Compounds and methods for transmembrane delivery of molecules

Abstract

To provide novel chemical conjugates for delivery of drugs across biological membranes.SOLUTION: The present invention provides a conjugate, having a structure as set forth in a Formula (I) (where D is a nucleic acid; E, E', and E'' independently represent a structure as set forth in a Formula (II)) or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] The present invention relates to delivery systems, cargo compounds, and polymers, as well as compounds and conjugates, including polymers, for use in biological purposes in vitro and in vivo, and to methods for delivering molecules and polymers across biological membranes to cells. [Background technology]

[0002] Oligonucleotide drugs (ODs) are macromolecular drugs containing sequences of nucleosides or nucleotides. ODs hold the potential for innovative treatments for a wide range of medical disorders. As is known in the art, ODs are single-stranded or double-stranded, natural or modified RNA or DNA molecules, or combinations thereof. Examples of ODs include, in particular, siRNA (small interfering RNA) sequences that are substrates for RNA-induced silencing complexes (RISCs); siRNA sequences that are substrates for Dicer endonucleases (dsiRNAs); microRNAs (miRNAs); messenger RNA (mRNA) drugs; or DNA sequences designed to function as antisense oligonucleotides (ASOs), all of which are active in the downstream regulation of target gene expression.

[0003] Due to the large, heavily charged structure of orbital dolphin (OD), there is an unmet need for delivery systems that can deliver OD across hydrophobic phospholipid membranes to cells. For the purpose of utilizing OD in a clinical setting, some features of the OD conjugate bound to a delivery system may include redox-sensitive cleavable groups that are advantageous, such as activity in the presence or absence of plasma proteins, or that are stable in the extracellular compartment but undergo efficient cleavage under reducing conditions that are dominant in the cytoplasm, thus enabling cargo drugs such as OD to exert their activity against cytoplasmic targets such as Dicer or RISC. [Overview of the project] [Means for solving the problem]

[0004] The present invention is based on a novel molecular delivery system (MDS) comprising a chemical moiety having the structure shown in formula (II), which is conjugated to cargo drugs such as high molecular weight orthosteroids (ODs), and thus, after the preparation of the conjugate according to formula (I), deliveries of the OD across the phospholipid membrane to cells, where they exert their respective biological activities, such as gene silencing. The present invention is based on the discovery and development of novel compounds by the inventors, which enable overcoming the enormous delivery barrier for large, heavily charged high molecular weight drugs across lipophilic cell membranes. The present invention provides, in particular, MDSs, conjugates containing the same, methods for synthesizing MDSs and their conjugates, and methods for utilizing MDSs for in vitro, ex vivo, and in vivo delivery of gene drugs to tissues and cells for the treatment of various medical disorders.

[0005] One embodiment of the present invention provides a conjugate having the structure shown in formula (I), or pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of a compound represented by the structure shown in formula (I), as well as solvates and hydrates of the salt.

[0006] [ka]

[0007] During the ceremony, D is a drug delivered across biological membranes (i.e., a cargo drug) and is selected from the group consisting of small molecule drugs, peptides, proteins, and ODs (i.e., natural or modified, single-stranded or double-stranded, DNA or RNA, siRNA, dsiRNA, or ASO). y, z, and w are integers independently selected from the group consisting of 0, 1, 2, 3, or 4, and if any of y, z, or w is 0, it means that each E part (or more) is null, and at least one of y, z, or w is not 0. E, E', or E'' may be the same or different, each independently having the structure shown in general formula (II), or comprising pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds represented by the structure shown in formula (II), as well as solvates and hydrates of salts.

[0008] [ka]

[0009] During the ceremony, M1, M2, M3, and M4 are independently selected from the group consisting of N', N'', null, ether, amide, ester, thioether, and thioester, respectively, and N' and N'' are independently selected from the group consisting of -N(CH3)-, -NH-, and -N(X)-, respectively, where X is an amine protecting group, and M1, M2, M3, and M4 may be the same or different, and N' and N'' may be the same or different. L is a linker selected from the group consisting of null, C1, C2, C3, C4, C5, C6 alkylene or heteroalkylene; C5 or C6 aryl or heteroaryl, or hydroxyl group (multiple) optionally substituted with fluorine atoms; and combinations thereof. G1, G2, G3, and G4 independently represent a hydrogen atom or a methyl group, the G groups may be the same or different, and at least two of the G1, G2, G3, or G4 groups are hydrogen atoms. a, b, c, d, and e are integers, each independently selected from the group consisting of 0, 1, 2, 3, 4, 5, or 6, where 0 = null, and a, b, c, d, and e can be the same or different. g represents an integer, selected from 0, 1, 2, 3, 4, or 5. W is a null, hydroxyl, dihydroxyl, amide, natural or modified nucleoside, formula (II 1 ), (II 2 ), and (II 3It is selected from the group consisting of residues of any of the structures shown in , and combinations thereof.

[0010] [Chemical formula]

[0011] In the formula, J is selected from the group consisting of null, -CH2-, secondary or tertiary amines, and oxygen. E, E', or E'' can be selected from the group consisting of D; a protecting group as defined herein (e.g., a protecting group for an alcohol); a hydrogen, phosphate, sulfate, and carboxyl group; an R or R' group; and can be attached to any part of the group consisting of a solid support. In the context of the present invention, the E, E', or E'' moiety can be attached to one D moiety via one or more points, and W can be attached to both D and R or R' simultaneously.

[0012] In one embodiment of the present invention, g is an integer of 0, 1, or 2.

[0013] In one embodiment, c and d independently of each other represent an integer of 1, 2, or 3, and c and d can be the same or different.

[0014] In one embodiment of the present invention, G1, G2, G3, G4 are all hydrogen atoms.

[0015] In one embodiment, L is difluorobenzylamine.

[0016] In another embodiment, X is a protecting group for an amine, TEOC [2-(trimethylsilyl)ethyl carbamate], or Fmoc.

[0017] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (V) or represented by the structure shown in formula (V), as well as solvates and hydrates of salts. Wherein, N' and N'' are independent of each other and have the same meaning as defined in formula (II).

[0018] [ka]

[0019] In another embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (VII) or represented by the structure shown in formula (VII), as well as solvates and hydrates of salts. Wherein, N' and N'' are independent of each other and have the same meaning as defined in formula (II).

[0020] [ka]

[0021] In another embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (VIII) or represented by the structure shown in formula (VIII), as well as solvates and hydrates of salts. Wherein, N' has the same meaning as in formula (II).

[0022] [ka]

[0023] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (VIII-H) or represented by the structure shown in formula (VIII-H), as well as solvates and hydrates of salts.

[0024] [ka]

[0025] In related embodiments, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (VIII-M) or represented by the structure shown in formula (VIII-M), as well as solvates and hydrates of salts.

[0026] [ka]

[0027] In another embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formulas (VIII-F) or represented by the structure shown in formulas (VIII-F), as well as solvates and hydrates of salts, where X is an amine protecting group.

[0028] [ka]

[0029] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XIV) or represented by the structure shown in formula (XIV), as well as solvates and hydrates of salts. In the formulas, p and q are independently integers of 0, 1, 2, 3, 4, 5, or 6, and N' has the same meaning as in formula (II).

[0030] [ka]

[0031] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XIV-H) or represented by the structure shown in formula (XIV-H), as well as solvates and hydrates of salts.

[0032] [ka]

[0033] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XIV-M) or represented by the structure shown in formula (XIV-M), as well as solvates and hydrates of salts.

[0034] [ka]

[0035] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XIV-F) or represented by the structure shown in formula (XIV-F), as well as solvates and hydrates of salts, where X is an amine protecting group.

[0036] [ka]

[0037] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where L is a difluorobenzylamine, and therefore E, E', or E'' each comprises pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XV) or represented by the structure shown in formula (XV), as well as solvates and hydrates of salts, where N' has the same meaning as in formula (II).

[0038] [ka]

[0039] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XV-H) or represented by the structure shown in formula (XV-H), as well as solvates and hydrates of salts.

[0040] [ka]

[0041] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XV-M) or represented by the structure shown in formula (XV-M), as well as solvates and hydrates of salts.

[0042] [ka]

[0043] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XV-F) or represented by the structure shown in formula (XV-F), as well as solvates and hydrates of salts, where X is an amine protecting group.

[0044] [ka]

[0045] In another embodiment, the present invention provides conjugates according to formulas (I) and (II), where the L portion is a tetrafluorobenzylamine, and therefore E, E', or E'' each comprises pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XVI) or represented by the structure shown in formula (XVI), as well as solvates and hydrates of salts, where N' has the same meaning as in formula (II).

[0046] [ka]

[0047] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XVI-H) or represented by the structure shown in formula (XVI-H), as well as solvates and hydrates of salts.

[0048] [ka]

[0049] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XVI-M) or represented by the structure shown in formula (XVI-M), as well as solvates and hydrates of salts.

[0050] [ka]

[0051] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XVI-F) or represented by the structure shown in formula (XVI-F), as well as solvates and hydrates of salts, where X is an amine protecting group.

[0052] [ka]

[0053] The present invention also provides a precursor molecule which is any E, E', or E'' moiety(s) of the present invention having a structure represented by any of the formulas (II), (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H), (XVI-M), bonded to an alcohol protecting group and / or an amine protecting group, wherein the protecting group is destined to be removed during chemical treatment of the molecule, for example, during conjugation to an oligonucleotide chain.

[0054] Some embodiments of the present invention relate to a method for delivering a drug across a biological membrane to a cell, either in vitro or in vivo, the method comprising bringing the cell into contact with a conjugate described herein.

[0055] Another embodiment of the present invention relates to a method for treating a medical disorder in a patient in need, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a conjugate of the present invention, drug D useful for treating the patient's disease, and a pharmaceutically acceptable salt or carrier.

[0056] To illustrate how embodiments of the present invention can be put into practice, non-limiting examples illustrating the invention are also provided. The examples describe various compounds and conjugates of the present invention. All described conjugates include an E, E', or E'' moiety according to formula (Cn-1) or (Cn-2), each having a structure according to general formula (II). The examples provide various E, E', or E'' moieties representing structures shown in formulas (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H), (XVI-M). All of these conjugates, in contrast to the control compounds which may share structural similarities with the compounds described above, exhibit biological performance in gene silencing; however, they do not fully adhere to the structural motif according to formula (II) and do not exhibit the desired biological activity. The performance profiles demonstrated by the conjugates of the present invention according to formulas (I) and (II) thus represent a general and specific structural framework that enables the associated compounds to be useful in the delivery of high molecular weight ODs across phospholipid membranes to cells, with the resulting useful biological activity performance both in vitro and in vivo.

[0057] In addition, the examples describe methods for the chemical synthesis of portion E of the present invention, their precursors, and their assemblies into useful conjugates. [Brief explanation of the drawing]

[0058] [Figure 1A] The binding mode of the E portion of the present invention to the oligonucleotide chain according to formula (III), and the redox-mediated cleavage of each E portion are illustrated. Figure 1A shows an RNA chain to which the E portion according to formula (III) is bound at an internal position. [Figure 1B]Figure 1B illustrates the redox-mediated cleavage of the disulfide group in this E portion by formula (III) under reducing conditions, such as when the dominant RNA drug is released in the cytoplasm. [Figure 2A] The mechanism of action (MOA) of the conjugate of the present invention is illustrated, and the conjugate is given by formula [Cn-2-(III)]. The RNA double strand is a 25 / 27 nucleotide length Dicer substrate, and each strand has a phosphate group attached at the 5' end. The W group of the internally located E portion follows formula (II1), and J is -CH2-. Figure 2A shows the intact conjugate. [Figure 2B] Figure 2B shows the cleavage and removal of the E, E', and E'' portions under reducing conditions, which are dominant in the cytoplasm, leaving short residual edges on each of the E, E', or E'' portions. [Figure 2C] Figure 2C illustrates the interaction between the RNA double strand and Dicer endonuclease, resulting in a double-strand break that leaves the 21 / 21 RNA double strand, as well as the removal of the E'' portion, leaving residual E and E' fragments joined at the 5' end and within the passenger strand. [Figure 2D] Figure 2D shows the removal of the sense (passenger) strand by an enzymatic helicase (i.e., a cytoplasmic enzyme capable of separating RNA strands), along with the simultaneous removal of residual fragments of the E and E' portions bound to the passenger strand. [Figure 2E] As a result, intact antisense strands are released and enter the RNA-induced silencing complex (RISC) to induce the desired gene silencing [Figure 2E]. [Figure 3A] The performance of the present invention's formula [Cn-1-(IX)], i.e., the conjugate having E and E' portions according to formula (IX), in silencing ApoC3 gene expression after intravenous administration in a mouse model in vivo is described (Figure 3A). [Figure 3B]Gene silencing in the liver (Figure 3B). The experimental groups were: (i) Vehicle: 5% glucose in water for injection, (ii) "naked" dsiRNA of ApoC3 (without binding of the molecular nanomotor portion), (iii) [Cn-1-(IX)]-Kras dsiRNA conjugate (unrelated RNA sequence conjugated to the Apo-Si molecular nanomotor delivery system), and (iv) Targeted [Cn-1-(IX)]-ApoC3 dsiRNA conjugate. [Figure 4A] In vitro silencing of EGFP gene expression involves [Cn-2-(VIII)], i.e., having E, E', and E'' portions according to formula (VIII), respectively. Figure 4A shows cultured HeLa cells. [Figure 4B] Figure 4B shows dose-response curves for cultured 3T3 cells in the concentration range of 0–300 nM. [Figure 5] The biological performance of two conjugates of the present invention, conjugate [Cn-1-(VIII-M)] and conjugate [Cn-2-(VIII-M)], in silencing EGFP gene expression in vitro in 3T3 cells is described. A clear dose / response was observed with a very significant logarithmic decay and curve fitting of R20.97 for both cell lines. For conjugate [Cn-1-(VIII-M)] (dotted line), which has two E moieties, the IC50 was found to be 2.2 nM, while for conjugate [Cn-2-(VIII-M)] (solid line), which has three E moieties, the IC50 was found to be 0.8 nM. [Modes for carrying out the invention]

[0059] This invention relates to a conjugate and its precursor, comprising a macromolecular drug such as OD, conjugated to a novel drug delivery system (MDS) capable of delivering cargo drugs across phospholipid biological membranes to cells to exert biological activity such as silencing the expression of target genes. This delivery system enables transmembrane delivery of macromolecular drugs such as gene drugs, e.g., siRNA or dsiRNA, antisense oligonucleotides (ASOs), or therapeutic proteins. This invention is based on the discovery and development of novel compounds by the inventors that exhibit advantageous performance in gene silencing by combining effective transmembrane delivery across phospholipid membranes with subsequent release based on robust reduction of cargo OD into the cytoplasm, thereby exerting their biological effects.

[0060] One embodiment of the present invention provides a conjugate having the structure shown in formula (I), or pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of a compound represented by the structure shown in formula (I), as well as solvates and hydrates of the salt.

[0061] [ka]

[0062] During the ceremony, D is a drug delivered across biological membranes (i.e., a cargo drug) and is selected from the group consisting of small molecule drugs, peptides, proteins, and ODs (i.e., natural or modified, single-stranded or double-stranded, DNA or RNA, siRNA, dsiRNA, or ASO). y, z, and w are integers independently selected from 0, 1, 2, 3, or 4, and if any of y, z, or w is 0, it means that each E portion (or more) is null, and at least one of y, z, or w is not 0. E, E', or E'' may be the same or different, each independently having the structure shown in general formula (II), or comprising pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds represented by the structure shown in formula (II), as well as solvates and hydrates of salts.

[0063] [ka]

[0064] During the ceremony, M1, M2, M3, and M4 are independently selected from the group consisting of N', N'', null, ether, amide, ester, thioether, and thioester, respectively, and N' and N'' are independently selected from the group consisting of -N(CH3)-, -NH-, and -N(X)-, respectively, where X is an amine protecting group, and M1, M2, M3, and M4 may be the same or different, and N' and N'' may be the same or different. L is a linker selected from the group consisting of null, C1, C2, C3, C4, C5, C6 alkylene or heteroalkylene; C5 or C6 aryl or heteroaryl, or hydroxyl group (multiple) optionally substituted with fluorine atoms; and combinations thereof. G1, G2, G3, and G4 independently represent a hydrogen atom or a methyl group, the G groups may be the same or different, and at least two of the G1, G2, G3, or G4 groups are hydrogen atoms. a, b, c, d, and e are integers, each independently selected from the group consisting of 0, 1, 2, 3, 4, 5, or 6, where 0 = null, and a, b, c, d, and e can be the same or different. g represents an integer, selected from 0, 1, 2, 3, 4, or 5. W is a null, hydroxyl, dihydroxyl, amide, natural or modified nucleoside, and formula (II) 1 ), (II 2 ), and (II 3The residues are selected from any of the structures shown in ) and from the group consisting of combinations thereof.

[0065] [ka]

[0066] During the ceremony, J is selected from the group consisting of null, -CH2-, secondary or tertiary amines, and oxygen. E, E', or E'' can be bonded to any portion of the group consisting of D; a protecting group as defined herein (e.g., an alcohol protecting group); an R or R' group selected from the group consisting of hydrogen, phosphoric acid, sulfuric acid, and carboxyl groups; and a solid support. In the context of the present invention, an E, E', or E'' portion may be bonded to a single D portion via one or more points, and W may be bonded to both D and R or R' simultaneously.

[0067] In one embodiment of the present invention, g is an integer of 0, 1, or 2.

[0068] In one embodiment, c and d independently represent integers 1, 2, or 3, and c and d may be the same or different.

[0069] In one embodiment of the present invention, G1, G2, G3, and G4 are all hydrogen atoms.

[0070] In one embodiment, L is difluorobenzylamine.

[0071] In another embodiment, X is TEOC [2-(trimethylsilyl)ethyl carbamate] or Fmoc.

[0072] Accordingly, in one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (III) or represented by the structure shown in formula (III), as well as solvates and hydrates of salts. Wherein, N' has the same meaning as defined in formula (II).

[0073] [ka]

[0074] In another embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (IV) or represented by the structure shown in formula (IV), as well as solvates and hydrates of salts.

[0075] [ka]

[0076] In another embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (V) or represented by the structure shown in formula (V), as well as solvates and hydrates of salts. Wherein, N' and N'' are independent of each other and have the same meaning as defined in formula (II).

[0077] [ka]

[0078] In another embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (VI) or represented by the structure shown in formula (VI), as well as solvates and hydrates of salts. Wherein, N' and N'' are independent of each other and have the same meaning as defined in formula (II).

[0079] [ka]

[0080] In another embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (VII) or represented by the structure shown in formula (VII), as well as solvates and hydrates of salts. Wherein, N' and N'' are independent of each other and have the same meaning as defined in formula (II).

[0081] [ka]

[0082] In another embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (VIII) or represented by the structure shown in formula (VIII), as well as solvates and hydrates of salts. Wherein, N' has the same meaning as in formula (II).

[0083] [ka]

[0084] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (VIII-H) or represented by the structure shown in formula (VIII-H), as well as solvates and hydrates of salts.

[0085] [ka]

[0086] In related embodiments, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (VIII-M) or represented by the structure shown in formula (VIII-M), as well as solvates and hydrates of salts.

[0087] [ka]

[0088] In another embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formulas (VIII-F) or represented by the structure shown in formulas (VIII-F), as well as solvates and hydrates of salts, where X is an amine protecting group.

[0089] [ka]

[0090] In another embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (IX) or represented by the structure shown in formula (IX), as well as solvates and hydrates of salts. Wherein, N' has the same meaning as in formula (II).

[0091] [ka]

[0092] In yet another embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (X) or represented by the structure shown in formula (X), as well as solvates and hydrates of salts. Wherein, N' has the same meaning as in formula (II).

[0093] [ka]

[0094] In yet another embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XI) or represented by the structure shown in formula (XI), as well as solvates and hydrates of salts, where N' has the same meaning as in formula (II).

[0095] [ka]

[0096] In another embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XII) or represented by the structure shown in formula (XII), as well as solvates and hydrates of salts.

[0097] [ka]

[0098] In the formula, p and q independently represent integers 0, 1, 2, 3, 4, 5, or 6, where 0 means null and p and q may be the same or different; k and g independently represent integers 0, 1, 2, 3, 4, 5, or 6, where 0 means null and k and g may be the same or different; Z is selected from the group consisting of null, -O-, and N'', where N'' and N'' have the same meanings as in formula (II).

[0099] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XIIa) or represented by the structure shown in formula (XIIa), as well as solvates and hydrates of salts. In the formulas, q is an integer between 0 and 1, and N' has the same meaning as in formula (II).

[0100] [ka]

[0101] The present invention also provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XIII) or represented by the structure shown in formula (XIII), as well as solvates and hydrates of salts.

[0102] [ka]

[0103] In the formula, a, b, c, d, and e are independent integers of 0, 1, 2, or 3, where 0 means null; a, b, c, d, and e can be the same or different; Q is selected from the group consisting of null and -O-; and N' has the same meaning as in formula (II).

[0104] In another embodiment, the present invention provides conjugates according to formulas (I) and (XIII), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XIIIa) or represented by the structure shown in formula (XIIIa), as well as solvates and hydrates of salts, where a is an integer of 1 or 2, and N' has the same meaning as in formula (II).

[0105] [ka]

[0106] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XIV) or represented by the structure shown in formula (XIV), as well as solvates and hydrates of salts. In the formulas, p and q are independently integers of 0, 1, 2, 3, 4, 5, or 6, and N' has the same meaning as in formula (II).

[0107] [ka]

[0108] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XIV-H) or represented by the structure shown in formula (XIV-H), as well as solvates and hydrates of salts.

[0109] [ka]

[0110] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XIV-M) or represented by the structure shown in formula (XIV-M), as well as solvates and hydrates of salts.

[0111] [ka]

[0112] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XIV-F) or represented by the structure shown in formula (XIV-F), as well as solvates and hydrates of salts, where X is an amine protecting group.

[0113] [ka]

[0114] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where the L portion is a difluorobenzylamine, and therefore E, E', or E'' each comprises pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XV) or represented by the structure shown in formula (XV), as well as solvates and hydrates of salts. In the formulas, p and q are independently integers of 0, 1, 2, 3, 4, 5, or 6, and N' has the same meaning as in formula (II).

[0115] [ka]

[0116] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XV-H) or represented by the structure shown in formula (XV-H), as well as solvates and hydrates of salts.

[0117] [ka]

[0118] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XV-M) or represented by the structure shown in formula (XV-M), as well as solvates and hydrates of salts.

[0119] [ka]

[0120] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XV-F) or represented by the structure shown in formula (XV-F), as well as solvates and hydrates of salts, where X is an amine protecting group.

[0121] [ka]

[0122] In another embodiment, the present invention provides conjugates according to formulas (I) and (II), where the L portion is a tetrafluorobenzylamine, and therefore E, E', or E'' each comprises pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XVI) or represented by the structure shown in formula (XVI), as well as solvates and hydrates of salts, where N' has the same meaning as in formula (II).

[0123] [ka]

[0124] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XVI-H) or represented by the structure shown in formula (XVI-H), as well as solvates and hydrates of salts.

[0125] [ka]

[0126] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XVI-M) or represented by the structure shown in formula (XVI-M), as well as solvates and hydrates of salts. [ka]

[0127] In one embodiment, the present invention provides conjugates according to formulas (I) and (II), where E, E', or E'' are pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds having the structure shown in formula (XVI-F) or represented by the structure shown in formula (XVI-F), as well as solvates and hydrates of salts, where X is an amine protecting group.

[0128] [ka]

[0129] If E, E', or E'' has a structure represented by any of the formulas (II), (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H), (XVI-M), it can be bonded to an alcohol or amine protecting group, the alcohol protecting group being, in many cases, without limitation, dimethoxytrityl[bis-(4-methoxyphenyl)phenylmethyl](DMT) and phosphoramidites. These parts are useful, among other things, in the conjugation process of the E, E', or E'' parts of the present invention to an oligonucleotide chain during the construction process of oligonucleotide drugs (ODs).

[0130] In addition, commonly used protecting groups for amines are Fmoc and TEOC [2-(trimethylsilyl)ethyl carbamate], which can be used to protect various functional groups bonded to E, E', or E'' during OD synthesis. Since they are removed under basic conditions, such protecting groups can therefore be effectively removed at the end of OD synthesis during the protecting group removal step, thus exposing the desired functional group on the final OD conjugate that was previously masked by the protecting group(s).

[0131] In the context of the present invention, precursor molecules are any E, E', or E'' moieties having the structure shown in any of formulas (II), (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H), (XVI-M), where the protecting group is destined to be removed during the chemical treatment of the molecule, for example, during conjugation to an oligonucleotide chain, and include pharmaceutically acceptable salts, hydrates, solvates, and metal chelates, solvates and hydrates of the salts.

[0132] Examples of precursor molecules of the present invention, provided in no particular order, are based on the structure of formula (VIII) and have the structure shown in formula (VIII-F)-precursors below, or include pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds represented by the structure shown in formula (VIII-F)-precursors, as well as solvates and hydrates of salts.

[0133] [ka]

[0134] Another example of a precursor molecule of the present invention, still based on the structure of formula (VIII), includes having the structure shown in formula (VIII-M)-precursor below, or pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of the compound represented by the structure shown in formula (VIII-M)-precursor, as well as solvates and hydrates of the salt.

[0135] [ka]

[0136] Another example of a precursor molecule of the present invention is based on the structure of formula (XIV) and includes the following formula (XIV-F)-precursor, or pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of the compound represented by the structure shown in formula (XIV-F)-precursor, as well as solvates and hydrates of the salt.

[0137] [ka]

[0138] Another example of a precursor molecule of the present invention still based on the structure of formula (XIV) includes having the following formula (XIV-M)-precursor, or pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of the compound represented by the structure shown in formula (XIV-M)-precursor, as well as solvates and hydrates of the salt.

[0139] [ka]

[0140] Another example of the precursor molecule of the present invention is based on the structure of formula (XV) and includes the following formula (XV-F)-precursor, or pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of the compound represented by the structure shown in formula (XV-F)-precursor, as well as solvates and hydrates of the salt.

[0141] [ka]

[0142] Another example of a precursor molecule of the present invention is one which still has the structure of formula (XV) and has the following formula (XV-M)-precursor, or includes pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of the compound represented by the structure shown in formula (XV-M)-precursor, as well as solvates and hydrates of the salt.

[0143] [ka]

[0144] Another example of the precursor molecule of the present invention is based on the structure of formula (XVI) and includes the following formula (XVI-F)-precursor, or pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of the compound represented by the structure shown in formula (XVI-F)-precursor, as well as solvates and hydrates of the salt.

[0145] [ka]

[0146] Another example of a precursor molecule of the present invention is one which still has the structure of formula (XVI) and has the following formula (XVI-M)-precursor, or includes pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of the compound represented by the structure shown in formula (XVI-M)-precursor, as well as solvates and hydrates of the salt.

[0147] [ka]

[0148] Compounds(s) according to any(s) of formulas (II), (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H), (XVI-M) can function as E, E', or E'' moieties for binding to the drug D, and thus can form the desired conjugates of the present invention for the purpose of biological performance in transmembrane delivery of D to cells.

[0149] One embodiment of the present invention provides a conjugate in which D is an OD such as an siRNA or Dicer substrate, each conjugated to the E, E', or E'' portion according to any of the formulas (II), (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H), (XVI-M), and comprises pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of each conjugate.

[0150] In this case, D is an oligonucleotide drug (OD), and the conjugate may, among other things, follow one of the following options: (i)OD is joined to a single E, E', or E'' part. (ii) The OD is bonded to two E-subunits, which may be the same or different, each bonded to one end of each oligonucleotide chain (e.g., the 5' end). (iii) The OD is bound to three identical or different E moieties, where the E and E' moieties are bound at the end of each oligonucleotide chain (e.g., the 5' end), while the E'' is bound at an internal position within the oligonucleotide chain. (iv) The OD is bound to several (n>3)E moieties, which are the same or different, and the E moieties are bound at the end of each oligonucleotide chain (e.g., the 5' end), while some other E moieties are bound at some internal positions along the oligonucleotide chain.

[0151] If the E portion is inserted at an internal position along the oligonucleotide chain, it can be located at any desired point. If D is OD, which is an siRNA RNA double strand, it is preferable to insert the internal E portion onto the passenger strand. Potentially beneficial positions along the passenger strand that do not interfere with the construct's activity in gene silencing may be positions 12 or 14. The E portion either replaces a nucleotide along the chain or adds to the sequence, and thus can form a "bulge" in the RNA double strand structure after the annealing process that generates the siRNA double strand.

[0152] For each conjugate, E, E', or E'' can also be attached to the R or R' portion, each being a phosphoric acid, sulfuric acid, or carboxyl group as defined by formula (II). The E, E', and E'' portions may be the same or different, and the R and R' portions may be the same or different.

[0153] For example, the conjugates of the present invention may have structures represented by the following formulas (Cn-1), (Cn-2), and (Cn-3), comprising E, E', or E'' portions, each having a structure according to any of the formulas (II), (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H), and (XVI-M), where R and R' are as defined in formula (II), and each conjugate comprises pharmaceutically acceptable salts, hydrates, solvates, and metal chelates.

[0154] [ka]

[0155] One embodiment of the present invention provides a conjugate comprising D, an antisense oligonucleotide (ASO) as defined above, comprising a single-stranded oligonucleotide of 15 to 25 nucleotides in length. This ASO is selected from the group consisting of natural or modified DNA, RNA, locked nucleic acid nucleotides (LNAs) (where the nucleotides are bound via phosphotryester groups, phosphorothioate groups, other nucleic acid binding strategies known in the art), or combinations thereof. The conjugate comprises binding to the E, E' portion as shown in formula (Cn-3).

[0156] [ka]

[0157] In the formula, T and T' are independently selected from the group consisting of null and 1',2'-dideoxyribose, nucleotides, or combinations thereof, T and T' may be the same or different, and the R and R' portions are as defined in formula (II), and include pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of the compound represented by the structure shown in formula (Cn-3), as well as solvates and hydrates of the salts.

[0158] In one embodiment, the present invention provides a conjugate according to formula (Cn-1), where R and R' are each phosphate groups, and E and E' are each according to formula (VIII-H), and the conjugate thus has the structure shown in the following formula [Cn-1-(VIII-H)].

[0159] [ka]

[0160] This includes pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds represented by the structure shown in formula [Cn-1-(VIII-H)], as well as solvates and hydrates of salts.

[0161] In another embodiment, the present invention provides a conjugate according to formula (Cn-1), where R and R' are each phosphate groups, and E and E' each have a structure according to formula (VIII-M), as shown in the following formula [Cn-1-(VIII-M)].

[0162] [ka]

[0163] This includes pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of the compound represented by the structure shown in formula [Cn-1-(VIII-M)], as well as solvates and hydrates of the salts.

[0164] In another embodiment, the present invention provides a conjugate according to formula (Cn-2), where R and R' are each phosphate groups, and E and E' each have the structure shown in the following formula [Cn-2-(VIII-H)], or comprises pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds represented by the structure shown in formula [Cn-2-(VIII-H)], as well as solvates and hydrates of salts.

[0165] [ka]

[0166] In another embodiment, the present invention provides a conjugate according to formula (Cn-2), where R and R' are each phosphate groups, and E and E' each have the structure shown in the following formula [Cn-2-(VIII-M)], or comprises pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds represented by the structure shown in formula [Cn-2-(VIII-M)], as well as solvates and hydrates of salts.

[0167] [ka]

[0168] In another embodiment, the present invention provides a conjugate according to formula (Cn-1), where R and R' are each phosphate groups, and E and E' each have the structure shown in the following formula [Cn-1-(XV-H)], or comprises pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds represented by the structure shown in formula [Cn-1-(XV-H)], as well as solvates and hydrates of salts.

[0169] [ka]

[0170] In another embodiment, the present invention provides a conjugate according to formula (Cn-1), where R and R' are each phosphate groups, and E and E' each have the structure shown in the following formula [Cn-1-(XV-M)], or comprises pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds represented by the structure shown in formula [Cn-1-(XV-M)], as well as solvates and hydrates of salts.

[0171] [ka]

[0172] In another embodiment, the present invention provides a conjugate according to formula (Cn-2), where R and R' are each phosphate groups, and E and E' each have the structure shown in the following formula [Cn-2-(XV-H)], or comprises pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of compounds represented by the structure shown in formula [Cn-2-(XV-H)], as well as solvates and hydrates of salts.

[0173] [ka]

[0174] In another embodiment, the present invention provides a conjugate according to formula (Cn-2), wherein R and R' are each a phosphate group, and E and E' each have a structure represented by the following formula [Cn-2-(XV-M)] according to formula (XV-M), or a pharmaceutically acceptable salt, hydrate, solvate, and metal chelate of a compound represented by the structure shown in formula [Cn-2-(XIV-M)], as well as solvates and hydrates of salts.

[0175]

Chemical formula

[0176] In one embodiment, the present invention provides a conjugate according to formula (Cn-1), wherein R and R' are each a phosphate group, and E and E' each have a structure represented by the following formula [Cn-1-(XVI-H)] according to formula (XVI-H),

[0177]

Chemical formula

[0178] including pharmaceutically acceptable salts, hydrates, solvates, and metal chelates of the compound represented by the structure shown in formula [Cn-1-(XVI-H)], as well as solvates and hydrates of salts.

[0179] In another embodiment, the present invention provides a conjugate according to formula (Cn-1), wherein R and R' are each a phosphate group, and E and E' each have a structure represented by the following formula [Cn-1-(XVI-M)] according to formula (XVI-M), or a pharmaceutically acceptable salt, hydrate, solvate, and metal chelate of a compound represented by the structure shown in formula [Cn-1-(XVI-M)], as well as solvates and hydrates of salts.

[0180]

Chemical formula

[0181] In another embodiment, the present invention provides a conjugate according to formula (Cn-2), wherein R and R' are each a phosphate group, and E and E' each have the structure shown in the following formula [Cn-2-(XVI-H)] according to formula (XVI-H), or a pharmaceutically acceptable salt, hydrate, solvate, and metal chelate of a compound represented by the structure shown in formula [Cn-2-(XVI-H)], as well as solvates and hydrates of the salts.

[0182]

Chemical formula

[0183] In another embodiment, the present invention provides a conjugate according to formula (Cn-2), wherein R and R' are each a phosphate group, and E and E' each have the structure shown in the following formula [Cn-2-(XVI-M)] according to formula (XVI-M), or a pharmaceutically acceptable salt, hydrate, solvate, and metal chelate of a compound represented by the structure shown in formula [Cn-2-(XVI-M)], as well as solvates and hydrates of the salts.

[0184]

Chemical formula

[0185] "Drug" or "cargo drug" (i.e., moiety D) in the context of the present invention refers to a molecule intended to be delivered into cells across the phospholipid membrane by the conjugate of the present invention, and D is either a small molecule drug or a macromolecule such as a peptide, protein, or oligonucleotide drug (OD).

[0186] In the context of this invention, the terms “drug” or “pharmaceutical” refer to chemical substances that, when administered to a patient suffering from a disease, can exert beneficial effects on the patient. Beneficial effects can range from improvement of symptoms to counteracting the effects of agents or substances (e.g., proteins) that play a role in the disease process. Drugs may include small molecules, or they may be proteins, or macromolecules such as single-stranded or double-stranded RNA or DNA, administered to inhibit gene expression. In particular, drugs may include siRNA, dsiRNA, or ASOs. In some embodiments, drugs are intended to treat degenerative disorders, cancer, ischemic, infectious, toxic, or traumatic seizures, hereditary or acquired metabolic disorders, or immune-mediated disorders.

[0187] The term “oligonucleotide drug,” also hereafter referred to as “OD,” in the context of this invention refers to a drug comprising a nucleoside or nucleotide. Examples of oligonucleotide drugs (ODs) are single-stranded or double-stranded, native or modified RNA or DNA. An OD may be a DNA sequence designed to function as an siRNA (small interfering RNA), a substrate for the Dicer enzyme (dsiRNA), a microRNA (miRNA), a messenger RNA (mRNA), or an antisense oligonucleotide (ASO). The binding between the nucleotide building blocks of an OD may, among other things, be via phosphate-tryester crosslinks, phosphorothioate bonds, or any other method known in the art. The nucleotides that function as building blocks of an OD may be either native or modified nucleotides, such as locked nucleic acids (LNAs).

[0188] A more specific embodiment of the present invention is a double-stranded RNA substrate, "siRNA," in which each RNA strand is 19-21 nucleotides long, intended to silence gene expression via RISC (RNA-induced silencing complex) cytoplasmic proteins, and is Dicer's siRNA substrate, "dsiRNA," in which each RNA strand is 24-30 nucleotides long. In one embodiment, the dsiRNA double-strand consists of one strand of 25 nucleotides and a second strand of 27 nucleotides. In another embodiment, the dsiRNA double-strand consists of one strand of 24 nucleotides and a second strand of 27 nucleotides. In yet another embodiment, the dsiRNA double-strand contains RNA strands of equal length, each consisting of 27 nucleotides.

[0189] Antisense oligonucleotides (ASOs) are synthetic, single-stranded, native, or modified DNA or RNA oligonucleotides, typically 15–20 nucleotides long. The sequence of an ASO is antisense, meaning it is complementary to the sense sequence of a specific mRNA that codes for a protein whose synthesis is to be inhibited. Binding of the ASO to this complementary sequence blocks the ribosome's ability to move along the mRNA, thereby preventing protein synthesis or accelerating the mRNA's degradation rate.

[0190] In the context of this invention, “nucleoside” is defined as a chemical moiety comprising a nitrogenous base (nucleic acid base) and a sugar with five or six carbon atoms (e.g., ribose or deoxyribose). The nucleic acid base is selected from natural or modified purines (e.g., adenine, guanine) and natural or modified pyrimidines (e.g., thymine, cytosine, uracil). The nucleic acid base can be modified by various modifications known in the art (e.g., methylation, acetylation). In addition, the sugar moiety of the nucleoside can also be modified as known in the art [e.g., 2'-deoxy derivatives, methylation at the 2' position of ribose, introduction of a 2'-fluoro atom or 2'-O-methoxyethyl, or having a bridge connecting the 2' oxygen and 4' carbon atoms to produce locked nucleic acid (LNA)]. Therefore, the use of such modified nucleosides conjugated to part E of this invention is also within the scope of this invention. In one embodiment, the nucleoside comprises a pyrimidine derivative selected from natural or modified cytosine, thymine, and uracil, and the sugar moiety is either ribose or deoxyribose.

[0191] In the context of this invention, a "nucleotide" is a nucleoside as defined above, bonded to a phosphate group. A nucleotide is the building block of an oligonucleotide.

[0192] In the context of the present invention, “precursor molecule” is defined as an E, E', or E'' moiety having a structure represented by any of the following formulas, (II), (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H), (XVI-M), (XVI), (XVI-H), or (XVI-M), bonded to an alcohol or amine protecting group, as defined below.

[0193] In the context of the present invention, a “protecting group” is defined as a chemical group that is destined to be removed or modified during the synthesis of the conjugate of the present invention. Such removal or modification may occur at various stages of synthesis, for example, without limitation, during the bonding of E, E', or E'' moieties to D, if D is a polymeric drug such as an oligonucleotide drug (OD). In preferred embodiments of the present invention, the protecting group is an alcohol protecting group as defined below.

[0194] In the context of this invention, “alcohol protecting group” refers to a chemical group that is bonded to a hydroxyl group in order to “mask” it during a particular chemical reaction and is subsequently potentially removed, as is known in the art. Examples of such protecting groups include acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl [bis-(4-methoxyphenyl)phenylmethyl] (DMT), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-methoxy-benzyl ether (PMB), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ethers [e.g., trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), triisopropylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers], ethoxyethyl ether (EE), phosphoramidite, and N-hydroxysuccinimide (NHS). Commonly used protecting groups for alcohols for partial conjugation of macromolecular drugs such as orally disintegrating compounds (ODs) are dimethoxytrityl[bis-(4-methoxyphenyl)phenylmethyl](DMT) and phosphoramidites.

[0195] The term "amine protecting group" [the X moiety according to formula (II)] in the context of the present invention refers to a chemical group that binds to an amine group to "mask" it during a particular chemical reaction and that can potentially be removed thereafter, as is known in the art. Examples of amine protecting groups within the scope of the present invention, provided without limitation, are the carbobenzyloxy (Cbz) group, the p-methoxybenzylcarbonyl (Moz or MeOZ) group, the tert-butyloxycarbonyl (BOC) group, the 9-fluorenylmethyloxycarbonyl (FMOC) group, the phenoxyacetyl (PAC) group, the 4-tert-butylphenoxyacetyl (t-PAC) group, the acetyl (Ac) group, the benzoyl (Bz) group, the benzyl (Bn) group, the carbamate group, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), the p-methoxyphenyl (PMP) group, the tosyl (Ts) group, the Troc (trichloroethyl chloroformate) group, and 2-(trimethylsilyl)ethyl carbamate (TEOC).

[0196] The term "point of attachment to a solid support" in the context of the present invention means the point of attachment of the E, E', or E'' moiety to a solid support during chemical synthesis. For example, controlled pore glass (CPG) can be used as a solid support for the attachment of the 3'-end of an oligonucleotide during the synthesis of an oligonucleotide chain of the present invention.

[0197] The term "biological membrane" according to the present invention refers to any phospholipid membrane related to a biological system. Examples of such phospholipid membranes are the plasma membranes of cells, intracellular membranes, or phospholipid membranes associated with biological barriers such as the blood-brain barrier (BBB), the blood-ocular barrier (BOB), or the blood-placenta barrier.

[0198] The term "flip-flop" according to the present invention refers to the movement of an amphiphilic compound (i.e., a molecule having both hydrophobic and hydrophilic elements) from one leaflet of a phospholipid membrane bilayer to the other.

[0199] The term "endocytosis" according to the present invention refers to the process by which a living cell takes up a molecule bound to its surface, and this process includes folding the plasma membrane inward, thereby bringing the molecule into the cell.

[0200] Embodiments of the present invention further relate to the use of conjugates according to the present invention, comprising proteins or therapeutically useful drugs such as ODs (e.g., siRNA, dsiRNA, or ASO) for the treatment of medical disorders in subjects requiring such treatment. Medical disorders may be, without limitation, degenerative disorders, cancer, vascular disorders, metabolic disorders, traumatic, toxic, or ischemic attacks, infections (e.g., viral or bacterial), or immune-mediated disorders, and certain proteins may play a role in either the etiology or pathogenesis of the disease. For such medical disorders, regulation of the expression of genes encoding these disease-related proteins via siRNA or antisense mechanisms, or regulation of the activity of each disease-related protein by therapeutic proteins, such as antibodies, or proteins that function in signaling, or by protein replacement therapy, may have beneficial effects in inhibiting disease-related processes or treating the underlying causes of the disease.

[0201] For example, a conjugate according to an embodiment of the present invention may be used as an antisense, siRNA, or dsiRNA therapy, which is a form of therapy comprising a single-stranded or double-stranded nucleic acid sequence (DNA, RNA, or a chemical analog thereof) and binding to either a DNA sequence encoding a specific protein or messenger RNA (mRNA) translated into a protein. This therapy may act to inhibit the expression of disease-related genes, thereby preventing the production of disease-related proteins that may play a role in the etiology or pathogenesis of the disease. Alternatively, the conjugate of the present invention may comprise a therapeutic protein or a protein / nucleic acid complex, such as a Cas9-RNA complex, that can perform gene editing.

[0202] Embodiments of the present invention also provide pharmaceutical compositions comprising the conjugates described herein and pharmaceutically acceptable carriers or salts. According to some embodiments, the conjugates and pharmaceutical compositions of the present invention may be used in vitro (e.g., in cell culture), ex vivo, or in vivo in living subjects, including in clinical settings.

[0203] Other embodiments of the present invention include the conjugate of the present invention, or a pharmaceutical composition containing the conjugate of the present invention, for use in the treatment of a medical disorder in a patient who requires it. Further embodiments of the present invention include the use of the conjugate of the present invention in the preparation of a pharmaceutical composition for the treatment of a medical disorder in a patient who requires it. In some embodiments, the medical disorder is cancer, metabolic disease, infectious disease, degenerative disease, vascular disease, trauma, or immune-mediated disease. The pharmaceutical composition may include pharmaceutically acceptable components known in the art, which are included to enable beneficial properties of the composition, such as sustained release, extended residence time, dispersion, or safety.

[0204] Conjugates according to embodiments of the present invention may be advantageous in improving the effectiveness of delivery of therapeutic proteins such as siRNA, dsiRNA, ASO, or antibodies across cell membranes or additional biological barriers such as the blood-brain barrier (BBB), compared to the effectiveness of delivery of the same therapeutic agent lacking portions E, E', or E'' of the present invention. Thus, conjugates of the present invention may improve the performance of macromolecular drugs in one or more embodiments, in addition to efficacy, e.g., safety or pharmacokinetics. Conjugates of the present invention may be administered via any mode of administration known in the art, including, in particular, oral, intravenous, intramuscular, subcutaneous, intratracheal, intrabronchial, intraperitoneal, or intrathecal.

[0205] The conjugate of the present invention, in which D is OD, can be synthesized non-limitingly according to the following methods: First, the gene to be silenced is selected based on its role in the etiology or pathogenesis of the disease. Then, the nucleotide sequence to be incorporated into the conjugate is designed and determined based on bioinformatics methodologies known in the art [typically, 19-21 base pair double-stranded siRNA on a RISC substrate, or 24-29 base pair double-stranded RNA (dsiRNA) on a Dicer substrate]. Synthesis is carried out in the 3' to 5' direction of the oligonucleotide. Solid-phase synthesis is performed using protected building blocks such as protected 2'-deoxynucleosides (dA, dC, dG, and dT), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, e.g., [LNA (locked nucleic acid) or BNA (crosslinked nucleic acid)]. The building blocks are provided as nucleoside precursors, with the 5'- and 3'-hydroxyl groups protected by DMT and phosphoramidite, respectively. These groups are sequentially removed in an order determined by the desired nucleotide sequence during the reaction in which the nucleotides are coupled to the growing oligonucleotide chain during synthesis.

[0206] For the purpose of synthesizing the conjugates of the present invention, the E group is provided as a precursor molecule and is the E, E', or E'' moiety of the present invention, each bonded to a protecting group(s) as described above. The protecting group(s) may be any hydroxyl protecting group known in the art, but phosphoramidites and DMT [dimethoxytritylbis-(4-methoxyphenyl)phenylmethyl] are often used conventionally in oligonucleotide synthesis. The main advantage of the conjugates of the present invention is that, as exemplified by the conjugates (Cn-1) and (Cn-2) above, the E, E', or E'' moiety is also bonded to the 5' end of the oligonucleotide chain, the 3' end of the oligonucleotide chain, or to an internal position(s) along the oligonucleotide chain. Thereafter, the E moiety of the present invention can be incorporated into the oligonucleotide chain, similar to intrinsic, natural oligonucleotide building blocks. The nucleotide binding can be via standard phosphotriester bonds, or via synthetic phosphorothioate bonds that may offer advantages such as stability in blood or preferred binding to blood proteins, or via any other nucleotide binding strategy known in the art. After completion of the strand assembly, the product is released from the solid support into solution, deprotected, and collected. To obtain the desired conjugate of the present invention in high purity, the desired conjugate is then isolated by high-performance liquid chromatography (HPLC). In the case of siRNA or dsiRNA, each of the complementary RNA strands is synthesized separately, and then the annealing of the two strands is carried out as known in the art to yield the desired double-stranded siRNA or dsiRNA, which is then subjected to purification and Ali coating.

[0207] One embodiment of the present invention provides a method for delivering a drug across a phospholipid biological membrane, selected from the group consisting of cell membranes and biological barriers, wherein the biological barrier is selected from the blood-brain barrier, the blood-eye barrier, or the blood-fetal barrier, and the method comprises bringing a cell or the respective biological barrier into contact with the conjugate of the present invention.

[0208] Embodiments of the present invention provide a method for delivering a drug to living cells, where the cells are in culture or in a living animal or human subject, and the method comprises contacting the cells with a conjugate or a pharmaceutical composition comprising the conjugate of the present invention. In the case of in vivo administration, contact with cells can be achieved through any route of drug administration known in the art, such as oral, intravenous, subcutaneous, or intramuscular administration.

[0209] One embodiment of the present invention provides a pharmaceutical composition comprising the conjugate of the present invention or a conjugate according to formula (I), where each of E, E', or E'' independently has a structure represented by any of the following formulas: (II), (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H), (XVI-M). The present invention also includes a method for the specific inhibition of gene expression in vitro or in vivo. In one embodiment of the present invention, the method may include the use of a conjugate of any of the formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H), (XVI-M), (Cn-1), (Cn-2), (Cn-3), or a pharmaceutical composition containing such conjugate, where D is an siRNA, dsiRNA, or ASO designed to silence the expression of a particular gene. In some embodiments, the gene encodes a pathogenic protein that plays a role in the etiology or pathogenesis of the disease. In some embodiments, D is a therapeutic protein.

[0210] In yet another embodiment of the present invention, a method for inducing endocytosis and / or flip-flop in a biological membrane is provided, the method comprising contacting a biological membrane with a conjugate of the present invention or a pharmaceutical composition comprising the conjugate, the conjugate comprising OD and E, E', or E'' portions, each having a structure represented by any of formulas (II), (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H), (XVI-M), thereby achieving endocytosis and / or flip-flop of the conjugate in a biological membrane.

[0211] In a non-limiting hypothesis, the basis for the induction of endocytosis or flip-flop lies in the structure of the conjugate of the present invention. When OD is siRNA or dsiRNA bound to the E portion, the conjugate approaches the outer membrane lobe, its cylindrical RNA double strand parallel to the membrane surface, and its E portion oriented toward the membrane core perpendicular to the membrane surface. This orientation thus anchors the RNA double strand to the outer membrane lobe. The resulting forced proximity of the highly negatively charged RNA to the outer membrane lobe causes energetically unfavorable focal strain, along with an expansion of the surface area of ​​the outer phospholipid lobe, disturbance of the hydration shell around the phospholipid head group, and focal bending of the membrane. The relaxation of this bending energy can then be achieved through either endocytosis and / or flip-flop. Both processes support the initiation and / or propagation of transmembrane delivery of the conjugate of the present invention to cells, including its polymeric cargo drug. Therefore, a method for inducing endocytosis or flip-flop in a phospholipid membrane falls within the scope of the present invention, and such method includes bringing the membrane into contact with the conjugate of the present invention.

[0212] Conjugates according to embodiments of the present invention may be used for the treatment of medical disorders. Embodiments of the present invention include a medical treatment method comprising administering a therapeutically effective amount of a pharmaceutical composition containing a conjugate according to any of the formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H), (XVI-M), (Cn-1), (Cn-2), (Cn-3), to a patient in need, wherein D is a drug useful for the treatment of each medical disorder.

[0213] In one embodiment, the method is for gene therapy using siRNA, dsiRNA, or ASO as a therapeutic agent. The method comprises administering to a patient in need a therapeutically effective amount of a pharmaceutical composition comprising a conjugate of the present invention according to any of the formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H), (XVI-M), (Cn-1), (Cn-2), (Cn-3), where D is an siRNA, dsiRNA, mRNA, miRNA, ASO, or therapeutic protein useful in inhibiting gene expression or blocking the activity of the respective protein that plays a role in the etiology or pathogenesis of the disease in a particular patient.

[0214] The treatment may involve the delivery of a drug to cells in culture either in vitro or ex vivo (i.e., cells removed from living tissue to be optionally returned to the patient after the treatment procedure), or to cells in a living animal or human subject in vivo. In some embodiments, the cells are neogeneic cells. In some embodiments, the neogeneic cells are tumor cells. In some embodiments, the neogeneic cells are cells within metastases. The cells may be eukaryotic cells, eukaryotic cells transfected with a carcinogen, human cells, cell lines, cells that are precancerous cells, or any combination thereof.

[0215] In yet another embodiment of the present invention, D is a protein administered as a replacement therapy, i.e., to replace a mutant or dysfunctional protein and thus address a physiological need. In another embodiment, D is a protein that plays a role in gene regulation, including, among other things, a protein that plays a role in DNA or RNA editing (addition, disruption, or modification of the sequence of a particular gene). In one embodiment, the protein may be a member of CRISPR (clustered, regularly spaced, short palindromic repetition) related proteins. Specifically, the protein may be a Cas9 protein (CRISPR-related protein 9), an RNA-guided DNA nuclease enzyme, or an analog thereof, potentially loaded with its guide oligonucleotide sequence.

[0216] One embodiment of the present invention describes a method for gene therapy for a medical disorder, the method being derived from formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H) The treatment involves administering a therapeutically effective amount of a pharmaceutical composition containing a conjugate of one of (XVI-M), (Cn-1), (Cn-2), or (Cn-3) to a patient in need, where D is a CRISPR protein such as Cas9, which is administered together with an appropriate guide oligonucleotide to achieve delivery of the guide oligonucleotide-loaded protein to the cell, and the CRISPR protein can exert its genome editing activity. In this context, the guide oligonucleotide is an RNA or DNA sequence that guides the Cas9 protein to a specific locus (location) on genomic DNA to induce a double-strand DNA break at that site, thereby enabling repair of a local defect in the genome. In the case of Cas9, the guide oligonucleotide is a short segment of RNA whose sequence is complementary to the sequence of the target DNA locus.

[0217] Accordingly, the conjugates and their respective pharmaceutical compositions, as well as their respective methods, according to embodiments of the present invention, may be beneficial in the treatment of medical disorders selected from, in particular, cancer, toxic seizures, metabolic diseases, ischemic diseases, infectious diseases, vascular disorders, protein accumulation diseases, trauma, immune-mediated diseases, degenerative diseases, and hereditary or acquired medical disorders.

[0218] Accordingly, embodiments of the present invention provide a method for treating a medical disorder, the method comprising administering to a patient in need a therapeutically effective amount of a pharmaceutical composition containing a conjugate of any of the formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), (XIV-M), (XV), (XV-H), (XV-M), (XVI), (XVI-H), (XVI-M), (Cn-1), (Cn-2), (Cn-3), where D is a drug useful for treating the medical disorder.

[0219] According to some embodiments, the medical disorder is cancer. As used herein, the term “cancer” means the presence of cells exhibiting characteristics typical of cancer-causing cells, such as uncontrolled growth, loss of specific function, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rates, or certain characteristic morphological and cellular markers known to be associated with cancer. Typically, cancer cells are tumor morphologies, either occurring locally within an animal or circulating in the bloodstream as independent cells, such as leukemia cells.

[0220] In the field of neurological disorders, the conjugates according to embodiments of the present invention may be particularly useful in the treatment of neurodegenerative disorders such as Alzheimer's disease, motor neuron disease, Parkinson's disease, Huntington's disease, multiple sclerosis, and Creutzfeldt-Jakob disease.

[0221] In the field of infectious disorders, the conjugates according to embodiments of the present invention may be useful, in particular, for the delivery of antibiotics to combat bacterial, fungal, or other parasitic infections, or for the delivery of antiviral agents to combat viral infections. Therefore, the D of the conjugate of the present invention may have anti-infective properties and thus be useful in the treatment of infectious diseases such as bacterial or viral infections. Examples of viral infections for which the conjugate of the present invention may be useful include, without limitation, human immunodeficiency virus (HIV); hepatotropic viruses such as hepatitis C virus (HCV) or hepatitis B virus (HBV); and infections by orthomyxoviridae such as influenza virus A, influenza virus B, influenza virus C, or parainfluenza virus. Therefore, embodiments of the present invention are conjugates of the E, E', or E'' portion(s) conjugated to an antiviral or antibacterial agent. Such agents may, in particular, be OD, whose sequence is intended to interact with the genetic material of the infectious agent and thus interfere with genetic processes that play a role in the replication, metabolism, infectivity, or survival of the pathogen. Such gene sequences may be siRNA or dsiRNA specifically designed to silence the expression of the gene(s) of an infectious agent (e.g., a virus).

[0222] The usefulness of the conjugate of the present invention in combating infection may be in at least one of the following uses: delivery of therapeutically useful drugs across biological membranes to host (e.g., human patient) cells, or delivery of pathogen (e.g., bacteria or viruses) cells across biological membranes.

[0223] In the field of metabolic disorders, the conjugates according to embodiments of the present invention may be particularly useful for the delivery of gene therapy aimed at downregulating the expression of the gene(s) causing the metabolic disorder, or for the administration of proteins to replace defective mutant proteins that play a role in the etiology or pathogenesis of the disease.

[0224] In other embodiments, the present invention relates to the potential use of the compounds of the present invention to enhance the delivery of compounds across phospholipid membranes to plant cells, and thus potentially beneficial for agricultural use. Depending on the conjugated compound and the desired indication, such delivery can have a variety of useful applications in agriculture. For example, such delivery in plants can help improve crop quality and quantity, among other things, by improving plant genetics or by eradicating various pathogens: insects, bacteria, or fungi. [Examples]

[0225] The following examples illustrate the present invention in a non-limiting manner to demonstrate how embodiments of the present invention can be actually carried out. The examples describe various compounds and conjugates of the present invention. All described conjugates contain an E, E', or E'' moiety according to formula (Cn-1) or (Cn-2), each having a structure according to general formula (II). The examples provide various E, E', or E'' moieties selected from the structures shown in formulas (III), (IV), (V), (VI), (VII), (VIII), (VIII-H), (VIII-M), (IX), (X), (XI), (XII), (XIIa), (XIII), (XIIIa), (XIV), (XIV-H), or (XIV-M). These conjugates exhibit biological activity in gene silencing. This performance supports the concept that conjugates comprising formulas (I) and (II) represent a common and unified structural motif that enables the useful delivery of high molecular weight OD across phospholipid membranes to cells, along with the resulting useful biological performance (in this case, gene silencing). In addition, the examples describe methods for the chemical synthesis of the E portion of the present invention, their precursors, and their assembly into useful conjugates.

[0226] Example 1: A general method for synthesizing a conjugate according to an embodiment of the present invention, in which the D portion is an oligonucleotide:

[0227] First, the gene to be silenced is selected based on its role in the etiology or pathogenesis of the disease. Then, the nucleotide sequence to be incorporated into the conjugate is designed and determined based on bioinformatics methodologies known in the art [typically, a 19-21 base pair double-stranded siRNA on a RISC substrate, or a 24-29 base pair double-stranded RNA (dsiRNA) on a Dicer substrate].

[0228] The synthesis is carried out in the 3' to 5' direction of the oligonucleotide. Solid-phase synthesis is performed using protected building blocks derived from protected 2'-deoxynucleosides (dA, dC, dG, and dT), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, e.g., [LNA (Locked Nucleic Acid) or BNA (Bridged Nucleic Acid)]. The building blocks are provided as nucleoside precursors, and the 5'- and 3'-hydroxyl groups are protected by DMT and phosphoramidite, respectively. These groups are sequentially removed in an order determined by the desired nucleotide sequence during the reaction in which the nucleotide is coupled to the growing oligonucleotide chain.

[0229] For the purpose of synthesizing the conjugates of the present invention, the E group is provided as a precursor molecule and is the E, E', or E'' moiety of the present invention, each bonded to a protecting group as described above. The protecting group may be any hydroxyl protecting group known in the art, but phosphoramidites and DMT [dimethoxytritylbis-(4-methoxyphenyl)phenylmethyl] are often used conventionally in oligonucleotide synthesis. The main advantage of the conjugates of the present invention is to provide the option of bonding the E, E', or E'' moiety at the 5' end of the oligonucleotide chain, the 3' end of the oligonucleotide chain, or at an internal position along the oligonucleotide chain, as exemplified by conjugates (Cn-1) and (Cn-2). Thereafter, the E moiety of the present invention can be incorporated into the oligonucleotide chain, as with intrinsic, natural oligonucleotide building blocks. Bonding between nucleotides may be via standard phosphotriester bonds, or via synthetic phosphorothioate bonds, which may offer advantages such as stability in blood or binding to blood proteins, or via any other nucleotide bonding methodology known in the art. After the assembly of the strands is complete, the product is released from the solid support into a solution, deprotected, and collected. The desired conjugate is then isolated by high-performance liquid chromatography (HPLC) to obtain the desired conjugate of the present invention in high purity. In the case of siRNA or dsiRNA, each complementary RNA strand is synthesized separately, and then the annealing of the two strands is carried out under standard conditions as known in the art to yield the desired double-stranded siRNA or dsiRNA, which is then subjected to purification and Ali coating.

[0230] Example 2: Method for chemical synthesis of a precursor molecule containing the E, E', or E'' portion of the present invention:

[0231] Example 2A: Synthesis of the main intermediate phenol 1:

[0232] [ka]

[0233] Estradiol was treated with excess sodium hydride, followed by the addition of allyl bromide, resulting in a clean conversion to compound 3. Subsequent hydroboration with 1.5 equivalents of 9-BBN yielded only terminal hydroxyl groups, while hydroboration with BH3 was far less selective and provided a mixture of adducts. Alcohol 5 was subjected to Mitsunobu reaction conditions and combined with perfluorinated tert-butanol to obtain compound 6. Hydrolysis of the benzyl group of compound 8 supplied phenol 1. In conclusion, phenol 1 was prepared from estradiol in 45% total yield via five synthetic steps.

[0234] 2bA1.(8R,9S,13S,14S,17S)-3-benzyloxy-17-hydroxyestra-1,3,5(10)-triene(2):

[0235] The synthesis of (8R,9S,13S,14S,17S)-3-benzyloxy-17-hydroxyestra-1,3,5(10)-triene(2) is disclosed herein in Section 2aA1 above.

[0236] 2bA2.(8R,9S,13S,14S,17S)-17-allyloxy-3-benzyloxyestra-1,3,5(10)-triene(3):

[0237] The synthesis of (8R,9S,13S,14S,17S)-17-allyloxy-3-benzyloxyestra-1,3,5(10)-triene(3) is disclosed herein in Section 2aA2 above.

[0238] 2bA3.(8R,9S,13S,14S,17S)-3-benzyloxy-17-(3-hydroxypropoxy)estra-1,3,5(10)-triene(7):

[0239] 9-Borabicyclo[3.3.1]nonane (800 mL, 0.5 M solution in THF, stabilized, 400 mmol) was added dropwise to a solution of crude alkene 3 (101.2 g, 251 mmol) in THF (1 L) at 0°C. After complete addition, the mixture was stirred overnight at room temperature. The solution was cooled to 0°C, and 30% aqueous NaOH (150 mL, 1.3 mol) and 35% aqueous NaOH (120 mL, 1.3 mol) were slowly and simultaneously added dropwise. The resulting heterogeneous mixture was vigorously stirred at room temperature for about 1 hour. The reaction mixture was then partitioned between HCl (2 L) and brine (500 mL). The organic phase was further washed with 500 mL of brine, dried over Na₂SO₄, and concentrated under vacuum. This procedure was repeated in a similar manner, and both parts were combined. Further purification of the concentrate by flash chromatography (25%–35% ethyl acetate gradient in silica gel and heptane) yielded alcohol 5 (130 g, 310 mmol) as a white solid in 61% yield (3 steps).

[0240] 2bA4.(8R,9S,13S,14S,17S)-3-benzyloxy-17-[3-(perfluoro-tert-butyloxy)propoxy]estra-1,3,5(10)-triene(8):

[0241] Diisopropyl azodicarboxylate (80 mL, 407 mmol) was added dropwise under a nitrogen atmosphere to a stirred mixture of alcohol 7 (130 g, 301 mmol), triphenylphosphine (162 g, 618 mmol), perfluoro-tert-butanol (70 mL, 497 mmol), and dry THF (2 L). The mixture was stirred at room temperature for approximately 18 hours. The reaction mixture was partially concentrated and heptane (1 L) was added. Precipitation began after complete removal of THF. The solid was removed by filtration and the filtrate was concentrated. Acetonitrile (1.5 L) was added and the mixture was stirred for 30 minutes until precipitation began. The solid was collected by filtration and dried under vacuum. Compound 8 (160 g, 251 mmol) was isolated as a white solid in 81% yield.

[0242] 2bA5.(8R,9S,13S,14S,17S)-3-hydroxy-17-[3-(perfluoro-tert-butyloxy)propoxy]estra-1,3,5(10)-triene(phenol 1)

[0243] Benzyl ether 8 (160g, 251 mmol) in SiO2 (1L) was placed in a Parr container, and 10% palladium (4g) on ​​carbon was added. The mixture was stirred at room temperature under hydrogen pressure (5 bar). The reaction was carried out. 1 The reaction was monitored by 1H NMR. After approximately 72 hours, the reaction mixture was filtered through a Celite pad (washed with ethylethanol) and re-transmitted to a hydrogen atmosphere (5 bar) with fresh 10% charcoal-supported palladium (4 g). After approximately 16 hours, the reaction mixture was filtered through a Celite pad (washed with ethylethanol) and concentrated to provide phenol 1 (125 g, 228 mmol) as a grayish solid in 91% yield.

[0244] Example 2b: Synthesis of the main building block K-93-A-1:

[0245] The synthesis is carried out according to the following synthesis scheme.

[0246] [ka]

[0247] Example 2c: Synthesis of the precursor of formula (III):

[0248] [ka]

[0249] [ka]

[0250] Intermediate K-93-A-1 is synthesized as described above, but the synthesis of intermediate K-103-5 is carried out according to the following synthesis scheme.

[0251] [ka]

[0252] Example 2d: Synthesis of the precursor of formula (IV):

[0253] [ka]

[0254] The synthesis of the precursor of formula (IV) is carried out by the conjugation of K-103A-2, a derivative of phenol 1, with the main building block K-93-A-1. The synthesis is carried out according to the following synthetic scheme.

[0255] [ka]

[0256] Example 2e: Method for synthesizing the precursor of formula (V):

[0257] [ka]

[0258] Synthesis of thioacetate 11:

[0259] [ka]

[0260] The synthesis began with the protection of the ketone with 1,3-propanediol, yielding compound 2 in good purity. Ring-opening of the acetal LiAlH4 and AlCl3 gave a mixture of compound 3 and estradiol in a ratio of approximately 85:15, the latter being less reactive and removed in the next step.

[0261] Phenol was alkylated with methyl bromoacetate to obtain compound 4. The perfluoro-tert-butanol moiety was introduced using Mitsunobu conditions (compound 5). Compound 5 was treated with methylamine to provide amide 6. Reduction of the amide using BH3.DMS provided amine 7. The amine was alkylated with bromide 8 to provide ester 9. The ester was reduced to the corresponding alcohol (10) with LiAlH4. Finally, a thioacetate was introduced using Mitsunobu conditions to provide the desired building block 11.

[0262] The alkylation of amine 7 appears to be less straightforward than expected. The conversion is not achieved at room temperature, suggesting the need for a base (such as Et3N or K2CO3). However, the yield obtained was typically in the range of 40%. Subsequent reduction of the ester to alcohol 10 in LiAlH4 resulted in a good conversion and a simple workup. The alumina salt is usually destroyed by adding 20% ​​aqueous KOH (160 mL per mole), which, after simple filtration, yields the desired material in THF. The conversion of the alcohol to thioacetate is achieved via Mitsunobu conditions. Regarding the order of addition, it should be noted that thioacetic acid should be added as the last component. After workup and careful purification, thioester 11 could be obtained.

[0263] Synthesis of thiotosylate 18:

[0264] [ka]

[0265] During the synthesis of previous compounds, it was observed that the presence of amines in the thiotosylate building block was the cause of very low yields. Therefore, a Boc-protected thiotosylate 18 was required. The Boc group was removed after disulfide formation, and the amine was subsequently alkylated. Aminopropanol was protected with a Boc group and then reduced to the corresponding methylamine 13 with LiAlH4. Here, the secondary amine was then protected with a new Boc group to provide compound 14. The alcohol was reacted with ethyl diazoacetate to introduce an ether functional group (15). The ester was reduced using LiAlH4 to provide alcohol 16. Bromination with NBS provided bromide 17. Substitution of the bromide with potassium thiotosylate yielded the desired thiotosylate building block 18.

[0266] Synthesis of iodide 22:

[0267] [ka]

[0268] Diethyl malonate was alkylated with methyl 4-bromobutyrate to provide triester 19. All three esters were simultaneously reduced by treatment with LiAlH4 to provide triol 20. An acetonide protecting group was introduced by reaction with dimethoxypropane to yield alcohol 21. The alcohol was converted to an iodide to provide the desired building block 22.

[0269] Formula (V) - Integration of precursor synthesis:

[0270] [ka]

[0271] [ka]

[0272] Disulfide formation between thioacetate 11 and thiotosylate 18 provided disulfide 23 in good yield. Treatment with TFA to remove the Boc group provided amine 24. Alkylation of the amine with iodide 22 provided 25 in 40% yield. Removal of the acetonide by acid treatment provided diol 26. Introduction of a DMT group yielded compound 27. Final phosphoramidite formation provided the precursor of formula (V).

[0273] Experiment section:

[0274] Synthesis of thioacetate 11:

[0275] (8R,9S,13S,14S)-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydrospiro[cyclopenta[a]phenanthrene-17,2'-[1,3]dioxane]-3-ol(2)

[0276] To a suspension of estrone (252 g, 0.93 mol) in toluene (1.5 L), trimethoxymethane (297 g, 350 mL, 2.80 mol), propane-1,3-diol (213 g, 250 mL, 2.80 mol), and pTsOH (2 g, 10 mmol) were added. The mixture was heated to 60°C and stirred for 16 hours. Triethylamine (6 mL) and water (600 mL) were added, and stirring was continued for a further 1 hour. The phases were separated, and the organic layer was washed with water (3 × 400 ml) and brine. It was dried over Na₂SO₄ and partially concentrated to approximately 1 L. The mixture was poured into heptane (4 L), and the white solid was filtered off. It was washed with heptane and vacuum dried. Compound 2 (271 g, 825 mmol) was isolated as a white solid in 88.5% yield.

[0277] (8R,9S,13S,14S,17S)-17-(3-hydroxypropoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-ol(3)

[0278] To a solution of (13S)-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydrospiro[cyclopenta[a]phenanthrene-17,2'-[1,3]dioxane]-3-ol (2, 60.7 g, 185 mmol) in THF at 0°C, lithium aluminum hydride (8.42 g, 222 mmol) was carefully added, followed by aluminum chloride (98.6 g, 739 mmol) (highly exothermic!) in small amounts. The mixture was stirred at 0°C for 15 minutes, then warmed to 50°C. (Due to clogging of the rotary evaporator) the mixture was stirred at 50°C for 2 hours, then cooled to 0°C, and quenching was started with dropwise addition of NH4Cl (aqueous) (500 mL). The mixture was stirred at room temperature for 1 hour. The phases were separated, the organic layer was washed with brine, and concentrated. A white solid (65 grams) contaminated with estradiol (approximately 15%) was obtained.

[0279] 2-(((8R,9S,13S,14S,17S)-17-(3-hydroxypropoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)methyl acetate(4)

[0280] Crude material 3 (89.4 g) was dissolved in acetone (1.25 L) and MeOH (0.2 L) and treated with potassium carbonate (60 g, 435 mmol) and methyl bromoacetate (50 mL, 435 mmol). The suspension was heated to 60°C and stirred for 16 hours. Based on TLC, all phenolic moieties were alkylated. The mixture was cooled to room temperature and filtered. The filtrate was concentrated and further purified using flash chromatography (to remove all impurities, 20-30% ethyl acetate in heptane, and 100% ethyl acetate as the eluent to obtain the desired material).

[0281] Compound 4 (56.7 g, 140.3 mmol) was isolated as a yellow oil in 65% yield.

[0282] 2-((((8R,9S,13S,14S,17S)-17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy))propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)methyl acetate(5)

[0283] To a solution of compound 4 (56.7 g, 140.3 mmol) in THF (1 L), triphenylphosphine (55.4 g, 211 mmol), nonafluoro-tert-butyl alcohol (30 mL), and azodicarboxylic acid di-tert-butyl (38.5 g, 167 mmol) were added. The mixture was stirred for 30 minutes when TLC showed complete conversion. Heptane (500 mL) was added, and the mixture was partially concentrated to approximately 500 mL. Further heptane (1 L) was added, and the mixture was stirred overnight at room temperature. A precipitate formed, which was filtered off, and the filtrate was concentrated to provide compound 5 as a yellow syrup, although it was contaminated with trace amounts of DBAD and triphenylphosphine.

[0284] 2-(((8R,9S,13S,14S,17S)-17-(3-((1,1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propan-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)-N-methylacetamide(6)

[0285] Crude syrup of compound 5 was diluted with MeOH (250 mL) and 40% aqueous methylamine (350 mL) was added. When TLC showed complete conversion, the white precipitate was stirred for 1 hour. Water (1 L) was added and the solid was filtered off. The residue was washed with water and absorbed in dichloromethane (1 L). The organic layer was washed with brine, dried over Na2SO4, and concentrated. Further purification was performed using the initial elution with 15% ethyl acetate in approximately 7 cm of silica and heptane. When all impurities had been removed from the column, compound 6 was eluted with 100% ethyl acetate. Compound 6 (82.0 g, 132 mmol) was isolated as a white solid in 94% yield, although trace amounts of triphenylphosphooxide remained.

[0286] 2-(((8R,9S,13S,14S,17S)-17-(3-((1,1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)-N-methylethane-1-amine(7)

[0287] To a solution of compound 6 (56.0 g, 90.5 mmol) in 500 mL of THF at 65°C, BH3.DMS (56 mL, 590 mmol) was added dropwise. Reflux was continued for a further 5 hours, and the mixture was then cooled to room temperature. The mixture was carefully dissolved in 300 mL of MeOH, and 4 M dioxane in 50 mL of HCl was added. The solution was refluxed for 30 minutes, then cooled to room temperature and concentrated. The mixture was dissolved in 300 mL of MeOH and refluxed for 30 minutes. After cooling and concentration, the syrup was absorbed in 1 L of CH2Cl2 and washed with aqueous saturated sodium bicarbonate (2x). The organic layer was dried over Na2SO4 and concentrated. Compound 7 (50.0 g, 82.6 mmol) was isolated as a clear oil that slowly solidified in 91% yield. The impurity profile with trace amounts of triphenyl phosphooxide was similar to that of compound 6.

[0288] 2-(3-bromopropoxy)ethyl acetate (8)

[0289] To a solution of 2-ethyl diazoethyl (100 g, 0.74 mol) and 3-bromopropan-1-ol (0.10 kg, 74 mL, 0.74 mol) in DCM (100 mL), BF3.OEt2 (1.1 g, 0.94 mL, 7.4 mmol) was added at 0°C. The reaction was stirred at 0°C for 15 minutes and at room temperature for 3 hours until no gas was observed. The mixture was diluted with DCM (500 mL), washed with H2O (500 mL) and brine (500 mL), and dried over Na2SO4. The solvent was removed under vacuum, and 2-(3-bromopropoxy)ethyl acetate (8, 180 g, 0.80 mol, 110%) was provided as a clear yellow oil.

[0290] 2-(3-((2-(((8R,9S,13S,14S,17S)-17-(3-((1,1,1,3,3,3-Hexafluoro-2-(fluoromethyl)propan-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Deca-hydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)ethyl)(methyl)amino)propoxy)ethyl acetate(9)

[0291] A suspension of 2-(3-bromopropoxy)ethyl acetate (11 g, 50 mmol), compound 7 (25 g, 41 mmol), potassium carbonate (11 g, 83 mmol), and potassium iodide (0.69 g, 4.1 mmol) in acetonitrile (300 mL) was heated at 70°C for 16 hours. The mixture was cooled to room temperature, diluted with SiO2 (100 mL), filtered, and concentrated. Further purification using flash chromatography (gradient of 20%-30% acetone + 1% Et3N in heptane) yielded compound 8 (12.0 g, 16 mmol) at 39% as a slowly solidified clear oil.

[0292] 2-((3-((2-(((8R,9S,13S,14S,17S)-17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propane)-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-Cyclopenta[a]phenanthrene-3-yl)oxy)ethyl)(methyl)amino)propoxy)ethane-1-ol(10)

[0293] To a solution of compound 9 (12 g, 16 mmol) in 0°C THF (200 mL), lithium aluminum hydride (0.91 g, 24 mmol) was added. The reaction mixture was warmed to room temperature and stirred for 1 hour and 30 minutes. TLC gave complete consumption. The reaction mixture was quenched with 20% KOH (160 mL / mol, V=4.2 mL), stirred for 1 hour, filtered, and concentrated under vacuum. Compound 10 (10.2 g, 14.4 mmol) was isolated as a clear solution.

[0294] S-(2-(3-((2-(((8R,9S,13S,14S,17S)-17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-Cyclopenta[a]phenanthrene-3-yl)oxy)ethyl)(methyl)amino)propoxy)ethyl)ethanethioate(11)

[0295] To a solution of compound 10 (20.2 g, 28.5 mmol) in THF, triphenylphosphine (9.73 g, 37.1 mmol) and DIAD (6.93 g, 6.66 mL, 34.3 mmol) were added, followed by thioacetic acid (3.26 g, 3.07 mL, 42.8 mmol) after 5 minutes. The mixture was stirred for 3 hours, then heptane (20 mL) was added and the solution was concentrated.

[0296] The compound was purified using flash chromatography (10% acetone + 1% Et3N in heptane). Compound 11 (15.4 g, 20.1 mmol) was isolated as a clear viscous oil in 71% yield.

[0297] Synthesis of thiotosylate 18:

[0298] (3-hydroxypropyl)carbamate tert-butyl(12)

[0299] To a solution of 3-aminopropan-1-ol (28.5 g, 379 mmol) and triethylamine (38.4 g, 53 mL, 379 mmol) in DCM (500 mL), di-tert-butyl dicarbonate (91.1 g, 417 mmol) was slowly added, and the resulting mixture was stirred at room temperature for 2 hours. The mixture was quenched with imidazole (scoop) and stirred for 5 minutes. The mixture was washed with 1 M HCl (300 mL), dried over Na2SO4, and concentrated to provide compound 12 (55 g, 0.31 mol, 83%) as a clear oil.

[0300] 3-(methylamino)propan-1-ol(13)

[0301] A solution of 12, 88 g, 0.34 mol tert-butyl(3-hydroxypropyl)carbamate in 250 mL of THF was added dropwise to an ice-cold suspension of lithium aluminum hydride (26 g, 0.67 mol) in 500 mL of THF at 0°C. After complete addition, the mixture was stirred at room temperature for 30 minutes and at 70°C for 16 hours. The mixture was cooled to room temperature and then quenched by the dropwise addition of KOH (20%, 107 mL) at 0°C. The resulting mixture was stirred at room temperature for 2 hours. The mixture was filtered through Celite, the filtrate was dried over Na2SO4 and concentrated. NMR showed that some Boc was still intact. The material was dissolved in 500 mL of THF and LiAlH4 (13 g, 0.341 eq.) was added. The mixture was stirred at 80°C for 16 hours. The mixture was cooled to room temperature. At 0°C, the reaction was quenched by slowly adding KOH (20%, 54 mL), and the resulting mixture was stirred at room temperature for 2 hours. The mixture was filtered through Celite, the filtrate was dried over Na2SO4, and concentrated to provide Boc-methylaminopropanol (22.6 g, 170 mmol, 50%) as a clear oil.

[0302] (3-hydroxypropyl)(methyl)carbamate tert-butyl(14)

[0303] To a solution of 3-(methylamino)propan-1-ol (13, 22.6 g, 254 mmol) and triethylamine (25.7 g, 35 mL, 254 mmol) in DCM (500 mL), di-tert-butyl dicarbonate (55.3 g, 254 mmol) was gradually added. The resulting mixture was stirred at room temperature for 1 hour until no gas generation was observed. The mixture was washed with 1 M HCl, dried over Na2SO4, and concentrated to provide Boc-protected amine 14 (43 g, 230 mmol, 90%) as a clear oil.

[0304] 2-(3-((tert-butoxycarbonyl)(methyl)amino)propoxy)ethyl acetate (15)

[0305] To a solution of alcohol 14 (36 g, 0.19 mol) in DCM (500 mL), ethyl 2-diazoethyl acetate (25 g, 23 mL, 0.19 mol) and boron trifluoride etherate (2.7 g, 2.4 mL, 19 mmol) were added, and the resulting mixture was stirred for 16 hours. The mixture was washed with water and brine, dried over Na2SO4, and concentrated. The crude material was purified by column chromatography (20% SiO in heptane) to provide ester 15 (11 g, 40 mmol, 21%) as a clear oil.

[0306] (3-(2-hydroxyethoxy)propyl)(methyl)carbamate tert-butyl(16)

[0307] A solution of ester 15 (13 g, 47 mmol) in THF (50 mL) was added dropwise to an ice-cold suspension of lithium aluminum hydride (2.7 g, 71 mmol) in THF at 0°C. The mixture was stirred at room temperature for 1 hour. The reaction was quenched by adding KOH (20%, 11 mL) dropwise at 0°C. The resulting mixture was stirred at room temperature for 30 minutes. The mixture was filtered through Celite, dried over Na2SO, and concentrated to provide alcohol 16 (8.5 g, 36 mmol, 77%) as a clear oil.

[0308] (3-(2-bromoethoxy)propyl)(methyl)carbamate tert-butyl(17)

[0309] To a solution of alcohol 16 (8.5 g, 36 mmol) in DCM (300 mL), triphenylphosphine (13 g, 51 mmol) and NBS (7.8 g, 44 mmol) were added, and the resulting mixture was stirred at room temperature for 16 hours. The mixture was washed with NaHCO3 and brine, dried over Na2SO4, and filtered. Heptane was added, and DCM was removed by evaporation. The formed solid was filtered off, and the filtrate was concentrated. The crude material was purified using column chromatography (20% SiO2 / heptane) to provide tert-butyl 17 bromide (7.6 g, 26 mmol, 70%) as a clear oil.

[0310] S-(2-(3-((tert-butoxycarbonyl)(methyl)amino)propoxy)ethyl)4-methylbenzene sulfonothioate (18)

[0311] A solution of bromide 17 (7.6 g, 26 mmol) and potassium 4-methylbenzene sulfonothioate (8.7 g, 38 mmol) in DMF (200 mL) was stirred at 50°C for 16 hours. The mixture was cooled to room temperature and diluted with water (1 L). The mixture was extracted with toluene / heptane (3 x 200 mL 1:1), the combined organic layer was washed with brine, dried over Na2SO4, and concentrated. The crude material was purified by column chromatography (20% toluene / heptane) to provide thiotosylate 18 (9.1 g, 23 mmol, 88%) as a clear oil.

[0312] Synthesis of iodide 22:

[0313] Butane-1,1,4-tricarboxylic acid 1,1-diethyl-4-methyl(19)

[0314] To an ice-cold suspension of sodium hydride (10 g, 0.26 mol) in DMF (500 mL), diethyl malonate (42 g, 40 mL, 0.26 mol) was slowly added, and the reaction mixture was stirred at room temperature for 1 hour until the mixture was purified. Methyl 4-bromobutanoate (47 g, 0.26 mol) was added at 0°C, and the resulting mixture was stirred at room temperature for 18 hours. The mixture was partially concentrated, and the residue was quenched with 1N HCl (300 mL) and water (1.3 L). The mixture was extracted with Hept / HCl (1 / 1, 2 x 250 mL). The combined organic layer was washed with brine (500 mL), dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (20% HCl / heptane) to provide triester 19 (63 g, 0.24 mol, 92%) as a clear oil.

[0315] 2-(hydroxymethyl)hexane-1,6-diol(20)

[0316] To an ice-cold suspension of lithium aluminum hydride (25 g, 0.66 mol) in THF (500 mL), a solution of triester 19 (63 g, 0.24 mol) in THF (100 mL) was slowly added at 0°C, and the resulting mixture was stirred at room temperature for 16 hours. The reaction was quenched by slowly adding KOH (20% aqueous solution, 106 mL) at 0°C. The resulting mixture was stirred at room temperature for 1 hour. The mixture was filtered through Celite, dried over Na2SO4, and concentrated to provide triol 20 (16 g, 0.11 mol, 45%) as a clear yellow oil.

[0317] 4-(2,2-dimethyl-1,3-dioxan-5-yl)butan-1-ol(21)

[0318] To a solution of triol 20 (16 g, 0.11 mol) in THF (200 mL), p-toluenesulfonic acid (5.1 g, 27 mmol) and 2,2-dimethoxypropane (34 g, 40 mL, 0.32 mol) were added, and the resulting mixture was stirred at room temperature for 1 hour. The mixture was concentrated, and the residue was dissolved in DCM (200 mL). The mixture was washed with NaHCO3 (200 mL saturated aqueous solution) and brine (200 mL), dried over Na2SO4, and concentrated to acetonide 21 (9.0 g, 48 mmol, 44%) as a brown oil.

[0319] 5-(4-iodobutyl)-2,2-dimethyl-1,3-dioxane(22)

[0320] Iodine (5.5 g, 22 mmol) was added to a solution of alcohol 21 (3.7 g, 20 mmol), triphenylphosphine (6.2 g, 24 mmol), and imidazole (1.6 g, 24 mmol) in DCM (250 mL), and the resulting mixture was stirred at room temperature for 1 hour. The mixture was washed with sodium thiosulfate (2 × 100 mL saturated aqueous solution) and brine, dried over Na₂SO₄, and concentrated. The crude material was purified by column chromatography (15% SiO₂ in heptane) to provide iodide 22 (3.7 g, 12 mmol, 63%) as a clear yellow oil.

[0321] Synthesis of the precursor of formula (V):

[0322] (1-(((8R,9S,13S,14S,17S)-17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)-3-methyl-7,14-dioxa-10,11-dithia-3-azaheptadecane-17-yl)(methyl)carbamate tert-butyl(23)

[0323] To a solution of thiotosylate 18 (3.4 g, 8.5 mmol) and thioacetate 11 (5.0 g, 6.5 mmol) in DCM (200 mL) and MeOH (20 mL), a solution of sodium methoxide (1.1 g, 3.6 mL, 20 mmol) in MeOH (5.4 M) was added, and the resulting mixture was stirred at room temperature for 1 hour. The mixture was diluted with 200 mL of DCM, washed with NaHCO3 and brine, dried over Na2SO4, and concentrated. The crude material was purified by column chromatography (10% acetone + 1% NET3 in heptane) to provide disulfide 23 (6.5 g, 6.7 mmol, quantitatively) as a clear oil.

[0324] N-(2-(((8R,9S,13S,14S,17S)-17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propan-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-Cyclopenta[a]phenanthrene-3-yl)oxy)ethyl)-N-methyl-3-(2-((2-(3-(methylamino)propoxy)ethyl)disulfanyl)ethoxy)propan-1-amine(24)

[0325] To a solution of tert-butyl disulfide 23 (6.5 g, 6.7 mmol) in DCM (200 mL), TFA (15 g, 10 mL, 0.13 mol) was added, and the resulting mixture was stirred at room temperature for 1 hour. An additional 5 mL of TFA was added, and the mixture was stirred for 30 minutes. The mixture was concentrated, and the residue was dissolved in DCM (250 mL). The mixture was washed with NaHCO3 and brine, dried over Na2SO4, and concentrated to provide amine 24 (5.3 g, 6.1 mmol, 91%) as a slightly yellow, clear oil.

[0326] 4-(2,2-dimethyl-1,3-dioxan-5-yl)-N-(1-(((8R,9S,13S,14S,17S)-17-(3-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)-3-methyl-7,14-dioxa-10,11-dithia-3-azaheptadecane-17-yl)-N-methylbutan-1-amine(25)

[0327] To a solution of amine 24 (5.3 g, 6.1 mmol) in acetonitrile (200 mL), potassium carbonate (0.84 g, 6.1 mmol) and iodide 22 (1.8 g, 6.1 mmol) were added, and the resulting mixture was stirred at 50°C for 16 hours. The mixture was concentrated, and the residue was dissolved in DCM (300 mL). The mixture was washed with NaHCO3 (250 mL of semi-saturated aqueous solution) and brine, dried over Na2SO4, and concentrated. The crude material was purified using column chromatography (25% acetone + 1% NET3 in heptane) to provide acetonide 25 (2.5 g, 39%) as a clear oil.

[0328] 2-(1-(((8R,9S,13S,14S,17S)-17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)-3,18-dimethyl-7,14-dioxa-10,11-dithia-3,18-diazadocosan-22-yl)propane-1,3-diol(26)

[0329] To a solution of acetonide 25 in MeOH (100 mL), p-toluenesulfonic acid (1.1 g, 6.0 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated, and the residue was dissolved in DCM (200 mL). The mixture was washed with NaHCO3 and brine, dried over Na2SO4, and concentrated to provide diol 26 (2.1 g, 2.1 mmol, 87%) as a clear oil.

[0330] 23-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(((8R,9S,13S,14S,17S)-17-(3-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)-3,18-dimethyl-7,14-dioxa-10,11-dithia-3,18-diazatetracosan-24-ol(27)

[0331] To a solution of diol 26 (2.1 g, 2.1 mmol) in DCM (200 mL), triethylamine (0.42 g, 0.58 mL, 4.2 mmol), DMAP (26 mg, 0.21 mmol), and 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (0.71 g, 2.1 mmol) were added, and the resulting mixture was stirred at room temperature for 16 hours. The mixture was washed with NaHCO3 and brine, dried over Na2SO4, and concentrated. The crude material was purified by column chromatography (25-40% acetone + 1% NEt3 in heptane, NEt3-pretreated silica) to provide DMT-protecting compound 27 (2.3 g, 1.8 mmol, 84%) as a yellowish oil.

[0332] 23-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(((8R,9S,13S,14S,17S)-17-(3-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)-3,18-dimethyl-7,14-dioxa-10,11-dithia-3,18-diazatetracosan-24-yl(2-cyanoethyl)diisopropylphosphoramidite(Apo-Si-K-105-B)

[0333] To a solution of compound 27 (2.3 g, 1.8 mmol) in DCM (100 mL), 3-((bis(diisopropylamino)phosphine)oxy)propanenitrile (0.69 g, 0.72 mL, 2.3 mmol) and a solution of NMM and TFA (4.6 mL, 0.5 M NMM and 0.25 M TFA, 1.3 eq. NMM) in DCM were added. The resulting mixture was stirred at room temperature for 3 hours. The mixture was washed with NaHCO3 and brine, dried over Na2SO4, and concentrated. The crude material was purified by column chromatography (30% acetone + 1% NEt3 in heptane, silica pretreated with heptane / NEt3 to inactivate) to provide formula (V)-precursor (2.2 g, 83%) as a clear oil.

[0334] Example 2f: Method for synthesizing the precursor of formula (VII):

[0335] [ka]

[0336] The synthesis of the precursor of formula (VII) is carried out according to the following synthesis scheme. The synthesis focuses on three main building blocks.

[0337] Building block K-105-6:

[0338] [ka]

[0339] Building block K-109B-1:

[0340] [ka]

[0341] Building block K-53-2: [ka]

[0342] Next, all building blocks are integrated into the final compound formula (VII)-precursor.

[0343] [ka]

[0344] Example 2g: Synthesis method of the precursor of formula (VIII-F):

[0345] Starting from estrone, a known intermediate, methyl ester 5, can be prepared using known chemicals. Nitrogen can then be introduced by reaction with aminopropanol. This may be possible directly, as previously done for methylamine. Otherwise, the methyl ester can be hydrolyzed and nitrogen introduced using a standard peptide coupling protocol. The amide can then be reduced to yield the desired secondary amine K-93-F-2. Subsequent introduction of a Boc group and a thioacetate functional group by Mitsunobu will provide the desired building block K-93-F-4.

[0346] [ka]

[0347] [ka]

[0348] Final formula (VIII-F) - Integration of fragments into precursor:

[0349] The assembly of the (VIII-F) precursor begins with disulfide formation between building block K-93-F-4 and thiotosylate 6. Subsequently, the acetonide moiety and Boc group are simultaneously removed by acid treatment. The free secondary amine is then protected with an Fmoc group. Subsequent DMT bonding and phosphoramidite formation provide the (VIII-F) precursor (Scheme 16).

[0350] [ka]

[0351] Example 2h: Synthesis method of the precursor of formula (XIIa):

[0352] [ka]

[0353] This synthesis is shown in the following scheme. The synthesis converges both sides of the disulfide moiety, with the left portion functionalized as a thioacetate and the steroid-containing right portion as a thiotosylate. These are merged and require minor modifications to provide the final compound.

[0354] Synthesis of thiotosylate 8:

[0355] [ka]

[0356] This synthesis began with the protection of the ketone with 1,3-propanediol to provide compound 2 in good purity. Ring-opening of the acetal with LiAlH4 and AlCl3 yielded a mixture of compound 3 and estradiol in a ratio of approximately 85:15, the latter being less reactive and removed in the next step. The phenol was alkylated with methyl bromoacetate to provide methyl ester 4. The perfluorobutanol moiety was then introduced using Mitsunobu conditions to provide compound 5. The methyl ester was reduced with lithium aluminum hydride, and the resulting alcohol 6 was brominated to provide bromide 7. Treatment with potassium thiotosylate yielded the desired building block thiotosylate 8.

[0357] Synthesis of thioacetate 16:

[0358] [ka]

[0359] The synthesis of building block 16 began with 4-aminobutan-1-ol. The amine was protected with a Boc group. The alcohol was then converted to the corresponding bromide (10), which was subsequently alkylated to diethyl malonate to provide bisester 11. When compound 11 was treated with LiAlH4 at room temperature, the ester was reduced, but the Boc group remained intact. The diol 12 was then protected by treatment with dimethoxypropane in the presence of a catalytic amount of acid to provide acetonide 13. Treatment with LiAlH4 at high temperature reduced the Boc moiety to the desired methylamine (14). Finally, reductive amination with aldehyde 15 provided thioacetate 16. Aldehyde 15 was readily available through a one-step reaction, namely the Michael addition of thioacetic acid to acrolein.

[0360] Formula (XIIa) - Integration of precursors:

[0361] [ka]

[0362] Disulfide formation between thiotosylate 8 and thioacetate 16 was carried out under known reaction conditions, in which the acetate was removed in situ with NaOMe and the resulting thiol attacked the thiotosylate. Removal of the acetonide and subsequent bonding of DMT-Cl provided 18. The final phosphoramidite formation yielded the (XIIa)-precursor.

[0363] Experiment section:

[0364] Synthesis of thiotosylate 8:

[0365] (8R,9S,13S,14S)-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydrospiro[cyclopenta[a]phenanthrene-17,2'-[1,3]dioxane]-3-ol(2)

[0366] To a suspension of estrone (252 g, 0.93 mol) in toluene (1.5 L), trimethoxymethane (297 g, 350 mL, 2.80 mol), propane-1,3-diol (213 g, 250 mL, 2.80 mol), and pTsOH (2 g, 10 mmol) were added. The mixture was heated to 60°C and stirred for 16 hours. Triethylamine (6 mL) and water (600 mL) were added, and stirring was continued for a further 1 hour. The phases were separated, and the organic layer was washed with water (3 x 400 mL) and brine. It was dried over Na2SO4 and partially concentrated to approximately 1 L. The mixture was poured into heptane (4 L), and the white solid was filtered off. It was washed with heptane and vacuum dried. Compound 2 (271 g, 825 mmol) was isolated as a white solid in 88.5% yield.

[0367] (8R,9S,13S,14S,17S)-17-(3-hydroxypropoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-ol(3)

[0368] To a solution of (13S)-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydrospiro[cyclopenta[a]phenanthrene-17,2'-[1,3]dioxane]-3-ol (2, 60.7 g, 185 mmol) in THF at 0°C, lithium aluminum hydride (8.42 g, 222 mmol) was carefully added, followed by aluminum chloride (98.6 g, 739 mmol) (highly exothermic!) in small amounts. The mixture was stirred at 0°C for 15 minutes, then warmed to 50°C. (Due to clogging of the rotary evaporator) the mixture was stirred at 50°C for 2 hours, then cooled to 0°C, and quenching was started with dropwise addition of NH4Cl (aqueous) (500 mL). The mixture was stirred at room temperature for 1 hour. The phases were separated, the organic layer was washed with brine, and concentrated. A white solid (65 grams) contaminated with estradiol (approximately 15%) was obtained.

[0369] 2-(((8R,9S,13S,14S,17S)-17-(3-hydroxypropoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)methyl acetate(4)

[0370] Crude material 3 (89.4 g) was dissolved in acetone (1.25 L) and MeOH (0.2 L) and treated with potassium carbonate (60 g, 435 mmol) and methyl bromoacetate (50 mL, 435 mmol). The suspension was heated to 60°C and stirred for 16 hours. Based on TLC, all phenolic moieties were alkylated. The mixture was cooled to room temperature and filtered. The filtrate was concentrated and further purified using flash chromatography (to remove all impurities, 20-30% HCl in heptane, and 100% HCl as the eluent to obtain the desired material). Compound 4 (56.7 g, 140.3 mmol) was isolated as a yellow oil in 65% yield.

[0371] 2-((((8R,9S,13S,14S,17S)-17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy))propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)methyl acetate(5)

[0372] To a solution of compound 4 (56.7 g, 140.3 mmol) in THF (1 L), triphenylphosphine (55.4 g, 211 mmol), nonafluoro-tert-butyl alcohol (30 mL), and azodicarboxylic acid di-tert-butyl (38.5 g, 167 mmol) were added. When TLC showed complete conversion, the mixture was stirred for 30 minutes. Heptane (500 mL) was added, and the mixture was partially concentrated to approximately 500 mL. Further heptane (1 L) was added, and the mixture was stirred overnight at room temperature. The precipitate was filtered off, and the filtrate was concentrated to provide compound 5 as a yellow syrup.

[0373] 2-(((13S,17S)-17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)ethane-1-ol(6)

[0374] To an ice-cold suspension of lithium aluminum hydride (2.0 g, 53 mmol) in THF (300 mL), a solution of methyl ester 5 (17 g, 27 mmol) in THF (100 mL) was slowly added, and the resulting mixture was stirred at room temperature for 2 hours. KOH (8.5 mL, 20% aqueous solution 160 mL / mol LiAlH4) was slowly added at 0°C, and the resulting mixture was stirred at room temperature for 30 minutes. The mixture was filtered through Celite, dried over Na2SO4, and concentrated to provide a clear oil in which alcohol 6 (15 g, 94%) slowly crystallized.

[0375] (13S,17S)-3-(2-bromoethoxy)-17-(3-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propan-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene(7)

[0376] To a solution of compound 6 (15 g, 25 mmol) in DCM (300 mL), triphenylphosphine (9.8 g, 38 mmol) and NBS (5.3 g, 30 mmol) were added, and the resulting mixture was stirred at room temperature for 16 hours. The mixture was washed with NaHCO3, dried over Na2SO4, and concentrated. Heptane (500 mL) was added to the dark green residue, and the resulting mixture was stirred for 30 minutes. The solid was removed by filtration, and the filtrate was concentrated. The mixture was absorbed into heptane (250 mL) and stirred for 1 hour. The mixture was filtered, and the filtrate was concentrated to provide bromide 7 (15.4 g, 94.1%) as a clear, slightly yellow oil. To remove residual PPh3, the material was dissolved in Et2O (300 mL) and washed with aqueous KMnO4 solution, water, and brine. The organic layer was dried over Na2SO4 and concentrated. P(O)Ph3 was then removed by column chromatography (5% butyl in heptane) to provide the product as a clear oil.

[0377] S-(2-(((13S,17S)-17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)ethyl)4-methylbenzenesulfonothioate(8)

[0378] To a solution of bromide 7 (2.9 g, 4.4 mmol) in DMF (150 mL), potassium 4-methylbenzene sulfonothioate (2.0 g, 8.8 mmol) was added, and the resulting mixture was stirred at 50°C for 16 hours and at room temperature for 128 hours. Water (500 mL) was added, and the mixture was extracted with SiO2 / heptane (1 / 1, 2 × 250 mL). The combined organic layer was washed with brine, dried over Na2SO4, and concentrated to provide thiotosylate 8 (2.2 g, 65%).

[0379] Synthesis of thioacetate 14:

[0380] (4-hydroxybutyl)carbamate tert-butyl(9)

[0381] A solution of 4-aminobutan-1-ol (25 g, 0.28 mol) in DCM (50 mL) was slowly added to a solution of di-tert-butyl dicarbonate (61 g, 0.28 mol) in DCM (150 mL). The resulting mixture was stirred for 90 minutes, after which no further gas generation was observed. The mixture was diluted with DCM (200 mL), washed with 1 M HCl (400 mL), dried over Na2SO4, and concentrated to provide tert-butyl (4-hydroxybutyl)carbamate (43 g, 230 mmol, 82%) as a clear oil.

[0382] (4-bromobutyl)carbamate tert-butyl(10)

[0383] (4-hydroxybutyl)carbamate tert-butyl (7, 43 g, 230 mmol) was dissolved in DCM (500 mL), and triphenylphosphine (90 g, 340 mmol) and NBS (45 g, 250 mmol) were slowly added while cooling with water. The resulting mixture was stirred at room temperature for 90 minutes. NMR showed complete conversion. The mixture was washed with saturated aqueous sodium bicarbonate (300 mL), dried over Na2SO4, and concentrated to one-third of the volume. Heptane (500 mL) was added, and the remaining DCM was removed under vacuum. Additional heptane (200 mL) was added, and the mixture was stirred at room temperature overnight. The mixture was filtered and concentrated to provide crude (4-bromobutyl)carbamate tert-butyl (10, 75 g, 74%) as a yellow oil. NMR showed 43% PPh3 and 57% correct product.

[0384] 2-(4-((tert-butoxycarbonyl)amino)butyl)diethyl malonate (11)

[0385] To an ice-cold suspension of sodium hydride (6.8 g, 0.17 mol) in DMF (500 mL), diethyl malonate (27 g, 26 mL, 0.17 mol) was slowly added, and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was cooled again to 0°C, and tert-butyl (4-bromobutyl)carbamate (10 g, 75 g, 0.17 mol) was added. The reaction mixture was allowed to reach room temperature and stirred for 18 hours. The reaction mixture was quenched with 1N HCl (300 mL) and water (1.3 L). The mixture was extracted with Hept / HCl (1 / 1) (2 x 250 mL). The combined organic layer was washed with brine (500 mL), dried over sodium sulfate, filtered, and concentrated. The crude material was purified by column chromatography (20% HCl / heptane) to provide compound 11 (48 g, 85%) as a clear oil. NMR showed that a small amount of diethyl malonate was still present. The materials were used directly in the reduction process.

[0386] (6-hydroxy-5-(hydroxymethyl)hexyl)carbamate tert-butyl(12)

[0387] To an ice-cold solution of LiAlH4 (25 g, 0.66 mol) in THF (750 mL), 2-(4-((tert-butoxycarbonyl)amino)butyl)diethyl malonate (11, 48 g, 0.14 mol) in THF (100 mL) was added dropwise. The reaction mixture was stirred at 0°C for 2.5 hours. The reaction was quenched by slowly adding 20% ​​KOH (106 mL) at 0°C. The resulting mixture was stirred at room temperature for 1 hour, and then filtered through Celite. The filtrate was dried over Na2SO4 and concentrated to provide (6-hydroxy-5-(hydroxymethyl)hexyl)carbamate tert-butyl (12, 22.3 g, 62%) as a clear oil.

[0388] (4-(2,2-dimethyl-1,3-dioxan-5-yl)butyl)carbamate tert-butyl(13)

[0389] To a solution of tert-butyl (6-hydroxy-5-(hydroxymethyl)hexyl)carbamate (12, 22.3 g, 90.2 mmol) in THF (300 mL), 2,2-dimethoxypropane (28.2 g, 34 mL, 270 mmol) and 4-methylbenzenesulfonic acid hydrate (3.43 g, 18.0 mmol) were added. The reaction mixture was stirred at room temperature for 30 minutes, after which NMR showed complete conversion. The mixture was diluted with RINKAN (300 mL) and washed with a saturated solution of sodium bicarbonate (300 mL). The aqueous layer was extracted again with RINKAN. The combined organic layer was dried over Na2SO4, filtered, and concentrated to provide tert-butyl carbamate (4-(2,2-dimethyl-1,3-dioxan-5-yl)butyl) (13, 21.6 g, 83.4%) as a clear oil. 4-(2,2-dimethyl-1,3-dioxan-5-yl)-N-methylbutan-1-amine(14)

[0390] A suspension of LiAlH4 (4.28 g, 113 mmol) in THF (300 mL) was slowly added to a solution of (4-(2,2-dimethyl-1,3-dioxan-5-yl)butyl)carbamate tert-butyl (13 g, 21.6 g, 75.2 mmol) in THF (100 mL). The resulting mixture was refluxed at 70°C for 16 hours. The mixture was cooled to 0°C, and the reaction was quenched by slowly adding KOH (20% aqueous solution, 18 mL). The resulting mixture was stirred at room temperature for 1 hour. The mixture was filtered through Celite, and the filtrate was dried over Na2SO4 before being concentrated under vacuum to obtain compound 14 (13 g, 86%) as a slightly yellowish clear oil.

[0391] S-(3-oxopropyl)ethanethioate (15)

[0392] To a solution of acrylaldehyde (15 g, 18 mL, 0.27 mol) in DCM (200 mL), triethylamine (6.8 g, 9.3 mL, 67 mmol) and thioacetic acid (20 g, 19 mL, 0.27 mol) were slowly added. The resulting mixture was stirred at room temperature for 18 hours. The mixture was concentrated, and the crude material was purified by column chromatography (15% HCl / heptane) to provide S-(3-oxopropyl)ethanethioate (15, 26.3 g, 74%) as a dark yellow / orange oil.

[0393] S-(3-((4-(2,2-dimethyl-1,3-dioxan-5-yl)butyl)(methyl)amino)propyl)ethanethioate(16)

[0394] A solution of 4-(2,2-dimethyl-1,3-dioxan-5-yl)-N-methylbutan-1-amine (14, 9.2 g, 46 mmol) in 1,2-dichloroethane (400 mL) and acetic acid (11 g, 10 mL, 0.18 mol) was mixed with S-(3-oxopropyl)ethanethioate (15, 7.2 g, 55 mmol), and the resulting mixture was stirred for 5 minutes. Then, sodium triacetoxyborohydride (39 g, 180 mmol) was added, and the resulting mixture was stirred at room temperature for 90 minutes. The reaction was quenched by adding aqueous saturated sodium bicarbonate (100 mL), and the mixture was stirred for 15 minutes. The mixture was diluted with aqueous saturated sodium bicarbonate (300 mL), and the phases were separated. The aqueous phase was extracted with dichloromethane, and the combined organic phase was washed with brine, dried over Na2SO4, and concentrated. The crude material was purified using column chromatography (40-50% SiO + 1% NEt3 in heptane) to provide S-(3-((4-(2,2-dimethyl-1,3-dioxan-5-yl)butyl)(methyl)amino)propyl)ethanethioate (16, 11.5 g, 79%) as an orange oil.

[0395] Synthesis of the precursor of formula (XIIa):

[0396] 4-(2,2-dimethyl-1,3-dioxan-5-yl)-N-(3-((2-(((13S,17S)-17-(3-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)ethyl)disulfaneyl)propyl)-N-methylbutan-1-amine(17)

[0397] To a solution of thiotosylate 8 (3.3 g, 4.3 mmol) and thioacetate 16 (2.3 g, 7.4 mmol) in DCM (200 mL) and MeOH (20 mL), sodium methoxide (0.47 g, 1.6 mL, 8.7 mmol) in MeOH was added. The resulting mixture was stirred for 2 hours. NMR showed complete consumption of the thiotosylate. The mixture was diluted with DCM (200 mL), washed with NaHCO3, dried over Na2SO4, and concentrated. The crude material was purified by column chromatography (10% acetone + 1% NET3 in heptane) to provide disulfide 17 (0.85 g, 22%) as a clear oil.

[0398] 2-(4-((3-((2-(((13S,17S)-17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-Cyclopenta[a]phenanthrene-3-yl)oxy)ethyl)disulfane)propyl)(methyl)amino)butyl)propane-1,3-diol

[0399] To a solution of acetonide 17 (2.1 g, 2.3 mmol) in MeOH (100 mL), tosylic acid (530 mg, 2.8 mmol) was added, and the resulting mixture was stirred at room temperature for 1 hour, after which TLC showed complete conversion. Volatile substances were removed under vacuum. The crude material was dissolved in DCM (100 mL), washed with NaHCO3, dried over Na2SO4, and concentrated to provide the product (1.7 g, 2.0 mmol, 87%) as a white solid.

[0400] 2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-((3-((2-(((13S,17S)-17-(3-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propan-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)ethyl)disulfaneyl)propyl)(methyl)amino)hexane-1-ol(18)

[0401] To a solution of diol (1.7 g, 2.0 mmol), triethylamine (0.25 g, 0.34 mL, 2.4 mmol), and DMAP (25 mg, 0.20 mmol) in DCM (100 mL), 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (0.68 g, 2.0 mmol) was added, and the resulting yellow mixture was stirred at room temperature for 16 hours. The mixture was washed with NaHCO3 and brine, dried over Na2SO4, and concentrated. The crude material was purified by column chromatography (25% acetone / heptane + 1% NET3) to provide product 18 (1.3 g, 1.1 mmol, 56%) as a clear oil.

[0402] 2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-((3-((2-(((8R,9S,13S,14S,17S)-17-(3-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propan-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-l)oxy)ethyl)disulfaneyl)propyl)(methyl)amino)hexyl(2-cyanoethyl)diisopropylphosphorumidite(Apo-Si-K-113)

[0403] To a solution of compound 18 (1.3 g, 1.1 mmol) in DCM (100 mL), solutions of N-methylmorpholine (0.15 g, 3.0 mL, 1.5 mmol) and TFA (84 mg, 3.0 mL, 0.74 mmol) in DCM (0.5 M NMM, 0.25 M TFA) were added. Then, 3-((bis(diisopropylamino)phosphanail)oxy)propanenitrile (0.45 g, 0.47 mL, 1.5 mmol) was added, and the resulting mixture was stirred at room temperature for 2 hours, with the reaction monitored by TLC. After complete conversion, the mixture was washed with NaHCO3, dried over Na2SO4, and concentrated. The crude material was purified by column chromatography (15% acetone + 1% NET3 in heptane) to provide Apo-Si-K-113 (1.5 g, 1.1 mmol, 98%) as a clear oil.

[0404] Example 2i: Method for synthesizing the precursor of formula (XIIb):

[0405] [ka]

[0406] The synthesis of the precursor of formula (XIIb) is carried out according to the following synthesis scheme.

[0407] [ka]

[0408] [ka]

[0409] Example 2j: Synthesis method of the precursor of formula (XIIIa):

[0410] [ka]

[0411] The synthesis of the precursor of formula (XIIIa) is carried out according to the following synthesis scheme.

[0412] [ka]

[0413] [ka]

[0414] Example 2k: Synthesis method of the precursor of formula (XIIIb): [ka]

[0415] The main feature of the (XIIIb)-precursor is the ether group between the disulfide and the steroid moiety. On the other side of the disulfide, there is another ether and a tertiary amine.

[0416] Synthesis of thiotosylate 10: [ka]

[0417] The synthesis began with the protection of the ketone with 1,3-propanediol, yielding compound 2 in good purity. Ring-opening of the acetal with LiAlH4 and AlCl3 gave a mixture of compound 3 and estradiol in a ratio of approximately 85:15, the latter being less reactive and removed in the next step. Allyl bromide was used as a reactive electrophile. Selective alkylation of the phenol, using Mitsunobu conditions, allowed for the subsequent bonding of perfluorinated tert-butanol to an aliphatic alcohol, yielding compound 5. Further functionalization of the allyl by 9-BBN treatment and subsequent reduction yielded alcohol 6. This alcohol could be extended with ethyl diazoethyl to yield compound 7. Reduction to an alcohol, conversion to the corresponding iodide, and subsequent thiotosylation yielded compound 10.

[0418] Synthesis of thioacetate 18:

[0419] [ka]

[0420] The first step in the synthesis of building block 18 was to protect the amine of aminobutanol to provide alcohol 11. The alcohol was then converted to bromide 12 by treatment with NBS and PPh3. The bromide was then alkylated to diethyl malonate to provide diester 13. Treatment with LiAlH4 at room temperature reduced the ester but not the Boc group, thus yielding diol 14. The diol was then protected as acetonide (15). The Boc group was reduced by treatment with LiAlH4 at high temperature to provide amine 16. Alkylation of the amine with a suitable chloride provided compound 17. Using Mitsunobu conditions, the hydroxyl moiety was converted to thioacetate to provide building block 18.

[0421] Synthesis of the precursor of formula (XIIIb):

[0422] [ka]

[0423] A mixture of thiotosylate 10 and thioacetate 18 was treated with NaOMe to provide disulfide 19. The acetonide group was removed by treatment with acid to provide diol 20. The DMT group was bonded to one of the alcohols to yield compound 21. Finally, phosphoramidite formation provided the precursor of formula (XIIIb).

[0424] Experiment section:

[0425] Synthesis of thiotosylate 10:

[0426] (8R,9S,13S,14S)-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydrospiro[cyclopenta[a]phenanthrene-17,2'-[1,3]dioxane]-3-ol(2)

[0427] To a suspension of estrone (252 g, 0.93 mol) in toluene (1.5 L), trimethoxymethane (297 g, 350 mL, 2.80 mol), propane-1,3-diol (213 g, 250 mL, 2.80 mol), and pTsOH (2 g, 10 mmol) were added. The mixture was heated to 60°C and stirred for 16 hours. Triethylamine (6 mL) and water (600 mL) were added, and stirring was continued for a further 1 hour. The phases were separated, and the organic layer was washed with water (3 × 400 mL) and brine. The mixture was dried over Na₂SO₄ and partially concentrated to approximately 1 L. The mixture was poured into heptane (4 L), the white solid was filtered off, washed with heptane, and dried under vacuum. Compound 2 (271 g, 825 mmol) was isolated as a white solid in 88.5% yield.

[0428] (8R,9S,13S,14S,17S)-17-(3-hydroxypropoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-ol(3)

[0429] To a solution of (13S)-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydrospiro[cyclopenta[a]phenanthrene-17,2'-[1,3]dioxane]-3-ol (2, 60.7 g, 185 mmol) in THF at 0°C, lithium aluminum hydride (8.42 g, 222 mmol) was carefully added, followed by aluminum chloride (98.6 g, 739 mmol) (highly exothermic!) in small amounts. The mixture was stirred at 0°C for 15 minutes, then warmed to 50°C. (Due to clogging of the rotary evaporator) the mixture was stirred at 50°C for 2 hours, then cooled to 0°C, and quenching was started with dropwise addition of NH4Cl (aqueous) (500 mL). The mixture was stirred at room temperature for 1 hour. The phases were separated, the organic layer was washed with brine, and concentrated. A white solid (65 grams) contaminated with estradiol (approximately 15%) was obtained.

[0430] 3-(((8R,9S,13S,14S,17S)-3-(allyloxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-17-yl)oxy)propane-1-ol(4)

[0431] A suspension of compound 3 (10.1 g, 30.6 mmol) and potassium carbonate (8.45 g, 61.1 mmol) in MeOH / acetone was treated with allyl bromide (7.39 g, 5.28 mL, 61.1 mmol). The mixture was heated under reflux for 4 hours, after which complete conversion was observed. The mixture was cooled to room temperature, filtered, and concentrated. Dichloromethane (250 mL) was added, the mixture was washed with brine, dried over Na2SO4, and concentrated. The crude material (12.8 grams, 34.5 mmol) was used directly in follow-up chemistry.

[0432] (8R,9S,13S,14S,17S)-3-(allyloxy)-17-(3-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propan-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene(5)

[0433] To a solution of compound 4 (12.78 g, 34.49 mmol) and triphenylphosphine (13.57 g, 51.74 mmol) in THF (300 mL), 1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propan-2-ol (12.21 g, 7.23 mL, 51.74 mmol) and (E)-diazene-1,2-dicarboxylate di-tert-butyl (10.32 g, 44.84 mmol) were added. The mixture was stirred at room temperature for 16 hours and then concentrated.

[0434] After purification using flash chromatography (a gradient of 5% to 10% toluene in heptane), compound 5 (16.7 g, 28.4 mmol) was isolated as a yellowish oil in 82% yield.

[0435] 3-(((8R,9S,13S,14S,17S)-17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propan-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)propan-1-ol(6)

[0436] 9-BBN (5.54 g, 90 mL, 45.4 mmol) was added dropwise to a solution of crude compound 5 (16.7 g, 28.4 mmol) in THF (250 mL) at 0°C. After complete addition, the mixture was stirred overnight at room temperature. The solution was cooled to 0°C, and sodium hydroxide (20 g, 16 mL, 148 mmol) and hydrogen peroxide (14 g, 13 mL, 148 mmol) were slowly added dropwise simultaneously. The resulting heterogeneous mixture was vigorously stirred at room temperature for about 1 hour. The reaction mixture was decanted and partitioned between HCl (700 mL) and brine (100 mL). The organic phase was washed with an additional 100 mL of brine, dried over Na₂SO₄, and concentrated under vacuum. Further purification of the concentrate by flash chromatography (silica gel, gradient of 25%–35% HCl in heptane) yielded compound 6 (13.6 g, 22.4 mmol) in 79.0% yield.

[0437] 2-(3-(((8R,9S,13S,14S,17S)17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)propoxy)ethyl acetate(7)

[0438] Compound 6 (13.6 g, 22.4 mmol) and 2-ethyl diazoethyl acetate (3.0 g, 2.8 mL, 22.4 mmol) were dissolved in DCM (250 mL), and BF3.OEt2 (31.8 mg, 28.4 μL, 224 μmol) was added at 0°C. The reaction was stirred at 0°C for 15 minutes and at room temperature for 3 hours until no gas generation was observed. Based on TLC, no complete conversion was observed. Further 2-ethyl diazoethyl acetate (5 mL) and BF3.OEt2 were added, and stirring was continued for 2 hours. The mixture was diluted with DCM (200 mL), triethylamine (1 mL) was added, and the mixture was washed with water (100 mL) and brine (50 mL) and dried over Na2SO4. After removing the solvent under vacuum, further purification using flash chromatography (a gradient of 5%–10% ethyl acetate in heptane) yielded compound 7 (5.5 g, 7.9 mmol) as a clear, yellowish oil in 35% yield.

[0439] 2-((3-(((8R,9S,13S,14S,17S))-17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)propoxy)ethane-1-ol (8) A solution of compound 7 (5.5 g, 7.9 mmol) was added dropwise to a suspension of LiAlH4 (0.36 g, 9.5 mmol) in THF (100 mL) at 0°C. The mixture was stirred at room temperature for 2 hours and then quenched by adding 20% ​​KOH in water (1.7 mL). The suspension was stirred for a further 1 hour and then filtered through a short channel of Celite. The compound was concentrated. Compound 8 (4.81 g, 7.4 mmol) was isolated in 93% yield and used directly for follow-up chemistry.

[0440] (8R,9S,13S,14S,17S)-3-(3-(2-bromoethoxy)propoxy)-17-(3-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propan-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene(9)

[0441] To a solution of compound 8 (4.81 g, 7.39 mmol), imidazole (604 mg, 8.87 mmol), and triphenylphosphine (2.33 g, 8.87 mmol) in dichloromethane (250 mL), iodine (2.06 g, 8.13 mmol) was added at 0°C. The mixture was stirred for 16 hours, then aqueous saturated sodium thiosulfate was added to separate the phases. The organic layer was washed with brine, dried over sodium sulfate, and concentrated. The residue was dissolved in heptane, allowed to stand for precipitation, and the solid was filtered off. The mixture was concentrated.

[0442] Further purification using flash chromatography (5% alkyl hydroxide in heptane) yielded compound 9 (3.90 g, 5.13 mmol) as a clear oil in 69% yield.

[0443] S-(2-(3-(((8R,9S,13S,14S,17S)17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)propoxy)ethyl)4-methylbenzene sulfonothioate (10)

[0444] Compound 9 (3.90 g, 5.13 mmol) was dissolved in DMF, and potassium 4-methylbenzene sulfonothioate (1.72 g, 7.6 mmol) was added. The suspension was stirred at room temperature for 16 hours. TLC showed only partial conversion. The mixture was heated to 50°C for 4 hours and then cooled to room temperature. HCl (50 mL), followed by heptane (100 mL), was added, and the mixture was washed three times with water, then once with brine, dried over sodium sulfate, and concentrated. Further purification was performed using flash chromatography (5%–15% HCl in heptane). Compound 10 (3.45 g, 4.2 mmol) was isolated as clean oil in 83% yield.

[0445] Synthesis of thioacetate 18:

[0446] (4-hydroxybutyl)carbamate tert-butyl(11) A solution of 4-aminobutan-1-ol (25 g, 0.28 mol) in DCM (50 mL) was slowly added to a solution of di-tert-butyl dicarbonate (61 g, 0.28 mol) in DCM (150 mL). The resulting mixture was stirred for 90 minutes, after which no further gas generation was observed. The mixture was diluted with DCM (200 mL), washed with 1 M HCl (400 mL), dried over Na2SO4, and concentrated to provide tert-butyl (4-hydroxybutyl)carbamate (11, 43 g, 82%) as a clear oil.

[0447] (4-bromobutyl)carbamate tert-butyl(12)

[0448] To a solution of tert-butyl (4-hydroxybutyl)carbamate (11, 23 g, 120 mmol) in DCM (500 mL), triphenylphosphine (44 g, 170 mmol) and NBS (25 g, 143 mmol) were slowly added. The resulting mixture was stirred at room temperature for 2 hours. The mixture was washed with NaHCO3, dried over Na2SO4, and concentrated. Heptane (500 mL) was added, and the mixture was stirred at room temperature for 1 hour. The formed solid was removed by filtration, and the filtrate was concentrated to provide tert-butyl (4-bromobutyl)carbamate (12, 32.8 g, 130 mmol, quantitatively) as a clear oil.

[0449] 2-(4-((tert-butoxycarbonyl)amino)butyl) diethyl malonate (13)

[0450] To an ice-cold suspension of sodium hydride (6.8 g, 0.17 mol) in DMF (500 mL), diethyl malonate (27 g, 26 mL, 0.17 mol) was slowly added, and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was cooled again to 0°C, and tert-butyl (4-bromobutyl)carbamate (12 g, 75 g, 0.17 mol) was added. The reaction mixture was allowed to reach room temperature and stirred for 18 hours. The reaction mixture was quenched with 1N HCl (300 mL) and water (1.3 L). The mixture was extracted with heptane / HCl (1 / 1, 2 x 250 mL). The combined organic layer was washed with brine (500 mL), dried over sodium sulfate, filtered, and concentrated. The crude material was purified by column chromatography (20% HCl / heptane) to provide diester 13 (48 g, 0.14 mol, 85%) as a clear oil.

[0451] (6-hydroxy-5-(hydroxymethyl)hexyl)carbamate tert-butyl(14)

[0452] Diester 13 (19 g, 57 mmol) in THF (100 mL) was added dropwise to an ice-cold solution of LiAlH4 (8.7 g, 0.23 mol) in THF (750 mL). The reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by slowly adding 20% ​​KOH (37 mL aqueous solution) at 0°C. The resulting mixture was stirred at room temperature for 1 hour, and then filtered through Celite. The filtrate was dried over Na2SO4 and concentrated to provide diol 14 (12 g, 50 mmol, 86%) as a clear oil.

[0453] (4-(2,2-dimethyl-1,3-dioxan-5-yl)butyl)carbamate tert-butyl(15)

[0454] To a solution of diol 14 (22 g, 90 mmol) in THF (300 mL), 2,2-dimethoxypropane (28 g, 34 mL, 270 mmol) and 4-methylbenzenesulfonic acid hydrate (3.4 g, 18 mmol) were added. The reaction mixture was stirred at room temperature for 30 minutes. The mixture was diluted with HCl (300 mL) and washed with a saturated solution of sodium bicarbonate (300 mL). The aqueous layer was extracted again with HCl. The combined organic layer was dried over Na2SO4, filtered, and concentrated to provide acetonide 15 (21.6 g, 83.4%) as a clear oil.

[0455] 4-(2,2-dimethyl-1,3-dioxan-5-yl)-N-methylbutan-1-amine(16)

[0456] To a suspension of LiAlH4 (2.2 g, 58 mmol) in THF (300 mL), acetonide 15 (11 g, 39 mmol) was added, and the resulting mixture was stirred at 70°C for 16 hours. The mixture was allowed to cool to room temperature. The mixture was then cooled to 0°C, and the reaction was quenched by slowly adding KOH (20%, 9.3 mL) at 0°C. The resulting mixture was stirred at room temperature for 1 hour. The mixture was filtered through Celite, dried over Na2SO4, and concentrated to provide methylamine 16 (7.8 g, 39 mmol, quantitatively) as a clear oil.

[0457] 2-(2-((4-(2,2-dimethyl-1,3-dioxan-5-yl)butyl)(methyl)amino)ethoxy)ethane-1-ol(17)

[0458] To a solution of amine 16 (4.1 g, 20 mmol) in MeCN (150 mL), 2-(2-chloroethoxy)ethane-1-ol (2.5 g, 2.1 mL, 20 mmol), potassium iodide (0.34 g, 2.0 mmol), and potassium carbonate (5.6 g, 41 mmol) were added, and the resulting mixture was stirred at 80°C for 40 hours. The mixture was diluted with water (100 mL) and NaHCO3 (100 mL) and extracted with DCM (2 × 200 mL). The combined organic layer was washed with brine, dried over Na2SO4, and concentrated. The crude material was purified by column chromatography (3% 7M NH3 in MeOH: 97% DCM) to provide alcohol 17 (2.9 g, 10 mmol, 49%) as a yellowish oil.

[0459] S-(2-(2-((4-(2,2-dimethyl-1,3-dioxan-5-yl)butyl)(methyl)amino)ethoxy)ethyl)ethanethioate(18)

[0460] To a solution of alcohol 17 (2.9 g, 10 mmol) in THF (100 mL), triphenylphosphine (4.2 g, 16 mmol) and (E)-diazene-1,2-dicarboxylate di-tert-butyl (3.0 g, 13 mmol) were added, and the resulting mixture was stirred for 5 minutes. Then, thioacetic acid (0.99 g, 0.94 mL, 13 mmol) was added, and most of the mixture was decolorized. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated, and the crude material was purified by column chromatography (20% acetone + 1% NEt3 in heptane) to provide thioacetate 18 (3.5 g, 10 mmol, quantitative) as a yellow oil.

[0461] Formula (XIIIb) - Integration of precursors:

[0462] 4-(2,2-dimethyl-1,3-dioxan-5-yl)-N-(2-(2-((2-((3-(((13S,17S)-17-(3-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)propoxy)ethyl)disulfane)ethoxy)ethyl)N-methylbutan-1-amine(19)

[0463] To a solution of thiotosylate 10 (1.1 g, 1.3 mmol) and thioacetate 18 (0.70 g, 2.0 mmol) in DCM (100 mL) and MeOH (10 mL), a solution of sodium methoxide (5.4 M, 0.50 mL, 2.7 mmol) was added. The resulting mixture was stirred for 1 hour. The mixture was diluted with DCM (100 mL), washed with NaHCO3 and brine, dried over Na2SO4, and concentrated. The crude material was purified by column chromatography (10-20% acetone / heptane + 1% NET3) to provide disulfide 19 (0.58 g, 0.6 mmol, 45%) as a clear oil.

[0464] 2-(1-(((13S,17S)-17-(3-((1,1,1,3,3,3-Hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-Decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)-14-methyl-4,11-dioxa-7,8-dithia-14-azaoctadecane-18-yl)propane-1,3-diol(20)

[0465] To a solution of disulfide 20 (0.58 g, 0.60 mmol) in MeOH (50 mL) (with additional DCM added for solubility), p-toluenesulfonic acid (0.14 g, 0.72 mmol) was added, and the resulting mixture was stirred at room temperature for 1 hour. The mixture was concentrated, and the residue was dissolved in DCM (100 mL). The mixture was washed with NaHCO3 and brine, dried over Na2SO4, and concentrated to provide diol 20 (0.53 g, 0.57 mmol, 95%) as a clear oil.

[0466] 19-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(((13S,17S)-17-(3-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)-14-methyl-4,11-dioxa-7,8-dithia-14-azaicosan-20-ol(21)

[0467] To a solution of diol 20 (1.0 g, 1.1 mmol) in DCM (50 mL), triethylamine (0.22 g, 0.30 mL, 2.2 mmol), DMAP (13 mg, 0.11 mmol), and finally 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (0.36 g, 1.1 mmol) were added, and the resulting mixture was stirred at room temperature for 16 hours. The mixture was washed with NaHCO3 and brine, dried over Na2SO4, and concentrated. The crude material was purified by column chromatography (using silica pretreated with NEt3, 20-25% acetone + 1% NEt3 in heptane) to provide compound 21 (0.96 g, 0.78 mmol, 72%) as a clear yellowish oil.

[0468] 19-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(((13S,17S)-17-(3-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propane-2-yl)oxy)propoxy)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3-yl)oxy)-14-methyl-4,11-dioxa-7,8-dithia-14-azaicosan-20-yl(2-cyanoethyl)diisopropylphosphoramidite[formula (XIIIb)-precursor]

[0469] To a solution of alcohol 21 (0.96 g, 0.78 mmol) in DCM (50 mL), 3-((bis(diisopropylamino)phosphanail)oxy)propanenitrile (0.31 g, 0.32 mL, 1.0 mmol) and a solution of NMM and TFA (2.0 mL, 0.5 M NMM and 0.25 M TFA, 1.3 eq. NMM) in DCM were added. The resulting mixture was stirred at room temperature for 2 hours, after which TLC showed partial conversion. Additional phosphanail (0.5 eq.) and NMM / TFA solution (0.5 eq. NMM) were added, and the mixture was stirred for 1 hour. TLC showed complete conversion. The mixture was washed with NaHCO3 and brine, dried over Na2SO4, and concentrated. The crude material was purified using column chromatography (using silica pretreated with NEt3, 20% acetone + 1% NEt3 in heptane) to provide the formula (XIIIb)-precursor (0.96 g, 0.67 mmol, 86%) as a clear oil.

[0470] Example 2L: Synthesis of the precursor of formula (XIV-F):

[0471] [ka]

[0472] Starting from compound 3, the phenol can be alkylated using bromide K-105-F-1 (see below). The perfluoro moiety can then be introduced using Mitsunobu conditions (K-150-F-3). The amide and ester are simultaneously reduced, and the resulting tertiary amine can then be protected with a Boc group. The final introduction of the thioacetate group using Mitsunobu conditions provides building block K-150-F-6.

[0473] [ka]

[0474] The shortest route to the desired building block K-150-F is ring-opening of 1,5-dioxepan-2-one at beta-alanine. Ring-opening at beta-alanine provides amide K-150-F-7. Esterification at EtOH protects the carboxylic acid. Subsequent conversion of the alcohol to bromide m provides building block K-105-F-1, which is then alkylated to phenol.

[0475] [ka]

[0476] Integration of the precursor of formula (XIV-F)

[0477] The assembly begins with disulfide formation between building block K-150-F-6 and thiotosylate 6. The acetonide moiety and Boc group will then be simultaneously removed by treatment with acid. The free tertiary amine can then be protected with the Fmoc group. Subsequent DMT bonding and phosphoramidite formation will provide the (XIV-F) precursor.

[0478] Example 2m: Synthesis of the precursor of formula (XV-F):

[0479] [ka]

[0480] The (XV-F) precursor is prepared from thiotosylate 13 (Scheme 22) and building block 7 (Scheme 23).

[0481] [ka]

[0482] The second building block, compound 7, is synthesized as shown in scheme 23. [ka]

[0483] Integration and completion of the synthesis of the precursor of formula (XV-F):

[0484] [ka]

[0485] Example 2n: Method for synthesizing the precursor of formula (VIII-F):

[0486] [ka]

[0487] The synthesis begins with the synthesis of thiotosylate 16, the main building block, according to Scheme 25 below.

[0488] [ka]

[0489] The synthesis proceeds according to the following synthesis scheme 26, which involves the synthesis of thioacetate K-93-F-4.

[0490] [ka]

[0491] Compound F-2 was Teoc-protected to provide compound F-3 in 95% yield. The synthesis of thioacetate F-4 was carried out in approximately 60% yield. 1.76 grams were isolated. Having the main fragments thiotosylate 16 and thioacetate K-93-F-4, the synthesis then proceeded according to scheme 27 below.

[0492] [ka]

[0493] First, the fragments thiotosylate 16 and thioacetate K-93-F-4 were combined to form a disulfide bond, yielding the intermediate K-95-F-5. F-5 was treated with pTsOH in MeOH to liberate the diol, thus producing F-6 (93% yield, 1.3 g). F-6 was then monoprotected with DMT-Cl to provide compound F-8. A phosphoramidite was then attached, and the compound according to formula (VIII-F)-precursor (Teoc) was isolated in 1.004 g and stored in a vial for shipment. It is noteworthy that Teoc as a protecting group is relatively stable to acids, bases, and reducing and oxidizing agents, yet can be readily removed by the addition of fluoride, thus proving useful as an amine protecting group in oligonucleotide synthesis. In the synthesis of the E portion of the present invention, this protecting group can therefore play a role in the formation of tertiary amines introduced into compounds of formulas (VIII-H), (XIV-H), and (XV-H).

[0494] Example 2p: Method for synthesizing the precursor of formula (XVI-F):

[0495] [ka]

[0496] The synthesis is identical to that of the precursors of formula (XV-F), with two exceptions, as described in Example 2m: (i) The starting material for the L portion is pentafluorobenzoate (A) and not difluorobenzoate (B). (ii) The protecting group of the amine is TEOC, as introduced as described in Example 2n.

[0497] [ka]

[0498] Example 3: Potential mechanism of action (MOA) of the Dicer substrate conjugate of the present invention in transmembrane delivery:

[0499] The potential mechanism of action of the conjugate of the present invention, including OD, is described non-limitingly herein. The MOA comprises four steps.

[0500] 1. Interaction between conjugates and outer leaflets:

[0501] When the conjugate of the present invention is an siRNA or dsiRNA bound to two or three E portions, the conjugate is positioned so that the cylindrical RNA double strand is parallel to the membrane surface and the E, E', or E'' portions are oriented toward the membrane core perpendicular to the membrane surface, in proximity to the outer membrane lobe. This approach acts to fix the OD to the membrane surface. The resulting forced proximity of the highly negatively charged RNA to the outer membrane lobe leads to energetically unfavorable focal membrane strain, expansion of the surface area of ​​the outer phospholipid lobe, and disturbance of the hydrated shell of the phospholipid head group, all of which cause focal bending of the membrane.

[0502] 2. Relaxation of bending energy:

[0503] The relaxation of unfavorable membrane bending can be achieved through endocytosis, flip-flop, or both. Both processes support the initiation and / or propagation of transmembrane delivery of the conjugate of the present invention, including macromolecular drugs into the cytoplasm, either directly or through endosomal compartments.

[0504] 3. Separation of intracytoplasmic E, E', or E'' portions in redox-mediated processes involving the release of cargo drugs.

[0505] As described above, one or more E, E', or E'' portions are necessary for transmembrane transport of siRNA or dsiRNA conjugates. However, once the conjugate reaches the cytoplasm, it is desirable to remove these delivery portions and excrete them from the body, thereby freeing the cargo drug and allowing it to approach its cytoplasmic site of action. When the cargo drug is siRNA or dsiRNA, this cleavage allows for avoidance of steric hindrance in the interaction between siRNA or dsiRNA and gene silencing protein complexes (Dicer and RISC). In addition, such separation of the cargo drug from the E portion minimizes the burden of the conjugate on the cellular phospholipid membrane, which is advantageous from a safety standpoint. For this purpose, the E portion of the present invention includes a disulfide portion. Under oxidative conditions, which are prevalent in the extracellular environment, the disulfide exhibits high stability and therefore allows the conjugate of the present invention to be widely distributed throughout the body after systemic administration and to come into contact with the large cellular membrane pool.

[0506] In contrast, the cytoplasm, which is continuously generated primarily within the cytoplasm of any living cell, is a highly reductive environment due to its high concentration of reduced glutathione (GSH), which is approximately 3-4 orders of magnitude higher than that of the extracellular space (1-5 mM vs. 3-5 μM).

[0507] Due to these significant reducing conditions in the cytoplasm, the disulfide group of the E moiety undergoes robust reduction in the cytoplasmic environment. As a result, a cargo drug (e.g., dsiRNA) is released to exert its pharmacological effect at its target site in the cytoplasm (e.g., in Dicer and / or RISC protein complexes for gene silencing). After disulfide cleavage, the E moiety of the present invention is excreted from the body via bile and / or urine, either directly or after metabolism (e.g., cytochrome-P-450-mediated hydroxylation or glucuronization in the liver), similar to other sterol molecules (e.g., estrogen).

[0508] 4. Interaction between released OD and cytoplasmic sites for gene silencing

[0509] The above MOA of the conjugate of the present invention is illustrated without limitation in Figures 2A, 2B, 2C, 2D, and 2E. An example is a conjugate according to [Cn-1-(III)] having E and E', each having a structure according to formula (III). W is defined by formula (II 1) follows. The exemplary RNA duplex is a 25 / 27 nucleotide long Dicer substrate with a phosphate group attached to the 5' end of each strand. After transmembrane delivery, upon reaching the cytoplasm, due to the significantly reductive ambient conditions, cleavage and removal of the E, E', and E'' portions occur, leaving short fragments for each E portion, still bound to the RNA duplex containing a thiol group attached to a short hydrocarbon chain (Figure 2B). The RNA duplex then interacts with Dicer endonuclease. This interaction is initiated by the binding of the 3' end of the guide (antisense) strand duplex, which has a 2-nucleotide overhang, to the hydrophobic pocket of the Dicer protein, and the interaction of the phosphate group of the passenger (sense) strand with the respective positively charged pockets on the protein surface. This fixation positions the RNA double strand on the protein surface, allowing the enzyme to perform precise double-strand cleavage of the RNA double strand, leaving a 21 / 21-nucleotide double helix with two E-stops remaining on the passenger (sense) strand (Figure 2C). Figure 2D shows the subsequent removal of the sense strand by the enzyme helicase (a cytoplasmic enzyme capable of separating RNA strands). This action removes the residual E-stops, thus releasing an intact antisense strand to enter the RNA-induced silencing complex (RISC) (Figure 2E), in order to induce desired gene silencing.

[0510] Example 4: Biological performance of the conjugate of the present invention in inducing gene silencing after intravenous administration in an in vivo mouse model:

[0511] the purpose:

[0512] Demonstration of the ability of the conjugate of the present invention, comprising dsiRNA bound to two E-parts of the present invention, to induce gene silencing after intravenous administration.

[0513] method:

[0514] (1) A dsi-RNA double strand conjugated to the E portion of the present invention:

[0515] A 25 / 27 nucleotide-long dsiRNA double-stranded design was used to silence the APOC3 gene. This RNA double-stranded design was ligated at the 5' end of each strand to the E portion of the present invention, according to formula (IX), which has the structure described below. The APOC3 gene encodes the APOC3 protein, a protein synthesized by the liver that plays a crucial role in triglyceride distribution and metabolism. APOC3 expression is primarily limited to the liver and kidneys.

[0516] The E moiety used in the experiment followed formula (IX). It was synthesized by Syncom BV (Netherlands) as a precursor molecule and has DMT and phosphoramidite as protecting groups. The Apo-Si-APOC3-dsiRNA conjugate was synthesized by Integrated DNA Technologies (IDT, Leuven, Belgium) as described in Example 1 above. It was a [Cn-1-(IX)]-APOC3-dsiRNA conjugate, that is, it contained two E moieties having the structure shown in formula (IX), each conjugated to a dsiRNA to silence the APOC3 gene. Thus, the conjugate had the following structure.

[0517] [ka]

[0518] The gene sequences used in this study were as follows: Targeted ApoC3-dsiRNA conjugate: Sense chain arrangement: / 5´-(IX) / mGmGrAmUrGmGrArCrArArUmCrAmCrUmUrCmArGrArUrCr CCT Antisense chain sequence: / 5´(IX) / rArGmGrGrArUrCrUmGrAmArGmUrGrArUrUrGrUrCrCrAmUrCmCmAmG m stands for methylation, and r stands for ribonucleotide.

[0519] Control #1: Naked ApoC3-dsiRNA conjugate (a double-stranded RNA sequence that is specific to silencing the ApoC3 gene but lacks the Apo-Si molecular nanomotor delivery system). Sense chain arrangement: mGmGrAmUrGmGrArCrA rArUmCrAmCrUmUmUrCmA rGrArU rCrCCT Antisense chain sequence: rArGmG rGrArU rCrUmG rAmArG mUrGrA rUrUrG rUrCrCrAmUrCmCmAmG

[0520] Control #2: [Cn-1-(IX)]-KRAS-dsiRNA conjugate (unrelated RNA sequence conjugated to an Apo-Si molecular nanomotor delivery system). Sense chain arrangement: / 5´-(IX) / mAmArGmGrUmGrUrArCrArGmUrUmArU mGrUmGrArArUrArCTT Antisense chain sequence: / 5´-(IX) / rArAmGrUrArUrUrCmArCmArUmArArCrUrGrUrArCrArCmCrUmU mGmU

[0521] Control #3: Vehicle, 5% glucose solution in water for injection.

[0522] (2) Formulation:

[0523] All test and control samples(s) were dissolved in RNase-free sterile water for injection containing 5% glucose. A fresh 5 mg / ml stock solution was prepared immediately before administration.

[0524] (3) Animal husbandry and housing:

[0525] BALB / c mice (male, 22-29g) were obtained from Envigo (Israel Ltd.). The mice were kept under a 12-hour day / night cycle, given free feed, and allowed to acclimate for at least 3 days before the experiment. Each experimental group consisted of 7 animals.

[0526] (4).Research design:

[0527] The study included the following four treatment groups: Vehicles: (i) 5% glucose in water for injection, (ii) "naked" dsiRNA of ApoC3 (without binding of the molecular nanomotor portion), (iii) [Cn-1-(IX)]-KRAS-dsiRNA conjugate (unrelated RNA sequence conjugated to an Apo-Si molecular nanomotor delivery system), and (iv) targeted [Cn-1-(IX)]-ApoC3 dsiRNA conjugate.

[0528] Each study group contained 5-7 mice. Mice received three consecutive once-daily doses of 50 mg / kg intravenously via the tail vein at a dose volume of 10 ml / kg. The protocol was adopted without modification from Wolfram, C, et al., (Nat Biotechnol. 25:1149-57, 2007). On day 4, 24 hours after the last dose, the mice were weighed, sacrificed using CO2 narcosis, and their livers were collected and immediately subjected to RNA extraction.

[0529] (5) RNA extraction and qRT-PCR: Liver samples of 10–100 mg were extracted and immersed in TRIzol reagent. The tissue was then homogenized using a bullet blender homogenizer (Next Advance) in the presence of stainless steel beads (0.9–2.0 mm). The mixture was briefly centrifuged to remove debris and bullets. Total RNA was extracted using a PureLink RNA mini-kit (Invitrogen) according to the manufacturer's instructions. RNA was quantified using an Infinite M200-Pro Multimode Reader (Tecan). RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (ABI), and ApoC3 mRNA expression was measured using the Taqman qRT-PCR procedure, normalized to beta-actin (Step-one-Plus, ABI).

[0530] (6) Data Analysis

[0531] Data analysis was performed using Microsoft Excel software. Statistical significance between groups was assessed using an unpaired Student's t-test (two-tailed), with significance defined as p<0.05. Data are presented as mean ± SD.

[0532] result:

[0533] As shown in Figure 3A, the [Cn-1-(IX)]-ApoC3 dsiRNA conjugate induced a significant knockdown of ApoC3 gene expression (40%). This reduction in gene expression was highly statistically significant compared to all other experimental groups.

[0534] As shown in Figure 3B, a statistically significant knockdown of ApoC3 gene expression was also observed in the kidneys (21%). Importantly, even in the kidneys, this difference was statistically significant compared to all three control groups.

[0535] Conclusion:

[0536] The present invention, a conjugate comprising two E-molets bound to a dsiRNA designed to silencing the expression of the ApoC3 gene, demonstrated significant activity upon in vivo systemic intravenous administration in inducing selective knockdown of the target gene expression in both the liver and kidneys. These results support the concept that the Apo-Si drug delivery system, having a systemic mode of activity and conjugated to dsiRNA after intravenous administration, enables systemic distribution of gene drugs as well as silencing of each multi-organ and specific gene.

[0537] Example 5: Biological performance of the conjugate of the present invention in in vitro gene silencing, where E and E' are each given by formula (IV):

[0538] Research purpose: Evaluation of the performance of the present invention in silencing EGFP gene expression in vitro, with each conjugate having the structure shown in formula (IV) and containing E and E' moieties, [Cn-1-(IV)]-EGFP-dsiRNA bound to an EGFP-specific dsiRNA.

[0539] method:

[0540] dsiRNA double strand:

[0541] The siRNA double-stranded was a Dicer substrate designed to silence the EGFP gene. The E portion of the structure shown in formula (IV) was bound to each RNA strand at its 5' end. Therefore, the conjugate used in this study was [Cn-1-(IV)]-EGFP-dsiRNA, having the following structure.

[0542] [ka]

[0543] The nucleotide sequence was as follows: Sense: 5'-phos / iApo-Si-K103A / mAmCrCmCrUmGrArArGrUrUmCrAmUrCmUrGmCrArCrCrArCmCGrUrCrA Antisense: 5'-phos / iApo-Si-K103A / rCrGmGrUrGrGrUrGmCrAmGrAmUrGrArArCrUrGmGrGmUmCmA

[0544] In vitro study: The heLa-GFP cell line was obtained from Cell Biolabs. Cells were grown in Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% FBS (Gibco), 100 U / ml penicillin, 100 mg / ml streptomycin (Biological Industries, Israel), and 10 μg / ml blastosidine. Cells were maintained in a 37°C incubator with 5% CO2 humidified air. One day before transfection, cells were seeded into 24-well black glass-bottom plates (40,000 cells / well). The following day, cells were exposed to conjugated [Cn-1-(IV)]-EGFP-dsiRNA in the presence of 10% serum. For serum-free transfection, the culture medium was aspirated, cells were washed with Hanks equilibrium salt solution (HBSS), the medium was replaced with serum-free Opti-MEM (Thermo Fisher Scientific) for 24 hours, and then serum was added for a further 48-hour incubation. Downregulation of protein expression was measured 72 hours after transfection. For this purpose, the culture medium was aspirated and cells were washed with HBSS. EGFP fluorescence intensity was quantified using an Infinite M200-Pro Multimode Reader (Tecan), excitation wavelength 488 nm, emission wavelength 535 nm. Untreated cells were used as a control. The experiment was performed in triple replication, and results are shown as mean ± SD. Differences between groups were evaluated by two-sided t-tests, and statistical significance was defined as p<0.05.

[0545] result:

[0546] The [Cn-1-(IV)]-EGFP-dsiRNA conjugate effectively induced EGFP expression knockdown as follows:

[0547] In the presence of serum, a 600 nM conjugate reduced EGFP expression to 73.7% ± 1.3% (mean ± SD) of the control. Under serum-free conditions, robust EGFP knockdown was dose-dependently induced by the conjugate, and when cells were treated with 10, 40, and 150 nM conjugates, respectively (p<0.001 in all intergroup comparisons), EGFP expression was reduced to 55.0% ± 2.6%, 29.9% ± 0.5%, and 7.4% ± 0.6% of the control.

[0548] Conclusion:

[0549] The conjugate [Cn-1-(IV)]-EGFP-dsiRNA of the present invention is a potent portion for the delivery of high molecular weight dsiRNA constructs across phospholipid membranes to cells and for inducing significant gene silencing in each.

[0550] Example 6: Biological performance and dose / response studies of the conjugates of the present invention, comprising E, E', and E'' according to formula (VIII-M), respectively, in in vitro gene silencing:

[0551] Research purpose:

[0552] Evaluation of the in vitro biological performance of the conjugate [Cn-2-(VIII-M)] of the present invention, i.e., dsiRNA having E, E', and E'' moieties, each conjugated to a 25 / 27 nucleotide Dicer substrate double strand, designed to silence the EGFP gene according to formula (VIII-M).

[0553] method:

[0554] The structure of the conjugate [Cn-2-(VIII-M)] is as follows:

[0555] [Chemical]

[0556] Cell culture:

[0557] The HeLa-GFP and 3T3 cell lines were obtained from Cell Biolabs. Cells of each cell type were grown in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% FBS (Gibco), 100 U / ml penicillin, 100 mg / ml streptomycin (Biological Industries, Israel), and 10 μg / ml blasticidin. The cells were maintained in a 37 °C incubator with 5% CO2 humidified air. The day before transfection, cells were seeded onto 24-well black plate glass bottom (40,000 cells / well). The next day, the cells were initiated with a 72-hour incubation with conjugate [Cn-2-(VIII-M)] at concentrations of 2 nM, 10 nM, 20 nM, 40 nM, 80 nM, 150 nM, and 300 nM. The first 24 hours were in serum-free Opti-MEM medium (Thermo Fisher Scientific), followed by a further 48-hour incubation in medium containing 10% serum. Downregulation of protein expression was measured 72 hours after transfection. For this purpose, the medium was aspirated and the cells were washed with HBSS. The EGFP fluorescence intensity was quantified by an Infinite M200-Pro Multimode Reader (Tecan) at an excitation wavelength of 488 nm and an emission wavelength of 535 nm. Untreated cells were used as controls. The experiments were performed in triplicate and the results were shown as mean ± SD. A curve of gene expression vs conjugate concentration was plotted and curve fitting was calculated using GraphPad, Prism-5 software.

[0558] Results:

[0559] As shown in FIGS. 4A and 4B, for both HeLa cells and 3T3 cells, there was a highly significant logarithmic decay, and for both cell lines, R 2A clear dose / response was observed with a curve fit of 0.95. For heLa cells, IC 50 It is 13.54 nM, and the IC is 19.25 nM. 50 It was found to be similar to 3T3 cells, which showed the same characteristics.

[0560] Conclusion:

[0561] As investigated in vitro in two cell lines, the conjugate [Cn-2-(VIII-M)]-EGFP exhibits robust delivery to cells in culture and potent induction of gene silencing, as assessed through protein level evaluation. Low nanomolar EC2 was observed. 50 The values ​​support the concept that designing conjugates with three E-molecule bonds per double helix is ​​useful for enabling effective biological performance in gene silencing.

[0562] Example 7: Biological performance of the conjugates of the present invention, each containing E and E' according to formula (X), in in vitro gene silencing.

[0563] Research purpose:

[0564] Evaluation of the in vitro biological performance of the conjugate [Cn-1-(X)] of the present invention, i.e., dsiRNA having E and E' moieties, each bound to a 25 / 27 nucleotide Dicer substrate double strand, designed to silence the EGFP gene according to formula (X).

[0565] method:

[0566] The structure of the conjugate [Cn-1-(X)]-EGFP-dsiRNA is as follows:

[0567] [ka]

[0568] The EGFP-dsiRNA sequence, cell culture of both HeLa and 3T3 cells, and processing protocol were all as described in Example 5. EGFP fluorescence intensity was quantified using an Infinite M200-Pro Multimode Reader (Tecan), with an excitation wavelength of 488 nm and an emission wavelength of 535 nm. Untreated cells were used as a control. The experiment was performed in triplicate, and the results are shown as mean ± SD. Differences between groups were evaluated by a two-sided t-test, and statistical significance was defined as p<0.05.

[0569] result:

[0570] The [Cn-1-(X)]-EGFP-dsiRNA conjugate effectively induced EGFP expression knockdown as follows:

[0571] In heLa cells: In the presence of serum, a 600 nM conjugate reduced EGFP expression to 76% ± 2 (mean ± SD) of the control. Under serum-free conditions, robust knockdown of EGFP expression was dose-dependently induced by the conjugate, and when cells were treated with 10, 40, and 150 nM conjugates, respectively (all between-group comparisons p<0.001), EGFP expression was reduced to 57.4% ± 4.8%, 32.1% ± 5.0%, and 15.9% ± 4.2% of the control.

[0572] In 3T3 cells: Under serum-free conditions, a significant knockdown of EGFP expression was dose-dependently induced by the conjugate. When cells were treated with 10, 40, and 150 nM conjugates, respectively (all between-group comparisons, p<0.001), EGFP expression was reduced to 91%±2.0%, 50.0%±5.0%, and 27%±4.0% of the control.

[0573] Conclusion:

[0574] The conjugate [Cn-1-(X)]-EGFP-dsiRNA of the present invention is a potent component for the delivery of high molecular weight dsiRNA constructs across phospholipid membranes to cells and for the resulting induction of significant gene silencing in each.

[0575] Example 8: Biological performance of the conjugates of the present invention, each containing E and E' according to formula (XI), in in vitro gene silencing.

[0576] Research purpose:

[0577] Evaluation of the in vitro biological performance of the conjugate [Cn-1-(XI)] of the present invention, i.e., dsiRNA having E and E' moieties, each conjugated to a substrate double strand of 25 / 27 nucleotide Dicer, designed to silence the EGFP gene according to formula (XI).

[0578] method:

[0579] The structure of the conjugate [Cn-1-(XI)]-EGFP-dsiRNA is as follows:

[0580] [ka]

[0581] The study was conducted using heLa cells. The EGFP-dsiRNA sequence, cell culture, and processing protocol were all as described in Example 5. EGFP fluorescence intensity was quantified using an Infinite M200-Pro Multimode Reader (Tecan), with an excitation wavelength of 488 nm and an emission wavelength of 535 nm. Untreated cells were used as a control. The experiment was performed in triplicate, and the results are presented as mean ± SD. Differences between groups were evaluated by a two-sided t-test, and statistical significance was defined as p<0.05.

[0582] result:

[0583] The [Cn-1-(XI)]-EGFP-dsiRNA conjugate effectively induced knockdown of EGFP expression as follows: In the presence of serum, a 600 nM conjugate reduced EGFP expression to 83% ± 4% (mean ± SD) of the control. Under serum-free conditions, significant knockdown of EGFP expression was dose-dependently induced by the conjugate, and when cells were treated with 10, 40, and 150 nM conjugates, respectively (p<0.01 in all intergroup comparisons), EGFP expression was reduced to 55.2% ± 10%, 34.5% ± 8%, and 15.9% ± 4.2% of the control.

[0584] Conclusion:

[0585] The conjugate [Cn-1-(XI)]-EGFP-dsiRNA of the present invention is a potent component for the delivery of high molecular weight dsiRNA constructs across phospholipid membranes to cells and for the resulting induction of significant gene silencing in each respective gene.

[0586] Example 9: Biological performance of the (Cn-1) and (Cn-2) conjugates of the present invention having various E, E', and E'' moieties in silencing EGFP gene expression in vitro.

[0587] Research purpose:

[0588] All relating to general formulas (I) and (II), and thus formulas (I) and (II) exemplify gene silencing exerted in vitro by various conjugates of the present invention having various E, E', or E'' moieties, enabling transmembrane delivery of dsiRNA across the phospholipid membrane to cells and consequently exerting biological activity.

[0589] method:

[0590] The studies were conducted in cultured 3T3 cells, and EFGP gene silencing was evaluated after incubation with various conjugates of the present invention. The methodology of the studies was as described in Examples 5-8 above. Incubation was performed for 72 hours, with the first 24 hours of incubation being serum-free, followed by the addition of 10% serum. In all evaluations, the conjugate concentration was either 40 nM or 150 nM. EGFP expression was evaluated by measuring the fluorescence of the protein via ELISA, as described in the examples above. EGFP expression was also measured in untreated cells that served as controls. The E-parts of the present invention evaluated were (II), (III), (IV), (V), (VI), (VII), (VIII-M), and (IX). As identified above, all these parts relate to general formula (II). In addition, six control E-parts were synthesized and conjugated to dsiRNA for EGFP gene silencing. The structural features of the control portion (Table 3) were similar to those of portion E, which was constructed according to general formula (II). However, they lacked complete alignment with the main structural formula (II).

[0591] result:

[0592] The results are shown in Tables 1, 2, and 3 below.

[0593] [Table 1]

[0594] [Table 2]

[0595] [Table 3]

[0596] Summary of results:

[0597] As shown in Tables 1 and 2, all conjugates whose structures conform to general formulas (I) and (II), while possessing various conjugate backbones and E-molets, exhibited robust silencing of the investigated gene EGFP at the nanomolar concentrations (40 and 150 nM) investigated. In contrast, all control conjugates (Table 3) did not exhibit gene silencing, even when tested at relatively high concentrations (up to 400 nM).

[0598] Conclusion:

[0599] This performance profile therefore supports the concept that the conjugates according to equations (I) and (II) possess common and characteristic structural motifs that enable the effective delivery of high molecular weight ODs across phospholipid membranes to cells, along with the resulting useful biological performance (in this case, gene silencing).

[0600] Example 10: Biological performance of the conjugates of the present invention, each containing the E portion according to formula (VIII-M), in in vitro gene silencing; dose / response studies of the (Cn-1) and (Cn-2) conjugates; and comparative analysis:

[0601] Research purpose:

[0602] Evaluation of the in vitro biological performance of the conjugates [Cn-1-(VIII-M)] and [Cn-2-(VIII-M)] of the present invention, i.e., dsiRNAs having either two or three E-molets, each E-molet, attached to a 25 / 27 nucleotide Dicer substrate double strand, designed to silence the EGFP gene according to formula (VIII-M).

[0603] method:

[0604] Cell culture:

[0605] The 3T3 cell line was obtained from Cell Biolabs. Cells of each cell type were grown in Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% FBS (Gibco), 100 U / ml penicillin, 100 mg / ml streptomycin (Biological Industries, Israel), and 10 μg / ml blastosidine. Cells were maintained in a 37°C incubator with 5% CO2 humidified air. The day before transfection, cells were seeded onto the glass bottom of 24-well black plates (40,000 cells / well). The following day, cells were incubated with conjugate [Cn-1-(VIII-M)] or conjugate [Cn-2-(VIII-M)] at concentrations of 1 nM, 2 nM, 5 nM, 10 nM, and 40 nM for 48 hours, the first 24 hours in serum-free Opti-MEM medium (Thermo Fisher Scientific), followed by a further 24 hours of incubation in medium containing 10% serum. Downregulation of protein expression was measured 48 hours after transfection. For this purpose, the medium was aspirated and the cells were washed with HBSS.

[0606] RNA extraction and qRT-PCR:

[0607] Total RNA was extracted using the PureLink RNA Mini Kit (Invitrogen) according to the manufacturer's instructions. RNA was quantified using the Infinite M200-Pro Multimode Reader (Tecan). RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (ABI), and ApoC3 mRNA expression was measured using the Syber qRT-PCR procedure, normalized to beta-actin (Step-one-Plus, ABI).

[0608] result:

[0609] As shown in Figure 5, both conjugate [Cn-1-(VIII-M)] and conjugate [Cn-2-(VIII-M)] induced robust silencing of EGFP gene expression, as assessed in vitro in 3T3 cells. Both cell lines exhibited a very significant logarithmic decay, and R 2 A clear dose / response was observed with a curve fit of 0.97. For the conjugate [Cn-1-(VIII-M)] (dotted line) having two E-parts, IC 50 While the current is 2.2nM, for the conjugate [Cn-2-(VIII-M)] (solid line) which has three E portions, IC 50 It was found to be 0.8 nM (Figure 5).

[0610] Conclusion:

[0611] As investigated for two conjugates: conjugate [Cn-1-(VIII)]-EGFP and conjugate [Cn-2-(VIII-M)], the conjugates of the present invention exhibit very low IC50, as can be assessed through RNA level measurements. 50 In terms of value, it exhibits robust gene silencing. Interestingly, the conjugate [Cn-2-(VIII-M)] containing three E-molets showed higher efficacy in gene silencing and lower IC50 compared to the conjugate containing two E-molets. 50 The values ​​(0.8 vs. 2.2 nM) were reflected. This observation supports the concept that the delivery system of the present invention exhibits a positive synergistic effect, with each E portion contributing to and enhancing the overall performance of the conjugate in gene silencing.

Claims

1. A conjugate having the structure shown in formula (I) below, or a pharmaceutically acceptable salt thereof. During the ceremony, D is RNA, DNA, or any combination thereof, selected from single-stranded or double-stranded, natural or modified RNA or DNA; y, z, and w are integers independently selected from the group consisting of 0, 1, 2, 3, and 4, and at least two of y, z, and w are non-zero; E, E', or E'' may be identical or different from each other, and independently have the structure shown in formula (II) below. During the ceremony, M １ 、M ２ 、M ３ 、M ４ are each independently selected from the group consisting of N′, N″, null, and ether; N′ and N″ are each independently selected from the group consisting of —N(CH ３ ), and —NH—; M １ 、M ２ 、M ３ 、M ４ may be the same as or different from each other; N′, N″ may be the same as or different from each other; L is null, C １ , C ２ , C ３ , C ４ , C ５ , and C ６ A linker selected from the group consisting of alkylenes; G １ G ２ G ３ G ４ Each represents a hydrogen atom or a methyl group independently of the other; G １ G ２ G ３ , or G ４ At least two of the groups are hydrogen atoms; a, b, c, d, and e are integers independently selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6, where 0 = null; a, b, c, d, and e may be identical or different from each other; g represents an integer selected from 0, 1, 2, 3, 4, or 5; W is given by the following equation (II １ This is the structure shown in the image. During the ceremony, J is null, -CH ２ - and selected from the group consisting of oxygen, (i) W is bonded to D via one -O- group, and another -O- group is further bonded to an alcohol protecting group, hydrogen, or phosphoric acid, or (ii) W is bonded to D via two -O- groups, The aforementioned alcohol protecting groups are acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl [bis-(4-methoxyphenyl)phenylmethyl] (DMT), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-methoxybenzyl ether (PMB), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ether, ethoxyethyl ether (EE), phosphoramidite, and N-hydroxysuccinimide (NHS).

2. g is 1, c and d independently represent integers 1, 2, or 3. c and d may be the same or different from each other. The conjugate according to claim 1.

3. The formula (II) representing W １ ) in The aforementioned J is null or -CH ２ - and The aforementioned W is bonded to the aforementioned D, and further bonded to hydrogen or phosphoric acid, The aforementioned G １ G ２ G ３ , or G ４ All of the elements are hydrogen atoms, The conjugate according to claim 1, wherein D is coupled to at least two of E, E', or E''.

4. Each of the above N' and N'' is -N(CH ３ ) - and The conjugate according to claim 1, wherein D is an oligonucleotide, and E, E', or E'' have the same structure.

5. The conjugate according to claim 1, wherein each of y, z, and w is 1.

6. The aforementioned parts E, E', or E'' are Is it a compound having the structure shown in formula (V) below? Is it a compound having the structure shown in formula (VI) below? Is it a compound having the structure shown in formula (VII) below? Is it a compound having the structure shown in formula (XIIa) below? or containing a pharmaceutically acceptable salt thereof, The conjugate according to claim 1, wherein q is an integer of 1, 2, 3, 4, 5, or 6.

7. A precursor molecule of the conjugate according to claim 1, It consists of a compound having the structure of E in formula (II), In formula (II) above, W is a precursor molecule which is a structure shown in the following formula. In the formula, J is null, and the -O- group of W is bonded to a protecting group of (i) a phosphoramidite or (ii) an alcohol. The aforementioned alcohol protecting groups are acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl [bis-(4-methoxyphenyl)phenylmethyl] (DMT), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-methoxybenzyl ether (PMB), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ether, ethoxyethyl ether (EE), phosphoramidite, and N-hydroxysuccinimide (NHS). M １ M ２ M ３ M ４ When one of them is a secondary amine, the secondary amine is bonded to an amine protecting group, and the amine protecting group is a carbobenzyloxy (Cbz) group, a p-methoxybenzylcarbonyl (Moz or MeOZ) group, a tert-butyloxycarbonyl (BOC) group, a 9-fluorenylmethyloxycarbonyl (FMOC) group, a phenoxyacetyl (PAC) group, a 4-tert-butylphenoxyacetyl (t-PAC) group, an acetyl (Ac) group, a benzoyl (Bz) group, a benzyl (Bn) group, a carbamate group, a p-methoxybenzyl (PMB), a 3,4-dimethoxybenzyl (DMPM), a p-methoxyphenyl (PMP) group, a tosyl (Ts) group, a Troc (trichloroethyl chloroformate) group, or a 2-(trimethylsilyl)ethyl carbamate (TEOC).

8. A pharmaceutical composition, The conjugate described in claim 1, A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

9. Use of the pharmaceutical composition according to claim 8 for manufacturing a medicine for the treatment of a patient's illness.

10. The use according to claim 9, wherein the treatment is a treatment that involves downregulating the expression of genes related to the disease.

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