WO2008051017A1 - A cleavage agent selectively acting on soluble assembly of amyloidogenic peptide or protein - Google Patents

A cleavage agent selectively acting on soluble assembly of amyloidogenic peptide or protein Download PDF

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Publication number
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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French (fr)
Inventor
Jung Hun Suh
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Seoul National University Industry Foundation
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Priority claimed from KR1020070075809A external-priority patent/KR20080036916A/en
Application filed by Seoul National University Industry Foundation filed Critical Seoul National University Industry Foundation
Priority to US12/446,905 priority Critical patent/US20100036122A1/en
Priority to JP2009534493A priority patent/JP2010507652A/en
Publication of WO2008051017A1 publication Critical patent/WO2008051017A1/en

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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

The present invention relates to a cleavage agent and a cleavage method selectively acting on soluble assembly of amyloidogenic peptide or protein.

Description

A CLEAVAGE AGENT SELECTIVELY ACTING ON SOLUBLE ASSEMBLY OF AMYLOIDOGENIC PEPTIDE OR PROTEIN
[Technical Field]
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.
[Background Art]
Amyloidosis refers to a variety of conditions in which insoluble amyloid proteins
are abnormally deposited in organs and/or tissues, causing disease (Bittan, G.; Fradinger,
E. A.; Spring, S. M.; Teplow, D. B. Amyloid 2005, 12, 88). Various amyloidogenic
peptides or proteins to generate amyloidosis are known in the art (Kelly, J. F. Curr. Opin.
Struct. Biol. 1996, 6, 11). The amyloids formed from various amyloidogenic peptides
or proteins maintain their intrinsic structure and function, and the amyloids have in
common a fibrous form and cross beta-sheet structure.
Amyloidogenic peptides or proteins can form soluble assemblies including various
oligomers and protofibrils which are converted to insoluble fibrils. According to past
studies, it is believed that the soluble oligomer of amyloidogenic peptide or protein is the
cause 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, α-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).
In the present invention, 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,
81, 741).
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 γ-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 A1S4O or AjS42, respectively. The amino acid sequence of A/342 is shown in Figure 1. The amino acid sequence of AjS40 can be obtained by removing the two C-terminal amino acids from the amino acid sequence of AjS42. AjS42, the major component of amyloid plaque, is more prone to aggregation than AjS40.
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 AjS42. Formation of fibrous aggregation and neuritic plaques according to the increasement of producing AjS42 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.
Although Katzman et al have identified only a weak correlation between dementia and amyloid plaques in the Alzheimer's disease patients (Katzman, R.; Terry, R.; De Teresa, R.; Brown, T.; Davies, P.; FuId, P.; Renbing, X.; Peck, A. Ann. Neurol. 1988, 23, 138: Naslund, J.; Haroutunian, V.; Mohs, R.; Davis, K L.; Davies, P.; Greengard, P.; Buxbaum J. D. J. Am. Med. Ass. 2000, 283, 1571), the Amyloid Cascade Hypothesis has been supported. Soluble AjS42 has also been detected in the brain of Alzheimer's disease patients (Kuo, Y. M.; Emmerling, M. R.; Vigo-Pelfrey, C; Kasunic, T. C; Kirkpatrick, J. B.; Murdoch, G. H.; Ball, M. J.; Roher, A. E. J. Biol. Chem. 1996, 271, 4077). Lue et al reported that Alzheimer's disease is related with the amount of soluble AjS42 rather than the amount of amyloid plaques (Lue, L. F.; 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: McLean, C. A.; Cherny, R. A.; Fraser, F. W.; Fuller, S. J.; Smith, M. J.; Beyreuther, K.; Bush, A. L; Masters, C. L. Ann. Neurol. 1999, 46, 860). Thus, increasing attention has been turned towards soluble AjS42 and the above hypothesis has been revised.
According to the revised hypothesis (Hardy, J.; Selkoe, D. J. Science 2002, 297, 353), the reason why synaptic dysfunction occurs in the brain of Alzheimer's disease patients is not due to the insoluble amyloid fibril or A/3 monomer, but due to the soluble oligomer of A/3. Recent studies disclosed that the soluble oligomer of A/342; such as the dodecamer, plays a role as a neurotoxic intermediate in Alzheimer's disease (Tanzi, R. E. Nature Neurosci. 2005, 8, 977: Snyder, E. M.; Nong, Y.; Almeida, C. G.; Paul, P.; Moran, T.; Choi, E. Y.; Nairn, A. C; Salter, M. W.; Lombroso, P. J.; Gouras, G. K.; Greengard, P. Nature Neurosci. 2005, 8, 1051: Barghorn, S.; Nimmrich, V.; Striebinger, A.; Krantz, C; Keller, P.; Janson, B.; Bahr, M.; Schmidt, M.; Bitner, R. S.; Harlan, J.; Barlow, E.; Ebert, R.; Hillen. H. J. Neurochem. 2005, 95, 834: Lesne S.; Koh, M. T.; Kotilinek, L.; Kayed, R.; Glabe, C. G.; Yang, A.; Gallagher, M.; Ashe, K. H. Nature 2006, 440, 352).
The soluble assemblies, including several oligomers or protofibrils, are formed reversibly, or partially irreversibly during the assembly process of A/342, 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. T.; Kotilinek, L.; Kayed, R.; Glabe, C. G.; Yang, A.; Gallagher, M.; Ashe, K. H. Nature 2006, 440, 352). The variable oligomers of A1S42 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/342 from the brain can be a candidate for the development of a method for relieving the neurotoxicity caused by A/342. To achieve this, Hardy et al. suggested a method which inhibits the activity of either β- or γ-secretase to prevent production of A/3 from APP (Hardy, J.; Selkoe, D. J. Science 2002, 297, 353). It is also possible to inhibit the oligomerization of AjS by using AjS immune agents (Schenk, 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. Nature 1999, 400, 173: DeMattos, R. B.; Bales, K. R.; Cummins, D. J.; Dodart, J.-C; Paul, S. M.; Holtzman. D. M. Proc. Natl. Acad. U.S.A. 2001, 98, 8850). A1S oligomerization can be inhibited by using small molecules which have high affinity for A1S (Cohen, T.; Frydman-Marom, A.; Rechter, M.; Gazit, E. Biochemistry 2006, 45, 4727). The amount of oligomer of A/342 in the brain can be reduced by stimulating the AjS
degradation enzyme, such as endothelin converting enzyme, insulin-degrading enzyme
and neprilysin (Choi, D. S.; Wang, D.; Yu, G. Q.; Zhu, G.; Kharazia, V. N.; Paredes, J.
P.; Chang, W. S.; Deitchman, J. K.; Mucke, L.; Messing, R. O. Proc. Natl. Acad. Sci.
2006, 703, 8215).
As illustrated by the soluble oligomer of A/342 related to the Alzheimer's disease,
various strategies are being adopted for treating the different types of amyloidosis
(Dobson, C. M. Science 2004, 304, 1259). Examples of such strategies include methods
for stabilizing the amyloidogenic peptide or protein itself, inhibiting the enzyme activity
which produces amyloidogenic peptide or protein from the precursor, modulating the
synthesis process of amyloidogenic peptide or protein or the precursor, stimulating the
elimination of the amyloidogenic peptide or protein, inhibiting the formation of fibrous
plaques, preventing the accumulation of fibrous plaques precursor, and the like.
[Disclosure]
[Technical Problem]
The present inventors have discovered a new method of cleaving the soluble
assembly of the amyloidogenic peptide or protein to reduce the amount of the soluble
oligomer. 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.
[Brief Description of the Drawings]
Fig. 1 shows the amino acid sequence of A/542,
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 Aβ4o (O) or Aβ42 (•) (initial concentration: 4.0 μM) passing the membrane with cut-off molecular weight (MW) of 10000 after incubation at pH 7.50 and 370C 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 AjS40 (4.0 μM) with cleavage agent A (3.0 μM) of Example 1 at 37 °C and pH 7.50 for 36 hours,
Fig. 7 shows MALDI-TOF mass spectrum taken after incubation of AjS42 (4.0 μM) with cleavage agent A (1.0 μM) 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 AjS40 (o) or A/342 (•) (4.0 μM) 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 Aβ4o (gray bars) or Aβ42 (dark bars) (4.0 μM) on cleavage yield by cleavage agent A (3.0 μM) of Example 1 measured after reacting for 36 hours at 370C and pH 7.50,
Fig. 10 shows the plot of the cleavage yield against reaction time for cleavage of Ap40 (O) or Aβ42 (•) (4.0 μM) by cleavage agent A (3.0 μM) of Example 1 at 370C and pH 7.50,
Fig. 11 shows the fraction of Am (initial concentration: 4.0 μM) passing the membrane with cut-off MW of 10000 after incubation at pH 7.50 and 370C for various period of time,
Fig. 12 shows MALDI-TOF mass spectrum of the products purified with HPLC after incubating Am (4.0 μM) with cleavage agent A (3.2 μM) of Example 1 at 37 °C and pH 7.50 for 36 hours,
Fig. 13 shows the plot of the cleavage yield against log C0ZM for cleavage of Am (4.0 μM) by cleavage agent A of Example 1 measured after reacting for 36 hours at 370C and pH 7.50,
Fig. 14 shows the fraction of Syn (initial concentration: 70 μM) passing a 0.22 mm Millipore filter (Millipore Millex-GV 4MM) after self-assembly during incubation at pH 7.50 and 370C for various period of time, Fig. 15 shows the plot of the cleavage yield against log CJM for cleavage of Syn
(70 μM) by cleavage agent A of Example 1 measured after reacting for 3 days at 370C and pH 7.50,
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/34o (4.0 μM) with cleavage agent B (3.0 μM) 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/342 (4.0 μM) with cleavage agent B (0.50 μM) 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 Aβ40 (o) or A/342 (•) (4.0 μM) 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 μM) with cleavage agent B (1.0 μM) 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
(4.0 μM) by cleavage agent B of Example 2 measured after reacting for 36 hours at 370C and pH 7.50,
Fig. 22 shows effects of period of preincubation of Am (4.0 μM) on cleavage yield by cleavage agent B (1.0 μM) of Example 2 measured after reacting for 36 hours at 370C and pH 7.50,
Fig. 23 shows the plot of the cleavage yield against reaction time for cleavage of Am (4.0 μM) by cleavage agent B (1.0 μM) of Example 2 at 370C and pH 7.50,
Fig. 24 shows the plot of the cleavage yield against log C0IM for cleavage of Syn (70 μM) by cleavage agent B of Example 2 measured after reacting for 3 days at 370C 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 AjS42 (4.0 μM) with cleavage agent C (1.00 μM) of Example 3 at 37 °C and pH 7.50 for 36 hours. Fig. 27 shows the plot of cleavage yield against log C0IM for cleavage of A1S40 (o) or A/342 (•) (4.0 μM) 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 μM) with cleavage agent C (3.2 μM) of Example 3 at 37 °C and pH 7.50 for 36 hours,
Fig. 29 shows the plot of the cleavage yield against log C0IM. for cleavage of Am (4.0 μM) by cleavage agent C of Example 3 measured after reacting for 36 hours at 370C 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/342 (4.0 μM) with cleavage agent D (1.00 μM) of Example 4 at 37 °C and pH 7.50 for 36 hours,
Fig. 32 shows the plot of cleavage yield against log C0IM for cleavage of A(S40 (o) or A/342 (•) (4.0 μM) 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 μM) with cleavage agent D (0.38 μM) 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 μM) by cleavage agent D of Example 4 measured after reacting for 36 hours at 370C 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 μM) with cleavage agent E (0.89 μM) 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 μM) by cleavage agent E of Example 5 measured after reacting for 36 hours at 370C 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 μM) with cleavage agent F (1.6 μM) of Example 6 at 37 "C and pH 7.50 for 36 hours,
Fig. 40 shows the plot of the cleavage yield against log C0ZM for cleavage of Am (4.0 μM) by cleavage agent F of Example 6 measured after reacting for 36 hours at 370C 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 μM) with cleavage agent G (0.89 μM) of Example 7 at 37 "C and pH 7.50 for 36 hours,
Fig. 43 shows the plot of the cleavage yield against log Co/M for cleavage of Am (4.0 μM) by cleavage agent G of Example 7 measured after reacting for 36 hours at 370C 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 μM) with cleavage agent H (7.1 μM) 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 Co/M for cleavage of Am (4.0 μM) by cleavage agent H of Example 8 measured after reacting for 36 hours at 370C and pH 7.50.
[Technical Solution]
The present invention relates to cleavage agent of formula 1 which selectively cleaves the soluble assembly of amyloidogenic peptide or protein: [formula 1]
(R)n-(L)111-Z wherein,
R refers to target recognition site selected from the group consisting of A, A-(Y)0- (CH2)P-(Y)0-A, A-(CH=CH)-A, A-(Y)o-(CH2)p-(Y)o-A-(Y)o-(CH2)p-(Y)o-A and A-(Y)0- (CH2)p-(Y)o-A-(Y)o-(CH2)p-(Y)o-A-(O)o-(CH2)p-(Y)o-A,
A represents independently C6-i4aryl, 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 C1-15alkyl, hydroxy, C1-15alkoxy, Ci-15alkylcarbonyloxy, Ci_i5alkylsulfonyloxy, amino, mono or diCi. 15alkylamino, Ci-isalkylcarbonylamino, Ci.i5alkylsulfonylamino, C^iscycloalkylamino, formyl, Ci-i5alkylcarbonyl, carboxy, Ci-isalkyloxycarbonyl, carbamoyl, mono or diCj. i5alkylcarbamoyl, Ci-isalkylsulfanylcarbonyl, d-isalkylsulfanylthiocarbonyl, Ci- i5alkoxycarbonyloxy, carbamoyloxy, mono or diCi-isalkylcarbamoyloxy, Ci- i5alkylsulfanylcarbonyloxy, Ci-isalkoxycarbonylamino, ureido, mono or di or triCi. i5alkylureido, Ci-isalkylsulfanylcarbonylamino, mercapto, Ci_i5alkylsulfanyl, Ci- i5alkyldisulfanyl, sulfo, Ci-i5alkoxysulfonyl, sulfamoyl, mono or
Figure imgf000015_0001
triCi-i5alkylsilanyl and halogen; Y is O or N-Z, wherein Z represents hydrogen or Ci_9alkyl; 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. [Advantageous Effects]
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. Thus, the cleavage agents have the capacity to recognize the soluble assembly of amyloidogenic peptide or protein and the capacity for cleaving peptide bonds.
Accordingly, 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.
[Mode of Invention]
The cleavage agents according to the present invention are specifically indicated as follows. As explained above during the process of oligomerization and fibril formation of AjS42 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. A.; Nichols, M. R.; Reed, D. K.; Hoh, J. H.; Rosenberry, T. L. MoI. Pharmacol. 2003, 64, 1160: Hull, R.; Westermark, G. T.; Westermark, P.; Kahn, S. J. CHn. Endicrinol. Metab. 204, 89, 3629). Therefore, if one or more soluble assemblies of the amyloidogenic peptide or protein are eliminated from the various soluble assemblies, the total amount of soluble assembly of amyloidogenic peptide or protein is reduced, suppressing fibril formation.
The effective cleavage agent can be obtained by mimicking the principle of enzyme's catalytic activity. In enzymatic reactions, the substrate forms a complex with the enzyme, and the enzyme converts the complex ed substrate into the product. Through formation of the enzyme-substrate complex, 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, according to the present invention, 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.
As suggested for A/342, the generation process of various kinds of soluble assemblies and insoluble fibrils are shown as Figure 2 (Bitan, G.; Kirkitadze, M. D.;
Lomakin, A.; Vollers, S. S.; Benedek, G. B.; Teplow, D. B. Proc. Natl. Acad. Sci. USA 2003, 100, 330). In Figure 2, 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.
Res. 2006, 39, 671). As shown in Figure 2, reducing the concentration of one kind of assembly through cleavage can reduce the concentrations of the other assemblies which can easily be transformed into the assembly cleaved. 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. Through complex formation between the recognition site of the cleavage agent and the soluble assembly, 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.
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]
„ , cleavage product m "cat
(Aβ42)ass.m + (R) -<L)m-Z ======= (Aβ42)ass-rr,- (R)n-(L)m-Z - +
(R)π-(L)m-Z
As reported for various derivatives of Co111 complex, if the cleavage agent acts as a catalyst and hydrolyzes the peptide bonds, the cleavage agent will be generated after cleaving the target (Jeon, J. W.; Son, S. J.; Yoo, C. E.; Hong, I. S.; Song, J. B.; Suh, J. Org. Lett. 2002, 4, 4155: Chae, P. S.; Kim, M.; Jeung, C; Lee, S. D.; Park, H.; Lee, S.;
Suh, J. J. Am. Chem. Soc. 2005, 127, 2396).
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. When the 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. However, the amounts of the assemblies including protofibrils or fibrils which cannot be easily converted to the (AP)aSs-m are not effectively reduced. Instead, reduction of the concentration of (AP)ass-m slows down the formation of protofibrils or fibrils from
(Ar)ass-m.
Target Recognition Sites To be effective, 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.
The interaction among aromatic side chains of amyloidogenic peptide or protein has a central role in the assembly formation (Cohen, T.; Frydman-Marom, A.; Rechter, M.; Gazit, E. Biochemistry 2006, 45, 4727). Kayed et al. reported that the soluble oligomers of various kinds of amyloidogenic peptide or protein have a common conformation-dependent structure (Kayed, R.; Head, E.; Thompson, J. L.; Mclntire, T.
M.; Milton, S. C; Cotman, C. W.; Glabe, C. G. Science 2003, 300, 486). Therefore, the organic groups having high affinity to microdomains formed by aromatic side chains can be used as the target recognition sites of the present invention without any restriction.
Preferably, the target recognition sites according to the present invention should be selected from the group consisting of A, A-(Y)O-(CH2)P-(Y)O-A, A-(CH=CH)-A, A-(Y)0- (CH2)P-(Y)0-A-(Y)0-(CH2)P-(Y)0-A and A-(Y)0-(CH2)P-(Y)0-A-(Y)0-(CH2)P-(Y)0-A-(O)0- (CH2)P-(Y)0-A.
Wherein, A represents independently C6-i4aryl, or 5- to 14-membered heteroaryl having one or more hetero atom selected from the group consisting of oxygen, sulfur and nitrogen,
Preferably, A should be selected from the group consisting of the compounds of following formulas:
Figure imgf000021_0001
wherein, X is independently selected from the group consisting of C, N, NH, O and S.
More preferably, A should be selected from the group consisting of the compounds of following formulas:
Figure imgf000022_0001
wherein,
X is NH, O, or S.
Wherein, A is unsubstituted or substituted by one or more substituent(s) independently selected from the group consisting of Ci.isalkyl, hydroxy,
Figure imgf000022_0002
C1- i5alkylcarbonyloxy, C1-15alkylsulfonyloxy, amino, mono or diCi-i5alkylamino, Ci- i5alkylcarbonylamino, Ci-isalkylsulfonylamino, C3-i5cycloalkylamino, formyl, Ci- i5alkylcarbonyl, carboxy, Ci-isalkyloxycarbonyl, carbamoyl, mono or diCi. i5alkylcarbamoyl, Ci-i5alkylsulfanylcarbonyl, Ci-i5alkylsulfanylthiocarbonyl, C1-
I5 alkoxycarbonyloxy, carbamoyloxy, mono or diCi-i5alkylcarbamoyloxy, C1- i5alkylsulfanylcarbonyloxy, Ci-isalkoxycarbonylamino, ureido, mono or di or triCi- i5alkylureido, Ci-isalkylsulfanylcarbonylamino, mercapto, Ci_i5alkylsulfanyl, Ci- i5alkyldisulfanyl, sulfo, Ci_i5alkoxysulfonyl, sulfamoyl, mono or diCi-isalkylsulfamoyl, triC i -i 5alkylsilanyl and halogen.
Preferably, A is unsubstituted or substituted by the substituents selected from the group consisting of Ci-6alkyl, Ci-6alkoxy, amino, mono or diCi-i2alkylamino, C1- 6alkylcarbonylamino, Ci-βalkylsulfonylamino, Cό-iscycloalkylamino, Ci-βalkylcarbonyl. carbamoyl, mono or diC1-6alkylcarbamoyl, Ci-6alkoxycarbonylamino, ureido, mono or di or triCi-6alkylureido, d-ealkylsulfanylcarbonylamino, C1-6alkylsulfanyl, C1- 6alkyldisulfanyl, sulfamoyl, mono or diCi-6alkylsulfamoyl, triC1-6alkylsilanyl and halogen.
More preferably, A is unsubstituted or substituted by the substituents selected from the group consisting of C1-4alkyl, C1-4alkoxy, amino, mono or diCi-salkylamino, C6- 12cycloalkylamino, C1-4alkylcarbonyl and halogen. Wherein, Y is O or N-Z,
Z is Ci-galkyl, preferably C1-4alkyl.
Wherein, p is independently an integer from 0 to 5, preferably 0 to 2.
Wherein, 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).
Catalytic Site
Several metal ions display catalytic activity in the hydrolysis of peptide bonds without the help of the organic functional groups or other metal ions (Suh, J. Ace. Chem. Res. 1992, 25, 273: Suh, J. Ace. Chem. Res. 2003, 36, 562).
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. By combining the cleavage agent of the present invention with the soluble assembly of amyloidogenic peptide or protein via the target recognition site, the effective concentration between the catalytic site of the cleavage agent and the cleavage site of the target molecule is greatly increased. Therefore, the cleavage agent of the present invention can cleave the peptide bonds of the target molecules effectively. Several metal ions that display activity for cleavage of peptides or proteins have been reported. The metal ion according to the present invention to be used in catalytic sites should be preferably selected from the group consisting of Co111, Cu1, Cu11, CeIV, Cev, Cr111, Fe", Fe111, MoIV, Ni11, Pd", Pt11, Vv and ZrIV, more preferably Co111, Cu11 or Pd", and most preferably Co111. 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:
Figure imgf000025_0001
Wherein, 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 C6-i4aryl or 5- to 14-membered heteroaryl.
Preferably, the ligand to be used in the catalytic site is selected from the group consisting of the following formulas:
Figure imgf000026_0001
More preferably, the ligand is a cycle consisting of 12 atoms, and selected from the group consisting of the following formulas:
Figure imgf000026_0002
wherein, the ligand is unsubstituted or substituted with one or more substituent(s) independently selected from the group consisting of Ci-i5alkyl, hydroxy, Ci-i5alkoxy, C1. i5alkylcarbonyloxy, Ci-isalkylsulfonyloxy, amino, mono or diCi-isalkylamino, C1-
15 alkylcarbonylamino, Ci-i5alkylsulfonylamino, formyl, Ci-i5alkylcarbonyl, carboxy, C1-
15 alkyloxycarbonyl, carbamoyl, mono or diCi_i5alkylcarbamoyl, C1- i5alkylsulfanylcarbonyl, Ci-15alkylsulfanylthiocarbonyl, Ci-isalkoxycarbonyloxy, carbamoyloxy, mono or diCi.isalkylcarbamoyloxy, Ci-isalkylsulfanylcarbonyloxy, Ci- i5alkoxycarbonylamino, ureido, mono or di or triCi-isalkylureido, C1- 15alkylsulfanylcarbonylamino, mercapto, Ci_i5alkylsulfanyl, Ci-isalkyldisulfanyl, sulfo, C1-15alkoxysulfonyl, sulfamoyl, mono or diCi_i5alkylsulfamoyl, triCi-isalkylsilanyl and halogen.
Preferably, the ligand is unsubstituted or substituted with one or more substituent(s) selected from the group consisting of C1-6alkyl, Ci-6alkoxy, C1. 6alkylcarbonyloxy and halogen.
More preferably, the ligand is unsubstituted or substituted with one or more substituent(s) selected from the group consisting of Ci-4alkyl, C1-4alkoxy and halogen.
Linker
In the cleavage agent of the present invention, 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. Or, 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:
Figure imgf000028_0001
wherein, vvo represents linker,
R represents a target recognition site, and
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, C1- galkylcarbonyloxy, Q.galkylsulfonyloxy, amino, mono or diCi.galkylamino, Ci- 9alkylcarbonylamino, Ci.galkylsulfonylamino, formyl, Cj.galkylcarbonyl, carboxy, C1- galkyloxycarbonyl, carbamoyl, mono or diCi.galkylcarbamoyl, Ci.galkylsulfanylcarbonyl, Ci-galkylsulfanylthiocarbonyl, Ci-galkoxycarbonyloxy, carbamoyloxy, mono or (IiC1- galkylcarbamoyloxy, Ci.galkylsulfanylcarbonyloxy, Ci.galkoxycarbonylamino, ureido, mono or di
Figure imgf000029_0001
Ci-galkylsulfanylcarbonylamino, mercapto, Ci- 9alkylsulfanyl, Ci^alkyldisulfanyl, sulfo, Ci_c>alkoxysulfonyl, sulfamoyl, mono or diCi. 9alkylsulfamoyl, triCi-galkylsilanyl and halogen.
Preferably, the linker is unsubstituted or substituted with one or more substituent(s) independently selected from the group consisting of C1-6alkyl, Ci-6alkoxy, mono or diCi_ 6alkylamino, C1-6alkylcarbonylamino, C1-6alkylsulfonylamino, Ci-6alkylcarbonyl, carbamoyl, mono or diCi-6alkylcarbamoyl, Ci-6alkoxycarbonylamino, ureido, mono or di ortriCi-όalkylureido, Ci-όalkylsulfanylcarbonylamino, Ci-6alkylsulfanyl, Ci- 6alkyldisulfanyl, sulfamoyl, mono or diCi-6alkylsulfamoyl, triCi_6alkylsilanyl and halogen.
More preferably, the linker is unsubstituted or substituted with one or more substituent(s) independently selected from the group consisting of Ci-4alkyl, Ci-4alkoxy, mono or diCi-4alkylamino, Ci_4alkylcarbonylamino, Ci-4alkylsulfonylamino, Ci-
4alkylcarbonyl, carbamoyl, mono or diCi-4alkylcarbamoyl, Ci-4alkoxycarbonylamino, ureido, mono or di ortriCi_4alkylureido, Ci-4alkylsulfanylcarbonylamino, Ci-
4alkylsulfanyl, Ci-4alkyldisulfanyl, sulfamoyl, mono or diCi-4alkylsulfamoyl, triCi. 4alkylsilanyl and halogen.
Persons skilled in the relevant arts should be able to design a combinatorial chemical experiment to select the linker structure suitable for modulating or changing the effective concentration between catalytic site of the synthetic catalyst and the cleavage site of the target protein. 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. hi other words, 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.
(1) Oligomers OfAjS4O and AjS42 associated with Alzheimer's disease AjS4O and AjS42 form various oligomers, protofibrils, and fibrils by self-assembly as shown in Figure 2. The aggregation Of A1S42 is faster than that Of AjS40. Therefore, in cases where the concentration of A1S42 monomer is higher than several μM, A1S42 is oligomerized in a few minutes. It is then converted to protofibrils with sizes smaller than 0.1 μm in a solvent or on a solid surface in a few hours (Kowalewski, T.; Holtzman, D. M. Proc. Natl. Acad. Sci. USA. 1999, 96, 3688: Bitan, G.; Kirkitadze, M. D.; Lomakin, A.; Vollers, S. S.; Benedek, G. B.; Teplow, D. B. Proc. Natl. Acad. Sci. USA 2003, 100, 330). It is reported that A1S4O forms dimer, trimer, tetramer and the like in the equilibrium process, and A1S42 forms mostly pentamer and hexamer (Bitan, G; Kirkitadze, M. D.; Lomakin, A.; Vollers, S. S.; Benedek, G. B.; Teplow, D. B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 330-335). Recently, reports have shown that A/342 usually exists as a mixture of its monomer and large oligomer, and A1S4O as its monomer and dimer in equilibrium (Hepler, R. W.; Grimm, K. M.; Nahas, D. D.; Breese, R.; Dodson, E. C; Acton, P.; Keller, P. M.; Yeager, M.; Wang, H.; Shughrue, P.; Kinney, G; Joyce, J. G. Biochemistry 2006, 45, 15157-15167).
The aggregation process of A/?40 and A1S42 is sensitive to experimental conditions (Hepler, R. W.; Grimm, K. M.; Nahas, D. D.; Breese, R.; Dodson, E. C; Acton, P.; Keller, P. M.; Yeager, M.; Wang, H.; Shughrue, P.; Kinney, G; Joyce, J. G. Biochemistry 2006, 45, 15157-15167). At this stage, it is not clear what kinds of oligomers among the various kinds of oligomers formed by amyloidogenic peptide or protein are cleaved by the cleavage agent of the present invention. Nontheless, when the concentration of the target oligomer is reduced due to cleavage by the cleavage agent, the concentrations of other oligomers which are in equilibrium with the target oligomer also decrease. Accordingly, the amount of oligomer which is the cause of amyloidosis will also be reduced.
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 AjS40 as well as those of AjS42 as in Example 1. AJS40 are mainly generated by proteolytic cleavage of the /3-amyloid precursor proteins. AjS40 is responsible for various physiological functions, and therefore, if AjS40 is drastically cleaved, its normal functions would be inhibited. However, the amount of
AjS40 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,
853-862). Therefore, partial cleavage of A/340 during cleavage of A1S42 may not cause considerably side effects in Alzheimer's disease patients.
The antibody raised against A/342 oligomer can recognize the AjS4O oligomer as well as the A1S42 oligomer, and oligomers of other amyloidogenic proteins or peptides, such as α-synuclein, amylin, polyglutamine, lysozyme, insulin, prion peptide 106-126 (Kayed,
R.; Head, E.; Thompson, J. L.; Mclntire, T. M.; Milton, S. C; Cotman, C. W.; Glabe, C.
G. Science 2003, 300, 486-489). This was taken to indicate that different types of soluble amyloid oligomers have a common conformation-dependent structure, and has prompted the speculation that different types of amyloidosis may be blocked by one single drug.
Some 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. (2) Oligomer of amylin associated with type 2 diabetes mellitus
The oligomer of amylin (Am; human islet amyloid polypeptide) has been reported as one of the causes of type 2 diabetes mellitus (Janson, J.; Ashley, R. H.; Harrison, D.;
Mclntyre, S.; Butler, P. C. Diabetes 1999, 48, 491-498: Kayed, R.; Head, E.; Thompson,
J. L.; Mclntire, T. M.; Milton, S. C; Cotman, C. W.; Glabe, C. G. Science 2003, 300, 486-489: Kayed, R.; Sokolov, Y.; Edmonds, B.; Mclntire, T. M.; Milton, S. C; Hall, J.
E.; Glabe, C. G. J. Biol. Chem. 2004, 279, 46363-46366:Meier, J. J.; Kayed, R.; Lin, C-
Y.; Gurlo, T.; Haataja, L.; Jayasinghe, S.; Langen, R.; Glabe, C. G.; Butler, P. C. Am. J.
Endicrinol. Metab. 2006, 291, E1317-E1324: Ritzel, R. A.; Meier, J. J.; Lin, C-Y.;
Veldhuis, J. D.; Butler, P. C Diabetes 2007, 56, 65-71 : Lin, C Y.; Gurlo, T.; Kayed, R.; Butler, A. E.; Haataja, L.; Glabe, C. G.; Butler, P. C. Diabetes 2007, 56, 1324-1332). Am is a cyclic oligopeptide consisting of 37 amino acid residues, and is capable of forming amyloids by self-assembly.
As shown in the Examples, 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 α-synuclein associated with Parkinson's disease
The oligomer of α-synuclein has been reported as one of the causes of Parkinson's disease (Giasson, B. L; Murry, I. V. J.; Trojanowski, J. Q.; Lee, V. M. J. Biol. Chem. 2001, 276, 2380-2386: Vladimir N.; Nversky, N.; Li, J.; Fink, A. L. J. Biol. Chem. 2001, 276, 10737-10744). α-synuclein (Syn) is a protein consisting of 140 amino acids and is capable of forming amyloids by self-assembly.
Pharmaceutical Compositions
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.
How and when the 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, can be prepared through conventional methods in the art, by using suitable dispersing agents, humectants or suspension.
Water, Ringer's solution and isotonic NaCl solution can be used to prepare the above formulation, and 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.
[Examples]
Having given a general description of the invention, the same will be more readily understood by reference to the following examples which are provided by way of illustration and in no way are intended to limit the present invention.
Example 1
Cleavage agent A was synthesized through the pathway shown in the Figure 4.
Figure imgf000038_0001
Synthesis of compound of formula Ia
4-Aminomethyl-benzoic acid (1.0 g, 6.8 mmol) and 2-aminophenol (0.69 g, 7.4 mmol) were mixed together with polyphosphoric acid (10 g) and heated to 17O0C under N2 atmosphere for 1.5 hours. The reaction mixture was cooled to room temperature and poured into 10 % K2CO3 solution. The precipitate was filtered under reduced pressure. The precipitate was recrystallized from acetone-water followed by treatment with activated charcoal in THF-water to obtain 4-benzooxazol-2-yl-benzylamine (Ia).
R/ 0.65 (EtOAc/hexane 1 :2); 1H NMR (CDCl3): δ 8.20 (d, 2 H), 7.76 (dd, 1 H), 7.66 (dd, 1 H), 7.55 (d, 2 H), 7.41 (m, 2 H), 3.90 (s, 2 H); MS (MALDI-TOF) m/z 225.33 (M+H)+ calcd for Ci4H13N2Oi 225.09.
Synthesis of compound of formula Ic
Cyanuric chloride (Ib) (0.20 g, 1.1 mmol.), the compound of formula Ia (0.20 g 0.90 mmol), and diisopropylethylamine (DIEA) (0.38 mL, 2.7 mmol) were mixed together in THF (50 niL), and the mixture was stirred for 4 hours in an ice bath. The residue obtained by evaporation of the mixture was purified by column chromatography to obtain (4-benzooxazol-2-yl-benzyl)-(4,6-dichloro-[l,3,5]triazin-2-yl)-amine (Ic).
R/ 0.7 (EtOAc/hexane 1 :4); 1H NMR (CDCl3): δ 8.13 (d, 2 H), 7.70 (d, 1 H), 7.51 (d, 1 H), 7.40 (d, 2 H), 7.27 (d, 2 H), 4.58 (d, 2 H); 13C NMR (300 CDCl3): δ 171.26, 170.17, 166.04, 162.43, 150.72, 141.96, 139.84, 128.161, 128.09, 126.96, 125.34, 124.73, 120.06, 110.65, 76.60, 45.05; MS (MALDI-TOF) m/z 372.28 (M+H)+, calcd for C17H12Cl2N5O 372.03.
Synthesis of resins of formula Id and If
To a THF solution (1.5 mL) of the compound of formula Ic (55 mg, 0.15 mmol) were added PS-Thiophenol resin (purchased from Argonaut Technologies) (50 mg, 0.074 mmol) and DIEA (0.10 mL, 0.74 mmol). The mixture was heated at 650C and left overnight. After filtration, the resulting resin was washed with N5N- dimethylformamide (DMF), methylene chloride(MC), MeOH and MC, and dried under nitrogen gas to give a resin of formula Id.
To the suspension of the resin of formula Id in jV-methyl-2-pyrrolidinone (NMP; 1 mL) were added rø-butanol (1 mL) and butylamine (49 μL, 0.58 mmol), followed by DIEA (120 μL, 0.87 mmol). The mixture was heated at 12O 0C for 8 hours. After filtration, the resin was washed with DMF, MC, MeOH and MC, and then dried under nitrogen gas to give a resin of formula Ie.
To the resin of formula Ie was added the mixture of a solution of m- chlororoperoxybenzoic acid (m-CPBA; 130 mg, 0.74 mmol) in 1,4-dioxane (1.8 mL) and aqueous 1 N NaOH (160 /zL). The mixture was stirred for 4 hours at room temperature.
After filtration, the resulting resin was washed with 1 ,4-dioxane and MC, and then dried under nitrogen gas to give a resin of formula If.
Synthesis of compound of formula Ih To the suspension of the resin of formula If in acetonitrile (1.5 mL), PS-DIEA resin (purchased from Argonaut Technologies) (30 mg, 0.12 mmol) and compound of formula Ig (P. S. Chae, M. Kim, C. Jeung, S. D. Lee, H. Park, S. Lee, J. Suh, J. Am. Chem. Soc. 2005, 127, 2396-2397) (39 mg, 0.074 mmol) were added. The reaction mixture was heated at 800C for 8 hours. The mixture was filtered and the resin was washed with MC (1 mL x 3). The combined filtrate and washing were evaporated in vacuo and the residue was purified by column chromatography to obtain 10-{3-[4-(4- benzooxazol-2-yl-benzylamino)-6-butylamino-[l,3,5]triazin-2-ylamino]-propyl}- l,4,7,10tetraaza-cyclododecane-l,4,7-tricarboxylic acid tri-tert-butyl ester (Ih). Rf 0.5 (EA only); 1H NMR (CDCl3): δ 8.13 (d, 2H), 7.70 (d, IH), 7.51 (d, IH), 7.40 (d, 2H), 7.27 (d, 2H), 4.58 (d, 2H), 3.8-3.2(br, 14H), 2.5 (br, 6H), 2.1 (br, 2H), 1.63 (br, 2H), 1.5-1.3 (m, 31H), 0.9-0.8 (dd 3H); 13C NMR (CDCl3): δ 164.89, 161.91, 154.33, 149.67, 142.56, 141.06, 126.72, 124.82, 124.01, 123.53, 118.89, 109.53, 78.51, 78.31, 75.58, 53.92, 52.91, 48.92, 48.08, 43.31, 39.35, 37.97, 30.86, 28.67, 27.65, 19.03, 13.10; MS (MALDI-TOF) m/z 902.99 (M+H)+, calcd for C47H72NnO7 902.55.
The synthesis of triazine derivatives by using resin in the Examples is according to the reference (Khersonsky, S. M.; Chang, Y. T. J. Comb. Chem. 2004, 6, 474) unless specifically cited in the specification.
Synthesis of compound of formula Ii and cleavage agent A The compound of formula Ih (5 mg) was treated with 50 % trifluoroacetic acid (TFA) in MC (50 μL) for 5 hours and diethyl ether (1 mL) was added to the mixture. The precipitate was separated by centrifugation, washed with diethyl ether several times, and dried under nitrogen gas to obtain the TFA salt of ./V-^-benzooxazol^-yl-benzyrj-iV- butyl-Λ^^MMJJOtetraaza-cyclododec-l-y^-propylHlAS^riazine^Aό-tri^ (Ii). The TFA salt of the compound of formula Ii was used for NMR and MS characterization; 1H NMR (CDCl3): δ 8.17 (d, 2 H), 7.73 (d, 1 H), 7.55 (d, IH), 7.43 (d, 2H), 7.33 (d, 2H), 4.42 (br, 2H), 3.4-3.0 (br, 14H), 2.85 (br, 6H), 2.62 (br, 2H), 1.68 (br, 2H), 1.36- 1.23 (m, 4H), 0.9-0.8 (dd 3H); 13C NMR (CDCl3): δ 164.89, 162.67, 150.62, 141.76, 127.80, 126.12, 125.30, 124.73, 119.83, 110.65, 76.60, 51, 48.90, 44.87, 42.80, 42.32, 38.07, 30.89, 29.70, 23.71, 19.83, 14.07; MS (MALDI-TOF) MS (MALDI-TOF) m/z 602.73. (M+H)+, calcd for C32H48NnO 602.40; HRMS m/z 602.4043. (M+H)+, calcd for C32H48NnO 602.4038.
To the solution obtained by dissolving the TFA salt of the compound of formula Ii in methanol in a concentration of about 5 mg/50 μL, 5-7 equivalents of LiOH, followed by an equivalent amount of CoCl2-H2O were added according to the reference (Kim, M. G.; Kim, M.-s.; Lee, S. D.; Suh, J. J. Inorg. Biol. Chem. 2006, 11. 867) to prepare the corresponding Co11 complex. The complex was stirred for 1 day in the air to oxidize the Co11 complex to the Co111 complex.
Oxidation of Co11 to Co111 was accompanied by appearance of deep violet color. The Co111 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.
Activity test of cleavage agent A (1) Cleavage of oligomers of A/34o and A/342 associated with Alzheimer's disease
The activity of 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.
To collect quantitative information regarding decreases in the amounts of monomer and small oligomers of A/34o and A/342, the following filtration experiment was conducted.
To generate the monomeric form of A/340 or Aβ42 in the early reaction stage, A/34o or A/342 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). The results for measurement of the fraction of Aβ40 (O) or Aβ42 (•) (initial concentration: 4.0 μM) passing the membrane with cut-off MW of 10000 after incubation at pH 7.50 and 370C for various periods of self-assembly are illustrated in Figure 5. According to the results, most (>80 %) of Aβ42 (MW 4514) passes the filter immediately after exposure to the pH 7.50 medium. During the filtration through the membrane which takes about 10 minutes, large oligomers which cannot pass the membrane may have been generated. Therefore, it appears that most of the Aβ42 passed the membrane.
It is well known in the art that the dimer and trimer of A/342 are produced in much lower concentrations than the monomer (Bitan, G.; Kirkitadze, M. D.; Lomakin, A.; Vollers, S. S.; Benedek, G. B.; Teplow, D. B. Proc. Natl Acad. ScL USA 2003, 100, 330; Hepler, R. W.; Grimm, K. M.; Nahas, D. D.; Breese, R.; Dodson, E. C; Acton, P.; Keller, P. M.; Yeager, M.; Wang, H.; Shughrue, P.; Kinney, G; Joyce, J. G. Biochemistry 2006, 45, 15157-15167), and thus AjS42 exists mostly as the monomer in the early reaction stage. After 3 or 36 hours, 2/3 or 90 %, respectively, of AjS42 is converted to large assemblies which cannot pass the membrane. However, in case of AjS40, more than 90 % of the AjS40 passed the membrane in the early reaction state, and after 24 hours, 50 % Of AjS40 passed the membrane.
The MALDI-TOF mass spectrum obtained by reacting AjS40 or A/342 (4.0 μM) with cleavage agent A are illustrated in Figure 6 or Figure 7, respectively. As shown in these Figures, AjS40 and AjS42 are cleaved by cleavage agent A. AjS1-20 and AjSi-21 were included in the cleavage products (in the Examples herein, Aβ fragments are named according to the amino acid sequence of Aβ42, 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.
Unless specifically described in the Examples, cleavage reaction was initiated by adding A/34o or AjS42 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 AjS40 or AjS42.
Thereafter, 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 by alkaline hydrolysis, and then, the total amount of amino acids was estimated with fluorescamine to determine 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 the AjS40 or AjS42. The cleavage yield measured by incubating A/34o or AjS42 (4.0 μM) with various concentrations of cleavage agent A at pH 7.50 and 370C for 36 hours is illustrated in
Figure 8. The cleavage yields of A/34O or A/342 in the Examples are the mean value measured by using 4-6 different reaction mixtures. The relative standard deviation
(%RSD) of each cleavage yield is 5-15%. Aβ4o or Aβ42 was incubated in the buffer solution for various periods of time for self-assembly before reacting with cleavage agent A. The cleavage yield was again measured and the results are summarized in Figure 9.
To obtain information on the progress of the cleavage reaction, the cleavage yield was measured by reacting Aβ4o or Aβ42 with cleavage agent A for various period of time at 370C and pH 7.50 and the results are summarized in Figure 10.
According to the results of Figure 9, when cleavage agent A was added to the reaction mixture after preincubation of Aβ42 for 24 hours, little cleavage was observed apparently due to polymerization of Aβ42 leading to formation of protofibrils or fibrils. When cleavage agent A was added to the reaction mixture after preincubation of Aβ42 for
3-6 hours, the cleavage yields were not much smaller than that obtained with cleavage agent A added initially without preincubation of Aβ42. This stands in contrast with the results of Figure 5, which show considerable reduction of the amount of Aβ42 monomer during the initial 3-6 hour period. These results indicated that the oligomer is cleaved by the cleavage agent A and not the A/342 monomer, protofibril, or fibril.
Addition of cleavage agent A after preincubation of Aβ40 for 24 hours leads to considerable peptide cleavage and the preincubation for longer periods reduces the cleavage yield. This is consistent with the slower formation of protofibrils and fibrils by Aβ4o compared with Aβ42. In addition, it reveals that the protofibrils or fibrils of Aβ40 are not cleaved by cleavage agent A. Since the yield for cleavage of Aβ40 by cleavage agent A does not decrease considerably by preincubation for 3-18 hours, the monomer of Aβ4o is not the main source of the fragments in view of results of the filtration experiment.
The plateau value of the cleavage yield of the cleavage agent A obtained at high concentration of the cleavage agent is about 30%. As Aβ42 oligomers exist as transient intermediates, the cleavage of an Aβ42 oligomer by a cleavage agent competes with the polymerization reaction of the oligomer. Since cleavage of Aβ42 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 Aβ42 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).
In the Example, cleavage reaction occurred at the concentration of 100 nM of the cleavage agent when the concentration of AjS42 was 4.0 μM. Significant cleavage reaction would occur even at concentrations of the cleavage agent considerably lower than 100 nM when the concentrations OfAjS42 are lowered to the in vivo level.
(2) Cleavage of oligomers of amylin associated with type 2 diabetes mellitus The fractions of Am passing the membrane with cut-off MW of 10000 after incubation at pH 7.50 and 370C for various period of time are illustrated in Figure 11. Am monomer and small oligomers such as dimer or trimer can pass the above membrane. The amount of Am passing the filter is reduced to half or less in a few hours.
MALDI-TOF mass spectrum of the products purified with HPLC after incubating Am (4.0 μM) with cleavage agent A is illustrated in Figure 12. In this Example and hereafter, MALDI-TOF mass spectra of cleavage products for Am were taken after purification with HPLC by the method described below.
As shown in Figure 12, Am is cleaved by cleavage agent A. The cleavage products include Am20-37 and AmIg-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).
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.
Unless specifically described in the Examples, 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 product solution obtained from the cleavage reaction was passed through the membrane (cut-off MW = 10000) to remove Am aggregates, and then the cleavage product was separated by HPLC. 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 μM) with various concentrations of cleavage agent A at 370C 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%. (3) Cleavage of oligomer of α-synuclein associated with Parkinson's disease
In the Examples, the slightly modified derivatives of α-synuclein (Syn) were used as a substrate to obtain α-synuclein (Syn) by gene recombination. To facilitate purification by the nickel chelate method, histidine tag (LEHHHHHH) was adhered to C- terminus. To avoid interference in the transcription, leucine instead of methionine was incorporated as the 5th amino acid residue.
To obtain information on rates for formation of large assemblies of Syn by self- assembly, the amounts of Syn passing a 0.22 mm Millipore filter after self-assembly during incubation at pH 7.50 and 370C for various period of time were measured and the results are summarized in Figure 14. The results indicated that a significant amount of Syn was absorbed on the reactant container or formed protofibrils or fibrils that could not pass through the filter within a few days.
Unless the context clearly indicates otherwise, 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 μM) with various concentrations of cleavage agent A at pH 7.50 and 370C 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.
(4) Reaction with control peptides or proteins The following control experiment was performed on cleavage agent A. The peptides or proteins used in the control experiment are not amyloidogenic. This control experiment was carried out to confirm that cleavage agent A did not cleave such peptides or proteins under the conditions of the Example. Two kinds of scrambled Aβ42, having the same 42 amino acids as Aβ42 in a scrambled sequence (KVKGLIDGAHIGDLVYEFMDSNSAIFREGVGAGHVHVAQVEF,
AIAEGDSHVLKEGAYMEIFDVQGHVFGGKIFRVVDLGSHNVA) (4.0 μM), were not cleaved by incubation with cleavage agent A (3.0 μM) at pH 7.50 and 370C for 36 hours.
When horse heart myoglobin, bovine serum γ-globulin, bovine serum albumin, human serum albumin, egg white lysozyme, egg white ovalbumin, or bovine pancreas insulin (each 2-7 μM) was incubated with cleavage agent A (5.0 μM) at pH 7.50 and 370C for 36 hours, cleavage reaction was not detected.
In order to check whether the activity of cleavage agent A to cleave oligomers of
Aj84o, Aβ42, Am, and Syn is lost when the recognition site of cleavage agent A is removed, the Co1" complex of cyclen (20 μM) was incubated with A/540 (4.0 μM), A/342 (4.0 μM), Am (4.0 μM), or Syn (70 μM) at pH 7.50 and 370C for 36 hours. No peptide cleavage was observed.
Example 2
Cleavage agent B was synthesized according to the pathway shown in Figure 16.
Figure imgf000052_0001
Synthesis of compound of formula 2a
A mixture of 2-aminothiophenol (1.3 g, 10 mmol) and TV-methyl -7V-(2- hydroxyethyl)-4-aminobenzaldehyde (1.8 g, 10 mmol) in dimethyl sulfoxide (10 rnL) was heated to 17O 0C for 1.5 hours. After cooling to room temperature, the reaction mixture was poured into water. The resulting mixture was extracted with ethyl acetate (EA) (50 mL x 2). The combined organic layers were dried over Na2SO4. The residue obtained by evaporation of the solvent was recrystallized from acetonitrile to afford 2- [(4-benzothiazol-2-yl-phenyl)-methyl-amino]-ethanol (2a) as a yellow solid.
Rf 0.20 (EA/hexane 1 :1); 1U NMR (CDCl3): δ 7.97 (d, 1 H), 7.95 (d, 2 H), 7.85 (d, 1 H), 7.45 (t, 1 H), 7.32 (t, 1 H), 6.81 (d, 2 H), 3.88 (t, 2 H), 3.60 (t, 2 H), 1.80 (br s, 3 H); 13C NMR (CDCl3): δ 154.09, 151.61, 134.38, 129.03, 126.10, 124.34, 122.23, 121.67, 111.96, 77.02, 60.19, 54.66, 39.03; MS (MALDI-TOF) 285.35 m/z (M+H)+ calcd for Ci6H17NOS 285.10.
Synthesis of compounds of formulas 2b and 2c
To the stirred solution of the compound of formula Ig (2.9 g, 5.5 mmol) in acetonitrile (100 mL), iV-α-Cbz-L-alanine (1.2 g, 5.5 mmol) and DIEA (2.9 mL, 17 mmol) were added. To the reaction mixture 2-(lH-benzotriazol-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate (HBTU; 2.1 g, 5.5 mmol) was added and the mixture was stirred for 1 hour. The residue obtained by evaporation of the solution was dissolved in EA (100 mL). The EA solution was washed with 5% aq. citric acid (50 mL), 5% aq. Na2CO3 (50 mL), and brine (50 mL), and dried over Na2SO4. The solvent was evaporated off and column chromatography afforded 10-[(S)-3-(2- benzyloxycarbonylamino-propionylamino)-propyl]- 1 ,4,7, 10-tetraaza-cyclododecane-
1,4,7-tricarboxylic acid tή-tert-buty\ ester (2b) as a colorless oil.
Rf 0.2 (EA/hexane 1 : 1). 1H NMR (CDCl3): δ 7.30 (s, 5H), 5.02 (s, 2), 3.50-3.10 (br, 15H) 2.60-2.30 (br, 6H), 1.57-1.49 (br, 2H), 1.39-1.36 (m, 27H), 1.18 (s, 3H); 13C NMR (CDCl3): δ 171.58, 154.82, 154.79, 154.22, 135.42, 127.55, 78.49, 65.71, 53.42, 49.56, 48.92, 47.57, 47.18, 46.53, 45.25, 37.68, 29.92, 27.64, 18.32; MS (MALDI-TOF) m/z 735.88 (M+H)+ calcd for C37H63N6O9735.46. A suspension of the compound of formula 2b (2.Og, 2.7 mmol) and 1.0 g of 10 %
Pd/C in 80 raL of EtOH was stirred under 1 atm of H2 for 24 hours. The catalyst was filtered off on Celite, and the solvent was evaporated off to afford \0-[(S)-3-(2- benzyloxycarbonylamino-propionylamino)-propyl] - 1 ,4,7, 1 O-tetraaza-cyclododecane- 1,4,7-tricarboxylic acid tri-tert-butyl ester (2c) as a solid. 1H NMR (CDCl3): δ 7.49 (s, IH), 3.63-3.18 (br, 15H), 2.72-2.52 (br, 6H), 1.88-
1.75 (br, 2H), 1.73-1.60 (m, 2H), 1.50-1.40 (m, 27H), 1.40-1.30 (d, 3H); 13C NMR (CDCl3): δ 171.98, 155.03, 154.69, 154.25, 78.57, 78.42, 78.28, 53.60, 52.95, 49.72, 49.01, 47.80, 47.07, 46.54, 46.18, 37.56, 29.92, 27.64, 22.99; MS (MALDI-TOF) 601.58 m/z (M+H)+ calcd for C29H57N6O7601.42.
Synthesis of compound of formula 2d
The compound of formula Ib (0.20 g 1.1 mmol), the compound of formula 2a (0.20 g 0.70 mmol) and DIEA (0.38 mL, 2.7 mmol) were mixed together in THF (50 mL) and the mixture was stirred for 8 hours at room temperature. The residue obtained by evaporation of the solvent was purified by column chromatography to obtain (4- benzothiazol-2-yl-phenyl)-[2-(4,6-dichloro-[l,3,5]triazin-2-yloxy)-ethyl]-methyl-amine
(2d).
Rf 0.7 (EA/hexane 1 :4); 1H NMR (CDCl3): δ 7.97 (t, 3H), 7.85 (d, IH), 7.45 (t, IH), 7.36 (t, IH), 6.78 (d, 2H), 4.67 (t, 2H), 3.84 (t, 2H), 3.12 (br, 3H); 13C NMR
(CDCl3): δ 172.53, 171.84, 168.15, 155.13, 151.39, 135.02, 129.21, 126.27, 124.59,
122.66, 122.46, 121.74, 112.14, 67.78, 50.58, 38.89; MS (MALDI-TOF) m/z 432.29
(M+H)+calcd for Ci9H16Cl2N5OS 432.04.
Synthesis of resins of formula 2e and 2g
To a THF solution (1.5 mL) of the compound of formula 2d (62 mg, 0.15 mmol),
PS-Thiophenol resin (purchased from Argonaut Technologies) (50 mg, 0.074 mmol) and
DIEA (0.10 mL 0.74 mmol) were added. The mixture was heated at 650C overnight.
After filtration, the resulting resin (2e) was washed with DMF, MC, MeOH, and MC (each 3 mL x 3) and dried under nitrogen gas.
To a suspension of the resin of formula 2e in NMP (1 mL), w-butanol (1 mL) and A- chlorobenzylamine (71 μL, 0.58 mmol) were added followed by DIEA (120 μL, 0.87mmol). The mixture was heated at 120 0C for 8 hours. After filtration, the resulting resin (2f) was washed with DMF, MC, MeOH, and MC (each 3 mL x 3) and dried under nitrogen gas.
The resin of formula 2f was added to the mixture of a solution of m-CPBA (130 mg,
0.74 mmol) in 1,4-dioxane (1.8 mL) and aqueous 1 N NaOH (160 μ,L). The mixture was shaken for 4 hours at room temperature. After filtration, the resulting resin (2g) was washed with 1,4-dioxane and MC (each 3 mL x 3) and was dried under nitrogen gas.
Synthesis of compounds of formulas 2h and 2i and cleavage agent B
To the suspension of the resin of formula 2g in acetonitrile (1.5 mL), PS-DIEA resin (purchased from Argonaut Technologies) (30 mg, 0.12 mmol) and the compound of formula 2c (39 mg, 0.074 mmol) were added. The reaction tube was heated at 800C for
8 hours. The mixture was filtered and the resin was washed with MC (1 mL x 3). The combined filtrate and washing were evaporated in vacuo and the resulting residue was purified by column chromatography to obtain 10-(3-{2-[4-{2-[(4-benzothiazol-2-yl- phenyl)-methyl-amino]-ethoxy} -6-(4-chloro-benzylamino)-[ 1 ,3,5]triazin-2-ylamino]-(S)- propionylamino} -propyl)- 1 ,4,7, 1 Otetraaza-cyclododecane- 1 ,4,7-tricarboxylic acid tri- tert-butyl ester (2h).
Rf 0.2 (EA only); 1B NMR (CDCl3): δ 8.13 (d, 2 H), 7.70 (d, 1 H), 7.51 (d, 1 H), 7.40 (d, 2 H), 7.27 (d, 2 H), 4.58 (d, 2 H), 3.8-3.2 (br, 12H), 2.5 (br, 6H), 2.1 (br, 2H), 1.63 (br, 4H), 1.5-1.3 (m, 31H), 0.9-0.8 (dd 3H); 13C NMR (CDCl3): δ 164.89, 161.91, 154.33, 149.67, 142.56, 141.06, 126.72, 124.82, 124.01, 123.53, 118.89, 109.53, 78.51, 78.31, 75.58, 53.92, 52.91, 48.92, 48.08, 43.31, 39.35, 37.97, 30.86, 28.67, 27.65, 19.03, 13.10; MS (MALDI-TOF) m/z 1102.59 (M+H)+, calcd for C55H78ClN12O8S 1102.54. The compound of formula 2h was treated with TFA as described above in Example
1 for Ih to obtain the TFA salt of 2-[4-{2-[(4-benzothiazol-2-yl-phenyl)-methyl-amino]- ethoxy} -6-(4-chloro-benzylamino)-[ 1 ,3,5]triazin-2-ylamino]-iV-[3-(l ,4,7, 10-tetraaza- cyclododec-l-yl)-propyl]-(S)-propionamide (2i). The TFA salt of 2i was used for NMR and MS characterization. 1H NMR (CDCl3): δ 7.95 (s, IH), 7.92-7.85 (q, 3H), 7.45 (t, IH), 7.37-7.28 (m,
5H), 6.79 (t, 2H), 4.70-4.40 (br, 2H), 3.78 (br, 2H), 3.13-2.70 (br, 15H), 2.74 (s, 3H), 2.58-2.44 (br, 6H), 1.93 (m, IH), 1.66-1.55(br, 2H), 1.44-1.19 (m, 5H); 13C NMR (CDCl3): δ 173.57, 169.69, 168.84, 163.26, 162.01, 155.42, 152.65, 138.23, 135.63, 132.63, 130.76, 130.52, 130.07, 127.63, 125.90, 123.26, 123.08, 122.51, 113.29, 69.08, 67.48, 52.18, 51.49, 50.44, 45.22, 43.73, 43.18, 39.98, 31.51, 24.91; MS (MALDI-TOF) m/z 801.57 (M+H)+, calcd for C40H54ClNi2O2S 801.39; HRMS m/z 801.3907. (M+H)+, calcd for C40H54ClNi2O2S 801.3896.
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
(1) Cleavage of oligomers of Aβ4o and A/342 associated with Alzheimer's disease The MALDI-TOF mass spectrum obtained by reacting A/340 or AjS42 (4.0 μM) with cleavage agent B are illustrated in Figure 17 or Figure 18, respectively. As shown by the Figures, AjS40 and Aj342 were cleaved by using cleavage agent B, and AjS1 -2o and AjS1, 21were included in the cleavage products.
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 AjS4O or AjS42 (4.0 μM) with various concentrations of cleavage agent B at pH 7.50 and 370C for 36 hours is illustrated in
Figure 19. The plateau value of the yield for cleavage of A/340 and A/342 by cleavage agent B obtained at high concentration of the cleavage agent is about 12% and 17%, respectively.
When the concentration Of AjS42 was 4.0 μM, cleavage reaction was detected with 30-50 nM of cleavage agent B. If the concentration of Aj342 is lowered to the level in a living human body, significant cleavage reaction would occur even at the concentration of cleavage agent B much lower than 30-50 nM, as explained in Example 1. (2) Cleavage of oligomers of amylin associated with type 2 diabetes mellitus MALDI-TOF mass spectrum of the products purified with HPLC after incubating Am (4.0 μM) with cleavage agent B is illustrated in Figure 20. The spectrum shows that Am was cleaved by cleavage agent B and, Am20-3?, Am19-37 and AnIi7-37 were included in the cleavage products.
The cleavage yields measured after reacting Am (4.0 μM) with various concentrations of cleavage agent B at 370C and pH 7.50 for 36 hours are illustrated in Figure, 21. Cleavage yields measured after preincubation of Am (4.0 μM) over various periods before reacting with cleavage agent B (1.0 μM) are illustrated in Figure 22.
When cleavage agent B was added to the reaction mixture after preincubation of Am for 36 hours or longer, little cleavage reaction occurred apparently due to polymerization of Am leading to formation of protofibrils or fibrils.
When cleavage agent B was added to the reaction mixture after preincubation of Am for 6 hours, the amounts of products formed by cleavage of Am were not much smaller than that obtained with B added initially without preincubation of Am. This stands in contrast with the considerable reduction of the amount of the monomer and small oligomers of Am during the initial 6 hour period shown in Figure 11. These results indicate that the oligomers of Am instead of the monomer, protofibrils, and fibrils of Am are cleaved by cleavage agent B.
To examine the progress of the cleavage reaction, the cleavage yields measured by reacting Am with cleavage agent B for various period of time at 370C and pH 7.50 are summarized in Figure 23. The results reveal that the cleavage yield does not increase even if the reaction period is increase beyond 36 hours.
(3) Cleavage of oligomer of α-synuclein associated with Parkinson's disease The cleavage yields measured by incubating Syn (70 μM) with various concentrations of cleavage agent B at pH 7.50 and 370C for 3 days are illustrated in Figure 24.
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 24 are fairly large. (4) Reaction with control peptides or proteins
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
Cleavage agent C was synthesized according to the pathway shown in Figure 25.
Figure imgf000061_0001
Synthesis of resins of formulas 3a and 3b To a suspension of the resin of formula 2e (50 mg, 0.046 mmol) in NMP (1 mL), n- butanol (1 mL) and cyclododecylamine (66 μL, 0.51 mmol) were added followed by DIEA (63 μL, 0.36 mmol). The mixture was heated at 120 0C for 8 hours. After filtration, the resulting resin (3a) was washed with DMF, MC, MeOH, and MC (each 3 mL x 3) and dried under nitrogen gas. To the resin of formula 3a, the mixture of a solution of m-CPBA (80 mg, 0.46 mmol) in 1,4-dioxane (1.8 mL) and aqueous 1 N NaOH (93 μL) were added. The mixture was shaken for 4 hours at room temperature. After filtration, the resulting resin (3b) was washed with 1,4-dioxane and MC (each 3 mL x 3) and was dried under nitrogen gas. Synthesis of compounds of formulas 3d and 3e and cleavage agent C
To the suspension of the resin of formula 3b in acetonitrile (1.5 mL), 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.
Suh, J. Am. Chem. Soc. 2005, 127, 2396-2397) (28 mg, 0.046 mmol). The reaction mixture was heated at 800C for 8 hours. The mixture was filtered and the resin was washed with MC (1 mL x 3). The combined filtrate and washing were evaporated in vacuo and the residue was purified by column chromatography to obtain 10-{3-[3-(4-{2- [(4-benzothiazol-2-yl-phenyl)-methyl-amino]-ethoxy}-6-cyclododecylamino-
[ 1 ,3,5]triazin-2-ylamino)-propionylamino]-propyl} -1 ,4,7, 1 O-tetraaza-cyclododecane- 1,4,7-tricarboxylic acid tri-tert-butyl ester (3d).
Rf 0.3 (EA only); 1H NMR (CDCl3): δ 7.95 (t, 3H), 7.83 (d, IH), 7.41 (t, IH), 7.33 (br, IH), 6.78 (m, 2H), 4.67 (br, 2H), 3.75 (br, 2H), 3.67 (br, 2H), 3.55-3.18 (br, 14H), 3.10 (s, 3H), 2.60 (br, 5H), 2.50 (br, 4H), 1.61-1.58 (br, 6H), 1.45-1.41 (m, 27H), 1.33 br, 18H); 13C NMR (CDCl3): δ 184.41, 173.21, 171.70, 169.97, 168.67, 156.28, 155.60, 155.24, 154.34, 150.84, 134.47, 129.75, 128.97, 125.99, 125.01, 124.21, 122.21, 121.35, 111.57, 111.46, 79.46, 62.94, 56.52, 55.11, 51.07, 49.97, 48.36, 47.41, 46.54, 40.92, 39.27, 36.76, 35.63, 29.69, 28.62, 23.50, 21.32; MS (MALDI-TOF) m/z 1144.02 (M+H)+, calcd for C60H95Ni2O8S 1143.70.
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
1H NMR (CDCl3): δ 7.94 (br, 3H), 7.83 (d, IH), 7.45 (br, IH), 7.35 (br, IH), 6.75 (d, 2H), 4.57 (br, 2H), 3.81 (br, 2H), 3.63 (br, 2H), 3.3-2.9 (br, 17H), 2.76 (br, 5H), 2.44 (br, 4H), 1.65-1.61 (br, 6H), 1.31 (br, 18H); 13C NMR (CDCl3): δ 188.78, 188.78, 172.02, 169.91, 169.79, 169.44, 157.24, 151.78, 133.60, 129.56, 126.89, 125.41, 125.16, 121.81, 120.57, 112.28, 112.02, 66.26, 65.57, 60.62, 50.69, 49.67, 48.68, 44.63, 42.51, 39.33, 39.16, 37.01, 30.20, 29.94, 23.82, 23.51, 21.48; MS (MALDI-TOF) m/z 843.79 (M+H)+, calcd for C45H7iNi202S 843.55; HRMS m/z 843.5547. (M+H)+, calcd for C45H7iNi202S 843.5538. The stock solution of cleavage agent C was obtained from 3e as described for cleavage agent A in Example 1.
Activity test of cleavage agent C
(1) Cleavage of oligomers of A/34o and Aβm associated with Alzheimer's disease When cleavage agent C (0.1-10 μM) was incubated with AjS40 (4.0 μM) at pH 7.50 and 370C for 36 hours, the MALDI-TOF MS data did not reveal any evidence for cleavage of A/340.
MALDI-TOF MS mass spectrum obtained by reacting A/342 (4.0 μM) with cleavage agent C is illustrated in Figure 26. As shown in the Figure 26, A/342 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 Aj340 or A/342 (4.0 μM) with various concentrations of cleavage agent C at pH 7.50 and 370C for 36 hours is illustrated in Figure 27. The plateau value of the yield for cleavage of A/342 by cleavage agent C obtained at high concentration of the cleavage agent is about 12%. When the concentration of A/342 was 4.0 μM, 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.
(2) Cleavage of oligomers of amylin associated with type 2 diabetes mellitus
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 Am17-37 was included in the cleavage products.
The cleavage yields measured after reacting Am (4.0 μM) with various concentrations of cleavage agent C at 370C and pH 7.50 for 36 hours are illustrated in Figure 29.
(3) Reaction with control peptides or proteins
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.
Example 4
Cleavage agent D was synthesized according to the pathway shown in Figure 30.
Figure imgf000065_0001
Synthesis of resins of formulas 4a and 4b
To a suspension of the resin of formula 2e (50 mg, 0.046 mmol) in NMP (1 mL), n- butanol (1 mL) and 2-methylbenzylamine (63 μL, 0.51 mmol) were added followed by
DIEA (63 μL, 0.36 mmol). The mixture was heated at 120 0C for 8 hours. After filtration, the resulting resin (4a) was washed with DMF, MC, MeOH, and MC (each 3 mL x 3) and dried under nitrogen gas.
To the resin of formula 4a, the mixture of a solution of m-CPBA (80 mg, 0.46 mmol) in 1,4-dioxane (1.8 mL) and aqueous 1 N NaOH (93 μL) were added. The mixture was shaken for 4 hours at room temperature. After filtration, the resulting resin (4b) was washed with 1,4-dioxane and MC (each 3 mL x 3) and was dried under nitrogen gas.
Synthesis of compounds of formulas 4c and 4d and cleavage agent D
To the suspension of the resin of formula 4b in acetonitrile (1.5 mL), 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 0C for 8 hours. The mixture was filtered and the resin was washed with MC (1 mL x 3). The combined filtrate and washing were evaporated in vacuo and the residue was purified by column chromatography to obtain 10-(3-{3-[4-{2-[(4-benzothiazol-2-yl- phenyl)-methyl-amino]-ethoxy} -6-(2-methyl-benzylamino)-[ 1 ,3,5]triazin-2-ylamino]- propionylamino} -propyl)- 1 ,4,7,10-tetraaza-cyclododecane- 1 ,4,7-tricarboxylic acid tri- tert-butyl ester (4c).
Rf 0.4 (EA only); 1H NMR (CDCl3): δ 7.96 (t, 3H), 7.83 (d, IH), 7.43 (t, IH), 7.32 (br, IH), 7.15 (br, 4H), 6.77 (d, 2H), 4.53 (br, 4H), 3.75 (br, 2H), 3.66 (br, 2H), 3.5-3.1
(br, 14H), 3.09 (s, 3H), 2.58 (br, 4H), 2.49 (br, 4H), 2.32 (s, 3H), 1.60 (br, 2H), 1.46-1.41
(m, 27H); 13C NMR (CDCl3): δ 186.87, 179.99, 173.28, 171.18, 168.68, 155.26, 154.35,
150.86, 136.88, 136.10, 134.50, 130.27, 128.99, 127.27, 126.09, 126.01, 125.02, 124.24,
122.24, 121.45, 121.38, 111.55, 80.31, 79.51, 63.54, 56.56, 55.10, 54.95, 50.98, 49.95, 48.35, 47.65, 40.01, 39.03, 36.93, 29.71, 28.65, 19.10; MS (MALDI-TOF) m/z 1081.89
(M+H)+, calcd for C56H8iNi208S 1081.59.
Compound of formula 4c was treated with TFA as described above in Example 1 for Ih to obtain the TFA salt of 3-[4-{2-[(4-benzothiazol-2-yl-phenyl)-methyl-amino]- ethoxy}-6-(2-methyl-benzylamino)-[l,3,5]triazin-2-ylamino]-N-[3-(l, 4,7,10-tetraaza- cyclododec-l-yl)-propyl]-propionamide (4d). The TFA salt of 4d was used for NMR and MS characterization.
]R NMR (CDCl3): δ 7.96 (t, 3H), 7.85 (d, IH), 7.49 (br, IH), 7.37 (br, IH), 7.15 (br, 4H), 6.80 (d, 2H), 4.56 (m, 4H), 3.58 (br, 2H), 3.3-2.8 (br, 17H), 2.33 (br, 8H), 2.31 (s, 3H), 1.48 (br, 2H); 13C NMR (CDCl3): δ 186.11, 180.06, 171.65, 169.85, 169.01, 151.51, 151.17, 136.08, 134.83, 132.77, 130.66, 129.65, 129.42, 127.99, 127.93, 127.82, 126.31, 126.13, 125.33, 121.76, 121.05, 112.09, 111.90, 66.12, 62.87, 53.89, 50.44, 49.72, 44.38, 44.22, 42.34, 42.09, 39.36, 36.63, 29.71, 19.07; MS (MALDI-TOF) m/z 781.73 (M+H)+, calcd for C4iH57Ni2O2S 781.44; HRMS m/z 781.4457. (M+H)+, calcd for C4IH57N12O2S 781.4443.
The stock solution of cleavage agent D was obtained from the compound of formula 4d as described for cleavage agent A in Example 1.
Activity test of cleavage agent D (1) Cleavage of oligomers of AjS40 and AjS42 associated with Alzheimer's disease
When cleavage agent D (0.1-10 μM) was incubated with A(S40 (4.0 μM) at pH 7.50 and 370C for 36 hours, the MALDI-TOF MS data did not reveal any evidence of cleavage OfAjS40.
MALDI-TOF MS mass spectrum obtained by reacting Aβ42 (4.0 μM) with cleavage agent D is illustrated in Figure 31. As shown in the Figure 31, A/342 was cleaved by cleavage agent D and AjSi -20 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 AjS40 or AjS42 (4.0 μM) with various concentrations of cleavage agent D at pH 7.50 and 370C for 36 hours is illustrated in Figure 32. The plateau value of the yield for cleavage of AjS42 by cleavage agent D obtained at high concentration of the cleavage agent is about 12%. When the concentration of Aβ42 was 4.0 μM, cleavage reaction was detected with 50-100 nM of cleavage agent D. If the concentration of Aj342 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.
(2) Cleavage of oligomers of amylin associated with type 2 diabetes mellitus MALDI-TOF mass spectrum of the products purified with HPLC after incubating
Am (4.0 μM) with cleavage agent D is illustrated in Figure 33. As shown in Figure 33, Am was cleaved by cleavage agent D, and Am20-37 and Ami9_37 were included in the cleavage products.
The cleavage yields measured after reacting Am (4.0 μM) with various concentrations of cleavage agent D at 370C and pH 7.50 for 36 hours are illustrated in Figure 34.
(3) Reaction with control peptides or proteins
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.
Example 5
Cleavage agent E was synthesized according to the pathway shown in Figure 35.
Figure imgf000070_0001
Synthesis of resins of formulas 5a and 5b
To the suspension of the resin of formula 2e (55 mg, 0.15 mmol) in NMP (1 mL), n-butanol (1 mL) and butylamine (49 μL, 0.58 mmol) were added followed by DIEA
(120 μL, 0.87 mmol). The mixture was heated at 12O 0C for 8 hours. After filtration, the resulting resin (5a) was washed with DMF, MC, MeOH, and MC (each 3 mL x 3) and dried under nitrogen gas.
To the resin of formula 5a, the mixture of a solution of m-CPBA (130 mg, 0.74 mmol) in 1,4-dioxane (1.8 mL) and aqueous 1 N NaOH (160 μL) were added. The mixture was shaken for 4 hours at room temperature. After filtration, the resulting resin (5b) was washed with 1,4-dioxane and MC (each 3 niL x 3) and was dried under nitrogen gas.
Synthesis of compounds of formulas 5c and 5d and cleavage agent E To the suspension of the resin of formula 5b in acetonitrile (1.5 mL), PS-DIEA resin (30 mg, 0.12 mmol) and the compound of formula Ig (39 mg, 0.074 mmol) were added. The mixture was heated at 800C for 8 hours. The mixture was filtered and the resin was washed with MC (1 mL x 3). The combined filtrate and washing were evaporated in vacuo and the residue was purified by column chromatography to obtain 10-[3-(4-{2-[(4-benzothiazol-2-yl-phenyl)-methyl-amino]-ethoxy}-6-butylamino-
[l,3,5]triazin-2-ylamino)-propyl]-l,4,7,10-tetraaza-cyclododecane-l,4,7-tricarboxylic acid tri-tert-butyl ester (5 c).
Rf 0.2 (EA/hexane 1 :1); 1H NMR (CDCl3): δ. 7.99 (t, 2H), 7.86 (d, IH), 7.44 (t, IH), 7.31 (t, IH), 6.79 (d, 2H), 4.48 (t, 2H), 3.80 (t, 2H), 3.54-3.11 (br, 16H), 3.11 (s, 3H), 2.61 (br, 6H), 1.73 (m, 2H), 1.53 (m, 2H), 1.46-1.38 (br, 27H), 1.29 (m, 2H), 0.90 (t, 3H); 13C NMR (CDCl3): δ. 170.47, 168.58, 167.25, 156.09, 155.69, 155.35, 154.43, 154.40, 150.92, 134.53, 128.97, 125.97, 124.19, 122.29, 121.56, 121.33, 111.57, 79.55, 79.34, 76.61, 62.54, 55.04, 54.16, 51.13, 50.02, 47.97, 40.52, 39.09, 31.75, 29.68, 28.66, 28.50, 24.93, 20.01, 13.79; MS (MALDI-TOF) m/z 962.33 (M+H)+, calcd for C49H75NnO7S 962.28.
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;
1H NMR (MeOD): δ 7.90 (q, 4 H), 7.49 (t, 1 H), 7.37 (t, IH), 6.85 (d, 2H), 4.73 (t, 2H), 3.89 (t, 2H), 3.2-3.0 (br, 12H), 2.98-2.86 (br, 4H), 2.84-2.75 (br, 4H), 2.68 (t, 3H), 1.76 (q, 2H), 1.52 (q, 2H), 1.31 (q, 2H), 0.89 (t, 3H); 13C NMR (CDCl3): δ 169.61, 161.13, 160.64, 160.32, 152.72, 151.72, 151.55, 133.49, 128.69, 128.64, 126.35, 124.67, 121.48, 120.93, 120.16, 118.24, 114.36, 111.84, 66.28, 49.98, 46.76, 44.31, 42.01, 41.86, 40.69, 38.95, 38.65, 37.84, 37.52, 30.51, 22.62, 19.55, 12.63; HRMS m/z 662.4073 (M+H)+, calcd for C34H52NnOS 662.4077.
To the solution obtained by dissolving the TFA salt of the compound of formula 5d in methanol in a concentration of about 3 mg/50 μL, 5-7 equivalents of LiOH was added followed by an equivalent amount of CoCl2-H2O to prepare the Co11 complex of the compound of formula 5d. The complex was stirred for 1 day in the air to oxidize the Co" complex to the Co111 complex. Oxidation of Co" to Co"1 was accompanied by appearance of deep violet color. The Co"1 complex was isolated with HPLC by detecting at 545 nm, and evaporated to produce a solid. The solid was dissolved in water and left at room temperature for several days to obtain the stock solution of cleavage agent E. The cobalt content was measured by ICP to determine the concentration of the cleavage agent in the solution.
Activity test of cleavage agent E
(1) Cleavage of oligomers of amylin associated with type 2 diabetes mellitus
MALDI-TOF mass spectrum of the products purified with HPLC after incubating Am (4.0 μM) with cleavage agent E is illustrated in Figure 36. As shown in Figure 36, Am was cleaved by cleavage agent E, and Ami7-37, AmJ6-37 and Am13-37 were included in the cleavage products.
The cleavage yields measured after reacting Am (4.0 μM) with various concentrations of cleavage agent E at 370C and pH 7.50 for 36 hours are illustrated in Figure 37. (2) Reaction with control peptides or proteins
The control experiment, identical to that of Example 1, was carried out for cleavage agent E. The results of the control experiment were the same as those obtained in Example 1. Example 6
Cleavage agent F synthesized according to the pathway shown in Figure 38.
Figure imgf000074_0001
Synthesis of compounds of formulas 6a and 6b To the stirred solution of the compound of formula Ig (2.9 g, 5.5 mmol) in acetonitrile (100 mL), N-α-Cbz-L-leucine (0.8 g, 5.5 mmol) and DIEA (2.9 mL, 17 mmol) were added. To the reaction mixture, HBTU (2.1 g, 5.5 mmol) was added and the mixture was stirred for 1 hour. The residue obtained by evaporation of the solution was dissolved in EA (100 mL). The EA solution was washed with 5% aq. citric acid (50 mL), 5% aq. Na2CO3 (50 mL), and brine (50 mL), and dried over Na2SO4. The solvent was evaporated off and column chromatography afforded 10-[(R)-3-(4-methyl-2- phenoxycarbonylamino-pentanoylamino)-propyl]- 1 ,4,7,10-tetraaza-cyclododecane- 1 ,4,7- tricarboxylic acid tri-tert-butyl ester (6a) as a colorless oil.
Rf0.5 (EA/hexane 2:1); 1H NMR (CDCl3): δ 7.31-7.26 (br, 5H), 4.75-4.33(br, 2H), 3.63-3.38 (br, 15H), 2.67-2.29 (br, 6H), 1.71-1.58 (m, 5H), 1.50-1.29 (br, 27H), 0.96-0.92 (t, 6H) MS (MALDI-TOF) m/z MS (MALDI-TOF) m/z 776.63 (M+H)+, calcd for C40H68N6O9 776.50.
A suspension of the compound of formula 6a (1.8g, 2.7 mmol) and 1.0 g of 10 % Pd/C in 80 mL of EA was stirred under 1 atm of H2 for 24 hours. The catalyst was filtered off on Celite, and the solvent was evaporated off to afford 10-[3-(2-amino-4- methyl-pentanoylarnino)-propyl] - 1 ,4,7, 10-tetraaza-cyclododecane- 1 ,4,7-tricarboxylic acid tri-tert-butyl ester (6b) as a solid
1H NMR (CDCl3): δ 7.72 (s, IH), 4.60-4.20(br, 2H), 3.70-3.13 (br, 15H), 2.75-2.50 (br, 6H), 1.86-1.62 (m, 4H), 1.54-1.36 (m, 28H), 1.05-0.84 (t, 6H); 13C NMR (MeOD):
5D 176.12, 156.31, 156.11, 155.91, 79.70, 54.16, 53.91, 53.18, 49.23, 46.84, 43.98,
37.53, 37.70, 27.70, 27.52, 24.51, 22.84, 22.02, 21.26; MS (MALDI-TOF) m/z 643.57
(M+H)+, calcd for C32H62N6O7 643.47.
Synthesis of resins of formulas 6c and 6d
To the suspension of the resin of formula 2e (55 mg, 0.15 mmol) in NMP (1 mL), n-butanol (1 mL) and piperidine (57 μL, 0.58 mmol) were added followed by DIEA (120 μL, 0.87 mmol). The mixture was heated at 12O 0C for 8 hours. After filtration, the resulting resin (6c) was washed with DMF, MC, MeOH, and MC (each 3 niL x 3) and dried under nitrogen gas.
To the resin of formula 6c, the mixture of a solution of m-CPBA (130 mg, 0.74 mmol) in 1,4-dioxane (1.8 mL) and aqueous 1 N NaOH (160 μL) were added. The mixture was shaken for 4 hours at room temperature. After filtration, the resulting resin
(6d) was washed with 1,4-dioxane and MC (each 3 mL x 3) and was dried under nitrogen gas.
Synthesis of compounds of formulas 6e and 6f and cleavage agent F To the suspension of the resin of formula 6d in acetonitrile (1.5 mL), PS-DIEA resin (30 mg, 0.12 mmol) and the compound of formula 6b (48 mg, 0.074 mmol) were added. The mixture was heated at 800C for 8 hours. The mixture was filtered and the resin was washed with MC (1 mL χ 3). The combined filtrate and washing were evaporated in vacuo and the residue was purified by column chromatography to obtain 10-{(R)-3-[2-(4-{2-[(4-benzothiazol-2-yl-phenyl)-methyl-amino]-ethoxy}-6-piperidin-l- yl-[ 1 ,3,5]triazin-2-ylamino)-4-methyl-pentanoylamino]-propyl} - 1 ,4,7,10-tetraaza- cyclodode-cane-l,4,7-tricarboxylic acid tri-tert-butyl ester (6e).
Rf 0.2 (EA/hexane 2:1); 1H NMR (CDCl3): δ. 7.96 (q, 3H), 7.84 (d, IH), 7.45 (t, IH), 7.30 (t, IH), 6.78 (d, 2H), 4.56-4.55 (br, IH), 4.45 (t, 2H), 3.78 (t, 2H), 3.69-3.67 (br, 4H), 3.61-3.21(br, 14H), 3.11(s, 3H), 2.75-2.31(br, 6H), 1.72 (m, IH), 1.72-1.50 (br, 10H), 1.49-1.29 (br, 27H), 0.93 (m, 6H); 13C NMR (CDCl3): δ 173.17, 170.46, 168.57, 166.78, 165.42, 156.13, 155.78, 155.30, 154.41, 150.89, 134.53, 129.00, 125.99, 124.23, 122.30, 121.60, 121.36, 111.56, 79.44, 76.61, 62.79, 54.88, 53.95, 51.10, 49.96, 49.15, 48.14, 44.46, 41.68, 39.10, 36.99, 29.69, 28.66, 28.51, 25.77, 24.87, 24.72, 24.46, 23.25, 21.80; MS (MALDI-TOF) m/z 1087.60 (M+H)+, calcd for C56H86N12O8S 1087.64.
The compound of formula 6e 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- amino]-ethoxy}-6-piperidin-l-yl-[l,3,5]triazin-2-ylamino)-4-methyl-pentanoic acid [3- (1, 4,7, lO-tetraaza-cyclododec-l-y^-propyl] -amide (6f). The TFA salt of the compound of formula 6f was used for NMR and MS characterization.
1H NMR (MeOD): δ. 7.90 (q, 4H), 7.48 (t, IH), 7.37 (t, IH), 6.88 (d, 2H), 4.70- 4.68 (br, IH), 4.52 (t, 2H), 3.89 (t, 2H), 3.82-3.68 (br, 4H), 3.20-3.05 (br, 13H), 3.02-2.93 (br, 4H), 2.85-2.80 (br, 4H), 2.59 (t, 2H), 1.75-1.50 (br, 7H), 0.89 (t, 6H) ; 13C NMR (MeOD): δ 173.16, 169.35, 161.64, 161.45, 158.40, 157.85, 157.30, 153.47, 151.32, 133.84, 128.59, 126.14, 124.46, 121.35, 120.62, 116.53, 111.61, 65.65, 65.47, 53.59, 50.18, 50.01, 46.77, 45.25, 44.06, 42.06, 41.84, 41.09, 37.83, 36.60, 25.09, 24.54, 23.81, 21.90, 20.64, 14.04; HRMS m/z 787.4913 (M+H)+, calcd for C4IH63Ni2O2S 787.4918.
The stock solution of cleavage agent F was obtained from the compound of formula 6f as described for cleavage agent E in Example 5.
Activity test of cleavage agent F
(1) Cleavage of oligomers of amylin associated with type 2 diabetes mellitus MALDI-TOF mass spectrum of the products purified with HPLC after incubating
Am (4.0 μM) with cleavage agent F is illustrated in Figure 39. As shown in Figure 39, Am was cleaved by cleavage agent F, and Ami9-37 and Am)7-37 were included in the cleavage products.
The cleavage yields measured after reacting Am (4.0 μM) with various concentrations of cleavage agent F at 370C and pH 7.50 for 36 hours are illustrated in Figure 40.
(2) Reaction with control peptides or proteins
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.
Example 7
Cleavage agent G was synthesized according to the pathway shown in Figure 41.
Figure imgf000079_0001
Synthesis of resins of formulas 7a and 7b
To the suspension of 2e (55 mg, 0.15 mmol) in NMP (1 mL), n-butanol (1 mL) and cyclododecylamine (75 μL, 0.58 mmol), followed by DIEA (120 μL, 0.87 mmol) were added. The mixture was heated at 120 0C for 8 hours. After filtration, the resulting resin (7a) was washed with DMF, MC, MeOH, and MC (each 3 mL x 3) and dried under nitrogen gas.
To the resin of formula 7a was added the mixture of a solution of m-CPBA (130 mg, 0.74 mmol) in 1,4-dioxane (1.8 mL) and aqueous 1 N NaOH (160 μL). The mixture was shaken for 4 hours at room temperature. After filtration, the resulting resin
(7b) was washed with 1,4-dioxane and MC (each 3 mL x 3) and was dried under nitrogen gas. Synthesis of compounds of formulas 7c and 7d and cleavage agent F
To the suspension of the resin of formula 7b in acetonitrile (1.5 mL) were added PS-DIEA resin (30 mg, 0.12 mmol) and the compound of formula 6b (48 mg, 0.074 mmol). The mixture was heated at 800C for 8 hours. The mixture was filtered and the resin was washed with MC (1 mL x 3). The combined filtrate and washing were evaporated in vacuo and the residue was purified by column chromatography to obtain 10-{(R)-3-[2-(4-{2-[(4-benzothiazol-2-yl-ρhenyl)-methyl-amino]-ethoxy}-6- cyclododecylamino-[l,3,5]triazin-2-ylamino)-4-methyl-pentanoylamino]-propyl}- l,4,7,10-tetraaza-cyclododecane-l,4,7-tricarboxylic acid tri-tert-butyl ester (7c). Rf 0.2 (EA/hexane 2:1); 1H NMR (CDCl3): δ. 7.96 (q, 3H), 7.84 (d, IH), 7.43 (t,
IH), 7.30 (t, IH), 6.77 (d, 2H), 4.47-4.41 (br, 3H), 4.14-4.04 (br, IH), 3.77 (t, 2H), 3.68- 21 (br, 14H), 3.12 (s, 3H), 2.75-2.34 (br, 6H), 1.72-1.55 (br, 5H), 1.52-1.40 (br, 27H), 1.38-1.28 (br, 22H), 0.87 (m, 6H); 13C NMR (CDCl3): δ 173.56, 170.42, 168.50, 166.90, 165.93, 156.07, 155.74, 155.26, 154.39, 150.91, 134.50, 128.97, 125.95, 124.20, 122.26, 121.62, 121.53, 121.32, 111.59, 111.49, 79.49, 79.34, 79.23, 76.73, 63.17, 63.06, 51.05, 49.87, 47.99, 47.55, 47.32, 39.21, 38.83, 30.58, 29.65, 28.64, 28.49, 25.00, 24.83, 24.10, 23.95, 23.73, 23.51, 23.32, 23.08, 22.15, 21.74, 21.17 ; MS (MALDI-TOF) m/z 1185.53 (M+H)+, calcd for C63H100Ni2O8S 1185.75.
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-
4.53 (br, 3H), 4.12-4.03 (br, IH), 3.89 (t, 2H), 3.22-3.12 (br, 10H), 3.10-3.00 (br, 3H), 2.97-2.92 (br, 4H), 2.90-2.73 (br, 4H), 2.63 (t, 2H), 1.76-1.60 (br, 5H), 1.40-1.12 (br, 22H), 0.72 (t, 6H); 13C NMR (MeOD): δ 172.79, 169.50, 161.97, 161.41, 160.99, 159.38, 157.83, 157.28, 151.60, 133.48, 128.80, 126.32, 124.63, 121.45, 120.93, 116.52, 111.63, 66.45, 65.47, 53.76, 49.94, 46.79, 44.15, 41.96, 41.92, 37.52, 24.66, 23.61, 23.53, 23.37, 23.52, 23.10, 22.90, 22.10, 20.86, 20.46, 14.06; HRMS m/z 885.5991 (M+H)+, calcd for C48H77Nj2O2S 885.6013.
The stock solution of cleavage agent G was obtained from the compound of formula 7d as described for cleavage agent E in Example 5.
Activity test of cleavage agent G
(1) Cleavage of oligomers of amylin associated with type 2 diabetes mellitus MALDI-TOF mass spectrum of the products purified with HPLC after incubating Am (4.0 μM) with cleavage agent G is illustrated in Figure 42. As shown in Figure 42, Am was cleaved by cleavage agent F, and Am2O-37, Am]7-37 and Am14-37 were included in the cleavage products.
The cleavage yields measured after reacting Am (4.0 μM) with various concentrations of cleavage agent G at 370C and pH 7.50 for 36 hours are illustrated in Figure 43.
(2) Reaction with control peptides or proteins
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.
Example 8
Cleavage agent H was synthesized according to the pathway shown in Figure 44.
Figure imgf000082_0001
Synthesis of compounds of formulas 8a and 8b
To the stirred solution of the compound of formula Ig (2.9 g, 5.5 mmol) in acetonitrile (100 mL), N-α-Cbz-L-tyrosine (1.1 g, 5.5 mmol) and DIEA (2.9 mL, 17 mmol) were added. To the reaction mixture was added HBTU (2.1 g, 5.5 mmol) and the mixture was stirred for 1 hour. The residue obtained by evaporation of the solution was dissolved in EA (100 mL). The EA solution was washed with 5% aq. citric acid (50 mL), 5% aq. Na2CO3 (50 mL), and brine (50 mL), and dried over Na2SO4. The solvent was evaporated off and column chromatography afforded 10-{(S)-3-[3-(4- hydroxy-phenyl)-2-phenoxycarbonylamino-propionylamino]-propyl} - 1 ,4,7, 10-tetraaza- cyclododecane-l,4,7-tricarboxylic acid tri-tert-butyl ester (8a) as a colorless oil.
Rf 0.5 (EA/hexane 3:1). 1H NMR (CDCl3): δ 7.42-7.23 (br, 5H), 7.07-6.85 (d, 2H), 6.77-6.62 (d, 2H), 4.60-4.13 (br, 4H), 3.77-2.92 (br, 16H), 2.69-2.54 (br, 4H), 2.50-2.37 (br, 2H), 1.65-1.31 (br, 29H); MS (MALDI-TOF) m/z 827.75 (M+H)+, calcd for C43H66N6O10 827.04. A suspension of the compound of formula 8a (2.Og, 2.7 mmol) and 1.0 g of 10 %
Pd/C in 80 mL of EA was stirred under 1 atm of H2 for 24 hours. The catalyst was filtered off on Celite, and the solvent was evaporated off to afford 10-{(R)-3-[2-amino-3- (4-hydroxy-phenyl)-propionylamino] -propyl} - 1 ,4,7, 10-tetraaza-cyclododecane- 1 ,4,7- tricarboxylic acid tri-tert-butyl ester (8b) as a solid. 1H NMR (CDCl3): δ 7.2 (s, IH), 7.05-6.97 (d, 2H), 6.81-6.72 (d, 2H), 3.65-3.58 (m, IH), 3.53-3.20 (br, 14H), 3.12-3.00(m, 2H), 2.75-2.48 (br, 8H), 1.76-1.64 (m, 2H), 1.54- 1.38 (m, 27H); 13C NMR (CDCl3): δ. 173.51, 155.91, 155.51, 130.40, 127.44, 115.62, 79.91, 79.65, 76.58, 56.12, 54.55, 49.76, 47.73, 39.63, 38.56, 37.13, 29.62, 28.61, 28.45, 24.23; MS (MALDI-TOF) m/z 692.84 (M+H)+, calcd for C35H60N6O8 692.90. Synthesis of resins of formulas 8c and 8d
To the suspension of the resin of formula 2e (55 mg, 0.15 mmol) in NMP (1 niL), n-butanol (1 niL) and dicyclohexylamine (115 μL, 0.58 mmol) were added followed by
DIEA (120 μL, 0.87 mmol). The mixture was heated at 120 0C for 8 hours. After filtration, the resulting resin (8c) was washed with DMF, MC, MeOH, and MC (each 3 mL x 3) and dried under nitrogen gas.
To the resin of formula 8c, the mixture of a solution of m-CPBA (130 mg, 0.74 mmol) in 1,4-dioxane (1.8 mL) and aqueous 1 N NaOH (160 μL) were added. The mixture was shaken for 4 hours at room temperature. After filtration, the resulting resin (8d) was washed with 1,4-dioxane and MC (each 3 mL χ 3) and was dried under nitrogen gas.
Synthesis of compounds of formulas 8e and 8f and cleavage agent H
To the suspension of 8d in acetonitrile (1.5 mL), 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 800C for 8 hours. The mixture was filtered and the resin was washed with MC (1 mL x 3). The combined filtrate and washing were evaporated in vacuo and the residue was purified by column chromatography to obtain 10-{(S)-3-[2- (4-{2-[(4-benzothiazol-2-yl-phenyl)-methyl-amino]-ethoxy}-6-dicyclohexylami-no- [ 1 ,3 ,5 ]triazin-2-ylamino)-3 -(4-hydroxy-phenyl)-propionylamino] -propyl} - 1 ,4,7, 10-tetra- aza-cyclododecane-l,4,7-tricarboxylic acid tri-tert-butyl ester (8e).
Rf 0.2 (EA:hexane 1 :4); 1H NMR (CDCl3): δ 7.87(t, 3H), 7.73 (d, 2H), 7.35 (t, 2H), 7.20 (m, 2H), 6.75-6.48 (br, 4H), 4.45-4.41(m, 3H), 3.73-3.68(m, 2H), 3.62-3.08 (br, 18H), 3.05-2.65 (br, 6H), 2.19-2.17 (m, 2H), 1.93-1.85(m, 2H), 1.75-1.62 (br, 4H), 1.60- 1.42 (br, 31H), 1.58-1.20 (br, 12H); 13C NMR (CDCl3): δ 169.55, 168.64, 165.09, 154.35, 151.53, 150.73, 135.81, 134.52, 129.04, 128.26, 125.53, 124.28, 122.30, 121.75, 121.38, 111.68, 76.71, 64.43, 64.24, 56.28, 56.16, 50.96, 47.21, 39.29, 34.24, 30.35, 29.99, 29.80, 29.72, 28.58, 28.46, 26.12, 25.58, 25.37, 21.22, 20.25, 20.18, 20.09; HRMS m/z 1232.7149 (M+H)+, calcd for C66H96N12O9S 1232.7144.
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)-τV-[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
1H NMR (CDCl3): δ 8.04 (t, 3H), 7.75 (d, 2H), 7.53 (t, 2H), 7.42 (m, 2H), 6.78- 6.64 (br, 4H), 4.48-4.45 (m, 3H), 3.81-3.74 (m, 2H), 3.63-2.75 (br, 24H), 2.25-2.15 (m, 3H), 1.85-1.63 (br, 12H), 1.60-1.42 (br, 10H); 13C NMR (CDCl3): Dδ 169.55, 168.64, 164.59, 160.23, 153.72, 142.86, 130.91, 128.97, 128.26, 125.52, 122.10, 118.18, 117.51, 113.69, 79.80, 74.60, 73.43, 56.70, 56.53, 39.59, 34.22, 30.88, 30.47, 30.31, 29.56, 27.98, 25.25, 24.93, 21.19, 19.92; HRMS m/z 932.5573 (M+H)+, calcd for C51H72Ni2O3S 932.5571.
The stock solution of cleavage agent H was obtained from the compound of formula 8f as described for cleavage agent E in Example 5.
Activity test of cleavage agent H
(1) Cleavage of oligomers of amylin associated with type 2 diabetes mellitus MALDI-TOF mass spectrum of the products purified with HPLC after incubating Am (4.0 μM) with cleavage agent H is illustrated in Figure 45. As shown in Figure 45, Am was cleaved by cleavage agent H, and Am1-^ was included in the cleavage products. The cleavage yields measured after reacting Am (4.0 μM) with various concentrations of cleavage agent H at 370C and pH 7.50 for 36 hours are illustrated in Figure 46. (2) Reaction with control peptides or proteins
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.

Claims

[Claim 1 ]
A cleavage agent of formula 1 selectively acting on soluble assembly of amyloidogenic peptide or protein: [formula 1]
(R)n-(L)m-Z wherein,
R is a target recognition site independently selected from the group consisting of A, A-(Y)O-(CH2)P-(Y)O-A, A-(CH=CH)-A, A-(Y)0-(CH2)P-(Y)0-A-(Y)0-(CH2)P-(Y)0-A and A-(Y)0-(CH2)P-(Y)0-A-(Y)0-(CH2)P-(Y)0-A-(Y)0-(CH2)P-(Y)0-A,
A is independently C6-i4aryl, or 5- to 14-membered heteroaryl having one or more hetero atom(s) selected from the group consisting of oxygen, sulfur and nitrogen, wherein, aryl or heteroaryl is unsubstituted or substituted with one or more substituent(s) independently selected from the group consisting of Ci-i5alkyl, hydroxy, Ci_i5alkoxy, Ci-15alkylcarbonyloxy, Ci-i5alkylsulfonyloxy, amino, mono or diQ. i5alkylamino, Ci-isalkylcarbonylamino, Ci.i5alkylsulfonylamino, C3.15cycloalkylam.ino, formyl, Q-isalkylcarbonyl, carboxy, Ci.isalkyloxycarbonyl, carbamoyl, mono or diCi_ i5alkylcarbamoyl, Ci-isalkylsulfanylcarbonyl, Ci-isalkylsulfanylthiocarbonyl, Ci- i5alkoxycarbonyloxy, carbamoyloxy, mono or diCi-isalkylcarbamoyloxy, Ci- i5alkylsulfanylcarbonyloxy, Ci-isalkoxycarbonylamino, ureido, mono or di or triCi. i5alkylureido, Ci-^alkylsulfanylcarbonylamino, mercapto, Ci.isalkylsulfanyl, C1- i5alkyldisulfanyl, sulfo, Ci-i5alkoxysulfonyl, sulfamoyl, mono or did-isalkylsulfamoyl, triC1-15alkylsilanyl and halogen; Y is O or N-Z, wherein Z is hydrogen or Ci.galkyl;
L is a linker;
Z is a metal ion-ligand complex as a catalytic site; n is an independent integer from 1 to 6; m and o are independently 0 or 1 ; p is an integer from 0 to 5.
[Claim 2]
The cleavage agent of claim 1, wherein A is selected from the group consisting of the following formulas; and p is independently 0, 1 or 2:
Figure imgf000089_0001
wherein,
X is independently selected from the group consisting of C, N, NH, O and S, A is unsubstituted or substituted with one or more substituent(s) independently selected from the group consisting of C1-4alkyl, Ci-4alkoxy, amino, mono or OiC1- i2alkylamino, C1-6alkylcarbonylamino, Ci-6alkylsulfonylamino, Cs-iscycloalkylamino, C1- 6alkylcarbonyl, carbamoyl, mono or diCi-6alkylcarbamoyl, Ci-6alkoxycarbonylamino, ureido, mono or di or triC1-6alkylureido, C1-6alkylsulfanylcarbonylamino, C1- 6alkylsulfanyl, C1-6alkyldisulfanyl, sulfamoyl, mono or diC1-6alkylsulfamoyl, MC1- i5alkylsilanyl and halogen.
[Claim 3]
The cleavage agent of claim 2, wherein A is selected from the group consisting of the following formulas:
Figure imgf000090_0001
wherein,
X is NH, O, or S,
A is unsubstituted or substituted with one or more substituent(s) independently selected from the group consisting of Ci-4alkyl, Ci^alkoxy, amino, mono or diQ. galkylamino,
Figure imgf000090_0002
and halogen.
[Claim 4]
The cleavage agent of claim 1, wherein the ligand is selected from the group consisting of the following formulas:
Figure imgf000091_0001
ΛO CO CO Λ> <C v
Figure imgf000091_0002
wherein, the nitrogen atom in the ligand may be replaced with an atom selected from the group consisting of oxygen, sulfur and phosphorous; the ligand may be fused with C6-i4aryl or 5- to 14-membered heteroaryl; the ligand is unsubstituted or substituted with one or more substituent(s) independently selected from the group consisting of Ci-i5alkyl, hydroxy, Ci-15alkoxy, Ci- i5alkylcarbonyloxy, Ci_i5alkylsulfonyloxy, amino, mono or diCi-i5alkylamino, Q. i5alkylcarbonylamino, Ci-isalkylsulfonylamino, formyl, Ci-isalkylcarbonyl, carboxy, Q- i5alkyloxycarbonyl, carbamoyl, mono or diCi-isalkylcarbamoyl, Ci. i salkylsulfanylcarbonyl, C \ . i salkylsulfanylthiocarbonyl, CM salkoxycarbonyloxy, carbamoyloxy, mono or diCi-isalkylcarbamoyloxy, Ci-isalkylsulfanylcarbonyloxy, C1- i5alkoxycarbonylamino, ureido, mono or di or trid-^alkylureido, Ci- i5alkylsulfanylcarbonylamino, mercapto, Ci.i5alkylsulfanyl, Ci-i5alkyldisulfanyl, sulfo, C1-15alkoxysulfonyl, sulfamoyl, mono or diCi-i5alkylsulfamoyl, triCi-i5alkylsilanyl and halogen.
[Claim 5]
The cleavage agent of claim 1, wherein the ligand is selected from the group consisting of the following formulas:
Figure imgf000092_0001
|— nH N 1 I N nH N L H HN~] ' LR HN
Figure imgf000092_0002
Figure imgf000092_0003
wherein, the ligand is unsubstituted or substituted with one or more substituent(s) independently selected from the group consisting of d^alkyl, Ci-6alkoxy, Ci- 6alkylcarbonyloxy and halogen.
[Claim 6] The cleavage agent of claim 1, wherein the metal ion is selected from the group consisting of Co111, Cu1, Cu", CeIV, Cev, Cr111, Fe11, Fe111, MoIV, Ni", Pd11, Pt11, Vv and ZrIV
[Claim 7]
The cleavage agent of claim 6, wherein the metal ion is Co111, Cu11 or Pd11.
[Claim 8]
The cleavage agent of claim 1, wherein the linker(L) is comprised of a backbone comprising 1 to 30 atom(s) independently selected from the group consisting of carbon, nitrogen, oxygen, silicon, sulfur and phosphorous, wherein, the atom in the backbone is present as the form of a functional group 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, and wherein the linker is unsubstituted or substituted with one or more substituent(s) independently selected from the group consisting of Ci-9alkyl, hydroxy, Ci^alkoxy, Q- 9alkylcarbonyloxy, Ci^alkylsulfonyloxy, amino, mono or diCi^alkylamino, Ci- 9alkylcarbonylamino, Ci-9alkylsulfonylamino, formyl, Ci_9alkylcarbonyl, carboxy, Ci. 9alkyloxycarbonyl, carbamoyl, mono or diCi_9alkylcarbamoyl, Ci-9alkylsulfanylcarbonyl, C i-galkylsulfanyl thiocarbonyl, Ci.galkoxycarbonyloxy, carbamoyloxy, mono or diCi- galkylcarbamoyloxy, Ci-galkylsulfanylcarbonyloxy, Ci-galkoxycarbonylamino, ureido, mono or di or triCi.galkylureido, Q.galkylsulfanylcarbonylamino, mercapto, C1- 9alkylsulfanyl, Ci-galkyldisulfanyl, sulfo, Ci-9alkoxysulfonyl, sulfamoyl, mono or diCi_ 9alkylsulfamoyl, triCi^alkylsilanyl and halogen.
[Claim 9] The cleavage agent of claim 8, wherein the number of atoms in the backbone is 1 to 20, and wherein the linker is unsubstituted or substituted with one or more substituent(s) independently selected from the group consisting of
Figure imgf000094_0001
Ci-6alkoxy, mono or diQ. 6alkylamino, Ci-6alkylcarbonylamino, Ci-6alkylsulfonylamino, C1-6alkylcarbonyl, carbamoyl, mono or diCi-6alkylcarbamoyl, Ci-6alkoxycarbonylamino, ureido, mono or di or triCi_6alkylureido, Ci-6alkylsulfanylcarbonylamino, Ci-6alkylsulfanyl, Ci- 6alkyldisulfanyl, sulfamoyl, mono or diCi-6alkylsulfamoyl, triCi.6alkylsilanyl and halogen.
[Claim 10] The cleavage agent of claim 1, wherein one or more of R, L and Z of the compound of formula 1 is further substituted with -(L)111-(R)n , wherein R, Z, L, m and n are the same as defined in claim 1. [Claim 11 ]
The cleavage agent of claim 1, wherein the agent cleaves oligomer of A/34o or AjS42.
[Claim 12]
The cleavage agent of claim 1, wherein the agent cleaves oligomer of amylin.
[Claim 13]
The cleavage agent of claim 1, wherein the agent cleaves oligomer of α-synuclein.
[Claim 14]
A pharmaceutical composition for prevention or treatment of amyloidosis, comprising the cleavage agent defined in claim 1 and pharmaceutically acceptable salts.
[Claim 15] The pharmaceutical composition of claim 14, wherein the amyloidosis is
Alzheimer's disease, type 2 diabetes mellitus, Parkinson's disease, spongiform encepahlopathies or Huntington's disease.
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