Crystal of amide-crosslinked compound and method for producing same

Abstract

The purpose of the present disclosure is to provide an amide-crosslinked amidite monomer which contains a guanine base and can contribute to a reduction in the amount of the amidite monomer used during oligonucleotide synthesis. The present disclosure relates to a crystal of an amide-crosslinked compound represented by formula (1). (In the formula, P1 represents a protecting group for a hydroxyl group and P2 represents a protecting group for an amino group.

Description

Crystal of amide-bridged compound and method for producing the same

[0001] The present disclosure relates to an amidite monomer, which is an effective raw material for synthesizing nucleic acid drugs, and a method for producing the same.

[0002] Nucleic acid drugs generally have a single-stranded or double-stranded DNA structure or RNA structure, and are classified according to their function and structure into antisense, siRNA, miRNA, aptamer, decoy, CpG oligodeoxynucleotide, etc. Among such nucleic acid drugs, those suitable for antisense include, for example, oligonucleotides having an amide bridge structure introduced into the five-membered ring of the sugar moiety, oligonucleotides having a guanidine bridge structure introduced into the five-membered ring of the sugar moiety, oligonucleotides having a spirocyclopropylene bridge structure introduced into the five-membered ring of the sugar moiety, and oligonucleotides having a cyclopropane ring introduced into the methylene group at the 5'-position of the sugar moiety.

[0003] Patent Document 1 discloses an amidite monomer represented by the following formula A-12 as a raw material for an oligonucleotide having an amide bridge structure introduced therein, and the amidite monomer of formula A-12 is produced by treating the hydroxyl group at the 3'-position of a nucleotide represented by the following formula A-11 with a phosphorodiamidite.

[0004]

[0005] For example, Example 6 of Patent Document 1 discloses a method for obtaining a compound represented by the following formula 24 from a compound represented by the following formula 23a.

[0006]

[0007] International Publication No. 2014 / 109384

[0008] However, in the synthesis of amide-bridged oligonucleotides using the amidite monomer represented by Formula A-12 above, it is necessary to use an excess amount of the amidite monomer in the coupling step to improve the reaction conversion rate, which poses a problem of increased synthesis costs. In particular, the inventors' studies have revealed that in oligonucleotide synthesis using an amide-bridged amidite monomer containing a guanine base, such as that represented by Formula 24 above, it is necessary to use a large amount of the amidite monomer to improve the reaction conversion rate, resulting in increased costs. The present disclosure has been made in light of the above-mentioned circumstances, and its main object is to provide an amide-bridged amidite monomer containing a guanine base that can contribute to reducing the amount of amidite monomer used in oligonucleotide synthesis.

[0009] Furthermore, according to the studies of the present inventors, it has been found that the amide-bridged amidite monomer containing a guanine base represented by the above formula 24 is a compound with a low diastereomeric excess, containing a substantial amount of both of the two diastereomers derived from the asymmetric phosphorus atom. Because the asymmetry of the amidite monomer is lost during oligonucleotide synthesis, the two diastereomers are equivalent and both are useful as raw materials for nucleic acid drug synthesis. Therefore, a preferred object of the present disclosure is to provide an amide-bridged amidite monomer containing a guanine base, which not only contributes to reducing the amount of amidite monomer used during oligonucleotide synthesis but also allows the effective use of both of the two diastereomers.

[0010] As a result of extensive research to solve the above problems, the present inventors have discovered that a specific method can be used to produce crystals of amide-bridged amidite monomers containing guanine bases, and that crystals of amide-bridged amidite monomers containing guanine bases can reduce the amount of amidite monomer used in oligonucleotide synthesis, thereby completing the present disclosure.

[0011] The gist of the present disclosure is as follows: [1] A crystal of an amide-bridged compound represented by the following formula (1):

[0012] (In the formula, P １ represents a protecting group for a hydroxyl group, and P ２ represents a protecting group for an amino group.) [2] The crystal according to [1], which contains both a compound represented by the following formula (1a) and a compound represented by the following formula (1b), which are in a diastereomeric relationship.

[0013] (In the formula, P １ and P ２ are the same as above.) [3] The crystal according to [2], wherein the diastereomeric excess calculated by the following formula is more than 60%. Diastereomeric excess (%) = | (amount of one diastereomer - amount of the other diastereomer) / (amount of one diastereomer + amount of the other diastereomer) | × 100 [4] The crystal according to [2], wherein the diastereomeric excess calculated by the following formula is 60% or less. Diastereomeric excess (%) = | (amount of one diastereomer - amount of the other diastereomer) / (amount of one diastereomer + amount of the other diastereomer) | × 100 [5] The crystal according to [2], wherein the diastereomeric excess calculated by the following formula is 60% or less. Diastereomeric excess (%) = | (amount of one diastereomer - amount of the other diastereomer) / (amount of one diastereomer + amount of the other diastereomer) | × 100 １ is a 4,4'-dimethoxytriphenylmethyl group, a tert-butyldimethylsilyl group, or a triisopropylsiloxymethyl group, ２is a benzoyl group, an acetyl group, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, an isobutyryl group, or a dimethylformamidinyl group. [6] The crystal according to any of [1] to [5], which has peaks at diffraction angles (2θ) of 5.6±0.2°, 13.6±0.2°, 14.0±0.2°, and 16.9±0.2° in a powder X-ray diffraction pattern measured using Cu-Kα radiation as an X-ray source. [7] A method for producing a crystal of an amide-bridged compound, the method comprising: dissolving an amide-bridged compound represented by the following formula (1), the compound having a diastereomeric excess calculated by the following formula of more than 60%, in a mixed solvent of ethyl acetate as a good solvent and methyl tert-butyl ether as a poor solvent; and allowing heptane as a poor solvent to be absorbed as vapor into the solution to increase the heptane concentration in the solution, thereby obtaining a crystal of the amide-bridged compound represented by the following formula (1), the diastereomeric excess calculated by the following formula of more than 60%.

[0014] (In the formula, P １ represents a protecting group for a hydroxyl group, and P ２represents a protecting group for an amino group.) Diastereomeric excess (%) = |(amount of one diastereomer - amount of the other diastereomer) / (amount of one diastereomer + amount of the other diastereomer) | × 100 [8] The production method according to [7], further comprising a step of preparing an amide-bridged compound represented by formula (1) having a diastereomeric excess calculated by the formula of more than 60% by separating the diastereomers from the amide-bridged compound represented by formula (1) having a diastereomeric excess calculated by the formula of 60% or less by high performance liquid chromatography or medium pressure preparative liquid chromatography using a chiral normal phase column. [9] The production method according to [7] or [8], wherein the mixing ratio of ethyl acetate to methyl tert butyl ether in the mixed solvent is 10 / 90 to 55 / 45 by volume (ethyl acetate / methyl tert butyl ether).

[10] The method for producing a crystal of an amide-bridged compound according to any one of [7] to [9], wherein the content ratio of the amide-bridged compound represented by formula (1), which has a diastereomeric excess calculated by the formula, to the mixed solvent (amide-bridged compound / mixed solvent) is 1 / 100 to 1 / 5 (mg / μL).

[11] A method for producing a crystal of an amide-bridged compound, which comprises dissolving an amide-bridged compound represented by formula (1), which has a diastereomeric excess calculated by the formula, of 60% or less in a solvent, adding crystals of the amide-bridged compound represented by formula (1), which has a diastereomeric excess calculated by the formula, of more than 60%, as seed crystals, to obtain a crystal of the amide-bridged compound represented by formula (1), which has a diastereomeric excess calculated by the formula, of 60% or less.

[0015] (In the formula, P １ represents a protecting group for a hydroxyl group, and P ２represents a protecting group for an amino group.) Diastereomeric excess (%) = |(amount of one diastereomer - amount of the other diastereomer) / (amount of one diastereomer + amount of the other diastereomer)| × 100

[12] A method for producing a crystal of an amide-bridged compound represented by formula (1), having a diastereomeric excess of 60% or less as calculated by the formula, by a crystallization method including a vapor diffusion method.

[13] A method for producing a crystal of an amide-bridged compound, comprising dissolving an amide-bridged compound represented by formula (1), having a diastereomeric excess of 60% or less as calculated by the formula, in a solvent, adding crystals of an amide-bridged compound represented by formula (1), having a diastereomeric excess of 60% or less as calculated by the formula, as seed crystals, to obtain a crystal of an amide-bridged compound represented by formula (1), having a diastereomeric excess of 60% or less as calculated by the formula.

[0016] (In the formula, P １ represents a protecting group for a hydroxyl group, and P ２ represents a protecting group for an amino group.) Diastereomeric excess (%) = | (amount of one diastereomer - amount of the other diastereomer) / (amount of one diastereomer + amount of the other diastereomer) | × 100

[14] The production method according to

[13] , wherein after the seed crystals are added, precipitation of crystals from the solution is initiated without cooling or adding a poor solvent.

[0017] According to the present disclosure, it is possible to provide crystals of amide-bridged compounds that can reduce the amount of amidite monomer used in oligonucleotide synthesis, and a method for producing the same.

[0018] Fig. 1 is a powder X-ray diffraction pattern of the crystals obtained in Example 5. Fig. 2 is a powder X-ray diffraction pattern of the crystals obtained in Example 6. Fig. 3 is a powder X-ray diffraction pattern of the crystals obtained in Example 7. Fig. 4 is a powder X-ray diffraction pattern of the crystals obtained in Example 8.

[0019] The crystal of the amide-bridged compound according to the present disclosure is a crystal having a chemical structure represented by the following formula (1): Hereinafter, the amide-bridged compound represented by formula (1) is also referred to as "amide-bridged compound (1)."

[0020] (In the formula, P １ represents a protecting group for a hydroxyl group, and P ２ represents a protecting group for an amino group.)

[0021] The amide-bridged compound (1) has two diastereomers derived from the asymmetric phosphorus atom, represented by the following formulae (1a) and (1b).

[0022] (In the formula, P １ and P ２ is the same as above.)

[0023] P １ Examples of the hydroxyl-protecting group represented by the formula (I) can include known protecting groups known as hydroxyl-protecting groups, such as ether-based protecting groups such as methyl, ethyl, tert-butyl, octyl, allyl, triphenylmethyl, benzyl, p-methoxybenzyl, fluorenyl, triphenylmethyl, 4-monomethoxytriphenylmethyl, and 4,4'-dimethoxytriphenylmethyl; acetal-based protecting groups such as methoxymethyl, methoxyethoxymethyl, 1-ethoxyethyl, tetrahydropyran-2-yl, and tetrahydrofuran-2-yl; acyl-based protecting groups such as acetyl and benzoyl; and silyl-based protecting groups such as trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and triisopropylsiloxymethyl. １ From the viewpoint of stability in the crystalline structure, the protecting group is preferably an ether-based protecting group (preferably a monoalkoxytriphenylmethyl group or a dialkoxytriphenylmethyl group) or a silyl-based protecting group, more preferably a 4,4'-dimethoxytriphenylmethyl group, a tert-butyldimethylsilyl group, or a triisopropylsiloxymethyl group, and even more preferably a 4,4'-dimethoxytriphenylmethyl group.

[0024] P ２As the protecting group for the amino group represented by the formula (I), a known protecting group known as a protecting group for an amino group can be used, and examples thereof include acyl protecting groups such as an acetyl group, a pivaloyl group, a benzoyl group, an isobutyryl group, a dimethylformamidinyl group, a trifluoroacetyl group, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, and a 4-tert-butylphenoxyacetyl group; and carbamate protecting groups such as a tert-butoxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group, a benzyloxycarbonyl group, and an allyloxycarbonyl group. ２ From the viewpoint of stability in the crystal structure, the protecting group is preferably an acyl-based protecting group, and more preferably a benzoyl group, an acetyl group, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, an isobutyryl group, or a dimethylformamidinyl group.

[0025] The amide-bridged compound (1) can be produced by a known method. For example, it can be produced by the method described in WO 2014 / 109384.

[0026] The solid amide-bridged compound (1) obtained by a conventionally known production method has an amorphous structure and does not have a crystalline structure. Furthermore, the amorphous amide-bridged compound (1) obtained by a conventionally known production method is a diastereomeric mixture consisting of the diastereomer represented by formula (1a) and the diastereomer represented by formula (1b), and there is no substantial difference in the amount of the diastereomer represented by formula (1a) and the diastereomer represented by formula (1b), and the diastereomeric excess is 60% or less. Specifically, the solid amide-bridged compound (1) produced by the method described in WO 2014 / 109384 is amorphous, and the diastereomeric ratio is approximately 0.5:1.0 to 2.0:1.0. Furthermore, even when the amorphous amide-bridged compound (1) having a low diastereomeric excess obtained by the conventionally known production method was crystallized by a common crystallization method such as a cooling method, a concentration method, a neutralization method, a poor solvent addition method, or a vapor diffusion method, it was not possible to obtain a compound having a crystalline structure (for example, Reference Examples 1 to 10 described below).

[0027] Whether a solid compound is crystalline or amorphous can be determined by powder X-ray diffraction. Specifically, a crystalline compound is one for which a clear diffraction peak is obtained in X-ray measurement, and an amorphous compound is one for which a clear diffraction peak is not obtained in X-ray measurement. Whether a compound is crystalline or amorphous can also be determined by observation with a polarizing microscope.

[0028] Specifically, in powder X-ray diffraction using Cu-Kα radiation, the crystals of the amide-bridged compound (1) preferably have peaks at diffraction angles (2θ) of 5.6±0.2°, 13.6±0.2°, 14.0±0.2°, and 16.9±0.2°, but may also have peaks at diffraction angles other than these four. In powder X-ray diffraction, "having a diffraction peak" means the presence of a peak that can be clearly distinguished from background noise. In the present disclosure, the diffraction peak preferably has a half-width of 3.0° or less, more preferably 2.0° or less. The half-width refers to the width on the horizontal axis at a position corresponding to half the maximum intensity of the diffraction peak (the difference between the diffraction angles (2θ) on both sides). Furthermore, in powder X-ray diffraction measurements, variations in the intensity of the diffraction peak may occur depending on the powder particle size, packing state, sample pretreatment method, sample installation state, etc. However, if the positions of the characteristic peaks, i.e., the values ​​(°) of the characteristic diffraction angles (2θ), are the same, they are considered to be essentially the same crystals even if there are changes in the peak intensities or the relative intensity ratios with other peaks.

[0029] The diastereomeric excess is a value calculated by the following formula, and the diastereomeric ratio and diastereomeric excess can be determined, for example, by nuclear magnetic resonance spectroscopy or high performance liquid chromatography: Diastereomeric excess (%) = |(amount of one diastereomer - amount of the other diastereomer) / (amount of one diastereomer + amount of the other diastereomer)| x 100

[0030] The present disclosure encompasses, as one embodiment, a method for producing crystals of amide-bridged compound (1) having a diastereomeric excess of more than 60% by dissolving amide-bridged compound (1) having a diastereomeric excess of more than 60% in a mixed solvent of ethyl acetate as a good solvent and methyl tert butyl ether as a poor solvent, and then allowing heptane as a poor solvent to be absorbed as vapor into the solution to increase the heptane concentration in the solution (this production method is also referred to as "production method (A)" hereinafter). The present disclosure also encompasses, as another embodiment, a method for producing crystals of amide-bridged compound (1) having a diastereomeric excess of 60% or less by dissolving amide-bridged compound (1) having a diastereomeric excess of 60% or less in a solvent, adding crystals of amide-bridged compound (1) having a diastereomeric excess of more than 60% as seed crystals, and thereby obtaining crystals of amide-bridged compound (1) having a diastereomeric excess of 60% or less (this production method is also referred to as "production method (B)" hereinafter). As yet another embodiment, the present disclosure encompasses a method for producing a crystal of the amide-bridged compound (1) having a diastereomeric excess of 60% or less, by dissolving the amide-bridged compound (1) having a diastereomeric excess of 60% or less in a solvent, adding a crystal of the amide-bridged compound (1) having a diastereomeric excess of 60% or less as a seed crystal, and obtaining a crystal of the amide-bridged compound (1) having a diastereomeric excess of 60% or less (hereinafter, this production method is also referred to as "production method (C)"). According to production methods (A) to (C) of the present disclosure, the amide-bridged compound (1) having a crystalline structure can be obtained.

[0031] The crystal of the amide-bridged compound (1) according to the present disclosure preferably contains both the compound represented by the formula (1a) and the compound represented by the formula (1b), which are in a diastereomeric relationship. The crystal of the amide-bridged compound (1) according to the present disclosure includes both crystals having a diastereomeric excess of more than 60% and crystals having a diastereomeric excess of 60% or less.

[0032] [Production Method (A)] The production method (A) is significant in that it can provide crystals of the amide-bridged compound (1) even in the absence of seed crystals.

[0033] An amide-bridged compound (1) having a diastereomeric excess of more than 60% can be prepared, for example, by separating diastereomers of an amide-bridged compound (1) having an excess of 60% or less (hereinafter, this preparation method is also referred to as "preparation method (A1)"). Specifically, it can be prepared by separating diastereomers by high-performance liquid chromatography (HPLC) or medium-pressure preparative liquid chromatography using a chiral normal-phase column. Because the diastereomers of the amide-bridged compound (1) are structurally similar to each other, separation using an achiral column is difficult.

[0034] The amide-bridged compound (1) having a diastereomeric excess of 60% or less used to prepare the amide-bridged compound (1) having a diastereomeric excess of more than 60% is preferably a liquid or amorphous solid. The amide-bridged compound (1) having a diastereomeric excess of 60% or less may be an oil that has become viscous during a concentration operation or the like. Due to properties such as low crystallinity, the amide-bridged compound (1) may become significantly more viscous in the latter half of the concentration operation, particularly when the production scale is scaled up, and may become oily without solidifying.

[0035] As the chiral normal phase column, a commercially available column may be used, for example, CHIRALPAK ID, CHIRALFLASH ID, etc. manufactured by Daicel Corporation.

[0036] The eluent used in the chromatography for diastereomer separation is preferably a non-aqueous solvent, and examples thereof include aliphatic hydrocarbons such as hexane, heptane, octane, nonane, decane, and 2-ethylhexane; alicyclic hydrocarbons such as cyclopentane, cyclohexane, and methylcyclohexane; aromatic hydrocarbons such as benzene, toluene, and xylene; and halogenated hydrocarbons such as dichloromethane, trichloromethane, tetrachloromethane, and dichloroethane. These solvents can be used alone or in combination of two or more. The eluent preferably contains a halogenated hydrocarbon, and more preferably contains an aliphatic hydrocarbon and a halogenated hydrocarbon. When the eluent contains an aliphatic hydrocarbon and a halogenated hydrocarbon, it is more preferable that the total content of the aliphatic hydrocarbon and the halogenated hydrocarbon in the eluent is 60 to 100% by volume. When the eluent contains aliphatic hydrocarbons and halogenated hydrocarbons, the volume ratio of the aliphatic hydrocarbons to the halogenated hydrocarbons (aliphatic hydrocarbons / halogenated hydrocarbons) is preferably 30 / 70 to 70 / 30, and more preferably 40 / 60 to 60 / 40. To improve separation ability, the eluent may contain one or more selected from the group consisting of acids such as trifluoroacetic acid and acetic acid, dialkylamines such as diethylamine, and trialkylamines such as triethylamine, and it is preferable to contain an acid and / or a dialkylamine. The amount of acid added is preferably such that the volume ratio to the eluent (acid / eluent) is 0.001 / 100 to 0.5 / 100, and more preferably 0.005 / 100 to 0.1 / 100. The amount of dialkylamine added is preferably such that the volume ratio to the eluent (dialkylamine / eluent) is 0.01 / 100 to 1.0 / 100, more preferably 0.05 / 100 to 0.5 / 100.

[0037] The column temperature and the flow rate of the eluent in the chromatographic separation can be selected appropriately.

[0038] The fraction containing the amide-bridged compound (1) or the amide-bridged compound (1) separated by chromatography may be washed, if necessary. A washing solution may be used appropriately for the washing, and examples of the washing solution include an aqueous solution containing one or more inorganic salts selected from the group consisting of sodium chloride, potassium chloride, sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate. The concentration of the aqueous solution is preferably 1 to 15% by mass. After washing, the compound may be dried or concentrated, if necessary.

[0039] The amide-bridged compound (1) with a diastereomeric excess of more than 60% obtained by chromatographic separation is in the form of a liquid or amorphous solid, or may be in the form of a viscous oil.

[0040] In the production method (A), the amide-bridged compound (1) having a diastereomeric excess of more than 60% used as a crystallization raw material (particularly, an amorphous raw material (hereinafter also referred to as "raw material amorphous")) preferably has an excess of 65% or more, more preferably 70% or more, even more preferably 73% or more, and even more preferably 75% or more. The excess may be 100%, but is preferably 99% or less, and more preferably 97% or less. That is, the excess is preferably more than 60% and 100% or less, more preferably 65 to 100%, even more preferably 70 to 100%, even more preferably 73 to 99%, and even more preferably 75 to 97%. In the production method (A), when the diastereomeric excess of the amide-bridged compound (1) dissolved in the mixed solvent is equal to or greater than the lower limit, crystals can be obtained by carrying out a specific crystallization procedure.

[0041] In production method (A), crystals can be obtained by crystallization using a vapor diffusion method, using a mixed solvent of ethyl acetate as a good solvent and methyl tert-butyl ether as a poor solvent, and heptane as a poor solvent. In the vapor diffusion method, the poor solvent is vaporized and absorbed into a solvent (good solvent) in which a compound is dissolved, thereby increasing the concentration of the poor solvent in the solution, thereby obtaining crystals of the compound. The vapor diffusion method does not require the application of shear energy due to stirring during nucleation, making it easier to maintain unstable primary crystal nuclei and facilitate nucleus growth compared to other crystallization methods. Furthermore, the solubility is reduced more gradually compared to other crystallization methods, thereby suppressing disordered arrangement (amorphization). Therefore, it is believed that crystals of amide-bridged compound (1) can be obtained.

[0042] The mixing ratio of ethyl acetate to methyl tert-butyl ether in the mixed solvent is preferably 10 / 90 to 55 / 45, more preferably 15 / 85 to 50 / 50, even more preferably 20 / 80 to 45 / 55, still more preferably 25 / 75 to 40 / 60, and even more preferably 30 / 70 to 35 / 65, in volume ratio (ethyl acetate / methyl tert-butyl ether).

[0043] The concentration of the amide-bridged compound (1) in the mixed solvent in production method (A) may be equal to or less than the saturated solubility, or may be supersaturated. The content ratio of the amide-bridged compound (1) to the mixed solvent in the solution in production method (A) (amide-bridged compound (1) / mixed solvent) is preferably 1 / 100 to 1 / 5 (mg / μL), more preferably 1 / 80 to 1 / 8 (mg / μL), even more preferably 1 / 60 to 1 / 10 (mg / μL), and even more preferably 1 / 40 to 1 / 13 (mg / μL).

[0044] From the viewpoint of improving the efficiency of crystal production, it is preferable that the content of protic solvents such as alcohols (e.g., methanol, ethanol, etc.); carboxylic acids (e.g., formic acid, acetic acid, etc.); nitromethane; water; and amines (e.g., triethylamine, pyridine, etc.) in the solution in which the amide-bridged compound (1) is dissolved is low. Specifically, the amount of protic solvent used in the solution is preferably 1.0 v / v% or less, more preferably 0.5 v / v% or less, even more preferably 0.1 v / v% or less, and may even be 0 v / v% relative to the total amount of all solvents in the solution. Furthermore, the amount of amine used in the solution is preferably 1.0 part by mass or less, more preferably 0.5 parts by mass or less, even more preferably 0.1 parts by mass or less, and may even be 0 parts by mass, relative to 100 parts by mass of the amide-bridged compound (1) in the solution.

[0045] The crystallization temperature in the production method (A) is preferably 15°C or lower, more preferably 0 to 13°C, and even more preferably 0 to 10°C, from the viewpoint of accelerating the crystallization of the amide-bridged compound (1).

[0046] The crystallization time in production method (A) can be set appropriately, and is, for example, preferably 0.5 to 96 hours, more preferably 1 to 72 hours, even more preferably 2 to 48 hours, and even more preferably 4 to 24 hours.

[0047] The crystals obtained by production method (A) can be isolated by a general solid-liquid separation procedure. Examples of the solid-liquid separation method include natural filtration, vacuum filtration, pressure filtration, centrifugal filtration, etc. Furthermore, the crystals obtained by production method (A) may be subjected to treatments such as washing and drying, if necessary.

[0048] The diastereomeric excess of the crystals obtained by production method (A) is more than 60%, preferably 65% ​​or more, more preferably 70% or more, even more preferably 73% or more, and still more preferably 75% or more, and may be 100%, or may be 99% or less, or 97% or less. That is, the excess is preferably more than 60% and 100% or less, more preferably 65 to 100%, even more preferably 70 to 100%, even more preferably 73 to 99%, and even more preferably 75 to 97%.

[0049] [Production Method (B)] Production Method (B) is characterized in that crystals can be obtained even when an amide-bridged compound (1) having a diastereomeric excess as low as 60% or less is used as a crystallization raw material (particularly, an amorphous raw material), and is a method in which crystals of an amide-bridged compound (1) having a diastereomeric excess of more than 60%, such as those obtained by Production Method (A), are used as seed crystals.

[0050] In production method (B), the amide-bridged compound (1) having a diastereomeric excess of 60% or less that dissolves in a solvent as a crystallization raw material is not particularly limited as long as the excess is 60% or less. Amorphous amide-bridged compound (1) obtained by a conventionally known production method may be used, or a liquid or oily compound that has been made viscous by a concentration operation may be used. The diastereomeric excess of the amide-bridged compound (1) used as a crystallization raw material is preferably 50% or less, more preferably 40% or less, and even more preferably 30% or less. If the amide-bridged compound (1) can be used as a crystallization raw material without increasing its diastereomeric excess, the use efficiency of the amide-bridged compound (1) can be increased.

[0051] In the production method (B), the solvent (X) for dissolving the amide-bridged compound (1) is preferably a solvent that does not decompose the amide-bridged compound (1), from the viewpoint of improving the efficiency of crystal production. The solvent (X) preferably contains a good solvent (X1) that does not decompose the amide-bridged compound (1) and is a good solvent for the amide-bridged compound (1). Examples of the good solvent (X1) include aromatic hydrocarbons such as benzene, toluene, and xylene; halogenated hydrocarbons such as dichloromethane, trichloromethane, tetrachloromethane, and dichloroethane; fatty acid esters such as ethyl acetate, butyl acetate, and ethyl butyrate; nitriles such as acetonitrile, propionitrile, and benzonitrile; and cyclic ethers such as dioxane, tetrahydrofuran, and 2-methyltetrahydrofuran. From the viewpoint of improving the efficiency of crystal production, it is more preferable that the solvent contains one or more solvents selected from the group consisting of toluene, dichloromethane, ethyl acetate, acetonitrile, and tetrahydrofuran. Furthermore, from the viewpoint of operational stability on an industrial scale, toluene and dichloromethane are preferred. The solvent (X) that dissolves the amide-bridged compound (1) is not limited to the good solvent (X1) and may be a mixed solvent of the good solvent (X1) and a poor solvent (X2). Examples of the poor solvent (X2) include aliphatic hydrocarbons such as hexane, heptane, octane, nonane, decane, and 2-ethylhexane; and acyclic ethers such as diethyl ether, diisopropyl ether, and methyl tert-butyl ether. The poor solvent (X2) preferably contains one or more solvents selected from the group consisting of n-hexane, n-heptane, methyl tert-butyl ether, and diisopropyl ether. When the amide-bridged compound (1) is dissolved in a mixed solvent of a good solvent (X1) and a poor solvent (X2), the mixing ratio of the good solvent (X1) to the poor solvent (X2) is preferably 10 / 90 to 55 / 45, more preferably 15 / 85 to 50 / 50, even more preferably 20 / 80 to 45 / 55, still more preferably 25 / 75 to 40 / 60, and even more preferably 30 / 70 to 35 / 65, by volume (good solvent (X1) / poor solvent (X2)).

[0052] From the viewpoint of improving the efficiency of crystal production, it is preferable that the content of protic solvents such as alcohols (e.g., methanol, ethanol, etc.); carboxylic acids (e.g., formic acid, acetic acid, etc.); nitromethane; water; and amines (e.g., triethylamine, pyridine, etc.) in the solution in which the amide-bridged compound (1) is dissolved is low. Specifically, the amount of protic solvent used in the solution is preferably 1.0 v / v% or less, more preferably 0.5 v / v% or less, even more preferably 0.1 v / v% or less, and may even be 0 v / v% relative to the total amount of all solvents in the solution. Furthermore, the amount of amine used in the solution is preferably 1.0 part by mass or less, more preferably 0.5 parts by mass or less, even more preferably 0.1 parts by mass or less, and may even be 0 parts by mass, relative to 100 parts by mass of the amide-bridged compound (1) in the solution.

[0053] The concentration of the amide-bridged compound (1) in the solution in which the amide-bridged compound (1) is dissolved may be equal to or less than the saturated solubility or may be supersaturated. The content ratio of the amide-bridged compound (1) (excluding the content of seed crystals) to the solvent (X) (amide-bridged compound (1) / solvent (X)) is preferably 1.0 / 100 to 20 / 100 (g / mL), more preferably 1.3 / 100 to 15 / 100 (g / mL), even more preferably 1.8 / 100 to 10 / 100 (g / mL), and even more preferably 2.0 / 100 to 8 / 100 (g / mL).

[0054] The seed crystals used in the production method (B) are not particularly limited as long as they have a diastereomeric excess of more than 60%, and for example, the crystals obtained by the above production method (A) can be preferably used.

[0055] The seed crystals having a diastereomeric excess of more than 60% used in production method (B) preferably have an excess of 65% or more, more preferably 70% or more, even more preferably 73% or more, and even more preferably 75% or more. The excess may be 100%, but is preferably 99% or less. That is, the excess is preferably more than 60% and 100% or less, more preferably 65 to 100%, even more preferably 70 to 100%, even more preferably 73 to 100%, and even more preferably 75 to 99%.

[0056] The amount of seed crystals added in production method (B) may be appropriately set depending on the crystallization method, the type of solvent used, and the like. From the viewpoints of improving production efficiency and economy, the amount of seed crystals added is preferably 0.01 to 20 parts by mass, more preferably 0.05 to 15 parts by mass, even more preferably 0.1 to 10 parts by mass, and even more preferably 0.3 to 5 parts by mass, relative to 100 parts by mass of the amide-bridged compound (1) dissolved in solvent (X).

[0057] In the production method (B), the crystals are obtained by adding seed crystals to the solution of the amide-bridged compound (1) dissolved in the solvent (X), and known methods such as a cooling method, a concentration method, a neutralization method, a poor solvent addition method, and a poor solvent vapor diffusion method (vapor diffusion method) can be used, and these methods may be combined. As the crystallization method in the production method (B), the vapor diffusion method is preferred from the viewpoint of ease of obtaining crystals.

[0058] In the production method (B), the poor solvent used in the poor solvent addition method or vapor diffusion method can be the same as the poor solvent (X2) exemplified as the mixed solvent for dissolving the amide-bridged compound (1). However, a solvent with lower solubility is preferred, and for example, aliphatic hydrocarbons such as hexane, heptane, octane, nonane, decane, and 2-ethylhexane are preferred, with hexane and heptane being more preferred.

[0059] In production method (B), the crystallization conditions such as the crystallization temperature and crystallization time at the crystallization stage can be appropriately set depending on the crystallization method employed. The crystallization temperature is preferably 0 to 40°C, more preferably 0 to 35°C, and even more preferably 0 to 30°C. The crystallization time is preferably 0.5 to 96 hours, more preferably 1 to 72 hours, even more preferably 2 to 48 hours, and even more preferably 4 to 24 hours.

[0060] The crystals obtained by production method (B) can be isolated by a general solid-liquid separation procedure. Examples of the solid-liquid separation method include natural filtration, vacuum filtration, pressure filtration, centrifugal filtration, etc. Furthermore, the crystals obtained by production method (B) may be subjected to treatments such as washing and drying, if necessary.

[0061] The diastereomeric excess of the crystals obtained by production method (B) is 60% or less, preferably 58% or less, more preferably 55% or less, even more preferably 50% or less, still more preferably 45% or less, even more preferably 40% or less, even more preferably 35% or less, or even 0%. Crystals with a low diastereomeric excess are mixed crystals in which both diastereomers of the crystallization raw material have been successfully crystallized effectively, and are superior in use efficiency of the compound (crystallization raw material) compared to when one diastereomer is discarded by preferential crystallization.

[0062] [Production Method (C)] Production Method (C) is characterized in that crystals can be obtained even when an amide-bridged compound (1) having a diastereomeric excess as low as 60% or less is used as a crystallization raw material (particularly, an amorphous or oily raw material (hereinafter also referred to as "raw material oil")), and further characterized in that crystals of an amide-bridged compound (1) having a diastereomeric excess of 60% or less, which can be obtained without column purification, are used as seed crystals.

[0063] In production method (C), the amide-bridged compound (1) having a diastereomeric excess of 60% or less that dissolves in a solvent as a crystallization raw material is not particularly limited as long as the excess is 60% or less. Amorphous amide-bridged compound (1) obtained by a conventionally known production method may be used, or a liquid or oily compound that has been made viscous by a concentration operation may be used. The diastereomeric excess of the amide-bridged compound (1) used as a crystallization raw material is preferably 50% or less, more preferably 40% or less, even more preferably 35% or less, and even more preferably 30% or less. If the amide-bridged compound (1) can be used as a crystallization raw material without increasing its diastereomeric excess, the use efficiency of the amide-bridged compound (1) can be increased.

[0064] In the production method (C), the solvent (Y) for dissolving the amide-bridged compound (1) preferably contains a solvent that is a good solvent for the amide-bridged compound (1) and does not decompose the amide-bridged compound (1). Examples of such a solvent include the same solvents as the good solvent (X1) in the production method (B), and the same preferred embodiments apply. The solvent (Y) may be a mixed solvent of the good solvent (X1) and the poor solvent (X2). Examples of the poor solvent (X2) include the same solvents as those described above, and the same preferred embodiments apply. Furthermore, the poor solvent (X2) in the solvent (Y) is preferably a solvent with a lower solubility. For example, aliphatic hydrocarbons such as hexane, heptane, octane, nonane, decane, and 2-ethylhexane are preferred, and hexane and heptane are more preferred. When the amide-bridged compound (1) is dissolved in a mixed solvent of a good solvent (X1) and a poor solvent (X2) as the solvent (Y), the mixing ratio of the good solvent (X1) to the poor solvent (X2) is preferably 10 / 90 to 90 / 10, more preferably 15 / 85 to 80 / 20, by volume (good solvent (X1) / poor solvent (X2)). In the case of the crystallization method (C1) described below or the vapor diffusion method, the mixing ratio of the good solvent (X1) to the poor solvent (X2) is preferably 10 / 90 to 55 / 45, more preferably 15 / 85 to 45 / 55, even more preferably 20 / 80 to 40 / 60, and even more preferably 25 / 75 to 35 / 65, by volume (good solvent (X1) / poor solvent (X2)). In the case of the poor solvent addition method described below, the mixing ratio of the good solvent (X1) to the poor solvent (X2) is preferably 15 / 85 to 90 / 10, more preferably 20 / 80 to 85 / 15, and even more preferably 30 / 70 to 80 / 20, in volume ratio (good solvent (X1) / poor solvent (X2)).

[0065] From the viewpoint of improving the efficiency of crystal production, it is preferable that the content of protic solvents such as alcohols (e.g., methanol, ethanol, etc.); carboxylic acids (e.g., formic acid, acetic acid, etc.); nitromethane; water; and amines (e.g., triethylamine, pyridine, etc.) in the solution in which the amide-bridged compound (1) is dissolved is low. Specifically, the amount of protic solvent used in the solution is preferably 1.0 v / v% or less, more preferably 0.5 v / v% or less, even more preferably 0.1 v / v% or less, and may even be 0 v / v% relative to the total amount of all solvents in the solution. Furthermore, the amount of amine used in the solution is preferably 1.0 part by mass or less, more preferably 0.5 parts by mass or less, even more preferably 0.1 parts by mass or less, and may even be 0 parts by mass, relative to 100 parts by mass of the amide-bridged compound (1) in the solution.

[0066] In the solution in which the amide-bridged compound (1) is dissolved, the content ratio of the amide-bridged compound (1) (excluding the content of seed crystals) to the solvent (Y) (amide-bridged compound (1) / solvent (Y)) is preferably 1.0 / 100 to 20 / 100 (g / mL), more preferably 1.3 / 100 to 15 / 100 (g / mL), even more preferably 1.8 / 100 to 10 / 100 (g / mL), and still more preferably 2.0 / 100 to 8 / 100 (g / mL).

[0067] The seed crystals used in Production Method (C) are not particularly limited as long as they have a diastereomeric excess of 60% or less, and for example, crystals obtained by Production Method (B) can be preferably used. Crystals with a high diastereomeric excess can be obtained from a crystallization raw material whose diastereomeric excess has been increased by column purification or the like, as in Production Method (A), whereas crystals with a low diastereomeric excess can be obtained without column purification or the like. Therefore, they can be easily obtained in large quantities industrially and are highly productive.

[0068] The seed crystals used in production method (C) having a diastereomeric excess of 60% or less preferably have an excess of 50% or less, more preferably 40% or less, and even more preferably 35% or less. The excess may be 0%, and is preferably 0.1% or more. That is, the excess is preferably 0 to 60%, more preferably 0 to 50%, even more preferably 0 to 40%, and even more preferably 0.1 to 35%.

[0069] The amount of seed crystals added in production method (C) may be appropriately set depending on the crystallization method, the type of solvent used, and the like. From the viewpoints of improving production efficiency and economy, the amount of seed crystals added is preferably 0.01 to 20 parts by mass, more preferably 0.05 to 15 parts by mass, even more preferably 0.1 to 10 parts by mass, and even more preferably 0.3 to 5 parts by mass, relative to 100 parts by mass of the amide-bridged compound (1) dissolved in the solvent.

[0070] In the production method (C), the addition of seed crystals to the solution of the amide-bridged compound (1) dissolved in the solvent (Y) to obtain crystals may be performed using a method that does not involve any particular operation to reduce the amount of dissolution (hereinafter also referred to as "crystallization method (C1)"), or may be performed using a known method for reducing the amount of dissolution, such as a cooling method, a concentration method, a neutralization method, a poor solvent addition method, or a poor solvent vapor diffusion method (vapor diffusion method) (hereinafter also referred to as "crystallization method (C2)"). The crystallization method (C1) utilizes the difference in solubility between the amorphous and crystalline forms of the amide-bridged compound (1). More specifically, when the amide-bridged compound (1) crystallizes, its solubility in the solvent becomes lower than that of the amorphous solid. Therefore, when seed crystals are added to an amorphous solution of the amide-bridged compound (1), crystals can be obtained from the solution without performing operations to reduce the amount of dissolution, such as cooling, the addition of a poor solvent, or concentration.

[0071] The crystallization temperature in the crystallization method (C1) is preferably 0 to 50°C, more preferably 1 to 40°C, and even more preferably 1 to 30°C. The crystallization temperature may be kept constant during crystallization or may be lowered after the start of crystal precipitation. In addition, in the crystallization method (C1), crystallization may be performed at room temperature without temperature control.

[0072] In the crystallization method (C1), it is preferable to carry out the crystallization while stirring in order to obtain crystals more efficiently. The strength of the stirring is not particularly limited and may be set appropriately.

[0073] The crystallization time in the crystallization method (C1) may be set appropriately, and is, for example, preferably 0.2 to 72 hours, more preferably 0.5 to 48 hours, even more preferably 0.8 to 24 hours, and even more preferably 1 to 12 hours.

[0074] In the crystallization method (C1), crystals can be obtained from the solution without cooling or adding a poor solvent. However, after the start of crystal precipitation, cooling or addition of a poor solvent may be carried out. If cooling or addition of a poor solvent is carried out, crystals can be obtained more efficiently.

[0075] The crystallization method (C2) for reducing the amount of dissolved solids is preferably a cooling method, a concentration method, a poor solvent addition method, or a vapor diffusion method, and these methods may be combined as appropriate, but it is preferable to at least perform a method using a raw material solution and a poor solvent (poor solvent addition method, vapor diffusion method). As the poor solvent, the same solvents as the poor solvent (X2) can be used, but a solvent with a lower solubility is preferred, and for example, aliphatic hydrocarbons such as hexane, heptane, octane, nonane, decane, and 2-ethylhexane are preferred, with hexane and heptane being more preferred.

[0076] The amount of the poor solvent added in the poor solvent addition method is preferably 0.01 to 200 v / v %, more preferably 0.1 to 200 v / v %, even more preferably 0.5 to 200 v / v %, still more preferably 1 to 200 v / v %, still more preferably 3 to 200 v / v %, still more preferably 30 to 200 v / v %, still more preferably 40 to 180 v / v %, and particularly preferably 50 to 150 v / v %, relative to the amount of the solvent (Y) used to dissolve the amide-bridged compound (1). Furthermore, when toluene or dichloromethane is used as the main component (specifically, more than 50 mass % in the good solvent (X1)) as the good solvent (X1) in the solvent (Y), the amount of the poor solvent added by the poor solvent addition method is preferably 0.01 to 150 v / v %, more preferably 0.1 to 100 v / v %, even more preferably 0.5 to 70 v / v %, still more preferably 1 to 50 v / v %, and even more preferably 3 to 30 v / v %, relative to the amount of the solvent (Y).

[0077] The crystallization temperature is preferably 0 to 50°C, more preferably 1 to 40°C, and even more preferably 1 to 30°C. The crystallization temperature may be kept constant during crystallization or may be lowered after the start of crystal precipitation. In addition, in the poor solvent addition method or vapor diffusion method, crystallization may be carried out at room temperature without temperature control.

[0078] In crystallization, crystals are usually precipitated while stirring. The strength of the stirring is not particularly limited and may be set appropriately.

[0079] The rate of addition of the poor solvent in the poor solvent addition method is not particularly limited and may be appropriately set depending on the amount of solvent used. After the addition of the poor solvent is completed, stirring may be continued. For example, after the addition is completed, stirring is preferably continued for 0.2 to 24 hours (more preferably 0.5 to 12 hours).

[0080] The crystallization time in the vapor diffusion method is, for example, 0.5 to 96 hours, preferably 1 to 72 hours, more preferably 2 to 48 hours, and even more preferably 4 to 24 hours.

[0081] The crystals obtained by production method (C) can be isolated by a general solid-liquid separation procedure. Examples of the solid-liquid separation method include natural filtration, reduced pressure filtration, pressure filtration, centrifugal filtration, etc. Furthermore, the crystals obtained by production method (C) may be subjected to treatments such as washing and drying, if necessary.

[0082] The diastereomeric excess of the crystals obtained by production method (C) is 60% or less, preferably 58% or less, more preferably 55% or less, even more preferably 50% or less, still more preferably 45% or less, even more preferably 40% or less, even more preferably 35% or less, or even 0%. Crystals with a low diastereomeric excess are mixed crystals in which both diastereomers of the crystallization raw material have been successfully crystallized effectively, and are superior in use efficiency of the compound (crystallization raw material) compared to when one diastereomer is discarded by preferential crystallization.

[0083] Amorphous amide-bridged compound (1) is highly hygroscopic, easily hydrolyzed and oxidized, and has poor stability. Furthermore, hydrolyzed or oxidized amide-bridged compound (1) cannot be used as a synthetic raw material for nucleic acid drugs (oligonucleotides), and is therefore unsuitable for long-term storage or preservation. Furthermore, due to poor stability during oligonucleotide synthesis, amorphous amide-bridged compound (1) may need to be added in excess relative to the substrate. In contrast, crystalline amide-bridged compound (1) according to the present disclosure has low hygroscopicity and high stability, making it suitable for long-term storage or preservation, and allowing for a reduction in the amount of amidite monomer used during oligonucleotide synthesis.

[0084] According to the production method of the present disclosure, it is possible to obtain crystals of amide-bridged compound (1), which have been difficult to obtain in the past. The production method of the present disclosure is also useful for industrial production. Furthermore, even when using crystals of amide-bridged compound (1) with a diastereomeric excess of more than 60% as seed crystals, surprisingly, crystals with an excess of 60% or less can be obtained as long as the excess of amide-bridged compound (1) in the mother liquor used for crystallization is 60% or less (production method (B)). Here, in oligonucleotide synthesis, the asymmetry of the amide-bridged compound (1) used as a raw material is lost, so that the desired oligonucleotide can be synthesized using either diastereomer. As described above, the production method of the present disclosure can crystallize either diastereomeric form of amide-bridged compound (1) in the mother liquor, which is the raw material solution for crystallization, regardless of the diastereomeric excess of the seed crystals. Furthermore, since either diastereomer can be used as a raw material for oligonucleotide synthesis, the amide-bridged compound (1) in the mother liquor can be more effectively utilized. Furthermore, both crystals of the amide-bridged compound (1) according to the present disclosure having a diastereomeric excess of 60% or less and crystals of the amide-bridged compound (1) having a diastereomeric excess of more than 60% are useful for the synthesis of oligonucleotides.

[0085] This application claims the benefit of priority based on Japanese Patent Application No. 2024-130715, filed on August 7, 2024. The entire contents of the specification of Japanese Patent Application No. 2024-130715, filed on August 7, 2024, are incorporated herein by reference.

[0086] The contents of the present disclosure will be explained in more detail below using examples, but the contents of the present disclosure are not limited to the examples below, and can be implemented with appropriate modifications within the scope that can conform to the intent described above and below, and all of these modifications are included in the technical scope of the present disclosure.

[0087] The structure of the compound was identified by measuring the X-ray diffraction pattern of the sample using powder X-ray diffraction. The conditions for powder X-ray measurement are as follows: Apparatus: Rigaku MiniFlex II (Dtex Ultra) X-ray used: CuKα Scanning speed: 2.0° / min Scanning axis: 2θ / θ Scanning range: 5 to 40° Scanning width: 0.020° Tube voltage: 30 kV Tube current: 15 mA

[0088] The diastereomeric ratio and diastereomeric excess of the compounds were calculated from the values ​​measured by HPLC. The HPLC conditions were as follows: Column: L-column 3 C18 (150 mm x 2.0 mm, 3 μm) manufactured by Chemicals Evaluation and Research Institute, Japan Mobile phase A: 5 mM ammonium bicarbonate aqueous solution Mobile phase B: acetonitrile Gradient conditions:

[0089] Flow rate: 0.30 mL / min Column temperature: 30°C Injection volume: 1 μL Sample concentration: 1 mg / mL Detection: UV (254 nm) Detected compounds: Compound represented by the above formula (1a), compound represented by the above formula (1b) Retention time: 7.3 min, 8.3 min

[0090] The diastereomeric ratio was calculated by the following formula: Diastereomeric ratio = amount of one diastereomer (peak area value) / amount of the other diastereomer (peak area value).

[0091] Synthesis Example 1: A compound represented by the following formula (1-1) was obtained as an amorphous compound by synthesis in accordance with the method described in Example 6 of WO 2014 / 109384. The diastereomeric ratio of the obtained amorphous compound was 1.1:1.0, and the diastereomeric excess was 4.8%. This amorphous compound is also referred to as "amorphous (1-1a)" hereinafter.

[0092]

[0093] Using amorphous (1-1a), an attempt was made to obtain a crystal of the compound represented by formula (1-1) (hereinafter also referred to as "crystal (1-1)") by a vapor diffusion method, but no crystal could be obtained (Reference Examples 1 to 10).

[0094] (Reference Example 1) A 6.0 mL screw tube containing a solution of 200 mg of amorphous (1-1a) dissolved in 800 μL of ethyl acetate was placed in a 61.0 mL standard bottle filled with n-hexane vapor and sealed. The solution was left to stand overnight at room temperature, but no crystals (1-1) were obtained.

[0095] Reference Example 2: A 0.3 mL vial insert containing a solution of 50 mg of amorphous (1-1a) dissolved in 100 μL of dichloromethane was placed in a 6.0 mL screw tube filled with n-hexane vapor and sealed. The solution was allowed to stand overnight at room temperature, but no crystals (1-1) were obtained.

[0096] Reference Example 3: A 0.3 mL vial insert containing a solution of 50 mg of amorphous (1-1a) dissolved in 100 μL of acetonitrile was placed in a 6.0 mL screw tube filled with n-hexane vapor and sealed. The solution was allowed to stand overnight at room temperature, but no crystals (1-1) were obtained.

[0097] Reference Example 4: A 6.0 mL screw tube containing a solution of 50 mg of amorphous (1-1a) in 250 μL of toluene was placed in a 28.0 mL standard bottle filled with n-hexane vapor and sealed. The solution was allowed to stand overnight at room temperature, but no crystals (1-1) were obtained.

[0098] Reference Example 5 A 6.0 mL screw tube containing a solution of 50 mg of amorphous (1-1a) in 250 μL of tetrahydrofuran was placed in a 28.0 mL standard bottle filled with n-hexane vapor and sealed. The solution was allowed to stand overnight at room temperature, but no crystals (1-1) were obtained.

[0099] (Reference Example 6) A 0.3 mL vial insert containing a solution of 50 mg of amorphous (1-1a) dissolved in 100 μL of ethyl acetate was placed in a 6.0 mL screw tube filled with methyl t-butyl ether vapor and sealed. The solution was allowed to stand overnight at room temperature, but no crystals (1-1) were obtained.

[0100] Reference Example 7: A 0.3 mL vial insert containing a solution of 50 mg of amorphous (1-1a) dissolved in 100 μL of dichloromethane was placed in a 6.0 mL screw tube filled with methyl t-butyl ether vapor and sealed. The solution was allowed to stand overnight at room temperature, but no crystals (1-1) were obtained.

[0101] Reference Example 8: A 0.3 mL vial insert containing a solution of 50 mg of amorphous (1-1a) dissolved in 100 μL of acetonitrile was placed in a 6.0 mL screw tube filled with methyl t-butyl ether vapor and sealed. The solution was allowed to stand overnight at room temperature, but no crystals (1-1) were obtained.

[0102] Reference Example 9 A 6.0 mL screw tube containing a solution of 50 mg of amorphous (1-1a) in 250 μL of toluene was placed in a 28.0 mL standard bottle filled with methyl t-butyl ether vapor and sealed. The solution was allowed to stand overnight at room temperature, but no crystals (1-1) were obtained.

[0103] Reference Example 10: A 6.0 mL screw tube containing a solution of 50 mg of amorphous (1-1a) in 250 μL of tetrahydrofuran was placed in a 28.0 mL standard bottle filled with methyl t-butyl ether vapor and sealed. The solution was allowed to stand overnight at room temperature, but no crystals (1-1) were obtained.

[0104] (Separation of diastereomers) Using the amorphous (1-1a), diastereomers were separated by HPLC under the following conditions. HPLC conditions: Column: CHIRALFLASH ID (3 cm ID x 10 cm L) manufactured by Daicel Corporation; Eluent: n-hexane / dichloromethane / diethylamine / acetic acid = 50 / 50 / 0.1 / 0.02 (volume ratio); Flow rate: 12 mL / min; Column temperature: room temperature; Injection volume: 1 mL (sample concentration 100 mg / mL); Detection: UV (254 nm).

[0105] The separated diastereomeric fractions were washed twice with a 5% aqueous solution of sodium bicarbonate and once with 10% saline, and then the organic layer was dried over anhydrous sodium sulfate. After drying, the mixture was filtered and concentrated to obtain amorphous compounds represented by formula (1-1) having a diastereomeric excess of more than 60%. Specifically, an amorphous compound represented by formula (1-1) having a diastereomeric ratio of 14:1.0 and a diastereomeric excess of 87% (hereinafter also referred to as "amorphous (1-1b)"), an amorphous compound represented by formula (1-1) having a diastereomeric ratio of 18:1.0 and a diastereomeric excess of 89% (hereinafter also referred to as "amorphous (1-1c)"), and an amorphous compound represented by formula (1-1) having a diastereomeric ratio of 7.9:1.0 and a diastereomeric excess of 78% (hereinafter also referred to as "amorphous (1-1d)") were obtained. In the amorphous compounds (1-1b) to (1-1d), the diastereomer with a higher proportion was the diastereomer with a retention time of 7.3 min by HPLC in the above-mentioned diastereomeric ratio and diastereomeric excess measurements. Similarly, in the amide-bridged compounds of the following Examples, the diastereomer with a higher proportion was the diastereomer with a retention time of 7.3 min by HPLC in the above-mentioned diastereomeric ratio and diastereomeric excess measurements.

[0106] When an achiral column was used to separate the diastereomers, separation was not possible.

[0107] Example 1 A 6.0 mL screw tube containing a solution of 20 mg of amorphous (1-1b) dissolved in a mixed solvent of 200 μL of ethyl acetate and 400 μL of methyl t-butyl ether was placed in a 28.0 mL standard bottle filled with n-heptane vapor and sealed. After standing overnight at 5°C, a crystal represented by the above formula (1-1) (hereinafter also referred to as "crystal (1-11)") was obtained, which had a diastereomeric ratio of 14:1.0 and a diastereomeric excess of 87%.

[0108] Example 2 A 6.0 mL screw tube containing a solution of 30 mg of amorphous (1-1d) dissolved in a mixed solvent of 200 μL of ethyl acetate and 400 μL of methyl t-butyl ether was placed in a 28.0 mL standard bottle filled with n-heptane vapor and sealed. After standing overnight at 5°C, a crystal represented by the above formula (1-1) (hereinafter also referred to as "crystal (1-12)") was obtained, which had a diastereomeric ratio of 9.1:1.0 and a diastereomeric excess of 80%.

[0109] Comparative Example 1: A 6.0 mL screw tube containing a solution of 20 mg of amorphous (1-1b) dissolved in 100 μL of dichloromethane was placed in a 28.0 mL standard bottle filled with n-hexane vapor and sealed. The solution was left to stand overnight at 5° C., but no crystals (1-1) were obtained.

[0110] Comparative Example 2: A 6.0 mL screw tube containing a solution of 20 mg of amorphous (1-1c) in 1 mL of acetone was placed in a 28.0 mL standard bottle filled with isopropyl ether vapor and sealed. The mixture was left to stand overnight at 5°C, but no crystals of (1-1) were obtained.

[0111] Synthesis Example 2 The compound represented by the formula (1-1) was obtained as an amorphous compound in the same manner as in Synthesis Example 1. The diastereomeric ratio of the obtained amorphous compound was 1.4:1.0, and the diastereomeric excess was 17%. This amorphous compound is also referred to as "amorphous (1-1e)" hereinafter.

[0112] Example 3: A solution of 100 mg of amorphous (1-1e) in a mixed solvent of 600 μL of ethyl acetate and 1200 μL of methyl t-butyl ether and a trace amount of crystalline (1-11) as seed crystals in a 6.0 mL screw tube were placed in a 28.0 mL standard bottle filled with n-heptane vapor and sealed. After standing overnight at 5°C, a crystalline (1-1) having a diastereomeric ratio of 1.6:1.0 and a diastereomeric excess of 23% (hereinafter also referred to as "crystalline (1-13)") represented by the above formula (1-1) was obtained.

[0113] Example 4: A solution of 100 mg of amorphous (1-1e) in a mixed solvent of 600 μL of ethyl acetate and 1200 μL of methyl t-butyl ether and a trace amount of crystal (1-13) as seed crystals in a 6.0 mL screw tube were placed in a 28.0 mL standard bottle filled with n-heptane vapor and sealed. After standing overnight at room temperature, crystals represented by the above formula (1-1) (hereinafter also referred to as "crystal (1-14)") were obtained, which had a diastereomeric ratio of 1.6:1.0 and a diastereomeric excess of 23%.

[0114] Comparative Example 3: A 6.0 mL screw tube containing a solution of 100 mg of amorphous (1-1e) dissolved in a mixed solvent of 600 μL of ethyl acetate and 1200 μL of methyl t-butyl ether was placed in a 28.0 mL standard bottle filled with n-heptane vapor and sealed. The solution was left to stand overnight at room temperature, but no crystals (1-1) were obtained.

[0115] Example 5 300 mg of amorphous (1-1e) was dissolved in a mixed solvent of 3 mL of ethyl acetate and 8.4 mL of methyl t-butyl ether, and 3 mg of crystalline (1-13) was added as seed crystals. The mixture was then stirred at room temperature for 3 hours. After filtration and drying under reduced pressure, 199.4 mg of crystalline (1-1) (hereinafter also referred to as "crystalline (1-15)") was obtained, which had a diastereomeric ratio of 2.4:1.0 and a diastereomeric excess of 41%. The powder X-ray diffraction pattern of crystalline (1-15) is shown in Figure 1. Powder X-ray diffraction analysis confirmed characteristic peaks at 2θ (±0.2°) = 5.6°, 13.6°, 14.0°, and 16.9°. The peak observed near a diffraction angle (2θ ±0.2°) = 38° is due to the sample plate used to hold the sample, and is not a peak due to the sample itself (the same applies to Figures 2 to 4).

[0116] Example 6 300 mg of amorphous (1-1e) was dissolved in a mixed solvent of 3 mL of ethyl acetate and 3 mL of methyl t-butyl ether, and 3 mg of crystalline (1-13) was added as seed crystals. 6 mL of n-heptane was added dropwise at room temperature over 1 hour, and the mixture was stirred for 2 hours after the completion of the dropwise addition. After filtration and drying under reduced pressure, 256.5 mg of crystalline (1-1) (hereinafter also referred to as "crystalline (1-16)") having a diastereomeric ratio of 3.4:1.0 and a diastereomeric excess of 55% was obtained. The powder X-ray diffraction pattern of crystalline (1-16) is shown in FIG. 2. Powder X-ray diffraction analysis confirmed characteristic peaks at 2θ (±0.2°) = 5.6°, 13.6°, 14.0°, and 16.9°.

[0117] Example 7 300 mg of amorphous (1-1e) was dissolved in a mixed solvent of 4.5 mL of ethyl acetate and 1.5 mL of n-heptane, and 3 mg of crystalline (1-13) was added as seed crystals. 6 mL of n-heptane was then added dropwise at room temperature over 1 hour, and the mixture was stirred for 2 hours after the dropwise addition. After filtration and drying under reduced pressure, 238.3 mg of crystalline (1-1) (hereinafter also referred to as "crystalline (1-17)") having a diastereomeric ratio of 1.6:1.0 and a diastereomeric excess of 23% was obtained. The powder X-ray diffraction pattern of crystalline (1-17) is shown in FIG. 3. Powder X-ray diffraction analysis confirmed characteristic peaks at 2θ (±0.2°) = 5.7°, 13.7°, 14.1°, and 17.0°.

[0118] (Synthesis Example 3) Synthesis was carried out in the same manner as in Synthesis Example 1, but on a scale approximately 1000 times larger, to obtain the compound represented by formula (1-1) as a viscous oil (solid content concentration 62% by mass). Note that due to the scale-up, the viscosity increased significantly in the latter half of the concentration operation, making it difficult to isolate the compound as a solid (amorphous), and as a result, the compound was obtained as an oil. The diastereomeric ratio of the obtained oil was 2.0:1.0, and the diastereomeric excess was 34%. This oil will hereinafter also be referred to as "oil (1-1)."

[0119] Example 8: 223 g of oil (1-1) was dissolved in a mixed solvent of 650 mL of toluene and 650 mL of methyl t-butyl ether, and 63 mg of crystals (1-13) were added as seed crystals. After overnight stirring, 65 mL of n-heptane was added dropwise at room temperature over 30 minutes, and the mixture was stirred for 1 hour after the completion of the dropwise addition. After filtration and drying under reduced pressure, 52.9 g of crystals represented by the above formula (1-1) (hereinafter also referred to as "crystals (1-18)") having a diastereomeric ratio of 3.9:1.0 and a diastereomeric excess of 59% was obtained. The powder X-ray diffraction pattern of crystals (1-18) is shown in FIG. 4. As a result of powder X-ray diffraction analysis, characteristic peaks were confirmed at 2θ (±0.2°) = 5.4°, 13.5°, 14.0°, and 16.8°.

[0120] The results of the above Examples, Comparative Examples, and Reference Examples are shown in Tables 2 and 3 below.

[0121]

[0122]

[0123] Crystallization using a conventional crystallization method using a good solvent and a poor solvent from amorphous starting material failed. This result was similar to that achieved by the vapor diffusion method, which is believed to be easier to obtain crystals from than the poor solvent addition method (Reference Examples 1-10, Comparative Example 3). Even when the diastereomeric excess of the amorphous starting material was increased, crystals could not be obtained by the poor solvent vapor diffusion method using a solution of the starting material dissolved in a good solvent (Comparative Examples 1 and 2). Crystals were only obtained by increasing the diastereomeric excess of the amorphous starting material, dissolving the amorphous starting material in a mixed solvent of a good solvent (AcOEt) and a poor solvent (MTBE), and then adding another poor solvent (n-HEP) to the mixture using the vapor diffusion method (Examples 1 and 2). This crystallization is also characterized by the absence of preferential crystallization, and the diastereomeric excess of the amorphous starting material is maintained in the obtained crystals. When the obtained crystals were used as seed crystals, crystals were obtained even from amorphous starting material with a low diastereomeric excess (Example 3). In this crystallization, preferential crystallization did not occur, and the diastereomeric excess of the starting amorphous material was maintained in the resulting crystals, allowing both diastereomers to be obtained in high yields. Furthermore, even when seed crystals with a low diastereomeric excess were used, crystals were obtained from the amorphous material with a low diastereomeric excess (Example 4). In this crystallization, preferential crystallization did not occur, and the diastereomeric excess of the starting amorphous material was maintained in the resulting crystals, allowing both diastereomers to be obtained in high yields. Furthermore, when the starting amorphous material was dissolved in a solvent and seed crystals were added, the solubility of the crystals generated in the solution was low, allowing spontaneous precipitation without the need for procedures to reduce the amount of solubility, such as adding a poor solvent, lowering the temperature, or concentrating (Example 5). When seed crystals were added, crystals were also obtained using a conventional crystallization method using a good solvent and a poor solvent (Examples 6 and 7). Similarly, when an oily starting material was dissolved in a solvent and seed crystals were added, crystals were also obtained using a conventional crystallization method using a good solvent and a poor solvent (Example 8). In the crystallizations of Examples 5 to 8, no strong preferential crystallization occurred, and the diastereomeric excess of the starting amorphous material tended to be maintained in the obtained crystals, allowing both diastereomers to be obtained in high yields.

Claims

1. A crystal of an amide-bridged compound represented by the following formula (1): (In the formula, P １ represents a protecting group for a hydroxyl group, and P ２ represents a protecting group for an amino group.) 2. The crystal according to claim 1, which contains both a compound represented by the following formula (1a) and a compound represented by the following formula (1b), which are in a diastereomeric relationship. (In the formula, P １ and P ２ is the same as above.) 3. The crystal according to claim 2, wherein the diastereomeric excess calculated by the following formula is greater than 60%: Diastereomeric excess (%) = |(amount of one diastereomer - amount of the other diastereomer) / (amount of one diastereomer + amount of the other diastereomer)| x 100 4. The crystal according to claim 2, wherein the diastereomeric excess calculated by the following formula is 60% or less: Diastereomeric excess (%) = |(amount of one diastereomer - amount of the other diastereomer) / (amount of one diastereomer + amount of the other diastereomer)| x 100 5. The above P １ is a 4,4'-dimethoxytriphenylmethyl group, a tert-butyldimethylsilyl group, or a triisopropylsiloxymethyl group, ２ The crystal according to claim 1, wherein is a benzoyl group, an acetyl group, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, an isobutyryl group, or a dimethylformamidinyl group.

6. The crystal according to any one of claims 1 to 5, which has peaks at diffraction angles (2θ) of 5.6±0.2°, 13.6±0.2°, 14.0±0.2°, and 16.9±0.2° in a powder X-ray diffraction pattern measured using Cu-Kα radiation as an X-ray source.

7. A method for producing a crystal of an amide-bridged compound, comprising dissolving an amide-bridged compound represented by the following formula (1), wherein the compound has a diastereomeric excess of more than 60% as determined by the following formula, in a mixed solvent of ethyl acetate as a good solvent and methyl tert-butyl ether as a poor solvent, and allowing heptane as a poor solvent to be absorbed as vapor into the solution to increase the heptane concentration in the solution, thereby obtaining a crystal of the amide-bridged compound represented by the following formula (1), wherein the diastereomeric excess is more than 60% as determined by the following formula: (In the formula, P １ represents a protecting group for a hydroxyl group, and P ２ indicates a protecting group for an amino group.) Diastereomeric excess (%) = |(amount of one diastereomer - amount of the other diastereomer) / (amount of one diastereomer + amount of the other diastereomer)| x 100 8. The production method according to claim 7, further comprising a step of separating the diastereomers of the amide-bridged compound represented by formula (1), which has a diastereomeric excess of 60% or less as determined by the formula, by high performance liquid chromatography or medium pressure preparative liquid chromatography using a chiral normal phase column, thereby preparing an amide-bridged compound represented by formula (1), which has a diastereomeric excess of more than 60% as determined by the formula.

9. The method according to claim 7, wherein the mixing ratio of ethyl acetate to methyl tert-butyl ether in the mixed solvent is 10 / 90 to 55 / 45 by volume (ethyl acetate / methyl tert-butyl ether).

10. The production method according to claim 7, wherein the content ratio of the amide-bridged compound represented by formula (1), which has a diastereomeric excess of more than 60% as determined by the formula, to the mixed solvent (amide-bridged compound / mixed solvent) is 1 / 100 to 1 / 5 (mg / μL).

11. A method for producing a crystal of an amide-bridged compound, comprising dissolving an amide-bridged compound represented by the following formula (1), which has a diastereomeric excess of 60% or less as determined by the following formula, in a solvent, adding crystals of an amide-bridged compound represented by the following formula (1), which has a diastereomeric excess of more than 60% as determined by the following formula, as seed crystals, and obtaining a crystal of the amide-bridged compound represented by the following formula (1), which has a diastereomeric excess of 60% or less as determined by the following formula. (In the formula, P １ represents a protecting group for a hydroxyl group, and P ２ indicates a protecting group for an amino group.) Diastereomeric excess (%) = |(amount of one diastereomer - amount of the other diastereomer) / (amount of one diastereomer + amount of the other diastereomer)| x 100 12. The method according to claim 11, wherein crystals of the amide-bridged compound represented by formula (1) having a diastereomeric excess of 60% or less as determined by the formula are obtained by a crystallization method including a vapor diffusion method.

13. A method for producing a crystal of an amide-bridged compound, comprising dissolving an amide-bridged compound represented by the following formula (1), which has a diastereomeric excess of 60% or less as determined by the following formula, in a solvent, adding crystals of an amide-bridged compound represented by the following formula (1), which has a diastereomeric excess of 60% or less as determined by the following formula, as seed crystals, and obtaining a crystal of the amide-bridged compound represented by the following formula (1), which has a diastereomeric excess of 60% or less as determined by the following formula. (In the formula, P １ represents a protecting group for a hydroxyl group, and P ２ indicates a protecting group for an amino group.) Diastereomeric excess (%) = |(amount of one diastereomer - amount of the other diastereomer) / (amount of one diastereomer + amount of the other diastereomer)| x 100 14. The method of claim 13, wherein after the seed crystals are added, crystals are allowed to precipitate from the solution without cooling or adding an antisolvent.

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