Heparan sulfate sugar mimic compounds, their uses, and intermediates for their preparation.

Abstract

This invention relates to heparan sulfate sugar mimics, which are mimics of heparan sulfate, and to the use of these compounds as pharmaceuticals, cosmeceuticals, and molecular probes. The invention also relates to intermediate compounds useful for the preparation of a wide range of sugar mimics. The invention is particularly relevant for the treatment or prevention of diseases such as cancer, inflammation, diabetic nephropathy, and neurodegenerative disorders, for bone healing or wound healing, for cosmeceutical applications, and as molecular probes including imaging agents.

Description

[Technical Field]

[0001] This invention generally relates to heparan sulfate sugar mimics, which are mimics of heparan sulfate, and to the use of these compounds as pharmaceuticals, cosmeceuticals, and molecular probes. The invention also relates to intermediate compounds useful for the preparation of a wide range of sugar mimics. The invention is particularly relevant for the treatment or prevention of diseases such as cancer, inflammation, diabetic nephropathy, and neurodegenerative disorders, for bone healing or wound healing, for cosmeceutical applications, and as molecular probes including imaging agents. Background

[0002] Heparanase is an endo-β-D-glucuronidase that degrades the heparan sulfate glycosaminoglycan side chains of proteoglycans in the extracellular matrix and basement membrane. Heparanase appears to regulate syndecan clustering, detachment, and mitogen binding. Heparanase enzymatic activity is known to be important in promoting tumor angiogenesis, primary tumor growth, invasion, and metastasis. Heparanase cleaves heparan sulfate side chains at low-sulfation sites, thereby promoting structural changes in the extracellular matrix and basement membrane beneath epithelial and endothelial cells. Importantly, heparanase activity correlates with the metastatic potential of cancer cells. The interaction between heparanase and its substrate, heparan sulfate, is well-characterized. Heparanase has been identified as the only major heparan sulfate-degrading enzyme in human cancer, and there is growing interest in the development of heparanase inhibitors for potential therapeutic applications, including cancer, inflammation, and diabetic nephropathy. The function of heparanase and its therapeutic potential are discussed in Fux, L. et al., Trends Biochem. Sci., 2009 Oct, 34(10):511~519.

[0003] As the population ages, neurodegenerative disorders such as Alzheimer's disease, multiple sclerosis, Parkinson's disease, meningitis, schizophrenia, and traumatic brain injury become more widespread. Alzheimer's disease is a common form of dementia and is progressive and irreversible. The pathogenesis of this disease is thought to involve the deposition of aggregated amyloid-beta peptides in the brain. The first and rate-limiting step in amyloid-beta peptide production is the cleavage of the amyloid precursor protein by β-secretase (β-cytoamyloid precursor protein cleavage enzyme-1, β-secretase-1, hereinafter "BACE-1"). This makes BACE-1 a noteworthy target in new Alzheimer's disease therapies.

[0004] Heparan sulfate and its highly sulfated analog, heparin, have been shown to inhibit BACE-1 activity. Both heparan sulfate and heparin are glycosaminoglycans containing β-D-glucuronic acid or α-L-iduronic acid, a 1,4-linked disaccharide unit of N-acetyl-α-D-glucosamine (predominant in the case of heparan sulfate) or N-sulfo-α-D-glucosamine (predominant in the case of heparin), and additional O-sulfate ester substituents. Heparin is a well-known pharmaceutical with anticoagulant activity. However, when heparin is used for other pharmaceutical purposes, its anticoagulant properties must be weakened. Otherwise, potential side effects such as internal bleeding and blood coagulation disorders may become a problem.

[0005] Multiple sclerosis (MS) is the most common neurodegenerative disorder. Neuroinflammation is a major contributing factor to this disease, and the enzyme heparanase is a key regulator of neuroinflammation. Overexpression of heparanase is closely associated with uncontrolled inflammation that destroys myelin, the insulating layer covering nerve fibers, leading to MS. Onset usually occurs in young adults and is characterized by progressive impairment of motor function, vision, and coordination, eventually leading to paralysis. MS affects 2.5 million people worldwide, and there is currently no cure. There is an urgent need for new drugs that can effectively halt or slow the progression of MS. Heparan sulfate-based neuroprotective drugs can reduce inflammation and promote central nervous system (CNS) repair.

[0006] Heparan sulfate is used in several cosmeceutical and dermatological formulations. Heparan sulfate oligosaccharides are highly sulfated glycosaminoglycans that play a crucial role in a wide range of essential physiological processes. Heparan sulfate has the ability to bind to collagen (the major protein of connective tissue), control its synthesis, and further arrange water molecules, filling the gaps between them. Collagen is responsible for the strength, texture, and elasticity of tissues, but the amount of collagen in the skin decreases by approximately 1% per year over time. Various glycosaminoglycan compounds, such as hyaluronic acid and low molecular weight heparin, have already been used in several cosmeceutical formulations, providing drug-like efficacy and skin rejuvenation effects. However, the use of heparan sulfate oligosaccharides has been limited by the complexity of their synthesis. The applicant has recently developed a robust and mass-producible method for preparing novel polyvalent presentations of small, specific heparan sulfate fragments into a chemical structure known as a dendritic core. This method significantly simplifies the synthesis requirements while retaining the desirable biological activity of the heparan sulfate structure. Biologically active heparan sulfate sugar mimics have the potential to dramatically delay, and in some cases reverse, the signs and symptoms of skin aging.

[0007] In the pursuit of improved treatments and therapies for the aforementioned diseases and disorders, the applicant has studied sugar mimetic compounds of heparan sulfate. Specific compounds based on heparan sulfate have been prepared by the applicant and described in International Publication 2014 / 084744 as potentially effective in treating BACE-1-related disorders. These compounds are heparan sulfate mimetic compounds in which disaccharide and tetrasaccharide fragments of heparan sulfate are bound to a dendritic core. These fragments were prepared from glucose and glucosamine monosaccharides by a multi-step synthesis involving glycosylation reactions and selective protection / deprotection reactions using an orthogonal protecting strategy. By binding appropriately protected heparan sulfate fragments to a core and performing selective deprotection, selective sulfation leading to the target compound became possible. While this method reduced the number of reaction steps compared to other methods for heparan sulfate oligosaccharide synthesis, the synthetic process remained lengthy.

[0008] The applicant has developed a new class of heparan sulfate mimic compounds. Some of these compounds have potential as therapeutic agents, such as anticancer agents, cosmeceuticals, or treatments for neurodegenerative disorders.

[0009] Furthermore, the applicant has discovered simple and effective synthetic routes for various similar compounds, "tagged" with chemical groups that enable their use in different applications. For example, some of these compounds are useful as molecular probes and imaging agents for in vivo distribution studies.

[0010] The present invention provides novel heparan sulfate mimic compounds with potential as therapeutic agents or cosmeceutical agents, or compounds useful as molecular probes or imaging agents. [Overview of the project]

[0011] In the first aspect, the present invention relates to a compound of formula (I). [ka] (In the formula, X is (CH2) p And p is an integer from 1 to 20, R1 is either H or CH3. R2 is [ka] Either R2 is, [ka] (In the formula, • is the bond point of R2 to the O atom, Y is (CH2) n And n is an integer from 1 to 3. R3 is H or C1-C3 alkyl. Z is (CH2) m And m is an integer from 1 to 6, M is [ka] (In the formula, R4 is H or SO3H, and · is the bond point to Z.) Or provide the salt.

[0012] In certain embodiments of the present invention, p is an integer between 5 and 12. In some embodiments, p is 5. In other embodiments, p is 6. In other embodiments, p is 12.

[0013] In a particular embodiment of the present invention, n is 2.

[0014] In certain embodiments of the present invention, R1 is H. In other embodiments, R1 is methyl.

[0015] In certain embodiments of the present invention, R3 is H. In other embodiments, R3 is methyl or ethyl.

[0016] In certain embodiments of the present invention, m is 4, 5, or 6. Preferably, m is 6.

[0017] In certain embodiments of the present invention, one or more of the R4 groups are SO3H or SO3Na. In some embodiments, all of the R4 groups are SO3H or SO3Na.

[0018] In a particular embodiment of the present invention, the compound of formula (I) is [ka] Selected from the group that includes JPEG2026521543000006.jpg119149.

[0019] In a further embodiment, the present invention relates to the compound of formula (II). [ka] (In the formula, R1 is either H or CH3. R2 is [ka] is R2 [ka] (In the formula, • is the bond point of R2 to the O atom, Y is (CH2) n And n is an integer from 1 to 3. R3 is H or C1-C3 alkyl. Z is (CH2) m And m is an integer from 1 to 6, M is [ka] (In the formula, R4 is H or SO3H, and · is the bond point to Z) A is a C1-C6 alkyl group substituted with -NH2, -N3, or -NH(C=O)R5 (wherein R5 is a) C1~C 12 alkyl group, b) Biotinyl substituent, c) Groups containing fluorescent labels, d) Groups containing fluorine-18 labeling, e) Groups containing fluorine-19 labeling, f) A group comprising a crown ether-type cage ligand for rhodium, iridium, actinium-225, or thorium-227, or g) A group containing N-acetate or C-14 radiolabeled N-acetate. Or provide the salt.

[0020] In a particular embodiment of the present invention, n is 2.

[0021] In certain embodiments of the present invention, R1 is H. In other embodiments, R1 is methyl.

[0022] In certain embodiments of the present invention, R3 is H. In other embodiments, R3 is methyl or ethyl.

[0023] In certain embodiments of the present invention, m is 4, 5, or 6. Preferably, m is 6.

[0024] In certain embodiments of the present invention, A is a C1-C6 alkylamine, for example, pentylamine. In other embodiments, A is a C1-C6 alkyl azide, for example, pentyl azide.

[0025] In a particular embodiment of the present invention, A is a C1-C6 alkyl substituted with NH(C=O)R4, and R4 is a C6-C6 alkyl 12 It is an alkyl group.

[0026] In certain embodiments of the present invention, A is a C1-C6 alkyl group substituted with NH(C=O)R4, where R4 is a biotinyl substituent. For example, A may be biotinyl.

[0027] In a particular embodiment of the present invention, A is a C1-C6 alkyl group substituted with NH(C=O)R4, and R4 is a C6-C6 alkyl group containing a fluorescent label. 12It is an alkyl group. Preferably, the fluorescent label is a BODIPY group or a cyanine 7.5 NIR dye.

[0028] In a particular embodiment of the present invention, A is a C1-C6 alkyl group substituted with NH(C=O)R4 (wherein, R4 is a fluorine-18 label, for example, a C6-C 18 alkyl group containing (1-[3-(2- 12 fluoro pyridin-3-yl oxy)propyl]pyrrole-2,5-dione)).

[0029] In a particular embodiment of the present invention, A is a C1-C6 alkyl group substituted with NH(C=O)R5 (wherein, R5 is a fluorine-19 label, for example, a C6-C 12 alkyl group containing an F-19 3,5-bis(trifluoromethyl)benzyl label).

[0030] In a particular embodiment of the present invention, A is a C1-C6 alkyl group substituted with NH(C=O)R4 (wherein, R4 is a C6-C 12 alkyl group containing 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 (H2 macroPa), or H2 macroPa-NCS).

[0031] In a particular embodiment of the present invention, one or more of the R4 groups are SO3H or SO3Na. In some embodiments, all of the R4 groups are SO3H or SO3Na.

[0032] In a particular embodiment of the present invention, the compound of formula (II) is

Chemical formula

[0033] In a particular embodiment of the present invention, A is a C3-C6 alkylamine, for example, pentylamine.

[0034] In another embodiment, the present invention provides a composition comprising an effective amount of a compound of formula (I) or formula (II) and a suitable carrier, diluent, or excipient. The composition may be a pharmaceutical composition or a cosmeceutical composition.

[0035] In another aspect, the present invention provides a method for treating or preventing one or more of cancer, inflammation, diabetic nephropathy, neurodegenerative disorders, or skin diseases, or for promoting bone healing or wound healing, comprising administering a pharmaceutically effective amount of a compound of formula (I) or formula (II) to a patient in need of treatment.

[0036] In another aspect, the present invention provides a method for rejuvenating skin or preventing skin aging, comprising administering an effective amount of a compound of formula (I) or formula (II) to human skin.

[0037] In another embodiment, the present invention provides a method for using a compound of formula (II) as an imaging agent in an in vitro or in vivo imaging technique. In a particular embodiment of the present invention, the imaging technique is fluorescence imaging. In another embodiment, the imaging technique is magnetic resonance imaging. In yet another embodiment, the imaging technique is positron emission tomography (PET) imaging.

[0038] In another embodiment, the present invention provides a method for using a compound of formula (II) as a molecular probe. In a particular embodiment of the present invention, the molecular probe is used for in vivo distribution surveys.

[0039] In another aspect, the present invention provides for the use of compounds of formula (I) or formula (II) to treat or prevent one or more of cancer, inflammation, diabetic nephropathy, neurodegenerative disorders, and skin diseases, or to promote bone healing or wound healing.

[0040] In another aspect, the present invention provides the use of a compound of formula (I) or formula (II) to rejuvenate the skin or prevent skin aging.

[0041] In another aspect, the present invention provides for the use of compounds of formula (I) or formula (II) in the manufacture of pharmaceuticals for treating or preventing one or more of cancer, inflammation, diabetic nephropathy, neurodegenerative disorders, and skin diseases, or for promoting bone healing or wound healing.

[0042] In another aspect, the present invention provides for the use of a compound of formula (I) or formula (II) in the manufacture of a pharmaceutical product for treating or preventing skin aging.

[0043] In another embodiment, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or formula (II) for treating or preventing one or more of cancer, inflammation, diabetic nephropathy, neurodegenerative disorders, and skin diseases, or for promoting bone healing or wound healing.

[0044] Inflammation can include neuroinflammatory diseases such as multiple sclerosis.

[0045] Neurodegenerative disorders include, but are not limited to, senile dementia, presenile dementia, multiple infarct dementia, and Alzheimer's disease.

[0046] In another embodiment, the present invention provides a compound of formula (I) or formula (II) in combination with at least one other compound, for example, a second drug compound. The other compound may be, for example, an oligosaccharide compound, a cyclitol such as scyllo-inositol or D-chiro-inositol, an acetylcholinesterase inhibitor, a nicotinic agonist, an antibody targeting β-amyloid, a β-amyloid inhibitor, a tau agglutination inhibitor, or memantine.

[0047] In another aspect, the present invention provides for the use of a compound of formula (I) or formula (II) in combination with at least one other compound, for example, a second drug compound, for example, an oligosaccharide compound, a cyclitol such as scyllo-inositol or D-chiro-inositol, an acetylcholinesterase inhibitor, a nicotinic agonist, an antibody targeting β-amyloid, a tau aggregation inhibitor, or memantine, for the treatment or prevention of one or more of cancer, inflammation, diabetic nephropathy, neurodegenerative disorders, and skin diseases, or for promoting bone healing or wound healing.

[0048] In another embodiment, the present invention provides a method for treating or preventing one or more of cancer, inflammation, diabetic nephropathy, neurodegenerative disorders, and skin diseases, or for promoting bone healing or wound healing, comprising administering a pharmaceutically effective amount of a compound of formula (I) or formula (II) in combination with at least one other compound, for example, a second drug compound, such as an oligosaccharide compound, a cyclitol such as scyllo-inositol or D-chiro-inositol, an acetylcholinesterase inhibitor, a nicotinic agonist, an antibody targeting β-amyloid, a β-amyloid inhibitor, a tau aggregation inhibitor, or memantine. The compound of formula (I) or formula (II) and the other compounds may be administered separately, simultaneously, or sequentially.

[0049] In another aspect, the present invention provides the use of a compound of formula (II) as a molecular probe.

[0050] In another embodiment, the present invention provides the use of a compound of formula (II) as an imaging agent in in vitro or in vivo imaging techniques. In a particular embodiment of the present invention, the imaging technique is fluorescence imaging. In another embodiment, the imaging technique is magnetic resonance imaging. In yet another embodiment, the imaging technique is positron emission tomography (PET) imaging. [Brief explanation of the drawing]

[0051] [Figure 1] This graph shows the binding of sulfated maltose tetramer BODIPY amide 37 to CD4 helper T immune cells over 12 and 24 hours in cell culture. [Figure 2]This figure includes graphs a-e, which compare the binding of sulfated maltose tetramer BODIPY amide 37 to immune cells in the blood of healthy animals and animals with EAE. Immune cells include CD45+ cells (a), CD4 helper T cells (b), CD8 cytotoxic T cells (c), monocytes (d), and neutrophils (e). [Figure 3] This figure includes graphs a-e, which compare the binding of sulfated maltose tetramer BODIPY amide 37 to immune cells isolated from the brains of healthy animals and animals with EAE. The immune cells include CD45+ cells (a), CD4 helper T cells (b), CD8 cytotoxic T cells (c), monocytes (d), and neutrophils (e). [Figure 4] This figure includes confocal microscopy images of mouse cerebral cortex sections from animals that were untreated, treated with compound 36 (non-sulfated maltose tetramer BODIPY amide), or treated with compound 37 (sulfated maltose tetramer BODIPY amide). The brain slices are stained to show the nuclei (blue), endothelial cells (red), and compound 36 or 37 (green). [Figure 5A] This image shows the binding of sulfated maltose tetramer BODIPY amide 37 to bEnd.3 cells incubated with the amide 37. [Figure 5B] This graph shows a comparison of the bonding between the sulfated maltose tetramer BODIPY amide 36 and the non-sulfated maltose tetramer BODIPY amide 37. [Figure 5C] This graph shows a comparison of the bonding between the sulfated maltose tetramer BODIPY amide 36 and the non-sulfated maltose tetramer BODIPY amide 37. [Figure 6] This figure includes confocal microscopy images of brain slices from healthy mice that were untreated, incubated with compound 36 (non-sulfated maltose tetramer BODIPY amide), or incubated with compound 37 (sulfated maltose tetramer BODIPY amide). [Figure 7]This figure includes confocal microscopy images of the cerebral cortex (A, B, E, F) and cerebellum (C, D, G, H) of mouse brains from healthy animals and EAE animals. [Figure 8A] This graph shows a comparison of the binding of compound 37 (sulfated maltose tetramer BODIPY amide) to immune cells in the blood of animals that were administered 60 μg or 10 μg of compound 37 by intraperitoneal injection (ip) or oral administration (po) 45 minutes prior to the administration. Immune cells include CD45+ cells. [Figure 8B] This graph shows a comparison of the binding of compound 37 (sulfated maltose tetramer BODIPY amide) to immune cells in the blood of animals that were administered 60 μg or 10 μg of compound 37 by intraperitoneal injection (ip) or oral administration (po) 45 minutes prior to the administration. Immune cells include monocytes. [Figure 8C] This graph compares the binding of compound 37 (sulfated maltose tetramer BODIPY amide) to free compound 37(C) in the blood of animals that received 60 μg or 10 μg of compound 37 (sulfated maltose tetramer BODIPY amide) by intraperitoneal injection (ip) or oral administration (po) 45 minutes prior to the administration. The graph shows the fluorescence levels of compound 37 in plasma 45 minutes after the administration of 10 μg and 60 μg doses by ip or oral administration. [Figure 9A] This graph shows the fluorescence levels of compound 37 (sulfated maltose tetramer BODIPY amide) in plasma 12 hours after 60 μg was administered intravenously or orally. [Figure 9B] This graph shows the fluorescence levels of compound 37 (sulfated maltose tetramer BODIPY amide) in plasma 12 hours after 60 μg was administered intravenously or orally. [Figure 9C] This graph shows the binding of compound 37 to effector memory CD4 T cells in the blood and brain over 12 hours after 60 μg of compound 37 was administered via intravenous injection (ip). [Figure 9D] This graph shows the binding of compound 37 to effector memory CD4 T cells in the blood and brain over 12 hours after 60 μg of compound 37 was administered via intravenous injection (ip). [Figure 10]This figure shows fluorescence microscopy images of fluorescently labeled mimetic bodies. Red fluorescence (phaloidin) highlights the cytoskeleton, blue fluorescence (Hoechst) visualizes the cell nucleus, and green fluorescence indicates sulfated maltose tetramer BODIPY amide 37. [Figure 11] This figure includes 19F-MRI images showing the detection of 19F-labeled compounds 20 and 24 during 480 digit additions and acquisition times between 6 and 213 minutes. [Figure 12] This graph shows the signal-to-noise ratio (SNR) of 19F-labeled compounds 20 and 24 at different concentrations. [Figure 13] This figure shows a tube containing compound 24 placed next to a mouse cadaver, along with MRI images showing a 1H anatomical scan (left) and an overlaid 19F signal (right). [Figure 14] This graph shows the heparanase inhibitory activity of compound 7a. [Figure 15] This graph shows the heparanase inhibitory activity of compound 7c. Detailed explanation

[0052] definition The term "C1-C6 alkyl" refers to any saturated hydrocarbon group having up to six carbon atoms, and is intended to include both linear and branched alkyl groups. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-ethylpropyl, n-hexyl, and 1-methyl-2-ethylpropyl.

[0053] The term "prodrug" refers to a pharmaceutically acceptable derivative of a compound of formula (I) such that the derivative undergoes metabolic transformation in vivo to produce the compound defined by formula (I). Prodrugs of compounds of formula (I) may be prepared by modifying functional groups present in the compound in such a way that they are cleaved in vivo to give the parent compound. Typically, prodrugs of compounds of formula (I) are in ester-type prodrug form.

[0054] The term "cosmeceutical" refers to a combination of pharmaceuticals and cosmetics, and generally refers to cosmetic products that contain one or more biologically active ingredients that have or are believed to have pharmacological effects.

[0055] The term "pharmaceutically acceptable salt" is intended to apply to non-toxic salts such as ammonium salts, metal salts, such as sodium salts, or salts of organic cations, or mixtures thereof.

[0056] The term "protecting group" refers to a group that selectively protects an organic functional group, temporarily masking the chemical properties of that functional group, thereby enabling manipulation of other parts of the molecule without affecting that functional group. Suitable protecting groups are known to those skilled in the art and are described, for example, in Protective Groups in Organic Synthesis (3rd edition), TW Greene and PGMWuts, John Wiley & Sons Inc, (1999). Examples of protecting groups include, but are not limited to, O-benzyl, O-benzhydryl, O-trityl, O-tert-butyldimethylsilyl, O-tert-butyldiphenylsilyl, O-4-methylbenzyl, O-acetyl, O-chloroacetyl, O-methoxyacetyl, O-benzoyl, O-4-bromobenzoyl, O-4-methylbenzoyl, O-fluorenylmethoxycarbonyl, and O-levlinoyl.

[0057] The term "experimental autoimmune encephalomyelitis" or "EAE" refers to an animal model of MS in which myelin-specific immune cells are immunized and these immune cells invade the central nervous system (CNS), causing demyelination and MS-like symptoms (i.e., paralysis).

[0058] The term "patient" includes both human and non-human animals.

[0059] Terms such as "treatment" and "to treat" include the alleviation of one or more symptoms, or improvement of conditions associated with a disease or disorder, such as improvement of cognitive function or improvement of memory function.

[0060] Terms such as "prevent" and "prevention" include the prevention of one or more symptoms associated with a disease or disorder.

[0061] The compounds of the present invention are useful in both free base form and salt and / or solvate form.

[0062] Compound of the present invention Compounds of formula (I) and formula (II) are referred to herein as “compounds of the present invention.” Compounds of the present invention include all forms of compounds, such as free forms, salt forms, or solvates.

[0063] The compound of the present invention is a compound of formula (I). [ka] (In the formula, X is (CH2) p And p is an integer from 1 to 20, R1 is either H or CH3. R2 is [ka] Either R2 is, [ka] (In the formula, • is the bond point of R2 to the O atom, Y is (CH2) n And n is an integer from 1 to 3. R3 is H or C1-C3 alkyl. Z is (CH2) m And m is an integer from 1 to 6, M is [ka] (In the formula, R4 is H or SO3H, and · is the bond point to Z.) or including its salt.

[0064] The compound of the present invention is a compound of formula (II). [ka] (In the formula, R1 is either H or CH3. R2 is [ka] is R2 [ka] (In the formula, • is the bond point of R2 to the O atom, Y is (CH2) n And n is an integer from 1 to 3. R3 is H or C1-C3 alkyl. Z is (CH2) m And m is an integer from 1 to 6, M is [ka] (In the formula, R4 is H or SO3H, and · is the bond point to Z) A is a C1-C6 alkyl group substituted with -NH2, -N3, or -NH(C=O)R5 (wherein R5 is a) C1~C 12alkyl group, b) Biotinyl substituent, c) Groups containing fluorescent labels, d) Groups containing fluorine-18 labeling, e) Groups containing fluorine-19 labeling, f) A group comprising a crown ether-type cage ligand for rhodium, iridium, actinium-225, or thorium-227, or g) A group containing N-acetate or C-14 radiolabeled N-acetate. Or it may include the salt.

[0065] Synthesis of the Compound of the Present Invention The compounds of the present invention may be prepared by a variety of different methods. The following are representative and non-limiting general methods for synthesizing the compounds of the present invention.

[0066] The compound of formula (I) may be prepared according to the procedure outlined in Scheme 1 and Scheme 2.

[0067] Scheme 1 shows a method for preparing useful intermediate tetraethyl ester compounds (3a,b,c) from known diesters (1) and dicarboxylic acids (2a,b,c) via peptide coupling using the peptide coupling agent HATU. Subsequently, compounds (3a,b,c) can be deesterified to their respective tetracarboxylic acids (4a,b,c). [ka]

[0068] Scheme 2 shows the coupling of tetracarboxylic acid (4a,b,c) with maltose alkylamine (5) using the peptide coupling agent PyBOP to obtain tetramer compounds (6a,b,c). Sulfated tetramers (7a,b,c) are obtained by persulfation with a sulfur trioxide-trimethylamine complex in DMF or DMF / toluene. [ka]

[0069] The compound of formula (II) may also be prepared using the intermediate compound (14). The method for preparing this compound is shown in Scheme 3. β-glutamate diethyl ester (10) is prepared from diethyl 3-oxoglutarate (8). Coupling of diester (10) with azidic acid (9) yields diester azidoamide (11). Deesterification yields dicarboxylic acid (12). Coupling with diester (1) using HATU yields tetraethyl ester azide (13). Deesterification yields tetracarboxylic acid azide (14). [ka]

[0070] As shown in Scheme 4, maltose tetramer azide (15) is obtained by treating tetracarboxylic acid (14) with maltose alkylamine (5). Azide (15) can be reduced to maltose tetramer amine (16), as shown in Scheme 5, or sulfated to form sulfated maltose tetramer azide (17). Sulfated azide (17) can be reduced to sulfated maltose tetramer amine (18), as shown in Scheme 6. Azides (15) and (17), as well as amines (16) and (18), have been shown to be versatile intermediates for synthesizing a variety of compounds with interesting potential applications. For example, the primary amine of compound (16) provides a functional group useful for addition to alkyl chains to produce isotopically labeled moieties useful as imaging agents for glycolipids and biodistribution studies. Furthermore, the primary amine of compound (18) provides a functional group useful for adding to a phosphor or biotin moiety to produce a fluorescently labeled compound or biotinylated compound useful as an imaging agent for in vivo distribution studies or as a vehicle for drug delivery. [ka] [ka] [ka]

[0071] Maltose tetrameramine (16) can be treated with alkyl or aromatic carboxylic acids having different alkyl chain lengths or aromatic ring arrangements to produce glycolipids. For example, treatment of amine (16) with undecanoic acid yields maltose tetramer undecanoamide (25), which can then be persulfated to obtain sulfated maltose tetramer undecanoamide (26). These types of glycolipids have potential as therapeutic agents or cosmeceuticals.

[0072] Furthermore, maltose tetrameramine (16) can be modified to introduce F-19 labeling or BODIPY labeling for use in imaging techniques. For example, treatment of amine (16) with F-19 labeled 3,5-bis(trifluoromethyl)benzoic acid yields an F6 labeled compound (19), which can then be persulfated to obtain compound (20). In another example, the F-19 labeled compound (23) yields an F27 labeled compound (24) after persulfation. BODIPY labeling is achieved by reacting amine (16) with BDP-COOH and then persulfating to obtain a BODIPY labeled compound (37).

[0073] Biotinylated compounds may be useful in drug delivery and in vivo distribution studies. Amine (16) can be biotinylated by treatment with biotin-N-hydroxysuccinimide ester, and compound (34) is obtained after persulfation. In another example, biotinylated compound (35) can be prepared from sulfated maltose tetrameramine (18). Biotin is a compound named 5-[3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazole-4-yl]pentanoic acid and has the following chemical structure. [ka]

[0074] therapeutic use Compounds of formula (I) and certain compounds of formula (II) are potential inhibitors of heparanase and therefore may have potential in the treatment or prevention of various diseases, including cancer, inflammation, diabetic nephropathy, and heart disease, and may be useful in bone healing or wound healing.

[0075] Furthermore, these compounds are also expected to be inhibitors of BACE-1, and therefore may be useful in the treatment or prevention of neurodegenerative disorders, senile dementia, presenile dementia, multiple infarct dementia, or Alzheimer's disease.

[0076] The glycolipid compounds of the present invention according to formula (II) were prepared with the aim of improving their efficacy and bioavailability while maintaining ease of manufacture and safety. This new class of glycolipid compounds was prepared by "decorating" sugar fragments with lipophilic "tails" and various other parts. Glycolipid molecules have a water-soluble "polar" sugar terminus and a fat-soluble "nonpolar" tail. This contributes to the orientation and assembly of glycolipid compounds in a specific manner within biological systems, maximizing their interactions. The lipid portion of the glycolipid is embedded in the cell membrane bilayer, while the sugar portion is prominently exposed on the surface of living cells and is recognized by proteins.

[0077] Examples 36-41 described in the following section, "Use as Imaging Agent and Molecular Probe," demonstrate the potential of compounds structurally related to sulfated maltose tetramer BODIPY amide (37) as therapeutic agents for diseases associated with inflammatory processes and neurodegeneration, due to BODIPY amide (37) having sustained interactions with immune cells in the blood and brain, and the ability to migrate to the CNS within 45 minutes after administration.

[0078] Cosmeceutical applications In addition to the therapeutic applications mentioned above, the compounds of the present invention have also been shown to have potential for cosmeceutical applications.

[0079] The decrease in fibrous type I collagen is a characteristic feature of aging or "unhealthy" skin over time, and there is no doubt that the breakdown of existing collagen is a central factor in the harmful changes observed in aging skin. The inability to replace damaged collagen with newly synthesized substances is also important in the overall pathophysiology. These observations provide the skincare industry with strong justification for developing topical formulations that can promote the production of more collagen in the skin and slow or reverse the pathophysiology of skin aging.

[0080] A decrease in extracellular matrix is ​​a common phenomenon in the aging process of connective tissue. Human dermal fibroblasts from aged donors have higher levels of collagenase mRNA and protein compared to younger donors, while the expression of type I and type III collagen genes decreases in an age-dependent manner. Replicational senescence of human dermal fibroblasts appears to correlate with loss of control and overexpression of collagenase activity. As a result of the increase in dermal collagenase with aging, skincare companies are continuously trying to develop compounds that can inhibit collagenase with the aim of increasing the dermal collagen content and providing the aesthetic appearance of "youthful skin."

[0081] Skin aging is also associated with the loss of skin moisture. A key molecule involved in skin moisture is hyaluronan or hyaluronic acid (HA), a type of glycosaminoglycan (GAG) that possesses the unique ability to bind to and retain water molecules. Epidermal HA synthesis is influenced by the underlying dermis and is regulated separately from dermal HA synthesis. The progressive decrease in the size of HA polymers in the skin is a consequence of aging. Therefore, the epidermis loses the key molecule responsible for binding to and retaining water molecules, resulting in skin moisture loss. A major age-related change in the dermis is the increased affinity of HA to tissue structures, leading to a loss of HA extractability. This occurs in parallel with the progressive cross-linking of collagen and the steady decline in collagen extractability with age. All of these age-related phenomena contribute to the obvious dehydration, atrophy, and loss of elasticity that characterize aged skin.

[0082] Abnormal pigmentation such as melasma, freckles, and senile lentigines, as well as other forms of melanin pigmentation, show satisfactory subjective improvement when treated with whitening agents such as hydroquinone, ascorbic acid derivatives, kojic acid, azelaic acid, electron-rich phenols, corticosteroids, and retinoids. Among these reagents, hydroquinone agents are effective in up to 80% of users. Because they can cause irritation and dermatitis, they are often formulated with steroids. Therefore, the search for safe compounds that can reduce skin pigmentation is ongoing. The rate-limiting step in mammalian melanin synthesis is controlled by tyrosinase, which catalyzes the conversion of L-tyrosine to L-dopa and L-dopa to L-dopaquinone. Therefore, determining the effect of a compound on tyrosinase activity can provide a strong indicator of whether that compound has the ability to act as a "whitening" ingredient.

[0083] Use as a drug delivery agent The compounds of the present invention may be useful as vitamin-inducible anticancer drug delivery vehicles. They guide drugs to the appropriate sites. Vitamins are required for specific metabolic processes in all mammalian cells. The water-soluble vitamins biotin (B7) and folic acid (B9) are essential for normal cell function, growth, and development. Biotin functions as a coenzyme in metabolic reactions. Exogenous biotin is taken up via the sodium-dependent multivitamin transporter (SMVT). Various cancer cells overexpress SMVT, making biotin a tumor target vector.

[0084] Tetramer egg white avidin and its bacterial counterpart streptavidin have the highest affinity for biotin known in nature (Kd approximately 10 13 ~10 15 M -1 These have the following properties. Because the avidin-biotin complex elicits only a low immune response, they can be used for drug delivery. The biotin demand in rapidly growing tumors is higher than in normal tissues. Biotin on various constructs can enhance the intracellular uptake of therapeutic molecules through active recognition by transporters. Therefore, biotinylation of anticancer drugs can improve selective delivery to cancer cells.

[0085] Examples 31-33 describe the synthesis of the biotinylated compounds of the present invention.

[0086] Use as imaging agents and molecular probes Fluorescently labeled derivatives of heparan sulfate sugar mimetic compounds can be used to study the biodistribution of these compounds as heparanase inhibitors. This could help to better understand the mechanism by which the compounds acquire their anticancer activity. Gaining insight into how the compounds are distributed at the living cell level, and more importantly, whether they are taken up by cells, could narrow down their potential interactome and lead to the identification of lectin targets. This could enable the development of biological assays, screening of potential protein targets, and rational drug design approaches to the synthesis of therapeutically effective compounds. A better understanding of the mechanism of action could lead to improvements in the efficacy, bioavailability, and specificity of sugar mimetic compounds.

[0087] Fluorescent labeling may also be useful in identifying whether a compound is tissue-specific or cell-type-specific in vitro and in vivo. Such selectivity for specific tissues, or possibly inflammatory regions, may provide a basis for utilizing sugar-mimicking compounds as drug delivery systems.

[0088] BODIPY compounds are chemically stable and possess excellent fluorescence properties. Examples of the synthesis of the BODIPY compounds of the present invention are described in Examples 34 and 35. Examples 36 to 41 describe studies on the cell interactions and in vivo distribution of sulfated maltose tetramer BODIPY amide (37) after in vivo administration.

[0089] Example 36 shows that cell-bound BODIPY amide (37) was detected on immune cells in the blood within 45 minutes after IP injection, and that its binding was similar in healthy mice and EAE mice. Mice were either immunized for experimental autoimmune encephalomyelitis (EAE) or healthy. Twenty-four days after EAE immunization, mice received a single IP dose (60 μg in 100 μl PBS) of either sulfated maltose tetramer BODIPY amide (37) or unsulfated maltose tetramer BODIPY amide (36). After circulating the mimetic for 45 minutes, the mice were sacrificially killed, and tissues (blood and brain, Figures 2 and 3, respectively) were collected for analysis by flow cytometry. BODIPY expression is shown as BODIPY MFI (Figures 2a-2c) as a percentage of the mean fluorescence intensity (MFI) of BODIPY detected in Siam-injected control animals, or as raw MFI values ​​(Figures 2d, 2e, and 3a-e). Data were combined from two independent experiments (n=5-8 in each group) and shown as mean ± SEM. *p<0.05, **p<0.01 are determined by two-way ANOVA using the Holm-Sidak multiple comparison test. This example demonstrates not only that sulfated BODIPY amide (37) binds to immune cells in the blood and neutrophils in the brain, but also that unsulfated BODIPY amide (36) does not bind to immune cells in the blood or brain. Therefore, binding depends on highly sulfated glycan dendrites that interact with various extracellular proteins and components found on immune cells. This embodiment also demonstrates that sulfated bodily amide (37) binds to and interacts with immune cells in the blood and neutrophils in the brain within 45 minutes after IP injection, and can bind to and interact with a variety of immune cells in the blood, including helper T cells, cytotoxic T cells, monocytes, and neutrophils. This embodiment also demonstrates that sulfated bodily amide (37) can bind to immune cells in the blood and brain of healthy animals and EAE animals.

[0090] Example 37 demonstrates that sulfated BODIPY amide (37) readily and stably binds to immune cells in vitro. Binding remained stable for at least 24 hours, with more cells becoming BODIPY-positive after 24 hours compared to 12 hours. Splenocytes were isolated from healthy mice and cultured in vitro for 12 or 24 hours in the presence of sulfated BODIPY amide (37) (0, 1.2, 4, 12 μM). The cells were then evaluated by flow cytometry to determine the percentage of sulfated BODIPY amide (37)-positive immune cells. BDP + CD4 + The average values ​​and SEM results for T cells are shown in Figure 1. These results are consistent with the finding that sulfated bodily amide (37) binds to a variety of immune cells in the blood of mice administered sulfated bodily amide (37) via ip administration (Figure 2).

[0091] Example 38 demonstrates that sulfated bodily amide (37) can be detected in the blood and brain bound to immune cells, as well as in a free state in plasma (see Figures 8 and 9). Mice were injected with 60 μg (Figures 8 and 9) or 10 μg (Figure 8) of sulfated bodily amide (37) via ip injection. Furthermore, the mice were treated with 60 μg (Figures 8 and 9) or 10 μg (Figure 8) of sulfated bodily amide (37) via po treatment. Control mice were left untreated. Treatment with sulfated bodily amide (37) was performed 45 minutes (Figures 8 and 9), 4 hours (Figure 9), or 12 hours (Figure 9) before euthanasia. Plasma was collected and fluorescence units were measured using a fluorescence plate reader (Figures 8C and 9A, n=3-11 mice in each group). The binding of sulfated BODIPY amide (37) to immune cells in the blood (Figures 8A, 8B, and 9B) and brain (Figure 9B) was evaluated by flow cytometry (n=3-11 mice in each group).

[0092] Example 38 further supports Example 36 by demonstrating that sulfated bodily amide (37) is detectable on peripheral blood immune cells (CD45+ cells (Figure 8A) and monocytes (Figure 8B)). Example 38 further demonstrates that sulfated bodily amide (37) is detectable on immune cells in the blood when administered via ip or orally (Figure 8), and is also detectable 45 minutes after 60 and 10 μg ip doses (*p<0.05 by one-sided Student's t-test). Example 38 also shows that sulfated bodily amide (37) is detectable in a free state in plasma 45 minutes after 60 μg ip administration (Figure 8C).

[0093] The graph in Figure 8A shows that sulfated bodily amide (37) was detected on immune cells (BDP+CD45+ cells) in the blood and is expressed as a percentage relative to immune cells (CD45+ cells) in the peripheral blood. While a 60 μg intravenous injection (IP) dose resulted in the maximum number of immune cells to which compound 36 bound, 10 μg IP and 60 μg po administrations of sulfated bodily amide (37) resulted in similar binding levels. Therefore, it can be concluded that a 10 μg IP dose delivers a similar amount of sulfated bodily amide (37) into the bloodstream 45 minutes after administration as a 60 μg oral administration.

[0094] The graph in Figure 8B shows the detection of sulfated bodily amide (37) on monocytes (BDP+ monocytes) in the blood, expressed as a percentage of monocytes. While 60 μg of intravenous immunotherapy (IP) resulted in the highest number of monocytes bound to sulfated bodily amide (37), 10 μg IP and 60 μg po administrations resulted in similar binding levels. Therefore, it can be concluded that a 10 μg IP dose delivers the same level of sulfated bodily amide (37) into the bloodstream 45 minutes after oral administration as a 60 μg dose.

[0095] The graph in Figure 8C shows that sulfated bodily amide (37) is detectable in plasma at the highest iP dose (60 μg). Sulfated bodily amide (37) is not detected in plasma at lower iP doses (10 μg) or oral doses, but it is detectable in the blood and bound to immune cells in the brain (Figures 3, 8A, and 8B). The detection of sulfated bodily amide (37) in plasma, which does not contain cells, indicates the presence of a free, i.e., "non-cell-bound" compound 36.

[0096] Furthermore, Example 38 demonstrates that sulfated BODIPY amide (37) is detectable on immune cells (CD4 effector memory helper T cells) in the blood for at least 12 hours after 60 μg of ip administration or oral administration (Figure 9B) (***p<0.001, **p<0.01, *p<0.05 are based on two-way ANOVA) with n=4 mice in each group. Example 38 also shows that sulfated BODIPY amide (37) is detectable in a free state in plasma for up to 4 hours after 60 μg of ip administration (Figure 9A).

[0097] The graph in Figure 9A shows that sulfated bodily amide (37) is detectable in plasma for up to 4 hours after administration of 60 μg of ip, but is not detectable thereafter. This suggests that the "non-cell-bound" compound 36 either binds to intracellular or extracellular components or is removed from the plasma within 4 hours after administration.

[0098] The graph in Figure 9B shows that sulfated bodily amide (37) is detectable on immune cells (CD4 effector memory T cells) in the blood for at least 12 hours after administration of 60 μg of ip, suggesting that "non-cell-bound" sulfated bodily amide (37) is no longer detectable, while cell-bound sulfated bodily amide (37) maintains a stable interaction. This finding is supported by Example 37 (Figure 1), which shows that sulfated bodily amide (37) stably binds to CD4 T cells for at least 24 hours in in vitro cell culture. Example 38 (Figure 9B) shows an increase in the number of sulfated bodily amide (37) + effector memory CD4 T cells in the brain at later time points (4 and 12 hours), suggesting recent infiltration into the CNS. These examples (36 and 38) provide clear evidence that sulfated bodily amide (37) can bind to immune cells and cross the blood-brain barrier. This is a mechanism that allows for passive diffusion and attachment to moving cells.

[0099] In summary, Example 38 demonstrates that sulfated bodily amide (37) interacts with multiple cell types in vivo. Furthermore, this binding is specific, mediated by the sulfated region of sulfated bodily amide (37), as non-specific binding was not observed with non-sulfated sulfated bodily amide (36). Sulfated bodily amide (37) can be delivered in multiple doses via intravenous and oral administration. Free sulfated bodily amide (37) is no longer detectable within 4 hours after administration, while sulfated bodily amide (37) forms stable interactions with immune cells that persist for more than 12 hours in vivo. The use of sulfated bodily amide (37) provides insights into the mechanism in a mouse model of MS disease.

[0100] Example 39 (Figure 4) demonstrates that sulfated BODIPY amide (37) is detectable in the brain parenchyma of EAE mice 45 minutes after IP treatment. EAE mouse brains treated with BODIPY-labeled Tet-29 or unsulfated BODIPY amide (36) (60 μg / mouse, both green), or untreated, were fixed with 4% PFA and sectioned into 20 μm sagittal sections. The sections were stained with collagen IV (red, blood vessels) and DAPI (blue, cell nuclei) and imaged at 20x magnification using a confocal microscope. Figure 4 shows that unsulfated BODIPY amide (36) was not detected in the brain parenchyma, leading to the conclusion that binding depends on highly sulfated glycosylated dendrites. In these animals, sulfated BODIPY amide (37) was not associated with CNS blood vessels and preferentially bound to neuronal and immune cell bodies. Furthermore, the detection of sulfated bodily amide (37) in the brain parenchyma within 45 minutes after IP administration indicates that sulfated bodily amide (37) can be transferred to the CNS, and that this transfer is sulfate-dependent.

[0101] Example 40 (Figure 6) demonstrates that sulfated bodily amide (37) binds to cells in brain slices via its sulfated region. Brain slices from healthy mice were stained with DAPI (blue, cell nuclei), collagen IV (red, blood vessels), and sulfated or unsulfated bodily amide (green). After washing, brain sections were imaged at 20x magnification using a confocal microscope. Scale bar = 50 μm. Figure 6A is the control, showing that no green fluorescence is detected in the brain without sulfated bodily amide (37). Figure 6B shows that no green fluorescence is detected in brain slices when unsulfated bodily amide (36) is added. Therefore, unsulfated bodily amide (36) does not bind to brain tissue. Figure 6C shows that when sulfated bodily amide (37) is incubated with brain tissue, green fluorescence is detectable in brain slices. Therefore, compound 36 binds to brain tissue.

[0102] Example 40 (Figure 7) also shows that sulfated bodily amide (37) specifically binds to the cell bodies of healthy and EAE brains. Sagittal sections were collected from the brains of healthy and EAE mice and stained with DAPI (blue, cell nuclei), collagen IV (red, blood vessels), and sulfated bodily amide (37) (green). Images of the cerebral cortex (A, B, E, F) and cerebellum (C, D, G, H) were acquired at 20x magnification.

[0103] Example 41 (Figure 5) demonstrates that sulfated bodily amide (37) binds to bEnd.3 cells in a concentration-dependent manner. bEND.3 brain endothelial cells were seeded on coverslips and incubated with different concentrations of sulfated bodily amide (37) and unsulfated bodily amide (36). Binding of sulfated bodily amide (37) is dependent on highly sulfated glycan dendrites that interact with a variety of proteins and ECM components. Both MFI (b) and area (c) show concentration-dependent adhesion of sulfated bodily amide (37), while adhesion of unsulfated bodily amide (36) is constant across concentrations. Results were analyzed using two-way ANOVA with Sidak multiple comparison test, with ****p<0.0001 and *p<0.05 compared to unsulfated Tet-29. There were n=3-4 technical replicates for each group. This finding indicates that sulfated BODIPY amide (37) binds to brain endothelial cells.

[0104] Example 43 describes the use of magnetic resonance imaging (MRI) to detect compounds labeled with fluorine-19. 19 F) is a naturally occurring, non-radioactive fluorine isotope and is virtually absent in living tissues, making it an excellent contrast agent for biological probes. 19Examples of F-tagged HS sugar mimetic compounds include compounds (20) and (24), which are suitable for in vivo biodistribution studies. Because these compounds retain their pharmacokinetic activity, their usefulness as "theranostics" may be extended by preferential localization to tumor tissue, and they could function as therapeutic agents for cancer treatment and as probes for detecting metastatic diseases. As shown in Example 43, both compounds were detected with a linear signal-to-noise ratio (SNR) up to a concentration of 5 mM. As expected, at high densities... 19 The 19F-labeled compound (24) showed higher sensitivity than compound (20). See Figures 12 and 13. These data confirm that the 19F-labeled mimetic can be detected without problems using MRI and demonstrate its potential application as a molecular probe, such as a molecular probe for cancer detection.

[0105] Figure 13 shows a tube containing 10 mM of compound (24) placed next to a mouse carcass. 1 H Anatomical scan (left), and superimposed 19 This shows an F signal. A strong signal from compound (24) 19 F signaling was detected from the tissue. 19 No F signal was detected. The compound is intended to be detected internally when injected into tissue. 19 It is expected to reduce background noise in F-scans. Imaging in anesthetized, living mice has revealed a novel approach. 19 It is expected that the in vivo detection of the 1F-labeled molecular probe will be demonstrated. Studies evaluating the pharmacokinetics in normal healthy mice and tumor-carrying mice, as well as whether the compound selectively accumulates in tumor tissue, are expected to provide useful information regarding the in vivo distribution of the compound of the present invention, which is essential for future clinical development.

[0106] Fluorine 18 (F-18) 18Radiolabeled sugar mimetics with F-18 may be useful imaging agents for in vivo distribution studies. Typically, one of the disaccharide arms of the sugar mime is substituted with a hydrophobic silicon-fluorine acceptor (SiFA) group. This hydrophobic SiFA arm acts similarly to the cholestanol aglycone in PG545, and the added lipophilicity may improve the efficacy and pharmacokinetics of the inhibitor. Fluorine-18 (F-18) can be readily introduced into the SiFA group using well-established isotope exchange protocols, potentially forming a PET tracer. F-18-labeled heparanase inhibitors may hold promise in oncological diagnostic PET imaging.

[0107] Formulation and administration The compounds of the present invention may be administered to patients by various routes, including orally, parenterally, as an inhalation spray, topically, rectally, intranasally, via the buccal mucosa, or via an implanted reservoir. The compounds may also be administered intracerebral, intraventricular, or subarachnoid delivery. In the case of parenteral administration, injections may be administered intravenously, intraarterially, intramuscularly, or subcutaneously.

[0108] The amount of the compound of the present invention administered to a patient varies considerably depending on the patient's characteristics and the nature and severity of the disorder being treated. Typically, the dose for adult humans ranges from about 0.01 μg / kg to about 1 g / kg, preferably from about 0.01 mg / kg to about 100 mg / kg. The specific dose required for a particular patient depends on various factors such as the patient's age, weight, overall health, sex, and diet. The optimal dose depends on other factors such as the method of administration and the progression of the disease or disorder. Administration may be once daily, or it may be necessary to administer it twice or more daily. For example, in a treatment plan for an Alzheimer's disease patient, administration may be required once in the morning and once in the evening. Alternatively, in a treatment plan for such a patient, administration may be required every four hours.

[0109] In the case of oral administration, the compounds can be formulated into solid or liquid preparations, such as tablets, capsules, granules, powders, solutions, suspensions, syrups, elixirs, and dispersions. Such preparations are well-known in the art, as are other oral dosage forms not described herein.

[0110] In the case of parenteral administration, the compounds of the present invention can be formulated into sterile solutions, emulsions, and suspensions.

[0111] The compounds of the present invention may be compressed into the desired shape and size after being mixed with a suitable vehicle. The compounds may be tableted using conventional tablet bases such as lactose, sucrose, and corn starch together with binders, disintegrants, and lubricants. The binder may be, for example, corn starch or gelatin, the disintegrant may be potato starch or alginic acid, and the lubricant may be magnesium stearate. For oral administration in capsule form, diluents such as lactose and dried corn starch may be used. Other ingredients such as coloring agents, sweeteners, or flavorings may be added. Tablets, capsules, or powders for oral administration may contain up to about 99% of the compounds of the present invention.

[0112] If a liquid preparation is required for oral use, the compounds of the present invention may be combined with a pharmaceutically acceptable carrier such as water, an organic solvent such as ethanol, or a mixture thereof, and optionally other additives such as emulsifying agents, suspending agents, buffering agents, preservatives, and / or surfactants may be used. Colorants, sweeteners, or flavoring agents may also be added.

[0113] The compounds may also be administered by injection in a pharmaceutically acceptable diluent such as water or physiological saline. The diluent may contain one or more other ingredients such as ethanol, propylene glycol, oil, or a pharmaceutically acceptable surfactant.

[0114] The compounds of the present invention may also be administered topically. Suitable carriers for topical administration of the compounds include mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying waxes, and water. The compounds may be present as ingredients in lotions or creams for topical administration to the skin or mucous membranes. Such creams may contain the active compounds suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0115] The compounds of the present invention may further be administered by a sustained-release system. For example, the compounds may be incorporated into slow-dissolving tablets or capsules. [Examples]

[0116] The following examples further illustrate the present invention. It should be understood that the present invention is not limited to these examples.

[0117] Abbreviation NMR nuclear magnetic resonance HRMS high resolution mass spectrometry ESI Electrospray Ionization TLC (Thin-Layer Chromatography) RT room temperature DCM Dichloromethane THF (Tetrahydrofuran) DMF Dimethylformamide BODIPY 4,4-difluoro-1,3-dimethyl-4-bora-3a,4a-diaza-s-indacene PyBOP Benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate)

[0118] general Air-sensitive reactions were carried out under an argon atmosphere. Organic solutions were dehydrated with anhydrous MgSO4, and the solvent was evaporated under reduced pressure. Anhydrous solvents and chromatography solvents were commercially available and used without further purification. Thin-layer chromatography was performed at 60°F. 254 The work was performed on glass or aluminum plates coated with silica gel. Organic compounds were visualized under ultraviolet light or by immersing ammonium molybdate (5 wt%) and cerium(IV) sulfate 4H2O (0.2 wt%) in an aqueous H2SO4 solution (2M). Chromatography (using a flash column or an automated system with continuous gradient functionality) was performed on silica gel (40-63 μm). 1 The 1H NMR spectrum was measured using CDCl3, CD3OD, or D2O (HOD, δ4.79). 13 13C NMR spectra were measured using CDCl3 (centerline, δ77.0), CD3OD (centerline, δ49.0), or D2O (no internal reference, δ1.47 if specified). 1 H and 13 The assignment of 1H-C resonances was based on 2D (1H-1H DQF-COSY, 1H-13C HSQC, HMBC) and DEPT experiments. NMR abbreviations used: b, broad; s, singlet; d, doublet; t, triplet; m, multiplet. High-resolution electrospray mass spectra (ESI-HRMS) were recorded using a Q-TOF tandem mass spectrometer.

[0119] Example 1: Tetraethyl ester 3a [ka] Glutaric acid 2a (55 mg, 0.42 mmol), ethyl 3-[2-amino-3-(3-ethoxy-3-oxo-propoxy)-2-methylpropoxy]propanoate 1 (O'Donovan, L.; De Bank, PA; Org. Biomol. Chem. 2014, 12, 7290~7296) (50 mg, 1.6 mmol, 3.9 equivalents), and HATU (440 mg, 1.16 mmol, 2.8 equivalents) were placed under argon. Then, anhydrous DMF (5 mL) and DIPEA (400 μL, 2.3 mmol, 5.6 equivalents) were added, and the reaction mixture was stirred at room temperature for 4 hours. After that, the reaction mixture was diluted with ethyl acetate and washed with saturated NH4Cl, water, and saline solution. Next, the organic phase was dehydrated with MgSO4, concentrated under vacuum, and then purified via flash chromatography (0% to 100% siRNA in hexane) to obtain tetraethyl ester 3a (270 mg, 0.380 mmol, 92% yield) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ = 6.07 (s, 2H, NH), 4.13 (q, J = 7.1 Hz, 8H, C(10)H2), 3.69 (t, J = 6.3 Hz, 8H, C(7)H2), 3.54 (dd, J = 81.7, 9.1 Hz, 8H, C(6)H2), 2.53 (t, J = 6.3 Hz, 8H, C(8)H2), 2.16 (t, J = 7.2 Hz, 4H, C(2)H2), 1.86 (p, J = 7.2 Hz, 2H, C(1)H), 1.31 (s, 6H, C(5)H2), 1.25 (t, J = 7.2 Hz, 12H, C(11)H2). 13C{1H} NMR (125 MHz, CDCl3) δ = 172.8 (C3), 171.7 (C9), 73.0 (C6), 66.9 (C7), 60.6 (C10), 56.7 (C4), 36.2 (C8), 35.1 (C2), 22.0 (C1), 19.2 (C5), 14.4 (C11). HRMS (ESI-TOF) m / z C 33 H 58 N2O 14 Na [M + Na] +Calculated value: 729.3780, Measured value: 729.3780.

[0120] Example 2: Maltose tetramer 6a [ka] Tetraethyl ester 3a (255 mg, 0.361 mmol, 1.00 equivalent) was dissolved in MeOH (2 mL), and NaOH (2.7 mL, 2.0 mol / L, 15 equivalents) was added. The reaction mixture was stirred at room temperature for 16 hours. The solvent was then removed under vacuum, and the substance was co-evaporated again with water. The white solid was dissolved in H2O (5 mL), and HCl (2.2 mL, 2.5 mol / L, 15 equivalents) was added. The mixture was concentrated again under vacuum and co-evaporated three times with acetone. The white solid was resuspended in acetone and filtered through a sintered glass funnel. The filtrate was concentrated under vacuum to obtain tetracarboxylic acid 4a (214 mg, 0.361 mmol, 99% yield) as a cream-colored amorphous solid. This substance was used without further purification. Tetracarboxylic acid 4a (214 mg, 0.360 mmol, 1.00 equivalent), PyBOP (930 mg, 1.80 mmol, 5.00 equivalent), and maltose alkylamine 5 (950 mg, 2.16 mmol, 6.00 equivalent) were placed under an argon atmosphere, then anhydrous DMF (10 mL) and DIPEA (0.5 mL, 3 mmol, 8 equivalents) were added, and the reaction mixture was stirred at room temperature for 5 hours. The mixture was diluted with MeOH, concentrated under vacuum, and co-evaporated five times with toluene. The viscous crude product was dissolved in 50% H2O in ACN, purified by flash chromatography (0%~50% H2O in ACN), and lyophilized to obtain maltose tetramer 6a (405 mg, 0.177 mmol, 49% yield) as a fluffy white solid.1H NMR (500 MHz, D2O) δ = 5.45 (d, J = 3.9 Hz, 4H, C(1'')H), 4.52 (d, J = 7.9 Hz, 4H C(1')H), 4.05 - 3.55 (m, 64H), 3.48 (t, J = 9.5 Hz, 4H), 3.36 (dd, J = 9.5, 8.0 Hz, 4H, C(2')H), 3.26 (t, J = 6.9 Hz, 8H, C(10)H2), 2.55 (t, J = 5.9 Hz, 8H, C(8)H2), 2.28 (t, J = 7.3 Hz, 4H, C(2)H2), 1.87 (p, J = 7.6 Hz, 2H, C(1)H), 1.69 (p, J = 6.8 Hz, 8H, C(14)H2), 1.58 (p, J = 7.1 Hz, 8H, C(11)H2), 1.49 - 1.38 (m, 16H, C(12)H2) and びC(13)H2), 1.34 (s, 6H, C(5)H3). 13C{1H} NMR (125 MHz, D2O) δ = 175.5 (C3), 173.9 (C9), 102.1 (C1'), 99.7 (C1''), 77.0, 76.3, 74.6, 73.1 (C2'), 72.9, 72.8, 72.3 (C6), 71.7, 70.5 (C15), 69.4, 67.6 (C7), 60.8, 60.6, 57.0 (C4), 39.4 (C10), 36.3 (C8), 35.7 (C2), 28.8 (C14), 28.4 (C11), 25.9 (C12 and 13), 24.8 (C12 and 13), 22.0 (C1), 18.9 (C5). HRMS (ESI-TOF) m / z C. 97 H 175 N6O 54 [M + H] + Calculated value: 2288.1132, measured value 2288.1155.

[0121] Example 3: sulfated マルトーステトラマー7a

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[0122] Example 4: Tutor 3b

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[0123] Example 5: Maltose tetramer 6b [ka] A solution of tetraethyl ester 3b (72 mg, 0.243 mmol) in MeOH (1.5 mL) and 2 M NaOH (1.5 mL) was stirred at 60°C for 72 hours. After cooling to 0°C, the reaction mixture was diluted with water (3 mL) and acidified to approximately pH 3 with 2 M HCl. The solvent was removed under vacuum, the aqueous layer was extracted twice with ethyl acetate, dried over magnesium sulfate, and concentrated to obtain tetracarboxylic acid 4b (60 mg, 0.099 mmol, 98%), which was used in the next step without further purification. Tetramer carboxylic acid 4b (59 mg, 0.097 mmol, 1.00 equivalent), PyBOP (250 mg, 0.480 mmol, 5.00 equivalent), and maltose alkylamine 5 (340 mg, 0.780 mmol, 8.00 equivalent) were placed under an argon atmosphere. Then, anhydrous DMF (10 mL) and DIPEA (0.13 mL, 0.78 mmol, 8.0 equivalent) were added, and the reaction mixture was stirred at room temperature for 5 hours. The mixture was diluted with MeOH, concentrated under vacuum, and co-evaporated five times with toluene. The viscous crude product was dissolved in 50% H2O in ACN, purified by flash chromatography (0%~50% H2O in ACN), and after lyophilization, maltose tetramer 6b (80 mg, 0.034 mmol, 36% yield) was obtained as a fluffy white solid. 1 H NMR (500 MHz, D2O) δ 5.31 (d, J = 3.8 Hz, 4H), 4.37 (d, J = 7.9 Hz, 4H), 3.89 - 3.73 (m, 14H), 3.74 - 3.44 (m, 49H), 3.34 (t, J = 9.5 Hz, 16H), 3.21 (t, J = 9.5, 8.0 Hz, 8H), 3.11 (t, J = 6.9 Hz, 8H), 2.40 (t, J = 5.9 Hz, 8H), 2.18 - 2.11 (m, 8H), 1.60 - 1.40 (m, 24H), 1.35 - 1.22 (m, J = 5.3, 3.8 Hz, 22H), 1.20 (s, 6H). 13C NMR (126 MHz, D2O) δ 176.1, 173.9, 102.1, 99.7, 77.0, 76.3, 74.6, 73.1, 72.9, 72.8, 72.3, 71.7, 70.5, 69.4, 67.6, 60.8, 60.6, 57.0, 39.4, 36.3, 36.2, 28.8, 28.4, 26.0, 24.8, 18.9. HRMS (ESI-TOF) m / z C 98 H 177 N6O 54 [M + H] + Calculated value: 2302.1289, Measured value: 2302.1274.

[0124] Example 6: Sulfated maltose tetramer 7b [ka] Maltose tetramer 6b (80 mg, 34.7 μmol) was dissolved in anhydrous DMF (4 mL), and the reaction mixture was stirred under argon at room temperature. Sulfur trioxide trimethylamine complex (713 mg, 4.86 mmol, 140 equivalents) was added, and the reaction mixture was stirred at 60°C for 72 hours. MeOH (1 mL) was added, and the mixture was stirred for 30 minutes and concentrated under vacuum. The product was obtained as an ammonium salt by chromatography (acetonitrile:water:ammonia water, 6:2:1 → 5:2:1 → 4:2:1 → 3:2:1). This was dissolved in water and Dowex 50WX8-200 (Na + The solution was passed through a resin column. The desired product was eluted with water to obtain sulfated maltose tetramer 7b (151 mg, 34 μmol, 84%) as the sodium salt. 1H NMR (500 MHz, D2O) δ 5.59 (d, J = 3.5 Hz, 4H), 4.97 (d, J = 4.2 Hz, 4H), 4.86 - 4.79 (m, 4H), 4.77 (t, J = 4.5 Hz, 4H), 4.56 (dd, J = 7.9, 3.8 Hz, 4H), 4.53 - 4.41 (m, 8H), 4.45 - 4.31 (m, 4H), 4.36 - 4.26 (m, 12H), 4.22 - 4.16 (m, 8H), 4.15 - 4.08 (m, 4H), 3.89 -3.84 (dd, J = 10.0, 6.8 Hz, 4H), 3.74 (t, J = 6.0 Hz, 8H), 3.73 - 3.61 (m, 12H), 3.56 - 3.19 (m, 12H), 2.49 (t, J = 5.9 Hz, 8H), 2.21 (d, J = 6.4 Hz, 4H), 2.09 - 1.98 (m, 4H), 1.63 - 1.55 (m, 8H), 1.59 - 1.48 (m, 16H), 1.44 - 1.33 (m, 8H). 13 C NMR (125 MHz, D2O) δ 175.20, 172.93, 118.44, 99.00, 93.16, 76.19, 75.19, 74.07, 72.61, 72.30, 71.15, 70.91, 69.12, 66.75, 66.55, 65.20, 55.99, 38.48, 35.28, 35.21, 27.55, 27.32, 24.91, 23.79, 17.91. HRMS (ESI-TOF) m / z C 98 H 177 N6O 54 [M + H] + Calculated value: 2302.1289, measured value 2302.1274.

[0125] Example 7: Tutor 3c

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[0126] Example 8: Maltose tetramer 6c [ka] A solution of tetraethyl ester 3c (195 mg, 0.242 mmol) in MeOH (5 mL) and 2 M NaOH (1.5 mL) was stirred at 60°C for 72 hours. After cooling to 0°C, the reaction mixture was diluted with water (3 mL) and acidified to approximately pH 3 with 2 M HCl. The solvent was removed under vacuum, the aqueous layer was extracted twice with butyl, dried over magnesium sulfate, and concentrated. The crude tetraic acid product was used in the next step without further purification. This substance (82 mg, 2.08 mmol, 4 equivalents) was dissolved in anhydrous DMF (2.5 mL), and the reaction mixture was stirred under argon at room temperature. PyBOP (311 mg, 591 mmol, 5 equivalents) was added, and the reaction mixture was stirred at room temperature for 20 minutes. Maltose alkylamine 5 (313 mg, 0.710 mmol, 6 equivalents) was added, followed by DIPEA (165 μL, 0.947 mmol, 8 equivalents). The reaction mixture was stirred at room temperature for 17 hours, after which DMF was removed under vacuum. The residue was purified by flash chromatography (70% ethanol in water) to obtain maltose tetramer 6c (175 mg, 0.118 mmol, 62%) as a solidified syrup. 1 H NMR (500 MHz, D2O) δ 5.32 (d, J = 4.0 Hz, 4H), 4.39 (d, J = 7.8 Hz, 4H), 3.90 - 3.83 (m, 12H), 3.82 - 3.70 (m, 40H), 3.69 - 3.52 (m, 8H), 3.54 - 3.47 (m, 4H), 3.34 (t, J = 9.3 Hz, 4H), 3.27 - 3.18 (m, 8H), 2.48 (t, J = 6.0 Hz, 8H), 2.19 - 2.11 (m, 6H), 1.60 - 1.54 (m, 22H), 1.33 - 1.20 (m, 28H) 1.12 (s, 6H). 13C NMR (125 MHz, D2O) δ 175.4, 173.7, 102.1, 99.7, 77.0, 76.3, 74.6, 73.1, 72.9, 72.7, 71.7, 70.4, 69.4, 62.5, 60.8, 60.5, 57.4, 39.8, 28.5, 25.5, 24.7, 16.8. HRMS (ESI) C 104 H 188 N6O 54 Na [M+Na] + Calculated value in m / z: 2408.2047; Measured value: 2408.2053.

[0127] Example 9: Sulfated maltose tetramer 7c [ka] Maltose tetramer 6c (170 mg, 71.2 μmol) was dissolved in anhydrous DMF (5 mL), and the reaction mixture was stirred under argon at room temperature. Sulfur trioxide trimethylamine complex (1.46 g, 9.97 mmol, 140 equivalents) was added, and the reaction mixture was stirred at 60°C for 72 hours. MeOH (1 mL) was added, and the mixture was stirred for 30 minutes and concentrated under vacuum. The product was obtained as an ammonium salt by chromatography (acetonitrile:water:aqueous ammonia, 6:2:1 → 5:2:1 → 4:2:1 → 3:2:1). This was dissolved in water and Dowex 50WX8-200 (Na + The solution was passed through a resin column. The desired product was eluted with water to obtain sulfated maltose tetramer 7c (310 mg, 71.2 μmol, 83%) as the sodium salt. 1H NMR (500 MHz, D2O) δ 5.60 (d, J = 3.5 Hz, 4H), 4.97 (d, J = 4.2 Hz, 4H), 4.85 - 4.74 (m, 4H), 4.61 - 4.52 (m, 4H), 4.48 (dd, J = 8.8, 6.9 Hz, 4H), 4.53 - 4.41 (m, 8H), 4.42 (dd, J = 11.2, 2.8 Hz, 4H), 4.40 - 4.30 (m, 12H), 4.22 - 4.10 (m, 8H), 4.0 - 3.95 (m, 4H), 3.90 (dd, J = 10.1, 6.8 Hz, 4H), 3.75 (t, J = 5.9 Hz, 8H), 3.72 - 3.61 (m, 12H), 3.56 - 3.19 (m, 12H), 2.50 (t, J = 5.9 Hz, 8H), 2.20 (d, J = 7.3 Hz, 8H), 2.09-1.97 (m, 16H), 1.63 -1.54 (m, 24H), 1.38-1.28 (m, 8H). 13 C NMR (125 MHz, D2O) δ 175.6, 173.1, 155.97, 100.06, 99.28, 94.23, 77.87, 77.43, 76.41, 76.35, 75.21, 75.09, 73.78, 73.61, 73.46, 73.36, 72.34, 72.07, 71.78, 70.30, 70.08, 69.67, 67.89, 67.82, 66.33, 47.01, 43.69, 42.89, 39.65, 35.63, 29.43, 28.76, 28.60, 28.40, 28.30, 26.59, 26.09, 25.19, 25.00, 24.92, 24.65. HRMS (ESI) C 112 H 160 F6N8Na 28 O 140 S 28 [M-8Na] 8- The calculated value of m / z is 635.4516; the measured value: 635.4538.

[0128] Example 10: β-Glutamate Diethyl Ester 10 [ka] A solution of diethyl 3-oxoglutarate 8 (0.500 mL, 2.75 mmol) and ammonium acetate (3.00 g, 38.9 mmol) in methanol (10 mL) was stirred on a molecular sieve (3 Å, approximately 1 g) at room temperature for 18 hours. The mixture was then acidified to approximately pH 3 by adding methanolic HCl (3 M, approximately 3.5 mL). Sodium borohydride cyanohydride (237 mg, 3.58 mmol) was then added in a single addition, and the resulting mixture was stirred for a further 1 hour. The mixture was then filtered on Celite, the filter cake was washed with excess methanol, and the filtrate was concentrated under vacuum. Saturated NaHCO3 aqueous solution (40 mL) and dichloromethane (40 mL) were added to the residual oil to separate the layers. The aqueous phase was further extracted with dichloromethane (40 mL), the combined organic extract was washed with saline solution, dehydrated with MgSO4, and concentrated under vacuum to obtain β-glutamate diethyl ester as a colorless oil (542 mg, 2.67 mmol, 97%), which was used directly without further purification. Reference: J. Chem. Soc., Perkin Trans. 1, 1990, 2363-2369.

[0129] Example 11: Diethyl 3-(6-azidohexaneamide)pentanedioate 11 [ka] A solution of DMF (5 mL) containing β-glutamic acid diethyl ester 10 (400 mg, 1.97 mmol) and PyBOP (1.50 g, 2.88 mmol) was added with 6-azidohexanoic acid 9 (0.45 mL, 3.08 mmol) and DIPEA (1.40 mL, 8.18 mmol), and the resulting mixture was stirred at room temperature for 16 hours. Then, the reaction mixture was concentrated under vacuum, ethyl acetate (30 mL) and saturated aqueous NH4Cl solution (30 mL) were added, and the layers were separated. The organic phase was further washed with saturated aqueous NaHCO3 solution (30 mL) and brine (30 mL), dehydrated with MgSO4 and concentrated under vacuum to obtain the crude product. Purification by flash column chromatography eluting with ethyl acetate - light petroleum (ethyl acetate 0% → 50%) using silica gel gave diethyl ester 11 as a yellow viscous oil (605 mg, 1.77 mmol, 90%). HRMS [ESI, M + Na] + Found: 365.1801 [C 15 H 26 N4O5+ Na] + Calculated 365.1804; δ H (500 MHz, methanol-d4) 4.58 - 4.53 (1 H, m, C H ), 4.12 (4 H, q, J 7.1, 2 x C H 2), 3.29 (2 H, t, J 6.9, C H 2), 2.64 - 2.55 (4 H, m, 2 x C H 2), 2.16 (2 H, t, J 7.4, C H 2), 1.64 - 1.56 (4 H, m, 2 x C H 2), 1.41 - 1.35 (2 H, m, C H 2), 1.25 (6 H, t, J 7.1, 2 x Me ); δ C(125 MHz, methanol-d4) 175.3 (C=O), 172.4 (2 x C=O), 61.7 (2 x CH2), 52.3 (CH2), 45.0 (CH), 40.0 (2 x CH2), 36.8 (CH2), 29.6 (CH2), 27.2 (CH2), 26.4 (CH2), 14.5 (2 x Me).

[0130] Example 12: 3-(6-azidohexaneamide)pentanedicarboxylic acid 12 [ka] To a 10 mL methanol solution containing diethyl 3-(6-azidohexaneamide)pentanedicarboxylic acid 12 (493 mg, 1.72 mmol) was added, and the resulting mixture was stirred at room temperature for 2 hours. The methanol was then removed under vacuum, and the resulting solution was acidified to approximately pH 2-3 using a 2 M HCl aqueous solution, followed by dilution with water (45 mL). Ethyl acetate (45 mL) was then added, the layers were separated, and the aqueous layer was further extracted with ethyl acetate (2 × 45 mL). The combined organic extract was dehydrated with MgSO4 and concentrated under vacuum to obtain 3-(6-azidohexaneamide)pentanedicarboxylic acid 12 as a white solid (493 mg, 1.72 mmol, 91%).

[0131] Example 13: Tetraethyl ester azide 13 [ka] Subsequently, 3-(6-azidohexamide)pentanedicarboxylic acid was dissolved in DMF (8 mL), and DIPEA (0.65 mL, 3.80 mmol) and HATU (1.469 g, 3.86 mmol) were added sequentially. To the resulting bright orange solution, diethyl ester 1 (1.118 g, 3.66 mmol) was added as a solution in DMF (7.5 mL), and the mixture was stirred at room temperature for 2 hours. Then, ethyl acetate (100 mL) and saturated NaHCO3 aqueous solution (100 mL) were added to separate the layers, the organic phase was further washed with saline solution (100 mL), dehydrated with MgSO4, and concentrated under vacuum. The crude substance was purified by flash column chromatography using silica gel and elution with DCM-methanol (0 → 10% methanol) to obtain tetraethyl ester azide 13 as a golden oily substance (1.288 g, 1.50 mmol, 88%). HRMS [ESI, M + Na] + Actual value: 883.4633 [C 39 H 68 N6O 15 + Na] + Calculated value 883.4640; δ H (500 MHz, CDCl3) 7.04 (1 H, d, J 8.0, N H ), 6.44 (2 H, s, 2 x N H ), 4.41-4.29 (1 H, m, C H ), 4.13 (8 H, q, J 7.1, 4 x C H 2), 3.70 (8 H, t, J 6.3, 4 x C H 2), 3.63-3.46 (8 H, m, 4 x C H 2), 3.25 (2 H, t, J 6.9, C H 2), 2.56-2.50 (10 H, m, 4 x C H 2, 2 x 1 / 2C H 2), 2.29 (2 H, dd, J 14.0, 6.9, 2 x 1 / 2C H 2), 2.16 (2 H, t, J 7.6, C H 2), 1.67-1.57 (4 H, m, 2 x C H2), 1.43-1.36 (2 H, m, C H 2), 1.30 (6 H, s, 2 x Me ), 1.25 (12 H, t J 7.1, 4 x Me ); δ C (125 MHz, CDCl3) 172.1 (C=O), 171.7 (4 x C=O), 171.1 (2 x C=O), 72.83 (2 x CH2), 72.76 (2 x CH2), 67.0 (4 x CH2), 60.7 (4 x CH2), 57.0 (2 x C), 51.4 (CH2), 45.0 (CH), 40.0 (2 x CH2), 36.6 (CH2), 35.1 (4 x CH2), 28.7 (CH2), 26.5 (CH2), 25.2 (CH2), 19.3 (2 x Me), 14.4 (4 x Me).

[0132] Example 14: Tetracarboxylic acid azide 14 [ka] To a methanol (4 mL) solution containing tetraethyl ester azide 13 (200 mg, 0.232 mmol), 1.4 mL of 2 M NaOH aqueous solution was added, and the resulting mixture was stirred at room temperature for 2 hours. The methanol was then removed under vacuum, and the resulting solution was acidified to approximately pH 2-3 using 2 M HCl aqueous solution, followed by dilution with water (30 mL). Next, ethyl acetate (30 mL) was added, the layers were separated, and the aqueous layer was further extracted with ethyl acetate (2 × 30 mL). The combined organic extract was dehydrated with MgSO4, concentrated under vacuum, and tetracarboxylic acid azide 14 was obtained as a colorless thin film (169 mg, 0.226 mmol, 97%). 1H NMR (500 MHz, MeOD) δ = 4.45 (p, J = 6.7 Hz, 1H, C(7)H2), 3.70 (t, J = 6.1 Hz, 8H, C(13)H2), 3.63 - 3.50 (m, 8H, C(12)H2), 3.29 (d, J = 6.9 Hz, 2H, C(1)H2), 2.54 (t, J = 6.1 Hz, 8H, C(14)H2), 2.41 (d, J = 6.8 Hz, 4H, C(8)H2), 2.20 (t, J = 7.6 Hz, 2H, C(5)H2), 1.68 - 1.56 (m, 4H, C(2)H2 and C(4)H2), 1.46 - 1.38 (m, 2H, C(3)H2), 1.30 (s, 6H, C(11)H3). 13C{1H} NMR (125 MHz, MeOD) δ = 175.8 (C15), 175.1 (C6), 172.7 (C9), 73.65 (C12), 72.59 (C12), 68.2 (C13), 58.3 (C10), 52.3 (C1), 46.4 (C7), 42.0 (C8), 37.1 (C5), 35.9 (C14), 29.6 (C2), 27.4 (C3), 26.4 (C4), 19.5 (C11). HRMS (ESI) C 31 H 53 N6O 15 Na [M+Na] + Calculated value in m / z: 771.3388; Measured value: 771.3382.

[0133] Example 15: Maltose tetramer azide 15 [ka] Tetracarboxylic acid azide 14 was dissolved in DMF (4 mL), and DIPEA (0.31 mL, 1.81 mmol) and HATU (447 mg, 1.14 mmol) were added sequentially. The resulting mixture was stirred at room temperature for 15 minutes, during which time it gradually turned dark brown. Next, this mixture was added all at once to a solution of maltose alkylamine 5 (OV Zubkova et al., ACS Chem. Biol., 2018, 13(12), 3236~3242) (565 mg, 1.28 mmol) in DMF (4 mL), and stirring was continued for 4 hours. After adding methanol (approximately 2 mL), the mixture was concentrated under vacuum to obtain the crude substance as an orange oily substance. This was purified by flash column chromatography using silica gel and acetonitrile-water (0 → 45% water) elution, and maltose tetramer azide 15 was obtained as a glassy white solid (364 mg, 0.149 mmol, 66%). HRMS [ESI, M + 2H] 2+ Actual value: 1221.6053 [C 103 H 184 N 10 O 55 + 2H] 2+ Calculated value 1221.6033; δ H (500 MHz, D2O) 5.41 (4 H, d, J 3.9, 4 x C H ), 4.51-4.48 (1 H, m, C H ), 4.47 (4 H, d, J 8.0, 4 x C H ), 3.96-3.54 (64 H, m, 24 x C H + 20 x C H 2), 3.44 (4 H, t, J 9.5, 4 x C H ), 3.36 (2 H, t, J 6.8, C H 2), 3.31 (4 H, dd, J 9.4, 8.1, 4 x C H), 3.21 (8 H, t, J 6.9, 4 x C H 2), 2.50 (8 H, t, J 5.7, 4 x C H 2), 2.47-2.40 (4 H, m, 2 x C H 2), 2.24 (2 H, d, J 7.5, C H 2), 1.68-1.38 (38 H, m, 19 x C H 2), 1.28 (6 H, s, 2 x Me ); 13C{1H} NMR δ C (125 MHz, D2O) 175.5 (C=O), 173.8 (4 x C=O), 172.0 (2 x C=O), 102.1 (4 x CH), 99.6 (4 x CH), 76.9 (4 x CH), 76.3 (4 x CH), 74.6 (4 x CH), 73.1 (4 x CH), 72.9 (4 x CH), 72.7 (4 x CH), 72.15 (2 x CH2), 72.06 (2 x CH2), 71.7 (4 x CH), 70.5 (4 x CH2), 69.3 (4 x CH), 67.5 (4 x CH2), 60.8 (4 x CH2), 60.5 (4 x CH2), 57.1 (2 x C), 51.0 (CH2), 45.0 (CH), 41.3 (2 x CH2), 39.4 (4 x CH2), 36.3 (4 x CH2), 35.8 (CH2), 28.7 (4 x CH2), 28.4 (4 x CH2), 27.8 (CH2), 25.9 (4 x CH2), 25.7 (CH2), 25.1 (CH2), 24.8 (4 x CH2), 18.7 (2 x Me).

[0134] Example 16: Marigold 16

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[0135] Example 17: Sulfated maltose tetramer azide 17 [ka] Maltose tetramer azide 15 (211 mg, 86.3 μmol) was dissolved in anhydrous DMF (4 mL), and the reaction mixture was stirred under argon at room temperature. Sulfur trioxide trimethylamine complex (1.7 g, 12 mmol, 140 equivalents) was added, and the reaction mixture was stirred at 60°C for 72 hours. MeOH (1 mL) was added, and the mixture was stirred for 30 minutes and concentrated under vacuum. The product was obtained as an ammonium salt by chromatography (acetonitrile:water:ammonia water, 6:2:1 → 5:2:1 → 4:2:1 → 3:2:1). This was dissolved in water and Dowex 50WX8-200 (Na + The solution was passed through a resin column. The desired product was eluted with water to obtain sulfated maltose tetramer azide 17 (365 mg, 86 μmol, 90%) as the sodium salt.1 H NMR (500 MHz, D2O) δ 5.58 (d, J = 3.5 Hz, 4H), 4.96 (d, J = 4.3 Hz, 4H), 4.82 (q, J = 7.5 Hz, 4H), 4.76 (t, J = 4.6 Hz, 4H), 4.95 - 4.8 (m, 4 H), 4.55 (dd, J = 13.1, 3.9 Hz, 4H), 4.52 - 4.39 (m, 10H), 4.31 - 4.09 (m, 28H), 3.89 (dd, J = 10.1, 6.8 Hz, 4H), 3.81-3.73 (m, 8H), 3.78 - 3.59 (m, 12H), 3.50 (d, J = 9.7 Hz, 4H), 3.33 (t, J = 6.8 Hz, 8H), 3.18 (t, J = 7.0 Hz, 2H), 2.48 (t, J = 5.9 Hz, 8H), 2.52 - 2.36 (m, 4H), 2.28 - 2.22 (t, J = 7.4 Hz, 4H), 2.07 (s, 4H), 1.56 - 1.36 (m, 50H), 1.25 (s, 6H). 13 C NMR (126 MHz, D2O) δ 177.1, 172.9, 171.2, 99.1, 93.2, 76.3, 75.3, 74.1, 72.6, 72.3, 71.2, 71.0, 69.2, 69.2, 66.9, 66.6, 65.3, 56.2, 50.0, 40.6, 38.6, 35.3, 34.9, 27.6, 27.4, 26.9, 25.0, 24.8, 24.1, 23.9, 17.8, 0.0. HRMS (ESI) C 112 H 160 F6N8Na 28 O 140 S 28 [M-8Na] 8- The calculated value of m / z is 635.4516; the measured value: 635.4538.

[0136] Example 18: sulfated マルトーステトラマーアミン 18

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[0137] Example 19: Maltose tetramarvis (trifluoromethyl)benzamide 19 [ka] To a solution of commercially available 3,5-bis(trifluoromethyl)benzoic acid (10.2 mg, 0.040 mmol) in DMF (0.5 mL), HATU (13.4 mg, 0.035 mmol) and DIPEA (9 μL, 0.051 mmol) were added. The resulting mixture was stirred at room temperature for 10 minutes, and then maltose tetramaramine 16 (47 mg, 0.019 mmol) was added dropwise as a solution in DMF (1 mL). After stirring at room temperature for 4 hours, the DMF was removed under vacuum, and the resulting crude substance was purified by flash column chromatography using silica gel and acetonitrile-water (0 → 50% water) elution to obtain maltose tetramarbis(trifluoromethyl)benzamide 19 as a fluffy white solid (12.0 mg, 0.005 mmol, 23%). HRMS [ESI, M + 2H] 2+ Actual value: 1328.6074 [C 112 H 188 F6N8O 56 + 2H] 2+ Calculated value 1328.6085; δ H (600 MHz, D2O) 8.29 (2 H, s, 2 x Ar H ), 8.27 (1 H, s, Ar H ), 5.35 (4 H, d, J 3.9, 4 x C H ), 4.44-4.40 (1 H, m, C H ), 4.39 (4 H, d, J 8.0, 4 x C H ), 3.89-3.44 (64 H, m, 24 x C H + 20 x C H 2), 3.39 (2 H, t, J 7.0, C H 2), 3.37 (4 H, t, J 9.4, 4 x C H ), 3.24 (4 H, dd, J 9.4, 8.1, 4 x C H ), 3.21 (8 H, td, J 6.9, 2.2, 4 x C H 2), 2.42 (8 H, t, J 5.8, 4 x C H 2), 2.39-2.32 (4 H, m, 2 x CH 2), 2.19 (2 H, t, J 7.3, C H 2), 1.64-1.29 (38 H, m, 19 x C H 2), 1.19 (6 H, s, 2 x Me ); δ C (150 MHz, D2O) 175.6 (C=O), 173.8 (4 x C=O), 172.0 (2 x C=O), 167.2 (C=O), 135.8 (C), 131.5 (q, J 33.5, 2 x C), 127.8 (2 x CH), 125.8 (CH), 123.1 (q, J 272.3, 2 x C) 102.1 (4 x CH), 99.6 (4 x CH), 76.8 (4 x CH), 76.3 (4 x CH), 74.5 (4 x CH), 73.0 (4 x CH), 72.8 (4 x CH), 72.7 (4 x CH), 72.1 (2 x CH2), 72.0 (2 x CH2), 71.7 (4 x CH), 70.5 (4 x CH2), 69.3 (4 x CH), 67.5 (4 x CH2), 60.7 (4 x CH2), 60.5 (4 x CH2), 57.0 (2 x C), 45.0 (CH), 41.3 (2 x CH2), 40.0 (CH2), 39.4 (4 x CH2), 36.2 (4 x CH2), 35.8 (CH2), 28.7 (4 x CH2), 28.4 (4 x CH2), 28.2 (CH2), 25.9 (4 x CH2), 25.8 (CH2), 25.2 (CH2), 24.8 (4 x CH2), 18.6 (2 x Me); δ F (564 MHz, D2O) -62.6 (2 x CF3).

[0138] Example 20: Sulfated Manitoba 20

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[0139] Example 21: Tris(perfluoro-t-butoxy)carboxylic acid 22 [ka] To acetone (4.8 mL) containing tris(perfluoro-t-butoxy) alcohol 21 (Jiang, ZX. and Yu, YB, Tetrahedron 2007, 63, 3982~3988) (120 mg, 0.152 mmol) at 0°C, Jones' reagent (2.5 M, 0.15 mL) was added, and the resulting mixture was slowly warmed to room temperature over 18 hours. Then, ethyl acetate (10 mL) and water (10 mL) were added, and the phases were separated. The aqueous phase was further extracted with ethyl acetate, and the combined organic extract was dehydrated with MgSO4 and concentrated under vacuum to obtain carboxylic acid 22 as a white solid (115 mg, 0.143 mmol, 94%). HRMS [ESI, M + CF3O2Na - H] - Actual value: 938.9540 [C 17 H7F 27 O5+ CF3O2Na - H] - Calculated value 938.9532; δ H (500 MHz, acetone-d6) 4.47 (2 H, s, C H 2); δ C (125 MHz, acetone-d6) 169.8 (C=O), 121.2 (q, J 292.3, 9 x C), 81.2-80.2 (m, 3 x C), 66.6 (3 x CH2), 54.1 (C); δ F (470 MHz, D2O) -71.1 (9 x CF3).

[0140] Example 22: Maltose tetramethyls(perfluoro-t-butoxy)amide 23 [ka] To a solution of DMF:THF (1:1, 1 mL) containing tris(perfluoro-t-butoxy)carboxylic acid 22 (17.9 mg, 0.022 mmol), HATU (23.8 mg, 0.024 mmol) and DIPEA (7 μL, 0.037 mmol) were added, and the resulting mixture was stirred at room temperature for 10 minutes. This mixture was then added dropwise to a solution of DMF:THF (2:1, 1.5 mL) containing maltose tetrameramine 16 (36.0 mg, 0.015 mmol). After stirring at 40°C for 4 hours, the mixture was concentrated under vacuum to obtain the crude substance. This was purified by flash column chromatography using silica gel and acetonitrile-water (0 → 50% water) elution, and amide 23 was obtained as a fluffy white solid (30.0 mg, 0.009 mmol, 63%). HRMS [ESI, M + 2Na] 2+ Actual value: 1624.0745 [C 120 H 191 F 27 N8O 59 + 2Na] 2+ Calculated value 1624.0795; δ H (500 MHz, methanol-d4) 5.17 (4 H, d, J 3.8, 4 x C) H ), 4.44 (1 H, quintet, J 6.8, C H ), 4.34 (6 H, s, 3 x C H 2), 4.28 (4 H, d, J 7.8, 4 x C H ), 3.92-3.50 (56 H, m, 16 x C H , 20 x C H 2), 3.45 (4 H, dd, J 9.7, 3.8, 4 x C H ), 3.37 (4 H, ddd, J 9.5, 4.5, 2.0, 4 x C H ), 3.30-3.15 (18 H, m, 8 x C H , 5 x C H 2), 2.45-2.40 (12 H, m, 6 x C H 2), 2.18 (2 H, t, J 7.6, C H2), 1.67-1.29 (38 H, m, 19 x C H 2), 1.29 (6 H, s, 2 x Me ); δ C (125 MHz, メタノール-d4) , 175.1 (C=O) , 173.8 (4 x C=O) , 172.6 (2 x C=O) , 169.1 (C=O) , 121.5 (q, J 292.8, 9 x C) , 104.3 (4 x CH) , 102.9 (4 x CH) , 81.3 (4 x CH) , 80.9 (3 x C) * , 77.9 (4 x CH) , 76.6 (4 x CH) , 75.1 (4 x CH) , 74.8 (4 x CH) , 74.7 (4 x CH) , 74.2 (4 x CH) , 73.6 (4 x CH2) , 71.5 (4 x CH) , 70.8 (4 x CH2) , 68.7 (4 x CH2) , 66.7 (3 x CH2) , 62.8 (4 x CH2) , 62.2 (4 x CH2) , 58.2 (2 x C) , 54.5 (C) , 46.5 (CH) , 42.2 (2 x CH2) , 41.0 (CH2) , 40.4 (4 x CH2) , 37.6 (4 x CH2) , 37.2 (CH2) , 30.7 (4 x CH2) , 30.5 (4 x CH2) , 30.2 (CH2) ,27.8 (5 x CH2) , 26.8 (4 x CH2) , 26.7 (CH2) , 19.7 (2 x Me); δ F (470 MHz, methanol-d4) -71.4 (9 x CF3). *Chemical shift was determined by HMBC.

[0141] Example 23: Sulfated maltose tetramethyls(perfluoro-t-butoxy)amide 24 [ka] Maltose tetramethyls(perfluoro-t-butoxy)amide 23 (30.0 mg, 0.009 mmol) and sulfur trioxide trimethylamine complex (192 mg, 1.31 mmol) were mixed with DMF (1.5 mL) and toluene (1.5 mL). The resulting mixture was heated to 60°C and stirred for 50 hours. After cooling to room temperature, the reaction mixture was diluted with aqueous ammonium hydroxide solution (5% w / w, 2 mL) and concentrated under vacuum. The crude substance was then dissolved in water (approximately 10 mL) and transferred to a dialysis cassette (molecular weight cutoff 1000 Da). The cassette was placed in 1 L of ammonium bicarbonate dialysate (7 μM), and the dialysate was changed at time intervals of 1, 2, 3, 16, and 2 hours. The sample was then removed from the cassette and replaced with Amberlist® exchange resin (Na + The sulfate amide 24 was passed through a ferrochemical solution and freeze-dried to obtain a fluffy white solid (52.7 mg, 0.009 mmol, 93%). HRMS [ESI, M - 8Na] 8- Actual value: 734.4438 [C 120 H 163 F 27 N8Na 28 O 143 S 28 - 8Na] 8- Calculated value 734.4430; δ H (500 MHz, D2O) 5.63 (4 H, d, J 3.4, 4 x C H), 5.00 (4 H, d, J 4.4, 4 x C H ), 4.91-4.88 (4 H, m, 4 x C H ), 4.82-4.79 (4 H, 4 x C H ) * , 4.63 (4 H, dd, J 8.0, 3.4, 4 x C H ), 4.58-4.52 (9 H, m, 9 x C H ), 4.48-4.15 (34 H, m, 12 x C H + 11 x C H 2), 3.97-3.91 (4 H, m, 2 x C H 2), 3.77 (8 H, t, J 6.0, 4 x C H 2), 3.74-3.53 (12 H, m, 6 x C H 2), 3.27-3.22 (2 H, m, C H 2), 3.22 (8 H, t, J 6.9, 4 x C H 2), 2.53 (8 H, t, J 5.8, 4 x C H 2), 2.49-2.40 (4 H, m, 2 x C H 2), 2.23 (2 H, t, J 7.6, C H 2), 1.69-1.32 (38 H, m, 19 x C H 2), 1.28 (6 H, s, 2 x Me ); δ C (125 MHz, D2O) 175.6 (C=O), 173.7 (4 x C=O), 172.2 (2 x C=O), 169.8 (C=O), 119.9 (q, J 292.6, 9 x C), 100.0 (4 x CH), 94.1 (4 x CH), 79.0 (3 x C) **, 77.4 (4 x CH), 76.3 (4 x CH), 75.0 (4 x CH), 73.5 (4 x CH), 73.2 (4 x CH), 72.2 (4 x CH), 72.1 (2 x CH2), 71.9 (4 x CH, 2 x CH2), 70.2 (4 x CH, 4 x CH2), 67.8 (4 x CH2), 67.6 (4 x CH2), 66.2 (4 x CH2), 65.0 (3 x CH2), 57.1 (2 x C), 53.1 (C), 44.9 (CH), 41.6 (2 x CH2), 39.5 (5 x CH2), 36.2 (4 x CH2), 35.9 (CH2) , 28.6 (4 x CH2), 28.3 (4 x CH2), 28.2 (CH2), 26.0 (4 x CH2), 25.9 (CH2), 25.3 (CH2), 24.8 (4 x CH2), 18.6 (2 x Me); δ F (470 MHz, methanol-d4) -70.4 (9 x CF3). *The chemical shift was unclear due to residual solvent peaks and was determined by HSQC. **The chemical shift was determined by HMBC.**

[0142] Example 24: Maltose tetramer undecaneamide 25 [ka] To a solution of DMF (0.5 mL) containing undecanoic acid (1.9 mg, 0.011 mmol), HATU (4.2 mg, 0.011 mmol) and DIPEA (3 μL, 0.018 mmol) were added, and the resulting mixture was stirred at room temperature for 10 minutes. This mixture was then added dropwise to a solution of maltose tetrameramine 16 (17.0 mg, 0.007 mmol) in DMF (0.5 mL). After stirring at room temperature for 4 hours, the mixture was concentrated under vacuum to obtain the crude substance. This was purified by flash column chromatography using silica gel and acetonitrile-water (0 → 40% water) elution, yielding maltose tetramer undecanamide 25 as a fluffy white solid (12.4 mg, 0.005 mmol, 68%). HRMS [ESI, M + 2Na] 2+ Actual value: 1314.6675 [C 114 H 206 N8O 56 + 2Na] 2+ Calculated value 1314.6650; δ H (500 MHz, D2O) 5.42 (4 H, d, J 3.9, 4 x C H ), 4.52-4.47 (1 H, m, C H ), 4.47 (4 H, d, J 8.0, 4 x C H ), 3.96-3.54 (64 H, m, 24 x C H + 20 x C H 2), 3.45 (4 H, t, J 9.5, 4 x C H ), 3.32 (4 H, dd, J 9.4, 8.1, 4 x C H ), 3.22 (8 H, t, J 7.0, 4 x C H 2), 3.22-3.19 (2 H, m, C H 2), 2.51 (8 H, t, J 5.7, 4 x C H 2), 2.47-2.41 (4 H, m, 2 x C H 2), 2.27-2.22 (4 H, m, 2 x C H 2), 1.69-1.30 (54 H, m, 27 x C H2), 1.29 (6 H, s, 2 x Me ), 0.91 (3 H, t, J 6.9, Me ); δ C (125 MHz, D2O) 176.6 (C=O), 175.4 (C=O), 173.7 (4 x C=O), 172.0 (2 x C=O), 102.1 (4 x CH), 99.7 (4 x CH), 77.0 (4 x CH), 76.3 (4 x CH), 74.6 (4 x CH), 73.0 (4 x CH), 72.9 (4 x CH), 72.7 (4 x CH), 72.2 (2 x CH2), 72.1 (2 x CH2), 71.7 (4 x CH), 70.5 (4 x CH2), 69.3 (4 x CH), 67.5 (4 x CH2), 60.8 (4 x CH2), 60.5 (4 x CH2), 57.1 (2 x C), 45.0 (CH), 41.3 (2 x CH2), 39.4 (4 x CH2), 39.0 (CH2), 36.3 (4 x CH2), 35.9 (2 x CH2), 31.4 (CH2), 28.9 (2 x CH2), 28.8 (4 x CH2), 28.7 (CH2), 28.5 (CH2), 28.4 (4 x CH2), 28.3 (CH2), 28.2 (CH2), 26.0 (4 x CH2), 25.8 (CH2), 25.5 (CH2), 25.2 (CH2), 24.9 (4 x CH2), 22.2 (CH2), 18.7 (2 x Me), 13.7 (Me).

[0143] Example 25: sulfated マルトーステトラマーウンデカンアミド 26

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[0144] Example 26: Triundecanyl ester 28 [ka] Undecanoic acid (165 mg, 0.886 mmol) was mixed with thionyl chloride (1 mL), and the resulting mixture was heated to 75°C and stirred for 2 hours. After cooling to room temperature, the thionyl chloride was removed under vacuum to obtain undecanoyl chloride, which was dissolved in dichloromethane (2 mL) and cooled to 0°C. Next, a solution of dichloromethane (2 mL) containing benzyl ether 27 (52 mg, 0.215 mmol) was added, followed by the dropwise addition of triethylamine (0.14 mL, 0.972 mmol). Finally, 4-dimethylaminopyridine (10.7 mg, 0.088 mmol) was added, and the reaction mixture was warmed to room temperature. After stirring for 16 hours, the mixture was concentrated under vacuum, followed by the addition of hexane (20 mL) and water (20 mL) to separate the resulting phases. The organic phase was further washed with saline solution (20 mL), dehydrated with MgSO4, and concentrated under vacuum to obtain triundecanyl ester benzyl ether 28 as a pale yellow oily substance (157 mg, 0.215 mmol, 93%). HRMS [ESI, M + Na] + Actual value: 753.5652 [C 45 H 78 O7+ Na] + Calculated value 753.5640; δ H (500 MHz, CDCl3) 7.34-7.27 (5 H, m, 5 x Ar H ), 4.47 (2 H, s, C H 2), 4.13 (6 H, s, 3 x C H 2), 3.43 (2 H, s, C H 2), 2.25 (6 H, t, J 7.6, 3 x C H 2), 1.59-1.54 (6 H, m, 3 x C H 2), 1.31-1.26 (42 H, m, 21 x C H 2), 0.88 (9 H, t, J 7.0, 3 x Me ); δ C(125 MHz, CDCl3) 173.5 (3 x C=O), 138.1 (C), 128.5 (2 x CH), 127.8 (CH), 127.7 (2 x CH), 73.6 (CH2), 68.4 (CH2), 62.8 (3 x CH2), 42.8 (C), 34.3 (3 x CH2), 32.1 (3 x CH2), 29.7 (3 x CH2), 29.6 (3 x CH2), 29.5 (3 x CH2), 29.4 (3 x CH2), 29.3 (3 x CH2), 25.1 (3 x CH2), 22.8 (3 x CH2), 14.3 (3 x Me).

[0145] Example 27: Triundecanyl ester alcohol 29 [ka] Triundecanyl ester benzyl ether 28 (23.0 mg, 0.031 mmol) was added to THF (3 mL) and Pd(OH)2 / C (20% Pdw / w, 2.4 mg) was added. The resulting mixture was stirred under a hydrogen atmosphere for 17 hours. The reaction mixture was then filtered through Celite (trademark), the filter cake was washed with dichloromethane (5 mL), and the filtrate was concentrated under vacuum. The crude residue was purified by flash column chromatography using silica gel and elution with hexane-ethyl acetate (0 → 40% ethyl acetate) to obtain triundecanyl alcohol 29 as a colorless oil (13.0 mg, 0.020 mmol, 65%). HRMS [ESI, M + Na] + Actual value: 663.5170 [C 38 H 72 O7+ Na] + Calculated value 663.5170; δ H (500 MHz, CDCl3) 4.11 (6 H, s, 3 x C H 2), 3.49 (2 H, d, J 6.9, C H 2), 2.52 (1 H, t, J 6.9, O H ), 2.32 (6 H, t, J 7.6, 3 x CH 2), 1.63-1.58 (6 H, m, 3 x C H 2), 1.31-1.26 (42 H, m, 21 x C H 2), 0.88 (9 H, t, J 7.0, 3 x Me ); δ C (125 MHz, CDCl3) 174.0 (3 x C=O), 62.2 (3 x CH2), 60.9 (CH2), 44.1 (C), 34.3 (3 x CH2), 32.0 (3 x CH2), 29.7 (3 x CH2), 29.6 (3 x CH2), 29.44 (3 x CH2), 29.39 (3 x CH2), 29.3 (3 x CH2), 25.1 (3 x CH2), 22.8 (3 x CH2), 14.2 (3 x Me).

[0146] Example 28: Maltose tetramer undecylamide 30 [ka] To acetone (2 mL) containing triundecanyl alcohol 29 (12.1 mg, 0.019 mmol) at 0°C, Jones' reagent (2.5 M, 50 μL) was added, and the resulting mixture was slowly warmed to room temperature over 17 hours. Then, ethyl acetate (5 mL) and water (5 mL) were added, and the phases were separated. The organic phase was further washed with water (5 mL), dehydrated with MgSO4, and concentrated under vacuum to obtain triundecanyl carboxylic acid 30 as a white solid (10.4 mg, 0.016 mmol, 84%). HRMS [ESI, M - H] - Actual value: 653.4996 [C 38 H 70 O8- H] - Calculated value 653.4998; δ H (500 MHz, CDCl3) 4.31 (6 H, s, 3 x C H 2), 2.30 (6 H, t, J 7.6, 3 x C H 2), 1.60-1.57 (6 H, m, 3 x C H2), 1.33-1.25 (42 H, m, 21 x C H 2), 0.88 (9 H, t, J 7.0, 3 x Me ); δ C (125 MHz, CDCl3) 174.4 (C=O), 173.2 (3 x C=O), 61.5 (3 x CH2), 50.2 (C), 34.2 (3 x CH2), 32.0 (3 x CH2), 29.7 (3 x CH2), 29.6 (3 x CH2), 29.5 (3 x CH2), 29.4 (3 x CH2), 29.3 (3 x CH2), 25.0 (3 x CH2), 22.8 (3 x CH2), 14.3 (3 x Me).

[0147] Example 29: Maltose tetramer undecylamide 31 [ka] To a solution of triundecylcarboxylic acid 30 (6.2 mg, 0.010 mmol) in DMF (0.5 mL), HATU (3.8 mg, 0.010 mmol) and DIPEA (3 μL, 0.018 mmol) were added, and the resulting mixture was stirred at room temperature for 10 minutes. This mixture was then added dropwise to a solution of maltose tetrameramide 16 (15.3 mg, 0.006 mmol) in DMF (0.5 mL). After stirring at room temperature for 16 hours, the mixture was concentrated under vacuum to obtain the crude substance. This was purified by flash column chromatography using silica gel and acetonitrile-water (0 → 40% water) elution, yielding maltose tetramerundecylamide 31 as a fluffy white solid (11.6 mg, 0.004 mmol, 60%). HRMS [ESI, M + CH2O2- 2H] 2- Actual value: 1548.3481 [C 141 H 254 N8O 62 + CH2O2- 2H] 2- Calculated value 1548.3450; δ H (500 MHz, methanol-d4) 5.17 (4 H, d, J 3.8, 4 x C)H ), 4.44 (1 H, quintet, J 6.9, C H ), 4.31 (6 H, s, 3 x C H 2), 4.28 (4 H, d, J 7.8, 4 x C H ), 3.92 - 3.80 (16 H, m, 8 x C H 2), 3.70 - 3.51 (40 H, m, 16 x C H , 12 x C H 2), 3.45 (4 H, dd, J 9.7, 3.7, 4 x C H ), 3.37 (4 H, ddd, J 9.6, 4.5, 1.9, 4 x C H ), 3.29 - 3.18 (18 H, m, 8 x C H , 5 x C H 2), 2.45 - 2.40 (12 H, m, 6 x C H 2), 2.33 (6 H, t, J 7.3, 3 x C H 2), 2.18 (2 H, t, J 7.5, C H 2), 1.65 - 1.30 (92 H, m, 43 x C H 2, 2 x Me ), 0.90 (9 H, t, J 6.9, 3 x Me ); δ C(125 MHz, methanol-d4) 176.3 (3 x C=O), 176.1 (C=O), 175.3 (C=O), 173.9 (4 x C=O), 172.7 (2 x C=O), 104.3 (4 x CH), 102.9 (4 x CH), 81.3 (4 x CH), 77.9 (4 x CH), 76.6 (4 x CH), 75.1 (4 x CH), 74.8 (4 x CH), 74.7 (4 x CH), 74.2 (4 x CH), 73.6 (4 x CH2), 71.5 (4 x CH), 70.8 (4 x CH2), 68.7 (4 x CH2), 63.3 (3 x CH2), 62.8 (4 x CH2), 62.2 (4 x CH2), 58.2 (2 x C), 54.8 (C), 46.5 (CH), 42.3 (2 x CH2), 40.4 (4 x CH2), 40.1 (CH2), 37.6 (4 x CH2), 37.2 (CH2), 34.8 (3 x CH2), 33.0 (3 x CH2), 30.70 (4 x CH2), 30.65 (3 x CH2), 30.57 (3 x CH2), 30.5 (4 x CH2), 30.41 (3 x CH2), 30.36 (3 x CH2), 30.2 (3 x CH2), 30.1 (CH2), 27.8 (4 x CH2), 27.6 (CH2), 26.8 (4 x CH2), 26.7 (CH2), 26.0 (3 x CH2), 23.7 (3 x CH2), 19.7 (2 x Me), 14.4 (3 x Me).

[0148] Example 30: Sulfated maltose tetramer undecyl sulfonate 32 [ka] Maltose tetramer undecylamide 31 (5.6 mg, 0.002 mmol) and sulfur trioxide trimethylamine complex (36 mg, 0.259 mmol) were mixed with DMF (0.3 mL) and toluene (0.3 mL). The resulting mixture was heated to 60°C and stirred for 46 hours. After cooling to room temperature, the reaction mixture was diluted with aqueous ammonium hydroxide solution (5% w / w, 1 mL) and concentrated under vacuum. The crude substance was then dissolved in water (approximately 5 mL) and transferred to a dialysis cassette (molecular weight cutoff 1000 Da). The cassette was placed in 1 L of ammonium bicarbonate dialysate (7 μM), and the dialysate was changed at time intervals of 1, 2, 3, 16, and 2 hours. The sample was then removed from the cassette and replaced with Amberlist® exchange resin (Na + The mixture was passed through a ferrochemical solution (HMS) and freeze-dried to obtain sulfated maltose tetramethylundecylsulfonate 32 as a fluffy white solid (9.9 mg, 0.002 mmol, 95%). HRMS [ESI, M - 6Na] 6- Actual value: 929.0663 [C 108 H 163 N8Na 31 O 149 S 31 - 6Na] 6- Calculated value 929.0676; δ H (500 MHz, D2O) 5.61 (4 H, d, J 3.4, 4 x C H ), 4.98 (4 H, d, J 4.6, 4 x C H ), 4.91-4.89 (4 H, m, 4 x C H ), 4.80 (4 H, 4 x C H ) * , 4.63 (4 H, dd, J 7.8, 3.5, 4 x C H ), 4.55-4.14 (43 H, m, 21 x C H + 11 x C H 2), 3.96-3.91 (4 H, m, 2 x C H 2), 3.77 (8 H, t, J 6.0, 4 x C H 2), 3.73-3.54 (12 H, m, 6 x C H2), 3.30 (2 H, t, J 6.8, C H 2), 3.22 (8 H, t, J 7.0, 4 x C H 2), 2.53 (8 H, it, J 5.9, 4 x C H 2), 2.48-2.40 (4 H, m, 2 x C H 2), 2.24 (2 H, t, J 7.6, C H 2), 1.68-1.53 ​​(20 H, m, 10 x C H 2), 1.43-1.38 (18 H, m, 9 x C H 2), 1.28 (6 H, s, 2 x Me ); δ C (125 MHz, D2O) 175.9 (C=O), 173.8 (4 x C=O), 172.2 (2 x C=O), 170.8 (C=O), 100.1 (4 x CH), 94.0 (4 x CH), 77.6 (4 x CH), 76.4 (4 x CH), 74.9 (4 x CH), 73.3 (4 x CH), 73.2 (4 x CH), 72.2 (4 x CH), 72.1 (2 x CH2), 71.9 (4 x CH, 2 x CH2), 70.34 (4 x CH2), 70.27 (4 x CH), 67.8 (4 x CH2), 67.6 (4 x CH2), 66.2 (4 x CH2), 65.8 (3 x CH2), 57.1 (2 x C), 50.4 (C), 44.8 (CH), 41.5 (2 x CH2), 39.6 (4 x CH2), 39.4 (CH2), 36.2 (4 x CH2), 35.9 (CH2), 28.6 (4 x CH2), 28.3 (4 x CH2), 28.0 (CH2), 26.0 (4 x CH2), 25.7 (CH2), 25.4 (CH2), 24.8 (4 x CH2), 18.7 (2 x Me). *The chemical shift was unclear due to residual solvent peaks and was determined by HSQC.

[0149] Example 31: Maltose tetramarch biotinylamide 33 [ka] A mixture of DMF (1 mL) containing maltose tetrameramine 16 (14.5 mg, 0.006 mmol) and commercially available biotin-N-hydroxysuccinimide ester (3.1 mg, 0.009 mmol) was mixed with DIPEA (3 μL, 0.0150 mmol). After stirring at room temperature for 4 hours, the mixture was concentrated under vacuum to obtain the crude substance. This crude substance was purified by flash column chromatography using silica gel and acetonitrile-water (0 → 55% water) elution to obtain maltose tetramer biotinylamide 33 as a fluffy white solid (15.2 mg, 0.006 mmol, 96%). HRMS [ESI, M + 2H] 2+ Actual value: 1322.1489 [C 113 H 200 N 10 O 57 S + 2H] 2+ Calculated value 1322.1484; δ H (500 MHz, D2O) 5.42 (4 H, d, J 3.9, 4 x C H ), 4.65 (1 H, dd, J 7.8, 4.9, C H ), 4.51-4.44 (2 H, m, 2 x C H ), 4.48 (4 H, d, J 8.0, 4 x C H ), 3.97-3.54 (64 H, m, 24 x C H + 20 x C H 2), 3.45 (4 H, t, J 9.5, 4 x C H ), 3.38-3.30 (5 H, m, 5 x C H ), 3.22 (8 H, t, J 6.9, 4 x C H 2), 3.22-3.19 (2 H, m, C H 2), 3.03 (1 H, dd, J 13.0, 4.9, 1 / 2C H2), 2.82 (1 H, d, J 13.0, 1 / 2C H 2), 2.51 (8 H, t, J 5.6, 4 x C H 2), 2.48-2.41 (4 H, m, 2 x C H 2), 2.29-2.23 (4 H, m, 2 x C H 2), 1.78-1.39 (44 H, m, 23 x C H 2), 1.29 (6 H, s, 2 x Me ); δ C (125 MHz, D2O) 176.4 (C=O), 175.5 (C=O), 173.8 (4 x C=O), 172.0 (2 x C=O), 165.3 (C=O), 102.1 (4 x CH), 99.7 (4 x CH), 76.9 (4 x CH), 76.3 (4 x CH), 74.6 (4 x CH), 73.1 (4 x CH), 72.9 (4 x CH), 72.7 (4 x CH), 72.2 (2 x CH2), 72.1 (2 x CH2), 71.7 (4 x CH), 70.5 (4 x CH2), 69.3 (4 x CH), 67.5 (4 x CH2), 62.1 (CH), 60.8 (4 x CH2), 60.5 (4 x CH2), 60.3 (CH), 57.1 (2 x C), 55.5 (CH), 45.0 (CH), 41.3 (2 x CH2), 39.8 (CH2), 39.4 (4 x CH2), 39.1 (CH2), 36.3 (4 x CH2), 35.8 (CH2), 35.6 (CH2), 28.8 (4 x CH2), 28.4 (4 x CH2), 28.2 (CH2), 28.0 (CH2), 27.8 (CH2), 25.9 (4 x CH2), 25.8 (CH2), 25.3 (CH2), 25.2 (CH2), 24.8 (4 x CH2), 18.7 (2 x Me).

[0150] Example 32: Sulfated Marinated Materials Material 34 [ka] Maltose tetramarch biotinylamide 33 (10.5 mg, 0.004 mmol) and sulfur trioxide trimethylamine complex (82 mg, 0.556 mmol) were mixed with DMF (0.5 mL) and toluene (0.5 mL). The resulting mixture was heated to 60°C and stirred for 48 hours. After cooling to room temperature, the reaction mixture was diluted with aqueous ammonium hydroxide solution (5% w / w, 1 mL) and concentrated under vacuum. The crude substance was then dissolved in water (approximately 5 mL) and transferred to a dialysis cassette (molecular weight cutoff 1000 Da). The cassette was placed in 1 L of ammonium bicarbonate dialysate (7 μM), and the dialysate was changed at time intervals of 1, 2, 3, 16, and 2 hours. The sample was then removed from the cassette and replaced with Amberlist® exchange resin (Na + Biotinylamide sulfonate 34 was passed through a ferrochemical solution (HRMS) and freeze-dried to obtain a fluffy white solid (16.9 mg, 0.003 mmol, 76%). HRMS [ESI, M - 8Na] 8- Actual value: 677.1984 [C 113 H 171 N 10 Na 29 O 144 S 30 - 8Na] 8- Calculated value 677.1985; δ H (500 MHz, D2O) 5.62 (4 H, d, J 3.4, 4 x C H ), 4.99 (4 H, d, J 4.5, 4 x C H ), 4.93-4.88 (1 H, m, C H ), 4.90 (4 H, t, J 7.2, 4 x C H ), 4.81-4.79 (4 H, 4 x C H ) * , 4.63 (4 H, dd, J 7.8, 3.2, 4 x C H ), 4.57-4.16 (38 H, m, 22 x C H , 8 x C H 2), 3.96-3.92 (4 H, m, 2 x C H2), 3.77 (8 H, t, J 6.0, 4 x C H 2), 3.73-3.54 (12 H, m, 6 x C H 2), 3.41-3.37 (1 H, m, C H ), 3.26-3.09 (12 H, m, 6 x C H 2), 2.53 (8 H, t, J 5.7, 4 x C H 2), 2.49-2.41 (4 H, m, 2 x C H 2), 2.29 (2 H, t, J 7.2, C H 2), 2.25 (2 H, t, J 7.4, C H 2), 1.83-1.35 (44 H, m, 22 x C H 2), 1.29 (6 H, s, 2 x Me ); δ C(125 MHz, D2O) 176.5 (C=O), 175.6 (C=O), 173.8 (4 x C=O), 172.2 (2 x C=O), 159.8 (C=O), 99.9 (4 x CH), 94.1 (4 x CH), 77.6 (4 x CH), 76.4 (4 x CH), 75.0 (4 x CH), 73.4 (4 x CH), 73.2 (4 x CH), 72.2 (4 x CH), 72.1 (2 x CH2), 72.0 (2 x CH2), 71.9 (4 x CH), 70.2 (4 x CH, 4 x CH2), 67.8 (4 x CH2), 67.6 (4 x CH2), 66.2 (4 x CH2), 65.2 (CH), 59.3 (CH), 57.1 (2 x C), 54.8 (CH), 44.9 (CH), 41.5 (2 x CH2), 39.6 (4 x CH2), 39.2 (CH2), 38.7 (CH2), 36.2 (4 x CH2), 35.9 (CH2), 35.6 (CH2), 28.6 (4 x CH2), 28.3 (4 x CH2), 28.2 (CH2), 27.9 (CH2), 27.8 (CH2), 26.0 (4 x CH2), 25.9 (CH2), 25.3 (CH2), 25.2 (CH2), 24.8 (4 x CH2), 18.7 (2 x Me). *The chemical shift was unclear due to residual solvent peaks and was determined by HSQC.

[0151] Example 33: Sulfated maltose tetramarch biotinylamide 35 [ka] Sulfated maltose tetrameramine 18(Na + First, replace the salt with Amberlist (trademark) replacement resin (NH4 + The amine 18 (21.9 mg, 0.003 mmol) was passed through a solution, and then repeatedly evaporated using water:methanol:triethylamine (2:1:0.1, 4 × 3 mL) to convert it to the triethylamine salt form.+ Triethylamine (4 μL, 0.029 mmol) was added to DMF (1 mL) containing salt and biotin-N-hydroxysuccinimide ester (9.9 mg, 0.029 mmol), and the resulting mixture was stirred at room temperature for 42 hours. The reaction mixture was then diluted with aqueous ammonium hydroxide solution (5% w / w, 1 mL) and concentrated under vacuum. The crude substance was then dissolved in water (approximately 5 mL) and transferred to a dialysis cassette (molecular weight cutoff 1000 Da). The cassette was placed in 1 L of ammonium bicarbonate dialysate (7 μM), and the dialysate was changed at time intervals of 1, 2, 3, 16, and 2 hours (this cycle was repeated twice). The sample was then removed from the cassette and dialysis was performed using amberlist exchange resin (Na + Biotinylamide 35 was passed through a ferrochemical solution and freeze-dried to obtain a fluffy white solid (14.5 mg, 0.003 mmol, 90%). HRMS [ESI, M - 6Na] 6- Actual value: 893.6034 [C 113 H 172 N 10 Na 28 O 141 S 29 - 6Na] 6- Calculated value 893.6042; δ H (400 MHz, D2O) 5.61 (4 H, d, J 3.4, 4 x C H ), 4.98 (4 H, d, J 4.6, 4 x C H ), 4.91-4.88 (4 H, m, 4 x C H ), 4.82-4.79 (4 H, 4 x C H ) * , 4.67 (1 H, dd, J 8.0, 4.9, C H ), 4.63 (4 H, dd, J 7.8, 3.4, 4 x C H ), 4.57-4.15 (38 H, m, 22 x C H + 8 x C H 2), 3.96-3.91 (4 H, m, 2 x C H 2), 3.77 (8 H, t, J 6.0, 4 x C H2), 3.73-3.53 (12 H, m, 6 x C H 2), 3.41-3.36 (1 H, m, C H ), 3.23-3.20 (10 H, m, 5 x C H 2), 3.05 (1 H, dd, J 13.1, 4.9, 1 / 2C H 2), 2.83 (1 H, d, J 13.1, 1 / 2C H 2), 2.53 (8 H, t, J 5.7, 4 x C H 2), 2.49-2.40 (4 H, m, 2 x C H 2), 2.34-2.23 (4 H, m, 2 x C H 2), 1.80-1.38 (44 H, m, 22 x C H 2), 1.28 (6 H, s, 2 x Me ); δ H(100 MHz, D2O) 176.5 (C=O), 175.6 (C=O), 173.8 (4 x C=O), 172.2 (2 x C=O), 165.3 (C=O), 99.9 (4 x CH), 94.1 (4 x CH), 77.6 (4 x CH), 76.4 (4 x CH), 75.0 (4 x CH), 73.4 (4 x CH), 73.2 (4 x CH), 72.2 (4 x CH), 72.1 (2 x CH2), 72.0 (2 x CH2), 71.9 (4 x CH), 70.2 (4 x CH, 4 x CH2), 67.8 (4 x CH2), 67.6 (4 x CH2), 66.2 (4 x CH2), 62.2 (CH), 60.3 (CH), 57.1 (2 x C), 55.5 (CH), 44.9 (CH), 41.5 (2 x CH2), 39.8 (CH2), 39.6 (4 x CH2), 39.1 (CH2), 36.2 (4 x CH2), 35.9 (CH2), 35.6 (CH2), 28.6 (4 x CH2), 28.3 (4 x CH2), 28.2 (CH2), 28.0 (CH2), 27.8 (CH2), 26.0 (4 x CH2), 25.8 (CH2), 25.3 (CH2), 25.2 (CH2), 24.8 (4 x CH2), 18.7 (2 x Me). *The chemical shift was unclear due to residual solvent peaks and was determined by HSQC.

[0152] Example 34: Maltose tetramer BODIPY amide 36 [ka] Maltose tetrameramine 16 (113 mg, 0.047 mmol), 4,4-difluoro-1,3-dimethyl-4-bora-5-carboxyethyl-3a,4a-diaza-s-indacene (19 mg, 0.051 mmol, 1.1 equivalents), and HATU (21 mg, 0.055 mmol, 1.2 equivalents) were dried together under vacuum and then placed under an argon atmosphere. Anhydrous DMF (2.5 mL) and DIPEA (10 μL, 0.058 mmol, 1.2 equivalents) were then added, and the reaction mixture was stirred at room temperature for 1.5 hours. The mixture was then transferred to a Falcon tube with a minimum amount of methanol, and 30 mL of siRNA was added. The mixture was centrifuged at 4000 rpm for 4 minutes, resulting in a dark orange residue at the bottom of the tube. The supernatant was discarded, and the residue was resuspended in 50% H2O in ACN. After purification by flash chromatography (0%-50% H2O in ACN), lyophilization was performed to obtain maltose tetramer BODIPY amide 36 (108 mg, 0.040 mmol, 85% yield) as a fluffy orange solid. TLC Rf = 0.2 (25% H2O in ACN).1H NMR (500 MHz, D2O) δ = 7.48 (s, 1H, CH), 7.08 (d, J = 4.3 Hz, 1H CH), 6.39 (d, J = 3.9 Hz, 1H CH), 6.33 (s, 1H, CH), 5.39 (d, J = 3.9 Hz, 4H, CH), 4.48 (p, J = 7.3 Hz, 1H, CH), 4.42 (d, J = 8.0 Hz, 4H, CH), 3.96 - 3.48 (m, 64H), 3.45 (t, J = 9.5 Hz, 4H), 3.30 (dd, J = 9.4, 8.0 Hz, 4H, CH), 3.18 (m, 12H, CH2, CH2 and CH2), 2.68 (t, J = 7.5 Hz, 2H, CH2), 2.54 (s, 3H, CH3), 2.51 - 2.37 (m, 12H, CH2 and CH2), 2.28 (s, 3H, CH3), 2.18 (t, J = 7.5 Hz, 2H, CH2), 1.68 - 1.43 (m, 20H), 1.41 - 1.29 (m, 18H), 1.27 (s, 6H, CH3). 13C{1H} NMR (125 MHz, D2O) δ = 175.3, 174.3, 173.73, 173.70, 172.0, 161.3, 156.1, 146.2, 135.3, 33.2, 128.9, 124.9, 121.2, 116.8, 102.1, 99.8, 77.1, 76.3, 74.6, 73.0, 72.9, 72.7, 72.1, 72.0, 71.7, 70.5, 69.3, 67.5, 60.7, 60.5, 57.0, 45.0, 41.3, 39.4, 39.1, 36.2, 35.8, 34.7, 28.8, 28.4, 28.1, 26.0, 25.7, 25.2, 24.8, 24.5, 18.6, 14.4, 10.7. HRMS (ESI-TOF) m / z [C。 117 H 198 BF2N 10 NaO 57 2+ [M + H + Na] 2+ ​Calculated value: 1365.1531, Measured value: 1365.1464.

[0153] Example 35: Sulfated maltose tetramer BODIPY amide 37 [ka] Maltose tetramer BODIPY amide 36 (32 mg, 0.012 mmol) and sulfur trioxide trimethylamine (232 mg, 1.67 mmol, 140 equivalents) were dried together under vacuum and then placed under an argon atmosphere. Next, anhydrous DMF (1 mL) and toluene (1.5 mL) were added, and the reaction mixture was stirred at 60°C for 16 hours. Then, the reaction mixture was cooled to room temperature, diluted with NH4OH (5% w / w), and concentrated under vacuum. The substance was evaporated four times with NH4OH (5% w / w), dissolved in H2O, and transferred to a dialysis cassette with a molecular weight cutoff of 1000 g / mol. The cassette was placed in 1 L of (NH4)2CO3 dialysate (7 μM), and the dialysate was changed at time intervals of 1, 2, 3, 10, and 2 hours. The sample was then removed from the cassette and freeze-dried to obtain sulfated maltose tetramer BODIPY amide 37 (57 mg, 0.010 mmol, yield 87%) as a fluffy orange solid. 1HNMR (600 MHz, D2O) δ = 7.54 (s, 1H, C(7)H), 7.11 (d, J = 4.0 Hz, 1H, CH), 6.40-6.32 (m, 2H, CH and CH), 5.56 (d, J = 3.5 Hz, 4H, CH), 4.88 (d, J = 4.6 Hz, 4H, CH), 4.84 (t, J = 8.1 Hz, 4H, CH), 4.73 (t, J = 5.0 Hz, 4H, CH), 4.57 (dd, J = 8.1, 3.3 Hz, 4H, CH), 4.53-4.42 (m, 9H, CH, CH and CH), 4.42-4.22 (m, 16H, CH and CH), 4.22-4.13 (m, 8H, CH), 4.13-4.04 (m, 4H, CH and CH), 3.84 (dt, J = 9.8, 6.9 Hz, 4H, 1 / 2 CH2), 3.68 (q, J = 7.8,5.6 Hz, 8H, CH2), 3.63-3.41 (m, 12H, CH2 and 1 / 2 CH2), 3.22-3.05 (m, 12H, CH2, CH2 and CH2), 2.65 (t, J = 7.3 Hz, 2H, CH2), 2.52 (s, 3H,CH3), 2.48-2.32 (m, 12H, CH2 and CH2), 2.30 (s, 3H, CH3), 2.11 (t, J= 7.4 Hz, 2H, CH2), 1.64-1.37 (m, 20H, CH2, CH2, CH2, CH2), 1.37-1.23 (m, 16H, C(32)H2, CH2), 1.20 (s, 8H, CH3 and CH3). 13C{1H} NMR (150 MHz, D2O) δ = 175.6, 174.6, 173.7, 172.2, 161.7, 155.8, 146.7, 135.5, 133.2, 129.0, 125.1, 121.4, 116.8, 99.9, 94.1, 77.5, 76.3, 74.9, 73.4, 73.2, 72.14, 72.05, 71.95, 71.87, 70.2, 70.1, 67.7, 67.5, 66.2, 57.1, 44.9, 41.5, 39.6, 39.1, 36.1, 35.8, 34.7, 28.6, 28.3, 28.0, 26.0, 25.6, 25.2, 24.8, 24.5, 18.6, 14.4, 10.8.

[0154] Example 36: Detection of cell-bound sulfated maltose tetramer BODIPY amide 37 on immune cells in the blood and brain after EAE induction. Female C57Bl / 6J mice were given 50-100 μg of MOG per mouse for EAE. 35-55(Genscript, Piscataway, NJ USA) mice were immunized in a complete Freund's adjuvant (Sigma, St. Louis, MO, USA), which contained 500 μg of heat-killed Mycobacterium tuberculosis (Difco Laboratories, Detroit, USA) and 200 ng of pertussis toxin (List Biological Laboratories, Campbell, CA USA) per mouse. Two days later, the mice received a second dose of pertussis toxin at a dose of 200 ng per mouse. Twenty-four days after EAE immunization, the mice received a single intravenous dose of sulfated BODIPY amide 37 or unsulfated BODIPY amide 36 (60 μg in 100 μl PBS). After circulating this compound for 45 minutes, the mice were sacrificially killed.

[0155] After euthanasia, blood was collected from the heart and lysed by incubation in 2 mL of RBC lysis buffer (Sigma-Aldrich, MA) to lyse red blood cells. The remaining blood cells were then washed, pelletized by centrifugation, resuspended in flow cytometry (FACs) buffer, and evaluated by flow cytometry. Subsequently, residual blood in the cerebral blood vessels was flushed with 20 mL of PBS by cardiac perfusion, and the brain was collected. The brain was divided into two sagittal sections and prepared for both flow cytometry and confocal microscopy analysis. For flow cytometry analysis, half of the brain was treated as a single-cell suspension. The sample was suspended in 37% Percoll solution (Sigma-Aldrich, MA), and CNS cells were separated from myelin by low-acceleration centrifugation. After myelin removal, the cells were resuspended in FACs buffer.

[0156] Flow cytometry data were collected using BD FACS Canto II (BD Biosciences, NJ) and analyzed with FlowJo software (Treestar, Ashland, OR USA). Immune cell types were identified as CD45+ (global immune cells), CD45+CD4+ (CD4 helper T cells), CD45+CD8+ (CD8 cytotoxic T cells), CD45+Ly6C+ (monocytes), and CD45+Ly6G+ (neutrophils). BODIPY expression was expressed as BODIPY MFI as a percentage of the mean BODIPY fluorescence intensity (MFI) detected in sham-injected control animals, or as raw MFI values. Data were combined from two independent experiments (n=2-15 in each group) and are shown as mean ± SEM. *p<0.05, **p<0.01 are based on two-way ANOVA with Holm-Sidak multiple comparison tests. All in vivo BODIPY amide 37 treatment experiments included control animals that did not receive the injection. Results are shown in Figures 2 and 3.

[0157] Example 37: In vitro analysis of the adhesion of sulfated maltose tetramer BODIPY amide 37 to immune cells Spleens were harvested from healthy C57BL / 6J mice and treated with single-cell suspension. Splenocytes were cultured at 37°C and 5% CO2 for 12 or 24 hours in the presence of BODIPY amide 37 (0, 1.2, 4, 12 ng / ml). After incubation, BODIPY amide 37 was washed off, and cells were stained with fluorescently tagged antibodies for flow cytometry analysis. CD4 helper T cells were identified as CD45+CD4+. Flow cytometry of splenocytes was performed using BD FACS Canto II (BD Biosciences, NJ), and the percentage of BODIPY amide 37-positive immune cells was determined using FlowJo software (Treestar Inc., Ashland, OR, USA). ***p<0.001 for both time and concentration was determined by two-way ANOVA. The results are shown in Figure 1.

[0158] Example 38: Detection of sulfated maltose tetramer BODIPY amide 37 in tissue after in vivo treatment Female C57Bl / 6J mice were immunized for EAE (same as in Example 36) or kept in a healthy state. 24–28 days after immunization, BODIPY amide 37 was administered to healthy or EAE mice either in 100 μL of PBS at a dose of 10 μg or 60 μg per mouse, either in ip or po, and circulated for 45 minutes, 4 hours, or 12 hours before euthanasia. After euthanasia, blood was collected from the heart and the animals were perfused. The brain was then collected after flushing out residual blood from the CNS vessels with 20 mL of PBS perfusion. Red blood cells in the blood were lysed by incubation in 2 mL of RBC lysis buffer (Sigma-Aldrich, MA). After washing the remaining blood cells, they were pelletized by centrifugation and resuspended in flow cytometry staining (FACs) buffer. Half of the brain was treated as a single-cell suspension for flow cytometry analysis. CNS cells were separated from myelin by suspending the sample in 37% Percoll solution (Sigma-Aldrich, MA) and centrifugating at low acceleration. After myelin removal, the cells were resuspended in FACs buffer. Single-cell suspensions of brain and blood were stained with fluorescently labeled antibodies for flow cytometry analysis as described in Example 36.

[0159] In some experiments, blood was collected and processed to obtain plasma for analysis of free BODIPY amide 37 levels. Here, blood was collected in a tube containing EDTA to prevent coagulation and lysed twice by incubation with RBC lysis buffer as described above. The sample was then centrifuged at 10,000 × g for 10 minutes, and the plasma was collected carefully to avoid disturbing the cell pellet. BODIPY amide 37 levels in the plasma were estimated by relative fluorescence units collected with an EnSpire plate reader (PerkinElmer). Data are shown as mean ± SEM. All in vivo BODIPY amide 37 treatment experiments included non-injection control animals. The results are shown in Figures 8 and 9.

[0160] Example 39: Visualization of sulfated maltose tetramer BODIPY amide 37 in brain parenchyma after in vivo treatment Female C57Bl / 6J mice were immunized for EAE (same as in Example 36) or kept in a healthy state. 24–28 days after immunization, sulfated bodily amide 37 or unsulfated bodily amide 36 was administered to healthy or EAE mice at a dose of 60 μg per mouse in 100 μl of PBS, circulated for 45 minutes, and then euthanized. After euthanasia, blood was collected from the heart and the animals were perfused. Subsequently, residual blood in the CNS blood vessels was flushed with 20 mL of PBS by cardiac perfusion, and the brain was collected. The brain was cut in half sagittally and prepared for both flow cytometry and confocal microscopy analysis. The cerebral hemispheres were rapidly frozen with Polyfreeze OCT compound (Sigma, USA) and stored at -80°C until sectioning.

[0161] The cerebral hemispheres were cut into 20 μm sagittal sections using a Leica CM3050S cryostat microtome (Leica Biosystems, Wetzlar, Germany) and placed on Superfrost plus slides (Thermo Fisher Scientific, MA). Immediately before staining, the slides were thawed at 37°C for 5 minutes, and the sections were fixed with 4% paraformaldehyde (PFA) at room temperature for 10 minutes. Tissue autofluorescence was quenched by immersion in fresh sodium boride solution (1 mg / ml in ddH2O) for 30 minutes. Subsequently, the tissue was blocked with 5% donkey serum (Sigma-Aldrich, MA) at room temperature for 2 hours. After blocking, the tissue was incubated overnight at 4°C in Tris-buffered saline (TBS) with rabbit anti-mouse collagen IV antibody (Abcam, Cambridge, UK). Sections were placed in 2.5 × 10⁶ TBS. -4After thorough washing with % Triton-X-100, the sections were incubated with donkey anti-rabbit AF647 antibody (Abcam, Cambridge, UK) for 2 hours at room temperature. Finally, the sections were washed twice with PBS and ddH2O, and then mounted with anti-bleeding glycerol mounting medium containing DAPI (Abcam, Cambridge, UK) and 0.17 nm coverslips. The penetration of BODIPY amide 37 into the brain parenchyma was visualized using a confocal microscope. The results are shown in Figure 4.

[0162] Example 40: Adhesion of sulfated maltose tetramer BODIPY amide 37 to brain sections As described in Example 39, the entire brain was collected from a healthy C57BL / 6J mouse, fixed overnight with 4% PFA, and then cryoprotected in sucrose solution for 48 hours. The tissue was embedded in Polyfreeze OCT compound (Sigma, USA), rapidly frozen, and then cut into 20 μm sagittal sections. The sections were subjected to antigen retrieval in sodium citrate buffer at 80-95°C for 15 minutes before quenching, blocking, and primary antibody incubation. Sulfated BODIPY amide 37 or unsulfated BODIPY amide 36 was added to the secondary antibody solution at 1 μg / mL and left on the sections at room temperature for 3 hours. The sections were then washed with PBS and ddH2O and mounted in a fading-preventing glycerol mounting medium containing DAPI (Abcam, Cambridge, UK) as described above. The sections were analyzed by confocal microscopy. The results are shown in Figures 6 and 7.

[0163] Example 41: Adhesion of sulfated maltose tetramer BODIPY amide 37 to brain endothelial (bEnd.3) cells While the coverslips were rinsed with 70% ethanol and allowed to dry in the 6-well plate, a 0.5 mg / ml solution of collagen I (Sigma-Aldrich, USA) was prepared. The collagen solution was then applied to the coverslips of the 6-well plate and left on a shaker at room temperature for 2 hours. Excess collagen was aspirated, and the wells were washed twice with 1 × dPBS (Gibco, USA). After washing, 3 × 10⁶ ions were placed in each coverslip. 5Nine bEnd.3 cells (ATCC, USA) were seeded. The cells were incubated in an incubator for 48–72 hours until confluence. Once confluence was reached, the medium was aspirated and replaced with preheated medium containing different concentrations of sulfated BODIPY amide 37 or unsulfated BODIPY amide 36. After 1 hour, the medium was removed and the cells were washed twice with 1× dPBS. The bEnd.3 cells were then fixed in 4% PFA (Sigma-Aldrich, USA) at room temperature for 15 minutes. The PFA was aspirated, the coverslip was washed twice with 1× dPBS, and the cells were mounted on Superfrost microscope slides (Fisherbrand, USA) using a DAPI-containing mounting medium (Abcam, UK). The results are shown in Figure 5. ****p<0.0001 and *p<0.05 are determined by two-way ANOVA with comparison to unsulfated BODIPY amide 36.

[0164] Example 42: Detection of fluorescently labeled mimics MC38 mouse colon adenocarcinoma cells were placed in DMEM medium (Gibco, Waltham, MA, USA) supplemented with 10% thermally inactivated FCS (Moregate Biotech, Hamilton, NZ), 100 U / mL penicillin, and 100 mg / mL streptomycin (Gibco) in a 2 × 10⁶ dose. 5The cells were suspended at a density of individual cells / mL. 1.5 mL of cell suspension (300,000 cells) was seeded into the wells of a 12-well plate containing poly-D-lysine coated coverslips (12-13 mm in diameter, 1.5H) and incubated at 37°C + 5% CO2 for 16 hours to allow cell adhesion. 5 μM sulfated BODIPY amide 37 was added to the cells, incubated for 1 hour, then the medium was removed from the wells and the cells were washed twice with PBS. Subsequently, the cells were fixed in 4% PFA (w / v) solution in PBS at room temperature for 15 minutes, washed three times with PBS, and then permeabilized with 0.1% Triton (Triton) X-100 in PBS for 5 minutes. After washing the cells three times with PBS, they were stained with Alexa Fluor Plus 647 Phalloidin (Invitrogen, Carlsbad, CA, USA) in PBS containing 1% bovine serum albumin (BSA) at room temperature for 60 minutes. Following incubation, cells were stained with 2 μg / mL Hoechst 33342 at room temperature for 30 minutes, washed three times with PBS, and then mounted on glass slides using ProLong® Glass Antifade Mountant (Invitrogen). After drying the slides for 48 hours, they were imaged using a Nikon Ni-E fluorescence microscope (Nikon, Tokyo, Japan). The results are shown in Figure 10. These data demonstrate that the heparin sulfate mimetic selectively binds to tumor cells in vitro, and that detection using fluorescence-based imaging was successful.

[0165] Example 43: By magnetic resonance imaging (MRI) 19 Detection of fluorine-labeled maltose tetramers Compound F6 20 (sulfated maltose tetramarvis (trifluoromethyl)benzamide) and Compound F27 24 (sulfated maltose tetramarvis (perfluoro-t-butoxy)amide) were sequentially diluted with PBS to prepare 40 mM, 20 mM, 10 mM, and 5 mM solutions, and each 19The F concentrations for compound 24 were 1080 mM, 540 mM, 270 mM, and 135 mM, and for compound 20, they were 240 mM, 120 mM, 60 mM, and 30 mM. 150 μL of each concentration was transferred to a 0.5 mL polypropylene tube for phantom imaging. 19 F images were acquired using a 2D FLASH sequence with the following parameters on an MRS DRYMAG7.0T MRI system (MRS7024, MR Solutions, Guildford, UK): Field of View (FOV) = 50 × 50 mm, Matrix = 64 × 64, Slice Thickness = 5 mm, TR = 200 ms, TE = 2.8 ms, Flip Angle (FA) = 20°, Bandwidth (BW) = 33 kHz, Number of Additions (NA) = 480, Acquisition Time = 102 minutes. DICOM images were exported using OsiriX Lite software (Pixmeo SARL, Bernex, Switzerland) and processed using ImageJ. The signal-to-noise ratio was calculated by normalizing the mean intensity value of the region of interest by the standard deviation of the signal-to-noise ratio. 19 The fluorine-labeled compound was detectable with a linear signal-to-noise ratio (SNR) up to a concentration of 5 mM. As expected, at high densities... 19 Compound 24, labeled with 1F, is more sensitive than compound 20. The results are shown in Figures 11-13. These data are 19 This demonstrates that fluorine-labeled maltose tetramer can be detected without problems using MRI.

[0166] Example 44: Heparanase Inhibition Assay The in vitro heparanase inhibition of compounds was evaluated using a colorimetric heparanase assay. In this assay, fondaparinux was used as the enzyme substrate, and WST-1 was used for color development. Recombinant human heparanase was purchased from R&D Systems. The assay was performed in a 96-well plate, with a total volume of assay solution in each well of 100 μL. The final concentrations in each well were as follows: 40 mM sodium acetate buffer (pH 5.0), μM 100 fondaparinux (molecular weight = 1726.77 g / mol, 12.5 mg / ml), 1 nM heparanase, and the desired concentration of sulfated maltose tetramer 7a or sulfated maltose tetramer 7c. The values ​​shown for each inhibitor concentration are the mean ± SD of independent experiments (n=3), and each independent experiment included three technical replicates. The wells were pre-treated with BSA (4% w / v) in 0.05% Tween 20 phosphate-buffered saline (PBST) at 37°C for 2 hours. The plate was then washed three times with PBST, shaken, and dried. Unused wells on the plate were filled with water to prevent excessive evaporation during incubation. Heparanase was added as the final component of the solution. The plate was then sealed with tape and aluminum foil and incubated at 37°C for 6 hours. The assay was stopped by adding 100 μL of 1.69 mM (WST-1)(4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate) in 0.1 M NaOH (1.1 mg / ml). The plate was covered and incubated at 60°C for 1 hour, and absorbance at 584 nm was measured. IC 50 The values ​​were calculated using Prism 9.0.1 software (GraphPad Software, La Jolla, CA, USA). The results are shown in Figures 14 and 15. [Industrial applicability]

[0167] This invention relates to compounds useful for the treatment or prevention of diseases including cancer, inflammation, diabetic nephropathy, and neurodegenerative disorders, as well as for specific cosmeceutical and dermatological applications.

Claims

1. Compound of formula (I) 【Chemistry 1】 (In the formula, X is (CH ２ ) ｐ And p is an integer from 1 to 20, R １ is H or CH ３ And, R ２ teeth, 【Chemistry 2】 is or R ２ teeth, 【Transformation 3】 (In the formula, ・ is R ２ It is the bonding point to the O atom, Y is (CH ２ ) ｎ and n is an integer from 1 to 3, R ３ is H or C １ ~C ３ It is alkyl, Z is (CH ２ ) ｍ And m is an integer from 1 to 6. M is, 【Chemistry 4】 (In the formula, R ４ is H or SO ３ H is the connection point to Z.))) Or its salt.

2. The compound according to claim 1, wherein p is an integer from 5 to 12.

3. The compound according to claim 1 or claim 2, wherein p is 5, 6, or 12.

4. The compound according to any one of claims 1 to 3, wherein n is 2.

5. R ３ The compound according to any one of claims 1 to 4, wherein is H.

6. R ３ The compound according to any one of claims 1 to 4, wherein the compound is methyl or ethyl.

7. The compound according to any one of claims 1 to 6, wherein m is 4, 5, or 6.

8. The aforementioned R ４ One or more of the elements are SO ３ H or SO ３ A compound according to any one of claims 1 to 7, wherein the compound is Na. 【Request Item 9】 【Chemistry 5】 【change】 A compound according to claim 1, selected from the group including the compound.

10. Compound of formula (II) 【Transformation 6】 (In the formula, R １ is H or CH ３ And, R ２ teeth 【Transformation 7】 is or R ２ teeth 【Transformation 8】 (In the formula, ・ is R ２ It is the bonding point to the O atom, Y is (CH ２ ) ｎ And n is an integer from 1 to 3, R ３ is H or C １ ~C ３ It is alkyl, Z is (CH ２ ) ｍ And m is an integer from 1 to 6. M is, 【Chemistry 9】 (In the formula, R ４ is H or SO ３ H is the connection point to Z.) A is -NH ２ , -N ３ , or -NH(C=O)R ５ C is replaced by １ ~C ６ It is an alkyl group (wherein R is R in the formula) ５ teeth, a) C １ ~C １２ alkyl group, b) Biothinyl substituent, c) Groups containing fluorescent labels, d) Groups containing fluorine-18 labeling, e) Groups containing fluorine-19 labeling, f) A group comprising a crown ether-type cage ligand for rhodium, iridium, actinium-225, or thorium-227, or g) A group containing N-acetate or C-14 radiolabeled N-acetate. Or its salt.

11. The compound according to claim 10, wherein n is 2.

12. R １ The compound according to claim 10 or claim 11, wherein is H.

13. R １ The compound according to claim 10 or claim 11, wherein is methyl.

14. R ３ The compound according to any one of claims 10 to 13, wherein is H.

15. R ３ The compound according to any one of claims 10 to 13, wherein the compound is methyl or ethyl.

16. The compound according to any one of claims 10 to 15, wherein m is 4, 5, or 6.

17. A is C １ ~C ６ A compound according to any one of claims 10 to 16, wherein the compound is an alkylamine.

18. A is C １ ~C ６ The compound according to any one of claims 10 to 16, which is an alkyl azide.

19. A is NH(C=O)R ４ C is replaced by １ ~C ６ Alkyl alkyl groups (wherein R ４ C ６ ~C １２ C containing alkyl and biotin substituents ６ ~C １２ C containing alkyl groups and fluorine-labeled BODIPY groups ６ ~C １２ C containing alkyl groups and fluorescent labels ６ ~C １２ C containing alkyl groups and fluorine-18 labeling ６ ~C １２ C containing alkyl groups or fluorine-19 labeling ６ ~C １２ The compound according to any one of claims 10 to 16, which includes an alkyl group.

20. The aforementioned R ４ One or more of the elements are SO ３ H or SO ３ The compound according to any one of claims 10 to 19, wherein the compound is Na. 【Request Item 21】 【Chemistry 10】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 A compound according to claim 10, selected from the group including the compound.

22. A pharmaceutical composition or cosmeceutical composition comprising an effective amount of the compound described in any one of claims 1 to 21 and a suitable carrier, diluent, or excipient.

23. A method for treating or preventing one or more of the following: cancer, inflammation, diabetic nephropathy, neurodegenerative disorders, multiple sclerosis, and skin diseases, comprising the step of administering to a patient in need of treatment a pharmaceutically effective amount of a compound described in any one of claims 1 to 21.

24. The method according to claim 23, wherein the neurodegenerative disorder is senile dementia, presenile dementia, multiple infarct dementia, or Alzheimer's disease.

25. A method for rejuvenating or preventing skin aging, comprising the step of administering an effective amount of a compound according to any one of claims 1 to 21 to human skin.

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