Skeletal muscle delivery vehicles and methods of use thereof

Abstract

This invention relates to delivery vectors for the specific and efficient delivery of payloads to skeletal muscle cells in an individual in vivo. The delivery vectors disclosed herein include targeting ligands (such as compounds with affinity for integrins including α-v-β-6) and pharmacokinetic / pharmacodynamic (PK / PD) modulators to facilitate payload delivery to cells, including skeletal muscle cells. Payloads suitable for the delivery vectors disclosed herein include RNAi agents that can be linked to or bound to the delivery vector and, upon delivery in vivo, provide inhibition of gene expression in skeletal muscle cells. Pharmaceutical compositions including skeletal muscle cell delivery vectors are also described, as well as methods for treating various diseases and conditions requiring the delivery of therapeutic payloads to skeletal muscle cells.

Description

Technical field

[0001] The present invention relates to delivery vehicles for in vivo delivery of payloads, such as RNA interference (RNAi) agents, eg, double-stranded RNAi agents, to skeletal muscle cells. Delivery of RNAi agents using the delivery vehicles disclosed herein provides repression of genes expressed in skeletal muscle cells.

Prior technology

[0002] Directing a therapeutic or diagnostic payload to a specific tissue of interest in vivo in an individual continues to be a great challenge in the field of medicine. This includes enabling specific and selective delivery to skeletal muscle cells from which a variety of diseases and disorders originate. The inability to selectively and efficiently deliver payloads, such as therapeutic drugs, into skeletal muscle cells prevents the proper treatment and resolution of various diseases and conditions.

[0003] Oligonucleotide-based agents such as antisense oligonucleotide compounds (ASO) and double-stranded RNA interference (RNAi) agents have shown great promise and the potential to revolutionize the field of medicine and provide effective treatment options. However, the delivery of oligonucleotide-based agents and especially double-stranded therapeutic RNAi agents has long been a challenge in the development of viable therapeutic agents. This is especially the case when attempting to achieve specific and selective delivery of oligonucleotide-based agents to non-hepatic cells such as skeletal muscle cells.

[0004] Although various attempts have been made over the past few years to direct oligonucleotide-based agents to skeletal muscle cells, for example using cholesterol conjugates (nonspecific and with known disadvantages of distribution to various undesired tissues and organs ) and lipid nanoparticles (LNP), which have frequently been reported to have toxicity issues, but neither have been able to achieve suitable delivery so far. Therefore, there remains a need for delivery vehicles that specifically and efficiently direct oligonucleotide-based agents, and especially RNAi agents, into skeletal muscle cells.

Content of invention

[0005] Disclosed herein is a delivery vehicle that incorporates a payload, such as an oligo-based oligonucleotide comprising an RNA interference (RNAi) agent (also referred to herein as an RNAi agent, RNAi trigger, or trigger; for example, a double-stranded RNAi agent). Agents of nucleotides that direct to skeletal muscle cells and facilitate selective and efficient inhibition of the expression of genes present in skeletal muscle cells. Further disclosed herein are compositions comprising the delivery vehicle comprising an RNAi agent for inhibiting expression of a gene of interest, wherein the RNAi agent is covalently linked to at least one targeting ligand having affinity for a cellular receptor present on the target cell body, and at least one pharmacokinetic and / or pharmacodynamic (PK / PD) modulator. The delivery vectors disclosed herein can selectively and effectively reduce or inhibit the expression of a gene of interest in an individual, such as a human or animal individual.

[0006] The delivery vectors described are useful in methods of therapeutic treatment (including prophylactic, interventional and prophylactic treatments) of conditions and diseases that may be mediated at least in part by a reduction in the expression of a gene of interest, including Examples include muscular dystrophy, including Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, myotonic muscular dystrophy, and facial scapulohumeral (FSHD). The delivery vehicles disclosed herein comprising RNAi agents can selectively reduce the expression of a target gene in cells of an individual. The methods disclosed herein include administering to a subject, eg, a human or animal subject, one or more delivery vehicles comprising an RNAi agent using any suitable method known in the art, such as intravenous infusion, intravenous injection, or subcutaneous injection.

[0007] Also described herein are pharmaceutical compositions comprising a delivery vehicle comprising an RNAi agent capable of inhibiting expression of a target gene, wherein the composition further comprises at least one pharmaceutically acceptable excipient. Pharmaceutical compositions comprising one or more delivery vehicles comprising RNAi agents are capable of selectively and effectively reducing or inhibiting the expression of target genes in vivo. Compositions comprising one or more of the delivery platforms described herein comprising RNAi agents can be administered to an individual, such as a human or animal individual, for treatment (including prophylactic treatment or inhibition) that can be mediated at least in part by a reduction in the expression of a gene of interest leading conditions and diseases, including, for example, muscular dystrophy.

[0008] One aspect described herein is a delivery vector for inhibiting the expression of a gene expressed in skeletal muscle cells, comprising: (a) an RNAi agent comprising: (i) comprising 17-49 An antisense strand of 15 nucleotides, wherein at least 15 nucleotides is complementary to the mRNA sequence of a gene expressed in skeletal muscle cells; and a sense strand of 16-49 nucleotides long, which is at least complementary to the antisense strand Partially complementary; (b) a targeting ligand having affinity for a receptor present on the surface of a skeletal muscle cell; wherein the targeting ligand is a polypeptide; and (c) a PK / PD modulator; wherein the RNAi agent Covalently linked to the targeting ligand and the PK / PD modulator.

[0009] In some embodiments, the targeting ligand has an affinity for an integrin receptor. In some embodiments, the targeting ligand has affinity for the αvβ6 integrin receptor.

[0010] In some embodiments, the polypeptide targeting ligand is a polypeptide of formula (P): or a pharmaceutically acceptable salt thereof, wherein Xaa1 is L-arginine with an N-terminal cap as appropriate, or, wherein each indicates the point of attachment to G'; G' is L-glycine or N-methyl-L-glycine; D is L-aspartic acid (L-aspartic acid ester); L is L-leucine; Xaa2 is L-α amino acid, L-β amino acid or α,α-disubstituted amino acid; Xaa3 is L-α amino acid, L-β amino acid Or α, α-disubstituted amino acid; Xaa4 is L-α amino acid, L-β amino acid or α, α-disubstituted amino acid; Xaa5 is L-α amino acid, L- β amino acid or α,α-disubstituted amino acid; and indicates the point of attachment to the RNAi agent.

[0011] In some embodiments, Xaa2 is L-alanine or L-glycine. In some embodiments, Xaa2 is L-alanine.

[0012] In some embodiments, Xaa3 is a non-standard amino acid. In some embodiments, Xaa3 is L-alanine, L-glycine, L-valine, L-leucine, L-isoleucine, or Lαamino-butyric acid. In some embodiments, Xaa3 is Lα amino-butyric acid.

[0013] In some embodiments, Xaa4 is L-arginine, L-citrulline, or L-glutamine. In some embodiments, Xaa4 is L-citrulline.

[0014] In some embodiments, Xaa5 is L-glycine, L-alanine, L-valine, L-leucine, L-isoleucine, or α-amino-isobutyric acid. In some embodiments, Xaa5 is alpha-amino-isobutyric acid.

[0015] In some embodiments, Xaal is N-acetyl-L-arginine. In some embodiments, Xaa1 is where the point of attachment to G' is indicated. In some embodiments, Xaa1 is where the point of attachment to G' is indicated.

[0016] In some embodiments, the targeting ligand has the formula: or a pharmaceutically acceptable salt thereof, wherein the point of attachment to the remainder of the delivery vehicle is indicated.

[0017] In some embodiments, the targeting ligand has the formula: or a pharmaceutically acceptable salt thereof, wherein the point of attachment to the remainder of the delivery vehicle is indicated.

[0018] In some embodiments, the targeting ligand has the formula: or a pharmaceutically acceptable salt thereof, wherein the point of attachment to the remainder of the delivery vehicle is indicated.

[0019] In some embodiments, the targeting ligand has the formula: or a pharmaceutically acceptable salt thereof, wherein the point of attachment to the remainder of the delivery vehicle is indicated.

[0020] In some embodiments, the targeting ligand has the formula: or a pharmaceutically acceptable salt thereof, wherein the point of attachment to the remainder of the delivery vehicle is indicated.

[0021] In some embodiments, the targeting ligand has the formula: or a pharmaceutically acceptable salt thereof, wherein the point of attachment to the remainder of the delivery vehicle is indicated.

[0022] In some embodiments, the PK / PD modulator comprises at least one polyethylene glycol (PEG) unit. In some embodiments, the PK / PD modulator comprises at least ten PEG units.

[0023] In some embodiments, the PK / PD regulator is a PK / PD regulator of formula (I): or a pharmaceutically acceptable salt thereof, wherein LA is a bond or Z is connected to the RNAi agent A bivalent moiety; Z is CH, phenyl, or N; L1 and L2 are each independently a linker comprising at least about 5 PEG units; X and Y are each independently a lipid comprising about 10 to about 50 carbon atoms ; and indicates the point of attachment to the RNAi agent.

[0024] In some embodiments, wherein L1 and L2 each independently comprise from about 15 to about 100 PEG units. In some embodiments, L1 and L2 each independently comprise from about 20 to about 60 PEG units. In some embodiments, L1 and L2 each independently comprise about 20 to about 30 PEG units. In some embodiments, L1 and L2 each independently comprise about 40 to about 60 PEG units. In some embodiments, one of L1 and L2 comprises about 20 to about 30 PEG units and the other comprises about 40 to about 60 PEG units. Each of L1 and L2 is independently selected from the group consisting of Part of the group identified in Table 2.

[0025] In some embodiments, at least one of X and Y is an unsaturated lipid. In some embodiments, at least one of X and Y is a saturated lipid. In some embodiments, at least one of X and Y is a branched lipid. In some embodiments, at least one of X and Y is a linear lipid. In some embodiments, at least one of X and Y is a lipid comprising about 10 to about 25 carbon atoms. In some embodiments, at least one of X and Y is cholesteryl. In some embodiments, at least one of X and Y is selected from the group consisting of the moieties identified in Table 4. In some embodiments, each of X and Y is independently selected from the group consisting of the moieties identified in Table 4.

[0026] In some embodiments, the LA is selected from the group consisting of the moieties identified in Table 5.

[0027] In some embodiments, the RNAi agent inhibits the expression of mRNA of a human gene in skeletal muscle cells.

[0028] In some embodiments, the pharmaceutically acceptable salt is the sodium salt. In some embodiments, the pharmaceutically acceptable salt is a potassium salt.

[0029] In some embodiments, the PK / PD regulator is a PK / PD regulator of formula (Ia): or a pharmaceutically acceptable salt thereof, wherein LA, L1, L2, X and Y are as in formula (I ) as defined in any of the embodiments of the lipid PK / PD modulator; and indicates the point of attachment to the RNAi agent.

[0030] In some embodiments, the PK / PD modulator is a PK / PD modulator of formula (Ib):

[0031] or a pharmaceutically acceptable salt thereof, wherein LA, L1, L2, X and Y are as defined in any embodiment of the lipid PK / PD regulator of formula (I) or (Ia), and Points of attachment to RNAi agents are indicated.

[0032] In some embodiments, the PK / PD modulator is a PK / PD modulator of formula (Ic): or a pharmaceutically acceptable salt thereof, wherein LA, L1, L2, X and Y are as in formula (I ), (Ia) or (Ib) as defined in any one of the embodiments of the lipid PK / PD modulator of (Ia) or (Ib), and indicates the point of attachment to the RNAi agent.

[0033] In some embodiments, the PK / PD modulator is a PK / PD modulator selected from the group consisting of lipid PK / PD modulators identified in Table 15. In some embodiments, the PK / PD modulator is a PK / PD modulator selected from the group consisting of lipid PK / PD modulators identified in Table 17.

[0034] Another aspect of the present invention provides a pharmaceutical composition comprising a delivery vehicle or a pharmaceutically acceptable salt thereof and a pharmaceutical excipient.

[0035] Another aspect of the invention provides a method of treating a disease or disorder of skeletal muscle cells in a subject.

[0036] The present invention also provides a method for synthesizing a delivery vehicle or a pharmaceutically acceptable salt thereof.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present disclosure, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0038] Other objects, features, aspects and advantages of the present invention will become apparent from the following embodiments, drawings and claims.

Implementation

[0041] CROSS-REFERENCE TO RELATED APPLICATIONS

[0042] This PCT application claims U.S. Provisional Application No. 63 / 077,141 filed on September 11, 2020, U.S. Provisional Application No. 63 / 214,747 filed on June 24, 2021, and U.S. Provisional Application No. 63 / 214,747 filed on August 2021 Benefit of U.S. Provisional Application No. 63 / 230,381 filed on June 6. Each of these documents is incorporated herein by reference in its entirety.

[0043] definition

[0044] As used herein, the terms "oligonucleotide" and "polynucleotide" mean a polymer of linked nucleosides, each of which may be independently modified or unmodified.

[0045] As used herein, "RNAi agent" (also referred to as "RNAi trigger") means a composition comprising RNA or RNA-like (eg, chemically modified RNA) oligonucleotide molecules capable of sequence-specific Reduce or inhibit (eg, reduce or inhibit under appropriate conditions) translation of a messenger RNA (mRNA) transcript of a target mRNA in a selective manner. As used herein, the RNAi agent may induce RNA interference via the RNA interference mechanism (i.e., via interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells) or by any alternative mechanism or path to work. Although it is believed that RNAi agents (as that term is used herein) act primarily via the mechanism of RNA interference, the disclosed RNAi agents are tied to or restricted to any particular pathway or mechanism of action. The RNAi agents disclosed herein are composed of a sense strand and an antisense strand, and include (but are not limited to): short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpins RNA (shRNA) and dicer substrate. The antisense strands of the RNAi agents described herein are at least partially complementary to the targeted mRNA. An RNAi agent can include one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0046] As used herein, the terms "silencing", "reducing", "suppressing", "down-regulating" or "blocking expression of a gene" when referring to the expression of a given gene mean the As measured by the amount of RNA transcribed from a gene or the amount of a polypeptide, protein, or protein subunit translated from mRNA in a cell, cell population, tissue, organ, or individual population, when the cell, cell population, When a tissue, organ or individual is treated, the expression of the gene is reduced compared to a second cell, cell population, tissue, organ or individual that has not undergone the treatment.

[0047] As used herein, the terms "sequence" and "nucleotide sequence" mean a series or order of nucleobases or nucleotides described by a series of letters using standard nomenclature.

[0048] As used herein, a "base", "nucleotide base" or "nucleobase" is a heterocyclic pyrimidine or purine compound as a component of a nucleotide, and includes the major purine bases adenine and guanine Purines and the major pyrimidine bases cytosine, thymine, and uracil. Nucleobases can be further modified to include, without limitation, universal bases, hydrophobic bases, promiscuous bases, size-extending bases, and fluorinated bases. (See e.g. Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008). The synthesis of such modified nucleobases, including phosphoramidate compounds containing modified nucleobases, is known in the art.

[0049] As used herein and unless otherwise indicated, the term "complementary" is used to describe a second nucleobase or nucleotide sequence (such as an RNAi agent antisense strand or a single-stranded antisense oligonucleotide) relative to a second nucleobase or nucleotide sequence. When a nucleobase or nucleotide sequence (such as the RNAi agent's sense strand or targeted mRNA), means that the oligonucleotide or polynucleotide comprising the first nucleotide sequence is under certain standard conditions Hybridizes (forms base-pair hydrogen bonds under mammalian physiological conditions (or similar in vitro conditions)) to an oligonucleotide or polynucleotide comprising a second nucleotide sequence and forms a double helix or double helix structure ability. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleosides, at least to the extent that the above hybridization requirements are met. acid or nucleotide mimetics. Sequence identity or complementarity is independent of modification. For example, a and Af, as defined herein, are complementary to U (or T) and identical to A for purposes of determining identity or complementarity.

[0050] As used herein, "perfectly complementary" or "fully complementary" means that in a hybridized pair of nucleobase or nucleotide sequence molecules, all (100%) bases in the contiguous sequence of the first oligonucleotide base will hybridize to the same number of bases in the contiguous sequence of the second oligonucleotide. A contiguous sequence may comprise all or a portion of the first or second nucleotide sequence.

[0051] As used herein, "partially complementary" means that in a hybridized pair of nucleobase or nucleotide sequence molecules, at least 70% (but not all) of the bases in the contiguous sequence of the first oligonucleotide will be Hybridizes to the same number of bases in the contiguous sequence of the second oligonucleotide. A contiguous sequence may comprise all or a portion of the first or second nucleotide sequence.

[0052] As used herein, "substantially complementary" means that in a hybridized pair of nucleobase or nucleotide sequence molecules, at least 85% (but not all) of the bases in the contiguous sequence of the first oligonucleotide Will hybridize to the same number of bases in the contiguous sequence of the second oligonucleotide. A contiguous sequence may comprise all or a portion of the first or second nucleotide sequence.

[0053] As used herein, the terms "complementary", "fully complementary", "partially complementary" and "substantially complementary" relate to between the sense strand and the antisense strand of the RNAi agent or between the antisense strand of the RNAi agent and the target mRNA used for nucleobase or nucleotide matching between sequences.

[0054] As used herein, an "oligonucleotide-based agent" is one containing about 10-50 (e.g., 10-48, 10-46, 10-44, 10-42, 10-40, 10-38, 10-50 36, 10 to 34, 10 to 32, 10 to 30, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 50, 12 to 48, 12 to 46, 12 to 44, 12 to 42, 12 to 40, 12 to 38, 12 to 36, 12 to 34, 12 to 32, 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 50, 14 to 48, 14 to 46, 14 to 44, 14 to 42, 14 to 40, 14 to 38, 14 to 36, 14 to 34, 14 to 32, 14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 16 to 50, 16 to 48, 16 to 46, 16 to 44, 16 to 42, 16 to 40, 16 to 38, 16 to 36, 16 to 34, 16 to 32, 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to 50, 18 to 48, 18 to 46, 18 to 44, 18 to 42, 18 to 40, 18 to 38, 18 to 36, 18 to 34, 18 to 32, 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, 18 to 20, 20 to 50, 20 to 48, 20 to 46, 20 to 44, 20 to 42, 20 to 40, 20 to 38, 20 to 36, 20 to 34, 20 to 32, 20 to 30, 20 to 28, 20 to 26, 20 to 24, 20 to 22, 22 to 50, 22 to 48, 22 to 46, 22 to 44, 22 to 42, 22 to 40, 22 to 38, 22 to 36, 22 to 34, 22 to 32, 22 to 30, 22 to 28, 22 to 26, 22 to 24, 24 to 50, 24 to 48, 24 to 46, 24 to 44, 24 to 42, 24 to 40, 24 to 38, 24 to 36, 24 to 34, 24 to 32, 24 to 30, 24 to 28, 24 to 26, 26 to 50, 26 to 48, 26 to 46, 26 to 44, 26 to 42, 26 to 40, 26 to 38, 26 to 36, 26 to 34, 26 to 32, 26 to 30, 26 to 28, 28 to 50, 28 to 48, 28 to 46, 28 to 44, 28 to 42, 28 to 40, 28 to 38, 28 to 36, 28 to 34, 28 to 32, to 28 to 30, 30 to 50, 30 to 48, 30 to 46, 30 to 44, 30 to 42, 30 to 40, 30 to 38, 30 to 36, 30 to 34, 30 to 32, 32 to 50, 32 to 48, 32 to 46, 32 to 44, 32 to 42 , 32 to 40, 32 to 38, 32 to 36, 32 to 34, 34 to 50, 34 to 48, 34 to 46, 34 to 44, 34 to 42, 34 to 40, 34 to 38, 34 to 36, 36 to 50, 36 to 48, 36 to 46, 36 to 44, 36 to 42, 36 to 40, 36 to 38, 38 to 50, 38 to 48, 38 to 46, 38 to 44, 38 to 42, 38 to 40 , 40 to 50, 40 to 48, 40 to 46, 40 to 44, 40 to 42, 42 to 50, 42 to 48, 42 to 46, 42 to 44, 44 to 50, 44 to 48, 44 to 46, 46 to 50, 46 to 48, or 48 to 50) nucleotides or nucleotide base pairs. In some embodiments, the oligonucleotide-based agent has a nucleobase sequence that is at least partially complementary to a coding sequence in a target nucleic acid or gene of interest expressed in the cell. In some embodiments, oligonucleotide-based agents are capable of inhibiting the expression of a latent gene upon delivery to cells expressing the gene, and are referred to herein as "expression-inhibiting oligonucleotide-based agents." Gene expression can be inhibited in vitro or in vivo.

[0055] "Oligonucleotide-based agents" include (but are not limited to): single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro RNA (miRNA), short hairpin RNA (shRNA), ribonucleases, interfering RNA molecules, and Dicer substrates. In some embodiments, oligonucleotide-based agents are single-stranded oligonucleotides, such as antisense oligonucleotides. In some embodiments, oligonucleotide-based agents are double-stranded oligonucleotides. In some embodiments, oligonucleotide-based agents are double-stranded oligonucleotides that are RNAi agents.

[0056] As used herein, the term "standard amino acid" refers to the following twenty (20) amino acids: alanine, arginine, asparagine, aspartic acid (aspartic acid ester ), cysteine, glutamine, glutamic acid (glutamate), glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine , proline, serine, threonine, tryptophan, tyrosine and valine.

[0057] As used herein, the term "non-standard amino acid" refers to an amino acid other than a "standard amino acid" as defined herein. "Non-standard amino acids" include but are not limited to selenocysteine, pyrrolysine, N-formylmethionine, hydroxyproline, selenomethionine, α-amino-isobutyric acid (Aib), L-α-amino-butyric acid (Abu), α,γ-diaminobutyric acid, dehydroalanine, norleucine, allisoleucine, t-leucine, α -Amino-n-heptanoic acid, α,β-diaminopropionic acid, β-N-oxalyl-α,β-diaminopropionic acid, allostreonine, homocysteine, homoserine , β-homoalanine (β3-hA), isovaline, norvaline (Nva), citrulline (Cit), ornithine, α-methyl-aspartate (αMeD), α-methyl-leucine (αMeL), N-methylalanine, N-methyl-glycine (NMeG), N-methylleucine (NMeL), O-cyclohexyl-alanine ( Cha), N-ethylalanine, N,N-ε-dimethyllysine (K(Me)2), dimethylarginine (R(Me)2), Dap(Ac), n- Alkylated L-alpha amino acids and other amino acid analogs or amino acid mimetics that function in a manner similar to naturally occurring amino acids.

[0058] As used herein and as understood by those skilled in the art, polyethylene glycol (PEG) units refer to repeating units of the formula -(CH2CH2O)-. It is understood that in the chemical structures disclosed herein, PEG units can be depicted as -(CH2CH2O)-, -(OCH2CH2)- or -(CH2OCH2)-. It should also be understood that numbers indicating the number of repeating PEG units can be placed on either side of the parentheses depicting the PEG units. It is further understood that the terminal PEG unit may be capped by an atom, such as a hydrogen atom, or some other moiety.

[0059] As used herein, the term "substantially identical" or "substantial identity" applied to nucleic acid sequences means that a nucleotide sequence (or a portion of a nucleotide sequence) has at least about 85% sequence compared to a reference sequence Concordance or higher, such as at least 90%, at least 95% or at least 99% concordance. The percent sequence identity is determined by comparing the two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions where the same type of nucleic acid base is present in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain Percentage of sequence identity. The invention disclosed herein encompasses nucleotide sequences substantially identical to those disclosed herein.

[0060] As used herein, the terms "treat", "treatment" and similar terms mean the number, severity and / or frequency of treatment for alleviating or alleviating one or more symptoms of a disease in an individual method or steps. As used herein, "treatment" may include prophylactic treatment, management, prophylactic treatment and / or inhibition or reduction of the number, severity and / or frequency of one or more symptoms of a disease in an individual.

[0061] As used herein, the phrase "introducing into a cell" when referring to an RNAi agent means functionally delivering the RNAi agent into the cell. The phrase "functional delivery" means delivering an RNAi agent into a cell in a manner that enables the RNAi agent to have a desired biological activity (eg, sequence-specific inhibition of gene expression).

[0062] As used herein, the term "isomer" refers to compounds having the same molecular formula, but differing in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers". Stereoisomers that are not mirror images of each other are termed "diastereoisomers" and stereoisomers that are non-superimposable mirror images are termed "enantiomers" or sometimes "optical isomers". A carbon atom bonded to four different substituents is called a "chiral center".

[0063] As used herein, unless specifically identified in a structure as having a particular configuration, for each asymmetric center in which there exists an enantiomer, diastereoisomer, or other stereoisomeric configuration Structures, each structure disclosed herein is intended to represent all such possible isomers, including optically pure and racemic forms thereof. For example, structures disclosed herein are intended to encompass mixtures of diastereoisomers as well as single stereoisomers.

[0064] As used in the technical solution herein, the phrase "consisting of ..." does not include any elements, steps or components not described in the technical solution. When applied to the technical solution of the present invention, the phrase "consisting essentially of" limits the scope of the technical solution to the specified materials or steps and materials or steps that do not substantially affect the basic and novel characteristics of the claimed invention.

[0065] Those of ordinary skill will readily understand and appreciate that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the compound or combination It depends on the environment where the object is placed. Thus, as used herein, the structures disclosed herein envisage that certain functional groups such as OH, SH or NH can be protonated or deprotonated. The disclosure herein is intended to cover the disclosed compounds and compositions regardless of their protonation state based on the environment, such as pH, as will be readily understood by one of ordinary skill.

[0066] As used herein, the term "lipid" refers to moieties and molecules that are soluble in non-polar solvents. The term lipid includes amphiphilic molecules comprising a polar water-soluble headgroup and a hydrophobic tail. Lipids can be of natural or synthetic origin. Non-limiting examples of lipids include fatty acids (such as saturated, monounsaturated, and polyunsaturated fatty acids), glycerolipids (such as monoacylglycerols, diacylglycerols, and triacylglycerols), phospholipids (such as phosphatidylethanolamine , phosphatidylcholine and phosphatidylserine), sphingolipids (such as sphingomyelin) and cholesterol esters. As used herein, the term "saturated lipid" refers to a lipid without any unsaturation. As used herein, the term "unsaturated lipid" refers to a lipid comprising at least one (1) degree of unsaturation. As used herein, the term "branched lipid" refers to a lipid comprising more than one linear chain, wherein each linear chain is covalently linked to at least one other linear chain. As used herein, the term "linear lipid" refers to a lipid without any branching.

[0067] As used herein, the term "link" or "bond" when referring to a link between two compounds or two molecules means that the two molecules are joined by a covalent bond or by a non-covalent bond (such as hydrogen bond or ionic bond) association. In some instances, where the term "link" or "bond" refers to an association between two molecules via a non-covalent bond, two different molecules in a physiologically acceptable buffer (such as buffered saline) The association between molecules has a KD of less than 1×10-4M (eg, less than 1×10-5M, less than 1×10-6M, or less than 1×10-7M). Unless stated otherwise, the terms "link" and "bond" as used herein may refer to a connection between a first compound and a second compound with or without any intervening atoms or groups of atoms.

[0068] As used herein, a linking group is one or more atoms that link one molecule or part of a molecule to another to a second molecule or part of a molecule. Similarly, as used in the art, the term backbone is sometimes used interchangeably with linking group. A linking group can comprise any number of atoms or functional groups. In some embodiments, a linking group may not facilitate any biological or pharmaceutical reaction, and is only used to link two biologically active molecules.

[0069] Unless otherwise stated, a symbol as used herein means that any group can be attached thereto in accordance with the scope of the invention described herein.

[0070] As used herein, the term "comprising" is used herein to mean and is used interchangeably with the phrase "including (but not limited to)". The term "or" is used herein to mean and is used interchangeably with the term "and / or" unless the context clearly indicates otherwise.

[0071] As used in the technical solution herein, the phrase "consisting of ..." does not include any elements, steps or components not described in the technical solution. When applied to the technical solution of the present invention, the phrase "consisting essentially of" limits the scope of the technical solution to the specified materials or steps and materials or steps that do not substantially affect the basic and novel characteristics of the claimed invention.

[0072] Modified Nucleotides

[0073] In some embodiments, the RNAi agent comprises one or more modified nucleotides. As used herein, "modified nucleotides" are nucleotides other than ribonucleotides (2'-hydroxy nucleotides). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides may include, but are not limited to: deoxyribonucleotides, nucleotide mimetics, abasic nucleotides (denoted herein as Ab), 2'-modified Nucleotides, 3' to 3' linking (reverse) nucleotides (denoted herein as invdN, invN, invn), nucleotides comprising modified nucleobases, bridging nucleotides, peptide nucleic acids (PNA), 2',3'-opened nucleotide mimetic (unlocked nucleobase analog, denoted herein as NUNA or NUNA), locked nucleotide (herein denoted as NLNA or NLNA), 3' - O-methoxy (2' internucleoside linkage) nucleotides (denoted herein as 3'-OMen), 2'-F-arabinonucleotides (herein denoted as NfANA or NfANA), 5 '-Me, 2'-fluoronucleotides (denoted herein as 5Me-Nf), N-morpholinyl nucleotides, phosphonate vinyl ester deoxyribonucleotides (herein denoted vpdN), phosphine-containing Vinyl phosphonate-containing nucleotides and cyclopropyl phosphonate-containing nucleotides (cPrpN). 2'-modified nucleotides (that is, nucleotides containing groups other than hydroxyl at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides ( Denoted herein as a lowercase letter "n" in a nucleotide sequence), 2'-deoxy-2'-fluoronucleotides (also referred to herein as 2'-fluoronucleotides, and denoted herein is Nf), 2'-deoxynucleotide (denoted herein as dN), 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotide (herein Also known as 2'-MOE, and denoted herein as NM), 2'-amino nucleotides and 2'-alkyl nucleotides. All positions of a given compound need not be uniformly modified. Conversely, more than one modification can be incorporated into a single RNAi agent or even into a single nucleotide thereof. RNAi agent sense and antisense strands can be synthesized and / or modified by methods known in the art. A modification on one nucleotide is independent of a modification on another nucleotide.

[0074] Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines, and purines substituted by N-2, N-6, and O-6 (e.g., 2-amine propyladenine, 5-propynyluracil or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine, xanthine , hypoxanthine, 2-aminoadenine, 6-alkyl (such as 6-methyl, 6-ethyl, 6-isopropyl or 6-n-butyl) derivatives of adenine and guanine, adenine 2-Alkyl (such as 2-methyl, 2-ethyl, 2-isopropyl or 2-n-butyl) and other alkyl derivatives of purine and guanine, 2-thiouracil, 2-thio Thymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine , 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-sulfhydryl, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo (such as 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7- Methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

[0075] In some embodiments, all or substantially all of the nucleotides of the RNAi agent are modified nucleotides. As used herein, an RNAi agent in which substantially all nucleotides are modified nucleotides is four or fewer (i.e., 0, 1, 2, 3 or 4) nucleotides are ribonucleotides (ie, unmodified) RNAi agents. As used herein, a strand in which substantially all of the nucleotides present are modified nucleotides is one in which two or fewer (i.e., 0, 1, or 2) nucleotides are modified. Sense shares of unmodified ribonucleotides. As used herein, an antisense strand in which substantially all nucleotides are modified nucleotides is one in which there are two or fewer (i.e., 0, 1, or 2) in the sense strand Nucleotides are the antisense strands of unmodified ribonucleotides. In some embodiments, one or more nucleotides of the RNAi agent is an unmodified ribonucleotide.

[0076] Modified internucleoside linkages

[0077] In some embodiments, one or more nucleotides of the RNAi agent are linked by a non-standard linkage or backbone (ie, a modified internucleoside linkage or a modified backbone). Modified internucleoside linkages or backbones include, but are not limited to, phosphorothioate groups (denoted herein as a lowercase "s"), chiral phosphorothioate, phosphorothioate, dithio Phosphate esters, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g. methyl phosphonate or 3'-alkylene phosphonate), chiral phosphonates, phosphonites , phosphoramidate (eg, 3'-aminophosphoramidate, aminoalkylphosphoramidate or thiocarbonylphosphorylamidate), thiocarbonylalkyl-phosphonate, thiocarbonylalkylphosphonate triester , N-morpholino linkage, borane phosphate with normal 3'-5' linkage, analogue of borane phosphate with 2'-5' linkage or borane phosphate with reversed polarity , wherein adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, the modified internucleoside linkage or backbone lacks a phosphorus atom. Modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl intersaccharide linkages, mixed heteroatom and alkyl or cycloalkyl intersaccharide linkages, or one or more A short-chain heteroatom or heterocyclic intersaccharide linkage. In some embodiments, modified internucleoside backbones include, but are not limited to, siloxane backbones, thioether backbones, sulfide backbones, sulfide backbones, formyl and thioformyl backbones, sulfide backbones, Methylformyl and thioformyl main chains, olefin-containing main chains, sulfamate main chains, methyleneimino and methylenehydrazine main chains, sulfonate and sulfonamide main chains chain, amide backbone, and other backbones with mixed N, O, S, and CH2 components.

[0078] In some embodiments, the sense strand of an RNAi agent may contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, and the antisense strand of an RNAi agent may contain 1, 2, 3, 4, 5 or 6 phosphorothioate linkages, or both the sense and antisense strands can independently contain 1, 2, 3, 4, 5 or 6 phosphorothioate linkages. In some embodiments, the sense strand of the RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, and the antisense strand of the RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages linkage, or both the sense and antisense strands may independently contain 1, 2, 3 or 4 phosphorothioate linkages.

[0079] In some embodiments, the RNAi agent's sense strand contains at least two phosphorothioate internucleoside linkages. In some embodiments, at least two phosphorothioate internucleoside linkages are between nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate internucleoside linkage is at the 5' end of the sense strand and the other phosphorothioate linkage is at the 3' end of the sense strand. In some embodiments, two phosphorothioate internucleoside linkages are at the 5' end of the sense strand and another phosphorothioate linkage is at the 3' end of the sense strand. In some embodiments, the sense strand does not include any phosphorothioate internucleoside linkages between nucleotides, but contains one, two, or Three phosphorothioate linkages and optionally inverted abasic residue end caps. In some embodiments, the targeting ligand is attached to the sense strand via a phosphorothioate linkage.

[0080] In some embodiments, the RNAi agent antisense strand contains four phosphorothioate internucleoside linkages. In some embodiments, four phosphorothioate internucleoside linkages are between nucleotides at positions 1-3 from the 5' end of the antisense strand and at positions 19-21 from the 5' end. Between nucleotides at 20-22, 21-23, 22-24, 23-25 ​​or 24-26. In some embodiments, three phosphorothioate internucleoside linkages are located between positions 1-4 from the 5' end of the antisense strand and a fourth phosphorothioate internucleoside linkage is located from The 5' end of the antisense strand is between position 20-21. In some embodiments, the RNAi agent contains at least three or four phosphorothioate internucleoside linkages in the antisense strand.

[0081] In some embodiments, the RNAi agent contains one or more modified nucleotides and one or more modified internucleoside linkages. In some embodiments, 2'-modified nucleosides are combined with modified internucleoside linkages.

[0082] targeting ligand and targeting group

[0083] The targeting group or targeting moiety enhances the pharmacokinetic or biodistribution properties of the conjugate or the RNAi agent to which it is attached, to improve the cell specificity (including in some cases organ specificity) of the conjugate or RNAi agent sex) distribution and cell-specific (or organ-specific) uptake. A targeting group can be monovalent, divalent, trivalent, tetravalent, or have a higher valency with respect to the target it targets. Representative targeting groups include, without limitation, compounds with affinity for cell surface molecules, cellular receptor ligands with affinity for cell surface molecules, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimetics. In some embodiments, the targeting group is attached to the RNAi agent using a linker, such as a PEG linker or one, two or three abasic and / or ribitol (abasic ribose) residues, These residues may in some cases act as linkers. In some embodiments, the targeting group comprises an integrin targeting ligand.

[0084] In some embodiments, an RNAi agent described herein is bound to a targeting group. In some embodiments, the targeting ligand enhances the ability of the RNAi agent to bind to a specific cellular receptor on the cell of interest. In some embodiments, a targeting ligand that binds to an RNAi agent described herein has an affinity for an integrin receptor. In some embodiments, targeting ligands suitable for use in the RNAi agents disclosed herein have affinity for integrin α-v-β6. A targeting group comprises two or more targeting ligands.

[0085] In some embodiments, the RNAi agents disclosed herein are linked to one or more integrin targeting ligands comprising a compound of formula (P): or a pharmaceutically acceptable salt thereof, wherein Xaa1 is viewed as L-arginine with an N-terminal cap, or where the point of attachment to G' is indicated; G' is L-glycine or N-methyl-L-glycine; D is L-asparagine Acid (L-aspartic acid ester); L is L-leucine; Xaa2 is L-α amino acid, L-β amino acid or α, α-disubstituted amino acid; Xaa3 is L- α amino acid, L-β amino acid or α,α-disubstituted amino acid; Xaa4 is L-α amino acid, L-β amino acid or α,α-disubstituted amino acid; Xaa5 is an L-alpha amino acid, an L-beta amino acid, or an α,α-disubstituted amino acid; and indicates the point of attachment to the RNAi agent.

[0086] In some embodiments, Xaa2 is L-alanine or L-glycine. In some embodiments, Xaa2 is L-alanine. In some embodiments, Xaa2 is L-glycine.

[0087] In some embodiments, Xaa3 is a non-standard amino acid. In some embodiments, Xaa3 is L-alanine, L-glycine, L-valine, L-leucine, L-isoleucine, or Lαamino-butyric acid. In some embodiments, Xaa3 is Lα amino-butyric acid. In some embodiments, Xaa3 is L-alanine. In some embodiments, Xaa3 is L-glycine. In some embodiments, Xaa3 is L-valine. In some embodiments, Xaa3 is L-leucine. In some embodiments, Xaa3 is L-isoleucine.

[0088] In some embodiments, Xaa4 is L-arginine, L-citrulline, or L-glutamine. In some embodiments, Xaa4 is L-citrulline. In some embodiments, Xaa4 is L-arginine. In some embodiments, Xaa4 is L-glutamine.

[0089] In some embodiments, Xaa5 is L-glycine, L-alanine, L-valine, L-leucine, L-isoleucine, or alpha-amino-isobutyric acid. In some embodiments, Xaa5 is alpha-amino-isobutyric acid. In some embodiments, Xaa5 is L-glycine. In some embodiments, Xaa5 is L-alanine. In some embodiments, Xaa5 is L-valine. In some embodiments, Xaa5 is L-leucine. In some embodiments, Xaa5 is L-isoleucine.

[0090] In some embodiments, Xaal is N-acetyl-L-arginine. In some embodiments, Xaa1 is where the point of attachment to G' is indicated. In some embodiments of Formula P, Xaa1 is where the point of attachment to G' is indicated.

[0091] In some embodiments, the targeting ligand has the formula: or a pharmaceutically acceptable salt thereof, wherein the point of attachment to the remainder of the delivery vehicle is indicated.

[0092] In some embodiments, the targeting ligand has the formula: or a pharmaceutically acceptable salt thereof, wherein the point of attachment to the remainder of the delivery vehicle is indicated.

[0093] In some embodiments, the targeting ligand has the formula: or a pharmaceutically acceptable salt thereof, wherein the point of attachment to the remainder of the delivery vehicle is indicated.

[0094] In some embodiments, the targeting ligand has the formula: or a pharmaceutically acceptable salt thereof, wherein the point of attachment to the remainder of the delivery vehicle is indicated.

[0095] In some embodiments, the targeting ligand has the formula: or a pharmaceutically acceptable salt thereof, wherein the point of attachment to the remainder of the delivery vehicle is indicated.

[0096] In some embodiments, the targeting ligand has the formula: or a pharmaceutically acceptable salt thereof, wherein the point of attachment to the remainder of the delivery vehicle is indicated.

[0097] The RNAi agent may comprise more than one targeting ligand. In some embodiments, the RNAi agent comprises 1-20 targeting ligands. In some embodiments, the RNAi agent comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 targeting ligands ligands to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 targeting ligands. In some embodiments, the targeting ligand can be bound at the 5' or 3' end of the sense strand of the RNAi agent. In some embodiments, targeting ligands can bind to internal nucleotides on the RNAi agent.

[0098] In some embodiments, the RNAi agent comprises a targeting group that includes 2 or more targeting ligands. In some embodiments, a targeting group can be bound to the 5' or 3' end of the sense strand of the RNAi agent. In some embodiments, targeting groups can bind to internal nucleotides on the RNAi agent. In some embodiments, a targeting group may consist of two targeting ligands linked together, referred to as a "bidentate" targeting group. In some embodiments, a targeting group may consist of three targeting ligands linked together, referred to as a "tridentate" targeting group. In some embodiments, a targeting group may consist of four targeting ligands linked together, referred to as a "tetradentate" targeting group.

[0099] In some embodiments, the RNAi agent may comprise a targeting group bound to the 3' or 5' end of the sense strand and additionally comprise a targeting ligand bound to an internal nucleotide. In some embodiments, the tridentate targeting group is bound to the 5' end of the sense strand of the RNAi agent, and at least one targeting ligand is bound to an internal nucleotide of the sense strand. In other embodiments, the tridentate targeting group is bound to the 5' end of the sense strand of the RNAi agent, and the four targeting ligands are bound to internal nucleotides of the sense strand.

[0100] As mentioned above, in some embodiments, the RNAi agents disclosed herein can be linked to one or more targeting ligands on internal nucleotides of the sense or antisense strand of the RNAi agent And / or one or more targeting groups to facilitate the delivery of RNAi agents in vivo. In some embodiments, the targeting ligand or targeting group is linked or bound to one or more internal nucleotides of the sense strand of the RNAi agent. For example, the targeting ligand can be attached to the 2' position of the ribose ring, the 3' position of the ribose ring, the individual nucleotide or the nucleobase of the nucleotide at the G position of the ribose ring, the 4' position of the ribose ring. ' position, the 5' position of a nucleotide, or the oxygen atom on the ribose ring. The following depicts hypothetical ribonucleotides, where the carbons are numbered:

[0101] In some embodiments, to facilitate linkage of one or more targeting ligands and / or targeting groups to internal nucleotides, 2'-O-propargyl-modified nucleotides Incorporated into the nucleotide sequence (see eg Table 23). After synthesis of the individual strands, the 2'-O-propargyl modified nucleotides can be linked or conjugated at the 2' position to a targeting ligand and / or targeting ligand using standard coupling techniques as known in the art. group.

[0102] Pharmacokinetic and / or pharmacodynamic modifiers

[0103] The delivery vehicles disclosed herein comprise pharmacokinetic and / or pharmacodynamic (also referred to herein as "PK / PD") modulators linked to the RNAi agent to facilitate delivery of the RNAi agent to the cells or tissues are required. PK / PD modulator precursors can be synthesized with reactive groups, such as maleimide or azido groups, to facilitate linkage to one or more linking groups on the RNAi agent. The synthesis of chemical reactions linking such PK / PD modulator precursors to RNAi agents is generally known in the art. The terms "PK / PD modulator" and "lipid PK / PD modulator" are used interchangeably herein.

[0104] In some embodiments, PK / PD modulators may include molecules that are fatty acids, lipids, albumin binding agents, antibody binding agents, polyesters, polyacrylates, polyamino acids, and molecules having about 20 - Linear or branched polyethylene glycol (PEG) moieties of 2000 PEG-(CH2CH2O)- units.

[0105] Table 1 shows certain exemplary PK / PD modulator precursors that can be used as starting materials for linking to the RNAi agents disclosed herein. The PK / PD modulator precursor can be covalently attached to the RNAi agent using any method known in the art. In some embodiments, the maleimide-containing PK / PD modulator precursor can react with a disulfide bond-containing moiety at the 3' end of the sense strand of the RNAi agent.

[0106] Table 1: Exemplary PK / PD modulator precursors suitable for linking to RNAi agents PEG40K (2×2 arms), Wherein n and m are each independently an integer, and the molecular weight of the sum of all PEG units is about 40 kilodaltons (kilodalton) NOF, Sunbright ® GL4-400MA PEG40K (4 arms), where n is an integer and the sum of all PEG units has a molecular weight of about 40 kilodaltons NOF, Sunbright® XY4-400MA PEG40K (2 arms), where n is an integer and the sum of all PEG units has a molecular weight of about 40 kilodaltons NOF, Sunbright® GL2-400MA PEG40K, where n is an integer and the sum of all PEG units has a molecular weight of about 40 kilodaltons NOF, Sunbright® ME-400MA PEG10K, where n is an integer and the sum of all PEG units has a molecular weight of about 10 kilodaltons NOF, Sunbright® ME-100MA PEG5K, where n is an integer and the sum of all PEG units has a molecular weight of about 5 kilodaltons NOF, Sunbright® ME-050MA DSPE-PEG5K-NHS (Naonsoft Polymers TM #SKU 1544) (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[succimidyl (polyethylene glycol)]), where n is an integer and the molecular weight of the sum of all PEG units is about 5 kilodaltons DSPE-PEG5K-MAL (Naonsoft Polymers TM SKU #2049) 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)], where n is an integer and the sum of all PEG units has a molecular weight of about 5 kilodaltons DSPE-PEG5K-N3 (Naonsoft Polymers TM SKU #2274) 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)], where n is an integer and the molecular weight of the sum of all PEG units is about 5 kilomolar Leon PEG47+C22 PEG47+CLS (Cholesterol) PEG23+C22 Double (PEG23+C14) Double (PEG23+C22) Double (PEG47+C22) PEG48+C22 PEG71+C22 PEG95+C22 PEG71+CLS PEG95+CLS Double (PEG23+C18) Reference (PEG23+C22) Reference (PEG23+CLS) Double (PEG23+CLS) PEG5K+C22 where n is an integer and the sum of all PEG units has a molecular weight of about 5 kilodaltons C18 (NHS)-PEG1K+C18 (Naonsoft Polymers TM SKU #10668-1000) where n is an integer and the sum of all PEG units has a molecular weight of about 1 kilodalton (NHS)-PEG2K+C18 (Naonsoft Polymers TM SKU #10668-2000) where n is an integer and the sum of all PEG units has a molecular weight of about 2 kilodaltons (NHS)-PEG5K+C18 (Naonsoft Polymers TM SKU#10668-5000) where n is an integer and the sum of all PEG units has a molecular weight of about 5 kilodaltons (MAL)-PEG5K+C18 (Naonsoft Polymers TM SKU #10647) where n is an integer and the sum of all PEG units has a molecular weight of about 5 kilodaltons PEG48+C18

[0107] In some embodiments, the RNAi agent can be bound to a lipid PK / PD modulator of formula (I): or a pharmaceutically acceptable salt thereof, wherein LA is a bond or Z is linked to the RNAi agent A bivalent moiety; Z is CH, phenyl, or N; L1 and L2 are each independently a linker comprising at least about 5 polyethylene glycol (PEG) units; X and Y are each independently comprising about 10 to about 50 carbon atoms of the lipid; and indicates the point of attachment to the RNAi agent.

[0108] In some embodiments, L1 and L2 each independently comprise from about 15 to about 100 PEG units. In some embodiments, L1 and L2 each independently comprise from about 20 to about 60 PEG units. In some embodiments, L1 and L2 each independently comprise about 20 to about 30 PEG units. In some embodiments, L1 and L2 each independently comprise about 40 to about 60 PEG units. In some embodiments, one of L1 and L2 comprises about 20 to about 30 PEG units and the other comprises about 40 to about 60 PEG units. For example, L1 and L2 can each independently comprise 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, 43, 44, 45, 46, 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, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 PEG units. In some embodiments, each of L1 and L2 comprises one or more additional divalent moieties linking the two PEG units in the linker (e.g., -C(O)-, -N(H)-, - N(H)-C(O)-, -C(O)-N(H)-, -S(O)2-, -S- and other divalent moieties other than PEG). For example, each of L1 and L2 comprises the structure or, wherein each X' is independently a divalent moiety other than a PEG unit, and each PEG is a PEG unit.

[0109] In some embodiments, each of L1 and L2 is independently selected from the group consisting of the moieties identified in Table 2.

[0110] Table 2: Example L1 and L2 parts of the present invention name structure linker 1 linker 2 linker 3 linker 4 linker 5 linker 6 connexon 7 connexon 8 connexon 9 connexon 10 connexon 11 linker 12 Wherein, each p is independently 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 or 30; each q is independently 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 or 30; each r is independently 1, 2, 3, 4, 5, 6, 7 , 8, 9, or 10; and each indicates the point of attachment to X, Y, or Z, provided that: (i) in linkers 1, 6, and 11, p+q+r≥5; (ii) in In linkers 2, 3, 7, 8, 9 and 10, p+q > 5; and (iii) in linkers 4 and 5, p > 5.

[0111] In some embodiments, each p is independently 20, 21, 22, 23, 24, or 25; each q is independently 20, 21, 22, 23, 24, or 25; and each r is independently 2 , 3, 4, 5 or 6. In some embodiments, each p is independently 23 or 24. In some embodiments, each q is independently 23 or 24. In some embodiments, each r is 4.

[0112] In some embodiments, each of L1 and L2 is independently selected from the group consisting of the moieties identified in Table 3.

[0113] Table 3: Example L1 and L2 sections of the present invention where the point of attachment to X, Y or Z is indicated.

[0114] In some embodiments, L1 is the same as L2. In other embodiments, L1 is different from L2.

[0115] In some embodiments, at least one of X and Y is an unsaturated lipid. In some embodiments, each of X and Y is an unsaturated lipid. In some embodiments, at least one of X and Y is a saturated lipid. In some embodiments, each of X and Y is a saturated lipid. In some embodiments, at least one of X and Y is a branched lipid. In some embodiments, each of X and Y is a branched lipid. In some embodiments, at least one of X and Y is a linear lipid. In some embodiments, each of X and Y is a linear lipid. In some embodiments, at least one of X and Y is cholesteryl. In some embodiments, each of X and Y is cholesteryl. In some embodiments, X and Y are the same. In other embodiments, X and Y are different.

[0116] In some embodiments, at least one of X and Y comprises from about 10 to about 45 carbon atoms. In some embodiments, at least one of X and Y comprises about 10 to about 40 carbon atoms. In some embodiments, at least one of X and Y comprises from about 10 to about 35 carbon atoms. In some embodiments, at least one of X and Y comprises about 10 to about 30 carbon atoms. In some embodiments, at least one of X comprises about 10 to about 25 carbon atoms. In some embodiments, at least one of X and Y comprises about 10 to about 20 carbon atoms.

[0117] In some embodiments, X and Y each independently comprise from about 10 to about 45 carbon atoms. In some embodiments, X and Y each independently comprise from about 10 to about 40 carbon atoms. In some embodiments, X and Y each independently comprise from about 10 to about 35 carbon atoms. In some embodiments, X and Y each independently comprise from about 10 to about 30 carbon atoms. In some embodiments, X and Y each independently comprise from about 10 to about 25 carbon atoms. In some embodiments, X and Y each independently comprise from about 10 to about 20 carbon atoms. For example, X and Y may each independently comprise 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, 43, 44, 45, 46, 47, 48, 49 or 50 carbon atoms.

[0118] In some embodiments, at least one of X and Y is selected from the group consisting of moieties identified in Table 4. In some embodiments, each of X and Y is independently selected from the group consisting of the moieties identified in Table 4.

[0119] Table 4: Example X and Y parts of the present invention name structure Lipid 1 Lipid 2 Lipid 3 Lipid 4 (cholesteryl) Lipid 5 Lipid 6 Lipid 7 Lipid 8 Lipid 9 Lipid 10 Lipid 11 Lipid 12 Lipid 14 Lipid 15 Lipid 16 Lipid 17 Lipid 18 Lipid 19 Lipid 20 Lipid 21 Lipid 22 Lipid 23 Lipid 24 where the point of connection to L1 or L2 is indicated.

[0120] In some embodiments, the LA comprises at least one PEG unit. In some embodiments, LA does not contain any PEG units. In some embodiments, LA comprises -C(O)-, -C(O)N(H)-, optionally substituted alkoxy, or optionally substituted alkylene heterocyclyl. In some embodiments, LA is a bond.

[0121] In some embodiments, the LA is selected from the group consisting of the moieties identified in Table 5.

[0122] Table 5: Example LA Sections of the Invention name structure tether 1 tether 2 tether 3 tether 4 tether 5 tether 6 tether 7 tether 8 tether 9 tether 10 Tether 11 tether 12 Tether 13 Tether 14 Wherein, each of m, n, o and a is independently 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 or 30, and each indicates the point of attachment to Z or the RNAi agent.

[0123] In some embodiments, each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21, 22, 23, or 25; each n is independently 2 , 3, 4 or 5; each a is independently 2, 3 or 4; In some embodiments, each m is independently 2, 4, 8 or 24. In some embodiments, each n is 3. In some embodiments, each o is independently 4, 8 or 12. In some embodiments, each a is 3.

[0124] Another aspect of the present invention provides a lipid PK / PD regulator of formula (Ia): or a pharmaceutically acceptable salt thereof, wherein LA, L1, L2, X and Y are as in formula (I) as defined in any of the embodiments of a lipid PK / PD modulator; and indicates the point of attachment to the RNAi agent.

[0125] In some embodiments, X and Y are each independently selected from lipid 3, lipid 4, lipid 5, lipid 6, lipid 7, lipid 10, lipid Cluster of lipid 12 and lipid 19, each of which indicates the point of attachment to L1 or L2.

[0126] In some embodiments, each of L1 and L2 is independently selected from the group consisting of Linker 2, Linker 3, Linker 4, and Linker 5 as set forth in Table 2, wherein each indicates The point of attachment to X, Y or CH of formula (Ia). In some embodiments, each p is 23. In some embodiments, each q is 24.

[0127] In some embodiments, LA is selected from the group consisting of tether2, tether3, and tether4 as set forth in Table 5. In some embodiments, each m is independently 2, 4, 8 or 24. In some embodiments, each n is 4. In some embodiments, each o is independently 4, 8 or 12.

[0128] In some embodiments, L1 and L2 are independently selected from the group consisting of and; wherein each p is independently 20, 21, 22, 23, 24 or 25; each q is independently 20, 21, 22 , 23, 24, or 25; and each indicates the point of attachment to X, Y, or CH of formula (Ia). In some embodiments, each p is 24. In some embodiments, each q is 24.

[0129] In some embodiments, LA is and each indicates the point of attachment to the RNAi agent or CH of Formula (Ia).

[0130] In some embodiments, each of X and Y is; wherein indicates a point of attachment to L1 or L2.

[0131] In some embodiments, the lipid PK / PD modulator of formula (Ia) is selected from the group consisting of LP 210a or LP 217a as set forth in Table 15, or such lipid PK / PD A pharmaceutically acceptable salt of any of the modulators, wherein each LAA is a bond or a divalent moiety linking the RNAi agent to the rest of the lipid PK / PD modulator, and each indicated with the RNAi agent Junction.

[0132] In some embodiments, the lipid PK / PD modulator of formula (Ia) is selected from the group consisting of LP 210b and LP 217b as set forth in Table 17, or such lipid PK / PD A pharmaceutically acceptable salt of any of the modulators, wherein each indicates a point of attachment to the RNAi agent.

[0133] Another aspect of the present invention provides a lipid PK / PD regulator of formula (Ib): or a pharmaceutically acceptable salt thereof, wherein LA, L1, L2, X and Y are as in formula (I) or The lipid PK / PD modulator of (Ia) is defined in any of the embodiments and indicates the point of attachment to the RNAi agent.

[0134] In some embodiments, X and Y are each independently selected from the group consisting of Lipid 3 and Lipid 19 as set forth in Table 4, wherein each indicates a point of attachment to L1 or L2. In some embodiments, X and Y are each lipid 3. In some embodiments, each of X and Y is lipid 19.

[0135] In some embodiments, each of L1 and L2 is independently selected from the group consisting of Linker 3, Linker 5, and Linker 9 as set forth in Table 2, wherein each indicates the same relationship as X, Y Or the connection point of the benzene ring of formula (Ib). In some embodiments, each p is 23 or 24. In some embodiments, each q is 24.

[0136] In some embodiments, the LA is selected from the group consisting of Tembron 5, Thong 6, Thong 7, Thong 8, and Thong 14 as set forth in Table 5, wherein each The point of attachment to the RNAi agent or the phenyl ring of formula (Ib) is indicated. In some embodiments, each m is 2 or 4. In some embodiments, each a is 3.

[0137] Another aspect of the present invention provides a lipid PK / PD regulator of formula (Ib1): or a pharmaceutically acceptable salt thereof, wherein LA, L1, L2, X and Y are as in formula (I), The lipid PK / PD modulator of (Ia) or (Ib) is defined in either embodiment and indicates the point of attachment to the RNAi agent.

[0138] Another aspect of the present invention provides a lipid PK / PD modulator of formula (Ic): or a pharmaceutically acceptable salt thereof, wherein LA, L1, L2, X and Y are as in formula (I), (Ia), (Ib) or (Ib1) as defined in any of the embodiments of the lipid PK / PD modulator and indicates the point of attachment to the RNAi agent.

[0139] In some embodiments, X and Y are each independently selected from lipid 1, lipid 2, lipid 3, lipid 5, lipid 8, lipid 9, lipid 11. Lipid 12, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, Lipid 19, Lipid 20, Lipid 21, Lipid 22, Lipid 23 and Lipid 24 of groups, each of which indicates a connection point with L1 and L2. In some embodiments, each of X and Y is Lipid 1, Lipid 2, Lipid 3, Lipid 5, Lipid 8, Lipid 9, Lipid 11, Lipid 12, Lipid 14 , Lipid 15, Lipid 16, Lipid 17, Lipid 18, Lipid 19, Lipid 20, Lipid 21, Lipid 22, Lipid 23 or Lipid 24.

[0140] In some embodiments, each of L1 and L2 is independently selected from the group consisting of Linker 1, Linker 6, Linker 10, Linker 11, and Linker 12 as set forth in Table 2 , wherein each indicates the point of attachment to X, Y, or N of formula (Ic). In some embodiments, each p is independently 23 or 24. In some embodiments, each q is independently 23 or 24. In some embodiments, each r is 4.

[0141] In some embodiments, the LA is selected from the group consisting of tether 1, tether 9, tether 10, tether 11, tether 12, and tether 13 as set forth in Table 5 wherein each indicates a point of attachment to the RNAi agent or N of formula (Ic).

[0142] Another aspect of the present invention provides a lipid PK / PD modulator of formula (Id): or a pharmaceutically acceptable salt thereof, wherein Z, L1, L2, X and Y are as in formula (I), (Ia), (Ib), (Ibl) or (Ic) as defined in any of the Examples of the lipid PK / PD modulator and indicates the point of attachment to the RNAi agent.

[0143] Another aspect of the present invention provides a lipid PK / PD modulator of formula (II): or a pharmaceutically acceptable salt thereof, wherein X and Y are as in formulas (I), (Ia), (Ib ), (Ib1), (Ic) or (Id) as defined in any embodiment of the lipid PK / PD modulator; L12 is L1 such as formula (I), (Ia), (Ib), (Ib1), (Ic) or (Id) as defined in any embodiment of the lipid PK / PD modulator; L22 is L2 such as formula (I), (Ia), (Ib), (Ib1), (Ic) or (Id ) as defined in any embodiment of the lipid PK / PD modulator; LA2 is LA such as the lipid PK / PD modulator of formula (I), (Ia), (Ib), (Ib1) or (Ic) As defined in any one of the embodiments; R1, R2 and R3 are each independently hydrogen or C1-6 alkyl; and indicate the connection point with the RNAi agent.

[0144] In some embodiments; LA2 is a bond or a divalent moiety linking the RNAi agent to -C(O)-; R1, R2, and R3 are each independently hydrogen or C1-6 alkyl; L12 and L22 each independently is a linker comprising at least about 5 PEG units; X and Y are each independently a lipid comprising about 10 to about 50 carbon atoms; and indicate a point of attachment to the RNAi agent.

[0145] In some embodiments, each of L12 and L22 is independently selected from the group consisting of the moieties identified in Table 6.

[0146] Table 6: Example L12 and L22 parts of the present invention name structure Linker 1-2 Linker 2-2 Wherein, p and q are each independently 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 or 30; and each indicates the point of attachment to X, Y, -NR2- or -NR3-, subject to the following restrictions: (i) at linker 1 In -2, p+q≥5; and (ii) in linker 2-2, p≥5.

[0147] In some embodiments, each p is independently 20, 21, 22, 23, 24, or 25. In some embodiments, each q is independently 20, 21, 22, 23, 24 or 25. In some embodiments, each p is independently 23 or 24. In some embodiments, each p is 23. In some embodiments, each q is 24.

[0148] In some embodiments, L12 is the same as L22. In other embodiments, L12 is different from L22.

[0149] In some embodiments, at least one of X and Y is selected from the group consisting of moieties identified in Table 4, wherein each indicates a point of attachment to L12 or L22. In some embodiments, each of X and Y is independently selected from the group consisting of the moieties identified in Table 4, wherein each indicates a point of attachment to L12 or L22.

[0150] In some embodiments, at least one of X and Y is selected from the group consisting of moieties identified in Table 7. In some embodiments, each of X and Y is independently selected from the group consisting of the moieties identified in Table 7.

[0151] Table 7: Exemplary Parts X and Y of Lipid PK / PD Modulators of Formula (II) name structure Lipid 3 Lipid 4 Lipid 5 Lipid 6 Lipid 7 Lipid 10 Lipid 12 Lipid 19 Which indicates the connection point with L21 or L22.

[0152] In some embodiments, LA2 comprises at least one PEG unit. In some embodiments, LA2 does not contain any PEG units. In some embodiments, LA2 comprises -C(O)-, -C(O)NH-, optionally substituted alkoxy, or optionally substituted alkylene heterocyclyl. In some embodiments, LA2 is a key.

[0153] In some embodiments, LA2 is selected from the group consisting of the moieties identified in Table 8.

[0154] Table 8: Example LA2 portions of the present invention name structure Tether 1-2 Tether 2-2 Tether 3-2 wherein each of m, n and o is independently 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 or 30, and each indicates the point of attachment to the RNAi agent or -C(O)-.

[0155] In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21, 22, 23, or 25. In some embodiments, m is 2, 4, 8 or 24. In some embodiments, each n is 2, 3, 4 or 5. In some embodiments, n is 4. In some embodiments, o is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13. In some embodiments, o is 4, 8 or 12.

[0156] In some embodiments, each of R1, R2, and R3 is independently hydrogen or C1-3 alkyl. In some embodiments, each of R1, R2, and R3 is hydrogen.

[0157] In some embodiments, the lipid PK / PD modulator of formula (II) is selected from the group consisting of LP 38a, LP 39a, LP 43a, LP 44a, LP 45a as set forth in Table 15 , LP 47a, LP 53a, LP 54a, LP 55a, LP 57a, LP 58a, LP 59a, LP 62a, LP 101a, LP 104a, and LP 111a or any of these lipid PK / PD modulators A pharmaceutically acceptable salt wherein each LAA is a bond or a divalent moiety linking the RNAi agent to the rest of the lipid PK / PD modulator, and each indicates the point of attachment to the RNAi agent.

[0158] In some embodiments, the lipid PK / PD modulator of formula (II) is selected from the group consisting of LP 38b, LP 39b, LP 41b, LP 42b, LP 43b as set forth in Table 17 , LP 44b, LP 45b, LP 47b, LP 53b, LP 54b, LP 55b, LP 57b, LP 58b, LP 59b, LP 60b, LP 62b, LP 101b, LP 104b, LP 106b, LP 107b, LP 108b, LP 109b and LP 111b or a pharmaceutically acceptable salt of any of these lipid PK / PD modulators, wherein each indicates a point of attachment to the RNAi agent.

[0159] Another aspect of the present invention provides a lipid PK / PD modulator of formula (III): or a pharmaceutically acceptable salt thereof, wherein X and Y are as in formulas (I), (Ia), (Ib ), (Ib1), (Ic), (Id) or any embodiment of the lipid PK / PD regulator of (II) is defined; L13 is such as formula (I), (Ia), (Ib), ( L1 or L13 as defined in any embodiment of the lipid PK / PD modulator of Ib1), (Ic) or (Id) is as defined in any embodiment of the lipid PK / PD modulator of formula (II) L12; L23 is L2 as defined in any one embodiment of the lipid PK / PD modulator of formula (I), (Ia), (Ib), (Ib1), (Ic) or (Id), or L23 L22 as defined in any one embodiment of the lipid PK / PD modulator of formula (II); W1 is -C(O)NR1- or -OCH2CH2NR1C(O)-, wherein R1 is hydrogen or C1-6 alkane base; W2 is -C (O) NR2- or -OCH2CH2NR2C (O)-, wherein R2 is hydrogen or C1-6 alkyl; LA3 is such as formula (I), (Ia), (Ib), (Ib1) or LA as defined in any of the embodiments of the lipid PK / PD modulator of (Ic), or LA3 is LA2 as defined in any of the embodiments of the lipid PK / PD modulator of formula (II); Attachment points for RNAi agents.

[0160] In some embodiments, LA3 is a bond or a bivalent moiety linking the RNAi agent to a benzene ring; W1 is -C(O)NR1- or -OCH2CH2NR1C(O)-, wherein R1 is hydrogen or C1- 6 alkyl; W2 is -C(O)NR2- or -OCH2CH2NR2C(O)-, wherein R2 is hydrogen or C1-6 alkyl; L13 and L23 are each independently a linker comprising at least about 5 PEG units; X and Y are each independently a lipid comprising about 10 to about 50 carbon atoms; and indicate a point of attachment to the RNAi agent

[0161] In some embodiments, each of L13 and L23 is independently selected from the group consisting of the moieties identified in Table 9.

[0162] Table 9: Example L13 and L23 parts of the present invention name structure Linker 1-3 Linker 2-3 Linker 3-3 Wherein, p and q are each independently 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 or 30; and each indicates the connection point with X, Y, W1 or W2; In linker 3-3, p+q≥5; and (ii) in linker 2-3, p≥5.

[0163] In some embodiments, each p is independently 20, 21, 22, 23, 24, or 25. In some embodiments, each p is independently 23 or 24. In some embodiments, each p is 23. In some embodiments, each p is 24. In some embodiments, each q is independently 20, 21, 22, 23, 24 or 25. In some embodiments, each q is 24.

[0164] In some embodiments, at least one of X and Y is selected from the group consisting of moieties identified in Table 4, wherein each indicates a point of attachment to L13 or L23. In some embodiments, each of X and Y is independently selected from the group consisting of the moieties identified in Table 4, wherein each indicates a point of attachment to L13 or L23.

[0165] In some embodiments, at least one of X and Y is selected from the group consisting of moieties identified in Table 10. In some embodiments, each of X and Y is independently selected from the group consisting of the moieties identified in Table 10.

[0166] Table 10: Exemplary Parts X and Y of Lipid PK / PD Modulators of Formula (III) name structure Lipid 3 Lipid 19 where the point of connection to L13 or L23 is indicated.

[0167] In some embodiments, LA3 comprises at least one PEG unit. In some embodiments, LA3 does not contain any PEG units. In some embodiments, LA3 comprises -C(O)-, -C(O)NH-, optionally substituted alkoxy, or optionally substituted alkylene heterocyclyl. In some embodiments, LA3 is a key.

[0168] In some embodiments, LA3 is selected from the group consisting of the moieties identified in Table 11.

[0169] Table 11: Example LA3 portion of the present invention name structure Tether 1-3 Tether 2-3 Tether 3-3 Tether 4-3 Tether 5-3 Wherein, each of m and a is independently 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 or 30, and each indicates the point of attachment to the RNAi agent or the benzene ring of formula (III).

[0170] In some embodiments, m is 1, 2, 3, 4, 5, 20, 21, 22, 23, or 25. In some embodiments, m is 1, 2, 3, 4 or 5. In some embodiments, m is 2 or 4. In some embodiments, a is 2, 3, 4 or 5. In some embodiments, a is 3.

[0171] In some embodiments, each of R1 and R2 is independently hydrogen or C1-3 alkyl (eg, methyl, ethyl, or n-propyl). In some embodiments, both R1 and R2 are hydrogen.

[0172] In some embodiments, the lipid PK / PD modulator of formula (III) is selected from the group consisting of LP 110a, LP 124a, LP 130a and LP 220a as set forth in Table 15 or A pharmaceutically acceptable salt of any of the lipid PK / PD modulators, wherein each LAA is a bond or bivalent moiety linking the RNAi agent to the remainder of the lipid PK / PD modulator; and each Points of attachment to RNAi agents are indicated.

[0173] In some embodiments, the lipid PK / PD modulator of formula (III) is selected from the group consisting of LP 110b, LP 124b, LP 130b, LP 143b, LP 220b as set forth in Table 17 , LP 221b, and LP 240b, or a pharmaceutically acceptable salt of any of these lipid PK / PD modulators, wherein each indicates a point of attachment to the RNAi agent.

[0174] Another aspect of the present invention provides a lipid PK / PD modulator of formula (IIIa): or a pharmaceutically acceptable salt thereof, wherein X and Y are as in formulas (I), (Ia), (Ib ), (Ib1), (Ic), (Id), (II) or (III) as defined in any embodiment of the lipid PK / PD modulator; L13 is such as formula (I), (Ia), ( Ib), (Ib1), (Ic) or (Id) lipid PK / PD modulator defined in any embodiment of L1, L13 is any one of the lipid PK / PD modulator such as formula (II) L12 as defined in the embodiment, or L13 is as defined in any embodiment of the lipid PK / PD regulator of formula (III); L23 is as defined in formula (I), (Ia), (Ib), (Ib1) , L2 as defined in any embodiment of the lipid PK / PD modulator of (Ic) or (Id), L23 is L22 as defined in any embodiment of the lipid PK / PD modulator of formula (II) , or L13 is as defined in any of the embodiments of the lipid PK / PD modulator of formula (III); LA3 is a lipid such as formula (I), (Ia), (Ib), (Ib1) or (Ic) LA as defined in any of the embodiments of lipid PK / PD modulators, LA3 is LA2 as defined in any of the embodiments of lipid PK / PD modulators of formula (II), or LA3 is lipids of formula (III) each of R1 and R2 is as defined in any one of the embodiments of lipid PK / PD modulators of formula (II) or (III); and indicates The point of attachment to the RNAi agent.

[0175] In some embodiments, LA3 is a bond or a divalent moiety linking the RNAi agent to a benzene ring; R1 and R2 are each independently hydrogen or C1-6 alkyl (e.g., methyl, ethyl, n-propyl L, n-butyl or n-pentyl); L13 and L23 are each independently a linker comprising at least about 5 PEG units; X and Y are each independently a lipid comprising about 10 to about 50 carbon atoms; and Points of attachment to RNAi agents are indicated.

[0176] In some embodiments, each of L13 and L23 is selected from the group consisting of Linkers 1-3 and Linkers 2-3 as set forth in Table 9, wherein each indicates the same as formula (IIIa) The connection point of X, Y, -NR1- or -NR2-, the restriction conditions are: (i) in the linker 1-3, p+q≥5; and (ii) in the linker 2-3 , p≥5.

[0177] In some embodiments, one of L13 and L23 is linker 1-3 and the other is linker 2-3. In some embodiments, each of L13 and L23 is linker 1-3. In some embodiments, each of L13 and L23 is linker 2-3.

[0178] In some embodiments, each p is independently 23 or 24. In some embodiments, each p is 23. In some embodiments, each p is 24. In some embodiments, q is 24.

[0179] In some embodiments, at least one of X and Y is selected from the group consisting of lipid 3 and lipid 19 as set forth in Table 10, wherein each indicates the same as L13 in formula (IIIa) or Junction point of L23. In some embodiments, each of X and Y is independently selected from the group consisting of Lipid 3 and Lipid 19. In some embodiments, one of X and Y is lipid 3 and the other is lipid 19. In some embodiments, each of X and Y is lipid 3. In some embodiments, each of X and Y is lipid 19.

[0180] In some embodiments, LA3 is selected from the group consisting of Embron 1-3, Ebolion 2-3, and Ebolion 5-3 as set forth in Table 11, wherein each is indicated with an RNAi agent Or the connection point of the benzene ring of formula (IIIa). In some embodiments, LA3 is tether 1-3. In some embodiments, LA3 is tether 2-3. In some embodiments, LA3 is tether 5-3.

[0181] In some embodiments, m is 1, 2, 3, 4, 5, 20, 21, 22, 23, or 25. In some embodiments, m is 1, 2, 3, 4 or 5. In some embodiments, m is 2 or 4. In some embodiments, a is 2, 3, 4 or 5. In some embodiments, a is 3.

[0182] In some embodiments, each of R1 and R2 is independently hydrogen or C1-3 alkyl. In some embodiments, each of R1 and R2 is hydrogen.

[0183] In some embodiments, the lipid PK / PD modulator of formula (IIIa) is selected from the group consisting of LP 110a, LP 124a, and LP 130a as set forth in Table 15 or such lipid PK / A pharmaceutically acceptable salt of any one of the PD modulators, wherein each LAA is a bond or a bivalent moiety linking the RNAi agent to the remainder of the lipid PK / PD modulator; and each indicates the same as RNAi The connection point of the agent.

[0184] In some embodiments, the lipid PK / PD modulator of Formula (IIIa) is selected from the group consisting of LP 110b, LP 124b, LP 130b, LP 143b, and LP 240b as set forth in Table 17 or a pharmaceutically acceptable salt of any of these lipid PK / PD modulators, wherein each indicates a point of attachment to the RNAi agent.

[0185] Another aspect of the present invention provides a lipid PK / PD modulator of formula (IIIb): or a pharmaceutically acceptable salt thereof, wherein X and Y are as in formulas (I), (Ia), (Ib ), (Ib1), (Ic), (Id), (II), (III) or (IIIa) as defined in any embodiment of the lipid PK / PD regulator; L13 is such as formula (I), ( Ia), (Ib), (Ib1), (Ic) or (Id) lipid PK / PD modulator defined in any embodiment of L1, L13 is the lipid PK / PD regulator such as formula (II) L12 as defined in any embodiment of the agent, or L13 as defined in any embodiment of the lipid PK / PD modulator of formula (III) or (IIIa); L23 is as defined in any embodiment of the lipid PK / PD modulator of formula (I), (Ia) , (Ib), (Ib1), (Ic) or L2 as defined in any embodiment of the lipid PK / PD regulator of (Id), L23 is the lipid PK / PD regulator of formula (II) L22 as defined in any embodiment, or L13 is as defined in any embodiment of the lipid PK / PD regulator of formula (III) or (IIIa); LA3 is as defined in formula (I), (Ia), ( LA as defined in any one embodiment of the lipid PK / PD modulator of Ib), (Ib1) or (Ic), LA3 is as defined in any embodiment of the lipid PK / PD modulator of formula (II) LA2, or LA3 is as defined in any of the embodiments of the lipid PK / PD modulator of formula (III) or (IIIa); each of R1 and R2 is as defined in formula (II), (III) or (IIIa ) as defined in any of the embodiments of the lipid PK / PD modulator; and indicates the point of attachment to the RNAi agent.

[0186] In some embodiments, LA3 is a bond or a divalent moiety that connects the RNAi agent to a benzene ring; R1 and R2 are each independently selected from hydrogen or C1-6 alkyl; L13 and L23 are each independently comprising A linker of at least about 5 PEG units; X and Y are each independently a lipid comprising about 10 to about 50 carbon atoms; and indicate a point of attachment to an RNAi agent.

[0187] In some embodiments, each of L13 and L23 is Linker 3-3 as set forth in Table 9, wherein each indicates a point of attachment to X, Y, or -C(O)-, which The restriction is that p+q > 5 in linker 3-3.

[0188] In some embodiments, p is 23 or 24. In some embodiments, p is 23. In some embodiments, p is 24. In some embodiments, q is 24.

[0189] In some embodiments, each of X and Y is Lipid 3 as set forth in Table 10, wherein each indicates a point of attachment to L13 or L23.

[0190] In some embodiments, LA3 is selected from the group consisting of Tembon 3-3 and Tecton 4-3 as set forth in Table 11, wherein each is indicated with an RNAi agent or a benzene of formula (IIIb) The junction of the ring. In some embodiments, LA3 is tether 3-3. In some embodiments, LA3 is tether 4-3.

[0191] In some embodiments, each of R1 and R2 is independently hydrogen or C1-3 alkyl. In some embodiments, each of R1 and R2 is hydrogen.

[0192] In some embodiments, the lipid PK / PD modulator of formula (IIIb) is LP 220a or a pharmaceutically acceptable salt thereof as set forth in Table 15, wherein LAA is a bond or the RNAi agent A bivalent moiety attached to the rest of the lipid PK / PD modulator; and indicates the point of attachment to the RNAi agent.

[0193] In some embodiments, the lipid PK / PD modulator of formula (IIIb) is selected from the group consisting of LP 220b and LP 221b as set forth in Table 17 or such lipid PK / PD modulators A pharmaceutically acceptable salt of any of the agents, wherein each indicates a point of attachment to the RNAi agent.

[0194] Another aspect of the present invention provides a lipid PK / PD regulator of formula (IV): or a pharmaceutically acceptable salt thereof, wherein X and Y are as in formulas (I), (Ia), (Ib ), (Ib1), (Ic), (Id), (II), (III), (IIIa) or (IIIb) lipid PK / PD modulator as defined in any one of the embodiments; L14 is such as formula ( L1 as defined in any embodiment of the lipid PK / PD regulator of I), (Ia), (Ib), (Ib1), (Ic) or (Id), L14 is a lipid such as formula (II) L12 as defined in any of the embodiments of the PK / PD modulator, or L14 is L13 as defined in any of the embodiments of the lipid PK / PD modulator of formula (III), (IIIa) or (IIIb); L24 is L2 as defined in any one embodiment of the lipid PK / PD modulator of formula (I), (Ia), (Ib), (Ib1), (Ic) or (Id), and L24 is as defined in formula ( II) L22 as defined in any embodiment of the lipid PK / PD modulator, or L24 is any embodiment of the lipid PK / PD modulator of formula (III), (IIIa) or (IIIb) L23 as defined; LA4 is LA as defined in any embodiment of the lipid PK / PD modulator of formula (I), (Ia), (Ib), (Ib1) or (Ic), LA4 is as defined in the formula LA2 as defined in any embodiment of the lipid PK / PD modulator of (II), or LA4 is any embodiment of the lipid PK / PD modulator of formula (III), (IIIa) or (IIIb) LA3 as defined; and indicates the point of attachment to the RNAi agent.

[0195] In some embodiments, LA4 is a bond or bivalent moiety linking the RNAi agent to -C(O)-; L14 and L24 are each independently a linker comprising at least about 5 PEG units; X and Each Y is independently a lipid comprising about 10 to about 50 carbon atoms; and indicates a point of attachment to the RNAi agent.

[0196] In some embodiments, each of L14 and L24 is independently selected from the group consisting of the moieties identified in Table 12.

[0197] Table 12: Example L14 and L24 parts of the present invention name structure Linker 1-4 Linker 2-4 Linker 3-4 Linker 4-4 Linker 5-4 wherein each p is independently 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 or 30; each q is independently 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 or 30; each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and each indicates the point of attachment to X, Y or formula (IV), wherein each * indicates the point of attachment to L14 or L24; the constraints are: (i) at linker 1-4, In linker 2-4 and linker 4-4, p+q+r≥5; and (ii) in linker 3-4, p+q≥5.

[0198] In some embodiments, each p is independently 20, 21, 22, 23, 24, or 25. In some embodiments, each p is independently 23 or 24. In some embodiments, each p is 23. In some embodiments, each p is 24. In some embodiments, each q is independently 20, 21, 22, 23, 24 or 25. In some embodiments, each q is independently 23 or 24. In some embodiments, each q is 24. In some embodiments, each q is 23. In some embodiments, r is 2, 3, 4, 5 or 6. In some embodiments, each r is 4.

[0199] In some embodiments, at least one of X and Y is selected from the group consisting of moieties identified in Table 4, wherein each indicates a point of attachment to L14 or L24. In some embodiments, each of X and Y is independently selected from the group consisting of the moieties identified in Table 4, wherein each indicates a point of attachment to L14 or L24.

[0200] In some embodiments, at least one of X and Y is selected from the group consisting of moieties identified in Table 13. In some embodiments, each of X and Y is independently selected from the group consisting of the moieties identified in Table 13.

[0201] Table 13: Exemplary Parts X and Y of Lipid PK / PD Modulators of Formula (IV) name structure Lipid 1 Lipid 2 Lipid 3 Lipid 5 Lipid 8 Lipid 9 Lipid 10 Lipid 11 Lipid 12 Lipid 15 Lipid 16 Lipid 17 Lipid 18 Lipid 19 Lipid 20 Lipid 21 Lipid 22 Lipid 23 Lipid 24 where the point of connection to L14 or L24 is indicated.

[0202] In some embodiments, LA4 comprises at least one PEG unit. In some embodiments, LA4 does not contain any PEG units. In some embodiments, LA4 comprises -C(O)-, -C(O)NH-, optionally substituted alkoxy, or optionally substituted alkylene heterocyclyl. In some embodiments, LA4 is a key.

[0203] In some embodiments, LA4 is selected from the group consisting of the moieties identified in Table 14.

[0204] Table 14: Example LA4 sections of the present invention name structure Tether 1-4 Tether 2-4 Tether 3-4 Tether 4-4 Tether 5-4 Tether 6-4 where each indicates the point of attachment to the RNAi agent or -C(O)- of formula (IV).

[0205] In some embodiments, the lipid PK / PD modulator of formula (IV) is selected from the group consisting of LP 1a, LP 28a, LP 29a, LP 48a, LP 49a as set forth in Table 15 , LP 56a, LP 61a, LP 87a, LP 89a, LP 90a, LP 92a, LP 93a, LP 94a, LP 95a, LP 102a, LP 103a, LP 223a, LP 225a, LP 246a, LP 339a, LP 340a, LP 357a and LP 358a or a pharmaceutically acceptable salt of any of these lipid PK / PD modulators, wherein each LAA is a bond or the rest of the lipid PK / PD modulator linking the RNAi agent and each indicates a point of attachment to the RNAi agent.

[0206] In some embodiments, the lipid PK / PD modulator of formula (IV) is selected from the group consisting of LP 1b, LP 28b, LP 29b, LP 48b, LP 49b as set forth in Table 17 , LP 56b, LP 61b, LP 87b, LP 89b, LP 90b, LP 92b, LP 93b, LP 94b, LP 95b, LP 102b, LP 103b, LP 223b, LP 224b, LP 225b, LP 226b, LP 238b, LP 246b, LP 247b, LP 339b, LP 340b, LP 357b, and LP 358b, or a pharmaceutically acceptable salt of any of these lipid PK / PD modulators, wherein each indicates a point of attachment to the RNAi agent.

[0207] Another aspect of the present invention provides a compound of formula (IVa): or a pharmaceutically acceptable salt thereof, wherein X and Y are as in formulas (I), (Ia), (Ib), (Ib1), (Ic), (II), (III), (IIIa), (IIIb) or (IV) as defined in any embodiment of the compound; L14 and L24 are as defined in any embodiment of the compound of formula (IV) defines; and RZ comprises oligonucleotide-based agents.

[0208] In some embodiments, RZ comprises an oligonucleotide-based agent; each of L14 and L24 is independently selected from the group consisting of and wherein each indicates the same as X, Y, or A connection point, each * indicates a point of attachment to L14 or L24, each p is independently 20, 21, 22, 23, 24 or 25, each q is independently 20, 21, 22, 23, 24 or 25, and each r is independently 2, 3, 4, 5, or 6; and each of X and Y is independently selected from the group consisting of, wherein the point of attachment to L14 or L24 is indicated.

[0209] In some embodiments, each p is independently 23 or 24. In some embodiments, each p is 23. In some embodiments, each p is 24. In some embodiments, each q is independently 23 or 24. In some embodiments, each q is 24. In some embodiments, each q is 23. In some embodiments, each r is 4.

[0210] In some embodiments, the compound of Formula (IVa) is selected from the group consisting of LP 339b, LP 340b, LP 357b, and LP 358b as set forth in Table 16, or any of these compounds wherein each RZ comprises an oligonucleotide-based agent.

[0211] In another aspect of the invention, the RNAi agent can bind to a lipid PK / PD modulator selected from the group consisting of the lipid PK / PD modulators identified in Table 15.

[0212] Table 15: Exemplary lipid PK / PD modulators of the invention (compound number appears before structure). Or a pharmaceutically acceptable salt of any one of these lipid PK / PD modulators, wherein each LAA is of formula (I), (Ia), (Ib), (Ib1), (Ic) LA as defined in any embodiment of the lipid PK / PD modulator, LAA is LA2 as defined in any embodiment of the lipid PK / PD modulator of formula (II), LAA is as defined in formula (III ), (IIIa) or LA3 as defined in any embodiment of the lipid PK / PD regulator of (IIIb), or LAA is in any embodiment of the lipid PK / PD regulator of formula (IV) LA4 as defined; and each indicates the point of attachment to the RNAi agent.

[0213] In some embodiments, each LAA is a bond or bivalent moiety linking the RNAi agent to the rest of the lipid PK / PD modulator; and each indicates the point of attachment to the RNAi agent.

[0214] In another aspect of the invention, the RNAi agent can be conjugated to a lipid PK / PD modulator selected from the group consisting of the lipid PK / PD modulators identified in Table 16.

[0215] Table 16: Exemplary lipid PK / PD modulators of the invention (compound number appears before structure). Or a pharmaceutically acceptable salt of any one of these lipid PK / PD modulators, wherein each LAA is of formula (I), (Ia), (Ib), (Ib1), (Ic) LA as defined in any embodiment of the lipid PK / PD modulator, LAA is LA2 as defined in any embodiment of the lipid PK / PD modulator of formula (II), LAA is as defined in formula (III ), (IIIa) or LA3 as defined in any embodiment of the lipid PK / PD regulator of (IIIb), or LAA is in any embodiment of the lipid PK / PD regulator of formula (IV) LA4 as defined; and each indicates the point of attachment to the RNAi agent.

[0216] In some embodiments, each LAA is a bond or bivalent moiety linking the RNAi agent to the rest of the lipid PK / PD modulator; and each indicates the point of attachment to the RNAi agent.

[0217] In some embodiments, the RNAi agent can bind to a lipid PK / PD modulator selected from the group consisting of the lipid PK / PD modulators identified in Table 17.

[0218] Table 17: Exemplary lipid PK / PD modulators of the invention (compound number appears before structure). or a pharmaceutically acceptable salt of any of these lipid PK / PD modulators, wherein each indicates a point of attachment to the RNAi agent.

[0219] In another aspect of the invention, the RNAi agent can bind to a lipid PK / PD modulator selected from the group consisting of the lipid PK / PD modulators identified in Table 1.

[0220] Table 18: Exemplary lipid PK / PD modulators of the invention (compound number appears before structure). or a pharmaceutically acceptable salt of any of these lipid PK / PD modulators, wherein each indicates a point of attachment to the RNAi agent.

[0221] In some embodiments, a lipid PK / PD modulator precursor suitable for linking to an RNAi agent may be a lipid PK / PD modulator precursor of formula (V): or a pharmaceutically acceptable Salt, wherein Z, L1, L2, X and Y are as defined in any embodiment of the lipid PK / PD modulator of formula (I), (Ia), (Ib), (Ib1) or (Ic); J is LA5-RX; LA5 is a bond or a bivalent moiety linking RX to Z; and RX is a reactive moiety for binding to an RNAi agent.

[0222] In some embodiments, J is LA5-RX; LA5 is a bond or a bivalent moiety linking RX to Z; RX is a reactive moiety for binding to an RNAi agent; Z is CH, phenyl, or N; L1 and L2 are each independently a linker comprising at least about 5 PEG units; and X and Y are each independently a lipid comprising about 10 to about 50 carbon atoms.

[0223] In some embodiments, LA5 is LA as defined in any of the embodiments of the lipid PK / PD modulator of formula (I), (Ia), (Ib), (Ib1) or (Ic) . In some embodiments, LA5 is selected from the group consisting of moieties identified in Table 19.

[0224] Table 19: Example LA5 portions of the present invention name structure Tether 1-5 Tether 2-5 Tether 3-5 Tether 4-5 Tether 5-5 Tether 6-5 Tether 7-5 Tether 8-5 Tether 9-5 Tether 10-5 Tether 11-5 Tether 12-5 Tether 13-5 wherein each of m, n, o and a is independently 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 or 30 and each of them indicates the connection point to Z or RX.

[0225] In some embodiments, each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21, 22, 23, or 25; each n is independently 2 , 3, 4 or 5; each a is independently 2, 3 or 4;

[0226] In some embodiments, each m is independently 2, 4, 8, or 24. In some embodiments, each n is 4. In some embodiments, each o is independently 4, 8 or 12. In some embodiments, each a is 3.

[0227] In some embodiments, RX is selected from the group consisting of, wherein each indicates a point of attachment to LA5. In some embodiments, RX is . In some embodiments, RX is . In some embodiments, RX is . In some embodiments, RX is .

[0228] In some embodiments, J is selected from the group consisting of moieties identified in Table 20.

[0229] Table 20: Example J Parts of the Invention where each indicates the point of attachment to Z.

[0230] Another aspect of the present invention provides the lipid PK / PD regulator precursor of formula (Va): or a pharmaceutically acceptable salt thereof, wherein J, L1, L2, X and Y are as in formula (V ) as defined in any of the embodiments of the lipid PK / PD modulator precursor.

[0231] In some embodiments, X and Y are each independently selected from lipid 3, lipid 4, lipid 5, lipid 6, lipid 7, lipid 10, lipid Cluster of lipid 12 and lipid 19, each of which indicates the point of attachment to L1 or L2.

[0232] In some embodiments, each of L1 and L2 is independently selected from the group consisting of Linker 2, Linker 3, Linker 4, and Linker 5 as set forth in Table 2, wherein each indicates The point of attachment to X, Y or CH of formula (Va). In some embodiments, each p is 23. In some embodiments, each q is 24.

[0233] In some embodiments, LA5 is selected from the group consisting of Tether2-5, Tether3-5, and Tether4-5 as set forth in Table 19, wherein each is indicated with RX or The point of attachment of CH of formula (Va). In some embodiments, m is 2, 4, 8 or 24. In some embodiments, n is 4. In some embodiments, o is 4, 8 or 12.

[0234] In some embodiments, each of L1 and L2 is independently selected from the group consisting of and; wherein each p is independently 20, 21, 22, 23, 24, or 25; each q is independently 20 , 21, 22, 23, 24, or 25; and each indicates a point of attachment to X, Y, or CH of formula (Va). In some embodiments, each p is 24. In some embodiments, each q is 24.

[0235] In some embodiments, LA5 is; wherein each indicates the point of attachment to RX or CH of formula (Va).

[0236] In some embodiments, each of X and Y is , where indicates a point of attachment to L1 or L2.

[0237] In some embodiments, the lipid PK / PD modulator precursor system of formula (Va) is selected from the group consisting of LP210-p or LP 217-p or such lipids as set forth in Table 21 A pharmaceutically acceptable salt of any of the PK / PD modulator precursors.

[0238] Another aspect of the present invention provides the lipid PK / PD regulator precursor of formula (Vb): or a pharmaceutically acceptable salt thereof, wherein J, L1, L2, X and Y are as in formula (V ) or (Va) as defined in any of the embodiments of the lipid PK / PD modulator precursor.

[0239] In some embodiments, X and Y are each independently selected from the group consisting of Lipid 3 and Lipid 19 as set forth in Table 4, wherein each indicates a point of attachment to L1 or L2. In some embodiments, X and Y are each lipid 3. In some embodiments, X and Y are each lipid 19.

[0240] In some embodiments, each of L1 and L2 is independently selected from the group consisting of Linker 3, Linker 5, and Linker 9 as set forth in Table 2, wherein each indicates the same value as that of formula (Vb ) X, Y or the point of attachment of the benzene ring. In some embodiments, p is 23 or 24. In some embodiments, q is 24.

[0241] In some embodiments, LA5 is selected from the group consisting of tether 5-5, tether 6-5, tether 7-5, tether 8-5, and tether Groups of 13-5, each of which indicates the point of attachment to RX or the benzene ring of formula (Vb). In some embodiments, m is 2 or 4. In some embodiments, a is 3.

[0242] Another aspect of the present invention provides the lipid PK / PD regulator precursor of formula (Vb1): or a pharmaceutically acceptable salt thereof, wherein J, L1, L2, X and Y are as in formula (V ), (Va) or (Vb) as defined in any of the embodiments of the lipid PK / PD modulator precursor.

[0243] Another aspect of the present invention provides the lipid PK / PD regulator precursor of formula (Vc): or a pharmaceutically acceptable salt thereof, wherein J, L1, L2, X and Y are as in formula (V ), (Va), (Vb) or (Vb1) as defined in any of the embodiments of the lipid PK / PD modulator precursor.

[0244] In some embodiments, X and Y are each independently selected from lipid 1, lipid 2, lipid 3, lipid 5, lipid 8, lipid 9, lipid 11. Composition of Lipid 12, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, Lipid 19, Lipid 20, Lipid 21, Lipid 22, Lipid 23 and Lipid 24 of groups, each of which indicates a connection point with L1 and L2. In some embodiments, each of X and Y is Lipid 1, Lipid 2, Lipid 3, Lipid 5, Lipid 8, Lipid 9, Lipid 11, Lipid 12, Lipid 14 , Lipid 15, Lipid 16, Lipid 17, Lipid 18, Lipid 19, Lipid 20, Lipid 21, Lipid 22, Lipid 23 or Lipid 24.

[0245] In some embodiments, each of L1 and L2 is independently selected from the group consisting of Linker 1, Linker 6, Linker 10, Linker 11, and Linker 12 as set forth in Table 2 , wherein each indicates the point of attachment to X, Y or N of formula (Vc). In some embodiments, p is 23 or 24. In some embodiments, q is 24. In some embodiments, r is 4.

[0246] In some embodiments, LA5 is selected from tethers 1-5, tethers 9-5, tethers 10-5, tethers 11-5, or tethers as set forth in Table 19 A group consisting of 12-5, each of which indicates the point of attachment to the RNAi agent or N of formula (Vc).

[0247] Another aspect of the present invention provides the lipid PK / PD regulator precursor of formula (Vd): or a pharmaceutically acceptable salt thereof, wherein Z, L1, L2, X and Y are as in formula (V ), (Va), (Vb), (Vb1 ) or (Vc) lipid PK / PD modulator precursor defined in any one of the embodiments.

[0248] Another aspect of the present invention provides the lipid PK / PD regulator precursor of formula (Ve): or a pharmaceutically acceptable salt thereof, wherein Z, L1, L2, RX, LA5, X and Y As defined in any of the embodiments of the lipid PK / PD modulator precursor of formula (V), (Va), (Vb), (Vbl), (Vc) or (Vd).

[0249] Another aspect of the present invention provides the lipid PK / PD regulator precursor of formula (Ve1): or a pharmaceutically acceptable salt thereof, wherein Z, L1, L2, LA5, X and Y are as in the formula (V), (Va), (Vb), (Vb1), (Vc), (Vd) or (Ve) as defined in any of the embodiments of the lipid PK / PD modulator precursor.

[0250] Another aspect of the present invention provides the lipid PK / PD modulator precursor of formula (Ve2): or a pharmaceutically acceptable salt thereof, wherein Z, L1, L2, LA5, X and Y are as in the formula (V), (Va), (Vb), (Vb1), (Vc), (Vd), (Ve) or (Ve1) as defined in any of the embodiments of the lipid PK / PD modulator precursor.

[0251] Another aspect of the present invention provides the lipid PK / PD regulator precursor of formula (Ve3): or a pharmaceutically acceptable salt thereof, wherein Z, L1, L2, LA5, X and Y are as in the formula (V), (Va), (Vb) (Vb1), (Vc), (Vd), (Ve), (Ve1) or (Ve2) in any embodiment of the lipid PK / PD regulator precursor defined.

[0252] Another aspect of the present invention provides the lipid PK / PD regulator precursor of formula (Ve4): or a pharmaceutically acceptable salt thereof, wherein Z, L1, L2, LA5, X and Y are as in the formula (V), (Va), (Vb), (Vb1), (Vc), (Vd), (Ve), (Ve1), (Ve2) or (Ve3) lipid PK / PD modulator precursor defined in any of the examples.

[0253] In some embodiments, the lipid PK / PD modulator precursor is selected from the group consisting of the lipid PK / PD modulator precursors identified in Table 21.

[0254] Table 21: Exemplary lipid PK / PD modulator precursors of the invention (compound number appears before structure). Or a pharmaceutically acceptable salt precursor of any one of these lipid PK / PD modulators.

[0255] In another aspect of the invention, the lipid PK / PD modulator precursor may be selected from the group consisting of the lipid PK / PD modulator precursors identified in Table 22.

[0256] Table 22: Exemplary lipid PK / PD modulator precursors of the invention (compound name appears before structure). Or a pharmaceutically acceptable salt precursor of any one of these lipid PK / PD modulators.

[0257] In some embodiments, the delivery vehicle may comprise one or more PK / PD modulators. In some embodiments, the delivery vehicle comprises one, two, three, four, five, six, seven or more PK / PD modulators.

[0258] PK / PD modulator precursors can be conjugated to RNAi agents using any method known in the art. In some embodiments, a PK / PD modulator precursor comprising a maleimide moiety can be reacted with an RNAi agent comprising a disulfide linkage to form a compound comprising a PK / PD modulator bound to the RNAi agent. The disulfide can be reduced and added to maleimide by means of a Michael-Addition reaction. An exemplary reaction scheme is shown below: wherein compound A is a PK / PD modulator precursor comprising a maleimide moiety, RNAi comprises an RNAi agent, and points of attachment to any suitable group known in the art are indicated. In some embodiments of the above reaction schemes, an alkyl group such as hexyl (C6H13) is attached.

[0259] In some embodiments, a PK / PD modulator precursor can comprise a sulfide moiety and can react with a disulfide. An exemplary reaction scheme is shown below: wherein compound B is a PK / PD modulator precursor comprising a phosphonium moiety, RNAi comprises an RNAi agent, and points of attachment to any suitable group known in the art are indicated. In some cases of the above reaction schemes, attachment is to an alkyl group such as hexyl (C6H13).

[0260] In some embodiments, a PK / PD modulator precursor can comprise an azide moiety and react with an RNAi agent comprising an alkyne to form a compound comprising a PK / PD modulator bound to an RNAi agent according to the following general reaction scheme : wherein compound C is a PK / PD modulator precursor comprising an azide moiety, and the RNAi comprises an RNAi agent.

[0261] In some embodiments, a PK / PD modulator precursor can comprise an alkyne moiety and react with an RNAi agent comprising a disulfide bond to form a compound comprising a PK / PD modulator bound to an RNAi agent according to the following general reaction scheme : wherein compound D is a PK / PD modulator precursor comprising an alkyne, RNAi comprises an RNAi agent, and indicates a point of attachment to any suitable group known in the art. In some cases of the above reaction schemes, attachment is to an alkyl group such as hexyl (C6H13).

[0262] In some embodiments, the PK / PD modulator can bind to the 5' end of the sense or antisense strand, the 3' end of the sense or antisense strand, or an internal nucleotide of the RNAi agent. In some embodiments, the RNAi agent is synthesized with a disulfide bond-containing moiety at the 3' end of the sense strand, and a PK / PD modulator precursor can be incorporated into the sense strand using the appropriate general synthetic scheme shown above. 3' end.

[0263] Examples of PK / PD modulators covalently linked to RNAi agents are shown below: PEG40K (2×2 arms), wherein n and m are each independently an integer, and the sum of all PEG units has a molecular weight of about 40 kilodaltons PEG40K (4 arms), where n is an integer and the sum of all PEG units has a molecular weight of about 40 kilodaltons PEG40K (2 arms), where n is an integer and the sum of all PEG units has a molecular weight of about 40 kilodaltons PEG40K, where n is an integer and the sum of all PEG units has a molecular weight of about 40 kilodaltons PEG10K, where n is an integer and the sum of all PEG units has a molecular weight of about 10 kilodaltons PEG5K, wherein n is an integer and the sum of all PEG units has a molecular weight of about 5 kilodaltons DSPE-PEG5K-NHS wherein n is an integer and the sum of all PEG units has a molecular weight of about 5 kilodaltons DSPE-PEG5K-MAL wherein n is an integer and the sum of all PEG units has a molecular weight of about 5 kilodaltons DSPE-PEG5K-N3 wherein n is an integer and the sum of all PEG units has a molecular weight of about 5 kilodaltons PEG47+C22 PEG47+CLS (Cholesterol) PEG23+C22 Double (PEG23+C14) Double (PEG23+C22) Double (PEG47+C22) PEG48+C22 PEG71+C22 PEG95+C22 PEG71+CLS PEG95+CLS Double (PEG23+C18) Reference (PEG23+C22) Reference (PEG23+CLS) Double (PEG23+CLS) PEG5K+C22 wherein n is an integer and the sum of all PEG units has a molecular weight of about 5 kilodaltons C18 (NHS)-PEG1K+C18 where n is an integer and the sum of all PEG units has a molecular weight of about 1 kilodalton (NHS)-PEG2K+C18 where n is an integer and the sum of all PEG units has a molecular weight of about 2 kilodaltons (NHS)-PEG5K+C18 wherein n is an integer and the sum of all PEG units has a molecular weight of about 5 kilodaltons (MAL)-PEG5K+C18 wherein n is an integer and the sum of all PEG units has a molecular weight of about 5 kilodaltons PEG48+C18 or a pharmaceutically acceptable salt of any of these PK / PD modulators, wherein the point of attachment to the RNAi agent is indicated.

[0264] Linking groups and delivery agents

[0265] In some embodiments, the RNAi agent contains or is bound to one or more non-nucleotide groups, including but not limited to linking groups or delivery agents. Non-nucleotide groups can enhance targeting, delivery or attachment of RNAi agents. Examples of linking groups are provided in Table 23. Non-nucleotide groups can be covalently linked to the 3' and / or 5' ends of the sense and / or antisense strands. In some embodiments, the RNAi agent contains a non-nucleotide group attached to the 3' and / or 5' end of the sense strand. In some embodiments, a non-nucleotide group is attached to the 5' end of the RNAi agent's sense strand. The non-nucleotide group can be linked to the RNAi agent directly or indirectly via a linker / linking group. In some embodiments, the non-nucleotide group is linked to the RNAi agent via a labile, cleavable or reversible bond or linker.

[0266] In some embodiments, the non-nucleotide group enhances the pharmacokinetic or biodistribution properties of the RNAi agent, or the conjugate to which it is attached, to improve the cell- or tissue-specific distribution and cell-specificity of the conjugate. sexual absorption. In some embodiments, the non-nucleotide group enhances endocytosis of the RNAi agent.

[0267] The RNAi agents described herein can be synthesized with reactive groups, such as amine groups (also referred to herein as amines), at the 5' and / or 3' ends. The reactive group can then be used to attach targeting moieties using methods typical in the art.

[0268] For example, in some embodiments, the RNAi agents disclosed herein are synthesized with an NH2-C6 group at the 5' end of the sense strand of the RNAi agent. The terminal amine group can then be reacted with, for example, a group comprising a compound having affinity for one or more integrins (ie, and an integrin targeting ligand) or a PK / PD modulator to form a conjugate. In some embodiments, the RNAi agents disclosed herein are synthesized with one or more alkyne groups at the 5' end of the sense strand of the RNAi agent. The terminal alkynyl group can then be reacted with, for example, a group including a targeting ligand to form a conjugate.

[0269] In some embodiments, the targeting group comprises an integrin targeting ligand. In some embodiments, integrin targeting ligands include compounds that have affinity for integrin alpha-v-beta6. The use of integrin-targeting ligands facilitates cell-specific targeting of cells with individual integrins on their respective surfaces, and binding of integrin-targeting ligands facilitates entry of RNAi agents to which they are attached, such as in skeletal muscle cell of cell. Targeting ligands, targeting groups, and / or PK / PD modulators can be attached to the 3' and / or 5' ends of the RNAi agent, and / or to the end of the RNAi agent using methods generally known in the art. internal nucleotides. The preparation of targeting ligands and targeting groups such as integrin αvβ6 is described in Example 3 below.

[0270] Embodiments of the invention include pharmaceutical compositions for delivering RNAi agents to skeletal muscle cells in vivo. Such pharmaceutical compositions may include, for example, an RNAi agent conjugated to a targeting group comprising an integrin targeting ligand with affinity for integrin αvβ6. In some embodiments, the targeting ligand consists of a compound that has an affinity for integrin αvβ6.

[0271] In some embodiments, the RNAi agents disclosed herein reduce gene expression in one or more of the following tissues: triceps, biceps, quadriceps, gastrocnemius, soleus, EDL (extensor digitorum longus), TA (tibialis anterior), and / or diaphragm.

[0272] In some embodiments, the RNAi agent is synthesized with a linking group that can then facilitate the covalent attachment of the RNAi agent to a targeting ligand, targeting group, PK / PD modulator, or another type of Delivery polymers or delivery vehicles. Linking groups can be attached to the 3' and / or 5' ends of the sense or antisense strand of the RNAi agent. In some embodiments, the linking group is attached to the RNAi agent's sense strand. In some embodiments, the linking group is bound to the 5' or 3' end of the RNAi agent's sense strand. In some embodiments, the linking group is bound to the 5' end of the RNAi agent's sense strand. Examples of linking groups include, but are not limited to: Alk-SMPT-C6, Alk-SS-C6, DBCO-TEG, Me-Alk-SS-C6, and C6-SS-Alk-Me; reactive groups, such as primary Amines and alkynes, alkyls, abasic residues / nucleotides, amino acids, triyne functionalization groups, ribitol and / or PEG units.

[0273] A linker or linking group is a divalent linkage between two atoms that connects one chemical group of interest (such as an RNAi agent) or segment to another via one or more covalent bonds. Chemical groups of interest (such as targeting ligands, targeting groups, PK / PD modulators or delivery agents) or segments. Unstable linkages contain unstable bonds. A linkage can optionally include a spacer that increases the distance between the two joined atoms. Spacers can further increase the flexibility and / or length of the linkage. Spacers include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, and aralkynyl; each of which may contain one or more heteroatoms, heterocycles, amino acids, nucleosides acids and sugars. Spacer groups are well known in the art and the foregoing list is not intended to limit the scope of the invention.

[0274] In some embodiments, the targeting group is attached to the RNAi agent without the use of additional linkers. In some embodiments, targeting groups are designed with readily available linkers to facilitate linkage to the RNAi agent. In some embodiments, when two or more RNAi agents are included in the composition, the two or more RNAi agents can be attached to their respective targeting groups using the same linker. In some embodiments, when two or more RNAi agents are included in the composition, the two or more RNAi agents are attached to their corresponding targeting groups using different linkers.

[0275] In some embodiments, a linking group can be synthetically attached to the 5' or 3' end of the sense strand of an RNAi agent described herein. In some embodiments, the linking group is synthetically attached to the 5' end of the sense strand of the RNAi agent. In some embodiments, the linking group that binds to the RNAi agent may be a triyne linking group.

[0276] Examples of certain modified nucleotides and linking groups are provided in Table 23.

[0277] Table 23. Structures representing various modified nucleotides and linking groups cP When inside an oligo: (invAb) When inside an oligo: (invAb)s When located at the 3' end of an oligonucleotide: (invAb) When located at the 3' end of an oligonucleotide: When inside an oligo: (C6-SS-C6) When located at the 3' end of an oligonucleotide: When inside an oligo: (6-SS-6) (C6-SS-Alk) or (Alk-SS-C6) DBCO-NHS (BroadPharm® BP-22231) L5 (Activate Scientific® AS28942) L6 (BroadPharm® BP-20907)

[0278] Alternatively, other linking groups known in the art may be used.

[0279] In addition or instead of linking the RNAi agent to one or more targeting ligands, targeting groups and / or PK / PD modulators, in some embodiments, the delivery agent can be used to deliver the RNAi agent to the cell or organization. A delivery agent is a compound that can improve the delivery of an RNAi agent to a cell or tissue, and can include, but not be limited to, or consist of: polymers such as amphiphilic polymers, membrane active polymers, peptides, melittin peptides , melittin-like peptide (MLP), lipid, reversibly modified polymer or peptide or reversibly modified membrane active polyamine.

[0280] In some embodiments, RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPC, or other delivery systems available in the art. RNAi agents can also be chemically conjugated to targeting moieties, lipids (including but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, e.g., WO 2000 / 053722, WO 2008 / 022309, WO 2011 / 104169 and WO 2012 / 083185, WO 2013 / 032829, WO 2013 / 158141, each of which is incorporated herein by reference) or other delivery available in the art system.

[0281] pharmaceutical composition

[0282] In some embodiments, the invention provides pharmaceutical compositions comprising, consisting of, or consisting essentially of one or more delivery vehicles comprising an RNAi agent disclosed herein.

[0283] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of an active pharmaceutical ingredient (API) and optionally one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than an active pharmaceutical ingredient (API, therapeutic product) intended to be included in a drug delivery system. An excipient does not exert or is not intended to exert a therapeutic effect at the intended dosage. Excipients can be used to a) aid in the processing of drug delivery systems during manufacturing, b) protect, support or enhance the stability, bioavailability or patient acceptance of the API, c) aid in product identification, and / or d) in storage Or any other attribute that enhances the overall security and effectiveness of API delivery during use. Pharmaceutically acceptable excipients may or may not be inert substances.

[0284] Excipients include, but are not limited to: absorption enhancers, antiadherents, antifoaming agents, antioxidants, binders, buffers, carriers, coatings, pigments, delivery enhancers, delivery polymers , dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, sliding agents, humectants, lubricants, oils, polymers, preservatives, saline, Salts, solvents, sugars, suspending agents, sustained release bases, sweeteners, thickeners, tonicity agents, vehicles, water repellents and wetting agents.

[0285] The pharmaceutical compositions described herein may contain other additional components commonly found in pharmaceutical compositions. In some embodiments, the additional component is a pharmaceutically active substance. Pharmaceutically active substances include (but are not limited to): antipruritics, astringents, local anesthetics or anti-inflammatory agents (such as antihistamines, diphenhydramine, etc.), small molecule drugs, antibodies, antibody fragments, appropriate body and / or vaccines.

[0286] The pharmaceutical composition may also contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for changing osmotic pressure, buffers, coatings or Antioxidants. It may also contain other agents with known therapeutic benefits.

[0287] The pharmaceutical compositions are administered in a variety of ways depending upon whether local or systemic treatment is desired and the area to be treated. Administration can be by any means generally known in the art, such as, but not limited to, topical (e.g., by transdermal patches), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by By nebulizer, intratracheal, intranasal), epidermal, transdermal, oral or parenteral. Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous (eg, via an implanted device), intracranial, intraparenchymal, intrathecal, and intraventricular administration. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection. Pharmaceutical compositions can be administered orally, for example, in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions. Administration can also be rectal, eg, using suppositories; topical or transdermal, eg, using ointments, creams, gels, or solutions; or parenteral, eg, using injectable solutions.

[0288] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL (BASF, Parsippany, N.J.) or phosphate-buffered saline. It should be stable under the conditions of manufacture and storage and should be resistant to the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol such as glycerol, propylene glycol, and liquid polyethylene glycol, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the desired particle size in the case of dispersions and by the use of surfactants. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol), and sodium chloride in the compositions. Prolonged absorption in injectable compositions can be brought about by including in the compositions absorption delaying agents, for example, aluminum monostearate and gelatin.

[0289] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization . Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0290] Formulations suitable for intra-articular administration may be in the form of a sterile aqueous preparation of any of the ligands described herein, which may be in microcrystalline form, eg, in the form of an aqueous microcrystalline suspension. Lipid formulations or biodegradable polymer systems can also be used to deliver any of the ligands described herein for intra-articular and ophthalmic administration.

[0291] The active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These materials can be prepared according to methods known to those skilled in the art, for example as described in US Patent No. 4,522,811.

[0292] The pharmaceutical compositions may contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to, antipruritics, astringents, local anesthetics, or anti-inflammatory agents (eg, antihistamines, diphenhydramine, etc.). As used herein, a "pharmacologically effective amount", "therapeutically effective amount" or simply "effective amount" refers to the amount of a pharmaceutically active agent that produces a pharmacological, therapeutic or prophylactic result.

[0293] Medicaments containing delivery vehicles comprising RNAi agents are also an object of the present invention, as are methods for the manufacture of such medicaments, which methods comprise making RNAi agents and, if necessary, one or more agents with One or more delivery vehicles for other substances of known therapeutic benefit in a pharmaceutically acceptable form.

[0294] The RNAi agent-comprising delivery vehicles and pharmaceutical compositions comprising the RNAi agent-comprising delivery vehicles disclosed herein may be packaged or included in a kit, container, package or dispenser. The delivery vehicle comprising the RNAi agent and the pharmaceutical composition comprising the delivery vehicle comprising the RNAi agent can be packaged in prefilled syringes or vials.

[0295] Therapeutic Methods and Performance Inhibition

[0296] The delivery vehicles disclosed herein comprising an RNAi agent can be used to treat an individual (eg, a human or other mammal) suffering from a disease or condition that would benefit from administration of an RNAi agent. In some embodiments, the delivery vehicles disclosed herein comprising RNAi agents can be used to treat individuals (e.g., humans) who would benefit from a reduction and / or inhibition of mRNA and / or target protein expression, such as those diagnosed with or suffering from Individuals with symptoms associated with muscular dystrophy.

[0297] In some embodiments, a therapeutically effective amount of one or more delivery vehicles disclosed herein comprising an RNAi agent is administered to an individual. Treatment of an individual may include curative and / or prophylactic treatment. An individual can be a human, a patient, or a human patient. An individual can be an adult, adolescent, child or infant. The pharmaceutical compositions described herein can be administered to humans or animals.

[0298] The delivery vehicles described herein comprising an RNAi agent can be used to treat at least one symptom of an individual suffering from a disease or condition associated with a gene of interest or having a disease or condition mediated at least in part by expression of the gene of interest. In some embodiments, a delivery vehicle comprising an RNAi agent is used to treat or manage the clinical manifestations of an individual suffering from a disease or condition that would benefit from, or be at least partially mediated by, a reduction in a target mRNA. A therapeutically effective amount of one or more delivery vehicles comprising an RNAi agent or a composition comprising a delivery vehicle described herein is administered to an individual. In some embodiments, the methods disclosed herein comprise administering to an individual to be treated a composition comprising a delivery vehicle described herein comprising an RNAi agent. In some embodiments, a prophylactically effective amount of any one or more of the described delivery vehicles comprising an RNAi agent is administered to an individual, thereby treating the individual by preventing or inhibiting at least one symptom.

[0299] In certain embodiments, the present invention provides methods for treating a disease, disorder, condition or pathological condition mediated at least in part by expression of a gene of interest in a patient in need thereof, wherein the methods comprise administering the The patient is administered any one of the delivery vehicles described herein comprising an RNAi agent.

[0300] In some embodiments, the gene expression level and / or mRNA level of the gene of interest in the individual to whom the delivery vehicle is administered is reduced by at least about 30%, relative to the individual prior to administration of the delivery vehicle or an individual who did not receive the delivery vehicle. %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or more than 99% . In cells, cell populations and / or tissues of an individual, gene expression and / or mRNA levels in the individual may be reduced.

[0301] In some embodiments, the protein content in the individual to whom the delivery vehicle is administered is reduced by at least about 30%, 35%, 40%, 45%, 50% relative to the individual prior to administration of the delivery vehicle or to an individual who did not receive the delivery vehicle. %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more than 99%. The protein content in a subject may be reduced in cells, cell populations, tissues, blood and / or other bodily fluids of the subject.

[0302] Reduction of mRNA levels and protein levels can be assessed by any method known in the art. As used herein, reduction or reduction of mRNA level and / or protein level is collectively referred to herein as reduction or reduction of target gene or inhibition or reduction of target gene expression. The examples set forth herein illustrate known methods for assessing inhibition of gene expression.

[0303] In some embodiments, a delivery vehicle comprising an RNAi agent may be used in the preparation of a pharmaceutical composition for the treatment of a disease, disorder or condition mediated at least in part by expression of a gene of interest. In some embodiments, the disease, disorder or condition mediated at least in part by expression of a gene of interest is muscular dystrophy.

[0304] In some embodiments, the method of treating an individual depends on the individual's body weight. In some embodiments, the delivery vehicle comprising the RNAi agent can be administered at a dose of about 0.05 mg to about 40.0 mg per kilogram of body weight of the subject. In other embodiments, the delivery vehicle comprising the RNAi agent may be administered at a dose of about 5 mg to about 20 mg per kilogram of body weight of the subject.

[0305] In some embodiments, a delivery vehicle comprising an RNAi agent may be administered in split doses, meaning that two doses are given to an individual within a short (eg, less than 24 hours) period of time. In some embodiments, about half of the required daily dose is administered in the initial dose, and the remaining about half of the required daily dose is administered approximately four hours after the initial dose.

[0306] In some embodiments, a delivery vehicle comprising an RNAi agent can be administered once a week (ie, once a week). In other embodiments, the delivery vehicle comprising the RNAi agent can be administered biweekly (once every other week).

[0307] In some embodiments, a delivery vehicle comprising an RNAi agent or a composition comprising a delivery vehicle comprising an RNAi agent may be used to treat a disease, disorder or condition mediated at least in part by expression of a gene of interest. In some embodiments, the disease, disorder or condition mediated at least in part by expression of a gene of interest is muscular dystrophy.

[0308] cells, tissues and non-human organisms

[0309] Cells, tissues and non-human organisms comprising at least one RNAi agent described herein are contemplated. A cell, tissue or non-human organism is made by delivering an RNAi agent to the cell, tissue or non-human organism by any means available in the art. In some embodiments, the cells are mammalian cells, including but not limited to human cells.

[0310] The embodiments and projects provided above are now illustrated by the following non-limiting examples.

[0311] Example

[0312] The following examples are non-limiting and are intended to illustrate certain embodiments disclosed herein.

[0313] Unless expressly stated otherwise, compounds used to refer to a given Example and / or Reaction Scheme are made only when referring to that particular Example and / or Reaction Scheme, and not to any other Example and / or Reaction Scheme disclosed herein . For example, Compound 1 in "Synthesis of LP1-p" in Example 4 is different from and does not refer to Compound 1 in "Synthesis of LP-5p" in Example 4. Similarly, it should be understood that particular compounds disclosed herein may be identified by different numbers in different Examples and / or Reaction Schemes. For example, compound 12 of "Synthesis of LP223-p" in Example 4 is the same as Compound 3 of "Synthesis of LP224-p" in Example 4.

[0314] Table 24: Some common abbreviations used in the examples name abbreviation Triethylamine TEA, NEt 3 Dichloromethane DCM, CH 2 Cl 2 ethyl acetate EA, EtOAc Hexane Hex Methanol MeOH Acetonitrile ACN, MeCN Trifluoroacetate TFA Acetic acid AcOH Perylenemethoxycarbonyl FMOC Tertiary butoxycarbonyl BOC Dimethylformamide DMF toluene PhMe, Tol. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC Triisopropylsilane TIS, TIPS 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate TBTU N,N-Diisopropylethylamine DIPEA, DIEA, i-Pr 2 NET 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethylhexafluorophosphate HBTU 1-cyano-2-ethoxy-2-oxoethyleneamineoxy)dimethylamino-N-morpholino-carbenium hexafluorophosphate COMU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HATU N-Hydroxybutanediamide NHS Dibenzocyclooctyne DBCO Tris(2-furyl)phosphine TFP Tetrahydrofuran THF hydrochloric acid HCl Iodomethane MeI, CH 3 I 4-Dimethylaminopyridine DMAP m-chloroperoxybenzoic acid mCPBA carbon disulfide CS 2 sodium hydroxide NaOH equivalent Eq, Equiv Anhydrous Anhyd aqueous solution Q

[0315] It should be understood that use of the term "EDC" in the examples herein refers to commercially available EDC hydrochloride, unless expressly stated otherwise.

[0316] Example 1. Synthesis of RNAi agents and compositions

[0317] The following describe general procedures for the synthesis of certain RNAi agents and conjugates thereof illustrated in the non-limiting examples set forth herein.

[0318] Synthesis of RNAi agents. RNAi agents can be synthesized using methods generally known in the art. For the synthesis of RNAi agents illustrated in the Examples set forth herein, the sense and antisense strands of the RNAi agents were synthesized according to solid-phase phosphoramidate-based techniques used in oligonucleotide synthesis. Depending on scale, use MerMade96E® (Bioautomation), MerMade12® (Bioautomation), or Oligopilot 100 (GE Healthcare). Performed on solid supports made of controlled pore glass (CPG, 500 Å or 600 Å, from Prime Synthesis, Aston, PA, USA) or polystyrene (from Kinovate, Oceanside, CA, USA) synthesis. All RNA and 2'-modified RNA phosphoramidates were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA), ChemGenes (Wilmington, MA, USA) or Honggene Biotech (Morrisville, NC, USA). Specifically, the following 2'-O-methylphosphites used include the following: (5'-O-dimethoxytrityl-N6-(benzoyl)-2'- O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphite amido, 5'-O-dimethoxy-trityl -N4-(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphite amido 、(5'-O-Dimethoxytrityl-N2-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N -Diisopropylamino) aminophosphite and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl- N,N-diisopropylamino) amino phosphite. 2'-deoxy-2'-fluoro-amido phosphite and 2'-O-propargyl amino phosphite carry and 2' -O-Methylaminophosphite same protecting group. 5'-Dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl -N,N-Diisopropylamino)aminophosphite was purchased from Glen Research (Virginia). Reverse abasic (3'-O-dimethoxytrityl-2'-deoxyribose -5'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from ChemGenes. The following UNA phosphoramidites used included the following: 5'-(4 ,4'-dimethoxytrityl)-N6-(benzoyl)-2',3'-cyclo-adenosine, 2'-benzoyl-3'-[(2- Cyanoethyl)-(N,N-diisopropyl)]-aminophosphite, 5'-(4,4'-dimethoxytrityl)-N-acetyl-2 ',3'-opening-cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-amidophosphite, 5'-(4,4'-dimethoxytrityl)-N-isobutyryl-2',3'-cyclo-guanosine, 2'-benzoyl-3'-[(2 -cyanoethyl)-(N,N-diisopropyl)]-amidophosphite and 5'-(4,4'-dimethoxy-trityl)-2',3' -opening-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-amidophosphite. For the introduction of thio Phosphate linkage using 3-phenyl 1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) at 100 mM in anhydrous acetonitrile or hydrogenation Flavonoids (TCI America, Portland, OR, USA) 200 mM solution in pyridine.

[0319] TFA amine-linked phosphoramidate (ThermoFisher) was also purchased commercially to introduce the (NH2-C6) reactive group linker. The TFA amine-linked aminophosphite was dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3Å) were added. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used as activator solution. Coupling times were 10 minutes (RNA), 90 seconds (2'O-Me) and 60 seconds (2'F). Triyne-containing phosphoramidates were synthesized to introduce the respective (TriAlk#) linkers. When used in conjunction with the RNAi agents presented in certain examples herein, the triyne-containing aminophosphite was dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amines The amino acid ester was dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3Å) were added. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used as activator solution. Coupling times were 10 minutes (RNA), 90 seconds (2'O-Me) and 60 seconds (2'F).

[0320] For some RNAi agents, a linker, such as a C6-SS-C6 or 6-SS-6 group, is introduced at the 3' end of the sense strand. Preloaded resins with respective linkers are commercially available. Alternatively, for some sense strands, dT resin was used and the respective linkers were then added via standard phosphoramidate synthesis.

[0321] Cleavage and deprotection of support-bound oligomers. After completion of solid-phase synthesis, the dried solid support was treated with 40% by weight (wt.)% methylamine aqueous solution and 28% to 31 % ammonium hydroxide solution (Aldrich) in a 1:1 volume solution for 1.5 hours. The solution was evaporated and the solid residue reconstituted in water (see below).

[0322] Purification. Crude oligomers were purified by anion exchange HPLC using a TSKgel® SuperQ-5PW 13 µm column (available from Tosoh Biosciences) and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA pH 9.0 and contained 20% acetonitrile, and buffer B was the same as buffer A with the addition of 1.5 M sodium chloride. UV traces were recorded at 260 nm. Appropriate fractions were pooled, followed by size exclusion HPLC using a GE Healthcare XK 16 / 40 column packed with Sephadex® G25 fine particles, with an operating buffer of 100 mM ammonium bicarbonate pH 6.7 and 20% acetonitrile or filtered water liquid to operate.

[0323] Bonding. Complementary strands are formed by combining equimolar RNA solutions (sense and antisense strands) in 1×PBS (Phosphate Buffered Saline, 1×, Corning®, Cellgro®) RNAi agent. Some RNAi agents were lyophilized and stored at -15°C to -25°C. The double helix concentration was determined by measuring the absorbance of the solution in 1×PBS by UV-Vis spectrometer. The absorbance at 260 nm of the solution was then multiplied by the conversion factor and the dilution factor to determine the double helix concentration. The conversion factor used is 0.037 mg / (mL∙cm) or calculated from the experimentally determined extinction coefficient.

[0324] Example 2. Synthetic linking group

[0325] Synthesis of L1

[0326] Compound 1 (423 mg) and Compound 2 (516 mg) were mixed together in DMF and DIPEA (0.26 ml) was added. The reaction mixture was stirred overnight. The product was separated by normal phase column chromatography to obtain 450 mg of compound 3.

[0327] Compound 3 (450 mg, 1 equiv) and compound 4 (0.12 ml, 1.2 equiv), TBTU (248 mg, 1.1 equiv) and DIPEA (0.183 ml, 1.5 equiv) were mixed together in DMF. The reaction mixture was stirred overnight. The product was separated via normal phase column chromatography to afford compound 5.

[0328] Compound 5 was treated with 20% piperidine in DMF for half an hour. The product was separated via normal phase column chromatography to afford compound 6.

[0329] Compounds 6 (93 mg, 1 equiv) and 7 (25.9 mg, 1.3 equiv), TEA (0.045 ml, 2 equiv) were mixed together in DCM. The reaction mixture was stirred overnight. To this mixture was added compound 9 (57 mg) and EDC (72 mg). The reaction mixture was stirred overnight. The product was separated via normal phase column chromatography to afford compound L1 (100 mg).

[0330] Synthetic L2

[0331] To a solution of compound 1 (1.69 g, 6.3 mmol) and propargyl bromide (1.499 g, 1.4 mL, d = 1.57 g / mL, 12.6 mmol) in acetone (50 mL) was added K2CO3 ( 3.477 g, 25.2 mmol). The reaction mixture was stirred at reflux for 3 hours (hrs). After the starting material was consumed, the reaction mixture was concentrated in vacuo and taken up with EA / hexane / DCM (30 mL each) and filtered. The mother liquor was concentrated and the residue was purified by CombiFlash® using silica gel as stationary phase and eluted with a gradient of EtOAc (0-50%) in hexane. Product yield: 0.438 g (23%). [M-H] calculated for C16H18NO5: 304.12, found: 304.46.

[0332] The product of the above reaction (438 mg) was dissolved in 4 M HCl in dioxane at room temperature for 5 hours and the reaction mixture was monitored by LC-MS for only 50% conversion. The mixture was centrifuged briefly and filtered. Then, 2 mL of TFA was added to the solid, and the starting material was consumed after 2 hours as monitored by LC-MS. The mixture was concentrated in vacuo. Compound 2 yield: 333 mg, solid, 96%. [M+H] calculated for C11H12NO3: 206.08, found: 206.26.

[0333] To TBTU (22.5 mg, 0.07 mmol), DBCO-PEG5-acid 3 (50 mg, 0.084 mmol), N,N-diisopropylethylamine (27 mg, 36 μL, d = 0.742 g / mL , 0.21 mmol) in DMF (0.8 mL) was added 2 (16.8 mg, 0.07 mmol). The reaction mixture was stirred at room temperature. After all starting material was consumed by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL x 3). The combined organic layers were washed sequentially with HCl(aq) and brine, dried over Na2SO4, and concentrated in vacuo. The crude product was loaded onto a silica column and purified (MPA:DCM, MPB:10% MeOH in DCM, 0-30% ramp over 30 minutes (min)) to give compound 4. Yield: 76.5 mg, 99%. [M+H] calculated for C43H50N3O11: 784.34, found: 784.83.

[0334] Compound 4 was dissolved in 0.3 mL THF / H2O (2:1 v / v) and 55.6 mg LiOH was added to the reaction mixture. After stirring overnight at room temperature, the reaction mixture was filtered through a short pad of silica gel. The filtrate was collected and concentrated in vacuo. The crude product was loaded onto a silica column and purified (MPA: DCM, MPB: 10% MeOH in DCM, 0-50% ramp in 30 minutes) to give the product. Yield: 42.9 mg. [M+H] calculated for C42H48N3O11: 770.33, found: 770.91.

[0335] To 5 (43 mg, 0.056 mmol), 2,3,5,6-tetrafluorophenol (46.5 mg, 0.28 mmol) and N,N-diisopropylethylamine (144.5 mg, 0.19 mL, d = 0.742 g / mL, 1.12 mmol) in DCM (2 mL) was added EDC hydrochloride (53.5 mg, 0.28 mmol). The reaction mixture was stirred at room temperature. After all starting material was confirmed to be consumed by LC-MS, the reaction mixture was concentrated by lyophilization, loaded onto a silica column and purified (MPA: DCM, MPB: 20% MeOH in DCM, within 30 minutes 0-30% rise), the product L2 was obtained. Yield: 12 mg, 23%. [M+H] calculated for C48H48F4N3O11: 918.32, found: 918.89.

[0336] Synthetic L3

[0337] Tonic acid 1 (1.2461 g, 4.9591 mmol), HATU (2.2613 g, 5.9509 mmol) and DIPEA (2.3030 g, 3.1 mL, d=0.742 g / mL, 17.8527 mmol, 3 equivalents) in DMF (4 mL) To the solution in amine 2 (1 g, 4.9591 mmol) / DMF (1 mL) was added. The reaction mixture was stirred at room temperature. After all starting material was confirmed to be consumed by LC-MS, the reaction mixture was concentrated by lyophilization, loaded onto a silica column, and purified (MPA: DCM, MPB: 10% MeOH in DCM, within 30 minutes 0-30% rise), to obtain compound 3. Yield: 1.3269 g, 67%. [M+H] calculated for C22H27N2O5: 399.19, found: 399.39.

[0338] To a solution of 4 M HCl in dioxane was added compound 3 (1.3269 g). After stirring at room temperature for 1 hour, the starting material was completely consumed. Compound 4 was obtained as a white solid by simple filtration. Yield, 630 mg, 63%. [M+H] calculated for C17H19N2O3: 299.14, found: 299.34.

[0339] To DBCO-acid 5 (0.1993 g, 0.5979 mmol), HATU (0.2726 g, 0.7175 mmol, 1.2 equivalents), DIPEA (0.1851 g, 0.249 mL, d=0.742 g / mL, 1.435 mmol, 2 equivalents) in A solution in DMF (0.3 mL) was added Compound 4 (0.2 g, 0.5979 mmol) / DMF (0.3 mL). The reaction mixture was stirred at room temperature. After confirming the consumption of all starting material by LC-MS, the reaction mixture was concentrated under lyophilization, loaded onto a silica column, and purified (MPA: DCM, MPB: 20% MeOH in DCM, within 30 minutes 0-30% rise), to obtain compound 6. Yield: 0.3297 g, 90%. [M+H] calculated for C38H36N3O5: 614.26, found: 614.51.

[0340] To a solution of compound 6 in THF / water (4 mL, 1:1 v / v) was added LiOH (0.0387 g, 1.6117 mmol). The reaction mixture was stirred at room temperature. After all starting material was consumed by LC-MS, a solution of HCl (1.6 mmol, 4M, 0.4 mL) in dioxane was added to neutralize the base. The reaction mixture was concentrated by lyophilization, loaded onto a silica column, and purified (MPA:DCM, MPB:10% MeOH in DCM, 0-50% ramp in 30 minutes) to afford compound 7. Yield: 0.2575 g, 80%. [M+H] calculated for C37H34N3O5: 600.25, found: 600.46.

[0341] Acid 7 (0.1241 g, 0.2069 mmol), amine 8 (0.05 g, 0.2069 mmol) and DIPEA (0.0961 g, 0.129 mL, 0.7448 mmol, 3 equivalents, d=0.742 g / mL) in DMF (1.5 mL ) was added HATU (0.0943 g, 0.2483 mmol) / DMF (0.5 mL). The reaction mixture was stirred at room temperature. After the starting material was consumed, the reaction mixture was concentrated in vacuo. After removal of DMF, the crude product was dissolved in 5 mL DCM and loaded on a column using silica gel as stationary phase (MPA: DCM; MPB: 20% MeOH / DCM; 0-100% ramp in 30 minutes). Yield of Compound 9: 0.1109 g, 68%. [M+H] calculated for C48H43N4O7: 787.31, found: 787.44.

[0342] To a solution of DBCO-ester 9 in THF / water (1 mL, 1:1 v / v) was added LiOH (0.0169 g, 0.7047 mmol). The reaction mixture was stirred overnight at room temperature. After complete consumption of starting material, the residue was neutralized by HCl(aq) and concentrated in vacuo. Compound 10 was purified by CombiFlash®. (MPA: DCM; MPB: 20% MeOH / DCM; 0-100% ramp in 30 minutes), yield: 0.0321 g, solid, 29%. [M+H] calculated for C47H41N4O7: 773.30, found: 773.49.

[0343] To a solution of acid 10 (0.0321 g, 0.0415 mmol), TFP (0.0103 g, 1.5 equiv, 0.0623 mmol) and DMAP (3 mg, 0.0249 mmol) in DMF (0.5 mL) was added EDC·HCl (0.0239 g , 0.1246 mmol). The reaction mixture was stirred at room temperature. After the starting material was consumed, the reaction mixture was concentrated in vacuo. After removal of DMF, the crude product was dissolved in 5 mL of DCM and loaded on a column using silica gel as stationary phase. (MPA: DCM; MPB: 20% MeOH / DCM; 0-100% ramp in 30 minutes). L3 yield: 20 mg, oily, 50%. [M+H] calculated for C53H41F4N4O7: 921.29, found: 921.85.

[0344] synthetic L4

[0345] To a solution of Compound 1 (3.00 g) in DMF was added Cs2CO3 (7.71 g) at room temperature. Then compound 2 (1.85 mL) was added slowly. The resulting reaction mixture was stirred overnight under N2 (g). Almost complete conversion to the desired product was then confirmed by LC-MS. The reaction mixture was quenched with NaHCO3 (10 mL). The product was extracted with EtOAc (5 x 10 mL) and then washed with water (3 x 8 mL) and brine (8 mL). The combined org. phases were dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of hexane to EtOAc (0-30%), where the product was eluted at 14% B. Compound 3 was concentrated in vacuo to give a white solid. LC-MS: [M+H]+ Calculated 191.06 m / z, Observed 191.23 m / z.

[0346] To a solution of compound 3 (2.87 g) in 1:1 THF / water was added LiOH (1.08 g) at room temperature under normal atmospheric pressure. The reaction mixture was stirred until complete conversion of compound 3 was observed by LC-MS. Residual starting material was extracted via EtOAc, and the aqueous phase was then acidified to ~pH 3 with 6 N HCl. Compound 4 precipitated out as a white solid and was dried in vacuo and washed with water. Due to its wetness / viscosity, a solvent was needed to transfer the solid to an in situ flask; material was transferred via MeOH and DCM. The material could not be dried over Na2SO4 due to poor solvation in either solvent and combination. Compound 4 was concentrated in vacuo to give a white fluffy crystalline solid and used without further purification. LC-MS: [M+H]+ Calculated 177.05 m / z, Observed 177.19 m / z.

[0347] To a solution of compounds 4 (1.00 g) and 5 (1.04 g) in DMF (10.0 mL) was added EDC (1.20 g) at room temperature under N2 (g). The reaction mixture was stirred until complete conversion was observed by LC-MS. Due to failure to observe product after stirring overnight, the reaction mixture was quenched with NaHCO3. The resulting precipitate was confirmed via LC-MS to contain starting material and was dried in vacuo, attempted to be resuspended in MeOH / DCM, and then concentrated in vacuo. The mixture was then redissolved in DMF, dried over Na2SO4, vacuum filtered, and rinsed with DMF. EDC was added back to the filtrate in DMF (ie, compounds 4 and 5), and the resulting mixture was stirred at room temperature overnight. The reaction mixture was directly concentrated and azeotroped with MeOH and PhMe for separation. The residue was purified by CombiFlash® using silica gel as stationary phase and eluting with 0-20% MeOH in DCM. L4 was eluted at 0% B to give a white solid. LC-MS: [M+H]+ Calculated 325.04 m / z, Observed 325.35 m / z.

[0348] Synthetic L7

[0349] To a solution of Compound 1 (0.300 g) and 2 (0.231 g) in DMF was added EDC (0.160 g) at ambient conditions. The reaction mixture was stirred for 2 hours until complete conversion was observed by LC-MS. The reaction mixture was then concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of hexane to EtOAc (0-30%), where the product was eluted at 10% B. Fractions containing product were concentrated in vacuo to afford L7 as a white oily residue. Yield: 0.329 g (81.6%). LC-MS: [M+H]+ Calculated 580.12 m / z, Observed 580.56 m / z.

[0350] synthetic L8

[0351] To a solution of compound 1 (500 mg, 3.286 mmol, 1.0 equiv) and potassium carbonate (908 mg, 6.572 mmol, 2.0 equiv) in anhydrous acetone (5 mL) was added compound 2 (0.549 mL, 4.929 mmol, 1.5 equiv). The reaction mixture was kept at 50°C for 3 hours. The reaction mixture was quenched with saturated sodium bicarbonate solution (5 mL). The aqueous phase was extracted with ethyl acetate (3 x 5 mL). The combined org. phases were dried over Na2SO4 and concentrated. Product 3 was purified by CombiFlash® eluting with 5-10% ethyl acetate in hexane. LC-MS: Calculated for [M+H]+ 191.06, found 191.19.

[0352] To a solution of compound 3 (584 mg, 3.070 mmol, 1.0 equiv) in THF (5 mL) and water (5 mL) was added lithium hydroxide (220 mg, 9.211 mmol, 3.0 equiv) at room temperature. The reaction mixture was kept at 40°C for 1 hour. The reaction mixture was quenched with HCl solution and the pH was adjusted to 3.0. The aqueous phase was extracted with ethyl acetate (3 x 10 mL). The combined org. phases were dried over Na2SO4 and concentrated. Product 4 was used without further purification. LC-MS: Calculated for [M+H]+ 177.17, found 177.37.

[0353] To a solution of compound 4 (185 mg, 1.050 mmol, 1.0 equiv), compound 5 (218 mg, 1.312 mmol, 1.25 equiv) in anhydrous DMF (2 mL) was added EDC HCl (251 mg, 1.312 mmol, 1.25 equiv). The reaction mixture was kept at room temperature for 2 hours. The reaction mixture was quenched with saturated sodium bicarbonate solution (5 mL). The aqueous phase was extracted with ethyl acetate (3 x 5 mL). The combined org. phases were dried over Na2SO4 and concentrated. Product L8 was purified by CombiFlash® and eluted with 5-10% ethyl acetate in hexanes. LC-MS: Calculated for [M+H]+ 325.04, found 325.26.

[0354] Synthetic L9

[0355] To a solution of compound 1 (200 mg, 1.368 mmol, 1.0 equiv), compound 2 (284 mg, 1.710 mmol, 1.25 equiv) in anhydrous DMF (2 mL) was added EDC HCl (327 mg, 1.710 mmol, 1.25 equiv). The reaction mixture was kept at room temperature for 2 hours. The reaction mixture was quenched with saturated sodium bicarbonate solution (5 mL). The aqueous phase was extracted with ethyl acetate (3 x 5 mL). The combined org. phases were dried over Na2SO4 and concentrated. Product L9 was purified by CombiFlash® and eluted with 5-10% ethyl acetate in hexanes. LC-MS: Calculated for [M+H]+ 295.03, found 294.69.

[0356] Synthetic L10

[0357] To a solution of Compound 1 (0.200 g) in DCM was added TFA (1.99 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour until complete conversion was observed by LC-MS. The reaction mixture was azeotroped with PhMe and concentrated in vacuo to afford 2 as a brown oil. Yield: 0.309 g (146%). LC-MS: [M+H]+ Calculated 132.07 m / z, Observed 132.10 m / z.

[0358] To a solution of compound 2 (0.212 g) in DCM was added 3 (0.0865 g) at 0°C. The mixture was stirred for 1.5 hours and then allowed to warm to room temperature with stirring. After 0.5 h, NEt3 was added and within 0.5 h, complete conversion was confirmed by LC-MS. The reaction mixture was concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase and eluted with a gradient of DCM to 20% MeOH (0-25% B) in DCM. The product eluted at 9% B. Concentration afforded 4 as a purple solid. Yield: 0.171 g (85.5%). LC-MS: [M+H]+ Calculated 232.09 m / z, Observed 232.28 m / z.

[0359] To a solution of compounds 4 (0.0400 g) and 5 (0.0316 g) in DMF was added EDC (0.0398 g) at room temperature. The reaction mixture was stirred for 1 hour until complete conversion was observed by LC-MS. After 1 hour, complete conversion was observed by LC-MS. The reaction mixture was quenched with NaHCO3 (15 mL). The product was extracted with EtOAc (3x8 mL) and washed with water (3x8 mL). The combined org. phases were dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase and eluted with a gradient of DCM to 20% MeOH / DCM (0-25% B). The product eluted at 6% B to afford L10 as a white solid. Yield: 0.0255 g (38.9%). LC-MS: [M+H]+ Calculated 380.08 m / z, Observed 380.35 m / z.

[0360] Example 3. Synthesis of Targeting Ligands

[0361] The peptides in this example were synthesized using standard peptide synthesis. ChemMatrix® Rink amide resin was placed in a sintered polypropylene syringe and stirred in DCM for 30 minutes prior to use. Use the following standard solid-phase peptide synthesis conditions. Deprotection of the Fmoc group was performed by soaking 40 ml of piperidine:DMF solution (20:80 v / v) per 1 millimolar of resin for 20 minutes. Amide coupling was performed by soaking the resin with 0.1 M concentration of Fmoc-amino acid in DMF for 40 minutes with 4 molar equivalents of Fmoc-amino acid, 4 molar equivalents of HBTU and 10 molar equivalents of diisopropylethylamine in DMF for 40 minutes . The DNP chromophore was attached to the resin using Fmoc-Dap(DNP)-OH, and the peptide was synthesized from the Dap α-amine. Cleavage from the resin was performed in TFA solution over 2 hours. The solvent was reduced to 10% of the initial volume via pressurized air and precipitated with Et2O. Product identity was verified by microlysis by TFA and analytical HPLC-MS. The peptide was then purified to > 95% pure. Purity was assessed using an analytical Shimadzu HPLC equipped with a Waters® XBridge BEH130 C18 column (250 mm x 6.6 mm, 5 μm particles) using 10-90% B solvent over 50 minutes. A solvent represents H2O:F3CCO2H 100:0.1 v / v, B solvent represents CH3CN: F3CCO2H 100:0.1 v / v. Fmoc-Dap(DNP)-OH

[0362] Synthesis of αvβ6 peptide 1

[0363] αvβ6 peptide 1 is prepared by modifying Arg-Gly-Asp(tBu)-Leu-Ala-Abu-Leu-Cit-Aib-Leu-Peg5-CO2-2-Cl-Trt resin 1-1, which is Obtained at 4.1 mmol scale using Fmoc-Peg5-CO2H preloaded 2-Cl-Trt resin (0.79 mmol / g) on ​​a CS Bio peptide synthesizer using general Fmoc peptide chemistry as described above. After cleavage from the resin, peptide 1-2 was converted to tetrafluorophenyl ester 1-3, and the crude product was used in the next step without purification.

[0364] Final deprotection was performed by treating the crude peptide with the deprotection mixture TFA / TIS / H2O=90:5:5 (80 mL) for 1-31.5 hours. The reaction mixture was added dropwise to methyl tert-butyl ether (700 mL), and the resulting precipitate was collected by centrifugation. Wash the pellet with additional methyl tertiary butyl ether (500 mL). The residue was analyzed by reverse phase (RP)-HPLC (Phenomenex Gemini C18 250×50 mm, 10 μm, 60 mL / min, gradient of 30-45% ACN in water containing 0.1% TFA, approximately 1 g of crude material per run) Purification afforded 4.25 g of pure peptide 1-4 (αvβ6 peptide 1).

[0365] Synthesis of αvβ6 peptide 5

[0366] αvβ6 peptide 5 is prepared by modifying H-Gly-Asp(tBu)-Leu-Ala-Abu-Leu-Cit-Aib-Leu-Peg5-CO2-2-Cl-Trt resin 5-1, which is Obtained on a Symphony peptide synthesizer at 0.2 mmol scale using general Fmoc peptide chemistry with Fmoc-Peg5-CO2H preloaded 2-Cl-Trt resin (0.85 mmol / g). The coupling step was performed by treating the resin with 3 equivalents of Fmoc-AA-OH, 3 equivalents of HBTU and 6 equivalents of DIEA for 2 hours. In the deprotection step, the resin was treated with 20% piperidine in DMF for 5 minutes and then 20 minutes. After completion of the automated synthesis, peptide resin 5-1 was transferred from the Symphony reaction vessel to the SPPS vessel for manual modification, washed with DMF (6 mL - 1 min x 4 times) and using the standard described above for Step 1 of Scheme 2 Coupling Procedure Coupling with 5-(N-Boc-amino)-5-(4-methylpyridin-2-yl)pentanoic acid.

[0367] The resulting peptide-resin was treated 5-23 times over 15 minutes with 3 portions of cleavage solution (20% hexafluoroisopropanol (HFIP) in DCM, 6 ml). The solution of cleaved protected peptide 5-3 was diluted with 20 ml toluene, concentrated and dried under vacuum. Residual HFIP was removed by additional evaporation of toluene from the product, and the product was dried under vacuum for 2 hours.

[0368] A portion of crude peptide 5-3 (133 mg) was dissolved in DCM (4 mL) and cooled to 0°C. Tetrafluorophenol (22 mg, 0.133 mmol) and EDC hydrochloride (26 mg, 0.133 mmol) were added, the cooling bath was removed, and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated and dried under vacuum, and the crude peptide was purified on Combiflash® using the system DCM:20% MeOH in DCM, gradient 0%-100%, 25 minutes to obtain 74 mg of pure peptide 5-4.

[0369] Final deprotection was performed by treating the purified peptide with the deprotection mixture TFA / TIS / H2O=95:2.5:2.5 (4 mL) for 5-41.5 hours. The reaction mixture was concentrated and dried under vacuum. Residual toluene was removed by coevaporation with toluene. Crude peptide 5-5 (αvβ6 peptide 5) was purified by HPLC using the following column: Syncronis™aQ 250×20 (Thermo Scientific), ACN (TFA 0.1%) in HO (TFA 0.1%) 20-30%, 25 minutes Inside. Conditions: ACN (TFA 0.1%) 35-60% in H2O (TFA 0.1%) within 25 minutes. Yield 55 mg. Calculated molecular weight (MW) 1556.81, 1 / 2M=778.40. Found: MS (ES, positive ion): 1557.52 [M+1]+; 780.39 [M+2]2+.

[0370] Synthetic αvβ6 peptide 6

[0371] αvβ6 peptide 6 is prepared by modifying GBA-Gly-Asp(tBu)-Leu-Ala-Abu-Leu-Cit-Aib-Leu-Peg5-CO2-2-Cl-Trt resin 6-1, which is It was obtained on a Symphony peptide synthesizer at a 0.2 mmol scale with Fmoc-Peg5-CO2H preloaded 2-Cl-Trt resin (0.85 mmol / g) using general Fmoc peptide chemistry as described above. After cleavage from the resin, peptide 6-2 was converted to tetrafluorophenyl ester 6-3 and purified on Combiflash® using system DCM: 20% MeOH in DCM, gradient 15% to 100%, 25 minutes to obtain 160 mg Pure peptides 6-3. Final deprotection was performed by treating the crude peptide with the deprotection mixture TFA / TIS / H2O = 90:5:5 (80 mL) for 6-31.5 hours. The reaction mixture was added dropwise to methyl tert-butyl ether (700 mL), and the resulting precipitate was collected by centrifugation. Wash the pellet with additional methyl tertiary butyl ether (500 mL). The residue was purified by HPLC using the following conditions: ACN (TFA 0.1%) 27-57% in H2O (TFA 0.1%) for 25 minutes. Yield 94 mg. Calculated value MW 1527.76, 1 / 2M=763.88. Found: MS (ES, positive ion): 1529.48 [M+1]+; 765.39 [M+2]2+.

[0372] Example 4. Synthesis of PK / PD modulator precursors

[0373] Some of the PK / PD modulator precursors of Table 1 were purchased from commercial suppliers as indicated in Table 1. The following procedure was used to prepare the remaining PK / PD modulator precursors.

[0374] Synthetic double (PEG47+C22)

[0375] Solid TBTU (1.68 g, 5.22 mmol) was added to behenic acid (1.486 g, 4.36 mmol), Boc-protected PEG-amine 1 (Quanta Biodesign Limited, 10 g, 4.35 mmol) and DIPEA (2.27 mL, 13.03 mmol) solution. The reaction mixture was sonicated to dissolve the solids and stirred at room temperature for 16 hours. Water (3 mL) was added and the solvent was removed under vacuum. The resulting residue was dissolved in chloroform (300 mL) and washed with NaHCO3 (2 x 75 mL) and brine (50 mL). Product 2 was dried (Na2SO4), concentrated in vacuo, and purified on a Combiflash® using the system DCM:20% MeOH in DCM, gradient 0-80% over 25 minutes. Yield 10 g (88%). Calculated MW 2623.72, (M +2×18) / 2=1329.86, (M +3H) / 3=875.57 Found: MS (ES, positive ion): 1330.58 [M+2NH4]2+, 875.93 [M+ 3H ]3+.

[0376] Synthesis of C18

[0377] Compound 1 (Sigma S4751) (0.125 g) was dissolved in DCM (2.0 mL). Then HATU (0.249 g) and DIEA (0.263 mL) were added to the mixture. The reaction mixture was stirred for 15 minutes and 0.265 g of Compound 2 (BroadPharm® BP-22226) was added. The reaction mixture was stirred for 1 hour.

[0378] The reaction mixture was then diluted with DCM (40 mL) and washed with H2O (2 x 7 mL), dried over Na2SO4, filtered and concentrated in vacuo. The organic layer was dissolved in 2 mL DCM and purified on a column (CombiFlash® in DCM: 20% MeOH in DCM; RediSeprf Gold® column; 0-40% mobile phase B over 30 min). Fractions containing product were collected and concentrated in vacuo to afford compound 3. Yield 223 mg (56%).

[0379] Synthesis of C22-PEG5K-Mal

[0380] Compound 1 (Sigma-Aldrich® 216941 ) (0.300 g) was dissolved in 4.5 mL of DCM. Then EDC (Oakwood Chemical 024810) (0.211 g) was added to the solution. Then NHS (Sigma-Aldrich® 130672) (0.203 g) was added to the solution. Finally, DMAP (Sigma-Aldrich® 107700) (0.0215 g) was added. The reaction mixture was stirred overnight. The solution was diluted with 40 mL DCM and washed with acidic H2O (3 x 7 mL), dried over Na2SO4, filtered and concentrated in vacuo. The concentrated product was dry loaded (3 mL silica) onto a 12 G Redi-Sep rf Gold® column over 25 minutes (mobile phase A:mobile phase B) in Hex:EtOAc 0->50%. Fractions containing product 2 were collected and concentrated. Yield 283 g (73%).

[0381] Compound 3 (Creative PEGWorks PHB-942) (0.100 g) was dissolved in 2 mL of DCM. Compound 2 (0.0438 g) was then added. Then 0.042 mL of TEA was added to the mixture. The reaction mixture was stirred for 2 hours. The reaction mixture was concentrated in vacuo. The concentrate was then dissolved in 1 mL of DCM and loaded onto a 4 G Redi-Sep rf Gold® column for 20 minutes DCM:DCM 0 -> 100% with 20% MeOH. Fractions containing product 4 were collected and concentrated on a rotary evaporator. Yield 41 mg (38%).

[0382] Synthetic PEG48+C22 To compound 1 (350 mg, 1.027 mmol, 1.0 equiv), compound 2 (181 mg, 1.130 mmol, 1.1 equiv) and DIPEA (0.537 mL, 3.082 mmol, 3.0 equiv) at room temperature in anhydrous DMF (3 mL) was added TBTU (396 mg, 1.233 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 2 hours. The reaction mixture was quenched with saturated aqueous NaHCO 3 (20 mL) and the aqueous phase was extracted with dichloromethane (3×10 mL). The combined org. phases were dried over anhydrous Na2SO4 and concentrated. Product 3 was purified by CombiFlash® and eluted with 4-5% methanol in dichloromethane. LC-MS: Calculated for [M+H]+ 483.44, found 483.67.

[0384] To a solution of compound 3 (290 mg, 0.600 mmol, 1.0 equiv) in dry 1,4-dioxane (1 mL) was added a solution of HCl in dioxane (0.751 mL, 3.003 mmol, 5.0 equiv). The reaction mixture was kept at room temperature for 3 hours and the solvent was removed in vacuo. Product 4 was used without further purification. LC-MS: Calculated for [M+H]+ 383.39, found 383.57.

[0385] To a solution of compound 5 (83 mg, 0.0322 mmol, 1.0 equiv) and compound 4 (13.5 mg, 0.322 mmol, 1.0 equiv) in anhydrous DMF (2 mL) was added TEA (0.014 mL, 0.0967 mmol, 3.0 equivalents). The reaction mixture was kept at room temperature for 3 hours and the solvent was removed in vacuo. Product 6 was purified by CombiFlash® and eluted with 10-15% methanol in dichloromethane. LC-MS: Calculated for [M+4H]+ / 4 698.18, found 698.49, calculated for [M+3H]+ / 3 930.58, found 930.61.

[0386] Synthetic PEG48+C18 To compound 1 (1437 mg, 5.051 mmol, 1.0 equiv), compound 2 (890 mg, 5.556 mmol, 1.1 equiv) and DIPEA (2.639 mL, 15.154 mmol, 3.0 equiv) at room temperature in anhydrous DMF (10 mL) was added TBTU (1946 mg, 6.061 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 2 hours. The reaction mixture was quenched with saturated aqueous NaHCO 3 (20 mL) and the aqueous phase was extracted with dichloromethane (3×10 mL). The combined org. phases were dried over anhydrous Na2SO4 and concentrated. Product 3 was purified by CombiFlash® and eluted with 4-5% methanol in dichloromethane. LC-MS: Calculated for [M+H]+ 427.38, found 427.74.

[0388] To a solution of compound 3 (445 mg, 1.042 mmol, 1.0 equiv) in dry 1,4-dioxane (1 mL) was added HCl in dioxane (1.304 mL, 5.214 mmol, 5.0 equiv). The reaction mixture was kept at room temperature for 3 hours and the solvent was removed in vacuo. Product 4 was used without further purification. LC-MS: [M+H]+ calcd. 327.33, found 327.48.

[0389] To a solution of compound 5 (90 mg, 0.035 mmol, 1.0 equiv) and compound 4 (13.3 mg, 0.0367 mmol, 1.05 equiv) in anhydrous DCM (2 mL) was added TEA (0.015 mL, 0.104 mmol, 3.0 equivalents). The reaction mixture was kept at room temperature for 1 hour and the solvent was removed in vacuo. Product 6 was purified by CombiFlash® and eluted with 12-18% methanol in dichloromethane. LC-MS: Calculated for [M+3H]+ / 3 911.90, found 912.65, [M+4H]+ / 4 684.17, found 685.21.

[0390] Synthetic PEG23+C22

[0391] To a solution of Compound 1 (0.0700 g) in DCM was added Compound 2 (0.0251 g) and TEA (0.0148 g) at room temperature. The reaction mixture was stirred for 0.5 h until complete conversion was confirmed by LC-MS. The reaction mixture was concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase and eluted with a gradient of DCM to 20% MeOH (0-100% B) in DCM. The product eluted at 60% B. Concentration afforded 3 as a white solid. LC-MS: [M+H]+ Calculated 1794.16 m / z, Observed 898.01 (+2 / 2) m / z. Yield: 0.0784 g (89.4%).

[0392] synthetic double (PEG23+C14)

[0393] To a solution of compounds 1 (0.0430 g) and 2 (0.221 g) in DCM was added TBTU (0.0725 g) followed by DIPEA (0.098 mL) at room temperature. The reaction mixture was stirred for 1 hour until complete conversion was observed by LC-MS. The reaction mixture was then concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of DCM to 20% MeOH in DCM (0-100%), where the product was eluted at 31% B. Product 3 was concentrated in vacuo to give a white solid. LC-MS: [M+H]+calculated 1383.92 m / z, observed 693.02 (+2 / 2) m / z. Yield: 0.253 g (97.0%).

[0394] To compound 3 (0.253 g) was added 4 M HCl (2.74 mL) in 1,4-dioxane at room temperature. The reaction mixture was stirred at room temperature for 1 hour until complete conversion was observed by LC-MS. The reaction mixture was concentrated in vacuo to afford 4 as a white solid. No further purification was required. LC-MS: [M+H]+calculated 1319.85 m / z, observed 642.97 (+2 / 2) m / z. Yield: 0.241 g (99.7%.).

[0395] To a solution of compound 4 (0.169 g) in DCM was added compound 5 (0.0500) followed by TEA (0.049 mL) at room temperature. The mixture was stirred overnight until complete conversion was confirmed by LC-MS. The reaction mixture was concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase and eluted with a gradient of DCM to 20% MeOH (0-100% B) in DCM. The product eluted at 95% B. Concentration afforded 6 as a white solid. LC-MS: [M+H]+calculated 3195.02 m / z, observed 800.43 (+4 / 4) m / z. Yield 0.0674 (36.2%).

[0396] synthetic double (PEG23+C18) To compound 1 (130 mg, 0.457 mmol, 1.0 equivalent), compound 2 (536 mg, 0.457 mmol, 1.0 equivalent) and diisopropylethylamine (0.239 mL, 1.370 mmol, 3.0 equivalent) at room temperature A solution in anhydrous DMF (3 mL) was added TBTU (176 mg, 0.548 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 2 hours. The reaction mixture was quenched with saturated aqueous NaHCO 3 (5 mL) and the aqueous phase was extracted with ethyl acetate (6×5 mL). The combined org. phases were dried over anhydrous Na2SO4 and concentrated. The product was purified by CombiFlash® and eluted with 4-8% methanol in dichloromethane. LC-MS: Calculated for [M+H]+ 1439.99, found 1440.53.

[0398] To a solution of compound 3 (445 mg, 0.309 mmol, 1.0 equiv) in anhydrous 1,4-dioxane (1 mL) was added HCl in dioxane (0.386 mL, 1.545 mmol, 5.0 equiv). The reaction mixture was kept at room temperature for 3 hours and the solvent was removed in vacuo. Product 4 was used without further purification. LC-MS: Calculated for [M+2H]+ / 2 670.46, found 670.93.

[0399] To a solution of compound 1 (100 mg, 0.116 mmol, 1.0 equiv) and compound 2 (352 mg, 0.256 mmol, 2.2 equiv) in anhydrous DMF (5 mL) was added TEA (0.082 mL, 0.582 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature for 3 hours and the solvent was removed in vacuo. The product was purified by CombiFlash® and eluted with 10-17% methanol in dichloromethane. LC-MS: Calculated for [M+4H]+ / 4 827.53, found 828.16, calculated for [M+3H]+ / 3 1103.05, found 1104.21.

[0400] synthetic double (PEG23+C22)

[0401] A solution of C22-PEG23-amine hydrochloride (2) (Quanta Biodesign Limited, 183 mg, 0.128 mmol) and bis-NHS ester 1 (BroadPharm, 50 mg, 0.058 mmol) in DMF (5 mL) Stir in the presence of TEA (50 uL, 0.35 mmol) at room temperature for 3 hours. The reaction mixture was concentrated and dried in vacuo. Residual DMF was removed by co-evaporation of toluene under vacuum and product 3 was purified on the Combiflash® using the system DCM:20% MeOH in DCM, gradient 15-80%, 25 minutes. Yield 84 mg (45%). Calculated value MW 3419.28, 1 / 2M=1709.64, (M +2×18) / 2=1727.64, (M+18+2) / 3=1146.42. Found: MS (ES, positive ion): 1727.73 [M+2NH4]2+, 1146.94 [M+NH4+ 2H]3+.

[0402] synthetic double (PEG23+CLS)

[0403] To a solution of compound 1 (0.158 g) in 1:1 THF / water was added LiOH (0.0473 g) at room temperature under normal atmospheric pressure. The reaction mixture was stirred at room temperature for 1 hour and then heated to 50 °C and stirred overnight until complete conversion was observed by LC-MS. The reaction mixture was acidified to about pH 3 with 6 N HCl. The product was extracted with EtOAc (3 x 5 mL). The combined org. phases were dried over Na2SO4, filtered, and concentrated to afford 2 as a white solid. LC-MS: [M+H]+ Calculated 227.06 m / z, Observed 227.06 m / z. Yield: 152 mg (102%).

[0404] To a solution of compound 4 (0.0709 g) in DCM was added compound 3 (0.0200 g) followed by TEA (0.019 mL) at room temperature. The reaction mixture was stirred until complete conversion was confirmed by LC-MS. The reaction mixture was concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase and eluted with a gradient of DCM to 20% MeOH (0-100% B) in DCM. The product eluted at 82% B. Concentration afforded 5 as a white solid. LC-MS: [M+H]+calculated 3599.30 m / z, observed 1200.23 (+3 / 3) m / z. Yield 0.0211 g (25.2%)

[0405] synthetic ginseng (PEG23+C22) To compound 1 (290 mg, 0.851 mmol, 1.0 equivalent), compound 2 (999 mg, 0.851 mmol, 1.0 equivalent) and diisopropylethylamine (0.445 mL, 2.554 mmol, 3.0 equivalent) at room temperature A solution in anhydrous DMF (3 mL) was added TBTU (328 mg, 1.021 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 2 hours. The reaction mixture was quenched with saturated aqueous NaHCO 3 (10 mL) and the aqueous phase was extracted with dichloromethane (3×10 mL). The combined org. phases were dried over anhydrous Na2SO4 and concentrated. Product 3 was purified by CombiFlash® and eluted with 7-16% methanol in dichloromethane. LC-MS: Calculated for [M+H]+ 1496.05, found 1496.59.

[0407] To a solution of compound 1 (642 mg, 0.429 mmol, 1.0 equiv) in dry 1,4-dioxane (0.5 mL) was added a solution of HCl in dioxane (2.146 mL, 8.582 mmol, 20 equivalents). The reaction mixture was kept at room temperature for 30 minutes and the solvent was removed in vacuo. The product was used directly without further purification. LC-MS: Calculated for [M+H]+ 1396.00, found 1396.60.

[0408] To a solution of compound 5 (24 mg, 0.0203 mmol, 1.0 equiv) and compound 4 (94 mg, 0.062 mmol, 3.05 equiv) in anhydrous DMF (2 mL) was added TEA (0.014 mL, 0.101 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature for 3 hours and the solvent was removed in vacuo. Product 6 was purified by CombiFlash® and eluted with 13-16% methanol in dichloromethane. LC-MS: Calcd. for [M+5H] / 5 974.25, found 975.18.

[0409] synthetic ginseng (PEG23+CLS)

[0410] To a solution of compound 1 (100 mg, 0.222 mmol, 1.0 equiv) and compound 2 (274 mg, 0.233 mmol, 1.05 equiv) in anhydrous DCM (2 mL) was added TEA (0.094 mL, 0.668 mmol, 3.0 equivalents). The reaction mixture was kept at room temperature for 2 hours and then concentrated in vacuo. Product 3 was purified by CombiFlash® and eluted with 8-15% methanol in dichloromethane. LC-MS: Calculated for [M+H2O]+ 1603.17, found 1603.18.

[0411] To a solution of compound 3 (353 mg, 0.222 mmol, 1.0 equiv) in anhydrous 1,4-dioxane (0.5 mL) was added a solution of HCl in dioxane (1.11 mL, 4.451 mmol, 20 equivalents). The reaction mixture was kept at room temperature for 30 minutes and the solvent was removed in vacuo. Product 4 was used without further purification. LC-MS: Calculated for [M+H]+ 1486.01, found 1486.50.

[0412] To a solution of compound 5 (24 mg, 0.0203 mmol, 1.0 equiv) and compound 4 (94 mg, 0.062 mmol, 3.05 equiv) in anhydrous DMF (2 mL) was added TEA (0.014 mL, 0.101 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature for 3 hours and the solvent was removed in vacuo. Product 6 was purified by CombiFlash® and eluted with 13-16% methanol in dichloromethane.

[0413] Synthetic PEG95+C22 To compound 1 (60 mg, 0.0419 mmol, 1.0 equivalent), compound 2 (52 mg, 0.0419 mmol, 1.0 equivalent) and DIPEA (0.022 mL, 0.125 mmol, 3.0 equivalent) at room temperature in anhydrous DMF (3 mL) was added TBTU (16 mg, 0.0503 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 2 hours. The reaction mixture was concentrated. The product was purified by CombiFlash® and eluted with 6-8% methanol in dichloromethane. LC-MS: Calculated for [M+4H]+ / 4 656.66, Found 656.17. Yield: 0.063 g (57.3%).

[0415] To a solution of compound 1 (60 mg, 0.0229 mmol, 1.0 equiv) in anhydrous 1,4-dioxane (0.5 mL) was added a solution of HCl in dioxane (0.286 mL, 1.143 mmol, 50 equivalents). The reaction mixture was kept at room temperature for 30 minutes and the solvent was removed in vacuo. The product was used directly without further purification. LC-MS: Calculated for [M+3H]+ / 3 841.88, found 841.48, calculated for [M+4H]+ / 4 631.66, found 632.41.

[0416] To a solution of compound 5 (55 mg, 0.0214 mmol, 1.0 equiv) and compound 4 (54.7 mg, 0.0214 mmol, 1.0 equiv) in anhydrous DMF (2 mL) was added TEA (0.009 mL, 0.0641 mmol, 3.0 equivalents). The reaction mixture was kept at room temperature for 2 hours and the solvent was removed in vacuo. Product 6 was purified by CombiFlash® and eluted with 15-20% methanol in dichloromethane. LC-MS: [M+5H]+ / 5 calculated 986.80, found 987.19, [M+6H]+ / 6 calculated 822.50, found 822.64.

[0417] Synthetic PEG47+C22

[0418] To a solution of compounds 1 (0.200 g) and 2 (0.0580 g) in DMF was added TBTU (0.0657 g) followed by DIPEA (0.089 mL) at room temperature. The reaction mixture was stirred for 2 hours until complete conversion was observed by LC-MS. The reaction mixture was concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of DCM to 20% MeOH in DCM (0-50%), where the product was eluted at 83% B. The product was concentrated in vacuo to afford 3 as a clear colorless oil. Yield: 0.161 g (63.0%). LC-MS: [M+H]+ Calculated 1495.05 m / z, Observed 1494.30 m / z.

[0419] To Compound 1 (0.161 g) was added 4 M HCl in dioxane (0.805 mL) at room temperature. The reaction mixture was stirred at room temperature. After 10 minutes, complete conversion was confirmed via LC-MS. The reaction mixture was concentrated in vacuo to afford the product as a white solid. No further purification was required. Yield: 0.156 g (101%). LC-MS: [M+H]+ Calculated 1396.00 m / z, Observed 1396.48 m / z.

[0420] To a solution of compounds 5 (0.152 g) and 4 (0.156 g) in DMF was added TEA (0.046 mL) at room temperature. The reaction mixture was stirred at room temperature. After 1.5 hours, complete conversion was confirmed by LC-MS. The reaction mixture was concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase and eluted with a gradient of DCM to 20% MeOH (0-100% B) in DCM. The product eluted at 100% B. Concentration of the fractions afforded 6 as a white solid. Yield: 0.216 g (74.0%). LC-MS: [M+H]+ calcd. 2674.69 m / z, 687.67 m / z (water adduct); observed 686.89 m / z.

[0421] Synthetic PEG47+CLS

[0422] To a solution of Compounds 1 (0.200 g) and 2 (0.0765 g) in DCM at 0°C in an ice-water bath was added TEA under normal atmospheric pressure. The reaction mixture was stirred in an ice-water bath for 10 minutes and then at room temperature for 2 hours until complete conversion was observed by LC-MS. The reaction mixture was concentrated for separation. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient from DCM to 20% MeOH / DCM (0-50%), where the product was eluted at 23% B. Product 3 was concentrated in vacuo to give a white solid. Yield: 0.156 g (57.8%). LC-MS: [M+H]+ Calculated 1586.06 m / z, Observed 1604.14 m / z.

[0423] To compound 3 (0.161 g) was added 4 M HCl in dioxane (0.759 mL) at room temperature. The reaction mixture was stirred at room temperature. After 10 minutes, complete conversion was confirmed via LC-MS. The reaction mixture was concentrated in vacuo to afford 4 as a white solid. No further purification was required. Yield: 0.157 g (102%). LC-MS: [M+H]+ Calculated 1486.01 m / z, Observed 1487.58 m / z.

[0424] To a solution of compounds 5 (0.144 g) and 4 (0.157 g) in DMF was added NEt3 (0.043 mL) at room temperature. The reaction mixture was stirred at room temperature. After 1.5 hours, complete conversion was confirmed by LC-MS. The reaction mixture was concentrated in vacuo. The residue was purified by CombiFlash® using silica gel as stationary phase and eluted with a gradient of DCM to 20% MeOH (0-100% B) in DCM. The product eluted at 37% B. Concentration of the fractions afforded 6 as a white solid. Yield: 0.225 g (79.0%). LC-MS: [M+H]+ calcd. 2764.70 m / z, 710.17 m / z (water adduct); observed 709.46 m / z.

[0425] Synthetic PEG71+C22 To compound 1 (50 mg, 0.146 mmol, 1.0 equivalent), compound 2 (172 mg, 0.146 mmol, 1.0 equivalent) and diisopropylethylamine (0.077 mL, 0.440 mmol, 3.0 equivalent) at room temperature A solution in anhydrous DMF (3 mL) was added TBTU (56 mg, 0.176 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 2 hours. The reaction mixture was quenched with saturated aqueous NaHCO 3 (10 mL) and the aqueous phase was extracted with dichloromethane (3×10 mL). The combined org. phases were dried over anhydrous Na2SO4 and concentrated. The product was purified by CombiFlash® and eluted with 6-8% methanol in dichloromethane. LC-MS: Calculated for [M+H]+ 1496.05, found 1496.23.

[0427] To a solution of compound 1 (120 mg, 0.0802 mmol, 1.0 equiv) in dry 1,4-dioxane (0.5 mL) was added a solution of HCl in dioxane (1.00 mL, 4.010 mmol, 50 equivalents). The reaction mixture was kept at room temperature for 30 minutes and the solvent was removed in vacuo. The product was used directly without further purification. LC-MS: Calculated for [M+H]+ 1396.00, found 1396.60.

[0428] To a solution of compound 5 (98 mg, 0.0381 mmol, 1.0 equiv) and compound 4 (54.5 mg, 0.0381 mmol, 1.0 equiv) in anhydrous DMF (5 mL) was added TEA (0.016 mL, 0.114 mmol, 3.0 equivalents). The reaction mixture was kept at room temperature for 3 hours and the solvent was removed in vacuo. Product 6 was purified by CombiFlash® and eluted with 15-20% methanol in dichloromethane. LC-MS: Calculated for [M+4H]+ / 4 951.25, found 952.14, calculated for [M+5H]+ / 5 761.20, found 761.67.

[0429] Synthetic PEG71+CLS

[0430] To a solution of compound 1 (100 mg, 0.222 mmol, 1.0 equiv) and compound 2 (274 mg, 0.233 mmol, 1.05 equiv) in anhydrous DCM (2 mL) was added TEA (0.094 mL, 0.668 mmol, 3.0 equivalents). The reaction mixture was kept at room temperature for 2 hours and the reaction mixture was concentrated. Product 3 was purified by CombiFlash® and eluted with 8-15% methanol in dichloromethane. LC-MS: Calculated for [M+H2O]+ 1603.17, found 1603.18.

[0431] To a solution of compound 3 (353 mg, 0.222 mmol, 1.0 equiv) in anhydrous 1,4-dioxane (0.5 mL) was added a solution of HCl in dioxane (1.11 mL, 4.451 mmol, 20 equivalents). The reaction mixture was kept at room temperature for 30 minutes and then the solvent was removed in vacuo. Product 4 was used without further purification. LC-MS: Calculated for [M+H]+ 1486.01, found 1486.50.

[0432] To a solution of compound 5 (70 mg, 0.0272 mmol, 1.0 equiv) and compound 4 (41.4 mg, 0.0272 mmol, 1.0 equiv) in anhydrous DMF (2 mL) was added TEA (0.012 mL, 0.0816 mmol, 3.0 equivalents). The reaction mixture was kept at room temperature for 3 hours and then the solvent was removed in vacuo. Product 6 was purified by CombiFlash® and eluted with 13-19% methanol in dichloromethane. LC-MS: Calculated for [M+4H]+ / 4 973.84, found 974.58, [M+5H]+ / 5 779.27, found 779.79.

[0433] Synthetic PEG95+CLS To compound 1 (60 mg, 0.0419 mmol, 1.0 equivalent), compound 2 (52 mg, 0.0419 mmol, 1.0 equivalent) and DIPEA (0.022 mL, 0.125 mmol, 3.0 equivalent) at room temperature in anhydrous DMF (3 mL) was added TBTU (16 mg, 0.0503 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 2 hours. The reaction mixture was then concentrated in vacuo. Product 3 was purified by CombiFlash® and eluted with 12-18% methanol in dichloromethane. LC-MS: Calculated for [M+4H]+ / 4 679.18, found 679.93, [M+3H]+ / 3 905.24, found 905.81. Yield: 0.082 g (76.7%).

[0435] To a solution of compound 3 (85 mg, 0.0313 mmol, 1.0 equiv) in dry 1,4-dioxane (0.3 mL) was added a solution of HCl in dioxane (0.391 mL, 1.565 mmol, 50 equivalents). The reaction mixture was kept at room temperature for 30 minutes and the solvent was removed in vacuo. Product 4 was used without further purification. LC-MS: Calculated for [M+3H]+ / 3 871.89, found 871.72, [M+4H]+ / 4 654.17, found 654.97.

[0436] To a solution of compound 5 (80 mg, 0.0311 mmol, 1.0 equiv) and compound 4 (82 mg, 0.0311 mmol, 1.0 equiv) in anhydrous DMF (2 mL) was added TEA (0.013 mL, 0.0932 mmol, 3.0 equivalents). The reaction mixture was kept at room temperature for 3 hours and then the solvent was removed in vacuo. Product 6 was purified by CombiFlash® and eluted with 13-19% methanol in dichloromethane. LC-MS: Calculated for [M+4H]+ / 4 1255.76, found 1255.57, [M+5H]+ / 5 1004.81, found 1005.79.

[0437] Synthesis of LP1-p

[0438] To compound 1 (2630 mg, 1.142 mmol, 1.0 equivalents), compound 2 (428 mg, 1.256 mmol, 1.1 equivalents) and diisopropylethylamine (0.597 mL, 3.427 mmol, 3.0 equivalents) at room temperature A solution in anhydrous DMF (10 mL) was added TBTU (440 mg, 1.371 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 2 hours and then concentrated. Compound 3 was purified by CombiFlash® eluting with 12-17% MeOH in DCM. LC-MS: Calculated for [M+4H]+ / 4 656.66, Found 656.65.

[0439] To Compound 3 solid (1150 mg, 0.438 mmol, 1.0 equiv) was added a solution of HCl in dioxane (5.478 mL, 21.910 mmol, 50 equiv) at room temperature. The reaction mixture was kept at room temperature for 30 minutes and then concentrated. Compound 4 was used without further purification. LC-MS: [M+3H]+ / 3 calculated 841.88, found 842.56, [M+4H]+ / 4 calculated 631.66, found 632.41.

[0440] To a solution of compound 5 (175 mg, 0.203 mmol, 1.0 equiv) and compound 4 (1095 mg, 0.427 mmol, 2.1 equiv) in anhydrous DCM (10 mL) was added TEA (0.144 mL, 1.018 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature for 3 hours and the solvent was removed under vacuum. LP1-p was purified by CombiFlash® eluting with 10-17% MeOH in DCM. LC-MS: Calculated for [M+6H]+ / 6 946.60, Found 947.10, Calculated for [M+7H]+ / 7 811.51, Found 811.35.

[0441] Synthetic LP5-p

[0442] Compound 1 (105 mg, 0.198 mmol) in DMF was treated with TBTU (4 equiv) and stirred for 5 minutes. DIEA (8 equiv) was then added and the mixture was added to 1 molar equivalent of ethylenediamine on pre-swelled 2-chlorotrityl resin. After 30 minutes of agitation, the resin was washed three times with DMF and then treated with 2% hydrazine in DMF for 10 minutes. The coupling of palmitic acid (202 mg, 0.789 mmol) was repeated using the same procedure as for the coupling of compound 1 . Upon completion, the resin was washed with 3 portions of DCM and treated with 1% TFA in DCM for 10 minutes. The TFA treatment was repeated and the resin was washed with 3 portions of DCM. All volatiles were removed and crude compound 2 was used without further purification. Yield 126 mg (81%).

[0443] To a mixture containing Compound 2 (23 mg, 37 µmol) and DIEA (14.1 uL, 81 µmol) in DMF (1 mL) was added NHS-PEG24-MAL (Compound 3, 61.5 mg, 0.0441 mmol) and The reaction mixture was stirred for 30 minutes. After completion, crude LP5-p was dry-loaded onto silica and isolated with gradient elution of MeOH in DCM. Yield 15 mg (21%).

[0444] Synthetic LP28-p

[0445] To a solution of compounds 1 (80 mg) and 2 (60.2 mg) in DMF was added TBTU (90.3 mg) followed by DIPEA (0.147 mL) at room temperature. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-80%, isocratic, and then to 100%) in DCM over 20-30 minutes, where compound 3 was in Eluted at 68% B. Compound 3 was concentrated in vacuo to give a white oily residue. LC-MS: Calculated for [M+H]+ 2567.65 m / z, Observed 1301.78 (+2 / 2, +H2O) m / z.

[0446] To compound 3 (100.4 mg) was added 4 M HCl in dioxane (14.3 mg) at room temperature. The reaction mixture was stirred at room temperature. The reaction mixture was stirred overnight until complete conversion was confirmed by LC-MS. The reaction mixture was azeotroped with PhMe and concentrated in vacuo overnight to afford compound 4 as an oil. LC-MS: [M+H]+ Calculated 2467.60 m / z, Observed 1243.32 m / z.

[0447] A solution of compound 4 (97.9 mg) and TEA (0.016 mL) in anhydrous DCM was prepared and stirred under an atmosphere of nitrogen. Compound 5 (15.8 mg) was then added to the reaction mixture. The reaction mixture was stirred at room temperature until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase and eluted with a gradient of 0-20% MeOH (0-100% B) in DCM. LP28-p elutes at 67% B. LC-MS: [M+H]+ calculated 5562.48 m / z, observed 1409.68 (+4 / 4, +H2O) m / z.

[0448] Synthetic LP29-p

[0449] To a solution of compounds 1 (40 mg) and 2 (334 mg) in DMF was added TBTU (50.1 mg) followed by DIPEA (0.082 mL) at room temperature. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-80%) in DCM over 20-30 minutes, where compound 3 was eluted at 71% B. Compound 3 was concentrated in vacuo to give a white oily residue. LC-MS: [M+H]+ calculated 2539.62 m / z, observed 1288.21 (+2 / 2, +H2O) m / z.

[0450] To compound 3 (147 mg) was added 4 M HCl in dioxane (21.2 mg) at room temperature. The reaction mixture was stirred at room temperature. The reaction mixture was stirred overnight until complete conversion was confirmed by LC-MS. The reaction mixture was azeotroped with PhMe and concentrated in vacuo overnight to afford compound 4 as an oil. LC-MS: [M+H]+ Calculated 2439.57 m / z, Observed 611.16 (+4 / 4) m / z.

[0451] A solution of compound 4 (143 mg) and TEA (0.024 mL) in anhydrous DCM was prepared and stirred under an atmosphere of nitrogen. Compound 5 (23.4 mg) was then added to the reaction mixture. The reaction mixture was stirred at room temperature until complete conversion was observed by LC-MS.

[0452] The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase and eluting with a gradient of 0-20% MeOH (0-100% B) in DCM. LP29-p elutes at 54% B. LC-MS: [M+H]+ calculated 5506.42 m / z, observed 1854.41 (+3 / 3, +H2O) m / z.

[0453] Synthetic LP33-p

[0454] To a solution of compound 1 (2.00 g, 4.45 mmol) and 2 (1.07 g, 6.68 mmol) in anhydrous DCM was added NEt3 (1.86 mL, 13.4 mmol) at room temperature. The reaction was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-100%) in DCM over 45 minutes, where compound 3 was eluted at 8% B. Compound 3 was concentrated to give a white solid. LC-MS: [M+H]+ Calculated 573.46 m / z, Observed 573.60 m / z.

[0455] To compound 3 (317 mg, 0.553 mmol) was added 4 M HCl in dioxane (1.383 mL) at room temperature. The reaction mixture was stirred at room temperature. The reaction mixture was stirred overnight until complete conversion was confirmed by LC-MS. The reaction mixture was concentrated under high vacuum overnight to afford compound 4 as a clear and colorless oily residue. LC-MS: [M+H]+ calculated 473.40 m / z, observed 473.58 m / z.

[0456] To a solution of compounds 4 (282 mg, 0.553 mmol) and 5 (1.35 g, 0.526 mmol) in anhydrous DCM was added NEt3 (0.386 mL) under N2 (g). The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-100%) in DCM over 45 minutes, with LP33-p eluting at 46% B. Concentration of LP33-p afforded a white solid. LC-MS: [M+H]+ calculated 2879.76 m / z, observed 960.98 (+3 / 3) m / z.

[0457] Synthetic LP38-p

[0458] To a solution of compounds 1 (35 mg) and 2 (299 mg) in DMF was added TBTU (43.8 mg) followed by DIPEA (0.071 mL) at room temperature. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-100%) in DCM over 20-30 minutes, where compound 3 was eluted at 56% B. Compound 3 was concentrated in vacuo to give a white oily residue. LC-MS: [M+H]+ calculated 2539.62 m / z, observed 1288.07 (+2 / 2, +H2O) m / z.

[0459] To compound 3 (186 mg) was added 4 M HCl in dioxane (26.7 mg) at room temperature. The reaction mixture was stirred at room temperature. The reaction mixture was stirred overnight until complete conversion was confirmed by LC-MS. The reaction mixture was azeotroped with PhMe and concentrated in vacuo overnight to afford compound 4 as an oil. LC-MS: [M+H]+calculated 2439.57 m / z, observed 1220.97 (+2 / 2) m / z.

[0460] To a solution of compound 4 (181 mg), TBTU (24 mg) and DIEA (0.033 mL) in DMF was added compound 5 (8.7 mg) at room temperature. The reaction was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-100%) in DCM over 20-30 minutes, where compound 6 was eluted at 65% B. Compound 6 was concentrated in vacuo to give a white oily residue. LC-MS: [M+H]+ calculated 5089.22 m / z, observed 1036.24 (+5 / 5, +H2O) m / z.

[0461] To compound 6 (130 mg) was added 4 M HCl / dioxane (9.3 mg) at room temperature. The reaction mixture was stirred at room temperature. The reaction mixture was stirred overnight until complete conversion was confirmed by LC-MS. The reaction mixture was azeotroped with PhMe and concentrated in vacuo overnight to afford compound 7 as an oil. LC-MS: [M+H]+ calculated value 4989.17 m / z, observed value 1248.58 (+4 / 4) m / z.

[0462] A solution of Compound 7 (128 mg) and NEt3 (0.018 mL) in anhydrous DCM was prepared at room temperature under N2 (g). Then compound 8 (10.3 mg) was added slowly. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-100%) in DCM over 30 minutes, with LP38-p eluting at 100% B. Concentration of LP38-p afforded a white solid. LC-MS: [M+H]+ calculated 5299.28 m / z, observed 1786.62 (+3 / 3, +H2O) m / z.

[0463] Synthetic LP39-p

[0464] Boc-protected PEG23-amine 1 (Quanta Biodesign Limited, 200 mg, 0.17 mmol) was mixed with cholesterol chloroformate 2 (77 mg, 0.17 mmol) and Et3N (48 uL, 0.341 mmol) in 5 mL Stir in DCM for 1.5 hours. The solvent was removed in vacuo, the residue was mixed with SiO2 (1 g) and loaded on a CombiFlash®. Compound 3 was purified using the system 0-20% MeOH in DCM, gradient 0-80% over 40 minutes. Calculated MW 1586.09, M + 18=1604.09, (M +2×18) / 2=811.05 Found: MS (ES, positive ion): 1603.55 [M+NH4]+, 811.07 [M+2NH4]2+.

[0465] Product 3 was deprotected from the Boc group and the resulting hydrochloride 4 (62 mg, 0.04 mmol) was dissolved in DCM with pentafluorophenyl ester 5 (24 mg, 0.04 mmol) and Et3N (14 uL, 0.1 mmol) (5 mL) was stirred for 1.5 hours. The solvent was removed in vacuo, the residue was mixed with SiO2 (400 mg) and loaded on a CombiFlash®. Product 6 was purified using the system 0-20% MeOH in DCM, gradient 0-70% over 30 minutes. Yield 57 mg. Calculated MW 1893.44, M + 18=1911.44, (M +2×18) / 2=964.72 Found: MS (ES, positive ion): 1911.00 [M+NH4]+, 964.46 [M+2NH4]2+.

[0466] The product 6 was treated with 4M HCl in bisoxane (10 mL) for 4 hours at room temperature. The solvent was removed in vacuo, the toluene was evaporated 2 times from the residue, the product 7 was dried and used directly in the next step.

[0467] Solid TBTU (50 mg, 0.156 mmol) was added to Boc-protected PEG23-amine 1 (Quanta Biodesign Limited, 152 mg, 0.13 mmol), palmitic acid 8 (33 mg, 0.13 mmol) and DIEA (68 uL , 0.39 mmol) in DMF (9 mL). The reaction mixture was sonicated to dissolve the solids and stirred at room temperature for 16 hours. The solvent was removed in vacuo, toluene was evaporated from the residue twice, the residue was dissolved in chloroform (50 mL), washed with NaHCO3 (2 x 10 mL) and brine (10 mL). Compound 9 was dried (Na2SO4), concentrated in vacuo, and purified on a Combiflash® (SiO2) using the system DCM:20% MeOH in DCM, gradient 0-80% over 20 minutes. Calculated MW 1411.85, M + 18=1429.85, (M +1+ 18) / 2=715.43 Found: MS (ES, positive ion): 1429.24 [M+NH4]+, 715.41 [M+H+NH4]2 +.

[0468] 9 was deprotected from the Boc protecting group under HCl / dioxane solution and compound 10 was used directly in the next step.

[0469] Derivative 7 (60 mg, 0.028 mmol) was prepared in DCM:DMF= 1:1 (8 mL) and stirred for 3 hours. The solvent was removed in vacuo, toluene was evaporated from the residue twice, and the solid was suspended in CHCl3 (50 mL). The suspension was washed twice with 2% NaHCO3 and brine. After concentration in vacuo, product 11 was purified on Combiflash® (0-20% MeOH in DCM, gradient 0-70%, 35 minutes).

[0470] The product 11 (51 mg, 0.0162 mmol) was stirred with Et3N in DMF (20%, 3 mL) for 16 hours, the solvent containing Et3N was removed in vacuo, and toluene was evaporated 3 times from the residue to obtain deprotected The base of the amine 12. Calculated MW 2908.81, (M +1+18) / 2=1463.91, (M +1+18 × 2) / 3=981.94 Found: MS (ES, positive ion): 1463.69 [M+ H +NH4]2+ , 981.99 [M+H+2NH4]3+.

[0471] Amine 12 (47 mg, 0.0162 mmol) was stirred with a mixture of NHS ester 13 (21 mg, 0.0147 mmol) and Et3N (6 uL, 0.041 mmol) in DCM (4 mL) for 16 h. The solvent was removed in vacuo and the product LP39-p was purified on the Combiflash® using the system 0-20% MeOH in DCM, gradient 0-100% over 40 minutes. Calculated MW 4188.28, (M +2+18) / 3=1402.76, (M +3+18×2) / 4=1052.32 Found: MS (ES, positive ion): 1402.71 [M+ 2H +NH4]3+ , 1052.32 [M+3H+NH4]4+.

[0472] Synthetic LP41-p

[0473] To a solution of Compound 1 (40.0 mg), TBTU (50.1 mg) and DIEA (0.098 mL) in DMF was added Compound 2 (298 mg) at room temperature. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (10-100% B) in DCM over 20-30 minutes, where compound 3 was eluted at 43% B. Compound 3 was concentrated in vacuo to give a white oily residue. LC-MS: [M+H]+ Calcd. 2539.62 m / z, Observed 1287.83 (+2 / 2, +H2O) m / z.

[0474] To compound 1 (260 mg) was added 4 M HCl in dioxane (37.4 mg) at room temperature. The reaction mixture was stirred at room temperature. The reaction mixture was stirred overnight until complete conversion was confirmed by LC-MS. The reaction mixture was azeotroped with PhMe and concentrated in vacuo overnight to afford compound 4 as an oil. LC-MS: [M+H]+ Calculated 2439.57 m / z, Observed 1220.61 (+2 / 2) m / z.

[0475] To a solution of Compound 4 (253 mg), TBTU (36.1 mg) and DIEA (0.045 mL) in DMF was added Compound 5 (11.9 mg) at room temperature. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase using a gradient of 0-20% MeOH (10-30, 35, then 100%) in DCM over 30 minutes, where compound 6 was eluted at 35% B . Compound 6 was concentrated in vacuo to give a white oily residue. LC-MS: [M+H]+ calculated 5089.22 m / z, observed 1715.43 (+3 / 3, +H2O) m / z.

[0476] To compound 6 (35.4 mg) was added 4 M HCl in dioxane (2.5 mg) at room temperature. The reaction mixture was stirred at room temperature. The reaction mixture was stirred overnight until complete conversion was confirmed by LC-MS. The reaction mixture was azeotroped with PhMe / MeOH and concentrated under high vacuum overnight to afford compound 7 as an oil. LC-MS: [M+H]+calculated 4989.17 m / z, observed 1676.42 (+HCl, +3 / 3) m / z.

[0477] A solution of Compound 7 (35 mg) and NEt3 (0.005 mL) in anhydrous DCM was prepared at room temperature under N2 (g). Then compound 8 (3.2 mg) was added slowly. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as the stationary phase using a gradient of 0-20% MeOH / DCM (10 to 30%, 40%, 50%, 70%, then 100% B) over 30 minutes, where LP41-p was eluted at 100% B. LC-MS: [M+H]+calculated 5837.84 m / z, observed 1079.90 (+5 / 5) m / z.

[0478] Synthetic LP42-p

[0479] To a solution of Compound 1 (40 mg), TBTU (50.1 mg) and DIEA (0.098 mL) in DMF was added Compound 2 (298 mg) at room temperature. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (10-100% B) in DCM over 20-30 minutes, where compound 3 was eluted at 43% B. Compound 3 was concentrated in vacuo to give a white oily residue. LC-MS: [M+H]+ Calcd. 2539.62 m / z, Observed 1287.83 (+2 / 2, +H2O) m / z.

[0480] To compound 3 (260 mg) was added 4 M HCl in dioxane (37.4 mg) at room temperature. The reaction mixture was stirred at room temperature. The reaction was stirred overnight until complete conversion was confirmed by LC-MS. The reaction mixture was azeotroped with PhMe and concentrated in vacuo overnight to afford compound 4 as an oil. LC-MS: [M+H]+ Calculated 2439.57 m / z, Observed 1220.61 (+2 / 2) m / z.

[0481] To a solution of Compound 4 (253 mg), TBTU (36.1 mg) and DIEA (0.045 mL) in DMF was added Compound 5 (11.9 mg) at room temperature. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase using a gradient of 0-20% MeOH (10-30, 35, then 100%) in DCM over 30 minutes, where compound 6 was eluted at 35% B . Compound 6 was concentrated in vacuo to give a white oily residue. LC-MS: [M+H]+ calculated 5089.22 m / z, observed 1715.43 (+3 / 3, +H2O) m / z.

[0482] To compound 6 (28.2 mg) was added 4 M HCl in dioxane (2.0 mg) at room temperature. The reaction mixture was stirred at room temperature. The reaction mixture was stirred overnight until complete conversion was confirmed by LC-MS. The reaction mixture was azeotroped with PhMe / MeOH and concentrated under high vacuum overnight to give an oil. LC-MS: [M+H]+ calculated 4989.17 m / z, observed 1000.21 (+5 / 5) m / z.

[0483] A solution of Compound 7 (27.9 mg) and NEt3 (0.004 mL) in anhydrous DCM was prepared at room temperature under N2 (g). Then compound 8 (3.4 mg) was added slowly. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as the stationary phase using a gradient of 0-20% MeOH (25 to 50% followed by 100% B) in DCM over 30 minutes, where after 5 minutes at 100% B LP42 - p is eluted at 100% B. LC-MS: [M+H]+calculated 5563.44 m / z, observed 946.45 (+6 / 6, +water) m / z.

[0484] Synthetic LP43-p To compound 1 (3.0 g, 1.303 mmol, 1.0 equivalent), compound 2 (0.401 g, 1.564 mmol, 1.2 equivalent) and diisopropylethylamine (0.681 mL, 3.91 mmol, 3.0 equivalent) at room temperature To a solution in DMF (20 mL) was added TBTU (0.502 g, 1.564 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 3 hours. The reaction mixture was concentrated. Compound 3 was purified by CombiFlash® eluting with 12-18% methanol in dichloromethane. The structure was confirmed by H-NMR.

[0486] To Compound 3 solid (2060 mg, 0.811 mmol, 1.0 equiv) was added a solution of HCl in dioxane (4.055 mL, 16.219 mmol, 20 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour and the solvent was removed under vacuum. Compound 4 was used without further purification. The structure was confirmed by H-NMR.

[0487] To compound 4 (2030 mg, 0.819 mmol, 1.0 equivalents), compound 5 (257 mg, 0.983 mmol, 1.2 equivalents) and diisopropylethylamine (0.428 mL, 2.459 mmol, 3.0 equivalents) at room temperature To a solution in dry DMF (10 mL) was added TBTU (315 mg, 0.983 mmol, 1.2 equiv). The reaction mixture was kept at room temperature overnight. The reaction mixture was concentrated. Compound 6 was purified by CombiFlash® eluting with 12-20% methanol in dichloromethane. LC-MS: [M+2H] / 2, calculated 1341.84, found 1342.69.

[0488] To a solution of compound 6 (1430 mg, 0.530 mmol, 1.0 equiv) in THF (20 mL) and water (20 mL) was added lithium hydroxide (63.8 mg, 2.664 mmol, 5.0 equiv) at room temperature. The reaction mixture was kept at room temperature for 3 hours. The reaction mixture was quenched with HCl solution and the pH was adjusted to 3.0. The aqueous phase was extracted with DCM (3 x 20 mL). The combined org. phases were dried over Na2SO4 and concentrated. Compound 7 was used without further purification. LC-MS: Calcd. for [M+2H] / 2 1334.83, found 1335.49.

[0489] To compound 7 (110 mg, 0.0412 mmol, 1.0 equivalent), compound 8 (103 mg, 0.0412 mmol, 1.00 equivalent) and diisopropylethylamine (0.022 mL, 0.123 mmol, 3.0 equivalent) at room temperature To a solution in DMF (2 mL) was added TBTU (15.9 mg, 0.0495 mmol, 1.2 equiv). The reaction mixture was kept at room temperature overnight and then concentrated. Compound 9 was purified by CombiFlash® eluting with 16-20% methanol in dichloromethane. LC-MS: Calcd. for [M+5H] / 5 1023.44, found 1024.00.

[0490] To compound 9 (84 mg, 0.0164 mmol, 1.0 equiv) was added 4M HCl in dioxane (0.205 mL, 0.0821 mmol, 50 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour and then concentrated. Compound 10 was used without further purification. LC-MS: Calcd. for [M+5H] / 5 1003.44, found 1004.07.

[0491] To a solution of compound 10 (125 mg, 0.0247 mmol, 1.0 equiv) and compound 11 (116 mg, 0.0272 mmol, 1.10 equiv) in anhydrous DCM (2 mL) was added triethylamine (0.017 mL) at room temperature , 0.123 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature overnight and then concentrated. LP43-p was purified by CombiFlash® eluting with 18-20% methanol in dichloromethane. LC-MS: Calcd. for [M+5H] / 5 1065.46, found 1066.13.

[0492] Synthetic LP44-p

[0493] Compound 1 was synthesized as shown in the above steps in the synthesis of LP43-p (compound 7 in the synthesis of LP43-p). Add compound 1 (135 mg, 0.0506 mmol, 1.0 equiv), compound 2 (129 mg, 0.0506 mmol, 1.00 equiv) and diisopropylethylamine (0.026 mL, 0.151 mmol, 3.0 equiv) in DMF ( 2 mL) was added TBTU (19.5 mg, 0.0607 mmol, 1.2 equiv). The reaction mixture was kept at room temperature overnight and then concentrated. Compound 3 was purified by CombiFlash® eluting with 12-20% methanol in dichloromethane. LC-MS: Calcd. for [M+5H] / 5 1035.06, found 1035.40.

[0494] To compound 3 (100 mg, 0.0193 mmol, 1.0 equiv) was added 4M HCl in dioxane (0.242 mL, 0.966 mmol, 50 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour and then concentrated. Compound 4 was used without further purification. LC-MS: Calculated for [M+5H] / 5 1015.05, found 1015.71.

[0495] To a solution of compound 4 (95 mg, 0.0186 mmol, 1.0 equiv) and compound 5 (8 mg, 0.0186 mmol, 1.0 equiv) in anhydrous DCM (2 mL) was added triethylamine (0.013 mL) at room temperature , 0.0930 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature overnight and then the solvent was removed under vacuum. LP44-p was purified by CombiFlash® eluting with 12-20% methanol in dichloromethane. LC-MS: Calculated for [M+5H] / 5 1077.74, found 1079.

[0496] Synthetic LP45-p

[0497] To a solution (2.0 mL) of palmitic acid 1 (30 mg, 0.1170 mmol) in DMF with Boc-PEG47-NH22 (269 mg, 0.1170 mmol) was added TBTU (45.1 mg, 0.1404 mmol) and DIPEA (60 uL ). After stirring the reaction mixture overnight, water was added and compound 3 was extracted using DCM:20% TFE and dried over Na2SO4. After filtration, the solvent was removed in vacuo to dryness and compound 3 was purified by flash chromatography (DCM: 20% MeOH).

[0498] To compound 3, 2 mL of 4N HCl:dioxane was added and the reaction mixture was stirred under anhydrous conditions until complete as determined by LC-MS: [M+H]+ calculated for C16-PEG47-NH2301 m / z , the experimental value is 2302.

[0499] Add NEt3 (29 uL) to a solution of Fmoc-Glu(OtBu)-Opfp5 (50 mg, 0.0845 mmol) in C16-PEG47-NH24 (206 mg, 0.0.0845 mmol), while in DCM (5.0 mL) and stirred. When the reaction mixture was determined to be complete, the solvent was removed in vacuo to dryness and crude compound 6 was purified by flash chromatography (DCM: 20% MeOH).

[0500] To Compound 6 was added 2 mL of 4N HCl:dioxane and stirred under anhydrous conditions until complete by LC-MS: [M+H]+calculated 2866.0 found 2867.

[0501] In a solution of Boc-PEG47-NH29 (269 mg, 0.1170 mmol) with TBTU (45.1 mg, 0.1404 mmol) and DIPEA (60 uL), while stirring in DMF (2.0 mL), compound 8 ( 30 mg, 0.1170 mmol). After stirring the resulting suspension overnight, water was added and the product was extracted using DCM:20% TFE and dried over Na2SO4. After filtration, the solvent was removed in vacuo to dryness and compound 10 was purified by flash chromatography (DCM: 20% MeOH). [M+H]+ Calculated value 2614.32 m / z, experimental value 2615.32.

[0502] To Compound 10 was added 2 mL of 4N HCl:dioxane. The reaction mixture was stirred under anhydrous conditions until complete. Product 11 was used in the next step without further purification.

[0503] To a solution of compound 7 (100 mg, 0.0375 mmol) in DMF (5.0 mL) with compound 11 (98 mg, 0.1914 mmol) was added TBTU (14.4 mg, 0.045 mmol) and DIPEA (20 uL). After stirring the resulting suspension overnight, water was added and extracted with DCM:20% TFE and dried over Na2SO4. After filtration, the solvent was removed in vacuo to dryness and purified by flash chromatography (DCM: 20% MeOH). To this was added 2 mL of 4N HCl:dioxane and the reaction mixture was stirred under anhydrous conditions until complete by LC-MS to afford compound 12. LC-MS: [M+H]+ calcd. 5134.26 m / z, found 5135. To a solution of compound 13 (10 mg, 0.0235 mmol, 1.0 equiv) and compound 12 (120 mg, 0.0235 mmol, 1.0 equiv) in anhydrous DCM (2 mL) was added triethylamine (17 uL) at room temperature , 0.1175 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature overnight and the solvent was removed under vacuum. LP45-p was purified by CombiFlash® eluting with 10-17% methanol in dichloromethane. LC-MS: Calculated [M+6H]+ 5474.38, found 5475.01.

[0505] Synthetic LP47-p

[0506] Solid TBTU (50 mg, 0.156 mmol) was added to Boc-protected Peg23-amine 2 (Quanta Biodesign Limited, 150 mg, 0.13 mmol), eicosapentaenoic acid 1 (39 mg, 0.13 mmol) and A solution of DIEA (68 µL mL, 0.39 mmol) in DMF (9 mL). The reaction mixture was sonicated to dissolve the solids and stirred at room temperature for 16 hours. The solvent was removed in vacuo, toluene was evaporated from the residue twice, the residue was dissolved in chloroform (50 mL), washed with NaHCO3 (2 x 10 mL) and brine (10 mL). The product was dried (Na2SO4), concentrated in vacuo, and purified on a Combiflash® (SiO2) using a system 0-20% MeOH in DCM, gradient 0-80% over 20 minutes. Removal of the Boc group with 4M HCl in dioxane gave hydrochloride 4. Calculated MW 1357.76, (M +2) / 2=679.88 Found: MS (ES, positive ion): 1358.29 [M+H]+, 679.77 [M+2H]2+.

[0507] The hydrochloride salt 4 (167 mg, 0.123 mmol) was stirred with pentafluorophenyl ester 5 (73 mg, 0.123 mmol) and Et3N (43 uL, 0.31 mmol) in DCM (5 mL) for 2 hours. The solvent was removed in vacuo, the residue was mixed with SiO2 (1 g) and loaded on a CombiFlash®. Product 6 was purified using the system 0-20% MeOH in DCM, gradient 0-50% over 25 minutes. Yield 169 mg. Calculated MW 1765.23, M +18=1783.23, (M +1+18) / 2=892.12 Found: MS (ES, positive ion): 1782.78 [M+NH4 ]+, 891.97 [M+H+NH4]2 +.

[0508] The product 6 was treated with HCl in dioxane to obtain the free acid 7 and used directly in the next step. Calculated value MW 3002.84, (M +2×18) / 2=1519.42, (M+3x18) / 3=1018.95. Found: MS (ES, positive ion): 1519.39 [M+2NH4]2+, 1019.17 [M+H+2NH4]3+.

[0509] Derivative 7 (47 mg, 0.028 mmol) was prepared in DCM:DMF= 1:1 (8 mL) and stirred for 3 hours. The solvent was removed in vacuo, toluene was evaporated from the residue twice, and the solid was suspended in CHCl3 (50 mL). The suspension was washed twice with 2% NaHCO3 and brine, and after concentration in vacuo, product 9 was purified on Combiflash® (0-20% MeOH in DCM, gradient 0-70%, 35 min).

[0510] The product 9 (49 mg, 0.0162 mmol) was stirred with Et3N in DMF (20%, 3 mL) for 16 hours, the solvent containing Et3N was removed in vacuo, and toluene was evaporated 3 times from the residue to obtain deprotected The base amine 10 was used directly in the next step.

[0511] Amine 10 (45 mg, 0.0162 mmol) was stirred with a mixture of NHS ester 11 (21 mg, 0.0147 mmol) and Et3N (6 uL, 0.041 mmol) in DCM (4 mL) for 16 h. The solvent was removed in vacuo and the product LP47-p was purified on the Combiflash® using the system DCM:20% MeOH in DCM, gradient 0-100% over 40 minutes. Calculated MW 4060.07, (M +3×18) / 3=1371.36, (M+4×18) / 4=1033.02 Experimental value: MS (ES, positive ion): 1371.76 [M+3NH4]3+, 1033.70 [ M+4NH4]4+.

[0512] Synthetic LP48-p

[0513] To a solution of Compound 1 (27.5 mg), TBTU (26.6 mg) and DIEA (0.022 mL) in DMF was added Compound 2 (173 mg) at room temperature. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using a 12 g silica gel column as stationary phase with a gradient of 0-20% MeOH (10-100%) in DCM over 20 minutes, where compound 3 was eluted at 66% B. Compound 3 was concentrated in vacuo to give a white oily residue. LC-MS: [M+H]+ calculated 2615.65 m / z, observed 1326.52 (+2 / 2, +H2O) m / z.

[0514] To compound 3 (56.7 mg) was added 4 M HCl in dioxane (7.9 mg) at room temperature. The reaction mixture was stirred at room temperature. The reaction was stirred overnight until complete conversion was confirmed by LC-MS. The reaction mixture was azeotroped with PhMe / MeOH and concentrated under high vacuum overnight to afford compound 4 as a white solid. LC-MS: [M+H]+calculated 2515.60 m / z, observed 1259.91 (+2 / 2) m / z.

[0515] A solution of Compound 4 (55.4 mg) and NEt3 (0.015 mL) in anhydrous DCM was prepared at room temperature under N2 (g). Then compound 5 (8.9 mg) was added slowly. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® through a 4 g silica gel column as stationary phase using a gradient of 0-20% MeOH / DCM (10% B to 100% B) over 20 minutes, where LP48-p was at 100% B Dissolution. Concentration of LP48-p gave a white oily residue. LC-MS: [M+H]+ calculated 5558.48 m / z, observed 1152.98 (+5 / 5, +H2O) m / z.

[0516] Synthetic LP49-p

[0517] To a solution of Compound 1 (31.3 mg), TBTU (33.4 mg) and DIEA (0.023 mL) in DMF was added Compound 2 (199 mg) at room temperature. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (10-100%) in DCM over 30 minutes, where compound 3 was eluted at 57% B. Compound 3 was concentrated in vacuo to give a white oily residue. LC-MS: [M+H]+ calculated 2583.65 m / z, observed 1311.03 (+2 / 2, +H2O) m / z.

[0518] To Compound 3 (70 mg) was added 4 M HCl in dioxane (9.9 mg) at room temperature. The reaction mixture was stirred overnight at room temperature until complete conversion was confirmed by LC-MS. The reaction mixture was azeotroped with PhMe and concentrated in vacuo overnight to afford compound 4 as an oil. LC-MS: Calculated for [M+H]+ 2483.59 m / z, observed 841.32 (+2 / 2, +H2O) m / z.

[0519] A solution of Compound 4 (68.3 mg) and NEt3 (13.7 mg) in anhydrous DCM was prepared at room temperature under N2 (g). Then compound 5 (11.2 mg) was added slowly. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® through a 4 g silica gel column as stationary phase using a gradient of 0-20% MeOH (10% B to 100% B) in DCM over 20 minutes, where LP49-p was at 100% B Dissolution. LC-MS: [M+H]+ calculated 5594.97 m / z, observed 1418.68 (+4 / 4, +H2O) m / z.

[0520] Synthetic LP53-p

[0521] To a solution of compound 1 (706 mg) and 2 (4.00 g) in DCM was added TBTU (670 mg) followed by DIPEA (0.908 mL) at room temperature. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated for isolation. The residue was purified by CombiFlash® using liquid injection using a gradient of 0-20% MeOH (0-100%) in DCM over 40 minutes. Compound 3 was concentrated in vacuo to give a white oily residue.

[0522] To compound 3 (4.00 g) was added 25 mL of 4 M HCl / dioxane at room temperature. The reaction mixture was stirred at room temperature for 1.5 hours until complete conversion was confirmed by LC-MS. The reaction mixture was then concentrated in vacuo. The residue was dissolved in DCM, then compound 5 (189 mg), HBTU (588 mg) and DIPEA (0.797 mL) were added. The reaction mixture was stirred at room temperature until complete conversion was observed by LC-MS.

[0523] The reaction mixture was directly concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-100% B) in DCM to afford compound 6.

[0524] To compound 6 (2.00 g) was added 20 mL of 4 M HCl / dioxane at room temperature. The reaction mixture was stirred at room temperature for 1.5 h until complete conversion was confirmed by LC-MS. The reaction was concentrated in vacuo. The residue was dissolved in DCM, then compound 7 (170 mg) and DIPEA (148 mg) were added. The reaction mixture was stirred at room temperature until complete conversion was observed by TLC.

[0525] The product LP53-p was extracted by standard workup (1 N HCl, saturated NaHCO3, brine). The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-100% B) in DCM.

[0526] Synthetic LP54-p

[0527] Oleic acid 1 (491 mg, 1.736 mmol) was mixed with Boc-amino-PEG47 derivative 2, TBTU (670 mg, 2.086 mmol) and DIEA (908 uL, 5.21 mmol) in DMF (50 mL) Stir for 4 hours. The solvent was removed in vacuo, toluene was evaporated from the residue 3 times and the residue was suspended in CHCl3 (150 mL). The resulting suspension was washed twice with H2O, 2% NaHCO3, brine, and treated with anhydrous Na2SO4. The mixture was concentrated to give product 3, which was dried in vacuo. Yield 4.391 g. Calculated MW 2566.24, (M +2×18) / 2=1301.12, (M +3×18) / 3=873.41 Experimental value: MS (ES, positive ion): 1301.79 [M+2NH4 ]2+, 874.08 [ M+3NH4]3+.

[0528] Compound 3 was converted to amine hydrochloride 4 by treatment with ice-cold 4M HCl / dioxane solution (5 mL), followed by stirring at room temperature for 1 hour. The reaction mixture was concentrated and dried in vacuo, residual HCl was removed by 2x evaporation of toluene from the product. The resulting amine hydrochloride 4 was dissolved in DMF:DCM=1:1 (60 mL ) and stirred for 16 hours. The solvent was removed in vacuo, toluene was evaporated from the residue 3 times and the residue was suspended in CHCl3 (300 mL). The suspension was washed twice with H2O, 2% NaHCO3, brine and dried over anhydrous Na2SO4. Product 5 was purified on the Combiflash® using the system 0-20% MeOH in DCM, gradient 0-100% over 45 minutes. Yield 2.72 g. Calculated MW 5143.46, (M +3×18) / 3=1732.49, (M +4×18) / 4=1303.87 Experimental value: MS (ES, positive ion): 1733.46 [M+3NH4]3+, 1304.55 [ M+4NH4]4+.

[0529] Compound 5 (2.72 g, 0.529 mmol) was stirred in 4M HCl / dioxane solution (30 mL) for 1 hour, the solvent was removed in vacuo, toluene was evaporated from the residue twice and the resulting dry hydrochloride 6 Stir with NHS-ester 7 (212 mg, 0.5 mmol) and Et3N in DCM (45 mL) for 16 h. The reaction mixture was diluted 3 times with CHCl, washed with H2O and brine, dried (Na2SO4), concentrated and the product LP54-p was purified on a Combiflash® using a system 0-20% MeOH in DCM, gradient 0-100% over 55 minutes . Yield 440 mg. Calculated MW 5353.65, (M +3×18) / 3=1802.55, (M +4×18) / 4=1356.41 Experimental value: MS (ES, positive ion): 1803.19 [M+3NH4]3+, 1357.24 [ M+4NH4]4+.

[0530] Synthetic LP55-p

[0531] To a solution of compounds 1 (297 mg) and 2 (2.00 g) in DCM was added TBTU (307 mg) followed by DIPEA (0.454 mL) at room temperature. The reaction mixture was stirred until complete conversion was observed by LC-MS. The product was extracted by standard workup (1N HCl, sat. NaHCO3, brine washes) and dried over Na2SO4. Crude compound 3 was used directly in the next step.

[0532] To compound 3 (2.00 g) was added 20 mL of 4 M HCl / dioxane at room temperature. The reaction mixture was stirred at room temperature for 1.5 hours until complete conversion was confirmed by LC-MS. The reaction mixture was concentrated in vacuo. The residue was dissolved in DCM, followed by the addition of DIPEA (0.0403 mL). Compound 5 (160 mg in DCM) was then added slowly (over 2-3 hours) using a syringe pump. The reaction mixture was stirred at room temperature until complete conversion was observed by TLC.

[0533] The product was extracted using standard workup (1N HCl, saturated NaHCO3, brine). The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-100% B) in DCM to afford compound 6.

[0534] To compound 6 (1.22 g) was added 10 mL of 4 M HCl / dioxane at room temperature. The reaction mixture was stirred at room temperature for 1.5 h until complete conversion was confirmed by LC-MS. The reaction mixture was concentrated in vacuo. The residue was dissolved in DCM, followed by the addition of compound 7 (105 mg) and DIPEA (148 mg). The reaction mixture was stirred at room temperature until complete conversion was observed by TLC.

[0535] The product LP55-p was extracted using standard workup (1N HCl, saturated NaHCO3, brine). The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-100% B) in DCM.

[0536] Synthetic LP56-p To compound 1 (150 mg, 0.0652 mmol, 1.0 equivalent), compound 2 (20 mg, 0.0717 mmol, 1.1 equivalent) and diisopropylethylamine (0.034 mL, 0.195 mmol, 3.0 equivalent) at room temperature A solution in anhydrous DMF (3 mL) was added TBTU (25.1 mg, 0.0782 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 2 hours and then concentrated. Compound 3 was purified by CombiFlash® eluting with 12-18% methanol in dichloromethane. LC-MS: Calculated for [M+2H]+ / 2 1283.32, found 1283.87.

[0538] To Compound 3 solid (82 mg, 0.0320 mmol, 1.0 equiv) was added a solution of HCl in dioxane (0.4 mL, 1.597 mmol, 50 equiv) at room temperature. The reaction mixture was kept at room temperature for 30 minutes and the solvent was removed under vacuum. Compound 4 was used without further purification. LC-MS: Calculated for [M+2H]+ / 2 1233.29, found 1233.69.

[0539] To a solution of compound 5 (13 mg, 0.0151 mmol, 1.0 equiv) and compound 4 (77.7 mg, 0.0310 mmol, 2.05 equiv) in anhydrous DCM (2 mL) was added triethylamine (0.011 mL) at room temperature , 0.0757 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature for 1 hour and the solvent was concentrated. LP56-p was purified by CombiFlash® eluting with 12-18% methanol in dichloromethane. LC-MS: [M+5H]+ / 5 Calcd 1112.49, Experimental 1112.34, [M+6H]+ / 6 Calcd 927.24, Experimental 927.97.

[0540] Synthetic LP57-p

[0541] To a solution of Compound 1 (787 mg), TBTU (985 mg) and DIEA (662 mg) in DMF was added Compound 2 (3.06 g) at room temperature. The reaction mixture was stirred overnight until complete conversion was observed by LC-MS. The reaction mixture was then washed with NaHCO3 and extracted with 20% trifluoroethanol / DCM. The residue was purified by CombiFlash® using a 80 g silica gel column as stationary phase with a gradient of DCM to 20% MeOH in DCM (0-100%) over 45 minutes, where compound 3 was eluted at 28% B. Compound 3 was concentrated in vacuo to give a white oily residue. LC-MS: Calculated for [M+H]+ 1411.95 m / z, observed 724.80 (+2 / 2, +H2O) m / z.

[0542] To compound 3 (1.27 g) was added 4 M HCl in dioxane (329 mg) at room temperature. The reaction mixture was stirred at room temperature until complete conversion was confirmed by LC-MS. The reaction mixture was azeotroped with PhMe / MeOH and concentrated under high vacuum overnight to afford compound 4 as a white solid. LC-MS: [M+H]+calculated 1311.90 m / z, observed 657.59 (+2 / 2) m / z.

[0543] To a solution of compound 4 (1.22 g), TBTU (348 mg) and DIEA (0.3825 mL) in DMF was added compound 5 (109 mg) at room temperature. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then washed with NaHCO3, extracted with 20% 2,2,2-trifluoroethanol (TFE) / DCM, washed with NH4Cl solution, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-100%) in DCM over 30 minutes, where compound 6 was eluted at 51% B. Pure and impure fractions were collected and concentrated. The impure fraction was re-isolated via DCM to 20% MeOH / DCM (0-100% B), where compound 6 eluted at 54% B and was collected as the pure fraction and concentrated. Concentration in vacuo afforded compound 6 as a white oily residue. LC-MS: [M+H]+ calculated 2833.89 m / z, observed 727.56 (+4 / 4, +H2O) m / z.

[0544] To compound 6 (130 mg) was added 4 M HCl / dioxane (16.7 mg) at room temperature. The reaction mixture was stirred at room temperature until complete conversion was confirmed by LC-MS. The reaction mixture was azeotroped with PhMe / MeOH and concentrated under high vacuum overnight to afford compound 7 as a white solid. LC-MS: [M+H]+ calcd. 2769.81 m / z, observed 694.07 (+ HCl, +4 / 4) m / z.

[0545] A solution of Compound 7 (127 mg) and TEA (0.026 mL) in anhydrous DCM was prepared at room temperature under N2 (g). Then compound 8 (24.8 mg) was added slowly. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® through a 12 g silica gel column as stationary phase using a gradient of 0-20% MeOH (0% B to 100% B) in DCM over 20 minutes with LP57-p at 100% B Dissolution. LC-MS: [M+H]+calculated 3132.00 m / z, observed 1584.89 (+3 / 3, +H2O) m / z.

[0546] Synthetic LP58-p

[0547] To a solution of compounds 1 (606 mg) and 2 (2.00 g) in DCM was added TBTU (657 mg) followed by DIPEA (0.891 mL) at room temperature. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® using liquid injection using a gradient of 0-20% MeOH (0-100%) in DCM over 40 minutes. Compound 3 was concentrated in vacuo to give a white oily residue.

[0548] To compound 3 (2.20 g) was added 5 mL of 4 M HCl / dioxane at room temperature. The reaction mixture was stirred at room temperature for 1.5 hours until complete conversion was confirmed by LC-MS. The reaction was concentrated in vacuo. The residue was dissolved in DCM, then compound 4 (171 mg), TBTU (567 mg) and DIPEA (0.770 mL) were added. The reaction mixture was stirred at room temperature until complete conversion was observed by TLC.

[0549] The product was extracted using standard workup (1N HCl, saturated NaHCO3, brine). The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-100% B) in DCM.

[0550] To compound 5 (1.34 g) was added 10 mL of 4 M HCl / dioxane at room temperature. The reaction mixture was stirred at room temperature for 1.5 hours until complete conversion was confirmed by LC-MS. The reaction was concentrated in vacuo. The residue was dissolved in DCM, then compound 6, TBTU (172 mg) and DIPEA (0.234 mL) were added. The reaction mixture was stirred at room temperature until complete conversion was observed by TLC.

[0551] The product LP58-p was extracted using standard workup (1 N HCl, saturated NaHCO3, brine). The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-100% B) in DCM.

[0552] Synthetic LP59-p Sinapinic acid 2f (587 mg, 1.736 mmol) was stirred in DMF (50 mL) with Boc-amino peg47 derivative 1b, TBTU (670 mg, 2.086 mmol) and DIEA (908 uL, 5.21 mmol) 4 hours. The solvent was removed in vacuo, toluene was evaporated from the residue 3 times, and the residue was suspended in CHCl3 (150 mL). The resulting suspension was washed twice with H2O, 2% NaHCO3, brine, and treated with anhydrous Na2SO4. The product 3f was isolated, concentrated and dried in vacuo. Yield 4.391 g. Calculated value MW 1494.00, M+18=1512.00, (M+2×18) / 2=765.00. Found: MS (ES, positive ion): 1512.53 [M+NH4]+, 765.72 [M+2NH4]2+.

[0554] The Boc protecting group was removed with 4M HCl in dioxane to afford the hydrochloride 4f (1.192 g, .834 mmol), which was used directly in the next step without purification. Pentafluorophenyl ester 10 (493 mg, 0.834 mmol) and Et3N (290 uL, 2.084 mmol) were mixed with hydrochloride 4f in DCM (30 mL). After stirring for 2 h, the reaction mixture was diluted with CHCl3 (150 mL), washed with H2O, 3% aqueous NaHCO3 and brine. The dried product 11c 1.539 g was used directly in the following step.

[0555] Compound 11c (1.539 g, 0.834 mmol) was stirred in 4M HCl / dioxane solution (20 mL) for 4 hours. The solvent was removed in vacuo and toluene was evaporated 2 times from the residue to obtain dry deprotected acid 12c (1.52 g, 0.827 mmol). This acid was combined with amine hydrochloride 4c (1.114 g, 0.827 mmol, synthesized as shown in the synthesis of LP39 above), TBTU (318.6 mg, 0.992 mmol) and DIEA (532 uL, 3.05 mmol) in DCM:DMF=1 : 2 mixture (30 mL) was stirred for 16 hours. The solvent was removed in vacuo and residual DMF was removed by evaporation of toluene an additional 3 times. The residue was suspended in CHCl3 (150 mL), washed with H2O, 3% NaHCO3 twice and brine. After drying over Na2SO4, the product 13e was concentrated and purified on a Combiflash® using the system DCM:20% MeOH in DCM, gradient 0-100%, 55 minutes. Yield 1.429 g. Calculated MW 3038.96, (M +2×18) / 2=1537.48, (M +3×18) / 3=1030.99 Experimental value: MS (ES, positive ion): 1537.97 [M+2NH4]2+, 1031.66 [ M+3NH4]3+.

[0556] The product 13e was deprotected with the Fmoc group as described above in the procedure for LP39. Product 14e was dried and reacted with NHS-ester 15c as described above in the procedure for LP39. Product 16e (LP59-p) was purified and isolated using CombiFlash®. Calculated value MW 3215.13, (M +2×18) / 2=1625.57, (M +3×18) / 4=1089.71. Found: MS (ES, positive ion): 1626.30 [M+2NH4]2+, 1090.58 [M+3NH4]3+.

[0557] Synthetic LP60-p

[0558] To a solution of Compound 1 (278 mg) and 2 (1.00 g) in DCM was added Compound 3 (DIPEA, 0.223 mL). The reaction mixture was stirred until complete conversion of 2 was observed by TLC. The product was extracted using standard workup (1N HCl, sat. NaHCO3, brine) and dried over Na2SO4. Crude compound 4 was used directly in the next step. To a solution of compound 5 (2500 mg, 2.130 mmol, 1.0 equiv) and compound 6 (655 mg, 2.556 mmol, 1.2 equiv) in anhydrous DCM (10 mL) was added EDC HCl (630 mg, 3.195 mmol, 1.5 equiv). The reaction mixture was kept at room temperature overnight. The reaction mixture was concentrated. The product was purified by CombiFlash® eluting with 12-20% methanol in dichloromethane. LC-MS: Calculated for [M+H]+ 1411.95, found 1413.64.

[0560] To Compound 7 solid (2100 mg, 1.487 mmol, 1.0 equiv) was added a solution of HCl in dioxane (7.438 mL, 29.75 mmol, 20 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour and the solvent was concentrated. Compound 8 was used without further purification. LC-MS: Calculated for [M+H]+ 1311.90, found 1312.95. To a solution of compound 8 (1210 mg, 0.897 mmol, 1.0 equiv) and compound 9 (539 mg, 1.032 mmol, 1.15 equiv) in anhydrous DCM (10 mL) was added triethylamine (0.381 mL) at room temperature , 2.692 mmol, 3.0 equivalents). The reaction mixture was kept at room temperature for 2 hours. The organic phase was washed with saturated NH4Cl and saturated aqueous NaHCO3. The organic phase was dried over Na2SO4 and concentrated. Compound 10 was isolated by CombiFlash® and eluted with 12-20% methanol in dichloromethane. LC-MS: Calculated for [M+H]+ 1719.07, found 1719.42.

[0562] To compound 10 (1100 mg, 0.639 mmol, 1.0 equiv) was added 4M HCl in dioxane (3.199 mL, 12.796 mmol, 20 equiv) at room temperature. The reaction mixture was kept at room temperature for 8 hours. The reaction mixture was concentrated. Compound 11 was used without further purification. LC-MS: Calcd. for [M+H]+ 1663.01, found 1664.00. To compound 11 (1060 mg, 0.637 mmol, 1.0 equivalent), compound 12 (970 mg, 0.637 mmol, 1.00 equivalent) and diisopropylethylamine (0.444 mL, 2.549 mmol, 4.0 equivalent) at room temperature To a solution in DMF (10 mL) was added TBTU (245 mg, 0.764 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 2 hours. The reaction mixture was concentrated. The residue was washed with saturated ammonium chloride and aqueous sodium bicarbonate. Compound 13 was purified by CombiFlash® eluting with 10-20% methanol in dichloromethane. LC-MS: Calculated for [M+2H] / 2 1565.50, found 1567.13.

[0564] To a solution of compound 13 (1.05 g) in 4 mL DMF was added 1 mL TEA at room temperature. The reaction mixture was stirred overnight and the solvent was removed in vacuo to afford compound 14. Compound 14 was used without further purification.

[0565] To a solution of compound 14 (585 mg) in 6 mL of DCM was added compound 15 (124 mg) and TEA (0.085 mL) at room temperature. The reaction mixture was stirred overnight. The product was extracted using standard workup (1N HCl, sat. NaHCO3, brine) and dried over Na2SO4. LP60-p was further purified by column chromatography.

[0566] Synthetic LP61-p To compound 1 (124 mg, 0.0539 mmol, 1.0 equivalent), compound 2 (19.5 mg, 0.0646 mmol, 1.2 equivalent) and diisopropylethylamine (0.028 mL, 0.161 mmol, 3.0 equivalent) at room temperature A solution in anhydrous DMF (2 mL) was added TBTU (20.8 mg, 0.0646 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 1 hour. The reaction mixture was quenched with saturated aqueous sodium bicarbonate. The aqueous phase was extracted with DCM (3 x 10 mL), and the combined org. phases were dried over Na2SO4 and concentrated. Compound 3 was purified by CombiFlash® eluting with 10-12% methanol in dichloromethane. LC-MS: Calculated for [M+2H]+ / 2 1270.31, found 1269.15.

[0568] To compound 3 (56 mg, 0.0220 mmol, 1.0 equiv) was added 4M HCl in dioxane (0.276 mL, 1.102 mmol, 50 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour and then concentrated. Compound 4 was used without further purification. LC-MS: Calcd. for [M+2H] / 2 1220.28, found 1221.63. To a solution of compound 5 (10 mg, 0.0116 mmol, 1.0 equiv) and compound 6 (59.1 mg, 0.0239 mmol, 2.05 equiv) in anhydrous DCM (2 mL) was added triethylamine (0.008 mL) at room temperature , 0.0931 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature for 4 hours and the solvent was removed under vacuum. LP61-p was purified by CombiFlash® eluting with 12-15% methanol in dichloromethane. LC-MS: Calculated for [M+6H]+ / 6 918.57, found 919.69.

[0570] Synthetic LP62-p

[0571] To a solution of compound 1 (1500 mg, 0.6517 mmol, 1.0 equiv) and compound 2 (200 mg, 0.782 mmol, 1.2 equiv) in anhydrous DCM (10 mL) was added EDC HCl (192 mg, 0.997 mmol, 1.5 equiv). The reaction mixture was kept at room temperature overnight and then concentrated. Compound 3 was purified by CombiFlash® eluting with 12-20% methanol in dichloromethane. LC-MS: Calculated for [M+2H]+ / 2 1270.31, found 1271.43.

[0572] To Compound 3 (1300 mg, 0.511 mmol, 1.0 equiv) was added 4M HCl in dioxane (6.397 mL, 25.588 mmol, 50 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour and then concentrated. Compound 4 was used without further purification. LC-MS: Calculated for [M+2H] / 2 1220.28, found 1221.87. To a solution of compound 4 (1350 mg, 0.5451 mmol, 1.0 equiv) and compound 5 (327 mg, 0.626 mmol, 1.15 equiv) in anhydrous DCM (10 mL) was added triethylamine (0.231 mL) at room temperature , 1.625 mmol, 3.0 equivalents). The reaction mixture was kept at room temperature overnight and then concentrated. Compound 6 was purified by CombiFlash® eluting with 12-20% methanol in dichloromethane. LC-MS: Calculated [M+3H] / 3 949.58, found 950.77. To a solution of compound 1 (1500 mg, 0.6517 mmol, 1.0 equiv) and compound 7 (265 mg, 0.782 mmol, 1.2 equiv) in anhydrous DCM (10 mL) was added EDC HCl (192 mg, 0.997 mmol, 1.5 equiv). The reaction mixture was kept at room temperature for 3 hours and then concentrated. The product compound 8 was purified by CombiFlash® eluting with 12-20% methanol in dichloromethane. LC-MS: Calculated for [M+2H]+ / 2 1311.35, found 1311.87.

[0575] To compound 6 (1220 mg, 0.428 mmol, 1.0 equiv) was added 4M HCl in dioxane (2.142 mL, 8.568 mmol, 20 equiv) at room temperature. The reaction mixture was kept at room temperature for 5 hours. The reaction mixture was then concentrated. Compound 9 was used without further purification. LC-MS: Calcd. for [M+3H] / 3 930.89, found 932.29.

[0576] To compound 9 (800 mg, 0.286 mmol, 1.0 equivalent), compound 10 (prepared from compound 8 under conventional deprotection conditions; 733 mg, 0.286 mmol, 1.00 equivalent) and diisopropyl To a solution of ethylethylamine (0.150 mL, 0.859 mmol, 3.0 equiv) in DMF (10 mL) was added TBTU (110 mg, 0.344 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 3 hours. The reaction mixture was then concentrated. Compound 11 was purified by CombiFlash® eluting with 10-20% methanol in dichloromethane. LC-MS: Calcd. for [M+5H] / 5 1059.46, found 1060.94.

[0577] To a solution of compound 11 (914 mg, 0.172 mmol, 1.0 equiv) in anhydrous DMF (4 mL) was added triethylamine (1 mL) at room temperature. The reaction mixture was kept at room temperature overnight and the solvent was removed under vacuum. Compound 12 was used without further purification. LC-MS: Calcd. for [M+5H] / 5 1015.05, found 1016.41. To a solution of compound 12 (875 mg, 0.172 mmol, 1.0 equiv) and compound 13 (97.5 mg, 0.189 mmol, 1.1 equiv) in anhydrous DCM (20 mL) was added triethylamine (0.073 mL) at room temperature , 0.517 mmol, 3.0 equivalents). The reaction mixture was kept at room temperature overnight and the solvent was concentrated. LP62-p was purified by CombiFlash® eluting with 12-20% methanol in dichloromethane. LC-MS: Calculated for [M+5H] / 5 1094.68, found 1095.98.

[0579] Synthetic LP87-p

[0580] Solid TBTU (50 mg, 0.156 mmol) was added to Boc-protected PEG47-amine 1a (Quanta Biodesign Limited, 300 mg, 0.13 mmol), linoleic acid 2a (37 mg, 0.13 mmol) and DIEA (68 µL mL, 0.39 mmol) in DMF (9 mL). The reaction mixture was sonicated to dissolve the solids and stirred at room temperature for 16 hours. The solvent was removed in vacuo, and toluene was evaporated twice from the residue. The residue was dissolved in chloroform (50 mL), washed with NaHCO3 (2 x 10 mL) and brine (10 mL). The product was dried (Na2SO4), concentrated in vacuo, and purified on a Combiflash® (SiO2) using a system 0-20% MeOH in DCM, gradient 0-80% over 20 minutes. Calculated MW 2564.22, (M +2×18) / 2=1300.1, (M +3×18) / 3=872.74 Experimental value: MS (ES, positive ion): 1299.74 [M+2NH4]2+, 873.04 [ M+3NH4]3+. Compound 3a (195 mg, 0.0764 mmol) was converted to amine hydrochloride 4b by treatment with ice-cold 4M HCl / dioxane solution (5 mL), followed by stirring at room temperature for 1 hour. The reaction mixture was concentrated and dried in vacuo, residual HCl was removed by 2x evaporation of toluene from the product. The dry amine hydrochloride was dissolved in anhydrous DMF (5 mL), bis-NHS ester 5 (28 mg, 0.033 mmol) and Et3N (28 uL, 0.198 mmol) were added and stirred at room temperature for 3 hours. The solvent was removed in vacuo, the toluene was evaporated twice from the residue and the product 6a (LP87-p) was purified on the Combiflash® using the system 0-20% MeOH in DCM, gradient 0-100% over 30 minutes. Calculated MW 5556.9, (M +3×18) / 3=1870.50, (M +4×18) / 4=1407.23 Experimental value: MS (ES, positive ion): 1870.50 [M+3NH4]3+, 1407.40 [ M+4NH4]4+.

[0581] Synthetic LP89-p

[0582] To a 25 mL sintered peptide synthesis vessel was added 2-chlorotrityl chloride resin 1 (0.4589 g, 1.46 mmol / g, 0.670 mmol). The resin was swelled in DCM and drained, followed by the addition of Fmoc-N-amido-PEG24-acid (0.9170 g, 0.670 mmol, 1 equiv) and diisopropylethylamine (DIEA) (0.584 mL, 3.35 mmol, 5 equivalents). The flask was shaken for 1 hour, followed by the addition of methanol (0.367 mL, 0.8 mL / g resin) to cap any remaining trityl resin. After 40 minutes, the flask was drained and washed three times with DCM, twice with DMF, twice with DCM and three times with MeOH (approximately 5 mL for each wash). The resin was dried overnight under high vacuum.

[0583] Resin loading: Suspend 11.5 mg resin in 0.8 mL DMF and swell for 15 minutes. 0.2 mL of piperidine was added and the mixture was allowed to stand for 15 minutes. The 10-fold dilution was dissolved in DMF and a UV-vis spectrum was obtained, A = 2.66 (approximately). The resin loading was calculated to be 0.297 mmol / g for a total of 919 mg resin for the 0.273 mmol scale.

[0584] Resin 2 was suspended in 9.6 mL 1:1:2 DCM / DMF / piperidine. After shaking for 30 minutes, the solution was drained and the resin was washed with DMF (4 x 9.2 mL).

[0585] Fmoc-N-amido-PEG24-acid (0.7473 g, 0.5460 mmol, 2 equiv), TBTU (0.1753 g, 0.5460 mmol, 2 equiv) and DIEA (0.190 mL, 1.092 mmol, 4 equiv) were combined in DMF (7.6 mL) and mix for 2-3 minutes, then add the solution to the resin in the synthesis flask. The flask was shaken for 1 hour, after which time the yellow-orange solution was drained from the orange resin. The resin was washed with DMF and MeOH (3 x 8.6 mL each), then dried under high vacuum overnight. 1.277 g resin, 1.227 g theoretical. Product mass was observed by LC-MS after microlysis.

[0586] The resin was treated with 20% piperidine in DMF (12.3 mL) for 30 minutes, followed by washing with DMF (4 x 12.3 mL).

[0587] Behenic acid (0.186 g, 0.546 mmol, 2.0 equiv), TBTU (0.175 g, 0.546 mmol, 2 equiv) and DIEA (0.190 mL, 1.092 mmol, 4 equiv) were dissolved in DMF (10.7 mL) . Add the solution to the resin. The solution vial was rinsed with DMF and added to the resin (2 x 1 mL). The mixture was shaken for 75 min, then drained and washed with DMF, THF and MeOH (3 x 13 mL each). The resin was dried under high vacuum (90 minutes). Obtained 1.351 g, theoretical 1.254 g. Product mass (and no starting material mass) was observed in LC-MS after microlysis.

[0588] The resin was treated with DCM (11 mL) and AcOH (1.1 mL) for 30 minutes, then drained. This cleavage was repeated a total of 4 times, then the resin was treated with 8 mL CH2Cl2, 1 mL AcOH, and 1 mL 2,2,2-trifluoroethanol, shaken for 30 minutes, and drained. Repeat this lysis a second time. The solutions from all cleavages were combined and concentrated to give 530.8 mg, which was purified by column chromatography.

[0589] The crude compound was loaded onto a silica column (24 g) and eluted with 0-20% MeOH in CH2Cl2. The pure fractions were combined to give 69.9 mg of the title compound.

[0590] Add N-mal-N-bis(PEG4)amine TFA salt (10.7 mg, 0.0128 mmol, 1 eq), acid-PEG24-amido-PEG24-C22 (69.9 mg, 0.0269 mmol, 2.1 eq) to the vial ), TBTU (10.3 mg, 0.0320 mmol, 2.5 eq), NEt3 (5.4 uL, 0.0385 mmol, 3 eq) and CH2Cl2 (1 mL). The reaction mixture was stirred for 24 hours, then NEt3 (5.4 uL, 0.0385 mmol, 3 equiv) was added. After approximately 50 hours, the reaction mixture was concentrated and purified by column chromatography, 0-30% MeOH in DCM to obtain 32.8 mg of LP89-p (44%).

[0591] Synthetic LP90-p

[0592] Solid TBTU (50 mg, 0.156 mmol) was added to Boc-protected PEG-amine 1a (Quanta Biodesign Limited, 300 mg, 0.13 mmol), mono-protected docosanedioic acid 2b (56 mg, 0.13 mmol) and DIEA (68 µL mL, 0.39 mmol) in DMF (9 mL). The reaction mixture was stirred at room temperature for 16 hours. The solvent was removed in vacuo and toluene was evaporated 3 times from the residue. The residue was dissolved in DCM 30 (mL), mixed with SiO2 (1.6 g), and loaded on CombiFlash®. The product was purified using the system 0-20% MeOH in DCM, gradient 0-100% over 45 minutes. Calculated MW 2710.45, (M +2×18) / 2=1373.22, (M +3×18) / 3=921.48 Experimental value: MS (ES, positive ion): 1373.18 [M+2NH4]2+, 921.37 [ M+3NH4]3+.

[0593] Compound 3b (238 mg, 0.088 mmol) was converted to the amino acid hydrochloride 4b by treatment with ice-cold 4M HCl / dioxane solution (6 mL), followed by stirring at room temperature for 4 hours. The reaction mixture was concentrated and dried in vacuo, residual HCl was removed by 2x evaporation of toluene from the residue.

[0594] The dry amine hydrochloride 4b was dissolved in anhydrous DCM (5 mL), bis-NHS ester 5 (34.2 mg, 0.04 mmol) and Et3N (55 uL, 0.4 mmol) were added and stirred at room temperature for 3 hours . The solvent was removed in vacuo and the product 6b (LP90-p) was purified on the Combiflash® using the system 0-20% MeOH in DCM, gradient 0-100% over 40 minutes. Calculated MW 5737.13, (M +3×18) / 3=1930.38, (M +4×18) / 4=1452.28 Experimental value: MS (ES, positive ion): 1930.45 [M+3NH4]3+, 1452.29 [ M+4NH4]4+.

[0595] Synthetic LP91-p

[0596] Solid TBTU (335 mg, 1.043 mmol) was added to Boc-protected PEG47-amine 1a (2 g, 0.869 mmol), behenic acid 2g (296 mg, 0.87 mmol) and DIEA (454 µL mL, 2.067 mmol) in DMF (16 mL). The reaction mixture was sonicated to dissolve the solids and stirred at room temperature for 16 hours. The solvent was removed in vacuo and toluene was evaporated twice from the residue. The residue was dissolved in chloroform (150 mL) and washed with NaHCO3 (2 x 30 mL) and brine (30 mL). Product 3g was dried (Na2SO4), concentrated in vacuo, and purified on a Combiflash® (SiO2) using the system 0-20% MeOH in DCM, gradient 0-80% over 35 minutes. Calculated value MW 2624.36, (M +2×18) / 2=1330.18, (M +3×18) / 4=892.79. Found: MS (ES, positive ion): 1330.58 [M+2NH4]2+, 893.21 [M+3NH4]3+.

[0597] Compound 3g (1.862 g) was converted to the amine hydrochloride salt 4g by treatment with 4 M HCl in dioxane (10 mL) as described above in the procedure for LP39-p.

[0598] As described in the preparation of LP54-p, an aliquot of 4 g of dry salt (227 mg, 0.089 mmol) was mixed with Boc-Asp-OH (10 mg, 0.043 mmol), TBTU (32 mg, 0.099 mmol) and DIEA (96 uL, 0.55 mmol) were combined to obtain compound 17 with a yield of 152 mg (0.029 mmol). This product was treated with HCl / dioxane solution as described above in the synthesis of LP54-p as in Preparation 14c to afford the hydrochloride salt 18 (100% yield), which was used directly in the following step. Calculated MW 5145.57, (M +3) / 3=1716.19, (M +4) / 4=1287.23. Found: MS (ES, positive ion): 1715.91 [M+3H]3+, 1287.23 [M+4H]4+.

[0599] As described above for 16c in the synthesis of LP54-p, hydrochloride 18 (0.029 mmol) was mixed with tetrafluorophenyl ester 20 (Quanta Biodesign, 15 mg, 0.032 mmol) and Et3N (12 uL, 0.087 mmol) combination. Product 21 (LP91-p) was purified on Combiflash®. Yield 40 mg. Calculated MW 5455.87, (M +4) / 4=1364.97, (M +5) / 5=1092.17. Found: MS (ES, positive ion): 1364.66 [M+4H]4+, 1092.05 [M+4H]4+.

[0600] Synthetic LP92-p

[0601] To compound 1 (140 mg, 0.0608 mmol, 1.0 equivalent), compound 2 (20.8 mg, 0.0669 mmol, 1.1 equivalent) and diisopropylethylamine (0.032 mL, 0.182 mmol, 3.0 equivalent) at room temperature A solution in anhydrous DMF (3 mL) was added TBTU (23.4 mg, 0.073 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 2 hours. The reaction mixture was concentrated. Compound 3 was purified by CombiFlash® eluting with 12-18% methanol in dichloromethane. LC-MS: Calculated for [M+2H]+ / 2 1297.33, Found 1297.19.

[0602] To Compound 3 solid (90 mg, 0.0347 mmol, 1.0 equiv) was added a solution of HCl in dioxane (0.434 mL, 1.734 mmol, 50 equiv) at room temperature. The reaction mixture was kept at room temperature for 30 minutes and then concentrated. Compound 4 was used without further purification. LC-MS: Calculated for [M+2H]+ / 2 1247.30, found 1247.98.

[0603] To a solution of compound 5 (14 mg, 0.0163 mmol, 1.0 equiv) and compound 4 (84.6 mg, 0.0334 mmol, 2.05 equiv) in anhydrous DCM (2 mL) was added triethylamine (0.012 mL) at room temperature , 0.0815 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature for 1 hour and the solvent was removed under vacuum. LP92-p was purified by CombiFlash® eluting with 12-18% methanol in dichloromethane. LC-MS: Calculated for [M+5H]+ / 5 1123.70, found 1124.10, calculated for [M+6H]+ / 6 936.58, found 937.22.

[0604] Synthetic LP93-p

[0605] To a solution of Boc-PEG47-NH22 (223 mg, 0.1 mmol) in DMF (2.0 mL) containing cis-11-eicosenoic acid 1 (30 mg, 0.0979 mmol) was added TBTU (37.2 mg, 0.115 mmol) and DIPEA (50uL). After stirring the resulting suspension overnight, water was added. The mixture was extracted with DCM:20% TFE and the combined organic phases were dried over Na2SO4. After filtration, the solvent was removed in vacuo to dryness and the crude product was purified by flash chromatography (20% MeOH in DCM). 2 mL of 4N HCl:dioxane was added to the product under anhydrous conditions until deprotection was complete as determined by LC-MS: [M+H]+ calcd. 2550.28 m / z, found 2551.

[0606] To a solution of compound 4 (19 mg, 0.0221 mmol, 1.0 equiv) and compound 3 (16 mg, 0.0454 mmol, 2.05 equiv) in anhydrous DCM (2 mL) was added triethylamine (16 uL, 0.1106 mmol, 5.0 equiv). The reaction mixture was kept at room temperature overnight and the solvent was removed under vacuum. LP93-p was purified by CombiFlash® eluting with 10-17% methanol in dichloromethane.

[0607] Synthetic LP94-p

[0608] To a solution of dihomo-γ-linolenic acid 1 (30 mg, 0.0979 mmol) in DMF (2.0 mL) was added Boc-PEG47-NH22 (225 mg, 0.1 mmol), TBTU (37.7 mg, 0.117 mmol) and DIPEA (50uL). After stirring the resulting suspension overnight, water was added. The mixture was extracted with DCM:20% TFE and the combined organic phases were dried over Na2SO4. After filtration, the solvent was concentrated to dryness and the crude product was purified by flash chromatography (DCM: 20% MeOH). 2 mL of 4N HCl:dioxane was added to the product under anhydrous conditions until complete deprotection as determined by LC-MS: [M+H]+ calcd. 2560.28 m / z, found 2561.01.

[0609] To a solution of Compound 4 (19 mg, 0.0221 mmol, 1.0 equiv) and Compound 3 (112 mg, 0.0454 mmol, 2.05 equiv) in anhydrous DCM (2 mL) was added triethylamine (16 uL, 0.1106 mmol, 5.0 equiv). The reaction mixture was kept at room temperature overnight and the solvent was removed under vacuum. LP94-p was isolated by CombiFlash® elution with 10-17% methanol in dichloromethane.

[0610] Synthetic LP95-p To compound 1 (150 mg, 0.0652 mmol, 1.0 equivalent), compound 2 (20 mg, 0.0717 mmol, 1.1 equivalent) and diisopropylethylamine (0.034 mL, 0.195 mmol, 3.0 equivalent) at room temperature A solution in anhydrous DMF (3 mL) was added TBTU (25.1 mg, 0.0782 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 2 hours. The reaction mixture was then concentrated. Compound 3 was purified by CombiFlash® eluting with 12-18% methanol in dichloromethane. LC-MS: Calculated for [M+2H]+ / 2 1281.30, found 1281.71.

[0612] To Compound 3 (80 mg, 0.0312 mmol, 1.0 equiv) was added a solution of HCl in dioxane (0.390 mL, 1.561 mmol, 50 equiv) at room temperature. The reaction mixture was kept at room temperature for 30 minutes and the solvent was removed under vacuum. Compound 4 was used without further purification. LC-MS: Calculated for [M+2H]+ / 2 1231.27, found 1231.65.

[0613] To a solution of compound 5 (13 mg, 0.0151 mmol, 1.0 equiv) and compound 4 (77.5 mg, 0.0310 mmol, 2.05 equiv) in anhydrous DCM (2 mL) was added triethylamine (0.011 mL) at room temperature , 0.0757 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature for 1 hour and the solvent was removed under vacuum. LP95-p was purified by CombiFlash® eluting with 12-18% methanol in dichloromethane. LC-MS: [M+5H]+ / 5 calculated value 1110.88, found value 1111.62, [M+6H]+ / 6 calculated value 925.90, found value 926.41.

[0614] Synthetic LP101-p To compound 1 (250 mg, 0.213 mmol, 1.0 equivalent), compound 2 (65 mg, 0.255 mmol, 1.20 equivalent) and diisopropylethylamine (0.111 mL, 0.629 mmol, 3.0 equivalent) at room temperature A solution in anhydrous DMF (3 mL) was added TBTU (102 mg, 0.319 mmol, 1.2 equiv). The reaction mixture was kept at room temperature overnight. Compound 3 was purified by CombiFlash® eluting with 6-12% methanol in dichloromethane. LC-MS: Calculated for [M+H]+ 1411.95, found 1411.95.

[0616] To Compound 3 solid (200 mg, 0.141 mmol, 1.0 equiv) was added a solution of HCl in dioxane (0.708 mL, 2.833 mmol, 20 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour and the solvent was removed in vacuo. The product was used directly without further purification. LC-MS: Calculated for [M+H]+ 1311.90, found 1312.32.

[0617] To compound 5 (100 mg, 0.0404 mmol, 1.0 equivalent), compound 4 (111 mg, 0.0829 mmol, 2.05 equivalent) and diisopropylethylamine (35 mL, 0.202 mmol, 3.0 equivalent) at room temperature To a solution in anhydrous DMF (3 mL) was added TBTU (32.5 mg, 0.101 mmol, 2.5 equiv). The reaction mixture was kept at room temperature overnight and then concentrated. Compound 6 was purified by CombiFlash® eluting with 6-10% methanol in dichloromethane. LC-MS: Calculated for [M+2H]+ / 2 1417.44, found 1418.19.

[0618] To compound 6 (80 mg, 0.0282 mmol, 1.0 equiv) was added 4M HCl in dioxane (0.353 mL, 1.411 mmol, 50 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour and then concentrated. Compound 7 was used without further purification. LC-MS: Calcd. for [M+2H] / 2 1367.41, found 1368.26.

[0619] To a solution of compound 7 (78 mg, 0.0281 mmol, 1.0 equiv) and compound 8 (12 mg, 0.0281 mmol, 1.0 equiv) in anhydrous DCM (2 mL) was added triethylamine (0.020 mL) at room temperature , 0.140 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature overnight and the solvent was concentrated. LP101-p was isolated by CombiFlash® elution with 12-20% methanol in dichloromethane. LC-MS: Calculated [M+3H] / 3 1015.31, Found 1015.71.

[0620] Synthetic LP102-p

[0621] To compound 1 (124 mg, 0.0539 mmol, 1.0 equivalent), compound 2 (19.5 mg, 0.0646 mmol, 1.2 equivalent) and diisopropylethylamine (0.028 mL, 0.161 mmol, 3.0 equivalent) at room temperature A solution in anhydrous DMF (2 mL) was added TBTU (20.8 mg, 0.0646 mmol, 1.2 equiv). The reaction mixture was kept at room temperature for 1 hour. The reaction mixture was quenched with saturated aqueous sodium bicarbonate. The aqueous phase was extracted with DCM (3 x 10 mL), and the combined org. phases were dried over Na2SO4 and concentrated. Compound 3 was purified by CombiFlash® eluting with 10-12% methanol in dichloromethane. LC-MS: Calculated for [M+2H]+ / 2 1281.76, found 1282.19.

[0622] To Compound 3 (66 mg, 0.0257 mmol, 1.0 equiv) was added 4M HCl in dioxane (0.322 mL, 1.287 mmol, 50 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour and then concentrated. Compound 4 was used without further purification. LC-MS: Calculated for [M+2H] / 2 1231.75, found 1232.01.

[0623] To a solution of compound 5 (11 mg, 0.0128 mmol, 1.0 equiv) and compound 4 (64 mg, 0.0256 mmol, 2.00 equiv) in anhydrous DCM (2 mL) was added triethylamine (0.009 mL) at room temperature , 0.064 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature for 1 hour and the solvent was concentrated. LP102-p was isolated by CombiFlash® elution with 12-18% methanol in dichloromethane. LC-MS: Calcd. for [M+6H]+ / 6 926.20, found 926.41.

[0624] Synthetic LP103-p

[0625] To a solution of Compound 1 (35 mg, 0.1170 mmol) in DMF (2.0 mL) was added Boc-PEG47-NH22 (269 mg, 0.1170 mmol), TBTU (45.1 mg, 0.1404 mmol) and DIPEA (60 uL) . After stirring the resulting suspension overnight, water was added. The mixture was extracted with DCM:20% TFE and the combined organic phases were dried over Na2SO4. After filtration, the solvent was removed in vacuo to dryness and crude compound 3 was purified by flash chromatography (DCM: 20% MeOH). 2 mL of 4N HCl:dioxane was added to the product under anhydrous conditions until deprotection was complete as determined by LC-MS: [M+H]+ calcd. 2483.59 m / z, found 2484.01.

[0626] To a solution of Compound 4 (10 mg, 0.0116 mmol, 1.0 equiv) and Compound 5 (59.3 mg, 0.0239 mmol, 2.05 equiv) in anhydrous DCM (2 mL) was added triethylamine (8 uL , 0.0582 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature overnight and the solvent was removed under vacuum. LP103-p was isolated by CombiFlash® elution with 10-17% methanol in dichloromethane. LC-MS: Calculated for [M+6H]+ / 6 933, found 934, calculated for [M+7H]+ / 7 800, found 801.

[0627] Synthetic LP104-p

[0628] Compound 1 (synthesized as shown above in the procedure for LP87) was conjugated to Fmoc-Glu-OH as described above in the procedure for LP54-p. Calculated MW 5261.56, (M +3×18) / 3=1771.86, (M +4×18) / 4=1333.39 Experimental value: MS (ES, positive ion): 1771.98 [M+3NH4]3+, 1333.57 [ M+4NH4]4+.

[0629] Compound 2 was deprotected with Fmoc as described above for compound 11 in the synthesis of LP39-p. The resulting product 3 was combined with activated ester compound 4 as described above in the procedure for the synthesis of LP39-p. LP104-p was isolated after CombiFlash® purification. Calculated value MW 5349.62, (M +3×18) / 3=1801.21, (M +4×18) / 4=1355.41. Found: MS (ES, positive ion): 1801.87 [M+3NH4]3+, 1355.92 [M+4NH4]4+.

[0630] Synthetic LP106-p

[0631] To compound 1 (200 mg, 0.676 mmol) in DCM (4 mL) was added TEA (218 uL, 1.56 mmol), followed by compound 2 (198 mg, 0.879 mmol) and the mixture was stirred at room temperature for 1 hour . After completion, all volatiles were removed and crude compound 3 was deprotected using 4N HCl to afford acid 5, which was then used without further purification.

[0632] Crude compound 5 (60 mg, assumed 0.1014 mmol) was dissolved in DMF (1 mL), treated with TBTU (71.6 mg, 0.223 mmol) and stirred for 5 minutes. Compound 4 (668 mg, 0.273 mmol) and DIEA (91.8 uL, 0.527 mmol) in DMF (1 mL) were then added and the mixture was stirred at room temperature for 16 hours. Upon completion, all volatiles were removed and compound 6 was isolated using a gradient elution of MeOH (0.1% TFA) in water (0.1% TFA) using a Phenomenex C18 Gemini® column (10u, 50 mm x 250 mm).

[0633] Compound 6 (23.5 mg, 0.0532 mmol) and Compound 7 (29.9 mg, 0.0586 mmol) were dissolved in 12.0 mL of DMF and the vessel was flooded with N2 for 5 minutes. Next, solidified copper (337 mg, 0.0532 mmol) and sodium ascorbate (31.6 mg, 0.1597 mmol) were added and the reaction mixture was stirred at 40 °C overnight.

[0634] Resin and other solids were filtered off. The filtrate was concentrated in vacuo and purified by HPLC to afford LP106-p.

[0635] Synthetic LP107-p

[0636] Compound 1 (982 mg, synthesized as shown in the procedure for LP38-p above) was dissolved in 10 mL of DCM. Compound 2 (90 mg) and triethylamine (0.081 mL) were added. The reaction mixture was stirred at room temperature for 5-8 hours until completion. The product was extracted using 1N HCl, then saturated NaHCO3, then washed with brine, and finally dried over Na2SO4. LP107-p was further purified using column chromatography.

[0637] Synthetic LP108-p

[0638] To compound 1 (595 mg, 1.610 mmol, 1.0 equivalents), compound 2 (8377 mg, 3.382 mmol, 2.10 equivalents) and diisopropylethylamine (1.122 mL, 6.443 mmol, 4.0 equivalents) at room temperature A solution in anhydrous DMF (100 mL) was added TBTU (1241 mg, 3.865 mmol, 2.4 equiv). The reaction mixture was kept at room temperature for 3 hours. The reaction mixture was then concentrated. The residue was washed with saturated ammonium chloride and aqueous sodium bicarbonate. Compound 3 was purified by CombiFlash® eluting with 12-20% methanol in dichloromethane. LC-MS: [M+5H] / 5, calculated 1043.05, found 1044.38.

[0639] To a solution of Compound 1 (104 mg, 0.0199 mmol, 1.0 equiv) in anhydrous DMF (1.6 mL) was added TEA (0.4 mL) at room temperature. The reaction mixture was kept at room temperature overnight and the solvent was removed under vacuum. Compound 4 was used without further purification. LC-MS: Calcd. for [M+5H] / 5 998.63, found 999.97.

[0640] To a solution of compound 4 (99 mg, 0.198 mmol, 1.0 equiv) and compound 5 (134 mg, 0.238 mmol, 1.2 equiv) in anhydrous DCM (3 mL) was added triethylamine (0.006 mL) at room temperature , 0.0397 mmol, 2.0 equivalents). The reaction mixture was kept at room temperature overnight and the solvent was removed under vacuum. LP108-p was isolated by CombiFlash® elution with 12-20% methanol in dichloromethane. LC-MS: Calculated for [M+5H] / 5 1088.48, found 1089.86.

[0641] Synthetic LP109-p

[0642] To compound 1 (595 mg, 1.610 mmol, 1.0 equivalents), compound 2 (8377 mg, 3.382 mmol, 2.10 equivalents) and diisopropylethylamine (1.122 mL, 6.443 mmol, 4.0 equivalents) at room temperature A solution in anhydrous DMF (100 mL) was added TBTU (1241 mg, 3.865 mmol, 2.4 equiv). The reaction mixture was kept at room temperature for 3 hours. The reaction mixture was then concentrated. The residue was washed with saturated ammonium chloride and aqueous sodium bicarbonate. Compound 3 was purified by CombiFlash® eluting with 12-20% methanol in dichloromethane. LC-MS: [M+5H] / 5, calculated 1043.05, found 1044.38.

[0643] To Compound 3 (100 mg) was added 20% NEt3 (0.053 mL) in DMF at room temperature. The reaction mixture was stirred at room temperature until complete conversion was confirmed by LC-MS. The reaction mixture was azeotroped with PhMe / MeOH and concentrated under high vacuum overnight to afford crude compound 4. LC-MS: [M+H]+ calculated 4989.17 m / z, observed 1262.31 (+4 / 4, +H2O) m / z.

[0644] A solution of Compound 4 (95.7 mg) and NEt3 in anhydrous DCM (0.008 mL) was prepared at room temperature under N2 (g). Then compound 5 (14.2 mg) was added slowly. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was then directly concentrated. The residue was purified by CombiFlash® through a 12 g silica gel column as stationary phase using a gradient of 0-20% MeOH (0% B to 100% B) in DCM over 20 minutes with LP109-p at 100% B Dissolution to obtain pure and impure fractions. Two clean fractions were collected and concentrated. The impure fractions were concentrated and resubjected to reaction conditions to drive further conversion. Separation via a gradient of 0-20% MeOH (0% B to 100% B) in DCM gave improved but slightly impure LP109-p, eluting at 88% B. LC-MS: Calculated [M+H]+5614.51 m / z, Observed 1422.64 (+4 / 4, +H2O) m / z.

[0645] Synthetic LP110-p

[0646] To a solution of Compound 1 (4.00 g) in 20 mL DMF was added Compound 2 (4.50 g) and 3 (11.6 g) at room temperature. The reaction mixture was stirred overnight. The product was extracted by standard workup (1 N NaOH, brine) and dried over Na2SO4. TLC showed removal of compound 2 by NaOH. Compound 4 was used directly in the next step.

[0647] To a solution of compound 4 (3.04 g) in 100 mL of MeOH was added a solution of NaOH (1.03 g) at room temperature. The reaction mixture was stirred overnight. The reaction mixture was concentrated to remove MeOH. The aqueous phase was extracted with ethyl acetate to remove any unreacted starting material. The mixture was acidified to pH 3, then extracted with ethyl acetate, dried over Na2SO4, and concentrated to yield compound 5 as a white solid. Compound 5 was used directly in the next step.

[0648] To compound 1 (2.9 mg) in DCM was added 2 equivalents of DIPEA (0.006 mL) at room temperature. Compound 6 (45 mg), TBTU (6.3 mg) and 2 equivalents of DIPEA (0.006 mL) were stirred at room temperature for 30 minutes. Slowly add the activated acid mixture to the PEG solution using a syringe pump (over 2-3 hours). The reaction mixture was stirred at room temperature. Until complete conversion was observed by TLC.

[0649] The product was extracted using standard workup (1N HCl, saturated NaHCO3, brine). The residue was purified by CombiFlash® using silica gel as stationary phase with a gradient of 0-20% MeOH (0-100% B) in DCM.

[0650] To Compound 7 (27 mg) was added 1.5 mL of 4 M HCl / dioxane at room temperature. The reaction mixture was stirred at room temperature for 1.5 hours until complete conversion was confirmed by LC-MS. The reaction mixture was concentrated in vacuo. Crude compound 8 was dissolved in DCM, and compound 9 (2.7 mg) and TEA (1.1 mg) were added. The reaction mixture was stirred at room temperature until complete conversion was observed by TLC.

[0651] LP110-p was purified by CombiFlash® using a gradient of DCM to 20% MeOH (0-100% B) in DCM using silica gel as the stationary phase.

[0652] Synthetic LP111-p

[0653] To a solution of compound 1 (2500 mg, 2.130 mmol, 1.0 equiv) and compound 2 (655 mg, 2.556 mmol, 1.2 equiv) in anhydrous DCM (10 mL) was added EDC HCl (630 mg, 3.195 mmol, 1.5 equiv). The reaction mixture was kept at room temperature overnight. The reaction mixture was concentrated. Compound 3 was purified by CombiFlash® eluting with 8-18% methanol in dichloromethane. LC-MS: Calculated for [M+H]+ 1411.95, found 1412.80.

[0654] To compound 3 (2400 mg, 1.699 mmol, 1.0 equiv) was added 4M HCl in dioxane (8.499 mL, 33.997 mmol, 20 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour and then concentrated. Compound 4 was used without further purification. LC-MS: Calcd. for [M+H] / + 1311.90, found 1312.95.

[0655] To compound 5 (300 mg, 0.812 mmol, 1.0 equivalents), compound 4 (2.299 g, 1.705 mmol, 2.10 equivalents) and diisopropylethylamine (0.566 mL, 3.248 mmol, 4.0 equivalents) at room temperature A solution in anhydrous DMF (10 mL) was added TBTU (625 mg, 1.949 mmol, 2.4 equiv). The reaction mixture was kept at room temperature for 1 hour. The reaction mixture was then concentrated. The residue was washed with saturated ammonium chloride and aqueous sodium bicarbonate. Compound 6 was purified by CombiFlash® eluting with 10-18% methanol in dichloromethane. LC-MS: [M+2H] / 2, calculated 1478.45, found 1479.89.

[0656] To a solution of compound 6 (1690 mg, 0.571 mmol, 1.0 equiv) in anhydrous DMF (8 mL) was added triethylamine (2 mL) at room temperature. The reaction mixture was kept at room temperature overnight and the solvent was removed under vacuum. Compound 7 was used without further purification. LC-MS: Calcd. for [M+2H] / 2 1367.41, found 1368.88.

[0657] To a solution of compound 7 (1563 mg, 0.571 mmol, 1.0 equiv) and compound 2 (381 mg, 0.743 mmol, 1.3 equiv) in anhydrous DCM (10 mL) was added TEA (0.162 mL, 1.143 mmol, 2.0 equivalents). The reaction mixture was kept at room temperature overnight and the solvent was removed under vacuum. LP111-p was purified by CombiFlash® eluting with 8-16% methanol in dichloromethane. LC-MS: Calculated [M+3H] / 3 1044.67, Found 1046.18.

[0658] Synthetic LP124-p

[0659] To Compound 1 (760 mg) was added 2 mL of 4 M HCl / dioxane at room temperature. The reaction mixture was stirred at room temperature. The reaction mixture was stirred for 1.5 hours until complete conversion was confirmed by LC-MS. The reaction mixture was concentrated in vacuo. The residue was dissolved in DCM, and compounds 3 (84.1 mg), 4 (207 mg) and 5 (0.281 mL) were added. The reaction mixture was stirred at room temperature until complete conversion was observed by TLC.

[0660] The product was extracted by standard workup (1N HCl, saturated NaHCO3, brine). Compound 6 was purified by CombiFlash® using silica gel as the stationary phase with a gradient of 0-20% MeOH (0-100% B) in DCM.

[0661] To compound 6 (250 mg) was added 4 mL of 4 M HCl / dioxane at room temperature. The reaction mixture was stirred at room temperature for 2 hours until complete conversion was confirmed by LC-MS. The reaction mixture was concentrated in vacuo. The residue was dissolved in DCM, then compounds 7 (52.9 mg) and 8 (0.036 mL) were added. The reaction mixture was stirred at room temperature until complete conversion was observed by TLC.

[0662] LP124-p was purified by CombiFlash® using a gradient of 0-20% MeOH (0-100% B) in DCM using silica gel as the stationary phase.

[0663] Synthetic LP130-p

[0664] To Compound 1 (1.89 g) was added 5 mL of 4 M HCl / dioxane at room temperature. The reaction mixture was stirred at room temperature for 1.5 hours until complete conversion was confirmed by LC-MS. The reaction mixture was then concentrated in vacuo. The residue was dissolved in DCM, and compounds 2 (209 mg), 3 (516 mg) and 4 (0.70 mL) were added. The reaction mixture was stirred at room temperature until complete conversion was observed by TLC.

[0665] The product was extracted by standard workup (1N HCl, saturated NaHCO3, brine). Compound 5 was purified by CombiFlash® using silica gel as the stationary phase with a gradient of 0-20% MeOH (0-100% B) in DCM.

[0666] To compound 5 (800 mg) was added 5 mL of 4 N HCl / dioxane at room temperature. The reaction mixture was stirred at room temperature for 2 hours until complete conversion was confirmed by LC-MS. The reaction mixture was then concentrated in vacuo. The residue was dissolved in DCM, then compound 2 (169 mg) and 3 (0.116 mL) were added. The reaction mixture was stirred at room temperature until complete conversion was observed by TLC.

[0667] LP130-p was purified by CombiFlash® using a gradient of DCM to 20% MeOH (0-100% B) in DCM using silica gel as the stationary phase.

[0668] Synthetic LP143-p

[0669] Compound 1 (500 mg) was dissolved in 10 mL of anhydrous THF in a pressure vessel and K2CO3 (398 mg) was added. Compound 2 (983 mg) was added as a solution in a small amount of DMF and the vessel was capped and the reaction mixture was left to stir at 40 °C overnight. Next, the reaction mixture was cooled to room temperature. The solid was filtered off and the reaction mixture was concentrated in vacuo. Compound 3 was purified using flash chromatography eluting with 0-100% EtOAc in hexanes.

[0670] Compound 3 (1070 mg) was dissolved in 4 mL of 4 M HCl in dioxane and stirred until all Boc was removed. The reaction mixture was then concentrated. Compound 4 was purified using flash chromatography eluting with 0-20% MeOH in DCM.

[0671] Compound 5 (1000 mg) was dissolved in 5 mL of anhydrous DMF in a pressure vessel and K2CO3 (1.315 g) was added. Then, compound 6 (850 mg) in a small amount of DMF was added and the reaction mixture was capped and stirred at 40 °C. Next, the reaction mixture was cooled to room temperature. The solid was filtered off and then the reaction mixture was concentrated in vacuo. Compound 7 was purified using flash chromatography eluting with 0-100% EtOAc in hexanes.

[0672] H3PO4 (0.594 mL) was added to a stirred solution of compound 7 (900 mg) in 20 mL of toluene. The reaction mixture was stirred overnight at room temperature. The reaction mixture was then diluted with water (30 mL) and washed 3 times with ethyl acetate (30 mL). The combined organic layers were dried over sodium sulfate and concentrated.

[0673] Compound 8 (100 mg) and TBTU (149 mg) were dissolved in 2 mL of DMF and stirred for 5 minutes. Then, TEA (0.152 mL) and compound 4 (142 mg) were added to the mixture and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate (10 mL) and washed with saturated ammonium chloride (3 x 10 mL). The organic layer was dried over sodium sulfate and concentrated. Compound 9 was purified using flash chromatography eluting with 0-100% hexane-ethyl acetate followed by DCM / MeOH 0-20%.

[0674] Compound 9 (197 mg) was dissolved in 4 mL THF. Then, LiOH (43 mg) and water (0.4 mL) were added. The reaction mixture was stirred until deprotection was confirmed by LC-MS. The reaction mixture was quenched with Amberlyst® 15. Amberlyst was filtered off and the reaction mixture was concentrated. Compound 10 was purified using flash chromatography eluting with 0-100% ethyl acetate in hexane with 0.1% HOAc additive.

[0675] Compound 10 (380 mg) was mixed with TBTU (424 mg) in 4 mL of DMF for five minutes. Next, compound 11 (2.12 g) was added followed by DIPEA (0.542 mL). The reaction mixture was stirred at room temperature and the following co-promoters were added: 50% TBTU and 50% DIPEA at 2 hours, 25% TBTU and 50% DIPEA at 3 hours, 50% DIPEA at 4 hours, 50% DIPEA at 5 hours. The reaction mixture was quenched after 6.5 hours. The reaction mixture was diluted with 20% TFE in DCM (15 mL) and washed twice with saturated ammonium chloride (15 mL). The organic layer was dried over sodium sulfate and concentrated. Compound 12 was then purified by HPLC.

[0676] mCPBA (70% pure, 12 mg) was added to a stirred solution of compound 12 (28 mg) in 1 mL of DCM at 0°C. After stirring overnight, the reaction mixture was allowed to warm to room temperature and monitored by LCMS. The mixture was diluted with 20% TFE in DCM (5 mL), then washed with saturated sodium sulfite (2×5 mL) and once with saturated sodium bicarbonate (5 mL). The organic layer was dried over sodium sulfate. The correct mass was confirmed to belong to LP143-p by LC-MS.

[0677] Synthetic LP210-p

[0678] Compound 1 (0.2 g, 0.08 mmol) and TBTU (0.0542 g, 0.735 mmol) were dissolved in DCM (5 mL) and NEt3 (0.0244 mL, 0.175 mmol) was added. In another vial, compound 2 (0.007 g, 0.037 mmol) and NEt3 (0.0244 mL, 0.175 mmol) were stirred together in DCM (1 mL). The resulting solution was stirred for 10 minutes. Compound 2 solution was added to Compound 1 solution after 10 minutes. The resulting mixture was stirred for 90 minutes and then checked by LC-MS. The reaction mixture was quenched with 5 mL of water and stirred for 5 minutes. The layers were separated, and the organic layer was washed with saturated NaHCO3(aq) (2×20 mL), water (20 mL), saturated NH4Cl(aq) (2×20 mL), saturated NaCl(aq) (2×20 mL) Washing, drying over Na2SO4 and concentration gave crude compound 3 (ca. 200 mg) as a waxy off-white solid. The crude product was purified by silica gel chromatography eluting with 0-20% MeOH in DCM. The pure fractions were combined to afford 50 (27% yield) of compound 3 as a white solid.

[0679] Compound 3 (0.05 g, 0.010 mmol) was dissolved in 1:1 MeOH / THF (5 mL), and LiOH (0.042 g, 1.74 mmol) and water (100 μL, 5.55 mmol) were added. The reaction mixture was stirred overnight at room temperature and checked by LC-MS. The organics were evaporated and the resulting suspension was diluted with approximately 10 mL of water. The resulting suspension was acidified to pH 1 with 3 M HCl(aq) and extracted with DCM (3 x 25 mL). The combined organics were washed with brine, dried over Na2SO4, concentrated, and dried in vacuo to afford 49 mg (98% yield) of compound 4 as an off-white solid. The product was used without further purification.

[0680] Compound 4 (0.05 g, 0.010 mmol) and COMU (0.0063 g, 0.015 mmol) were dissolved in DCM (1 mL) and NEt3 (13.7 μL, 0.098 mmol) was added and the resulting solution was stirred for 10 minutes. In another vial, Compound 5 was dissolved in DCM (0.3 mL). After 10 minutes, the compound 5 solution was added to the solution containing 1807-019. The resulting solution was stirred for 2 hours. The reaction mixture was quenched with 1 M HCl(aq) (10 mL) and the organic layer was diluted with 10 mL DCM. The layers were separated, and the organic layer was further washed with 1M HCl(aq) (20 mL), saturated NaHCO3(aq) (1 x 20 mL), saturated NaCl(aq) (1 x 20 mL), dried over Na2SO4, concentrated , and dried in vacuo to afford 94 mg of crude LP210-p as an off-white solid. The crude product was purified by silica gel chromatography eluting with 0-20% MeOH in DCM. Fractions containing pure LP210-p were combined and concentrated to give 7 mg (13.3% yield).

[0681] Synthetic LP217-p

[0682] Compound 1 (0.265 g, 0.105 mmol) and COMU (0.0542 g, 0.735 mmol) were dissolved in DCM (5 mL) and NEt3 (0.1 mL, 0.74 mmol) was added. The resulting solution was stirred for 10 minutes. After 10 minutes, compound 2 (0.010 g, 0.049 mmol) was added to the reaction. The resulting mixture was stirred overnight and checked by LC-MS. The reaction mixture was quenched with 5 mL of water and stirred for 5 minutes. The layers were separated and the organic layer was washed with sat. NaHCO3(aq) (2x20 mL), water (20 mL), 2 M HCl(aq) (2x20 mL), sat. NaCl(aq) (20 mL) , dried over Na2SO4, and concentrated to afford crude compound 3 (ca. 350 mg) as a waxy off-white solid. Crude compound 3 was purified by silica gel chromatography 2-20% MeOH in DCM. Fractions 3 containing compound were combined to afford 89 mg (36% yield) of an off-white solid.

[0683] Compound 3 (0.089 g, 0.017 mmol) was dissolved in 1:1 MeOH / THF (5 mL) and LiOH (0.042 g, 1.74 mmol) and water (180 μL, 9.85 mmol) were added. The reaction mixture was stirred overnight at room temperature and checked by LC-MS. The organics were evaporated and the resulting suspension was diluted with approximately 10 mL of water. The suspension was acidified to pH 1 with 3 M HCl(aq) and extracted with DCM (3 x 25 mL). The combined organic layers were washed with brine, dried over Na2SO4, concentrated, and dried in vacuo to afford 81 mg (91% yield) of compound 4 as an off-white solid. The product was used without further purification.

[0684] Compound 4 (0.081 g, 0.016 mmol) and COMU (0.010 g, 0.024 mmol) were dissolved in DCM (1 mL) and NEt3 (44.2 μL, 0.32 mmol) was added. The resulting solution was stirred for 10 minutes. In another vial, Compound 5 was dissolved in DCM (0.3 mL). After 10 minutes, the compound 5 solution was added to the compound 4 containing solution. The resulting mixture was stirred for 2 hours. The reaction mixture was quenched with 1 M HCl(aq) (10 mL) and the organic layer was diluted with 10 mL DCM. The layers were separated, and the organic layer was further washed with 1M HCl(aq) (20 mL), saturated NaHCO3(aq) (1 x 20 mL), saturated NaCl(aq) (1 x 20 mL), dried over Na2SO4, concentrated, and dried in vacuo to afford 94 mg of crude LP217-p as an off-white solid. The crude product was purified by silica gel chromatography with 0-20% MeOH in DCM. Fractions containing pure LP217-p were combined and concentrated to give 24 mg (28% yield).

[0685] Synthetic LP220-p

[0686] To a solution of Compound 2 (3.3381 mmol, 4.0140 g) and TEA (4.0058 mmol, 0.4054 g, 0.558 mL) in DCM was added Compound 1 (3.5050 mmol, 0.9634 g, 1.059 mL). The reaction mixture was stirred until complete conversion of compound 2 was observed by LC-MS. The residue was purified by standard workup (1N HCl, sat. NaHCO3, brine washes, and dried over Na2SO4). Compound 3 was used without further purification. Yield: 4.5 g.

[0687] To a solution of compound 5 (29.7354 mmol, 5.0000 g) in 50 mL of DMF was added compound 4 (65.4178 mmol, 13.7502 g) and Cs2CO3 (118.9414 mmol, 38.7535 g) at room temperature. The reaction mixture was stirred overnight at 60 °C. The reaction mixture was purified by standard workup (IN NaOH, brine washes, and dried over Na2SO4). Compound 6 was purified by silica gel chromatography and concentrated to afford 6.0 g.

[0688] Compound 3 (1.0500 mmol, 1.5129 g) was dissolved in 8 mL 4N HCl / dioxane and stirred at room temperature for 5 hours. After removal of HCl, compound 2 (1.0000 mmol, 1.2020 g), COMU (1.2000 mmol, 0.5139 g) and TEA (3.0000 mmol, 0.3035 g, 0.418 mL) in DCM was added. The reaction mixture was stirred until complete conversion of compound 2 was observed by TLC. The residue was purified by standard workup (1N HCl, sat. NaHCO3, brine washes, and dried over Na2SO4). Compound 7 was purified by silica gel chromatography and concentrated to afford 2.28 g.

[0689] To a solution of NaOH in 5 mL MeOH was added compound 6 (1.0000 mmol, 0.4545 g) in 20 mL DCM at room temperature. The reaction mixture was stirred overnight at room temperature. The reaction mixture was acidified to pH 3. The product was dried over Na2SO4 to yield 0.200 g of compound 8 which was used without further purification.

[0690] Compound 7 (0.7707 mmol, 1.9800 g) was dissolved in 10 mL 4N HCl / dioxane overnight at room temperature. The solvent was removed and the product was placed under vacuum for 2 hours, yielding 1.50 g of compound 9 which was used without further purification.

[0691] Compound 10 (0.0782 mmol, 0.0300 g) was dissolved in 1 mL of DCM, and 0.5 mL of TFA was added and the mixture was stirred for 2 hours. TFA was removed and compound 11 was dried under vacuum for 1 hour. Compound 8 (0.0822 mmol, 0.0362 g), COMU (0.0939 mmol, 0.0402 g) and TEA (0.2347 mmol, 0.0237 g, 0.033 mL) were dissolved in 5 mL of DCM over 5 min, followed by the addition of compound 11 in DCM. The reaction mixture was stirred until complete conversion of compound 11 was observed by TLC. Compound 12 was purified by silica gel chromatography to afford 0.0135 g.

[0692] Compound 12 (0.0191 mmol, 0.0135 g) was dissolved in 1 mL of DCM, 0.5 mL of TFA was added and the mixture was stirred for 1 hour. TFA was removed and compound 13 was dried under vacuum for 1 hour. Compound 9 (0.0398 mmol, 0.1000 g), COMU (0.0477 mmol, 0.0204 g) and TEA (0.1194 mmol, 0.0121 g, 0.017 mL) were dissolved in 3 mL of DCM for 5 min, followed by the addition of compound 13 in DCM. The mixture was stirred until complete conversion of compound 13 was observed by TLC. LP220-p was purified by silica gel chromatography to yield 0.0400 g.

[0693] Synthetic LP221-p

[0694] Carbon disulfide (75.0045 mmol, 5.7101 g, 4.532 mL) was slowly added to a solution of compound 1 (25 mmol, 4.20 g) and potassium hydroxide in EtOH (150 mL). The reaction mixture was refluxed for 24 hours. After completion, the solvent was evaporated under reduced pressure and the residue was dissolved in water. The aqueous solution was acidified to pH 2 using HCl. The product was extracted with EtOAc and purified by silica gel chromatography using EtOAc / hexanes. After purification, 3.5 g of compound 2 were obtained as an orange solid.

[0695] Compound 2 (10.0000 mmol, 2.1021 g) in THF (40 mL) was cooled to 0°C. CH3I (11.0000 mmol, 1.5609 g, 0.685 mL) was added followed by TEA (10.1000 mmol, 1.0221 g, 1.408 mL). The reaction mixture was stirred for 4 hours. After completion, the solvent was quenched by NH4Cl. The organic phase was washed with brine, dried and purified by silica gel chromatography to yield 1.5 g of compound 3.

[0696] To a solution of compound 4 (6.8679 mmol, 1.5391 g) in 10 mL of DMF was added compound 3 (3.1218 mmol, 0.7000 g) and Cs2CO3 (9.3654 mmol, 3.0514 g) at room temperature. The reaction mixture was stirred overnight at 60 °C. The reaction mixture was purified by standard workup (IN NaOH, brine washes, and dried over Na2SO4) and silica gel chromatography to afford 1.0 g of compound 5.

[0697] A mixture of compound 5 (0.2000 mmol, 0.1021 g) and mCPBA (0.9998 mmol, 0.1725 g) in DCM was stirred until complete conversion of mCPBA was observed by TLC. The reaction mixture was purified by standard workup (1N HCl, sat. NaHCO3, washed with brine, and dried over Na2SO4) and silica gel chromatography to afford 0.05 g of compound 6.

[0698] Compound 6 (0.0191 mmol, 0.0104 g) was dissolved in 1 mL of DCM, and 0.5 mL of TFA was added and the mixture was stirred for 1 hour. All TFA was removed, and compound 7 was dried under vacuum for 1 hour. Compound 8 (0.0398 mmol, 0.1000 g), COMU (0.0477 mmol, 0.0204 g) and TEA (0.1990 mmol, 0.0201 g, 0.028 mL) were dissolved in 3 mL of DCM over 5 min, followed by the addition of compound 7 in DCM. The reaction mixture was stirred until complete conversion of compound 7 was observed by TLC. The residue was purified by silica gel chromatography to yield 0.016 gLP221-p.

[0699] Synthetic LP223-p To compound 1 (741 mg, 2.442 mmol, 1.0 equivalent), compound 2 (528 mg, 2.930 mmol, 1.20 equivalent) and diisopropylethylamine (1.276 mL, 7.327 mmol, 3.0 equivalent) at room temperature To a solution in anhydrous DMF (10 mL) was added TBTU (980 mg, 3.052 mmol, 1.25 equiv). The reaction mixture was kept at room temperature for 2 hours. The organic phase was quenched with saturated aqueous sodium bicarbonate (10 mL) and extracted with EtOAc (2 x 10 mL). The organic phases were combined, dried over anhydrous Na2SO4, and concentrated. Compound 3 was purified by CombiFlash® and eluted with 40-80% EtOAc in hexanes. LC-MS: [M+H]+, calculated 466.25, found 466.72.

[0701] To Compound 3 (990 mg, 2.126 mmol, 1.0 equiv) was added 4M HCl in dioxane (6.38 mL, 25.518 mmol, 12 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour and then concentrated. Compound 4 was used without further purification. LC-MS: Calcd. for [M+H] / + 266.14, found 266.43. To compound 4 (100 mg, 0.295 mmol, 1.0 equivalent), compound 5 (755 mg, 0.606 mmol, 2.05 equivalent) and diisopropylethylamine (0.257 mL, 0.025 mmol, 5.0 equivalent) at room temperature A solution in dry DCM (10 mL) was added with COMU (278 mg, 0.650 mmol, 2.20 equiv). The reaction mixture was kept at room temperature for 1 hour. The reaction mixture was washed with saturated ammonium chloride (10 mL) and aqueous sodium bicarbonate (10 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated. Compound 6 was purified by CombiFlash® eluting with 8-18% MeOH in DCM. LC-MS: [M+3H] / 3, calculated 907.86, found 907.61.

[0703] To compound 6 (550 mg, 0.202 mmol, 1.0 equiv) was added 4M HCl in dioxane (1.01 mL, 4.040 mmol, 20 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour and then concentrated. Compound 7 was used without further purification. LC-MS: Calcd. for [M+H] / + 841.16, found 842.20. To compound 1 (490 mg, 0.188 mmol, 1.0 equivalent), compound 5 (482 mg, 0.387 mmol, 2.05 equivalent) and diisopropylethylamine (0.164 mL, 0.944 mmol, 5.0 equivalent) at room temperature A solution in anhydrous DCM (10 mL) was added COMU (177 mg, 0.415 mmol, 2.20 equiv). The reaction mixture was kept at room temperature for 1 hour. The reaction mixture was washed with saturated ammonium chloride (10 mL) and aqueous sodium bicarbonate (10 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated. Compound 8 was purified by CombiFlash® eluting with 8-20% MeOH in DCM. LC-MS: [M+5H] / 5, calculated 960.18, found 961.74.

[0705] To Compound 1 (670 mg, 0.134 mmol, 1.0 equiv) was added 4M HCl in dioxane (0.673 mL, 2.691 mmol, 20 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour and then concentrated. Compound 9 was used without further purification. LC-MS: Calcd. for [M+5H] / 5 956.16, found 957.66. To a solution of Compound 9 (650 mg, 0.134 mmol, 1.0 equiv) and Compound 10 (106 mg, 0.301 mmol, 2.25 equiv) in anhydrous DCM (20 mL) was added TEA (0.095 mL, 0.669 mmol, 5.0 equivalents). The reaction mixture was kept at room temperature for 2 hours and the solvent was concentrated. Compound 11 was isolated by CombiFlash® eluting with 8-20% MeOH in DCM. LC-MS: Calculated for [M+5H] / 5 1051.45, found 1053.44.

[0707] To a solution of compound 11 (460 mg, 0.0875 mmol, 1.0 equiv) in THF (5 mL) and water (5 mL) was added LiOH (10.5 mg, 0.437 mmol, 5.0 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour. The pH of the reaction mixture was adjusted to 3.0 by adding HCl and extracted with DCM (2 x 10 mL). The combined organic phases were dried over anhydrous Na2SO4 and concentrated. Compound 12 was used without further purification. LC-MS: Calculated for [M+5H]+ / 5 1048.65, found 1050.68. To compound 12 (100 mg, 0.0191 mmol, 1.0 equivalent), compound 13 (4.8 mg, 0.021 mmol, 1.1 equivalent) and diisopropylethylamine (0.010 mL, 0.0572 mmol, 3.0 equivalent) at room temperature A solution in anhydrous DCM (3 mL) was added COMU (10.2 mg, 0.0238 mmol, 1.25 equiv). The reaction mixture was kept at room temperature for 1 hour. The reaction mixture was washed with saturated aqueous sodium bicarbonate (5 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated. LP223-p was purified by CombiFlash® eluting with 8-20% MeOH in DCM. LC-MS: [M+5H] / 5, calculated 1090.47, found 1091.85.

[0709] Synthetic LP224-p

[0710] To a solution of compound 1 (12 mg, 0.0313 mmol, 1.0 equiv) in DCM (1 mL) was added TFA (0.5 mL) at room temperature. The reaction mixture was kept at room temperature for 30 minutes and then concentrated. Compound 2 was used without further purification. LC-MS: Calcd. for [M+H]+ 284.06, found 284.26.

[0711] To compound 3 (150 mg, 0.0286 mmol, 1.0 equivalent, compound 12, synthesized from LP223-p), compound 2 (12.5 mg, 0.0315 mmol, 1.1 equivalent) and diisopropylethylamine (0.015 mL, 0.0859 mmol, 3.0 equiv) in anhydrous DCM (3 mL) was added COMU (15.3 mg, 0.0358 mmol, 1.25 equiv). The reaction mixture was kept at room temperature for 1 hour. The reaction mixture was washed with saturated aqueous sodium bicarbonate (5 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated. LP224-p was purified by CombiFlash® eluting with 8-16% MeOH in DCM. LC-MS: [M+5H] / 5, calculated 1101.66, found 1103.13.

[0712] Synthetic LP225-p To compound 1 (80 mg, 0.130 mmol, 1.0 equivalent), compound 2 (652 mg, 0.267 mmol, 2.05 equivalent) and diisopropylethylamine (0.068 mL, 0.391 mmol, 3.0 equivalent) at room temperature A solution in anhydrous DCM (10 mL) was added COMU (134 mg, 0.312 mmol, 2.40 equiv). The reaction mixture was kept at room temperature overnight. Compound 3 was purified by CombiFlash® eluting with 8-16% MeOH in DCM. LC-MS: [M+5H] / 5, calculated 1091.89, found 1093.41.

[0714] To Compound 3 (340 mg, 0.0623 mmol, 1.0 equiv) was added 4M HCl in dioxane (0.311 mL, 1.245 mmol, 20 equiv) at room temperature. The reaction mixture was kept at room temperature for 1 hour and then concentrated. Compound 4 was used without further purification. LC-MS: Calcd. for [M+5H] / 5 1071.88, found 1073.36.

[0715] To a solution of compound 4 (100 mg, 0.0185 mmol, 1.0 equiv) and compound 5 (3.9 mg, 0.0204 mmol, 1.10 equiv) in anhydrous DCM (2 mL) was added TEA (0.008 mL, 0.0556 mmol, 3.0 equivalents). The reaction mixture was kept at room temperature for 2 hours and the solvent was concentrated. LP225-p was isolated by CombiFlash® eluting with 13-20% MeOH in DCM. LC-MS: Calculated for [M+5H] / 5 1102.48, found 1104.45.

[0716] Synthetic LP226-p To compound 1 (80 mg, 0.130 mmol, 1.0 equivalent), compound 2 (652 mg, 0.267 mmol, 2.05 equivalent) and diisopropylethylamine (0.068 mL, 0.391 mmol, 3.0 equivalent) at room temperature A solution in anhydrous DCM (10 mL) was added COMU (134 mg, 0.312 mmol, 2.40 equiv). The reaction mixture was kept a...

Claims

1. A delivery vector for inhibiting the expression of a gene expressed in skeletal muscle cells, comprising: (a) an RNAi agent comprising: (i) an antisense strand of 17-49 nucleotides, wherein at least 15 nucleotides are complementary to the mRNA sequence of a gene expressed in skeletal muscle cells; (ii) a sense strand of 16-49 nucleotides, which is at least partially complementary to the antisense strand; (b) a targeting ligand having affinity for a receptor present on the surface of skeletal muscle cells, wherein the targeting ligand has the following formula: , , , , or, or a pharmaceutically acceptable salt thereof, wherein an indication is made for the connection point with the remainder of the delivery vector; and (c) a PK / PD regulator of formula (I): or a pharmaceutically acceptable salt thereof, wherein LA is a single bond or a divalent portion of the RNAi agent linked to Z; Z is CH, phenyl, or N; L1 and L2 are each independently a linker comprising at least 5 PEG units; X and Y are each independently lipids containing 10 to 50 carbon atoms; and indicate the connection point with the RNAi agent; wherein the RNAi agent is connected to the targeting ligand and the PK / PD modulator.

2. The delivery vector of claim 1, wherein the RNAi agent is linked to the target ligand and the PK / PD regulator via a linker.

3. The delivery vector of claim 1, wherein the sense strand further comprises an inverted baseless residue at the 5' and / or 3' ends.

4. The delivery vector of claim 1, wherein the targeting ligand has the following formula: or a medically acceptable salt thereof, wherein the connection point with the remainder of the delivery vector is indicated.

5. The delivery vector of claim 1, wherein the targeting ligand has the following formula: or a medically acceptable salt thereof, wherein the connection point with the remainder of the delivery vector is indicated.

6. The delivery vector of claim 1, wherein the PK / PD modulator is: or a pharmaceutically acceptable salt of any of such PK / PD modulators, wherein a connection point with the RNAi agent is indicated, and wherein the connection point comprises a linker.

7. The delivery carrier of Request 1, wherein at least one of X and Y is selected from the group consisting of: wherein the connection point with L1 or L2 is indicated.

8. As in the delivery carrier of request item 1, wherein both X and Y are each independently selected from the group consisting of: wherein the connection point with L1 or L2 is indicated.

9. As in request item 1, the delivery carrier, wherein LA is selected from the group consisting of: where, Each of m, n, o, and a is independently 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, or 30, and each indicates a connection point with Z or the RNAi agent.

10. The delivery vector of claim 1, wherein the PK / PD modulator is selected from the group consisting of: or a pharmaceutically acceptable salt of any of these PK / PD modulators, wherein each indicates a linking site with the RNAi agent, and wherein the linking site contains a linker.

11. The delivery vector of any one of claims 1 to 10, wherein the RNAi agent inhibits the expression of human gene mRNA in skeletal muscle cells.

12. The delivery carrier as requested in item 4 or 5, wherein the medically acceptable salt is a sodium salt.

13. The delivery carrier as requested in item 4 or 5, wherein the medically acceptable salt is a potassium salt.

14. A composition comprising a delivery carrier as claimed in any one of claims 1 to 13.

15. A pharmaceutical composition comprising the composition of claim 14 and a pharmaceutical excipient.

16. The pharmaceutical composition of claim 15, wherein the pharmaceutical excipient is selected from water for injection and saline solution.

17. The pharmaceutical composition of claim 16, wherein the pharmaceutical excipient is a saline solution.

18. Use of a delivery carrier as claimed in any one of claims 1 to 13, a composition as claimed in claim 14, or a pharmaceutical composition as claimed in any one of claims 15 to 17, for the preparation of a medicament for treating muscular dystrophy.

19. As claimed in claim 18, wherein the muscular atrophy is selected from the group consisting of: Duchenne muscular atrophy, ankylosing spondylitis, Behringer's muscular atrophy, limb-girdle muscular atrophy, facioscapulohumeral muscular atrophy, congenital muscular atrophy, oculopharyngeal muscular atrophy, distal muscular atrophy, and Edwin's muscular atrophy.

20. A method of manufacturing a delivery carrier as claimed in any one of claims 1 to 13, the method comprising: (i) synthesizing the positive term; (ii) synthesizing the antisense term; (iii) bonding the positive term and the antisense term; (iv) binding a targeting ligand to the positive term or the antisense term before or after bonding the positive term and the antisense term; and (v) binding a PK / PD modulator to the positive term or the antisense term before or after bonding the positive term and the antisense term, and before or after binding the targeting ligand to the positive term or the antisense term.

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