Method for manufacturing pharmaceuticals including a freeze-drying step with a mixed solvent

Abstract

The present invention addresses the problem of providing a medicine production method including a freeze-drying method by which the concentration of a residual solvent can be reduced with respect to a wide range of substrate concentrations ranging from a low to a high concentration. The medicine production method according to the present invention comprises (1) a step in which a liquid mixture comprising water, an organic solvent, and a substance to be freeze-dried is prepared and (2) a step in which the liquid mixture is subjected to freeze-drying, the proportion of the mass of the organic solvent to the total mass of the water and the organic solvent in step (1) being 68-99 mass%.

Description

[Technical Field]

[0001] This invention relates to a method for producing pharmaceuticals, which includes a freeze-drying step using a mixed solvent. [Background technology]

[0002] Freeze-drying is an effective method not only for the extraction of amorphous compounds but also for handling unstable compounds and designing injectable formulations (Non-Patent Documents 1 and 2). Among these, freeze-drying of aqueous solutions is a well-known method. However, freeze-drying of aqueous solutions is costly due to the low sublimation rate of ice and the time required (Non-Patent Document 2). Freeze-drying using a mixed solvent system of organic solvents and water is also attracting attention. Some organic solvents have a higher sublimation pressure than water and a lower latent heat of sublimation than water, so improvements in the sublimation rate can lead to shorter drying times, and in the case of compounds unstable in water, improvements in the stability of bulk solutions and dried products can be expected (Non-Patent Document 3). For example, there have been reports of the use of solvents such as t-butanol, dimethyl sulfoxide, 1,4-dioxane, and acetonitrile as organic solvents (Non-Patent Documents 3, 4, 5, and 6). Among these, the most widely evaluated organic solvent / water mixed solvent system is the t-butanol / water mixed system (Non-Patent Document 3). The t-butanol / water mixture can be completely frozen using a commercial freeze-dryer. Furthermore, because t-butanol has a higher vapor pressure and lower latent heat of sublimation compared to water, it sublimes easily during primary drying, allowing for an improved sublimation rate. In addition, it has relatively low toxicity compared to other organic solvents.

[0003] On the other hand, in freeze-drying using a mixed solvent of organic solvent and water, controlling the concentration of toxic residual solvents is a crucial challenge. t-butanol (TBA), one of the solvents used in freeze-drying, is classified as a Class 2 solvent in the ICH Q3C guidelines and is a solvent that should be regulated as a residual solvent in pharmaceuticals (Non-Patent Literature 7).

[0004] Here, it is known that in freeze-drying, the higher the concentration of the substrate to be freeze-dried, the more difficult it becomes to control the residual solvent concentration (Non-Patent Literature 4). For example, Non-Patent Literature 8 reports that if the compound subjected to freeze-drying is not uniformly dissolved in the solvent, it becomes difficult to remove TBA by distillation. That is, when freeze-drying is performed using TBA-water, the mixture separates into a substrate-rich layer and a TBA-rich layer, and it is thought that this layer separation of the solution is the cause of the high residual TBA value. Furthermore, a graph shows that layer separation is more likely to occur at higher substrate concentrations. In addition, Non-Patent Literature 8 discloses that when tobramycin is freeze-dried using 6-9% TBA, the residual TBA concentration tends to increase as the TBA concentration increases, and that the reason for the high residual TBA concentration is the layer separation of the solution. Furthermore, Non-Patent Document 9 describes the dissolution of PC-1, a peptide synthesized by Bristol-Myers Squibb (BMS), in a 20-70% tBuOH / H2O mixed solvent and lyophilization at a substrate concentration of 5-10 mg / mL, but lyophilization at high substrate concentrations is not performed. Non-Patent Document 10 states that to reduce residual TBA concentration, a single-layer solution (monophase solution) should be prepared, the TBA concentration should be higher than the concentration at which crystals form, and an appropriate type of cyclodextrin and lyophilization process should be selected, but there is no disclosure or suggestion regarding substrate concentration. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Biopolymers. 2000; 55(3):227-250. [Non-Patent Document 2] Journal of the Japanese Society of Pharmaceutical Machinery and Technology, 2015, Vol. 24, No. 2, pages 15, 39. [Non-Patent Document 3] European Journal of Pharmaceutical Sciences 15 (2002) 115-133. [Non-Patent Document 4] Journal of Pharmaceutical Sciences 107 (2018) 2005-2012. [Non-Patent Document 5] Journal of Pharmaceutical Sciences 107 (2018) 887-896. [Non-Patent Document 6] Journal of Pharmaceutical Sciences 108 (2019) 399-415. [Non-Patent Document 7] ICH Q3C Guidelines; Q3C(R8) impurities: Guideline for Residual Solvents, Endorsed on 25 March 2020. [Non-Patent Document 8] Journal of Pharmaceutical Sciences, Vol. 91,(2002) 1147-1155. [Non-Patent Document 9] Pharmaceutical Research, Vol. 25, No. 12, December 2008, 2799-2806. [Non-Patent Document 10] Acta Pharmaceutical Sinica 2007, 42(3) 314-317. [Overview of the project] [Problems that the invention aims to solve]

[0006] As mentioned above, it is known that in conventional freeze-drying, controlling the residual solvent concentration becomes more difficult as the substrate concentration increases (Non-Patent Literature 4). In fact, when the inventors applied a lipid-soluble peptide to an existing TBA-aqueous freeze-drying method, they found that there was room for improvement in the residual solvent concentration. However, in the case of freeze-drying of active pharmaceutical ingredients considering large-scale production, the efficiency of freeze-drying decreases when the substrate concentration is low, so it is desirable to be able to perform freeze-drying under conditions of higher substrate concentrations, especially on a large industrial scale. The present invention aims to provide a method for producing pharmaceuticals that includes a freeze-drying method that can reduce the residual solvent concentration over a wide range of substrate concentrations, from low to high. [Means for solving the problem]

[0007] The inventors diligently investigated ways to reduce residual solvent concentration and, to their surprise, discovered that by performing freeze-drying using a mixed solvent consisting of an organic solvent and water in a specific ratio, the residual solvent concentration can be significantly reduced, even when the concentration of the freeze-dried substance ranges from low to high.

[0008] In other words, the present invention relates to the following: [1] A method for manufacturing a pharmaceutical product, including the following steps: (1) A step of preparing a mixed solution containing water, an organic solvent, and a substance to be freeze-dried, (2) A step of subjecting the mixture to freeze-drying, The method wherein, in step (1), the mass ratio of the organic solvent to the total mass of water and the organic solvent is 68% by mass or more and 97% by mass or less. [2] A freeze-drying method for a substance to be freeze-dried, including the following steps: (1) A step of preparing a mixed solution containing water, an organic solvent, and a substance to be freeze-dried, (2) A step of subjecting the mixture to freeze-drying, The method wherein the mass ratio of the organic solvent to the total mass of water and the organic solvent in step (1) is 68% by mass or more and 99% by mass or less. [3] The method according to [1] or [2], wherein step (1) includes preparing a mixed solvent containing water and the organic solvent, and mixing the mixed solvent and the substance to be lyophilized. [4] The method according to any one of [1] to [3], wherein the mass ratio of the substance to be lyophilized in the volume of the mixed solution containing water, an organic solvent, and the substance to be lyophilized is 2 w / v% or more and 20 w / v% or less. [5] The method according to any one of [1] to [4], wherein step (2) includes a preliminary freezing step. [6] The method according to any one of [1] to [5], wherein step (2) includes a multi-stage drying step. [7] The method according to any one of [1] to [6], wherein the freezing point of the mixed solvent containing the organic solvent and water is -60°C or higher. [8] The method according to any one of [1] to [7], wherein the organic solvent is t-butanol. [9] The method according to any one of [1] to [8], wherein the substance to be lyophilized is a lipophilic peptide.

[10] The method according to [9], wherein the lipophilic peptide has a cyclic structure.

[11] The method according to [9] or

[10] , wherein the CLogP of the lipophilic peptide is 25 or less.

[12] The method according to any one of [9] to

[11] , wherein the molecular weight of the lipophilic peptide is 5000 or less. [(13)] The method according to any one of [9] to

[12] , wherein the number of amino acid residues of the lipophilic peptide is 5 or more and 30 or less. [(14)] The method according to any one of [9] to

[13] , wherein the lipophilic peptide contains non-natural amino acid residues. [(15)] The method according to

[14] , wherein the non-natural amino acid residue is a non-natural N-substituted amino acid residue.

[16] The method according to any one of [1] to

[15] , wherein the residual solvent concentration in the freeze-dried powder obtained by freeze-drying is 1.2% or less. [Effects of the Invention]

[0009] According to the method of the present invention, by performing freeze-drying using a mixed solvent obtained by mixing an organic solvent and water in a specific ratio, the residual solvent concentration can be significantly reduced over a wide range of substrate concentrations, from low to high. [Modes for carrying out the invention]

[0010] Unless otherwise specified herein, the abbreviations used herein are as follows: 2-MeTHF:2-methyltetrahydrofuran BEP: 2-bromo-1-ethylpyridinium tetrafluoroborate CPME: Cyclopentyl methyl ether DIPEA: N,N-diisopropylethylamine DMAP: 4-dimethylaminopyridine DMC: Dimethyl Carbonate HATU:O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HMDS: 1,1,1,3,3,3-Hexamethyldisilazane LiBH4: Lithium boron hydride MeCN: Acetonitrile MeOH: methanol MTBE: Methyl tert-butyl ether NMI: N-methylimidazole Pd / C: Palladium Carbon PyBOP: 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate T3P: Propylphosphonic anhydride TBAF: Tetrabutylammonium fluoride TFA: Trifluoroacetic acid THF: Tetrahydrofuran TMSOTf: Trimethylsilyl trifluoromethanesulfonate

[0011] Terms used in this specification In this specification, "lyophilized substance" refers to a compound that is to be freeze-dried. In the present invention, the lyophilized compound is preferably an active ingredient of a pharmaceutical as defined herein, and more preferably a low molecular weight compound or a peptide compound. In this specification, the term "substrate" is used in the same sense as "lyophilized substance." In this specification, the composition to be freeze-dried and / or dried may be referred to as a "sample."

[0012] In this specification, "low molecular weight compounds" refers to compounds, preferably with a molecular weight of 2000 or less, excluding the peptide compounds described later. Examples include natural compounds and compounds whose manufacturing process involves chemical synthesis. The molecular weight of low molecular weight compounds is more preferably 1000 or less, even more preferably 800 or less, and particularly preferably 500 or less. Furthermore, the CLogP of low molecular weight compounds is preferably 16 or less, more preferably 10 or less, even more preferably 7 or less, even more preferably 6 or less, particularly preferably 5 or less, and most preferably 4 or less. ClogP is the partition coefficient calculated by computer after dividing the compound into substructures (calculation software is publicly known; for example, it can be calculated using software from Daylight Chemical Information Systems, Inc., etc.).

[0013] The term "peptide compound" as used herein is not particularly limited as long as it is a peptide compound in which natural amino acids and / or non-natural amino acids are linked by amide bonds or ester bonds, but preferably has 5 or more residues, more preferably 7 or more residues, even more preferably 8 or more residues, and particularly preferably 9 or more residues, and preferably 30 or fewer residues, more preferably 25 or fewer residues, even more preferably 15 or fewer residues, and particularly preferably 13 or fewer residues. Furthermore, the peptide compound may preferably have 5 or more residues, 30 or fewer residues, more preferably 7 or more residues, 25 or fewer residues, even more preferably 8 or more residues, 15 or fewer residues, and particularly preferably 9 or more residues, 13 or fewer residues.

[0014] The peptide compounds that can be used in the present invention preferably contain at least three N-substituted amino acids in one peptide, and more preferably at least five or more N-substituted amino acids. These N-substituted amino acids may be present continuously or discontinuously in the peptide compound. The peptide compounds in the present invention may be linear or cyclic, and cyclic peptide compounds are preferred. They may also have a branched structure. Among peptide compounds having unnatural amino acid residues, it is further preferable that the unnatural amino acid residues are unnatural N-substituted amino acid residues (also referred to as "unnatural N-substituted amino acid residues").

[0015] In this specification, "cyclic peptide compound" is not particularly limited as long as it is a peptide compound having a cyclic portion composed of 5 or more amino acid residues, nor is the method of cyclization limited, but it is a cyclic peptide compound that can be obtained by cyclizing the N-terminal group and the C-terminal group of a linear peptide compound. The cyclization can take any form, such as cyclization via a carbon-nitrogen bond like an amide bond, cyclization via a carbon-oxygen bond like an ester bond or ether bond, cyclization via a carbon-sulfur bond like a thioether bond, cyclization via a carbon-carbon bond, or cyclization by heterocycle construction. Of these, cyclization via a covalent bond such as an amide bond or carbon-carbon bond is preferred, and cyclization via an amide bond between a carboxyl group of a side chain and an amino group of the N-terminal main chain is more preferred. The position of the carboxyl group, amino group, etc. used in cyclization may be on the main chain or on the side chain, and is not particularly limited as long as it is in a position where cyclization is possible.

[0016] In this specification, "amino acids" include natural amino acids and non-natural amino acids (sometimes referred to as amino acid derivatives). In this specification, "natural amino acids" refer to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro. Non-natural amino acids (amino acid derivatives) are not particularly limited, but examples include β-amino acids, D-type amino acids, N-substituted amino acids (excluding Pro), α,α-disubstituted amino acids, amino acids with side chains different from natural amino acids, and hydroxycarboxylic acids. Any stereochemistry is permitted for amino acids in this specification. There are no particular restrictions on the selection of amino acid side chains, but in addition to hydrogen atoms, they 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-bonded cycloalkyl groups. Each of these groups may be substituted, and these substituents are not limited; for example, one or more substituents may be freely selected independently from any group containing halogen atoms, oxygen atoms, sulfur atoms, nitrogen atoms, boron atoms, silicon atoms, or phosphorus atoms. Examples include substituted alkyl groups, alkoxy groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, cycloalkyl groups, or oxo, aminocarbonyl, halogen atoms. In one non-limiting embodiment, the amino acids in this specification may be compounds having both a carboxyl group and an amino group within the same molecule (even in this case, imino acids such as proline and hydroxyproline are also included as amino acids).

[0017] Examples of halogen-derived substituents include fluoro(-F), chloro(-Cl), bromo(-Br), and iod(-I).

[0018] Examples of substituents derived from the oxygen atom include hydroxy(-OH), oxy(-OR), carbonyl(-C=OR), carboxy(-CO2H), oxycarbonyl(-C=O-OR), carbonyloxy(-OC=OR), thiocarbonyl(-C=O-SR), carbonylthio(-SC=OR), aminocarbonyl(-C=O-NHR), carbonylamino(-NH-C=OR), oxycarbonylamino(-NH-C=O-OR), sulfonylamino(-NH-SO2-R), aminosulfonyl(-SO2-NHR), sulfamoylamino(-NH-SO2-NHR), thiocarboxy(-C(=O)-SH), and carboxylcarbonyl(-C(=O)-CO2H).

[0019] Examples of oxy (-OR) compounds include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, and aralkyloxy.

[0020] Examples of carbonyl (-C=OR) include formyl (-C=OH), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, and aralkylcarbonyl.

[0021] Examples of oxycarbonyl (-C=O-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, and aralkyloxycarbonyl.

[0022] Examples of carbonyloxy (-OC=OR) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, and aralkylcarbonyloxy.

[0023] Examples of thiocarbonyl (-C=O-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, and aralkylthiocarbonyl.

[0024] Examples of carbonylthio (-SC=OR) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, and aralkylcarbonylthio.

[0025] Examples of aminocarbonyl (-C=O-NHR) include alkylaminocarbonyl, cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, and aralkylaminocarbonyl. In addition to these, compounds in which the hydrogen atom bonded to the nitrogen atom in -C=O-NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl compounds are also included.

[0026] Examples of carbonylamino (-NH-C=OR) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, and aralkylcarbonylamino. In addition to these, compounds in which the H atom bonded to the N atom in -NH-C=OR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl compounds are also included.

[0027] Examples of oxycarbonylaminos (-NH-C=O-OR) include alkoxycarbonylaminos, cycloalkoxycarbonylaminos, alkenyloxycarbonylaminos, alkynyloxycarbonylaminos, aryloxycarbonylaminos, heteroaryloxycarbonylaminos, and aralkyloxycarbonylaminos. In addition to these, compounds in which the hydrogen atom bonded to the nitrogen atom in the -NH-C=O-OR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl groups are also included.

[0028] Examples of sulfonylaminos (-NH-SO2-R) include alkylsulfonylaminos, cycloalkylsulfonylaminos, alkenylsulfonylaminos, alkynylsulfonylaminos, arylsulfonylaminos, heteroarylsulfonylaminos, and aralkylsulfonylaminos. In addition to these, compounds in which the H atom bonded to the N atom in -NH-SO2-R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl compounds are also included.

[0029] Examples of aminosulfonyl (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, and aralkylaminosulfonyl. In addition to these, compounds in which the H atom bonded to the N atom in -SO2-NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl compounds are also included.

[0030] Examples of sulfamoylamino (-NH-SO2-NHR) include alkyl sulfamoylamino, cycloalkyl sulfamoylamino, alkenyl sulfamoylamino, alkynyl sulfamoylamino, aryl sulfamoylamino, heteroaryl sulfamoylamino, and aralkyl sulfamoylamino. Furthermore, the two H atoms bonded to the N atom in -NH-SO2-NHR may be substituted with substituents independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, and these two substituents may form a ring.

[0031] Examples of substituents derived from the sulfur atom include thiol (-SH), thio (-SR), sulfinyl (-S=OR), sulfonyl (-S(O)2-R), sulfo (-SO3H), and pentafluorosulfanil (-SF5).

[0032] Examples of thio(-SR) are selected from alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, and aralkylthio.

[0033] Examples of sulfinyl (-S=OR) include alkylsulfinyl, cycloalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, arylsulfinyl, heteroarylsulfinyl, and aralkylsulfinyl.

[0034] Examples of sulfonyl (-S(O)2-R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, and aralkylsulfonyl.

[0035] Examples of substituents derived from the N atom include azide (-N3, also called the "azide group"), cyano (-CN), primary amino (-NH2), secondary amino (-NH-R), tertiary amino (-NR(R')), amidino (-C(=NH)-NH2), substituted amidino (-C(=NR)-NR'R''), guanidino (-NH-C(=NH)-NH2), substituted guanidino (-NR-C(=NR''')-NR'R''), and aminocarbonylamino (-NR-CO-NR'R'').

[0036] Examples of secondary amino acids (-NH-R) include alkylaminos, cycloalkylaminos, alkenylaminos, alkynylaminos, arylaminos, heteroarylaminos, and aralkylaminos.

[0037] Examples of tertiary aminos (-NR(R')) include alkyl(aralkyl)aminos, which are amino groups having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, and these two substituents may form a ring.

[0038] Examples of substituted amidinos (-C(=NR)-NR'R'') include groups in which the three substituents R, R', and R'' on the N atom are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, such as alkyl(aralkyl)(aryl)amidinos.

[0039] Examples of substituted guanidinos (-NR-C(=NR''')-NR'R'') include groups where R, R', R'', and R''' are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, or groups in which these groups form a ring.

[0040] Examples of aminocarbonylamino (-NR-CO-NR'R'') include groups in which R, R', and R'' are independently selected from hydrogen atoms, alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, or groups that form a ring.

[0041] Examples of substituents derived from the B atom include boryl (-BR(R')) and dioxyboryl (-B(OR)(OR')). These two substituents R and R' can be independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, or they may form a ring. Specifically, examples include cyclic boryl groups, and more specifically, pinacolate boryl groups, neopentanediolate boryl groups, and catecholate boryl groups.

[0042] Specifically, substituents on the nitrogen atom of N-substituted amino acids in this specification include alkyl, C1-C6 alkyl, C1-C4 alkyl, methyl, and C7-C 14 Examples include aralkyl, benzyl, and phenethyl.

[0043] The main amino group of an amino acid may be unsubstituted (-NH2) or substituted (i.e., -NHR, where R represents an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl group, and may also have a ring formed between the carbon chain bonded to the N atom and the carbon atom at the α-position, as in proline). Amino acids in which such main amino groups are substituted are sometimes referred to as "N-substituted amino acids" in this specification. Preferred "N-substituted amino acids" in this specification are N-alkyl amino acids, N-C1-C6 alkyl amino acids, N-C1-C4 alkyl amino acids, N-methyl amino acids, and N-C7-C 14 Examples include aralkyl amino acids, N-benzyl amino acids, and N-phenethyl amino acids, but are not limited to these.

[0044] As used herein, "amino acid" includes all corresponding isotopes. An isotope of an "amino acid" is one in which at least one atom is replaced by an atom having the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons). Examples of isotopes included in the "amino acid" of this specification include hydrogen atom, carbon atom, nitrogen atom, oxygen atom, phosphorus atom, sulfur atom, fluorine atom, chlorine atom, etc., respectively, 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, 32 P, 35 S, 18 F, <00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ In the method of the present invention, low molecular weight compounds or peptide compounds can be used as the substance to be freeze-dried. Of these, lipid-soluble peptide compounds can be preferably used. Furthermore, among lipid-soluble peptides, lipid-soluble peptides having a cyclic structure can be more preferably used.

[0049] The CLogP of lipid-soluble peptides is preferably 25 or less, more preferably 22 or less, even more preferably 20 or less, even more preferably 18 or less, even more preferably 16 or less, even more preferably 15 or less, preferably 5 or more, more preferably 10 or more, even more preferably 12 or more. The CLogP of lipid-soluble peptide compounds may preferably be 5 or more, 25 or less, more preferably 10 or more, 20 or less, even more preferably 12 or more, and 18 or less.

[0050] The molecular weight of the lipid-soluble peptide is preferably 5000 or less, more preferably 3000 or less, even more preferably 2000 or less, preferably 500 or more, more preferably 800 or more, and even more preferably 1000 or more. Furthermore, the molecular weight of the lipid-soluble peptide compound may be preferably 500 or more, 5000 or less, more preferably 800 or more, 3000 or less, and even more preferably 1000 or more, and 2000 or less.

[0051] The "organic solvent" used in the present invention is preferably one that has a vapor pressure higher than that of water and a latent heat of sublimation lower than that of water. Preferably, the organic solvent is at least one solvent selected from the group consisting of t-butanol, dimethyl sulfoxide, 1,4-dioxane, acetonitrile, acetic acid, cyclohexane, and dimethyl carbonate, more preferably at least one solvent selected from the group consisting of t-butanol, dimethyl sulfoxide, and acetonitrile, and even more preferably t-butanol.

[0052] The freezing point of the organic solvent under atmospheric pressure is preferably -50°C or higher, more preferably 0°C or higher, even more preferably 10°C or higher, particularly preferably 20°C or higher, preferably 50°C or lower, more preferably 40°C or lower, and even more preferably 30°C or lower.

[0053] The freezing point of a mixed solvent containing an organic solvent and water under atmospheric pressure is preferably -60°C or higher, more preferably -40°C or higher, even more preferably -20°C or higher, particularly preferably -10°C or higher, preferably 10°C or lower, more preferably 0°C or lower, and even more preferably -5°C or lower. Furthermore, the freezing point of a mixed solvent containing an organic solvent and water under atmospheric pressure may more preferably be -60°C or higher, 10°C or lower, even more preferably -20°C or higher, 0°C or lower, and particularly preferably -10°C or higher and -5°C or lower.

[0054] Furthermore, if the mixed solvent has multiple freezing points, the above range is a preferred range for the lowest freezing point. The freezing point can also be determined by subjecting a mixed solvent of organic solvent and water, prepared in a predetermined ratio, to measurement using a DSC (Differential Scanning Calorimeter). Specifically, it can be determined by the method described in the examples.

[0055] In step (1) above, the order in which the water, organic solvent, and freeze-drying target substance are mixed is not particularly limited when preparing the mixed solution containing water, organic solvent, and freeze-drying target substance. Preferably, a mixed solvent containing water and organic solvent can be prepared, and the mixed solution can be prepared by mixing the mixed solvent with the freeze-drying target substance. Alternatively, the freeze-drying target substance can be added to the organic solvent to prepare an organic solvent-target substance mixture, and water can be added to the organic solvent-target substance mixture to prepare the mixed solution.

[0056] The mass ratio of the freeze-drying target substance to the volume of the mixed solution containing water, an organic solvent, and the freeze-drying target substance can be preferably set to a lower limit of 2.0 w / v% or more, 5.0 w / v% or more, 7.0 w / v% or more, 9.0 w / v% or more, or 10.0 w / v% or more, and preferably to an upper limit of 20.0 w / v% or less, 15.0 w / v% or less, or 12.0 w / v% or less. The mass ratio of the freeze-drying target substance can be within any combination of these ranges, for example, preferably 2.0 w / v% or more and 20.0 w / v% or less, more preferably 5.0 w / v% or more and 15.0 w / v% or less, and even more preferably 9.0 w / v% or more and 12.0 w / v% or less.

[0057] The mass ratio of the organic solvent to the total mass of water and the organic solvent in step (1) can be preferably set to a lower limit of 68% by mass or more, 70% by mass or more, 75% by mass or more, 80% by mass or more, 85% by mass or more, 88% by mass or more, or 90% by mass or more, and preferably to an upper limit of 99% by mass or less, 97% by mass or less, 95% by mass or less, 92% by mass or less, or 90% by mass or less. The mass ratio of the organic solvent to the total mass of water and the organic solvent in step (1) can be within any combination of these ranges. For example, the mass ratio can be preferably 68% by mass or more and 99% by mass or less, more preferably 68% by mass or more and 97% by mass or less, even more preferably 80% by mass or more and 95% by mass or less, and even more preferably 88% by mass or more and 92% by mass or less. By performing freeze-drying using water and the organic solvent in such mass ratios, a freeze-dried product with a significantly reduced residual solvent concentration can be obtained not only when the freeze-drying target substance is at a low concentration, but also when the freeze-drying target substance is at a high concentration.

[0058] The "freeze-drying" used in step (2) is a process of removing the solvent in the sample by sublimation while it is frozen. More specifically, freeze-drying in the present invention is carried out by freezing the mixture and then reducing the ambient pressure so that the frozen water and frozen organic solvent in the sample sublimate directly from the solid phase to the gas phase. Methods for performing freeze-drying are known to those skilled in the art, and known freeze-drying apparatus can be used. For example, shelf freeze-dryers, spray freeze-dryers, and manifold freeze-dryers can be used, and shelf freeze-dryers are preferred.

[0059] In the present invention, freeze-drying may include a pre-freezing step. Furthermore, it may include multiple drying steps such as primary drying and secondary drying.

[0060] In the pre-freezing step, the mixture is cooled and frozen. The pre-freezing step can preferably be carried out under atmospheric pressure.

[0061] The cooling temperature in the pre-freezing step is not particularly limited, depending on the type and proportion of organic solvent used, but is preferably -80°C or higher, more preferably -60°C or higher, more preferably -10°C or lower, and more preferably -30°C or lower. Alternatively, the cooling temperature can be preferably -80°C or higher, -10°C or lower, more preferably -60°C or higher, and -30°C or lower.

[0062] The cooling rate during cooling is not particularly limited, but preferably a cooling rate of 0.1°C / min or more and 10°C / min or less, more preferably 1°C / min or more and 5°C / min or less, is sufficient to cool the sample to a predetermined cooling temperature.

[0063] After reaching a predetermined cooling temperature, the sample can be frozen by maintaining the predetermined temperature for a period of preferably 30 minutes or more and 10 hours or less, more preferably 1 hour or more and 5 hours or less.

[0064] In the drying process, the frozen sample is dried under reduced pressure. The reduced pressure lowers the boiling point of the solvent, causing the solvent in the sample to sublimate and be removed, thus drying the sample. To supply the energy required for sublimation, the sample can be heated after the reduced pressure is applied.

[0065] The pressure applied to the sample in the freeze-drying apparatus during the drying process is preferably 1 Pa or more and 100 Pa or less, more preferably 10 Pa or more and 50 Pa or less.

[0066] The drying temperature for drying the sample after reduced pressure is not particularly limited, depending on the type and proportion of organic solvent used, but is preferably -60°C or higher, more preferably -50°C or higher, preferably 60°C or lower, and more preferably 50°C or lower. For example, the temperature during drying after reduced pressure can preferably be in the temperature range of -60°C or higher, 60°C or lower, and more preferably -50°C or higher, and 50°C or lower.

[0067] The heating rate (rate of temperature increase) when heating the sample to the predetermined drying temperature is not particularly limited, depending on the type and proportion of organic solvent used, but is preferably 0.1°C / min or more, more preferably 1°C / min or more, and can be a heating rate of about 10°C / min or less, and more preferably 5°C / min or less. For example, the sample can be heated to the predetermined drying temperature at a heating rate of preferably 0.1°C / min or more, 10°C / min or less, and more preferably 1°C / min or more, and about 5°C / min or less.

[0068] The drying process can be carried out in multiple stages. If the drying process is divided into two stages, the first stage can be performed at a temperature preferably of -60°C or higher, more preferably of -30°C or higher, and preferably of 0°C or lower. For example, the drying temperature in the first stage can be in the temperature range of preferably -60°C or higher and 0°C or lower, more preferably -30°C or higher and 0°C or lower.

[0069] Furthermore, in the second stage, drying can be performed at a temperature preferably above 0°C, more preferably above 20°C, and preferably below 60°C, more preferably below 50°C. For example, the drying temperature in the second stage can be in the range of preferably above 0°C, below 60°C, and more preferably above 20°C and below 50°C.

[0070] In the first and second stages, the heating rate for drying the sample is not particularly limited, but preferably the sample can be heated to a predetermined drying temperature at a heating rate of 0.1°C / min or more, more preferably 1°C / min or more, preferably 10°C / min or less, and more preferably 5°C / min or less. For example, the sample can be heated to a predetermined drying temperature at a heating rate of preferably 0.1°C / min or more, 10°C / min or less, more preferably 1°C / min or more, and 5°C / min or less.

[0071] After reaching a predetermined temperature, the sample can be dried by maintaining the predetermined drying temperature for a period of preferably 12 hours or more and 72 hours or less, and more preferably 24 hours or more and 50 hours or less, in each of the first and second stages.

[0072] The residual solvent concentration in the freeze-dried powder obtained by freeze-drying is preferably 1.2% or less, more preferably 1% or less, even more preferably 0.8% or less, and particularly preferably 0.6% (w / w) or less. The residual solvent concentration in the freeze-dried powder can be measured by dissolving the freeze-dried powder sample in a suitable solvent to make a sample solution, and then subjecting the sample solution to gas chromatography. Specifically, it can be measured by the method described in the examples.

[0073] All prior art documents cited herein are incorporated herein by reference. [Examples]

[0074] The following describes preferred specific embodiments of the present invention as examples, but the present invention is not limited thereto.

[0075] [Example 1] [Preparation of substrate solution] 1.0 g of cyclosporine A ("Cyclosporine A", manufactured by ZHEJIANG RUIBANG LABORATORIES; molecular weight 1202.61, CLogP: 14.36, number of amino acid residues 11) and 8 mL of a mixed solvent of tert-butyl alcohol (TBA, "t-butanol", manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., freezing point approximately 25°C) and water (TBA:water = 68:32 mass ratio) were mixed at room temperature. The mixture was then shaken at 100 rpm for 30 minutes at room temperature using a shaking device ("Small Rotary Shaker", manufactured by Nisshin Rika Co., Ltd.) to dissolve the cyclosporine A. The resulting mixture was diluted with the TBA-water mixed solvent to 10 mL to prepare a 10% (w / v) mixture.

[0076] [Pre-freeze] 6.5 mL of the prepared mixture was poured into a stainless steel tray (W23mm, D32mm, H38mm) and then placed on a shelf inside the drying chamber of a freeze-dryer ("VirTis AdVantage Pro ADP-B2EL-EOG-X", manufactured by SP SCIENTIFIC) under atmospheric pressure. Subsequently, the temperature inside the freeze-dryer was cooled to -45°C at a cooling rate of 1°C / min and held there for 2 hours.

[0077] [Primary drying] After pre-freezing, the pressure inside the freeze dryer was reduced to 150 mTorr (approximately 20 Pa), and then the temperature was raised from -45°C to -25°C at a heating rate of 1°C / min, where it was held. The total time, including the holding period at -25°C from the start of heating, was 48 hours.

[0078] [Secondary drying] After primary drying, while maintaining the pressure from the primary drying, the temperature inside the freeze dryer was raised from -25°C to 40°C at a heating rate of 1°C / min and held there. The total time, including the time from the start of heating to holding at 40°C, was 48 hours.

[0079] [Pretreatment for measuring residual solvent concentration] Nitrogen gas was introduced into the freeze-dryer, and the pressure was restored to atmospheric pressure to obtain freeze-dried powder, which was to be used as a sample for measuring the residual solvent concentration. 20.5 mg was weighed out from the obtained freeze-dried powder, and a 2 mL solution was prepared using 1,3-dimethyl-2-imidazolidinone (DMI, "1,3-dimethyl-2-imidazolidinone", manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to serve as the sample solution for measuring the residual solvent concentration.

[0080] [Measurement of residual solvent concentration] The residual solvent concentration in the sample solution was measured by gas chromatography under the following conditions.

[0081] For the residual solvent standard solution, a solution of 26.7 mg of TBA diluted in 2 mL of DMI was used.

[0082] ·Equipment: GC-2010 Plus (SHIMADZU) • Column: DB-624 (Agilent Technologies) 0.53mm ID x 30m, 3μm film thickness ·Injection volume: 2 μL • Column temperature: After maintaining at 40°C for 5 minutes, the temperature was increased to 220°C at a heating rate of 10°C / min. It was then maintained at the same temperature for another 5 minutes. ·Inlet temperature: 200℃ • Detector: FID Detector temperature: 230℃ • Carrier gas: Helium (linear velocity: 35 cm / s) Split ratio: 1:10 • TBA retention time: 1.30 min

[0083] The residual solvent concentration was calculated using the peak area of ​​the chromatogram obtained by gas chromatography, according to the following equations 1 and 2.

[0084] (Formula 1) TIFF0007897151000001.tif19152

[0085] (Formula 2) TIFF0007897151000002.tif13145

[0086] The residual solvent concentrations obtained by Equation 2 are shown in Table 1.

[0087] [Examples 2-11, Comparative Examples 1-4] The residual solvent concentration was measured in the same manner as in Example 1, except that the amount of cyclosporine A used in the preparation of the substrate solution, the mass ratio of TBA to the total mass of TBA and water, the freezing point of the TBA-water mixed solvent at each TBA mass ratio (the lowest freezing point if the mixed solvent has multiple freezing points), the substrate concentration when diluted to 10 mL with the TBA-water mixed solvent, and the amount of freeze-dried powder weighed out in the pretreatment for measuring the residual solvent concentration were as shown in Table 1. The results are shown in Table 1.

[0088] The freezing points of the mixed solvent of TBA and water at each TBA mass ratio were measured using the following method (however, the freezing point for 100% TBA mass ratio is the manufacturer's catalog value).

[0089] [Measurement of freezing point] Approximately 3 μL of a mixed solvent of TBA and water, prepared in advance to each TBA mass ratio, was dispensed into a SUS sealed container (15 μL SUS sealed sample container, Hitachi High-Tech Science) using a micropipette (Rainin Pos-D positive displacement pipette, METTLER TOLEDO). The SUS container was then sealed using a sample sealer (electric sample sealer, Hitachi High-Tech Science), and the freezing point was measured using a thermal analyzer (DSC 3+, METTLER TOLEDO).

[0090] Equipment used: Thermal analyzer DSC 3+, manufactured by METTLER TOLEDO Measurement conditions: The sample was cooled from 30°C to -60°C at a cooling rate of -5°C / min and maintained at the same temperature for 20 minutes. Subsequently, the temperature was increased from -60°C to 30°C at a heating rate of 1°C / min.

[0091] [Table 1]

[0092] [Synthesis Example] Synthesis of Cyclic Peptide Compound 1 The analytical conditions for HPLC are shown below. HPLC analysis conditions method 1 Equipment: Waters ACQUITY UPLC H-Class Column: Ascentis Express 90A C18 (Sigma-Aldrich), 2.1 mm ID × 50 mm, 2.7 μm Mobile phase: 0.05% TFA / water (A), 0.05% TFA / MeCN (B) Elution method: B) 5%(0 min)→100%(5 min)→5%(5.1 min)→5%(7 min) Flow rate: 0.5 mL / min Column temperature: 35°C Detection wavelength: 210nm (PDA (Photodiode Array))

[0093] HPLC analysis conditions method 3 Equipment: Waters ACQUITY UPLC H-Class Column: CAPCELL CORE ADME (OSAKA SODA), 2.1 mm ID×50 mm, 2.7 μm Mobile phase: 0.05% TFA / water (A), 0.05% TFA / MeCN (B) Elution method: B): 5% (0 min) → 100% (5 min) → 5% (5.1 min) → 5% (7 min) Flow rate: 0.5 mL / min Column temperature: 35℃ Detection wavelength: 210nm (PDA)

[0094] HPLC analysis conditions method 4 Equipment: Waters ACQUITY UPLC H-Class Column: ACQUITY UPLC CSH C18 (Waters), 2.1 mm ID × 100 mm, 1.7 μm Mobile phase: 0.05% TFA / water (A), 0.05% TFA / MeCN (B) Elution method: B) 20%(0 min)→100%(10 min)→100%(13.5 min)→20%(13.6 min)→20%(18.0 min) Flow rate: 0.3 mL / min Column temperature: 50°C Detection wavelength: 210 nm (PDA)

[0095] HPLC analysis conditions method 5 Equipment: Waters ACQUITY UPLC H-Class Column: ACQUITY UPLC CSH C18 (Waters), 2.1 mm ID × 150 mm, 1.7 μm Mobile phase: 0.05% TFA / water (A), 0.05% TFA / MeCN (B) Elution method: B) 20%(0 min)→100%(24 min)→100%(29 min)→20%(29.1 min)→20%(34 min) Flow rate: 0.3 mL / min Column temperature: 50°C Detection wavelength: 220 nm (PDA)

[0096] Table 2 shows the HPLC analysis conditions used for each compound. [Table 2] TIFF0007897151000005.tif82170

[0097] Synthesis Example 1 Step H'1 Synthesis of compound a03:tert-butyl 2-[[(2S)-2-[benzyloxycarbonyl(methyl)amino]-3-cyclohexyl-propanoyl]-methyl-amino]acetate TIFF0007897151000006.tif90170

[0098] Compound a01 (2.00 g) and compound a02 (1.37 g) were added to the reaction vessel, then 2-MeTHF (19.0 mL) was added and stirred. After adding DIPEA (5.5 mL), T3P (50 w / w% 2-MeTHF solution, 11.7 mL) was added dropwise while maintaining the internal temperature of the reaction mixture below 32°C, and the mixture was stirred at room temperature for 1 hour. 5% sodium carbonate aqueous solution (12 mL) was added dropwise while maintaining the internal temperature of the reaction mixture below 36°C and stirred, after which the aqueous layer was drained. The obtained organic layer was washed with 5% sodium carbonate aqueous solution (12 mL x 1), 5% sodium bisulfate monohydrate aqueous solution (12 mL x 1), and 10% sodium chloride aqueous solution (50 mL x 2). The obtained organic layer was concentrated under reduced pressure to obtain a residue (2.72 g) containing compound a03. Retention time by HPLC analysis: 4.499 minutes (HPLC analysis conditions: method 3)

[0099] Synthesis Example 2 Step H'2-1 Synthesis of compound a04:tert-butyl 2-[[(2S)-3-cyclohexyl-2-(methylamino)propanoyl]-methyl-amino]acetate TIFF0007897151000007.tif35170

[0100] To the residue (2.70 g) containing compound a03 obtained in Step H'1, 2-MeTHF (18 mL) was added, followed by 5% Pd / C (1.27 g, N.E. Chemcat, STD type 50% hydrated). Degassing with hydrogen gas was performed three times, and then the mixture was stirred for 2 hours. The reaction mixture was filtered by suction using filter paper (Kiriyama Seisakusho, 40 mm, No. 5C), and the residue was washed with 2-MeTHF solution (18 mL x 3). The obtained filtrate and washing solution were combined and concentrated under reduced pressure to obtain a residue (1.77 g) containing compound a04. Retention time by HPLC analysis: 2.419 minutes (HPLC analysis conditions: method 3)

[0101] Synthesis Example 3 Step H'2-2 Synthesis of compound a06:tert-butyl 2-[[(2S)-2-[[2-[benzyloxycarbonyl(methyl)amino]acetyl]-methyl-amino]-3-cyclohexyl-propanoyl]-methyl-amino]acetate TIFF0007897151000008.tif37170

[0102] The residue (1.71 g) containing compound a04 obtained in Step H'2-1, and compound a05 (1.29 g) were dissolved in 2-MeTHF (11.6 mL) and stirred. After adding DIPEA (3.4 mL), T3P (50 w / w% 2-MeTHF solution, 7.2 mL) was added dropwise while maintaining the internal temperature of the reaction mixture below 27°C, and the mixture was stirred at room temperature for 2 hours. 5% sodium carbonate aqueous solution (7.2 mL) was added dropwise while maintaining the internal temperature of the reaction mixture below 29°C and stirred, after which the aqueous layer was drained. The obtained organic layer was washed with 5% sodium carbonate aqueous solution (7.2 mL x 1), 5% sodium bisulfate monohydrate aqueous solution (7.2 mL x 1), and 10% sodium chloride aqueous solution (7.2 mL x 2). The obtained organic layer was concentrated under reduced pressure to obtain a residue (2.70 g) containing compound a06. Retention time by HPLC analysis: 4.458 minutes (HPLC analysis conditions: method 3)

[0103] Synthesis Example 4 Step H'3-1 Synthesis of compound a07:tert-butyl 2-[[(2S)-3-cyclohexyl-2-[methyl-[2-(methylamino)acetyl]amino]propanoyl]-methyl-amino]acetate TIFF0007897151000009.tif35170

[0104] To the residue (2.70 g) containing compound a06 obtained in Step H'2-2, 2-MeTHF (10 mL) was added, followed by 5% Pd / C (0.71 g, N.E. Chemcat, STD type 50% hydrated). Degassing with hydrogen gas was performed three times, and then the mixture was stirred for 2 hours. The reaction mixture was filtered by suction using filter paper (Kiriyama Seisakusho, 40 mm, No. 5C), and the residue was washed with 2-MeTHF solution (10 mL x 3). The obtained filtrate and washing solution were combined and concentrated under reduced pressure to obtain a residue (1.82 g) containing compound a07. Retention time by HPLC analysis: 2.848 minutes (HPLC analysis conditions: method 3)

[0105] Synthesis Example 5 Step H'3-2 Synthesis of compound a09:tert-butyl 2-[[(2S)-2-[[2-[[2-[[benzyloxycarbonyl(methyl)amino]acetyl]-methyl-amino]acetyl]-methyl-amino]-3-cyclohexyl-propanoyl]-methyl-amino]acetate TIFF0007897151000010.tif81170

[0106] The residue (1.80 g) containing compound a07 obtained in Step H'3-1 and compound a08 (1.57 g) were dissolved in 2-MeTHF (14.2 mL) and stirred. After adding DIPEA (4.1 mL), T3P (50 w / w% 2-MeTHF solution, 8.8 mL) was added dropwise while maintaining the internal temperature of the reaction mixture below 30°C, and the mixture was stirred at room temperature for 2 hours. 5% sodium carbonate aqueous solution (10.8 mL) was added dropwise while maintaining the internal temperature of the reaction mixture below 33°C and stirred, after which the aqueous layer was drained. The obtained organic layer was washed with 5% sodium carbonate aqueous solution (10.8 mL x 1), 5% sodium bisulfate monohydrate aqueous solution (10.8 mL x 1), and 5% sodium carbonate aqueous solution (10.8 mL x 1). The obtained organic layer was concentrated under reduced pressure to obtain a residue (2.61 g) containing compound a09. Retention time by HPLC analysis: 4.055 minutes (HPLC analysis conditions: method 3)

[0107] Synthesis Example 6 Step H'4-1 Synthesis of compound a10:tert-butyl 2-[[(2S)-3-cyclohexyl-2-[methyl-[2-[methyl-[2-(methylamino)acetyl]amino]acetyl]amino]propanoyl]-methyl-amino]acetate TIFF0007897151000011.tif46170

[0108] To the residue (2.40 g) containing compound a09 obtained in Step H'3-2, 2-MeTHF (12.3 mL) was added, followed by 5% Pd / C (0.44 g, N.E. Chemcat, STD type 50% hydrated). Degassing with hydrogen gas was performed three times, and then the mixture was stirred for 1 hour. The reaction mixture was filtered by suction using filter paper (Kiriyama Seisakusho, 40 mm, No. 5C), and the residue was washed with 2-MeTHF solution (12 mL x 3). The obtained filtrate and washing solution were combined and concentrated under reduced pressure to obtain a residue (1.97 g) containing compound a10. Retention time by HPLC analysis: 2.521 minutes (Analysis conditions: method 3)

[0109] Synthesis Example 7 Step H'4-2 Synthesis of compound a12:tert-butyl 2-[[(2S)-2-[[2-[[2-[[(2S,3S)-2-(benzyloxycarbonylamino)-3-methylpentanoyl]-methylamino]acetyl]-methylamino]acetyl]-methylamino]-3-cyclohexyl-propanoyl]-methylamino]acetate TIFF0007897151000012.tif73170

[0110] The residue (1.92 g) containing compound a10 obtained in Step H'4-1 and compound a11 (1.69 g) were dissolved in 2-MeTHF (12.8 mL) and stirred. After adding DIPEA (3.7 mL), T3P (50 w / w% 2-MeTHF solution, 7.8 mL) was added dropwise while maintaining the internal temperature of the reaction mixture below 29°C, and the mixture was stirred at room temperature for 2 hours. 5% sodium carbonate aqueous solution (14.4 mL) was added dropwise while maintaining the internal temperature of the reaction mixture below 33°C and stirred, after which the aqueous layer was drained. The resulting organic layer was washed with 5% sodium carbonate aqueous solution (14.4 mL x 1), 5% sodium bisulfate monohydrate aqueous solution (14.4 mL x 1), and 5% sodium carbonate aqueous solution (14.4 mL x 1). The obtained organic layer was further washed twice with 5% sodium bisulfate monohydrate aqueous solution (14.4 mL x 1) and 5% sodium carbonate aqueous solution (14.4 mL x 1). 2-MeTHF (14.4 mL) was added, and this was washed with 5% sodium bisulfate monohydrate aqueous solution (14.4 mL x 1) and 5% sodium carbonate aqueous solution (14.4 mL x 1). Further washing was performed with 1% sodium carbonate aqueous solution (14.4 mL x 3), 5% sodium carbonate aqueous solution (14.4 mL x 5), 5% sodium bisulfate monohydrate aqueous solution (14.4 mL x 1), 5% sodium carbonate aqueous solution (14.4 mL x 1), and 5% sodium carbonate aqueous solution (14.4 mL x 1, 7.2 mL x 10). Finally, washing was performed with 2.5% ammonia water (7.2 mL x 3) and 10% sodium chloride aqueous solution (1 mL x 1). The resulting organic layer was concentrated under reduced pressure to obtain a residue (2.39 g) containing compound a12. Retention time by HPLC analysis: 4.006 minutes (HPLC analysis conditions: method 1)

[0111] Synthesis Example 8 Step H'5-1 Synthesis of compound a13:tert-butyl 2-[[(2S)-2-[[2-[[2-[[(2S,3S)-2-amino-3-methylpentanoyl]-methyl-amino]acetyl]-methyl-amino]acetyl]-methyl-amino]-3-cyclohexyl-propanoyl]-methyl-amino]acetate TIFF0007897151000013.tif44170

[0112] To the residue (2.15 g) containing compound a12 obtained in Step H'4-2, 2-MeTHF (9.3 mL) was added, followed by 5% Pd / C (0.66 g, N.E. Chemcat, STD type 50% hydrated). Degassing with hydrogen gas was performed three times, and then the mixture was stirred for 2 hours. The reaction mixture was filtered by suction using filter paper (Kiriyama Seisakusho, 40 mm, No. 5C), and the residue was washed with 2-MeTHF solution (10 mL x 3). The obtained filtrate and washing solution were combined and concentrated under reduced pressure to obtain a residue (1.95 g) containing compound a13. Retention time by HPLC analysis: 2.776 minutes (HPLC analysis conditions: method 1)

[0113] Synthesis Example 9 Step H'5-2 Synthesis of compound a15: tert-butyl 2-[[(2S)-3-cyclohexyl-2-[methyl-[2-[methyl-[2-[methyl-[(2S,3S)-3-methyl-2-[[(2S)-4-methyl-2-[methyl(2-trimethylsilylethoxycarbonyl)amino]pentanoyl]amino]pentanoyl]amino]acetyl]amino]acetyl]amino]propanoyl]-methyl-amino]acetate TIFF0007897151000014.tif54170

[0114] The residue (1.94 g) containing compound a13 obtained in Step H'5-1 and compound a14 (1.49 g) were dissolved in 2-MeTHF (10.3 mL) and stirred. The reaction vessel was cooled in an ice bath, DIPEA (3.0 mL) was added, and T3P (50 w / w% 2-MeTHF solution, 6.3 mL) was added dropwise while maintaining the internal temperature of the reaction mixture below 9°C. Then the ice bath was removed and the mixture was stirred at room temperature for 1 hour. 5% sodium carbonate aqueous solution (12 mL) was added dropwise while maintaining the internal temperature of the reaction mixture below 22°C and stirred, and the aqueous layer was drained. The resulting organic layer was washed with 5% sodium carbonate aqueous solution (12 mL x 1), 5% sodium bisulfate monohydrate aqueous solution (12 mL x 1), and 5% sodium carbonate aqueous solution (12 mL x 1), and the resulting organic layer was concentrated under reduced pressure. The compound was redissolved in 20 mL of 2-MeTHF and washed with 12 mL x 2 of 5% sodium bisulfate monohydrate aqueous solution and 12 mL x 2 of 5% sodium carbonate aqueous solution. N-methylimidazole (0.3 mL) and 12 mL of 5% sodium carbonate aqueous solution were added to the organic layer and stirred for 6.5 hours, after which the aqueous layer was drained. The mixture was washed with 12 mL x 1 of 5% sodium carbonate aqueous solution, 12 mL x 2 of 5% sodium bisulfate monohydrate aqueous solution and 12 mL x 2 of 5% sodium carbonate aqueous solution, and concentrated under reduced pressure. The compound was redissolved in 20 mL of 2-MeTHF, and 20 mL of heptane:MTBE mixture (1.5:1) was added, washed with 20 mL x 2 of 5% sodium carbonate aqueous solution, and concentrated under reduced pressure to obtain a residue (2.38 g) containing compound a15. Retention time by HPLC analysis: 4.919 minutes (HPLC analysis conditions: method 1)

[0115] Synthesis Example 10 Step H'6 Synthesis of compound a16:tert-butyl 2-[[(2S)-3-cyclohexyl-2-[methyl-[2-[methyl-[2-[methyl-[(2S,3S)-3-methyl-2-[[(2S)-4-methyl-2-(methylamino)pentanoyl]amino]pentanoyl]amino]acetyl]amino]acetyl]amino]propanoyl]-methyl-amino]acetate TIFF0007897151000015.tif36170

[0116] After adding 2-MeTHF (14 mL) to the residue (2.35 g) containing compound a15 obtained in Step H'5-2, the external temperature of the reaction vessel was set to 50°C, and tetrabutylammonium fluoride (1 M THF solution, 7.0 mL) was added. The reaction mixture was stirred for 2 hours. After cooling to room temperature, isopropyl acetate (7 mL) was added, and after washing with 5% potassium carbonate aqueous solution (7 mL x 6), the mixture was concentrated under reduced pressure to obtain a residue (1.92 g) containing compound a16. Retention time by HPLC analysis: 2.909 minutes (HPLC analysis conditions: method 1)

[0117] Synthesis Example 11 StepS'0 Synthesis of compound a19:tert-butyl (3S)-3-[benzyloxycarbonyl(methyl)amino]-4-oxo-4-(1-piperidyl)butanoate TIFF0007897151000016.tif37170

[0118] Compound a17 (2.01 g) and 2-MeTHF (11.7 mL) were added to the reaction vessel and stirred. After adding DIPEA (1.8 mL) and compound a18 (0.53 mL), T3P (3.61 mL) was added at room temperature and the mixture was stirred for 1 hour. 10% citric acid aqueous solution (12 mL) was added while stirring, and the aqueous layer was drained. The resulting organic layer was washed with 10% citric acid aqueous solution (12 mL x 1) and 5% sodium carbonate aqueous solution (12 mL x 2). The resulting organic layer was concentrated under reduced pressure to obtain a residue (1.56 g) containing compound a19. Retention time by HPLC analysis: 3.934 minutes (HPLC analysis conditions: method 3)

[0119] Synthesis Example 12 Step S'1-1 Synthesis of compound a20:tert-butyl (3S)-3-(methylamino)-4-oxo-4-(1-piperidyl)butanoate TIFF0007897151000017.tif29170

[0120] To the residue (3.08 g) containing compound a19 obtained in Step S'0, 2-MeTHF (21.3 mL) was added, followed by the addition of 5% Pd / C (4.09 g). Degassing with hydrogen gas was performed three times, and the mixture was then stirred for 2 hours. The reaction mixture was filtered by suction using filter paper (Kiriyama Seisakusho Co., Ltd., 40 mm, No. 5C), and the residue was washed with 2-MeTHF solution (21.3 mL x 3). The obtained filtrate and washing solution were combined and concentrated under reduced pressure to obtain a residue (2.09 g) containing compound a20. Retention time by HPLC analysis: 2.058 minutes (HPLC analysis conditions: method 3)

[0121] Synthesis Example 13 Step S'1-2 Synthesis of compound a22:tert-butyl (3S)-3-[[(2S)-2-[benzyloxycarbonyl(methyl)amino]-3-methyl-butanoyl]-methyl-amino]-4-oxo-4-(1-piperidyl)butanoate TIFF0007897151000018.tif38170

[0122] The residue (2.04 g) containing compound a20 obtained in Step 1-1 and compound a21 (2.14 g) were dissolved in 2-MeTHF (6.3 mL) and stirred. After adding DIPEA (5.3 mL), HATU (3.95 g) dissolved in 2-MeTHF (5.9 mL) and MeCN (4.1 mL) at room temperature was added, and the mixture was stirred at 50°C for 5 hours. After adding CPME (5.3 mL) at room temperature, 5% potassium carbonate aqueous solution (4.1 mL) and NMI (0.55 mL) were added, and the mixture was stirred for 1 hour and 30 minutes. After adding 2.5% ammonium aqueous solution (16.3 mL) while stirring, the aqueous layer was drained. The obtained organic layer was washed with 2.5% ammonium aqueous solution (16.3 mL × 1), 10% sodium bisulfate monohydrate aqueous solution (20.4 mL × 4), 5% potassium carbonate aqueous solution (20.4 mL × 1), 10% sodium bisulfate monohydrate aqueous solution (20.4 mL × 3), and 5% potassium carbonate aqueous solution (20.4 mL × 1). The obtained organic layer was concentrated under reduced pressure to obtain a residue (3.66 g) containing compound a22. Retention time by HPLC analysis: 4.428 minutes (HPLC analysis conditions: method 3)

[0123] Synthesis Example 14 Step S'2-1 Synthesis of compound a23:tert-butyl (3S)-3-[methyl-[(2S)-3-methyl-2-(methylamino)butanoyl]amino]-4-oxo-4-(1-piperidyl)butanoate TIFF0007897151000019.tif34170

[0124] To the residue (3.56 g) containing compound a22 obtained in Step S'1-2, 2-MeTHF (18.3 mL) was added, followed by the addition of 5% Pd / C (2.10 g). Degassing with hydrogen gas was performed three times, and the mixture was stirred for 2 hours and 30 minutes. The reaction mixture was filtered by suction using filter paper (Kiriyama Seisakusho Co., Ltd., 40 mm, No. 5C), and the residue was washed with 2-MeTHF (18.3 mL x 3). The obtained filtrate and washing solution were combined and concentrated under reduced pressure to obtain a residue (2.58 g) containing compound a23. Retention time by HPLC analysis: 2.393 minutes (HPLC analysis conditions: method 3)

[0125] Synthesis Example 15 Step S'2-2 Synthesis of compound a25:tert-butyl (3S)-3-[methyl-[(2S)-3-methyl-2-[methyl-[1-[(2,2,2-trifluoroacetyl)amino]cyclopentanecarbonyl]amino]butanoyl]amino]-4-oxo-4-(1-piperidyl)butanoate TIFF0007897151000020.tif36170

[0126] Compound a24 (2.90 g) was dissolved in 2-MeTHF (18.6 mL) and stirred. Then, DIPEA (5.4 mL) and the residue (2.50 g) containing compound a23 obtained in Step S'2-1 were added, followed by the addition of T3P (10.4 mL) and DMAP (1.59 g) at room temperature, and the mixture was stirred for 8 hours. Compound a24 (1.47 g), DMAP (0.80 g), T3P (5.5 mL), and DIPEA (2.8 mL) were added, and the mixture was stirred for 2 hours. 5% sodium carbonate aqueous solution (20.5 mL) was added while stirring, and the aqueous layer was drained. The obtained organic layer was washed with 5% sodium bisulfate monohydrate aqueous solution (20.5 mL x 4), 5% sodium carbonate aqueous solution (20.5 mL x 2), 5% sodium bisulfate monohydrate aqueous solution (20.5 mL x 2), and 5% sodium carbonate aqueous solution (20.5 mL x 1). The obtained organic layer was concentrated under reduced pressure to obtain a residue (3.45 g) containing compound a25. Retention time by HPLC analysis: 4.002 minutes (HPLC analysis conditions: method 3)

[0127] Synthesis Example 16 Step S'3-1 Synthesis of compound a26:tert-butyl (3S)-3-[[(2S)-2-[(1-aminocyclopentanecarbonyl)-methyl-amino]-3-methyl-butanoyl]-methyl-amino]-4-oxo-4-(1-piperidyl)butanoate TIFF0007897151000021.tif37170

[0128] The residue (3.41 g) containing compound a25 obtained in Step S'2-2 was dissolved in 2-MeTHF (1362 mL) and MeOH (1.4 mL) and stirred. LiBH4 (ALDRICH, 2M THF solution, 4.5 mL) was added dropwise at -20°C and the mixture was stirred for 2 hours. 2,2,2-trifluoroethanol (6.4 mL) was added dropwise, the temperature was raised to 0°C, and the mixture was stirred for 20 minutes. 20% ammonium chloride aqueous solution (10.2 mL) was added dropwise, and the aqueous layer was drained. Trifluoroacetic acid (0.69 mL) was added to the resulting organic layer at room temperature and the mixture was stirred for 10 minutes. The reaction solution containing compound a26 was added dropwise to a reaction vessel containing 2M sodium hydroxide aqueous solution (44.3 mL). The aqueous layer was drained, and the mixture was then washed with 2M sodium hydroxide aqueous solution (34.1 mL × 2) and 10% dipotassium hydrogen phosphate aqueous solution (17.0 mL × 1). The resulting organic layer was concentrated under reduced pressure to obtain a residue (2.90 g) containing compound a26. Retention time by HPLC analysis: 2.868 minutes (HPLC analysis conditions: method 3)

[0129] Synthesis Example 17 Step S'3-2 Synthesis of compound a28: benzyl (2S)-2-[[1-[[(1S)-1-[[(1S)-3-tert-butoxy-3-oxo-1-(piperidine-1-carbonyl)propyl]-methyl-carbamoyl]-2-methyl-propyl]-methyl-carbamoyl]cyclopentyl]carbamoyl]pyrrolidine-1-carboxylate TIFF0007897151000022.tif32170

[0130] The residue (2.90 g) containing compound a26 obtained in Step S'3-1 and compound a27 (1.66 g) were dissolved in MeCN (14.5 mL) and stirred. After adding DIPEA (2.67 mL), BEP (2.11 g) was added at room temperature and the mixture was stirred for 3 hours. After adding CPME (29.3 mL), 5% potassium carbonate aqueous solution (17.4 mL) and N-methylimidazole (0.41 mL) were added and the mixture was stirred at room temperature for 30 minutes. After draining the aqueous layer, the obtained organic layer was washed with 5% sodium bisulfate monohydrate aqueous solution (17.4 mL × 5), 5% sodium carbonate aqueous solution (17.4 mL × 2), 5% sodium bisulfate monohydrate aqueous solution (17.4 mL × 3), and 5% sodium carbonate aqueous solution (17.4 mL × 2). The obtained organic layer was concentrated under reduced pressure to obtain a residue (3.86 g) containing compound a28. Retention time by HPLC analysis: 4.323 minutes (HPLC analysis conditions: method 3)

[0131] Synthesis Example 18 Step S'4-1 Synthesis of compound a29:tert-butyl (3S)-3-[methyl-[(2S)-3-methyl-2-[methyl-[1-[[(2S)-pyrrolidine-2-carbonyl]amino]cyclopentanecarbonyl]amino]butanoyl]amino]-4-oxo-4-(1-piperidyl)butanoate TIFF0007897151000023.tif33170

[0132] To the residue (3.81 g) containing compound a28 obtained in Step S'3-2, THF (16.8 mL) was added, followed by the addition of 5% Pd / C (0.40 g). Degassing with hydrogen gas was performed three times, and the mixture was then stirred for 4 hours and 30 minutes. 5% Pd / C (0.20 g) was added, and the mixture was then stirred for 1 hour and 30 minutes. The reaction mixture was filtered by suction using filter paper (Kiriyama Seisakusho Co., Ltd., 40 mm, No. 5C), and the residue was washed with 2-MeTHF solution (6.6 mL x 3). The obtained filtrate and washing solution were combined and concentrated under reduced pressure to obtain a residue (3.12 g) containing compound a29. Retention time by HPLC analysis: 2.970 minutes (HPLC analysis conditions: method 3)

[0133] Synthesis Example 19 Step S'4-2 Synthesis of compound a31:tert-butyl (3S)-3-[[(2S)-2-[[1-[[(2S)-1-[(2S)-2-(benzyloxycarbonylamino)-4-phenyl-butanoyl]pyrrolidine-2-carbonyl]amino]cyclopentanecarbonyl]-methyl-amino]-3-methyl-butanoyl]-methyl-amino]-4-oxo-4-(1-piperidyl)butanoate TIFF0007897151000024.tif37170

[0134] The residue (2.06 g) containing compound a29 obtained in Step S'4-1 and compound a30 (1.10 g) were dissolved in 2-MeTHF (10.3 mL) and stirred. After adding DIPEA (2.8 mL), T3P (5.4 mL) was added at room temperature and the mixture was stirred for 2 hours. Compound a30 (0.55 g), DIPEA (1.1 mL), and T3P (2.2 mL) were added separately and the mixture was stirred for 5 hours. Compound a30 (0.57 g), DIPEA (1.1 mL), and T3P (2.2 mL) were added and the mixture was left to stand overnight, then stirred for 2 hours the next day. 5% potassium carbonate aqueous solution (12.4 mL) and N-methylimidazole (0.29 mL) were added and the mixture was stirred at room temperature for 3 hours. N-methylimidazole (0.23 mL) was added and the mixture was stirred for 1 hour, after which the aqueous layer was drained. 12.4 mL of 2-MeTHF, 0.23 mL of N-methylimidazole, and 12.4 mL of 5% potassium carbonate aqueous solution were added and stirred for 1 hour, after which the aqueous layer was drained. The resulting organic layer was washed with 12.4 mL x 2 of 10% sodium bisulfate monohydrate aqueous solution and 12.4 mL x 1 of 5% potassium carbonate aqueous solution. A mixed solution of heptane and MTBE (heptane / MTBE = 1.5:1, 12.4 mL) and MeCN (4.7 mL) were added to the organic layer, and the aqueous layer was drained. 2.1 mL of 2-MeTHF was added to the organic layer, and then it was washed seven times with MeCN (7.0 mL) and 17.7 mL of 5% potassium carbonate aqueous solution. Isopropyl acetate (7.6 mL) was added to the organic layer, and it was concentrated under reduced pressure. Isopropyl acetate (7.6 mL) was added to the resulting residue to obtain a solution containing compound a31 (10.31 g). Retention time by HPLC analysis: 4.794 minutes (HPLC analysis conditions: method 3)

[0135] Synthesis Example 20 Step 1' Synthesis of compound a32: (3S)-3-[[(2S)-2-[[1-[[(2S)-1-[(2S)-2-(benzyloxycarbonylamino)-4-phenyl-butanoyl]pyrrolidine-2-carbonyl]amino]cyclopentanecarbonyl]-methyl-amino]-3-methyl-butanoyl]-methyl-amino]-4-oxo-4-(1-piperidyl)butanoic acid TIFF0007897151000025.tif42170

[0136] The residue (10.27 g) containing compound a31 obtained in Step S'4-2 was dissolved in isopropyl acetate (51.4 mL) and stirred. After adding HMDS (2.1 mL), TMSOTf (1.4 mL) was added dropwise at 0°C, and the mixture was stirred at room temperature for 2 hours and 30 minutes. 2-MeTHF (51.4 mL) and 5% dipotassium hydrogen phosphate aqueous solution (102.8 mL) were added at room temperature, and the aqueous layer was drained. The organic layer was washed with 5% sodium dihydrogen phosphate aqueous solution (102.8 mL), and the resulting organic layer was concentrated under reduced pressure by adding DIPEA (3.0 mL), and isopropyl acetate (7.6 mL) was added to obtain a solution (7.92 g) containing compound a32. Retention time by HPLC analysis: 4.001 minutes (HPLC analysis conditions: method 3)

[0137] Synthesis Example 21 Step 2' Synthesis of compound a33: tert-butyl 2-[[(2S)-2-[[2-[[2-[[(2S,3S)-2-[[(2S)-2-[[(3S)-3-[[(2S)-2-[[1-[[(2S)-1-[(2S)-2-(benzyloxycarbonylamino)-4-phenyl-butanoyl]pyrrolidine-2-carbonyl]amino]cyclopentanecarbonyl]-methyl-amino]-3-methyl-butanoyl]-methyl-amino]-4-oxo-4-(1-piperidyl)butanoyl]-methyl-amino]-4-methyl-pentanoyl]amino]-3-methyl-pentanoyl]-methyl-amino]acetyl]-methyl-amino]acetyl]-methyl-amino]-3-cyclohexyl-propanoyl]-methyl-amino]acetate TIFF0007897151000026.tif62170

[0138] The residue containing compound a32 obtained in Step 1' (7.92 g) and the residue containing compound a16 obtained in Step H'6 (1.33 g) were dissolved in 2-MeTHF (4.6 mL) and stirred. DIPEA (1.6 mL) and HATU (1.43 g) were added at room temperature, and the mixture was stirred for 2 hours. The residue containing compound a16 (approximately 300 mg) was added, and the mixture was stirred for another 2 hours. Then, the residue containing compound a16 (approximately 300 mg) was added, and the mixture was stirred for 1 hour and 30 minutes. After adding HATU (0.79 g), the mixture was stirred for 1 hour. CPME (3.5 mL), N-methylimidazole (0.13 mL), and 5% potassium carbonate aqueous solution (2.7 mL) were added, and the mixture was stirred at room temperature for 3 hours. After draining the aqueous layer, the mixture was washed with 2.5% ammonia aqueous solution (9.2 mL x 1), 10% sodium bisulfate monohydrate aqueous solution (9.2 mL x 1), 5% sodium carbonate aqueous solution (9.2 mL x 1), 10% sodium bisulfate monohydrate aqueous solution (9.2 mL x 3), and 5% sodium bicarbonate aqueous solution (9.2 mL x 2). Heptane / MTBE (1.5:1, 9.2 mL) was added to the organic layer, and it was washed with 5% sodium carbonate aqueous solution (9.2 mL x 2). 2-MeTHF (9.2 mL) was added to the resulting organic layer, and it was concentrated under reduced pressure to obtain a residue (4.49 g) containing compound a33. Retention time by HPLC analysis: 10.65 minutes (Analysis conditions: method 4)

[0139] Synthesis Example 22 Step 3' Synthesis of compound a34: 2-[[(2S)-2-[[2-[[2-[[(2S,3S)-2-[[(2S)-2-[[(3S)-3-[[(2S)-2-[[1-[[(2S)-1-[(2S)-2-(benzyloxycarbonylamino)-4-phenyl-butanoyl]pyrrolidine-2-carbonyl]amino]cyclopentanecarbonyl]-methyl-amino]-3-methyl-butanoyl]-methyl-amino]-4-oxo-4-(1-piperidyl)butanoyl]-methyl-amino]-4-methyl-pentanoyl]amino]-3-methyl-pentanoyl]-methyl-amino]acetyl]-methyl-amino]acetyl]-methyl-amino]-3-cyclohexyl-propanoyl]-methyl-amino]acetic acid TIFF0007897151000027.tif58170

[0140] The residue (4.49 g) containing compound a33 obtained in Step 2' was dissolved in 2-MeTHF (49.5 mL) and stirred. HMDS (2.9 mL) and TMSOTf (2.1 mL) were added at room temperature, and the mixture was stirred for 2 hours. After adding 5% dipotassium hydrogen phosphate aqueous solution (14.2 mL), the aqueous layer was drained. The obtained organic layer was washed three times with a mixed aqueous solution of 10% citric acid aqueous solution (3.4 mL) and 5% dipotassium hydrogen phosphate aqueous solution (10.5 mL), and once with 5% sodium carbonate aqueous solution. THF (20.9 mL) was added to the organic layer, and azeotropic dehydration was performed three times. Then, THF (5.9 mL) was added to the resulting residue to obtain a solution (9.59 g) containing compound a34. Retention time by HPLC analysis: 9.26 minutes (Analysis conditions: method 4)

[0141] Synthesis Example 23 Step 4' Synthesis of compound a35: 2-[[(2S)-2-[[2-[[2-[[(2S,3S)-2-[[(2S)-2-[[(3S)-3-[[(2S)-2-[[1-[[(2S)-1-[(2S)-2-amino-4-phenyl-butanoyl]pyrrolidine-2-carbonyl]amino]cyclopentanecarbonyl]-methyl-amino]-3-methyl-butanoyl]-methyl-amino]-4-oxo-4-(1-piperidyl)butanoyl]-methyl-amino]-4-methyl-pentanoyl]amino]-3-methyl-pentanoyl]-methyl-amino]acetyl]-methyl-amino]acetyl]-methyl-amino]-3-cyclohexyl-propanoyl]-methyl-amino]acetic acid TIFF0007897151000028.tif82170

[0142] 5% Pd / C (0.49 g, manufactured by N.E. Chemcat, STD type 50% hydrated) was added to the reaction vessel, suspended in THF (8 mL), and stirred under a hydrogen atmosphere for 30 minutes. Then, nitrogen was purged, and a solution of the residue (9.3 g) containing compound a34 obtained in Step 3' dissolved in THF (8 mL) was added, and the mixture was stirred under a hydrogen atmosphere for 6 hours, resulting in a reaction conversion rate of 76%. After purging with nitrogen and storing in a refrigerator overnight, the mixture was returned to room temperature the next day and stirred for 2 hours, resulting in a reaction conversion rate of 87%. After purging with nitrogen, a suspension of 5% Pd / C (0.24 g) in THF (4 mL) was added to the reaction mixture, hydrogen was purged, and the mixture was stirred for 4 hours (reaction conversion rate 99.0%). After purging with nitrogen and storing in a refrigerator overnight, the mixture was returned to room temperature the next day and measured, resulting in a reaction conversion rate of 99.4%. The reaction mixture was filtered by suction using filter paper (Kiriyama Seisakusho Co., Ltd., 40 mm, No. 5C), and the residue was washed with 2-MeTHF solution (6.5 mL x 10). The obtained filtrate and washing solution were combined and concentrated under reduced pressure. The obtained residue was dissolved in acetonitrile (16.3 mL) and 2-MeTHF (6.5 mL), washed with heptane (37.1 mL), and concentrated. The obtained residue was again dissolved in acetonitrile (16.3 mL) and 2-MeTHF (6.5 mL), washed with heptane (37.1 mL), and concentrated to obtain a residue (2.96 g) containing compound a35. Retention time by HPLC analysis: 12.39 minutes (HPLC analysis conditions: method 4)

[0143] Synthesis Example 24 Step 5' Synthesis of compound a36: (3S,9S,18S,21S,25S,28S,34S)-9-(cyclohexylmethyl)-21-isobutyl-28-isopropyl-7,10,13,16,22,26,29-heptamethyl-18-[(1S)-1-methylpropyl]-3-(2-phenylethyl)-25-(piperidine-1-carbonyl)spiro[1,4,7,10,13,16,19,22,26,29,32-undecazabicyclo[32.3.0]heptatriacontane-31,1'-cyclopentane]-2,5,8,11,14,17,20,23,27,30,33-undecaone TIFF0007897151000029.tif88170

[0144] A dimethyl carbonate solution (72.5 mL) containing compound a35 (2.90 g) obtained in the previous step and DIPEA (1.44 mL) was prepared, and this was added dropwise to a dimethyl carbonate solution (72.5 mL) of PyBOP (4.34 g) over 3 hours. After sampling 30 minutes after the end of the dropwise addition, the reaction was confirmed, and insoluble matter was removed by suction filtration using filter paper (Kiriyama Seisakusho Co., Ltd., 40 mm, No. 5C), and the residue was washed with dimethyl carbonate (15 mL). The solution of the filtrate and washing solution was washed with 2.5% aqueous ammonia solution (58 mL), 5% aqueous potassium bisulfate solution (58 mL), 5% aqueous disodium hydrogen phosphate solution (58 mL), 5% aqueous sodium chloride solution (58 mL), and 0.5% aqueous sodium chloride solution (58 mL). The resulting organic layer was concentrated under reduced pressure to obtain a residue (2.72 g) containing compound a36. Retention time by HPLC analysis: 18.69 minutes (HPLC analysis conditions: method 5)

[0145] The obtained residue (2.72 g) was purified by silica gel column chromatography (dichloromethane / methanol) to obtain compound a36 (1.1 g). The molecular weight of the obtained compound a36 was 1299.71, the ClogP was 13.64, and the number of amino acid residues was 11.

[0146] [Examples 12-16] Compound a36 was used instead of cyclosporine A, and the residual solvent concentration was measured in the same manner as in Example 1, except that the amount of compound a36 used in the preparation of the substrate solution, the mass ratio of TBA to the total mass of TBA and water, the freezing point of the TBA-water mixed solvent at each TBA mass ratio (the lowest freezing point if the mixed solvent has multiple freezing points), the substrate concentration when diluted to 10 mL with the TBA-water mixed solvent, and the amount of freeze-dried powder weighed out in the pretreatment for measuring the residual solvent concentration were as shown in Table 3. The results are shown in Table 3.

[0147] [Table 3]

[0148] As shown in the results above, by performing freeze-drying using water and organic solvents in a specific mass ratio, it is possible to obtain a freeze-dried product with significantly reduced residual solvent concentration, even when the substance to be freeze-dried is at a high concentration. For example, when comparing Example 1 and Comparative Example 3, and Example 6 and Comparative Example 4, it can be seen that the residual solvent concentration differs significantly despite only a slight difference in the TBA / water ratio. Such results in residual solvent concentration were previously unknown and are a surprising result that was revealed for the first time through the inventors' research. [Industrial applicability]

[0149] In this invention, by performing freeze-drying using a mixed solvent obtained by mixing an organic solvent and water in a specific ratio, the residual solvent concentration can be significantly reduced across a wide range of substrate concentrations, from low to high. In other words, according to the method of this invention, the residual solvent concentration can be reduced even when freeze-drying is performed under high substrate concentration conditions, so freeze-drying can be efficiently carried out even on a large industrial scale, for example.

Claims

1. A pharmaceutical manufacturing method that includes the following steps: (1) A step of preparing a mixed solution containing water, an organic solvent, and a substance to be freeze-dried, (2) A step of subjecting the mixture to freeze-drying, Here, the mass ratio of the organic solvent to the total mass of water and organic solvent in step (1) is 68% by mass or more and 99% by mass or less. The mass ratio of the freeze-drying target substance to the volume of the mixture containing water, an organic solvent, and the freeze-drying target substance is 20 w / v% or less. The substance to be freeze-dried is a lipid-soluble peptide having a cyclic structure and containing non-natural amino acid residues. The CLogP of the lipid-soluble peptide is 5 or more and 25 or less. The number of amino acid residues of the lipid-soluble peptide is 5 or more and 30 or less. A method wherein the organic solvent is t-butanol.

2. The method according to claim 1, wherein step (1) includes preparing a mixed solvent containing water and the organic solvent, and mixing the mixed solvent with the substance to be freeze-dried.

3. The method according to claim 1, wherein the mass ratio of the freeze-drying target substance to the volume of the mixture containing water, an organic solvent, and the freeze-drying target substance is 2 w / v% or more and 20 w / v% or less.

4. The method according to claim 1, wherein step (2) includes a pre-freezing step.

5. The method according to claim 1, wherein step (2) includes a multi-stage drying step.

6. The method according to claim 1, wherein the freezing point of the mixed solvent containing the organic solvent and water is -60°C or higher.

7. The method according to claim 1, wherein the residual solvent concentration in the freeze-dried powder obtained by freeze-drying is 1.2% or less.

8. The method according to claim 1, wherein the CLogP of the lipid-soluble peptide is 10 or more and 25 or less.

9. The method according to claim 1, wherein the molecular weight of the lipid-soluble peptide is 5000 or less.

10. The method according to claim 1, wherein the non-natural amino acid residue is a non-natural N-substituted amino acid residue.

11. The method according to any one of claims 1 to 10, wherein the pharmaceutical product contains the freeze-dried substance as an active ingredient.

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