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  • A61P5/18—Drugs for disorders of the endocrine system of the parathyroid hormones
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  • A61P5/18—Drugs for disorders of the endocrine system of the parathyroid hormones
  • A61P5/20—Drugs for disorders of the endocrine system of the parathyroid hormones for decreasing, blocking or antagonising the activity of PTH
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  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
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  • C07C2602/14—All rings being cycloaliphatic
  • C07C2602/24—All rings being cycloaliphatic the ring system containing nine carbon atoms, e.g. perhydroindane

Definitions

• This disclosure relates generally to novel prodrugs of vitamin D-related compounds.
• the disclosure includes 1-deoxy prohormones of active Vitamin D hormones, e.g.
• Vitamin D is a term that refers broadly to the organic substances named Vitamin D 2 , Vitamin D 3 , Vitamin D 4 , etc., and to their metabolites and hormonal forms that influence calcium and phosphorus homeostasis.
• Vitamin D 2 is produced in plants from ergosterol during sunlight exposure and is present, to a limited extent, in the human diet. Vitamin D is generated from 7- dehydrocholesterol in human skin during exposure to sunlight and also is found, to a greater extent than Vitamin D 2 , in the human diet, principally in dairy products (milk and butter), certain fish and fish oils, and egg yolk. Vitamin D supplements for human use consist of either Vitamin D 2 or Vitamin D .
• Vitamin D 2 and Vitamin D are metabolized into prohormones by one or more enzymes located in the liver.
• the involved enzymes are mitochondrial and microsomal cytochrome P450 (CYP) isoforms, including CYP27A1, CYP2R1, CYP3A4, CYP2J3 and possibly others.
• CYP mitochondrial and microsomal cytochrome P450
• These enzymes metabolize Vitamin D 2 into two prohormones known as 25 -hydroxy vitamin D 2 and 24(S)-hydroxyvitamin D 2 , and Vitamin D into a prohormone known as 25 -hydroxy vitamin D .
• the two 25-hydroxylated prohormones are more prominent in the blood, and can be collectively referred to as "25 -hydroxy vitamin D.”
• Vitamin D 2 and Vitamin D 3 can be metabolized into their respective prohormones outside of the liver in certain epithelial cells, such as enterocytes, which contain the same (or similar) enzymes, but extrahepatic prohormone production probably contributes little to blood levels of 25 -hydroxy vitamin D.
• the rates of hepatic and extrahepatic production of the Vitamin D prohormones are not tightly regulated, and they vary mainly with intracellular concentrations of the precursors (Vitamin D 2 and Vitamin D ). Higher concentrations of either precursor increase prohormone production, while lower concentrations decrease production. Hepatic production of prohormones is inhibited by high levels of 25 -hydroxy vitamin D via a poorly understood mechanism apparently directed to prevention of excessive blood prohormone levels.
• Vitamin D prohormones are further metabolized in the kidneys into potent hormones by an enzyme known as CYP27B1 (or 25 -hydroxy vitamin D -l -hydroxylase) located in the proximal kidney tubule.
• the prohormones 25 -hydroxy vitamin D 2 and 24(S)-hydroxyvitamin D 2 are metabolized into hormones known as l ,25-dihydroxyvitamin D 2 and l ,24(S)-dihydroxyvitamin D 2 .
• 25 -hydroxy vitamin D is metabolized into a hormone known as l ,25- dihydroxy vitamin D (or calcitriol). These hormones are released by the kidneys into the blood for systemic delivery.
• Vitamin D prohormones can be metabolized into hormones outside of the kidneys in keratinocytes, lung epithelial cells, enterocytes, cells of the immune system (e.g., macrophages) and certain other cells containing CYP27B1 or similar enzymes, but such extrarenal hormone production is incapable of sustaining normal blood levels of 1,25-dihydroxyvitamin D in advanced chronic kidney disease (CKD).
• CKD advanced chronic kidney disease
• Blood levels of 1,25-dihydroxyvitamin D are precisely regulated by a feedback mechanism which involves parathyroid hormone (PTH).
• PTH parathyroid hormone
• the renal lcc-hydroxylase (or CYP27B1) is stimulated by PTH and inhibited by 1,25-dihydroxyvitamin D.
• VDR Vitamin D receptors
• the secreted PTH stimulates expression of renal CYP27B1 and, thereby, increases production of Vitamin D hormones.
• As blood PTH levels fall renal production of Vitamin D hormones decreases. Rising blood levels of 1,25- dihydroxyvitamin D also directly inhibit further Vitamin D hormone production by CYP27B 1.
• PTH secretion can be abnormally suppressed in situations in which blood 1,25- dihydroxyvitamin D concentrations become excessively elevated, as can occur in certain disorders such as sarcoidosis or as a result of bolus doses of Vitamin D hormone replacement therapies.
• Blood levels of 1,25-dihydroxyvitamin D and substrate 25 -hydroxy vitamin D prohormone, and regulation thereof, can also be affected by vitamin D hormone analogs, such as 19-nor-l,25 dihydroxyvitamin D 2 and 22-oxacalcitriol, the prodrugs l -hydroxy vitamin D 2 and la- hydroxyvitamin D 2 , 24-sulfoximine vitamin D 3 compounds, oxime analogs of la,25- dihydroxyvitamin D 3 , and 25-S0 2 substituted analogs of la,25-dihydroxyvitamin D 3 , as disclosed in U.S. Patent No. 7,101,865, U.S. Patent No. 6,982,258, and U.S. Patent Application No.
• Vitamin D hormones have essential roles in human health which are mediated by the intracellular VDR.
• the Vitamin D hormones regulate blood calcium levels by controlling intestinal absorption of dietary calcium and reabsorption of calcium by the kidneys. Excessive hormone levels can lead to abnormally elevated urine calcium (hypercalciuria), blood calcium (hypercalcemia) and blood phosphorus (hyperphosphatemia).
• Vitamin D deficiency is associated with secondary hyperparathyroidism, parathyroid gland hyperplasia, hypocalcemia, CKD, and metabolic bone diseases such as osteitis fibrosa cystica, osteomalacia, rickets, osteoporosis, and extraskeletal calcification.
• Vitamin D hormones are required for the normal functioning of the musculoskeletal, immune and renin- angiotensin systems. Numerous other roles for Vitamin D hormones are being postulated and elucidated, based on the documented presence of intracellular VDR in nearly every human tissue. For example, vitamin D has been postulated to play a role in cellular differentiation and cancer, in regulation of the immune system (immune enhancing or immune suppressing effects, depending on the situation), atherosclerosis, growth and normal bone formation and metabolism. Vitamin D deficiency increases the risk of many common cancers, multiple sclerosis, rheumatoid arthritis, hypertension, cardiovascular heart disease, blood pressure, antifibrosis, red blood cell formation, hair growth, and type I diabetes.
• Vitamin D hormones on specific tissues depend on the degree to which they bind to (or occupy) the intracellular VDR in those tissues. VDR binding increases as the intracellular concentrations of the hormones rise, and decreases as the intracellular concentrations fall. In all cells, intracellular concentrations of the Vitamin D hormones change in direct proportion to changes in blood hormone concentrations. In cells containing CYP27B1 (or similar enzymes), intracellular concentrations of the Vitamin D hormones also change in direct proportion to changes in blood and/or intracellular prohormone concentrations, as discussed above.
• Vitamin D 2 , Vitamin D 3 and their prohormonal forms have affinities for the VDR which are estimated to be at least 100-fold lower than those of the active Vitamin D hormones and do not effectively activate the receptor.
• physiological concentrations of these hormone precursors exert little, if any, biological actions without prior metabolism to active Vitamin D hormones.
• Vitamin D 2 and Vitamin D 3 Blood levels of Vitamin D 2 and Vitamin D 3 are normally present at stable concentrations in human blood, given a sustained, adequate supply of Vitamin D from sunlight exposure and an unsupplemented diet. Slight, if any, increases in blood Vitamin D levels occur after meals since unsupplemented diets have low Vitamin D content, even those containing foods fortified with Vitamin D. The Vitamin D content of the human diet is so low that the National Institutes of Health (NIH) cautions "it can be difficult to obtain enough Vitamin D from natural food sources" [NIH, Office of Dietary Supplements, Dietary Supplement Fact Sheet: Vitamin D (2005)]. Almost all human Vitamin D supply comes from fortified foods, exposure to sunlight or from dietary supplements, with the last source becoming increasingly important.
• NASH National Institutes of Health
• Vitamin D hormone concentrations also remain generally constant through the day in healthy individuals, but can vary significantly over longer periods of time in response to seasonal changes in sunlight exposure or sustained alterations in Vitamin D intake. Marked differences in normal Vitamin D hormone levels are commonly observed between healthy individuals, with some individuals having stable concentrations as low as approximately 20 pg/mL and others as high as approximately 70 pg/mL. Due to this wide normal range, medical professionals have difficulty interpreting isolated laboratory determinations of serum total 1,25-dihydroxyvitamin D; a value of 25 pg/mL may represent a normal value for one individual or a relative deficiency in another.
• hyperparathyroidism progressively increases in severity, causing debilitating metabolic bone diseases, including osteoporosis and renal osteodystrophy.
• Kidney Disease Outcomes Quality Initiative (K/DOQI) Clinical Practice Guidelines for Bone Metabolism and Disease in Chronic Kidney Disease [Am. J. Kidney Dis. 42:S1-S202, 2003)].
• K/DOQI Guidelines identified the primary etiology of secondary hyperparathyroidism as chronically low blood levels of 1,25-dihydroxyvitamin D and recommended regular screening in CKD Stages 3 through 5 for elevated blood PTH levels relative to Stage- specific PTH target ranges.
• Stage 3 was defined as moderately decreased kidney function (GFR of 30-59 mL/min/1.73 m ) with an intact PTH (iPTH) target range of 35-70 pg/mL;
• Stage 4 was defined as severely decreased kidney function (GFR of 15-29 mL/min/1.73 m ), with an iPTH target range of 70-110 pg/mL; and
• Stage 5 was defined as kidney failure (GFR of ⁇ 15 mL/min/1.73 m or dialysis) with an iPTH target range of 150-300 pg/mL.
• GFR kidney failure
• iPTH target range 150-300 pg/mL.
• the Guidelines recommended a follow-up evaluation of serum total 25 -hydroxy vitamin D to detect possible Vitamin D insufficiency or deficiency. If 25- hydroxyvitamin D levels below 30 ng/mL was observed, the recommended intervention was Vitamin D repletion therapy using orally administered ergocalciferol. If 25 -hydroxy vitamin D levels above 30 ng/mL was observed, the recommended intervention was Vitamin D hormone replacement therapy using known oral or intravenous Vitamin D hormones or analogs.
• Vitamin D sufficiency as serum 25-hydroxyvitamin D levels > 30 ng/mL.
• Vitamin D hormone replacement therapy is used to treat or prevent vitamin D insufficiency or deficiency in patients.
• Activated Vitamin D particularly la,25- dihydroxyvitamin D 3 (calcitriol)
• CYP24 carries out the conversion of 25 -hydroxy vitamin D 3 to 24,25- dihydroxyvitamin D 3 and the conversion of 1,25-dihydroxyvitamin D 3 (calcitriol) to 1,24,25- trihydroxyvitamin D eventually giving rise to calcitroic acid.
• CYP24 can also hydroxylate at the 23 position, resulting in the production of the terminal metabolite 1,25-dihydroxyvitamin D - 26,23- lactone. Further processing by Phase II catabolic enzymes ultimately leads to clearance of vitamin D compounds from the body. Inhibiting catabolism by CYP24 is expected to lengthen the biological lifetime of the Vitamin D hormones and thus to allow smaller amounts of them to be used. Further, inhibition of catabolism by CYP24 increases the endogenous levels of vitamin D hormones, which provides beneficial therapeutic effects.
• This present disclosure is directed to novel prodrugs of vitamin D-related compounds.
• the prodrugs can have one or more additional benefits, including selective inhibition of the enzyme CYP24, low calcemic profile, and anti-proliferative.
• This disclosure is also directed to pharmaceutical and diagnostic compositions containing the prodrugs of the invention, and to their medical use, particularly as prodrugs in the treatment and/or prevention of diseases.
• the invention provides a compound of Formula I, or a pharmaceutically acceptable salt, solvate, or hydrate thereof:
• each— independently is a single bond or a double bond
• n 0, 1 or 2;
• R 1 is selected from the group consisting of OH, OCi-ealkyl, and halo;
• R 4 is Ci-ealk l;
• R 5 and R 6 are each independently H, halo, C 1-4 alkyl, or can be taken, together with the carbon atom to which they are bound, to form a C 3 _ 6 cycloalkyl ring, with the proviso that when— between carbon-23 and carbon-24 is a double bond, then R 5 is absent;
• R 7 is selected from the group consisting of O, NH, N(C 1-6 alkyl), and NC(0)R 9 ;
• R is selected from the group consisting of Ci- 6 alkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl, wherein each of Ci- 6 alkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl are either unsubstituted or substituted with 1 to
• substituents independently selected from the group consisting of C 1 _ 4 alkyl, OC 1-4 alkyl, CF 3 , N0 2 , halo, OH, OCF 3 , SH, SCi_ 4 alkyl, NH 2 , NHC ⁇ alkyl, N(Ci- 4 alk l)(Ci- 4 alk l), and CN; and
• R 9 is selected from the group consisting of Ci- 6 alkyl, C 3 _ 6 cycloalkyl, aryl-Ci_ 4 alkyl, aryl and heteroaryl, wherein each of C 1-6 alkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl are either unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1-4 alkyl,
• OCi_ 4 alkyl CF 3 , N0 2 , halo.
• the invention provides a compound of Formula II, or a pharmaceutically acceptable salt, solvate, or hydrate thereof:
• each— independently is a single bond or a double bond
• R 1 is selected from the group consisting of OH, OCi- 6 alkyl, and halo;
• R 4 is Ci-ealkyl
• R 5 and R 6 are each independently H, halo, Ci_ 4 alkyl, or can be taken, together with the carbon atom to which they are bound, to form a C 3 _ 6 cycloalkyl ring, with the proviso that when— between carbon-23 and carbon-24 is a double bond, then R 5 is absent;
• R is selected from the group consisting of H, Ci_ 6 alkyl, C 2 _ 6 alkenyl, aryl and heteroaryl, with Ci- ealkyl and C 2 - 6 alkenyl being unsubstituted or substituted with 1 to 4 groups independently selected from C 1-4 alkyl, C 2 _ 4 alkenyl, OCi_ 4 alkyl, OC 2 _ 4 alkenyl, OH, halo, NH 2 , NHCi_ 4 alkyl, NHC 2 _ 4 alkenyl, N(C 2 _ 4 alkenyl)(C 1 _ 4 alkyl), and with aryl and heteroaryl being unsubstituted or substituted with 1 to 5 groups independently selected from C ⁇ alkyl, C 2 _ 4 alkenyl, OC 1-4 alkyl, OC 2 _ 4 alkenyl, OH, CF 3 , OCF 3 , halo, SH, SC ⁇ alkyl, SC 2 _
• R is selected from the group consisting of C 1-6 alkyl, C 2 _ 6 alkenyl, cyclo(C 3 -C 6 )alkyl, cyclo(Cs- C 6 )alkenyl, aryl, heteroaryl, aryl-Ci ⁇ alkyl, aryl-C 2 _ 6 alkenyl, heteroaryl-Ci-ealkyl, and heteroaryl-C 2 _ 6 alkenyl with Ci- 6 alkyl and C 2 _ 6 alkenyl being unsubstituted or substituted with 1-4 groups independently selected from C ⁇ alkyl, C 2 _ 4 alkenyl, OC 1-4 alkyl, OC 2 _ 4 alkenyl, OH, halo, NH 2 , NHQ.
• the invention provides a compound of Formula III, or a
• each— independently is a single bond or a double bond
• R 4 is Ci-ealkyl
• R 5 and R 6 are each independently H, halo, C 1-4 alkyl, or can be taken, together with the carbon atom to which they are bound, to form a C 3 _ 6 cycloalkyl ring;
• R 7 is selected from the group consisting of O, NH, N(C 1-6 alkyl), and NC(0)R 9 ;
• R is selected from the group consisting of C 1-6 alkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl, wherein each of C 1-6 alkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl are either unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1 _ 4 alkyl, OC 1-4 alkyl, CF 3 , N0 2 , halo, OH, OCF 3 , SH, SC alk l, NH 2 , NHC M alk l, N(Ci_ 4 alkyl)(Ci_ 4 alkyl), and CN; and,
• R 9 is selected from the group consisting of C 1-6 alkyl, C 3-6 cycloalkyl, aryl-C 1-4 alkyl, aryl and heteroaryl, wherein each of C 1-6 alkyl, C 3-6 cycloalkyl, aryl and heteroaryl are either unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1-4 alkyl, OCi_ 4 alkyl, CF 3 , N0 2 , halo.
• a pharmaceutical composition comprising a prodrug of the invention in an admixture with a pharmaceutically- acceptable excipient, e.g. a diluent or carrier.
• a pharmaceutically- acceptable excipient e.g. a diluent or carrier.
• a method for treating diseases which benefit from a modulation of the levels of la,25 -dihydroxyvitamin D 3 comprising administering an effective amount of a compound of Formula I or II to a cell or animal in need thereof.
• the invention also includes the use of a compound of Formula I or II to modulate the levels of la,25-dihydroxyvitamin D 3 .
• a method for treating diseases which benefit from inhibiting the catabolism of CYP24 substrates comprising administering an effective amount of a CYP-24 inhibiting prodrug described herein (e.g., a compound of Formula I or II) to a cell or animal in need thereof.
• a CYP-24 inhibiting prodrug described herein e.g., a compound of Formula I or II
• the invention also includes the use of such compounds to inhibit the catabolism of CYP24 substrates (e.g., la,25 -dihydroxyvitamin D 3 ).
• a method of inhibiting the proliferation of a cancer cell comprising administering an effective amount of a vitamin D receptor agonist prodrug described herein (e.g., a compound of Formula I or II) to a cell or animal in need thereof.
• the invention also includes a use of such compounds to inhibit cancer cell proliferation.
• a method of modulating CYP24 activity in a cell or animal by administering an effective amount of a CYP-24 inhibiting prodrug described herein (e.g., a compound of Formula I or II).
• the invention provides a method of modulating CYP24 activity, preferably inhibiting CYP24 activity by administering an effective amount of a compound of a CYP-24 inhibiting prodrug described herein (e.g., a compound of Formula I or II) to a cell or animal in need thereof.
• a compound of a CYP-24 inhibiting prodrug described herein e.g., a compound of Formula I or II
• the present invention also provides a use of such compounds to modulate, preferably to inhibit, CYP24 activity.
• a method of increasing the efficacy of a vitamin D receptor agonist comprising co-administering an effective amount of a CYP24 inhibitor prodrug described herein (e.g. a compound of Formula I or II) and an effective amount of the vitamin D receptor agonist, preferably lcc,25-dihydroxy vitamin D (calcitriol).
• a CYP24 inhibitor prodrug described herein e.g. a compound of Formula I or II
• an effective amount of the vitamin D receptor agonist preferably lcc,25-dihydroxy vitamin D (calcitriol).
• Figure 1 is a graph showing the binding of Prodrug Ibii to the VDR compared to 1,25- dihydroxyvitamin D .
• Figures 2a-2d are graphs showing the induction of transcription of CYP24 in HPKlaRas cells by Prodrug Ibii.
• Figure 2a shows the relative CYP24 expression in HPKlaRas cells treated with Prodrug Ibii compared to calcitriol at 100 nM.
• Figure 2b shows the relative CYP24 expression in HPKlaRas cells treated with Prodrug Ibii compared to vehicle treated cells.
• Figure 2c shows the relative CYP24 transcription in HPKlaRas cells treated with Prodrug Ibii compared to calcitriol, 25 -hydroxy vitamin D , and the 1-hydroxy active form of Prodrug Ibii.
• Figure 2d shows the relative CYP24 transcription in HPKlaRas cells treated with Prodrug Ibii compared to 25 -hydroxy vitamin D 3 and the 1 -hydroxy active form of Prodrug Ibii.
• Figure 3 shows the relative CYP27B1 transcription in HPKlaRas cells treated with Prodrug Ibii compared to 25 -hydroxy vitamin D 3 and the 1 -hydroxy active form of Prodrug Ibii.
• Figure 4 shows results of serum PTH suppression in uremic rats by Prodrug Ibii and the 1- hydroxy active form of Prodrug lib, demonstrating that Prodrug Ibii can be transformed in vivo and suppress PTH in uremic rats.
• Figure 5 shows the effect on serum calcium in uremic rats by Prodrug Ibii and the 1- hydroxy active form of Prodrug lib, demonstrating that Prodrug Ilbii is a non-calcemic vitamin D analog prodrug.
• Figure 6 shows the effect on serum FGF23 levels in uremic rats by Prodrug Ibii and the 1- hydroxy active form of Prodrug lib, demonstrating that Prodrug Ilbii does not increase serum FGF23 levels in uremic rats.
• Figure 7 shows the effect on PTH by Prodrug Ibii in vitamin D deficient animals treated 3x/week for five days via oral and IV dosing, demonstrating that both IV and orally-administered Prodrug Ivii can effectively lower PTH.
• Figure 8 shows the effect on body weight by Prodrug Ibii in Vitamin D deficient rats, demonstrating that Prodrug Ibii does not exhibit overt toxicity.
• Figure 9 shows the relative CYP27B1 mRNA expression in U937 and HPKla-ras cells, demonstrating that HPKla-ras cells exhibit a higher expression of CYP27B1 mRNA compared to U937 cells.
• Figures 10-12 show relative CYP24 expression (compared to calcitriol) in PMA-U927 cells treated with calcitriol and Prodrugs Ibii, Iaii and Ilaii described herein at concentrations up to ⁇ ( Figure 10) and with calcitriol and Prodrugs Ibii, Ieii, Icii, and Idii described herein at concentrations up to lOOnm ( Figures 11 and 12), demonstrating that in cells which do not substantially express CYP27B1, the Prodrugs are inactive.
• the y-axis on Figure 12 has been segmented to show detail at lower levels of relateive CYP24 expression for the Prodrugs.
• analogs of la,25-dihydroxyvitamin D 3 such as sulfoximine, oxime, and sulfone analogs of l ,25-dihydroxyvitamin D can have beneficial therapeutic effects, some can also cause an unphysiologically rapid increase in the blood level of both calcium and activated vitamin D hormone analog, followed by an almost as rapid decrease in the blood level of activated vitamin D hormone analog. Such rapid peaks and valleys of either the blood calcium or the activated vitamin D hormone analog are undesirable and can be harmful.
• prodrugs that would allow the slow or "on-demand” release analogs of la,25-dihydroxyvitamin D , for example low calcemic, anti-proliferative, selective CYP24 inhibitor analogs of l ,25-dihydroxyvitamin D 3 , to the body.
• This present disclosure is directed to novel prodrugs of vitamin D-related compounds, and preferably those that show selective inhibition of the enzyme CYP24, and which are low calcemic and anti-proliferative. This disclosure is also directed to pharmaceutical and diagnostic
• compositions containing the prodrugs of the invention and to their medical use, particularly as prodrugs in the treatment and/or prevention of diseases.
• the compounds described herein can be described as synthetic prohormones, and can be activated by CYP27B1, for example by CYP27B1 expressed in the kidney, or by extrarenal CYP27B1.
• CYP24 is involved in causing vitamin D insufficiency and/or resistance to vitamin D therapy in CKD
• the regulation of CYP24 and CYP27B1 was determined in normal rats and rats treated with adenine to induce CKD. Helvig et al. Kidney International 78, 463-472 (September 2010). As expected, CYP24 decreased whereas CYP27B1 increased when normal animals were rendered vitamin D deficient. Unexpectedly, renal CYP24 mPvNA and protein expression were markedly elevated, irrespective of the vitamin D status of the rats.
• CYP27B1 is expressed in uremic kidneys
• the use of compounds described herein can be used as prodrugs even in CKD patients.
• some of the active l ,25-dihydroxy vitamin D 3 analogs produced by the prodrugs described herein are more potent than l ,25-dihydroxy vitamin D 3
• the prodrugs can find utility in cases of End Stage Renal Disease where there is, in fact, very little renal CYP27B1 expression, but sufficient remaining renal CYP27B1 expression combined with extrarenal CYP27B1 expression to yield potent la,25-dihydroxyvitamin D analogs.
• extrarenal CYP27B may be sufficient to activate a prodrug described herein in target tissues.
• activation could occur in the parathyroid gland which contains CYP27B. This activation would present active hormone to the parathyroid VDR thereby reducing PTH gene transcription
• certain of the 1-deoxy compounds described herein will be 1 -alpha hydroxylated by CYP27B 1 to result in active compounds which are selective CYP24 inhibitors having little or no vitamin D agonist activity.
• Such compounds can have utility, for example, by selective suppression of CYP24 activity in cells in which they are activated. For example, in certain tumor cells (e.g., prostate cancer), CYP27B1 is overexpressed and CYP24 is overexpressed.
• a CYP24 inhibitor prodrug as described herein would be more selectively activated in the tumor cell via overexpressed CYP27B1, and the activated form would selectively inhibit CYP24 activity in such cells.
• compositions includes both methods practiced on the human body and also the foregoing activities.
• Vitamin D toxicity is meant to refer to the side effects suffered from excessively elevated Vitamin D blood levels, including one or more of nausea, vomiting, polyuria, hypercalciuria, hypercalcemia and hyperphosphatemia.
• Vitamin D insufficiency and deficiency is generally defined as having serum 25- hydroxyvitamin D levels below 30 ng/mL (see National Kidney Foundation guidelines, NKF, Am. J. Kidney Dis. 42:S1-S202 (2003), incorporated herein by reference).
• hypocalcemia refers to condition in a patient wherein the patient has corrected serum levels of calcium above 10.2 mg/dL. Normal corrected serum levels of calcium for a human are between about 8.6 to 10.2 mg/dL.
• hypophosphatemia refers to a condition in a patient having normal kidney function, or Stage 3-4 CKD, wherein the patient has serum phosphorous levels above 4.6 mg/dL. In a patient who has Stage 5 CKD, hyperphosphatemia occurs when the patient has serum levels above 5.5 mg/dL. Normal values for serum phosphorous in a human are 2.5-4.5 mg/dL.
• over suppression of plasma iPTH refers to a condition in a patient having normal kidney function, or Stage 1-3 CKD, wherein the patient has levels of plasma iPTH below 15 pg/mL.
• over suppression of plasma iPTH occurs when the patient has levels of plasma iPTH below 30 pg/mL.
• over suppression of plasma iPTH occurs when the patient has levels of plasma iPTH below 100 pg/mL.
• Vitamin D hormone replacement therapy refers to the administration to a patient of an effective amount of an active vitamin D hormone such as 1,25- dihydroxyvitamin D 3 and/or 1,25-dihydroxyvitamin D 2 , optionally together with or other metabolites and analogs of Vitamin D which can substantially occupy the intracellular VDR.
• an active vitamin D hormone such as 1,25- dihydroxyvitamin D 3 and/or 1,25-dihydroxyvitamin D 2 , optionally together with or other metabolites and analogs of Vitamin D which can substantially occupy the intracellular VDR.
• Vitamin D in reference to intraluminal, intracellular and blood levels of Vitamin D refers to a total concentration of the vitamin D compound markedly greater than the generally stable levels observed in a Vitamin D-replete subject, animal or human patient over the course of any 24-hour period by laboratory measurement when Vitamin D supplementation has been withheld for at least 30 days.
• “Adverse supraphysiologic surge” refers to a local or serum concentration of a vitamin D compound that elicits an adverse effect, such as excessive extrarenal hormone production, leading to a local adverse effect, for example on calcium or phosphorus metabolism, inhibition of hepatic 25-hydroxylation of vitamin D, increased catabolism of both Vitamin D and 25- hydroxyvitamin D, hypercalciuria, hypercalcemia and/or hyperphosphatemia, with possible cardiovascular sequelae.
• terapéuticaally effective amount depends on the patient's condition and is an amount effective to achieve a desired clinical effect, e.g. to maintain a laboratory test value within the normal range or the recommended range for that patient's condition, or an amount effective to reduce the occurrence or severity of a clinical sign or symptom of disease.
• a desired clinical effect e.g. to maintain a laboratory test value within the normal range or the recommended range for that patient's condition, or an amount effective to reduce the occurrence or severity of a clinical sign or symptom of disease.
• a therapeutically effective amount is an amount effective on average to achieve at least a 15%, 20%, 25% or 30% reduction in serum parathyroid hormone levels (iPTH) from baseline levels without treatment.
• a therapeutically effective amount is an amount effective on average to reach CKD Stage-specific iPTH target ranges, which for Stage 3 is 35-70 pg/mL (equivalent to 3.85-7.7 pmol/L), for Stage 4 is 70-110 pg/mL (equivalent to 7.7-12.1 pmol/L), and for Stage 5 is 150-300 pg/mL (equivalent to 16.5-33.0 pmol/L) (defined in K/DOQI Guideline No. 1).
• hypoparathyroidism refers to primary hyperparathyroidism, secondary hyperparathyroidism and hyperparathyroidism secondary to chronic kidney disease (Stage 3, 4 or 5).
• subject generally includes humans, mammals (e.g., dogs, cats, rodents, sheep, horses, cows, goats), veterinary animals and zoo animals.
• the term "pharmaceutically acceptable salt” refers to an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.
• hydrate refers to a salt of a compound of the invention, wherein molecules of water are incorporated in the crystal lattice.
• solvate refers to a compound of the invention wherein molecules of a suitable solvent are incorporated in the crystal lattice.
• a suitable solvent is physiologically tolerable at the dosage administered.
• An example of a suitable solvent is ethanol.
• esters refers to esters that are formed with one or more available hydroxyl groups of the prodrug compounds described herein.
• Contemplated esters include phenyl esters, aliphatic (C8-C24) esters, acyloxymethyl esters, carbamates, and amino acid esters.
• R 1 when R 1 is OH in a prodrug compound described herein, it may be acylated using a carboxylic acid or an activated form of a carboxylic acid in the presence of a base, and optionally, in an inert solvent (e.g. an acid chloride in pyridine).
• an inert solvent e.g. an acid chloride in pyridine
• alkyl refers to straight chained and branched saturated hydrocarbon groups, nonlimiting examples of which include methyl, ethyl, and straight chain and branched propyl and butyl groups.
• Ci- 6 alkyl refers to alkyl groups that can have, for example, from 1 to 6 carbon atoms encompassing the entire range (i.e., 1 to 6 carbon atoms), as well as all subgroups (e.g., 1-5, 2-6, 1-4, 3-6, 1, 2, 3, 4, 5, and 6 carbon atoms).
• C 1 _ 4 alkyl refers to alky groups that can have, for example, from 1 to 4 carbon atoms encompassing the entire range (i.e., 1 to 4 carbon atoms), as well as all subgroups (e.g., 1-3, 2-4, 2-3, 3-4, 1, 2, 3, and 4 carbon atoms).
• Ci_ 2 alkyl refers to alky groups that can have 1 or 2 carbon atoms).
• Ci-ealkyl and C ⁇ alkyl can be unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1-4 alkyl, C 2 _ 4 alkenyl, OC 1-4 alkyl, OC 2 _ 4 alkenyl, CF 3 , N0 2 , halo, OH, OCF 3 , SH, SCi_ 4 alkyl, NH 2 , NHC M alkyl, NHC 2 _ 4 alkenyl, N(Ci_ 4 alkyl)(Ci_ 4 alkyl), N(C 2 _ 4 alkenyl)(C 1 _ 4 alkyl) and CN.
• alkenyl refers to straight and/or branched chain unsaturated alkenyl radicals.
• C 2 _ 6 alkenyl refers to alkenyl groups that can have, for example, from 2 to 6 carbon atoms encompassing the entire range (i.e., 2 to 6 carbon atoms), as well as all subgroups (e.g., 2-5, 3-6, 2-4, 4-6, 2, 3, 4, 5, and 6 carbon atoms).
• C 2 _ 4 alkenyl refers to alkeny groups that can have, for example, from 2 to 4 carbon atoms encompassing the entire range (i.e., 2 to 4 carbon atoms), as well as all subgroups (e.g., 2-3, 3-4, 2, 3, and 4 carbon atoms).
• C 2 _ 6 alkenyl and C 2 _ 4 alkenyl can be unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1-4 alkyl, C 2 _ 4 alkenyl, OC 1-4 alkyl, OC 2 _ 4 alkenyl, CF 3 , N0 2 , halo, OH, OCF 3 , SH, SC 1-4 alkyl, NH 2 , NHC 1-4 alkyl, NHC 2-4 alkenyl, NiC ⁇ alkylXC ⁇ alkyl), N(C 2 _
• cycloalkyl refers to saturated, non-aromatic cyclic alkyl radicals.
• C 3 _ 6 cycloalkyl refers to cycloalkyl groups containing from three to six carbon atoms and includes cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
• Heterocycloalkyl is defined similarly as cycloalkyl, except the ring contains one or more heteroatoms, for example, one to three heteroatoms, independently selected from the group consisting of oxygen, nitrogen, and sulfur.
• Nonlimiting examples of heterocycloalkyl groups include piperdinyl, tetrahydrofuranyl, tetrahydropyranyl, dihydrofuranyl, and the like.
• Cycloalkyl and heterocycloalkyl groups can optionally substituted with 1 to 5 substituents independently selected from the group consisting of C 1-4 alkyl, C 2 _ 4 alkenyl, OC 1-4 alkyl, OC 2 _ 4 alkenyl, CF 3 , N0 2 , halo, OH, OCF3, SH, SC 1-4 alkyl, NH 2 , NHC 1-4 alkyl, NHC 2-4 alkenyl, N(Ci- 4 alkyl)(Ci- 4 alkyl), N(C 2 _ 4 alkenyl)(C 1 _ 4 alkyl) and CN.
• cycloalkenyl refers to unsaturated, non-aromatic cyclic alkenyl radicals.
• cyclo(C 3 -C6)alkenyl refers to cyclic alkenyl radicals containing from three to six carbon atoms and includes cyclopropenyl, cylobutenyl, cyclopentenyl and cyclohexenyl.
• halo refers to the halogens of Group VIIA of the periodic table, such as F, CI, Br, and I.
• aryl refers to a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group, e.g., phenyl or naphthyl. Unless otherwise indicated, an aryl group can be unsubstituted or substituted with 1 to 5 groups independently selected from C ⁇ alkyl, C 2 - 4 alkenyl, OC ⁇ alkyl, OC 2 _ 4 alkenyl, OH, CF 3 , OCF 3 , halo, SH, SC ⁇ alkyl, SC ⁇ alkenyl NH 2 , NHC 1-4 alkyl, NHC 2-4 alkenyl, NCC ⁇ alkylXC M alkyl), N(C M alkenyl)(Ci- 4 alkyl)CN, C(0)OH, C(0)OC 1-4 alkyl, C(0)OC 2 _ 4 alkenyl, C(0)NHC 1-4 alkyl, C(0)NHC 2 _ 4 alkenyl, C(0)NHC 1-4 alky
• aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, chlorophenyl, methylphenyl, methoxyphenyl, trifluoromethylphenyl, nitrophenyl, and 2,4- methoxychlorophenyl .
• heteroaryl refers to a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group, containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring.
• a heteroaryl group can be unsubstituted or substituted with 1 to 5 groups independently selected from C 1-4 alkyl, C 2 _ 4 alkenyl, OC 1-4 alkyl, OC 2 _ 4 alkenyl, OH, CF 3 , OCF 3 , halo, SH, SC ⁇ alkyl, SC 2 _ 4 alkenyl NH 2 , NHCi_ 4 alkyl, NHC 2 _ 4 alkenyl, NCC ⁇ alkylXC ⁇ alkyl), N(C M alkenyl)(Ci- 4 alk l)CN, C(0)OH, C(0)OC 1-4 alkyl, C(0)OC 2 _ 4 alkenyl, C(0)NHC 1-4 alkyl, C(0)NHC 2 _ 4 alkenyl, NHC(0)C 1-4 alkyl, NHC(0)C 2 _ 4 alkenyl, OC(0)Ci_ 4 alkyl, OC(0)C 2 _ 4 alkenyl, S
• heteroaryl groups include, but are not limited to, thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.
• the term “modulate” includes the inhibition or suppression of a function or activity (such as CYP24 activity) as well as the enhancement of a function or activity.
• a function or activity such as CYP24 activity
• CYP24 activity is to reduce the function of activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition.
• animal includes all members of the animal kingdom including human.
• the animal is preferably a human.
• the term "cell” includes a plurality of cells. Administering a compound to a cell includes in vivo, ex vivo, and in vitro treatment.
• cancer cells includes all forms of cancer or neoplastic disease.
• catabolism refers to the metabolic process by which organisms convert substances into compounds for excretion.
• any numerical value recited herein includes all values from the lower value to the upper value, i.e., all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.
• a concentration range or a beneficial effect range is stated as 1% to 50%, it is intended that intermediate values and ranges, such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended.
• the invention provides compounds of Formula I, or a pharmaceutically acceptable salt, solvate, or hydrate thereof:
• each— independently is a single bond or a double bond
• n 0, 1 or 2;
• R 1 is selected from the group consisting of OH, OCi-ealkyl, and halo;
• R 4 is Ci-ealkyl
• R 5 and R 6 are each independently H, halo, Ci ⁇ alkyl, or can be taken, together with the carbon atom to which they are bound, to form a C3_ 6 cycloalkyl ring, with the proviso that when— between carbon-23 and carbon-24 is a double bond, then R 5 is absent;
• R 7 is selected from the group consisting of O, NH, N(C 1-6 alkyl), and NC(0)R 9 ;
• R is selected from the group consisting of C 1-6 alkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl, wherein each of C 1-6 alkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl are either unsubstituted or substituted with 1 to
• substituents independently selected from the group consisting of C 1 _ 4 alkyl, OC 1-4 alkyl, CF 3 , N0 2 , halo, OH, OCF 3 , SH, SC ⁇ alkyl, NH 2 , NHC ⁇ alkyl, N(Ci- 4 alk l)(Ci- 4 alk l), and CN; and
• R 9 is selected from the group consisting of C 1-6 alkyl, C 3 _ 6 cycloalkyl, aryl-Ci ⁇ alkyl, aryl and heteroaryl, wherein each of C 1-6 alkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl are either unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1-4 alkyl,
• OCi_ 4 alkyl CF 3 , N0 2 , halo.
• n 0 or 1.
• R 1 being OH or halo, more preferably OH or F, and further preferably OH.
• R 4 being C 1-4 alkyl, and more preferably CH .
• R 5 and R 6 each independently being H, C 1 _ 2 alkyl, or halo, and more preferably H, CH 3 , or halo, with the proviso that when— between carbon-23 and carbon-24 is a double bond, then R 5 is absent.
• R 7 being selected from the group consisting of O, NH, and N(C 1-6 alkyl), and more preferably O or NH.
• R 8 being selected from the group consisting of C 1-6 alkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl, wherein each of C 1-6 alkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl are either unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1-4 alkyl, OC 1-4 alkyl, CF 3 , N0 2 , and halo; more preferably R is selected from the group consisting of C 1-4 alkyl, aryl and heteroaryl, wherein each of aryl and heteroaryl are either unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1-4 alkyl, OC 1-4 alkyl, CF , N0 2 , and halo;
• R is selected from the group consisting of C 1-4 alkyl and aryl, wherein aryl is either unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1-4 alkyl, OC 1-4 alkyl, CF 3 , N0 2 , and halo.
• R 24 is a double bond, then R 5 is absent; R 7 is O or NH; and R 8 is selected from the group consisting of C 1-4 alkyl, aryl and heteroaryl, wherein each of aryl and heteroaryl are either unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1-4 alkyl, OC 1-4 alkyl, CF 3 , N0 2 , and halo.
• aryl is either unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1-4 alkyl, OC 1-4 alkyl, CF , N0 2 , and halo.
• the compounds of Formula I include:
• All of the compounds of Formula I have more than one chiral center. Where the compounds according to the invention possess more than one chiral center, they may exist as stereoisomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present invention.
• the stereochemistry of the A, C and D rings and at the C20 position of the compounds of the invention is preferably that of natural 25- dihydroxy vitamin D 3 . When R is not O, the stereochemistry at the sulfur atom may be either R or S. Therefore the present invention preferably provides compounds of Formula I, and
• n, and R -R are as defined above.
• One class of embodiments is characterized by— being a double bond between carbon-23 and carbon-24 with '£" stereochemistry.
• such compounds of Formula I may also contain certain amounts (e.g. less than 20%, preferably less than 10%, more preferably less than 5%) of compounds of Formula I having alternate stereochemistry.
• a compound of Formula I having the 3 ⁇ - stereochemistry of natural 25 -dihydroxy vitamin D 3 shown above, may contain less then 20%, preferably less then 10%, more preferably less then 5%, of a compound of Formula I having the unnatural 3cc-stereochemistry.
• the compounds of Formula I include:
• the compounds of Formula I include:
• the invention provides a compound of Formula II, or a pharmaceutically acceptable salt, solvate or hydrate thereof:
• each— independently is a single bond or a double bond
• R 1 is selected from the group consisting of OH, OCi-ealkyl, and halo;
• R 4 is Ci-ealkyl
• R 5 and R 6 are each independently H, halo, C 1 _ 4 alkyl, or can be taken, together with the carbon atom to which they are bound, to form a C3_ 6 cycloalkyl ring, with the proviso that when— between carbon-23 and carbon-24 is a double bond, then R 5 is absent;
• R is selected from the group consisting of H, Ci-ealkyl, C 2 - 6 alkenyl, aryl and heteroaryl, with Ci_ 6 alkyl and C 2 - 6 alkenyl being unsubstituted or substituted with 1 to 4 groups independently selected from C 1-4 alkyl, C 2 - 4 alkenyl, OC 1-4 alkyl, OC 2 - alkenyl, OH, halo, NH 2 , NHC 1-4 alkyl, NHC 2 - alkenyl, N(C 2 - 4 alkenyl)(C 1 _ 4 alkyl), and with aryl and heteroaryl being
• R is selected from the group consisting of Ci- 6 alkyl, C 2 - 6 alkenyl, cyclo(C 3 -C 6 )alkyl, cyclo(Cs- C 6 )alkenyl, aryl, heteroaryl, aryl-Ci-ealkyl, aryl-C 2 - 6 alkenyl, heteroaryl-Ci-ealkyl, and heteroaryl-C 2 _ 6 alkenyl with Ci-ealkyl and C 2 _ 6 alkenyl being unsubstituted or substituted with 1-4 groups independently selected from Ci ⁇ alkyl, C 2 - 4 alkenyl, OC 1-4 alkyl, OC 2 - 4 alkenyl, OH, halo, NH 2 , NHQ- 4 alkyl, NHC 2 _ 4 alkenyl, and N(C 2 - 4 alkenyl)(C 1 _ 4 alkyl), and with cyclo(C 3 - C 6
• R 1 being OH or F, and more preferably OH.
• R 4 being C 1-4 alkyl, and more preferably CH .
• each R 5 and R 6 independently being H, C 1 _ 2 alkyl, or halo, more preferably H, CH 3 ,C1, or F, and further preferably both H, with the proviso that when— between carbon-23 and carbon-24 is a double bond, then R 5 is absent.
• R 7 being selected from the group consisting of H, C 1 _ 4 alkyl, C 2 - 5 alkenyl, aryl and heteroaryl, with C 1-4 alkyl and C 2 - 4 alkenyl being unsubstituted or substituted with 1 to 4 groups independently selected from C 1-4 alkyl, C 2 - 4 alkenyl, OC 1-4 alkyl, OC 2 - 4 alkenyl, OH, and halo, and with aryl and heteroaryl being unsubstituted or substituted with 1 to 5 groups independently selected from C 1-4 alkyl, C 2 - 4 alkenyl, OC 1-4 alkyl, OC 2 - 4 alkenyl, OH, CF 3 , OCF , and halo.
• R is selected from the group consisting of H, C 1-4 alkyl, C 2 - 5 alkenyl, aryl and heteroaryl, with aryl and heteroaryl being unsubstituted or substituted with 1 to 5 groups independently selected from C 1-4 alkyl, OC 1-4 alkyl, OH, CF 3 , OCF 3 , and halo.
• R 8 being selected from the group consisting of C 1-4 alkyl, C 2 - 4 alkenyl, cyclo(C 3 -C 6 )alkyl, cyclo(Cs-C 6 )alkenyl, aryl, heteroaryl, with C 1-4 alkyl and C 2 - 4 alkenyl being unsubstituted or substituted with 1-4 groups independently selected from Ci ⁇ alkyl, C 2 - 4 alkenyl, OC 1-4 alkyl, OC 2 - 4 alkenyl, OH, and halo, and with cyclo(C 3 -C 6 )alkyl, cyclo(Cs-C 6 )alkenyl aryl, and heteroaryl, being unsubstituted or substituted with 1-5 groups independently selected from C 1-4 alkyl, C 2 - 4 alkenyl, OC 1-4 alkyl, OC 2 _ 4 alken
• R is selected from the group consisting of C 1-4 alkyl, aryl, and heteroaryl, with aryl, and heteroaryl, being unsubstituted or substituted with 1-5 groups independently selected from C 1-4 aLkyl, OC 1-4 alkyl, OH, CF , OCF , and halo. Further preferably, R is selected from the group consisting of C 1-4 alkyl, aryl, and heteroaryl, with aryl, and heteroaryl, being unsubstituted or substituted with 1-5 groups independently selected from C 1-4 alkyl, OCi- 4 alkyl, OH, CF 3 , OCF 3 , and halo.
• a compound of Formula II includes:
• the compounds of Formula II have more than one chiral center. Where the compounds according to the invention possess more than one chiral center, they may exist as stereoisomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present invention.
• C20 position of the compounds of the invention is preferably that of natural 25 -dihydroxy vitamin D 3 . Therefore the present invention preferably provides compounds of Formula I, and
• R -R are as defined above.
• One class of embodiments is characterized by— being a double bond between carbon-23 and carbon-24 with '£" stereochemistry.
• such compounds of Formula II may also contain certain amounts (e.g. less than 20%, preferably less than 10%, more preferably less than 5%) of compounds of Formula II having alternate stereochemistry.
• a compound of Formula II having the 3 -stereochemistry of natural 25 -dihydroxy vitamin D 3 shown above, may contain less then 20%, preferably less then 10%, more preferably less then 5%, of a compound of Formula II having the unnatural 3 a- stereochemistry.
• Another class of embodiments of the present invention is characterized by a compound of Formula II:
• a compound of Formula II includes:
• the invention provides compounds of Formula III, or a pharmaceutically acceptable salt, solvate, or hydrate thereof:
• each— independently is a single bond or a double bond
• R 1 is selected from the group consisting of OH, OCi_ 6 alkyl, and halo;
• R 4 is Ci-ealk l
• R 5 and R 6 are each independently H, halo, C 1-4 alkyl, or can be taken, together with the carbon atom to which they are bound, to form a C 3 _ 6 cycloalkyl ring;
• R 7 is selected from the group consisting of O, NH, N(C 1-6 alkyl), and NC(0)R 9 ;
• R is selected from the group consisting of Ci-ealkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl, wherein each of Ci_ 6 alkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl are either unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1-4 alkyl, OC 1-4 alkyl, CF 3 , N0 2 , halo, OH, OCF 3 , SH, SC alk l, NH 2 , NHC M alk l, N(Ci_ 4 alkyl)(Ci_ 4 alkyl), and CN; and,
• R 9 is selected from the group consisting of C 1-6 alkyl, C 3 _ 6 cycloalkyl, aryl-C 1-4 alkyl, aryl and heteroaryl, wherein each of C 1-6 alkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl are either unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of Ci_ 4 alkyl, OCi_ 4 alkyl, CF 3 , N0 2 , halo.
• R 1 being OH or halo, more preferably OH or F, and further preferably OH.
• R 4 being Ci_ 4 alkyl, and more preferably CH 3 .
• R 7 being selected from the group consisting of O, NH, and N(C 1-6 alkyl), and more preferably.
• R 8 being selected from the group consisting of C 1-6 alkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl, wherein each of C 1-6 alkyl, C 3 _ 6 cycloalkyl, aryl and heteroaryl are either unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1-4 alkyl, OC 1-4 alkyl, CF , N0 2 , and halo; more preferably R is selected from the group consisting of C 1-4 alkyl, aryl and heteroaryl, wherein each of aryl and heteroaryl are either unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1 _ 4 alkyl, OC 1-4 alkyl, CF 3 , N0 2 , and halo; further preferablyR is selected from the group consisting of C 1-4 alkyl
• R 7 is O or NH
• R 8 is selected from the group consisting of C 1-4 alkyl and aryl, wherein aryl is either unsubstituted or substituted with 1 to 5 substituents independently selected from the group consisting of C 1-4 alkyl, OC 1-4 alkyl, CF 3 , N0 2 , and halo.
• spe la III include:
• All of the compounds of Formula III have more than one chiral center. Where the compounds according to the invention possess more than one chiral center, they may exist as stereoisomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present invention.
• the stereochemistry of the A, C and D rings and at the C20 position of the compounds of the invention is preferably that of natural 25- dihydroxy vitamin D 3 . When R is not O, the stereochemistry at the sulfur atom may be either R or S. Therefore the present invention preferably provides compounds of Formula I, and
• R -R are as defined above.
• One class of embodiments is characterized by— being a double bond between carbon-22 and carbon-23 with '£" stereochemistry.
• such compounds of Formula III may also contain certain amounts (e.g. less than 20%, preferably less than 10%, more preferably less than 5%) of compounds of Formula III having alternate stereochemistry.
• a compound of Formula III having the 3 -stereochemistry of natural 25 -dihydroxy vitamin D shown above, may contain less then 20%, preferably less then 10%, more preferably less then 5%, of a compound of Formula III having the unnatural 3 a- stereochemistry.
• the compounds of Formula III include:
• the compounds of Formula III include:
• the 1-nor compounds of the invention include a double bond on their side chain, such as when— between carbon-23 and carbon-24 in Formula I is a double bond (e.g., compound lb), or when— between carbon-22 and carbon-23 in Formula III is a double bond (e.g., compound Ilia).
• a double bond incorporated into the side chain introduces planarity to the side chain of the prodrug, which increases the ability of the prodrug to bind to the active site of the enzyme, relative to a prodrug that does not have a planarity side chain.
• the 1-nor compounds of the invention include a side chain with a low degree of steric hindrance.
• Side chains with a low degree of steric hindrance include those that are unsubstituted at the 22-, 23- and/or 24-positions; contain a double bond between the 22-position and the 23-position; or a double bond between the 23-position and the 24-position; contain 10 or fewer atoms in the longest linear chain of the side chain, or a combination thereof.
• a less sterically hindered side chain increases the ability of the prodrug to bind to the active site of the enzyme, relative to a prodrug with more sterically hindered side chain.
• prodrugs that have a less sterically hindered side chain include compound lb and Ig.
• the 1-nor compounds of the invention include a less sterically hindered side chain that itself has a double bond. Examples of these compounds include compound lb, If, and Hi.
• the present invention also encompasses 1-nor analogs of other known approved or experimental active Vitamin D compounds, such as, for example, 1-nor prohormone forms of paricalcitol, alfacacidiol, 22-oxacalcitriol (OCT), calcipotril (i.e., DOVONEX), falecalcitriol, tacalcitol, EB1089, KH1060, ED-71, gemini Vitamin D analogs (e.g., BXL024), la,25(OH) 2 -16- ene-20-cyclopropylvitamin D 3 (e.g., BXL-62), and others. Examples of these compounds are shown below:
• the present invention includes ester derivatives of the prodrug compounds of the invention.
• ester derivatives are esters that are formed with one or more available hydroxyl groups of the prodrug compounds described herein.
• Contemplated esters include phenyl esters, aliphatic (C8-C24) esters, acyloxymethyl esters, carbamates, and amino acid esters.
• R 1 is OH in a prodrug compound described herein, it may be acylated using a carboxylic acid or an activated form of a carboxylic acid in the presence of a base, and optionally, in an inert solvent (e.g. an acid chloride in pyridine).
• X is a leaving group (e.g. N-hydroxysuccinimide, halogen, alcohol, sulfonate ester, carboxylate).
• Contemplated activated forms of carboxylic acids include acyl chlorides, anhydrides, and esters.
• the present invention also includes radiolabeled forms of compounds of the invention, for example, compounds of the invention labeled by incorporation within the structure 3 H or 14 C or a radioactive halogen such as 125 I.
• a radiolabeled compound of the invention may be prepared using standard methods known in the art.
• tritium may be incorporated into a compound of the invention using standard techniques, for example by hydrogenation of a suitable precursor to a compound of the invention using tritium gas and a catalyst.
• a compound of the invention containing radioactive iodo may be prepared from the corresponding trialkyltin (suitably trimethyltin) derivative using standard iodination conditions, such as [ 125 I] sodium iodide in the presence of chloramine-T in a suitable solvent, such as dimethylformamide.
• the trialkyltin compound may be prepared from the corresponding non-radioactive halo, suitably iodo, compound using standard palladium-catalyzed stannylation conditions, for example hexamethylditin in the presence of tetrakis(triphenylphosphine) palladium (0) in an inert solvent, such as dioxane, and at elevated temperatures, suitably 50-100°C.
• Vitamin D prohormones e.g. 25 -hydroxy vitamin D 3
• active hormones e.g. l ,25-dihydroxy vitamin D 3
• the blood levels of 1,25-dihydroxyvitamin D 3 and the substrate 25 -hydroxy vitamin D prohormone, and regulation thereof, can be affected by vitamin D hormone analogs, 24-sulfoximine vitamin D 3 compounds, oxime analogs of la,25-dihydroxyvitamin D 3 , and 25-S0 2 substituted analogs of l ,25-dihydroxyvitamin D .
• the compounds of the invention will be metabolized into active hormones by CYP27B1 as shown, for example, below for compound Ibii.
• the 1-deoxy compounds of Formulas I and II do not substantially bind to the VDR (see Figure 1).
• physiological concentrations of these hormone precursors exert little, if any, biological actions without metabolism by CYP27B 1. Therefore, the 1-deoxy compounds represented by Formulas I and II can act as effective prodrugs of their 1-hydroxylated, active counterparts, e.g. such as 24-sulfoximine, oxime, and 25-S0 2 substituted analogs of la,25-dihydroxyvitamin D .
• the compounds will provide slower, "on- demand” introduction of these low calcemic, anti-proliferative analogs of l ,25-dihydroxy vitamin D 3 to the body.
• Administration of the compounds of Formulas I and II as prodrugs of the low calcemic, anti-proliferative, CYP24 inibitory analogs of la,25-dihydroxyvitamin D 3 e.g. 24-sulfoximine, oxime, and 25-S0 2 substituted analogs
• Direct administration of 25 -hydroxy vitamin D can produce surges or spikes in blood and intracellular 25-hydroxyvitamin D levels, thereby promoting toxicity manifesting as hypercalcemia and hypercalciuria.
• surges or spikes in blood and intracellular 25-hydroxyvitamin D levels can promote one or more disadvantages, including (a) competitive displacement of Vitamin D hormones from the serum Vitamin D Binding Protein (DBP) and excessive delivery of the displaced hormones to tissues containing VDR, and (b) transiently excessive renal and extrarenal production of Vitamin D hormones, which together can lead to local aberrations in calcium and phosphorus metabolism.
• DBP serum Vitamin D Binding Protein
• 25-hydroxyvitamin D levels can promote catabolism of both Vitamin D and 25-hydroxyvitamin D by 24-and/or 26-hydroxylation in the kidney and other tissues, and down-regulation of hepatic production of Vitamin D prohormones, unnecessarily impeding the efficient repletion of Vitamin D insufficiency or deficiency, and, additional local aberrations in calcium and phosphorus homeostasis mediated by direct binding to VDR.
• 25- hydroxyvitamin D is believed to promote its intestinal absorption via a mechanism substantially involving transport to the liver in chylomicrons, rather than bound to the serum DBP.
• the invention is related to a method of treating or preventing vitamin D deficiency by administering a compound of Formula I or II to a subject in need of vitamin D supplementation, either prophylactically to prevent vitamin D insufficiency or deficiency, or therapeutically to supplement low serum vitamin 25(OH)D levels with a prodrug of the invention to provide a sufficient pool of prohormone and prohormone analog for conversion to active vitamin D (native) and an analog thereof.
• the prodrugs of the invention are also useful for preventing or treating hyperparathyroidism, for example hyperparathyroidism secondary to CKD.
• serum 25(OH)D values less than 5 ng/mL indicate severe deficiency associated with rickets and osteomalacia. Although 30 ng/mL has been suggested as the low end of the normal range, more recent research suggests that PTH levels and calcium absorption are not optimized until serum total 25(OH)D levels reach approximately 40 ng/mL. [See also Vieth, R. Prog Biophys Mol Biol. 2006 Sep;92(l):26-32.]
• Patients in need of vitamin D supplementation include healthy subjects and subjects at risk for vitamin D insufficiency or deficiency, for example, subjects with stage 1, 2, 3, 4 or 5 chronic kidney disease; infants, children and adults that do not drink vitamin D fortified milk (e.g.
• lactose intolerant subjects subjects with milk allergy, vegetarians who do not consume milk, and breast fed infants
• subjects with rickets subjects with dark skin (e.g., in the U.S., 42% of African American women between 15 and 49 years of age were vitamin D deficient compared to 4% of white women); the elderly (who have a reduced ability to synthesize vitamin D in skin during exposure to sunlight and also are more likely to stay indoors); institutionalized adults (who are likely to stay indoors, including subjects with Alzheimer's disease or mentally ill); subjects who cover all exposed skin (such as members of certain religions or cultures); subjects who always use sunscreen (e.g., the application of sunscreen with an Sun Protection Factor (SPF) of 8 reduces production of vitamin D by 95%, and higher SPFs may further reduce cutaneous vitamin D production); subjects with fat malabsorption syndromes (including but not limited to cystic fibrosis, cholestatic liver disease, other liver disease, gallbladder disease, pancreatic enzyme deficiency, Crohn's disease,
• carbamazepine, and rifampin subjects taking medications that reduce absorption of vitamin D, including cholestyramine, colestipol, orlistat, mineral oil, and fat substitutes; subjects taking medications that inhibit activation of vitamin D, including ketoconazole; subjects taking
• the prodrugs of the invention are useful for the prophylactic or therapeutic treatment of vitamin D-responsive diseases, i.e., diseases where vitamin D, 25(OH)D or active vitamin D (e.g., 1, 25(OH) 2 D) prevents onset or progression of disease, or reduces signs or symptoms of disease.
• vitamin D-responsive diseases include cancer, for example (e.g., breast, lung, skin, melanoma, colon, colorectal, rectal, prostate and bone cancer).
• l,25(OH) 2 D has been observed to induce cell differentiation and/or inhibit cell proliferation in vitro for a number of cells.
• Vitamin D-responsive diseases also include autoimmune diseases, for example, type I diabetes, multiple sclerosis, rheumatoid arthritis, polymyositis, dermatomyositis, scleroderma, fibrosis, Grave's disease, Hashimoto's disease, acute or chronic transplant rejection, acute or chronic graft versus host disease, inflammatory bowel disease, Crohn's disease, systemic lupus erythematosis, Sjogren's Syndrome, eczema and psoriasis, dermatitis, including atopic dermatitis, contact dermatitis, allergic dermatitis and/or chronic dermatitis.
• autoimmune diseases for example, type I diabetes, multiple sclerosis, rheumatoid arthritis, polymyositis, dermatomyositis, scleroderma, fibrosis, Grave's disease, Hashimoto's disease, acute or chronic transplant rejection, acute or chronic graft
• Vitamin D-responsive diseases also include other inflammatory diseases, for example, asthma, chronic obstructive pulmonary disease, polycystic kidney disease (PKD), polycystic ovary syndrome, pancreatitis, nephritis, hepatitis, and/or infection. Vitamin D-responsive diseases have also been reported to include hypertension and cardiovascular diseases.
• cardiovascular diseases for example, subjects with atherosclerosis, arteriosclerosis, coronary artery disease, cerebrovascular disease, peripheral vascular disease, myocardial infarction, myocardial ischemia, cerebral ischemia, stroke, congestive heart failure, cardiomyopathy, obesity or other weight disorders, lipid disorders (e.g. hyperlipidemia, dyslipidemia including associated diabetic dyslipidemia and mixed dyslipidemia
• hypoalphalipoproteinemia hypertriglyceridemia, hypercholesterolemia, and low HDL (high density lipoprotein)
• metabolic disorders e.g. Metabolic Syndrome, Type II diabetes mellitus, Type I diabetes mellitus, hyperinsulinemia, impaired glucose tolerance, insulin resistance, diabetic complication including neuropathy, nephropathy, retinopathy, diabetic foot ulcer and cataracts
• thrombosis e.g. Metabolic Syndrome, Type II diabetes mellitus, Type I diabetes mellitus, hyperinsulinemia, impaired glucose tolerance, insulin resistance, diabetic complication including neuropathy, nephropathy, retinopathy, diabetic foot ulcer and cataracts
• the compounds of Formulas I and II are prodrugs of hormones that selectively modulate CYP24, the enzyme that metabolizes lcc,25-dihydroxy vitamin D 3 . Therefore, the levels of lcc,25- dihydroxy vitamin D 3 (either endogenous or administered as part of a chemo therapeutic regimen), or analogs thereof, may also be modulated with the prodrugs of Formulas I and II. Diseases that benefit from a modulation, in particular an increase, of the levels of lcc,25-dihydroxyvitamin D 3 can therefore be treated using a prodrug of a modulator of CYP24. By acting preferentially on CYP24, side effects caused by interaction with other enzymes and receptors may be reduced.
• the present invention provides a method for treating diseases which benefit from a modulation, preferably an increase, of the levels of lcc,25-dihydroxyvitamin D 3 , or an analog of lcc,25- dihydroxy vitamin D 3 , comprising administering an effective amount of a compound of Formula I or II to a cell or animal in need thereof.
• the invention also includes the use of a compound of Formula I or II to treat diseases which benefit from a modulation, preferably an increase, of the levels of lcc,25-dihydroxy vitamin D , or an analog of lcc,25-dihydroxyvitamin D .
• the invention includes a use of a compound of the invention to prepare a medicament to treat diseases which benefit from a modulation, preferably an increase, of the levels of lcc,25-dihydroxy vitamin D , or an analog of lcc,25-dihydroxy vitamin D .
• Inhibition of CYP24 will inhibit the catabolism of lcc,25-dihydroxy vitamin D 3 , or its analogs, which is expected to lengthen the biological lifetime of these compounds and thus allow smaller amounts of them to be used for effective disease treatment. Such smaller dosing is expected to avoid, or at least minimize, the hypercalcemic toxicity associated with medicinal use of lcc,25- dihydroxyvitamin D and its analogs. Further, by inhibiting the catabolism of lcc,25- dihydroxy vitamin D 3 , the prodrugs of the invention will increase the endogenous levels of this hormone, which will have similar beneficial therapeutic effects.
• the present invention provides a method for treating diseases which benefit from inhibiting the catabolism of lcc,25-dihydroxyvitamin D 3 , or an analog of lcc,25-dihydroxy vitamin D 3 , comprising administering an effective amount of the prodrug of the invention to a cell or animal in need thereof.
• the invention also includes the use of a prodrug of the invention to treat diseases which benefit from inhibiting the catabolism of lcc,25-dihydroxy vitamin D , or an analog of lcc,25-dihydroxyvitamin D 3 .
• the invention includes a use of a prodrug of the invention to prepare a medicament to treat diseases which benefit from inhibiting the catabolism of lcc,25-dihydroxyvitamin D , or an analog of lcc,25-dihydroxy vitamin D .
• VDDR hypophosphtatemic vitamin D resistant rickets
• the disease that benefits from a modulation in the levels of lcc,25- dihydroxy vitamin D 3 , or an analog of lcc,25-dihydroxyvitamin D can be selected from cancer, dermatological disorders (for example psoriasis), parathyroid disorders (for example
• hyperparathyroidism and secondary hyperparathyroidism for example
• osteoporosis and autoimmune disorders.
• the disease that benefits from a modulation is a cell proliferative disorder.
• a method for modulating cell proliferation (preferably inhibiting cell proliferation) and/or promoting cell differentiation comprising administering an effective amount of a prodrug of the invention to a cell or animal in need thereof.
• the invention also includes a use of a prodrug of the invention to modulate cell proliferation (preferably to inhibit cell proliferation) and/or to promote cell differentiation.
• the invention further includes a use of a prodrug of the invention to prepare a medicament to modulate cell proliferation (preferably to inhibit cell proliferation) and/or to promote cell differentiation.
• the method of the invention is useful in inhibiting the proliferation of abnormal but not normal cells.
• Abnormal cells include any type of cell that is causative of or involved in a disease or condition and wherein it is desirable to modulate or to inhibit the proliferation of the abnormal cell, or to promote its differentiation, in order to treat the disease or condition.
• Examples of abnormal cells include malignant or cancerous cells as well as cells that over-proliferate in inflammatory conditions such as psoriasis.
• the disease that benefits from a modulation in particular an increase, in the levels of lcc,25-dihydroxy vitamin D 3 , or an analog of lcc,25-dihydroxy vitamin D , is cancer.
• the present invention provides a method of treating cancer comprising administering an effective amount of a prodrug of the invention to a cell or animal in need thereof.
• the invention also includes a use of a prodrug of the invention to treat cancer.
• the invention further includes a use of a prodrug of the invention to prepare a medicament to treat cancer.
• the cancer is selected from the group consisting of breast cancer, lung cancer, prostate cancer, colon and colorectal cancer, kidney cancer, head and neck cancer, pancreatic cancer, skin cancer, Kaposi's sarcoma and leukemia.
• the invention provides a method of modulating CYP24 activity in a cell by administering an effective amount of a prodrug of the invention.
• the invention provides a method of inhibiting CYP24 activity in a cell by administering an effective amount of a prodrug of the invention.
• the present invention also provides a use of a prodrug of the invention to modulate, preferably to inhibit, CYP24 activity.
• the present invention further provides a use of a prodrug of the invention to prepare a medicament to modulate CYP24 activity, preferably to inhibit, CYP24 activity.
• the prodrugs of the invention can be used alone or in combination with other agents that modulate CYP24 activity, or in combination with other types of treatment (which may or may not modulate CYP24) for diseases that benefit from a modulation, preferably an increase, in the levels of lcc,25-dihydroxyvitamin D 3 , or analogs thereof, and/or an inhibition of the catabolism of lcc,25- dihydroxy vitamin D , or an analog thereof.
• the compounds of the invention can be administered in combination with lcc,25-dihydroxyvitamin D (calcitriol), an analog of lcc,25-dihydroxyvitamin D or other vitamin D receptor agonists.
• vitamin D receptor agonists such as lcc,25-dihydroxy vitamin D , or analogs thereof, will lengthen the biological lifetime or efficacy of these therapies and thus allow smaller amounts of the drug to be used for effective human chemotherapy; such smaller dosing will avoid, or at least reduce or minimize, the side effects, for example the hypercalcemic toxicity, associated with medicinal use of vitamin D agonist compounds.
• the present invention therefore provides a method of increasing the efficacy of a vitamin D receptor agonist comprising co-administering an effective amount of a prodrug of the invention and an effective amount of the vitamin D receptor agonist.
• the invention includes the use of a prodrug of the invention to increase the efficacy of a vitamin D receptor agonist and a use of a prodrug of the invention to prepare a medicament to increase the efficacy of a vitamin D receptor agonist.
• the vitamin D receptor agonist is lcc,25- dihydroxyvitamin D 3 , or an analog thereof.
• analog of lcc,25-dihydroxy vitamin D 3 it is meant a chemically modified analog of la,25 -dihydroxy vitamin D 3 which is a vitamin D receptor agonist, and preferably one which exhibits a therapeutic profile similar to la,25 -dihydroxy vitamin D 3 .
• Treatments used in combination with the compounds of the present invention may be based on the disease type and do not have to specifically target CYP24 activity or the VDR.
• the prodrugs of the invention are used in combination with other therapies and therapeutics to treat dermatological disorders, bone disorders, cancer and autoimmune disorders.
• Such therapies include, but are not limited to the following: for cancer: surgery, radiation, chemotherapies and biotherapies; for psoriasis: ultraviolet B radiation, chemotherapy and biotherapies.
• prodrugs of the invention would have therapeutic utility, for example, in inhibiting cell proliferation in any type of cancer or cell proliferative disorder.
• Prodrugs may be examined for their potency in inhibiting cell growth in cell proliferation assays such as inhibition of growth of murine keratinocyte cells (cell line PE) and for the inhibition of TPA-induced ornithine decarboxylase (ODC) activity as described in US. Patent No. 5,830,885, the contents of which are incorporated herein by reference.
• the prodrugs of the invention are useful in treating other conditions involving aberrant or abnormal cell proliferation.
• Other cell proliferative disorders that may be treated by the present invention include inflammatory diseases, allergies, autoimmune disease, graft rejection, psoriasis, restenosis, artherosclerosis, and any other disorder wherein it is desirable to inhibit, prevent or suppress cell growth.
• Prodrugs of the invention may be tested for their potency in a particular cell proliferation disorder using assays and techniques known to those of skill in the art.
• the prodrugs of the invention are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.
• the present invention provides a pharmaceutical composition comprising a prodrug of the invention in admixture with a pharmaceutically- acceptable excipient, e.g. a diluent or carrier.
• the present invention can further comprise a pharmaceutical composition comprising a prodrug of the invention and a vitamin D receptor agonist in admixture with a suitable a pharmaceutically-acceptable excipient.
• the vitamin D receptor agonist is lcc,25-dihydroxyvitamin D 3 , or an analog thereof.
• compositions containing the prodrugs of the invention can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
• suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
• the compositions can include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the
• the prodrugs of the invention may be used in the form of the free base, as ester prodrugs of the prodrugs of the invention, and in the form of solvates and as hydrates. All forms are within the scope of the invention.
• the described prodrugs or solvates thereof may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art.
• the compositions of the invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal (topical) administration and the pharmaceutical compositions formulated
• Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.
• a prodrug of the invention thereof may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
• the compound of the invention may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• a prodrug of the invention may also be administered parenterally.
• Solutions of a compound of the invention can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
• Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• a person skilled in the art would know how to prepare suitable formulations. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (1990 - 18th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
• compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device.
• the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use.
• the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon.
• the aerosol dosage forms can also take the form of a pump-atomizer.
• compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine.
• Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.
• compositions for topical administration may include, for example, propylene glycol, isopropyl alcohol, mineral oil and glycerin.
• Preparations suitable for topical administration include liquid or semi-liquid preparations such as liniments, lotions, applicants, oil-in- water or water-in-oil emulsions such as creams, ointments or pastes; or solutions or suspensions such as drops.
• the topical preparations may include one or more additional ingredients such as diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, preservatives, e.g. methyl hydroxybenzoate (including anti-oxidants), emulsifying agents and the like.
• Time-release compositions e.g. sustained- or extendedrelease
• directed (e.g., delayed) release compositions can be formulated, e.g. liposomes or those wherein the active compound is protected with differentially degradable coatings, such as by microencapsulation, multiple coatings, etc. It is also possible to freeze-dry the compounds of the invention and use the lypolizates obtained, for example, for the preparation of products for injection.
• the prodrugs of the invention may be administered to a subject alone or in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.
• the dosage of the prodrugs and/or compositions of the invention can vary depending on many factors such as the pharmacodynamic properties of the compound, the mode of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment and the type of concurrent treatment, if any, and the clearance rate of the compound in the animal to be treated.
• One of skill in the art can determine the appropriate dosage based on the above factors.
• ointments, creams, or lotions containing from 1-1000 ⁇ g/g of a compound of the invention may be administered.
• Oral preparations may be formulated, preferably as tablets, capsules, or drops, containing from 0.5-1000 ⁇ g of a prodrug of the invention, per dosage unit.
• the compounds of the invention may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. For ex vivo treatment of cells over a short period, for example for 30 minutes to 1 hour or longer, higher doses of compound may be used than for long term in vivo therapy.
• the prodrugs of the invention are also useful in diagnostic assays, screening assays and as research tools.
• the prodrugs of the invention may be useful in identifying or detecting a cell proliferative disorder.
• the prodrugs of the invention may be radiolabeled (as hereinbefore described) and contacted with a population of cells. The presence of the radiolabel on the cells may indicate a cell proliferative disorder.
• the prodrugs of the invention may be used to identify other compounds that modulate cell proliferation or CYP24 activity.
• the compounds of the invention may be used in receptor binding assays and assays to study the localization of CYP24. In such assays, the compounds may also be radiolabeled.
• NMR spectra were recorded on a Bruker 400 MHz spectrometer. Chemical shift values are recorded in ⁇ units (ppm). Solvents and chemicals were obtained from either the Aldrich Chemical Company or Acros Organics.
• IR spectroscopy was performed on a Perkin-Elmer series FT-IR instrument.
• UV spectroscopy was performed on a Varian Cary 50 Cone UV-Vis spectrophotometer.
• Mass spectrometry was performed on a VG-70S magnetic sector mass spectrometer. Optical rotation was determined using Jasco P-1010.
• the 1-nor compounds of the invention can be synthesized by reacting either 1- deoxy-A-ring-phosphine oxide or l-deoxy-19-nor-A-ring-phosphine oxide with a ketone precursor having a desired D-ring and a desired side chain by using n-butyllithium and hydrofluoric acid or camphorsulfonic acid.
• Enantiomerically pure 1-deoxy-A-ring-phosphine oxide can be prepared using a procedure by Wilson, S. R. et. al. in Bioorganic Chemistry, 1995, 23, 22-32, incorporated herein by reference. Kutner et al., Bioorganic Chemistry, 23:22-32 (1995), and Toh and Okamura, J. Org. Chem.
• the l-deoxy-19-nor-A-ring-phosphine oxide precursor can be prepared according to procedures described in Perlman et al., Tetrahedron Letters 32(52):7663-7666 (1991), incorporated herein by reference, as shown in the scheme shown below.
• compound a is esterified and the hydroxyl groups are protected to result in compound b (p-TsOH, MeOH, rt, 24 h, 92%; TBDMSCl, TEA, DMF, rt, 18 h, 70%).
• the thioimidazolide, c is prepared through reaction of b with 1,1 '-thiocarbonyl-diimidazole in methylene chloride (60 h, rt, 90%).
• Ester d is reduced to the alcohol, e (DIBAL-H, toluene, -78 °C, 2 h, 60%), which then undergoes oxidation to form cyclohexanone derivative f (saturated NaI0 4 in water, MeOH, 0 °C, 30 min, 78%), Reaction of f with ethyl (trimethylsilyl)acetate in the presence of lithium diisopropylamide (LDA) in THF (-78 °C, 2 h, 86%) produces the cyclohexyldiene ester, g.
• e DIBAL-H, toluene, -78 °C, 2 h, 60%
• f saturated NaI0 4 in water, MeOH, 0 °C, 30 min, 78%)
• Reaction of f with ethyl (trimethylsilyl)acetate in the presence of lithium diisopropylamide (LDA) in THF (-
• the method of synthesizing the ketone precursor is dependent on the exact composition of the ketone (e.g., D-ring and side chain compositions).
• Methods of preparing Vitamin D ketone precursors having saturated or unsaturated D-rings and variable side chains are known to one skilled in the art.
• U.S. Patent No. 7,101865 incorporated herein by reference, describes the synthesis of a ketone precursor of compound Ieii in Example 2.
• Example 1 Synthesis of 3-(2- ⁇ l-r4,4-Difluoro-l-methyl-4-(2-methyl-propane-2-sulfonyl)-butyll-7 a -methyl-3,3o 5,6,7 Ja-hexahydro-inden-4-ylidene
• Compound 2 was synthesized according to the procedure in Grzywacz et al. Archives of Biochemistry and Biophysics, 2007, 460, 274-284.
• a flame dried, three-neck 1000 mL round bottom flask was connected to an ozonolyzer at the first opening, and to a gas adaptor at the third opening with a tygon tube that was dipped in 1000 mL of a saturated solution of sodium bisulfite.
• the central opening of the round bottom flask was plugged with a glass stopper.
• the flask was then transferred to a 0 °C ice water bath and NaBH 4 (4.30 g, 113.45 mmol, 9.00 equiv.) was added in five, separate portions to minimize the exothermic effects.
• the reaction mixture was then stirred at 0 °C for 5 hours. TLC analysis determined that the intermediate material had been consumed.
• the clear reaction solution was then was acidified to a pH of 6 with 30% acetic acid in methanol.
• the crude material was concentrated under reduced pressure, taken up in CH 2 C1 2 (300 mL) and washed with saturated NaHC0 3 (4 x 200 mL), brine (2 x 200 mL) and water (2 x 200 mL).
• the clear reaction solution was quenched with ammonium chloride (10 mL).
• the reaction mixture was taken up in CH 2 CI 2 (20 mL), washed with brine (2 x 10 mL), water (2 x 10 mL), dried over MgS0 4 , and the solvent was reduced under vacuum.
• Purification was performed using a silica gel column with a solvent system of ethyl acetate/petroleum ether (3:7) to yield pure product 4 as an clear oil in (90 % yield, 0.25 g, 0.76 mmol).
• reaction mixture was stirred at -78 °C for an additional 30 minutes and then allowed to warm to room temperature. After 4 hours, TLC analysis indicated that starting material was consumed.
• the reaction was quenched with buffer (pH 7), extracted with ethyl acetate, washed with brine, dried over anhydrous MgS0 4 , concentrated in vacuo, and then purified by silica gel column chromatography (20% ethyl acetate/petroleum ether) to afford compound 13 (0.06 g, 0.14 mmol, 85% yield) as a colorless oil.
• a flame-dried 10-mL recovery flask equipped with a magnetic stir bar, a septum, and an argon balloon was charged with compound 16 (0.02 g, 0.05 mmol, 1.00 equiv.) dissolved in 1 mL of freshly distilled THF and cooled to -78 °C in a 2-propanol/dry ice bath.
• a solution of compound 17 was transferred dropwise into the flask containing the phosphine oxide anion at -78 °C via cannula over several minutes. After the addition was complete, a deep red color persisted and the mixture was allowed to stir at -78 °C for about 8 hours. During this time, the color of the solution was monitored.
• the reaction was quenched at -78 °C by addition of 5 mL of buffer (pH 7) and warmed to room temperature. The mixture was then rinsed into a separatory funnel with ethyl acetate and extracted with ethyl acetate (3 x 25 mL). The extracts were combined, washed with water (1 x 25 mL), washed with brine solution (1 x 25 mL), dried over Na 2 S0 4 , and filtered. The filtrate was concentrated in vacuo to give crude product.
• buffer pH 7
• the product was purified by column chromatography using an eluent of 50% ethyl acetate in hexanes in the presence of 1% triethylamine to afford a coupled product.
• This coupled product (0.02 g, 0.04 mmol, 91% yield) was charged into a 5 mL argon-purged polypropylene vial equipped with a magnetic stir bar and then dissolved in 2.5 mL of acetonitrile to result in a 0.02 M solution. This solution was stirred and HF was added (2.50 mmol, 8.6 mL) via syringe at room temperature. The mixture was stirred at room temperature in the dark for 1 hour. TLC showed the completion of the reaction.
• This reaction mixture was diluted with ether (25 mL), and a saturated solution of NaHC0 3 was added until the liberation of carbon dioxide ceased.
• the reaction mixture was then rinsed into a separatory funnel with ethyl acetate and extracted with ethyl acetate (5 x 25 mL). The extracts were combined, washed with water (1 x 25 mL), washed with brine solution (1 x 25 mL), dried over Na 2 SC"4, and filtered. The filtrate was concentrated in vacuo to give the crude product.
• the product was purified by column flash chromatography using 99% ethyl acetate in the presence of 1% triethylamine as the eluent to afford (0.01 g, 0.03 mmol, 70% yield) of Iaii as a single diastereomer.
• Enantiomerically pure 1-deoxy-phosphine oxide 1 was prepared according to Wilson, et al. Bioorganic Chemistry 1995, 23, 22-32) and compound 2 was prepared according to Posner et al. J. Med. Chem. 1999, 42, 3425-3435). These compounds were separately azeotropically dried with anhydrous benzene (3 x 4 mL) on a rotary evaporator and held under vacuum for 120 hours prior to use.
• the organic extracts were combined, dried over MgS0 4 , and filtered. The filtrate was concentrated in vacuo to give crude product.
• the product was purified by column chromatography (20 % ethyl acetate/hexanes with 1 % triethylamine) to affording the protected analog (24 mg, 0.022 mmol, 93 % yield).
• the protected analog was dissolved in acetonitrile (2 mL) and hydrofluoric acid (8.0 mL, 2.2 mmol, 49% aqueous solution) was added to the solution. The reaction was stirred for 4 hours, quenched with a saturated aqueous solution of sodium bicarbonate (10 mL), and stirred until gas ceased to evolve.
• ketone 4 (6.28 g, 18.55 mmol, 1:2 cis-trans mix at side chain olefin) was dissolved in anhydrous THF (50 mL) and slowly transferred via cannula to the reaction flask under argon pressure. The resulting mixture was stirred for another 45 minutes at -78 °C. TLC (in methylene chloride) indicated that the starting materials had been completely consumed. A saturated solution of ammonium chloride was added to the reaction flask and extraction was performed with 200 mL of ethyl acetate. The organic layer was washed with brine, water, dried over anhydrous Na 2 S0 4 , filtered, and concentrated. The crude product was purified by gradient column chromatography using 0 to 100% methylene chloride in hexane. The pure trans olefin fractions were concentrated to yield the protected product (65%) as a white solid.
• Enantiomerically pure 1-deoxy-phosphine oxide 1 was prepared according to Wilson, et al. Bioorganic Chemistry 1995, 23, 22-32) and compound 2 was prepared according to Posner et. al, J. Steroid Biochem. Mol. Biol., 2005, 89-90, 5-12. These compounds were separately azeotropically dried with anhydrous benzene (3 x 4 mL) on a rotary evaporator and held under vacuum (less than 0.5 mmHg) for 96 hours prior to use.
• Phosphine oxide 1 (63 mg, 0.14 mmol) was added to a flame dried 10 mL round bottom flask equipped with a magnetic stir bar and an argon balloon, and dissolved in 1 mL of freshly dried tetrahydrofuran (THF), distilled from sodium-benzophenone, and cooled -78 °C.
• THF tetrahydrofuran
• Enantiomerically pure 1-deoxy-phosphine oxide 19 was prepared according to Wilson, et al. Bioorganic Chemistry 1995, 23, 22-32). Compounds 19 and 18 were separately azeotropically dried with anhydrous benzene (3 x 3 mL) on a rotary evaporator and held under vacuum (less than 0.5 mmHg) for 120 hours prior to use.
• Compound 2 was synthesized according to the procedure in Grzywacz et al. Archives of Biochemistry and Biophysics, 2007, 460, 274-284. A flame dried three neck 1000 mL round bottom flask was connected at the first opening to an ozonolyzer and at the third opening to a gas adaptor with a tygon tube attached to it dipped in a saturated solution of sodium bisulfite (1000 mL).
• the clear reaction solution was purged with 0 2 for 1 hour and the solution turned to a light blue color.
• the flask was then transferred to a 0 °C ice water bath and NaBH 4 (4.30 g, 113.45 mmol, 9.00 equiv.) was added in five portions to minimize the exothermic effects.
• the reaction mixture was then stirred at 0 °C for 5 hours. TLC analysis indicated that the intermediate material had been consumed.
• the clear reaction solution was acidified to pH 6 with 30% acetic acic /MeOH.
• the clear reaction solution was quenched with ammonium chloride (10 mL).
• the reaction mixture was taken up in CH 2 CI 2 (20 mL), washed with brine (2 x 10 mL), water (2 x 10 mL), dried over MgS0 4 , and the solvent was reduced under vacuum.
• Purification was performed using a silica gel column with a solvent system of ethyl acetate/petroleum ether (3:7) to yield pure product 4 as an clear oil in (90 % yield, 0.25 g, 0.76 mmol).
• Compound 5 was synthesized according to the procedure in U.S. Patent Application No. US/2007/238702. A flame dried 50 mL single neck round bottom flask was charged with argon gas, triphenylphosphine (0.77 g, 2.95 mmol, 3.60 equiv.) and anhydrous CH 2 CI 2 (20 mL). The mixture was stirred at 0 °C and I 2 (0.83 g, 3.28 mmol, 4.00 equiv.) and imidazole (0.46 g, 6.72 mmol, 8.20 equiv.) were added to the stirring solution in one portion. The deep red mixture was stirred at 0 °C for 10 min.
• n-BuLi (2.10 mmol, 1.31 mL, 1.6 M solution in hexanes, 6.00 equiv.) dropwise over several minutes and a pale yellow color developed. This mixture was stirred at -78 °C for an additional 30 minutes.
• the reaction was quenched by addition of 6 mL of buffer solution (pH 7), and rinsed into a separatory funnel with ethyl acetate.
• the mixture was extracted with ethyl acetate (3 x 25 mL).
• the extracts were combined, washed with water (1 x 25 mL), washed with brine solution (1 x 25 mL), dried over Na 2 S0 4 , and filtered.
• the filtrate was concentrated in vacuo and the crude product was purified by flash column chromatography (1: 1 ethyl acetate/hexanes) to afford pure product (0.08 g, 0.13 mmol, 60% yield).
• the reaction was quenched at -78 °C by addition of 5 mL of buffer (pH 7) and allowed to warm to room temperature. The mixture was then rinsed into a separatory funnel with ethyl acetate and extracted with ethyl acetate (3 x 25 mL). The extracts were combined, washed with water (1 x 25 mL), washed with brine solution (1 x 25 mL), dried over Na 2 S0 4 , and filtered.
• buffer pH 7
• the mixture was stirred at room temperature in the dark for 5 hours. TLC indicated that completion of the reaction had occurred.
• the reaction mixture was diluted with ether (25 mL), and a saturated solution of NaHCC"3 was added until the liberation of carbon dioxide ceased.
• the reaction mixture was then rinsed into a separatory funnel with ethyl acetate, and extracted with ethyl acetate (5 x 25 mL). The extracts were combined, washed with water (1 x 25 mL), washed with brine solution (1 x 25 mL), dried over Na 2 S0 4 , and filtered.
• the radioactivity in 100 mL of the supernatant was measured using a scintillation counter.
• the control reactions contained either no VDR protein (background) or no competing ligand (maximum binding).
• the mean background binding was subtracted and the data divided by the mean maximum binding to yield B/B max values.
• the concentration necessary to displace 50% of [26,27-methyl- ⁇ H]- 1 ,25(OH)2D3 from VDR was calculated as the B50.
• Figure 1 is a graph showing the binding of Prodrug Ibii to the VDR compared to 1,25- dihydroxyvitamin D 3 .
• HPKla-ras cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum and plated at 1 x 10 5 cells/well in 24- well plates. The media was exchanged after 24 hours for DMEM containing 1% Bovine Serum Albumin. Cells were treated in duplicate with Ibii and control compounds at various concentrations (10 ⁇ 6 to 10 "9 M) for 6 or 7 hours. Extraction of cellular RNA was performed using Trizol ® (Invitrogen). Aliquots of RNA were reverse-transcribed to cDNA using random hexamers and Thermoscript reverse transcriptase according to the manufacturer's instructions (Invitrogen).
• Quantitative real-time PCR was performed using an ABI StepOnePlus system using the Taqman Universal PCR Master Mix. PCR reaction volumes of 20 were used with 50 cycles of amplification. Each cDNA sample was tested in duplicate using TaqMan® gene expression assays with the following ID numbers: human GAPDH (Hs99999905_ml), human CYP24 (Hs00167999_ml). The real-time PCR results were analyzed using the StepOne system software V2.1. Gene expression levels were calculated using the comparative CT method, and normalized to the GAPDH expression levels.
• Prodrug Ibii reproducibly and significantly induces transcription of CYP24 in HPKlaRas cells ( Figures 2a- 2d).
• Prodrug Ibii induced transcription of CYP24 about 100-fold at 100 nM and about 500-fold at 1 ⁇ , when compared to vehicle.
• Prodrug Ibii was about 1/100 as potent as its 1- hydroxylated analog.
• Lower concentrations of Prodrug Ibii did not significantly induce the transcription of CYP24A1.
• Prodrug Ibii does not bind to the VDR, transcription upregulation of CYP24 by Prodrug Ibii is likely occuring through its 1-hydroxylated analog, which is expected to result from hydroxylation of Prodrug Ibii at position- 1 by CYP27M.
• HPKla-ras cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum and plated at 1 x 10 5 cells/well in 24- well plates. The media was exchanged after 24 hours for DMEM containing 1% Bovine Serum Albumin. Cells were treated in duplicate with Ibii and control compounds at various concentrations (10 ⁇ 6 to 10 "9 M) for 6 or 7 hours. Extraction of cellular RNA was performed using Trizol ® (Invitrogen). Aliquots of RNA were reverse-transcribed to cDNA using random hexamers and Thermoscript reverse transcriptase according to the manufacturer's instructions (Invitrogen).
• DMEM Dulbecco's Modified Eagle's medium
• Quantitative real-time PCR was performed using an ABI StepOnePlus system using the Taqman Universal PCR Master Mix. PCR reaction volumes of 20 were used with 50 cycles of amplification. Each cDNA sample was tested in duplicate. The real-time PCR results were analyzed using the StepOne system software V2.1. Gene expression levels were calculated using the comparative CT method, and normalized to the GAPDH expression levels.
• Example 9 Comparison of Prodrug Ibii and its 1 -hydroxy active metabolite on PTH, calcium, and FGF23 levels in adenine-induced uremic rats
• the body weight of each animal is determined prior to dosing and every week thereafter.
• the animals are fed Labdiet 5002 (see htt :// www .1 abdiet . com/pdf/5002.pdf) .
• Day 0 is the first day of dosing.
• Approximately 1 mL of blood was collected from each animal at day 1 (24 hours), day 7, and day 14 to be used for the preparation of serum.
• Serum PTH, calcium, FGF23 and phosphate were measured using the blood samples.
• the animals were sacrificed and serum was collected.
• the kidneys, intestine, parathyroid glands, liver and bone from each animal were also collected. The levels of CYP24 and other genes of interest were measured.
• Ibii can lower circulating iPTH levels in the serum of adenine treated rats and that the potency of Ibii that decreases circulating iPTH levels is comparable to but lower than the 1 -hydroxy active form of Ibii administered directly ( Figure 4); (ii) Ibii is non- calcemic and displays a comparable or lesser effect on serum calcium compared to the 1 -hydroxy active form of Ibii administered directly ( Figure 5); (iii) Ibii does not increase serum FGF23 levels, and has less effect on serum FGF23 levels than the 1 -hydroxy active form of Ibii administered directly ( Figure 6).
• Vitamin D deficient Sprague Dawley rats were treated with Ibii 3x/week for five days by IV and oral routes, according to the Table below: Dose Volume No. of
• Vehicle Corn oil ethanol - 8.0 20
• Example 11 Effect on CYP24 expression in cells which do not substantially express CYP27B1
• PMA- stimulated U937 cells were maintained in RPMI media supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids and 10% Fetal Bovine Serum at optimal cell density (between 1x105 cells/mL and 2x106 cells/mL). Prior to treatment, cells were pelleted by centrifugation and resuspended to a concentration of 5 x 105 cells/mL. Cells were then treated with phorbol myristate acetate (PMA) at a final concentration of 20 ng/ml. To each well of a 24-well plate, 1 ml of cell suspension was added, which was incubated overnight at 37°C + 5% C02.
• PMA phorbol myristate acetate
• Figures 10-12 show relative CYP24 expression (compared to calcitriol) in PMA-U927 cells treated with calcitriol and Prodrugs Ibii, Iaii and Ilaii described herein at concentrations up to ⁇ ( Figure 10) and with calcitriol and Prodrugs Ibii, Ieii, Icii, and Idii described herein at concentrations up to lOOnm ( Figures 11 and 12).
• the y-axis on Figure 12 has been segmented to show detail at lower levels of relateive CYP24 expression for the Prodrugs. The results demonstrate that in cells which do not substantially express CYP27B1, the Prodrugs are inactive.
• Example 12 Transcriptional activity of Prodrugs Iaii and Ilaii in HPKA1 A-ras cells
• HPKla-ras cells exhibit a significantly higher expression of
• CYP27B1 mRNA compared to U937 cells. See Figure 9. CYP24al transcription activity in HPKla-ras cells was determined for prodrugs Iaii and Ilaii. As with the protocol described in Example 7, HPKla-ras cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum and plated at 1 x 10 5 cells/well in 24- well plates. Prior to treatment with compounds, cells were washed with PBS and replaced with DMEM containing 1% Bovine Serum Albumin. Cells were treated in duplicate with vehicle or compounds at various concentrations (10-6 to 10-9 M) for 6 hr at 37 °C. Media was removed and extraction of cellular RNA was performed using Trizol® (In vitro gen). Aliquots of RNA were reverse-transcribed to cDNA using oligo dT primers and Thermoscript reverse transcriptase according to the
• Figure 13 shows CYP24 transcription activity in HPKla-ras cells for calcitriol, Prodrug Iaii, and Prodrug Ilaii described herein, demonstrating that Prodrug Iaii, and Prodrug Ilaii (in cells which express CYP27B1) are active. These prodrugs are less potent than Prodrug Ibii in these cells because they display significant transcriptional activity only when used at ⁇ compared to at ⁇ for Prodrug Ibii.
• Prodrugs Iaii and Ilaii do not bind to the VDR, transcription upregulation of CYP24 by Prodrugs Iaii and Ilaii is likely occuring through its 1-hydroxylated activated form, which is expected to result from hydroxylation of Prodrug Iaii and Ilaii at their respective position- 1 by CYP27M.
• Example 12 Transcriptional activity and CYP24 inhibitory activity of Prodrugs Icii, Idii, and leii in HPKlA-ros cells
• Transcriptional activity of calcitriol and Prodrugs was determined by CYP24al relative transcription in HPKla-ras cells treated for 6 hours with calcitriol, combinations of calcitriol with Prodrugs Icii, Idii, and leii, and combinations of calcitriol with 1-hydroxylated active forms of Prodrugs Icii, Idii, and leii which are known CYP24 inhibitors. Results are shown in Figures 14 and 15.
• Figures 14 and 15 show CYP24 transcription activity in HPKla-ras cells for calcitriol alone and calcitriol together with each of Prodrug Icii, Prodrug leii, Prodrug Idii, and the 1 -hydroxy activated forms thereof at ⁇ concentrations of the analogs and O. lnM calcitriol ( Figure 14) and InM calcitriol ( Figure 15).
• the results demonstrate the CYP24 inhibitory effect by administration of all test compounds in these CYP27B1 -expressing cells.
• Increased CYP24al transcription activity when the test compounds are administered together with calcitriol is a response to the increased halflife of calcitriol achieved via CYP24 inhibition by the test compounds.
• compositions are described as including components or materials, it is contemplated that the compositions can also consist essentially of, or consist of, any combination of the recited components or materials, unless described otherwise.
• the invention illustratively disclosed herein suitably may be practiced in the absence of any element or step which is not specifically disclosed herein.

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