Carbocyclic derivatives and conjugated derivatives thereof, and their use in vaccines

Abstract

To provide carba analogue polysaccharide derivatives of the Neisseria meningitidis capsular polysaccharides, having good stability, exhibiting a good immunogenic profile, and suited for the preparation of a vaccine, preferably to be formulated in liquid form, against meningitis.SOLUTION: The invention provides an oligomer of formula (Ia) or (Ib).SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the field of vaccines and pertains to oligomers having a selected degree of polymerization obtained by linking a plurality of carbocyclic repeating units, as well as conjugate derivatives thereof. The oligomers of the present invention and their conjugate derivatives also have a selected degree of acetylation. The derivatives of the present invention are useful, for example, in the preparation of immunogenic compositions in the form of vaccines.

Background Art

[0002] Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide and can cause the occurrence and epidemic of invasive diseases. Invasive meningococcal diseases occur worldwide. The incidence rates vary by region of the world, but infants, children, and young people are the most vulnerable to the onset of invasive diseases. The symptoms of the disease progress rapidly and often result in a devastating outcome. Based on the antigenic differences of their capsular polysaccharides, 12 serotypes of Neisseria meningitidis have been identified. Virtually all disease-related isolates are encapsulated, and serotypes A, B, C, W, X, and Y are involved in more than 90% of invasive meningococcal infections worldwide. There are geographical and temporal variations in the distribution of these serotypes.

[0003] Generally, the capsular polysaccharide (CPS) of Neisseria meningitidis is a T-cell independent antigen, which means that it can elicit an immune response without the involvement of T cells. This response lacks some important characteristics that characterize T-cell dependent immune responses, such as immune memory, class switching from IgM to IgG, and affinity maturation. However, when the polysaccharide moiety is linked to a carrier protein, it elicits a cellular immune response that produces a memory effect and protects infants. Such polysaccharides linked to carrier proteins are often referred to as glycoconjugates and are particularly valuable as vaccines. In this regard, particularly efficient vaccines (glycoconjugate vaccines) can be made by connecting the sugar to the carrier protein via a linker moiety (or spacer), or even by direct coupling of the selected sugar of the carrier protein, or by connecting the sugar to the carrier protein by direct coupling. In any case, glycoconjugates can elicit T-cell dependent immune responses with memory and effect even in infants, while non-conjugated CPS generally does not provide either a memory effect in adults or a substantial immunogenic effect in infants.

[0004] Among Neisseria meningitidis capsular polysaccharides, Neisseria meningitidis serogroup A capsular polysaccharide (MenA CPS) is known to be affected by its inherent chemical instability in water (see, for example, Frasch et al., Adv. Biotechnol. Processes, 1990, 12, 123-145). MenA CPS is composed of (1→6)-linked 2-acetamido-2-deoxy-α-D-mannopyranosyl phosphate repeating units, and the hydrolytic instability of MenA polysaccharide is mainly due to hydrolysis of the phosphodiester linkage promoted by the ring oxygen and N-acetamide. In fact, both the oxygen within the ring and the N-acetyl group destabilize the phosphodiester glycoside linkage, and it has been recognized that the axial position of NHAc also contributes to this mechanism, as shown in Scheme A below (Berti et al., Vaccine, 2012, 30, 6409-6415).

Chemical formula

[0005] If a MenA polysaccharide mimetic resistant to hydrolysis is available, it is very attractive for developing more stable conjugate vaccines. The stabilization of CPS can be achieved in various ways, and MenA CPS analogs in which the epoxy group is replaced by a methylene group have been reported in the prior art. In particular, in this regard, as shown in Scheme B, the replacement of the oxygen in the ring with carbon prevents the destabilization described in Scheme A.

Chemical formula

[0006] Toma et al. Org. Biomol. Chem., 2009, 7, 3734 - 3740 describes the production of O-(2-acetamido-2-deoxy-5a-carba-α-D-mannopyranosyl) phosphate, a monomer in which the pyranose oxygen of the repeating unit of MenA CPS is replaced by a methylene group. This document only mentions the chemical synthesis production of the monomer itself.

[0007] Gao et al. (Org. Biomol. Chem. 2012, 10(33), 6673, and ACS Chem. Biol. 2013, 8(11), 2561) and Ramella D. et al. (Eur J. Org. Chem, 2014, 5915 - 5924) explain the stabilization of glycosyl 1-O-phosphate by using carba sugars in which the pyranose oxygen atom is replaced by a methylene group. They also report the conjugation of synthetic carba trimers to protein carriers, but have not further investigated the behavior of carba analogs with higher degrees of polymerization. There is also no mention of carba analogs with a specific level of acetylation and / or a specific acetylation pattern. Furthermore, the trimers examined are unlikely to inhibit the binding of anti-MenA CPS antibodies, indicating that the described derivatives are relatively weak conjugate antigens.

[0008] Therefore, there is a need to find a carbapenem derivative that has good stability, also shows a good immunogenic profile, can be obtained according to a reliable and convenient synthetic method, and is preferably formulated in liquid form for the production of a vaccine against meningitis. SUMMARY OF THE INVENTION

[0009] In a first aspect, the present invention relates to an oligomer of formula (Ia) or (Ib). [Chemical formula] Wherein, n is ≧ 6; R is H or -P(O)(OR″)2, and R″ is H or a pharmaceutically acceptable phosphate counterion; R′ is H or a pharmaceutically acceptable phosphate counterion; R x is H or -C(O)CH3, and may be the same or different in each repeating unit; R y is H or -C(O)CH3, and may be the same or different in each repeating unit; R x or R y at least one of which is -C(O)CH3 in at least one repeating unit, and overall, about 50 - 90% of R x and R y in the oligomer is -C(O)CH3; Az is an aza substituent selected from the group consisting of -NH(CO)R 1 , -N(R 1 )2 and -N3, and R 1 is independently selected from the group consisting of H, linear or branched C1-C6-alkyl and linear or branched C1-C6-haloalkyl; Z is (i) a protecting group, (ii) a functional linker for conjugation to a protein, or (iii) a straight-chain or branched C1-C6 alkyl, optionally substituted phenyl, -C(O)Y, or a straight-chain or branched C1-C6-alkyl-X wherein Y is H, a straight-chain or branched C1-C6-alkyl or a protecting group; X is -NH2, -N3, -C≡CH, -CH=CH2, -SH or -S-C≡N.

[0010] In a second aspect, the present invention relates to an oligomeric conjugate antigen of formula (IIa) or (IIb). [Chemical formula] wherein n, R, R′, R x and R y are as defined above in relation to the first aspect; Z is a linker or a bond; P is a protein.

[0011] In a third aspect, the present invention relates to an immunogenic composition comprising (a) the conjugate as described above according to the second aspect of the invention; and (b) at least one pharmaceutically acceptable excipient.

[0012] In a fourth aspect, the present invention relates to a vaccine comprising the conjugate as described above according to the second aspect of the invention, or the immunogenic composition as described above according to the third aspect of the invention.

[0013] In a fifth aspect, the present invention relates to a method for treating or preventing meningitis A, C, W135 or Y in a subject, the method comprising administering to the subject a therapeutically or prophylactically effective amount of the conjugate according to the second aspect of the invention, or the immunogenic composition according to the third aspect of the invention, or the vaccine according to the fourth aspect of the invention.

[0014] In a sixth aspect, the present invention relates to a method of immunizing a subject against meningitis A, C, W135 or Y, the method comprising administering to the subject an immunogenic composition according to the third aspect of the present invention or a vaccine according to the fourth aspect of the present invention in an immunologically effective amount.

[0015] In a seventh aspect, the present invention relates to a method of inducing an immune response in a subject against meningitis A, C, W135 or Y, the method comprising administering to the subject an immunogenic composition according to the third aspect of the present invention or a vaccine according to the fourth aspect of the present invention in an immunologically effective amount.

[0016] In an eighth aspect, the present invention relates to the use of an immunogenic composition according to the third aspect of the present invention or a vaccine according to the fourth aspect of the present invention in the manufacture of a medicament for the treatment or prevention of meningitis A, C, Wl35 or Y.

[0017] In a ninth aspect, the present invention relates to an immunogenic composition according to the third aspect of the present invention or a vaccine according to the fourth aspect of the present invention for use in the prevention or treatment of meningitis A, C, W135 or Y.

[0018] In a tenth aspect, the present invention relates to an immunogenic composition according to the third aspect of the present invention or a vaccine according to the fourth aspect of the present invention for use in inducing an immune response against meningitis A, C, W135 or Y. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5a

Figure 5b

Figure 6

Figure 7

Figure 8

Figure 9

DETAILED DESCRIPTION OF THE INVENTION

[0020] To facilitate understanding of the present invention, many terms and phrases are defined below. Even where not specifically described, synonyms or alternative words recognized in the art for the following terms and phrases (including tenses such as past, present, etc.) are contemplated.

[0021] As used in this disclosure and the claims, the singular forms "a", "an", and "the" include the plural unless the context clearly dictates otherwise. That is, "a" means "one or more" unless otherwise stated.

[0022] The term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A", and "B". Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0023] Unless otherwise stated, the designations "A%-B%", "A-B%", "A% to B%", "A to B%", "A%-B", "A% to B" are all given their ordinary and customary meanings. In some embodiments, these designations are synonyms.

[0024] The terms "substantially" or "substantial" mean that the described or claimed state functions in all important aspects as the described standard. Thus, "substantially free" means, even if a numerical value indicates the presence of some impurities or substances, a state that functions as a free state in all important aspects. "Substantial" generally means a value exceeding 90%, preferably exceeding 95%, and most preferably exceeding 99%. When a specific value is used in the specification and claims, unless otherwise indicated, the term "substantially" means the acceptable error range for that specific value.

[0025] "Effective amount" means an amount sufficient to cause the referenced effect or outcome. An "effective amount" can be determined empirically and by conventional methods using known techniques relevant to the stated purpose.

[0026] An "immunologically effective amount" or "therapeutically effective amount" means that administration of that amount to an individual, either as a single dose or as part of a series of administrations, is effective for treatment or prevention. This amount can vary depending on the health and condition of the individual being treated, age, taxonomic group of the individual being treated (e.g., non-human primates, primates, etc.), the ability of the immune system of the individual synthesizing the antibody, the degree of protection desired, the formulation of the vaccine, the medical situation as evaluated by the physician administering the treatment, and other relevant factors. It is expected to fall within a relatively wide range that can be determined by routine testing.

[0027] The term "treatment" means any one of the following: (i) prevention of infection or reinfection, as in the case of conventional vaccines; (ii) reduction in the severity of symptoms or elimination of symptoms; (iii) delay in the recurrence of symptoms; and (iv) substantial or complete elimination of the causative agent or disorder of the problem in the subject. Thus, treatment can be affected prophylactically (before infection) or therapeutically (after infection).

[0028] The term "weight percent (% w / w)" indicates, as shown, the weight percentage of a given compound relative to different compounds or relative to the total content of the composition.

[0029] Similarly, the term "volume % (% v / v)" indicates, as shown, the volume percentage of a given compound relative to the total content of different compounds or compositions.

[0030] The term "oligosaccharide" includes, in its meaning, polysaccharides having 3 to 10 monosaccharide units, as generally known in the art (see, for example, https: / / en.wikipedia.org / wiki / Oligosugar).

[0031] The term "oligomer" refers to carba-related polysaccharides in which the oxygen within the ring is replaced by a methylene (-CH2-) group to provide a cyclohexane skeleton.

[0032] The "degree of polymerization" (DP) indicates the number of monomers linked together to provide the final oligomer. In the present invention, unless otherwise specified, DP is represented by "n" in formulas (I) and (II).

[0033] The "average degree of polymerization" (avDP) indicates the average number of repeating units constituting the oligomer.

[0034] The term "capsular polysaccharide / saccharide" (CPS) refers to saccharides that can be seen in a layer that is part of the outer envelope outside the bacterial cell envelope and thus outside the bacterial cell itself. CPS is expressed on the outermost surface of a wide range of bacteria and, in some cases, also in fungi.

[0035] Unless otherwise defined, the term "conjugation" refers to the connection or linkage of a target entity, particularly an oligomer of the present invention having n (i.e., DP) ≧ 6 and a selected protein.

[0036] As used herein, the term "alkyl" represents a saturated, straight-chain, or branched hydrocarbon moiety. The term "C1-C6-alkyl" refers to an alkyl moiety containing 1 to 6 carbon atoms.

[0037] As used herein, the term "haloalkyl" represents a saturated, straight-chain or branched hydrocarbon moiety in which one or more hydrogen atoms are replaced by halogen atoms. In particular, when referring to "haloalkyl", it is a reference to "fluoroalkyl" in which the halogen is fluoro. The term "C1-C6-haloalkyl" refers to an alkyl moiety containing 1 to 6 carbon atoms in which one or more hydrogen atoms are replaced by halogen atoms. Examples include -CF3, -CH2F, -CH2CF3, and the like.

[0038] As used herein, particularly according to the definition of Z, phenyl may be substituted. The phenyl group may be substituted with one or more reactive functional groups to enable conjugation such as N3, NH2, SH, etc. Other suitable groups are well known to those skilled in the art.

[0039] As used herein, the term "protecting group" is any suitable protecting group for a given purpose. The selection and use of such protecting groups, and the details of their use, can be those described, for example, in Greene, T. W. and Wuts, P. G. M., "Protective Groups in Organic Synthesis". Suitable protecting groups are known to those skilled in the art.

[0040] As used herein, the term "pharmaceutically acceptable phosphate counterion" is any counterion suitable for a phosphate group, i.e., within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation or other problems or complications, and is a metal cation commensurate with a reasonable benefit / risk ratio. The pharmaceutically acceptable phosphate counterion can be a Group 1 or Group 2 metal. Specific examples of such pharmaceutically acceptable phosphate counterions are sodium (Na + ) and potassium (K + ). For example, when the oligomer or conjugate of the present invention is in a buffer solution, the counterion is preferably sodium.

[0041] As described above, the present invention relates to a polysaccharide carb analogue having a degree of polymerization of at least 6 and a first analogue monomer connected to a second analogue monomer via a 1,6-linkage connecting C-1 of the first unit to C-6 of the second unit, wherein the 1,6-linkage contains a phosphonate moiety (i.e., a mannosamine unit in which the ring oxygen is replaced by methylene). Notably, the derivatives of the present invention are expected not only to be able to mimic natural polysaccharides from the MenA serotype but also to have improved stability compared to native CPS.

[0042] In certain embodiments, the oligomers of the present invention are defined by formula (Ia). In certain embodiments, the oligomer conjugate antigen of the present invention is defined by formula (IIa).

[0043] As defined above, n is ≧6. In certain embodiments, n is 8 - 30. In another embodiment, n is 8 - 20. In a particular embodiment, n is 8 - 15. In certain embodiments, n is ≦15. In particular, n is 8 or 10. In certain embodiments, n is 8.

[0044] In certain embodiments, R is H or -P(O)(OR″)2, where at least one R″ is Na + and. In certain embodiments, R is H.

[0045] In certain embodiments, R′ is Na + and the oligomers of the present invention are defined by formula (Ia′) or (Ib′), preferably formula (Ia′).

Chemical formula

[0046] Thus, in certain embodiments, the oligomer conjugate antigen of the present invention is defined by formula (IIa′) or formula (IIb′), preferably formula (IIa′).

Chemical formula

[0047] As defined above, R x is H or -C(O)CH3, and may be the same or different in each repeating unit. R y is H or -C(O)CH3, and may be the same or different in each repeating unit. R x or R y at least one of which is -C(O)CH3 in at least one repeating unit, and overall, about 50 - 90% of R x and R y in the oligomer is -C(O)CH3. Therefore, it should be understood that the formulas defined within the brackets according to formulas (Ia), (IIa), (Ib) and (IIb) have this backbone for each unit of the oligomer, but considering that different options for R x and R y can be selected for each repeating unit defined within the brackets, the monomer units defined by the brackets are not necessarily identical. Therefore, depending on the selection of H or -C(O)CH3 for n and R x and R y , it will be understood that different acetylation rates (%) can be achieved. For example, each repeating unit of the oligomer defined by the brackets may be the same or different depending on the level of acetylation, i.e., depending on the selection of H or -C(O)CH3 for each of R x and R y .

[0048] As defined above, overall, about 50 - 90% of R x and R y in the oligomer is -C(O)CH3. In other words, the total amount of acetylation of the oligomer is about 50 - 90%. In other words, in the oligomer of the present invention, at least one of R x and one of R y is -C(O)CH3 in the same or different repeating units, and at the 3-position (R y is -C(O)CH3) and at the 4-position (R xThe total degree of acetylation with -C(O)CH3 is about 50 - 90%. To avoid misunderstanding, as described above, R x and R y may be the same or different in each repeating unit of the oligomer.

[0049] In another embodiment, overall, about 60 - 80% of R x and R y in the oligomer is -C(O)CH3. In other words, the total amount of acetylation of the oligomer is about 60 - 80%. To avoid misunderstanding, as described above, R x and R y may be the same or different in each repeating unit of the oligomer.

[0050] In one embodiment, both R x and R y are -C(O)CH3 in at least one same repeating unit of the oligomer, preferably in about 40 - 50% of the repeating units of the oligomer; about 10 - 30% of the remaining repeating units are R x or R y having one of them, and the remaining repeating units in the oligomer have R x =R y =H.

[0051] As defined above, Az is an aza substituent selected from the group consisting of -NH(CO)R 1 , -N(R 1 )2 and -N3, and R 1 is independently selected from the group consisting of H, linear or branched C1 - C6 - alkyl and linear or branched C1 - C6 - haloalkyl. The nitrogen atom is directly connected to the carbacycle repeating unit.

[0052] Examples of such Az substituents include -N3, -NH2, -NH-C1-C6 alkyl, -N-(C1-C6 alkyl)2, and -NH(CO)-C1-C6 alkyl. In certain embodiments, the -C1-C6 alkyl is -C1-C4 alkyl, particularly -CH3. Thus, according to certain embodiments, Az is -NH(CO)-C1-C6 alkyl, particularly -NH(CO)-CH3, and is also represented as -NHAc (Ac represents acetate, i.e., -C(O)CH3).

[0053] Z can have different meanings depending on whether the oligomer of the present invention is conjugated to a protein or not.

[0054] According to formula (Ia) or (Ib), the oligomer of the present invention is not conjugated to a protein. Thus, according to formula (Ia) or (Ib), as defined above, Z is one of the following. (i) A protecting group, (ii) A linear or branched C1-C6 alkyl, an optionally substituted aryl, -C(O)Y, or a linear or branched C1-C6-alkyl-X, or (iii) A functional linker for conjugation to a protein.

[0055] Thus, according to certain embodiments, Z is, for example, a means for capping the terminal sugar unit so that it can be non-reactive or reactive for further chain extension or for subsequent modification.

[0056] When Z is intended to be a means for capping the terminal carb analogue unit, it can include a protecting group or a capping group, such as a linear or branched C1-C6 alkyl, an optionally substituted phenyl, C(O)-Y, or a linear or branched -C1-C6 alkyl-X, where X is -NH2, -N3, -C≡CH, -CH=CH2, -SH or -SC≡N, and Y is H, a linear or branched C1-C6-alkyl or a protecting group.

[0057] As defined herein, Z can be a functional linker for conjugation to a protein. In this case, a "functional linker" refers to any linker known in the art to be used for conjugating a sugar to a protein.

[0058] In certain embodiments, X is -NH2.

[0059] In certain embodiments, Z according to formula (Ia) or (Ib) is selected from -(CH2)6-NH2, -(CH2)4-NH2, -(CH2)3-NH2, and -(CH2)2-NH2, and the amino group may be protected by a suitable protecting group, such as -C(O)CH3 (for the selection and use of such protecting groups and details of their usage, for example, those described in Greene, T. W. and Wuts, P. G. M., "protective groups in organic synthesis" can be used).

[0060] The oligomers of the present invention can be prepared according to synthetic methods known in organic synthesis for the production of polysaccharide carb analogs. Generally, the production of the oligomers of the present invention can be achieved by linking at least six mannosamine carb analog building blocks in a desired manner by forming a 1,6-alpha linkage between repeating units, thereby providing an oligomer having a degree of polymerization of at least six. As shown in formula (I), those monomers are linked via an alpha-(1→6) phosphate linkage, and such a connection can be made using standard polymerization techniques such as those described, inter alia, in Gao et al., Org. Biomol. Chem., 2012, 10, 6673.

[0061] The mannosamine carb analog building blocks can have acetate at position 3 and / or 4, or a protecting group that can be replaced by acetate at any step of the synthesis.

[0062] Alternatively, according to an embodiment, the present invention relates to a method for producing an oligomer of formula (I) comprising the following steps. a. Production of a monomer having a phosphodiester linkage; b. Extension reaction of the obtained monomer, for example using phosphoramidite; c. O-acetylation of the oligomer.

[0063] In one embodiment, when R y is C(O)CH3, steps (b) and (c) may be reversed so that O-acetylation is performed before the extension reaction.

[0064] More specifically, the process may include the steps shown in Scheme 1.

Chemical formula

[0065] To avoid misunderstanding, Ac is intended to refer to the acetyl group, i.e., -C(O)CH3.

[0066] Scheme 1: Method for producing the oligosaccharide of the present invention (a) TBAF, THF, 0 °C → room temperature, 92%. (b) MeONa, MeOH, room temperature, 85%. (c) DMTrCl, Et3N, DCM, room temperature, 91%. (d) 2-Cyanoethyl N,N-diisopropyl-chlorophosphoramidite, N,N-diisopropylethylamine, DCM, room temperature, 9 (94%). (e) I. 11, DCI, MeCN, II. CSO, MeCN, III. TCA, DCM, H2O, 94%. (f) I. 9, DCI, MeCN, II. CSO, MeCN, III. TCA, DCM, H2O, 16 (82%), 17 (95%), 18 (90%), 19 (92%), 20 (88%), 21 (86%), 22 (87%). (g) NH4OH, H2O, dioxane. (h) H2, Pd black, H2O, AcOH, 1 (99%), 2 (76%), 3 (69%), 4 (39%), 5 (88%), 6 (83%), 7 (77%), 8 (44%), (i): (Boc)2O, NaHCO3, room temperature, 16 h; (l): Ac2O / imidazole, 40 °C, ~9 d; (NS); TFA, room temperature, 1 h.

[0067] In particular, the use of phosphoramidite building blocks is effective in forming phosphodiester linkages. The inventors selected the use of dimethoxytrityl (DMTr) ethers to temporarily mask the functionality of the primary alcohol to be extended. Each extension step is based on the repetition of a three-step procedure involving the coupling of the phosphoramidite with the growing chain alcohol, the oxidation of the corresponding phosphite intermediate to the phosphodiester, and the unmasking of the primary hydroxyl on the (n + 1) oligomer. As shown in Scheme 1, the important building block 9 is obtained from intermediate 10, and intermediate 10 is obtained in three steps from the known carbosugar 12 (see, for example, Q. Gao et al., Org. Biomol. Chem., 2012, 10, 6673-6681). The latter carbamannose building block can be prepared from commercially available 3,4,6-tri-O-acetyl-D-glucal according to prior art methodologies. Thus, the primary silyl ether and acetyl ester were removed from compound 12 by the successive action of tetrabutylammonium fluoride (TBAF) and NaOMe to give diol 14 in 85% yield. Next, the DMTr group was introduced regioselectively to give alcohol 10 in 91% yield. This compound was converted to the extension block phosphoramidite 9 by reaction with 2-cyanoethyl-N,N-diisopropyl-chlorophosphoramidite. The target oligomer was assembled with the available building blocks. The synthesis was initiated by attaching an aminohexanol spacer to alcohol 10 using the known phosphoramidite 11. The building blocks were coupled in a two-step one-pot reaction using dicyanoimidazole (DCI) as the activator for the activation of the phosphoramidite. The phosphite formed in situ was oxidized using (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). DCI (pKa 5.2) has a low acidity and is suitable for use in combination with the acid-labile DMTr group, and thus has been conventionally used instead of tetrazole (pK aIt was more preferable than (4.9). Since the solubility in a non-aqueous solvent such as acetonitrile was high, CSO was used instead of iodine. The crude phosphodiester product was treated with TCA to cleave the DMTr group. The product was purified by size exclusion chromatography (Sephadex LH-20) to obtain monomer 15 equipped with a spacer in a yield of 94%. All subsequent couplings were carried out according to the above procedure until the desired degree of polymerization of 8 or more was reached. For the elongation of longer oligomers, a larger amount of phosphoramidite 9 was used and the coupling reaction time was increased to ensure complete conversion of the alcohol. The yield for each elongation cycle was in the range of 82% - 95%, which was good to excellent. Octamer 22 was obtained in a total yield of 40% starting from 10. The fragments 16 - 22 were deprotected using a two-step procedure. First, the cyanoethyl group (CE) was removed using an aqueous ammonia solution (33%). Next, all the remaining protecting groups (benzyl ether and carboxybenzyl carbamate) on the phosphodiester thus formed were cleaved by hydrogenolysis with palladium black to obtain the target non-acetylated oligomers 1 - 8.

[0068] The non-acetylated oligomers 1 - 8 can be randomly O-acetylated at the 3- and / or 4-positions, that is, taken together, about 50 - 90% of R x and R y in the oligomer is -C(O)CH3. This can be achieved by (i) BOC protection of the free amine group; (ii) O-acetylation using, for example, Ac2O / imidazole; and (iii) obtaining the acetylated oligomers 1c - 8c or 1d - 8d by deprotection. Next, such acetylated oligomers can be activated with a linker group such as bis-succinimidyl adipate (also known as SIDEA) and conjugated to a protein such as CRM 197 .

Chemical Structure

[0069] Process leading to the production of the 2.3-O-acetylated monomer building block (a) K2CO3, MeOH; (b) PMBCH(OMe)2, PPTS; (c) BnBr, NaH; (d) DIBAL-H, DCM; (e) DMP, DCM; (f) PPh3CH3I, KHMDS, THF, -78 °C; (g) m-dichlorobenzene, t, μ-wave; (h) NaBH4, EtOH / THF; (i) TDSCl, Im, DCM; (j) OsO4, TMANO, 3:1 acetone-H2O; (l) (MeO)3Cme, PTSA, CAN, then 80% AcOH; (m) Tf2O, DCM / py, -20 °C to room temperature; then NaN3, 19:1 DMF-H2O; (n) PPh3, THF, 60 °C, H2O; then Ac2O, MeOH; (o) NaOMe / MeOH; (p) TBSOTf, 2,6-lutidine, DCM; (q) DDQ, then Ac2O, py; (r) HF / pyridine, THF; (s) DMTrCl, pyridine, DCM.

[0070] Alternatively, the 3-O-acetylated monomer building block and the 4-O-acetylated building block can be produced by the process shown in Scheme 3 below. [Chemical formula]

[0071] Process leading to the production of the 3.3-O-acetylated and 4-O-acetylated monomer building blocks (a′) K2CO3, MeOH; (b′) TDSCl, imidazole, DMF, -30 °C; (c′) BnBr, NaH, DMF, 0 °C; (d′) TBAF, THF; (e′) IBX, AcOEt; (f′) PPh3CH3I, KHMDS, THF, from -78 °C to room temperature; (g′) 1,3-dichlorobenzene, NaBH4, EtOH / THF, 230 °C; (h′) TIPSCl, imidazole, DMF; (i′) TiCl4, DCM / toluene 2:8, -70 °C; (l′) NapBr, NaH, DMF, 0 °C; (m′) Me3NO·2H2O, acetone / H2O 3:1, OsO4; (n′) (MeO)3CMe, PTSA, ACN; (o′) Tf2O, DCM / Py, from -20 °C to room temperature; then NaN3, 19:1 DMF-H2O; (p′) NaOMe, MeOH; (q′) TBSOTf, -10 °C to 70 °C, Pyr, DMAP; (r′) Pd / C, H2, AcOH, then Ac2O, Pyr; (s′) HFpyr, Pyr; (t′) DMTrCl, Pyr, 0 °C; (r″) DDQ, DCM, H2O; (s″) PPh3, H2O, THF, then DMTrCl, Pyr.

[0072] The acetylated building blocks 38, 55a, 55b and the fully acetylated building blocks (i.e., having O-Ac groups at both the C3 and C4 positions of the same unit) can be converted to their oligomeric versions by conversion to phosphorimidates and subsequent coupling as described above in connection with compound 9.

[0073] An important prerequisite for the immunogenicity of the carba analogs of the present invention is their ability to mimic the corresponding MenA capsular sugars. To examine this, competitive ELISAs were performed using carba analogs of different degrees of polymerization.

[0074] The oligomers of the present invention can be introduced into a host, such as a mammalian host and preferably a human host, alone, or linked to a carrier protein, or as a homopolymer or heteropolymer of mannosylcarbohydrate analog units. In certain embodiments, the oligomers of the present invention are used as protein conjugates. Accordingly, in a further aspect, the present invention includes conjugate derivatives comprising an oligomer of the present invention of formula (I) connected to a protein according to general formula (IIa) or (IIb).

Chemical formula

[0075] Oligomers of general formula (Ia) or (Ib) are particularly useful when conjugated to a protein via a Z moiety connected to the C-1 carbon of the first repeating unit, preferably via a phosphate moiety. The thus obtained oligomer-protein conjugate derivative of formula (IIa) or (IIb) can induce an immunogenic response in infants and may be useful in the preparation of a composition that can induce a cellular response that provides a memory effect, perhaps to extend the effectiveness of vaccination.

[0076] In one embodiment, the oligomer conjugate is preferably defined by formula (IIa), i.e., the protein conjugates at the 1-position rather than the 6-position of the carbohydrate analog.

[0077] A protein (or carrier protein) can affect the immunogenic response and even affect the exact nature of the antibodies resulting from treating a mammal with one or more compounds of the present invention when delivered as a conjugate. Suitable proteins have functional groups that can react with the terminal portion of the Z moiety to form the conjugate derivatives of the present invention. Preferably, the functional group is selected from -NH2 and -SH and can be connected to the Z moiety that forms an amide bond or a thioether. More preferably, the protein has an -NH2 group suitable for forming an amide bond when reacted with Z.

[0078] Useful proteins are known in the art. However, in certain embodiments, P is an inactivated bacterial toxin selected from diphtheria toxoid (DT), tetanus toxoid (TT), CRM 197 , Escherichia coli (E. coli) ST and Pseudomonas aeruginosa exotoxin (rEPA), or P is a polyamino acid such as poly(lysine:glutamic acid), or P is a hepatitis B virus core protein or SPR96-2021, or a Neisseria meningitidis serogroup B antigen fHbp-231 (i.e., a fusion protein of variants 2, 3 and variant I of factor H binding protein (fHbp) as defined in WO2015 / 128480, which is incorporated herein by reference).

[0079] In certain embodiments, P is TT, DT or CRM 197 is.

[0080] In certain embodiments, P is CRM 197 is.

[0081] As defined above, according to formula (IIa) or (IIb), Z is a linker or a bond. When Z is a linker, it can be derived from any suitable linker known in the art suitable for the conjugation of oligosaccharides to proteins.

[0082] In other words, Z in the unreacted form, i.e., when not linked to the oligomer and the protein, can be a functional linker (defined according to formula (Ia) and formula (Ib)) by having a functional group that enables it to act as a linker between the oligomer and the protein of the present invention. Preferably, Z is derived from a compound containing an amine, carboxylate, or hydroxyl group for coupling with a complementary group on the protein carrier, but other groups known in the art for providing methods of conjugating oligosaccharides to proteins are also contemplated.

[0083] When the oligomer of the present invention is conjugated to a protein, the preferred Z moiety in formula (IIa) or (IIb) is derived from a linker that is an amine-substituted alkoxy group which may be in a protected form. In this form, the amine is acetylated or alkylated with a bifunctional reagent and the other end thereof is similarly connected to the protein.

[0084] In certain embodiments, according to formula (IIa) or (IIb), Z is derived from a linker that is either homo-bifunctional or hetero-bifunctional and capable of linking the oligomer of the present invention to a protein. In this regard, bifunctional linkers suitable for use in the conjugates of the present invention include those known in the art such as dicarboxylic acids, preferably malonic acid, succinic acid, adipic acid and suberic acid, or their activated forms. Alternatively, squarate esters can be used. These types of reagents are particularly convenient for linking a compound containing an amine to a protein for the spacer moiety. Preferably, the bifunctional linker is derived from adipic acid N-hydroxysuccinimide diester (SIDEA), and BS(PEG)5.

[0085] In some embodiments, Z is at least 2 or 3 atoms in length. Some non-limiting examples of linkers include -(CH2) m A, -Ph-A, -(CH2) a -Ph-(CH2) a-A and its substituted forms etc. exist, each Ph represents a phenyl group which may be substituted, and a and m each independently represent an integer from 1 to 10. "A" represents -NH2, -OH or -SH, an ester, an amide, or other carboxyl-containing group, a diene, or a dienophile, a maleimide, an alkyne, a cycloalkyne, etc. which are capable of proteins, or a functional group or its residue that can link proteins or link them. Z can include OR′, SR′ or N(R′)2, each R′ is independently H or a C1-C6-alkyl, acyl, aryl, arylalkyl, heteroacyl, heteroaryl, or heteroarylalkyl group, and can further include A.

[0086] In certain embodiments, Z in formula (IIa) or (IIb) is a heterobifunctional linker having the following formula.

Chemical formula

[0087] In certain embodiments, Z has the formula * -(CH2)6NHCO(CH2)4CO * having.

[0088] In another embodiment, Z is a linker having the following formula.

Chemical formula

[0089] In another embodiment, Z has the following formula.

Chemical formula

[0090] The Z linker is usually introduced into the monomer that is linked to the protein before the monomer to be elongated is connected, and optionally introduced in a protected form, so as not to affect or participate in the subsequent elongation reaction.

[0091] Thus, in certain embodiments, Z is a divalent linker having the following general formula. [Chemical formula] In the formula, r is an integer from 2 to 6, ( * ) represents the connection point to the oligomer, PG represents hydrogen or a protecting group, preferably selected from alkoxycarbonyl, methoxycarbonyl, t-butyloxycarbonyl or benzyloxycarbonyl. The protein is connected via an amine.

[0092] When present, PG can be preferably removed to enable the Z moiety to react with the protein to obtain its conjugate. Alternatively, PG can be removed, and the free amino group thus obtained can be further functionalized, for example, by introducing a further spacer moiety suitable for linking to the protein.

[0093] In certain embodiments, an oligomer conjugate according to the following formula is provided. [Chemical formula] In the formula, n, R, R′, R x , and R y are as defined above.

[0094] In certain embodiments of the present invention, an oligomer conjugate according to the following formula, i.e., where R′ is Na + is provided. [Chemical formula]

[0095] In the formula, n, R, Rx and R y is as defined above.

[0096] When the randomly acetylated oligomer conjugate is incorporated into a vaccine composition, it exhibits a higher percentage of acetylated stability than the native MenA conjugate and is less than 5% of the acetylation that may be lost when the carb analogue is formulated into the vaccine.

[0097] To avoid misunderstanding, it should be noted that the oligomers of the present invention can be conjugated to a protein by any suitable method known in the art, for example, according to the methods reported in "The design of semi - synthetic and synthetic glycoconjugate vaccines", P. Constantino et al., Expert Opin. Drug. Discov.

[0098] The conjugation reaction can also be carried out using conjugation methods similar to those used for the conjugation of MenA sugars to carrier proteins and described, for example, in WO2004 / 067030. In certain embodiments, the oligomers of the present invention use a conjugation procedure that utilizes a di - N - hydroxysuccinimidyl adipate linker reported, for example, in Berti et al., ACS Chem. Biol., 2012, 7, 1420 - 1428, to couple with CRM 197 and can be purified by co - precipitation with acetone after treatment with the selected linker in DMSO containing trimethylamine and used for conjugation. Thus, CRM at a 100:1 oligomer / protein molar ratio 197By incubating overnight, the desired neoconjugate can be obtained. The conjugation can be envisioned as the activation of the oligomer of formula (Ia) / (Ib), followed by conjugation to the selected protein, or the activation of a related protein functional group, and then followed by conjugation, typically via the Z moiety, to the oligosaccharide of the present invention. Thus, according to one embodiment, the oligomer of the present invention is first activated with a suitable activator according to methods known in the art and then coupled to the -NH2 residue of the selected protein.

[0099] In one embodiment, the Z group is activated by reaction with the first terminal portion of the linker, whereby the other end of the linker can be linked to the selected protein. For example, and according to one embodiment, the process can include obtaining an activated ester of the starting oligomer by activation of the oligomer of the present invention with SIDEA in the presence of triethylamine. Next, such an activated ester can be reacted with CRM 197 in the presence of a phosphate buffer to obtain the desired conjugate.

[0100] After conjugation, the oligomer-protein conjugate can be purified by a variety of techniques known in the art. One goal of the purification process is to remove unbound oligomers from the oligomer-protein conjugate. Typically, the conjugate of the present invention can be purified from unreacted proteins and oligomers by any number of standard techniques such as size exclusion chromatography, density gradient centrifugation, hydrophobic interaction chromatography, or ammonium sulfate fractionation, as described, for example, in Anderson, P. W., et al. J. Immunol. (1986)137:1181-1186, and Jennings, H. J. et al., J. Immunol. (1981)127:1011-1018.

[0101] In another embodiment, Z can be a monosaccharide, preferably mannosamine as described below. Thus, in a further embodiment, the invention also relates to an oligomer having the following formula (III).

Chemical formula

Chemical formula

[0102] For example, one example of the conjugate thus defined is as follows.

Chemical formula

[0103] According to this embodiment, the derivative of the present invention can produce a conjugate derivative having an -O-linker-P moiety directly connected to the carbon atom of the terminal monomer by directly linking to a selected protein via the -O-linker Z moiety. As far as the linker is concerned, this can be any suitable divalent linker by the linker Z shown above. Alternatively, Z can be considered to be an amine for conjugating to a protein derivatized with a linker having a keto or aldehyde group.

[0104] According to a further aspect of the invention, there is provided an immunogenic composition comprising (a) the conjugate as described above; and (b) at least one pharmaceutically acceptable excipient.

[0105] Generally, a pharmaceutically acceptable excipient can be any substance that does not itself induce the production of antibodies, is not harmful to the patient to whom the composition is administered, and can be administered without undue toxicity. Pharmaceutically acceptable carriers and excipients are those used in the art and can include liquids such as water, saline, glycerol, and ethanol. According to the prior art, auxiliary substances such as wetting or emulsifying agents, pH buffering substances, etc. can also be present in such media.

[0106] The immunogenic composition can further comprise an adjuvant. The adjuvant can be an aluminum-based adjuvant such as aluminum hydroxide or aluminum phosphate.

[0107] The immunogenic composition can further comprise at least one antigen derived from one of Neisseria meningitidis serogroups C, W135, Y, and optionally A.

[0108] The immunogenic composition of the present invention is often administered in combination with other pharmaceutically active substances or other vaccines. The composition for administration may, for example, contain other types of immunogenic compounds such as glycoconjugates that can induce an immune response to provide protection against other meningitis pathogens.

[0109] According to a further aspect of the present invention, there is provided a conjugate as described above, or a vaccine comprising an immunogenic composition as described above.

[0110] The vaccine can be formulated as a sterile, substantially aqueous mixture, a pyrogen-free buffered saline, or a phosphate-containing solution that can or cannot contain a preservative. The solution can be approximately isotonic, and its isotonicity can be adjusted with agents such as sodium tartrate, sodium chloride, and propylene glycol. The concentration of the immunogenic oligomer conjugate of the present invention in the formulation can vary widely from less than about 0.1% by weight to up to 20% to 50% by weight or more, and is selected primarily by the fluid volume, viscosity, etc., and according to the specific mode of administration selected.

[0111] The present invention also includes a method of enhancing an immune response in a vertebrate, preferably a mammal, comprising administering the oligomer conjugate of the present invention or the immunogenic composition of the present invention to the mammal or other vertebrate. The immune response is preferably protective and preferably involves antibodies. This method can increase the booster response.

[0112] In one aspect, the present invention relates to a method for treating or preventing meningitis A, C, W135, or Y in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of the oligomer conjugate according to the present invention, or the immunogenic composition according to the present invention, or the vaccine according to the present invention. Such a method can further include administration in combination with at least one serotype selected from C, W135, Y, and optionally A.

[0113] As used herein, the term "derivative of the present invention" refers to both oligomers and their oligomer conjugates. Derivatives of the present invention can also be used to immunize other mammals, such as cows, sheep, and pigs, as well as other non-mammalian vertebrates such as fish and poultry.

[0114] In another aspect, the present invention relates to a method of immunizing a subject against meningitis A, C, W135 or Y, the method comprising administering to the subject an immunologically effective amount of an immunogenic composition according to the present invention or a vaccine according to the present invention.

[0115] In another aspect, the present invention relates to a method of inducing an immune response against meningitis A, C, W135 or Y in a subject, the method comprising administering to the subject an immunologically effective amount of an immunogenic composition according to the present invention or a vaccine according to the present invention.

[0116] In certain embodiments, the subject is a human.

[0117] In a further aspect, the present invention relates to the use of an immunogenic composition according to the present invention or a vaccine according to the present invention in the manufacture of a medicament for the treatment or prevention of meningitis A, C, W135 or Y.

[0118] In another aspect, the present invention relates to an immunogenic composition according to the present invention or a vaccine according to the present invention for use in the prophylaxis of meningitis A, C, W135 or Y or for use in inducing an immune response against meningitis A, C, W135 or Y.

[0119] The immunogenic compositions of the present invention are generally administered directly to the subject. Direct delivery can be achieved by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or into the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, nasal, ocular, otic, pulmonary, or other mucosal administration. For example, intramuscular administration to the thigh or upper arm is preferred. The injection can be carried out via a needle (e.g., a hypodermic needle), although alternatively, needle-free injection can also be used.

[0120] The present invention can also be used to induce systemic and / or mucosal immunity. The dosing regimen can be a single-dose schedule or a multiple-dose schedule. Multiple doses can be used in a prime immunization schedule and / or a booster immunization schedule. A booster immunization dose can be administered after the prime dose schedule. Appropriate timings between prime doses (e.g., 4 to 16 weeks), and between the prime and booster immunizations can be determined by conventional methods. Since infectious diseases affect various regions of the body, the compositions of the present invention can be prepared in various forms. For example, the composition can be prepared as an injection, either as a solution or a suspension. Solid forms suitable for dissolution or suspension in a liquid medium prior to injection can also be prepared. The composition can be prepared for topical administration as an ointment, cream or powder. The composition can be prepared for oral administration as a tablet or capsule, or as a syrup (optionally flavored). The composition can be prepared for pulmonary administration using a fine powder or aerosol, for example as an inhaler. The composition can be prepared as a suppository or pessary. The composition can be prepared for nasal, ear or ocular administration, for example as drops. Compositions suitable for parenteral injection are most preferred. The composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered, for example, to a pH of from 6 to 8, generally around pH 7. The compositions of the present invention can be isotonic with respect to humans.

[0121] The immunogenic composition comprises an immunologically effective amount of the conjugate of the present invention and, optionally, any other specific components as required. The dosing regimen can be a single-dose schedule or a multiple-dose schedule (including, for example, booster immunization doses). The composition can be administered in combination with other immunomodulators. Adjuvants that can be used in the composition of the present invention include insoluble metal salts, oil-in-water emulsions (such as MF59 or AS03, both containing squalene), saponins, non-toxic derivatives of LPS (such as monophosphoryl lipid A or 3-O-deacylated MPL), immunostimulatory oligonucleotides, detoxified bacterial ADP-ribosylating toxins, microparticles, liposomes, imidazoquinolones, or mixtures thereof, preferably aluminum hydroxide, phosphate, or mixtures thereof, but not limited thereto. Other substances that act as immunostimulants are disclosed, for example, in Watson, Pediatr. Infect. Dis. J. (2000) 19:331-332. These salts include oxyhydroxides and hydroxyphosphates. The salts can take any suitable form (such as gel, crystal, amorphous, etc.).

[0122] Numbered embodiments Embodiment 1 An oligomer of the following formula (Ia) or (Ib).

Chemical formula

[0123] Embodiment 2 The oligomer of Embodiment 1 as defined by formula (Ia).

[0124] Embodiment 3 The oligomer of Embodiment 1 or Embodiment 2, wherein n is 8 - 30.

[0125] Embodiment 4 The oligomer of Embodiment 1 or Embodiment 2, wherein n is 8 - 20.

[0126] Embodiment 5 The oligomer of Embodiment 1 or Embodiment 2, wherein n is 8 - 15.

[0127] Embodiment 6 The oligomer according to any one of the preceding embodiments, wherein Az is -NHC(O)CH3.

[0128] Embodiment 7 The oligomer of any one of the preceding embodiments, wherein n is 8.

[0129] Embodiment 8 R x and R y The oligomer of any one of embodiments 1-7, wherein both of are —C(O)CH 3 in at least one same repeat unit.

[0130] Embodiment 9 R x and R y The oligomer of any one of embodiments 1-8, wherein both of the repeat units of the oligomer are —C(O)CH 3 in 40-50% of the repeat units of the oligomer.

[0131] Embodiment 10 In 10-20% of the remaining repeat units of the oligomer, R x or R y One of the repeating units is -C(O)CH3, and the remaining repeating units in the oligomer are R x =R y 10. The oligomer of embodiment 9, having =H.

[0132] Embodiment 11 An oligomeric conjugate antigen of formula (IIa) or (IIb). [ka] During the ceremony, n, R, R′, R x and R y is as defined in any one of embodiments 1 to 10; Z is a linker or a bond; P is a protein.

[0133] Embodiment 12 The conjugate of embodiment 11, defined by formula (IIa):

[0134] Embodiment 13 P is diphtheria toxoid (DT), tetanus toxoid (TT), CRM 197 , E. coli ST, and Pseudomonas aeruginosa exotoxin (rEPA), or P is a polyamino acid such as poly(lysine:glutamic acid), or P is Hepatitis B virus core protein or SPR96-2021.

[0135] Embodiment 14 P is CRM 197 14. The conjugate of any one of embodiments 11 to 13, wherein

[0136] Embodiment 15 15. The conjugate of any one of embodiments 11 to 14, wherein Z is a linker having the formula: [ka] During the ceremony, * represents the connection point, p is independently selected from 1 to 10; X is selected from -O-, -S- and -NH-.

[0137] Embodiment 16 15. The conjugate of any one of embodiments 11 to 14, wherein Z is a linker having the formula: [ka] During the ceremony, m is independently selected from 1 to 10;

[0138] Embodiment 17 17. The conjugate of any one of embodiments 11 to 16, having the structure: [ka] During the ceremony, n, R, R x and R yIt is defined by any one of Embodiments 1 to 10.

[0139] Embodiment 18 (a) A conjugate according to any one of Embodiments 11 to 17; and (b) An immunogenic composition according to any one of Embodiments 11 to 17, comprising at least one pharmaceutically acceptable excipient.

[0140] Embodiment 19 An immunogenic composition according to Embodiment 18, further comprising an adjuvant.

[0141] Embodiment 20 An immunogenic composition according to Embodiment 18 or Embodiment 19, further comprising at least one antigen derived from one of Neisseria meningitidis serogroups C, W135, Y, and optionally A.

[0142] Embodiment 21 A vaccine comprising a conjugate according to any one of Embodiments 11 to 17, or an immunogenic composition according to any one of Embodiments 17 to 18.

[0143] Embodiment 22 A method for treating or preventing Meningitis A, C, W135, or Y in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a conjugate according to any one of Embodiments 11 to 17, or an immunogenic composition according to any one of Embodiments 18 to 20, or a vaccine according to Embodiment 21.

[0144] Embodiment 23 A method for immunizing a subject against Meningitis A, C, W135, or Y, comprising administering to the subject an immunologically effective amount of an immunogenic composition according to any one of Embodiments 18 to 20 or a vaccine according to Embodiment 21.

[0145] Embodiment 24 A method for inducing an immune response against meningitis A, C, W135 or Y in a subject, comprising administering to the subject an immunogenic composition according to any one of embodiments 18 to 20 or a vaccine according to embodiment 21 in an immunologically effective amount.

[0146] Embodiment 25 The method according to any one of embodiments 22 to 24, wherein the subject is a human.

[0147] Embodiment 26 Use of an immunogenic composition according to any one of embodiments 18 to 20 or a vaccine according to embodiment 21 in the manufacture of a medicament for the treatment or prevention of meningitis A, C, W135 or Y.

[0148] Embodiment 27 An immunogenic composition according to any one of embodiments 18 to 20 or a vaccine according to embodiment 21 for use in the treatment or prevention of meningitis A, C, W135 or Y.

[0149] Embodiment 28 An immunogenic composition according to any one of embodiments 18 to 20 or a vaccine according to embodiment 21 for use in inducing an immune response against meningitis A, C, W135 or Y.

[0150] The present invention will be described in more detail in the following experimental section, which is intended to better explain the present invention without limiting the scope of the present invention.

Examples

[0151] Experimental section General procedures and materials All chemicals (Acros, Biosolve, Sigma-Aldrich, and TCI) were used as received, and all reactions were carried out under an argon atmosphere at ambient temperature (22 °C) unless otherwise noted. For TLC analysis, aluminum sheets (Merck, TLC silica gel 60 F254) were used and sprayed with a solution in H2SO4 / EtOH (20%), or a solution in 10% aqueous H2SO4 of (NH4)6Mo7O 24 ·4H2O (25 g / L) and (NH4))4Ce(SO4)4·2H2O (10 g / L), or a solution in aqueous H2O of KMnO4 (2%) and K2CO3 (1%), and heated at approximately 140 °C. For column chromatography, 40 - 63 μm 60 Å silica gel (SD Screening Devices) was used. NMR spectra (1H, 13 C and 31 P) were recorded on a Bruker AV-400liq or Bruker AV-500 or Bruker AV-600. High-resolution mass spectra were recorded on a mass spectrometer (Thermo Finnigan LTQ Orbitrap) equipped with an electrospray ion source by direct injection in positive mode (source voltage 3.5 kV, sheath gas flow 10, capillary temperature 250 °C) at m / z 400 (mass range m / z = 150 - 2000) and with dibutyl phthalate (m / z = 391.28428) as lock mass at a resolution R = 60000.

[0152] Abbreviations AcOH = acetic acid ACN = acetonitrile DCM = dichloromethane DMTrCl = 4,4′-dimethoxytrityl chloride EtOAc = ethyl acetate THF = tetrahydrofuran TBAF = tetrabutylammonium fluoride

[0153] Example 1: Preparation of the oligomers of the invention of formula (Ia) according to Scheme 1 Acetamido-3,4-di-O-benzyl-2-deoxy-6-O-tert-butyldimethylsilyl-5a-carba-α-D-mannopyranose (13) Silyl ether 12 can be prepared according to the procedure described in Q. Gao et al. Org. Biomol. Chem., 2012, 10, 6673.

[0154] Silyl ether 12 (1.6 g, 2.7 mmol) was dissolved in dry THF (20 mL). The mixture was cooled to 0 °C. 0.1 M TBAF / THF solution (4.1 mL, 4.1 mmol) was added slowly. The reaction solution was warmed to room temperature and stirred for 3 h. AcOH (0.31 mL) was added to the reaction solution. The solution was extracted three times with DCM and washed once with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (EtOAc / hexane) to give product 13 in 92% yield (1.1 g, 2.52 mmol). The spectral data were consistent with the reported data.

[0155] 2-Acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranose (14) Alcohol 13 (1.12 g, 2.5 mmol) was dissolved in MeOH (32 mL). NaOMe (0.03 g, 0.5 mmol) was added to the mixture. The reaction solution was stirred at room temperature for 3 h. Amberlite H+ resin was added until neutral pH was reached. The suspension was filtered and concentrated under reduced pressure. 1 H NMR (400 MHz, CDCl3) δ = 1.70 - 1.85 (m, 2H, H-5a), 1.90 (s, 3H, AcNH), 2.19 - 2.23 (m, 1H, H-5), 3.60 - 3.79 (m, 3H, H-6, H-1), 3.83 - 3.90 (m, 1H, H-2), 3.91 - 3.99 (m, 1H, H-4), 4.14 - 4.23 (m, 1H, H-3), 4.33 - 4.41 (m, 1H, CHH Bn), 4.54 - 4.72 (m, 3H, CH2Bn, CHH Bn), 5.79 (m, 1H, NHAc), 7.22 - 7.42 (m, 10H, H arom )13 13C NMR (100 MHz, CDCl3) δ = 23.5 (CH3AcNH), 30.6 (CH2C-5a), 39.5 (CH C-5), 53.5 (CH C-3), 64.1 (CH2C-6), 67.9 (CH C-4), 72.4 (CH2Bn), 73.8 (CH2Bn), 75.5 (CH C-1), 79.0 (CH C-4), 127.3 - 128.9 (CH arom ), 171.8 (C=OAcNH). HRMS: [C 23 H 29 NO5 + H] + requires 400.21251, found 400.21179.

[0156] 2-Acetamido-3,4-di-O-benzyl-2-deoxy-6-O-(bis(4-methoxyphenyl)(phenyl))-5-carba-α-D-mannopyranose (10) Diol 14 (0.9 g, 2.25 mmol) was dissolved in dry DCM (30 mL). Et3N (1.9 mL, 13.5 mmol) was added to the mixture. DMTrCl (1.16 g, 3.38 mmol) was added. The reaction was stirred for 2 h. H2O was added to the reaction and washed once with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (EtOAc / hexane) to give product 10 in 91% yield (1.6 g, 2.04 mmol). 1 1H NMR (400 MHz, CD3CN) δ = 1.70 - 1.85 (m, 1H, 5a′-H), 1.91 (s, 3H, AcNH), 2.00 - 2.21 (m, 2H, 5a-H, 5-H), 3.01 - 3.19 (m, 1H, 6′-H), 3.27 - 3.37 (m, 1H, 6-H), 3.51 - 3.67 (m, 1H, H-4), 3.73 (s, 7H, H-3, 2×OMe), 4.06 - 4.20 (m, 1H, H-1), 4.22 - 4.32 (m, 1H, CHH Bn), 4.40 - 4.62 (m, 3H, CH2Bn, H-2), 4.65 - 4.73 (m, 1H, CHH Bn), 6.35 - 6.44 (m, 1H, NHAc), 6.78 - 7.47 (m, 23H, H arom ). 1313C NMR (100 MHz, CD3CN) δ = 23.2 (CH3AcNH), 31.6 (CH2C-5a), 38.6 (CH C-5), 53.3 (CH C-2), 55.8 (2×CH3OMe), 64.6 (CH2C-6), 67.6 (CH C-1), 72.1 (CH2Bn), 73.8 (CH2Bn), 77.2 (CH C-4), 79.8 (CH C-3), 86.5 (Cq DMTr), 113.9 (CH arom ), 127.3 - 130.7 (CH arom ), 137.2 - 159.4 (5×Cq DMTr), 171.1 (C=OAcNH). HRMS: [C 44 H 47 NO7+Na] + requires 724.32501, found 724.32483.

[0157] 1-O-((N,N-Diisopropylamino)-O-2-cyanoethyl-phosphoramidite))-2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-(bis(4-methoxyphenyl)(phenyl))-5a-carba-α-D-mannopyranose (9) Alcohol 10 (1.5 g, 2.14 mmol) was co-evaporated with ACN three times and dissolved in dry DCM (22 mL). Fresh activated MS 3Å and DIPEA (0.6 mL, 3.2 mmol) were added to the mixture. 2-Cyanoethyl N,N-diisopropyl-chlorophosphoramidite (0.6 mL, 2.6 mmol) was added to the mixture. The reaction was stirred for 2 h. H2O was added to this solution and it was washed once with a 1:1 solution of brine / NaHCO3. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (DCM / acetone / Et3N) to give product 9 in 94% yield (1.81 g, 2.0 mmol) (a mixture of diastereomers). 11H NMR (400 MHz, CD3CN) δ = 1.04 - 1.24 (m, 12H, 4×isopropylamino), 1.70 - 1.85 (m, 1H, 5a′-H), 1.92 (s, 3H, AcNH), 2.00 - 2.21 (m, 2H, 5a-H, 5-H), 2.55 - 2.75 (m, 2H, CH2cyanoethyl), 2.98 - 3.10 (m, 1H, 6′-H), 3.27 - 3.37 (m, 1H, 6-H), 3.47 - 3.70 (m, 3H, 2×CH isopropylamino, H-4), 3.70 - 3.88 (m, 9H, H-3, CH2cyanoethyl, 2×OMe), 4.06 - 4.20 (m, 1H, H-1), 4.22 - 4.32 (m, 1H, CHH Bn), 4.40 - 4.62 (m, 3H, CH2Bn, H-2), 4.65 - 4.73 (m, 1H, CHH Bn), 6.35 - 6.44 (m, 1H, NHAc), 6.78 - 7.47 (m, 23H, H arom )。 13 13C NMR (100 MHz, CD3CN) δ = 20.7 (CH2cyanoethyl), 22.9 (CH,3AcNH), 24.5 - 24.7 (2×CH,3isopropylamino), 30.6 (CH2C-5a), 38.5 (CH C-5), 43.7 (2×CH isopropylamino), 51.7 (CH C-2), 55.5 (2×CH,3OMe), 59.1 (CH2cyanoethyl), 64.2 (CH2C-6), 70.5 (CH C-1), 71.5 (CH2Bn), 74.3 (CH2Bn), 77.8 (CH C-4), 79.5 (CH C-3), 86.2 (Cq DMTr), 113.6 (CH arom )、127.3 - 130.7 (CH arom )、136.8 - 159.2 (5×Cq DMTr), 170 (C=O AcNH)。 31 31P NMR (162 MHz, CD3CN) δ = 146.9, 147.26。

[0158] General procedures for phosphoramidite coupling, oxidation, and detritylation on a normal scale (0.03 - 0.3 mmol) The starting alcohol was co-evaporated with ACN three times, and freshly activated MS 3 Å and DCI (0.25 M / ACN solution, 1.5 equiv) were added. The solution was stirred for 15 minutes. A phosphoramidite reagent (0.1 - 0.16 M / ACN solution, 1.3 - 3 equiv) was added to the mixture and stirred until the starting material was completely converted (about 2 hours). Subsequently, CSO (0.5 M / ACN solution, 2 equiv) was added to the reaction mixture and stirred for 15 minutes. The mixture was diluted with EtOAc and washed with a 1:1 solution of brine / NaHCO3. The aqueous layer was extracted twice with EtOAc. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was co-evaporated with ACN three times and dissolved in DCM (5 - 10 mL). TCA (0.18 M DCM solution) was added to this solution and stirred for 1 hour. H2O was added to the reaction mixture and stirred for 15 minutes. The reaction was washed with a 1:1 solution of brine / NaHCO3. The aqueous layer was extracted three times with DCM. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (DCM / acetone) or size exclusion chromatography (sephadex LH-20, MeOH / DCM 1:1).

[0159] 1-O-((2-Acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzyl-carbamate (15) Using the general procedure described above, alcohol 10 (0.21 g, 0.3 mmol) was coupled with phosphoramidite 11 (2.5 mL 0.16 M / ACN solution, 0.45 mmol), oxidized, and detritylated. The crude product was purified by flash chromatography (DCM / acetone) to give product 15 in 94% yield (0.216 g, 0.282 mmol). 11H NMR (400 MHz, CD3CN) δ = 1.24 - 1.40 (m, 4H, 2×CH2 hexyl spacer), 1.40 - 1.51 (m, 2H, CH2 hexyl spacer), 1.58 - 1.70 (m, 2H, CH2 hexyl spacer), 1.80 - 1.92 (m, 4H, 5a'-H, AcNH), 1.96 - 2.02 (m, 2H, 5a-H, 5-H), 2.72 - 2.82 (m, 2H, CH2 cyanoethyl), 2.96 (bs, 1H, OH), 3.02 - 3.12 (m, 2H, CH2 hexyl spacer), 3.56 - 3.74 (m, 3H, H-6, H-4), 3.76 - 3.84 (m, 1H, H-3), 3.95 - 4.07 (m, 2H, CH2 hexyl spacer), 4.08 - 4.20 (m, 2H, CH2 cyanoethyl), 4.44 - 4.63 (m, 5H, H-1, H-2, CH2Bn, CHH Bn), 4.72 - 4.80 (m, 1H, CHH Bn), 5.03 (s, 2H, CH2Bn spacer), 5.70 (bs, 1H, NH), 6.49 - 6.60 (m, 1H, NHAc), 7.23 - 7.44 (m, 15H, H arom ). 13 13C NMR (100 MHz, CD3CN) δ = 19.9 (CH2 cyanoethyl), 22.8 (CH3 AcNH), 25.4 (CH2 hexyl spacer), 26.4 (CH2 hexyl spacer), 30.0 (CH2C-5a), 30.4 (CH2 hexyl spacer), 30.5 (CH2 hexyl spacer), 40.0 (CH C-5), 41.0 (CH2 hexyl spacer), 51.1 (CH C-2), 62.9 (CH2C-6), 63.0 (CH2 cyanoethyl), 66.3 (CH2Bn spacer), 68.8 (CH2 hexyl spacer), 72.2 (CH2Bn), 74.0 (CH2Bn), 75.1 (CH C-1), 76.7 (CH C-4), 79.3 (CHC-3), 128.1 - 129.1 (CH arom ), 138.9 - 139.7 (3×Cq Bn), 170.8 (C=O AcNH). 31 31P NMR (162 MHz, CD3CN) δ = -2.40, -2.36. HRMS: [C 40 H 52 N3O 10 P+H] +766.34707 was required, and the measured value was 766.34707.

[0160] 1-O-Di-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate (16) Using the general procedure above, alcohol 15 (0.186 g, 0.24 mmol) was coupled with phosphoramidite 9 (2.3 mL 0.16 M / ACN solution, 0.37 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to give product 16 in 82% yield (0.255 g, 0.199 mmol). 1 H NMR (400 MHz, CD3CN) δ = 1.25 - 1.40 (m, 4H, 2×CH2 hexyl spacer), 1.40 - 1.51 (m, 2H, CH2 hexyl spacer), 1.58 - 1.71 (m, 2H, CH2 hexyl spacer), 1.80 - 1.92 (m, 8H, 2×5a'-H, 2×AcNH), 1.96 - 2.02 (m, 4H, 2×5a-H, 2×5-H), 2.70 - 2.81 (m, 4H, 2×CH2 cyanoethyl), 2.96 (bs, 1H, OH), 3.01 - 3.12 (m, 2H, CH2 hexyl spacer), 3.56 - 3.87 (m, 6H, 2×H-6, 2×H-4), 3.94 - 4.28 (m, 8H, 2×H-3, CH2 hexyl spacer, 2×CH2 cyanoethyl), 4.29 - 4.85 (m, 12H, 2×H-1, 2×H-2, 4×CH2Bn), 5.03 (s, 2H, CH2Bn spacer), 5.75 (bs, 1H, NH), 6.52 - 6.62 (m, 1H, NHAc), 6.85 - 6.99 (m, 1H, NHAc), 7.21 - 7.41 (m, 25H, H arom ) 1313C NMR (100 MHz, CD3CN) δ = 19.9 - 20.0 (2 × CH2 cyanoethyl), 22.9 - 23.0 (2 × CH3 AcNH), 25.5 (CH2 hexyl spacer), 26.5 (CH2 hexyl spacer), 29.1 - 29.2 (2 × CH2 C-5a), 30.1 (CH2 hexyl spacer), 30.5 (CH2 hexyl spacer), 38.1 - 40.0 (2 × CH C-5), 41.1 (CH2 hexyl spacer), 50.9 - 51.4 (2 × CH C-2), 62.5 - 62.6 (2 × CH2 C-6), 63.0 - 63.2 (2 × CH2 cyanoethyl), 66.3 (CH2 Bn spacer), 68.9 (CH2 hexyl spacer), 72.1 - 72.3 (4 × CH2 Bn), 75.0 - 75.4 (2 × CHC-1), 75.5 - 76.9 (2 × CHC-4), 79.2 - 79.5 (2 × CH C-3), 128.2 - 129.1 (CH arom ), 138.9 - 139.6 (5 × Cq Bn), 170.8 (2 × C=O AcNH). 31 31P NMR (162 MHz, CD3CN) δ = -2.60, -2.58, -2.34, -2.32, -2.22, -2.17. HRMS: [C 66 H 83 N5O 17 P2 + H] + requires 1280.53320, found 1280.53320.

[0161] 1-O-Tris-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate (17) Using the general procedure above, alcohol 16 (0.215 g, 0.167 mmol) was coupled with phosphoramidite 9 (1.6 mL 0.16 M / ACN solution, 0.25 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to give product 17 in 95% yield (0.285 g, 0.158 mmol). 11H NMR (400 MHz, CD3CN) δ = 1.25 - 1.40 (m, 4H, 2×CH2 hexyl spacer), 1.40 - 1.51 (m, 2H, CH2 hexyl spacer), 1.58 - 1.71 (m, 2H, CH2 hexyl spacer), 1.80 - 1.92 (m, 12H, 3×5a′-H, 3×AcNH), 1.96 - 2.30 (m, 6H, 3×5a-H, 3×5-H), 2.68 - 2.83 (m, 6H, 3×CH2 cyanoethyl), 2.93 (bs, 1H, OH), 3.00 - 3.11 (m, 2H, CH2 hexyl spacer), 3.59 - 3.89 (m, 9H, 3×H-6, 3×H-4), 3.96 - 4.22 (m, 11H, 3×H-3, CH2 hexyl spacer, 3×CH2 cyanoethyl), 4.31 - 4.86 (m, 18H, 3×H-1, 3×H-2, 6×CH2Bn), 5.03 (s, 2H, CH2Bn spacer), 5.78 (bs, 1H, NH), 6.55 - 6.65 (m, 1H, NHAc), 6.9 - 7.15 (m, 2H, 2×NHAc), 7.19 - 7.40 (m, 35H, H arom ). 13 13C NMR (100 MHz, CD3CN) δ = 20.0 - 20.1 (3×CH2 cyanoethyl), 22.9 - 23.0 (3×CH3 AcNH), 25.5 (CH2 hexyl spacer), 26.5 (CH2 hexyl spacer), 28.9 - 29.2 (3×CH2C-5a), 30.1 (CH2 hexyl spacer), 30.5 (CH2 hexyl spacer), 38.0 - 40.0 (3×CH C-5), 41.1 (CH2 hexyl spacer), 50.8 - 51.4 (3×CH C-2), 62.5 - 63.0 (3×CH2C-6), 63.0 - 63.3 (3×CH2 cyanoethyl), 66.3 (CH2Bn spacer), 68.4 (CH2 hexyl spacer), 72.1 - 74.1 (6×CH2Bn), 75.2 - 75.5 (3×CH C-1), 75.5 - 76.1 (3×CH C-4), 79.3 - 79.5 (3×CH C-3), 128.2 - 129.1 (CH arom ), 138.9 - 139.7 (7×Cq Bn), 170.9 - 171.2 (3×C=O AcNH). 31162 MHz, CD3CN) δ = -2.82, -2.77, -2.62, -2.58, -2.36, -2.33, -2.24, -2.20, -2.16. HRMS: [C 92 H 11 4N7O 24 P3+H] + requires 1795.72333, found 1795.22333.

[0162] 1-O-Tetra-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carbamoyl-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate (18) Using the general procedure above, alcohol 17 (0.267 g, 0.148 mmol) was coupled with phosphoramidite 9 (1.4 mL 0.16 M / ACN solution, 0.22 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to give product 18 in 87% yield (0.299 g, 0.129 mmol). 1 1H NMR (400 MHz, (CD3)2CO) δ = 1.31 - 1.47 (m, 4H, 2×CH2 hexyl spacer), 1.47 - 1.57 (m, 2H, CH2 hexyl spacer), 1.62 - 1.75 (m, 2H, CH2 hexyl spacer), 1.85 - 2.02 (m, 16H, 4×5a′-H, 4×AcNH), 2.07 - 2.17 (m, 8H, 4×5a-H, 4×5-H), 2.82 - 3.00 (m, 8H, 4×CH2 cyanoethyl), 3.08 - 3.18 (m, 2H, CH2 hexyl spacer), 3.66 - 4.01 (m, 12H, 4×H-6, 4×H-4), 4.04 - 4.36 (m, 14H, 4×H-3, CH2 hexyl spacer, 4×CH2 cyanoethyl), 4.40 - 4.94 (m, 24H, 4×H-1, 4×H-2, 8×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.39 (bs, 1H, NH), 7.17 - 7.42 (m, 45H, H arom ), 7.42 - 7.80 (m, 4H, NHAc). 1313C NMR (100 MHz, (CD3)2CO) δ = 20.0 - 20.1 (4 × CH2 cyanoethyl), 23.1 - 23.2 (4 × CH3 AcNH), 25.8 (CH2 hexyl spacer), 26.8 (CH2 hexyl spacer), 29.2 - 29.8 (4 × CH2 C-5a), 30.8 (CH2 hexyl spacer), 30.8 (CH2 hexyl spacer), 38.3 - 40.3 (4 × CH C-5), 41.4 (CH2 hexyl spacer), 51.2 - 51.5 (4 × CH C-2), 62.6 - 63.4 (4 × CH2 C-6), 63.4 - 63.6 (4 × CH2 cyanoethyl), 66.2 (CH2 Bn spacer), 68.8 (CH2 hexyl spacer), 72.0 - 75.0 (8 × CH2 Bn), 75.6 - 75.8 (4 × CH C-1), 76.5 - 77.2 (4 × CH C-4), 79.7 - 79.8 (4 × CH C-3), 128.1 - 129.1 (CH arom ), 139.3 - 140.1 (9xCq Bn), 170.7 - 171.2 (4 × C=O AcNH). 31 31P NMR (162 MHz, (CD3)2CO) δ = -2.84, -2.77, -2.68, -2.47, -2.42, -2.37, -2.30, -1.96, -1.91, -1.89. HRMS: [C 118 H 145 N9O 31 P4+2H] ++ requires 1155.45892, and the measured value was 1155.45892.

[0163] 1-O-Penta-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate (19) Using the general procedure above, alcohol 18 (0.277 g, 0.120 mmol) was coupled with phosphoramidite 9 (1.1 mL 0.16 M / ACN solution, 0.18 mmol) and oxidized and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to give product 19 in 92% yield (0.31 g, 0.110 mmol).1 1H NMR (400 MHz, (CD3)2CO) δ = 1.31 - 1.46 (m, 4H, 2×CH2 hexyl spacer), 1.46 - 1.58 (m, 2H, CH2 hexyl spacer), 1.62 - 1.75 (m, 2H, CH2 hexyl spacer), 1.84 - 2.02 (m, 20H, 5×5a′-H, 5×AcNH), 2.07 - 2.19 (m, 10H, 5×5a-H, 5×5-H), 2.82 - 2.97 (m, 10H, 5×CH2 cyanoethyl), 3.08 - 3.18 (m, 2H, CH2 hexyl spacer), 3.67 - 4.02 (m, 15H, 5×H-6, 5×H-4), 4.04 - 4.36 (m, 17H, 5×H-3, CH2 hexyl spacer, 5×CH2 cyanoethyl), 4.38 - 4.95 (m, 30H, 5×H-1, 5×H-2, 10×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.43 (bs, 1H, NH), 7.16 - 7.41 (m, 55H, H arom ), 7.42 - 7.86 (m, 5H, NHAc). 13 13C NMR (100 MHz, (CD3)2CO) δ = 19.8 - 20.0 (5×CH2 cyanoethyl), 23.0 - 23.1 (5×CH3 AcNH), 25.7 (CH2 hexyl spacer), 26.7 (CH2 hexyl spacer), 29.2 - 30.0 (5×CH2C-5a), 30.7 (CH2 hexyl spacer), 30.7 (CH2 hexyl spacer), 38.2 - 40.2 (5×CH C-5), 41.2 (CH2 hexyl spacer), 51.0 - 51.4 (5×CH C-2), 62.5 - 63.2 (5×CH2C-6), 63.3 - 63.5 (5×CH2 cyanoethyl), 66.1 (CH2Bn spacer), 68.7 (CH2 hexyl spacer), 72.0 - 75.0 (10xCH2Bn), 75.6 - 75.8 (5×CH C-1), 76.5 - 77.2 (5×CH C-4), 79.7 - 79.8 (5×CH C-3), 128.0 - 129.0 (CH arom ), 139.2 - 140.0 (11×Cq Bn), 170.7 - 171.2 (5×C=O AcNH). 31P NMR (162 MHz, (CD3)2CO) δ = -2.84, -2.77, -2.68, -2.47, -2.42, -2.37, -2.30, -1.96, -1.88, -1.89, -1.86, -1.84, -1.79. HRMS: [C 144 H 176 N 11 O 38 P5+2H] ++ requires 1412.55219 and the measured value was 1412.55219.

[0164] 1-O-Hexa-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate (20) Using the above general procedure, alcohol 19 (0.280 g, 0.099 mmol) was coupled with phosphoramidite 9 (1.24 mL 0.16 M / ACN solution, 0.20 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to give product 20 in 88% yield (0.29 g, 0.087 mmol). 1 H NMR (500 MHz, (CD3)2CO) δ = 1.31 - 1.46 (m, 4H, 2×CH2 hexyl spacer), 1.46 - 1.57 (m, 2H, CH2 hexyl spacer), 1.63 - 1.74 (m, 2H, CH2 hexyl spacer), 1.84 - 2.02 (m, 24H, 6×'5a-H, 6×AcNH), 2.07 - 2.30 (m, 12H, 6×5a-H, 6×5-H), 2.82 - 2.97 (m, 12H, 6×CH2 cyanoethyl), 3.09 - 3.18 (m, 2H, CH2 hexyl spacer), 3.67 - 4.04 (m, 18H, 6×H-6, 6×H-4), 4.04 - 4.38 (m, 20H, 6×H-3, CH2 hexyl spacer, 6×CH2 cyanoethyl), 4.38 - 5.00 (m, 36H, 6×H-1, 6×H-2, 12×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.42 (bs, 1H, NH), 7.16 - 7.41 (m, 65H, H arom), 7.42 - 7.89 (m, 6H, NHAc). 13 C NMR (100 MHz, (CD3)2CO) δ = 19.9 - 20.0 (6 × CH2 cyanoethyl), 23.0 - 23.1 (6 × CH3 AcNH), 25.7 (CH2 hexyl spacer), 26.8 (CH2 hexyl spacer), 29.2 - 30.2 (6 × CH2 C-5a), 30.4 (CH2 hexyl spacer), 30.7 (CH2 hexyl spacer), 38.2 - 40.2 (6 × CH C-5), 41.3 (CH2 hexyl spacer), 51.0 - 51.4 (6 × CH C-2), 62.5 - 63.4 (6 × CH2 C-6), 63.4 - 63.5 (6 × CH2 cyanoethyl), 66.2 (CH2 Bn spacer), 68.7 (CH2 hexyl spacer), 72.2 - 75.6 (12 × CH2 Bn), 75.6 - 75.8 (6 × CH C-1), 76.5 - 77.2 (6 × CH C-4), 79.7 - 79.8 (6 × CHC-3), 128.1 - 129.1 (CH arom ), 139.2 - 140.0 (13 × Cq Bn), 170.7 - 171.2 (6 × C=O AcNH). 31 P NMR (162 MHz, CD3)2CO) δ = -2.84, -2.77, -2.68, -2.45, -2.42, -2.37, -2.31, -1.94, -1.81, -1.78. HRMS: [C 170 H 207 N 13 O 45 P6+NH4] + requires 3356.312, found 3357.010.

[0165] To produce the oligomer with n = 6, the general deprotection procedure described below can be carried out after the above steps.

[0166] 1-O-Epta-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate (21) Using the general procedure described above, alcohol 20 (0.140 g, 0.042 mmol) was coupled with phosphoramidite 9 (0.8 mL of 0.1 M / ACN solution, 0.84 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to give product 21 in 86% yield (0.139 g, 0.036 mmol). 1 H NMR (500 MHz, (CD3)2CO) δ = 1.31 - 1.46 (m, 4H, 2×CH2 hexyl spacer), 1.46 - 1.57 (m, 2H, CH2 hexyl spacer), 1.63 - 1.74 (m, 2H, CH2 hexyl spacer), 1.84 - 2.02 (m, 28H, 7×5a′-H, 7×AcNH), 2.07 - 2.30 (m, 14H, 7×5a-H, 7×5-H), 2.82 - 2.97 (m, 14H, 7×CH2 cyanoethyl), 3.09 - 3.18 (m, 2H, CH2 hexyl spacer), 3.67 - 4.04 (m, 21H, 7×H-6, 7×H-4), 4.04 - 4.38 (m, 23H, 7×H-3, CH2 hexyl spacer, 7×CH2 cyanoethyl), 4.38 - 5.00 (m, 42H, 7×H-1, 7×H-2, 14×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.42 (bs, 1H, NH), 7.16 - 7.41 (m, 75H, H arom )、7.42 - 7.89 (m, 7H, NHAc). 1313C NMR (125 MHz, (CD3)2CO) δ = 19.9 - 20.0 (7 × CH2 cyanoethyl), 23.0 - 23.1 (7 × CH3 AcNH), 25.7 (CH2 hexyl spacer), 26.8 (CH2 hexyl spacer), 29.2 - 30.2 (7 × CH2 C-5a), 30.4 (CH2 hexyl spacer), 30.7 (CH2 hexyl spacer), 38.2 - 40.2 (7 × CH C-5), 41.3 (CH2 hexyl spacer), 51.0 - 51.4 (7 × CH C-2), 62.5 - 63.4 (7 × CH2 C-6), 63.4 - 63.5 (7 × CH2 cyanoethyl), 66.2 (CH2 Bn spacer), 68.7 (CH2 hexyl spacer), 72.2 - 75.6 (14 × CH2 Bn), 75.6 - 75.8 (7 × CH C-1), 76.5 - 77.2 (7 × CH C-4), 79.7 - 79.8 (7 × CH C-3), 128.1 - 129.1 (CH arom ), 139.2 - 140.0 (15 × Cq Bn), 170.7 - 171.2 (7 × C=O AcNH). 31 31P NMR (202 MHz, (CD3)2CO) δ = -2.84, -2.77, -2.68, -2.45, -2.42, -2.37, -2.31, -1.94, -1.81, -1.78. HRMS: [C 196 H 238 N 15 O 52 P7 + 2H] ++ requires 1926.73908, and the measured value was 1926.73908.

[0167] 1 - O - Octa - ((2 - acetamido - 3,4 - di - O - benzyl - 2 - deoxy - 5a - carba - α - D - manno - pyranosyl - 1 - O - phosphoryl)2 - cyanoethyl) - 6 - hexyl - benzylcarbamate (22) n = 8 Using the general procedure described above, alcohol 22 (0.105 g, 0.027 mmol) was coupled with phosphoramidite 9 (0.7 mL of 0.1 M / ACN solution, 0.68 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to afford product 22 in 87% yield (0.103 g, 0.023 mmol). 1 H NMR (500 MHz, (CD3)2CO) δ = 1.31 - 1.46 (m, 4H, 2×CH2 hexyl spacer), 1.46 - 1.57 (m, 2H, CH2 hexyl spacer), 1.63 - 1.74 (m, 2H, CH2 hexyl spacer), 1.84 - 2.02 (m, 32H, 8×5a′-H, 8×AcNH), 2.07 - 2.30 (m, 16H, 8×5a-H, 8×5-H), 2.82 - 2.97 (m, 16H, 8×CH2 cyanoethyl), 3.09 - 3.18 (m, 2H, CH2 hexyl spacer), 3.67 - 4.04 (m, 24H, 8×H-6, 8×H-4), 4.04 - 4.38 (m, 26H, 8×H-3, CH2 hexyl spacer, 8×CH2 cyanoethyl), 4.38 - 5.00 (m, 48H, 8×H-1, 8×H-2, 16×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.42 (bs, 1H, NH), 7.16 - 7.41 (m, 85H, H arom )、7.42 - 7.89 (m, 8H, NHAc). 1313C NMR (125 MHz, (CD3)2CO) δ = 19.9 - 20.0 (8 × CH2 cyanoethyl), 23.0 - 23.1 (8 × CH3 AcNH), 25.7 (CH2 hexyl spacer), 26.8 (CH2 hexyl spacer), 29.2 - 30.2 (8 × CH2 C-5a), 30.4 (CH2 hexyl spacer), 30.7 (CH2 hexyl spacer), 38.2 - 40.2 (8 × CH C-5), 41.3 (CH2 hexyl spacer), 51.0 - 51.4 (8 × CH C-2), 62.5 - 63.4 (8 × CH2 C-6), 63.4 - 63.5 (8 × CH2 cyanoethyl), 66.2 (CH2 Bn spacer), 68.7 (CH2 hexyl spacer), 72.2 - 75.6 (16 × CH2 Bn), 75.6 - 75.8 (8 × CH C-1), 76.5 - 77.2 (8 × CH C-4), 79.7 - 79.8 (8 × CH C-3), 128.1 - 129.1 (CH arom ), 139.2 - 140.0 (17 × Cq Bn), 170.7 - 171.2 (8 × C=O AcNH). 31 31P NMR (202 MHz, (CD3)2CO) δ = -2.84, -2.77, -2.68, -2.45, -2.42, -2.37, -2.31, -1.94, -1.81, -1.78. HRMS: [C 222 H 269 N 17 O 59 P8 + 2H] ++ requires 2184.33410, found 2184.33410.

[0168] General procedure for deprotection on a normal scale (5 - 40 μmol) The starting alcohol was dissolved in NH3 (30 - 33% aqueous solution, 1 mL per 10 μmol) and dioxane until completely dissolved. The reaction mixture was stirred for 2 hours. The mixture was concentrated under reduced pressure. 1 1H NMR and 31The ³¹P NMR analysis showed complete conversion to the semi-protected intermediate. The crude product was dissolved in MilliQ H2O and eluted through a column containing Dowex Na⁺ cation exchange resin (type: 50WX4-200, stored in 0.5 M NaOH / H2O solution, rinsed with MilliQ H2O and MeOH before use). The crude product was dissolved in MilliQ H2O (2 mL per 10 μmol). 4 - 5 drops of glacial acetic acid were added to the reaction mixture. The mixture was purged with Ar. 1 cup of Pd black was added to the solution. The reaction mixture was purged with H2 for several seconds and stirred under a H2 atmosphere for 3 days. Celite was added to the mixture. The solution was filtered and concentrated under reduced pressure. The crude product was purified by size exclusion chromatography (Toyopearl HW-40). The pure compound was dissolved in MilliQ H2O, eluted through a column containing Dowex Na⁺ cation exchange resin (type: 50WX4-200, stored in 0.5 M NaOH / H2O solution, rinsed with MilliQ H2O and MeOH before use), and lyophilized.

[0169] 1-O-octa-(2-acetamido-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)-6-hexylamine (8) n = 8 Alcohol 22 (23.2 μmol) was deprotected using the general procedure described above. Pure oligomer 8 was obtained in 44% yield (25.9 mg, 10.2 μmol). 1 H NMR (500 MHz, D2O) δ = 1.33 - 1.43 (m, 4H, 2×CH2 hexyl spacer), 1.57 - 1.69 (m, 4H, 2×CH2 hexyl spacer), 1.73 - 2.08 (m, 48H, 8×5a′-H, 8×5a-H, 8×5-H, 8×AcNH), 2.92 - 3.00 (m, 2H, CH2 hexyl spacer), 3.48 - 3.68 (m, 8H, 8×H-4), 3.68 - 3.76 (m, 2H, CH2 hexyl spacer), 3.81 - 4.22 (m, 24H, 8×H-3, 8×H-6), 4.25 - 4.36 (m, 8H, 8×H-1), 4.37 - 4.53 (m, 8H, 8×H-2). 13C NMR (126 MHz, DO) δ = 21.9 (8 × CHAcNH), 24.4 (CHhexyl spacer), 25.1 (CHhexyl spacer), 26.6 (CHhexyl spacer), 28.0 (8 × CHC-5a), 29.5 (CHhexyl spacer), 38.6 (8 × CHC-5), 39.4 (CHhexyl spacer), 53.5 (8 × CHC-2), 61.9 (8 × CHC-6), 66.2 (CHhexyl spacer), 70.1 (8 × CHC-1), 70.4 (8 × CHC-4), 71.9 (8 × CHC-3), 174.7 (8 × C=OAcNH). 31 P NMR (202MHz, D2O) δ=0.25, 0.37, 0.41, 0.44, 0.48. HRMS:[C 78 H 145 N9O 57 P8+H] ++ The required value was 1183.83071, and the actual value was 1183.83071.

[0170] Preparation of Randomly Acetylated Carba-Oligomers According to the Invention 1. Amine protection as Boc derivative The dried carba analogs DP6 (n = 6), DP7 (n = 7), and DP8 (n = 8) were dissolved in HO:dioxane 1:1 (volume ratio), and then NaHCO (2.95 equiv.) and (Boc)O (1.13 equiv.) were added at 4 ° C. The reaction was then maintained overnight under magnetic stirring at room temperature, and the product was then purified by Sephadex G10 column (eluent: HO), and the compound-containing fractions were dried.

[0171] 2. Random O-acetylation The dried Boc-protected carba analog from step 1 was resuspended in acetonitrile, and acetic anhydride (3.6 equivalents for each -OH group in the molecule) and imidazole (1.8 equivalents) were added. The reaction mixture was kept at 40°C, and the acetylation reaction time was extended until the target acetylation rate (%) (approximately 75%) was reached. 1 The acetylation was monitored by H-NMR. The crude acetylated compound was then dried.

[0172] To avoid misunderstanding, "random O-acetylation" means that the final control is not on how many of R x and R y are -C(O)CH3. However, using NMR technology, the total O-acetylation rate (%) in the oligomer can be determined.

[0173] 3. Boc Deprotection The dried crude O-acetylated carbacyclin analog from Step 2 was dissolved in CH2Cl2:TFA 4:1 (volume ratio), and the reaction solution was maintained under magnetic stirring at room temperature for 1 hour. Next, the crude reaction solution was dried, redissolved in H2O, and purified by a Sephadex G10 column (eluent: H2O).

[0174] NMR Protocol for Measuring Acetylation Rate (%) The sample was dried under vacuum, regenerated with 0.6 mL of D2O, and transferred into a 5 mm NMR tube. A proton NMR spectrum was obtained by a standard one-dimensional pulse program at 400 MHz and 25 °C. The acquisition and processing of the spectrum were performed by TopSpin Bruker software.

[0175] The measurement of the O-acetylation rate (%) in the carbacyclin analog was carried out by integrating the peak of H3+H4O-Ac (i.e., the H of the acetate group) at 5 - 5.4 ppm and the triplet of CH2 adjacent to the NH2 of the linker at approximately 3 ppm with a given value of 2. Looking at Figure 1, assuming that when the O-acetylation is 100%, the integration value of H3+H4O-Ac should be 12 (14 for DP7 and 16 for DP8) for DP6, the following ratio is applied. 12:100 = 9.04:X (X = acetylation rate (%))

[0176] The final product was 1 characterized by 1H-NMR to confirm the identical structure and determine the O-acetylation rate (%) of the synthesized sugar (Figure 2 and Table 1).

[0177] Figure 2 shows the final randomly acetylated carbamate analogs 1 1H NMR, and also shows the integration for the measurement of the acetylation rate (%) (n = 8).

[0178]

Table 1

[0179] For the same randomly acetylated carbamate analog of formula (Ia) with n = 8, the distribution of acetyl groups between the 3- and 4-positions was 31 determined by 31P NMR spectroscopy (101 MHz, D2O). The recorded spectrum is shown in Figure 3. This shows a simultaneous acetylation occurring to the extent of about 44% at C3 and C4 (i.e., R x and R y are both -C(O)CH3.) and acetylation at C3 or C4 up to about 28% (i.e., in the same repeating unit, R x is -C(O)CH3 and R y is H, or R x is H and R y is -C(O)CH3.), indicating that 27% of the repeating units are not acetylated.

[0180] Generation of the selectively acetylated carbamonomer building block according to Scheme 2 (i.e., R x is H and R y is -C(O)CH3) D-glucal (23)

Chemical Structure

[0181] 4,6 - O - (4 - Methoxybenzylidene) - D - glucal (24)

Chemical Structure

[0182] δ 1 H (400 MHz; CDCl3) 7.43 (2H, td, J8.6, J4.7, 8 - H), 6.90 (2H, dt, J8.8, J4.9, 9 - H), 6.33 (1H, ddd, J6.1, J1.6, J0.4, 1 - H), 5.55 (1H, s, 7 - H), 4.76 (1H, dd, J6.1, J2.0, 2 - H), 4.49 (1H, brd, J7.3, 3 - H), 4.35 (1H, dd, J10.3, J5.0, 5 - H), 3.93 - 3.87 (1H, m, 6 - H), 3.83 - 3.79 (1H, m, 6 - H), 3.80 (3H, s, - OMe), 3.77 - 3.75 (1H, m, 4 - H), 2.47 (1H, s, - OH).

[0183] δ 13 C (100 MHz; CDCl3) 159.4 (11-C), 143.3 (1-C), 128.6 (8-C), 126.7 (9-C), 112.8 (10-C), 102.7 (2-C), 100.9 (7-C), 79.8 (4-C), 68.9 (5-C), 67.6 (6-C), 65.7 (3-C), 54.4 (OMe).

[0184] 3-O-Benzyl-oxy-4,6-O-(4-methoxy-benzylidene)-D-glucal (25)

Chemical formula

[0185] δ 1 H (400 MHz; CDCl3) 7.42 (2H, dt, J8.5, J4.6, 8-H), 7.37 - 7.23 (7H, m, H arom)、6.90 (2H, dt, J 8.9, J 4.9, 9-H), 6.34 (1H, dd, J 6.2, J 1.4, 1-H), 5.58 (1H, s, 7-H), 4.81 (1H, dd, J 6.17, J 2.06, 2-H), 4.79 (1H, d, J 12.1, 10-H CH2Ph), 4.70 (1H, d, J 12.1, 10-H CH2Ph), 4.36 - 4.32 (2H, m, 3-H, 6a-H), 4.00 (1H, dd, J 9.8, J 7.4, 6b-H), 3.88 (1H, td, J 10.1, J 4.7, 5-H), 3.81 (1H, t, J 10.1, 4-H), 3.80 (3H, s, -OMe).

[0186] δ 13 C (100 MHz; CDCl3) 160.2 (11-C), 144.5 (1-C), 138.6 (13-C), 129.9 (8-C), 129.9 - 127.2 (C arom 9, 14, 15, 16-C), 113.7 (10-C), 102.4 (2-C), 101.3 (7-C), 80.1 (5-C), 73.2 (4-C), 72.1 (6-C), 68.8 (3-C), 68.4 (12-C), 55.4 (-OMe).

[0187] 3-O-Benzyl-oxy-4-O-(4-methoxybenzyl-oxy)-D-glucal (26)

Chemical Structure

[0188] δ 1 H (400 MHz; CDCl3) 7.34-7.20 (7H, m, H arom ), 6.83(2H, dt, J8.7, J4.8, 9-H), 6.34(1H, dd, J6.1, J1.2, 1-H), 4.82(1H, dd, J6.1, J2.6, 2-H), 4.75(1H, d, J11.1, 10-H CH2Ph), 4.63(1H, d, J11.1, 10-H CH2Ph), 4.61(1H, d, J11.8, 7-H CH2Ph(4-OMe)), 4.52(1H, d, J11.8, 7-H CH2Ph(4-OMe)), 4.19(1H, ddd, J6.3, J2.4, J2.3, 3-H), 3.87(1H, dt, J8.8, J4.2, 5-H), 3. 81-3.79(2H, m, 6-H), 3.77(1H, dd, J8.7, J6.3, 4-H), 3.71(3H, s, -OMe), 2.65(1H, s, -OH).

[0189] δ 13 C (100 MHz; CDCl3) 159.2(11-C), 144.4(1-C), 138.1(13-C), 130.1(8-C), 129.7-127.6(C arom9, 14, 15, 16 - C), 113.7(10 - C), 100.1(2 - C), 77.5(5 - C), 75.6(3 - C), 74.1(4 - C), 73.3(12 - C), 70.4(7 - C), 61.4(6 - C), 55.1(-OMe).

[0190] 1,5 - anhydro - 3 - O - benzyloxy - 4 - O - (4 - methoxybenzyloxy) - 2,6,7 - trideoxy - D - arabino - hept - 1,6 - dienitol(28)

Chemical formula

[0191] On the other hand, at - 78 °C, an ylide was prepared with a dry THF solution (12.0 mL) of fresh PPh3CH3I (1.48 g, 3.65 mmol) and stirred for 25 minutes. Next, KHMDS (7.3 mL, 3.65 mmol, 0.5 M / toluene solution) was added dropwise at - 78 °C. The mixture was stirred in the order of 20 minutes at - 78 °C, 50 minutes at 0 °C, and finally 30 minutes at - 78 °C to form the ylide.

[0192] Furthermore, the oxidation reaction was quenched for 10 minutes with a solution of Na2S2O3 (30 mL) and NaHCO3 (30 mL). Next, the aldehyde was post - treated with DCM (3 times with 40 mL), dried over Na2SO4, and DCM was distilled off under reduced pressure.

[0193] Next, the aldehyde (11.0 mL) in dry THF was added dropwise to the aforementioned ylide at - 78 °C. The reaction mixture was stirred overnight. The mixture was treated with NH4Cl (20 mL) and DCM (50 mL). Next, the organic layer was extracted again with DCM (2 times with 30 mL), washed with NaCl (80 mL), and dried over Na2SO4. The residue was purified by flash chromatography (nHexane / EtOAc = 7:3) to obtain the alkene as a yellow oil in a two - step yield of 83%.

[0194] δ 1 H (400 MHz; CDCl3) 7.37 - 7.27 (4H, m, H arom )、7.24 (2H, dt, J 8.6, J 5.5, 9 - H)、6.86 (2H, td, J 8.7, J 5.5, 10 - H)、6.41 (1H, dd, J 6.1, J 1.3, 1 - H)、6.04 (1H, ddd, J 17.2, J 10.6, J 6.6, 6 - H)、5.43 (1H, dt, J 2.9, J 17.3, 7b - H)、5.31 (1H, dt, J 2.6, J 10.6, 7a - H)、4.88 (1H, dd, J 6.2, J 2.7, 2 - H)、4.70 (1H, d, J 10.9, 11 - H, CH2Ph)、4.64 (1H, d, J 11.7, 8 - H, CH2Ph(4 - OMe))、4.62 (1H, d, J 10.9, 11 - H CH2Ph)、4.58 (1H, d, J 11.7, 8 - H CH2Ph(4 - OMe))、4.31 (1H, dd, J 7.1, J 8.0, 5 - H)、4.19 (1H, ddd, J 6.2, J 2.5, J 1.5, 3 - H)、3.79 (3H, s, - OMe)、3.59 (1H, dd, J 8.6, J 6.2, 4 - H)。

[0195] δ 13 C (100 MHz; CDCl3) 159.4 (12 - C)、144.6 (1 - C)、138.5 (14 - C)、134.5 (6 - C)、130.3 (9 - C)、129.8 - 127.8 (C arom 10、15、16、17 - C)、118.4 (7 - C)、113.9 (11 - C)、100.5 (2 - C)、78.2 (5 - C)、78.0 (4 - C)、75.5 (3 - C)、73.6 (8 - C)、70.8 (13 - C)、55.4 (- OMe)。

[0196] (3R, 4R, 5R) - 4 - O - Benzyloxy - 3 - O - (4 - methoxybenzyloxy) - 5 - (hydroxymethyl) cyclohexene (29)

Chemical Structure

[0197] δ 1 H (400 MHz; CDCl3) 7.28 - 7.16 (7H, m, H arom )、6.79 (2H, brd, J 8.3, 14-H), 5.67 - 5.64 (1H, m, 1-H), 5.64 - 5.59 (1H, m, 2-H), 4.88 (1H, d, J 11.3, 8-H CH2Ph), 4.64 (1H, d, J 11.3, 8-H CH2Ph), 4.56 (1H, d, J 11.2, 12-H CH2Ph(4-OMe)), 4.48 (1H, d, J 11.7, 12-H CH2Ph(4-OMe)), 4.12 (1H, brd, 4-H), 3.71 (3H, s, -OMe), 3.57 - 3.47 (3H, m, 3-H, 6-H), 2.35 (1H, s, -OH), 2.07 - 2.00 (1H, m, 7-H), 1.97 - 1.88 (1H, m, 5-H), 1.82 - 1.75 (1H, m, 7-H) δ 13 C (100 MHz; CDCl3).

[0198] δ 13 C (100 MHz; CDCl3) 159.4 (17-C), 138.5 (9-C), 132.1 (14-C), 130.5 - 128.0 (C arom10, 11, 12, 15 - C), 127.7(1 - C), 126.1(2 - C), 114.0(16 - C), 82.3(3 - C), 80.9(4 - C), 74.4(8 - C), 71.1(13 - C), 65.9(6 - C), 55.4(-OMe), 40.7(5 - C), 28.1(7 - C).

[0199] 4 - O - Benzyl - 3 - O - (4 - methoxybenzyloxy) - 6 - O - tert - butyldimethylsilyl - 5 - methylcyclohexene (30) [Chemical formula] Alcohol 29 (715 mg, 2.02 mmol) was dissolved in dry THF (17 mL) at room temperature. Imidazole (125 mg, 1.83 mmol) was added and the mixture was stirred at room temperature for 5 minutes and then at 0 °C for 10 minutes. Next, tert - butyldimethylsilyl chloride (1.19 mL, 6.05 mmol) was carefully added dropwise to form a white precipitate. The ice bath was removed at the first precipitation and the remaining TDSCl was slowly added to the mixture, which was then warmed to room temperature and stirred overnight. The reaction was monitored by TLC (Pent / AcOEt 3:1). The organic phase was extracted with EtOAc and then washed with distilled water (5 times). The residue was purified by flash chromatography (nHex / AcOEt 95:5) to form compound 30 as a yellow oil in quantitative yield.

[0200] δ 1 H(400 MHz; CDCl3) 7.37 - 7.16 (7H, m, H arom)、6.88 - 6.84 (2H, m, 14 - H), 5.75 (1H, ddq, J9.0, J4.3, J2.4, 1 - H), 5.64 (1H, brd, 2 - H), 4.91 (1H, d, J11.0, 8 - H CH2Ph), 4.68 (1H, d, J11.0, 8 - H CH2Ph), 4.64 (1H, d, J11.3, 12 - H CH2Ph(4 - OMe)), 4.60 (1H, d, J11.3, 12 - H CH2Ph(4 - OMe)), 4.16 (1H, ddq, J7.1, J3.6, J1.8, 3 - H), 3.86 (1H, dd, J9.8, J4.8, 6 - H), 3.79 (3H, s, - OMe), 3.64 (1H, dd, J10.0, J6.6, 4 - H), 3.63 - 3.58 (1H, m, 6 - H), 2.28 - 2.16 (1H, m, 7 - H), 2.10 (1H, dt, J18.4, J5.3, 7 - H), 1.91 (1H, ttd, J10.5, J5.1, J2.7, 5 - H), 1.64 (1H, hept, J6.9, 17 - H), 0.90 (6H, d, J6.9, 18 - H), 0.87 (6H, s, 16 - H), 0.13 (6H, s, 15 - H).

[0201] δ 13 C(100MHz; CDCl3) 159.3 (14 - C), 139.3 (9 - C), 133.8 (17 - C), 131.0 - 128.0 (C arom 10, 11, 12, 15 - C), 127.6 (1 - C), 126.3 (2 - C), 113.9 (16 - C), 81.5 (3 - C), 79.7 (4 - C), 74.7 (8 - C), 71.5 (13 - C), 62.6 (6 - C), 55.4 (- OMe), 41.4 (5 - C), 34.3 (21 - C), 28.7 (7 - C), 25.3 (19 - C), 20.5 - 20.3 (20 - C), 18.8 - 18.7 (22 - C), - 3.27 - - 3.46 (18 - C).

[0202] 4 - O - Benzyl - 3 - O - (4 - methoxybenzyloxy) - 6 - O - tert - butyldimethylsilyl - 5a - carba - α - D - glucopyranose (31)

Chem.

[0203] δ 1 H (400 MHz; CDCl3) 7.37 - 7.15 (7H, m, H arom )、6.87 (2H, brd, J 8.7, 14-H), 4.90 (1H, d, J 12, 8-H CH2Ph), 4.88 (1H, d, J 8, 12-H CH2Ph(4-OMe)), 4.69 (1H, d, J 10.9, 8-H CH2Ph), 4.61 (1H, d, J 11.1, 12-H CH2Ph(4-OMe)), 4.05 (1H, brd, J 2.7, 1-H), 3.96 (1H, dd, J 10.0, J 3.3, 6-H), 3.78 (3H, s, -OMe), 3.71 (1H, t, J 9.4, 3-H), 3.48 (2H, t, J 10.0, 6-H, 4-H), 3.43 (1H, dd, J 2.3, J 9.4, 2-H), 2.64 (1H, s, -OH), 2.58 (1H, s, -OH), 2.09 - 2.03 (1H, m, 5-H), 1.77 (1H, dt, J 14.5, J 3.6, 7-H), 1.62 (1H, hept, J 6.9, 17-H), 1.59 - 1.52 (1H, m, 7-H), 0.88 (6H, d, J 6.9, 18-H), 0.85 (6H, d, d 1.2, 16-H), 0.07 (6H, s, 15-H).

[0204] δ 13 C (100 MHz; CDCl3) 159.5(14-C), 138.9(9-C), 130.9(17-C), 129.7-127.7(C arom 10, 11, 12, 15-C), 114.2(16-C), 83.4(3-C), 81.0(4-C), 75.1(13-C), 74.9(8-C), 74.6(2-C), 68.5(1-C), 62.1(6-C), 55 .3(-OMe), 38.9(5-C), 34.3(21-C), 30.4(7-C), 25.2(19-C), 20.5-20.4(20-C), 18.8-18.7(22-C), -3.35--3.56(18-C).

[0205] 1-O-acetyl-4-O-benzyl-3-O-(4-methoxybenzyloxy)-6-O-thexyldimethylsilyl-5a-carba-α-D-glucopyranose (32) [ka] Compound 31 (155 mg, 0.29 mmol) was dissolved in acetonitrile (2.9 mL) at room temperature under nitrogen. Trimethyl orthoacetate (115 μL, 0.88 mmol) and PTSA (5 mg, 0.03 mmol) were added sequentially to the mixture, which was then stirred at room temperature under nitrogen for 60 min. After the reaction was complete, an 80% AcOH solution (2.32 mL AcOH + 0.58 mL HO) was added. The subsequent acetylation reaction was complete in 60 min. The organic phase was extracted with DCM (5 mL), washed with water (5 mL) and NaHCO3 (5 mL), and finally dried over Na2SO4. The residue was purified by flash chromatography (nHex / AcOEt) to give compound 32, selectively acetylated at the pseudoanomeric position, as a colorless oil in quantitative yield.

[0206] δ 1 H (400 MHz; CDCl3) 7.39-7.13 (7H, m, H arom)、6.87 (2H, dt, J 8.7, J 5.0, 14-H), 5.26 (1H, dd, J 5.7, J 3.0, 1-H), 4.91 (1H, d, J 10.6, 8-H CH2Ph), 4.90 (1H, d, J 10.9, 12-H CH2Ph(4-OMe)), 4.70 (1H, d, J 10.0, 8-H CH2Ph), 4.68 (1H, d, J 10.5, 12-H CH2Ph(4-OMe)), 3.95 (1H, dd, J 10.0, J 3.5, 6-H), 3.80 (3H, s, -OMe), 3.75 (1H, t, J 9.3, 3-H), 3.58 (1H, brd, J 9.6, 2-H), 3.53 (1H, dd, J 9.1, J 10.1, 4-H), 3.50 (1H, dd, J 9.8, J 2.4, 6-H), 2.28 (1H, s, -OH), 2.08 (3H, s, -OAc), 1.95 - 1.88 (1H, m, 5-H), 1.85 (1H, dt, J 14.8, J 7.6, 7-H), 1.61 (1H, dt, J 13.8, J 6.9, 7-H), 1.61 (1H, hept, J 6.9, 17-H), 0.88 (6H, d, J 6.8, 18-H), 0.84 (6H, d, J 1.7, 16-H), 0.07 (6H, d, J 4.4, 15-H).

[0207] δ 13 C (100 MHz; CDCl3) 170.9 (C(O), -OAc), 159.5 (14-C), 138.7 (9-C), 130.8 (17-C), 129.8 - 127.9 (C arom 10, 11, 12, 15-C), 114.8 (16-C), 84.0 (3-C), 80.5 (4-C), 75.4 (13-C), 75.3 (8-C), 73.4 (2-C), 71.8 (1-C), 61.8 (6-C), 55.4 (-OMe), 39.6 (5-C), 34.3 (21-C), 28.8 (7-C), 25.3 (19-C), 21.4 (CH3, -OAc), 20.5 - 20.4 (20-C), 18.8 - 18.7 (22-C), -3.28 - -3.53 (18-C).

[0208] 1-O-Acetyl-2-azido-4-O-benzyloxy-3-O-(4-methoxybenzyloxy)-6-O-tert-butyldimethylsilyl-5a-carba-α-D-mannopyranose [Chemical Formula] Compound 32 (220 mg, 0.38 mmol) was dissolved in a mixture of DCM / pyridine (5:1, 0.05 M) and stirred at 10 °C for 10 min under nitrogen. Trifluoromethanesulfonic anhydride (355 μL, 2.11 mmol) was added dropwise at -10 °C. The mixture was stirred successively for 30 min to reach 0 °C slowly and then stirred at 0 °C for 30 min. After completion of the reaction, the organic phase was washed with NaHCO3 and brine. The organic layer was dried over Na2SO4, and the resulting crude product was used directly in the next step after azeotropic distillation with toluene (3 times). Next, the dried crude product was dissolved in DMF / H2O (19:1, 0.2 M) at 40 °C. Sodium azide (125 mg, 1.92 mmol) and 15-crown-5 (15.2 μL, 0.08 mmol) were added at room temperature, and the reaction was allowed to proceed at 40 °C overnight. After the triflate intermediate had completely disappeared, the solvent was distilled off, and the residue was finally purified by flash chromatography (nHex / EtOAc) to form the title compound azide as a colorless oil in 82% yield.

[0209] δ 1 H (400 MHz; CDCl3) 7.38 - 7.14 (7H, m, H arom)、6.86 (2H, dt, J 8.6, J 4.9, 14-H), 4.98 - 4.94 (1H, m, 1-H), 4.88 (1H, d, J 10.7, 8-H CH2Ph), 4.66 (1H, d, J 19.1, 12-H CH2Ph(4-OMe)), 4.63 (1H, d, J 19.5, 12-H CH2Ph(4-OMe)), 4.59 (1H, d, J 10.9, 8-H CH2Ph), 3.87 - 3.84 (1H, m, 2-H), 3.84 (1H, dd, J 6.3, J 2.7, 6-H), 3.80 (3H, s, -OMe), 3.82 - 3.75 (2H, m, 4-H, 3-H), 3.52 (1H, dd, J 9.9, J 2.1, 6-H), 2.00 (3H, s, -OAc), 1.91 - 1.82 (2H, m, 5-H, 7-H), 1.65 - 1.57 (2H, m, 7-H, 17-H), 0.89 (6H, d, J 6.9, 18-H), 0.85 (6H, d, J 1.2, 16-H), 0.07 (6H, d, J 4.1, 15-H).

[0210] δ 13 C (100 MHz; CDCl3) 169.8 (C(O), -OAc), 159.6 (14-C), 138.9 (9-C), 130.2 (17-C), 129.8 - 127.8 (C arom 10, 11, 12, 15-C), 114.0 (16-C), 81.1 (4-C), 77.0 (3-C), 75.4 (8-C), 72.9 (13-C), 70.6 (1-C), 62.2 (6-C), 61.4 (2-C), 55.4 (-OMe), 39.8 (5-C), 34.4 (21-C), 27.1 (7-C), 25.3 (19-C), 21.2 (CH3, -OAc), 20.6 - 20.5 (20-C), 18.8 - 18.7 (22-C), -3.35 - -3.52 (18-C).

[0211] 1-O-acetyl-2-acetamido-4-O-benzyloxy-3-O-(4-methoxybenzyloxy)-6-O-tert-butyldimethylsilyl-5a-carba-α-D-mannopyranose (33)

Chemical formula

[0212] δ 1 H (400 MHz; CDCl3) 7.39 - 7.28 (5H, m, H arom )、7.19 (2H, dt, J9.4, J4.6, 13-H), 6.86 (2H, dt, J9.4, J4.8, 14-H), 5.59 (1H, d, J8.1, NHAc), 5.12 (1H, td, J7.2, J3.9, 1-H), 4.71 (1H, d, J11.3, 8-H CH2Ph), 4.56 (1H, d, J11.3, 8-H CH2Ph), 4.50 (1H, d, J11.2, 12-H CH2Ph(4-OMe)), 4.42 (1H, td, J7.7, J4.1, 2-H), 4.36 (1H, d, J11.2, 12-H CH2Ph(4-OMe)), 3.84 (1H, dd, J2.4, J4.0, 3-H), 3.85 - 3.82 (1H, m, 6-H), 3.80 (3H, s, -OMe), 3.72 (1H, t, J6.3, 4-H), 3.60 (1H, dd, J9.9, J5.5, 6-H), 2.09 - 2.02 (1H, m, 5-H), 2.01 (3H, s, -OAc), 1.90 (3H, s, -NHAc), 1.82 (2H, tdd, J14.2, J7.4, J4.6, 7-H), 1.66 - 1.57 (1H, hept, J6.9, 17-H), 0.89 (6H, d, J6.9, 18-H), 0.84 (6H, s, 16-H), 0.08 (6H, d, J6.2, 15-H).

[0213] δ 13 C (100 MHz; CDCl3) 170.7 (C(O), -NHAc), 170.1 (C(O), -OAc), 159.6 (14-C), 138.6 (9-C), 130.0 (15-C), 129.9 (17-C), 128.6 - 127.8 (C arom 10, 11, 12-C), 114.1 (16-C), 78.7 (3-C), 74.4 (4-C), 73.6 (8-C), 71.9 (13-C), 69.6 (1-C), 62.5 (6-C), 55.4 (-OMe), 50.6 (2-C), 39.9 (5-C), 34.4 (21-C), 27.1 (7-C), 25.2 (19-C), 23.5 (CH3, -NHAc), 21.3 (CH3, -OAc), 20.5 (20-C), 18.8 (22-C), -3.37 - -3.48 (18-C).

[0214] 1-O-tert-Butyldimethylsilyl-2-acetamido-4-O-benzyl-2-deoxy-3-O-(4-methoxybenzyloxy)-6-O-tert-butyldimethylsilyl-5a-carba-α-D-mannopyranose (35)

Chemical Structure

[0215] Under a stream of N2 gas, 34 (0.95 mmol) of a DCM solution (4 mL) was placed in a flask. At 0 °C, 2,6-lutidine (2.37 mmol) was added dropwise, followed by TBSOTf (437 μL, 1.9 mmol). The mixture was stirred and warmed to room temperature. After completion, the reaction was cooled to room temperature, quenched with MeOH, and the mixture was diluted with chloroform. The mixture was washed with 10% aqueous CuSO4 (twice), H2O, and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification by column chromatography (nHex / EtOAc) gave the title compound 35 as an orange-red oil in 83% yield over two steps.

[0216] J. D. C. Codee et al., J. Org. Chem, 2017, 82, 2, 848-868

[0217] δ 1 H(400MHz;CDCl3) 7.41-7.24(5H, m, H arom)、7.19 (2H, dt, J 9.5, J 4.6, 13-H), 6.86 (2H, dt, J 9.4, J 4.8, 14-H), 5.57 (1H, d, J 5.7, NHAc), 4.93 (1H, d, J 10.6, 8-H CH2Ph), 4.58 (1H, d, J 10.5, 8-H CH2Ph), 4.56 (1H, d, J 11.1, 12-H CH2Ph(4-OMe)), 4.48 (1H, d, J 11.1, 12-H CH2Ph(4-OMe)), 4.27 (1H, dd, J 5.2, J 2.3, 2-H), 4.25 - 4.21 (1H, m, 1-H), 4.03 (1H, dd, J 9.6, J 4.5, 3-H), 3.97 (1H, dd, J 9.7, J 3.6, 6-H), 3.81 (3H, s, -OMe), 3.54 (1H, t, J 9.9, 4-H), 3.48 (1H, dd, J 9.7, J 2.2, 6-H), 2.09 - 2.02 (1H, m, 5-H), 2.01 (3H, s, -NHAc), 1.78 - 1.69 (1H, m, 7-H), 1.69 - 1.59 (1H, m, 17-H), 1.52 - 1.45 (1H, m, 7-H), 0.93 (6H, d, J 6.9, 18-H), 0.87 (6H, s, 16-H), 0.86 (6H, s, 20-H), 0.84 (6H, s, 16-H), 0.12 (6H, d, J 12.0, 19-H), 0.09 (6H, d, J 9.4, 15-H).

[0218] δ 13 C (100 MHz; CDCl3) 170.7 (C(O), -NHAc), 159.5 (14-C), 139.1 (9-C), 130.2 (17-C), 130.0 (15-C), 128.6 - 127.7 (C arom 10, 11, 12-C), 114.0 (16-C), 78.5 (3-C), 77.6 (4-C), 75.5 (8-C), 71.4 (13-C), 67.7 (2-C), 62.6 (6-C), 55.4 (-OMe), 53.4 (1-C), 38.6 (5-C), 34.6 (21-C), 30.4 (7-C), 25.9 (25-C), 25.2 (19-C), 23.6 (CH3-NHAc), 20.7 - 20.6 (20-C), 18.9 - 18.8 (22-C), 18.0 (24-C), -3.37 - -3.58 (18-C), -4.82 - -4.92 (23-NS).

[0219] 1-O-tert-Butyldimethylsilyl-2-acetamido-4-O-benzyloxy-3-O-(4-methoxybenzyloxy)-6-O-tert-butyldimethylsilyl-5a-carba-α-D-mannopyranose

Chem.

[0220] Dan Van Der Es, Thesis, 2016, Universiteit Leiden, pp160.

[0221] δ 1 H (400 MHz; CDCl3) 7.41 - 7.27 (5H, m, H arom)、5.52 (1H, d, J 5.4, NHAc), 4.73 (2H, s, 8-H CH2Ph), 4.26 (1H, brd, J 2.7, 1-H), 4.16 (1H, dt, J 9.0, J 3.8, 3-H), 4.06 (1H, dd, J 9.0, J 4.5, 2-H), 3.94 (1H, dd, J 9.9, J 3.7, 6-H), 3.53 (1H, dd, J 10.0, J 2.1, 6-H), 3.46 (1H, t, J 9.5, 4-H), 2.73 (1H, s, -OH), 2.10 - 2.03 (1H, m, 5-H), 2.00 (3H, s, -NHAc), 1.81 - 1.69 (1H, m, 7-H), 1.69 - 1.59 (1H, m, 14-H), 1.51 (1H, dt, J 13.7, J 3.2, 7-H), 0.93 (6H, d, J 6.9, 15-H), 0.88 (6H, s, 17-H), 0.87 (6H, s, 13-H), 0.14 - 0.04 (12H, m, 16-H, 12-H).

[0222] δ 13 C (100 MHz; CDCl3) 170.1 (C(O), -NHAc), 138.8 (9-C), 128.7 - 127.7 (C arom 10, 11, 12-C), 79.6 (4-C), 74.8 (8-C), 70.7 (3-C), 67.6 (1-C), 62.9 (6-C), 56.5 (2-C), 38.5 (5-C), 34.6 (16-C), 31.2 (7-C), 25.9 (20-C), 25.3 (14-C), 23.6 (CH3, -NHAc), 20.7 - 20.6 (15-C), 18.9 - 18.8 (17-C), 18.0 (19-C), -3.37 - -3.53 (13-C), -4.80 - -4.90 (18-C).

[0223] 1-O-tert-butylsilyl-2-acetamido-4-O-benzyloxy-6-O-tert-butyldimethylsilyl-5a-carba-α-D-mannopyranose (36)

Chem.

[0224] δ 1 H (400 MHz; CDCl3) 7.37 - 7.13 (5H, m, H arom )、5.44 (1H, dd, J10.3, J4.5, 3-H)、5.27 (1H, d, J7.4, NHAc)、4.70 (2H, d, J10.9, 8-H CH2Ph)、4.61 (1H, d, J10.9, 8-H CH2Ph)、4.31 (1H, dt, J7.3, J3.8, 2-H)、4.10 (1H, brd, J2.7, 1-H)、3.97 (1H, dd, J9.8, J3.2, 6-H)、3.61 (1H, t, J10.3, 4-H)、3.46 (1H, dd, J9.8, J2.0, 6-H)、2.18 - 2.11 (1H, m, 5-H)、2.00 (3H, s, -NHAc)、1.98 (3H, s, -OAc)、1.79 - 1.70 (1H, m, 7-H)、1.70 - 1.61 (1H, m, 14-H)、1.52 (1H, dt, J14.3, J2.8, 7-H)、0.95 (6H, d, J6.9, 15-H)、0.90 (6H, s, 17-H)、0.88 (6H, s, 13-H)、0.13 (6H, d, J15.1, 16-H)、0.09 (6H, d, J14.8, 12-H).

[0225] δ 13 C (100 MHz; CDCl3) 170.0 (C(O), -NHAc)、169.8 (C(O), -OAc)、138.7 (9-C)、128.6 - 127.6 (C arom10, 11, 12-C), 76.2 (4-C), 75.1 (8-C), 73.2 (3-C), 68.1 (1-C), 62.3 (6-C), 54.0 (2-C), 38.7 (5-C), 34.6 (16-C), 30.6 (7-C), 25.8 (20-C), 25.3 (14-C), 23.6 (CH3, -NHAc), 21.2 (CH3, -OAc), 20.7 - 20.6 (15-C), 19.0 - 18.9 (17-C), 18.1 (19-C), -3.41 - -3.62 (13-C), -4.90 - -4.99 (18-C).

[0226] 2-Acetamido-4-O-benzyloxy-5a-carba-α-D-mannopyranose (37) [Chemical formula] Compound 36 (120 mg, 0.20 mmol) was dissolved in dry THF (2.0 mL) at 0 °C. A solution of 30% HF / Py (420 μL) was added dropwise, and the reaction mixture was slowly warmed from 0 °C to room temperature while stirring overnight. Next, the mixture was quenched with NaHCO3 (3 mL). The organic layer was extracted twice with EtOAc, washed with brine, and dried over Na2SO4. The resulting crude compound 37 was filtered through silica to give a white solid in 60% yield.

[0227] δ 1 H (400 MHz; CD3OD) 7.37 - 7.26 (5H, m, H arom ), 5.33 (1H, dd, J8.4, J4.4, 3-H), 4.72 (2H, d, J11.4, 8-H CH2Ph), 4.66 (1H, d, J11.4, 8-H CH2Ph), 4.45 (1H, t, J4.8, 2-H), 4.10 (1H, brd, J2.7, 1-H), 3.87 (1H, q, J4.5, 1-H), 3.78 - 3.73 (2H, m, 4-H, 6-H), 3.68 (1H, dd, J10.6, J4.2, 6-H), 2.17 - 2.09 (1H, m, 5-H), 2.04 (1H, s, -OH), 2.03 (1H, s -OH), 2.02 (3H, s, -NHAc), 1.98 (3H, s, -OAc), 1.83 (2H, dd, J7.8, J3.8, 7-H).

[0228] δ 13 C (100 MHz; CDCl3) 173.6 (C(O), -NHAc), 172.0 (C(O), -OAc), 140.0 (9-C), 129.3 - 128.6 (C arom 10, 11, 12-C), 77.2 (4-C), 74.9 (8-C), 74.7 (3-C), 68.2 (1-C), 63.1 (6-C), 54.0 (2-C), 40.7 (5-C), 30.9 (7-C), 22.5 (CH3, -NHAc), 21.1 (CH3, -OAc).

[0229] 2-Acetamido-4-O-benzyloxy-6-O-dimethoxytrityl-5a-carba-α-D-mannopyranose (38)

Chem.

[0230] δ 1 H (400 MHz; CD3OD) 7.40 - 7.05 (14H, m, H arom)、6.79 (4H, dd, J8.9, J1.7, 13-H), 5.24 (1H, dd, J7.9, J4.3, 3-H), 4.53 (1H, d, J11.3, 8-H CH2Ph), 4.38 (1H, t, J4.8, 2-H), 4.31 (1H, d, J11.3, 8-H CH2Ph), 3.79 (1H, q, J5.2, 1-H), 3.72 (3H, s, -OMe), 3.72 (3H, s, -OMe), 3.61 (1H, t, J8.1, 4-H), 3.34 - 3.26 (1H, m, 6-H), 3.05 (1H, t, J8.3, 6-H), 2.34 - 2.24 (1H, m, 5-H), 2.08 - 1.98 (1H, m, 7-H), 1.95 (3H, s, -NHAc), 1.86 (3H, s, -OAc), 1.85 - 1.79 (1H, m, 7-H).

[0231] δ 13 C (100 MHz; CD3OD) 173.6 (C(O), -NHAc), 172.0 (C(O), -OAc), 160.0 (17-C), 146.7 (9-C), 137.6 (14-C), 137.5 (14-C), 137.3 (9-C), 131.4 (18-C), 129.9 - 126.3 (C arom 10, 11, 12, 15, 19, 20, 21-C), 114.0 (16-C), 87.2 (13-C), 77.3 (4-C), 74.5 (3-C), 74.4 (8-C), 68.0 (1-C), 65.0 (6-C), 55.7 (-OMe), 54.1 (2-C), 39.2 (5-C), 31.9 (7-C), 22.5 (CH3, -NHAc), 21.1 (CH3, -OAc).

[0232] Reference Example: Oligomer Conjugate - CRM without Acetylation 197 -MenA DP6 (without OAC) and CRM 197 -Preparation of MenA DP8 (without OAc) The starting oligomers (DP6 and DP8) were vacuum dried, dissolved in a 1:9 H2O:DMSO solution to a final amino group concentration of 40 mmol / mL, and reacted with a 12-fold molar excess of adipic acid di-N-hydroxysuccinimidyl linker (SIDEA) in the presence of a 5-fold molar excess of triethylamine compared to the amino groups. The reaction was maintained at room temperature for 3 hours with gentle stirring. The activated oligosaccharides were precipitated with 4 volumes of ethyl acetate and purified by washing the pellet 10 times with 1 mL of the same solvent. Finally, the pellet was vacuum dried and the content of the introduced N-hydroxysuccinimide ester groups was determined.

[0233] The conjugate was prepared at pH 7 in 50 mM NaH2PO4 using a molar ratio of active ester (AE):protein of 40:1 and allowed to proceed overnight at room temperature with gentle stirring. The conjugate was purified by tangential flow filtration (Vivaspin) using a 30 kDa cut-off and PBS pH 7.2 as the buffer. The conjugate was characterized by SDS-page for total protein content using micro BCA 2 and by MALDI analysis for total sugar content.

[0234] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-Page). SDS-Page was performed using a precast 3-8% polyacrylamide gel (NuPAGE® Invitrogen). Electrophoresis was carried out by loading 5 μg of protein for each sample and using an electrophoresis chamber at a voltage of 150 V in Tris-acetate SDS running buffer (NuPAGE® Invitrogen) for approximately 40 minutes. The samples were prepared by adding 3 μL of NuPAGE® LDS sample buffer. After electrophoresis, the gel was washed 3 times with H2O and stained with Coomassie.

[0235] Example 2: Preparation of the oligomer conjugate of the invention according to formula (IIa) The randomly O-acetylated carbacyclin analogs produced as described above were activated with di-N-hydroxysuccinimidyl adipate linker (SIDEA), and the activation rates (%) obtained for the oligosaccharides were estimated to be 56% for DP6OAc, 79% for DP7OAc, and 84% for DP8OAc.

[0236] The activated oligosaccharides (i.e., the activated O-acetylated carbacyclin analogs) were lyophilized and prepared for the conjugation step. Conjugates were obtained by applying the chemistry of the report in the same figure shown in Figure 4 and SDS-page characterization, and the smear of the conjugate can be observed.

[0237] As shown in Table 2, the purified glycoconjugates (i.e., those containing randomly O-acetylated carbacyclin analogs) were characterized for protein content by MicroBCA and sugar content by HPAEC-PAD.

[0238]

Table 2

[0239] In vitro analysis of antibody responses by mouse immunization and ELISA and serum bactericidal assays (rSBA and hSBA) The antigen formulations were prepared under aseptic conditions. A group of 10 mice (BALB / c) was immunized on days 1, 14, and 28. Blood was collected on day 0 (before immunization), day 27 (2 days after immunization), and day 42 (3 days after immunization). The vaccine was administered at a saccharide dose and at a dose of 2 μg / mouse / dose with respect to the saccharide. The adjuvant AlPO4 was used at a dose of 0.12 mg of Al 3+ of dose.

[0240] The vaccine formulations used for the Carbamen A conjugate were as follows. 324.96 μL of AlPO4 (4.43 mg / mL containing 2 mg / mL NaCl) was added to the conjugate of interest. By adding PBS buffer at pH 7.2, the volume was adjusted to 1.2 mL with an AlPO4 concentration of 1.2 mg / mL. Finally, the solution was diluted 1:1 (v / v) with PBS to a final volume of 2.4 mL with a final AlPO4 concentration of 0.6 mg / mL. 200 μL of the formulation was injected per mouse. This procedure was also used for the formulation of MenA-CRM 197 from the stock solution.

[0241] ELISA of serum. The antibody response induced by the glycoconjugate was measured by ELISA. In this analysis, pre-immune serum was used as a negative control. To the plates, 100 μL / well of a 5 μg / mL polysaccharide solution in PBS buffer at pH 8.2 was added and then incubated overnight at 4 °C to coat with HSA-DeOAc (manufactured according to the method described in the literature 21 or MenA CPS). HSA-DeOAc MenA CPS, CRM 197 conjugate and CRM 197It was coated with a protein concentration of 2 μg / mL in pH 7.2 PBS buffer. The coating solution was removed from the plate by washing three times with PBS buffer (TPBS) containing 0.05% Tween 20 (Sigma). Next, a blocking step was performed by adding 100 μL / well of 3% BSA / TPBS solution and incubating the plate at 37 °C for 1 hour. The blocking solution was removed from the plate by washing three times with TPBS. 200 μL / well of pre-diluted serum (1:25 for pre-immune negative control, 1:200 - 1:500 for reference serum, 1:25 to 1:200 for test serum) was added to the first well of each column of the plate, and 100 μL of TPBS was dispensed into the other wells. Next, 8 consecutive two-fold dilutions were performed along each column by transferring 100 μL of the serum solution from well to well. After the primary antibody dilution, the plate was incubated at 3,7 °C for 2 hours. It was washed three times with TPBS, 100 μL / well of a TPBS solution of the secondary antibody alkaline phosphatase conjugate (anti-mouse IgG 1:10000, Sigma-Aldrich) was added, and the plate was incubated at 37 °C for 1 hour. After further washing three times with TPBS, 100 μL / well of 1 mg / mL p-NPP (Sigma) in 0.5 M diethanolamine buffer pH 9.6 was added. Finally, the plate was incubated at room temperature for 30 minutes and read at 405 nm using a plate reader Spectramax190. The serum titer was expressed as the reciprocal of the serum dilution corresponding to a cut-off OD = 1.

[0242] Each immunized group is represented as the geometric mean (GMT) in the 95% CI of the single mouse titer. Statistical analysis and graph analysis were performed by GraphPad Prism7 software.

[0243] Immunological evaluation To examine the immunogenicity of conjugate carb DP6 and DP8 analogs with and without random acetylation, groups of 8 BALB / c female mice were immunized with neo-glycoconjugates. Conjugated MenA polysaccharide of a combined size was used as a control. The mice were immunized with 3 subcutaneous (s.c.) administrations (2 μg based on carbohydrate) at 2-week intervals. When evaluating the anti-MenA CPS response, the data showed no response for the conjugates obtained with the carb MenA sugar antigen without O-acetylation for both sugar chain lengths 6 (n = 6) and 8 (n = 8). Conversely, the carb MenA conjugates obtained after random O-acetylation of the oligomers induced a significantly higher response against native MenA CPS compared to the non-acetylated vaccine (Table 3 and Figure 5). In comparison, the response induced by the O-acetylated vaccine was only 2-fold lower than the benchmark MenA-CRM 197 conjugate for DP8, which gave a better response among the vaccines examined.

[0244] The vaccine formulations used for the carb MenA conjugates were as follows. 324.96 μL of AlPO4 (4.43 mg / mL containing 2 mg / mL NaCl) was added to the conjugate of interest. The volume was adjusted to 1.2 mL with a concentration of AlPO4 of 1.2 mg / mL by adding PBS buffer at pH 7.2. Finally, the solution was diluted 1:1 (volume ratio) with PBS to 2.4 mL so that the final concentration of AlPO4 was 0.6 mg / mL. 200 μL / mouse of the formulation was injected. This procedure was also used for the formulation of MenA-CRM 197 from the stock solution.

[0245] The ELISA responses after the 2nd and 3rd administrations are reported in Table 3. As can be seen from Table 3, groups 2 and 3 are according to the present invention. For group 2, an oligomer conjugate with n = 6 and random acetylation as described above was used. For group 3, an oligomer conjugate with n = 8 and random acetylation as described above was used. The acetylation levels of the conjugates in groups 2 and 3 were approximately 75%.

[0246]

Table 3

[0247] Figures 5a and 5b provide the ELISA titers after two and three administrations. The p-value refers to the comparison between the benchmark native MenA-CRM 197 and the other groups.

[0248] By comparing the above-mentioned random O-acetylated carbapenem MenA DP8 analogs of the present invention with carbapenem MenA DP8 selectively O-acetylated only at position 3 with an O-acetylation rate of about 70%, and with a MenA vaccine as a positive control, a second immunological test was conducted according to the method described below, and all of these were CRM 197 conjugated.

[0249] Three groups of 10 Balb / C mice were immunized with the above conjugate. The mice were immunized with three subcutaneous (s.c.) administrations at two-week intervals (2 μg based on carbohydrate, 200 μL of formulation / mouse). The vaccine formulation used for the carbapenem MenA conjugate was the same as that reported above for the first immunological study. The anti-MenA CPS response was evaluated, and the data showed that the total IgG response after the third immunization with 3-O-acetylated carbapenem MenA DP8 was about one-tenth of the MenA vaccine benchmark. Conversely, the random O-acetylated carbapenem MenA DP8 conjugate of the present invention induced a significantly higher response against native MenA CPS compared to the 3-O-acetylated conjugate and was substantially equivalent to the response of the MenA vaccine benchmark (Figure 6).

[0250] TIFF2025111445000044.tif42153

[0251] in vitro bactericidal assay Functional antibodies induced by vaccination were analyzed by measuring complement-mediated lysis of meningococci in an in vitro bactericidal assay.

[0252] Commercially available lots of infant rabbit complement were used as a source of active complement for rSBA, and human plasma obtained from volunteer donors under informed consent was used as a complement source for hSBA. That is, meningococcal strains were grown overnight at 37 °C in 5% CO2 on chocolate agar plates. Colonies were inoculated into Mueller-Hinton broth containing 0.25% glucose to reach an OD600 of 0.05 - 0.08 and incubated at 37 °C with shaking. When the OD600 of the bacterial suspension reached 0.25 - 0.27, the bacteria were diluted with assay buffer (DPBS containing 1% BSA and 0.1% glucose) at a working dilution rate (about 10 4 CFU / mL). The total volume in each well was 50 μL, with 25 μL of serial two-fold dilutions of test serum, 12.5 μL of bacteria at the working dilution rate, and 12.5 μL of the complement source. Test sera were pooled and heat-inactivated at 56 °C for 30 minutes. Negative controls included complement sera without test sera and complement sera with test sera, as well as heat-inactivated complement, along with bacteria incubated separately. Immediately after the addition of infant rabbit complement, the negative controls were plated onto Mueller-Hinton agar plates using the pour plate method (time 0). The microtiter plates were incubated at 37 °C for 1 hour, and then each sample was spotted in duplicate onto Mueller-Hinton agar plates, with the controls plated using the pour plate method (time 1). The agar plates were incubated overnight at 37 °C, and the colonies (viable bacteria) corresponding to time 0 and time 1 were counted. Serum bactericidal titers were defined as the serum dilution at which colony-forming units (CFU) / mL decreased by 50% compared to the control CFU / mL at time 0 after incubating the bacteria in the reaction mixture for 60 minutes. Typically, bacteria incubated without test serum in the presence of complement (negative control) showed a 150 - 200% increase in CFU / mL during the 60-minute incubation period. The reference strain for Neisseria meningitidis serogroup A was F8238.

[0253] The results reported in Figure 7 and Table 4 show the ability of anti-MenA antibodies to be bactericidal against MenA strains. In particular, native MenA-CRM 197 vaccines and vaccines obtained with random O-acetylated synthetic carbamoyl analogs (Groups 2 and 3) were able to maintain significant bactericidal activity even when tested with human complement. Figure 7 depicts the SBA titers after two and three administrations obtained with rabbit (rSBA) and human (hSBA) complement.

[0254]

Table 4

[0255] Figure 8 shows the human complement-mediated serum bactericidal titers induced by the selective 3-O-acetylated carbamoyl MenA DP8 and random acetylated carbamoyl MenA DP8 of the present invention after three administrations. Still, the MenA-CRM 197 vaccine was the positive control. 197 The SBA titers induced by the random O-acetylated carbamoyl MenA-CRM

[0256] conjugates were statistically equivalent to the MenA vaccine benchmark after three administrations, but the 3O-acetylated carbamoyl MenA-CRM 197 conjugates induced significantly lower SBA titers in serum compared to the vaccine benchmark, and these were measured with both neonatal rabbit complement and human complement. 197 Statistical methods

[0257] For the data obtained from ELISA, a non-parametric t-test was performed, and the Mann-Whitney was performed on two subject groups (CRM -MenA avDP15 and CRM 197 -MenA avDP15 and CRM 197GraphPad software was applied and run to compare the ranks between MenA DP6OAc or DP8OAc). ELISA data were reported as geometric mean with CI95%. Furthermore, as fixed effects, groups (all except 4 and 5), time, and log of group / time interaction, etc. 10 An analysis of variance (ANOVA) model was fitted to the antibody titers. A heteroscedastic model was used because equal variance was not assumed between groups. For each endpoint, this model was used to estimate the geometric mean of the groups and its 95% CI, as well as the geometric mean ratio (O-acetylated formulation vs. benchmark) and 95% CI. In contrast, for the SBA data, since there was a single observation for each group at each time point (pool of sera), only graphical analysis was performed.

[0258] Protocol for the quantification of hydrolyzed MenA and carbMenA oligomers in the final conjugate The amount of monomers released over time from the MenA and carbMenA conjugates of the present invention was quantified using HPAEC-PAD. The titers reported in Figure 9 were obtained by hydrolyzing the samples in an oven at 110 °C for 2 hours with 6 M HCl at the final concentration. After incubation, the samples were dried in a Speedvac system, redissolved in water, and filtered through 0.45 μm. Quantification was performed by NMR using a standard curve prepared in the range of 0.5 - 5.0 μg / mL with carbMenA DP7 treated as a sample. The analysis was performed on an ICS5000 system (Dionex - Thermo Fisher) equipped with a guarded CarboPac PA1 column. Elution was carried out using a sodium acetate gradient in the presence of 100 mM sodium hydroxide at 1.0 mL / min, and peaks were detected by pulsed amperometric detection using a quadruple waveform for carbohydrates. The results were generated using Chromeleon (trademark) 7.2 chromatography data system (CDS) software. Conclusion Based on the obtained data, it can be concluded that the MenA oligomers of the present invention can be used to develop a more stable version of the MenA vaccine, and that the OAc moiety in combination with the oligomer length is key in inducing a functional immune response against MenA strains.

Claims

1. An oligomer of formula (Ia) or (Ib). 【Chemistry 1】 [In the formula, n is ≧6; R is H or -P(O)(OR"). ２ wherein R″ is H or a pharmaceutically acceptable phosphate counterion; R' is H or a pharmaceutically acceptable phosphate counterion; R ｘ is H or -C(O)CH ３ may be the same or different in each repeat unit; R ｙ is H or -C(O)CH ３ may be the same or different in each repeat unit; R ｘ or R ｙ At least one of the repeating units is —C(O)CH ３ In summary, R in the oligomer ｘ and R ｙ Approximately 50 to 90% of the ３ and Az is —NH(CO)R １ , -N(R １ ) ２ and -N ３ and R is an aza substituent selected from the group consisting of １ is H, linear or branched C １ -C ６ - alkyl and linear or branched C １ -C ６ -haloalkyl; Z is (i) a protecting group; (ii) a functional linker for conjugation to a protein, or (iii) Linear or branched C １ -C ６ alkyl, optionally substituted phenyl, —C(O)Y, or straight or branched C １ -C ６ -Alkyl-X and Y is H, straight or branched C １ -C ６ - alkyl or a protecting group, X is -NH ２ , -N ３ , -C≡CH, -CH=CH ２ , —SH, or —SC≡N.]

2. 2. The oligomer of claim 1 defined by formula (Ia):

3. 3. The oligomer of claim 1 or claim 2, wherein n is 8.

4. 3. The oligomer according to claim 1, wherein n is 8 to 15.

5. Az is -NHC(O)CH ３ The oligomer according to any one of claims 1 to 4,

6. R ｘ and R ｙ are both —C(O)CH in 40-50% of the repeat units of the oligomer. ３ The oligomer according to any one of claims 1 to 5,

7. In 10 to 30% of the remaining repeat units of the oligomer, R ｘ or R ｙ One of the is -C(O)CH ３ and the remaining repeat units in the oligomer are R ｘ =R ｙ 7. The oligomer of claim 6 having =H.

8. The oligomer according to any one of claims 1 to 7 for use as a vaccine.

9. The oligomer according to any one of claims 1 to 7, for use in preparing an antigen conjugated with a protein.

10. 10. The oligomer according to claim 9 for use in preparing a conjugated antigen of formula (IIa) or (IIb): 【Chemistry 2】 [In the formula, n, R, R', R ｘ and R ｙ is as defined in any one of claims 1 to 7; Z is a linker or a bond; P is diphtheria toxoid (DT), tetanus toxoid (TT), CRM １９７ , an inactivated bacterial toxin selected from Escherichia coli ST and Pseudomonas aeruginosa exotoxin (rEPA); P is a polyamino acid such as poly(lysine:glutamic acid); P is hepatitis B virus core protein or SPR96-2021, or meningococcal serogroup B antigen fHbp-231.

11. The P is CRM １９７ 11. The oligomer according to claim 10, for use in preparing a conjugate antigen of formula (IIa) or (IIb), wherein:

12. 12. The oligomer of claim 10 or 11 for use in preparing a conjugated antigen having the structure: 【Transformation 3】 [In the formula, n, R, R ｘ and R ｙ is defined in any one of claims 1 to 7.

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