AU2012268858B2 - Crystallization of 1a-hydroxy-2-methylene-18,19-dinor-homopregnacalciferol - Google Patents

Abstract

A method of purifying 1a-hydroxy-2-methylene-18,19-dinor-homopregnacalciferol to obtain lIa ydroxy-2-methylene-1819-dinor-homopregnacalciferol in crystalline form using precipitation with exane from ethyl acetate. A method of preparing la-hydroxy-2-methylene-18,19-dinor omopregnacalciferol crystals acceptable for X-ray experiment using precipitation with hexane from enzene by diffusive exchange of the solvents is also described.

Description

CRYSTALLIZATION OF la-HYDROXY-2-MEIHYLENE-18,19-DINOR-FIOMOPREGNACALCIFEROL

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under DK047814 awarded by the National Institutes of Flealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to purification of organic compounds, and more particularly to the purification of la-hydroxy-2-methylene-18,19-dinor-homopregnacalciferol (referred to herein as "051810") by preparing it in crystalline form.

[0003] Purification of organic compounds, especially those designated for pharmaceutical use, is of considerable importance for chemists synthesizing such compounds. Preparation of the compound usually requires many synthetic steps and, therefore, the final product can be contaminated not only with side-products derived from the last synthetic step of the procedure but also with compounds that were formed in previous steps. Even chromatographic purification, which is a very efficient but relatively time-consuming process, does not usually provide compounds which are sufficiently pure to be used as drugs, [0004] Depending on the method used to synthesize ία-hydroxy vitamin D compounds, different minor undesirable compounds can accompany the final product. Thus, for example, if direct C-l hydroxylation of 5,6-trans geometric isomer of vitamin D is performed, followed by SeC^/NMO oxidation and photochemical irradiation [see Andrews et ah, J. Org. Chem, 51, 1635 (1986); Calverley et al., Tetrahedron 43, 4609 (1987); Choudry et al,,/. Org. Chem. 58, 1496 (1993)], the final Ια-hydroxyvitamin D product can be contaminated with 1β-hydroxy- as well as 5,6-trans isomers. If the method consists of C-l allylic oxidation of the 4-phenyl-l,2,4-triazoline-3,5-dione adduct of the previtamin D compound, followed by cycioreversion of the modified adduct under basic conditions [Nevinckx el al,, Tetrahedron 47, 9419 (199F); Vanmaele et al. Tetrahedron 41, 541 (1985) and 40, 1179 (1994); Vanmaele et al., Tetrahedron Lett. 23. 995 (1982)], one can expect that the desired Ια-hydroxyvitamin can be contaminated with the previtamin 5(10), 6,8-triene and Ιβ-hydroxy isomer. One of the most useful C-l hydroxylation methods, of very broad scope and numerous applications, is the experimentally simple procedure elaborated by Paaren et al. [see J. Org. Chem. 45, 3253 (1980) and Proc. Natl Acad, Sci U.S.A. 75, 2080 (1978)]. This method consists of allylic oxidation of 3,5-cyclovitamin D derivatives, readily obtained from the buffered solvolysis of vitamin D tosylates, with SetT/t-BuOOH and subsequent acid-catalyzed cycloreversion to the desired la-hydroxy compounds. Taking into account this synthetic path it is reasonable to assume that the final product can be contaminated with ία-hydroxy epimer, 5,6-trans isomer and the previtamin D form. 1 α-hydroxyvitamin D4 is another undesirable contaminant found in Ια-hydroxyvitamin D compounds synthesized from vitamin D2 or from ergosterol. la-hydroxyvitamin D4 results from C-l oxidation of vitamin D4, which in turn is derived from contamination of the commercial ergosterol material. Typically, the final product may contain up to about 1.5% by-weight Ια-hydroxyvitamin D4. Thus, a purification technique that would eliminate or substantially reduce the amount of Ια-hydroxyvitamin D4 in the final product to less than about 0.1-0.2% would be highly desirable.

[0005] The vitamin D conjugated triene system is not only heat- and light- sensitive but it is also prone to oxidation, leading to the complex mixture of very polar compounds. Oxidation usually happens when a vitamin D compound has been stored for a prolonged time. Other types of processes that can lead to a partial decomposition of vitamin D compounds consist of some water-elimination reactions; their driving force is allylic (la-) and homoallylic (3β-) position of the hydroxy groups. The presence of such above-mentioned oxidation and elimination products can be easily detected by thin-layer chromatography.

[0006j Usually, al! Ια-hydroxylation procedures require at least one chromatographic purification. However, even chromatographically purified la-hydroxyvitamin D compounds, although showing consistent spectroscopic data, suggesting homogeneity, do not meet the purity criteria required for therapeutic agents that can be orally, parenterally or transdennally administered. Therefore, it was evident that a suitable method of purification of the 1 α-hydroxylated vitamin D compound 051810 is required.

[0006A] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

[0006B] Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment, or any form of suggestion, that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

SUMMARY OF THE INVENTION

[0007] The presen t invention relates to a method of purifying 051810 by means of crystallization to obtain 051810 in crystalline form. The solvent plays a crucial role in the crystallization process, and is typically an individual liquid substance or a suitable mixture of different liquids. For crystallizing 051810, the most appropriate solvent and/or solvent system is characterized by the following factors: (1) low toxicity; (2) low boiling point; (3) significant dependence of solubility properties with regard to temperature (condition necessary for providing satisfactory crystallization yield); and (4) relatively low cost.

[0008] Interestingly, hexane, so frequently used for crystallization purposes, was found less suitable as the sole solvent for crystallization of 051810. However, it was found that a mixture of ethyl acetate and hexane, was most useful for the crystallization of 051810. In particular, it was determined that a mixture of about 1% ethyl acetate with about 99% hexane (by volume) performed well. The ethyl acetate/hexane solvent mixture was also easy to remove by evaporation or other well known methods. In all cases the crystallization process occurred easily and efficiently; and the precipitated crystals were sufficiently large to assure their recovery by filtration or other means.

[0009] Accordingly, there is obtained la-hydroxy-2-methylene-l8,l9-dinor- homopregnacalciferol in crystalline form. The crystalline form and three dimensional structure of la-hydroxy-2-methylene-18,19-di nor-homo pregnacalciferol has a molecular packing arrangement defined by space group P2 and unit cell dimensions a=4.8A, b=22.9A, e=36.lA, α=90°, β=90° and γ=90°.

[00010] In one embodiment, there is described a method of purifying la-hydroxy-2-methylene-18,19-dmor-homopregnacalciferol, comprising the steps of: (a) dissolving a product containing 1 α-h yd roxy-2-methylene-! 8,19-dinor-homopregnacalciferol to be purified in a solvent comprising ethyl acetate; (b) adding hexane to said solvent and dissolved product to form a mixture; (c) cooling said mixture containing said dissolved product below ambient temperature for a sufficient amount of time to form a precipitate of 1 a-hydroxy-2-methyIene-18,19-dinor-homopregnacalcifero! crystals; and (d) separating the la-hydroxy-2-methylene-l 8,19-dinor-homopregnacalciferol crystals from the mixture.

[00011] In another embodiment, there is described a method of preparing la-hydroxy-2-methylene-18,19-dinor-homopregnacalciferol crystals by diffusive exchange of solvents, comprising the steps of: (a) dissolving a product containing la-hydroxy-2-methylene-18,19-dinor-homopregnacalciferol in a first solvent comprising benzene; (b) providing a second solvent comprising hexane; (c) allowing said first solvent with dissolved product and said second solvent to diffuse together for a sufficient amount of time to form a precipitate of 1 a-hydroxy-2-methylene-18,19-dinor-homopregnacalciferol crystals; and (d) recovering the 1 a-hydroxy-2-methyelene-18,19-dinor-homopregnacalciferof crystals.

BRIEF DESCRIPTION OF THE DRAWINGS

[00012| FIG. 1 is an illustration of the three dimensional structure of the first crystallographic asymmetric molecule for 051810 as defined by the atomic positional parameters discovered and set forth herein; [00013] FIG. 2a is an illustration of the three dimensional structure of the second crystallographic asymmetric molecule for 051810 in the absence of the benzene molecule and as defined by the atomic positional parameters discovered and set forth herein; [00014] FIG. 2b is an illustration of the three dimensional structure of the second asymmetric molecule for 051810 in the presence of the benzene molecule and as defined by the atomic positional parameters discovered and set forth herein; and [00015} FIG. 3 is an illustration of a ball-and-stick representation of the entire asymmetric unit for 051810. DETAILED DESCRIPTION OF THE INVENTION [00016} The present invention provides la-hydroxy-2-methy!ene-18,19-dinor-homopregnacalciferol (051810) in crystalline form, a pharmacologically important compound, characterized by the formula I shown below:

[00017] The present invention also provides a valuable method of purification of 051810. The purification technique involves obtaining the 05! 810 product in crystalline form by utilizing a crystallization procedure wherein the 051810 material to be purified is dissolved using ethyl acetate as the solvent and further precipitation with hexane. Preferably a ratio of ethyl acetate and hexane is about 1:99 (by volume). Thereafter, the solvent can be removed by evaporation, with or without vacuum, or other means as is well known, or the resultant crystals may be filtered from the mother liquor. The technique can be used to purify a wide range of final products containing 051810 obtained from any known synthesis thereof, and in varying concentrations, i.e. from microgram amounts to kilogram amounts. As is well known to those skilled in this ait, the amount of solvent utilized should be minimized and/or adjusted according to the amount of 051810 to be purified. {00018] The usefulness and advantages of the present crystallization procedure is shown in the following specific Example 1. After crystallization, the precipitated material was observed under a microscope to conitrm its crystalline form. Yields of crystals were relatively high and the obtained crystals showed a relatively sharp melting point of 140-145°C.

[00019] The described crystallization process of the synthetic 051810 product represents a valuable purification method, which can remove most side products derived from the synthetic path. Such impurity is the result of the contamination of starting raw materials. The crystallization process occurred easily and efficiently; and the precipitated crystals were sufficiently large to assure their recovery by filtration, or other means. {00020] Crystallization of 1a-hydroxy-2-methylene-18,19-(linor-homopreg- nacalciferol (051810). EXAMPLE 1

Crystallization from ethyl acetate /hexane [00021] 1.25 g of prepurified (see U.S. Pat. No. 7,238,681) crude la-hydroxy-2-methylene-18,19-dinor-homopregnacalciferol was dissolved in ethyl acetate (3 ml) at room temperature and hexane (300 ml) was poured into the vigorously shaken solution. The mixture was kept in a refrigerator (at 4°C) overnight and the resulted crystals were filtered off, washed with one portion (30 ml) of cooled (4°C) hexane and dried under reduced pressure for 3h giving 1.10 g of a pure product.

[00022] In order to obtain crystals acceptable for the X-ray experiment, la-Hydroxy-2-methylene-18,19~dinor-homopregnacalciferol (12 mg) was placed in an inner tube of a vessel and dissolved in 300 μ! of benzene. To an outer tube of the vessel hexane (2 ml) was poured so that a benzene to hexane ratio of about 13:87, by volume, is obtained, and the whole system was carefully purged with argon, and then maintained as a closed system. The vessel was kept tightly closed for 4 days at room temperature. Crystals were grown employing diffusive exchange of the two solvents.

[00023] A colorless rod-shaped crystal of dimensions 0.73 x 0.08 x 0.02 mm was selected for structural analysis, intensity data were collected using a Bruker AXS Platinum 135 CCD detector controlled with the PROTEEJM software suite (Bruker AXS Inc., Madison. WI).

The x-ray source was CuKa radiation (1.54178 A) from a Rigaku RU200 x-ray generator equipped with Montel optics, operated at 50 kV and 90 mA. The x-ray data were processed with SAINT version 7.06A (Bruker AXS Inc.) and internally scaled with SADABS version 2005/1 (Bruker AXS Inc.). The sample was mounted on a glass fiber using vacuum grease and cooled to 100 K. The intensity data were measured as a series of phi and omega oscillation frames each of 1° for 60-120 sec/frame. The detector was operated in 1024 x 1024 mode and was positioned 5.0 cm from the sample. Cell parameters were determined from a non-iinear least squares lit of 9999 peaks in the range of 3.0 < theta < 50.8°. The data were merged to form a set of 4693 independent data with R(int)=0.0884, [00024J The orthorhombic space group P2( 1)2( 1)2(1) was determined by systematic absences and statistical tests and verified by subsequent refinement. The structure was solved by direct methods and refined by full-matrix least-squares methods on F , (a) G.M. Sheldrick (1994), SHELXTL Version 5 Reference Manual, Bruker AXS Ine.; (b) International Tables for Crystallography. Vol. C, Kluwer; Boston (1995). The asymmetric unit is comprised of two molecules of 051810 and a benzene molecule. Molecule “A” is shown in Figure 1, with thermal ellipsoids drawn at the 40% probability level. Based on lattice packing, the benzene molecule could not be present at full occupancy, and refinement of its occupancy led to a value of approximately 0.5. The presence of the benzene molecule lead to a disorder in a portion of molecule “B” Refinement of the occupancies of the two disordered configurations independently of the occupancy of the benzene converged also at a value of about 0.5 for each. Figure 2a shows molecule B in its configuration in the absence of the benzene molecule, and Figure 2b shows its configuration in the presence of the benzene molecule; both figures are drawn with thermal ellipsoids at the 40% probability level. Figure 3 shows a ball-and-stick representation of the entire asymmetric unit, with the hollow bonds showing the conformation in the presence of the benzene molecule. Hydrogen atom positions were refined by a riding model with idealized geometry. Non-hydrogen atoms were refined with anisotropic displacement parameters. The benzene molecule was refined with idealized geometry. A total of 559 parameters were refined against 26 restraints and 4693 data to give wR2 = 0.2524 and S = 0.951 for weights of w = 1/[$2(FZ) + (0.1719P)2], where P = [F,2 + 2Fe2]/3. The final R(F) was 0.0910 for the 6781 observed data. The largest shift/s.u. was 0.001 in the final refinement cycle and the final difference map had maxima and minima of 0.366 and -0.305 e/AJ, respectively. The absolute structure was determined by refinement of the Flack parameter, H.D. Flack, Acta Cryst. A, vol. 39,876-881 (1983).

[00025] The three dimensional structure of 051810 as defined by the following physical data and atomic positional parameters described and calculated herein is illustrated in Figures 1,2a, 2b and 3.

Table 1. Crystal data and structure refinement for 051810.

Identification Code 051810

Empirical formula C45 H67 04

Formula weight 671,99

Temperature 100(1) K

Wavelength 1.54178 A

Crystal system Orthorhombic

Space group P2(1)2(1)2(1)

Unit cell dimensions a = 4.8020(10)A a = 90° b = 22.94 0(5) A β = 90° c = 36.124(7)A γ = 90°

Volume 3979.3(14)A3 Z 4

Density (calculated) 1.122 Mg/m3

Absorption coefficient 0.533 mm" F (000) 1476

Crystal 0.73 x 0.08 x 0.02 nun

Theta range for data collection 2.28 to 54.17°

Limiting indices -4<h<4,-24<k<23,-37<1< 37

Reflections collected 12592

Independent reflections 4693 [R(int) = 0.0884]

Completeness to Theta = 54.17° 99.2 %

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 4693 / 26 / 559 Goodness-of-fit on F2 0,951

Final R indices [Ι>2σ(Ι)] R1 = 0.0910, wR2 = 0.2214 R indices (all data) R1 = 0.1268, wR2 = 0.2524

Extinction coefficient 0.0124(12)

Largest diff. peak and hole 0.366 and -0,305 e/A3

Melting Point 140-145“C

Table 2. Atomic coordinates (x 101) and equivalent isotropic displacement parameters (A2 x 103] for 051810. U(eq) is defined as one third of the trace of the orthogonalized Ui;j tensor.

Table 3. Bond lengths [A] for 051810.

Table 4. Bond angles [°] for 051810.

Table 5. Anisotropic displacement parameters [A2 x 10J] for 051810. The anisotropic displacement factor exponent takes the form: -2π2 [h2a*2U11 +...+ 2hka*b*U12]

Table 6. Hydrogen coordinates (x 104) and isotropic displacement parameters (A"1 x 103) for 051810.

EXAMPLE 2 Synthesis of 051810 [00026] The preparation of 051810 having the basic structure 1 can be accomplished by a ommon general method, i.e. the condensation of a bicyciic Windaus-Grundmann type ketone II with he allylic phosphine oxide III to the corresponding 2-methylene-19-nor-vitamm D analog IV followed iy deprotection at C-l and C-3 in the latter compound IV to obtain compound L i.e, 051810.

[00027] In phosphine oxide ill, Y| and Y? are preferably hydroxv-proiecting groups such is silyl protecting groups. The t-butyldimethylsilyl (TMDMS) group is an example of a particularly iseful hydroxy-protecting group. The process described above represents an application of the convergent synthesis concept, which has been applied effectively to the preparation of numerous vitamin 9 compounds (see Lythgoe et al.,./ Chem. Sue. Perkin Trans, L 590 (1978); Lythgoe. Chem. Sac. Rev. / 449 (1983); Toh et al, ,/. Org. Chem. 48, 1414 (1983); Baggio 1 ini et al, J. Org. Chem. 51, 3098 1986); Sardina et al,./ Org. Chem, 51, 1264 (1986);,/ Org. Chem, 51, 1269 (1986); DeLuca et al., J.S. Pat, No. 5,086,191; DeLuca et al, U.S. Pal. No. 5,536,713; and DeLuca et al, U.S. Pat. No. 1843,928 all of which are hereby incorporated by reference in their entirety and for all purposes as if fully set forth herein.

[0(1028] Phosphine oxide II! is a convenient reagent that can he used to prepare a large number of 19-nor-vitamin D compounds and is prepared according to the procedures described by Sicinski et al, J. Med. Chem., 41, 4662 (1998), DeLuca et al. U.S. Pat. No. 5,843,928; Perlman ct al.. Tetrahedron Lett. 32, 7663 (1991); and DeLuca et al, U.S. Pal. No. 5,086,191 which are hereby incorporated by reference in their entirety as if fully set forth herein.

[00029) The overall process of the synthesis of compound I is illustrated and described more completely in U.S. Pat. No. 5,843,928 entitled "2-Alkylidene-19-Nor-Vitamin D Compounds" and in U.S. Pat. No. 7,238,681, entitled "2-Methylene-18,19-Dinor-ta-Hydroxy-Homopregnacalciferol and Its Uses" the specifications of which are specifically incorporated herein by reference.

Claims (13)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: We claim; 1. 1 a-hydroxy-2-methylene-18,19-dinor-homopregnacalciferol in crystalline form.
2. 2. A crystalline form of la-hydroxy-2-methylene-18,19-dinor-homopregnacalciferol having molecular packing arrangement defined by space group P2 and unit cell dimensions a=4.8A, b=22.9A, c=36.lA, .oi—900, β=90° and γ=90°.
3. 3. A three dimensional structure for the compound la-hydroxy-2-methylene-18,19-dinor-homopregnacalciferol as defined by the molecular packing arrangement set forth in claim 2.
4. 4. A method of purifying la-hydroxy-2-methylene-l8,19-dinor-homopregnacalciferol, comprising the steps of: (a) dissolving a product containing la-hydroxy-2-methylene-18,19-dinor-homopregnacaleiferol to be purified in a solvent comprising ethyl acetate; (b) adding hexane to said solvent and dissolved product to form a mixture; (c) cooling said mixture containing said dissolved product below ambient temperature for a sufficient amount of time to form a precipitate of 1 u-hydroxy-2-methylene-l8,19-dinor-homopregnacalciferol crystals; and (d) separating the la-hydroxy-2-methylene-18,19-dinor-hornopregnaealciferol crystals from the mixture.
5. 5. The method of claim 4 wherein the step of separating comprises filtering the mixture and precipitate to obtain the crystals.
6. 6. The method of claim 4 or claim 5 including a further step (d) comprising repeating steps (a) through (c) using the recovered crystals from step (c) as the product of step (a).
7. 7. The method of any one of claims 4 to 6 wherein a ratio of ethyl acetate and hexane is about 1:99, by volume.
8. 8. A method of preparing la-hydroxy-2-methylene-18,19-dinor-homopregnacalciferol crystals by diffusive exchange of solvents, comprising the steps of: (a) dissolving a product containing la-hydroxy-2-methylene-18,19-dinor-homopregnacalciferol in a first solvent comprising benzene; (b) providing a second solvent comprising hexane; (c) allowing said first solvent with dissolved product and said second solvent to diffuse together for a sufficient amount of time to form a precipitate of 1 a-hydroxy-2-methylene-18,19-dinor-homopregnacalciferol crystals; and (d) recovering the 1 a-hydroxy-2-methyelene-18,19-dinor-homopregnacalciferol crystals.
9. 9. The method of claim 8 wherein a ratio of benzene and hexane is about 13:87, by volume.
10. 10. The method of claim 8 or claim 9 wherein the step of recovering comprises filtering to obtain the crystals.
11. 11. The method of any one of claims 8 to 10 wherein the step of allowing said first solvent with dissolved product and said second solvent to diffuse together takes place in a closed system purged with argon.
12. 12. The method of any one of claims 8 to 11 wherein the step of allowing said first solvent with dissolved product and said second solvent to diffuse together takes place at room temperature.
13. 13. la-hydro xy-2-methyelene-18,19-dinor-homopregnacalciferol, when prepared by the method of any one of claims 4 to 12,