Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
  • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
  • C07K1/063—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups for alpha-amino functions
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
  • C07K5/06147—Dipeptides with the first amino acid being heterocyclic and His-amino acid; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/08—Tripeptides
  • C07K5/0802—Tripeptides with the first amino acid being neutral
  • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/10—Tetrapeptides
  • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic

Definitions

• the present invention relates to a method for removing dibenzofulvene (DBF) or dibenzofulvene derivatives.
• DPF dibenzofulvene
• the Fmoc (9-fluorenylmethoxycarbonyl) group is widely used in peptide synthesis as a protecting group for the amino groups of amino acids and peptides.
• Peptide synthesis can be performed using solid-phase synthesis or liquid-phase synthesis, with liquid-phase synthesis often being used for mass production of pharmaceuticals and other products.
• the Fmoc group can be easily deprotected under basic conditions, so it is also used as a protecting group for amino groups in liquid-phase synthesis.
• dibenzofulvene or a compound in which the amine used in the deprotection is added to dibenzofulvene (hereinafter also referred to as the "amine adduct") is generated as a by-product. If peptide synthesis is continued while the by-product remains, it may cause side reactions such as 9-fluorenylmethylation, so the dibenzofulvene or amine adduct must be removed in this deprotection step.
• Patent Document 1 describes a method of removing the amine adduct as a carbonate by contacting a reaction mixture containing the amine adduct with carbon dioxide.
• the object of the present invention is to provide a new method for removing dibenzofulvene or dibenzofulvene derivatives that can capture dibenzofulvene or dibenzofulvene derivatives and remove them without regenerating them.
• the present inventors have investigated a new method for removing dibenzofulvene or a dibenzofulvene derivative, and have found that by using sulfite ions or hydrogen sulfite ions or a compound that generates these ions as a scavenger, dibenzofulvene or a dibenzofulvene derivative can be captured and removed without regeneration, leading to the completion of the present invention.
• the present inventors have found that the present invention can be applied to a wide variety of synthetic processes, particularly the synthesis of amino group-containing compounds, for example, the synthesis of peptides.
• the present invention also provides a new method for deprotecting a protecting group having an Fmoc skeleton.
• the present invention provides a new method for producing an amino group-containing compound such as a peptide.
• the present invention includes the following.
• a method for removing dibenzofulvene or a dibenzofulvene derivative comprising a step of mixing the following (i) and (ii): (i) dibenzofulvene or a dibenzofulvene derivative, (ii) Sulfite or bisulfite ions or compounds which generate sulfite or bisulfite ions.
• a method for removing dibenzofulvene or a dibenzofulvene derivative comprising the following steps (1), (1'), or (1''): (1) mixing the following (i) and (ii): (i) dibenzofulvene or a dibenzofulvene derivative, (ii) a sulfite ion or hydrogen sulfite ion, or a compound (1') capable of generating a sulfite ion or hydrogen sulfite ion, comprising mixing the following (i) and (ii): (i) dibenzofulvene or a dibenzofulvene derivative, (ii) At least one member selected from the group consisting of hydrogen sulfite, disulfite and its salts, sulfurous acid and its salts, dithionous acid and its salts, and solvates thereof.
• (1′′) A step of forming (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof.
• (1') A method for removing dibenzofulvene or a dibenzofulvene derivative, comprising a step of mixing the following (i) and (ii): (i) dibenzofulvene or a dibenzofulvene derivative, (ii) At least one member selected from the group consisting of hydrogen sulfite, disulfite and its salts, sulfurous acid and its salts, dithionous acid and its salts, and solvates thereof.
• a method for removing dibenzofulvene or a dibenzofulvene derivative from a mixture containing dibenzofulvene or a dibenzofulvene derivative comprising a step of mixing (i) dibenzofulvene or a dibenzofulvene derivative with (ii) sulfite ion or hydrogen sulfite ion, or a compound capable of generating sulfite ion or hydrogen sulfite ion, to form (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof.
• a method for removing dibenzofulvene or a dibenzofulvene derivative from a mixture containing dibenzofulvene or a dibenzofulvene derivative comprising a step of mixing (i) dibenzofulvene or a dibenzofulvene derivative with (ii) at least one selected from the group consisting of hydrogensulfite, disulfite and its salts, sulfurous acid and its salts, dithionous acid and its salts, and solvates thereof, to form (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof.
• [6-1](2) The method according to any one of [1] to [5], comprising a step of mixing a first amino-group-containing compound, in which an amino group is protected with a protecting group having an Fmoc skeleton, with a deprotecting agent capable of deprotecting the protecting group having an Fmoc skeleton, before the step (1), (1'), (1'', (1''') or (1'''').
• a deprotecting agent capable of deprotecting the protecting group having an Fmoc skeleton
• a method for removing dibenzofulvene or a dibenzofulvene derivative comprising a step of mixing the following (i) to (iii): (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent, (iii) Sulfite or bisulfite ions or compounds which generate sulfite or bisulfite ions.
• a method for removing dibenzofulvene or a dibenzofulvene derivative comprising a step of mixing the following (i) to (iii): (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) Sulfite or bisulfite ions or compounds which generate sulfite or bisulfite ions.
• a method for deprotecting a protecting group having an Fmoc skeleton comprising mixing the following (i) to (iii): (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) Sulfite or bisulfite ions or compounds which generate sulfite or bisulfite ions.
• a method for removing dibenzofulvene or a dibenzofulvene derivative comprising a step of mixing the following (i) to (iii): (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent, (iii) At least one member selected from the group consisting of hydrogen sulfite, disulfite and its salts, sulfurous acid and its salts, dithionous acid and its salts, and solvates thereof.
• a method for removing dibenzofulvene or a dibenzofulvene derivative comprising a step of mixing the following (i) to (iii): (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) At least one member selected from the group consisting of hydrogen sulfite, disulfite and its salts, sulfurous acid and its salts, dithionous acid and its salts, and solvates thereof.
• [B1-1] (3') A method for deprotecting a protecting group having an Fmoc skeleton, comprising a step of mixing the following (i) to (iii): (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) At least one member selected from the group consisting of hydrogen sulfite, disulfite and its salts, sulfurous acid and its salts, dithionous acid and its salts, and solvates thereof.
• a method for removing dibenzofulvene or a dibenzofulvene derivative comprising the following steps (3) or (3'): (3) A step of mixing the following (i) to (iii): (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) a step of mixing sulfite ion or hydrogen sulfite ion, or a compound capable of generating sulfite ion or hydrogen sulfite ion (3') with the following (i) to (iii): (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) At
• a method for deprotecting a protecting group having an Fmoc skeleton comprising the following step (3) or (3′): (3) A step of mixing the following (i) to (iii): (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) a step of mixing sulfite ion or hydrogen sulfite ion, or a compound capable of generating sulfite ion or hydrogen sulfite ion (3') with the following (i) to (iii): (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) At least one
• a method for removing dibenzofulvene or a dibenzofulvene derivative from a mixture containing dibenzofulvene or a dibenzofulvene derivative comprising the steps of mixing the following (i) to (iii) to form (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof: (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) Sulfite or bisulfite ions or compounds which generate sulfite or bisulfite ions.
• a method for removing dibenzofulvene or a dibenzofulvene derivative from a mixture containing dibenzofulvene or a dibenzofulvene derivative comprising the step of mixing the following (i) to (iii) to form (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof: (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) At least one member selected from the group consisting of hydrogen sulfite, disulfite and its salts, sulfurous acid and its salts, dithionous acid and its salts, and solvates thereof.
• a method for removing dibenzofulvene or a dibenzofulvene derivative from a mixture containing dibenzofulvene or a dibenzofulvene derivative comprising the following steps (3) or (3'): (3) A step of mixing the following (i) to (iii): (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) a step of mixing sulfite ion or hydrogen sulfite ion, or a compound capable of generating sulfite ion or hydrogen sulfite ion (3') with the following (i) to (iii): (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of de
• a method for deprotecting a protecting group having an Fmoc skeleton comprising a step of mixing the following (i) to (iii) to form (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof: (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) Sulfite or bisulfite ions or compounds which generate sulfite or bisulfite ions.
• [B1-4] (3') A method for deprotecting a protecting group having an Fmoc skeleton, comprising a step of mixing the following (i) to (iii) to form (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof: (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) At least one member selected from the group consisting of hydrogen sulfite, disulfite and its salts, sulfurous acid and its salts, dithionous acid and its salts, and solvates thereof.
• a method for deprotecting a protecting group having an Fmoc skeleton comprising the following step (3) or (3′): (3) A step of mixing the following (i) to (iii): (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) sulfite ion or bisulfite ion or a compound which generates sulfite ion or bisulfite ion; (3') A step of mixing the following (i) to (iii): (i) a first amino group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton; (ii) a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton; (iii) At least one member selected from the group consist
• R 1 -R 8 are independently selected from the group consisting of hydrogen, C 1 -C 8 alkyl, C 1 -C 8 fluoroalkyl, halogen, sulfo, and trimethylsilyl, and R 9 -R 10 are independently hydrogen or methyl.
• [17-1] The method according to any one of [1-1], [3] to [16], and [B1], wherein the (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof is a compound represented by the following formula (2) or a salt thereof, or a compound represented by the following formula (3):
• R 1 -R 8 are independently selected from the group consisting of hydrogen, C 1 -C 8 alkyl, C 1 -C 8 fluoroalkyl, halogen, sulfo, and trimethylsilyl
• R 9 -R 10 are independently hydrogen or methyl, provided that no two of R 1 -R 8 are hydrogen.
• R 1 -R 8 are independently selected from the group consisting of hydrogen, C 1 -C 8 alkyl, C 1 -C 8 fluoroalkyl, halogen, sulfo, and trimethylsilyl, and R 9 -R 10 are independently hydrogen or methyl, provided that no two of R 1 -R 8 are hydrogen.
• [18-1] The method according to any one of [6] to [17] or [B1], wherein the protecting group having an Fmoc skeleton is a compound represented by the following formula (4): (Wherein, R 1 -R 8 are independently selected from the group consisting of hydrogen, C 1 -C 8 alkyl, C 1 -C 8 fluoroalkyl, halogen, sulfo, and trimethylsilyl, R 9 -R 10 are independently hydrogen or methyl, and no two of R 1 -R 8 are hydrogen. The wavy line represents the point of attachment to the amino group.
• the protecting group having an Fmoc skeleton is a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a 2,7-di-tert-butyl-Fmoc (Fmoc(2,7tb)) group, a 1-methyl-Fmoc (Fmoc(1Me)) group, a 2-fluoro-Fmoc (Fmoc(2F)) group, a 2,7-dibromo-Fmoc (Fmoc(2,7Br)) group, a 2-monoisooctyl-Fmoc (mio-Fmoc) group, a 2,7-diisooctyl-Fmoc (dio-Fmoc) group, a 2,7-(3,3,4,4,5,5,6,6,7,7,8
• [20] The method according to any one of [6] to [19] or [B1], wherein the protecting group having an Fmoc skeleton is a 9-fluorenylmethyloxycarbonyl group.
• the first amino group-containing compound is a peptide, an amino acid, or an amino acid amide.
• the first amino group-containing compound is a peptide or an amino acid.
• the first amino group-containing compound is a peptide.
• [22-1] The method according to [21], wherein the first amino group-containing compound is an amino acid.
• [26-1] The method according to any one of [1], [4] to [23], and [B1], wherein the sulfite ion or hydrogen sulfite ion, or the compound capable of generating sulfite ion or hydrogen sulfite ion, is at least one selected from the group consisting of hydrogen sulfite, disulfite and its salts, sulfurous acid and its salts, dithionous acid and its salts, and solvates thereof.
• the compound capable of generating sulfite ions or hydrogen sulfite ions is at least one selected from the group consisting of sodium hydrogen sulfite, sodium metabisulfite, potassium hydrogen sulfite, sodium dithionite, ammonium sulfite monohydrate, calcium sulfite 0.5 hydrate, sodium sulfite, and potassium sulfite.
• [31] The method according to [25], wherein the compound generating sulfite ions or hydrogen sulfite ions is at least one selected from the group consisting of sodium hydrogen sulfite, potassium hydrogen sulfite, and sodium dithionite.
• [31-1] The method according to any one of [1], [4] to [23], and [B1], wherein the sulfite ion or hydrogen sulfite ion, or the compound that generates the sulfite ion or hydrogen sulfite ion, is at least one selected from the group consisting of sodium hydrogen sulfite, potassium hydrogen sulfite, and sodium dithionite.
• [32] The method according to any one of [1] to [31] or [B1], further comprising mixing an additive in the step (1), (1'), (1'', (1'''), (1'''', (3) or (3').
• the additive is a first base.
• the first base is a tertiary amine.
• the tertiary amine is at least one selected from the group consisting of triethylamine, N,N-diisopropylethylamine, and 2,6-lutidine.
• [36] The method according to any one of [1] to [35], further comprising mixing water in the step (1), (1'), (1'', (1'''), (1'''', (3) or (3').
• [36-1] The method according to any one of [1] to [35], wherein the step (1), (1'), (1'', (1'''), (1'''', (3) or (3') does not include further mixing with water.
• [36-2] The method according to [36], wherein the water is not water contained in an organic solvent.
• [37] The method according to any one of [6] to [36] or [B1], wherein the deprotecting agent is a second base.
• the second base is at least one selected from the group consisting of an organic base having an amidine skeleton, a primary amine, a secondary amine, a tertiary amine, and an inorganic base (with the proviso that none of the primary amine, secondary amine, and tertiary amine has an amidine skeleton in the molecule).
• the second base is an organic base having an amidine skeleton.
• the organic base having an amidine skeleton is at least one selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]-5-nonene (DBN), and 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD).
• DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
• DBU 1,5-diazabicyclo[4.3.0]-5-nonene
• MTBD 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene
• DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
• the second base is a primary amine.
• [54] The method according to any one of [6] to [51] or [B1], wherein one or more of steps (1), (1'), (1'', (1'''), (1'''), and (2) are carried out in the presence of a solvent containing at least one selected from the group consisting of acetonitrile, dimethylacetamide, 2-methyltetrahydrofuran, and methanol.
• a solvent containing at least one selected from the group consisting of acetonitrile, dimethylacetamide, 2-methyltetrahydrofuran, and methanol [54-1] The method according to any one of [6] to [51] or [B1], wherein one or more of steps (1), (1'), (1'', (1'''), (1''''), and (2) are carried out in the presence of a solvent containing acetonitrile.
• [54-2] The method according to any one of [6] to [51] or [B1], wherein one or more steps among (3) and (3') are carried out in the presence of a solvent containing at least one selected from the group consisting of acetonitrile, methanol, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide.
• a solvent containing at least one selected from the group consisting of acetonitrile, methanol, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide [54-1] The method according to any one of [6] to [51] or [B1], wherein one or more steps among (3) or (3') are carried out in the presence of a solvent containing 1,3-dimethyl-2-imidazolidinone.
• [55] The method according to any one of [13] to [54], wherein the cleaning solution has a pH of 10 to 14.
• a method for producing a peptide compound comprising the following steps: 1) treating a peptide protected with a protecting group having an Fmoc skeleton with a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton in the presence of sulfite ions or hydrogen sulfite ions to obtain a deprotected peptide by removing the protecting group; 2) elongating the deprotected peptide, optionally with one or more amino acids, to obtain an elongated peptide.
• a method for producing a peptide compound comprising the following steps: 1) treating a protected peptide, in which an amino group is protected with a protecting group having an Fmoc skeleton, with a deprotecting agent capable of deprotecting the protecting group having an Fmoc skeleton to obtain (a) dibenzofulvene or a dibenzofulvene derivative, and (b) a first mixture of deprotected peptide from which the protecting group has been removed; 2) treating the first mixture with sulfite or bisulfite ions to remove the dibenzofulvene or dibenzofulvene derivative to obtain a second mixture comprising the deprotected peptide; 3) elongating the deprotected peptide, optionally with one or more amino acids, to obtain an elongated peptide.
• [A3] The method of [A1] or [A2], wherein the deprotected peptide or the extended peptide has an amino acid residue having one reactive site on the C-terminal side and an amino acid residue having another reactive site on the N-terminal side, and further comprises a step of bonding the one reactive site and the other reactive site to form a cyclic peptide compound.
• the first compound is an amino acid or a peptide.
• the first compound is (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof.
• the present invention provides a new method for removing dibenzofulvene that can capture dibenzofulvene generated during the deprotection process of a protecting group having an Fmoc skeleton and can remove the dibenzofulvene without regenerating it.
• dibenzofulvene derivative refers to a compound in which an arbitrary substituent is introduced at an arbitrary position on the fluorene ring of dibenzofulvene.
• dibenzofulvene derivative include compounds represented by the following formula: (Wherein, R 1 -R 8 are independently selected from the group consisting of hydrogen, C 1 -C 8 alkyl, C 1 -C 8 fluoroalkyl, halogen, sulfo, and trimethylsilyl, wherein at least one of R 1 -R 8 is other than hydrogen, and R 9 -R 10 are independently hydrogen or methyl.
• the dibenzofulvene derivative is preferably one in which only one, two, three or four of R 1 to R 8 are not hydrogen, more preferably one in which only one or two of R 1 to R 8 are not hydrogen, and most preferably one in which only two of R 1 to R 8 are not hydrogen.
• (9H-fluoren-9-yl)methanesulfonic acid and “(9H-fluoren-9-yl)methanesulfonic acid derivative” refer to compounds in which a group represented by "-SO 3 H” has been introduced at the end of the exo-olefin in dibenzofulvene and dibenzofulvene derivatives.
• (9H-fluoren-9-yl)methanesulfonic acid and (9H-fluoren-9-yl)methanesulfonic acid derivatives include compounds represented by the following formula: (Wherein, R 1 -R 8 are independently selected from the group consisting of hydrogen, C 1 -C 8 alkyl, C 1 -C 8 fluoroalkyl, halogen, sulfo, and trimethylsilyl, and R 9 -R 10 are independently hydrogen or methyl.
• the (9H-fluoren-9-yl)methanesulfonic acid derivative is preferably one in which only one, two, three or four of R 1 to R 8 are not hydrogen, more preferably one in which only one or two of R 1 to R 8 are not hydrogen, and most preferably one in which only two of R 1 to R 8 are not hydrogen.
• salts of (9H-fluoren-9-yl)methanesulfonic acid and “salts of (9H-fluoren-9-yl)methanesulfonic acid derivatives” refer to salts of compounds in which a group represented by "-SO 3 - " has been introduced onto the exo-olefin in dibenzofulvene and dibenzofulvene derivatives, or salts of compounds in which a group represented by "-SO 3 H” has been introduced onto the terminal of the exo-olefin in dibenzofulvene and dibenzofulvene derivatives.
• Salts of the compounds described herein can be, for example, salts with at least one selected from the group consisting of alkali metals, alkaline earth metals, and ammonium (NH 4 + ).
• Examples of salts of (9H-fluoren-9-yl)methanesulfonic acid and salts of (9H-fluoren-9-yl)methanesulfonic acid derivatives include compounds represented by the following formula: (Wherein, R 1 -R 8 are independently selected from the group consisting of hydrogen, C 1 -C 8 alkyl, C 1 -C 8 fluoroalkyl, halogen, sulfo, and trimethylsilyl, and R 9 -R 10 are independently hydrogen or methyl.
• the salt of the (9H-fluoren-9-yl)methanesulfonic acid derivative is preferably one in which only one, two, three or four of R 1 to R 8 are not hydrogen, more preferably one in which only one or two of R 1 to R 8 are not hydrogen, and most preferably one in which only two of R 1 to R 8 are not hydrogen.
• protecting group having an Fmoc skeleton refers to an Fmoc group or a group in which an arbitrary substituent has been introduced at an arbitrary position of the structural skeleton of an Fmoc group.
• Specific examples of such a protecting group containing an Fmoc skeleton include the protecting group represented by the following formula: (Wherein, R 1 -R 8 are independently selected from the group consisting of hydrogen, C 1 -C 8 alkyl, C 1 -C 8 fluoroalkyl, halogen, sulfo, and trimethylsilyl; R 9 -R 10 are independently hydrogen or methyl.
• the wavy line represents the point of attachment to the amino group.
• R 1 to R 8 are not hydrogen, more preferably only one or two of R 1 to R 8 are not hydrogen, and most preferably only two of R 1 to R 8 are not hydrogen.
• protective groups having an Fmoc skeleton include 9-fluorenylmethyloxycarbonyl (Fmoc) group, 2,7-di-tert-butyl-Fmoc (Fmoc(2,7tb)) group, 1-methyl-Fmoc (Fmoc(1Me)) group, 2-fluoro-Fmoc (Fmoc(2F)) group, 2,7-dibromo-Fmoc (Fmoc(2,7Br)) group, 2-monoisooctyl-Fmoc (mio-Fmoc) group, 2,7-diisooctyl-Fmoc (dio-Fmoc) group, 2,7-(3,3, 4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-Fmoc (tdf-Fmoc) group, 2,7-bis(trimethylsilyl)-Fmoc (t
• removing a protecting group is also referred to as “deprotecting a protecting group.”
• capturing dibenzofulvene or a dibenzofulvene derivative means forming (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof through the coexistence of dibenzofulvene or a dibenzofulvene derivative and sulfite ions or hydrogen sulfite ions.
• the term “sulfite ion” refers to an ion represented by “SO 3 2- "
• the term “hydrogen sulfite ion” refers to an ion represented by "HSO 3 - "
• the term “compound that generates sulfite ion or hydrogen sulfite ion” is not particularly limited as long as it is a compound that generates sulfite ion or hydrogen sulfite ion by ionization in a solution (preferably in an aqueous solution), and examples thereof include hydrogen sulfite, disulfite (H 2 S 2 O 5 ) and its salts, sulfurous acid (H 2 SO 3 ) and its salts, dithionous acid (H 2 S 2 O 4 ) and its salts, and solvates thereof.
• Examples of hydrogen sulfite, disulfite, sulfite, and dithionite include salts with at least one selected from the group consisting of alkali metals such as sodium and potassium; alkaline earth metals such as calcium, and ammonium.
• Specific examples of hydrogen sulfite include sodium hydrogen sulfite and potassium hydrogen sulfite.
• Specific examples of disulfites include sodium disulfite (sodium metabisulfite), potassium disulfite (potassium metabisulfite), etc.
• Specific examples of sulfites include sodium sulfite, potassium sulfite, ammonium sulfite, calcium sulfite, etc.
• Specific examples of dithionites include sodium dithionite, potassium dithionite, etc.
• halogen examples include F, Cl, Br, and I.
• alkyl refers to a monovalent group derived by removing any one hydrogen atom from an aliphatic hydrocarbon, and is a group having a hydrocarbyl or hydrocarbon group structure subset containing hydrogen and carbon atoms, without containing heteroatoms (meaning atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton.
• the alkyl includes not only linear ones but also branched ones.
• alkyl examples include alkyl having 1 to 20 carbon atoms (C 1 -C 20 , hereinafter "C p -C q " means that the number of carbon atoms is p to q), preferably C 1 -C 10 alkyl, more preferably C 1 -C 8 alkyl, and even more preferably C 1 -C 6 alkyl.
• alkyl examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, isobutyl (2-methylpropyl), n-pentyl, s-pentyl (1-methylbutyl), t-pentyl (1,1-dimethylpropyl), neopentyl (2,2-dimethylpropyl), isopentyl (3-methylbutyl), 3-pentyl (1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, n-hexyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylmethylprop
• fluoroalkyl refers to a group in which one or more hydrogen atoms of the "alkyl” defined above have been substituted with fluorine atoms, and is preferably a C 1 -C 8 fluoroalkyl.
• fluoroalkyl include monofluoromethyl, difluoromethyl, trifluoromethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3-difluoropropyl, 4,4-difluorobutyl, 5,5-difluoropentyl, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl, and the like.
• sulfo refers to a monovalent group represented by -SO3H .
• alkenyl refers to a monovalent group having at least one double bond (two adjacent SP 2 carbon atoms). Depending on the arrangement of the double bond and the substituents (if any), the geometry of the double bond can be in an Entadel (E) or Entumble (Z), cis or trans configuration. Alkenyl includes not only linear but also branched chains.
• Alkenyl is preferably C 2 -C 10 alkenyl, more preferably C 2 -C 6 alkenyl, and specifically includes, for example, vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, hexenyl, and the like.
• alkynyl refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Alkynyl includes not only straight chain but also branched chain. Preferred examples of alkynyl include C 2 -C 10 alkynyl, more preferably C 2 -C 6 alkynyl, and specific examples thereof include ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propynyl, 3-(2'-fluorophenyl)-2-propynyl, 2-hydroxy-2-propynyl, 3-(3-fluorophenyl)-2-propynyl, and 3-methyl-(5-phenyl)-4-pentynyl.
• aryl refers to a monovalent aromatic hydrocarbon ring, preferably C 6 -C 10 aryl. Specific examples of aryl include phenyl and naphthyl (e.g., 1-naphthyl, 2-naphthyl).
• heteroaryl refers to an aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms.
• the ring may be a single ring or a condensed ring with other rings, and may be partially saturated.
• the number of atoms constituting the ring is preferably 5 to 10 (5- to 10-membered heteroaryl), and more preferably 5 to 7 (5- to 7-membered heteroaryl).
• heteroaryl examples include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, indolizinyl, and imidazopyridyl.
• aralkyl refers to a group in which at least one hydrogen atom of an "alkyl” as defined above is substituted with an "aryl” as defined above, preferably a C 7 -C 14 aralkyl, more preferably a C 7 -C 10 aralkyl.
• aryl as defined above
• Specific examples of aralkyl include benzyl, phenethyl, and 3-phenylpropyl.
• heteroarylalkyl refers to a group in which at least one hydrogen atom of an "alkyl” as defined above is substituted with a “heteroaryl” as defined above, and is preferably a 5- to 10-membered heteroaryl C 1 -C 6 alkyl, and more preferably a 5- to 10-membered heteroaryl C 1 -C 2 alkyl.
• heteroarylalkyl examples include 3-thienylmethyl, 4-thiazolylmethyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-(2-pyridyl)ethyl, 2-(3-pyridyl)ethyl, 2-(4-pyridyl)ethyl, 2-(6-quinolyl)ethyl, 2-(7-quinolyl)ethyl, 2-(6-indolyl)ethyl, 2-(5-indolyl)ethyl, and 2-(5-benzofuranyl)ethyl.
• cycloalkyl refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic (fused, bridged, bicyclic spiro, etc.) and other polycyclic rings.
• Preferred cycloalkyls include C3 - C8 cycloalkyls, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, spiro[3.3]heptyl, and the like.
• alkoxy refers to an oxy group bonded to the above-defined “alkyl”, and is preferably a C 1 -C 6 alkoxy. Specific examples of alkoxy include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, pentyloxy, and 3-methylbutoxy.
• fluoroalkoxy refers to a group in which one or more hydrogen atoms of the "alkoxy” defined above are substituted with fluorine atoms, and is preferably a C 1 -C 8 fluoroalkoxy. Specific examples of fluoroalkoxy include monofluoromethoxy, difluoromethoxy, and trifluoromethoxy.
• amino refers to -NRR', where R and R' are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or R and R' are taken together with the nitrogen atom to which they are attached to form a ring.
• Preferred examples of amino include -NH 2 , mono-C 1 -C 6 alkylamino, di-C 1 -C 6 alkylamino, and 4- to 8-membered cyclic amino.
• aminocarbonyl refers to a carbonyl group bonded to the above-defined “amino”, and preferably includes -CONH 2 , mono-C 1 -C 6 alkylaminocarbonyl, di-C 1 -C 6 alkylaminocarbonyl, and 4- to 8-membered cyclic aminocarbonyl.
• aminocarbonyl examples include -CONH 2 , dimethylaminocarbonyl, 1-azetidinylcarbonyl, 1-pyrrolidinylcarbonyl, 1-piperidinylcarbonyl, 1-piperazinylcarbonyl, 4-morpholinylcarbonyl, 3-oxazolidinylcarbonyl, 1,1-dioxidethiomorpholinyl-4-ylcarbonyl, and 3-oxa-8-azabicyclo[3.2.1]octan-8-ylcarbonyl.
• alkylsulfonyl refers to a sulfonyl group having an “alkyl” bonded thereto as defined above, and is preferably C 1 -C 6 alkylsulfonyl. Specific examples of alkylsulfonyl include methylsulfonyl.
• alkylsulfonylamino refers to an amino group having the above-defined “alkylsulfonyl” bonded thereto, and preferably includes C 1 -C 6 alkylsulfonylamino. Specific examples of alkylsulfonylamino include methylsulfonylamino.
• heterocyclyl refers to a non-aromatic cyclic monovalent group containing 1 to 5 heteroatoms in addition to carbon atoms. Heterocyclyl may have double and/or triple bonds in the ring, and the carbon atoms in the ring may be oxidized to form a carbonyl, and may be a single ring or a condensed ring.
• the number of atoms constituting the ring is preferably 4 to 10 (4- to 10-membered heterocyclyl), and more preferably 4 to 7 (4- to 7-membered heterocyclyl).
• heterocyclyl examples include azetidinyl, oxiranyl, oxetanyl, azetidinyl, dihydrofuryl, tetrahydrofuryl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridyl, tetrahydropyrimidyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, piperidinyl, piperazinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, and cyclohexyl.
• solvate refers to a compound that forms a molecular group together with a solvent.
• examples include solvates with a single solvent such as hydrates, alcohol solvates (ethanol solvates, methanol solvates, 1-propanol solvates, 2-propanol solvates, etc.), and dimethyl sulfoxide, as well as solvates with multiple solvents per molecule of a compound, or solvates with multiple types of solvents per molecule of a compound.
• the solvent is water, it is called a hydrate.
• a hydrate is preferred, and specific examples of such hydrates include 0.5 to 10 hydrates, preferably 0.5 to 5 hydrates, and more preferably 0.5 to 3 hydrates.
• amino acid includes natural amino acids and unnatural amino acids.
• amino acid may refer to amino acid residues.
• natural amino acids refer to Gly, L-Ala, L-Ser, L-Thr, L-Val, L-Leu, L-Ile, L-Phe, L-Tyr, L-Trp, L-His, L-Glu, L-Asp, L-Gln, L-Asn, L-Cys, L-Met, L-Lys, L-Arg, and L-Pro.
• unnatural amino acids include, but are not limited to, ⁇ -amino acids, D-amino acids, N-substituted amino acids, ⁇ , ⁇ -disubstituted amino acids, amino acids whose side chains are different from those of natural amino acids, and hydroxycarboxylic acids.
• amino acids are permitted to have any stereoconfiguration.
• the side chain of the amino acid can be freely selected from, for example, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, heteroaralkyl groups, cycloalkyl groups, and spiro-linked cycloalkyl groups.
• alkyl groups alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, heteroaralkyl groups, cycloalkyl groups, and spiro-linked cycloalkyl groups.
• substituents are not limited, and for example, one or more of any substituents including a halogen atom, an O atom, a S atom, a N atom, a B atom, a Si atom, or a P atom may be independently selected.
• the amino acid in this specification may be a compound having a carboxyl group and an amino group in the same molecule (even in this case, proline, hydroxyproline, azetidine-2-carboxylic acid, etc., in which the nitrogen atom of the amino group and any atom of the side chain are combined to form a ring, are also included in the amino acid).
• amino acid amide refers to a compound in which at least one carboxyl group of a natural or unnatural amino acid has been converted to an amide group.
• peptide refers to a compound in which two or more amino acids are linked by amide bonds. Peptides having an ester bond in part of the main chain, such as depsipeptides, are also included in the peptides used herein. Peptides are preferably peptides of 2 to 29 residues, more preferably 3 to 20 residues, and even more preferably 4 to 14 residues. Peptides and peptide compounds may be linear or cyclic peptides.
• amino group-containing compound means a compound having at least one primary amino group and/or secondary amino group.
• amino group-containing compound in which the amino group is protected by a protecting group means a compound in which at least one of the primary amino group and/or secondary amino group contained in the amino group-containing compound is protected by a protecting group.
• substituents are not limited, and may be independently selected from any substituents including, for example, a halogen atom, an oxygen atom, a sulfur atom, a nitrogen atom, a boron atom, a silicon atom, or a phosphorus atom.
• substituents examples include alkyl, alkoxy, fluoroalkyl, fluoroalkoxy, oxo, aminocarbonyl, alkylsulfonyl, alkylsulfonylamino, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, halogen, nitro, amino, monoalkylamino, dialkylamino, cyano, carboxyl, and alkoxycarbonyl.
• nitrile solvents examples include acetonitrile and propionitrile.
• amide solvents examples include dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone, etc.
• examples of alcohol-based solvents include methanol, ethanol, n-propanol, and 2-propanol.
• examples of benzene-based solvents include benzene, toluene, xylene, fluorobenzene, chlorobenzene, 1,2-dichlorobenzene, bromobenzene, anisole, ethylbenzene, nitrobenzene, cumene, and benzotrifluoride.
• ester solvents include methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, and isobutyl acetate.
• one or more means one or more than one.
• the term means a number from one to the maximum number of substituents permitted by that group. Specific examples of "one or more” include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and/or more.
• A, B, and/or C includes the following seven variations: (i) A, (ii) B, (iii) C, (iv) A and B, (v) A and C, (vi) B and C, (vii) A, B, and C.
• dibenzofulvene or a dibenzofulvene derivative can be removed by any one of the following steps (1), (1') or (1'').
• "removing dibenzofulvene or a dibenzofulvene derivative” includes converting dibenzofulvene or a dibenzofulvene derivative to (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof, and then removing it.
• the method for removing dibenzofulvene or a dibenzofulvene derivative of the present invention may be a method for removing dibenzofulvene or a dibenzofulvene derivative from a mixture containing dibenzofulvene or a dibenzofulvene derivative.
• the term “remove” is not particularly limited, but means, for example, reducing the amount of a target to 1% or less, 0.5% or less, 0.1% or less, or an undetectable amount based on the total amount of the entire mixture containing the target.
• the term “remove dibenzofulvene or a dibenzofulvene derivative” means, for example, reducing the value represented by the following formula A, calculated from the UVarea value at 210 nm by HPLC analysis, to 1% or less, 0.5% or less, 0.1% or less, or an undetectable amount.
• Steps (1) and (1') Step (1) is a step of mixing (i) dibenzofulvene or a dibenzofulvene derivative, and (ii) sulfite ion or hydrogen sulfite ion, or a compound that generates sulfite ion or hydrogen sulfite ion, and step (1') is a step of mixing (i) dibenzofulvene or a dibenzofulvene derivative, and (ii) at least one selected from the group consisting of hydrogen sulfite, disulfite and its salts, sulfurous acid and its salts, dithionous acid and its salts, and solvates thereof.
• sulfite ions or hydrogen sulfite ions or compounds that generate them are used as a scavenger to convert dibenzofulvene or a dibenzofulvene derivative to (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof, thereby making it possible to remove dibenzofulvene or a dibenzofulvene derivative.
• steps (1) and (1') may be mixed as they are, or solutions of (i) and/or (ii) dissolved in a solvent described below may be mixed.
• mixing (i) and (ii) includes any of the following aspects: adding (i) to (ii), adding (ii) to (i), and adding (i) and (ii) simultaneously.
• mixing does not require that a homogeneous mixture be obtained when mixing.
• Steps (1) and (1') can be carried out in the presence or absence of a solvent, at a temperature of preferably -20°C to 80°C, more preferably 10°C to 80°C, for a period of preferably 0.1 hours to 48 hours, more preferably 1 hour to 24 hours.
• dibenzofulvene is preferably used.
• hydrogen sulfite, disulfite, sulfite, dithionite, and solvates thereof are preferably used.
• salts of hydrogen sulfite, disulfite, sulfite, and dithionite and at least one selected from the group consisting of alkali metals, alkaline earth metals, and ammonium are preferably used, and salts of hydrogen sulfite, disulfite, sulfite, and dithionite and at least one selected from the group consisting of sodium, potassium, calcium, and ammonium are more preferably used, and sodium hydrogen sulfite, sodium metabisulfite, potassium hydrogen sulfite, sodium dithionite, ammonium sulfite monohydrate, calcium sulfite 0.5 hydrate, sodium sulfite, and potassium sulfite are further preferably used, and sodium hydrogen sulfite
• the amount of (ii) used is not particularly limited, but is preferably 1 molar equivalent or more and 10 molar equivalents or less relative to (i), and more preferably 1 molar equivalent or more and 5 molar equivalents or less.
• the solvent may be, for example, at least one selected from the group consisting of nitrile solvents, amide solvents, sulfoxide solvents, alcohol solvents, and ether solvents.
• Steps (1) and (1') can be carried out in the presence of an additive.
• a base can be used, and for example, a tertiary amine such as triethylamine, N,N-diisopropylethylamine, or 2,6-lutidine is preferably used, and triethylamine is more preferably used.
• the amount of additive used is not particularly limited, but is preferably 1 molar equivalent or more and 20 molar equivalents or less relative to (i), and more preferably 1 molar equivalent or more and 10 molar equivalents or less.
• Steps (1) and (1') can be carried out in the presence or absence of water, but are preferably carried out in the presence of water.
• the amount of water used is not particularly limited, but is preferably 1 molar equivalent or more and 300 molar equivalents or less, and more preferably 1 molar equivalent or more and 100 molar equivalents or less, relative to (i).
• the water used is different from the water contained in the organic solvent.
• Step (1′′) is a step of forming (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof.
• the method for forming (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof is not particularly limited, and can be formed, for example, by carrying out the above-mentioned step (1) or (1′).
• Step (1''') is a step of reacting dibenzofulvene or a dibenzofulvene derivative with sulfite ion or hydrogen sulfite ion to form (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof.
• Step (1"" is a step of mixing dibenzofulvene or a dibenzofulvene derivative with at least one member selected from the group consisting of hydrogensulfite, disulfite and its salts, sulfurous acid and its salts, dithionous acid and its salts, and solvates thereof, to form (9H-fluoren-9-yl)methanesulfonic acid or its salt, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or its salt.
• step (2) may be included before any of steps (1), (1'), (1'', (1''') or (1'''').
• Step (2) is a step, prior to the above step (1), (1'), (1'', (1''') or (1''''), of mixing a first amino-group-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton with a deprotecting agent.
• the "protecting group having an Fmoc skeleton" in the first amino group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton is preferably a 9-fluorenylmethyloxycarbonyl (Fmoc) group.
• the "first amino group-containing compound” in the first amino group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton is preferably a peptide, an amino acid, or an amino acid amide, and more preferably a peptide.
• the peptide, amino acid, or amino acid amide includes a peptide, amino acid, or amino acid amide bound to a resin for solid-phase synthesis or a support for liquid-phase peptide synthesis (e.g., a hydrophobic tag).
• carriers for liquid phase peptide synthesis include, for example, compounds described in Patent Document 4, Japanese Patent No. 5113118, Japanese Patent No. 5929756, Japanese Patent No. 6092513, Japanese Patent No. 5768712, Japanese Patent No. 5803674, Japanese Patent No. 6116782, Japanese Patent No. 6201076, Japanese Patent No. 6283774, Japanese Patent No. 6283775, Japanese Patent No. 6322350, Japanese Patent No. 6393857, Japanese Patent No. 6531235, International Publication No. 2020/175472, and International Publication No. 2020/175473.
• Step (2) can be carried out in the presence or absence of a solvent, at a temperature of preferably -20°C to 80°C, more preferably 10°C to 80°C, for preferably 0.1 hours to 48 hours, more preferably 0.5 hours to 24 hours.
• the solvent may be, for example, at least one selected from the group consisting of nitrile solvents, amide solvents, sulfoxide solvents, alcohol solvents, and ether solvents.
• a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton is used.
• a base can be used as the deprotecting agent, and examples of the base include organic bases having an amidine skeleton, primary amines, secondary amines, tertiary amines, and inorganic bases.
• Examples of organic bases having an amidine skeleton include 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]-5-nonene (DBN), and 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), and among these, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) is preferably used.
• Examples of primary amines include propane-1-amine.
• Examples of secondary amines include morpholine, diethylamine, dicyclohexylamine, 1,1,1,3,3,3-hexamethyldisilazane, piperidine, pyrrolidine, and piperazine, among which morpholine, diethylamine, dicyclohexylamine, and 1,1,1,3,3,3-hexamethyldisilazane are preferably used.
• Examples of tertiary amines include triethylamine.
• Examples of inorganic bases include carbonates such as sodium carbonate and potassium carbonate; metal alkoxides such as sodium tert-butoxide and potassium tert-butoxide, among which sodium carbonate and potassium tert-butoxide are preferably used.
• the amount of the deprotecting agent used is not particularly limited, but is preferably 0.5 to 10 molar equivalents, and more preferably 1 to 5 molar equivalents, relative to the first amino group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton.
• dibenzofulvene or a dibenzofulvene derivative can be removed by any of the following steps (3) or (3').
• dibenzofulvene or a dibenzofulvene derivative can be removed from the mixture produced by any of the following steps (3) or (3').
• Steps (3) and (3') Step (3) is a step of mixing (i) a first amino-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton, (ii) a deprotecting agent, and (iii) a sulfite ion or a hydrogen sulfite ion, or a compound that generates a sulfite ion or a hydrogen sulfite ion.
• Step (3') is a step of mixing (i) a first amino-containing compound in which an amino group is protected with a protecting group having an Fmoc skeleton, (ii) a deprotecting agent, and (iii) at least one selected from the group consisting of hydrogen sulfite, disulfite and its salts, sulfurous acid and its salts, dithionous acid and its salts, and solvates thereof.
• steps (3) and (3') (i), (ii) and (iii) may be mixed as they are, or solutions of (i), (ii) and/or (iii) dissolved in a solvent as described below may be mixed.
• “mixing (i) to (iii)” includes both the act of sequentially adding one of the components (i) to (iii) to the other, and the act of simultaneously adding (i) to (iii).
• mixing does not require that a homogeneous mixture be obtained when mixing.
• a 9-fluorenylmethyloxycarbonyl (Fmoc) group is preferred.
• a peptide, an amino acid, or an amino acid amide is preferred, and a peptide or an amino acid is more preferred.
• the peptide, amino acid, or amino acid amide includes a peptide, amino acid, or amino acid amide bound to a resin for solid-phase synthesis or a support for liquid-phase peptide synthesis (e.g., a hydrophobic tag).
• the liquid phase peptide synthesis carrier include compounds described in Patent Document 4, Japanese Patent No. 5113118, Japanese Patent No.
• a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton is used.
• a base can be used, and examples thereof include organic bases having an amidine skeleton, primary amines, secondary amines, tertiary amines, and inorganic bases.
• Examples of organic bases having an amidine skeleton include 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]-5-nonene (DBN), and 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), and among these, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) is preferably used.
• Examples of primary amines include propan-1-amine.
• Examples of secondary amines include morpholine, diethylamine, dicyclohexylamine, 1,1,1,3,3,3-hexamethyldisilazane, piperidine, pyrrolidine, and piperazine, among which morpholine, diethylamine, dicyclohexylamine, and 1,1,1,3,3,3-hexamethyldisilazane are preferably used.
• Examples of tertiary amines include triethylamine.
• Examples of inorganic bases include carbonates such as sodium carbonate and potassium carbonate; metal alkoxides such as sodium tert-butoxide and potassium tert-butoxide, among which sodium carbonate and potassium tert-butoxide are preferably used.
• the amount of (ii) used is not particularly limited, but is preferably 0.5 to 10 molar equivalents relative to (i), and more preferably 1 to 5 molar equivalents.
• hydrogen sulfite, disulfite, sulfite, dithionite, and solvates thereof are preferably used.
• salts of hydrogen sulfite, disulfite, sulfurous acid, and dithionous acid with at least one selected from the group consisting of alkali metals, alkaline earth metals, and ammonium are preferably used
• salts of hydrogen sulfite, disulfite, sulfurous acid, and dithionous acid with at least one selected from the group consisting of sodium, potassium, calcium, and ammonium are more preferably used
• sodium hydrogen sulfite, sodium metabisulfite, potassium hydrogen sulfite, sodium dithionite, ammonium sulfite monohydrate, calcium sulfite 0.5 hydrate, sodium sulfite, and potassium sulfite are further preferably used, and sodium hydrogen sulfite, potassium hydrogen sulfite, and sodium dithionite are particularly
• the amount of (iii) used is not particularly limited, but is preferably 1 molar equivalent or more and 10 molar equivalents or less relative to (i), and more preferably 1 molar equivalent or more and 5 molar equivalents or less.
• Steps (3) and (3') can be carried out in the presence or absence of a solvent, at a temperature of preferably -20°C to 80°C, more preferably 10°C to 80°C, for a period of preferably 1 hour to 48 hours, more preferably 1 hour to 24 hours.
• the solvent may be, for example, at least one selected from the group consisting of nitrile solvents, amide solvents, sulfoxide solvents, alcohol solvents, and ether solvents.
• Steps (3) and (3') can be carried out in the presence of an additive. In some embodiments, steps (3) and (3') are carried out without an additive.
• a base can be used as the additive, and for example, a tertiary amine such as triethylamine, N,N-diisopropylethylamine, 2,6-lutidine, etc. is preferably used, and triethylamine is more preferably used.
• the amount of additive used is not particularly limited, but is preferably 1 molar equivalent or more and 20 molar equivalents or less relative to (i), and more preferably 1 molar equivalent or more and 10 molar equivalents or less.
• Steps (3) and (3') can be carried out in the presence or absence of water, but are preferably carried out in the presence of water.
• the amount of water used is not particularly limited, but is preferably 1 molar equivalent or more and 300 molar equivalents or less, and more preferably 1 molar equivalent or more and 100 molar equivalents or less, relative to (i).
• the water is not water contained in an organic solvent.
• the method may include the following step (4) after one or more of steps (1), (1'), (1'', (1'''), (1'''), (3) or (3').
• Step (4) is a step of washing the mixture after one or more steps among (1), (1'), (1'', (1''', (1''',), (3) or (3') with a washing solution.
• This step makes it possible to efficiently remove dibenzofulvene or a dibenzofulvene derivative (preferably (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof) in the mixture from the organic solution that is the mixture after one or more steps among (1), (1'), (1'', (1'''', (1'''',), (3) or (3').
• dibenzofulvene or a dibenzofulvene derivative preferably (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof
• washing means removing substances other than the target substance that may be impurities from a solution containing the target substance by a liquid separation operation using a solution that does not contain the target substance.
• the target substance is usually present in the organic layer, and in this case, the organic layer can be washed with an aqueous solution to extract and remove the substances that may be impurities in the aqueous layer.
• the cleaning solution it is preferable to use a basic solution (pH 10-14), usually a basic aqueous solution with a pH of 10-14.
• a basic solution pH 10-14
• the cleaning solution it is preferable to use at least one selected from the group consisting of an aqueous ammonia solution, an aqueous carbonate solution, and an aqueous phosphate solution, and more preferably an aqueous ammonia solution.
• the carbonate solution may be, for example, an aqueous sodium carbonate solution.
• the phosphate solution may be, for example, an aqueous potassium phosphate solution.
• step (4) it is preferable to use an extraction solvent in order to efficiently extract the amino group-containing compound from the mixture in which the protecting group having the Fmoc skeleton has been deprotected.
• an extraction solvent for example, at least one solvent selected from the group consisting of benzene-based solvents, ester-based solvents, and ether-based solvents can be used as such an extraction solvent.
• the present invention provides a method for producing a first compound, comprising the above-mentioned method for removing dibenzofulvene or a dibenzofulvene derivative.
• the first compound produced by the method is not particularly limited, and examples thereof include amino acids and peptides.
• the production method of the present invention can produce (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof.
• the present invention provides a method for producing a peptide compound, comprising the steps of: 1) treating a peptide protected with a protecting group having an Fmoc skeleton with a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton in the presence of sulfite ions or hydrogen sulfite ions to obtain a deprotected peptide by removing the protecting group; 2) optionally extending the deprotected peptide with one or more amino acids to obtain an extended peptide.
• the present invention provides a method for producing a peptide compound, comprising the steps of: 1) treating a protected peptide, in which an amino group is protected with a protecting group having an Fmoc skeleton, with a deprotecting agent capable of deprotecting the protecting group having an Fmoc skeleton to obtain (a) dibenzofulvene or a dibenzofulvene derivative, and (b) a first mixture of deprotected peptide from which the protecting group has been removed; 2) treating the first mixture with sulfite or bisulfite ions to remove the dibenzofulvene or dibenzofulvene derivative to obtain a second mixture comprising the deprotected peptide; 3) optionally extending the deprotected peptide with one or more amino acids to obtain an extended peptide.
• the deprotected peptide or extended peptide may have an amino acid residue having one reactive site on the C-terminal side and may have an amino acid residue having another reactive site on the N-terminal side, and the production method of the present invention may further include a step of bonding the one reactive site and the other reactive site to form a cyclic peptide compound.
• the manufacturing method of the present invention may be either liquid phase synthesis or solid phase synthesis, and is not limited to these, but liquid phase synthesis is preferred. Liquid phase synthesis and solid phase synthesis can be carried out by methods well known to those skilled in the art.
• the solid phase synthesis method is a method in which a compound is bound to a solid phase (a resin for synthesis) and the compound is chemically reacted with a reagent on the solid phase to synthesize the target compound.
• a desired amino acid or peptide is bound to a solid phase, and then further desired amino acids or peptides are successively linked to the amino acids or peptides bound to the solid phase to elongate the peptide chain, and the peptide is synthesized while still bound to the solid phase.
• the peptide bound to the solid phase can be separated from the solid phase to obtain the desired peptide.
• Liquid phase synthesis is a method of synthesizing the desired compound by chemically reacting compounds in the liquid phase (solution).
• Liquid phase peptide synthesis includes homogeneous reactions in which all reagents, including amino acids and peptides, are dissolved in a solvent, as well as heterogeneous reactions in which all or part of the reagents are not dissolved in the solvent but are dispersed or suspended.
• the manufacturing method of the present invention may further include, before one or more of steps (2), (3) and (3') in the above removal method, a step of condensing a second amino group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton with a third amino group-containing compound to obtain the first amino group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton.
• the "protecting group having an Fmoc skeleton" in the second amino group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton is preferably a 9-fluorenylmethyloxycarbonyl (Fmoc) group.
• One embodiment of the "second amino group-containing compound” in the second amino group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton is preferably an amino group-containing compound having a carboxyl group.
• Another embodiment of the "second amino group-containing compound” in the second amino group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton is preferably a peptide or an amino acid, more preferably an amino acid.
• an amino group-containing compound that is not bound to a support for liquid-phase peptide synthesis is preferred, and more preferably, the third amino group-containing compound is an amino group-containing compound that is not bound to a support for liquid-phase peptide synthesis, and the support for liquid-phase peptide synthesis is a compound that is bound to the third amino group-containing compound directly or via a linker to increase their solubility in organic solvents and increase their insolubility in water.
• the support for liquid-phase peptide synthesis include the compounds described in Patent Document 4.
• a peptide, an amino acid, or an amino acid amide is preferred.
• the process for obtaining the first amino group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton can be carried out in the presence or absence of a solvent and in the presence or absence of a condensing agent, at a temperature preferably in the range of -50°C to near the boiling point of the solvent, more preferably in the range of -20°C to 80°C, for preferably 0.1 hours to 48 hours, more preferably 0.5 hours to 24 hours.
• Solvents that can be used include, for example, acetonitrile, 2-MeTHF, THF, dichloromethane, toluene, ethyl acetate, isopropyl acetate, DMF, DMA, and NMP.
• Condensing agents include, for example, HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, CAS RN: 148893-10-1), T3P (2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide (50% solution in ethyl acetate, approximately 1.7 mol/L), CAS RN: 68957-94-8), HBTU (1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate , CAS RN: 94790-37-1), COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate, CAS RN: 1075
• the process of obtaining the first amino group-containing compound by such a condensation reaction can be carried out with reference to methods described in the literature.
• the manufacturing method of the present invention may include repeating the above-mentioned method for removing dibenzofulvene or a dibenzofulvene derivative two or more times, or three or more times.
• One specific order of the method of the present invention is to carry out step (2), followed by steps (1), (1'), (1'', (1'") or (1''"), followed by (4).
• a second amino-group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton and a third amino-group-containing compound are condensed in a reaction vessel to obtain the first amino-group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton.
• the first amino-group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton in the reaction vessel is mixed with a deprotecting agent to deprotect the protecting group having an Fmoc skeleton.
• dibenzofulvene or a dibenzofulvene derivative is produced.
• Sulfite ions or hydrogen sulfite ions, or a compound that generates sulfite ions or hydrogen sulfite ions are then added to the reaction vessel and mixed with dibenzofulvene or a dibenzofulvene derivative to form (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof.
• An organic solvent is then added to the reaction vessel, and the organic layer is washed with a washing solution.
• step (3) is performed followed by step (4).
• a second amino group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton is condensed with a third amino group-containing compound in a reaction vessel to obtain the first amino group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton.
• the first amino group-containing compound in which the amino group is protected with a protecting group having an Fmoc skeleton is mixed with (ii) a deprotecting agent and (iii) sulfite ions or hydrogen sulfite ions or a compound that generates sulfite ions or hydrogen sulfite ions in the reaction vessel to form (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof.
• an organic solvent is added to the reaction vessel, and the organic layer is washed with a washing solution.
• the deprotection of the protecting group having the Fmoc skeleton and the formation of (9H-fluoren-9-yl)methanesulfonic acid or its salt, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or its salt are carried out in one step.
• the present invention provides a method for deprotecting a protecting group having an Fmoc skeleton, comprising the step of treating a first amino group-containing compound protected by a protecting group having an Fmoc skeleton with a deprotecting agent capable of deprotecting the protecting group having an Fmoc skeleton in the presence of a sulfite ion or a bisulfite ion.
• Another aspect of the present invention is the use of (a) and/or (b) below for removing dibenzofulvene or a dibenzofulvene derivative: (a) sulfite ion or bisulfite ion, or a compound that generates sulfite ion or bisulfite ion; (b) at least one compound selected from the group consisting of hydrogen sulfite, disulfite and its salts, sulfurous acid and its salts, dithionous acid and its salts, and solvates thereof;
• Another aspect of the present invention is the combined use of the following (a) and/or (b) for deprotecting a protecting group having an Fmoc skeleton and a deprotecting agent capable of deprotecting a protecting group having an Fmoc skeleton: (a) sulfite ion or bisulfite ion, or a compound that generates sulfite ion or bisulfite ion; (b) at least one compound selected from the group consisting of hydrogen sulfite, disulfite and its salts, sulfurous acid and its salts, dithionous acid and its salts, and solvates thereof;
• composition comprising an amino group-containing compound, and (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof, wherein the value represented by the following formula A, calculated from the UVarea value at 210 nm by HPLC analysis, is 1% or less.
• the value represented by Formula A is less than 1%, less than 0.5%, or undetectable.
• the present invention provides a composition comprising an amino group-containing compound and (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof, wherein the content of (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof is 0.01 or less relative to the amino group-containing compound and (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or a (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof.
• the content is calculated by measuring the UVarea value at 210 nm by HPLC analysis. In one embodiment, the content is 0.01 or less, 0.005 or less, or undetectable.
• the amino group-containing compound contained in the composition of the present invention is a peptide, an amino acid, or an amino acid amide.
• the (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof, or the (9H-fluoren-9-yl)methanesulfonic acid derivative or a salt thereof contained in the composition of the present invention is (9H-fluoren-9-yl)methanesulfonic acid or a salt thereof.
• a Waters H-Class system was used for HPLC analysis, with measurements performed using a PDA detector and analysis at a wavelength of 210 nm.
• the analytical conditions are shown below.
• a Waters H-Class system was used for LCMS analysis, and a QDa detector or SQD2 detector was used for MS analysis.
• the analytical conditions are shown below.
• Sample preparation method 1 A mixture containing the target compound was diluted with MeCN.
• Sample preparation method 2 A mixture containing the target compound was diluted with a mixture of MeCN and H 2 N n Pr in a 9:1 ratio.
• Sample preparation method 3 The mixture containing the target substance was diluted with a mixture of MeOH and water in a ratio of 4:1.
• reaction conversion rate was calculated by one of the following formulas using the area values of the raw materials and the target product calculated by HPLC analysis, or the area values of the raw materials, the area values of the propyl amide of the raw materials, and the area values of the target product.
• Reaction conversion rate (%) area value of target substance / (area value of raw material + area value of propyl amide of raw material + area value of target substance) x 100
• the deprotection reaction conversion rate of the Fmoc group was calculated according to the following formula using the area value of the Fmoc form and the area value of the de-Fmoc form calculated by HPLC analysis.
• the capture reaction conversion rate of DBF was calculated according to the following formula using the area value of DBF calculated by HPLC analysis and the area value of FMSA or FMSA Salt, or the area value of compound 35, or the area value of compound 36, or the area value of compound 37.
• Formula 2 for calculating the capture reaction conversion rate of DBF: Capture reaction conversion rate of DBF (%) area value of FMSA or FMSA Salt / (area value of DBF + area value of compound 35 + area value of FMSA or FMSA Salt) ⁇ 100
• Formula 3 for calculating the capture reaction conversion rate of DBF: Capture reaction conversion rate of DBF (%) area value of compound 36/(area value of DBF + area value of compound 36) ⁇ 100
• the residual rate of FMSA or FMSA Salt was calculated according to the following formula using the area value of the de-Fmoc form calculated by HPLC analysis and the area value of FMSA or FMSA Salt.
• Residual rate of FMSA or FMSA Salt (%) Area value of FMSA or FMSA Salt / (Area value of de-Fmoc product + Area value of FMSA or FMSA Salt) ⁇ 100
• reaction mixture was stirred while T3P (50 w/w% 2-MeTHF solution, 2.68 mL, 1.5 eq.) was added, and the mixture was stirred at room temperature for 1 hour. Then, DIPEA (1.70 mL, 3.4 eq.) and T3P (50 w/w% 2-MeTHF solution, 2.68 mL, 1.5 eq.) were added at room temperature while stirring, and the mixture was stirred for another 0.5 hours.
• the reaction mixture was sampled and prepared as a sample (sample preparation method 1), and the reaction conversion rate was confirmed to be 99.9% by HPLC analysis (calculation formula for reaction conversion rate 1).
• a 5% aqueous sodium carbonate solution (15 mL) was added to the reaction vessel and stirred for 10 minutes. After the aqueous layer was discharged, the obtained organic layer was washed with a 5% aqueous sodium hydrogen sulfate monohydrate solution (15 mL x 2), a 5% aqueous sodium carbonate solution (15 mL), and a 5% aqueous sodium chloride solution (15 mL). The obtained organic layer was dehydrated with sodium sulfate and filtered to remove sodium sulfate. The mixture was concentrated under reduced pressure at an external temperature of 40°C to obtain a residue containing compound 3 (1.43 g).
• sample preparation method 1 The reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis, and it was confirmed that the reaction conversion rate had progressed to 99.9% or more (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• triethylamine 96.0 ⁇ L, 4.0 eq.
• water 31.3 ⁇ L, 10 eq.
• sodium hydrogen sulfite 45.6 mg, 2.5 eq.
• Example preparation method 1 After stirring the reaction mixture for 1 hour, the reaction mixture was sampled and prepared as a sample (sample preparation method 1), and the DBF capture reaction was evaluated by HPLC analysis, confirming that the reaction conversion rate was 99.9% or more (calculation formula 1 for DBF capture reaction conversion rate).
• IPAc (0.50 mL), toluene (0.50 mL), and 20% aqueous ammonia solution (1.0 mL) were added to the reaction vessel and stirred for 5 minutes. After discharging the aqueous layer, the obtained organic layer was washed again with 20% aqueous ammonia solution (1.0 mL).
• Example preparation method 1 The reaction mixture was sampled and prepared as a sample (sample preparation method 1), and the residual rate of FMSA or FMSA Salt was confirmed to be 0.39% by HPLC analysis (calculation formula for residual rate of FMSA or FMSA Salt).
• the obtained organic layer was concentrated under reduced pressure at an external temperature of 40 ° C. MeCN (50 ⁇ L) was added to obtain a MeCN solution (124 mg) containing compound 4, which was subjected to the following analysis.
• Example 2 Structural identification of (9H-fluoren-9-yl)methanesulfonic acid (FMSA or FMSA salt)
• the aqueous layer obtained in the separation washing step with 20% aqueous ammonia solution in Example 1-(iii) was freeze-dried under reduced pressure to remove water and other volatile components, thereby obtaining FMSA as a 1:1 mixture with DBU.
• Example 3 Compound 6: Synthesis of tert-butyl (2S)-2-[[(2S)-1-[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-(p-tolyl)propanoyl]pyrrolidine-2-carbonyl]amino]-3-phenyl-propanoate
• Compound 4 (177 mg) and compound 5 (245 mg, 1.1 eq.) obtained in Example 1-(iii) were dissolved in MeCN (1.0 mL) in a flask, and the atmosphere of the reaction vessel was replaced with nitrogen.
• NMM 183 ⁇ L, 3.0 eq.
• HATU 317 mg, 1.5 eq.
• reaction conversion rate calculation formula 1 Cyclopentyl methyl ether (2.0 mL), 5% aqueous sodium carbonate solution (1.0 mL), and N-methylimidazole (44.5 ⁇ L, 1.0 eq.) were added in sequence.
• the reaction mixture was stirred for 1 hour, then allowed to stand, and the aqueous layer was removed. 20% aqueous ammonia solution (1.0 mL) was added to the reaction vessel, and the mixture was stirred for 5 minutes, then allowed to stand, and the aqueous layer was removed.
• Example 4 (i) Synthesis of Compound 8: (tert-butyl (3S)-3-[benzyloxycarbonyl(methyl)amino]-4-(dimethylamino)-4-oxo-butanoate)
• Compound 7 (62.8 kg) and 2-MeTHF (310 kg) were added in sequence at room temperature to a reaction vessel purged with nitrogen.
• the external temperature of the reaction vessel was set to 10°C, and DIPEA (89.5 kg) and dimethylamine-THF solution (2 M, THF solution, 71.4 kg) were added in sequence while stirring the reaction mixture, and the mixture was stirred for 30 minutes.
• reaction conversion rate 2 After adding T3P (50% w/w, 2-MeTHF solution, 151 kg), the external temperature of the reaction vessel was set to 25°C and the mixture was stirred for 2 hours. The reaction mixture was sampled and sample preparation (sample preparation method 2), and the reaction conversion rate was confirmed to be 90.2% by HPLC analysis (calculation formula for reaction conversion rate 2). The external temperature of the reaction vessel was set to 10°C, and dimethylamine-THF solution (2 M, THF solution, 9.80 kg) was added while stirring, and the external temperature of the reaction vessel was set to 25°C and the mixture was stirred for 20 minutes.
• reaction conversion rate 2 was confirmed to be 99.4% by HPLC analysis (calculation formula for reaction conversion rate 2).
• the external temperature of the reaction vessel was set to 10°C, and 10% aqueous citric acid monohydrate solution (380 kg) was added to the reaction mixture.
• the external temperature of the reaction vessel was set to 25°C, and after stirring for 10 minutes, the stirring was stopped and the aqueous layer was discharged from the reaction vessel.
• the obtained organic layer was washed with 10% aqueous citric acid monohydrate solution (380 kg x 2) and 5% aqueous sodium carbonate solution (380 kg x 2).
• Example preparation method 1 the reaction conversion rate was confirmed to be 99.9% by HPLC analysis (reaction conversion rate calculation formula 1). After replacing the inside of the reaction vessel with nitrogen, the reaction mixture was pressure filtered.
• HATU 67.7 kg
• the reaction mixture was stirred at 25°C for 3 hours, and then sampled and sampled (sample preparation method 2).
• the reaction conversion was confirmed to be 98.5% by HPLC analysis (calculation formula 2 for reaction conversion).
• CPME 54 kg
• 5% aqueous potassium carbonate solution 48 kg
• N-methylimidazole 9.70 kg
• the obtained organic layer was washed with 2.5% aqueous ammonia solution (240 kg), 10% aqueous sodium hydrogen sulfate monohydrate solution (240 kg x 2), and 5% aqueous potassium carbonate solution (240 kg x 2).
• 2-MeTHF 86 kg was added to the obtained organic layer, and the mixture was concentrated under reduced pressure while stirring at an external temperature of 60°C until the liquid volume was about 100 L, and a solution containing compound 11 (98.1 kg) was obtained.
• Example preparation method 1 The solution containing compound 11 obtained in Example 4-(iii) (97.1 kg) and 2-MeTHF (160 kg) were added in sequence at room temperature.
• the external temperature of the reaction vessel was set to 25°C, and the internal pressure of the reaction vessel was pressurized with hydrogen until it reached 0.18 MPaG (2.8 atm). After stirring for 2 hours, it was confirmed that the internal pressure did not fluctuate, and then the reaction vessel was pressurized with hydrogen to 0.18 MPaG (2.8 atm), and further stirred for 1 hour.
• the reaction mixture was sampled and prepared (sample preparation method 1), and the reaction conversion rate was confirmed to be 99.9% by HPLC analysis (reaction conversion rate calculation formula 1).
• the reaction vessel was pressurized to 0.18 MPaG (2.8 atm) with hydrogen and stirred for 1 hour.
• the reaction mixture was sampled and prepared (sample preparation method 1), and the reaction conversion rate was confirmed to be 99.9% or more by HPLC analysis (reaction conversion rate calculation formula 1).
• the reaction mixture was pressure filtered.
• the inside of the reaction vessel and the filter were washed with 2-MeTHF (85 kg x 2), and the obtained filtrate and washing liquid were combined and concentrated under reduced pressure while stirring at an external temperature of 60 ° C until the liquid volume was about 100 L. This concentrated liquid and the washing liquid obtained by washing the reaction vessel with 2-MeTHF (43 kg) were combined to obtain a solution containing compound 12 (133 kg).
• the external temperature of the reaction vessel was cooled to 10°C, and DIPEA (77.1 kg), a solution containing compound 12 obtained in Example 4-(iv) (133 kg), T3P (50% w/w, 2-MeTHF solution, 194 kg), and DMAP (28.1 kg) were added in sequence.
• the external temperature of the reaction vessel was set to 50°C, and stirring was continued for 5 hours.
• the reaction mixture was sampled to prepare a sample (sample preparation method 2), and the reaction conversion rate was confirmed to be 99.3% by HPLC analysis (calculation formula 2 for reaction conversion rate).
• the external temperature of the reaction vessel was set to 10°C, and a 5% aqueous sodium carbonate solution (350 kg) was added.
• the external temperature of the reaction vessel was set to 25°C, and stirring was continued for 30 minutes, after which the stirring was stopped and the aqueous layer was discharged from the reaction vessel.
• 5% sodium hydrogen sulfate monohydrate aqueous solution (350 L) was added, and after stirring for 10 minutes, the stirring was stopped and the aqueous layer was discharged from the reaction vessel.
• the obtained organic layer was washed with 5% sodium hydrogen sulfate monohydrate aqueous solution (350 kg) and 5% sodium carbonate aqueous solution (350 kg).
• LiBH4 (4 M, THF solution, 0.6 kg) was added while stirring, and the reaction mixture was sampled for sample preparation (sample preparation method 3), and the reaction conversion rate was confirmed to be 99.5% by HPLC analysis (calculation formula 1 for reaction conversion rate).
• TFE 228 kg
• the external temperature of the reaction vessel was raised to 0°C over 1 hour, and the mixture was stirred at 0°C for 1 hour.
• 20% aqueous ammonium chloride solution 200 kg was added dropwise over 2 hours, and the mixture was stirred for 30 minutes at an external temperature of 25°C, after which the stirring was stopped and the aqueous layer was discharged from the reaction vessel.
• the external temperature of the reaction vessel was set to 10°C, and trifluoroacetic acid (26.0 kg) was added.
• the external temperature of the reaction vessel was set to 25°C, and the mixture was stirred for 1 hour.
• the obtained reaction mixture and the washing liquid obtained by washing the reaction vessel with 2-MeTHF (84.7 kg x 2) were mixed to obtain a mixture.
• 2 M aqueous sodium hydroxide solution (630 kg) was added to another reaction vessel purged with nitrogen at room temperature, and the external temperature of the reaction vessel was set to 10°C.
• the above mixture was added dropwise over 1.5 hours, and the external temperature of the reaction vessel was set to 25°C. After stirring for 10 minutes, the stirring was stopped, and the aqueous layer was discharged from the reaction vessel.
• the obtained organic layer was washed with 2 M aqueous sodium hydroxide solution (630 kg x 3) and 10% aqueous dipotassium hydrogen phosphate solution (330 kg).
• the obtained organic layer was concentrated under reduced pressure at an external temperature of 40°C until the liquid volume was about 220 L.
• 2-MeTHF (85 kg) was added again, and the mixture was concentrated under reduced pressure at an external temperature of 40°C until the liquid volume was about 100 L. This concentration procedure was repeated 10 times, and the concentrated solution and the washings obtained by washing the reaction vessel with acetonitrile (56.2 kg) were combined to obtain a solution containing compound 15 (151 kg).
• the external temperature of the reaction vessel was set to 25°C, and the mixture was stirred for 40 minutes, and the aqueous layer was discharged from the reaction vessel.
• the obtained organic layer was washed with 5% aqueous sodium hydrogen sulfate monohydrate solution (320 kg ⁇ 2) and 5% aqueous sodium carbonate solution (320 kg ⁇ 2).
• the obtained organic layer was concentrated under reduced pressure at an external temperature of 60° C. until the liquid volume was about 500 L.
• 2-MeTHF (86 kg) the mixture was concentrated under reduced pressure at an external temperature of 40° C. until the liquid volume was about 200 L, and a solution containing compound 17 (176 kg) was obtained.
• the mixture was stirred for 2 hours while maintaining the internal pressure pressurized to 0.40 MPaG (5.0 atm).
• the solution containing compound 17 obtained in Example 4-(vii) (176 kg) and THF (230 kg) were added in sequence at room temperature.
• the external temperature of the reaction vessel was set to 25°C, and the reaction vessel was pressurized with hydrogen until the internal pressure reached 0.18 MPaG (2.8 atm). After 4 hours, it was confirmed that the internal pressure did not change, and then the reaction vessel was pressurized with hydrogen to 0.18 MPaG (2.8 atm) after nitrogen replacement, and further stirred for 1 hour.
• the reaction mixture was sampled and prepared (sample preparation method 1), and the reaction conversion rate was confirmed to be 99.6% by HPLC analysis (calculation formula for reaction conversion rate 1).
• the reaction mixture was pressure filtered.
• the reaction vessel and the filter were washed with 2-MeTHF (85 kg x 2).
• the obtained filtrate and washing liquid were concentrated under reduced pressure at an external temperature of 50 ° C until the liquid volume was about 600 L.
• 2-MeTHF 160 kg
• the mixture was concentrated under reduced pressure at an external temperature of 50 ° C until the liquid volume was about 230 L. This concentration operation was repeated three times.
• the external temperature of the reaction vessel was set to 50 ° C, heptane (100 kg) was added, and then seed crystals of compound 18 (116 g) and heptane (5.3 kg) were added in sequence.
• 2-MeTHF (12 mL, 12 v/w) and DIPEA (1.70 mL, 3.4 eq.) were added in sequence at room temperature.
• T3P 50 w/w% 2-MeTHF solution, 2.68 mL, 1.5 eq.
• the reaction mixture was sampled and prepared as a sample (sample preparation method 1), and the reaction conversion rate was confirmed to be 99.9% or more by HPLC analysis (calculation formula for reaction conversion rate 1).
• 5% aqueous sodium carbonate solution (15 mL) was added to the reaction vessel and stirred for 5 minutes.
• the obtained organic layer was washed with 5% aqueous sodium hydrogen sulfate monohydrate solution (15 mL x 4), 5% aqueous sodium carbonate solution (15 mL), and 5% aqueous sodium chloride solution (15 mL).
• the obtained organic layer was dehydrated with sodium sulfate, filtered to remove sodium sulfate, and concentrated under reduced pressure at an external temperature of 40° C. to obtain a residue containing compound 20 (2.42 g).
• Example preparation method 1 DBU (25.9 ⁇ L, 1.0 eq.) was added to the reaction mixture while stirring at room temperature, and the mixture was stirred at room temperature for 30 minutes.
• the reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis, and it was confirmed that the reaction conversion rate had progressed to 99.9% or more (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• triethylamine 96.0 ⁇ L, 4.0 eq.
• water 31.3 ⁇ L, 10 eq.
• sodium hydrogen sulfite 44.5 mg, 2.5 eq.
• Example preparation method 1 After stirring for 1 hour, the reaction mixture was sampled and prepared as a sample (sample preparation method 1), and the DBF capture reaction was evaluated by HPLC analysis, confirming that the reaction conversion rate was 99.9% or more (calculation formula 1 for DBF capture reaction conversion rate).
• IPAc (0.75 mL), toluene (0.75 mL), and 20% aqueous ammonia solution (1.5 mL) were added to the reaction vessel and stirred for 5 minutes. After discharging the aqueous layer, the obtained organic layer was washed again with 20% aqueous ammonia solution (1.5 mL).
• Example preparation method 1 Samples were sampled and prepared as a sample (sample preparation method 1), and HPLC analysis confirmed that the residual rate of FMSA or FMSA Salt was 0.33% (calculation formula for the residual rate of FMSA or FMSA Salt).
• the obtained organic layer was concentrated under reduced pressure at an external temperature of 40 ° C. MeCN (100 ⁇ L) was added to obtain a MeCN solution (211 mg) containing compound 21, which was subjected to the following analysis.
• reaction vessel After the reaction vessel was substituted with nitrogen, it was pressurized with hydrogen to 0.40 MPaG (5.0 atm), and the internal temperature of the reaction vessel was set to 25 ° C. The mixture was stirred for 30 minutes while maintaining the internal pressure pressurized to 0.40 MPaG (5.0 atm).
• a solution containing compound 22 synthesized according to the method described in Example 21 of International Publication WO2022/234864 (7.22 g, content 42.4 wt%, actual mass 3.06 g) and 2-MeTHF (6.0 mL) were added in sequence at room temperature. The external temperature of the reaction vessel was set to 25 ° C., and the internal pressure of the reaction vessel was pressurized with hydrogen until it reached 0.20 MPaG (3.0 atm).
• reaction mixture was sampled and prepared (sample preparation method 1), and the reaction conversion rate was confirmed to be 97.3% by HPLC analysis (calculation formula for reaction conversion rate 1). After replacing the inside of the reaction vessel with nitrogen, the reaction mixture was pressure filtered. The reaction vessel and the filter were washed with 2-MeTHF (12 mL x 3).
• reaction vessel was replaced with nitrogen, and HATU (378 mg) was added.
• the external temperature was set to 25°C, and after stirring for 3 hours, the reaction mixture was sampled and sample preparation (sample preparation method 1) was performed, and it was confirmed that the reaction conversion rate was 96.1% by HPLC analysis (calculation formula 1 for reaction conversion rate).
• 2-MeTHF (3.0 mL), 5% potassium carbonate aqueous solution (6.0 mL), and N-methylimidazole (52.9 ⁇ L) were added in sequence, and after stirring for 30 minutes, the aqueous layer was discharged.
• the obtained organic layer was washed with 10% ammonia aqueous solution (6.0 mL ⁇ 2), 5% sulfuric acid aqueous solution (6.0 mL), and 5% potassium carbonate aqueous solution (6.0 mL).
• the organic layer was concentrated under reduced pressure at an external temperature of 40° C., and the residue was purified by silica gel chromatography (eluent: MeOH/ethyl acetate 0:100 to 8:92).
• the eluate containing compound 25 was concentrated under reduced pressure at 30° C. to give compound 25 (1.04 g).
• Example preparation method 1 DBU (22.8 ⁇ L, 1.0 eq.) was added to the reaction mixture while stirring at room temperature, and the mixture was stirred at room temperature for 30 minutes.
• the reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis to confirm that the reaction was complete (meaning that the peak of the deprotected Fmoc body was confirmed and that the peak of the Fmoc body was not confirmed. The same applies below.) (Calculation formula for the conversion rate of the deprotection reaction of the Fmoc group).
• the obtained organic layer was washed again with 20% aqueous ammonia solution (3.0 mL).
• the reaction mixture was sampled and prepared as a sample (sample preparation method 1), and the residual rate of FMSA or FMSA Salt was confirmed to be 0.11% by HPLC analysis (calculation formula for residual rate of FMSA or FMSA Salt).
• the obtained organic layer was concentrated under reduced pressure at an external temperature of 40 ° C. to obtain a residue containing compound 26 (261 mg), which was subjected to the following analysis.
• reaction mixture was sampled and prepared as a sample (sample preparation method 1), and the reaction conversion rate was confirmed to be 81.7% by HPLC analysis (calculation formula for reaction conversion rate 1).
• Triethylamine (0.217 mL) was added, and the resulting slurry was filtered, and the solid residue was washed with cyclohexane (20 mL ⁇ 3).
• reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis to confirm that the reaction was complete (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• sample preparation method 1 The reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis to confirm that the reaction was complete (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• triethylamine (279 ⁇ L, 4.0 eq.
• water 90.1 ⁇ L, 10 eq.
• sodium hydrogen sulfite 130 mg, 2.5 eq.
• Example preparation method 1 After stirring for 1 hour, the reaction mixture was sampled and prepared as a sample (sample preparation method 1), and the DBF capture reaction was evaluated by HPLC analysis, confirming that the reaction conversion rate was 99.9% (calculation formula 1 for DBF capture reaction conversion rate).
• IPAc 1.5 mL
• toluene 1.5 mL
• 20% aqueous ammonia 3.0 mL
• Example preparation method 1 Samples were sampled and prepared as a sample (sample preparation method 1), and HPLC analysis confirmed that the residual rate of FMSA or FMSA Salt was 1.09% (calculation formula for the residual rate of FMSA or FMSA Salt).
• the obtained organic layer was concentrated under reduced pressure at an external temperature of 40 ° C. to obtain a residue (175 mg) containing compound 29, which was subjected to the following analysis.
• LC purity of compound 29 93.3% (HPLC analysis conditions: method 1) Content: 93.7 wt% (The obtained residue and 3,5-bis(trifluoromethyl)benzoic acid were dissolved in DMSO-d 6 and subjected to qNMR analysis.) Yield: 92.7%
• Example 7 Synthesis of Compound 31: 5-methyl-4-phenylthiazol-2-amine
• Compound 30 300 mg
• MeCN 0.60 mL, 2 v/w
• DBU 109 ⁇ L, 1.0 eq.
• the reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis to confirm that the reaction was complete (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• sample preparation method 1 After the aqueous layer was discharged, the obtained organic layer was washed again with 20% aqueous ammonia (1.0 mL). Sampling was performed to prepare a sample (sample preparation method 1), and it was confirmed by HPLC analysis that the residual rate of FMSA or FMSA Salt was 0.65% (calculation formula for the residual rate of FMSA or FMSA Salt).
• the obtained organic layer was concentrated under reduced pressure at an external temperature of 40°C to obtain a residue (130 mg) containing compound 31, which was subjected to the following analysis.
• Example 8 (i) Synthesis of Compound 33: tert-Butyl (2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)pent-4-enoyl]amino]-3-phenyl-propanoate
• Compound 32 (0.964 g) and compound 2 (0.809 g, 1.1 eq.) were added to the reaction vessel, and the atmosphere in the reaction vessel was replaced with nitrogen.
• 2-MeTHF (12 mL, 12 v/w) and DIPEA (1.70 mL, 3.4 eq.) were added to the reaction mixture at room temperature in that order.
• T3P 50 w/w% 2-MeTHF solution, 2.68 mL, 1.5 eq.
• the reaction mixture was sampled to prepare a sample (sample preparation method 1), and the reaction conversion rate was confirmed to be 99.9% by HPLC analysis (calculation formula for reaction conversion rate 1).
• 5% aqueous sodium carbonate solution (15 mL) was added to the reaction vessel and stirred for 5 minutes.
• the obtained organic layer was washed with 5% aqueous sodium hydrogen sulfate monohydrate solution (15 mL x 2), 5% aqueous sodium carbonate solution (15 mL), and 5% aqueous sodium chloride solution (15 mL).
• the obtained organic layer was dehydrated with sodium sulfate, filtered to remove sodium sulfate, and concentrated under reduced pressure at an external temperature of 40° C. to obtain a residue containing compound 33 (0.794 g).
• the reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis, and it was confirmed that the reaction conversion rate had progressed to 99.9% or more (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• sample preparation method 1 The reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis, and it was confirmed that the reaction conversion rate had progressed to 99.9% or more (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• triethylamine (96.0 ⁇ L, 4.0 eq.
• water 31.3 ⁇ L, 10 eq.
• sodium hydrogen sulfite 45.4 mg, 2.5 eq.
• Example preparation method 1 After stirring for 1 hour, the reaction mixture was sampled and prepared as a sample (sample preparation method 1), and the DBF capture reaction was evaluated by HPLC analysis, confirming that the reaction conversion rate was 99.9% or more (calculation formula 1 for DBF capture reaction conversion rate).
• IPAc (0.50 mL), toluene (0.50 mL), and 20% aqueous ammonia solution (1.0 mL) were added to the reaction vessel and stirred for 5 minutes. After discharging the aqueous layer, the obtained organic layer was washed again with 20% aqueous ammonia solution (1.0 mL).
• Example preparation method 1 Samples were sampled and prepared as a sample (sample preparation method 1), and HPLC analysis confirmed that the residual rate of FMSA or FMSA Salt was 0.30% (calculation formula for the residual rate of FMSA or FMSA Salt).
• the obtained organic layer was concentrated under reduced pressure at an external temperature of 40 ° C. MeCN (50 ⁇ L) was added to obtain a MeCN solution (121 mg) containing compound 34, which was subjected to the following analysis.
• LC purity of compound 34 99.1% (HPLC analysis conditions: method 1) Content: 44.9 wt% (The obtained residue and 3,5-bis(trifluoromethyl)benzoic acid were dissolved in DMSO-d 6 and subjected to qNMR analysis.) Yield: 98.6%
• Example 9 Using compound 33 for which reaction conditions were examined , deprotection of the Fmoc group and subsequent capture reaction of DBF were carried out under the reaction conditions shown in the table below in the same manner as in Example 8-(ii), and the deprotection rate of the Fmoc group and the capture rate of DBF were measured (calculation formula for the deprotection reaction conversion rate of the Fmoc group, and calculation formula 1 for the capture reaction conversion rate of DBF).
• Example 9-1 the molar equivalents of each reagent were increased, and it was confirmed that the reaction conversion rate of the DBF capture reaction was 99.9% or more.
• Example 9-2 when the reaction was carried out without adding triethylamine, the reaction conversion rate of the DBF capture reaction decreased to 15.9%.
• Example 9-3 in which the reaction temperature was heated to 50°C, the conversion rate increased to 88.9% 6 hours after the start of the reaction
• Example 9-4 in which the reaction temperature was heated to 80°C, the conversion rate reached 100% 6 hours after the start of the reaction.
• Examples 9-5, 9-6, 9-7, 9-8, 9-9, and 9-10 potassium sulfite, potassium hydrogen sulfite, ammonium sulfite monohydrate, calcium sulfite 0.5 hydrate, and sodium dithionite were used as scavengers, respectively, and it was confirmed that DBF capture proceeded as FMSA or FMSA Salt under all conditions.
• Examples 9-11 and 9-12 the amide solvent DMA and the alcohol solvent MeOH were used as the solvent, and it was confirmed that the conversion rates of the DBF capture reaction were 99.1% and 99.9%, respectively.
• Examples 9-13 and 9-14 DIPEA and 2,6-lutidine were used as the bases added under DBF capture conditions, and the conversion rates of the DBF capture reaction were confirmed to be 99.8% and 67.4%, respectively.
• Example 10 Considering cleaning conditions Compound 33 (32.0 mg, content 79.4%, actual mass 25.4 mg) and MeCN (540 ⁇ L, 21 v/w) were added to the reaction vessel, and the atmosphere in the reaction vessel was replaced with nitrogen. Next, DBU (7.0 ⁇ L, 1.0 eq.) was added to the reaction mixture while stirring at room temperature, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis, confirming that the reaction conversion rate was 99.3% (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• reaction solution was divided into three, and 2-MeTHF (300 ⁇ L) was added to each, followed by washing under the conditions shown in the table below, and the residual rate of FMSA or FMSA Salt was confirmed by HPLC analysis (calculation formula for FMSA or FMSA Salt residual rate).
• Example 11 Example using morpholine as a deprotecting agent Compound 33 (32.5 mg, content 79.4%, actual mass 25.8 mg) and MeCN (0.127 mL, 5 v/w) were added to the reaction vessel, and the atmosphere in the reaction vessel was replaced with nitrogen. Then, morpholine (20.2 ⁇ L, 5.0 eq.) was added at room temperature while stirring the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. Then, morpholine (20.2 ⁇ L, 5.0 eq.) was added at room temperature while stirring the reaction mixture, and the reaction mixture was stirred for another 1 hour.
• morpholine 20.2 ⁇ L, 5.0 eq.
• reaction mixture was sampled and prepared as a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis, and the reaction conversion rate was confirmed to be 91.8% (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• DBF and compound 35 which is a morpholine adduct of DBF, were observed, and the ratio of DBF to compound 35 was 6.5:93.5.
• the deprotection reaction of the Fmoc group and the capture reaction of DBF were evaluated by HPLC analysis, and it was confirmed that the reaction conversion rate of the deprotection reaction of the Fmoc group was 100% and the reaction conversion rate of the capture reaction of DBF was 15.6% (calculation formula for the deprotection reaction conversion rate of the Fmoc group, calculation formula for the capture reaction conversion rate of DBF 2).
• the external temperature of the reaction vessel was heated to 50 ° C., and after stirring for 2 hours, the reaction mixture was sampled and prepared as a sample (sample preparation method 1).
• the capture reaction of DBF was evaluated by HPLC analysis, and it was confirmed that the reaction conversion rate was 44.3% (calculation formula for the capture reaction conversion rate of DBF 2).
• reaction vessel was raised to 78°C, and the reaction mixture was stirred for 2 hours.
• the reaction mixture was then sampled and prepared as a sample (sample preparation method 1).
• the DBF capture reaction was evaluated by HPLC analysis, and the reaction conversion rate was confirmed to be 95.7% (Equation 2 for calculating the DBF capture reaction conversion rate).
• Example 12 Example compound 33 using a primary amine or a secondary amine as a deprotecting agent was used to carry out the deprotection reaction of the Fmoc group and the subsequent capture reaction of DBF using the amines shown in the table below in the same manner as in Example 11, and the deprotection rate of the Fmoc group and the capture rate of DBF were measured (calculation formula for the deprotection reaction conversion rate of the Fmoc group, calculation formula 1 for the capture reaction conversion rate of DBF).
• Examples 12-1, 12-2, 12-3, and 12-4 unlike the investigation of the addition of morpholine in Example 11, no compounds in which the corresponding amine was added to DBF were observed, but it was confirmed that DBF was captured as FMSA or FMSA Salt. In addition, it was confirmed that the deprotection reaction of the Fmoc group proceeded even after the DBF capture conditions were applied, and that the deprotection reaction and the DBF capture reaction proceeded simultaneously.
• Example 13 Example of simultaneous deprotection and DBF capture Compound 33 (32.0 mg, content 79.4%, actual mass 25.4 mg), MeCN (110 ⁇ L, 4.3 v/w), and 2-MeTHF (110 ⁇ L, 4.3 v/w) were added to a reaction vessel, and the atmosphere in the reaction vessel was replaced with nitrogen. Next, triethylamine (65.2 ⁇ L, 10 eq.), water (8.4 ⁇ L, 10 eq.), sodium hydrogen sulfite (16.5 mg, 3.4 eq.), and DBU (20.9 ⁇ L, 3.0 eq.) were added successively at room temperature while stirring the reaction mixture, and the mixture was stirred for 1 hour.
• reaction mixture was sampled to prepare a sample (sample preparation method 1).
• HPLC analysis confirmed that the reaction conversion rate of the Fmoc group deprotection reaction was 100% (calculation formula for the Fmoc group deprotection conversion rate), and the reaction conversion rate of the dibenzofulvene capture reaction was 99.9% or more (calculation formula 1 for the DBF capture reaction conversion rate).
• Example 14 Using compound 33 for reaction condition investigation , deprotection of the Fmoc group and capture of DBF were simultaneously carried out under the reaction conditions shown in the table below in the same manner as in Example 13, and the deprotection rate of the Fmoc group and the capture rate of DBF were measured (calculation formula for the deprotection reaction conversion rate of the Fmoc group, calculation formula 1 for the capture reaction conversion rate of DBF).
• Examples 14-1, 14-2, 14-3, and 14-4 MeOH solvent was used, and it was confirmed that the deprotection reaction and the DBF capture reaction proceeded at 25°C regardless of the presence or absence of water, and that the deprotection reaction and the DBF capture reaction were completed in 1 hour when heated to 50°C.
• Example 14-8 When the DMI solvent of Example 14-5 was used, the deprotection reaction and the DBF capture reaction were completed in 1 hour even at a reaction temperature of 25° C. In Example 14-6, the reaction was carried out in the presence of DMI solvent without adding water, and a delay in the DBF capture reaction was confirmed. On the other hand, under conditions without adding triethylamine as in Example 14-17, the deprotection reaction and the DBF capture reaction were confirmed to be completed in 1 hour at 50° C. In Examples 14-8 and 14-9, DMSO solvent was used, and both were carried out under conditions without the addition of triethylamine. In Example 14-8, under conditions with the addition of water, the deprotection reaction and the DBF capture reaction were completed in 1 hour, and it was confirmed that even under conditions without the addition of water in Example 14-9, both reactions were completed by extending the reaction time to 4 hours.
• Example 15 Confirmation of DBF regeneration during base washing removal when sodium bisulfite is used as a scavenger Compound 33 (32.0 mg, content 79.4%, actual mass 25.4 mg) and MeCN (540 ⁇ L, 21 v/w) were added to the reaction vessel, and the atmosphere in the reaction vessel was replaced with nitrogen. Next, DBU (7.0 ⁇ L, 1.0 eq.) was added to the reaction mixture while stirring at room temperature, and the mixture was stirred at room temperature for 1 hour.
• DBU 7.0 ⁇ L, 1.0 eq.
• reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis, confirming that the reaction conversion rate was 99.4% (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• sample preparation method 1 The reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis, confirming that the reaction conversion rate was 99.4% (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• triethylamine (65.5 ⁇ L, 10 eq.)
• water 85 ⁇ L, 100 eq.
• sodium hydrogen sulfite 27.6 mg, 5.6 eq.
• Example preparation method 1 After stirring for 4 hours, the reaction mixture was sampled to prepare a sample (sample preparation method 1), and the DBF capture reaction was evaluated by HPLC analysis, confirming that the reaction conversion rate was 99.9% or more (calculation formula 1 for DBF capture reaction conversion rate).
• ethyl acetate 0.5 mL was added to the reaction mixture, and the mixture was washed four times with 2.5% aqueous ammonia solution (0.5 mL). It was confirmed that almost no compound 34 was present in each aqueous layer after washing, and the area ratio of FMSA or FMSA Salt to compound 34 and the area ratio of DBF to compound 34 were measured by HPLC analysis using compound 34 as an internal standard.
• Table 7 As the number of washes increased, the area ratio of FMSA or FMSA Salt gradually decreased, while there was no change in DBF, that is, it was confirmed that DBF was not regenerated from FMSA or FMSA salt.
• reaction mixture was sampled and prepared as a sample (sample preparation method 1), and HPLC analysis confirmed that the reaction conversion rate of the Fmoc group deprotection reaction was 100% (calculation formula for the Fmoc group deprotection reaction conversion rate) and the reaction conversion rate of the DBF capture reaction was 84.8% (calculation formula for the DBF capture reaction conversion rate 3).
• the reaction mixture was washed four times with 5% tripotassium phosphate aqueous solution (1 mL). It was confirmed that almost no compound 34 was present in each aqueous layer after washing, and the area ratios of compound 36 to compound 34 and the area ratios of DBF to compound 34 were measured by HPLC analysis using compound 34 as an internal standard. The results are shown in Table 8. It was confirmed that the area ratio of compound 36 gradually decreased while the area ratio of DBF gradually increased with increasing number of washings, that is, DBF was regenerated from compound 36.
• reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis to confirm that the reaction was complete (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• sample preparation method 1 The reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis to confirm that the reaction was complete (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• triethylamine 240 ⁇ L, 10 eq.
• water 310 ⁇ L, 100 eq.
• sodium 2-mercaptoethanesulfonate 160 mg, 5.6 eq.
• Example preparation method 1 After stirring for 1 hour, the reaction mixture was sampled to prepare a sample (sample preparation method 1), and the capture reaction of DBF was evaluated by HPLC analysis, and it was confirmed that the reaction conversion rate was 100% (calculation formula 3 for the capture reaction conversion rate of DBF).
• ethyl acetate 2.0 mL was added to the reaction mixture, and the mixture was washed four times with 2.5% aqueous ammonia solution (2.0 mL). It was confirmed that almost no compound 34 was present in each aqueous layer after washing, and the area ratio of FMSA or FMSA Salt to compound 34 and the area ratio of DBF to compound 34 were measured by HPLC analysis using compound 34 as an internal standard.
• Table 9 It was confirmed that the area ratio of compound 36 gradually decreased with increasing number of washings, while DBF gradually increased, that is, DBF was regenerated from compound 36.
• reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis to confirm that the reaction was complete (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• sample preparation method 1 The reaction mixture was sampled to prepare a sample (sample preparation method 1), and the deprotection reaction of the Fmoc group was evaluated by HPLC analysis to confirm that the reaction was complete (calculation formula for the deprotection reaction conversion rate of the Fmoc group).
• triethylamine 69.7 ⁇ L, 10 eq.
• water 90 ⁇ L, 100 eq.
• (3-mercaptopropyl)phosphonic acid 43.7 mg, 5.6 eq.
• the present invention provides a new method for removing dibenzofulvene that can capture dibenzofulvene produced during the deprotection process of a protecting group having an Fmoc skeleton and remove the dibenzofulvene without regenerating it.

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