Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/64—Cyclic peptides containing only normal peptide links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K4/00—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
  • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
  • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a cyclic peptide having cell membrane permeability and an MDMX inhibitor.
• the human transcription factor protein p53 plays an important role in protecting cells from malignant transformation by inducing cell growth arrest or apoptosis in response to DNA damage and cellular stress.
• MDM2 and MDMX are known as oncoproteins that interact with p53, negatively regulate p53 function, and inhibit its transcriptional activity. MDM2 and MDMX are found to be amplified or overexpressed in many cancers and are involved in the development and progression of human cancer. Inhibitors of p53-MDM2 interaction and p53-MDMX interaction are expected to be anticancer agents because they can restore p53 activity.
• MDMX2 inhibitors such as Nutlin-3a, which is a low molecular weight compound, efficiently kill cancer cells having the wild-type p53 gene.
• MDMX inhibitors no effective compound has been reported regarding MDMX inhibitors, and a treatment method for cancer cells overexpressing MDMX has not yet been established.
• peptide compounds (molecular weight 500-2000) can interact with a target called protein-protein interaction (PPI) and have the potential to impart cell membrane permeability. Attention has been paid.
• cyclic peptides have advantages over linear peptides, such as improved target binding with target proteins, specificity, cell membrane permeability, and metabolic stability.
• cyclic peptides derived from natural products such as cyclosporin are commercially available as pharmaceuticals.
• cyclic peptides with 10 amino acid residues or more are preferable, but cyclic peptides with 10 amino acid residues or more generally have high cell membrane permeability. I know there isn't.
• Patent Documents 1 and 2 describe dual MDM2/MDMX inhibitors that are cyclic peptides that utilize staple crosslinking using the ⁇ -helical region of p53 as a drug discovery seed.
• Patent Document 3 and Non-Patent Document 1 describe an MDM2/MDMX dual inhibitor that is a cyclic peptide that mimics the TA domain of p53 by combining ⁇ -turn sequences to form a ⁇ -sheet structure through cyclosporin-like molecular design. is listed.
• Patent Document 4 reports a design in which hydrophobicity is improved by introducing a long alkyl side chain in a thioether cyclic peptide compound, and cell membrane permeability is imparted by utilizing structural changes in a hydrophilic and hydrophobic environment. There is.
• Non-Patent Document 2 describes an MDMX inhibitor that utilizes a low-molecular compound that can bind to the MDMX pocket instead of the TA domain of p53, in addition to the conformationally controlled cyclic peptide described above.
• Cyclic peptides have high target binding properties, but low cell membrane permeability.
• Patent Documents 1 to 3 and Non-Patent Document 1 attempt to improve cell membrane permeability by controlling a cyclic peptide to a conformation desirable for cell membrane permeation.
• no matter which approach is used problems still remain, and new designs are required to impart cell membrane permeability.
• An object of the present invention is to provide a cyclic peptide or a salt thereof having excellent cell membrane permeability.
• a further object of the present invention is to provide an MDMX inhibitor containing the above-mentioned cyclic peptide or a salt thereof.
• a cyclic peptide or a salt thereof which is represented by the following formula (1) and has the following characteristics (a) to (c).
• Y represents a divalent group represented by *-CR 1 -S-CR 2 -*
• R 1 and R 2 represent a hydrogen atom or a substituent, and * bonds with Xaa and Xbb.
• n Xaa each independently represent any amino acid residue or any amino acid analog residue
• m Xbb each independently represent any amino acid residue or any amino acid analog residue
• n+m represents an integer from 5 to 50;
• the molecular shape factor r calculated by the following formula (2) is within the range of 0.4 to 0.6;
• the main chain structure of the peptide contains a sulfur atom;
• the peptide is nonionic under physiological circumstances;
• X is an oxygen atom or a sulfur atom
• R1 , R2 , R3 , R4 , R5, R6 , R7, R8 , Q1, Q2 , Q3 , Q4 , Q5 , Q6 , Q7 , Q8 , A and B each independently represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, or a substituent.
• a non-aromatic heterocyclic group which may have P 1 , P 2 , P 3 , P 4 , P 5 , P 6 , P 7 , and P 8 each independently represent a hydrogen atom, an alkyl group that may have a substituent, or a substituent-containing alkyl group.
• an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aromatic carbocyclic group, an optionally substituted non-aromatic carbocyclic group is a formula group, an aromatic heterocyclic group which may have a substituent, or a non-aromatic heterocyclic group which may have a substituent, or
• the carbon atoms to which R1 , R2 , R3 , R4 , R5 , R6 , R7, R8 , A and B are bonded are P1 , P2 , P3 , P4 , P5 , P 6 , P 7 and P 8 may form a heterocycle together with the nitrogen atom to which they are bonded,
• m Xaa each independently represent any amino acid residue or any amino acid analog residue
• n is an integer from 0 to 5
• m is an integer from 0 to 2.
• ⁇ 3> The cyclic peptide or salt thereof according to ⁇ 2>, wherein n is 0 or 1.
• ⁇ 4> The cyclic peptide or a salt thereof according to ⁇ 2>, wherein m is 1.
• At least one of R 3 to R 8 or Q 3 to Q 8 is an aromatic carbocyclic group that may have a substituent or an aromatic heterocyclic group that may have a substituent
• R 3 , R 5 , R 6 , R 7 , R 8 or Q 3 , Q 5 , Q 6 , Q 7 , Q 8 is an aromatic group which may have a substituent
• the cyclic peptide or salt thereof according to ⁇ 2> which is a carbocyclic group or an aromatic heterocyclic group which may have a substituent.
• R 1 and Q 1 or A and B, and P 1 are combined with the carbon atom to which R 1 and Q 1 or A and B are bonded, and the nitrogen atom to which P 1 is bonded.
• R 1 and Q 1 or A and B, and P 1 are combined with the carbon atom to which R 1 and Q 1 or A and B are bonded, and the nitrogen atom to which P 1 is bonded. may form a heterocycle, and the carbon atom to which R 2 and Q 2 are bonded may form a heterocycle together with the nitrogen atom to which P 2 is bonded,
• the cyclic peptide or a salt thereof according to ⁇ 2>, wherein the Xaa may be proline, and the structure formed from these contains at least one heterocycle.
• R 1 and Q 1 or A and B, and P 1 are combined with the carbon atom to which R 1 and Q 1 or A and B are bonded, and the nitrogen atom to which P 1 is bonded.
• An MDMX inhibitor comprising the cyclic peptide or salt thereof according to any one of ⁇ 1> to ⁇ 10>.
• ⁇ A> A method for inhibiting MDMX, comprising administering to a subject the cyclic peptide or salt thereof according to any one of ⁇ 1> to ⁇ 10>.
• ⁇ B> The cyclic peptide or a salt thereof according to any one of ⁇ 1> to ⁇ 10> for use in a treatment that inhibits MDMX.
• ⁇ C> Use of the cyclic peptide or a salt thereof according to any one of ⁇ 1> to ⁇ 19> for producing an MDMX inhibitor.
• the cyclic peptide or its salt and MDMX inhibitor of the present invention have excellent cell membrane permeability.
• FIG. 1 shows a three-dimensional structure of a two-dimensionally drawn structural formula of a cyclic peptide.
• Figure 2 shows an ellipsoidal approximation of the structure of a cyclic peptide.
• ⁇ indicates a range that includes the numerical values described before and after it as the minimum and maximum values, respectively.
• Amino acid refers to a molecule containing both an amino group and a carboxyl group.
• the amino acid may be either a natural amino acid or a non-natural amino acid, and may be either a D- or L-isomer.
• the amino acid may be an ⁇ -amino acid.
• ⁇ -Amino acid refers to a molecule containing an amino group and a carboxyl group attached to a carbon designated as the ⁇ -carbon.
• Natural amino acids include alanine (A), arginine (R), asparagine (N), cysteine (C), aspartic acid (D), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), Isorosine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and Indicates either valine (V).
• Unnatural amino acids refer to amino acids other than the 20 types of natural amino acids mentioned above.
• Amino acid analog means a molecule that is structurally similar to an amino acid and can be used in place of an amino acid in the production of cyclic peptides.
• Amino acid analogs include ⁇ -amino acids and amino acids whose amino or carboxyl groups are substituted by similarly reactive groups (e.g., substitution of a primary amine with a secondary or tertiary amine, or a carboxyl group). substitution with an ester), but is not particularly limited.
• ⁇ -amino acid refers to a molecule containing both an amino group and a carboxyl group in the ⁇ configuration.
• unnatural amino acid residues and amino acid analog residues include the following, but are not particularly limited.
• Amino acid analogs include ⁇ -amino acid analogs.
• ⁇ -amino acid analogs include, but are not limited to: cyclic ⁇ -amino acid analogs; ⁇ -alanine; (R)- ⁇ -phenylalanine; (R)-1,2,3 ,4-tetrahydro-isoquinoline-3-acetic acid; (R)-3-amino-4-(1-naphthyl)-butyric acid; (R)-3-amino-4-(2,4-dichlorophenyl)butyric acid; (R) )-3-amino-4-(2-chlorophenyl)-butyric acid; (R)-3-amino-4-(2-cyanophenyl)-butyric acid; (R)-3-amino-4-(2-fluorophenyl )-butyric acid; (R)-3-amino-4-(2-furyl)-butyric acid; (R)-3-a
• Amino acid analogs include analogs of alanine, valine, glycine or leucine.
• Examples of amino acid analogs of alanine, valine, glycine, and leucine include, but are not limited to: ⁇ -methoxyglycine; ⁇ -allyl-L-alanine; ⁇ -aminoisobutyric acid; ⁇ -methyl -Leucine; ⁇ -(1-naphthyl)-D-alanine; ⁇ -(1-naphthyl)-L-alanine; ⁇ -(2-naphthyl)-D-alanine; ⁇ -(2-naphthyl)-L-alanine ; ⁇ -(2-pyridyl)-D-alanine; ⁇ -(2-pyridyl)-L-alanine; ⁇ -(2-thienyl)-D-alanine; ⁇ -(2-thienyl)-L-alanine;
• Amino acid analogs include phenylalanine and tyrosine analogs.
• amino acid analogs of phenylalanine and tyrosine include: ⁇ -methyl-phenylalanine, ⁇ -hydroxyphenylalanine, ⁇ -methyl-3-methoxy-DL-phenylalanine, ⁇ -methyl-D-phenylalanine, ⁇ -Methyl-L-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 2,4-dichloro-phenylalanine, 2-(trifluoromethyl)-D-phenylalanine, 2-(trifluoromethyl) -L-phenylalanine, 2-bromo-D-phenylalanine, 2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine, 2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine, 2-cyano-L -
• Amino acid analogs include proline analogs.
• Examples of amino acid analogs of proline include, but are not limited to: 3,4-dehydro-proline, 4-fluoro-proline, cis-4-hydroxy-proline, thiazolidine-2-carboxylic acid. , and trans-4-fluoro-proline.
• Amino acid analogs include serine and threonine analogs.
• Examples of amino acid analogs of serine and threonine include, but are not limited to: 3-amino-2-hydroxy-5-methylhexanoic acid, 2-amino-3-hydroxy-4-methylpentane. acid, 2-amino-3-ethoxybutanoic acid, 2-amino-3-methoxybutanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-amino-3-benzyloxypropionic acid, 2-amino -3-benzyloxypropionic acid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutanoic acid, and ⁇ -methylserine.
• Amino acid analogs include tryptophan analogs.
• Examples of amino acid analogs of tryptophan include, but are not limited to: ⁇ -methyl-tryptophan; ⁇ -(3-benzothienyl)-D-alanine; ⁇ -(3-benzothienyl)- L-alanine; 1-methyl-tryptophan; 4-methyl-tryptophan; 5-benzyloxy-tryptophan; 5-bromo-tryptophan; 5-chloro-tryptophan; 5-fluoro-tryptophan; 5-hydroxy-tryptophan; 5-hydroxy -L-tryptophan; 5-methoxy-tryptophan; 5-methoxy-L-tryptophan; 5-methyl-tryptophan; 6-bromo-tryptophan; 6-chloro-D-tryptophan; 6-chloro-tryptophan; 6-fluoro-tryptophan ; 6-Methyl-tryptophan; 7-benzyloxy-tryp
• the amino acid analog is racemic.
• the D isomer of the amino acid analog may be used, or the L isomer of the amino acid analog may be used.
• Amino acid analogs may also contain chiral centers that are in the R or S configuration.
• the amino group(s) of the ⁇ -amino acid analogs may be substituted with protecting groups such as tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), tosyl, etc. good.
• protecting groups such as tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), tosyl, etc. good.
• the carboxylic acid functionality of the ⁇ -amino acid analog may be protected, for example as an ester derivative thereof.
• salts of amino acid analogs may also be used.
• the alkyl group includes a straight chain or branched hydrocarbon group having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, and more preferably 1 to 6 carbon atoms.
• alkyl groups may also have at least one carbon atom substituted with a heteroatom, such as oxygen, nitrogen, sulfur, phosphorus or silicon.
• An alkenyl group is a straight chain or branched chain having 2 to 15 carbon atoms, preferably 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and having one or more carbon-carbon double bonds at any position. It includes a hydrocarbon group of the form.
• at least one carbon atom may be substituted with a heteroatom.
• Alkynyl group refers to a straight chain or branched group having 2 to 10 carbon atoms, preferably 2 to 8 carbon atoms, more preferably 2 to 6 carbon atoms, and having one or more carbon-carbon triple bonds at any position. hydrocarbon groups. Furthermore, it may have a double bond at any position. Examples include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, and the like. Further, in these alkynyl groups, at least one carbon atom may be substituted with a heteroatom.
• An aromatic carbocyclic group is a carbocyclic group containing a single aromatic ring or multiple aromatic rings.
• the aromatic carbocyclic group preferably has 5 to 24 carbon atoms, more preferably 5 to 18 carbon atoms, and still more preferably 5 to 14 carbon atoms.
• Aromatic carbocyclic groups include phenyl, naphthyl, and the like.
• a non-aromatic carbocyclic group is a carbocyclic group containing a single non-aromatic ring or multiple non-aromatic rings.
• the number of carbon atoms in the non-aromatic carbocyclic group is preferably 3 to 24, more preferably 3 to 18, and still more preferably 3 to 14.
• Non-aromatic carbocyclic groups include saturated and unsaturated cyclic hydrocarbon groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
• An aromatic heterocyclic group has 1 to 3 heteroatoms if monocyclic, 1 to 6 heteroatoms if bicyclic, or 1 to 9 heteroatoms if tricyclic. , represents an aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system. Heteroatoms can be selected from O, N or S.
• aromatic heterocyclic groups include pyridyl group, pyrimidinyl group, pyrrolyl group, furyl group, furanyl group, thiophenyl group, thienyl group, indolyl group, isoindolyl group, indolizinyl group, imidazolyl group, pyridonyl group, pyrimidyl group, and pyrazinyl group.
• oxazolyl group, thiazolyl group, purinyl group, quinolinyl group, isoquinolinyl group, benzimidazolyl group, benzofuranyl group, and benzoxazolyl group but are not limited thereto.
• a non-aromatic heterocyclic group is one having 1 to 3 heteroatoms in the case of a monocyclic type, 1 to 6 heteroatoms in the case of a bicyclic type, or 1 to 9 heteroatoms in the case of a tricyclic type. represents a non-aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system. Heteroatoms can be selected from O, N or S.
• Non-aromatic heterocyclic groups include, but are not limited to, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.
• the heterocycle includes the above-mentioned aromatic heterocyclic groups and non-aromatic heterocyclic groups.
• substituent means any of the chemical groups listed above (alkyl group, alkenyl group, alkynyl group, aromatic carbocyclic group, non-aromatic carbocyclic group, aromatic heterocyclic group)
• one or more hydrogen atoms are substituted with an atom or chemical group other than hydrogen atom.
• Substituents include halogen (fluorine, chlorine, bromine or iodine, etc.), hydroxy, mercapto, oxo, nitro, haloalkyl having 1 to 10 carbon atoms, alkyl having 1 to 10 carbon atoms, aryl having 6 to 20 carbon atoms, carbon Aralkyl having 7 to 20 carbon atoms, alkoxy having 1 to 10 carbon atoms, thioalkoxy having 1 to 10 carbon atoms, aryloxy having 6 to 20 carbon atoms, amino, alkoxycarbonyl having 2 to 10 carbon atoms, amide, carboxy, carbon number Examples include, but are not limited to, alkanesulfonyl having 1 to 10 carbon atoms, alkylcarbonyl having 2 to 10 carbon atoms, and a cyano group.
• R 1 and R 2 in the divalent group represented by *-CR 1 -S-CR 2 -* may each represent a substituent, and the substituent may include halogen (fluorine, chlorine, bromine or iodine, etc.), hydroxy, mercapto, oxo, nitro, haloalkyl having 1 to 10 carbons, alkyl having 1 to 10 carbons, aryl having 6 to 20 carbons, aralkyl having 7 to 20 carbons, aralkyl having 1 to 10 carbons, Alkoxy, thioalkoxy having 1 to 10 carbon atoms, aryloxy having 6 to 20 carbon atoms, amino, alkoxycarbonyl having 2 to 10 carbon atoms, amido, carboxy, alkanesulfonyl having 1 to 10 carbon atoms, having 2 to 10 carbon atoms Examples include, but are not limited to, alkylcarbonyl and cyano groups.
• the cyclic peptide of the present invention is represented by the following formula (1) and has the following characteristics (a) to (c).
• Y represents a divalent group represented by *-CR 1 -S-CR 2 -*
• R 1 and R 2 represent a hydrogen atom or a substituent
• * bonds with Xaa and Xbb. indicates the location
• n Xaa each independently represent any amino acid residue or any amino acid analog residue
• m Xbb each independently represent any amino acid residue or any amino acid analog residue
• n+m represents an integer from 5 to 50.
• n+m preferably represents an integer of 5 to 50, more preferably an integer of 5 to 20, and still more preferably an integer of 9 to 11.
• the cyclic peptide of the present invention has high permeability into cells. Since the cyclic peptide of the present invention can inhibit MDMX-p53 interaction, it is useful in the treatment of cancers such as solid tumors in which MDMX is overexpressed.
• the molecular shape factor r calculated by formula (2) is preferably 0.4 to 0.6, more preferably 0.4 to 0.55.
• the peptide is nonionic under physiological circumstances.
• Non-ionic under physiological circumstances means that the peptide does not have substituents that are charged under physiological circumstances.
• the cyclic peptide of the present invention is preferably represented by the following formula (3).
• X is an oxygen atom or a sulfur atom
• a and B each independently represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, or a substituent.
• An aromatic carbocyclic group that may have a substituent a non-aromatic carbocyclic group that may have a substituent, an aromatic heterocyclic group that may have a substituent, or a substituent.
• an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aromatic carbocyclic group, an optionally substituted non-aromatic carbocyclic group is a formula group, an aromatic heterocyclic group which may have a substituent, or a non-aromatic heterocyclic group which may have a substituent, or
• the carbon atoms to which R1 , R2 , R3 , R4 , R5 , R6 , R7, R8 , A and B are bonded are P1 , P2 , P3 , P4 , P5 , P 6 , P 7 and P 8 may form a heterocycle together with the nitrogen atom to which they are bonded,
• m Xaa each independently represent any amino acid residue or any amino acid analog residue
• n is an integer from 0 to 5
• m is an integer from 0 to 2.
• n is 0 or 1.
• m is 1.
• At least one of R 3 to R 8 or Q 3 to Q 8 is an aromatic carbocyclic group that may have a substituent or an aromatic heterocyclic group that may have a substituent. It is a formula group.
• At least one of R 3 , R 5 , R 6 , R 7 , R 8 or Q 3 , Q 5 , Q 6 , Q 7 , and Q 8 is an aromatic carbon which may have a substituent.
• R 1 and Q 1 or A and B, and P 1 together with the carbon atom to which R 1 and Q 1 or A and B are bonded and the nitrogen atom to which P 1 is bonded form a hetero They may form a ring, and the above R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , Q 2 , Q 3 , Q 4 , Q 5 , Q 6 , Q 7 and the carbon atom to which Q 8 is bonded forms a heterocycle together with the nitrogen atom to which P 2 , P 3 , P 4 , P 5 , P 6 , P 7 and P 8 are bonded.
• the Xaa may be proline, and the structure formed from these may contain at least one heterocycle.
• R 1 and Q 1 or A and B, and P 1 together with the carbon atom to which R 1 and Q 1 or A and B are bonded and the nitrogen atom to which P 1 is bonded form a hetero
• the carbon atom to which R 2 and Q 2 are bonded may form a heterocycle together with the nitrogen atom to which P 2 is bonded, and the carbon atom to which R 2 and Q 2 are bonded may form a heterocyclic ring.
• Xaa may be proline, and the structure formed therefrom contains at least one heterocycle.
• R 1 and Q 1 or A and B, and P 1 together with the carbon atom to which R 1 and Q 1 or A and B are bonded and the nitrogen atom to which P 1 is bonded form pyrrolidine.
• the carbon atom to which R 2 and Q 2 are bonded may form a pyrrolidine ring together with the nitrogen atom to which P 2 is bonded.
• Xaa may be proline, and the structure formed therefrom contains at least one pyrrolidine ring.
• R 1 and Q 1 or A and B and Xaa are each any D-amino acid residue or any D-amino acid analog residue.
• the cyclic peptide of the present invention may be modified such as phosphorylation, methylation, acetylation, adenylylation, ADP ribosylation, and glycosylation depending on the purpose.
• the cyclic peptide of the present invention may be a salt. Salts are preferably salts with physiologically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include: acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecyl sulfate, formate, fumarate.
• Acid salt glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate , 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate , tosylate, and undecanoate.
• Salts derived from appropriate bases include alkali metal (eg, sodium) salts, alkaline earth metal (eg, magnesium) salts, ammonium salts, and N-(alkyl)4+ salts.
• the method for producing the cyclic peptide of the present invention is not particularly limited.
• the cyclic peptide of the present invention may be produced by a method using a cell-free translation system or by a chemical peptide synthesis method. Chemical synthesis of peptides can generally be performed using an automatic peptide synthesizer.
• Peptides may be synthesized by solid phase synthesis or liquid phase synthesis, but preferably solid phase synthesis.
• Solid-phase synthesis of peptides is known to those skilled in the art, and includes, for example, combining the hydroxyl group of a resin with a hydroxyl group and the first amino acid whose ⁇ -amino group is protected with a protecting group (usually the C-terminus of the desired peptide).
• Carboxy group of amino acid undergoes esterification reaction.
• dehydration condensation agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIPCDI) can be used.
• MSNT 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole
• DCC dicyclohexylcarbodiimide
• DIPCDI diisopropylcarbodiimide
• the ⁇ -amino group of the second amino acid is deprotected, a third amino acid with all functional groups other than the carboxy group of the main chain protected is added, the carboxy group is activated, and the second and Attach a third amino acid.
• resins for solid phase synthesis include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), HMPA-PEGA resin (Merck), etc. It will be done.
• These resins may be used after being washed with a solvent (dimethylformamide (DMF), 2-propanol, methylene chloride, etc.).
• a solvent dimethylformamide (DMF), 2-propanol, methylene chloride, etc.
• protecting groups for ⁇ -amino groups benzyloxycarbonyl (Cbz or Z) group, tert-butoxycarbonyl (Boc) group, fluorenylmethoxycarbonyl (Fmoc) group, benzyl group, allyl group, allyloxycarbonyl (Alloc) group, etc. ) groups, etc.
• Cbz groups can be deprotected by hydrofluoric acid, hydrogenation, etc.
• Boc groups can be deprotected by trifluoroacetic acid (TFA)
• Fmoc groups can be deprotected by treatment with piperidine.
• methyl ester, ethyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester, etc. can be used.
• the hydroxyl group of serine and threonine can be protected with a benzyl group or tert-butyl group, and the hydroxyl group of tyrosine can be protected with a 2-bromobenzyloxycarbonyl group or tert-butyl group.
• the amino group of the lysine side chain and the carboxy group of glutamic acid and aspartic acid can be protected in the same way as the ⁇ -amino group and ⁇ -carboxy group.
• Activation of the carboxy group can be performed using a condensing agent.
• condensing agent examples include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H benzotriazol-1-yloxy)tris( Examples include dimethylamino)phosphonium hexafluorophosphate (BOP), 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU), and the like. Cleavage of the peptide chain from the resin can be performed by treatment with an acid such as TFA or hydrogen fluoride (HF).
• an acid such as TFA or hydrogen fluoride (HF).
• the method for cyclizing a peptide with a thioether is not particularly limited, but, for example, cyclization can be carried out by including the following functional groups in the side chain or main chain of the peptide.
• the positions of functional groups 1 and 2 are not particularly limited, but either functional group 1 or 2 may be located at the N-terminus or C-terminus of the peptide, both may be located at the terminal, or one may be located at the terminal, The other may be non-terminal, or both may be non-terminal.
• the peptide compound synthesis step and the cyclization reaction step may be separated or may proceed continuously. Cyclization can be performed by methods known to those skilled in the art, such as those described in WO2013/100132, WO2008/117833, WO2012/074129, and the like.
• X1 represents chlorine, bromine or iodine.
• an MDMX inhibitor comprising the cyclic peptide of the present invention or a salt thereof.
• the cyclic peptide of the present invention or a salt thereof has an MDMX inhibitory effect.
• MDMX inhibitory effect refers to the effect of inhibiting the binding between MDMX and p53.
• NCBI National Center for Biotechnology Information
• the cyclic peptide of the present invention can be used in a pharmaceutical composition for the treatment of diseases involving MDMX inhibitors.
• the administration form of the pharmaceutical composition is not particularly limited, and may be administered orally or parenterally.
• parenteral administration include injection administration such as intramuscular injection, intravenous injection, and subcutaneous injection, transdermal administration, and transmucosal administration (nasal, oral, ophthalmic, pulmonary, vaginal, and rectal). administration, etc.
• the cyclic peptide in the pharmaceutical composition can be subjected to various modifications in view of its tendency to be easily metabolized and excreted.
• polyethylene glycol (PEG) or a sugar chain can be added to a cyclic polypeptide to increase its residence time in blood and reduce its antigenicity.
• biodegradable polymer compounds such as polylactic acid, polyglycolic acid, and PLGA, porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc. with sustained release groups. It may be used as an agent and encapsulate a cyclic peptide therein.
• a weak electrical current can be applied to the skin surface to penetrate the stratum corneum.
• the pharmaceutical composition may use the active ingredient as it is, or may be formulated by adding a pharmaceutically acceptable carrier, excipient, additive, etc.
• Dosage forms include, for example, liquids (e.g. injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, troches, Examples include inhalants, ointments, eye drops, nasal drops, ear drops, poultices, and the like.
• Formulation includes, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavoring agents, stabilizers, emulsifiers, absorption enhancers, surfactants, and pH adjustment. It can be carried out by a conventional method using appropriate agents, preservatives, antioxidants, etc.
• ingredients used in formulation include purified water, saline, phosphate buffer, dextrose, glycerol, pharmaceutically acceptable organic solvents such as ethanol, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystals.
• Absorption enhancers that improve the absorption of cyclic peptides include surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponin; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; EDTA and salicylic acids.
• Chelating agents such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, mixed micelles and other fatty acids; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosan, monoxide A nitrogen donor or the like may also be used.
• Pills or tablets can also be coated with sugar-coated, gastric or enteric substances.
• Injectables can include distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, and the like.
• wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubilizers, preservatives, etc. can be added.
• the cyclic peptide and MDMX inhibitor of the present invention can be used as pharmaceuticals, cosmetics, DDS (Drug Delivery System) materials, etc., but are not limited thereto.
• the cyclic peptides and MDMX inhibitors of the present invention are useful in competitive binding assays to identify drugs that bind to MDMX.
• labeled cyclic peptides of the invention can be used in MDMX binding assays with small molecules that competitively bind MDMX.
• Competitive binding assays allow rapid in vitro evaluation and determination of drug candidates specific for the p53/MDMX system. Such binding studies can be performed using the cyclic peptides of the invention.
• the cyclic peptide of the present invention can also be used for producing antibodies against the cyclic peptide of the present invention.
• the cyclic peptides and MDMX inhibitors of the invention can be used to treat subjects with disorders associated with aberrant (eg, insufficient or excessive) expression or activity of p53 or MDMX.
• the disorder is caused by abnormal levels (eg, overexpression or underexpression) of p53 or MDMX, or by the presence of p53 or MDMX that exhibit abnormal activity.
• the cyclic peptides and MDMX inhibitors of the present invention can be used to treat or prevent diseases such as hyperproliferative diseases and inflammatory disorders by interfering with the interaction or binding between p53 and MDMX.
• an effective amount of the cyclic peptide or MDMX inhibitor of the present invention can be administered to mammals including humans.
• the cyclic peptides and MDMX inhibitors of the present invention can be used to treat, prevent, and/or diagnose cancer and neoplastic diseases.
• cancer, hyperproliferative and neoplastic refer to abnormal conditions or diseases characterized by cells with the capacity for autonomous proliferation, ie, rapidly proliferating cell proliferation.
• cancer or neoplastic diseases include, but are not limited to: fibrosarcoma, sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial Sarcoma, lymphangiosarcoma, intralymphatic sarcoma, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer , head and neck cancer, skin cancer, brain cancer, squamous cell carcinoma, sebaceous carcinoma, breast cancer (papillary carcinoma, papillary adenocarcinoma, etc.), cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, Hepatocellular carcinoma, cholangiocarcinoma,
• proliferative disorders include hematopoietic neoplastic disorders.
• Hematopoietic neoplastic disorders include diseases involving hyperplastic/neoplastic cells of hematopoietic origin, eg, originating from the myeloid, lymphoid or erythroid lineages, or their progenitors.
• the disease can be caused by undifferentiated acute leukemias, such as erythroblastic leukemia and acute megakaryoblastic leukemia.
• Mass spectra were measured using ACQUITY SQD LC/MS System (Waters, Inc., ionization method: ESI (ElectroSpray Ionization) method). Retention time (RT) was measured using ACQUITY SQD LC/MS System (Waters) and expressed in minutes (min).
• ⁇ Example 1> 1 shows a synthetic route for the cyclic peptide of the present invention.
• the cyclic peptide of the present invention can be produced, for example, by the general synthesis method shown below.
• R 1 , Q 1 , A and B in general formula (4) are each independently a hydrogen atom, an alkyl group which may have a substituent, an alkenyl group which may have a substituent, Alkynyl group that may have a substituent, aromatic carbocyclic group that may have a substituent, non-aromatic carbocyclic group that may have a substituent, an aromatic heterocyclic group which may have a substituent, and a non-aromatic heterocyclic group which may have a substituent, and n represents an integer of 0 to 5.
• PG represents a protecting group, such as a fluorenylmethoxycarbonyl (Fmoc) group or a tert-butoxycarbonyl (Boc) group.
• Step 1 Supporting thiol group-containing compounds on 2-chlorotrityl chloride resin
• 2-chlorotrityl chloride 100-200 mesh, 1% DVB, purchased from Watanabe Chemical Co., Ltd., 100 mg, 0.137 mmol
• dehydrated dichloromethane (2 mL)
• a mixture of 4 molar equivalents of a dichloromethane (0.5 M) solution of a thiol group-containing compound represented by general formula (3) and 5 molar equivalents of diisopropylethylamine was added to the resin. Added to container and shaken for 2 hours.
• Step 2 Peptide solid-phase synthesis using an automatic peptide synthesizer
• Peptide solid-phase synthesis was performed using an automatic peptide synthesizer (Syro I manufactured by Biotage). For detailed operating procedures, the manual provided with the synthesizer was followed.
• One cycle consisted of Fmoc deprotection (20 minutes), washing with NMP, condensation of Fmoc amino acids (1 hour), and washing with NMP, and by repeating this cycle, the peptide chain was elongated. After condensing the N-terminal amino acid, chloroacetic acid was condensed to the N-terminal amino group using the same procedure.
• Step 3 Cutting out from the resin and deprotecting the protecting group of the side chain functional group Because cutting out the linear peptide from the resin and deprotecting the protecting group of the side chain functional group are performed simultaneously, the amount is equivalent to 5 times the resin.
• TCEP solution 0.5 M was added and stirred at room temperature for 30 minutes to 48 hours. After confirming that the raw linear peptide had disappeared by LC/MS mass spectrometry (Acquity UPLC/SQD manufactured by Waters), the solvent was distilled off under reduced pressure to obtain a crudely purified cyclic peptide.
• Step 5 Cyclic peptide purification The obtained crude product was purified by liquid chromatography. Finally, the desired cyclic peptide was obtained as a lyophilized powder.
• Fmoc-amino acids were used in the synthesis of the cyclic peptides described herein. Furthermore, in the sequences shown in this specification, the following abbreviations are used. Fmoc-amino acids were purchased from Watanabe Chemical Co., Ltd., CHEM-IMPEX, Acrotein, or Amatek Chemical.
• reaction solution was cooled to below -30°C, and isobutyl chloroformate (0.36 mL, 2.79 mmol) was added. After stirring the reaction solution at -30°C for 30 minutes, an aqueous solution (3 M, 3 mL, 0.28 g) of oily sodium hydride (60%) was added dropwise over 5 minutes. The mixture was stirred at -30°C for 30 minutes. Then, using an ice bath, saturated citric acid water (20 mL) was slowly added dropwise to the reaction mixture, and the mixture was stirred for 30 minutes. After warming to room temperature, ethyl acetate (20 mL) was added to the reaction mixture.
• a dimethylformamide solution (1 mL/g, 0.2 mL) of compound aa6 (0.20 g, 0.41 mmol) was added dropwise to the reaction solution, and the mixture was stirred at room temperature for 1 hour. Then, pure water (10 mL) was slowly added dropwise and stirred for 30 minutes. Extracted twice with ethyl acetate (10 mL), and the obtained organic layers were combined and washed with saturated brine.
• Step 1 of the general method the thiol group of compound aa3 was supported on 2-chlorotrityl chloride resin.
• Fmoc-C2S-2-chlorotrityl resin (1.01 mmol/g, 0.05 mmol, 50 mg) was suspended in NMP, allowed to swell for 1 hour, and NMP was removed by filtration.
• Fmoc-NMe-Ala, Fmoc-Hse(Me)-OH, Fmoc-D-Pro-OH, Fmoc-NMe-Phe-OH, Fmoc-Phe(4-Cl)-OH, Fmoc-Thr(Trt)-OH , Fmoc-Phe(4-F)-OH, Fmoc-NMe-Ala-OH, and chloroacetic acid were condensed in this order. After the extension was completed, the resin was washed with NMP and dichloromethane, and then the solvent was distilled off under reduced pressure.
• Step 1 of the general method the thiol group of compound aa13 was supported on 2-chlorotrityl chloride resin.
• Fmoc-D-Pro-S-2-chlorotrityl resin (0.99 mmol/g, 0.05 mmol, 51 mg) was suspended in NMP, allowed to swell for 1 hour, and NMP was removed by filtration.
• Fmoc-NMe-Phe-OH, Fmoc-Phe(4-Cl)-OH, Fmoc-Thr(Trt)-OH, Fmoc-Phe(4-F)-OH, Fmoc-NMe-Ala-OH, Fmoc-D -NMe-Ala, Fmoc-Ala(4-Thz)-OH, Fmoc-NMe-Ala, and chloroacetic acid were condensed in this order. After the extension was completed, the resin was washed with NMP and dichloromethane, and then the solvent was distilled off under reduced pressure.
• Cyclic Peptides 3 to 6 and Comparative Examples 1 and 2 Cyclic peptides 3 to 6 were synthesized in the same manner as cyclic peptide 1. Comparative Example 1 was synthesized by the method described in WO2021102322. Comparative Example 2 was synthesized by the method described in WO2013123266.
• Table 1 shows the sequences of the cyclic peptides.
• the left side shows the N-terminal chloroacetylated amino acid residue
• the right side shows the thiol group-containing compound. forming a peptide.
• Comparative Example 1 the left side shows the N-terminal amino acid residue
• the right side shows the C-terminal amino acid residue
• both ends of the peptide form a cyclic peptide by forming an amide bond.
• Example 2 Alpha Screen assay for MDMX of the cyclic peptide according to the present invention was performed.
• MDMX MDMX (24-108, His-tag at the N-terminus; PDB ID: 2MWY, https://www.rcsb.org/structure/2MWY) synthesized by solid phase synthesis was used.
• a p53 fragment peptide [manufactured by AnaSpec, p53(17-26) FITC-labeled] was used as a binding partner for MDMX.
• Anti-FITC AlphaScreen was used as donor beads.
• Anti-His tag AlphaLISA was used as the acceptor beads.
• the composition of the buffer used for the reaction was PBS, 0.1% BSA, and 0.01% Tween 20.
• DMSO final concentration 1-5% adjusted compound
• MDMX 5 ⁇ L final concentration 1 ⁇ M
• p53 peptide 5 ⁇ L final concentration 10 nM
• a total of 10 ⁇ L of acceptor beads modified with anti-His tag antibody and donor beads modified with anti-FITC antibody were added at a final concentration of 20 ⁇ g/mL, and incubated at room temperature for 1 hour.
• the fluorescence signal was measured using Envision. In order to prevent signal attenuation due to light during liquid preparation, the experiment was conducted under a light source equipped with a green filter.
• Example 3 Cell membrane permeability evaluation of the cyclic peptide according to the present invention was carried out. 300 ⁇ L of 1.0 ⁇ 10 6 cells/mL MDCKII cells (ECACC standard cell line) were seeded in an insert (for 24 well plate, pore size 3.0 ⁇ m, manufactured by CORNING) and cultured at 37° C. in a 5% CO 2 environment. After 3 days, the electrical resistance value (measuring device) of the cell layer was measured and it was confirmed that it had high barrier properties (>100 ⁇ cm2).
• ⁇ Permeation test> After washing the insert by immersing it in HBSS (without phenol red, the same applies hereafter), 200 ⁇ L of the sample prepared at 10 ⁇ M/HBSS was added, and the insert was placed in a low-adsorption 24-well plate containing 900 ⁇ L of HBSS ( 37°C 5% CO2 ). After 2 hours, the upper (apical) and lower (basal) layer solutions (10 ⁇ L for apical and 500 ⁇ L for basal) of the insert were collected. After testing, we confirmed that there was no leakage using Lucifer Yellow, an opaque fluorescent dye.
• V basal volume (0.9 mL)
• C0 initial concentration (10 ⁇ M)
• S Surface area of monolayer film (0.33 cm2)
• dC/dt basal concentration change [ ⁇ M/s]
• Example 4 Calculating the molecular shape factor r value using MD calculation
• the structural formula drawn in two dimensions of a cyclic peptide is input into Chem3D and structured in three dimensions ( Figure 1).
• the structure is optimized using a quantum chemical calculation method (B3LYP/6-31G*, software is Gaussian) to obtain a locally stable structure.
• the electrostatic field generated by the cyclic peptide is determined using a quantum chemical calculation method (B3LYP/6-31G*, software is Gaussian), and a point charge (RESP) is placed on each atom to reproduce the above electrostatic field. charge).
• the state of covalent bonds between each atom is analyzed (Amber) and van der Waals parameters (gaff2) are assigned to each atom.
• the book charge and the book van der Waals parameters together are called a force field.
• a molecular dynamics (MD) simulation is performed in chloroform using the above locally stable structure as the initial structure (software is Gromacs and plumed).
• MD simulations we use the replica exchange MD method, which uses not only room temperature but also higher temperatures during simulation, as an efficient method to efficiently search a wide conformational space. Six temperatures were used (six types of replicas), as shown in the table below. Note that this temperature is applied only to the cyclic peptide, and 298 K is always applied to the chloroform present around the cyclic peptide.
• the three-dimensional coordinates of atoms belonging to the main chain of the above-mentioned most stable cyclic peptide are (X a,1 , X a,2 , X a,3 )
• a is a label that identifies an atom belonging to the main chain, and is an integer from 1 to N.
• N is the total number of atoms belonging to the main chain of the cyclic peptide.
• Calculate the r value for this three-dimensional coordinate Calculation of the r value can be performed using the following steps.
• the main chain structure of the peptide contains a sulfur atom.
• the peptide is nonionic under physiological circumstances.

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