Protein fucosylation inhibitors and their applications

Abstract

This provides an inhibitor of protein fucosylation. [Solution] A method for inhibiting the fucosylation of a protein, or a fragment or derivative thereof, comprising contacting a eukaryotic cell or mammal with a specific carbocyclic compound or a salt thereof.

Description

Field of Invention

[0001] This invention relates to inhibitors of protein fucosylation. More specifically, this invention relates to carbocyclic compounds useful as inhibitors of protein fucosylation. Background of the Invention

[0002] Fucosylation is a common modification involving the transfer of fucose residues from GDP-fucose to N-glycans, O-glycans, or glycolipids, catalyzed by fucosyltransferases (FUTs). FUTs catalyze fucose transfer as follows: FUT1 and 2 catalyze α1,2-linking; FUT3-7 and FUT9-11 catalyze α1,3-linking; FUT3 and 5 catalyze α1,4-linking; FUT8 catalyzes α1,6-linking ("core fucosylation"); POFUT1 catalyzes the direct binding of EGF-like repeats to serine / threonine residues; and POFUT1 also catalyzes the direct binding of thrombospongin repeats to serine / threonine residues. Fucosylation is involved in various cellular processes.

[0003] Antibody-dependent cell-mediated cytotoxicity (ADCC) involves the selective targeting and lysis of cells by effector cells of the immune system. ADCC is centrally important for the efficacy of anti-cancer antibodies and is mediated by the binding of the antibody's Fc region to the Fc receptor (FcyR) on effector-natural killer (NK) cells and the antibody to the membrane-exposed antigen on cancer cells. The majority of monoclonal antibodies approved by the Food and Drug Administration (FDA) are of the IgG1 isotype, incorporating two N-linked complex oligosaccharides into their Fc region. ADCC of anti-cancer antibodies is attenuated by the circulation of IgG, which nonspecifically competes for binding to FcyR on effector-NK cells. To overcome this limitation, several strategies have been explored that focus on modifying the N-glycan structure in the antibody's Fc region. Most notably, the production of fucose-deficient (FD) antibodies has been found to improve binding to FcyR, resulting in a significant increase (approximately 50-fold) in ADCC. Therefore, there is interest in developing fucose-deficient (FD) antibodies. FD antibody production can be achieved by small molecule inhibition or gene knockout of key enzymes involved in fucosylation, such as GDP-mannose dehydration enzyme (GMD), GDP-fucose synthase (GFS), or FUT8. Small molecule inhibitors of one or more of these enzymes have clear advantages: they eliminate the need for expensive and time-consuming cell line manipulation and minimize disruption to existing manufacturing processes.

[0004] While several carbohydrate inhibitors of antibody fucosylation have demonstrated usefulness as additives in the production of FD antibodies, these carbohydrate inhibitors are incorporated into antibody N-glycans to varying degrees. Such inhibitors may pose a significant drug consistency risk and may be immunogenic. [Overview of the project]

[0005] This invention relates to inhibitors of protein fucosylation. More specifically, this invention relates to carbocyclic compounds useful as inhibitors of protein fucosylation.

[0006] In one aspect, the present invention provides a method for inhibiting the fucosylation of a protein, or a fragment or derivative thereof, by contacting a eukaryotic cell or a mammal with a compound of formula (I) or a salt thereof. JPEG2026136206000001.jpg6369Wherein, R 1 is optionally substituted C1-C 10 alkyl, optionally substituted C1-C 10 alkenyl, or optionally substituted C1-C 10 alkynyl, R 2 is H or -C(=O)(C1-C6)alkyl, R 3 is halo, OH, or O-C(=O)(C1-C6)alkyl, and R 4 is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ), wherein R 5 and R 6 are each independently H, (C1-C6)alkyl, (CH2)2SC(=O)CH3, CH2OC(=O)OR 7 , or CH2OC(=O)R 7 , wherein R 7 is C1-C8alkyl, or, wherein R 5 and R 6 are linked to form a ring, wherein the fucosylation of the protein is reduced by at least 5% compared to the amount of fucosylation of the protein in the eukaryotic cell or mammal when the compound is not administered.

[0007] In some embodiments, the protein may be an N-glycan.

[0008] In some embodiments, the compound may not be incorporated into the N-glycan.

[0009] In some embodiments, the protein may be an antibody.

[0010] In some embodiments, R 1 R may be CH3 or CHCH2, 2 It may be H, and R 3 R may be OH, 4 is H or -P (=O) (OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 H may also be used.

[0011] In some embodiments, the compound is The filename JPEG2026136206000002.jpg5049 is also acceptable.

[0012] In some embodiments, the mammal may have cancer, an autoimmune disease, an inflammatory disease, or an infection.

[0013] In some embodiments, the method may further include administering a cancer-related antigen or an antigenic fragment thereof as an immunogen to a mammal having cancer.

[0014] In some embodiments, the mammal may be a human.

[0015] In some embodiments, the salt may be a pharmaceutically acceptable salt.

[0016] In another embodiment, the present invention provides a culture medium for mammalian cells containing an effective amount of the compound of formula (I) or a salt thereof. JPEG2026136206000003.jpg6369In formula, R 1 C1-C may be substituted. 10 Alkyl, possibly substituted C1-C 10 Alkenyl or possibly substituted C1-C 10 It is alkinyl, R 2 is H or -C(=O)(C1-C6)alkyl, R 3 These are halo, OH, or OC(=O)(C1-C6)alkyl, and also R 4 This is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) and in the formula, R 5 and R 6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR 7 , or CH2OC(=O)R 7 And in the formula, R 7 is a C1-C8 alkyl group, or in the formula, R 5 and R 6 They are connected to form a ring.

[0017] In some embodiments, the culture medium may be useful for producing fucose-deficient proteins, or fragments or derivatives thereof.

[0018] In some embodiments, the effective amount may be an amount of the compound sufficient to reduce the fucose incorporation into the glycans of the fucose-deficient protein, or a fragment or derivative thereof, by at least 50%.

[0019] In some embodiments, the culture medium may be a culture medium for Chinese hamster ovary cells.

[0020] In another embodiment, the present invention provides a method for treating cancer, autoimmune diseases, infections, inflammatory diseases, or sickle cell disease by administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a mammal in need. JPEG2026136206000004.jpg6369In formula, R 1 C1-C may be substituted. 10 Alkyl, possibly substituted C1-C10 Alkenyl or possibly substituted C1-C 10 It is alkinyl, R 2 is H or -C(=O)(C1-C6)alkyl, R 3 These are halo, OH, or OC(=O)(C1-C6)alkyl, and also R 4 This is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) and in the formula, R 5 and R 6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR 7 , or CH2OC(=O)R 7 And in the formula, R 7 is a C1-C8 alkyl group, or in the formula, R 5 and R 6 They are connected to form a ring.

[0021] In another aspect, the present invention provides uses for inhibiting the fucosylation of a compound of formula (I) or a pharmaceutically acceptable salt thereof of a protein, or a fragment or derivative thereof (where the fucosylation of the protein is reduced by at least 5% compared to the amount of fucosylation of the protein in eukaryotic cells or mammals in the absence of the compound), or for treating cancer, autoimmune diseases, infections, inflammatory diseases, or sickle cell disease. JPEG2026136206000005.jpg6369In formula, R 1 C1-C may be substituted. 10 Alkyl, possibly substituted C1-C 10 Alkenyl or possibly substituted C1-C 10 It is alkinyl, R 2 is H or -C(=O)(C1-C6)alkyl, R 3These are halo, OH, or OC(=O)(C1-C6)alkyl, and also R 4 This is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) and in the formula, R 5 and R 6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR 7 , or CH2OC(=O)R 7 And in the formula, R 7 is a C1-C8 alkyl group, or in the formula, R 5 and R 6 They are connected to form a ring.

[0022] In another embodiment, the present invention provides a compound of formula (II) or a pharmaceutically acceptable salt thereof. JPEG2026136206000006.jpg6669In formula, R 1 C1-C may be substituted. 10 Alkyl, possibly substituted C1-C 10 Alkenyl or possibly substituted C1-C 10 It is alkinyl, R 2 is H or -C(=O)(C1-C6)alkyl, R 3 These are halo, OH, or OC(=O)(C1-C6)alkyl, and also R 4 This is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) and in the formula, R 5 and R 6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR 7 , or CH2OC(=O)R 7 And in the formula, R 7 is a C1-C8 alkyl group, or in the formula, R 5 and R6 is linked to form a ring, wherein R 1 when is CH3, R 2 is not H, R 3 is not OH, and R 4 is not H.

[0023] In another aspect, the present invention provides a composition comprising a compound of formula (II).

[0024] The summary of the present invention does not necessarily describe all features of the present invention.

Brief Description of Drawings

[0025] These and other features of the present invention will become more apparent from the following description with reference to the accompanying drawings.

[0026] Figures 1A - 1D show the results of cell - based assays against carbafucose and carbafucose analogs according to one embodiment of the present invention. (A) 2 - deoxy - 2 - fluoro - L - fucose (2FFuc), (B) carbafucose (1 - 21), (C) carbafucose analog 2 - 3 (phosphate), and (D) carbafucose analog 1 - 42 (alkene). Detailed description

[0027] The present disclosure provides, in part, the generation of carbacyclic analogues of sugars and their use in modifying the incorporation of carbohydrates into proteins such as antibodies during the generation of such proteins. In some embodiments, the present disclosure provides the generation and use of carbafucose and carbafucose analogues that can be useful, for example, in inhibiting the amount of core fucosylation of antibodies and proteins. In some embodiments, the compounds according to the present disclosure reduce protein (e.g., antibody) fucosylation. In some embodiments, the compounds according to the present disclosure are not incorporated into proteins (e.g., antibodies, N - glycans).

[0028] In some embodiments, the present disclosure provides carbafucose and its analogs, as well as methods for manufacturing them. "Carbafucose" means a compound having the following structure. JPEG2026136206000007.jpg5561

[0029] In one aspect, the present disclosure provides a compound of formula I or a salt thereof. JPEG2026136206000008.jpg6369Where, R 1 may be optionally substituted C1-C 10 alkyl, optionally substituted C1-C 10 alkenyl, or optionally substituted C1-C 10 alkynyl, R 2 may be H or -C(=O)(C1-C6)alkyl, R 3 may be halo, OH, or O-C(=O)(C1-C6)alkyl, and also R 4 may be H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ), where, R 5 and R 6 are each independently H, (C1-C6)alkyl, (CH2)2SC(=O)CH3, CH2OC(=O)OR 7 , or CH2OC(=O)R 7 , where, R 7 is C1-C8 alkyl, or, where, R 5 and R 6 may be linked to form a ring.

[0030] In an alternative aspect, the present disclosure provides a compound of formula II or a salt thereof. JPEG2026136206000009.jpg6669Where, R 1 may be optionally substituted C1-C 10Alkyl, possibly substituted C1-C 10 Alkenyl or possibly substituted C1-C 10 It may also be alkinyl. R 2 This may be H or -C(=O)(C1-C6) alkyl, R 3 This may be a halo, OH, or OC(=O)(C1-C6)alkyl, and R 4 This is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR 7 , or CH2OC(=O)R 7 It may be so, and in the formula, R 7 is a C1-C8 alkyl group, or in the formula, R 5 and R 6 They may be connected to form a ring. In the formula, R 1 If CH3, R 2 It's R, not H. 3 It is not OH, and R 4 It is not H.

[0031] In some embodiments, R 1 C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkinyl, Perhalo (C1-C 10 ) alkyl, or halo(C1-C 10 ) It may also be alkyl.

[0032] In some embodiments, R 1 This may be a C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, perhalo(C1-C6)alkyl, or halo(C1-C6)alkyl.

[0033] In some embodiments, R 1 This may be an acyl or an ester.

[0034] In some embodiments, R 1 The group may be selected from the group consisting of CH3, CH2F, CHF2, CF3, CCH, CCCH3, COCH3, COCH2CH3, CHCH2, CO2CH3, and CH2Br.

[0035] In some embodiments, R 1 CH3 may also be used.

[0036] In some embodiments, R 1 CHCH2 may also be used.

[0037] In some embodiments, R 2 This can be H or -C(=O)(C1-C6)alkyl.

[0038] In some embodiments, R 2 H may also be used.

[0039] In some embodiments, R 2 This may also be a -C(=O)(C1-C6) alkyl group.

[0040] In some embodiments, R 3 This can be a halo, an OH group, or an OC(=O)(C1-C6) alkyl group.

[0041] In some embodiments, R 3 It may also be OH.

[0042] In some embodiments, R 3 It may also be F.

[0043] In some embodiments, R 3 This can be an OH or OC(=O)(C1-C6)alkyl group.

[0044] In some embodiments, R 4 These are H, OC(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 Each of these may independently be H, (C1-C6) alkyl, or (CH2)2SC(=O)CH3.

[0045] In some embodiments, R 4 H may also be used.

[0046] In some embodiments, R 4 is -P(=O)(OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 Each of these can be H independently.

[0047] In some embodiments, R 4 is -P(=O)(OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 Both can be H.

[0048] In some embodiments, R 1 This may be CH3, and R 2 R may be H or -C(=O)CH3, 3 R may be OH or OC(=O)CH3, and 4 This can be H or -C(=O)CH3.

[0049] In some embodiments, R 1 This may be CH3, and R 2 It may be H, and R 3 R may be OH, and 4 H may also be used.

[0050] In some embodiments, R 1 may be CH3, and R 2 may be H, and R 3 may be OH, and R 4 may be -P(=O)(OR 5 )(OR 6 ), where R 5 and R 6 may be H.

[0051] In some embodiments, R 1 may be CH2F, R 2 may be H or -C(=O)CH3, R 3 may be OH or O-C(=O)CH3, and R 4 may be H or -C(=O)CH3.

[0052] In some embodiments, R 1 may be CHF2, R 2 may be H or -C(=O)CH3, R 3 may be OH or O-C(=O)CH3, and R 4 may be H or -C(=O)CH3.

[0053] In some embodiments, R 1 may be CF3, R 2 may be H or -C(=O)CH3, R 3 may be OH or O-C(=O)CH3, and R 4 may be H or -C(=O)CH3.

[0054] In some embodiments, R 1 may be CCH, R 2 may be H or -C(=O)CH3, R 3 may be OH or O-C(=O)CH3, and R 4 may be H or -C(=O)CH3.

[0055] In some embodiments, R 1 This may be CCCH3, and R 2 R may be H or -C(=O)CH3, 3 R may be OH or OC(=O)CH3, and 4 This can be H or -C(=O)CH3.

[0056] In some embodiments, R 1 This may be COCH3, and R 2 R may be H or -C(=O)CH3, 3 R may be OH or OC(=O)CH3, and 4 This can be H or -C(=O)CH3.

[0057] In some embodiments, R 1 This may also be COCH2CH3, and R 2 R may be H or -C(=O)CH3, 3 R may be OH or OC(=O)CH3, and 4 This can be H or -C(=O)CH3.

[0058] In some embodiments, R 1 This may be CHCH2, and R 2 R may be H or -C(=O)CH3, 3 R may be H or -C(=O)CH3, and R 4 This can be H or -C(=O)CH3.

[0059] In some embodiments, R 1 This may be CHCH2, and R 2 It may be H, and R 3 R may be OH, and 4 H may also be used.

[0060] In some embodiments, R 1 This may be CO2CH3, and R2 R may be H or -C(=O)CH3, 3 R may be OH or OC(=O)CH3, and 4 This can be H or -C(=O)CH3.

[0061] In some embodiments, R 1 This may be CH2Br, and R 2 R may be H or -C(=O)CH3, 3 R may be OH or OC(=O)CH3, and 4 This can be H or -C(=O)CH3.

[0062] In some embodiments, R 1 R may be CH3 or CHCH2, 2 It may be H, and R 3 R may be OH, and 4 is H or -P (=O) (OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 H may also be used.

[0063] In some embodiments, the present disclosure provides compounds of formula (III) or salts thereof. JPEG2026136206000010.jpg6064In formula, R 2 is -C(=O)(C1-C 10 ) It may also be alkyl, R 3 , OC(=O)(C1-C 10 ) It may also be alkyl, and R 4 This is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR7 , or CH2OC(=O)R 7 It may be so, and in the formula, R 7 is a C1-C8 alkyl group, or R 5 and R 6 They may be connected to form a ring.

[0064] In some embodiments, the present disclosure provides compounds of formula (IV) or salts thereof. JPEG2026136206000011.jpg6062In formula, R 2 is H or -C(=O)(C1-C 10 ) It may also be alkyl, R 3 This is OH or OC (=O) (C1-C 10 ) It may also be alkyl, and R 4 This is -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR 7 , or CH2OC(=O)R 7 It may be so, and in the formula, R 7 is a C1-C8 alkyl group, or R 5 and R 6 They may be connected to form a ring.

[0065] In some embodiments, the present disclosure provides compounds of formula (V) or salts thereof. JPEG2026136206000012.jpg7066In formula, R 2 is H or -C(=O)(C1-C 10 ) It may also be alkyl, R 3 This is OH or OC (=O) (C1-C 10 ) It may also be alkyl, and R 4This is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR 7 , or CH2OC(=O)R 7 It may be so, and in the formula, R 7 is a C1-C8 alkyl group, or R 5 and R 6 They may be connected to form a ring.

[0066] In some embodiments, the present disclosure provides compounds of formula (VI) or salts thereof. JPEG2026136206000013.jpg6360In formula, R 2 is H or -C(=O)(C1-C 10 ) It may also be alkyl, R 3 This is OH or OC (=O) (C1-C 10 ) It may also be alkyl, and R 4 This is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR 7 , or CH2OC(=O)R 7 It may be so, and in the formula, R 7 is a C1-C8 alkyl group, or R 5 and R 6 They may be connected to form a ring.

[0067] In some embodiments, the present disclosure provides compounds of formula (VII) or salts thereof. JPEG2026136206000014.jpg6964In formula, R 2 is optionally H or -C(=O)(C1-C 10 )alkyl, R 3 is optionally OH or O-C(=O)(C1-C 10 )alkyl, and also R 4 is optionally H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ), where R 5 and R 6 are each independently optionally H, (C1-C6)alkyl, (CH2)2SC(=O)CH3, CH2OC(=O)OR 7 , or CH2OC(=O)R 7 , where R 7 is C1-C8 alkyl, or R 5 and R 6 are optionally linked to form a ring.

[0068] In some embodiments, the disclosure provides a compound of formula (VIII) or a salt thereof. JPEG2026136206000015.jpg6169where R 2 is optionally H or -C(=O)(C1-C 10 )alkyl, R 3 is optionally OH or O-C(=O)(C1-C 10 )alkyl, and also R 4 is optionally H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ), where R 5 and R 6 are each independently optionally H, (C1-C6)alkyl, (CH2)2SC(=O)CH3, CH2OC(=O)OR 7 , or CH2OC(=O)R 7 , where R 7 is C1-C8 alkyl, or R 5 and R6 They may be connected to form a ring.

[0069] In some embodiments, the present disclosure provides compounds of formula (IX) or salts thereof. JPEG2026136206000016.jpg7072In formula, R 2 is H or -C(=O)(C1-C 10 ) It may also be alkyl, R 3 This is OH or OC (=O) (C1-C 10 ) It may also be alkyl, and R 4 This is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR 7 , or CH2OC(=O)R 7 It may be so, and in the formula, R 7 is a C1-C8 alkyl group, or R 5 and R 6 They may be connected to form a ring.

[0070] In some embodiments, the present disclosure provides compounds of formula (X) or salts thereof. JPEG2026136206000017.jpg6264In formula, R 2 is H or -C(=O)(C1-C 10 ) It may also be alkyl, R 3 This is OH or OC (=O) (C1-C 10 ) It may also be alkyl, and R 4 This is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR 7 , or CH2OC(=O)R 7 It may be so, and in the formula, R 7 is a C1-C8 alkyl group, or R 5 and R 6 They may be connected to form a ring.

[0071] In some embodiments, the present disclosure provides compounds of formula (XI) or salts thereof. JPEG2026136206000018.jpg7578In formula, R 2 is H or -C(=O)(C1-C 10 ) It may also be alkyl, R 3 This is OH or OC (=O) (C1-C 10 ) It may also be alkyl, and R 4 This is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR 7 , or CH2OC(=O)R 7 It may be so, and in the formula, R 7 is a C1-C8 alkyl group, or R 5 and R 6 They may be connected to form a ring.

[0072] In some embodiments, the present disclosure provides compounds of formula (XII) or salts thereof. JPEG2026136206000019.jpg6370In formula, R 2 is H or -C(=O)(C1-C 10 ) It may also be alkyl, R 3This is OH or OC (=O) (C1-C 10 ) It may also be alkyl, and R 4 This is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR 7 , or CH2OC(=O)R 7 It may be so, and in the formula, R 7 is a C1-C8 alkyl group, or R 5 and R 6 They may be connected to form a ring.

[0073] In some embodiments, the present disclosure provides compounds of formula (XIII) or salts thereof. JPEG2026136206000020.jpg7171In formula, R 2 is H or -C(=O)(C1-C 10 ) It may also be alkyl, R 3 This is OH or OC (=O) (C1-C 10 ) It may also be alkyl, and R 4 This is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR 7 , or CH2OC(=O)R 7 It may be so, and in the formula, R 7 is a C1-C8 alkyl group, or R 5 and R 6 They may be connected to form a ring.

[0074] In some embodiments, the present disclosure provides compounds of formula (XIV) or salts thereof. JPEG2026136206000021.jpg6867In formula, R 2 is H or -C(=O)(C1-C 10 ) It may also be alkyl, R 3 This is OH or OC (=O) (C1-C 10 ) It may also be alkyl, and R 4 This is H, -C(=O)(C1-C6)alkyl, or -P(=O)(OR 5 )(OR 6 ) may also be, and in the formula, R 5 and R 6 These are, independently, H, (C1-C6) alkyl, (CH2)2SC(=O)CH3, and CH2OC(=O)OR 7 , or CH2OC(=O)R 7 It may be so, and in the formula, R 7 is a C1-C8 alkyl group, or R 5 and R 6 They may be connected to form a ring.

[0075] "Alkyl" refers to a linear or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, without unsaturation, comprising, for example, 1 to 10 carbon atoms, or any value in between, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, bonded to the rest of the molecule by single bonds. In some embodiments, alkyl refers to a linear or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, without unsaturation, comprising, for example, 1 to 6 carbon atoms, or any value in between, e.g., 1, 2, 3, 4, 5, or 6 carbon atoms, bonded to the rest of the molecule by single bonds. Unless specifically described herein, alkyl groups may be optionally substituted with one or more substituents described herein. Unless specifically described herein, such substitutions are understood to occur on any carbon of the alkyl group.

[0076] "Alkenyl" refers to a linear or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, comprising, for example, 1 to 10 carbon atoms, or any value in between, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, bonded to the rest of the molecule by single bonds, and containing at least one double bond. In some embodiments, alkenyl refers to a linear or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, comprising, for example, 1 to 6 carbon atoms, or any value in between, e.g., 1, 2, 3, 4, 5, or 6 carbon atoms, bonded to the rest of the molecule by single bonds, and containing at least one double bond. Unless specifically described herein, the alkenyl group may be optionally substituted with one or more substituents described herein. Unless specifically described herein, such substitution may occur on any carbon of the alkenyl group.

[0077] "Alkynyl" refers to a linear or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, comprising, for example, 2 to 10 carbon atoms, or any value in between, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, bonded to the rest of the molecule by single bonds, and containing at least one triple bond. In some embodiments, alkynyl refers to a linear or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, comprising, for example, 2 to 6 carbon atoms, or any value in between, e.g., 2, 3, 4, 5, or 6 carbon atoms, bonded to the rest of the molecule by single bonds, and containing at least one triple bond. Unless specifically described herein, the alkynyl group may be optionally substituted with one or more substituents described herein.

[0078] "Acyl" is expressed as formula -C(O)R a It refers to the base of, and in the formula, R a As described herein, C 1-10 Alkyl or C 1-6 It is an alkyl group. The alkyl group may be optionally substituted as described herein.

[0079] "Optional" or "may" means that the event or situation described thereafter may or may not occur, and that the description includes cases where the event or situation occurs once, several times, or does not occur. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and that the description includes both substituted alkyl groups and unsubstituted alkyl groups, and that the alkyl group may be substituted once or more times. Suitable optional substituents include, but are not limited to, halos, oxos, or esters. In some embodiments, the oxo group may be at the C1 position of R1 in formula I, as described herein. In some embodiments, the ester group may be at the C1 position of R1 in formula I, as described herein.

[0080] "Halo" refers to bromo, chloro, fluoro, iodine, etc. In some embodiments, suitable halogens include fluorine or bromine.

[0081] The terms “inhibit,” “to inhibit,” or “inhibit of” mean to reduce by a measurable amount or to prevent completely.

[0082] In some embodiments, the compounds according to this disclosure may be perhalo compounds.

[0083] In some embodiments, R5 and R6 in the compounds according to this disclosure may be linked to form a ring. In such embodiments, “ring” refers to a monocyclic phosphate ester having up to seven members, which may be saturated or monounsaturated. In some embodiments, R5 and R6 may be linked to form an aryl phosphate ester.

[0084] In some embodiments, the salt of the compound according to the Disclosure may be any suitable salt, such as a sodium salt, a potassium salt, or an ammonium salt, without limitation. In some embodiments, the salt of the compound according to the Disclosure may be a pharmaceutically acceptable salt.

[0085] FD-protein production In some embodiments, the compounds of this disclosure can be used, for example, to produce "fucos-deficient" or "FD" proteins or antibodies using standard techniques described herein, or U.S. Patent No. 9,816,069 granted November 14, 2017, or PCT Publication WO / 2012 / 019165 published February 9, 2012, or known in the art. Accordingly, this disclosure provides, in part, methods for inhibiting the fucosylation of proteins or fragments or derivatives of proteins by contacting eukaryotic cells or mammals with the compounds described herein, for example, compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, such as carbafucose or a salt thereof, using standard techniques described herein or known in the art. These methods can be carried out in vitro or in vivo.

[0086] Generally, fucosylation refers to the transfer of fucose from GDP to glycans via α(1,2)-, α(1,3)-, α(1,4)-, and / or α(1,6)- links. Core fucosylation refers to the transfer of fucose from GDP to the innermost N-acetylglucosamine (GlcNAc) residue (reducing end) of N-linked glycans in proteins such as antibodies, via α1,6- links or α-1,6-fucosyltransferase 8 (FUT 8).

[0087] A "fucosylated" or "FD" protein means a polypeptide, such as an antibody, in which fucosylation is reduced compared to a polypeptide, such as an antibody, produced in the absence of a carbocyclic compound, for example, one of the compounds described herein, such as compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, for example, carbafucose. The reduction in fucosylation may be at least about 5% to 100%, or any value in between, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the reduction of fucosylation may be at least about 50% to 100%, or any value in between, for example, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the reduction of fucosylation may be a reduction of "core fucosylation".

[0088] In some embodiments, after administration of a carbocyclic compound described herein, such as carbafucose, only a small amount of fucose can be incorporated into a sugar chain (e.g., a glycan such as N-glycan). For example, in various embodiments, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of antibodies in the serum of an animal (e.g., a mammal such as a human) is fucosylated compared to cells or animals that have not received a carbocyclic compound described herein (e.g., carbafucose). In some embodiments, compared to animals that have not received a carbocyclic compound described herein (e.g., carbafucose), the antibodies in the serum are substantially unfucosylated (i.e., less than 0.5%).

[0089] The proteins or antibodies for use described herein can be produced using recombinant techniques. Recombinant expression of a protein or antibody, or a fragment or derivative thereof, that binds to a target antigen typically involves constructing an expression vector containing a nucleic acid that codes for the antibody or its derivative. Once such a nucleic acid that codes for a protein is obtained, a vector for the production of the protein molecule can be produced by recombinant DNA techniques using techniques well known in the art. Standard techniques such as those described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 3rd edition, 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2nd edition, 1989); Ausubel et al., Short Protocols for Molecular Biology (John Wiley & Sons, New York, 4th edition, 1999); and Glick & Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA (ASM Press, Washington, DC, 2nd edition, 1998) can be used for recombinant nucleic acid methods, nucleic acid synthesis, cell culture, transgene integration, and recombinant protein expression.

[0090] Therefore, in some embodiments, antibodies or fragments or derivatives thereof can be produced using recombinant expression techniques from hybridomas, bone marrow, or other suitable cells (including mammalian cells).

[0091] For example, for the recombinant expression of an antibody or a fragment or derivative thereof, an expression vector may code its heavy chain or light chain, or a heavy chain or light chain variable domain operably linked to a promoter. The expression vector may, for example, contain a nucleotide sequence coding the constant region of an antibody molecule (see, e.g., PCT publications WO86 / 05807 or WO89 / 01036; or U.S. 5,122,464), and the variable domain of the antibody may be cloned into such a vector for the expression of the entire heavy chain or light chain. The expression vector can be transmitted to host cells by techniques known in the art, and the transgenic cells can then be cultured in the presence of a carbocyclic compound described herein (e.g., carbaf course) by techniques known in the art to produce antibodies. Typically, for the expression of a double-chain antibody, a vector coding both the heavy and light chains can be co-expressed in host cells for the expression of the entire immunoglobulin molecule.

[0092] Antibodies or fragments or derivatives thereof can be expressed using any suitable mammalian cells or cell lines. For example, mammalian cells such as Chinese hamster ovary cells (CHO) (e.g., DG44, Dxb11, CHO-K, CHO-K1, and CHO-S) can be used. In some embodiments, human cell lines can be used. Suitable myeloma cell lines include, but are not limited to, human myeloma cell lines such as SP2 / 0 and IR983F, and Namalwa. Other suitable cells include, but are not limited to, human embryonic kidney cells (e.g., HEK293), monkey kidney cells (e.g., COS), and human epithelial cells (e.g., HeLa, PERC6, Wil-2, Jurkat, Vero, Molt-4, BHK, and K6H6). Other suitable host cells include, but are not limited to, YB2 / 0 cells.

[0093] In some embodiments, the host cells may be derived from a hybridoma.

[0094] In some embodiments, the host cells do not contain a fucose transporter gene knockout. In some embodiments, the host cells do not contain a fucosyltransferase (e.g., FUT8) gene knockout. In some embodiments, the host cells do not contain a GnTIII knock-in that codes for nucleic acids. In some embodiments, the host cells do not contain a Golgi α-mannosidase II knock-in that codes for nucleic acids.

[0095] Antibodies or fragments or derivatives thereof can be expressed using various mammalian host expression vector systems. For example, mammalian cells such as Chinese hamster ovary cells (CHO) (DG44, Dxb11, CHO-K1, and CHO-S, etc.) combined with vectors such as major intermediate early gene promoter elements derived from human cytomegalovirus or the Chinese hamster ovary EF-1α promoter are effective expression systems for the production of antibodies and their derivatives (see, e.g., Foecking et al., 1986, Gene 45:101; Collett et al., 1990, Bio / Technology 8:2; Allison, U.S. Patent No. 5,888,809). In some embodiments, the appropriate cell line may be a Chinese hamster ovary (CHO) cell line or a CHO-derived cell line.

[0096] Cell lines can be cultured in appropriate media. Appropriate media include, for example, those containing the necessary salts for growth, carbon sources (such as sugars), nitrogen sources, amino acids, trace elements, antibiotics, and selective agents. Examples include appropriate Ham's F10 (Sigma), basal media (MEM, Sigma), RPMI-1640 (Sigma), Dulbecco's modified Eagle medium (DMEM, Sigma), PowerCHO® cell medium (Lonza Group Ltd.), and hybridoma serum-free medium. Commercial culture media such as (HSFM)(GIBCO) may be suitable for culturing host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphates), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin®), trace elements (usually defined as inorganic compounds present in the micromolar range at final concentrations), and glucose or corresponding energy sources. Any other necessary supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH may be those previously used with host cells selected for expression, as known in the art. In some embodiments, the medium is not supplemented with fucose. In some embodiments, the medium may be serum-free. In some embodiments, the medium may be animal-derived protein.

[0097] In some embodiments, an effective amount of the compounds described herein, for example, compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, such as carbafcose or a salt thereof, may be added to the culture medium. In alternative embodiments, the culture medium may contain an effective amount of the compounds described herein, for example, compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, such as carbafcose or a salt thereof. Accordingly, in some embodiments, the compounds described herein, for example, compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, such as carbafcose or a salt thereof, may be provided with instructions in a kit for use in inhibiting the fucosylation of proteins such as antibodies, or fragments or derivatives thereof. The kit may further include, but are not limited to, cell culture media, cell lines, etc.

[0098] To produce FD-proteins or FD-antibodies, or fragments or derivatives thereof, an effective amount of a carbocyclic compound, for example, a compound described herein, for example, a compound of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, such as carbafucose, can be added to the culture medium, or the cell culture medium may contain an effective amount of the carbocyclic compound. In this context, “effective amount” means an amount of a carbocyclic compound described herein that is sufficient to reduce the fucose incorporation into the sugar chain of the protein or antibody, or fragment or derivative thereof, by at least about 5% to 100%, or any value in between, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, an effective amount of the carbocyclic compound described herein may be sufficient to reduce the fucose incorporation into the sugar chain of the protein or antibody, or a fragment or derivative thereof, by at least about 50%.

[0099] Cells expressing proteins or antibodies, or fragments or derivatives thereof, can be cultured by growing host cells in any appropriate volume of medium supplemented with carbocyclic compounds as described herein. The cells can be cultured in any suitable culture system, including T-flasks, spinner and shaker flasks, WaveBag® bags, roller bottles, bioreactors, and agitated bioreactors, according to any method known in the art. Scaffold-dependent cells can also be cultured on microcarriers maintained in a suspension within an agitated bioreactor, such as polymer spheres. Alternatively, cells can be grown in a single-cell suspension. The medium may be added in a batch process, for example, by adding the medium to the cells once in a single batch, or by a fed-batch process in which small batches of medium are added periodically. The medium can be harvested several times at the end of culture or during culture. Continuous perfusion production processes are also known in the art, in which fresh medium is continuously supplied to the culture while the same volume is continuously withdrawn from the reactor. Perfusion culture typically achieves higher cell densities than batch culture and can be maintained for several weeks or months by repeating harvests.

[0100] For cells grown in batch culture, the volume of the culture medium may be at least 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 10 liters, 15 liters, 20 liters, or more. For industrial applications, the volume of the culture medium may be at least 100 liters, at least 200 liters, at least 250 liters, at least 500 liters, at least 750 liters, at least 1000 liters, at least 2000 liters, at least 5000 liters, or at least 10,000 liters. The carbocyclic compounds described herein, for example, carbafcose, can be added to seed trains, initial batch media, after the rapid growth phase, or continuously with the medium (e.g., during continuous feeding). For example, the carbocyclic compounds described herein, for example, carbafcose, can be added to the initial seed train or feed stock at a 10-fold or 100-fold concentration, thereby changing the concentration of the carbocyclic compound to an effective level by subsequent addition of the medium. Alternatively, the carbocyclic compounds described herein, such as carbafcose, can be added directly to the culture medium without the need for dilution. In some embodiments, to optimize the production of a desired protein or antibody, or a fragment or derivative thereof, the carbocyclic compounds, such as the compounds described herein, for example, compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, such as carbafcose, can be added relatively early in the cell culture process and an effective concentration can be maintained throughout the culture process.

[0101] In some embodiments, the proteins or antibodies, or fragments or derivatives thereof produced as described herein, exhibit at least a 10% reduction in fucosylation compared to antibodies or antibody derivatives produced from host cells cultured in the absence of the carbocyclic compounds described herein, e.g., carbafco. In some embodiments, the proteins or antibodies, or fragments or derivatives thereof produced by this method, exhibit at least a 50% reduction in fucosylation compared to antibodies or antibody derivatives produced from host cells cultured in the absence of the carbocyclic compounds described herein, e.g., carbafco.

[0102] The amount of the carbocyclic compounds described herein (e.g., any of the compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, e.g., carbafcose) can be measured by standard cell culture methods. For example, a cell culture assay can be performed to determine the optimal dose range. The precise amount used may depend on the administration time, host cell line, cell density, etc. The effective dose may be extrapolated from dose-response curves from in vitro models or test systems.

[0103] In some embodiments, the carbocyclic compounds described herein, for example, carbafcose, can be present in the culture medium at a concentration of 10 nM to 50 mM. In some embodiments, the carbocyclic compounds described herein, for example, carbafcose, can be present in the culture medium at a concentration of 10 nM to 10 mM. In some embodiments, the carbocyclic compounds described herein, for example, carbafcose, can be present in the culture medium at a concentration of 100 nM to 5 mM. In some embodiments, the carbocyclic compounds described herein, for example, carbafcose, can be present in the culture medium at a concentration of 100 nM to 3 mM. In some embodiments, the carbocyclic compounds described herein, for example, carbafcose, can be present in the culture medium at a concentration of 100 nM to 2 mM. In some embodiments, the carbocyclic compounds described herein, for example, carbafcose, can be present in the culture medium at a concentration of 100 nM to 1 mM. In some embodiments, the carbocyclic compounds described herein, for example, carbafcose, can be present in the culture medium at a concentration of 1 μM to 1 mM. In some embodiments, the carbocyclic compounds described herein, such as carbafcose, can be present in the culture medium at a concentration of 10 nM to 1 mM. In some embodiments, the carbocyclic compounds described herein, such as carbafcose, can be present in the culture medium at a concentration of 10 nM to 500 μM. In some embodiments, the carbocyclic compounds described herein, such as carbafcose, can be present in the culture medium at a concentration of 1 μM to 500 μM. In some embodiments, the carbocyclic compounds described herein, such as carbafcose, can be present in the culture medium at a concentration of 1 μM to 250 μM. In some embodiments, the carbocyclic compounds described herein, such as carbafcose, can be present in the culture medium at a concentration of 10 μM to 100 μM. In some embodiments, the carbocyclic compounds described herein, such as carbafcose, may be soluble in the culture medium at a concentration of at least 100 nM (at a temperature suitable for host cell maintenance / growth).

[0104] The content (e.g., ratio) of glycans in which fucose is not bound to the N-acetylglucosamine at the reducing end and glycans in which fucose is bound to the N-acetylglucosamine at the reducing end can be measured, for example, as described in U.S. Patent Application Publication No. 2004-0110282. That is, the free glycans are subjected to hydrazine degradation or enzymatic digestion (see, for example, Biochemical Experimental Methods 23: Methods for Studying Glycoprotein Glycans (Japan Society Press), edited by Reiko Takahashi (1989)), fluorescent labeling or radioisotope labeling, and the labeled glycans are separated by chromatography or other appropriate technique. The composition of the free glycans can be measured by analyzing the chains using the HPAEC-PAD method (see, for example, J. Liq Chromatogr. 6:1557 (1983)) or any other appropriate technique.

[0105] In some embodiments, antibodies, or fragments or derivatives thereof, prepared as described herein may have higher effector activity (e.g., ADCC activity) than antibodies, or fragments or derivatives thereof, prepared in the absence of the carbocyclic compounds described herein. Such effector activity can be modulated by changing the concentration of the carbocyclic compounds described herein in the culture medium and / or the exposure time to the carbocyclic compounds described herein. ADCC activity may be measured using assays known in the art, and in exemplary embodiments, it increases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x compared to the fucosylated parent antibody. Cytotoxic activity against antigen-positive cultured cell lines can be evaluated by measuring effector activity (e.g., as described in Cancer Immunol. Immunother. 36:373 (1993)).

[0106] Proteins or antibodies, or fragments or derivatives thereof, can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. In some embodiments, proteins or antibodies, or fragments or derivatives thereof, can be purified using affinity chromatography.

[0107] In some embodiments, protein A can be used to purify antibodies, or fragments or derivatives thereof, based on human IgG1, IgG2, or quadruple chains. The suitability of protein A as an affinity ligand may depend on the species and isotype of any immunoglobulin Fc domain present in the antibody, or fragment or derivative thereof.

[0108] In some embodiments, protein G can be a mouse isotype, or several human antibodies, or fragments or derivatives thereof. The matrix to which the affinity ligand is attached may be agarose or any other suitable matrix. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than those achievable with agarose. The antibody or its fragment or derivative may be C H3 If the antibody contains a domain, Bakerbond ABX® resin (JTBaker, Phillipsburg, NJ) may be useful for purification. Depending on the antibody to be recovered or its fragments or derivatives, other techniques for protein purification may also be used, such as fractionation on ion exchange columns (cationic or anion exchange), ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE®, chromatography on anion or cation exchange resins (e.g., polyaspartate columns), isoelectric focusing, SDS-PAGE, and ammonium sulfate precipitation.

[0109] Following any purification step, the mixture containing the antibody of interest or a fragment or derivative thereof, and contaminants, can be subjected to hydrophobic interaction chromatography at a low pH (for example, using an elution buffer with a pH of about 2.5–4.5, preferably at a low salt concentration (e.g., about 0–0.25 M salt)).

[0110] Generally, it should be understood that any suitable method described herein or known in the art can be used in combination with the carbocyclic compounds described herein to produce FD proteins and / or FD antibodies, or fragments or derivatives thereof.

[0111] Antibodies, or fragments or derivatives thereof, that can be manufactured as described herein may be monoclonal, chimeric, humanized (including veneers), or human antibodies. Suitable antibodies also include antibody fragments such as single-chain antibodies having an Fc region or domain having a complex N-glycosidic sugar chain (e.g., human IgG 1Fc region or domain). The Fc region or domain may include an FCγ receptor binding site. The antibody may be human or humanized. In some embodiments, the antibody may be from rodents (e.g., mice and rats), donkeys, sheep, rabbits, goats, guinea pigs, pigs, camels, horses, or chickens.

[0112] Antibodies, or fragments or derivatives thereof, may be monospecific, bispecific, tripspecific, or more highly multispecific. Multispecific antibodies may be specific to different epitopes of different target antigens, or to different epitopes on the same target antigen (see, for example, WO93 / 17715; WO92 / 08802; WO91 / 00360; WO92 / 05793; Tutt et al., 1991, J. Immunol. 147:60-69; U.S. Patents Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; Kostelny et al., 1992, J. Immunol. 148:1547-1553).

[0113] The antibody, or a fragment or derivative thereof, may also be described in terms of its specificity in binding to the target antigen.

[0114] "Antibody" means a polypeptide or fragment (such as an antigen-binding fragment) belonging to the immunoglobulin family, as well as conservative substitutions of polypeptides and fragments as further discussed herein. Antibodies are outlined in Harlow & Lane's "Antibodies: A Laboratory Manual" (Cold Spring Harbor Laboratory Press, 1988). Unless otherwise stated, "antibody" includes antibody derivatives as described herein.

[0115] "Natural antibodies" or "natural immunoglobulins" are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is typically linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also contains intrachain disulfide bridges arranged at regular intervals. Each heavy chain has one variable domain ("V") at one end. H ) and several subsequent constant domains ("C H Each light chain has a variable domain ("V") at one end. L ) and a steady domain ("C L The constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. The "variable domains" have widely different sequences and are involved in the binding and specificity of antibodies to their antigens. Variability is concentrated in three segments called "hypervariable regions" in both the light chain and heavy chain variable domains, while less variable sequences are called "framework regions" or "FRs". The constant domains do not directly participate in antigen binding, but instead exhibit various effector functions.

[0116] The term "Fc region" refers to the constant region of an antibody (e.g., the C region which may have a CH4 domain).H L-Hinge-C H 2-C H This refers to derivatives with three domains or conservatively substituted Fc regions.

[0117] The term "Fc domain" refers to the constant region domain of an antibody (e.g., C H 1. Hinge, C H 2, C H 3, or C H This refers to a 4-domain (or a conservatively substituted derivative of such an Fc domain).

[0118] The terms "antigen-binding portion," "antigen-binding fragment," or "antigen-binding domain," "antibody fragment," or "functional fragment of an antibody" refer to antibody fragments that retain the ability to specifically bind to an antigen (see Holliger et al., Nature Biotech. 23(9)1126-1129 (2005) in general). Examples of antibody fragments include, but are not limited to, Fab fragments, V L , V H , C L and C H A monovalent fragment consisting of domains, an F(ab')2 fragment, a divalent fragment containing two Fab fragments linked by disulfide bridges in the hinge region, V H and C H Fd fragment consisting of domains, V of a single arm of antibody H and C H Fv fragments consisting of domains, V H (v)dAb fragments consisting of domains, synthetically concatenated V L and V H A single-chain Fv (scFv) molecule consisting of domains, synthetically bound to a short linker that requires pairing with complementary domains of different antibody chains, produces two antigen-binding sites. L and V H These include bispecific antibodies consisting of domains, isolated complementarity-determining regions (CDRs), etc. Generally, antigen-binding fragments contain complex N-glycosidic sugar chains.

[0119] In some embodiments, the antibody may be a monoclonal antibody. The term "monoclonal antibody" refers to an antibody derived from a single-cell clone or phaziclon, including any eukaryotic or prokaryotic cell clone, and not the method by which it is produced. Therefore, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology.

[0120] In some embodiments, the antibody may be a chimeric antibody. A chimeric antibody is a molecule derived from different animal species, such as an antibody in which different parts of the antibody consist of a variable region derived from a mouse monoclonal antibody and a constant region from a human immunoglobulin. Methods for producing chimeric antibodies are known in the art (see, for example, Morrison, Science, 1985, 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J.Immunol. Methods 125:191-202; U.S. Patents 5,807,715, 4,816,567, and 4,816,397).

[0121] In some embodiments, the antibody may be a humanized antibody containing a benya antibody. A humanized antibody is an antibody molecule that binds to a desired antigen and has one or more complementarity-determining regions (CDRs) from a non-human species, and a framework and constant region from a human immunoglobulin molecule. Often, framework residues within the human framework region are substituted with corresponding residues from the CDR donor antibody to modify, or preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art. For example, the interaction between the CDR and the framework residue is modeled to identify framework residues important for antigen binding and sequence comparison, and to identify abnormal framework residues at specific locations (see, for example, Queen et al., U.S. Patent No. 5,585,089; Riecbmann et al., 1988, Nature 332:323). Antibodies are used in various techniques known in the art, such as CDR transplantation (EP 0 239 400; WO 91 / 09967; U.S. Patent No. 5,225,539; No. 5,530,101; and No. 5,585,089), Humanization can be performed using veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973), and chain shuffling (U.S. Patent No. 5,565,332).

[0122] In some embodiments, the antibody may be a human antibody. Human antibodies can be produced by various methods known in the art, such as phage display using an antibody library derived from human immunoglobulin sequences. See, for example, U.S. Patents 4,444,887 and 4,716,111, WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741. Human antibodies that recognize a selected epitope can also be produced using a technique called "inducible selection," where a selected non-human monoclonal antibody, such as a mouse antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (see, for example, Jespers et al., 1994, Biotechnology 12:899-903). Human antibodies can also be produced using genetically modified mice that express human immunoglobulin genes. Monoclonal antibodies against antigens can be obtained from immunized genetically modified mice using conventional hybridoma technology. For an overview of the techniques for producing human antibodies, see Lonberg and Huszar, 1995, Int. Rev. Immunol. 13:65-93. For detailed descriptions of techniques and protocols for producing human antibodies and human monoclonal antibodies, see, for example, PCT publications WO98 / 24893; WO92 / 01047; WO96 / 34096; WO96 / 33735; European Patent No. 0 598,877; U.S. Patents No. 5,413,923; No. 5,625,126; No. 5,633,425; No. 5,569,825; No. 5,661,016; No. 5,545,806; No. 5,814,318; No. 5,885,793; No. 5,916,771; and No. 5,939,598.

[0123] Examples of antibodies include Herceptin® (trastuzumab; Genentech), Rituxan® (rituximab; Genentech), Lintuzumab (Seattle Genetics, Inc.), Palivizumab (Medimmune), Alemtuzumab (BTG), and Epratuzumab (Immunomedics).

[0124] "Antibody derivative" refers to an antibody or fragment that has been modified by covalent bonding of heterologous molecules, such as by the binding of heterologous polypeptides (e.g., ligand-binding domains of heterologous proteins), or by glycosylation (other than fucosylation), deglycosylation (other than defucosylation), acetylation, phosphorylation, or other modifications not usually related to the antibody or fragment.

[0125] Examples of antibody derivatives include, but are not limited to, binding domain-Ig fusions. The binding domain may be, for example, a ligand, the extracellular domain of a receptor, a peptide, a non-natural peptide, etc. Exemplary immunoglobulin or Fc domain-containing fusions include, but are not limited to, etanercept (US Patent No. 5,605,690), a fusion protein of sTNFRII and an Fc domain; alefacept (US Patent No. 5,914,111), a fusion protein of LFA-3 expressed on antigen-presenting cells and an Fc domain; cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and an Fc domain (J. Exp. Med. 181:1869 (1995)); interleukin-15 and an Fc domain (J. Immunol. 160:5742 (1998)); and factor VII and an Fc domain (Proc. Natl. Acad. Sci. USA 98:12180 (2001)). Fusion proteins of interleukin 10 and Fc region (J. Immunol. 154:5590 (1995)), fusion proteins of interleukin 2 and Fc region (J. Immunol. 146:915 (1991)), fusion proteins of CD40 and Fc region (Surgery 132:149 (2002)), Fusion proteins of Flt-3 (fms-like tyrosine kinase) and antibody Fc domain (Acta. Haemato. 95:218 (1996)), fusion proteins of OX40 and antibody Fc domain (J. Leu. Biol. 72:522 (2002)), and fusion proteins with other CD molecules (e.g., CD2, CD30 (TNFRSF8), CD95 (Fas), CD106 (VCAM-I), CD137), adhesion molecules (e.g., ALCAM (activated leukocyte adhesion molecule), cadherins, ICAM (intercellular adhesion molecule)-1, ICAM-2, ICAM-3), cytokine receptors (e.g., interleukin-4R, interleukin-5R, interleukin-6R, interleukin-9R, interleukin-10R, interleukin-12R, interleukin-13Rα1, interleukin-13Rα2, interleukin-15R, interleukin-21Rα), chemokines, cell death induction signaling molecules (e.g., B7-H1, DR6 (cell death receptor 6), PD-1 (programmed death-1), TRAIL) This includes R1), co-stimulatory molecules (e.g., B7-1, B7-2, B7-H2, ICOS (inducing co-stimulatory factors)), growth factors (e.g., ErbB2, ErbB3, ErbB4, HGFR), differentiation-inducing factors (e.g., B7-H3), activators (e.g., NKG2D), signaling molecules (e.g., gp130), BCMA, and TACI.

[0126] An "antigen" is a molecule to which an antibody, or a fragment or derivative thereof, specifically binds.

[0127] The term "specifically binding" means that an antibody, or fragment or derivative thereof, binds very selectively to its corresponding target antigen and does not bind to a large number of other antigens. For example, an antibody, or fragment or derivative thereof, binds at least about 1 x 10⁶ times. -7 M, preferably 10 -8 M~10 -9 M, 10 -10 M, 10 -11 M, or 10 -12It can bind with affinity M, and binds to a given antigen with an affinity at least twice as great as its affinity for binding to nonspecific antigens other than the closely related antigen (e.g., BSA, casein).

[0128] Antibodies can be measured for specific binding to target antigens by conventional methods such as competitive and non-competitive immunoassay systems using techniques such as Western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, precipitation immunoassay, immunoradioquantification, fluorescence immunoassay, and protein A immunoassay (see, for example, Ausubel et al., Short Protocols in Molecular Biology (John Wiley & Sons, Inc., New York, 4th edition, 1999); Harlow & Lane, Antibody Use: Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999)). Furthermore, the binding affinity of an antibody to a target antigen and the dissociation rate of the antibody-antigen interaction can be measured by surface plasmon resonance, competitive FACS using labeled antibodies, or other competitive binding assays. An example of a competitive binding assay is radioimmunoassay, which measures the presence of a labeled antigen (e.g., in the presence of an increasing amount of unlabeled antibody) in the presence of a labeled antigen (e.g., 3 H or 125 I) is incubated with the target antibody and the antibody bound to the labeled antigen is detected. The affinity and binding / dissociation rate of the antibody can then be measured from data obtained by Scatchard plot analysis. Competition with the second antibody can also be measured using radioimmunoassay. In this case, the antigen is labeled with the antigen in the presence of an increased amount of the unlabeled second antibody (e.g., 3 H or 125 I) is incubated with the target antibody bound to it. Alternatively, the binding affinity of the antibody and the binding-dissociation rate of the antibody-antigen interaction can be measured by surface plasmon resonance.

[0129] In some embodiments, the antibody, or a fragment or derivative thereof, specifically binds to CD19, CD20, CD21, CD22, CD30, CD33, CD38, CD40, CD70, CD133, CD138, or CD276. In other embodiments, the antibody, or fragment or derivative thereof, specifically binds to BMPR1B, LAT1(SLC7A5), STEAP1, MUC16, megakaryocyte-enhancing factor (MPF), Napi3b, Sema 5b, PSCA hlg, ETBR (endothelin B receptor), STEAP2, TrpM4, CRIPTO, CD21, CD79a, CD79b, FcRH2, HER2, HER3, HER4, NCA, MDP, IL20R.α, Brevican, Ephb2R, ASLG659, PSCA, PSMA, GEDA, BAFF-R, CXCR5, HLA-DOB, P2X5, CD72, LY64, FCRH1, or IRTA2.

[0130] Antibodies can be prepared from antigen-containing fragments of the target antigen using standard methods appropriate to the type of antibody (see, for example, Kohler et al., Nature, 256:495, (1975); Harlow & Lane, Antibodies, Laboratory Manual (CSHP, NY, 1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); and WO90 / 07861; Dower et al., WO91 / 17271; and McCafferty et al., WO92 / 01047, each incorporated by reference for all purposes). As an example, monoclonal antibodies can be prepared using a wide variety of techniques, including, for example, the use of hybridomas, recombinants, and phage display techniques, or combinations thereof. Hybridoma technology is outlined, for example, in Harlow et al., supra, and Hammerling et al., Monoclonal Antibodies and T Cell Hybridomas, pp. 563-681 (Elsevier, NY, 1981).Examples of phage display methods that can be used for antibody production include, for example, Briinnan et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Progress in Immunology 57:191-280; PCT application No. PCT / GB91 / 01 134; PCT publications WO90 / 02809; WO 91 / 10737; WO92 / 01047; WO92 / 18619; WO93 / 11236; This includes what is disclosed in WO95 / 15982; WO95 / 20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743; and 5,969,108. Examples of techniques that can be used for the production of single-chain Fv and antibodies include those described in U.S. Patents 4,946,778 and 5,258,498; Huston et al., 1991, Methods of Enzymology 203:46-88; Shu et al., 1993, Proc. Natl. Acad. Sci. USA 90:7995-7999; and Skerra et al., 1988, Science 240:1038-1040.

[0131] Direct administration

[0132] In some embodiments, the carbocyclic compounds described herein, such as carbafucose, can be administered to a subject, such as a mammal such as a human, to reduce the fucosylation of proteins. Accordingly, this disclosure provides, in part, a method for reducing or inhibiting the fucosylation of proteins, or fragments or derivatives thereof, by administering the compounds described herein, such as compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, such as carbafucose, or pharmaceutically acceptable salts thereof, to a subject, such as a mammal such as a human.

[0133] As used herein, subjects may be mammals such as humans, non-human primates, rats, mice, cattle, horses, pigs, sheep, goats, dogs, and cats. Such subjects may be clinical patients, clinical trial volunteers, laboratory animals, etc.

[0134] In some embodiments, after administration of a carbocyclic compound described herein (e.g., carbafucose), only a small amount of fucose is incorporated into a glycan (e.g., N-glycan or complex N-glycosidic glycan). For example, in various embodiments, compared to animals that have not received a carbocyclic compound described herein (e.g., carbafucose), less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the proteins in the animal (e.g., mammals such as humans) are corefucosylated. In some embodiments, compared to animals that have not received a carbocyclic compound described herein (e.g., carbafucose), the antibodies in the animal serum are substantially not corefucosylated (i.e., less than 0.5%).

[0135] In some embodiments, protein fucosylation is reduced by about 60%, about 50%, about 40%, about 30%, about 20%, about 15%, about 10%, about 5%, or about 1% in animal cell surface proteins (e.g., mammals such as humans) compared to animals that have not received the carbocyclic compounds described herein (e.g., carbafucose). In some embodiments, α(1,2) bond-mediated protein fucosylation is reduced by about 60%, about 50%, about 40%, about 30%, about 20%, about 15%, about 10%, about 5%, or about 1% in animal cell surface proteins (e.g., mammals such as humans) compared to animals that have not received the carbocyclic compounds described herein (e.g., carbafucose).

[0136] In some embodiments, α(1,3) linkage-mediated protein fucosylation is reduced by about 60%, about 50%, about 40%, about 30%, about 20%, about 15%, about 10%, about 5%, or about 1% in animal cell surface proteins (e.g., mammals such as humans) compared to animals that have not received the carbocyclic compounds described herein (e.g., carbafucose). In some embodiments, α(1,4) linkage-mediated protein fucosylation is reduced by about 60%, about 50%, about 40%, about 30%, about 20%, about 15%, about 10%, about 5%, or about 1% in animal cell surface proteins (e.g., mammals such as humans) compared to animals that have not received the carbocyclic compounds described herein (e.g., carbafucose).

[0137] In some embodiments, α(1,6) linkage-mediated protein fucosylation is reduced by about 60%, about 50%, about 40%, about 30%, about 20%, about 15%, about 10%, about 5%, or about 1% in animal (e.g., mammals such as humans) cell surface proteins compared to animals that have not received the carbocyclic compounds described herein (e.g., carbafucose).

[0138] In some embodiments, fucosylation of leukocytes in the serum of animals (e.g., mammals such as humans) is reduced by at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5% compared to animals that have not received the carbocyclic compounds described herein (e.g., carbafucose). In some embodiments, α(1,3) bond-mediated fucosylation of leukocytes in the serum of animals (e.g., mammals such as humans) is reduced by at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5% compared to animals that have not received the carbocyclic compounds described herein (e.g., carbafucose). In some embodiments, α(1,4) bond-mediated fucosylation of leukocytes in the serum of animals (e.g., mammals such as humans) is reduced by at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 15%, at least about 10%, or at least about 5% compared to animals that have not received the carbocyclic compounds described herein (e.g., carbafucose).

[0139] In certain embodiments, only small amounts of fucose analogs (or metabolites or products of the carbocyclic compounds described herein, e.g., carbafucose) are incorporated into the glycans of the antibody or antibody derivative (e.g., N-glycans or complex N-glycosidic glycans), or other glycans of the protein. For example, in various embodiments, less than about 60%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than 1% of the carbocyclic compounds described herein, e.g., carbafucose (or metabolites or products of the carbocyclic compounds described herein), are incorporated into the antibody glycans in the animal serum, compared to animals that have not received the carbocyclic compounds described herein. In some embodiments, less than 60%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the carbocyclic compounds described herein, e.g., carbafcose (or metabolites or products of the carbocyclic compounds described herein), are incorporated into the glycans of animal cell surface proteins, compared to animals that have not received the carbocyclic compounds described herein. In some embodiments, none of the carbocyclic compounds described herein, e.g., carbafcose (or metabolites or products of the carbocyclic compounds described herein), are incorporated into the glycans of antibodies or antibody derivatives (e.g., N-glycans or complex N-glycosidic sugar chains), or into other glycans of proteins.

[0140] In some embodiments, compared to animals that have not received the carbocyclic compounds described herein, less than 60%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the carbocyclic compounds described herein, such as carbafcose (or metabolites or products of the carbocyclic compounds described herein), are incorporated into the glycans of leukocytes in animal serum. In some embodiments, compared to animals that have not received the carbocyclic compounds described herein, none of the carbocyclic compounds described herein, such as carbafcose (or metabolites or products of the carbocyclic compounds described herein), are incorporated into the glycans of leukocytes in animal serum.

[0141] Carbocyclic compounds and their uses

[0142] In some embodiments, the carbocyclic compounds described herein, such as carbafcose, may be useful in a variety of therapeutic and non-therapeutic applications. For example, the carbocyclic compounds and / or antibodies may be used as therapeutic agents. Antibody derivatives (e.g., receptor-Fc fusions) may be used as therapeutic molecules. In some embodiments, the antibody or antibody derivative is not conjugated with other molecules. In some embodiments, the antibody is conjugated with a suitable drug (e.g., an antibody-drug conjugate) or other activator. The antibody and antibody derivative may also be used for non-therapeutic purposes such as diagnostic assays, prognostic assays, and release assays. Accordingly, this disclosure provides, in part, a kit containing the compounds described herein, such as compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, such as carbafcose, along with instructions for use in diagnostic assays, prognostic assays, release assays, etc., or for inhibiting fucosylation in animals or cells.

[0143] In some embodiments, the carbocyclic compounds can provide therapeutic benefits to patients suffering from cancer, autoimmune diseases, infections, inflammatory diseases, or sickle cell disease. Accordingly, this disclosure provides, in part, a method for treating cancer, autoimmune diseases, infections, inflammatory diseases, or sickle cell disease by administering the compounds described herein, for example, compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, for example, carbafcose, or a pharmaceutically acceptable salt thereof, to mammals such as humans. Accordingly, this disclosure provides, in part, a pharmaceutical composition for mammals such as humans, comprising the compounds described herein, for example, compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV, for example, carbafcose, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.

[0144] The subjects may have cancer, autoimmune disease, infectious disease, inflammatory disease, or sickle cell disease, or may have been diagnosed with cancer, autoimmune disease, infectious disease, inflammatory disease, or sickle cell disease, or may be control subjects confirmed not to have cancer, autoimmune disease, infectious disease, inflammatory disease, or sickle cell disease. Methods for diagnosing cancer, autoimmune disease, infectious disease, inflammatory disease, and sickle cell disease, and clinical descriptions of such diagnoses are known to those skilled in the art.

[0145] cancer

[0146] The carbocyclic compounds described herein may be useful in the treatment of cancer in patients. Administration of the carbocyclic compounds described herein to animals in need (e.g., mammals such as humans) can lead to inhibition of the proliferation of tumor or cancer cells or treatment of cancer in animals (e.g., human patients). Therefore, the carbocyclic compounds described herein can be used in a variety of situations for the treatment of animal cancers.

[0147] The carbocyclic compounds described herein may also be useful for enhancing the in vivo production of fucosylated antibodies. By increasing the proportion of such antibodies against cancer targets in patients, it may be possible to inhibit the proliferation of tumor or cancer cells or treat cancer in animals (e.g., human patients). Therefore, the carbocyclic compounds described herein can be used in a variety of situations for the treatment of animal cancers.

[0148] Certain types of cancer that can be treated with the carbocyclic compounds described herein include solid tumors and hematological malignancies. Such cancers include, but are not limited to, (1) fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangiosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, and sweat gland cancer. Cancer, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial carcinoma, glioma, glioblastoma, pleomorphic astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, and retinal cancer. Solid tumors including but not limited to blastomas, (2) acute lymphoblastic leukemia "ALL", acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia "AML", acute promyelocytic leukemia "APL", acute monoblastic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphoblastic leukemia, acute anaplastic leukemia, chronic myeloid leukemia (3) Lymphomas such as leukemia "CML", chronic lymphocytic leukemia "CLL", hairy cell leukemia, multiple myeloma, acute and chronic leukemias, including but not limited to hematological cancers such as lymphoblastic myeloid and lymphocytic myeloid leukemia, and (4) lymphomas such as Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, Waldenström hypergammaglobulinemia, heavy chain disease, and polycythemia vera.

[0149] Cancers, including but not limited to tumors, metastases, or any disease or disorder characterized by uncontrolled cell proliferation, can be treated or prevented by administering the carbocyclic compounds described herein to animals in need (e.g., mammals such as humans). In some embodiments, the present invention provides a method for treating or preventing cancer, the method comprising administering an effective amount of the carbocyclic compounds described herein and optionally a chemotherapeutic agent to an animal in need. In one embodiment, the chemotherapeutic agent is one in which the cancer has not been found to be refractory to treatment. In another embodiment, the chemotherapeutic agent is one in which the cancer has been found to be refractory to treatment. The carbocyclic compounds described herein can also be administered to animals that have also undergone surgery as treatment for cancer.

[0150] In one embodiment, the other treatment method may be radiation therapy.

[0151] In certain embodiments, the carbocyclic compounds described herein are administered concurrently with chemotherapeutic agents or radiotherapy. In other specific embodiments, the chemotherapeutic agents or radiotherapy are administered before or after the administration of the carbocyclic compounds described herein, preferably at least 1 hour, 5 hours, 12 hours, 1 day, 1 week, 2 weeks, 3 weeks, 1 month, or several months (e.g., up to 3 months) before or after the administration of the carbocyclic compounds described herein.

[0152] Chemotherapy agents can be administered over a series of sessions and may be any one or a combination of the chemotherapeutic agents provided herein. With regard to radiation, any radiotherapy protocol may be used depending on the type of cancer being treated. For example, but not limited to, X-ray radiation can be administered, particularly high-energy megavoltage (radiation with energy greater than 1 MeV) for deep tumors, and electron beam and orthovoltage X-ray radiation can be used for skin cancer. Gamma-ray radioisotopes, such as radium, cobalt, and other elemental radioisotopes, may also be administered.

[0153] Furthermore, the present invention provides a method for treating cancer with the carbocyclic compounds described herein as an alternative to chemotherapy or radiotherapy when chemotherapy or radiotherapy has been proven, or may be proven, to be too toxic, for example, when it causes unacceptable or unbearable side effects for the subject receiving treatment. The animal being treated may, at its discretion, be treated with another cancer treatment such as surgery, radiotherapy, or chemotherapy, depending on which treatment is acceptable or tolerable.

[0154] This disclosure also provides a method for treating cancer, the method comprising administering an effective amount of a therapeutic agent, which is a carbocyclic compound and an anticancer agent described herein, to an animal in need thereof. Suitable anticancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cytoxane, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. In preferred embodiments, the anticancer agent may include, but is not limited to, alkylating agents, nitrogen mustards (cyclophosphamide, ifosfamide, trophosfamide, chlorambucil), nitrosourea (carmustine (BCNU), lomustine (CCNU)), alkylsulfonic acids (busulfan, treosulfan), triazenes (dacarbazine), platinum-containing compounds (cisplatin, oxaliplatin, carboplatin), plant alkaloids (vinca alkaloids - vincristine, vinblastine, vindesine, vinorelbine), taxoids (paclitaxel, docetaxol), and DNA topoisomers. - Dehydrogenase inhibitors, epipodophilins (etoposide, teniposide, topotecan, 9-aminocamptothecin, camptothecin), cristol, mitomycin (mitomycin C); antimetabolites such as antifolic acid agents: DHFR inhibitors: methotrexate, trimethrexate; IMP dehydrogenase inhibitors: mycophenol, thiazophrine, ribavirin, EICAR; ribonucleotide reductase inhibitors: hydroxyurea deferoxamine; pyrimidine analogs: uracil analogs: 5-fluorouracil, phloxuridine, doxyfluridine, lacitrexed; cytosine analogs: cytarabine (ara C), cytosine arabinoside, fludarabine; purine analogs: mercaptopurine, thioguanine;Hormone therapy: Receptor antagonists: Anti-estrogens: Tamoxifen, raloxifene, megestrol; LHRH agonists: Goscucrine, leuprolide acetate; Anti-androgens: Flutamide, bicalutamide; Retinoids / deltoids: Vitamin D3 analogs: EB 1089, CB 1093, KH 1060; Photodynamic therapy: Bertholorfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxyhypocrelin A (2BA-2-DMHA); Cytokines: Interferon-alpha, interferon-gamma; Tumor necrosis factor: Others: Isoprenylation inhibitors: Lovastatin; Dopaminergic neurotoxins: 1-methyl-4-phenylpyridinium ion; Cell cycle inhibitors: Staurosporine; Actinomycin: Actinomycin D, dactinomycin; Bleomycin: Bleomycin A2, Bleomycin B2, Peplomycin; Anthracyclines: Daunorubicin, Doxorubicin (Adriamycin), Idarubicin, Epirubicin, Pirarubicin, Zolubicin, Mitoxantrone; MDR inhibitors: Verapamil; and Ca; 2+ ATPase inhibitors include thapsigargin.

[0155] In some embodiments, the carbocyclic compounds described herein can be used as immunostimulants in combination with cancer vaccines. As used herein, the term “cancer vaccine” means a compound that selectively damages tumor cells by inducing and / or enhancing a specific immune response against tumor cells. Cancer vaccines may be, for example, agents comprising peptides, polypeptides, or proteins of TAA or TSA, and pharmaceutical compositions comprising peptides, polypeptides, or proteins of TAA or TSA. As used herein, TSA refers to “tumor-specific antigen,” and TAA refers to tumor-associated antigen. TSA is a molecule unique to cancer cells. TAA is a molecule shared by cancer cells and normal cells, but expressed differently.

[0156] The dosage of the cancer vaccine can be determined by appropriate modification depending on the degree of stimulation of the immune response to the vaccine. Generally, it is 0.01 to 100 mg / day per adult, preferably 0.1 to 10 mg / day per adult, as the active ingredient. The cancer vaccine can be administered once every few days to once every few months. Administration can be carried out according to well-known methods for administering medical peptides, polypeptides, or proteins, such as percutaneous, intravenous, or intramuscular. The peptides, polypeptides, or proteins can be used in the presence or absence of a suitable immunostimulant, or linked to or unlinked to a carrier, in order to induce and / or enhance the immune response during administration. The carrier is not particularly limited, as long as it does not adversely affect the human body and can enhance antigenicity. Examples of carriers include cellulose, high molecular weight amino acids, and albumin. The immunostimulant may be one that is commonly used for peptide vaccination. Examples include Freund's incomplete immunostimulant (FIA), aluminum immunostimulant (ALUM), Bordetella pertussis vaccine, and mineral oil. Furthermore, appropriate formulations can be selected by applying appropriate and well-known methods for formulating peptides, polypeptides, or proteins.

[0157] Alternatively, an effective cancer vaccine effect can be obtained by collecting a fraction of mononuclear cells from the patient's peripheral blood, incubating it with the peptide, polypeptide, or protein of the present invention, and then returning the fraction of mononuclear cells in which CTL induction and / or CTL activation is observed back into the patient's blood. The carbocyclic compounds described herein can be administered simultaneously during or after the re-administration of mononuclear cells. Culture conditions such as the concentration of mononuclear cells, the concentration of the peptide, polypeptide, or protein, and the culture time can be determined simply by repeating the test. During culture, substances having the ability to enhance lymphocyte proliferation, such as interleukin-2, may be added.

[0158] autoimmune disease

[0159] The carbocyclic compounds described herein, for example, carbafcose, may be useful in modulating or treating autoimmune diseases, thereby reducing symptoms and / or autoimmune responses. The carbocyclic compounds described herein can be used in a variety of situations for the treatment of autoimmune diseases in animals.

[0160] In one embodiment, the carbocyclic compounds described herein can downcontrol or downregulate autoimmune antibodies associated with certain autoimmune diseases.

[0161] Specific types of autoimmune diseases that can be treated with the carbocyclic compounds described herein include, but are not limited to, Th2 lymphocyte-related disorders (e.g., atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omen syndrome, systemic sclerosis, and graft-versus-host disease); Th1 lymphocyte-related disorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome, Hashimoto's thyroiditis, Grebe's disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis); and activated B lymphocyte-related disorders (e.g., systemic lupus erythematosus, Goodpasture syndrome, rheumatoid arthritis, type 1 diabetes mellitus);Active chronic hepatitis, Addison's disease, allergic alveolitis, allergic reaction, allergic rhinitis, Alport syndrome, anaphylaxis, ankylosing spondylitis, antiphospholipid syndrome, arthritis, roundworm infection, aspergillosis, atopic allergy, atopic dermatitis, atopic rhinitis, Behçet's disease, bird flu, bronchial asthma, Kaplan syndrome, cardiomyopathy, celiac disease, Chagas disease, chronic glomerulonephritis, Kogan syndrome, cold agglutinin disease, congenital rubella infection, CREST syndrome, Crohn's disease, cryoglobulinemia, Cushing's syndrome, dermatomyositis, lupus discoid, Dressler syndrome, Eaton-Lambert syndrome, Echovirus infection, encephalomyelitis, endocrine ophthalmopathy, Epstein-Barr virus infection, equine hee, Erythema, Evans syndrome, Felty syndrome, fibromyalgia, Fuchs ring inflammation, gastric atrophy, gastrointestinal allergy, giant cell arteritis, glomerulonephritis, Goodpasture syndrome, graft-versus-host disease, Graves' disease, Guillain-Barré disease, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein syndrome, idiopathic purpura, adrenal atrophy, idiopathic pulmonary fibrosis, IgA nephropathy, inflammatory bowel disease, insulin-dependent diabetes mellitus, juvenile arthritis, juvenile diabetes mellitus (Type 1), Lambert-Eaton syndrome, laminitis, lichen planus, lupoid hepatitis, lupus lymphopenia, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polycystic dysplasia This includes syndromes, presenile dementia, primary aganglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, recurrent miscarriage, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, Sumpter's syndrome, schistosomiasis, Schmidt's syndrome, scleroderma, Schulmann syndrome, Scheergen syndrome, Stifmann syndrome, sympathetic ophthalmitis, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, thyroiditis, thrombocytopenia, thyroidopathy, toxic epidermal necrolysis type B, insulin-resistant type 1 diabetes, ulcerative colitis, uveitis, vitiligo, Waldenström macroglobulinemia, and Wegener's granulomatosis.

[0162] This disclosure also provides a method for treating autoimmune diseases, the method comprising administering an effective amount of a carbocyclic compound described herein, and optionally, a known anti-autoimmune disease therapeutic agent, to an animal (e.g., a mammal) in need thereof. In one embodiment, the anti-autoimmune disease therapeutic agent includes, but is not limited to, cyclosporine, cyclosporine A, mycophenylate, mofetil, sirolimus, tacrolimus, enanercept, prednisone, azathioprine, methotrexate, cyclophosphamide, prednisone, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil, DHEA, danazol, bromocriptine, meloxicam, or infliximab.

[0163] infectious disease

[0164] The carbocyclic compounds described herein, such as carbafcose, may be useful for enhancing immune responses that increase the killing or inhibition of the proliferation of infection-causing cells, or for treating infections. Accordingly, the carbocyclic compounds described herein can be used in a variety of situations for the treatment of infections in animals.

[0165] In one embodiment, the carbocyclic compounds described herein can enhance the immune response, resulting in the death or inhibition, or increased death or inhibition, of the proliferation of cells that produce specific infections.

[0166] The specific types of infections that can be treated with the carbocyclic compounds described herein include, but are not limited to, (1) bacterial diseases: dipteria, pertussis, latent bacteremia, urinary tract infections, gastroenteritis, cellulitis, epiglottitis, tracheitis, adenoid hypertrophy, reposterior pharyngeal abscess, impetigo, eczema, pneumonia, endocarditis, septic arthritis, pneumococcal infections, peritonitis, bacterial meningitis, acute suppurative meningitis, urethritis, cervicitis, proctitis, pharyngitis, salpingitis, epididymitis, (1) Gonorrhea, syphilis, listeriosis, anthrax, nocardiosis, salmonella, typhoid fever, dysentery, conjunctivitis, sinusitis, brucellosis, talaremia, cholera, bubonic plague, tetanus, necrotizing enterocolitis, mixed actinomycete anaerobic infection, syphilis, relapsing fever, leptospirosis, Lyme disease, rat bite fever, tuberculosis, lymphadenitis, leprosy, chlamydia, chlamydial pneumonia, trachoma, inclusion conjunctivitis, systemic; (2) Fungal diseases: histoplasmosis, coccidiosis, blastomycosis, sporotrichosis, cryptococcosis, (3) Systemic candidiasis, aspergillosis, mucolitis, mycetoma, chromomycetis; (4) Rickettsial diseases: typhus, Rocky Mountain spotted fever, errlichiosis, Eastern tick-borne rickettsial disease, rickettsialpox, Q fever, and bartonellosis; (5) Parasitic diseases: malaria, babesiosis, African sleeping sickness, Chagas disease, leishmaniasis, dum-dum fever, toxoplasmosis, meningoencephalitis, keratitis, enterobacteriaceae, giardiasis Cryptosporidiasis, isosporiasis, cyclosporiasis, microsporidiasis, roundworm infection, whipworm infection, hookworm infection, filarial infection, ocular larval migratory disease, trichinellosis, guinea worm disease, lymphatic filariasis, roasis, river blindness, canine heartworm infection, schistosomiasis, swimmer's pruritus, Oriental lung fluke, Oriental liver fluke, fascial claudication, fascial illusion, opisthochiosis, tapeworm infection, hydatidosis, alveolar hydatidosis;(5) Viral diseases: Measles, subacute sclerosing panencephalitis, common cold, mumps, rubella, rash, fifth disease, chickenpox, respiratory syncytial virus infection, crow, bronchiolitis, infectious mononucleosis, polio, herpangina, hand-foot-and-mouth disease, Bornholm's disease, genital herpes, genital warts, aseptic meningitis, myocarditis, pericarditis, gastroenteritis, acquired immunodeficiency syndrome (AIDS), Reye's syndrome, Kawasaki syndrome, influenza, bronchitis, viral pneumonia, acute febrile respiratory illness, acute pharyngoconjunctival fever, epidemic keratoconjunctivitis, herpes simplex virus 1 (HSV-1), herpes simplex This includes Herpes Zoster 2 virus (HSV-2), shingles, cytoplasmic inclusion disease, rabies, progressive multiple leukoencephalopathy, Koo's disease, fatal familial insomnia, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, tropical spastic paralysis, Western equine encephalitis, California encephalitis, St. Louis encephalitis, yellow fever, dengulymphocytic choroidal meningitis, Lassa fever, hemorrhagic fever, Hunt virus, pulmonary syndrome, Marburg virus infection, Ebola virus infection, smallpox, and COVID-19.

[0167] This disclosure also provides a method for treating an infectious disease, the method comprising administering a carbocyclic compound described herein and, optionally, a therapeutic agent which is an anti-infective agent, to an animal (e.g., a mammal) in need thereof. In one embodiment, the anti-infective agent is, without limitation, (1) antibacterial agents: β-lactam antibiotics: penicillin G, penicillin V, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, bacampicillin, azurocillin, carbenicillin, mezlocillin, piperacillin, ticalcillin; aminoglycosides: amikacin, gentamicin, kanamycin, neomycin, netylmycin, streptomycin, tobramycin; macrolides: azithromycin, clarithromycin, erythromycin, lincomycin, clindamycin; Tetracyclines: Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline; Quinolones: Synoxacin, Nalidixic acid; Fluoroquinolones: Ciprofloxacin, Enoxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxicin; Polypeptides: Bacitracin, Colistin, Polymyxin B; Sulfonamides: sulfisoxasol, sulfamethoxasol, sulfadiazine, sulfamethizol, sulfacetamide; Other antibacterial agents: trimethoprim, sulfametasol, chloramphenicol, vancomycin, metronidazole, quinupristin, dalfopristin, rifampin, spectinomycin, nitrofurantoin; Antiviral agents: common antiviral agents: idoxrazine, vidarabine, trifluridine, acyclovir, famcicyclovir, pencicyclovir, valacyclovir, gancicyclovir, foscarnet, ribavirin, amantadine, rimantadine, cidofovir; Antisense oligonucleotides; Immunoglobulins; Interferon; The drugs used to treat HIV infection are zidovudine, didanosine, zalcitabine, stabudine, lamivudine, nevirapine, delavirudine, saquinavir, ritonavir, indinavir, and nelfinavir.

[0168] inflammatory diseases

[0169] Inflammation involves the vascularized living tissue's response to injury. For example, fucose containing cell adhesion-mediated epitopes is important in the body's anti-infective immune response, but in other situations, fucose containing cell adhesion-mediated epitopes can be undesirable or excessive, leading to tissue damage instead of repair. For instance, many medical conditions (autoimmune and inflammatory diseases, shock, and reperfusion injury, etc.) involve abnormal adhesion of leukocytes mediated by fucose-containing epitopes. Thus, inflammation affects blood vessels and adjacent tissues in response to damage or abnormal stimuli caused by physical, chemical, or biological factors. Examples of inflammatory diseases or disorders include, but are not limited to, vascular inflammatory diseases, dermatitis, chronic eczema, psoriasis, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, graft-versus-host disease, sepsis, diabetes mellitus, atherosclerosis, Sjögren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary syndrome, ischemic reperfusion, Crohn's disease, inflammatory bowel disease, endometriosis, glomerulonephritis, myasthenia gravis, idiopathic pulmonary fibrosis, asthma, allergic reactions, acute respiratory distress syndrome (ARDS) or other acute leukocytosis-mediated lung injury, vasculitis, or inflammatory autoimmune myositis. Other diseases and disorders for which the carbocyclic compounds described herein may be useful for treatment and / or prevention include hypercoronary circulation, microbial infections, cancer metastasis, thrombosis, wounds, burns, spinal cord injury, gastrointestinal mucosal disorders (e.g., gastritis, ulcers), osteoporosis, osteoarthritis, septic shock, traumatic shock, stroke, nephritis, atopic dermatitis, frostbite, adult dyspnea syndrome, ulcerative colitis, diabetes and reperfusion injury after ischemic episodes, prevention of restenosis associated with vascular stent placement, and prevention of undesirable angiogenesis, such as angiogenesis associated with tumor growth.

[0170] Other medications

[0171] The methods provided in accordance with this disclosure may further include the administration of a carbocyclic compound described herein, for example, carbafcose, and a therapeutic agent or a pharmaceutically acceptable salt or solvate thereof. The carbocyclic compounds and therapeutic agents described herein may act additionally or, more preferably, synergistically. In a preferred embodiment, a composition comprising a carbocyclic compound described herein is administered concurrently with the administration of one or more therapeutic agents, which may be part of or different from the same composition comprising a carbocyclic compound described herein. In another embodiment, the carbocyclic compound described herein is administered before or after the administration of the therapeutic agent.

[0172] In the present invention, a method for treating cancer, autoimmune disease, or infection, the therapeutic agent may be an antiemetic. Suitable antiemetics include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acetylleucine monoethanolamine, arizaprid, azasetron, benzquinamide, vietanautin, bromoprid, buclidine, clevoprid, cyclidine, dimenhydrinate, diphenidol, drasetron, meclizine, metalatarl, metopimazine, nabilone, oxypendyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinol, thiethylperazine, thioproperazine, and tropisetron.

[0173] In another embodiment, the therapeutic agent may be a hematopoietic colony-stimulating factor. Suitable hematopoietic colony-stimulating factors include, but are not limited to, filgrastim, salglamostim, morglamostim, and erythropoietin alfa.

[0174] In yet another embodiment, the therapeutic agent may be an opioid or non-opioid analgesic. Suitable opioid analgesics include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, methopone, apomorphine, normorphine, etorphine, buprenorphine, meperidine, lopermid, anilelysine, etheptadine, pimidine, betaprozine, diphenoxylate, fentanyl, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazosine, pentazosine, cyclazosine, methadone, isomethadone, and propoxifene. Appropriate non-opioid analgesics include, but are not limited to, celecoxib, rofecoxib, diclofinac, diflucinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamete, mefenamic acid, nabumetone, naproxen, piroxicam, and sulindac.

[0175] Composition, dosage, and administration

[0176] The carbocyclic compounds described herein (e.g., carbafcose) can be formulated for therapeutic use. The carbocyclic compounds can be formulated as pharmaceutical compositions comprising a therapeutically or prophylactically effective amount of antibody or derivative and one or more pharmaceutically compatible (acceptable) components.

[0177] The “effective dose” of a carbocyclic compound (e.g., carbafcose) as described herein includes a therapeutic effective dose, a prophylactic effective dose, or a nutritional effective dose. “Therapeutic effective dose” refers to the effective dose in the amount and duration required to achieve the desired therapeutic outcome. The therapeutic effective dose of a compound may vary depending on factors such as the individual’s disease state, age, sex, and weight, as well as the compound’s ability to induce the desired response in the individual. The dosage regimen can be adjusted to provide the optimal therapeutic response. The therapeutic effective dose is also the amount in which the toxic or adverse effects of the compound outweigh the therapeutically beneficial effects. “Prophylactic effective dose” refers to the effective dose in the amount and duration required to achieve the desired prophylactic outcome. Typically, a prophylactic effective dose may be less than a therapeutic effective dose, as prophylactic doses are used in subjects before or at an early stage of the disease. An exemplary range for the therapeutic or prophylactic effective dose of a compound may be approximately 5 to approximately 50 mg / day per kg of body weight of a subject, e.g., a human.

[0178] It should be noted that the dosage may vary depending on the severity of the condition to be alleviated. For any particular subject, a specific dosing plan may be adjusted over time according to the individual needs and the professional judgment of the administerer or supervisor of the composition. The range of dosages described herein is illustrative and does not limit the range of dosages that can be selected by a practicing physician. The amount of the active compound in the composition may vary depending on factors such as the individual's disease state, age, sex, and weight. The dosing plan can be adjusted to provide an optimal therapeutic response. For example, it may be administered as a single rapid dose, or in several divided doses over time, or the dosage may be proportionally reduced or increased depending on the urgency of the treatment situation. For ease of administration and uniformity of dosage, it may be advantageous to formulate the parenteral composition in dosing unit form.

[0179] In general, the carbocyclic compounds should be used without causing substantial toxicity. The toxicity of the carbocyclic compounds can be measured using standard techniques, for example, by testing the therapeutic index in cell cultures, experimental animals, or subjects. Here, the therapeutic index is the ratio of LD50 (lethal dose for 50% of the population) to ED50 (minimum effective dose for 50% of the population) for non-human animals, and the ratio of TD50 (toxic dose for 50% of the population) to ED50 (minimum effective dose for 50% of the population) for humans. The maximum tolerated dose (MTD) is the highest dose of a compound or composition that is administered regularly over a period of time in a subject study without causing obvious toxicity (e.g., without causing unacceptable side effects). The subject may be a human or an animal such as a mouse or rat.

[0180] The regularly administered dose may be a daily dose, a single rapid dose, or the daily dose may be divided into two or more partial doses so that the subject receives the total daily dose over time. The study period may vary from several days to several months, for example, about 10, 20, 30, 60, 90, or 120 days, or any value in between. Examples of apparent toxicity may include, but are not limited to, significant cell death or organ dysfunction, toxic symptoms predicted to significantly shorten the subject's lifespan, or a delay of 10% or more in weight gain. In some embodiments, the carbocyclic compound may be provided in a form suitable for administration to humans or animals in the presence of liposomes, immunostimulants, or any pharmaceutically or physiologically acceptable carrier, together with other compounds (e.g., nucleic acid molecules, small molecules, peptides, or peptide analogs). If necessary, therapies using the carbocyclic compounds according to the present invention may be combined with more traditional and existing therapies for the condition being treated.

[0181] A formulation or composition suitable for administering a carbocyclic compound to a patient suffering from a condition to be treated may be provided using conventional pharmaceutical or non-pharmaceutical methods. For example, a pharmaceutical or non-pharmaceutical composition typically comprises one or more carriers (e.g., water and a sterile liquid such as oil (including petroleum, animal, plant, or synthetically derived oils such as peanut oil, soybean oil, mineral oil, sesame oil, etc.)). When the pharmaceutical composition is administered intravenously, water is a more typical carrier. Saline solution, aqueous dextrose, and glycerol solution can also be used as liquid carriers, especially for injectable solutions. Suitable excipients include, for example, amino acids, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. If necessary, the composition may also contain a small amount of wetting agent or emulsifier, or a pH buffer. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmacology" by E.W. Martin. Such compositions typically contain a therapeutically effective amount of protein, typically in a purified form, together with an appropriate amount of carrier to provide a form for appropriate administration to a patient. The formulation corresponds to the mode of administration.

[0182] Any suitable route of administration may be employed, such as parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intrasacral, intraspinal, intracisional, intraperitoneal, intranasal, aerosol, or oral administration. The therapeutic formulation may be in the form of a liquid solution or suspension; in the case of oral administration, the formulation may be in the form of a tablet or capsule; and in the case of an intranasal formulation, it may be in the form of a powder, nasal drop, or aerosol.

[0183] Methods for producing formulations known in the art can be found, for example, in Remington's Pharmacology. Parenteral formulations may contain, for example, excipients, sterile water, or saline, polyalkylene glycosides such as polyethylene glycoside, plant-derived oils, or hydrogenated naphthalene. The release of the compound may be controlled using biocompatible or biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers. Other potentially useful parenteral delivery systems for moduloable compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Inhalation formulations may contain excipients, such as lactose, and may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholine salts, and deoxycholate salts, or oily solutions for administration in the form of nasal drops or gels. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the drug may also contain a solubilizer and a local anesthetic (such as lignocaine) to relieve pain at the injection site. Generally, the components are supplied separately or mixed together in a unit dosage form, for example, as a lyophilized powder or water-free concentrate in a sealed container such as an ampoule or pouch indicating the amount of the active ingredient. When the drug is administered by infusion, it can be prepared in an infusion bottle containing sterile pharmaceutical-grade water or saline. When the drug is administered by injection, an ampoule of sterile water or saline for injection can be provided so that the components can be mixed before administration.

[0184] The present invention will be further explained in the following embodiments.

[0185] [Examples]

[0186] General details of the experiment

[0187] All described anhydrous reactions were carried out under a nitrogen atmosphere using flame-dried glassware. Normal-phase column chromatography was performed using 230-400 mesh silica gel (Silicycle, SiliaFlash® P60). Trace amounts of solvent were concentrated and removed under vacuum provided by a Buchi rotary evaporator with a dry ice / acetone condenser and a Buchi V-500 pump.

[0188] All reagents and starting materials were purchased from Sigma Aldrich, Alfa Aesar, TCI America, Arcos, or Carbosynth and used without repurification. All solvents were purchased from Sigma Aldrich, EMD, Anachemia, Caledon, Fisher, or ACP and used without repurification unless otherwise noted. CH2Cl2 was freshly distilled over CaH2, and tetrahydrofuran (THF) was freshly distilled over Na metal / benzophenone. Low temperature The reaction was maintained under the following conditions: 0°C, ice bath, -78°C, acetone-dry ice bath. The temperature of -78°C to 0°C, which is required for longer reaction times, was maintained in a 2-propanol bath using a Neslab Cryocool Immersion Cooler (CC-100 II).

[0189] Nuclear magnetic resonance (NMR) spectra were recorded using CDCl3 or CD3OD. Signal position (δ) is shown in ppm from tetramethylsilane (δ 0) and measured relative to the solvent signal. 1 H NMR: CDCl3: δ 7.26, CD3OD: δ 3.31, D2O: δ 4.79; 13 ¹³C NMR: CDCl3: δ 77.16, CD3OD: δ 49.00). The coupling constant (J value) is shown in Hertz (Hz), and reported at the nearest 0.1 Hz. 1 The 1H NMR spectral data were presented in the following order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br., broad), binding constant, and proton number.

[0190] Infrared (IR) spectra were recorded using a Perkin Elmer Spectrum Two® Fourier transform spectrometer with a suitable sample. For each compound, only selected characteristic absorption data are provided.

[0191] High-resolution mass spectra were obtained using an Agilent 6210 TOF LC / MS with ESI-MS technology.

[0192] Optical rotation was measured at 589 nm using a Perkin Elmer 341 polarimeter. JPEG2026136206000022.jpg97161

[0193] Synthesis of diene 4: Selectfluo® (2.0 mmol, 1 equivalent) and (L)-proline (2.0 mmol, 1 equivalent) were added to a 20 mL solution of DMF (2.0 mmol, 1 equivalent) at 5°C. The mixture was stirred at 5°C for 1 hour, treated with H2O, and then extracted with Et2O. The combined organic layer was washed with brine and then dried over Na2SO4. The solvent was removed under vacuum, and the residue was redissolved in CH2Cl2 (10 mL). Subsequently, (L)-proline (1.6 mmol, 0.8 Equivalents of 5-(methanesulfonyl)-1-phenyl-1H-tetrazole (5.0 mmol, 2.5 equivalents) were added dropwise to 14 mL of cooled (-78°C) 5-(methanesulfonyl)-1-phenyl-1H-tetrazole (5.0 mmol, 2.5 equivalents) in THF solution, and the mixture was stirred at 0°C. The resulting mixture was treated with H2O and extracted with Et2O. The combined organic layers were washed with brine and dried over Na2SO4. The solvent was removed under vacuum and the residue was dissolved in THF (6 mL). In a separate flask, LiHMDS (1.0 M THF solution, 5.0 mmol, 2.5 equivalents) was added dropwise to 14 mL of cooled (-78°C) THF solution of 5-(methanesulfonyl)-1-phenyl-1H-tetrazole (5.0 mmol, 2.5 equivalents) and stirred at -78°C for 30 minutes. Subsequently, a THF solution (6 mL) of ketone 3 was added dropwise at -78°C, and the mixture was stirred for a further 1 hour before being quenched with H2O. The mixture was extracted with Et2O, and the combined organic layer was washed with brine and dried with Na2SO4. The solvent was removed under vacuum, and the residue was purified by flash column chromatography (pentane:ethyl ether, 25:1) to obtain compound 4 as a yellow oil (276 mg, 30% in 2 steps). [α] D = -14.5 (c = 1.1, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 5.94 (ddd, J = 6.5, 10.5, 17.1 Hz, 1 H), 5.40 (dt, J = 1.4, 17.3 Hz, 1 H), 5.23 (m, 1 H), 5.01 (m, 1 H), 4.68-4.46 (m, 2 H), 4.13 (d, J = 8.8 Hz, 1 H), 3.56 (ddd, J = 5.4, 8.9, 27.8 Hz, 1 H), 1.79 (d, J = 5.4 Hz, 1 H), 1.71 (s, 3 H), 1.07 (s, 12 H), 1.06 (s, 6 H), 0.88 (s, 9 H), 0.08 (s, 3 H), 0.02 (s, 3 H); 13C NMR (101 MHz, CDCl3) δ 145.2, 137.2 (d, J = 7.0 Hz), 117.4, 115.3, 93.3 (d, J = 183.0 Hz), 76.4 (d, J = 4.2 Hz), 74.5 (d, J = 22.6 Hz), 70.5 (d, J = 18.2 Hz), 25.9, 18.2, 18.1, 18.1, 18.1, 16.4, 12.6, -4.6, -5.3, -5.3; 19 F NMR (377 MHz, CDCl3) δ -208.7 (ddd, J = 12.6, 27.8, 46.3 Hz); IR (neat) ν 3570, 2939, 1641, 1466, 1087, 829 cm -1 HRMS (ESI-TOF) m / z: [M + H] + C 24 H 50 Calculated value for FO3Si2: 461.3277; measured value: 461.3274.

[0194] Synthesis of Triol 5: TBAF (1.5 mmol, 3 equivalents) was added to a THF solution (5 mL) of 4 (0.5 mmol, 1 equivalent) at 0°C. The mixture was stirred at 0°C for 2 hours. The solvent was removed under vacuum, and the residue was purified by flash column chromatography (pentane:ethyl acetate, 1:1) to obtain compound 5 as a colorless oil (85 mg, 90%).

[0195] Synthesis of fluorotriol 6: Grubbs' II catalyst (0.01 mmol, 0.1 equivalent) was added to a solution of 5 (0.1 mmol, 1 equivalent) of CH2Cl2 at room temperature (5 mL). The mixture was heated to 40°C under argon and held at 40°C for 2 hours. The reaction was cooled to room temperature and concentrated under vacuum. The residue was then purified by flash column chromatography (pentane:ethyl acetate, 6:1 then 1:1) to obtain compound 6 as a colorless oil (12.1 mg, 71%). [α] D = -79.5 (c = 0.5, CH3OH); 1H NMR (400 MHz, CD3OD) δ 5.36 (m, 1 H), 4.44 (ddd, J = 7.2, 10.4, 54.3 Hz, 1 H), 4.16 (m, 1 H), 3.99 (t, J = 4.7 Hz, 1 H), 3.63 (ddd, J = 4.4, 8.5, 10.5 Hz, 1 H), 1.83 (t, J = 1.8 Hz, 3 H); 13 C NMR (151 MHz, CD3OD) δ 136.9 (d, J = 2.4 Hz), 126.9 (d, J = 9.7 Hz), 96.0 (d, J = 177.7 Hz), 72.4 (d, J = 7.7 Hz), 71.7 (d, J = 20.6 Hz), 71.1 (d, J = 16.0 Hz), 20.6; 19 F NMR (377 MHz, CD3OD) δ -205.2; IR (cast film) ν 3728, 3691, 2919, 1718, 1443, 967 cm -1 HRMS (ESI-TOF) m / z: [M - H] - C7H 10 Calculated value for FO3: 161.0619; measured value: 161.0617.

[0196] Synthesis of acetate 7: 6 (0.01 mmol) was dissolved in Ac20 (25 μL) and pyridine (25 μL) and stirred at room temperature for 12 hours. The solvent was removed under vacuum, and the residue was purified by flash column chromatography (pentane:ethyl acetate, 2:1) to obtain compound 7 as a colorless oil (2.3 mg, 78%).

[0197] Synthesis of Fluorotriol 8: A mixture of 6 (0.04 mmol, 1 equivalent) and 10% Pd / C (0.004 mmol, 0.1 equivalent) in EtOH (0.4 mL) was stirred at room temperature under H2 (2 atm). After 12 hours, the reaction mixture was filtered through Celite, and the filtrate was evaporated under vacuum. The residue was purified by flash column chromatography (pentane:ethyl acetate, 2:1) to obtain compound 8 as a colorless oil (5.6 g, 85%, a mixture of diastereomers in a ratio of approximately 1.5:1). 19 F NMR (377 MHz, CD3OD): δ -203.7, -203.8. JPEG2026136206000023.jpg43149

[0198] Synthesis of acetate 9: 8 (0.01 mmol) was dissolved in Ac2O (25 μL) and pyridine (25 μL) and stirred at room temperature for 12 hours. The solvent was removed under vacuum, and the residue was purified by flash column chromatography (pentane:ethyl acetate, 2:1) to obtain compound 9 as a colorless oil (2.2 mg, 77%). JPEG2026136206000024.jpg96161

[0199] Synthesis of Ketone 10: To a CH2Cl2 solution (5 mL) of aldehyde 1 (2.0 mmol, 1 equivalent) at 0°C, NCS (2.2 mmol, 1.1 equivalents) and L-proline (1.6 mmol, 0.8 equivalents) were added. The reaction mixture was left at 0°C for 30 minutes, and a DMSO solution (5.0 mL) of ketone 2 (4 mmol, 2 equivalents) was added at 0°C. The reaction mixture was heated to room temperature and stirred for 12 hours. The reaction mixture was diluted with CH2Cl2, washed with water, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (pentane:ethyl ether, 8:1) to obtain compound 10 as a yellow oil (536 mg, 56%). [α] D = -25.3 (c = 1.19, CHCl3); 1H NMR (600 MHz, CDCl3) δ 5.66-5.58 (m, 4 H), 5.36-5.27 (m, 2 H), 4.83 (dd, J = 10.5, 3.0 Hz, 1 H), 4.41 (m, 1 H), 2.14 (s, 3 H), 2.08 (s, 3 H), 2.06 (m, 1 H), 1.97 (s, 3 H), 1.92-1.78 (m, 2 H), 1.24 (s, 18 H), 0.95 (d, J = 6.6 Hz, 3 H); 13 IR (neat) ν 3522, 2994, 1721, 1099, 838cm -1 ; HRMS (ESI-TOF) m / z: [M + NH4] + C 23 H 51 Calculated value for NClO4Si2: 496.3040; measured value: 496.3045.

[0200] Synthesis of diene 11: LiHMDS (1.0 M THF solution, 2.7 mmol, 2 equivalents) was added dropwise to a cooled (-78°C) THF solution (9.5 mL) of 5-(methanesulfonyl)-1-phenyl-1H-tetrazole (2.7 mmol, 2 equivalents), and the mixture was stirred at -78°C for 30 minutes. Then, 1.35 mmol, 1 equivalent of 10 (4 mL) THF solution was added dropwise at -78°C, and the mixture was stirred for a further 1 hour, followed by quenching with H2O. The mixture was extracted with Et2O, and the combined organic layers were washed with brine and dried over Na2SO4. The solvent was removed under vacuum, and the residue was purified by flash column chromatography (pentane:ethyl ether, 20:1) to obtain compound 11 as a yellow oil (464 mg, 72%). [α] D = -15.1 (c = 0.96, CHCl3); 1H NMR (500 MHz, CDCl3) δ 6.09 (ddd, J = 7.2, 10.3, 17.4 Hz, 1 H), 5.31 (dt, J = 1.3, 17.3 Hz, 1 H), 5.22 (dt, J = 1.0, 10.3 Hz, 1 H), 5.0 (m, 1 H), 4.54 (m, 1 H), 4.33 (d, J = 5.4 Hz, 1 H), 4.04 (d, J = 8.6 Hz, 1 H), 3.98 (dd, J = 5.6, 8.7 Hz, 1 H), 2.12 (d, J = 5.5 Hz, 1 H), 1.73 (s, 3 H), 1.07 (s, 12 H), 1.06 (s, 6H), 0.88 (s, 9H), 0.10 (s, 3H), 0.03 (s, 3H); 13 IR (neat) ν 3661, 2957, 1572, 1462. 1084, 836 cm -1 HRMS (ESI-TOF) m / z: [M + H] + C 24 H 50 Calculated value for ClO3Si2: 477.2982; measured value: 477.2977.

[0201] Synthesis of triol 12: TBAF (3.0 mmol, 3.0 equivalents) was added to a THF solution (10 mL) of 11 (1.0 mmol, 1 equivalent) at 0°C. The mixture was stirred at 0°C for 2 hours. The solvent was removed under vacuum, and the residue was purified by flash column chromatography (pentane:ethyl acetate, 1:1) to obtain compound 12 as a yellow oil (169 mg, 82%).

[0202] Synthesis of Tetraol 13: Grubbs' II catalyst (0.05 mmol, 0.1 equivalent) was added to a solution of 12 (0.5 mmol, 1 equivalent) of CH2Cl2 (25 mL) at room temperature. The mixture was heated to 40°C under argon and held at 40°C for 1 hour. The reaction was cooled to room temperature, and the reaction mixture was filtered through Celite. The filtrate was evaporated under vacuum. The mixture was redissolved in THF (2.5 mL) / NaOH (2.0 M, 0.5 mL). The reaction was heated and refluxed for 12 hours. The reaction was cooled to room temperature. The solvent was removed under vacuum, and the residue was purified by flash column chromatography (CH2Cl2:MeOH, 20:1 then 4:1) to obtain compound 13 as a colorless oil (8.0 mg, 10%). [α] D = -61.5 (c = 0.9, CH3OH); 1 H NMR (400 MHz, CD3OD) δ 5.40 (m, 1 H), 3.95 (d, J = 4.3 Hz, 1 H), 3.88 (m, 1 H), 3.57 (dd, J = 7.6, 10.5 Hz, 1 H), 3.38 (dd, J = 4.3, 10.5 Hz, 1 H), 1.82 (t, J = 1.7 Hz, 1 H); 13 C NMR (151 MHz, CD3OD) δ 136.3, 128.3, 74.1, 73.6, 72.8, 72.0, 20.8; IR (cast film) ν 3724, 3698, 2349, 1664, 1436, 1084 cm -1 ; HRMS (ESI-TOF) m / z: [M + NH4] + C7H 16 Calculated value for NO4: 178.1074; measured value: 178.1075.

[0203] Synthesis of acetate 14: 13 (0.01 mmol) was dissolved in Ac2O (25 μL) and pyridine (25 μL) and stirred at room temperature for 12 hours. The solvent was removed under vacuum, and the residue was purified by flash column chromatography (pentane:ethyl acetate, 3:1) to obtain compound 14 as a colorless oil (2.6 mg, 79%).

[0204] Synthesis of 15: A mixed solution of 13 (0.025 mmol, 1 equivalent) and 10% Pd / c (0.0025 mmol, 0.1 equivalent) in EtOH (0.25 mL) was stirred at room temperature under H2 (2 atm). After 12 hours, the reaction mixture was filtered through Celite, and the filtrate was evaporated under vacuum. The residue was purified by flash column chromatography (CH2Cl2:MeOH, 20:1) to obtain 15 as a colorless oil (3.0 mg, 78%, a mixture of 1.5:1 diastereomers, mainly 1-21). The diastereomers were separated by column chromatography (a solution of ethyl acetate in 20% MeOH) to obtain 1-21. [α] D 20 = +22.3 (c = 0.05, CH3OH); 1 H NMR (600 MHz, CD3OD) δ 3.68 (brs, 1 H), 3.48-3.45 (m, 1 H), 3.39-3.35 (m, 1 H), 3.27 (dd, J = 9.6, 3.0 Hz, 1 H), 1.65-1.60 (m, 1 H), 1.57-1.48 (m, 2 H), 1.02 (d, J = 6.8 Hz, 3 H); 13 C NMR (151 MHz, CD3OD): δ 76.5, 76.4, 74.9, 73.8, 35.8, 33.1, 17.8; IR (neat) ν 3368, 2958, 2928, 2857, 1731, 1668, 1462, 1261, 1067, 1022, 799cm -1 ; HRMS (ESI-TOF) m / z: [M + Na] + C7H 14 Calculated value for NaO4: 185.0784; measured value: 185.0784.

[0205] Synthesis of acetate 16: 0.01 mmol of 15 was dissolved in Ac2O (25 μL) and pyridine (25 μL) and stirred at room temperature for 12 hours. The solvent was removed under vacuum, and the residue was purified by flash column chromatography (pentane:ethyl acetate, 3:1) to obtain compound 16 as a colorless oil (2.6 mg, 80%).

[0206] Glycan analysis

[0207] One day after gene transfer using HER2 antibody, Expi-CHO cells were treated with compounds 16, 7, 9, 13, 6, 8, 14, and 15 at a final concentration of 0.1 mM. Eight days later, the gene-transferred cells were collected, and the purified HER2 antibody was deglycosylated using PNGaseF. The released glycans were purified and analyzed by capillary electrophoresis-laser-induced fluorescence (CE-LIF). The results are shown in Table 1. Table 1 JPEG2026136206000025.jpg116138JPEG2026136206000026.jpg63154

[0208] Epoxides 1-2 were prepared according to the literature protocol (Hudlicky, T.;Price, JD;Rulin, F.;Tsunoda, TJ Am. Chem. Soc. 1990, 112, 9439-9440). Acetates 1-3 were prepared according to the literature protocol (Banwell, MG;Ma, X.;Karunaratne, OP;Willis, AC Aust. J. Chem. 2010, 63, 1437-1447).

[0209] Synthesis of vinyl bromide 1-4: Potassium carbonate (200 mg, 1.45 mmol) was added to a methyl solution (5 mL) of viriacetate 1-3 (402 mg, 1.31 mmol) that had been stirred, and the mixture was stirred for 1 hour. The reaction mixture was concentrated under reduced pressure. The resulting solid mass was treated with a saturated aqueous solution of NH4Cl (10 mL) and extracted with ethyl acetate (20 mL). The aqueous layer was separated and further extracted with ELISA (2 x 20 mL). The combined organic phases were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain a crude diol as a pale yellow viscous liquid. Without further purification, this crude diol was dissolved in CH2Cl2 (5 mL) and cooled to 0°C. Triethylamine (1.25 mL, 8.97 mmol), followed by tert-butyldimethylsilyltrifluoromethanesulfonate (0.90 mL, 3.92 mmol) was added dropwise. The ice bath was removed, and the reaction mixture was heated to room temperature. After stirring for 10 hours, the reaction mixture was treated with a saturated aqueous solution of NH4Cl (10 mL) and extracted with Et20 (20 mL). The aqueous layer was separated and further extracted with Et20 (2 × 20 mL). The combined organic phases were washed with brine (20 mL), dried over anhydrous Na2SiO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (10% Et20 in hexane solution) to obtain 1-4 as a white solid (491 mg, 76%). 1 H NMR (500 MHz, CDCl3) δ 6.06 (d, J = 3.4 Hz, 1 H), 4.61 (d, J = 6.4 Hz, 1 H), 4.16 (t, J = 6.2 Hz, 1 H), 4.03 (m, 1 H), 3.81 (t, J = 5.6 Hz, 1 H), 1.49 (s, 3 H), 1.39 (s, 3 H), 0.90 (s, 9 H), 0.89 (s, 9 H), 0.12 (s, 6 H), 0.10 (s, 3 H), 0.09 (s, 3 H).

[0210] Synthesis of Ketones 1-5: A flame-dried 50-mL flask equipped with a magnetic stirring rod, argon inlet, and septum was filled with acetonide 1-4 (565 mg, 1.14 mmol) and THF (6 mL). The mixture was cooled to -55°C using a dry ice and acetone bath, and then tert-butyllithium (1.45 M pentane solution, 1.25 mL, 1.81 mmol) was added dropwise. The reaction mixture was stirred at -55°C for 30 minutes, and then triisopropyl borate (neat, 1.10 mL, 4.77 mmol) was added dropwise. The dry ice and acetone bath was removed, and the reaction mixture was slowly heated to room temperature and stirred for 60 hours. Then, NaBO3·4H2O (1.77 g, 11.5 mmol) and H2O (6 mL) were added. The mixture was stirred at room temperature for 24 hours, then poured into water (20 mL) and extracted with Et20 (30 mL). The aqueous layer was separated and further extracted with Et20 (2 x 30 mL). The combined organic phase was washed with brine (20 mL), dried over anhydrous Na2SiO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (20% Et20 in hexane solution) to obtain 1-5 as a white solid (386 mg, 78% in 3 steps). 1 H NMR (500 MHz, CDCl3) δ 4.42 (m, 2 H), 4.19 (m, 1 H), 4.12 (m, 1 H), 2.94 (dd, J = 13.6, 2.9 Hz, 1 H), 2.38 (dd, J = 13.6, 3.8 Hz, 1 H), 1.41 (s, 3 H), 1.36 (s, 3 H), 0.92 (s, 9 H), 0.86 (s, 9 H), 0.16 (s, 3 H), 0.13 (s, 3 H), 0.11 (s, 3 H), 0.10 (s, 3 H); 13 C NMR (126 MHz, CDCl3) δ 206.2, 110.6, 82.3, 78.1, 74.6, 70.3, 42.8, 27.3, 25.9, 25.7, 25.6, 18.0, 17.9, -4.88, -4.91, -5.1. JPEG2026136206000027.jpg71152

[0211] Synthesis of Alkynes 1-6: To a THF solution (1 mL) of stirred alkynyltrimethylsilane (0.25 mL, 1.77 mmol), n-butyllithium (2.41 M hexane solution, 600 μL, 1.45 mmol) was added at -15°C under an Ar atmosphere. The reaction mixture was stirred at -15°C for 30 minutes and then cooled to -50°C. A THF solution (2 mL) of ketones 1-5 (201 mg, 0.467 mmol) was added dropwise to the reaction mixture and stirred at -50°C to -30°C for 3 hours. A saturated aqueous solution of NH4Cl (10 mL) was added to quench the excess lithium trimethylsilyl etanide, and the mixture was extracted with toluene (20 mL). The aqueous layer was separated and further extracted with toluene (2 x 20 mL). The combined organic phases were washed with brine (20 mL), dried over anhydrous Na₂SiO₄, filtered, concentrated, and the resulting crude product was purified by column chromatography (in a hexane solution of 20% Et₂O) to obtain 1-6 (235 mg, 95%). 1 H NMR (500 MHz, CDCl3) δ 4.23 (d, J = 5.4 Hz, 1 H), 3.99 (t, J = 5.6 Hz, 1 H), 3.77 (m, 1 H), 3.65 (dd, J = 8.0, 5.9 Hz, 1 H), 2.09 (dd, J = 13.0, 3.8 Hz, 1 H), 2.00 (dd, J = 13.0, 9.7 Hz, 1 H), 1.54 (s, 3 H), 1.37 (s, 3 H), 0.91 (s, 9 H), 0.90 (s, 9 H), 0.18 (s, 9 H), 0.13 (s, 3 H), 0.12 (s, 3H), 0.09 (s, 3H), 0.08 (s, 3 H); 13 C NMR (126 MHz, CDCl3) δ 110.0, 90.8, 81.1, 79.6, 77.8, 77.4, 71.2, 67.7, 27.9, 26.5, 26.4, 26.3, 18.3, 18.2, -0.1, -3.7, -3.7, -4.1, -4.4.

[0212] Synthesis of Alkynes 1-7: N,N-dimethylaminopyridine (280 mg, 2.29 mmol) was added to a stirred 0°C solution of alcohols 1-6 (203 mg, 0.384 mmol) in CH2Cl2 (3 mL), followed by dropwise addition of methyl 2-chloro-2-oxoacetate (220 μL, 2.39 mmol). The ice bath was removed, and the reaction mixture was heated to room temperature and stirred for 12 hours. The reaction mixture was treated with a cold saturated aqueous solution of NaHCO3 (5 mL) and extracted with ethyl acetate (20 mL). The aqueous layer was separated and further extracted with ethyl acetate (2 x 20 mL). The combined organic phases were dried over anhydrous Na2SiO4, filtered, concentrated, and the resulting crude product was rapidly purified by column chromatography (in a hexane solution of 15% Et20) to obtain the corresponding esters. The esters were dissolved in toluene (1 mL) in a microwave-safe glass tube. 2,2'-azobis(2-methylpropionitrile) (75 mg, 0.46 mmol) was added to the reaction mixture, and N2 gas was passed through the solution for 30 minutes. In a separate microwave-safe glass tube, nit-n-tributyltin hydride (1.20 mL, 4.46 mmol) was added, and N2 gas was passed through the solution for 30 minutes, after which the reaction mixture was heated at 120°C for 10 minutes. The crude reaction mixture was added dropwise over 2 minutes, stirred at 120°C for 2 hours, and then the solution was cooled to room temperature over 20 minutes. The reaction mixture was treated with a saturated aqueous solution of NH4Cl (10 mL) and extracted with ELISA (20 mL). The aqueous layer was separated and further extracted with ELISA (2 x 20 mL). The combined organic phases were washed with brine (20 mL), dried over anhydrous Na2SiO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (5% Et2O hexane solution) to obtain 1-7 as a colorless viscous liquid (117 mg, 59% in 2 steps). This was found in CDCl3 at 27°C. 1 Results measured by 1H NMR spectroscopy showed a diastereomer ratio of approximately 3:1.

[0213] Synthesis of alkynes 1-8 and 1-9: Potassium carbonate (18 mg, 0.13 mmol) was added to a stirred solution of alkyne 1-7 (44.9 mg, 87.5 μmol) in CH3OH (1 mL) and CH2Cl2 (0.2 mL), and the mixture was stirred for 23 hours. The reaction mixture was concentrated under reduced pressure. The resulting solid mass was treated with a saturated aqueous solution of NH4Cl (5 mL) and extracted with ethyl acetate (10 mL). The aqueous layer was separated and further extracted with SiO2 (2 x 10 mL). The combined organic phases were washed with brine (10 mL), dried over anhydrous Na2SiO4, filtered, and concentrated. The resulting crude product was purified by column chromatography (in a hexane solution of 5% Et20) to obtain pseudo-D-altroisomers 1-8 as a colorless viscous liquid (25.3 mg, 66%), and pseudo-L-fucoisomers 1-9 as a colorless viscous liquid (9 mg, 23%). Data for 1-8: 1 H NMR (500 MHz, CDCl3) δ 4.10 (dd, J = 8.5, 5.2 Hz, 1 H), 4.00 (dd, J = 5.1, 2.7 Hz, 1 H), 3.88 (m, 1 H), 3.75 (m, 1 H), 3.00 (m, 1 H), 2.10 (d, J = 2.5 Hz, 1 H), 1.88 (m, 1 H), 1.72 (m, 1 H), 1.49 (s, 3 H), 1.34 (s, 3 H), 0.90 (s, 9 H), 0.89 (s, 9 H), 0.10 (s, 3 H), 0.08 (s, 6 H), 0.07 (s, 3H); 13 ¹³C NMR (126 MHz, CDCl₃) δ 108.9, 86.6, 78.5, 77.5, 72.1, 69.7, 69.1, 32.2, 28.5, 27.1, 26.2, 25.9, 18.1, -4.5, -4.6, -4.7, -4.8. Data for 1-9: 1H NMR (500 MHz, CDCl3) δ 4.30 (m, 1 H), 3.98 (m, 1 H), 3.61 (m, 1 H), 3.51 (m, 1 H), 2.82 (m, 1 H), 2.17 (d, J = 2.5 Hz, 1 H), 1.97-1.85 (m, 2 H), 1.54 (s, 3 H), 1.38 (s, 3 H), 0.89 (s, 9 H), 0.88 (s, 9 H), 0.11 (s, 3 H), 0.08 (s, 6 H), 0.07 (s, 3 H); 13 C NMR (126 MHz, CDCl3) δ 109.5, 83.9, 80.4, 77.2, 74.9, 72.7, 69.8, 33.2, 27.6, 27.5, 26.2, 26.1, 25.8, 18.2, -3.8, -3.9, -4.2, -4.3.

[0214] Synthesis of tetraol 1-10: To a 1 mL solution of stirred alkyne 1-9 (15 mg, 34 μmol) in CH3OH, 1 M HCl (0.4 mL, 0.4 mmol) was added and the mixture was stirred for 14 hours. The reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3 x 2 mL). The resulting crude product was purified by column chromatography (5% CH3OH solution of siRNA) to obtain 1-10 as a colorless viscous liquid (4.7 mg, 80%). 1 H NMR (500 MHz, CD3OD) δ 3.93 (m, 1 H), 3.49 (m, 1 H), 3.35 (m, 1 H), 3.25 (dd, J = 9.6, 3.0 Hz, 1 H), 2.55 (m, 1 H), 2.37 (d, J = 2.5 Hz, 1 H), 1.93-1.83 (m, 2H); 13 C NMR (126 MHz, CD3OD) δ 85.2, 76.0, 75.5, 73.0, 72.9, 70.7, 34.1, 31.8; HRMS (ESI-TOF) m / z: [M + NH4] + C8H 12 Calculated value for O4: 190.1074; measured value: 190.1074. JPEG2026136206000028.jpg91155

[0215] Synthesis of trifluoromethyl compounds 1-11: Trimethyl(trifluoromethyl)silane (2.00 M THF solution, 180 μL, 360 μmol) was added to a stirred 0°C solution of ketones 1-5 (141 mg, 0.327 mmol) in THF (2 mL), followed by the addition of tetra-n-butylammonium fluoride (1.0 M THF solution, 10 μL, 10 μmol). The ice bath was removed, and the reaction mixture was heated to room temperature and stirred for 30 minutes. The reaction mixture was treated with a saturated aqueous solution of NH4Cl (5 mL) and extracted with siRNA (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered, concentrated, and the resulting crude product was dissolved in CH3OH (2 mL). Potassium carbonate (50 mg, 0.36 mmol) was added to the solution and stirred for 1 hour. The reaction mixture was concentrated under reduced pressure. The obtained solid mass was treated with a saturated aqueous solution of NH4Cl (5 mL) and extracted with ethyl acetate (20 mL). The aqueous layer was separated and further extracted with siRNA (2 x 20 mL). The combined organic phase was washed with brine (10 mL), dried over anhydrous Na2SiO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (10% Et2O hexane solution) to obtain 1-11 as a colorless viscous liquid (110 mg, 67%). 1 H NMR (500 MHz, CDCl3) δ 4.3 (d, J = 6.4 Hz, 1 H), 4.17-4.11 (m, 2 H), 3.92 (m, 1 H), 2.00 (m, 2 H), 1.56 (s, 3 H), 1.38 (s, 1 H), 0.91 (s, 9 H), 0.88 (s, 9 H), 0.12 (s, 12 H).

[0216] Synthesis of trifluoromethyl compound 1-12: To a stirred solution of alcohol 1-11 (352 mg, 0.703 mmol) in CH2Cl2 (3 mL) at 0°C, N,N-dimethylaminopyridine (515 mg, 4.22 mmol) was added, followed by the dropwise addition of methyl 2-chloro-2-oxoacetate (0.40 mL, 4.4 mmol). The ice bath was removed, and the reaction mixture was heated to room temperature and stirred for 14 hours. The reaction mixture was treated with a cold saturated aqueous solution of NaHC3 (5 mL) and extracted with ELISA (30 mL). The aqueous layer was separated, the organic phase was dried over anhydrous Na2SiO4, filtered, and concentrated. The resulting crude product was rapidly purified by column chromatography (in a hexane solution of 10% Et20) to obtain the corresponding ester along with the hydrolyzed ester (starting material). The crude product was dissolved in toluene (1 mL) in a microwave-safe glass tube. 2,2'-azobis(2-methylpropionitrile) (60 mg, 0.37 mmol) was added to the reaction mixture, and N2 gas was passed through the solution for 30 minutes. In a separate microwave-safe glass tube, nit-n-tributyltin hydride (900 μL, 3.35 mmol) was added, and N2 gas was passed through the solution for 30 minutes, after which the reaction mixture was heated at 120°C for 10 minutes. The crude reaction mixture was added dropwise over 2 minutes, stirred at 120°C for 2 hours, and then the solution was cooled to room temperature for 20 minutes. The reaction mixture was treated with a saturated aqueous solution of NH4Cl (10 mL) and extracted with ELISA (30 mL). The aqueous layer was separated and further extracted with ELISA (2 x 20 mL). The combined organic phases were washed with brine (20 mL), dried over anhydrous Na2SiO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (2.5% Et2O hexane solution) to obtain 1-12 as a colorless viscous liquid (119 mg, 35% in two steps) along with 1-11 (65 mg, 18%). This was found in CDCl3 at 27°C. 1 Results measured by 1H NMR spectroscopy showed a diastereomer ratio of approximately 1.2:1.

[0217] Synthesis of diols 1-13 and 1-14: To a 2 mL THF solution of stirred acetonide 1-12 (100 mg, 0.206 mmol), tetra-n-butylammonium fluoride (1.0 mL, 1.0 mmol in 1.0 M THF solution) was added and stirred for 10 hours. The reaction mixture was treated with H2O and extracted with ethyl acetate (20 mL). The aqueous layer was separated and further extracted with siRNA (2 x 20 mL). The combined organic phases were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (65% siRNA in hexane solution) to obtain 1-13 as a colorless viscous liquid (24 mg, 46%) and 1-14 as a white solid (80% siRNA in hexane solution, 20 mg, 38%). Data for 1-13: 1 H NMR (500 MHz, CDCl3) δ 4.35 (t, J = 5.3 Hz, 1 H), 4.03 (t, J = 6.8 Hz, 1 H), 3.78 (m, 1 H), 3.67 (t, J = 7.7 Hz, 1 H), 2.81 (m, 1 H), 2.04 (m, 1 H), 1.88 (m, 1 H), 1.53 (s, 3 H), 1.38 (s, 3 H); 13 ¹³C NMR (126 MHz, CDCl₃): δ 126.9, 109.8, 78.6, 75.6, 72.2, 68.0, 40.2, 28.1, 26.2, 25.9. Data for 1-14: 1 H NMR (500 MHz, CDCl3) δ 4.37 (m, 1 H), 3.95 (m, 1 H), 3.56 (m, 1 H), 3.48 (m, 1 H), 2.54 (m, 1 H), 2.06 (m, 1 H), 1.85 (m, 1 H), 1.55 (s, 3 H), 1.38 (s, 3 H); 13 C NMR (126 MHz, CDCl3) δ 127.0, 110.9, 80.5, 77.8, 71.5, 70.4, 40.1, 28.4, 26.4, 25.9.

[0218] Synthesis of Tetraol 1-15: To a solution of stirred diol 1-13 (10.1 mg, 39.4 μmol) in CH3OH (0.9 mL), 1 M HCl (0.2 mL, 0.2 mmol) was added and the mixture was stirred for 12 hours. The reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3 x 2 mL). The resulting crude product was purified by column chromatography (a solution of 3% CH3OH in siRNA) to obtain 1-15 as a colorless viscous liquid (6.1 mg, 72%). 1 H NMR (500 MHz, CD3OD) δ 4.02 (dd, J = 7.1, 3.1 Hz, 1 H), 3.80-3.72 (m, 2 H), 3.70 (m, 1 H), 2.72 (m, 1 H), 1.99-1.88 (m, 2 H); 13 C NMR (126 MHz, CD3OD) δ 129.0, 74.7, 73.4, 70.6, 68.2, 41.8, 27.7; 19 F NMR (377 MHz, CD3OD) δ -68.7; HRMS (ESI-TOF) m / z: [M + NH4] + C7H 11 Calculated value for F3O4: 234.0948; measured value: 234.0951.

[0219] Synthesis of Tetraol 1-16: To a 0.9 mL solution of stirred diol 1-14 (7.40 mg, 26.9 μmol) in CH3OH, 1 M HCl (0.2 mL, 0.2 mmol) was added and the mixture was stirred for 12 hours. The reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3 x 2 mL). The resulting crude product was purified by column chromatography (a 3% CH3OH solution with siRNA) to obtain 1-16 as a colorless viscous liquid (5.1 mg, 82%). 1 H NMR (500 MHz, CD3OD) δ 4.13 (m, 1 H), 3.53 (m, 1 H), 3.43 (m, 1 H), 3.28 (dd, J = 9.6, 3.0 Hz, 1 H), 2.35 (m, 1 H), 1.93-1.82 (m, 2 H); 13C NMR (126 MHz, CD3OD) δ 128.2, 76.1, 75.4, 72.5, 68.8, 42.7, 27.2; HRMS (ESI-TOF) m / z: [M + NH4] + C7H 11 Calculated value for F3O4: 234.0948; measured value: 234.0952. JPEG2026136206000029.jpg103151

[0220] Synthesis of Alkene 1-17: A flame-dried 50-mL flask equipped with a magnetic stirring rod, argon inlet, and septum was packed with acetonide 1-4 (199 mg, 0.403 mmol) and THF (3 mL). The mixture was cooled to -55°C using a dry ice and acetone bath, and then tert-butyllithium (1.55 M pentane solution, 410 μL, 0.636 mmol) was added dropwise. The reaction mixture was stirred at -55°C for 30 minutes, and then methyl iodide (0.10 mL, 1.6 mmol) was added dropwise. The dry ice and acetone bath was removed, and the reaction mixture was slowly heated to room temperature and stirred for 2 hours. The reaction mixture was treated with a saturated aqueous solution of NH4Cl (10 mL) and extracted with Et2O (30 mL). The aqueous layer was separated and further extracted with Et2O (2 x 20 mL). The combined organic phases were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (in a hexane solution of 2% Et2O) to obtain 1-17 as a colorless viscous liquid (104 mg, 60% in 2 steps). 1 H NMR (500 MHz, CDCl3) δ 5.36 (m, 1 H), 4.42 (d, J = 7.0 Hz, 1 H), 4.02 (m, 1 H), 3.99 (m, 1 H), 3.56 (m, 1 H), 1.80 (s, 3 H), 1.45 (s, 3 H), 1.35 (s, 3 H), 0.91 (s, 9 H), 0.90 (s, 9 H), 0.13 (s, 3 H), 0.09 (s, 6 H), 0.08 (s, 3 H); 13C NMR (126 MHz, CDCl3) δ 131.6, 129.0, 109.5, 78.8, 76.1, 75.8, 71.7, 28.0, 26.3, 26.2, 25.8, 19.9, 18.4, 18.3, -3.5, -3.7, -3.9, -4.4.

[0221] Synthesis of Diol 1-18: To a 2 mL THF solution of stirred acetonide 1-17 (119.5 mg, 0.2787 mmol), tetra-n-butylammonium fluoride (1.4 mL, 1.4 mmol in a 1.0 M THF solution) was added and stirred for 10 hours. The reaction mixture was then treated with H2O and extracted with ethyl acetate (20 mL). The aqueous layer was separated and further extracted with siRNA (2 x 20 mL). The combined organic phases were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (95% siRNA in hexane solution) to obtain 1-18 as a colorless viscous liquid (50 mg, 90%). 1 H NMR (500 MHz, CDCl3) δ 5.50 (s, 1 H), 4.42 (d, J = 6.5 Hz, 1 H), 4.07 (dd, J = 8.9, 6.6 Hz, 1 H), 4.03 (m, 1 H), 3.52 (t, J = 8.5 Hz, 1 H), 1.82 (s, 3 H), 1.48 (s, 3 H), 1.37 (s, 3 H); 13 C NMR (126 MHz, CDCl3) δ 131.9, 128.0, 110.3, 77.7, 76.1, 75.4, 70.5, 28.3, 25.9, 20.1.

[0222] Synthesis of Diol 1-20: A solution of Diol 1-18 (216 mg, 1.08 mmol) in CH3OH (6 mL) was stirred under an argon balloon. Potassium carbonate (15 mg, 0.11 mmol) was added to the solution, followed by PtO2 (26 wt%, 56 mg, 0.25 mmol) under an argon atmosphere. The argon balloon was replaced with an H2 balloon (1 atm), and the solution was passed through H2 for 10 seconds. The reaction mixture was stirred under an H2 balloon for 24 hours, then the H2 balloon was replaced with an argon balloon, and the mixture was passed through argon for 1 minute. The reaction mixture was diluted with CH3OH (30 mL) and filtered through a Celite-545 pad. The filtrate was concentrated, and the resulting crude product was purified by column chromatography (95% siRNA in hexane solution). D-altro isomers 1-19 (26 mg, 12%) and 1-20 were obtained as a colorless viscous liquid (126 mg, 58%). Data for 1-20: 1 H NMR (500 MHz, CDCl3) δ 4.07 (m, 1 H), 3.86 (m, 1 H), 3.48 (m, 1 H), 3.44 (m, 1 H), 1.92 (m, 1 H), 1.80-1.69 (m, 2 H), 1.51 (s, 3 H), 1.35 (s, 3 H), 1.12 (d, J = 6.9 Hz, 3 H); 13 C NMR (126 MHz, CDCl3) δ 109.2, 81.0, 78.6, 78.3, 71.4, 34.5, 30.0, 28.6, 26.5, 17.4; HRMS (ESI-TOF) m / z: [M + NH4] + C 10 H 18 Calculated value for O4: 203.1278; measured value: 203.1275.

[0223] Synthesis of Carbafco-se 1-21: To a solution of stirred diol 1-20 (62.0 mg, 0.307 mmol) in CH3OH (8.3 mL), 1 M HCl (1.7 mL, 1.7 mmol) was added and the mixture was stirred for 12 hours. The reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3 x 3 mL). The resulting crude product was purified by column chromatography (5% CH3OH solution of siRNA) to obtain 1-21 as a white solid (42 mg, 85%). [α] D 20 = +22.3 (c = 0.05, CH3OH); 1 H NMR (600 MHz, CD3OD) δ 3.68 (brs, 1 H), 3.48-3.45 (m, 1 H), 3.39-3.35 (m, 1 H), 3.27 (dd, J = 9.6, 3.0 Hz, 1 H), 1.65-1.60 (m, 1 H), 1.57-1.48 (m, 2 H), 1.02 (d, J = 6.8 Hz, 3 H); 13 C NMR (151 MHz, CD3OD): δ 76.5, 76.4, 74.9, 73.8, 35.8, 33.1, 17.8; IR (neat): ν 3368, 2958, 2928, 2857, 1731, 1668, 1462, 1261, 1067, 1022, 799cm -1 ; HRMS (ESI-TOF) m / z: [M + Na] + C7H 14 Calculated value for NaO4: 185.0784; measured value: 185.0784.

[0224] Synthesis of acetate 1-20a: To a 1 mL solution of stirred diol 1-20 (6 mg, 29 μmol) in CH3OH, 1 M HCl (0.2 mL, 0.4 mmol) was added and the mixture was stirred for 12 hours. The reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3 x 2 mL), and the resulting crude product was dissolved in pyridine (1 mL). Ac20 (0.10 mL, 1.1 μmol) was added and the mixture was heated at 50°C for 12 hours. The reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3 x 2 mL), and the resulting crude product was purified by column chromatography (20% siRNA solution in hexane) to obtain 1-20a as a white solid (7.8 mg, 80% in 2 steps). 1 H NMR (500 MHz, CDCl3) δ 5.36 (t, J = 10.1 Hz, 1 H), 5.31 (m, 1 H), 4.92-4.86 (m, 2 H), 2.14 (s, 3 H), 2.03 (s, 3 H), 2.02 (s, 3 H), 1.97 (s, 3 H), 1.92 (m, 1 H), 1.89 (m, 1 H), 1.64 (m, 1 H); HRMS (ESI-TOF) m / z: [M + NH4] + C 15 H 26 Calculated value for NO8: 348.1653; measured value: 348.1650. JPEG2026136206000030.jpg74158

[0225] Synthesis of acetates 1-22a and 1-22b: To a THF solution (1.5 mL) of stirred diol 1-20 (63.5 mg, 0.314 mmol), CeCl3·7H2O (11 mg, 30 μmol) was added, followed by acetic anhydride (150 μL, 1.59 mmol), and the mixture was stirred for 24 hours. The reaction mixture was treated with a saturated aqueous solution of NaHC3 (10 mL) and extracted with siRNA (20 mL). The aqueous layer was separated and further extracted with siRNA (2 x 20 mL). The combined organic phases were washed with brine (20 mL), dried over anhydrous Na2SiO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (40% siRNA solution in hexane) to obtain unseparable 1-22a and 1-22b as colorless viscous liquids (60 mg, 78%). This was found in CDCl3 at 27°C. 1 Results measured by 1H NMR spectroscopy showed a positional isomer ratio of approximately 5.6:1. 1 H NMR (400 MHz, CDCl3, Major isomer) δ 4.88 (dd, J = 10.1, 7.7 Hz, 1 H), 4.08 (m, 1 H), 3.98 (dd, J = 7.7, 4.8 Hz, 1 H), 3.48 (m, 1 H), 2.14 (s, 3 H), 1.91 (m, 1 H), 1.81 (m, 1 H), 1.61 (m, 1 H), 1.53 (s, 3 H), 1.34 (s, 3 H), 1.13 (d, J = 7.0 Hz, 3 H); 13 C NMR (101 MHz, CDCl3) δ 172.2, 109.5, 79.8, 78.4, 78.1, 70.9, 35.8, 29.8, 28.1, 26.4, 21.3, 17.3; HRMS (ESI-TOF) m / z: [M + NH4] + C 12 H 20 Calculated value for O5: 245.1384; measured value: 245.1387.

[0226] Synthesis of phosphate 1-24: Ac20 (40 μL, 0.42 mmol) was added to a stirred CH2Cl2 solution (2.7 mL) of tetra-n-butylammonium methyl H-phosphate 1-23 (125 mg, 0.389 mmol) and pyridine (0.3 mL). The reaction mixture was stirred at 55°C for 1 hour and then cooled to room temperature over 5 minutes using an ice bath. A CH2Cl2 solution (1.5 mL) of acetate 1-22 (12.0 mg, 37.3 μmmol) was added dropwise to the cooled reaction mixture over 5 minutes, and the reaction mixture was stirred at 55°C for 15 hours and then cooled to room temperature over 15 minutes. The reaction mixture was concentrated, and the resulting crude product was rapidly purified by column chromatography (75% Âxane solution) to obtain the desired 2-O-acetyl H-phosphate. The H-phosphate (8.0 mg, 24.8 μmmol) was dissolved in CH2Cl2 (1 mL) and cooled to 0°C. Et3N (20 μL, 0.18 mmol) was added, followed by a pyridine solution of l2 (16 mg, 63 μmmol) (1 mL). The ice bath was removed, and the reaction mixture was heated to room temperature over 4 hours. A saturated aqueous solution of Na2SiO4 (5 mL) was added to quench the excess l2, and the reaction mixture was extracted with ELISA (20 mL). The aqueous layer was separated and further extracted with ELISA (2 x 20 mL). The combined organic phase was washed with brine (10 mL), dried over anhydrous Na2SiO4, filtered, concentrated, and the resulting crude product was rapidly purified by column chromatography (a solution of ELISA in 20% CH3OH) to obtain the desired methyl H-phosphate. This was dissolved in CH3CN (1.5 mL). Et3N (35 μL, 0.25 mmol), NaI (18 mg, 0.12 mmol), followed by POM-Cl (35 μL, 0.24 mmol), were added sequentially, and the mixture was heated at 70°C for 14 hours. The reaction mixture was treated with a saturated aqueous solution of Na2S2O4 (10 mL) and extracted with SiO2 (20 mL). The aqueous layer was separated and further extracted with SiO2 (2 x 20 mL).The combined organic phases were washed with brine (20 mL), dried over anhydrous Na2SiO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (30% siRNA in hexane solution) to obtain 1-24 as a colorless viscous liquid (2.7 mg, 10% in 3 steps). 1 H NMR (400 MHz, CDCl3) δ 5.66-5.56 (m, 4 H), 5.12 (dd, J = 10.3, 7.9 Hz, 1 H), 4.28 (m, 1 H), 4.08 (m, 1 H), 3.98 (dd, J = 7.7, 4.8 Hz, 1 H), 2.12 (s, 3 H), 2.07-1.91 (m, 3 H), 1.55 (s, 3 H), 1.35 (s, 3 H), 1.24 (s, 18 H), 1.15 (d, J = 6.7 Hz, 3H); 31 P NMR (162 MHz, CDCl3) δ -5.08; HRMS (ESI-TOF) m / z: [M + NH4] + C 24 H 41 O 12 The calculated value for P is 570.2674; the measured value is 570.2675.

[0227] Synthesis of 1-25: A cooled TFA:H2O (9:1, 0.5 mL) solution was added to acetonide 1-24 (2.0 mg, 3.6 μmol) and stirred for 3 hours. The reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3 x 2 mL), and the resulting crude product was dissolved in pyridine (1 mL). Ac20 (0.10 mL, 1.1 μmol) was added and the mixture was heated at 50°C for 24 hours. The reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3 x 2 mL), and the resulting crude product was purified by column chromatography (30% ELISA in hexane solution) to obtain 1-25 as a colorless viscous liquid (1.5 mg, 69% in 2 steps). 1H NMR (400 MHz, CDCl3) δ 5.66-5.58 (m, 4 H), 5.36-5.27 (m, 2 H), 4.83 (dd, J = 10.5, 3.0 Hz, 1 H), 4.41 (m, 1 H), 2.14 (s, 3 H), 2.08 (s, 3 H), 2.06 (m, 1 H), 1.97 (s, 3 H), 1.92-1.78 (m, 2 H), 1.24 (s, 18 H), 0.95 (d, J = 6.6 Hz, 3 H); 31 P NMR (162 MHz, CDCl3) δ -5.11. HRMS (ESI-TOF) m / z: [M + NH4] + C 25 H 41 O 14 The calculated value for P is 614.2572; the measured value is 614.2575. JPEG2026136206000031.jpg83165

[0228] Synthesis of dibenzyl phosphate 2-1: To a 4 mL solution (CH2Cl2) of stirred acetates 1-22a and 1-22b (233 mg, 0.954 mmol), 1H tetrazole (0.40 M acetonitrile solution, 6.0 mL, 2.4 mmol) was added, followed by the addition of dibenzyl diisopropyl phosphoramidite (0.80 mL, 2.4 mmol) at room temperature. After 1 hour, m-CPBA (77%, 854 mg, 3.81 mmol) was added to the mixture at 0°C. After stirring at 0°C for 1 hour, the mixture was diluted with CH2Cl2 (30 mL) and washed with saturated aqueous solution of Na2SiO3 (10 mL) and saturated aqueous solution of NaHCO3 (10 mL). The aqueous layer was separated and further extracted with CH2Cl2 (2 x 20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (25% siRNA in hexane solution) to obtain 2-1 as a white solid (337 mg, 70%), along with undesirable isomers (12%). Data for 2-1: 1H NMR (500 MHz, CDCl3) δ 7.38-7.28 (m, 10 H), 5.13 (dd, J = 10.2, 7.7 Hz, 1 H), 5.03-4.92 (m, 4 H), 4.24 (dddd, J = 11.4, 10.2, 7.5, 4.0 Hz, 1 H), 4.09-4.03 (m, 1 H), 3.96 (dd, J = 7.8, 4.7 Hz, 1 H), 1.92 (s, 3 H), 1.91-1.82 (m, 2 H), 1.77-1.66 (m, 1 H), 1.56 (s, 3 H), 1.34 (s, 3 H), 1.10 (d, J = 6.7 Hz, 3 H); 13 C NMR (125 MHz, CDCl3) δ 170.5, 135.8 (2C), 128.7, 128.7, 128.1, 128.0, 109.8, 78.4, 77.7, 76.3, 76.3, 75.8, 75.7, 69.5, 69.4, 69.4, 33.7, 29.4, 28.0, 26.5, 21.1, 17.1; 31 P NMR (162 MHz, CDCl3) δ -1.79.

[0229] Synthesis of Acetonide 2-2: To a THF solution (3 mL) of cooled (0°C) acetate 2-1 (85 mg, 0.17 mmol), EtMgBr (3.0 M Et2O solution, 0.95 mL, 2.9 mmol) was added and the mixture was stirred at 0°C for 1.5 hours. A saturated aqueous solution of NH4Cl (10 mL) was added, and the excess Grignard reagent was quenched. The mixture was extracted with siRNA (2 x 20 mL). The aqueous layer was separated and further extracted with siRNA (2 x 20 mL). The combined organic phases were washed with brine (15 mL), dried over anhydrous Na2SiO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (45% siRNA solution in hexane) to obtain 2-2 as a colorless viscous liquid (65 mg, 85%). 1H NMR (600 MHz, CDCl3) δ 7.41-7.28 (m, 11 H), 5.13-4.90 (m, 4 H), 4.11-3.98 (m, 2 H), 3.87 (dd, J = 7.3, 4.9 Hz, 1 H), 3.61 (dd, J = 9.7, 7.3 Hz, 1 H), 1.82 (ddq, J = 16.2, 6.9, 3.5 Hz, 1 H), 1.72 (dt, J = 12.7, 4.0 Hz, 1 H), 1.66-1.54 (m, 1 H), 1.50 (s, 3 H), 1.34 (s, 3 H), 1.08 (d, J = 6.9 Hz, 3 H); 13 C NMR (151 MHz, CDCl3) δ 135.81, 135.80, 135.76, 135.75, 128.8, 128.74, 128.72, 128.2, 128.18, 128.15, 128.13, 109.4, 80.7, 79.7, 79.66, 77.5, 76.5, 76.4, 69.81, 69.77, 69.72, 69.68, 33.22, 33.20, 29.6, 28.5, 26.4, 17.2; 31 P NMR (162 MHz, CDCl3) δ -0.82。

[0230] Synthesis of 2-3: To a 1.2 mL solution of stirred acetonide 2-2 (30.3 mg, 65.5 μmol) in CH3OH, 1 M HCl (0.3 mL, 0.3 mmol) was added and the mixture was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3 x 2 mL). The resulting crude product was rapidly purified by column chromatography (5% CH3OH in siRNA solution) to obtain the corresponding triol as a colorless viscous liquid (22.7 mg, 82%). A 3 mL solution of the triol (22.7 mg, 53.7 μmol) in CH3OH was stirred under argon. Pd(OH)2-C (20% by wt, 17 mg, 0.024 mmol) was added to the reaction mixture, and the argon balloon was replaced with an H2 balloon (1 atm), and the mixture was stirred for 5 hours. At this point, the H2 balloon was replaced with an argon balloon, and the mixture was run with argon for 1 minute. The reaction mixture was diluted with CH3OH (20 mL), and the solution was filtered through a Celite-545 pad. The filtrate was concentrated, and the resulting crude product was purified by column chromatography (50% CH3OH in CH2Cl2 solution) using spherical silica gel (particle size 40-75 μm) to obtain 2-3 as a white solid (9.5 mg, 60% in 2 steps). 1 H NMR (500 MHz, D2O) δ 3.98-3.91 (m, 1 H), 3.79 (t, J = 2.8 Hz, 1 H), 3.60 (dd, J = 9.5, 9.5 Hz, 1 H), 3.49 (dd, J = 10.0, 3.1 Hz, 1 H), 1.89 (dt, J = 13.0, 4.2 Hz, 1 H), 1.78-1.69 (m, 1 H), 1.48 (td, J = 13.0, 11.5 Hz, 1 H), 0.99 (d, J = 6.9 Hz, 3 H). JPEG2026136206000032.jpg71159

[0231] Synthesis of dibenzyl phosphate 2-4: Imidazole (55 mg, 0.81 mmol) was added to a 2 mL solution of cooled (0°C) alcohol 2-2 (37 mg, 80 μmol) in CH2Cl2, followed by TESCl (0.10 mL, 0.74 mmol). The ice bath was removed, and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with CH2Cl2, washed with a saturated aqueous solution of NaHCO3 (10 mL), and extracted with CH2Cl2 (20 mL). The aqueous layer was separated and further extracted with CH2Cl2 (2 x 20 mL). The combined organic phases were washed with brine (15 mL), dried over anhydrous Na2SO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (20% siRNA solution in hexane) to obtain 2-4 as a colorless viscous liquid (44.6 mg, 97%). 1 H NMR (400 MHz, CDCl3) δ 7.38-7.28 (m, 10 H), 5.14-4.94 (m, 4 H), 4.13-3.96 (m, 2 H), 3.82 (dd, J = 6.6, 5.1 Hz, 1 H), 3.63 (dd, J = 9.1, 6.6 Hz, 1 H), 1.95 (dt, J = 12.6, 4.0 Hz, 1 H), 1.89-1.77 (m, 1 H), 1.68-1.53 ​​(m, 1 H), 1.49 (s, 3 H), 1.33 (s, 3 H), 1.05 (d, J = 6.9 Hz, 3 H), 0.93 (t, J = 7.9 Hz, 9 H), 0.69-0.58 (m, 6 H); 31 P NMR (162 MHz, CDCl3) δ -1.49.

[0232] Synthesis of POM-phosphate 2-5: NaHCO3 (76 mg, 0.90 mmol) was added to a stirred CH3CN solution (3 mL) of dibenzyl phosphate 2-4 (46 mg, 80 μmol), followed by Pd(OH)2-C (20% by wt, 23 mg, 33 μmol) added to the reaction mixture under argon. The argon balloon was replaced with an H2 balloon (1 atm), and the mixture was stirred for 4 hours. At this point, the H2 balloon was replaced with an argon balloon, and the mixture was run under argon for 1 minute. The reaction mixture was diluted with CH3OH (20 mL), and the solution was filtered through a Celite-545 pad. The filtrate was concentrated, and the resulting crude product was dissolved in CH3CN (2 mL) and CH2Cl2 (0.5 mL). DIPEA (0.14 mL, 0.80 mmol) was added sequentially, followed by POM-I (0.15 mL, 0.99 mmol), and the mixture was stirred for 36 hours. The reaction mixture was treated with a saturated aqueous solution of Na2S2O3 (10 mL) and extracted with ethyl acetate (20 mL). The aqueous layer was separated and further extracted with ethyl acetate (2 x 20 mL). The combined organic phase was washed with brine (15 mL), dried over anhydrous Na2S2O4, filtered, concentrated, and the resulting crude product was purified by column chromatography (13% ethyl acetate in hexane solution) to obtain 2-5 as a pale yellow viscous liquid (7 mg, 14% in 2 steps). 1 H NMR (600 MHz, CDCl3) δ 5.71-5.57 (m, 4 H), 4.15-4.05 (m, 1 H), 4.04-4.00 (m, 1 H), 3.87-3.75 (m, 1 H), 3.61 (dd, J = 9.1, 6.7 Hz, 1 H), 1.99 (dt, J = 12.9, 4.2 Hz, 1 H), 1.94-1.82 (m, 1 H), 1.70-1.57 (m, 1 H), 1.47 (s, 3 H), 1.33 (s, 3 H), 1.23 (s, 18 H), 1.10 (d, J = 7.0 Hz, 3 H), 0.94 (t, J = 8.0 Hz, 9H), 0.70-0.57 (m, 6H); 13C NMR (150 MHz, CDCl3) δ 176.8 (2C), 109.0, 83.0, 82.9, 82.81, 82.77, 81.9, 81.8, 80.3, 80.2, 77.8, 77.2, 77.1, 38.9, 33.0, 29.5, 28.4, 26.99, 26.97, 26.5, 17.3, 6.9 (3C), 5.0 (3C); 31 P NMR (162 MHz, CDCl3) δ -4.67.

[0233] Synthesis of POM-phosphate triol 2-6: A solution of TFA:H2O (9:1, 0.7 mL) was added to acetonide 2-6 (7.6 mg, 12 μmol) and stirred for 4 hours. The reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3 x 2 mL). The resulting crude product was purified by column chromatography (90% siRNA solution in hexane) to obtain triol 2-6 as a pale yellow viscous liquid (4.0 mg, 70%). 1 H NMR (600 MHz, CDCl3) δ 5.75-5.57 (m, 4 H), 4.33-4.19 (m, 1 H), 3.86-3.83 (m, 1 H), 3.80 (app t, J = 9.4 Hz, 1 H), 3.46 (dd, J = 9.4, 2.9 Hz, 1 H), 1.90-1.73 (m, 2 H), 1.72-1.62 (m, 1 H), 1.24 (s, 18 H), 1.08 (d, J = 6.8 Hz, 3 H); 13 C NMR (150 MHz, CDCl3) δ 177.4, 177.0, 83.0, 82.99, 82.97, 82.95, 81.4, 81.3, 74.8, 73.63, 73.60, 71.9, 38.94, 38.91, 32.93, 32.91, 31.2, 29.9, 26.97, 26.96, 17.1; 31 P NMR (162 MHz, CDCl3) δ -4.17; HRMS (ESI-TOF) m / z: [M + NH4] + C 19 H 39 NO11 The calculated value for P is 488.2255; the measured value is 488.2174. JPEG2026136206000033.jpg91162

[0234] Synthesis of Alcohol 3-1: To a 17 mL THF / Et20 (1:1) solution of stirred ketones 1-5 (464 mg, 1.077 mmol), iodine fluoromethane (0.086 M pentane solution, 8.37 mL, 0.72 mmol) was added at -78°C, followed by the addition of MeLi.LiBr (1:1) (1.6 M Et20 solution, 1.34 mL, 2.15 mmol). The reaction mixture was stirred at -78°C for 5 minutes. The reaction mixture was then treated with a saturated aqueous solution of NH4Cl and extracted with Et20. The organic layer was dried over anhydrous MgSiO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (5% Et20 hexane solution) to obtain 3-1 as a colorless viscous liquid (207 mg, 62%). [α] D = +7.5 (c = 0.96, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 4.23 (dd, J = 8.9, 59.9 Hz, 1 H), 4.13-4.11 (m, 2 H), 4.10 (dd, J = 8.9, 60.2 Hz, 1 H), 4.08 (d, J = 2.6 Hz, 1 H), 3.90-3.85 (m, 1 H), 2.04 (ddd, J = 2.1, 4.1, 14.6 Hz, 1 H), 1.78 (dd, J = 4.9, 14.6 Hz, 1 H), 1.55 (s, 3 H), 1.37 (s, 1 H), 0.91 (s, 9 H), 0.89 (s, 9 H), 0.12 (s, 9 H), 0.10 (s, 3H); 13C NMR (101 MHz, CDCl3): δ 109.4, 84.7 (d, J = 174.9 Hz), 78.5, 73.5 (d, J = 3.0 Hz), 72.4, 71.5, 70.8 (d, J = 18.1 Hz), 33.3 (d, J = 2.5 Hz), 26.3, 26.0, 25.9, 25.5, 18.1 (d, J = 13.3 Hz), -4.3, -4.4, -4.5, -4.8; 19 F NMR (377 MHz, CDCl3): δ -228.3 (t, J = 12.7, 47.4 Hz); IR (neat) ν 3468, 2930, 2370, 1255, 1082, 837 cm -1 HRMS (ESI-TOF) m / z: [M + H] + C 22 H 45 Calculated value for FO5Si2: 465.2862; measured value: 465.2858.

[0235] Synthesis of fluoromethyl compound 3-2: N,N-dimethylaminopyridine (63.2 mg, 0.516 mmol) was added to a stirred solution of alcohol 3-1 (40 mg, 0.086 mmol) in CH2Cl2 (1.2 mL) at -17°C, followed by dropwise addition of methyl 2-chloro-2-oxoacetate (47.5 μL, 0.516 mmol). After 2 hours, the ice bath was removed, and the reaction mixture was heated to room temperature and stirred for 1 hour. The reaction mixture was treated with a cooled saturated aqueous solution of NaHC3 and extracted with ethyl acetate. The aqueous layer was separated, the organic phase was dried over anhydrous MgSiO4, filtered, and concentrated. The resulting crude product was rapidly purified by column chromatography (in a hexane solution of 33% Et20) to obtain the corresponding ester. 1H NMR (400 MHz, CDCl3) δ 5.04 (dd, J = 10.3, 48.1 Hz, 1 H), 4.58 (dd, J = 10.3, 45.7 Hz, 1 H), 4.50 (d, J = 6.7 Hz, 1 H), 3.99 (t, J = 6.5 Hz, 1 H), 3.89 (s, 3 H), 3.81 (dd, J = 6.7, 7.9 Hz, 1 H), 3.65 (m, 1 H), 2.25 (m, 2 H), 1.51 (s, 3 H), 1.32 (s, 3 H), 0.90 (s, 9 H), 0.89 (s, 9 H), 0.13 (s, 3 H), 0.10 (s, 3 H), 0.09 (s, 3 H), 0.08 (s, 3 H). The crude product was dissolved in toluene (1 mL) in a microwave-safe glass tube. 2,2'-azobis(2-methylpropionitrile) (28.2 mg, 0.172 mmol) was added to the reaction mixture, and N2 gas was passed through the solution for 30 minutes. In a microwave-safe glass tube, nete n-tributyltin hydride (463 μL, 1.72 mmol) was added, and N2 gas was passed through the solution for 30 minutes. The reaction mixture was then heated at 120°C. The crude reaction mixture was rapidly added to the crude reaction mixture and stirred at 120°C for 2 hours, after which the solution was cooled to room temperature. The reaction mixture was treated with a saturated aqueous solution of NH4Cl and extracted with siRNA. The aqueous layer was separated and further extracted with EtOA. The combined organic phases were washed with brine (20 mL), dried over anhydrous MgSiO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (5% Et20 in hexane solution) to obtain 3-2 as a colorless viscous liquid (34 mg, 88% in 2 steps). This was found in CDCl3 at 25°C. 1 Results measured by 1H NMR spectroscopy showed a diastereomer ratio of approximately 2:1. 1H NMR (400 MHz, CDCl3) δ 4.67–4.26 (m, 2 H), 4.24 (m, 0.6 H), 4.07–3.94 (m, 1.7 H), 3.81 (m, 0.4 H), 3.61–3.48 (m, 1.3 H), 2.38–2.13 (m, 1). H), 1.81–1.68(m,1H), 1.64–1.43(m,1H), 1.50(s,2H), 1.47(s,1H), 1.36(s,1H), 1.35(s,18H), 0.93–0.90(s,18H), 0.15–0.07(s,12). H); 13 C NMR (101 MHz, CDCl3): δ 109.2, 108.5, 85.4 (d, J = 167.5 Hz), 84.6 (d, J = 167.9 Hz), 81.7, 79.0, 78.4, 77.4, 73.3, 73.2, 71.8,000; 70.2, 35.9 (d, J = 18.9 Hz), 35.2 (d, J = 18.6 Hz), 29.6 (d, J = 18.9 Hz), 29.6 (d, J = 3.1 Hz), 28.6, 28.0, 26.3, 26.2, 25.9, 25.8, 18.4,000; 18.3, 18.1, -3.5, -3.7, -4.0, -4.3, -4.6, -4.7, -4.8, -4.8; 19 F NMR (377 MHz, CDCl3): δ-222.7 (dt, J = 12.7, 46.9 Hz), -231.1 (dt, J = 29.9, 48.0 Hz); IR (neat) ν 3698, 3621, 2929, 2369, 1470, 1245, 829 cm -1 ; HRMS (ESI-TOF) m / z: [M + H] + C 22 H 45 FO4Si2 and its resolution 449.2913; Label 449.2912.

[0236] Synthesis of diols 3-3 and 3-4: To a 3 mL THF solution of stirred acetonide 3-2 (129 mg, 0.288 mmol), tetra-n-butylammonium fluoride (1.15 mL, 1.15 mmol in 1.0 M THF solution) was added and stirred for 12 hours. The reaction mixture was then treated with a saturated aqueous solution of NH4Cl and extracted with ethyl acetate. The aqueous layer was separated and further extracted with siRNA. The combined organic phases were washed with brine, dried over anhydrous MgSiO4, filtered, concentrated, and the resulting crude product was purified by column chromatography (20% acetone in DCM solution) to obtain isomer 3-4 as a white solid (21.8 mg, 30%) and isomer 3-3 as a white solid (37.0 mg, 51%). Data for 3-3: [α] D = + 30.7 (c = 1.4, acetone); 1 H NMR (400 MHz, CDCl3) δ 4.59 (ddd, J = 7.6, 8.9, 46.7 Hz, 1 H), 4.42 (ddd, J = 6.9, 8.9, 47.1 Hz, 1 H), 4.26 (t, J = 4.3 Hz, 1 H), 3.93 (m, 1 H), 3.57-3.44 (m, 2 H), 2.50 (d, J = 2.7 Hz, 1 H), 2.34 (d, J = 2.4 Hz, 1 H), 2.29 (m, 1 H), 1.88 (m, 1 H), 1.51 (s, 3 H), 1.48 (m, 1 H), 1.36 (s, 3 H); 13 C NMR (101 MHz, CDCl3) δ 109.9, 85.0, 83.4, 80.7, 78.7, 73.7 (d, J = 6.1 Hz), 71.0 (d, J = 1.5 Hz), 36.3 (d, J = 19.1 Hz), 28.6 (d, J = 5.7 Hz), 28.5, 26.5; 19 F NMR (377 MHz, CDCl3): δ -222.6 (dt, J = 12.8, 46.7 Hz); IR (neat) ν 3721, 3694, 3624, 3597, 3214, 2369, 1366, 1218 1024 cm-1 HRMS (ESI-TOF) m / z: [M + H] + C 10 H 17 Calculated value for FO4: 221.1184; measured value: 221.1183. Data from 3-4: [α] D = + 17.4 (c = 1.1, acetone); 1 H NMR (500 MHz, CDCl3) δ 4.56 (ddd, J = 4.5, 9.3, 36.7 Hz, 1 H), 4.46 (ddd, J = 4.0, 9.3, 36.0 Hz, 1 H), 4.20 (t, J = 5.7 Hz, 1 H), 4.00 (t, J = 6.6 Hz, 1 H), 3.77-3.55 (m, 2 H), 2.52 (s, 1 H), 2.47 (s, 1 H), 2.34 (m, 1 H), 1.87 (m, 2 H), 1.52 (s, 3 H), 1.37 (s, 3 H); 13 C NMR (101 MHz, CDCl3): δ 109.3, 85.6 (d, J = 170.0 Hz), 79.0 (d, J = 1.1 Hz), 76.5, 74.8 (d, J = 4.8 Hz), 68.8 (d, J = 1.9 Hz), 36.3 (d, J = 18.8 Hz), 29.8 (d, J = 3.1 Hz), 28.2, 25.9; 19 F NMR (377 MHz, CDCl3): δ -225.0 (dt, J = 31.3, 47.5 Hz); IR (neat) ν 3712, 3695, 3677, 3665, 2919, 2852, 1446, 1369, 1004 cm -1 HRMS (ESI-TOF) m / z: [M + H] + The calculated value for C10H17FO4 is 221.1184; the measured value is 221.1182.

[0237] Synthesis of Tetraol 3-5: To a stirred solution of diol 3-3 (24 mg, 93.7 μmol) in CH3OH (4.8 mL), 1 M HCl (1.2 mL) was added and the mixture was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography (in a 20% CH3OH solution of Â) to obtain 3-5 as a colorless viscous liquid (15.4 mg, 91%). [α] D = +2.8 (c = 1.4, CH3OH); 1 H NMR (400 MHz, CD3OD) δ 4.50 (dt, J = 8.5, 47.5 Hz, 1 H), 4.32 (ddd, J = 6.5, 8.7, 47.3 Hz, 1 H), 3.94 (m, 1 H), 3.53 (t, J = 9.3 Hz, 1 H), 3.50-3.39 (m, 1H), 3.29 (dd, J = 3.0, 9.5 Hz, 1 H), 1.92 (m, 1 H), 1.64 (dt, J = 4.2, 12.5 Hz, 1 H), 1.47 (q, J = 12.4 Hz, 1 H), 1.02 (d, J = 6.7 Hz, 1 H); 13 C NMR (151 MHz, CD3OD) δ 83.7 (d, J = 166.7 Hz), 75.2, 74.7, 72.0, 68.5 (d, J = 5.3 Hz), 37.9 (d, J = 18.6 Hz), 28.0 (d, J = 6.5 Hz); 19 F NMR (377 MHz, CD3OD) δ -225.0 (dt, J = 12.4, 47.4 Hz); IR (neat) ν 3728, 3694, 2345, 1598, 1044 cm -1 ; HRMS (ESI-TOF) m / z: [M + NH4] + C7H 17 Calculated value as NFO4: 198.1136; measured value: 198.1139.

[0238] Synthesis of Tetraol 3-6: To a 4.8 mL solution of stirred diol 3-4 (18 mg, 70.3 μmol) in CH3OH, 1.2 mL of 1 M HCl was added and the mixture was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography (in a 20% CH3OH solution of siRNA) to obtain 3-6 as a colorless viscous liquid (12.2 mg, 96%). [α] D = +5.8 (c = 0.3, CH3OH); 1 H NMR (500 MHz, CD3OD) δ 4.59 (ddd, J = 5.2, 9.2, 47.8 Hz, 1 H), 4.47 (ddd, J = 4.4, 9.2, 47.7 Hz, 1 H), 3.85-3.78 (m, 2 H), 3.75-3.68 (m, 2 H), 2.21 (m, 1 H), 1.87-1.83 (m, 2 H); 13 C NMR (151 MHz, CD3OD) δ 85.9 (d, J = 167 Hz), 74.8, 73.9, 71.4, 69.7 (d, J = 3.8 Hz), 37.8 (d, J = 17.9 Hz), 30.5 (d, J = 3.5 Hz); 19 F NMR (377 MHz, CD3OD) δ -229.0 (broad signal); IR (neat) ν 3731, 3691, 2346, 1645, 1064 cm -1 ; HRMS (ESI-TOF) m / z: [M + NH4] + C7H 17 Calculated value as NFO4: 198.1136; measured value: 198.1141.

[0239] Synthesis of acetate 3-7: Ac20 (0.02 mL, 0.2 mmol) was added to a pyridine solution (0.6 mL) of stirred tetraol 3-6 (3 mg, 16.7 μmol), and the mixture was stirred at 50°C for 12 hours. The reaction mixture was concentrated under reduced pressure and co-evaporated with toluene. The resulting crude product was purified by column chromatography (30% siRNA solution) to obtain 3-7 as a colorless viscous liquid (5.2 mg, 89%). [α]D = +12.6 (c = 0.6, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 5.32-5.25 (m, 2 H), 5.18 (d, J = 3.3, 7.5 Hz, 1 H), 5.08-5.02 (m, 1 H), 4.56 (ddd, J = 4.4, 9.7, 47.5 Hz, 1 H), 4.49 (ddd, J = 4.4, 9.7, 47.1 Hz, 1 H), 2.37 (m, 1 H), 2.09 (s, 3 H), 2.06 (s, 3 H), 2.05 (s, 3 H), 2.04 (s, 3 H), 2.05-2.01 (m, 2 H); 13 C NMR (151 MHz, CDCl3) δ 170.0, 170.0, 169.9, 169.7, 84.0 (d, J = 171.2 Hz), 70.2, 69.6 (d, J = 3.1 Hz), 69.5 (d, J = 2.5 Hz), 69.4 (d, J = 1.8 Hz), 35.9 (d, J = 19.5 Hz), 27.6 (d, J = 3.1 Hz, 21.1, 21.0, 20.9, 20.8; 19 F NMR (377 MHz, CDCl3) δ -223.9 (broad signal); IR (cast film) ν 2919, 2849, 1748, 1369, 1218, 1040 cm -1 ; HRMS (ESI-TOF) m / z: [M + NH4] + C 15 H 25 Calculated value for NO8: 366.1559; measured value: 366.1557.

[0240] Aldehydes 1-29 were prepared from L-quebrachitol according to methods reported in the literature. JPEG2026136206000034.jpg86158

[0241] Synthesis of methyl ester 1-33: To a CH2Cl2:Et3N (4:1, c=0.1 M) solution of aldehyde 1-29 (Monda, S.; Sureshan, KMJ Org. Chem. 2016, 81, 11635, 0.161 mmol, 34.5 mg, 1.0 equivalent) at 0°C, tert-butyldimethylsilyltrifluoromethanesulfonate (4.0 equivalents) was added. After stirring at room temperature for 1 hour, the reaction mixture was quenched with a saturated aqueous solution of NaHCO3. The aqueous layer was extracted with Et2O. The combined organic layers were washed with brine, dried over Na2SO4, and evaporated under vacuum. The residue (1.0 equivalent) was... t The reaction mixture was dissolved in BuOH:H2O (4:1, c=0.01 M). 2-methyl-2-butene (100 equivalents), NaH2PO4 (11 equivalents), and NaCl2 (10 equivalents) were added at 0°C. After stirring at 0°C for 10 minutes, the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with H2O. The aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, and evaporated under vacuum. The residue (1.0 equivalent) was redissolved in CH2Cl2:MeOH (4:1, c=0.01 M), and (trimethylsilyl)diazomethane (5.0 equivalents) was added. The reaction mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue (1.0 equivalent) was dissolved in EtOH (c=0.1 M) and Rh / Al2O3 (0.5 equivalents). The reaction mixture was stirred at room temperature and under H2 (50 atm). After 12 hours, the reaction mixture was filtered through Celite, and the filtrate was evaporated under vacuum. The residue was subjected to silica gel column chromatography. n Purified with hexane:Et20 (10:1), 1-33 was obtained as a white solid (52 mg, isolation yield 68%). mp:105-106℃. 1H NMR (600 MHz, CDCl3): δ 4.53 (dd, J = 4.5, 4.1 Hz, 1H), 4.01 (t, J = 6.8 Hz, 1H), 3.73 (s, 3H), 3.59 (dd, J = 6.9, 6.5 Hz, 1H), 3.52-3.48 (m, 1H), 2.74 (dt, J = 13.4, 3.5 Hz, 1H), 1.98 (dt, J = 13.4, 3.8 Hz, 1H), 1.91-1.85 (m, 1H), 1.48 (s, 3H), 1.32 (s, 3H), 0.89 (d, J = 3.0 Hz, 18H), 0.11-0.07 (m, 12H); 13 C NMR (151 MHz, CDCl3): δ 172.1, 109.4, 80.9, 77.7, 74.1, 72.9, 52.2, 40.4, 28.8, 27.6, 26.20, 26.18, 25.9, 18.3, 18.2, -3.7, -3.8, -4.2, -4.3; IR (neat): ν 3182, 2950, ​​2931, 2857, 1747, 1306, 1255, 1108, 1046, 827, 780 cm -1 HRMS (ESI): m / z [M + H] + C 23 H 47 Calculated value for O6Si2: 475.2911; Measured value: 475.2910; [α] D 20 = 29.3 (c = 0.1, MeOH).

[0242] Synthesis of Tetrole 1-34

[0243] JPEG2026136206000035.jpg57441-33 (0.025 mmol, 12.0 mg) was dissolved in methanol:1N HCl (4:1, c=0.05M). After stirring at room temperature for 5 hours, the solvent was removed under vacuum. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH, 4:1) to obtain 1-34 as a white solid (5.2 mg, isolation yield 76%). 1H NMR (400 MHz, MeOD): δ 4.26 (t, J = 2.7 Hz, 1H), 3.70 (s, 3H), 3.52-3.47 (m, 1H), 3.41-3.37 (m, 1H), 3.33-3.32 (m, 1H), 2.61-2.56 (m, 1H), 1.94-1.89 (m, 2H); 13 C NMR (151 MHz, MeOD): δ 174.5, 76.1, 75.7, 72.9, 71.8, 52.3, 44.3, 29.6; IR (neat): ν 3178, 2921, 2853, 1718, 1578, 1421, 1298, 847cm -1 HRMS (ESI): m / z [M + H] + C8H 15 Calculated value for O6: 207.0863; measured value: 207.0861; [α] D 20 = -14.0 (c = 0.1, MeOH). JPEG2026136206000036.jpg99140

[0244] Synthesis of alkyl bromides 1-38: LiAlH4 (3.0 equivalents) was added in several portions to a solution of 1-33 (0.058 mmol, 27.4 mg, 1.0 equivalent) in Et20 (c=0.03 M). After heating under reflux for 16 hours, the reaction mixture was quenched with wet Et20. The aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, and evaporated. The residue (1.0 equivalent) was dissolved in CH2Cl2 (c=0.05 M). PPh3 (1.0 equivalent), CBr4 (1.0 equivalent), and Et3N (1.0 equivalent) were added. After stirring at room temperature for 48 hours, the solvent was removed under vacuum. The residue was subjected to silica gel column chromatography. n Purified with hexane:Et20, 40:1), 1-38 was obtained as a foamy solid (21.2 mg, isolation yield 72%). Data for 1-38: 1H NMR (600 MHz, CDCl3): δ 4.29 (dd, J = 4.3, 4.1 Hz, 1H), 3.92 (t, J = 5.8 Hz, 1H), 3.54-3.50 (m, 2H), 3.47-3.43 (m, 1H), 3.34 (dd, J = 9.8, 6.9 Hz, 1H), 2.12-2.06 (m, 1H), 1.83 (dt, J = 12.8, 3.6 Hz, 1H), 1.51-1.47 (m, 1H), 1.47 (s, 3H), 1.33 (s, 3H), 0.90 (s, 18H), 0.12-0.08 (m, 12H); 13 C NMR (151 MHz, CDCl3): δ 109.0, 81.6, 78.9, 74.5, 73.2, 38.0, 34.5, 32.8, 29.9, 28.0, 26.3, 26.2, 18.34, 18.32, -3.6, -3.7, -4.1, -4.3; IR (neat): ν 2954, 2932, 2861, 1474, 1384, 1257, 1097, 1052, 1034, 803 cm -1 HRMS (ESI): m / z [M + H] + C 22 H 46 Calculated value for BrO4Si2: 509.2113; Measured value: 509.2113; [α] D 20 = -6.3 (c = 0.3, MeOH).

[0245] Synthesis of tetraol 1-39: 1-38 (0.029 mmol, 14.9 mg) was dissolved in methanol:1N HCl (4:1, c=0.05 M). After stirring at room temperature for 3 hours, the solvent was removed under vacuum. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH, 15:1 then 2:1) to obtain 1-39 as a colorless oil (5.0 mg, isolation yield 71%). 1H NMR (600 MHz, MeOD): δ 4.02 (brs, 1H), 3.55-3.49 (m, 2H), 3.42-3.35 (m, 2H), 3.27 (dd, J = 9.6, 2.9 Hz, 1H), 1.87-1.79 (m, 2H), 1.53-1.47 (m, 1H); 13 C NMR (151 MHz, MeOD): δ 76.4, 76.1, 73.2, 71.3, 41.6, 35.2, 32.9; IR (neat): ν 3372, 2924, 2857, 1451, 1257, 1116, 1072, 1049, 989 cm -1 HRMS (ESI): m / z [M + Na] + C7H 13 Calculated value for BrNaO4: 262.9889; measured value: 262.9886; [α] D 20 = 9.2 (c = 0.12, MeOH). JPEG2026136206000037.jpg74153

[0246] Synthesis of Alkene 1-42: LiAlH4 (3.0 equivalents) was added in several portions to a solution of 1-33 (0.066 mmol, 31.1 mg, 1.0 equivalent) in Et20 (c=0.03 M). After heating under reflux for 16 hours, the reaction mixture was quenched with wet Et20. The aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, and evaporated under vacuum. The residue (1.0 equivalent) was dissolved in CH2Cl2 (c=0.1 M), and Dess-Martin-Periodinane (1.2 equivalents) was added. After stirring at room temperature for 30 minutes, the solvent was removed under vacuum to obtain crude aldehyde 1-40. Under an N2 atmosphere, PPh3MeBr (3.0 equivalents) was added to a solution of THF. nBuLi (3.0 equivalents in a 2.5 M hexane solution) was added at -78°C. The mixture was heated to room temperature for 30 minutes, then cooled to -78°C, and the crude aldehyde 1-40 (1.0 equivalent) was added. The reaction mixture was heated to room temperature for 30 minutes. The reaction mixture was quenched with water and extracted with Et20. The organic layer was dried over Na2SO4 and concentrated to obtain crude alkene 1-41. The residue (0.055 mmol) was dissolved in methanol:1N HCl (4:1, c=0.05 M). After stirring at room temperature for 3 hours, the solvent was removed under vacuum. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH, 15:1 then 2:1) to obtain 1-42 as a colorless oil (4.0 mg, isolation yield 35%). 1 H NMR (600 MHz, MeOD): δ 5.98-5.90 (m, 1H), 5.08 (dt, J = 17.3, 1.6 Hz, 1H), 5.04 (dt, J = 10.4, 1.5 Hz, 1H), 3.81 (brs, 1H), 3.53-3.49 (m, 1H), 3.45-3.39 (m, 1H), 3.33-3.31 (m, 1H), 2.24-2.19 (m, 1H), 1.78-1.63 (m, 2H); 13 C NMR (151 MHz, MeOD): δ 140.8, 115.1, 76.5, 76.2, 74.1, 73.7, 42.8, 33.2; IR (neat): ν 3318, 2932, 2876, 1645, 1421, 1257, 1123, 1071, 1049, 989, 903cm -1 HRMS (ESI): m / z [M + Na] + C8H 14 Calculated value as NaO4: 197.0784; measured value: 197.0787; [α] D 20 = -5.4 (c = 0.3, MeOH). JPEG2026136206000038.jpg67151

[0247] Synthesis of Nitrile 1-43: To a solution of aldehyde 1-29 (0.115 mmol, 24.6 mg, 1.0 equivalent) in CH2Cl2:Et3N (4:1, c=0.1 M) at 0°C, tert-butyldimethylsilyltrifluoromethanesulfonate (1.0 equivalent) was added. After stirring at room temperature for 1 hour, the reaction mixture was quenched with a saturated aqueous solution of NaHCO3. The aqueous layer was extracted with Et20. The combined organic layers were washed with brine, dried over Na2SO4, and evaporated under vacuum to obtain crude 1-30. A solution of dimethylhydrazine hydrochloride (1.5 equivalents) and Et3N (1.5 equivalents) in MeOH (c=0.175 M) was stirred at room temperature for 10 minutes. Crude 1-30 (1.0 equivalent) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. Next, the reaction solution was added dropwise at 0°C to a solution of magnesium monoperoxyphthalic acid hexahydrate (2.5 equivalents) in MeOH (c=0.7 M). After stirring at 0°C for 10 minutes, the reaction mixture was quenched with H2O. The aqueous layer was extracted with Et2O. The combined organic layers were washed with brine, dried with Na2SiO4, and evaporated under vacuum. The residue was analyzed by silica gel column chromatography. n The solution was purified with hexane:Et20 (12:1) to obtain 1-43 as a foamy solid (22.8 mg, isolation yield 45%). 1 H NMR (600 MHz, CDCl3): δ 6.46 (dd, J = 3.2, 0.8 Hz, 1H), 4.62 (d, J = 6.4 Hz, 1H), 4.18 (dd, J = 6.0, 6.0 Hz, 1H), 4.09-4.07 (m, 1H), 3.80 (dd, J = 5.8, 5.7 Hz, 1H), 1.47 (s, 3H), 1.37 (s, 3H), 0.90 (s, 9H), 0.88 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H), 0.095 (s, 3H), 0.088 (s, 3H); 13C NMR (151 MHz, CDCl3): δ 146.5, 117.0, 113.1, 111.6, 77.2, 72.5, 71.4, 69.7, 29.8, 27.7, 26.00, 25.99, 25.9, 25.8, 18.2, 18.1, -4.1, -4.3, -4.4; IR (neat): ν 2929, 2859, 1679, 1463, 1378, 1258, 1096, 837, 779 cm -1 HRMS (ESI): m / z [M + H] + C 22 H 42 Calculated value for NO4Si2: 440.2652; Measured value: 440.2650; [α] D 20 = 50.1 (c = 0.6, CH2Cl2).

[0248] Synthesis of Tetraol 1-45: Rh / Al2O3 (0.5 equivalents) was added to a solution of 1-43 (0.048 mmol, 21.2 mg, 1.0 equivalent) in EtOH (c=0.05 M). The reaction mixture was stirred at room temperature under H2 (50 atm). After 12 hours, the reaction mixture was filtered through Celite, and the filtrate was evaporated under vacuum. The residue was dissolved in methanol:1N HCl (4:1, c=0.05 M). After stirring at room temperature for 3 hours, the solvent was removed under vacuum. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH, 15:1) to obtain 1-45 as a colorless oil (2.5 mg, isolation yield 30%). 1 H NMR (600 MHz, MeOD): δ 4.05 (dd, J = 2.4, 2.4 Hz, 1H), 3.51-3.48 (m, 1H), 3.38-3.35 (m, 1H), 3.26 (dd, J = 9.6, 2.8 Hz, 1H), 2.94-2.91 (m, 1H), 2.00-1.97 (m, 2H); 13C NMR (151 MHz, MeOD): δ 121.4, 75.6, 74.9, 72.2, 70.7, 31.4, 31.3; IR (neat): ν 2928, 2859, 1679, 1463, 1378, 1258, 1196, 1100, 837, 776, 683cm -1 HRMS (ESI): m / z [M + Na] + C7H 11 Calculated value for NNaO4: 196.0586; measured value: 196.0590; [α] D 20 = 2.9 (c = 0.1, MeOH). JPEG2026136206000039.jpg105164

[0249] Synthesis of ethyl ketone 1-47: To a CH2Cl2:Et3N (4:1, c=0.1 M) solution of aldehyde 1-29 (0.1 mmol, 21.4 mg, 1.0 equivalent) at 0°C, tert-butyldimethylsilyltrifluoromethanesulfonate (1.0 equivalent) was added. After stirring at room temperature for 1 hour, the reaction mixture was quenched with a saturated aqueous solution of NaHCO3. The aqueous layer was extracted with Et2O. The combined organic layers were washed with brine, dried over Na2SiO4, and evaporated under vacuum. The residue (1.0 equivalent) was dissolved in THF (c=0.1 M), and EtMgBr (3.0 equivalents) was added at 0°C. After stirring at 0°C for 2 hours, the reaction mixture was quenched with a saturated aqueous solution of NH4Cl. The aqueous layer was extracted with Et2O. The combined organic layers were washed with brine, dried over Na2SiO4, and evaporated under vacuum. The residue (1.0 equivalent) was dissolved in CH2Cl2 (c=0.1 M). Dess-Martin-Periodinane (1.2 equivalents) and NaHCO3 (3.0 equivalents) were added. After stirring at room temperature for 30 minutes, the solvent was removed under vacuum. The residue was subjected to silica gel column chromatography. n The solution was purified with hexane:Et2O (15:1), and 1-47 was obtained as a foamy solid (31.8 mg, isolation yield 66%). 1H NMR (400 MHz, CDCl3): δ 6.64 (d, J = 2.3 Hz, 1H), 5.01 (d, J = 6.6 Hz, 1H), 4.16 (ddd, J = 7.2, 2.2, 1.2 Hz, 1H), 4.07 (dd, J = 7.6, 6.6 Hz, 1H), 3.69 (t, J = 7.5 Hz, 1H), 2.79 (dq, J = 17.7, 7.2 Hz, 1H), 2.63 (dq, J = 17.7, 7.2 Hz, 1H), 1.45 (s, 3H), 1.40 (s, 3H), 1.12 (t, J = 7.2 Hz, 3H), 0.94 (s, 9H), 0.90 (s, 9H), 0.14-0.09 (m, 12H); 13 C NMR (101 MHz, CDCl3): δ 200.3, 141.5, 134.9, 110.3, 78.5, 74.7, 71.7, 70.8, 31.9, 29.8, 28.2, 26.3, 26.2, 26.1, 18.4, 18.3, 8.0, -3.7, -3.8, -3.9, -4.3; IR (neat): ν 2930, 2855, 1715, 1682, 1462, 1381, 1250, 1202, 1145, 1115, 1073, 838, 779 cm -1 HRMS (ESI): m / z [M + H] + C 24 H 47 Calculated value for O5Si2: 471.2962; Measured value: 471.2961; [α] D 20 = 15.2 (c = 1.0, CH2Cl2).

[0250] Synthesis of ketones 1-49 JPEG2026136206000040.jpg37146

[0251] Synthesis of ketone 1-49: Rh / Al2O3 (0.5 equivalents) was added to a solution of 1-47 (0.05 mmol, 23.6 mg, 1.0 equivalent) in EtOH (c=0.05 M). The reaction mixture was stirred at room temperature under H2 (50 atm). After 12 hours, the reaction mixture was filtered through Celite, and the filtrate was evaporated under vacuum. The residue was dissolved in methanol:1N HCl (4:1, c=0.05 M). After stirring at room temperature for 3 hours, the solvent was removed under vacuum. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH, 15:1 then 2:1) to obtain 1-49 as a colorless oil (4.1 mg, isolation yield 40%). 1 H NMR (600 MHz, MeOD): δ 4.32 (dd, J = 2.7, 2.4 Hz, 1H), 3.50-3.47 (m, 1H), 3.41-3.38 (m, 1H), 3.36 (dd, J = 9.5, 2.9 Hz, 1H), 2.68-2.51 (m, 3H), 1.87-1.84 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H); 13 C NMR (151 MHz, MeOD): δ 212.8, 76.2, 76.0, 73.2, 71.7, 50.9, 34.2, 29.1, 7.9; IR (neat): ν 2939, 2852, 1713, 1672, 1256, 1202, 1070, 841, 789cm -1 HRMS (ESI): m / z [M + H] + C9H 17 Calculated value for O5: 205.1076; Measured value: 205.1072; [α] D 20 = -24.1 (c = 0.1, MeOH).

[0252] Cell-based assays

[0253] CHO K1 cells were grown in a T175 flask in F12 medium supplemented with 10% PBS under conditions of 37°C and 5% CO2. 350 cells were seeded into each well of a 384-well plate (Corning 4680, final volume 45 μL) and incubated overnight at 37°C. The inhibitor was stored as a 20 mM DMSO stock solution. The inhibitor was diluted to the desired concentration in the CHO cell culture medium and dispensed into the wells of the plate (final well volume 50 mL). An equal volume of DMSO (medium) was dispensed into the control wells. The cells were incubated in the presence of the inhibitor for 5 days. After treatment, the culture medium was removed and the cells were washed four times with 60 μL of PBS-T using a wash / dispenser (EL406, Biotek). Next, 50 μL of 4% PFA in PBS-T solution was dispensed onto the plate, and the cells were incubated at room temperature for 15 minutes, followed by four washes with 60 μL of PBS-T. Subsequently, to prevent nonspecific lectin binding, the cells were incubated at room temperature for 45 minutes with 5% bovine serum albumin (BSA) in PBS-T solution. Then, the BSA solution was replaced with 50 μL of 5 μg / mL fluorescein-conjugated Alluria orentia lectin (AAL) (Vector Laboratories) solution, and the cells were kept in the dark at room temperature for 1 hour. Negative controls were co-cultured with AAL and 100 mM fucose as a lectin antagonist. Subsequently, the cells were washed four times with 60 μL of PBS-T. Subsequently, 50 μL of PBS solution containing 1 μg / mL Hoechst was dispensed into each well, and cells were directly imaged using an ImageXpress MicroXLS high-content microscope (Molecular Device). Four sites were imaged per well using DAPI and FITC channels (50 ms and 300 ms exposures, respectively). Images were processed using MetaXpress software. Briefly, for each site, the integrated mean fluorescence was quantified and normalized for the corresponding number of cells measured by nuclear staining. The results from each site were combined to provide a single value for each well. At least four independent wells were imaged for each condition providing four biological replicates. IC 50The curves were plotted using GraphPad Prism. The results were normalized by the mean value of the medium well (DMSO treated).

[0254] The inventors evaluated the efficacy of carbafucose and selected analogues in cell-based assays through dose-response experiments over a concentration range of 3.2 nM to 100 μM. The cell-based fucosylation antagonist 2-deoxy-2-fluoro-L-fucose (2FFuc) was used as a control (Figure 1A-D).

[0255] Under the conditions of the assay described above, IC at 2FFuc 50 It was measured at 137±79 μM, and the most potent compound was IC, as follows: 50 This was a 16.1 ± 7.7 μM Carbaff course. JPEG2026136206000041.jpg135135

[0256] The following compounds did not show favorable results under the conditions of the assay described above. JPEG2026136206000042.jpg98133

[0257] All quotations are incorporated herein by reference.

[0258] Other Embodiments

[0259] The present invention has been described in relation to one or more embodiments. However, it will be apparent to those skilled in the art that numerous changes and modifications can be made without departing from the scope of the invention as defined in the claims. Thus, although various embodiments of the invention are disclosed herein, many adaptations and modifications can be made within the scope of the invention according to the common general knowledge of those skilled in the art. Such modifications include replacing any aspect of the invention with known equivalents to achieve the same results in substantially the same way. It should be understood that particular embodiments can be combined in any way and in any number to create additional embodiments, and any permutations and combinations of embodiments should be considered disclosed by the specification of this application unless the context indicates otherwise. Numerical ranges include numerical values ​​that define the range. The enumeration of numerical ranges of values ​​herein is intended merely to serve as a simple way to refer individually to the individual values ​​that fall within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were described separately herein. "Approximately" means a difference (plus or minus) from a value or range of 5% or less, e.g., 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, etc. The terms "a," "an," and "the" and similar references used in the context describing this invention should be interpreted as covering both singular and plural forms unless otherwise indicated herein or unless the context clearly contradicts this interpretation. In this specification, the word "including" is used as an open-end term substantially equivalent to the phrase "including, but not limited to," and the word "including" has the corresponding meaning.However, it should be understood that when the words “including” or “including,” or variations of the same etymology, are used herein, variations or modifications to “consisting of…” or “consisting of…” that exclude unspecified elements, steps, or components, or variations or modifications to “essentially consisting of…” or “essentially consisting of…” that limit the claimed invention to specific materials or enumerated steps, as well as those that do not substantially affect the fundamental and novel features of the claimed invention. References made herein should not be construed as an admission that such references are prior art to the present invention. All publications are incorporated herein by reference in such a manner as if each individual publication were specifically and individually indicated to be incorporated herein by reference and as if it were fully described herein. The present invention includes substantially all embodiments and variations described herein by reference.

Claims

1. A method for inhibiting the fucosylation of a protein, or a fragment or derivative thereof, comprising contacting a eukaryotic cell or mammal with a compound of formula (I) or a salt thereof, During the ceremony, R 1 C may be substituted. 1 -C 10 Alkyl, possibly substituted C 1 -C 10 Alkenyl, or possibly substituted C 1 -C 10 It is alkinyl, R 2 is H or -C(=O)(C 1 -C 6 )alkyl, and R 3 This is a halo, OH, or OC (=O)(C 1 -C 6 ) is alkyl, and R 4 H, -C(=O)(C 1 -C 6 )alkyl, or -P(=O)(OR 5 )(OR 6 ) and in the formula, R 5 and R 6 These are H and (C) respectively, independently. 1 -C 6 ) alkyl, (CH 2 ) 2 SC(=O)CH 3 CH 2 OC(=O)OR 7 , or CH 2 OC(=O)R 7 And in the formula, R 7 C 1 -C 8 It is alkyl, or in the formula, R 5 and R 6 They are connected to form a ring, The method wherein the fucosylation of the protein is reduced by at least 5% compared to the amount of fucosylation of the protein in eukaryotic cells or mammals when the compound is not administered.

2. The method according to claim 1, wherein the protein comprises an N-glycan.

3. The method according to claim 2, wherein the compound is not incorporated into the N-glycan.

4. The method according to any one of claims 1 to 3, wherein the protein is an antibody.

5. R 1 However, CH 3 or CHCH 2 And R 2 However, H is R 3 However, it is OH, and R 4 However, H or -P (=O) (OR 5 )(OR 6 ) and in the formula, R 5 and R 6 The method according to any one of claims 1 to 4, wherein H is present.

6. The aforementioned compound, The method according to any one of claims 1 to 4.

7. The method according to any one of claims 3 to 6, wherein the mammal has cancer, an autoimmune disease, an inflammatory disease, or an infection.

8. The method according to claim 7, further comprising administering a cancer-related antigen or an antigenic fragment thereof as an immunogen to a mammal having cancer.

9. The method according to any one of claims 3 to 8, wherein the mammal is a human.

10. The method according to any one of claims 3 to 9, wherein the salt is a pharmaceutically acceptable salt.

11. A culture medium for mammalian cells comprising an effective amount of the compound of formula (I) or a salt thereof, During the ceremony, R 1 C may be substituted. 1 -C 10 Alkyl, possibly substituted C 1 -C 10 Alkenyl, or possibly substituted C 1 -C 10 It is alkinyl, R 2 is H or -C(=O)(C 1 -C 6 ) is alkyl, R 3 This is a halo, OH, or OC (=O)(C 1 -C 6 ) is alkyl, and R 4 H, -C(=O)(C 1 -C 6 )alkyl, or -P(=O)(OR 5 )(OR 6 ) and in the formula, R 5 and R 6 These are H and (C) respectively, independently. 1 -C 6 ) alkyl, (CH 2 ) 2 SC(=O)CH 3 CH 2 OC(=O)OR 7 , or CH 2 OC(=O)R 7 And in the formula, R 7 C 1 -C 8 It is alkyl, or in the formula, R 5 and R 6 This is a culture medium for mammalian cells, where cells are linked together to form a ring.

12. The mammalian culture medium according to claim 11, wherein the culture medium is useful for producing fucose-deficient proteins, or fragments or derivatives thereof.

13. The mammalian culture medium according to claim 12, wherein the effective amount is an amount of the compound sufficient to reduce the fucose incorporation into the sugar chain of the fucose-deficient protein, or a fragment or derivative thereof, by at least 50%.

14. The mammalian culture medium according to any one of claims 11 to 13, wherein the culture medium for the mammalian cells is a culture medium for Chinese hamster ovary cells.

15. A method for treating cancer, autoimmune disease, infection, inflammatory disease, or sickle cell disease, comprising administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a mammal in need thereof. During the ceremony, R 1 C may be substituted. 1 -C 10 Alkyl, possibly substituted C 1 -C 10 Alkenyl, or possibly substituted C 1 -C 10 It is alkinyl, R 2 is H or -C(=O)(C 1 -C 6 ) is alkyl, R 3 is halo, OH, or O-C(=O)(C 1 -C 6 )alkyl, and R 4 is H, -C(=O)(C 1 -C 6 )alkyl, or -P(=O)(OR 5 )(OR 6 ), wherein R 5 and R 6 are each independently H, (C 1 -C 6 )alkyl, (CH 2 ) 2 SC(=O)CH 3 、CH 2 OC(=O)OR 7 、or CH 2 OC(=O)R 7 , wherein R 7 is C 1 -C 8 alkyl, or, wherein R 5 and R 6 are linked to form a ring, said method.

16. To inhibit the fucosylation of a protein, or a fragment or derivative thereof, of a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the fucosylation of the protein is reduced by at least 5% compared to the amount of fucosylation of the protein in eukaryotic cells or mammals in the absence of the compound, or for use in the treatment of cancer, autoimmune diseases, infections, inflammatory diseases, or sickle cell disease, During the ceremony, R 1 C may be substituted. 1 -C 10 Alkyl, possibly substituted C 1 -C 10 Alkenyl, or possibly substituted C 1 -C 10 It is alkinyl, R 2 is H or -C(=O)(C 1 -C 6 ) is alkyl, R 3 This is a halo, OH, or OC (=O)(C 1 -C 6 ) is alkyl, and R 4 H, -C(=O)(C 1 -C 6 )alkyl, or -P(=O)(OR 5 )(OR 6 ) and in the formula, R 5 and R 6 These are H and (C) respectively, independently. 1 -C 6 ) alkyl, (CH 2 ) 2 SC(=O)CH 3 CH 2 OC(=O)OR 7 , or CH 2 OC(=O)R 7 And in the formula, R 7 C 1 -C 8 It is alkyl, or in the formula, R 5 and R 6 The aforementioned use involves connecting them to form a ring.

17. A compound of formula (II) or a pharmaceutically acceptable salt thereof, During the ceremony, R 1 C may be substituted. 1 -C 10 Alkyl, possibly substituted C 1 -C 10 Alkenyl, or possibly substituted C 1 -C 10 It is alkinyl, R 2 is H or -C(=O)(C 1 -C 6 ) is alkyl, R 3 This is a halo, OH, or OC (=O)(C 1 -C 6 ) is alkyl, and R 4 H, -C(=O)(C 1 -C 6 )alkyl, or -P(=O)(OR 5 )(OR 6 ) and in the formula, R 5 and R 6 These are H and (C) respectively, independently. 1 -C 6 ) alkyl, (CH 2 ) 2 SC(=O)CH 3 CH 2 OC(=O)OR 7 , or CH 2 OC(=O)R 7 And in the formula, R 7 C 1 -C 8 It is alkyl, or in the formula, R 5 and R 6 They are connected to form a ring, In the formula, R 1 CH 3 In the case of R 2 It's R, not H. 3 It is not OH, and R 4 A compound of formula (II) or a pharmaceutically acceptable salt thereof, wherein the compound is not H.

18. A composition comprising the compound described in claim 17.

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