WO2024259930A9 - Phosphonate compound and medical use thereof - Google Patents

Abstract

A phosphonate compound or a pharmaceutically acceptable salt or ester thereof, and the use thereof in the preparation of a drug for treating, inhibiting or preventing a disease caused by a viral infection or cell proliferation, particularly for treating, inhibiting or preventing monkeypox virus infection or a disease caused thereby in mammals, such as human.

Description

A phosphonate compound and its medical use

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on the Chinese patent application with application number 202310745552.4 and application date June 21, 2023, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is hereby introduced into this application as a reference.

Technical Field

The present invention relates to a phosphonate compound or a pharmaceutically acceptable salt or ester thereof, and use thereof in preparing a medicine for treating, inhibiting or preventing a virus infection disease or a cell proliferation disease.

Background Art

Cidofovir is an acyclic nucleoside phosphonate (ANP) analog, which is essentially a nucleoside monophosphate analog with the advantage of metabolic stability. Current studies have shown that cidofovir has broad-spectrum activity against all DNA viruses, including adenovirus, polyomavirus, papillomavirus and poxvirus. Among poxviruses, vaccinia, smallpox, cowpox, monkeypox, camelpox, molluscum contagiosum and sheeppox virus (orf) [De Clercq, Antiviral Research 2002, 55, 2002, 113].

Monkeypox is a viral zoonosis that causes symptoms in humans similar to those seen in smallpox patients in the past. While smallpox no longer exists in the world after it was eradicated in 1980, monkeypox still occurs in parts of Africa. Monkeypox occurs in monkeys in the rainforests of central and western Africa, and can also infect other animals and occasionally humans. The clinical manifestations are similar to smallpox, but the condition is milder. The disease is caused by the monkeypox virus, which can be transmitted from animals to humans through direct close contact, and can also be spread from person to person. The 2022 monkeypox outbreak was first discovered in the UK on May 7, 2022 local time. On May 20, local time, with more than 100 confirmed and suspected cases of monkeypox in Europe.

Since the phosphonate portion of cidofovir is negatively charged at physiological pH, cidofovir cannot cross lipid-rich cell membranes, which hinders its antiviral activity and bioavailability [De Clercq, Antiviral Research 2002, 55, 2002, 113]. Cidofovir can also cause renal insufficiency [De Clercq & Holy, Nat. Rev. Drug Discov. 2005, 4, 928-940]. In order to reduce the nephrotoxicity of cidofovir, researchers synthesized cyclic cidofovir (cHPMPC) [Bischofberger et al. Antimicrob. Agents Chemother. 1994, 38, 2387-2391]. Unfortunately, due to the problem that the remaining phosphonate is negatively charged at physiological pH, the bioavailability of cHPMPC is still very low. Based on cidofovir, researchers have designed a number of analogs or prodrugs, such as Brincidofovir (BCV, US8962829). However, how to design a cidofovir prodrug with better ADMET (absorption, distribution, metabolism, excretion and toxicity of the drug) is still a problem that needs to be solved.

Summary of the invention

The main technical problem solved by the present application is to provide a phosphonate prodrug, which can have at least one or more of the following effects:

1) Change the pharmacokinetic properties in the body and adjust the absorption and distribution of drugs in the body;

2) Improve the stability and solubility of drugs;

3) Reduce toxicity and adverse reactions;

4) improve transport and distribution to specific sites; and

5) Improve the sustained-release effect and prolong the duration of action.

In one aspect, the present invention provides a phosphonate compound represented by formula (I) or a pharmaceutically acceptable salt or ester thereof:

Wherein, X is selected from -OR ⁶ , or X and R ³ are combined to form a chemical bond;

^R1 is selected from H or C4-C30 carbonyl, the C4-C30 carbonyl includes substituted or unsubstituted hydrocarbylcarbonyl, substituted or unsubstituted arylcarbonyl or heterocyclic carbonyl and substituted or unsubstituted hydrocarbyloxycarbonyl, and the carbon number of the C4-C30 carbonyl is 4 to 30, which can be 4 to 10, 10 to 30 or 20 to 30, and specifically can be 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30;

R ² and R ⁶ are independently selected from H, wherein R ⁷ is selected from substituted or unsubstituted C15-C30 hydrocarbon groups, specifically C16, C17, C18, C19, C20 alkyl groups, and the hydrocarbon group may be a straight-chain hydrocarbon group or a branched hydrocarbon group, especially a straight-chain hydrocarbon group;

^R3 is selected from: H, formyl, C4-C30 carbonyl, wherein the C4-C30 carbonyl includes substituted or unsubstituted alkylcarbonyl, substituted or unsubstituted arylcarbonyl or heterocyclic carbonyl, and substituted or unsubstituted alkoxycarbonyl, and the carbonyl has no more than 30 carbon atoms, or an amino acid residue;

R ⁴ and R ⁵ are independently selected from hydrogen (H) or deuterium (D);

As a limitation, when X is -OR ⁶ , R ¹ , R ² , R ³ , and R ⁶ are not H at the same time, and the compound is not brincidofovir; when X and R ³ are combined to form a chemical bond, R ¹ and R ² are not H at the same time.

Furthermore, the above compound is a compound represented by formula (II):

In some preferred embodiments, in formula (II), R ¹ is selected from substituted or unsubstituted arylcarbonyl, especially substituted or unsubstituted phenylcarbonyl; C1-C6 alkylcarbonyl, especially C1-C4 alkylcarbonyl; and

^R2 is Wherein ^R7 is selected from substituted or unsubstituted C15-C20, especially C16-C20 straight chain hydrocarbon groups.

Furthermore, the above compound is a compound represented by formula (III):

As a limitation, when X is -OR ⁶ , R ¹ , R ³ , and R ⁶ are not H at the same time; when X and R ³ are combined to form a chemical bond, R ¹ is not H.

In some embodiments, in formula III, R ¹ is selected from H; substituted or unsubstituted arylcarbonyl, especially substituted or unsubstituted phenylcarbonyl; CH ₃ OCH ₂ CH ₂ Ocarbonyl; C1-C5 alkyloxycarbonyl; CH ₃ OCH ₂ CH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ Ocarbonyl; CH ₃ CH ₂ CH ₂ COOCH(CH ₃ )Ocarbonyl; substituted or unsubstituted heteroarylalkyloxycarbonyl, especially substituted or unsubstituted five-membered ring carbonate C1-C2 alkoxycarbonyl, such as substituted or unsubstituted heteroarylalkyloxycarbonyl, (CH ₃ ) ₂ CHCOOCH(CH ₃ )Ocarbonyl, CH ₃ COOCH(CH ₃ )Ocarbonyl;

^R3 _is selected from H; formyl, substituted or unsubstituted arylcarbonyl, especially substituted or unsubstituted phenylcarbonyl; C1- _C5 alkyloxycarbonyl; _{CH3OCH2CH2OCH2CH2OCH2CH2Ocarbonyl ; C1} - _C8 alkylcarbonyl; ( _CH3 ) _2CHCOOCH ( _CH3 )Ocarbonyl; substituted or unsubstituted heteroarylalkyloxycarbonyl, especially substituted or unsubstituted _five -membered ring carbonate C1-C2 alkoxycarbonyl, such as (CH ₃ ) ₂ CHCOOCH(CH ₃ )Ocarbonyl; and

X is a hydroxyl group.

In some embodiments, the above compound is a compound represented by (IIIa) or (IIIb):

wherein X, R ¹ or R ³ involved are as defined above.

Preferably, in formula (IIIa), R ³ is H;

In some embodiments, ^R3 is

In some embodiments, the above compound is a compound represented by formula (IV):

As a limitation, when X is -OR ⁶ , R ¹ , R ³ , and R ⁶ are not H at the same time; when X and R ³ are combined to form a chemical bond, R ¹ is not H.

In some embodiments, the above compound is a compound represented by (IVa) or (IVb):

wherein X, R ¹ or R ³ involved are as defined above.

Furthermore, in all the above general formulae, ^R1 is selected from substituted or unsubstituted hydrocarbon carbonyl groups, specifically, including the groups shown below:

Furthermore, the above ^R1 is selected from substituted or unsubstituted arylcarbonyl or heterocyclic carbonyl, specifically, including the following groups:

wherein Y is selected from H, F, Cl, Br, Me, -OMe, -OEt, _-CF3 or -CN.

Furthermore, the above ^R1 is selected from substituted or unsubstituted hydrocarbonoxycarbonyl groups, specifically, including the following groups:

Furthermore, the above ^R7 includes the following groups:

Specifically, R ³ includes the following groups;

In some embodiments, ^R3 is

In some embodiments, ^R3 is

Furthermore, the above compounds include compounds represented by formula (Va) to formula (Vd):

The compounds include the compounds shown in Table 1a and Table 1b below:

[Corrected 08.01.2025 in accordance with Rule 26]
Table 1b

Table 1b

The compounds provided herein can be used to prepare drugs for treating, inhibiting or preventing viral infections or diseases caused by viral infections. In some embodiments, viral infections include hepatitis B virus (HBV), new coronavirus (SARS-COV-2), human immunodeficiency virus (HIV), varicella zoster virus (VZV), cytomegalovirus (CMV), herpes simplex virus (HSV), BK virus, JC virus, Epstein-Barr virus (EBV), Ebola virus, polyomavirus, papillomavirus, orthopoxvirus, hepatitis C virus (HCV), respiratory syncytial virus (RSV), dengue virus, influenza virus, adenovirus, parainfluenza virus and/or infection caused by rhinovirus.

Furthermore, the above-mentioned orthopoxviruses include severe and mild smallpox viruses, monkeypox virus, cowpox virus, camelpox virus, molluscum contagiosum, sheeppox virus, aractuba virus (ARAV), BeAn 58058 virus (BAV), cantagalo orthopoxvirus (CTGV), mousepox virus, elephantpox virus, vaccinia virus (VV), rabbitpox virus, raccoonpox virus, skunkpox virus, gerbilpox virus and volepox virus. Preferably, the compounds disclosed in the present application can be used to prepare drugs for treating, inhibiting or preventing diseases caused by monkeypox virus or smallpox virus infection in mammals.

The compounds provided in the present application can be used to prepare drugs for treating, inhibiting or preventing diseases caused by viral infection. Further, the diseases caused by viral infection include diseases caused by DNA virus infection, specifically, the diseases are selected from retinitis, pneumonia, cystitis, proteinopathy, etc.

The compounds provided herein can also be used to prepare drugs for treating, inhibiting or preventing cell proliferation-induced diseases. Further, the cell proliferation-induced disease is a tumor or cancer, specifically, the tumor or cancer is selected from multiple myeloma (MM), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), solid tumors, refractory solid tumors, non-Hodgkin's lymphoma, hematological tumors, neuroblastoma, colorectal cancer, cervical cancer, lung cancer, leukemia, breast cancer, pancreatic cancer, B-cell malignancies, metastatic tumors and colon cancer.

The present application also provides a pharmaceutical composition, comprising one or more of the above compounds and at least one pharmaceutically acceptable carrier or excipient. Specifically, the pharmaceutically acceptable carrier includes one or more of creams, emulsions, gels, liposomes and nanoparticles; the pharmaceutically acceptable excipient includes one or more of binders, fillers, disintegrants, lubricants and glidants.

Furthermore, the above pharmaceutical composition is suitable for oral administration or injection administration.

The present application also provides a kit, which includes any one or more of the above-mentioned compounds or pharmaceutically acceptable salts or esters or any one or more of the pharmaceutical compositions.

The specific compounds or pharmaceutically acceptable salts or esters thereof provided in the present application, or pharmaceutical compositions containing the same, can effectively treat, inhibit or prevent viral infections and/or cell proliferation diseases in mammals, especially smallpox and monkeypox, and have at least one or more of the effects of changing the pharmacokinetic properties in the body, adjusting the absorption and distribution of the drug in the body, improving the stability and solubility of the drug, reducing toxicity and adverse reactions, improving the transport and distribution to specific parts, improving the sustained release effect, and prolonging the duration of action.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the inhibitory activity-drug concentration curve of the test compound.

FIG. 2 shows a death curve diagram of a surrogate endpoint of mouse death, wherein FIG. 2a is a graph of body weight change, and FIG. 2b is a surrogate endpoint death curve.

FIG. 3 shows a graph of the actual mortality of mice, wherein FIG. 3a is a graph of weight changes, and FIG. 3b is an actual mortality curve.

DETAILED DESCRIPTION

In order to provide a clear and consistent understanding of the terms used in the specification of the present invention, some definitions are provided below. In addition, unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by ordinary technicians in the field to which the present invention belongs.

When used in conjunction with the term "comprising" in the claims and/or the specification, the use of the word "a" can mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Similarly, the word "another" can mean at least a second or many.

As used in this specification and claims, the words "comprising", "having" and synonyms are inclusive and open-ended and do not exclude additional unlisted elements or processing steps. The term "about" or "approximately" is used to indicate that the value includes the error caused by the instruments and methods used in determining the value.

The term "pharmaceutically acceptable" as used herein refers to drugs, medicines, inert ingredients, etc. described by the term, which are suitable for contact with the tissues of humans and lower animals without abnormal toxicity, incompatibility, instability, irritation, allergic reaction, etc., commensurate with a reasonable benefit/risk ratio. It is preferably a compound, composition, and preparation, etc. listed in the pharmacopoeia or other recognized pharmacopoeia for use in animals, more particularly in humans.

A "pharmaceutically acceptable salt" of a compound refers to a salt of a compound that is pharmaceutically acceptable. Desirable salts of compounds (basic, acidic or charged functional groups) can retain or improve the biological activity and properties of the parent compound as defined herein and are not biologically undesirable.

The term "ester" refers to esters derived from the compounds of the general formulae herein, including physiologically hydrolyzable esters (which can be hydrolyzed under physiological conditions to release the compounds of the present invention in free acid or alcohol form). The compounds of the present invention themselves may also be esters.

The term "prodrug" or its equivalent refers to a pharmaceutical agent that is directly or indirectly converted into an active form in vitro or in vivo (see, for example, R.B.Silverman, 1992, "The Organic Chemistry of Drug Design and Drug Action," Academic Press, Chap. 8; Bundgaard, Hans; Editor. Neth. (1985), "Design of Prodrugs". 360pp. Elsevier, Amsterdam; Stella, V.; Borchardt, R.; Hageman, M.; Oliyai, R.; Maag, H.; Tilley, J. (Eds.)(2007), "Prodrugs: Challenges and Rewards, XVIII, 1470p. Springer). Prodrugs can be used to alter the biodistribution (e.g., so that the agent does not normally enter the protease reaction site) or pharmacokinetics of a particular drug. A variety of groups such as esters, ethers, phosphates/salts, etc. have been used to modify compounds to form prodrugs. When the prodrug is administered to a subject, the group is cleaved off enzymatically or non-enzymatically, reduced, oxidatively or hydrolytically, or otherwise releases the active compound. As used herein, "prodrug" includes pharmaceutically acceptable salts, or pharmaceutically acceptable solvates, and any crystalline forms of the above. Prodrugs are generally (although not necessarily) pharmaceutically inactive until they are converted to an active form.

It should be understood that the term "substituted" or "substituted" as used in the present invention includes an implicit condition, that is, such substitution changes with the valence of the substituted atom and the substituent, and the substitution produces a stable compound (for example, the compound cannot spontaneously undergo rearrangement, cyclization, elimination, etc.). As used in the present invention, the term "substituted" includes all permissible substituents of organic compounds. In a broad sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic organic compounds. The substituent can be one or more. For example, the term "substituted" means that when the above groups are substituted at one or more positions, the substituents include acylamino (including carbamoyl and ureido), alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, alkoxycarbonyl, carboxyl, carboxyl, aminocarbonyl, mono- and dialkylaminocarbonyl, cyano, azido, halogen (F, Cl, Br and I), hydroxy, nitro, trifluoromethyl, thio, alkylthio, arylthio, alkylthiocarbonyl, thiocarboxylate, lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, lower alkoxy, aryloxy, aryloxycarbonyloxy, benzyloxy, benzyl, sulfinyl, alkylsulfinyl, sulfonyl, sulfate, sulfonate, sulfonamide, phosphate, phosphonate, imino, formyl, etc. If permitted, any of the above substituents can be further substituted, for example, by alkyl, aryl or other groups.

The term "alkylcarbonyl", "arylcarbonyl", "heterocycliccarbonyl" or "alkyloxycarbonyl" refers to -C(=O) _Ra , where _Ra is a hydrocarbon, aryl, heteroaryl, heterocyclic or alkyloxy. The term "alkyloxy" refers to _-ORb , where _Rb is a hydrocarbon. For example, the expression "C4-C30carbonyl" refers to "-C(=O)Ra" where _Ra has 3 to 29 carbon atoms, specifically, the number of carbon atoms may be 3, 9, 11, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and _Ra may be a substituted or unsubstituted hydrocarbon, a substituted or unsubstituted aryl, a substituted or unsubstituted _three -membered ring, a four-membered ring, a five-membered ring, a substituted or unsubstituted alkyloxy.

The term "C15-C30 hydrocarbon group" means having 15 to 30 carbon atoms in the hydrocarbon structure, specifically, the number of carbon atoms can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and the term "C1-C16 hydrocarbon group" means having 1 to 16 carbon atoms in the hydrocarbon structure, specifically, the number of carbon atoms can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16.

The term "aryl" or "aryl ring" used in the present invention refers to an aromatic group having "4n+2" (π) electrons in a conjugated monocyclic or polycyclic system (condensed or non-condensed), and having 6 to 14 ring atoms, wherein n is an integer from 1 to 3. The polycyclic system includes at least one aromatic ring. The aryl group can be directly connected or connected through a C1-C3 alkyl group (also referred to as an arylalkyl group or an aralkyl group). Examples of aryl groups include, but are not limited to, phenyl, benzyl, phenethyl, 1-phenylethyl, tolyl, naphthyl, biphenyl, terphenyl, indenyl, benzocyclooctenyl, benzocycloheptenyl, azulenyl, acenaphthenyl, fluorenyl, phenanthrenyl, anthracenyl, etc. The term "aryl" includes unsubstituted aryl and substituted aryl.

The term "heteroaryl" or "heteroaryl ring" as used herein refers to an aromatic group having "4n+2" (π) electrons in a conjugated monocyclic or polycyclic system (fused or non-fused), wherein n is an integer from 1 to 3 and includes one to six heteroatoms (e.g., N, O, S, P) or a group including heteroatoms (e.g., NH, NRx (Rx is alkyl, acyl, aryl, heteroaryl or cycloalkyl), ^PO2 , SO, _SO2 , etc.). The polycyclic system includes at least one heteroaromatic ring. The heteroaryl group can be directly attached or attached through a C1-C3 alkyl group (also called heteroarylalkyl or heteroaralkyl). The heteroaryl group can be attached to carbon or to a heteroatom (e.g., through a nitrogen atom). Examples of heteroaryl groups include, but are not limited to, pyridyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, tetrazolyl, furanyl, thienyl; isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolidinyl, quinolyl, isoquinolyl, indolyl, isoindolyl, chromenyl, isochromenyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl , pyrazinyl, triazine, isoindolyl, pteridinyl, furanyl, benzofuranyl, benzothiazolyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinolinone, isoquinolinone, quinoxalinyl, naphthyridinyl, furopyridinyl, carbazolyl, phenanthridinyl, acridinyl, peryl, phenanthroline, phenazinyl, phenothiazinyl, phenoxazinyl, dibenzofuranyl, etc. The term "heteroaryl" includes unsubstituted heteroaryl and substituted heteroaryl. The term "C5-Cn heteroaryl", wherein n is an integer from 6 to 15, means a heteroaryl having from 5 to "n" atoms in the ring structure, including at least one heterocyclic group or atom as defined above.

In some embodiments, the present invention provides a phosphonate compound, or a pharmaceutically acceptable salt or ester thereof, for use in the preparation of a medicament for treating, inhibiting or preventing a viral infection or a cell proliferation disease, wherein the phosphonate compound is selected from but not limited to the compounds listed in Table 1a and Table 1b.

The term "amino acid residue" refers to the major portion of an amino acid after removal of the carboxyl group.

As used herein, the term "amino acid" generally refers to an organic compound that contains both a carboxylic acid group and an amino group. The term "amino acid" includes "natural" and "unnatural" amino acids. In addition, the term "amino acid" includes O-alkylated or N-alkylated amino acids, as well as amino acids with nitrogen-, sulfur- or oxygen-containing side chains (e.g., Lys, Cys or Ser), wherein the nitrogen, sulfur or oxygen atom may or may not be acylated or alkylated. The amino acid may be an L-amino acid, a D-amino acid or a mixed L- and D-amino acid, including (but not limited to) a racemic mixture.

The term "natural amino acid" and equivalent expressions used in the present invention refer to L-amino acids that are usually found in naturally occurring proteins. Examples of natural amino acids include, but are not limited to, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), β-alanine (β-Ala) and γ-aminobutyric acid (GABA), etc.

The term "non-natural amino acid" as used herein refers to any derivative of a natural amino acid, including D-amino acids and their derivatives, as well as α- and β-amino acid derivatives. It should be noted that certain non-natural amino acids (e.g., hydroxyproline) in the present invention may exist in certain biological tissues or specific proteins in nature. Amino acids with many different protecting groups suitable for direct use in solid phase peptide synthesis are commercially available. In addition to the twenty most common natural amino acids, the following examples of unnatural amino acids and amino acid derivatives can be used according to the present invention (common abbreviations are in parentheses): 2-aminoadipic acid (Aad), 3-aminoadipic acid (β-Aad), 2-aminobutyric acid (2-Abu), α,β-dehydro-2-aminobutyric acid (8-AU), 1-aminocyclopropane-1-carboxylic acid (ACPC), aminoisobutyric acid (Aib), 3-aminoisobutyric acid (β-Aib), 2-amino-thiazoline-4-carboxylic acid, 5-aminopentanoic acid (5-Ava), 6-aminohexanoic acid (6-Ahx), 2-aminoheptanoic acid (Ahe), 8-aminooctanoic acid (8-Aoc), 11-aminoundecanoic acid (11-Aun), 12-aminododecanoic acid (12-Ado), 2-aminobenzoic acid (2-Abz), 3-aminobenzoic acid (3-Abz), 4-aminobenzoic acid (4-Abz), 4-amino-3-hydroxy-6-methylheptanoic acid (Statine, Sta), aminooxyacetic acid (Aoa), 2-aminotetralin-2-carboxylic acid (ATC), 4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA), p-aminophenylalanine (4-NH2-Phe), 2-aminopimelic acid (Apm), biphenylalanine (Bip), p-bromophenylalanine (4-Br-Phe), o-chlorophenylalanine (2 -Cl-Phe), m-chlorophenylalanine (3-Cl-Phe), p-chlorophenylalanine (3-Cl-Phe), m-chlorotyrosine (3-Cl-Tyr), p-benzoylphenylalanine (Bpa), tert-butylglycine (TLG), cyclohexylalanine (Cha), cyclohexylglycine (Chg), desmosine (Des), 2,2-diaminopimelic acid (Dpm), 2,3-diaminopropionic acid (Dpr), 2,4- diaminobutyric acid (Dbu), 3,4-dichlorophenylalanine (3,4-Cl2-Phe), 3,4-difluorophenylalanine (3,4-F2-Phe), 3,5-diiodotyrosine (3,5-I2-Tyr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), o-fluorophenylalanine (2-F-Phe), m-fluorophenylalanine (3-F-Phe), p-fluorophenylalanine (4-F-Phe), he), m-fluorotyrosine (3-F-Tyr), homoserine (Hse), homophenylalanine (Hfe), homotyrosine hydroxylysine (Hyl), isohydroxylysine (aHyl), 5-hydroxytryptophan (5-OH-Trp), 3- or 4-hydroxyproline (3- or 4-Hyp), p-iodophenylalanine-isotrosine (3-I-Tyr), indoline-2-carboxylic acid (Idc), isoiduromycin (Ide), iso Leucine (α-Ile), isopenipecolic acid (Inp), N-methylisoleucine (MeLys), m-methyltyrosine (3-Me-Tyr), N-methylvaline (MeVal), 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), p-nitrophenylalanine (4-NO2-Phe), 3-nitrotyrosine (3-NO2-Tyr), norleucine (Nle), norvaline (Nva ), ornithine (Orn), o-phosphotyrosine (H2PO3-Tyr), octahydroindole-2-carboxylic acid (Penicillamine), pentafluorophenylalanine (F5-Phe), phenylglycine (Phg), pipecolic acid (Pip), propargylglycine (Pra), pyroglutamic acid (PGLU), sarcosine (Sar), tetrahydroisoquinoline-3-carboxylic acid (Tic), thiazolidine-4-carboxylic acid (thioproline, Th).

The present invention provides methods for treating mammalian diseases associated with viral infection, inappropriate cell proliferation, etc. These methods specifically comprise administering to a human or other mammal in need of treatment a therapeutically effective amount of a compound of the present invention.

In some embodiments, the compounds provided herein can be used to prepare a drug that can be used to treat, inhibit or prevent viral infection or viral infection-induced diseases. In some embodiments, the compounds provided herein and their pharmaceutically acceptable salts or esters are used to prepare drugs for treating smallpox virus infection or diseases caused by smallpox virus. In some embodiments, the compounds provided herein and their pharmaceutically acceptable salts or esters are used to prepare drugs for treating monkeypox virus infection or diseases caused by monkeypox virus.

The compounds provided herein have good effects in treating, inhibiting or preventing viral infections and diseases caused by viral infections. At the same time, the applicant has found that the compounds provided herein also have good effects in treating cancer or tumors. In some embodiments, the compounds provided herein and their pharmaceutically acceptable salts or esters can be used to prepare drugs, which can be used to treat, inhibit or prevent tumors or cancers caused by cell proliferation.

In some embodiments, the drug provided herein further comprises at least one pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutically acceptable carrier or diluent is selected from creams, emulsions, gels, liposomes or nanoparticles.

The compound or medicine provided by the present invention can be applied to the subject in any appropriate manner known in the art. Suitable routes of administration include, but are not limited to, oral; parenteral, such as intramuscular, intravenous, subcutaneous (such as injection or implantation), intraperitoneal, intracisternal, intraarticular, intracerebral (intracerebral parenchyma and intraventricular); nasal; vaginal; sublingual; intraocular; rectal; topical (such as transdermal); oral and inhaled. The accumulation injection method generally administered subcutaneously or intramuscularly can also be used to release the compound or medicine disclosed in the present application within a limited time period. In some embodiments, the medicine is an injectable preparation. In other embodiments, the medicine is formulated for oral administration to the subject.

The present invention also provides a kit comprising an antiviral infection compound or drug. The kit is generally in the form of a physical structure that accommodates various components, and can be used, for example, to implement the method provided herein. For example, the kit may include one or more compounds or drugs disclosed in the present invention (e.g., provided in a sterile container), which may be in the form of a pharmaceutical composition suitable for administration to a subject. The compound may be provided in a ready-to-use form (e.g., tablet or capsule) or in a form that requires, for example, reconstruction or dilution (e.g., powder) before administration. When the compound is in a form that requires the user to be reconstructed or diluted, the kit may also include a diluent (e.g., sterile water), a buffer, a pharmaceutically acceptable excipient, etc. that is packaged together with the compound or packaged separately. When a combination therapy is used, the kit may contain several therapeutic agents independently, or they may have been combined in the kit. Each component of the kit may be encapsulated in a separate container, and all the various containers may be in a single package. The kit of the present invention may be designed to appropriately maintain the conditions required for the components contained therein (e.g., refrigeration or freezing).

The medicaments or pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, the method of such preparation comprises the steps of combining a compound described herein ("active ingredient") with a carrier and/or one or more other auxiliary ingredients, and then, if necessary and/or desired, forming and/or packaging the product into a desired single-dose unit or multiple-dose units.

Wherein the drug or pharmaceutical composition can be prepared, packaged and/or sold in batches as a single unit dose and/or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and/or a convenient fraction of such a dose, such as half or one-third of such a dose.

In order to better understand the present invention and to more clearly show how to implement the present invention, the features of the embodiments according to the present invention are now described by way of examples.

Example

The present invention will be more readily understood by reference to the following examples, which are provided to illustrate the present invention and are not to be construed as limiting the scope of the present invention in any way.

The compounds provided by the present invention can be synthesized according to the following general formula, wherein all reagents used to prepare the compounds of the present invention are commercially available or prepared according to preparation methods disclosed in the literature.

Compound A is used as a starting material, and under the action of a condensation agent 1H-benzotriazole-1-yloxytripyrrolidino hexafluorophosphate (PYBOP), it undergoes cyclization and condensation reaction with different alcohols to obtain compound B; under the action of a base such as potassium carbonate, the amino group on compound B reacts with an acyl chloride to obtain compound C, which is then hydrolyzed with a sodium hydroxide solution and acidified to obtain compound D;

The amino group on the intermediate compound B is protected by Boc anhydride to obtain compound E, which is then hydrolyzed with sodium hydroxide solution to obtain a ring-opened compound F. Compound F is condensed with an acyl chloride to obtain compound G, which is then deprotected to obtain compound H.

All R groups such as R ¹ , R ² , R ³ , R ⁴ in the general formula are as defined in the specification and claims.

Unless otherwise defined or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

Synthesis of intermediates:

Step A: Sodium hydride (4.91 g, 122.82 mmol, 1.5 eq.) and DMF (150 mL) were added to the reaction flask. Under stirring in an ice-water bath, a DMF solution of 1,3-propylene glycol (28.04 g, 368.45 mmol, 26.63 mL, 4.5 eq.; dissolved in 150 mL DMF) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 10 minutes. 1-bromohexadecane (25 g, 81.88 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the reaction was stirred at 95°C for 5 hours. The mixture was cooled to room temperature, ethyl acetate (400 mL) was added, and the layers were separated and washed with water (300 mL). The organic layer was washed three times with brine (300 mL*3). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was separated and purified by silica gel column (petroleum ether: ethyl acetate = 100: 0-80: 20) to obtain product b (12.5 g, yield 50.80%).

Step B: Add [rac-(1S)-1-[(4-amino-2-oxopyrimidin-1-yl)methyl]-2-hydroxyethoxy]methylphosphonic acid (1.5 g, 5.37 mmol, 1 eq.), DMF (25 mL) and DIPEA (10 mL) to the reaction flask. Stir the reaction at 45°C for 2 hours. Concentrate and remove the solvent to obtain a white solid residue, add DMF (25 mL), b (2.4 g, 8.06 mmol, 1.5 eq.), DIPEA (4.17 g, 32.24 mmol, 5.61 mL, 6.0 eq.) and pyBOP (8.39 g, 16.12 mmol, 3.0 eq.). Stir the reaction at 45°C for 16 hours, concentrate and remove the reaction solvent, and separate and purify the residue with a silica gel column (dichloromethane: methanol = 100: 0-90: 10) to obtain intermediate 1 (2 g, yield 68.47%).

Step C: Add intermediate 1 (100 mg, 0.18 mmol, 1 eq.) and sodium hydroxide aqueous solution (0.5 M, 3.68 mmol, 10 eq.) to the reaction flask. Stir the reaction for 4 hours at room temperature. The reaction system becomes clear. In an ice-water bath, slowly add 1N HCl solution dropwise to adjust the pH to about 1. A large amount of solid precipitates, which is filtered and dried in vacuo to obtain intermediate 2 (50 mg, yield 47.09%). ¹ H NMR (500MHz, CD ₃ Cl ₃ )δppm:0.84(t,J＝6.6Hz,3H),1.21(s,26H),1.49(s,2H),1.74-1.91(m,2H),3.28-3.86(m,10 H), 3.93 (d, J = 6.4Hz, 2H), 4.22 (d, J = 13.9Hz, 1H), 6.11 (s, 1H), 7.70 (d, J = 7.2Hz, 1H); m/z (ESI ⁺ ):562.6(M+H).

Synthesis of compounds

Example 1: Synthesis of Compound 1b

Step A: Add intermediate 1 (100 mg, 183.93 μmol, 1 eq.), DCM (15 mL), and benzoyl chloride (33.61 mg, 239.11 μmol, 27.76 μL, 1.3 eq.) to the reaction flask. Add triethylamine (55.84 mg, 551.80 μmol, 76.70 μL, 3.0 eq.) dropwise under stirring at 0°C. React at room temperature for 16 hours, concentrate to remove the reaction solvent, and separate and purify the residue with a silica gel column (dichloromethane: methanol = 100: 0-97: 3) to obtain compound 1a (60 mg, yield 49.61%). ¹ H NMR (500MHz, CDCl ₃ )δppm:0.87(t,J＝6.8Hz,3H),1.26(d,J＝15.6Hz,26H),1.54(s,2H),1.85-2 .04(m,2H),3.39(dd,J＝12.0,6.3Hz,2H),3.49(dt,J＝18.5,5.9Hz,2H),3.6 2(dd,J＝14.1,7.9Hz,1H),3.87(dd,J＝27.5,14.4Hz,1H),4.10-4.48(m,7H) ,7.53(t,J＝7.5Hz,3H),7.64(dd,J＝15.7,7.1Hz,2H),7.89(d,J＝6.7Hz,2H); ³¹ P NMR (203MHz, CDCl ₃ ) δppm: 10.01, 11.97; m/z (ESI ⁺ ): 648.6 (M+H).

Example 2: Synthesis of Compound 2b

Step A: Add intermediate 2 (600 mg, 1.07 mmol, 1.0 eq.), DMF (10 mL) and triethylamine (5 mL) to a reaction flask, stir at room temperature for half an hour, concentrate to remove the solvent, add DMF (10 mL), 1-[chloro-(4-methoxyphenyl)-phenylmethyl]-4-methoxybenzene (545 mg, 1.6 mmol, 1.5 eq.) and triethylamine (325 mg, 3.2 mmol, 3.0 eq.). Stir at room temperature for one hour, concentrate to remove the solvent. Add DMF (20 mL) to the residue and prepare the intermediate 2-1 reaction reagent (46 mg/mL) for standby use.

Step B: Add intermediate 2-1 reaction reagent (300 mg, 46 mg/mL, 7 mL, 1.0 eq.), DMF (2 mL) and DIPEA (225 mg, 1.74 mmol, 3.0 eq.) to the reaction flask. Slowly add benzoyl chloride (98 mg, 0.7 mmol, 2.0 eq.) under ice-water bath. Stir the reaction at room temperature for 16 hours. Concentrate to remove the reaction solvent, and the residue is separated and purified by silica gel column (dichloromethane: methanol = 100: 0-92: 8) to obtain crude product 2-2 (200 mg, yield 59.5%).

Step C: Add crude product 2-2 (200 mg, 0.2 mmol, 1.0 eq.), acetonitrile (2 mL) and acetic acid (4 mL) to the reaction flask. Stir the reaction at room temperature for 16 hours. The reaction solution is directly injected into a reverse phase C-18 silica gel column for separation and purification (water: acetonitrile = 100: 0-35: 65), and lyophilized to obtain a crude product. Add water (2 mL) to the crude product, then slowly add sodium bicarbonate aqueous solution to adjust the pH to 7-8, and then add acetonitrile until the system is clear. The clarified system is prepared by a preparative column (water: acetonitrile = 100: 0-20: 80) to obtain compound 2b. ¹ H NMR (500MHz, CDCl ₃ )δppm:0.87(t,J＝6.7Hz,3H),1.10-1.32(m,26H),1.36(s,2H),1.66(s,2H),3.20-3.28(m,6H),3.40-4.29(m,8H ),6.11(s,1H),7.38(t,J＝7.4Hz,2H),7.49(dd,J＝18.4,10.7Hz,2H),7.89(dd,J＝51.9,6.8Hz,3H),10.28(s,1H); ³¹ P NMR (203MHz, CDCl ₃ ) δppm: 16.29; m/z (ESI ⁺ ): 666.6 (M+H).

Example 3: Synthesis of Compound 3b

Step B: Add intermediate 1 (110 mg, 202.33 μmol, 1 eq.), DIPEA (78.45 mg, 606.98 μmol, 3 eq.), 2-methoxyethyl (4-nitrophenyl) carbonate (97.6 mg, 404.65 μmol, 2 eq.) and DMF (2 mL) to the reaction flask. Stir the reaction at 50 °C for 16 hours. TLC column chromatography detected that the reaction raw materials were completely consumed. The reaction system was restored to room temperature, water (40 mL) was added, ethyl acetate was extracted (20 mL*2), the organic layer was washed once with saturated brine (40 mL), the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and after concentration, the residue was separated and purified by silica gel column (dichloromethane: methanol = 100: 0-94: 6) to obtain product 3-1 (80 mg, yield 61.23%).

Step C: Add 3-1 (80 mg, 123.89 μmol, 1 eq.) and THF (4 mL) to the reaction flask, add sodium hydroxide aqueous solution (0.5 mol/L, 743.31 μL, 3 eq.), and stir at room temperature for one hour. LC-MS monitored the complete consumption of the reaction raw materials, and adjusted the pH of the reaction system to 2 with 1 mol/L hydrochloric acid aqueous solution under ice-water bath conditions, and then concentrated to remove most of the ^solvent . Then the crude product was directly separated and purified using a C-18 reverse phase column (water: acetonitrile = 100: 0-60: 40), and lyophilized to obtain compound 3b (53.1 mg, yield 64.60%). NMR(500MHz,MeOD)δ0.93(t,J＝6.7Hz,3H),1.32(s,26H),1.54-1.62(m,2H),1.86-1.94(m,2H) ,3.41(s,3H),3.45(t,J＝6.6Hz,2H),3.53(t,J＝6.1Hz,2H),3.63(dd,J＝12.2,3.8Hz,1H),3.66 -3.71(m,2H),3.73-3.86(m,3H),3.93(ddd,J＝21.8,13.6,8.6Hz,2H),4.07(q,J＝6.5Hz,2H),4 .29(d,J＝11.4Hz,1H),4.34-4.40(m,2H),7.22(d,J＝7.3Hz,1H),8.03(d,J＝7.3Hz,1H); m/z(ESI ⁺ ):664.5(M+H).

Example 4: Synthesis of Compound 4b

Step B: Add intermediate 1 (102 mg, 187.61 μmol, 1 eq.), DIPEA (72.74 mg, 562.84 μmol, 3 eq.) and DMF (2 mL) to the reaction flask. Add a solution of n-pentyl chloroformate (56.51 mg, 375.22 μmol, 2 eq.) in DMF (0.5 mL) dropwise to the reaction system under stirring, and stir at 50°C for 16 hours. TLC column chromatography detected that the reaction raw materials were completely consumed. The reaction system was restored to room temperature, water (40 mL) was added, ethyl acetate was extracted (20 mL*2), the organic layer was washed once with saturated brine (40 mL), the organic layer was separated, dried and filtered with anhydrous sodium sulfate, and after concentration, the residue was separated and purified by silica gel column (dichloromethane: methanol = 100: 0-94: 6) to obtain product 4-1 (64 mg, yield 51.86%). Step C: Add 4-1 (64 mg, 97.29 μmol, 1 eq.) and THF (3 mL) to the reaction flask, add sodium hydroxide aqueous solution (0.5 mol/L, 486.46 μL, 2.5 eq.), and stir at room temperature for one hour. LC-MS monitoring shows that the reaction raw materials have been consumed. Adjust the pH of the reaction system to 2 with 1 mol/L hydrochloric acid aqueous solution under ice-water bath conditions, and then concentrate to remove most of the ^solvent . Then the crude product is directly separated and purified using a C-18 reverse phase column (water: acetonitrile = 100: 0-50: 50), and freeze-dried to obtain compound 4b (17.7 mg, yield 27.23%). NMR(500MHz,MeOD)δ0.93(t,J＝6.8Hz,3H),0.97(t,J＝6.7Hz,3H),1.30-1.36(m,26H),1.40-1.47(m,4H),1. 55-1.61(m,2H),1.69-1.79(m,2H),1.85-1.92(m,2H),3.44(t,J＝6.6Hz,2H),3.52(t,J＝6.2Hz,2H),3.57-3. 63(m,1H),3.70(dd,J＝13.2,9.5Hz,1H),3.80(d,J＝10.0Hz,2H),3.90(td,J＝13.9,8.4Hz,2H),4.03(q,J＝6.2 Hz,2H),4.22(t,J＝6.6Hz,2H),4.27(d,J＝14.2Hz,1H),7.25(d,J＝7.3Hz,1H),8.05(d,J＝7.3Hz,1H); m/z(ESI ⁺ ):676.6(M+H).

Example 5: Synthesis of Compound 5b

Step A: Add p-chlorobenzoic acid (0.5 g, 3.19 mmol, 1.0 eq.), dichloromethane (15 mL), p-nitrophenol (533 mg, 3.83 mmol, 1.2 eq.) and DCC (0.79 g, 3.83 mmol, 1.2 eq.) to the reaction flask. Stir overnight at room temperature, filter to remove solids, concentrate the mother liquor, and purify the residue by silica gel column separation (petroleum ether: ethyl acetate = 100: 0-75: 25) to obtain the product 4-nitrophenyl-4-chlorobenzoate (0.8 g, yield 92%)

Step B: Add intermediate 1 (0.15 g, 0.275 mmol, 1 eq.), 4-nitrophenyl-4-chlorobenzoate (0.191 g, 0.689 mmol, 2.5 eq.), DMF (5 mL) and DIPEA (0.11 g, 0.827 mmol, 3.0 eq.) to the reaction flask. Stir the reaction at 50°C for 8 hours, add ethyl acetate (30 mL) and water (15 mL), extract and wash the layers, and wash the organic layer with brine 3 times. Concentrate to remove the solvent, and the residue is separated and purified by silica gel column (dichloromethane: methanol = 100: 0-95: 5) to obtain product 5-1 (130 mg, yield 69.1%).

Step C: Add 5-1 (80 mg, 0.12 mmol, 1 eq.), tetrahydrofuran (5 mL) and sodium hydroxide aqueous solution (0.5 M, 469 ul, 2.0 eq.) to the reaction flask. Stir the reaction for 1.5 hours at room temperature. The reaction system becomes clear. In an ice-water bath, slowly add 1N HCl solution to adjust the pH to about 1. After adding water and freeze-drying, the crude product is separated and purified by reverse phase C-18 silica gel column (water: acetonitrile = 100: 0-30: 70), and freeze-dried to obtain compound 5b (45 mg, yield 54.1%). ¹ H NMR (500MHz, CD ₃ OD)δppm:0.89(t,J＝6.7Hz,3H),1.26(d,J＝10.9Hz,26H),1.48(s,2H),1.76-1.89(m,2H),3.36(t,J＝6.5Hz,2H),3.47(t,J＝6.2Hz,2H),3 .54-3.70(m,2H),3.75-4.05(m,6H),4.28(d,J＝13.6Hz,1H),7.55(t,J＝8.8Hz,3H),7.98(d,J＝8.4Hz,2H),8.10(d,J＝7.0Hz,1H); m/z(ESI ⁺ ):700.5(M+H).

Example 6: Synthesis of Compound 6b

The synthetic steps were the same as in Example 5 except that m-chlorobenzoic acid was used to obtain the product compound 6b (35 mg, yield 38.02%). ¹ H NMR (500 MHz, MeOD) δ 0.93 (t, J = 6.8 Hz, 3H), 1.30 (d, J = 7.0 Hz, 26H), 1.50-1.55 (m, 2H), 1.87-1.93 (m, 2H), 3.41 (t, J = 6.6 Hz, 2H), 3.51 (t, J = 6.1 Hz, 2H), 3.65 (dd, J = 12.0, 3.4 Hz, 1H), 3.75 (dd, J = 13.4, 9.7 Hz,1H),3.80-3.90(m,2H),3.91-3.99(m,2H),4.06(q,J＝6.4Hz,2H),4.34(d,J＝11.7Hz,1H),7.54-7 .62(m,2H),7.70(d,J＝7.8Hz,1H),7.96(d,J＝7.7Hz,1H),8.04(s,1H),8.13(d,J＝7.2Hz,1H); m/z(ESI ⁺ ):700.5(M+H).

Example 7: Synthesis of Compound 7b

Step A: Compound 5b (84 mg, 0.12 mmol, 1.0 eq.) DMF (4 mL) and NaH (47.98 mg, 1.20 mmol, 60% purity, 10.0 eq.) were added to the reaction flask. Stir at room temperature for 20 minutes. 4-nitrophenyl-4-chlorobenzoate (66.7 mg, 0.239 mmol, 2.1 eq.) DMF (0.5 mL) solution was added dropwise to the reaction. Stir the reaction at room temperature for 3 hours, add ethyl acetate (25 mL) to dilute, and add 1N HCl dropwise to quench the reaction at low temperature to ensure that the pH of the aqueous phase is 1-2. The organic layer was washed twice with brine, dried and concentrated, and the residue was separated and purified by silica gel column (dichloromethane: methanol = 100: 0-80: 20) to obtain compound 7b (35 mg, yield 33.32%). ¹ H NMR (500MHz, CDCl ₃ )δppm:0.84(t,J＝6.8Hz,3H),1.19(d,J＝17.3Hz,25H),1.42(s,2H),1.75(s,2H),3.25(s,2H),3.37(s,2H),3.84(t,J＝51.6H z,5H),4.28(s,2H),4.44(s,1H),4.53(s,1H),7.39(t,J＝13.0Hz,4H),7.53(s,1H),7.80(s,1H),7.85-8.07(m,4H); m/z(ESI ⁺ ):838.5(M+H).

Example 8: Synthesis of Compound 8b

Step B: n-pentanol (0.5 g, 3.32 mmol, 1 eq.) and DCM (20 mL) were added to the reaction flask, and pyridine (0.31 g, 3.98 mmol, 1.2 eq.) was added dropwise under ice bath conditions. A solution of 4-nitrophenyl phosgene (0.46 g, 3.32 mmol, 1 eq.) in DCM (15 mL) was added dropwise, and the reaction was stirred at room temperature for 2 hours after the addition. TLC monitored the complete consumption of the reaction raw materials, and water (30 mL) was added, and dichloromethane was extracted and washed (30 mL*2). The organic layer was washed once with saturated brine (60 mL), and the organic layer was separated, dried over anhydrous sodium sulfate, and filtered. After concentration, the residue was separated and purified by silica gel column (petroleum ether: ethyl acetate = 100: 0-75: 25) to obtain the product (4-nitrophenyl) pentyl carbonate (0.8 g, yield 95.15%). Intermediate 2 (175 mg, 258.94 μmol, 1 eq.) and THF (6 mL) were added to the reaction flask, and LiHMDS (1 M, 1.04 mL, 4 eq.) was slowly added at 0 ° C. Ten minutes later, (4-nitrophenyl) pentyl carbonate (196.73 mg, 776.82 μmol, 3 eq.) was dissolved in THF (0.3 mL) and added to the above reaction and reacted at room temperature for 3 hours. TLC detected that the reaction was complete, and then the reaction was quenched with 1 M hydrochloric acid. The organic phase was extracted with dichloromethane, filtered and the solvent was removed. The residue was prepared by column chromatography (dichloromethane/methanol=100:0-80:20) and preparation plate to obtain compound 8b (97 mg, yield 47.42%). ¹ H NMR (500MHz, CDCl ₃ and MeOD)δppm:0.78-0.82(d,9H),1.16-1.21(d,34H),1.44(s,2H),1.58(s,4H),1.73(s,2H),3.28(s,2H ),3.37(s,2H),3.52(m,3H),3.78(s,3H),4.07(m,6H),4.28(s,1H),7.11(s,1H),7.72(s,1H); m/z(ESI ⁺ ):790.7(M+H).

Example 9: Synthesis of Compound 9b

Step B: Add intermediate 1 (150 mg, 275.90 μmol, 1 eq.), dichloromethane (6 mL), DIPEA (106.97 mg, 827.70 μmol, 144.17 μL, 3 eq.), DMAP (16.85 mg, 137.95 μmol, 0.5 eq.) and CbzCl (141.20 mg, 827.70 μmol, 116.50 μL, 3 eq.) to the reaction flask. Stir and react at room temperature for 16 hours. After the reaction system was diluted with dichloromethane (20 ml), it was washed once with 0.1 M dilute hydrochloric acid (10 mL), washed with concentrated brine, the organic phase was dried and concentrated, and the residue was separated and purified by silica gel column (dichloromethane: methanol = 100: 0-95: 5) to obtain product 9-1 (148 mg, yield 79.14%)

Step C: Add 9-1 (30 mg, 44.26 μmol, 1 eq.), tetrahydrofuran (2 mL) and 0.5 M NaOH aqueous solution (0.5 M, 619.65 μL, 7 eq.) to the reaction flask. Stir at room temperature for 1 hour and directly concentrate to obtain product 9-2 (30.7 mg, yield 99.94%).

Step D: Add 2-(2-(2-methoxymethoxy)ethoxy)ethanol-1-ol (2.0 g, 12.18 mmol, 1 eq.) and DCM (20 mL) into a reaction flask, add triethylamine (1.85 g, 18.27 mmol, 1.5 eq.), add 4-nitrophenylphosgene (2.46 g, 12.18 mmol, 1 eq.) in DCM (15 mL) dropwise under ice bath conditions, and stir the reaction at room temperature for 16 hours. The reaction raw materials were consumed as monitored by TLC, and water (100 mL) was added, and the mixture was washed with dichloromethane (30 mL*2). The organic layer was washed once with saturated brine (60 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and filtered. After concentration, the residue was separated and purified by silica gel column (petroleum ether: ethyl acetate = 100: 0-60: 40) to obtain the product 2-(2-(2-methoxymethoxy)ethoxy)ethyl (4-nitrophenyl) carbonate (2.1 g, yield 52.36%). 9-2 (400 mg, 574.86 μmol, 1 eq.) and DMF (5 mL) were added to the reaction flask, and NaH (183.96 mg, 4.60 mmol, 60% purity, 8 eq.) was added under ice bath. The reaction was stirred at 0°C for 20 min, and then a solution of 2-(2-(2-methoxymethoxy)ethoxy)ethyl(4-nitrophenyl)carbonate (378.61 mg, 1.15 mmol, 2 eq.) in DMF (1 mL) was added dropwise. After the addition, the reaction was stirred at room temperature for 16 hours. TLC monitoring showed that most of the raw materials were consumed. Under ice-water bath conditions, the reaction system was adjusted to pH 2 with 1 mol/L hydrochloric acid aqueous solution, water (50 mL) was added, and ethyl acetate was extracted and washed (20 mL*4). The organic layer was washed once with saturated brine (60 mL), and the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified using a preparative plate (dichloromethane: methanol = 10: 1) to obtain 9-3 (180 mg, yield 35.34%).

Step E: Add 9-3 (155 mg, 174.94 μmol, 1 eq.), tetrahydrofuran/methanol (V/V=5:1, 15 mL) and palladium carbon (25 mg, 10% carbon adsorption, containing 55% water) to the reaction flask. Stir the reaction at room temperature overnight under hydrogen atmosphere. LC-MS monitoring showed that most of the raw materials were consumed, and the reaction was filtered and the filter cake was washed with methanol (50 mL). The mother liquor was concentrated to obtain a crude product, which was separated and purified by reverse ^phase preparative column (acetonitrile, water, ammonium acetate system) and freeze-dried to obtain compound 9b (50 mg, yield 35.61%). NMR(500MHz,MeOD)δ0.93(t,J＝6.6Hz,3H),1.32(s,26H),1.55-1.60(m,2H),1.84-1 .91(m,2H),3.38(s,3H),3.45(t,J＝6.4Hz,2H),3.52-3.61(m,5H),3.64-3.69(m,6H ),3.72-3.76(m,2H),3.88(dd,J＝12.6,8.9Hz,1H),3.95-4.00(m,4H),4.18-4.34(m ,4H),4.43(d,J＝11.7Hz,1H),6.01(d,J＝7.4Hz,1H),7.99(d,J＝7.3Hz,1H); m/z(ESI ⁺ ):752.7(M+H).

Example 10: Synthesis of Compound 10b

Step A: Add 4-(nitro)phenol (2.51 g, 18.01 mmol, 1.1 eq.), dichloromethane (80 mL), benzoic acid (2 g, 16.38 mmol, 1.0 eq.) and DCC (4.05 g, 19.65 mmol, 1.2 eq.) to the reaction flask. React at room temperature for 16 hours under stirring. Filter to remove solids, concentrate the mother liquor to remove dichloromethane, add ethyl acetate (100 mL) and water (70 mL) to the residue, extract and wash the layers, and wash the organic layer with brine (70 mL). Concentrate the organic phase to remove the solvent, and pass through a silica gel column (petroleum ether: ethyl acetate = 100:0-75:25) to obtain the product (4-nitrophenyl) benzoate (2.1 g, yield: 46.18%).

Step B: Add 9-2 (0.2 g, 0.287 mmol, 1 eq.) and tetrahydrofuran (5 mL) to the reaction flask. Add LiHMDs (1 M, 1.15 mL, 4 eq.) dropwise under ice-water bath conditions, stir for 20 minutes after the addition is complete. Add a tetrahydrofuran solution of (4-nitrophenyl) benzoate (0.21 g, 0.862 mmol, 3.0 eq. dissolved in 1 mL tetrahydrofuran) dropwise to the reaction system. React at room temperature for 4 hours under stirring. Add ethyl acetate (30 mL), then add 1N hydrochloric acid dropwise to quench the reaction, ensure that the pH of the aqueous phase is about 1-2, and wash the organic layer with saturated brine (30 mL). After the organic layer is concentrated, pass it through a silica gel column (dichloromethane: methanol = 100:0-85:15) to obtain the product 10-1 (0.11 g, yield: 47.84%)

Step C: Add 10-1 (140 mg, 0.175 mmol, 1 eq.), tetrahydrofuran (20 mL), methanol (5 mL) and Pd/C (25 mg) to the reaction flask. React at room temperature for 16 hours under hydrogen atmosphere, filter to remove palladium carbon, concentrate the mother liquor and purify it by preparative column to obtain compound 10b (23 mg, yield 19.55%). ¹ H NMR (500 MHz, CD ₃ OD)δppm:0.89(t,J＝6.8Hz,3H),1.26(s,26H),1.48(d,J＝6.4Hz,2H),1.73-1.82(m,2H),3.34(t,J＝6.6H z,2H),3.44(t,J＝6.4Hz,2H),3.58-3.72(m,1H),3.92(dd,J＝11.9,8.4Hz,3H),3.98(dd,J＝14.1,7.1Hz, 1H),4.07(s,1H),4.26(d,J＝14.1Hz,1H),4.37(dd,J＝12.0,3.9Hz,1H),4.55(dd,J＝11.8,4.3Hz,1H),5. 97(d,J＝7.5Hz,1H),7.47(t,J＝7.6Hz,2H),7.60(t,J＝7.4Hz,1H),8.01(dd,J＝21.9,7.7Hz,3H); m/z(ESI ⁺ ):666.5(M+H).

Example 11: Synthesis of Compound 11b

The preceding synthesis steps are the same as those in Example 9.

Step D: Nonanoic acid (1.0 g, 6.32 mmol, 1 eq.), 4-nitrophenol (879.11 mg, 6.32 mmol, 1 eq.) and DCM (30 mL) were added to the reaction flask, and N, N'-dicyclohexylcarbodiimide (1.56 g, 7.58 mmol, 1.2 eq.) was added, and the reaction was stirred at room temperature for 6 hours. TLC monitoring showed that most of the reaction raw materials were consumed, and water (60 mL) was added, and dichloromethane was extracted and washed (30 mL*2). The organic layer was washed once with saturated salt water (60 mL), and the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column (petroleum ether: ethyl acetate = 100: 0-80: 20) to obtain the product 4-nitrophenyl nonanoate (670 mg, yield 37.95%). 9-2 (175 mg, 251.50 μmol, 1 eq.) and THF (2 mL) were added to the reaction flask, and LiHMDS (1 M, 1.01 mL, 4 eq.) was added dropwise under an ice bath. The reaction was stirred at 0°C for 20 min, and then a solution of 4-nitrophenyl nonanoate (210.76 mg, 754.50 μmol, 3 eq.) in THF (0.5 mL) was added dropwise. After the addition, the reaction was stirred at room temperature for 3 hours. TLC monitoring showed that most of the raw materials were consumed. The reaction system was adjusted to pH 2 with 1 mol/L hydrochloric acid aqueous solution under ice-water bath conditions. Water (50 mL) was added and washed with ethyl acetate (20 mL*4). The organic layer was washed once with saturated brine (60 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column (dichloromethane: methanol = 100: 0-85: 15) to obtain the product 11-1 (120 mg, yield 57.07%).

Step E: Add 11-1 (100 mg, 119.61 μmol, 1 eq.), tetrahydrofuran/methanol (V/V=5:1, 15 mL) and palladium carbon (20 mg, 10% carbon adsorption, containing 55% water) to the reaction flask. Stir the reaction at room temperature overnight under hydrogen atmosphere. LC-MS monitored the complete consumption of the reaction raw materials, filtered, and the filter cake was washed with methanol (50 mL). The mother liquor was concentrated to obtain a crude product, which was separated and purified by reverse phase preparative column (acetonitrile, water, ammonium acetate system), and lyophilized to obtain compound 11b (15 mg, yield 17.72%). ¹ H NMR(500MHz, CDCl3)δ0.86(t,J=6.0Hz,6H),1.24(s,36H),1.52(s,2H),1.61(s,2H),1.85(s,2H),2.34(s,2H) ,3.34-3.68(m,6H),3.90-3.94(m,4H),4.07-4.39(m,3H),5.46-6.25(m,1H),7.56(d,J＝145.0Hz,1H); m/z(ESI ⁺ ):702.6(M+H).

Example 12: Synthesis of Compound 12b

Step B: Add 2-(2-(2-methoxymethoxy)ethoxy)ethanol-1-ol (2.0 g, 12.18 mmol, 1 eq.) and DCM (20 mL) into a reaction flask, add triethylamine (1.85 g, 18.27 mmol, 1.5 eq.), add 4-nitrophenylphosgene (2.46 g, 12.18 mmol, 1 eq.) in DCM (15 mL) dropwise under ice bath conditions, and stir the reaction at room temperature for 16 hours. After TLC monitoring, the reaction raw materials were consumed, water (100 mL) was added, and dichloromethane was extracted and washed (30 mL*2). The organic layer was washed once with saturated brine (60 mL), the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column (petroleum ether: ethyl acetate = 100: 0-60: 40) to obtain the product 2-(2-(2-methoxymethoxy)ethoxy)ethyl(4-nitrophenyl)carbonate (2.1 g, yield 52.36%). Intermediate 1 (130 mg, 239.11 μmol, 1 eq.), DIPEA (92.71 mg, 717.34 μmol, 3 eq.) and DMF (2 mL) were added to the reaction flask. Under stirring, add 2-(2-(2-methoxymethoxy)ethoxy)ethyl(4-nitrophenyl)carbonate (157.48 mg, 478.23 μmol, 2 eq.) in DMF (1 mL) to the reaction system, and stir at 50°C for 16 hours. TLC column chromatography detected that the reaction raw materials were completely consumed. The reaction system was restored to room temperature, and water (40 mL) was added, and ethyl acetate was extracted (20 mL*2). The organic layer was washed once with saturated brine (40 mL), the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column (dichloromethane: methanol = 100: 0-96: 4) to obtain 12-1 (110 mg, yield 62.69%).

Step C: Add 12-1 (110 mg, 149.89 μmol, 1 eq.) and THF (5 mL) to the reaction flask, add sodium hydroxide aqueous solution (0.5 mol/L, 749.45 μL, 2.5 eq.), and stir at room temperature for one hour. LC-MS monitoring shows that the reaction raw materials have been consumed. Adjust the reaction system pH to 2 with 1 mol/L hydrochloric acid aqueous solution under ice-water bath conditions, and then concentrate to remove most of the ^solvent . Then the crude product is directly separated and purified using a C-18 reverse phase column (water: acetonitrile = 100: 0-55: 45), and lyophilized to obtain 12-2 (50 mg, yield 45.12%). NMR(500MHz,MeOD)δ0.93(t,J＝6.7Hz,3H),1.32(s,26H),1.55-1.60(m,2H),1.87 -1.94(m,2H),3.38(s,3H),3.45(t,J＝6.5Hz,2H),3.51-3.58(m,4H),3.61-3.70(m ,7H),3.74-3.87(m,5H),3.88-4.00(m,2H),4.03-4.12(m,2H),4.28(d,J＝12.1Hz ,1H),4.37(d,J＝2.4Hz,2H),7.22(d,J＝7.2Hz,1H),8.04(d,J＝7.2Hz,1H); m/z(ESI ⁺ ):752.7(M+H).

Step D: 12-2 (235 mg, 312.55 μmol, 1 eq.) and DMF (4 mL) were added to the reaction flask, and NaH (100.02 mg, 2.50 mmol, 60% purity, 8 eq.) was added under ice bath, and the reaction was stirred at 0°C for 20 min. Then, a solution of 2-(2-(2-methoxymethoxy)ethoxy)ethyl(4-nitrophenyl)carbonate (205.85 mg, 625.10 μmol, 2 eq.) in DMF (1 mL) was added dropwise, and the reaction was stirred at 50°C for 2 hours. TLC monitoring showed that most of the raw materials were consumed. The reaction system was adjusted to pH 2 with 1 mol/L hydrochloric acid aqueous solution under ice-water bath conditions. Water (50 mL) was added and washed with ethyl acetate (20 mL*4). The organic layer was washed once with saturated brine (60 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column (dichloromethane: methanol = 100: 0-85: 15) to obtain compound 12b (50 mg, yield 15.90%). ¹ H NMR(500MHz, CDCl3)δ0.79(t,J=6.2Hz,3H),1.17(s,26H),1.44(s,2H),1.70-1.74(m,2H),3.30(s,4H),3.34-3.38(m,2H),3.48(s,4H),3.5 2-3.60(m,17H),3.63-3.69(m,5H),3.74(s,2H),3.86-4.08(m,3H),4.15-4.60(m,6H),7.16(d,J＝5.1Hz,1H),7.71(d,J＝6.9Hz,1H); m/z(ESI ⁺ ):942.99(M+H).

Example 13: Synthesis of Compound 13b

Step A: Methanol (0.5 g, 315.6 mmol, 1.0 eq.), dichloromethane (40 mL), (4-nitrophenyl) carbonyl chloride (3.15 g, 15.6 mmol, 1.0 eq.) and DIPEA (3.03 g, 23.41 mmol, 1.5 eq.) were added to the reaction flask. The reaction was stirred at room temperature for 4 hours. The solvent was removed by concentration, and ethyl acetate (30 mL) and water (20 mL) were added to extract and separate the layers. The organic layer was washed with saturated brine (30 mL). After the organic layer was concentrated, it was passed through a silica gel column (petroleum ether: ethyl acetate = 100: 0-75: 50) to obtain the product (4-nitrophenyl) methyl carbonate (1.6 g, yield 52.01%).

Step B: Add intermediate 1 (0.15 g, 0.275 mmol, 1 eq.), (4-nitrophenyl) methyl carbonate (0.108 g, 0.551 mmol, 2.0 eq.), DMF (5 mL) and DIPEA (0.11 g, 0.827 mmol, 3.0 eq.) to the reaction flask. Stir the reaction at 50°C for 16 hours, add ethyl acetate (30 mL) and water (15 mL), extract and wash the layers, and wash the organic layer with brine three times. Concentrate to remove the solvent, and the residue is separated and purified by silica gel column (dichloromethane: methanol = 100: 0-95: 5) to obtain 13-1 (108 mg, yield 65.06%).

Step C: 13-1 (110 mg, 0.182 mmol, 1 eq.), tetrahydrofuran (7 mL) and sodium hydroxide aqueous solution (0.5 M, 731 ul, 2.0 eq.). Stir the reaction at room temperature for 1 hour. The reaction system becomes clear. In an ice-water bath, slowly drop 1N HCl solution to adjust the pH to about 1. After adding water and freeze-drying, the crude product is separated and purified by reverse phase C-18 silica gel column (water: acetonitrile = 70:30-30:70), and freeze-dried to obtain compound 13b (53 mg, yield 47.89%). ¹ H NMR (500 MHz, CD ₃ OD) δppm: 0.89 (t, J＝6.8Hz, 3H), 1.26 (d, J＝17.7Hz, 26H), 1.53 (d, J＝6.8Hz, 2H), 1.79- 1.90(m,2H),3.41(t,J＝6.6Hz,2H),3.49(t,J＝6.2Hz,2H),3.57(d,J＝8.3Hz,1H),3.69( dd,J＝13.4,9.5Hz,1H),3.73-3.82(m,5H),3.88(dt,J＝14.8,7.6Hz,2H),4.01(q,J＝6. 5Hz,2H),4.24(d,J＝13.7Hz,1H),7.20(d,J＝7.3Hz,1H),8.00(d,J＝7.3Hz,1H); m/z(ESI ⁺ ):620.7(M+H).

Example 14: Synthesis of Compound 14b

The preceding synthesis steps are the same as those in Example 9.

Step D: Add 9-2 (150 mg, 215.57 μmol, 1 eq.) and THF (2 mL) to the reaction flask, add LiHMDS (1 M, 862.29 μL, 4 eq.) dropwise under ice bath, stir and react at 0°C for 20 min, then add 4-nitrophenylpentyl carbonate (163.78 mg, 646.72 μmol, 3 eq.) in THF (0.5 mL) dropwise, and stir and react at room temperature for 16 hours. LC-MS monitoring showed that most of the reaction raw materials were consumed. The reaction system was adjusted to pH 2 with 1 mol/L hydrochloric acid aqueous solution under ice-water bath conditions. Water (50 mL) was added and washed with ethyl acetate (20 mL*4). The organic layer was washed once with saturated brine (60 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column (dichloromethane: methanol = 100: 0-85: 15) to obtain 14-1 (100 mg, yield 57.27%).

Step E: Add 14-1 (100 mg, 123.46 μmol, 1 eq.), tetrahydrofuran/methanol (V/V=5:1, 10 mL) and palladium carbon (20 mg, 10% carbon adsorption, containing 55% water) to the reaction flask. Stir the reaction at room temperature overnight under hydrogen atmosphere. LC-MS monitoring showed that most of the reaction raw materials were consumed, filtered, and the filter cake was washed with methanol (50 mL). The mother liquor was concentrated to obtain a crude product, which was separated and purified by reverse phase preparative column (acetonitrile, water, ammonium acetate system), and lyophilized to obtain compound 14b (30 mg, yield 44.45%). ¹ H NMR(500MHz, CDCl3)δ0.84-0.89(m,6H),1.22(s,28H),1.32(s,4H),1.50(s,2H),1.64(s,2H),1.82(s,2H),3.34(s,2H),3.43(s,2H), 3.48-3.61(m,2H),3.83(s,1H),3.90(s,2H),4.11(s,3H),4.17-4.30(m,2H),4.44(d,J＝9.8Hz,1H),6.09(s,1H),7.65(s,1H); m/z(ESI ⁺ ):676.6(M+H).

Example 15: Synthesis of Compound 15b

Step A: Add intermediate 2 (100 mg, 0.053 mmol, 1 eq.) and formic acid (2.5 mL) to the reaction flask. Stir the reaction at 60°C for 16 hours, concentrate to remove formic acid, and purify the residual crude product by reverse phase C-18 silica gel column (water: acetonitrile = 70:30-30:70), and freeze-dry to obtain compound 15b (20 mg, yield 17.77%). ¹ H NMR (500 MHz, CD ₃ OD)δppm:0.89(t,J＝6.8Hz,3H),1.28(s,26H),1.54(s,2H),1.77-1.90(m,2H),3 .41(t,J＝6.5Hz,2H),3.50(t,J＝6.4Hz,2H),3.54-3.62(m,1H),3.84(dd,J＝21.9, 14.1Hz,2H),3.92(d,J＝6.0Hz,2H),3.98(s,1H),4.17(d,J＝12.5Hz,2H),4.40(d, J＝7.7Hz,1H),5.96(d,J＝7.4Hz,1H),7.89(d,J＝7.1Hz,1H),8.13(s,1H); m/z(ESI ⁺ ):590.5(M+H).

Example 16: Synthesis of Compound 16b

Step C: 4-nitrophenol (2.0 g, 14.38 mmol, 1 eq.) and Py (4 mL) were added to the reaction flask, and acetic anhydride (2.20 g, 21.57 mmol, 1.5 eq.) was added dropwise under ice bath conditions. The reaction was stirred at room temperature for 16 hours after the addition was completed. TLC monitoring showed that the reaction raw materials were completely consumed. Under ice water bath conditions, 1 mol/L hydrochloric acid aqueous solution was used to adjust the reaction system to a weak acidity. Water (80 mL) and dichloromethane were added for extraction and washing (30 mL*2). The organic layer was washed once with saturated brine (60 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column (dichloromethane) to obtain the product 4-nitrophenyl acetate (2.2 g, yield 84.47%).

The intermediate 2 (90 mg, 160.23 μmol, 1 eq.) and DMF (2 mL) were added to the reaction flask, and NaH (51.27 mg, 1.28 mmol, 60% purity, 8 eq.) was added under ice bath, and the reaction was stirred at 0°C for 20 min, and then a solution of 4-nitrophenyl acetate (116.10 mg, 640.92 μmol, 4 eq.) in DMF (0.5 mL) was added dropwise, and the reaction was stirred at 50°C for 2 hours after the addition. After the reaction raw materials were consumed, the reaction system was adjusted to pH 2 with 1 mol/L hydrochloric acid aqueous solution under ice-water bath conditions, water (50 mL) was added, and ethyl acetate was extracted and washed (20 mL*4), and the organic layer was washed once with saturated brine (60 mL), the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by preparative plate to obtain compound 16b (16.6 mg, yield 15.56%). ¹ H NMR(500MHz, CDCl3)δ0.87(s,3H),1.24(s,26H),1.49(s,2H),1.72(s,2H),2.06(s,3H),2.20(s,3H),3.03-3. 38(m,4H),3.60-3.78(m,4H),4.02-4.08(m,3H),4.30(s,2H),7.48(s,1H),7.56(s,1H),11.55(s,1H); m/z(ESI ⁺ ):646.5(M+H).

Example 17: Synthesis of Compound 17b

Step A: Add intermediate 2 (120 mg, 0.213 mmol, 1 eq.), DMF (5 mL), ethyl 1-(4-nitrophenoxy)carbonyloxybutyrate (95.26 mg, 0.32 mmol, 1.5 eq.) and DIPEA (110.44 mg, 0.85 mmol, 1 eq.) to the reaction flask. Stir the reaction at 80 °C for 1 hour. Add ethyl acetate (50 mL) to dilute, add 1N HCl dropwise at low temperature to quench the reaction, and ensure that the pH of the aqueous phase is 1-2. Wash the organic layer twice with brine, dry and concentrate, and separate and purify the residue with a silica gel column (dichloromethane: methanol = 100: 0-75: 25) to obtain a crude product. The crude product was prepared, purified and freeze-dried to obtain compound 17b (15 mg, yield 11.32%). ¹ H NMR (500 MHz, CD ₃ OD)δppm:0.89(t,J＝6.7Hz,3H),0.95(dd,J＝9.8,4.7Hz,6H),1.28(s,26H),1.47(d,J＝5.2Hz,3H),1.52(t ,J＝7.2Hz,5H),1.58-1.70(m,4H),1.81(s,2H),2.32(t,J＝7.2Hz,4H),3.40(t,J＝6.4Hz,2H),3.48(t,J＝6. 2Hz,2H),3.57(s,1H),3.81(d,J＝8.8Hz,1H),3.88-4.02(m,4H),4.13-4.29(m,2H),4.41(dd,J＝37.2,9.9H z,1H),6.71(d,J＝5.0Hz,1H),6.86(d,J＝5.4Hz,1H),7.21(d,J＝7.2Hz,1H),8.10(t,J＝6.9Hz,1H); m/z(ESI ⁺ ):878.5(M+H).

Example 18: Synthesis of Compound 18b

Step A: Add intermediate 1 (200 mg, 367.87 μmol, 1 eq.), DCM (15 mL), and n-butyryl chloride (50.96 mg, 478.23 μmol, 49.66 μL, 1.3 eq.) to the reaction flask. Add triethylamine (111.67 mg, 1.10 mmol, 153.40 μL, 3.0 eq.) dropwise under stirring at 0°C. React at room temperature for 16 hours, concentrate to remove the reaction solvent, and separate and purify the residue with a silica gel column (dichloromethane: methanol = 100: 0-97: 3) to obtain compound 18b (125 mg, yield 54.39%). ¹ H NMR (500 MHz, CDCl ₃ )δppm:0.88(t,J＝6.9Hz,3H),0.99(t,J＝7.4Hz,3H),1.27(d,J＝14.9Hz,26H),1 .50-1.60(m,2H),1.73(d,J＝7.3Hz,2H),1.87-2.07(m,2H),2.42(s,2H),3.39(q ,J＝6.8Hz,2H),3.49(dt,J＝18.3,5.9Hz,2H),3.54-3.68(m,1H),3.86(dd,J＝32 .2,14.5Hz,1H),4.05-4.48(m,7H),7.41(d,J＝7.3Hz,1H),7.62(s,1H); m/z(ESI ⁺ ):614.6(M+H).

Example 19: Synthesis of Compound 19b

The specific synthesis steps are shown in Example 12, and compound 19b (50 mg, yield 45.12%) was obtained. ¹ H NMR (500 MHz, MeOD) δ 0.93 (t, J = 6.7 Hz, 3H), 1.32 (s, 26H), 1.55-1.60 (m, 2H), 1.87-1.94 (m, 2H), 3.38 (s, 3H), 3.45 (t, J = 6.5 Hz, 2H), 3.51-3.58 (m, 4H), 3.61-3.70 (m ,7H),3.74-3.87(m,5H),3.88-4.00(m,2H),4.03-4.12(m,2H),4.28(d,J＝12.1Hz ,1H),4.37(d,J＝2.4Hz,2H),7.22(d,J＝7.2Hz,1H),8.04(d,J＝7.2Hz,1H); m/z(ESI ⁺ ):752.7(M+H).

Example 20: Synthesis of Compound 20b

Step B: Nicotinic acid (500 mg, 4.06 mmol, 1 eq.), 4-nitrophenol (677.98 mg, 4.87 mmol, 1.2 eq.) and DCM (30 mL) were added to the reaction flask, and N, N'-dicyclohexylcarbodiimide (1.01 g, 4.87 mmol, 1.2 eq.) was added, and the reaction was stirred at room temperature for 16 hours. TLC monitoring showed that the reaction raw materials were completely consumed, and water (70 mL) was added, and dichloromethane was extracted and washed (30 mL*2). The organic layer was washed once with saturated brine (60 mL), and the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column (dichloromethane: ethyl acetate = 100: 0-99: 1) to obtain the product 4-nitrophenyl nicotinate (400 mg, yield 40.33%). The intermediate 1 (170 mg, 312.69 μmol, 1 eq.), DIPEA (121.24 mg, 938.07 μmol, 3 eq.) and DMF (2 mL) were added to the reaction flask. A solution of 4-nitrophenyl nicotinate (152.72 mg, 625.38 μmol, 2 eq.) in DMF (1 mL) was added to the reaction system and stirred at 50°C for 16 hours. The reaction raw materials were completely consumed by TLC column chromatography. The reaction system was restored to room temperature, water (50 mL) was added, ethyl acetate was extracted (25 mL*2), the organic layer was washed once with saturated brine (50 mL), the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column (dichloromethane: methanol = 100: 0-95: 5) to obtain the product 20-1 (120 mg, yield 59.15%).

Step C: Add 20-1 (120 mg, 184.97 μmol, 1 eq.) and THF (5 mL) to the reaction flask, add sodium hydroxide aqueous solution (0.5 mol/L, 739.86 μL, 2 eq.), and stir at room temperature for one hour. LC-MS monitored the complete consumption of the reaction raw materials, and adjusted the reaction system pH to 2 with 1 mol/L hydrochloric acid aqueous solution under ice-water bath conditions, and then concentrated to remove most of the ^solvent . Then the crude product was directly separated and purified using a C-18 reverse phase column (water: acetonitrile = 100: 0-60: 40), and lyophilized to obtain compound 20b (20 mg, yield 16.67%). NMR(500MHz, CDCl3)δ0.91(t,J=6.5Hz,3H),1.28(s,26H),1.55-1.59(m,2H),1.90-1.97(m, 2H),3.39-3.45(m,2H),3.53(t,J＝5.7Hz,2H),3.62(d,J＝12.0Hz,1H),3.73-3.93(m,4H),3.9 9(dd,J＝13.3,7.2Hz,1H),4.10-4.15(m,2H),4.29(d,J＝13.5Hz,1H),7.49(d,J＝6.4Hz,1H), 7.61(s,1H),7.96(d,J＝7.1Hz,1H),8.48(d,J＝6.9Hz,1H),8.85(s,1H),9.30(s,1H); m/z(ESI ⁺ ):667.5(M+H).

Example 21: Synthesis of Compound 21b

Step A: Compound 5b (87 mg, 0.124 mmol, 1.0 eq.) DMF (3 mL) and NaH (50 mg, 1.24 mmol, 60% purity, 10.0 eq.) were added to the reaction flask. Stir at room temperature for 20 minutes. 4-Nitrophenyl-4-butyrate (52 mg, 0.248 mmol, 2.0 eq.) DMF (0.5 mL) solution was added dropwise to the reaction. Stir the reaction at room temperature for 3 hours, add ethyl acetate (25 mL) to dilute, and add 1N HCl dropwise to quench the reaction at low temperature to ensure that the pH of the aqueous phase is 1-2. The organic layer was washed twice with brine, dried and concentrated, and the residue was separated and purified by silica gel column (dichloromethane: methanol = 100: 0-85: 15) to obtain compound 21b (30 mg, yield 29.43%). ¹ H NMR (500 MHz, CDCl ₃ )δppm:0.78(t,J＝6.8Hz,3H),0.86(t,J＝7.4Hz,3H),1.15(d,J＝5.5Hz,26H),1.39(s,2H),1.55(dt,J＝14.8 ,7.5Hz,2H),1.62-1.75(m,2H),2.23(t,J＝7.4Hz,2H),3.23(t,J＝6.6Hz,2H),3.33(t,J＝5.9Hz,2H),3.55( d,J＝7.3Hz,1H),3.70(d,J＝14.1Hz,1H),3.77(d,J＝6.3Hz,3H),3.98(d,J＝11.2Hz,1H),4.13(s,2H),4.25( d,J＝9.3Hz,1H),7.39(d,J＝8.2Hz,2H),7.47(s,1H),7.79(d,J＝6.9Hz,1H),7.87(d,J＝8.1Hz,2H); m/z(ESI ⁺ ):770.5(M+H).

Example 22: Synthesis of Compound 22b

Step A: Add 4-(hydroxymethyl)phenol (2.82 g, 22.70 mmol, 1 eq.), dichloromethane (100 mL), n-butyric acid (2 g, 22.70 mmol, 1.0 eq.), EDCI (5.22 g, 27.24 mmol, 1.2 eq.) and DMAP (277.33 mg, 2.27 mmol, 0.1 eq.) to the reaction flask. Under stirring, react at room temperature for 16 hours. Concentrate to remove the solvent, and pass through a silica gel column (petroleum ether: ethyl acetate = 100: 0-66: 34) to obtain the product [4-(hydroxymethyl)phenyl] n-butyrate (2 g, yield: 45.36%).

Step B: Add [4-(Hydroxymethyl)phenyl]butyrate (2g, 10.30mmol, 1eq.), dichloromethane (30mL), (4-nitrophenyl)carbonyl chloride (2.08g, 10.3mmol, 1.1eq.) and DIPEA (2g, 15.45mmol, 1.5eq.) to the reaction flask. React at room temperature for 4 hours under stirring. Concentrate to remove the solvent, add ethyl acetate (30mL) and water (20mL) to extract and wash the layers, and wash the organic layer with saturated brine (30mL). After the organic layer is concentrated, pass it through a silica gel column (petroleum ether: ethyl acetate = 100:0-50:50) to obtain the product [4-[(4-nitrophenoxy)carbonylmethyl]phenyl]butyrate (1.7g, yield: 45.94%)

Step C: Add intermediate 2 (30 mg, 0.053 mmol, 1 eq.), DMF (3 mL), [4-[(4-nitrophenoxy)carbonylmethyl]phenyl]butyrate (28.79 mg, 0.080 mmol, 1.5 eq.) and DIPEA (27.61 mg, 0.21 mmol, 1 eq.) to the reaction flask. Stir the reaction at 80 °C for 16 hours. Add ethyl acetate (25 mL) to dilute, add 1N HCl dropwise at low temperature to quench the reaction, and ensure that the pH of the aqueous phase is 1-2. Wash the organic layer twice with brine, dry and concentrate, and separate and purify the residue with a silica gel column (dichloromethane: methanol = 100: 0-75: 25) to obtain compound 22b (15 mg, yield 33.78%). ¹ H NMR (500 MHz, CD ₃ OD)δppm:0.89(t,J＝6.8Hz,3H),1.03(t,J＝7.4Hz,3H),1.28(d,J＝9.8Hz,26H),1.5 0(d,J＝6.5Hz,2H),1.68-1.79(m,4H),2.55(t,J＝7.3Hz,2H),3.36(d,J＝6.7Hz,2H), 3.42(t,J＝6.2Hz,2H),3.58(t,J＝12.0Hz,2H),3.69-4.2(m,6H),4.61(s,1H),5.23( s,2H),7.10(d,J＝8.4Hz,3H),7.46(d,J＝8.3Hz,2H),8.03(d,J＝7.2Hz,1H); m/z(ESI ⁺ ):782.6(M+H).

Example 23: Synthesis of Compound 23b

Step B: Add ethanol (457.11 mg, 9.92 mmol, 2 eq.) and DCM (10 mL) to the reaction flask, add triethylamine (753.04 mg, 7.44 mmol, 1.5 eq.), add 4-nitrophenylphosgene (1.0 g, 4.96 mmol, 1 eq.) in DCM (10 mL) dropwise under ice bath conditions, and stir and react at room temperature for 16 hours. TLC monitoring shows that the reaction raw materials are completely consumed, add water (50 mL), wash with dichloromethane (25 mL*2), wash the organic layer with saturated brine once (50 mL), separate the organic layer, dry and filter with anhydrous sodium sulfate, and after concentration, the residue is separated and purified by silica gel column (petroleum ether: ethyl acetate = 100: 0-85: 15) to obtain the product (4-nitrophenyl) ethyl carbonate (750 mg, yield 71.59%). The intermediate 1 (130 mg, 203.25 μmol, 85% purity, 1 eq.), DIPEA (78.80 mg, 609.74 μmol, 3 eq.) and DMF (2 mL) were added to the reaction flask. A solution of (4-nitrophenyl) ethyl carbonate (85.84 mg, 406.49 μmol, 2 eq.) in DMF (0.5 mL) was added to the reaction system under stirring, and the reaction was stirred at 50°C for 16 hours. The reaction raw materials were completely consumed by TLC column chromatography. The reaction system was restored to room temperature, water (40 mL) was added, and ethyl acetate was extracted (20 mL*2). The organic layer was washed once with saturated brine (40 mL), the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column (dichloromethane: methanol = 100: 0-95: 5) to obtain product 23-1 (75 mg, yield 59.93%).

Step C: Add 23-1 (75 mg, 121.81 μmol, 1 eq.) and THF (4 mL) to the reaction flask, add sodium hydroxide aqueous solution (0.5 mol/L, 609.03 μL, 2.5 eq.), and stir at room temperature for one hour. LC-MS monitored the complete consumption of the reaction raw materials, and adjusted the reaction system pH to 2 with 1 mol/L hydrochloric acid aqueous solution under ice-water bath conditions, and then concentrated to remove most of the ^solvent . The crude product was then separated and purified by reverse phase preparative column (acetonitrile, water, ammonium acetate system), and lyophilized to obtain compound 23b (39.1 mg, yield 50.53%). NMR(500MHz, CDCl3)δ0.88(t,J＝6.7Hz,3H),1.25(s,26H),1.32(t,J＝7.1Hz,3H),1. 50-1.54(m,2H),1.76-1.85(m,2H),3.36(t,J＝6.8Hz,2H),3.44(t,J＝6.1Hz,2H),3.5 3-3.62(m,2H),3.69-3.78(m,3H),3.83-3.89(m,2H),3.95-4.02(m,1H),4.05-4.12 (m,1H),4.22(q,J＝6.9Hz,2H),7.23(d,J＝7.2Hz,1H),7.86(d,J＝7.2Hz,1H); m/z(ESI ⁺ ):634.6(M+H).

Example 24: Synthesis of Compound 24b

Step C: Add intermediate 2 (100 mg, 0.053 mmol, 1 eq.), DMF (3 mL), (5-methyl-2-oxo-1,3-dioxo-4-yl)methyl (4-nitrophenyl) carbonate (105.11 mg, 0.356 mmol, 2.0 eq.) and DIPEA (92.04 mg, 0.712 mmol, 4 eq.) to the reaction flask. Stir the reaction at 80 °C for 7 hours, and then stir the reaction at 50 °C for 16 hours. Add ethyl acetate (25 mL) to dilute, add 1N HCl dropwise at low temperature to quench the reaction, and ensure that the pH of the aqueous phase is 1-2. Wash the organic layer twice with brine, dry and concentrate, and separate and purify the residue with a silica gel column (dichloromethane: methanol = 100: 0-75: 25) to obtain a crude product. The crude product was prepared and separated to obtain compound 24b (20 mg, yield 15.51%). ¹ H NMR (500 MHz, CD ₃ OD) δppm: 0.89 (t, J＝6.8Hz, 3H), 1.27 (s, 26H), 1.52 (d, J＝6.8Hz, 2H), 1.82 (dd, J＝12. 8,6.5Hz,2H),2.19(d,J＝11.5Hz,6H),3.39(t,J＝6.6Hz,2H),3.47(t,J＝6.3Hz,2H),3 .55-3.62(m,1H),3.79-4.03(m,5H),4.22(dd,J＝15.0,9.5Hz,2H),4.44(d,J＝11.7Hz ,1H),4.96(s,2H),5.03(s,2H),7.22(d,J＝7.3Hz,1H),8.09(d,J＝7.2Hz,1H); m/z(ESI ⁺ ):874.7(M+H).

Example 25: Synthesis of Compound 25b

Step A: Add 1-butyric acid (600 mg, 6.81 mmol, 1 eq.), water (20 mL) and sodium hydroxide (272 mg, 6.81 mmol, 1 eq.) to the reaction flask. Stir the reaction at room temperature for 10 minutes, and the system becomes clear. Add silver nitrate (1.16 g, 6.81 mmol, 1 eq.) and stir at room temperature for 30 minutes. A large amount of solid is generated, which is filtered and vacuum dried to obtain the product 1-butyric acid silver (1.1 g, 82.85%).

Step B: Add silver 1-butyrate (500 mg, 895.93 umol, 1 eq.), toluene (20 mL) and 1-iodoethyl (4-nitrophenyl) carbonate (1.04 g, 3.08 mmol, 1 eq.) into a reaction flask. Stir and react at 50 degrees for 5 hours, and concentrate through a silica gel column (petroleum ether: ethyl acetate = 100: 0-95: 5) to obtain the product 1-(4-nitrophenoxy)carbonyloxybutyric acid ethyl ester (450 mg, 59.03%).

Step C: Add intermediate 2 (120 mg, 0.213 mmol, 1 eq.), DMF (5 mL), ethyl 1-(4-nitrophenoxy)carbonyloxybutyrate (95.26 mg, 0.32 mmol, 1.5 eq.) and DIPEA (110.44 mg, 0.85 mmol, 1 eq.) to the reaction flask. Stir the reaction at 80 °C for 1 hour. Add ethyl acetate (50 mL) to dilute, add 1N HCl dropwise at low temperature to quench the reaction, and ensure that the pH of the aqueous phase is 1-2. Wash the organic layer twice with brine, dry and concentrate, and separate and purify the residue with a silica gel column (dichloromethane: methanol = 100: 0-75: 25) to obtain compound 25b (40 mg, yield 26.1%). ¹ H NMR (500 MHz, CD ₃ OD)δppm:0.89(t,J＝6.7Hz,3H),0.95(t,J＝7.4Hz,3H),1.28(s,26H),1.52(d,J＝5.4Hz,5H),1.63( dd,J＝14.7,7.3Hz,2H),1.74-1.82(m,2H),2.32(t,J＝7.3Hz,2H),3.39(t,J＝6.5Hz,2H),3.46(t,J ＝5.9Hz,2H),3.61(dd,J＝26.6,10.7Hz,2H),3.71-3.82(m,2H),3.88(dd,J＝14.1,7.6Hz,3H),4.08 (s,1H),4.17(d,J＝11.4Hz,1H),6.88(d,J＝5.3Hz,1H),7.09(s,1H),8.06(d,J＝7.0Hz,1H); m/z(ESI ⁺ ):720.5(M+H).

Example 26: Synthesis of Compound 26b and Compound 27b

Step C: Add isobutyric acid (1.0 g, 11.35 mmol, 1 eq.) and H ₂ O (10 mL) to a reaction flask, and dropwise add an aqueous solution (10 mL) of NaOH (454.0 mg, 11.35 mmol, 1 eq.) under ice bath conditions, and stir and react at room temperature for half an hour. Then dropwise add an aqueous solution (10 mL) of AgNO ₃ (1.93 g, 11.35 mmol, 1 eq.), and stir and react at room temperature for another hour. The reaction solution is directly filtered, and the filter cake is washed with a small amount of water and dried to obtain the product (isobutyryloxy)silver (1.2 g, yield 54.23%).

Step D: 1-iodoethyl (4-nitrophenyl) carbonate (1.3 g, 3.86 mmol, 1 eq.), (isobutyryloxy)silver (902.33 mg, 4.63 mmol, 1.2 eq.) and toluene (50 mL) were added to the reaction flask, and the reaction solution was stirred at 50° C. for 16 hours. TLC monitoring showed that most of the reaction raw materials were consumed, the reaction solution was cooled and filtered, the filtrate was concentrated, and the residue was separated and purified by silica gel column (petroleum ether: ethyl acetate = 100: 0-95: 5) to obtain the product 1-(((4-nitrophenoxy)carbonyl)oxy)isobutyric acid ethyl ester (690 mg, yield 60.18%).

Step E: Intermediate 2 (110 mg, 195.84 μmol, 1 eq.), DIPEA (101.24 mg, 783.35 μmol, 4 eq.) and DMF (3 mL) were added to a reaction flask, and then a solution of 1-(((4-nitrophenoxy)carbonyl)oxy)isobutyric acid ethyl ester (87.33 mg, 293.76 μmol, 1.5 eq.) in DMF (0.5 mL) was added dropwise. The reaction was stirred at 80°C for 1 hour. LC-MS monitored the complete consumption of the reaction raw materials. The reaction system was adjusted to pH 2 with 1 mol/L hydrochloric acid aqueous solution under ice-water bath conditions. Water (50 mL) was added and washed with ethyl acetate (20 mL*4). The organic layer was washed once with saturated brine (60 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated and purified by reverse phase preparative column (acetonitrile, water, ammonium acetate system) and freeze-dried to obtain compound 26b (45.5 mg, yield 32.28%) ^. NMR(500MHz,MeOD)δ0.91-0.95(m,3H),1.16-1.21(m,6H),1.31(s,26H),1 .56(s,5H),1.85(s,2H),2.57-2.63(m,1H),3.42-3.46(m,2H),3.50-3.60( m,4H),3.70-3.75(m,2H),3.82(d,J＝11.1Hz,1H),3.94(s,3H),4.24(d,J＝1 2.8Hz,1H),6.89(s,1H),7.22-7.26(m,1H),8.09(d,J＝6.1Hz,1H); m/z(ESI ⁺ ):720.6 (M+H); and compound 27b (18 mg, yield 12.77%). ¹ H NMR (500 MHz, MeOD) δ0.93 (t, J=6.7 Hz, 3H), 1.16-1.22 (m, 12H), 1.32 (s, 26H), 1.51 (d, J=5.2 Hz, 3H), 1.53-1.60 (m, 5H), 1.84-1.88 (m, 2H), 2.60 (dt, J=13.6, 6.6 Hz, 2H), 3.44 (t, J=6.5 Hz, 2H), 3.52 (t, J=5.9 Hz, 2H). z,2H),3.56-3.66(m,1H),3.84-3.92(m,1H),3.93-4.03(m,4H),4.18-4.30(m,2H),4.46(dd,J＝40.6,1 0.5Hz,1H),6.71-6.76(m,1H),6.86-6.92(m,1H),7.25(d,J＝7.2Hz,1H),8.14(t,J＝7.0Hz,1H); m/z(ESI ⁺ ):878.7(M+H).

Example 27: Synthesis of Compound 28b

Step E: Intermediate 2 (120 mg, 213.64 μmol, 1 eq.), DIPEA (110.44 mg, 854.56 μmol, 4 eq.) and DMF (3 mL) were added to a reaction flask, and then a solution of ethyl 1-(((4-nitrophenoxy)carbonyl)oxy)acetate (86.27 mg, 320.46 μmol, 1.5 eq.) in DMF (1 mL) was added dropwise. The mixture was stirred at 80°C for 1 hour. LC-MS monitored that the reaction raw materials were consumed. The reaction system was adjusted to pH 2 with 1 mol/L hydrochloric acid aqueous solution under ice-water bath conditions. Water (50 mL) was added, and ethyl acetate was extracted and washed (20 mL*4). The organic layer was washed once with saturated brine (60 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated and purified by reverse phase preparative column (acetonitrile, water, ammonium acetate system) and freeze-dried to obtain compound ^28b (42 mg, yield 26.18%). NMR(500MHz,MeOD)δ0.93(t,J＝6.6Hz,3H),1.32(s,26H),1.53-1.59(m,5H),1.85 (t,J＝6.0Hz,2H),2.10(s,3H),3.44(t,J＝6.3Hz,2H),3.51-3.61(m,4H),3.70-3.7 6(m,2H),3.82(dd,J＝12.3,3.0Hz,1H),3.91-3.98(m,3H),4.25(dd,J＝13.6,2.4Hz ,1H),6.89(d,J＝5.3Hz,1H),7.24(d,J＝7.2Hz,1H),8.10(d,J＝7.2Hz,1H); m/z(ESI ⁺ ):692.5(M+H).

Effect Example

1. Biological assay

General approach to pharmacokinetic studies:

The test compound is dissolved in PBS (pH = 8) at a concentration determined by the desired dose and dosing volume of the specific animal to which the compound is administered. The animal is administered a measured volume of dosing solution by oral administration. After administration of the test compound, blood samples are collected at specific time points (e.g., 0.5, 1, 2, 4, 8, 24, 32, and 48 hours). The blood samples are converted into plasma samples using standard techniques. LC-MS/MS analysis is performed to obtain the concentration of the test compound and Brincidofovir (BCV, comparison compound) in plasma.

1.1 Pharmacokinetic study of the compounds of the present invention in mice

After each compound and the control compound were injected intravenously (iv)/orally (po) to fasted ICR male mice, blood samples were collected at 0.083h (IV), 0.25h (IV), 0.5h, 1h, 2h, 4h, 8h, 24h, 32h and 48h after administration. Plasma was separated by centrifugation (3200rpm, 10min, 4°C) and frozen (-80°C) until used for analysis. The concentration of the compound in mouse plasma was determined by UPLC-MS/MS. Plasma was distributed to appropriate tubes containing internal standards and precipitants, and the tubes were shaken vigorously for 1 minute to achieve deproteinization, followed by centrifugation at 12000rmp for 5 minutes. After the supernatant was transferred to a 96-well plate for dilution, the mixture was shaken and centrifuged again (4100rpm, 5min, 4°C), and quantitative analysis was performed by LC-MS/MS. DAS 3.2.8 software was used to calculate pharmacokinetic parameters such as AUC0-t, AUC0-∞, Cmax, tmax, t1/2, MRT, CL and Vd. The absolute bioavailability was calculated as follows: F = [AUC (i.g.) × dose (i.v.)] / [AUC (i.v.) × dose (i.g.)] × 100%.

Compound 2b and the control compound were orally administered to ICR male mice at an equimolar amount of 20 mg/kg. The experimental method was as described above. The obtained pharmacokinetic parameters are shown in Table 2.

Table 2

The remaining compounds and comparative examples were orally administered to ICR male mice at an equimolar amount of 40 mg/kg. The experimental method was as described above. The obtained pharmacokinetic parameters are shown in Table 3.

Table 3

2. Efficacy test

2.1. In vitro efficacy test

Vero cells (ATCC CRL-1587) were inoculated in 96-well plates in a medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in MEM (Gibco Cat#41500034), and 0.05 PFU/cell of vaccinia virus, Tiantan strain was added. After incubation at 37°C in a 5% _CO2 incubator for 2 hours, 10 series of half-diluted drugs BCV (Brincidofovir), compound 15b, HPMPA prodrug 3b or HPMPA prodrug 6b (10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.63 μM, 0.32 μM, 0.16 μM, 0.08 μM, 0.04 μM, 0.02 μM) were added, and each concentration had 3 replicate wells. The cells were cultured continuously for 3 days, and the remaining cells were fixed with 4% neutral paraformaldehyde and stained with 0.1% crystal violet to count the number of virus plaques. Vero cells not infected with the virus were used as controls. Virus inhibition activity (%) = (number of virus plaques in the drug group/number of virus plaques in the control group) × 100.

The results of the in vitro test are shown in FIG1 . The inhibitory activities of BCV and compound 15b are similar, with EC50 values of 1.944 μM and 2.403 μM, respectively.

2.2 In vivo efficacy test

60 male Balb/c nude mice were randomly divided into 5 groups, namely blank control group, BCV administration group, compound 15b administration group, HPMPA prodrug 3b administration group and HPMPA prodrug 6b administration group. The experimental mice were inoculated with vaccinia virus on D1 of the experiment, and then gavaged with the corresponding drugs on D2, D4 and D6 of the experiment (the blank control group was only given the same volume of solvent), and the three administration doses were 20 mg/kg, 5 mg/kg and 5 mg/kg respectively. The weight changes of the animals before and after administration were recorded during the experiment to evaluate the protective effect of the test substance on the animals, and blood and tissues were taken for standby use.

The weight of each group of mice during the experiment was recorded, and the weight of mice on D0 was used as the benchmark, and the weight loss of mice > 30% of the initial weight was used as the surrogate endpoint of death. The results are shown in Figures 2a and 2b. It can be clearly observed that the mice in the blank control group gradually reached the surrogate endpoint from D3, a large number of mice reached the surrogate endpoint on D6 and D7, and all mice reached the surrogate endpoint on D8; the BCV administration group failed to improve the situation of mice reaching the surrogate endpoint, and all mice in this group also reached the surrogate endpoint on D8; while the situation of the compound 15b administration group was significantly improved.

The weight of each group of mice during the experiment was recorded, and the weight of mice on D0 was used as the benchmark to obtain the weight change graph and the actual death curve, as shown in Figures 3a and 3b. It can be clearly observed from the weight change graph that the weight of mice in the blank control group decreased significantly, and D14 was only 70%-80% of the weight on D0. The weight of mice in the BCV administration group decreased significantly in the early stage, and recovered in the later stage, and D14 was close to the level of D0; the weight of mice in the compound 15b administration group was always higher than that in the BCV administration group. From the actual death curve, it can be observed that only 2 mice in the blank control group survived on D14, 10 mice in the BCV administration group survived on D14, and all mice in the compound 15b administration group survived on D14.

Whether it is weight change, alternative endpoint death curve or actual death curve, the efficacy of compound 15b is better than BCV. It can delay the virus-induced weight loss in mice and even restore the weight growth of mice. It can also reduce the death of mice caused by the virus.

Although the present invention has been described in detail with reference to the embodiments of the present invention, these embodiments are provided to illustrate rather than limit the present invention. Other embodiments that can be obtained according to the principles of the present invention all belong to the scope defined by the claims of the present invention.

Claims (20)

A compound represented by formula (I) or a pharmaceutically acceptable salt or ester thereof: Wherein, X is selected from -OR ⁶ , or X and R ³ are combined to form a chemical bond; ^R1 is selected from H or C4-C30 carbonyl, wherein the C4-C30 carbonyl includes substituted or unsubstituted hydrocarbon carbonyl, substituted or unsubstituted aryl carbonyl or heterocyclic carbonyl, and substituted or unsubstituted hydrocarbonoxy carbonyl, and the carbonyl has no more than 30 carbon atoms; R ² and R ⁶ are independently selected from H, Wherein R ⁷ is selected from substituted or unsubstituted C15-C30 hydrocarbon groups, and the hydrocarbon group may be a straight-chain hydrocarbon group or a branched hydrocarbon group; ^R3 is selected from: H, formyl, C4-C30 carbonyl, wherein the C4-C30 carbonyl includes substituted or unsubstituted alkylcarbonyl, substituted or unsubstituted arylcarbonyl or heterocyclic carbonyl, and substituted or unsubstituted alkoxycarbonyl, and the carbon number of the carbonyl is not more than 30; or an amino acid residue; R ⁴ and R ⁵ are independently selected from hydrogen (H) or deuterium (D); As a limitation, when X is -OR ⁶ , R ¹ , R ² , R ³ , and R ⁶ are not H at the same time, and the compound is not brincidofovir; when X and R ³ are combined to form a chemical bond, R ¹ and R ² are not H at the same time. The compound according to claim 1, wherein the compound is a compound represented by formula (II): Wherein, R ¹ and R ² are not H at the same time. The compound according to claim 1, wherein the compound is a compound represented by formula (III): As a limitation, when X is -OR ⁶ , R ¹ , R ³ , and R ⁶ are not H at the same time; when X and R ³ are combined to form a chemical bond, R ¹ is not H. The compound according to claim 2, wherein the compound is a compound represented by formula (IIIa): The compound according to any one of claims 1 to 4, wherein the R ¹ is a substituted or unsubstituted hydrocarbylcarbonyl group, and the substituted or unsubstituted hydrocarbylcarbonyl group includes the groups shown below: The compound according to any one of claims 1 to 4, wherein the R ¹ is a substituted or unsubstituted arylcarbonyl or heterocyclic carbonyl group, and the substituted or unsubstituted arylcarbonyl or heterocyclic carbonyl group includes the groups shown below: wherein Y is selected from H, F, Cl, Br, Me, -OMe, -OEt, _-CF3 or -CN. The compound according to any one of claims 1 to 4, wherein the R ¹ is a substituted or unsubstituted alkoxycarbonyl group, and the substituted or unsubstituted alkoxycarbonyl group includes the groups shown below: The compound according to any one of claims 1 to 4, wherein the R ⁷ comprises the following groups: The compound according to any one of claims 1 to 4, wherein the R ³ comprises the following groups: Preferably, ^R3 is selected from [Corrected 08.01.2025 in accordance with Rule 26]
A compound selected from the following and a pharmaceutically acceptable salt thereof: A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 10, and at least one pharmaceutically acceptable carrier or excipient. The pharmaceutical composition according to claim 11, wherein the pharmaceutically acceptable carrier comprises one or more of a cream, an emulsion, a gel, a liposome and nanoparticles; and the pharmaceutically acceptable excipient comprises one or more of a binder, a filler, a disintegrant, a lubricant and a glidant. The pharmaceutical composition according to claim 11 or 12, wherein the pharmaceutical composition is suitable for oral administration or injection administration. Use of the compound according to any one of claims 1 to 10 or the pharmaceutical composition according to any one of claims 11 to 13 in the preparation of a medicament for treating, inhibiting or preventing viral infection or a disease caused by viral infection. The use according to claim 14, wherein the viral infection comprises infection caused by hepatitis B virus (HBV), new coronavirus (SARS-COV-2), human immunodeficiency virus (HIV), varicella zoster virus (VZV), cytomegalovirus (CMV), herpes simplex virus (HSV), BK virus, JC virus, Epstein-Barr virus (EBV), Ebola virus, polyomavirus, papillomavirus, orthopoxvirus, hepatitis C virus (HCV), respiratory syncytial virus (RSV), dengue virus, influenza virus, adenovirus, parainfluenza virus and/or rhinovirus. The use according to claim 15, wherein the orthopoxvirus includes severe and mild smallpox viruses, monkeypox virus, cowpox virus, camelpox virus, molluscum contagiosum, sheeppox virus, aractuba virus, BeAn 58058 virus, cantagalo orthopoxvirus, mousepox virus, elephantpox virus, vaccinia virus (VV), rabbitpox virus, raccoonpox virus, skunkpox virus, gerbilpox virus and volepox virus. The use according to claim 16, wherein the viral infection includes infection caused by smallpox virus and monkeypox virus. Use of the compound according to any one of claims 1 to 10 or the pharmaceutical composition according to any one of claims 11 to 13 in the preparation of a medicament for treating, inhibiting or preventing diseases caused by cell proliferation. The use according to claim 18, wherein the cell proliferation-induced disease includes a tumor, a cancer or other viral infection disease, and the tumor or cancer is selected from multiple myeloma (MM), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), solid tumors, refractory solid tumors, non-Hodgkin's lymphoma, hematological tumors, neuroblastoma, colorectal cancer, cervical cancer, lung cancer, leukemia, breast cancer, pancreatic cancer, B-cell malignancies, tumors, metastatic tumors, and colon cancer; the disease caused by viral infection is selected from diseases caused by DNA virus infection, including retinitis, pneumonia, cystitis, proteinopathy, etc. A kit comprising a compound or a pharmaceutically acceptable salt or ester as defined in any one of claims 1 to 10 or a pharmaceutical composition as defined in any one of claims 11 to 13.