Estradiol crystallization

This controlled-release subdermal implant is fabricated by dispersing micronized estradiol crystals in a viscous mixture of silicone elastomer and catalyst and then coating the estradiol-polymer dispersion around a rigid (drug-free) silicone rod by an extrusion technique to form a cylinder-shaped implant (Fig. 11). This implant is designed for subcutaneous implantation in the steer s ear flap for a duration of 200 or 400 days, during which a controlled quantity of estradiol is released daily for growth promotion. 

Fig. 8.3 P-estradiol crystallized from isopropyl alcohol. Polarized light microscopy.

The general reaction procedure and apparatus used are exactly as described in Procedure 2. Ammonia (465 ml) is distilled into a 2-liter reaction flask and to this is added 165mlofisopropylalcoholandasolutionof30g(0.195 mole) of 17/ -estradiol 3-methyl ether (mp 118.5-120°) in 180 ml of tetrahydrofuran. The steroid is only partially soluble in the mixture. A 5 g portion of sodium (26 g, 1.13 g-atoms total) is added to the stirred mixture and the solid dissolves in the light blue solution within several min. As additional metal is added, the mixture becomes dark blue and a solid (matted needles) separates. Stirring is inefficient for a few minutes until the mass of crystals breaks down. All of the sodium is consumed after 1 hr and 120 ml of methanol is then added to the mixture with care. The product is isolated as in Procedure 4h 2. After being air-dried, the solid weighs 32.5 g (ca. 100% for a monohydrate). A sample of the material is dried for analysis and analyzed as described in Procedure 2 enol ether, 91% unreduced aromatics, 0.3%. The crude product may be crystallized from acetone-water or preferably from hexane. 

Members of the different SERM families bind to the ligand-binding domain (LBD) of the ER, whose particular crystal structure has been revealed for estradiol and for raloxifene (Brzozowski et al. 1997). Once inserted into the binding cavity, estradiol makes direct hydrogen bonds bewteen its A-ring and... 

The implant consists of a water-swellable Hydron (cross-linked ethylene glycomethacrylate) polymer matrix in which estradiol valerate (Norgestomet) crystals are dispersed. It is used for the synchronization of estrus/ovulation in cycling heifers. Once implanted in the animal s ear, the implant delivers estradiol valerate at the rate of 504 ug cm-2 day-172 over a period of 16 days. 

Estradiol is the natural agonist ligand of the estrogen receptors (ER). There are two isotypes of the estrogen receptor, ER-a and ER-y . There are four structures of the complex between the LED of ER-a and estradiol in the PBD. Expression, purification and crystallization of LEDs can often be problematic. Thus, the first structure solved (PDE entry lERE) [6] used protein in which the free cysteines were carboxymethylated. The refined structure showed at least one of the cysteines was modified. Another structure (PDE entry 1A52) [7] used protein that was refolded. The structure of this protein showed an artifact where two LEDs were connected by an intermolecular disulfide bond. Through much experimentation, conditions were found to express, purify and crystallize ER-a LED without modification or refolding. The protein structure was deposited as PDB entry IQKU [8] and it is this protein structure that will be discussed. 

There also exist a class of ligands referred to as SERMs (selective estrogen receptor modulators) that display tissue-selective pharmacology [13]. Raloxifene and tamoxifen are two clinically used SERMs for which structures are available. Crystal structures of the estrogen receptor bound to different ligands (estradiol, tamoxifen, or raloxifene) reveal that ligands of different sizes and shapes induce a spectrum of receptor conformational states. These states can be interpreted by the cellular complexion of co-regulators and the environment of the local promoter of... 

Figure 7.1. Comparison oftheX-ray structures ofthe estrogen receptor bound to estradiol (dark-gray) and diethylstilbestrol (gray). The two crystal structures were overlaid using the backbone atoms. Only the amino acid residues in proximity to the binding pocket are shown for clarity.

In addition to these SBD methods the ERa crystal structure has recently been used in evaluating linear interaction energy (LIE) methodology for lead optimization [125], and in calculating ab initio molecular interactions between ERa and 17p-estradiol [126],... 

J -3-Keto isomerase catalyzes the isomerization of J -3-ketosteroids to zl -3-ketosteroids by stereospecific transfer of a hydrogen atom from C(4) to C(6). There is considerable evidence that it is the 40- and 6/5-hydrogens that are involved and that the reaction proceeds via an enolic intermediate. A low resolution (6 A) crystal structure determination has been published and the probable steroid-binding site identified via a bound inhibitor, 4-acetoxy-mercuric estradiol. The results of a higher resolution study (2.5 A) combined with the results of NMR studies and analysis of activity of mutant forms of the enzyme have helped to further define the probable active site of the enzyme [64]. 

Acetate C H CIO, SH 714 CPA, Androcur, Cyprostat. Crystals from diisopropyl ether mp 200-201°. uv max (methanol) 281 nm (e 17280). Mixture with ethinyl estradiol Dianette, Diane 35 ... 

Crystals, mp 109-110. [ Jo + 55° (chloroform). Mixture with ethinyl estradiol, q.v.. Cyclosa, Dicromil, Marveton 150/30, Mercilon, Oviol, Vartoline. 

Crystals, mp 102. [a]+10" (c = 1 in chloroform), uv max (isopropanol) 240 nm (Ej, 450). Practically insol in water. Sol in ethanol. Slightly sol in acetone. chloroform. Mixture with ethinyl estradiol, g.v., Oracon, Ovin, Tova. therap CAT Progestogen. 

Heptanoate, C2SH34Oy estradiol enauthate, SQ 16150. Crystals from diisopropyl ether, inp 94-96. See Gauthier et al. Ann. Phartn. Franc. 16, 757 (1958). 

Crystals from acetone-hexane, mp 197 9". Mixture with ethinyl estradiol, Femodene, Femovati, Ginoden, Gynera, Minulet, Monedte, Phaeva. 

Crystals from methanol, mp 214-216. Soly at 37 in water (2 g/ml) in plasma (24 eg/ml), fojjj +5" (chloroform). nv max (ethanol) 287 nm (log 4.40), Mixture with ethinyl estradiol, q.v., Co-Ervonum, Kombiquens, Noval, Nuvacon, Ovex, Planovtn, Tri-Ervonum, Volidan, Weradys. Mixture with mestranol, q.r Delpregnin. 

Acetate, CjjHjgOj, Aygestin, Norlutate, Norlutin-A, Pri-malut-Nor. Crystals from methylene chloride + hexane, mp 161-162. uv max 240 nm (e 18690). Mixture with ethinyl estradiol, Etalondn, Primosiston, Anovlar 21, Gynovlar, Locstrin. Minovlar. Norlestrin. 

Brzozowski AM, Pike ACW, Dauter Z et al. The crystallization of the ligand-binding domain (LBD) of the estrogen receptor (ER) with 17p-estradiol and raloxifene. Nature 1997 389 753-758. 

The sublimation temperature and the distance of the collecting surface from the material undergoing sublimation have a great influence on the form and size of the crystals produced. The occurrence of polymorphic modifications depends on the temperature of sublimation. In general, it may be assumed that unstable crystals form preferentially at lower temperatures, while at higher temperatures stable forms are to be expected. Nevertheless, mixtures consisting of several modifications are frequently found together. This is the case for barbital and for estradiol benzoate. It should be obvious that the sublimation technique is applicable only to those compounds that are thermally stable. 

---