alkoxy group, an allyl group, a benzyl group, a C ₆ -C ₁₂ aryl group, a C ₁ -C ₆ alkoxy-C ₁ -C ₆ alkyl group, a C ₁ -C ₆ alkoxy-carbonyl group, a phenoxycarbonyl group, a C ₂ -C ₆ alkynyl-carbonyl group, a C ₂ -C ₆ alkenyl-carbonyl group, a C ₃ -C 6 C ₆ cycloalkyl-carbonyl, C ₁ -C ₆ alkyl-sulfonyl, phenyl, 5-7 membered heteroaryl, C ₃ -C ₈ cycloalkyl, 3-12 membered heterocyclyl, or C(O)NH—L ₂ H. The compound of formula (I) according to claim 4, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that: The _L2 is a group selected from the group consisting of optionally substituted -(CHR) _m1- , optionally substituted -(CHR) _m1 -C(O)NH-( _CH2CH2O ) _n3- (CHR) _m2- , _{and optionally substituted -(CHR)m1-(CH2CH2O ) n3- ( CHR} ) _m2- ; p1 is 0; The L ₄ is a chemical bond. The compound of formula (I) according to claim 4, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that: The _L2 is a group selected from the group consisting of optionally substituted -(CHR) _m1 -C(O)NH-( _CH2CH2O ) _n3- (CHR _{) m2-} ; p1 is 0; The L ₄ is a chemical bond. The compound of formula (I) according to claim 4, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that: The _L2 is a group selected from the group consisting of optionally substituted -(CHR) _m1- , optionally substituted _- (CHR) _m1- ( _CH2CH2O ) _n3- (CHR) _m2- ; p1 is 0; The L ₄ is a chemical bond. The compound of formula (I) according to claim 4, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that: The L ₂ is a group selected from the group consisting of optionally substituted -(CHR) _m1 -C(O)-; p1 is 1, 2, or 3; The _L4 is an optionally substituted group selected from the following group: The compound of formula (I) according to claim 4, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that: The L ₂ is a group selected from the group consisting of optionally substituted (CHR) _m1 -C(O)-; p1 is 1, 2, or 3; The _L4 is an optionally substituted group selected from the following group: The compound of formula (I) as claimed in claim 4, or its stereoisomer, enantiomer, or pharmaceutically acceptable Acceptable salts, characterized in that: The _L2 is a group selected from the following group: optionally substituted -( _CH2 ) _m1 -C(O)-, optionally substituted -( _CH2CH2O ) _n3- (CHR) _m2 -C(O)-, optionally substituted -(CHR) _m1 - _C3 - _{C9cycloalkyl-} (CHR) _m2- ; preferably, m1 and m2 are each independently selected from 0 _, 1, 2, 3, 4 or 5; n3 is selected from 0, 1 or 2; p1 is 0; The L ₄ is a chemical bond, or an optionally substituted group selected from the following group: The compound of formula (I) according to claim 4, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that: The _L2 is a group selected from the following group: optionally substituted -(CHR) _m1 -C(O)-L ₄ -NH(RCH) _m1 -(OCH ₂ CH ₂ ) _n3 -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C(O)NH-(CHR) _m1 -5-10 membered aromatic heterocycle -(CH ₂ CH ₂ O) _n3 -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C(O)-L ₄ -NH-(CH ₂ CH ₂ O) _n3 -L ₄ -(CHR) _m2 -, optionally substituted -(CHR) _m1 -C(O)-L ₄ -NH(RCH) _m1 -(OCH ₂ CH ₂ ) _n3 -L ₄ -(CHR) _m2 -; preferably, the 5- to 10-membered aromatic heterocycle is a 5- or 6-membered aromatic heterocycle. p1 is 0; The L ₄ is a chemical bond, or an optionally substituted group selected from the following group: The compound of formula (I) according to claim 4, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that _L3 is a peptide residue consisting of an amino acid selected from the following group: phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine. The compound of formula (I) according to claim 4, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that _L3 is a peptide residue consisting of an amino acid selected from the following group: glycine, alanine, lysine, phenylalanine, valine and citrulline. The compound of formula (I) according to claim 4, or a stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that _L3 is a peptide residue selected from the following group: -glycine-phenylalanine-glycine-(-Gly-Phe-Gly-), -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-), -valine-citrulline-(-Val-Cit-), -citrulline-valine-(-Cit-Val-), -citrulline-alanine-(-Cit-Ala-), -valine-alanine-(-Val-Ala-), -valine-arginine-(-Val-Arg-), -valine-lysine-(-Val-Lys-), -valine-lysine (Ac)-(-Val-Lys (Ac)-), -lysine-valine-(-Lys-Val-), -leucine -Citrulline-(-Leu-Cit-), -Isoleucine-Citrulline-(-Ile-Cit-), -Tryptophan-Citrulline-(-Trp-Cit-), -Phenylalanine-Lysine-(-Phe-Lys-), -Phenylalanine-Lysine(Ac)-(-Phe-Lys(Ac)-), -Phenylalanine-Citrulline-(-Phe-Cit-), -Phenylalanine-Alanine-(-Phe-Ala-), -Phenylalanine-Arginine Acid-(-Phe-Arg-), -Alanine-Lysine-(-Ala-Lys-), -Alanine-Alanine-(-Ala-Ala-), -Alanine-Alanine-Alanine-(-Ala-Ala-Ala-), -Alanine-Alanine-Asparagine-(-Ala-Ala-Asn-), -Alanine-Alanine-Aspartic Acid-(Ala-Ala-Asp-), -Lysine-Alanine-Alanine-Asparagine-(-Ly s-Ala-Ala-Asn-), -Lysine-Alanine-Alanine-Aspartic Acid-(-Lys-Ala-Ala-Asp-), -(D)-Valine-Leucine-Lysine-(-D-Val-Leu-Lys-), -Glycine-Glycine-Arginine-(-Gly-Gly-Arg-), -Glycine-Glycine-Asparagine-(-Gly-Gly-Asn-), -Glycine-Glycine-Phenylalanine-(-Gl -y-Gly-Phe-), -valine-lysine-glycine-(-Val-Lys-Gly-), -glutamic acid-alanine-alanine-(-Glu-Ala-Ala-), -aspartic acid-alanine-alanine-(-Asp-Ala-Ala-), -valine-lysine-glycine-glycine-(-Val-Lys-Gly-Gly-), and -lysine-alanine-asparagine-(-Lys-Ala-Asn-). The compound of formula (I) according to claim 4, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that _L3 is a structure selected from the following group:

The compound of formula (I) as claimed in claim 4, or its stereoisomer, enantiomer, or pharmaceutically acceptable The acceptable salt is characterized in that the L ₄ is a chemical bond, or is an optionally substituted group selected from the following group: Wherein, the ^Ra and ^Rb are each independently selected from the following group: hydrogen, optionally substituted _C1 - _C4 alkyl, and optionally substituted _C1 - _C4 deuterated alkyl. The compound of formula (I) according to claim 4, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that L ₄ is a chemical bond, or is an optionally substituted structure selected from the following group: The compound of formula (I) according to claim 4, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that Y is -C(O)-, -OC(O)-, -NHC(O)-, or a chemical bond. The compound of formula (I) according to claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that L ₂₀₁ , L ₂₀₂ and L ₂₀₃ are each independently a structure selected from the following group:

wherein each u, v and w are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; _u1 and _u2 are independently selected from 0 or 1; Glu is an unmodified or modified 5-6-membered glycosyl; X and _Y2 are each independently selected from the group consisting of -NH-(C ₁ -C ₆ alkyl)-NH-, -NH-(C ₃ -C ₈ cycloalkyl)-NH-, -NH-(4-10 membered heterocyclyl)-NH-, Wherein, the ring D is a 4-15 membered heterocyclic group; The cycloalkyl and heterocyclic groups may be monocyclic, condensed, bridged or spirocyclic; the heterocyclic group may be saturated or partially unsaturated (but not aromatic). The compound of formula (I) according to claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that X and _Y2 each independently have a structure selected from the following group: -NH-(C ₁ -C ₆ alkyl)-NH-, -NH-(C ₃ -C ₈ cycloalkyl)-NH-, -NH-(4-10 membered heterocyclyl)-NH-, or a structure selected from the following group:
The compound of formula (I) as claimed in claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable The acceptable salt is characterized in that the sugar group has a structure shown in the following formula III:
Wherein, the R ₂₁₁ is selected from the following group: -NH(C ₂ -C ₆ acyl), -NH(halogenated C ₂ -C ₆ acyl), -NH(C ₂ -C ₆ sulfonyl), -NH(halogenated C ₂ -C ₆ sulfonyl); R ₂₁₂ , R ₂₁₃ and R ₂₁₄ are each independently selected from the group consisting of H or C ₁ -C ₆ acyl. The compound of formula (I) according to claim 21, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that R ₂₁₁ is selected from the group consisting of -NH (C ₂ -C ₄ acyl); R ₂₁₂ , R ₂₁₃ and R ₂₁₄ are each independently selected from the group consisting of H or C ₁ -C ₄ acyl. The compound of formula (I) according to claim 21, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that R ₂₁₁ is -NHAc; R ₂₁₂ , R ₂₁₃ and R ₂₁₄ are each independently selected from the following group: H or Ac. The compound of formula (I) according to claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that L ₁₁ is selected from the group consisting of -O-, -NH- or -S-. The compound of formula (I) as claimed in claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that R ₁ is selected from the following group: C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _3-12 cycloalkyl or 4-12 membered heterocycloalkyl; and the R ₁ may be further substituted by one or more R ^1a substituents, and the R ^1a is selected from the following group: halogen, hydroxyl, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _3-6 cycloalkyl, -NR ^a1 R ^a2 , -NHC(＝O)-R ^a3 , 5-6 membered heteroaryl substituted by one or more R ^a4 , -OC(＝O)R ^a5 , -C(＝O)R ^a5 , -OC(＝O)OR ^a5 , -C(＝O)OR ^a5 . The compound of formula (I) as claimed in claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that the R ₁ is selected from the following group: C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _3-12 cycloalkyl or 4-12 membered heterocycloalkyl; and the R ₁ may be further substituted by one or more R ^1a substituents, and the R ^1a is selected from the following group: halogen, hydroxyl, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _3-6 cycloalkyl. The compound of formula (I) as claimed in claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that R ₁ is selected from the following group: H, C _1-8 alkyl, C _1-8 alkoxy, C _2-8 alkenyl, C _2-8 alkynyl, C _3-8 cycloalkyl or 4-8 membered heterocycloalkyl; wherein the R ₁ may be further substituted by one or more R ^1a substituents, and the R ^1a is selected from the following group: hydrogen, halogen, hydroxyl, cyano, C _1-4 alkyl, C _1-4 alkoxy. The compound of formula (I) according to claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that B is absent, or B is selected from the following group: C _3-8 cycloalkyl, 4-7 membered heterocyclyl, C _6-10 aryl, or The compound of formula (I) according to claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that L ₁₂ is selected from the following groups: -(CR ^1b R ^1c ) _p -(NR ^1d ) _q -, -O-, -S-, -(CR ^1b R ^1c ) _p -C(＝O)-, -(CR 1b R 1c ) p -C(＝O)-, -(CR ^1b R ^1c ) _p -C(＝O)NH-, -(CR ^1b R ^1c ) _p -NHC(＝O)-, -S(＝O)- or -S(＝O) ₂ -; wherein R ^1b , R ^1c are selected from the following groups: hydrogen, halogen, C _1-6 alkyl; R ^1d is hydrogen or C _1-6 alkyl; p is 0, 1, 2 or 3, and q is 0 or 1. The compound of formula (I) according to claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that R ₅ is selected from the group consisting of halogen, hydroxy, cyano, amino, C _1-6 alkyl, C _1-6 alkoxy C _3-6 cycloalkyl. The compound of formula (I) according to claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that the toxin is selected from the group consisting of:












The compound of formula (I) according to claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that the R ₂₀₁ -NHC(O) has a structure selected from the group consisting of:

The compound of formula (I) according to claim 1, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that the compound is selected from the following group:






A compound represented by the following formula Ia, Ib, Ic or Id, or a stereoisomer, enantiomer, or a pharmaceutically acceptable salt thereof
in: R ₄ is selected from the group consisting of hydrogen, halogen, cyano, amino, hydroxy, C _1-12 alkyl, C _1-12 alkoxy, C _2-12 alkenyl, C _2-12 alkynyl, C _3-12 cycloalkyl, 4-12 heterocycloalkyl, C _6-12 aryl, or 5-12 heteroaryl; and said R ₄ may be optionally substituted by one or more R ^1e substituents, wherein R ^1e is selected from the group consisting of hydrogen, halogen, hydroxy, carboxylic acid, amino, C _1-6 alkyl, one or more R ^e1 substituted C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkoxy, C _3-12 cycloalkyl, 4-12 heterocycloalkyl, C _6-12 aryl, 5-12 heteroaryl, -N(R ^e2 R ^e3 ), -C(＝O)OR ^e2 , -C(＝O)NH-R ^e2 , -S(＝O) ₂ -R ^e2 ; And the R ₄ is substituted by at least one R ^1e substituent, one; wherein the R ^1e substituent has the following structure:
Wherein, Q ₂ is -CR ₃₀₄ R ₃₀₅ -; R ₃₀₄ and R ₃₀₅ are the same or different, and are independently selected from the following group: hydrogen, halogen, cyano, substituted or unsubstituted C _1-6 alkyl, substituted or unsubstituted C _1-6 alkoxy, substituted or unsubstituted C _3-6 cycloalkyl, substituted or unsubstituted 4-6 membered heterocycloalkyl; preferably, R ₃₀₄ and R ₃₀₅ are: H or F. The compound according to claim 34, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof, characterized in that the compound is selected from the group consisting of:

A compound selected from the group consisting of:

A pharmaceutical composition comprising the compound according to any one of claims 1 to 36, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt, and optionally a pharmaceutically acceptable carrier, diluent or excipient. Use of the compound according to any one of claims 1 to 36, or its stereoisomer, enantiomer, or pharmaceutically acceptable salt, and/or the pharmaceutical composition according to claim 37 in the preparation of a medicament for treating or preventing viral infection or tumor. A combined adjuvant, characterized in that the combined adjuvant comprises: an aluminum adjuvant, and a TLR7/8 agonist adsorbed on the aluminum adjuvant; wherein the TLR7/8 agonist comprises a compound as described in any one of claims 1 to 36, or a stereoisomer, an enantiomer, or a pharmaceutically acceptable salt thereof.