Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/02—Nutrients, e.g. vitamins, minerals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates generally to the vitamin D endocrine 15 system. More particularly, the present invention relates to a method for controlling genomic and nongenomic cellular responses which are mediated by 1 ⁇ ,25-(OH) 2 vitamin D 3 [1 ⁇ ,25-(OH) 2 D 3 ].
• Vitamin D 3 is a secosteroid which is responsible for a wide variety of biological responses in higher animals. These biological responses include maintenance of calcium homeostasis, immunomodulation and selected cell differentiation. Vitamin D 3 , itself, is biologically inert. However, metabolism of vitamin D 3 to metabolites such as 1 ⁇ ,25-(OH) 2 D 3 results in the formation
• 1 ⁇ ,25-(OH) 2 D 3 has been shown to be present in 30 different tissues and it belongs to the same super family of proteins which includes receptors for the steroid hormones, retinoic acid and thyroxine (1 ,4,5).
• genomic responses it has become apparent that a subset of biological responses are mediated by 1 ⁇ ,25-(OH) 2 D 3 via a nongenomic mechanism (3,6).
• These biological responses include the rapid stimulation of intestinal Ca 2+ transport known as transcaltachia (7-9).
• Transcaltachia involves the opening of Ca 2+ channels (10).
• nongenomic cellular responses which are mediated by 1 ⁇ ,25-(OH) 2 D 3 include opening of voltage- gated Ca 2+ channels in rat osteosarcoma cells (11 ,12) as well as other rapid effects in kidney (13), liver (14), parathyroid cells (15) and intestine (16).
• analogues of 1 ⁇ ,25-(OH) 2 D 3 are effective in controlling nongenomic cellular responses which are mediated by 1cr,25-(OH) 2 D 3 .
• specific analogues are selected which function as either antagonists or agonists of the nongenomic cellular response.
• the cellular responses mediated by 1 ⁇ ,25-(OH) 2 D 3 can be controlled.
• Transcaltachia is a particular nongenomic cellular response which can be controlled in accordance with the present invention.
• Vitamin D analogues which can be used as agonists of transcaltachia include 1 ⁇ ,25- (OH) 2 -previtamin D 3 .
• 1 ?,25- (OH) 2 vitamin D 3 [1 ?,25-(OH) 2 D 3 ] is used as an antagonist.
• control procedures provided in accordance with the present invention are effective in limiting or increasing transcaltachia and other nongenomic responses of 1 ⁇ ,25-(OH) 2 D 3 which are exhibited by a variety of cells.
• the present invention is useful in controlling nongenomic responses both in vivo and in vitro.
• fifteen analogues are provided which are effective in controlling either genomic or nongenomic cellular responses. The fifteen analogues are described in the following detailed description. The above described and many other features and attendant advan ⁇ tages of the present invention will become better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.
• FIG. 1A shows the metabolic scheme for producing vitamin D 3 including the formation of previtamin D 3 .
• FIG. 1 B shows the two conformations of the hormonally active form of vitamin D 3 , namely 1 ⁇ ,25-(OH) 2 D 3 .
• FIG. 2 shows the equilibrium relationship between 1 ,25-(OH) 2 -pre- vitamin D and 1 ⁇ ,25-(OH) 2 D 3 forms.
• FIG. 3 depicts the structures of 1 ⁇ ,25-(OH) 2 D 3 , 1/?,25-(OH) 2 D 3 and other related analogues.
• FIG. 4 depicts the structures of 1 ⁇ ,25-(OH) 2 D 3 , 1£,25-(OH) 2 D 3 and other related analogues.
• FIG. 5A depicts the effect of 1 ,25-(OH) 2 -d 5 -pre-D 3 and 1 ,25-(OH) 2 D 3 on the appearance of 45 Ca 2+ in the venous effluent of perfused duodena from vitamin D-replete chicks.
• FIG. 5B shows the dose-response analysis of 1 ,25-(OH) 2 D 3 and 1 ,25- (OH) 2 -d 5 -pre-D 3 in stimulating transcaltachia in the perfused duodena.
• the experimental conditions were as described in FIG. 5A. Values are the mean ⁇ S.E. at 30 minutes for three to five duodena perfused with each concentration of agonist. * p ⁇ 0.02; **p ⁇ 0.01 ; ***p ⁇ 0.005 with respect to duodena not exposed to agonist.
• FIG. 6 shows the result of evaluation of 45 Ca 2+ uptake in osteosarcoma cells stimulated by 1 ,25-(OH) 2 D 3 or 1 ,25-(OH) 2 -d 5 -pre-D 3 .
• the left inset displays the 45 Ca 2+ uptake properties of the cells in resting buffer (R), stimulating buffer (S), and when exposed to the optimal con ⁇ centration of 1 ,25-(OH) 2 D 3 , designated as C.
• the ROS 17/2.8 cells were assayed for 45 Ca 2+ as described in Example 1. In all cases, additions of the secosteroids were made to cells exposed to the resting buffer.
• 1 ,25-(OH) 2 D 3 stimulated 45 Ca 2+ uptake with a maximum response occurring between 1.0 and 5.0 nM (see Re. 28). Data points represent the mean of triplicate measurements ⁇ S.D.
• FIG. 7A shows the affinity of the 1 ,25-(OH) 2 D 3 nuclear receptor from pig intestinal mucosa for 1 ,25-(OH) 2 D 3 (•), 1 ,25-(OH) 2 -d 5 -pre-D 3 ( ⁇ ), and
• FIG. 7B shows the determination of the RCI for the chick intestinal receptor and the pig receptor for 1 ,25-(OH) 2 D 3 (analog C), 1 ,25-(OH) 2 -d 5 - pre-D 3 (analog HF), and 1 ,25-(OH) 2 -d 5 -D 3 (analog HG).
• FIG. 8 shows the affinity of purified human DBP for 1 ,25-(OH) 2 D 3 (o),
• FIG. 9 shows the effect of 1 ,25-(OH) 2 D 3 and its analogues on the serum concentration of osteocalcin after a single 400-ng intramuscular injection in vitamin D-deficient chicks. •, 1 ,25-(OH) 2 D 3 ; ⁇ , 1 ,25-(OH) 2 -d 5 -
• FIG. 10A shows the effect of chronic administration to mice of 1 ,25-
• mice were administered the indicated daily intraperitoneal dose of the indicated secosteroid for 7 days. Values represent the mean ⁇ S.E. (six mice/group). * p ⁇ 0.01 in comparison with the control group.
• FIG. 10B shows the effect of chronic administration to mice of 1 ,25-
• mice were administered the indicated daily intraperitoneal dose of the indicated secosteroid for 7 days. Values represent the mean ⁇ S.E. (six mice/group). *p ⁇ 0.01 in comparison with the control group.
• FIG. 11 shows the effect of 1 ,25-(OH) 2 D 3 and its analogues in MG-63 cells on the inhibition of proliferation (FIG. 11 A) and induction of osteocalcin
• FIG. 11 B •, 1 ,25-(OH) 2 D 3 ; ⁇ , 1 ,25-(OH) 2 -d 5 -pre-D 3 ; A , 1 ,25-(OH) 2 -d 5 -D 3 .
• FIG. 12 shows the effect of 1 ,25-(OH) 2 D 3 and its analogues on differentiation of HL-60 cells.
• the differentiating effect was evaluated by nitro blue tetrazolium (NBT) reduction. •, 1 ,25-(OH) 2 D 3 ; ⁇ , 1 ,25-(OH) 2 -d 5 -
• FIG. 13 shows the results of screening of the four A-ring diastereomers of 1 ⁇ ,25-(OH) 2 D 3 in the transcaltachia assay.
• the effect of each analog on the appearance of 45 Ca 2+ (5 //Ci/ml) in GBSS was vascularly perfused (25°C) for the first 20 minutes with control medium (GBSS containing 0.125% bovine serum albumin and 0.05 ⁇ of ethanol/ml) and then at time zero with the indicated concentration of the stipulated analog or control medium.
• Values are the mean ⁇ S.E. for five duodena within each experimental group. •, indicated analog; o, control.
• FIG. 14 depicts the antagonistic action of 1 ?,25-(OH) 2 D 3 -stimulated intestinal 45 Ca 2+ transport activity in the perfused chick duodena.
• FIG. 15 depicts the results of the evaluation of the ability of 1 ?,25- (OH) 2 D 3 to inhibit the agonist actions of 1 ⁇ ,25-(OH) 2 D 3 on transcaltachia.
• the 1 ?,25-(OH) 2 D 3 (HL) was presented to the perfused duodenum either in advance or simultaneously with 1 ⁇ ,25-(OH) 2 D 3 at varying concentrations.
• the data presented represent the mean ⁇ S.E. from four or five duodena. •, analog HL and C [1 ⁇ ,25-(OH) 2 D 3 ]; o, control.
• FIG. 15A the 1 ?,25-(OH) 2 D 3 (HL) was presented to the perfused duodenum either in advance or simultaneously with 1 ⁇ ,25-(OH) 2 D 3 at varying concentrations.
• the data presented represent the mean ⁇ S.E. from four or five duodena. •, analog HL and C [1 ⁇ ,25-(OH) 2 D 3 ];
• FIG. 16 shows the inhibition of 5 Ca 2+ uptake in osteosarcoma cells stimulated by 1 ⁇ ,25-(OH) 2 D 3 (C) by 1/?,25-(OH) 2 D 3 (HL).
• the left side displays the 45 Ca 2+ uptake properties of the cells in resting buffer (/ ⁇ ?), stimulating buffer (S), and when exposed to the optimal concentration of 1 ⁇ ,25-(OH) 2 D 3 designated as C.
• the concentration of HL is the upper value and C the lower value.
• the ROS 17/2.8 cells were assayed for 45 Ca 2+ as described in Example 2. In all cases, additions of the secosteroids were made to cells exposed to the resting buffer.
• FIG. 17 shows the dose-response effects of 1 ⁇ ,25-(OH) 2 D 3 , 1 ?,25- (OH) 2 D 3 , and 1 ⁇ ,25-(OH) 2 -3-epi-D 3 on ICA (FIG. 17A) and BCM (FIG. 17B) in the vitamin D-deficient chick.
• the analogues and 1 ⁇ ,25-(OH) 2 D 3 were given intramuscularly to vitamin D-deficient chicks 12 hours before assay; the control D 3 was 48 hours before assay.
• Results are expressed as mean ⁇ S.E. of groups of five to seven chicks. Each assay included a negative control, open bar (-D) and a positive control of vitamin D 3 3.25 mnol, solid black bar; the difference between these two groups was significant at p ⁇ 0.01. A detailed evaluation was carried out for the four diastereomers in separate bioassays; the results are summarized in Table I.
• FIG. 18 shows the effect of the four A-ring diastereomers in MG-63 cells on the induction of osteocalcin.
• Both 1 ⁇ ,25-(OH) 2 D 3 and 1 ?,25-(OH) 2 D 3 were evaluated independently of one another for their ability to induce osteocalcin (FIG. 18A).
• the ability of 10,25-(OH) 2 D 3 to antagonize the actions of 1 ⁇ ,25-(OH) 2 D 3 on induction of osteocalcin was assessed (FIG. 18B).
• the concentration of 1 ?,25-(OH) 2 D 3 was held constant at 10 "9 -10 "7 M, whereas the concentration of
• 1 ⁇ ,25-(OH) 2 D 3 was varied from 10 "11 to 10 ° M. V, 10 "9 M 1/?,25-(OH) 2 D 3 + varying concentrations of 1 ⁇ ,25-(OH) 2 D 3 ; D , 1 ?,25-(OH) 2 D 3 , 10 "8 M + varying concentrations of 1 ⁇ ,25-(OH) 2 D 3 ; ⁇ , 1 ?,25-(OH) 2 D 3 , 10 "7 M + varying concentrations of 1 ⁇ ,25-(OH) 2 D 3 .
• 1 ⁇ ,25-(OH) 2 D 3 (•) and 1 ?,25-(OH) 2 D 3 (o) were evaluated alone, i.e. in the absence of the other secosteroid. For details, see Example 2. The data presented are from a representative experiment; three experiments were conducted.
• FIG. 19 shows the effect of 1 ⁇ ,25-(OH) 2 D 3 and 1 ?,25-(OH) 2 D 3 on dif ⁇ ferentiation of HL-60 cells.
• the differentiating effect of the secosteroids was evaluated by NBT reduction as described in Example 2. The data presented are from a representative experiment that was repeated twice.
• FIG. 20 shows the effects of 1cr,25-(OH) 2 D 3 A-ring analogues on keratinocyte differentiation.
• Human skin keratinocytes were grown in tissue culture in 96-well plates as described in Example 2.
• the A-ring analogues were added at the indicated concentrations, and then the rate of cell proliferation was assessed for 3 hours by the addition of [ 3 H]thymidine.
• FIG. 21 sets forth the structural formulas for analogues GE, GF, HS, IB, JD, JM, JN, JO and JP in accordance with the present invention.
• FIG. 22 sets forth the structural formulas for analogues JR, JS, JV, JW, JX and JY in accordance with the present invention.
• FIG. 23 depicts the synthesis pathway for making analogues GE and
• FIGS. 24(A) and (B) show the results of testing of analogues JM and JN, respectively, as set forth in Example 6.
• augmented 5 Ca 2+ transport in duodenal loops was vascularly perfused with 1 ⁇ ,25(OH) 2 D 3 , or 1 ⁇ ,25(OH) 2 -7-dehydrocholesterol (JM), or 1 ⁇ ,25(OH) 2 -lumisterol 3 45 Ca 2+ (5 //Ci/ml of buffer), and vascularly perfused with control medium for the first 20 minutes with collection of the venous effluent occurring at 2-minute intervals during the final 10 minutes to establish basal transport rates.
• duodena were then either re-exposed to control medium containing the vehicle ethanol (0.005%, final concentration) through the celiac artery, or vascularly perfused with 300 pM agonist, or 650 pM agonist.
• the venous effluent was again collected at 2 minute intervals for liquid scintillation spectrophotometry.
• FIGS. 25(A) and (B) show the results of testing of analogues JM and JN, respectively, as set forth in Example 6.
• the tests involved dose- response analyses of JM and JN as agonists for transcaltachia.
• Duodena which were perfused as described in FIG. 24 with vehicle or a range of JM or JN concentrations. Normalized transport after 40 minutes of perfusion is depicted for the indicated concentrations of (A) JM; (B) JN.
• FIG. 26 is a chart showing the various diseases which may be treated using the analogous of the present invention.
• FIG. 27 is a schematic representation of the synthesis of analogue IB.
• the present invention provides methods for controlling transcaltachia and other nongenomic cellular responses which are mediated by 1 ⁇ ,25- (OH) 2 D 3 .
• Activation of the nongenomic cellular response is accomplished in accordance with the present invention by treating cells with 1 ,25-(OH) 2 - previtamin D 3 (compound BC in FIG. 2).
• the 1 ,25-(OH) 2 -previtamin D 3 has been found to be an agonist of transcaltachia as is described in detail in Example 1.
• Another aspect of the present invention involves antagonizing the nongenomic responses mediated by 1 ,25-(OH) 2 D 3 by treating cells with 1/?,25-(OH) 2 D 3 as described in Example 2.
• Treatment with 1 ⁇ ,25-(OH) 2 -previtamin D 3 or 1 ?,25-(OH) 2 D 3 can be accomplished as set forth in the examples, i.e. by direct injection or perfusion of the analog in an appropriate pharmaceutical carrier.
• the dosage levels are preferably similar to the dosage levels described in the examples.
• the type of cells treated can be any of those which are known to undergo nongenomic cellular responses which are mediated by 1 ⁇ ,25-(OH) 2 D 3 .
• the cells may be treated either in vivo or in vitro.
• the preferred nongenomic mediated response which can be con- trolled in accordance with the present invention is transcaltachia which involves opening of the Ca 2+ channels in the intestines.
• Control of caltachia is preferably accomplished by selecting the desired agonist or antagonist analog described above and introducing it by way of perfusion into the intestine.
• previtamin D 3 is effective in stimulating two nongenomic cellular responses which are mediated by 1 ⁇ ,25-(OH) 2 D 3 .
• transcaltachia in isolated perfused chicken duodenum is stimulated and Ca + channel opening in rat osteogenic sarcoma cells is also stimulated.
• FIG. 1A shows the metabolic scheme for production of vitamin D 3 .
• the provitamin, 7-dehydrocholesterol, present in the skin is converted by ultraviolet irradiation into the secosteroid vitamin D 3 .
• Previtamin D 3 is in thermal equilibrium with vitamin D 3 ; the conversion involves a [1 ,7]-sigmatropic shift, i.e. the intramolecular migration of a hydrogen from carbon-19 to carbon-9.
• the resulting product vitamin D 3 is a conformationally mobile molecule with respect to the orientation of the A ring in relation to the C/D ring structure.
• the seco-B ring can assume one of two conformations as a consequence of rotation about the carbon 6-7 single bond; in the 6-s-c/s orientation the A ring is related to the C/D rings as in the conventional steroid orientation, referred to here as the "steroid-like conformation" and when the conformation is in the 6-s-trans orientation, the A ring is present in an "extended conformation.”
• FIG 1 B depicts two orientations of 1 ,25-(OH) 2 D 3 .
• 1 ,25-(OH) 2 D 3 has free rotation about the single bond between carbon-6 and carbon-7; accordingly it can assume in solution the steroid-like conformation (6-s-c/s) or the extended conformation (6-s-frans) orientation.
• Analogues HF and 1 ,25-(OH) 2 -d 5 -D 3 were synthesized according to the method of Curtin and Okamura (19).
• 1 ,25-(OH) 2 -d 5 - pre-D 3 which had been stored at -60°C for about 1 year with occasional warming to ambient temperatures for withdrawal of samples for biological evaluation, the sample analyzed to be comprised of 4.4% of the vitamin and 95.6% of the previtamin form of analog HF.
• the composition determinations were carried out by analytical high performance liquid chromatography on a normal phase column (Whatman Partisil column using 90% ethyl acetate, 10% hexane as solvent; 5 ml/min flow rate) using a Waters photodiode array detector.
• a separate comparison using cut and weigh integrated peak areas was used as a cross-check, and the overall agreement was estimated to be ⁇ 0.7%.
• the retention times were as follows: 1 ,25-(OH) 2 -d 5 -D 3 , about 18 minutes; 1 ,25-(OH) 2 -d 5 -pre-D 3 , 24.5 minutes. These retention times are essentially identical to those of the undeuteriated forms of these two secosteroids.
• the human promyelocytic leukemia cell line (HL-60) and the MG-63 cells were obtained from the American Type Culture Collection (Rockville, MD).
• One-day-old RIR chicks were housed in a windowless room and raised on a vitamin D-replete diet for 1 week followed by a vitamin D- deficient diet (Hope Farms, Woerden, The Netherlands) for the next 5 weeks. After a total of 6 weeks, they were divided into groups and received a single intramuscular injection of 400 ng of 1 ,25-(OH) 2 D 3 or analogues HF or HG solubilized in 10:10:80 v/v/v ethanol, Tween 80, NaCI, 0.9%.
• Serum osteocalcin was measured by radioimmunoassay using specific anti-chick antisera raised against these chick proteins.
• Mice, strain NMRI were fed a normal diet (Hope Farms) for 40-60 days. They received a daily subcutaneous dose of 1 ,25-(OH) 2 D 3 or analogues HF and HG for 7 days.
• Serum Ca 2+ was determined via atomic absorption spectrophotometry and serum osteocalcin levels via radioimmunoassay.
• the pig intestinal mucosa was obtained from a normal 20-kg pig under Ketalar anesthesia. The mucosa was scraped and stored at -80°C until time of preparation of the 1 ,25-(OH) 2 D 3 nuclear receptor (see below).
• the ROS 17/2.8 cells obtained from Merck, Sharp and Dohme (West Point, PA) were cultured in Dulbecco's modified Eagle's medium: Ham's F- 12 medium 1 :1 containing 10% fetal calf serum (GIBCO-BRL). The medium was supplemented with 1.1 mM CaCI 2 as described (24). For 45 Ca 2+ uptake experiments, cells were seeded at a density of 30,000 cells/ml into 3.5-cm dishes and grown to approximately 50% confluence. Calcium Uptake Assays ROS 17/2.8 cells were assayed for Ca 2+ uptake using procedures described previously (20). Assays were standardized to 1 minute, which preliminary experiments demonstrated to be within the interval of linear uptake.
• Intestinal 45 Ca 2+ Transport Measurements of 45 Ca 2+ transport were carried out in perfused chick duodena as described previously (21-23). Normal vitamin D-replete chicks weighing approximately 500 g were anesthetized with Chloropent (Fort Dodge, IA; 0.3 ml/100 g), and the duodenal loop was surgically exposed. Blood vessels branching off from the celiac artery were lighted before cannulation of the celiac artery itself and simultaneous initiation of vascular perfusion. The duodenal loop was then excised and, after cannulation of the celiac vein, placed between layers of saline-moistened cheesecloth at 24°C.
• the arterial perfusion was initiated during cannulation with modified Grey's balanced salt solution (GBSS) modified to contain 0.9 mM CaCI 2 and oxygenated with 95% 02 and 5% CO 2 at a flow rate of 2 ml/min.
• GBSS Grey's balanced salt solution
• An auxiliary pump was used for the introduction of vehicle (ethanol) or test substances plus albumin (0.125% w/v final concentration) to the vascular perfusate at a rate of 0.25 ml/min.
• the intestinal lumen was then flushed and filled with GBSS containing 45 Ca 2+ (5 /Ci/MI) but without bicarbonate or glucose.
• a basal transport rate was established by perfusion with control medium for 20 minutes after the lumen was filled with 45 Ca 2+ .
• the tissue was then exposed to 1 ,25-(OH) 2 D 3 or 1 ,25-(OH) 2 -d 5 -pre-D 3 or reexposed to control medium for an additional 40 minutes.
• the vascular perfusate was collected at 2-minute intervals during the last 10 minutes of the basal and during the entire treatment period.
• Duplicate 100-//I aliquots were taken for determination of the 5 Ca 2+ levels by liquid scintillation spectrometry. The results are expressed as the ratio of the 45 Ca 2+ appearing in the 40-minute test period over the average initial basal transport period.
• each analog to compete with [ 3 H]1,25-(OH) 2 D 3 for binding to the chick intestinal nuclear receptor for 1 ,25-(OH) 2 D 3 was carried out under in vitro conditions according to standard procedures (24,25).
• increasing concentrations of nonradioactive 1 ,25-(OH) 2 D 3 or the test analog are incubated with a fixed saturating amount of [ 3 H]1 ,25-(OH) 2 D 3 and chick intestinal nuclear extract obtained from vitamin D-deficient chicks; the reciprocal of the percentage of maximal binding of [ 3 H]1 ,25-(OH) 2 D 3 was then calculated and plotted as a function of the relative concentration of the analog and [ 3 H]1 ,25-(OH) 2 D 3 .
• the plots give linear curves characteristic for each analog, the slopes of which are equal to the analog's competitive index value (24).
• the competitive index value for each analog was then normalized to a standard curve obtained with nonradioactive 1 ,25-(OH) 2 D 3 as the competing steroid and placed on a linear scale of relative competitive index (RCI), where the RCI of 1 ,25-(OH) 2 D 3 by definition is 100. Binding to the 1 ,25-(OH) 2 D 3 receptor was determined in mucosa obtained from a vitamin D-replete pig.
• Frozen (- 80°C) duodenal mucosa was sonicated in 4 volumes of buffer (0.5 M Tris- HCI, 0.5 M KCl, 5 mM dithiothreitol, 10 mM Na 2 MoO 4 , 1.5 mM EDTA, pH 7.5). The high speed supernatant was then incubated with 0.2 nM [ 3 H]1 ,25- (OH) 2 D 3 and increasing concentrations of nonradioactive 1 ,25-(OH) 2 D 3 or its analogues in a final volume of 0.3 ml overnight at 25°C followed by 5 minutes at 4°C. Phase separation was then obtained by the addition of cold dextran-coated charcoal.
• Binding of the 1 ,25-(OH) 2 D 3 and its analogues to hDBP was performed at 4°C essentially as described previously (26).
• [ 3 H]1 ,25-(OH) 2 D 3 and 1 ,25-(OH) 2 D 3 or its analogues were added in 5 ⁇ of ethanol into glass tubes and incubated with hDBP (0.18 ⁇ u) in a final volume of 1 ml (0.01 M Tris-HCl, 0.154 M NaCI, pH 7.4) for 4 h at 4°C. Phase separation was then obtained by the addition of 0.5 ml of cold dextran-coated charcoal.
• HL-60 cells were seeded at 1.2 x 10 5 cells/ml, and 1 ,25-(OH) 2 D 3 or its analogues were added in ethanol (final concentration ⁇ 0.2%) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (GIBCO), 100 units/ml penicillin, and 100 //g/ml streptomycin (Boehringer).
• RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (GIBCO), 100 units/ml penicillin, and 100 //g/ml streptomycin (Boehringer).
• the MG-63 cells were seeded at 5 x 10 3 cells/ml in 96-well flat bottomed culture plates (Falcon, Becton Dickinson, NJ) in a volume of 200 ⁇ of Dulecco's modified Eagle's medium containing 2% of heat-inactivated charcoal-treated fetal calf serum, and 1 ,25-(OH) 2 D 3 or its analogues were added in ethanol (final concentration ⁇ 0.2%). After 72 hours of culture in a humidified atmosphere of 5% CO 2 in air at 37°C the inhibition of proliferation by [ 3 H] thymidine incorporation and measurement in the medium of osteocalcin concentration using a homologous human radioimmunoassay were performed (26).
• Nitro Blue Tetrazolium Reduction Assay Superoxide production was assayed by nitro blue tetrazolium- reducing activity as described previously (26).
• HL-60 cells at 1.0 x 10 5 cells/ml were mixed with an equal volume of freshly prepared solution of phorbol 12-myristate 13-acetate (200 ng/ml) and nitro blue tetrazolium (2 mg/ml) and incubated for 30 minutes at 37°C. The percentage of cells containing black formazan deposits were determined using a hemacytometer.
• This example compares the biological profile of the two deuteriated analogues, 1 ,25-(OH) 2 d 5 -pre-D 3 (HF) and 1 ,25-(OH) 2 -d 5 -D 3 (HG) in relation to 1 ,25-(OH) 2 D 3 .
• the structures of the analogues are given in FIG. 2.
• the 1 ,25-(OH) 2 -d 5 -pre-D 3 , analog HF, is kinetically suppressed (21) in its previtamin form (because of a primary deuterium kinetic isotope effect) and thus can function as an analog only of the 6-s-c/s form of 1 ,25-(OH) 2 D 3 (FIG. 1).
• FIG. 5A shows the results of an evaluation of the relative ability of 1 ,25-(OH) 2 D 3 and 1 ,25-(OH) 2 -d 5 -pre-D 3 to stimulate the nongenomic biological response of transcaltachia.
• Vascular perfusion with the physiological concentration of 60 pM 1 ,25-(OH) 2 -d 5 -pre-D 3 for 34 minutes yielded a 4.5-fold increase in 45 Ca + transport over control levels.
• the stimulatory effect of both secosteroids on 45 Ca 2+ becomes significant within 2-8 minutes as observed previously (7,9).
• FIG. 5B shows the dose- responsive relationship for each secosteroid in terms of its ability to stimulate transcaltachia.
• the analog 1 ,25-(OH) 2 -d 5 -pre-D 3 was able to stimulate transcaltachia significantly at a dose of 10 pM, and the maximal response was attained at 60 pM secosteroid.
• the dose-response for 1 ,25-(OH) 2 -d 5 -pre-D 3 is biphasic (7-9).
• 1 ,25-(OH) 2 D 3 is active at the low concentration of 25 pM, and the maximum stimulation is achieved over the range of 60-650 pM 1 ,25-(OH) 2 D 3 (FIG. 5B).
• the typical biphasic dose response is apparent.
• FIG. 6 shows the results of the evaluation of the ability of 1 ,25-(OH) 2 - d 5 -pre-D 3 to stimulate 5 Ca 2+ uptake into ROS 17/2.8 cells.
• concentration range of 1-10 x 10 "9 M 1 ,25-(OH) 2 -d 5 -pre-D 3 produced a maximum uptake of 45 Ca 2+ within 1 minute of the application of the secosteroids. Previous studies have established that this is the range of maximum response to 1 ,25-(OH) 2 D 3 (15,27).
• the voltage-gated Ca 2+ channel can either be opened by exposure to appropriate agonists (vitamin D analogues or the dihydropyridine BAY K- 8644 (15)) or by depolarization of the cell membrane, which is achieved by the 132 mM external KCl (stimulating buffer; see "Experimental Proce ⁇ dures”).
• appropriate agonists vitamin D analogues or the dihydropyridine BAY K- 8644 (15)
• depolarization of the cell membrane which is achieved by the 132 mM external KCl (stimulating buffer; see "Experimental Proce ⁇ dures”).
• Such stimulated uptake of 45 Ca 2+ in the presence of depolarizing extracellular solutions is characteristic of cells expressing voltage-gated Ca 2+ channels, and the level of this stimulation is directly related to the concentration of the Ca 2+ channels on the cell surface.
• the maximum influx of 45 Ca 2+ which can be achieved (see inset of FIG. 6) occurs in the presence of high external K + .
• the level of 45 Ca 2+ uptake occurring in low K + represents the basal uptake, which is a reflection of the Ca 2+ permeability of the resting membrane.
• S/R the maximum ratio of (stimulated)/( resting), S/R is approximately 2.4-fold, which represents 100% Ca 2+ channel opening.
• Both the analog 1 ,25-(OH) 2 -d 5 -pre- D 3 and 1 ,25-(OH) 2 D 3 achieve a 2-fold stimulation of 45 Ca 2+ uptake over that which occurs in the low K + environment.
• FIG. 7 shows the results of the determination of the RCI for binding to the intestinal nuclear 1 ,25-(OH) 2 D 3 receptor from both the chick and pig, as determined under in vitro conditions.
• the steroid that is kinetically repressed in the previtamin form has a reduction in its RCI from 90-100% to 12-14%, which shows that the nuclear 1 ,25-(OH) 2 D 3 receptor can discriminate between the previtamin 6-s-c/s and the vitamin D form, which may exist either as the 6-s-c/s or 6-s-trans forms, with the latter predominating.
• the principal carrier of vitamin D secosteroids in the blood compart ⁇ ment is the plasma DBP.
• This protein has a binding domain that tightly binds its ligand with a K,, of 5 x 10 "9 M and 5 x 10 "8 M for 25-(OH)D 3 and 1 ,25-(0H) 2 D 3 , respectively (29); thus the affinity of any ligand for DBP will effectively determine its "free" concentration in the plasma and perhaps influence its relative availability to target cells.
• FIG. 7-10 show the evaluation of the biological efficacy of the previtamin form of the pentadeuteriated analog of 1 ,25-(OH) 2 D 3 under in vivo conditions as well as in cultured cells.
• FIG. 9 shows the levels of serum osteocalcin which are achieved after a single intramuscular injection of vitamin D-deficient chick with 400 ng of either 1 ,25-(OH) 2 D 3 , 1 ,25-(OH) 2 -d 5 -D 3 , or 1,25-(OH) 2 -d 5 -pre-D 3 .
• 1 ,25-(OH) 2 D 3 has been shown to induce via interaction with a nuclear 1 ,25-(OH) 2 D 3 receptor present in bone osteoblast cells the de novo biosynthesis of osteocalcin; small amounts of the osteocalcin are released into the blood (as a consequence of bone remodeling) where it may be conveniently determined via a radioimmunoassay (26). It is apparent that the 1 ,25-(OH) 2 -d 5 -pre-D 3 , when administered as a single dose under in vivo conditions, has little ability to interact effectively with the nuclear 1 ,25-(OH) 2 D 3 receptor to induce osteocalcin.
• both 1 ,25-(OH) 2 D 3 and 1 ,25-(OH) 2 -d 5 -D 3 caused a significant increase in the plasma levels of osteocalcin.
• treatment with the deuteriated analog resulted in a consistently higher induction of the plasma osteocalcin levels.
• FIG. 10 shows the levels of serum Ca 2+ and osteocalcin achieved after 1 week of daily treatment with doses of 1 ,25-(OH) 2 D 3 or the analogues 1 ,25-(OH) 2 -d 5 -pre-D 3 and 1 ,25-(OH) 2 -d 5 -D 3 .
• Both 1 ,25-(OH) 2 D 3 and 1 ,25-(OH) 2 -d 5 -D 3 were virtually equipotent in regard to evaluation of serum Ca 2+ and osteocalcin.
• both of these responses are mediated by the nuclear 1 ,25-(OH) 2 D 3 receptor.
• the 1 ,25-(OH) 2 -d 5 -pre-D 3 had only approximately 1% of the activity of 1 ,25-(OH) 2 D 3 or 1 ,25-(OH) 2 -d 5 -D 3 .
• the 1 ,25-(OH) 2 -d 5 -D 3 was indistinguishable from 1 ,25-(OH) 2 D 3 in its ability to induce osteocalcin and displayed approximately 90% of the activity of 1 ,25-(OH) 2 D 3 in terms of its ability to inhibit cell proliferation.
• the differentiation of HL-60 cells was markedly enhanced by the pre ⁇ sence of 1 ,25-(OH) 2 D 3 or 1 ,25-(OH) 2 -d 5 -D 3 (FIG. 12).
• the 1 ,25- (OH) 2 -d 5 -pre-D 3 displayed only 1 -4% of the potency of 1 ,25-(OH) 2 D 3 , which again shows that the HL-60 nuclear 1 ,25-(OH) 2 D 3 receptor does not effi ⁇ ciently bind this ligand.
• the biological profile of 1 ,25-(OH) 2 - d 5 -pre-D 3 has been compared with that of the pair of rapidly interconverting 6-s conformers of 1 ,25-(OH) 2 D 3 (FIGS. 1 and 2).
• the example demonstrates that two nongenomic biological systems are fully responsive to the 1 ,25-(OH) 2 -d 5 -pre-D 3 analog. Both the process of transcaltachia as studied in the isolated perfused chick duodenum (FIG. 5) and the process of Ca 2+ channel opening in the rat osteogenic sarcoma cell line, ROS 17/2.8 cells (FIG.
• FIG. 8 indicates that both the pig and chick intestinal 1 ,25-(OH) 2 D 3 nuclear receptors discriminate against the previtamin form of the secosteroid.
• the RCI of 1 ,25-(OH) 2 -d 5 -pre-D 3 for binding to the chick intestinal receptor was 10% and for the pig intestinal receptor was 4%. Accordingly, the nuclear 1 ,25-(OH) 2 D 3 receptor's ligand binding domain favors the 6-s-trans conformer (extended steroid conformation) over the 6-s- c/s (steroid-like conformation).
• FIGS. 9-12 show the evaluation of 1,25-(OH) 2 -d 5 -pre-D 3 , 1 ,25-(OH) 2 -d 5 -D 3 , and 1 ,25-(OH) 2 D 3 in four systems that all generate biological effects via a nuclear receptor- mediated regulation of gene transcription.
• FIG. 9-12 show the evaluation of 1,25-(OH) 2 -d 5 -pre-D 3 , 1 ,25-(OH) 2 -d 5 -D 3 , and 1 ,25-(OH) 2 D 3 in four systems that all generate biological effects via a nuclear receptor- mediated regulation of gene transcription.
• FIG. 9 shows the in vivo measurement of serum osteocalcin levels, after a single dose of 1 ,25-(OH) 2 - d 5 -pre-D 3 to chicks.
• FIG. 10 shows the results of daily administration of 1 ,25-(OH) 2 -d 5 -pre-D 3 on serum Ca 2+ and osteocalcin levels in mice.
• FIG. 11 shows, using MG-63 cells in cell culture, the inhibition of cell proliferation and the induction of osteocalcin.
• FIG. 12 shows, using HL-60 cells in culture, the inhibition of cell proliferation. The relative inability of analog 1 ,25-(OH) 2 -d 5 -pre-D 3 (less than 2% for osteocalcin induction in vivo, FIG.
• mice 7 days (mice, in FIG. 10), and 96 h (MG-63 cells, in FIG. 11 ; HL-60 cell, in FIG. 12), and if the vitamin form, 1 ,25-(OH) 2 d- 5 -D 3 had been generated there would have been ample time for appearance of detectable manifestations of the various genomic responses.
• the results from the chronic dosing of mice with 1 ,25-(OH) 2 -d 5 -pre-D 3 show that the previtamin form is subject to metabolic clearance before it has had an opportunity to isomerize thermally into the biologically active 1 ,25-(OH) 2 d- 5 -D 3 .
• the previtamin D form of 1 ,25- (OH) 2 D 3 is effective as an agonist of the nongenomic receptor for 1 ,25- (OH) 2 D 3 and therefore may be used to initiate biological responses which utilize the nongenomic receptor.
• White Leghorn cockerels (Lakeview Farms, Lakeview, CA) were obtained on the day of hatch and maintained on a vitamin D-supplemented diet (1.2% calcium and 0.7% phosphorous; O.H. Kruse Grain and Milling, Ontario, CA) for 5-6 weeks to prepare normal vitamin D 3 -replete chicks. All experiments employing animals were approved by the University of California-Riverside Chancellor's Committee on Animals in Research. The human promyelocytic leukemia cell line (HL-60) and the human osteoblast MG-63 cells were obtained from the American Type Culture Collection (Rockville, MD).
• Both uptake solutions contained 12.5 /Ci/ml 45 Ca + (Du Pont-New England Nuclear) and the concentrations of vitamin D agonists indicated in FIG. 13. Uptake was terminated by aspiration of the labeling solution, followed by three washes with ice-cold resting buffer. Cell-associated 45 Ca 2+ was extracted by a 2- hour incubation with 0.5 M NaOH and measured by liquid scintillation counting. It was found that 5 Ca 2+ uptake by monolayer cultures of ROS 17/2.8 cells was density-dependent. Maximal uptake rates were consis ⁇ tently found for cultures that were between 50 and 80% confluent.
• Intestinal 45 Ca 2+ Transport Measurements of 45 Ca 2+ transport were carried out in perfused chick duodena as described previously (32-34). Normal vitamin D-replete chicks weighing approximately 500 g were anesthetized with Chloropent (Fort Dodge, IA; 0.3 ml/100 g), and the duodenal loop was surgically exposed. Blood vessels branching off from the celiac artery were lighted before cannulation of the celiac artery itself and simultaneous initiation of vascular perfusion. The duodenal loop was then excised and, after cannulation of the celiac vein, placed between layers of saline-moistened cheesecloth at 24°C.
• the arterial perfusate consisted of Grey's balanced salt solution (GBSS) modified to contain 0.9 mM CaCI 2 and oxygenated with 95% 0 2 and 5% CO 2 at a flow rate of 2 ml/min.
• GBSS Grey's balanced salt solution
• An auxiliary pump was used for the introduction of vehicle (ethanol) or test substances plus albumin (0.125% w/v final concentration) to the vascular perfusate at a rate of 0.25 ml/min.
• the intestinal lumen was then flushed and filled with GBSS containing 5 Ca 2+ (5 Ci/MI) but without bicarbonate or glucose and perfused at a flow rate of 0.2 ml/min.
• a basal transport rate was established by perfusion with control medium for 20 minutes after the lumen was filled with 45 Ca 2+ .
• the tissue was then exposed to 1 ⁇ ,25-(OH) 2 D 3 or 1 ?,25-(OH) 2 D 3 or reexposed to control medium for an additional 40 minutes.
• the vascular perfusate was collected at 2-minute intervals during the last 10 minutes of the basal period and during the entire treatment period.
• Duplicate 100- /I aliquots were taken for determination of the 45 Ca 2+ levels by liquid scintillation spectrometry. The results are expressed as the ratio of the 5 Ca 2+ appearing in the 40-minute test period over the average initial basal transport period.
• each analog to compete with [ 3 H]1 ⁇ ,25-(OH) 2 D 3 for binding to the chick intestinal nuclear receptor for 1 ⁇ ,25-(OH) 2 D 3 was carried out in vitro according to the procedure set forth in Example 1.
• increasing concentrations of nonradioactive 1 ,25-(OH) 2 D 3 or the test analog were incubated with a fixed saturating amount of [ 3 H]1 ⁇ ,25-(OH) 2 D 3 and chick intestinal nuclear extract obtained from vitamin D-deficient chicks; the reciprocal of the percentage of maximal binding of [ 3 H]1 ⁇ ,25-(OH) 2 D 3 was then calculated and plotted as a function of the relative concentration of the analog and [ 3 H]1cr,25-(OH) 2 D 3 .
• Such plots give linear curves characteristic for each analog, the slopes of which are equal to the analog's competitive index value (25).
• the competitive index value for each analog is then normalized to a standard curve obtained with nonradioactive 1 ⁇ ,25-(OH) 2 D 3 as the competing steroid and placed on a linear scale of relative competitive index (RCI), where the RCI of 1 ⁇ ,25- (OH) 2 D 3 by definition is 100.
• HL-60 cells were seeded at 1.2 x 10 5 cells/ml, and 1 ⁇ ,25-(OH) 2 D 3 or its analogues were added in ethanol (final concentration ⁇ 0.2%) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 //g/ml streptomycin (Boehringer Mannheim). After 4 days of culture in a humidified atmosphere of 5% CO 2 in air at 37°C the dishes were shaken to loosen any adherent cells, and all cells were then assayed for differentiation by NBT reduction assay and for proliferation by [ 3 H]thymidine incorporation.
• the MG-63 cells were seeded at 5 x 10 3 cells/ml in 96-well flat bottomed culture plates (Falcon, Becton Dickinson, NJ) in a volume of 200 ⁇ of Dulecco's modified Eagle's medium containing 2% of heat-inactivated charcoal-treated fetal calf serum, and 1 ⁇ ,25-(OH) 2 D 3 or its analogues were added in ethanol (final concentration ⁇ 0.2%).
• Human skin keratinocytes were isolated and cultured using a modifi ⁇ cation of the method of Kitano and Okada (36). Briefly, skin from biopsies of patients with breast tumors was cut into pieces measuring 3-5 mm and soaked overnight at 4°C in a solution of dispase (20 Boehringer units/ml). The epidermis was peeled from the dermis, washed with calcium- and magnesium-free phosphate-buffered saline, and incubated and shaken in a 0.25% trypsin solution for 10 minutes at room temperature. The reaction was then stopped by the addition of phosphate-buffered saline containing 10% fetal calf serum.
• the cells were collected after centrifugation at 4°C for 10 minutes at 800 rpm. After an additional washing with phosphate- buffered saline, the pellet was suspended in culture medium into 25-cm 2 primaria flasks from Becton Dickinson. The keratinocytes were cultivated at 37°C in an atmosphere of 5% CO 2 in air. A few hours later the medium was replaced by a new one. The medium (keratinocyte medium from Life Technologies, Inc., containing epidermal growth factor (5 ng/ml), bovine pituitary extract (35-50 /g/ml), and antibiotics) was renewed every other day until confluence.
• epidermal growth factor 5 ng/ml
• bovine pituitary extract 35-50 /g/ml
• antibiotics was renewed every other day until confluence.
• keratinocytes were cultured in 96-well plates and after 24 hours were treated with various concentrations of the vitamin D analogues followed by pulse labeling with 1 ⁇ C ⁇ of [ 3 H]thymidine for 3 hours. Cultures were washed three times with phosphate-buffered saline and twice with 10% (v/v) ice-cold trichloroacetic acid. Cells were solubilized with 1 M NaOH and the radioactivity determined via liquid scintillation measurement. Statistics Statistical evaluation of the data was performed in the same manner as Example 1.
• This example provides a comparison of the biological profile of the three A-ring diastereomers of 1 ⁇ ,25-(OH) 2 D 3 with respect to their ability to act as agonists for nongenomic and genomic responses of various components of the vitamin D endocrine system.
• the structures of these secosteroids are presented in FIGS. 3 and 4.
• the two asymmetric centers are located at carbons-1 and -3.
• the orientation of the two hydroxyl groups on the A-ring of the naturally occurring hormone 1 ⁇ ,25-(OH) 2 D 3 are 1 ⁇ and 3/?.
• transcaltachia A biological response of 1 ⁇ ,25-(OH) 2 D 3 which has been shown to occur via a nongenomic mechanism is the rapid hormonal stimulation of intestinal Ca 2+ , termed transcaltachia (22).
• FIG. 13 presents the results showing the relative ability of the four A-ring diastereomers of the secosteroid hormone to stimulate transcaltachia.
• the optimal agonist is the naturally occurring hormone 1 ⁇ ,25-(OH) 2 D 3 (designated C).
• the onset of stimulation of 45 Ca + transport occurs within 4 minutes of introduction of the hormone.
• the dose-response curve for transcaltachia is biphasic, with a maximal stimulation occurring at 650 pM 1 ⁇ ,25-(OH) 2 D 3 (21 ,22).
• FIG. 14 illustrates the ability of 1/?,25-(OH) 2 D 3 to block the action of 1 ⁇ ,25-(OH) 2 D 3 to stimulate transcaltachia.
• the duodena are simulta ⁇ neously perfused with both 1 ?,25-(OH) 2 D 3 and 1 ⁇ ,25-(OH) 2 D 3 the characteri- stic stimulation of transcaltachia was absent; this shows that 1 ?,25-(OH) 2 D 3 can function as an antagonist of 1 ⁇ ,25-(OH) 2 D 3 to control nongenomic responses in accordance with the present invention.
• FIG. 14 illustrates the ability of 1/?,25-(OH) 2 D 3 to block the action of 1 ⁇ ,25-(OH) 2 D 3 to stimulate transcaltachia.
• FIG. 15A there is presented an evaluation of the different concen ⁇ trations of 1 ?,25-(OH) 2 D 3 which are effective at inhibiting 300 pM 1 ⁇ ,25- (OH) 2 D 3 -stimulated transcaltachia.
• FIG. 15B summarizes the dose response of the inhibition of 1 ⁇ ,25-(OH) 2 D 3 -stimulated transcaltachia by varying concentrations of 1 ?,25-(OH) 2 D 3 . It is apparent that a concentration as low as 60 pM 1/?,25-(OH) 2 D 3 can inhibit 300 pM 1 ⁇ ,25-(OH) 2 D 3 .
• FIG. 16 presents an evaluation of the ability of 1 ?,25-(OH) 2 D 3 to function as an agonist or antagonist of 45 Ca + uptake into ROS 17/2.8 cells.
• this response occurs as a consequence of the ability of 1 ⁇ ,25-(OH) 2 D 3 or its analogues to open dihydropyridine-sensitive Ca 2+ channels via a nongenomic mechanism (20).
• 1 ,25-(OH) 2 D 3 is the most potent agonist in this system.
• Concentrations as high as 10 "8 M 1 ?,25-(OH) 2 D 3 is the most potent agonist in this system.
• FIG. 17 presents the ICA and BCM results for 1 ⁇ ,25-(OH) 2 D 3 , 1 ?,25-(OH) 2 D 3 (HL) and 1 ⁇ ,25-(OH) 2 -3-epi-D 3 (HJ).
• Table I summarizes the ICA and BCM results for all four diastereomers.
• the most potent stimulator of ICA and BCM was the reference compound 1 ⁇ ,25-(OH) 2 D 3 ; the activity produced by 100 pmol of 1 ⁇ ,25-(OH) 2 D 3 was set to 100% for both ICA and BCM. Then, the dose of the comparison analogues required to achieve a biological response of either ICA or BCM equivalent to the 100-pmol dose of 1 ⁇ ,25-(OH) 2 D 3 was calculated and converted to a percentage.
• the analog 1 ⁇ ,25-(OH) 2 D-3-epi- 3 was the only diastereomer to have detectable ICA or BCM, which was only 1.5-2.8% of that of the reference 1cr,25-(OH) 2 D 3 .
• the two diastereomers, 1 ?,25-(OH) 2 D 3 and 1 ?,25-(OH) 2 D-3-epi-D 3 had less than 0.1% ICA and BCM.
• the alteration of the orientation of either the 3 ?-hydroxyl group or the 1 ⁇ -hydroxyl group greatly diminishes the biological activity in the vitamin D- deficient chick in vivo.
• results presented for the bioassay ICA and BCM are derived from dose- response studies like that presented in FIG. 6. The results are expressed in terms of the dose of analog required to achieve an ICA or BCM response equivalent to that achieved by a 100-pmol dose of 1 ⁇ ,25-(OH) 2 D 3 , calculated as a percentage, i.e. [1 ⁇ ,25-(OH) 2 D 3 dose]/[analog dose] x 100.
• Table I also summarizes the relative ability of the four diastereomers to bind in vitro to the chick intestinal 1 ⁇ ,25-(OH) 2 D 3 nuclear receptor as well as to the DBP.
• the 1 ⁇ ,25-(OH) 2 D 3 nuclear receptor is the presumed media ⁇ tor of genomic responses to 1or,25-(OH) 2 D 3 in vivo.
• 1 ⁇ ,25-(OH) 2 D 3 is the reference compound, and its RCI is by definition 100%. Inversion of the orientation of the 3 ?-hydroxyl to 3 ⁇ -hydroxyl, as in analog 1 ⁇ ,25-(OH) 2 -3- epi-D 3 , resulted only in a reduction of RCI to 24%.
• the reference analog has been defined to be 1 ⁇ ,25-(OH) 2 D 3 ; but it is important to realize that the optimal ligand for DBP is 25-(OH)D 3 and that is has an RCI of 66,700. If the RCI of 25-(OH)D 3 were set to 100%, then the RCI for 1 ⁇ ,25-(OH) 2 D 3 would only be 0.15%; this is a reflection of the fact that the presence of an ⁇ -hydroxyl on carbon-1 results in a marked reduction in affinity of the ligand for DBP. Inversion of the 1 ⁇ -hydroxyl to the 1/?
• calbindin-D28k levels present in the chick intestine 12 hours after dosing vitamin D-deficient chicks with either 1 ⁇ ,25-(OH) 2 D 3 alone or in the presence of 1 ? ,25-(OH) 2 D 3 were determined via enzyme-linked immunosorbent assay, as described under "Experimental Procedures.”
• FIG. 18A results are presented describing the potency of the four diastereomers to induce osteocalcin in MG-63 cells; in addition, the ability of 1/?,25-(OH) 2 D 3 to function as an antagonist of 1 ⁇ ,25-(OH) 2 D 3 - induced osteocalcin is presented (FIG. 18B).
• 1 ⁇ ,25-(OH) 2 D 3 is a potent agonist for osteocalcin in the MG-63 cell line (35); half-maximal induction occurs at a concentration of 3.8 x 10 *9 M 1 ⁇ ,25-(OH) 2 D 3 (FIG. 18A).
• 1 ⁇ ,25-(OH) 2 D 3 is a potent stimulator of HL-60 cell differentiation; the half-maximal concentration was 1.5 x 10 "8 M. The concentration of 1 ?,25-(OH) 2 D 3 which achieved half- maximal stimulation of cell differentiation was 2.5 x 10 ⁇ 7 M, some 10 times higher. Again there was no evidence that 1 ?,25-(OH) 2 D 3 could antagonize the cell differentiation actions of 1 ⁇ ,25-(OH) 2 D 3 .
• FIG. 20 presents the evaluation of the potencies of 1 ⁇ ,25-(OH) 2 D 3 and the three A-ring diastereomers in inhibiting the proliferation of human keratinocytes.
• the relative order of potency was 1 ⁇ ,25-(OH) 2 D 3 , 1 ⁇ ,25-(OH) 2 -3-epi-D 3 , 1 ?,25-(OH) 2 D 3 , and 1 ?,25-(OH) 2 -3-epi-D 3 , 1 :6.2:27:75.
• the most potent inhibitor of cell proliferation was 1 ⁇ ,25-(OH) 2 D 3
• the least potent was 1/?,25-(OH) 2 D 3 .
• the potential of 1/?,25-(OH) 2 D 3 to antagonize 1 ⁇ ,25-(OH) 2 D 3 -mediated inhibition of keratinocyte was tested.
• This example demonstrates the biological profile of the four A-ring diastereomers of the hormonally active form of vitamin D 3 (see FIG. 3). Only 1 ⁇ ,25-(OH) 2 D 3 is known to occur naturally in biologically systems. The only difference in structure of these four compounds is the orientation of the hydroxyl groups on carbons-1 and -3.
• the nuclear 1 ⁇ ,25-(OH) 2 D 3 receptor's ligand binding domain clearly prefers the 1 ⁇ , 3 ⁇ orientation of the naturally occurring hormone and that it can also discern differences among the three other A- ring diastereomers (see the RCI values of Table I).
• the correct orientation of the hydroxyl on carbon-1 is more critical than the orientation of the hydroxyl on carbon-3.
• inversion of the 1 ⁇ -hydroxyl to the . ⁇ orientation results in a change in RCI from 100 to 0.8%
• inversion of the 3 .-hydroxyl to a 3 ⁇ -hydroxyl only results in a reduction from 100 to 24%.
• DBP is the principal plasma transport protein for vitamin D metabolites; the ligand with highest affinity is 25-(OH)D 3 ; however, it also binds 1 ⁇ ,25-(OH) 2 D 3 , 24R,25-(OH) 2 D 3 , and the parent vitamin D 3 with a significant affinity.
• the addition of a 1 ⁇ -hydroxyl to 25-(OH)D 3 results in a 666-fold reduction in RCI (3).
• 1 ⁇ ,25-(OH) 2 D 3 has the lowest RCI; this implies that 1 ⁇ ,25-(OH) 2 D 3 would have, under in vivo circumstances, the highest "free” concentration or greatest "availability" of the four diastereomers.
• ICA and BCM responses occur under in vivo conditions and both likely represent a response to an integrated set of components that respond to vitamin D ligands, it is not known which proportion of the responding elements is comprised of genomic and nongenomic responses. However, both, ICA and BCM responses can be blocked by administration of actinomycin D, an inhibitor of DNA-directed RNA synthesis (38,39), and a good correlation between binding to the nuclear 1 ⁇ ,25-(OH) 2 D 3 receptor and ICA and BCM has been shown (3).
• hormone response element(s) associated with the nongenomic response of transcaltachia clearly have a ligand specificity different from that of the nuclear 1 ⁇ ,25-(OH) 2 D 3 receptor (compare with RCI results in Table I).
• the putative transcaltachic membrane response element (40) is more tolerant of the presence of 3 ⁇ -hydroxyl.
• this example shows that the analog 10,25-(OH) 2 D 3 (HL) is a potent antagonist of both 1 ⁇ ,25-(OH) 2 D 3 - stimulated transcaltachia (FIG. 15) and rat osteoblast 5 Ca 2+ uptake (FIG. 16).
• 1/?,25-(OH) 2 D 3 and 1 ⁇ ,25-(OH) 2 D 3 were perfused simultaneously and, even in some circumstances when the intestine was preexposed only to the 1 ?,25-(OH) 2 D 3 for 8 minutes and then followed by perfusion with only 1 ⁇ ,25-(OH) 2 D 3 , there was a clear inhibition by exposure of the intestine to 1 ?,25-(OH) 2 D 3 .
• This example demonstrates that the analog of 1 ⁇ ,25-(OH) 2 D 3 is able to function as an antagonist of a biological response stimulated by 1 ⁇ ,25-(OH) 2 D 3 .
• analogues of vitamin D 3 have been synthesized and demonstrated to be active in controlling genomic and/or nongenomic responses in the vitamin D endocrine system.
• the fifteen analogues are set forth in FIGS. 21 and 22 and tabulated in Table III.
• the analogues may be administered in the same manner as the previously described two analogues. They are effective in controlling a wide variety of responses within the vitamin D endocrine system including the genomic mechanisms which are controlled by mechanisms similar to that of other steroid or steroid like hormones (e.g. estradiol, testosterone, stanolone, progesterone, cortisol, aldosterone, retinoic acid and thyroxine).
• the analogues are useful in treating a variety of diseases associated with malfunction of the vitamin D endocrine system including skin conditions (e.g. psoriasis), bone conditions (e.g. osteoporosis, venal osteodystrophy), and oncologic diseases such as breast, colon and prostate cancers and leukemia, induction of hey proteins like nerve growth factor and other brain proteins which may be involved in Alzheimer's disease.
• skin conditions e.g. psoriasis
• bone conditions e.g. osteoporosis, venal osteodystrophy
• oncologic diseases such as breast, colon and prostate cancers and leukemia, induction of hey proteins like nerve growth factor and other brain proteins which may be involved in Alzheimer's disease.
• the various diseases associated with vitamin D metabolism are set forth in FIG. 26.
• the analogues are useful in treating and diagnosing this group of diseases. The synthesis and usefulness of these analogues will be further described in the following examples.
• Example 3
• analogues GE and GF were prepared from the known A-ring phosphine oxide 10(41) and the appropriate (CD) ketone 14.
• Grundmann's ketone 11(42) readily available from the ozonolysis of vitamin D 3 , was selectively oxidized at C-25 to alcohol 12 as previously described (43). Epimerization of the latter to the c/s-fused hydrindanone 13 was accomplished with base.
• the crude mixture consisted of a 71/29 ratio of 13/12 whereas a 49% yield (66% based on recovered 12) of purified 13 was actually isolated by HPLC(44,45,46).
• Intestinal calcium absorption (ICA) and bone calcium mobilization (BCM) were measured in vivo to compare analogues GE and GF to 1 ⁇ ,25- (OH) 2 -D 3 (3) in the vitamin D deficient chick system previously described(37).
• the results in this standard rachitic chick assay can be reported as the percentage of activity observed for ICA and BCM in comparison to standard doses of 1 ⁇ ,25-(OH) 2 -D 3 (47).
• the two analogues GE and GF exhibited 3.9% and ⁇ 0.1%, respectively of the activity as compared to 1 ⁇ ,25-(OH) 2 -D 3 . Similar results ( ⁇ 0.01% and 2%, respectively) were obtained with respect to the BCM determination.
• the GE and GF analogues were evaluated in vitro in terms of their ability to bind to the chick intestinal nuclear receptor.
• the analogues were evaluated in terms of their chick intestinal receptor relative competitive indices (RCIs) wherein the value for 1 ⁇ ,25-(OH) 2 -D 3 is 100 by definition (48).
• the RCI values for GE and GF were 15.0 ⁇ 2.0 and 1.6 ⁇ 0.9, respectively.
• the lack of in vivo calcemic activity observed for GE is somewhat at variance with its RCI value of 15. It is believed that GE binds to the chick intestinal receptor without inducing its necessary activation, which is required of steroid hormone receptors prior to stimulation of transcription.
• the human DBP RCI values for analogues GE and GF were 12.1 ⁇ 2.1 and 2.2 ⁇ 0.7, respectively.
• the above test results show that GE and GF are useful respectively for biological responses involving the nuclear VDR (GE) and regulation of gene transcription (GE) and the membrane VDR anovated with nongenomic rapid actions (GF).
• BCM BCM Mobilization
• ICA and BCM were determined in vivo in vitamin D deficient chicks as described previously (37, 47). Twelve hours before assay, the chicks, which had been placed on a zero-calcium diet 48 hours before assay, were injected intramuscularly with the vitamin D metabolite or analogue in 0.1 mL of ethanol/1 ,2-propanediol (1 :1 , v/v) or with vehicle. At the time of assay, 4.0 mg of 40 Ca 2+ + 5 ⁇ C ⁇ of 45 Ca 2+ (New England Nuclear) were placed in the duodenum of the animals anesthetized with ether. After 30 min, the birds were decapitated and the blood collected.
• the radioactivity content of 0.2 mL of serum was measured in a liquid scintillation counter (Beckman LS8000) to determine the amount of 45 Ca 2+ absorbed (which is a measure of ICA).
• BCM activity was estimated from the increase of total serum calcium as measured by atomic absorption spectrophotometry.
• Chick Intestinal Receptor Steroid Competition Assay A measure of competitive binding to the chick intestinal 1 ⁇ ,25- (OH) 2 -D 3 receptor was performed by using the hydroxylapatite batch assay (48). Increasing amounts of non radioactive 1 ⁇ ,25-(OH) 2 -D 3 or analogue were added to a standard amount of [ 3 H]-1 ⁇ ,25-(OH) 2 -D 3 and incubated with chick intestinal cytosol.
• the relative competitive index (RCI) for the analogues was determined by plotting the percent maximum 1 ⁇ ,25-(OH) 2 - [ 3 H]-D 3 bound x 100 on the ordinate versus [competitor]/[1 ⁇ ,25-(OH) 2 -[ 3 H]- D 3 ] on the abscissa.
• the slope of the line obtained for a particular analogue is divided by the slope of the line obtained for 1 ⁇ ,25-(OH) 2 -D 3 ; multiplication of this value by 100 gives the RCI value.
• the RCI for 1 ⁇ ,25- (OH) 2 -D 3 is 100.
• DBP human vitamin D binding protein
• Example 4 Synthesis and Biological Activity of 1 ⁇ , 18,25-(OH 3 )-D 3 (HS) and 1 ⁇ ,25-Dihydroxy-.rat.s-isotachysterol (1 ,25-fratvs-lso-T) (JD) HS is prepared as follows: The protected alcohol precursor of HS was first prepared as follows: 18-Acetoxy-25-[(trimethylsilyl)oxy]-1 ⁇ -[(fetf-Butyldimethylsilyloxy)
• Vitamin D 3 tert-butyldimethylsilyl] ether (Compound A) was prepared according to the procedure described in Maynard et al., J. Org. Chem., 1992, v. 57, pp. 3214-17.
• JD was synthesized by preparing and reacting precursors A-D as follows:
• precursor D (4.5 mg, 43% yield).
• the analytical data for precursor D is 1 H-NMR: ⁇ 0.88 (3H, C 18 -CH 3 , s), 0.96 (3H, C 21 -CH 3 , d, J -6.6 Hz), 1.21 (6H, C 2627 -2CH 3 , s), 1.69 (3H, C 19 -CH 3 , s), 2.52 (1 H, dd, J - 16.5 Hz, 4.2 Hz), 4.07 (1 H, C 3 -H, m), 4.19 (1 H, C H, br s), 5.78 and 5.92 (2H, C 6 -H and C7-H, AB pattern, J - 12.1 Hz).
• JJV (100% EtOH) ⁇ max 256 nm (e 11 ,000); ⁇ mn
• a solution of precursor D (7.5 mg, 0.0180 mmol) was dissolved in ether (1 ml) under argon.
• the solution was then condensed to leave a crude oily residue.
• the crude residue was subjected to HPLC (Rainin Microsorb, 5 ⁇ m silica, 10 mm x 25 cm, 11% isopropanol/hexanes) to produce JD (3.9 mg, 52%).
• HS and JD will have the same biological activity as described in Example 2.
• HS biological responses will mimic 1 ,25(OH) 2 D 3 in that it can assume both 6-s-cis and 6-s-trans.
• IB was prepared according to the procedure set forth in FIG. 27. In step 1 of the synthesis, 3-iodobenzoic acid is refluxed for 14 hours in 80 ml
• step 3 the product of step 2 was reacted with 55 mg OH, 183 mg pyridinium chlorochromate (PDC), 12 mg pyridinium trifluoroacetate (PTFA) and 100 ml CH 2 CI 2 according to the procedure set forth in S.A. Barrack et al., J. Org. Chem. 1988, 53, 1790. The reaction was carried out at room temperature for 5 hours. The resulting black mixture was filtered and washed with CH 2 CR 2 and extracted with ethyl acetate to produce pale yellow oil. This oil was flash chromatographed to produced C 22 H 26 O 3 .
• PDC pyridinium chlorochromate
• PTFA pyridinium trifluoroacetate
• step 4 the product of step 3 was reacted with 70 mg phosphine oxide, 82 ⁇ n-Butyl, 35 mg of the CD ketone in 2 ml of a solution of THF.
• the n-Butyl was added dropwise to the solution of phosphine oxide in THF.
• the resulting orange colored solution was stirred at -78°C for 10 minutes and the CD ring ketone in THF was added dropwise.
• the reaction mixture was stirred at -78°C for 4 hours. At this point the solution turned pale yellow.
• the solution was quenched with H 2 O, extracted with ethyl acetate and dried over Na 2 SO 4 .
• the solvent was vacuum evaporated and the resulting product purified by flash chromatography.
• 10 mg of the product of step 4 reacted with 53 ⁇ MeLi in
• step 6 10 mg of the product of step 5 was dissolved in 1 ml THF.
• Example 6 Synthesis and Biological Activity of 1 ⁇ ,25-(OH) 2 -7-DHC (JM) and 1 ⁇ ,25-(OH) 2 -Lumisterol 3 (JN).
• JM and JN are closed B-ring analogues which both stimulate transcaltachia while neither competes with 1 ⁇ ,25(OH) 2 D 3 for binding to either the nuclear vitamin D receptor (N-VDR) or the serum vitamin D transport protein (DBP).
• N-VDR nuclear vitamin D receptor
• DBP serum vitamin D transport protein
• JM [1 ⁇ ,25-(OH) 2 -7-Dehydrocholesterol] and JN [1 ⁇ ,25-(OH) 2 -7-Lumi- sterol 3 ] were synthesized as follows.
• a solution of the known 1 ⁇ ,25-(OH) 2 - previtamin D 3 (120 mg) in methanol was irradiated (Hanovia 450 watt medium pressure mercury lamp, pyrex filter, ⁇ > 300 nm) for 3 hours at room temperature.
• JN The identifying characteristics of JN are: 1 H-NMR (CDCI 3 ): ⁇ 0.61 (3H, C 18 -CH 3 , s), 0.78 (3H, C 19 -CH 3 , s), 0.91 (3H, C 21 -CH 3 , d, J -5.2 (Hz), 1.21 (6H, C 2627 -CH 3) s), 2.50 (2H, m), 4.10 (1 H, H,, dd, J -9.2 Hz, 4.8 Hz), 4.14 (1 H, H3, dd, J - 3.0 Hz, 3.0 Hz), 5.45 (1 H, H 6or7 , m), 5.75 (1 H, H 7or6 , dd, J - 5.1 Hz, 1.7 Hz).
• auxiliary pump was used for the introduction of vehicle (0.005% ethanol v/v, final concentration) or test analogues plus albumin (0.125% w/v final concentration) to the vascular perfusate at a rate of 0.25 ml/min.
• vehicle 0.005% ethanol v/v, final concentration
• test analogues plus albumin 0.125% w/v final concentration
• the intestinal loop was then excised and the lumen flushed and filled with GBSS (lacking NaHCO 3 and glucose) containing 45 Ca 2+ (5 /Ci/ml).
• the lumenal solution was renewed constantly at a rate of 0.25 ml/min to insure a steady concentration of 45 Ca 2+ at the brush border of the epithelia.
• the intestinal preparation at 27°C was kept moist under layers of saline-dampened cheese cloth.
• duodenum was perfused with control medium (vehicle) for 20 min after filling the lumen with 45 Ca 2+ to establish the basal transport rate.
• the tissue was then either exposed to the test analog or continued on vehicle for an additional 40 minutes.
• the competitive index value for each analog is then normalized to a standard curve obtained with nonradioactive 1 ⁇ ,25(OH) 2 D 3 as the competitive steroid and placed on a linear scale of Relative Competitive lndex(s) (RCI), where the RCI of 1 ⁇ ,25(OH) 2 D 3 is by definition 100.
• RCI Relative Competitive lndex(s)
• the relative ability of vitamin D analogues to bind to the plasma transport protein, the vitamin D binding protein (DBP) were carried out in a similar fashion (52). JM and JN differ in the fixed orientations of the two A-ring hydroxyls;
• JM is 1 ⁇ -axial and 3 ?-equatorial
• JN is 1 ⁇ -equatorial and 3 ?-axial.
• JM and JN are analogues of the 6-s-c/s form of 1 ⁇ ,25(OH) 2 D 3 ; they cannot exist in the extended 6-s-trans conformation.
• FIGS. 24A and 24B illustrate the appearance of 5 Ca 2+ in the venous effluent mediated by the two different concentrations of analogues, JM and JN, respectively, vehicle control (ethanol only) and 650 pM 1 ⁇ ,25(OH) 2 D 3 as positive control.
• the efficacy of 300 pM JM in initiating transcaltachia is not significantly greater than the control, and the response elicited at 650 pM JM is only 60% of that induced by the natural metabolite.
• Perfusion with JN produced a stimulation nearly identical to that of 1 ⁇ ,25(OH) 2 D 3 with JN achieving only a slightly lower ratio of transport 45 Ca 2+ than that achieved by 1 ⁇ ,25(OH) 2 D 3 .
• FIGS. 25A and 25B present the dose response curves for JM and JN, respectively.
• Each bar represents the 40 minute data point of FIGS. 25A and 25B which is taken as the maximum response elicited by the analog at that concentration.
• Analog JN eventually reaches the 4-fold plateau at 1300 pM which is the equivalent of the maximum stimulation achieved by 1 ⁇ ,25(OH) 2 D 3 at 650 pM.
• the 5 Ca 2+ transport ratio for analog JM at 650 pM peaked at 2.5 and was not further increased as a consequence of increasing the JM concentration of 1300 pM.
• JM and JN are useful in controlling nongenomic mechanisms, such as transcaltachia. These two analogues can be used to act as agonists for nongenomic responses as previously described.
• Example 7 Synthesis and Biological Activity of (9 ⁇ ,10 ⁇ )-and (9/?, 10/?)- 1 ⁇ ,25-Dihydroxy-7-dehydrocholesterol-1 ⁇ ,25(OH) 2 -Pyrocalciferol (JO) and 1 ⁇ ,25(OH) 2 -lsopropylcalciferol 3 (JP).
• JO and JP were prepared according to the following procedures: An argon flushed solution of 1 ⁇ ,25-(OH) 2 -previtamin D 3 (54.2 mg) dis ⁇ solved in DMF (15 mL) containing a drop of 2,4,6-trimethylpyridine was heated in a screw cap vial (156°C) for 18 hours.
• Both JO and JP will have the same nongenomic actions achieved by analogues JM and JN since JO and JP locked in the 6-s-cis conformation.
• Example 8 Synthesis and Biological Activity of (1S, 3R, 6S)-7,19-Retro- 1 ,25-(OH) 2 -D 3 (JV) and (1S, 3R, 6S)-7,19-Retro-1 ,25-(OH) 2 -D 3 (JW)
• JV The analog JV was synthesized as follows:
• (1S,3R,6S)-1,3-Di(tert-butyldimethylsilyloxy)-25-trimethylsilyloxy- 9,10-secocholesta-5(10),6,7-triene (A) is a starting material that was prepared first as follows: Freshly purified 1 ,2-diiodoethane (412 mg, 1.46 mmol) and samarium metal (286 mg, 1.90 mmol) were dried under vacuum and suspended in 4 mL THF under an argon atmosphere. This solution was stirred for 2 hours until it became deep blue.
• the product was purified by flash chromatography (silica gel, 2% EtOAc/hexanes) followed by HPLC (2% EtOAc/hexanes, Rainin Dynamax column, 8 mL/min flow rate) to afford vinylallene A (0.3085 g, 75.5%).
• the product was identified by 1 H-NMR analysis. This material is more stable as the triol.
• JW which is also known as (1S,3R,6R)-1,3,25-Trihydroxy-9,10- secocholesta-5(10),6,7-triene was isolated from the above solution as follows: A solution of (6S/6R)-vinylallenes JV and JW (2.6 mg, 0.0062 mmol, -92:8 ratio of 6S:6R) in methanol-d 4 (1 mL) was prepared in a quartz NMR tube. The solution was saturated with argon for 30 minutes and then the NMR tube was capped and then irradiated with ultraviolet light from a Hanovia 450 watt medium pressure lamp for 30 minutes.
• Example 9 Synthesis and Biological Activity of 1 ,25-(OH) 2 7, 8-c/s-D 3 (JR) and 1 ,25-(OH) 2 -5,6-fra ⁇ s-7,8-c/s-D 3 (JS). JR was synthesize from JV as follows:
• Acetone was removed under reduced pres ⁇ sure and the product was purified by flash chromatography (silica gel, 80% EtOAc/hexanes) followed by separation by HPLC (80% EtOAc/hexanes, Rainin Microsorb column, 4.0 mL/min flow rate) to afford three components in the following order of elution: major product JR (17.0 mg, 86.4%), recovered starting material JV (1.4 mg, 7.1%), and a minor amount of cis- isotachysterol (1.5 mg, 7.6%).
• the first separation (80% ethyl acetate/ hexanes, Rainin Microsorb column, 4 mL/min flow rate) gave two fractions, each of which was subjected to NMR analysis.
• Fraction I contained products A, B, and C and fraction II contained products B, C, and D.
• JR and JS were determined by the chick intestinal receptor steroid competition assay which has been described in the preceding examples. Both JR and JS significantly suppress the ability of the natural hormone to bind receptor. These results show that JR and JS are also useful for regulating nongenomic mechanisms such as transcaltachia.
• Example 10 Synthesis and Biological Activity of 22-(p-Hydroxyphenyl)- 23,24,25,26,27,pentanor-D 3 (JX) and 22-(m-Hydroxyphenyl-23,24,25,26,27- pentanor-D 3 (JY)
• the A-ring phosphine oxide (48 mg, 0.11 mmol) in dry THF (1.8 mL) was cooled to -78°C and n-butyllithium (1.5 M in hexanes, 0.074 mL, 0.11 mmol) was added dropwise via a syringe.
• the resulting deep red solution was stirred for 10 minutes and then treated with a solution of the appropriate CD-ring ketone (28 mg, 0.070 mmol) in dry THF (0.6 mL) via cannula.
• the mixture was stirred 2 hours at -78°C, warmed to room temperature and quenched with water (5 mL).
• the aqueous layer was separated and extracted with EtOAc (3 x 5 mL).
• the protected vitamin (19.2 mg, 0.03 mmol) in dry THL (1 mL) was placed under argon and TBAF (1 M in THF, 0.30 mn, 0.30 mmol) was added dropwise. After stirring 18 hours, the solvent was partially evaporated and diluted with water (5 mL). After extracting the aqueous layer with EtOAc (3 x 5 mL), the combined organic layers were washed with brine and dried over Na 2 SO 4 . The residue was purified by HPLC (20% EtOAc/hexanes) and after vacuum drying afforded 2.8 mg (23%) of JY.
• Vitamin D Gene Regulation, Structure-Function Analysis and Clinical Application (Norman, A.W., Bouillon, R., and Thomasset, M., eds) pp. 146-154, Walter de Gruyter, Berlin.

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