Classifications

• • 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
  • C07K5/12—Cyclic peptides with only normal peptide bonds in the ring
• • 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
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/30—Extraction; Separation; Purification by precipitation
• • 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
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/30—Extraction; Separation; Purification by precipitation
  • C07K1/32—Extraction; Separation; Purification by precipitation as complexes

Definitions

• the present invention relates to a method for purifying a cyclic peptide and a method for producing a cyclic peptide using the same.
• Non-Patent Document 1 purification by column chromatography (Non-Patent Document 1) has been known as a method for purifying cyclic peptides.
• Patent Document 1 describes that "a cyclic peptide compound produced without isolating and purifying an intermediate can be isolated and purified by crystallization without relying on column chromatography to obtain crystals of the cyclic peptide compound.”
• the present invention relates to, for example, each of the following inventions [1] to [150].
• [1] 1.
• the purification method includes the following separation step (i) or (ii): (i) separating the cyclic peptide to be purified from a mixture containing the cyclic peptide to be purified as a complex with a metal atom or a metal ion; (ii) separating the cyclic peptide to be purified or the peptide to be an impurity from a mixture containing the cyclic peptide to be purified and a peptide to be an impurity as a complex with a metal atom or a metal ion, wherein the metal atom is at least one selected from the group consisting of an alkali metal atom, an alkaline earth metal atom, a transition metal atom, a poor metal atom, and a rare earth metal atom;
• the method for purifying wherein the metal atom is at least one selected from the group consist
• a method for purifying a cyclic peptide comprising the steps of: (1)' comprising a step of mixing a mixture containing a cyclic peptide to be purified, or a mixture containing a cyclic peptide to be purified and a peptide as an impurity, with a metal atom or a metal ion in a first solvent;
• the metal atom is at least one selected from the group consisting of an alkali metal atom, an alkaline earth metal atom, a transition metal atom, a poor metal atom, and a rare earth metal atom;
• the method for purifying, wherein the metal ion is at least one selected from the group consisting of an alkali metal ion, an alkaline earth metal ion, a transition metal ion, a poor metal ion and a rare earth metal ion.
• a method for purifying a cyclic peptide comprising the steps of: (4) mixing the cyclic peptide to be purified or the peptide as an impurity with a complex of a metal atom or a metal ion, and a second solvent; the metal atom is at least one selected from the group consisting of an alkali metal atom, an alkaline earth metal atom, a transition metal atom, a poor metal atom, and a rare earth metal atom; The method for purifying, wherein the metal ion is at least one selected from the group consisting of an alkali metal ion, an alkaline earth metal ion, a transition metal ion, a poor metal ion and a rare earth metal ion.
• step (6) further comprises a step of removing the third solvent from the organic phase to obtain the cyclic peptide as the purification target.
• step (6) further comprises a step of removing the third solvent from the organic phase to obtain the cyclic peptide as the purification target.
• the removal of the third solvent from the organic phase is carried out by vacuum distillation.
• step (6) further comprises a step of mixing the separated complex, a fourth solvent, and a ligand or a compound that generates a ligand, or an anion or a compound that generates an anion, to form a first complex or a first metal salt between the metal atom or the metal ion and the ligand or the anion.
• the complex is a complex obtained by contacting the cyclic peptide as a purification target or the peptide as an impurity with the metal atom or the metal ion.
• the poor metal is at least one selected from the group consisting of bismuth and indium.
• the metal atom is at least one selected from the group consisting of magnesium atoms, scandium atoms, and samarium atoms, The purification method according to any one of [1] to [57], wherein the metal ion is at least one selected from the group consisting of magnesium ions, scandium ions, and samarium ions.
• the metal atoms include magnesium atoms, The purification method according to any one of [1] to [57], wherein the metal ion includes a magnesium ion.
• metal salt is at least one selected from the group consisting of iodide salts, bromide salts, chloride salts, perchlorate salts, oxide salts, trifluoromethanesulfonate salts, toluenesulfonate salts, isopropylsulfonate salts, methanesulfonate salts, carbonate salts, acetate salts, bis(trifluoromethanesulfonic acid)imide salts, ethylmalonate salts, and nitrite salts.
• the metal salt is at least one selected from the group consisting of iodide salts, bromide salts, chloride salts, perchlorate salts, oxide salts, trifluoromethanesulfonate salts, toluenesulfonate salts, isopropylsulfonate salts, methanesulfonate salts, carbonate salts, acetate salts, bis(tri
• the bis(trifluoromethanesulfonyl)imide salt is at least one selected from the group consisting of magnesium bis(trifluoromethanesulfonyl)imide, zinc bis(trifluoromethanesulfonyl)imide, and iron(II) bis(trifluoromethanesulfonyl)imide.
• the ethyl malonate is magnesium ethyl malonate.
• the metal atom or the metal ion is selected from the group consisting of lithium iodide, lithium perchlorate, lithium tetrafluoroborate, lithium bromide, lithium chloride, potassium iodide, lithium fluoride, potassium carbonate, potassium nitrite, potassium acetate, potassium tetrafluoroborate, potassium hexafluorophosphate, barium iodide, barium perchlorate, magnesium ethylmalonate, magnesium bis(trifluoromethanesulfonic acid)imide, magnesium oxide, magnesium bromide, magnesium perchlorate, magnesium iodide, magnesium trifluoromethanesulfonate, magnesium sulfate, magnesium acetate, magnesium chloride, magnesium fluoride, calcium iodide, calcium perchlorate, calcium bromide, calcium trifluoromethanesulfonate, calcium carbonate, strontium iodide, scandium trifluoromethanesulfon
• [112] The purification method according to any one of [73] and [109] to [111], wherein the second complex is at least one selected from the group consisting of iron(III) tris(acetylacetonate), ferrocene, zinc(II) acetylacetonate zinc(II) and tungsten hexacarbonyl.
• the first solvent is a solvent capable of forming a complex between the cyclic peptide as a purification target or the peptide as an impurity and the metal atom or the metal ion.
• the third solvent is at least one selected from the group consisting of 2-methyltetrahydrofuran, tetrahydrofuran, dimethyl carbonate, anisole, isopropyl acetate, ethyl acetate, MTBE (tert-butyl methyl ether), diethyl ether, dichloromethane, chloroform, DME (dimethyl ether), CPME (cyclopentyl methyl ether), 4-methyltetrahydropyran, heptane, and toluene.
• the third solvent is at least one selected from the group consisting of 2-methyltetrahydrofuran, tetrahydrofuran, dimethyl carbonate, anisole, isopropyl acetate, ethyl acetate, MTBE (tert-butyl methyl ether), diethyl ether, dichloromethane, chloroform, DME (dimethyl ether), CPME (cyclopentyl methyl ether), 4-
• mixing may mean the “operation” of mixing a substance with another substance, and does not necessarily mean only bringing a substance into a mixed state with another substance.
• mixing (i) and (ii) includes any of the following: adding (i) to (ii), adding (ii) to (i), and adding (i) and (ii) simultaneously.
• the term “to” indicating a range includes both ends of the range, for example, “A to B” means a range equal to or greater than A and equal to or less than B.
• the term “about” when used in combination with a numerical value means a range of +10% and -10% of the numerical value.
• 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, and (vii) A, B, and C.
• the metal atom or the metal ion is preferably 0.2 molar equivalents to 12 molar equivalents, more preferably 0.5 molar equivalents to 4 molar equivalents, and most preferably 0.8 molar equivalents to 1.2 molar equivalents.
• the first complex may mean a complex (complex) between the cyclic peptide to be purified or the peptide that is an impurity and a metal atom or a metal ion, and a complex different from the second complex, and may be the same complex as the complex between the cyclic peptide to be purified or the peptide that is an impurity and a metal atom or a metal ion, and the second complex.
• a solution in which the compound to be measured is dissolved in a solvent suitable for NMR measurement can be added to an NMR measurement sample tube and set in a measurement device for measurement.
• the solvent suitable for NMR measurement is preferably a solvent capable of dissolving the compound to be measured, and a commercially available deuterated solvent can also be used.
• the NMR measurement conditions can be conditions known to those skilled in the art or conditions described in the manual of the measurement device.
• the measurement conditions are not particularly limited as long as the target peak component can be measured, but for example, the measurement temperature can be in the range of 273 K to 320 K, the integration time can be in the range of 1 second to 7 days, and the rotation speed of the sample tube can be in the range of 0 to 20 Hz.
• Each may have a substituent, and the substituents are not limited, and may be independently selected from any substituents including, for example, a halogen atom, an oxygen atom, a nitrogen atom, a sulfur atom, a boron atom, a silicon atom, or a phosphorus atom. That is, examples include alkyl groups, alkoxy groups, alkoxyalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, cycloalkyl groups, etc., which may be substituted, or oxo, aminocarbonyl, halogen atoms, etc.
• side chain of an amino acid means, in the case of an ⁇ -amino acid, the atomic group attached to the carbon to which the amino group and carboxyl group are attached ( ⁇ -carbon).
• ⁇ -amino acid the atomic group attached to the carbon to which the amino group and carboxyl group are attached
• the methyl group of Ala is the side chain of an amino acid.
• the atomic group attached to the ⁇ -carbon and/or ⁇ -carbon can be the side chain of the amino acid
• the atomic group attached to the ⁇ -carbon, ⁇ -carbon, and/or ⁇ -carbon can be the side chain of the amino acid.
• the "main chain of an amino acid” refers to the chain portion consisting of an amino group, an ⁇ -carbon, and a carboxyl group in the case of an ⁇ -amino acid, the chain portion consisting of an amino group, a ⁇ -carbon, an ⁇ -carbon, and a carboxyl group in the case of a ⁇ -amino acid, and the chain portion consisting of an amino group, a ⁇ -carbon, a ⁇ -carbon, an ⁇ -carbon, and a carboxyl group in the case of a ⁇ -amino acid.
• the main chain amino group of an amino acid may be unsubstituted (-NH 2 ) or substituted (i.e., -NHR, where R is, for example, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a cycloalkyl group, or the like, which may have a substituent, and the carbon chain bonded to the N atom and the carbon atom at the ⁇ -position may form a ring, as in proline).
• R is, for example, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a cycloalkyl group, or the like, which may have a substituent, and the carbon chain bonded to the N atom and the carbon atom at the ⁇ -position may form a ring, as in proline).
• substituteduents containing halogen atoms include fluoro (-F), chloro (-Cl), bromo (-Br), and iodine (-I).
• examples of a "substituent containing a phosphorus atom” include phosphoryl (-P(O)-R 1 R 2 ), phosphonyl (-O-P(O)-R 1 R 2 ) and phospho (-PO 3 H 2 ).
• the cyclic peptide to be purified may contain at least one or more unnatural amino acids.
• the cyclic peptide to be purified may contain an N-substituted amino acid.
• the cyclic peptide to be purified may contain 3 or more, 4 or more, 5 or more, or 6 or more N-substituted amino acids, and may contain 15 or less, 13 or less, or 10 or less.
• the number of N-substituted amino acid residues in the cyclic peptide to be purified may be 45% or more, 50% or more, 55% or more, or 60% or more of the number of amino acid residues in the cyclic portion of the cyclic peptide, and may be 80% or less, 75% or less, 70% or less, or 65% or less, with 45% to 80% being preferred.
• an amino acid whose main chain amino group is unsubstituted is referred to as an "N-unsubstituted amino acid".
• the cyclic peptide to be purified may contain an N-unsubstituted amino acid.
• the N-unsubstituted amino acid may be a non-natural amino acid.
• the ratio of the number of the N-unsubstituted amino acids in the cyclic peptide to be purified to the total number of amino acid residues in the cyclic peptide to be purified may be 55% or less, 50% or less, 45% or less, or 40% or less, and may be 20% or more, 25% or more, 30% or more, or 35% or more, with 20% to 55% being preferred.
• the compounds described herein may contain non-natural isotope atoms in one or more atoms constituting such compounds.
• the present invention also includes compounds in which any atom in a compound is replaced with another isotope atom having the same atomic number (proton number) and a different mass number (sum of the number of protons and neutrons), thereby replacing the isotope with an abundance ratio different from that of the natural isotope, that is, a compound labeled with an isotope atom.
• Examples of isotope elements contained in the compounds of the present specification include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, and each of these includes 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, 32 P, 35 S, 18 F, and 36 Cl.
• Compounds labeled with isotope atoms are useful as therapeutic agents, preventive agents, research reagents (e.g., assay reagents), or diagnostic agents (e.g., in vivo imaging diagnostic agents).
• Compounds herein containing radioactive or non-radioactive isotopes in all proportions are within the scope of the present invention.
• Compounds labeled with isotope atoms can be prepared in a similar manner to the preparation of unlabeled compounds, using reagents and solvents containing the corresponding isotope atoms.
• the peptide that is an impurity may be a peptide produced during the synthesis process of the cyclic peptide that is the target of purification.
• the peptide that is an impurity may be a cyclic peptide different from the cyclic peptide that is the target of purification.
• the peptide that is an impurity may be a cyclic peptide having twice the number of amino acids as the cyclic peptide that is the target of purification, a cyclic peptide having three times the number of amino acids as the cyclic peptide that is the target of purification, or an isomer of the cyclic peptide that is the target of purification.
• a preferred example of an isomer is a diastereomer.
• the metal atom or metal ion may be an atom or ion of an alkali metal, alkaline earth metal, transition metal, rare earth metal, or poor metal.
• the alkali metal is preferably at least one selected from the group consisting of lithium, sodium, potassium, rubidium and cesium, and more preferably lithium or potassium.
• the alkaline earth metal is preferably at least one selected from the group consisting of magnesium, calcium, strontium, and barium, and more preferably at least one selected from the group consisting of magnesium, calcium, and barium.
• the metal atom or metal ion may be an atom or ion resulting from a metal salt or a solvate thereof, or a second complex, or may be an atom or ion resulting from a metal salt or a solvate thereof.
• the second complex may mean a complex (complex) between the cyclic peptide to be purified or the peptide to be an impurity and a metal atom or a metal ion, or a complex different from the first complex, or a complex between the cyclic peptide to be purified or the peptide to be an impurity and a metal atom or a metal ion, or the same complex as the first complex may be used.
• the metal salt may mean a metal salt different from the first metal salt, or may be the same metal salt as the first metal salt.
• the bromide salt may be at least one selected from the group consisting of lithium bromide, magnesium bromide, calcium bromide, samarium(III) bromide, zinc bromide, and indium bromide.
• the trifluoromethanesulfonate may be at least one selected from the group consisting of magnesium trifluoromethanesulfonate, calcium trifluoromethanesulfonate, scandium trifluoromethanesulfonate, samarium (III) trifluoromethanesulfonate, cerium trifluoromethanesulfonate, ytterbium trifluoromethanesulfonate, zinc trifluoromethanesulfonate, manganese trifluoromethanesulfonate, indium trifluoromethanesulfonate, iron (III) trifluoromethanesulfonate, and copper trifluoromethanesulfonate.
• the toluenesulfonate may be zinc(II) toluenesulfonate.
• the isopropyl sulfonate may be zinc(II) isopropyl sulfonate.
• the methanesulfonate may be at least one selected from the group consisting of cerium(III) methanesulfonate and zinc difluoromethanesulfonate.
• the carbonate may be at least one selected from the group consisting of potassium carbonate, calcium carbonate, and zinc carbonate.
• the bis(trifluoromethanesulfonyl)imide salt may be at least one selected from the group consisting of magnesium bis(trifluoromethanesulfonyl)imide, zinc bis(trifluoromethanesulfonyl)imide, and iron(II) bis(trifluoromethanesulfonyl)imide.
• the ethyl malonate may be magnesium ethyl malonate.
• Metal atoms or metal ions include lithium iodide, lithium perchlorate, lithium tetrafluoroborate, lithium bromide, lithium chloride, potassium iodide, lithium fluoride, potassium carbonate, potassium nitrite, potassium acetate, potassium tetrafluoroborate, potassium hexafluorophosphate, barium iodide, barium perchlorate, magnesium ethylmalonate, magnesium bis(trifluoromethanesulfonic acid)imide, magnesium oxide, magnesium bromide, magnesium perchlorate, magnesium iodide, magnesium trifluoromethanesulfonate, magnesium sulfate, magnesium acetate, magnesium chloride, magnesium fluoride, calcium iodide, calcium perchlorate, calcium bromide, calcium trifluoromethanesulfonate, calcium carbonate, strontium iodide, scandium trifluoromethanesulfonate, scandium chloride, sama
• the metal atom or metal ion is preferably an atom or ion derived from at least one metal salt or a solvate thereof selected from the group consisting of lithium iodide, lithium perchlorate, potassium iodide, barium iodide, barium perchlorate, magnesium bromide, magnesium perchlorate, magnesium iodide, magnesium trifluoromethanesulfonate, calcium bromide, scandium trifluoromethanesulfonate, samarium trifluoromethanesulfonate, cerium trifluoromethanesulfonate, ytterbium trifluoromethanesulfonate, zinc trifluoromethanesulfonate, manganese trifluoromethanesulfonate, indium trifluoromethanesulfonate, magnesium sulfate, magnesium acetate, and magnesium chloride.
• the first solvent may be a solvent capable of forming a complex between the cyclic peptide to be purified or the peptide to be an impurity and the metal atom or the metal ion, and may include at least one solvent selected from the group consisting of alcohol-based solvents, nitrile-based solvents, benzene-based solvents, ether-based solvents, ketone-based solvents, halogen-based solvents, ester-based solvents, sulfoxide-based solvents, and amide-based solvents.
• the first solvent is a solvent described in this paragraph, a complex between the cyclic peptide to be purified or the peptide to be an impurity and the metal atom or metal ion is easily formed.
• alcohol-based solvents include methanol, ethanol, 1-propanol, 2-propanol, tert-butanol, 2,2,2-trifluoroethanol, ethylene glycol, etc.
• nitrile-based solvents include chain nitriles such as acetonitrile, propionitrile, and acrylonitrile; and cyclic nitriles such as benzonitrile.
• benzene-based solvents include toluene, o-dichlorobenzene, 1,2,4-trichlorobenzene, and xylene.
• the ether-based solvent as the first solvent is preferably at least one selected from the group consisting of tetrahydrofuran, 1,4-dioxane, 2-methyltetrahydrofuran, MTBE (tert-butyl methyl ether), DME (dimethyl ether), and CPME (cyclopentyl methyl ether).
• the ketone-based solvent as the first solvent is preferably acetone or methyl ethyl ketone.
• the halogen-based solvent as the first solvent is preferably dichloromethane.
• the sulfoxide-based solvent as the first solvent is preferably dimethyl sulfoxide.
• the amide-based solvent as the first solvent is preferably dimethylformamide or dimethylacetamide.
• the second solvent may include at least one selected from the group consisting of ether-based solvents, benzene-based solvents, ketone-based solvents, halogen-based solvents, ester-based solvents, nitrile-based solvents, hydrocarbon-based solvents, alcohol-based solvents, sulfoxide-based solvents, and amide-based solvents.
• the ether-based solvent as the second solvent is preferably at least one selected from the group consisting of 2-methyltetrahydrofuran, tetrahydrofuran, 1,4-dioxane, MTBE (tert-butyl methyl ether), DME (dimethyl ether), and CPME (cyclopentyl methyl ether).
• the benzene-based solvent as the second solvent is preferably toluene or xylene.
• the ketone-based solvent as the second solvent is preferably acetone or methyl ethyl ketone.
• the halogen-based solvent as the second solvent is preferably dichloromethane.
• the hydrocarbon-based solvent as the second solvent is preferably at least one selected from the group consisting of hexane, heptane, cyclohexane, methylcyclohexane, and isooctane.
• the ester-based solvent as the second solvent is preferably isopropyl acetate or ethyl acetate.
• the nitrile-based solvent as the second solvent is preferably acetonitrile.
• the alcohol-based solvent as the second solvent is preferably at least one selected from the group consisting of methanol, ethanol, butanol, or benzyl alcohol.
• the sulfoxide-based solvent as the second solvent is preferably dimethyl sulfoxide.
• the amide solvent used as the second solvent is preferably dimethylformamide.
• the second solvent is preferably a combination of isopropyl acetate or methyl ethyl ketone and heptane, a combination of dichloromethane and hexane, or a combination of acetonitrile and MTBE.
• the third solvent may be a solvent that is immiscible with water (e.g., a solvent that has low solubility in water, a solvent that has a high octanol/water partition coefficient (log Kow), or a solvent that has a high predicted octanol/water partition coefficient).
• a solvent that is immiscible with water e.g., a solvent that has low solubility in water, a solvent that has a high octanol/water partition coefficient (log Kow), or a solvent that has a high predicted octanol/water partition coefficient).
• the octanol/water partition coefficient may be determined by any method known in the art or described herein.
• the predicted octanol/water partition coefficient (Log Kow) may be determined by known means in separate explicit measurements, such as, but not limited to, a database search or literature search.
• the method for measuring the octanol/water partition coefficient includes, but is not limited to, a method in accordance with Japanese Industrial Standard JIS 7260-107:2000 Determination of partition coefficient (1-octanol/water) - Shake flask method (https://kikakurui.com/z7/Z7260-107-2000-01.html [Accessed: December 25, 2023]).
• the water-immiscible organic solvent includes, but is not limited to, an organic solvent having low water solubility (e.g., solubility in water of 200 g/L or less, preferably 150 g/L or less).
• the water-immiscible organic solvent may contain other water-miscible organic solvents in trace amounts, e.g., 0.01 wt % or less.
• the water solubility may be determined by any method known in the art or described herein. Exemplary methods for determining the solubility include, but are not limited to, gas chromatography, which may be determined by measuring the concentration of the organic solvent in water prepared by mixing equal volumes of the organic solvent and water at room temperature (e.g., 15° C. to 40° C., preferably 20° C. to 30° C.).
• the water immiscible solvent can be characterized as an ester having 3 to 10 carbon atoms, examples of which include ethyl acetate, isopropyl acetate, n-propyl acetate, t-butyl acetate, methyl propionate, or ethyl propionate.
• NMR measurements were performed at absolute temperatures of 298K or 278K using a Bruker AVANCE III 600 Cryo-TCI, AVANCE III HD 600 SMART-BBFO, AVANCE NEO 600 iProbeTBO, or AVANCE NEO 600 Cryo-TCI-H&F.
• N-alkylamino acid An amino acid having an alkylated N-terminus (N-alkylamino acid) can be produced by reacting an amino acid having a non-alkylated N-terminus with an aldehyde in accordance with the method of Freidinger et al. (J. Org. Chem., 1983, 48(1), 77-81) to obtain an oxazolidinone having a cyclic protecting group introduced therein, and then carrying out a ring-opening reaction of the cyclic protecting group.
• compound aa01 was purchased from a commercial supplier.
• the synthesis method of compound aa02 will be described in detail.
• EDCI.HCl (67.1 g, 350 mmol)
• HOBt (43.4 g, 321 mmol)
• Fmoc-Asp(OtBu)-OH (compound aa01, CAS: 71989-14-5) (120 g, 292 mmol) were mixed in order in DMF (600 mL) at 0° C.
• the resulting mixture was stirred at 0° C. for 1 hour.
• Pyrrolidine (26.3 mL, 321 mmol) was slowly added to the resulting reaction solution, and the mixture was stirred at 0° C.
• dichloromethane 350 mL was added and the reaction vessel was shaken for 5 minutes. After applying nitrogen pressure to remove the liquid components in the reaction vessel, dichloromethane (350 mL) was added and the reaction vessel was shaken for 5 minutes. In this way, washing of the resin with dichloromethane was repeated five times.
• compound aa05 Fmoc-Ile-OH, CAS: 71989-23-6
• Fmoc-Pro-OH CAS: 71989-31-6
• Fmoc-Asp(OAl)-OH CAS: 146982-24-3
• peptide elongation was performed by the basic synthesis method of a cyclic peptide shown in Figure 1.
• the basic synthesis method of a cyclic peptide shown in Figure 1 is as follows: 1) A peptide elongation reaction from the N-terminus of an amino acid by the Fmoc method using a carboxyl group of the Asp side chain or a carboxyl group of a peptide main chain supported on a 2-chlorotrityl resin (a peptide chain elongation reaction using an amino acid protected with an Fmoc group as a raw material); 2) Cleavage process of peptide from 2-chlorotrityl resin; 3) Amide cyclization by condensation of the carboxyl group of the Asp side chain or the carboxyl group of the peptide main chain generated by the cleavage process from the 2-chlorotrityl resin with the amino group of the N-terminus of the peptide chain (triangle unit).
• Peptide compounds containing N-alkylamino acids can be synthesized using the N-alkylamino acids protected by Fmoc groups shown in Tables 2 to 4 as starting materials according to " ⁇ General peptide synthesis method>" described in the present Examples and Reference Examples.
• the number of molar equivalents was calculated based on the amino acid loading rate (mmol/g) of the resin used as the raw material multiplied by the amount of resin used (usually 100 mg).
• the resin was washed 4 times with DMF (0.7 mL per reaction vessel) and 4 times with DCM (0.7 mL per reaction vessel), and dried to obtain Fmoc-MeAla-Pro-MeAla-Cha-Leu-Pro-MePhe(34-F2)-Ala(cPent)-MePhe(4-F)-MeVal-Asp(O-Trt(2-Cl)-resin)-pyrro (compound 3).
• the number of molar equivalents was calculated based on the amino acid loading rate (mmol/g) of the resin used as the raw material multiplied by the amount of resin used (usually 100 mg).
• the production of the desired cyclic peptide was confirmed by LC/MS measurement (Waters SQ Detector2), and the reaction solution was distilled under reduced pressure using a Genevac high-throughput centrifugal evaporator (HT-12).
• the compound was extracted with 1,2-dimethyl-2,5-dihydro-1,2,3-tetrahydrofuran-2,6-dihydro-2,7-trimethyl-4-(2-fluorophenyl)methyl-3-isobutyl-25-isopropyl-9,10,18,19,26,29,35-heptamethyl-22-(pyrrolidine-1-carbonyl)-1,4,7,10,16,19,23,26,29,32,35-undecazatricyclo[35.3.0.012,16]tetracontane-2,5,8,11,17,20,24,27,30,33,36-undecaone) (2.3 g, 68%, purity 84% [peak area percentage]).
• isomer 1 and isomer 2, which are isomers of compound 6 were confirmed from the results of accurate mass analysis before and after the retention time of compound 6 (around 11.1 minutes) (around retention time 10.8 minutes and around retention time 11.4 minutes).
• isomer 1 of compound 6 observed around retention time 10.8 minutes was present at a peak area percentage of 2.0%
• isomer 2 of compound 6 observed around retention time 11.4 minutes was present at a peak area percentage of 6.8%.
• isomer 1 and/or isomer 2 are presumed to be diastereomers of compound 6.
• the relative retention time refers to the value obtained by dividing the retention time of each peak by the retention time of the target product (compound 6 in this case).
• the 1 H-NMR spectrum of compound 6 without the addition of magnesium perchlorate was compared with the 1 H-NMR spectrum of compound 6 with the addition of about 1 molar equivalent of magnesium perchlorate relative to compound 6.
• the spectrum was marked as "not formed” if this did not suggest the formation of a complex.
• Example 1-1 The sample for HPLC measurement in Example 1-1 was prepared to have an initial concentration of about 0.3 mg/mL.
• HPLC was performed on a sample prepared under the same conditions (purity analysis conditions A) as the purity confirmation conditions for compound 6 in Reference Example 1-2.
• purity analysis conditions A purity analysis conditions
• an accurately prepared sample of about 0.3 mg/mL was subjected to HPLC measurement under the above conditions, and the purity and recovery rate were calculated based on the peak area percentage and peak area (measured at 220 nm).
• the ratio of isomer 1 and isomer 2 to compound 6 and the purification efficiency were calculated.
• Recovery rate calculation method 1 For example, if an experiment was conducted using about a mg of raw material, the solid and solution were centrifuged, the solvent was removed by vacuum reduction or nitrogen spraying, and the supernatant was vacuum dried. b mL of acetonitrile was added to the solid, and (150 b/a) ⁇ L of the resulting solution was sampled, and then a sample was prepared so that the entire solution was 0.5 mL of 50% v/v acetonitrile aqueous solution. The HPLC of the prepared sample and an accurately prepared 0.30 mg/mL standard were measured, and if the peak areas (220 nm) of the target substance were c, d, and d, respectively, the recovery rate was expressed by the following formula. In addition, correction was performed based on the weighed value in consideration of the case where the actual weighed value (e mg) does not match the assumed weight a mg.
• Recovery rate calculation method 3 In addition, if an experiment was performed using a mg of raw material, the supernatant and solid were centrifuged, the solvent contained in the supernatant or solid was removed by vacuum distillation and dried, and then 0.5 mL (or 1.0 mL) of acetonitrile was added, and 5 ⁇ L (or 10 ⁇ L) of the resulting solution was mixed with 495 ⁇ L (or 490 ⁇ L) of acetonitrile to prepare a sample. Meanwhile, d mg of raw material was weighed and a standard was prepared in a 10 mL measuring flask. The HPLC of the prepared sample and standard were measured, and if the peak areas (220 nm) of the target product were f and e, respectively, the recovery rate was expressed by the following formula.
• the obtained solid was dried for 6 hours and then dissolved in acetonitrile (0.5 mL), and the purity and recovery rate were confirmed by HPLC, showing that the purity was 90% and the recovery rate was 76%. It was confirmed that the precipitated solid was purified by these operations.
• Methyl ethyl ketone (0.33 mL) was further added and stirred for 6 hours, and the resulting solid and solution were centrifuged. After distilling off the solvent, the vacuum-dried supernatant and solid were redissolved in acetonitrile (1.0 mL). From the obtained supernatant and acetonitrile solution of the solid (1.0 mL), 15 ⁇ L was sampled, and the whole solution was adjusted to 0.5 mL of 50% v/v acetonitrile aqueous solution. The purity and recovery rate were confirmed by HPLC, and it was confirmed that the solid was purified.
• the masses of the inorganic salts in Runs 27 to 31 were calculated from the volume of the solution obtained by preparing an acetonitrile solution of each inorganic salt, dispensing it, and mixing it with compound 6.
• the obtained solid was washed with a mixed solution of dichloromethane (1 mL) and hexane (0.5 mL), and the solid obtained by centrifugation was vacuum dried overnight, and 13.64 mg of compound 6-Mg(ClO 4 ) 2 complex was obtained as a solid.
• the purity and recovery rate of the obtained complex were confirmed by HPLC, and it was found that the purity was 90% and the recovery rate was 82%. It was confirmed that the precipitated solid was purified by these operations.
• Table 11 shows the calculation results of the purity of the supernatant and solid, the recovery rate, the ratio of compound 6 to isomer 1 or isomer 2 given by the following formula, and the purification efficiency for confirming the degree to which compound 6, isomer 1, and isomer 2 were separated from compound 6.
• Ratio of isomer 1 to compound 6 x 100 (Peak area of isomer 1) ⁇ (Peak area of compound 6) ⁇ 100
• Runs 27-31 ethanol (or 60% ethanol in water) was used as the solvent for complex formation, and a purification effect was confirmed in the supernatant in Runs 27 and 28, and in the solid in Runs 30 and 31. Although no significant improvement in purity was observed in Run 29, the purification efficiency was calculated by focusing on the peak at relative retention time 0.71 as shown in Figure 8, and the purification efficiency in the solid was -131%, while in the supernatant it was 100% (not observed), confirming the purification effect.
• the reason why a purification effect was observed in Runs 27-31 is thought to be that the metal salt and compound 6 (or impurities) were dissolved and mixed together in ethanol (or 60% ethanol in water), allowing the formation of a complex.
• Hexane (0.5 mL) was added to each solution and stirred for 1 minute, after which the resulting solid and liquid were quickly centrifuged, and 15 ⁇ L was sampled from the supernatant.
• the solution was stirred again, and 10 minutes after the addition of hexane, centrifuged again, and the supernatant was sampled (15 ⁇ L).
• 30 minutes, 1 hour, 2 hours, 6 hours, and 2 days after the addition of hexane each solution was centrifuged and the supernatant was sampled (15 ⁇ L).
• the solvent was removed from the samples at each stage, and the entire solution was prepared to be 50 ⁇ L of 50% v/v acetonitrile aqueous solution.
• the solvent was removed from the samples sampled at each stage, and the entire solution was prepared to be 0.5 mL of 50% v/v acetonitrile aqueous solution.
• the purity and recovery rate of the supernatant were confirmed by HPLC, and the purity of the standard was 80%, while the purity of the supernatant was 62% and the recovery rate was 21%, indicating that there was a purification effect.
• the solvent was removed from the samples sampled at each stage, and the samples were prepared so that the total solution was 250 ⁇ L and 1.5 mL of 50% v/v acetonitrile aqueous solution, respectively, to match the concentration.
• the purity of the supernatant decreased in both cases, and a purification effect was observed.
• Ratio of Impurity 1 to Compound 9 x 100 (Peak area of impurity 1) ⁇ (Peak area of compound 9) ⁇ 100
• Ratio of impurity 2 to compound 9 x 100 (Peak area of impurity 2) ⁇ (Peak area of compound 9) ⁇ 100
• FIG. 2-a is a diagram of the 1 H-NMR spectrum of compound 6 in an acetonitrile-d3 (CD 3 CN) solution (1.4 mM) without the addition of magnesium perchlorate (Mg(ClO 4 ) 2 ).
• FIG. 2-b is a diagram of the 1 H-NMR spectrum of compound 6 in an acetonitrile-d3 (CD 3 CN) solution (1.4 mM) when magnesium perchlorate (Mg(ClO 4 ) 2 ) was added in an amount of 1 molar equivalent to compound 6.
• FIG. 2-d is a diagram of the 1 H-NMR spectrum of compound 6 in an acetonitrile-d3 (CD 3 CN) solution (1.4 mM) when magnesium perchlorate (Mg(ClO 4 ) 2 ) was added in an amount of 6 molar equivalents relative to compound 6.
• Figure 3 shows 1H-NMR spectra obtained by measuring 1H-NMR at 298K for a solution obtained by dissolving compound 6 in acetonitrile-d3 ( CD3CN ) (cyclic peptide concentration: 1.4 mM) and for solutions obtained by adding 1, 3, and 6 molar equivalents of magnesium iodide ( MgI2 ) to the cyclic peptide (cyclic peptide concentration: 1.4 mM, metal salt addition experiment).
• CD3CN acetonitrile-d3
• MgI2 magnesium iodide
• FIG. 4 shows 1H-NMR spectra obtained by measuring 1H-NMR at 298K for a solution obtained by dissolving compound 6 in acetonitrile-d3 ( CD3CN ) (cyclic peptide concentration: 1.4 mM) and for solutions obtained by adding 1, 3, and 6 molar equivalents of magnesium trifluorosulfonate (Mg(OTf) 2 ) to the cyclic peptide (cyclic peptide concentration: 1.4 mM, metal salt addition experiment).
• CD3CN acetonitrile-d3
• Mg(OTf) 2 magnesium trifluorosulfonate
• FIG. 4-b is a diagram of the 1 H-NMR spectrum of compound 6 in an acetonitrile-d3 (CD 3 CN) solution (1.4 mM) when magnesium trifluorosulfonate (Mg(OTf) 2 ) was added in an amount of 1 molar equivalent to compound 6.
• FIG. 5-b is a diagram of the 1 H-NMR spectrum of compound 6 in an acetonitrile-d3 (CD 3 CN) solution (1.4 mM) when calcium iodide (CaI 2 ) was added in an amount of 1 molar equivalent relative to compound 6.
• FIG. 6 shows 1H-NMR spectra obtained by measuring 1H-NMR at 298K for a solution obtained by dissolving compound 6 in acetonitrile-d3 (CD 3 CN) (cyclic peptide concentration: 1.4 mM) and for solutions obtained by adding 1, 3, and 6 molar equivalents of scandium(III) trifluorosulfonate (Sc(OTf) 3 ) to the cyclic peptide (cyclic peptide concentration: 1.4 mM, metal salt addition experiment) .
• CD 3 CN acetonitrile-d3
• Sc(OTf) 3 scandium(OTf) trifluorosulfonate
• FIG. 6-a is a diagram of the 1 H-NMR spectrum of compound 6 in an acetonitrile-d3 (CD 3 CN) solution (1.4 mM) without the addition of scandium (III) trifluorosulfonate (Sc(OTf) 3 ).
• FIG. 6-b is a diagram of the 1 H-NMR spectrum of compound 6 in an acetonitrile-d3 (CD 3 CN) solution (1.4 mM) when scandium(III) trifluorosulfonate (Sc(OTf) 3 ) was added in an amount of 1 molar equivalent to compound 6.
• FIG. 6-d is a diagram of the 1 H-NMR spectrum of compound 6 in an acetonitrile-d3 (CD 3 CN) solution (1.4 mM) when 6 molar equivalents of scandium(III) trifluorosulfonate (Sc(OTf) 3 ) were added to compound 6.
• FIG. 7 shows 1H-NMR spectra obtained by measuring 1H-NMR at 298K for a solution obtained by dissolving compound 6 in acetonitrile-d3 ( CD3CN ) (cyclic peptide concentration: 1.4 mM) and for solutions obtained by adding 1, 3, and 6 molar equivalents of silver trifluorosulfonate (AgOTf) to the cyclic peptide (cyclic peptide concentration: 1.4 mM, metal salt addition experiment).
• CD3CN acetonitrile-d3
• AgOTf silver trifluorosulfonate
• FIG. 7-b is a diagram of the 1 H-NMR spectrum of compound 6 in an acetonitrile-d3 (CD 3 CN) solution (1.4 mM) when 1 molar equivalent of silver trifluorosulfonate (AgOTf) was added to compound 6.
• FIG. 7-c) is a diagram of the 1 H-NMR spectrum of compound 6 in an acetonitrile-d3 (CD 3 CN) solution (1.4 mM) when 3 molar equivalents of silver trifluorosulfonate (AgOTf) were added to compound 6.
• FIG. 8 shows the LC chart measured in Run 29 of Example 2-1.
• Figure 9 shows the LC chart measured in Run 2 of Example 2-3-1.
• FIG. 10-a shows a 1 H-NMR spectrum obtained by adding deuterated acetonitrile (CD 3 CN, 0.6 mL) to 1.13 mg of the solid obtained in Experimental Procedure 1, and measuring 1 H-NMR using 0.5 mL of the resulting solution.
• FIG. 10-b shows the 1 H-NMR spectrum obtained by adding 1 mL of dichloromethane to 1.02 mg of the solid obtained in Experimental Procedure 1, leaving it overnight, drying it, and then adding deuterated acetonitrile (CD 3 CN, 0.5 mL) and measuring the 1 H-NMR.
• FIG. 10-c shows a 1H-NMR spectrum obtained by adding 1 mL of dichloromethane to 1.14 mg of the solid obtained in Experimental Procedure 1 , and then performing a separation operation according to Experimental Procedure 3, to the solid obtained, and adding deuterated acetonitrile (CD 3 CN, 0.5 mL) to measure H-NMR.

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