WO2008051017A1 - Agent de clivage agissant sélectivement sur un assemblage soluble de peptide ou protéine amyloïdogénique - Google Patents

Agent de clivage agissant sélectivement sur un assemblage soluble de peptide ou protéine amyloïdogénique Download PDF

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WO2008051017A1
WO2008051017A1 PCT/KR2007/005247 KR2007005247W WO2008051017A1 WO 2008051017 A1 WO2008051017 A1 WO 2008051017A1 KR 2007005247 W KR2007005247 W KR 2007005247W WO 2008051017 A1 WO2008051017 A1 WO 2008051017A1
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cleavage
cleavage agent
mono
group
dici
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PCT/KR2007/005247
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Jung Hun Suh
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Seoul National University Industry Foundation
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Priority claimed from KR1020070075809A external-priority patent/KR20080036916A/ko
Application filed by Seoul National University Industry Foundation filed Critical Seoul National University Industry Foundation
Priority to JP2009534493A priority Critical patent/JP2010507652A/ja
Priority to US12/446,905 priority patent/US20100036122A1/en
Publication of WO2008051017A1 publication Critical patent/WO2008051017A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

  • the present invention relates to a cleavage agent and a cleavage method selectively acting on soluble assembly of amyloidogenic peptide or protein.
  • the cleavage agent of the present invention inhibits biological activity of the amyloidogenic peptide or protein by cleaving the soluble assembly of amyloidogenic peptide or protein.
  • Amyloidosis refers to a variety of conditions in which insoluble amyloid proteins
  • amyloids maintain their intrinsic structure and function, and the amyloids have in
  • Amyloidogenic peptides or proteins can form soluble assemblies including various
  • amyloidosis causes of the pathogeneses of amyloidosis (Bittan, G.; Fradinger, E. A.; Spring, S. M.; Teplow, D. B. Amyloid 2005, 12, 88).
  • the soluble oligomer of the amyloidogenic peptide or protein for example amyloid beta (A/3) peptide, amylin, ⁇ -synuclein, prion, or polyglutamine, can cause Alzheimer's disease, type 2 diabetes mellitus, Parkinson's disease, spongiform encepahlopathies, or Huntington's disease (Demuro, A. G.; Mina, E.; Kayed, R.; Milton, S.; Parker, L; Glabe, C. G. J. Biol. Chem. 2005, 280, 17294).
  • the chemical and biological properties of the soluble oligomer of amyloidogenic peptide or protein are explained by using the Alzheimer's disease as a model.
  • Alzheimer's disease is major cause of senile dementia.
  • Alzheimer's disease is a degenerative brain disorder that is characterized clinically by the progressive loss of neuronal cells. Plaques consisting of A/3 peptide and neurofibrillary tangles are detected in the brains of Alzheimer's disease patients (Selkoe, D. J. Physiol. Rev. 2001,
  • the A/3 peptide is formed after sequential cleavage of the amyloid precursor protein (APP).
  • A/3 protein is generated by successive action of /3- and ⁇ -secretases, and these secretases mainly generate oligopeptides of 40 and 42 amino acid residues in length (Sambamurti, K.; Greig, N. H.; Lahiri, D. K. Neuromol. Med. 2002, 1, 1). These oligopeptides of 40 and 42 amino acid residues in length are referred to as A 1 S 4O or AjS 42 , respectively.
  • the amino acid sequence of A/3 42 is shown in Figure 1.
  • the amino acid sequence of AjS 40 can be obtained by removing the two C-terminal amino acids from the amino acid sequence of AjS 42 .
  • AjS 42 the major component of amyloid plaque, is more prone to aggregation than AjS 40 .
  • the Amyloid Cascade Hypothesis was proposed in 1992 (Hardy, J. A.; Higgins, G. A. Science 1992, 256, 184). This hypothesis suggested that the mismetabolism of APP was the initiating event in AD pathogenesis, subsequently leading to the aggregation of AjS 42 . Formation of fibrous aggregation and neuritic plaques according to the increasement of producing AjS 42 would set off further pathological events, including disruption of synaptic connections, which would lead to a reduction in neurotransmitters, and the death of tangle-bearing neurons and dementia.
  • the soluble assemblies including several oligomers or protofibrils, are formed reversibly, or partially irreversibly during the assembly process of A/3 42 , and then the insoluble fibril is formed irreversibly (Bitan, G.; Kirkitadze, M. D.; Lomakin, A.; Vollers, S. S.; Benedek, G. B.; Teplow, D. B. Proc. Natl. Acad. ScL USA 2003, 100, 330: Moss, M. A.; Nichols, M. R.; Reed, D. K.; Hoh, J. H.; Rosenberry, T. L. MoI. Pharmacol. 2003, 64, 1160: Lesne S.; Koh, M.
  • variable oligomers of A 1 S 42 have their own unique structures (Urbanic, B.; Cruz, L.; Yun, S.; Buldyrev, S. V.; Bitan, G.; Teplow, D. B.; Stanley. H. E. Proc. Natl. Acad. ScL USA 2004, 101, 17345).
  • a method of stimulating the removal of the oligomer of A/3 42 from the brain can be a candidate for the development of a method for relieving the neurotoxicity caused by A/3 42 .
  • Hardy et al. suggested a method which inhibits the activity of either ⁇ - or ⁇ -secretase to prevent production of A/3 from APP (Hardy, J.; Selkoe, D. J. Science 2002, 297, 353).
  • AjS immune agents Schoenk, D.; Barbour, R.; Dunn, W.; Gordon, G.; Grajeda, H.; Guido, T.; Hu, K.; Huang, J.; Johnson- Wood, K.; Khan, K.; Kholodenko, D.; Lee, M.; Liao, Z.; Lieberburg, I.; Motter, R.; Mutter, L.; Soriano, F.; Shopp, G.; Vasquez, N.; Vandevert, C; Walker, S.; Wogulis, M.; Yednock, T.; Games, D.; Seubert, P.
  • a 1 S oligomerization can be inhibited by using small molecules which have high affinity for A 1 S (Cohen, T.; Frydman-Marom, A.; Israeler, M.; Gazit, E. Biochemistry 2006, 45, 4727).
  • the amount of oligomer of A/3 42 in the brain can be reduced by stimulating the AjS
  • degradation enzyme such as endothelin converting enzyme, insulin-degrading enzyme
  • plaques preventing the accumulation of fibrous plaques precursor, and the like.
  • the present inventors have discovered a new method of cleaving the soluble
  • the synthetic cleavage molecule which selectively acts on the soluble assembly of amyloidogenic peptide or protein (hereinafter referred to as the, "cleavage agent") is an artificial enzyme for eliminating the soluble assembly of amyloidogenic peptide or protein.
  • the inventors of the present invention have researched to find cleavage agents that have the above properties. They have found cleavage agents by connecting the sites that selectively recognize the soluble assembly of amyloidogenic peptide or protein with the reactive portions that cleave peptide bonds. They confirmed accomplishment of the object of the present invention to reduce the amount of soluble oligomers of the amyloidogenic peptide or protein by using the cleavage agents to complete the present invention. Accordingly, the present invention provides cleavage agents which eliminate the soluble assembly of amyloidogenic peptide or protein.
  • the present invention also provides a pharmaceutical composition for treatment or prevention of amyloidosis comprising the above cleavage agents and a pharmaceutically acceptable carrier.
  • Fig. 1 shows the amino acid sequence of A/5 42 .
  • Fig. 2 schematically shows the formation process for soluble and insoluble assemblies of amyloidogenic peptides or proteins
  • Fig. 3 schematically shows the process of reduction of the amounts of the soluble and insoluble assemblies of amyloidogenic peptides or proteins by the cleavage agent
  • Fig. 4 shows the synthesis pathway of cleavage agent A in Example 1 of the present invention
  • Fig. 5 shows the fraction of A ⁇ 4 o (O) or A ⁇ 42 (•) (initial concentration: 4.0 ⁇ M) passing the membrane with cut-off molecular weight (MW) of 10000 after incubation at pH 7.50 and 37 0 C for various periods of time (each data represents the mean value from at least 5 measurements),
  • Fig. 6 shows MALDI-TOF mass spectrum taken after incubation of AjS 40 (4.0 ⁇ M) with cleavage agent A (3.0 ⁇ M) of Example 1 at 37 ° C and pH 7.50 for 36 hours,
  • Fig. 7 shows MALDI-TOF mass spectrum taken after incubation of AjS 42 (4.0 ⁇ M) with cleavage agent A (1.0 ⁇ M) of Example 1 at 37 ° C and pH 7.50 for 36 hours,
  • Fig. 8 shows the plot of cleavage yield against log CJM for cleavage of AjS 40 (o) or A/3 42 (•) (4.0 ⁇ M) by cleavage agent A of Example 1 measured after reacting for 36 hours at 37 ° C and pH 7.50,
  • Fig. 9 shows effects of the period of preincubation of A ⁇ 4 o (gray bars) or A ⁇ 42 (dark bars) (4.0 ⁇ M) on cleavage yield by cleavage agent A (3.0 ⁇ M) of Example 1 measured after reacting for 36 hours at 37 0 C and pH 7.50,
  • Fig. 10 shows the plot of the cleavage yield against reaction time for cleavage of Ap 40 (O) or A ⁇ 42 (•) (4.0 ⁇ M) by cleavage agent A (3.0 ⁇ M) of Example 1 at 37 0 C and pH 7.50,
  • Fig. 11 shows the fraction of Am (initial concentration: 4.0 ⁇ M) passing the membrane with cut-off MW of 10000 after incubation at pH 7.50 and 37 0 C for various period of time,
  • Fig. 12 shows MALDI-TOF mass spectrum of the products purified with HPLC after incubating Am (4.0 ⁇ M) with cleavage agent A (3.2 ⁇ M) of Example 1 at 37 ° C and pH 7.50 for 36 hours,
  • Fig. 13 shows the plot of the cleavage yield against log C 0 ZM for cleavage of Am (4.0 ⁇ M) by cleavage agent A of Example 1 measured after reacting for 36 hours at 37 0 C and pH 7.50,
  • Fig. 14 shows the fraction of Syn (initial concentration: 70 ⁇ M) passing a 0.22 mm Millipore filter (Millipore Millex-GV 4MM) after self-assembly during incubation at pH 7.50 and 37 0 C for various period of time
  • Fig. 15 shows the plot of the cleavage yield against log CJM for cleavage of Syn
  • Fig. 16 shows the synthesis pathway of cleavage agent B in Example 2,
  • Fig. 17 shows MALDI-TOF mass spectrum taken after incubation of A/3 4 o (4.0 ⁇ M) with cleavage agent B (3.0 ⁇ M) of Example 2 at 37 ° C and pH 7.50 for 36 hours,
  • Fig. 18 shows MALDI-TOF mass spectrum taken after incubation of A/3 42 (4.0 ⁇ M) with cleavage agent B (0.50 ⁇ M) of Example 2 at 37 ° C and pH 7.50 for 36 hours.
  • Fig. 19 shows the plot of cleavage yield against log CJM for cleavage of A ⁇ 40 (o) or A/3 42 (•) (4.0 ⁇ M) by cleavage agent B of Example 2 measured after reacting for 36 hours at 37 ° C and pH 7.50,
  • Fig. 20 shows MALDI-TOF mass spectrum of the products purified by HPLC after incubating Am (4.0 ⁇ M) with cleavage agent B (1.0 ⁇ M) of Example 2 at 37 ° C and pH 7.50 for 36 hour.
  • Fig. 21 shows the plot of the cleavage yield against log CJM for cleavage of Am
  • Fig. 22 shows effects of period of preincubation of Am (4.0 ⁇ M) on cleavage yield by cleavage agent B (1.0 ⁇ M) of Example 2 measured after reacting for 36 hours at 37 0 C and pH 7.50,
  • Fig. 23 shows the plot of the cleavage yield against reaction time for cleavage of Am (4.0 ⁇ M) by cleavage agent B (1.0 ⁇ M) of Example 2 at 37 0 C and pH 7.50,
  • Fig. 24 shows the plot of the cleavage yield against log C 0 IM for cleavage of Syn (70 ⁇ M) by cleavage agent B of Example 2 measured after reacting for 3 days at 37 0 C and pH 7.50,
  • Fig. 25 shows the synthesis pathway of cleavage agent C in Example 3
  • Fig. 26 shows MALDI-TOF mass spectrum taken after incubating AjS 42 (4.0 ⁇ M) with cleavage agent C (1.00 ⁇ M) of Example 3 at 37 ° C and pH 7.50 for 36 hours.
  • Fig. 27 shows the plot of cleavage yield against log C 0 IM for cleavage of A 1 S 40 (o) or A/3 42 (•) (4.0 ⁇ M) by cleavage agent C of Example 3 measured after reacting for 36 hours at 37 ° C and pH 7.50,
  • Fig. 28 shows MALDI-TOF mass spectrum of the products purified by HPLC after incubating Am (4.0 ⁇ M) with cleavage agent C (3.2 ⁇ M) of Example 3 at 37 ° C and pH 7.50 for 36 hours,
  • Fig. 29 shows the plot of the cleavage yield against log C 0 IM. for cleavage of Am (4.0 ⁇ M) by cleavage agent C of Example 3 measured after reacting for 36 hours at 37 0 C and pH 7.50,
  • Fig. 30 shows the synthesis pathway of cleavage agent D in Example 4
  • Fig. 31 shows MALDI-TOF mass spectrum taken after incubating A/3 42 (4.0 ⁇ M) with cleavage agent D (1.00 ⁇ M) of Example 4 at 37 ° C and pH 7.50 for 36 hours,
  • Fig. 32 shows the plot of cleavage yield against log C 0 IM for cleavage of A(S 40 (o) or A/3 42 (•) (4.0 ⁇ M) by cleavage agent D of Example 4 measured after reacting for 36 hours at 37 ° C and pH 7.50
  • Fig. 33 shows MALDI-TOF mass spectrum of the products purified by HPLC after incubating Am (4.0 ⁇ M) with cleavage agent D (0.38 ⁇ M) of Example 4 at 37 ° C and pH 7.50 for 36 hours,
  • Fig. 34 shows the plot of the cleavage yield against log CJM for cleavage of Am (4.0 ⁇ M) by cleavage agent D of Example 4 measured after reacting for 36 hours at 37 0 C and pH 7.50,
  • Fig. 35 shows the synthesis pathway of cleavage agent E in Example 5
  • Fig. 36 shows MALDI-TOF mass spectrum of the products purified by HPLC after incubating Am (4.0 ⁇ M) with cleavage agent E (0.89 ⁇ M) of Example 5 at 37 ° C and pH 7.50 for 36 hours,
  • Fig. 37 shows the plot of the cleavage yield against log CJM for cleavage of Am (4.0 ⁇ M) by cleavage agent E of Example 5 measured after reacting for 36 hours at 37 0 C and pH 7.50,
  • Fig. 38 shows the synthesis pathway of cleavage agent F in Example 6
  • Fig. 39 show MALDI-TOF mass spectrum of the products purified by HPLC after incubating Am (4.0 ⁇ M) with cleavage agent F (1.6 ⁇ M) of Example 6 at 37 " C and pH 7.50 for 36 hours,
  • Fig. 40 shows the plot of the cleavage yield against log C 0 ZM for cleavage of Am (4.0 ⁇ M) by cleavage agent F of Example 6 measured after reacting for 36 hours at 37 0 C and pH 7.50,
  • Fig. 41 shows the synthesis pathway of cleavage agent G in Example 7,
  • Fig. 42 shows MALDI-TOF mass spectrum of the products purified by HPLC after incubating Am (4.0 ⁇ M) with cleavage agent G (0.89 ⁇ M) of Example 7 at 37 "C and pH 7.50 for 36 hours,
  • Fig. 43 shows the plot of the cleavage yield against log C o /M for cleavage of Am (4.0 ⁇ M) by cleavage agent G of Example 7 measured after reacting for 36 hours at 37 0 C and pH 7.50,
  • Fig. 44 shows the synthesis pathway of cleavage agent H in Example 8
  • Fig. 45 shows MALDI-TOF mass spectrum of the products purified by HPLC after incubating Am (4.0 ⁇ M) with cleavage agent H (7.1 ⁇ M) of Example 8 at 37 ° C and pH 7.50 for 36 hours, and
  • Fig. 46 shows the plot of the cleavage yield against log C o /M for cleavage of Am (4.0 ⁇ M) by cleavage agent H of Example 8 measured after reacting for 36 hours at 37 0 C and pH 7.50.
  • the present invention relates to cleavage agent of formula 1 which selectively cleaves the soluble assembly of amyloidogenic peptide or protein: [formula 1]
  • A represents independently C 6- i 4 aryl, or 5- to 14-membered heteroaryl having one or more hetero atom selected from the group consisting of oxygen, sulfur and nitrogen, wherein, aryl or heteroaryl is unsubstituted or substituted by one or more substituent(s) independently selected from the group consisting of C 1-15 alkyl, hydroxy, C 1-15 alkoxy, Ci -15 alkylcarbonyloxy, Ci_i 5 alkylsulfonyloxy, amino, mono or diCi.
  • Y is O or N-Z, wherein Z represents hydrogen or Ci_ 9 alkyl;
  • L is linker;
  • Z is a metal ion-ligand complex which acts as a catalytic site; n is independently an integer from 1 to 6; m and o are independently 0 or 1 ; p is an integer from 0 to 5.
  • the cleavage agents according to the present invention comprise of target recognition sites which recognize the soluble assembly of amyloidogenic peptide or protein and catalytic sites which display cleavage activity, specifically cleaving peptide bonds.
  • the cleavage agents have the capacity to recognize the soluble assembly of amyloidogenic peptide or protein and the capacity for cleaving peptide bonds.
  • the cleavage agents of the present invention are effective for the selective inhibition of bioactivity of soluble oligomers of amyloidogenic peptide or protein in the presence of various kinds of biomolecules.
  • cleavage agents according to the present invention are specifically indicated as follows. As explained above during the process of oligomerization and fibril formation of AjS 42 as an example, various soluble oligomers and protofibrils are formed reversibly or partially irreversibly, and fibril is formed therefrom irreversibly during the assembly processes of the amyloidogenic peptide or protein (Bitan, G.; Kirkitadze, M. D.; Lomakin, A.; Vollers, S. S.; Benedek, G. B.; Teplow, D. B. Proc. Natl. Acad. ScL USA 2003, 100, 330). Protofibril can be regarded as soluble or insoluble polymer (Moss, M.
  • the effective cleavage agent can be obtained by mimicking the principle of enzyme's catalytic activity.
  • the substrate forms a complex with the enzyme, and the enzyme converts the complex ed substrate into the product.
  • highly effective molarity of the catalytic functional groups of the enzyme toward the substrate is attained, leading to a very high reaction rate.
  • the cleavage agent of formula 1 according to the present invention is an artificial enzyme.
  • the cleavage agent has the target recognition site which recognizes the soluble assembly of amyloidogenic peptide or protein, and thus can selectively combine with one or more soluble assembly.
  • the target recognition site and the soluble assembly are combined, and then the catalytic site of the cleavage agent, according to the present invention, cleaves the peptide bond in the soluble assembly.
  • the species placed in the rectangle represents soluble assemblies.
  • the soluble assemblies include soluble oligomers and soluble protofibrils.
  • the soluble protofibrils can be regarded as very large soluble oligomers. Conversion of large assemblies, such as protofibrils and fibrils, into smaller ones is slow, and formation of the large assemblies can be considered as irreversible or partially irreversible. For some amyloidogenic peptides or proteins, it is suggested that the fibril formation is reversible with the fibrils and the monomer being in equilibrium (Wetzel, R. Ace. Chem.
  • the cleavage agent of the present invention combines with one or more species shown in the rectangle of Figure 2, and then cleaves the peptide bonds of the amyloidogenic peptide or protein to achieve its function.
  • the catalytic site of the cleavage agent is located in proximity to the peptide bonds of the amyloidogenic peptide or protein. Amyloidogenic peptide or protein is then effectively cleaved by the attack of the catalytic sites.
  • reaction 1 The reaction of cleavage agent with the target is summarized as Reaction 1 which is similar to the Michaelis-Menten equation applied to the enzyme reaction: [Reaction 1]
  • Figure 3 (which is the combination of Figure 2 and Reaction 1) shows the process of reducing the amounts of the soluble and insoluble assemblies of amyloidogenic peptide or protein by the action of the cleavage agent.
  • (AP) ass-m in Figure 3 represents cleaved assembly.
  • concentration of the (AP) ass-m> which is cleaved by the cleavage agent of the present invention is reduced, the concentrations of other assemblies which can be easily converted to (AP) ass-m are also reduced.
  • the amounts of the assemblies including protofibrils or fibrils which cannot be easily converted to the (AP) aS s-m are not effectively reduced. Instead, reduction of the concentration of (AP) ass-m slows down the formation of protofibrils or fibrils from
  • the cleavage agent needs to form a complex with the soluble assembly of amyloido genie peptide or protein in a very low concentration of the cleavage agent. Therefore, the cleavage agent of the present invention is comprised of a target recognition site, which can selectively and strongly combine with the soluble assembly.
  • A represents independently C 6- i 4 aryl, or 5- to 14-membered heteroaryl having one or more hetero atom selected from the group consisting of oxygen, sulfur and nitrogen,
  • A should be selected from the group consisting of the compounds of following formulas:
  • X is independently selected from the group consisting of C, N, NH, O and S.
  • A should be selected from the group consisting of the compounds of following formulas:
  • X is NH, O, or S.
  • A is unsubstituted or substituted by one or more substituent(s) independently selected from the group consisting of Ci.isalkyl, hydroxy, C 1- i 5 alkylcarbonyloxy, C 1-15 alkylsulfonyloxy, amino, mono or diCi-i 5 alkylamino, Ci- i 5 alkylcarbonylamino, Ci-isalkylsulfonylamino, C 3- i 5 cycloalkylamino, formyl, Ci- i 5 alkylcarbonyl, carboxy, Ci-isalkyloxycarbonyl, carbamoyl, mono or diCi. i 5 alkylcarbamoyl, Ci-i 5 alkylsulfanylcarbonyl, Ci-i 5 alkylsulfanylthiocarbonyl, C 1-
  • A is unsubstituted or substituted by the substituents selected from the group consisting of Ci -6 alkyl, Ci -6 alkoxy, amino, mono or diCi-i 2 alkylamino, C 1- 6 alkylcarbonylamino, Ci- ⁇ alkylsulfonylamino, C ⁇ -iscycloalkylamino, Ci- ⁇ alkylcarbonyl.
  • A is unsubstituted or substituted by the substituents selected from the group consisting of C 1-4 alkyl, C 1-4 alkoxy, amino, mono or diCi-salkylamino, C 6- 12 cycloalkylamino, C 1-4 alkylcarbonyl and halogen.
  • substituents selected from the group consisting of C 1-4 alkyl, C 1-4 alkoxy, amino, mono or diCi-salkylamino, C 6- 12 cycloalkylamino, C 1-4 alkylcarbonyl and halogen.
  • Y is O or N-Z
  • Z is Ci-galkyl, preferably C 1-4 alkyl.
  • p is independently an integer from 0 to 5, preferably 0 to 2.
  • o is independently 0 or 1.
  • the cleavage agent according to the present invention preferably includes 1 to 6, more preferably 1 to 4, 1 or 2 of target recognition site(s).
  • the metal ion can be used as a key component in the catalytic site of the present invention based upon its catalytic activity in peptide hydrolysis.
  • the catalytic site of the present invention is comprised of metal ion-ligand complex.
  • the metal ion according to the present invention to be used in catalytic sites should be preferably selected from the group consisting of Co 111 , Cu 1 , Cu 11 , Ce IV , Ce v , Cr 111 , Fe", Fe 111 , Mo IV , Ni 11 , Pd", Pt 11 , V v and Zr IV , more preferably Co 111 , Cu 11 or Pd", and most preferably Co 111 .
  • the present inventors found that in selectively cleaving soluble oligomers of amyloidogenic peptide or protein, restricting the ligand in the catalytic site to a specific structure is important in inhibiting their biological activity.
  • the ligand to be used in the catalytic site of the present invention is selected from the group consisting of the following compounds:
  • nitrogen atom included in ligand is independently replaced with the atom selected from the group consisting of oxygen, sulfur and phosphorous;
  • the ligand may be fused with C 6- i 4 aryl or 5- to 14-membered heteroaryl.
  • the ligand to be used in the catalytic site is selected from the group consisting of the following formulas:
  • the ligand is a cycle consisting of 12 atoms, and selected from the group consisting of the following formulas:
  • the ligand is unsubstituted or substituted with one or more substituent(s) independently selected from the group consisting of Ci-i 5 alkyl, hydroxy, Ci-i 5 alkoxy, C 1 . i 5 alkylcarbonyloxy, Ci-isalkylsulfonyloxy, amino, mono or diCi-isalkylamino, C 1-
  • the ligand is unsubstituted or substituted with one or more substituent(s) selected from the group consisting of C 1-6 alkyl, Ci -6 alkoxy, C 1 . 6 alkylcarbonyloxy and halogen.
  • the ligand is unsubstituted or substituted with one or more substituent(s) selected from the group consisting of Ci -4 alkyl, C 1-4 alkoxy and halogen.
  • the target recognition site (R) is connected through the linker, or directly to catalytic site (Z).
  • the modes of connection between the target recognition site and the catalytic site through the linker include the connection between one target recognition site and one catalytic site through the linker, the parallel connection of two or more target recognition sites to a catalytic site through separate linkers, the parallel connection of two or more target recognition sites to a catalytic site through a linker having a branched structure, a series connection in which two or more target recognition sites are connected to one another through a linker and one of the target recognition sites is connected to the catalytic site through a separate linker.
  • the cleavage agent can be formed by combining connection modes listed above to connect a multiple number of target recognition sites to the catalytic site:
  • R represents a target recognition site
  • Z represents a catalytic site.
  • the linker includes a main chain which connects the target recognition site and the catalytic site directly or connects two target recognition sites, and a substituent optionally attached to the main chain.
  • the target recognition site binds to the target protein, and then the catalytic site cleaves one or more of the peptide bonds in the target protein.
  • the reactivity of the catalytic site is increased by increasing the effective concentration between the cleavage site on the protein and the catalytic site.
  • the efficient way to modulate the effective concentration is by adjusting the relative positions between the target recognition site and the catalytic site in the cleavage agent.
  • the length and shape of the linker can be used to modulate the relative positions.
  • the linker of the present invention is used to connect the target recognition site and the catalytic site.
  • the linker of the present invention is comprised of the backbone comprising one or more atoms which is independently selected from the group consisting of carbon, nitrogen, oxygen, silicon, and phosphorous.
  • the number of atoms included in the backbone should be between 1 and 30 but, preferably between 1 and 20, and more preferably between 1 and 15.
  • the atoms included in the backbone of the linker are present as members of functional groups independently selected from the group consisting of alkane, alkene, alkyne, carbonyl, thiocarbonyl, amine, ether, silyl, sulfide, disulfide, sulfonyl, sulfmyl, phosphoryl, phosphinyl, amide, imide, ester and thioester.
  • the linker is unsubstituted or substituted with one or more substituent(s) independently selected from the group consisting of Ci.galkyl, hydroxy, Ci-galkoxy, C 1- galkylcarbonyloxy, Q.galkylsulfonyloxy, amino, mono or diCi.galkylamino, Ci- 9 alkylcarbonylamino, Ci.galkylsulfonylamino, formyl, Cj.galkylcarbonyl, carboxy, C 1- galkyloxycarbonyl, carbamoyl, mono or diCi.galkylcarbamoyl, Ci.galkylsulfanylcarbonyl, Ci-galkylsulfanylthiocarbonyl, Ci-galkoxycarbonyloxy, carbamoyloxy, mono or (IiC 1- galkylcarbamoyloxy, Ci.galkylsulfanylcarbonyloxy, Ci.galkoxycarbonylamino, ureido,
  • the linker is unsubstituted or substituted with one or more substituent(s) independently selected from the group consisting of C 1-6 alkyl, Ci -6 alkoxy, mono or diCi_ 6 alkylamino, C 1-6 alkylcarbonylamino, C 1-6 alkylsulfonylamino, Ci -6 alkylcarbonyl, carbamoyl, mono or diCi -6 alkylcarbamoyl, Ci -6 alkoxycarbonylamino, ureido, mono or di ortriCi- ⁇ alkylureido, Ci- ⁇ alkylsulfanylcarbonylamino, Ci -6 alkylsulfanyl, Ci- 6 alkyldisulfanyl, sulfamoyl, mono or diCi -6 alkylsulfamoyl, triCi_ 6 alkylsilanyl and halogen.
  • substituent(s) independently selected from the group consisting
  • the linker is unsubstituted or substituted with one or more substituent(s) independently selected from the group consisting of Ci -4 alkyl, Ci -4 alkoxy, mono or diCi -4 alkylamino, Ci_ 4 alkylcarbonylamino, Ci -4 alkylsulfonylamino, Ci-
  • the cleavage agent of the present invention recognizes its target via the interaction between the aromatic microdomains included in the soluble assembly of amyloidogenic peptide or protein and the aromatic component included in the target recognition site of the cleavage agent. Therefore, how many different kinds of amyloidogenic peptide or protein are used to form the soluble assembly is not important as long as the soluble assembly includes the aromatic microdomains.
  • the soluble assembly formed by one kind of amyloidogenic peptide or protein, as well as the soluble assembly formed by two or more kinds of amyloidogenic peptide or protein can both be the target for the cleavage agent of the present invention. Meanwhile, during the formation process of the soluble assembly, any kind of biomolecules can be incorporated into the assembly. Even when those biomolecules are present in the assembly, the soluble assembly can still be the target of the cleavage agent of the present invention.
  • the cleavage agent of the present invention can selectively cleave the soluble assembly of peptide or protein associated with one kind of amyloidosis, or cleave soluble assemblies of peptides or proteins associated with two or more kinds of amyloidosis.
  • the cleavage agent of the present invention is specifically effective for cleaving the following, but not limited to the following oligomers.
  • Oligomers OfAjS 4O and AjS 42 associated with Alzheimer's disease AjS 4 O and AjS 42 form various oligomers, protofibrils, and fibrils by self-assembly as shown in Figure 2.
  • the aggregation Of A 1 S 42 is faster than that Of AjS 40 . Therefore, in cases where the concentration of A 1 S 42 monomer is higher than several ⁇ M, A 1 S 42 is oligomerized in a few minutes. It is then converted to protofibrils with sizes smaller than 0.1 ⁇ m in a solvent or on a solid surface in a few hours (Kowalewski, T.; Holtzman, D. M. Proc. Natl. Acad. Sci. USA.
  • Some cleavage agents among the cleavage agents as shown in the Examples are capable of cleaving oligomers of various kinds of amyloidogenic peptide or protein.
  • Cleavage agent A cleaves oligomers of AjS 40 as well as those of AjS 42 as in Example 1.
  • AJS 40 are mainly generated by proteolytic cleavage of the /3-amyloid precursor proteins. AjS 40 is responsible for various physiological functions, and therefore, if AjS 40 is drastically cleaved, its normal functions would be inhibited. However, the amount of
  • AjS 40 in the brain of Alzheimer's disease patients is 30 to 40 times higher than those of nondemented elderly controls (Lue, L. R; Kuo, Y. M.; Roher, A. E.; Brachova, L.; Shen, Y.; Sue, L.; Beach, T.; Kurth, J. H.; Rydel, R. E.; Rogers, J. Am. J. Pathol. 1999, 155,
  • the antibody raised against A/3 42 oligomer can recognize the AjS 4O oligomer as well as the A 1 S 42 oligomer, and oligomers of other amyloidogenic proteins or peptides, such as ⁇ -synuclein, amylin, polyglutamine, lysozyme, insulin, prion peptide 106-126 (Kayed,
  • cleavage agents in the Examples are capable of cleaving oligomers of two or more kinds of amyloidogenic peptides or proteins in agreement with the antibody study.
  • amylin human islet amyloid polypeptide
  • Am is a cyclic oligopeptide consisting of 37 amino acid residues, and is capable of forming amyloids by self-assembly.
  • the cleavage agents of the present invention cleave the oligomer of Am. It is not clear which oligomers among the various oligomers of Am are cleaved by the cleavage agents of the present invention. However, the concentrations of the other oligomers which are equilibrium with the target decrease in accordance with the reduction of the target oligomer's concentration. Accordingly, the amount of oligomers which cause type 2 diabetes mellitus will also be reduced. (3) Oligomer of ⁇ -synuclein associated with Parkinson's disease
  • ⁇ -synuclein is a protein consisting of 140 amino acids and is capable of forming amyloids by self-assembly.
  • the cleavage agents of the present invention cleave the soluble assembly formed by amyloidogenic peptide or protein, and inhibit the biological activity of the soluble assembly to prevent or treat amyloidosis.
  • the present invention relates to a pharmaceutical composition for preventing or treating amyloidosis, comprising cleavage agent of formula 1 and pharmaceutically acceptable salts.
  • Amyloidosis includes, but is not limited to, Alzheimer's disease, type 2 diabetes mellitus, Parkinson's disease, spongiform encephalopathy or Huntington's disease.
  • cleavage agent can be administered to a patient can be modified according to the patient's weight, sex, overall health, diet, the severity of the disease, and other drugs being taken by the patient.
  • the cleavage agent of the present invention can be administered by any route dictated by the targets of the cleavage agent. Accordingly, the cleavage agent of the present invention can be administered intravenously, orally, intranasally, subcutaneously, peritoneally, retroperitoneally, rectally, etc, However, the intravenous, oral, and intranasal methods are preferred.
  • Injection formulation for example, sterile injection aqueous or oleaginous suspension
  • sterile injection aqueous or oleaginous suspension can be prepared through conventional methods in the art, by using suitable dispersing agents, humectants or suspension.
  • sterile fixing oil can be used conventionally as a solvent or suspension media.
  • Any nonirritant fixing oil including mono-, di-glyceride can be used, and fatty acids, such as oleic acid can be used in the injection formulation.
  • the agent of the present invention can also be formulated in oral preparation including capsules, tablets, pills, powders, granules, and the like. However, tablets and capsules are preferred, such as a enteric coated tablet or pill.
  • the solid administration formulations can be prepared by mixing the cleavage agent of the present invention of formula 1 with inactive diluents, such as sucrose, lactose, starch, and the like; and pharmaceutically acceptable carriers, such as, lubricants such as magnesium stearate, disintegrants, and binders.
  • inactive diluents such as sucrose, lactose, starch, and the like
  • pharmaceutically acceptable carriers such as, lubricants such as magnesium stearate, disintegrants, and binders.
  • Cleavage agent A was synthesized through the pathway shown in the Figure 4.
  • Oxidation of Co 11 to Co 111 was accompanied by appearance of deep violet color.
  • the Co 111 complex was isolated with HPLC by detecting at 545 nm, and evaporated to produce a solid.
  • the solid was dissolved in 0.1 M NaOH solution, and left at 37 ° C for 1 hour.
  • the solution was neutralized with HCl to pH 6-8, and left at room temperature for several days to obtain the stock solution of cleavage agent A.
  • the cobalt content was measured by ICP to determine the concentration of the cleavage agent in the solution.
  • each cleavage agent was tested at 37 ° C and pH 7.50 (0.050 M phosphoric acid) in Eppendorf tubes unless indicated otherwise in the Examples.
  • A/3 4 o or A/3 42 was treated with NaOH prior to exposure to the pH 7.50 reaction medium (Fezoui, Y; Hartley, D. M.; Harper, J. D.; Khurana, R.; Walsh, D. M.; Condron, M. M.; Selkoe, D. J.; Lansbury, RT. Jr.; Fink, A. L.; Teplow, D. B. Amyloid 2000, 7,166-178).
  • AjS 42 exists mostly as the monomer in the early reaction stage. After 3 or 36 hours, 2/3 or 90 %, respectively, of AjS 42 is converted to large assemblies which cannot pass the membrane. However, in case of AjS 40 , more than 90 % of the AjS 40 passed the membrane in the early reaction state, and after 24 hours, 50 % Of AjS 40 passed the membrane.
  • the MALDI-TOF mass spectrum obtained by reacting AjS 40 or A/3 42 (4.0 ⁇ M) with cleavage agent A are illustrated in Figure 6 or Figure 7, respectively.
  • AjS 40 and AjS 42 are cleaved by cleavage agent A.
  • AjS 1-20 and AjSi -21 were included in the cleavage products (in the Examples herein, A ⁇ fragments are named according to the amino acid sequence of A ⁇ 42 , and the structures of the cleavage products with the m/z values assigned to the peaks were confirmed with MALDI LIFT-TOF/TOF MS). Since the intensity of a MALDI-TOF MS peak does not stand for the relative concentration, some oligopeptide fragments may be present in significant concentrations without showing strong MALDI-TOF MS peaks.
  • cleavage reaction was initiated by adding A/3 4 o or AjS 42 to the solution containing the cleavage agent, and the cleavage yield was estimated through the following process.
  • a product solution formed by the cleavage reaction was passed through the membrane with a cut-off MW of 10000 to remove aggregates of AjS 40 or AjS 42 .
  • the cleavage yield was calculated by comparing the amount of the cleavage product with the initially added amount of the AjS 40 or AjS 42 .
  • the cleavage yield measured by incubating A/3 4 o or AjS 42 (4.0 ⁇ M) with various concentrations of cleavage agent A at pH 7.50 and 37 0 C for 36 hours is illustrated in
  • the plateau value of the cleavage yield of the cleavage agent A obtained at high concentration of the cleavage agent is about 30%.
  • a ⁇ 42 oligomers exist as transient intermediates, the cleavage of an A ⁇ 42 oligomer by a cleavage agent competes with the polymerization reaction of the oligomer. Since cleavage of A ⁇ 42 with a cleavage agent is first order in the concentration of the oligomer, the half-life of the target oligomer due to cleavage is not affected by the concentration of the peptide as far as the concentration of the cleavage agent is fixed.
  • the polymerization reaction of the oligomer is at least second-order in peptide concentration, and the half life is increased by decreasing the concentration of peptide.
  • the total concentration of A ⁇ 42 is much lower than 1 nM in the brains of patients of Alzheimer's disease (Lue, L: R; Kuo, Y. M.; Roher, A. E.; Brachova, L.; Shen, Y.; Sue, L.; Beach, T.; Kurth, J. H.; Rydel, R. E.; Rogers, J. Am. J. Pathol. 1999, 155, 853-862).
  • cleavage reaction occurred at the concentration of 100 nM of the cleavage agent when the concentration of AjS 42 was 4.0 ⁇ M. Significant cleavage reaction would occur even at concentrations of the cleavage agent considerably lower than 100 nM when the concentrations OfAjS 42 are lowered to the in vivo level.
  • MALDI-TOF mass spectrum of the products purified with HPLC after incubating Am (4.0 ⁇ M) with cleavage agent A is illustrated in Figure 12.
  • MALDI-TOF mass spectra of cleavage products for Am were taken after purification with HPLC by the method described below.
  • Am is cleaved by cleavage agent A.
  • the cleavage products include Am 20-37 and Am I g -37 (the Am fragments are named according to the amino acid sequence of Am, and the structures of the cleavage products with the m/z values assigned to the peaks were confirmed with MALDI LIFT-TOF/TOF MS).
  • cleavage reaction of Am was initiated by adding Am to the solution containing the cleavage agent, and the cleavage yield was estimated through the following process.
  • the total amount of the cleavage product was quantified.
  • the cleavage product was converted to amino acids by alkaline hydrolysis, and the total amount of amino acids was estimated by using fluorescamine to determine the amount of the cleavage product.
  • the cleavage yield was calculated by comparing the amount of cleavage product with that of the initial amount of Am.
  • the cleavage yields measured after reacting Am (4.0 ⁇ M) with various concentrations of cleavage agent A at 37 0 C and pH 7.50 for 36 hours are illustrated in Figure 13.
  • the cleavage yields of Am in the Examples are the mean value measured by using 4-6 different reaction mixtures.
  • the relative standard deviation of each cleavage yield is 5-15%.
  • Syn cleavage was initiated by adding Syn to the solution of the cleavage agent.
  • the cleavage yield was calculated according to the following method.
  • a product solution formed by the cleavage reaction was passed through the membrane with the cut-off MW of 10000 to remove Syn and its assemblies. Then, the cleavage products were separated by HPLC, and the total amount of the cleavage products was estimated. The cleavage product was degraded to amino acids through alkaline hydrolysis. The total amount of the amino acids was then estimated with fluorescamine to quantify the total amount of the cleavage product. The cleavage yield was calculated by comparing the amount of the cleavage product with the initially added amount of Syn.
  • the cleavage yields measured by incubating Syn (70 ⁇ M) with various concentrations of cleavage agent A at pH 7.50 and 37 0 C for 3 days are illustrated in Figure 15.
  • the cleavage yields in the Examples are the mean value measured by using 4 ⁇ 6 different reaction solutions. Since the MW of Syn used in the Examples is about 15000, some of the protein fragments formed by the cleavage of Syn might have been too large to pass through the membrane with cut-off MW of 10000. Considering this possible cause for underestimation, the cleavage yields summarized in Figure 15 are fairly large.
  • AIAEGDSHVLKEGAYMEIFDVQGHVFGGKIFRVVDLGSHNVA (4.0 ⁇ M), were not cleaved by incubation with cleavage agent A (3.0 ⁇ M) at pH 7.50 and 37 0 C for 36 hours.
  • bovine pancreas insulin (each 2-7 ⁇ M) was incubated with cleavage agent A (5.0 ⁇ M) at pH 7.50 and 37 0 C for 36 hours, cleavage reaction was not detected.
  • Cleavage agent B was synthesized according to the pathway shown in Figure 16.
  • PS-Thiophenol resin purchased from Argonaut Technologies (50 mg, 0.074 mmol)
  • the resin of formula 2f was added to the mixture of a solution of m-CPBA (130 mg,
  • PS-DIEA resin purchased from Argonaut Technologies
  • the stock solution of cleavage agent B was obtained from 2i as described for cleavage agent A in Example 1. Activity test of cleavage agent B
  • Example 3 The control experiment, identical to that of Example 1, was carried out for cleavage agent B. The results of the control experiment were the same as those obtained in Example 1.
  • Example 3 The control experiment, identical to that of Example 1, was carried out for cleavage agent B. The results of the control experiment were the same as those obtained in Example 1.
  • Cleavage agent C was synthesized according to the pathway shown in Figure 25.
  • PS-DIEA resin purchased from Argonaut Technologies (19 mg, 0.075 mmol) and the compound of formula 3c were added (P. S. Chae, M. Kim, C. Jeung, S. D. Lee, H. Park, S. Lee, J.
  • the compound of formula 3d was treated with TFA as described above for the compound of Ih in Example 1 to obtain the TFA salt of 3-(4- ⁇ 2-[(4-benzothiazol-2-yl- phenyl)-methyl-amino]-ethoxy ⁇ -6-cyclododecylamino-[ 1 ,3,5]triazin-2-ylamino)-./V-[3- (l ⁇ lO-tetraaza-cyclododec-l-yFj-propylj-propionamide (3e).
  • the TFA salt of 3e was used for NMR and MS characterization
  • MALDI-TOF MS mass spectrum obtained by reacting A/3 42 (4.0 ⁇ M) with cleavage agent C is illustrated in Figure 26.
  • A/3 42 was cleaved by cleavage agent C and AjSi -2O was included in the cleavage product. Since the intensity of a MALDI-TOF MS peak does not stand for the relative concentration, some oligopeptide fragments may be present in significant concentrations without showing strong MALDI-TOF MS peaks.
  • the cleavage yield measured by incubating Aj3 40 or A/3 42 (4.0 ⁇ M) with various concentrations of cleavage agent C at pH 7.50 and 37 0 C for 36 hours is illustrated in Figure 27.
  • the plateau value of the yield for cleavage of A / 3 42 by cleavage agent C obtained at high concentration of the cleavage agent is about 12%.
  • concentration of A/3 42 was 4.0 ⁇ M
  • cleavage reaction was detected with 100 nM of cleavage agent C. If the concentration of A/? 42 is lowered to the level in a living human body, significant cleavage reaction would occur even at concentrations of cleavage agent C much lower than 100 nM, as explained in Example 1.
  • MALDI-TOF mass spectrum of the products purified with HPLC after incubating Am (4.0 /xM) with cleavage agent C is illustrated in Figure 28. As shown in Figure 28, Am was cleaved by cleavage agent C, and Am 17-37 was included in the cleavage products.
  • Example 1 The control experiment, identical to that of Example 1, was carried out for cleavage agent C. The results of the control experiment were the same as those obtained in Example 1.
  • Cleavage agent D was synthesized according to the pathway shown in Figure 30.
  • PS-DIEA resin purchased from Argonaut Technologies (19 mg, 0.075 mmol) and the compound of formula 3c (28 mg, 0.046 mmol) were added.
  • the reaction mixture was heated at 80 0 C for 8 hours.
  • the mixture was filtered and the resin was washed with MC (1 mL x 3).
  • the TFA salt of 4d was used for NMR and MS characterization.
  • the stock solution of cleavage agent D was obtained from the compound of formula 4d as described for cleavage agent A in Example 1.
  • MALDI-TOF MS mass spectrum obtained by reacting A ⁇ 42 (4.0 ⁇ M) with cleavage agent D is illustrated in Figure 31.
  • a / 3 42 was cleaved by cleavage agent D and AjSi -2 0 was included in the cleavage product. Since the intensity of a MALDI-TOF MS peak does not stand for the relative concentration, some oligopeptide fragments may be present in significant concentrations without showing strong MALDI-TOF MS peaks.
  • the cleavage yield measured by incubating AjS 40 or AjS 42 (4.0 ⁇ M) with various concentrations of cleavage agent D at pH 7.50 and 37 0 C for 36 hours is illustrated in Figure 32.
  • the plateau value of the yield for cleavage of AjS 42 by cleavage agent D obtained at high concentration of the cleavage agent is about 12%.
  • concentration of A ⁇ 42 was 4.0 ⁇ M
  • cleavage reaction was detected with 50-100 nM of cleavage agent D. If the concentration of Aj3 42 is lowered to the level in a living human body, significant cleavage reaction would occur even at concentrations of cleavage agent D much lower than 50-100 nM, as explained in Example 1.
  • Am (4.0 ⁇ M) with cleavage agent D is illustrated in Figure 33. As shown in Figure 33, Am was cleaved by cleavage agent D, and Am 20-37 and Ami 9 _ 37 were included in the cleavage products.
  • Example 1 The control experiment, identical to that of Example 1, was carried out for cleavage agent D. The results of the control experiment were the same as those obtained in Example 1.
  • Cleavage agent E was synthesized according to the pathway shown in Figure 35.
  • the compound of formula 5c (5 mg) was treated with TFA as described above in Example 1 for Ih to obtain the TFA salt of 6- ⁇ 2-[(4-benzothiazol-2-yl-phenyl)-methyl- amino]-ethoxy ⁇ -N-butyl-N '-[3-(I ,4,7,10-tetraaza-cyclododec- 1 -yl)-propyl]- [l,3,5]triazine-2,4-diamine (5d).
  • the TFA salt of 5d was used for NMR and MS characterization;
  • MALDI-TOF mass spectrum of the products purified with HPLC after incubating Am (4.0 ⁇ M) with cleavage agent E is illustrated in Figure 36. As shown in Figure 36, Am was cleaved by cleavage agent E, and Ami 7-37 , Am J6-37 and Am 13-37 were included in the cleavage products.
  • the stock solution of cleavage agent F was obtained from the compound of formula 6f as described for cleavage agent E in Example 5.
  • Am (4.0 ⁇ M) with cleavage agent F is illustrated in Figure 39. As shown in Figure 39, Am was cleaved by cleavage agent F, and Ami 9-37 and Am) 7-37 were included in the cleavage products.
  • Example 1 The control experiment, identical to that of Example 1, was carried out for cleavage agent F. The results of the control experiment were the same as those obtained in Example 1.
  • Cleavage agent G was synthesized according to the pathway shown in Figure 41.
  • the compound of formula 7c was treated with TFA as described above in Example 1 for Ih to obtain the TFA salt of 2-((S)-4- ⁇ 2-[(4-benzothiazol-2-yl-phenyl)-methyl- aminoj-ethoxy ⁇ -6-cyclododecylamino-[ 1 ,3,5]triazine-2-ylamino)-4-methyl-pentanoic acid [3-(l,4,7,10-tetraaza-cyclodode-l-sil)-propyl]-amide (7d).
  • the TFA salt of the compound of formula 7d was used for NMR and MS characterization. 1H NMR (MeOD): 6 7.90 (q, 4H), 7.47 (t, IH), 7.35 (t, IH), 6.81 (d, 2H), 4.80-
  • the stock solution of cleavage agent G was obtained from the compound of formula 7d as described for cleavage agent E in Example 5.
  • Example 1 The control experiment, identical to that of Example 1, was carried out for cleavage agent G. The results of the control experiment were the same as those obtained in Example 1.
  • Cleavage agent H was synthesized according to the pathway shown in Figure 44.
  • PS-DIEA resin (30 mg, 0.12 mmol) and the compound of formula 8b (51 mg, 0.074 mmol) were added.
  • the reaction mixture was heated at 80 0 C for 8 hours.
  • the mixture was filtered and the resin was washed with MC (1 mL x 3).
  • the compound of formula 8e was treated with TFA as described above in Example 1 for Ih to obtain the TFA salt of 2-(4- ⁇ (S)-2-[(4-benzothiazol-2-yl-phenyl)- methyl-amino]-ethoxy ⁇ -6-dicyclohexylamino-[l,3,5]triazine-2-ylamino)-3-(4-hydroxy- phenyl)- ⁇ V-[3-(l,4,7,10-tetraaza-cyclodode-l-sil)-propyl]-propionamide (8f).
  • the TFA salt of the compound of formula 8f was used for NMR and MS characterization
  • the stock solution of cleavage agent H was obtained from the compound of formula 8f as described for cleavage agent E in Example 5.
  • Example 1 The control experiment, identical to that of Example 1, was carried out for cleavage agent H. The results of the control experiment were the same as those obtained in Example 1.

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Abstract

La présente invention concerne un agent de clivage ainsi qu'un procédé de clivage agissant sélectivement sur un assemblage soluble de peptide ou protéine amyloïdogénique.
PCT/KR2007/005247 2006-10-24 2007-10-24 Agent de clivage agissant sélectivement sur un assemblage soluble de peptide ou protéine amyloïdogénique WO2008051017A1 (fr)

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US8877190B2 (en) 2006-11-30 2014-11-04 Abbvie Inc. Aβ conformer selective anti-Aβ globulomer monoclonal antibodies
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CN107739369A (zh) * 2017-09-11 2018-02-27 陕西莱特光电材料股份有限公司 一种三嗪类衍生物及其制备方法

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