KR20000022113A - Crystals of vitamin d derivatives and process for the preparation thereof - Google Patents

Abstract

PURPOSE: Crystals of vitamin D derivatives and a process for the preparation thereof are provided, which enables the constant and mass supply of high-purity vitamin D derivatives, particularly ED-71. CONSTITUTION: Crystals of vitamin D derivatives of formula I are prepared by purifying unpurified or roughly purified vitamin D derivatives by reversed phase chromatography and crystallizing the purified derivatives in organic solvents. Novel by-products are formed in the synthesis of vitamin D derivatives.

Description

Vitamin D Derivative Crystal and Its Manufacturing Method

Various vitamin D derivatives are known to have useful physiological activity. For example, JP 6-23185 B / 1994 discloses that 1 α-hydroxyvitamin D ₃ derivative represented by the following formula is useful as a therapeutic agent for diseases caused by calcium metabolism or as an anti-tumor agent:

[Wherein, R ₁ represents an amino group or a formula OR ′ (R ′ represents a lower alkyl group of 1 to 7 carbon atoms unsubstituted or substituted with a hydroxyl group, a halogen atom, a cyano group or an acylamino group), and R ₂ represents a halogen An atom or a hydroxyl group.

One of the compounds represented by the above formula, 1α, 25-dihydroxy-2β- (3-hydroxypropoxy) vitamin D ₃ (also called ED-71), is an active form of a vitamin D derivative having a bone forming action. It is under development as a treatment for osteoporosis.

If the vitamin D derivative is established as a therapeutic agent, it must be highly purified and continuously supplied in large quantities. Therefore, there is a need to establish a method for preparing vitamin D derivatives as soon as possible.

In particular, ED-71 is obtained only in amorphous form and has not been reported for the isolation of ED-71 in crystalline form.

The present invention relates to novel vitamin D derivative crystals and more particularly to novel vitamin D derivative crystals obtained by purifying vitamin D derivatives via reverse phase chromatography and then crystallizing the purified derivatives from organic solvents. The present invention also relates to a method for purifying a vitamin D derivative consisting of a crystallization step.

1 is a molecular structure projection showing the structure of the ED-71 crystal.

Fig. 2 is a stereoscopic projection view of the molecular structure showing the structure of the ED-71 crystal.

3 is a molecular structure projection showing the structure of ED-71 crystals focused on hydrogen bonding.

Fig. 4 is a stereoscopic projection view of the molecular structure showing the structure of the ED-71 crystal focused on hydrogen bonding.

Preferred Embodiments of the Invention

As used herein, the term "vitamin D derivative" means a compound having the structure of Formula V in partial structure:

Vitamin D derivatives are preferably compounds represented by the formula VIA, VIB or VIC:

[Wherein, R ₁ represents (1) an amino group;

(2) -OR ₅ (wherein R ₅ is a lower alkyl group, lower alkenyl group or lower alkynyl group which may be substituted with a hydroxyl group, a halogen atom, a cyano group, an amino group, an acylamino group or a lower alkoxy group; or

(3) a lower alkyl group, lower alkenyl group or lower alkynyl group, which may be substituted with a hydroxyl group, a halogen atom, a cyano group, an amino group, an acylamino group or a lower alkoxy group;

R ₂ , R ₃ and R ₄ each represent an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an alkynyl group having 2 to 10 carbon atoms, each of which may be substituted with one or more hydroxyl groups; And

A represents a sulfur atom or an oxygen atom.

In the above definition of a substituent, the term "lower" means that the number of carbon atoms contained in the functional group represented by lower is 1 to 7 for an alkyl group, 2 to 7 alkenyl and alkynyl groups, and 1 to 7 alkoxy groups Means that.

The vitamin D derivative is more preferably 1α-hydroxyvitamin D ₃ , 1α, 25-dihydroxyvitamin D ₃ , 24,25-dihydroxyvitamin D ₃ , or a compound represented by the following formula VIIA or VIIB:

[Wherein n represents an integer of 1 to 7],

Or a compound represented by the formula VIIIA or VIIIB:

[Wherein, A represents a sulfur atom or an oxygen atom].

Particularly preferred vitamin D derivatives are those represented by the formula VIIA:

[VIIA]

[Wherein n is an integer of 1 to 7].

The most preferred vitamin D derivative is a compound represented by the following formula (IX) called ED-71:

As used herein, the term "crystal" is used in its broadest sense and therefore is not limited to crystalline forms, crystal systems, and the like.

The determination of ED-71, the most preferred vitamin D derivative of the present invention, is not limited by any physical properties as described earlier. However, it is particularly preferred to have the following properties:

(1) Appearance: white crystalline powder when visual or fluorescence-microscopy observation;

(2) solubility: complete dissolution at a concentration of 1 mg / mL in ethanol;

(3) confirmation method: IR or NMR method;

(4) melting point: 130 ° C. or higher as measured by DSC;

(5) absorbance index: ε = 16000 or greater as measured at 265 nm at a concentration of 40 μg / mL in ethanol; And

(6) HPLC purity: 97% or more based on the area of the peak for ED-71 relative to the area of the total peak recorded in HPLC under the following conditions: DIACHROMA ODS N-20 5 μm 4.6 × 250 mm, 45% acetonitrile- Water, flow rate of 1 mL / min, 220 nm, 10 μL of 1 mg / mL, 4-90 min.

According to another aspect of the present invention, it is not only to provide a vitamin D derivative crystal obtainable by purifying a crude product or a preliminary purified product of a vitamin D derivative through reverse phase chromatography and then crystallizing the purified derivative from an organic solvent. Provided are a method for purifying a vitamin D derivative consisting of performing reverse phase chromatography with a vitamin D derivative and / or crystallizing the purified vitamin D derivative from an organic solvent.

As used herein, the term “crude or prepurified product” refers to a vitamin D derivative product obtained from the synthesis of a vitamin D derivative, without purification immediately after the synthesis reaction or by purification in a conventional manner, which generally In amorphous form.

As used herein, the term "reverse phase chromatography" refers to a chromatography system in which the stationary phase has a lower polarity than the mobile phase. High performance liquid chromatography (HPLC) is preferred as reversed phase chromatography.

In order to effectively separate the desired material, it will be necessary to properly select the eluent, the column packing and the load to the column.

Examples of eluates include, but are not limited to, acetonitrile / water and acetonitrile / methanol / water. The mixing ratio of the solvent used as the above-mentioned eluate varies depending on factors such as the material to be purified and the column packing used, and the titration ratio of the solvent for a specific application can be determined by a person skilled in the art. The ratio of acetonitrile / methanol / water is generally within 20-60 / 0-40 / 0-80 parts by weight.

The column packing can be selected according to its particle diameter and porosity, taking into account the material to be purified and the compatibility of the column and column packing used.

The loading to the column also varies depending on the internal diameter of the column and the like. However, the loading can be, for example, about 25 μg to 10 g, preferably about 25 μg to 3 g when the internal diameter is 50 mm.

Fractions obtained by performing chromatography as described above must be treated to isolate the solutes contained in the fractions prior to crystallization. Isolation processes include evaporation, freeze-drying, extraction and filtration. From the above processes for isolation, one or more processes suitable for the material may be selected in consideration of the nature of the material to be purified. For example, the evaporation method is operationally advantageous for the purification of ED-71 because it is renewable and does not degrade ED-71.

As an organic solvent that can be used for crystallization of the vitamin D derivative, an aprotic organic solvent is preferable. Examples of aprotic organic solvents include esters such as ethyl acetate, ketones such as acetone, ethers such as diethyl ether and diisopropyl ether, acetonitrile, and mixtures thereof, preferably ethyl acetate, Acetone, acetonitrile, and a mixture thereof.

Crystallization conditions vary depending on factors such as the material to be purified and the solvent used, so that conditions suitable for a particular application can be determined by one skilled in the art. However, crystallization is generally carried out using a solvent in an amount of 1-100 times higher, preferably 5-10 times higher than the crude vitamin D derivative, at a temperature of 30 ° C. or lower, preferably -10 ° C. or lower.

According to another aspect of the present invention, there is provided a process for purifying a compound of formula (II) consisting of recrystallizing a crude or prepurified product of a compound represented by formula (II) from an alcohol, as well as Provide the compound:

[Formula II]

The alcohol used for the recrystallization is methanol.

The physical properties of the compound represented by formula (II) purified by recrystallization from alcohol as above are not limited to any numerical values. However, it is particularly preferable to have the following properties:

(1) Appearance: white to yellow crystalline powder when visual or fluorescence-microscopy observation;

(2) Solubility: complete dissolution at a concentration of 2 mg / mL in ethanol (solution may be water-white to yellow);

(3) confirmation method: IR or NMR method;

(4) water content: 3.0% or less as measured by Karl-Fischer method using 100 mg of sample;

(5) absorbance index: ε = 10000 or more as measured at 282 nm at a concentration of 100 μg / mL in ethanol; And

(6) HPLC purity: There is no peak observable between the proform and unP ₄ peaks in HPLC under the following conditions, 85% based on the area of the peak for the compound of formula II relative to the area of the total peak recorded in HPLC. Above: DIACHROMA ODS N-20 5 μm 4.6 × 250 mm, 55% acetonitrile-water, flow rate of 1 mL / min, 220 nm, 1 mg / mL 10 μL, 4-70 min; And

(7) content; 85% or more in HPLC performed using internal standard under the following conditions: YMC Pack ODS A-303 5 μm 4.6 × 250 mm, 55% acetonitrile-water, flow rate of 1 mL / min, 220 nm.

The following examples are provided to further illustrate the invention but are not limited thereto.

It is an object of the present invention to establish a method for the preparation of highly purified vitamin D derivatives, in particular ED-71, so that the product can be continuously supplied in large quantities.

It is another object of the present invention to provide vitamin D derivative crystals that can be obtained by purifying crude or preliminary purified products of vitamin D derivatives.

Another object of the present invention to provide a method for purifying a vitamin D derivative consisting of a crystallization step.

It is a further object of the present invention to provide a process for purifying the precursor compound of ED-71 consisting of a crystallization step, and a purified precursor compound obtained by this method.

It is another object of the present invention to provide novel compounds which are formed secondary during the synthesis and purification of vitamin D derivatives.

The inventors have conducted extensive studies on the following points appearing in the synthesis and purification of ED-71 from provitamin D derivatives (proform): (1) Impurities in proformation for HPLC preliminary purification of ED-71 Influence of; (2) the stability of ED-71 and its precursor vitamin D derivatives (preforms) against heat, light and oxygen; (3) manipulation of ED-71, which exhibits high physiological action even in very small amounts; (4) possibility of purification of ED-71 by crystallization. As a result of the study, recrystallization of the proform from methanol, photoreaction and thermal isomerization of the recrystallized proform at low temperature, purification of the isomerized product by reverse phase HPLC, concentration of the eluate, and The present invention was completed by finding that ED-71 crystals could be obtained in grams in the order of determining the residue from ethyl acetate. Moreover, the structure of the byproducts originally contained in the pro form or formed during the photo-reaction were revealed and some of them were new.

According to one aspect of the present invention, a compound crystal represented by Formula I is provided:

According to another aspect of the present invention, vitamin D derivative crystals are obtained by purifying crude or pre-purified products of vitamin D derivatives via reverse phase chromatography and then crystallizing the purified derivatives from organic solvents.

According to another aspect of the present invention, there is provided a method for purifying a vitamin D derivative consisting of performing reverse phase chromatography on the vitamin D derivative.

According to another aspect of the present invention, there is provided a method for purifying a vitamin D derivative consisting of crystallizing the vitamin D derivative from an organic solvent.

According to another aspect of the present invention, there is provided a method for purifying a vitamin D derivative consisting of purifying a crude or preliminary purified product of a vitamin D derivative via reverse phase chromatography and then crystallizing the purified derivative from an organic solvent.

According to another aspect of the present invention there is provided a process for purifying a compound represented by formula (II) consisting of recrystallizing a crude or preliminary purification product of the compound represented by formula (II) from an alcohol:

According to another aspect of the present invention there is provided a purification product of a compound represented by formula (II) obtained by recrystallizing a crude or prepurified product of a compound represented by formula (II) from an alcohol.

[Formula II]

According to another embodiment of the present invention, formula (II):

[Formula II]

The crude or prepurified product of the compound represented by is recrystallized from alcohol and subjected to ultraviolet irradiation and thermal isomerization reaction to the recrystallized compound of formula II to produce a vitamin D derivative represented by formula I, reversed phase chromatography Formula I consisting of purifying the initial or preliminarily purified vitamin D derivative of formula I and crystallizing the vitamin D derivative of formula I from an organic solvent:

[Formula I]

Provided is a method for preparing a purified product of a vitamin D derivative.

According to another aspect of the invention, there is provided a compound represented by formula III and a compound represented by formula IV:

The compounds are contained in the reaction mixture obtained by ultraviolet irradiation of the pro form of ED-71 followed by thermal isomerization reaction.

Example 1 Synthesis and Purification of 2β- (3'-hydroxypropoxy) -5,7-cholestadiene-1α, 3β-triol (Proform)

A mixture of epoxy compound (1) (1.00 g, 2.41 mmol), tert-potassium butoxide (0.75 g, 6.68 mmol) and 1,3-propanediol (20 ml) is stirred at room temperature for 10 minutes. The reaction mixture is then heated to an internal temperature of 95 ° C. and stirred at this temperature for 5 hours. The reaction mixture is poured into saturated aqueous ammonia (40 ml) solution under stirring. After stirring for 10 minutes at room temperature (25-35 ° C.), the formed crystals are collected on a filter and washed three times with distilled water (20 ml). Crude crystals containing water (6.3 g) are stirred in acetonitrile (20 ml) at room temperature (27-32 ° C.) for 1 hour. The crystals are collected on a filter, washed twice with acetonitrile (5 ml) and then dried to give the compound (2) in proform (0.96 g, 81% yield).

The obtained proform compound (2) (29.0 g) was heated, dissolved in argon gas, and dissolved in methanol (290 ml), which was pretreated, and the resulting solution was heated at a high temperature. Filter through 4). After cooling to room temperature, seed crystals are added to the solution to induce crystallization. After further cooling to −10 ° C. or lower, the formed crystals are collected on a filter and washed twice with 29 ml of cold methanol. The crystals are then vacuum dried at room temperature to give 22.9 g of purified proform (79.1% recovery, 92.1% recycle). Physical data of the purified proforms are as follows:

NMR (CD ₃ OD) and IR (KBr): refer to the title compound;

TLC (CH ₂ Cl ₂ : EtOH = 9: 1): only one point developed (Rf 0.5);

HPLC purity (220 nm): 98.7%;

Content: 97.1% (internal standard method); And

DSC: lowest peak 95.6 ° C. and 163.2 ° C., highest peak 120.2 ° C.

Example 2: (1R, 2R) -1,25-dihydroxy-2- (3'-hydroxypropoxy) -cholecalciferol; 2β- (3'-hydroxypropoxy)-(1α, 3β, 5Z, 7E) -9,10-secocholester-5,7,10 (19) -triens-1,3,25-triol Synthesis and Purification of (ED-71)

Purified Proform 2 (6.02 g) obtained in Example 1 was dissolved in THF (1 L) in a 1 liter container and the solution was rained for 150 minutes under argon atmosphere under cooling conditions (internal temperature -13 ° C or lower). UV light is irradiated with a 400 W lamp containing high pressure mercury vapor through a Vycor filter. After warming to room temperature, the vessel is washed with fresh THF (100 mL) while pouring the reaction solution from the vessel into a 2 liter eggplant type flask. The combined solution is heated at reflux for 180 minutes. After the reaction mixture is concentrated, the resulting residue is dissolved in methanol (80 mL) to form a separate sample. Using a pump, 20 mL of a sample containing 1.5 g of solute calculated in pro-formation was prepared with DIACHROMA ODS N-20 (Mitsubishi kakouki) having an internal diameter of 50 mm, a length of 300 mm and a particle diameter of 5 μm on the market. Co., Ltd.) was added dropwise to the purification chromatography column filled with. 45% acetonitrile in water was passed through the column at a flow rate of 60 ml / min to confirm the eluate with UV-rays of 220 and 305 nm. About 2.4 L ED-71-containing fractions are collected for a time between about 130 and 170 minutes after the start of chromatography. This series of processes is repeated three more times and the fractions of ED-71 total about 9 liters. The combined fractions are then concentrated on a 10 L rotary evaporator. The residue is dissolved in ethanol and the solution is evaporated to dryness again. The resulting residue is collected using ethyl acetate (20 mL) and the solution is stirred at room temperature to precipitate crystals. The suspension is further cooled down to -10 ° C and stirred at this temperature for 15 minutes. The crystalline material is filtered off, washed three times with cold ethyl acetate (6 ml) and vacuum dried overnight at room temperature to give ED-71 (2.17 g, 36.1% yield).

HPLC purity: 99.8% (220 nm), 99.9% (265 nm)

UV (EtOH): λmax 265.4 nm (ε17100)

DSC: 135.3 ° C. (minimum peak), 122 mJ / mg

Residual Solvent (GC Method): 1.24% (EtOAc), 0.24% (ETOH)

IR (cm ^-1 ): 3533, 3417, 3336, 2943, 2918, 2862, 1649, 1470, 1444, 1416, 1381, 1377, 1342, 1232, 1113, 1078, 1072, 1045, 999, 974, 957, 955 , 924, 910, 895, 868, 833, 796, 764, 663, 634, 594, 472

Example 3: Physical Data of Related Compounds

Some analogs formed during the photo- and thermal isomerization reactions are isolated to reveal the structure and then characterized. The ED-71 and proforms thereof obtained in Examples 1 and 2 are also characterized in detail. Note that the physical data reported below are for samples that were further purified by recrystallization of analogues and the like.

Melting point is not accurate. The IR spectrum is determined by JEOL JIR-6000 by the KBr tablet method. ¹ H-NMR and ¹³ C-NMR spectra are determined using JEOL JNM-270EX. TMS is used as internal standard for ¹ H-NMR, and the peak of CHCl ₃ is used as internal standard for ¹³ C-NMR. UV is determined using HITACHI U-3210 in ethanol at room temperature.

Physical data of the proform of ED-71 obtained in Example 1:

¹ H-NMR (ppm): 0.63 (3H, s), 0.96 (3H, d, J ^20-21 = 6.3 Hz), 1.07 (3H, s),

1.22 (6H, s), 3.6-4.0 (7H, m), 5.36-5.40 (lH, m), 5.70-5.73 (lH, m)

¹³ C-NMR (ppm): 141.1, 136.6, 120.8, 115.1, 82.2, 71.0, 70.9, 68.3, 66.7,

59.8, 55.7, 54.4, 44.1, 42.9, 41.3, 39.0, 38.3, 36.3, 36.0, 34.6,

32.0, 28.8, 28.7, 27.9, 22.9, 20.7, 20.5, 18.6, 15.8, 11.7

UV; λmax (ε): 294.2 nm (6550), 282.2 nm (11300),

271.9 nm (10500), 204.7 nm (2420)

IR (cm ^-1 ): 3385, 2941, 2872, 1471, 1468, 1381, 1379,

1327, 1138, 1082, 1080, 1053

Physical data of ED-71:

¹ H-NMR (ppm): 6.37 (lH, d; 11.4 Hz), 6.05 (lH, d; 11.4 Hz), 5.50 (lH, t; 2.l Hz),

5.08 (lH, t; 2.l Hz), 4.32 (lH, d; 8.9 Hz), 4.26 (lH, m), 3.88-

3.96 (lH, m), 3.85 (2H, t; 5.7 Hz), 3.69-3.77 (lH, m), 3.27 (lH, dd;

9.OHz, 2.8 Hz), 2.78-2.83 (lH, m), 2.55 (lH, dd; 10.6 Hz, 4.OHz),

2.42 (lH, bd; 13.6 Hz), 1.8-2.1 (5H, m), 1.22 (6H, s), 1.2-1.7 (11H,

m), 0.94 (3H, d; 6.3 Hz), 0.9-1.1 (lH, m), 0.55 (3H, s)

¹³ C-NMR (ppm): 144.2, 143.0, 132.2, 124.9, 117.2, 111.8, 85.4, 71.6, 71.1,

68.3, 66.6, 61.1, 56.6, 56.4, 45.9, 44.4, 40.5, 36.4, 36.1,

31.9, 29.3, 29.2, 29.1, 27.7, 23.7, 22.4, 20.8, 18.8, 11.9

UV; λmax: 265.4 nm (ε17900)

Melting point: 134.8-135.8 ° C (l ° C / min),

DSC: 137 ° C. (minimum peak, 115 mJ / mg),

TG / DTA: 138 ° C (minimum peak, loss on melting dry weight: about 1%, test sample 1.96 mg)

IR (cm ^-1 ): 3533, 3417, 3336, 2943, 2918, 2862, 1649, 1470, 1444, 1416, 1381,

1377, 1342, 1232, 1113, 1078, 1072, 1045, 999, 974, 957, 955, 924,

910, 895, 868, 833, 796, 764, 663, 634, 594, 472

Lumi form of ED-71 represented by Formula IV:

[Formula IV]

HPLC purity: 97.5% (220 nm)

¹ H-NMR (ppm): 5.75 (lH, d, J = 5.3 Hz), 5.42-5.44 (lH, m), 4.19 (lH, q, J = 2.9 Hz),

3.8-4.0 (4H, m), 3.6-3.7 (lH, m), 3.25 (lH, dd, J = 2.6 Hz, 9.6 Hz),

1.21 (6H, s), 0.90 (3H, d, J = 5.6 Hz), 0.82 (3H, s), 0.58 (3H, s)

¹³ C-NMR (ppm): 141.9, 136.2, 123.3, 115.5, 82.8, 77.9, 71.1, 67.4, 64.9, 61.1,

57.2, 49.5, 46.7, 44.4, 43.8, 41.4, 37.5, 36.2, 35.9, 32.0,

29.4, 29.2, 28.8, 22.6, 21.4, 20.9, 18.5, 18.3, 8.5

UV; λmax: 273.5 nm (ε9010)

IR (cm ^-1 ): 3437, 3383, 3309, 3041, 2960, 2935, 2872, 2787, 1657, 1641, 1470,

1441, 1375, 1257, 1205, 1203, 1167, 1128, 1097, 1074, 1039, 1011,

980, 935, 908, 885, 820, 781, 779, 723, 671, 613

Taki form of ED-71 represented by Formula III:

[Formula III]

HPLC purity: 97.6% (220 nm)

¹ H-NMR (ppm): 6.65 (lH, d, J = 16.lHz), 6.10 (lH, d, J = 16.lHz), 5.73 (lH, d, J = 2.8

Hz), 4.21-4.25 (2H, m), 3.70-3.90 (4H, m), 3.45 (lH, dd, J = 2.4 Hz, 6.OHz),

1.91 (3H, s), 1.22 (6H, s), 0.98 (3H, d, J = 6.5 Hz), 0.69 (3H, s)

¹³ C-NMR (ppm): 138.1, 130.9, 129.5, 127.8, 126.0, 124.5, 83.1, 72.4, 71.1,

68.5, 65.3, 61.1, 54.0, 50.0, 44.4, 42.8, 36.4, 36.0, 35.9,

31.9, 31.4, 29.4, 29.2, 28.7, 25.1, 24.3, 20.8, 18.7, 15.1, 11.2

UV; λmax: 281.4 nm (ε26100)

IR (cm ^-1 ): 3375, 2945, 2875, 1664, 1632, 1612, 1468, 1429, 1377, 1215, 1157,

1095,1068, 957, 908, 879, 764, 710, 646

Precursor form of ED-71 represented by the following formula:

HPLC purity: 97.2% (220 nm)

¹ H-NMR (ppm): 5.91, 5.78 (1H × 2, d, J = 12 Hz), 5.52 (1H, d, J = 3.3 Hz), 4.0-4.2

(2H, m), 3.7-4.0 (4H, m), 3.43 (lH, dd), 1.76 (3H, s), 1.22 (6H, s),

0.96 (3H, d, J = 6.6 Hz), 0.70 (3H, s)

UV; λmax: 206 nm (ε10300)

IR (cm ^-1 ): 3377, 2949, 2947, 2872, 1643, 1470, 1435, 1406, 1379, 1377, 1263,

1215, 1140, 1119, 1088, 1063, 1047, 1032, 1030, 962, 937, 935,

756, 735, 542

Example 4 X-ray Crystal Structure Analysis of ED-71

X-ray diffraction experiments of ED-71 were performed using crystalline powders selected from sample powders, some of which were also used in Example 3. As a result, the crystal is a rhombic system with a space group of P2 ₁ 2 ₁ 2 ₁ , with lattice constants a = 10.352 (2), b = 34.058 (2) and c = 8.231 (1) Was found to be 4. From the above experiments, 2520 reflection data were obtained.

The structural analysis is as follows. A direct method using SHELXS86 is used for the determinant and then each position of the non-hydrogen atoms is identified by Fourier mapping. For carbon-bonded hydrogen atoms, their respective positions are determined by calculating the position of the carbon atoms. For oxygen-bonded hydrogen atoms, their respective positions are determined by D mapping after determining each position of the other atoms.

After improving the accuracy of the temperature factor of the anisotropy for the non-hydrogen atoms by the position and non-square method of non-hydrogen and oxygen-bonded hydrogen atoms, the reliability factor (R value) of the analytical results converges to 3.9%. However, the absolute structure of the crystal is not determined directly from the result but rather by calculating the result on the condition that the coordination of positions 13, 14, 17 and 20 of ED-71 is equal to the corresponding coordination of cholesterol.

1 to 4 show the structure of ED-71 and the hydrogen bonds therein, respectively, which are determined based on the analysis results.

Reference Example 1 Stability of ED-71 Crystalline Compound

Amorphous crystalline ED-71 compounds are tested for stability at 10 ° C, 25 ° C and 40 ° C. To measure stability, HPLC quantitative assays and absorbance and purity measurements are used. Purity refers to the peak area ratio in percentage (% P.A.R.) in HPLC. The test is carried out using the following process:

(1) Method of stability test

Accurately weigh about 2 mg portions of each amorphous crystalline sample and place each in a clear 10 ml test tube with screw cap.

The test tubes are purged with argon using a vacuum desiccator and glove box. The purge process does not apply to test tubes designated as "1M (air)" in the table below.

The test tubes were placed in a group and fixed in a thermostatically controlled thermostat and left in a dark state. After 1 week (1W) or 2 weeks (2W) or 1 month (1M), the test tubes are removed from the thermostat to perform the following assays and measurements:

(2) HPLC quantitative assay

Sample solution is prepared by accurately adding 5 ml of anhydrous alcohol to the test tube. Both 1 ml of sample solution and 1 ml of internal standard solution are correctly added to another test tube to dilute the resulting mixture with methylene chloride to give a total volume of 20 ml. This diluted solution is referred to as solution 1. Internal standard solutions are formed by dissolving 2-aminopyrimidine in methanol at a concentration of 0.6 mg / ml.

Next, add another 1 ml of the standard solution and 1 ml of the internal standard solution to another test tube accurately for the quantitative assay formed by dissolving crystalline ED-71 in anhydrous ethanol at a concentration of 0.4 mg / ml, and the resulting mixture Is diluted with methylene chloride to give a total volume of 20 ml. This diluted solution is referred to as solution 2.

HPLC is performed using 20 μl of Solutions 1 and 2 respectively. For each solution, the area ratio of the peak of ED-71 to the peak of the internal standard is determined by HPLC data. The content of ED-71 in the sample is determined as the ratio of the area ratio of solution 1 to the area ratio of solution 2.

Survival (%) is determined by dividing the obtained ED-71 content by the ED-71 content in the same sample before the treatment.

HPLC usage conditions:

column; YMC A-004SIL (4.6 × 300 mm)

Mobile phase; Methylene chloride / methanol mixtures (95/5)

Flow rate; 1 ml / min

detection; UV 265 nm.

(3) absorbance measurement

1 ml of the sample solution prepared in the “HPLC quantitative assay” was diluted with anhydrous ethanol to give a total volume of 10 ml, and the absorbed solution was measured at 265 nm using an ultraviolet spectrophotometer. Absorbance is converted to an E 1% value derived from Lambert-Beer Law, which conversion is according to the following equation:

E 1% = A / cd

[Wherein A is absorbance, c is concentration (g / 100ml), and b is the length of light intensity (cm) passing through the test solution, usually 1].

(4) purity measurement

(4-1) Normal Phase HPLC

1 ml of the sample solution prepared in the “HPLC Quantitative Assay” is vacuum dried to remove the solvent (anhydrous ethanol). The residue is dissolved in 1 ml of methylene chloride. HPLC assays are performed using 25 μl of the solution.

HPLC usage conditions:

column; YMC A-004SIL (4.6 × 300 mm)

Mobile phase; Methylene chloride / methanol mixtures (96/4)

Flow rate; 1.8 ml / min

detection; UV 265 nm.

(4-2) Reversed Phase HPLC

HPLC is performed using 25 μl of sample solution prepared in “HPLC Quantitative Assay”.

HPLC usage conditions:

column; Inertsil ODS-2 (5 × 250 mm)

Mobile phase; Acetonitrile / water mixtures (55/45)

Flow rate; 1 ml / min

detection; UV 265 and 220 nm.

The values of HPLC quantitative assay and purity measurements are averaged over two runs. The results obtained are shown in Tables 1 to 5 below.

Results of HPLC Quantitative Assay (% Survival) 10 ℃ 25 ℃ 40 ℃ Amorphous crystalline form Amorphous crystalline form Amorphous crystalline form 01 W2 W1 M1 M (Air) 100 10098.5 96.894.8 97.097.4 95.0 100 10099.5 99.494.7 98.8 100 10094.4 99.588.8 102.9 Note: All samples are purged with argon except 1 M (air).

E 1% 10 ℃ 25 ℃ 40 ℃ Amorphous crystalline form Amorphous crystalline form Amorphous crystalline form 01 W2 W1 M1 M (Air) 327.5 350.2324.9 343.8320.5 342.7316.3 341.7 327.5 350.2323.0 349.5316.9 341.6 327.5 350.2313.2 349.8304.0 348.7 Note: All samples are purged with argon except 1 M (air).

Purity measurement using normal phase HPLC (% P.A.R at 265 nm) 10 ℃ 25 ℃ 40 ℃ Amorphous crystalline form Amorphous crystalline form Amorphous crystalline form 0ED-71 pioneer form different peak 96.06 99.151.67 0.212.27 0.63 96.06 99.151.67 0.212.27 0.63 96.06 99.151.67 0.212.27 0.63 1WED-71 precursor form different peak 95.97 99.561.88 0.142.14 0.30 94.47 99.552.53 0.143.00 0.31 2WED-71 precursor form different peak 95.46 99.062.27 0.662.28 0.27 94.74 98.952.25 0.673.01 0.38 92.12 98.922.99 0.674.89 0.41 1MED-71 precursor form different peak 94.58 98.912.33 0.653.09 0.44 1M (air) ED-71 precursor form different peak 95.26 98.882.31 0.662.42 0.46 Note: All samples are purged with argon except 1 M (air).

Purity measurement using reverse phase HPLC (% P.A.R at 265 nm) 10 ℃ 25 ℃ 40 ℃ Amorphous crystalline form Amorphous crystalline form Amorphous crystalline form 0ED-71 pioneer form different peak 95.38 99.041.16 0.363.47 0.60 95.38 99.041.16 0.363.47 0.60 95.38 99.041.16 0.363.47 0.60 1WED-71 precursor form different peak 94.78 99.351.27 0.283.95 0.38 92.47 99.401.98 0.295.55 0.31 2WED-71 precursor form different peak 94.48 98.841.75 0.833.77 0.32 94.20 98.781.72 0.854.08 0.37 90.68 98.752.53 0.856.78 0.40 1MED-71 precursor form different peak 93.05 98.771.88 0.865.07 0.37 1M (air) ED-71 precursor form different peak 94.11 98.721.82 0.864.07 0.42 Note: All samples are purged with argon except 1 M (air).

220 nm (%) 10 ℃ 25 ℃ 40 ℃ Amorphous crystalline form Amorphous crystalline form Amorphous crystalline form 0ED-71 pioneer form different peak 93.95 97.441.35 0.524.70 2.04 93.95 97.441.35 0.524.70 2.04 93.95 97.441.35 0.524.70 2.04 1WED-71 precursor form different peak 93.37 98.171.50 0.445.13 1.40 90.00 98.182.29 0.477.71 1.35 2WED-71 precursor form different peak 92.85 97.912.02 1.015.13 1.09 92.39 97.561.93 1.015.68 1.43 87.40 97.652.88 1.029.72 1.33 1MED-71 precursor form different peak 92.85 97.442.02 1.055.13 1.51 1M (air) ED-71 precursor form different peak 92.07 97.092.04 1.095.88 1.82 Note: All samples are purged with argon except 1 M (air).

As can be seen from the table, the crystalline form was more stable for at least 2 weeks at 25 ° C and 40 ° C than amorphous.

Since the vitamin D derivative crystal of the present invention has improved purity, stability and continuous properties than the vitamin D derivative, it is useful for preparing a pharmaceutical containing a vitamin D derivative. Moreover, the method for purifying vitamin D derivatives of the present invention makes it possible to supply high purity vitamin D derivatives in large quantities (in grams) and continuously.

Moreover, the tachy and rumi forms, analogs of ED-71, and the proforms of ED-71 are novel compounds, respectively, and are useful for tests or assays that can be performed in the synthesis of vitamin D derivatives.

Claims (31)

Compound determination represented by formula (I): [Formula I] Vitamin D derivative crystals obtained by purifying crude or prepurified product of vitamin D derivatives using reverse phase chromatography and then crystallizing the purified derivative from an organic solvent. The crystal of claim 2, wherein the organic solvent is an aprotic organic solvent. 4. The crystal of claim 3 wherein the aprotic organic solvent is ethyl acetate, acetone, acetonitrile, or a mixture thereof. The crystal according to claim 2, wherein the vitamin D derivative is a compound represented by the formula: [Formula I] The crystal of claim 5, wherein the organic solvent is an aprotic organic solvent. The crystal according to claim 6, wherein the aprotic organic solvent is ethyl acetate, acetone, acetonitrile or a mixture thereof. Purification method of the vitamin D derivative, characterized in that the reverse phase chromatography of the vitamin D derivative. The method of claim 8, wherein the vitamin D derivative is a compound represented by Formula I: [Formula I] A method for purifying a vitamin D derivative, characterized in that the vitamin D derivative is crystallized from an organic solvent. The method of claim 10, wherein the organic solvent is an aprotic organic solvent. 12. The process of claim 11 wherein the aprotic organic solvent is ethyl acetate, acetone, acetonitrile, or mixtures thereof. The method of claim 10 wherein the vitamin D derivative is a compound represented by formula (I): [Formula I] The method of claim 13, wherein the organic solvent is an aprotic organic solvent. 15. The method of claim 14, wherein the aprotic organic solvent is ethyl acetate, acetone, acetonitrile, or a mixture thereof. A process for purifying a vitamin D derivative, characterized in that the crude product or the preliminary purified product of the vitamin D derivative is purified via reverse phase chromatography and then the purified derivative is crystallized from an organic solvent. The method of claim 16, wherein the organic solvent is an aprotic organic solvent. 18. The process of claim 17, wherein the aprotic organic solvent is ethyl acetate, acetone, acetonitrile, or a mixture thereof. The method of claim 16, wherein the vitamin D derivative is a compound represented by Formula I: [Formula I] The method of claim 19, wherein the organic solvent is an aprotic organic solvent. The method of claim 20, wherein the aprotic organic solvent is ethyl acetate, acetone, acetonitrile, or a mixture thereof. A process for purifying a compound represented by formula (II) characterized in that the crude or prepurified product of the compound represented by formula (II) is recrystallized from alcohol: [Formula II] The method of claim 22, wherein the alcohol is methanol. Purified product of compound represented by formula II obtained by recrystallization of a crude or preliminary product of a compound represented by formula II from an alcohol: [Formula II] The method of claim 24, wherein the alcohol is methanol. A process for preparing a purified product of a vitamin D derivative represented by formula I, consisting of: [Formula I] Recrystallising the crude or prepurified product of the compound represented by Formula II from alcohol; [Formula II] Performing ultraviolet irradiation and thermal isomerization reaction on the recrystallized compound of formula II to obtain a vitamin D derivative represented by formula I; Purifying initial or pre-purified vitamin D derivatives of formula (I) via reverse phase chromatography; And The vitamin D derivatives of formula I are crystallized from organic solvents. 27. The method of claim 26, wherein the organic solvent is an aprotic organic solvent. 28. The method of claim 27, wherein the aprotic organic solvent is ethyl acetate, acetone, acetonitrile, or a mixture thereof. 30. The method of any of claims 26-29, wherein the alcohol is methanol. Compound represented by formula III: [Formula III] Compound represented by formula IV: [Formula IV]