Cytosine-type cross-linked nucleoside amidite crystals and method for producing the same

Abstract

This invention provides a cytosine-type cross-linked nucleoside amidite crystal that exhibits excellent stability and can be stably stored even under high temperature conditions of 50°C, as well as a method for producing the same. [Solution] The specification describes a cytosine-type cross-linked nucleoside amidite crystal and a method for producing the same.

Description

Technical Field

[0001] The present invention relates to cytosine-type crosslinked nucleoside amidites and a method for producing the same.

Background Art

[0002] As treatment methods for diseases using nucleic acid pharmaceuticals, there are antisense methods, antigene methods, aptamer methods, siRNA methods, and the like.

[0003] Normally, the furanose ring of the nucleoside sugar moiety does not have a planar structure but takes a distorted conformation called N-type or S-type, and is biased towards a specific conformation by the cyclic substituents. For example, in the case of ribonucleosides having a hydroxyl group at the 2'-position, the N-type conformation is predominant. Imase et al. succeeded in forcibly fixing the conformation of nucleosides to the N-type by crosslinking the 4'-position and the 2'-hydroxyl group of the nucleoside sugar moiety. As a result, it has been revealed that LNA (Locked Nucleic Acids) containing this crosslinked nucleoside forms an extremely stable double strand with nucleic acids having complementary sequences (see Patent Document 1). Due to the above characteristics and the like, in recent years, expectations for LNA as a material for nucleic acid pharmaceuticals have been increasing.

[0004] The synthesis of LNA generally uses a solid-phase synthesis method using an amidite form of a crosslinked nucleoside called the phosphoramidite method (see Non-Patent Document 1). In this method, LNA is synthesized by repeating the cycle of detritylation → coupling reaction of the amidite form → cap reaction → oxidation (or sulfurization) reaction until the target chain length is reached. That is, the amidite form of the crosslinked nucleoside is industrially useful because it is used as a raw material for LNA synthesis.

[0005] The amidite form of the crosslinked nucleoside is distributed in the form of a foamy amorphous (see Non-Patent Documents 2, 3, and 4). On the other hand, there is no report that the amidite form of the crosslinked nucleoside crystallizes. [Preliminary Technology Documents] [License]

[0006] [License 1] Special Announcement No. 10-304889 [Non-licensed literature]

[0007] [Non-licensed Document 1] T. Uemoto, No. 8, "Direct and practical synthesis of 2'-O,4'-C-aminomethylene-bridged nucleic acid purine derivatives by transglycosylation", Tetrahedron, 2017, vol.73, p.1211-1218 [Non-licensed Document 2] L. Takeshita, third name, "Synthesis of Deoxypseudouridine 5'-Triphosphate Bearing the Photoremovable Protecting Group at the N1 Position Capable of Enzymatic Incorporation to DNA", J. Org. Chem., 2020, vol.85, p.1861-1870 [Non-licensed Document 3] M. Horiba, 2 outsiders, "Synthesis of scpBNA-mC, -A, and -G Monomers and Evaluation of the Binding Affinities of scpBNA-Modified Oligonucleotides toward Complementary ssRNA and ssDNA", J. Org. Chem., 2016, vol.81, p.11000-11008 [Non-licensed Document 4] C. Riml and 1 others, “Synthesis of 5-Hydroxymethylcytidine- and 5-Hydroxymethyl-uridine-Modified RNA”, Synthesis, 2016, vol.48, p.1108-1116 [Overview of the project] [Problems that the invention aims to solve]

[0008] During their research into the synthesis of LNA, the inventors discovered that the amorphous form of cross-linked nucleoside amidites has low stability. The present invention aims to solve the problem of the amorphous form present in cross-linked nucleoside amidites. [Means for solving the problem]

[0009] The inventors, through diligent research, have obtained crystals of the amidite form of cytosine-type cross-linked nucleoside for the first time. They have found that these crystals can solve the problems of the amorphous form described above, and through further investigation, have completed the present invention. In this specification, the amidite form of cytosine-type cross-linked nucleoside may be abbreviated as "cytosine-type cross-linked nucleoside amidite," its crystal as "cytosine-type cross-linked nucleoside amidite crystal," and the amorphous form of cytosine-type cross-linked nucleoside amidite as "cytosine-type cross-linked nucleoside amorphous."

[0010] In other words, the present invention is a cytosine-type cross-linked nucleoside amidite crystal represented by the following structural formula.

[0011] [ka] In the above formula, R 1 , R 2 R is a substituent. 3 This indicates a protecting group.

[0012] Furthermore, the present invention relates to a method for producing cytosine-type cross-linked nucleoside midite crystals, comprising the step of dissolving cytosine-type cross-linked nucleoside midite amorphous material in a nitrile-based solvent and obtaining precipitated crystals.

[0013] Furthermore, the present invention is a method for producing cytosine-type cross-linked nucleoside midite crystals, comprising the step of dissolving cytosine-type cross-linked nucleoside midite amorphous in an easily soluble solvent, and then adding a sparingly soluble solvent. [Effects of the Invention]

[0014] According to the present invention, it is possible to provide cytosine-type cross-linked nucleoside amidite crystals and a method for producing the same, which solve the problems of amorphous material present in the amidite form of cross-linked nucleosides. Specifically, the cytosine-type cross-linked nucleoside amidite crystals of the present invention have excellent stability and can be stored stably even under high temperature conditions of 50°C. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 shows the appearance of crystal A of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-4'-C-methylene-5-methylcytidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite] obtained in Example 1. [Figure 2] Figure 2 shows the appearance of crystal B of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-4'-C-methylene-5-methylcytidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite] obtained in Example 2. [Figure 3] Figure 3 shows the appearance of crystal C of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-4'-C-methylene-5-methylcytidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite] obtained in Example 3. [Figure 4]Figure 4 shows the X-ray diffraction spectrum of crystal A of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-4'-C-methylene-5-methylcytidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite] obtained in Example 1. The vertical axis represents diffraction intensity (CPS), and the horizontal axis represents diffraction angle (2θ). [Figure 5] Figure 5 shows the X-ray diffraction spectrum of crystal B of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-4'-C-methylene-5-methylcytidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite] obtained in Example 2. The vertical axis represents diffraction intensity (CPS), and the horizontal axis represents diffraction angle (2θ). [Figure 6] Figure 6 shows the X-ray diffraction spectrum of crystal C of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-4'-C-methylene-5-methylcytidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite] obtained in Example 3. The vertical axis represents diffraction intensity (CPS), and the horizontal axis represents diffraction angle (2θ). [Figure 7] Figure 7 shows the differential scanning calorimetry results for crystal A of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-4'-C-methylene-5-methylcytidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite] obtained in Example 1. In the figure, the vertical axis "DSC mW" represents the change in calorimetry, and the horizontal axis "Temp °C" represents the change in temperature. [Figure 8] Figure 8 shows the differential scanning calorimetry results for crystal B of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-4'-C-methylene-5-methylcytidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite] obtained in Example 2. In the figure, the vertical axis "DSC mW" represents the change in calorimetry, and the horizontal axis "Temp °C" represents the change in temperature. [Figure 9]Figure 9 shows the differential scanning calorimetry results for crystal C of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-4'-C-methylene-5-methylcytidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite] obtained in Example 3. In the figure, the vertical axis "DSC mW" represents the change in calorimetry, and the horizontal axis "Temp °C" represents the change in temperature. [Figure 10] Figure 10 shows the differential scanning calorimetry results for the amorphous N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-4'-C-methylene-5-methylcytidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite] obtained in the comparative example. In the figure, the vertical axis "DSC mW" represents the change in calorimetry, and the horizontal axis "Temp °C" represents the change in temperature. [Figure 11] Figure 11 shows the stability test results for N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-4'-C-methylene-5-methylcytidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite] crystals A-C and the control amorphous form obtained in Examples 1-3. In the figure, the vertical axis represents HPLC (%) and the horizontal axis represents the number of days elapsed. [Modes for carrying out the invention]

[0016] In one embodiment, the present invention provides a cytosine-type cross-linked nucleoside amidite crystal represented by the following structural formula.

[0017] [ka]

[0018] In the above formula, R 1 , R 2 R is a substituent. 3 R indicates a protecting group. 1The substituent represented by is an acyl group such as an acetyl group, a propionyl group, an isobutyryl group, or a benzoyl group, an amino group protected with a dimethylaminomethylene group, or an amino group. R 2 The substituent represented by is a methyl group or a hydrogen atom. R 3 The protecting group represented by is a trityl group, a monomethoxytrityl group, a dimethoxytrityl group, or a pixyl group. Among these, from the viewpoint of versatility, R 1 is a benzamide group, R 2 is a methyl group, R 3 is preferably a dimethoxytrityl group.

[0019] The cytosine-type crosslinked nucleoside amidite crystal of the present invention can take different crystal forms depending on the crystal acquisition conditions. In this specification, these crystal polymorphs are described for convenience as crystal A, crystal B, and crystal C.

[0020] The cytosine-type crosslinked nucleoside amidite crystal of the present invention is obtained as columnar or dendritic crystals. An external appearance photograph of crystal A is shown in FIG. 1, an external appearance photograph of crystal B is shown in FIG. 2, and an external appearance photograph of crystal C is shown in FIG. 3.

[0021] When the cytosine-type crosslinked nucleoside amidite crystal of the present invention is analyzed with a powder X-ray diffractometer using Cu-Kα rays, as shown in the examples described later, it has any one of the characteristic peak patterns of crystal A, crystal B, and crystal C shown below for the diffraction angle (2θ). (Crystal A) 7.96, 8.94, 9.50, 10.49, 11.00, 11.12, 13.45, 13.82, 16.79, 17.76, 18.22, 19.14, 19.27, 19.84, 20.31, 20.60, 21.38, 22.65, 24.99, 25.33, 26.42 (°) (Crystal B) 8.09, 10.28, 10.59, 11.07, 11.38, 12.93, 13.50, 15.01, 16.73, 16.92, 17.43, 17.74, 18.77, 19.71, 20.51, 21.50, 21.74, 22.00, 22.22, 22.72, 23.43, 23.80, 24.13, 24.78, 28.10 (°) (Crystal C)8.06,10.20,10.52,11.01,11.35,11.63,12.92,13.48,14.93,15.95,16.68,16.89,17 .33,17.61,18.50,18.72,19.70,19.90,20.49,21.43,21.85,22.16,22.57,23.76,27.96(°) The peak patterns of crystal A are shown in Figure 4, crystal B in Figure 5, and crystal C in Figure 6.

[0022] Generally, the diffraction angle (2θ) in powder X-ray diffraction may include an error range of less than 5%. Therefore, the cytosine-type cross-linked nucleoside amidite crystals of the present invention include not only crystals whose peak diffraction angles perfectly match those of crystals in powder X-ray diffraction, but also crystals whose peak diffraction angles match with an error of less than 5%. For example, the cytosine-type cross-linked nucleoside amidite crystal of the present invention has one of the characteristic peak patterns of crystals A, B, and C shown below as the diffraction angle (2θ) in powder X-ray diffraction. (Crystal A) 7.96±0.40, 8.94±0.45, 9.50±0.48, 10.49±0.52, 11.00±0.55, 11.12±0.56, 13.45±0.67, 13.82±0.69, 16.79±0.84, 17.76±0.89, 18.22±0.91, 19.14±0.96, 19.27±0.96, 19.84±0.99, 20.31±1.02, 20.60±1.03, 21.38±1.07, 22.65±1.13, 24.99±1.25, 25.33±1.27, 26.42±1.32 (°) (Crystal B)8.09±0.41,10.28±0.51,10.59±0.53,11.07±0.55,11.38±0.57,12.93±0.65, 13.50±0.68,15.01±0.75,16.73±0.84,16.92±0.85,17.43±0.87,17.74±0.89,18.7 7±0.94, 19.71±0.99, 20.51±1.03, 21.50±1.08, 21.74±1.09, 22.00±1.10, 22.22±1.11, 22.72±1.14, 23.43±1.17, 23.80±1.19, 24.13±1.21, 24.78±1.24, 28.10±1.41(°) (Crystal C)8.06±0.40,10.20±0.51,10.52±0.53,11.01±0.55,11.35±0.57,11.63±0.58, 12.92±0.65,13.48±0.67,14.93±0.75,15.95±0.80,16.68±0.83,16.89±0.85,17.3 3±0.87, 17.61±0.88, 18.50±0.93, 18.72±0.94, 19.70±0.99, 19.90±1.00, 20.49±1.02, 21.43±1.07, 21.85±1.09, 22.16±1.11, 22.57±1.13, 23.76±1.19, 27.96±1.40(°)

[0023] In this specification, powder X-ray diffraction shall be performed under the following conditions. Equipment used: X-ray diffractometer X'Pert PRO MPD (Spectris) Target: Cu X-ray tube current: 40mA X-ray tube voltage: 45kV Scanning range: 2θ = 4.0~40.0°

[0024] When the cytosine-type cross-linked nucleoside amidite crystals of the present invention were analyzed using a differential scanning calorimetry analyzer (Shimadzu Corporation) (heating rate 5°C / min), crystal A showed endothermic peaks due to melting at around 96, 176, and 184°C (±2°C error) as shown in Figure 7, crystal B showed endothermic peaks at around 148 and 183°C (±2°C error) as shown in Figure 8, and crystal C showed endothermic peaks at around 177 and 183°C (±2°C error) as shown in Figure 9. On the other hand, the amorphous crystals did not show endothermic peaks, as shown in Figure 10.

[0025] The cytosine-type cross-linked nucleoside amidite crystals of the present invention are extremely stable, and as demonstrated in the examples described later, even when stored for 20 days under conditions of 50°C, for example, the degree of degradation can be kept to within 1%.

[0026] In this specification, "degree of degradation" is defined as the difference between the purity of the cytosine-type cross-linked nucleoside amidite crystal at the start of a stability test and the purity after storage, when a stability test is performed for a certain period under the following conditions. Purity can be determined by HPLC. A high degree of degradation means that degradation has progressed and the purity has decreased. (Stability test conditions) Temperature: 50℃ Storage conditions: Place 20 mg of crystals in a glass vial and store in a sealed container. (HPLC conditions) Column: YMC-Triart C18, 150×4.6mm I.D. (manufactured by YMC) Elution: 5 mM triethylammonium acetate (pH 7.0) containing 80% by volume acetonitrile. Detection method: Detection using UV280nm

[0027] In another embodiment, the present invention provides a method for producing cytosine-type cross-linked nucleoside amidite crystals.

[0028] The cytosine-type cross-linked nucleoside amidite crystals of the present invention can be obtained by utilizing the low affinity of phosphoramidite groups to nitrile solvents. Specifically, the cytosine-type cross-linked nucleoside amidite crystals of the present invention can be obtained by dissolving cytosine-type cross-linked nucleoside amidite amorphous material in a nitrile solvent and precipitating the crystals (Method A). Alternatively, cytosine-type cross-linked nucleoside amidite crystals can also be obtained by dissolving cytosine-type cross-linked nucleoside amidite amorphous material in an easily soluble solvent and then adding a sparingly soluble solvent (Method B).

[0029] A specific example of Method A is a method in which, after the addition reaction of the phosphoramidite group shown in Chemical Formula 3 below and the usual purification procedure, the obtained cytosine-type cross-linked nucleoside amidite amorphous is dissolved once in a nitrile solvent and then precipitated as crystals. In this case, stirring and cooling are not particularly necessary, but these operations may be performed.

[0030] [ka] In the above formula, R 1 , R 2 R is a substituent. 3 This indicates a protecting group.

[0031] In this case, nitrile solvents such as acetonitrile, propionitrile, butyronitrile, valeronitrile, and benzonitrile can be used. Among these, acetonitrile and propionitrile are preferred from the standpoint of safety and handling, and acetonitrile is more preferred from the standpoint of cost. On the other hand, in this method A, it is important not to include protic polar solvents such as methanol and ethanol, or nonpolar solvents such as dichloromethane, ethyl acetate, chloroform, and tetrahydrofuran, due to the high solubility of the compound.

[0032] A specific example of Method B is a method in which the cytosine-type cross-linked nucleoside amidite amorphous material obtained in the same manner as in Method A is dissolved in an easily soluble solvent, and then precipitated as crystals by adding a sparingly soluble solvent. Easily soluble solvents that can be used include alcohol-based solvents such as methanol, ethanol, isopropanol, tert-butanol, and n-butanol; ketone-based solvents such as acetone and butanone; halogen-based solvents such as dichloromethane and chloroform; ester-based solvents such as ethyl acetate and butyl acetate; and ether-based solvents such as tetrahydrofuran and tert-butyl methyl ether. Sparingly soluble solvents that can be used include hydrocarbon-based solvents such as pentane, hexane, and heptane.

[0033] In the crystal acquisition process described above, additional operations such as cooling the solution, adding seed crystals, and ultrasonic irradiation may be performed to acquire crystals more efficiently.

[0034] This synthesis process can be carried out by those skilled in the art by appropriately referring to known literature (Tetrahedron 1998, 54, 3607-3630, etc.). For example, a combination of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphoramidite and tetrazole or substituted tetrazole, or a combination of 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite and diisopropylethylamine can be reacted in an organic solvent such as acetonitrile or dichloromethane at room temperature for 2 to 3 hours. After confirming the disappearance of the starting materials by thin-layer chromatography (TLC) or HPLC, the usual post-processing can be carried out.

[0035] The cytosine-type cross-linked nucleoside amidite synthesized in this manner can be purified by chromatography using silica gel as a support, dissolved in a nitrile solvent, and further precipitated as crystals by adding a sparingly soluble solvent, seed crystals, cooling, and stirring, depending on the circumstances.

[0036] The cytosine-type cross-linked nucleoside amidite crystals obtained by the above manufacturing method can be collected by filtration methods such as pressure filtration, vacuum filtration, basket separation, and filter pressing, and then dried to produce the final product. For drying, methods such as conical drying, vacuum drying including shelf drying, fluidized bed drying, forced-air drying including shelf drying, and spray drying can be used as appropriate. [Examples]

[0037] The present invention will be specifically described below with reference to examples, but it is clear that the present invention is not limited thereto.

[0038] (Example 1: Crystal A) 5-methylcytosine-type cross-linked nucleoside amidite (R 1 = benzamide group, R 2 =methyl group, R 3 Preparation of crystals (=dimethoxytrityl group) According to the method described in the publicly available literature (Tetrahedron 1998, 54, 3607-3630), 5-methylcytosine type cross-linked nucleoside midite (R 1 = benzamide group, R 2 =methyl group, R 3 A dimethoxytrityl group was prepared. The obtained amorphous 5-methylcytosine-type crosslinked nucleoside amidite (R 1 = benzamide group, R 2 =methyl group, R 3 Crystals precipitated when 0.65 g of (dimethoxytrityl group) was dissolved in 5 mL of acetonitrile and allowed to stand at -20°C for 7 days.

[0039] The obtained crystals are collected by suction filtration and then vacuum-dried to produce 5-methylcytosine-type cross-linked nucleoside midites (R 1 = benzamide group, R 2 =methyl group, R 3 Crystals of the dimethoxytrityl group were obtained (0.35g).

[0040] (Example 2: Crystal B) 5-methylcytosine-type cross-linked nucleoside amidite (R 1 = benzamide group, R 2 =methyl group, R 3 Preparation of crystals (=dimethoxytrityl group) According to the method described in the publicly available literature (Tetrahedron 1998, 54, 3607-3630), 5-methylcytosine type cross-linked nucleoside midite (R 1 = benzamide group, R 2 =methyl group, R 3 A dimethoxytrityl group was prepared. The obtained amorphous 5-methylcytosine-type crosslinked nucleoside amidite (R 1 = benzamide group, R 2 =methyl group, R 3 1.00 g of (dimethoxytrityl group) was dissolved in 1 mL of dichloromethane, and then 15 mL of hexane was gradually added. Crystals precipitated when this solution was irradiated with ultrasound. After adding another 10 mL of hexane to this solution, it was allowed to stand for 3 hours. The crystals precipitated in the solution were collected by suction filtration and then vacuum dried to obtain 5-methylcytosine type cross-linked nucleoside midite (R 1 = benzamide group, R 2 =methyl group, R 3 Crystals of the dimethoxytrityl group were obtained (0.81g).

[0041] (Example 3: Crystal C) 5-methylcytosine-type cross-linked nucleoside amidite (R 1 = benzamide group, R 2 =methyl group, R 3 Preparation of crystals (=dimethoxytrityl group) According to the method described in the publicly available literature (Tetrahedron 1998, 54, 3607-3630), 5-methylcytosine type cross-linked nucleoside midite (R 1 = benzamide group, R 2 =methyl group, R 3 A dimethoxytrityl group was prepared. The obtained amorphous 5-methylcytosine-type crosslinked nucleoside amidite (R 1= benzamide group, R 2 =methyl group, R 3 1.00 g of (dimethoxytrityl group) was dissolved in 1 mL of acetonitrile and allowed to stand at -20°C for 2 hours, after which crystals precipitated (0.80 g).

[0042] (Comparative Example) When dichloromethane alone or dichloromethane-acetonitrile (1:1) is used as the crystallization solvent. Amorphous 5-methylcytosine-type crosslinked nucleoside amidite was obtained using the method described in Example 1. Crystallization was investigated by changing the solvent from acetonitrile to dichloromethane alone, or dichloromethane-acetonitrile (1:1), while keeping all other conditions the same as in Example 1, but no crystals precipitated.

[0043] (Example 4) Analysis of various physical properties of crystals 5-methylcytosine-type crosslinked nucleoside amidite (R) obtained in Examples 1, 2, and 3 1 = benzamide group, R 2 =methyl group, R 3 Various physical properties of the crystal (=dimethoxytrityl group) were analyzed.

[0044] (a) Crystal form The 5-methylcytosine-type crosslinked nucleoside amidite (R) obtained in Example 1 1 = benzamide group, R 2 =methyl group, R 3 Figure 1 shows a photograph of crystal A (=dimethoxytrityl group). As shown in Figure 1, it was revealed that the 5-methylcytosine type cross-linked nucleoside amidite crystal exhibits a columnar crystal shape.

[0045] The 5-methylcytosine-type cross-linked nucleoside amidites obtained in Examples 2 and 3 also exhibited similar columnar or dendritic appearances, although their crystal sizes differed. Figure 2 shows a photograph of crystal B obtained in Example 2, and Figure 3 shows a photograph of crystal C obtained in Example 3.

[0046] (i) Purity measurement The 5-methylcytosine-type crosslinked nucleoside amidite (R) obtained in Example 1 above 1 = benzamide group, R 2 =methyl group, R 3 The purity of crystal A (=dimethoxytrityl group) was analyzed by HPLC, and the purity of 5-methylcytosine-type cross-linked nucleoside amidite crystal A was found to be 98.8%. The HPLC method was performed under the following conditions. (HPLC conditions) Column: YMC-Triart C18, 150×4.6mm I.D. (manufactured by YMC) Elution: 5 mM triethylammonium acetate (pH 7.0) containing 80% by volume acetonitrile. Detection method: Detection using UV280nm

[0047] The purity of 5-methylcytosine-type cross-linked nucleoside amidite crystals B and C obtained in Examples 2 and 3 above was measured under the same conditions. The purity of 5-methylcytosine-type cross-linked nucleoside amidite crystal B obtained in Example 2 was 98.7%, and the purity of 5-methylcytosine-type cross-linked nucleoside amidite crystal C obtained in Example 3 was 98.6%.

[0048] (c) Powder X-ray diffraction 5-methylcytosine-type crosslinked nucleoside amidite (R) obtained in Examples 1-3 1 = benzamide group, R 2 =methyl group, R 3 The X-ray diffraction spectra of crystals A, B, and C (which contain the dimethoxytrityl group) were measured using an X-ray diffractometer X'Pert PRO MPD (Spectris) under the following measurement conditions. (Measurement conditions) Target: Cu X-ray tube current: 40mA X-ray tube voltage: 45kV Scanning range: 2θ = 4.0~40.0°

[0049] As shown in Figure 4 and Table 1, 5-methylcytosine type cross-linked nucleoside amidite (R 1= benzamide group, R 2 =methyl group, R 3 =dimethoxytrityl group) Crystal A has a diffraction angle (2θ), (Crystal A) shows peaks around 7.96, 8.94, 9.50, 10.49, 11.00, 11.12, 13.45, 13.82, 16.79, 17.76, 18.22, 19.14, 19.27, 19.84, 20.31, 20.60, 21.38, 22.65, 24.99, 25.33, and 26.42 (°), particularly around 7.96, 11.00, 13.45, 13.82, 16.79, 17.76, and 20.60 (°). A distinctive peak was observed in the vicinity.

[0050] [Table 1]

[0051] As shown in Figure 5 and Table 2, the 5-methylcytosine type crosslinked nucleoside amidite (R) obtained in Example 2 1 = benzamide group, R 2 =methyl group, R 3 (Dimethoxytrityl group) Crystal B has a diffraction angle (2θ), (Crystal B)8.09,10.28,10.59,11.07,11.38,12.93,13.50,15.01,16.73,16.92,17.43,17.74,18 .77,19.71,20.51,21.50,21.74,22.00,22.22,22.72,23.43,23.80,24.13,24.78,28.10(°) A distinctive peak was observed in the vicinity.

[0052] [Table 2]

[0053] As shown in Figure 6 and Table 3, the 5-methylcytosine-type crosslinked nucleoside amidite (R) obtained in Example 3 1 = benzamide group, R 2 =methyl group, R 3=dimethoxytrityl group) Crystal C has a diffraction angle (2θ), (Crystal C)8.06,10.20,10.52,11.01,11.35,11.63,12.92,13.48,14.93,15.95,16.68,16.89,17 .33,17.61,18.50,18.72,19.70,19.90,20.49,21.43,21.85,22.16,22.57,23.76,27.96(°) A distinctive peak was observed in the vicinity.

[0054] [Table 3]

[0055] (e) Differential scanning calorimetry 5-methylcytosine-type cross-linked nucleoside amidite (R 1 = benzamide group, R 2 =methyl group, R 3 Crystals A-C (containing the dimethoxytrityl group) were analyzed using differential scanning calorimetry (DSC) at a heating rate of 5°C / min. Crystal A showed endothermic peaks due to melting at approximately 96, 176, and 184°C (±2°C error), as shown in Figure 7; Crystal B showed endothermic peaks due to melting at approximately 148 and 183°C (±2°C error), as shown in Figure 8; and Crystal C showed endothermic peaks due to melting at approximately 177 and 183°C (±2°C error), as shown in Figure 9. On the other hand, the amorphous crystal did not have an endothermic peak, as shown in Figure 10.

[0056] (Example 5) Stability comparison between 5-methylcytosine-type crosslinked nucleoside amidite crystals and known amorphous materials The 5-methylcytosine-type crosslinked nucleoside amidite (R) obtained in Example 1, Example 2, and Example 3 1 = benzamide group, R 2 =methyl group, R 3 The stability of crystals A-C (=dimethoxytrityl group) and the amorphous material obtained in the comparative example was compared using the method described below.

[0057] (Stability test conditions) Temperature: 50℃ Storage conditions: Place 20 mg of crystals A-C or amorphous crystals into a glass vial and store in a sealed container. Sampling time: After 1, 2, 3, 4, 6, and 20 days, each sample was collected and subjected to HPLC analysis. (HPLC conditions) Column: YMC-Triart C18, 150×4.6mm I.D. (manufactured by YMC) Elution: 5 mM triethylammonium acetate (pH 7.0) containing 80% by volume acetonitrile. Detection method: Detection using UV280nm

[0058] The results of the stability test are shown in Figure 11 and Table 4. In the table, the leftmost column represents the number of days elapsed, and the column "Example 1 (Crystal A)" represents the 5-methylcytosine-type cross-linked nucleoside amidite (R) obtained in Example 1. 1 = benzamide group, R 2 =methyl group, R 3 =dimethoxytrityl group) The HPLC purity of crystal A after the corresponding number of days is shown in the "Example 2 (crystal B)" column, which is the 5-methylcytosine type cross-linked nucleoside amidite (R) obtained in Example 2. 1 = benzamide group, R 2 =methyl group, R 3 =dimethoxytrityl group) The HPLC purity of crystal B after the corresponding number of days is shown in the "Example 3 (crystal C)" column, which is the 5-methylcytosine type cross-linked nucleoside amidite (R) obtained in Example 3. 1 = benzamide group, R 2 =methyl group, R 3 The HPLC purity of crystal C (=dimethoxytrityl group) after the corresponding number of days is shown in the "Control" column, and the HPLC purity of the amorphous material used as a control after the corresponding number of days is shown in the "Control" column. Cells marked "-" in the table indicate that no measurement was performed. The "Degradation (Δ%)" at the bottom shows the difference when the HPLC purity after 20 days is subtracted from the HPLC purity on day 0, and a larger value means that more degradation has occurred.

[0059] [Table 4]

[0060] Based on the above results, the 5-methylcytosine-type crosslinked nucleoside amidite (R) obtained in Examples 1-3 1 = benzamide group, R 2 =methyl group, R 3 Crystals A-C (=dimethoxytrityl group) showed no degradation of more than 1% in HPLC purity even after being stored at 50°C for 20 days, indicating extremely high stability. Furthermore, since the degree of degradation in the control amorphous material was 6.6%, it was understood that the stability of the 5-methylcytosine-type cross-linked nucleoside amidite crystals of the present invention is significantly higher than that of existing 5-methylcytosine-type cross-linked nucleoside amidite products.

Claims

[Claim 1] Cytosine-type cross-linked nucleoside amidite crystals and methods for producing the same as described in the specification.

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