CN120092088A

CN120092088A - Artificial nucleic acid molecules

Info

Publication number: CN120092088A
Application number: CN202280100916.7A
Authority: CN
Inventors: 张军; 王泰云; 李航文; 董慧芳
Original assignee: Siwei Shanghai Biotechnology Co ltd
Current assignee: Siwei Shanghai Biotechnology Co ltd
Priority date: 2022-11-11
Filing date: 2022-11-11
Publication date: 2025-06-03
Also published as: WO2024098361A1

Abstract

The invention relates to the field of biomedicine, and specifically to an artificial nucleic acid molecule, which comprises at least one open reading frame and at least one 3′-untranslated region element, and the artificial nucleic acid molecule has high translation efficiency.

Description

Artificial nucleic acid molecules

Technical Field

The invention belongs to the field of biological medicine. The present invention relates to artificial nucleic acid molecules comprising an open reading frame, a 3 '-untranslated region element (3' -UTR element) and/or a5 '-untranslated region element (5' -UTR element). The invention also relates to a vector comprising a 3'-UTR element and/or a 5' -UTR element, to a cell comprising said artificial nucleic acid molecule or said vector, to a lipid composition or a pharmaceutical composition comprising said artificial nucleic acid molecule or said vector, and to a kit comprising said artificial nucleic acid molecule, said vector, said lipid composition and/or said pharmaceutical composition, preferably for use in the field of gene therapy and/or genetic vaccination.

Background

For gene therapy and gene vaccination, stable RNAs are often required. Stable RNAs allow the accumulation of products encoded by RNA sequences in vivo and maintain their structural and functional integrity during their storage, preparation and administration. Thus, there is a need to provide stable RNA molecules for use in gene therapy or gene vaccination to prevent them from undergoing early degradation or decay. As a scheme for mRNA stabilization, however, it has been found that naturally occurring eukaryotic mRNA molecules contain specific stabilizing elements. For example, its 3 '-untranslated region (3' -UTR) and/or 5 '-untranslated region (5' -UTR). Both 3'-UTR and 5' -UTR are typical pre-maturation (premature) mRNA elements.

An mRNA molecule carries a gene encoding the corresponding protein. The gene is flanked by specific untranslated regions, called the 5 '-untranslated region and the 3' -untranslated region, 5 'to the AUG start codon and 3' to the stop codon. Typically, the 3' -UTR is a portion of the sequence between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the polyadenylation sequence that can regulate mRNA stability, localization, and expression.

Many untranslated region sequences have been designed and experimentally developed in the art based on the untranslated regions of some naturally occurring proteins to facilitate enhanced mRNA stability and expression levels. There remains a need in the art for more untranslated region sequences that are useful for improving the stability and safety of gene therapy and gene vaccination.

Disclosure of Invention

In one aspect, the present invention provides an artificial nucleic acid molecule comprising

A. at least one Open Reading Frame (ORF), and

B. at least one 3' -untranslated region element (3 ' -UTR element), said 3' -UTR element comprising a nucleic acid sequence selected from the group consisting of:

(i) wherein the 3'-UTR element comprises a variant of the nucleic acid sequence shown in SEQ ID NO. 44 which comprises a truncation, terminal extension and/or 1,2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence shown in SEQ ID NO. 44, or (ii) wherein the 3' -UTR element comprises a nucleic acid sequence of a 3'-UTR derived from a transcript of HCV, coV2, CVB3, AES and AAT or variants thereof which comprises a truncation, terminal extension and/or 1,2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence derived from, or (iii) wherein the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 16, or the corresponding RNA sequence of the above nucleic acid sequence.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 3.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 4.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 44 and further comprises the nucleic acid sequence of SEQ ID NO. 90, 91 or 93.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 1,2 or 5.

In one embodiment, the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of transcripts of the viral genes HCV, coV2 and CVB3 or variants thereof, or from the 3' -UTR of transcripts of the mouse gene AES or from the 3'-UTR of transcripts of the human gene AAT or variants thereof, or from the 3' -UTR of transcripts of the bovine gene AES or variants thereof, wherein the variants comprise truncations, terminal extensions and/or 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence from which they were derived.

In one embodiment, the 3' -UTR element exhibits a length of 3-500 nucleotides, preferably 5-250 nucleotides, more preferably 90-215 nucleotides.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 6,7,8, 9 or 12.

In one embodiment, the 3' -UTR element further comprises the nucleic acid sequence of SEQ ID NO. 92.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 10, 14 or 15.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 12 and further comprises the nucleic acid sequence of SEQ ID NO. 9 or 94.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 11 or 13.

In one embodiment, the artificial nucleic acid molecule further comprises at least one 5 '-untranslated region element (5' -UTR element).

In one embodiment, the 5' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 45.

In one embodiment, the artificial nucleic acid molecule further comprises a 5' cap structure, a polycytidylic acid sequence, a polyadenylation sequence, or a histone stem loop.

In one embodiment, the artificial nucleic acid molecule further comprises a 5' cap structure, a polycytidylic acid sequence, a polyadenylation sequence, and a histone stem loop.

In one embodiment, the ORF is codon optimized.

In one embodiment, the artificial nucleic acid molecule is RNA, preferably mRNA.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62.

In yet another aspect, the invention also provides a vector comprising an artificial nucleic acid molecule of the invention.

In another aspect. The invention also provides a cell comprising an artificial nucleic acid molecule of the invention or a vector of the invention.

In another aspect, the invention also provides a lipid composition comprising an artificial nucleic acid molecule of the invention and a lipid encapsulating the artificial nucleic acid molecule, wherein the lipid encapsulating the artificial nucleic acid molecule comprises a cationic lipid, a phospholipid, a steroid, and a polyethylene glycol modified lipid, and a cationic polymer, wherein the cationic polymer associates with the artificial nucleic acid molecule as a complex, and is co-encapsulated in the lipid to form a lipid-multimeric complex.

In one embodiment, the cationic lipid comprises a lipid compound of formula (I), (II), (III), (IV), or a pharmaceutically acceptable salt thereof, as defined herein. In a preferred embodiment, the cationic lipid is M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.

In one embodiment, the lipid composition comprises 10-70 mole% cationic lipid, 10-70 mole% phospholipid, 10-70 mole% steroid, and 0.05-20 mole% polyethylene glycol modified lipid.

In one embodiment, the lipid composition comprises 35-50 mole% cationic lipid, 10-30 mole% phospholipid, 24-44 mole% fusoid alcohol, and 1-1.5 mole% polyethylene glycol modified lipid.

In a preferred embodiment, the lipid composition comprises 35-50 mole% cationic lipid, 10-30 mole% DOPE, 24-44 mole% cholesterol, and 1-1.5 mole% DMG-PEG.

In a preferred embodiment, the lipid composition comprises 50 mole% cationic lipid, 10 mole% DOPE, 38.5 mole% cholesterol, and 1.5 mole% DMG-PEG.

In a preferred embodiment, the lipid composition comprises 40 mole% cationic lipid, 15 mole% DOPE, 43.5 mole% cholesterol, and 1.5 mole% DMG-PEG.

In yet another aspect, the invention also provides a pharmaceutical composition comprising an artificial nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a lipid composition of the invention, and a pharmaceutically acceptable carrier, excipient or diluent.

In a further aspect, the invention also provides the use of an artificial nucleic acid molecule of the invention, a vector of the invention, a cell of the invention, a lipid composition of the invention or a pharmaceutical composition of the invention for the preparation of a vaccine or a medicament for gene therapy.

In a further aspect, the invention also provides the use of an artificial nucleic acid molecule of the invention, a vector of the invention, a cell of the invention, a lipid composition of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prophylaxis of a disease.

In a further aspect, the invention also provides a method for increasing the efficiency of translation of an artificial nucleic acid molecule, preferably an mRNA molecule or vector, comprising ligating an open reading frame with a 3' -UTR element as defined herein.

In a further aspect, the invention also provides a kit comprising an artificial nucleic acid molecule of the invention, a vector of the invention, a cell of the invention, a lipid composition of the invention or a pharmaceutical composition of the invention.

In yet another aspect, the invention also provides a method of producing an artificial nucleic acid molecule, the method comprising:

a) Synthesis of the Artificial nucleic acid molecules of the invention or

B) An artificial nucleic acid molecule is synthesized by the vector of the present invention.

Drawings

FIG. 1 shows pUC57-Luc plasmid map.

FIG. 2 shows exemplary sequences of test artificial nucleic acid molecules.

FIG. 3 shows the effect of different 3' -UTR elements on reporter luciferase expression. The ordinate indicates the sequence numbers of the corresponding 3' -UTR elements contained in the different artificial nucleic acid molecules, wherein the artificial nucleic acid molecule comprising only the poly (A) sequence after the stop codon of the luciferase gene is a reference nucleic acid molecule (ctrl) relative to the artificial nucleic acid molecule under test.

Detailed Description

Definition of the definition

All patents, patent applications, scientific publications, manufacturer's instructions and guidelines, and the like, cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, "at least one" or "one or more" may mean 1,2,3, 4, 5, 6, 7, 8 or more.

As used herein, the terms "comprises," "comprising," "includes," "including," "having" and "containing" are open-ended, meaning the inclusion of the stated elements, steps or components, but not the exclusion of other non-recited elements, steps or components. The expression "consisting of" does not include any element not specified steps or components. The expression "consisting essentially of means that the scope is limited to the specified elements, steps or components, plus any optional elements, steps or components that do not significantly affect the basic and novel properties of the claimed subject matter. It is to be understood that the expression "consisting essentially of the expression" comprising "and" consisting of the expression "comprising" is encompassed within the meaning of the expression "comprising".

As used herein, the term "and/or" in connection with a plurality of recited elements should be understood to include both individual and combined options. In other words, "and/or" includes "and" as well as "or". For example, a and/or B includes A, B and a+b. A. B and/or C include A, B, C and any combination thereof, such as A+ B, A + C, B +C and A+B+C. Further elements defined by "and/or" are to be understood in a similar manner and include any one of, and any combination of, these.

Any numerical value or range of numerical values, such as concentration or range of concentration, should be construed as modified by the term "about" in any event, unless otherwise indicated. Thus, a numerical value typically includes ±10% of the value. For example, a concentration of 1mg/mL includes 0.9mg/mL to 1.1mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a numerical range explicitly includes all possible subranges, all individual values within the range including integers and fractions within the range unless the context clearly indicates otherwise.

Herein, "nucleotide" includes deoxyribonucleotides and ribonucleotides and derivatives thereof. As used herein, a "ribonucleotide" is a constituent material of ribonucleic acid (RNA) and consists of one molecule of base, one molecule of pentose, and one molecule of phosphate, which refers to a nucleotide having a hydroxyl group at the 2' -position of the β -D-ribofuranose (β -D-ribofuranosyl) group. The "deoxyribonucleotide" is a constituent substance of deoxyribonucleic acid (DNA), and also comprises one molecule of base, one molecule of pentose and one molecule of phosphoric acid, and refers to a nucleotide in which the hydroxyl group at the 2' -position of the beta-D-ribofuranose (beta-D-ribofuranosyl) group is replaced by hydrogen, and is a main chemical component of a chromosome. "nucleotide" is generally referred to by a single letter representing a base therein, "A (a)" means deoxyadenylate or adenylate containing adenine, "C (C)" means deoxycytidylate or cytidylate containing cytosine, "G (G)" means deoxyguanylate or guanylate containing guanine, "U (U)" means uridylate containing uracil, and "T (T)" means deoxythymidylate containing thymine.

As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Polynucleotide sequence", "nucleic acid sequence" and "nucleotide sequence" are used interchangeably to refer to the ordering of nucleotides in a polynucleotide. It will be appreciated by those skilled in the art that the coding strand (sense strand) of DNA can be considered to have the same nucleotide sequence as the RNA it encodes, with deoxythymidylate in the sequence of the coding strand of DNA corresponding to uridylate in the sequence of the RNA it encodes.

As used herein, an "artificial nucleic acid molecule" may be understood as a non-natural nucleic acid molecule. Such nucleic acid molecules may be non-natural due to their individual sequences (which do not naturally occur) and/or due to other modifications (e.g., structural modifications of naturally occurring nucleotides). The artificial nucleic acid molecule may be a DNA molecule, an RNA molecule or a hybrid molecule comprising DNA and RNA portions. Typically, artificial nucleic acid molecules can be designed and/or produced by genetic engineering methods. In this case, the artificial nucleic acid molecule comprises an artificial sequence which is not normally naturally occurring and which differs from the wild type sequence by at least one nucleotide. The term "wild-type sequence" may be understood as a naturally occurring sequence.

As used herein, "modified" refers to non-natural. For example, the RNA may be modified RNA. That is, the RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linking groups. "modified" groups may also be referred to herein as "altered" groups. The groups may be chemically, structurally or functionally modified or altered. For example, the modified nucleobase may comprise one or more non-naturally occurring substitutions.

As used herein, the term "transfection" refers to the introduction of a nucleic acid molecule, such as a DNA or RNA (e.g., mRNA) molecule, into a cell, preferably into a eukaryotic cell. Within the scope of the present invention, the term "transfection" includes any method known to the person skilled in the art for introducing nucleic acid molecules into cells, preferably eukaryotic cells, such as mammalian cells. Such methods include, for example, electroporation, lipofection based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers (such as DEAE-dextran or polyethylenimine), and the like. Preferably, the method is lipofection.

As used herein, the term "expression" includes transcription and/or translation of a nucleotide sequence. Thus, expression may involve the production of transcripts and/or polypeptides.

As used herein, the term "translation efficiency" relates to a nucleic acid molecule (e.g., mRNA) comprising an Open Reading Frame (ORF). Translation efficiency is experimentally measurable. Translation efficiency is typically measured by determining the amount of protein translated from the ORF. For experimental measurement of translation efficiency, the ORF preferably encodes a reporter protein or any other protein that can be quantified. In the context of the present invention, the translation efficiency is particularly useful for nucleic acid molecules in which, in addition to the ORF, at least one 3' -UTR element is comprised, preferably as defined herein. It should be appreciated that in the present invention, high translation efficiency is typically provided by a particular UTR element (a particular 3' -UTR element). Although the ORF suitably encodes a reporter protein or any other protein that can be quantified in terms of experimentally quantified translation efficiency, the invention is not limited to such a purpose, and thus, at least one 3' -UTR element of the invention (which provides high translation efficiency) may be contained in a nucleic acid molecule containing an ORF that does not encode a reporter protein.

Translation efficiency is a relative term that is determined and compared by determining the translation efficiency of a plurality (e.g., two or more) nucleic acid molecules, e.g., by experimentation to quantify the protein encoded by an ORF. One of the nucleic acid molecules may be referred to as a "reference nucleic acid molecule" or "reference construct" and the other as a "test nucleic acid molecule" or "test construct", the test nucleic acid molecule may be an artificial nucleic acid molecule according to the invention. For this purpose, the reference nucleic acid molecule and the test nucleic acid molecule share the same ORF (same nucleic acid sequence), and preferably the nucleic acid sequence of the test nucleic acid molecule is identical to the nucleic acid sequence of the reference nucleic acid molecule, with the difference that the UTR element tested, i.e.the 3' -UTR element, in other words, the test nucleic acid molecule and the reference nucleic acid molecule preferably differ from each other only in that the 3' -UTR element has a different nucleic acid sequence, such that the 3' -UTR element becomes the only structural feature distinguishing between the test nucleic acid molecule and the reference nucleic acid molecule.

As used herein, a "vector" is a vehicle for introducing an exogenous polynucleotide into a host cell, which exogenous polynucleotide is amplified or expressed when the vector is transformed into an appropriate host cell. Vectors typically remain episomal, but may be designed to integrate a gene or portion thereof into the chromosome of the genome. As used herein, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), and the like. Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, poxviral vectors, and baculovirus vectors, among others.

As used herein, a "cell" is a cell that is used to receive, hold, replicate, and amplify a vector. Cells may also be used to express polypeptides encoded by the vectors. When the cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The cells may be eukaryotic or prokaryotic cells. Suitable cells include, but are not limited to, CHO cells, various COS cells, heLa cells, HEK cells such as HEK 293 cells.

As used herein, an "aliphatic" group is a non-aromatic group in which carbon atoms are linked in a chain, and may be saturated or unsaturated.

As used herein, the term "alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon comprising one or more carbon atoms. The term "C ₁-C ₁₂ alkyl" or "C _1-12 alkyl" refers to optionally substituted straight or branched chain saturated hydrocarbons comprising 1 to 12 carbon atoms. As used herein, the term "alkoxy" refers to an alkyl group as described herein that is attached to the remainder of the molecule through an oxygen atom. The term "alkylene" refers to a divalent group formed by the corresponding alkyl group losing one hydrogen atom. The term "C ₁-C ₁₂ alkylene" or "C _1-12 alkylene" refers to an optionally substituted straight or branched chain alkylene group comprising 1 to 12 carbon atoms.

As used herein, the term "alkenyl" refers to an optionally substituted straight or branched chain hydrocarbon comprising two or more carbon atoms and at least one double bond. The term "C ₂-C ₁₂ alkenyl" or "C _2-12 alkenyl" refers to optionally substituted straight or branched chain hydrocarbons comprising 2 to 12 carbon atoms and at least one carbon-carbon double bond. Alkenyl groups may include one, two, three, four or more carbon-carbon double bonds.

As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine.

As used herein, the term "carbocyclic" refers to a monocyclic or polycyclic non-aromatic system comprising one or more rings of carbon atoms. The term "C _3-8 carbocycle" means a carbocycle comprising 3 to 8 carbon atoms. Carbocycles may include one or more carbon-carbon double or triple bonds. Examples of carbocycles include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, and the like. As used herein, when a carbocycle is saturated (i.e., free of unsaturation), the corresponding cycloalkyl group may also be referred to. Unless specifically stated otherwise, carbocycles as described herein refer to unsubstituted and substituted, i.e., optionally substituted carbocycles.

As used herein, the term "heterocycle" refers to a single or multiple ring system comprising one or more rings and including at least one heteroatom. The heteroatom may be, for example, a nitrogen, oxygen, phosphorus or sulfur atom. The heterocycle may include one or more double or triple bonds and may be non-aromatic. Examples of heterocycles include, but are not limited to, imidazolidinyl, oxazolidinyl, thiazolidinyl, pyrazolidinyl, isoxazolidinyl, isothiazolidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuranyl, and piperidinyl. The heterocyclic ring may comprise, for example, 3-10 atoms (other than hydrogen), i.e., a 3-10 membered heterocyclic ring (e.g., 3,4, 5, 6, 7, 8, 9, or 10 membered), wherein one or more of the atoms is a heteroatom (e.g., N, O, S or P). When the heterocycle is saturated (i.e., does not contain an unsaturated bond), the corresponding heterocycloalkyl group may also be referred to. Unless specifically stated otherwise, a heterocycle as described herein refers to both unsubstituted and substituted heterocyclic groups, i.e., an optionally substituted heterocycle.

As used herein, the term "aryl" refers to an all-carbon monocyclic or fused-polycyclic aromatic ring radical having a conjugated pi-electron system. For example, a C ₆-C ₁₀ alkylaryl group can have from 6 to 10 carbon atoms, such as 6, 7, 8, 9, 10 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and the like.

As used herein, the term "heteroaryl" refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, S, the remaining ring atoms being C, and having at least one aromatic ring. Heteroaryl groups may have 5 to 10 ring atoms (5 to 10 membered heteroaryl groups) including 5, 6, 7, 8, 9 or 10 membered, especially 5 or 6 membered heteroaryl. Examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, and the like.

As used herein, the term "interrupted by one or more groups" means that the one or more groups are present on the carbon chain and the remainder of the carbon chain is attached to both ends of the one or more groups.

Unless specifically stated otherwise, a group described herein (e.g., any of R ₁-R ₇, such as alkyl, alkylene, alkenyl, aryl, amino, etc.) may be optionally substituted. The optional substituents may be selected from, but are not limited to, halogen atoms (e.g., chloro, bromo, fluoro, OR iodo), carboxylic acids (e.g., -C (O) OH), alcohols (e.g., hydroxy, -OH), esters (e.g., -C (O) OR OR-OC (O) R), aldehydes (e.g., -C (O) H), carbonyl groups (e.g., -C (O) R, OR represented by C=O), acyl halides (e.g., -C (O) X, where X is a halogen selected from bromo, fluoro, chloro, and iodo), carbonate groups (e.g., -OC (O) OR), alkoxy groups (e.g., -OR), acetals (e.g., -C (OR) ₂ R ', where each OR is the same OR different alkoxy group and R' "is alkyl OR alkenyl), acyl halides (e.g., -C (O) X, wherein X is a halogen selected from bromo, fluoro, chloro, and iodo), Phosphate (e.g., P (O) ₄ ^3-), thiol (e.g., -SH), sulfoxide (e.g., -S (O) R), sulfinic acid (e.g., -S (O) OH), sulfonic acid (e.g., -S (O) ₂ OH), thioaldehyde (e.g., -C (S) H), thiol (e.g., thiol) and thiol (e.g., thiol and thiol), Sulfate (e.g., S (O) ₄ ^2-), sulfonyl (e.g., -S (O) ₂ -), amide (e.g., -C (O) NR ₂ or-N (R) C (O) R), Azido (e.g., -N ₃), nitro (e.g., -NO ₂), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC (O) R), amino (e.g., -NR ₂, NRH or-NH ₂), amino, carbamoyl (e.g., -OC (O) NR ₂, -OC (O) NRH or-OC (O) NH ₂), sulfonamide (e.g., -S(O) ₂NR ₂、-S (O) ₂NRH、-S(O) ₂NH ₂、-N(R)S(O) ₂R、-N(H)S(O) ₂R、-N(R)S(O) ₂H、-N(H)S(O) ₂H)、C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl), C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, or 3-10 membered heterocycle. In any of the foregoing, each R independently can be a substituent as defined herein, such as alkyl, alkoxy, alkylene, halo, carbocycle, heterocycle, aryl, heteroaryl, alkenyl. In some embodiments, the substituents themselves may be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein. For example, an alkyl group may be further substituted with one, two, three, four, five, or six substituents as described herein.

As used herein, the term "compound" is intended to include isotopic compounds of the depicted structure. "isotope" refers to an atom having the same atomic number but different mass numbers due to the number of neutrons in the core, such as a deuterium isotope. Isotopes of hydrogen include, for example, tritium and deuterium. In addition, the compounds, salts or complexes of the invention may be prepared in combination with solvents or water molecules to form solvates and hydrates by conventional methods.

The term "optionally" or "optionally" (e.g., optionally substituted) means that the subsequently described event may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and the description includes both substituted and unsubstituted alkyl radicals.

It is understood that when chemical groups are written in a particular order, the reverse order is also contemplated unless otherwise indicated. For example, in the general formula- (R) _i-(M1) _k-(R) _m - (i.e., - (R) _i-C(O)-NH-(R) _m -) where M ₁ is defined as-C (O) NH-, compounds where M ₁ is-NHC (O) -, i.e., - (R) _i-NHC(O)-(R) _m -, are also contemplated unless otherwise indicated.

As used herein, the term "contacting" refers to establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a lipid composition means that the mammalian cell and the lipid nanoparticle are physically linked together. Methods for contacting cells with external entities in vivo and ex vivo are well known in the biological arts. For example, contacting the lipid composition with mammalian cells in a mammalian body may be performed by different routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and may involve different amounts of the lipid composition. Furthermore, the lipid composition may be contacted with more than one mammalian cell.

As used herein, the term "delivering" refers to providing an entity to a target. For example, delivering an artificial nucleic acid molecule to a subject may involve administering a lipid composition comprising the artificial nucleic acid molecule to the subject.

As used herein, a "lipid component" is a component of a composition that includes one or more lipids. For example, the lipid component may include one or more cationic lipids, pegylated lipids, structural lipids, or helper lipids.

The phrase "pharmaceutically acceptable" is used herein to refer to compounds, salts, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is altered by converting the existing acid or base moiety to its salt form (e.g., by reacting a free basic group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, alkali metal or organic salts of acidic residues such as carboxylic acids, and the like. Representative acid addition salts include, but are not limited to, acetates, adipates, alginates, ascorbates, aspartate, benzenesulfonates, benzoates, bisulphates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptonates, glycerophosphate, hemisulfates, heptanoates, caprates, hydrobromides, hydrochlorides, hydroiodides, 2-hydroxy-ethane sulfonates, lactobionic aldehyde, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalene sulfonates, nicotinates, nitrates, oleates, oxalates, palmates, pamonates, pectates, persulfates, 3-phenylpropionates, phosphates, bitrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, toluenesulfonates, undecanoates, valerates, and the like. Representative alkali or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, magnesium salts, and the like, and non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethyl ammonium, tetraethyl ammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the invention include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the invention can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. In general, these salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two, with non-aqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile being generally preferred.

As used herein, "gene therapy" is understood to mean the treatment of a patient's body or an isolated component of a patient's body, such as an isolated tissue/cell, by a nucleic acid encoding a peptide or protein. Which typically may comprise at least one of a) directly administering a nucleic acid (preferably an artificial nucleic acid molecule as defined herein) to a patient by any route of administration or in vitro to isolated cells/tissues of a patient resulting in vivo/ex vivo or in vitro transfection of cells of the patient, b) transcribing and/or translating the introduced nucleic acid molecule, and optionally c) re-administering the isolated, transfected cells to the patient if the nucleic acid is not directly administered to the patient.

As used herein, "genetic vaccination" may be typically understood as vaccination by administration of a nucleic acid molecule encoding an antigen or immunogen or a fragment thereof. The nucleic acid molecule may be administered to the body of the subject or to an isolated cell of the subject. When transfected into certain cells of the body or when transfected into isolated cells, the antigen or immunogen may be expressed by those cells and subsequently presented to the immune system, eliciting an adaptive (i.e., antigen-specific) immune response. Thus, genetic vaccination typically comprises at least one of the steps of a) administering a nucleic acid (preferably an artificial nucleic acid molecule as defined herein) to a subject (preferably a patient), or to isolated cells of a subject (preferably a patient), which generally result in transfection of cells of the subject in vivo or in vitro, b) transcribing and/or translating the introduced nucleic acid molecule, and optionally c) re-administering the isolated, transfected cells to the subject (preferably a patient) if the nucleic acid is not directly administered to the patient.

As used herein, "vaccine" refers to a composition comprising an active ingredient (e.g., an artificial nucleic acid molecule of the invention) that is capable of eliciting an immune response in an vaccinated subject upon vaccination. In particular embodiments, the immune response it induces provides immune protection and is sufficient to prevent and/or ameliorate at least one symptom associated with a pathogen or disease infection

As used herein, the term "treatment" refers to the partial or complete alleviation, amelioration, alleviation of one or more symptoms or features of a particular infection, disease, disorder or condition, delay of its onset, inhibition of its progression, reduction of its severity or reduction of its occurrence. "preventing" means preventing an underlying disease or preventing worsening of symptoms or disease progression.

The term "prophylactically or therapeutically effective amount" refers to an amount of an agent (e.g., nucleic acid, drug, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) sufficient to prevent or inhibit the occurrence of a disease or symptom and/or to slow, alleviate, delay the progression or severity of a disease or symptom. The prophylactically or therapeutically effective amount is affected by factors including, but not limited to, the rate and severity of the disease or condition development, the age, sex, weight and physiological condition of the subject, the duration of the treatment, and the particular route of administration. A prophylactically or therapeutically effective amount may be administered in one or more doses. A prophylactically or therapeutically effective amount may be achieved by continuous or intermittent administration.

Artificial nucleic acid molecules

Provided herein is an artificial nucleic acid molecule comprising

A. at least one Open Reading Frame (ORF), and

B. at least one 3 '-untranslated region element (3' -UTR element).

As used herein, an "Open Reading Frame (ORF)" is a sequence of some nucleotide triplets that can be translated into a peptide or protein. The open reading frame preferably contains an initiation codon, i.e., a combination of three consecutive nucleotides (ATG) typically encoding the amino acid methionine, at the end of its 5' -untranslated region element, and an immediate region typically exhibiting a multiple of 3 nucleotides in length. The open reading frame of the invention is preferably a nucleotide sequence consisting of a very polynucleotide that can be divided by three, which starts with a start codon (e.g., ATG) and which preferably ends with a stop codon (e.g., TAA, TGA, or TAG). The open reading frame may be isolated or it may be integrated into a longer nucleic acid sequence, such as a vector or mRNA. The open reading frame may also be referred to as a "protein coding region"

The untranslated region (UTR) may have features that provide regulatory effects, such as increased or decreased stability, localization, and/or translation efficiency. The UTR-containing polynucleotide may be administered to a cell, tissue, or organism, and one or more regulatory characteristics may be measured using conventional methods.

As used herein, the term "3'-UTR" refers to a portion of an artificial nucleic acid molecule that is located 3' (i.e., "downstream") of the open reading frame and that is not translated into a protein. Typically, the 3' -UTR is a portion of the mRNA between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the polyadenylation sequence of the mRNA. The 3' -UTR of mRNA is not translated into an amino acid sequence. 3 '-UTRs play an important role in the regulation of biological complexity, which can regulate the localized expression of mRNA, can regulate the translation of mRNA, and can also regulate protein-protein interactions (see, for example, mayr C.what Are 3'UTRs DoingCold Spring Harb Perspect Biol.2019Oct 1;11 (10): a 034728).

In one embodiment, the artificial nucleic acid molecule comprises

A. at least one Open Reading Frame (ORF), and

B. At least one 3 '-untranslated region element (3' -UTR element) comprising a variant of the nucleic acid sequence depicted in SEQ ID No. 44, said variant comprising a truncation, a terminal extension and/or 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence depicted in SEQ ID No. 44.

In one embodiment, the 3' -UTR element comprises T87C or T94C as compared to SEQ ID NO 44.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 3 and also comprises the nucleic acid sequence of SEQ ID NO. 92.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 44 and further comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO. 6,7, 9, 12, 90, 91, 92, 93, 95 and 96.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 44 and further comprises the nucleic acid sequence of SEQ ID NO. 90.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 1.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 44 and also comprises the nucleic acid sequence of SEQ ID NO. 91.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 2.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 44 and further comprises the nucleic acid sequence of SEQ ID NO. 93.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 5.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 44 and further comprises the nucleic acid sequence of SEQ ID NO. 92.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 17.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 44 and further comprises the nucleic acid sequence of SEQ ID NO. 95.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 18.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 44 and also comprises the nucleic acid sequence of SEQ ID NO. 7.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 25.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 44 and also comprises the nucleic acid sequences of SEQ ID NO. 96 and 92.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 26.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 44 and further comprises the nucleic acid sequences of SEQ ID NO. 9 and SEQ ID NO. 92.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO 27 or 38.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 44 and also comprises the nucleic acid sequences of SEQ ID NO. 12 and 92.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 29 or 40.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 44 and also comprises the nucleic acid sequence of SEQ ID NO. 9.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 37.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 44 and also comprises the nucleic acid sequence of SEQ ID NO. 12.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO: 39.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 44 and also comprises the nucleic acid sequence of SEQ ID NO. 6.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 41.

In one embodiment, the artificial nucleic acid molecule comprises

A. at least one Open Reading Frame (ORF), and

B. at least one 3 '-untranslated region element (3' -UTR element) comprising a nucleic acid sequence of SEQ ID NO:1, 2,3, 4,5, 17, 18, 25, 26, 27, 29, 37, 38, 39, 40 or 41.

In a preferred embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 1,2,3, 4 or 5.

In one embodiment, the artificial nucleic acid molecule comprises

A. at least one Open Reading Frame (ORF), and

B. At least one 3' -untranslated region element (3 ' -UTR element) comprising a nucleic acid sequence of the 3' -UTR derived from a transcript of HCV, coV2, DENV2, TCV, CYBA, BYDA, CVB, AES and AAT or a variant thereof comprising a truncation, terminal extension and/or 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence derived from.

The term "nucleic acid sequence derived from the 3'-UTR of a gene transcript" refers to a nucleic acid sequence based on the 3' -UTR sequence of a gene transcript or fragment or portion thereof (preferably a naturally occurring gene or fragment or portion thereof). "nucleic acid sequences derived from the 3'-UTR of a gene" includes sequences corresponding to the entire 3' -UTR sequence, i.e., the full length 3'-UTR sequence of the transcript of the gene, and sequences corresponding to fragments of the 3' -UTR sequence of the transcript of the gene. Preferably, a fragment of the 3' -UTR of a transcript of a gene comprises a stretch of contiguous nucleotides corresponding to a stretch of contiguous nucleotides in the full length 3' -UTR of the transcript of the gene representing at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% |, at least 60%, at least 70%, at least 80% or at least 90% of the full length 3' -UTR of the transcript of the gene. In this context, it is preferred that the fragment retains regulatory functions for translation of the ORF linked to the 3' -UTR or fragment thereof.

The term "truncated" refers to a fragment or portion of a nucleic acid sequence of the 3' -UTR sequence of a gene-based transcript. Preferably, the truncated 3' -UTR sequence comprises a stretch of contiguous nucleotides corresponding to a stretch of contiguous nucleotides in a full length 3' -UTR of a transcript of a gene representing at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% |, at least 60%, at least 70%, at least 80% or at least 90% of the full length 3' -UTR of the transcript of the gene.

The term "variant" refers to a variant of the 3' -UTR of a transcript of a naturally occurring gene, preferably a variant of the 3' -UTR of a transcript of a viral gene, more preferably a variant of the 3' -UTR of a transcript of a mammalian gene. The variant may be a modified 3' -UTR of a transcript of a gene. For example, a variant of a 3'-UTR may exhibit truncations, terminal extensions, or one or more nucleotide deletions, additions, and/or mutations compared to the naturally occurring 3' -UTR from which the variant is derived. Preferably, the variant of the 3'-UTR of the transcript of the gene is at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% identical to the naturally occurring 3' -UTR from which the variant is derived.

As used herein, the term "terminal extension" refers to the addition of a variant of a 3'-UTR with one or more nucleotides at its N-or C-terminus as compared to the naturally occurring or modified 3' -UTR from which the variant is derived.

In one embodiment, the 3' -UTR element exhibits a length of at least about 3 nucleotides, preferably at least about 5 nucleotides, more preferably at least about 10, 15, 20, 25 or 30 nucleotides, even more preferably at least about 50 nucleotides, and most preferably at least about 90 nucleotides. In a preferred embodiment, the 3' -UTR element exhibits a length of 3 to about 500 nucleotides, preferably 5 to about 250 nucleotides, more preferably 90 to 215 nucleotides.

In one embodiment, the nucleic acid sequence is derived from the nucleic acid sequence of the 3' -UTR of transcripts of the viral genes HCV, coV2, DENV2, TCV, CYBA, BYDA and CVB3, and is truncated.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 8, 19, 21 or 23.

In one embodiment, the nucleic acid sequence is derived from the nucleic acid sequence of the 3' -UTR of the transcript of the mouse gene AES and is truncated.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 9.

In one embodiment, the nucleic acid sequence is derived from the nucleic acid sequence of the 3' -UTR of a transcript of the human gene AAT or AES and is truncated.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 12 or 96.

In one embodiment, the nucleic acid sequence is derived from the nucleic acid sequence of the 3' -UTR of the transcript of bovine gene AES and is truncated.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 98.

In one embodiment, the nucleic acid sequence is derived from the nucleic acid sequence of the 3' -UTR of transcripts of the viral genes HCV, coV2, DENV2, TCV, CYBA, BYDA and CVB3 or variants thereof, wherein the variants comprise truncations and 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence from which they were derived.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 6,7, 20, 22, 30, 31 or 32.

In one embodiment, the nucleic acid sequence is derived from a nucleic acid sequence of the 3' -UTR of a transcript of bovine gene AES or a variant thereof, wherein the variant comprises a truncation and 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence derived from.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 94.

In one embodiment, the nucleic acid sequence is derived from a nucleic acid sequence of the 3' -UTR of a transcript of the mouse gene AES or a variant thereof, wherein the variant comprises a truncation and 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it was derived.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 97.

In one embodiment, the nucleic acid sequence is derived from a nucleic acid sequence of the 3' -UTR of a transcript of the mouse gene AES or a variant thereof, wherein the variant comprises a truncation and a terminal extension compared to the nucleic acid sequence derived from.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 9 and further comprises the nucleic acid sequence of SEQ ID NO. 92 or 12.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 10, 13 or 28.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 9 and also comprises the nucleic acid sequences of SEQ ID NO. 92 and 12.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 33.

In one embodiment, the nucleic acid sequence is derived from a nucleic acid sequence of the 3' -UTR of a transcript of a human gene AAT or a variant thereof, wherein said variant comprises a truncation and a terminal extension compared to the nucleic acid sequence derived from.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 12 and further comprises the nucleic acid sequence of SEQ ID NO. 6, 9, 92, 94, 97 or 98.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 11, 13, 14, 28, 34, 35 or 36.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 12 and also comprises the nucleic acid sequences of SEQ ID NO. 9 and 92.

In one embodiment, the nucleic acid sequence is derived from a nucleic acid sequence of the 3' -UTR of a transcript of bovine gene AES or a variant thereof, wherein the variant comprises a truncation and a terminal extension compared to the nucleic acid sequence derived.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 98 and also comprises the nucleic acid sequence of SEQ ID NO. 12.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 35.

In one embodiment, the nucleic acid sequence is derived from a nucleic acid sequence of the 3' -UTR of a transcript of bovine gene AES or a variant thereof, wherein the variant comprises a truncation, terminal extension, and 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it was derived.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 94 and also comprises the nucleic acid sequence of SEQ ID NO. 12.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 11.

In one embodiment, the nucleic acid sequence is derived from the nucleic acid sequence of the 3' -UTR of transcripts of the viral genes HCV, coV2, DENV2, TCV, CYBA, BYDA and CVB3 or variants thereof, wherein the variants comprise truncations, terminal elongations and 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence from which they were derived.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 6 and also comprises the nucleic acid sequence of SEQ ID NO. 92.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 15.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 7 and also comprises the nucleic acid sequence of SEQ ID NO. 92.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 24.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 6 and also comprises the nucleic acid sequence of SEQ ID NO. 12.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 36.

In one embodiment, the nucleic acid sequence is derived from a nucleic acid sequence of the 3' -UTR of a transcript of the mouse gene AES or a variant thereof, wherein the variant comprises a truncation, terminal extension and 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it was derived.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 97 and also comprises the nucleic acid sequence of SEQ ID NO. 12.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 34.

In one embodiment, the artificial nucleic acid molecule comprises

A. at least one Open Reading Frame (ORF), and

B. At least one 3 '-untranslated region element (3' -UTR element) comprising a nucleic acid sequence of SEQ ID NO 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 19, 20, 21, 22, 23, 24, 28, 30, 31, 32, 33, 34, 35, 36, 94, 96, 97 or 98.

In a preferred embodiment, the artificial nucleic acid molecule comprises

A. at least one Open Reading Frame (ORF), and

B. At least one 3' -untranslated region element (3 ' -UTR element) comprising a nucleic acid sequence of the 3' -UTR derived from transcripts of HCV, coV2, CVB3, AES and AAT or variants thereof comprising truncations, terminal extensions and/or 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence derived from.

In a preferred embodiment, the nucleic acid sequence is derived from the nucleic acid sequence of the 3' -UTR of transcripts of the viral genes HCV, coV2 and CVB3 or variants thereof, wherein said variants comprise truncations, terminal elongations and/or 1, 2,3 or more mutations, additions or deletions compared to the nucleic acid sequence from which they were derived.

In a preferred embodiment, the nucleic acid sequence is derived from a nucleic acid sequence of the 3' -UTR of a transcript of the mouse gene AES or a variant thereof, wherein the variant comprises a truncation, terminal extension and/or 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it was derived.

In a preferred embodiment, the nucleic acid sequence is derived from a nucleic acid sequence of the 3' -UTR of a transcript of a human gene AAT or a variant thereof, wherein said variant comprises a truncation, terminal extension and/or 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it was derived.

In a preferred embodiment, the nucleic acid sequence is derived from a nucleic acid sequence of the 3' -UTR of a transcript of bovine gene AES or a variant thereof, wherein the variant comprises a truncation, terminal extension and/or 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it was derived.

In a preferred embodiment, the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of transcripts of the viral genes HCV, coV2 and CVB3 or variants thereof, or from the 3' -UTR of transcripts of the mouse gene AES or from the 3'-UTR of transcripts of the human gene AAT or variants thereof, or from the 3' -UTR of transcripts of the bovine gene AES or variants thereof, wherein the variants comprise truncations, terminal extensions and/or 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence from which they were derived.

In one embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

In one embodiment, the artificial nucleic acid molecule comprises

A. at least one Open Reading Frame (ORF), and

B. At least one 3 '-untranslated region element (3' -UTR element) comprising the nucleic acid sequence of SEQ ID NO. 16, 42 or 43.

In a preferred embodiment, the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 16.

In one embodiment, the artificial nucleic acid molecule comprises

A. at least one Open Reading Frame (ORF), and

(i) Wherein the 3' -UTR element comprises a variant of the nucleic acid sequence shown in SEQ ID NO. 44 which comprises a truncation, terminal extension and/or 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence shown in SEQ ID NO. 44, or

(Ii) Wherein the 3'-UTR element comprises a nucleic acid sequence of the 3' -UTR derived from transcripts of HCV, coV2, DENV2, TCV, CYBA, BYDA, CVB, AES and AAT or variants thereof comprising truncations, terminal extensions and/or 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it was derived, or

(Iii) Wherein the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 16, 42 or 43.

In one embodiment, the artificial nucleic acid molecule comprises

A. at least one Open Reading Frame (ORF), and

SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42 Or 43.

The nucleic acid sequences of specific 3' -UTR elements are shown in Table 1.

TABLE 1 nucleic acid sequences of 3' -UTR elements

In a preferred embodiment, the artificial nucleic acid molecule comprises

A. at least one Open Reading Frame (ORF), and

(Ii) Wherein the 3'-UTR element comprises a nucleic acid sequence of the 3' -UTR derived from transcripts of HCV, coV2, CVB3, AES and AAT or variants thereof comprising truncations, terminal elongations and/or 1,2,3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it was derived, or

(Iii) Wherein the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 16.

In a particularly preferred embodiment, the artificial nucleic acid molecule comprises

A. at least one Open Reading Frame (ORF), and

(Iii) Wherein the 3' -UTR element comprises the nucleic acid sequence of SEQ ID NO. 16;

Or the corresponding RNA sequence of the above nucleic acid sequence.

In a specific embodiment, the artificial nucleic acid molecule comprises

A. at least one Open Reading Frame (ORF), and

1,2,3, 4,5,6, 7,8, 9, 10, 11, 12, 13, 14, 15 or 16.

As used herein, the term "5' -untranslated region element (5 ' -UTR element)" refers to a portion of an artificial nucleic acid molecule that is located at the 5' end of the open reading frame (i.e., "upstream") and that is not translated into a protein. Typically, the 5' -UTR starts at the transcription initiation site and terminates one nucleotide before the start codon of the open reading frame. 5' -UTRs play a key role in regulating gene expression. Many cis-regulatory elements may be included in the DNA sequence of the 5' -UTR, which may interact with transcription mechanisms to regulate the abundance of messenger RNA (mRNA). In addition, transcribed 5 '-UTRs consist of a variety of RNA-based regulatory elements including 5' cap structures, secondary structures, RNA binding protein motifs, upstream open reading frames (uofs), internal ribosome entry sites, terminal Oligopyrimidine (TOP) bundles, and G-quadruplexes. These elements can alter the efficiency of mRNA translation, and some can also affect mRNA transcription levels through changes in stability or degradation (see, e.g.) Lim,Y.,et al.Multiplexed functional genomic analysis of 5'untranslated region mutations across the spectrum of prostate cancer.Nat Commun 12,4217(2021)).

In a preferred embodiment, the artificial nucleic acid molecule further comprises at least one 5 '-untranslated region element (5' -UTR element).

In a preferred embodiment, the 5 '-untranslated region element (5' -UTR element) comprises the nucleic acid sequence of SEQ ID NO. 45.

As used herein, the term "poly (a) sequence" or "poly (a) tail" refers to a nucleotide sequence comprising continuous or discontinuous adenylates. The poly (A) sequence is typically located at the 3' end of the RNA, e.g., 3' end (downstream) of the 3' -UTR. In some embodiments, the poly (a) sequence does not comprise nucleotides other than adenylate at its 3' end. Poly (A) sequences can be transcribed from the coding sequence of a DNA template by a DNA-dependent RNA polymerase during the preparation of IVT-RNA or can be linked to the free 3' end of IVT-RNA, e.g., the 3' end of the 3' -UTR, by a DNA-independent RNA polymerase (Poly (A) polymerase). In one embodiment, the artificial nucleic acid molecule further comprises a polyadenylation sequence.

As used herein, the term "polycytidylic acid sequence" refers to a nucleotide sequence comprising continuous or discontinuous cytidylic acids. The polycytidylic acid sequence is typically located at the 3' end of the RNA, e.g. 3' end (downstream) of the 3' -UTR. In some embodiments, the polycytidylic acid sequence does not contain nucleotides other than cytidylic acid at its 3' end. In one embodiment, the artificial nucleic acid molecule further comprises a polycytidylic acid sequence.

As used herein, the term "5 'cap" generally refers to an N7-methylguanosine structure (also known as "m7G cap", "m7 Gppp-") attached to the 5' end of an mRNA by a 5 'to 5' triphosphate bond. The 5' cap may be co-transcribed into the RNA in vitro transcription (e.g., using an anti-reverse cap analogue "ARCA") or may be post-transcriptionally linked to the RNA using a capping enzyme. In one embodiment, the artificial nucleic acid molecule further comprises a 5' cap structure.

As used herein, a "stem loop" (whether it is a histone or not) may typically be present in single stranded DNA, or more commonly in RNA. This structure is also known as a hairpin or hairpin loop and is typically composed of a stem and a (terminal) loop within a continuous sequence, wherein the stem is formed of two adjacent fully or partially reverse complementary sequences separated by a short sequence that acts as a spacer, which becomes the loop of the stem-loop structure. These two adjacent fully or partially reverse complementary sequences can be defined as, for example, stem loop elements stem 1 and stem 2. When two adjacent fully or partially reverse complementary sequences, e.g. stem loop element stem 1 and stem 2, form a base pair with each other, a double stranded nucleic acid sequence fragment is formed which comprises at its ends the unpaired loop formed by the short sequence between stem loop element stem 1 and stem 2 located on the consecutive sequence, thereby forming a stem loop.

As used herein, a "histone stem loop" is derived from a histone gene (e.g., a gene derived from histone families H1, H2A, H2B, H3, H4) and comprises the intramolecular base pairing of two adjacent fully or partially reverse complementary sequences, thereby forming a stem loop. In one embodiment, the artificial nucleic acid molecule further comprises a histone stem loop.

In one embodiment, the artificial nucleic acid molecule further comprises a 5' cap structure and a polycytidylic acid sequence.

In one embodiment, the artificial nucleic acid molecule further comprises a 5' cap structure and a polyadenylation sequence.

In one embodiment, the artificial nucleic acid molecule further comprises a 5' cap structure, a polycytidylic acid sequence, and a histone stem loop.

In one embodiment, the artificial nucleic acid molecule further comprises a 5' cap structure, a polyadenylation sequence and a histone stem loop.

In a preferred embodiment, the artificial nucleic acid molecule comprises a polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 46.

The artificial nucleic acid molecules defined herein may be prepared using any method known in the art, including synthetic methods, e.g., solid phase synthesis, and in vitro methods, e.g., in vitro transcription reactions or in vivo reactions, e.g., in vivo propagation of DNA plasmids in bacteria. The artificial nucleic acid molecules of the invention may be codon optimized for the host cell used for expression.

In a preferred embodiment, the Open Reading Frame (ORF) is codon optimized.

In one embodiment, the artificial nucleic acid molecule is RNA.

In a preferred embodiment, the artificial nucleic acid molecule is an mRNA.

In one embodiment, the 3' -UTR element comprises a nucleic acid sequence of SEQ ID NO:47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88 or 89.

Modified nucleotides

In some embodiments, the mRNA comprises modified nucleotides, wherein the modified nucleotides are selected from one or more of 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deadenosine, 7-deazaguanosine, 8-oxoguanosine, O (6) -methylguanosine, pseudouridine, N-1-methyl-pseudouridine, 2-thiouridine, and 2-thiocytidine, methylated bases, intercalating bases, 2' -fluororibose, ribose, 2' -deoxyribose, arabinose, and hexose, and 5' -phosphoramidite. Modified nucleotides as described in PCT/CN2020/074825, PCT/CN 2020/106696.

Vectors and host cells

In yet another aspect, the invention also provides an expression vector comprising an artificial nucleic acid molecule of the invention. The expression vector may further comprise additional nucleic acid sequences, such as regulatory sequences and antibiotic resistance genes. The artificial nucleic acid molecules of the invention may be present in one or more expression vectors.

In one embodiment, the vector is a DNA vector.

In one embodiment, the vector is a plasmid vector or a viral vector.

In a preferred embodiment, the vector is a plasmid vector. Such as pUC57 plasmid vectors.

In one embodiment, the carrier is a circular molecule.

In one embodiment, the artificial nucleic acid molecules of the invention are prepared as recombinant nucleic acids. Recombinant nucleic acids can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination techniques (e.g., polymerase Chain Reaction (PCR) techniques), and the like.

In yet another aspect, the invention also provides a method of producing an artificial nucleic acid molecule of the invention, the method comprising:

a) Synthesis of the Artificial nucleic acid molecules of the invention or

The invention also provides a cell comprising an artificial nucleic acid molecule or vector of the invention. The artificial nucleic acid molecules or vectors of the invention may be introduced into suitable cells using a variety of methods known in the art. Such methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection, among others.

In a preferred embodiment, the cells are used to express a peptide or protein encoded by an ORF in an artificial nucleic acid molecule of the invention. Examples of cells include, but are not limited to, prokaryotic cells (e.g., bacteria, e.g., E.coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Suitable mammalian host cells include, but are not limited to, external human cervical cancer cells (HeLa cells), human embryonic kidney cells (HEK cells, e.g., HEK 293 cells), chinese Hamster Ovary (CHO) cells, and other mammalian cells.

In one embodiment, the cell is a mammalian cell.

In a preferred embodiment, the cell is an isolated cell of a human subject.

Lipid composition

In yet another aspect, the invention also provides a lipid composition. The lipid composition is a lipid delivery vehicle, and the lipid can encapsulate the artificial nucleic acid molecule of the invention to form a nanoparticle for delivery into an organism.

As used herein, the term "lipid" refers to an organic compound comprising a hydrophobic moiety and optionally also a hydrophilic moiety. Lipids are generally poorly soluble in water but soluble in many organic solvents. Generally, amphiphilic lipids comprising a hydrophobic portion and a hydrophilic portion may be organized in an aqueous environment as a lipid bilayer structure, for example in the form of vesicles. Lipids may include, but are not limited to, fatty acids, glycerides, phospholipids, sphingolipids, glycolipids, and steroids, cholesterol esters, and the like.

As used herein, "lipid nanoparticle" or "LNP" refers to a lipid vesicle with a homogeneous lipid core, which is a particle formed from lipids, the lipid components undergoing intermolecular interactions to form a nanostructure entity. Nucleic acids (e.g., mRNA) are encapsulated in lipids.

Particularly preferred lipid compositions may be, for example, lipid multimeric complexes (LPPs) as described herein. Methods of preparing such compositions may be as described herein. LPP is a particle having a core-shell structure in which nucleic acids are contained in a multimeric complex, which itself is encapsulated in a biocompatible lipid bilayer shell to constitute the lipid nanoparticle of the present invention. In some embodiments, the lipid composition of the invention is a lipid multimeric complex (LPP). In some embodiments, the lipid composition of the invention is a lipid multimeric complex (LPP) comprising an artificial nucleic acid molecule.

In some embodiments, the lipid encapsulating the artificial nucleic acid molecule of the invention is selected from one or more of a cationic lipid, a phospholipid, a steroid and/or a polyethylene glycol modified lipid. In a preferred embodiment, the cationic lipid is an ionizable cationic lipid.

The lipid composition of the present invention comprises the artificial nucleic acid molecule of the present invention and a lipid encapsulating the artificial nucleic acid molecule. The lipids encapsulating the artificial nucleic acid molecules comprise cationic lipids, phospholipids, steroids, and polyethylene glycol modified lipids.

In one embodiment, the lipid composition comprises a cationic lipid, wherein the cationic lipid comprises DOTMA、DOTAP、DDAB、DOSPA、DODAC、DODAP、DC-Chol、DMRIE、DMOBA、DLinDMA、DLenDMA、CLinDMA、DMORIE、DLDMA、DMDMA、DOGS、N4- cholesteryl-spermine, DLin-KC2-DMA, DLin-MC3-DMA, a compound of formula (I), (II), (III), or (IV) as described herein, or a combination thereof. In a preferred embodiment, the cationic lipid comprises M5, MC3, ALC-0315, SM-102. In a preferred embodiment, the cationic lipid comprises SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2. In a preferred embodiment, the cationic lipid comprises M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.

In one embodiment, the lipid composition comprises a phospholipid and/or a steroid. In one embodiment, the lipid composition comprises a phospholipid as described herein, wherein the phospholipid comprises 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-bisundecanoyl-sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-di-O-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphorylcholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphorylcholine (C16 Lyso PC), 1, 2-di-linolenoyl-sn-glycero-3-phosphorylcholine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphorylcholine, 1, 2-di-docosahexaenoic acyl-sn-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), 1, 2-di-phytanoyl-sn-glycero-3-phosphate ethanolamine (ME 16.0 PE), 1, 2-di-stearoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-oleoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-linolenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-docosahexaenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPG), Dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-based phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or combinations thereof. In one embodiment, the lipid composition comprises a steroid as described herein, wherein the steroid comprises cholesterol, fecal sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycopersicin, ursolic acid, alpha-tocopherol, and derivatives thereof. In one embodiment, the lipid composition comprises a phospholipid and a steroid as described herein. In one embodiment, the lipid composition comprises DOPE. In one embodiment, the lipid composition comprises DSPC. In one embodiment, the lipid composition comprises cholesterol. In one embodiment, the lipid composition comprises DOPE and cholesterol. In one embodiment, the lipid composition comprises DSPC and cholesterol.

In one embodiment, the lipid composition comprises cationic lipids M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, phospholipid DOPE and cholesterol. In one embodiment, the lipid composition comprises cationic lipids M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, phospholipid DSPC and cholesterol.

In some embodiments, the lipid encapsulating the artificial nucleic acid molecule of the invention further comprises a polyethylene glycol modified lipid. In one embodiment, the polyethylene glycol modified lipid comprises DMG-PEG (e.g., DMG-PEG 2000), DOG-PEG, and DSPE-PEG, or a combination thereof. In one embodiment, the polyethylene glycol modified lipid is DSPE-PEG. In one embodiment, the polyethylene glycol modified lipid is DMG-PEG (e.g., DMG-PEG 2000).

In one embodiment, the lipid composition comprises a cationic lipid, DOPE, cholesterol, and DSPE-PEG.

In one embodiment, the lipid composition comprises a cationic lipid, DSPC, cholesterol, and DSPE-PEG.

In one embodiment, the lipid composition comprises a cationic lipid, DSPC, cholesterol, and DMG-PEG.

In a preferred embodiment, the lipid composition comprises a cationic lipid, DOPE, cholesterol, and DMG-PEG.

In a preferred embodiment, the lipid composition comprises the cationic lipids M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, DOPE, cholesterol and DMG-PEG.

In some embodiments, the lipid composition of the present invention further comprises a cationic polymer associated with the artificial nucleic acid molecule as a complex, co-encapsulated in the lipid.

In one embodiment, the cationic polymer comprises poly-L-lysine, protamine, polyethylenimine (PEI), or a combination thereof. In one embodiment, the cationic polymer is protamine. In one embodiment, the cationic polymer is a polyethyleneimine.

In one embodiment, the amount of lipid in the lipid composition is calculated as mole percent (mole%) based on the total moles of lipid in the composition.

In one embodiment, the amount of cationic lipid in the lipid composition is from about 10 to about 70 mole%. In some embodiments, the amount of cationic lipid in the lipid composition is from about 20 to about 60 mole%, from about 30 to about 50 mole%, from about 35 to about 45 mole%, from about 38 to about 45 mole%, from about 40 to about 50 mole%, or from about 45 to about 50 mole%.

In one embodiment, the amount of phospholipid in the lipid composition is from about 10 to about 70 mole%. In one embodiment, the amount of phospholipid in the lipid composition is from about 20 to about 60 mole%, from about 30 to about 50 mole%, from about 10 to about 30 mole%, from about 10 to about 20 mole%, or from about 10 to about 15 mole%.

In one embodiment, the amount of cholesterol in the lipid composition is from about 10 to about 70 mole%. In one embodiment, the amount of cholesterol in the lipid composition is from about 20 to about 60 mole%, from about 24 to about 44 mole%, from about 30 to about 50 mole%, from about 35 to about 40 mole%, from about 35 to about 45 mole%, from about 40 to about 45 mole%, or from about 45 to about 50 mole%.

In one embodiment, the amount of polyethylene glycol modified lipid in the lipid composition is from about 0.05 to about 20 mole%. In one embodiment, the amount of polyethylene glycol modified lipid in the lipid composition is from about 0.5 to about 15 mole%, from about 1 to about 10 mole%, from about 5 to about 15 mole%, from about 1 to about 5 mole%, from about 1 to about 1.5 mole%, from about 1.5 to about 3 mole%, or from about 2 to 5 mole%.

In one embodiment, the lipid composition comprises 10-70 mole% cationic lipid, 10-70 mole% phospholipid, 10-70 mole% steroid, and 0.05-20 mole% polyethylene glycol modified lipid. In a preferred embodiment, the lipid composition comprises 35-50 mole% cationic lipid, 10-30 mole% phospholipid, 24-44 mole% fusoid alcohol, and 1-1.5 mole% polyethylene glycol modified lipid.

In one embodiment, the LPP comprises an artificial nucleic acid molecule of the present invention and a lipid encapsulating the artificial nucleic acid molecule, wherein the lipid encapsulating the artificial nucleic acid molecule comprises a cationic lipid, a phospholipid, a steroid, and a polyethylene glycol modified lipid, and further comprises a cationic polymer, wherein the cationic polymer associates with the artificial nucleic acid molecule as a complex. In one embodiment, the lipid composition of the present invention comprises the artificial nucleic acid molecule of the present invention and a lipid encapsulating the artificial nucleic acid molecule, wherein the lipid encapsulating the artificial nucleic acid molecule comprises a cationic lipid, a phospholipid, a steroid, and a polyethylene glycol modified lipid, and further comprises a cationic polymer, wherein the cationic polymer associates with the artificial nucleic acid molecule as a complex, and is co-encapsulated in the lipid to form a lipid-multimeric complex. In one embodiment, the lipid composition comprises 2.5 to 20 mole% of polyethylene glycol modified lipids, based on the total amount of all lipids in the lipid composition. In one embodiment, the phospholipid is selected from 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoyl phosphatidylcholine (DSPC), or a combination thereof. In one embodiment, the steroid is cholesterol. In one embodiment, the cationic polymer is protamine. In one embodiment, the polyethylene glycol modified lipid is selected from 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (DMG-PEG), 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-poly (ethylene glycol) (DSPE-PEG), or a combination thereof. In one embodiment, the cationic lipid is selected from M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.

In one embodiment, the lipid of the encapsulation complex comprises 50 mole% M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2,10 mole% DOPE,38.5 mole% cholesterol and 1.5 mole% DMG-PEG. In one embodiment, the lipid of the encapsulation complex comprises 40 mole% M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2,15 mole% DOPE,43.5 mole% cholesterol and 1.5 mole% DMG-PEG.

Cationic lipids

Cationic lipids are lipids that can carry a net positive charge at a given pH. Lipids with a net positive charge can associate with nucleic acids through electrostatic interactions.

Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium propane (1, 2-di-O-octadecenyl-3-trimethylammonium-propane, DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (1, 2-dioleoyl-3-trimethylammonium-propane, DOTAP), bisdecanyl dimethylammonium bromide (Didecyldimethylammonium bromide, DDAB), 2, 3-Dioleoyloxy-N- [2 (spermine carboxamide) ethyl ] -N, N-dimethyl-l-propylamine onium trifluoroacetate (2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate,DOSPA)、 dioctadecyl dimethyl ammonium chloride (dioctadecyldimethyl ammonium chloride, DODAC), 1,2-dioleoyl-3-dimethyl ammonium-propane (1, 2-dioleoyl-3-dimethylammonium-propane, DODAP), 3- (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol (3- (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol, DC-Chol), 2, 3-bis (tetradecyloxy) propyl- (2-hydroxyethyl) -dimethylaminoonium (2, 3-di (tetradecoxy) propyl- (2-hydroxyethyl) -dimethylazanium, DMRIE), N-dimethyl-3, 4-dioleoxybenzylamine (N, N-dimethyl-3,4-dioleyloxybenzylamine, DMOBA), and, 1, 2-Dialkenyloxy-N, N-dimethylaminopropane (1, 2-dilinoleyloxy-N, N-dimethylaminopropane, DLinDMA), 1, 2-Dialkenyloxy-N, N-dimethylaminopropane (1, 2-dilinolenyloxy-N, N-dimethylaminopropane, DLinDMA), 3-dimethylamino-2- (cholest-5-en-3- β -oxybutan-4-oxy) -1- (cis, cis-9, 12-octadecadienyloxy) propane (3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane,CLinDMA)、N-(2- aminoethyl) -N, N-dimethyl-2,3-bis (tetradecyloxy) propane-1-aminium bromide (N- (2-aminoethyl) -N, N-dimethyl-2,3-bis (tetradecyloxy) propan-1-aminium bromide, DMORIE), N, N-dimethyl-2,3-bis (dodecyloxy) propan-1-amine (N, N-dimethyl-2,3-bis (dodecyloxy) propan-1-amine, DLDMA), N-dimethyl-2,3-bis (tetradecyloxy) propan-1-amine (N, N-dimethyl-2,3-bis (tetradecyloxy) propan-1-amine, DMDMA), dioctadecyl amidoglycyl spermine (dioctadecylamidoglycyl spermine, DOGS), N4-cholesteryl-spermine (N4-cholesteryl-spermine), 2-diiodol-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (2, 2-dilinoleyl-4- (2-dimethylaminoethyl) - [1,3] -dioxane, DLin-KC 2-DMA), triacontan-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butanoate (heptatriaconta-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butanoate, DLin-MC 3-DMA), a compound of formula (I), (II), (III), or (IV) as described herein, or a combination thereof.

In some embodiments, the cationic lipid is preferably an ionizable cationic lipid. The ionizable cationic lipid carries a net positive charge at, for example, an acidic pH, and is neutral at a higher pH (e.g., physiological pH). Examples of ionizable cationic lipids include, but are not limited to, dioctadecyl amidoglycyl spermine (dioctadecylamidoglycyl spermine, DOGS), N4-cholesteryl-spermine (N4-cholesteryl-spermine), 2-diiodol-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (2, 2-dilinoleyl-4- (2-dimethylaminoethyl) - [1,3] -dioline, DLin-KC 2-DMA), triacontanyl-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butyrate (heptatriaconta-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butanoate butanoate, DLin-MC 3-DMA), compounds of formula (I), (II), (III) or (IV) as described herein, or combinations thereof.

In one embodiment, the cationic lipid comprises a compound of formula (I):

Wherein,

R ₁ and R ₂ are each independently selected from the group consisting of a bond, C ₁-C ₁₂ alkyl and C ₂-C ₁₂ alkenyl;

R ₃ and R ₄ are each independently selected from C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₆-C ₁₀ aryl, and 5-10 membered heteroaryl, and R ₃ and R ₄ are each independently optionally substituted with t R ₆, t being an integer selected from 1-5;

Each R ₆ is independently selected from C ₁-C ₁₂ alkyl and C ₂-C ₁₂ alkenyl;

m ₁ and M ₂ are each independently selected from the group consisting of bond, H, -O-, -S-, -C (O) -, -OC (O) -, -C (O) O-, -OC (O) O-, -SC (S) -, -C (S) S-, 3-10 membered heterocycle, -NR ₇ -, or

R ₅ together with one of M ₁ and M ₂ together with the N atom to which they are attached form a 3-to 10-membered heterocyclic ring, and the corresponding R ₁/R ₃ or

R ₂/R ₄ is absent, said heterocycle optionally substituted with R ₇;

r ₅ is selected from C _3-8 carbocycle, -C _1-12 alkylene-Q, Q is selected from H、-OR ₇、-SR ₇、-OC(O)R ₇、-C(O)OR ₇、-N(R ₇)C(O)R ₇、-N(R ₇)S(O) ₂R ₇、-N(R ₇)C(S)R ₇、-N(R ₇) ₂、 cyano, C _3-8 carbocycle, 3-10 membered heterocycle, C ₆-C ₁₀ aryl, each optionally substituted with one or more C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O);

m and n are each independently an integer selected from 0 to 12;

The alkyl, alkenyl and alkylene groups each optionally being independently interrupted by one or more groups selected from the group consisting of-O-, -S-, -NR ₇-、-C(O)-、-OC(O)-、-C(O)O-、-SC(S)-、-C(S)S-、C _3-8 carbocycle, and the alkyl, alkenyl and alkylene groups each optionally being substituted by one or more R ₇;

R ₇ is each independently selected from H, C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C _3-8 carbocycle, each of which is optionally substituted with one or more C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O).

In one embodiment, R ₁ and R ₂ are each independently selected from C ₁-C ₁₂ alkyl and C ₂-C ₁₂ alkenyl, such as C ₁-C ₁₂ alkyl. In yet another embodiment, one of R ₁ and R ₂ is a bond and the other is independently selected from C ₁-C ₁₂ alkyl and C ₂-C ₁₂ alkenyl, such as C ₁-C ₁₂ alkyl.

In one embodiment, R ₃ and R ₄ are each independently selected from the group consisting of C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₆-C ₁₀ aryl, and 5-to 10-membered heteroaryl. In yet another embodiment, R ₃ and R ₄ are each independently selected from C ₁-C ₁₂ alkyl and C ₂-C ₁₂ alkenyl.

R ₃ and R ₄ are each independently optionally substituted by t R ₆, t being 1,2, 3,4, 5. In one embodiment, each R ₆ is independently selected from C ₁-C ₁₂ alkyl.

In yet another embodiment, at least one of R ₃ and R ₄ is a C ₆-C ₁₀ aryl or a 5-10 membered heteroaryl, such as a C ₆-C ₁₀ aryl.

In one embodiment, R ₅ is selected from the group consisting of C _3-8 carbocycle, -C _1-12 alkylene-Q. Q may be selected from H、-OR ₇、-SR ₇、-OC(O)R ₇、-C(O)OR ₇、-N(R ₇)C(O)R ₇、-N(R ₇)S(O) ₂R ₇、-N(R ₇)C(S)R ₇、-N(R ₇) ₂、 cyano, C _3-8 carbocycle, 3-10 membered heterocycle, C ₆-C ₁₀ aryl. The above groups, including groups covering the options of Q, may each be optionally substituted, where appropriate, with one or more C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=o).

In yet another embodiment, R ₅ is selected from C _3-8 carbocycle, -C _1-12 alkylene-Q, Q is selected from H、-OR ₇、-SR ₇、-OC(O)R ₇、-C(O)OR ₇、-N(R ₇)C(O)R ₇、-N(R ₇)S(O) ₂R ₇、-N(R ₇)C(S)R ₇、-N(R ₇) ₂、 cyano, C _3-8 carbocycle, 3-10 membered heterocycle, C ₆-C ₁₀ aryl. The above groups, including groups covering the options of Q, may each be optionally substituted, where appropriate, with one or more C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=o).

In the compounds of formula (I), R ₇ may each be independently selected from H, C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C _3-8 carbocycle, preferably selected from H, C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C ₆-C ₁₀ aryl and 5-10 membered heteroaryl. Such groups (e.g., H, C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, amino, carbamoyl, sulfonamide, C ₆-C ₁₀ aryl, when appropriate), 5-10 membered heteroaryl, 3-10 membered heterocycle, C _3-8 carbocycle) are each optionally substituted with one or more C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=o) substitution.

In one embodiment, each of the groups in the above description, e.g., C _3-8 carbocycle, -C _1-12 alkylene-Q, including -OR ₇、-SR ₇、-OC(O)R ₇、-C(O)OR ₇、-N(R ₇)C(O)R ₇、-N(R ₇)S(O) ₂R ₇、-N(R ₇)C(S)R ₇、-N(R ₇) ₂、C _3-8 carbocycle, 3-10 membered heterocycle, C ₆-C ₁₀ aryl, C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, amino, carbamoyl, sulfonamide, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C _3-8 carbocycle, etc., covering the Q option may each be optionally substituted with one or more C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=o).

In one embodiment, the alkyl, alkenyl and alkylene groups (as mentioned in R ₁-R ₇) in the compound of formula (I) may each optionally be independently interrupted by one or more groups selected from the group consisting of-O-, -S-, -NR ₇-、-C(O)-、-OC(O)-、-C(O)O-、-SC(S)-、-C(S)S-、C _3-8 carbocycles, and each optionally substituted by one or more R ₇ groups. That is, the alkyl, alkenyl and alkylene chains (straight or branched) may each optionally contain one or more groups selected from the group consisting of-O-, -S-, -NR ₇-、-C(O)-、-OC(O)-、-C(O)O-、-SC(S)-、-C(S)S-、C _3-8 carbocycles.

R ₇ is each independently selected from H, C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C _3-8 carbocycle; preferably, R ₇ is independently selected from H, C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C ₆-C ₁₀ aryl, and 5-10 membered heteroaryl. Such groups (e.g., H, C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, amino, carbamoyl, sulfonamide, C ₆-C ₁₀ aryl, when appropriate), 5-10 membered heteroaryl, 3-10 membered heterocycle, C _3-8 carbocycle) are each optionally substituted with one or more C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=o) substitution.

In the compounds of formula (I), m and n may each independently be an integer selected from 0 to 12, for example, 0, 1,2,3,4, 5,6, 7,8, 9, 10, 11, 12. When 0 is taken, it means that the corresponding group is absent.

In one embodiment, M ₁ or M ₂ is a bond, the corresponding M or n is not 0, and the carbon chain preceding M ₁ or M ₂ is attached to the corresponding R ₁ or R ₂.

In one embodiment, M or N is 0, the corresponding M ₁ or M ₂ is not a bond, and the N atom is directly attached to M ₁ or M ₂.

In one embodiment M ₁ or M ₂ are bonds, the corresponding M or n is 0, and the N atom is directly attached to the corresponding R ₁ or R ₂.

In one embodiment, M ₁ and M ₂ are each independently selected from the group consisting of-C (O) -, -OC (O) -, -C (O) O-, and-OC (O) O-. In yet another embodiment, each of M ₁ and M ₂ is independently selected from-NR ₇-,R ₇ as described above.

In another embodiment, R ₅ forms a 3-10 membered heterocyclic ring with one of M ₁ and M ₂ together with the attached N atom, and the corresponding R ₁/R ₃ or R ₂/R ₄ is absent, the heterocyclic ring optionally being substituted with R ₇, R ₇ as described above.

In one embodiment, R ₅ is selected from-C _1-12 alkylene-Q, Q is selected from H、-OR ₇、-OC(O)R ₇、-C(O)OR ₇、-N(R ₇)C(O)R ₇、-N(R ₇) ₂、 cyano, and R ₇ is as described above.

In a preferred embodiment, R ₁ and R ₂ are each independently selected from C ₁-C ₁₂ alkyl and C ₂-C ₁₂ alkenyl;

Wherein R ₃ and R ₄ are each independently selected from C ₁-C ₁₂ alkyl and C ₂-C ₁₂ alkenyl, and R ₃ and R ₄ are each independently optionally substituted with t R ₆, t is an integer selected from 1-5, and R ₆ are each independently selected from C ₁-C ₁₂ alkyl and C ₂-C ₁₂ alkenyl.

M ₁ and M ₂ are each independently selected from the group consisting of-OC (O) -, -C (O) O-, -OC (O) O-, -SC (S) -and-C (S) S-;

R ₅ is selected from-C ₁- ₁₂ alkylene-Q, Q is selected from-OR ₇ and-SR ₇,R ₇ is independently selected from H, C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C ₆-C ₁₀ aryl and 5-10 membered heteroaryl;

m and n are each independently an integer selected from 1 to 12.

In a preferred embodiment, the cationic lipid comprises a lipid compound having the structure shown below, or a pharmaceutically acceptable salt thereof:

in a preferred embodiment, the cationic lipid comprises M5 or SM-102.

In a preferred embodiment, the cationic lipid comprises MC3.

In a preferred embodiment, the cationic lipid comprises ALC-0315.

In one embodiment, the cationic lipid comprises a compound of formula (I):

R ₁ and R ₂ are each independently selected from C ₁-C ₁₂ alkyl and C ₂-C ₁₂ alkenyl;

R ₃ and R ₄ are each independently selected from the group consisting of C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₆-C ₁₀ aryl, and 5-to 10-membered heteroaryl;

Provided that at least one of R ₃ and R ₄ is a C ₆-C ₁₀ aryl or a 5-10 membered heteroaryl, and R ₃ and R ₄ are each independently optionally substituted with t R ₆, t is an integer selected from 1-5, R ₆ are each independently selected from C ₁-C ₁₂ alkyl and C ₂-C ₁₂ alkenyl;

R ₅ is selected from-C _1-12 alkylene-Q, Q is selected from-OR ₇ and-SR ₇,R ₇ is independently selected from H, C ₁-C ₁₂ alkyl, C ₂-C ₁₂ alkenyl, C ₁-C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C ₆-C ₁₀ aryl and 5-10 membered heteroaryl;

m and n are each independently an integer selected from 1 to 12.

In one embodiment, R ₂ is selected from C ₁-C ₁₂ alkyl. In another embodiment, R ₂ is selected from C ₁-C ₆ alkyl.

In one embodiment, one of R ₃ and R ₄ is C ₆-C ₁₀ aryl or 5-10 membered heteroaryl, the other is C ₁-C ₁₂ alkyl or C ₂-C ₁₂ alkenyl.

In a specific embodiment, R ₃ and R ₄ are each independently selected from C ₁-C ₁₂ alkyl and phenyl, provided that at least one of R ₃ and R ₄ is phenyl. In another embodiment, one of R ₃ and R ₄ is phenyl and the other is C ₁-C ₁₂ alkyl.

In yet another embodiment, R ₃ and R ₄ are each independently substituted with t R ₆, t is an integer selected from 1-5, for example 1,2,3,4, or 5. Preferably, t is an integer from 1 to 3, for example 1,2 or 3, in particular 1 or 2.

In one embodiment, each R ₆ is independently selected from C ₁-C ₁₂ alkyl, such as C ₁-C ₁₀ alkyl.

In one embodiment, t is 1 and R ₆ is substituted on the phenyl ring in the meta or para position relative to R ₁ or R ₂.

In another embodiment, t is 2 and R ₆ is substituted on the phenyl ring in the meta and para positions relative to R ₁ or R ₂.

In one embodiment, R ₄ is substituted at the 1-or last position of R ₂. The 1-position refers to the position of the C atom in R ₂ directly connected with M ₂. The last position refers to the position of the C atom in R ₂ that is furthest from M ₂. In a specific embodiment, R ₄ is selected from C ₁-C ₁₂ alkyl and R ₃ is phenyl.

In one embodiment, R ₃ is substituted at the 1-or last position of R ₁. The 1-position refers to the position of the C atom in R ₁ directly connected with M ₁. The last position refers to the position of the C atom in R ₁ that is furthest from M ₁. In a specific embodiment, R ₃ is selected from C ₁-C ₁₂ alkyl and R ₄ is phenyl.

In one embodiment, M ₁ and M ₂ are each independently selected from the group consisting of-OC (O) -, -C (O) O-and-OC (O) O-.

In one embodiment, R ₅ is selected from-C _1-5 alkylene-Q, such as C ₁、C ₂、C ₃、C ₄ or C ₅ alkylene-Q. In exemplary embodiments, R ₅ is selected from-C _1-3 alkylene-Q, e.g., C ₁、C ₂ or C ₃ alkylene-Q.

In another embodiment, Q is selected from the group consisting of-OH and-SH, especially-OH.

In some embodiments, m and n are each independently an integer selected from 2-9, such as 2,3, 4,5, 6, 7, 8, or 9. Preferably, m and n are each independently an integer selected from 2-7, such as 2,3, 4,5, 6 or 7, more preferably, m and n are each independently an integer selected from 5-7, such as 5, 6 or 7.

In certain embodiments, the compound of formula (I) comprises a compound of formula (II):

or a pharmaceutically acceptable salt thereof, wherein the groups are as defined herein.

In one embodiment,

R ₁ is selected from C ₁-C ₆ alkyl;

R ₂ is selected from C ₁-C ₁₀ alkyl;

R ₄ is selected from C ₁-C ₁₀ alkyl;

M ₁ and M ₂ are each independently selected from the group consisting of-OC (O) -, -C (O) O-and-OC (O) O-;

R ₅ is selected from-C _1-5 alkylene-Q, Q is selected from-OR ₇ and-SR ₇,R ₇ is independently selected from H, C ₁-C ₁₂ alkyl and C ₂-C ₁₂ alkenyl;

Each R ₆ is independently selected from C ₁-C ₁₂ alkyl and C ₂-C ₁₂ alkenyl, particularly C ₁-C ₁₂ alkyl;

m and n are each independently an integer selected from 2-9, for example 2,3, 4,5, 6,7,8 or 9;

t is an integer selected from 1-3.

In one embodiment, R ₅ is selected from the group consisting of-C _1-3 alkylene-Q, Q is selected from the group consisting of-OH and-SH, and especially-OH.

In one embodiment, m and n are each independently integers selected from 2-7, such as 2,3, 4,5, 6, or 7.

In some embodiments, t is 1 or 2.

In one embodiment, R ₄ is substituted at the 1-or last position of R ₂. The 1-position refers to the position of the C atom in R ₂ directly connected with M ₂. The last position refers to the position of the C atom in R ₂ that is furthest from M ₂.

In one embodiment, t is 1 and R ₆ is substituted on the phenyl ring in the meta or para position relative to R ₁.

In another embodiment, t is 2 and R ₆ is substituted on the phenyl ring in the meta and para positions relative to R ₁.

In certain embodiments, the compound of formula (I) comprises a compound of formula (III):

In one embodiment,

R ₁ is selected from C ₁-C ₆ alkyl;

R ₂ is selected from C ₁-C ₁₀ alkyl;

R ₄ is selected from C ₁-C ₁₀ alkyl;

R ₅ is selected from the group consisting of-C _1-3 alkylene-Q, Q is selected from the group consisting of-OH and-SH, especially-OH;

t is 1 or 2;

R ₆ is selected from the group consisting of C ₁-C ₁₂ alkyl and C ₂-C ₁₂ alkenyl, particularly C ₁-C ₁₂ alkyl;

m and n are each independently an integer selected from 2-7, for example 2,3, 4,5, 6 or 7.

In one embodiment, R ₄ is substituted at the 1-or last position of R ₂. The 1-bit refers to the R ₂ andThe position of the partially directly attached C atom. The last position refers to the sum in R ₂ The position of the C atom that is partially furthest from.

In certain embodiments, the compound of formula (I) comprises a compound of formula (IV):

In one embodiment,

R ₁ is selected from C ₁-C ₆ alkyl;

R ₂ is selected from C ₁-C ₁₀ alkyl;

R ₄ is selected from C ₁-C ₁₀ alkyl;

t is 1 or 2;

In a particular embodiment, the lipid compounds of the present invention do not contain alkenyl groups in the substituents (e.g., R ₁-R ₇).

In a preferred embodiment, the cationic lipid comprises a lipid compound of SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.

In a preferred embodiment, the cationic lipid comprises a lipid compound selected from the group consisting of M5, MC3, ALC-0315, SM-102, SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2, and SW-II-140-2.

Phospholipid

The lipid composition of the present invention comprises phospholipids which can assist in cell penetration of the lipid composition.

Examples of phospholipids include, but are not limited to, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-di-O-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphorylcholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (QC 16), 1, 2-dioleoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-glycero-3-phosphorylcholine (POPC) 1, 2-dioleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), 1, 2-di-phytanoyl-sn-glycero-3-phosphate ethanolamine (ME 16.0 PE), 1, 2-di-stearoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-oleoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-linolenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-docosahexaenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPG) dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-based phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) or a combination thereof.

Steroid compounds

The lipid composition of the present invention comprises a steroid which may act as a structural component of the lipid composition.

Examples of steroids include, but are not limited to, for example, cholesterol, fecal sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassinosteroids, lycorine, ursolic acid, alpha-tocopherol, and derivatives thereof.

Polyethylene glycol modified lipids

As used herein, the term "polyethylene glycol modified lipid" or "PEG lipid" refers to a molecule comprising a polyethylene glycol moiety and a lipid moiety, which is a lipid modified with polyethylene glycol. The PEG lipid may be selected from the non-limiting group consisting of PEG modified phosphatidylethanolamine, PEG modified phosphatidic acid, PEG modified ceramide (PEG-CER), PEG modified dialkylamine, PEG modified diacylglycerol (PEG-DEG), PEG modified dialkylglycerol, or combinations thereof. For example, examples of polyethylene glycol modified lipids include, but are not limited to, 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol, DMG-PEG), 1,2-dioleoyl-rac-glycerol, methoxy-polyethylene glycol (1, 2-Dioleoyl-rac-glycol, methoxypolyethylene Glycol, DOGPEG) and 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-poly (ethylene glycol) (1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly (ethylene glycol), DSPE-PEG).

In one embodiment, the polyethylene glycol modified lipid is DMG-PEG, such as DMG-PEG 2000. In one embodiment, the DMG-PEG 2000 has the following structure:

Wherein n has an average value of 44.

Cationic polymers

As used herein, the term "cationic polymer" refers to any ionic polymer capable of carrying a net positive charge at a specified pH to electrostatically bind nucleic acids. Examples of cationic polymers include, but are not limited to, poly-L-lysine, protamine, polyethylenimine (PEI), or combinations thereof. The polyethyleneimine may be a linear or branched polyethyleneimine.

The term "protamine" refers to arginine-rich low molecular weight basic proteins that are present in sperm cells of various animals (particularly fish) and bind to DNA in place of histones. In a preferred embodiment, the cationic polymer is protamine (e.g., protamine sulfate).

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising an artificial nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a lipid composition of the invention, and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers can include, but are not limited to, diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, glidants, sweeteners, coatings, excipients, preservatives, antioxidants (such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated Hydroxyanisole (BHA), butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like), solubilizing agents, gelling agents, softeners, solvents (such as water, alcohols, acetic acid, and syrups), buffers (such as phosphate buffers, histidine buffers, and acetate buffers), surfactants (such as nonionic surfactants, such as polysorbate 80, polysorbate 20, poloxamers or polyethylene glycols), antibacterial agents, antifungal agents, isotonic agents (such as trehalose, sucrose, mannitol, sorbitol, lactose, glucose), absorption delaying agents, chelating agents, and emulsifying agents. For pharmaceutical compositions comprising an artificial nucleic acid molecule, carrier, cell or lipid composition, a suitable carrier may be selected from buffers (e.g., citrate buffer, acetate buffer, phosphate buffer, histidine salt buffer), isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamers), or combinations thereof.

The pharmaceutical compositions provided herein may be in a variety of dosage forms including, but not limited to, solid, semi-solid, liquid, powder, or lyophilized forms. For pharmaceutical compositions comprising artificial nucleic acid molecules, vectors, cells or lipid compositions, preferred dosage forms may generally be, for example, injections and lyophilized powders.

The pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, for example, by systemic or topical administration. Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavity), topical (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or topical). Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion). The method of administration may be, for example, injection or infusion.

Treatment of

In a further aspect, the invention relates to the use of an artificial nucleic acid molecule of the invention, a vector of the invention, a cell of the invention, a lipid composition of the invention or a pharmaceutical composition of the invention for the preparation of a vaccine or a medicament for gene therapy.

In a further aspect, the invention relates to the use of an artificial nucleic acid molecule according to the invention, a vector according to the invention, a cell according to the invention, a lipid composition according to the invention or a pharmaceutical composition according to the invention for the preparation of a medicament for the treatment or prophylaxis of a disease.

The artificial nucleic acid molecules of the invention, the vectors of the invention, the cells of the invention, the lipid compositions of the invention or the pharmaceutical compositions of the invention may be used for the treatment of diseases, disorders or conditions. In particular, the artificial nucleic acid molecules of the invention, the vectors of the invention, the cells of the invention, the lipid compositions of the invention or the pharmaceutical compositions of the invention may be used for the treatment of diseases, disorders or conditions characterized by lost or abnormal protein or polypeptide activity. For example, an artificial nucleic acid molecule, vector, cell, lipid composition, or pharmaceutical composition comprising an mRNA encoding a missing or aberrant polypeptide may be administered or delivered to a cell. Subsequent translation of the mRNA can produce the polypeptide, thereby reducing or eliminating problems caused by the absence or aberrant activity of the polypeptide. Since translation can occur rapidly, these methods and artificial nucleic acid molecules, vectors, cells, lipid compositions or pharmaceutical compositions are useful for treating acute diseases, disorders or conditions such as sepsis, stroke and myocardial infarction.

Diseases, disorders or conditions characterized by dysfunctional or aberrant protein or polypeptide activity that may be administered an artificial nucleic acid molecule, vector, cell, lipid composition or pharmaceutical composition of the invention include, but are not limited to, rare diseases, infectious diseases (in vaccine and therapeutic forms), cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases. There are a variety of diseases, disorders or conditions that can be characterized by a loss of protein activity (or a substantial decrease in protein activity such that proper protein function cannot occur). These proteins may not be present or they may be substantially nonfunctional. Specific examples of dysfunctional proteins are missense mutant variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produce dysfunctional protein variants of the CFTR protein, thereby causing cystic fibrosis. The present invention provides a method of treating such a disease, disorder or condition in a subject by administering an artificial nucleic acid molecule, vector, cell, lipid composition or pharmaceutical composition of the invention, wherein the RNA can be mRNA encoding a polypeptide that antagonizes or otherwise overcomes the aberrant protein activity present in the cells of the subject.

The artificial nucleic acid molecules, vectors, cells, lipid compositions or pharmaceutical compositions of the invention may be administered to a subject using any reasonable amount and any route of administration that is effective to effect prevention, treatment, diagnosis or for any other purpose of a disease, disorder or condition. The particular amount administered to a given subject may vary depending on the species, age, and general condition of the subject, the purpose of administration, the particular composition, the mode of administration, and the like.

In some embodiments, the artificial nucleic acid molecules, vectors, cells, lipid compositions or pharmaceutical compositions of the invention may be administered to a subject by any method known to those of skill in the art, such as parenterally, orally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously or intraperitoneally.

Kit for detecting a substance in a sample

The invention also provides a kit comprising an artificial nucleic acid molecule, vector, cell, lipid composition or pharmaceutical composition of the invention, and instructions for use. The kit may also comprise a suitable container. In certain embodiments, the kit further comprises a device for administering the drug. Kits generally include a label that indicates the intended use and/or method of use of the kit contents. The term "label" includes any written or recorded material provided on or with or otherwise with the kit.

Advantageous effects

The artificial nucleic acid molecules, vectors, cells, lipid compositions or pharmaceutical compositions of the invention may exhibit excellent effects such as, but not limited to, 1) increased translation efficiency of the contained mRNA, and/or 2) high stability of the contained mRNA.

Examples

A further understanding of the present application may be obtained by reference to the specific examples which are set forth to illustrate, but are not intended to limit the scope of the present application. It will be apparent that various modifications and variations can be made to the present application without departing from the spirit of the application, and therefore, such modifications and variations are also within the scope of the application as claimed. The proportions used herein include percentages, by weight unless otherwise indicated.

Experimental materials

Cationic lipids according to formula (I) are prepared for Sterculia synthesis or reference, for example, CN110520409A, WO2018081480A1 or US11,246,933B1, phospholipid (DOPE) is purchased from CordenPharma, cholesterol is purchased from Sigma-Aldrich, mPEG2000-DMG (i.e., DMG-PEG 2000) is purchased from Avanti Polar Lipids, inc., PBS is purchased from Invitrogen, protamine sulfate is purchased from North Beijing Lian pharmaceutical Co., ltd, mPEG2000-DSPE is purchased from lipoid GmbH, DSPC is purchased from Avanti Polar Lipids, inc.

EXAMPLE 1 Synthesis of Compounds according to formula (I)

General considerations

All solvents and reagents used were commercially available and used as received unless otherwise indicated. ¹ H NMR spectra were recorded in CDCl ₃ using Bruker Ultrashield MHz instrument at 300K. Chemical shifts are reported in parts per million (ppm) relative to TMS (0.00) for ¹ H. Silica gel chromatography was performed on ISCO CombiFlash Rf +lumen instruments using ISCO REDISEP RF Gold flash column (particle size: 20-40 microns).

The procedure described below can be used to synthesize compounds SW-II-115 to SW-II-140-2.

The following abbreviations are used herein:

THF tetrahydrofuran

MeCN acetonitrile

LAH lithium aluminum hydride

DCM: dichloromethane

DMAP 4-dimethylaminopyridine

LDA lithium diisopropylamide

Rt, room temperature

DME 1, 2-Dimethoxyethane

N-BuLi n-butyllithium

CPME cyclopentyl methyl ether

EDCI N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide

DIEA N, N-diisopropylethylamine

PE Petroleum ether

EA ethyl acetate

A. Compounds SW-II-115

1. Synthesis of intermediate 3

To a solution of compound 1 (10 g,45mmol,1 eq.) and compound 2 (7.8 g,54mmol,1.2 eq.) in DCM (100 mL) were added EDCI (17.3 g,90mmol,2 eq.) and DMAP (2.2 g,18mmol,0.4 eq.) followed by DIEA (23.2 g,180mmol,4 eq.). The reaction mixture was stirred at room temperature under protection of N ₂ for 16 hours. TLC (petroleum ether: ethyl acetate=30:1) showed that compound 1 was consumed and the desired product formed. The reaction mixture was diluted with DCM (20 mL) and washed with H ₂ O (40 mL), dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether in ethyl acetate (1:0-20:1) to give compound 3 (4.365 g, 28%) as a colourless oil.

2. Synthesis of intermediate 5

A solution of compound 3 (500 mg, 1.433 mmol,1 eq.) and compound 4 (2.63 g,43.103mmol,30 eq.) in EtOH is stirred under protection of N ₂ at 60℃for 16 hours. TLC (DCM: meoh=10:1) showed compound 3 was consumed and TLC (DCM/meoh=10/1) showed new principal spots were observed. The reaction mixture was concentrated under reduced pressure. The residue was diluted with EtOAc (50 mL) and washed with H ₂ O (3X 50 mL). The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1:0-10:1, v/v) to give compound 5 as a yellow oil (264 mg, 56%).

3. Synthesis of intermediate 8

To a mixed solvent of compound 6 (500 mg,1.712mmol,1 eq.) and compound 7 (1.113 g,8.562mmol,5 eq.) in dioxane/water (5 mL/0.5 mL) was added Pd (dppf) Cl ₂ (112 mg,0.171mmol,0.1 eq.) and potassium carbonate (709 mg,5.136mmol,3 eq.). The mixture was stirred at 100 ℃ overnight under N ₂. TLC (PE: ea=15:1) showed the reaction was complete and new major spots were observed. The mixture was extracted with EA and washed with water, and the organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE:EA (1:0-10:1) to give compound 8 (45 mg, 88%) as a colourless oil.

4. Synthesis of intermediate 9

To a solution of compound 8 (45 mg,1.497mmol,1 eq.) in THF (5 mL) under protection of N ₂ at 0 ℃ was added LiAlH ₄ (1.5 mL,1.497mmol,1m in THF, 1 eq.). The mixture was stirred at room temperature under N ₂ for 2 hours. TLC (PE: etoac=5:1) showed the reaction was complete and new major spots were observed. The mixture was quenched with water (1.5 mL) and treated with 2N HCl to adjust the PH between 6 and 7, extracted with EA and washed with brine. The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo to give crude compound 9 (419 mg, > 100%) as a colorless oil without further purification.

5. Synthesis of intermediate 10

To a solution of compound 1 (399 mg,1.518mmol,1 eq.) and compound 9 (319 mg,1.518mmol,1 eq.) in DCM (4 mL) were added EDCI (803 mg,3.036mmol,2 eq.) and DMAP (74 mg, 0.603 mmol,0.4 eq.) followed by DIEA (783 mg,6.072mmol,4 eq.). The reaction mixture was stirred at room temperature under protection of N ₂ for 16 hours. TLC (petroleum ether: ethyl acetate=10:1) showed formation of the desired product. The reaction mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether:ethyl acetate (1:0-10:1) to give compound 10 (447 mg, 60.7%) as a colorless oil.

6. Synthesis of end product SW-II-115

To a mixed solvent CPME/CH ₃ CN (3 mL/3 mL) containing compound 10 (307 mg,0.64mmol,1 eq.) and compound 5 (210 mg,0.64mmol,1 eq.) was added K ₂CO ₃ (530 mg,3.84mmol,6 eq.) and KI (212 mg,1.28mmol,2 eq.). After the addition was complete, the mixture was stirred at 90 ℃ under N ₂ overnight. TLC (DCM: meoh=10:1) showed the reaction was complete and new major spots were observed. The mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM: meOH (1:0-10:1, v/v) to give compound SW-II-115 (266 mg, 57%) as a yellow oil.

LCMS:Rt:1.293min;MS m/z(ELSD):730.5[M+H] ⁺;

HPLC 99.472% purity, ELSD, rt=4.895 min.

¹H NMR(400MHz,CDCl ₃)δ7.21–6.99(m,3H),5.05(s,2H),4.05(t,J＝6.8Hz,2H),3.58(t,J＝5.3Hz,2H),2.69–2.46(m,10H),2.31(dt,J＝20.0,7.5Hz,4H),1.69–1.18(m, 51H),0.89(dt,J＝12.4,6.3Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.90(s),173.68(s),140.80(d,J＝13.0Hz),133.31(s),129.25(d,J＝16.2Hz),128.30(s),125.75(s),77.30(d,J＝11.5Hz),77.04(s),76.72(s),66.22(s),64.43(s),58.12(s),55.72(s),53.90(s),34.32(d,J＝1.9Hz),32.69(s),32.48(s),31.81(d,J＝11.2Hz),31.25(s),29.59–28.91(m),28.66(s),27.17(s),26.64(s),25.94(s),24.91(d,J＝5.1Hz),22.65(d,J＝3.3Hz),14.10(s).

B. Compounds SW-II-118

1. Synthesis of intermediate 3

A solution of compound 1 (1.22 g,5.0mmol,1.0 eq.) and compound 2 (765 mg,7.5mmol,1.5 eq.) in toluene (10 mL) and H ₂ O (1 mL) at 110 ℃, stirring for 1 hour under N ₂ protection, TLC (petroleum ether: ethyl acetate=19:1) showed that compound 1 was consumed and a new spot was observed, the reaction mixture was diluted with DCM (50 mL) and washed with H ₂ O (40 mL), dried over anhydrous Na ₂SO ₄, filtered and concentrated, and the residue was purified by silica gel column chromatography eluting with petroleum ether: ethyl acetate (1:0-10:1) to give colorless compound 3 (0.5 g, 45%).

¹H NMR(400MHz,CDCl ₃)δ7.16(dd,J＝23.5,8.1Hz,4H),4.14(q,J＝7.1Hz,2H),3.57(s,2H),2.64–2.48(m,2H),1.66–1.51(m,2H),1.35(dd,J＝15.0,7.4Hz,2H),1.25(t,J＝7.1Hz,3H),0.92(t,J＝7.3Hz,3H).

2. Synthesis of intermediate 4

LiAlH ₄ (193 mg,5.09mmol,4.0 eq.) was added to a solution containing compound 3 (280 mg,1.27mmol,1.0 eq.) in THF (10 mL) at-78℃and the reaction was then reacted at 10℃for 3 hours. TLC showed good reaction, the reaction was concentrated and diluted with Na ₂SO ₄ (20 mL) and extracted with EA (30 mL x 2), the organic phase was dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure to give compound 4 (3.12 g, crude) as a yellow oil.

3. Synthesis of intermediate 6

A solution of compound 4 (215 mg,1.2mmol,1.0 eq.) compound 5 (404 mg,1.8mmol,1.5 eq.) EDCI (1.15 g,6.0mmol,5.0 eq.), DMAP (730 mg,1.8 eq.), DIEA (1.29 g,12.0mmol,10.0 eq.) and DIEA (1.29 g,12.0mmol,10.0 eq.) in DCM (5 mL) was stirred under N ₂ for 16h at 10 ℃. TLC (DCM: meoh=10:1) showed the reaction was complete and new major spots were observed. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with PE:EA (1:0-10:1, v/v) to give compound 6 (145 mg, 31%) as a colorless oil.

¹H NMR(400MHz,CDCl ₃)δ7.12(s,4H),4.27(t,J＝7.1Hz,2H),3.52(t,J＝6.7Hz,1H),3.40(t,J＝6.8Hz,1H),2.90(t,J＝7.1Hz,2H),2.65–2.50(m,2H),2.28(t,J＝7.5Hz,2H),1.93–1.70(m,2H),1.64–1.56(m,4H),1.44–1.27(m,8H),0.92(t,J＝7.3Hz,3H).

4. Synthesis of end product SW-II-118

A mixture containing compound 6 (140 mg,0.37mmol,1.0 eq.) compound 7 (243 mg,0.55mmol,1.5 eq.) K ₂CO ₃ (153 mg,1.11mmol,3.0 eq.) and KI (123 mg,0.74mmol,2.0 eq.) was stirred at 90℃for 16 hours in a mixed solvent of CPME (1 mL) and CH ₃ CN (1 mL) under N ₂. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with EtOAc (50 mL) and washed with NaHCO ₃ (30 mL). The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM: meOH (1:0-10:1, v/v) to give SW-II-118 as a yellow oil (105 mg, 61%).

LCMS:Rt:1.946min;MS m/z(ELSD):744.4[M+H] ⁺;

HPLC 99.64% purity, ELSD, rt=5.875 min.

¹H NMR(400MHz,CDCl ₃)δ7.11(s,4H),4.91–4.79(m,1H),4.26(t,J＝7.2Hz,2H),3.80–3.68(m,2H),2.90(t,J＝7.1Hz,4H),2.81–2.67(m,4H),2.62–2.52(m,2H),2.28(td,J＝7.5,2.6Hz,4H),1.64–1.51(m,11H),1.38–1.17(m,42H),0.93–0.82(m,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.61(d,J＝11.7Hz),141.11(s),134.90(s),128.74(s),128.51(s),77.40(s),77.08(s),76.77(s),74.17(s),64.90(s),57.48(s),56.24(s),53.98(s),35.25(s),34.66(d,J＝14.4Hz),34.16(d,J＝5.1Hz),33.67(s),31.86(s),29.52(d,J＝2.4Hz),29.24(s),29.21–28.74(m),26.90(d,J＝4.9Hz),25.42–24.92(m),24.92–24.88(m),24.74(s),22.67(s),22.37(s),14.04(d,J＝15.7Hz).

C. Compounds SW-II-120

1. Synthesis of intermediate 3

Containing compound 1 (1.22 g,5.0mmol,1.0 eq.) compound 2 (1.30 mg,10.0mmol,2.0 eq.) Pd (PPh ₃) ₄ (289 mg,0.25mmol,0.05 eq.) and K ₂CO ₃ (1.38 g,10.0mmol,2.0 eq.) in a mixed solution of toluene (10 mL) and H ₂ O (1 mL) under N ₂ protection stirring for 1 hour at 110 ℃, TLC (petroleum ether: ethyl acetate=19:1) showed that compound 1 was consumed and a new spot was observed, the reaction mixture was diluted with DCM (50 mL) and washed with H ₂ O (40 mL), dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure the residue was purified by silica gel column chromatography eluting with petroleum ether: ethyl acetate (1:0-10:1) to give colorless compound 3 (0.78 g, 62%).

¹H NMR(400MHz,CDCl ₃)δ7.19(d,J＝8.1Hz,2H),7.13(d,J＝8.1Hz,2H),4.14(q,J＝7.1Hz,2H),3.57(s,2H),2.62–2.51(m,2H),1.58(d,J＝11.1Hz,2H),1.35–1.21(m,9H),0.88(t,J＝6.7Hz,3H).

2. Synthesis of intermediate 4

LiAlH ₄ (477 mg,12.56mmol,4.0 eq.) was added to a solution of compound 3 (780 mg,3.14mmol,1.0 eq.) in THF (10 mL) at-78℃and the reaction was stirred at 10℃for 3 hours. Thin layer chromatography showed that the reaction performed well. The reaction was concentrated and diluted with Na ₂SO ₄ (20 mL) and extracted with EA (30 mL x 2), the organic phase was dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure to give compound 4 (640 mg, crude) as a colourless oil.

3. Synthesis of intermediate 6

A solution of compound 4 (640 mg,3.10mmol,1.0 eq.), compound 5 (1.06 g,4.70mmol,1.5 eq.), EDCI (2.98 g,15.5mmol,5.0 eq.), DMAP (1.85 g,15.0 eq.) and DIEA (4.0 g,31.0mmol,10.0 eq.) in DCM (10 mL) was stirred under N ₂ at 10℃for 16h. TLC (DCM: meoh=10:1) showed the reaction was complete and new major spots were observed. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with PE:EA (1:0-10:1, v/v) to give compound 6 (460 mg, 36%) as a colourless oil.

4. Synthesis of end product SW-II-120

A mixture containing compound 6 (100 mg,0.25mmol,1.0 eq.), compound 7 (161 mg,0.36mmol,1.5 eq.), K ₂CO ₃ (104 mg,0.75mmol,3.0 eq.) and KI (83 mg,0.50mmol,2.0 eq.) was stirred at 90℃for 16 hours under N ₂ in CPME (1 mL) and CH ₃ CN (1 mL). The reaction mixture was concentrated under reduced pressure, and the residue was diluted with EtOAc (50 mL) and washed with NaHCO ₃ (30 mL). The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM: meOH (1:0-10:1, v/v) to give SW-II-120 as a yellow oil (100 mg, 52%).

LCMS:Rt:2.500min;MS m/z(ELSD):772.4[M+H] ⁺;

HPLC 99.70% purity, ELSD, rt=8.675 min.

¹H NMR(400MHz,CDCl ₃)δ7.07(d,J＝8.9Hz,4H),4.89–4.73(m,1H),4.23(t,J＝7.2Hz,2H),3.83–3.65(m,2H),2.87(t,J＝7.2Hz,4H),2.82–2.67(m,4H),2.61–2.45(m,2H),2.25(td,J＝7.5,2.5Hz,4H),1.65–1.44(m,15H),1.27(dd,J＝13.2,11.3Hz,42H),0.85(t,J＝6.8Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.57(d,J＝11.5Hz),141.13(s),134.88(s),128.73(s),128.48(s),77.45(s),77.13(s),76.81(s),74.14(s),64.89(s),57.34(s),56.17(s),53.92(s),35.57(s),34.64(d,J＝16.1Hz),34.14(d,J＝3.3Hz),31.79(d,J＝13.4Hz),31.49(s),29.50(d,J＝2.2Hz),29.23(s),29.10–28.71(m),26.85(d,J＝5.0Hz),25.49–25.38(m),25.13(d,J＝35.4Hz),24.72(s),22.63(d,J＝5.8Hz),14.11(s).

D. Compounds SW-II-121

1. Synthesis of intermediate 3

To a solution of compound 1 (1.3 g,5.86mmol,1.5 eq.) and compound 2 (1 g,3.9mmol,1.0 eq.) in DCM (20 mL) were added EDCI (1.495 g,7.8mmol,2.0 eq.), DMAP (0.19 g,1.56mmol,0.4 eq.) and DIEA (2.57 mL,15.6mmol,4.0 eq.). The reaction mixture was stirred at room temperature under N ₂ for 16 hours. TLC (petroleum ether: ethyl acetate=19:1) showed that compound 2 was consumed and the desired product formed. The reaction mixture was diluted with DCM (20 mL) and washed with H ₂ O (40 mL), dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with petroleum ether in ethyl acetate (1:0-10:1) to give compound 3 (1.2 g, 66.9%) as a yellow oil.

¹H NMR(400MHz,CDCl ₃)δ4.92–4.82(m,1H),3.42(t,J＝6.8Hz,2H),2.31(t,J＝7.5Hz,2H),1.95–1.82(m,2H),1.70–1.19(m,36H),0.90(t,J＝6.8Hz,6H).

2. Synthesis of intermediate 5

A solution of compound 3 (5.2 g,11.30mmol,1.0 eq.) and compound 4 (20.6 g, 319 mmol,30 eq.) in EtOH (5 mL) under N ₂ is stirred at 60℃for 16 h. TLC (petroleum ether: ethyl acetate=19:1) showed that compound 3 was consumed and TLC (DCM/meoh=10/1) showed that new principal points were observed. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with EtOAc (50 mL) and washed with H ₂ O (3×50 mL). The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with DCM: meOH (1:0-10:1, v/v) to give compound 5 as a yellow oil (3 g, 60%).

3. Synthesis of intermediate 8

Pd (pph ₃) ₄(238mg,0.206mmol,0.05eq.)、K ₂CO ₃ (1.7 g,12.35mmol,3 eq.) was added to a toluene/water (10 mL) mixture containing compound 6 (1 g,4.115mmol,1 eq.) and compound 7 (889 mg,6.173mmol,1.5 eq.) and the mixture was stirred at N ₂ and 110 ℃ for 2 hours.

4. Synthesis of intermediate 9

To a mixture of compound 8 (710 mg,2.725mmol,1 eq.) in THF (7 mL) at 0 ℃ under N ₂ protection was added LiAlH ₄ (2.7 mL,2.725mmol,1m in THF, 1 eq.) and the mixture stirred at room temperature for 2 hours. TLC (PE: etoac=10:1) showed the reaction was complete and new major spots were observed. The mixture was quenched with water (2.7 mL) and treated with 2N HCl to adjust the PH between 6 and 7, extracted with EA and washed with brine. The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE:EA (1:0-10:1) to give compound 9 (103 mg, 63%) as a colourless oil.

5. Synthesis of intermediate 11

To DCM (3 mL) containing compound 9 (300 mg, 1.264 mmol,1 eq.) and compound 10 (803 mg,1.64mmol,1.2 eq.) were added EDCI (254 mg, 2.428 mmol,2 eq.), DMAP (67 mg,0.546mmol,0.4 eq.) and DIEA (704 mg, 5.458 mmol,4 eq.). The reaction mixture was stirred at room temperature under N ₂ for 16 hours. TLC (petroleum ether: ethyl acetate=10:1) showed formation of the desired product. The reaction mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1:0-10:1) to give compound 11 (169 mg, 29%) as a colorless oil.

6. Synthesis of end product SW-II-121

To a mixed solvent of CPME/CH ₃ CN (2 mL/2 mL) containing compound 11 (169 mg,0.399mmol,1 eq.) and compound 5 (176 mg,0.399mmol,1 eq.) were added K ₂CO ₃ (330 mg, 2.284 mmol,6 eq.) and KI (132 mg,0.798mmol,2 eq.). After the addition was complete, the mixture was stirred at 90 ℃ under N ₂ overnight. TLC (DCM: meoh=10:1) showed the reaction was complete and new major spots were observed. The mixture was extracted with EA and washed with water, and the organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM: meOH (1:0-10:1, v/v) to give compound SW-II-121 (145 mg, 46%) as a yellow oil.

LCMS:Rt:1.493min;MS m/z(ELSD):786.5[M+H] ⁺;

HPLC 99.869% purity, ELSD, rt= 10.655min.

¹H NMR(400MHz,CDCl ₃)δ7.11(s,4H),4.92–4.80(m,1H),4.26(t,J＝7.2Hz,2H),3.80(s,2H),2.87(dd,J＝26.6,19.4Hz,7H),2.62–2.51(m,2H),2.28(td,J＝7.2,3.6Hz,4H),1.75–1.45(m,14H),1.42–1.09(m,45H),0.88(t,J＝6.8Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.61(d,J＝12.3Hz),141.20(s),134.90(s),128.75(s),128.51(s),77.35(s),77.03(s),76.72(s),74.21(s),64.93(s),54.15(s),35.59(s),34.66(d,J＝16.6Hz),34.16(d,J＝3.0Hz),31.85(d,J＝4.4Hz),31.55(s),29.64–29.15(m),29.15–28.78(m),26.85(d,J＝4.5Hz),25.33(s),24.95(s),24.72(s),22.68(s),14.12(s).

E. Compounds SW-II-122

1. Synthesis of Compound 3

Compound 1 (1 g,4.65mmol,1 eq.) and compound 2 (726 mg,5.58mmol,1.2 eq.) were dissolved in toluene/water (10/1, 20 mL.) then K ₂CO ₃ (1.92 g,13.9mmol,3 eq.) and Pd (pph ₃) ₄ (269 mg,0.23mmol,0.05 eq.) were added to the mixture, the reaction mixture was heated to 110 ℃ for 2 hours with stirring, TLC (petroleum ether/ethyl acetate=19/1) showed that compound 1 was consumed and a new major spot was observed, the reaction mixture was quenched with H ₂ O (80 mL) and extracted with ethyl acetate (60 ml×3), the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, the residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-10/1) to give yellow oil compound 3 (800 mg, 78%).

2. Synthesis of Compound 4

To compound 3 (700 mg,3.18mmol,1.0 eq.) dissolved in THF (14 mL) was added LiAlH ₄ (3.2 mL,3.18mmol,1 eq.) under nitrogen at 0 ℃. The reaction was allowed to warm to room temperature under nitrogen and stirred for 2 hours. TLC (PE/etoac=10/1) showed the reaction was complete and a new major spot was observed. The mixture was quenched with water (3.2 mL) and 1M HCl (3.2 mL), respectively. Water (6 mL) was then added to the mixture, and extraction was performed with ethyl acetate (60 mL. Times.3). The organic layer was washed with brine (30 ml×2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with ethyl acetate/petroleum ether=1/10 to give compound 4 (600 mg, 98%) as a yellow oil.

3. Synthesis of Compound 6

Compound 4 (680 mg,3.5mmol,1.0 eq.) and compound 5 (1.13 g,5.1mmol,1.5 eq.) were dissolved in DCM (10 mL), and EDCI (1.20 g,6.25mmol,2.0 eq.), DMAP (166 mg,1.36mmol,0.4 eq.) and DIEA (1.78 g,13.8mmol,4.0 eq.) were added to the mixture. After the addition was complete, the reaction mixture was stirred overnight at room temperature under nitrogen. TLC (DCM/meoh=30/1) showed that the starting material was consumed and a new spot formed. The mixture was quenched with water (70 mL) and extracted with DCM (80 mL. Times.3). The combined organic layers were washed with brine (2×20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with ethyl acetate/petroleum ether=3/97 to give compound 6 (680 mg, 48.5%) as a yellow oil.

4. Synthesis of SW-II-122

Compound 6 (108 mg,0.27mmol,1.2 eq) and compound 7 (100 mg,0.23mmol,1 eq.) were dissolved in CPME (2 mL) and CH ₃ CN (2 mL), and potassium carbonate (157 mg,1.14mmol,5.0 eq) and potassium iodide (75 mg,0.45mmol,2.0 eq) were added to the mixture. After the addition was complete, the reaction mixture was stirred under nitrogen at 90 ℃ for 16 hours. TLC (DCM/meoh=10/1) showed the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give SW-II-122 (68 mg, 40%) as a colorless oil.

LCMS:Rt:1.487min;MS m/z(ELSD):758.5[M+H] ⁺;

HPLC 97.3% purity, ELSD, rt= 7.622min.

¹H NMR(400MHz,CDCl ₃)δ7.32(d,J＝26.4Hz,1H),7.17(dd,J＝27.2,21.1Hz,3H),5.09(s,2H),4.91–4.79(m,1H),3.85(s,2H),2.98(s,2H),2.87(s,4H),2.65–2.54(m,2H),2.35(t,J＝7.6Hz,2H),2.28(t,J＝7.6Hz,2H),1.74–1.57(m,9H),1.50(d,J＝5.6Hz,4H),1.37–1.15(m,43H),0.94–0.80(m,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.55(d,J＝2.4Hz),143.35(s),135.92(s),128.67–128.19(m),125.47(s),77.36(s),77.04(s),76.73(s),74.22(s),66.27(s),57.15(s),56.74(s),54.14(s),35.88(s),34.55(s),34.15(d,J＝3.6Hz),31.79(d,J＝15.2Hz),31.43(s),29.52(d,J＝2.8Hz),29.25(s),28.92(dd,J＝14.2,5.8Hz),26.77(d,J＝4.8Hz),25.33(s),24.92(s),24.71(s),24.48(s),22.64(d,J＝6.8Hz),14.12(s).

F. Compounds SW-II-127

1. Synthesis of Compound 3

Compound 1 (1.3 g,5.86mmol,1.5 eq.) and Compound 2 (1 g,3.9mmol,1.0 eq.) are dissolved in DCM (20 mL), EDCI (1.495 g,7.8mmol,2.0 eq.) and DMAP (0.19 g,1.56mmol,0.4 eq.) are added to the mixture, followed by DIEA (2.57 mL,15.6mmol,4.0 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether/ethyl acetate=19/1) showed that compound 2 was consumed and the desired product formed. The reaction mixture was diluted with DCM (20 mL) and washed with H ₂ O (40 mL), dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-10/1) to give compound 3 (1.2 g, 66.9%) as a yellow oil.

2. Synthesis of Compound 5

Compound 3 (5.2 g,11.30mmol,1.0 eq.) and compound 4 (20.6 g, 319 mmol,30 eq.) were added to EtOH (5 mL) and the mixture was stirred under nitrogen for 16 hours at 60 ℃. TLC (petroleum ether/ethyl acetate=19/1) showed that compound 3 was consumed and TLC (DCM/meoh=10/1) showed that a new major spot was observed. The reaction mixture was concentrated under reduced pressure. The residue was diluted with EtOAc (50 mL) and washed with H ₂ O (3X 50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound 5 as a yellow oil (3 g, 60%).

¹H NMR(400MHz,CDCl ₃)δ4.95–4.75(m,1H),3.74–3.58(m,2H),2.87–2.74(m,2H),2.69–2.56(m,2H),2.36(s,2H),2.28(t,J＝7.5Hz,2H),1.65–1.42(m,8H),1.38–1.17(m,30H),0.88(t,J＝6.8Hz,6H).

3. Synthesis of Compound 8

Compound 7 (522 mg,2.5mmol,1.2 eq.) and compound 6 (400 mg,2.083mmol,1 eq.) were dissolved in DCM (4 mL), and EDCI (800 mg,4.166mmol,2 eq.) and DMAP (102 mg,0.833mmol,0.4 eq.) and DIEA (1.075 mg,8.332mmol,4 eq.) were added to the mixture. After the addition was complete, the reaction mixture was stirred overnight at room temperature under nitrogen. TLC (PE: ea=10:1) showed that the starting material was consumed and a new spot formed. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-10/1) to give compound 8 (454 mg, 57%) as a colourless oil.

4. Synthesis of SW-II-127

Compound 8 (100 mg,0.262mmol,1 eq.) and compound 5 (139 mg,0.314mmol,1.2 eq.) were dissolved in CPME/CH3CN (1 mL/1 mL), and potassium carbonate (217 mg, 1.282 mmol,6 eq.) and potassium iodide (87 mg,0.524mmol,2 eq.) were added to the mixture. After the addition was complete, the reaction mixture was stirred overnight at 90 ℃ under nitrogen. TLC (DCM/meoh=10/1) showed the reaction was complete and the desired product was formed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound SW-II-127 (42.49 mg, 22%) as a yellow oil.

LCMS:Rt:1.323min;MS m/z(ELSD):744.5[M+H] ⁺;

HPLC 99.742% purity, ELSD, rt= 7.339min.

¹H NMR(400MHz,CDCl ₃)δ7.25(s,2H),7.17(d,J＝8.0Hz,2H),5.07(s,2H),4.91–4.82(m,1H),3.83(s,2H),2.90(d,J＝44.8Hz,5H),2.64–2.55(m,2H),2.35(t,J＝7.4Hz,2H),2.28(t,J＝7.5Hz,2H),1.76–1.46(m,14H),1.42–1.19(m,41H),0.88(t,J＝6.8Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.50(d,J＝8.5Hz),133.17(s),128.61(s),128.34(s),77.29(d,J＝11.4Hz),77.03(s),76.71(s),74.23(s),66.19(s),54.20(s),35.71(s),34.56(s),34.10(d,J＝8.8Hz),31.80(d,J＝15.4Hz),31.43(s),29.53(d,J＝2.5Hz),29.25(s),28.95(d,J＝10.5Hz),28.63(s),26.71(d,J＝18.2Hz),25.33(s),24.93(s),24.62(s),22.65(d,J＝6.6Hz),14.13(s).

G. Compounds SW-II-134-1

1. Synthesis of Compound 3

To a mixture of compound 1 (500 mg,2.283mmol,1 eq.) and compound 2 (890 mg,6.849mmol,3 eq.) in toluene/water (5 mL/1 mL) were added palladium acetate (51 mg,0.228mmol,0.1 eq.) Ruphos (213 mg,0.457mmol,0.2 eq.) and potassium carbonate (945 mg,6.849mmol,3 eq.). The mixture was stirred under nitrogen at 110 ℃ overnight. TLC (PE/ea=20/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-20/1) to give compound 3 (323 mg, 99.6%) as a colourless oil.

2. Synthesis of Compound 4

Lithium aluminum hydride (2.3 mL,2.27mmol,1M in THF, 1 eq.) was added to a mixture of compound 3 (323 mg,2.27mmol,1 eq.) in THF (8 mL) at 0deg.C under nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE/ea=5/1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (2.3 mL) and treated with 2N hydrochloric acid to adjust the PH between 6 and 7, extracted with ethyl acetate and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give compound 4 (383mg, 58%) as a colourless oil without further purification.

3. Synthesis of Compound 6

To a mixture of compound 4 (383mg, 1.3mmol,1 eq.) and compound 5 (352 mg,1.6mmol,1.2 eq.) in DCM (4 mL) were added EDCI (499 mg,2.6mmol,2 eq.) and DMAP (63 mg,0.52mmol,0.4 eq.) followed by DIEA (671 mg,5.2mmol,4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether/ethyl acetate=20/1) showed formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-20/1) to give compound 6 (272 mg, 44%) as a colourless oil.

4. Synthesis of SW-II-134-1

To a mixture of compound 6 (150 mg,0.303mmol,1 eq.) and compound 7 (110 mg,0.333mmol,1.1 eq.) in CPME/CH ₃ CN (2 mL/2 mL) was added potassium carbonate (251 mg, 1.812 mmol,6 eq.) and potassium iodide (101 mg,0.61mmol,2 eq.). After addition, the mixture was stirred under nitrogen at 90 ℃ overnight. TLC (DCM/meoh=15/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound SW-II-134-1 (168 mg, 75%) as a yellow oil.

LCMS:Rt:1.276min;MS m/z(ELSD):744.4[M+H] ⁺;

HPLC 98.481% purity, ELSD, rt= 10.724min.

¹H NMR(400MHz,CDCl ₃)δ7.06(d,J＝7.6Hz,1H),7.01–6.93(m,2H),4.25(t,J＝7.3Hz,2H),4.05(t,J＝6.8Hz,2H),3.85–3.72(m,2H),2.98–2.69(m,8H),2.62–2.48(m,4H),2.29(t,J＝7.5Hz,4H),1.72–1.48(m,14H),1.45–1.17(m,36H),0.89(dt,J＝11.9,6.0Hz,9H).

¹³CNMR(101MHz,CDCl ₃)δ173.78(d,J＝16.7Hz),140.72(s),138.81(s),134.91(s), 129.70(s),129.22(s),126.19(s),77.30(d,J＝11.4Hz),77.03(s),76.72(s),65.02(s),64.49(s),57.42(s),56.36(s),54.08(s),34.76(s),34.22(d,J＝4.2Hz),32.74(s),32.36(s),31.81(d,J＝9.1Hz),31.35(d,J＝5.3Hz),29.49(d,J＝2.8Hz),29.24(d,J＝2.2Hz),28.92(s),28.66(s),26.86(s),25.93(s),25.04(s),24.78(d,J＝6.6Hz),22.65(d,J＝2.6Hz),14.10(s).

H. compounds SW-II-134-2

1. Synthesis of Compound 3

To a mixture of compound 1 (500 mg,2.283mmol,1 eq.) and compound 2 (1.08 g,6.849mmol,3 eq.) in toluene/water (5 mL/1 mL) were added palladium acetate (51 mg,0.228mmol,0.1 eq.) Ruphos (213 mg,0.457mmol,0.2 eq.) and potassium carbonate (945 mg,6.849mmol,3 eq.). The mixture was stirred under nitrogen at 110 ℃ overnight. TLC (PE/ea=20/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-20/1) to give compound 3 (254 mg, 100%) as a colourless oil.

2. Synthesis of Compound 4

Lithium aluminum hydride (2.3 mL,2.28mmol,1M in THF, 1 eq.) was added to a mixture of compound 3 (254 mg,2.28mmol,1 eq.) in THF (9 mL) at 0deg.C under nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE/ea=5/1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (2.3 mL) and treated with 2N hydrochloric acid to adjust the PH between 6 and 7, extracted with ethyl acetate and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give compound 4 (724 mg, 92%) as a colourless oil, without further purification.

3. Synthesis of Compound 6

To a mixture of compound 4 (244 mg,2.09mmol,1 eq.) and compound 5 (560 mg,2.51mmol,1.2 eq.) in DCM (8 mL) were added EDCI (803 mg,4.18mmol,2 eq.) and DMAP (102 mg,0.84mmol,0.4 eq.) followed by DIEA (1.078 g,8.36mmol,4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether/ethyl acetate=20/1) showed formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-20/1) to give compound 6 (473 mg, 41%) as a colourless oil.

4. Synthesis of SW-II-134-2

To a mixture of compound 6 (150 mg,0.27mmol,1 eq.) and compound 7 (108 mg,0.33mmol,1.1 eq.) in CPME/CH ₃ CN (2 mL/2 mL) was added potassium carbonate (225 mg,1.63mmol,6 eq.) and potassium iodide (90 mg,0.54mmol,2 eq.). After addition, the mixture was stirred under nitrogen at 90 ℃ overnight. TLC (DCM/meoh=15/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound SW-II-134-2 (71.77 mg, 33%) as a yellow oil.

LCMS:Rt:1.527min;MS m/z(ELSD):800.4[M+H] ⁺;

HPLC 97.311% purity, ELSD, rt= 9.025min.

¹H NMR(400MHz,CDCl ₃)δ7.06(d,J＝7.6Hz,1H),6.96(d,J＝9.6Hz,2H),4.25(t,J＝7.3Hz,2H),4.05(t,J＝6.8Hz,2H),3.80–3.66(m,2H),2.86(dd,J＝12.8,5.6Hz,4H),2.78–2.67(m,4H),2.60–2.52(m,4H),2.29(t,J＝7.5Hz,4H),1.57(dt,J＝15.8,7.3Hz,14H),1.30(d,J＝20.3Hz,45H),0.88(t,J＝6.7Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.82(d,J＝16.9Hz),140.73(s),138.82(s),134.91(s),129.71(s),129.23(s),126.19(s),77.36(s),77.14(d,J＝20.4Hz),76.72(s),65.03(s),64.49(s),57.57(s),56.13(s),54.02(s),34.76(s),34.25(d,J＝4.2Hz),32.76(s),32.37(s),31.89(d,J＝5.3Hz),31.40(d,J＝6.0Hz),29.84(d,J＝3.7Hz),29.63–29.14(m),28.97(s),28.65(s),26.93(s),25.66(d,J＝54.4Hz),24.80(d,J＝6.6Hz),22.68(d,J＝1.8Hz),14.12(s).

I. compounds SW-II-134-3

1. Synthesis of Compound 3

To a mixture of compound 1 (10 g,45mmol,1 eq.) and compound 2 (7.8 g,54mmol,1.2 eq.) in DCM (100 mL) were added EDCI (17.3 g,90mmol,2 eq.) and DMAP (2.2 g,18mmol,0.4 eq.) followed by DIEA (23.2 g,180mmol,4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether/ethyl acetate=30/1) showed that compound 1 was consumed and the desired product was formed. The reaction mixture was extracted with ethyl acetate (20 mL) and washed with water (40 ml×3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-20/1) to give compound 3 (4.365 g, 28%) as a colourless oil.

2. Synthesis of Compound 5

A mixture of compound 3 (5 g,14.38mmol,1 eq.) and compound 4 (8.8 g,143.7mmol,10 eq.) in ethanol (2 mL) was stirred under nitrogen at 55deg.C for 16 hours. TLC (DCM/meoh=10/1) showed that a new major spot was observed. The reaction mixture was extracted with ethyl acetate (50 mL) and washed with water (3X 50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound 5 as a yellow oil (1.008 g, 21%).

3. Synthesis of Compound 8

To a mixture of compound 6 (500 mg,2.283mmol,1 eq.) and compound 7 (699 mg,6.849mmol,3 eq.) in toluene/water (5 mL/1 mL) were added palladium acetate (51 mg,0.228mmol,0.1 eq.) Ruphos (213 mg,0.457mmol,0.2 eq.) and potassium carbonate (945 mg,6.849mmol,3 eq.). The mixture was stirred under nitrogen at 110 ℃ overnight. TLC (PE/ea=20/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-20/1) to give compound 8 (507 mg, 85%) as a colourless oil.

4. Synthesis of Compound 9

Lithium aluminum hydride (2 mL,1.935mmol,1M in THF, 1 eq.) was added to a mixture of compound 8 (507 mg,1.935mmol,1 eq.) in THF (5 mL) under nitrogen at 0deg.C. The mixture was stirred at room temperature for 3 hours. TLC (PE/ea=5/1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (2 mL) and treated with 2N hydrochloric acid to adjust the PH between 6 and 7, extracted with ethyl acetate and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give compound 9 (492 mg, > 100%) as a colourless oil, without further purification.

5. Synthesis of Compound 10

To a mixture of compound 9 (492 mg,2.103mmol,1 eq.) and compound 1 (563 mg, 2.803 mmol,1.2 eq.) in DCM (5 mL) were added EDCI (806 mg,4.206mmol,2 eq.) and DMAP (103 mg,0.84mmol,0.4 eq.) followed by DIEA (1.085 g,8.412mmol,4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether/ethyl acetate=15/1) showed formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-10/1) to give compound 10 (399 mg, 36%) as a colorless oil.

6. Synthesis of SW-II-134-3

To a mixture of compound 10 (150 mg,0.34mmol,1 eq.) and compound 5 (134 mg,0.41mmol,1.2 eq.) in CPME/CH ₃ CN (2 mL/2 mL) was added potassium carbonate (282 mg,2.04mmol,6 eq.) and potassium iodide (113 mg,0.68mmol,2 eq.). After addition, the mixture was stirred under nitrogen at 90 ℃ overnight. TLC (DCM/meoh=10/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound SW-II-134-3 (63.59 mg, 25%) as a yellow oil.

LCMS:Rt:1.247min;MS m/z(ELSD):688.3[M+H] ⁺;

HPLC 95.945% purity, ELSD, rt= 6.186min.

¹H NMR(400MHz,CDCl ₃)δ7.07(d,J＝7.6Hz,1H),6.97(dd,J＝9.9,2.2Hz,2H),4.26(t,J＝7.2Hz,2H),4.05(t,J＝6.8Hz,2H),2.88(dd,J＝14.8,7.6Hz,4H),2.78–2.74(m,2H),2.67–2.54(m,8H),2.29(t,J＝7.5Hz,4H),1.68–1.47(m,15H),1.37–1.22(m,27H),0.98–0.86(m,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.86(d,J＝17.1Hz),140.66(s),138.76(s),134.93(s),129.74(s),129.24(s),126.19(s),77.36(s),77.04(s),76.72(s),65.01(s),64.48(s),57.73(s),55.73(s),53.93(s),34.76(s),34.28(d,J＝3.9Hz),33.54(d,J＝4.5Hz),32.41(s),31.95(d,J＝16.5Hz),29.49(s),29.15(dd,J＝21.1,2.4Hz),28.66(s),27.04(s),25.95(d,J＝3.3Hz), 24.85(d,J＝6.6Hz),22.98–22.58(m),14.08(d,J＝7.5Hz).

J.SW-II-135-1

1. Synthesis of Compound 3

Compound 1 (500 mg,2.16mmol,1.0 eq.) and Compound 2 (750 mg,6.46mmol,3.0 eq.) were dissolved in toluene/H ₂ O (5 mL/1 mL), and Ruphos (201 mg,0.43mmol,0.2 eq.), pd (OAc) ₂ (48.5 mg,0.22mmol,0.1 eq.) and Cs ₂CO ₃ (2.10 g,6.46mmol,3.0 eq.) were added to the mixture. The reaction mixture was heated at 110 ℃ under nitrogen reflux for 16 hours. TLC (petroleum ether/ethyl acetate=10/1) showed the reaction was complete and the desired product was formed. The reaction mixture was washed with H ₂ O (40 mL) and extracted 3 times with EA (50 mL), and the resulting organic phase was washed twice with brine (20 mL), dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-30/1) to give compound 3 (540 mg, 82.44%) as a yellow oil.

2. Synthesis of Compound 4

To compound 3 (540 mg,1.78mmol,1.0 eq.) dissolved in THF (5 mL) was added LiAlH ₄ (3.55mL,3.55mmol,1M THF, 2 eq.) under nitrogen blanket at 0 ℃. The reaction was allowed to warm to room temperature under nitrogen and stirred for 2 hours. TLC (PE/etoac=10/1) showed the reaction was complete and a new major spot was observed. The mixture was quenched with water (10 mL), then ph=6-7 was adjusted with 1M hydrochloric acid and extracted 3 times with ethyl acetate (50 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-10/1) to give compound 4 (442 mg, 90.2%) as a colourless oil.

3. Synthesis of Compound 6

Compound 4 (442 mg,1.60mmol,1.0 eq.) and compound 5 (428.5 mg,1.92mmol,1.2 eq.) were dissolved in DCM (5 mL), EDCI (612 mg,3.2mmol,2.0 eq.) and DMAP (78.2 mg,0.64mmol,0.4 eq.) were added to the mixture, followed by DIEA (706 mg,6.4mmol,4.0 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether/ethyl acetate=10/1) showed that compound 4 was consumed and the desired product formed. The reaction mixture was washed with H ₂ O (40 mL) and extracted 3 times with EA (50 mL), and the resulting organic phase was washed twice with brine (20 mL), dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-10/1) to give compound 3 (349mg, 44.5%) as a yellow oil.

4. Synthesis of SW-II-135-1

Compound 6 (175 mg,0.365mmol,1.2 eq.) and compound 7 (100 mg,0.304mmol,1.0 eq.) were dissolved in CPME/CH ₃ CN (1 mL/1 mL) and potassium carbonate (210 mg,1.52mmol,5.0 eq.) and potassium iodide (101 mg,0.61mmol,2.0 eq.) were added to the mixture. After the addition was complete, the reaction mixture was stirred overnight at 90 ℃ under nitrogen. TLC (DCM/meoh=10/1) showed the reaction was complete and the desired product was formed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound SW-II-135-1 (83.89 mg, 55.6%) as a yellow oil.

LCMS:Rt:1.356min;MS m/z(ELSD):730.5[M+H] ⁺;

HPLC:100%purity at ELSD;RT=12.614min.

¹H NMR(400MHz,CDCl3)δ6.97(d,J＝7.6Hz,1H),6.91–6.74(m,2H),4.76(s,1H),3.99(dt,J＝13.6,6.4Hz,4H),3.72–3.58(m,2H),2.85–2.73(m,2H),2.72–2.61(m,4H),2.59–2.41(m,6H),2.22(dd,J＝13.2,7.2Hz,4H),1.93–1.79(m,2H),1.62–1.41(m,14H),1.23(d,J＝24.4Hz,32H),0.82(ddd,J＝13.6,8.0,5.6Hz,9H).

¹³C NMR(101MHz,CDCl3)δ172.81(d,J＝6.4Hz),139.55(s),137.38(s),137.14(s),128.16(d,J＝2.4Hz),124.69(s),76.51(s),76.19(s),75.88(s),63.43(s),62.78(s),56.53(s),54.90(s),52.84(s),33.23(d,J＝2.4Hz),31.73(s),31.28(s),30.91(dd,J＝20.0,6.4Hz),30.10(d,J＝3.2Hz),29.29(s),28.36(d,J＝22.8Hz),28.23(s),27.97(s),27.64(s),25.92(s),24.92(s),24.34(s),23.84(s),21.62(d,J＝7.6Hz),13.08(d,J＝4.7Hz).

K. Compounds SW-II-135-2

SW-II-135-2

1. Synthesis of Compound 3

Compound 1 (500 mg,2.16mmol,1.0 eq.) and Compound 2 (931 mg,6.46mmol,3.0 eq.) are dissolved in toluene/H ₂ O (5 mL/1 mL), and Ruphos (201 mg,0.43mmol,0.2 eq.), pd (OAc) ₂ (48.5 mg,0.22mmol,0.1 eq.) and Cs ₂CO ₃ (2.10 g,6.46mmol,3.0 eq.) are added to the mixture. The reaction mixture was heated at 110 ℃ under nitrogen reflux for 16 hours. TLC (petroleum ether/ethyl acetate=10/1) showed the reaction was complete and the desired product was formed. The reaction mixture was washed with H ₂ O (40 mL) and extracted 3 times with EA (50 mL), and the resulting organic phase was washed twice with brine (20 mL), dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-30/1) to give compound 3 (651 mg, 84%) as a yellow oil.

2. Synthesis of Compound 4

To compound 3 (651 mg,1.81mmol,1.0 eq.) dissolved in THF (7 mL) was added LiAlH ₄ (3.62 mL,3.62mmol,1m, in THF, 2 eq.) under nitrogen blanket at 0 ℃. The reaction was allowed to warm to room temperature under nitrogen and stirred for 2 hours. TLC (PE/etoac=10/1) showed the reaction was complete and a new major spot was observed. The mixture was quenched with water (10 mL), then ph=6-7 was adjusted with 1M hydrochloric acid and extracted 3 times with ethyl acetate (50 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-10/1) to give compound 4 (571 mg, 95.2%) as a colourless oil.

3. Synthesis of Compound 6

Compound 4 (571 mg,1.72mmol,1.0 eq.) and compound 5 (459 mg,2.06mmol,1.2 eq.) were dissolved in DCM (6 mL), EDCI (657 mg,3.44mmol,2.0 eq.) and DMAP (84 mg,0.68mmol,0.4 eq.) were added to the mixture, followed by DIEA (887.5 mg,6.88mmol,4.0 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether/ethyl acetate=10/1) showed that compound 4 was consumed and the desired product formed. The reaction mixture was washed with H ₂ O (50 mL) and extracted 3 times with EA (60 mL), and the resulting organic phase was washed twice with brine (25 mL), dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-10/1) to give compound 3 (248 mg, 26.5%) as a yellow oil.

4. Synthesis of SW-II-135-2

Compound 6 (245 mg, 0.458 mmol,1.5 eq.) and compound 7 (100 mg,0.3mmol,1.0 eq.) were dissolved in CPME/CH ₃ CN (1 mL/1 mL) and potassium carbonate (210 mg,1.52mmol,5.0 eq.) and potassium iodide (101 mg,0.61mmol,2.0 eq) were added to the mixture. After the addition was complete, the reaction mixture was stirred overnight at 90 ℃ under nitrogen. TLC (DCM/meoh=10/1) showed the reaction was complete and the desired product was formed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound SW-II-135-2 (31.41 mg, 21.9%) as a yellow oil.

LCMS:Rt:1.608min;MS m/z(ELSD):786.4[M+H] ⁺;

HPLC 95.16% purity, ELSD, rt= 7.919min.

¹H NMR(400MHz,CDCl ₃)δ6.98(d,J＝7.6Hz,1H),6.87(d,J＝2.4Hz,2H),4.28–4.13(m,1H),4.04–3.95(m,4H),3.94–3.84(m,2H),3.14–2.89(m,6H),2.59–2.43(m,6H),2.23(dd,J＝13.8,7.2Hz,4H),1.88–1.82(m,2H),1.70(s,4H),1.57–1.46(m,10H),1.33–1.16(m,40H),0.90–0.72(m,9H).

¹³C NMR(100MHz,CDCl ₃)δ172.82(d,J＝6.8Hz),139.61(s),137.29(d,J＝16.4Hz),128.15(s),124.67(s),76.41(s),76.09(s),75.77(s),63.50(s),62.87(s),55.49(s),54.92(s),52.98(s),33.16(d,J＝2.4Hz),31.77(s),31.33(s),30.80(d,J＝6.5Hz),30.42(d,J＝3.6Hz),29.29(s),28.99–28.66(m),28.47(s),28.23(d,J＝2.8Hz),28.06–27.45(m),25.58(s),24.91(s),23.71(s),22.79(s),21.66(s),13.10(s).

L. Compounds SW-II-136-2

SW-II-136-2

1. Synthesis of Compound 3

Compound 1 (3 g,13.70mmol,1.0 eq.) and Compound 2 (5.34 g,41.09mmol,3.0 eq.) are dissolved in toluene/H ₂ O (30 mL/3 mL), and Ruphos (1.28 g,2.74mmol,0.2 eq.), pd (OAc) ₂ (308.3 mg,1.37mmol,0.1 eq.) and K ₂CO ₃ (5.67 g,41.10mmol,3.0 eq.) are added to the mixture. The reaction mixture was heated at 110 ℃ under nitrogen reflux for 16 hours. TLC (PE/ea=10/1) showed the reaction was complete and the desired product was formed. The reaction mixture was washed with H ₂ O (90 mL) and extracted 3 times with EA (110 mL), and the resulting organic phase was washed twice with brine (40 mL), dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-30/1) to give compound 3 (1.98 g, 45.5%) as a yellow oil.

2. Synthesis of Compound 4

To compound 3 (1.98 g,6.23mmol,1.0 eq.) dissolved in THF (20 mL) was added LiAlH ₄ (1 m,12.45mL,2.0 eq.) under nitrogen blanket at 0 ℃. The reaction was allowed to warm to room temperature under nitrogen and stirred for 2 hours. TLC (PE/etoac=10/1) showed the reaction was complete and a new major spot was observed. The mixture was quenched with H ₂ O (70 mL), then ph=6-7 was adjusted with 1M hydrochloric acid and extracted 3 times with EA (80 mL). The organic layer was washed with brine, dried over anhydrous Na ₂SO ₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-10/1) to give compound 4 (1.28 g, 71.1%) as a colourless oil.

3. Synthesis of Compound 7

To compound 4 (900 g,3.1mmol,1.0 eq.) dissolved in DCM (9 mL) was added DMSO (3.63 g,51.72mmol,15 eq.), TEA (1.25 g,12.4mmol,4.0 eq.) and PySO ₃ (1.27 g,7.97mmol,2.57 eq.) under nitrogen. The mixture was stirred at 0 ℃ for 30 minutes and then warmed to room temperature under nitrogen for 90 minutes. Compound 6 (4.74 g,13.62mmol,3.0 eq.) was then added to the mixture and the reaction mixture was reacted under nitrogen for 2 hours at 25 ℃. TLC (PE/ea=10/1) showed the reaction was complete and the desired product was formed. The reaction mixture was washed with H ₂ O (60 mL) and extracted 3 times with EA (70 mL), and the resulting organic phase was washed twice with brine (40 mL), dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-10/1) to give compound 7 (345 mg, 27.9%) as a yellow oil.

4. Synthesis of Compound 8

Compound 7 (340 mg,0.95mmol,1.0 eq.) and Pd/C (100 mg) were added to MeOH (4 ml) and the reaction mixture stirred at room temperature under hydrogen protection for 16h. TLC (PE/ea=10/1) showed complete consumption of starting material and produced the desired product. The reaction mixture was filtered through celite and washed with MeOH (40 ml×2), dried over anhydrous Na ₂SO ₄ and the filtrate concentrated under reduced pressure to give compound 8 (298 mg, 88.2%) as a pale yellow oil.

5. Synthesis of Compound 9

To compound 8 (298 mg,0.83mmol,1.0 eq.) dissolved in THF (3 mL) was added LiAlH ₄ (1 m,1.66mL,2.0 eq.) under nitrogen at 0 ℃. The reaction was allowed to warm to room temperature under nitrogen and stirred for 2 hours. TLC (PE/etoac=10/1) showed the reaction was complete and a new major spot was observed. The mixture was quenched with H ₂ O (20 mL), then ph=6-7 was adjusted with 1M hydrochloric acid and extracted 3 times with EA (30 mL). The organic layer was washed with brine, dried over anhydrous Na ₂SO ₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-10/1) to give compound 9 (254 mg, 98.3%) as a colourless oil.

6. Synthesis of Compound 11

Compound 9 (254 mg,0.80mmol,1.0 eq.) and compound 10 (214 mg,0.96mmol,1.2 eq.) were dissolved in DCM (3 mL), EDCI (305.6 mg,1.6mmol,2.0 eq.) and DMAP (39 mg,0.32mmol,0.4 eq.) were added to the mixture, followed by DIEA (412.8 mg,3.2mmol,4.0 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (PE/ea=10/1) showed that compound 9 was consumed and the desired product formed. The reaction mixture was adjusted ph=4-6 with 1M hydrochloric acid and extracted 3 times with EA (30 mL), and the resulting organic phase was washed twice with brine (15 mL), dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-7/1) to give compound 11 (210 mg, 50.5%) as a yellow oil.

7. Synthesis of SW-II-136-2

Compound 11 (200 mg,0.38mmol,1.2 eq.) and compound 12 (105 mg,0.32mmol,1.0 eq.) were dissolved in CPME/CH ₃ CN (1.5 mL/1.5 mL) and K ₂CO ₃ (220.2 mg,1.60mmol,5.0 eq.) and KI (106 mg,0.64mmol,2.0 eq.) were added to the mixture. After the addition was complete, the reaction mixture was stirred overnight at 90 ℃ under nitrogen. TLC (DCM/meoh=10/1) showed the reaction was complete and the desired product was formed. The mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na ₂SO ₄, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound SW-II-136-2 (208 mg, 90.4%) as a yellow oil.

LCMS:Rt:2.146min;MS m/z(ELSD):773.3[M+H] ⁺;

HPLC 99.49% purity, ELSD, rt= 8.055min.

¹H NMR(400MHz,CDCl ₃)δ7.04(d,J＝7.6Hz,1H),6.92(d,J＝9.6Hz,2H),4.45(s,1H),4.06(dd,J＝12.0,5.2Hz,4H),3.64(t,J＝5.2Hz,2H),2.72(t,J＝5.2Hz,2H),2.65–2.50(m,10H),2.29(t,J＝7.6Hz,4H),1.69–1.48(m,18H),1.41–1.24(m,36H),0.95–0.78(m,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.86(d,J＝2.8Hz),140.48(s),139.24(s),138.01(s),129.13(d,J＝14.8Hz),125.67(s),77.37(s),77.05(s),76.73(s),64.45(s),64.23(s),57.88(s),55.91(s),53.94(s),35.07(s),34.29(d,J＝3.2Hz),32.79(s),32.35(s),31.82(d,J＝8.4Hz),31.38(s),29.50(d,J＝2.4Hz),29.16(dd,J＝18.0,2.0Hz),28.66(s),28.35(s),27.78(s),27.08(s),26.02(d,J＝17.2Hz),24.89(d,J＝1.6Hz),22.65(s),14.10(s).

M. Compounds SW-II-137-1

1. Synthesis of Compound 3

Compound 1 (500 mg,1.95mmol,1.0 eq.) was dissolved in toluene (5.0 mL) and then compound 2 (239 mg,2.34mmol,1.2 eq.) was added, pd (PPh ₃) ₄ (225 mg,0.19mmol,0.1 eq.), water (1 mL) and K ₂CO ₃ (258 g,5.85mmol,3.0 eq.) were reacted under nitrogen for 3 hours.TLC (PE/EA=5/1) indicating that the starting material had reacted and the desired product formed, reaction plus H ₂ O (70 mL), EA (80 mL. Times.3) was extracted, combined with saturated brine (2X 30 mL), dried over anhydrous Na ₂SO ₄, filtered and spin-dried.

2. Synthesis of Compound 4

Compound 3 (300 mg,1.28mmol,1.0 eq.) was dissolved in THF (4.0 mL) and LAH (97 mg,2.56mmol,2.0 eq.) was added under nitrogen. Then reacted at room temperature for 2 hours. TLC (PE/ea=10/1) showed complete reaction of starting material and formation of the desired product. Quenched with HCl (1M, 4 mL) and H ₂ O (10 mL), extracted with EA (50 mL. Times.3). The organic phase was washed with saturated brine (2×30 mL), dried over anhydrous Na ₂SO ₄, filtered and spun dry under reduced pressure. The residue was purified by a silica gel column eluting with PE/EA (1/0-10/1, v/v) to give compound 4 (224 mg, 84.8%) as a yellow oil.

3. Synthesis of Compound 6

Compound 4 (90 mg,0.47mmol,1.0 eq.) was dissolved in DCM (3.0 mL) and compound 5 (127 mg,0.56mmol,1.2 eq.) EDCI (180 mg,0.94mmol,2.0 eq.), DIEA (242 mg,1.88mmol,4.0 eq.) and DMAP (23 mg,0.18mmol,0.4 eq.) were added. Then, the reaction was carried out at room temperature under nitrogen atmosphere overnight. TLC (PE/ea=20/1) showed that starting material had been reflected and the desired product formed. The reaction was quenched with HCl (1M) solution, ph=4 to 6, and extracted with EA (40 ml×3). The organic phases were combined and washed with saturated brine (2×30 mL), dried over anhydrous Na ₂SO ₄, filtered and spun dry under reduced pressure. The residue was purified by using a silica gel column, eluting with PE/EA (1/0-20/1, v/v) to give Compound 6 (90 mg, 48.6%) as a colourless oil.

4. Synthesis of SW-II-137-1

Compound 6 (90 mg,0.25mmol,1.0 eq.) was dissolved in MeCN (2 mL) and compound 7 (110 mg,0.25mmol,1.0 eq.), KI (76 mg,0.50mmol,2.0 eq.), CPME (2 mL) and K ₂CO ₃ (157 mg,1.25mmol,5.0 eq) were added. The reaction was carried out overnight at 90℃under nitrogen. TLC (DCM/meoh=10/1) showed complete reaction of starting material and formation of the desired product. Quench with water (50 mL), extract EA (40 mL. Times.3). The organic phases were combined and washed with saturated brine (2×30 mL), dried over anhydrous Na ₂SO ₄, filtered and spun dry under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10/1, v/v) to give the compound (98 mg,52.12%, SW-II-137-1) as a yellow oil.

LCMS:Rt:1.596min;MS m/z(ELSD):758.4[M+H] ⁺;

HPLC 98.02% purity, ELSD, rt= 5.993min.

¹H NMR(400MHz,CDCl ₃)δ7.02(d,J＝8.8Hz,4H),4.92–4.71(m,1H),4.01(t,J＝6.4Hz,2H),3.78(s,1H),3.55(t,J＝5.2Hz,2H),2.76–2.40(m,10H),2.21(dd,J＝15.6,7.7Hz,4H),1.95–1.80(m,2H),1.49(ddd,J＝24.4,15.8,6.2Hz,15H),1.34–1.13(m,37H),0.82(dt,J＝13.6,7.2Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.79(s),173.57(s),140.49(s),138.30(s),128.43(s),128.22(s),77.43(s),77.11(s),76.79(s),74.11(s),63.66(s),57.96(s),55.75(s),53.90(s),35.22(s),34.63(s),34.20(d,J＝11.6Hz),33.70(s),31.80(d,J＝11.2Hz),30.30(s),29.51(d,J＝2.8Hz),29.13(dd,J＝9.6,6.8Hz),27.12(d,J＝2.8Hz),26.29(s),25.31(s),24.97(d,J＝15.6Hz),22.66(s),22.37(s),14.02(d,J＝15.2Hz) ^.

N. Compounds SW-II-137-2

1. Synthesis of Compound 3

Compound 1 (500 mg,2.06mmol,1.0 eq.), compound 2 (284 mg,2.47mmol,1.2 eq.), pd (PPh ₃) ₄ (119 mg,0.1mmol,0.1 eq.) and K ₂CO ₃ (851 mg,6.21mmol,3.0 eq.) were dissolved in toluene (5.0 mL), water (0.5 mL.) was added and then reacted under nitrogen for 3 hours at 110 ℃ for a.tlc (PE/ea=5/1) showed complete reaction of starting material and formation of the desired compound the reaction was quenched with H ₂ O (70 mL), EA (80 ml×3) was extracted, the organic phase was washed with saturated brine (2×30 mL), dried over anhydrous Na ₂SO ₄, filtered and the residue was dried under reduced pressure, purified with a silica gel column, eluting with (PE/ea=5/1, v/v) to give compound 3 (420 mg, 87.5%).

2. Synthesis of Compound 4

Compound 3 (420 mg,1.78mmol,1.0 eq.) was dissolved in THF (3.0 mL) and LAH (1M, 7mL,2.0 eq.) was added dropwise at 0deg.C under nitrogen. Then, the reaction was carried out at room temperature for 2 hours. TLC (PE/ea=5/1) showed that the starting material was reacted and the desired product was formed. Quenched with HCl (1M, 4 mL) solution and H ₂ O (10 mL), extracted with EA (50 mL. Times.3). The organic phase was washed with saturated brine (2×30 mL), dried over anhydrous Na ₂SO ₄, filtered and spun dry under reduced pressure. The residue was purified by a silica gel column and eluted with (PE/ea=5/1, v/v) to give compound 4 (320 mg, 94%) as a colorless oil.

3. Synthesis of Compound 6

Compound 4 (320 mg,1.55mmol,1.0 eq.) was dissolved in DCM (4.0 mL), and Compound 5 (416 mg,1.86mmol,1.2 eq.) EDCI (594 mg,3.11mmol,2.0 eq.), DIEA (803 mg,6.21mmol,4.0 eq.) and DMAP (76 mg,0.62mmol,0.4 eq.) were added. Then, the reaction was carried out at room temperature under nitrogen atmosphere overnight. TLC (PE/ea=20/1) showed that the starting material was reacted and the desired product was formed. The reaction was quenched with HCl (1M) solution and ph=4-6, extracted with dcm (60 ml×3). The organic phase was washed with saturated brine (2×35 mL), dried over anhydrous Na ₂SO ₄, filtered and dried under reduced pressure. The residue was purified by a silica gel column and eluted with (PE/ea=5/1, v/v) to give compound 6 (300 mg, 47.17%) as a colorless oil.

4. Synthesis of SW-II-137-2

Compound 6 (167 mg,0.41mmol,1.2 eq.), compound 7 (150 mg,0.34mmol,1.0 eq.), KI (113 mg,0.68mmol,2.0 eq.) and CPME (2 mL) were dissolved in MeCN (2 mL) and K ₂CO ₃ (235 mg,1.70mmol,5.0 eq.) was added. The reaction was carried out overnight at 90℃under nitrogen. TLC (DCM/meoh=10/1) showed complete reaction of starting material and formation of desired product. The reaction was quenched with water (50 mL) and extracted with EA (60 mL x 3). The organic phase was dried over anhydrous Na ₂SO ₄, filtered and dried under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10/1, v/v) to give the compound (105 mg,40.3%, SW-II-137-2) as a pale yellow oil.

LCMS:Rt:1.660min;MS m/z(ELSD):772.4[M+H] ⁺;

HPLC 98.38% purity, ELSD, rt= 8.743min.

¹H NMR(400MHz,CDCl ₃)δ7.10(d,J＝8.8Hz,4H),5.04–4.74(m,1H),4.08(t,J＝6.4Hz,2H),3.58(t,J＝5.2Hz,2H),2.65(dd,J＝9.6,5.6Hz,4H),2.60–2.44(m,6H),2.29(dd,J＝16.4,7.6Hz,4H),2.01–1.88(m,2H),1.59(dt,J＝9.2,7.2Hz,6H),1.54–1.42(m, 8H),1.39–1.11(m,41H),0.88(dt,J＝11.8,6.0Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.86(s),173.63(s),140.59(s),138.34(s),128.45(s),128.24(s),77.36(s),77.04(s),76.72(s),74.14(s),63.69(s),58.11(s),55.71(s),53.90(s),35.53(s),34.68(s),34.23(d,J＝14.8Hz),31.82(d,J＝11.6Hz),31.56(s),31.26(s),30.32(s),29.53(d,J＝2.8Hz),29.19(dd,J＝8.0,4.4Hz),27.20(d,J＝2.4Hz),26.64(s),25.33(s),25.02(d,J＝15.6Hz),22.62(d,J＝11.6Hz),14.08(d,J＝8.0Hz).

O. Compound SW-II-137-3

1. Synthesis of Compound 3

To a mixture of compound 1 (11.8 g,53mmol,1.2 eq.) and compound 2 (11.2 g,44mmol,1 eq.) in DCM (110 mL) were added EDCI (16.9 g,88mmol,2 eq.) and DMAP (2.1 g,18mmol,0.4 eq.) followed by DIEA (22.7 g,176mmol,4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether/ethyl acetate=30/1) showed that compound 1 was consumed and the desired product was formed. The reaction mixture was extracted with ethyl acetate (200 mL) and washed with water (200 ml×3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-20/1) to give compound 3 (7.3991 g, 37%) as a colourless oil.

2. Synthesis of Compound 5

A mixture of compound 3 (7.399mg, 16.07mmol,1 eq.) and compound 4 (29.4 g,482.02mmol,30 eq.) in ethanol (2 mL) was stirred under nitrogen at 55deg.C for 16 hours. TLC (DCM/meoh=10/1) showed that a new major spot was observed. The reaction mixture was extracted with ethyl acetate (100 mL) and washed with water (3X 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound 5 as a yellow oil (3.695 g, 52%).

3. Synthesis of Compound 8

To a mixture of compound 6 (1 g,4.12mmol,1 eq.) and compound 7 (803 g,6.17mmol,1.5 eq.) in 1, 4-dioxane/water (10 mL/1 mL) was added Pd (dtbpf) Cl ₂ (399 mg,0.41mmol,0.1 eq.) and potassium carbonate (1.7 g,12.36mmol,3 eq). The mixture was stirred under nitrogen at 100 ℃ overnight. TLC (PE/ea=20/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-20/1) to give compound 8 (218 mg, 56%) as a colourless oil.

4. Synthesis of Compound 9

Lithium aluminum hydride (2.3 mL,2.29mmol,1M in THF, 1 eq.) was added to a mixture of compound 8 (618 mg,2.29mmol,1 eq.) in THF (6 mL) at 0deg.C under nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE/ea=5/1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (2.3 mL) and treated with 2N hydrochloric acid to adjust the PH between 6 and 7, extracted with ethyl acetate and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give compound 9 (541 mg, > 100%) as a colourless oil, without further purification.

5. Synthesis of Compound 10

To a mixture of compound 9 (441 mg,2mmol,1 eq.) and compound 1 (536 mg,2.4mmol,1.2 eq.) in DCM (5 mL) were added EDCI (768 mg,4mmol,2 eq.) and DMAP (98 mg,0.8mmol,0.4 eq.) followed by DIEA (1.032 g,8mmol,4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether/ethyl acetate=10/1) showed formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-10/1) to give compound 10 (372 mg, 44%) as a colourless oil.

6. Synthesis of SW-II-137-3

To a mixture of compound 10 (150 mg,0.353mmol,1 eq.) and compound 5 (156 mg, 0.356 mmol,1 eq.) in CPME/CH ₃ CN (2 mL/2 mL) was added potassium carbonate (244 mg,1.765mmol,6 eq.) and potassium iodide (117 mg,0.706mmol,2 eq.). After addition, the mixture was stirred under nitrogen at 90 ℃ overnight. TLC (DCM/meoh=10/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound SW-II-137-3 (56.17 mg, 20%) as a yellow oil.

LCMS:Rt:1.550min;MS m/z(ELSD):786.4[M+H] ⁺;

HPLC 98.597% purity, ELSD, rt= 13.153min.

¹H NMR(400MHz,CDCl ₃)δ7.09(s,4H),4.92–4.78(m,1H),4.08(t,J＝6.6Hz,2H),3.62(t,J＝5.2Hz,2H),2.78–2.50(m,10H),2.35–2.22(m,4H),2.00–1.88(m,2H),1.57(ddd,J＝28.9,13.5,4.5Hz,14H),1.38–1.20(m,42H),0.88(t,J＝6.8Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.83(s),173.60(s),140.60(s),138.33(s),128.44(s),128.24(s),77.36(s),77.04(s),76.72(s),74.15(s),63.69(s),57.95(s),55.83(s),53.95(s),35.56(s),34.65(s),34.21(d,J＝13.0Hz),31.81(d,J＝12.3Hz),31.54(s),30.32(s),29.52(d,J＝3.1Hz),29.34–28.94(m),27.13(d,J＝2.5Hz),26.31(s),25.33(s),24.99(d,J＝15.7Hz),22.64(d,J＝5.7Hz),14.11(s).

P. Compounds SW-II-138-1

1. Synthesis of Compound 2

Compound 1 (4 g,16.46mmol,1.0 eq.) is dissolved in MeOH (40 mL), cooled to 0deg.C and SOCl ₂ (3.9 g,32.92mmol,2.0 eq.) is added dropwise. Then, the reaction was carried out at room temperature for 1 hour. TLC (PE/ea=5/1) showed that the starting material was consumed and the desired product formed. The system was dried directly under reduced pressure, and NaHCO ₃ (70 mL) solution was added to the residue, which was extracted with EA (80 mL. Times.3). The organic phases were combined and washed with saturated brine (2×30 mL), dried over anhydrous Na ₂SO ₄, filtered and spun dry under reduced pressure. The residue was purified by column chromatography on silica gel eluting with PE/EA (1/0-5:1, v/v) to give compound 2 (4.1 mg, 95%) as a yellow oil.

2. Synthesis of Compound 4

Compound 2 (500 mg,1.95mmol,1.0 eq.) compound 3 (239 mg,2.34mmol,1.2 eq.) Pd (PPh ₃) ₄ (225 mg,0.19mmol,0.1 eq.) and K ₂CO ₃ (806 g,5.85mmol,3.0 eq.) were dissolved in toluene (5.0 mL.) and reacted with water (1 mL.) then under nitrogen for 3 hours.TLC (PE/EA=5/1) showed complete consumption of starting material and formation of the desired product reaction was quenched with H ₂ O (70 mL.) EA (80 mL. Times.3) was extracted, the organic phases were combined and washed with saturated brine (2X 30 mL), dried over anhydrous Na ₂SO ₄, filtered and the residue was dried under reduced pressure using a silica gel column, eluting with PE/EA (1/0-5:1, v/v) to give compound 4 (320 mg, 70%) as a yellow oil.

3. Synthesis of Compound 5

Compound 4 (300 mg,1.28mmol,1.0 eq.) was dissolved in THF (4.0 mL) and LAH (97 mg,2.56mmol,2.0 eq.) was added at 0deg.C. Then the reaction was carried out at room temperature for 2 hours under nitrogen protection. TLC (PE/ea=5/1) showed that the starting material was consumed and the desired product formed. The reaction was quenched with HCl (1M, 4 mL) and H ₂ O (10 mL) and extracted with EA (50 mL. Times.3). The organic phase was washed with saturated brine (2×30 mL), dried over anhydrous Na ₂SO ₄, filtered and dried under reduced pressure. The residue was purified by column chromatography on silica gel eluting with PE/EA (1/0-5:1, v/v) to give compound 5 (224 mg, 84.8%) as a yellow oil.

4. Synthesis of Compound 7

Compound 7 (224 mg,1.09mmol,1.0 eq.) was dissolved in DCM (3.0 mL) and compound 6 (290 mg,1.30mmol,1.2 eq.) EDCI (418 mg,2.17mmol,2.0 eq.), DIEA (560 mg,4.35mmol,4.0 eq.) and DMAP (53 mg,0.43mmol,0.4 eq.) were added. Then, the reaction was carried out at room temperature under nitrogen atmosphere overnight. TLC (PE/ea=30/1) showed that the starting material was consumed and the desired product formed. The reaction was quenched with HCl (1M) solution and ph=4-6 was adjusted and extracted with DCM (80 ml×3). The combined organic phases were washed with saturated brine (2×30 mL), dried over anhydrous Na ₂SO ₄, filtered and dried under reduced pressure. The residue was purified by a silica gel column eluting with PE/EA (1/0-30:1, v/v) to give compound 7 (208 mg, 46.7%) as a colourless oil.

5. Synthesis of SW-II-138-1

Compound 10 (110 mg,0.25mmol,1 eq.), compound 7 (153 mg,0.37mmol,1.5 eq.), KI (83 mg, 0.50mmol,2.0 eq.) and CPME (2 mL) were dissolved in MeCN (2 mL) plus K ₂CO ₃ (172 mg,1.25mmol,5.0 eq). The reaction was carried out overnight at 90℃under nitrogen. TLC (DCM/meoh=10/1) showed complete consumption of starting material and formation of the desired product. The reaction was directly dried under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give the compound as a pale yellow oil (65 mg,32%, SW-II-138-1).

LCMS:Rt:1.684min;MS m/z(ELSD):772.4[M+H] ⁺;

HPLC 96.56% purity, ELSD, rt= 6.346min.

¹H NMR(400MHz,CDCl ₃)δ7.09(s,4H),4.86(s,1H),4.09(d,J＝6.0Hz,2H),3.97(s, 2H),3.07(d,J＝38.8Hz,6H),2.69–2.51(m,4H),2.28(td,J＝7.3,3.6Hz,4H),1.79(s,4H),1.70–1.46(m,16H),1.42–1.17(m,37H),0.90(dt,J＝13.6,7.2Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.80(s),173.53(s),140.32(s),139.13(s),128.28(d,J＝13.6Hz),77.43(s),77.11(s),76.80(s),74.21(s),64.22(s),56.85(s),55.98(s),53.93(s),35.22(s),35.01(s),34.54(s),34.14(d,J＝5.6Hz),33.71(s),31.85(s),29.50(d,J＝2.8Hz),29.22(s),29.12–28.60(m),28.26(s),27.78(s),26.70(d,J＝4.4Hz),25.31(s),24.82(d,J＝17.6Hz),24.28(s),22.65(s),22.37(s),14.03(d,J＝15.2Hz).

Q.SW-II-138-2

1. Synthesis of Compound 3

Compound 1 (500 mg,1.95mmol,1.0 eq.) compound 2 (271 mg,2.34mmol,1.2 eq.) Pd (PPh ₃) ₄ (225 mg,0.20mmol,0.1 eq.) and K ₂CO ₃ (809 g,5.86mmol,3.0 eq.) were dissolved in toluene (5.0 mL) and water (1 mL) was added, then reacted under nitrogen for 3 hours at 110℃TLC (PE/EA=5/1) showing the starting material was consumed and the desired product formed, quenched with water (70 mL), extracted with EA (80 mL. Times.3), the organic phases were combined and washed with saturated brine (2X 30 mL), dried over anhydrous Na ₂SO ₄, filtered and dried under reduced pressure, the residue was purified on a silica gel column eluting with PE/EA (1/0-30:1, v/v) to give compound 3 (320 mg, 70%) as a colourless oil.

2. Synthesis of Compound 4

Compound 3 (320 mg,1.29mmol,1.0 eq.) was dissolved in THF (3.0 mL), LAH (67 mg,1.77mmol,2.0 eq.) was added at 0deg.C, and then reacted at room temperature under nitrogen for 2 hours. TLC (PE/ea=5/1) showed that the starting material was consumed and the desired product formed. The reaction was quenched with HCl (1M, 2 mL) solution and H ₂ O (10 mL), and extracted with EA (50 mL. Times.3). The combined organic phases were washed with saturated brine (2×30 mL), dried over anhydrous Na ₂SO ₄, filtered and dried under reduced pressure. The residue was purified by a silica gel column eluting with PE/EA (1/0-30:1, v/v) to give Compound 4 (180 mg, 64%) as a colourless oil.

3. Synthesis of Compound 6

Compound 4 (180 mg,0.82mmol,1.0 eq.) was dissolved in DCM (3.0 mL) and compound 5 (245 mg, 1.10mmol,1.2 eq.), EDCI (457 mg,1.82mmol,2.0 eq.), DIEA (470 mg,3.63mmol,4.0 eq.) and DMAP (45 mg,0.36mmol,0.4 eq.) were added. The reaction was then allowed to stand at room temperature overnight under nitrogen. TLC (PE/ea=30/1) showed complete consumption of starting material and formation of the desired product. The reaction was quenched with HCl (1M) solution and ph=5-6 was adjusted and extracted with DCM (80 ml×3). The combined organic phases were washed with saturated brine (2×30 mL), dried over anhydrous Na ₂SO ₄, filtered and dried under reduced pressure. The residue was purified by a silica gel column eluting with PE/EA (1/0-30:1, v/v) to give Compound 6 (220 mg, 63.6%) as a colourless oil.

4. Synthesis of SW-II-138-2

Compound 6 (158 mg,0.37mmol,1.5 eq.) and compound 7 (110 mg,0.25mmol,1.0 eq.), KI (83 mg,0.50mmol,2.0 eq.) and CPME (2 mL) were dissolved in MeCN (2 mL) and K ₂CO ₃ (172 mg,1.25mmol,5.0 eq.) was added. The reaction was then allowed to react overnight at 90 ℃ under nitrogen. TLC (DCM/meoh=10/1) showed complete consumption of starting material and formation of the desired product. The reaction was directly dried under reduced pressure and the residue was purified on a silica gel column eluting with DCM/MeOH (1/0-10:1, v/v) to give the desired product (100 mg,51%, SW-II-138-2) as a colourless oil.

LCMS:Rt:1.834min;MS m/z(ELSD):786.4[M+H] ⁺;

HPLC 99.20% purity, ELSD, rt= 7.990min.

¹H NMR(400MHz,CDCl ₃)δ7.00(s,4H),4.88–4.73(m,2H),4.00(t,J＝5.6Hz,2H),3.81–3.54(m,2H),3.00–2.81(m,2H),2.81–2.65(m,4H),2.50(dd,J＝16.4,8.4Hz,4H),2.20(td,J＝7.6,3.2Hz,4H),1.56(ddd,J＝18.4,10.4,5.2Hz,13H),1.43(d,J＝5.6Hz,4H),1.34–1.07(m,40H),0.81(dt,J＝11.2,5.6Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.78(s),173.52(s),140.32(s),139.11(s),128.25(d,J＝11.6Hz),77.49(s),77.17(s),76.85(s),74.14(s),64.17(s),57.25(s),55.82(s),53.85(s),35.50(s),35.01(s),34.56(s),34.14(d,J＝7.2Hz),31.84(s),31.53(s),31.23(s),29.49(d,J＝2.8Hz),29.21(s),28.94(dd,J＝6.4,4.4Hz),28.25(s),27.77(s),26.84(d,J＝4.4Hz),25.30(s),25.25–24.59(m),22.59(d,J＝11.2Hz),14.05(d,J＝7.6Hz).

R.SW-II-138-3

1. Synthesis of Compound 3

Compound 1 (500 mg,1.95mmol,1.0 eq.) compound 2 (305 mg,2.34mmol,1.2 eq.) Pd (PPh ₃) ₄ (225 mg,0.20mmol,0.1 eq.) and K ₂CO ₃ (809 g,5.86mmol,3.0 eq.) were dissolved in toluene (5.0 mL.) then the reaction was reacted under nitrogen for 3 hours at 110 ℃ after which time TLC (PE/ea=5/1) showed complete consumption of starting material and formed the desired compound, the reaction was quenched with water (80 mL) and extracted with EA (80 ml×3), the organic phases were combined and washed with saturated brine (2×40 mL), dried over anhydrous Na ₂SO ₄, filtered and the residue was dried under reduced pressure with a silica gel column eluting with PE/EA (1/0-5:1, v/v) to give compound 3 (260 mg, 51.3%) as a colorless oil.

2. Synthesis of Compound 4

Compound 3 (260 mg,0.99mmol,1.0 eq.) was dissolved in THF (4.0 mL) and LAH (75 mg,1.98mmol,2.0 eq.) was added at 0deg.C. Then, the reaction was carried out at room temperature under nitrogen atmosphere for 2 hours. TLC (PE/ea=5/1) showed that the starting material was reacted and the desired compound was formed. The reaction was quenched with HCl (1M, 4 mL) solution and H ₂ O (20 mL), and extracted with EA (50 mL. Times.3). The combined organic phases were washed with saturated brine (2×30 mL), dried over anhydrous Na ₂SO ₄, filtered and spun dry under reduced pressure. The residue was purified by a silica gel column eluting with PE/EA (1/0-5:1, v/v) to give Compound 4 (230 mg, 98%) as a colourless oil.

3. Synthesis of Compound 6

Compound 4 (240 mg,1.03mmol,1.0 eq.) was dissolved in DCM (4.0 mL) and compound 5 (275 mg,1.23mmol,1.2 eq.), EDCI (390 mg,2.07mmol,2.0 eq.), DIEA (530 mg,4.10mmol,4.0 eq.) and DMAP (50 mg,0.41mmol,0.4 eq.) were added sequentially. Then the reaction was carried out overnight at room temperature under nitrogen. TLC (PE/ea=20/1) showed that the starting material was consumed and the desired compound was formed. The reaction was quenched with HCl (1M) and ph=5-6 was adjusted and extracted with dcm (80 ml×3). The combined organic phases were washed with saturated brine (2×30 mL), dried over anhydrous Na ₂SO ₄, filtered and spun dry under reduced pressure. The residue was purified by a silica gel column eluting with PE/EA (1/0-20:1, v/v) to give Compound 6 (180 mg, 40.9%) as a colourless oil.

4. Synthesis of SW-II-138-3

Compound 6 (164 mg,0.37mmol,1 eq.) and compound 7 (110 mg,0.24mmol,1.0 eq.), KI (83 mg,0.49mmol,2.0 eq.) and CPME (2 mL) were dissolved in MeCN (2 mL) and K ₂CO ₃ (172 mg,1.24mmol,5.0 eq.) was added. Then, the reaction was carried out at 90℃overnight under nitrogen. TLC (DCM/meoh=10/1) showed the starting material was consumed and the desired product formed. The reaction was directly dried under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give the desired product (108 mg,52.76%, SW-II-138-3) as a colourless oil.

LCMS:Rt:2.007min;MS m/z(ELSD):800.4[M+H] ⁺;

HPLC 97.95% purity, ELSD, rt= 9.455min.

¹H NMR(400MHz,CDCl ₃)δ7.08(s,4H),4.86(p,J＝6.4Hz,1H),4.08(s,2H),3.60(t,J＝5.2Hz,3H),2.76–2.42(m,10H),2.28(td,J＝7.6,2.8Hz,4H),1.70–1.42(m,18H),1.28(d,J＝20.0Hz,41H),0.88(t,J＝6.8Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.85(s),173.59(s),140.39(s),139.15(s),128.27(d,J＝12.0Hz),77.38(s),77.07(s),76.75(s),74.13(s),64.17(s),58.06(s),55.75(s),53.92(s),35.57(s),35.03(s),34.66(s),34.22(d,J＝13.2Hz),31.81(d,J＝12.4Hz),31.54(s),29.52(d,J＝2.9Hz),29.34–28.95(m),28.29(s),27.79(s),27.16(d,J＝3.6Hz),26.50(s),25.32(s),24.99(d,J＝17.6Hz),22.64(d,J＝5.6Hz),14.10(s).

S. Compounds SW-II-139-1

1. Synthesis of Compound 3

To a mixture of compound 1 (1 g,4.37mmol,1 eq.) and compound 2 (412 g,6.55mmol,1.5 eq.) in 1, 4-dioxane/water (10 mL/1 mL) was added Pd (dtbpf) Cl ₂ (284 mg,0.437mmol,0.1 eq.) and potassium carbonate (1.8 g,13.11mmol,3 eq.). The mixture was stirred under nitrogen at 100 ℃ overnight. TLC (PE/ea=20/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-20/1) to give compound 3 (691 mg, 68%) as a colourless oil.

2. Synthesis of Compound 4

Lithium aluminum hydride (3 mL,2.95mmol,1M in THF, 1 eq.) was added to a mixture of compound 3 (691 mg,2.95mmol,1 eq.) in THF (7 mL) at 0deg.C under nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE/ea=5/1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (3 mL) and treated with 2N hydrochloric acid to adjust the PH between 6 and 7, extracted with ethyl acetate and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give compound 4 (547 mg, 90%) as a colourless oil without further purification.

3. Synthesis of Compound 6

To a mixture of compound 4 (447 mg,2.17mmol,1 eq.) and compound 5 (581 mg,2.6mmol,1.2 eq.) in DCM (5 mL) were added EDCI (833 mg,4.34mmol,2 eq.) and DMAP (106 mg,0.87mmol,0.4 eq.) followed by DIEA (1.12 g,8.68mmol,4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether/ethyl acetate=15/1) showed formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-20/1) to give compound 6 (45 mg, 51%) as a colourless oil.

4. Synthesis of SW-II-139-1

To a mixture of compound 6 (150 mg,0.365mmol,1 eq.) and compound 7 (161 mg,0.365mmol,1 eq.) in CPME/CH ₃ CN (2 mL/2 mL) was added potassium carbonate (252 mg, 1.823mmol, 6 eq.) and potassium iodide (121 mg,0.73mmol,2 eq.). After addition, the mixture was stirred under nitrogen at 90 ℃ overnight. TLC (DCM/meoh=10/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound SW-II-139-1 (54.53 mg, 19%) as a yellow oil.

LCMS:Rt:1.521min;MS m/z(ELSD):772.4[M+H] ⁺;

HPLC 99.637% purity, ELSD, rt= 12.347min.

¹H NMR(400MHz,CDCl ₃)δ7.20(t,J＝7.7Hz,1H),7.03(t,J＝6.8Hz,3H),4.94–4.78(m,1H),4.27(t,J＝7.2Hz,2H),3.65(t,J＝5.1Hz,2H),2.90(t,J＝7.2Hz,2H),2.73(t,J＝4.9Hz,2H),2.67–2.41(m,6H),2.28(td,J＝7.5,2.7Hz,4H),1.67–1.45(m,14H),1.41–1.19(m,42H),0.88(dd,J＝7.9,5.7Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.65(d,J＝11.3Hz),143.17(s),137.67(s),129.04(s),128.34(s),126.61(s),126.11(s),77.30(d,J＝11.6Hz),77.04(s),76.72(s),74.16(s),64.85(s),57.88(s),55.93(s),53.97(s),35.94(s),35.13(s),34.64(s),34.20(d,J＝10.5Hz),31.80(d,J＝13.7Hz),31.50(s),29.52(d,J＝2.9Hz),29.34–28.92(m),27.08(d,J＝3.9Hz),26.10(s),25.33(s),25.05(s),24.82(s),22.64(d,J＝6.5Hz),14.11(s).

T, compounds SW-II-139-2

1. Synthesis of Compound 3

To a mixture of compound 1 (1 g,4.37mmol,1 eq.) and compound 2 (668 g,6.55mmol,1.5 eq.) in 1, 4-dioxane/water (10 mL/1 mL) was added Pd (dtbpf) Cl ₂ (284 mg,0.437mmol,0.1 eq.) and potassium carbonate (1.8 g,13.11mmol,3 eq). The mixture was stirred under nitrogen at 100 ℃ overnight. TLC (PE/ea=20/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-20/1) to give compound 3 (605 mg, 67%) as a colourless oil.

2. Synthesis of Compound 4

Lithium aluminum hydride (3 mL,2.94mmol,1M in THF, 1 eq.) was added to a mixture of compound 3 (605 mg,2.94mmol,1 eq.) in THF (7 mL) at 0deg.C under nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE/ea=5/1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (3 mL) and treated with 2N hydrochloric acid to adjust the PH between 6 and 7, extracted with ethyl acetate and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give compound 4 (534 mg, > 100%) as a colourless oil, without further purification.

3. Synthesis of Compound 6

To a mixture of compound 4 (433 mg,2.44mmol,1 eq.) and compound 5 (652 mg,2.93mmol,1.2 eq.) in DCM (5 mL) were added EDCI (937 mg,4.88mmol,2 eq.) and DMAP (119 mg,0.976mmol,0.4 eq.) followed by DIEA (1.399 g,9.76mmol,4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether/ethyl acetate=15/1) showed formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-20/1) to give compound 6 (355 mg, 38%) as a colourless oil.

4. Synthesis of SW-II-139-2

To a mixture of compound 6 (122 mg,0.319mmol,1 eq.) and compound 7 (140 mg,0.319mmol,1 eq.) in CPME/CH ₃ CN (2 mL/2 mL) was added potassium carbonate (220 mg,1.595mmol,5 eq.) and potassium iodide (106 mg, 0.428 mmol,2 eq.). After addition, the mixture was stirred under nitrogen at 90 ℃ overnight. TLC (DCM/meoh=10/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound SW-II-139-2 (45.48 mg, 19%) as a yellow oil.

LCMS:Rt:1.346min;MS m/z(ELSD):744.3[M+H] ⁺;

HPLC 97.994% purity, ELSD, rt= 11.235min.

¹H NMR(400MHz,CDCl ₃)δ7.20(t,J＝7.8Hz,1H),7.03(t,J＝7.6Hz,3H),4.91–4.81(m,1H),4.27(t,J＝7.2Hz,2H),3.89–3.75(m,2H),2.99–2.79(m,7H),2.64–2.48(m,2H),2.28(td,J＝7.5,3.1Hz,4H),1.74–1.08(m,53H),0.90(dt,J＝13.6,7.2Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.60(d,J＝11.7Hz),143.13(s),137.65(s),129.06(s),128.34(s),126.64(s),126.11(s),77.30(d,J＝11.4Hz),77.04(s),76.72(s),74.22(s),64.88(s),57.28(s),56.55(s),54.11(s),35.60(s),35.12(s),34.56(s),34.15(d,J＝4.0Hz),33.68(s),31.86(s),29.52(d,J＝2.8Hz),29.24(s),28.91(dd,J＝7.0,4.2Hz),26.81(d,J＝3.9Hz),25.33(s),25.12–24.98(m),24.83(d,J＝22.2Hz),22.67(s),22.40(s),14.04(d,J＝14.4Hz).

U.Compounds SW-II-140-1

1. Synthesis of Compound 3

To a mixture of compound 1 (1 g,4.37mmol,1 eq.) and compound 2 (412 g,6.55mmol,1.5 eq.) in 1, 4-dioxane/water (10 mL/1 mL) was added Pd (dppf) Cl ₂ (284 mg,0.437mmol,0.1 eq.) and potassium carbonate (1.8 g,13.11mmol,3 eq). The mixture was stirred under nitrogen at 100 ℃ overnight. TLC (PE/ea=20/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-20/1) to give compound 3 (748 mg, 73%) as a colourless oil.

2. Synthesis of Compound 4

Lithium aluminum hydride (3.2 mL,3.2mmol,1M in THF, 1 eq.) was added to a mixture of compound 3 (748 mg,3.2mmol,1 eq.) in THF (8 mL) at 0deg.C under nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE/ea=5/1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (3 mL) and treated with 2N hydrochloric acid to adjust the PH between 6 and 7, extracted with ethyl acetate and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give compound 4 (493 mg, 75%) as a colourless oil without further purification.

3. Synthesis of Compound 6

To a mixture of compound 4 (393 mg,1.91mmol,1 eq.) and compound 5 (511 mg,2.29mmol,1.2 eq.) in DCM (5 mL) were added EDCI (733 mg,3.82mmol,2 eq.) and DMAP (93 mg,0.76mmol,0.4 eq.) followed by DIEA (986 mg,7.64mmol,4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether/ethyl acetate=15/1) showed formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-20/1) to give compound 6 (327 mg, 42%) as a colourless oil.

4. Synthesis of SW-II-140-1

To a mixture of compound 6 (150 mg,0.365mmol,1 eq.) and compound 7 (161 mg,0.365mmol,1 eq.) in CPME/CH ₃ CN (2 mL/2 mL) was added potassium carbonate (302 mg,2.19mmol,6 eq.) and potassium iodide (121 mg,0.73mmol,2 eq.). After addition, the mixture was stirred under nitrogen at 90 ℃ overnight. TLC (DCM/meoh=10/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound SW-II-140-1 as a yellow oil (180 mg, 64%).

LCMS:Rt:1.568min;MS m/z(ELSD):772.4[M+H] ⁺;

HPLC 98.053% purity, ELSD, rt=8.702 min.

¹H NMR(400MHz,CDCl ₃)δ7.23–7.05(m,4H),4.95–4.79(m,1H),4.25(t,J＝7.4Hz,2H),3.62(t,J＝4.8Hz,2H),2.96(dd,J＝15.4,8.0Hz,2H),2.74–2.49(m,8H),2.28(dd,J＝14.2,7.2Hz,4H),1.67–1.44(m,14H),1.41–1.20(m,42H),0.90(dt,J＝13.2,7.1Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.68(d,J＝10.2Hz),141.26(s),135.23(s),129.73(s),129.37(s),126.72(s),125.92(s),77.35(s),77.03(s),76.71(s),74.17(s),64.52(s),57.99(s),55.87(s),53.94(s),34.66(s),34.21(d,J＝11.5Hz),32.75(s),31.83(d,J＝9.8Hz),31.32(s),29.65–28.88(m),27.15(d,J＝3.7Hz),26.35(s),25.33(s),25.07(s),24.83(s),22.66(d,J＝3.4Hz),14.12(s).

V.SW-II-140-2

1. Synthesis of Compound 3

To a mixture of compound 1 (1 g,4.37mmol,1 eq.) and compound 2 (668 g,6.55mmol,1.5 eq.) in 1, 4-dioxane/water (10 mL/1 mL) was added Pd (dppf) Cl ₂ (284 mg,0.437mmol,0.1 eq.) and potassium carbonate (1.8 g,13.11mmol,3 eq). The mixture was stirred under nitrogen at 100 ℃ overnight. TLC (PE/ea=20/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE/EA (1/0-20/1) to give compound 3 (406 mg, 45%) as a colourless oil.

2. Synthesis of Compound 4

Lithium aluminum hydride (2 mL,1.97mmol,1M in THF, 1 eq.) was added to a mixture of compound 3 (406 mg,1.97mmol,1 eq.) in THF (5 mL) at 0deg.C under nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE/ea=5/1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (2 mL) and treated with 2N hydrochloric acid to adjust the PH between 6 and 7, extracted with ethyl acetate and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give compound 4 (3411 mg, 97%) as a colourless oil without further purification.

3. Synthesis of Compound 6

To a mixture of compound 4 (241 mg,1.35mmol,1 eq.) and compound 5 (361 mg,1.62mmol,1.2 eq.) in DCM (3 mL) were added EDCI (518 mg,2.7mmol,2 eq.) and DMAP (66 mg,0.54mmol,0.4 eq.) followed by DIEA (697 mg,5.4mmol,4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether/ethyl acetate=15/1) showed formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (1/0-20/1) to give compound 6 (185 mg, 32%) as a colourless oil.

4. Synthesis of SW-II-140-2

To a mixture of compound 6 (185 mg, 0.4813 mmol,1 eq.) and compound 7 (213 mg, 0.4813 mmol,1 eq.) in CPME/CH ₃ CN (2 mL/2 mL) was added potassium carbonate (400 mg,2.898mmol,6 eq.) and potassium iodide (160 mg,0.966mmol,2 eq.). After addition, the mixture was stirred under nitrogen at 90 ℃ overnight. TLC (DCM/meoh=10/1) showed the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with DCM/MeOH (1/0-10:1, v/v) to give compound SW-II-140-2 as a yellow oil (161 mg, 45%).

LCMS:Rt:1.696min;MS m/z(ELSD):744.3[M+H] ⁺;

HPLC 94.658% purity, ELSD, rt= 5.938min.

¹H NMR(400MHz,CDCl ₃)δ7.22–7.03(m,4H),4.94–4.78(m,1H),4.25(t,J＝7.3Hz,2H),3.70–3.54(m,2H),2.96(t,J＝7.4Hz,2H),2.77–2.41(m,8H),2.28(dd,J＝14.3,7.1Hz,4H),1.65–1.18(m,52H),0.91(dt,J＝13.3,7.1Hz,9H).

¹³C NMR(101MHz,CDCl ₃)δ173.67(d,J＝10.8Hz),141.22(s),135.23(s),129.73(s),129.39(s),126.72(s),125.92(s),77.36(s),77.04(s),76.72(s),74.17(s),64.52(s),57.92(s),55.92(s),53.96(s),34.66(s),34.21(d,J＝11.2Hz),33.51(s),32.44(s),31.83(d,J＝9.3Hz),29.53(d,J＝2.9Hz),29.14(dd,J＝11.3,8.5Hz),27.12(d,J＝4.1Hz),26.23(s),25.33(s),25.06(s),24.82(s),22.73(d,J＝9.9Hz),14.08(d,J＝8.8Hz).

EXAMPLE 2 Gene cloning of plasmids containing different 3' -UTR elements and reference control plasmids

The plasmids containing the different 3' -UTR elements were obtained by cloning the different 3' -UTR elements (sequences shown in SEQ ID NOS: 1-43) into the middle region of the reporter firefly luciferase (Firefly Luciferase) downstream termination codon and poly (A) sequence, i.e., the 3' -UTR region shown in FIG. 1, by homologous recombination, the remainder being identical. A plasmid containing only the poly (A) sequence after the stop codon downstream of firefly luciferase and no 3' -UTR element tested was used as a reference construct. FIG. 2 is an exemplary test artificial nucleic acid molecule (SEQ ID NO:99, wherein exemplary 3'-UTR elements are SEQ ID NO: 1), underlined is the 3' -UTR element tested. All elements of the sequence shown in FIG. 2 are identical to the reference artificial nucleic acid molecule (SEQ ID NO: 100), except for the underlined elements. Thus, SEQ ID NO 99 differs from SEQ ID NO 100 only in that there is a different 3' -UTR element tested before the poly (A) sequence.

The reference plasmid is prepared by replacing reporter gene firefly luciferase (Firefly Luciferase) with Renilla luciferase gene (renilla luciferase, rluc), and other sequences of the plasmid are identical.

Plasmids containing different 3' -UTR elements and reference control plasmids were all synthesized by Shanghai Biotechnology.

The DNA template was obtained by PCR amplification using a pair of primers (upstream universal primer: 5'TTGGACCCTCGTACAGAAGCTAATACG 3'; and downstream specific complementary long primer carrying poly (T)) and a high-fidelity DNA polymerase-based PCR amplification kit (Noruzan Biotechnology Co., ltd.).

EXAMPLE 3 preparation of mRNA

Using the PCR product purified (Takara purification kit) as prepared in example 2 as a template, a co-transcription capping reaction was performed using T7RNA polymerase, and in vitro transcription of RNA was performed, thereby producing Cap1mRNA. 1-methyl-pseudouridine triphosphate was added in place of Uridine Triphosphate (UTP) in vitro transcription, so that the modification ratio of 1-methyl-pseudouracil in vitro transcribed Cap1mRNA was 100%. After transcription, the DNA template was digested with dnaseli (sameil technologies limited) to reduce the risk of residual DNA template. mRNA was purified using DynabeadsMyone (Semer Feishul technologies Co., ltd.). Purified mRNA was dissolved in 1mM sodium citrate buffer (pH 6.5+/-0.1), sterile filtered, and cryopreserved at-80℃until use.

Example 4 cell expression validation of candidate 3' -UTR elements on reporter Gene influence

HEK-293 cells with good growth state are inoculated into a 96-well cell culture plate with the inoculation density of 3.5X10 ⁴ cells/well, and then placed into a 37 ℃ cell culture box for culture for 18-24 hours. Three multiplex wells were placed per sample in HEK293 cells of the 96-well plate using Lipofectamine Messenger MAX reagents (Thermo Fisher) simultaneously transfected with mRNA from example 3 plasmid containing reporter firefly luciferase and mRNA from reference control plasmid containing reporter trepang luciferase (100 ng total mRNA) at a 2:1 mRNA mass ratio per well. The cell plates after mRNA transfection were incubated in a 37℃5% CO ₂ cell incubator for 24 hours.

The transfected cell samples were tested using a double luciferase reporter assay kit (Northenzan, DL 101-01). First, pretreatment was performed, and the cell lysate was added to a 96-well cell plate at 100. Mu.L/well. Then, 14. Mu.L of the lysed cell supernatant was carefully aspirated, transferred to a 96 Kong Quanhei ELISA plate, 70. Mu.L of firefly luciferase substrate equilibrated to room temperature was added to the ELISA plate, immediately after rapid mixing, the Relative Light Unit (RLU) value of firefly luciferase was detected at 560nm wavelength after shaking the plate for 10 seconds, 70. Mu.L of freshly prepared working solution of Renilla luciferase substrate was continuously added to the above reaction solution after detection, immediately after rapid mixing, the Relative Light Unit (RLU) value of Renilla luciferase was detected at 480nm wavelength using an ELISA plate (BioTek). Each well is actually reported as the ratio (Fluc/Rluc) of firefly luciferase RLU (Fluc) to Renilla luciferase RLU (Rluc) in each well. In the data analysis, the ratio of the Fluc/Rluc ratio of the artificial nucleic acid molecule comprising the different 3' -UTR elements tested relative to the reference is the final relative expression level, with Fluc/Rluc of the artificial nucleic acid molecule comprising only the poly (a) sequence as reference nucleic acid molecule. And analyzing the influence of different 3' -UTR elements on the expression of the reporter gene according to the results of three independent repeated experiments, drawing a statistical histogram, and performing statistical analysis.

As shown in FIG. 3 and Table 2, most of the artificial nucleic acid molecules comprising the 3' -UTR elements tested had higher translational efficiencies than the reference nucleic acid molecules. Wherein the relative expression level of the artificial nucleic acid molecule comprising the 3' -UTR elements (SEQ ID NOS: 1-16) numbered U3006, U3008, U3009, U3010, U3011, U3016, U3020, U3030, U3051, U3053, U3055, U3056, U3057, U3058, U3060 and U3067 is greater than 1.9, and has higher translation efficiency.

TABLE 2 cell expression validation results

EXAMPLE 5 preparation of LPP formulation

5.1. Experimental materials

Cationic lipid SW-II-140-2 was synthesized by Sterculia, helper phospholipid (DOPE) purchased from CordenPharma, cholesterol purchased from Sigma-Aldrich, mPEG2000-DMG (i.e., DMG-PEG 2000) purchased from Avanti Polar Lipids, inc., PBS purchased from Invitrogen, protamine sulfate purchased from Beijing Lian pharmaceutical Co.

Preparation of lipid multimeric complexes (LPP) of mRNA

Preparation of nucleic acid aqueous solution Each mRNA prepared as in example 3 was diluted to 0.2mg/mL of mRNA solution with 10mM citric acid-sodium citrate buffer (pH 4.0).

Preparation of lipid solution SW-II-140-2:DOPE cholesterol mPEG2000-DMG was dissolved in ethanol solution at a molar ratio of 40:15:43.5:1.5 to prepare 10mg/mL lipid solution.

The preparation of the protamine sulfate solution comprises the step of dissolving protamine sulfate in water without a nuclease to prepare the protamine sulfate solution with the working concentration of 0.25 mg/mL.

Preparation of a Nuclear nanoparticle (core nanoparticle) solution A solution of Nuclear nanoparticle formed from protamine and artificial nucleic acid molecules was obtained by mixing a solution of protamine sulfate with a nucleic acid solution using microfluidic technology (Michain technologies Co., ltd.; model: inano D) under the following conditions: volume=4.0 mL, flow rate ratio=5 (mRNA): 1 (protamine solution), total flow rate=12 mL/min, pre-waste (START WASTE) =0.35 mL, post-waste (end waste) =0.1 mL, room temperature.

Preparation of LPP the core nanoparticle solution was mixed twice with the lipid solution at volume=4.0 mL, flow rate ratio=3 (lipid solution): 1 (core nanoparticle solution), total flow rate=12 mL/min, front waste=0.35 mL, rear waste=0.1 mL, room temperature to obtain LPP solution.

Centrifugal ultrafiltration, namely, removing ethanol from the LPP solution by ultrafiltration centrifugation (centrifugal force 3000g, centrifugal time 60min, and temperature 4 ℃) to obtain 0.1mg/ml LPP preparation containing different artificial nucleic acid molecules.

It will be apparent to those skilled in the art that many modifications and variations of the present invention can be made without departing from its spirit and scope. The specific embodiments described herein are offered by way of example only and are not meant to be limiting in any way. The true scope and spirit of the invention is indicated by the following claims, which are exemplary only.

Sequence listing

Claims

An artificial nucleic acid molecule comprising

a. at least one open reading frame (ORF); and

b. at least one 3'-untranslated region element (3'-UTR element), the 3'-UTR element comprising a nucleic acid sequence selected from the group consisting of:

(i) wherein the 3'-UTR element comprises a variant of the nucleic acid sequence shown in SEQ ID NO:44, wherein the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence shown in SEQ ID NO:44; or

(ii) wherein the 3'-UTR element comprises a nucleic acid sequence derived from the 3'-UTR of a transcript of the following genes or a variant thereof: HCV, CoV2, CVB3, AES and AAT, wherein the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence derived therefrom; or

(iii) wherein the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO: 16;

Or the corresponding RNA sequence of the above nucleic acid sequence.

The artificial nucleic acid molecule of claim 1, wherein the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO: 3.

The artificial nucleic acid molecule of claim 2, wherein the 3'-UTR element further comprises the nucleic acid sequence of SEQ ID NO: 92.

The artificial nucleic acid molecule of claim 3, wherein the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO: 4.

The artificial nucleic acid molecule of claim 1, wherein the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO: 44, and further comprises the nucleic acid sequence of SEQ ID NO: 90, 91 or 93.

The artificial nucleic acid molecule of claim 5, wherein the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO: 1, 2 or 5.

An artificial nucleic acid molecule according to claim 1, wherein the nucleic acid sequence is derived from the nucleic acid sequence of the 3'-UTR of the transcript of the following viral genes or a variant thereof: HCV, CoV2 and CVB3; or from the nucleic acid sequence of the 3'-UTR of the transcript of the mouse gene AES or a variant thereof; or from the nucleic acid sequence of the 3'-UTR of the transcript of the human gene AAT or a variant thereof; or from the nucleic acid sequence of the 3'-UTR of the transcript of the bovine gene AES or a variant thereof, wherein the variant comprises truncation, terminal extension and/or 1, 2, 3 or more mutations, additions or deletions compared to the nucleic acid sequence from which it is derived.

The artificial nucleic acid molecule according to claim 7, wherein the 3'-UTR element exhibits a length of 3-500 nucleotides, preferably 5-250 nucleotides, more preferably 90-215 nucleotides.

The artificial nucleic acid molecule of claim 8, wherein the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO: 6, 7, 8, 9 or 12.

The artificial nucleic acid molecule of claim 9, wherein the 3'-UTR element further comprises the nucleic acid sequence of SEQ ID NO:92.

The artificial nucleic acid molecule of claim 10, wherein the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO: 10, 14 or 15.

The artificial nucleic acid molecule of claim 9, wherein the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO: 12, and further comprises the nucleic acid sequence of SEQ ID NO: 9 or 94.

The artificial nucleic acid molecule of claim 12, wherein the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO: 11 or 13.

The artificial nucleic acid molecule according to any one of claims 1 to 13, further comprising at least one 5'-untranslated region element (5'-UTR element).

The artificial nucleic acid molecule of claim 14, wherein the 5'-UTR element comprises the nucleic acid sequence of SEQ ID NO: 45.

The artificial nucleic acid molecule according to any one of claims 1 to 15, further comprising a 5' cap structure, a polycytidylic acid sequence, a polyadenylic acid sequence and/or a histone stem-loop; preferably, the artificial nucleic acid molecule comprises a polyadenylic acid sequence; preferably, the polyadenylic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 46.

The artificial nucleic acid molecule according to any one of claims 1 to 16, wherein the ORF is codon-optimized.

The artificial nucleic acid molecule according to any one of claims 1 to 17, which is RNA, preferably mRNA.

The artificial nucleic acid molecule of claim 18, wherein the 3'-UTR element comprises the nucleic acid sequence of SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62.

A vector comprising the artificial nucleic acid molecule according to any one of claims 1 to 19.

The vector of claim 20, which is a DNA vector.

The vector of claim 20 or 21, which is a plasmid vector or a viral vector, preferably a plasmid vector.

The vector according to any one of claims 20 to 22, which is a circular molecule.

A cell comprising the artificial nucleic acid molecule according to any one of claims 1 to 19 or the vector according to any one of claims 20 to 23.

The cell of claim 24, which is a mammalian cell; preferably a cell isolated from a human subject.

A lipid composition, comprising an artificial nucleic acid molecule according to any one of claims 1 to 19 and a lipid that encapsulates the artificial nucleic acid molecule, wherein the lipid that encapsulates the artificial nucleic acid molecule comprises a cationic lipid, a phospholipid, a steroid and a polyethylene glycol-modified lipid; the lipid composition further comprises a cationic polymer, wherein the cationic polymer is associated with the artificial nucleic acid molecule as a complex and is co-encapsulated in the lipid to form a lipid polymer complex.

The lipid composition of claim 26, wherein the cationic lipid comprises a compound of formula (I), or a pharmaceutically acceptable salt thereof

in,

_R1 and _R2 are each independently selected from a bond, a _C1 - _C12 alkyl group, and a _C2 - _C12 alkenyl group;

_R3 and _R4 are each independently selected from _C1 - _C12 alkyl, C2- _C12 _alkenyl , _C6 - _C10 aryl and 5-10 membered heteroaryl; and _R3 and _R4 are each independently optionally substituted by t _R6 , t being an integer selected from 1-5;

R ₆ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl;

_M1 and _M2 are each independently selected from a bond, H, -O-, -S-, -C(O)-, -OC(O)-, -C(O)O-, -OC(O)O-, -SC(S)-, -C(S)S-, a 3-10 membered heterocycle, _-NR7- , or

R _5, together with one of M ₁ and M ₂ and the N atom to which they are attached, form a 3-10 membered heterocyclic ring, and the corresponding R ₁ /R ₃ or R ₂ /R ₄ do not exist, and the heterocyclic ring is optionally substituted by R ₇ ;

R ₅ is selected from C _3-8 carbocycle, -C _1-12 alkylene-Q, Q is selected from H, -OR ₇ , -SR ₇ , -OC(O)R ₇ , -C(O)OR ₇ , -N(R ₇ )C(O)R ₇ , -N(R ₇ )S(O) ₂ R ₇ , -N(R ₇ )C(S)R ₇ , -N(R ₇ ) ₂ , cyano, C _3-8 carbocycle, 3-10 membered heterocycle, C ₆ -C ₁₀ aryl, each of the above groups is optionally substituted with one or more C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, oxo (＝O);

m and n are each independently an integer selected from 0-12;

The alkyl, alkenyl and alkylene groups are each optionally and independently interrupted by one or more groups selected from: -O-, -S-, -NR ₇ -, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C _3-8 carbocycle, and the alkyl, alkenyl and alkylene groups are each optionally substituted by one or more R ₇ ;

_R7 is each independently selected from H, _C1 - _C12 alkyl, _C2 - _C12 alkenyl, _C1 - _C12 alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, _C6 - _C10 aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, _C3-8 carbocycle, and each of the above groups is optionally substituted with one or more _C1 - _C12 alkyl, _C2 - _C12 alkenyl, _C1 - _C12 alkoxy, _C6 - _C10 aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, oxo (=O).

The lipid composition of claim 27, wherein

_R1 and _R2 are each independently selected from _C1 - _C12 alkyl and _C2 - _C12 alkenyl;

wherein R ₃ and R ₄ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl; and R ₃ and R ₄ are each independently optionally substituted by t R ₆ , t being an integer selected from 1-5; and R ₆ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl.

_M1 and _M2 are each independently selected from -OC(O)-, -C(O)O-, -OC(O)O-, -SC(S)- and -C(S)S-;

R ₅ is selected from -C _1-12 alkylene-Q, Q is selected from -OR ₇ and -SR ₇ , R ₇ is independently selected from H, C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C ₆ -C ₁₀ aryl and 5-10 membered heteroaryl;

m and n are each independently an integer selected from 1-12.

The lipid composition of claim 27, wherein the cationic lipid comprises a lipid compound having the structure shown below or a pharmaceutically acceptable salt thereof:

The lipid composition of claim 27, wherein the cationic lipid comprises a lipid compound having the structure shown below or a pharmaceutically acceptable salt thereof

The lipid composition of claim 27, wherein

R ₃ and R ₄ are each independently selected from C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₆ -C ₁₀ aryl and 5-10 membered heteroaryl;

Provided that at least one of R ₃ and R ₄ is C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl, and R ₃ and R ₄ are each independently optionally substituted by t R ₆ , t being an integer selected from 1-5; R ₆ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl;

m and n are each independently an integer selected from 1-12.

The lipid composition of claim 32, wherein

_R1 and _R2 are each independently selected from _C1 - _C12 alkyl groups.

The lipid composition of claim 32 or 33, wherein

R ₃ and R ₄ are each independently selected from C ₁ -C ₁₂ alkyl and C ₆ -C ₁₀ aryl;

Provided that one of R ₃ and R ₄ is a C ₆ -C ₁₀ aryl group and the other is a C ₁ -C ₁₂ alkyl group;

R ₃ and R ₄ are each independently substituted by t R ₆ , where t is an integer selected from 1-3;

R ₆ are each independently selected from C ₁ -C ₁₂ alkyl groups.

The lipid composition according to any one of claims 32 to 34, wherein

_M1 and _M2 are each independently selected from: -OC(O)-, -C(O)O- and -OC(O)O-.

The lipid composition according to any one of claims 32 to 35, wherein

_R5 is selected from _-C1-5 alkylene-Q, where Q is -OH.

The lipid composition according to any one of claims 32 to 36, wherein

m and n are each independently an integer selected from 2-7.

The lipid composition according to any one of claims 32 to 37, wherein

R ₄ is substituted at the 1st or last position of R ₂ ; and/or

R ₃ is substituted at the 1st position or the last position of R ₁ .

The lipid composition according to any one of claims 32 to 38, wherein

t is 1 or 2, and R ₆ is substituted at the meta and/or para position relative to R ₁ or R ₂ on the benzene ring.

The lipid composition according to any one of claims 32 to 39, wherein

t is 1 or 2, and R ₆ is each independently selected from a C ₁ -C ₁₀ alkyl group.

The lipid composition of any one of claims 32 to 40, wherein the cationic lipid comprises a compound of formula (II), or a pharmaceutically acceptable salt thereof:

wherein R ₁ , R ₂ , R ₄ , R ₅ , R ₆ , M ₁ , M ₂ , t, m and n are as defined in any one of claims 32-40;

Preferably, in formula (II)

R ₁ is selected from C ₁ -C ₆ alkyl;

R ₂ is selected from C ₁ -C ₁₀ alkyl;

R ₄ is selected from C ₁ -C ₁₀ alkyl;

_M1 and _M2 are each independently selected from: -OC(O)-, -C(O)O- and -OC(O)O-;

R ₅ is selected from -C _1-5 alkylene-Q, Q is selected from -OR ₇ and -SR ₇ , R ₇ is independently selected from H, C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl;

m and n are each independently an integer selected from 2-9;

t is an integer selected from 1-3;

R ₆ is each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl.

The lipid composition of any one of claims 32 to 40, wherein the cationic lipid comprises a compound of formula (III), or a pharmaceutically acceptable salt thereof:

wherein R ₁ , R ₂ , R ₄ , R ₅ , R ₆ , t, m and n are as defined in any one of claims 32-40;

Preferably, in formula (III),

R ₁ is selected from C ₁ -C ₆ alkyl;

R ₂ is selected from C ₁ -C ₁₀ alkyl;

R ₄ is selected from C ₁ -C ₁₀ alkyl;

R ₅ is selected from -C _1-3 alkylene-Q, Q is selected from -OH and -SH;

t is 1 or 2;

R ₆ is selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl;

m and n are each independently an integer selected from 2-7.

The lipid composition of any one of claims 32 to 40, wherein the cationic lipid comprises a compound of formula (IV), or a pharmaceutically acceptable salt thereof:

wherein R ₁ , R ₂ , R ₄ , R ₆ , t, m and n are as defined in any one of claims 32-40;

Preferably, in formula (IV),

R ₁ is selected from C ₁ -C ₆ alkyl;

R ₂ is selected from C ₁ -C ₁₀ alkyl;

R ₄ is selected from C ₁ -C ₁₀ alkyl;

t is 1 or 2;

m and n are each independently an integer selected from 2-7.

The lipid composition of claim 32, wherein the cationic lipid comprises a lipid compound having the structure shown below or a pharmaceutically acceptable salt thereof:

Preferably, the cationic lipid is SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2.

The lipid composition of any one of claims 27 to 44, wherein the phospholipids comprise 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl Phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or a combination thereof;

DSPC, DOPE or a combination thereof is preferred.

The lipid composition of claim 45, wherein the steroid comprises cholesterol, coproposterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and derivatives thereof; preferably, the steroid is cholesterol.

The lipid composition of claim 46, wherein the polyethylene glycol-modified lipid comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-dioleoyl-rac-glycero, methoxy-polyethylene glycol (DOGPEG) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG); preferably DSPE-PEG, DMG-PEG or a combination thereof.

The lipid composition of claim 47, comprising

10-70 mol% of a cationic lipid, 10-70 mol% of a phospholipid, 10-70 mol% of a steroid, and 0.05-20 mol% of a polyethylene glycol-modified lipid;

Preferably comprising 35-50 mol % of cationic lipids, 10-30 mol % of phospholipids, 24-44 mol % of steroids and 1-1.5 mol % of polyethylene glycol-modified lipids; and/or

cationic lipids, DOPE, cholesterol, and DMG-PEG;

Preferably, it contains 50 mol% of cationic lipid, 10 mol% of DOPE, 38.5 mol% of cholesterol and 1.5 mol% of DMG-PEG or 40 mol% of cationic lipid, 15 mol% of DOPE, 43.5 mol% of cholesterol and 1.5 mol% of DMG-PEG.

The lipid composition of claim 48, wherein the cationic polymer comprises poly-L-lysine, protamine, polyethyleneimine (PEI) or a combination thereof; preferably, the cationic polymer is protamine.

A pharmaceutical composition comprising the artificial nucleic acid molecule according to any one of claims 1 to 19, the vector according to any one of claims 20 to 23, the cell according to claim 24 or 25 or the lipid composition according to any one of claims 26 to 49, and a pharmaceutically acceptable carrier.

Use of the artificial nucleic acid molecule of any one of claims 1 to 19, the vector of any one of claims 20 to 23, the cell of claim 24 or 25, or the lipid composition of any one of claims 26 to 49, or the pharmaceutical composition of claim 50 in the preparation of a vaccine or a medicament for gene therapy.

Use of the artificial nucleic acid molecule of any one of claims 1 to 19, the vector of any one of claims 20 to 23, the cell of claim 24 or 25, or the lipid composition of any one of claims 26 to 49, or the pharmaceutical composition of claim 50 in the preparation of a medicament for treating or preventing a disease.

A method for increasing the translation efficiency of an artificial nucleic acid molecule, preferably an mRNA molecule or a vector, the method comprising linking an open reading frame to a 3'-UTR element as defined in any one of claims 1 to 19.

A kit comprising the artificial nucleic acid molecule according to any one of claims 1 to 19, the vector according to any one of claims 20 to 23, the cell according to claim 24 or 25, or the lipid composition according to any one of claims 26 to 49, or the pharmaceutical composition according to claim 50.

A method for producing an artificial nucleic acid molecule, the method comprising:

a) synthesizing an artificial nucleic acid molecule according to any one of claims 1 to 19; or

b) synthesizing an artificial nucleic acid molecule using the vector according to any one of 20 to 23.