JP2007532512A - Tripartite complex containing structures interacting with cell membrane rafts and uses thereof - Google Patents

Abstract

The present invention relates to the case where moiety A and moiety A ′ are raft affinity moieties, moiety B and moiety B ′ are linkers, moiety C and moiety C ′ are pharmacophores, and moiety B and moiety B ′ are A linker having a skeleton of at least 8 carbon atoms, wherein one or more of the carbon atoms may be substituted with nitrogen, oxygen or sulfur, of the CBA or C′-B′-A ′ type It relates to compounds containing a tripartite structure. In addition, certain pharmaceutical uses of the compounds of the invention are disclosed.

Description

  The present invention relates to the case where moiety A and moiety A ′ are raft affinity moieties, moiety B and moiety B ′ are linkers, moiety C and moiety C ′ are pharmacophores, and moiety B and moiety B ′ are A linker having a skeleton of at least 8 carbon atoms, wherein one or more of the carbon atoms may be substituted with nitrogen, oxygen or sulfur, of the CBA or C′-B′-A ′ type It relates to compounds containing a tripartite structure. In addition, certain pharmaceutical uses of the compounds of the invention are disclosed.

The lipid bilayer that forms the cell membrane is a two-dimensional liquid whose organization has been the object of thorough investigation by biochemists and biophysicists for decades. Although the bilayer bulk is considered to be a homogeneous fluid, repeated attempts have been made to introduce lateral heterogeneity, lipid microdomains into our bilayer liquid structure and kinetic model. (Glaser, Curr. Opin. Struct. Biol. 3 (1993), 475-481; Jacobson, Comments Mol. Cell Biophys. 8 (1992), 1-144; Jain, Adv. Lipid Res. 15 (1977). ), 1-60; Vaz, Curr. Opin. Struct. Biol. 3 (1993)). The understanding that epithelial cells polarize their cell surface into apical and basolateral with different protein and lipid compositions in each part has led to new developments that lead to the concept of “lipid rafts” (Simons Biochemistry 27 (1988), 6197-6202; Simons, Nature 387 (1997), 569-572). The concept of the assembly of sphingolipids and cholesterol that function as a membrane protein platform is facilitated by the observation that these assemblies survive surfactant extraction, and the surfactant-resistant membrane DRM (Brown, Cell 68 (1992), 533- 544). This was a dramatic breakthrough in operations in which the raft association was regarded as equivalent to resistance to Triton-X100 extraction at 4 ° C. Depletion of cholesterol using methyl-β-cyclodextrin resulting in loss of surfactant resistance (Ilangumaran, Biochem. J. 335 (1998), 433-440; Scheiffele, Embo J. 16 (1997), 5501-5508) With the addition of this second criterion, several groups in the field have turned to exploring the role of lipid microdomains in a wide range of biological reactions. There is now a lot of support for the role of lipid aggregates in regulating many cellular processes such as cell polarity, protein transport and signal transduction. The cell membrane is a two-dimensional liquid. Thus, lateral inhomogeneity means liquid-liquid immiscibility at the membrane plane. It is well known that hydrated lipid bilayers undergo a phase transition as a function of temperature. These transitions that occur at a fixed temperature for each lipid type always involve several changes in the order of the system. The most important of these transitions is the so-called “main” or “chain melting” transition where the bilayer is converted from a highly ordered quasi-two-dimensional crystal solid to a quasi-two-dimensional liquid. It involves dramatic changes in the order of the system, in particular the translational (positional) order in the bilayer plane and the lipid chain conformation order perpendicular to this plane. The translation order is related to the lateral diffusion coefficient in the plane of the membrane, and the conformation order is related to the trans / Gauche ratio in the acyl chain. The main transition is described as an order-disorder phase transition in which the two phases can be labeled as an ordered solid (s _o ) below the transition temperature and a disordered liquid (l _d ) above the transition temperature. Cholesterol and phospholipids are capable of forming an ordered liquid phase (l _o) that can tolerate phase coexistence in a perfectly liquid phase film by coexisting with a disordered liquid (l _d ) phase that is poor in cholesterol (Ipsen Biochem. Biophys. Acta 905 (1987) 162-172; Ipsen, Biophys. J. 56 (1989), 661-667). As a result of its flat and strict molecular structure that can force conformation order to adjacent aliphatic chains, sterols dramatically increase the translational mobility of the corresponding lipid when the sterol is the closest neighbor to the chain. (Nielsen, Phys. Rev. E. Stat. Phys. Plasmas Fluids Relat. Interdiscip. Topics 59 (1999), 5790-5803), and so on (Sankaram, Biochemistry 29 (1990), 10676). −10684). Due to the fact that the sterol is s _o (gel) does not fit exactly into the lipid bilayer of the crystal lattice, if it dissolves in this phase, it is not possible to significantly disrupting the conformation order, crystal translated sequence Would be confusing. Therefore, cholesterol in the appropriate mole fraction can be converted to l _d or s _o lipid bilayer phase ordered liquid (l _o) phase.

Rafts are lipid platforms of specific chemical components (rich in sphingomyelin and cholesterol in the outer leaves of the cell membrane) that function to sequester membrane components within the cell membrane. Rafts are understood to be relatively small (diameter 30-50 nm, size estimates vary significantly depending on the probe used and the cell type assayed). The selectivity with respect to lipid composition implies a phase separation behavior in a heterogeneous model membrane system. In fact, many properties in chemical composition and surfactant solubility are identical to those observed in model systems consisting of unsaturated phosphatidylcholine, sphingomyelin (or long chain saturated phosphatidylcholine) and cholesterol ternary mixtures (de Almeida, Biophys. J. 85 (2003), 2406-2416). Rafts could be considered to be _lo phase domains in the heterogeneous l-phase lipid bilayers that make up the plasma membrane. The other coexisting phase (s) are not currently clear. Biomembrane has consensus that is liquid, thus, s _o phase coexistence may be ignored in most cases. Whether the other phase (s) is the l _d or l _o phase depends on the chemical identity of the phospholipids that make up this phase (s) and the mole fraction of cholesterol therein It depends on. Rafts can be considered equivalent to ordered liquid phases, and the rest of the film is referred to as the non-raft liquid phase. From a thermodynamic point of view, a phase is always a macroscopic system consisting of a large number of molecules. However, in lipid bilayers, phases often tend to be subdivided into small domains (often just a few thousand molecules), each of which has its own thermodynamic determination of the phase. There are not enough molecules to be strictly satisfied. In the absence of a better representation for this kind of mesoscopic state and the assumption that there are multiple domains in a particular system, the domain is part of the macroscopic phase, as if the same property was due to the domain representing the phase. That domain can be processed as if This definition is probably appropriate because the domain does not get too small. Thus, the ordered liquid raft phase contains all the domains (small or clustered) of the raft phase in the membrane. The liquid phase that is the remainder of the membrane surrounding the raft may be a homogeneous osmotic liquid phase or may be further subdivided into liquid domains that have not yet been characterized.

  The prior art hypothesizes that some pharmaceuticals are active on biological membranes such as cell membranes or viral envelopes. For example, it was self-evident that the anti-HIV agent cosalane acts by inhibiting gp120 binding to CD4, as well as by inhibiting post-binding events prior to reverse transcription; Cushman, J. Chem. 37 (1994), 3040. It is speculated that the cholestane portion of cosaran is embedded in the lipid bilayer, and “Golebiewski, Bioorg. & Med. Chemistry 4 (1996), 1637” obtained incorporation of a phosphate group into the cosaran linker. It is speculated that the resulting phosphodiester resembles the structure of polylipids.

  In "Ruell, J. Org. Chem. 64 (1999), 5858", research on cosaran compounds was expanded. In particular, it is suggested that cosaran pharmacophore analogs have short amide and methylene linkers attached to their terminally substituted benzoic acid rings instead of the originally proposed benzyl ether linkage. This study demonstrated that the proposed cosaran-type compound is accessible by a route that does not utilize cosaran itself as an intermediate or starting material, indicating an in vitro effect in inhibiting the cytopathic action of HIV-I . In “Casimiro-Garcia, J. Bioorg. Med. Biochem. 8 (2000), 191”, a cosaran analog in which an amide group or an amino moiety is introduced into the alkenyl-linker chain of cosalane is proposed. Again, cosaran analogs inhibited the cytopathic effects of HIV-1 and HIV-2 in vitro. In addition, cosaran analogs are known from US 5,439,899, US 6,562,805 and US 2003/0212045. All these cosaran compounds / analogs contain modifications in their linker structure. Furthermore, in particular the pharmacological part of cosaran was modified in this study. These modifications were made in an attempt to enhance the effectiveness of membrane incorporation, but decreased in all cases. Again, all known cosaran and cosaran analogs include structures that are not capable of distinguishing different biological membranes and / or capable of distributing different membranes. “2001, Hussey Organic Letters 4, 415-418” described the synthesis of chimeric estradiol derivatives conjugated to cholesterol and cholestyramine designed for delivery of estradiol to cells by internalization. Similarly, “Hussey, J. Am. Chem. Soc. 123 (2001), 1271-1273” proposes a synthetic streptavidin protein complex for intracellular delivery of macromolecules to mammalian cells. Yes. “Hussey, J. Am. Chem. Soc. 124 (2002), 6265-6273” includes additional synthetic molecules that allow cellular uptake of streptavidin through non-covalent interactions with cholesterol and sphingolipids. Described and lipid rafts are discussed. Corresponding compounds include derivatives of cholesterylamine linked to D-biotin via an 11 atom tether. “Martin, Bioconjugate Chem. 14 (2003), 67-74” proposes a non-natural cell surface receptor comprising a peptide capped with cholesterylamine. Ligands for these “non-natural receptors” are presumed to bind non-covalently to the peptide moiety, and proposed ligands include anti-HA, anti-Flag or streptavidin. Again, non-native cell membrane receptors have been proposed as delivery strategies for macromolecular uptake into cells.

The problem underlying the present invention is due to biochemical interactions or processes occurring in the sphingolipid / cholesterol microdomains of mammalian cells or compounds for medical / pharmaceutical intervention in disorders associated therewith, and It was the provision of a method.
The solution to this technical problem is achieved by providing the specific examples described in the claims.
Thus, the present invention provides that moiety A and moiety A ′ are raft affinity moieties, moiety B and moiety B ′ are linkers, moiety C and moiety C ′ are pharmacophores, and moiety A and moiety A A linker with a raft affinity of 'includes partitioning into lipid membranes characterized by insolubility to nonionic surfactants at 4 ° C, with moieties B and B' having a backbone of at least 8 carbon atoms Wherein one or more of the carbon atoms may be substituted with nitrogen, oxygen or sulfur, comprising a ternary structure: CBA or C′-B′-A ′.

  The term “ternary structure” relates to a compound comprising a covalently linked raft affinity moiety, a linker and a pharmacophore, and thus the individual parts of the tripartite structure are referred to herein as a raft affinity moiety. “Part A and Part A ′” for the linker, “Part B and Part B ′” for the linker and “Part C and Part C ′” for the specific pharmacophore. Furthermore, it should be noted that the “three-way structure” of the compounds of the present invention may also contain additional structural or functional moieties. These can be attached to the N- or C-terminus of the construct of the invention, or can be attached to the linkers “C” and “C ′”, eg via side chains (eg radiolabels, fluorescent labels). , Purification tags, etc.), but is not limited thereto. Additional functional or structural domains of the constructs of the present invention can be charged groups, polar groups capable of accepting or donating hydrogen bonds, lipophilic or to thermodynamically support the accumulation of the constructs of the present invention in lipid rafts. It contains non-covalently crosslinkable functional groups such as amphiphilic groups that mediate between the hydrophilic compartments and groups that can interact with each other. It is preferred that the further functional or structural domain does not bind directly to the pharmacophore moiety. Most preferably, the additional domain or moiety contacts the linker B / B ′ either directly or indirectly.

The term “raft affinity moiety” relates to a compound capable of interacting with membrane rafts. Rafts are known in the art, see especially Simons, (1988) (supra), or Danielson, Biochem. Biophys. 1617 (2003), 1-9. A “raft affinity moiety” includes not only natural compounds but also synthetic compounds described in detail below. The “raft-affinity moiety” included in the tripartite structure of the present invention has a high affinity for the ordered liquid (l _o ) phase (referred to herein as raft) of the membrane bilayer, and the disordered liquid (l _d ) More time is spent in this phase compared to the phase (meaning non-raft herein). Distribution to the raft occurs either directly from the extracellular or vesicle lumen space or laterally from the bilayer. Thus, one of the characteristics of “part A and part A ′” of the constructs of the present invention relates to its ability to partition into lipid membranes, preferably cellular lipid membranes, whereby the lipid membranes are non- Characterized by insolubility in ionic surfactants such as 1.0% Triton X-100, 0.5% Lubrol WX or 0.5% Brij 96. The characteristics of “portion A and portion A ′” correspond to the fact that “raft affinity moieties” have the ability to insert into or interact with sphingolipid and cholesterol rich microdomains. Thus, as defined above and as taught by Simons (1988, 1997), (supra) and Brown (1992), (supra), rafts are (nonionic) surfactant resistant. It can be defined as a membrane (DRM) structure. Thus, whether a particular compound (having a tripartite structure as defined herein) comprises a “moiety A” or “moiety A ′” as defined herein or a particular molecule herein One possibility to demonstrate whether it can function as a defined "Part A / Part A '" is disclosed in the prior art and is surfactant resistant as detailed in the experimental part of this specification. It is a membrane (DRM) test. In summary, the accumulation of test compounds in cell membrane fractions derived from non-raft and raft membranes is determined in a DRM assay. The test system includes treating cultured cells with a test compound. Following incubation, the cells are lysed in detergent solution and the DRM fraction (raft) is isolated on a sucrose gradient. The DRM fraction is collected and the test compound is measured by fluorescence analysis or quantitative mass spectrometry. Raft affinity is determined as the proportion of test compound recovered in the DRM fraction compared to the amount of total membrane. A better comparison of the results of the different experiments is achieved by comparing the raft affinity of the test compound with that of a known raft affinity standard. Corresponding examples include cholesteryl 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-dodecanoate (cholesteryl BODIPY® FL C ₁₂ , Molecular Probes (Eugene , US)) or [ ³ H] cholesterol. More specifically, the DRM test is performed as follows. Cultured cells are incubated with the test compound, for example at 37 ° C. for 1 hour, then the cells are washed and extracted with cold detergent, usually cold (4 ° C.) 1% Triton X-100. The cell lysate is centrifuged through a sucrose density gradient to create a floating layer containing a surfactant resistant membrane. For the purpose of determining raft affinity, they can be identical to the raft. Rafts and other materials are collected and analyzed by, for example, mass spectrometry or fluorescence analysis (if the test compound is fluorescent) to determine the amount of test compound in each raft. The relative abundance in the raft (raft affinity) is then calculated. Corresponding examples are provided in the experimental part.

For example, raft substituent lipids such as cholesterol (sterols), sphingolipids (ceramides), GPI anchors or saturated fatty acids can be considered to be natural raft affinity moieties. In particular, as provided herein, derivatization of these types of compounds is not presumed to interfere with their association with rafts, as determined by raft assays, and is at least the same as the parent compound. Strong to the extent.
As discussed above, examples of such natural raft affinity moieties are derivatives of cholesterol having functional groups attached to hydroxyl groups, sterol rings or side chains. Further, a corresponding example is as described later.

  The linker (B / B ′) links the raft affinity moiety (A / A ′) and the pharmacophore (C / C ′). The raft affinity moiety and the precursor of the linker contain functional groups that allow covalent bonding between them. The nature of the functional group is not particularly limited, and corresponding examples are as described later in this specification. The functional group of the raft affinity moiety (A / A ′) used to covalently attach the raft affinity moiety (A / A ′) to the linker (B / B ′) is referred to herein as “hook Also called. The chemical structure of these hooks is not particularly limited and the only requirement is that the hooks do not interfere with the dissection of the tripartite into the raft. In a preferred embodiment, the raft affinity moiety (A / A ′) raft affinity and the raft affinity of the ternary structure of the invention are enhanced by appropriate selection of hooks. The effect of hooks on the raft affinity of the raft affinity moiety (A / A ′) is demonstrated in the Examples section below.

In the case of the ternary structure C′-B′-A ′, the raft affinity moiety A ′ is attached to the nucleophilic group on the linker B ′, ie its N-terminus. As can be clearly seen from the specific structure described below, the N-terminus of the linker does not necessarily contain a nitrogen atom, and may contain an oxygen atom, such as a linker 22 in which X ²²¹ is oxygen. Examples of hooks that can be used to attach raft affinity moiety A ′ to the N-terminus of linker B ′ are succinyl and acetyl groups, where the N-terminus of linker B ′ is the succinyl or acetyl group. Binds to the carbonyl group. Particularly preferred is a hook comprising an ether bond such as an acetyl group which is bonded to the oxygen atom of the raft affinity moiety A ′ via the α carbon atom of the acetyl group. Suitable amino acid hooks in the raft affinity moiety A ′ are aspartic acid and glutamic acid, where the amino acid residue is attached to the raft affinity moiety via the side chain carboxylic acid group of the amino acid residue, and the linker is To the α-carboxylic acid group of the same amino acid residue. The α-amino group of the amino acid residue can be protected, for example, as its acetate.

In the case of the ternary structure C-B-A, the raft affinity moiety A is attached to the electrophilic group on linker B, ie its C-terminus. As can be clearly seen from the specific structures described below, the C-terminus of the linker does not necessarily have to be a C═O group (eg, as in linkers 20, 21, and 22), for example, a sulfonyl (SO ₂ ) group. (Eg, linker 24). Raft affinity moiety A can be directly coupled to the C-terminus of linker B by use of a terminal heteroatom of raft affinity moiety A. Alternatively, if direct coupling is not appropriate or feasible, for example, an amino acid can be used as a hook to attach raft affinity moiety A to linker B: eg, raft affinity Portion A can be coupled to the ε-amino acid of the lysine residue, and the C-terminus of linker B can be coupled to the α-amino group of the same lysine residue. Other suitable amino acid hooks on the raft affinity moiety A are aspartic acid and glutamic acid, where the amino acid residue binds to the raft affinity moiety via the side chain carboxylic acid group of the amino acid residue, The linker is attached to the α-amino group of the same amino acid residue. In the amino acid hook, the alpha carboxylic acid group can be protected, for example, as a primary amide.

A synthetic raft affinity moiety is a moiety or precursor thereof that has a high affinity for rafts but is not an analog or derivative of a natural raft lipid substituent. Again, examples of such synthetic raft affinity moieties are provided herein.
As pointed out above, the tendency of compounds to partition from aqueous phase into raft domains or laterally separate from surrounding non-raft bulk lipids into raft domains allows for efficient accumulation or packing of compounds with raft lipids. Be in the particular nature of its structure. In particular, raft affinity is determined by the compound's interaction with the lipid component of the raft or the transmembrane portion of the raft-associated membrane protein, in particular the DRM assay outlined above or the LRA described below and in the examples. As determined by the assays provided herein.

  Features related to raft affinity include, alone or in combination, the hydrophobicity and degree of branching of chains containing hydrocarbon or transunsaturation, the ability of hydrogen bonding at the top of the raft, as demonstrated by sphingolipids and cholesterol, An almost flat carbocyclic structure, a multi-hydrocarbon chain, a structure that efficiently packs with sphingolipids and cholesterol, and a structure that is thermodynamically advantageous.

In a preferred raft affinity moiety “A / A ′”, the hydrocarbon chain uses the full length corresponding to the hydrocarbon chain found in the natural components of rafts such as sphingolipids and cholesterol. For example, a hydrocarbon chain having a length of about 8-12 carbon atoms in the portion represented by Formulas 2 and 3 below is preferred. In the moieties represented by formulas 4a and 5a below, hydrocarbon chains having a length of about 18-24 carbon atoms are preferred. In addition, efficient packing with sphingolipids and cholesterol in rafts is facilitated by selecting saturated linear hydrocarbon chains. In the raft affinity moiety “A / A ′” having one or more long chain substituents, the difference in the number of carbon atoms between the long chain substituents is preferably 4 or less, more preferably 2 or less. . For example, if the raft affinity moiety “A / A ′” has a first long chain substituent that is a straight chain C ₁₈ alkyl group, the second long chain substituent is a straight chain C _14-22 alkyl group. Preferably, it is a straight chain C _16-20 alkyl group. By minimizing the difference in the number of carbon atoms between two or more long chain substituents on the raft affinity moiety “A / A ′”, the overall cone of this part can be avoided, The disraft effect of “A / A ′” on the raft affinity moiety incorporated into the raft can be minimized.
Certain structural features are excluded from the raft and therefore cannot be included in the raft affinity moiety. Such features include hydrocarbon chains with cis-unsaturation (eg, dioleyl phosphatidylcholine), orthogonal heterocyclic structures and nucleosides.

  The tendency of a compound to partition into a raft domain is determined in an assay that measures the concentration of the compound in the raft and non-raft domains after a certain incubation time with the lipid membrane system under consideration. In addition to the DRM tests discussed above, a liposomal raft affinity assay (LRA) can be used. Briefly, monolayer liposomes composed of non-raft lipids (such as phosphatidylcholine and phosphatidylethanolamine) or liposomes composed of raft lipids (such as sphingolipid, phosphatidylcholine and cholesterol) are converted into the tripartite compound of the present invention or the present invention. Incubate in aqueous suspension, for example, at 37 ° C. for 1 hour with a test compound such as a precursor of the moiety predicted to be capable of functioning as “Part A / A ′” of the inventive tripartite compound . Fractions are separated and the amount of each test compound is measured. For each liposome type, the lipophilicity value is determined from the amount of test compound incorporated into the liposome. Raft affinity is defined as the ratio of lipophilicity for raft to non-raft liposomes in a particular compound. Again, a corresponding example is shown in the experimental part. Furthermore, those skilled in the art can easily perform LRA by performing the protocol summarized below. The lipophilicity of the compound is measured in particular by the LRA. For each liposome type (raft or non-raft), lipophilicity is between the aqueous phase (ie, concentration in the supernatant) versus the lipid layer (raft or non-raft) (ie, concentration in the lipids that make up the liposome). Defined as distribution.

The test system includes three components in which the test compound is found: a lipid membrane, an aqueous supernatant, and a test tube wall. Following incubation, the liposomes are removed from the system and the test compound in the aqueous and test tube wall fractions is measured by fluorometry or quantitative mass spectrometry. Data is computerized to obtain partition coefficients and raft affinities.
Thus, LRA as described herein and also known in the art is defined as raft affinity of a compound comprising the tripartite structure described herein or defined herein and utilized in the compounds of the present invention. Further test systems are provided to elucidate the raft affinity of the precursors of “Part A” as well as “Part A ′”.

  In the present invention, it should be noted that those skilled in the art can easily prepare liposomes. These liposomes include the aforementioned “raft” liposomes as well as “mixed” and “non-raft” liposomes. Corresponding lipids are known in the art. A “liposome-forming lipid” has a hydrophobic and polar head group portion and can (a) spontaneously form bilayer vesicles in water, such as phospholipids, or (b) a bilayer membrane Means an amphiphilic lipid that is stably incorporated into a lipid bilayer having a hydrophobic portion in contact with the inner hydrophobic region of the membrane and a polar head group portion directed toward the outer polar surface of the membrane. This type of liposome-forming lipid typically contains one or two hydrophobic acyl hydrocarbon chains or steroid groups, and a chemically reactive group such as an amine, acid, ester, aldehyde or alcohol in the polar head group. Can be included. This type of lipid includes phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI) and sphingomyelin (SM), where two hydrocarbons The chains are typically about 14-22 carbon atoms long and have varying degrees of unsaturation. Raft lipids are defined herein.

  The term “linker (linker structure)” used in the tripartite structure of the present invention is used to link the raft affinity moiety A or A ′ to the pharmacophore C or C ′. These subunits should not compete in terms of raft affinity by the raft affinity moiety A or A ′ nor in terms of drug activity by the pharmacophore C or C ′. In accordance with the present invention, the linker rather provides covalent attachment of the raft affinity moiety to the pharmacophore, allowing the raft affinity moiety to perform its functions such as enrichment and anchoring in lipid rafts, for example. And provides an ideal distance between the raft affinity moiety and the pharmacophore to allow the pharmacophore to perform its function, such as inhibition of the enzyme. For this purpose, the length of the linker is adapted to the situation from case to case by means of modular assembly.

The subunits of the linker (part B and part B ′) can be amino acids, derivatized or functionalized amino acids, polyethers, ureas, carbamates, sulfonates or meet the above requirements, ie, raft affinity moieties (“parts Other subunits that provide the distance between A and portion A ′ ”) and the pharmacophore (“ portion C and portion C ′ ”).
As noted above, moiety B and moiety B ′ are linkers having at least 8 carbon atom (C) skeletons, wherein one or more of the carbon atoms are nitrogen (N), oxygen ( O) or sulfur (S) may be substituted. Preferably the skeleton has at least 8 and at most 390 atoms, more preferably the skeleton has at least 9 and at most 385 atoms, and the skeleton has at least 10 and at most Even more preferably, it has 320 atoms.

If the linker B or B ′ comprises a sequence of α or β amino acids covalently linked, it is preferred that there are at least 9 and at most 320 atoms mentioned above in the backbone, more preferably 10-80 A linker composed of an amino acid having a skeleton having 20 to 70, more preferably 30 to 65, and most preferably 34 to 60 carbon atoms is more preferable. When the linker B or B ′ includes a sequence of polyether (amino acid having a polyether skeleton), the linker preferably has 9 to 285 atoms in the skeleton. When the linker B or B ′ contains urea, the preferred number of atoms in the skeleton is 10 to 381. The backbone structure made of carbamate has 10 to 381 atoms in part B or part B ′, and the linker part B or part B ′ consisting of the sulfonamide has at least 8 and at most 339 atoms Is preferred.
Accordingly, the total length of portion B or portion B ′ is 1 nm to 50 nm, more preferably 5 to 40 nm, more preferably 8 nm to 30 nm and most preferably 10 nm to 25 nm.

Structures / portions as defined herein, particularly linker lengths, are known in the art such as (but not limited to) molecular modeling (preferably using standard software such as Hyperchem®) And can be determined by a known method. Furthermore, the corresponding length (or the distance between part A / A ′ and part C / C ′) can also be estimated by crystal analysis methods, in particular X-ray crystal analysis. Such X-ray crystal analysis are known in the art [particularly, McRee (1999), Practical Protein Crystallography, 2 nd edition, see "X-ray chrystallography methods and interpretation" in the Academic Press], Corresponding information is also available on the Internet [especially available from the National Center for Biotechnology Information, US National Library of Medicine at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi See X-ray crystallographic structure and protein / peptide sequence information].

One function of the linker is to link the raft affinity moiety to the pharmacophore (such as an inhibitor) in such a way that the raft affinity moiety can accumulate in the bilayer lipid raft subcompartment (raft). Can bind to and / or interact with a specific site of action (eg, an inhibitor binding site and / or interaction site) in a target molecule.
The linker corresponds at least to the length of the backbone structure having at least 8 carbon atoms, the phosphoryl head group of the raft lipid or other equivalent head group and a pharmacophore (preferably inhibitor) linkage in the target molecule and Select to have a length corresponding to the distance between the interaction sites. The binding and / or initiation site is a site targeted by the virus to bind to a natural ligand binding site such as an enzyme active site, protein-protein docking site, ligand receptor binding site or cell membrane protein. Furthermore, the present invention is not limited to the target molecules / sites described above. The length of the linker can be determined by information and methods known in the art such as X-ray crystallography, molecular modeling or screening with different linker lengths.

An example of how to determine the length of the linker is obtained by considering the BACE-1 β-secretase protein. Examples are also provided in the experimental part. The distance between the transmembrane sequence and the cleavage site of the BACE-1 substrate amyloid-precursor protein (APP) is known to be 29 amino acids (De Strooper, B., Annaert, W. (2000) Proteolytic processing and cell biological functions of the amyloid precursor protein, J. Cell Sci. 113, 1857-70). Given a simple α-helix conformation, this is, in this particular example, 29 amino acids in length or 10 nm to bridge the distance between the raft affinity moiety and the inhibitor binding site. A linker is meant to be necessary.
Inhibitor III is a known inhibitor of the BACE-1 β-secretase protein. Inhibitor III sequence (Capell, J. Biol. Chem. 277 (2002), 5637; Tung, J. Med. Chem. 45 (2002), 259) replaces the first β-cleavage bond with a non-hydrolyzable statin , Containing 4 or more residues at the C-terminus. 24 additional amino acids from inhibitor III to the membrane are required. These amino acids correspond to the linkers defined for delivery of inhibitor III to rafts in the examples described herein for the tripartite compounds herein.

Thus, a suitable ternary structure of the present invention is the pharmacophore-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala- Glu-Asp-Val-Gly-Ser-Asn-Lys-raft affinity moiety, where pharmacophore is, for example, Glu-Val-Asn-Sta-Val-Ala-Glu-Phe (Sta is a statin Is). For example, cholesteryl glycolic acid can be used as a raft affinity moiety. See also the compound of formula 24 below.
Alternatively, the 10 nm distance in the above example can be bridged by a linker containing an appropriate number of polyethylene glycol units. See also compounds of formulas 25 and 25b below, in particular 25b. This type of linker is particularly preferred as it increases the solubility of the tripartite structure in aqueous media.

  Since the target of the raft affinity moiety-linked inhibitor is likely an enzyme or receptor with a membrane proximal inhibitor-binding site, the range of lengths expected to be bridged by the linker is Thus, 1 nm to 50 nm, preferably 8 to 30 nm. 1 nm to 50 nm corresponds to about 8 to 390 carbon atoms in the skeleton. One skilled in the art will know that the length of a linker as defined herein is determined not only by its primary structure, but also by its secondary structure (eg, peptide linker α-helix and / or β-sheet). Take that into account. In addition, some natural amino acids such as Pro, Met, Cys, etc. may be included in the linker, but they can induce a turn in the shape of the linker construct, so Appropriateness as a block is considered low. This can lead to reduced flexibility or sensitivity to oxidation during in vitro synthesis. Thus, in view of the above, a 50 nm long (peptide) linker does not necessarily contain only about 80 amino acids, but may contain more amino acids.

  In the examples and preferred embodiments of the invention, the bridged range is a polypeptide length of 3 to 80 amino acids corresponding to 9 to 240 carbon atoms or of 3 to 95 (ethylene) glycol units. Corresponds to polyglycol length. However, it is preferred that the linker comprises at least 3, more preferably at least 10, even more preferably at least 15 amino acids or (ethylene) glycol units. Most preferred are linkers consisting of 15-30 amino acids or (ethylene) glycol units. However, the present invention is not limited to linkers consisting of amino acids or (ethylene) glycol units. It should be noted that the upper limit of 80 units mentioned above does not limit the inventive construct. Longer linkers containing more than 80 units are expected. As pointed out above, the corresponding distance is the phosphoryl head group or the corresponding head group contained in the raft lipid as described above and below and the pharmacophore (preferably inhibitor molecule) binding and / or interaction site. Should be defined by the distance / length between.

  Accordingly, the linker of the present invention preferably comprises 3 to 80 or more amino acids, where the amino acids are α and β amino acids (eg, His, Arg, Lys, Phe, Leu, Ala, Asn, Natural amino acids such as Val, Gly, Ser, Gln, Tyr, Asp, Glu, Thr and β-Ala) and one amino acid side chain (eg Glu or Lys) can be assayed (Dye) labels for detection in (such as rhodamine or synthetically modified derivatives thereof) or other labels known in the art. Possible compounds of the invention include, for example, their three-part structure, but also include additional functional groups, ie additional labels.

  Other functions of the linker are to keep pharmacophores, such as inhibitors, away from the hydrophobic lipid bilayer and to improve the solubility of the entire compound in aqueous media. Thus, the linker is most preferably polar. This is accomplished through the use of amphiphilic subunits or the introduction of polar functional groups into the linker. As one example, the introduction of one or more arginine residues into a polypeptide linker increases polarity and solubility. In one preferred embodiment, the linker comprises polyethylene glycol units known to increase solubility in aqueous media.

  Another linker function that is expected is that the raft affinity moiety can itself be optimally located for accumulation into the raft, and because the pharmacophore interacts with the inhibitor binding site It also allows for lateral movement of the raft affinity moiety in the lipid bilayer and rotational movement of the raft affinity moiety and pharmacophore so that it can be optimally located by itself.

  The aforementioned biological assays, such as DRM and LRA provided herein, allow the binding of fluorescent, radioactive or dye labels (eg, fluorescein, Mca, rhodamine B or synthetically modified derivatives thereof) to the compounds of the invention. Will be advantageous. It is preferred that the label is attached to the linker structure. A label is covalently attached to the linker (eg, on the side chain of an amino acid such as glutamic acid or lysine) in order to keep the raft affinity moiety and the respective interaction of the pharmacophore and its surroundings undisturbed. ). Thus, if necessary, labeling for detection can be another linker function. However, the (detectable) label may be part of the “part A / A ′” or “part C / C ′” of the tripartite compound of the invention.

  A linker may comprise subunits that can be referred to as linker building blocks (or units) of the linker. They are specifically described below and can contain a carboxylic acid or sulfonic acid functional group (referred to as “acceptor end”) at one end and an amino or hydroxy functional group (“donor end”) at the other end. be able to. Depending on the choice of synthetic route and the type of pharmacophore used, the pharmacophore can be attached to the donor end of the linker via a carboxyl group (eg, the C-terminus of the inhibitor peptide) and has a raft affinity The moiety may be, for example, a linker, either via a lysine unit that couples by a ε-amino group to the carboxy terminus of the heteroatom or raft affinity moiety and by its α-amino function to the acceptor terminus of the linker building block. To the acceptor end of

In the present invention, the term “pharmacophore” relates to the covalently linked active site contained in the ternary compound of the present invention, whereby the pharmacophore is molecular and / or biogenic that occurs in rafts. Inhibitory units that are capable of interfering with chemical processes are preferred.
Apart from the active moiety, the pharmacophore also includes a hook moiety (eg, succinyl, acetyl) that binds to the linker. Furthermore, a dye label, preferably a fluorescent dye label such as rhodamine, Mca, fluorescein or synthetically modified derivatives thereof, may be attached to the pharmacophore.

A pharmacophore may be, among other things, a small molecule drug with specificity for a binding site (eg, enzyme active site, protein-protein docking site, ligand-receptor binding site or viral protein attachment site, etc.). Furthermore, the pharmacophore may also be a peptidomimetic or a peptide transition state inhibitor or a polypeptide or (nucleic acid) aptamer. As detailed below, one example of a peptide transition state inhibitor is a commercially available β-secretase inhibitor III (Glu-Val-Asn-Sta-Val-Ala-) that inhibits BACE-1 cleavage of APP at the β-cleavage site. Glu-Phe-CONH ₂ , where Sta is a statin) (Calbiochem). Other examples include EGF receptor (heregulin) inhibitor A30, nucleic acid (RNA) aptamer (Chen, Proc. Natl. Acad. Sci. (USA) 100 (2003), 9226-31) or anti-EGF receptor blocking (monoclonal). ) An antibody (eg, trastuzumab (Herceptin), etc.). It is also envisaged that rifamycin analogs (see US 6,143,740) may be used in the present invention as small molecule EGF receptor antagonists. Furthermore, an anti-HER2 / novel peptidomimetic (AHNP) small molecule inhibitor (Park, Nat. Biotech. 18 (2000), 1948) may be a pharmacophore used for the compound of the present invention. It is further envisaged to use influenza virus neuraminidase inhibitors such as zanamivir (relenza) and oseltamivir (Tamiflu) that bind to the active site of neuraminidase.

The primary target for a pharmacophore, preferably an inhibitor, is a protein whose (inhibitor) binding site is accessible to the raft affinity moiety-linker-pharmacophore compound of the invention. Thus, these are, for example, proteins located in rafts or moved to rafts to perform functions.
The pharmacophore interaction sites on such target proteins are usually phosphoryl head groups or other equivalents of raft lipids in the extracellular space in the case of the plasma membrane or in the luminal space in the case of the vesicle membrane. 1 nm to 50 nm from the head group.

  In accordance with the present invention, surprisingly, the novel compounds described herein and having the tripartite structure described above occur in certain pharmacophores, particularly in / on the plasma membrane and / or vesicular raft Inhibitors of biological / biochemical processes) have been found to be capable of linking to the corresponding target. Of particular importance to the present invention is only to provide an accurate distance between the head group of the raft lipid and the binding and / or interaction site of the pharmacophore and its corresponding target molecule as defined herein. Rather, it is a linker structure that also provides unambiguous enrichment of the pharmacophore in the raft along with the raft affinity moiety “Part A / A ′”. In this context, it is important that the term “raft” as used herein is not limited to rafts on the plasma membrane of a cell, but also relates to the inner membrane and vesicular rafts. Enrichment of pharmacophores in rafts leads to an unexpected increase in potency over fold enrichment based on its concentration. Thus, if the raft affinity of the tripartite compound is, for example, 10, the increase in potency is on the order of 100. This is the result of an increased number of productive interactions between the pharmacophore and the target active site due to the longer residence time of the pharmacophore in the vicinity of the target.

In a more preferred embodiment of the invention, the tripartite compound comprises cholesterol and / or a functional component of cholesterol, a sphingolipid and / or a functional analog of sphingolipid, a lipid component such as glycolipid and glycerophospholipid. Contains "Part A / A '" capable of partitioning within the lipid membrane.
In the present invention, the cholesterol analog is ergosterol, 7-dihydrocholesterol or stigmasterol. Cholesterol analogs may be used in “rafts” to test the compounds of the invention. The preferred sphingolipid is sphingomyelin, the preferred sphingolipid analog is ceramide, the preferred glycolipid is ganglioside or cerebroside or globoside or sulfatide, the preferred glycerophospholipid is preferably saturated or monounsaturated (fatty acid acylated) phosphatidylcholine and phosphatidylethanolamine or phosphatidyl Serine.
“Functional analogs” of cholesterol or sphingolipid mean, inter alia, lipid structures containing structural features that allow the formation of corresponding steroids or rafts (Xu, J. Biol. Chem. 276, (2001) 33540. -33546, Wang, Biochemistry 43, (2004) 1010-8).

  In a more preferred embodiment of the tripartite compound of the invention, the lipid component (part A / A ′ is distributed) comprises a glycolipid that is a ganglioside or cerebroside. It is also envisaged that globoside is included in the lipid component. The lipid component is considered to be a “raft” lipid component as opposed to a “non-raft” lipid component. Thus, it is most preferred that the lipid component is rich in cholesterol and sphingolipids. Furthermore, as mentioned above, gangliosides may also be included. These gangliosides may be GM1, GD1a, GD1b, GD3, GM2, GM3, GQ1a or GQ1b, among others. These gangliosides are known in the art, see in particular Svennerholm, Asbury, Reisfeld, “Biological Function of Gangliosides”, Elsevier Science Ltd, 1994.

  The raft affinity and biological, biopharmaceutical and / or pharmaceutical properties of the compounds of the invention described above may be tested in vitro. The appended examples provide further indications for that. In addition, cholesterol in the range of 5-60% and / or functional analogues of cholesterol, sphingolipids in the range of 5-40% and / or functional analogues of sphingolipids and glycerophospholipids in the range of 20-80%, etc. Corresponding tests are preferably carried out with “rafts” containing the lipid components of Most preferably, the raft lipid membrane comprises cholesterol, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine and ganglioside (bovine brain, type III, Sigma-Aldrich Co.). Raft lipid membranes contain 40-60% cholesterol, 10-20% sphingomyelin, 10-20% phosphatidylcholine, 10-20% phosphatidylethanolamine and 1-10% More preferably, it includes a range of gangliosides. A good example and most preferred "artificial" raft comprises a lipid membrane consisting of 50% cholesterol, 15% sphingomyelin, 15% phosphatidylcholine, 15% phosphatidylethanolamine and 5% ganglioside.

Further, the lipid membrane used to test the compounds of the present invention and in particular the precursor of “Part A” of the tripartite compound comprises cholesterol, sphingolipid and / or functional analogues and phospholipids in equal parts. It is also assumed. For example, an “artificial” raft can also consist of a lipid membrane comprising 33% cholesterol, 33% sphingomyelin / ceramide and 33% phosphatidylcholine. Examples of “non-raft” lipid structures and liposomes are also given herein and in the appended examples.
The following are examples of the tripartite compound of the present invention. Particular examples of the compounds according to the invention are also given in the appended claims.

は、単結合または二重結合を表すために用い、

は、単結合、二重結合または三重結合を表すために用いる。 In the following equation:

Is used to represent a single bond or a double bond,

Is used to represent a single, double or triple bond.

  “Hydrocarbon” is used to represent a straight or branched, saturated or unsaturated, acyclic or cyclic, but non-aromatic group based on carbon and hydrogen atoms. The hydrocarbon group can also contain a combination of these groups. If necessary, some of the hydrogen atoms can be replaced with fluorine atoms. For example, hydrocarbon groups include, among others, alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, cycloalkenyl groups, alkylene-cycloalkyl groups, cycloalkylene-alkyl groups, alkylene-cycloalkenyl groups, and cycloalkenylene-alkyl groups. Can be mentioned. Cycloalkyl and cycloalkenylene groups preferably have from 3 to 8 carbon atoms in the ring. Cycloalkenyl and cycloalkenylene groups preferably have 5 to 8 carbon atoms in the ring.

The present invention is meant to include pharmaceutically acceptable salts of the compounds of the present invention. Pharmaceutically acceptable salts of the compounds of this invention can be formed with a variety of organic and inorganic acids and bases. Examples of acid addition salts include acetate, adipate, alginate, ascorbic acid, benzoate, benzenesulfonate, hydrogen sulfate, borate, butyrate, citrate, camphorate, camphorsulfonate , Cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfonate, heptanoate, hexanoate, hydrochloride, bromide Hydronate, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectate , Persulfate, 3-phenylsulfonate, phosphate, picrate, pivalate, propionate, salicylate, sulfate, sulfo , Tartrate, thiocyanate, toluenesulfonate, such as tosylate, undecanoate, and the like can be mentioned. Examples of base addition salts include ammonium salts, alkali metal salts such as sodium, lithium and potassium; alkaline earth metal salts such as calcium and magnesium; benzazetin, dicyclohexylamine, hydrabine, N-methyl-D-glucamine, N-methyl -Salts with organic bases such as D-glucamide and t-butylamine (organic amines), and salts with amino acids such as arginine and lysine.
Furthermore, the general formula in the present invention is intended to encompass all possible stereoisomers and diastereomers of the compounds it represents.

三者構造がC−B−Aである場合、X²¹およびX³¹は、NH、O、S、NH(CH₂)_cOPO₃ ⁻、NH(CH₂)_cSO₂CF₂、NH(CH₂)_cSO₂NH、NHCONH、NHCOO、NHCH(CONH₂)(CH₂)_dCOO、NHCH(COOH)(CH₂)_dCOO、NHCH(CONH₂)(CH₂)_dCONH、NHCH(COOH)(CH₂)_dCONH、NHCH(CONH₂)(CH₂)₄NH((CO)CH₂O)_fおよびNHCH(COOH)(CH₂)₄NH((CO)CH₂O)_f、好ましくは、NH、NH(CH₂)_cOPO₃ ⁻およびNHCONHから指向的に選ばれる［ここで、リンカーは、X²¹またはX³¹に結合する］。もう1つの好ましい実施態様において、X²¹およびX³¹は、NHCH(CONH₂)(CH₂)_dCOOである。本発明において、「指向的に」は、X²¹およびX³¹で示される部分が、指示された方向においてリンカーおよび隣接構造に結合することを意味する。たとえば、NH(CH₂)_cOPO₃ ⁻の場合、NHはリンカーに結合し、OPO₃ ⁻はステロイド構造に結合する。cは2〜3の整数であり、2が好ましい。dは1〜2の整数であり、1が好ましい。fは0〜1の整数であり、0が好ましい。三者構造がC'−B'−A'である場合、X²¹およびX³¹は、CO(CH₂)_b(CO)_aNH、CO(CH₂)_b(CO)_aO、CO(CH₂)_bS、CO(CH₂)_bOPO₃ ⁻、CO(CH₂)_bSO₂CF₂、CO(CH₂)_bSO₂NH、CO(CH₂)_bNHCONH、CO(CH₂)_bOCONH、CO(CH₂)_eCH(CONH₂)NHCO(CH₂)_b(CO)_aNH、CO(CH₂)_eCH(COOH)NHCO(CH₂)_b(CO)_aNH、CO(CH₂)_eCH(CONH₂)NHCO(CH₂)_b(CO)_aO、CO(CH₂)_eCH(COOH)NHCO(CH₂)_b(CO)_aO、COCH(NH₂)(CH₂)_eCOOまたはCOCH(NHCOCH₃)(CH₂)_eCOOであり、好ましくは、CO(CH₂)_b(CO)_aNH、CO(CH₂)_b(CO)_aO、CO(CH₂)_bSO₂NH、CO(CH₂)_bNHCONHまたはCO(CH₂)_bOCONH、より好ましくは、CO(CH₂)_b(CO)_aNHまたはCO(CH₂)_b(CO)_aOである［ここで、リンカーは、X²¹またはX³¹の末端カルボニル基に結合する］。もう1つの好ましい実施態様において、X²¹およびX³¹は、CO(CH₂)_eCH(CONH₂)NHCO(CH₂)_b(CO)_aNH、CO(CH₂)_eCH(COOH)NHCO(CH₂)_b(CO)_aNH、CO(CH₂)_eCH(CONH₂)NHCO(CH₂)_b(CO)_aO、CO(CH₂)_eCH(COOH)NHCO(CH₂)_b(CO)_aO、COCH(NH₂)(CH₂)_eCOOまたはCOCH(NHCOCH₃)(CH₂)_eCOOである。aは0〜1の整数である。bは1〜3の整数である。aが0ならば、bは1であるのが好ましい。aが1ならば、bは2であるのが好ましい。eは1〜2の整数であり、1が好ましい。 The moieties represented by the following formulas 2 and 3 are useful as raft affinity moieties A or A ′ in the present invention:

When the ternary structure is C-B-A, X ²¹ and X ³¹ are NH, O, S, NH (CH ₂ ) _c OPO ₃ ⁻ , NH (CH ₂ ) _c SO ₂ CF ₂ , NH (CH ₂ ) _c SO ₂ NH, NHCONH, NHCOO, NHCH (CONH ₂ ) (CH ₂ ) _d COO, NHCH (COOH) (CH ₂ ) _d COO, NHCH (CONH ₂ ) (CH ₂ ) _d CONH, NHCH (COOH) (CH ₂ ) _d CONH, NHCH (CONH ₂ ) (CH ₂ ) ₄ NH ((CO) CH ₂ O) _f and NHCH (COOH) (CH ₂ ) ₄ NH ((CO) CH ₂ O) _f , preferably , NH, NH (CH ₂ ) _c OPO ₃ ^— and NHCONH [wherein the linker binds to X ²¹ or X ³¹ ]. In another preferred embodiment, X ²¹ and X ³¹ are NHCH (CONH ₂ ) (CH ₂ ) _d COO. In the present invention, “directly” means that the moieties represented by X ²¹ and X ³¹ bind to the linker and adjacent structure in the indicated direction. For example, in the case of NH (CH ₂ ) _c OPO ₃ ^— , NH binds to the linker and OPO ₃ ^— binds to the steroid structure. c is an integer of 2 to 3, and 2 is preferable. d is an integer of 1 to 2, and 1 is preferable. f is an integer of 0 to 1, and 0 is preferable. When the tripartite structure is C′-B′-A ′, X ²¹ and X ³¹ are CO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _b S, CO (CH ₂ ) _b OPO ₃ ⁻ , CO (CH ₂ ) _b SO ₂ CF ₂ , CO (CH ₂ ) _b SO ₂ NH, CO (CH ₂ ) _b NHCONH, CO (CH ₂ ) _b OCONH, CO (CH ₂ ) _e CH (CONH ₂ ) NHCO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _e CH (COOH) NHCO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _e CH (CONH ₂ ) NHCO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _e CH (COOH) NHCO (CH ₂ ) _b (CO) _a O, COCH (NH ₂ ) (CH ₂ ) _e COO or COCH (NHCOCH ₃ ) (CH ₂ ) _e COO, preferably CO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _b SO ₂ NH, CO (CH ₂ ) _b NHCONH or CO (CH ₂ ) _b OCONH, more preferably CO (CH ₂ ) _b (CO) _a NH or CO (CH ₂ ) _b (CO) _a O [wherein the linker is bonded to the terminal carbonyl group of X ²¹ or X ^31]. In another preferred embodiment, X ²¹ and X ³¹ are CO (CH ₂ ) _e CH (CONH ₂ ) NHCO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _e CH (COOH) NHCO ( CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _e CH (CONH ₂ ) NHCO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _e CH (COOH) NHCO (CH ₂ ) _b ( CO) _a O, COCH (NH ₂ ) (CH ₂ ) _e COO or COCH (NHCOCH ₃ ) (CH ₂ ) _e COO. a is an integer of 0 to 1. b is an integer of 1 to 3. If a is 0, b is preferably 1. If a is 1, b is preferably 2. e is an integer of 1 to 2, and 1 is preferable.

は、単結合である。ステロイド骨格の炭素5および6の間の単結合の組み込みはより容易な合成を可能にする。さらに、ステロイド骨格の炭素5および6の間の単結合を有する誘導体はしばしば、対応する不飽和誘導体よりも、より高いラフト親和性を示す。 R ²¹ and R ³¹ are C _4-20 hydrocarbon groups, wherein one or more hydrogens are optionally replaced with fluorine. R ²¹ and R ³¹ are preferably in the range of C _4-20 hydrocarbon group containing one or more trans double bonds optionally and more preferably C _4-20 alkyl group. Even more preferably, R ²¹ and R ³¹ are C _8-12 alkyl groups. Most preferably, R ²¹ and R ³¹ are branched C ₈ H ₁₇ alkyl groups present in natural cholesterol.
It is preferred that the stereocenter at C3 of moiety 2 appears to be found in natural cholesterol.
In a preferred embodiment,

Is a single bond. Incorporation of a single bond between carbons 5 and 6 of the steroid skeleton allows for easier synthesis. In addition, derivatives with a single bond between carbons 5 and 6 of the steroid skeleton often exhibit higher raft affinity than the corresponding unsaturated derivatives.

部分200a、200b、200c、200e、200f、200j、200kおよび200lは、ラフト親和性部分A'の好ましい例である。部分200bおよび200fは、ラフト親和性部分A'のより好ましい例である。部分300aも、ラフト親和性部分A'の好ましい例である。部分200mは、ラフト親和性部分Aの特に好ましい例である。 The following parts 200a-200m and 300a-300g are preferred examples of parts 2 and 3 for the raft affinity part A ′:

Portions 200a, 200b, 200c, 200e, 200f, 200j, 200k and 200l are preferred examples of raft affinity moieties A ′. Portions 200b and 200f are more preferred examples of raft affinity moieties A ′. Portion 300a is also a preferred example of raft affinity portion A ′. The portion 200m is a particularly preferred example of the raft affinity portion A.

三者構造がC−B−Aである場合、X^41a、X^41b、X^51aおよびX^51bは、NH、O、NH(CH₂)_cOPO₃ ⁻、NH(CH₂)_cSO₂NH、NHCONH、NHCOO、NHCH(CONH₂)(CH₂)_dCOO、NHCH(COOH)(CH₂)_dCOO、NH(CH₂)₄CH(CONH₂)NH、NH(CH₂)₄CH(COOH)NH、NHCH(CONH₂)(CH₂)₄NHおよびNHCH(COOH)(CH₂)₄NH、好ましくは、O、NH(CH₂)_cOPO₃ ⁻およびNHCOOから指向的に選ばれる［ここで、リンカーは、X^41a、X^41b、X^51aまたはX^51bに結合する］。もう1つの好ましい実施態様において、X^41a、X^41b、X^51aおよびX^51bは、NHCH(CONH₂)(CH₂)_dCOOである。cは2〜3の整数であり、2が好ましい。dは1〜2の整数であり、1が好ましい。三者構造がC'−B'−A'である場合、X^41a、X^41b、X^51aおよびX^51bは、CO(CH₂)_b(CO)_aNH、CO(CH₂)_b(CO)_aO、CO(CH₂)_bS、CO(CH₂)_bOPO₃ ⁻、CO(CH₂)_bSO₂NH、CO(CH₂)_bNHCONH、CO(CH₂)_bOCONH、CO(CH₂)_bOSO₃、CO(CH₂)_bNHCO₂、CO(CH₂)_eCH(CONH₂)NH、CO(CH₂)_eCH(COOH)NH、COCH(NH₂)(CH₂)_eCOOまたはCOCH(NHCOCH₃)(CH₂)_eCOO、好ましくは、CO(CH₂)_b(CO)_aNHまたはCO(CH₂)_b(CO)_aOである［ここで、リンカーは、X^41a、X^41b、X^51aまたはX^51bの末端カルボニル基に結合する］。aは0〜1の整数である。bは1〜3の整数である。aが0ならば、bは1であるのが好ましい。aが1ならば、bは2であるのが好ましい。eは1〜2の整数であり、1が好ましい。 The moieties represented by the following formulas 4a, 4b, 5a and 5b are useful as raft affinity moieties A or A ′ of the present invention:

When the ternary structure is C-B-A, X ^41a , X ^41b , X ^51a and X ^51b are NH, O, NH (CH ₂ ) _c OPO ₃ ⁻ , NH (CH ₂ ) _c SO ₂ NH, NHCONH, NHCOO, NHCH (CONH ₂ ) (CH ₂ ) _d COO, NHCH (COOH) (CH ₂ ) _d COO, NH (CH ₂ ) ₄ CH (CONH ₂ ) NH, NH (CH ₂ ) ₄ CH (COOH) NH, NHCH (CONH ₂ ) (CH ₂ ) ₄ NH and NHCH (COOH) (CH ₂ ) ₄ NH, preferably selected from O, NH (CH ₂ ) _c OPO ₃ ⁻ and NHCOO [where The linker is linked to X ^41a , X ^41b , X ^51a or X ^51b ]. In another preferred embodiment, X ^41a , X ^41b , X ^51a and X ^51b are NHCH (CONH ₂ ) (CH ₂ ) _d COO. c is an integer of 2 to 3, and 2 is preferable. d is an integer of 1 to 2, and 1 is preferable. When the ternary structure is C′-B′-A ′, X ^41a , X ^41b , X ^51a and X ^51b are CO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _b S, CO (CH ₂ ) _b OPO ₃ ⁻ , CO (CH ₂ ) _b SO ₂ NH, CO (CH ₂ ) _b NHCONH, CO (CH ₂ ) _b OCONH, CO (CH ₂ ) _b OSO ₃ , CO (CH ₂ ) _b NHCO ₂ , CO (CH ₂ ) _e CH (CONH ₂ ) NH, CO (CH ₂ ) _e CH (COOH) NH, COCH (NH ₂ ) (CH ₂ ) _e COO or COCH (NHCOCH ₃ ) (CH ₂ ) _e COO, preferably CO (CH ₂ ) _b (CO) _a NH or CO (CH ₂ ) _b (CO) _a O [where the linker is X ^41a , X ^41b , X ^51a or X ^51b binds to the terminal carbonyl group]. a is an integer of 0 to 1. b is an integer of 1 to 3. If a is 0, b is preferably 1. If a is 1, b is preferably 2. e is an integer of 1 to 2, and 1 is preferable.

X ^42a , X ^42b , each X ^52a and each X ^52b are independently NH, O, S, OCO, NHCO, NHCONH, NHCO ₂ or NHSO ₂ , preferably NH, O, NHCO, NHCONH, NHSO ₂ or OCO, more preferably NHCO or NHSO ₂ , even more preferably NHCO.

は単結合であり、Y^41aまたはY^41bがOである場合、

は二重結合である。Y^41aおよびY^41bは好ましくは、OHまたはO、さらにより好ましくは、OHである。 Y ^41a and Y ^41b are NH ₂ , NHCH ₃ , OH, H, halogen or O, provided that when Y ^41a or Y ^41b is NH ₂ , NHCH ₃ , OH, H or halogen,

Is a single bond and when Y ^41a or Y ^41b is O,

Is a double bond. Y ^41a and Y ^41b are preferably OH or O, even more preferably OH.

が三重結合である場合、各Y^42aは存在しない。各Y^42aは好ましくはHである。 Each Y ^42a is independently H or OH, provided that

When is a triple bond, each Y ^42a is absent. Each Y ^42a is preferably H.

R ^41a is a C _10-30 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine. Preferably, R ^41a is a C _10-30 hydrocarbon group, optionally containing one or more trans double bonds. More preferably, R ^41a is a C _13-19 alkyl group.

R ^42a and each R ^52a are independently a C _14-30 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine. Preferably, R ^42a and each R ^52a are independently a C _14-30 alkyl group, optionally containing one or more trans double bonds. More preferably, R ^42a and each R ^52a are independently a C _14-30 alkyl group. More preferred groups for R ^42a and each R ^52a are C _16-26 alkyl groups, C _18-24 alkyl groups and C _18-20 alkyl groups.
L ^41b and L ^51b are a C _24-40 alkylene group, a C _24-40 alkenylene group or a C _24-40 alkynylene group, wherein one or more hydrogens are optionally replaced by fluorine. .

For the side chains of moieties 4a and 5a, ie R ^41a , R ^42a and each R ^52a, it is preferred that these groups do not contain double or triple bonds. Furthermore, it is preferred that these groups are straight-chain, i.e. free of branching. In a particularly preferred embodiment, the difference in the number of carbon atoms between the R ^41a and R ^42a groups is 2 or less, more preferably 1 or less, and the carbon atom between two ^a R ^52a groups. The difference in the number of is 4 or less, more preferably 2 or less. These preferred groups are chosen in view of optimizing the geometric conformation of the raft affinity moiety to match the overall structure of the raft affinity moiety. Saturated straight side chains are thought to provide the highest degree of conformational flexibility to the side chains to facilitate incorporation into the raft. When incorporated in raft assembly by choosing the difference in the number of carbon atoms in the two side chains of one raft affinity moiety as small as possible, i.e. avoiding the entire raft affinity moiety from becoming conical. It is believed that the potential destabilizing effect of the raft affinity portion of is minimal.

が二重結合である場合、それはシス立体配置またはトランス立体配置のいずれかである。部分4aおよび4bにおいて、

が二重結合である場合、それはトランス立体配置であるのが好ましい。 The stereocenter in the parts 4a, 4b, 5a and 5b is preferably the same as that of natural sphingosine.
In parts 4a and 4b,

When is a double bond, it is either in the cis or trans configuration. In parts 4a and 4b,

When is a double bond, it is preferably in the trans configuration.

部分400aa、400ab、and 400ad〜400apにおいて、Y^41aは、単結合を介して炭素骨格に結合する。部分400acにおいて、Y^41aは、二重結合を介して炭素骨格に結合する。
部分400aa、400ad、400af、400aj、400ak、400alおよび400apは、ラフト親和性部分A'の好ましい例である。 The following parts 400aa to 400ap, 400ba, 500aa to 500ae and 500ba are examples of parts 4a, 4b, 5a and 5b for the raft affinity part A ′:

In the portions 400aa, 400ab, and 400ad to 400ap, Y ^41a is bonded to the carbon skeleton through a single bond. In the portion 400ac, Y ^41a is bonded to the carbon skeleton via a double bond.
The moieties 400aa, 400ad, 400af, 400aj, 400ak, 400al and 400ap are preferred examples of the raft affinity moiety A ′.

部分500aaおよび500aeは、ラフト親和性部分A'の好ましい例である。部分500aeが特に好ましい。

Portions 500aa and 500ae are preferred examples of raft affinity moieties A ′. Part 500ae is particularly preferred.

以下の式6および7で表される部分は、本発明におけるラフト親和性部分AまたはA'として有用である：

三者構造がC−B−Aである場合、X⁶¹およびX⁷¹は、Oである［ここで、リンカーは、X⁶¹またはX⁷¹に結合する］。三者構造がC'−B'−A'である場合、X⁶¹およびX⁷¹は、CO(CH₂)_b(CO)_aOである［ここで、リンカーは、X⁶¹またはX⁷¹の末端カルボニル基に結合する］。aは0〜1の整数である。bは1〜3の整数である。aが0ならば、bは1であるのが好ましい。aが1ならば、bは2であるのが好ましい。

The moieties represented by the following formulas 6 and 7 are useful as raft affinity moieties A or A ′ in the present invention:

When the tripartite structure is CBA, X ⁶¹ and X ⁷¹ are O [wherein the linker binds to X ⁶¹ or X ⁷¹ ]. When the tripartite structure is C′-B′-A ′, X ⁶¹ and X ⁷¹ are CO (CH ₂ ) _b (CO) _a O [where the linker is the end of X ⁶¹ or X ⁷¹ To carbonyl group]. a is an integer of 0 to 1. b is an integer of 1 to 3. If a is 0, b is preferably 1. If a is 1, b is preferably 2.

または以下の式の基：

である。 Each X ⁷⁵ is independently a CO—C _13-25 hydrocarbon group, wherein one or more hydrogens are optionally substituted with fluorine, and is a group of the following formula:

Or a group of the following formula:

It is.

ここで、

は単結合であるのが好ましい。
X⁶²および各X⁷²は独立して、OまたはOCO、好ましくは、OCOである。
X⁶³およびX⁷³は、PO₃ ⁻CH₂、SO₃CH₂、CH₂、CO₂CH₂および直接結合、好ましくは、PO₃ ⁻CH₂から指向的に選ばれる。
X⁶⁴およびX⁷⁴は、NH、O、S、OCO、NHCO、NHCONH、NHCO₂またはNHSO₂である。
X⁷⁶は、CO(CH₂)_b(CO)_aOおよびCO(CH₂)_b(CO)_aNH、好ましくは、CO(CH₂)_b(CO)_aOから指向的に選ばれる。aは0〜1の整数である。bは1〜3の整数である。aが0ならば、bは1であるのが好ましい。aが1ならば、bは2であるのが好ましい。最も好ましくは、X⁷⁶は、COCH₂Oである。
Y⁶¹は、NH₂、NHCH₃、OH、H、ハロゲンまたはOであるが、ただし、Y⁶¹が、NH₂、NHCH₃、OH、Hまたはハロゲンである場合、

は、単結合であり、Y⁶¹がOである場合、

は二重結合である。Y⁶¹がOHであるのが好ましい。
各R⁶¹および各R⁷¹は独立して、C₁₆₋₃₀炭化水素基であり、ここで、1つまたはそれ以上の水素は必要に応じてフッ素で置換される。好ましくは、各R⁶¹および各R⁷¹は独立して、C₁₆₋₂₄炭化水素基であり、必要に応じて1つまたはそれ以上のトランス二重結合を含む。より好ましくは、各R⁶¹および各R⁷¹は独立して、C₁₆₋₂₀アルキル基である。 Preferably, X ⁷⁵ is a CO—C _13-25 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine, and even more preferably, CO—C _{18 -20} alkyl group. In an even more preferred embodiment, X ⁷⁵ is a group represented by the following formula:

here,

Is preferably a single bond.
X ⁶² and each X ⁷² are independently O or OCO, preferably OCO.
X ⁶³ and X ^{73 _{are, PO 3 - CH 2, SO}} 3 CH 2, CH 2, CO 2 CH 2 , and a direct bond, preferably, PO ₃ ^- is selected from CH ₂ directionally.
X ⁶⁴ and X ⁷⁴ are NH, O, S, OCO, NHCO, NHCONH, NHCO ₂ or NHSO ₂ .
X ⁷⁶ is directionally selected from CO (CH ₂ ) _b (CO) _a O and CO (CH ₂ ) _b (CO) _a NH, preferably CO (CH ₂ ) _b (CO) _a O. a is an integer of 0 to 1. b is an integer of 1 to 3. If a is 0, b is preferably 1. If a is 1, b is preferably 2. Most preferably, X ⁷⁶ is COCH ₂ O.
Y ⁶¹ is NH ₂ , NHCH ₃ , OH, H, halogen or O, provided that when Y ⁶¹ is NH ₂ , NHCH ₃ , OH, H or halogen,

Is a single bond and when Y ⁶¹ is O,

Is a double bond. Preferably Y ⁶¹ is OH.
Each R ⁶¹ and each R ⁷¹ is independently a C _16-30 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine. Preferably, each R ⁶¹ and each R ⁷¹ are independently a C _16-24 hydrocarbon group, optionally including one or more trans double bonds. More preferably, each R ⁶¹ and each R ⁷¹ are independently a C _16-20 alkyl group.

が二重結合である場合、それはシス立体配置またはトランス立体配置のいずれかである。部分6において、

が二重結合である場合、それはトランス立体配置であるのが好ましい。 R ⁶² is a C _13-25 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine. Preferably, R ⁶² is a C _13-25 hydrocarbon group, containing one or more trans double bonds as necessary. More preferably, R ⁶² is a C _13-19 alkyl group.
R ⁷² is a C _4-20 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine. Preferably, R ⁷² is a C _4-20 hydrocarbon group optionally containing one or more trans double bonds, more preferably a C _4-20 alkyl group. Even more preferably, R ⁷² is a C _8-12 alkyl group. Most preferably, R ⁷² is a branched C ₈ H ₁₇ alkyl group present in natural cholesterol.
In part 6,

When is a double bond, it is either in the cis or trans configuration. In part 6,

When is a double bond, it is preferably in the trans configuration.

With respect to the side chains of moieties 6 and 7, ie R ⁶¹ , R ⁶² and R ⁷¹ , it is preferred that these groups do not contain either double or triple bonds. Furthermore, it is preferred that these groups are straight-chain, ie do not contain any branches. In a particularly preferred embodiment, the difference in the number of carbon atoms between the R ⁶¹ and R ⁶² groups or the three R ⁷¹ groups is 4 or less, more preferably 2 or less. When X ⁷⁵ is a CO—C _13-25 hydrocarbon group, the difference in the number of carbon atoms between R ⁷¹ and the X ⁷⁵ group is 4 or less, more preferably 2 or less. These preferred groups are chosen in view of optimizing the geometric conformation of the raft affinity moiety to match the overall structure of the raft affinity moiety. Saturated straight side chains are thought to provide the highest degree of conformational flexibility to the side chains to facilitate incorporation into the raft. When incorporated in raft assembly by choosing the difference in the number of carbon atoms in the two side chains of one raft affinity moiety as small as possible, i.e. avoiding the entire raft affinity moiety from becoming conical. It is believed that the potential destabilizing effect of the raft affinity portion of is minimal.

The following parts 600 and 700 are preferred examples of parts 6 and 7 for the raft affinity part A ′:

The following portions 700a, 700b and 700c are particularly preferred examples of portion 7 for raft affinity portion A ′:

三者構造がC−B−Aである場合、X^81a、X^81b、X⁹¹およびX¹⁰¹は、NH、O、NH(CH₂)_cOPO₃ ⁻、NH(CH₂)_cSO₂NH、NHCONHおよびNHCOO、好ましくは、NHおよびNHCONHから指向的に選ばれる［ここで、リンカーは、X^81a、X^81b、X⁹¹またはX¹⁰¹に結合する］。cは2〜3の整数、好ましくは、2である。三者構造がC'−B'−A'である場合、X^81a、X^81b、X⁹¹およびX¹⁰¹は、CO(CH₂)_b(CO)_aNH、CO(CH₂)_b(CO)_aO、CO(CH₂)_bS、CO(CH₂)_bOPO₃ ⁻、CO(CH₂)_bSO₂NH、CO(CH₂)_bNHCONH、CO(CH₂)_bOCONH、CO(CH₂)_bOSO₃またはCO(CH₂)_bNHCO₂、好ましくは、CO(CH₂)_b(CO)_aNHまたはCO(CH₂)_b(CO)_aOである［ここで、リンカーは、X^81a、X^81b、X⁹¹またはX¹⁰¹の末端カルボニル基に結合する。aは0〜1の整数である。bは1〜3の整数である。aが0ならば、bは1であるのが好ましい。aが1ならば、bは2であるのが好ましい。 Represented by the following formulas 8a, 8b, 9 and 10 are useful as raft affinity moieties A or A ′ of the present invention:

When the tripartite structure is C-B-A, X ^81a , X ^81b , X ⁹¹ and X ¹⁰¹ are NH, O, NH (CH ₂ ) _c OPO ₃ ⁻ , NH (CH ₂ ) _c SO ₂ NH, Directly selected from NHCONH and NHCOO, preferably NH and NHCONH [wherein the linker binds to X ^81a , X ^81b , X ⁹¹ or X ¹⁰¹ ]. c is an integer of 2 to 3, preferably 2. When the ternary structure is C′-B′-A ′, X ^81a , X ^81b , X ⁹¹ and X ¹⁰¹ are CO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _b S, CO (CH ₂ ) _b OPO ₃ ⁻ , CO (CH ₂ ) _b SO ₂ NH, CO (CH ₂ ) _b NHCONH, CO (CH ₂ ) _b OCONH, CO (CH ₂ ) _b OSO ₃ or CO (CH ₂ ) _b NHCO ₂ , preferably CO (CH ₂ ) _b (CO) _a NH or CO (CH ₂ ) _b (CO) _a O [where the linker is Bonded to the terminal carbonyl group of X ^81a , X ^81b , X ⁹¹ or X ¹⁰¹ . a is an integer of 0 to 1. b is an integer of 1 to 3. If a is 0, b is preferably 1. If a is 1, b is preferably 2.

Each X ^82a , each X ^82b , each X ⁹² and X ¹⁰² is independently CH ₂ or O, preferably CH ₂ .
n ⁹ is an integer of 1 to 2.
Each R ^81a , each R ^81b and each R ⁹¹ is independently H or a C _16-30 hydrocarbon group, wherein one or more hydrogens are optionally substituted with fluorine, provided that , At least one R ^81a , at least one R ^81b and at least one R ⁹¹ are C _16-30 hydrocarbon groups, wherein one or more hydrogens are optionally substituted with fluorine . Preferably, each R ^81a , each R ^81b and each R ⁹¹ is independently H or a C _16-30 hydrocarbon group, optionally with one or more trans double bonds or one or more The above triple bond is included, provided that at least one R ^81a , at least one R ^81b and at least one R ⁹¹ are a C _16-30 hydrocarbon group. More preferably, each R ^81a , each R ^81b and each R ⁹¹ is independently H or a C _16-30 alkyl group provided that at least one R ^81a , at least one R ^81b and at least one R ⁹¹ Is a C _16-30 alkyl group.

R ¹⁰¹ is a C _16-30 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine. Preferably, R ¹⁰¹ is a C _16-30 hydrocarbon group, optionally containing one or more trans double bonds or one or more triple bonds. More preferably, R ¹⁰¹ is a C _16-30 alkyl group.

R ^82a , R ^82b and R ¹⁰² are a C _1-15 hydrocarbon group (where one or more hydrogens are optionally replaced by fluorine) or a C _1-15 hydrocarbon group (wherein , One or more hydrogens are optionally replaced with fluorine). Preferably, R ^82a , R ^82b and R ¹⁰² are a C _1-15 alkyl group or a C _1-15 alkoxy group.
Preferably, X ^81a is bonded to the benzo ring at the 6-position. Preferably, X ^81b is bonded to the benzo ring at the 7-position. Preferably, X ⁹¹ — (CH ₂ ) _n9 — is bonded to the pyrrole ring at the 3-position. Preferably, X ¹⁰¹ is bonded to the benzo ring at the 3 position.

The following parts 800a, 900 and 1000 are preferred examples of parts 8a, 9 and 10 for the raft affinity part A ′:

三者構造がC−B−Aである場合、X¹¹¹は、O、NH、O(CH₂)_cOおよびNH(CH₂)_cSO₂NHから指向的に選ばれる［ここで、リンカーは、X¹¹¹に結合する］。cは2〜3の整数である。三者構造がC'−B'−A'である場合、X¹¹¹は、CO(CH₂)_b(CO)_aOまたはCO(CH₂)_b(CO)_aNHである［ここで、リンカーは、X¹¹¹の末端カルボニル基に結合する］。aは0〜1の整数である。bは1〜3の整数である。aが0ならば、bは1であるのが好ましい。aが1ならば、bは2であるのが好ましい。
三者構造がC−B−Aである場合、X¹¹²は、(CH₂)_cNHまたは直接結合から指向的に選ばれる［ここで、リンカーは、X¹¹²に結合する］。cは2〜3の整数、好ましくは、2である。三者構造がC'−B'−A'である場合、X¹¹²は、CO(CH₂)_bO(CO)またはCO(CH₂)_bである［ここで、リンカーは、X¹¹²のCO(CH₂)_b部分のカルボニル基に結合する］。bは1〜3の整数、好ましくは、2である。
各R¹¹¹および各R¹²¹は独立して、C₁₆₋₃₀炭化水素基であり、ここで、1つまたはそれ以上の水素は必要に応じてフッ素で置換される。好ましくは、各R¹¹¹および各R¹²¹は独立して、C₁₆₋₃₀炭化水素基であり、必要に応じて1つまたはそれ以上のトランス二重結合または1つまたはそれ以上の三重結合を含む。より好ましくは、各R¹¹¹および各R¹²¹は独立して、C₁₆₋₃₀アルキル基である。 The moieties represented by the following formulas 11 and 12 are useful as raft affinity moieties A or A ′ of the present invention:

When the tripartite structure is C-B-A, X ¹¹¹ is directionally selected from O, NH, O (CH ₂ ) _c O and NH (CH ₂ ) _c SO ₂ NH [wherein the linker is , ^Binds to X111]. c is an integer of 2 to 3. When the tripartite structure is C′-B′-A ′, X ¹¹¹ is CO (CH ₂ ) _b (CO) _a O or CO (CH ₂ ) _b (CO) _a NH [wherein the linker It is bonded to the terminal carbonyl group of X ^111]. a is an integer of 0 to 1. b is an integer of 1 to 3. If a is 0, b is preferably 1. If a is 1, b is preferably 2.
When the tripartite structure is C-B-A, X ¹¹² is selected from (CH ₂ ) _c NH or a direct bond [where the linker binds to X ¹¹² ]. c is an integer of 2 to 3, preferably 2. When the tripartite structure is C′-B′-A ′, X ¹¹² is CO (CH ₂ ) _b O (CO) or CO (CH ₂ ) _b [wherein the linker is the CO of X ¹¹² (CH ₂ ) bonded to the carbonyl group of the _b moiety]. b is an integer of 1 to 3, preferably 2.
Each R ¹¹¹ and each R ¹²¹ is independently a C _16-30 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine. Preferably, each R ¹¹¹ and each R ¹²¹ is independently a C _16-30 hydrocarbon group, optionally containing one or more trans double bonds or one or more triple bonds. . More preferably, each R ¹¹¹ and each R ¹²¹ are independently a C _16-30 alkyl group.

The following parts 1100a, 1100b, 1200a and 1200b are preferred examples of parts 11 and 12 for the raft affinity part A ′:

三者構造がC−B−Aである場合、X^131aおよびX^131bは、NH、O、NH(CH₂)_cOPO₃ ⁻、NH(CH₂)_cSO₂NH、NHCONHおよびNHCOOから指向的に選ばれる［ここで、リンカーは、X^131aまたはX^131bに結合する］。cは2〜3の整数、好ましくは、2である。三者構造がC'−B'−A'である場合、X^131aおよびX^131bは、CO(CH₂)_b(CO)_aNH、CO(CH₂)_b(CO)_aO、CO(CH₂)_bS、CO(CH₂)_bOPO₃ ⁻、CO(CH₂)_bSO₂NH、CO(CH₂)_bNHCONH、CO(CH₂)_bOCONH、CO(CH₂)_bOSO₃またはCO(CH₂)_bNHCO₂、好ましくは、CO(CH₂)_b(CO)_aOである［ここで、リンカーは、X^131aまたはX^131bの末端カルボニル基に結合する］。aは0〜1の整数である。bは1〜3の整数である。aが0ならば、bは1であるのが好ましい。aが1ならば、bは2であるのが好ましい。X^132aは、NH、OまたはSO₂、好ましくは、NHまたはO、より好ましくは、Oである。 The moiety represented by formula 13 below is useful as the raft affinity moiety A or A ′ of the present invention:

When the tripartite structure is C-B-A, X ^131a and X ^131b are directed from NH, O, NH (CH ₂ ) _c OPO ₃ ⁻ , NH (CH ₂ ) _c SO ₂ NH, NHCONH and NHCOO. [Wherein the linker binds to X ^131a or X ^131b ]. c is an integer of 2 to 3, preferably 2. When the tripartite structure is C′-B′-A ′, X ^131a and X ^131b are CO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _b S, CO (CH ₂ ) _b OPO ₃ ⁻ , CO (CH ₂ ) _b SO ₂ NH, CO (CH ₂ ) _b NHCONH, CO (CH ₂ ) _b OCONH, CO (CH ₂ ) _b OSO ₃ or CO (CH ₂ ) _b NHCO ₂ , preferably CO (CH ₂ ) _b (CO) _a O [wherein the linker is attached to the terminal carbonyl group of X ^131a or X ^131b ]. a is an integer of 0 to 1. b is an integer of 1 to 3. If a is 0, b is preferably 1. If a is 1, b is preferably 2. X ^132a is NH, O or SO ₂ , preferably NH or O, more preferably O.

Each X ^133a and each X ^133b is independently O, NH, CH ₂ , OCO or NHCO, preferably OCO or NHCO.
Y ^131a is NH ₂ , NHCH ₃ , OH, H or halogen, preferably H or OH.
Each R ^131a and each R ^131b is independently a C _16-30 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine. Preferably, each R ^131a and each R ^131b is independently a C _16-30 hydrocarbon group, optionally including one or more trans double bonds or one or more triple bonds. . More preferably, each R ^131a and each R ^131b is independently a C _16-30 alkyl group.

以下の部分1300bは、ラフト親和性部分A'のための部分13bの好ましい例である：

The following portions 1300aa-1300ac are preferred examples of portion 13a for raft affinity portion A ′:

The following portion 1300b is a preferred example of portion 13b for raft affinity portion A ′:

および1つの−X¹⁴²−R¹⁴¹によって置換された（ただし、

および−X¹⁴²−R¹⁴¹が結合する炭素原子は、少なくとも2つの炭素原子によって分離される）ナフタレン部分14a、フェナントレン部分14bおよびクリセン部分14cは、本発明におけるラフト親和性部分AまたはA'として有用である。
ここで、非置換ナフタレン、非置換フェナントレンおよび非置換クリセンは、それぞれ以下の式14a−1、14b−1および14c−1によって表される：

用語「少なくとも2つの炭素原子によって分離される」は、

および−X¹⁴²−R¹⁴¹が結合する炭素原子間の最短経路が、

および−X¹⁴²−R¹⁴¹が結合する炭素原子を数えずに、少なくとも2つの炭素原子を含むことを意味する。 One of each available location

And one -X ¹⁴² -R ¹⁴¹

And the carbon atom to which -X ¹⁴² -R ¹⁴¹ is bound is separated by at least two carbon atoms) Naphthalene moiety 14a, phenanthrene moiety 14b and chrysene moiety 14c are useful as raft affinity moieties A or A 'in the present invention It is.
Here, unsubstituted naphthalene, unsubstituted phenanthrene and unsubstituted chrysene are represented by the following formulas 14a-1, 14b-1 and 14c-1, respectively:

The term “separated by at least two carbon atoms”

And the shortest path between the carbon atoms to which -X ¹⁴² -R ¹⁴¹ is bonded,

And -X ¹⁴² -R ¹⁴¹ means that it contains at least two carbon atoms without counting the carbon atoms to which it is bonded.

When the tripartite structure is C-B-A, X ¹⁴¹ is NH, O, NH (CH ₂ ) _c OPO ₃ ⁻ , NH (CH ₂ ) _c SO ₂ NH, NHCONH and NHCOO, preferably NH and chosen directionally from NHCONH [wherein the linker is bonded to X ^141]. c is an integer of 2 to 3. When the tripartite structure is C′-B′-A ′, X ¹⁴¹ represents CO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _b S, CO (CH ₂ ) _b OPO ₃ ⁻ , CO (CH ₂ ) _b SO ₂ NH, CO (CH ₂ ) _b NHCONH, CO (CH ₂ ) _b OCONH, CO (CH ₂ ) _b OSO ₃ or CO (CH ₂ ) _b NHCO ₂ , preferably CO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _b (CO) _a O or CO (CH ₂ ) _b SO ₂ NH [wherein the linker is , bonded to the terminal carbonyl group of X ^141]. a is an integer of 0 to 1. b is an integer of 1 to 3. If a is 0, b is preferably 1. If a is 1, b is preferably 2.
X ¹⁴² is O or CH ₂ .
R ¹⁴¹ is a C _12-30 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine. Preferably, R ¹⁴¹ is a C _12-30 hydrocarbon group, optionally containing one or more trans double bonds or one or more triple bonds. More preferably, R ¹⁴¹ is a C _12-30 alkyl group.

The following moieties 1400aa-1400ae are preferred examples of naphthalene moieties for the raft affinity moiety A ′:

The following compound 1400b is a preferred example of a phenanthrene moiety for the raft affinity moiety A ′:

三者構造がC−B−Aである場合、X¹⁵¹およびX¹⁶¹は、NH、O、NH(CH₂)_cOPO₃ ⁻、NH(CH₂)_cSO₂NH、NHCONHおよびNHCOO、好ましくは、NHおよびNHCONHから指向的に選ばれる［ここで、リンカーは、X¹⁵¹またはX¹⁶¹に結合する］。cは2〜3の整数、好ましくは、2である。三者構造がC'−B'−A'である場合、X¹⁵¹およびX¹⁶¹は、CO(CH₂)_b(CO)_aNH、CO(CH₂)_b(CO)_aO、CO(CH₂)_bS、CO(CH₂)_bOPO₃ ⁻、CO(CH₂)_bSO₂NH、CO(CH₂)_bNHCONH、CO(CH₂)_bOCONH、CO(CH₂)_bOSO₃またはCO(CH₂)_bNHCO₂、好ましくは、CO(CH₂)_b(CO)_aNHまたはCO(CH₂)_b(CO)_aOである［ここで、リンカーは、X¹⁵¹またはX¹⁶¹の末端カルボニル基に結合する］。aは0〜1の整数である。bは1〜3の整数である。aが0ならば、bは1であるのが好ましい。aが1ならば、bは2であるのが好ましい。
X¹⁵²およびX¹⁶²は、CH₂またはOである。
R¹⁵¹およびR¹⁶¹は、C₁₄₋₃₀炭化水素基であり、ここで、1つまたはそれ以上の水素は必要に応じてフッ素で置換される。好ましくは、R¹⁵¹およびR¹⁶¹は、C₁₄₋₃₀炭化水素基であり、必要に応じて1つまたはそれ以上のトランス二重結合または1つまたはそれ以上の三重結合を含む。より好ましくは、R¹⁵¹およびR¹⁶¹は、C₁₄₋₃₀アルキル基である。
各R¹⁵²および各R¹⁶²は独立して、水素、CH₃またはCH₂CH₃、好ましくは、水素である。 The moieties represented by the following formulas 15 and 16 are useful as raft affinity moieties A or A ′ of the present invention:

When the tripartite structure is C-B-A, X ¹⁵¹ and X ¹⁶¹ are NH, O, NH (CH ₂ ) _c OPO ₃ ⁻ , NH (CH ₂ ) _c SO ₂ NH, NHCONH and NHCOO, preferably , NH, and NHCONH [wherein the linker binds to X ¹⁵¹ or X ¹⁶¹ ]. c is an integer of 2 to 3, preferably 2. When the tripartite structure is C′-B′-A ′, X ¹⁵¹ and X ¹⁶¹ are CO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _b S, CO (CH ₂ ) _b OPO ₃ ⁻ , CO (CH ₂ ) _b SO ₂ NH, CO (CH ₂ ) _b NHCONH, CO (CH ₂ ) _b OCONH, CO (CH ₂ ) _b OSO ₃ or CO (CH ₂ ) _b NHCO ₂ , preferably CO (CH ₂ ) _b (CO) _a NH or CO (CH ₂ ) _b (CO) _a O [wherein the linker is X ¹⁵¹ or X ¹⁶¹ To the terminal carbonyl group]. a is an integer of 0 to 1. b is an integer of 1 to 3. If a is 0, b is preferably 1. If a is 1, b is preferably 2.
X ¹⁵² and X ¹⁶² are CH ₂ or O.
R ¹⁵¹ and R ¹⁶¹ are C _14-30 hydrocarbon groups, wherein one or more hydrogens are optionally replaced with fluorine. Preferably, R ¹⁵¹ and R ¹⁶¹ are C _14-30 hydrocarbon groups, optionally including one or more trans double bonds or one or more triple bonds. More preferably, R ¹⁵¹ and R ¹⁶¹ are C _14-30 alkyl groups.
Each R ¹⁵² and each R ¹⁶² are independently hydrogen, CH ₃ or CH ₂ CH ₃ , preferably hydrogen.

The following portions 1500a and 1600a are preferred examples of portions 15 and 16 for the raft affinity portion A ′:

三者構造がC−B−Aである場合、X^181aおよびX^181bは、NH、O、NH(CH₂)_cOPO₃ ⁻、NH(CH₂)_cSO₂NH、NHCONHおよびNHCOO、好ましくは、OおよびNH(CH₂)_cOPO₃ ⁻から指向的に選ばれる［ここで、リンカーは、X^181aまたはX^181bに結合する］。cは2〜3の整数、好ましくは、2である。三者構造がC'−B'−A'である場合、X^181aおよびX^181bは、CO(CH₂)_b(CO)_aNH、CO(CH₂)_b(CO)_aO、CO(CH₂)_bS、CO(CH₂)_bOPO₃ ⁻、CO(CH₂)_bSO₂NH、CO(CH₂)_bNHCONH、CO(CH₂)_bOCONH、CO(CH₂)_bOSO₃またはCO(CH₂)_bNHCO₂、好ましくは、CO(CH₂)_b(CO)_aOである［ここで、リンカーは、X^181aまたはX^181bの末端カルボニル基に結合する］。aは0〜1の整数である。bは1〜3の整数である。aが0ならば、bは1であるのが好ましい。aが1ならば、bは2であるのが好ましい。
各Y^181aおよび各Y^181bは独立して、NH₂、NHCH₃、OH、Hまたはハロゲン、好ましくは、OHである。
各X^182aおよび各X^182bは独立して、O、NH、OCOまたはNHCO、好ましくは、OCOである。
各R^181aおよび各R^181bは独立して、C₁₅₋₃₀炭化水素基であり、ここで、1つまたはそれ以上の水素は必要に応じてフッ素で置換される。好ましくは、各R^181aおよび各R^181bは独立して、C₁₅₋₃₀炭化水素基であり、必要に応じて1つまたはそれ以上のトランス二重結合を含む。より好ましくは、各R^181aおよび各R^181bは独立して、C₁₅₋₂₄アルキル基である。 The moieties represented by the following formulas 18a and 18b are useful as raft affinity moieties A or A ′ of the present invention:

When the ternary structure is C-B-A, X ^181a and X ^181b are NH, O, NH (CH ₂ ) _c OPO ₃ ⁻ , NH (CH ₂ ) _c SO ₂ NH, NHCONH and NHCOO, preferably , O and NH (CH ₂ ) _c OPO ₃ ^— [wherein the linker binds to X ^181a or X ^181b ]. c is an integer of 2 to 3, preferably 2. When the tripartite structure is C′-B′-A ′, X ^181a and X ^181b are CO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _b S, CO (CH ₂ ) _b OPO ₃ ⁻ , CO (CH ₂ ) _b SO ₂ NH, CO (CH ₂ ) _b NHCONH, CO (CH ₂ ) _b OCONH, CO (CH ₂ ) _b OSO ₃ or CO (CH ₂ ) _b NHCO ₂ , preferably CO (CH ₂ ) _b (CO) _a O [wherein the linker is attached to the terminal carbonyl group of X ^181a or X ^181b ]. a is an integer of 0 to 1. b is an integer of 1 to 3. If a is 0, b is preferably 1. If a is 1, b is preferably 2.
Each Y ^181a and each Y ^181b is independently NH ₂ , NHCH ₃ , OH, H or halogen, preferably OH.
Each X ^182a and each X ^182b is independently O, NH, OCO or NHCO, preferably OCO.
Each R ^181a and each R ^181b is independently a C _15-30 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine. Preferably, each R ^181a and each R ^181b is independently a C _15-30 hydrocarbon group, optionally including one or more trans double bonds. More preferably, each R ^181a and each R ^181b is independently a C _15-24 alkyl group.

With respect to the side chains of moieties 18a and 18b, ie R ^181a and R ^181b, it is preferred that these groups do not contain double or triple bonds. Furthermore, it is preferred that these groups are straight-chain, i.e. free of branching. In particularly preferred embodiments, the difference in the number of carbon atoms between each R ^181a group or between each R ^181b group is 4 or less, more preferably 2 or less. These preferred groups are chosen in view of optimizing the geometric conformation of the raft affinity moiety to match the overall structure of the raft affinity moiety. Saturated straight side chains are thought to provide the highest degree of conformational flexibility to the side chains to facilitate incorporation into the raft. When incorporated in raft assembly by choosing the difference in the number of carbon atoms in the two side chains of one raft affinity moiety as small as possible, i.e. avoiding the entire raft affinity moiety from becoming conical. It is believed that the potential destabilizing effect of the raft affinity portion of is minimal.

The following portions 1800a-1800c are preferred examples of portion 18a for raft affinity portion A ′:

The following portion 1800d is a preferred example of portion 18d for raft affinity portion A ′:

三者構造がC−B−Aである場合、X^191aは、NH、O、NH(CH₂)_cOPO₃ ⁻、NH(CH₂)_cSO₂NH、NHCONH、NHCOO、NHCH(CONH₂)(CH₂)_dCOO、NHCH(COOH)(CH₂)_dCOO、NH(CH₂)₄CH(CONH₂)NH、NH(CH₂)₄CH(COOH)NH、NHCH(CONH₂)(CH₂)₄NHおよびNHCH(COOH)(CH₂)₄NH、好ましくは、OおよびNHCOOから指向的に選ばれる［ここで、リンカーは、X^191aに結合する］。もう1つの好ましい実施態様において、X^191aは、NHCH(CONH₂)(CH₂)_dCOOである。cは2〜3の整数であり、2が好ましい。dは1〜2の整数であり、1が好ましい。三者構造がC'−B'−A'である場合、X^191aは、CO(CH₂)_b(CO)_aNH、CO(CH₂)_b(CO)_aO、CO(CH₂)_bS、CO(CH₂)_bOPO₃ ⁻、CO(CH₂)_bSO₂NH、CO(CH₂)_bNHCONH、CO(CH₂)_bOCONH、CO(CH₂)_bOSO₃、CO(CH₂)_bNHCO₂、CO(CH₂)_eCH(CONH₂)NH、CO(CH₂)_eCH(COOH)NH、COCH(NH₂)(CH₂)_eCOOまたはCOCH(NHCOCH₃)(CH₂)_eCOO、好ましくは、CO(CH₂)_b(CO)_aOである［ここで、リンカーは、X^191aの末端カルボニル基に結合する］。aは0〜1の整数である。bは1〜3の整数である。aが0ならば、bは1であるのが好ましい。aが1ならば、bは2であるのが好ましい。eは1〜2の整数であり、1が好ましい。 The moieties represented by the following formulas 19a and 19b are useful as raft affinity moieties A or A ′ of the present invention:

When the ternary structure is C-B-A, X ^191a is NH, O, NH (CH ₂ ) _c OPO ₃ ⁻ , NH (CH ₂ ) _c SO ₂ NH, NHCONH, NHCOO, NHCH (CONH ₂ ) (CH ₂ ) _d COO, NHCH (COOH) (CH ₂ ) _d COO, NH (CH ₂ ) ₄ CH (CONH ₂ ) NH, NH (CH ₂ ) ₄ CH (COOH) NH, NHCH (CONH ₂ ) (CH ₂ ) ₄ NH and NHCH (COOH) (CH ₂ ) ₄ NH, preferably selected from O and NHCOO [wherein the linker binds to X ^191a ]. In another preferred embodiment, X ^191a is NHCH (CONH ₂ ) (CH ₂ ) _d COO. c is an integer of 2 to 3, and 2 is preferable. d is an integer of 1 to 2, and 1 is preferable. When the tripartite structure is C′-B′-A ′, X ^191a is CO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _b S, CO (CH ₂ ) _b OPO ₃ ⁻ , CO (CH ₂ ) _b SO ₂ NH, CO (CH ₂ ) _b NHCONH, CO (CH ₂ ) _b OCONH, CO (CH ₂ ) _b OSO ₃ , CO (CH ₂ ) _b NHCO ₂ , CO (CH ₂ ) _e CH (CONH ₂ ) NH, CO (CH ₂ ) _e CH (COOH) NH, COCH (NH ₂ ) (CH ₂ ) _e COO or COCH (NHCOCH ₃ ) (CH _{2) e} COO, preferably a _{CO (CH 2) b (CO} ) a O [ wherein the linker is bonded to the terminal carbonyl group of X ^191a]. a is an integer of 0 to 1. b is an integer of 1 to 3. If a is 0, b is preferably 1. If a is 1, b is preferably 2. e is an integer of 1 to 2, and 1 is preferable.

When the tripartite structure is C—B—A, X ^191b is NH (CH ₂ ) _c NHCO [wherein the linker is attached to the terminal amino group of X ^191b ]. c is an integer of 2 to 3, preferably 2. If the tripartite structure is C'-B'-A ', X 191b is CO [wherein the linker is bonded to X ^191b].
X ^192a is directionally selected from NHCOCH ₂ NH or NHCOCH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ NH.
X ^192b is selected directionally from COCH ₂ CH ₂ NHCOCH ₂ or COCH ₂ .
X ^193a and each X ^193b are independently selected from O, NH, C _1-8 alkylene-O and C _1-8 alkylene-NH.
Y ^191a is NH ₂ , OH or H, preferably OH.
R ^191a and each R ^191b are independently a C _4-18 hydrocarbon group. Preferably, R ^191a and each R ^191b are independently a C _4-18 hydrocarbon group, optionally containing one or more trans double bonds. More preferably, R ^191a and each R ^191b are independently a C _4-18 alkyl group. Most preferably, R ^191a and each R ^191b is a branched C ₈ H ₁₇ alkyl group present in natural cholesterol.
R ^192a is a C _13-25 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine. Preferably, R ^192a is C _13-25 hydrocarbon group, containing one or more trans double bonds as necessary. More preferably, R ^192a is a C _13-19 alkyl group.

が二重結合である場合、それはシス立体配置またはトランス立体配置のいずれかである。部分19aにおいて、環系の一部ではない

が二重結合である場合、それはトランス立体配置であるのが好ましい。
部分19aおよび19bにおいて、環系の一部ではない

が単結合であるのが好ましい。この状況において、部分2について上述した同じ注意が適用される。 In part 19a, not part of the ring system

When is a double bond, it is either in the cis or trans configuration. In part 19a, not part of the ring system

When is a double bond, it is preferably in the trans configuration.
In parts 19a and 19b not part of the ring system

Is preferably a single bond. In this situation, the same precautions described above for part 2 apply.

The following portions 1900a and 1900b are preferred examples of portions 19a and 19b for the raft affinity portion A ′.

The following describes the synthesis of a precursor that, when coupled to a linker, yields a moiety as described above that is useful as a raft affinity moiety A or A ′ in a compound of the invention.
The synthesis of cholesteryl glycolic acid, 3-cholesterylamine and cholesteryl glycerol has been described in the literature (SL Hussey, E. He, BR Peterson, J. Am. Chem. Soc. 2001, 123, 12712-12713; SL Hussey E. He, BR Peterson, Org. Lett. 2002, 4, 415-418; SE Martin, BR Peterson, Bioconjugate Chem. 2003, 14, 67-74). A precursor of moiety 2 having an amide, sulfonamide, urea or carbamate functional group at the 3-position of the steroid concept can be prepared from 3-cholesterylamine. For example, 3-cholesterylamine can be reacted with succinic anhydride in the presence of DMAP to give the corresponding succinyl substituted compound. Reaction of 3-cholesterylamine with chlorosulfonylacetic acid can give the corresponding sulfonamide, which can be prepared as described in the literature (RL Hinman, L. Locatell, J. Am. Chem. Soc. 1959 81, 5655-5658). The corresponding urea or carbamate can be prepared via the corresponding isocyanate according to literature procedures (H.-J. Knolker, T. Braxmeier, G. Schlechtingen, Angew. Chem. Int. Ed. 1995, 34, 2497; H.-J. Knolker, T. Braxmeier, G. Schlechtingen, Synlett 1996, 502; H.-J. Knolker, T. Braxmeier, Tetrahedron Lett. 1996, 37, 5861). Precursors of moiety 2 having a phosphate or carboxymethylated phosphate at position 3 of the steroid structure can be prepared as described in the literature (Golebriewski, Keyes, Cushman, Bioorg. Med. Chem. 1996, 4, 1637- 1648; Cusinato, Habeler et al., J. Lipid Res. 1998, 39, 1844-1851; Himber, Missano et al., J. Lipid Res. 1995, 36, 1567-1585). The precursor of moiety 2 having a thiol at the 3-position of the steroid structure can be prepared as described in the literature (JG Parkes, H.R.Watson, A. Joyce, R. Phadke, ICP Smith, Biochim. Biophys Acta 1982, 691, 24-29), the simple alkylation described for the corresponding amine and alcohol gives the corresponding carboxymethylthiol. The precursor of moiety 2 having a difluoromethylene sulfone derivative at position 3 of the steroid structure can be prepared as described in the literature (J. Lapierre, V. Ahmed, M.-J. Chen, M. Ispahany, JG Guillemette, S. D. Taylor, Bioorg. Med. Chem. Lett. 2004, 14, 151-155). Introduction of various side chains at position 17 of the precursor of moiety 2 can be achieved by the use of procedures described in the literature starting from dehydroisoandrosterone or pregnenolone (ED Bergmann, M. Rabinovitz, ZH Levinson, J. Am. Chem. Soc. 1959, 81, 129-1243 and references cited therein). The precursor of moiety 2 derived from cholestane is a counterpart derived from cholesterol by reduction of 5,6-double bonds using, for example, literature procedures such as hydrogenation in the presence of various transition metal catalysts. Can be obtained from the precursor of part 2.

  In a manner similar to that described for the precursor of moiety 2 starting from estrone, the precursor of moiety 3 having an oxygen-derived substituent at the 3-position is prepared. In analogy to the method described for the precursor of moiety 2 starting from 3-aminoestrone, the precursor of moiety 3 having a nitrogen-induced substitution at the 3-position can be prepared according to the literature description (X. Zhang, Z. Sui, Tetrahedron Lett. 2003, 44, 3071-3073; L. W. L. Woo, M. Lightowler, A. Purohit, MJ Reed, BVL Potter, J. Steroid Biochem. Molec. Biol. 1996, 57, 79-88). In analogy to the method described for the precursor of moiety 2 starting from 3-thioestrone, the precursor of moiety 3 with a sulfur-derived substituent at the 3-position can be prepared according to the literature description (L W. L. Woo, M. Lightowler, A. Purohit, MJ Reed, BVL Potter, J. Steroid Biochem. Molec. Biol. 1996, 57, 79-88). Introduction of various side chains at position 17 of the estrone structure can be achieved by the Wittig method followed by hydrogenation of the resulting double bond, as described in the literature (RH Peters, DF Crowe, M. A. Avery, WKM Chong, M. Tanabe, J. Med. Chem. 1989, 32, 1642-1652; AM Krubiner, EP Oliveto, J. Org. Chem. 1966, 31, 24-26). Further manipulations within the side chain (eg, double bond construction, cycloalkyl modification, etc.) can be achieved by standard procedures (eg, Suzuki coupling).

Precursors of moieties 4a belonging to the class of ceramides, dehydroceramides and dihydroceramides with different hydrocarbon groups are obtained as outlined in the literature (AH Merrill, Jr., YA Hannun (Eds.), Methods in Enzymology, Vol. 311, Academic Press, 1999; PM Koskinen, AMP Koskinen, Synthesis 1998, 1075). In particular, the sphingosine base can be used as an important intermediate for all precursors of the moiety 4a having an oxygen-induced substitution at position 1 of the sphingosine backbone. The corresponding amino derivative can be obtained by substitution of the sulfonate and can be prepared from the alcohol according to known procedures. Alkylation and acylation of 1-amino or 1-hydroxy derivatives can be achieved by reaction with bromoacetic acid and succinic anhydride, respectively. Thioacetylated derivatives can be prepared by substitution of sulfonate and mercaptoacetic acid. Phosphate and sulfate derivatives are obtained as described in the literature (AH Merrill, Jr., YA Hannun (Eds.), Methods in Enzymology, Vol. 311, Academic Press, 1999; PM Koskinen, AMP Koskinen, Synthesis 1998, 1075). Acylation, sulfonylation, urea and carbamate formation can be achieved by standard procedures. The precursor of moiety 4a where X ^42a is an amino or amino derived functional group is prepared using standard procedures and starting from a sphingosine base available by Koskinen's presentation (PM Koskinen, AMP Koskinen, Synthesis 1998, 1075) can do. The corresponding 2-oxygen-substituted sphingolipid can be obtained by the strategy published by Yamanoi (T. Yamanoi et al., Chem. Lett. 1989, 335). The precursor of moiety 4a in which both Y ^42a represent a hydroxyl group can be obtained by bishydroxylation of the corresponding alkene using known procedures. The corresponding monohydroxy derivative can be prepared as described in the literature (AR Howell, AJ Ndakala, Curr. Org. Chem. 2002, 6, 365-391). Precursors of moiety 4a having a triple bond incorporated at the 4-position of the sphingosine skeleton can be obtained as described in the literature (P. Garner et al., J. Org. Chem. 1988, 53, 4395; P. Herold Helv. Chim. Acta 1988, 74, 354; H.-E. Radunz et al., Liebigs Ann. Chem. 1988, 1103). Modification of the substituents R ^41a and R ^42a in the precursor of moiety 4a can be accomplished by procedures and strategies outlined in various reviews (HJ Harwood, Chem. Rev. 1962, 62, 99-154; WJ Gensler, Chem. Rev. 1957, 57, 191-280).
The precursor of part 4b is a procedure described in the literature (S. Muller et al., J. Prakt. Chem. 2000, 342, 779) as well as the procedure described for the preparation of the precursor of part 4a. It can be obtained by combination.

The precursor of moiety 5a where X ^51a and X ^52a are oxygen-derived substituents can be found in Fraser-Reid (U. Schlueter, J. Lu, B. Fraser-Reid, Org. Lett. 2003, 5, 255-257). As outlined, it can be prepared starting from commercially available (R)-(−)-2,2-dimethyl-1,3-dioxolane-4-methanol. Variation of substituent R ^52a in compound 5a can be achieved by procedures and strategies outlined in various reviews (HJ Harwood, Chem. Rev. 1962, 62, 99-154; WJ Gensler, Chem. Rev. 1957, 57, 191-280). Precursors of moiety 5a where X ^51a and X ^52a are nitrogen-derived substituents start from the corresponding oxygen substitution system by nucleophilic substitution and further modification of the corresponding sulfonate as outlined above or can be obtained as described in the literature. (K. Henrick, M. McPartlin, S. Munjoma, PG Owston, R. Peters, SA Sangokoya, PA Tasker, J. Chem. Soc. Dalton Trans. 1982, 225-227).
The precursor of moiety 5b is obtained in a manner similar to the precursor of moiety 4b or alternatively by ring-closing metathesis of the ω-ethenylated precursor of moiety 5a.

The precursors of moieties 6 and 7 are synthesized according to synthetic strategies described in the literature (J. Xue, Z. Guo, Bioorg. Med. Chem. Lett. 2002, 12, 2015-2018; J. Xue, Z. Guo, J. et al. Am. Chem. Soc. 2003, 16334-16339; J. Xue, N. Shao, Z. Guo, J. Org. Chem. 2003, 68, 4020-4029; N. Shao, J. Xue, Z. Guo, Angew. Chem. Int. Ed. 2004, 43, 1569-1573) and a combination of this strategy and the method described above for the preparation of the precursors of parts 4a and 5a.
The precursors of moieties 8a, 8b and 10 are obtained by total synthesis according to synthetic strategies described in the literature (H.-J. Knolker, Chem. Soc. Rev. 1999, 28, 151-157; H.-J. Knolker, KR Reddy, Chem. Rev. 2002, 102, 4303-4427; H.-J. Knolker, J. Knoll, Chem. Commun. 2003, 1170-1171; H.-J. Knolker, Curr. Org. Synthesis 2004, 1, in preparation).

  The precursor of moiety 9 can be prepared by Nenitzescu-type indole synthesis starting from 4-methoxy-3-methylbenzaldehyde to give 6-methoxy-5-methylindole. Ether cleavage, triflate formation and Sonogashira coupling gives the corresponding 6-alkynyl substituted 5-methylindole. Vilsmeier formylation followed by nitromethane addition provides a 3-nitrovinyl substituted indole derivative, which is subjected to total hydrogenation to form 6-alkyl substituted 5-methyltryptamine. Production is completed by acylation of the amino group with succinic anhydride.

The precursor of moiety 11 can be prepared analogously to the structure reported in the literature (NK Djedovic, R. Ferdani, PH Schlesinger, GW Gokel, Tetrahedron 2002, 58, 10263-10268).
The precursor of moiety 12 can be prepared by performing the known guanidine formation via the corresponding thiourea followed by simple alkylation or acylation.
The precursor of moiety 13a is a method published in Grinstaff (GS Hird, TJ McIntosh, MW Grinstaff, J. Am. Chem. Soc. 2000, 122, 8097-8098) starting from ribose or azaribose derivatives, respectively. It can be manufactured in the same manner.
The precursor of portion 13b can be prepared starting from cyclopentadiene. Monoepoxidation followed by treatment with lithium aluminum hydride yields 3-cyclopentene-1, which is silyl protected. Bishydroxylation yields the corresponding diol, which is then acylated with a fatty acid. After desilylation, the hydroxy function is alkylated or acylated.

The precursor of moiety 14a can be prepared from the corresponding commercially available bromo and nitro substituted naphthalenes by palladium mediated coupling to introduce alkyl substituted alkynes. Subsequent reduction of the nitro to the amino functional group and the alkyne to the alkyl group, followed by either acylation of the amino group with succinic anhydride or alkylation with bromoacetic acid, yields the desired compound.
The precursor of part 15 can be prepared in the same way as described in the literature (J. G. Witteveen, A. J. A. Van der Weerdt, Rec. Trav. Chim. Pays-Bas 1987, 106. 29-34).
The precursor of moiety 14b can be prepared starting from 2,7-phenanthrenediol, monoprotection and subsequent triflate formation, then Sonogashira coupling, alkyne to alkyl reduction, deprotection and acyl respectively By synthesis or alkylation, it is synthesized as described in the literature (MS Newman, RL Childers, J. Org. Chem 1967, 32, 62-66).
The precursor of portion 16 can be prepared in a manner similar to that described in the literature (W. Sucrow, H. Minas, H. Stegemeyer, P. Geschwinder, HR Murawski, C. Krueger, Chem. Ber. 1985). 118, 3332-3349; H. Minas, HR Murawski, H. Stegemeyer, W. Sucrow, J. Chem. Soc. Chem. Commun. 1982, 308-309).

The precursor of moiety 18 can be prepared starting from myo- or scyllo-inositol by combining procedures outlined in the literature (N. Shao, J. Xue, Z. Guo, Angew. Chem. Int. Ed. 2004, 43, 1569-1573 and references cited therein; D.-S. Wang, C.-S. Chen, J. Org. Chem. 1996, 61, 5905-5910, and references cited therein) .
The precursor of moiety 19a is acylated with either glycine or 2- (2-aminoethoxy) ethoxyacetic acid on the free amino function of the sphingosine base in a manner similar to that described for the precursor of moiety 4a. The free N-terminus can then be acylated with the corresponding cholesteryl or cholestanyl derivative, which can be prepared as described above.
The precursor of moiety 19b includes acylation of the ε-amino function with a cholesteryl or cholestanyl derivative prepared as described above, and acylation of an α-amino function with either a cholesteryl or cholestanyl derivative, or β-alanine, It can then be prepared by N-terminal acylation with cholesteryl or cholestanyl derivatives.

m²⁰は3〜80の整数であり、好ましくは、5〜80、より好ましくは、5〜40、最も好ましくは、5〜20である。各n²⁰は独立して、0〜1の整数であり、より好ましくは、0である。各R^aaは独立して、天然アミノ酸の側鎖のいずれかであり、必要に応じて、色素標識で置換され、蛍光色素標識が好ましい。色素標識は、ローダミン、Mca、フルオレセインまたはその合成的に修飾された誘導体である。三者構造C−B−Aにおいて、C末端は、ラフト親和性部分Aに結合し、N末端は、ファーマコフォアCに結合する。三者構造C'−B'−A'において、N末端は、ラフト親和性部分A'に結合し、C末端は、ファーマコフォアC'に結合する。 The moiety represented by the following formula 20 is useful as linker B or B ′ of the present invention:

m ²⁰ is an integer of 3 to 80, preferably 5 to 80, more preferably 5 to 40, and most preferably 5 to 20. Each n ²⁰ is independently an integer of 0 to 1, more preferably 0. Each R ^aa is independently any of the side chains of natural amino acids and is optionally substituted with a dye label, preferably a fluorescent dye label. The dye label is rhodamine, Mca, fluorescein or a synthetically modified derivative thereof. In the ternary structure C-B-A, the C-terminus binds to the raft affinity moiety A and the N-terminus binds to pharmacophore C. In the ternary structure C′-B′-A ′, the N-terminus binds to the raft affinity moiety A ′ and the C-terminus binds to the pharmacophore C ′.

以下の部分2001は、リンカーBおよびB'のための部分20の好ましい例である。リンカー2001は、BACE−1 βセクレターゼタンパク質の阻害のための三者構造を含む化合物にとって特に適当である。

The following part 2000 is an example of part 20 for linkers B and B ′:

The following part 2001 is a preferred example of part 20 for linkers B and B ′. Linker 2001 is particularly suitable for compounds containing a tripartite structure for inhibition of BACE-1 β-secretase protein.

各n²¹は独立して、1〜2の整数であり、好ましくは、1である。各o²¹は独立して、1〜3の整数であり、好ましくは、1〜2、より好ましくは、1である。各p²¹は独立して、0〜1の整数である。k²¹および各m²¹は独立して、0〜5の整数であり、好ましくは、1〜4、より好ましくは、1〜3である。l²¹は0〜10の整数であり、好ましくは、1〜5、より好ましくは、2〜3であり、ただし、k²¹およびl²¹の合計は、少なくとも1である。各R^aaは独立して、天然アミノ酸の側鎖のいずれかであり、必要に応じて、色素標識で置換され、蛍光色素標識が好ましい。色素標識は、ローダミン、Mca、フルオレセインまたはその合成的に修飾された誘導体である。三者構造C−B−Aにおいて、C末端は、ラフト親和性部分Aに結合し、N末端は、ファーマコフォアCに結合する。三者構造C'−B'−A'において、N末端は、ラフト親和性部分A'に結合し、C末端は、ファーマコフォアC'に結合する。リンカーBおよびB'のための部分21の好ましい例は、ポリグリコールユニット、すなわち、各n²¹が1であることを含む。 The moiety represented by formula 21 below is useful as linker B or B ′ of the present invention:

Each n ²¹ is independently an integer of 1 to 2, preferably 1. Each o ²¹ is independently an integer of 1 to 3, preferably 1 to 2, more preferably 1. Each p ²¹ is independently an integer from 0 to 1. k ²¹ and each m ²¹ are each independently an integer of 0 to 5, preferably 1 to 4, more preferably 1 to 3. l ²¹ is an integer of 0 to 10, preferably 1 to 5, more preferably 2 to 3, provided that the sum of k ²¹ and l ²¹ is at least 1. Each R ^aa is independently any of the side chains of natural amino acids and is optionally substituted with a dye label, preferably a fluorescent dye label. The dye label is rhodamine, Mca, fluorescein or a synthetically modified derivative thereof. In the ternary structure C-B-A, the C-terminus binds to the raft affinity moiety A and the N-terminus binds to pharmacophore C. In the ternary structure C′-B′-A ′, the N-terminus binds to the raft affinity moiety A ′ and the C-terminus binds to the pharmacophore C ′. Preferred examples of moieties 21 for linkers B and B ′ include polyglycol units, ie each n ²¹ is 1.

特に、リンカーBおよびB'のそれぞれまたはどれかのための部分21の好ましい例において、好ましくはそれぞれのn²¹は1、それぞれまたはどれか、好ましくはそれぞれのo²¹は2およびそれぞれまたはどれか、好ましくは、それぞれのp²¹は0である。このタイプの部分の1つの例は、以下の部分2001である：

リンカーBとして式2101を有する部分の有用性は、添付の実施例において実証される。 The following part 2100 is a preferred example of part 21 for linkers B and B ′:

In particular, in preferred examples of moieties 21 for each or any of linkers B and B ′, preferably each n ²¹ is 1, each or any, preferably each o ²¹ is 2 and each or any, Preferably, each p ²¹ is 0. One example of this type of part is the following part 2001:

The utility of the moiety having formula 2101 as linker B is demonstrated in the accompanying examples.

m²²は0〜40の整数、好ましくは、2〜30、より好ましくは、4〜20である。n²²は0〜1の整数である。各o²²は独立して、1〜5の整数、好ましくは、1〜3である。各X²²¹は独立して、NHまたはOである。各R^aaは独立して、必要に応じて好ましくは蛍光色素標識である色素標識で置換された天然アミノ酸の側鎖のいずれかである。色素標識は、ローダミン、Mca、フルオレセインまたは合成によって修飾されたその誘導体であってよい。三者構造C−B−Aにおいて、C末端は、ラフト親和性部分Aに結合し、X²²¹末端は、ファーマコフォアCに結合する。三者構造C'−B'−A'において、X²²¹末端は、ラフト親和性部分A'に結合し、C末端は、ファーマコフォアC'に結合する。 The moiety represented by formula 22 below is useful as linker B or B ′ of the present invention:

m ²² is an integer of 0 to 40, preferably 2 to 30, and more preferably 4 to 20. n ²² is an integer of 0 to 1. Each o ²² is independently an integer of 1 to 5, preferably 1 to 3. Each X ²²¹ is independently NH or O. Each R ^aa is independently any of the natural amino acid side chains substituted with a dye label, preferably a fluorescent dye label, as required. The dye label may be rhodamine, Mca, fluorescein or a synthetically modified derivative thereof. In the ternary structure C-B-A, the C-terminus binds to the raft affinity moiety A and the X ²²¹ terminus binds to pharmacophore C. In the ternary structure C′-B′-A ′, the X ²²¹ terminus binds to the raft affinity moiety A ′ and the C terminus binds to the pharmacophore C ′.

m²³は0〜40の整数、好ましくは、2〜30、より好ましくは、4〜20である。n²³は0〜1の整数である。各o²³は独立して、1〜5の整数、好ましくは、1〜3である。各R^aaは独立して、必要に応じて好ましくは蛍光色素標識である色素標識で置換された天然アミノ酸の側鎖のいずれかである。色素標識は、ローダミン、Mca、フルオレセインまたは合成によって修飾されたその誘導体であってよい。三者構造C−B−Aにおいて、SO₂末端は、ラフト親和性部分Aに結合し、N末端は、ファーマコフォアCに結合する。三者構造C'−B'−A'において、N末端は、ラフト親和性部分A'に結合し、SO₂末端は、ファーマコフォアC'に結合する。
リンカーBおよびB'として用いることができる種々の部分のうち、一般式21によって表される部分が好ましい。たとえば、各n²¹が1、各o²¹が2および各p²¹が0である一般式21によって表される部分などのポリグリコールユニットを含む部分が特に好ましい。 The moiety represented by formula 23 below is useful as linker B or B ′ of the present invention:

m ²³ represents an integer of 0 to 40, preferably 2 to 30, more preferably 4 to 20. n ²³ is an integer of 0 to 1. Each o ²³ is independently an integer of 1 to 5, preferably 1 to 3. Each R ^aa is independently any of the natural amino acid side chains substituted with a dye label, preferably a fluorescent dye label, as required. The dye label may be rhodamine, Mca, fluorescein or a synthetically modified derivative thereof. In the ternary structure CBA, the SO ₂ terminus binds to the raft affinity moiety A and the N terminus binds to pharmacophore C. In the ternary structure C′-B′-A ′, the N-terminus binds to the raft affinity moiety A ′ and the SO ₂ terminus binds to the pharmacophore C ′.
Of the various moieties that can be used as the linkers B and B ′, the moiety represented by general formula 21 is preferred. For example, a portion containing a polyglycol unit such as a portion represented by the general formula 21 in which each n ²¹ is 1, each o ²¹ is 2 and each p ²¹ is 0 is particularly preferable.

As pointed out above, the pharmacophore included in the tripartite compound of the present invention has a binding or interaction site (enzyme active site, protein-protein interaction site, receptor-ligand elicitor site, or more particularly, A molecule, preferably a small molecule, containing specificity for the virus, bacteria or parasite attachment site. Thus, the pharmacophore is a molecule capable of interacting with the aforementioned biological system, eg, signaling molecule interactions or receptor-ligand-interactions (eg, EGF-receptor and its corresponding Most preferably, it is capable of interfering with the system, such as a ligand.
Accordingly, the pharmacophore “C” or “C ′” included in the three-component compound of the present invention is an enzyme, an enzyme inhibitor, a receptor inhibitor, an antibody or a fragment or derivative thereof, an aptamer, a peptide, a fusion protein, a small molecule. Selected from inhibitors, heterocyclic or carbocyclic compounds and nucleoside derivatives.

  As described above, “part C” and “part C ′” of the tripartite compound of the present invention may be an antibody or a fragment or derivative thereof. For example, a known anti-HER2 (herceptin) (or functional fragment or derivative thereof) antibody used for the management of breast cancer can be used. The term “antibody” also includes derivatives or fragments thereof that still retain binding specificity. These are considered “functional fragments or derivatives”. Techniques for the production of antibodies are known in the art and are described, for example, in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988.

  Thus, the present invention encompasses compounds comprising, as “part C / part C ′”, chimeric single chain and humanized antibodies and, inter alia, antibody fragments such as Fab fragments. Antibody fragments or derivatives further include F (ab ′) 2, Fv or scFv fragments; see, for example, Harlow and Lane, supra. Various procedures are known in the art and can be used to produce antibodies and / or fragments. Thus, (antibody) derivatives can be produced by peptidomimetic molecules. In addition, techniques described for the production of single chain antibodies (see in particular US Pat. No. 4,946,778) can be adapted to produce single chain antibodies for the polypeptides of the invention. In addition, transgenic animals can be used to express humanized antibodies for the polypeptides of the invention. Antibodies useful in the present invention are monoclonal antibodies. For the production of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. Examples of such techniques include trioma technology, human B cell hybridoma technology and EBV hybridoma technology for producing human monoclonal antibodies. Techniques describing the production of single chain antibodies (eg, US Pat. No. 4,946,778) can be adapted to the production of single chain antibodies for the immunogenic polypeptides described above. It is particularly preferred that the antibody / antibody constructs and antibody fragments or derivatives used in the present invention are capable of expression in cells. This is achieved in particular by direct injection of the corresponding proteinaceous molecule or by injection of a nucleic acid molecule that encodes a leak. In the present invention, the term “antibody molecule” included in the tripartite construct as “Part C / Part C ′” also relates to whole immunoglobulin molecules as well as parts of such immunoglobulin molecules. Furthermore, the term relates to modified and / or altered antibody molecules such as chimeric and humanized antibodies, as described above. The term also relates to monoclonal or polyclonal antibodies as well as recombinantly or synthetically produced / synthesized antibodies. The term also relates to intact antibodies and antibody fragments thereof such as isolated light and heavy chains, Fab, Fab / c, Fv, Fab ', F (ab') 2. The term “antibody molecule” also includes bifunctional antibodies and antibody constructs such as single chain Fvs (scFv) or antibody-fusion proteins.

  Aptamers or aptamer moieties are also considered pharmacophores included in the compounds of the present invention. According to the present invention, the term “aptamer” means a nucleic acid molecule capable of binding to a target molecule. Aptamers typically include RNA, single stranded DNA, modified RNA or modified DNA molecules. Aptamer production is known in the art and includes, inter alia, the use of combinatorial RNA libraries to identify binding sides (Gold, Ann. Rev. Biochem. 64 (1995), 763-797). Examples of aptamers used in the tripartite compound of the present invention include those described herein and the following.

The pharmacophores “C” and “C ′” may be enzyme inhibitors. Most preferably, the enzyme inhibitor is β-secretase inhibitor III, as described herein.
As described above, the pharmacophore C / C ′ may be a receptor inhibitor, such as, for example, a receptor inhibitor that interferes with the interaction between the receptor and its corresponding ligand. Such receptor inhibitors include EGF receptor inhibitor herstatin (Azios, Oncogene, 20, (2001) 5199-5209) or aptamer A30 (Chen, Proc. Natl. Acad. Sci. USA, 100 (2003) 9226- 9231).

  In a preferred embodiment, the pharmacophore C / C ′ included in the present invention is an antiviral agent. Antiviral agents are known in the art and include zanamivir (2,4-dideoxy-2,3-didehydro-4-guanidinosialic acid); von Itzstein M. Nature. (1993) 363, 418-23; Woods JM . Antimicrob Agents Chemother. (1993) 37, 1473-9.) Or oseltamivir (ethyl (3R, 4R, 5S) -4-acetamido-5-amino-3- (1-ylpropoxy) -1-cyclohexene-1-carboxy Rate); Eisenberg EJ. Antimicrob Agents Chemother. (1997) 41, 1949-52; Kati WM. Biochem Biophys Res Commun. (1998) 244, 408-13.), But is not limited thereto. These compounds are particularly useful in the treatment or amelioration of influenza infection. Influenza virus binding agent, RWJ-270201 (peramivir), BCX-1812, BCX-1827, BCX-1898 and BCX-1923 (Babu YS, J Med Chem. (2000) 43, 3482-6; Smee DF. Antimicrob Agents Chemother (2001) 45, 743-8.), Noraquine (1-tricyclo- (2,2,1,0) -heptyl- (2) -1-phenyl-3-piperidine-propanol; triperidene), aquinone (α Also preferred are -5-norbornen-2-yl-α-phenyl-1-piperidinepropanol; biperidene), antiparkin (ethylbenzhydramine) or parcopan (trihexyphenidyl). Antiviral drugs were selected from Fuzeon (Hartt JK. Biochem Biophys Res Commun. (2000) 272, 699-704; Tremblay CL. J Acquir Immune Defic Syndr. (2000) 25, 99-102.), T1249 (39-mer; Trimeris Inc.), Cocelan, AMD3100, AMD070, SCH351125, AD101 (all bicyclams; De Clercq. E Antimicrob Agents Chemother. (1994) 38, 668-74; Palani A. J Med Chem. (2001) 44, 3339-42 .) Can also be selected. In this situation, cyclopentane neuraminidase inhibitors are also envisaged as pharmacophores; inter alia, Smee, Antimicrob. See Agents Chemother. 45 (2001), 743-748. These corresponding tripartite compounds may be used in the management of HIV infection and AIDS. Anilino-naphthalene compounds such as ANS, AmNS or bis-ANS are also used as pharmacophore C / C ′. The corresponding tripartite compounds of the invention are particularly useful in the treatment or prevention of PvP-related diseases such as infectious spongiform encephalopathy. ANS, AmNS and bis-ANS are defined as follows in their pharmaceutical use in prion-related diseases.

  Accordingly, the compounds of the present invention, i.e., the tripartite compounds described herein, are particularly useful in medical settings involving not only use as pharmaceuticals but also as comparative test substances. For example, as described herein, a tripartite compound, such as a compound of formula 24, can include additional functional moieties or structures, such as a labeled structure. The corresponding compounds can be used in the raft affinity assays described herein and can be used in comparative as well as non-comparative test settings. However, the most important use of the compounds of the invention provided in the present invention is their pharmaceutical use. Accordingly, the present invention also relates to pharmaceutical compositions comprising any of the tripartite compounds described herein.

The compounds of the present invention may be administered as compounds by themselves or, if necessary, carriers, diluents, extenders, disintegrants, lubricants, binders, colorants, dyes, stabilizers, storage May be formulated as a pharmaceutical composition comprising a pharmaceutically acceptable excipient such as an agent or an antioxidant.
The pharmaceutical composition can be formulated by techniques known to those skilled in the art, such as those described in Remington's Pharmaceutical Sciences, 20th edition. The pharmaceutical composition can be formulated in parenteral dosage forms such as oral, intramuscular, intravenous, subcutaneous, intraarterial, rectal, nasal, topical or intravaginal administration. As oral dosage forms, coated and uncoated tablets, soft gelatin capsules, hard gelatin capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders And granules, medicinal gums, chewing tablets and foamed tablets. Dosage forms for parenteral administration include solutions, emulsions, suspensions, reconstitution dispersants and powders and granules. Emulsions are a preferred dosage form for parenteral administration. Dosage forms for rectal and vaginal administration include suppositories and ovula. Dosage forms for nasal administration can be administered via inhalation or insufflation, for example, by the inhaler method. Dosage forms for topical administration include creams, gels, ointments, salves, patches and transdermal delivery systems.

The present invention also provides methods for the treatment, amelioration or prevention of disorders or diseases due to (or associated with) biochemical and / or biophysiological processes that occur on or in the raft structure of mammalian cells. In the most preferred settings, the compounds of the invention are used in these methods of treatment by administration of the compounds to patients in need of treatment, particularly human patients.
In addition to liquid cluster formation, the tripartite compounds of the present invention are particularly useful in medical settings because several specific cellular proteins partition into the liquid-ordered raft phase (Simons, Annu. Rev. Biophys. Biomol). Struct. 33 (2004), 269-295). For example, the transferrin receptor is typically excluded from rafts and marks the liquid disordered phase, whereas GPI-anchored proteins are commonly used as markers for lipid rafts (Harder, J. Cell Biol. 141 (1998 ), 929-942). Such partitioning can also be regulated, thereby modulating activity and raft protein complex formation (Harder, Curr. Opin. Cell Biol. 9 (1997), 534-542). For example, H-Ras results in a function in signaling at the exit from these microdomains and an inactive state in rafts. On the other hand, APP processing with β-secretase requires partitioning into rafts (see below). The importance of lipid rafts in membrane compartmentalization and cellular physiology is understood by their involvement in pathological processes. The following are some examples of the role of lipid rafts and their regulation in important human diseases.

  Alzheimer's disease (AD) is dependent on the formation of senile plaques containing amyloid-β-peptide (Aβ), a fragment derived from the large type 1 transmembrane protein APP (amyloid precursor protein) (London, Curr. Opin. Struct Biol. 12 (2002), 480-486). Aβ fragments are sequentially cleaved by enzymes called β-secretase (BACE) and beta-secretase. BACE is an aspartyl protease that cleaves APP in its luminal domain to produce a secreted extracellular domain. The resulting 10-kDa C-terminal fragment is sequentially cleaved by beta-secretase acting in the transmembrane domain of APP, releasing Aβ. A third enzyme activity, beta-secretase, is also cleaved by products that cleave APP in the central part of the Aβ region and are non-amyloidogenic (the beta fragment (secreted extracellular domain) and beta-secretase) Offset BACE activity by obtaining short C-terminal stubs. Thus, beta cleavage competes directly with beta cleavage for their usual substrate APP.

  Lipid rafts play a role in regulating beta and beta secretase access to the substrate APP. Cholesterol depletion inhibits beta cleavage and Aβ formation in nerves and other cells, leading to a higher rate of beta cleavage (London, Biochim. Biophys. Acta 1508 (2000), 182-195). APP and BACE co-patch with another antibody that crosslinks within the lipid raft (Ehehalt, J. Cell Biol. 160 (2003), 113-123). APP and BACE fractions are found in DRM, a biochemical feature of localization to lipid rafts (Simons, Proc. Natl. Acad. Sci. USA 95 (1998), 6460-6464; Riddell, Curr Biol. 11 (2001) 1288-1293). Aβ production is strongly stimulated in raft cluster formation that brings together surface rafts including APP and BACE (Ehehalt, (2003), supra). In demonstrating the causal relationship between raft partitioning and Aβ production, these data show that 1) interference with APP and BACE partitioning in rafts; 2) within the same raft to inhibit Aβ fragment formation and the development of Alzheimer's Provides a means of intracellular transport to gather; and 3) activity of BACE in the raft. Corresponding preferred constructs for intervention in Alzheimer's disease are provided herein; see, for example, Formulas 24 and 25 and Formula 25b, which are particularly preferred embodiments of the present invention. It is also envisaged that the corresponding compounds are used for the treatment of Down's syndrome.

  In addition, infectious diseases can be treated or even prevented by the use of the tripartite compounds described herein. These include measles virus, respiratory syncytial cell virus, Ebola virus, Marburg virus, Epstein-Barr virus, Echovirus 1, papilloma virus (eg, simian virus 40, etc.), polyoma virus or mycobacterial infection, especially Myco Including, but not limited to, infection by bacterial infections such as bacterial tuberculosis, Mycobacterium kansasii or Mycobacterium bovis infection. Infection with E. coli, Campylobacter jejuni, Vibrio cholerae, Clostridium difficile, tetanus, Salmonella or Shigella is also contemplated to be treated or prevented by the compounds provided herein. Several viruses and bacteria use lipid rafts for infection to the host. The aforementioned pathogens and the specific examples described below are related by their raft requirements during the infection cycle.

The first example of a virus characterized for raft requirements was influenza virus (Ipsen, (1987), supra). Rafts play a role in the virus assembly process. The virus contains two endogenous glycoproteins, hemagglutinin and neuraminidase. Both glycoproteins are raft-related as judged by cholesterol-dependent surfactant resistance (Ipsen, (1987), supra). Influenza viruses bud from the apical membrane of epithelial cells and are abundant in raft lipids. Influenza viruses preferentially contain raft lipids in their envelope during budding via polymerization of M proteins that drive raft cluster formation (Ipsen, (1987), supra).
The tripartite compounds described herein provide a medical tool for treatment in influenza infection. The specific corresponding pharmacophore is as described above.

  Rafts are also linked to four important events in the HIV infection cycle. 1) Passing across the host mucosa. HIV binds to the glycosphingolipid galactosylceramide at the apical surface of the mucosal epithelial cells and is then transcellularly transported across the epithelium and released to the basolateral side. Disrupting raft association blocks viral transcellular transport (Israelachvili, Biochim. Biophys. Acta 469 (1977), 221-225). 2) Virus entry into immune cells. During the infection of target cells, viral envelope components as well as internal Gag proteins (essential for viral envelope assembly) (Jacobson (1992), supra) are all first Related to rafts. In fact, viral glycoproteins can be co-patched with known raft-related proteins on the surface of living cells after cross-linking with specific antibodies (Jain (1977), supra). Interestingly, viral receptors on the host cell surface are also raft-related. The HIV glycoprotein gp120 co-patches with the cell surface receptor CD40 and the co-receptors chemokine receptors CCR5 and CXLR4. CD4, CCR5 and CXLR4 are found in DRM. Binding of the virus, which first binds to CD4 and then to the chemokine receptor, to its surface receptor appears to result in raft cluster formation in the membrane and lateral assembly of protein complexes to initiate fusion of the viral envelope and cell membrane It seems to be. Both cholesterol and specific glycosphingolipid species serve as critical elements in the construction of the fusion complex (Jorgensen, J. Phys. Chem. 104 (2000), 11763-11773; Keller, Phys. Rev. Lett. 81 (1998), 5019-5022). 3) Changes in signal transduction in the host cell. HIV produces host cells by changing the intracellular state of signaling. Nef, an early HIV gene product, promotes viral infectivity via lipid rafts (Kenworthy, Mol. Biol. Cell 11 (2000), 1645–1644); Nef-deficient HIV-1 virion infections can reach AIDS Does not progress (Kholodenko, Trends Cell Biol. 10 (2000), 173-178). Nef proteins are peripheral myristoylated membrane proteins with proline-rich repeats that can bind to the Src family of raft-related non-receptor tyrosine kinases. It is related to DRM and appears to stimulate host cells against HIV infection by reducing the threshold required for T cell activation (Kenworthy (2000), supra). Resting T cells do not assist in productive HIV infection, but Nef activates T cells by IL-2 secretion, eliminating the need for costimulatory signals. By clustering lipid rafts with associated host surface proteins, Nef oligomerization can assist in the construction of T cell signaling complexes and HIV budding sites (Kenworthy (2000), supra); Kurzchalia, Curr. Opin. Cell Biol. 11 (1999), 424-431). 4) Escape virus from cells and disperse through host vasculature. Another raft-dependent step, HIV escape from cells, is critically dependent on viral Gag protein (Jorgensen (2000), supra); Lipowsky, J. Biol. Phys. 28 (2002), 195 −210). The virus contains 1,200-1,500 Gag molecules that form multimers that drive virus assembly and budding on the cytosolic leaflet of the membrane. In this process, the Gag-Gag interaction collects viral spike proteins at the budding site. This process requires palmitoylation of gp120 and myristoylation of Gag and can be blocked by cholesterol depletion (Jorgensen (2000), supra). Thus, it can be assumed that the Gag protein specifically binds to rafts containing HIV spike proteins and clusters the rafts together to promote virus assembly. The interaction of HIV-1 protein and lipid rafts can cause conformational changes in Gag that are required for envelope assembly.

  Recent studies have demonstrated that HIV virion budding and fusion with target cells occurs through lipid rafts. Budding probably occurs through preferential sorting of HIV Gag into lipid rafts (Nguyen, J. Virol. 74 (2000) 3264-72). HIV-1 particles produced by infected T cell lines acquire raft components such as GPI-binding proteins Thy-1 and CD59 and ganglioside GM1, which is known to be preferentially distributed within lipid rafts . Infectious type 1 human immunodeficiency virus (HIV-1) virions require the incorporation of the viral envelope glycoproteins gp41 and gp120. The HIV envelope glycoprotein gp41 also plays an important role in the fusion of the virus with the target cell membrane. The extracellular domain of gp41 contains three important functional regions: a fusion peptide (FP), an N-terminal amino acid repeat (NHR) and a C-terminal amino acid repeat (CHR). During the process of fusion of HIV with the target cell membrane, FP inserts into the target, and then the NHR and CHR regions change conformation and associate with each other to form a fusion active gp41 core. Peptides derived from the NHR and CHR regions, referred to as N and C peptides, respectively, have potent inhibitory activity against HIV fusion by binding to the CHR and NHR regions, respectively, and preventing the formation of a fusion active gp41 core. Small molecule non-peptide HIV fusion inhibitors with a mechanism of action similar to C-peptide have recently been revealed (Jiang, Curr. Pharm. Des. 8 (2002) 563-80. Wadia, Nat, Med. 10 ( 2004) 310-315). Therefore, these peptides and non-peptide inhibitors can be used as the pharmacophore C / C ′ of the compound of the present invention.

  Accordingly, the present invention also provides the aforementioned tripartite compound comprising a specific compound that inhibits the life cycle of HIV as pharmacophore “C / C ′”. Examples of such pharmacophores include, but are not limited to, Cosalan, AMD3100, AMD070, Fuzeon (registered trademark), T20, T1249, DP178, and the like. As described herein, a particularly preferred pharmacophore C / C ′ in the present invention is the peptide analog T20 / T1249 / fuzeon® or enfuvirtide.

  As described above and below, pharmacophore C / C ′ may also include peptides or peptide derivatives. A corresponding non-limiting example is an inhibitory “HR2 peptide” known in the art as “T20”. The peptide has been shown to be effective in the medical management of HIV / AIDS. “T20” is also known as “DP178” and its related peptides and / or derivatives are well known in the art and have been described for their antiretroviral activity; in particular, Wild (1992) PNAS 91 9770; WO 94/282920, US 5,464,933. The peptide “T1249” is also known in the art and can be used for medical intervention. T1249 can be included as a pharmacophore C / C ′ in the tripartite structure of the present invention. Again, such a tripartite raft affinity portion of the present invention is particularly useful in the treatment and / or medical intervention of retroviral infections, particularly in the management of AIDS and / or HIV infection. T20 and T1249 are also known and may be included in the constructs of the present invention, particularly in the form of PEGylated products described in WO2004013165. The manufacture of T1249 is described, inter alia, in US 5,955,422 or US 6,348,568. Further details of the corresponding ternary constructs of the invention are disclosed in the examples below and in FIG. The corresponding inventive construct is described in particular as formula 25c.

  Tuberculosis is a further example of an infectious disease caused by bacteria involving rafts. First, type 3 complement receptor (CR3) is a receptor that can internalize zymosan and C3bi-coated particles and is responsible for the non-opsonophagocytosis of Mycobacterium kansasii in human neutrophils. In these cells, CR3 has been found to associate with several GPI-anchored proteins that are located in lipid rafts of the plasma membrane. Cholesterol depletion significantly inhibits the phagocytosis of Mycobacterium kansasii without affecting the phagocytosis of zymosan or serum opsonized Mycobacterium kansasii. When CR3 associates with the GPI protein, it is rearranged into a cholesterol-rich domain that is followed by Mycobacterium kansasii. When CR3 does not associate with GPI proteins, it is a mycobacterial antigen that remains outside of these domains, mediates phagocytosis of zymosan and opsonized particles, but specifically stimulates V gamma 9V delta 2 T cells Does not mediate the phagocytosis of Mycobacterium kansasii isopentenyl pyrophosphate (IPP). This delay, which appears to be responsible for the observed delay in TNF-α production, is discussed in terms of the ability of antigens to cross-link and replenish transfer molecules largely anchored to lipid rafts (Peyron, J , Immunol. 165 (2000), 5186-5191). Therefore, the ternary structure compounds of the present invention are also useful in the prevention, amelioration and / or treatment of tuberculosis and / or other diseases caused by Mycobacteria such as Mycobacterium tuberculosis, Mycobacterium bovis.

  Furthermore, malaria infection of human erythrocytes by Plasmodium can be blocked by raft-cholesterol depletion. Erythrocyte rafts serve as a path for entry into protozoa (Samuel, J. Biol. Chem. 276 (2001), 29319-29329). Therefore, the compound of the present invention is useful in inhibiting the infection route of P. falciparum. For example, it is envisaged that anti-CD36 antibodies or functional fragments thereof can be used as pharmacophores in the compounds of the present invention. Such antibodies are known in the art, see for example Alessio, Blood 82 (1993), 3637-3647.

  Furthermore, in a further embodiment of the invention, the tripartite compound of the invention can be used as a medicament in the management of prion diseases. The conformational changes that lead to amyloid formation are also involved in the pathogenesis of prion disease. Prion disease is thought to be promoted by an abnormal form (PrPsc) of host-encoded protein (PrPc). PrPsc can interact with its normal PrPc and change the conformation of PrPc so that it changes to PrPsc. PrPsc then self-associates in the brain, and these aggregates are thought to cause disorders that appear in humans as Creutzfeldt-Jakob disease, Kul or Gerstmann-Stroisler-Scheinker syndrome (McConnell, Annu. Rev. Biophys. Biomol. Struct. 32 (2003), 469-492). Although the mechanism by which PrPc is converted to PrPsc is not known, several lines of evidence suggest that lipid rafts are involved (McLaughlin, Annu. Rev. Biophys. Biomol. Struct. 31 (2002), 151 -175; Milhiet, Single Mol. 2 (2001), 119-121).

  PrP is a GPI-anchored protein. Both PrPc and PrPsc associate with DRM in a cholesterol-dependent manner. Cellular cholesterol depletion results in decreased formation of PrPsc from PrPc. GPI anchors require conversion. When a GPI anchor is exchanged for a transmembrane domain, conversion to an abnormal protein is blocked. In vitro, PrPc to PrPsc conversion occurs when microsomes containing PrPsc fuse with DRM containing PrP, as monitored by PrP protease resistance (McLaughlin (2002), supra). Extraction with surfactant results in raft cluster formation in DRM. Fusion of microsomes and DRM was necessary in this experiment because simply mixing the membranes did not result in measurable production of new PrPsc. On the other hand, the release of PrP extracellular domain from PrPsc by phospholipase C treatment also stimulated the conversion of PrP to PrPsc in this system. Baron et al. (McLaughlin (2002), supra) assume that membrane components exchange between juxtaposed cells; a possible mechanism for such exchange is for cells to fuse with neighboring cells. The release of membrane vesicles containing PrPsc. Indeed, it has been found that a similar process mediates the migration of the raft-associated chemokine receptor CCR5 (Murata, Proc. Nat. Acad. Sci. USA 92 (1995), 10339-10343). Alternatively, the GPI-anchored PrPsc could itself be released from one cell and migrate across the extracellular water phase to be inserted into another cell. Recently, it has been discovered that direct cell-cell contact is required for PrPsc infectious transmission in cell culture (Nielsen (1999), supra). Therefore, the construct of the present invention is useful for the management of PrPsc infection.

  Prion protein (PrP) is a protein that is considered to be involved in the pathogenic mechanisms underlying infectious spongiform encephalopathy. The conformational change from PrP (C) to pathogenic PrP (Sc) involves a transformation from an α-helix structure to a β-sheet rich structure. Bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-sulfonate), ANS (1-anilinonaphthalene-8-sulfonate) and AmNS (1-amino-5-naphthalenesulfonate) Anilino-naphthalene compounds such as inhibit prion peptide aggregation by directly interacting with PrP (Cordeiro, J. Biol. Chem. 279 (7) (2004), 5346-5352). Since PrP is a GPI-anchored protein and both PrPc and PrPsc associate with lipid rafts, the activity of anilino-naphthalene compounds is enhanced through such preferential targeting from pharmacophores to rafts.

  Asthma is also a target disease for use of the tripartite compounds of the present invention. The most intensively used cell to study the role of lipid rafts in FcεRI-mediated signaling is rat basophilic leukemia cells (RBL). The role of rafts in interactions following FcεRI aggregation, primarily in the signaling complex, assembled around a linker (LAT) for T cell activation. Raft involvement in immediate events following antigen-mediated FcεRI clustering has been described. Rafts are important in the control and integration of signal progression following FcεRI activation in mast cell lines. Therefore, the tripartite compound of the present invention prevents this signal progression.

  Furthermore, since a number of signaling components are regulated through distribution to rafts, the compounds of the present invention are useful for the management of proliferative disorders. For example, the tyrosine kinase activity of the EGF receptor is suppressed in rafts, and cholesterol plays a regulatory role in this process (Ringerike, J. Cell Sci. 115 (2002), 1331-1340). Similarly, H-Ras is inactive in rafts and its signaling activity occurs upon escape from rafts (Parton, Trends Cell Biol. 14 (2004), 141-147). The list of signaling factors whose activity depends on rafts is extended to various types of ligand-receptor complexes and downstream signaling components (Simons (2004), supra); Miaczynska, Curr. Opin. Cell Biol. 16 (2004), ongoing). Thus, as described above, specific pharmacophores capable of interfering with these signaling properties may be introduced into the tripartite compounds of the present invention. The compounds of the present invention are preferably used in the treatment of skin cancer such as breast cancer, colon cancer, gastric cancer, urogenital cancer, lung cancer or black species. For the treatment / prevention of breast cancer, it is also envisaged that antiestrogens such as tamoxifen, fulvestrant or anastrol are used as the pharmacophore C / C ′ of the compounds of the present invention.

  For example, the peptide hormone endothelin transmits proliferative signals through the G protein coupled receptors, endothelin type A (ETAR) and type B (ETBR) receptors. These molecules are therefore important therapeutic targets in the development of anti-tumor therapy. ETAR and ETBR are important in the development of melanoma. ETBR forms a complex with caveolin-1 and therefore localizes to a special form of lipid raft called caveolae (Yamaguchi, Eur. J. Biochem. 270 (2003) 1816-1827). Small molecule A-192621 is a non-peptide ETBR antagonist that significantly inhibits melanoma growth in nude mice by blocking downstream ETBR signaling pathways that are important in host-tumor interactions and cancer progression (Bagnato Cancer Res. 64, (2004) 1436-1443). Therefore, A-192621 and similar derivatives can be used as pharmacophores in the compounds of the present invention.

Recent studies have revealed that insulin signaling leading to GLUT-4 translocation depends on insulin receptor signaling emanating from caveolae or lipid rafts in the plasma membrane (Khan, Diabetologia 45 (2002), 1475). −1483). Accordingly, the described tripartite compounds are also useful for medical management of diabetes.
In a further embodiment, the tripartite compound can be used in medical / pharmaceutical interventions for parasitic infections as described above for malaria. Furthermore, it is envisioned that other parasitic infections such as trypanosoma, leishmania or Toxoplasma gondii infection are treated by administration of the tripartite compounds of the invention.

  It is also envisioned that the compounds of the present invention are used for medical management of hypertension and / or congestive heart failure. The corresponding pharmacophore C / C ′ is losartan, valsartan, candesartan cilexetil or irbesartan or TCV-116 (2-ethoxy-1- [2 ′-(1H-tetrazol-5-yl) biphenyl-4-yl] 1-benzimidazole-7-carboxylate). In accordance with the teachings of the present invention, the compounds disclosed herein have hyperallergic and asthma, T and B cell responses, autoimmune diseases, chronic inflammation, atherosclerosis, lysosomal storage diseases, Niemann-Pick disease, It is also useful for the treatment, amelioration and / or prevention of disorders and diseases such as Tay-Sachs disease, Fabry disease, metachromatic leukodystrophy, hypertension, Parkinson's disease, polyneuropathy, demyelinating disease and Kindystrophy.

  As mentioned above, the present invention also preferably comprises: a) the binding and / or interaction site of the phosphoryl head group or equivalent head group of the raft lipid and the pharmacophore C / C ′ on the raft-associated target molecule. B) Select a linker B / B ′ capable of bridging the distance determined in a); then c) Raft affinity moiety A / A with the linker selected in b) Provided are methods of making the compounds described herein, comprising the step of binding 'and pharmacophore C / C'.

  Corresponding embodiments for such methods are described herein and are also described in the accompanying embodiments. One skilled in the art can deduce the relevant binding or interaction sites of the known or possible pharmacophore, and thus the raft lipid phosphoryl head group or equivalent head group and the raft association The distance between the binding and / or interaction site of the pharmacophore C / C ′ on the target molecule can be determined. Such methods include molecular modeling, in vitro and / or molecule-interaction or binding assays (eg, yeast two or three hybrid systems, peptide spotting, overlay assays, phage display, bacterial display, ribosome display, etc.), atomic force Including but not limited to microscopy and spectroscopy and X-ray crystallography. In addition, methods such as site-directed mutagenesis can be used to identify putative interaction sites of known or candidate pharmacophores and their corresponding targets.

  As mentioned above, the target molecule is most preferably a molecule involved in biological processes performed in lipid rafts (ie cholesterol-sphingolipid microdomains). Non-limiting examples of target molecules include beta secretase (BACE-1) and amyloid precursor protein (APP), raft-associated viral or bacterial receptor (described above), Prp / PrP (SC), hormone receptor ( For example, insulin receptor, endothelin receptor or angiotensin II receptor), growth factor receptor (for example, EGF receptor), Ig receptor (for example, IgE receptor FcεRI), cell surface protein (for example, surface) Glycoprotein CD36 (GPIV) etc.). It is preferred that the target molecule is an enzyme, a receptor molecule and / or a signaling molecule. Further examples of target molecules are as described above. In the present invention, the term “raft-associated target molecule” refers to whether a molecule is contained in a raft or translocated to a raft upon corresponding stimulation and / or modification (eg, secondary modification by phosphorylation). It means that it can be either.

The choice of linker is as described above and is also shown in the experimental part. Such selection includes the selection of linkers known in the art and, for example, molecular modeling and corresponding synthesis, or the production and use of new linkers by methods provided herein and methods known in the art.
The term `` bridging '' as used in step b) above refers to placing a specific pharmacophore (preferably an inhibitor) at the exact location on the target molecule, e.g. an enzyme, signaling molecule or receptor. , And choosing the length of the linker B / B ′ so that the raft affinity portion A / A ′ is in the lipid layer portion of the raft.

Because of the medical importance of the tripartite compounds of the present invention, the present invention also relates to a pharmaceutical composition comprising a mixture of a compound as defined herein and one or more pharmaceutically acceptable excipients. A manufacturing method is provided. Corresponding excipients are as described above and include, but are not limited to, cyclodextrins. As mentioned above, the pharmaceutical composition of the present invention should be administered by injection or infusion, and preferably the pharmaceutical composition is an emulsion.
It should be argued that one skilled in the art can easily estimate, confirm and / or evaluate the known tripartite structure as well as the raft affinity of the individual part A / A ′. Corresponding test assays are provided herein and are also described in the accompanying examples.

  For example, for the evaluation of the various raft affinity moieties described herein, rhodamine labeling complexes containing the raft affinity moieties to be evaluated and modified rhodamine dyes known in the literature described in Example 32 were prepared. . To simplify production and modularity, a modified rhodamine dye was attached to the side chain of glutamic acid and the resulting labeled amino acid was used as the dye marker. The raft affinity moiety and rhodamine labeled glutamic acid were coupled through a linker building block such as Arg-Arg-βAla or 3GI (12-amino-4,7,10-trioxadodecanoic acid).

In the case of steroid-derived raft affinity moieties, compounds containing a single bond between positions 5 and 6 of the steroid-derived backbone are preferred over compounds having a double bond at this position. For example, in the evaluation of the raft affinity moiety 200a in the LRA assay, the raft affinity was Rf16.5 (and relative raft affinity r _rel 0.493 in the DRM assay), while the same linker and dye-labeled substructure For the raft affinity portion 200b containing, the raft affinity in the LRA assay was Rf42.7 (and the relative raft affinity r _rel 0.536 in the DRM assay).
A raft affinity moiety comprising structure 19b is preferred, as demonstrated by comparison of moiety 19b and moiety 200b attached to the same linker and dye label substructure. In the LRA assay, the raft affinity Rf of portion 19b was calculated to be 76.3, while the Rf value of portion 200b was 58.6. Evaluation of the same structure in the DRM assay relative raftophilicity r _rel 0.503 for section 19b, resulting in a relative raftophilicity r _rel 0.336 for portion 200b.

In the case of the ceramide-derived raft affinity part of the general structure 400a, considering the chain length of the substituents R ^41a and R ^42a , a generally symmetrical form is preferred to obtain a high raft affinity value. For example, when comparing raft affinity moieties 400aa and 400af that contain the same linker and dye labeling substructure, in the DRM assay, for the more symmetrical moiety 400aa compared to the relative raft affinity r _rel 0.560 of moiety 400af A higher relative raft affinity r _rel 0.772 was obtained. Higher symmetry is obtained from the palmityl (C16) side chain at the portion 400aa as compared to the eicosanoyl (C20) side chain of the portion 400af.

In the case of raft affinity moiety 7, compounds containing steroid-derived substructures as side chains are preferred over compounds having simple acyl side chains. For example, raft affinity portion 700c is preferred over raft affinity portion 700b, which is itself preferred over raft affinity portion 700a, as demonstrated in both LRA and DRM evaluations. The raft affinity of 700c was calculated as the relative raft affinity r _rel 0.414 in the RRA assay in the Rf37.3 and DRM assays, while the 700b measurement was measured in the LRA, in the Rf28.8 and DRM assays. The relative r _{rel was} 0.403. Evaluation of the simple fatty acid modified moiety 700a resulted in a relative raft affinity r _rel 0.266 in the RRA, in the Rf18.6 and DRM assays.

In general, an acetic acid hook is preferred over a succinic acid hook for all raft affinity moieties A / A ′ described in the present invention. When comparing compounds that contain the same linker and dye-labeled substructure, for example, the raft affinity of raft affinity moiety 200e was measured to be Rf8.1 in the LRA assay, whereas in raft affinity moiety 200b Rf42.7 in the LRA assay. In a similar comparison using the DRM assay, relative raft affinity (r _rel ) of 0.468 and 0.536 were obtained, respectively. Thus, in particular, raft affinity moieties containing an ether or amine function at position 3 of the steroid-derived backbone or position 1 of the sphingosine-derived structure are preferred over similar moieties having amide or ester functions at these positions. . This is also true in terms of chemical stability, since ether and amine functional groups are more stable than amide and ester functional groups against solvolysis and enzyme-mediated cleavage, The group is more stable than the ester functionality.

  To evaluate the effect of the linker moiety on the raft affinity of a known raft affinity moiety, a 12-amino-4,7,10-trioxadodecanoic acid linker was added in a manner that conjugated 200b to the 12-amino functional group. Via a modified rhodamine dye, a raft affinity moiety 200b was attached, and the N-terminus of the modified dye building block was attached to the C-terminus. Raft affinity (Rf) 58.6 was calculated using the LRA assay. When testing a similar complex made with a peptide linker of the sequence Arg-Arg-βAla, Rf42.6 was obtained, and when testing a similar conjugate made with the peptide linker of the sequence Rf24.1 was obtained. Thus, to obtain high raft affinity, linkers made from polyethers are preferred over linkers made from peptides, and a linker containing an alginate unit in the vicinity of the raft affinity moiety is Is preferred over linkers having Furthermore, the qualitative solubility assessment of compounds 24b and 25b clearly demonstrated the higher solubility of compound 24b with a polyether linker in aqueous media.

The invention will also be described by reference to the following chemical, biological and biochemical examples, which are illustrative only and limit the scope of the invention. is not.
Also. The present invention will also be described in the following drawings. The attached drawings are as shown below.

3GI：

Glu(Rho)：

Abbreviation
DCC: N, N'-dicyclohexylcarbodiimide
Dde: 1- (4,4-dimethyl-2,6-dioxocyclohexylidene) ethyl
DIPEA: Diisopropylethylamine
DMAP: Dimethylaminopyridine
DMF: Dimethylformamide
Fmoc: N-α- (9-fluorenylmethyloxycarbonyl)
HATU: 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate
HBTU: 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate
NMO: N-methylmorpholine N-oxide
Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5ylsulfonyl
PE: Petroleum ether
RT: Retention time
TBDMS: tert-butyldimethylsilyl
TBDPS: tert-butyldiphenylsilyl
THF: tetrahydrofuran
Trt: Trityl βAla: β-alanine
Sta: statin
Chol: Cholesteryl
Dhc: Dihydrocholesteryl
Glc: -O-CH ₂ -CO-
_{Succ: -CO-CH 2 -CH 2} -CO-
4GI:

3GI:

Glu (Rho):

General procedure
Acylation to introduce side chains (ceramide)
To a solution of the corresponding acid (1.2 eq) and HATU (1.2 eq) in DMF / CH ₂ Cl ₂ (1: 1) is added DIPEA (2.55 eq) with stirring at room temperature for 5 min. The solution was then added to a solution of 3 (1.0 eq) in DCH ₂ Cl ₂ and stirred at room temperature for 2 hours. The reaction mixture is diluted with CH ₂ Cl ₂ (100 mL), washed with 1 N HCl solution and extracted with CH ₂ Cl ₂ (3 × 100 mL). The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by flash chromatography (silica, PE / EtOAc) to give the product.

Esterification (inositol and glycerol)
To a solution of alcohol (1.0 eq) in CH ₂ Cl ₂ (5 mL) was added DCC (1.4 eq), DMAP (0.66 eq) and the corresponding acid (1.4 eq) and 24 h at room temperature under argon atmosphere. Stir. The solvent is removed in vacuo and the residue is subjected to flash chromatography (silica, petroleum ether / EtOAc) to give the product.

Binding of succinic head group to ceramide Succinic anhydride (1.1 eq) is added to a stirred solution of ceramide (1.0 eq) in CH ₂ Cl ₂ (10 mL). After adding DMAP (1.2 eq), the reaction mixture is stirred at room temperature for 16 h. The mixture is diluted with 50 mL CH ₂ Cl ₂ , washed with 1 N HCl solution and extracted with CH ₂ Cl ₂ (3 × 100 mL). The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by flash chromatography (silica, hexane / EtOAc) to give the product.

Binding of succinic head group to glycerol Succinic anhydride (2.0 eq) is added to a stirred solution of alcohol (1.0 eq) in CH ₂ Cl ₂ (10 mL). After adding DMAP (2.0 eq), the reaction mixture is stirred at room temperature for 48 h. The mixture is diluted with CH ₂ Cl ₂ , washed with saturated NaCl solution and extracted with CH ₂ Cl ₂ .
The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by flash chromatography (silica, PE / EtOAc) to give the product.

Removal of TBDPS groups A solution of tetrabutylammonium fluoride (1 M in THF) (4.25 eq) is added to a solution of TBDPS protected ceramide (1.0 eq) in THF (15 mL) and heated at 60 ° C. for 3 h. The reaction mixture is cooled, diluted with CH ₂ Cl ₂ (100 mL), washed with 1 N HCl solution and extracted with CH ₂ Cl ₂ (3 × 100 mL). The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, hexane / EtOAc / MeOH) to give the product.

Removal of benzyl group
Add 10% palladium / carbon to a solution of benzyl protected inositol (1.0 eq) in a mixture of methanol (5 mL) and CH ₂ Cl ₂ (5 mL) and vigorously under H ₂ atmosphere (800-900 torr) for 24 h. Stir. The reaction mixture is filtered through a short celite pass (followed by washing with methanol / CH ₂ Cl ₂ ) and the solvent is evaporated. The residue is flash chromatographed on a silica gel column (silica, methanol / CH ₂ Cl ₂ ) to give the product.

Deallylation
To a solution of O-allyl-inositol (1.0 eq) in a mixture of CH ₂ Cl ₂ (10 mL), acetic acid (19 mL) and H ₂ O (1 mL) was added palladium (II) chloride (1.6 eq), acetic acid. Add sodium (4.0 eq) and stir at room temperature for 24 hours. The solvent is removed in vacuo and the residue is dissolved in EtOAc and washed with saturated NaHCO ₃ . The organic layers are combined, washed with brine, dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, petroleum ether / EtOAc) to give the product.

ジクロロメタン(5ml)中の3−O−^tブチルジフェニルシリル−D−エリスロ−C₁₆−セラミド(0.85g、1.09mmol)の溶液に、ジメチルアミノピリジン(0.25g、2.1mmol)および無水コハク酸(0.21g、2.1mmol)を加える。得られるスラリーを室温にて2日間撹拌して、明黄色溶液を得る。ジクロロメタン(50ml)で希釈した後、反応混合物を1M HClおよびH₂Oで洗浄し、乾燥(硫酸ナトリウム)する。粗収量：0.86g 無色油状物。
テトラヒドロフラン(4ml)中の上述の粗物質(0.82g、0.94mmol)およびフッ化テトラブチルアンモニウム(75%in H₂O、1.05g、3mmol)の明黄色溶液を60℃にて3時間撹拌する。室温に冷却した後、反応混合物に1M HCl(50ml)を加えて反応を停止し、酢酸エチル(50ml)で抽出する。有機相を分離し、1M HClおよびH₂Oで洗浄し、乾燥(硫酸ナトリウム)する。粗物質(0.69g、白色個体)をシリカゲルカラムクロマトグラフィー(石油エーテル/酢酸エチル/メタノール 10：10：1)により精製して、0.3gのコハク酸モノ(D−エリスロ−C₁₆−セラミジル)エステルを白色個体で得る。
¹H−NMR(300 MHz、CDCl₃)：d＝0.88(t、6H)、1.26(s、46H)、1.60(m、2H)、2.03(q、2H)、2.20(dt、2H)、2.64(m、4H)、4.19(m、3H)、4.33(m、1H)、5.46(dd、1H)、5.75(dt、1H)、6.10(d、1H). MS(ESI)：m/z＝660(M+Na)。 Example 1: Succinic acid mono (D-erythro -C ₁₆ - Seramijiru) esters, preparation of the precursor portions 400aa

Dichloromethane (5ml) solution of ^{3-O-t-butyldiphenylsilyl} -D- erythro -C ₁₆ - ceramide (0.85 g, 1.09 mmol) to a solution of dimethylaminopyridine (0.25 g, 2.1 mmol) and succinic anhydride (0.21 g, 2.1 mmol) is added. The resulting slurry is stirred at room temperature for 2 days to give a light yellow solution. After dilution with dichloromethane (50 ml), the reaction mixture is washed with 1M HCl and H ₂ O and dried (sodium sulfate). Crude yield: 0.86 g colorless oil.
A light yellow solution of the above crude material (0.82 g, 0.94 mmol) and tetrabutylammonium fluoride (75% in H ₂ O, 1.05 g, 3 mmol) in tetrahydrofuran (4 ml) is stirred at 60 ° C. for 3 hours. After cooling to room temperature, the reaction mixture is quenched with 1M HCl (50 ml) and extracted with ethyl acetate (50 ml). The organic phase is separated, washed with 1M HCl and H ₂ O and dried (sodium sulfate). The crude material (0.69 g, white solid) was purified by silica gel column chromatography (petroleum ether / ethyl acetate / methanol 10: 10: 1) to give 0.3 g of succinic acid mono (D-erythro-C ₁₆ -ceramidyl) ester. Is obtained as a white solid.
¹ H-NMR (300 MHz, CDCl ₃ ): d = 0.88 (t, 6H), 1.26 (s, 46H), 1.60 (m, 2H), 2.03 (q, 2H), 2.20 (dt, 2H), 2.64 (m, 4H), 4.19 (m, 3H), 4.33 (m, 1H), 5.46 (dd, 1H), 5.75 (dt, 1H), 6.10 (d, 1H). MS (ESI): m / z = 660 (M + Na).

ジクロロメタン(5ml)中の3−O−^tブチルジフェニルシリル−D−エリスロ−C₂₀−セラミド(1g、1.2mmol)の溶液に、ジメチルアミノピリジン(0.32g、2.6mmol)および無水コハク酸(0.22g、2.2mmol)を加える。得られるスラリーを室温にて3時間撹拌する。ジクロロメタン(50ml)で希釈した後、反応混合物を1M HClおよびH₂Oで洗浄し、乾燥(硫酸ナトリウム)する。粗収量：1.02g 無色油状物。
粗物質(1.02g、1.09mmol)およびTBAF(75%水溶液、1.05g、3mmol)をTHF(3ml)に溶解して、明黄色溶液を得、50℃にて3時間撹拌する。室温に冷却した後、反応混合物に1M HCl(50ml)を加えて反応を停止し、酢酸エチル(50ml)で抽出する。有機相を分離し、水性飽和塩化ナトリウム溶液で洗浄し、乾燥(硫酸ナトリウム)する。粗物質(0.89g 白色個体)をシリカゲルカラムクロマトグラフィー(石油エーテル/酢酸エチル/メタノール 10：10：1)により精製して、0.14gのコハク酸モノ(D−エリスロ−C₂₀−セラミジル)エステルを白色個体で得る。
¹H−NMR(300 MHz、CDCl₃)：d＝0.88(t、6H)、1.25(s、54H)、1.60(m、2H)、2.03(q、2H)、2.20(dt、2H)、2.64(m、4H)、4.18−4.33(m、4H)、5.46(dd,1H)、5.75(dt、1H)、6.05(d、1H)。
MS(ESI)：m/z＝716(M+Na)。 Example 2: Succinic acid mono (D-erythro -C ₂₀ - Seramijiru) esters, preparation of the precursor portion 400af

Dichloromethane (5ml) solution of ^{3-O-t-butyldiphenylsilyl} -D- erythro -C ₂₀ - ceramide (1 g, 1.2 mmol) to a solution of dimethylaminopyridine (0.32 g, 2.6 mmol) and succinic anhydride (0.22 g 2.2 mmol). The resulting slurry is stirred at room temperature for 3 hours. After dilution with dichloromethane (50 ml), the reaction mixture is washed with 1M HCl and H ₂ O and dried (sodium sulfate). Crude yield: 1.02 g colorless oil.
The crude material (1.02 g, 1.09 mmol) and TBAF (75% aqueous solution, 1.05 g, 3 mmol) are dissolved in THF (3 ml) to give a light yellow solution and stirred at 50 ° C. for 3 hours. After cooling to room temperature, the reaction mixture is quenched with 1M HCl (50 ml) and extracted with ethyl acetate (50 ml). The organic phase is separated, washed with aqueous saturated sodium chloride solution and dried (sodium sulfate). The crude material (0.89 g white solid) was purified by silica gel column chromatography (petroleum ether / ethyl acetate / methanol 10: 10: 1) to give 0.14 g succinic mono (D-erythro-C ₂₀ -ceramidyl) ester. Obtained in white individuals.
¹ H-NMR (300 MHz, CDCl ₃ ): d = 0.88 (t, 6H), 1.25 (s, 54H), 1.60 (m, 2H), 2.03 (q, 2H), 2.20 (dt, 2H), 2.64 (m, 4H), 4.18-4.33 (m, 4H), 5.46 (dd, 1H), 5.75 (dt, 1H), 6.05 (d, 1H).
MS (ESI): m / z = 716 (M + Na).

ジクロロメタン(5ml)中の3−O−^tブチルジフェニルシリル−4,5−デヒドロ−D−エリスロ−C₁₆−セラミド(0.84g、1.08 mmol)の溶液に、ジメチルアミノピリジン(0.33g、2.7mmol)および無水コハク酸(0.2g、2mmol)を加える。得られるスラリーを室温にて2時間撹拌する。ジクロロメタン(50ml)で希釈した後、反応混合物を1M HClおよびH₂Oで洗浄し、乾燥(硫酸ナトリウム)する。粗収量：0.83g 無色油状物。
粗物質およびフッ化テトラブチルアンモニウム(75%水溶液、1.1g、3.2mmol)をテトラヒドロフラン(4ml)に溶解して、明黄色溶液を得、60℃にて3.5時間撹拌する。室温に冷却した後、反応混合物にH₂O(50ml)を加えて反応を停止し、酢酸エチル(50ml)で抽出する。有機相を分離し、水性飽和塩化ナトリウム溶液で洗浄し、乾燥(硫酸ナトリウム)する。ロウ状の明黄色固体である粗物質(0.82g)をシリカゲルカラムクロマトグラフィー(石油エーテル/酢酸エチル/メタノール 10：10：1)により精製して、0.28gのコハク酸モノ(D−エリスロ−C₁₆−セラミジル)エステルを白色個体で得る。
¹H−NMR(300 MHz、CDCl₃)：d＝0.88(t、6H)、1.26(s、44H)、1.49(q、2H)、1.62(m、2H)、2.22(q、4H)、2.65(br s、4H)、4.27(t、1H)、4.36(m、2H)、4.53(br s、1H)、6.13(d、1H)。
MS(ESI)：m/z＝658(M+Na)。 Example 3: Succinic acid mono (D-erythro -C ₁₆ - Seramijiru) esters, preparation of the precursor portions 400ad

Dichloromethane ^{3-O-t-butyldiphenylsilyl-4,5-dehydro} -D- erythro -C ₁₆ in (5 ml) - ceramide (0.84 g, 1.08 mmol) to a solution of dimethylaminopyridine (0.33 g, 2.7 mmol) And succinic anhydride (0.2 g, 2 mmol) is added. The resulting slurry is stirred at room temperature for 2 hours. After dilution with dichloromethane (50 ml), the reaction mixture is washed with 1M HCl and H ₂ O and dried (sodium sulfate). Crude yield: 0.83 g colorless oil.
The crude material and tetrabutylammonium fluoride (75% aqueous solution, 1.1 g, 3.2 mmol) are dissolved in tetrahydrofuran (4 ml) to give a light yellow solution and stirred at 60 ° C. for 3.5 hours. After cooling to room temperature, the reaction mixture is quenched with H ₂ O (50 ml) and extracted with ethyl acetate (50 ml). The organic phase is separated, washed with aqueous saturated sodium chloride solution and dried (sodium sulfate). The crude material (0.82 g) as a waxy light yellow solid was purified by silica gel column chromatography (petroleum ether / ethyl acetate / methanol 10: 10: 1) to give 0.28 g of monosuccinic acid (D-erythro-C). ₁₆ -ceramidyl) ester is obtained in a white solid.
¹ H-NMR (300 MHz, CDCl ₃ ): d = 0.88 (t, 6H), 1.26 (s, 44H), 1.49 (q, 2H), 1.62 (m, 2H), 2.22 (q, 4H), 2.65 (br s, 4H), 4.27 (t, 1H), 4.36 (m, 2H), 4.53 (br s, 1H), 6.13 (d, 1H).
MS (ESI): m / z = 658 (M + Na).

文献の手順にしたがって、化合物1を得る。
DMF(25 mL)中の1(10.9 g、24.8 mmol)、イミダゾール(3.4 g、50 mmol)およびTBDPSCl(10.4 mL、40 mmol)の溶液を80℃にて3時間およびさらに100℃にて2時間撹拌する。反応混合物を室温に冷却し、H₂O(300 mL)を加えて反応を停止し、Et₂O(2x150 mL)で抽出する。有機層を合わせ、1 N HCl(100 mL)溶液、飽和NaHCO₃溶液(100 mL)およびH₂O(200 mL)で洗浄し、乾燥(硫酸ナトリウム)し、濃縮する。残渣をクロマトグラフィー(シリカ、PE/EtOAc 30：1)により精製して、化合物2を無色油状物で得る(13.7 g、81%)。
¹H−NMR(300 MHz、CDCl₃)：δ＝0.86(m、3H)、1.03(s、12H)、1.16(m、18H)、1.39(m、15H)、1.63(br s、2H)、3.85(m、2H)、4.12(m、2H)、4.90(m、1H)、5.18(m、1H)、7.34(m、6H)、7.61(m、4H)。 Example 4 Preparation of Precursor of Part 400al The following reaction sequence yields a precursor of compound 400al:

Compound 1 is obtained according to literature procedures.
A solution of 1 (10.9 g, 24.8 mmol), imidazole (3.4 g, 50 mmol) and TBDPSCl (10.4 mL, 40 mmol) in DMF (25 mL) at 80 ° C. for 3 hours and further at 100 ° C. for 2 hours. Stir. The reaction mixture is cooled to room temperature, quenched with H ₂ O (300 mL) and extracted with Et ₂ O ( ₂ × 150 mL). The organic layers are combined, washed with 1 N HCl (100 mL) solution, saturated NaHCO ₃ solution (100 mL) and H ₂ O (200 mL), dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, PE / EtOAc 30: 1) to give compound 2 as a colorless oil (13.7 g, 81%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 0.86 (m, 3H), 1.03 (s, 12H), 1.16 (m, 18H), 1.39 (m, 15H), 1.63 (br s, 2H), 3.85 (m, 2H), 4.12 (m, 2H), 4.90 (m, 1H), 5.18 (m, 1H), 7.34 (m, 6H), 7.61 (m, 4H).

1M HCl (25 mL) is added to a solution of 2 (13.7 g, 20.2 mmol) in 1,4-dioxane (150 mL) and heated at 100 ° C. for 1 h. The reaction is cooled to room temperature, quenched with saturated NaHCO ₃ (100 mL) solution and extracted with Et ₂ O ( ₂ × 150 mL). The organic layers are combined, washed with brine (100 mL), dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, CH ₂ Cl ₂ / MeOH 20: 1) to give 3 as a light yellow oil (7.97 g, 73%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 0.81 (m, 3H), 1.05 (s, 9H), 1.14 (m, 22H), 1.81 (m, 2H), 2.02 (br s, 3H), 2.80 (m, 1H), 3.42 (m, 1H), 3.59 (m, 1H), 4.01 (m, 1H), 5.21 (m, 2H), 7.31 (m, 6H), 7.62 (m, 4H).

To a solution of 3 (1.076 g, 2.0 mmol) in CH ₂ Cl ₂ (20 mL) is added DMAP (488.7 mg, 4.0 mmol) and TBDMSCl (0.603 g, 4.0 mmol). The mixture is stirred at room temperature for 16 hours. The reaction mixture is diluted with CH ₂ Cl ₂ (100 mL), washed with 1 N HCl solution and extracted with CH ₂ Cl ₂ (3 × 100 mL). The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, CH ₂ Cl ₂ / MeOH 20: 1) to give 4 as a light yellow oil (1.31 g, 100%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = −0.10 (m, 6H), 0.72-0.82 (m, 21H), 0.92-1.16 (s, 22H), 1.73 (m, 2H), 3.08 (m , 1H), 3.67 (d, J = 5.6 Hz, 2H), 4.23 (m, 1H), 5.16 (m, 2H), 7.22 (m, 6H), 7.51 (m, 4H).

To a solution of 4 (1.311 g, 2.01 mmol) in CH ₂ Cl ₂ (20 mL) is added DMAP (492 mg, 4.03 mmol) and 1-hexadecanesulfonyl chloride (1.334 g, 4.10 mmol). The mixture is heated to reflux for 20 hours. The reaction mixture is diluted with CH ₂ Cl ₂ (100 mL), quenched with H ₂ O (1000 mL), washed with NaCl solution and extracted with CH ₂ Cl ₂ (3 × 100 mL). The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, CH ₂ Cl ₂ / MeOH 20: 1) to give 5 as a light yellow oil (1.486 g, 79%). The crude product is subjected to the next step. The crude product is subjected to the next step.

1M HCl (10 mL) is added to a solution of 5 (1.486 g, 1.58 mmol) in dioxane (10 mL) and heated at 80 ° C. for 2 hours and further at 100 ° C. for 2 hours. The reaction is quenched with saturated NaHCO ₃ (50 mL) solution and extracted with CH ₂ Cl ₂ (3 × 100 mL). The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, hexane / EtOAc 4: 1) to give 6 as a waxy solid (642 mg, 49%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 0.86 (m, 6H), 1.07 (s, 9H), 1.26 (m, 46H), 1.73 (m, 5H), 2.22 (m, 1H), 2.90 (m, 1H), 3.34 (m, 1H), 3.62 (m, 1H), 3.70 (s, 2H), 3.83 (m, 1H), 4.32 (m, 1H), 4.59 (d, J = 8.06 Hz, 1H), 5.32 (m, 2H), 7.33 (m, 6H), 7.60 (m, 4H).
MS (ESI): m / z = 843.6 (M + NH ₄ )

Coupling the succinic head group as described in the general procedure gives compound 7 (598 mg; 89%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 0.86 (m, 6H), 1.05 (s, 9H), 1.26 (m, 46H), 1.70 (m, 5H), 2.09 (m, 1H), 2.22 (m, 1H), 2.58 (m, 5H), 2.89 (m, 2H), 3.65 (m, 1H), 4.22 (m, 2H), 4.41 (d, J = 8.7 Hz, 1H), 5.30 (m, 2H), 7.33 (m, 6H), 7.60 (m, 4H).
MS (ESI): m / z = 943.6 (M + NH ₄ )

The protecting group is removed according to the general procedure to give compound 400al (300 mg; 72%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 0.86 (m, 6H), 1.26 (s, 46H), 1.52 (m, 1H), 1.70 (m, 7H), 2.09 (m, 1H), 2.24 (m, 2H), 2.47 (m, 2H), 2.52 (br s, 2H), 3.03 (m, 1H), 3.39 (m, 4H), 4.21 (m, 2H), 4.85 (d, J = 8.7 Hz , 1H), 5.30 (m, 1H), 5.78 (m, 1H).
MS (ESI): m / z = 705.5 (M + NH ₄ )

CH₂Cl₂₍10 mL)中の実施例1で得た化合物(98 mg、0.154 mmol)の溶液に、NMO(19 mg、0.162 mmol)およびOsO₄(39 mg、0.154 mmol)を加える。混合物を室温にて3時間撹拌し、次いで、50 mL CH₂Cl₂で希釈し、H₂O(250 mL)、次いで、1N HClで洗浄し、CH₂Cl₂で抽出する。有機層を合わせ、乾燥(硫酸ナトリウム)し、濃縮する。残渣をクロマトグラフィ(シリカ、CH₂Cl₂/MeOH 1：1)ーにより精製して、部分400akの前駆体をロウ状固体で得る(106 mg、100%)。
MS(ESI)：m/z＝672.5(M+1) Example 5: Preparation of precursor of part 400ak

To a solution of the compound obtained in Example 1 (98 mg, 0.154 mmol) in CH ₂ Cl _{2 (} 10 mL) is added NMO (19 mg, 0.162 mmol) and OsO ₄ (39 mg, 0.154 mmol). The mixture is stirred at room temperature for 3 hours, then diluted with 50 mL CH ₂ Cl ₂ , washed with H ₂ O (250 mL), then 1N HCl, and extracted with CH ₂ Cl ₂ . The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, CH ₂ Cl ₂ / MeOH 1: 1) to give a partial 400 ak precursor as a waxy solid (106 mg, 100%).
MS (ESI): m / z = 672.5 (M + 1)

前記実施例4に記載のとおり、化合物3を製造する。
ヘキサデシルイソシアネート(0.81 mL、2.6 mmol)をCH₂Cl₂(5 mL)中の3の溶液に加え、室温にて2時間撹拌する。反応混合物をCH₂Cl₂(100 mL)で希釈し、1 N HCl溶液で洗浄し、CH₂Cl₂(3x100 mL)で抽出する。有機層を合わせ、乾燥(硫酸ナトリウム)し、濃縮する。残渣をクロマトグラフィー(シリカ、PE/EtOAc 3：1)により精製して、生成物13を無色油状物で得る(0.72 g、35%)。
一般手順に記載のとおり、コハク酸頭部基を結合して、化合物14を得る(780 mg；97%)。粗生成物を次工程に付す。
一般手順にしたがって、保護基を除去して、部分400apの前駆体(440 mg；76%)を得る。
¹H−NMR(300 MHz、CDCl₃)：δ＝0.76(m、6H)、1.14(s、49H)、1.35(m、1H)、1.90(m、2H)、2.50(m、4H)、2.96(m、2H)、3.81(m、1H)、3.97(m、1H)、4.04(m、1H)、4.11(m、1H)、5.33(m、1H)、5.58(m、1H)。
MS(ESI)：m/z＝667.5(M+1) Example 6: Preparation of precursor of part 400ap According to the reaction sequence, the precursor of part 400ap is obtained.

Compound 3 is prepared as described in Example 4 above.
Hexadecyl isocyanate (0.81 mL, 2.6 mmol) is added to a solution of 3 in CH ₂ Cl ₂ (5 mL) and stirred at room temperature for 2 hours. The reaction mixture is diluted with CH ₂ Cl ₂ (100 mL), washed with 1 N HCl solution and extracted with CH ₂ Cl ₂ (3 × 100 mL). The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, PE / EtOAc 3: 1) to give product 13 as a colorless oil (0.72 g, 35%).
Coupling the succinic head group as described in the general procedure gives compound 14 (780 mg; 97%). The crude product is subjected to the next step.
Following the general procedure, the protecting group is removed to give a precursor of the portion 400ap (440 mg; 76%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 0.76 (m, 6H), 1.14 (s, 49H), 1.35 (m, 1H), 1.90 (m, 2H), 2.50 (m, 4H), 2.96 (m, 2H), 3.81 (m, 1H), 3.97 (m, 1H), 4.04 (m, 1H), 4.11 (m, 1H), 5.33 (m, 1H), 5.58 (m, 1H).
MS (ESI): m / z = 667.5 (M + 1)

前記実施例4に記載のとおり、化合物3を製造する。
DMF/CH₂Cl₂(1：1)(10 mL)の混合物中のパルミチン酸(0.77 g、3.0 mmol)およびHATU(1.142 g、3.0 mmol)の溶液5分間を撹拌する。CH₂Cl₂(10 mL)中のDIPEA(0.825 g、6.38 mmol)および3(1.345 g、2.5 mmol)の溶液を反応混合物に加え、室温にて2時間撹拌し、次いで、100 mL CH₂Cl₂で希釈し、1 N HCl溶液で洗浄し、CH₂Cl₂(3x100 mL)で抽出する。有機層を合わせ、乾燥(硫酸ナトリウム)し、濃縮する。残渣をクロマトグラフィー(シリカ、ヘキサン/EtOAc 4：1)により精製して、16をロウ状固体で得る(1.685 g、87%)。
¹H−NMR(300 MHz、CDCl₃)：δ＝0.84(m、6H)、1.04(s、6H)、1.24(m、44H)、1.49(m、4H)、1.86−2.33(m、6H)、2.79−2.95(m、2H)、3.58(m、1H)、3.85(m、2H)、4.32(m、1H)、5.38(m、2H)、5.92(m、1H)、7.35(m、6H)、7.59(m、4H)。
MS(ESI)：m/z＝776(M+1)
無水THF(15 mL)中の13(217 mg、0.28 mmol)の溶液を0℃に冷却し、LiAlH₄(1M THF溶液)(0.842 mL、0.84 mmol)の溶液を滴下する。混合物を0℃にて2時間、室温にて16時間撹拌する。反応物に水(100 mL)を加えて反応を停止し、CH₂Cl₂(3x100 mL)で抽出する。有機層を合わせ、乾燥(硫酸ナトリウム)し、濃縮する。残渣をクロマトグラフィ(シリカ、EtOAc)ーにより精製して、17を白色個体で得る(83 mg、57%)。
¹H−NMR(300 MHz、CDCl₃)：δ＝0.86(m、6H)、1.26(s、46H)、1.52(m、5H)、2.05(m、2H)、2.43(m、2H)、2.70(m、2H)、3.42(m、1H)、3.73(br s、2H)、4.21(m、1H)、5.42(m、1H)、5.75(m、1H)。
MS(ESI)：m/z＝524.6(M+1)
一般手順に記載のとおり、コハク酸頭部基を結合して、化合物18を得る(98 mg；41%)。
¹H−NMR(300 MHz、CDCl₃)：δ＝0.86(m、6H)、1.26(s、46H)、1.52(m、5H)、2.05(m、2H)、2.54(m、6H)、2.95(m、2H)、3.25(m、1H)、3.97(m、2H)、4.72(m、1H)、5.42(m、1H)、5.75(m、1H)。
MS(ESI)：m/z＝624.5(M+1) Example 7: Preparation of precursor of part 400aj

Compound 3 is prepared as described in Example 4 above.
Stir a solution of palmitic acid (0.77 g, 3.0 mmol) and HATU (1.142 g, 3.0 mmol) in a mixture of DMF / CH ₂ Cl ₂ (1: 1) (10 mL) for 5 min. A solution of DIPEA (0.825 g, 6.38 mmol) and 3 (1.345 g, 2.5 mmol) in CH ₂ Cl ₂ (10 mL) was added to the reaction mixture and stirred at room temperature for 2 hours, then 100 mL CH ₂ Cl Dilute with ₂ and wash with 1 N HCl solution and extract with CH ₂ Cl ₂ (3 × 100 mL). The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, hexane / EtOAc 4: 1) to give 16 as a waxy solid (1.685 g, 87%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 0.84 (m, 6H), 1.04 (s, 6H), 1.24 (m, 44H), 1.49 (m, 4H), 1.86-2.33 (m, 6H) 2.79-2.95 (m, 2H), 3.58 (m, 1H), 3.85 (m, 2H), 4.32 (m, 1H), 5.38 (m, 2H), 5.92 (m, 1H), 7.35 (m, 6H) ), 7.59 (m, 4H).
MS (ESI): m / z = 776 (M + 1)
A solution of 13 (217 mg, 0.28 mmol) in anhydrous THF (15 mL) is cooled to 0 ° C. and a solution of LiAlH ₄ (1M in THF) (0.842 mL, 0.84 mmol) is added dropwise. The mixture is stirred at 0 ° C. for 2 hours and at room temperature for 16 hours. The reaction is quenched with water (100 mL) and extracted with CH ₂ Cl ₂ (3 × 100 mL). The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, EtOAc) to give 17 as a white solid (83 mg, 57%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 0.86 (m, 6H), 1.26 (s, 46H), 1.52 (m, 5H), 2.05 (m, 2H), 2.43 (m, 2H), 2.70 (m, 2H), 3.42 (m, 1H), 3.73 (br s, 2H), 4.21 (m, 1H), 5.42 (m, 1H), 5.75 (m, 1H).
MS (ESI): m / z = 524.6 (M + 1)
Coupling the succinic head group as described in the general procedure provides compound 18 (98 mg; 41%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 0.86 (m, 6H), 1.26 (s, 46H), 1.52 (m, 5H), 2.05 (m, 2H), 2.54 (m, 6H), 2.95 (m, 2H), 3.25 (m, 1H), 3.97 (m, 2H), 4.72 (m, 1H), 5.42 (m, 1H), 5.75 (m, 1H).
MS (ESI): m / z = 624.5 (M + 1)

3−O−p−メトキシベンジル−sn−グリセロール
無水ジメチルホルムアミド(60 ml)中の3.0 g(22.70 mmol)(R)−2,2−ジメチル−1,3−ジオキサラン−4−メタノールの溶液をアルゴン雰囲気下で0℃に冷却し、塩化p−メトキシベンジル(3.70 ml、4.27 g、27.24 mmol)を加える。15分後、NaH(0.625 g、26.05 mmol)をゆっくりと加える。反応混合物を室温まで温め、20時間撹拌する。
反応物に5 ml エタノールを加えて反応を停止する。混合物に水性飽和塩化ナトリウム溶液を注ぎ入れ、水性層を酢酸エチルで2回抽出する。合わせた有機層を水で洗浄し、乾燥(硫酸ナトリウム)し、濾過し、蒸発して、p−メトキシベンジル誘導体を得、さらに精製することなく次工程に用いる。
物質をメタノール(60 ml)および酢酸(50 ml)の混合物に溶解し、室温にて4日間撹拌する。ジオキサンとの連続的共蒸発により溶媒を除去する。残渣をシリカゲルフラッシュクロマトグラフィー(酢酸エチル)により精製して、3−O−p−メトキシベンジル−sn−グリセロール(2.57 g、12.10 mmol)を無色油状物で得る。
¹H−NMR(300 MHz、CDCl₃)：δ＝7.24(d、J＝8.6 Hz、2 H)、6.88(d、J＝8.6 Hz、2 H)、4.42(s、3 H)、3.85−3.94(m、1 H)、3.81(s、3 H)、3.64−3.70(m、2 H)、3.52−3.56(m、2 H)。
¹³C−NMR(125 MHz、CDCl₃)：δ＝159.40(C)、129.72(C)、129.45(CH)、113.90(CH)、73.27(CH₂)、71.53(CH₂)、70.53(CH)、64.12(CH₂)、55.30(CH₃)。 Example 8: Preparation of succinic acid mono (2,3-di-eicosyloxycarbonyl-propyl) ester, precursor of portion 500aa

3-O-p-methoxybenzyl-sn-glycerol A solution of 3.0 g (22.70 mmol) (R) -2,2-dimethyl-1,3-dioxalane-4-methanol in anhydrous dimethylformamide (60 ml) was added to argon. Cool to 0 ° C. under atmosphere and add p-methoxybenzyl chloride (3.70 ml, 4.27 g, 27.24 mmol). After 15 minutes, NaH (0.625 g, 26.05 mmol) is added slowly. The reaction mixture is warmed to room temperature and stirred for 20 hours.
Stop the reaction by adding 5 ml ethanol to the reaction. Pour aqueous saturated sodium chloride solution into the mixture and extract the aqueous layer twice with ethyl acetate. The combined organic layers are washed with water, dried (sodium sulfate), filtered and evaporated to give the p-methoxybenzyl derivative which is used in the next step without further purification.
The material is dissolved in a mixture of methanol (60 ml) and acetic acid (50 ml) and stirred at room temperature for 4 days. The solvent is removed by continuous co-evaporation with dioxane. The residue is purified by silica gel flash chromatography (ethyl acetate) to give 3-Op-methoxybenzyl-sn-glycerol (2.57 g, 12.10 mmol) as a colorless oil.
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 7.24 (d, J = 8.6 Hz, 2 H), 6.88 (d, J = 8.6 Hz, 2 H), 4.42 (s, 3 H), 3.85− 3.94 (m, 1 H), 3.81 (s, 3 H), 3.64-3.70 (m, 2 H), 3.52-3.56 (m, 2 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 159.40 (C), 129.72 (C), 129.45 (CH), 113.90 (CH), 73.27 (CH ₂ ), 71.53 (CH ₂ ), 70.53 (CH) 64.12 (CH ₂ ), 55.30 (CH ₃ ).

(R) -1,2-di-eicosyloxycarbonyl-3- (p-methoxybenzyl) -sn-glycerol
p-Methoxybenzylglycerol (212 mg, 1 mmol), eicosanoic acid (781 mg, 2.5 mmol) and dimethylaminopyridine (24 mg, 0.2 mmol) are dissolved in anhydrous dichloromethane (50 ml) and cooled to 0 ° C. Dicyclohexylcarbodiimide (516 mg, 2.5 mmol) is dissolved in 10 ml anhydrous dichloromethane and slowly added to the stirred reaction. The reaction mixture is warmed to room temperature and stirred for 22 hours. The solvent was removed under reduced pressure and the residue was purified by silica gel flash chromatography (petroleum ether / ethyl acetate: 6: 1) to give (R) -1,2-di-eicosyloxycarbonyl-3- (p-methoxybenzyl). ) -Sn-glycerol (724 mg, 90%) is obtained.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 7.22 (d, J = 8.6 Hz, 2 H), 6.86 (d, J = 8.6 Hz, 2 H), 5.21 (m, 1 H), 4.45 ( m, 2 H), 4.14-4.33 (m, 2 H), 3.79 (s, 3 H), 3.53-3.56 (m, 2 H), 2.30 (t, J = 7.5 Hz, 2 H), 2.28 (t , J = 7.5 Hz, 2 H), 1.54-1.62 (m, 4 H), 1.24 (s, br, 64 H), 0.87 (t, J = 6.8 Hz, 6 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 173.42 (C═O), 173.12 (C═O), 159.30 (C), 129.76 (C), 129.29 (2′CH), 113.80 (2′CH ), 72.95 (CH ₂ ), 70.00 (CH), 67.89 (CH ₂ ), 62.69 (CH ₂ ), 55.25 (CH ₃ ), 34.33 (CH ₂ ), 34.12 (CH ₂ ), 31.92 (CH ₂ ), 29.70 (CH ₂ ), 29.66 (CH ₂ ), 29.50 (CH ₂ ), 29.36 (CH ₂ ), 29.30 (CH ₂ ), 29.13 (CH ₂ ), 29.09 (CH ₂ ), 24.96 (CH ₂ ), 24.88 (CH _{2), 22.69 (CH 2)} , 14.12 (2xCH 3).
MS (ESI): 823.9 (M + Na ^< + ^> ).

(R) -1,2-di-eicosyloxycarbonyl-sn-glycerol
(R) -1,2-di-eicosyloxycarbonyl-3- (p-methoxybenzyl) -sn-glycerol (434 mg, 0.55 mmol) and DDQ 2,3-dichloro-5,6-dicyano-1, 4-Benzoquinone (160 mg, 0.70 mmol) is dissolved in 25 ml dichloromethane. Water (1.5 ml) is added and the mixture is stirred under air for 24 hours.
The solution is filtered and aqueous saturated sodium bicarbonate solution is added. The aqueous layer is extracted twice with dichloromethane (2x50 ml) and the combined organic layers are washed with aqueous saturated sodium bicarbonate solution and aqueous saturated sodium chloride solution, dried (sodium sulfate), filtered and the solvent removed in vacuo . The residue can be used without further purification.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 5.07 (m, 1 H), 4.21-4.33 (m, 2 H), 3.72 (d, J = 4.4 Hz, 2 H), 2.33 (t, J = 7.5 Hz, 2 H), 2.31 (t, J = 7.5 Hz, 2 H), 1.57-1.65 (m, 4 H), 1.26 (s, br, 64 H), 0.87 (t, J = 6.8 Hz, 6H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 173.80 (C═O), 173.43 (C═O), 72.09 (CH), 61.96 (CH ₂ ), 61.57 (CH ₂ ), 34.29 (CH ₂ ) , 34.10 (CH ₂ ), 31.92 (CH ₂ ), 29.70 (CH ₂ ), 29.66 (CH ₂ ), 29.62 (CH ₂ ), 29.48 (CH ₂ ), 29.36 (CH ₂ ), 29.27 (CH ₂ ), 29.12 _{(CH 2), 29.09 (CH} 2), 24.94 (CH 2), 24.89 (CH 2), 22.69 (CH 2), 14.12 (2xCH 3).
MS (ESI): 703.6 (M + Na ^< + ^> ).

Succinic acid mono (2,3-di-eicosyloxycarbonyl-propyl) ester
(R) -1,2-di-eicosyloxycarbonyl-sn-glycerol (272 mg, 0.4 mmol), succinic anhydride (50 mg, 0.5 mmol) and dimethylaminopyridine (61 mg, 0.5 mmol) in anhydrous dichloromethane Dissolve in (25 ml). The reaction mixture is stirred for 20 hours.
Aqueous saturated sodium chloride solution (5 ml) is added and the mixture is stirred for 5 minutes. The mixture is diluted with dichloromethane (50 ml) and water (20 ml). The aqueous layer is extracted twice with dichloromethane (25 ml). The combined organic layers are dried (sodium sulfate), filtered and the solvent is removed. The residue was purified by silica gel flash chromatography (petroleum ether / ethyl acetate: 1: 1) to give succinic acid mono (2,3-di-eicosyloxycarbonyl-propyl) ester (204 mg, 0.26 mmol) as a colorless powder. Get in.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 5.26 (m, 1 H), 4.26-4.33 (m, 2 H), 4.11-4.19 (m, 2 H), 2.62-2.69 (m, 4 H ), 2.31 (t, J = 7.5 Hz, 2 H), 2.30 (t, J = 7.5 Hz, 2 H), 1.56-1.63 (m, 4 H), 1.24 (s, br, 64 H), 0.87 ( t, J = 6.8 Hz, 6 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 175.68 (C = O), 173.37 (C = O), 172.95 (C = O), 171.58 (C = O), 68.71 (CH), 62.63 (CH _{2), 62.01 (CH 2)} , 34.17 (CH 2), 34.04 (CH 2), 31.92 (CH 2), 29.70 (CH 2), 29.66 (CH 2), 29.49 (CH 2), 29.36 (CH 2) 29.28 (CH ₂ ), 29.12 (CH ₂ ), 29.07 (CH ₂ ), 28.71 (CH ₂ ), 28.46 (CH ₂ ), 24.89 (CH ₂ ), 24.88 (CH ₂ ), 22.69 (CH ₂ ), 14.12 (2xCH ₃ ).
MS (ESI): 803.9 (M + Na ^< + ^> ).

(R)−1,2−O−ジ−エイコシル−3−O−ベンジル−sn−グリセロール(5)
無水DMF(20 mL)中の3−O−ベンジル−sn−グリセロール4(182 mg、1 mmol)の溶液に、エイコシルブロミド(904 mg、2.5 mmol)およびNaH(60%、100 mg、2.5 mmol)をアルゴン雰囲気下で加え、100℃にて22時間、さらに130℃にて24時間加熱する。反応混合物を室温に冷却し、CH₂Cl₂(50 mL)で希釈し、H₂Oで洗浄する。有機層を合わせ、乾燥(硫酸ナトリウム)し、濃縮する。残渣をクロマトグラフィー(シリカ、PE/EtOAc 20：1)により精製して、化合物5を無色ロウ状固体で得る(493 mg、66 %)。
¹H−NMR(300 MHz、CDCl₃)：δ＝7.28−7.34(m、5 H)、4.55(s、2 H)、3.48−3.59(m、5 H)、3.42(t、J＝6.6 Hz、2 H)、1.52−1.57(m、4 H)、1.20−1.35(m、68 H)、0.88(t、J＝6.7 Hz、6 H)。
MS(ESI)：743.7(M＋H⁺)、760.7(M＋NH₄ ⁺) Example 9 Preparation of Precursor for Part 500ae The following reaction sequence yields a precursor for compound 500ae.

(R) -1,2-O-di-eicosyl-3-O-benzyl-sn-glycerol (5)
To a solution of 3-O-benzyl-sn-glycerol 4 (182 mg, 1 mmol) in anhydrous DMF (20 mL) was added eicosyl bromide (904 mg, 2.5 mmol) and NaH (60%, 100 mg, 2.5 mmol). ) In an argon atmosphere and heated at 100 ° C. for 22 hours and further at 130 ° C. for 24 hours. The reaction mixture is cooled to room temperature, diluted with CH ₂ Cl ₂ (50 mL) and washed with H ₂ O. The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, PE / EtOAc 20: 1) to give compound 5 as a colorless waxy solid (493 mg, 66%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 7.28-7.34 (m, 5 H), 4.55 (s, 2 H), 3.48-3.59 (m, 5 H), 3.42 (t, J = 6.6 Hz , 2 H), 1.52-1.57 (m, 4 H), 1.20-1.35 (m, 68 H), 0.88 (t, J = 6.7 Hz, 6 H).
MS (ESI): 743.7 (M + H ⁺ ), 760.7 (M + NH ₄ ⁺ )

(R) -1,2-O-di-eicosyl-3-O-benzyl-sn-glycerol (6)
The benzyl group is removed as described in the general procedure to give compound 6 as a colorless waxy solid (349 mg, 81%)).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 3.41-3.71 (m, 9 H), 1.52-1.57 (m, 4 H), 1.20-1.39 (m, 68 H), 0.88 (t, J = 6.7 Hz, 6 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 78.24 (CH), 71.86 (CH ₂ ), 70.93 (CH ₂ ), 70.39 (CH ₂ ), 63.13 (CH ₂ ), 31.92 (CH ₂ ), 30.08 _{(CH 2), 29.70 (CH} 2), 29.62 (CH 2), 29.47 (CH 2), 29.36 (CH 2), 26.10 (CH 2), 22.68 (CH 2), 14.10 (2xCH 3).
MS (ESI): 670.7 (M + NH ₄ ⁺ ), 675.7 (M + Na ⁺ )

(R) -1,2-O-di-eicosyl-sn-glycerol-3-succinate (7)
Coupling the succinic head group as described in the general procedure gives compound 7 as a colorless waxy solid (345.7 mg, 90%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 4.25 (dd, J = 11.5, 4.3 Hz, 2 H), 3.60-3.63 (m, 1 H), 3.54 (t, J = 6.7 Hz, 2 H ), 3.39-3.47 (m, 4 H), 2.66 (t, J = 3.2 Hz, 4 H) 1.47-1.55 (m, 4 H), 1.20-1.39 (m, 68 H), 0.87 (t, J = 6.7 Hz, 6 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 171.99 (C═O), 171.16 (C═O), 77.43 (CH), 71.80 (CH ₂ ), 70.68 (CH ₂ ), 70.23 (CH ₂ ) , 64.41 (CH ₂ ), 60.39 (2xCH ₂ ), 31.92 (CH ₂ ), 29.99 (CH ₂ ), 29.70 (CH ₂ ), 29.64 (CH ₂ ), 29.48 (CH ₂ ), 29.35 (CH ₂ ), 28.87 _{(CH 2), 26.08 (CH} 2), 26.04 (CH 2), 22.68 (CH 2), 14.18 (CH 3), 14.10 (CH 3).
MS (ESI): 770.8 (M + NH ₄ ⁺ ), 775.7 (M + Na ⁺ )

文献に記載のとおり、化合物9を得る(J. Org. Chem. 2003、68、4020)。
一般手順に記載したとおり、エステル化を行って、化合物10をロウ状固体で得る(583 mg、98 %)。
¹H−NMR(500 MHz、CDCl₃)：δ＝7.25−7.34(m、17 H)、6.85(d、J＝8.6 Hz、2 H)、5.94−6.00(m、1 H)、5.82(t、J＝2.7 Hz、1 H)、5.26(dd、J＝17.2、1.6 Hz、1 H)、5.15(dd、J＝10.4、1.3 Hz、1 H)、4.80−4.87(m、3 H)、4.76(d、J＝10.6 Hz、1 H)、4.71(d、J＝11.2 Hz、1 H)、4.63(d、J＝10.8 Hz、1 H)、4.50(d、J＝11.2 Hz、1 H)、4.44(d、J＝10.8 Hz、1 H)、4.36(dd、J＝12.1、5.7 Hz、1 H)、4.25(dd、J＝12.1、5.7 Hz、1H)、3.81(t、J＝9.6 Hz、1 H)、3.80(s、3 H)、3.69(t、J＝9.5 Hz、1 H)、3.36−3.45(m、3 H)、2.36(t、J＝7.3 Hz、2 H)、1.58−1.63(m、2 H)、1.16−1.29(m、32 H)、0.87(t、J＝6.9 Hz、3 H)。
¹³C−NMR(125 MHz、CDCl₃)：δ＝173.28(C=O)、159.24(CH)、138.71(C)、138.52(C)、137.72(C)、135.30(CH)、129.93(C)、129.62(CH)、128.41(CH)、128.32(CH)、128.21(CH)、128.11(CH)、127.98(CH)、127.72(CH)、127.57(CH)、116.63(CH₂)、113.71(CH)、82.91(CH)、81.36(CH)、81.12(CH)、78.34(CH)、77.90(CH)、76.29(CH₂)、75.91(CH₂)、74.61(CH₂)、72.11(CH₂)、66.37(CH)、55.23(CH₃)、34.51(CH₂)、32.79(CH₂)、31.91(CH₂)、30.90(CH₂)、29.70(CH₂)、29.65(CH₂)、29.61(CH₂)、 29.50(CH₂)、29.47(CH₂)、29.40(CH₂)、29.35(CH₂)、29.27(CH₂)、28.97(CH₂)、26.41(CH₂)、25.51(CH₂)、25.45(CH₂)、25.32(CH₂)、24.70(CH₂)、22.68(CH₂)、14.12(CH₃)。 Example 10 Preparation of Precursor for Part 700a The following reaction sequence provides a precursor for compound 700a.

Compound 9 is obtained as described in the literature (J. Org. Chem. 2003, 68, 4020).
Esterification is performed as described in the general procedure to give compound 10 as a waxy solid (583 mg, 98%).
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 7.25-7.34 (m, 17 H), 6.85 (d, J = 8.6 Hz, 2 H), 5.94-6.00 (m, 1 H), 5.82 (t , J = 2.7 Hz, 1 H), 5.26 (dd, J = 17.2, 1.6 Hz, 1 H), 5.15 (dd, J = 10.4, 1.3 Hz, 1 H), 4.80-4.87 (m, 3 H), 4.76 (d, J = 10.6 Hz, 1 H), 4.71 (d, J = 11.2 Hz, 1 H), 4.63 (d, J = 10.8 Hz, 1 H), 4.50 (d, J = 11.2 Hz, 1 H) ), 4.44 (d, J = 10.8 Hz, 1 H), 4.36 (dd, J = 12.1, 5.7 Hz, 1 H), 4.25 (dd, J = 12.1, 5.7 Hz, 1 H), 3.81 (t, J = 9.6 Hz, 1 H), 3.80 (s, 3 H), 3.69 (t, J = 9.5 Hz, 1 H), 3.36-3.45 (m, 3 H), 2.36 (t, J = 7.3 Hz, 2 H) 1.58-1.63 (m, 2 H), 1.16-1.29 (m, 32 H), 0.87 (t, J = 6.9 Hz, 3 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 173.28 (C═O), 159.24 (CH), 138.71 (C), 138.52 (C), 137.72 (C), 135.30 (CH), 129.93 (C) , 129.62 (CH), 128.41 (CH), 128.32 (CH), 128.21 (CH), 128.11 (CH), 127.98 (CH), 127.72 (CH), 127.57 (CH), 116.63 (CH ₂ ), 113.71 (CH ), 82.91 (CH), 81.36 (CH), 81.12 (CH), 78.34 (CH), 77.90 (CH), 76.29 (CH ₂ ), 75.91 (CH ₂ ), 74.61 (CH ₂ ), 72.11 (CH ₂ ) , 66.37 (CH), 55.23 (CH ₃ ), 34.51 (CH ₂ ), 32.79 (CH ₂ ), 31.91 (CH ₂ ), 30.90 (CH ₂ ), 29.70 (CH ₂ ), 29.65 (CH ₂ ), 29.61 ( CH ₂ ), 29.50 (CH ₂ ), 29.47 (CH ₂ ), 29.40 (CH ₂ ), 29.35 (CH ₂ ), 29.27 (CH ₂ ), 28.97 (CH ₂ ), 26.41 (CH ₂ ), 25.51 (CH ₂ ) ), 25.45 (CH ₂ ), 25.32 (CH ₂ ), 24.70 (CH ₂ ), 22.68 (CH ₂ ), 14.12 (CH ₃ ).

To a solution of 10 (213 mg, 0.24 mmol) in a mixture of acetonitrile (23 mL), toluene (2 mL) and water (1 mL) was added ceric ammonium nitrate (645 mg, 1.18 mmol) at 0 ° C. Stir at 0 ° C. for 30 minutes and warm to room temperature and stir for 2 hours. The reaction mixture is diluted with EtOAc and washed with saturated NaHCO ₃ solution. The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, PE / EtOAc 5: 1) to give compound 11 (162 mg, 88%) as a colorless oil.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 7.25-7.34 (m, 15 H), 5.90-5.98 (m, 1 H), 5.74 (t, J = 2.6 Hz, 1 H), 5.27 (dd , J = 17.2, 1.5 Hz, 1 H), 5.18 (dd, J = 10.4, 1.3 Hz, 1 H), 4.89 (d, J = 10.6 Hz, 1 H), 4.88 (d, J = 10.5 Hz, 1 H), 4.72-4.79 (m, 3 H), 4.49 (d, J = 11.2 Hz, 1 H), 4.42 (dd, J = 12.5, 5.5 Hz, 1 H), 4.23 (dd, J = 12.5, 6.0 Hz, 1H), 3.82 (t, J = 9.5 Hz, 1 H), 3.58-3.64 (m, 2 H), 3.51 (dd, J = 9.6, 2.8 Hz, 1H), 3.46 (t, J = 9.2 Hz , 1 H), 2.39 (t, J = 7.4 Hz, 2 H), 2.29 (s, br 1 H) 1.60-1.67 (m, 2 H), 1.16-1.29 (m, 32 H), 0.87 (t, J = 6.9 Hz, 3 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 173.28 (C═O), 138.57 (C), 138.28 (C), 137.61 (C), 134.78 (CH), 129.45 (CH), 128.34 (CH) 128.21 (CH), 128.08 (CH), 127.93 (CH), 127.79 (CH), 127.76 (CH), 127.61 (CH), 117.39 (CH ₂ ), 83.12 (CH), 81.89 (CH), 81.17 (CH ), 78.50 (CH), 75.96 (CH ₂ ), 75.93 (CH ₂ ), 74.34 (CH ₂ ), 72.08 (CH ₂ ), 70.17 (CH), 68.85 (CH), 34.50 (CH ₂ ), 29.69 (CH _{2), 29.66 (CH 2)} , 29.52 (CH 2), 29.37 (CH 2), 29.35 (CH 2), 29.02 (CH 2), 25.24 (CH 2), 22.68 (CH 2), 14.12 (CH 3) .

Esterification is performed as described in the general procedure to give compound 12 (271 mg, 91%) as a waxy solid.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 7.26-7.32 (m, 15 H), 5.84-5.90 (m, 1 H), 5.73 (t, J = 2.7 Hz, 1 H), 5.22 (dd , J = 17.2, 1.5 Hz, 1 H), 5.12 (dd, J = 10.5, 1.3 Hz, 1 H), 4.80-4.88 (m, 4 H), 4.76 (d, J = 10.6 Hz, 1 H), 4.67 (d, J = 11.1 Hz, 1 H), 4.46 (d, J = 11.1 Hz, 1 H), 4.27 (dd, J = 12.5, 5.5 Hz, 1 H), 4.17 (dd, J = 12.5, 6.0 Hz, 1H), 3.81 (t, J = 9.6 Hz, 1 H), 3.77 (t, J = 9.8 Hz, 1 H), 3.58 (dd, J = 9.7, 2.7 Hz, 1 H), 3.49 (t, J = 9.4 Hz, 1 H) 2.35 (t, J = 7.3 Hz, 2 H), 2.27 (t, J = 7.4 Hz, 2 H) 1.60-1.67 (m, 4 H), 1.16-1.29 (m, 64 H), 0.87 (t, J = 6.9 Hz, 6 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 172.85 (C═O), 172.82 (C═O), 138.55 (C), 138.29 (C), 137.50 (C), 134.82 (CH), 128.44 ( CH), 128.33 (CH), 128.19 (CH), 128.11 (CH), 127.93 (CH), 127.80 (CH), 127.76 (CH), 127.61 (CH), 116.68 (CH ₂ ), 82.84 (CH), 81.30 (CH), 79.07 (CH), 78.10 (CH), 76.31 (CH ₂ ), 75.95 (CH ₂ ), 74.38 (CH ₂ ), 72.14 (CH ₂ ), 71.30 (CH), 67.37 (CH), 34.40 ( CH ₂ ), 34.26 (CH ₂ ), 31.91 (CH ₂ ), 29.71 (CH ₂ ), 29.66 (CH ₂ ), 29.58 (CH ₂ ), 29.48 (CH ₂ ), 29.41 (CH ₂ ), 29.36 (CH ₂ ) ), 29.32 (CH ₂ ), 29.13 (CH ₂ ), 29.04 (CH ₂ ), 25.34 (CH ₂ ), 24.77 (CH ₂ ), 14.12 (2 × CH ₃ ).

The allyl group is removed as described in the general procedure to give compound 13 (199 mg, 79%) as a colorless oil.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 7.26-7.35 (m, 15 H), 5.75 (t, J = 2.7 Hz, 1 H), 4.95 (d, J = 11.1 Hz, 1 H), 4.91 (d, J = 10.7 Hz, 1 H), 4.73-4.79 (m, 4 H), 4.68 (d, J = 11.1 Hz, 1 H), 4.47 (d, J = 11.1 Hz, 1 H), 3.98 (t, J = 10.0 Hz, 1 H), 3.84 (t, J = 9.5 Hz, 1 H), 3.60 (dd, J = 9.7, 2.8 Hz, 1 H), 3.40 (t, J = 9.3 Hz, 1 H) 2.35 (t, J = 7.3 Hz, 2 H), 2.28 (t, J = 7.4 Hz, 2 H) 1.55-1.64 (m, 4 H), 1.16-1.31 (m, 64 H), 0.87 (t , J = 6.9 Hz, 6 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 173.18 (C═O), 172.83 (C═O), 138.42 (C), 138.23 (C), 137.42 (C), 128.64 (CH), 128.38 ( CH), 128.36 (CH), 128.14 (CH), 128.06 (CH), 128.02 (CH), 127.98 (CH), 127.94 (CH), 127.81 (CH), 127.69 (CH), 82.72 (CH), 81.08 (CH) CH), 78.71 (CH), 75.90 (CH ₂ ), 75.80 (CH ₂ ), 72.12 (CH ₂ ), 71.05 (CH), 70.95 (CH), 67.18 (CH), 34.34 (CH ₂ ), 34.13 (CH ₂ ) ₂ ), 31.91 (CH ₂ ), 29.71 (CH ₂ ), 29.65 (CH ₂ ), 29.55 (CH ₂ ), 29.46 (CH ₂ ), 29.36 (CH ₂ ), 29.29 (CH ₂ ), 29.06 (CH ₂ ) 29.05 (CH ₂ ), 25.26 (CH ₂ ), 24.75 (CH ₂ ), 22.68 (CH ₂ ), 14.12 (2 x CH ₃ ).

Conjugation of the succinic head group as described in the general procedure gives compound 14 (126 mg, 62%) as a colorless oil.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 7.22-7.32 (m, 15 H), 5.73 (t, J = 2.7 Hz, 1 H), 5.48 (t, J = 10.2 Hz), 4.83-4.89 (m, 3 H), 4.73 (d, J = 10.6 Hz, 1 H), 4.69 (d, J = 11.1 Hz, 1 H), 4.62 (d, J = 11.3 Hz, 1 H), 4.47 (d, J = 10.1 Hz, 1 H), 3.91 (t, J = 9.5 Hz, 1 H), 3.60 (dd, J = 9.7, 2.8 Hz, 1 H), 3.53 (t, J = 9.5 Hz, 1 H) 2.32 -2.53 (m, 6 H), 2.21 (t, J = 7.4 Hz, 2 H) 1.50-1.70 (m, 4 H), 1.16-1.31 (m, 64 H), 0.87 (t, J = 6.9 Hz, 6H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 172.81 (C = O), 173.06 (C = O), 172.91 (C = O), 170.86 (C = O), 138.32 (C), 138.13 (C ), 137.36 (C), 128.42 (CH), 128.36 (CH), 128.11 (CH), 128.03 (CH), 127.91 (CH), 127.85 (CH), 127.78 (CH), 127.71 (CH), 81.30 (CH ), 80.56 (CH), 77.91 (CH), 76.00 (CH ₂ ), 75.74 (CH ₂ ), 72.19 (CH ₂ ), 71.63 (CH), 69.20 (CH), 66.96 (CH), 34.27 (CH ₂ ) 33.98 (CH ₂ ), 31.93 (CH ₂ ), 29.72 (CH ₂ ), 29.66 (CH ₂ ), 29.55 (CH ₂ ), 29.49 (CH ₂ ), 29.36 (CH ₂ ), 29.30 (CH ₂ ), 29.07 _{(CH 2), 29.05 (CH} 2), 28.75 (CH 2), 28.21 (CH 2), 25.20 (CH 2), 24.70 (CH 2), 22.67 (CH 2), 14.11 (2 x CH 3).

Removal of the benzyl group as described in the general procedure gives compound 15.
MS (ESI): 886.7 (M + NH ₄ ⁺ ), 867.4 (M−H ⁺ )

一般手順に記載したとおり、エステル化を行って、化合物11から化合物18(250 mg、99 %)をロウ状固体で得る。
¹H−NMR(500 MHz、CDCl₃)：δ＝7.26−7.32(m、15 H)、5.83−5.89(m、1 H)、5.72(t、J＝2.7 Hz、1 H)、5.21(dd、J＝17.2、1.6 Hz、1 H)、5.18(dd、J＝10.4、1.3 Hz、1 H)、4.90(dd、J＝10.3、2.8 Hz、1 H)、4.87(d、J＝10.7 Hz、1 H)、4.85(d、J＝10.5 Hz、1 H)、4.81(d、J＝10.4 Hz、1 H)、4.77(d、J＝10.6 Hz、1 H)、4.67(d、J＝11.1 Hz、1 H)、4.47(d、J＝11.1 Hz、1 H)、4.27(dd、J＝12.5、5.6 Hz、1 H)、4.15(dd、J＝12.3、5.3 Hz、1H)、4.07(d、J＝2.1 Hz、2 H)、3.82(t、J＝9.5 Hz、1 H)、3.77(t、J＝9.8 Hz、1 H)、3.58(dd、J＝9.7、2.7 Hz、1H)、3.50(t、J＝9.4 Hz、1 H)、3.30−3.40(m、1 H)、2.35(dt、J＝7.4、2.1 Hz、2 H)、2.30(m、1 H)、1.93−1.97(m、1 H)、1.78−1.86(m、2 H)、1.71(dt、J＝6.8、3.5 Hz、1 H)、1.58−1.66(m、4 H)、1.56(s、3 H)、1.45−1.54(m、4 H)、1.35−1.41(m、1 H)、1.19−1.33(m、32 H)、1.16(s、1 H)、0.93−1.15(m、11 H)、0.84−0.92(m、15 H)、0.79(s、3 H)、0.64(s、3 H)、0.60−0.62(m、1 H)。
¹³C−NMR(125 MHz、CDCl₃)：δ＝172.85(C=O)、170.32(C=O)、138.52(C)、138.24(C)、137.44(C)、134.81(CH)、128.44(CH)、128.35(CH)、128.19(CH)、128.09(CH)、127.92(CH)、127.82(CH)、127.80(CH)、127.63(CH)、116.66(CH₂)、82.74(CH)、81.97(CH)、79.40(CH)、78.97(CH)、76.30(CH₂)、75.95(CH₂)、74.34(CH₂)、72.19(CH₂)、71.83(CH)、67.34(CH)、65.19(CH₂)、60.39(CH₂)、56.46(CH)、56.27(CH)、54.33(CH)、44.75(CH)、42.57(C)、40.01(CH₂)、39.49(CH₂)、36.85(CH₂)、36.15(CH₂)、35.78(CH)、35.69(C)、35.46(CH)、34.41(CH₂)、34.35(CH₂)、32.07(CH₂)、31.92(CH₂)、29.75(CH₂)、29.72(CH₂)、29.66(CH₂)、29.62(CH₂)、29.45(CH₂)、29.36(CH₂)、29.08(CH₂)、28.77(CH₂)、28.00(CH₃)、27.78(CH₂)、25.29(CH₂)、24.20(CH₂)、23.82(CH₂)、22.81(CH₃)、22.69(CH₂)、22.55(CH₃)、21.23(CH₂)、21.05(CH)、18.65(CH₃)、14.13(CH₃)、12.26(CH₃)、12.05(CH₃)。
MS(ESI)：1230.9(M＋NH₄ ⁺)、1235.9(M＋Na⁺)。 Example 11 Preparation of Precursor for Part 700b The following reaction sequence provides a precursor for compound 700b.

Esterification is performed as described in the general procedure to give compound 11 to compound 18 (250 mg, 99%) as a waxy solid.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 7.26-7.32 (m, 15 H), 5.83-5.89 (m, 1 H), 5.72 (t, J = 2.7 Hz, 1 H), 5.21 (dd , J = 17.2, 1.6 Hz, 1 H), 5.18 (dd, J = 10.4, 1.3 Hz, 1 H), 4.90 (dd, J = 10.3, 2.8 Hz, 1 H), 4.87 (d, J = 10.7 Hz , 1 H), 4.85 (d, J = 10.5 Hz, 1 H), 4.81 (d, J = 10.4 Hz, 1 H), 4.77 (d, J = 10.6 Hz, 1 H), 4.67 (d, J = 11.1 Hz, 1 H), 4.47 (d, J = 11.1 Hz, 1 H), 4.27 (dd, J = 12.5, 5.6 Hz, 1 H), 4.15 (dd, J = 12.3, 5.3 Hz, 1H), 4.07 (d, J = 2.1 Hz, 2 H), 3.82 (t, J = 9.5 Hz, 1 H), 3.77 (t, J = 9.8 Hz, 1 H), 3.58 (dd, J = 9.7, 2.7 Hz, 1H ), 3.50 (t, J = 9.4 Hz, 1 H), 3.30-3.40 (m, 1 H), 2.35 (dt, J = 7.4, 2.1 Hz, 2 H), 2.30 (m, 1 H), 1.93- 1.97 (m, 1 H), 1.78-1.86 (m, 2 H), 1.71 (dt, J = 6.8, 3.5 Hz, 1 H), 1.58-1.66 (m, 4 H), 1.56 (s, 3 H) 1.45-1.54 (m, 4 H), 1.35-1.41 (m, 1 H), 1.19-1.33 (m, 32 H), 1.16 (s, 1 H), 0.93-1.15 (m, 11 H), 0.84 -0.92 (m, 15 H), 0.79 (s, 3 H), 0.64 (s, 3 H), 0.60-0.62 (m, 1 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 172.85 (C═O), 170.32 (C═O), 138.52 (C), 138.24 (C), 137.44 (C), 134.81 (CH), 128.44 ( CH), 128.35 (CH), 128.19 (CH), 128.09 (CH), 127.92 (CH), 127.82 (CH), 127.80 (CH), 127.63 (CH), 116.66 (CH ₂ ), 82.74 (CH), 81.97 (CH), 79.40 (CH), 78.97 (CH), 76.30 (CH ₂ ), 75.95 (CH ₂ ), 74.34 (CH ₂ ), 72.19 (CH ₂ ), 71.83 (CH), 67.34 (CH), 65.19 (CH) CH ₂ ), 60.39 (CH ₂ ), 56.46 (CH), 56.27 (CH), 54.33 (CH), 44.75 (CH), 42.57 (C), 40.01 (CH ₂ ), 39.49 (CH ₂ ), 36.85 (CH _{2), 36.15 (CH 2)} , 35.78 (CH), 35.69 (C), 35.46 (CH), 34.41 (CH 2), 34.35 (CH 2), 32.07 (CH 2), 31.92 (CH 2), 29.75 ( CH ₂ ), 29.72 (CH ₂ ), 29.66 (CH ₂ ), 29.62 (CH ₂ ), 29.45 (CH ₂ ), 29.36 (CH ₂ ), 29.08 (CH ₂ ), 28.77 (CH ₂ ), 28.00 (CH ₃ ) ), 27.78 (CH ₂ ), 25.29 (CH ₂ ), 24.20 (CH ₂ ), 23.82 (CH ₂ ), 22.81 (CH ₃ ), 22.69 (CH ₂ ), 22.55 (CH ₃ ), 21.23 (CH ₂ ), 21.05 (CH), 18.65 (CH ₃ ), 14.13 (CH ₃ ), 12.26 (CH ₃ ), 12.05 (CH ₃ ).
MS (ESI): 1230.9 (M + NH 4 +), 1235.9 (M + Na +).

The allyl group is removed as described in the general procedure to give compound 19 (190 mg, 86%) as a waxy solid.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 7.26-7.35 (m, 15 H), 5.74 (t, J = 2.7 Hz, 1 H), 4.95 (d, J = 11.1 Hz, 1 H), 4.91 (d, J = 10.7 Hz, 1 H), 4.84 (dd, J = 10.4, 2.8 Hz, 1 H), 4.76 (d, J = 10.7 Hz, 1 H), 4.73 (d, J = 11.1 Hz, 1 H), 4.68 (d, J = 11.1 Hz, 1 H), 4.48 (d, J = 11.1 Hz, 1 H), 4.03-4.14 (m, 2 H), 3.98 (t, J = 9.8 Hz, 1 H), 3.84 (t, J = 9.5 Hz, 1 H), 3.61 (dd, J = 9.7, 2.8 Hz, 1 H), 3.40 (t, J = 9.3 Hz, 1 H), 3.28-3.37 (m, 1 H), 2.35 (dt, J = 7.5, 3.6 Hz, 2 H), 2.25 (br, 1 H), 1.93-1.97 (m, 1 H), 1.74-1.86 (m, 2 H), 1.71 (m, 1 H), 1.43-1.68 (m, 11 H), 1.35-1.41 (m, 1 H), 1.19-1.33 (m, 34 H), 0.93-1.17 (m, 11 H), 0.84-0.92 (m, 15 H), 0.78 (s, 3 H), 0.63 (s, 3 H), 0.60-0.62 (m, 1 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 172.82 (C═O), 170.62 (C═O), 138.39 (C), 138.19 (C), 137.36 (C), 128.66 (CH), 128.39 ( CH), 128.12 (CH), 128.08 (CH), 127.98 (CH), 127.85 (CH), 127.71 (CH), 82.60 (CH), 81.05 (CH), 79.48 (CH), 79.18 (CH), 75.93 ( CH ₂ ), 75.82 (CH ₂ ), 72.17 (CH ₂ ), 71.60 (CH), 70.84 (CH), 67.15 (CH), 65.16 (CH ₂ ), 60.40 (C), 56.48 (CH), 56.28 (CH ), 54.31 (CH), 44.75 (CH), 42.58 (C), 40.02 (CH ₂ ), 39.50 (CH ₂ ), 36.85 (CH ₂ ), 36.16 (CH ₂ ), 35.79 (CH), 35.69 (C) , 35.46 (CH), 34.43 (CH ₂ ), 34.30 (CH ₂ ), 32.07 (CH ₂ ), 31.93 (CH ₂ ), 29.75 (CH ₂ ), 29.67 (CH ₂ ), 29.60 (CH ₂ ), 29.42 ( CH ₂ ), 29.37 (CH ₂ ), 29.09 (CH ₂ ), 28.77 (CH ₂ ), 28.25 (CH), 28.00 (CH ₂ ), 27.76 (CH ₂ ), 25.22 (CH ₂ ), 24.21 (CH ₂ ) , 22.81 (CH ₃ ), 22.70 (CH ₂ ), 22.55 (CH), 21.23 (CH ₂ ), 18.65 (CH ₃ ), 14.13 (CH ₃ ), 12.26 (CH ₃ ), 12.06 (CH ₃ ).
MS (ESI): 1190.9 (M + NH 4 +), 1195.9 (M + Na +).

Coupling the succinic head group as described in the general procedure provides compound 20 (150 mg, 90%) as a waxy solid.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 7.20-7.32 (m, 15 H), 5.70 (t, J = 2.7 Hz, 1 H), 5.48 (t, J = 10.1 Hz, 1 H), 4.97 (dd, J = 10.6, 2.8 Hz, 1 H), 4.87 (d, J = 10.6 Hz, 1 H), 4.83 (d, J = 11.4 Hz, 1 H), 4.74 (d, J = 10.6 Hz, 1 H), 4.67 (d, J = 11.2 Hz, 1 H), 4.60 (d, J = 11.4 Hz, 1 H), 4.49 (d, J = 11.2 Hz, 1 H), 4.00-4.14 (m, 2 H), 3.93 (t, J = 9.5 Hz, 1 H), 3.60 (dd, J = 9.5, 2.8 Hz, 1 H), 3.52 (t, J = 9.5 Hz, 1 H), 3.33-3.38 (m, 1 H), 2.29-2.54 (m, 6 H), 2.25 (br, 1 H), 1.93-1.97 (m, 1 H), 1.75-1.84 (m, 2 H), 1.69-1.73 (m, 1 H) 1.58-1.65 (m, 5 H), 1.42-1.57 (m, 5 H), 1.19-1.40 (m, 35 H), 0.93-1.17 (m, 11 H), 0.84-0.92 (m, 15 H) 0.77 (s, 3 H), 0.63 (s, 3 H), 0.56-0.61 (m, 1 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 173.28 (C = O), 173.03 (C = O), 170.75 (C = O), 170.11 (C = O), 138.24 (C), 138.02 (C ), 137.24 (C), 128.45 (CH), 128.40 (CH), 128.37 (CH), 128.08 (CH), 128.03 (CH), 127.98 (CH), 127.90 (CH), 127.81 (CH), 127.74 (CH) ), 81.25 (CH), 80.47 (CH), 80.14 (CH), 77.69 (CH), 76.02 (CH ₂ ), 75.78 (CH ₂ ), 72.24 (CH ₂ ), 71.09 (CH), 69.60 (CH), 67.02 (CH), 65.02 (CH ₂ ), 60.40 (C), 56.44 (CH), 56.28 (CH), 54.29 (CH), 44.76 (CH), 42.56 (C), 39.99 (CH ₂ ), 39.49 (CH _{2), 36.79 (CH 2)} , 36.15 (CH 2), 35.78 (CH), 35.64 (C), 35.44 (CH), 34.34 (CH 2), 34.25 (CH 2), 32.04 (CH 2), 31.92 ( _{CH 2), 29.74 (CH 2} ), 29.72 (CH 2), 29.66 (CH 2), 29.61 (CH 2), 29.42 (CH 2), 29.36 (CH 2), 29.08 (CH 2), 28.70 (CH 2 ), 28.23 (CH), 28.00 (CH ₂ ), 27.49 (CH ₂ ), 25.16 (CH ₂ ), 24.19 (CH ₂ ), 23.83 (CH ₂ ), 22.81 (CH ₃ ), 22.69 (CH ₂ ), 22.55 _{(CH), 21.22 (CH 2} ), 18.64 (CH 3), 14.12 (CH 3), 12.23 (CH 3), 12.05 (CH 3).
MS (ESI): 1290.8 (M + NH 4 +), 1295.9 (M + Na +), 1271.7 (M-H +).

Removal of the benzyl group as described in the general procedure gives compound 21 (81.9 mg, 80%) as a colorless solid.
MS (ESI): 1020.8 (M + NH 4 +), 1025.8 (M + Na +), 1001.5 (M-H +).

一般手順に記載したとおり、エステル化を行って、化合物23から化合物24(171.5 mg、80%)をロウ状固体で得る。
¹H−NMR(300 MHz、CDCl₃)：δ＝7.25−7.32(m、15 H)、5.79−5.92(m、1 H)、5.77(t、J＝2.6 Hz、1 H)、5.21(dd、J＝17.2、1.7 Hz、1 H)、5.12(d、J＝11.7 Hz、1 H)、4.93(dd、J＝9.9、2.6 Hz、1 H)、4.83−4.89(m、3 H)、4.78(d、J＝10.8 Hz、1 H)、4.67(d、J＝11.1 Hz、1 H)、4.50(d、J＝11.4 Hz、1 H)、4.24− 4.34(m、2 H)、4.08−4.17(m、4 H)、3.79−3.86(m、2 H)、3.60(dd、J＝10.1、3.0 Hz、1H)、3.52(t、J＝9.3 Hz、1 H)、3.28−3.37(m、2 H)、1.92−1.98(m、2 H)、1.76−1.85(m、4 H)、1.62−1.72(m、8 H)、1.36−1.56(m、10 H) 1.20−1.31(m、18 H)、1.00−1.15(m、16 H)、0.87−0.97(m、20 H)、0.78(d、J＝5.7 Hz、6 H)、0.63(s、6 H)、0.54−0.61(m、2 H)。
MS(ESI)：1365.0(M＋NH₄ ⁺)、1370.0(M＋Na⁺)。 Example 12 Preparation of Precursor for Part 700c The following reaction sequence provides a precursor for compound 700c.

Esterification is performed as described in the general procedure to give compound 24 to compound 24 (171.5 mg, 80%) as a waxy solid.
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 7.25-7.32 (m, 15 H), 5.79-5.92 (m, 1 H), 5.77 (t, J = 2.6 Hz, 1 H), 5.21 (dd , J = 17.2, 1.7 Hz, 1 H), 5.12 (d, J = 11.7 Hz, 1 H), 4.93 (dd, J = 9.9, 2.6 Hz, 1 H), 4.83-4.89 (m, 3 H), 4.78 (d, J = 10.8 Hz, 1 H), 4.67 (d, J = 11.1 Hz, 1 H), 4.50 (d, J = 11.4 Hz, 1 H), 4.24-4.34 (m, 2 H), 4.08 -4.17 (m, 4 H), 3.79-3.86 (m, 2 H), 3.60 (dd, J = 10.1, 3.0 Hz, 1 H), 3.52 (t, J = 9.3 Hz, 1 H), 3.28-3.37 ( m, 2 H), 1.92-1.98 (m, 2 H), 1.76-1.85 (m, 4 H), 1.62-1.72 (m, 8 H), 1.36-1.56 (m, 10 H) 1.20-1.31 (m , 18 H), 1.00-1.15 (m, 16 H), 0.87-0.97 (m, 20 H), 0.78 (d, J = 5.7 Hz, 6 H), 0.63 (s, 6 H), 0.54-0.61 ( m, 2 H).
MS (ESI): 1365.0 (M + NH 4 +), 1370.0 (M + Na +).

The allyl group is removed as described in the general procedure to give compound 25 (123.0 mg, 74%) as a waxy solid.
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 7.26-7.34 (m, 15 H), 5.78 (t, J = 2.5 Hz, 1 H), 4.94 (d, J = 11.1 Hz, 1 H), 4.90 (d, J = 10.7 Hz, 1 H), 4.86 (dd, J = 10.4, 2.6 Hz, 1 H), 4.77 (d, J = 10.9 Hz, 1 H), 4.74 (d, J = 11.2 Hz, 1 H), 4.67 (d, J = 11.1 Hz, 1 H), 4.50 (d, J = 11.1 Hz, 1 H), 4.04-4.21 (m, 4 H), 3.96 (t, J = 9.8 Hz, 1 H), 3.83 (t, J = 9.5 Hz, 1 H), 3.63 (dd, J = 9.7, 2.6 Hz, 1 H), 3.40 (t, J = 9.6 Hz, 1 H), 3.27-3.33 (m, 2 H), 1.92-1.98 (m, 2 H), 1.76-1.85 (m, 4 H), 1.62-1.72 (m, 8 H), 1.36-1.56 (m, 10 H) 1.20-1.31 (m, 18 H ), 1.00-1.15 (m, 16 H), 0.87-0.97 (m, 20 H), 0.78 (d, J = 5.7 Hz, 6 H), 0.63 (s, 6 H), 0.54-0.61 (m, 2 H).
MS (ESI): 1365.0 (M + NH 4 +), 1370.0 (M + Na +).

As described in the general procedure, coupling of the succinic head group followed by removal of the benzyl group affords compound 26 (80 mg, 93% in two steps) as a waxy solid.
MS (ESI): 1154.6 (M + NH ₄ ⁺ ), 1159.7 (M + Na ⁺ ), 1135.6 (M−H ⁺ )

文献に記載のとおり、化合物28を得る(Biochemistry、1994、33、11586)。 Example 13 Preparation of Precursor for Part 1800d The following reaction sequence provides a precursor for compound 1800d.

Compound 28 is obtained as described in the literature (Biochemistry, 1994, 33, 11586).

_{CH 2 Cl 2 (20 mL)} 28 (889.4 mg, 1.85 mmol) in a solution of 4-methoxybenzyl chloride (434.7 mg, 2.78 mmol) and sodium hydride (60%, 111 mg, 2.78 mmol) was added The mixture is stirred at -15 ° C for 1 hour and at room temperature for 18 hours. The reaction mixture is diluted with EtOAc (50 mL), washed with saturated NaCl solution and water, and extracted with EtOAc. The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, PE / EtOAc 6: 1) to give compound 29 (1.023 g, 92%) as a colorless oil.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 7.28-7.36 (m, 12 H), 6.84 (d, J = 8.7 Hz, 2 H), 5.87-6.03 (m, 3 H), 5.27-5.33 (m, 3 H), 5.14-5.18 (m, 3 H), 4.87 (d, J = 10.5 Hz, 1 H), 4.80-4.83 (m, 3 H), 4.65 (d, J = 11.8 Hz, 1 H), 4.58 (d, J = 11.7 Hz, 1 H), 4.27-4.37 (m, 4 H), 4.06-4.12 (m, 2 H), 3.95-4.05 (m, 2 H), 3.81-3.84 ( m, 1 H), 3.80 (s, 3 H), 3.30 (dd, J = 9.9, 2.3 Hz, 1 H), 3.26 (t, J = 9.3 Hz, 1 H), 3.14 (dd, J = 9.9, 2.3 Hz, 1 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 158.91 (C), 138.86 (C), 138.48 (C), 135.51 (CH), 135.40 (CH), 134.98 (CH), 131.03 (C) 129.38 ( CH), 128.28 (CH), 128.25 (CH), 128.13 (CH), 127.47 (CH), 127.44 (CH), 116.46 (CH 2), 116.40 (CH 2), 116.34 (CH 2), 113.43 (CH) , 83.28 (CH), 81.52 (CH), 81.33 (CH), 80.69 (CH), 80.44 (CH), 75.83 (CH ₂ ), 74.55 (CH ₂ ), 74.48 (CH ₂ ), 73.79 (CH), 73.52 _{(CH 2), 72.65 (CH} 2), 71.61 (CH 2), 55.21 (CH 3).
MS (ESI): 618.2 (M + NH ₄ ⁺ ), 623.2 (M + Na ⁺ )

To a solution of 29 (216.5 mg, 0.36 mmol) in ethanol (20 mL), 10% Pd / C (100 mg) and p-toluenesulfonic acid (180 mg, 0.95 mmol) are added and heated to reflux for 2 hours. The solvent is removed in vacuo and the residue is subjected to flash chromatography (silica, EtOAc) to give the product (97 mg, 56%) as a colorless oil.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 7.25-7.34 (m, 12 H), 6.86 (d, J = 8.6 Hz, 2 H), 4.97 (m, 2 H), 4.75 (d, J = 10.1 Hz, 1 H), 4.68 (s, 2 H), 4.60 (d, J = 10.2 Hz, 1 H), 3.99-4.01 (m, 1 H), 3.83 (t, J = 9.5 Hz, 1 H ), 3.79 (s, 3 H), 3.70 (t, J = 9.5 Hz, 1 H), 3.43-3.45 (m, 1 H), 3.32-3.38 (m, 2 H), 3.03 (s, br, 1 H), 2.77 (s, br, 1 H), 2.45 (s, br, 1 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 159.26 (C), 138.57 (C), 138.00 (C), 130.60 (C) 129.48 (CH), 128.48 (CH), 128.47 (CH), 128.39 ( CH), 128.00 (CH), 127.78 (CH), 127.70 (CH), 127.61 (CH), 113.62 (CH), 81.13 (CH), 80.80 (CH), 76.83 (CH), 75.43 (CH ₂ ), 74.53 _{(CH 2), 74.38 (CH} ), 73.59 (CH), 72.85 (CH 2), 72.06 (CH), 55.25 (CH 3).

Esterification is performed as described in the general procedure to give compound 30 to compound 31 (97 mg, 56%) as a waxy solid.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 7.20-7.33 (m, 12 H), 6.83 (d, J = 8.7 Hz, 2 H), 5.56 (t, J = 10.1 Hz, 1 H), 5.10 (t, J = 9.6 Hz, 1 H), 4.84 (d, J = 11.4 Hz, 1 H), 4.78 (dd, J = 10.5, 2.4 Hz, 1 H), 4.73 (d, J = 11.4 Hz, 1 H), 4.59-4.64 (m, 4 H), 4.10 (t, J = 2.2 Hz, 1 H), 4.06 (t, J = 9.7 Hz, 1 H), 3.79 (s, 3 H), 3.57 ( dd, J = 9.7, 2.1 Hz, 1 H), 2.10-2.30 (m, 6 H), 1.40-1.52 (m, 6 H), 1.15-1.30 (m, 96 H), 0.87 (t, J = 6.9 Hz, 9 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 172.95 (C═O), 172.68 (C═O), 172.63 (C═O), 159.20 (C), 138.37 (C), 137.89 (C), 130.24 (C) 129.51 (CH), 128.42 (CH), 128.26 (CH), 127.74 (CH), 127.60 (CH), 127.53 (CH), 127.50 (CH), 113.64 (CH), 80.46 (CH), 78.96 (CH), 75.45 (CH ₂ ), 74.37 (CH ₂ ), 74.09 (CH), 72.94 (CH ₂ ), 72.47 (CH), 71.52 (CH), 69.96 (CH), 55.23 (CH ₃ ), 34.19 ( CH ₂ ), 34.17 (CH ₂ ), 31.92 (CH ₂ ), 29.71 (CH ₂ ), 29.66 (CH ₂ ), 29.52 (CH ₂ ), 29.46 (CH ₂ ), 29.36 (CH ₂ ), 29.34 (CH ₂ ) ), 29.21 (CH ₂ ), 29.19 (CH ₂ ), 24.93 (CH ₂ ), 24.84 (CH ₂ ), 24.80 (CH ₂ ), 22.69 (CH ₂ ), 14.12 (CH ₃ ).

To a solution of 31 (100 mg, 0.073 mmol) in a mixture of CH ₂ Cl ₂ (10 mL) and water (0.2 mL) is added dichlorodicyanobenzoquinone (25 mg, 0.101 mmol) and stirred at room temperature for 5 hours. . The reaction mixture is diluted with CH ₂ Cl ₂ , washed with saturated NaHCO ₃ solution and extracted with CH ₂ Cl ₂ . The organic layers are combined, dried (sodium sulfate) and concentrated. The residue is purified by chromatography (silica, PE / EtOAc 8: 1) to give product 32 (88.6 mg, 97%) as a waxy solid.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 7.22-7.32 (m, 10 H), 5.55 (t, J = 10.2 Hz, 1 H), 5.12 (t, J = 9.9 Hz, 1 H), 4.88 (dd, J = 10.3, 2.8 Hz, 1 H), 4.83 (d, J = 11.4 Hz, 1 H), 4.67 (s, 2 H), 4.62 (d, J = 11.4 Hz, 1 H), 4.28 (t, J = 2.5 Hz, 1 H), 3.98 (t, J = 9.7 Hz, 1 H), 3.61 (dd, J = 9.5, 2.5 Hz, 1 H), 2.25-2.35 (m, 2 H), 2.09-2.23 (m, 4 H), 1.45-1.58 (m, 6 H), 1.09-1.39 (m, 96 H), 0.87 (t, J = 6.9 Hz, 9 H).
¹³ C-NMR (125 MHz, CDCl ₃ ): δ = 172.92 (C═O), 172.63 (C═O), 172.53 (C═O), 138.17 (C), 137.23 (C), 128.58 (CH), 128.34 (CH), 128.14 (CH), 127.94 (CH), 127.63 (CH), 127.58 (CH), 79.54 (CH), 78.64 (CH), 75.59 (CH ₂ ), 73.02 (CH ₂ ), 72.06 (CH ), 70.84 (CH), 69.21 (CH), 67.83 (CH), 34.17 (CH ₂ ), 33.79 (CH ₂ ), 31.92 (CH ₂ ), 29.72 (CH ₂ ), 29.52 (CH ₂ ), 29.49 (CH ₂ ), 29.38 (CH ₂ ), 29.33 (CH ₂ ), 29.28 (CH ₂ ), 29.21 (CH ₂ ), 29.19 (CH ₂ ), 29.12 (CH ₂ ), 28.40 (CH ₂ ), 24.94 (CH ₂ ) 24.92 (CH ₂ ), 24.79 (CH ₂ ), 22.69 (CH ₂ ), 14.12 (CH ₃ ).

Coupling the succinic head group as described in the general procedure provides compound 33 (213 mg, 78%) as a colorless solid.
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 7.19-7.32 (m, 10 H), 5.77 (t, J = 2.6 Hz, 1 H), 5.41 (t, J = 10.3 Hz, 1 H), 5.14 (t, J = 9.9 Hz, 1 H), 4.91 (dd, J = 10.4, 2.7 Hz, 1 H), 4.84 (d, J = 11.4 Hz, 1 H), 4.66 (d, J = 11.0 Hz, 1 H), 4.59 (d, J = 11.4 Hz, 1 H), 4.49 (d, J = 11.4 Hz, 1 H), 3.87 (t, J = 9.5 Hz, 1 H), 3.67 (dd, J = 10.1 3.1 Hz, 1 H), 2.77-2.81 (m, 2 H), 2.65-2.70 (m, 2 H), 2.12-2.28 (m, 6 H), 1.41-1.59 (m, 6 H), 1.16- 1.38 (m, 96 H), 0.87 (t, J = 6.8 Hz, 9 H).
MS (ESI): 1361.1 (M + NH ₄ ⁺ ), 1341.9 (M−H ⁺ )

Removal of the benzyl group as described in the general procedure gives compound 34 (160 mg, 97%) as a colorless solid.
¹ H-NMR (500 MHz, CDCl ₃ ): δ = 5.45 (t, J = 2.7 Hz, 1 H), 5.26 (t, J = 10.2 Hz, 1 H), 4.85-4.94 (m, 2 H), 3.69 (t, J = 9.8 Hz, 1 H), 3.56 (dd, J = 9.9, 2.8 Hz, 1 H), 2.59-2.62 (m, 2 H), 2.53-2.56 (m, 2 H), 2.16- 2.25 (m, 2 H), 2.06-2.12 (m, 4 H), 1.41-1.59 (m, 6 H), 1.16-1.38 (m, 96 H), 0.75 (t, J = 6.7 Hz, 9 H) .
¹³ C-NMR (125 MHz, CDCl ₃ ): 174.45 (C = O), 173.27 (C = O), 172.77 (C = O), 172.50 (C = O), 171.71 (C = O), 72.55 (CH ), 70.94 (CH), 70.81 (CH), 69.64 (CH), 69.41 (CH), 69.26 (CH), 44.34 (CH ₂ ), 33.95 (CH ₂ ), 33.91 (CH ₂ ), 33.66 (CH ₂ ) , 31.69 (CH ₂ ), 29.47 (CH ₂ ), 29.12 (CH ₂ ), 28.95 (CH ₂ ), 28.82 (CH ₂ ), 24.69 (CH ₂ ), 24.40 (CH ₂ ), 22.44 (CH ₂ ), 22.23 (CH ₂ ), 21.89 (CH ₂ ), 13.78 (CH ₃ ).
MS (ESI): 1186.0 (M + Na ⁺ ), 1161.7 (M−H ⁺ )

Example 14 Preparation of Precursor of Part 200a The free acid derivative of part 200a is prepared as follows.
Ethyl diazoacetate (1.93 g, 16.8 mmol) and a catalytic amount of boron trifluoride ether complex (670 μl) were added to a solution of commercially available cholesterol (5 g, 12.9 mmol) in dichloromethane (100 mL) under an argon atmosphere. Add continuously below. The resulting reaction mixture is stirred at room temperature for about 3 hours. After slowly adding saturated aqueous sodium bicarbonate solution (60 mL), the organic layer is separated and washed again with saturated sodium bicarbonate solution. The solvent is removed under reduced pressure and the crude material is purified by silica gel column chromatography (hexane / diethyl ether 5: 1). The corresponding alkylated cholesteryl derivative is obtained as a colorless solid (3.67 g, 60% yield).
The material thus obtained is dissolved in ethanol (200 mL) and the solution is heated to 50 ° C. After the addition of potassium hydroxide solid (1.2 g, 22.3 mmol), the resulting reaction mixture is stirred at 50 ° C. for about 1 hour. Acidify the mixture by adding hydrochloric acid and partition between dichloromethane (800 mL) and water (800 mL). The organic layer is separated, dried (sodium sulfate) and the solvent removed in vacuo. The crude product is purified by silica gel column chromatography (dichloromethane / methanol 95: 5). The free acid derivative of portion 200a is obtained as a colorless solid (3.04 g, 92% yield).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 5.36 (d, J = 5.1 Hz, 1 H), 4.14 (s, 2 H), 3.29 (m, 1 H), 2.22-2.38 (m, 3 H), 1.76-2.1 (m, 6 H), 0.84-1.67 (m, 28 H), 1.0 (s, 3 H), 0.67 (s, 3 H).
MS (ESI): m / z = 443.3 (M−H) ⁻

Example 15 Preparation of Precursor of Part 200b The free acid derivative of part 200b is prepared as follows.
Ethyl diazoacetate (3.73 g, 32.8 mmol) is added to a solution of commercial dihydrocholesterol (9.8 g, 25.2 mmol) in anhydrous dichloromethane (50 mL) under an argon atmosphere. After a catalytic amount of boron trifluoride ether (1 mL, 1M solution in diethyl ether) is added in portions, the resulting reaction mixture is stirred at room temperature for 36 hours. Pour a saturated aqueous solution of sodium bicarbonate (1 L) into the reaction mixture and extract with ethyl acetate (1 L). After washing with water (1 L), the organic layer is dried (magnesium sulfate) and the solvent is removed under reduced pressure. The crude product is purified by silica gel column chromatography (pure dichloromethane as eluent).
The resulting product is dissolved in dichloromethane (15 mL) and a 1M aqueous solution of potassium hydroxide (20 mL) is added. The resulting reaction mixture is stirred vigorously at room temperature for about 48 hours. Add 1 M aqueous hydrochloric acid until the pH of the aqueous layer is about pH 1-2. The mixture is partitioned between water (1 L) and ethyl acetate (900 mL). After separation, the organic layer was washed with water (1 L), dried (magnesium sulfate) and the solvent removed in vacuo to give analytically pure product as a colorless solid (5.39 g, 48% total yield). rate).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 3.75 (s, 2 H), 3.32 (m, 1 H), 0.85-2.1 (m, 40 H), 0.80 (s, 3 H), 0.64 ( s, 3 H).
MS (ESI): m / z = 445.2 (M−H) ⁻

Example 16 Preparation of Precursor of Part 200c The free acid derivative of part 200c is prepared as follows. In BR Peterson et al. (SL Hussey, E. He, BR Peterson, J. Am. Chem. Soc. 2001, 123, 12712-12713; SE Martin, BR Peterson, Bioconjugate Chem. 2003, 14, 67-74). The free acid derivative of moiety 200c is prepared according to the synthetic method described in detail. In fact, instead of the free acid derivative of moiety 200c, the corresponding N-nosyl protected derivative was incorporated by solid phase synthesis and the nosyl protecting group was removed after the complex construction by the experimental protocol described in the above BR Peterson literature To do. However, the final raft affinity partial building block is represented by the free acid derivative of portion 200c.

Example 17 Preparation of Precursor for Part 200e The free acid derivative of part 200e is prepared as follows.
Triethylamine (284 mg, 2.81 mmol) was added to a solution of commercially available dihydrocholesterol (840 mg, 2.16 mmol), succinic anhydride (281 mg, 2.81 mmol) and DMAP (342 mg, 2.81 mmol) in dichloromethane (10 mL). In addition, the resulting reaction mixture is stirred overnight at room temperature. After dilution with ethyl acetate (900 mL), the reaction mixture is washed successively with 0.1 M aqueous hydrochloric acid (1 L) and water (2 × 1 L). The organic layer is dried (sodium sulfate) and the solvent removed in vacuo to give analytically pure product as a colorless solid (926 mg, 87% yield).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 4.71 (m, 1 H), 2.67 (m, 2 H), 2.59 (m, 2 H), 1.96 (m, 1 H), 0.85-1.81 ( m, 39 H), 0.81 (s, 3 H), 0.64 (s, 3 H).

Example 18 Preparation of Precursor for Part 200f The free acid derivative of part 200f is prepared as follows.
A solution of commercial dihydrocholesterol (10 g, 25.7 mmol), triphenylphosphine (20.3 g, 77.2 mmol) and methanesulfonic acid (5.2 g, 53.9 mmol) in anhydrous THF (250 mL) at 42 ° C. under an argon atmosphere. Heat to. After the addition of diisopropyl azodicarboxylate (15.6 g, 77.2 mmol), the resulting reaction mixture is stirred at 40 ° C. for about 18 hours. Water (20 mL) is added and the reaction mixture is stirred at room temperature for 10 minutes. Add more water (400 mL) and dichloromethane (200 mL), then separate the organic layer. Extract the aqueous layer again with dichloromethane (200 mL), combine the organic layers and wash with brine (800 mL). After the organic layer is dried (sodium sulfate), the solvent is removed in vacuo and the crude material is subjected to silica gel column chromatography using gradient elution (petroleum ether / ethyl acetate 10: 1 to 6: 1). The expected mesylate is obtained in white solids (4.1 g, 34% yield).
The material thus obtained (4.1 g, 8.8 mmol) is dissolved in DMSO (80 mL) and sodium azide (5.7 g, 88 mmol) is added. The resulting reaction mixture is stirred at 90 ° C. for about 18 hours. After adding water (500 mL), the mixture is extracted with dichloromethane (400 mL). Separate the organic layer and wash thoroughly with water (4 x 700 mL). After drying (sodium sulfate), the solvent is removed under reduced pressure and the resulting analytically pure azide is dried in vacuo (2.7 g, 75% yield).
The azide so obtained (2.5 g, 6 mmol) was dissolved in anhydrous diethyl ether (25 mL), and the resulting solution was lithium aluminum hydride (690 mg, 18.3 mmol) in anhydrous diethyl ether (50 mL). Is added dropwise at 36 ° C. under an argon atmosphere. The resulting reaction mixture is stirred at reflux temperature for about 18 hours, then cooled in an ice-water bath and water is added dropwise until gas evolution ceases. 2M aqueous sodium hydroxide solution (1 L) is added and the mixture is extracted with diethyl ether (500 mL). Extract the aqueous layer again with dichloromethane (2 x 500 mL), combine the organic layers and dry (sodium sulfate). After removing the solvent under reduced pressure and drying under high vacuum, analytically pure expected amine is obtained (1.3 g, 55% yield).
A solution of the above amine (160 mg, 0.41 mmol), DMAP (125 mg, 1 mmol) and succinic anhydride (103 mg, 1 mmol) in dichloromethane (10 mL) is stirred at room temperature for about 48 hours. The solvent is removed under reduced pressure and the resulting solid residue is dissolved in ethyl acetate (20 mL). Saturated aqueous sodium bicarbonate solution (20 mL) and a catalytic amount of DMAP are added sequentially, and the resulting mixture is stirred at room temperature for 1 hour. 0.1M aqueous hydrochloric acid (500 mL) is added and the aqueous layer is extracted with ethyl acetate (2 x 400 mL). After the organic layer is dried (sodium sulfate), the solvent is removed in vacuo to give analytically pure portion 200f of the free acid derivative (78 mg, 40% yield).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 3.78 (m, 1 H), 2.66 (m, 4 H), 1.97 (m, 1 H), 0.85-1.81 (m, 40 H), 0.78 ( s, 3 H), 0.64 (s, 3 H).
MS (ESI): m / z = 488.4 (M + H) ⁺

Example 19 Preparation of Precursor of Part 200j Using the same protocol described above for the free acid derivative of part 200e, the free acid derivative of part 200j is prepared from commercially available cholesterol.
Part 200j of the free acid derivative is obtained as a colorless solid (1.2 g, 95% yield).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 5.37 (d, J = 4.1 Hz, 1 H), 4.63 (m, 1 H), 2.66-2.70 (m, 2 H), 2.58-2.62 (m , 2 H), 2.32 (d, J = 7.8 Hz, 2 H), 1.77-2.05 (m, 5 H), 0.85-1.65 (m, 30 H), 1.02 (s, 3 H), 0.68 (s, 3 H).
MS (ESI): m / z = 485.1 (M−H) ⁻

Example 20: Preparation of Compound Containing Portion 200k Using a standard esterification protocol known to those skilled in the art, the free carboxylic acid functionality of the side chain of commercially available Fmoc-Asp-OtBu is coupled with dihydrocholesterol. The resulting dihydrocholesteryl ester of Fmoc-Asp-OtBu is then subjected to cleavage of the OtBu ester using a standard trifluoroacetic acid protocol to give the corresponding dihydrocholesteryl ester of Fmoc-Asp.
This building block is then ligated to the N-terminus of the rhodamine labeled linker substructure, followed by standard Fmoc deprotection to yield a compound containing the moiety 200k.

Example 21: Preparation of compound containing part 200l The compound containing part 200l is obtained from the compound containing part 200k obtained in Example 20 by simple acetyl capping using standard protocols known in the literature.

Example 22 Preparation of Precursor 200m Partial Binding of Fmoc-Asp dihydrocholesteryl ester obtained in Example 20 to a solid support, followed by N-terminal Fmoc deprotection as described in the preparation of compound 25b And a compound containing the 200m portion by solid phase peptide chemistry to assemble a linker and pharmacophore substructure on the free N-terminus.

Example 23: Preparation of Precursor for Part 300a The free acid derivative of part 300a is prepared as follows.
A suspension of sodium hydride (500 mg mineral oil suspension, 12.25 mmol sodium hydride) in anhydrous DMSO (15 mL) is heated at 70 ° C. under argon atmosphere for about 45 minutes. After adding a solution of commercially available dodecylphosphonium bromide in anhydrous DMSO (20 mL), the resulting red solution is held at about 60-65 ° C. for about 10 minutes. A solution of commercially available estrone (668 mg, 2.47 mmol) in anhydrous DMSO (20 mL) is then added to the hot solution and the reaction mixture is stirred at 60 ° C. for 18 hours. Pour water (1 L) into the mixture and extract with diethyl ether (2 x 500 mL). The organic layers were combined, washed repeatedly with water (4 x 1 L), dried (sodium sulfate), and so on. After removing the solvent under reduced pressure, the crude material is purified by silica gel column chromatography (petroleum ether / ethyl acetate 4: 1). The expected 17-dodecylidenyl estrone is obtained in a white solid (531 mg, 51% yield).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 7.08 (d, J = 8.5 Hz, 1 H), 6.56 (dd, J = 2.7, 8.5 Hz, 1 H), 6.49 (d, J = 2.7 Hz , 1 H), 4.98 (t, J = 7.4 Hz, 1 H), 4.58 (s, 1 H), 2.75 (m, 2 H), 2.04-2.41 (m, 9 H), 1.15-1.87 (m, 24 H), 0.84 (s, 3 H), 0.82 (t, J = 6.9 Hz, 3 H).
MS (ESI): m / z = 422.6 (M ⁺ )

Hydrogenation of the 17,20 double bond in the above mentioned material is a solution of 17-dodecylidenylated estrone (475 mg, 1.12 mmol) in dichloromethane (10 mL) and palladium (120 mg 10% / carbon, 0.11 mmol). This is accomplished by treating for 36 hours at room temperature under a hydrogen atmosphere. The reaction mixture is filtered through a celite pad and the solvent removed in vacuo to give analytically pure 17β-dodecyl substituted estrone as a colorless solid (452 mg, 95% yield).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 7.09 (d, J = 8.4 Hz, 1 H), 6.55 (dd, J = 2.6, 8.4 Hz, 1 H), 6.49 (d, J = 2.6 Hz , 1 H), 4.51 (s, 1 H), 2.73-2.77 (m, 2 H), 2.15-2.19 (m, 2 H), 1.77-1.82 (m, 2 H), 1.03-1.65 (m, 16 H), 0.81 (t, J = 6.9 Hz, 3 H), 0.53 (s, 3 H).
MS (ESI): m / z = 424.7 (M ⁺ )

A solution of the above 17β-dodecyl substituted estrone (440 mg, 1.04 mmol) in DMF (6 mL) and dichloromethane (6 mL) was added to sodium hydride (50 mg mineral oil suspension, 1.14 mmol sodium hydride). In addition, the resulting suspension is heated to reflux for about 20 minutes. Add tert-butyl bromoacetate (242 mg, 1.24 mmol) and heat the reaction mixture to reflux for about 28 hours with stirring. Pour into water (1 L) and extract with dichloromethane (600 mL), then wash the organic layer with water (3 x 800 mL) and dry (magnesium sulfate). The solvent is removed in vacuo to give analytically pure product as a colorless solid.
The resulting material is dissolved in dichloromethane (10 mL), trifluoroacetic acid (2.5 mL) is added, and the resulting reaction mixture is stirred at room temperature for 3 hours. The solvent is removed under reduced pressure and the resulting material is dried under high vacuum for 18 hours. The free acid derivative of portion 300a is obtained as a pale yellow solid (394 mg, 79% overall yield).
MS (ESI): m / z = 481.3 (M−H) ⁻

N−メチル−2−ピロリドン(4 mL)中の1(447 mg、1 mmol)、HATU(380 mg、1 mmol)、H−Gly−2Cl−Trt樹脂(0.54 mmol)(Novabiochemから市販されている、カタログ番号04−12−2800)およびDIPEA(259 mg、2 mmol)の溶液をペプチド合成機で1時間振とうする。N−メチル−2−ピロリドン洗浄、次いで、CH₂Cl₂洗浄を行う。このようにして得られる樹脂をCH₂Cl₂中の1%トリフルオロ酢酸溶液で処理することにより切断する。生成物を水(100 mL)で洗浄し、CH₂Cl₂(3x100 mL)で抽出する。有機層を合わせ、乾燥(硫酸ナトリウム)し、減圧濃縮して、2を白色個体で得る(250 mg、100%)。
¹H−NMR(300 MHz、CDCl₃)：δ＝0.58(s、6H)、0.70−1.77(series of m、36H)、1.87(d、J＝12.3 Hz、2H)、3.09(m、2H)、3.26(m、1H)、3.98(m、2H)、4.04(m、2H)、7.23(m、1H)、9.7(br s、1H)。
MS(ESI)：m/z＝502(M−1)
一般手順に記載したとおり、ステロイド含有側鎖を導入して、化合物4を白色個体で得る(281 mg、62%)。
¹H−NMR(300 MHz、CDCl₃)：δ＝0.65(s、3H)、0.79(s、3H)、0.85−0.91(m、18H)、1.06−1.45(m、50H)、1.46−2.17(m、9H)、3.27(m、1H)、3.56−4.13(m、8H)、4.35(m、1H)、5.41(m、2H)、6.12(m、1H)、7.36(m、6H)、7.59(m、4H)。
MS(ESI)：m/z＝1023(M+1)
一般手順に記載したとおり、コハク酸頭部基を結合して、化合物5を得る(224 mg、73%)。
¹H−NMR(300 MHz、CDCl₃)：δ＝0.63(s、3H)、0.78(s、3H)、0.84−0.95(m、18H)、0.98−1.31(m、50H)、1.41−1.82(m、10H)、1.94(br d、J＝12.13 Hz、2H)、2.47(m、4H)、3.27(m、1H)、3.48(m、1H)、3.95(br s、2H)、4.19(m、4H)、5.28(m、2H)、7.29(m、6H)、7.59(m、4H)。
MS(ESI)：m/z＝1123.7(M+1)
一般手順に記載したとおり、保護基を除去して、化合物6を得る。
MS(ESI)：m/z＝885.6(M+1) Example 24 Preparation of Precursor for Part 1900a The free acid derivative of part 1900a is prepared as follows.

1 (447 mg, 1 mmol), HATU (380 mg, 1 mmol), H-Gly-2Cl-Trt resin (0.54 mmol) in N-methyl-2-pyrrolidone (4 mL) (commercially available from Novabiochem , Catalog number 04-12-2800) and DIPEA (259 mg, 2 mmol) are shaken in a peptide synthesizer for 1 hour. N- methyl-2-pyrrolidone washed and then performs the CH ₂ Cl ₂ wash. The resin thus obtained is cleaved by treatment with a 1% trifluoroacetic acid solution in CH ₂ Cl ₂ . The product is washed with water (100 mL) and extracted with CH ₂ Cl ₂ (3 × 100 mL). The organic layers are combined, dried (sodium sulfate) and concentrated in vacuo to give 2 as a white solid (250 mg, 100%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 0.58 (s, 6H), 0.70-1.77 (series of m, 36H), 1.87 (d, J = 12.3 Hz, 2H), 3.09 (m, 2H) 3.26 (m, 1H), 3.98 (m, 2H), 4.04 (m, 2H), 7.23 (m, 1H), 9.7 (brs, 1H).
MS (ESI): m / z = 502 (M-1)
As described in the general procedure, a steroid-containing side chain is introduced to give compound 4 in a white solid (281 mg, 62%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 0.65 (s, 3H), 0.79 (s, 3H), 0.85-0.91 (m, 18H), 1.06-1.45 (m, 50H), 1.46-2.17 ( m, 9H), 3.27 (m, 1H), 3.56-4.13 (m, 8H), 4.35 (m, 1H), 5.41 (m, 2H), 6.12 (m, 1H), 7.36 (m, 6H), 7.59 (m, 4H).
MS (ESI): m / z = 1023 (M + 1)
Coupling the succinic head group as described in the general procedure provides compound 5 (224 mg, 73%).
¹ H-NMR (300 MHz, CDCl ₃ ): δ = 0.63 (s, 3H), 0.78 (s, 3H), 0.84-0.95 (m, 18H), 0.98-1.31 (m, 50H), 1.41-1.82 ( m, 10H), 1.94 (br d, J = 12.13 Hz, 2H), 2.47 (m, 4H), 3.27 (m, 1H), 3.48 (m, 1H), 3.95 (br s, 2H), 4.19 (m , 4H), 5.28 (m, 2H), 7.29 (m, 6H), 7.59 (m, 4H).
MS (ESI): m / z = 1123.7 (M + 1)
Removal of the protecting group as described in the general procedure gives compound 6.
MS (ESI): m / z = 885.6 (M + 1)

Example 25 Preparation of Precursor for Part 1900b The free acid derivative of part 1900b is prepared starting from commercially available Fmoc-Lys (Dde) using solid phase peptide chemistry known to those skilled in the art. After first conjugating the amino acid described in Example 24, protected at right angles, to a solid support, the Dde protecting group is removed by protocols known in the literature, followed by partial peptide coupling using standard peptide coupling. Capping the free acid. The production of the free acid of portion 200b is as described above. The Fmoc protecting group is then removed and then the commercial Fmoc-β-Ala and the free acid of the raft affinity moiety 200b are coupled sequentially. Final cleavage from the solid support under standard conditions yields the free acid of moiety 1900b.

General Procedure for the Synthesis of the Compounds of the Invention The tripartite compounds described herein are based on an Applied Biosystems 433A peptide synthesizer (hereinafter also referred to as ABI 433A and ABI 433) equipped with a Series 200 UV / VIS detector. And can be synthesized on a solid support. All peptide synthesis can be performed using, for example, piperidine as a deprotection reagent and 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) as a coupling reagent or Performed using the Fmoc method with O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetra-methyluronium hexafluorophosphate (HATU). The principles of synthesis are described in general texts (eg, G.A. Grant, “Synthetic Peptides: A User's Guide”, WH Freeman & Co., New York 1992). A detailed description of the synthesis procedure used by ABI 433A can be found in the ABI 433A operating instructions, part numbers 904855 Rev. C and 904856C (copyright 2001, Applied Biosystems) and instructions, `` Running the 433A with the Series 200 UV Detector "(Copyright 2002, Applied Biosystems). The amide resin Fmoc-PAL-PEG-PS (Applied Biosystems, part number GEN913398) can be used as a solid support. Amino acid building blocks, coupling reagents and solvents are purchased ready for use from either Applied Biosystems or Novabiochem. Amino acids having a polyglycol skeleton are prepared according to the protocol described in the literature (D. Boumrah, MM Campbell, S. Fenner, RG Kinsman, tetrahedron 1997, 53, 6977-6992) or purchased from Novabiochem. Liquid building blocks that cannot be processed by ABI 433A (eg, due to low solubility) are, for example, manually coupled to the N-terminus of the peptide on the solid support generated as described above. After synthesis is complete, the final product is cleaved from the solid support. A typical procedure is as follows: a cleavage reaction involving trifluoroacetic acid (87%), water (4%), anisole (3%), thioanisole (3%) and triisopropylsilane (3%). A new mixture is produced. 4 ml of this mixture is cooled in an ice bath and 70 mg of resin bound peptide or lipopeptide is added. The mixture is stirred at 5 ± 2 ° C. for 90-120 minutes. The mixture is filtered into 100 ml diethyl ether and hexane (2: 1) ice-cold mixture and the resin is washed several times with the cleavage reaction mixture and filtered in the same manner. The diethyl ether / hexane mixture containing the combined filtrates is cooled in a freezer (−18 ° C.) and the crude or lipopeptide is isolated by membrane filtration. The crude product is washed with diethyl ether / hexane (2: 1), dried under high vacuum and purified by preparative reverse phase HPLC.

自動ペプチド合成機を用いて、Fmoc−PAL−PEG−PS樹脂(610 mg、0.25 mmol、ローディング：0.41 mmol/g)を反応器内で以下の操作に付す：ジクロロメタンで洗浄、N−メチル−2−ピロリドンで洗浄、N−メチル−2−ピロリドン中の20%ピペリジンを用いる末端Fmoc基の切断(controlled by UVモニターにより制御)、N−メチル−2−ピロリドンで洗浄。 Example 26

Using an automated peptide synthesizer, Fmoc-PAL-PEG-PS resin (610 mg, 0.25 mmol, loading: 0.41 mmol / g) is subjected to the following operations in the reactor: washed with dichloromethane, N-methyl-2 -Wash with pyrrolidone, cleavage of the terminal Fmoc group using 20% piperidine in N-methyl-2-pyrrolidone (controlled by UV monitor), wash with N-methyl-2-pyrrolidone.

Amino acid activation and coupling is achieved as follows: Fmoc-Phe (1 mmol) is added to HBTU (1 mmol, 2.2 mL of 0.45 MN-methyl-2-pyrrolidone solution) and diisopropylethylamine (1 mmol) in an airtight cartush. 2 mmol, 0.5 mL of 2 MN-methyl-2-pyrrolidone solution) and then passing the corresponding N-hydroxy-1H-benzotriazole ester (by passing nitrogen gas through the reaction mixture until a clear solution is obtained). Activation). Transfer the mixture to the reactor and shake with the resin for 30 minutes (coupling). Drain the resin and wash with N-methyl-2-pyrrolidone. The above sequence of operations is followed by the following amino acids: Repeat for Gly, Fmoc-βAla to obtain the protected derivative of inhibitor III available from Calbiochem, catalog number 565780.
After a final wash with dichloromethane, the resin is high vacuum dried and stored at -18 ° C. Continue production of 26 using part of the resin.

Coupling of rhodamine labeled glutamic acid is performed manually in a round bottom flask. A solution of neat diisopropylethylamine (78 mg, 0.6 mmol) in F-moc-Glu (Rho) (295 mg, 0.3 mmol) and HATU (115 mg, 0.3 mmol) in N-methyl-2-pyrrolidone (6 mL). And the resulting reaction mixture is stirred at room temperature for 10 minutes (activation). After this mixture is added to the aforementioned resin (341 mg, 0.1 mmol), the resulting heterogeneous reaction mixture is carefully stirred at room temperature for 1 hour. The resin is drained, transferred to the reactor and washed sequentially with N-methyl-2-pyrrolidone and dichloromethane using an automated peptide synthesizer.
Peptide chain completion is achieved by sequential activation and coupling of Fmoc-βAla, Fmoc-Arg (Pbf) and Fmoc-Arg (Pbf) in the same manner as described above using an automated peptide synthesizer.

  Coupling of cholesteryl glycolic acid (ie, the precursor of portion 200a) is again performed manually in a round bottom flask. The raw diisopropylethylamine (42 mg, 0.16 mmol) was added to cholesteryl glycolic acid (73 mg, 0.16 mmol), HBTU (62 mg, 0.16 mmol) and N-hydroxybenzotriazole (25 mg, 25 mL, 0.16 mmol) and the resulting reaction mixture is stirred for 10 minutes at room temperature (activation). After this mixture is added to the aforementioned resin (186 mg, 0.054 mmol), the resulting heterogeneous reaction mixture is carefully stirred at room temperature for 2 hours. The resin is drained, transferred to the reactor and washed sequentially with N-methyl-2-pyrrolidone and dichloromethane using an automated peptide synthesizer.

Cleavage from the resin as described in general section: Resin (134 mg) is trifluoroacetic acid (87%), water (4%), triisopropylsilane (3%), thioanisole (3%) and Suspend in a mixture of anisole (3%) and stir at 5 ° C. (± 2 ° C.) for 90 minutes. Drain the resin and wash repeatedly with the previously mentioned cutting reaction mixture (4x1 mL). Pour the filtrate into ice cold diethyl ether (40 mL) and dilute to 100 mL with hexane / diethyl ether (1: 2) to complete the precipitation. The product is separated by membrane filtration (PP membrane, 0.45 μm), washed with hexane / diethyl ether (1: 2) and dried under high vacuum (crude yield: 41 mg).
Preparative HPLC purification of crude product (Vydac-C8-column, 40 mL / min, A: water + 0.1% trifluoroacetic acid, B: acetonitrile + 0.1% trifluoroacetic acid, 51% to 63% B 15 Gradient elution over minutes, observed retention time: 9.8 min), and after removing the solvent in high vacuum and lyophilizing from acetic acid, 26 is obtained as a reddish pink foam.
HPLC analysis: Agilent Zorbax-C ₈ column 4.6x125 mm, flow rate 1 mL / min, A: water + 0.1% trifluoroacetic acid, B: acetonitrile + 0.1% trifluoroacetic acid, 10% to 100% B for 45 minutes Gradient elution over, retention time: 30.8 min, detected at 215 nm, 91% purity.
ESI-MS: 1262.4 [M + H] ²⁺ , 842.3 [M + 2H] ³⁺ .

コレステリルグリコール酸(の前駆体部分200a)の代わりに、コハク酸モノ(D−エリスロ−C₁₆−セラミジル)エステル(すなわち、部分400aaの前駆体)をN末端アルギニンへカップリングすることによって、化合物26の製造の記載と同様にして、化合物27の製造を達成する。化合物26の記載と同様にして、切断および精製を行う。化合物27を赤色粉末で得る(4.1 mg)。
HPLC分析：化合物26の記載と同様のプロトコルであるが、ただし、66%アセトニトリル＋0.1%トリフルオロ酢酸で45分間の定組成溶離を用いる；保持時間：13.5分；215 nmにて検出；90%純度。
ESI−MS：1358 [M+2H]²⁺、906 [M+2H]³⁺。 Example 27

Instead of cholesteryl glycolic acid (a precursor portion 200a), succinic acid mono (D-erythro -C ₁₆ - Seramijiru) ester (i.e., precursor portion 400Aa) by coupling to N-terminal arginine, compound 26 The preparation of compound 27 is achieved analogously to the description of the preparation of Cleavage and purification are performed as described for compound 26. Compound 27 is obtained as a red powder (4.1 mg).
HPLC analysis: protocol similar to that described for compound 26 but using isocratic elution with 66% acetonitrile + 0.1% trifluoroacetic acid for 45 minutes; retention time: 13.5 minutes; detection at 215 nm; 90 %purity.
ESI-MS: 1358 [M + 2H] ²⁺ , 906 [M + 2H] ³⁺ .

一般的記載において前述したとおり、24の製造を達成する。Fmoc−PAL−PEG−PSアミド樹脂および自動固相ペプチド合成プロトコルを用い、Fmoc−Lys(CholGlc)、Fmoc−Asn、Fmoc−Ser(tBu)、Fmoc−Gly、Fmoc−Val、Fmoc−Asp(OtBu)、Fmoc−Glu(Rho)、Fmoc−Ala、Fmoc−Phe、Fmoc−Phe、Fmoc−Val、Fmoc−Leu、Fmoc−Lys(Trt)、Fmoc−Gln、Fmoc−His、Fmoc−His、Fmoc−Val、Fmoc−Glu(OtBu)、Fmoc−Tyr、Fmoc−Gly、Fmoc−Ser、Fmoc−Asp(OtBu)、Fmoc−His、Fmoc−Arg(Pbf)、Fmoc−Phe、Fmoc−Glu(OtBu)、Fmoc−Ala、Fmoc−Val、Fmoc−Sta、Fmoc−Asn、Fmoc−Val、Fmoc−Glu(OtBu)の連続的カップリングを行い、固相上にファーマコフォア−ペプチドリンカー配列を得る。標準的ペプチドカップリング技術を用いるコレステリルグリコール酸(部分200aの前駆体)の連続的手動カップリングにより、C末端を介して固相支持体に固定された24を得る。
最終的に、前述の一般的切断手順にしたがって樹脂から切断し、プレパラティブHPLCにより精製した後、アミド24を得る。
リンカー長は、Hyperchem(登録商標)ソフトウェアを用いるMM＋力場最適化によって8.87 nmであると計算された。 Example 28: Preparation of a compound of the invention of formula 24

24 production is achieved as described above in the general description. Using Fmoc-PAL-PEG-PS amide resin and automated solid phase peptide synthesis protocol, Fmoc-Lys (CholGlc), Fmoc-Asn, Fmoc-Ser (tBu), Fmoc-Gly, Fmoc-Val, Fmoc-Asp (OtBu ), Fmoc-Glu (Rho), Fmoc-Ala, Fmoc-Phe, Fmoc-Phe, Fmoc-Val, Fmoc-Leu, Fmoc-Lys (Trt), Fmoc-Gln, Fmoc-His, Fmoc-His, Fmoc- Val, Fmoc-Glu (OtBu), Fmoc-Tyr, Fmoc-Gly, Fmoc-Ser, Fmoc-Asp (OtBu), Fmoc-His, Fmoc-Arg (Pbf), Fmoc-Phe, Fmoc-Glu (OtBu), Fmoc-Ala, Fmoc-Val, Fmoc-Sta, Fmoc-Asn, Fmoc-Val, and Fmoc-Glu (OtBu) are sequentially coupled to obtain a pharmacophore-peptide linker sequence on the solid phase. Sequential manual coupling of cholesteryl glycolic acid (precursor of moiety 200a) using standard peptide coupling techniques yields 24 immobilized to the solid support via the C-terminus.
Finally, amide 24 is obtained after cleavage from the resin according to the general cleavage procedure described above and purification by preparative HPLC.
The linker length was calculated to be 8.87 nm by MM + force field optimization using Hyperchem® software.

Glu(Rho)−βAla−Gly−Glu−Val−Asn−Sta−Val−Ala−Glu−Phe−Arg−His−Asp−Ser−Gly−Tyr−Glu−Val−His−His−Gln−Lys−Leu−Val−Phe−Phe−Ala−Glu−Asp−Val−Gly−Ser−Asn−Lys−Asp(ジヒドロコレステリル)−NH₂
化合物25bの記載にしたがって、Fmoc−Asp(ジヒドロコレステリル)(前記の部分200kの記載にしたがって製造)を0.1 mmolのPAL−PEG−PS樹脂にロードする。自動洗浄、キャッピングおよび脱保護の後、234 mg(0.5 mmol)のFmoc−Lys、190 mg(0.5 mmol)のHATU、190 μl(1.0 mmol)のDIPEA、前述の手順を用いて、次のアミノ酸、Fmoc−Lys(Boc)を手動でロードする。化合物25bの記載にしたがって、以下のとおり、ABI 433ペプチド合成機を用いて、βAlaまでの残りの配列を構築する。化合物25bと同様にして、244 mg(0.25 mmol)のFmoc−Glu(Rho)、95 mg(0.25 mmol)のHATU、84 μlのDIPEAおよび3 ml DMFを用いて、Glu(Rho)を手動で結合する。ABI 433を用いて樹脂を脱保護および洗浄し、減圧乾燥する。化合物25bの記載にしたがって、トリフルオロ酢酸/H₂O/トリイソプロピルシラン/アニソール/チオアニソール(87：4：3：3：3)を用いて切断を行う。他の条件は、化合物25bの記載と同様にして、30分間にわたる42〜46%Bの勾配を用いて、HPLC精製を行う(RT 約24分)。乾燥(硫酸マグネシウム)して、12.7 mgの赤色個体を得る。
C8カラム型Vydac 208TP104、プレパラティブ分離と同じ溶離液、1 ml/分の流速および35分間にわたる42〜56%Bの勾配を用い、分析的HPLC−MSを行う。合計純度は、MSトレースにより80.9%であることがわかる。生成物ピークの内部で共溶離する2つの不純物の量は12.3%である。ESI−MS：1246.1 [M]⁴⁺. MALDI：4977.7 [M]⁺。 Example 29: Preparation of the compounds of the invention of formula 24b

Glu (Rho) -βAla-Gly-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu -Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Asp (Dihydrocholesteryl) -NH ₂
Fmoc-Asp (dihydrocholesteryl) (prepared as described above in part 200k) is loaded onto 0.1 mmol PAL-PEG-PS resin as described for compound 25b. After automatic washing, capping and deprotection, 234 mg (0.5 mmol) Fmoc-Lys, 190 mg (0.5 mmol) HATU, 190 μl (1.0 mmol) DIPEA, using the above procedure, the following amino acids: Manually load Fmoc-Lys (Boc). As described for compound 25b, the remaining sequence up to βAla is constructed using an ABI 433 peptide synthesizer as follows. Glu (Rho) was coupled manually using 244 mg (0.25 mmol) Fmoc-Glu (Rho), 95 mg (0.25 mmol) HATU, 84 μl DIPEA and 3 ml DMF as in compound 25b. To do. The resin is deprotected and washed with ABI 433 and dried under vacuum. Cleavage is performed using trifluoroacetic acid / H ₂ O / triisopropylsilane / anisole / thioanisole (87: 4: 3: 3: 3) as described for compound 25b. Other conditions are similar to those described for compound 25b, with HPLC purification using a gradient of 42-46% B over 30 minutes (RT ˜24 minutes). Dry (magnesium sulfate) to obtain 12.7 mg of a red solid.
Analytical HPLC-MS is performed using a C8 column type Vydac 208TP104, the same eluent as the preparative separation, a flow rate of 1 ml / min and a gradient of 42-56% B over 35 min. The total purity is found to be 80.9% by MS trace. The amount of the two impurities co-eluting inside the product peak is 12.3%. ESI-MS: 1246.1 [M] ⁴⁺ . MALDI: 4977.7 [M] ⁺ .

一般的記載において前述したとおり、25の製造を達成する。リシンのε−アミノ基にコレステリルグリコール酸(部分200aの前駆体)を手動カップリングした後、得られるリシン誘導体をそのC末端を介してFmoc−PAL−PEG−PSアミド樹脂にカップリングし、次いで、2−[2−(2−アミノエトキシ)エトキシ]エトキシ酢酸2回、ローダミン標識グルタミン酸、2−[2−(2−アミノエトキシ)エトキシ]エトキシ酢酸2回、フェニルアラニン、グルタミン酸、アラニン、バリン、スタチン、アスパラギン、バリンおよびグルタミン酸の自動固相ペプチド合成カップリングを連続的に行って、固相支持体上にファーマコフォア−ポリグリコールリンカー−ラフト親和性部分複合体を得る。次いで、前述の一般的切断手順にしたがって樹脂から切断し、プレパラティブHPLCにより精製した後、25を得る。 Example 30: Preparation of a compound of the invention of formula 25

25 production is achieved as described above in the general description. After manual coupling of cholesteryl glycolic acid (precursor of moiety 200a) to the ε-amino group of lysine, the resulting lysine derivative is coupled to Fmoc-PAL-PEG-PS amide resin via its C-terminus, and then 2- [2- (2-aminoethoxy) ethoxy] ethoxyacetic acid, rhodamine-labeled glutamic acid, 2- [2- (2-aminoethoxy) ethoxy] ethoxyacetic acid, phenylalanine, glutamic acid, alanine, valine, statin Automated, solid phase peptide synthesis coupling of asparagine, valine and glutamic acid is performed sequentially to obtain a pharmacophore-polyglycol linker-raft affinity partial complex on a solid support. It is then cleaved from the resin according to the general cleavage procedure described above and 25 is obtained after purification by preparative HPLC.

Glu(Rho)−βAla−Gly−Glu−Val−Asn−Sta−Val−Ala−Glu−Phe−4Gl−3Gl−4Gl−3Gl−4Gl−Asp(ジヒドロコレステリル)−NH₂
363 mg(0.5 mmol)のFmoc−Asp(ジヒドロコレステリル)(部分200kのために前述したとおり製造)、190 mg(0.5 mmol)のHATU、190 μl(1.0 mmol)のDIPEA、2 mlのCH₂Cl₂および1 mlのDMFから活性エステル溶液を製造する。この溶液を100 μmolの脱保護CH₂Cl₂−湿潤PAL−PEG−PS−樹脂(ローディング：0.21 mmol/g)に加える。アミノ酸を1時間カップリングに付し、その間に沈澱を除去するために1 mlのDMFを加える。ABI−433合成機を用いて洗浄および脱保護を行う。Glu(Rho)を除いて、残りの配列をABI−433で構築する。樹脂ロードが少なく、処理する配列が長いので、より大きい反応量を可能にし、より多い洗浄溶媒を使用する0.25 mmolで行う化学反応プログラムを使用する。さらに、カップリング時間を50分間に延長し、第2の「残基」(4Gl)を、二重カップリングを介して結合する。最終的脱保護および洗浄の後、Fmoc−Asp(DHC)のカップリングのために前述した方法と同様にして、293 mg(0.3 mmol)のGlu(Rho)、114 mg(0.3 mmol)のHATU、102 μl(0.6 mmol)のDIPEA、2 mlのDMFおよび2 mlのCH₂Cl₂およびカップリング時間1.5時間を用いて、N末端Glu(Rho)を結合するABI−433を用いて。最終的脱保護および洗浄を行う。トリフルオロ酢酸/H₂O/anisol/トリイソプロピルシラン(90：4：3：3)および90 分間の反応時間を用いて、切断および脱保護を行う。エーテル/石油エーテル(30：70)で生成物を沈澱させ、MeCN/MeOH(1：1)を加え、回転蒸発乾固し、高真空乾燥する。25分間にわたる40〜57%B勾配を用いて、プレパラティブHPLC精製を行う(RT＝24.06分)。乾燥した後、34.1 mgの赤色固体を得る。分析的HPLCにより、保持時間(RT)32.1分および純度93%(215 nm)を得る。MALDI：3346.9 [M]⁺。 Example 31: Preparation of a compound of the invention of formula 25b

Glu (Rho) -βAla-Gly-Glu-Gal-Asn-Sta-Val-Ala-Glu-Phe-4Gl-3Gl-4Gl-3Gl-4Gl-Asp (dihydrocholesteryl) -NH ₂
363 mg (0.5 mmol) Fmoc-Asp (dihydrocholesteryl) (prepared as described above for portion 200k), 190 mg (0.5 mmol) HATU, 190 μl (1.0 mmol) DIPEA, 2 ml CH ₂ Cl _An active ester solution is prepared from ₂ and 1 ml DMF. This solution is added to 100 μmol of deprotected CH ₂ Cl ₂ -wet PAL-PEG-PS-resin (loading: 0.21 mmol / g). The amino acid is coupled for 1 hour during which 1 ml of DMF is added to remove the precipitate. Wash and deprotect using an ABI-433 synthesizer. The remaining sequence is constructed with ABI-433, except for Glu (Rho). Because of the low resin load and long processing sequence, a chemical reaction program is used that allows for larger reaction volumes and is performed at 0.25 mmol using more wash solvent. In addition, the coupling time is extended to 50 minutes and the second “residue” (4Gl) is attached via a double coupling. After final deprotection and washing, 293 mg (0.3 mmol) Glu (Rho), 114 mg (0.3 mmol) HATU, as described above for Fmoc-Asp (DHC) coupling, With ABI-433 binding N-terminal Glu (Rho) using 102 μl (0.6 mmol) DIPEA, 2 ml DMF and 2 ml CH ₂ Cl ₂ and coupling time 1.5 h. Perform final deprotection and washing. Cleavage and deprotection are performed using trifluoroacetic acid / H ₂ O / anisol / triisopropylsilane (90: 4: 3: 3) and a reaction time of 90 minutes. Precipitate the product with ether / petroleum ether (30:70), add MeCN / MeOH (1: 1), rotoevaporate to dryness and dry under high vacuum. Preparative HPLC purification is performed using a 40-57% B gradient over 25 minutes (RT = 24.06 min). After drying, 34.1 mg of a red solid is obtained. Analytical HPLC gives a retention time (RT) of 32.1 minutes and a purity of 93% (215 nm). MALDI: 3346.9 [M] ⁺ .

Example 32: Production of rhodamine labeled raft affinity moieties for assessment of raft affinity in LRA and DRM assays
Peptide coupling is performed on an ABI-433 synthesizer using the Fmoc protocol and HBTU as a coupling reagent. Typically, 4 equivalents of active ester and a coupling time of 1 hour are used for the resin. To maximize compound usage, use HATU instead of HBTU, use longer coupling times, optionally in smaller quantities (less than 2 equivalents of active ester), expensive amino acids or difficult couplings To do.
For example, to avoid side reactions / decomposition of ceramide during cleavage from the solid support, the use of a very acid labile Sieber resin is preferred. Amino acids such as Arg (Pbf) require 85% or more trifluoroacetic acid and a reaction time of 1 hour or more for deprotection. Since Sieber linkers cause side reactions in concentrated trifluoroacetic acid, the linker is preferably PAL-PEG-PS-resin in these cases.

DHC−Glc−Arg−Arg−βAla−Glu(Rho)−Gly−NH₂
前述したとおり、0.25 mmolのシーバーアミド樹脂をFmoc−Glyでロードし、ピペリジンで脱保護する。樹脂をN−メチル−2−ピロリドンおよびCH₂Cl₂で洗浄し、アルゴン入り口、隔壁および撹拌棒を備えたフラスコに移す。フラスコをすばやく脱気し、アルゴンを2回補充する。別のフラスコにて、590 mg(0.6 mmol)のFmoc−Glu(Rho)および229 mg(0.6 mmol)のHATUを、アルゴン下で撹拌しながら3 mlのDMFに懸濁する。208 μL(1.2 mmol)のDIPEAを加え、固体を撹拌および5−10分間の超音波処理により溶解する。得られる深赤色溶液を、シリンジを介して樹脂に加える。この溶液中で樹脂を緩やかに1時間撹拌する。液体を濾去し、樹脂をN−メチル−2−ピロリドンおよびCH₂Cl₂で簡単に洗浄し、合成機に戻し、洗液が無色になるまでN−メチル−2−ピロリドンで洗浄する。Fmoc−βAlaおよび2xFmoc−Argのカップリングによって自動合成を続ける。UVモニタリングを用いて、カップリングステップの完了を確認する。最終的脱保護の後、樹脂をN−メチル−2−ピロリドン およびCH₂Cl₂で洗浄し、約50 μmolずつに分割し、減圧乾燥する。ジヒドロコレステリルグリコール酸(142 mg、318 μmol)およびHATU(121 mg、318 μmol)をアルゴン下で加える。DMF(3 ml)、CH₂Cl₂(2 mL)およびDIPEA(104 μL、636 μmol)を加え、混合物を撹拌し、透明な溶液が得られるまで超音波処理する（約5分間)。この溶液を50 μMol部の前述の樹脂に移し、得られる懸濁液をアルゴン下で1時間緩やかに撹拌する。樹脂をDMFおよびCH₂Cl₂で簡単に手動洗浄し、ABI合成機に移し、N−メチル−2−ピロリドンおよびCH₂Cl₂で洗浄し、減圧乾燥する。乾燥した樹脂に、3−4 mlのトリフルオロ酢酸/H₂O/アニソール/チオアニソール/トリイソプロピルシラン(87：4：3：3：3)の混合物を加え、得られる懸濁液をアルゴン下で2時間緩やかに撹拌する。樹脂を濾去し、約2 mlの切断用反応混液で洗浄する。冷エーテル/石油エーテル(1：2、約100 mL)を加えることによって生成物を濾液から沈澱させ、遠心分離によって分離する。上清を捨て、油状赤色沈澱にMeCN/MeOH(2：1)を加え、回転蒸発乾固し、減圧乾燥する。
Vydac C8カラム(30x250 mm)タイプ208TP1030、溶離液AとしてH₂O/MeCN/MeOH(90：5：5)＋0.1%トリフルオロ酢酸、溶離液BとしてMeCN＋0.1%トリフルオロ酢酸、流速40 mL/分および30分間にわたる50〜65%Bの勾配を用いて、プレパラティブRP−HPLC精製を行う(RT＝14分、215 nmにおけるUV検出)。生成物画分を合わせ、回転蒸発乾固し、HV中で乾燥して、24.4 mgの暗紫色固体を得る。
上述と同じ溶離剤、Vydac 208TP104カラム(4.6x250 mm)および1 ml/分の流速での25分間にわたる45〜70%Bの勾配を用いて分析的HPLCを行う。RT＝18.3分、純度：99%(215 nm)。ESI−MS：754.5 [M+H]²⁺。 Typical Procedure Production of rhodamine labeled raft affinity moiety 200b with a short peptide linker between the raft affinity moiety and the dye label using a cleavage protocol using concentrated trifluoroacetic acid.

DHC-Glc-Arg-Arg-βAla-Glu (Rho) -Gly-NH ₂
As described above, 0.25 mmol of Cieberamide resin is loaded with Fmoc-Gly and deprotected with piperidine. The resin is washed with N-methyl-2-pyrrolidone and CH ₂ Cl ₂ and transferred to a flask equipped with an argon inlet, septum and stir bar. The flask is quickly evacuated and refilled with argon twice. In a separate flask, 590 mg (0.6 mmol) Fmoc-Glu (Rho) and 229 mg (0.6 mmol) HATU are suspended in 3 ml DMF with stirring under argon. 208 μL (1.2 mmol) of DIPEA is added and the solid is dissolved by stirring and sonicating for 5-10 minutes. The resulting deep red solution is added to the resin via a syringe. The resin is gently stirred in this solution for 1 hour. Filtration of the liquid, the resin was briefly washed with N- methyl-2-pyrrolidone and CH ₂ Cl _2, back to the synthesizer, washed with N- methyl-2-pyrrolidone until the washings are colorless. Automated synthesis continues by coupling Fmoc-βAla and 2xFmoc-Arg. Use UV monitoring to confirm completion of coupling step. After final deprotection, the resin is washed with N-methyl-2-pyrrolidone and CH ₂ Cl ₂ , divided into approximately 50 μmol portions and dried under reduced pressure. Dihydrocholesteryl glycolic acid (142 mg, 318 μmol) and HATU (121 mg, 318 μmol) are added under argon. DMF (3 ml), CH ₂ Cl ₂ (2 mL) and DIPEA (104 μL, 636 μmol) are added and the mixture is stirred and sonicated until a clear solution is obtained (about 5 minutes). This solution is transferred to 50 μMol of the aforementioned resin and the resulting suspension is gently stirred for 1 hour under argon. The resin was easily manually washed with DMF and CH ₂ Cl _2, transferred to a ABI synthesizer, washed with N- methyl-2-pyrrolidone and CH ₂ Cl _2, dried under reduced pressure. To the dried resin is added 3-4 ml of a mixture of trifluoroacetic acid / H ₂ O / anisole / thioanisole / triisopropylsilane (87: 4: 3: 3: 3) and the resulting suspension is under argon. Stir gently for 2 hours. The resin is filtered off and washed with about 2 ml of cutting reaction mixture. The product is precipitated from the filtrate by adding cold ether / petroleum ether (1: 2, ca. 100 mL) and separated by centrifugation. Discard the supernatant, add MeCN / MeOH (2: 1) to the oily red precipitate, rotary evaporate to dryness and dry under reduced pressure.
Vydac C8 column (30x250 mm) type 208TP1030, H ₂ O / MeCN / MeOH (90: 5: 5) + 0.1% trifluoroacetic acid as eluent A, MeCN + 0.1% trifluoroacetic acid as eluent B, flow rate 40 Preparative RP-HPLC purification is performed using mL / min and a gradient of 50-65% B over 30 min (RT = 14 min, UV detection at 215 nm). The product fractions are combined, rotoevaporated to dryness and dried in HV to give 24.4 mg of a dark purple solid.
Analytical HPLC is performed using the same eluent as above, a Vydac 208TP104 column (4.6 × 250 mm) and a 45-70% B gradient over 25 minutes at a flow rate of 1 ml / min. RT = 18.3 min, purity: 99% (215 nm). ESI-MS: 754.5 [M + H] < ²⁺ >.

Chol−Suc−3Gl−Glu(Rho)−NH₂
リンカービルディングブロックとして市販のFmoc−12−アミノ−4,7,10−トリオキサドデカン酸を用い、前述の方法と同様にして、250 μmolの3Gl−Glu(Rho)−NH−[シーバー樹脂]を製造する。50 μmol部のこの樹脂に、前述のとおり、コレステリルヘミコハク酸塩(97.4 mg、200 mmol)、HATU(76 mg、200 μmol)、CH₂Cl₂(1.5 mL)、DMF(0.5 mL)およびDIPEA(68 μL、400 μmol)から製造した活性エステル溶液を加える。2.5時間反応させた後、樹脂を前述のとおり洗浄する。酸溶液が無色になるまで、2−4 ml部の1%トリフルオロ酢酸／CH₂Cl₂とともにCH₂Cl₂−湿潤樹脂を2−3分間繰り返し振とうし、濾過する（約8−10回）。合わせた濾液を回転蒸発乾固し、28℃浴温にて減圧乾燥する。残渣にアセトニトリルを加え、より小さいフラスコに移し、再度回転蒸発し、減圧乾燥する。
上述のとおり、HPLC精製を行が、ただし、溶離液AとしてH₂O/MeCN/MeOH(85：10：5)＋0.1%トリフルオロ酢酸および20分間にわたる64〜74%Bの勾配を用いる(RT：14.5分)。同じ溶離液および45分間にわたる10〜100%Bの勾配を用いて、分析的HPLCを行う（他の条件は、先の実施例と同じ）。RT：38.4分、純度：96%(215 nm)。ESI−MS：1310.8 [M]⁺。 Production of rhodamine-labeled raft affinity moiety 200j with a short glycol linker between the raft affinity moiety and the dye label using a gentle cleavage protocol using 1% trifluoroacetic acid / dichloromethane

Chol-Suc-3Gl-Glu (Rho) -NH ₂
Using commercially available Fmoc-12-amino-4,7,10-trioxadodecanoic acid as the linker building block, 250 μmol of 3Gl-Glu (Rho) -NH- [Sieber resin] was obtained in the same manner as described above. To manufacture. To 50 μmol parts of this resin, as described above, cholesteryl hemisuccinate (97.4 mg, 200 mmol), HATU (76 mg, 200 μmol), CH ₂ Cl ₂ (1.5 mL), DMF (0.5 mL) and DIPEA Add the active ester solution prepared from (68 μL, 400 μmol). After reacting for 2.5 hours, the resin is washed as described above. Shake the CH ₂ Cl ₂ -wet resin with 2-4 ml portions of 1% trifluoroacetic acid / CH ₂ Cl ₂ repeatedly for 2-3 minutes and filter (about 8-10 times) until the acid solution is colorless. ). The combined filtrates are rotoevaporated to dryness and dried in vacuo at 28 ° C. bath temperature. Acetonitrile is added to the residue, transferred to a smaller flask, rotary evaporated again and dried in vacuo.
HPLC purification is performed as described above, but using H ₂ O / MeCN / MeOH (85: 10: 5) + 0.1% trifluoroacetic acid and a gradient of 64-74% B over 20 minutes as eluent A (RT: 14.5 minutes). Analytical HPLC is performed using the same eluent and a gradient of 10-100% B over 45 minutes (other conditions are the same as in the previous example). RT: 38.4 min, purity: 96% (215 nm). ESI-MS: 1310.8 [M] ^<+> .

Example 33: Liposome Raft Affinity Assay (LRA) and Surfactant Resistant Membrane Assay (DRM)
In accordance with the present invention, the raft affinity of the compounds of the invention can be quantified by in vitro testing of the synthesized compounds. Such in vitro tests include the tests provided herein. The assays provided herein and described in detail below can be used as a single assay or a combined assay.

A. LRA principle Quantifies the distribution of test compounds to liposomes presenting either non-raft or raft membranes. The test system includes three components where a test compound can be found, a lipid membrane (non-raft or raft), an aqueous supernatant and a test tube wall. Following incubation, liposomes are removed from the system and test compounds are measured in aqueous and tube wall fractions by fluorimetry or quantitative mass spectrometry using a Tecan Safire multifunctional double monochromator fluorescence intensity reader. Mass spectrometry is performed by a combination of HPLC and mass spectrometry (HPLC-MS) using Hewlett-Packard 1100 (HPLC) and Eskyya LC (mass spectrometry); the method used for mass spectrometry is also used for chemical reactions Spray ionization (ESI). Data is calculated to obtain partition coefficient and raft affinity.

Experimental protocol
1． Raft (R) or non-raft (N) liposomes (see liposome production; see below) are added to the replica tube and preincubated.
2． Test compounds (usually in dimethyl sulfoxide (DMSO)) are added and incubated for 1 hour. Remove the liposomes from one set of tubes and elute the attached compounds from the tube wall with 100 μl of 40 mM octyl-β-D-glucopyranoside (OG) / phosphate buffer (PBS) (A). According to reaction scheme 1, the second set of tubes is centrifuged at 400,000 × g and the supernatant (S) is collected.

蛍光定量法または定量的質量分析によって、水性上清および付着画分を検出する。 Reaction process formula 1

Aqueous supernatant and attached fractions are detected by fluorimetry or quantitative mass spectrometry.

Calculation of partition coefficient and raft affinity
Fraction
L: Liposomes (before centrifugation)
A: Tube deposit
S: Supernatant, free compound concentration
I: Input concentration = L + A
Concentration in membrane fraction (M)
M = L−S (1)
[M] = M ^* f (volume ratio factor) (2)
Aqueous: membrane volume ratio at f = 1 mg lipid / ml = 878.65
The partition coefficient and raft affinity partition coefficient Cp are the ratio of the compound concentration in the membrane and aqueous phase.
Cp = [M] / S (3)
Raft affinity (Rf)
Rf ＝ Cp (R) / Cp (N) (4)

Production and storage of liposome-produced lipid mixtures Lipid solutions and mixtures are usually at 10 mg / ml.
Example composition of liposome mixture.

Formation of liposomes by dialysis
1． Lipid is added to 600 μl of 400 mM 1-octyl-β-D-glucoside (OG) in PBS or other buffer at room temperature and rotary evaporated at 37 ° C. (non-raft lipid) or 50 ° C. (raft lipid) . Vortex for 10 seconds when dissolving. The surfactant concentration is changed in proportion to the lipid concentration.
2． Dilute lipid to 1 mg / ml. Add 5.4 ml buffer (cell culture quality) at room temperature and vortex for 10 seconds. If lipid residue remains, continue spinning at 37 ° C or 50 ° C for an additional 5-10 minutes. At the start of dialysis, the raft lipid: surfactant ratio should be 0.04.
3. Prepare a 5 liter glass beaker with 5 liters of PBS and 20 g of pre-treated Amberlite XAD-2 beads in a 22 ° C. room. Stir at 200-250 rpm.
Four. Aspirate the lipid into a 10-20 ml syringe and inject into the slide-A-riser cassette window. Carefully evacuate all air from the cassette. Dialyze for 8 hours, change and dialyze overnight.
Five. Liposome recovery: Amberlite beads adhering to the outside of the cassette are removed by rinsing with buffer. Fill the cassette with sufficient air from an unused window with a 10-20 ml syringe, tilt the cassette and collect the liposomes.
6． Transfer to a glass tube and store in ice in a dark room. Keep in a cool place until use and use within the next 3 days.

According to the invention, the compounds of the invention, in particular the ternary compounds, have a ratio of equilibrium constants as defined above of greater than 8, more preferably greater than 9, still more preferably greater than 10, even more preferably greater than 11. In some cases it is considered “raft affinity”. As described herein, more preferred compounds (either precursor A of the ternary compound of the invention and the precursor of the moiety A ′ or the complete ternary compound) have a ratio of equilibrium constants greater than 20, Preferably the compound is greater than 30, most preferably greater than 40.
This test / assay as well as the following DRM assay can be used to estimate, confirm and / or determine the raft affinity of a construct, eg, the tripartite construct of the invention and the raft affinity of the portion A / A 'of the compound of the invention. Useful.

B. Surfactant Resistant Membrane Assay (DRM) Principle Determine the accumulation of test compounds in non-raft and raft membrane derived cell membrane fractions. The test system includes treating cultured cells with a test compound. Following incubation, the cells are lysed in detergent solution and the DRM fraction (raft) is isolated on a sucrose gradient. The DRM fraction is collected and the test compound is measured by fluorimetry or quantitative mass spectrometry. Raft affinity is determined as the ratio of the test compound recovered in the DRM fraction to the test compound contained in the entire membrane.
A better comparison of the results of different experiments is achieved by comparing the raft affinity of the test compound with known raft affinity standards.

Experimental protocol
1． Cultured mammalian cells (eg, MDCK, NIH3T3, E6.1 Jurkat T, RBL-2H3, NK3.3, Ramos, Caco-2, BHK, etc.) are grown in sub-confluence.
2． Cells are incubated with test compounds (usually 1-10 μM in ethanol or DMSO) for 1 hour at 37 ° C .; 2 ml ice-cold TNE buffer (100 mM Tris (pH 7.5), 150 mM NaCl and 0.2 mM EGTA ( The dish is washed twice with ethylene glycol-bis- (beta-amino-ethyl ether) N, N, N ′, N′-tetra-acetic acid)) and cooled for 5 minutes. Cells are extracted with 0.5 ml of TNE buffer containing 1% (w / v) Triton X-100 for 30 minutes at 4 ° C.
3． Scrape the cells with a cell lifter and homogenize by passing 10 times through a 1 ml pipette tip. The lysate is transferred to an Eppendorf tube, sonicated in an ice-water bath, and then centrifuged at 3,000 rpm for 5 minutes at 4 ° C.
4. Transfer 0.3 ml of lysate to an Eppendorf tube containing 0.6 ml 65% (w / w) sucrose / TNE and add approximately 47% sucrose to the lysate by vortexing vigorously. Place 0.7 ml of this lysate / sucrose sample at the bottom of the SW-60 tube, add 2.7 ml of 35% (w / w) sucrose / TNE, then 0.8 ml of TNE. The gradient is centrifuged at 335,000 xg for 16 hours at 4 ° C in a SW-60 rotor.
Five. Collect 1040 μl of DRM and non-DRM fractions starting from the top of the gradient. DRM fractions are fractions 2 and 3, and non-DRM fractions are fractions 6, 7 and 8. Detect compounds in the fractions by fluorimetry or quantitative mass spectrometry.

Calculation of Raft Affinity Based on DRM The dimensionless raft affinity quotient rq is obtained from the following equation:
r _q =% DRM /% non-DRM
The relative raft affinity (r _rel ) of an unknown compound relative to a standard is calculated by the following formula:
r _rel = r _q (test compound) / r _q (standard)
A positive value indicates that the test compound has a higher raft affinity than the standard. As standards, cholesteryl 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-dodecanoate (cholesteryl BODIPY® FL C12; Molecular Probes, Eugene, USA) However, it is not limited to this.
According to the specification and the assays provided herein, a compound, particularly a compound of the invention, is judged to be “raft affinity” if the corresponding relative value (compared to a standard) is greater than 0.1. .

Example 34: Exemplary LRA Test This assay is used for all test compounds that are sufficiently water soluble to obtain a measurable concentration in water after incubation with liposomes. Other lipophilic test compounds (eg, compound 27, etc.) are measured in the DRM assay (see Example 33).
Raft (cholesterol: sphingomyelin: phosphatidylcholine: phosphatidylethanolamine: ganglioside (bovine brain, when compared with a lipid mixture representing non-raft (phosphatidylcholine: phosphatidylethanolamine (50:50))) The ability to partition at 37 ° C. into liposomes consisting of a lipid mixture representing type III, Sigma-Aldrich Co.) (50: 15: 15: 15: 5)) is evaluated. The aforementioned relative partition is defined as raft affinity in the LRA assay. Compounds are added to a double set of liposomes using the composition described above from DMSO or ethanol stock solutions at a final concentration of 0.2-2.0 μM. The maximum compound concentration is 2 mol% with respect to lipid concentration. Liposomes are pre-incubated in a thermomixer for 30 minutes in PBS, then compounds are added and further incubated at 37 ° C. for 1 hour. Liposomes are transferred quantitatively from one set of tubes and the remaining compound is eluted from the tube wall with 100 μl of 40 mM octyl-β-D-glucopyranoside in PBS. Centrifuge a second set of tubes at 400,000 xg and collect the supernatant. Determine compound concentration in total liposome solution, adherent fraction and aqueous supernatant by fluorimetry or quantitative mass spectrometry.

The partition coefficient of the compound in each liposome type is determined as the ratio of the concentration of the compound in the liposome membrane to the concentration in the aqueous supernatant. The volume of liposome membrane is calculated using the aqueous: membrane volume ratio at 1 mg lipid / ml of 878.65. Raft affinity (raft affinity) is calculated as the ratio of partition coefficients for raft and non-raft liposomes. The LRA raft affinity of cholesterol-based raft anchors alone is about 50 (ie, 50 times greater affinity for raft liposomes), and the affinity of the tripartite is 50 or greater.
As described above, according to the present invention, a value of 8 or more, preferably 9 or more is determined to be an index of raft affinity in LRA. When the corresponding LRA value is 10 or more, the preferred raft affinity compound is significantly raft affinity. Therefore, the tested ternary compounds are judged to be compounds with high raft affinity.

Example 35: Exemplary DRM assay / DRM test Sub-confluent MCDK grown in minimal essential medium (MEM-E) with Earl salt, 1x GlutaMax I (Invitrogen), 5% FCS, 250 μg / ml G418 (Canine kidney epithelium) or RBL-2H3 (rat B cell lymphocyte) cells were washed with MEM-E, 1x GlutaMax I, 10 mM HEPES, pH 7.3, in the same medium containing compound 27, DMSO or ethanol Incubate with a raft marker material such as 1.0 μM cholesteryl BODIPY-FL C12 (Molecular Probes, Inc) from both stock solutions for 1 hour at 37 ° C. at a final concentration of 1.0-10 μM. The cells were washed twice with 2 ml of ice-cold Dulbecco's PBS containing Ca2 + and Mg2 +, cooled at 4 ° C for 5 minutes, and then 0.5 ml of 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA (ethylenediamine). Extract with tetraacetic acid), 1% (w / v) Triton X-100 (TN-T) at 4 ° C. for 30 minutes. Cells are scraped off the plate and homogenized by passing 10 times through a 25G syringe needle. The lysate is sonicated with an Bandelin Sonoplus HD200 sonifier (MS73 chip, power setting MS72 / D, 60 sec, 10% cycle) in an ice-water bath, followed by centrifugation at 3000 xg for 5 min at 4 ° C. Transfer 0.3 ml of lysate to an eppendorf tube containing 0.6 ml 65% (w / w) sucrose / 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA (TNE). Add% sucrose and vortex vigorously. Place 0.7 ml of the lysate / sucrose sample on the bottom of the SW-60 tube, add 2.7 ml of 35% (w / w) sucrose / TNE, then 0.8 ml of TNE. The gradient is centrifuged for 16 hours at 45,000C in a Beckman LE 80K centrifuge with SW-60 rotor at 335,000xg. Collect each 520 μl fraction from the top to the bottom of the gradient. Collect pooled fractions 2 and 3. This represents the DRM fraction. Pooled fractions 6, 7 and 8 represent non-DRM fractions. Determine compound concentration in DRM and non-DRM fractions by quantitative fluorimetry or mass spectrometry. The dimensionless raft affinity quotient rq is obtained from the formula: rq =% DRM /% non-DRM, where DRM% and non-DRM% are the total fluorescence or mass of the compound in each fraction. The raft affinity rq of the test compound in the DRM assay is normalized to a standard raft affinity such as, for example, cholesterol BODIPY-FL C12 available from Molecular Probes, rrel = rq (test compound) / rq (standard) .
In this assay, the r _rel of tripartite 27 containing ceramide as a raft affinity moiety is 0.48.
Thus, if rrel (in this assay system) is 0.2 or greater, more preferably 0.3 or greater, even more preferably 0.4 or greater, the compounds defined herein, particularly the tripartite constructs / compounds and the individual moieties A and Portion A ′ can be determined to be “raft affinity”.

Example 36: Inhibition of BACE-1 by the tripartite compounds of the present invention For their ability to inhibit beta-secretase (BACE-1) and their potency when compared to the titer of free inhibitor III in a whole cell assay. Tripartite compounds of formula 24, 24b, 25 and 25b were tested. Infect mouse neuroblastoma cells (N2a) grown in DMEM (Dulbecco's Modified Eagle Medium), 1x glutamine, 10% FCS (fetal calf serum) with recombinant adenovirus containing amyloid precursor protein (APP) gene Let 75 minutes after infection, cells are washed, trypsinized, and subcultured. After about 20 hours (50% confluence), the medium is aspirated and replaced with fresh medium containing 10 nM to 10 μM test compound in DMSO or methanol, and the cells are incubated at 37 ° C. in 5% CO ₂ . Incubate for 3-4 hours. Following incubation, supernatant samples are collected and the production of β-cleaved extracellular domains (βAPPs) of APP is measured using an ELISA assay with a specific monoclonal antibody against βAPPs and a second antibody against the N-terminus of βAPPs. As demonstrated in the accompanying drawings, in this system, the inhibitor III alone is not inhibitory, but the tripartite compound 25, especially 25b, is a potent inhibitor of βAPPs and thus has a potent beta secretase activity. Inhibitor. Furthermore, tripartite compounds containing shorter linkers [see compounds 26 and 27] are less effective in this specific assay and demonstrate that linker length is critical for inhibition of beta-secretase by inhibitor III . Thus, compounds 26 and 27 probably do not place a specific pharmacophore inhibitor III at the exact locus on the BACE-1 enzyme. Furthermore, the linkers defined in compounds 26 and 27 are useful in other test systems for the inhibition of biomolecules where the corresponding binding / interaction site is located closer to the head of the raft phospholipid.

Example 37: Tripartite Compound of the Invention in Proteoliposome Assay In the following, the following additional non-limiting assay for confirmation and / or characterization is described using the three-part structure disclosed herein as a model To do. This assay is a proteoliposome assay.
Principle of BACE Proteoliposome Assay The tripartite raft affinity test compound is incorporated into a liposome representing the raft membrane and then reconstituted with recombinant BACE as described in A (BACE proteoliposome). BACE is a membrane anchor type with a transmembrane domain. At the same time as anchoring the lipid portion of the test compound to the membrane, a spacer and pharmacophore are introduced into the aqueous phase. In an optimal topology, the pharmacophore can block the BACE active site (FIG. 1: top). For activity and inhibition assays, BACE proteoliposomes are suspended in assay buffer and preincubated for 10 minutes at room temperature. The internal quenching fluorescent substrate analog FS-1 (Dabcyl- [Asn670, Leu671] -amyloid β / A4 protein precursor 770 fragment (661-675) -Edans; Sigma A 4972) was added, Cleavage reveals a fluorescent signal. This signal is recorded at a specified interval with a Thermoscan Ascent fluorimeter (see Figure 1: bottom).

Experimental protocol Proteoliposomes are produced in two steps:
1． Liposome formation by continuous dialysis and test compound incorporation;
2． Purification by BACE surfactant-accelerated membrane reconstruction and gel filtration and density gradient centrifugation.
Ad 1. Formation of liposomes incorporating test compounds by dialysis
1.1. 5 mg porcine brain lipid (Avanti 131101) in chloroform solution is applied to a round bottom flask in a rotary evaporator and evaporated overnight in a desiccator. To the lipid, add 0.5 ml of 400 mM 1-octyl-β-D-glucoside (OG) in water, rotate at 50 ° C., then 1.166 ml phosphate buffered saline (PBS), 0.02% sodium azide ( NaN ₃ ) is added to a final OG concentration of 120 mM and a lipid concentration of 3 mg / ml or 4.8 mM. Rotate the suspension again for about 5 minutes at 50 ° C. until homogeneous.
1.1.a Demonstration of raft characteristics of porcine brain lipid liposomes. Porcine brain lipid is qualitatively and quantitatively similar to the raft lipid mixture used in LRA, but is a more complex composition. Example 33, A.I. Raft (R) liposomes and non-raft (N) liposomes and porcine brain lipid liposomes (P) prepared according to the principles of LRA, liposome production, are tested with standard raft affinity tracers. Distribution to P liposomes is shown to be identical or somewhat greater than distribution to R liposomes; raft affinity (Rf) ≧ 40.

1.2. Aliquot the lipid suspension in a glass tube (0.35 ml for control and 0.26 ml for incorporation of test compound). To several aliquots, add test compound from a 100x stock solution in DMSO and vortex for 10 seconds. At the start of dialysis, the total lipid: surfactant ratio should be 0.04 and 1% DMSO. The test compound starting concentration is between 0.0005 and 0.05 mol%.
1. Aspirate the 0.25 ml (initial volume, v _i ) lipid mixture with a 1 ml syringe and inject into the window of a 0.5 ml slide-A-riser cassette (Pierce) predialyzed overnight at 10 kD exclusion. Carefully evacuate the cassette. Each cassette is transferred to a Petri dish containing 375 μl PBS / 0.02% NaN ₃ placed directly under the cassette and 375 μl PBS placed on top of the cassette. Dialyze for 3 hours and change twice using a new petri dish for each change. The third dialysis is performed overnight. Continue on day 2 with 3 changes of 2x2.5 ml PBS.

1.4. Prepare a 5 L glass beaker containing 5 L PBS and 100 ml of 20% pre-treated Amberlite XAD-2 beads (Supelco 20275) in an incubator at 22 ° C. Transfer the cassette to a beaker and dialyze for 16 hours. Stir at 200-250 rpm.
1.5. Amberlite beads adhering to the outside of the cassette are removed by rinsing with buffer. Fill the cassette with enough air from an unused window with a 1 ml syringe, tilt the cassette, and collect the liposomes.
1.6. The volume after dialysis (v _p ) is measured with a syringe and transferred to a brown glass tube. Dilute each sample to 3 times the initial volume. The test compound concentration after dialysis is determined by fluorimetry, mass spectrometry or other suitable method. Store on ice in the dark until use within.

Ad 2. Proteoliposome production
2.1. Liposomes are pelleted and added to 70 μl 10 mM Hepes / 150 mM NaCl pH 7.3 (buffer). Add 8 μl 10% decanoyl l-N-hydroxyethylglucamide (HEGA 10). 2 μg / 8 μl recombinant BACE (in 0.4% Triton X-100) is then added.
2.2. Gel filter with Sephadex G-50 in 10 mM Hepes / 150 mM NaCl pH 7.3.
2. Separating proteoliposomes from empty liposomes by floating on a 5% Optiprep gradient.
2.4. The roteoliposome band is collected, diluted and pelleted. Resuspend the pellet in 50-100 μl buffer and quantify the protein.

Examples of proteoliposome assays:
1． Formation of liposome-incorporated test compounds by dialysis
1.1. Lipid Application and Homogenization 4 mg porcine brain lipid (Avanti 131101) in chloroform is dried at 50 ° C. in a round bottom flask in a rotary evaporator. Add 1.5 ml tert-butanol to redissolve the lipids. Rotate the flask at 50 ° C. until the lipid forms a homogeneous film. Trace amounts of solvent are removed by drying the flask overnight in a desiccator. Add 0.5 ml of 400 mM 1-octyl-β-D-glucoside (OG) in water to the lipid, then rotate at 50 ° C., then 1.166 ml phosphate buffered saline (PBS), 0.02% sodium azide (NaN ₃ ) is added to a final OG concentration of 120 mM and a lipid concentration of 3 mg / ml or 4.8 mM. Rotate the suspension again for about 5 minutes at 50 ° C. until homogeneous.
1.2. Add Test Compound Lipid suspension is aliquoted into glass tube (0.35 ml for control and 0.26 ml for incorporation of test compound). To an aliquot, add test compound 25b from a 100x stock solution in DMSO and dilute 1: 100 (compare Table 1):
0.05 mol% 2.4 μM 2.4 μl (250 μM stock solution)
0.005 mol% 0.24 μM 2.4 μl (25 μM stock solution)
0.0005 mol% 0.024 μM 2.4 μl (2.5 μM stock solution)
The tube is then vortexed for 10 seconds.

1.3. Continuous dialysis
0.25 ml (initial value, v _i , Table 1) of the lipid mixture is transferred to a 1 ml syringe and injected into the window of a 0.5 ml slide-A-riser cassette (Pierce) pre-dialyzed overnight at 10 kD exclusion. The air is then evacuated from the cassette. Place each cassette in a separate Petri dish containing 375 μl PBS / 0.02% NaN ₃ pipetted directly under the cassette and 375 μl PBS transferred to the top of the cassette. After dialysis for 3 hours, transfer the cassette to a new petri dish and repeat the procedure. The third dialysis is performed overnight. On the second day, repeat the procedure with 3 changes of 2 × 2.5 ml PBS (2.5 ml PBS under the cassette and 2.5 ml PBS at the top) three times. Wrap the Petri dish with aluminum foil throughout the procedure to avoid fading.
1.4. Bulk dialysis
A 5 L glass beaker containing 5 L PBS and 100 ml of 20% pretreated Amberlite XAD-2 beads (Supelco 20275) and a magnetic stir bar is placed in a 22 ° C. incubator. All cassettes are inserted into a float (Pierce), placed in a beaker and dialyzed at 200-250 rpm for 16 hours. Wrap the beaker with aluminum foil.
1.5. Liposome recovery
Rinse off the amberlite beads adhering to the outside of the cassette.
Using a 1 ml syringe, fill the cassette with air from an unused window, tilt the cassette, and collect the liposomes with the syringe. The volume after dialysis (v _p ) is measured using a syringe and the liposomes are transferred to a brown glass tube with a screw cap. Each sample is diluted to 3 times the initial volume v _i with PBS (see Table 1).
1.6. Determination of final 25b concentration
25b concentration standards 25, 250 and 2500 nM are prepared in PBS / 40 mM OG and 100 μl of 4 samples of each standard are filled into wells of a 96 well plate (Nunc Maxisorb). 50 μl of each liposome preparation is diluted in 50 μl of 80 mM OG / PBS in a 96 well plate. PBS and OG controls are added and shaken briefly before recording fluorescence at 553/592 nm (excitation / emission wavelength) in a Tecan Safire fluorometer plate. Plot the baseline fluorescence reading, calculate the regression line (Excel), and then calculate the final 25b concentration in the liposome preparation (see Table 1).

2.Proteoliposome preparation
2.1. The liposomes are pelleted by treatment at 48,000 rpm for 20 minutes in a TLA-100 rotor and 70 μl of 10 mM Hepes / 150 mM NaCl pH 7.3 (buffer) is added. Add 8 μl of 10% decanoyl-N-hydroxyethylglucamide (HEGA 10). Finally, 2 μg / 8 μl of recombinant BACE (in 0.4% Triton X-100) is added and mixed by pipetting and draining.
2.2 Gel filtration on Sephadex G-50 in 10 mM Hepes / 150 mM NaCl pH 7.3. Pipet the sample into the gel filtration column. Collect the flow-through containing the proteoliposomes.
2.3. Pipet this material onto a 5% Optiprep gradient and centrifuge. Proteoliposome bands are collected, diluted and pelleted. Resuspend the pellet in 50-100 μl of buffer. Quantify the protein.
2.4. BACE assay
Add 10 μl proteoliposome (60 ng BACE, 4.5 μg lipid), 70 μl 80 mM NaOAc pH 5.1 and 20 μl 10 mM Hepes / 150 mM NaCl pH 7.3 to each well of a 96 well plate. The mixture is preincubated for 30 minutes at 37 ° C. Finally, 2 μl of substrate FS-1 in 1.5 M HAc (5 μM final concentration) is added. Shake for 8 seconds before each measurement and record fluorescence at 485 nm (excitation 340 nm) every 40 seconds.

¹ mol %は、脂質濃度に関して付与される。 table 1. Overview of lipid and test compound concentrations during preparation of proteoliposomes

¹ mol% is given in terms of lipid concentration.

Enrichment of inhibitors within the raft subcompartment by coupling to the raft affinity moiety not only results in an increase in intensity proportional to the inhibitor concentration, but also an unbalanced increase due to the reduced ability of the inhibitor to diffuse from the site of action. Bring. This “lock-in” effect takes advantage of the same phenomenon used by cells to increase protein-protein interactions. The results shown in FIG. 1 reveal that 25b is even more potent than inhibitor III. Measurements obtained from the graph, compared to ED ₅₀ inhibitor III is 500-1000 nM, indicating that 25b of the ED ₅₀ (the concentration of BACE activity is reduced to 50%) is about 1 nM . Thus, the inhibitor strength is increased by a factor of 500-1000 by incorporation into a type of tripartite structure exemplified by 25b.

  Inhibitors were also tested in a functional assay incorporating neurons expressing exogenous swAPP (highly cleavable from APP) as described in Example 36 (FIG. 2: see top). Cells are treated with 25b or inhibitor III and the release of beta cleavage products in the cell culture supernatant is measured.

  From the results shown in FIG. 2, it is clear that 25b is inhibitory in the whole cell assay, whereas the commercially available inhibitor III is completely inactive. 25b can reduce activity by 65% at 1 μM. BACE-1 is active in acidic endosomes, thus free inhibitor III is required to cross both the cell and endosomal membranes to reach the target. Without being bound by theory, 25b appears to be inserted into the raft membrane where BACE-1 is located at a specific location and taken up with the protein. Therefore, targeting and efficacy is ensured by the presence of the tripartite construct (Figure 2: top). Therefore, inhibitor III does not cross the cell membrane, and raft affinity partial binding inhibitor 25b gains entry into the cell and efficiently inhibits beta-secretase activity.

Example 38: Specific inhibition of HIV infection by a tripartite raft affinity structure
The early stages of HIV infection—from absorption to entry into host cells—include the following sequential stages (reviewed in Olson (2003) Infect. Disorders 3, 255): via its spike protein gp120, HIV Binds to the first receptor CD4 raft protein. Binding induces a conformational change in gp120, allowing it to bind to a co-receptor, one of several chemokine receptors, and recruit to rafts (Fantini (2001) Glycoconj. J. 17, 199-204). This in turn triggers a conformational change in gp41, a viral fusion protein closely linked to gp120. gp41 adopts two elongated pre-hairpin conformations in which the N-terminal fusion peptide protrudes into the plasma membrane and exposes the seven repeat regions HR1 and HR2. When three copies of HR2 fold back into a hairpin-forming HR1 trimer, the virus and plasma membrane-two juxtaposed raft domains-form and fuse together (Weissenhorn et (1997) Nature 387, 426 −430). The strong interaction between HR1 and HR2 is blocked by a soluble HR2 peptide analog (enfuvirtide (T20; DP178) is one of them) (Wild (1992) Proc Natl Acad Sci USA 91, 9770-9774). . Also, inter alia, PEGylated forms of T1249 and these peptide inhibitors are known.

  A hairpin is not formed, preventing fusion of the virus with the host membrane. It is clear that the soluble inhibitor can only bind to the virus after binding to its two receptors, i.e. it acts proximal to the membrane. In fact, T20 is also inhibitory when expressed on the cell membrane from an appropriate construct (Hildinger (2001) J. Virol. 75, 3038-3042). The pharmacophore (enfuvirtide) in the tripartite structure of the present invention is a raft anchor (raft affinity moiety) via a spacer element (hinge and linker) so that the inhibitor sticks out of the target membrane raft towards the infected virion. (See FIG. 3). The ternary drug pharmacophore (HR2 analog) binds to the HR1 element exposed during the gp41 conformational change, effectively locking the protein within its conformational transition state, as well as the protoplasm It can be physically fixed to the membrane. (1) Tripartite drugs are enriched in the raft domain about 10,000 times for media and about 50 times for raft membranes, and (2) necessary to irreversibly block infection and aim virions for destruction Since the number of tripartite drug molecules per virion is smaller, the drug concentration to achieve this is expected to be on the order of a lower scale than that of a soluble inhibitor such as enfuvirtide. In addition to inhibiting free virus entry, the same inhibitory mechanism blocks the fusion of infected and uninfected cells subject to the same event.

HIV invasion assay (after Salzwedel et al., 1999).
Human fetal kidney 293T cells are transfected with a proviral clone of the subject HIV strain. After 60-72 hours, the virus-containing cell culture supernatant is collected and filtered through a 0.45 μm pore size filter. The virus is then used to infect HeLa-CD4 / LTR-β-gal cells. The cells are stained in situ with X-gal, and the monolayer is imaged with a CCD camera (Fuji LAS), and the blue lesions are counted. As another reading, HIV gp24 expression can be monitored by ELISA (see, eg, Hildinger (2001), supra). Similarly, to measure cell-cell fusion, infected 293T cells are mixed with uninfected HeLa-CD4 / LTR-β-gal cells and evaluated in a similar manner; Virol. 73, see 2469-2480.

Ac−Tyr−Thr−Ser−Leu−Ile−His−Ser−Leu−Ile−Glu−Glu−Ser−Gln−Asn−Gln−Gln−Glu−Lys−Asn−Glu−Gln−Glu−Leu−Leu−Glu−Leu−Asp−Lys−Trp−Ala−Ser−Leu−Trp−Asn−Trp−Phe−4Gl−Glu(Rho)−4Gl−Lys(CO−CH₂−ジヒドロコレステリル)−NH₂
HBTUのABI−433の貯蔵溶液をと置き換えるか、または固体HATUおよびFmoc−アミノ酸(1 mmol each)の混合物を合成機のアミノ酸カートリッジに置き、それに合うように合成機のソフトウェアを変更するかのいずれかによって、HATUを用いるカップリングを行う。固相支持体としてPAL−PEG−PS樹脂(ローディング：0.21 mmol/g)を用いる。0.25 mmolで行う化学反応プログラムおよび0.25 mmolの反応器を用いて0.1 mmolの樹脂を処理して、合成中に相当な量の獲得を可能にする。Dde−Lys(Fmoc)を用いてラフト親和性部分をリシンの側鎖に結合する。[Novabiochemカタログ2004/5、p48；p4−12]。Ac₂Oによるキャッピングに続いて各カップリングを行う。 Synthesis of compound 25c of the present invention containing T20 as pharmacophore C

Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu-Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Leu- Glu-Leu-Asp-Lys- Trp-Ala-Ser-Leu-Trp-Asn-Trp-Phe-4Gl-Glu (Rho) -4Gl-Lys (CO-CH 2 - dihydrocholesteryl) -NH ₂
Either replace HBTU's ABI-433 stock solution or place a mixture of solid HATU and Fmoc-amino acids (1 mmol each) in the synthesizer amino acid cartridge and modify the synthesizer software to match. Depending on the type of coupling, use HATU. PAL-PEG-PS resin (loading: 0.21 mmol / g) is used as a solid support. The 0.1 mmol resin is processed using a 0.25 mmol chemical reaction program and a 0.25 mmol reactor to allow for the acquisition of significant amounts during synthesis. The raft affinity moiety is attached to the side chain of lysine using Dde-Lys (Fmoc). [Novabiochem catalog 2004/5, p48; p4-12]. Each coupling is performed following capping with Ac ₂ O.

Dde-Lys (Fmoc) is bound to the resin, deprotected and washed with an automated synthesizer. Dihydrocholesteryl-CH ₂ —COOH is coupled to the side chain and the Dde group is removed by treatment with 2% hydrazine hydrate (4 × 12 ml; 5 minutes each) in DMF. As above, the remaining sequences are coupled. Only 0.5 mmol (5 eq) of Glu (Rho) is used. UV monitoring shows that the coupling yield decreases towards the end of the sequence.

Prior to cleavage from the resin, the trityl group is removed by washing 5 times with CH ₂ Cl ₂ / triisopropylsilane / trifluoroacetic acid (94: 5: 1). The resin is washed with CH ₂ Cl ₂ (4x) and dried in vacuo. Cleavage and deprotection are carried out using trifluoroacetic acid / H ₂ O / dithiothreitol / triisopropylsilane (87: 5: 5: 3) and a reaction time of 2 hours. The solution is filtered, concentrated to <50% on a rotary evaporator (bath temperature 28 ° C.) and triturated with petroleum ether / methyl tert-butyl ether (3: 1). The crude oily product is separated by centrifugation and triturated with 4 parts petroleum ether / methyl tert-butyl ether (4: 1) to form a red semi-solid. This is dissolved in a mixture of acetonitrile (3.5 ml), H ₂ O (2.5 ml) and acetic acid (65 μl), degassed with a stream of argon and left at room temperature overnight.

A: H ₂ O / MeCN (85:15) + 0.1% trifluoroacetic acid, B: MeCN + 0.1% trifluoroacetic acid, Vydac-C8 column type 208TP104 and 10% over 45 minutes at a flow rate of 1 ml / min. Analytical HPLC of the crude mixture is performed using a 100% B gradient. ESI-MS shows product RT = 32.2 min t.

  Perform preparative purification in two stages. First, the material is chromatographed on a Vydac column type 208TP1030 using the same eluent as above and a gradient of 53% to 64% B at a flow rate of 20 ml /. Collect the fractions that elute at RT = 18.1-23.1 minutes. A flow rate of 40 ml / min and a gradient of 30% to 40% B over 5 minutes, then a gradient of 40% to 50% B over 100 minutes (eluent as above). The product (RT = 81.3 min) is separated, concentrated on a rotary evaporator and dried under reduced pressure. Yield: 4.5 mg red solid.

Proposed mechanism of upper, tripartite 25b and BACE inhibitor III; inhibition of BACE activity by lower, compound 25b and BACE inhibitor III. Control (no inhibitor) activity was set at 100%. Upper part, proposed mechanism of beta secretase (BACE) activity inhibitory action in whole neurons using compound 25b and BACE inhibitor III; lower part, inhibition of beta secretase (BACE) activity by compound 25b and BACE inhibitor III (see also Example 36) ) Upper, proposed mechanism of activity of a tripartite structure incorporating the HIV membrane fusion inhibitor enfuvirtide. HIV spike proteins bind to cell membrane receptors in rafts and promote membrane fusion. Enfuvirtide prevents changes in the conformation of the bound spike protein and prevents membrane fusion. The potency of the tripartite inhibitor is suggested to be 100-1000 times higher due to its richness in rafts.

Claims (58)

Tripartite structure: C-B-A or C'-B'-A '
[Wherein part A and part A ′ are raft-affinity moieties;
Where moieties B and B ′ are linkers;
Where parts C and C ′ are pharmacophores;
Here, the raft affinity of part A and part A ′ includes partitioning into lipid membranes characterized by insolubility in nonionic surfactants at 4 ° C .; and where parts B and B ′ are A linker having a skeleton of at least 8 carbon atoms, wherein one or more of the carbon atoms may be replaced with nitrogen, oxygen or sulfur]
A compound comprising   Lipids comprising a lipid composition wherein the raft affinity of part A and part A ′ comprises cholesterol and / or functional analogs of cholesterol, sphingolipids and / or functional analogs of sphingolipids, glycolipids and glycerophospholipids 2. A compound according to claim 1 comprising partitioning into a membrane.   The compound according to claim 2, wherein the lipid composition comprises a glycolipid selected from ganglioside, cerebroside, globoside and sulfatide.   The compound according to claim 3, wherein the ganglioside is GM1, GD1a, GD1b, GD3, GM2, GM3, GQ1a or GQ1b.   The compound according to any one of claims 2 to 4, wherein the sphingolipid and / or the functional analog of the sphingolipid is sphingomyelin or ceramide.   The compound according to any one of claims 2 to 4, wherein the glycerophospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine.   Claims wherein the lipid composition comprises 5-60% cholesterol and / or a functional analog of cholesterol, 5-40% sphingolipid and / or a functional analog of sphingolipid and 20-80% phospholipid. Item 7. The compound according to any one of Items 2 to 6.   The compound according to any one of claims 2 to 7, wherein the lipid membrane comprises cholesterol, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine and ganglioside.   9. A compound according to claim 8, wherein the lipid membrane comprises 40-60% cholesterol, 0-20% sphingomyelin, 10-20% phosphatidylcholine, 10-20% phosphatidylethanolamine and 1-10% ganglioside. .   10. A compound according to claim 9, wherein the lipid membrane consists of 50% cholesterol, 15% sphingomyelin, 15% phosphatidylcholine, 15% phosphatidylethanolamine and 5% ganglioside.   The lipid membrane comprises equal amounts of the cholesterol and / or functional analogue thereof, the sphingolipid and / or functional analogue thereof and the phospholipid and / or functional analogue thereof. 8. The compound according to any one of 7.   12. The compound of claim 11, wherein the lipid membrane comprises 33% cholesterol, 33% sphingomyelin / ceramide and 33% phosphatidylcholine.   The compound according to any one of claims 1 to 12, wherein the moiety B and the moiety B 'have a total length of 1 nm to 50 nm. The raft affinity moiety A or A ′ is represented by the following formulas 2 and 3:

[here,

Is a single bond or a double bond;
When the tripartite structure is C-B-A, X ²¹ and X ³¹ are NH, O, S, NH (CH ₂ ) _c OPO ₃ ⁻ , NH (CH ₂ ) _c SO ₂ CF ₂ , NH (CH ₂ ) _c SO ₂ NH, NHCONH, NHCOO, NHCH (CONH ₂ ) (CH ₂ ) _d COO, NHCH (COOH) (CH ₂ ) _d COO, NHCH (CONH ₂ ) (CH ₂ ) _d CONH, NHCH (COOH) Directed from (CH ₂ ) _d CONH, NHCH (CONH ₂ ) (CH ₂ ) ₄ NH ((CO) CH ₂ O) _f and NHCH (COOH) (CH ₂ ) ₄ NH ((CO) CH ₂ O) _f Where c is an integer from 2 to 3, d is an integer from 1 to 2, and f is an integer from 0 to 1, where the linker is attached to X ²¹ or X ³¹ Do;
When the tripartite structure is C′-B′-A ′, X ²¹ and X ³¹ are CO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _b S, CO (CH ₂ ) _b OPO ₃ ⁻ , CO (CH ₂ ) _b SO ₂ CF ₂ , CO (CH ₂ ) _b SO ₂ NH, CO (CH ₂ ) _b NHCONH, CO (CH ₂ ) _b OCONH, CO (CH ₂ ) _e CH (CONH ₂ ) NHCO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _e CH (COOH) NHCO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _e CH (CONH ₂ ) NHCO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _e CH (COOH) NHCO (CH ₂ ) _b (CO) _a O, COCH (NH ₂ ) (CH ₂ ) _e COO or COCH (NHCOCH ₃ ) (CH ₂ ) _e COO, where a is an integer from 0 to 1, b is an integer from 1 to 3, and e is an integer from 1 to 2. Where the linker is attached to the terminal carbonyl group of X ²¹ or X ³¹ ; and
R ²¹ and R ³¹ are C _4-20 hydrocarbon groups, wherein one or more hydrogens are optionally replaced by fluorine]
The compound according to claim 1, which is represented by one of the following: The raft affinity moiety A or A ′ is represented by the following formulas 4a and 5a:

[here,

Is a single bond, double bond or triple bond, provided that

Each Y ^42a is absent when is a triple bond;
When the ternary structure is C-B-A, X ^41a and X ^51a are NH, O, NH (CH ₂ ) _c OPO ₃ ⁻ , NH (CH ₂ ) _c SO ₂ NH, NHCONH, NHCOO, NHCH ( CONH ₂ ) (CH ₂ ) _d COO, NHCH (COOH) (CH ₂ ) _d COO, NH (CH ₂ ) ₄ CH (CONH ₂ ) NH, NH (CH ₂ ) ₄ CH (COOH) NH, NHCH (CONH ₂ ) (CH ₂ ) ₄ NH and NHCH (COOH) (CH ₂ ) ₄ NH, wherein c is an integer from 2 to 3 and d is an integer from 1 to 2, where The linker binds to X ^41a or X ^51a ;
When the tripartite structure is C′-B′-A ′, X ^41a and X ^51a are CO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _b S, CO (CH ₂ ) _b OPO ₃ ⁻ , CO (CH ₂ ) _b SO ₂ NH, CO (CH ₂ ) _b NHCONH, CO (CH ₂ ) _b OCONH, CO (CH ₂ ) _b OSO ₃ , _{CO (CH 2) b NHCO 2} , CO (CH 2) e CH (CONH 2) NH, CO (CH 2) e CH (COOH) NH, COCH (NH 2) (CH 2) e COO or COCH (NHCOCH ₃ ) (CH ₂ ) _e COO, where a is an integer from 0 to 1, b is an integer from 1 to 3, and e is an integer from 1 to 2, where the linker is X ^{Binds to} the terminal carbonyl group of ^41a or X ^51a ;
X ^42a and each X ^52a are independently NH, O, S, OCO, NHCO, NHCONH, NHCO ₂ or NHSO ₂ ;
Y ^41a is NH ₂ , NHCH ₃ , OH, H, halogen or O, provided that when Y ^41a is NH ₂ , NHCH ₃ , OH, H or halogen,

Is a single bond and when Y ^41a is O,

Is a double bond;
Each Y ^42a is independently H or OH, provided that when Y ^42a is OH,

Is a single bond;
R ^41a is a C _10-30 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine; and
R ^42a and each R ^52a are independently a C _14-30 hydrocarbon group, wherein one or more hydrogens are optionally replaced by fluorine]
The compound according to claim 1, which is represented by one of the following: The raft affinity moiety A or A ′ is represented by the following formulas 6 and 7:

[here,

Is a single bond, a double bond or a triple bond;
When the tripartite structure is CBA, X ⁶¹ and X ⁷¹ are O, where the linker is attached to X ⁶¹ or X ⁷¹ ;
When the tripartite structure is C′-B′-A ′, X ⁶¹ and X ⁷¹ are CO (CH ₂ ) _b (CO) _a O, where a is an integer from 0 to 1, b is an integer from 1 to 3, where the linker is attached to the terminal carbonyl group of X ⁶¹ or X ⁷¹ ;
Each X ⁷⁵ is independently a CO—C _13-25 hydrocarbon group, wherein one or more hydrogens are optionally fluorine, the following formula:

Or the following formula:

Substituted with a group represented by
X ⁶² and each X ⁷² are independently O or OCO;
X ⁶³ and X ⁷³ are, PO ₃ ^- chosen directed from _{CH 2, SO 3 CH 2,} CH 2, CO 2 CH 2 , and a direct bond;
X ⁶⁴ and X ⁷⁴ are NH, O, S, OCO, NHCO, NHCONH, NHCO ₂ or NHSO ₂ ;
X ⁷⁶ is selected from CO (CH ₂ ) _b (CO) _a O and CO (CH ₂ ) _b (CO) _a NH, where a is an integer from 0 to 1 and b is 1 An integer of ~ 3;
Y ⁶¹ is NH ₂ , NHCH ₃ , OH, H, halogen or O, provided that Y ⁶¹ is NH ₂ , NHCH ₃ , OH, H or halogen,

Is a single bond and when Y ⁶¹ is O,

Is a double bond;
Each R ⁶¹ and each R ⁷¹ is independently a C _16-30 hydrocarbon group, wherein one or more hydrogens are optionally replaced by fluorine;
R ⁶² is a C _13-25 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine; and
R ⁷² is a C _4-20 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine]
The compound according to claim 1, which is represented by one of the following: The raft affinity moiety A or A ′ is represented by the following formulas 18a and 18b:

[here,
When the ternary structure is C-B-A, X ^181a and X ^181b are directed from NH, O, NH (CH ₂ ) _c OPO ₃ ⁻ , NH (CH ₂ ) _c SO ₂ NH, NHCONH and NHCOO. Where c is an integer from 2 to 3, wherein the linker is attached to X ^181a or X ^181b ;
When the tripartite structure is C′-B′-A ′, X ^181a and X ^181b are CO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _b S, CO (CH ₂ ) _b OPO ₃ ⁻ , CO (CH ₂ ) _b SO ₂ NH, CO (CH ₂ ) _b NHCONH, CO (CH ₂ ) _b OCONH, CO (CH ₂ ) _b OSO ₃ or is _{CO (CH 2) b NHCO 2} , wherein, a is an integer from 0 to 1, b is an integer from 1 to 3, wherein the linker is a terminal carbonyl group of X ^181a or X ^181b Join;
Each Y ^181a and each Y ^181b is independently NH ₂ , NHCH ₃ , OH, H or halogen;
Each X ^182a and each X ^182b is independently O, NH, OCO or NHCO; and each R ^181a and each R ^181b is independently a C _15-30 hydrocarbon group, wherein one Or more hydrogen is optionally replaced by fluorine]
The compound in any one of Claims 1-13 represented by these. The raft affinity moiety A or A ′ is represented by the following formulas 19a and 19b:

[here,

Is a single bond or a double bond;
When the ternary structure is C-B-A, X ^191a is NH, O, NH (CH ₂ ) _c OPO ₃ ⁻ , NH (CH ₂ ) _c SO ₂ NH, NHCONH, NHCOO, NHCH (CONH ₂ ) (CH ₂ ) _d COO, NHCH (COOH) (CH ₂ ) _d COO, NH (CH ₂ ) ₄ CH (CONH ₂ ) NH, NH (CH ₂ ) ₄ CH (COOH) NH, NHCH (CONH ₂ ) (CH ₂ ) ₄ NH and NHCH (COOH) (CH ₂ ) ₄ NH, selected from directional, where c is an integer from 2 to 3 and d is an integer from 1 to 2, where the linker is , ^Binds to X ^191a ;
When the tripartite structure is C′-B′-A ′, X ^191a is CO (CH ₂ ) _b (CO) _a NH, CO (CH ₂ ) _b (CO) _a O, CO (CH ₂ ) _b S, CO (CH ₂ ) _b OPO ₃ ⁻ , CO (CH ₂ ) _b SO ₂ NH, CO (CH ₂ ) _b NHCONH, CO (CH ₂ ) _b OCONH, CO (CH ₂ ) _b OSO ₃ , CO (CH _{2) b NHCO 2, CO (} CH 2) e CH (CONH 2) NH, CO (CH 2) e CH (COOH) NH, COCH (NH 2) (CH 2) e COO or COCH (NHCOCH ₃₎ (CH _{2) e} COO, wherein, a is an integer from 0 to 1, b is an integer from 1 to 3, e is an integer of 1 to 2, wherein the linker is terminus of X ^191a Binds to a carbonyl group;
When the tripartite structure is C-B-A, X ^191b is NH (CH ₂ ) _c NHCO, where c is an integer from 2 to 3, where the linker is the end of X ^191b Binds to an amino group;
When the tripartite structure is C′-B′-A ′, X ^191b is CO, wherein the linker binds to X ^191b ;
X ^192a is directionally selected from NHCOCH ₂ NH or NHCOCH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ NH;
X ^192b is directionally selected from COCH ₂ CH ₂ NHCOCH ₂ or COCH ₂ ;
X ^193a and each X ^193b are independently selected from O, NH, C _1-8 alkylene-O and C _1-8 alkylene-NH;
Y ^191a is NH ₂ , OH or H;
R ^191a and each R ^191b are independently a C _4-18 hydrocarbon group, wherein one or more hydrogens are optionally replaced with fluorine; and
R ^192a is a C _13-25 hydrocarbon group, wherein one or more hydrogens are optionally replaced by fluorine]
The compound in any one of Claims 1-13 represented by these. Linker B or B ′ is represented by the following formula 20:

[here,
m ²⁰ is an integer from 3 to 80;
Each n ²⁰ is independently an integer from 0 to 1;
Each R ^aa is independently any of the natural amino acid side chains, optionally substituted with a dye label;
Where, in the ternary structure CBA, the C-terminus binds to the raft affinity moiety A and the N-terminus binds to pharmacophore C; and
Here, in the ternary structure C′-B′-A ′, the N-terminus binds to the raft affinity moiety A ′ and the C-terminus binds to the pharmacophore C ′]
The compound in any one of Claims 1-18 represented by these.
Linker B or B ′ is represented by the following formula 21:

[here,
Each n ²¹ is independently an integer from 1 to 2;
Each o ²¹ is independently an integer from 1 to 3;
Each p ²¹ is independently an integer from 0 to 1;
k ²¹ and each m ²¹ are independently an integer from 0 to 5;
l ²¹ is an integer from 0 to 10, provided that the sum of k ²¹ and l ²¹ is at least 1; and
R ^aa is as defined in claim 19;
Where, in the ternary structure CBA, the C-terminus binds to the raft affinity moiety A and the N-terminus binds to pharmacophore C; and
Here, in the ternary structure C′-B′-A ′, the N-terminus binds to the raft affinity moiety A ′ and the C-terminus binds to the pharmacophore C ′]
The compound in any one of Claims 1-18 represented by these. Linker B or B ′ is represented by the following formula 22:

[here,
m ²² is an integer from 0 to 40;
n ²² is an integer from 0 to 1;
Each o ²² is independently an integer from 1 to 5;
Each X ²²¹ is independently NH or O; and
R ^aa is as defined in claim 19;
Here, in the ternary structure C-B-A, the C-terminus binds to the raft affinity moiety A and the X ²²¹ terminus binds to the pharmacophore C; and where the ternary structure C′-B In '-A', the X ²²¹ terminus binds to the raft affinity moiety A 'and the C terminus binds to the pharmacophore C']
The compound in any one of Claims 1-18 represented by these. Linker B or B ′ is represented by the following formula 23:

[here,
m ²³ is an integer from 0 to 40;
n ²³ is an integer from 0 to 1;
Each o ²³ is independently an integer from 1 to 5; and
R ^aa is as defined in claim 19;
Here, in the ternary structure CBA, the SO ₂ terminus binds to the raft affinity moiety A and the N terminus binds to pharmacophore C; and
Here, in the ternary structure C′-B′-A ′, the N-terminus binds to the raft affinity moiety A ′ and the SO ₂ terminus binds to the pharmacophore C ′]
The compound in any one of Claims 1-18 represented by these.   Claims wherein pharmacophore C or C 'is selected from an enzyme, antibody or fragment or derivative thereof, aptamer, peptide, fusion protein, small molecule inhibitor, carbocyclic compound, heterocyclic compound, nucleoside derivative and anilinonaphthalene compound Item 23. The compound according to any one of Items 1 to 22.   23. A compound according to any one of claims 1 to 22, wherein pharmacophore C or C is an enzyme inhibitor.   The compound according to claim 24, wherein the enzyme inhibitor is beta-secretase inhibitor III.   23. A compound according to any one of claims 1 to 22, wherein pharmacophore C or C 'is a receptor inhibitor.   27. The compound of claim 26, wherein the receptor inhibitor is an EGF receptor inhibitor.   The EGF receptor inhibitor is aptamer A30, antibody IMC-C225 or a functional fragment thereof, antibody ABX-EGF or a functional fragment thereof, antibody EMD7200 or a functional fragment thereof, antibody hR3 or a functional fragment thereof or antibody trastuzumab or an antibody thereof 28. A compound according to claim 27, selected from functional fragments.   23. A compound according to any one of claims 1 to 22, wherein pharmacophore C or C 'is an ETBR antagonist.   30. The compound of claim 29, wherein the ETBR antagonist is A-192621.   The compound according to any one of claims 1 to 22, wherein pharmacophore C or C 'is losartan, valsartan, candesartan cilexetil (TCV-116) or irbesartan.   24. The anilinonaphthalene compound according to claim 23, wherein the anilinonaphthalene compound is selected from bis-ANS (bis 8-anilinona naphthalene sulfonate), ANS (8-anilinona naphthalene sulfonate) and AmNS (1-amino-5-naphthalene sulfonate). Compound.   The compound according to any one of claims 1 to 22, wherein pharmacophore C or C 'is an antiviral agent.   Antiviral drugs include zanamivir (2,4-dideoxy-2,3-didehydro-4-guanidinosialic acid), oseltamivir (ethyl (3R, 4R, 5S) -4-acetamido-5-amino-3- (1- Tilpropoxy) -1-cyclohexene-1-carboxylate), RWJ-270201 (peramivir), BCX-1812, BCX-1827, BCX-1898, BCX-1923, noraquine (1-tricyclo- (2,2,1, 0) -heptyl- (2) -1-phenyl-3-piperidine-propanol; triperidene), aquinone (α-5-norbornen-2-yl-α-phenyl-1-piperidinepropanol; biperidene), antiparkin (ethylbenz) 34. A compound according to claim 33 selected from hydramine) and parcopan (trihexyphenidyl).   34. The compound of claim 33, wherein the antiviral agent is selected from fuzeon, T20, T1249, cocelan, AMD3100, AMD070, SCH351125 and AD101. Equation 24b below:

The compound of Claim 25 represented by these, and its pharmaceutically acceptable salt. The following equation 25b:

The compound of Claim 25 represented by these, and its pharmaceutically acceptable salt. Raft affinity moiety A 'according to any of claims 14 to 18 and formula 28:

[here,
R ^28aa is the side chain of a natural amino acid substituted with a dye label;
R ^28bb is H or CH ₂ CONH ₂ ; and
m ²⁸ and n ²⁸ are independently an integer of 1 to 3;
Here, the N-terminus of the linker binds to the raft affinity moiety A ′]
The compound containing the linker represented by these. The linker is of formula 28a:

The compound of Claim 38 represented by these.   A pharmaceutical composition comprising the compound according to any one of claims 1 to 39.   Use of a compound according to any one of claims 1 to 25, 37 and 38 for the manufacture of a pharmaceutical composition for the treatment, prevention and / or amelioration of neurological disorders.   42. Use according to claim 41, wherein the neurological disorder is Alzheimer's disease or Down's syndrome.   Use of a compound according to any of claims 1 to 23, 27 and 28 for the manufacture of a pharmaceutical composition for the treatment, prevention and / or amelioration of a proliferative disorder or cancer.   44. Use according to claim 43, wherein the cancer is selected from breast cancer, colon cancer, stomach cancer, genitourinary cancer, liver cancer, head and neck cancer, lung cancer or skin cancer (black species).   29. Use of a compound according to any of claims 27 and 28 for the manufacture of a pharmaceutical composition for the treatment, prevention and / or amelioration of breast cancer.   Use of a compound according to any of claims 29 to 31 for the manufacture of a pharmaceutical composition for the treatment, prevention and / or amelioration of hypertension and / or congestive heart failure.   44. Use of a compound according to claim 43 for the manufacture of a pharmaceutical composition for the treatment, prevention and / or amelioration of prion (PrP) related diseases.   Use of a compound according to claims 1 to 23 and 33 to 35 for the manufacture of a pharmaceutical composition for the treatment, prevention and / or amelioration of infectious diseases.   49. Use according to claim 48, wherein the infectious disease is a viral infection.   50. Use according to claim 49, wherein the viral infection is an HIV infection.   50. Use according to claim 49, wherein the viral infection is an influenza infection.   49. Use according to claim 48, wherein the infectious disease is a bacterial infection.   53. Use according to claim 52, wherein the bacterial infection is a mycobacterial infection or an infection of Escherichia coli, Campylobacter jejuni, Vibrio cholerae, Clostridium difficile, tetanus, Salmonella or Shigella.   54. Use according to claim 53, wherein the mycobacterial infection is an infection of Mycobacterium tuberculosis or an infection of Mycobacterium kansasii or Mycobacterium bovis.   24. Use of a compound according to any one of claims 1 to 23 for the manufacture of a pharmaceutical composition for the treatment of parasitic infections.   56. Use according to claim 55, wherein the parasitic infection is a Plasmodium falciparum infection or an infection with Trypanosoma, Leishmania or Toxoplasma gondii. a) determining the distance between the phosphoryl head group or equivalent head group of the raft lipid and the binding and / or interaction site of the pharmacophore C / C ′ on the raft-associated target molecule;
b) select linker B / B ′ capable of bridging the distance determined in a);
c) linking the raft affinity moiety A / A ′ and pharmacophore C / C ′ with the linker selected in b);
The manufacturing method of the compound as described in any one of Claims 1-35 including a step.   36. A method of making a pharmaceutical composition comprising mixing a compound according to any one of claims 1-35 and one or more pharmaceutically acceptable excipients.

Images